Identification and structural characterization of the stress degradation products of omeprazole using Q-TOF-LC-ESI-MS/MS and NMR experiments: Evaluation of the toxicity of the degradation products

Article abstract of DOI:10.1039/c9nj00932a

Omeprazole (OMP), a prototype proton pump inhibitor used for the treatment of peptic ulcers and gastroesophageal reflux disease (GERD), was subjected to forced degradation studies as per ICH guidelines Q1A (R2). The drug undergoes degradation under acid, base, neutral hydrolysis and oxidative degradation conditions and forms a total of sixteen degradation products, which were characterized by LC-MS/MS experiments and accurate mass measurements. Oxidative degradation products (OMP-15 and OMP-16) were synthesized and confirmed by various NMR experiments. The cytotoxic effects of OMP-15 and OMP-16 were tested on normal human cells HEK 293 and NIH3T3 by MTT assay. Based on the cytotoxicity results, compared to the standard OMP, both OMP-15 and OMP-16 were found to have relatively weaker toxic effects towards normal cells. Further, the in silico toxicity of OMP and its degradation products (OMP-1 to OMP-16) was assessed by the ProTox-II prediction tool. OMP and OMP-8 are predicted to have carcinogenicity, OMP-7 to have hepatotoxicity and OMP-2, OMP-3, OMP-9, OMP-11, OMP-14 and OMP-16 to have immune system toxicity with a high confidence score. The drug, OMP-1, OMP-6, OMP-7, OMP-8, OMP-13 and OMP-15 are predicted to combine with the aryl hydrocarbon receptor (AhR) with a high probability score. Additionally, two different targets, amine oxidase A and prostaglandin G/H synthase 1, are predicted as toxicity targets for OMP, OMP-1, OMP-6, OMP-8, OMP-13, OMP-15 and OMP-16 with probable binding.

Full text of DOI:10.1039/c9nj00932a

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Identification and structural characterization of stress degradation products of omeprazole
using Q-TOF-LC-ESI-MS/MS and NMR experiments: Evaluation of toxicity of
degradation products
G. Shankara, Roshan M. Borkara, Suresh Uduthaa, M. Kanakarajub, G. Sai Charanb,
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S Misrac, R. Srinivas*a
a,bAnalytic chemistry, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500 007, India
cBiology Division, CSIR-Indian Institute of Chemical Technology, Hyderabad, 500007, India
Keywords: omeprazole, degradation products, accurate mass measurements, in vitro and in
silico toxicity
a,b* Corresponding author
:
srini@iict.res.in (R. Srinivas)
sragampeta@yahoo.co.in
: +91-40-27193122
Telephone Number
Fax Number
:
+91-40-27193156
Manuscript Number: IICT/Pubs./2018/388

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Abstract
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Omeprazole (OMP), a prototype proton pump inhibitor used for the treatment of peptic ulcers
and gastroesophageal reflux disease(GERD), was subjected to forced degradation studies as per
ICH guidelines Q1A (R2). The drug undergoes degradation under acid, base, neutral hydrolysis
and oxidative degradation conditions and forms a total of sixteen degradation products which
were characterized by LC-MS/MS experiments and accurate mass measurements. Oxidative
degradation products (OMP-15 and OMP-16) were synthesized and confirmed by various NMR
experiments. The cytotoxic effects of OMP-15 and OMP-16 were tested on normal human cells
such as HEK 293 and NIH3T3 by MTT assay. Based on the cytotoxicity results, compared to
the standard OMP, both OMP-15 and OMP-16 were found to have relatively lesser toxic effects
towards normal cells. Further, in silico toxicity of OMP and its degradation products (OMP-1 to
OMP-16) were assessed by ProTox-II prediction tool. OMP and OMP-8 are predicted for
carcinogenicity, OMP-7 for hepato toxicity and OMP-2, OMP-3, OMP-9, OMP-11, OMP-14
and OMP-16 for immune system with high confidence score. The drug, OMP-1, OMP-6, OMP-
7, OMP-8, OMP-13 and OMP-15 are predicted to combine with aryl hydro carbon receptor
(AhR) with high probability score. Additionally, two different targets, Amine Oxidase A and
Prostaglandin G/H Synthase 1 are predicted as toxicity targets for OMP, OMP-1, OMP-6, OMP-
8, OMP-13, OMP-15 and OMP-16 with probable binding.
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1. Introduction
Stress degradation is the process for examining the stability of drug molecules in the presence of
chemical and environmental conditions to determine the product breakdown levels. According to
the ICH guidelines stress testing is a process for identification of degradation products and it
will further help to determine the inherent stability of the drug molecule [1]. Stress testing

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provides information about degradation pathways and potential degradation products.
Identification and structural characterization of degradation products is useful to establish the
mechanism of formation of degradation products from the drug and to know the toxicity
information and side effects of drug [2]. Identification of low level of unknown degradation
products and impurities can be challenging [3]. There are many reports in the literature on
structural elucidation of degradation products of various pharmaceutical drugs using LC-MS/MS
experiments [4-8].
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All the drug substances and drug products tends to degrade over the timeframe affected by the
factor such as light, pH, moisture, heat etc. Considering these factors International Conference
on Harmonization (ICH) guideline has brought protocols in a harmonized way [9]. Formation of
degradation products can also be formed by hydrolysis, oxidation, dimerization, rearrangement,
adduct formation and the combination of these processes [10].
The identified and characterized degradation products can be subjected to toxicity studies for risk
assessment of potentially genotoxic degradation products. Evaluation of toxicity of degradation
products is important from the safety and efficacy point of view as per ICH guideline Q3A and
Q3B [11,12]. There are several examples in the literature on the adverse effects of degradation
products of various drugs. For example severe adverse effects of the degradation products of
tetracycline and aminopencillins have been reported [13,14]. Degradation products can lead to
carcinogenicity due to genetic mutations, chromosomal breaks, and/or chromosomal
rearrangements [15] Recently, we have reported in vitro and in silico toxicity of degradation
products of lansoprazole, rabeprazole and sumatriptan succinate [16-18].
Gastroesophageal reflux disease (GERD) is a common chronic disorder, which is characterized
by increased reflux of gastric contents into the lower esophagus [19,20]. The severity of GERD

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is directly correlated with the degree and duration of esophageal acid exposure and is highly pH
dependent [21,22]. Severe reflux esophagitis may convert to serious complications like
esophageal structure and Barrett’s esophagus in patients with GERD [23,24].
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Proton pump inhibitors (PPIs) are the most widely prescribed medication to reduce gastric acid
secretion. Omeprazole (OMP ), 5-methoxy-2-[[(4-methoxy-3,5-dimethyl-2-pyridinyl) methyl]
sulphinyl]-1H-benzo [d] imidazole, is widely used for the treatment of peptic ulcers,
Gastroesophageal Reflux Disease (GERD), dyspepsia and Zollinger–Ellison syndrome [25].
OMP is the first clinically used prototypical proton pump inhibitor, subsequently lansoprazole,
pantoprazole, rabeprazole and the stereo-isomeric drug substances esomeprazole and
dexlansoprazole were introduced [26]. Long-term OMP therapy is highly effective and more safe
for control of reflux esophagitis [27].
Several analytical methods have been reported for determination of OMP and its impuriies in
bulk drug and formulations [28-30]. Few HPLC and LC MS methods have been reported for
determination of OMP and its metabolites in plasma [31-33]. However, no study exists on
systematic stress degradation study of OMP as per ICH guidelines. Hence, the main aim of this
study is to identify and characterize the degradation products of OMP. This was done by
exposing the drug to ICH-recommended stress conditions of hydrolysis, oxidation, thermal and
photolysis. The resultant solutions were subjected to optimized LC-MS/MS experiments to
establish the fragmentation pattern of the drug and its degradation products. Further we
synthesized the oxidative degradation products and confirmed their structures by 1D, 2D NMR
experiments prior to evaluating their in vitro toxicity . In addition, we have studied the in silico
toxicities of all the degradation products.

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2. Experimental
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2.1 Chemicals and reagents
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Omeprazole (OMP), methanol-d4, tris hydrochloride (Tris-HCl), 3-(4,5-Dimethyl-2-thiazolyl)-
2,5-diphenyl-2H-tetrazolium bromide and Deoxyribonucleic acid from calf thymus (ctDNA) was
purchased from Sigma Aldrich, India. HPLC Grade acetonitrile (ACN) and methanol (MeOH)
were purchased from Merck, India. HPLC grade water was prepared by filtrating through a
Millipore Milli-Q- plus system (Millipore, Milford, MA, USA). Ammonium acetate of HPLC
grade was purchased from Finar Chemicals Pvt. Ltd. (Ahmedabad, India). All analytical grade
reagents, formic acid, sodium hydroxide, hydrochloric acid, dichloromethane (DCM) and 30%
hydrogen peroxide were purchased from Merck (Mumbai, India).
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2.2. Instrumentation
2.2.1. Liquid Chromatography-Mass spectrometry
The Agilent 1290 infinity series LC system (Agilent technologies, USA) consisting of an auto
sampler (G7129B), a quaternary pump (G7104A), a diode array detector (G7117A), a column
compartment (G7116B) and a degasser was employed for HPLC analysis. LC/MS analysis was
performed on a Ultra Performance liquid chromatography (UPLC) coupled to a quadrupole time-
of- flight mass spectrometer (Q-TOF LC/MS 6545 series- G6545A, Agilent Technologies,
USA). Acquisition of the data was under the control of Mass Hunter workstation software.
The fragmentation pathway of OMP was established by carrying out Q-TOF-MS/MS. ESI
source was operated in positive ionization mode with a capillary voltage of 3000-3500 V and
skimmer at 60 V. Collision gas used for mass experiments was ultrahigh pure nitrogen and 30 eV
energy was used for MS/MS studies. Nitrogen was used as the drying (325◦C, 10 Lmin−1) and
nebulizing (45 psi) gas. Mass-Hunter Workstation software was used for the data processing of

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total ion chromatograms (TICs) and to determine the elemental composition from accurate mass
measurements of m/z values.
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2.2.2. Nuclear magnetic resonance spectroscopy
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The degradation products (OMP-15 and OMP-16) were analysed by various NMR techniques
(1H,13C and 2D). Chemical shift values on δ scale in ppm were measured at 400MHz frequency
by using NMR spectroscopy (AVANCE III HD-400, Bruker, Billerica, Massachusetts, United
States). Tetra methyl silane (TMS) is used as internal standard and adjusted to 0 ppm on δ scale.
The data acquisition and processing of NMR spectra was done using Top spin software
(3.2version).
2.3. Stressed degradation conditions
Stress degradation studies of OMP were carried out on the bulk drug as per ICH guidelines Q1A
(R2). The drug solutions were prepared in 1.0 mg/mL concentration for all the stressed reactions.
Acidic hydrolysis of the drug was carried out by using 0.01N HCl at room temperature for 30
min. Whereas, basic and neutral hydrolysis were conducted by refluxing the drug in 2N NaOH
and water at 80°c under reflux for 48 h and 24 h, respectively. For oxidative degradation study,
the drug was subjected to 3% H2O2 at room temperature for 24 h. Photolytic studies were
performed by exposing solid and solution of the drug sample to 1.2×106 lux h of fluorescent light
and 200W h m-2 UV light in a photo stability chamber. For thermal degradation study, the drug
sample was sealed in glass vial and kept in a thermostatic block at 80oC for 1week. All stressed
samples were kept in a refrigerator at 4°C until analysis.
2.4. Sample preparation
All the stressed samples (hydrolytic, oxidative, thermal and photolytic) were collected and

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neutralized. All solutions were filtered through 0.22 μm pore size membrane syringe filter before
LC-MS/MS analysis.
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2.5. Analysis of stressed samples
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2.5.1. Method development and optimization of LC-MS conditions
The chromatographic conditions were optimized using Hiber Purospher, C18 (250 X 4.6mm, 5µ)
(Merck Lichro, Switzerland) column with a mobile phase composed of 10 mM ammonium
acetate(A) and ACN(B) in gradient elution mode. The linear gradient programme was set as
follows: (Tmin/% proportion of solvent B): 0-5/10, 5-7/30, 7-14/ 40, 14-20/45, 20-30/ 45, 30-35/ 50,
35-
40/85, 40-42/10, 42-44/10. The column temperature, flow rate, injection volume, and detector
wavelength were at 30 °C, 1.0 ml/min, 20.0 µl, and 302 nm, respectively. The typical operating
source conditions for MS scan in positive ion ESI mode were optimized as follows; the
fragmentor voltage was set at, 120 V; the capillary at, 3000–3500 V; sheath gas temp at, 350°C;
flow of sheath gas at, 11 L/min; nebulizing (45 psi) gas and nitrogen was used as the drying
(250°C; 13 L/min). For full scan MS mode, the mass range was set at m/z 50–1000.
For collision-induced dissociation( CID) experiments, keeping MS1 static, interested precursor
ion was selected using the quadrupole analyzer, and the product ions were analyzed by a time-of-
flight (TOF) analyzer. Ultrahigh pure nitrogen was used as collision gas.
2.6 Chemical synthesis
The mono and di oxidative degradation products of OMP (OMP-15, OMP-16) were obtained by
the synthesis. OMP was used as a starting material and meta- Chloroperoxybenzoic acid (m-
CPBA) as a reagent. The reagent m-CPBA (172mg, 1 mmol for OMP-15 and 2 mmol for OMP-
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16) was added to a solution of OMP (345 mg, 1.0 mmol) in 3 ml of DCM at 0 C and the
reaction mixture was stirred overnight at room temperature. The reaction mixture was quenched

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with sodium bicarbonate followed by extraction with ethyl acetate. After extraction, the residue
was purified by using silica gel column chromatography (DCM–MeOH, 9.5:0.5 %) to get the
pure products as white solid. The samples were characterized by 1D, 2D-NMR experiments
using deuterated methanol (CD3OD).
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2.7. In vitro toxicity evaluation
2.7.1. In vitro 3-(4, 5-dimethylthiazol-2-yl)-2, 5-diphenyl tetrazolium bromide (MTT) assay
Cytotoxicity assay (MTT) was evaluated for all the test compounds as per our earlier published
work (Bollu et al., 2016) [34]. Two different normal cell lines viz., Normal human embryonic
kidney cells (HEK-293) and Normal mouse embryo fibroblast cells (NIH3T3) were obtained
from the ATCC (Bethesda, MD, USA) and maintained in DMEM supplemented with 10 % FBS,
2 mM l-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37 °C in a 5 % CO2
incubator. After seeding of cells in 96 well flat bottom tissue culture plate and test compounds of
different concentrations ranging from 1 to 100 µg were added in triplicates and incubated for
48hr. The cells were then incubated with MTT (0.5 mg/mL) for 3h and 100 µl DMSO was
added to each well to dissolve the insoluble formazan crystals. Finally the absorbance of the
plates were measured using a Synergy H1 multi-mode plate reader, USA. OMP was used as
positive control for the comparison.
3. Results and discussion
3.1 Degradation behaviour of the OMP
The degradation behaviour of OMP was analyzed using LC/MS under stress degradation
conditions as per ICH guidelines. The optimized LC/MS method was used for identification of
OMP degradation products. The drug was found to be degraded in acid, base, neutral hydrolysis

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and oxidation degradation conditions. Whereas it was found to be stable under thermal and
photolytic conditions. Totally 16 degradation products of OMP were identified and characterized
by using LC-MS/MS. The elemental compositions of OMP degradation products and their
product ions have been confirmed by accurate mass measurements. Structure of the degradation
products was proposed based on the MS/MS fragmentation data of the drug and degradation
products. The proposed structures of degradation products of OMP and their elemental
compositions are given in scheme 1 and table 1, respectively.
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Scheme 1: Proposed structures of protonated degradation products of OMP.
3.2.1 Hydrolysis
The LC/ESI/HRMS analysis of OMP (Figure 1(a)), in acid hydrolysis showed an extensive
degradation and formed five degradation products (OMP-1, OMP-2, OMP-3, OMP-4, OMP-5)

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(Figure 1(b)). In the case of base hydrolysis (Figure 1(c)) six degradation products (OMP-1,
OMP-6, OMP-7, OMP-8, OMP-9, OMP-10) were formed, whereas in neutral condition (Figure
1(d)) seven degradation products (OMP-2, OMP-5, OMP-8, OMP-11, OMP-12, OMP-13, OMP-
14) formed, respectively.
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Table 1: Elemental composition of protonated degradation products of OMP
Degradation
Products
Rt
Formula
Observed
(m/z)
Calculated
(m/z)
Error
(ppm)
3.02
0.67
-0.60
-3.81
1.60
3.91
-2.20
4.74
1.66
3.53
-0.95
5.27
0.33
4.02
4.69
4.23
(min)
27.39
14.43
18.33
22.34
18.66
11.50
13.05
14.07
18.29
20.68
10.66
14.70
23.17
23.97
20.02
15.14
OMP-1
OMP-2
OMP-3
OMP-4
OMP-5
OMP-6
OMP-7
OMP-8
OMP-9
OMP-10
OMP-11
OMP-12
OMP-13
OMP-14
OMP-15
OMP-16
C17H20N3O2S+
330.1260
298.1528
328.1116
314.1511
312.1337
332.1052
181.0404
316.1094
180.1016
198.0576
314.1138
284.1378
300.1341
298.1174
362.1152
378.1102
330.1270
298.1550
328.1114
314.1499
312.1342
332.1063
181.0430
316.1114
180.1019
198.0583
314.1135
284.1393
300.1342
298.1186
362.1169
378.1118
+
C17H20N3O2
C17H18N3O2S+
+
C17H20N3O3
+
C17H18N3O3
C16H18N3O3S+
C8H9N2OS+
C16H18N3O2S+
+
C10H14NO2
C9H12NO2S+
+
C16H16N3O4
+
C16H18N3O2
+
C16H18N3O3
+
C16H16N3O3
C17H20N3O4S+
C17H20N3O5S+

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(a)
(b)
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(c)
(d)
Figure.1. UPLC/ESI/MS TICs of OMP(a) and its degradation products under acid (b), base (c),
and neutral (d) hydrolytic conditions.
3.2.2. Oxidation
A total of three degradation products (OMP-2, OMP-15, OMP-16) of OMP were formed, when
it was subjected to 3% H2O2 at room temperature for 24 hr. (Figure. 2(a)).

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3.2.3. Thermal degradation
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The OMP was found to be stable under thermal condition after the exposures of 1 week at 80
C. (Figure. 2(b))
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3.2.4. Photolytic degradation
The drug was found to be stable under UV and fluorescence degradation conditions after the
exposures of 1 week. (Figure.2(c))
(a)
(b)
(c)
Figure.2. UPLC/ESI/MS TICs of OMP degradation products under oxidation (a), thermal (b) and
photolytic (c) condition

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4. MS/MS study of the protonated drug and its degradation products
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4.1 OMP ([M+H] +, m/z 346.1233)
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The fragmentation pathway of the drug was established by using LC-MS/MS combined with
accurate mass measurements. The protonated drug at m/z 346 ([M+H]+) with an elemental
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+
composition C19H23FN3O4 was eluted at 17.36 min. (Figure.1(a)) The LC-MS/MS spectrum of
[M+H]+ of OMP showed high abundance product ion at m/z 198 (loss of C8H8N2O ) and low
abundance product ions at m/z 328 (loss of H2O), m/z 295 (loss of SH radical from m/z 328),
m/z 180 (loss of H2O from m/z 198), m/z 168 (loss of C8H6N2OS), m/z 151 (loss of OH radical
from m/z 168), m/z 149 (loss of C9H11NO2S), m/z 136 (loss of CH3OH from m/z 168), m/z 121
(loss of CH2O from m/z 151) and m/z 79 (loss of C2H4N radical). The product ion at m/z 149 is
characteristic ion for 5-methoxy-1H-benzo[d] imidazole skeleton and m/z168, m/z 151, m/z 136
and m/z 121 are significant ions for 4-methoxy-3,5-dimethylpyridine. (Supplementary Scheme
S1, Figure S1(a), see Supporting Information) The product ions at m/z 328, m/z 295, m/z 198
and m/z 180 indicates the presence of sulphur monoxide linked to 5-methoxy-1H-benzo[d]
imidazole skeleton and 4-methoxy-3,5-dimethylpyridine. The elemental compositions of all
these ions have been confirmed by accurate mass measurements. (Supplementary Table.S1, see
Supporting Information)
4.2 MS/MS study of degradation products
OMP-1 ([M+H]+, m/z 330.1260)
The degradation product OMP-1 at m/z 330 ([M+H]+) with an elemental composition
C17H20N3O2S+ detected at 27.39 min in acid and base hydrolysis conditions. The decrease of 16
Da in molecular weight compared to the [M+H]+ of the drug suggests that OMP-1 was formed
by loss of ‘O’ atom from the drug. The LC-MS/MS spectrum of OMP-1 showed high abundance

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product ion at m/z 182 (loss of 5-methoxy-1H-benzo[d]imidazole) and low abundance product
ions at m/z 297 (loss of SH radical), m/z 267 (loss of CH2O from m/z 297), m/z 166 (loss of CH4
from m/z 182), m/z 151 (loss of CH3 radical from m/z 166), m/z 150 (loss of C8H8N2OS), m/z
149 (loss of C9H11NOS), m/z 136 (loss of CH2S from m/z 182) and m/z 120 (loss of CH2O from
m/z 150). The diagnostic product ions at m/z 182 and m/z 149 supports the proposed structure of
OMP-1. The product ions at m/z 295, m/z 280 and m/z 180 are characteristic ions for OMP-1.
(Supplementary Scheme.S2, Figure S1(b) Table.S2, see Supporting Information) Based on
these data, OMP-1 was identified as 5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-
yl)methylthio)-1H-benzo[d]imidazole.
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OMP-2 ([M+H]+, m/z 298.1528)
The degradation product OMP-2 at m/z 298 ([M+H]+) with an elemental composition
+
C17H20N3O2 detected at 14.43 min in acidic, neutral hydrolytic and oxidation conditions. The
LC-MS/MS spectrum of [M+H]+ of OMP-2 showed high abundance product ion at m/z 268 (loss
of CH3 radical from m/z 283) and low abundance product ions at m/z 283 (loss of CH3 radical),
m/z 282 (loss of CH4), m/z 266 (loss of CH3OH), m/z 254 (loss of CO from m/z 282), m/z 253
(loss of CH3 radical from m/z 268), m/z 240 (loss of CO from m/z 268), m/z 151 (loss of
C8H7N2O radical), m/z 150 (loss of C8H8N2O) and m/z 147 (loss of C9H13NO). The diagnostic
product ions at m/z 147 and m/z 150 supports the proposed structure of OMP-2.
(Supplementary Scheme S3, Figure S1(c), Table.S2, see Supporting Information) Based on
these data OMP-2 was identified as 5-methoxy-2-(4-methoxy-3,5-dimethyl-2-methylenepyridin-
1(2H)-yl)-1H-benzo[d]imidazole.

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OMP-3 ([M+H]+, m/z 328.1116)
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The degradation product OMP-3 at m/z 328 ([M+H]+) with an elemental composition
C17H18N3O2S+ detected at 18.33 min in acid hydrolytic condition. The mass difference between
the protonated drug (m/z 346) and OMP-3 (m/z 328) is 18 u which suggests that OMP-3 was
formed by loss of H2O from the drug. The LC-MS/MS spectrum of [M+H]+ of OMP-3 showed
high abundance product ions at m/z 280 (loss of ‘S’ from m/z 312), m/z 120 (loss of CH2O from
m/z 150), m/z 106 (loss of CH2O from m/z 136) and low abundance ions at m/z 312 (loss of
CH4), m/z 298 (loss of C2H6), m/z 295 (loss of SH radical), m/z 284 (loss of CO from m/z 312),
m/z 280 (loss of ‘S’ from m/z 312), m/z 265 (loss of CH3 radical from m/z 280) and m/z 180
(loss of C8H8N2O). The characteristic product ions at m/z 295, m/z 280 and m/z 180 supports
the proposed structure of OMP-3. (Supplementary Scheme.S4, Figure S1(d), Table.S3, see
Supporting Information). Based on all these data, OMP-3 structure was proposed as shown in
Scheme. 1.
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OMP-4 ([M+H]+, m/z 314.1511)
The acid hydrolytic degradation product OMP-4 at m/z 314 ([M+H]+) with an elemental
+
composition C17H20N3O3 detected at 22.34 min. The LC-MS/MS spectrum of [M+H]+ of OMP-
4 showed high abundance product ion at m/z 120 (loss of CH2O from m/z 150) and low
abundance product ions at m/z 299 (loss of CH3 radical), m/z 284 (loss of CH3 radical from m/z
299), m/z 282 (loss of CH3OH from m/z 314), m/z 268 (loss of OCH3 radical from m/z 299), m/z
151 (loss of C8H7N2O2 radical), m/z 150 (loss of C8H8N2O2) and m/z 120 (loss of CH2O from
m/z 150). The product ions at m/z 268, m/z 149, m/z 151, m/z 150 and m/z 120 are diagnostic
ions for the proposed structure of OMP-4. (Supplementary Scheme. Scheme.S5, Figure S2(a),
Table. S3, see Supporting Information) Based on these data OMP-4 was identified as 5-

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methoxy-2-9(4-methoxy-3,5-dimethyl-2-methylenepyridin-2-yl)-methoxy)-1H-
benzo[d]imidazole.
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OMP-5 ([M+H]+, m/z 312.1337)
The acidic and neutral hydrolytic degradation product OMP-5 at m/z 312 ([M+H]+) with an
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+
elemental composition C17H18N3O3 was eluted at 18.66 min. The LC-MS/MS spectrum of
[M+H]+ of OMP-5 showed high abundance product ions at m/z 269 (loss of CH3 radical from
m/z 284), m/z 254 (loss of CH3 radical from m/z 269) and low abundant product ions at m/z 297
(loss of CH3 radical), m/z 296 (loss of CH4), m/z 284(loss of CO), m/z 280 (loss of CH3OH),
m/z 268 (loss of CH4 from m/z 284), m/z 252 (loss of CH3OH from m/z 284), m/z 241(loss of
CO from m/z 269), m/z 240 (loss of CO from m/z 268), m/z 226 (loss of CH4 from m/z 241),
m/z 215 (loss of C2H2 from m/z 241), m/z 198 (loss of CO from m/z 226), m/z 147 (loss of
C6H7N from m/z 240) and m/z 136 (loss of C7H4N2 from m/z 252). The product at m/z 147 is
diagnostic ion for the presence of 6-methoxy-1H-benzo[d]imidazole moiety and the product ion
at m/z 136, which authenticates presence of 4-methoxy-3,5-dimethylpyridine in OMP-5.
Formation of product ion at m/z 284 by the loss CO supports the proposed structure of OMP-5.
(Supplementary Scheme.S6, Figure. S2(b), Table.S4, see Supporting Information) Based on
all these data, the structure of OMP-5 was proposed as shown in Scheme. 1.
OMP-6 ([M+H]+, m/z 332.1052)
The base hydrolytic degradation product OMP-6 at m/z 332 ([M+H]+) with an elemental
composition C16H18N3O3S+ was detected at 11.50 min. The LC-MS/MS spectrum of [M+H]+ of
OMP-6 showed high abundant product ion at m/z 184 (loss of 5-methoxy-1H-
benzo[d]imidazole) and low abundance ions at m/z 314 (loss of H2O), m/z 238 (loss of C6H6O),
m/z 197 (loss of C8H9NO), m/z 179 (loss of C8H11NO2), m/z 166 (loss of H2O from m/z 184),

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m/z154 (loss of C8H6N2OS), m/z 149 (loss of C8H9NO2S) and m/z 137 (loss of OH radical from
m/z 154). Formation of product ion at m/z 314 clearly indicates presence of free -OH group in
OMP-6. The product ions at m/z 184 and m/z 149 were supports the proposed structure of OMP-
6. (Supplementary Scheme.S7, Figure S2(c), Table.S5, see Supporting Information) Based
on these data, OMP-6 was identified as 2-((5-methoxy-1H-benzo[d]imidazol-2-
ylsulfinyl)methyl)-3,5-dimethylpyridine-4-ol.
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OMP-7 ([M+H] +, m/z 181.0404)
The degradation product OMP-7 at m/z 181 ([M+H]+) with an elemental composition
+
C22H27FN3O6 was detected at 13.05 min in base hydrolytic condition. The LC-MS/MS
spectrum of [M+H]+ of OMP-7 showed high abundance product ions at m/z 166 (loss of CH3
radical), m/z 138(loss of CO from m/z 166) and low abundance product ions at m/z 165 (loss of
CH3OH), m/z 150 (loss of ‘O’ atom from m/z 166)), m/z 149 (loss of ‘S’ atom), m/z 148 (loss of
SH radical) and m/z 133 (loss of SH radical from m/z 166). The product ions at m/z 148 and m/z
149 are authenticate presence of free –SH group at imidazole carbon. Based on these data, OMP-
7 was identified as 5-methoxy-1H-benzo[d]imidazole-2-thiol. (Supplementary Scheme.S8,
Figure S2(d), Table.S5, see Supporting Information)
OMP-8 ([M+H]+, m/z 316.1094)
The degradation product OMP-8 at m/z 316 ([M+H]+) with an elemental composition
C16H18N3O2S+ detected at 14.07 min in base and neutral hydrolytic conditions. The decrease of
30 Da in molecular weight compared to [M+H]+ of the drug suggests that OMP-8 was formed by
loss of ‘CH2O’ molecule from the drug. The LC-MS/MS spectrum of [M+H]+ of OMP-8 showed
high abundance product ion at m/z 168 (loss of 5-methoxy-1H-benzo[d]imidazole) and low
abundance ions at m/z 301 (loss of CH3 radical), m/z 284 (loss of OH radical from m/z 301) and
m/z 149 (loss of C8H9NOS). The product ions at m/z 168 and m/z 149 are characteristic ion for

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the proposed structure of OMP-8. The diagnostic product ion at m/z 149 authenticates presence
of OCH3 group on imidazole ring. (Supplementary Scheme.S9, Figure S3(a), Table.S5, see
Supporting Information). Based on these data OMP-8 was identified as 5-methoxy-2-((3,5-
dimethylpyridin-2-yl)methylsulfinyl)-5-methoxy-1H-benzo[d]imidazole.
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OMP-9 ([M+H] +, m/z 180.1016)
The degradation product OMP-9 at m/z 180 ([M+H]+) with an elemental composition
+
C10H14NO2 was detected at 18.29 min in base hydrolysis conditions. The LC-MS/MS spectrum
of [M+H]+ of OMP-9 showed highly abundance product ions at m/z 137 (loss of CH3 radical
from m/z 152) and low abundance product ions at m/z 164 (loss of CH4), m/z 152(loss of CO) ,
m/z 150 (loss of CH2O), m/z 136 (loss of CO from m/z 164) and m/z 108 (loss of CO from m/z
136). The significant product ions at m/z 152 and m/z 108 indicate presence of free carbonyl
group at pyridine nitrogen. (Supplementary Scheme.S10, Figure S3(b), Table.S6, see
Supporting Information) Based on these data OMP-9 was identified as 4-methoxy-3,5-
dimethyl-2-methylene pyridine-1(2H)-carbaldehyde.
OMP-10 ([M+H]+, m/z 198.0576)
The degradation product OMP-10 at m/z 198([M+H]+) with an elemental composition
C9H12NO2S+ was detected at 20.68 min in base hydrolytic condition. The ESI/MS/MS spectrum
of [M+H]+ of OMP-10 showed intense product ions at m/z 180 (loss of H2O) and low intense
product ions at m/z 181 (loss of OH radical), m/z 166 (loss of CH3 radical from m/z 181) and
m/z 150 (loss of SO group). The product ions at m/z 181 and m/z 180 indicates presence of free
–OH group in OMP-10. The product ion at m/z 150 clearly indicates presence of free –S-OH
group in OMP-10. (Supplementary Scheme.S11, Figure S3(c), Table.S6, see Supporting

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Information). Based on these data OMP-10 was identified as (z)-((4-methoxy-5-methyl-3-
methylenepyridin-2(3H)-ylidene) methyl)sulfanol.
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OMP-11 ([M+H]+, m/z 314.1138)
The neutral hydrolysis degradation product OMP-11 at m/z 314 ([M+H]+) with an elemental
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+
composition C16H16N3O4 detected at 10.66 min. The LC-MS/MS spectrum of [M+H]+ of OMP-
11 showed high abundance product ion at m/z 270 (loss of CO2) and low abundance product ions
at m/z 296 (loss of H2O), m/z 268 (loss of CO from m/z 296), m/z 255 (loss of CH3 radical from
m/z 270), m/z 149 (loss of C8H7NO3) and m/z 122 (loss of C8H6N2O from m/z 268). Formation
of diagnostic product ion at m/z 270 by the loss of CO2 clearly indicates presence of –COOH
group on OMP-11. The product ions at m/z 268, m/z 149 and m/z 122 supports the proposed
structure of OMP-11. (Supplementary Scheme.S12, Figure S3(d), Table.S7, see Supporting
Information) Based on these data, OMP-11 was identified as 1-(6-methoxy-1H-
benzo[d]imidazole-2-yl)-5-methyl-3-methylene-4-oxo-1,2,3,4-tetrahydropyridine-2-carboxylic.
OMP-12 ([M+H]+, m/z 284.1378)
The degradation product OMP-12 at m/z 284 ([M+H]+) with an elemental composition
+
C16H18N3O2 detected at 14.70 min in neutral hydrolytic degradation condition. The LC-MS/MS
spectrum of [M+H]+ of OMP-12 showed high abundance product ions at m/z 269 (loss of CH3
radical), m/z 254(loss of CH3 radical from m/z 269) and low abundance product ion at m/z
268(loss of CH4), m/z 147 (loss of C8H11NO), m/z 136 (loss of C7H4N2O from m/z 268) and m/z
108 (loss of CO from m/z 136). The product ions at m/z 108, m/z 136 and m/z 147 clearly
indicates ‘O’ forms bridge between 6-methoxy-1H-benzo[d]imidazole ring and 2,3,5-
trimethylpyridine moiety in OMP-12. Formation of diagnostic product ion at m/z 147
authenticates presence of OCH3 group on imidazole ring. (Supplementary Scheme.S13, Figure

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S4(a), Table.S7, see Supporting Information) Based on these data, OMP-12 was identified as
2-((3,5-dimethylpyridin-2-yl)methoxy)-5-methoxy-1H-benzo[d]imidazole.
OMP-13 ([M+H]+, m/z 300.1341)
The neutral hydrolytic degradation product OMP-13 at m/z 300([M+H]+) with an elemental
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composition C16H18N3O3 was detected at 23.17 min. The LC-MS/MS spectrum of [M+H]+ of
OMP-13 showed high abundance product ion at m/z 270 (loss of CH3 radical from m/z 285) and
low abundance product ions at m/z 285 (loss of CH3 radical), m/z 272 (loss of CO), m/z 257
(loss of CO from m/z 285), m/z 242 (loss of CH3 from m/z 257), m/z 225 (loss of OH radical
from m/z 242) and 173 (loss of C4H4 from m/z 225). The diagnostic product ion at m/z 173
indicates ‘O’ forms bridge between 6-methoxy-1H-benzo[d]imidazole ring and 2,3,5-
trimethylpyridine-4-ol moiety in OMP-13. (Supplementary Scheme.S14, Figure S4(b),
Table.S8, see Supporting Information) Based on these data, OMP-13 was identified as 2-((6-
methoxy-1H-benzo[d]imidazol-2-yloxy) methyl)-3,5-dimethylpyridin-4-ol.
OMP-14 ([M+H]+, m/z 298.1174)
The degradation product OMP-14 at m/z 298 ([M+H]+) with an elemental composition
+
C16H16N3O3 detected at 23.97 min in neutral hydrolysis conditions. The LC-MS/MS spectrum
of [M+H]+ of OMP-14 showed high abundance product ion at m/z 254 (loss of CO from m/z
282) and low abundance product ions at m/z 283 (loss of CH3 radical), m/z 282 (loss of CH4),
m/z 270 (loss of CO), m/z 240 (loss of CH3 radical from m/z 255), m/z 224 (loss of ‘O’ from m/z
240) and m/z 173 (loss of C4H3 radical from m/z 224). The product ions at m/z 270 indicates
presence of carbonyl moiety in OMP-14 and the product ion at m/z 173 is diagnostic ion for the
proposed structure of OMP-14. (Supplementary Scheme.S15, Figure S4(c), Table.S8, see

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Supporting Information) Based on these data OMP-14 was identified as 2-((6-methoxy-1H-
benzo[d]imidazole-2-yloxy)methyl)-5-methyl-3-methylenepyridin-4(3H)-one.
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8
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OMP-15 ([M+H]+, m/z 362.1152)
The oxidative degradation product OMP-15 at m/z 362 ([M+H]+) with an elemental composition
C17H20N3O4S+ detected at 20.02 min. The increase of 16 Da in molecular weight compared to the
[M+H]+ of the drug suggests that OMP-15 was oxidative degradation product. The LC-MS/MS
spectrum of [M+H]+ of OMP-15 at m/z 362 showed high abundance product ion at m/z 150 (loss
of ‘O’ from m/z 166) and low abundant product ions at m/z 298 (loss of SO2), m/z 283 (loss of
CH3 radical from m/z 298), m/z 268 (loss of CH2O from m/z 298), m/z 268 (loss of CH3 radical
from m/z 283), m/z 266 (loss of OH radical from m/z 283), m/z 240 (loss of CO from m/z 268),
m/z 214 (loss of C8H8N2O), m/z 195 (loss of C9H13NO2), m/z 179 (loss of ‘O’ from m/ z 195)
and m/z 168 (loss of C8H6N2O2S). The product ions at m/z 214, m/z 195 and m/z 168 clearly
indicates oxidation takes place at sulphoxide of the drug. (Supplementary Scheme.S16, Figure
S4(d), Table.S9, see Supporting Information) Based on these data OMP-15 was identified as
5-methoxy-2-((4-methoxy-3,5-dimethylpyridin-2-yl) methylsulfonyl)-1H-benzo[d]imidazole..
OMP-16 ([M+H]+, m/z 378.1102)
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The oxidative degradation product OMP-16 at m/z 362 ([M+H]+) with an elemental composition
C17H20N3O4S+ detected at 15.14 min. The increase of 32 Da in molecular weight compared to the
[M+H]+ of the drug suggests that OMP-16 was di oxidative degradation product. The LC-
MS/MS spectrum of [M+H]+ of OMP-16 showed high abundance product ion at m/z 150 (loss of
‘O’ from m/z 166) and low abundance product ions at m/z 314 (loss of SO2), m/z 299 (loss of
CH3 radical from m/z 314), m/z 211 (loss of C9H13NO2), m/z 183 (loss of C10H14NO3) and m/z
168 (loss of C8H6N2O3S). The product ions at m/z 314, m/z 211 and m/z 168 support the

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7
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proposed structure of OMP-16. (Supplementary Scheme.S17, Figure S4(e), Table.S9, see
Supporting Information). All these data are consistent with the structure, 4-methoxy-2-((5-
methoxy-1H-benzo[d]imidazole-2-ylsulfonyl)methyl-3,5-dimethylpyridine 1-oxide which has
been proposed for OMP-16.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
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5. Mechanistic pathway for the formation of degradation products
5.1 Hydrolytic degradation condition
1. The degradation product OMP-1, formed by loss of ‘Ó’ from the drug under acidic conditions
may involve a radical cation mechanism [35] . Formation of OMP-2 and OMP-3 can be
explained by abstraction of an acidic proton by ‘N’ of the imidazole ring followed nucleophilic
attack of pyridine leading to cleavage of C-S bond to form an intermediate with free S-OH
group. The consecutive losses of ‘SO’ and H2O molecules can lead to the formation of OMP-1
and OMP-3, respectively. Formation of OMP-4 can be explained by sulfoxide oxygen forms
new O-C bond with the imidazole carbon followed by the loss of ‘S’ leads to from OMP-4. The
degradation product OMP-4 further abstracts acidic proton followed by participation of
rearrangement leads to form OMP-5. (Supplementary Scheme. S18, see Supporting
Information)
2. In the case of base hydrolysis the base OH- ion abstracts the proton from the OMP followed
by hydrolysis leads to form OMP-6, OMP-7 and OMP-8. Formation of OMP-9 can be explained
by OH- ion abstracts the proton followed by nucleophilic attack of pyridine aleading to cleavage
of C-S bond. After which may undergo hydrolysis leads to form OMP-9 and OMP-10.
(Supplementary Scheme. S18, see Supporting Information)
3. Loss of ‘S’ atom from the OMP followed by loss of CH2O and CH4 molecules leads to form
OMP-12 and OMP-14 in neutral hydrolytic reaction. The degradation product OMP-14, which
may participates in neutral hydrolysis reaction leads to form OMP-11. The degradation product

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2
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5
6
7
8
9
OMP-8 was formed by loss of CH2O molecule and formation of OMP-13 was explained by loss
of ‘O’ atom followed by loss of CH2O leads to form OMP-13. (Supplementary Scheme. S19,
see Supporting Information).
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
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60
5.2. Oxidative degradation condition
Formation of oxidative degradation product OMP-15 can be explained by nucleophilic attack of
peroxide on sulfoxide of the drug of the drug followed by loss of H2O molecule leads to form
OMP-15. This degradation product OMP-15 further oxidised by H2O2 leads to form OMP-16
(Supplementary Scheme. S19, see Supporting Information).
6. Synthesis and NMR studies of oxidative degradation products
6.1. Procedure for synthesis of OMP-15 and OMP-16
For the synthesis of OMP-15 and OMP-16, 1 mmol and 2 mmol m-CPBA (172mg, 344mg)
respectively was added separately to each of the two solutions of OMP (346 mg, 1 mmol) in 3ml
of DCM. Mixture of the reaction was stirred for overnight at room temperature. After the
completion of the reaction, the sample mixture was quenched with NaHCO3 followed by
extraction with ethyl acetate (15 ml) and separated organic layer to be collected. The collected
sample was dried over on anhydrous magnesium sulfate and concentrated under vacuum to get
dry product. The sample residue was purified with silica gel column chromatography (DCM–
MeOH, 9.5:0.5 %) to afford pure products OMP-15 and OMP-16 as solid. Then the pure solid
compounds were subjected to 1D and 2D NMR experiment studies. Based on the NMR
experiments, structures of OMP-15 and OMP-16 were confirmed (Supplementary Figure S5-
S13, see Supporting Information). (Scheme 1).
6.2. Structural elucidation of OMP-15 and OMP-16 by NMR experiments
1H NMR (1D and 2D) experiments, were performed on a 400 MHz (Avance 400 MHz, Bruker)

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spectrometer using CD3OD as a solvent. Chemical shift values were reported on delta scale in
ppm using solvent residual signal as internal standard.
7
8
9
Structures were fully characterized based on extensive analysis of 1D and 2D NMR experiments
including 2-D Double Quantum Filtered Correlation Spectroscopy (DQF-COSY), Nuclear
Overhauser Effect Spectroscopy (NOESY), Heteronuclear-Single Quantum correlation (HSQC)
and Heteronuclear-multiple Quantum correlation (HMBC) experiments. Molecular formula of
protonated OMP-15 was deduced as C17H19N3O4S by HRMS .
10
11
12
13
14
15
16
17
18
19
20
21
22
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53
54
55
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57
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59
60
The distinctive singlet at 7.98 ppm in OMP-15 due to H13 was used to initiate the assignments
with the help of DQF-COSY and NOESY experiments. The chemical shift at δ 2.20 ppm was
assigned to 14-CH3 with the help of strong nOe correlation, H13/14-CH3 (δ 2.20 ppm). The
remaining methyl, 16-CH3 and 15-OCH3 appeared at δ 2.19 and δ 3.69 ppm. In addition the nOe
correlations, 14-CH3/15-OCH3 and 15-OCH3/16-CH3 provide further affirmation. It was easy to
distinguish H1 and H6 protons observed as double doublet at δ 7.01 and doublet δ 7.55 ppm,
respectively. The close proximity of H1/2-OCH3, supporting to fixed as 2-OCH3 at δ 3.84 ppm.
The HRMS and the NMR spectroscopic data adequately support the proposed structure of OMP-
15. (Figure.3)
Figure.3: The structure of OMP-15 along with the characteristic NOE correlations represented as
double headed arrows.

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The molecular formula of OMP-16 (m/z 378 [M+H]+) was deduced as C17H19N3O5S by HRMS.
Its NMR data were similar to those of OMP-15, indicating the presence of an identical moiety.
The chemical shifts (Supplementary Table S10, see Supporting Information) of protons
varying due to the presence of additional oxygen atom and the HRMS and NMR spectroscopic
data adequately supports the proposed structure of OMP-16 as shown in the Figure.4.
(Supplementary Table S11, see Supporting Information)
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11
12
13
14
15
16
17
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20
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60
Figure.4: The structure of OMP-16 along with the characteristic NOE correlations represented as
double headed arrows.
7. MTT ASSAY
All the test compounds were treated to two different normal cells. The standard OMP was found
to be more toxic compared to OMP-15 and OMP-16. In standard OMP, where, 50% inhibition
of cells were noticed at 32.85 µg and 28.21 µg in HEK-29 and NIH3T3 cell lines. In case of
OMP-15, it was found to be at 62.70 µg and 56.40 µg and for OMP-16 at 57.05 µg and 63.76
µg in HEK-29 and NIH3T3 cell lines. However, when compared to the standard OMP, both
OMP-15 and OMP-16 were found to have relatively lesser toxic effects towards normal cells.
(Supplementary Table S12, see Supporting Information)

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8. In silico toxicity studies
5
6
7
8
The potential toxicity of OMP and its degradation products were assessed by ProTox-II [36]. It
predicts toxicity of chemicals into different levels such as oral toxicity, organ toxicity
(hepatotoxicity), toxicological endpoints (such as mutagenicity, carcinotoxicity, cytotoxicity and
immunotoxicity), toxicological pathways and toxicity targets.
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
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27
28
29
30
31
32
33
34
35
36
37
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The predicted toxicity of the molecule having data point with a confidence below 70% are
omitted whereas active predicted target with the input molecule are shown in bold predicted tag.
The toxicity results of input molecule of OMP and its degradation products are shown in the
Table.2((A),(B),(C)). Organ toxicity, hepato toxicity, of OMP-7 is predicted with confidence
score 0.74 and the toxicological endpoint, carcinogenicity, of OMP and OMP-8 are predicted
with confidence score 0.81 and 0.79. The degradation products OMP-2, OMP-3, OMP-9, OMP-
11, OMP-14 and OMP-16 have adverse effects on the immune system are predicted with
confidence score 0.99, 0.97, 0.86, 0.94, 0.98 and o.92. Tox21 Nuclear receptor signaling
pathways, aryl hydro carbon receptor(AhR), of OMP, OMP-1, OMP-6, OMP-7, OMP-8, OMP-
13 and OMP-15 are predicted with confidence score 1, 0.74, 0.83, 0.74, 0.98, 0.73 and 0.85.
Additionally, two different targets, Prostaglandin G/H Synthase 1 and Amine Oxidase A are
predicted as toxicity targets for OMP, OMP-1, OMP-6, OMP-8, OMP-13, OMP-15 and OMP-
16, DP-3, DP-4 and DP-6 with probable binding. ProTox-II provides insight that there is a
feasibility that OMP degradation products can be active for multiple toxicity endpoint and
thereby resulting in severe toxic effects.

Page 27 of 34
New Journal of Chemistry
Table 2(A): Toxicity prediction of OMP and its degradation products
1
2
3
OMP and its degradation products
Classification
Target
4
5
6
7
8
9
OMP
4
OMP-1
5
OMP-2
4
OMP-3
2
OMP-4
4
OMP-5
4
Toxicity Class
Predicted LD50
Oral toxicity
Hepatotoxicity
Carcinogenicity
Immunotoxicity
Mutagenicity
Cytotoxicity
5000mg/kg
Inactive
Inactive
Inactive
Inactive
Inactive
Active2
330mg/kg 33mg/kg
1250mg/kg
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
840mg/kg
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
1400mg/kg
Inactive
Active1a
Inactive
Inactive
Inactive
Active1b
Organ toxicity
Inactive
Inactive
Active3
Inactive
Inactive
Inactive
Inactive
Inactive
Active4
Inactive
Inactive
Inactive
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Toxicity end points
Toxicity end points
Toxicity end points
Toxicity end points
Aryl hydrocarbon Receptor
(AhR)
Tox21-Nuclear receptor signaling pathways
Tox21-Nuclear receptor signaling pathways
Tox21-Nuclear receptor signaling pathways
Androgen Receptor (AR)
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Androgen Receptor Ligand
Binding Domain (AR-LBD)
Tox21-Nuclear receptor signaling pathways
Tox21-Nuclear receptor signaling pathways
Aromatase
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Estrogen Receptor Alpha (ER)
Estrogen Receptor Ligand
Binding Domain (ER-LBD)
Tox21-Nuclear receptor signaling pathways
Tox21-Nuclear receptor signaling pathways
Peroxisome Proliferator
Activated Receptor Gamma
(PPAR-Gamma)
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Nuclear factor (erythroid-
derived 2)-like 2/antioxidant
responsive element (nrf2/ARE)
Tox21-Stress response pathways
Heat shock factor response
element (HSE)
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Tox21-Stress response pathways
Tox21-Stress response pathways
Mitochondrial Membrane

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Potential (MMP)
1
2
3
4
5
Phosphoprotein (Tumor
Supressor) p53
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Tox21-Stress response pathways
Tox21-Stress response pathways
ATPase family AAA domain-
containing protein 5 (ATAD5)
6
7
8
9
AOFA
PGH1
AOFA
PGH1
No
binding
No binding
No binding No binding
Toxicity Target
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Table 2(B): Toxicity prediction of OMP and its degradation products
OMP and its degradation products
Classification
Target
OMP-6
4
OMP-7
4
OMP-8
5
OMP-9
4
OMP-10
4
OMP-11
5
Toxicity Class
Predicted LD50
Oral toxicity
Hepatotoxicity
Carcinogenicity
Immunotoxicity
Mutagenicity
Cytotoxicity
500mg/kg
Active6a
Inactive
Inactive
Inactive
Inactive
Active6b
5000mg/kg 1000mg/kg 1000mg/kg 3200mg/kg
1400mg/kg
Inactive
Inactive
Inactive
Inactive
Inactive
Active5
Organ toxicity
Inactive
Active7a
Inactive
Inactive
Inactive
Active7b
Inactive
Inactive
Active8
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active9
Inactive
Inactive
Inactive
Toxicity end points
Toxicity end points
Toxicity end points
Toxicity end points
Tox21-Nuclear receptor signaling
pathways
Aryl hydrocarbon Receptor (AhR)
Androgen Receptor (AR)
Tox21-Nuclear receptor signaling
pathways
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Tox21-Nuclear receptor signaling
pathways
Androgen Receptor Ligand Binding
Domain (AR-LBD)
Tox21-Nuclear receptor signaling
pathways
Aromatase
Tox21-Nuclear receptor signaling
pathways
Estrogen Receptor Alpha (ER)

Page 29 of 34
New Journal of Chemistry
Tox21-Nuclear receptor signaling
pathways
Estrogen Receptor Ligand Binding
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
1
2
3
4
5
6
7
8
Domain (ER-LBD)
Tox21-Nuclear receptor signaling
pathways
Peroxisome Proliferator Activated
Receptor Gamma (PPAR-Gamma)
Nuclear factor (erythroid-derived 2)-like
2/antioxidant responsive element
(nrf2/ARE)
Tox21-Stress response pathways
Tox21-Stress response pathways
9
Heat shock factor response element
(HSE)
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
Mitochondrial Membrane Potential
(MMP)
Tox21-Stress response pathways
Tox21-Stress response pathways
Phosphoprotein (Tumor Supressor) p53
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
ATPase family AAA domain-containing
protein 5 (ATAD5)
Tox21-Stress response pathways
Toxicity Target
AOFA
PGH1
No binding
AOFA
PGH1
No binding No binding No binding
Table 2(C): Toxicity prediction of OMP and its degradation products
OMP and its degradation products
Classification
Target
OMP-12
4
OMP-13
5
OMP-14
4
OMP-15
4
OMP-16
4
Toxicity Class
Predicted LD50
Oral toxicity
Hepatotoxicity
5000mg/kg
Inactive
Inactive
Inactive
Inactive
Inactive
Active10
780mg/kg 1400mg/kg
1400mg/kg
Inactive
Inactive
Active13
Inactive
Inactive
Inactive
500mg/kg
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Organ toxicity
Inactive
Inactive
Active11
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Inactive
Active12
Toxicity end points
Toxicity end points
Toxicity end points
Toxicity end points
Carcinogenicity
Immunotoxicity
Mutagenicity
Cytotoxicity
Aryl hydrocarbon Receptor (AhR)
Tox21-Nuclear receptor signaling