A facile atom economic one pot multicomponent synthesis of bioactive spiro-indenoquinoxaline pyrrolizines as potent antioxidants and anti-cancer agents

Article abstract of DOI:10.1039/c7nj02993d

A competent and highly discriminating one-pot method for the synthesis of biologically active novel spiro-indenoquinoxaline pyrrolizines accumulating three pharmocophoric cores, heterocyclic quinoline scaffold, pyrrolizines and indeno-quinoxaline, in a single molecular framework by means of a four-component reaction between ninhydrin, o-phenylenediamine, l-proline and quinolinyl chalcones in methanol via [3+2] cycloaddition was developed. The structures of the compounds were well characterized by FT-IR, NMR, ESI-MS, XRD and elemental analysis. The synthesized compounds were screened for in vitro antioxidant activity using DPPH, nitric oxide, super oxide radical and in vitro cytotoxic activity against MCF-7 and A-549 cancer cell lines and visualized using the fluorescent microscopic technique. The newly synthesized compounds exhibited excellent antioxidant activity compared to the standard molecule, i.e., BHT. In terms of cytotoxic activity, compounds 5c, 5e, 6c and 6e were found to exhibit significant activity, when compared to standard doxorubicin against MCF-7 and A-549 cancer cells using the MTT assay method. The cell morphology analysis clearly indicates cell death was induced by apoptosis and necrosis pathways. Molecular docking studies were performed using an EGFR inhibitor to determine the molecular interactions between the compounds and proteins.

Full text of DOI:10.1039/c7nj02993d

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A facile atom economic one pot multicomponent synthesis of bioactive spiroꢀ
indenoquinoxaline pyrrolizines as potent antioxidant and anti cancer agents
Kailasam Saravana Mania, Werner Kaminskyb and Subramaniam Parameswaran Rajendrana*
aDepartment of Chemistry, Bharathiar University, Tamil Nadu, India
bDepartment of Chemistry, University of Washington, Seatle, WA 98195, USA
Corresponding author: chemsaran.mani@gmail.com, rajendransp@yahoo.com
Abstract
An competent and highly discriminating oneꢀpot synthesis of biologically active novel
spiroꢀindenoquinoxaline pyrrolizines accumulating three pharmocophoric cores, heterocyclic
quinoline scaffold, pyrrolizines and indenoꢀquinoxaline in a single molecular framework by
means of fourꢀcomponent reaction between ninhydrin, oꢀphenylenediamine, Lꢀproline and
quinolinyl chalcones in methanol via [3+2]cycloaddition. The structures of the compounds were
well characterized by FTꢀIR, NMR, ESIꢀMS, XRD and elemental analysis. The synthesized
compounds were screened for in vitro antioxidant activity using DPPH, nitric oxide, super oxide
radical and in vitro cytotoxic activity against MCFꢀ7 and Aꢀ549 cancer cell lines and were
visualized using Fluorescent microscopic technique. The newly synthesized compounds
exhibited excellent antioxidant activity when compared to the standard molecule ie., BHT. In
case of cytotoxic activity compounds 5c, 5e, 6c and 6e were found to be admirable activity when
compared to standard doxorubicin against MCFꢀ7 and Aꢀ549 cancer cells by MTT assay method.
From the cell morphology analysis we clearly indicate that induced cell death by apoptosis and
necrosis pathway. Molecular docking studies were performed out using EGFR inhibitor to
determine the molecular interactions between compounds and proteins.
Keywords
Spiroꢀindenoquinoxaline pyrrolizines, quinoline dipolarophile, Lꢀproline, antioxidants and
cytotoxic activity
1

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1. Introduction
In current trends, it is still a great challenge to our synthetic chemist for the development
of novel bioactive molecules for the treatment of cancer urging with paramount less side effects
and safety so far. However chemotherapy is the leading cancer treatment approach, its side
effects, including death on normal cells and time duration or dose level, shrink its clinical
applications1. Nitrogen containing heterocycles constructed through 1,3ꢀdipolar cycloaddition of
azomethine ylides2ꢀ5 have been documented as an important cluster of significant bioactive
compounds. It is a major class of heterocyclic systems plays an essential role in the field of
anticancer drug enlargement and it holds several applications in medicinal chemistry including
antibacterial,6 antimalarial,7 antiꢀinflammatory,8 apoptosis,9 DNA repair and tyrosine
kinases(TK) inhibitor.10 Tetracyclic Indeno Quinoxalines are also an another important class of
heterocycles used as intermediates in the synthesis of several types of spiro compounds11, 12
which possess potent αꢀglycosidase inhibitors.
Pyrrolizine derivatives represent a class of novel heterocycles which serves as promising
scaffolds for anticancer drugs. The unique antitumor properties of mitocin C inspired chemists to
develop different pyrrolizine systems and assess their potential antitumor activities against a
wide variety of cancer types.13
Fig. 1: Some important pyrrolizine antitumor candidates
Mitocin C isolated from Streptomyces caespitosus or Streptomyces laevendulae is used to
treat upper esophageal carcinoma, anal, breast cancers and superficial bladder tumors. 14ꢀ16 Further it
is believed to act as a good DNA alkylator which can crossꢀlink DNA with high efficiency and
specificity.17,18 Clazamycin A and Clazamycin B are naturally occurring pyrrolizines with quite
2

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interesting antiꢀtumor properties.19(Fig. 1). Recently Multicomponent reactions is a powerful
tool to construct molecules of structural diversity and complexity with minimum waste
generation, useful widely in both combinatorial and medicinal chemistry arena.20 The above
mentioned tremendous biological evaluation of quinolines, indeno quinoxaline and pyrrolizines
prompted us to develop a cluster of all three heterocyclic scaffold together by multicomponent
reaction and finding their biological evaluation.
In this paper, we report a new synthetic methodology for the synthesis of novel bioactive
spiroꢀindenoquinoxaline pyrrolizine molecules through multicomponent reactions, Herein we
would like to report an efficient, atom economic, regioselective and high yielding MCR protocol
for oneꢀpot facile synthesis of bioactive functionalized spiroꢀindenoquinoxaline pyrrolizine
heterocyclic
scaffold
by
the
reaction
of
ninhydrin,
oꢀphenylenediamine, proline, quinoline bearing chalcones on refluxing in methanol and the
evaluation of their in vitro antioxidant and in vitro anti tumor activity.
2. Results and Discussion
2.1. Chemistry
The synthesis of spiro [indeno[1,2ꢀb] quinoxaline pyrrolizines 5(aꢀf) and 6(aꢀf) were
achieved through a 1,3ꢀdipolar cycloaddition reaction protocol21. Initially, quinoline based
dipolarophiles 4(aꢀf) were synthesized by the Claizen Schmidt condensation reaction between 2ꢀ
chloroꢀ3ꢀformyl quinoline derivatives and acetophenone under alcoholic KOH condition. An
equimolar mixture of ninhydrin 1, oꢀphenylenediamine 2, ꢀproline 3 and dipolarophile 4a in
L
methanol was refluxed for 2 to 3 hours (Scheme 1). After completion of reaction was monitored
by TLC, a single product spiroꢀindenoquinoxaline pyrrolizines 5a was separated by evaporation
of solvent in good yields. The optimized reaction condition, time and yields of 5(aꢀf) were
mentioned in Table 1. The FTꢀIR spectrum of 5a displayed characteristic band at 1680 cmꢀ1,
1588 cmꢀ1 and 759 cmꢀ1 corresponding to C=O, ꢀC=N and CꢀCl in quinoline dipolarophile
1
moiety. H NMR spectrum of product 5a, showed the characteristic multiplets at δ 1.89ꢀ2.64
corresponding to pyrrolizine ring.
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Scheme 1: Synthesis of spiroꢀindenoquinoxaline pyrrolizines 5(aꢀf)
The proton attached to benzoyl group appeared at δ 4.99 as triplet with coupling constant
(J = 9.6 Hz) and pyrrolizine ring proton attached to quinoline moiety appeared at δ 5.70 as
doublet with coupling constant (J = 11.6 Hz). The proton attached to benzoyl moiety consistent
with trans disposition with proton attached to quinoline moiety. Similarly, the –NCH proton
attached to pyrrolizine ring is in consistent with cis position to the proton attached to benzoyl
group as shown XRD structure in Fig. 2 whereas the signals for all other protons in aromatic
region are in good agreement with proposed structure.
Fig. 2.Molecular structure of compound 5e, except hydrogen atoms in pyrrolizine ring are
omitted for clarity. (CCDC 1569306)
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In 13C NMR spectrum, the quaternary spiro carbon appears at δ 75.12 and carbonyl
carbon attached to phenyl ring is appeared at δ 197.34. Whereas, the signals for all other carbons
are located at appropriate chemical shifts were in perfect agreement with proposed structure. In
DEPTꢀ135 NMR, the appearance of three negative signals corresponding to methylene protons
in pyrrolizine core further strongly attested the formation of product. The observed mass
spectrum of the product HRMS (TOF ES+) m/z 593.2092 further confirmed the formation of
compound 5a.
In order to introduce the electron withdrawing substituent benzoyl group in quinoxaline
core, we bring in 3,4ꢀdiaminobenzophenone instead of oꢀphenylenediamine. With the optimized
condition in hand, we then explored the scope of this cycloaddition reaction using ninhydrin 1, 3,
4ꢀdiaminobenzophenone 2a, Lꢀproline 3 and quinoline dipolarophile 4(aꢀf) in refluxing methanol
(Scheme 2). After the completion of reaction, monitored by TLC, the precipitate obtained was
filtered and washed with water to afford the single product 6(aꢀf). In IR spectrum of compound
6a, characteristic peak at 1659 cmꢀ1, 1586 cmꢀ1 and 701 cmꢀ1 attributed to carbonyl stretching,
quinoline –C=N and CꢀCl respectively. The yield of compounds 6(aꢀf) will be slightly high when
compared to compounds 5(aꢀf).
Scheme 2: Synthesis of benzoyl substituted spiroꢀindenoquinoxaline pyrrolizines 6(aꢀf)
1
In H NMR spectrum of compound 6a, the sharp singlet appeared at δ 2.50 attributed to
–CH3 group in quinoline scaffold. Multiplets of the pyrrolizine ring proton appeared in the range
between δ 1.97 ꢀ δ 2.71. The proton attached to quinoline moiety will be slightly deshielded to
δ 5.69 as doublet (J = 11.6 Hz) and the proton attached to benzoyl group resonated at δ 4.96 as
triplet (J = 9.6 Hz). The chemical shifts at aromatic regions are in good agreement with proposed
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structure. The observed mass spectrum of the product HRMS (TOF ES+) m/z 697.2482 further
confirmed the formation of compound 6b.
Table 1: Reaction conditions for the synthesis of spiro indenoꢀquinoxaline 5, 6(aꢀf)
aIsolated yield, Reaction condition: ninhydrin 1 (1mmol), oꢀphenylenediamine 2 or 3,4ꢀdiamino
benzophenone 2a(1mmol), Lꢀproline 3(1mmol)quinoline chalcones4(1mmol), methanol(10ml)
6

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Fig. 3. 1H and 13C NMR chemical shift values of products 5c and 6c
In 13C NMR spectrum, the peak at δ 197.116 and δ 195.863 which is due to two carbonyl
carbons and peak at δ 75.181 attributed to quaternary spiro carbon. These observed chemical
shift values are in good agreement with the structure of the compound 6a. In DEPTꢀ135 NMR
spectrum the negative signals corresponding to three –CH2 protons in pyrrolizine core.
Furthermore, the presence of molecular ion peak at m/z 697.2476 in the mass spectrum
confirmed the formation of product 6c. The formation of spiro[indeno[1,2ꢀb]quinoxalineꢀ11,3'ꢀ
pyrrolizines scaffold probably involves a complex multistep sequence. Initially the
cyclocondensation reaction takes place between ninhydrin 1 and oꢀphenylenediamine 2 forms
indenoꢀquinoxalineꢀ11ꢀone which further condensed with proline 3 that undergoes
decaboxylation to give azomethine ylide 7 subsequently through cycloaddition reaction with
dipolarophile 4 to give final product. (Scheme 3)
2. Biological Evaluation of Synthesized Compounds
2.1. In Vitro Antioxidant Activity
The antioxidant effectiveness mainly involves scavenging radicals and inhibiting
oxidations of biological species. A number of quinoline derivatives were tested for anti oxidant
activity as reflected the ability to inhibit lipid perꢀoxidation in rat brain and kidney homogenates.
Free radical scavenging capacity can be wellꢀknown by the neutralization of electrons or
hydrogens. Compounds having low IC50 values having high hydrogen donating ability lead more
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antioxidant activity. The radical scavenging capacity of compounds 5(aꢀf) and 6(aꢀf) along with
standard BHT were tested against DPPH, nitric oxide and super oxide radicals and the results
were summarized in (Table 2).
Scheme 3: Proposed mechanism for the formation of spiro indenoꢀquinoxaline pyrrolizines
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Table 2. Radical scavenging activity of compounds 5(aꢀf) and 6(aꢀf) in IC50 ꢁg/mL
Compounds
DPPH
Nitric
oxide
5.87
4.56
2.68
5.51
1.34
9.52
6.95
5.98
5.24
5.43
3.43
6.32
7.19
Super
oxide
4.43
5.69
4.12
3.45
4.01
8.54
10.11
8.65
3.4
5a
5b
5c
6.13
5.82
3.42
3.32
2.96
7.23
8.4
5d
5e
5f
6a
6b
6c
6.71
3.6
6d
6e
3.2
3.9
3.56
8.24
7.93
3.8
6f
9.54
9.30
BHT
From the results obtained all the synthesized compounds (Fig. 4) exhibited excellent
activity when compared to that of the standard BHT. Interestingly compound 5e possess most
promising activity with the IC50 values of 2.96, 1.34 and 4.01 against DPPH, Nitric oxide and
super oxide radicals which outperformed standard BHT with the IC50 values of 7.93, 7.19 and
9.30 respectively. Next in the series, compounds 5c, 5d, 6c, 6d and 6e showed excellent activity
in all three radical scavenging assays. The compounds 5a, 5b, 5f, 6a, 6b and 6f displayed
equipotent activity with standard molecule BHT. The reason for high scavenging capacity of the
compounds may be nature of substituents in quinoline core. From the results, we clearly suggest
that compounds having electron donating groups22, 23 at in 6th and 8th position of quinoline
scaffold will be significantly more active than that of compound having electron donating group
at 7th position. The presence of –OCH324 in 6th position will be more active when compared to –
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CH3 group in 6th position. From the structural analysis, the absence of electron donating group in
quinoline moiety showed less activity when compared to rest of the compounds. (Scheme 4)
Fig. 4. Flow chart representing antioxidant activity of synthesized compounds
2.2. In Vitro Cytotoxic Activity
The use of drugs in different target, often discrete with multiple biological targets, with
the expectation of synergistic effects and low toxicity, safety becomes an ideal approach of
increasing interest in nowadays research. The anticancer properties of synthesized compounds
5(aꢀf) and 6(aꢀf) were screened against MCFꢀ7 and Aꢀ549 cancer cell lines by MTT [3ꢀ(4,5ꢀ
dimethylthiazoꢀ2ꢀyl)ꢀ2,5ꢀdiphentltetrazolium bromide] assay method. Doxorubicin can act as a
positive control for this study. Inhibition of cells was measured in terms of IC50 values after
48hrs of cell exposure. The results obtained were summarized in Table 3.
As listed in Table 3, the entire synthesized compounds exhibited good to moderate
activity against two cell lines while compared to Doxorubicin standard. Among the synthesized
compounds, compounds 5e and 6e were showed equipotent activity with IC50 values of 15±1.6
and 16±0.5 against MCFꢀ7 cell line and 16±1.8 and 17±1.8 against Aꢀ549 cell lines when
compared to that of positive control Doxorubicin 14±1.0 (MCFꢀ7) and 15±1.4 (Aꢀ549)
respectively. Increase in cytotoxic values of 5e and 6e suggests that electron donating methyl
group may be favorable at 6th and 8th position of quinoline core.
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Table 3: Cytotoxic activity of spiro indenoꢀquinoxaline pyrrolizines (5aꢀf) & (6aꢀf) in IC50 (µM)
Samples
5a
MCFꢀ7a
33± 1.4
23±0.5
17±1.6
28± 1.5
15± 1.9
35±1.5
25± 1.4
24± 1.5
18± 1.3
26± 1.3
16± 0.5
30±0.7
Aꢀ549b
24± 1.0
36± 1.5
19± 0.2
24± 1.5
16±1.8
37±1.7
32 ± 1.0
32± 0.9
20±1.5
24± 0.5
17± 1.8
33±1.8
15± 1.4
5b
5c
5d
5e
5f
6a
6b
6c
6d
6e
6f
Doxorubucin 14± 1.0
aMCFꢀ7 (breast cancer cell line)
bAꢀ549(lung cancer cell line)
On further investigation on methyl group in the position of quinoline core, surprisingly
methyl group at 8th position possess good activity with IC50 values of 17±1.6 and 18±1.3
(Compound 5c) against MCFꢀ7 and 19±0.2 and 20±1.8 (Compound 6c) against Aꢀ549 cancer
cell lines. Meanwhile rests of the compounds with electron donating groups at 6th and 7th position
showed moderate activity against both cancer cell lines. This clearly indicates that electron
donating group at 8th position increase the anticancer potency rather than other positions.
Absence of electron donating group in any of the position in quinoline core displayed week activity
towards MCFꢀ7 and Aꢀ549 cell lines. (Scheme 4)
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Scheme 4ꢀRole of electron donation substituents in quinoline core
2.3. Cell Morphology Analysis
To monitor the effect of synthesized compounds on cell morphology, treated cancer cells
were examined by inverted light microscopy and compared with untreated cells. Treated cells
showed significant changes compared with untreated cells. Cytological investigations elucidate
the anticancer effect routed through membrane blebbing, membrane instability and disturbing the
cytoskeleton of the cells by the compounds.
Fig. 5: Morphological analysis of treated MCFꢀ7 cancer cells and the arrow mark indicates
the formation of floating cells and appearance of membrane blebbing
Fig. 5 and Fig. 6 reveal the morphological changes in MCFꢀ7 and Aꢀ549 after treatment
with compounds with their respective inhibitory concentrations for 24 hrs. Phase contrast
micrographs revealed that the compounds 5c, 5e, 6c and 6e induced increase cell shrinkage,
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membrane blebbing and form floating cells in a dose independent manner.
Fig 6: Morphological analysis of treated Aꢀ549 cells and the arrow mark indicates the
formation of floating cells and appearance of membrane blebbing
2.4. Acridine OrangeꢀEthidium Bromide (AOꢀEB) Staining
Acridine orange/ Ethidium bromide (AO/EB) fluorescent staining assay were performed
to distinguish the pathway i.e apoptotic, necrotic and live cells. 25 AO permeates the intact cell
membrane and stains the nuclei green, whereas EB permeates only cells with loss in membrane
integrity and stain the nucleus orange. The compounds 5c, 5e, 6c and 6e untreated and treated
MCFꢀ7 and Aꢀ549 cells were stained with AOꢀEB and analyzed under fluorescent microscope.
Fig.7 and Fig. 8 shows that the untreated cancer cells (control) did not show any significant
adverse effects compared to the compounds treated cancer cells. It can be observed that with the
addition of compounds to the cancer cells, the green color of cells are converted into orange /red
color cells which is due to induced apoptosis and the nuclear condensation effects on the cells.
Fig.7 and Fig. 8 also shows that the compounds were significantly inducing the apoptosis in
performed cancer lines.
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Fig. 7: AO/EB apoptotic analysis of treated MCFꢀ7 cells and arrows indicate apoptotic cells
Fig. 8: AO/EB apoptotic analysis of treated Aꢀ549 cells and arrows indicate apoptotic cells
2.5. DAPI Straining method
4′,6ꢀDiamidinoꢀ2ꢀphenylindole (DAPI) is a nuclear stain which strongly binds to
adenineꢀthymine clusters of the minor groove of doubleꢀstranded DNA and detect the chromatin
condensation or nuclear damage. Binding of DAPI to DNA produces a ~20ꢀfold enhanced
fluorescence, apparently due to the displacement of water molecules from both DAPI and the
minor groove.26 DAPI stains the apoptotic cells as bright colored due to the condensed nucleus
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which is a typical apoptotic feature. Fluorescence microscopy images of lung and breast cancer
cells after 24 hrs stained with DAPI in the absence and presence of compounds are shown in
Fig. 9 and Fig. 10, which reveals that the untreated cells didn’t show any significant changes
whereas compounds treated cancer cells shows bright fetches which indicates the condensed
chromatins and nuclear fragmentations in the cancer cells. Thus, the results from MTT assay and
Fluorescence microscopy analysis, we confidently report here that the compounds may be used
as potent therapeutic agents.
Fig. 9: DAPI apoptotic analysis of treated MCFꢀ7 cells and arrows indicate apoptotic cells
Fig. 10: DAPI apoptotic analysis of treated Aꢀ549 cells and arrows indicate apoptotic cells
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2.6. Molecular Docking
Docking studies predicted the interaction of inhibitors with protein and residues involved
in this complex. The orientation and conformation of inhibitors bound in the active site of the
protein and formed protein–ligand complex with the highest binding energy value are the most
important requirement for docking studies. The inhibition potency of the newly synthesized
compounds 5e and 6e were subjected for further docking studies to explore the binding pattern
against Epidermal growth factor receptor (EGFR)(PDB code: 1M17)28 using Autodock 4.2
software.28 As depicted from Fig. 11, compound 5e is surrounded by hydrophobic amino acids
such as GLY 695, CYS 773, TYR 777, GLY 772, LEU 694, VAL 693, LYS 704 and LYS 692.
The key amino acid CYS 773 is considered to play a crucial role in the stabilization of the
receptor by compound 5e which can be observed in hydrophobic enclosure where nitrogen atom
in quinoxaline ring is mounted towards the CYS 773 hydrophobic amino acid (1.8 Å) with glide
score (ꢀ5.54 kcal/mol). Similarly the compound 6e is surrounded by hydrophobic amino acids
ARG 817, LEU 820, GLY 772, CYS 773, LYS 692, PHE 699 etc. As like observed in compound
5e, compound 6e too observed that key amino acid CYS 773 is considered to play a crucial role
in the stabilization of receptor through hydrophobic enclosure where nitrogen atom in
Fig.11. Docking studies on compounds 5e and 6e with EGF receptor and dotted lines shows
hydrogen bond
quinoxaline ring is hydrogen bonded with CYS 773 hydrophobic amino acid (2.2 Å) with glide
score (ꢀ6.05 kcal/mol). So it is assumed that hydrophobicity is one of the important
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physicochemical properties which impart lipophilicity to the molecule as well as membrane
permeability and their absorption in biological system.
3. Conclusion
In summary, we have developed a highly atom economic synthesis of spiro indenoꢀ
quinoxaline derivatives via one pot four component 1,3ꢀdipolar cycloaddition reactions of
azomethine ylide and quinoline derived dipolarophile in successful manner. The pure products
were isolated by recrystallization. The structures of the synthesized compounds were well
characterized by using single crystal XRD, spectral and elemental analysis. All the synthesized
compounds were successfully screened them against antioxidants and cytotoxic activity for two
cancer cell lines (MCFꢀ7 and Aꢀ549). Interestingly all the compounds exhibited excellent
activity against DPPH, nitric oxide and super oxide free radicals. Compounds 5c, 5e, 6c and 6e
found to be significantly active in this series and also induced cell death by apoptosis and
necrosis. The substituents of electron donating group in 6th and 8th positions turns out to be the
best candidates in this series screened.
Acknowledgement
We would like to thank SAIF, CUSAT, Cochin for NMR spectral analysis and University of
Washington, Seatle, US for Single crystal XRD data. We thank Department of Botany and
Zoology, Bharathiar University for evaluating antioxidant and anticancer studies.
Electronic Supplementary Information
X-ray crystal data and Spectral data of all the compounds associated with article will be
available as Supporting Information.
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A facile atom economic one pot multicomponent synthesis of bioactive spiro-
indenoquinoxaline pyrrolizines as potent antioxidant and anti cancer agents
Kailasam Saravana Mania, Werner Kaminskyb and Subramaniam Parameshwaran Rajendrana*
aDepartment of Chemistry, Bharathiar University, Tamil Nadu, India
bDepartment of Chemistry, University of Washington, Seatle, WA 98195, USA
Corresponding author: chemsaran.mani@gmail.com, rajendransp@yahoo.com
GRAPHICAL ABSTRACT