A diversity oriented synthesis of natural product inspired molecular libraries

Article abstract of DOI:10.1039/c7ob02230a

Natural products are the source of innumerable pharmaceutical drug candidates and also form an important aspect of herbal remedies. They are also a source of various bioactive compounds. Herein we have leveraged the structural attributes of several natural products in building a library of architecturally diverse chiral molecules by harnessing R-tryptophan as the chiral auxiliary. It is converted to its corresponding methyl ester 1 which in turn provided a bevy of 1-aryl-tetrahydro-β-carbolines 2a-d, which were then converted to chiral compounds via a diversity oriented synthetic strategy (DOS). In general, intermolecular and intramolecular ring rearrangements facilitated the formation of the final compounds. Four different classes of molecules with distinct architectures were generated, adding up to nearly twenty-two individual molecules. Phenotypic screening of a representative section of the library revealed two molecules that selectively inhibit MCF7 breast cancer cells with IC₅₀ of ～5 μg mL^-1 potency.

Full text of DOI:10.1039/c7ob02230a

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A diversity oriented synthesis of natural product inspired molecular libraries
Jyoti Chauhan,a Tania Luthra,a Rambabu Gundlab, Antonio Ferraro,c Ulrike Holzgrabec and
Subhabrata Sena, *
a Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Dadri, Chithera,
GautamBudh Nagar, UP 201314, India
subhabrata.sen@snu.edu.in
b Department of Chemistry, Gitam Institute of Technology, GITAM University, Hyderabad,
Telengana, India
cInstitute of Pharmacy and Food Chemistry, University of Würzburg, Am Hubland, Dꢀ97074
Würzburg, Germany
Abstract
Natural products are the origin for innumerable pharmaceutical drug candidates and also forms
an important aspect of herbal remedies. They are source of various bioactive compounds. Herein
we have leveraged structural attributes of several natural products in building a library of
architecturally diverse chiral molecules by harnessing Rꢀtryptophan, as the chiral auxiliary. It is
converted to its corresponding methyl ester 1 which in turn provided a bevy of 1ꢀarylꢀtetrahydroꢀ
βꢀcarbolines 2aꢀd which were then converted to chiral compounds via diversity oriented
synthetic strategy (DOS). In general intermolecular and intramolecular ring rearrangements
facilitated the formation of the final compounds. Four different classes of molecules with distinct
architecture were generated accounting to nearly twenty two individual molecules. Phenotypic
screening of a representative section of the library revealed two molecules that selectively inhibit
MCF7 breast cancer cells with IC50 of ~5 ꢀgm/mL potency.

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Introduction
To investigate complex biological system, small molecules have been applied to modulate
proteins by directly interacting with them.1 In conventional genetic approaches biological
systems were assessed by developing random mutations which were then screened in search of a
precise cellular phenotype.2 Analogous to the genetic approach, large random collections of
small molecules can be used to elucidate the roles of specific proteins in many biological
pathways.3 The essence of this chemical genetic approach is the design and synthesis of a library
of compounds which cover large areas of biologically relevant chemical space.4
By virtue of binding both their biosynthetic enzymes and their target molecules, natural products
necessarily reside in biologically relevant chemical space.5 Natural product families are libraries
of prevalidated, functionally diverse structures.6 Therefore developing library of molecules with
scaffolds inspired from natural products could provide biologically active novel molecules.7
There are quite a few reports of library of molecules inspired from natural products.8aꢀd For
example, Khan et al. reported synthesis of a molecular library inspired from marine natural
product ianthelliformisamines and their biological evaluation, in another report, Singh and coꢀ
workers generated a natural product inspired βꢀcarboline and γꢀlactones based molecular hybrids
and Waldmann reported stereoselective synthesis of natural product inspired tetrahydroindolo[2,
3-a]ꢀquinolizine compound library.8cꢀd
Additionally, to counter the challenge of gaining rapid access to structurally diverse natural
product inspired libraries, diversity oriented synthesis (DOS) is an ideal tool.9 This concept
discovered by Schreiber at the beginning of last decade, is a forward directional strategy that
involves facile preparation of libraries of architecturally complex and diverse compounds from
simple starting materials. DOS can be achieved through various approaches.10 One such strategy,
the reagent based approach, involves generation of a densely functionalized central building
block which in turn can be transformed into various scaffolds by subjecting them to varied
tranformations.11 Oxidative rearrangement chemistry extends interesting possibilities for DOS
application, especially in reagent based approaches. In this direction a bevy of diverse reactions
can be performed to generate scaffolds with diverse architecture.12
Here in we have reported the design and synthesis of a library of molecules based on natural
product Perophoramidine, Spirotryptostatin, Harmicin and Tryprostatin A and B (Figure 1a).13

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The resulting compounds were generated by modifying a central tetrahydroꢀβꢀcarboline moiety
under various reaction conditions which underwent amalgamation of the scaffolds of the
aforementioned natural products to generate 3, 4, 5 and 6 (Scheme 1b). Such an effort is purely
intuitive with the expectation that the process will provide novel biologically active molecules.
In general, this strategy involved a DOS, via a novel (oxidative) inter/ intramolecular ring
rearrangement. A representative section of the library was screened against MCF7 cell lines to
assess their ability to modulate a particular cancer phenotype.
Figure 1. Natural products and the library inspired from them
Results and discussion
Design
The design of this library is based on tetrahydroꢀβꢀcarbolines 2aꢀd. They were conceived as
central scaffolds because they contained several pluripotent reaction centers such as a, b, c, d and
e (Scheme1). Careful choice of reaction condition will enable us to harness these centers

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selectively to provide us the desired scaffolds 3ꢀ6. The design enabled the library to proliferate
from one scaffold to the other. For example, compound 2, could provide 3 and 5. 3 in turn could
furnish 4 and 6 (Scheme 1). Accordingly, we envisioned that compounds 3, could be accessed by
exploiting the reactivity of center a and b of 2, by sequential treatment of chloroacetyl chloride
and appropriate amines (Scheme 1). Next, compound 4 could be obtained from 3 by an oxidative
ring opening reaction involving centers c, e and f (Scheme 1). We further envisioned that 6 could
also be accessed from 3 via an intramolecular ring rearrangement involving centers c, d, e and f
(Scheme 1). A similar oxidative ring rearrangement of 2 again involving centers cꢀf could also
provide compound 5 (Scheme 1).
Scheme 1. Design of the hybrid library
Chemistry
With the design in place we embarked on the synthesis of the molecular library. To begin with,
the central tetrahydroꢀβꢀcarboline building blocks 2aꢀd were synthesized via a Pictet Spengler13f
reaction between 1 (obtained by methylation of Rꢀtryptophan) and appropriately substituted oꢀ
nitrobenzaldehydes (i.e. oꢀnitrobenzaldehyde, 4ꢀchlorobenzaldehyde, 5ꢀchlorobenzaldehyde and
veratraldehyde) (Scheme 2). A typical reaction involved refluxing 1, 1.5 equiv. of trifluoroacetic
acid and 1.2 equiv. of oꢀnitrobenzaldehyde in acetonitrile. The desired products were obtained as

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1:1 diasteromeric mixtures. From the desired products that where generated we opted to proceed
with the compound 2a (in a bid to prove this modular concept this was a random choice) which
was isolated via column chromatography. Accordingly, single crystal Xꢀray of 2a (refer SI)
indicated that, the methyl ester and the aryl moiety were in syn conformation. Compounds 2bꢀd
were generated in a similar fashion and were taken forward to proliferate the library (Scheme 2).
1.5 eq. TFA
Dichloromethane
rt, ~20h
CO2Me
NH2
CHO
NO2
+
Diastereomeric Ratio: 7:3
Average Yield: 68-87%
(both the diasteromers)
N
R
H
R = H; o-nitrobenzaldehyde
4-chloro-2-nitrobenzaldehyde
1
R = 4-Cl;
R = 5-Cl; 5-chloro-2-nitrobenzaldehyde
R = 3, 4-dimethoxy; Veratraldehyde
CO2Me
NH
NO2
N
H
R
(major)
2a
2a: R = H
2b
: R = 4-chloro
2c: R = 5-chloro
2d: R = 3, 4-dimethoxy
Scheme 2. Synthesis of central scaffolds 2aꢀd
The first set of molecules 3aꢀg, that we envisioned, possessed tetrahydroꢀβꢀcarboline
diketopiperazine (DKP) scaffolds. Accordingly 2a, was treated with chloroacetyl chloride to
provide amide 9a which was further treated with methyl amine to facilitate an in situ
nucleophilic substitution followed by lactamization(with the ester) to generate the desired hybrid
3a in 58% yield. In a similar fashion, 2a, after reaction with chloroacetyl chloride (ClCH2COCl)
was treated with various other amines i.e. ethyl, benzyl, hexyl, propyl and isopropyl to afford 3bꢀ
f. Accordingly 2d provided 3h.

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Scheme 3. Synthesis of tetrahydroꢀβꢀcarbolineꢀDKP hybrids 3aꢀg
Next, as envisioned during the design of the molecules, sodium dithionate mediated nitro
reduction of 3aꢀd, f and g facilitated the amine intermediates 10aꢀd, f and g (Scheme 4). The
reduction was extremely facile with no formation of any byꢀproducts. Consequently, amines 10aꢀ
d, f and g, were taken to the next step without further purification. To promote the intramolecular
oxidative ring rearrangement of these amines, variety of oxidants such as tꢀbutoxychloride (tꢀ
BuOCl), Nꢀbromosuccinimide (NBS), sodium tungstate (dehydrate) (Na2WO4.2H2O), lead
tetraacetate(Pb(OAc)4) and osmium tetraoxide (OsO4)were explored with 10a as the model
substrate. To our extreme gratification, NBS furnished the best yield (~91%) of the desired
hybrid 6a which is the major diastereomer in a 70:30 mixture of the products. It was separated

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from the mixture by column chromatography. With the optimized condition in hand, the other
hybrids, 6bꢀf were also synthesized in moderate to excellent yield (Scheme 4).
Scheme 4. Synthesis of hybrids 6aꢀf
The rational of transformation of the resulting hybrids 6aꢀf from the corresponding amine
intermediate could be explained by the putative mechanism of the intramolecular ring
rearrangement of 6f, as depicted in Scheme 5 below. The reaction involved treatment of 10f with
NBS as an oxidant, which brominates at CX of 10f to generate A. The bromination is expected to
occur from the β face to avoid the steric interaction from the DKP and 2ꢀnitroaryl

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Scheme 5. Putative mechanism of the intramolecular ring rearrangement to generate 6
moiety. A nucleophilic attack by the aromatic amine at CY generated B followed by a final bond
rearrangement prompted by the imine formation, where the bond shift happened from the

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opposite face of the bromide functionality (in a fashion similar to S
N
2
reactions) to afford the
desired products 6f/f´ as 70:30 mixture of diastereomers (Scheme 5) from which the major
diastereomer, 6f was isolated by column chromatography). 1DꢀNOESY experiment of 6f
provided the relative configuration of the stereocenters a and b as S and R respectively (Scheme
5).
In a bid to further diversify our scaffold library, 3aꢀg were treated with NBS in presence of
tetrahydrofuran: water: trifluoroacetic acid (1:1:1). Initially the bromination occurred at C3 of the
indole skeleton to afford the bromo iminium intermediate A΄, which subsequently leads to the
formation of enamine B΄. Elimination of the bromide on B΄generated C΄ followed by the attack
of the water molecule to afford hemiaminal D΄. D΄ then underwent ring opening to furnish the
desired hybrids 4 (scheme 6).
Scheme 6. Synthesis of hybrid 4aꢀg

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Finally compounds 5aꢀd were synthesized via a similar ring rearrangement as in compounds 6aꢀ
g from the starting substrate 2aꢀd, by their subsequent reduction with sodium dithionite (to
generate 9aꢀd) followed by treatment with NBS and acetic acid. Here too we obtained the major
diastereomers 5aꢀd from a 80:20 mixture, by column chromatography in 60ꢀ72% yield (Scheme
7).
Scheme 7. Synthesis of hybrid scaffolds 5
Phenotyping screening
The original natural products that inspired our molecules have a rich legacy as anticancer
compounds. Subsequently we decided to screen our molecules for anticancer activity. Due to
the paucity of the cell lines, a representative selection of the library of molecules were
screened against MCF7 (breast cancer cells; Table 1). Potent cancer drug Doxorubicin was
used as the positive control. From the screening it was evident that our library of molecules
was efficacious against MCF7 cells (Table 1). Among the compounds screened, 6a and 6e,
(comprised of indoloquinolone, spirooxindole and DKP scaffolds) inhibited the proliferation
of MCF7 cells with an IC50 of ~5 ꢀgm/mL. To investigate whether our compounds deploy
their anticancer activity through cytotoxicity, we screened them against MCF10A, a normal

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cell line. For 6a and 6e, the dose required to induce cytotoxicity was ~100 fold higher than
their respective IC50 concentration (Table 1). These anticancer hits at low ꢀgm/mL level,
fluently accentuates the merit of our DOS library, since the presumption from such unbiased,
early scaffold exploration are modest (double digit ꢀgm/mL potency). This further underlines
the “significance” of the hybrid aspect of our library and the power of the DOS approach to
deliver hit and sometimes lead structures.
Table 1. Inhibition of proliferation of MCF7 cancer cells
IC50
Entry
Compound #
(ꢀgm/mL)a
MCF7
16.28
9.21
1
2
3a
3b
3
12.88
9.21
3e
4
3f
5
15.95
5.01
4g
6
6a
7
9.58
6b
8
7.02
6c
9
8.32
6d
10
11
12
13
5.33
6e
14.01
28.91
1.81
6f
5a
Doxorubicin
aThe results showed are expressed as mean ± SEM of three independent assays.
Conclusion
We have reported a DOS strategy based primarily on oxidative ring rearrangement and ring
opening reactions to synthesize a library of chiral molecules with high architectural diversity. An
extremely versatile tetrahydroꢀβꢀcarboline motif was harnessed as the central building block. The
molecules inspired from diverse natural products are essentially comprised of four distinct
architectures. This modular strategy demonstrated an efficient 2 steps/scaffold ratio. Phenotypic

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screening against MCF7 breast cancer cells displayed selective low ꢀmolar inhibitory activity by
two compounds against MCF7 cell proliferation. This effort demonstrates the utility of an
unbiased approach like diversity oriented synthesis in discovering novel bioactive scaffolds. The
low ꢀmolar hits against MCF7 obtained from this endeavor can be utilized further to develop
drug candidates for breast cancer through protein deconvolution and subsequent structure
activity relationship studies.
EXPERIMENTAL SECTION
General. All reactions were carried out under N2 or O2 atmosphere as specified. Column
chromatography was performed on Silica gel (100ꢀ200 mesh), and reactions was monitored by
thin layer chromatography (TLC, Silica gel 60 F254), using UV light to visualize the course of the
1
reaction. H NMR and 13C NMR spectra were recorded with tetramethylsilane as an internal
standard at ambient temperature unless otherwise indicated with Bruker 400 MHz instruments at
1
400 MHz for H NMR and 125 MHz for 13C NMR spectroscopy. Splitting patterns are
designated as singlet (s), broad singlet (br. s), doublet (d), triplet (t). Splitting patterns that could
not be interpreted or easily visualized are designated as multiplet (m). Mass spectrometry
analysis was done with a 6540 UHD AccurateꢀMass QTOF LC–MS system (Agilent
Technologies) equipped with an Agilent 1290 LC system obtained by the Department of
Chemistry, School of Natural Sciences, Shiv Nadar University, Uttar Pradesh 201314,
India.HPLC experiments were carried out in Agilent Eclipse Plus C18 column. SEM and EDX
analysis was carried out in Zeiss EVO 18 Special system. ICPꢀMS experiment was carried in
Element XR system. TEM analysis was carried out in Tecnai G2 F30 (300kV) system.
Representative experimental procedures for Pictet Spengler reactions of Rꢀtrytptophan
and appropriate aldehydes (2aꢀd). To the solution of Lꢀtryptophan methyl ester in
dichloromethane (DCM) was added substituted 2ꢀnitrobenzaldehydes with trifluoroacetic acid
(TFA) and allowed to stir at room temperature for 24 h. After completion of reaction, reaction
mixture was concentrated under reduced pressure and quenched with saturated solution of
sodium bicarbonate (NaHCO3). It was then extracted with ethyl acetate (EtOAc) (3 X 20 mL).
The organic layer was washed with brine solution and dried over sodium sulphate (Na2SO4) and
concentrated over reduced pressure to obtain crude product. It is purified with column
chromatography.

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(1R, 3S)ꢀmethylꢀ1ꢀ(2ꢀnitrophenyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indoleꢀ3ꢀcarboxylate
(2a):Following the general procedure the desired compound 2a was generated as yellow solid
with yield of 89%(purified via flash column chromatography with ethyl acetateꢀhexane (2:8) as
eluent).1H NMR (400 MHz, CDCl3) δ 7.90 (s, 1H), 7.86 (dd, J = 1.2, 8 Hz, 1H), 7.73 (dd, J= 1.2,
8 Hz, 1H), 7.57ꢀ7.50 (m, 2H), 7.44 (m, 1H), 7.60ꢀ7.23 (m, 1H), 7.18ꢀ7.11 (m, 2H), 5.71 (s, 1H),
3.96 (dd, J= 4, 10.8 Hz,1H), 3.83 (s, 3H), 3.27 (dd, J= 2.4, 15.2 Hz, 1H), 3.06 (m, 1H), 2.95 (s,
1H) ;13C NMR (100 MHz, CDCl3) δ 172.92, 150.26, 136.45, 133.61, 133.40, 131.80, 129.03,
126.88, 123.77, 122.36, 119.86, 118.45, 111.17, 109.66, 56.58, 53.18, 52.48, 25.49. HRMS
(EI+) m/z calcd. for C19H17N3O4 [M]+: 352.1292, found: 352.1291.
(1R,3S)ꢀmethylꢀ1ꢀ(4ꢀchloroꢀ2ꢀnitrophenyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indoleꢀ3ꢀ
carboxylate (2b):Following the general procedure the desired compound 2b was generated as
light yellow solid with yield of 79% (purified via flash column chromatography with ethyl
acetateꢀhexane (1:9) as eluent).1H NMR (400 MHz; CDCl3): 8.21 (s, 1H); 7.79ꢀ7.78 (d, J= 4 Hz,
1H); 7.54ꢀ7.52 (d, J= 8 Hz, 1H); 7.50ꢀ7.48 (m, 2H); 7.24ꢀ7.15 (m, 3H); 5.94 (s, 1H); 4.09ꢀ4.05
(m, 1H); 3.83 (s, 3H); 3.31ꢀ3.26 (s, 1H); 3.17ꢀ3.10 (m, 1H). 13C NMR (100 MHz; CDCl3):
169.38, 149.62, 137.13, 136.88, 134.09, 133.63, 128.49, 126.20, 125.73, 125.58, 123.54, 120.35,
118.56, 111.70, 108.62, 53.32, 52.80, 50.57, 22.30. [M+H]+ calculated for (C19H16N3ClO4)
386.0902, found 386.0928
(1R,3S)ꢀmethylꢀ1ꢀ(5ꢀchloroꢀ2ꢀnitrophenyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indoleꢀ3ꢀ
carboxylate (2c):Following the general procedure the desired compound 2c was generated as
light yellow solid with yield of 72% (purified via flash column chromatography with ethyl
acetateꢀhexane (1:9) as eluent).1H NMR (400 MHz; CDCl3): 8.24 (s,1H); 7.68ꢀ7.65 (d, J= 12 Hz,
2H); 7.54ꢀ7.52 (d, J= 8 Hz,1H); 7.35ꢀ7.28 (m, 2H); 7.24ꢀ7.16 (m, 2H); 6.12 (s, 1H); 4.14ꢀ4.09
(m, 1H); 3.87 (s, 3H); 3.36ꢀ3.31 (s, 1H); 3.25ꢀ3.19 (m, 1H). 13C NMR (100 MHz; CDCl3):
169.68, 148.03, 140.30, 136.81, 130.72, 126.22, 125.81, 123.21, 120.34, 118.44, 117.38, 111.80,
108.77, 65.88, 55.86, 53.27, 23.04.[M+H]+ calculated for (C19H16N3ClO4) 386.0902, found
386.0929
Methylꢀ1ꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)ꢀ2,3,4,9ꢀtetrahydroꢀ1Hꢀpyrido[3,4ꢀb]indoleꢀ3
carboxylate (2d):Following the general procedure the desired compound 2d was generated as
yellow colored solid with yield of 87% (purified via flash column chromatography with ethyl

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acetateꢀhexane (3:7) as eluent). 1H NMR (400 MHz, CDCl3) δ 8.02 (s, 1H), 7.57 (d, J = 8.4 Hz,
1H), 7.48 (s, 1H), 7.28 (s, 1H), 7.22 (s, 1H), 7.20ꢀ7.13 (m, 2H), 5.86 (s, 1H), 4.01 (d, J = 3.6 Hz,
1H), 3.99 (d, J = 4 Hz, 1H), 3.96 (s, 3H), 3.86 (s, 3H), 3.80 (s, 3H), 3.30ꢀ3.25 (m, 1H), 3.12ꢀ3.05
(m, 1H) ;13C NMR (100 MHz, CDCl3) δ 172.99, 153.56, 148.55, 142.52, 136.38, 133.80, 131.48,
126.97, 122.25, 119.78, 118.38, 112.10, 111.19, 109.42, 107.05, 56.68, 56.56, 56.53, 53.34,
52.47, 25.38. HRMS (EI+) m/z calcd. for C21H21N3O6 [M]+: 412.1503, found: 412.1532.
Representative experimental procedures for the synthesis of tetrahydroꢀβꢀcarbolineꢀ
diketopiperazine hybrids (3aꢀh). To the stirred solution of compound 2a and d (1 equiv.) and
sodium bicarbonate (NaHCO3) (1.2 equiv.) in chloroform (CHCl3) was added dropwise
chloroacetyl chloride (2.4 equiv.) at 0 ºC. The reaction mixture was stirred at room temperature
under nitrogen atmosphere for 3 h. After completion of reaction which was monitored by TLC
the reaction mixture was diluted by CHCl3 and washed with saturated solution of NaHCO3,
brine, dried over Na2SO4, and evaporated under reduced pressure to obtained crude product
which was taken to the next step without purification.
To the solution of the above crude compound (1 equiv.) and appropriate amine (5 equiv.) in
ethanol was stir at room temperature for 24 h. It was then concentrated under reduced pressure
after completion of reaction. The crude product was purified with column chromatography with
ethyl acetateꢀhexane as eluent.
2ꢀmethylꢀ6ꢀ(2ꢀnitrophenyl)ꢀ2,3,12,12aꢀtetrahydropyrazino[1', 2': 1,6]pyrido[3,4ꢀb]indoleꢀ1,
4(6H, 7H)ꢀdione (3a):Following the general procedure the desired compound 3a was generated
as light yellow solid with yield of 58% over two steps (purified via flash column
chromatography with ethyl acetateꢀhexane (3:7) as eluent). 1H NMR (400 MHz, DMSO) δ 10.53
(s, 1H), 7.96 (d, J = 7.2 Hz, 1H), 7.58 (d, J = 8 Hz, 1H), 7.50 (t, 1H), 7.43ꢀ7.39 (m, 1H), 7.33 (s,
1H), 7.31 (s, 1H), 7.06 (t, J = 6.8, 16 Hz, 1H), 7.01 (t, J = 7.2, 14 Hz, 1H), 6.71 (s, 1H), 4.50 (d,
13
J = 7.2 Hz, 1H), 4.01 (s, 1H), 3.91 (s, 1H), 3.63ꢀ3.59 (m, 1H), 3.18 (m, 1H), 2.87 (s, 3H) ; C
NMR (100 MHz, DMSO) δ167.15, 165.70, 148.82, 138.23, 137.01, 133.81, 131.87, 128.91,
127.98, 125.70, 12359, 121.62, 118.98, 118.19, 111.82, 106.00, 55.55, 51.52, 50.91, 32.69,
24.08, 18.55. HRMS (EI+) m/z calcd. for C21H18N4O4 [M]+: 391.1401, found: 391.1403.
2ꢀethylꢀ6ꢀ(2ꢀnitrophenyl)ꢀ2,3,12,12aꢀtetrahydropyrazino[1', 2': 1, 6]pyrido[3,4ꢀb]indoleꢀ1,
4(6H, 7H)ꢀdione (3b): Following the general procedure the desired compound 3b was generated

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as light brown solid with yield of 62% over two steps (purified via flash column chromatography
with ethyl acetateꢀhexane (3:7) as eluent). 1H NMR (400 MHz, CDCl3) δ 8.60 (s, 1H), 7.85 (d, J
= 7.6 Hz, 1H), 7.61 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.2, 14.8 Hz, 1H), 7.33 (s, 1H), 7.32 (s, 1H),
7.30 (s, 1H), 7.21ꢀ7.13 (m 2H), 6.715 (s, 1H), 4.37 (dd, J = 3.6, 11.3 Hz, 1H), 4.12 (d, J = 7.2
Hz, 1H), 4.07 (d, J = 17.2 Hz, 1H), 3.86 (s, 1H), 3.83 (t, J = 4.4, 8 Hz, 1H), 3.70 (q, J = 6.8, 13.6
Hz, 1H), 3.39ꢀ3.32 (m, 2H), 1.19 (t, J = 7.2, 4.4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 166.78,
165.58, 149.27, 138.33, 136.54, 133.88, 131.45, 128.22, 127.98, 126.15, 124.07, 123.00, 120.23,
118.77, 111.50, 106.72, 60.54, 56.36, 52.36, 52.48, 49.37, 41.44, 24.48, 14.34, 12.09. HRMS
(EI+) m/z calcd. for C22H20N4O4 [M]+: 381.1557, found: 381.1570. M. P. 98 °C
2ꢀbenzylꢀ6ꢀ(2ꢀnitrophenyl)ꢀ2,3,12,12aꢀtetrahydropyrazino[1', 2': 1, 6]pyrido[3, 4ꢀb]indoleꢀ1,
4(6H, 7H)ꢀdione (3c): Following the general procedure the desired compound 3c was generated
as grey solid with yield of 56% over two (purified via flash column chromatography with ethyl
1
acetateꢀhexane (2:8) as eluent). H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.84 (d, J = 8 Hz,
1H), 7.62 (d, J = 7.6 Hz, 1H), 7.44 (t, J = 7.6, 14.8 Hz, 1H), 7.34ꢀ7.27 (m, 8H), 7.19 (t, J = 6.8,
14.4 Hz, 1H), 7.16 (t, J = 5.2, 12.8 Hz, 1H), 4.89 (d, J = 14.4 Hz, 1H), 4.89 (d, J = 14.4 Hz, 1H),
4.41 (d, J = 4 Hz, 1H), 4.12 (q, J = 7.2 Hz, 1H), 3.93 (d, J = 5.2 Hz, 1H), 3.89 (d, J = 4.4 Hz,
13
1H), 3.79 (d, J = 18 Hz, 1H), 3.38 (dd, J = 11.6, 16 Hz, 1H); C NMR (100 MHz, CDCl3) δ
166.54, 166.07, 149.19, 138.34, 136.51, 135.11, 133.87, 131.43, 129.16, 128.40, 128.23, 127.89,
126.12, 124.12, 123.01, 120.24, 118.74, 111.51, 106.63, 60.54, 56.34, 52.45, 49.96, 49.41, 24.59,
21.20, 14.34. HRMS (EI+) m/z calcd. for C27H22N4O4 [M]+: 467.1714, found: 467.1830.
2ꢀhexylꢀ6ꢀ(2ꢀnitrophenyl)ꢀ2,3,12,12aꢀtetrahydropyrazino[1', 2': 1, 6]pyrido[3, 4ꢀb]indoleꢀ1,
4(6H, 7H)ꢀdione (3d): Following the general procedure the desired compound 3c was generated
as grey solid with yield of 77% over two steps (purified via flash column chromatography with
1
ethyl acetateꢀhexane (3:7) as eluent). H NMR (400 MHz, CDCl3) δ 8.61 (s, 1H), 7.85 (d, J = 8
Hz, 1H), 7.61 (d, J = 8 Hz, 1H), 7.42 (d, J = 8 Hz, 1H), 7.33ꢀ7.30 (m, 3H), 7.21ꢀ7.130 (m, 2H),
6.72 (s, 1H), 4.38 (dd, J = 4.4, 11.2 Hz, 1H), 4.12 (d, J = 8 Hz, 1H), 4.06 (d, J = 8 Hz, 1H), 3.86
(s, 1H), 3.81 (s, 1H), 3.61ꢀ3.54 (m, 1H), 3.38ꢀ3.29 (m, 1H), 2.04 (s, 1H), 1.59 (s, 3H), 1.30 (s,
8H); 13C NMR (100 MHz, CDCl3) δ 166.84, 165.85, 149.25, 138.40, 136.54, 133.87, 131.44,
128.22, 127.95, 126.16, 124.10, 123.00, 120.23, 118.76, 111.50, 106.69, 56.26, 52.45, 49.91,

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46.57, 31.53, 26.82, 26.48, 24.43, 22.64, 14.11. HRMS (EI+) m/z calcd. for C26H28N4O4 [M]+ :
461.2183, found : 461.2209.
2ꢀisopropylꢀ6ꢀ(2ꢀnitrophenyl)ꢀ2, 3, 12, 12aꢀtetrahydropyrazino[1', 2': 1, 6]pyrido[3, 4ꢀ
b]indoleꢀ1, 4(6H, 7H)ꢀdione (3e): Following the general procedure the desired compound 3e
was generated as light yellow solid with yield of 81% over two steps (purified via flash column
chromatography with ethyl acetateꢀhexane (3:7) as eluent).1H NMR (400 MHz, CDCl3) δ 8.16 (s,
1H), 7.83ꢀ7.81 (m, 1H), 7.56 (d, J = 6.8 Hz, 1H), 7.48ꢀ7.46 (m, 2H), 7.34 (s, 1H), 7.32 (s, 1H),
7.23ꢀ7.15 (m, 2H), 4.83 (t, J = 6.8, 13.6 Hz, 1H), 4.47 (dd, J = 4, 11.6 Hz, 1H), 3.91 (s, 2H), 3.78
(s, 1H), 3.66 (dd, J= 4, 15.6 Hz, 1H), 2.99ꢀ2.92 (m, 1H), 1.25 (s, 6H); 13C NMR (100 MHz,
CDCl3) δ 164.52, 163.42, 150.57, 136.67, 132.94, 132.61, 130.07, 129.75, 126.26, 124.54,
123.18, 120.38, 118.69, 111.46, 109.23, 54.15, 53.90, 48.12, 44.58, 43.24, 29.40, 27.19, 19.04.
HRMS (EI+) m/z calcd. for C23H22N4O4 [M]+: 419.1714, found: 419.1736. M. P. 86 °C.
6ꢀ(2ꢀnitrophenyl)ꢀ2ꢀpropylꢀ2, 3, 12, 12aꢀtetrahydropyrazino[1', 2': 1, 6]pyrido[3, 4ꢀ
b]indoleꢀ1, 4(6H, 7H)ꢀdione (3f): Following the general procedure the desired compound 3f
was generated as yellow solid with yield of 85% over two steps (purified via flash column
1
chromatography with ethyl acetateꢀhexane (3:7) as eluent). H NMR (400 MHz, CDCl3) δ 8.18
(s, 1H), 7.82ꢀ7.79 (m, 1H), 7.56 (d, J = 8 Hz, 1H), 7.47ꢀ7.45 (m, 2H), 7.33 (d, J = 5.2 Hz, 2H),
7.23ꢀ7.17 (m, 2H), 5.29 (s, 1H), 4.48 (dd, J= 3.6, 11.6 Hz, 1H), 4.06 (d, J = 16 Hz, 1H), 3.94 (d,
J = 18 Hz, 2H), 3.66 (dd, J= 4, 15.6 Hz, 1H), 3.50ꢀ3.44 (m, 2H), 2.99ꢀ2.92 (m, 1H), 1.25 (t, J =
7.2, 14 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 164.97, 163.11, 150.53, 136.68, 132.94, 132.65,
130.03, 129.74, 128.91, 126.25, 124.52, 123.18, 120.39, 118.69, 111.48, 109.18, 54.05, 49.31,
48.20, 47.79, 27.33, 19.81, 11.24. HRMS (EI+) m/z calcd. for C23H22N4O4 [M]+: 419.1714,
found : 419.1732. M. P. 82 °C
6ꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)ꢀ2ꢀmethylꢀ2,3,12,12aꢀtetrahydropyrazino[1',
2':
1,
6]pyrido[3, 4ꢀb]indoleꢀ1, 4ꢀ(6H, 7H)ꢀdione (3g): Following the general procedure the desired
compound 3g was generated as yellow solid with yield of 79% over two steps (purified via flash
column chromatography with ethyl acetateꢀhexane (4:6) as eluent). 1H NMR (400 MHz, CDCl3)
δ 8.67 (s, 1H), 7.60 (d, J = 7.6 Hz, 1H), 7.41 (s, 1H), 7.30 (d, J = 8 Hz, 1H), 7.21ꢀ7.12 (m, 2H),
6.91 (s, 1H), 6.68 (s, 1H), 4.38 (dd, J= 4, 11.2 Hz, 1H), 4.13 (d, J = 4 Hz, 1H), 4.10 (d, J = 7.2
Hz, 1H), 3.92 (d, J = 3.2 Hz, 1H), 3.88 (d, J = 8 Hz, 1H), 3.85 (s, 3H), 3.75 (s, 3H), 3.27 (dd, J=

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16, 12.4 Hz, 1H), 3.07 (s, 3H) ;13C NMR (100 MHz, CDCl3) δ 166.52, 166.32, 153.81, 148.01,
141.37, 136.42, 133.27, 131.72, 126.01, 122.94, 120.15, 118.61, 111.57, 108.47, 107.29, 106.35,
56.46, 56.37, 56.28, 33.92, 24.48. HRMS (EI+) m/z calcd. for C23H22N4O6 [M]+ : 451.1612,
found : 451.1635. M. P. 123 °C
6ꢀ(4,5ꢀdimethoxyꢀ2ꢀnitrophenyl)ꢀ2ꢀbenzylꢀ2, 3, 12, 12a tetrahydropyrazino[1', 2':1,
6]pyrido[3, 4ꢀb]indoleꢀ1, 4ꢀ(6H, 7H)ꢀdione (3h): Following the general procedure the desired
compound 3h was generated as light yellow solid with yield of 88% over two steps (purified via
1
flash column chromatography with ethyl acetateꢀhexane (4:6) as eluent). H NMR (400 MHz,
CDCl3) δ 8.71 (s, 1H), 7.62 (d, J = 8.0 Hz, 1H), 7.43 (s, 1H), 7.33ꢀ7.27 (m, 6H), 7.25ꢀ7.14 (m,
2H), 6.95 (s, 1H), 6.70 (s, 1H), 4.74ꢀ4.63 (dd, J = 4, 11.2 Hz, 1H), 4.47ꢀ4.43 (m, 1H), 4.03ꢀ3.98
(m, 1H), 3.93ꢀ3.92 (m, 1H), 3.89ꢀ3.85 (d, J = 8 Hz, 1H), 3.87 (s, 3H), 3.72 (s, 3H), 3.41ꢀ3.33
(dd, J = 16, 12.4 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ 166.68, 166.47, 153.91, 148.02,
141.09, 136.34, 135.33, 133.59, 131.68, 129.17, 128.46, 128.28, 125.99, 122.97, 120.19, 118.59,
111.58, 108.21, 107.40, 106.17, 56.47, 56.24, 56.20, 52.42, 49.85, 49.60, 29.82, 24.36. HRMS
(EI+) m/z calcd. for C29H26N4O6 [M]+ : 527.1925, found : 527.1948.
Representative experimental procedures for the synthesis of hybrids (6aꢀg). To the stirred
solution of compound 3aꢀg (1 equiv) in ethanol, sodium dithionate (Na2S2O4) (10 equiv.) and
potassium carbonate (K2CO3) (2 equiv.) were added at 50 ºC under nitrogen atmosphere. After
five mintues water was added to dissolve the solid completely and reaction mixture was stirred
for thirty mintues. The reaction progress was monitored by thin layer chromatography (TLC),
after completion of reaction, the insoluble substance was remove by filtration and filtrate was
extracted with ethyl acetate (EtOAc) (3 X 30 mL), dried over sodium sulphate (Na2SO4) and
evaporated under reduced pressure to obtained reductive product, which was used in next step
without further purification.
The crude compound from the previous step was added to a mixture of tetrahydrofuran and
acetic acid (1:1) and the resulting solution was stirred for fifteen minutes after which Nꢀ
bromosuccinimide (NBS) (1 equiv) was added portionwise to stirred reaction mixture. Reaction
was monitored by TLC, after completion of which solid NaHCO3 was added to quench the
reaction and evaporated under reduced pressure to obtained solid. The solid was extracted with

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ethyl acetate (3 X 30 mL), dried over sodium sulphate and evaporated under reduced pressure to
obtain crude compound which was further purified by column chromatography.
3ꢀmethylꢀ2, 3, 4a, 5ꢀtetrahydroindolo[2, 3ꢀb]pyrazino[1', 2':1, 5]pyrrolo[3, 2ꢀc]quinolineꢀ1,
4(10H, 15bH)ꢀdione (6a): Following the general procedure the desired compound 6a was
generated as lemon yellow solid with yield of 70% over two steps via flash column
1
chromatography with ethyl acetateꢀhexane (1:1) as eluent. H NMR (400 MHz, CDCl3) δ 7.86
(d, J = 8 Hz, 1H), 7.72 (s, 1H), 7.45ꢀ7.41 (m, 3H), 7.36 (d, J = 8 Hz, 1H), 7.01 (s, 1H), 6.69 (d,
J= 4 Hz, 1H), 5.53 (s, 1H), 4.92 (m, 1H), 4.31 (d, J = 16 Hz, 2H), 4.03 (m, 1H), 3.04 (s, 3H),
2.49 (m, 1H), 2.33 (m, 1H); 13C NMR (100 MHz, CDCl3) δ 169.42, 167.13, 166.91, 154.91,
142.01, 134.88, 132.96, 130.98, 129.42, 126.86, 123.86, 122.85, 121.42, 120.90, 112.67, 65.67,
61.98, 54.74, 36.68, 33.95, 32.05. M. P. 142 °C
HRMS (EI+) m/z calcd. for C21H18N4O2 [M]+: 359.1503, found: 359.1506.
3ꢀethylꢀ2, 3, 4a, 5ꢀtetrahydroindolo[2, 3ꢀb]pyrazino[1', 2': 1, 5]pyrrolo[3, 2ꢀc]quinolineꢀ1,
4(10H, 15bH)ꢀdione (6b): Following the general procedure the desired compound 6b was
generated as yellow solid with yield of 72% over two steps (purified via flash column
1
chromatography with ethyl acetateꢀhexane (1:1) as eluent). H NMR (400 MHz, CDCl3) δ 7.87
(d, J = 7.2 Hz, 1H), 7.67 (s, 1H), 7.46ꢀ7.34 (m, 4H), 7.04ꢀ6.89 (m, 2H), 5.50 (s, 1H), 4.67 (s,
1H), 4.28 (d, J = 17.2 Hz, 2H), 4.02 (d, J = 16.8 Hz, 1H), 3.50 (d, J = 7.2 Hz, 2H), 2.7 (s, 3H);
13C NMR (100 MHz, CDCl3) δ 170.69, 167.55, 167.17, 156.39, 142.34, 134.85, 132.04, 129.77,
128.92, 127.96, 123.05, 121.30, 119.23, 118.62, 112.11, 65.12, 60.47, 56.47, 51.63, 35.63, 35.29,
29.51, 24.28, 12.61. HRMS (EI+) m/z calcd. for C22H20N4O2 [M]+: 373.1659, found: 373.1684.
M. P. 134 °C
3ꢀbenzylꢀ2, 3, 4a, 5ꢀtetrahydroindolo[2, 3ꢀb]pyrazino[1', 2': 1,5]pyrrolo[3, 2ꢀc]quinolineꢀ1,
4(10H, 15bH)ꢀdione (6c): Following the general procedure the desired compound 6c was
generated as yellow solid with yield of 80% over two steps (purified via flash column
chromatography with ethyl acetateꢀhexane (1:1) as eluent). 1H NMR (400 MHz, DMSO) δ 11.05
(s, 1H), 7.93ꢀ6.90 (m, 13H), 5.52 (d, J = 8 Hz, 1H), 4.33 (s, 2H), 3.93 (d, J = 15.6 Hz, 1H), 2.09
13
(d, J = 15.6 Hz, 2H); C NMR (100 MHz, CDCl3) δ 179.36, 167.65, 155.09, 141.94, 136.95,
136.60, 135.10, 132.26, 129.90, 128.88, 128.00, 127.44, 126.91, 125.07, 123.31, 119.32, 114.14,

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110.24, 65.82, 65.63, 60.44, 56.41, 51.86, 48.02, 29.50. HRMS (EI+) m/z calcd. for C27H22N4O2
[M]+: 435.1816, found : 435.1829. M. P. 125 °C
3ꢀhexylꢀ2, 3, 4a, 5ꢀtetrahydroindolo[2, 3ꢀb]pyrazino[1', 2': 1, 5]pyrrolo[3, 2ꢀc]quinolineꢀ1,
4(10H, 15bH)ꢀdione (6d): Following the general procedure the desired compound 6d was
generated as yellow solid with yield of 65% over two steps (purified via flash column
1
chromatography with ethyl acetateꢀhexane (1:1) as eluent). H NMR (400 MHz, DMSO) δ 7.79
(d, J = 7.6 Hz, 1H), 7.56 (s, 1H), 7.52 (d, J = 8.4 Hz, 1H), 7.42 (d, J = 7.6 Hz, 1H), 6.91 (t, J =
8.4, 16.4 Hz, 2H), 5.42 (s, 1H), 4.72ꢀ4.68 (m, 2H), 4.34 (d, J = 16.8 Hz, 1H), 4.06 (d, J = 16.8
Hz, 1H), 3.54ꢀ3.47 (m, 2H), 3.15 (t, J = 6.4, 13.2 Hz, 2H), 2.69 (s, 2H), 2.56 (s, 6H), 2.2ꢀ2.09
(m, 3H) HRMS (EI+) m/z calcd. for C26H28N4O2 [M]+: 429.2285, found: 429.2311. M. P. 76 °C
3ꢀpropylꢀ2, 3, 4a, 5ꢀtetrahydroindolo[2, 3ꢀb]pyrazino[1', 2':1, 5]pyrrolo[3, 2ꢀc]quinolineꢀ1,
4(10H, 15bH)ꢀdione (6e): Following the general procedure the desired compound 6e was
generated as light solid with yield of 56% over two steps (purified via flash column
1
chromatography with ethyl acetateꢀhexane (1:1) as eluent). H NMR (400 MHz, DMSO) δ 7.78
(s, 1H), 7.69 (d, J = 6.8 Hz, 1H), 7.37 (s, 1H), 7.33 (d, J = 8.4 Hz, 1H), 6.93ꢀ6.88 (m, 2H), 5.75
(s, 1H), 5.43 (s, 1H), 4.73ꢀ4.69 (m, 2H), 4.35 (d ,J = 16 Hz, 1H), 4.05 (d, J = 16.4 Hz, 1H), 3.51ꢀ
3.42 (m, 2H), 1.50ꢀ1.45 (m, 2H), 0.78 (t ,J = 7.2, 14.8 Hz, 3H) ; 13C NMR (100 MHz, CDCl3) δ
170.68, 167.68, 167.44, 156.39, 134.85, 129.84, 128.90, 127.97, 123.06, 121.31, 119.24, 118.66,
112.12, 74.58, 65.13, 60.47, 56.48, 52.00, 46.39, 35.32, 20.29, 10.63. HRMS (EI+) m/z calcd.
for C23H22N4O2 [M]+ : 387.1816, found : 387.1831. M. P. 95 °C
13,14ꢀdimethoxyꢀ3ꢀbenzylꢀ2, 3, 4a, 5ꢀtetrahydroindolo[2, 3ꢀb]pyrazino[1', 2': 1,
5]pyrrolo[3,2ꢀc]quinolineꢀ1, 4(10H, 15bH)ꢀdione (6f): Following the general procedure the
desired compound 6f was generated as white solid with yield of 80% over two steps (purified via
1
flash column chromatography with ethyl acetateꢀhexane (1:1) as eluent). H NMR (400 MHz,
DMSO) δ 7.64 (d, J = 7.6 Hz, 1H), 7.39 (t, J = 7.6, 14.8 Hz, 1H), 7.08 (s, 1H), 7.01 (s, 1H), 6.91ꢀ
6.86 (m, 1H), 5.75 (s, 1H), 4.70ꢀ4.65 (m, 1H), 4.05 (d, J = 17.2 Hz, 2H), 3.79 (s, 3H), 3.66 (s,
3H), 2.89 (s, 3H), 2.82 (s, 1H), 2.71 (s, 1H) ;13C NMR (100 MHz, DMSO) δ 168.07, 167.62,
167.62, 167.16, 155.68, 148.86, 147.42, 136.89, 134.01, 122.53, 121.73, 119.14, 117.27, 112.03,
110.61, 110.45, 65.19, 61.35, 56.20, 55.66, 55.52, 53.85, 35.74, 33.01. HRMS (EI+) m/z calcd.
for C29H26N4O4 [M]+: 495.2027, found : 495.2058. M. P. 103 °C

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Representative procedure for the synthesis of hybrids 4aꢀd. Compound 3aꢀc and g (1 equiv)
in a mixture of THF:AcOH:H2O (1:1:1) and was stirred for fifteen minutes and then Nꢀ
bromosuccinimide(1 equiv) was added portionꢀwise to stirred reaction mixture and continue stir
for 3h. Reaction was monitored by TLC, after completion of reaction solid sodium bicarbonate
(NaHCO3) was added to quench the reaction and evaporated under reduced pressure to obtaina
solid residue. The solid was dissolvedinEtOAc, dried over sodium sulphate (Na2SO4) and
evaporated under reduced pressure to obtained crude compound which was further purified by
column chromatography.
(R)ꢀ1ꢀmethylꢀ3ꢀ((2ꢀ(2ꢀnitrobenzoyl)ꢀ1Hꢀindolꢀ3ꢀyl)methyl)piperazineꢀ2
,
5ꢀdione (4a):
Following the general procedure the desired compound 4a was generated as white gummy liquid
with yield of 82% (purified via flash column chromatography with ethyl acetateꢀhexane (1:1) as
eluent). 1H NMR (400 MHz, DMSO) δ 7.97 (d, J = 3.2 Hz, 1H), 7.95 (s, 1H), 7.81 (s, 1H), 7.59
(d, J = 8.4 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.2, 14.8 Hz, 1H), 7.24 (s, 1H), 7.09 (t,
J = 7.2, 14.4 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 3.50 (d, J = 17.2 Hz, 1H), 3.40ꢀ3.35 (m, 2H),
2.88 (s, 3H), 2.51 (s, 2H) ; 13C NMR (100 MHz, DMSO) δ 166.00, 165.28, 162.30, 153.82,
149.26, 138.40, 136.89, 131.98, 130.36, 127.47, 125.89, 121.00, 120.09, 117.59, 112.71, 110.69,
107.31, 56.60, 56.37, 56.62, 50.51, 35.78, 32.92, 30.77. HRMS (EI+) m/z calcd. for C21H18N4O5
[M]+: 407.1350, found: 407.1372. M. P. 204 °C
(R)ꢀ1ꢀethylꢀ3ꢀ((2ꢀ(2ꢀnitrobenzoyl)ꢀ1Hꢀindolꢀ3ꢀyl)methyl)piperazineꢀ2,
5ꢀdione
(4b):
Following the general procedure the desired compound 4b was generated as colorless oil with
yield of 80% (purified via flash column chromatography with ethyl acetateꢀhexane (1:1) as
eluent). 1H NMR (400 MHz, CDCl3) δ 8.76 (s, 1H), 8.23ꢀ8.19 (m, 1H), 7.81ꢀ7.75 (m, 1H), 7.72ꢀ
7.67 (m, 3H), 6.32 (s, 1H), 4.35 (s, 1H), 3.86ꢀ3.81 (m, 1H), 3.45 (dd, J = 8, 16 Hz, 1H), 3.29ꢀ
3.24 (m, 1H), 3.11ꢀ3.04 (m, 2H), 0.87 (t, , J = 4 Hz, 3H); 13C NMR (100 MHz, CDCl3) δ 185.66,
165.72, 165.25, 146.34, 136.91, 135.47, 134.83, 131.22, 129.30, 127.98, 127.70, 125.04, 121.86,
121.81, 119.72, 112.38, 56.84, 48.40, 41.17, 29.47, 11.46. HRMS (EI+) m/z calcd. for
C22H20N4O5 [M]+: 421.1506, found : 421.1529. M. P. 175 °C
(R)ꢀ1ꢀbenzylꢀ3ꢀ((2ꢀ(2ꢀnitrobenzoyl)ꢀ1Hꢀindolꢀ3ꢀyl)methyl)piperazineꢀ2,
5ꢀdione
(4c):
Following the general procedure the desired compound 4c was generated as colorless oil with
yield of 80% (purified via flash column chromatography with ethyl acetateꢀhexane (1:1) as

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DOI: 10.1039/C7OB02230A
eluent).1H NMR (400 MHz, CDCl3) δ 8.56 (s, 1H), 8.22 (d, J = 8 Hz, 1H), 7.78ꢀ7.76 (m, 2H),
7.67 (d, J = 7.6 Hz, 2H), 7.41ꢀ7.32 (m, 3H), 7.24ꢀ7.19 (m, 3H), 7.03ꢀ7.00 (m, 2H), 6.29 (s, 1H),
4.63 (d, J = 14.4 Hz, 1H), 4.46 (s, 2H), 3.99 (d, J = 14.4 Hz, 1H), 3.90 (dd, J = 4.8, 14 Hz, 2H),
2.92 (d, J = 18 Hz, 1H) ; 13C NMR (100 MHz, CDCl3) δ 185.64, 165.61, 165.39, 146.35, 134.88,
134.78, 131.22, 129.26, 128.99, 128.49, 128.18, 127.76, 125.03, 121.89, 121.84, 112.47, 56.74,
49.63, 48.33, 29.84, 29.60. HRMS (EI+) m/z calcd. for C27H22N4O5 [M]+: 483.1663, found:
483.1689. M. P. 167 °C
(R)ꢀ3ꢀ((2ꢀ(4, 5ꢀdimethoxyꢀ2ꢀnitrobenzoyl)ꢀ1Hꢀindolꢀ3ꢀyl)methyl)ꢀ1ꢀmethylpiperazineꢀ2, 5ꢀ
dione (4d): Following the general procedure the desired compound 4d was generated as
colorless oil with yield of 71% (purified via flash column chromatography with ethyl acetateꢀ
1
hexane (1:1) as eluent). H NMR (400 MHz, DMSO) δ 7.97 (d, J = 3.2 Hz, 1H), 7.95 (s, 1H),
7.81 (s, 1H), 7.59 (d, J = 8.4 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H), 7.29 (t, J = 7.2, 14.8 Hz, 1H),
7.24 (s, 1H), 7.09 (t, J = 7.2, 14.4 Hz, 1H), 3.97 (s, 3H), 3.90 (s, 3H), 3.50 (d, J = 17.2 Hz, 1H),
13
3.40ꢀ3.35 (m, 2H), 2.88 (s, 3H), 2.51 (s, 2H) ; C NMR (100 MHz, DMSO) δ 166.00, 165.28,
162.30, 153.82, 149.26, 138.40, 136.89, 131.98, 130.36, 127.47, 125.89, 121.00, 120.09, 117.59,
112.71, 110.69, 107.31, 56.60, 56.37, 56.62, 50.51, 35.78, 32.92, 30.77. HRMS (EI+) m/z calcd.
for C23H22N4O7 [M]+: 467.1561, found: 467.1592. M. P. 109 °C
Representative experimental procedures for the synthesis of hybrids (5aꢀc). To the stirred
solution of compound 2aꢀc (1 equiv) in ethanol, sodium dithionate (Na2S2O4) (10 equiv.) and
potassium carbonate (K2CO3) (2 equiv.) were added at 50 ºC under nitrogen atmosphere. After
five mintues water was added to dissolve the solid completely and reaction mixture was stirred
for thirty mintues. The reaction progress was monitored by thin layer chromatography (TLC),
after completion of reaction, the insoluble substance was remove by filtration and filtrate was
extracted with ethyl acetate (EtOAc) (3 X 30 mL), dried over sodium sulphate (Na2SO4) and
evaporated under reduced pressure to obtained reductive product, which was used in next step
without further purification.
The crude compound from the previous step was added to a mixture of tetrahydrofuran and
acetic acid (1:1) and the resulting solution was stirred for fifteen minutes after which Nꢀ
bromosuccinimide (NBS) (1 equiv) was added portion wise to stirred reaction mixture. Reaction
was monitored by TLC, after completion of which solid NaHCO3 was added to quench the

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reaction and evaporated under reduced pressure to obtained solid. The solid was extracted with
ethyl acetate (3 X 30 mL), dried over sodium sulphate and evaporated under reduced pressure to
obtain crude compound which was further purified by column chromatography.
Methyl 2, 3, 8, 13bꢀtetrahydroꢀ1Hꢀindolo[2, 3ꢀb]pyrrolo[3, 2ꢀc]quinolineꢀ2ꢀcarboxylate (5a).
Following the general procedure the desired compound 5a was generated as colorless gummy
solid with yield of 64% over two steps (purified via flash column chromatography with ethyl
acetateꢀhexane (6:4) as eluent).1H NMR (400 MHz; CDCl3): 7.81(s,1H);7.43ꢀ7.41 (d, J = 8Hz,
1H); 7.37ꢀ7.34 (dd, J = 4Hz, 8Hz,1H); 7.29ꢀ7.25 (m,2H); 7.16ꢀ7.14 (d, J = 8 Hz, 1H); 7.04ꢀ7.01
(m, 1H); 6.94ꢀ6.92 (d, J = 8Hz, 1H); 4.67 (s,1H); 4.53(s, 1H); 3.91ꢀ3.87(m, 1H); 3.75(s, 3H);
2.70ꢀ2.63(m, 2H); 2.12ꢀ2.07(m, 1H). M. P. 72 °C
Methylꢀ11ꢀchloroꢀ2, 3, 8, 13bꢀtetrahydroꢀ1Hꢀindolo[2, 3ꢀb]pyrrolo[3, 2ꢀc]quinolineꢀ2ꢀ
carboxylate (5b). Following the general procedure, the desired compound 5b was generated as
light yellow oil with yield of 57% over two steps (purified via flash column chromatography
with ethyl acetateꢀhexane (7:3) as eluent). 1H NMR (400 MHz; CDCl3): 7.55ꢀ7.51(t,J=8Hz, 2H);
7.43ꢀ7.41(t, J = 8 Hz 1H); 7.37ꢀ7.35(d, J = 8 Hz, 1H); 7.29ꢀ7.23 (m, 3H); 4.75 (s, 1H); 4.35ꢀ4.30
(m, 1H); 3.61(s, 3H); 2.67ꢀ2.62 (m, 1H); 2.27ꢀ2.22(m,1H). 13C NMR (100 MHz; CDCl3):
173.18, 170.65, 140.49, 135.39, 133.88, 132.77, 130.05, 129.94, 126.91, 125.45, 123.01, 122.87,
119.37, 113.95, 113.95, 61.29, 57.29, 55.19, 52.72, 39.63. [M+H]+ calculated for
(C19H16N3ClO2) 354.1044 found 354.1028. M. P. 86 °C
Methylꢀ12ꢀchloroꢀ2, 3, 8, 13bꢀtetrahydroꢀ1Hꢀindolo[2, 3ꢀb]pyrrolo[3, 2ꢀc]quinolineꢀ2ꢀ
carboxylate (5c). Following the general procedure, the desired compound 5c was generated as
light yellow oil with yield of 75% over two steps (purified via flash column chromatography
with ethyl acetateꢀhexane (7:3) as eluent). 1H NMR (400 MHz; CDCl3): 7.49(s,1H); 7.43ꢀ7.41(d,
J = 8Hz,1H); 7.32ꢀ7.28(t, J = 8 Hz, 1H); 7.19ꢀ7.14 (m,2H); 7.07ꢀ7.04 (t, J = 8 Hz, 1H); 6.98ꢀ
6.96 (d, J = 8 Hz, 1H); 4.58 (s, 1H); 4.29ꢀ4.24 (m, 1H); 3.62 (s, 3H); 3.50ꢀ3.45 (m, 1H); 2.55ꢀ
2.50 (m, 1H). 13C NMR (100 MHz; CDCl3): 173.86, 171.36, 146.79, 136.89, 135.25, 129.18,
129.02, 128.89, 128.57, 126.52, 122.81, 122.20, 121.03, 113.86, 62.04, 57.70, 54.45, 52.46,
39.27. [M+H]+ calculated for (C19H16N3ClO2) 354.1004, found 354.1037. M. P. 84 °C
Biological assays

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Cell culture
MCFꢀ7 cell line derived from pleural effusion of breast adenocarcinoma from a female patient
was grown in Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen Bioservices India Pvt.
Ltd.) supplemented with 10% Fetal Bovine Serum (FBS, Gibco, USA) with 1% antibiotics
(pencillinꢀ10,000 units/ml, Streptomycinꢀ10,000 ꢀg/ml, Gibco, USA). Cells were confluent at
the time of experimentation (Cell confluency ~ 80%), then cells were dislodged from flasks by
trypsinization containing 0.25% Trypsin. All cell lines were purchased from NCSS
(www.nccs.res.in)
Compound exposure for doseꢀresponse curves
15000 cells/200 ꢀl of media were plated per well in 96ꢀwell plates and were allowed to adhere
for 18 hours. Adhere cells were then treated with 1ꢀ100 ꢀg/ml compounds 3a, b, eꢀf, 4g, 6aꢀg, 5a
and Doxorubicin in triplicates for 24 hours. All the solutions were prepared from concentrated
stock solutions (in DMSO) of the compounds.
Viability assays
After 24 hours of cell incubation in the presence or absence of each compound, cell viability was
evaluated by using MTT ((3ꢀ(4,5ꢀDimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀDiphenyltetrazolium Bromide))
(Sisco Research Laboratories Pvt. Ltd., India). In brief, this is homogeneous, colorimetric
method for determining the number of viable cells in proliferation, cytotoxicity or
chemosensitivity assays. MTT is bioreduced by cells into a formazan product that is soluble in
tissue culture medium. After 24 hours of compound treatment media was removed and 100ꢀl of
MTT (0.5 mg/ml in media) was added to the cells and was kept in dark for 2 hours at 37oC and
resulting formazan formed was dissolved in 100ꢀl DMSO (Dimethyl Sulfoxide, ACS).14ꢀ15 The
absorbance of the formazan product at 595 nm was measured directly from 96ꢀwell assay plates
without additional processing by a multimode Plate reader (BioꢀRad) (iMark, India), as
absorbance is directly proportional to the number of viable cells in culture. Percentage of viable
cells in each group is determined with respect to untreated control cells.
Acknowledgments

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We are grateful to the funds from Shiv Nadar University and Dr. Parthapratim Munshi for the
single crystal XꢀRay crystallographic analyses of our samples.
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