Synthesis and biological evaluation of isoindoloisoquinolinone, pyroloisoquinolinone and benzoquinazolinone derivatives as poly(ADP-ribose) polymerase-1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.12.017

A series of novel fused isoquinolinones with isoindoloisoquinolinone, pyroloisoquinolinone, and benzoquinalizinone skeletons were synthesized from corresponding phenethylimides. The isoquinolinone derivatives were evaluated for their protective effect on chicken erythrocytes subjected to oxidative damage. The effect of isoquinolinone derivatives were analysed by estimation of cell viability, antioxidant enzyme activities, DNA damage (comet assay), PARP-1 inhibition assay and molecular docking of the compounds with PARP-1 active site. The compounds CRR-271, CRR-288 and CRR-224+225 showed significant protective effect at 100 μM concentration. The PARP-1 inhibition assay revealed the IC₅₀ values of CRR-271, CRR-288 and CRR-224+225 as <200 nM, further molecular docking studies shows higher binding energies with PARP-1 active site. Interesting findings in this study suggest that the novel isoquinolinone derivatives inhibit PARP-1 activity and protect cells against oxidative DNA damage, which could be implemented in the treatment of inflammatory diseases.

Full text of DOI:10.1016/j.bmc.2014.12.017

Accepted Manuscript
Synthesis and biological evaluation of isoindoloisoquinolinone, pyroloisoqui-
nolinone and benzoquinazolinone derivatives as PolyADP-Ribose Polymer-
ase-1 inhibitors
Arumugam Suyavaran, Chitteti Ramamurthy, Ramachandran Mareeswaran,
Yagna Viswa Shanthi, Jayaraman Selvakumar, Selvaraj Mangalaraj, Muthuvel
Suresh Kumar, Chinnasamy Ramaraj Ramanathan, Chinnasamy
Thirunavukkarasu
PII:
S0968-0896(14)00865-7
DOI:
http://dx.doi.org/10.1016/j.bmc.2014.12.017
BMC 11949
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
1 October 2014
24 November 2014
11 December 2014
Please cite this article as: Suyavaran, A., Ramamurthy, C., Mareeswaran, R., Viswa Shanthi, Y., Selvakumar, J.,
Mangalaraj, S., Suresh Kumar, M., Ramaraj Ramanathan, C., Thirunavukkarasu, C., Synthesis and biological
evaluation of isoindoloisoquinolinone, pyroloisoquinolinone and benzoquinazolinone derivatives as PolyADP-
Ribose Polymerase-1 inhibitors, Bioorganic & Medicinal Chemistry (2014), doi: http://dx.doi.org/10.1016/j.bmc.
2014.12.017
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Synthesis and biological evaluation of isoindoloisoquinolinone, pyroloisoquinolinone
and benzoquinazolinone derivatives as PolyADP-Ribose Polymerase-1 inhibitors
Arumugam Suyavarana, Chitteti Ramamurthya, Ramachandran Mareeswarana, Yagna Viswa
Shanthi a, Jayaraman Selvakumarb, Selvaraj Mangalarajb, Muthuvel Suresh Kumarc
,
Chinnasamy Ramaraj Ramanathanb* and Chinnasamy Thirunavukkarasua*
aDepartment of Biochemistry and Molecular Biology, Pondicherry University, Puducherry
b
605 014, India. Department of Chemistry, Pondicherry University, Puducherry 605 014,
c
India. Centre for advance studies in Bioinformatics, School of Life Sciences, Pondicherry
University, Puducherry- 605 014
* Corresponding authors: Tel.:+91 413 2654872; Fax: +91 413 2655255; E-email address:
tchinnasamy@hotmail.com (C. Thirunavukkarasu); Tel.: +91 413 2654416; Fax: +91-413
2656740; Email: crrnath.che@pondiuni.edu.in (C. R. Ramanathan).
1

Abstract
A series of novel fused isoquinolinones with isoindoloisoquinolinone, pyroloisoquinolinone,
and benzoquinalizinone skeletons were synthesized from corresponding phenethylimides.
The isoquinolinone derivatives were evaluated for their protective effect on chicken
erythrocytes subjected to oxidative damage. The effect of isoquinolinone derivatives were
analysed by estimation of cell viability, antioxidant enzyme activities, DNA damage (comet
assay), PARP-1 inhibition assay and molecular docking of the compounds with PARP-1
active site. The compounds CRR-271, CRR-288 and CRR-224+225 showed significant
protective effect at 100 μM concentration. The PARP-1 inhibition assay revealed the IC50
values of CRR-271, CRR-288 and CRR-224+225 as <200 nM, further molecular docking
studies shows higher binding energies with PARP-1 active site. Interesting findings in this
study suggest that the novel isoquinolinone derivatives inhibit PARP-1 activity and protect
cells against oxidative DNA damage, which could be implemented in the treatment of
inflammatory diseases.
Keywords:
Isoquinolinones; Inflammation; DNA damage; Oxidative stress; PARP-1
2

1. Introduction
Cellular oxidative stress is one of the major causative in the development of
degenerative diseases. It results due to the imbalance between antioxidants and free radicals,
predominantly reactive oxygen species (ROS) within the cell.1 The ROS mainly comprise of
superoxide anions, hydroxyl radicals (OH˙) and hydrogen peroxide (H2O2). ROS interact
with all cellular macromolecules at various levels and impair their function which is the
mainstay for initiation and progression of several degenerative disorders.1,2 Among the ROS,
H2O2 is the most stable, freely diffusible and serves as the source for OH˙, hence it
contributes more to the oxidative insult than the rest of ROS. The oxidative stress in cells is
usually augmented by inflammatory mediators such as interleukins and cytokines leading to
different forms of cell death.1,3
Sustained oxidative stress in chronic inflammatory conditions would eventually lead
to necrosis, which is an uncontrolled and passive process of cellular degeneration resulting
from depletion of ATP content of a cell.4, 5 Damaged cells, further recruit the inflammatory
cells, by releasing the various pro-inflammatory mediators into the surrounding tissues,
which worsen the tissue injury.3 Several molecular mediators have been identified as culprit
for initiation and progression of cell degeneration during necrosis. Poly (ADP-ribose)
polymerases-1 (PARP-1) is one such molecule which has recently gained importance as a key
initiator for necrosis activated by oxidative stress.6
PARP-1 also known as NAD+ ADP-ribosyltransferase-1 or poly[ADP-ribose]
,
synthase-1, is a chromatin-associated, highly conserved enzyme with molecular weight of
116 kDa.7 It gets activated in response to DNA damage, which is initiated due to imbalance
in oxidative stress. The activated PARP-1 binds to damaged DNA and initiates the synthesis
of long branches of poly (ADP-ribose) polymers (PAR chains) using NAD+.8 In most severe
3

and sustained oxidative stress and inflammatory conditions, excessive DNA damage triggers
the hyper activation of PARP-1 and it leads to the depletion of NAD+. Depleted NAD+
eventually leads to exhaustion of ATP reserve of the cell, which results in necrotic cell death.
Inhibition of hyper activated PARP-1 would lead to reduced consumption of NAD+ and
restoration of ATP reserve which in turn prevents the cell death. Hence, PARP-1 inhibitors
are recently gaining more attention as novel therapeutic agents in the treatment of
inflammatory diseases.9-11
Extensive investigations have been conducted in the identification of novel PARP-1
inhibitors. Various approaches to design the scaffolds for PARP-1 inhibitors based on the
prototypical PARP-1 inhibitor benzamide as pharmacophore, which mimics the nicotinamide
moiety of NAD+.12-14 Five PARP-1 inhibitors are currently under the clinical oncology trials
(AG014699, KU59436, BSI-201, INO-1001 and GPI 21016).15,16 According to the structure-
activity relationship studies several molecules with benzamide backbone are enriching the
potent inhibitor for PARP-1 (Fig. 1).
[insert Figure 1]
Fused isoquinolinones are heterocyclic motifs that are formed by the fusion of
benzene ring with isoindolone or pyrolone or piperidone units. The derivatives of
isoquinolinones show diverse biological activities like inhibition of PARP-1,17-19 anti-
inflammatory activity,20 anti-cancer,21 and vaso relaxation22. The wide applicability of
isoquinolinone units against various biological targets prompted us to investigate PARP-1
inhibitory activity of the fused isoquninolinone derivatives. The isoquinolinone derivatives
used in this study are analogues of pharmacologically available alkaloids such as nuevamine
and crispine-A.23-25
4

In vitro exposure of cells to H2O2 for a short period has shown to implicate DNA
damage to the cells. Prolonged exposure to H2O2 has shown to induce irreversible damage to
the cellular DNA.26-28 Hence this in vitro model is considered to mimic cellular pathology
during oxidative stress. In the present study, we evaluated the ameliorative effect of 11
different isoquinolinone derivatives against H2O2 induced oxidative DNA damage in chick
red blood cells (RBCs).
2. Results and Discussion
The PARP-1 activity is increased during inflammatory conditions and leads to
depletion of energy reserves of the cell which results in cell death. Many novel
isoquinolinones have been discovered with anti-inflammatory activity and are gaining
importance due to their potent PARP-1 inhibitory activity.17,18 This is the due reason for
isoquinolinones to gain more attention over existing non-steroidal anti-inflammatory drugs.
Since inflammatory pathway is activated in various diseases, isoquinolinones would gain
importance in future as multi-therapeutic compound. Hence, it is essential that the interaction
of isoquinolinones at cellular and molecular level to be tested extensively.
Unlike regular eukaryotic nucleus, the chicken erythrocytes lack a intricate
nucleoprotein complex, making it a simpler system for investigation of drug interaction at
nuclear level.29 Moreover, the chicken erythrocytes contain active mitochondria which
significantly contribute to internal ROS generation.30 Hence in the current study we analyzed
the protective ability of the chosen isoquinolinone derivatives namely CRR-222, CRR-203,
CRR-211, CRR-271, CRR-204, CRR-228, CRR-227, CRR-213, CRR-238, CRR-267 and
CRR-224+225 (Fig. 1) against H2O2 induced oxidative damage in RBCs. H2O2 induced
cellular toxicity is a standard model to assess the protective effects of pharmacophores
against oxidative stress induced damage.31-33. Previous studies show that chick RBCs exposed
5

to oxidative damage is a suitable model for assessment of antioxidant status upon treatment
of pharmacophores.33-35
2.1. Synthesis of isoquinolinone derivatives
The isoquinolinone derivatives used in this study (isoindoloisoquinolinone,
pyroloisoquinolinone and benzoquinalizinone skeletons) were synthesized from
corresponding phenethylimides via electrophilic activation of imide carbonyl group by
trifluoromethanesulfonic acid (TfOH).36 Phenethylimides undergo intramolecular 6-exo-trig
cyclization with electrophilically activated imide carbonyl group (by TfOH) and generate
fused cyclic N-acyliminium ion. This N-acyliminium ion on treatment with oxygen
nucleophile (H2O) delivers fused hydroxy-isoquinolinones or with hydride nucleophile
(NaBH4) delivers fused isoquinolinones (Fig. 2).
[insert Figure 2]
The rigid bicyclic isoquinolinone CRR-267 synthesis starts from the preparation of
corresponding imide by condensation of 3,4-dimethoxy phenethylamine 11 and anhydride 12.
The anhydride 12 is in turn derived from anthracene and maleic anhydride through Diels-
Alder reaction. The imide 13 under established cyclization condition delivers the rigid
isoquinolinone CRR-267 in 87% yield (Fig. 3). The crystal structure of CRR-267 was
obtained and has been represented in Figure 4.
[insert Figure 3]
[insert Figure 4]
The molecules CRR-203, CRR-222, CRR-211, CRR-271, CRR-204, CRR-228 and CRR-
227 are composed of the essential PARP-1 inhibitory benzamide core fused with isoquinoline
6

skeleton as well as hydroxyl group. The molecules such as CRR-224 and CRR-225 contain
two methoxy substitutions in benzamide core. The molecules CRR-213, CRR-238 and
CRR-267 possess benzylamide unit instead of benzamide in addition to a rigid bicyclic unit.
The presence of these structural features prompted us to examine these benzamide fused
molecules for their activity against PARP-1. The regioisomeric mixtures CRR-224 and
CRR-225 were obtained in 1:1 ratio following the established cyclization procedure.
2.2. Acrindine orange (AO) and Ethidium bromide (EB) staining
AO and EB staining method is superior over other staining methods for detection of
cell viability upon oxidative damage. 37 The viability of RBCs after exposure to 100 µM H2O2
for 1 hour at 37 ºC, was assessed by AO and EB staining. The untreated RBCs and the RBCs
exposed to 0.5% DMSO alone showed viability of about 94%, while the viability of RBCs
exposed to H2O2 alone was reduced to 41%. We have not observed any change in normal
RBCs viability when pre-treated with various concentrations of different fused
isoquinolinone derivatives (25 µM to 100 µM – data not shown). The RBCs were pre-treated
with various concentrations (25 µM to 100 µM) of different fused isoquinolinone derivatives
º
º
for 1 hour at 37 C and then challenged against 100 µM H2O2 for 1 hour at 37 C. We have
observed increased viability of cells pre-treated with all the compounds, which was also dose
dependent. Interestingly the compounds CRR-271 and CRR-224+225 at the concentrations
of 100 μM (Other concentration data’s are not shown) showed viability of <90% (Table 1;
Fig. 5). Other compounds show moderate cell viability ranging from 75 – 87% and the least
(54%) viability was observed for CRR-204 (Table 1). Previous studies have also reported the
oxidative DNA damage by employing AO and EB staining method and have shown similar
results for other pharmacophores.38-39
[insert Table 1]
7

[insert Figure 5]
2.3. Antioxidants and Lipid peroxidation
In the current study, a significant decrease (P≤0.05) in the activities of catalase and
superoxide dismutase (SOD) was found in the RBCs treated with 100 µM H2O2 alone, when
compared to untreated RBCs. Pre-treatment of isoquinolinone derivatives demonstrated
restoration of the antioxidant enzyme activities. RBCs pre-treated with CRR-271, CRR-267
and CRR-224+225 at the concentration of 100 µM showed significant protection (P≤0.05) in
the superoxide dismutase and catalase activities when compared to RBCs treated with H2O2
alone (Table 2).
[insert Table 2]
A significant increase (P≤0.05) in the extent of lipid peroxidation was observed in the
H2O2 alone treated RBCs when compared to the untreated RBCs (Table 2). The RBCs pre-
treated with CRR-271, CRR-228, CRR-267 and CRR-224+225 at the concentration of 100
µM showed significant decrease in lipid peroxidation level when compared to RBCs treated
with H2O2 alone (P≤0.05). Other isoquinolinone derivatives did not show such restoration in
above enzyme activities and lowering of lipid peroxidation level. Vehicle control showed the
values similar to that of normal control in all assays. Previous works have shown that H2O2
induced damage to the membrane via lipid peroxidation can be suppressed by protective
effect of the pharmacophores with antioxidant properties.40 Lipid peroxidation is the one of
the main reasons for aggravation of oxidative damage, since it leads to a chain reaction
producing lipid peroxyl radicals, which ultimately results in altered membrane function and
leakage of cellular contents leading to further inflammatory progression.41-42
8

2.5. Isoquinolinone on genomic DNA damage
Single cell alkaline gel electrophoresis (Comet assay) in normal and DMSO treated
RBCs demonstrated comet pattern with higher percent of DNA in head region (>95%). RBCs
treated with H2O2 alone produced comet with about 45% of DNA in tail depicting damage to
the genomic DNA. Pre-treatment of cells with 100 μM concentration of CRR-222, CRR-
271, CRR-228 and CRR-224+225 showed comet with >90% DNA in head region (Table 3).
CRR-211, CRR-204 and CRR-213 showed negligible effect on DNA protection (P≤0.05).
Similar reports with comet assay on DNA damage and study of protective effects of
antioxidants at nuclear level have been reported earlier.43-45
[insert Table 3]
2.6. PARP-1 inhibition assay
The potential of PARP-1 inhibitory activity of CRR-271, CRR-228 and CRR-
224+225 were evident from the PARP-1 enzyme inhibition assay. CRR-228 and CRR-
224+225 have IC50 values of 138.5 nM and 186.5 nM respectively, while the IC50 value of
CRR-271 is 216.2 nM. Thus the IC50 values of these compounds are comparable to that of
the standard isoquinolinone, such as 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-
isoquinolinone (DPQ) whose IC50 is 53.6 nM. The IC50 value of CRR-238 is <500 nM while
the other isoquinolinone compound used in this study have IC50 values >800 nM making
them less suitable candidates as potent PARP-1 inhibitor. Though IC50 values of these
compounds are in nM concentrations, on the other hand µM concentrations (shows positive
effect in the present study) are necessary to counteract the oxidants such as H2O2 and to gain
access into cellular milieu and bind with active PARP-1 enzyme. Sakaue et al have reported
9

antioxidant activity of isoquinolinones in their study, where they have used 30 µM
concentration of DPQ (IC50-53.5 nM) for co-treatment of rat cerebellar granule cells with
methyl mercury.46
2.7. Interaction of isoquinolinone derivatives with active site of PARP-1
The docking studies revealed that CRR-271, CRR-228 and CRR-224+225 are the
isoquinolinones which form hydrogen bonds with the active site amino acids such Gly 863,
Lys 903, Tyr 907 and Tyr 896 and have binding energy >-7.3 kcal/mol (Table. 4). CRR-271
forms three hydrogen bonds, each with Lys 903, Tyr 907 and Tyr 896 respectively with a
binding energy of -7.88 kcal/mol (Table.4). CRR-228 and CRR-224+225 form hydrogen
bond with Tyr 896 and Gly 863 respectively with binding energy of -7.33 kcal/mol and -8.43
kcal/mol. CRR-267 has binding energy more >-8.00 kcal/mol, however it does not form any
hydrogen bonds with any of the amino acids in the active site, further it has an IC50 value of
>800 nM hence it may not be a suitable competitive inhibitor of PARP-1, though its
inhibition might be through some other mode. On the other hand CRR-271, CRR-228 and
CRR-224+225 have reasonable binding energy, forms hydrogen bonds with active site amino
acids and these compounds have IC50 values 138.5 nM – 216.2 nM, hence we suggest that
CRR-271, CRR-228 and CRR-224+225 may serve as potent PARP-1 inhibitor.
The binding score of DPQ – a standard isoquinolinone was found to be -8.67, which
is comparable to the binding scores of the above compounds. The docking results correlate
well with other biochemical results, suggesting the potency of these compounds to function
as antioxidants and PARP-1 inhibitors. Moreover, the anti-inflammatory property of indole
and pyrrole derivatives gives an added advantage to the isoquinolinones to act synergistically
and efficiently by preventing inflammatory degeneration of the cells in addition to blocking
PARP-1 as required in the treatment of several chronic inflammatory disease.47,48 Hence even
10

though the docking scores of isoquinolinones under study are lower than (but comparable to)
the DPQ, they are better candidates for clinical consideration because of their additional anti-
oxidant property.
[insert Table 4]
3. Conclusion
Present study concludes that the tetrahydroisoquinolinone derivatives CRR-271,
CRR-228 and CRR-224+225 which are novel derivatives of isoquinolinone, protect chick
RBCs against H2O2 induced damage at 100 μM concentration as evident from the cell
viability, antioxidant enzyme activities and lipid peroxidation at cellular and genomic DNA
damage protection at nuclear level. The potential inhibitory activity of CRR-271, CRR-228
and CRR-224+225 is evident from PARP-1 inhibition assay. The docking studies show that
CRR-271, CRR-228 and CRR-224+225 form hydrogen bonds with active site amino acids
suggesting competitive binding with PARP-1 substrate binding site. Further studies are
necessary to establish the molecular interactions and pharmacodynamics of these novel
isoquinolinone derivatives, which would see them as effective synergistic combinations along
with existing anti-inflammatory drugs.
4. Experimental Section
4.1. General
We performed Column chromatography on MerckTM silica gel 100-200 mesh. TLC
(Thin layer chromatography) analyses were facilitated using phosphomolybdic acid stain and
permanganate stain and visualized under UV light with Merck 60 F254 pre-coated silica
11

plates. Melting points reported here are uncorrected and were determined using EZ Melt,
Stanford Research Systems, USA. Infrared spectra were recorded on Thermo Nicolet 6700
FT-IR (Fourier transform infra-red) Spectrophotometer using KBr thin films, and are reported
in frequency of absorption (cm-1). Mass spectra, GC-MS (Gas chromatography – mass
spectrometry), were recorded on Varian GC-MS (CP-3800/Saturn 2200). LDI-MS (Laser
desorption ionization – mass spectrometry) were recorded on ABI Voyager DE-STR mass
spectrometer. HRMS (High resolution mass spectrometry) were measured with micro mass
1
Q-TOF (ESI-HRMS). H and 13C NMR were recorded on Brucker AVANCE 400
spectrometer. All the NMR (Nuclear magnetic resonance) spectra were recorded at room
temperature in CDCl3 or DMSO-d6 using TMS (Tetramethylsilane) as an internal standard.
The chemical shifts are expressed in δ ppm down field from the signal of internal TMS.
Coupling constant (J) values are given in Hz. The splittings in spectra are reported as: s =
singlet, d = doublet, t = triplet, q = quartet, qn = quintet, m = multiplet, dd = doublet of
doublet, ddd = doublet of doublet of doublets, dt = doublet of triplet, td = triplet of doublet, br
s = broad singlet. All the anhydrides are used as purchased. By following our earlier reported
procedure we have synthesized all the isoquinolinone molecules.36
4.2. Typical procedure to synthesize of isoquinolinone molecules
An oven dried two neck round bottom flask bearing septum in side arm was cooled to
room temperature under a steady stream of nitrogen gas flow. The flask was charged with
stirring bar, imide (0.5 mmol, 1 equiv) and dry dichloromethane (5 mL) and cooled down to 0
°C (using ice). To this solution TfOH (0.2 mL, 4 equiv.) was added with stirring. After
completion of reaction (monitored by TLC), the reaction mixture was quenched with water
(10 mL) followed by NaHCO3 (1g). The organic layer was separated and aqueous layer was
extracted with dichloromethane (2 x 15 mL). The combined organic extract was washed with
12

brine solution and dried over anhydrous Na2SO4. The extract was filtered and the solvent was
removed under vacuum to dryness on rotary evaporator which yielded the crude cyclized
product.
The dried compound was purified through the short silica gel column
chromatography using ethyl acetate and hexane (50:50) as eluent.
4.2.1. 12b-Hydroxy-2,3-dimethoxy-5,12b-dihydro-6H-isoindolo[1,2-a]isoquinolin-8-one
(CRR-203): Following the general procedure, N-[2-(3,4-dimethoxy-phenyl)ethyl]-
phthalimide furnished the cyclized product as colorless solid, 149 mg (96% yield), Mp 159
1
ºC, IR (KBr, cm-1): 3331, 1672, 1614, 1518. H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 7.6
Hz, 1H), 7.67 (d, J = 7.6 Hz, 1H), 7.63 (td, J = 7.6, 1.2 Hz, 1H), 7.47 (td, J = 7.6, 1.2 Hz,
1H), 7.41 (s, 1H), 6.56 (s, 1H), 4.16 (ddd, J = 13.2, 7.6, 1.6 Hz, 1H), 4.04 (s, 1H), 3.95 (s,
3H), 3.83 (s, 3H), 3.42-3.35 (m, 1H), 2.95-2.87 (m, 1H), 2.67 (ddd, J = 16.0, 8.0, 1.6 Hz,
1H).13C NMR (100 MHz, CDCl3): δ 167.4, 149.4, 148.2, 148.0, 132.7, 130.6, 129.5, 127.8,
127.6, 123.7, 123.0, 111.5, 110.4, 86.4, 56.2, 56.0, 34.9, 29.1. LDI-MS (m/z); Found
+
312.1248 and calculated 312.1236 for C18H17NO4 [M+H].
4.2.2.
12b-Hydroxy-3-methoxy-5,12b-dihydro-6H-isoindolo[1,2-a]isoquinolin-8-one
(CRR-204): Following the general procedure, N-[2-(3-methoxy-phenyl)ethyl]-phthalimide
furnished the cyclized product as colorless solid, 130 mg (93% yield), Mp 169 ºC. IR (KBr,
cm-1): 3265, 1681, 1613, 1580, 1407. 1H NMR (400 MHz, DMSO-d6):  8.12 (d, J = 8.0 Hz,
1H), 7.89 (d, J = 4.8 Hz, 1H), 7.63-7.68 (m, 2H), 7.51 (td, J = 7.6, 0.8 Hz, 1H), 6.89 (s, 1H),
6.84 (dd, J = 8.4, 2.4 Hz, 1H), 6.72 (d, J = 2.4 Hz, 1H), 4.23-4.17 (m, 1H), 3.71 (s, 3H), 3.45-
3.38 (m, 1H), 2.79 (dd, J = 7.6, 4.0 Hz, 2H). 13C NMR(100 MHz, DMSO-d6):  165.8, 158.5,
148.7, 135.9, 132.2, 130.2, 129.6, 129.2, 129.1, 123.7, 122.3, 113.0, 112.9, 85.4, 55.0, 29.0,
+
34.1. LDI-MS (m/z) Found 282.1112 and calculated 282.1130 for C17H15NO3 [M+H].
13

4.2.3. 12b-Hydroxy-3,4-dimethoxy-5,12b-dihydro-6H-isoindolo[1,2-a]isoquinolin-8 one
(CRR-211): Following the general procedure, N-[2-(2,3-dimethoxy-phenyl)ethyl]-
phthalimide furnished the cyclized product as colorless solid, 132 mg (85% yield), Mp 198
ºC. IR (KBr, cm-1): 3293, 1678, 1602, 1458. 1H NMR (400 MHz, CDCl3): δ 7.99 (d, J = 8.0
Hz, 1H), 7.69 (s, 1H), 7.69-7.68 (m, 1H), 7.62 (td, J = 7.6, 1.2 Hz, 1H), 7.46 (td, J = 7.2, 0.8
Hz, 1H), 6.87 (d, J = 8.8 Hz, 1H), 4.20 (ddd, J = 13.2, 6.4, 2.4 Hz, 1H), 3.83 (s, 3H), 3.81 (s,
1H), 3.73 (s, 3H), 3.41-3.34 (m, 1H), 3.02 (ddd, J = 16.8, 4.4, 2.0 Hz, 1H), 2.81–2.72 (m,
1H). 13C NMR (100 MHz, CDCl3): δ 167.1, 152.4, 147.9, 146.2, 132.5, 130.5, 129.6, 129.5,
128.9, 123.5, 123.4, 123.2, 110.8, 86.1, 60.1, 55.7, 34.2, 23.4. LDI-MS (m/z) Found 312.1257
+
and Calculated 312.1236 for C18H17NO4 [M+H].
4.2.4. 8,10-Dimethoxy-1,2,5,6-tetrahydropyrrolo[2,1-a]isoquinolin-3(10bH)-one (CRR-
213:): Cyclization using TfOH followed by reduction using NaBH4/ MeOH, N-[2-(3,5-
dimethoxyphenyl)-ethyl]succinimide furnished the cyclized product CRR-213 as colorless
1
solid, 109 mg (87% yield), Mp 121-122 ºC. IR (KBr, cm-1): 2974, 1689, 1586, 1442. H
NMR (400 MHz, CDCl3):  6.33 (d, J = 2.4 Hz, 1H), 6.24 (d, J = 2.4 Hz, 1H), 4.75-4.71 (m,
1H), 4.40-4.35 (m, 1H), 3.79 (s, 3H), 3.77 (s, 3H), 2.87-2.78 (m, 3H), 2.66-2.62 (m, 1H),
2.53-2.47 (m, 1H), 2.40-2.33 (m, 1H), 1.69-1.60 (m, 1H). 13C NMR (100 MHz, CDCl3): 
173.4, 159.2, 157.7, 135.8, 118.6, 104.6, 96.9, 55.3, 55.2, 54.9, 36.6, 31.6, 29.7, 28.1. LDI-
+
MS (m/z) Found 248.1300 and calculated 248.1287 for C14H17NO3 [M+H].
4.2.5. 12b-Hydroxy-1,4-dimethoxy-5,12b-dihydro-6H-isoindolo[1,2-a]isoquinolin-8-one
(CRR-222): Following the general procedure, N-[2-(2,5-dimethoxy-phenyl)ethyl]-
phthalimide furnished the cyclized product as colorless solid, 135 mg (87% yield), Mp 165-
166 ºC. IR (KBr, cm-1): 3398, 3054, 2925, 2854, 1717, 1675, 1586, 1458, 1290, 1063, 1020,
887, 739. 1H NMR (400 MHz, CDCl3):  8.16-8.14 (m, 1H), 7.79-7.77 (m, 1H), 7.51 (td, J =
7.5, 1.3 Hz, 1H), 7.45 (td, J = 7.5, 1.0 Hz, 1H), 6.84 (d, J = 8.9 Hz, 1H), 6.76 (d, J = 8.9 Hz,
14

1H), 4.53 (ddd, J = 13.0, 5.8, 1.0 Hz, 1H), 4.45 (s, 1H), 4.07 (s, 3H), 3.75 (s, 3H), 3.30 (td, J
= 13.0, 3.5 Hz, 1H), 2.91 (ddd, J = 17.2, 3.5, 1.0 Hz, 1H), 2.68-2.59 (m, 1H). 13C NMR (100
MHz, CDCl3): δ 167.8, 151.7, 151.2, 148.8, 132.4, 130.8, 129.4, 126.7, 125.6, 124.1, 123.2,
110.1, 108.9, 87.1, 55.8, 55.5, 34.7, 24.1. HRMS-ESI (m/z) found 334.1053 and calculated
334.1055 for C18H17NO4+ [M+Na].
4.2.6. 10,11-Dimethoxy-5,11b-dihydro-6H-1,3-dioxa-6a-aza-indeno[5,6-c]fluoren-7-one
and
8,9-Dimethoxy-5,11b-dihydro-6H-1,3-dioxa-6a-aza-indeno[5,6-c]fluoren-7-one
(CRR-224+CRR-225): Cyclisation using TfOH and reduction using NaBH4/ TFA, 2-[2-(1,3-
benzodioxol-5-yl)ethyl]-4,5-dimethoxy-1H-isoindole-1,3(2H)-dione furnished the cyclized
product as colorless solid, 145 mg (86% yield), IR (KBr, cm-1): 2972, 2940, 2841, 1681,
1620, 1446, 1408, 1273, 1220, 1035; 1H NMR (400 MHz, CDCl3): δ 7.58 (d, J = 8.0 Hz, 1H),
7.43 (dd, J = 0.8, 8.4 Hz, 1H), 7.32 (s, 1H), 7.14 (d, J = 8.0 Hz, 1H), 7.07 (d, J = 8.4, 1H),
7.03 (s, 1H), 6.66 (s, 1H), 6.65 (s, 1H), 5.95 (d, J = 1.2 Hz, 1H), 5.92 (d, J = 1.2 Hz, 1H),
5.89 (d, J = 1.2 Hz, 1H), 5.86 (d, J = 1.2 Hz, 1H), 5.63 (s, 1H), 5.45 (s, 1H), 4.34-4.28 (m,
1H), 4.07 (s, 3H), 4.0-4.09 (m, 1H), 3.99 (s, 3H), 3.97 (s, 3H), 3.90 (s, 3H), 3.59-3.53 (m,
13
1H), 3.42-3.36 (m, 1H), 3.05-2.94 (m, 2H), 2.90-2.82 (m, 1H), 2.79-2.75 (m, 1H). C NMR
(100 MHz, CDCl3): δ 167.6, 166.2, 155.5, 152.8, 146.7, 146.5, 146.4, 144.3, 137.6, 136.1,
128.8, 128.4, 128.3, 127.8, 126.6, 125.1, 119.7, 118.4, 116.2, 113.2, 109.0, 108.4, 107.5,
105.5, 101.1, 100.9, 62.5, 60.5, 58.4, 58.0, 56.7, 56.3, 38.8, 38.3, 29.3, 28.9.
4.2.7.
12b-Hydroxy-3-methyl-5,12b-dihydro-6H-isoindolo[1,2-a]isoquinolin-8-one
(CRR-227): Following the general procedure, N-[2-(3-methyl-phenyl)ethyl]-phthalimide,
furnished the cyclized product as colorless solid, 103 mg (78% yield), Mp 174-175 ºC. IR
(KBr, cm-1): 3269, 2947, 2893, 2840, 1695, 1614, 1576, 1417, 1294, 1107, 1027, 939, 824,
768, 701. 1H NMR (400 MHz, CDCl3): 8.01 (d, J = 8.0 Hz, 1H), 7.81 (d, J = 8.0 Hz, 1H),
7.62-7.58 (m, 2H), 7.42 (td, J = 7.6, 0.8 Hz, 1H), 7.09 (d, J = 12 Hz, 1H), 6.93 (s, 1H), 4.28
15

(s, 1H), 4.04 (ddd, J = 13.1, 6.0, 2.4 Hz, 1H), 3.36 (ddd, J = 13.1, 11.2, 4.4 Hz, 1H), 2.94-
13
2.85 (m, 1H), 2.74 (ddd, J = 12.4, 4.4, 2.4 Hz, 1H), 2.28 (s, 3H). C NMR (100 MHz,
CDCl3): δ 167.3, 147.9, 138.4, 134.5, 133.0, 132.5, 130.4, 129.7, 129.3, 127.6, 127.3, 123.4,
123.3, 86.4, 34.7, 29.2, 21.0. HRMS-ESI (m/z) found 288.1010 and calculated 288.1000 for
+
C17H15NO2 [M+Na].
4.2.8. 12b-Hydroxy-3-bromo-5,12b-dihydro-6H-isoindolo[1,2-a]isoquinolin-8-one (CRR-
228): Refluxing N-[2-(3-bromo-phenyl)ethyl]-phthalimide with TfOH under neat condition
followed by aqueous base work up furnished the cyclized product CRR-228 as colorless
solid, 109 mg (66% yield), Mp 163 ºC. IR (KBr, cm-1): 3246, 2951, 2894, 2838, 1683, 1593,
1472, 1414, 1181, 1114, 1040, 939, 883, 765, 697. 1H NMR(400 MHz, CDCl3):  7.98 (d, J =
7.5 Hz, 1H), 7.80 (d, J = 8.4 Hz, 1H), 7.70-7.68 (m, 1H), 7.65 (td, J = 7.5, 1.2 Hz, 1H), 7.49
(td, J = 7.5, 0.8 Hz, 1H), 7.42 (dd, J = 8.3, 1.8 Hz, 1H), 7.31-7.31 (m, 1H), 4.18 (ddd, J =
13.1, 6.1, 2.4 Hz, 1H), 3.71 (s, 1H), 3.42 (ddd, J = 13.1, 11.4, 4.5 Hz, 1H), 2.99-2.91 (m, 1H),
13
2.80 (ddd, J = 16.4, 4.2, 2.3 Hz, 1H). C NMR (100 MHz, CDCl3): δ 167.3, 147.4, 136.9,
134.9, 132.7, 132.0, 130.3, 130.0, 129.7, 129.1, 123.5, 123.2, 122.5, 86.2, 34.4, 29.0. HRMS-
+
ESI (m/z) found 351.9948 and calculated 351.9949 for C16H12BrNO2 [M+Na].
4.2.9.
8,9-Dimethoxy-2,3,6,7-tetrahydro-1H-pyrido[2,1-a]isoquinolin-4(11bH)-one
(CRR-238): Cyclization using TfOH followed by reduction using NaBH4/ MeOH, the N-[2-
(2,3-dimethoxyphenyl)-ethyl]glutarimide furnished the cyclized product CRR-238 as
colorless solid, 108 mg (83% yield), Mp 95-96 ºC. IR (KBr, cm-1): 2952, 2833, 1693, 1493,
1
1464, 1417, 1331, 1276, 1223, 1080, 1046, 1003, 861, 634. H NMR (400 MHz, CDCl3): δ
6.91 (d, J = 8.6 Hz, 1H), 6.81 (d, J = 8.6 Hz, 1H), 4.84-4.80 (m, 1H), 4.60 (dd, J = 10.4, 4.7
Hz, 1H), 3.85 (s, 3H), 3.79 (s, 3H), 2.92-2.73 (m, 3H), 2.56-2.48 (m, 2H), 2.39-2.30 (m, 1H),
1.97-1.77 (m, 2H), 1.73-1.63 (m, 1H). 13C NMR (100 MHz, CDCl3): δ 169.4, 151.1, 146.3,
16

130.8, 129.8, 120.4, 110.7, 60.4, 56.5, 55.9, 39.5, 32.4, 30.7, 23.1, 19.7. HRMS-ESI (m/z)
+
found 284.1261 and calculated 284.1263 for C15H19NO3 [M+Na].
4.2.10.
12b-Hydroxy-2-hydroxy-3-methoxy-5,12b-dihydro-6H-isoindolo[1,2-a]-
isoquinolin-8-one (CRR-271): Following the general procedure, N-[2-(3-methoxy-4-
hydroxy-phenyl)ethyl]phthalimide furnished the cyclized product as colorless solid, 123 mg
(83% yield), IR (KBr, cm-1): 3294, 2935, 2845, 1653, 1606, 1524, 1426, 1367, 1201, 1108,
1031, 761, 682. 1H NMR (400 MHz, CDCl3):δ 8.96 (s, 1H), 7.97 (d, J = 7.6 Hz, 1H), 7.68 (t,
J = 7.6 Hz, 1H), 7.64 (d, J = 7.4 Hz, 1H), 7.51 (t, J = 7.4 Hz, 1H), 7.34 (s, 1H), 6.85 (s, 1H),
13
6.66 (s, 1H), 4.21-4.17 (m, 1H), 3.71 (s, 3H), 3.42-3.35 (m, 1H), 2.77-2.67 (m, 2H). C
NMR (100 MHz, CDCl3): δ 165.8, 148.8, 147.5, 144.9, 132.2, 130.3, 129.3, 129.1, 125.0,
123.5, 122.4, 114.5, 111.8, 85.3, 55.5, 34.5, 28.3.
4.2.11. Synthesis of isoquinolinone derivative, CRR-267
(i) The imide 13 required to synthesize CRR-267 was prepared based on the
following procedure: 3,4-Dimethoxyphenethylamine 11 (250 mg, 1.4 mmol) and anhydride
12 (381 mg, 1.4 mmol) were taken in a round bottom flask containing toluene (25 mL) the
mixture was refluxed at 110 ˚C. After 12 h the solvent was evaporated and the mixture was
purified through silica gel column chromatography with hexane and ethyl acetate (1:4)
mixture as eluent to furnish the imide 13 as colorless solid, 490 mg, 81% yield. FT-IR (KBr,
cm-1): 3067, 3001, 2942, 2831, 1769, 1700, 1030.1H-NMR (400 MHz, CDCl3):  7.38 (dd, J
= 3.2, 2.0 Hz, 2H), 7.30 (dd, J = 3.2, 2.0 Hz, 2H), 7.16 (dd, J = 3.2, 2.4 Hz, 2H), 7.11 (dd, J =
3.2, 2.4 Hz, 2H), 6.73 (d, J = 8 Hz, 1H), 6.63-6.59 (m, 2H), 4.80 (s, 2H), 3.83 (s, 3H), 3.80 (s,
3H), 3.24 (m, 2H), 3.17 (s, 2H), 1.87 (m, 2H).13C-NMR (100 MHz, CDCl3):  176.65,
148.90, 147.69, 141.44, 138.72, 130.34, 126.98, 126.74, 125.04, 124.26, 120.65, 111.80,
111.27, 55.88, 55.86, 46.79, 45.64, 39.72, 32.53.
17

(ii) Cyclization of 13 to CRR-267: An oven dried two neck round bottom flask
bearing septum in side arm was cooled to room temperature under a steady stream of nitrogen
gas flow. The flask was charged with stirring bar, substrate (100 mg, 0.23 mmol) and dry
dichloromethane (10 mL) and cooled down to 0 °C (using ice). To this solution TfOH (0.2
mL, 8 equiv) was added with stirring. After 10 hours, the reaction mixture was quenched
with water (10 mL) followed by NaHCO3 (1g). The organic layer was separated and aqueous
layer was extracted with dichloromethane (2 x 15 mL). The combined organic extract was
washed with brine solution and dried over anhydrous Na2SO4. The extract was filtered and
the solvent was removed under vacuum to dryness on rotary evaporator which yielded the
cyclized product in 87 mg (87% yield).
CRR-267: Yield: 87 mg (87 %); M.p.: 162 ˚C. FT IR (KBr, cm-1): 3406, 2966, 1667, 1514,
1438, 1357, 1262, 1129, 1023, 764. 1H NMR (400 MHz, CDCl3 ): δ 7.34-7.32 (m, 1H), 7.26-
7.17 (m, 3H), 7.12-7.09 (m, 2H), 7.00 (td, J = 7.4, 0.96 Hz, 1H), 6.78 (td, J = 7.4, 0.96 Hz,
1H), 6.46 (s, 1H), 5.88 (d, J = 7.2 Hz, 1H), 4.74 (d, J = 3.8 Hz, 1H), 4.25 (d, J = 2.5, 1H),
4.08 (s, 1H), 3.93 (s, 1H), 3.72 (dd, J = 12.8, 4.7 Hz, 1H), 3.42 (dd, J = 8.4, 2.8 Hz, 1H),
3.16 (s, 1H), 3.08 (dd, J = 8.4, 2.6 Hz, 1H), 2.77 (td, J = 13.2, 2.7 Hz, 1H), 2.11 (dd, J =
15.5, 2.4 Hz, 1H), 1.26-1.18 (m, 1H).13C NMR (100 MHz, CDCl3): δ 172.8, 149.2, 147.9,
143.3, 142.0, 140.9, 137.7, 128.9, 126.6, 126.3, 126.1, 125.9, 125.3, 124.8, 124.5, 124.1,
123.5, 110.8, 110.6, 88.1, 77.3, 77.0, 76.7, 56.4, 56.0, 51.1, 48.6, 46.7, 45.6, 34.3, 27.9.
4.3. Chicken erythrocytes preparation
Unlike those of mammals, chick RBCs have intact but transcriptionally inactive
nucleus.49-50 Chick RBCs possess intact antioxidant systems as that of a normal eukaryotic
cell. Hence they are useful animal cell model for simulated oxidative damage in animal cells
18

and to monitor the response with treatment. They can be easily stored in PBS (phosphate
buffered saline) at physiological pH 7.4 at 4oC.
Blood from healthy chicken was collected, centrifuged and the pellet was re-
suspended in 40 mL of PBS. The number of cells in the stock was counted to be 5×106/mL,
from which working dilution of 1/10 was made such that the final cell count was 0.5×106/mL
and was used as working sample for all assays. Viability of the cells was checked each time
before assays by preparing a wet film and observing under light microscope at 40x for normal
cell and nuclear morphology.
The chick RBCs suspension in PBS were taken in separate tubes (1mL in each tube)
and pre-treated with 4 concentrations (25 μM, 50 μM, 75 μM & 100 μM) of each compound
(mentioned in Fig.2 & Fig.3) and exposed to 100 μM H2O2 for 1 hour at 37˚C. After the
incubation period, the samples were divided as follows: 20 µL of sample was taken for cell
viability assay by acridine orange & ethidium bromide staining method, 80 µL of the sample
was allotted for comet assay, and the remaining was reserved for analysis of antioxidant
status.
4.4. Analysis of protective properties
4.4.1. Acridine orange and ethidium bromide staining
AO is a cell permeable dye, when bound with double stranded DNA emits green
fluorescence (emission 540 nM) under UV excitation. The dye, when bound to single
stranded DNA emits orange fluorescence (emission 640 nM), thus marking damaged DNA.
The staining mixture contains AO (1 mg/mL) and ethidium bromide (EB) (5 mg/mL) in
phosphate buffered saline. AO is permeable in intact cell membrane where as EB is
impermeable. The normal cells appear green and cells with different grades of DNA damage
19

vary in range from yellowish green to bright orange. After incubation, exactly 20 µl of RBCs
suspensions were taken on a clean glass slides and 7 µl of staining solution was added, mixed
well and a cover slip was placed. The wet mount was then observed under fluorescent
microscope at 40 x magnification and the images were captured and processed with
ProgresTM software. The cells with intact double stranded DNA appeared green and the cells
with damaged DNA showed orange-red fluorescence.49
4.4.2. Antioxidant status
The compound pre-treated and H2O2 exposed RBCs samples were centrifuged at 2500
x g for 5 min. The RBCs pellet was subjected to hypotonic lysis and centrifuged at 1200 x g
for 10 minutes to remove cell debris. The supernatant was used for the analysis of
biochemical parameters such as catalase, superoxide dismutase and the extent of lipid
peroxidation. Catalase (EC 1.11.1.6) catalyses the breakdown of H2O2 to water and molecular
oxygen. UV absorption of H2O2 can be measured at 240 nm, whose absorption decreases
when degraded by the enzyme catalase.51 The SOD (superoxide dismutase) activity (EC
1.15.1.1) of the sample was determined by using Marklund and Marklund method.52 The
activity of SOD was quantitatively measured by the amount of pyrogallol oxidized. The
increase in the absorbance of pyrogallol was measured at 420 nm against blank containing
tris-HCl buffer.
4.4.3. Lipid peroxidation
The extent of lipid peroxidation in the controls and the drug treated samples was
quantified by the method of Ohkawa by thiobarbituric acid reaction.53 The RBCs samples
incubated were centrifuged to get the cell pellet. The supernatant was discarded and the
RBCs cell pellet was lysed using distilled water. The lysate was mixed with 10 % TCA to
20

precipitate the proteins. Then, the samples were centrifuged and the supernatant was
collected. Equal amount of supernatant and TBA were mixed and incubated in the boiling
water bath for 15 minutes and the colour developed was measured at 535 nm. The results
were expressed as nmols of malondialdehyde formed per minute per mg of protein.
4.4.5. Comet assay
Comet assay is a versatile technique, which is increasingly being used to quantitate
DNA damage within cells. The slides were coated with 1 % normal melting point (NMP)
agarose. The RBCs suspension was diluted with 0.5 % low melting point (LMP) agarose (1 in
100 dilution) and was layered on to the slide. The layer was covered with 1 % NMP. A
coverslip was placed and the cells with agarose are allowed to set rapidly by chilling on ice.
The slides were kept in lysis buffer (10 mM TrisHCl pH 10 with 100 mM EDTA, 2.5 M
NaCl, detergents and 10 % DMSO) for one hour and were later neutralized with 0.4 M Tris
pH 7.5. Electrophoresis (Dark room) was done at 50mA for 20min with alkaline buffer pH 13
and the slides were observed using fluorescent microscope at 40x objective after staining
with EB of 10 mg/mL concentration. The comet images were captured under the microscope
and processed using Tritek Comet Score freewareTM v1.5.
4.5. PARP-1 inhibition assay
Potential PARP-1 inhibitory activity of the novel isoquinolinone derivatives was
assayed by employing Homogenous PARP-1 inhibition assay kit from Trevigen. (Trevigen,
MD, USA). The reactions were performed in triplicates in a 96 well plate and the readings
were obtained from FLX800 fluorescence microtitre plate reader (Biotek instruments inc,
USA). The IC50 values were deduced from NAD+ standard graph for RFU (Relative
fluorescence units) and comparative derivation of PARP-1 inhibitory activity for various
21

dilutions as per the protocol in the Kit. A standard isoquinolinone 3, 4-dihydro-5-[4-(1-
piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) was used in the assay to compare its
inhibitory potency with that of the compounds under study.
4.6. Docking studies
The interaction of the newly synthesized isoquinolinone derivatives with the PARP-1
catalytic fragment of chicken was analyzed using docking studies with Autodock 4.0. The
structures of isoquinolinones were drafted using Chemsketch and converted to PDB (Protein
data bank) format using Pymol viewer. The PARP-1 sequence (PDB ID - 2PAW) in PDB text
format was retrieved from PDB website. The sequence was processed by removal of water
molecules and unwanted residues and by addition of missing residues using build/repair
model option available in whatif server. The PDB format of the fixed sequence of PARP-1
catalytic fragment was used in the docking studies. The grid was set by specifically choosing
the amino acids in the NAD+ binding pocket of PARP-1 catalytic fragment. A standard
isoquinolinone 3,4-dihydro-5-[4-(1-piperidinyl)butoxyl]-1(2H)-isoquinolinone (DPQ) was
used to compare the binding efficiency of novel isoquinolinone derivatives.54 The results of
the docking were documented in Pymol viewer Binding energy values for the isoquinolinone
derivatives were obtained from the RMSD (Root mean square deviation) table.
4.7. Statistical evaluation
Statistical analyses was performed using one-way analysis of variance (ANOVA) followed
by Tukey‘s Multiple Range test. Differences were considered to be significant at (P≤0.05)
against control group. Data were presented as mean ± S.D of three independent samples
(triplicate).
22

Acknowledgements
We duly acknowledge the funding support for the current study in the form of DST-
FIST to the Department of Biochemistry and Molecular Biology, Pondicherry University,
Puducherry. A. Suyavaran acknowledges the CSIR-UGC, New Delhi, India for junior
research fellowship. C.R.R thanks CSIR and DST, New Delhi for financial support. J.S.K
and S.M.R thanks respectively CSIR and UGC, New Delhi for SRF. We gratefully
acknowledge CIF, Pondicherry University for NMR/IR data and the single crystal XRD
facility (DST-FIST), Department of Chemistry, Pondicherry University for XRD data.
Supplementary Data
Supplementary data containing physical and spectral data of the isoquinolinones of all
the isoquinolinone derivatives synthesized and investigated in the current study, can be found
in the online version at (http link to be provided by editor).
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27

Tables
Table 1: Effect of H2O2, DMSO and isoquinolinone derivatives on RBCs viability by
acridine orange-ethidium bromide staining.
S.No
Sample
RBC control
Percentage of viability#
1
2
94±1.52
41±2.08
H2O2 control
3
DMSO control
93.6±1.52
72±2.64
4
CRR-222 100 µM
CRR-203 100 µM
CRR-211 100 µM
CRR-271 100 µM
CRR-204 100 µM
CRR-228 100 µM
CRR-227 100 µM
CRR-213 100 µM
CRR-238 100 µM
CRR-267 100 µM
CRR-224+225 100 µM
5
79.3±2.05
65.6±3.21
93.6±1.05
53.6±2.08
75.3±1.52
82.3±2.08
68.3±2.30
86.6±2.51
87.3±2.08
92.6±2.30
6
7
8
9
10
11
12
13
14
The samples represent RBCs suspended in PBS and treated with the isoquinolinone derivatives (CRR-227 to
CRR-224+225), each compound at the concentration of 100 µM, followed by H2O2 treatment for 1 hour.
#Values represent the percentage of viable cells (mean±S.D) counted at three different fields (under 40 x
magnification).

Table 2: Activities of some antioxidant enzymes and lipid peroxidation level in control,
H2O2, DMSO and isoquinolinone derivatives treated RBCs.
Samples
Catalase
Superoxide dismutase Lipid peroxidation level
RBC Control
H2O2 Control
DMSO Control
75.34 ± 1.24
15.97±2.12
31.54 ± 1.43
11.32 ± 1.96 *
12.35 ± 1.87
84.32 ± 12.04
69.37 ± 7.74
23.65 ± 1.76 *
31.76 ± 3.32 *
47.53 ± 2.87
28.43 ± 2.54
16.32 ±2.43 *
19.24 ± 2.25 *
23.76 ± 2.65
64.98 ± 2.46
36.23 ± 4.76 *
28.21 ± 1.96 *
14.24 ± 1.87
25
50
75
CRR-222
100 68.46 ± 5.08
27.76 ± 1.76
17.34 ± 3.65 *
19.35 ± 1.32 *
23.98 ± 2.23
28.45 ± 2.08
13.19 ± 2.45 *
15.96 ± 2.87 *
15.54 ± 1.96 *
18.13 ± 3.67 *
18.616±2.49*
21.63±1.82*
28.863±6.20*
30.94±2.20*
12.65 ± 1.54 *
11.87 ± 2.76 *
11.48 ± 2.54 *
11.37 ± 1.95 *
12.8667±2.24
14.05±3.56
20.14 ± 2.64 *
38.46 ± 1.28 *
29.27 ± 3.76 *
16.54 ± 2.64
18.32 ± 1.49
53.65 ± 1.25 *
46.37 ± 2.69 *
41.21 ± 1.54 *
48.32 ± 1.76 *
52.71±3.45*
47.15±1.29*
26.35±3.66
25
50
75
26.54 ± 2.43 *
43.65 ± 1.76 *
48.43 ± 1.98
CRR-203
CRR-211
CRR-271
CRR-204
CRR-228
CRR-227
CRR-213
CRR-238
CRR-267
CRR – 224+225
100 64.87 ± 1.25
25
50
75
15.31 ± 0.95 *
16.25 ± 2.54 *
16.46 ± 1.34 *
100 16.37 ± 0.36 *
25
50
75
29.52±3.09
51.49±3.40*
62.57±2.44*
100 70.66±1.87*
15.02±1.40
25
50
75
15.31 ± 0.95 *
16.25 ± 2.54 *
16.46 ± 1.34 *
62.98 ± 0.32 *
56.32 ± 1.47 *
48.12 ± 1.34 *
42.14 ± 1.57 *
70.74±1.86*
54.24±2.53*
34.52±2.07
100 16.37 ± 0.36 *
25
50
75
36.597±1.58
51.14±2.28*
56.05±3.24*
17.51±1.85*
21.58±2.65*
11.51±2.42*
14.56±1.45*
15.08±3.17
100 65.007±4.24*
16.75±2.16
25
50
75
31.71±2.29
50.35±1.84*
54.56±1.78*
53.66±1.29*
43.76±1.94*
26.52±1.92
100 65.35±3.32*
18.6±2.12
16.95±.62
25
50
75
38.65 ± 1.32 *
46.35 ± 1.37 *
58.28 ± 0.65
17.23 ± 0.76 *
19.43 ± 2.64 *
24.35 ± 1.98
29.34 ± 1.96
11.76 ± 2.58 *
11.48 ± 0.25 *
11.95 ± 0.76 *
12.98 ± 0.25 *
15.86±1.58
19.63±3.23*
23.58±1.94*
27.87±2.88*
13.1033±2.24
18.50±2.51*
22.79±4.18*
30.52±4.64*
28.38 ± 1.96 *
19.12 ± 1.47 *
16.87 ± 1.45
16.14 ± 1.37
61.45 ± 2.64 *
54.23 ± 2.76 *
48.23 ± 2.78 *
49.54 ± 1.46 *
51.48±2.53*
35.60±3.20*
24.44±1.90
100 70.43 ± 1.49
25
50
75
17.36 ± 2.35 *
25.95 ± 3.65 *
28.47 ± 1.54 *
100 31.43 ± 1.36 *
25
50
75
46.11±2.01*
55.89±2.02*
65.12±2.93*
100 67.91±5.49*
18.10±1.67
25
50
75
35.65±1.59
43.84±2.11*
53.92±2.42*
57.145±1.98*
46.08±3.55*
24.70±2.61
100 63.90±3.06*
18.90±1.42
RBC control – RBCs suspended in PBS; DMSO control – RBCs suspended in PBS with 0.5 % DMSO; H2O2
control – RBCs in PBS treated with 100 µM H2O2;
Values represents mean ± SD, * P≤0.005 against the RBC control group, where n=3. The values are expressed
as: Catalase: nmol of H2O2 utilized/min/mg of protein at 37°C; SOD: µmol of pyrogallol oxidized/min/mg of
protein at 37°C; LPO: nmol malondialdehyde formed/ mg protein at 37°C.