Design, synthesis and biological evaluation of a novel library of antimitotic C2-aroyl/arylimino tryptamine derivatives that are also potent inhibitors of indoleamine-2, 3-dioxygenase (IDO)

Article abstract of DOI:10.1016/j.ejps.2018.08.033

A novel library of C₂-substituted tryptamines (based on diverse C₂-aroyl/arylimino indoles and indole-diketopiperazine hybrids) possessing antimitotic properties were designed, synthesized and screened for their inhibitory activity against tubulin polymerization, and against proliferation of A549 lung cancer, HeLa cervical cancer, MCF7 breast cancer and HePG2 liver cancer cell lines. The design of molecules were inspired from known antimitotic compounds and natural products. The molecular docking of the designed compounds indicated that they bind to the colchicin binding site of tubulin. They were synthesized by a unique iodine catalysed oxidative ring opening reaction of 1-aryltetrahydro-β-carbolines. Among the compounds synthesized quite a few compounds induced cytotoxicity on the cancer cells by disrupting the tubulin polymerization. They were found to be non-toxic for healthy cells. Immuno Fluorescence study for the most active molecules (between ~6 μM concentration) against A549 and HeLa cells demonstrated complete disruption and shrinkage of the microtubule structures. These compounds also inhibited indoleamine-2, 3-dioxygenase with low micromolar IC₅₀.

Full text of DOI:10.1016/j.ejps.2018.08.033

Accepted Manuscript
Design, synthesis and biological evaluation of a novel library
of antimitotic C2-aroyl/arylimino tryptamine derivatives that are
also potent inhibitors of indoleamine-2, 3-dioxygenase (IDO)
Jyoti Chauhan, Moumita Dasgupta, Tania Luthra, Akanksha
Awasthi, Sayantan Tripathy, Anindyajit Banerjee, Santunu Paul,
Debashish Nag, Saikat Chakrabarti, Gopal Chakrabarti,
Subhabrata Sen
PII:
DOI:
Reference:
S0928-0987(18)30395-6
doi:10.1016/j.ejps.2018.08.033
PHASCI 4670
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European Journal of Pharmaceutical Sciences
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Revised date:
Accepted date:
23 June 2018
16 August 2018
23 August 2018
Please cite this article as: Jyoti Chauhan, Moumita Dasgupta, Tania Luthra, Akanksha
Awasthi, Sayantan Tripathy, Anindyajit Banerjee, Santunu Paul, Debashish Nag, Saikat
Chakrabarti, Gopal Chakrabarti, Subhabrata Sen , Design, synthesis and biological
evaluation of a novel library of antimitotic C2-aroyl/arylimino tryptamine derivatives
that are also potent inhibitors of indoleamine-2, 3-dioxygenase (IDO). Phasci (2018),
doi:10.1016/j.ejps.2018.08.033
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ACCEPTED MANUSCRIPT
Design, synthesis and biological evaluation of a novel library of antimitotic C2-
aroyl/arylimino tryptamine derivatives that are also potent inhibitors of indoleamine-2,
3-dioxygenase (IDO)
Jyoti Chauhan,1, § Moumita Dasgupta,2, § Tania Luthra,1 Akanksha Awasthi,1 Sayantan
Tripathy,1 Anindyajit Banerjee,3 Santunu Paul,2 Debashish Nag,2, Saikat Chakrabarti,3 Gopal
Chakrabarti2 and Subhabrata Sen*, 1,2
1 Department of Chemistry, School of Natural Sciences, Shiv Nadar University, Dadri,
Chithera, Gautambudh Nagar, UP 201314, India
2 Department of Chemistry, SRM University-AP, Amaravati, Andhra Pradesh 52202, India
subhabrata.sen@snu.edu.in/ subhabrata.s@srmap.edu.in
2 Department of Biotechnology and Dr B.C. Guha Centre for Genetic, Engineering and
Biotechnology, University of Calcutta,
35 Ballygunge Circular Road, Kolkata, WB 700019, India
3 Structural Biology and Bio-Informatics Division, CSIR-Indian Institute of Chemical
Biology, 4, Raja SC Mullick Road, Kolkata, West Bengal 700032, India
§ Equal contributors
Abstract
A novel library of C2-substituted tryptamines (based on diverse C2-aroyl/arylimino indoles and
indole-diketopiperazine hybrids) possessing antimitotic properties were designed, synthesized
and screened for their inhibitory activity against tubulin polymerization, and against
proliferation of A549 lung cancer, HeLa cervical cancer, MCF7 breast cancer and HePG2 liver
cancer cell lines. The design of molecules were inspired from known antimitotic compounds
and natural products. The molecular docking of the designed compounds indicated that they
bind to the colchicin binding site of tubulin. They were synthesized by a unique iodine
catalysed oxidative ring opening reaction of 1-aryltetrahydro--carbolines. Among the
compounds synthesized quite a few compounds induced cytotoxicity on the cancer cells by
disrupting the tubulin polymerization. They were found to be non-toxic for healthy cells.
Immuno Fluorescence study for the most active molecules (between ~6 M concentration)
against A549 and HeLa cells demonstrated complete disruption and shrinkage of the

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microtubule structures. These compounds also inhibited indoleamine-2, 3-dioxygenase with
low micromolar IC50.
Keywords: Tubulin, C2-aroyl/arylimino tryptamine, antimitotic, indoleamine-2, 3
dioxygenase
1. Introduction
Since the discovery of Taxol in 2001, Bristol Myers Squibbs’ (BMS) blockbuster drug for
ovarian and breast cancers, small molecules that interfere the cell cycle and inhibit microtubule
dynamics have been found to inhibit the proliferation of cancer cells (Liu et al. 2016;
Banaszynski et al., 2006; Lampson et al., 2006; Jordan et al. 2004). Hence they are successfully
used as cancer drugs. Many microtubule-targeting agents (also called anti-mitotic agents) have
been discovered via high throughput screening of diverse natural products (Kingston et al.,
2009). Nearly 75% of the available drugs between 1940 and 2010 were naturally derived
(Mishra et al. 2011). Due to the high potential of natural products to target microtubules, they
have been utilized as one of the major sources of inspiration in deriving novel anticancer agents
targeting microtubule, be it a hybrid, multifunctional or simple small molecules (Cragg et al.,
2009).
By virtue of their presence in innumerable natural products privileged scaffolds like indole and
diketopiperazines are crucial building blocks that are frequently utilized by the medicinal and
synthetic organic molecules to design biologically relevant chemical entities. Diverse indole
containing molecules have been identified as potential tubulin polymerization inhibitors. For
example, arylthioindole (ATI) analogs 1-6, which contain 3-(3, 4, 5-trimethoxyphenyl)thio
functionality at the C2 position inhibits tubulin assembly substantially (Galliford and Scheidt,
2007; De Martino et al., 2004; Kaur et al, 2014; La Regina et al., 2011; Wu et al., 2009; La
Regina et al. 2012). The sulphur bridging linker 1, 2 and 3 are superior or at par to the ketone
or the methylene analogue for the inhibition of proliferation of MCF7 cell lines (Galliford and
Scheidt, 2007; De Martino et al., 2004; Kaur et al, 2014). They also inhibited growth of HEK,
MI4 and U937 cells with IC50 of 13-220 nM. 1-3 (possessing 5-bromo or methoxy and 2-
carbomethoxy functionality at the indole) were also potent microtubule disrupting agent.
Newer ATIs like 4-6 where the C2-ester was replaced by 5-membered heterocycles improved
the biological profile of the resulting compounds than 1-3 (La Regina et al., 2011; Wu et al.,
2009; La Regina et al. 2012). These agents modulated Pgp-overexpression and human

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transformed cell lines with improved solubility. The sulphur bridged compound 4, displayed
decent metabolic stability and pk properties (Figure 1) (Chen et al., 2012).
C2-aroyl indoles, D-64131, 7, discovered from 3 via high throughput screening are known to
inhibit tubulin. It arrests tumour cells in G2/M phase and binds at the colchicine binding site of
the tubulin. In the later years Florent et al. discovered compound 8 a distant analogue of 7
which displayed moderate tubulin inhibition (IC50 ~ 4 M) (Figure 1) (Arthuis et al., 2011).
Compound 9-11 is a series of indoles fused with seven membered amide ring. They are
equipotent to colchicine while inhibiting tubulin polymerization and also interrupts the
proliferation of the cancer cells at the G2/M phase (Figure 1) (Putey et al., 2009; Keller et al,
2008; Singh et al., 2017).
There is also a range of antimitotic diketopiperazine derivatives that demonstrates anticancer
activity by cell cycle inhibition or by binding to specific enzymes. For example, phenylahistine,
12, a fungal metabolite isolated from Aspegillus Ulstus is a chiral isoprenylated DKP where (-
)-enantiomer is cytotoxic. Interestingly 13, the dehydrogenated analogue, is an antimitotic
agent and ~1000 times more active than 12, towards the cleavage of sea urchin embryo.
Additionally, 13 displayed substantial inhibition in the growth of various cancer cells.
Plinabulin, 14, a tert-butyl analogue of 12, is a colchicine like antimitotic compound with low
IC50s. They are also active against MDR tumor cells and is now a clinical candidate at phase
III, as a vascular disrupting anticancer agent (Figure 1) (Borthwick, 2012; Boulikas and Tsogas,
2008; Hayashi et al., 2014).
An interesting culmination of both DKP and indole moieties results in the natural product
tryprostatin A/B, 15 and 16 (Figure 1). They are isolated from aspergillus fumigates, inhibited
the cell cycle progression of rat normal fibroblast 3Y1 cells in M phase in a dose and time
dependent but non-specific manner. Additionally, 15 disassembles the microtubules and
interfere with the spindle apparatus, which is confirmed by in-vitro experiments. Its mechanism
of action is similar to anti-mitotic drugs such as Vinblastine and Rhizoxin, 15 and 16 disrupted
preformed microtubules in a dose dependent manner in-vitro. 15 is the first natural product that
inhibited the microtubule assembly by interfering with interaction between MPPs and the C-
terminal domain of tubulin (Figure 1) (Usui et al., 1998; Bhattacharya et al. 2008).

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Figure 1. Chemical structures of indoles, DKP and aroyl based tubulin binding inhibitors and
the molecules resulting from hybridizing relevant fragments (emboldened)
From various examples discussed in the aforementioned section and as depicted in figure 1, it
is quite obvious that indoles, DKP and aroyl fragments endorse the importance of their presence
in a molecule for imposing cytotoxicity towards cancer cells via modulating the tubulin
polymerization. Based on these compounds we envisaged three different classes of indole
derivatives, 17, 18 and 19 by hybridizing these three structural motifs. 17 are C2 aroyl
tryptamines, inspired from 1-8. 18 are C2-arylimino tryptamines, the functional variant of 17,
where the carbonyl is replaced by an imine. Finally, 19 are the DKP tethered C2-aroyl indoles
which were influenced from 1-6 and 12-14 and more so from the natural product tryprostatin
A/B, 15 and 16. The representative molecules from these individual classes were docked

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against tubulin to investigate the presence of any relevant binding interaction. Encouraging
results from the docking experiment prompted us to synthesize the library of molecules, via an
iodine oxidative ring opening of 1-aryltetrahydro--carbolines. In vitro screening of the
molecules demonstrated microtubule disruption by some of the molecules, which also induced
decent inhibition to the proliferation of A549, HeLa and MCF-7 cancer cell lines. Preliminary
investigation was conducted to understand their mechanism of action.
2. Materials and Methods
2.1 In silico studies
Crystal structure of tubulin complex (PDB ID: 4O2B) co-crystallized with colchicine from was
retrieved from the protein data bank (PDB) (Prota et al., 2014; Berman et al. 2000). Three-
dimensional (3D) coordinates and their respective conformers of 17e, 18a and 19a molecules
were generated via in-silico modeling using Maestro package from Schrödinger (Biologics
Suite, 2018). Molecular docking of 17e, 18a and 19a along with their different conformers
(Figure 1 in Supplementary Information) were performed at the colchicine, rhizoxin, taxol and
vinblastine binding sites (Figure 2 in Supplementary Information), respectively of the tubulin
structure using the GLIDE and GOLD (Genetic Optimization for Ligand Docking) packages
(Friesnar et al., 2004; Jones et al. 1997).
GLIDE provides a flexible docking protocol that uses incremental construction algorithm to
place the ligand into the active site of the macromolecule. Default parameters were used in
GLIDE docking. GOLD software optimizes the fitness score of many possible docking
solutions using a genetic algorithm. Following parameters were used in the docking cycles:
population size (10), selection pressure (1.100000), number of operations (100,000), number
of islands (5), niche size (2), crossover weight (95), mutate weight (95), and migrate weight
(10). The best docking solutions were identified from both GOLD and GLIDE docking studies,
based on the individual clustering analysis. In each docking study, all the docking solutions
were considered for clustering analysis based on their root mean square deviation (RMSD)
representing structural similarity. Sub-clusters with minimum of 3 solutions with RMSD < 3
Å with the highest docking scores were considered for further analysis. Both the GOLD score
and the GLIDE docking score (XP score) were further used to identify the lowest energy-
docking mode among the selected docking solutions. Representative solution (pose) from each
of these sub-clusters was selected based on the docking score. Figure 3 in supplementary
information provides a flowchart of the docking protocol.

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Protein-ligand interactions from the representative docking were identified by the Discovery
Studio package (Accelrys Software Inc. 2007).
2.2 Biological experiments
2.2.1 Materials
The cell culture materials DMEM (Dulbecco’s Modified Eagle Medium) powder and an
antibiotic-antimycotic solution,FBS(Fetal Bovine Serum) and 0.25% trypsin-EDTA solution
were purchased from HiMedia. DAPI (4’,6-diamidino-2-phenylindole) and Colchicine were
purchased from Sigma-Aldrich. The primary antibodies to α-tubulin and propidium iodide were
purchased from Santa Cruz Biotechnology (Santa Cruz, CA). The Bradford protein estimation
kit is from HiMedia and TRITC-conjugated secondary antibodies were from NOVEX by Life
Technologies. All the other chemicals and reagents were purchased from Sisco Research
Laboratories, India.
2.2.2 Methods/ experimental procedure
2.2.2.1 Cell culture
Human non-small lung adenocarcinoma (A549), cervical cancer (HeLa), breast cancer (MCF7)
and liver cancer (HePG2) cells were cultured in nutrient mixture DMEM which is
supplemented with 12% FBS, 3.7 g/L sodium bicarbonate, and a 1% antibacterial- antimycotic
solution comprised of penicillin, streptomycin, and amphotericin B. Cells were grown in a 37
°C incubator (constant temperature) that has a humidified atmosphere of 5% carbon dioxide
and 95% air in it.
2.2.2.2 Cytotoxicity assay
MTT assay (a colorimetric assay to assess cell metabolic activity) was performed to check the
growth inhibitory eff ects of the ligands on human cancer cell lines. Cultured A549, HeLa,
MCF7 and HepG2 cells were seeded on a 96-well microtiter plate (1 × 104 cells/mL) and treated
with varying concentrations (0 µM-50 µM) of the ligands (17a-n, 18a-d and 19a-e) for 48
hours. After 48 hours, 50 μL of the MTT solution (2 mg/mL) was added to each well followed
by the dissolution of the purple formazan crystals formed in 100 μL of DMSO. Then, the
absorbance was measured at 595 nm in an enzyme-linked immunosorbent assay reader
(Multiskan EX, Labsystems). Data were calculated as the percentage of inhibition by the
following formula (Mukherjee et al., 2010; Sladowski et al. 1993).

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% inhibition=(100-(At/As) * 100) %
where At and As are the absorbance of the test substances and solvent control, respectively.
2.2.2.3 Tubulin purification from goat brain
Tubulin was extracted from homogenized Goat brains via two cycles of temperature-dependent
assembly and disassembly in PEM buffer [50 mM PIPES (pH 6.9), 1 mM MgSO4, and 1 mM
EGTA] in the presence of 1 mM GTP (Sloboda et al., 1982; Das Mukherjee et al., 2016). Pure
tubulin was obtained by a third cycle of the temperature-dependent assembly process in
glutamate buffer [1M monosodium glutamate, 1 mM MgS04, and 1 mM EGTA (pH 6.8)] in
addition with 1.5 mM GTP (Hamel et al., 1981). Then the purified tubulin is kept at -80°C with
aliquots and protein content was estimated by Bradford protein estimation procedure and also
by the Ellman’s assay using DTNB (5,5’-dithiobis-(2-nitrobenzoic acid) (Bradford et al.,
1976).
2.2.2.4 Microtubule polymerization assay
Microtubule polymerization was done in the absence and presence of 17, 18 and 19 series
compounds, in microtubule assembly PIPES buffer upon addition of 1 mM GTP. The tubulin
polymer formation was monitored turbidimetrically at 350 nm for 15 min at 37°C using a
JASCO V-630 UV−visible spectrophotometer (Das Mukherjee et al., 2016; Gaskin et al.,
1974). Again, a similar experiment was done with different concentrations of 17, 18 and 19
series compounds to determine the concentration of the ligands required to inhibit tubulin
polymerization by 50% compared to the control (IC50 value). Colchicine is well-known tubulin
polymerization inhibitor, and Colchicine was used as positive control tubulin polymerization
inhibition study (Jordan et al., 2004).
2.2.2.5 Immuno-fluorescence microscopy
Cultured A549 cells and HeLa cells were grown as a monolayer on a glass cover slip in 35mm
tissue culture dish, were treated with different doses ofTLJC-8G (5µM, 10µM, 15µM) and
TLJC-8U(5µM, 7.5µM, 10µM) for 48 hours. Colchicine (50 nM) treated cell was kept as
positive control. Subsequently, the cells were washed with (1X PBS), fixed (3% glutaraldehyde
for 60 min), and permeabilized (0.1% sodium citrate and 0.1% Triton X for 45 min), and
nonspecific binding was blocked using 5% BSA. The cellular microtubule was stained with the
anti-α-tubulin primary antibody (1:100 dilution) overnight, followed by the TRITC-conjugated
secondary antibody (1:100 dilution) for 2 hours, and the nuclear DNA was counterstained by

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DAPI (50 µg/ mL) for 15 minutes (Das Mukherjee et al. 2016; Acharya et al., 2009). Then
slides were prepared with the mounting solution (66% glycerol in PBS) and sides of the cover
slips were sealed. Finally, the images (α-Tubulin, DNA and merged) were obtained by an
Olympus LSM IX81 confocal microscope and analyzed with the Olympus FLUOVIEW
Viewer Software.
2.2.2.6 IDO screening from MCF7
MCF7 Cells were grown in 80 mm culture plate with DMEM (HiMedia) supplemented with
10% FBS (HiMedia, US Origin), 1% Pen-Strep (HiMedia, 10000 U Penicillin, 5 mg
streptomycin, 0.09% saline) in 37°C incubator with 5% CO2. After drug treatment cells were
lysed with Ripa buffer (10^7 cells/ml) and centrifuged in 13,000 r.p.m for 5 mins in cold
condition (4 °C). Supernatant was collected and total protein estimation was done by Bradford
assay. Total IDO concentration is measured by IDO detection ELISA kit (Elabscience).
2.2.2.7 IDO screening from A549
A549 cells were grown in 80 mm culture plate with DMEM (HiMedia) supplemented with
10% FBS (HiMedia, US Origin), 1% Pen-Strep (HiMedia, 10,000U Penicillin, 5mg
streptomycin, 0.09% saline) in 37 °C incubator with 5% CO2. A549 cells were cultured with
IFN-ƴ (Sigma-Aldrich, 100units/ml) to generate the IDO in cells for 12 hours. After Drug
treatment cells were lysed with Ripa buffer (10^7 cells/ml) and centrifuged in 13,000 r.p.m for
5 minutes in cold condition (4°). Supernatant was collected and total protein estimation was
done by Bradford assay. Total IDO concentration is measured by IDO detection ELISA kit
(Elabscience).
2.3 Chemistry
2.3.1 Materials
All reactions were carried out under N2 atmosphere as specified. Column chromatography was
performed on Silica gel (100-200 mesh), and reactions was monitored by thin layer
chromatography (TLC, Silica gel 60 F254), using UV light to visualize the course of the
1
13
reaction. H NMR and C NMR spectra were recorded with tetramethylsilane as an internal
standard at ambient temperature unless otherwise indicated with Bruker 400 MHz instruments
1
13
at 400 MHz for H NMR and 100 MHz for C 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

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analysis was done with a 6540 UHD Accurate-Mass 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.
2.3.2 Methods and experimentals
2.3.2.1 Synthesis of tetrahydro--carbolines 20a-n
To the solution of tryptamine or S-tryptophan methyl ester in [bbim]BF4 was added substituted
benzaldehydes (1.1 equiv). The resulting mixture was allowed to stir at 100 °C for 24 h. Once
thin layer chromatography (TLC) confirmed complete consumption of the starting material,
the reaction mixture was extracted with ether and the organic layer was separated, washed with
water, brine and dried over anhydrous magnesium sulfate and the solvent was concentrated
under reduced pressure. The final product was obtained by recrystallization with minimum
amount of ethanol and was taken to the next step without further purification.
To a solution of the above compounds (1 equiv.) obtained from tryptamine, and 4N aq. sodium
hydroxide (NaOH) solution (1 equiv.) or triethylamine (3 equiv.) in DCM was added dropwise
methyl chloroformate (1 equiv.) or acetyl chloride (2 equiv.) or trifluoroacetic anhydride (2
equiv.) at 0 ºC. The reaction mixture was stirred at room temperature under nitrogen
atmosphere for 3 h. Once TLC confirms the complete consumption of the starting material the
reaction mixture was diluted with water and extracted with ethyl acetate. The organic layer was
washed with saturated solution of sodium bicarbonate (NaHCO3) and brine. The organic layer
was dried over anh. sodium sulfate (Na2SO4), and evaporated under reduced pressure to
obtained crude product (20a-n) which was taken to the next step without purification.
2.3.2.2 Oxidative ring opening for the synthesis of 17a-n
The above compounds 20a-n (1 equiv.), iodine (0.3 equiv.), TBHP (4 equiv.) was taken in
acetonitrile and was stirred at 60 ºC for 4 h. Once the reaction is complete it was evaporated
under reduced pressure to obtained crude product which was purified by column
chromatography with ethyl acetate-hexane as eluent.
2.3.2.2.1 Methyl (2-(2-(2-fluoro-4-methoxybenzoyl)-1H-indol-3-yl)ethyl)carbamate (17a)
Following the general procedure, the desired compound 17a was synthesized from 20a
(generated from tryptamine (500 mg, 3.12 mmol) and 2-fluoro-4-methoxybenzaldehyde (0.32

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ml, 2.49 mmol) followed by protection of carboline nitrogen with carbomethoxy group) and
purified via column chromatography with ethyl acetate-hexane (3:7) as eluent and was obtained
1
as grey solid with a yield of 65%. H NMR (400 MHz; DMSO): δ 11.47(s, 1H), 7.76-7.74 (d,
J = 8 Hz, 1H), 7.56-7.52 (t, J = 8 Hz, 1H), 7.42-7.40 (d, J = 8 Hz, 1H), 7.31-7.27(t, J = 8 Hz,
1H), 7.19-7.16 (m, 1H), 7.11-7.07 (t, J = 8 Hz, 1H), 7.07-6.93 (m, 2H), 3.88 (s, 3H), 3.48 (s,
3H), 3.18-3.13 (m, 2H), 2.98-2.94 (t, J = 8 Hz, 2H); 13C NMR (100 MHz; DMSO): δ 184.58,
163.68, 163.56, 162.44, 159.95, 157.07, 137.27, 132.62, 132.01, 128.04, 126.07, 121.28,
120.70, 120.55, 120.26, 113.19, 111.23, 102.72, 102.46, 56.50, 51.58, 41.80, 25.64. HRMS
(EI+) m/z calcd. for C20H19FN2O4 [M+H]+ 371.1402, found 371.1438. M.P. 180 ºC
2.3.2.2.2 Methyl (2-(2-(4-chlorobenzoyl)-1H-indol-3-yl)ethyl)carbamate (17b)
Following the general procedure, the desired compound 17b was synthesized from 20b
(generated from tryptamine (500 mg, 3.12 mmol) and 4-chlorobenzaldehyde (0.31 ml, 2.49
mmol) followed by protection of carboline nitrogen with carbomethoxy group) and was
purified with column chromatography with ethyl acetate-hexane (3:7) as eluent. It was
1
generated as yellow solid with yield of 95%. H NMR (400 MHz; DMSO): δ 11.48(s, 1H),
7.79-7.75 (m, 3H), 7.65 (d, J = 8 Hz, 2H), 7.43 (d, J = 8 Hz, 1H), 7.32-7.28 (m, 1H), 7.17 (t, J
13
= 6 Hz, 1H), 7.13-7.09 (m, 2H), 3.46 (s, 3H), 3.21-3.16 (m, 2H), 3.01 (t, J = 8 Hz,2H). C
NMR (100 MHz; DMSO): δ 187.77, 157.07, 138.01, 137.52, 137.29, 131.67, 131.30, 129.18,
127.99, 125.94, 121.59, 121.59, 121.16, 120.36, 113.24, 51.56, 41.79, 25.75. HRMS (EI+) m/z
calcd. for C19H17ClN2O3 [M+H]+ 357.1000, found 357.1034. M.P. 165 ºC
2.3.2.2.3 Methyl (2-(2-(4-nitrobenzoyl)-1H-indol-3-yl)ethyl)carbamate (17c)
Following the general procedure, the desired compound 17c was synthesized from 20c
(generated from tryptamine (500 mg, 3.12 mmol) and 4-nitrobenzaldehyde (378 mg, 2.49
mmol) and the corresponding carboline nitrogen was protected with carbomethoxy
functionality) was purified with column chromatography with ethyl acetate-hexane (3:7) as
1
eluent. It was generated as yellow solid with yield of 73%. H NMR (400 MHz; DMSO): δ
11.56 (s, 1H), 8.41-8.39(d, J = 9Hz, 2H), 7.98-7.96 (d, J = 9 Hz, 2H), 7.79-7.76 (d, J = 9 Hz,
1H), 7.45-7.43 (d, J = 8 Hz, 1H), 7.35-7.31 (m, 1H), 7.16-7.11 (m, 2H), 3.46 (s, 3H), 3.19-3.14
(m, 2H), 2.96-2.93 (m, 2H); 13C NMR (100 MHz; DMSO): δ 187.48, 157.07, 149.64, 145.04,
137.67, 131.40, 130.33, 128.06, 126.53, 124.27, 122.64, 121.40, 120.55, 113.32, 51.57, 41.66,
25.87. HRMS (EI+) m/z calcd. for C19H17N3O5 [M+H]+ 368.1241, found 368.1279. M.P. 157
ºC

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2.3.2.2.4 Methyl (2-(2-(3-fluorobenzoyl)-1H-indol-3-yl)ethyl)carbamate (17d)
Following the general procedure, the desired compound 17d was synthesized 20d (generated
from tryptamine (500 mg, 3.12 mmol) and 3-fluorobenzaldehyde (0.26 ml, 2.49 mmol) and the
nitrogen of the resulting carboline was protected by carbomethoxy group) and was purified
with column chromatography with ethyl acetate-hexane (3:7) as eluent and obtained as white
1
solid with yield of 81%. H NMR (400 MHz; DMSO): δ 11.49 (s, 1H), 7.77-7.75 (d, J= 8 Hz,
1H), 7.67-7.58 (m, 2H), 7.55-7.52 (d, J = 9 Hz, 2H), 7.45-7.43 (d, J = 8 Hz, 1H), 7.32-7.29 (m,
13
1H), 7.18-7.09 (m, 2H), 3.46 (s, 3H), 3.20-3.15 (m, 2H), 3.04-3.00(m, 2H); C NMR (100
MHz; DMSO): δ 187.48, 163.59, 161.15, 157.07, 141.56, 137.36, 131.53, 131.31, 131.23,
128.02, 126.04, 125.57, 121.97, 121.18, 120.38, 119.59, 119.39, 116.04, 115.81, 113.29, 51.54,
41.76, 25.70. HRMS (EI+) m/z calcd. for C19H17FN2O3 [M+H]+ 341.1296, found 341.1337.
M.P. 124 ºC
2.3.2.2.5 N-(2-(2-(4-chlorobenzoyl)-1H-indol-3-yl)ethyl)acetamide (17e)
Following the general procedure, the desired compound 17e was synthesized from 20e
(generated from tryptamine (500 mg, 3.12 mmol) and 4-chlorobenzaldehyde (0.31 ml, 2.49
mmol) and the nitrogen of the resulting carboline was protected with acetyl group) and purified
by column chromatography with ethyl acetate-hexane (1:1) as eluent and was obtained as white
solid with yield of 69%. 1H NMR (400 MHz, DMSO): δ 11.69 (s, 1H), 7.97 (s, 1H), 7.94 (d, J
= 8 Hz, 1H), 7.90 (t, J = 8 Hz, 1H), 7.79 (d, J = 8 Hz, 2H), 7.66 (d, J = 8 Hz, 2H), 7.60-7.56
13
(m, 1H), 7.40 (s, 2H), 3.22 (dd, J = 8, 12 Hz, 2H), 2.96 (t, J = 8 Hz, 2H), 1.68 (s, 3H); C
NMR (100 MHz, DMSO): δ 187.31, 169.04, 137.38, 137.23, 135.28, 132.21, 131.17, 130.94,
129.30, 128.81, 127.94, 123.04, 120.65, 114.88, 112.39, 24.79, 22.56. HRMS (EI+) m/z calcd.
for C19H17ClN2O2 [M]- 339.0966, found 339.0906. M.P. 139 ºC
2.3.2.2.6 Methyl (2-(2-(3-methoxybenzoyl)-1H-indol-3-yl)ethyl)carbamate (17f)
Following the general procedure, the desired compound 17f was synthesized from 20f
(generated from tryptamine (500 mg, 3.12 mmol) and 3-methoxybenzaldehyde (0.31 ml, 2.49
mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with
carbomethoxy group) and purified via column chromatography with ethyl acetate-hexane (3:7)
1
as eluent and was obtained as white solid with yield of 56%. H NMR (400 MHz; DMSO): δ
11.46(s, 1H), 7.77-7.75(d, J = 8 Hz, 1H), 7.51-7.44 (m, 2H), 7.34-7.23(m, 4H), 7.18-7.16 (t, J
= 4 Hz, 1H), 7.12-7.09 (m, 1H), 3.83 (s, 3H), 3.47 (s, 3H), 3.21-3.16 (m, 2H), 3.04-3.01 (m,
2H); 13C NMR (100 MHz; DMSO): δ 188.59, 159.62, 157.08, 140.64, 137.19, 131.81, 130.24,

ACCEPTED MANUSCRIPT
128.01, 125.77, 121.79, 121.41, 121.10, 120.27, 118.69, 114.06, 113.28, 55.77, 51.56, 41.85,
25.74. HRMS (EI+) m/z calcd. for C20H20N2O4 [M+H]+ 353.1496, found 353.1528. M.P. 107
ºC
2.3.2.2.7 Methyl (2-(2-(2,6-difluorobenzoyl)-1H-indol-3-yl)ethyl)carbamate (17g)
Following the general procedure, the desired compound 17g was synthesized from 20g
(generated from tryptamine (500 mg, 3.12 mmol) and 2,6-difluorobenzaldehyde (0.27 ml, 2.49
mmol) and and the nitrogen of the resulting tetrahydro--carboline was protected with
carbomethoxy group) and purified by column chromatography with ethyl acetate-hexane (3:7)
1
as eluent and generated as colorless gummy solid with yield of 77%. H NMR (400 MHz;
CDCl3): δ 9.01 (s, 1H), 7.77-7.75 (d, J = 8 Hz, 1H), 7.54-7.45 (m, 1H), 7.40-7.39 (d, J = 4 Hz,
2H), 7.18-7.14 (m, 1H), 7.07-7.03 (m, 2H), 4.68 (s, 1H), 3.63 (s, 3H), 3.34-3.30 (m, 2H), 2.93-
2.90 (t, J = 7 Hz, 2H); 13C NMR (100 MHz; CDCl3): δ 179.54, 160.68, 158.19, 156.91, 137.06,
132.33, 132.24, 132.09, 128.33, 127.76, 123.70, 121.83, 121.05, 117.65, 112.30, 112.24,
112.06, 52.04, 41.78, 25.39. HRMS (EI+) m/z calcd. for C19H16F2N2O3 [M+H]+ 359.1202,
found 359.1206. M.P. 102 ºC
2.3.2.2.8 Methyl (2-(2-(3,5-bis(trifluoromethyl)benzoyl)-1H-indol-3-yl)ethyl)carbamate (17h)
Following the general procedure, the desired compound 17h was synthesized from 20h
(generated from tryptamine (500 mg, 3.12 mmol) and 3,5-bis(trifluoromethyl)benzaldehyde
(0.41 ml, 2.49 mmol) and the nitrogen of the resulting tetrahydro--carboline was protected
with carbomethoxy group) and purified by column chromatography with ethyl acetate-hexane
1
(3:7) as eluent and was obtained as white gummy oil with yield of 79%. H NMR (400 MHz;
CDCl3): δ 8.73 (s, 1H), 8.23 (s, 2H), 8.12 (s, 1H), 7.83-7.81(d, J = 8 Hz, 1H), 7.44-7.43 (d, J
= 3 Hz, 1H), 7.24-7.21 (m, 1H), 4.93 (s, 1H), 3.61 (s, 3H), 3.43-3.39 (m, 2H), 3.08-3.04 (t, J =
13
6 Hz, 2H); C NMR (100 MHz; CDCl3): δ 184.56, 155.88, 139.57, 131.46, 129.61, 127.99,
126.60, 123.16, 122.73, 120.57, 120.40, 111.27, 50.99, 40.75, 28.68. HRMS (EI+) m/z calcd.
for C21H16F6N2O3 [M+H]+ 459.1138, found 459.1172. M.P. 159 ºC
2.3.2.2.9 N-(2-(2-benzoyl-1H-indol-3-yl)ethyl)acetamide (17i)
Following the general procedure, the desired compound 17i was synthesized from 20i
(generated from tryptamine (500 mg, 3.12 mmol) and benzaldehyde (0.25 ml, 2.49 mmol) and
the nitrogen of the resulting tetrahydro--carboline was protected with acetyl group) and
purified by column chromatography with ethyl acetate-hexane (1:1) as eluent and was obtained
as pale yellow solid with yield of 61%. 1H NMR (400 MHz, CD3OD): δ 7.82 (s, 1H), 7.76 (d,

ACCEPTED MANUSCRIPT
J = 8 Hz, 1H), 7.65 (t, J = 8 Hz, 1H), 7.56 (d, J = 8 Hz, 1H), 7.43 (d, J = 12 Hz, 1H), 7.31 (t, J
= 8 Hz, 1H), 7.12 (t, J = 8 Hz, 1H), 3.43 (t, J = 8 Hz, 2H), 3.16 (t, J = 8 Hz, 2H), 1.81 (s, 3H)
13
; C NMR (100 MHz, CD3OD): δ 191.19, 173.22, 140.56, 138.62, 133.49, 130.26, 129.71,
129.28, 126.92, 123.22, 121.73, 121.26, 113.57, 41.69, 25.74, 22.54. HRMS (EI+) m/z calcd.
for C19H18N2O2 [M]- 305.1299, found 305.1296. M.P. 102 ºC
2.3.2.2.10 N-(2-(2-(4-nitrobenzoyl)-1H-indol-3-yl)ethyl)acetamide (17j)
Following the general procedure, the desired compound 17j was synthesized from 20j
(generated from tryptamine (500 mg, 3.12 mmol) and 4-nitrobenzaldehyde (378 mg, 2.49
mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with acetyl
group) and purified by column chromatography with ethyl acetate-hexane (1:1) as eluent and
was obtained as yellow solid with yield of 72%. 1H NMR (400 MHz, DMSO): δ 11.55 (s, 1H),
8.41 (d, J = 8 Hz, 2H), 7.99 (d, J = 12 Hz, 2H), 7.87 (t, J = 4 Hz, 1H), 7.78 (d, J = 8 Hz, 1H),
7.44 (d, J = 8 Hz, 1H), 7.33 (t, J = 8 Hz, 1H), 7.12 (t, J = 8 Hz, 1H), 3.22 (dd, J = 8, 12 Hz,
13
2H), 2.96 (t, J = 4 Hz, 2H), 1.69 (s, 3H); C NMR (100 MHz, DMSO): δ 187.01, 168.99,
149.18, 144.58, 137.20, 130.86, 129.96, 127.60, 126.07, 123.81, 122.52, 121.01, 112.88, 25.11,
22.60. HRMS (EI+) m/z calcd. for C19H17N3O4 [M+H]+ 352.1292, found 352.1295. M.P. 158
ºC
2.3.2.2.11 Methyl (2-(2-benzoyl-1H-indol-3-yl)ethyl)carbamate (17k)
Following the general procedure, the desired compound 17k was synthesized from 20k
(generated from tryptamine (500 mg, 3.12 mmol) and benzaldehyde (0.25 ml, 2.49 mmol) and
the nitrogen of the resulting tetrahydro--carboline was protected with carbomethoxy group)
and purified by column chromatography with ethyl acetate-hexane (3:7) as eluent and was
1
obtained as pale yellow solid with yield of 51%. H NMR (400 MHz; DMSO): δ 11.47 (s, 1H),
7.78 (s, 1H), 7.76 (s, 2H), 7.68 (t, J = 8 Hz, 1H), 7.58 (t, J = 8 Hz, 2H), 7.43 (t, J = 8 Hz, 1H),
7.29 (t, J = 8 Hz, 1H), 7.17 (t, J = 4 Hz, 1H), 7.11 (t, J = 8 Hz, 1H), 3.47 (s, 3H), 3.19 (dd, J =
8, 12 Hz, 2H), 3.01 (t, J = 8 Hz, 2H); 13C NMR (100 MHz; DMSO): δ 188.52, 156.63, 138.88,
136.75, 132.26, 131.47, 128.60, 127.57, 125.28, 120.79, 120.64, 119.81, 112.80, 51.10, 41.41,
25.28. HRMS (EI+) m/z calcd. for C19H18N2O3 [M+H]+ 323.1390, found 323.1398. M.P. 105
ºC
2.3.2.2.12 Methyl (2-(2-(3,4-dimethoxybenzoyl)-1H-indol-3-yl)ethyl)carbamate (17l)

ACCEPTED MANUSCRIPT
Following the general procedure, the desired compound 17l was synthesized from 20l
(generated from tryptamine (500 mg, 3.12 mmol) and 3,4-dimethoxybenzaldehyde (414.59 mg,
2.49 mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with
carbomethoxy group) and purified by column chromatography with ethyl acetate-hexane (3:7)
1
as eluent and was obtained as pale yellow solid with yield of 55%. H NMR (400 MHz;
DMSO): δ 11.45 (s, 1H), 7.75 (d, J = 8 Hz, 1H), 7.43 (t, J = 8 Hz, 2H), 7.38 (s, 1H), 7.27 (t, J
= 8 Hz, 1H), 7.17 (t, J = 8 Hz, 1H), 7.13-7.08 (m, 2H), 3.88 (s, 3H), 3.83 (s, 3H), 3.47 (s, 3H),
13
3.21 (dd, J = 4, 8 Hz, 2H), 3.02 (t, J = 8 Hz, 2H); C NMR (100 MHz; DMSO): δ 187.16,
156.63, 152.63, 148.56, 136.48, 131.82, 131.05, 127.49, 124.02, 120.44, 119.66, 112.68,
111.70, 110.95, 55.80, 55.50, 51.11, 25.31. HRMS (EI+) m/z calcd. for C21H22N2O5 [M+H]+
383.1601, found 383.1637. M.P. 172 ºC
2.3.2.2.13 N-(2-(2-(2-nitrobenzoyl)-1H-indol-3-yl)ethyl)acetamide (17m)
Following the general procedure, the desired compound 17m was synthesized from 20m
(generated from tryptamine (500 mg, 3.12 mmol) and 2-nitrobenzaldehyde (376.28 mg, 2.49
mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with acetyl
group) and purified by column chromatography with ethyl acetate-hexane (3:7) as eluent and
1
was obtained as pale yellow solid with yield of 76%. H NMR (400 MHz; CDCl3): δ 8.88 (s,
1H), 8.27 (d, J = 8 Hz, 1H), 7.81 (t, J = 8 Hz, 1H), 7.72 (t, J = 8 Hz, 2H), 7.54 (d, J = 8 Hz,
1H), 7.39 (d, J = 4 Hz, 2H), 7.19-7.15 (m, 1H), 5.74 (s, 1H), 3.37 (q, J = 8, 12 Hz, 2H), 2.91
(t, J = 8 Hz, 2H), 1.83 (s, 3H) ; 13C NMR (100 MHz; CDCl3): δ 185.41, 170.46, 137.15, 136.05,
134.59, 131.23, 131.04, 128.62, 128.37, 127.66, 125.27, 123.47, 121.68, 121.27, 112.45, 40.51,
24.23, 23.30. HRMS (EI+) m/z calcd. for C19H17N3O4 [M+H]+ 352.1292, found 352.1298. M.P.
156 ºC
2.3.2.2.14 Methyl (2-(2-(2-nitrobenzoyl)-1H-indol-3-yl)ethyl)carbamate (17n)
Following the general procedure, the desired compound 17n was synthesized from 20n
(generated from tryptamine (500 mg, 3.12 mmol) and 2-nitrobenzaldehyde (376.28 mg, 2.49
mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with
carbomethoxy group) and purified by column chromatography with ethyl acetate-hexane (3:7)
1
as eluent and was obtained as pale yellow solid with yield of 82%. H NMR (400 MHz;
DMSO): δ 11.53 (s, 1H), 8.31 (d, J = 8 Hz, 1H), 7.94 (t, J = 8 Hz, 1H), 7.87 (t, J = 4 Hz, 1H),
7.72 (dd, J = 8, 16 Hz, 2H), 7.40 (d, J = 8 Hz, 1H), 7.32 (t, J = 8 Hz, 1H), 7.18-7.09 (m, 2H),
3.48 (s, 3H), 3.08 (q, J = 4, 12 Hz, 2H), 2.83 (t, J = 8 Hz, 2H); 13C NMR (100 MHz; DMSO):

ACCEPTED MANUSCRIPT
δ 185.33, 156.58, 146.04, 137.25, 135.85, 134.98, 131.34, 130.72, 128.83, 127.62, 125.01,
121.88, 120.99, 120.09, 112.82, 51.17, 41.09, 25.25. HRMS (EI+) m/z calcd. for C19H17N3O5
[M+H]+ 368.1241, found 368.1249. M.P. 162 ºC
2.3.2.3 Oxidative ring opening and in situ imination for the synthesis of 18a-d
Compounds 20a, l, k and h (1 equiv.), molecular iodine (0.3 equiv.) and TBHP (4 equiv.) was
taken in methylamine and was stirred at 60 °C temperature for 4-8 h. The reaction was
monitored by TLC, once the reaction is complete the reaction mixture was cooled to room
temperature and quenched with saturated solution of sodium thiosulphate in water and
extracted with ethyl acetate. The organic layer was dried over anhydrous sodium sulfate
(Na2SO4), and evaporated under reduced pressure. The crude product was purified by gradient
column chromatography with (30-50%) ethyl acetate-hexane as eluent.
2.3.2.3.1
(E)-methyl(2-(2-((2-fluoro-4-methoxyphenyl)(methylimino)methyl)-1H-indol-3-
yl)ethyl)carbamate (18a)
Following the general procedure, the desired compound 18a was synthesized from 20a
(prepared from tryptamine (500 mg, 3.12 mmol) and 2-fluoro-4-methoxybenzaldehyde (0.32
ml, 2.49 mmol)) and methyl amine (6 mL) was purified with column chromatography with
ethyl acetate-hexane (3:10) as eluent. It was generated as light yellow solid with yield of 76%.
1H NMR (400 MHz; DMSO): δ 10.99 (s, 1H), 7.59 (d, J= 7.92Hz, 1H), 7.36 (d, J = 8.16 Hz
,1H), 7.19 (t, J = 8.32 Hz, 1H), 7.15-7.11 (m, 2H),7.01-6.95(m,2H), 6.92 (dd, J1 = 2.04 Hz and
J2 = 6.36 Hz, 1H); 3.85 (s, 3H), 3.48 (s, 3H), 3.16 (s,3H), 2.97-2.92(m, 2H), 2.45(d, J= 7.28Hz,
2H). 13C NMR (100 MHz; DMSO): δ 161.96, 157.89, 156.96, 136.41, 133.51, 130.54, 128.97,
123.78, 119.85, 119.23, 115.17, 112.29, 111.64, 102.53, 56.21, 51.53, 42.10, 24.97. HRMS
(EI+) m/z calcd. for (C21H22FN3O3) [M+H]+ 384.1718 found 384.1765. M.P. 188 ºC
2.3.2.3.2 (E)-methyl(2-(2-((3,4-dimethoxyphenyl)(methylimino)methyl)-1H-indol-3-
yl)ethyl)carbamate (18b)
Following the general procedure, the desired compound 18b was synthesized from 20l
(generated from tryptamine (500 mg, 3.12 mmol) and 3,4-dimethoxybenzaldehyde (414.59 mg,
2.49 mmol) and methyl amine (6 mL) and was purified with column chromatography with
ethyl acetate-hexane (4:6) as eluent. It was generated as light yellow solid with yield of 65%.
1H NMR (400 MHz; DMSO): δ 11.46 (s, 1H), 7.75 (d, J= 8 Hz, 1H), 7.45-7.38 (m, 2H), 7.37
(s, 1H), 7.27 (t, J = 16 Hz, 1H), 7.19 (t, J = 12 Hz, 2H), 7.17-7.08 (m, 3H), 3.87 (s, 3H), 3.82

ACCEPTED MANUSCRIPT
(s, 3H), 3.47 (s, 3H), 3.34 (s, 1H), 3.21-3.19 (m, 2H), 3.03-3.01 (m, 2H). 13C NMR (100 MHz;
DMSO): δ 187.61, 157.07, 153.07, 149.00, 136.92, 132.26, 131.50, 127.94, 125.28, 124.47,
120.89, 120.13, 113.13, 112.15, 111.39, 56.25, 55.95, 51.56, 41.94, 25.77. HRMS (EI+) m/z
calcd. for (C22H25N3O4) [M+H]+ 396.1918 found 396.1923. M.P. 167 ºC
2.3.2.3.3 (E)-methyl (2-(2-((methylimino)(phenyl)methyl)-1H-indol-3-yl)ethyl)carbamate
(18c)
Following the general procedure, the desired compound 18c was synthesized from 20k
(prepared from tryptamine (500 mg, 3.12 mmol) and benzaldehyde (0.25 ml, 2.49 mmol)) and
methyl amine (6 mL) and was purified with column chromatography with ethyl acetate-hexane
1
(3:10) as eluent. It was generated as light yellow solid with yield of 71%. H NMR (400 MHz;
DMSO): δ 10.95 (s, 1H), 7.58-7.51 (m, 4H), 7.37 (d, J= 8 Hz, 1H), 7.28 (d, J = 4 Hz, 2H), 7.12
(t, J = 12 Hz, 1H), 7.03 (s, 1H), 6.97 (d, J = 8 Hz, 1H), 3.47 (s, 3H), 3.15 (s, 3H), 2.90 (s, 2H),
2.32 (s, 2H).13C NMR (100 MHz; DMSO): δ 163.49, 156.94, 136.38, 136.15, 133.87, 129.35,
129.30, 129.02, 127.79, 123.66, 119.83, 119.15, 115.34, 112.30, 55.38, 51.53, 42.22, 42.22,
24.811. HRMS (EI+) m/z calcd. for (C20H21N3O2) [M+H]+ 336.1707 found 336.1712. M.P.
163 ºC
2.3.2.3.4 (E)-methyl(2-(2-((3,5-bis(trifluoromethyl)phenyl)(methylimino)methyl)-1H-indol-3-
yl)ethyl) carbamate (18d)
Following the general procedure, the desired compound 18d was synthesized from 20h
(prepared from tryptamine (500 mg, 3.12 mmol) and 3,5-bis(trifluoromethyl)benzaldehyde
(0.41 ml, 2.49 mmol)) and methyl amine (6 mL) and was purified with column chromatography
with ethyl acetate-hexane (3:10) as eluent. It was generated as light yellow solid with yield of
1
67%. H NMR (400 MHz; DMSO): δ 11.25 (s, 1H), 8.26 (s, 1H), 8.08 (s, 2H), 7.68 (d, J= 7.84
Hz, 1H), 7.40 (d, J = 8.08 Hz, 1H), 7.22-7.18 (m, 1H), 7.12-7.08 (m, 1H), 7.04-7.01 (m, 1H),
3.38 (s, 3H), 3.33 (s, 3H), 3.12-3.08 (m, 2H), 2.62(t, J = 7.04 Hz, 2H). 13C NMR (100 MHz;
DMSO): δ 160.19, 156.86, 141.81, 136.89, 131.13, 130.80, 128.03, 127.39, 126.91, 124.17,
122.99, 122.26, 119.74, 119.67, 113.16, 112.26, 51.35, 42.61, 41.08, 25.46. HRMS (EI+) m/z
calcd. for (C22H19N3O2F6) [M+H]+ 472.1454 found 472.1489. M.P. 141 ºC
2.3.2.4 Synthesis of 19a-e
Representative experimental procedures for the synthesis of tetrahydro--carboline-
diketopiperazine hybrids (19a-e).

ACCEPTED MANUSCRIPT
To the solution of tryptophan methyl ester (1 equiv.) in dichloromethane (DCM) , substituted
benzaldehydes (0.8 equiv.) with trifluoroacetic acid (1.5equiv.) were added 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 5%sodium bicarbonate
(NaHCO3). It was then recrystallized with minimum amount of DCM to obtain crude product
which was taken to the next step without purification.
To the above crude compound (1 equiv.) and sodium bicarbonate (NaHCO3) (1.2 equiv.) in
chloroform (CHCl3) chloroacetyl chloride (2.4 equiv.) was added dropwise at 0 ºC. The
reaction mixture was stirred at room temperature under nitrogen atmosphere for 3 h. After
completion of reaction it was monitored by TLC and the reaction mixture was diluted by CHCl3
followed by washing with saturated solution of NaHCO3, brine, dried over Na2SO4, which was
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.) appropriate amine (5 equiv.) was added
in ethanol and was stirred at 50 ºC for 24 h. It was then concentrated under reduced pressure
after completion of reaction. Purification of the crude product was done with column
chromatography keeping ethyl acetate-hexane as eluent.
Oxidative ring opening for the synthesis of (19a-e).
The above compounds 21a-e (1 equiv.), iodine (0.3 equiv.), TBHP (4 equiv.) was taken in
acetonitrile and was stirred at 60 ºC for 4 h. Once the reaction is complete it was evaporated
under reduced pressure to obtained crude product which was purified by column
chromatography with ethyl acetate-hexane as eluent.
2.3.2.4.1 3-((2-benzoyl-1H-indol-3-yl)methyl)-1-benzylpiperazine-2,5-dione (19a)
Following the general procedure, the desired compound 19a was synthesized from 21a
(prepared from tryptophane methyl ester (500 mg, 2.29 mmol) and benzaldehyde (0.19 ml,
1.83 mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with DKP
group having bezyl protection of nitrogen of DKP) and purified by column chromatography
with ethyl acetate-hexane (3:7) as eluent and was obtained as pale yellow solid with yield of
1
73%. H NMR (400 MHz; CDCl3): δ 9.68 (s, 1H), 7.81 (d, J = 8 Hz, 1H), 7.52 (d, J = 8 Hz,
1H), 7.45 (d, J = 8 Hz, 2H), 7.39-7.33 (m, 3H), 7.29 (s, 1H), 7.27 (s, 1H), 7.24 (s, 1H), 7.22 (s,
1H), 7.07 (t, J = 4 Hz, 2H), 6.54 (s, 1H), 4.63 (d, J = 12 Hz, 1H), 4.32 (s, 1H), 4.11 (d, J = 16

ACCEPTED MANUSCRIPT
Hz, 1H), 3.66 (dd, J = 8, 16 Hz, 2H), 3.40 (d, J = 20 Hz, 1H), 2.98 (d, J = 20 Hz, 1H), 1.77 (s,
2H) ; 13C NMR (100 MHz; CDCl3): δ 188.75, 166.11, 165.82, 138.39, 136.94, 135.03, 132.66,
132.49, 129.41, 129.32, 129.02, 128.96, 128.64, 128.45, 128.11, 127.49, 126.77, 121.48,
121.29, 118.42, 112.89, 56.24, 49.68, 48.42, 28.84. HRMS (EI+) m/z calcd. for C27H23N3O3
[M+H]+ 438.1812, found 438.1819. M.P. 142 ºC
2.3.2.4.2 1-benzyl-3-((2-(3,5-bis(trifluoromethyl)benzoyl)-1H-indol-3-yl)methyl)piperazine-
2,5-dione (19b)
Following the general procedure, the desired compound 19b was synthesized from 21b
(prepared from tryptophane methyl ester (500 mg, 2.29 mmol) and 3,5-
bis(trifluoromethyl)benzaldehyde (0.30 ml, 1.83 mmol) and the nitrogen of the resulting
tetrahydro--carboline was protected with DKP group having bezyl protection of nitrogen of
DKP) and purified by column chromatography with ethyl acetate-hexane (3:7) as eluent and
was obtained as pale yellow solid with yield of 73%. 1H NMR (400 MHz; DMSO): δ 11.70
(s, 1H), 8.42 (s, 1H), 8.31 (s, 1H), 8.05 (s, 1H), 7.75 (d, J = 8 Hz, 1H), 7.48 (d, J = 8 Hz, 1H),
7.36 (t, J = 8 Hz, 1H), 7.27 (d, J = 8 Hz, 1H), 7.15 (t, J = 8 Hz, 1H), 7.03 (d, J = 8 Hz, 1H),
4.41 (d, J = 12 Hz, 1H), 4.20 (d, J = 12 Hz, 1H), 4.16-4.12 (m, 1H), 3.55 (t, J = 8 Hz, 1H),
3.50-3.42 (m, 1H), 3.24 (d, J = 16 Hz, 1H). 13C NMR (100 MHz; DMSO): δ 185.95, 165.77,
164.68, 140.93, 137.04, 135.91, 131.64, 130.58, 130.25, 129.53, 128.51, 127.64, 127.54,
127.36, 125.96, 124.41, 121.70, 121.03, 120.37, 118.53, 112.85, 55.54, 48.40, 48.18, 28.89.
HRMS (EI+) m/z calcd. for C29H21F6N3O3 [M+H]+ 574.1560, found 574.1563. M.P. 146 ºC
2.3.2.4.3 1-ethyl-3-((2-(2-fluoro-4-methoxybenzoyl)-1H-indol-3-yl)methyl)piperazine-2,5-
dione (19c)
Following the general procedure, the desired compound 19c was synthesized from 21c
(prepared from tryptophane methyl ester (500 mg, 2.29 mmol) and 2-fluoro-4-
methoxybenzaldehyde (282.07 mg, 1.83 mmol) and the nitrogen of the resulting tetrahydro--
carboline was protected with DKP group having ethyl protection of nitrogen of DKP) and
purified by column chromatography with ethyl acetate-hexane (3:7) as eluent and was obtained
as pale yellow solid with yield of 73%. 1H NMR (400 MHz; DMSO): δ 11.49 (s, 1H), 7.91 (s,
1H), 7.66 (d, J = 8 Hz, 1H), 7.53 (t, J = 8 Hz, 1H), 7.41 (d, J = 8 Hz, 1H), 7.07 (t, J = 8 Hz,
1H), 7.01 (t, J = 8 Hz, 1H), 6.96-6.93 (m, 2H), 4.05-4.02 (m, 1H), 3.88 (s, 3H), 3.50 (d, J = 20
Hz, 1H), 3.45-3.39 (m, 1H), 3.35 (d, J = 8 Hz, 1H), 3.16 (d, J = 20 Hz, 1H), 3.13-3.00 (m,
2H), 0.75 (t, J = 8 Hz, 3H). 13C NMR (100 MHz; DMSO): δ 184.07, 165.19, 164.98, 136.51,
132.98, 132.06, 127.53, 125.32, 120.97, 119.84, 116.97, 112.57, 110.57, 101.87, 56.01, 55.76,

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48.09, 28.94. HRMS (EI+) m/z calcd. for C23H22FN3O4 [M+H]+ 424.1667, found 424.1674.
M.P. 68 ºC
2.3.2.4.4 1-benzyl-3-((2-(3-fluorobenzoyl)-1H-indol-3-yl)methyl)piperazine-2,5-dione (19d)
Following the general procedure, the desired compound 19b was synthesized from 21b
(prepared from tryptophane methyl ester (500 mg, 2.29 mmol) and 3-fluorobenzaldehyde (0.19
ml, 1.83 mmol) and the nitrogen of the resulting tetrahydro--carboline was protected with
DKP group having bezyl protection of nitrogen of DKP) and purified by column
chromatography with ethyl acetate-hexane (3:7) as eluent and was obtained as pale yellow solid
with yield of 73%.1H NMR (400 MHz; DMSO): δ 11.59 (s, 1H), 8.05 (s, 1H), 7.72 (d, J = 8
Hz, 1H), 7.60 (s, 1H), 7.56 (d, J = 8 Hz, 1H), 7.49 (t, J = 12 Hz, 3H), 7.33 (t, J = 8 Hz, 2H),
7.27 (t, J = 8 Hz, 2H), 7.12 (t, J = 8 Hz, 1H), 7.04 (d, J = 8 Hz, 2H), 4.43 (d, J = 12 Hz, 1H),
4.15 (d, J = 20 Hz, 2H), 3.51 (dd, J = 4, 12 Hz, 1H), 3.44-3.40 (m, 2H), 3.21 (d, J = 20 Hz,
1H). 13C NMR (100 MHz; DMSO): δ 187.23, 165.86, 164.63, 136.73, 135.98, 132.09, 130.67,
128.53, 127.65, 127.48, 127.36, 125.44, 125.22, 120.85, 120.11, 119.09, 117.54, 115.81,
115.59, 112.79, 55.66, 48.43, 48.19, 29.04. HRMS (EI+) m/z calcd. for C27H22FN3O3 [M+H]+
456.1718, found 456.1723. M.P. 132 ºC
2.3.2.4.5 1-benzyl-3-((2-(4-chlorobenzoyl)-1H-indol-3-yl)methyl)piperazine-2,5-dione (19e)
Following the general procedure, the desired compound 19e was synthesized from 21e
(prepared from tryptophane methyl ester (500 mg, 2.29 mmol) and 4-chlorobenzaldehyde
(257.24 mg, 1.83 mmol) and the nitrogen of the resulting tetrahydro--carboline was protected
with DKP group having bezyl protection of nitrogen of DKP) and purified by column
chromatography with ethyl acetate-hexane (3:7) as eluent and was obtained as pale yellow solid
with yield of 73%. 1H NMR (400 MHz; CDCl3): δ 9.16 (s, 1H), 7.84 (d, J = 8 Hz, 1H), 7.60 (d,
J = 8 Hz, 2H),7.49-7.47 (m, 2H), 7.43-7.38 (m, 3H), 7.25 (d, J = 8 Hz, 2H), 7.06-7.04 (m, 2H),
6.58 (s, 1H), 4.66 (d, J = 12 Hz, 1H), 4.41 (s, 1H), 4.08 (d, J = 16 Hz, 1H), 3.78 (dd, J = 4, 12
Hz, 1H), 3.68 (dd, J = 4, 12 Hz, 1H), 3.45 (d, J = 20 Hz, 1H), 2.97 (d, J = 20 Hz, 1H), 2.75 (s,
1H). 13C NMR (100 MHz; CDCl3): δ 188.85, 166.14, 165.57, 138.58, 136.75, 135.07, 132.59,
129.27, 128.96, 128.44, 128.11, 127.68, 126.94, 121.72, 121.46, 118.84, 112.60, 56.43, 49.74,
48.51, 29.85, 28.89. HRMS (EI+) m/z calcd. for C27H22ClN3O3 [M+H]+ 472.1422, found
472.1425. M.P. 139 ºC
2.3.2.5 Synthesis of N-(2-(2-(4-chloro-benzyl)-1H-indol-3-yl)ethyl)acetamide (22)

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Compound 17e (500mg, 1.54 mmol) was dissolved in ethanol (EtOH) (10 mL) and was cooled
to 0 ºC. It was then treated with sodium borohydride (NaBH4) (63 mg, 1.7 mmol). The reaction
was stirred at this temperature and was monitored by TLC. After about ten minutes the starting
material was consumed. The reaction was quenched with brine and was extracted to DCM. The
organic layer was dried over Na2SO4 and was evaporated to afford the desired alcohol which
was pure enough to be taken to the final step.
The compound from the previous step, was dissolved in trifluoro acetic acid (5 mL) and was
treated with triethyl silane (200 mg, 1.7 mmol) at 0 ºC. The reaction was stirred and slowly
warmed to room temperature. Once the TLC indicates complete consumption of the starting
material the reaction was quenched with brine solution and was extracted with
dichloromethane. The organic layer dried over Na2SO4 and was evaporated to afford the crude
compound which was purified by column chromatography (Eluent: 50% EtOAc in hexane) to
generate 22 as faint yellow solid in 78% yield. 1H NMR (400 MHz, CD3OD) δ 7.626-7.623 (d,
J = 1.2 Hz, 0.5 H), 7.41-7.37 (m, 1H), 7.26-7.24 (d, J = 8 Hz, 3H), 7.18-7.16 (d, J = 8 Hz, 3H),
7.13 (s, 0.5H), 7.10-7.05 (m, 1H), 4.06-4.05 (d, J = 4 Hz, 2H), 3.289-3.285 (m, 2H), 2.91-2.89
(dd, J = 8 Hz, 2H), 1.83-1.82 (d, J = 4 Hz, 3H). 13C NMR (100 MHz, CD3OD) δ 173.33, 139.60,
138.31, 137.00, 136.31, 133.13, 131.06, 129.60, 124.59, 122.75, 121.56, 120.39, 115.25,
114.51, 113.30, 112.85, 110.35, 109.92, 41.54, 32.18, 25.09, 22.61. HRMS (EI+) m/z calcd.
for C19H19ClN2O [M-H]+ 327.1259, found 327.1262.
3. Results
3.1 Molecular docking
To corroborate our initial design, we docked three molecules 17e, 18a and 19a (Figure 2)
against tubulin complex (PDB ID: 4O2B) co-crystallized with colchicine (Prota et al., 2014).
The crystal structure was retrieved from the protein data bank (PDB) (Berman et al., 2000).
Figure 2. Representative compound class for newly designed compound library

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Figure 3 provides the result of the molecular docking analysis where 17e, 18a and 19a were
subjected to dock onto the crystal structure of tubulin complex (PDB ID: 4O2B). The average
and the top docking score derived from both GLIDE and GOLD sub-clusters docking solutions
revealed the overall preferential binding of the molecules at the colchicine binding site. The
preferential mode of binding of 17e, 18a and 19a onto the tubulin structure (PDB ID: 4O2B)
Figure 3: Docking analysis of the ligand (17e, 18a and 19a) with Tubulin (PDB ID: 4O2B).
Panel A, C and B, D represent the average and the top docking score of the best cluster derived
from GOLD and Schrödinger docking suit, GLIDE, respectively. Panel E, F and G depicts the

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probable binding poses of 17e, 19a and 18a onto the Tubulin structure (PDB ID: 4O2B) and
its corresponding types of interactions which are likely to be involved in stabilizing the protein-
ligand complexes.
and its corresponding types of interactions derived from GOLD docking solutions were shown
in Figure 3E-G. All the protein-ligand complexes were further considered for the interaction
analysis. 17e forms hydrogen bond interactions with LYS254, THR314 and ALA354 of tubulin
whereas the LEU248, ALA250, LEU255 and LYS352 are involved in hydrophobic
interactions. On the other hand, 18a forms hydrogen bond interactions with VAL238 and
ASN249 and hydrophobic interactions with ALA250, LEU248, LYS254, MET259, LYS352,
ALA316, CYS241, LEU255, respectively. 19a is involved in hydrophobic interactions with
LEU255, ASN258 and LYS352 (Figure 3).
3.2 Synthesis
We began our synthetic endeavor with the optimization of the catalytic oxidative ring opening
of 1-aryltetrahydro--carbolines 20a-n to afford the desired C2-aroyl tryptamine derivatives
17a-n. A thorough literature screening revealed that similar oxidative ring opening of
tetrahydro--carbolines had not been reported under catalytic condition until now. Hence to
identify an efficient catalytic procedure we explored iodine catalyzed ring opening reactions of
1-phenyl-tetrahydro--carboline, 20b in presence of variety of oxidants at diverse temperature
with acetonitrile as solvent (Table 1). The desired product 17b was obtained in poor to
moderate yield (16-45%), when the reaction was conducted at r.t., 40 and 60 °C in presence of
0.1 equiv. of iodine (I2) and four equivalent of tert-butylperoxide (TBHP) (Table 1, entry 1-3).
The next experiments in absence of iodine and TBHP (Table 1, entry 4-5), lead to no or
extremely poor yield of the desired product 17b (Table 1, entry 4 and 5). This further
emphasized the importance of iodine and TBHP in the reaction. Increment of the catalyst
quantity to 0.3 equiv. improved the yield of 17b to ~95% (Table 1, entry 6) at 60 ºC. Exploring
other iodine based catalysts such as tetrabutyl ammonium iodide (nBu4NI), sodium iodide
(NaI), magnesium iodide (MgI2) and potassium iodide (KI) could not improve the yields further
(Table 1, entry 7-10). Hence the optimized protocol for the synthesis of 17b involved heating
20b at 60 ºC in acetonitrile, in presence of 0.3 equiv. of iodine and 4 equiv. of TBHP for 6h.
Table 1. Optimization of organocatalytic oxidative ring opening of 1-phenyltetrahydro--
carboline, 20ba

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Entry Iodine source (equiv)
Temp. (°C) Yield of 2a (%)b
1
2
I2 (0.1)

r. t.
21
39
48
-
40
3
60

4
-
40
5c
I2 (0.1)
I2 (0.3)
Bu4NI (0.3)
NaI (0.3)
MgI2 (0.3)
KI (0.3)
̎
̎
̎
̎
̎
̎
8
6
95
68
14
62
12
7
8
9
10
a Reaction condition: 1a (1 mmol), iodine source, TBHP (2 equiv) for ~ 4h. b Isolated yields. c
The reaction was performed without TBHP
With the protocol for oxidative ring opening of 20b optimized, a diverse variety of C2-aroyl
tryptamine derivatives 17a, c-n, were generated (Scheme 1). The overall substitutions ranged
from electron withdrawing to electron donating functionalities were the desired products were
obtained in moderate to excellent yield (Scheme 1). In general the substrates with electron
withdrawing functionalities such as 17c-e, g-h, j and m-n provided the products in slightly
better yields than the substrates with electron donating functional groups. The tryptamine
nitrogen were protected with acetyl and carbomethoxy functionalities. It is noteworthy that the
Boc- protection of the carboline nitrogen in the substrate rendered it inactive towards oxidative
ring opening reaction. Deprotection of either acetyl or carbomethoxy group from the products
prompted the ring closing imination reaction to generate the dihydro--carbolines.

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Scheme 1. Library of C2 aroyl tryptamines

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Next, iminoaryl derivatives 18a-d, were accessed by the protocol optimized for the synthesis
of 17a-n in presence of appropriate amines. We envisaged that an in situ imination of the
corresponding C2-aroyl derivatives (that results from oxidative ring opening of 1-
aryltetrahydro--carbolines) could afford 18a-d. Accordingly, 20a, l, k and h were reacted with
four equiv. of TBHP, 0.3 equiv. of iodine in methylamine to provide the desired products in
excellent yields (Scheme 2).
Scheme 2. Library of C2 arylimino tryptamines
The diketopiperazinyl-C2-aroyl indole hybrid analogs 19a-e were synthesized from
diketopiperazinyl tetrahydro--carbolines 21a-e (Scheme 3). They in turn where synthesized
from L-tryptophan methyl ester. The initial step involved Pictet Spengler cyclization of L-
tryptophan methyl ester with appropriate aldehydes to generate tetrahydro-b-carbolines as
single diastereomers, which precipitated from dichloromethane. The crude were then treated
with chloroacetyl chloride to provide the corresponding amides which was further treated with
ethyl and benzyl amine to promote an in situ nucleophilic substitution and subsequent
lactamization (with the ester) to generate the desired compounds 21a-e in 58-84% yield. Iodine
catalysed oxidative ring opening of 21a-e provided the final compounds 19a-e (Scheme 3).

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Scheme 3. Synthesis of diketopiperazinyl-C2-aroyl indole hybrids
Next, we were also interested to investigate the inhibitory effect of an aryl substituent at the C2
position of the tryptamine derivatives towards the tubulin polymerization. Accordingly, we
synthesized compound 22 (Scheme 4) from the C2-aroyl derivative 17e where it was first
reduced at 0 ºC in ethanol and the crude intermediate was treated with triethyl silane in presence
of trifluoroacetic acid from 0 °C to r. t., to afford 22. It was then purified through flash column
chromatography, to provide the desired product in 86% yield over two steps (Scheme 4).
Scheme 4. Synthesis of 22
3.3 In vitro tubulin inhibition and anticancer activity
With the library in hand, next it was subjected to in vitro screening against tubulin and a panel
of cancer cells such as MCF7 (breast cancer), A549 (lung cancer), HeLa (cervical cancer cells)
and HePG2 (liver cancer cells) (Table 2).
To assess the antimitotic properties, the compounds were screened at 10 M concentration in
vitro on purified tubulin polymerization in cell free system which were monitored by light
scattering assay at 350 nM (Table 2). Among the compounds screened, the C2 aroyl compounds

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17d, g, h, m and n inhibited tubulin assembly at 55  79% (Table 2). Other compounds of the
series did not exhibit effective tubulin polymerization inhibitory activity. Positive control
colchicine (@ 1 M) exhibited 48% inhibition of tubulin assembly.
Cytotoxic screening of the library against the cancer cell lines revealed that 17m was most
potent and inhibited the proliferation of A549, HeLa and MCF7 cells with an IC50 of 6, 10 and
7.2 M respectively. 17h was next best potent molecule inhibiting selectively A549 and HeLa
cells over MCF7 and HepG2 with IC50 of 6 and 6.5 M respectively. This was encouraging as
they were also the most potent tubulin depolymerizing agent as observed from the tubulin
screening (Table 2). Compound 17a and f were specifically active against HeLa cell line.
Interestingly, their activity was modest against tubulin, thereby indicating an alternate pathway
of action for the inhibition of proliferation of HeLa cells by these molecules (Table 2). It is
noteworthy that the C2 aryl imino analogs 18a-d and the tryptamine-DKP hybrids 19a-e, were
completely inactive against tubulin polymerization and also failed to evoke any inhibitory
activity against the proliferation of the cancer cell lines (Table 2). The C2-aryl derivative 22,
was poorly active against tubulin and was inactive against the cancer cell lines (Table 2). To
make certain that our compounds were not exerting their anticancer activity through
cytotoxicity, we screened the most active compounds 17h and m against WI-38 a diploid
human cell line composed of fibroblasts from lung tissue. For each of the compounds, the dose
that was required to exhibit cytotoxicity was at least 10-fold higher than their respective IC50
concentrations (Figure 4 in Supporting Information).

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Table 2. In vitro screening of the library of molecules 17a-n, 18a-d and 19a-e
In vitro tubulin
A549 48 h HeLa 48h MCF7 48h HePG2 48h
assemby inhibition
@ 10 M drug
concentration (%ge)
Compound
name
(IC50 in
(IC50 in
(IC50 in
(IC50 in
Structures
M)
M)
M)
M)
6
> 50
> 50
> 50
> 50
17b
47
56
22
30
>50
>50
>50
>50
17c
17d
 45
> 50

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46
>50
3
>50
>50
17a
31
58
>50
8
>50
>50
>50
>50
17f
17g
35
50
65
>50
50
17h
6  1
6.5  2