Synthesis of tetrahydro-1: H -indolo[2,3- b] pyrrolo[3,2- c] quinolones via intramolecular oxidative ring rearrangement of tetrahydro-β-carbolines and their biological evaluation

Article abstract of DOI:10.1039/c7nj04616b

A simple oxidative ring rearrangement of diversely substituted 1-(2-amminoaryl)-tetrahydro-β-carbolines has been developed to generate architecturally interesting tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolones. This unique transformation involves fou

Full text of DOI:10.1039/c7nj04616b

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Synthesis of tetrahydro-1H-indolo[2, 3-b]pyrrolo[3, 2-c]quinolones via intramolecular
oxidative ring rearrangement of tetrahydro-β-carbolines and their biological evaluation
Chandramohan Bathula,1 Catarina RomaꢀRodrigues,2 Jyoti Chauhan,1 Alexandra R.Fernandes,2,*and Subhabrata
Sen1, *
1 Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Chithera, Dadri, GautamBudh
Nagar, Uttar Pradesh, India 201314
2UCIBIO,Departamento de Ciências da Vida, Faculdade de Ciências e Tecnologia, Universidade Nova de Lisboa,
Caparica, Portugal
*corresponding authors: Alexandra
R
Fernandes (ma.fernandes@fct.unl.pt) and Subhabrata Sen
(subhabrata.sen@snu.edu.in).
Abstract
A simple oxidative ring rearrangement of diversely substituted 1ꢀ(2ꢀamminoaryl)ꢀtetrahydroꢀβꢀ
carbolines has been developed to generate architecturally interesting tetrahydroꢀ1Hꢀindolo[2, 3ꢀ
b]pyrrolo[3, 2ꢀc]quinolones. This unique transformation involves four reaction center (aniline,
C1ꢀcarboline and C2/C3 of indole) and utilizes tertꢀbutylhypochlorite as the reagent. The generic
nature of the reaction was demonstrated by the synthesis of a wide variety of analogs 9aꢀj. A
putative reaction mechanism was proposed. Cytotoxicity screening of these compounds against
three human cancer cells (A2780 ovarian and HCT116 colorectal carcinoma cell lines and A549
lung adenocarcinoma cell line) revealed selective inhibition of proliferation of A2780 human
ovarian carcinoma cell line by one of the molecules 9a with an IC50 of 14 ꢀM. No cytotoxic
activity was observed in human normal fibroblasts for concentrations up to 100 ꢀM. Compound
9a induce hyperpolarization of mitochondrial membrane potential of A2780 cell line leading to
an increase of reactive oxygen species (ROS) that trigger cell death via apoptosis. Interestingly,
compound 9a was also able to induce cell death via autophagy. Compounds that induce
apoptosis and autophagy, thus leading to cancer cells’ death, are good candidates for cancer
therapy.

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Introduction
Spiropyrroloxindole and quinolino indole alkaloids are interesting classes of compounds due to
their abundance in nature and their intriguing biological activity.1 In general they have imparted
substantial inhibition against a bevy of cancer cells. For example Spirotryptostatin A and B, 1
and 2 (Scheme 1), isolated from fermentation broth of Aspergillus fumigatin inhibit G2/M
progression of mammalian tsFT210 cells and synthetic spiroindole, 3, a novel potent nonꢀ
peptidic inhibitor of p53ꢀMDM2 proteinꢀprotein interaction, critical for regulating p53 protein
tumor suppressing activity(Figure 1).2aꢀd Additionally, Perophoramidine, 4 and Communecin B, 5
are structurally concomitant indole alkaloids possessing a unique 6, 5, 6, 6 ring system belong to
a class of interesting natural products that elicit inhibitory activity in cancer cells by inducing
apoptosis via PARPꢀ1 cleavage (a signal irreparable DNA damage) and by disrupting
microfilament in cultured mammalian cell, respectively (Figure 1). Interestingly
Perophoramidine and dehalo analogs has exhibited inhibitory activity (IC50 ~ 60 – 70 ꢀM)
against a series of colorectal cancer cell lines (HCT116, HT29 and LoVo).3aꢀd Several procedures
have been reported in the literature for the synthesis of spiropyrrolooxindole and 6, 5, 6, 6 ring
systems belonging to Perophoramidine and Communecin B. These generally deploy,
intramolecular mannich reaction of oxytryptamine hydrochloride, oxidative rearrangement of
tetrahydroꢀβꢀcarbolines, 1, 3ꢀdipolar cycloaddition with azomethine ylides with oxindoylidine 3ꢀ
ylidene acetate, radical cyclization of 2ꢀbromoꢀ4ꢀmethoxyaniline with Cbzꢀprotected glycine
ethyl ester and etc. for the synthesis of the synthesis of spiropyrroloxindole. As for the 6,5,6,6ꢀ
ring system biosynthesis involving inverse electron demand hetero Diels Alder reaction of the
ergot alkaloid aurantioclavine with tryptamine derivatives, azaꢀoꢀxylylene cycloaddition (by
Funk and coꢀworkers) and interrupted Fischer indolization based reaction by Garg et al. are few
of the synthetic approaches.4ꢀ5
The anticancer legacy of these scaffolds prompted us to develop an efficient intramolecular ring
rearrangement of 1ꢀ(2ꢀaminoaryl)tetrahydroꢀβꢀcarbolines for the production of compounds
incorporating the two aforementioned heterocycles. The resulting compounds were subjected
through phenotypic screening against A2780 ovarian, HCT116 colorectal and A549 lung
adenocarcinoma cell lines followed by the phenotypic profiling of the most active compound.

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Figure 1. Design and proposed synthesis of hybrids inspired from representative natural
products
Results and discussion
Chemistry
In one of our recent works we have demonstrated oxidative ring rearrangement reactions of 1ꢀ
aryltetrahydroꢀβꢀcarbolines to provide spiropyrrolooxindole.6 In the reaction, water acted as the
active nucleophile that prompted the ring rearrangement which in turn was initiated by C2
bromination of carboline. Drawing inspiration from this results and similar oxidative ring
rearrangement we explored tertꢀBuOCl mediated intramolecular ring rearrangement of 1ꢀ
arylaminoꢀtetrahydroꢀβꢀcarbolines 8aꢀj to provide the desired compounds 9aꢀj (our initial
exploratory effort with NBS, yielded the desired product in moderate yield).7 tertꢀBuOCl is a
versatile and economical oxidizing reagent used in the oxidation of alcohol, aldehydes, sulfides
and hydroxylamines.8 Very recently it was reported to promote CꢀH functionalization of C1 of
tetrahydro carbazoles.9 Similar to these reported examples we assume that the initial step of this
reaction involved C3 chlorination of 1ꢀ(2ꢀaminophenyl)tetrahydroꢀβꢀcarboline 8a to provide A
(Scheme 1). The aniline present in the moiety prompted an intramolecular attack at C2 to afford
B. Subsequent imine formation in B promoted the migration of the C1ꢀC2 bond to C3, where the
chloride is eliminated to generate the desired product 9a.

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Scheme 1. Putative mechanism of the intramolecular oxidative ring rearrangement
This was demonstrated by reacting 1ꢀarylaminotetrahydroꢀβꢀcarboline 8a with 1.1 equiv of tertꢀ
BuOCl and 1.2 equiv of triethyl amine in dichloromethane. Once the starting material is
consumed the solvent was evaporated and the residue was refluxed in a 1:1 mixture of methanol
and acetic acid to provide the final compound 9a, which was isolated by column
chromatography. This strategy provided advantages of harnessing inexpensive, air stable
reagents like tertꢀBuOCl via an operationally simple procedure. Another noteworthy aspect is
that the substrate 8a could be synthesized in a three step sequence from economically viable
tryptamine ($ 500/kg) involving PictetꢀSpengler condensation with 2ꢀnitrobenzaldehyde, benzyl
protection of the carboline nitrogen and iron (Fe) calcium chloride (CaCl2) reduction of the aryl
nitro moiety. Interestingly 8a was obtained clean enough to be used as such in the oxidative ring
rearrangement (Scheme 2).
Scheme 2. Intramolecular oxidative rearrangement of 8a in presence of tertꢀBuOCl and TEA
along with AcOH

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The generic nature of this method was demonstrated by subjecting various 1ꢀaryltetrahydroꢀβꢀ
carbolines 8aꢀj to the conditions outlined in Scheme 3. The outcome illustrates that the R groups
substituting the aryl moiety as C1 of the carbolines, R1 functionality on the carboline nitrogen
and R2 on the indole benzene ring substantiated no significant effect on the yield of the reaction.
R included trifluoromethyl, chloro, fluoro and amine functionalities at various position of the C1
aromatic moiety, R1 typically encompassed methyl and benzyl functionality and R2 included
bromo, chloro and trifluromethoxy group at C5 position of the indole benzene ring. The average
yield of the desired products 9bꢀj ranged from 72ꢀ84% (Scheme 3). Interestingly the reaction
generates two stereogenic centers, a and b (refer structure 9b) within the molecules. The
compounds have [3,4,0] bicyclic system at the core, which we envision to favor a cis fusion. The
NMR spectra further indicates the presence of only one diastereomer.
F
Bn
N
Bn
N
Bn
N
N
Br
b
Cl
Cl
a
N
N
N
N
N
H
N
H
N
N
H
H
9e, 77%
9d, 85%
9c, 78%
9b, 81%
Bn
Cl
F
Bn
N
Bn
N
N
CF3
N
MeO
N
N
N
N
N
N
H
N
H
N
H
H
9g, 86%
9f, 80%
9i, 85%
9h, 75%
H
N
CF3
N
N
H
9j, 84%
Scheme 3. Library of natural product inspired hybrid molecules
In vitro screening

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As discussed in the introduction, both spiropyrrolooxindole and 6, 5, 6, 6, indoloquinolone
containing natural products have exhibited substantial inhibitory activity against cancer cells.
Consequently, it was pragmatic for us to investigate the inhibition induced by the library of our
molecules against the proliferation of several cancer cell lines such as A2780 ovarian cancer cell
line, HCT116 human colorectal carcinoma cell line and A549 lung adenocarcinoma cells and
normal human fibroblasts using the MTS assay (Table 1).
Compound 9a exhibited a high cytotoxic effect against A2780 cell line, with a low relative IC50
value (14.2 ± 0.3 µM), a moderate cytotoxicity against HCT116 cell line (relative IC50 37.3 ± 0.4
µM) and no cytotoxic activity in A549 ((relative IC50 > 100 µM) (Table 1). Of relevance,
compound 9a has no cytotoxic activity in human normal fibroblasts demonstrating its selectivity
towards ovarian carcinoma cell line (selectivity index – 7ꢀfold) (Table 1).
All the other compounds did not present any cytotoxic activity in HCT116, A549 cancer cell
lines or normal human fibroblasts (Table 1). However, they presented high to median
cytotoxicity in A2780 cell line (9d>9e>9h>9f>9g, 9i-j) (Table 1). A close scrutiny of the
structure activity relationship between the functionalities that encrusted our library of molecules
against the antiproliferative effects they impose against the cancer cells indicated not much scope
of modification. No functional groups be it electron donating or electron withdrawing at the
terminal aromatic rings of the molecules are tolerated, as they lead to the reduction of their
antagonistic activity on the cancer cells when compared to the unsubstituted analog 9a.
Additionally, the benzyl functionality at the pyrrolidine nitrogen of our compounds proved to be
the best substituent as deprotection of it to NHꢀ or replacement with a methyl functionality
proved to be detrimental to the antiproliferative effect of the analogs as illustrated by compounds
9h/ j (30.7 ꢀM versus no activity, respectively) and 9d/ 9f (23.6 ꢀM versus 38.9 ꢀM).
This selective antiproliferative effect of our molecules at low micromolar concentration,
accentuates the eminence of our natural product inspired compounds; usually the expected
outcome from such unbiased early explorations of molecules are poor (high micromolar). This
modest hit further highlights the importance of incorporating natural product scaffolds in
discovering novel bioactive molecules.

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Table 1. Relative IC50 values for compounds 9a-j in tumor cell lines (A2780, HCT116 and
A549) and normal fibroblasts
IC50 (ꢀM)
Entry
Compounds
A2780
14.2
59.6
72.3
23.6
26.4
38.9
100
HCT116
A549
Fibroblast
1
2
37.3
>100
>100
9a
9b
9c
9d
9e
9f
>100
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
̎
3
̎
4
̎
5
̎
6
̎
7
̎
9g
9h
9i
8
30.7
>100
̎
̎
9
93.3
>100
10
9j
Cells were treated with increasing concentrations of compound 9a for 48 h and cell viability was determined by
MTS assay. Data were normalized against the control treated with 0.1 % (v/v) DMSO. Results are the mean of at
least 3 independent biological MTS assays (±SEM).
Since 9a, was the most potent among our library of molecules we investigated into the
mechanism of cytotoxic action induced by compound 9a against A2780 ovarian carcinoma cell
line, apoptosis and autophagy were assessed. Hoechst nuclei staining of A2780 cells in absence
(DMSO as control) or presence of compound 9a at its IC50 (Table 1) was assessed (Figure 2).

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Figure 2. Hoechst staining (excitation and fluorescence emission 352 and 461 nm, respectively)
of A2780 cell line for visualization of apoptotic nuclei. Cells were grown in DMEM culture
medium supplemented with 10% fetal bovine serum in the presence of: A) DMSO control (0.1 %
v/v), B) compound9a (at IC50). Plates were photographed in an AXIO Scope (Carl Zeiss,
Oberkochen Germany). Three random microscopic fields per sample with circa 50 nuclei were
counted. White arrows indicate chromatin condensation. C) % of apoptotic cells after exposition
of A2780 cells to control vehicle (DMSO) or the compound 9a. *pꢀvalue < 0.05 relative to
apoptosis in cells incubated with DMSO.
Hoechst
33258
(2'ꢀ[4ꢀethoxyphenyl]ꢀ5ꢀ[4ꢀmethylꢀ1ꢀpiperazinyl]ꢀ2,5'ꢀbiꢀ1Hꢀbenzimidazole
trihydrochloride trihydrate) is a cellꢀpermeable DNA stain that binds preferentially to adenineꢀ
thymine (AꢀT) regions of the DNA and it can detect nuclear damage and apoptosis. Normal

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nuclei show nonꢀcondensed chromatin uniformly distributed over the entire nucleus, whereas
apoptotic nuclei show condensate or fragmented chromatin. Some cells form apoptotic bodies.10
Figure 2 shows a clear increase of apoptotic markers, such as chromatin condensation and
nuclear structure abnormalities (arrows in Figure 2) in A2780 cells incubated with the compound
9a. The level of cell death due to apoptosis observed in Figure 1 for compound 9a is 1.5 x higher
that control cells (DMSO).
Despite its ability to induce cell death via apoptosis, we also evaluated if this complex could
induce other types of programmed cell death (PCD) such as Type II ꢀ autophagic cell death.
Autophagy is a catabolic process that digests cellular contents within lysosomes and may be
accelerated by a variety of cellular stressors such as nutrient starvation, DNA damage, and
organelle damage.11
Figure 3 shows that compound 9a can also induce autophago lysosomes accumulation, a
characteristic of autophagy. Rapamycin was used as a positive marker for autophagy (Figure 3).

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Figure 3. Autophagic cell death evaluation using the CYTOꢀID Autophagy detection assay in
the presence of DMSO as vehicle control (0.1 % v/v) (A), compound 9a (B) andRapamycin as an
autophagy marker (C) and assessed by fluorescence microscopy. Merge images of nuclei that
were stained with DAPI (in blue; excitation and fluorescence emission 358 and 461 nm,
respectively) and autophagolysosomes that were stained in green (excitation and fluorescence
emission 463 and 534 nm, respectively). White arrows point to accumulation of
autophagolysosomes. Plates were photographed in an AXIO Scope (Carl Zeiss, Oberkochen
Germany). D) % of Autophagic A2780 cells in the presence of compound 9a. DMSO was used

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as vehicle control and Rapamycin as autophagic positive marker. *pꢀvalue < 0.05 relative to
DMSO autophagy.
Mitochondrial dysfunction has been demonstrated to participate in the induction of apoptosis and
has even been suggested to be central to the apoptoticꢀsignaling pathway.12 The membraneꢀ
permeant JCꢀ1 dye is widely used in apoptosis studies as an indicator of mitochondrial
membrane potential in a variety of cell types.13
In this regard, the effect of compound 9a in mitochondrial membrane potential (ΔΨM) of A2780
cell line was evaluated (Figure 4).
Figure 4. Mitochondrial membrane potential. Cells were incubated in the absence (cells) (A) or
presence of 0.1% (v/v) DMSO (control) (B) or compound 9a (at IC50) (C) for 48 h. D)
Fluorescence ratio red to green (JCꢀ1 aggregate/JCꢀ1 monomer).Plates were photographed in an
AXIO Scope (Carl Zeiss, Oberkochen Germany). Results are the mean ± SEM from three
independent biological assays. Statistical analysis was performed by OneꢀWay ANOVA (** p ≤
0.01 relative to DMSO).

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As we can observe in Figure 4, a high increase of the red versus green fluorescence in A2780
cells after exposure to compound 9a can be due to the hyperpolarization of the mitochondrial
membrane. Interestingly, mitochondrial hyperpolarization appears to be the earliest change
associated with apoptotic pathways and leads to uncoupling of oxidative phosphorylation (i.e.
continued reactive oxygen species (ROS) production in the absence of ATP synthesis), which
disrupts mitochondrial membrane potential (ꢁΨm) and damages integrity of the inner
mitochondrial membrane. Disruption of ꢁΨm has been proposed as the point of no return in cell
death signaling.14ꢀ15 These results are in agreement of the apoptosis induction in the presence of
compound 9a (Figure 2).
Taking into consideration these results, the levels of ROS in the absence or presence of
compound 9a was evaluated (Figure 5).
2.0
1.5
*
*
1.0
0.5
0.0
H2O2
Control
(DMSO)
9a
Figure 5. Quantification of the fluorescence intensity of H2DCFꢀDA dye in A2780 cells after
exposition to hydrogen peroxide (H2O2; 25 µM), 0.1 % v/v DMSO (control) or compound 9a (at
its IC50) for 48 h. Results are the mean ± SEM of three independent biological experiments and
were normalized to control cells (0.1 % v/v DMSO). H2O2 was used as a positive control for
ROS induction. Statistics analysis was performed by OneꢀWay ANOVA ((* p ≤ 0.05).

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The cellꢀpermeant 2',7'ꢀdichlorodihydrofluorescein diacetate (H2DCFDA) (also known as
dichlorofluorescin diacetate) is a chemically reduced form of fluorescein used as an indicator for
reactive oxygen species (ROS) in cells.16 This cellꢀpermeable nonꢀfluorescent compound
accumulates within cells upon deꢀacetylation reacting within tracellular ROS, turning highly
fluorescent.17
As observed in Figure 5 there is a statistical significant increase of ROS production both in the
presence of H2O2 or compound 9a. Increase in the rate of ROS production is responsible for the
accumulation of ROSꢀassociated damages in biological molecules such as DNA, proteins, and
lipids, and may result in progressive cell dysfunctions leading to apoptosis.18ꢀ19
Taken together these results conforms that compound 9a induce membrane hyperpolarization
and an increase of ROS production that lead to an increase of cell death through apoptosis.
Additionally, compound 9a is also able to induce cell death via autophagy.
Compounds that induce apoptosis and autophagy, thus leading to cancer cells’ death, are good
candidates for cancer therapy.20 Therefore, compound 9a is a promising early lead to ultimately
identify a suitable candidate for combinatory therapy modalities with standard therapies in
ovarian cancer potentiating cancer cell death.
Conclusions
Herein we have reported synthesis of tetrahydroꢀ1Hꢀindolo[2, 3ꢀb]pyrrolo[3, 2ꢀc]quinolones.
They are novel natural product inspired molecules, which were accessed through a four step
linear sequence starting from tryptamine and its derivatives. Interestingly the intermediates of
this sequence were clean enough to be taken forward and only the final compounds were
purified. The key step involved atypical intramolecular oxidative ring rearrangement of
substituted 1ꢀ(2ꢀnitroaryl)ꢀtetrahydroꢀβꢀcarbolines (8aꢀj). A focussed library of ten compounds
(9aꢀj) was prepared based on a putative mechanism of transformation supported by literature
evidence. Since the original natural products that inspired this strategy had an anticancer legacy,
the antiproliferative potential of the library was examined towards human ovarian carcinoma
(A2780), colorectal carcinoma (HCT116) and lung adenocarcinoma (A549) cell lines.
Compound 9a was found to have the highest cytotoxic effect compared to the other compounds
particularly in A2780 ovarian carcinoma (IC50 of 14 µM). Indeed, compound 9a exhibits a high

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selectivity (more than 7x) towards this type of cancer cells when compared to normal human
fibroblasts (no cytotoxic activity for concentrations up to 100 µM). against colorectal carcinoma
cells, with IC50 lower than that of cisplatin (10.5 ± 0.3 µM compared to 15.2 ± 0.55 µM,
respectively). The viability loss induced by compound 9a was due to the induction of autophagy
and it agrees with Hoechst 33258 staining and the typical morphological apoptotic characteristics
like chromatin condensation and nuclear fragmentation. Compound 9a was shown to
hyperpolarize the membrane potential leading to an increase of ROS production and finally a
pointꢀofꢀnoꢀreturn – cell death via apoptosis.
Taken together these results demonstrate that in comparison to the other compounds,
compound 9a provides several advantages namely its high cytotoxic potential in A2780 cell line
with the simultaneously no cytotoxicity in healthy cells, making this a positive feature for further
development particularly towards ovarian carcinoma.
We intend to realise the protein target of 9a, via deconvolution technique to develop a
much potent candidate through molecular docking and structure activity relationship campaign.
This is presently ongoing in our lab.
EXPERIMENTAL SECTION
Chemistry
General. All reactions were carried out in flameꢀdried glassware with magnetic stirring. Unless
otherwise noted, all experiments were performed under argon atmosphere. All reagents were
purchased from Sigma Aldrich, Acros organics or Alfa Aesar. Solvents were treated with 4 Å
molecular sieves or sodium and distilled prior to use. Purifications of reaction products were
1
carried out by column chromatography using Chem Lab silica gel (230ꢀ400 mesh). H NMR and
13C NMR spectra were recorded with tetramethylsilane (TMS) as internal standard at ambient
1
temperature unless otherwise indicated in Bruker AVHDN at 400 MHz for H NMR and 100
MHz for 13C NMR. Chemical shifts are reported in parts per million (ppm) and coupling
constants are reported as Hertz (Hz). Splitting patterns are designated as singlet (s), broad singlet
(bs), doublet (d), triplet (t). Splitting patterns that could not be interpreted or easily visualized are
designated as multiple (m). The Mass Spectrometry analysis was done on the 6540 UHD

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AccurateꢀMass QꢀTOF LC/MS system (Agilent Technologies) equipped with Agilent 1290 LC
system. 1H and 13C NMR data included in supplementary information
General experimental procedure for the synthesis of the final compounds
To a stirred solution of appropriate βꢀaryl amines (0.5 mmoles, 1 equiv.) and various oꢀ
nitrobenzaldehydes (0.5 mmoles, 1 equiv.) in dichloromethane (10 mL) was added trifluro acetic
acid (0.5 mmoles, 1 equiv.) at ambient temperature. The resulting mixture was then stirred under
argon for 10ꢀ16h. Once thin layer chromatography (TLC) confirms the complete consumption of
the starting materials, the reaction mixture was concentrated under reduced pressure and
minimum volume of dichloromethane was added to the resulting solution and stored at 0°C for
30 minutes. The products crystallized from the solution, filtered, washed with dichloromethane
and dried to provide the crude compounds which were used in the next step without purification.
The above compound was dissolved in dimethylformamide (DMF) (10 mL) and triethyl amine
(TEA) (1.5 mmoles, 3 equiv.) was added. Then the reaction mixture was then cooled to 0°C
before adding methyl iodide/benzyl bromide (0.75 mmoles, 1.5 equiv.). The resulting solution
was allowed to stir at room temperature for 30 minutes. Once thin layer chromatography (TLC)
confirms the complete consumption of the starting materials, the reaction mixture was quenched
with water and was extracted with ethyl acetate (2 ×10 mL). The organic layer was separated,
dried over anhydrous sodium sulphate (Na2SO4) and was evaporated to obtain the desired crude
compound. This crude compound was further used for next step without purification.
The crude intermediate from the previous step was dissolved in EtOH : Water (9.5 : 0.5 mL)
mixture and treated with Fe powder (0.75 mmoles, 1.5 equiv.) and CaCl2 (0.75 mmoles, 1.5
equiv.) at room temperature. The resulting mixture was warmed to 60°C and stirred for 45
minutes. Once thin layer chromatography (TLC) confirms the complete consumption of the
starting materials, the reaction was cooled to room temperature and concentrated under reduced
pressure to obtain the desired crude compounds 8a-j.
8a-j were dissolved in dichloromethane at room temperature and was further treated with triethyl
amine (1.2 equiv.) and tertꢀbutylhypochlorite (1.1 equiv). The resulting mixture was further
stirred at same temperature for about 4h. Once thin layer chromatography (TLC) confirms the
complete consumption of the starting materials, the reaction was quenched with water and the

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organic layer was isolated and evaporated. The crude reaction mixture was dissolved in 1:1
acetic acid: methanol (10 mL) mixture and was refluxed for ~ 3h. Then the reaction was
quenched with solid NaHCO3, filtered and evaporated to obtain the desired crude compounds.
The crude solids were purified by flash column chromatography (FCC) using ethyl acetateꢀ
hexane as the mobile phase, to provide the final compounds 9a-j as solid or semi solid form.
1-benzyl-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinoline (9a):
Following the general procedure tryptamine, oꢀnitrobenzaldehyde and benzyl bromide provided
1
9a (84% yield). H NMR (400 MHz; CDCl3): 7.57 (s, 1H), 7.55 (s, 1H), 7.47 (d, J = 4 Hz,
1H),7.41 (d, J = 8 Hz, 3H), 7.33 (d, J = 4 Hz, 3H), 7.19 (d, J = 8, 16 Hz, 1H), 7.06 (t, J = 4, 16
Hz, 1H), 7.00 (d, J = 8 Hz, 1H), 4.59ꢀ4.50 (m, 2H), 4.41 (t, J = 12 Hz, 1H), 3.13 (t, J = 4 Hz,
13
1H), 3.02 (d, J = 4 Hz, 1H), 2.55ꢀ2.45 (m, 1H), 1.79 (s, 1H). C NMR (100 MHz; DMSOꢀd6):
171.6, 140.3, 129.5, 128.4, 127.8, 127.7, 126.9, 125.9, 122.3, 122.3, 121.0, 66.9, 60.6, 49.9, 38.1.
IR (neat, ν cmꢀ1): 2936, 2864, 1648, 1638, 1628, 1599, 1564, 1465, 1202. HRMS (ESIꢀTOF)
m/z: [M + H]+calculated for (C24H22N3) 352.1808, found 352.1801. M.p.ꢀ 133 °C
1-benzyl-5-chloro-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9b):
Following the general procedure 5ꢀChlorotryptamine, oꢀnitrobenzaldehyde and benzyl bromide
provided 9b (81% yield).1H NMR (400 MHz; DMSOꢀd6): 7.51 (t, J = 4 Hz, 2H), 7.44ꢀ7.36 (m,
3H), 7.34ꢀ7.25 (m, 2H), 7.22 (d, J = 7.22, 2H), 7.18ꢀ7.12 (m, 2H), 6.99ꢀ6.88 (m, 2H), 4.58
(s,1H), 4.53ꢀ4.48 (m, 1H), 4.42ꢀ4.33 (m, 1H), 2.90ꢀ2.88 (m, 2H), 2.25ꢀ2.17 (m, 1H), 1.66ꢀ1.62
(m, 1H). 13C NMR (100 MHz; DMSOꢀd6): 179.6, 171.4, 159.7, 157.4, 140.8, 128.9, 128.9,
128.5, 127.5, 123.1, 121.4, 116.1, 115.9, 115.0, 114.8, 67.4, 61.0, 50.6, 38.4. IR (neat, ν cmꢀ1):
2931, 2827, 1654, 1609, 1573, 1462, 1123, 747. HRMS (ESIꢀTOF) m/z: [M + H]+calculated for
(C24H21N3Cl) 386.1419, 388.1398 found 386.1420, 388.1396 M.p.ꢀ 148 °C
5-chloro-1-methyl-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9c):
Following the general procedure 5ꢀChlorotryptamine, oꢀnitrobenzaldehyde and methyl iodide
provided 9c (78% yield).1H NMR (400 MHz; DMSOꢀd6): 7.44 (d, J = 8 Hz, 1H), 7.29ꢀ7.25 (m,
2H), 7.34ꢀ7.25 (m, 2H), 7.18ꢀ7.12 (m, 2H), 7.00ꢀ6.93 (m, 2H), 4.27 (s, 1H), 2.99 (s, 3H), 2.98ꢀ
2.91 (m, 2H), 2.22ꢀ2.15 (m, 1H), 1.66ꢀ1.61 (m, 1H). 13C NMR (100 MHz; DMSOꢀd6): 171.1,

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159.3, 157.0, 137.0, 136.0, 128.0, 122.6, 120.6, 120.8, 115.4, 115.2, 114.5, 114.3, 113.4, 67.5,
54.9, 53.27, 51.5, 43.2, 38.6. IR (neat, ν cmꢀ1): 2251, 2127, 1660, 1650, 1578, 1485, 831. HRMS
(ESIꢀTOF) m/z: [M + H]+calculated for (C18H17N3Cl) 310.1106, 312.1081 found 310.1109,
312.1084. M.p.ꢀ 123 °C
1-benzyl-12-fluoro-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9d):
Following the general procedure tryptamine, 5ꢀfluoroꢀ2ꢀnitrobenzaldehyde and benzyl bromide
provided 9d (85% yield).1H NMR (400 MHz; CDCl3): 7.55 (s,1H), 7.53 (s, 1H), 7.41 (dd, J = 4,
12 Hz, 3H), 7.31 (dd, J = 8, 20 Hz, 3H), 7.20 (d, J = 8 Hz, 1H), 7.12 (dd, J = 4, 12 Hz, 1H), 7.05
(t, J = 8 Hz, 2H), 6.90 (td, J = 4, 8 Hz, 1H), 4.51 (t, J = 16 Hz, 2H), 4.41 (d, J = 12 Hz, 1H), 3.14
13
(t, J = 8 Hz, 1H), 3.05ꢀ2.98 (m, 1H), 2.49ꢀ2.41 (m, 1H), 1.79 (dd, J = 8, 12 Hz, 1H). C NMR
(100 MHz; CDCl3): 179.1, 172.4, 160.7, 158.3, 148.7, 139.8, 136.6, 135.2, 128.8, 128.8, 128.6,
127.5, 123.0, 121.8, 121.0, 116.2, 115.9, 115.3, 114.0, 67.5, 61.4, 54.1, 50.4, 38.3. IR (neat, ν
cmꢀ1): 2920, 2850, 1640, 1577, 1505, 1433. HRMS (ESIꢀTOF) m/z: [M + H]+calculated for
(C24H21N3F) 370.1714, found 370.1710. M.p.ꢀ 158 °C
1-benzyl-5-bromo-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9e):
Following the general procedure 5ꢀbromotryptamine, oꢀnitrobenzaldehyde and benzyl bromide
provided 9e (77% yield).1H NMR (400 MHz; CDCl3): 7.55 (s, 1H), 7.53 (s, 1H), 7.41 (dd, J = 8,
12 Hz, 3H), 7.31 (dd, J = 4, 16 Hz, 3H), 7.20 (d, J = 8 Hz, 1H), 7.11 (dd, J = 4, 8 Hz, 1H), 7.05
(t, J = 4 Hz, 2H), 6.90 (td, J = 4, 8 Hz, 1H), 4.51 (t, J = 16 Hz, 2H), 4.41 (d, J = 12 Hz, 1H), 3.14
(t, J = 8 Hz, 1H), 3.05ꢀ2.99 (m, 1H), 2.49ꢀ2.41 (m, 1H), 1.79 (dd, J = 8, 12 Hz, 1H).13C NMR
(100 MHz; CDCl3): 179.3, 172.6, 160.9, 158.5, 148.9, 140.0, 136.8, 135.4, 129.0, 128.8, 127.7,
123.2, 122.0, 121.2, 116.4, 116.1, 115.5, 115.3, 114.2, 67.7, 61.6, 54.3, 50.6, 38.5. HRMS (ESIꢀ
TOF) m/z: [M + H]+calculated for (C24H21N3Br) 432.0896, 430.0913 found 432.0864, 430.0887.
M.p.ꢀ 168 °C
12-fluoro-1-methyl-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9f):
Following the general procedure tryptamine, 5ꢀfluoroꢀ2ꢀnitrobenzaldehyde and methyl iodide
provided 9f (80% yield).1H NMR (400 MHz; CDCl3): 7.33 (d, J = 8 Hz, 1H), 7.29 (d, J = 4 Hz,
1H), 7.23 (s, 1H), 7.21 (d, J = 4 Hz, 1H), 7.17 (d, J = 8 Hz, 1H), 7.02 (t, J = 8 Hz, 1H), 6.98 (t, J

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= 4 Hz, 1H), 6.90 (td, J = 4, 8 Hz, 1H), 4.18 (s, 1H), 3.21 (t, J = 8 Hz, 1H), 3.09 (s, 1H), 3.05ꢀ
13
3.00 (m, 1H), 2.49ꢀ2.41 (m, 1H), 1.78 (dd, J = 8, 12 Hz, 1H). C NMR (100 MHz; CDCl3):
173.0, 160.7, 158.3, 149.5, 137.3, 135.2, 129.2, 129.2, 128.5, 123.0, 121.6, 120.8, 115.6, 114.9,
114.0, 68.5, 54.5, 52.0, 43.7, 39.0. HRMS (ESIꢀTOF) m/z: [M + H]+calculated for (C18H17N3F)
294.2184, found 294.2180. M.p.ꢀ 133 °C
1-benzyl-5-methoxy-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9g):
Following the general procedure 5ꢀmethoxytryptamine, oꢀnitrobenzaldehyde and benzyl bromide
provided 9g (86% yield). 1H NMR (400 MHz; CDCl3): 7.53 (t, J = 8 Hz, 2H), 7.45 (d, J = 8 Hz,
1H), 7.39 (t, J = 8 Hz, 2H), 7.32 (d, J = 4 Hz, 1H), 7.19 (dd, J = 8, 16 Hz, 3H), 7.10 (d, J = 12
Hz, 1H), 7.02 (t, J = 8 Hz, 1H), 6.96 (d, J = 8 Hz, 1H), 6.85 (d, J = 8 Hz, 1H), 4.52 (s, 1H), 4.45
(dd, J = 4, 16 Hz, 2H), 3.86 (s, 1H), 3.08ꢀ3.01 (m, 2H), 2.51ꢀ2.40 (m, 1H), 1.81 (t, J = 8 Hz,
1H). 13C NMR (100 MHz; CDCl3): 173.2, 140.6, 139.6, 139.3, 137.0, 132.4, 131.3, 131.1, 129.5,
127.5, 127.3, 125.9, 125.4, 123.5, 117.8, 117.2, 114.6, 67.2, 66.8, 60.6, 56.0, 49.6, 38.1. HRMS
(ESIꢀTOF) m/z: [M + H]+calculated for (C25H24N3O) 382.1914, found 382.1915. M.p.ꢀ 149 °C
1-benzyl-11-(trifluoromethyl)-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-
c]quinolone (9h):
Following the general procedure tryptamine, 2ꢀnitroꢀ5ꢀ(trifluoromethyl) benzaldehyde and
benzyl bromide provided 9h (75% yield).1H NMR (400 MHz; CDCl3): 7.69 (s, 1H), 7.67 (s, 2H),
7.56 (s, 4H), 7.48 (s, 3H), 7.41 (d, J = 4 Hz, 2H), 7.24 (s, 1H), 4.70 (t, J = 16 Hz, 2H), 4.58 (d, J
13
= 16 Hz, 1H), 3.30 (d, J = 4 Hz, 1H), 2.64 (d, J = 8 Hz, 1H), 1.99 (d, J = 4 Hz, 1H). C NMR
(100 MHz; CDCl3): 173.4, 149.5, 139.9, 139.7, 137.4, 136.4, 130.6, 130.6, 129.0, 128.9, 128.7,
127.6, 123.0, 122.2, 120.1, 116.1, 114.6, 67.4, 61.6, 54.8, 50.6, 38.6. IR (neat, ν cmꢀ1): 2920,
2850, 1640, 1577, 1505, 1433. HRMS (ESIꢀTOF) m/z: [M + H]+calculated for (C25H21N3F3)
420.1682, found 420.1680. M.p.ꢀ 165 °C
1-benzyl-12-chloro-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9i):
Following the general procedure tryptamine, 5ꢀchloroꢀ2ꢀnitrobenzaldehyde and benzyl bromide
provided 9i (85% yield).1H NMR (400 MHz; CDCl3): 7.55 (s, 1H), 7.54 (s, 1H), 7.42 (t, J = 8
Hz, 3H), 7.32 (dd, J = 8, 16 Hz, 3H); 7.18 (d, J = 8 Hz, 1H), 7.13 (dd, J = 4, 12 Hz, 1H), 7.06 (t,

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J = 8 Hz, 1H), 6.98 (dd, J = 4, 8 Hz, 1H), 6.89 (t, J = 8 Hz, 1H), 4.52 (t, J = 8 Hz, 2H), 4.41 (d, J
= 16 Hz, 1H), 3.15 (t, J = 8 Hz, 1H), 3.04ꢀ2.91 (m, 1H), 2.51ꢀ2.43 (m, 1H), 1.82ꢀ1.78 (m, 1H).
13C NMR (100 MHz; CDCl3): 172.4, 160.7, 158.3, 148.7, 139.8, 136.6, 135.2, 128.8, 128.8,
128.6, 127.5, 123.0, 121.8, 121.0, 120.9, 116.2, 115.9, 115.3, 115.1, 114.0, 67.5, 61.4, 54.1, 50.4,
38.3. IR (neat, ν cmꢀ1): 2921, 2853, 1659, 1636, 1576, 1493, 795. HRMS (ESIꢀTOF) m/z: [M +
H]+ calculated for (C24H21N3Cl) 386.1419, 388.1398 found 386.1411, 388.1389. M.p.ꢀ 141 °C
12-(trifluoromethyl)-2,3,8,13b-tetrahydro-1H-indolo[2,3-b]pyrrolo[3,2-c]quinolone (9j):
Following the general procedure tryptamine and 2ꢀnitroꢀ5ꢀ(trifluoromethyl) benzaldehyde
provided 9j (84% yield).1H NMR (400 MHz; DMSOꢀd6): 10.53 (s, 1H), 7.75 (d, J = 8 Hz, 1H),
7.51 (d, J = 8 Hz, 1H), 7.30 (d, J = 8 Hz, 1H), 7.21 (t, J = 8 Hz, 1H), 7.05 (d, J = 8 Hz, 1H), 6.94
(t, J = 8 Hz, 1H), 4.49 (s, 1H), 3.29ꢀ3.18 (m, 1H), 2.79 (t, J = 12 Hz, 1H), 2.07ꢀ1.99 (m, 1H),
13
1.64 (t, J = 8 Hz, 1H). C NMR (100 MHz; CD3OD): 173.7, 170.2, 140.6, 134.2, 133.8, 131.9,
129.1, 126.1, 125.3, 124.7, 122.8, 117.9, 112.8, 62.2, 57.7, 51.4, 40.4. HRMS (ESIꢀTOF) m/z:
[M + H]+calculated for (C18H15N3F3) 330.1213, found 330.1214. M.p.ꢀ 145 °C
Biological assays
Cell culture. Human ovarian carcinoma (A2780), colorectal carcinoma (HCT116) and lung
adenocarcinoma (A549) cell lines were grown in Dulbecco’s modified Eagle’s medium
(DMEM) (Invitrogen Corp., Grand Island, NY, USA) supplemented with 10% fetal bovine
serum and 1% antibiotic/antimycotic solution (Invitrogen Corp.) and maintained at 37 ºC in a
humidified atmosphere of 5 % (v/v) CO2.17 and 21 MCF7 cell line derived from pleural effusion of
breast adenocarcinoma from a female patient was grown in similar conditions, supplemented
with
1
%
MEM nonꢀessential amino acids (Invitrogen Corp.).22 Normal
Humanfibroblastsweregrown in thesameconditions as MCF7 cell line.17, 21ꢀ22 All cell lines were
purchase from ATCC (www.atcc.org).
Compound exposure for dose-response curves. Cells were plated at 5000 cells/well in 96ꢀwell
plates. Media was removed 24 h after platting and replaced with fresh media containing: 0.1 ꢀ
100 µM of compounds 9a, 9d-j or 0.1 % (v/v) DMSO (vehicle control). All the previous
solutions were prepared from concentrated stock solutions (in DMSO) of the compounds.
Viability assays. After 48 h of cell incubation in the presence or absence of each compound, cell
viability was evaluated with CellTiter 96® AQueous NonꢀRadioactive Cell Proliferation Assay

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(Promega,
Madison,
WI,
USA),
using
3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ5ꢀ(3ꢀ
carboxymethoxyphenyl)ꢀ2ꢀ(4ꢀsulfophenyl)ꢀ2Hꢀtetrazolium, inner salt (MTS) as previously
described.17ꢀ24 In brief, this is a homogeneous, colorimetric method for determining the number
of viable cells in proliferation, cytotoxicity or chemosensitivity assays. The CellTiter 96®
AQueous Assay is composed of solutions of MTS and an electron coupling reagent
(phenazinemethosulfate, PMS). MTS is bioreduced by cells into a formazan product that is
soluble in tissue culture medium. The absorbance of the formazan product at 490 nm can be
measured directly from 96ꢀwell assay plates without additional processing. The conversion of
MTS into the aqueous soluble formazan product is accomplished by dehydrogenase enzymes
found in metabolically active cells. The quantity of formazan product was measured in a Bioꢀ
Rad microplate reader Model 680 (BioꢀRad, Hercules, CA, USA) at 490 nm, as absorbance is
directly proportional to the number of viable cells in culture.
Assessment of apoptosis through Hoechst 33258 staining. HCT116 cells grown as described
above were plated at 7500 cells/mL and incubated for 48 h in culture medium containing the
compound 9a or 0.1 % (v/v) DMSO (vehicle control). Hoechst staining (excitation and
fluorescence emission 352 and 461nm, respectively) was used to detect apoptotic nuclei as
previously described.16 Briefly, medium was removed, cells were washed with phosphateꢀ
buffered saline 1X (PBS) (Invitrogen), fixed with 4 % (v/v) paraformaldehyde in PBS 1X (10
min in the dark) and incubated with Hoechst dye 33258 (Sigma, Missouri, USA; 5 µg/mL in
PBS 1X) for another 10 min. After washed with PBS 1X, cells were mounted using 20 µL of
PBS: glycerol (3:1; v/v) solution. Fluorescent nuclei were sort out per the chromatin
condensation degree and characteristics. Normal nuclei showed nonꢀcondensed chromatin
uniformly distributed over the entire nucleus. Apoptotic nuclei showed condensate or fragmented
chromatin. Some cells formed apoptotic bodies. Plates were photographed in an AXIO Scope
(Carl Zeiss, Oberkochen Germany), and three random microscopic fields per sample with ca. 50
nuclei were counted. Mean values were expressed as the percentage of apoptotic nuclei.21
Autophagy. For autophagy analysis, 0.5x105 of HCT116 cells were seeded on top of a sterilized
cover slide and let to adhere for 24 h. The supernatant was substituted with fresh medium
containing compound 9a (at its IC50). For control purposes, one cover slide with cell monolayer
was treated with 0.1% (v/v) DMSO in fresh medium, and another cover slide was treated with
fresh medium for 24 h, when rapamycin was added to a final concentration of 50 µg/mL. After

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48 h incubation of cells with the compound 9a and the control compound, medium was removed
and cells were stained according to the instructions of CYTOꢀID Autophagy detection kit
(ENZO, NY, USA).19 and 25 Stained cells were imaged using a fluorescence microscope (Carl
Zeiss) and autophagy measured using the CYTOꢀID® Green dye (excitation and fluorescence
emission 463 and 534 nm, respectively; DAPI was used to counterstain the nucleus (excitation
and fluorescence emission 358 and 461 nm, respectively) and respective software (ZEN Blue
edition, 2011). The total number of cells and the number of cells with autophagolysosomes were
counted in at least 5 different images of each sample to calculate the % of cells in
autophagocytosis.
Evaluation of mitochondria membrane potential. Mitochondrial transmembrane potential was
analyzed using the dye 5,5’,6,6’ꢀtetrachloroꢀ1,1’,3,3’ꢀ tetraethylbenzimiꢀdazolylꢀcarbocyanine
iodide (JCꢀ1; Abnova Corporation, Walnut, CA, USA). A2780 cell line was seeded into 24ꢀwell
plates cells at 0.75 × 105 cells/well density and incubated during 24 h. After the incubation, cells
were treated with compound 9a (at the IC50) or 0.01% (v/v) DMSO (vehicle control) and
incubated for 48 h. For the measurement of the fluorescence intensity, cells were stained with
JCꢀ1 staining solution for 20 min at 37°C in the dark, followed by visualization with a
fluorescence Microscope Olympus Bx51 microscope equipped with Olympus DP50 camera
using the same exposition time for all samples. Green to red fluorescence ratio of each sample
was determined using ImageJ software (National Institutes of Health (NIH), Bethesda, MD,
USA).
Detection of reactive oxygen species (ROS). The detection of ROS was performed using the
ROS assay (Life Technologies, InvitrogenTM, USA). A2780 cell line was seeded and incubated
at 37ºC, 99% (v/v) humidity and 5% (v/v) CO2 with a cell density of 1 x 105 cells/mL. After 24
h, culture medium was removed and replaced by fresh medium containing compound 9a (at its
IC50) or and 0.01 % (v/v) DMSO (vehicle control). Hydrogen peroxide (H2O2) at a concentration
of 50 ꢂM was used as a positive control. Cells were harvested after 48 h of exposure to
compounds, washed two times with PBS 1X, reꢀsuspended in preꢀwarmed PBS 1X containing 10
ꢂM H2DCFꢀDA and incubated for 20 min at 37°C, in absence of light. The levels of DCF
positive cells was measured in an Attune cytometer (Applied Biosystems, Foster City, CA, USA)
and quantified with the Attune Cytometric Software (Applied Biosystems).
Statistical analysis

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All data were expressed as mean ± SEM from at least three independent experiments.
Statistical significance was evaluated using the Student’s tꢀtest; p < 0.05 was considered
statistically significant.
ASSOCIATED CONTENT
Supporting Information
1H and 13C NMR spectra of all products. This material is available free of charge via the Internet
at http://pubs.acs.org.
AUTHOR INFORMATION
Corresponding Author
Eꢀmail: subhabrata.sen@snu.edu.in; ma.fernandes@fct.unl.pt
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
Acknowledgements
This work was supported by Shiv Nadar University and The Unidade de Ciências
Biomoleculares Aplicadas ꢀ UCIBIO which is financed by national funds from FCT/MEC
(UID/Multi/04378/2013) and coꢀfinanced by the ERDF under the PT2020 Partnership
Agreement (POCIꢀ01ꢀ0145ꢀFEDERꢀ007728).
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