Discovery of tetrahydrocarbazoles as dual pERK and pRb inhibitors

Article abstract of DOI:10.1016/j.ejmech.2017.02.062

The extracellular signal-regulated kinase (ERK) is one of the most important molecular targets for cancer that controls diverse cellular processes such as proliferation, survival, differentiation and motility. Similarly, the Rb (retinoblastoma protein) is

Full text of DOI:10.1016/j.ejmech.2017.02.062

Accepted Manuscript
Discovery of tetrahydrocarbazoles as dual pERK and pRb inhibitors
Mahesh R. Kulkarni, Madhav S. Mane, Usha Ghosh, Rajiv Sharma, Nitin P. Lad,
Ankita Srivastava, Asha Kulkarni-Almeida, Prashant S. Kharkar, Vijay M. Khedkar,
S.S. Pandit, M. Sc., Ph. D.
PII:
S0223-5234(17)30147-2
10.1016/j.ejmech.2017.02.062
EJMECH 9258
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 October 2016
Revised Date: 25 February 2017
Accepted Date: 27 February 2017
Please cite this article as: M.R. Kulkarni, M.S. Mane, U. Ghosh, R. Sharma, N.P. Lad, A. Srivastava,
A. Kulkarni-Almeida, P.S. Kharkar, V.M. Khedkar, S.S. Pandit, Discovery of tetrahydrocarbazoles
as dual pERK and pRb inhibitors, European Journal of Medicinal Chemistry (2017), doi: 10.1016/
j.ejmech.2017.02.062.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT
Discovery of Tetrahydrocarbazoles as Dual pERK and pRb
Inhibitors
Mahesh R. Kulkarnia,b, Madhav S. Maneb, Usha Ghoshb, Rajiv Sharmab , Nitin P. Lada,b,
Ankita Srivastavac, Asha Kulkarni-Almeidac, Prashant S. Kharkard, Vijay M. Khedkare S. S.
Pandita*
aPost Graduate and Research centre, Department of Chemistry, Padmashri Vikhe Patil
College of Arts, Science and Commerce, Pravaranagar, A/P Loni, Tal. Rahata, Dist.
Ahmednagar – 413713. India.
bDepartment of Medicinal chemistry, Piramal Enterprises Limited 1, Nirlon Complex, Off
Western Exp. Highway, Near NSE Complex, Goregaon East, Mumbai, Maharashtra- 400
063. India.
cDepartment of Pharmacology, Piramal Enterprises Limited 1, Nirlon Complex, Off Western
Exp. Highway, Near NSE Complex, Goregaon East, Mumbai, Maharashtra- 400063. India.
dSPP School of Pharmacy and Technology Management, SVKM’s NMIMS, V. L. Mehta
Road, Vile Parle (West), Mumbai-400 056. India.
eSchool of Health Sciences, University of KwaZulu Natal, Westville Campus, Durban 4000,
South Africa
† Electronic Supplementary Information (ESI) available: spectral data
*Authors for correspondence
Dr. S. S. Pandit, M. Sc., Ph. D.,
Post Graduate and Research centre, Department of Chemistry, Padmashri Vikhe Patil College
of Arts, Science and Commerce, Pravaranagar, A/P Loni, Tal. Rahata, Dist. Ahmednagar –
413713. India.
E-mail: akankshapandit2002@yahoo.com

ACCEPTED MANUSCRIPT
Tel.; +91-9766959195
ABSTRACT:
The extracellular signal‐regulated kinase (ERK) is one of the most important molecular
targets for cancer that controls diverse cellular processes such as proliferation, survival,
differentiation and motility. Similarly, the Rb (retinoblastoma protein) is a tumor suppressor
protein and its function is to prevent excessive cell growth by inhibiting cell cycle
progression. When the cell is ready to divide, pRb is phosphorylated, becomes inactive and
allows cell cycle progression. Herein, we discovered a new series of tetrahydrocarbazoles as
dual inhibitors of pERK and pRb phosphorylation. The in-house small molecule library was
screened for inhibition of pERK and pRb phosphorylation, which led to the discovery of
tetrahydrocarbazole series of compounds as potential leads. N-(3-methylcyclopentyl)-6-nitro-
2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-amine (1) is the dual inhibitor lead identified through
screening, displaying inhibition of pERK and pRb phosphorylation with IC50 values of 5.5
and 4.8 µM, respectively. A short structure-activity relationship (SAR) study has been
performed, which identified another dual inhibitor 9-methyl-N-(4-methylbenzyl)-
2,3,4,4a,9,9a-hexahydro-1H-carbazol-2-amine (16) with IC50 values 4.4 and 3.5 µM for
inhibition of pERK and pRb phosphorylation, respectively. This compound has a potential for
further lead optimization to discover promising molecularly-targeted anticancer agents.
KEYWORDS
Tetrahydrocarbazole, pERK, pRb, MDR, MAPK, CDKs
1. Introduction
Despite significant advances and progress made in the area of cancer management, multi-
drug resistance (MDR) is a growing concern [1, 2]. Currently, molecularly-targeted therapy
promises to provide better therapeutic and toxicity profiles [3]. In addition, mechanism-based
targeting of the cancer survival and resistance pathways should translate into increased
efficacy and reduced MDR. Cancer is a disease that is characterized by uncontrolled cell
growth which is manifested due to cellular crosstalk or signaling between and within cells
[4]. The aberrations are pleiotropic, and occur due to effects on several different signaling
pathways; the most significant among these being mitogen-activated protein kinase (MAPK)
pathways, Ras-Raf pathways and cyclin dependent unrestrained cell proliferation [3]. The
balance and integration between these signals may widely vary in different tumors, but are
important for the outcome and the sensitivity to drug therapy.

ACCEPTED MANUSCRIPT
The MAPK pathway is a chain of proteins in the cell that communicates a signal from
a receptor on the surface of the cell to the DNA in the nucleus of the cell [5, 6]. The signal
starts when a signaling molecule binds to the receptor on the cell surface and ends when the
DNA in the nucleus expresses a protein resulting in cell division [7, 8]. The pathway includes
many proteins, including MAPKs, originally called ERKs (extracellular signal-regulated
kinases), which communicate by adding phosphate groups to a neighbouring protein, which
acts as an "on" or "off" switch. This pathway is also known as the Ras-Raf-MEK-ERK
pathway [8]. Drugs that reverse the "on" or "off" switch are being investigated for cancer
treatment [9]. The ERK, one of the key players in this pathway, is a major determinant in the
control of diverse cellular processes such as proliferation, survival, differentiation and
motility. This pathway is often upregulated in human tumors and as such represents an
attractive target for the development of anticancer drugs. Specific blockade of the ERK
pathway is expected to result in not only antiproliferative effect but also in antimetastatic and
antiangiogenic effects in tumor cells. Recently potent small‐molecule inhibitors targeting the
components of the ERK pathway have been developed. Among them, BAY 43‐900610 (Raf
inhibitor) [10], PD184352, PD032590111 [11] and ARRY‐14288612 (MEK1/2 inhibitors)
[12] have reached the clinical trial stages.
Similarly, the retinoblastoma protein (Rb) is a tumor-suppressor protein that is
dysfunctional in several major cancers [13]. The phosphorylated form of this protein (pRb)
functions to prevent excessive cell growth by inhibiting cell cycle progression thus
preventing cell division. It is also a recruiter of several chromatin-remodeling enzymes such
as methylases and acetylases which are implicated in signaling activation of several signaling
pathways. Rb is phosphorylated to pRb by certain cyclin-dependent kinases (CDKs). pRb
when hyper phosphorylated is unable to complex E2F and therefore, unable to restrict
progression from the G1 phase to the S phase of the cell cycle. During the M to G1 transition,
pRb is progressively dephosphorylated by PP1, returning to its growth-suppressive hypo
phosphorylated state Rb. When it is time for a cell to enter the S phase, complexes of CDKs
and cyclins phosphorylate Rb to pRb, inhibiting its activity. The initial phosphorylation is
performed by cyclin D/CDK4/CDK6 and followed by additional phosphorylation by cyclin
E/CDK2. pRb remains phosphorylated throughout S, G2 and M phases [14, 15]. Due to its
key role in the cell cycle progression, it is believed to be a good anticancer target [16, 17].
Modern approaches in cancer therapeutics include combination of ERK pathway inhibitors
(cytostatic agents) and conventional anticancer drugs (cytotoxic agents) which may provide

ACCEPTED MANUSCRIPT
an excellent basis for the development of new chemotherapeutic strategies against cancer
[18]. Thus, our study focuses on targeting primary signaling events in the Ras-Raf pathway
and cell cycle progression. We believe that mitigating both events simultaneously may lead to
improved efficacy in targeting cancer.
In this direction, we have screened our in-house small molecule library of compounds
(777 compounds) originally designed for inflammatory targets (TNF-α and IL-6) against
pERK and pRb signaling. We tested for pERK, pRb phosphorylation inhibition at 10 µM
concentration (n=3) and the compound which demonstrated >70% inhibition were considered
as active. 25 compounds showed >70% inhibition in the pERK assay, out of these 6
compound showed consistent activity in dose dependent manner in pERK assay. In pRb assay
14 compound showed >70% inhibition in pRb assay and only 2 compounds demonstrated
dual inhibition of pERK and pRb phosphorylation. The dose response effects of these two
hits confirmed the activity of compounds and led to the identification of tetrahydrocarbazole
based inhibitor 1 (Figure 1) as initial hit with pERK (IC50= 5.52 µM) and pRb (IC50 = 4.81
µM) inhibitory activity. Tetrahydrocarbazole core is known to possess a wide range of
biological activities such as, 5-HT6-receptor antagonists [19], antifungal [20], treatment of
human papilloma infection[21-23], treatment of viruses belonging to flaviviruses,
pestiviruses, hepaciviruses, prophylaxis dengue fever, yellow fever, west nile virus,
HCV[24], treatment of neurological, cardiac and metabolic diseases [25], BTK inhibitor [26],
antitumor agents [27], etc. We set out with the primary aim of improving the potency of this
core (1) as dual pathway inhibitor. The present study describes the investigations into the
SAR, molecular docking and further optimization of 1 as a dual inhibitor of pERK and pRb
phosphorylation.
<<<Figure 1 >>>
2. Result and Discusion
2.1 Chemistry. The synthetic route to 1-aminotetrahydrocarbazole analogues of
1
(pERK and pRb phosporylation inhibitor) is shown in Scheme 1. The
tetrahydrocarbazole core was assembled by cyclization of commercially available 4-
(1
corresponding 2,3,4,9-tetrahydro-1
2,3,4,9-tetrahydro-1 -carbazol-1-amine
H
-indol-3-yl)butanoic acid
2
in presence of polyphosphoric acid (PPA) to the
-carbazol-1-one which was converted to
by subsequent oxime formation followed by
H
3
H
5

ACCEPTED MANUSCRIPT
reduction of the oxime using LiAlH4. Further the amine was converted to the designed
analogues of
1 (6).
<<<Scheme 1 Here>>>
Syntheses of 2-amino-substituted tetrahydrocarbazole derivatives 13 were achieved by
sequential reactions of 1,3-cyclohexadione and phenylhydrazine or 4-
methylphenyhydrazine as shown in Scheme 2. The intermediate ketone 10 was
synthesized according to the reported procedure [28]. 1,3-cyclohexadione ( ) and
phenylhydrazine or 4-methylphenyhydrazine were condensed to give hydrazone 8
which on treatment with TSA and ethylene glycol in toluene gave protected ketone 9.
7
7
p
It was further deprotected to the corresponding ketone 10 using 10% aqueous H2SO4
or conc. HCl in methanol. Ketone 10 was further converted to oxime 11 by treatment
with NH2OH.HCl, which was reduced to the corresponding amine 12 by treatment
with LiAlH4. Amine 12 was converted to the designed compounds by reductive
amination with respective aldehyde or ketone or by coupling with respective benzoyl
chloride or sulfonyl chloride to yield amide or sulfonamide in presence of Et3N.
<<<Scheme2 Here>>>
In order to synthesize
Scheme 3 was followed.
using methyl iodide in presence of NaH led to 14, which was deprotected to
corresponding ketone 15 followed by its conversion to -methyl analogue 16
N-methyl analogues of 13l, the reaction sequence shown in
N
-Methylation of the protected ketone was carried out
9
N
.
<<<Scheme3 Here>>>
2.2 Biological activity
Our in-house efforts to discover a dual pERK and pRb phosphorylation inhibitor
yielded a moderately potent dual inhibitor with IC50 values of 5.5 and 4.8 µM,
1
respectively, for pERK and pRb. Initial SAR studies (Table 1) showed that only 3-
methylcyclopentyl substitution 6c retained activity compared to the initial hit in both
the assays. This led to obvious conclusion that the 6-NO2 group on the carbazole
nucleus contributed little to the biological activity. Substitutions with cyclohexyl and

ACCEPTED MANUSCRIPT
cyclopentyl led to decrease in the biological activity. Other substituents bearing
aromatic sulfonamide groups, were not tolerated (Table 1, compounds 6d-f). There
was no improvement in potency over 1. Hence, we investigated the effect of changing
the position of amine group from benzylic to homobenzylic 13 (Scheme 2).
Compound 13a bearing cyclohexyl substitution exhibited the IC50 of 7 µM and 11 µM
for inhibition of pERK and pRB phosphorylation, respectively. Compounds 13b, 13c
having cyclopentyl and 3-methylcyclopentyl substitutions showed moderate activity
(IC50 = 10–30 µM) in both the assays, and 13d having tetrahydropyran was inactive in
both the assays. This showed that the cycloalkyl groups were not favorable for
activity. Sulfonamide 13f having 4-fluorophenyl substitution showed good activity in
both the assays having IC50 7.5 µM and 9.5 µM in pERK and pRb, respectively,
indicated the presence of electron-withdrawing group was favorable but at the same
time 13g having electron-withdrawing cyano group retained the activity in pERK (IC50
= 7.0 µM) but loss in activity (IC50 = >100 µM) in pRb assay was observed. Other
benzamides (13h, 13i and 13j) and sulfonamides (13e) were shown to be good to
moderately active in pERK assay but were not tolerated in pRb assay.
When cycloalkyls were replaced by benzyl groups (benzyl, p-tolyl), 13k (pERK IC50 =
6.11 µM, pRb IC50 = 6.92 µM) and 13l (pERK IC50 = 3.62 µM, pRb IC50 = 4.40 µM)
showed good activity in both the assays. Compound 13l having
p-tolyl substitution
showed improved activity compared to the initial hit, . In order to synthesize benzyl-
1
substituted derivatives of 13, we tried direct reductive amination on ketone 10 with
respective benzylamine but we observed unexpected aromatized product as major one.
On investigation, we confirmed that it was known in literature [29]. Hence, the same
reaction sequence as depicted in Scheme 2 was followed. Furthermore, we were
curious to see effect of substitution on carbazole ring on the biological activity. Thus,
we synthesized compound with methyl substitution at position 6. These compounds
were synthesized according to Scheme 2, the only exception being p-tolylhydrazine,
which was used instead of phenylhydrazine. With 6-methyl substitution on
tetrahydrocarbazole, both the cycloalkyl and benzyl substitutions were active in both
the assays (compounds 13m-13o). Compound 13o having p-tolyl substitution showed
IC50 4.6 µM and 2.9 µM in pERK and pRb assays, respectively.
<<<Table 1 Here>>>

ACCEPTED MANUSCRIPT
The SAR showed that methyl substitutions at the 6-position of tetrahydrocarbazole
were quite well tolerated. Among the synthesized derivatives of 2-amino substituted
tetrahydrocarbazole, 13l and 13o were the most potent compounds. Further, we
investigated the role of NH of tetrahydrocarbazole on the activity by converting NH to
N-Me of the most potent compound 13l. We observed that the corresponding N-methyl
derivative 16 was found to be similar in potency for the inhibition of pERK and pRb
phosphorylation, compared to the parent 13l in both the assays. From this, we could
conclude that the activity was indifferent to either NH free or
N-methyl substitution.
<<<Table2 Here>>>
<<<Figure 2 Here>>>
2.3 Molecular Docking. We were further interested in understanding the binding mode of
these inhibitors in the ligand-binding pocket of ERK2 kinase. A recently deposited high-
resolution crystal structure of ERK2 kinase [30] (PDB ID: 5BVD) was used for performing
the docking studies of few select molecules (1, 13l, 13o and 16). All the molecules along with
the crystal structure ligand were docked in the ligand-binding pocket. One of these
molecules, compound 16, exhibited favorable binding with the ERK2 kinase (Figure 3,
Table 3) as seen from the XP GScore and the MMGBSA binding energy (Table 3). The
binding modes of other molecules 1, 13l, 13o and 16 along with the crystal structure ligand
17 can be found in the Electronic Supplementary Information (ESI) section. As seen from the
IC50 values for ERK (Figure 2 and Tables 2 and 3), the binding energy values are in
agreement with the experimental activity. This only increased our confidence in the
molecular docking studies.
The summary of molecular docking analyses (XP GScore) and the corresponding MMGBSA
binding energies are listed in Table 3. Compound 13o showed the highest XP GScore of -
6.956 while others such as 13l and 16 were comparable with 1 being the lowest (-5.397).
Similarly, the MMGBSA binding energy was found to be the highest for compound 13o
followed by 16, 1 and 13l, with the values ranging from -120 to -111 Kcal/mol). The binding
modes of these compounds are shown in Figures 4-7 (can be found in the Electronic
Supplementary Information (ESI) section).

ACCEPTED MANUSCRIPT
Compound 1 exhibited the binding mode similar to crystal structure ligand 17 (Figure 7).
Two H-bonds were seen with Met106 formed by the exo- and endocyclic Ns. Even though
the protonated N was in close proximity to Glu107, no salt-bridge interaction was seen. The
6-NO2 group did not make any interaction with the ERK2 kinase. In addition, the 3-methyl
substituent on the cyclopentyl ring was exposed to the solvent. The tetrahydrocarbazole ring
of 1 was found to occupy the same area occupied by the N-tetrahydropyranyl 2-
aminopyrimidine substructure of 17 (Figure 7, alignment not shown).
Unlike the binding mode exhibited by 1, compounds 13l, 13o and 16 showed quite
different binding modes, where a consistent salt-bridge interaction of exocyclic protonated N
was seen with Asp106 (Figures 4-6). In addition, the protonated exocyclic N in compound
13l also formed H-bond with Asn164. Another consistent cation-π interaction of Lys52 was
found with the benzylic aromatic ring (Figures 5-7). The tetrahydrocarbazole ring of 13l, 13o
and 16 was found to occupy same area of the ERK2 kinase ligand-binding pocket occupied
by the 1H,2H,3H,4H-pyrrolo[1,2-a]pyrazin-1-one substructure of 17 (Figure 7, alignments
not shown). We believe that the altered binding mode of 1 and its SAR compounds are
responsible for gain in potency over 1. The docking studies are likely to contribute to our
understanding of the inhibition of ERK2 kinase by the lead molecules described in this study.
<<<Table 3 Here>>>
3.0 Conclusions
In the present investigation, we have discovered tetrahydrocarbazoles as a new class of
pERK and pRb phosphorylation inhibitors. A series of 1-amino- and 2-amino
substituted tetrahydracarbazole derivatives were synthesized, where the initial SAR
suggested that the homobenzylamine with benzyl substitution was preferable for the
dual inhibition of pERK and pRB phosphorylation. The docking studies of few select
molecules with the ERK2 kinase shed some light on the binding modes, and
subsequently the binding potency of the SAR molecules compared to the initial hit 1.
The most potent dual inhibitor of pERK and pRb phosphorylation 16 can be a good
starting point for an extensive medicinal chemistry program, which may lead to further
identification of more potent pERK-pRb dual inhibitors.

ACCEPTED MANUSCRIPT
4. Experimental Section
4.1 General Chemistry Details.
All the chemicals (intermediates and reagents) and solvents (including dry solvents)
were procured from commercial suppliers such as Sigma-Aldrich Chemical Co.,
Fischer Scientific, VWR International, etc., and were used as received unless
otherwise indicated. All reactions were performed under an inert atmosphere (argon or
N2) unless otherwise noted. Analytical silica gel 60 F254-coated TLC plates were
purchased from Merck Chemicals, and were visualized with UV light or by treatment
with TLC reagents such as ninhydrin, Dragandorff’s or phosphomolybdic acid (PMA).
Flash-column chromatography was carried out on Combiflash Rf using silica gel (230–
1
400 mesh). H-NMR spectra were routinely recorded on Bruker 300 MHz FT NMR,
with tetramethylsilane (TMS) as an internal standard. The purity of all compounds was
determined by HPLC (Waters 2695 Alliance) system implementing either Method A, Method
B or Method C for chromatographic separation. HPLC solvents: A: Acetonitrile. B:
0.01MNH4OAc + 0.5% TEA, pH 5.0 with AcOH. HPLC Columns: Column 1: Ascentis TM
Express (50 x 4.6 mm I.D.), 2.7 µm operated at 1 mL/min, detection at 288 nm. Column 2:
Poroshell 120 EC-C18 (50 X 4.6mm I.D.), 2.7µm operated at 1 mL/min, detection at 276 nm.
HPLC Methods: Method A: Elution with 20-80% linear gradient of A in 6 min followed by
20-80% linear gradient of B in 1 min that is continued using an isocratic elution with 80% B
for 3 min using Column 1. Method B: Elution with 20-80% linear gradient of B in 6 min
followed by 20-80% linear gradient of A in 1 min that is continued using an isocratic elution
with 80% A for 3 min using Column 2.
4.1.1. 2,3,4,9-Tetrahydro-1H-carbazol-1-one (3): Polyphosphoric acid (20 mL) was added
to a stirred solution of 4-(1H-indol-3-yl)butanoic acid 2 (20g, 0.0984 mole) in toluene (200
mL) and the resulting reaction mixture was allowed to stir at reflux for 5 h. Progress of
reaction was monitored by tlc and after complete conversion of starting material reaction
mixture was cooled to rt. Toluene was decanted, and oily residue was washed with toluene
and discarded toluene layers. Oily residue was diluted with water (200 mL) and stirred at rt
for overnight. Reaction mixture was extracted with ethyl acetate (3 x 100 mL), combined
organic layers were washed with water (100 mL), brine (100 mL), dried over anhydrous
sodium sulphate and filter. Filtrate was concentrated to get crude solid which was purified by

ACCEPTED MANUSCRIPT
silica gel column chromatography in n- Hexane: Ethyl acetate; 7:3 as elute to get title
compound (12.5g, 68.60%) as an Off-White solid; 1H-NMR (300MHz, DMSO-d6): δ 2.11-
2.19 (m, 2H), 2.53-2.57 (m, 2H), 2.93-2.97 (m, 2H), 7.07 (t, J = 7.2 Hz, 1H), 7.30 (t, J = 7.2
Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.67(d, J = 7.8 Hz, 1H), 11.58 (s, 1H); MS (ESI) m/z:
186.5[M+H]+; HPLC Retention time: 3.764 min., Purity: 89.81%(Method A).
4.1.2. 2,3,4,9-tetrahydro-1H-carbazol-1-amine (5): Step-I (Z)-2,3,4,9-tetrahydro-1H-
carbazol-1-one oxime (4): To a stirred solution of 2,3, 4, 9-tetrahydro-1H-carbazol-1-one 3
(1.5 g, 8.10 mmol) in ethanol (20 mL) added subsequently solution of hydroxylamine
hydrochloride (1.13 g, 16.2 mmol) in water(10 mL) and a solution of sodium acetate (2.19 g,
26.7 mmol) in water (10 mL). After being stirred at 80°C for 2 h, cooled reaction mixture to
25 – 30°C. Concentrated reaction mixture under reduced pressure and resulting residue was
diluted with water (25 mL). Reaction mixture was extracted with ethyl acetate (2 x 50 mL),
combined organic layers were washed with brine (50 mL) dried over anhydrous sulphate and
filtered. Filtrate was concentrated under reduced pressure to get title compound as a brown
solid (1.58g, 97.53%). Step-II: 2,3,4,9-tetrahydro-1H-carbazol-1-amine (5): Lithium
aluminium hydride (1.0 M in THF, 70mL) was added drop wise to a stirred solution of (Z)-
2,3,4,9-tetrahydro-1H-carbazol-1-one oxime 4 (1.55g, 7.74 mmol) in THF (80 mL). After
being stirred reflux for 7 h. Cooled reaction mixture to 0°C and an excess of LiAlH4 was
quenched by addition of sodium sulphate decahydrate until bubbling ceased. Stirred resulting
reaction mixture for 0.5 h. precipitated inorganics were removed by filtration through celite
bed. Filtrate was concentrated to get crude product which was purified by flash
chromatography on silica (dichloromethane : Methanol; 9.5:0.5) to provide 2,3, 4, 9-
1
tetrahydro-1H-carbazol-1-amine 5 (1.09g, 85.15%) as a brown Solid; H-NMR (300MHz,
DMSO-d6): δ 1.49-1.58 (m, 1H), 1.62-1.74 (m, 1H), 1.89-2.00 (m, 2H), 2.04 - 2.08 (m, 2H),
2.59 (d, J = 6.9 Hz, 2H), 3.93(t, J = 6.9 Hz, 1H), 6.91 (t, J = 7.2 Hz, 1H), 6.99 (t, J = 7.5 Hz,
1H), 7.28 (d, J = 7.8 Hz, 1H), 7.33(d, J = 7.8 Hz, 1H), 10.67 (s, 1H); MS (ESI) m/z:
170.1[M+H]+; HPLC Retention time: 1.737 min., Purity: 98.34%(Method A).
4.1.3. N-cyclohexyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride (6a)
2,3,4,9-tetrahydro-1H-carbazol-1-amine(0.2 g, 1.074 mmol) was treated with cyclohexanone
(0.126 g, 1.289 mmol) in tetrahydrofuran (10 mL) at 25-30°C in the presence of
trifluoroacetic acid (0.041 mL, 0.537 mmol). After being stirred for 4h.added
sodiumcyanoborohydride (0.202 g, 3.22 mmol) and continued stirring at same temperature
for 16 h. Reaction mixture was diluted with cold water (50mL) and extracted with ethyl

ACCEPTED MANUSCRIPT
acetate (2 x 25mL). Combined organic layers were washed with water (25mL), brine (25mL),
dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated under
reduced pressure to get crude which was purified by silica gel(60/120) column
chromatography in dichloromethane: methanol; 9:1 as elute to get title compound (0.12g,
40.76%).Which was converted to the corresponding hydrochloride using IPA. HCl in ethyl
acetate using 0.1g of base (N-cyclohexyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine).
1
Yield: 40.76%; White Solid; mp: 119-121°C; H-NMR (300MHz, DMSO-d6):
δ 1.08-1.20
(m, 2H), 1.22-1.40 (m, 2H), 1.42-1.59 (m, 2H), 1.60-1.67 (m, 2H), 1.72-1.90 (m, 3H), 1.95-
2.22 (m, 5H), 2.69 (s, 2H), 7.02 (t, J = 7.2 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 8.1
Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 8.82 (s, 1H), 9.38 (s, 1H), 11.29 (s, 1H); MS (ESI) m/z:
269.1 [M+H]+; HPLC Retention time: 3.359 min., Purity: 99.41%(Method A).
4.1.4. N-cyclopentyl-2,3,4,9-tetrahydro-1H-carbazol-1-amine hydrochloride(6b)
2,3,4,9-tetrahydro-1H-carbazol-1-amine 5 (0.2 g, 1.074 mmol) was treated with
cyclopentanone (0.181 g, 2.148 mmol) in tetrahydrofuran (10 mL) in the presence of TFA
(6.12 mg, 0.054 mmol) at 25-30°C. The resulting reaction mixture was stirred for 4 h. at same
temperature, then added sodiumcyanoborohydride (0.202 g, 3.22 mmol) continued stirring for
16 h. The reaction mixture was diluted with cold water (50mL), extracted with ethyl acetate
(2 x 25 mL), combined organic layers were washed with water (25mL), brine (25mL). Dried
organic layers over anhydrous sodium sulphate and filtered. The filtrate was concentrated
under reduced pressure to get crude. Purification was done by silica gel (60/120) column
chromatography in dichloromethane: methanol; 9:1 as elute to get title compound (0.172g,
62.0%) and the base was converted to corresponding hydrochloride salt by using IPA-HCl in
ethyl acetate as solvent with 0.1g of free base to yield 0.032 g of hydrochloride 6b.
1
Yield: 62.0%; Off-white solid; mp: 114-116°C; H-NMR (300MHz, DMSO-d6):
δ 1.57 (s,
2H), 1.80 (s, 5H), 2.06-2.14 (m, 5H), 2.69 (s, 2H), 3.74 (s, 1H), 4.58 (s, 1H), 7.02 (t, J = 7.2
Hz, 1H), 7.14 (t, J = 7.5 Hz, 1H), 7.38 (d, J = 7.8 Hz, 1H), 7.48 (d, J = 7.5 Hz, 1H), 9.18 (s,
1H), 9.60 (s, 1H), 11.40 (s, 1H); MS (ESI) m/z: 255.1 [M+H]+; HPLC Retention time: 3.736
min., Purity: 97.91%(Method A).
4.1.5.
(6c)
N-(3-methylcyclopentyl)-2,3,4,9-tetrahydro-1H-carbazol-1-aminehydrochloride

ACCEPTED MANUSCRIPT
2,3,4,9-tetrahydro-1H-carbazol-1-amine 5 (0.2 g, 1.074 mmol) was treated with 3-
methylcyclopentanone (0.21 g, 2.148 mmol) in THF (10 mL) in the presence of TFA (9.18
mg, 0.081 mmol) at 25-30°C. After being stirred for 4 h. at 25 – 30 °C added
sodiumcyanoborohydride (0.202 g, 3.22 mmol) and continued stirring for 16 h. The reaction
mixture was diluted with cold water (50mL), extracted with ethyl acetate (2 x 25 mL).
Combined organic layers were washed with water (25mL), brine (25mL), dried over
anhydrous and filtered. The filtrate was concentrated under reduced pressure to get crude
which was purified by silica gel (60/120) column chromatography in PET. Ether: Ethyl
acetate; 7:3 as elute to get title compound (0.168g, 53.16%). Corresponding hydrochloride
salt 6c by using IPA-HCl in ethyl acetate as solvent with 0.1g of free base.
Yield: 53.16%; White solid;137-139°C; 1H-NMR (300MHz, DMSO-d6):
δ 0.97-1.01 (m, 2H),
1.05 (d, J = 6.3 Hz, 3H), 1.30-1.50 (m, 2H), 1.70-2.26 (m, 5H), 2.68-2.70 (m, 2H), 3.70-3.95
(m, 2H), 4.53-4.54 (m, 2H), 7.02 (t, J= 7.5 Hz, 1H), 7.15 (t, J = 7.5 Hz, 1H), 7.39 (d, J = 8.1
Hz, 1H), 7.49 (d, J = 7.5 Hz, 1H), 9.01 (s, 1H), 9.35 (s, 1H), 11.25 (s, 1H); MS (ESI) m/z:
269.1 [M+H]+; HPLC Retention time: 4.447 min., Purity: 98.79%(Method A).
4.1.6. N-(2,3,4,9-tetrahydro-1H-carbazol-1-yl)pyridine-3-sulfonamide (6d)
Pyridine-3-sulfonyl chloride (0.157g, 0.885 mmol) was added under inert atmosphere to a
stirred solution of 2,3,4,9-tetrahydro-1H-carbazol-1-amine 5 (0.15g,0.805 mmol) and
triethylamine (0.168mL,1.207mmol) in dry dichloromethane (10 mL) at 0°C. After being
stirred at 0°C for 0.5h warmed to rt and stirred for 16 h. After the complete disappearance of
starting material on TLC, the reaction mixture was quenched in sat. sodium bicarbonate
solution (10 mL). Layers separated and organic layer was washed with water (10 mL), brine
(10 mL), dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated to
get crude which was purified by column chromatography using n- hexane: ethyl acetate; 7:3
as elute to yield title compound (0.203g, 70.34%).
Yield: 70.34; Off-white solid; mp: 159-161°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.51-1.53
(m, 1H), 1.55-1.70 (m, 2H), 1.75-1.90 (m, 1H), 2.49-2.55 (m, 2H, merged with DMSO),
4.65-4.67 (m, 1H), 6.94 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.2 Hz, 1H), 7.36 (d, J = 8.1 Hz, 2H),
7.65-7.69 (m, 1H), 8.29(d, J = 7.8 Hz, 1H), 8.43 (d, J = 8.4 Hz, 1H), 8.54 (d, J = 4.2 Hz, 1H),
9.07 (s, 1H), 10.66 (s, 1H); MS (ESI) m/z: 328.0[M+H]+; HPLC Retention time: 5.149 min.,
Purity: 96.82%(Method A).
4.1.7. 4-fluoro-N-(2,3,4,9-tetrahydro-1H-carbazol-1-yl)benzenesulfonamide (6e)

ACCEPTED MANUSCRIPT
4-Fluorobenzenesulfonyl chloride (0.171g, 0.885 mmol) was added under inert atmosphere to
a stirred solution of 2,3,4,9-tetrahydro-1H-carbazol-1-amine 5 (0.15g,0.805 mmol) and
triethylamine(0.168mL,1.207mmol) in dry dichloromethane (10 mL) at 0°C. After being
stirred at 0°C for 0.5h reaction mixture was warmed to rt and stirred for 16h. After the
complete disappearance of starting material on TLC, the reaction mixture was quenched in
saturated sodium bicarbonate solution (10 mL). Layers separated and organic layers were
washed with water (10 mL), brine (10 mL), dried over anhydrous sodium sulphate and
filtered. The filtrate was concentrated to get crude which was purified by column
chromatography using n- Hexane: Ethyl acetate; 7:3 as elute to get title compound (0.167g,
55.18%).
Yield: 55.18%; Off-white solid; mp: 171-173°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.48-1.50
(m, 1H), 1.51-1.58 (m, 2H), 1.61-1.90 (m, 1H), 2.49-2.52 (m, 2H, merged with DMSO),
4.57-4.59 (m, 1H), 6.92 (t, J = 7.2 Hz, 1H), 7.04 (t, J = 7.5 Hz, 1H), 7.36-7.38 (m, 2H),
7.47(t, J = 8.7 Hz, 2H), 7.96-7.80 (m, 2H), 8.22 (d, J = 7.2 Hz, 1H), 10.61 (s, 1H); MS (ESI)
m/z: 343.1[M-H]+; HPLC Retention time; 6.406 min., Purity: 97.60%(Method A).
4.1.8. 4-Cyano-N-(2,3,4,9-tetrahydro-1H-carbazol-1-yl)benzenesulfonamide (6f)
4-Cyanobenzenesulfonyl chloride (0.178g, 0.885 mmol) was added under inert atmosphere to
a stirred solution of 2,3,4,9-tetrahydro-1H-carbazol-1-amine 5 (0.15g,0.805 mmol) and
triethylamine (0.168mL,1.207mmol) in dry dichloromethane (10 mL) at 0°C. After being
stirred at 0°C for 0.5h slowly warmed to 25-30°C and stirred for overnight. After the
complete disappearance of starting material on TLC, the reaction mixture was quenched in
saturated sodium bicarbonate solution (10 mL). Layers separated and organic layers were
washed with water (10 mL), brine (10 mL), dried over anhydrous sodium sulphate and
filtered. The filtrate was concentrated to get crude, which was purified by column
chromatography using n- Hexane: Ethyl acetate; 7:3 as elute to get 4-cyano-N-(2,3,4,9-
tetrahydro-1H-carbazol-1-yl)benzenesulfonamide 6f (0.235g, 75.64%).
Yield: 75.64%; White solid; mp: 200-202°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.17-1.24 (m,
1H), 1.49-1.60 (m, 2H), 1.61-1.79 (m, 1H), 2.50-2.54 (m, 2H, merged with DMSO), 4.60 (s,
1H), 6.94 (t, J = 7.2 Hz, 1H), 7.05 (t, J = 7.8 Hz, 1H), 7.36 (d, J = 7.8 Hz, 2H), 8.07 (d, J =
8.1 Hz, 2H), 8.12 (d, J = 8.4 Hz, 2H), 8.49 (s, 1H), 10.65 (s, 1H); MS (ESI) m/z:
352.1[M+H]+; HPLC Retention time: 6.105 min., Purity: 97.24%(Method A).
4.1.9. 3-(2-Phenylhydrazinyl)cyclohex-2-enone (8)

ACCEPTED MANUSCRIPT
Cyclohexane-1,3-dione (20.0 g, 178 mmol) was dissolved in 125 mL of water at 20°C in a
1.0 liter round bottom flask and apparatus was thereafter evacuated twice and decompressed
with nitrogen. A solution of phenylhydrazine (20.06 g, 186 mmol) in 250 mL of water was
allowed to run in via dropping funnel at an internal temperature of 20-25°C within the course
of 1h. Subsequently, the reaction mixture was further stirred for 3 h. The sandy-like
cyclohexane-1,3-dione monophenylhydrazone obtained was filtered off and washed with 200
mL of water. The crude product thus obtained was dried at 60°C under reduced pressure to
get hydrazone 8 (33.5g, 93.0%).
4.1.10. 1,3,4,9-tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] (9)
3-(2-phenylhydrazinyl)cyclohex-2-enone 8 (33 g, 163 mmol) and 4-methylbenzenesulfonic
acid hydrate (37.2 g, 196 mmol) was dissolved in a mixture of toluene (500 mL) and ethylene
glycol (80mL, 1435 mmol). The reaction mixture was refluxed with a dean stark trap for 24
h. Decanted toluene from reaction, washed out the residue with toluene (2 x 50 mL),
combined toluene layers were washed with 5% aq. sodium bicarbonate solution (2 x 100
mL), water (100mL) and with brine (100mL). Dried organic layer over anhydrous sodium
sulphate and filtered. The filtrate was concentrated to a get crude product which was purified
by trituration in 5% ethyl acetate in n-hexane to get title compound (23.5g, 59.7%) as Off-
white solid.
Yield: 59.7%; Off-white solid; 1H-NMR (300MHz, CDCl3):
δ 1.59 (s, 1H), 2.06 (t, J = 6.3
Hz, 2H), 2.38-2.47 (m, 1H), 2.90 (t, J = 6.0 Hz, 2H), 3.99 (s, 4H), 7.09 (t, J = 6.9 Hz, 1H),
7.16 (t, J = 6.6 Hz, 1H), 7.38(d, J = 7.5 Hz, 1H), 7.48(d, J = 7.2 Hz, 1H), 7.71 (s, 1H); MS
(ESI) m/z: 237.0[M+H]+.
4.1.11. 1,3,4,9-Tetrahydro-2H-carbazol-2-one (10)
1,3,4,9-Tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] 9 (23 g, 100 mmol) in methanol (330
mL) was treated with 10% aqueous sulphuric acid (100 mL, 100 mmol) for 6 h. at 25 - 30°C.
Evaporated methanol on rotavapor and the resulting residue was diluted with cold water (100
mL), the reaction mixture was extracted with ethyl acetate (2 x 100 mL), and combined
organic layers were washed with water (100 mL) and brine (100 mL). Dried organic layer
over anhydrous sodium sulphate and filtered. The filtrate was concentrated to get a crude
product which was purified by silica gel (60/120) column chromatography in Pet. Ether:
Ethyl acetate; 1:1 as elute to get title compound 10 (11.0g, 53.3%) as a white solid.

ACCEPTED MANUSCRIPT
Yield: 53.3%; White Solid: 1H-NMR (300MHz, CDCl3):
δ 2.80 (t, J = 6.6 Hz, 2H), 3.13 (t, J
= 6.6 Hz, 2H), 3.68 (s, 2H), 7.15 (t, J = 6.9 Hz, 1H), 7.21 (t, J = 7.2 Hz, 1H), 7.35(d, J = 7.8
Hz, 1H), 7.53(d, J = 7.2 Hz, 1H), 7.82 (s, 1H); MS (ESI) m/z: 186.1[M+H]+.
4.1.12. (E)-1,3,4,9-tetrahydro-2H-carbazol-2-one oxime(11)
Solution of sodium acetate (5.31 g, 64.8 mmol) in water (10 mL) and hydroxyl amine
hydrochloride (2.251 g, 32.4 mmol) in water (5 mL) were added to a stirred solution of 3,4-
dihydro-1H-carbazol-2(9H)-one 10 (3.0 g, 16.20 mmol) in ethanol (50 mL). After being
stirred at precipitated solid was collected by filtration and washed with water, ethanol and
then with n-hexane. Dried the solid under reduced pressure at 60°C to get intermediate oxime
as Off-white solid (9.23g, 70.3%).
Yield: 70.3%; Off-white solid; 1H-NMR (300MHz, CDCl3):
δ 2.68 (t, J = 6.0 Hz, 2H), 2.86
(t, J = 6.0 Hz, 2H), 3.84 (s, 2H), 7.06 (t, J = 6.9 Hz, 1H), 7.12 (t, J = 6.9 Hz, 1H), 7.30(d, J =
7.2 Hz, 1H), 7.45(d, J = 7.2 Hz, 1H), 8.40 (s, 1H), 8.94 (s, 1H); MS (ESI) m/z: 201.2[M+H]+.
4.1.13. 2,3,4,9-Tetrahydro-1H-carbazol-2-amine (12)
Lithium aluminium hydride 1M in THF (32.4 mL, 32.4 mmol) was added to a stirred solution
of (Z)-3,4-dihydro-1H-carbazol-2(9H)-one oxime (2.30 g, 11.39 mmol) in THF (50.0 mL) at
rt and stirred at the same temperature for 0.5 h. The resulting reaction mixture was heated to
reflux for 2 h. Progress of the reaction was monitored by TLC. Cooled reaction mixture to 0-
5°C an excess LiAlH4 was quenched by addition of the sodium sulphate decahydrate, stirred
reaction mixture for 1h., inorganic were removed by filtration through celite bed. The filtrate
was concentrated to get a crude which was purified by silica gel (60/120) column
chromatography in Chloroform: Methanol; 9.5:0.5 as elute to get title compound (0.75g,
22.66%).
Yield: 22.6%; Brown solid; 1H-NMR (300MHz, CDCl3):
δ 2.16-2.56 (m,2H), 2.68-2.88 (m,
2H), 3.04-3.17 (m, 2H), 3.56 (s, 1H), 6.94 (t, J = 6.9 Hz, 1H), 7.02 (t, J = 7.5 Hz, 1H), 7.27(d,
J = 7.8 Hz, 1H), 7.36(d, J = 7.5 Hz, 1H), 8.31 (s, 2H), 10.82 (s, 1H); MS (ESI) m/z:
187.1[M+H]+.
4.1.14. N-cyclohexyl-2,3,4,9-tetrahydro-1H-carbazol-2-amine (13a)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.074 mmol) was treated with
cyclohexanone(0.126 g, 1.289 mmol) in tetrahydrofuran (10 mL) at 25-30°C in the presence

ACCEPTED MANUSCRIPT
of trifluoroacetic acid (0.041 mL, 0.537 mmol) for 4 h. Then added sodiumcyanoborohydride
(0.202 g, 3.22 mmol) and continued stirring at same temperature for overnight. The reaction
mixture was diluted with cold water (50mL), extracted with ethyl acetate (2 x 25 mL),
combined organic layers were washed with water (25mL), brine (25mL), dried over
anhydrous sodium sulphate and filtered. The filtrate was concentrated to get crude which was
purified by silica gel (60/120) column chromatography in dichloromethane: methanol; 9:1 as
elute to get N-cyclohexyl-2,3,4,9-tetrahydro-1H-carbazol-2-amine 13a (0.03g, 10.19%).
1
Yield: 10.19%; White solid; mp: 172-174°C; H-NMR (300MHz, DMSO-d6):
δ 0.80-1.08
(m, 2H), 1.08-1.27 (m, 3H), 1.55-1.62 (m, 2H), 1.66-1.70 (m, 2H), 1.83-1.87 (m, 2H), 1.96-
2.09 (m, 1H), 2.40-2.49 (m, 1H), 2.50-2.73 (m, 3H), 2.92 2.97 (m, 1H), 3.14 (s, 1H), 6.88-
6.99 (m, 2H), 7.22 (d, J = 7.8 Hz, 1H), 7.31 (d, J = 7.2 Hz, 1H), 10.57 (s, 1H); MS (ESI) m/z:
269.1[M+H]+; HPLC Retention time; 3.422 min., Purity: 97.88%(Method A).
4.1.15. N-cyclopentyl-2,3,4,9-tetrahydro-1H-carbazol-2-amine (13b)
2,3,4,9-tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.074 mmol) was treated with
cyclopentanone (0.181 g, 2.148 mmol) in tetrahydrofuran (10 mL) in the presence of TFA
(6.12 mg, 0.054 mmol) at 25-30°C. After being stirred for 4 h. at the same temperature added
sodiumcyanoborohydride (0.202 g, 3.22 mmol) and continued stirring for 16 h. The reaction
mixture was diluted with cold water (50mL), extracted with ethyl acetate (2 x 25 ml),
combined organic layers were washed with water (25ml), brine (25ml) dried over anhydrous
sodium sulphate and filtered. The filtrate was concentrated to get crude. Purification was
done by silica gel (60/120) column chromatography in dichloromethane: methanol; 9:1 as
elute to get title compound 13b (0.134g, 48.5%).
1
Yield: 48.5%; White solid; mp: 180-182°C; H-NMR (300MHz, DMSO-d6):
δ 1.59-1.65 (m,
3H), 1.66-1.74 (m, 3H), 1.84-1.90 (m, 1H), 2.00-2.12 (m, 2H), 2.22-2.28 (m, 1H), 2.62-2.90
(m, 3H), 3.20-3.25 (m, 1H), 3.60 – 3.80 (m, 2H), 3.75 (s, 1H), 6.96 (t, J = 7.2 Hz, 1H), 7.04
(t, J = 7.8 Hz, 1H), 7.29 (d, J = 8.1 Hz, 1H), 7.38 (d, J = 7.5 Hz, 1H), 10.79 (s, 1H); MS
(ESI) m/z: 255. [M+H]+; HPLC retention time: 3.085 min., Purity: 99.28% (Method A).
4.1.16. 2,3,4,9-Tetrahydro-N-(3-methylcyclopentyl)-1H-carbazol-2-amine (13c)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine (0.3 g, 1.611 mmol) was treated with 3-
methylcyclopentanone (0.316 g, 3.22 mmol) in THF (10 mL) in presence of TFA (9.18 mg,
0.081 mmol) at 25-30°C. The reaction mixture stirred for 4 h. at 25 - 30°C. Charged

ACCEPTED MANUSCRIPT
sodiumcyanoborohydride (0.202 g, 3.22 mmol) and stirred at the same temperature for
overnight. The reaction mixture was diluted with cold water (50mL), extracted with ethyl
acetate (2 x 25 mL). Combined organic layers were washed with water (25mL) and brine
(25mL). Dried organic layers over anhydrous sodium sulphate and filtered. The filtrate was
concentrated to get crude product. Purification was done silica gel (60/120) column
chromatography in dichloromethane: methanol; 9:1 as elute to get title compound (0.168g,
38.9%). Hydrochloride salt was prepared in ethyl acetate with 140 mg of base by using
methanolic HCl to get hydrochloride 13c as white solid (0.11g, 57.89%).
1
Yield: 57.89%; White solid; mp: 213-215°C; H-NMR (300MHz, DMSO-d3):
δ 0.97 (d, J =
4.5 Hz, 3H), 1.17-1.34 (m, 1H), 1.32-1.50 (m, 1H), 1.54-1.66 (m, 2H), 1.74-1.88 (m, 2H),
2.01-2.05 (m, 2H), 2.41-2.42 (m, 1H), 2.67 -2.72 (m, 2H), 2.92-2.97 (m, 2H), 3.18-3.24 (m,
1H), 6.90 (t, J = 7.5 Hz, 1H), 6.96 (t, J = 6.9 Hz, 1H), 7.22 (d, J = 7.8 Hz, 1H), 7.30 (d, J =
7.5 Hz, 1H), 10.58 (s, 1H); MS (ESI) m/z: 269.1[M+H]+; HPLC Retention time: 3.481 min.,
Purity: 99.09%(Method A).
4.1.17. 2,3,4,9-tetrahydro-N-(tetrahydro-2H-pyran-4-yl)-1H-carbazol-2-amine (13d)
2,3,4,9-tetrahydro-1H-carbazol-2-amine 12 (0.3 g, 1.611 mmol) was treated with dihydro-
2Hpyran-4(3H)-one (0.242 g, 2.416 mmol) in THF (10 mL) in the presence of TFA (9.18 mg,
0.081mmol) at 25-30°C. The reaction mixture stirred for 4 hrs at 25 - 30°C. Charged
sodiumcyanoborohydride (0.202 g, 3.22 mmol) and stirred at the same temperature for
overnight. The reaction mixture was diluted with cold water (50mL), extracted with ethyl
acetate (2 x 25 mL), combined organic layers were washed with water (25mL) and brine
(25mL). Dried organic layers over anhydrous sodium sulphate and filtered. The filtrate was
concentrated to get which was purified by silica gel (60/120) column chromatography in
dichloromethane: methanol; 9:1 as elute to get title compound 13d (0.132g, 27.5%).
Hydrochloride salt was prepared in ethyl acetate by using methanolic HCl with 100 mg of
base (0.038g, 29.92%).
Yield: 29.92%; Brown solid; mp: 155-157°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.17-1.23
(m, 1H), 1.30-1.69 (m, 2H), 1.70-1.82 (m, 1H), 1.90-1.99 (m, 4H), 2.18-2.27 (m, 2H), 2.55-
2.73 (m, 4H), 3.87-3.90 (m, 2H), 6.95 (t, J = 7.2 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 7.25 (d, J
= 7.8 Hz, 1H), 7.34 (d, J = 7.8 Hz, 1H), 10.72 (s, 1H); MS (ESI) m/z: 271.2[M+H]+; HPLC
Retention time: 2.464 min., Purity: 90.82%(Method A).

ACCEPTED MANUSCRIPT
4.1.18. N-(2,3,4,9-tetrahydro-1H-carbazol-2-yl)pyridine-3-sulfonamide (13e)
2,3,4,9-tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.074 mmol) was treated with pyridine-3-
sulfonyl chloride (0.229 g, 1.289 mmol) in the presence of triethylamine (0.326 g, 3.22
mmol) at 25-30°C for 4 h. The reaction mixture was diluted with chloroform (25 mL),
washed with water (25mL), brine (25mL), dried over anhydrous sodium sulphate and filtered.
The filtrate was concentrated to get a crude product which was purified by silica gel (60/120)
column chromatography in Pet. Ether: Ethyl acetate; 8:2, as elute to get title compound 13e
(0.08g, 21.98%).
Yield: 21.98%; White solid; mp: 168-170°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.61-1.79 (m,
1H), 1.81-1.86 (m, 1H), 2.55-2.63 (m, 2H), 2.64-2.80 (m, 2H), 3.56-3.58 (m, 1H), 6.90 (t, J =
7.2 Hz, 1H), 6.97 (t, J = 6.9 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H), 7.66
(t, J = 7.5 Hz, 1H), 8.17 (d, J = 6.3 Hz, 1H), 8.25 (d, J = 7.8 Hz, 1H), 8.83 (d, J = 4.5 Hz,
1H), 9.12 (s, 1H), 10.59 (s, 1H); MS (ESI) m/z: 328.0[M+H]+; HPLC Retention time: 4.774
min., Purity: 96.58%(Method A).
4.1.20. 4-Fluoro-N-(2,3,4,9-tetrahydro-1H-carbazol-2-yl)benzenesulfonamide (13f)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.074 mmol) was treated with 4-
fluoroobenzene-1-sulfonyl chloride (0.230 g, 1.181 mmol) in dichloromethane (5 mL) in the
presence of triethylamine (0.449 mL, 3.22 mmol) at 25-30°C for 2 h. The progress of the
reaction was monitored by TLC. The reaction mixture was diluted with dichloromethane (25
mL), washed with water (25mL) and brine (25mL). The organic layer was dried over
anhydrous sodium sulphate and filtered. The filtrate was concentrated to a get crude product
which was purified by silica gel (60/120) column chromatography in Pet. Ether: Ethyl
acetate; 8:2, as elute to get title compound 13f (0.118g, 31.6%).
1
Yield: 31.6%; Off-white solid; mp:180-182°C; H-NMR (300MHz, DMSO-d6):
δ 1.20-1.70
(m, 1H), 1.80-1.86 (m, 1H), 2.54-2.62 (m, 2H), 2.68-2.76 (m, 2H), 3.41-3.49 (m, 1H), 6.90 (t,
J = 7.5 Hz, 1H), 6.97 (t, J = 7.2 Hz, 1H), 7.20 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 8.7 Hz, 1H),
7.46 (t, J = 8.7 Hz, 2H), 7.94 (t, J = 8.1 Hz, 3H), 10.59 (s, 1H); MS (ESI) m/z: 367.5[M+H]+;
HPLC Retention time: 5.924 min., Purity: 98.96%(Method A).
4.1.21. 4-Cyano-N-(2,3,4,9-tetrahydro-1H-carbazol-2-yl)benzenesulfonamide (13g)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine (0.2 g, 1.074 mmol) was treated with 4-
cyanobenzene-1-sulfonyl chloride (0.238 g, 1.181 mmol) in dichloromethane (5 mL) in

ACCEPTED MANUSCRIPT
presence of the triethylamine (0.326 g, 3.22 mmol) at 25-30°C for 2 h. The reaction mixture
was diluted with dichloromethane (25 mL), washed with water (25 mL) and brine (25 mL).
The organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was
concentrated to get crude product which was purified by silica gel (60/120) column
chromatography in Pet. Ether: Ethyl acetate; 8:2, as elute to get 4-cyano-N-(2,3,4,9-
tetrahydro-1H-carbazol-2-yl)benzenesulfonamide 13g (0.072g, 18.79%).
Yield: 18.79%; Off-white solid; mp: 93-95°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.61-1.69
(m, 1H), 1.80-1.90 (m, 1H), 2.56-2.61 (m, 2H), 2.68-2.71 (m, 2H), 3.51-3.61 (m, 1H), 6.90 (t,
J = 7.2 Hz, 1H), 6.98 (t, J = 6.9 Hz, 1H), 7.21 (d, J = 7.8 Hz, 1H), 7.30 (d, J = 7.5 Hz, 1H),
8.04 (d, J = 8.4 Hz, 2H), 8.11 (d, J = 8.1 Hz, 2H), 8.23 (s, 1H), 10.58 (s, 1H); MS (ESI) m/z:
352.0[M+H]+; HPLC Retention time: 5.692 min., Purity: 98.47%(Method A).
4.1.22. 3-Fluoro-N-(6,7,8,9-tetrahydro-5H-carbazol-7-yl)benzamide (13h)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.074 mmol) was treated with 3-
fluorobenzoyl chloride (0.204 g, 1.289 mmol) in dichloromethane (5 mL) in the presence of
triethyl amine (0.326 g,3.22 mmol) at 25-30°C for 1 h. The progress of reaction was
monitored by TLC. The reaction mixture was diluted with dichloromethane (25 mL), washed
with water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous sodium
sulphate and filtered. The filtrate was concentrated to get crude which was purified by silica
gel (60/120) column chromatography in Pet. Ether: Ethyl acetate; 8:2, as elute to get 3-fluoro-
N-(6,7,8,9-tetrahydro-5H-carbazol-7-yl)benzamide 13h (0.15g, 39.16%).
Yield: 39.16%; White solid; mp: 172-174 °C; 1H-NMR (300MHz, DMSO-d6):
δ 1.83-1.89
(m, 1H), 2.05-2.19 (m, 1H), 2.60-2.73 (m, 1H), 2.78-2.86 (m, 2H), 3.01-3.09 (m, 1H), 4.31-
4.40 (m, 1H), 6.93 (t, J = 7.2 Hz, 1H), 7.01 (t, J = 6.9 Hz, 1H), 7.26 (d, J = 7.8 Hz, 1H), 7.34-
7.42 (m, 2H), 7.54 (q, J = 7.5 Hz, 1H), 7.68 (s, 1H), 7.74 (t, J = 7.8 Hz, 1H), 8.56 (d, J = 7.2
Hz, 1H), 10.69 (s, 1H); MS (ESI) m/z: 331.1[M+H]+; HPLC Retention time: 5.652 min.,
Purity: 99.09% (Method A).
4.1.23. N-(6,7,8,9-tetrahydro-5H-carbazol-7-yl)-3-methoxybenzamide(13i)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.976 mmol) was treated with 3-
methoxybenzoyl chloride (0.371 g, 2.174 mmol) in Chloroform (5 mL) in the presence of
triethyl amine (1.104 g, 5.93 mmol) at 25-30°C for 1 h. The reaction mixture was diluted
with chloroform (25 mL), washed with water (25mL) and brine (25mL). The organic layer

ACCEPTED MANUSCRIPT
was dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated to get a
crude product which was purified by silica gel (60/120) column chromatography in Pet.
Ether: Ethyl acetate; 8:2, as elute to get title compound 13i (0.135g, 36.98%).
Yield: 36.98%; White solid; mp: 190-192°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.85-1.88 (m,
1H), 2.05-2.19 (m, 1H), 2.60-2.69 (m, 1H), 2.78-2.86 (m, 3H), 3.00-3.07 (m, 1H), 3.82 (s,
3H), 4.30-4.40 (m, 1H), 6.93 (t, J = 7.5 Hz, 1H), 7.00 (t, J = 6.9 Hz, 1H), 7.10 (d, J = 7.5 Hz,
1H), 7.26 (d, J = 7.8 Hz, 1H), 7.34-7.48 (m, 4H), 8.47 (d, J = 7.2 Hz, 1H), 10.68 (s, 1H); MS
(ESI) m/z: 343.1[M+H]+; HPLC Retention time: 5.341 min., Purity: 97.33% (Method A).
4.1.24. 2-Fluoro-N-(6,7,8,9-tetrahydro-5H-carbazol-7-yl)benzamide (13j)
2,3,4,9-Tetrahydro-1H-carbazol-2-amine 12 (0.2 g, 1.074 mmol) was treated with 2-
fluorobenzoyl chloride (0.187 g, 1.181 mmol) in Chloroform (10 mL) in the presence of
triethylamine (0.150 mL, 1.074 mmol) at 25-30°C for 1 h. The reaction mixture was diluted
with chloroform (25 mL), washed with water (25mL) and brine (25mL). The organic layer
was dried over anhydrous sulphate and filtered. The filtrate was concentrated under reduced
pressure to get crude product which was purified by silica gel (60/120) column
chromatography in Pet. Ether: Ethyl acetate; 8:2, as elute to get title compound 13j (0.14g,
39.54%).
Yield: 39.54%; Off-white solid; mp: 168-170°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.10-1.90
(m, 1H), 1.99-2.07 (m, 1H), 2.61-2.72 (m, 1H), 2.73-2.86 (m, 2H), 3.03-3.10 (m, 1H), 4.30-
4.40 (m, 1H), 6.93 (t, J = 7.5 Hz, 1H), 7.00 (t, J = 7.2 Hz, 1H), 7.27 (t, J = 8.7 Hz, 3H), 7.30
(d, J = 7.5 Hz, 1H), 7.51 (t, J = 6.3 Hz, 1H), 7.60 (t, J = 7.2 Hz, 1H), 8.44 (d, J = 7.2 Hz, 1H),
10.67 (s, 1H); MS (ESI) m/z: 331.1[M+H]+; HPLC Retention time: 5.596 min., Purity:
99.47%(Method A).
4.1.25. N-benzyl-2,3,4,9-tetrahydro-1H-carbazol-2-amine (13k)
Concentrated H2SO4 (7.94 mg, 0.081 mmol) was added to a stirred solution of 3,4-dihydro-
1H-carbazol-2(9H)-one 10 (0.3 g, 1.620 mmol) and phenylmethanamine (0.191 g, 1.782
mmol) in THF (2 mL) at 25-30°C. Being stirred at the same temperature for 3 h added
sodium cyanoborohydride (0.153 g, 2.430 mmol) and stirred for another 1.5 h. Diluted
reaction mixture with water (25 mL), extracted with ethyl acetate (2 x 25 mL), combined
organic layers were washed with water (25 mL) and brine (25 mL). The organic layer was
dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated to get crude

ACCEPTED MANUSCRIPT
which was purified by silica gel (60/120) column chromatography in PET. Ether: ethyl
acetate; 1:1 as elute to get N-benzyl-2,3,4,9-tetrahydro-1H-carbazol-2-amine 13k (0.032g,
7.08%).
Yield: 7.08%; Brown solid; 1H-NMR (300MHz, CDCl3):
δ 1.77-1.85 (m, 1H), 2.12-2.25 (m,
1H), 2.61-2.80 (m, 2H), 2.81-2.88 (m, 1H), 3.00-3.10 (m, 1H), 3.15-3.25 (m, 1H), 3.95 (s,
2H), 7.09-7.14 (m, 2H), 7.31-7.37 (m, 5H), 7.47 (d, J = 7.2 Hz, 1H), 7.72-7.74 (m, 1H); MS
(ESI) m/z: 277.2[M+H]+; HPLC Retention time: 3.183 min., Purity: 98.80%(Method B).
4.1.26. N-(4-methylbenzyl)-2,3,4,9-tetrahydro-1H-carbazol-2-amine (13l)
Concentrated H2SO4 (7.94 mg, 0.081 mmol) was added to a stirred solution of 3,4-dihydro-
1H-carbazol-2(9H)-one 10 (0.3 g, 1.620 mmol) and p-tolylmethanamine (0.216 g, 1.782
mmol) in THF (2 mL) at 25-30°C. after being stirred at same temperature for 3 h., added
sodium cyanoborohydride (0.153 g, 2.430 mmol) and stirred for another 1.5 h. Diluted
reaction mixture with water (25 mL), extracted with ethyl acetate (2 x 25 mL), combined
organic layers were washed with water (25mL) and brine (25 mL). The organic layer was
dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated under
reduced pressure to get crude which was purified by silica gel (60/120) column
chromatography in PET. Ether: ethyl acetate; 1:1 as elute to get title compound 13l (0.132g,
28.20%).
Yield: 28.2%; Off-white solid; 1H-NMR (300MHz, DMSO-d6):
δ 1.50-1.68 (m, 1H), 2.01-
2.11 (m, 2H), 2.27 (s, 3H), 2.66-2.80 (m, 2H), 2.90-3.00 (m, 2H), 3.78 (s, 2H), 6.89 (t, J = 7.5
Hz, 1H), 6.94 (t, J = 7.5 Hz, 1H), 7.11 (d, J = 7.8 Hz, 2H), 7.21 (d, J = 9.0 Hz, 1H), 7.26 (d, J
13
= 7.8 Hz, 2H), 7.30 (d, J = 9.0 Hz, 1H), 10.62 (s, 1H); C-NMR(100 MHz, DMSO-d6 ): δ
19.55, 21.16, 29.93, 30.58, 50.52, 53.40, 108.17, 111.01, 117.61, 118.46, 121.41, 127.39,
128.36(2C), 129.18(2C), 133.93, 135.82, 136.49, 138.64; MS (ESI) m/z: 291.2[M+H]+;
HPLC Retention time: 3.253 min., Purity: 98.03%(Method A).
4.1.27. 3-(2-p-Tolylhydrazinyl)cyclohex-2-enone (8a)
p-Tolylhydrazine (11.33 g, 93 mmol) was added to a stirred solution of cyclohexane-1,3-
dione(10 g, 89 mmol) in water (300 mL) at 15-20°C and resulting reaction mixture was
stirred at the same temperature for 1h. Precipitated solid was collected by filtration. Washed
solid with water and Pet. Ether, dried under reduced pressure in rotating flask at 50°C to get
title compound (16.0g, 83.0%).

ACCEPTED MANUSCRIPT
4.1.28. 6-Methyl-1,3,4,9-tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] (9a)
To a stirred suspension of 3-(2-(p-tolyl)hydrazinyl)cyclohex-2-enone 8a (15 g, 69.4 mmol) in
toluene (300 mL) charged 4-methylbenzenesulfonic acid hydrate (15.83 g, 83 mmol) and
ethane-1,2-diol (37.9 g, 610 mmol). The resulting mixture was refluxed with a dean stark trap
for 24 h. Decanted toluene from reaction, washed out the residue with toluene (2 x 50mL),
combined toluene layers were washed with 5% aq. sodium bicarbonate solution (2 x 100mL),
water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium
sulphate and filtered. The filtrate was concentrated under reduced pressure to get a crude
product which was purified by trituration in 5% ethyl acetate in n-hexane to get title
compound 9a (9.2g, 54.1%) as Off-white solid.
Yield: 54.1%; Off-white solid; 1H-NMR (300MHz, DMSO-d6):
δ 1.89 (t, J = 6.0 Hz, 2H),
2.34(s, 3H), 2.67 (t, J = 5.7 Hz, 2H), 2.87 (s, 2H), 3.95 (s, 4H), 6.80 (d, J = 8.1 Hz, 1H), 7.11-
7.13 (m, 2H), 10.49(s, 1H); MS (ESI) m/z: 244.1[M+H]+; HPLC Retention time: 4.815 min.,
Purity: 99.23%(Method A).
4.1.29. 6-Methyl-1,3,4,9-tetrahydro-2H-carbazol-2-one (10a)
6-Methyl-1,3,4,9-tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] 9a (3.0 g, 12.33 mmol) in
methanol (330 mL) was treated with concentrated hydrochloric acid (5 mL) for overnight at
25 - 30°C. Evaporated methanol on rotavapor, residue was diluted with cold water (100 mL)
and extracted with ethyl acetate (2 x 100mL). Combined organic layers were washed with
water (100 mL) and brine (100 mL). The organic layer was dried over anhydrous sodium
sulphate and filtered. The filtrate was concentrated under reduced pressure to get crude
product which was purified by silica gel (60/120) column chromatography in Pet. Ether:
Ethyl acetate; 1:1 as elute to get title compound 10a (1.3g, 53.06).
Yield: 53.06%; White solid; 1H-NMR (300MHz, DMSO-d6):
δ 2.36(s, 3H), 2.66 (t, J = 6.3
Hz, 2H), 2.92 (t, J = 6.0 Hz, 2H), 3.61 (s, 2H), 6.86 (d, J = 7.8 Hz, 1H), 7.16-7.19 (m, 2H),
10.67(s, 1H); MS (ESI) m/z: 200.1[M+H]+; HPLC Retention time: 4.196 min., Purity:
99.58%(Method A).
4.1.30. (Z)-6-methyl-1,3,4,9-tetrahydro-2H-carbazol-2-one oxime(11a)
To a stirred solution of 6-methyl-3,4-dihydro-1H-carbazol-2(9H)-one 10a (0.52 g, 2.61
mmol) in Ethanol (20 mL) charged solution of sodium acetate (0.856 g, 10.44 mmol) in
water (1 mL) and hydroxylamine hydrochloride (0.363 g, 5.22 mmol) in water (1 mL). The

ACCEPTED MANUSCRIPT
resulting reaction mixture was stirred at 25 -30°C for 0.5h. Precipitated solid was collected
by filtration, washed with water and ethanol and then with n-hexane. Dried solid under
vacuum at 60°C to get oxime 11a (0.55g, 98.38%).
4.1.31. 6-methyl-2,3,4,9-tetrahydro-1H-carbazol-2-amine (12a)
Lithium aluminium hydride (2.61 mL, 5.22 mmol) 1M in THF was added to a stirred solution
of (Z)-6-methyl-3,4-dihydro-1H-carbazol-2(9H)-one oxime 11a (0.55g, 2.56 mmol) in
THF(50 mL) at 25-30°C and stirred for 0.5h. Then heated resulting reaction mixture to
reflux for 2 h. Progress of the reaction was monitored by TLC. Cooled reaction mixture to 0-
5°C and quenched an excess of LiAlH4 with sodium sulphate decahydrate. Stirred reaction
mixture for 1h.at 25-30°C and inorganic were removed by filtration through celite bed.
Evaporated filtrate to get crude product which was purified by silica gel (60/120) column
chromatography using chloroform: Methanol; 9.5:0.5as elute to get 6-methyl-2,3,4,9-
tetrahydro-1H-carbazol-2-amine 12a (0.45g, 86.0%).
1
Yield: 86.0%; Brown solid; H-NMR (300MHz, DMSO-d6):
δ 1.35(s, 1H), 1.48-1.57 (m,
1H), 1.65-1.75 (m, 1H), 1.90-1.94 (m, 1H), 2.33 (s, 3H), 2.38-2.41(m, 1H), 2.55-2.70 (m,
2H), 2.82-2.89 (m, 1H), 3.05-3.12 (m, 1H), 6.78 (d, J = 8.1 Hz, 1H), 7.08-7.10 (m, 2H),
10.43(s, 1H); MS (ESI) m/z: 201.1[M+H]+; HPLC Retention time: 2.159 min., Purity:
94.55%(Method A).
4.1.32. 6-methyl-N-(3-methylcyclopentyl)-2,3,4,9-tetrahydro-1H-carbazol-2-amine (13m)
2,2,2-Trifluoroacetic acid (5.69 mg, 0.050 mmol) was added to a stirred solution 6-methyl-
2,3,4,9-tetrahydro-1Hcarbazol-2-amine 12a (0.2g, 0.999mmol) and 3-methylcyclopentanone
(0.118 g, 1.198 mmol) in THF ( 5 mL) and stirred for 1 h. Charged sodium
cyanotrihydroborate (0.126 g, 1.997 mmol) and the resulting reaction mixture was stirred
overnight. Diluted reaction mixture with water (25 mL), extracted with ethyl acetate (2 x 25
mL), combined organic layers were washed with water (25 mL) and brine (25 mL). The
organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was
concentrated under reduced pressure to get crude product which was purified by silica gel
(60/120) column chromatography in dichloromethane: methanol; 9:1 as elute to get title
compound 13m (0.15g, 53.1%).
1
Yield: 53.1%; White solid; mp: 214-216°C; H-NMR (300MHz, CDCl3):
δ 0.87-0.99 (m,
1H), 1.03 (d, J = 6.3 Hz, 3H), 1.26-1.36 (m, 2H), 1.43-1.47 (m, 2H), 1.72-1.81 (m, 2H), 1.89-

ACCEPTED MANUSCRIPT
2.00 (m, 2H), 2.09-2.18 (m, 2H), 2.44 (s, 3H), 2.54-2.62 (m, 1H), 2.71-2.74 (m, 1H), 2.78-
2.84 (m, 1H), 2.98-3.03 (m, 1H), 3.16 (s, 1H), 3.31 -3.45 (m, 1H), 6.95 (d, J = 7.8 Hz, 1H),
7.18 (d, J = 8.1 Hz, 1H), 7.27 (s, 1H), 7.60 (s, 1H); MS (ESI) m/z: 283.1[M+H]+; HPLC
Retention time: 3.221 min., Purity: 99.87%(Method A).
4.1.33. N-benzyl-2,3,4,9-tetrahydro-6-methyl-1H-carbazol-2-amine hydrochloride (13n)
2,2,2-Trifluoroacetic acid (4.27 mg, 0.037 mmol) was added to a stirred solution of 6-methyl-
2,3,4,9-tetrahydro-1H-carbazol-2-amine 12a (0.15 g, 0.749 mmol) and benzaldehyde (0.095
g, 0.899 mmol) After being stirred at rt for 1 h added sodium cyanotrihydroborate (0.094 g,
1.498 mmol) and continued stirring overnight at same temperature. Diluted reaction mixture
with water (25 mL), extracted with ethyl acetate (2x25 mL), combined organic layers were
washed with water (25 mL) and brine (25 mL). The organic layer was dried over anhydrous
sodium sulphate and filtered. The filtrate was concentrated under reduced pressure to get
crude product which was purified by making hydrochloride salt 13n (0.09g, 36.8%) by using
IPA-HCl in ethyl acetate.
Yield: 36.8%; Off-white solid; mp: 208-210°C; 1H-NMR (300MHz, DMSO-d6):
δ 1.91-1.98
(m, 1H), 2.34 (s, 3H), 2.40-2.44 (m, 1H), 2.55-2.65 (m, 1H), 2.78-2.83 (m, 1H), 3.01-3.04
(m, 1H), 3.22-3.28 (m, 1H), 3.56 (s, 1H), 4.29 (s, 2H), 6.84 (d, J = 8.1 Hz, 1H), 7.15 (d, J =
7.8 Hz, 2H), 7.44-7.63 (m, 5H), 9.40 (s, 1H), 9.59 (s, 1H), 10.68 (s, 1H); 13C-NMR(100
MHz, CDCl3): δ 19.18, 21.68, 25.98, 26.48, 48.03, 54.41, 107.47, 110.99, 117.69, 122.60,
127.15, 127.18, 129.11(2C), 129.32, 130.62(2C), 131.09, 132.75, 135.04; MS (ESI) m/z:
291.1[M+H]+; HPLC Retention time: 3.193 min., Purity: 98.79%(Method A).
4.1.34.
6-methyl-N-(4-methylbenzyl)-2,3,4,9-tetrahydro-1H-carbazol-2-amine
hydrochloride (13o)
2,2,2-trifluoroacetic acid (4.27 mg, 0.037 mmol) was added to a stirred solution of 6-methyl-
2,3,4,9-tetrahydro-1H-carbazol-2-amine 12a (0.15 g, 0.749 mmol) and 4-
methylbenzaldehyde (0.108g, 0.899 mmol) in tetrahydrofuran (5 mL) After being stirred for
stirred for 1h.at rt. Added sodium cyanotrihydroborate (0.094 g, 1.498 mmol) and continued
stirring for overnight. Diluted reaction mixture with water (25 mL), extracted with ethyl
acetate (2 x 25 mL), combined organic layers were washed with water (25 mL) and brine (25
mL). Organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was
concentrated under reduced pressure to get crude product which was purified by silica gel
column chromatography in Pet. Ether:Ethyl acetate; 8:2 as elute to get 6-methyl-N-(4-

ACCEPTED MANUSCRIPT
methylbenzyl)-2,3,4,9-tetrahydro-1H-carbazol-2-amine
which
was
converted
to
corresponding hydrochloride 13o (0108g, 36.8%) by using IPA-HCl and ethyl acetate as
solvent.
Yield: 36.8%; Off-white solid; mp: 210-212 °C; 1H-NMR (300MHz, DMSO-d6):
δ 1.91-1.92
(m, 1H), 2.32-2.33(m, 2H), 2.34 (s, 3H), 2.50-2.60 (m, 1H), 2.77-2.83 (m, 1H), 2.95-2.99 (m,
1H), 3.20-3.33 (m, 1H), 3.53 (s, 1H), 4.23 (s, 2H), 6.84 (d, J = 8.1 Hz, 1H), 7.15 (d, J = 8.1
Hz, 2H), 7.26 (d, J = 7.2 Hz, 2H), 7.49 (d, J = 7.2 Hz, 2H), 7.26-7.38 (bs, 2H), 10.62 (s, 1H);
13C-NMR(100 MHz, DMSO-d6): δ 19.18, 21.28, 21.70, 25.98, 26.46, 47.72, 54.16, 107.46,
111.00, 117.69, 122.60, 127.14, 129.64(3C), 130.59(3C), 131.1, 135.02, 138.75; MS (ESI)
m/z: 305.1[M+H]+; HPLC Retention time: 3.551 min., Purity: 93.05%(Method A).
4.1.35. 9-Methyl-1,3,4,9-tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] (14)
Sodium hydride (0.698 g, 17.45 mmol) was added to a stirred solution of 1,3,4,9-
tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] 9 (2.0 g, 8.72 mmol) in THF (50 mL) at 0-
5°C. Stirred reaction mixture for 10 min. and added slowly iodomethane (1.636 mL, 26.2
mmol). After being stirred at same temperature for 1 h stirred reaction mixture overnight at
25 – 30 °C. Quenched an excess of sodium hydride with methanol, evaporated solvent on
rotavapor to thick residue which was diluted with water (50 mL), extracted with ethyl acetate
(2 x 50mL). Combined organic layers were washed brine (50 mL), dried over anhydrous
sodium sulphate and filtered. The filtrate was concentrated under reduced pressure to get
crude product which was purified by silica gel (60/120) column chromatography in Pet.
Ether: Ethyl acetate; 1:1 as elute to get title compound 14 (1.7g, 80.0%).
Yield: 80.0%; Viscous Oil; 1H-NMR (300MHz, CDCl3):
δ 2.05 (t, J = 6.3 Hz, 2H), 2.91 (t, J
= 6.3 Hz, 2H), 3.00 (s, 2H), 3.62 (s, 3H), 4.09 (s, 4H), 7.08 (t, J = 6.9 Hz, 1H), 7.17 (t, J = 6.6
Hz, 1H), 7.26(d, J = 9.3 Hz, 1H), 7.49(d, J = 7.8 Hz, 1H); MS (ESI) m/z: 252.1[M+H]+.
4.1.36. 9-Methyl-1,3,4,9-tetrahydro-2H-carbazol-2-one (15)
To a stirred suspension of 9-methyl-1,3,4,9-tetrahydrospiro[carbazole-2,2'-[1,3]dioxolane] 14
(1.6g, 6.58 mmol) in methanol (30 mL) charged slowly 10% aqueous sulphuric acid (20 mL)
at 25 - 30°C and stirred resulting reaction mixture at same temperature for 30 h. Diluted
reaction mixture with water(150 mL) and extracted with ethyl acetate(2 x 100mL). Combined
organic layers were washed with water (100 mL) and brine (100 mL). The organic layer was
dried over anhydrous sodium sulphate and filtered. The filtrate was concentrated under
reduced pressure to get crude product which was purified silica gel (60/120) column

ACCEPTED MANUSCRIPT
chromatography in Pet. Ether: Ethyl acetate; 1:1 as elute to get 9-methyl-1,3,4,9-tetrahydro-
2H-carbazol-2-one 15 (0.4g, 30.5%).
Yield: 30.5%;1H-NMR (300MHz, CDCl3):
δ 2.80 (t, J = 6.3 Hz, 2H), 3.13 (t, J = 6.6 Hz,
2H), 3.63 (s, 2H), 3.64 (s, 3H), 7.15 (t, J = 6.9 Hz, 1H), 7.24 (t, J = 7.8 Hz, 1H), 7.32(d, J =
8.1 Hz, 1H), 7.53(d, J = 7.8 Hz, 1H); MS (ESI) m/z: 200.1[M+H]+.
4.1.37.
9-Methyl-N-(4-methylbenzyl)-2,3,4,9-tetrahydro-1H-carbazol-2-amine
hydrochloride 16)
Mixture of p-Tolylmethanamine (0.182 g, 1.506 mmol) and 9-methyl-3,4-dihydro-1H-
carbazol-2(9H)-one 15(0.3 g, 1.506 mmol) was heated to 100 °C in preheated oil bath for 30
min. Then resulting reaction mixture was cooled to 25-30°C and diluted with THF (10 mL)
followed by added sodium cyanoborohydride (0.189 g, 3.01 mmol) and continued stirring for
overnight at rt. Diluted reaction mixture with water(25 mL), extracted with ethyl acetate
(2x25 mL), combined organic layers were washed with water(25mL) and brine(25mL). The
organic layer was dried over anhydrous sodium sulphate and filtered. The filtrate was
concentrated under reduced pressure to get crude which was purified by silica gel column
chromatography in Chloroform: Methanol; 9:1 as elute to get base which was converted to
corresponding hydrochloride 16 (006g, 13.6%) by using IPA-HCl and ethyl acetate as
solvent.
1
Yield: 13.6%; White solid; mp: 222-224 °C; H-NMR (300MHz, DMSO-d6):
δ
1.86-1.99 (m,
1H), 2.33 (s, 3H), 2.40-2.50(m, 1H), 2.60 -2.73 (m, 1H), 2.83-2.99 (m, 3H), 3.55-3.56 (m,
1H), 3.65 (s, 3H), 4.21-4.39 (m, 2H), 6.99 (t, J = 7.2 Hz, 1H), 7.10 (t, J = 7.8 Hz, 1H), 7.27
(d, J = 7.8 Hz, 2H), 7.39 (d, J = 8.4 Hz, 2H), 7.51 (d, J = 7.8 Hz, 2H), 9.28 (s, H), 9.44 (s,
1H); 13C-NMR (100MHz, DMSO-D6): δ 19.15, 21.28, 25.12, 26.14, 29.53, 47.80, 54.13,
107.87, 109.65, 118.11, 119.06, 121.22, 126.36, 129.62, 129.69(2C), 130.59(2C), 132.51,
137.49, 138.84; MS (ESI) m/z: 305.8[M+H]+; HPLC Retention time: 6.156 min., Purity:
99.68%(Method A).
4.2. Biological Assay
4.2.1. pERK assay: Briefly, 25,000 MDAMB231 cells were grown in 96 well transparent
plate and kept in CO2 incubator for 24 h.. After 24 h. cells were serum starved for 6 h. and
later treated with 10µM concentration of drug for 1 h., after treatment cells were fixed with
3.7% formaldehyde and permeabilized with 0.1% triton X, then the cells were stained with P-

ACCEPTED MANUSCRIPT
p44/p42 MAPK (T202/Y204) antibody and secondary ECLTM Anti-Rabbit IgG antibody.
Color was developed with TMB substrate and read on SpectraMax.
4.2.2. pRB assay: Briefly, 20,000 MDAMB231 cells were grown in black 96-well
transparent bottom plates and kept in CO2 incubator for 24 h. After incubation the cells were
treated with 10µM concentration drug for 16 h.. and treated cells were fixed with 3.7%
formaldehyde and permeabilized with 0.1% triton X, then the cells were stained with anti-
pRb antibody and secondary Dylight 549 antibodies. Cells were fluorescently stained and
analyzed using the Cellomics ArrayScan HCS Reader (Cellomics/Thermo Fisher). Cell nuclei
were stained with Hoechst 33342 to identify individual cells and to optimize focusing.
4.3. Molecular Docking
4.3.1. Software. All the molecular modeling studies described herein were performed using
Small-Molecule Drug Discovery Suite 2015-4[31] and the modules included therein for
performing various molecular modeling operations are described below.
4.3.2. Protein Structure. The crystal structure of ERK2 kinase, retrieved from the Protein
Data Bank (PDB ID 5BVD [30]), was subjected to Protein Preparation wizard using default
settings as implemented in Schrödinger suite 2015-4 [32]. The termini were capped, missing
side chains were added using Prime version 4.2 and all the water molecules were deleted. H-
bonds were optimized and in the end, restrained minimization was performed wherein the
heavy atoms were converged to root mean square deviation (RMSD) 0.3 Å. The prepared
protein was then subjected to further minimization using Prime 4.2 (default settings) [33, 34].
Glide version 6.9[35], as implemented in Schrödinger suite 2015-4, was used for performing
docking studies of few select molecules 1 (Figure 1), 13l and 13o (Table 2) and 16 (Figure 2).
4.3.3. Ligand Structures. The ligand structures were built in Maestro 10.4. All the structures
were then optimized using the LigPrep (version 2.6) module, as implemented in Schrödinger
suite 2015-4 using the default settings. This set of molecules was then subjected to the
docking studies. The aim of the study was to investigate the binding mode of few synthesized
molecules in the binding site of the ERK2 kinase.

ACCEPTED MANUSCRIPT
4.3.4. Receptor Grid Generation. The refined protein structure was used to define the active
site using Receptor Grid Generation module. The native ligand present in the target structure
(compound 17, Figure 3) was used for placing the enclosing box. The grid was then used for
docking the ligands in the ligand-binding site.
For docking studies, Extra Precision (XP) (Ref. 35a) mode was used. The docked poses were
minimized and RMSD to input ligand geometries were calculated. Table 3 lists the docking
scores for all the molecules used in the docking studies. Figures 4-6 show the docking poses
of representative molecules into the binding cavity of the ERK2 kinase. After docking
studies, the pose viewer file was then used as input for Prime-MMGBSA calculations. The
MMGBSA binding energies of the top-ranked pose for each molecule are given in Table 3.
5. Acknowledgements
The support received from the analytical department of Piramal Enterprises Ltd.
Goregaon, Mumbai is gratefully acknowledged. Authors are also thankful to
Schrödinger Inc. for providing the Demo license of Schrödinger Suite and especially
Craig Coel for the valuable support that has helped in this work.
References
1. G. Housman, S. Byler, S. Heerboth, K. Lapinska, M. Longacre, N. Snyder, S. Sarkar,
Drug Resistance in Cancer: An Overview, Cancers 6 (2014) 1769-1792.
2. C. Holohan, S. V. Schaeybroeck, D. B. Longley and P. G. Johnston, Cancer drug
resistance: an evolving paradigm, Nat. Rev. Cancer 13 (2013) 714-726.
3. S. Faivre, S. Djelloul, E. Raymond, New Paradigms in Anticancer Therapy: Targeting
Multiple Signaling Pathways With Kinase Inhibitors, Semin. Oncol. 33 (2006) 407-
420.
4. M. D. Garrett, Cell cycle control and cancer, Curr. Sci. 81 (2001) 515-522.
5. E. K. Kim and E. J. Choi, Pathological roles of MAPK signaling pathways in human
diseases, Biochim. Biophys. Acta. 1802 (2010) 396-405.
6. M. D. Thakur, D. D. Stuart, Molecular Pathways: Response and Resistance to BRAF
and MEK Inhibitors in BRAFV600E Tumors, Clin. Cancer Res. 20 (2013) 1074-
1080.
7. C.H. Wong and C. Y. Cheng, Mitogen-activated protein kinases, adherens junction
dynamics, and spermatogenesis: A review of recent data, Dev. Biol. 186 (2005) 1-15.

ACCEPTED MANUSCRIPT
8. D. T. Leicht, V. Balan, A. Kaplun, V. Singh-Gupta, L. Kaplun, M. Dobson, G.
Tzivion, Raf kinases: Function, regulation and role in human cancer, Biochim.
Biophys. Acta 1773 (2007) 1196-1212.
9. R. Roskoski Jr., ERK1/2 MAP kinases: Structure, function, and regulation,
Pharmacol. Res. 66 (2012) 105-143.
10. S. M. Wilhelm, C. Carter, L. Tang, D. Wilkie, A. McNabola, Hong Rong, C. Chen, X.
Zhang, P. Vincent, M. McHugh, Y. Cao, J. Shujath, S. Gawlak, D. Eveleigh, B.
Rowley, L. Liu, L. Adnane, M. Lynch, D. Auclair, I. Taylor, R. Gedrich, A.
Voznesensky, B. Riedl, L., E. Post, G. Bollag, P. A. Trail, BAY 439006 Exhibits
Broad Spectrum Oral Antitumor Activity and Targets the RAF/MEK/ERK Pathway
and Receptor Tyrosine Kinases Involved in Tumor Progression and Angiogenesis,
Can. Res. 64 (2004) 7099-7109.
11. E. B. Haura, A. D. Ricart, T. G. Larson, P. J. Stella, L. Bazhenova, V. A. Miller, R. B.
Cohen, P. B. Eisenberg, P. Selaru, K. D. Wilner, S. M. Gadgeel, A Phase II Study of
PD-0325901, an Oral MEK Inhibitor, in Previously Treated Patients with Advanced
Non–Small Cell Lung Cancer, Clin. Cancer Res. 16 (2010) 2450-2457.
12. F. Catalanotti, D. B. Solit, M. P. Pulitzer, M. F. Berger, S. N. Scott, T. Iyriboz, M. E.
Lacouture, K. S. Panageas, J. D. Wolchok, R. D. Carvajal, G. K. Schwartz, N. Rosen,
P. B. Chapman, Phase II Trial of MEK Inhibitor Selumetinib (AZD6244, ARRY-
142886) in Patients with BRAFV600E/K-Mutated Melanoma, Clin. Cancer Res.19
(2013) 2257-2264.
13. R. A. Weinberg, The Retinoblastoma Protein and Cell Cycle Control, Cell 81 (1995)
323-330.
14. N. Nath, S. Wang, V. Betts, E. Knudsen, S. Chellappan, Apoptotic and mitogenic
stimuli inactivate Rb by differential utilization of p38 and cyclin-dependent kinases,
Oncogene 22 (2003) 5986-5994.
15. C. D. Davis, N. J. Emenaker, J. A. Milner, Cellular Proliferation, Apoptosis and
Angiogenesis: Molecular Targets for Nutritional Pre-emption of Cancer, Semin.
Oncol. 37 (2010) 243-257.
16. B. Popov, N. Petrov, pRb-E2F signaling in life of mesenchymal stem cells: Cell cycle,
cell fate, and cell differentiation, Genes & Diseases 1 (2014) 174-187.
17. E. S. Knudsen, J. Y. J. Wang, Targeting the RB-Pathway in Cancer Therapy, Clin.
Cancer Res. 16 (2010) 1094-1103.