Regio- and stereoselective synthesis of dispirooxindole-pyrrolocarbazole hybrids via 1,3-dipolar cycloaddition reactions: Cytotoxic activity and SAR studies

Article abstract of DOI:10.1016/j.ejmech.2017.11.039

A library of novel dispiro compounds containing oxindole-pyrrolo-carbazole hybrid frame works has been synthesized in a fully regio- and stereoselective fashion by the three-component 1,3-dipolar cycloaddition of azomethineylides generated in situ from th

Full text of DOI:10.1016/j.ejmech.2017.11.039

Accepted Manuscript
Regio- and stereoselective synthesis of dispirooxindole-pyrrolocarbazole hybrids via
1,3-dipolar cycloaddition reactions: Cytotoxic activity and SAR studies
Karunanidhi Murali, Hazel A. Sparkes, Karnam Jayarampillai Rajendra Prasad
PII:
S0223-5234(17)30942-X
10.1016/j.ejmech.2017.11.039
EJMECH 9914
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 March 2017
Revised Date: 6 November 2017
Accepted Date: 17 November 2017
Please cite this article as: K. Murali, H.A. Sparkes, K.J. Rajendra Prasad, Regio- and stereoselective
synthesis of dispirooxindole-pyrrolocarbazole hybrids via 1,3-dipolar cycloaddition reactions:
Cytotoxic activity and SAR studies, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.11.039.
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ACCEPTED MANUSCRIPT
Regioselective synthesis and stereochemical structure of anti-proliferative active
dispirooxindole-pyrrolo-carbazole hybrids via 1,3-dipolar cycloaddition reactions
Karunanidhi Murali a, Hazel A. Sparkes b, Karnam Jayarampillai Rajendra Prasad a*
a Department of Chemistry, Bharathiar University, Coimbatore 641046, India.
b School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, United Kingdom.
Graphical abstract
Corresponding author:
Dr. K. J. Rajendra Prasad
Emeritus Professor
Department of Chemistry
Bharathiar University
Coimbatore-641046,
India.
*Corresponding author e-mail: prasad_125@yahoo.com, Tel: +91-422-2423763, Fax: +91-422-2422387.

ACCEPTED MANUSCRIPT
Regio- and stereoselective synthesis of dispirooxindole-pyrrolocarbazole hybrids via 1,3-
dipolar cycloaddition reactions: Cytotoxic activity and SAR studies
Karunanidhi Murali a, Hazel A. Sparkes b, Karnam Jayarampillai Rajendra Prasad a*
a Department of Chemistry, Bharathiar University, Coimbatore 641 046, India.
b School of Chemistry, University of Bristol, Cantock's Close, Bristol, BS8 1TS, United Kingdom.
Abstract
A library of novel dispiro compounds containing oxindole-pyrrolo-carbazole hybrid frame works
has been synthesized in a fully regio- and stereoselective fashion by the three-component 1,3-
dipolar cycloaddition of azomethineylides generated in situ from the condensation of isatins and
benzylamine with 2-arylidene/heteroarylidene-2,3,4,9-tetrahydro-1H-carbazole-1-one. The
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13
structures of the compounds were established by FT-IR, H NMR, C NMR, X-ray diffraction
and elemental analysis. The synthesized dispiro heterocycles have been screened for in vitro
cytotoxic activity by MTT assay and displayed enviable growth inhibition on both the cancer cell
lines i.e. breast cancer cell line MCF-7 and lung cancer cell line A-549. Morphological changes
and apoptosis induction have been studied by inverted light microscopic, fluorescent microscopic
techniques and by flow cytometry analyses. The preliminary structure activity relationships were
also carried out. Data indicated that among dispiro-carbazole compounds,6-chloro-4'-(thiophen-
2-yl)-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-dione 7e could
be exploited as a significant therapeutic drug against breast cancer as well as lung cancer cell
proliferation.
Keywords
1,3-Dipolar cycloaddition
Azomethineylide
Anti-proliferative
Apoptosis
Flow cytometry
*Corresponding author e-mail: prasad_125@yahoo.com, Tel: +91-422-2423763, Fax: +91-422-2422387
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1. Introduction
Breast cancer and lung cancer are the two most recurrently diagnosed malignant tumors
worldwide. Variety of breast cancer drugs such as Tamoxife, Letrozole, Docetaxyl etc. are being
prescribed to the patients as preventive and curative treatments [1,2]. However undue toxicity
and side effects in these medicines spoil their efficacy [3-5]. Despite recent advances made in
anti lung cancer drug development, the presence of resistance to chemotherapeutic agents is a
major obstacle to the effective treatment of lung cancer [6]. Consequently, discovering novel,
puissant molecular entities as potential anti cancer drugs with improved efficacy and resistance
to complement the present chemotherapeutic strategies is highly desired. Interestingly natural
products provide a healthy source for such compounds. The spirooxindole-pyrrolidine nucleus is
often found in the molecular framework of many natural products, vizhorsfiline [7], coerulescine
[8], elacomine [9], isopteropodine [10], formosanine [11], rychnophyilline [12] and
spirotryprostatins A and B [13] etc. It possess a myriad of biological activities such as inhibition
of the mammalian cell cycle at G2/Mphase [14], inhibition of microtubule assembly [15],
modulation of the function of muscarinic serotonin receptors [16] and malignant glioma GAMG
[17], and anticancer [18], antimicrobial [19] and local anesthetic [20] activities.
Carbazole and its derivatives represent an important class of bio-active heterocycles.
Many natural products and drug molecules with the carbazole framework exhibit a broad range
of biological and pharmacological activities [21-28]. In particular, carbazole derivatives show
significant antitumor activity [29-31]. For example, glybomine B and C showed significant
antitumor-promoting activity, which was confirmed by the inhibiting effect of these alkaloids in
conjunction with the tumor promoter 12-o-tetradecanoylphorbol-13-acetate (TPA) [32], while
heptaphylline and 7-methoxyheptaphylline displayed strong cytotoxicity against NCI-H187 and
KB cell lines [33]. In addition carbazole arrests the tumor cell cycle at the M phase and induces
apoptotic cell death by increasing expression of p-53 and promoting bcl-2 phosphorylation
[34,35] (Figure 1).
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The dominance by these two pharmacophores (carbazole and spiro-pyrrolooxindole) in
nature and their impact in medicinal chemistry inspired us to develop new molecular hybrids by
incorporating both the frameworks for our anticancer studies. Mostly, design of the hybrid drugs
aims to circumvent the drug resistance, minimize the risk of drug-drug interactions,
counterbalance the known side effects associated with the other hybrid part and amplify the
activity through the interaction with multiple targets as one single molecule [36,37]. Now, hybrid
drug design has emerged as a premier tool for the discovery of innovative anticancer therapies
that can potentially overcome most of the pharmacokinetic drawbacks encountered when using
conventional anticancer drugs.
The 1,3-dipolar azomethineylide cycloaddition is a powerful tool for the construction of
five-membered heterocycles and spiro-compounds [38] in a highly regio- and stereoselective
manner [39]. Azomethineylides are reactive and versatile 1,3-dipoles, which readily react with
diverse dipolarophiles with exocyclic double bonds to afford spiro-heterocycles [40].
Herein, we report a regio- and stereoselective one-pot method for the synthesis of a novel
class of polynuclear dispiroheterocyclic structures comprising spiro-pyrrolooxindole and
carbazole moieties. The synthesis was accomplished by 1,3-dipolar cycloaddition of azomethine
ylides generated in situ from isatins and benzylamine to 2-arylidene/heteroarylidene-2,3,4,9-
tetrahydro-11H-carbazole-1-one. All the newly synthesized compounds were subjected to
cytotoxic screening against breast cancer cell line MCF-7 and lung cancer cell line A-549.
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Although there are reports available for the synthesis of substituted spiro-pyrrolooxindole, there
seems to be no reports to the best of our knowledge for the synthesis of a rare class of
dispiropyrrolooxindole having keto carbazole skeleton.
2. Results and discussion
2.1. Chemistry
The dipolarophiles 3 employed in the present work were synthesized by the reaction of
2,3,4,9-tetrahydro-1H-carbazole-1-one 1 with aryl/hetroaryl aldehydes 2 in the presence of
alcoholic KOH in good yields at room temperature (Scheme 1).
The structural elucidation of all the dipolarophiles 3 (a-i) was done with the help of
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13
spectroscopic (FT-IR, H-NMR & C-NMR) and elemental analysis data. The FT-IR spectrum
of 3a showed the characteristic absorptions of an indole NH at 3290 cm-1 and a carbonyl group at
1644 cm-1. The 1H NMR spectrum of 3a showed a broad singlet at δ 11.64 ppm attributed to the
indole NH proton and a singlet at δ 8.27 ppm due to the presence of C5-H. Multiplet signals in
the region of δ 7.68-7.34 ppm arise from the six aromatic protons, while a multiplet at δ 7.12-
6.98 ppm was assigned to the C7 and C2 aromatic and olefinic protons. The methylene protons of
C3 and C4 appeared as two multiplets centered at δ 3.04 and 3.01 ppm respectively. A singlet at δ
2.50 ppm accounts for three methyl protons at C6 position. The 13C NMR spectrum revealed the
presence of 20 carbons. A signal resonating at δ 179.5 was attributable to the carbonyl carbon.
The universal validity of the reaction was tested with other substituted derivatives 3 (b-i).
The choice of an appropriate reaction medium is of crucial importance for successful
synthesis. Initially, the three component reaction of 2-arylidene/heteroarylidene-2,3,4,9-
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tetrahydro-1H-carbazole-1-one 3a, isatin 4 and benzylamine 5, as a simple model substrate, was
investigated to establish the feasibility of the strategy and optimize the reaction conditions.
Various solvents such as EtOH, MeOH, 1,4-dioxane, acetonitrile, toluene, EtOH/dioxane and
MeOH/dioxane were screened. MeOH/dioxane (Table 1, entry 7) emerged as the solvent of
choice, producing the highest yield (87 %) of the target compound.
Table 1.Solvent screening for the Synthesis of dispiro compound 7a
Entry Solvent
Reaction time Yield (%)
1.
2.
3.
4.
5.
6.
7.
EtOH
7
56
64
70
52
38
73
87
MeOH
7
1,4-Dioxane
CH3CN
8
10
10
7
Toluene
EtOH/1,4-Dioxane
MeOH/1,4-Dioxane
5
The one pot regioselective synthesis of compounds 7 (a–i) has been performed by taking
2-arylidene/heteroarylidene-2,3,4,9-tetrahydro-1H-carbazole-1-one 3 (a–i) (0.001 mol), isatin 4
(0.001 mol) and benzylamine 5 (0.001 mol) in dioxane and methanol under reflux conditions.
After completion of the reaction as evident from TLC, the solvent was removed and the crude
product was purified by column chromatography to obtain pure 4′-arylidene/heteroarylidene-5'-
phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-dione 7. The reaction
proceeds through decarboxylative condensation of isatin 4 with benzyl amine 5 to generate
unstabilize azomethineylide, which subsequently undergoes 1,3-dipolar cycloaddition with the
dipolarophile 3 to afford novel cycloadduct 7 as a single regioisomer (Scheme 2).
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We next explored the scope and generality of this three component cycloaddition reaction
with substrates having (i) aryl rings bearing electron-withdrawing and electron releasing
substituents and (ii) heteroaryl rings at the arylidene side chain. Moreover, the carbazole core
was either unsubstituted or substituted with electron-releasing (methyl) or electron-withdrawing
(Cl) groups. These structurally and electronically varied starting materials reacted efficiently
with isatin and benzylamine, affording the corresponding cycloadducts 7 in good yields under
the same set of reaction conditions in all cases (Table 2).
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Table2. Synthesis of a library of spiropyrrolidinyloxindole-carbazole hybrids via 1,3-dipolar
cycloaddition reaction with variety of aromatic aldehydes were executed.
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The products 7(a-i) were characterized unambiguously by spectroscopic, crystallographic
and analytical studies. The FT-IR spectrum of 7a revealed prominent absorptions at 3419, 3322
and 3222 cm-1due to indole NH, carbazole NH and pyrrolo NH stretchings respectively. The
peak at 1696 cm-1 is due to the carbonyl of indole ring whilst a peak at 1617 cm-1 is assignable to
1
the carbazole carbonyl group. In the H NMR spectrum of 7a a doublet appeared at δ 4.69 ppm
(Jcis = 10.0 Hz) due to C4' proton, which confirms the formation of cycloadduct. If the other
possible regioisomer 8 had been formed then the 1H NMR spectrum would have shown a singlet
for C4' proton instead. A pair of broad singlets appeared at δ 11.18 and δ 10.18 ppm
corresponding to carbazole NH and oxindole NH respectively. A doublet at δ 7.50 ppm (Jo = 7.4
Hz) accounted for C8 and C7'' aromatic protons and the aromatic protons on C5'''' and C3''''
resonated as a multiplet at δ 7.38 ppm. A multiplet in the region of δ 7.26-7.22 ppm accounted
for the four aromatic protons on the substituted phenyl ring. The three aromatic protons in the
C6'', C6'''' and C2'''' positions showed a multiplet in the region between δ 7.18-7.13 ppm. The C5
proton appeared as a singlet at δ 7.09 ppm. Two doublets at δ 7.05 and δ 6.97 (Jo=7.4 Hz) ppm
corresponding to C4'' and C7 protons. Three triplets appeared at δ 6.84, 6.56 and 6.54 ppm (J =
7.4, 7.2 and 7.2 Hz) due to C5'', C4''' and C4'''' protons respectively. The C5' proton occurred as
doublet of doublet at δ 5.45 ppm (J1,2-cis = 5.6 Hz & J1,2-cis = 10.0 Hz). The –NH proton of pyrrolo
moiety resonated as a doublet at δ 3.61(J1,2-cis = 5.6 Hz) ppm. Methylene protons of C4 and C3
appeared as multiplets centered at δ 2.57 and 2.47 ppm respectively. A singlet at δ 2.22 ppm
assigned to the C6-CH3 protons. In 13CNMR spectrum of 7a, the peaks at δ 72.3 and δ 61.5 ppm
corresponded to the two spiro carbons. The oxindole ring carbonyl resonated at δ 180.5 ppm, and
benzoylcarbonyl resonated at δ 191.5 ppm. Scrutiny of all spectral data confirmed the formation
of
the
dispiropyrrolo
derivatives,
4′-arylidene/heteroarylidene-5'-phenyl-3,4-
dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-dione 7.
In order to verify the structure of 7a, a single crystal was obtained and analyzed by X-ray
crystallography (Figure 2), which clearly indicated the formation of dispiro scaffold. Similarly,
by straightforward considerations, the 1H and 13C chemical shifts of other derivatives 7(b-i) were
also assigned.
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Figure 2. Structure of 7a with the atomic numbering scheme shown. Hydrogen atoms omitted
and only one position of disorder phenyl ring (C29-C34) shown for clarity. Thermal ellipsoids
depicted at the 50% probability level.
A proposed mechanism for the regio- and stereoselective formation of the hybrid
heterocycles 7(a-i) is presented in Scheme 3. The azomethine ylide 6a generated in situ from the
reaction of isatin 4 with benzylamine 5, has one potential nucleophilic carbon, which
subsequently undergoes 1,3-dipolar cycloaddition with the electron deficient β-carbon of the
dipolarophile 3 to afford novel cycloadduct 7 as a single regioisomer. Control of the relative
stereochemistry at the spiro centre was observed. Accordingly the observed regioisomer 7 via
path A is more favorable because of the secondary orbital interaction [41] which is not possible
in path B for getting other isomer 8.
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2.2. Biological evaluation
2.2.1. In vitro cytotoxic activity
Anti-proliferative activity of the newly synthesized dispiro compounds 7(a-i) was
examined in two human cancer cell lines, MCF-7 breast cancer and A-549 lung cancer using
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MTT assay. Cisplatin is one of the most effective broad-spectrum anticancer drugs. Cisplatin
enters cells via multiple pathways and forms different DNA–platinum adducts while initiating a
cellular self-defense system by activating or silencing a variety of different genes [42]. Hence,
we chose cisplatin as reference drug. Three independent experiments in triplicate were
performed for the determination of sensitivity to each compound. The percentage cell viability
was determined using the following formula, and the relationship between percentage cell
viability and drug concentration was plotted to obtain the survival curve of the tested cancer cell
lines. The response parameter calculated was the IC50 value, which corresponds to the
concentration required for 50% inhibition of cell viability. The results are summarized in Table
3. The in vitro cytotoxic activity of the synthesized compounds (7e and 7d) (10-100 µM
concentrations) against both cancer cells has been presented in Figure 3. The experimental
results demonstrate that all the compounds have the ability to inhibit cell proliferation in a dose
dependent manner.
Table 3. In vitro cytotoxicity and IC50 (µM)
Compounds
MCF-7a
19±1.4
18±0.8
15±1.5
14±0.8
13±1.6
15±0.8
18±1.7
19±0.8
17±0.8
9±1.0
A-549b
18±1.5
15±1.4
17±0.5
15±1.4
14±1.2
16±1.5
20±1.4
18±1.4
21±1.4
10±1.5
7a
7b
7c
7d
7e
7f
7g
7h
7i
Cisplatin
Bold indicates more active compounds.
a breast cancer. b lung cancer.
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As shown in Table 3, it was obvious that the synthesized dispiro-carbazole compounds
displayed significant to modest growth inhibitory activity against the tested cancer cell lines.
Investigations of the cytotoxic activity revealed that majority of the synthesized compounds
exhibited potent anticancer activity against both the cancer cell lines, MCF-7 and A-549. Among
the newly synthesized spiro compounds, compound 7e was found to be the most promising
derivative against MCF-7 with IC50 values 13±1.6 µΜ compared to the IC50 value of cisplatin
(9±1.0 µΜ). The next most outstanding compounds are 7d and 7f which displayed stronger
cytotoxicity against MCF-7 cell line with IC50 values 14±0.8 and 15±0.8 µΜ respectively.
Furthermore, the screening against MCF-7 cell line, the compounds 7a, 7b and 7c showed
significant activity with IC50 value ranging from 15±1.5 to 19±1.4 µM. While compounds 7g, 7h
and 7i were moderately active with IC50 values of 18±1.7, 19±0.8 and 17±0.8 µΜ respectively.
Subsequently, those compounds which showed good activity with MCF-7 were screened against
A-549 cell line. Compounds 7e, 7f and 7d were the most active analogs through this study with
IC50 values of 14±1.2, 16±1.5 and 15±1.4 µM respectively. In addition, compounds 7a, 7b and
7c with 1C50 values ranging from 15±1.4 to 18±1.5 µΜ displayed substantial activity against A-
549 cell line whereas compounds 7g, 7h and 7i were moderately active with IC50 values of
20±1.4, 18±1.4 and 21±1.4 µΜ respectively. In general, it was found that all the synthesized
dispiro-carbazole derivatives displayed selective cytotoxicity against both the tested cell lines-
A-549 and MCF-7 and finally, compound 7e was emerged as the most promising anticancer
agent against MCF-7 and A-549 cell lines with IC50 values of 13±1.6 and 14±1.2 µM
respectively.
Figure 3. Cytotoxic effect of the compounds 7e & 7d on the viability of MCF-7 & A-549 cell lines
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2.2.2. Structure activity relationship (SAR)
Based on the aforementioned biological data, the following assumptions could be deduced
about the structural activity relationship (SAR):
∑
It is clear from the results summarized in Table 3 that the substitution attached to the
pyrrolo group oriented at the C4'-position of the synthesized spiro heterocycle plays an
important role in developing the observed anti-tumor properties. Among the synthesized
compounds, the compound 6-chloro-4'-(thiophen-2-yl)-5'-phenyl-3,4-dihydrodispiro
[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-dione 7e showed higher cytotoxic efficacy
with 1C50 value 13±1.6 µM for MCF-7 and 14±1.2 µΜ for A-549 cell line. Compounds
7d and 7f also exhibited stronger cytotoxic activity against both the tested cell lines. It
was due to the presence of thiophene moiety [43,44] which boots the cytotoxic activity.
On the other hand, replacing the thiophene group at the C4'-position of the pyrrolo moiety
by the electron donating methoxy group reduced the anticancer potency of the dispiro
compounds. The compounds 7g, 7h and 7i having methoxy substitution on the pyrrolo
ring showed moderate anticancer activities (Scheme. 4).
∑
∑
In general, it was observed that the substituent present in the carbazole ring plays a vital
role in determining the anticancer potency. Among the dispiro-carbazole compounds, the
compounds bearing electron withdrawing chloro group in the carbazole ring enhanced the
cytotoxic activity more than the electron donating methyl group and unsubstituted group
[45]. (Scheme. 5)
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H3C
Cl
H
6
S
S
H
S
5
7
8
H
H
4
9
3
H
O
H
H
O
HN
2
HN
HN
NH
NH
NH
NH
NH
1
O
O
O
O
NH
7e
7f
7d
Scheme 5. Role of C6- substituent in incresing the efficany of cytotooxicity
2.2.3. Apoptosis assay
Apoptosis, the process of programmed cell death (PCD), is an important therapy target
for cancer chemotherapy. Apoptotic cell death is morphologically defined by chromatin
condensation, nuclear fragmentation, shrinkage of cytoplasm and formation of apoptotic bodies.
The apoptotic events were analyzed using various staining procedures to study the cytotoxicity
and cell viability, extent of apoptosis, morphological changes, nuclear changes and DNA
fragmentation, which occurs at the final stage of apoptosis. The development of human cancers
is often mainly a consequence of deregulated cell cycle control and/or suppressed apoptosis.
2.2.3.1. Morphological changes of selected human cancer cell lines using
Inverted Light Microscopic analysis
.
The apoptogenic property of the active dispiro compounds was investigated through
morphological changes in MCF-7 and A-549 human cancer cells. Apoptotic cells displayed
typical common features such as cell shrinkage, nuclear condensation, membrane blebbing,
chromatin cleavage, and formation of pyknotic bodies of condensed chromatin. These distinctive
typical forms of morphological changes in apoptotic cells are widely used for the identification
and quantification of apoptosis. Thus, determination of the morphological changes to define
apoptosis was visualized using inverted light microscopic technique.
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Figure 4. Morphometric analysis of treated MCF-7 & A-549 cells, the arrows indicates the formation of
floating cells and appearance of membrane blebbing
Figure 4 shows the morphological changes in MCF-7 breast cancer and A-549 lung
cancer cells after treatment with compounds with their respective inhibitory concentration for 24
h in comparison to control cells. Visualization of the control (untreated) cells showed that the
cells maintained their original morphology form. Phase-contrast micrographs revealed that the
compounds 7e and 7d induces increased cell shrinkage, membrane blebbing and forms floating
cells, compared to compounds 7a, 7b, 7c, 7f, 7g and 7h in a dose-dependent manner.
2.2.3.2. Morphological observation of selected human cancer cell lines using Fluorescence
Microscopic analysis
.
Fluorescence light microscopy with differential uptake of fluorescent DNA binding dyes
(such as EB/AO, DAPI staining) is a method of choice for its simplicity, rapidity, and accuracy.
In such an assay, apoptotic index and cell membrane integrity can be assessed simultaneously
and there is no cell fixation step, thus avoiding a number of potential artifacts. Fluorescence
microscopy analysis revealed the effects of synthesized compounds to induce apoptosis in MCF-
7 breast cancer and A-549 lung cancer cells. Induction of apoptosis is the most important
mechanism for many anticancer agents. Fluorescence microscopic analysis of cell death showed
that treatment of cells with compounds induce more apoptotic cell death rather than necrotic
death. Mechanism of cell death was studied by nuclear staining methods such as AO/EB and
DAPI staining methods. Staining cells with fluorescent dye is used in evaluating the nuclear
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morphology of apoptotic cells. One of the characteristics of cells undergoing apoptosis is nuclear
chromatin condensation. The DNA in condensed chromatin stains strongly with fluorescent dyes
which allows for differentiation of apoptotic from non-apoptotic cells.
2.2.3.2.1. 4',6-Diamidino-2-phenylindole (DAPI) staining method
DAPI detection provides a rapid and convenient assay for apoptosis based upon
fluorescent detection. DAPI (4',6-diamidino-2-phenylindole) is a fluorescent stain that binds
strongly to A-T rich regions in DNA. Its high cell permeability allows efficient staining of
nuclei. Once it overpasses cell membranes of normal cells, the blue fluorescence will be
observed by fluorescent microscopy. With the process of apoptosis, the ability of permeability
for dye is improved and the apoptotic cells will produce high blue fluorescence. Fluorescence
microscopic images of breast cancer cells after 24 h stained with DAPI in the presence and
absence of compounds are shown in Figure 5. The compounds 7e, 7d, 7c and 7f displayed
higher level of nuclear fragmentation and the untreated cells did not show any significant
changes in the nuclear appearance, whereas the other compounds exhibited bright fetches when
treated with MCF-7 cancer cells, which indicates the condensed chromatins and nuclear
fragmentations in the cells. Figure 5 showed fluorescent DAPI analysis of compounds treated A-
549 lung cancer cells. Compounds 7d and 7e exhibit higher level of nuclear fragmentation in the
treated A-549 cells, rather than the remaining compounds 7a, 7b, 7c, 7f, 7h and 7g. The
apoptosis observed in compounds which are treated with MCF-7 breast cancer cells are higher
than that observed in A-549 lung cancer cells.
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Figure 5. DAPI apoptotic analysis of treated MCF-7 & A-549 cells the arrows indicate apoptotic cells
2.2.3.2.2. Acridine orange/ Ethidium bromide (AO/EB) staining method
To confirm the induction of apoptosis, treated cells were visualized by fluorescence
microscopy following treatment with 1:1 ratio of AO/EB, which allow differentiation of dead
and viable cells by staining DNA. Fluorescence microscopy images of MCF-7 and A-549 cancer
cells in the absence of compounds (control) and in the presence of compounds are shown Figure
6. The untreated MCF-7 cancer cells did not show any significant adverse effect compared to the
compounds treated cancer cells. It can be observed that the addition of compounds7e and 7d to
the MCF-7 cancer cells, the fluorescence green colour of cells are changed to orange/red colour,
which is due to induced apoptosis and nuclear condensation effect. The cells with intact
membranes shows fluorescence green due to AO staining while EtBr stains cells with damaged
membranes which exhibits orange/red fluorescence due to DNA intercalation of both stains. The
rest of the compounds showed weak fluorescence intensity owing to the reduced level of
induction of apoptosis in the MCF-7 cells. In the case of lung cancer cell, compounds 7d, 7e and
7f exhibited significant apoptotic induction rather than the compounds 7a, 7b, 7c, 7g and 7h. The
apoptotic induction values of the synthesized compounds were remarkable in MCF-7 cells
compared to the lung cancer cells.
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Figure 6. AO/EB apoptotic analysis of treated MCF-7 & A-549 cells the arrows indicate apoptotic cells
2.2.3.2.3. Apoptotic analysis of Human cancer cells by flow cytometry
To further confirm the dispiro hetrocycles-induced apoptosis, the apoptosis was detected
by flow cytometric technique using the Annexin V method, with the aid of an Annexin V-FITC
to perform double-staining with propidium iodide. Treatment with compound 7e shows a
significant population of annexin-V positive cells (pro and late apoptotic cells) in the right hand
quadrants of flow cytometric graphs in a dose dependent manner (Figure. 7). The compound 7e
has the potential to stimulate the apoptotic signals and activate subsequent cell death
mechanisms. Moreover the compound significantly active on MCF-7 cells rather than the A-549
cells (Figure. 8). This activity may be the marker receptor present on the cell surface and it
could be more active on breast cancer cells. These results indicate that dispiro heterocyclic
compound induced cell death is mediated by the induction of apoptotic pathways in selected
human cancer cells.
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Figure 7. Flow cytometry analysis of breast cancer cells (MCF-7). Quadrant 4 represents necrotic cells
(D-20
µ
M/ml), Quadrant 3 represents late apoptotic cells (C-10
µM/ml), Quadrant 2 represents
proapoptotic cells (B-5 M/ml), and Quadrant 1 represents live cells (A-Control).
µ
19

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Figure 8. Flow cytometry analysis of Lung cancer cells (A-549). Quadrant 4 represents necrotic cells (D-
20
µ
M/ml), Quadrant 3 represents late apoptotic cells (C-10
µM/ml), Quadrant 2 represents proapoptotic
cells (B-5 M/ml), and Quadrant 1 represents live cells (A-Control).
µ
3. Conclusions
The present investigation describes the synthesis of new and highly functionalized
carbazole containing dispiropyrrolooxindole hybrid molecules obtained through regio- and
stereoselective 1,3-dipolar cycloaddition reaction of azomethineylide with (E)-2-
arylidine/heteroarylidine-2,3-4,9-tetrahydro-1H-carbazole-1-one as dipolarophiles. Interestingly,
benzylamine played a critical role in the regioselectivity of this 1,3-cycloaddition to construct the
4'-arylidene/heteroarylidene-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-
9(H)-1,2''-dione ring system and it was obtained as a single regioisomer. The structural
assignments of the corresponding cycloadducts were confirmed by FT-IR, NMR spectroscopy
and the structure of compound 7a was further confirmed by X-ray crystallographic analysis. The
cytotoxic efficacy of these compounds was assessed against the human cancer cell line MCF-7
20

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and A-549 and all dispiro compounds were either equipotent or moderate to the positive control
cisplatin. Among which, compounds 7e, 7f and 7g having thiophene moiety showed potent in
vitro anti-proliferative activity. Moreover, the preliminary structure activity relationships of these
derivatives have been established. The mode of cell death assessed by inverted light microscopic,
fluorescence microscopic (DAPI staining and AO/EB dual staining procedures) techniques and
flow cytometry analyses revealed that the dispiro-carbzole compounds were able to trigger
apoptosis in both MCF-7 and A-549 human cancer cells lines.
4. Experimental protocols
4.1. Chemistry
4.1.1. General
All the chemicals were bought from Sigma-Aldrich and Merck and were utilized for the
process without further purification. Melting points (M.p.) were determined on a Mettler FP 51
apparatus (Mettler Instruments, Switzerland) and are uncorrected. They are expressed in degree
centigrade (°C). FT-IR spectra were recorded on Avatar Model FT-IR (4000–400 cm-1)
1
13
spectrophotometer. H NMR and C NMR spectra were recorded on a Agilent- 400 MHz (1H)
and 100 MHz (13C) spectrometers respectively in CDCl3 using TMS (tetramethylsilane) as
internal reference; chemical shifts are expressed in parts per million (ppm); coupling constants
(J) are reported in hertz (Hz) and the terms Jo and Jm refer to ortho coupling constant and meta
coupling constant. The signals were characterized as s (singlet), d (doublet), t (triplet), m
(multipiet), bs (broad singlet) and dd (doublet, and doublet). Microanalyses were carried out
using Vario EL III model CHNS analyzer (Vario, Germany). When known compounds had to be
prepared according to literature procedures and pertinent references are given. The purity of the
products was tested by TLC plates coated with silica gel-G using petroleum ether and ethyl
acetate in the ratio of 1:1 as developing solvents.
4.1.2. Synthesis
4.1.2.1. General procedure for the synthesis of 2-arylidene/heteroarylidene-2,3,4,9-
tetrahydrocarbazol-1-one 3.
An equimolar mixture of the 2,3,4,9-tetrahydrocarbazol-1-one 1 (1.0 mmol) and
aryl/heteroaryl aldehyde 2 (1.0 mmol) was treated with 5 % ethanolic KOH (25 mL) solution and
21

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stirred for 24 h at room temperature. The completion of the reaction was monitored by TLC.
After the completion of the reaction, the reaction mixture was cooled to room temperature and
poured into ice cold water and neutralized with 1:1 HCl. The precipitated solid was filtered and
purified by column chromatography over silica gel using petroleum ether: ethyl acetate (99:1)
mixture as eluant to afford the respective product, 2-arylidene/heteroarylidene-2,3,4,9-
tetrahydrocarbazol-1-one 3.
4.1.2.1.1. 2-Benzylidene-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one (3a). Yellow solid;
ͦ
1
yield: 244 mg (85%); m.p. 228-230 C; FT-IR (KBr, cm-1) ѵmax: 3290 (NH), 1644 (C=O); H
NMR (400 MHz, CDCl3) (ppm) δH: 11.64 (b s, 1H, N9-H), 8.27 (s, 1H, C5-H), 7.68-7.34 (m, 6H,
C8, C6', C5', C4', C3' & C2'-H), 7.12-6.98 (m, 2H, C7 & C2-2H), 3.05-3.03 (m, 2H, C3-2H), 3.02-
3.00 (m, 2H, C4-2H), 2.50 (s, 3H, C6-CH3); 13C NMR (100 MHz, CDCl3) (ppm) δC: 179.5
(C=O), 138.4 (C2), 136.9 (C8a), 135.6 (C2a), 133.6 (C1'), 132.1 (C9a), 129.5 (C6), 128.4 (C5' &
C3'), 128.1 (C6' & C2'), 127.7 (C4b), 126.7 (C4'), 124.9 (C4a), 122.4 (C7), 120.0 (C5), 118.5 (C8),
27.2 (C3), 20.3 (C4), 17.0 (CH3); Anal. calcd. for C20H17NO: C, 83.59; H, 5.96; N, 4.87; Found:
C, 83.50; H, 5.92; N, 4.93.
4.1.2.1.2. 2-Benzylidene-6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-one (3b). Yellow solid;
ͦ
1
yield: 224 mg (73%); m.p. 231-233 C; FT-IR (KBr, cm-1) ѵmax: 3324 (NH), 1652 (C=O); H
NMR (400 MHz, CDCl3) (ppm) δH: 9.43 (b s, 1H, N9-H), 7.82 (s, 1H, C5-H), 7.62 (s, 1H, C2-H),
7.44-7.30 (m, 7H, C8, C7, C6', C5', C4', C3' & C2'-H), 3.27-3.24 (m, 2H, C3-2H), 3.03-3.00 (m, 2H,
C4-2H); 13C NMR (100 MHz, CDCl3) (ppm) δC: 180.1 (C=O), 138.7 (C2), 137.3 (C8a), 135.9
(C2a), 134.1 (C1'), 132.8 (C9a), 129.7 (C6), 128.7 (C5' & C3'), 128.5 (C6' & C2'), 128.2 (C4b), 126.3
(C4'), 125.4 (C4a), 122.7 (C7), 120.3 (C5), 118.7 (C8), 27.3 (C3), 20.4 (C4); Anal. calcd. for
C19H14ClNO: C, 74.15; H, 4.58; N, 4.55; Found: C, 74.25; H, 4.54; N, 4.61 .
4.1.2.1.3. 2-Benzylidene-2,3,4,9-tetrahydro-1H-carbazol-1-one (3c). Yellow solid; yield: 227 mg
ͦ
1
(83%); m.p. 232-234 C; FT-IR (KBr, cm-1) ѵmax: 3262 (NH), 1647 (C=O); H NMR (400 MHz,
CDCl3) (ppm) δH: 9.51 (b s, 1H, N9-H), 7.84 (s, 1H, C2-H), 7.66 (d, 1H, C5-H, Jo = 7.8 Hz),
7.49-7.33 (m, 7H, C8, C6, C6', C5', C4', C3' & C2'-H), 7.15 (t, 1H, C7-H, J = 7.8 Hz), 3.28-3.25
22

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(m, 2H, C3-2H), 3.09-3.06 (m, 2H, C4-2H); 13C NMR (100 MHz, CDCl3) (ppm) δC: 179.3
(C=O), 138.2 (C2), 136.7 (C8a), 135.4 (C2a), 133.8 (C1'), 132.3 (C9a), 129.2 (C6), 128.6 (C5' &
C3'), 128.5 (C6' & C2'), 127.4 (C4b), 126.5 (C4'), 124.8 (C4a), 122.2 (C7), 120.3 (C5), 118.7 (C8),
27.1 (C3), 20.4 (C4); Anal calcd. for C19H15NO: C, 83.49; H, 5.53; N, 5.12; Found: C, 83.41; H,
5.58; N, 5.5 .
4.1.2.1.4. 6-Methyl-2-(thiophen-2-ylmethylene)-2,3,4,9-tetrahydro-1H-carbazol-1-one (3d).
ͦ
Yellow solid; yield: 243 mg (83%); m.p. 221-223 C; FT-IR (KBr, cm-1) ѵmax: 3242 (NH), 1631
(C=O); 1H NMR (400 MHz, CDCl3) (ppm) δH: 8.84 (b s, 1H, N9-NH), 7.96 (s, 1H, C2'-H), 7.50
(d, 1H, C4'-H, J4',3' = 5.2 Hz), 7.44 (s, 1H, C5-H), 7.37 (d, 1H, C2'-H, J2',3' = 4.0 Hz), 7.32 (d, 1H,
C8-H, Jo = 8.2 Hz), 7.21 (d, 1H, C7-H, Jo = 8.2 Hz), 7.13 (d d, 1H, C3'-H, J3',2' = 3.8 Hz & J3',4'
=
4.8 Hz), 3.37-3.33 (m, 2H, C3-2H), 3.15-3.13 (m, 2H, C4-2H), 2.45 (s, 3H, C6-CH3); Anal. calcd.
for C18H15NOS: C, 73.69; H, 5.15; N, 4.77; Found: C, 73.60; H, 5.19; N, 4.83 .
4.1.2.1.5. 6-Chloro-2-(thiophen-2-ylmethylene)-2,3,4,9-tetrahydro-1H-carbazol-1-one (3e).
ͦ
Yellow solid; yield: 223 mg (76%); m.p. 219-221 C; FT-IR (KBr, cm-1) ѵmax: 3231 (NH), 1632
(C=O); 1H NMR (400 MHz, CDCl3) (ppm) δH: 8.93 (b s, 1H, N9-H), 7.97 (s, 1H, C2-H), 7.64 (d,
1H, C4'-H, J4',3' = 5.2 Hz), 7.52 (d, 1H, C2'-H, J2',3' = 5.2 Hz), 7.42-7.28 (m, 2H, C3' & C5-H),
7.14-7.11 (m, 1H, C8-H), 7.06-7.04 (m, 1H, C7-H), 3.37-3.34 (m, 2H, C3-2H), 3.13-3.10 (m, 2H,
C4-2H); 13C NMR (100 MHz, CDCl3) (ppm) δC: 179.4 (C=O), 144.5 (C2a), 139.6 (C8a), 137.0
(C1'), 132.4 (C2), 131.7 (C4'), 130.7 (C2'), 130.5 (C9a), 127.3 (C6), 126.6 (C3'), 125.3 (C4b), 124.1
(C4a), 118.3 (C7), 116.6 (C5), 109.4 (C8), 24.6 (C3), 22.4 (C4); Anal. calcd. for C17H12ClNOS: C,
65.07; H, 3.85; N, 4.46; Found: C, 65.16; H, 3.89; N, 4.40.
4.1.2.1.6. 2-(thiophen-2-ylmethylene)-2,3,4,9-tetrahydro-1H-carbazol-1-one (3f). Yellow solid;
ͦ
1
yield: 223 mg (80%); m.p. 220--221 C; FT-IR (KBr, cm-1) ѵmax: 3235 (NH), 1633 (C=O); H
NMR (400 MHz, CDCl3) (ppm) δH: 8.96 (b s, 1H, N9-H), 7.97 (s, 1H, C2-H), 7.69-7.63 (m, 1H,
C4'-H), 7.49-7.33 (m, 2H, C2' & C5-H), 7.18-7.10 (m, 3H, C8, C7 & C6-H), 7.04 (t, 1H, C3'-H, J =
13
4.4 Hz), 3.18-3.07 (m, 4H, C3 & C4-2H); C NMR (100 MHz, CDCl3) (ppm) δC: 180.2 (C=O),
145.4 (C2a), 139.8 (C8a), 136.5 (C1'), 130.0 (C2), 127.9 (C4'), 125.8 (C4b), 124.0 (C2'), 123.1
23

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(C9a), 122.7 (C3'), 121.7 (C4a), 116.6 (C7), 116.4 (C6), 115.7 (C5), 107.6 (C8), 24.9 (C3), 22.7
(C4); Anal. calcd. for C17H13NOS: C, 73.09; H, 4.69; N, 5.01; Found: C, 73.01; H, 4.73; N, 5.07.
4.1.2.1.7. 2-(4'-methoxybenzylidene)-6-methyl-2,3,4,9-tetrahydro-1H-carbazol-1-one (3g).
ͦ
Yellow solid; yield: 265 mg (87%); m.p. 222-224 C; FT-IR (KBr, cm-1)ѵmax: 3321 (NH), 1630
1
(C=O); H NMR (400 MHz, CDCl3) (ppm) δH: 8.89 (b s, 1H, N9-H), 7.74 (s, 1H, C2-H), 7.43-
7.41 (m, 3H, C5, C6' & C5'-H), 7.33-7.27 (m, 1H, C8-H), 7.21-7.18 (m, 1H, C7-H), 6.95 (d, 2H,
C5' & C3'-H, Jo = 8.8 Hz), 3.85 (s, 3H, C4'-OCH3), 3.27-3.24 (m, 2H, C3-2H), 3.05-3.01 (m, 2H,
C4-2H), 2.44 (s, 3H, C6-CH3); 13C NMR (100 MHz, CDCl3) (ppm) δC: 179.8 (C=O), 159.5 (C4'),
138.7 (C8a), 135.5 (C2), 134.3 (C2a), 132.2 (C6' & C2'), 131.7 (C4b), 128.4 (C9a), 127.5 (C1'), 126.7
(C4a), 125.6 (C7), 121.5 (C6), 120.1 (C5), 114.0 (C5' & C3'), 112.4 (C8), 55.5 (OCH3), 27.7 (C3),
24.8 (C4), 18.7 (CH3); Anal. calcd. for C21H19NO2: C, 79.47; H, 6.03; N, 4.41; Found: C, 79.54;
H, 6.00; N, 4.47.
4.1.2.1.8.
2-(4'-methoxybenzylidene)-6-chloro-2,3,4,9-tetrahydro-1H-carbazol-1-one
(3h).
ͦ
Yellow solid; yield: 253 mg (78%); m.p. 220-222 C; FT-IR (KBr, cm-1) ѵmax: 3235 (NH), 1641
1
(C=O); H NMR (400 MHz, CDCl3) (ppm) δH: 9.87 (b s, 1H, N9-H), 7.81 (s, 1H, C2-H), 7.47-
7.45 (m, 3H, C5, C6' & C5'-H), 7.37-7.33 (m, 1H, C8-H), 7.26-7.22 (m, 1H, C7-H), 7.05 (d, 2H,
C5' & C3'-H, Jo = 8.8 Hz), 3.83 (s, 3H, C4'-OCH3), 3.25-3.21 (m, 2H, C3-2H), 3.07-3.05 (m, 2H,
C4-2H); Anal. calcd. for C20H16ClNO2: C, 71.11; H, 4.77; N, 4.15; Found: C, 71.11; H, 4.77; N,
4.15.
4.1.2.1.9. 2-(4'-methoxybenzylidene)-2,3,4,9-tetrahydro-1H-carbazol-1-one (3i). Yellow solid;
ͦ
1
yield: 241 mg (83%); m.p. 224-226 C; FT-IR (KBr, cm-1) ѵmax: 3267 (NH), 1642 (C=O); H
NMR (400 MHz, CDCl3) (ppm) δH: 9.57 (b s, 1H, N9-H), 7.81 (s, 1H, C2-H), 7.65 (d, 2H, C6' &
C2'-H, Jo = 8.4 Hz), 7.61-7.42 (m, 2H, C8 & C5-H), 7.38-7.34 (m, 1H, C6-H), 7.16-7.12 (m, 1H,
C7-H), 6.98-6.94 (m, 2H, C5' & C3'-H, ), 3.85 (s, 3H, C4'-OCH3), 3.30-3.26 (m, 2H, C3-2H), 3.08-
3.05 (m, 2H, C4-2H); 13C NMR (100 MHz, CDCl3) (ppm) δC: 181.0 (C=O), 159.7 (C4'), 138.6
(C8a), 135.2 (C2), 134.5 (C2a), 132.4 (C6' & C2'), 131.5 (C4b), 128.0 (C9a), 127.0 (C1'), 126.9 (C4a),
125.8 (C7), 121.3 (C6), 120.3 (C5), 113.9 (C5' & C3'), 112.6 (C8), 55.3 (OCH3), 27.6 (C3), 24.9
(C4); Anal. calcd. for C20H17NO2: C, 79.19; H, 5.65; N, 4.62; Found: C, 79.10; H, 5.69; N, 4.68.
24

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4.1.2.2. General procedure for the synthesis of 4'-arylidene/heteroarylidene-5'-phenyl-3,4-
dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-dione 7
An appropriate mixture of 2-arylidene/heterolidene-2,3,4,9-tetrahydrocarbazol-1-one 3
(1.0 mmol) isatin 4 (1.0 mmol) and bezylamine 5 (1.0 mmol) was refluxed in dioxane: methanol
(1:1) for 5 h. After completion of the reaction, the solvent was removed in vacuum and crude
product was subjected to silica gel column chromatography using petroleum ether: ethyl acetate
(91:9) as eluant to yield the respective 4'-arylidene/heteroarylidene-5'-phenyl-3,4-
dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-dione 7.
4.1.2.2.1. 6-Methyl-4',5'-diphenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-
ͦ
1,2''-dione (7a). White solid; yield: 455 mg (87%); m.p. 216-218 C; FT-IR (KBr, cm-1) ѵmax
:
3419 (Indole NH), 3322 (carbazole NH), 3222 (pyrrolo NH), 1696 (indole C=O), 1617
1
(carbazole C=O); H NMR (400 MHz, CDCl3) (ppm) δH: 11.18 (b s, 1H, N9-H), 10.18 (b s, 1H,
N1''-H), 7.50 (d, 2H, C8 & C7''-H, Jo = 7.4 Hz), 7.38 (m, 2H, C5'''' & C3''''-H), 7.26-7.22 (m, 4H,
C6''',C5''', C3''' & C2'''-H), 7.18-7.13 (m, 3H, C6'', C6'''' & C2''''-H), 7.09 (s, 1H, C5-H), 7.05 (d, 1H,
C4''-H, Jo = 7.4 Hz), 6.97 (d, 1H, C7-H, Jo = 7.4 Hz), 6.84 (t, 1H, C5''-H, J = 7.4 Hz), 6.56 (t, 1H,
C4'''-H, J = 7.2Hz), 6.54 (t, 1H, C4''''-H, J = 7.2 Hz), 5.45 (d d, 1H, C5'-H, J1,2-cis = 5.6 Hz & J1,2 -
cis = 10.0 Hz), 4.69 (d, 1H, C4'-H, Jcis = 10.0 Hz), 3.61 (d, 1H, pyrrolo NH, J1,2-cis = 5.6 Hz), 2.58-
2.56 (m, 2H, C4-H), 2.48-2.45 (m, 2H, C3-H) 2.22 (s, 3H, C6-CH3); 13C NMR (100 MHz, CDCl3)
(ppm) δC: 191.5 (C=O), 180.5 (C=O), 143.3 (C3a''), 142.2 (C7a''), 139.2 (C1'''), 137.2 (C8a), 131.9
(C1''''), 129.6 (C9a), 128.9 (C5''), 128.9 (C6), 128.5 (C5''' & C3'''), 128.4 (C5'''' & C3''''), 127.9 (C6'''
& C2'''), 127.5 (C6'''' & C2''''), 127.0 (C4b), 126.7 (C6''), 126.1 (C4''' & C4''''), 125.1 (C4''), 121.1
(C4a), 120.5 (C7), 112.7 (C5 & C8), 109.5 (C7''), 72.3 (spiro indole carbon C3''), 63.8 (C5'), 61.5
(spiro carbazole carbon C2), 58.3 (C4'), 32.1 (C4), 21.3 (CH3), 18.5 (C3); Anal. calcd. for
C35H29N3O2: C, 80.28; H, 5.58; N, 8.02; Found: C, 80.36; H, 5.53; N, 8.08.
4.1.2.2.2. 6-Chloro-4',5'-diphenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-
ͦ
1,2''-dione (7b). White solid; yield: 504 mg (93%); m.p. 212-214 C; FT-IR (KBr, cm-1) ѵmax
:
3416 (Indol NH), 3322 (carbazole NH), 3220 (pyrrolo NH), 1695 (indole C=O), 1622 (carbazole
1
C=O); H NMR (400 MHz, CDCl3) (ppm) δH: 10.85 (b s, 1H, N9-H), 10.02 (b s, 1H, N1''-H),
7.49 (d, 2H, C8 & C7''-H, Jo = 7.4 Hz), 7.40 (d, 2H, C5'''' & C3''''-H, Jo = 6.0 Hz), 7.19-7.04 (m,
25

ACCEPTED MANUSCRIPT
8H, C5, C5''', C3''', C4''', C6'''', C2'''', C6'' & C4'-H), 7.00 (d, 2H, C6''' & C2'''-H, Jo = 8.6 Hz), 6.76 (t,
1H, C6'-H, Jo = 7.4 Hz), 6.54-6.46 (m, 2H, C5'' & C7-H), 5.47 (d, 1H, C5'-H, J1,2-cis = 8.0 Hz),
13
4.75 (d, 1H, C4'-H, J1,2-cis = 10.0 Hz), 261-2.50 (m, 2H, C4-2H), 1.46-1.39 (m, 2H, C3-2H); C
NMR (100 MHz, CDCl3) (ppm) δC: 192.3 (C=O), 179.8 (C=O), 141.9 (C3a''), 141.7 (C7a''), 138.6
(C1'''), 136.9 (C1''''), 132.6 (C8a), 130.6 (C5''), 128.9 (C9a), 128.2 (C6''' & C2'''), 128.1 (C6'''' &
C2''''), 127.6 (C5''' & C3'''), 127.3 (C5'''' & C3''''), 126.8 (C4'''), 126.7 (C4''''), 126.3 (C4b), 125.3 (C6),
124.8 (C5 & C7), 121.3 (C6'' & C4''), 119.9 (C4a), 114.0 (C7''), 109.6 (C8), 78.0 (spiro indol carbon
C3''), 64.7 (C5'), 62.2 (spiro carbazole carbon C2), 58.5 (C4'), 31.8 (C4), 18.5 (C3); Anal. calcd. for
C34H26ClN3O2: C, 75.06; H, 4.82; N, 7.72; Found: C, 75.15; H, 4.78; N, 7.66.
4.1.2.2.3.
4',5'-Diphenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-9(H)-1,2''-
ͦ
dione (7c). White solid; yield: 417 mg (82%); m.p. 214-216 C; FT-IR (KBr, cm-1) ѵmax: 3374
(Indol NH), 3307 (carbazole NH), 3229 (pyrrolo NH), 1687 (indole C=O), 1636 (carbazole
C=O); 1H NMR (400 MHz, CDCl3) (ppm) δH: 10.50 (b s, 1H, N9-H), 9.98 (b s, 1H, N1''-H), 7.50
(t, 2H, C5'''' & C3''''-H, J = 7.2 Hz), 7.41 (d, 2H, C8 & C5-H, Jo = 7.2Hz), 7.24-7.04 (m, 11H, C7'',
C5''', C3''', C6''', C2''', C6'''', C2'''', C4'', C4''', C4'''' & C6''-H), 6.82 (t, 1H, C5''-H, J = 7.2 Hz), 6.78-
6.74 (m, 1H, C6-H), 6.50-6.48 (m, 1H, C7-H), 5.47 (d, 1H, C5'-H, Jo = 10.2 Hz), 4.76 (d, 1H, C4'-
H, J1,2-cis = 10.2 Hz), 2.60-2.46 (m, 2H, C4-2H), 1.48-1.43 (m, 2H, C3-2H); 13C NMR (100 MHz,
CDCl3) (ppm) δC: 197.0 (C=O), 184.6 (C=O), 146.7 (C3a''), 146.5 (C7a''), 143.5 (C1'''), 143.4
(C8a), 136.3 (C1''''), 135.4 (C5''), 133.6 (C9a), 133.2 (C5''' & C3'''), 133.0 (C5'''' & C3''''), 132.8 (C4b),
132.7 (C6''' & C2''') 132.4 (C6'''' & C2''''), 132.0 (C4'''), 131.4 (C4''''), 130.9 (C6'' & C4''), 130.1 (C7),
129.7 (C4a), 126.1 (C6), 125.6 (C5), 124.2 (C6''), 117.4 (C8), 114.3 (spiro indol carbon C3''), 69.6
(C5'), 67.1 (spiro carbazole carbon C2), 63.6 (C4'), 36.7 (C4), 23.4 (C3); Anal. calcd. for
C34H27N3O2: C, 80.13; H, 5.34; N, 8.25; O, 6.28; Found: C, 80.06; H, 5.38; N, 8.18.
4.1.2.2.4. 6-Methyl-4'-(thiophen-2-yl)-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-
ͦ
indole]- 9(H)-1,2''-dione (7d). White solid; yield: 423 mg (80%); m.p. 196-198 C; FT-IR (KBr,
cm-1) ѵmax: 3423 (Indole NH), 3392 (carbazole NH), 3263 (pyrrolo NH), 1697 (indol C=O), 1618
1
(carbazole C=O); H NMR (400 MHz, CDCl3) (ppm) δH: 8.65 (b s, 1H, N9-H), 7.79 (b s, 1H,
N1''-H), 7.67-7.65 (m, 2H, C5'''' & C3''''-H), 7.32-7.28 (m, 2H, C4''' & C7''), 7.24-7.21 (m, 1H, C8-
H), 7.18 (s, 1H, C5-H), 7.16-7.07 (m, 5H, C6'''',C2'''', C4'''', C4''& C6''-H), 6.93-6.89 (m, 2H, C5'' &
26

ACCEPTED MANUSCRIPT
C7-H), 6.69-6.65 (m, 1H, C3'''-H), 6.62 (d, 1H, C2'''-H, Jo = 7.6 Hz), 5.48 (d, 1H, C5'-H, J1,2-cis
=
9.0 Hz), 5.06 (d, 1H, C4'-H, J1,2-cis = 9.0 Hz), 2.70-2.68 (m,2H, C4-H), 2.65-2.64 (m, 1H, C3b-H),
13
2.33 (s, 3H, C6-CH3), 1.91-1.97 (m, 1H, C3a-H); C NMR (100 MHz, CDCl3) (ppm) δC: 191.7
(C=O), 179.6 (C=O), 141.3 (C3a''), 141.1 (C1'''), 140.2 (C7a''), 136.7 (C8a), 131.3 (C1''''), 129.5
(C5''), 129.2 (C9a), 129.1 (C6), 128.5 (C5'''' & C3''''), 128.3 (C6'''' & C2''''), 128.1 (C4b), 127.7 (C3'''),
127.6 (C6''), 126.7 (C4''''), 125.7 (C4''' & C2'''), 125.5 (C4''), 124.5 (C4a), 122.2 (C7), 120.5 (C5),
111.8 (C7''), 109.3 (C8), 73.2 (spiro indol carbon C3'), 66.9 (C5'), 62.1 (spiro carbazole carbon
C2), 53.9 (C4'), 30.8 (C4), 21.2 (C3), 18.6 (CH3); Anal. calcd. for C33H27N3O2S: C, 74.83; H,
5.14; N, 7.93; Found: C, 74.94; H, 5.10; N, 7.99.
4.1.2.2.5.
6-Chloro-4'-(thiophen-2-yl)-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-
ͦ
2',3''-indole]- 9(H)-1,2''-dione (7e). White solid; yield: 401 mg (73%); m.p. 194-196 C; FT-IR
(KBr, cm-1) ѵmax: 3427 (Indol NH), 3329 (carbazole NH), 3225 (pyrrolo NH), 1696 (indol C=O),
1620 (carbazole C=O); 1H NMR (400 MHz, CDCl3) (ppm) δH: 10.51 (b s, 1H, N9-H), 9.71 (b s,
1H, N1''-H), 7.43 (d, 2H, C9 & C7''-H, Jo = 6.8 Hz), 7.12-6.98 (m, 5H, C5, C5'''',C3'''', C4''' & C6''-
H), 6.94-6.88 (3H, C6'''', C2'''' & C4''''-H), 6.85 (d, 1H, C4''-H, Jo = 7.4 Hz), 6.72-6.70 (m, 1H, C7-
H), 6.66 (t, 1H, C5''-H, J = 7.4 Hz), 6.41 (d, 1H, C2'''-H, Jo = 8.0 Hz), 6.35 (t, 1H, C3'''-H, J = 8.0
Hz), 5.26 (d, 1H, C5'-H, J1,2-cis = 9.4 Hz), 4.80 (d, 1H, C4'-H, J1,2-cis = 9.4 Hz), 2.45-2.35 (m, 3H,
C3a & C4-H), 1.63-1.54 (m, 1H, C3b-H); 13C NMR (100 MHz, CDCl3) (ppm) δC: 192.0 (C=O),
179.3 (C=O), 141.5 (C3a''), 141.6 (C1'''), 140.4 (C7a''), 136.3 (C8a), 131.8 (C1''''), 129.7 (C5''),
129.4 (C9a), 129.2 (C6), 128.7 (C5'''' & C3''''), 128.5 (C6'''' & C2''''), 128.0 (C4b), 127.9 (C3'''), 127.3
(C6''), 126.4 (C4''''), 125.9 (C4''' & C2'''), 125.3 (C4''), 124.7 (C4a), 122.5 (C7), 120.1 (C5), 112.0
(C7''), 109.7 (C8), 73.4 (spiro indole carbon C3''), 67.1 (C5'), 62.3 (spiro carbazole carbon C2),
54.1 (C4'), 31.0 (C4), 21.3 (C3); Anal .calcd. for C32H24ClN3O2S: C, 69.87; H, 4.40; N, 7.64;
Found: C, 69.96; H, 4.44; N, 7.70.
4.1.2.2.6. 4'-(Thiophen-2-yl)-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-2',3''-indole]-
ͦ
9(H)-1,2''-dione (7f). White solid; yield: 406 mg (79%); m.p. 198-200 C; FT-IR (KBr, cm-1)
ѵmax: 3376 (Indole NH), 3314 (carbazole NH), 3275 (pyrrolo NH), 1685 (indol C=O), 1642
1
(carbazole C=O); H NMR (400 MHz, CDCl3) (ppm) δH: 8.69 (b s, 1H, N9-H), 7.78 (b s, 1H,
N1''-H), 7.67 (d, 2H, C5'''' & C3''''-H, Jo = 7.2 Hz), 7.37 (d, 1H, C5-H), 7.33-7.22 (m, 4H, C8, C4''',
27

ACCEPTED MANUSCRIPT
C7'' & C4''-H), 7.18-7.12 (m, 4H, C6'''', C2'''', C4'''' & C6''-H), 7.00 (t, 1H, C5''-H, J = 7.2 Hz), 6.94-
6.89 (m, 2H, C7 & C6-H), 6.67 (t, 1H, C3'''-H, J = 7.6 Hz), 6.62 (d, 1H, C2'''-H, Jo = 7.6 Hz), 5.50
(d, 1H, C5'-H, J1,2-cis = 9.4 Hz), 5.06 (d, 1H, C4'-H, J1,2-cis = 9.4 Hz), 2.73-2.63 (m, 3H, C3a & C4-
H), 1.91-1.88 (m, 1H, C3b-H); 13C NMR (100 MHz, CDCl3) (ppm) δC: 191.3 (C=O), 179.9
(C=O), 141.4 (C3a''), 141.8 (C1'''), 140.4 (C7a''), 135.2 (C8a), 131.5 (C1''''), 129.3 (C5''), 129.1
(C9a), 129.0 (C6), 128.7 (C5'''' & C3''''), 128.5 (C6'''' & C2''''), 128.3 (C4b), 127.5 (C3'''), 127.4 (C6''),
126.9 (C4''''), 125.6 (C4''' & C2'''), 125.7 (C4''), 124.3 (C4a), 122.6 (C7), 121.2 (C5), 111.6 (C7''),
109.5 (C8), 73.6 (spiro indole carbon C3'), 66.5 (C5'), 62.3 (spiro carbazole carbon C2), 53.7 (C4'),
30.6 (C4), 21.4 (C3); Anal. calcd. for C32H25N3O2S: C, 74.54; H, 4.89; N, 8.15; Found: C, 74.53;
H, 4.85; N, 8.21.
4.1.2.2.7.6-Methyl-4'-(4'''-methoxyphenyl)-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-
ͦ
2',3''-indole]-9(H)-1,2''-dione (7g). White solid; yield: 407 mg (85%); m.p. 200-202 C; FT-IR
(KBr, cm-1) ѵmax: 3420 (Indol NH), 3378 (carbazole NH), 3241 (pyrrolo NH), 1689 (indole
C=O), 1643 (carbazole C=O); 1H NMR (400 MHz, CDCl3) (ppm) δH: 8.78 (b s, 1H, N9-H), 7.93
(b s, 1H, N1''-H), 7.60 (d, 2H, C6''' & C2'''-H, Jo = 7.6 Hz), 7.43 (d, 2H, C8 & C7''-H, Jo = 7.6 Hz),
7.33-7.25 (m, 2H, C6'''' & C2''''-H), 7.20 (t, 3H, C5'''', C3'''' & C4''''-H, Jo =7.2 Hz), 7.11 (s, 1H, C5-
H), 7.07 (d, 1H, C4''-H, Jo = 7.6 Hz), 6.90 (t, 1H, C6''-H, J = 7.6 Hz), 6.80 (d, 2H, C5''' & C3'''-H,
Jo = 8.4 Hz), 6.68 (t, 1H,C5''-H, J=7.6 Hz), 6.61 (d, 1H, C7-H, Jo = 7.6 Hz), 5.52 (d, 1H, C5'-H,
J1,2-cis = 9.6 Hz), 4.80 (d, 1H, C4'-H, J1,2-cis = 9.6 Hz), 3.74 (s, 3H, OCH3), 2.69-2.53 (m, 3H, C3a
13
& C4-H), 2.31 (s, 3H, CH3), 1.69-1.63 (m, 1H, C3b-H); C NMR (100 MHz, CDCl3) (ppm) δC:
192.3 (C=O), 180.0 (C=O), 158.4 (C4'''), 141.6 (C3a''), 140.2 (C8a), 136.7 (C7a''), 131.6 (C1''''),
131.4 (C5''), 130.3 (C9a), 129.4 (C1'''), 129.1 (C6''' & C2'''), 128.9 (C6), 128.5 (C5'''' & C3''''), 128.4
(C6'''' & C2''''), 128.3 (C4b), 127.5 (C6''), 127.3 (C4''''), 125.8 (C4''), 125.5 (C4a), 122.2 (C7), 120.5
(C5), 113.6 (C7''), 111.8 (C5''' & C3'''), 109.3 (C8), 73.1 (spiro indole carbon C3''), 65.5 (C5'), 62.4
(spiro carbazole carbon C2), 58.1 (C4'), 55.0 (OCH3), 31.8 (C4), 21.2 (CH3), 18.7 (C3); Anal.
calcd. for C36H31N3O3: C, 78.10; H, 5.64; N, 7.59; O, 8.67; Found: C, 78.19; H, 5.59; N, 7.52.
4.1.2.2.8. 6-Chloro-4'-(4'''-methoxyphenyl)-5'-phenyl-3,4-dihydrodispiro[carbazole-2,3'-pyrrolo-
ͦ
2',3''-indole]-9(H)-1,2''-dione (7h). White solid; yield: 517 mg (90%); m.p. 201-203 C; FT-IR
(KBr, cm-1) ѵmax: 3410 (Indole NH), 3371 (carbazole NH), 3315 (pyrrolo NH), 1686 (indole
28