Synthesis, in vitro and structural aspects of benzothiazole analogs as anti-oxidants and potential neuroprotective agents

Article abstract of DOI:10.1016/j.etap.2020.103415

Catalase, an important antioxidant enzyme, is known to have a neuroprotective role against neurodegenerative disorder. Earlier study has focussed on benzothiazole-triazole hybrid molecules that are larger in size and molecular weight and inhibit the amyloid β (Aβ)-catalase interaction thus aid in neuroprotection. Here we have synthesized the novel benzothiazole molecules with low molecular weight using One-pot methodology and assayed the neuroprotective effects of the synthesized compounds in the U87 MG cell line under H₂O₂ induced stressed condition and compared with other cell lines such as breast cancer (MCF-7) and macrophage (RAW-264.7) using cell viability assay. These analogs were found to enhance the neuronal cell viability and protect neuronal cells from the ROS mediated neuronal damage induced by H₂O₂. Furthermore, compounds 6a, 6b, 6c, 6d, and 7a modulate catalase and enhanced the catalase activity up to 90 percent during the H₂O₂ exposure in the U87MG cell line. These analogs (6a, 6b, 6c and 6d) have exhibited strong binding energies of -7.39, -7.52, -6.5 and -7.1 as observed by molecular modeling studies using AutoDockTool-1.5.6. Lig Plot + program using potent analogs 6b and 6c and catalase enzyme indicated the presence of hydrophobic interactions in the catalytic site of catalase enzyme. Furthermore, a simulation study was conducted between ligand and catalase protein by DESMOND software that further strengthens these ligand and enzyme interactions. In silico ADMET study was conducted by the Swiss ADME program revealed the drug-likeliness of these analogs. The present study has identified benzothiazole analogs such as 6b, 6c and 6d have potential catalase modulating activity and is comparable with that of known drug Valproic acid, thus help in neuroprotection. This study can be further taken up for the in vivo animal model study for the possible therapy.

Full text of DOI:10.1016/j.etap.2020.103415

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Synthesis, in vitro and structural aspects of benzothiazole analogs as
anti-oxidants and potential neuroprotective agents
M. Janaki Ramaiah, Divyapriya Karthikeyan, Sivagami Mathavan,
Rajesh B.R.D. Yamajala, Srimathy Ramachandran, P.Jaya Vasavi,
Nuthakki Venkata Chandana
PII:
S1382-6689(20)30091-0
DOI:
https://doi.org/10.1016/j.etap.2020.103415
ENVTOX 103415
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Environmental Toxicology and Pharmacology
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19 May 2020
Please cite this article as: Ramaiah MJ, Karthikeyan D, Mathavan S, Yamajala RBRD,
Ramachandran S, Vasavi PJaya, Chandana NV, Synthesis, in vitro and structural aspects of
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Synthesis, in vitro and structural aspects of benzothiazole analogs as anti-oxidants and
potential neuroprotective agents
M. Janaki Ramaiaha*# Divyapriya Karthikeyana# Sivagami Mathavanb Rajesh B.R.D.
Yamajalab* Srimathy Ramachandrana, P. Jaya Vasavia, Nuthakki Venkata Chandanaa
aFunctional genomics and Disease Biology Laboratory, School of Chemical and
Biotechnology, SASTRA Deemed University, Thanjavur-613401, Tamil Nadu, India,
bOrganic Synthesis and catalysis Laboratory, School of Chemical and Biotechnology,
SASTRA Deemed University, Thanjavur-613401, Tamil Nadu, India
* Corresponding authors (Main): M. Janaki Ramaiah, E-mail: janakiramaiah@scbt.sastra.edu,
# Equal first authorship
phone No.: +91-8870948256
Graphical abstract

Catalase activity
Neuroprotection

Highlights
 Benzothiazole analogs modulate catalase activity and cause neuroprotection.
 Benzothiazole analogs bind to catalase enzyme and have shown drug-likeliness.
 Neuroprotection in H2O2 exposed U-87 MG cells occurs Via inhibition of ROS levels.
Abstract
Catalase, an important antioxidant enzyme, is known to have a neuroprotective role against
neurodegenerative disorder. Earlier study has focussed on benzothiazole-triazole hybrid
molecules that are larger in size and molecular weight and inhibit the amyloid β (Aβ)-catalase
interaction thus aid in neuroprotection. Here we have synthesized the novel benzothiazole
molecules with low molecular weight using One-pot methodology and assayed the
neuroprotective effects of the synthesized compounds in the U87 MG cell line under H2O2
induced stressed condition and compared with other cell lines such as breast cancer (MCF-7)
and macrophage (RAW-264.7) using cell viability assay. These analogs were found to enhance
the neuronal cell viability and protect neuronal cells from the ROS mediated neuronal damage
induced by H2O2. Furthermore, compounds 6a, 6b, 6c, 6d, and 7a modulate catalase and
enhanced the catalase activity up to 90% during the H2O2 exposure in the U87MG cell line.
These analogs (6a, 6b, 6c and 6d) have exhibited strong binding energies of -7.39, -7.52, -6.5
and -7.1 as observed by molecular modeling studies using AutoDockTool-1.5.6. Lig Plot +
program using potent analogs 6b and 6c and catalase enzyme indicated the presence of
hydrophobic interactions in the catalytic site of catalase enzyme. Furthermore, a simulation
study was conducted between ligand and catalase protein by DESMOND software that further
strengthens these ligand and enzyme interactions. In silico ADMET study was conducted by

the Swiss ADME program revealed the drug-likeliness of these analogs. The present study has
identified benzothiazole analogs such as 6b, 6c and 6d have potential catalase modulating
activity and is comparable with that of known drug Valproic acid, thus help in neuroprotection.
This study can be further taken up for the In vivo animal model study for the possible therapy.
KEYWORDS
Alzheimer’s disease; neuroprotection; catalase; benzothiazole; hydrogen peroxide
1. Introduction
Alzheimer’s disease (AD) as well as other neuronal disorders such as Parkinson’s, needs urgent
medical attention. The treatment options in AD include small molecules that can block
amyloid-beta (Aβ) interactions, reduction of the caspase protein expression, inhibition of the
reactive oxygen species (ROS) dependent enzyme activity (Longo & Massa, 2004). ROS
enhances the β-Amyloid protein level which in turn causes direct injury to the central nervous
system (CNS) and induce neuronal cell death (Guo, Sun, Chen, & Zhang, 2013). In order to
protect from the ill effects of ROS, cells can employ various antioxidant enzymes such as
catalase (CAT), superoxide dismutase (SOD), and non-enzymatic antioxidant factors such as
All-trans retinol 2, ascorbic acid, α-tocopherol, α-carotene, and glutathione, etc. (Birben,
Sahiner, Sackesen, Erzurum, & Kalayci, 2012).
Antioxidants are compounds inhibit the formation of peroxides or quench the peroxide or
scavenge the species that can generate peroxides. The production of antioxidant enzymes takes
place in glial cells and helps in the protection of the neuronal cells from oxidative stress
(Wilson, 1997; Peuchen et al., 1997). The high amount of ROS was generated when neuronal
cells were exposed to radiation or oxidative stress in SH-SY5Y neuronal cells. But the amount
of ROS was drastically reduced when SH-SY5Y cells co-cultured with U-87 MG glial cells.

This indicates that co-culturing of SH-SY5Y with U-87 MG glial cells can enhance the
mitochondrial membrane potential by upregulating the production of antioxidant enzymes
superoxide dismutase and antioxidant glutathione in SH-SY5Y cells (Saeed et al., 2015).
Catalase (CAT) is a common enzyme found in nearly all living organisms that are exposed to
oxygen, comprising of four protein subunits and four iron ions and has one of the highest
turnover numbers of all the enzymes (Nisticò et al., 1992). The catalase enzyme binds with
high affinity with the amyloid β plaques (Aβ plaques) and thereby making the catalase enzyme
inactive (Chilumuri, Odell, & Milton, 2013). H2O2-induced oxidative stress and cellular injury
is a well-established and useful model for studying The anti-oxidative, and cytoprotective
nature of compounds against neurodegenerative diseases such as Alzheimer’s disease (AD)
and Parkinson’s disease (PD). Studies have indicated that Xanthohumol (Xn) analogs and
Dithiolethiones were found to have neuroprotection nature against H2O2 induced reactive
oxygens species and apoptosis in PC12 cells (a neuronlike rat pheochromocytoma cell line)
and N2a cells (mouse neuroblastoma N2a cells) respectively (Bai et al., 2019; Song et al.,
2020). Thus, we have taken H2O as a neurotoxin in our experiments to mimic the condition of
AD-like cell death. Studies have indicated that inactivated catalase enzyme cannot breakdown
hydrogen peroxide (H2O2) which is generated in response to Aβ (Zhang et al., 1996).
Non-conventional compounds are exhibiting promising beneficial effects on Alzheimer´s
disease (AD). These include methylene blue, natural products, organic phosphorous
compounds, and Chinese medicine (de Castro et al., 2019). The symptomatic AD can be treated
up to one year with acetylcholinesterase (AChE) inhibitors such as tacrine, donepezil,
rivastigmine, and galanthamine. Thus, repositioning of available drugs can be considered as an
alternative strategy (de Castro et al., 2018). In addition metal-based drugs and several chelating
agents can also help in decreasing the symptomatic effects of several neurological disorders (
Sales et al., 2019). Interestingly, benzothiazole compounds such as phenyl benzothiazoles and

Manganese-benzothiazole can also be used as spectroscopic and MRI probes respectively
(Pereira, Goncalves, Mancini, Kuca, and Ramalho, 2019; Rosa, Silva Giacoppo, de Cunho,
Fernandes, and Horvath, 2019).
Natural compounds are found to possesses anti-oxidant compounds and regulate anti-oxidant
genes by various mechanisms such as direct radical scavenging and activation of antioxidant
enzymes (Fraunberger, Scola, Laliberté, Duong, & Andreazza, 2016). Benzothiazole aniline
(BTA) has been reported to inhibit beta-amyloid protein aggregations. A tetra (ethylene glycol)
derivative of benzothiazole induces the spinogenesis via a Ras-dependent pathway (Megill et
al., 2013). Compounds that are heterocyclic (aromatic) in nature have a good therapeutic role
in the field of drug discovery (Goyal, Kaur, & Goyal, 2018; Jouha et al. 2017; Sucheta, Tahlan,
& Verma, 2017; Hiremathad, & Piemontese, 2017). Benzothiazole (BT) and its derivatives are
found to have heterocyclic structures (Hutchinson, Jennings, Vishnuvajjala, Westwell, &
Stevens, 2002; Hutchinson et al., 2002) 6-trifluoromethoxy-2-benzothiazolamine, was able to
block excitatory amino acid-mediated neurotransmission (Evindar, & Batey, 2006; Sharma,
Sinhmar, Sharma, Rajak, & Pathak, 2013; Malik, Manvi, Nanjwade, Singh, & Purohit, 2010).
Compounds such as benzothiazole aniline tetra (ethylene glycol) (BTA-EG4) and BTA-EG6
have been known to specifically disrupt the interaction of catalase with Aβ (1-42) by binding
to multiple regions and eventually inhibiting the binding of catalase and also has the ability to
penetrate the blood-brain barrier. These compounds were also found to work better in the AD
animal model (Megill et al., 2013). Tacrine-allyl/propargylcysteine–benzothiazole trihybrids
cause the anti-ROS activity, acetylcholinesterase enzyme activity inhibition, and 5-fold
decrease in amyloid-beta (Aβ) peptide production (Hiremathad et al., 2016). The methods for
selecting docking studies are chemi-informatics techniques, AutoDock and QM calculations
(De Lima. Francisco, da Cunha, Radic, Taylor, and Franco, et al.2017). Also, AutoDock
version 3.0.5 was used for the prediction of binding conformations of flexible ligands.

Earlier studies by Kumbhare et al., 2012 have studied the anti-cancer activity of benzothiazoles
on breast and other cancer cell lines. Here we have synthesized various analogs of
benzimidazoles, benzothiazoles, and benzoxazole compounds. All of the benzimidazole,
benzothiazole, and benzoxazole derivatives (5a-5c, 6a-6d, 7a) were produced in pure form
without any column chromatography, characterized by 1H NMR spectra, and the known
compounds were confirmed by comparing their spectral data and melting points with those
reported in the literature (Xue, & Long, 2014; Yang et al. 2014; Ye et al. 2017; Gopalaiah, &
Chandrudu, 2015). The 1H NMR data were provided in the supplementary figures S1 and
Supplementary file S2 Here we have studied the possible role of these analogs on neuronal
protection by reducing ROS level and enhancing the catalase activity.
2. Materials and Methods
2.1. Chemistry
Heterocyclic compounds are part of many natural products and are vital in the pharmaceutical
industry, due to the presence of the structure of these heterocycles in different drugs consumed
worldwide. Among them, benzimidazoles, benzothiazoles, and benzoxazoles are of great
interest, since they are present as the core motif in many of the biologically active molecules.
A wide variety of methods have been developed for the synthesis of benzimidazoles,
benzothiazoles, and benzoxazoles. Usually, these compounds are prepared through the
condensation of 1, 2-diamines /2-aminothiols / 2-aminophenols with aldehydes or acyl
chlorides. These are also prepared by the condensation of 1, 2-diamines/2-aminothiols/2-
aminophenols with -phenyl glyoxylic acid.
As part of our continuing efforts to develop green synthetic methodologies, we report herein
the synthesis of these heterocycles by an efficient protocol under eco-friendly conditions. This

method involves the use of graphene oxide as a catalyst and water as a green solvent under
room temperature and heating conditions (Figure 1).
The condensation reaction of o-phenylenediamine / 2-aminothiols (1 mmol) with -phenyl
glyoxylic acid (1 mmol) was carried out in the presence of GO catalyst in the water at room
temperature. The catalyst loading of 5, 10, 20 mg was tested. Reactions with 10 mg and 20 mg
catalyst gave similar yields in 1h. In the absence of catalysts, the reaction did not proceed at all
even after a long reaction time. In the case of benzothiazoles, the condensation of 2-
aminophenols with -phenyl glyoxylic acid resulted in the product formation only in heating
condition and the reaction took 10h to get completion. All of the benzimidazole, benzothiazole,
and benzoxazole derivatives were produced in pure form without any column chromatography,
characterized by 1H NMR spectra, and the known compounds were confirmed by comparing
their spectral data and melting points with those reported in the literature. All reagents and
solvents were of analysis or synthesis grade and were used without further purification unless
otherwise indicated. 1H NMR was recorded on a Bruker AVANCE III 400 MHz NMR at 400
or on a Varian 300 instrument at 300 in CDCl3 or DMSO-d6. Chemical shift is reported in
ppm with the solvent residual peak as reference; CDCl3 (dH 7.26), DMSO-d6 (dH 2.50) and
CD3OD (dH 4.87). The reactions were monitored by thin-layer chromatography (TLC), on
silica plated (Silica gel 60 F254, E. Merck) aluminum sheets, detecting spots by UV (254 and
365 nm). All the analogs used in the study were represented in Figure 2 with the corresponding
codes.
2.2. Cell lines
Human malignant glioma (U-87 MG), human breast cancer cell line (MCF-7), human neuronal
cell line (SH-SY5Y) and mouse macrophage cell line (RAW 264.7) were obtained from NCCS,
INDIA. All the cell lines were cultured in DMEM supplemented with 10% fetal bovine serum

(FBS), 4 mM glutamine, 100 units/ml penicillin, and 100µg /ml streptomycin. All the cell lines
were maintained at 37 °C in the CO2 incubator.
2.3. Effects of compounds on neuronal cell viability
In order to choose the concentration of neurotoxin that is needed to produce adequate
neurotoxicity, various concentrations of H2O2 (425 μM - 9450 µM) were tested on U-87 MG
cell line. 1 x 104 cells / well of 96 well plate was incubated for 12 h with the H2O2 (i.e.
neurotoxin) and cell viability was determined by using MTT dye. Here the compounds were
incubated 24h before H2O2 addition. The 50 μL MTT solution (5 mg/mL in phosphate-buffered
saline (PBS)) was added to each well, the plates were incubated at 37°C for 4 h, the culture
medium containing MTT was removed and 100 μL dimethyl sulfoxide (DMSO) was added to
each well to dissolve the formazan. The absorbance was measured at 570 nm using a Multiplate
reader. Results were expressed as a percentage of the untreated cells.
Cell Viability % = (OD570 nm treated / OD570 nm untreated) x 100%
2.4. Combinatorial effect of benzothiazole analogs and H2O2 toxicity
Stock solutions of benzothiazole analogs were prepared in DMSO prior to dilution to the
required concentration in the culture medium. Synthesized compounds stock solutions were
dissolved in DMSO and varying concentrations ranging from 1μM to 64 μM were employed.
To produce oxidative stress, H2O2 of varying concentrations was freshly prepared from 30%
(w/v) H2O2 stock solution before each experiment. To study the neuroprotective effect of
benzothiazole analogs against H2O2 toxicity, 1 x 104 cells were seeded in a 96-well flat-bottom
plate and allowed to adhere for 24 h at 37 C in the CO2 incubator. After 24 h of incubation,
the culture medium was replaced with a fresh medium. For testing the effects of these
compounds, the cells were pre-treated with test compounds for 12 h before treatment with
H2O2. For testing the effects of H2O2 toxicity, cells were incubated for 4 h before the
determination of cell viability. Then MTT assay was performed.

2.5. Intracellular ROS detection using DCFH-DA assay
The degree of ROS generation in cells was measured using fluorescence assay with 2’, 7’-
dichlorodihydrofluorescein diacetates (H2DCFDA, Sigma). DCFH-DA is the most widely used
probe for detecting intracellular H2O2 and oxidative stress. After diffusion into the cell,
DCFDA / H2DCFDA / DCFH-DA is deacetylated by cellular esterases to a non-fluorescent
compound, which is later oxidized by ROS into 2’, 7’ –dichlorofluorescein (DCF). Briefly after
treatment, the cells were scraped and washed in PBS and the pellet re-suspended in 200 μl of
PBS loaded with 10 μM H2DCFH-DA at 37°C for 30 min and then the cells were washed twice
with PBS and centrifuged at 1000 rpm for 5 min. Finally, the pellet was re-suspended in 200
μl PBS and the fluorescence intensity of DCF was measured with Fluorescence Spectroscopy.
The measurement of ROS is calculated Excitation (Ex) / Emission (Em) at 470nm / 545nm.
2.6. Catalase activity assay
Catalase activity was measured using the manufacturer’s instructions (Catalase, ab83464)
(Abcam, USA). Here, 2 × 106 was treated with the potential compounds (6a-6d, 7a) for 24 h.
Then lysates were isolated using cold lysis buffer, and the samples were subjected to catalase
activity assay. Here the compound treated cell lysates were allowed to interact with 12 μL of
H2O2 (1 mM solution) and followed by the addition of stop solution (25ºC for 5 minutes), to
stop the reaction between catalase present in the lysate and H2O2 (represented as -H).
Similarly, the same amount of lysate was subjected to inhibition with stop solution (25ºC for 5
minutes) followed by the addition of H2O2 and is represented as +H. A master mix of
developer consists of 46 μL of assay buffer, 2 μL of OxiRed Probe, and 2 μL of HRP Solution
was prepared and added into each well. The plates were incubated at 25ºC for 10 min. The
catalase present in the sample reacts with hydrogen peroxide (H2O2) to produce water and
oxygen. The unconverted H2O2 reacts with the probe to produce a product that can be measured

calorimetrically at OD570 nm using a microplate reader. Therefore, the catalase activity present
in the sample is inversely proportional to the signal obtained.
2.7. In Silico docking studies
Docking studies will be employed at different stages of drug discovery to predict a ligand-
receptor interaction and also to rank the compounds based on their binding energies. The three-
dimensional structure of human erythrocyte catalase (ID: 1QQW), were obtained from the
protein data bank. All the non-protein molecules were removed. AutoDock 4.0 (Morris et al.,
2009) software was used for docking study. AutoDock tools 1.5.6 (Huey, Morris, Olson, &
Goodsell, 2007) were used to prepare, run, and analyze the docking simulations. Kollman
united atom charges and polar hydrogen was added to the protein. The grid box size was set at
65X65X65 Å (x, y, and z) centered on the protein with the spacing between grid points at 0.375
Å. Docking results were analyzed and visualized by AutoDock tools and PyMOL (DeLano,
2002).
3. Results and Discussion
3.1. Effect of hydrogen peroxide (H2O2) on the viability of neuroblastoma and
glioblastoma cancer cell lines (SH-SY5Y and U-87 MG)
Hydrogen peroxide (H2O2) is considered as one of the major neurotoxic compounds that
produce high amounts of free oxide radicals in the cells, especially in the neuronal cells. It is
associated with the genesis of neurodegenerative diseases such as Alzheimer’s, Multiple
sclerosis and Parkinson’s disease (Bayani, 2009). To understand the neurotoxic effect of H2O2
on cells that are of glial origin, we have taken U-87 MG cells. Various concentrations (425-
9500 μM) of H2O2 were added to the U-87 MG cells for a period of 12h. The concentration of
425 μM resulted in a 20% reduction in cell viability, whereas 950 μM resulted in 40% reduction
in cell viability. Thus, we considered 950 μM as an optimal concentration for inducing
oxidative stress (hypoxia condition) for further experiments on neuroprotective studies of

Benzothiazole analogs. Surprisingly, above 4750 μM concentration, the cell started a protective
mechanism against H2O2 to a certain extent which clearly indicates the cell adaption to H2O2
mediated toxic or hypoxic effects (Figure 3a).
A similar experiment was conducted in neuroblastoma (SH-SY5Y) cells with H2O2 ranging
from (0-256 μM). Here we observed 2-8 μM 30% reduction in cell viability whereas 40% cell
reduction was observed at concentrations ranging from 16-256 μM (Figure 3b). It is evident
from the studies of (Fan et al., 2012) that glial cells i.e. U-87 MG cells protect neuronal cells
from indirect effects of radiation and oxidative stress in the brain. Maybe due to these reasons,
we need to use higher concentration (950 μM) to understand H2O2 mediated cell death in U-87
MG (Saeed et al., 2015).
3.2. Neuroprotective effect of benzothiazole analogs on H2O2 induced neurotoxicity in U-
87 MG
The neuroprotective effect of benzothiazole analogs (5b, 5c, 6a-6d, and 7a) was studied on U-
87 MG cell lines exposed to oxidative stress (H2O2). Here 2 μM concentration of compound
produced effective cytotoxicity up to 30% whereas 4 μM concentration of compound had a
drastic change in the cell viability. The cells were incubated with benzothiazole analogs for 12
h and were followed by addition of H2O2 (950 μM as deduced from the above studies) for 4 h.
Surprisingly the combination of both compound and H2O2 did not cause any considerable
reduction in the cell death probably due to compounds may directly chelate H2O2 and nullify
its effects or compounds might modulate the catalase activity (Figure 4a). The standard
molecule such as Valproic acid (VA) also has shown neuroprotection when cells were treated
with H2O2. Similar experiments were conducted using Breast cancer cell line (MCF-7) (Figure
4b) and normal mouse macrophage cell line (RAW 264.7) (Figure 4c) and a similar trend was
observed.
3.3. Effect of benzothiazole analogs on H2O2 induced ROS in glioblastoma cells

ROS is modulated by antioxidant enzymes such as superoxide dismutase (SOD) and catalase
(Min et al., 2015). H2O2 generates highly reactive oxygen species (ROS) and cause apoptosis
of neuronal cells (Lee et al., 2008; Wang et al., 2008), leading to various neurodegenerative
diseases such as Alzheimer’s disease (Zhu, Su, Wang, Smith, & Perry, 2007), Parkinson’s
disease (Thomas, 2009), Huntington’s disease (Chen, 2011) and amyotrophic lateral sclerosis
(Barber, Mead, & Shaw, 2006). So, to understand the neuroprotective effect of benzothiazoles,
we have treated U-87 MG cells with compound alone (2 μM) as well as the combination of
both compound and H2O2 (950 μM as deduced from the cell viability experiment). The amount
of ROS generated was measured. Compounds induced ROS generation in U-87 MG cells, but
when compounds encountered H2O2 they have decreased the amounts of ROS in U-87 MG
cells, this shows that compounds binding to H2O2 and inhibiting the H2O2 mediated ROS
generation (Figure 5).
3.4. Effect of benzothiazole analogs on catalase activity
Catalase is the ubiquitous enzyme that prevents cell oxidative damage (Alfonso-Prieto,
Biarnés, Vidossich, & Rovira, 2009). Catalase degrades the H2O2 to give H2O and O2 with high
efficiency (George, 1947). Catalase binds to Aβ with high affinity and catalase-Aβ interaction
in Alzheimer's disease deactivates catalase enzyme function that leads to the high amounts of
H2O2 and high cytotoxicity in neuronal cells. A small molecule inhibitor of catalase amyloid
interaction protects the neuronal cells from the evil effects of amyloid induced cytotoxicity
(Habib, Lee, & Yang, 2010). Thus, we were interested in understanding the catalase activity in
cells treated with compounds and H2O2. Lysates were allowed to interact with H2O2 (12 mM)
and measured the changes in optical density (OD) values at 570 nm. The less OD indicates
more catalytic activity. Compounds 6b, 6c, and 6d have exhibited 88%, 90%, and 85% of
catalase activity respectively. The positive control has shown 94% of catalase activity (Figure

6). The catalytic activity of these compounds was found to be almost equal to the standard
neuroprotective compounds such as VA. This indicates that these compounds have promising
neuroprotective effects.
3.5. In silico docking studies with Human Erythrocyte Catalase (HEC)
We observed an enhanced catalase activity in cells treated with a combination of H2O2 and
benzothiazole analogs. This may be due to 2 possibilities. Either compound might chelate
H2O2 action and also enhance the catalase activity during the H2O2 treatment in U-87 MG cells.
Thus to understand the interaction between benzothiazole analogs and catalase was conducted
using template 3D structure PDB ID: 1QQW retrieved from the RCSB PDB database. The
incomplete, modified, missing, and cris-cross residues were studied by using AutoDockTools-
1.5.6. 3D docking interactions for benzothiazole analogs and VA inside the active site cavity
of the Human Erythrocyte Catalase (HEC) was performed. The compounds such as 5b, 5c, 6a,
6d, 7a, have exhibited efficient binding ability with catalase as shown in Figure 7. From the
molecular modeling studies, it was found compounds have exhibited higher binding energy
when compared with the standard neuroprotective agent [HDAC inhibitor (i.e.) Valproic acid
(VA)]. Among all, 5b (△G = -7.88), 5c (△G = -7.68), 6a (△G = -7.69), 6d (△G=-7.1), 7a (△=-
7.65) whereas VA has exhibited binding energy of -4.82 (Table 1). The above data indicates
these potential compounds need to be further studied in Alzheimer’s model [Tg2576 mouse
model – Aβ model or APPK670/671L].
Furthermore, 2-D representation of the ligands 6b, 6c in the active site of catalase generated
using LigPlot+. Ligplot + program designs for representing ligand-protein, ligand nucleic acid
interactions and generates hydrophobic contacts as represented by red arcs with radiating lines.
We could observe that the major interaction force for the ligands in hydrophobic. The chlorine
and methyl groups of 6b and 6c also contribute for the hydrophobic interactions with active

site residues, Val 73 and Val74 (Figure 8). Also, simulation study was conducted for the
protein ligand complex using DESMOND simulation package for duration of 1 ns. The RMSD
profile of the ligands does not deviate much and indicate a stable interaction of ligands with
the catalase docking during simulations (Supplementary figure 3 or Figure S3). Furthermore,
the druglikeliness of these compounds was tested using in silico ADMET analysis using
SwissADME prediction program. All these compounds have exhibited moderate solubility and
followed Lipinski rule (Table-2).
4. Conclusions
Drug development has been a principal driving force in the rapid maturation of the field of
medicinal chemistry during the past several decades (Khan, Ibrar, Abbas, & Saeed, 2014).
Here, we have synthesized a broad range of novel and efficient Benzothiazole analogs to inhibit
the Catalase-amyloid β interaction and thereby activating the efficiency of the antioxidant
enzyme, catalase. This article is also focused on reducing the loss or death of neuronal cells.
The structure-activity relationship of the reported parent compound (i.e.) azole, revealed that
the choice of a suitable substitution pattern on the basic skeleton plays a key role in regulating
the biological potential of the synthesized compounds.
Antioxidants are therapeutic drugs against neuronal loss, as they have the capability to combat
by inhibiting and chelating free radicals. Interestingly, antioxidants have a wide scope to
sequester metal ions involved in neuronal plaque formation and prevent oxidative stress (ROS).
In this context, antioxidant therapy is an urgent requirement for scavenging free radicals and
for protection against the neuronal degeneration in post-oxidative stress (Poulsen, Prieme, &
-
Loft, 1998). O2 may cause damage to neuronal cells ultimately resulting in loss of cell structure
and function of the brain and this is one of the main characteristics of neurological disorder. In
AD, the neuronal cells are filled with hyperphosphorylated Tau tangles and insoluble Aβ

proteins such as Aβ (1-42) resulting in the loss of neuronal function. Apart from Aβ (1-42), the
other most important problems include loss of function of anti-oxidant enzymes such as
catalase and SOD. Thus, modulating these enzymes will help in neuroprotection.
Benzothiazoles were found to be anti-cancerous, anti-diabetic, anti-leishmanial, etc. Chilumuri
et.al has found that the compound BTA-EG4 blocks the binding interaction of catalase to Aβ,
thereby helping in the neuroprotection. The main drawback of the compounds was found to
have higher molecular weight, which might hinder the passage of compounds through the
blood-brain barrier (BBB). Thus, we tried to synthesis low molecular weight compounds
namely benzothiazole analogs with various substitutions such as methyl, methoxy, phenyl,
chloro, and bromo in the benzothiazole skeleton (i.e. parent compound).
We initially initiated our work by creating a ROS condition using various concentrations of
H2O2, using SH-SY5Y and U-87 MG cell lines. To our surprise, the SH-SY5Y cells responded
at a concentration of 256μM whereas U-87 MG cells responded at a higher concentration of
1900 μM, this may be due to the glial cells have the potential to tolerate a high amount of stress
such as H2O2 and the function of glial cells is to protect the neuronal cells. Keeping this as an
optimal concentration, we tested the neuroprotective effect of the benzothiazole analogs and
found that the compounds 6a, 6b, and 6c are resistant to H2O2 mediated hypoxia and thus helps
in cell viability.
Some of the anti-inflammatory and antioxidant drugs such as coumarin derivative drugs
possess the capability to attenuate elevated levels of ROS in cells (Kontogiorgis, Xu,
Hadjipavlou-Litina, & Luo, 2007). Compounds that nullify the H2O2 mediated effects can have
the inhibitor activity on ROS. So, we tested the effect of the compound on ROS in U-87 MG
cells, in which we observed 6a, 6b, and 6c has caused the reduction in ROS level as indicated
by DCF-DA fluorometric assay.

Further, the activity of catalase was measured using Catalase Activity Colorimetric Assay Kit,
as catalase is the most important key enzyme that helps in cell viability during hypoxia, stress
at peroxisomes and mitochondria junction, in which the benzothiazole compounds 6a, 6b, and
6c showed high catalytic activity when compared with other analogs. Thus, these analogs have
found to have potent neuroprotective activity. To strengthen our results, we performed a
molecular docking study to predict the binding-conformation of small-molecule ligands to the
appropriate target protein, as these computational methodologies have become a crucial
component of many drug discovery events (Kitchen, Decornez, Furr, & Bajorath, 2004). The
molecular docking was performed with the help of AutoDockTools-1.5.6, in which the
compounds 6a, 6b, and 6c showed high binding energy. The △G values, simulations obtained
from desmond software, lig plot+ analysis revealed that the correlation between binding
energies as well as interaction of 6b, 6c compounds with catalase. Root mean square deviation
(RMSD) profile revealed the ligands does not deviate much and binding is specific between
6b, 6c with catalase. These theoretical findings as well as experimental catalase activity
indicated that benzothiazole based compounds especially 6b and 6c are effective in modulating
catalase. Therefore, the compounds need to be tested in animal models for further confirmation
as a potent drug for the treatment of Alzheimer’s disease by protecting the neuronal cells from
oxidative damage.
Statistical analysis
Statistical analysis was performed using the graph-pad software to evaluate the significant
difference between the control and treated samples. All variables were tested in three
independent experiments. The results were reported as mean ± SD. * represents p-value < 0.05,
** represents p-value < 0.01 and *** represents p-value < 0.001

Acknowledgments
Thanks to DST –SERB (EMR/2017/001201) for financial support and thanks to SASTRA
management for the infrastructure and constant encouragement.
Disclosure of statement
No potential conflict of interest was reported by the authors
ORCID ID: https://orcid.org/0000-0003-1814-2819 Janaki Ramaiah
http://orcid.org/0000-0002-2446-9503 Divyapriya Karthikeyan
https://orcid.org/0000-0002-2914-3617 Rajesh YBRD
NO ORCID for Srimathy Ramachandran,
NO ORCID P. Jaya vasavi
NO ORCID for Nuthakki Chandana
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Figure 1. Schematic representation of synthesis procedure of various Benzothiazole analogs
used in the study.
Figure 2. The chemical structures of various Benzothiazole analogs used.

Figure 3a. Effect of H2O2 on the viability of U87MG cells: Various concentrations of H2O2
(425 – 9500μM) was added to human glioblastoma cancer cells (U87MG) and cells were
incubated for 12h. The concentration of 950 µM H2O2 resulted in 40% reduction in the cell
viability. This was followed by MTT based cell viability assay. The OD570 nm value was
represented as percentage of cell viability. Three independent experiments were conducted and
GraphPad software was used to test statistical significance. ** represents p-value < 0.01 and
*** represents p-value < 0.001
Figure 3b. Effect of H2O2 on neuroblastoma SH-SY5Y cells.
The SH-SY5Y cells were treated with H2O2 at a concentration ranging from 0 – 256 μM and
incubated for 12 h. Forty percent reduction was observed from 16-256 µM concentration. This
is followed by MTT based cell viability assay. The OD570 value was converted to % values.
Three independent experiments were conducted and GraphPad software was used to test
statistical significance. ** represents p-value < 0.01 and *** represents p-value < 0.001

Figure 4a. Effect of Benzothiazole analogs on H2O2 induced neurotoxicity in U-87 MG
cells. U-87 MG cells were pre-treated with compounds (5b, 5c, 6a-6d, 7a, and VA) of
concentration 2 and 4 μM for 12h followed by 4h of H2O2 treatment (950 μM) as obtained in
the above studies. Then the cell viability was determined. Here, Valproic acid (VA) is the
standard neuro protective drug employed. Here 2 µMc indicates 2 µM of compound and 4 µMc
indicates 4 µM of BT compounds used in the study. Three independent experiments were
conducted and GraphPad software was used to test statistical significance. *** represents p-
value < 0.001