Synthesis and Biological Assessment of Pyrrolobenzoxazine Scaffold as a Potent Antioxidant

Article abstract of DOI:10.1021/acs.jmedchem.9b00717

Reduction of mitochondrial oxidative stress-mediated diseases is an important pharmaceutical objective in recent biomedical research. In this context, a series of novel pyrrolobenzoxazines (PyBs) framework with enormous diversity (compounds 5a-w) was synt

Full text of DOI:10.1021/acs.jmedchem.9b00717

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Article
Synthesis and Biological Assessment of
Pyrrolobenzoxazine Scaffold as a Potent Antioxidant
Tania Kundu, Bharati Bhattacharjee, Subhenjit Hazra, Arnab
K. Ghosh, Debasish Bandyopadhyay, and Animesh Pramanik
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00717 • Publication Date (Web): 17 Jun 2019
Downloaded from http://pubs.acs.org on June 17, 2019
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Synthesis and Biological Assessment of Pyrrolobenzoxazine Scaffold as a
Potent Antioxidant
†
‡
†
‡
Tania Kundu, Bharati Bhattacharjee, Subhenjit Hazra, Arnab K. Ghosh, Debasish
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‡
,†
Bandyopadhyay,* and Animesh Pramanik*
†Department of Chemistry, University of Calcutta, 92, A. P. C. Road, Kolkata-700 009,
India.
‡Department of Physiology, Oxidative Stress, and Free Radical Biology Laboratory, University
of Calcutta, 92, A. P. C. Road, Kolkata-700 009, India.
AUTHOR INFORMATION
*Corresponding Authors
Animesh Pramanik, Department of Chemistry, University of Calcutta, 92, A. P. C. Road,
Kolkata-700 009, India, Fax: +91-33-2351-9755; Tel: +91-33-2484-1647. E-mail:
animesh_in2001@yahoo.co.in.
Debashis Bandyopadhyay, Department of Physiology, University of Calcutta, 92 A. P. C. Road,
Kolkata, 700 009, India. E-mail: debasish63@gmail.com
ORCID
Animesh Pramanik: 0000-0002-0308-7186
Debasish Bandyopadhyay: 0000-0001-7993-0777
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ABSTRACT
Reduction of mitochondrial oxidative stress-mediated diseases is an important pharmaceutical
objective in recent biomedical research. In this context, a series of novel pyrrolobenzoxazines
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(PyBs) framework with enormous diversity (compounds 5a-w) was synthesized by employing a
low-temperature greener pathway and antioxidant property of the synthesized compounds was
successfully demonstrated on preclinical model goat heart mitochondria, in vitro. Copper-
ascorbate (Cu-As) was utilized as an oxidative stress generator. Out of screened PyBs,
compound possessing -OH and -OMe group on benzene nucleus along with pyrrolobenzoxazine
core moiety (compound 5w) displayed magnificent antioxidant property with a minimum
effective dose of 66 μM during the biochemical assessment. The ameliorative effect of
synthesized pyrrolobenzoxazine moiety on levels of biomarkers of oxidative stress, antioxidant
enzyme, activities of Krebs cycle and respiratory chain enzymes, mitochondrial morphology and
Ca2+ permeability of mitochondrial membrane was investigated in presence of Cu-As.
Furthermore, the binding mode of Cu-As by compound 5w was explored successfully using
isothermal titration calorimetry (ITC) analysis.
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INTRODUCTION
In past few decades, a large amount of attention has been paid over to defend against the extreme
assembly of reactive oxygen species (ROS) which interact rapidly with natural macromolecules
in meticulous tissues by chain process, thereby causing them incapable towards primary function
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due to oxidative damage. Irregular assembly of mitochondrial ROS and oxidative damage may
have a contribution in cardiovascular diseases (CVDs) like high blood pressure, coronary artery
disease, etc. as well as play a pivotal role for obesity, insulin resistance, and ageing. Oxidative
stress is first and foremost associated with annihilation of mitochondrial function by production
of numerous reactive species including superoxide anion radical (O2• −), hydroxyl radical ( OH)
and hydrogen peroxide (H O ) or a significantly decrease the effectiveness of natural antioxidant
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defenses, such as glutathione. Endogenous and exogenous sources of ROS are mainly side
products of mitochondrial respiration, Haber-Weiss reaction, Fenton reaction and also generated
due to ageing or different types of radiation, ultrasound, inflammation, drugs, human diet even
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,5
2+
3+
shear stress (homogenization). Transition metal ions (Cu and/or Fe ) interact with H O and
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O via Fenton and Haber Weiss reaction to generate a highly-reactive oxidizing species, mainly
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•
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OH which gets accumulated in organs. Biological system has a capability to provide a shield
against ROS with enzymatic and non-enzymatic antioxidants like superoxide dismutase,
glutathione peroxidase, catalase, vitamin C, vitamin E, glutathione, etc. by the generation of
hydrogen atom in stoichiometric fashion.7-9 Mitochondrion, being a semi-autonomous organelle
is well recognized to be implicated for irregular ROS assembly through one-electron carriers in
the respiratory chain due to its structural vulnerability towards oxidative stress which is
confirmed by enormous information on decreasing level of reduced glutathione (GSH) content,
peroxidation of lipids, especially phospholipids of mitochondrial membranes and oxidative
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deterioration of DNA as well as proteins. Oxidative stress is mainly responsible for different
types of apoptosis and mitochondria-generated ROS possess a major role in this phenomenon as
shown in Scheme 1, since releasing of cytochrome c in the cytoplasm and permeability transition
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pore opening are vital processes in the apoptotic cascade.11,12
Scheme 1. Possible Mechanistic Pathway of Mitochondrial Damage due to ROS Generation
The antioxidants are class of compounds which are able to regulate or restrain the
oxidative damage via interruption of the ROS formation or by the termination of the chain
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reaction via scavenging ROS. In modern research huge application of antioxidant plants are
well recognized for several health benefits, but research works on mitochondrial antioxidant
pharmacophore are in running track till now. The pyrrole fused heterocyclic scaffolds is a central
privileged guest of nature in a huge number of bioactive natural products such as lamellarins14
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and related pyrrole-derived alkaloids like lukianols A and B, ningalins A-D, acortatarins A
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and B , cyclooroidin etc. which possess antioxidant, cytotoxicity, antibiotic and antitumor
activity.15-17 In addition to that benzoxazine framework also serves as a spectrum of bioactivities
such as antioxidant, antihyperlipidemic agents, a potent inhibitor of hepatitis C virus and
antimicrobial activity.22-25 After critically reviewing both pyrrole and benzoxazine derivatives
having antioxidant property, our motto was to unite both the moiety to make a series of fused
pyrrolobenzoxazine compounds which may act as an antioxidant (Scheme 2).
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Scheme 2. Proposed fused pyrrolobenzoxazine moiety as an antioxidant.
Synthesis of such an important pyrrolobenzoxazine framework by employing a simple
eco-friendly methodology with minimum consumption of time, labor, cost as well as an
assessment of its pharmaceutical significance towards ROS evolves as a fascinating research
objective. Since lessening of mitochondrial oxidative stress becomes an important objective in
medicinal science, our research interest has been directed towards the synthesis of a ROS
mediated mitochondrial damage protecting pyrrolobenzoxazine (PyB) moiety in a regioselective
manner under greener route. It is well established that mitochondria generate ROS because of
incessant operation of electron transport chain (ETC) passing through several metabolic
pathways.26,27 Literature survey reveals that Cu-As incubated cardiac mitochondria gets ruptured
mainly by protein carbonylation of mitochondrial membrane, the elevation of lipid peroxidation,
reduction of mitochondrial GSH level, alteration of mitochondrial membrane architecture as well
as potential, even declining normal activities of Krebs cycle enzymes and respiratory chain
enzymes, which leads to cell death.28,29 In this context, synthesis of a novel mitochondriotropic
antioxidant is reported, along with the establishment of its antioxidant property against copper-
ascorbate (Cu-As) mediated oxidative damage in a preclinical biological model like goat heart
mitochondria.
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RESULTS AND DISCUSSION
Chemistry. There are a very small number of reports on the synthesis of pyrrolobenzoxazine
derivatives available in the literature till date.30,31 But those earlier reported methodologies have
some limitations, like prolong reaction time, employment of hazardous acid catalysts, requisite
of high reaction temperature, etc. Therefore, the implication of magnetically separable
Fe O @rGO carbocatalyst might be capable to offer a simple, competent and environmentally
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benign synthetic route towards the formation of pyrrolobenzoxazine derivatives (PyBs). The
synthesis of PyBs involved (a) the initial formation of benzoxazine intermediates 3 through
regioselective condensation of 2-aminophenols 1 with dialkylacetylenedicarboxylates 2 under
neat condition at room temperature upon stirring for 10 min,32,33 and then (b) Michael type
addition of benzoxazine intermediates 3 to β-nitrostyrene derivatives 4, followed by cyclisation
and aromatization (Scheme 3). The step (b) in the synthesis had been effortlessly accomplished
by Fe O @rGO in 1 h under solvent-free reaction condition at 80 ˚C in an open container. The
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utilization of magnetically separable and environment-friendly nanocatalyst Fe O @rGO
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(Supporting Information Figure S2) accelerated the reaction rate for the formation of diversely
substituted PyBs (Table 1, compounds 5a-w) with excellent yield (up to > 80%). Synthesis and
characterization of Fe O @rGO could be found in supporting information (Supporting
3
4
Information Figure S4-S7). All the synthesized compounds (5a-w) along with benzoxazine
intermediate 3 were characterized by using spectroscopic analysis and details could be found in
the experimental section. Formation of PyBs was further confirmed from single crystal X-ray
diffraction study of compound 5k (Supporting Information Figure S1).
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Scheme 3. Synthetic pathway for the formation of pyrrolobenzoxazines (PyBs). Reagents and
optimized reaction conditions are the following: (i) neat condition, 10 min stirring at room
temperature; (ii) Ar-CH=CH-NO / Fe O @rGO, 80 ˚C, 1 h.
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Table 1. One-pot Synthesis of Diversified Pyrrolobenzoxazines (5a-w)
Entry
R1
H
R2
Et
Ar
4-MePh
4-MePh
Ph
Compd
5a
% yielda
81
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H
Me
Et
5b
5c
83
H
87
H
Me
Et
Ph
5d
5e
90
H
4-OMePh
4-OMePh
4-ClPh
4-ClPh
4-FPh
85
H
Me
Et
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87
H
5g
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H
Me
Et
5h
5i
88
H
91
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H
Me
Me
Et
4-FPh
5j
87
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H
4-BrPh
2-ClPh
4-OMePh
5k
5l
82
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H
88
Me
Et
5m
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3-OMe-4-OHPh
(3,4,5-
triOMe)Ph
4-OHPh
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H
H
Me
Et
5s
5t
84
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(3,4,5-
triOMe)Ph
4-OHPh
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Me
H
Et
Me
Et
5u
5v
81
87
89
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3
3-ClPh
H
3-OMe-4-OHPh
5w
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isolated yield of products.
Biology. Evaluation of Antioxidant Properties of Pyrrolobenzoxazines (PyBs), an in vitro
Study. Diversely functionalized PyBs containing different substituents like -Me, -OMe, -OH,
halogens, etc. were synthesized (Table 1). Considering the electronic and steric factors of the
substituents, four representative compounds i.e. compounds 5a, 5t, 5v, and 5w (Figure 1) were
scrutinized for the antioxidant property. Antioxidant activity of PyBs was ascertained by
•
•−
performing several ROS ( OH, O ) scavenging assays, decomposition of H O , ferric reducing
2
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power assay in a chemically defined system. Chemically tested representative PyBs (compounds
a, 5t, 5v, and 5w) displayed notable antioxidant activity. In this present study, tested organic
5
compounds were dissolved in 2:3 ethanol:water (v/v) solution to prepare the stock solution (1
mM) which was further diluted with water as per requirement in due course of antioxidant study.
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Alcohol blank experiment using 2:3 ethanol:water (v/v) solution was performed in every
mentioned assay to analyze the interfering effects of alcohol to ROS scavenging activity. But no
such significant response to ROS scavenging activity was observed in alcohol blank
experiments.
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Figure 1. Structures of biologically screened pyrrolobenzoxazine derivatives.
•
The hydroxyl radical ( OH) scavenging activity was expressed in terms of optical density
at λmax = 532 nm which is the absorption maxima of deoxyribose degradation product. In this
assay, the tested compounds 5a, 5t, 5v, and 5w were employed in a dose-dependent manner and
an appreciable ROS quenching ability in a concentration-dependent pattern was also observed in
each case (Supporting Information Table S1). At higher applied concentration beyond 66 μM,
•
all these tested compounds behaved as a pro-oxidant and scavenged OH at a lesser extent than
the previously applied concentrations. All the screened compounds exhibited their significant
•OH scavenging capability, but compounds 5t and 5v remarkably scavenged about 92% and 90%
respectively against control (0.617 ± 0.02) (#p<0.001) at 66 µM (Supporting Information
Table S1).
•−
The effect of PyBs (compounds 5a, 5t, 5v, and 5w) on the superoxide anion radical (O )
2
scavenging ability was also studied by monitoring the rate of superoxide-mediated epinephrine
auto-oxidation. The O2•− quenching capacity of tested compounds was analyzed in a dose-
dependent way and a significant scavenging activity was noticed in each case (Supporting
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Information Table S1). Compound 5t at 10.72 µM was acting as a pro-oxidant by scavenging
O2•− smaller extent compared to the preceding applied concentrations. At the same time,
compounds 5t and 5v displayed their notable O2•− quenching efficiency about 83% at 8.04 and
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0.72 µM respectively against A.O. of epinephrine (0.0186 ± 0.004) (#p<0.001) (Supporting
Information Table S1).
The H O scavenging activity of PyBs, if any, was also examined in vitro by studying the
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decomposition of H O . There was no such positive response towards H O scavenging activity
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of the compounds 5a, 5t, 5v, and 5w at a concentration of 1.34, 2.68, 5.36, and 10.72 µM.
2
,2-diphenyl-1-picrylhydrazyl (DPPH) leads to the generation of a stable diamagnetic
•
radical in methanolic solution. DPPH scavenging ability of all the tested compounds was studied
by allowing 30 min incubation under the dark condition at room temperature. In this assay,
•
compounds 5a, 5t, and 5v exhibited feeble scavenging ability towards DPPH , but compound 5w
was able to scavenge this radical considerably in a dose-dependent manner (Supporting
Information Table S2) among all other tested compounds. Maximum scavenging ability was
•
observed by compound 5w about 82.3 ± 1.06 % (#p<0.001) at 64 µM against DPPH blank
(
0.811 ± 0.02), whereas rest of the tested contenders illustrated maximum up to 14% DPPH•
scavenging (Supporting Information Table S2).
Along with ROS quenching power, a potent antioxidant should have an effectual
reducing power which accelerates the rate of formation of reduced glutathione (GSH). For this
reason, the ferric reducing assay was carried out with the screened compounds, where only
compounds 5a and 5w reduced potassium ferricyanide in a dose-dependent manner (Supporting
Information Table S2). Compound 5a showed its highest reducing power at 68.48 µM while,
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compound 5w achieved the same activity at much lower concentration (34.24 µM) against blank
(0.05± 0.01) (Supporting Information Table S2).
•
•
Investigation of DPPH and OH scavenging activity is one of the most commonly used
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techniques for exploration of the antioxidant activities of synthesized compounds as well as
standard antioxidant. To have a comprehensive insight into the antioxidant capacity of PyBs with
respect to standard antioxidant Trolox, a vital approach was taken to evaluate IC , which is the
5
0
concentration of synthesized compounds required for 50% scavenging of radical. In the dose-
dependent study, compounds 5a, 5t, 5v, and 5w exhibited maximum ROS scavenging power at a
certain concentration followed by pro-oxidant nature of the respective compounds appeared. IC50
values of the screened compounds were calculated from the plot of % scavenging vs.
concentration of scavenger (Supporting Information Figure S3A and Figure S3B).
•
Noteworthy, in case of OH scavenging, compounds 5a and 5w showed IC almost close to
5
0
Trolox with a comparable TEAC (Trolox Equivalent Antioxidant Capacity) values, while
compounds 5t and 5v possessed the superior antioxidant property with higher TEAC values in
•
comparison with Trolox (Table 2). On the other hand, in case of DPPH scavenging assay, a
promising result was obtained only for compound 5w with a comparable TEAC (0.78) value
(Table 2), whereas such productive outcomes of other tested compounds were not found
(Supporting Information Table S2) which caused the calculation of IC irrelevant for those
5
0
compounds.
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Table 2. 50% Radical Scavenging Activity Concentrations (IC ) of Tested Compounds and
5
0
Their Trolox Equivalent Antioxidant Capacity (TEAC).
•OH
IC (μM)
DPPH•
Tested
0
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Compound
TEAC*
0.95
IC (μM)
TEAC*
--
5
0
50
5
a
10.4 ± 0.59
4.8 ± 0.33
8.4 ± 0.41
11.0 ± 0.36
9.9± 0.71
--
5t
2.06
--
--
5
v
1.18
--
--
5
w
0.90
46.4 ± 3.71
36.4 ± 2.87
0.78
1.00
Trolox
1.00
*
TEAC= Trolox Equivalent Antioxidant Capacity (i.e. IC Trolox/IC tested compound); All
50 50
data are expressed in mean ± SE; All experiments were performed three times (n = 3).
In the perspective of dose-dependent ROS scavenging activity of PyBs (Supporting
Information Table S1 and Table S2) and TEAC values (Table 2) strongly established the
antioxidant property of PyBs. A noticeable affirmative response of compound 5w assured its
•
promising antioxidant activity against a potent diamagnetic DPPH . Compound 5w also
•
displayed appreciable TEAC value in case of DPPH quenching. This observation was further
ensured by performing ferric reducing assay on tested candidates (Supporting Information
•
Table S2). Noteworthy, although the compounds 5t and 5v were a quite better scavenger of OH,
•−
•
O , but they were almost incapable towards DPPH scavenging as well as ferric reducing assay.
2
Compounds 5a and 5w exhibited a potent reducing power but compound 5w was superior
compared to compound 5a as effective concentration was lower in the case of compound 5w
(Supporting Information Table S2). From the standpoints of all performed ROS scavenging,
•
•
reducing power experiments and considering their relative TEAC values in OH and DPPH
scavenging, compound 5w emerges as a proficient scavenger for a wide range of radicals along
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with effective reducing power compared to other three assessed PyBs derivatives. So, after
scrutinizing antioxidant property of tested compounds by chemical assessment, compound 5w
had been further explored in detail biochemical analysis against Cu-As (0.2 mM CuCl , 1 mM
2
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ascorbic acid) induced oxidative damage in cardiac mitochondria (Cmt) by applying doses of
8
.25, 16.5, 33, and 66 μM. Since at higher doses (> 66 μM) all screened compounds appeared as
a pro-oxidant (Supporting Information Table S1), so further biochemical analysis using
compound 5w was not carried out beyond minimum effective dose i.e 66 μM.
Biomarkers of Oxidative Stress
Determination of Reduced Glutathione (GSH) Content, Mitochondrial Lipid Peroxidation
(LPO) Level, and Protein Carbonyl (PCO) Content. Glutathione, natural antioxidant of the cell is
basically tripeptide, ubiquitous and cysteine-rich non-protein thiol (-SH) compound. GSH, being a
vital redox buffer in cell, performs as cofactor and co-substrate of several antioxidant metabolizing
3
4
enzymes, helps in DNA repairing and also scavenges ROS. Cu-As is known to be involved in the
•
•-
generation of OH along with H O and O thereby leading to noticeably decrease in the level of
2
2
2
GSH as well as alterations in the level of lipid peroxidation and protein carbonyl content which
might be due to the inhibition of various antioxidant enzymes.35,36 So, Cu-As incubated
mitochondrial sample showed a significant reduction in the content of GSH level in Cmt (75.89%;
*p<0.001 vs. control). Meanwhile, in co-incubation of mitochondrial sample groups with Cu-As and
different doses of compound 5w, the reduced level of GSH content was found to be significantly
increased in a dose-dependent manner (79.6%, 3.06 fold, 4.66 fold, and 6.3 fold increase
respectively; #p<0.001 vs. Cu-As) (Figure 2A). However, only compound 5w with different doses
did not exhibit any change in comparison with the control. In the present study, replenishment of
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GSH level was observed appreciably when the goat heart mitochondrial sample was co-incubated
with Cu-As and compound 5w (66 µM), thereby curing Cu-As mediated oxidative damage in Cmt.
Most of the oxidized proteins become functionally inert and rapidly get eliminated from the
biological system, but a few numbers of oxidized proteins are regularly accumulated into the body
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which might have a contribution to oxidative stress. Therefore, administration of Cu-As led a
significant elevation in the levels of LPO and PCO content in Cmt (60.11% in LPO and 5.16 fold in
PCO; *p<0.001 vs. control). The elevated level of LPO and PCO content were found to decrease
significantly (12.26%, 19.87%, 28.19%, and 36.43% decrease respectively in LPO as well as
2
1.86%, 44.03%, 64.75%, and 72.13% decrease respectively in PCO; #p< 0.001 vs. Cu-As) when
the mitochondrial samples were co-incubated with Cu-As and different doses of compound 5w
Figure 2B and Figure 2C), whereas compound 5w, alone with elevated concentration was unable
(
to show any deflection on the level of LPO and PCO content compared to control value. From the
above set of experiments on biomarker (GSH, LPO, and PCO), it can be concluded that compound
5
w illustrates potent resistance against oxidative stress.
Figure 2. Ameliorative effect of compound 5w at the different doses against Cu-As induced
alterations in the levels of reduced (A) GSH, (B) LPO, and (C) PCO content in the incubated Cmt.
Samples were measured in control, positive control (5w), Cu-As incubated (Cu-As) and compound
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w protection (Cu-As+5w) groups. These values are expressed as mean ± SE of three independent
experiments; *p<0.001versus control; #p<0.001 versus Cu-As using one way ANOVA.
Determination of the Mn-Superoxide Dismutase (Mn-SOD) Activity in Cardiac
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+
+
Mitochondria. Rapid electron exchange reaction between reduced metals (Fe , Cu ) and
•-
molecular oxygen generates superoxide anions radicals (O ) which are short-lived paramagnetic
2
38
radicals. Superoxide anion radicals are generated by dismutation reaction which subsequently
produces cell-damaging nonradical oxidizing agent H O either spontaneously or catalyzed by
2
2
superoxide dismutase (SOD).39-41 The analysis of the altered activity of manganese superoxide
dismutase (Mn-SOD) was a crucial parameter as Mn-SOD is a first line defensive mitochondrial
antioxidant having active participation in protective mechanism to guard the cell against
oxidative injury. The activity of Mn-SOD was increased significantly to quench surplus O2•-
when the mitochondrial samples being treated with Cu-As (3.36 fold increased; *p<0.001 vs.
control), whereas, the boosted level of Mn-SOD activity was found to be significantly reduced
(decreases 25.72%, 34.06%, 41.59%, and 60.87% respectively; #p<0.001 vs. Cu-As) in a dose-
dependent fashion, when mitochondrial samples were co-incubated with Cu-As along with
different doses of compound 5w (Figure 3). However, compound 5w by itself did not possess
any alteration on the activity of the enzyme, compared to the respective control value.
Amazingly, it was noticed that the adverse effect of oxidative stress on the activity of Mn-SOD
was almost abolished when a dose of 66 µM of compound 5w was employed, due to inactivation
of Mn-SOD.
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Figure 3. Protective effect of compound 5w at the different doses against Cu-As induced
alterations in the activity of Mn-SOD in incubated heart mitochondrial samples. The sample
groups were measured in control, positive control (5w), Cu-As incubated sample groups (Cu-As)
and compound 5w protected (Cu-As+5w) groups. The values are expressed as mean ± SE of
three independent experiments; *p<0.001 versus control; #p<0.001 versus Cu-As using one way
ANOVA.
Measurement of Pyruvate Dehydrogenase and Other Krebs Cycle Enzymes Activities and
Inhibition Pattern. Notably, a miserable condition appears in Krebs cycle enzymes by declining
the activities of NAD-linked enzymes like pyruvate dehydrogenase (PDH), isocitrate
dehydrogenase (ICDH), α-ketoglutarate dehydrogenase (α-KGDH), and FAD-linked enzyme i.e.
succinate dehydrogenase (SDH) in Cu-As induced Cmt, due to the excessive accumulation of
ROS. Mitochondrial Krebs cycle enzymes play a vital role in cellular defense against oxidative
stress-mediated damages apart from playing a crucial role in ATP synthesis. So, incubation of
Cmt with Cu-As caused a significant reduction in the activity of PDH, ICDH, α-KGDH, and
SDH when compared with the control (reduction of 66.25%, 65.58%, 69.78%, and 71.51%
respectively; *p<0.001 vs. control) (Figure 4A, Figure 4B, Figure 4C, and Figure 4D). These
activities of the enzymes were found to be significantly elevated [(increment of 49.31%, 94.52%,
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.94 fold, and 3.82 fold in case of PDH), (increase of 37.73%, 75.47%, 2.4 fold, and 3.52 fold in
ICDH), (elevation of 43.09%, 85.36%, 2.74 fold, and 4.62 fold in α-KGDH) (increment of
51.66%, 2.08 fold, 3.06 fold, and 5 fold in SDH; #p<0.001 vs. Cu-As)] in Cmt samples co-
incubated with Cu-As and different doses of compound 5w (Figure 4A, Figure 4B, Figure 4C,
and Figure 4D). Remarkably compound 5w only remain ineffective towards the alteration of
these enzymatic activities. In order to examine the nature of inhibition pattern of the activities of
Krebs cycle enzymes the Lineweaver Burk Double Reciprocal Plot (LBDR) was conducted with
increasing concentration of substrate [0.0625, 0.125, 0.25, and 0.5 mM sodium pyruvate in case
of PDH; 0.0625, 0.125, 0.25, and 0.5 mM isocitrate in case of ICDH; 0.0625, 0.125, 0.25, and
0.5 mM α-ketoglutarate for α-KGDH and on the other hand 0.01, 0.02, 0.04, and 0.08 mM
concentration of succinate was used as substrate in case of SDH] by treating of mitochondrial
samples with Cu-As and compound 5w at 66 μM concentration. The LBDR plot of all Krebs
cycle enzymes showed that upon treatment of Cmt with Cu-As, a significant decrease in both
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Vmax and K as compared to control that was indicating towards Cu-As mediated uncompetitive
m
inhibition (Table 3). However, when the Cmt was co-incubated with compound 5w (66 µM) in
the presence of Cu-As, the Vmax and K of all these enzymes were significantly protected from
m
being altered. The only compound 5w treated mitochondria, by itself did not show any
significant decrease from the activities of these enzymes of the control group (Figure 4E, Figure
4
F, Figure 4G, and Figure 4H).
Analysis of the Mitochondrial Intactness Applying Janus Green B Stain. ROS induced lipid
peroxidation and protein carbonylation in mitochondrial membrane lead to an osmotic imbalance
which is associated with mitochondrial death. Furthermore, intensification of this studies on the
basis of the structural and functional viability of mitochondria by Janus green B staining and
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variation in SDH activity were monitored with a co-incubated system containing Cu-As and
compound 5w in Cmt. Janus green B, a basic dye fluoresces as green in case of oxygenated
living mitochondria.42 Mitochondrial intactness was examined by fluorescence intensity (FI)
measurement employing Janus green B staining. Significantly it was found that when compound
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w was solely incubated with Cmt, any obvious change in fluorescence intensity was not
observed (Figure 4I and Figure 4J) which may imply the possible bio-compatible characteristics
of compound 5w in Cmt. This beneficial finding could also be explained from SEM analysis
(Figure 6B) where any kind of rapture in mitochondrial surface topology was not noticed. A
significant reduced FI was noticed following incubation of Cmt with Cu-As (70.05% decreased;
*p<0.001 vs. control) and a significant shielding effect of compound 5w was observed when the
Cmt was co-incubated with Cu-As along with compound 5w at a concentration of 66 μM (4.42
fold increased; #p<0.001 vs. Cu-As) (Figure 4J). However, the Janus Green B staining images
indicated that a substantial decrease in fluorescence was observed when Cmt was treated with
Cu-As, whereas co-incubated system containing Cu-As and compound 5w possessed significant
fluorescence (Figure 4I). Therefore staining experiment, as well as monitoring SDH activity
(Figure 4D), support the shielding ability of compound 5w against oxidative damage.
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Figure 4. Protective effect of compound 5w at the different doses on Cmt against Cu-As induced
alterations on (A) PDH, (B) ICDH, (C) α-KGDH, and (D) SDH activities. Incubated Cmt
samples were measured in control, positive control (5w), Cu-As incubated mitochondrial sample
(Cu-As) and compound 5w protection (Cu-As+5w) groups. Inhibition pattern of Krebs cycle
enzyme activities (E) PDH, (F) ICDH, (G) α-KGDH, and (H) SDH in Cmt with Cu-As followed
by Lineweaver Burk Double Reciprocal Plot (LBDR) (n = 3 independent experiments). (I) Janus
Green B staining images of mitochondrial smears of control, the only compound 5w treated, Cu-
As treated and Cu-As and compound 5w treated groups at 20X magnification. All images are
represented by performing three independent experiments. (J) Fluorescent intensity of Cmt
samples stained with Janus Green B. The values are expressed as mean ± SE by performing three
independent experiments; *p<0.001 versus control; #p<0.001 versus Cu-As using one way
ANOVA
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Table 3. Kinetic Constants (Vmax and K ) of PDH and Other Krebs Cycle Enzymes (ICDH,
m
α-KGDH, SDH) Inhibition by LBDR Plot Using Straight Line Equation
Tissue
Variables
Groups
Cu-As
0
1
2
3
4
5
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9
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9
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0
Control
0.19
0.08
0.44
0.34
0.3
5w
Cu-As+5w
0.27
PDH
ICDH
Vmax
0.21
0.14
0.87
0.34
0.5
0.14
0.07
0.11
0.08
0.11
0.43
0.84
0.02
Km
Vmax
Km
0.08
Cardiac
0.39
Mitochondria
0.34
α-KGDH
SDH
Vmax
Km
0.25
0.59
3.76
0.06
0.12
7.19
0.06
0.05
Vmax
Km
3.21
0.06
Measurement of the Mitochondrial Respiratory Chain Enzymes Activity. One of the most
essential candidates of ETC is cytochrome c which is implanted in the phospholipid bilayers of
the mitochondrial inner membrane by the hydrophobic interactions. When ROS peroxidizes the
phospholipid molecules of the bilayer membrane, the apoptotic pathway gets activated by
releasing cytochrome c outside from the cell.12 Thus to explore the defending effect of
compound 5w by monitoring altered activities of cytochrome c oxidoreductase and cytochrome c
oxidase was an important object. So, administration of Cmt with Cu-As caused an apoptotic
alteration by declining the activities of cytochrome c oxidoreductase and cytochrome c oxidase
significantly (47.82% and 72.41% decrease in cytochrome c oxidoreductase and cytochrome c
oxidase respectively; *p<0.001 vs. control). Both the parameters illustrated a dose-dependent
increment in the activities level of cytochrome c oxidoreductase (23.5%, 35.17%, 49.83% and
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5.17% increased; #p< 0.001 vs. Cu-As) cytochrome c oxidase (increase of 56.25%, 2.36 fold,
.5 fold, and 5.12 fold; #p< 0.001 vs. Cu-As) in co-incubated Cmt samples with Cu-As and
different doses of compound 5w (Figure 5A and Figure 5B).
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Figure 5. Ameliorative effect of compound 5w at the different doses against Cu-As mediated
alterations in (A) cytochrome c oxidoreductase and cytochrome c oxidase (B) in incubated Cmt.
Sample groups were studied in control, positive control (5w), Cu-As incubated (Cu-As) and
compound 5w protected (Cu-As+5w) groups. The values are expressed as mean ± SE by
performing three independent experiments; *p<0.001 versus control; #p<0.001 versus Cu-As
using one way ANOVA.
However, compound 5w alone made no deflection on the activities of these respiratory
enzymes. Finally, apoptotic-like adaptation caused by oxidative damage in ETC linked enzymes
were eradicated during the administration of Cmt with Cu-As and compound 5w at 66 μM.
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Scanning Electron Microscopic Analysis. It is well established that incubation of mitochondria
with Cu-As emerges a pronounced harmful effect on activities of some Krebs cycle enzymes
including succinate dehydrogenase (SDH) which corresponds to a structural distortion in
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mitochondria. Such irregular surface topology of Cmt was further confirmed from scanning
electron microscopic (SEM) analysis. SEM images of Cmt depicted convoluted membranes and
perforated surface covering blebs when the Cmt samples were incubated with Cu-As (Figure
6
C). The reduced enzymatic activity and Cmt membrane morphology were found to be
ameliorated when the Cmt were co-incubated with Cu-As and 66 µM of compound 5w (Figure
D and Figure 6D), probably because of cytoprotection capability of compound 5w. This
4
phenomenon strongly designates that compound 5w is able to protect mitochondria from Cu-As
induced toxic injury by itself being a quencher of ROS.
Figure 6. SEM images of mitochondria in (A) control, (B) positive control of compound 5w, (C)
Cu-As incubated stressed Cmt and (D) protection of compound 5w (66 µM) in Cu-As treated
mitochondria. The images are representative of three independent experiments.
Measurement of Ca2+ Permeability of the Mitochondrial Membrane Through Flow
Cytometry. It is documented that mitochondrial membrane potential mainly is associated with
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ETC generated proton gradient in the inner mitochondrial membrane and ATP synthesis. The
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administration of Cu-As in Cmt causing alterations in the respiratory chain enzymes activity may
+
perturb the permeability of proton (H ) in the inner membrane of mitochondria, which leads to
short-circuiting of electrochemical gradient, confirmed by flow cytometry study. Calcein-AM is
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2+
2+
a dye that makes fluorescence upon binding with Ca . Permeability of Ca across the inner
mitochondrial membrane was expressed in terms of calcein FITC-A median. Fluorescence
2
+
intensity (FI) of calcein dye due to Ca accumulation in Cu-As incubated mitochondrial sample
for 30 min increased (25.32% increased vs. control) in comparison to control. But co-incubation
of compound 5w and Cu-As with Cmt for 60 min displayed a protective action against Cu-As
induced alteration at the control level (15% decreased vs Cu-As 30 min) (Figure 7A). In case of
Cu-As incubated Cmt (for 30 min) there was an elevation of FI in calcein compared to control,
that was confirmed by rightward shifting of mean fluorescence intensity (MFI) in the histogram
plot (Figure 7B), which was protected from being altered upon co-incubation of mitochondria
with compound 5w at 66 µM concentration. On the other hand, the mitochondrial sample when
incubated with Cu-As for 60 min showed a decline in the calcein fluorescence.
Incubation of Cmt only with compound 5w at the same concentration did not show any
adverse effect of MFI in the histogram. In case of Cu-As treated mitochondrial sample the higher
fluorescence of calcein was confirming about increasing concentration of Ca2+ in mitochondria,
also indicating towards the increased depolarization of mitochondria in presence of Cu-As,
which was prevented in presence of compound 5w (Figure 7A and Figure 7B). But in case of
Cu-As treated mitochondria for 60 min the major cause of drastic fall of calcein fluorescence
was possibly due to exhaustion of mitochondria.
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Figure 7. (A) Flow cytometric analysis of cardiac mitochondria upon calcein-AM treatment by
performing three independent experiments. Data were expressed as mean ± SE. (B) Histogram of
calcine-AM strained Cmt depicts control (C-P1), positive control (PC-P1), Cu-As 30 min
incubation (CUAS30′-P1), Cu-As 60 min incubation (CUAS60′-P1) and compound 5w along
with Cu-As (PROTECTION-P1).
2
+
Isothermal Titration Calorimetry (ITC) Study of Cu , Ascorbate, and Compound 5w.
Isothermal titration calorimetry (ITC) was carried out to analyze thermodynamic binding mode
2
+
of Cu , ascorbic acid and Cu-As with compound 5w through monitoring heat
released/absorption at a constant temperature. When Cu2+ was loaded in the ligand cell and
injected on the sample cell containing compound 5w at a concentration of 66 µM a constant
decrease in heat change was observed. But upon injection of only ascorbic acid in ligand cell and
compound 5w (66 µM) containing sample cell, a gradual decrease in heat change was observed,
that was tended towards saturation. When Cu2+ and ascorbic acid were taken together in the
ligand cell and injected into the sample cell containing compound 5w (66 µM), then there was a
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gradual decrease in heat change, which was reached to saturation gradually (Figure 8A, Figure
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B, and Figure 8C).
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Figure 8. A representative isothermal titration calorimetric (ITC) dataset of compound 5w at a
concentration of 66 µM in heat change vs. time titration curve. The each peak represents an
injection of the ligand into the sample cell containing compound 5w, using CuCl as a ligand in
2
(
A), employing ascorbic acid as a ligand in (B) and using CuCl and ascorbic acid together in
2
ligand cell (C). The area under the curve indicating the amount of heat change per second (ΔH)
in terms of kcal mol-1 of injectant against molar ratio is illustrated in bottom of each
representative curve (top curve) of the respective graphs.
2
+
ITC data expressed that an exothermic, sequential binding took place between Cu and
compound 5w as evident from Figure 8A. When the same experiment was performed using
2
+
ascorbic acid instead of Cu in the ligand cell, an exothermic, single-site binding profile was
also observed (Figure 8B). Finally, CuCl and ascorbic acid were taken together in ligand cell
2
and compound 5w (66 µM) in the sample cell, a strong binding profile was obtained which was
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supported from the formation of another exothermic and sequential binding spectrum (Figure
8
C). In each binding experiment net change in enthalpy was negative which implied a
spontaneous reaction, even sequential binding mode recommended that all binding sites of
compound 5w did not get saturated at a time. The probable reason behind this phenomenon is
supposed to be the binding of one molecule of Cu-As with compound 5w that leads to open up
another site of compound 5w for binding and the reaction continues subsequently.
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Although synthesized pyrrole and benzoxazine scaffolds illustrated their beneficial action
to defend oxidative stress according to literature survey,45,46 herein, a productive and promising
result was found against ROS mediated oxidative injury when pyrrole and benzoxazine moiety
were fused together to form compound 5.
CONCLUSIONS
A series of novel pyrrolobenzoxazine derivatives have been synthesized employing an efficient
low-temperature greener synthetic methodology and the mitochondriotropic antioxidant property
was also productively assessed with representative compounds. The presence of pyrrole fused
1
,4-benzoxazine as core moiety along with δ-lactone and various polar substituents in pyrrole
ring is the prime reason behind the ROS scavenging potency. From the thorough biochemical
analysis, pyrrolobenzoxazine compound 5w which possesses hydroxyl (-OH) and methoxy (-
OMe) substituents at benzene nucleus attached to pyrrole ring of pyrrolobenzoxazine core
moiety emerges as a potential contender for further significant therapeutic application in various
mitochondrial dysfunctional diseases.
Moreover, another extraordinary property of pyrrolobenzoxazine compound 5w was
revealed from ITC analysis, the obstruction of reactive oxygen species generation by stressor
molecules (Cu-As) took place through strong and sequential binding between compound 5w and
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Cu-As. Besides the inhibitory profile of compound 5w towards ROS generation, it also exhibited
ROS scavenging efficiency without alteration of mitochondrial membrane morphology as
evident from the microscopic analysis. Compound 5w also played a vital role in replenishment of
GSH level in Cmt, as well as it decreases the elevated level of LPO and PCO contents caused by
stressors. As per our literature survey, this is the very first example of the low-temperature
synthesis of pyrrolobenzoxazine derivatives (PyBs) using the environment-friendly solvent-free
methodology and in vitro study of its antioxidant property over preclinical model isolated goat
heart mitochondria. The underlying beneficial response of pyrrolobenzoxazine remains as an
excellent prospect in further biomedical research.
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EXPERIMENTAL SECTION
Chemical Analysis. All chemicals and reagents were purchased from Sigma-Aldrich, India and
used without further purification as well as standard antioxidant Trolox were purchased from
TCI, India. Melting points were measured in open capillary tubes and were uncorrected. IR
1
13
spectra were recorded using JASCO FTIR-6300 spectrometer. H (300 MHz, 500 MHz) and C
NMR (75 MHz, 126 MHz) spectra were performed on Bruker instrument (300 MHz, 500 MHz)
and 400 MHz (JEOL-JNM-ECZ400S/L1), in CDCl and DMSO-d relative to tetramethylsilane
3
6
(TMS) as an internal reference. HRMS spectra were recorded using Xevo G2-S QTof. Elemental
analyses (C, H, and N) were recorded using Perkin-Elmer CHN analyzer 2400 elemental
analyzer. The single crystal X-ray diffraction data for crystallized compounds were collected
with MoK radiation at 296 K using the Bruker APEX-II CCD System. The crystallinity of
α
synthesized Fe O @rGO nanocatalyst was determined by powder XRD analysis using
3
4
PANalytical, XPERT-PRO diffractometer with CuK (λ= 1.54060) as X-ray source. The
α
morphological analysis was performed by JEOL JSM-7600F Field Emission Scanning Electron
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Microscopy (FESEM) and Transmission Electron Microscopy (TEM) (JEOL JEM 2100 HR with
EELS) at an accelerating voltage of 80-200 kV. The purity of synthesized compounds was
determined by high-performance liquid chromatography (HPLC), employing an Agilent 1200
series HPLC with DAD detector Column C18 (150X4.6 mm) 5 µm. The purity of compounds
was found to be ≥ 96%. All optical densities were recorded using a UV-VIS spectrophotometer
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(Bio-Rad, Hercules, CA, USA).
General Synthetic Procedure for the Preparation of Pyrrolobenzoxazine 5a-w. Synthesis of
pyrrolobenzoxazine derivatives was accomplished by using a one-pot three component
condensation strategy. In the first step, an equimolar mixture of 2-aminophenols 1 (1.0 mmol)
and dialkylacetylene dicarboxylates 2 (1.0 mmol) was taken into a 50 mL round-bottomed flask
and the mixture was stirred at room temperature for 10 min, which led to the formation of
benzoxazines 3. In the final step, β-nitrostyrenes 4 (1.0 mmol), which was synthesized following
4
7
a previously reported method and nanocatalyst Fe O @rGO (30 mg) were added in the reaction
3
4
mixture. The resulting mixture, which was semisolid in nature, was stirred at 80 ˚C in an oil bath
for 1 h. The progress of the reaction was monitored by thin layer chromatography (TLC). After
completion of the reaction, 20 mL of ethyl acetate was added to the reaction mixture and
sonicated. Then Fe O @rGO nanocatalyst was separated by using an external magnet. The
3
4
organic layer was dried over anhydrous sodium sulfate (Na SO ) and concentrated by
2
4
evaporating the solvent in a vacuum to get the crude products. Finally, the crudes were purified
by column chromatography on silica gel (60-120 mesh) using ethyl acetate/hexane as the eluent
to obtain pure compounds 5 (PyBs).
Methyl (Z)-2-(2-oxo-2H-benzo[b][1,4]oxazin-3(4H)-ylidene)acetate (3). The title compound
1
was yellow solid and yield of 98%. H NMR (400 MHz, CDCl ) ppm δ 10.62 (s, 1H), 7.12 - 7.08
3
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(
m, 2H), 6.98 (t, J = 8 Hz, 1H), 6.94 - 6.92 (m, 1H), 5.89 (s, 1H), 3.75 (s, 3H); C NMR (100
MHz, CDCl ) ppm δ 170.33, 155.97, 140.08, 138.22, 125.76, 124.24, 122.88, 117.10, 114.91,
3
+
+
90.76, 51.55. HRMS: calcd for C H NO Na (MNa , ESI ) 242.0429, found 242.0427. mp 167 -
1
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68 ˚C.
Ethyl 4-oxo-2-(p-tolyl)-4H-benzo[b]pyrrolo[1,2-d][1,4]oxazine-3-carboxylate (5a). The title
compound was yellowish white solid and synthesized according to general procedure with a
-1 1
yield of 81%: IR (KBr) 2920, 1734, 1724, 1610, 1420, 1266, 1243 cm . H NMR (300 MHz,
CDCl ) ppm δ 7.59 (s, 1H), 7.57 - 7.54 (m, 1H), 7.33 - 7.25 (m, 3H), 7.24 - 7.22 (m, 2H), 7.15 -
3
13
7
.12 (m, 2H), 4.35 (q, J = 7.2 Hz, 2H), 2.31 (s, 3H), 1.25 (t, J = 7.2 Hz, 3H); C NMR (126
MHz, CDCl ) ppm δ 164.86 , 152.06, 143.04 , 137.76 , 129.41 , 129.19 , 129.08 , 127.60 ,
3
1
27.04 , 125.02 , 122.41, 121.62 , 118.31 , 115.59 , 115.53 , 114.45 , 61.95 , 21.19 , 13.97.
+
+
HRMS: calcd for C H NO Na (MNa , ESI ) 370.1056, found 370.1059. mp 163 ˚C.
2
1
17
4
Methyl 4-oxo-2-(p-tolyl)-4H-benzo[b]pyrrolo[1,2-d][1,4]oxazine-3-carboxylate (5b). The title
compound was yellowish white solid and synthesized according to general procedure with a
1
yield of 83%: H NMR (300 MHz, CDCl ) ppm δ 7.59 (s, 1H), 7.58 - 7.55 (m, 1H), 7.31 - 7.23
3
1
3
(
m, 5H), 7.19 - 7.13 (m, 3H), 3.85 (s, 3H), 2.31 (s, 3H); C NMR (75 MHz, CDCl ) ppm δ
3
1
1
7
65.16, 151.96, 142.99, 137.76, 129.48, 129.30, 128.95, 127.99, 127.49, 127.01, 124.93, 121.90,
21.53, 118.31, 115.38, 114.31, 52.73, 21.08. Anal Calcd for C H NO (MW 333.34 g/mol): C,
2
0
15
4
2.06; H, 4.54; N, 4.20. Found C, 72.01; H, 4.52; N, 4.18. mp 158 - 160 ˚C.
Ethyl 4-oxo-2-phenyl-4H-benzo[b]pyrrolo[1,2-d][1,4]oxazine-3-carboxylate (5c). The title
compound was grayish white solid and synthesized according to general procedure with a yield
1
of 87%: H NMR (300 MHz, CDCl ) ppm δ 7.62 (s, 1H), 7.56 (d, J = 8.7 Hz, 1H), 7.44 - 7.36
3
1
3
(
m, 2H), 7.34 - 7.25 (m, 6H), 4.34 (q, J = 7.2 Hz, 2H), 1.23 (t, J = 7.2 Hz, 3H); C NMR (75
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