Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor

Article abstract of DOI:10.1016/j.ejmech.2018.08.077

The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and replication in host cells and is a promising drug target. Through an enzymatic screen of the National Institute of Health clinical compound library, we report the discovery of zafirlukast, an FDA approved treatment for asthma, as an inhibitor for the WNV NS2B-NS3 protease. Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an IC₅₀ value of 32 μM. A structure activity relationship study of zafirlukast revealed the cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3 protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an IC₅₀ of 22 μM. Experimental and computational docking analysis support the inhibition model of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the NS2B cofactor from binding, resulting in protease inhibition.

Full text of DOI:10.1016/j.ejmech.2018.08.077

Accepted Manuscript
Breathing new life into West Nile virus therapeutics; discovery and study of zafirlukast
as an NS2B-NS3 protease inhibitor
Anastasia A. Martinez, Bianca A. Espinosa, Rebecca N. Adamek, Brent A. Thomas,
Jennifer Chau, Edwardo Gonzalez, Niroshika Keppetipola, Nicholas T. Salzameda
PII:
S0223-5234(18)30749-9
10.1016/j.ejmech.2018.08.077
EJMECH 10688
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 3 May 2018
Revised Date: 6 August 2018
Accepted Date: 27 August 2018
Please cite this article as: A.A. Martinez, B.A. Espinosa, R.N. Adamek, B.A. Thomas, J. Chau, E.
Gonzalez, N. Keppetipola, N.T. Salzameda, Breathing new life into West Nile virus therapeutics;
discovery and study of zafirlukast as an NS2B-NS3 protease inhibitor, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.08.077.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Breathing new life into West Nile virus therapeutics; discovery and study
of zafirlukast as an NS2B-NS3 protease inhibitor
Anastasia A. Martinez, Bianca A. Espinosa, Rebecca N. Adamek, Brent A. Thomas, Jennifer
Chau, Edwardo Gonzalez, Niroshika Keppetipola, Nicholas T. Salzameda*
Department of Chemistry & Biochemistry, California State University, Fullerton, 800 N. State
College, Fullerton, CA, 92831, USA
*Corresponding author. Tel.: +001-657-278-3594; fax: +001-657-278-5316; e-mail: nsalzameda@fullerton.edu
ABSTRACT
The West Nile virus (WNV) has spread throughout the world causing neuroinvasive diseases
with no treatments available. The viral NS2B-NS3 protease is essential for WNV survival and
replication in host cells and is a promising drug target. Through an enzymatic screen of the
National Institute of Health clinical compound library, we report the discovery of zafirlukast, an
FDA approved treatment for asthma as an inhibitor for the WNV NS2B-NS3 protease.
Zafirlukast was determined to inhibit the protease through a mixed mode mechanism with an
IC50 value of 32 µM. A structure activity relationship study of zafirlukast revealed the
cyclopentyl carbamate and N-aryl sulfonamide as structural elements crucial for NS2B-NS3
protease inhibition. Replacing the cyclopentyl with a phenyl improved inhibition, resulting in an
IC50 of 22 µM. Experimental and computational docking analysis support the inhibition model
of zafirlukast and analogs binding at an allosteric site on the NS3 protein, thereby disrupting the
NS2B cofactor from binding, resulting in protease inhibition.
Keywords: West Nile virus, NS2B-NS3 protease, Zafirlukast, Allosteric inhibitor, Serine
protease
1. Introduction
The West Nile virus (WNV) is a species of the Flaviviridae family and has been a global
epidemic since the 1990’s. The virus’ mode of transmission is between birds and mosquitoes
with humans as accidental hosts via mosquito blood meals.[1] Most cases of WNV infections
are asymptomatic, but 20% of infections can manifest into mild symptoms of fever and
headaches.[2, 3] In serious cases, infections progress into neurological diseases such as

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meningitis and encephalitis that can be fatal.[2, 3] There is currently no treatment or cure for
WNV infections.
The WNV is an enveloped single-stranded, positive-sense RNA virus. The 11 kb genome is
transcribed into a single viral polyprotein that contains three structural and seven nonstructural
proteins required for WNV replication from host cell to host cell [1]. The structural proteins are
the envelope, capsid, and premembrane, while the nonstructural (NS) proteins are designated
NS1 to NS5 [4, 5]. The viral polyprotein is processed by host cell furin and the WNV NS2B-
NS3 protease into the individual structural and nonstructural proteins needed for viral replication
in the endoplasmic reticulum (ER) [6].
From a therapeutic standpoint, the NS3 protein is of interest due to its required functions for
WNV infection and proliferation. The NS3 protein can function as a 5’-triphosphatase,
nucleoside triphosphatase, RNA helicase, and serine protease, which are all important for viral
replication [1]. The serine protease activity of the NS3 protein requires the NS2B protein as a
cofactor [7]. The NS2B protein has three hydrophobic and one hydrophilic domain [8]. The
hydrophobic domains are integrated into the ER membrane, while the hydrophilic domain non-
covalently binds to the NS3 protein to form the active serine protease complex [1, 8]. The WNV
NS2B-NS3 protease active site has the typical serine protease catalytic triad comprised of His51,
Asp75, and Ser135 near the N-terminus of the NS3 protein [9, 10]. The WNV NS2B-NS3
protease is known to cleave the viral polyprotein at basic residues, notably arginine and lysine
[10].
The NS2B-NS3 protease is a thoroughly studied target for pharmaceutical treatment of WNV
infections, due to the essential role of the protease in viral replication [11]. It has been shown
that mutating the NS3 protease into an inactive enzyme is deleterious for further WNV infection

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[12]. Small molecules featuring a variety of scaffolds, [13-15] along with peptides[16-21] have
been investigated as WNV NS2B-NS3 protease inhibitors.
The National Institute of Health clinical compound library was screened in search of NS2B-
NS3 protease inhibitors. The library screen identified Zafirlukast (Figure 1) as a promising
candidate for NS2B-NS3 protease inhibition.
Figure 1. Zafirlukast is a WNV NS2B-NS3 protease inhibitor.
Zafirlukast commonly known as Accolate is an FDA approved orally administered drug for
the effective inhibition of smooth muscle airway inflammation for the treatment of asthma [22].
It has also been reported to have antibacterial activity against two oral infections,
Porphyromonas gingivalis and Streptococcus mutants [23]. Zafirlukast contains both a toluic
acid-N-aryl sulfonamide and cyclopentyl carbamate domains linked via an indole. The large
molecular size of zafirlukast provided an ideal starting point for structure activity relationship
(SAR) studies to probe WNV NS2B-NS3 protease inhibitor activity. Here, we report the
inhibitor kinetic study of zafirlukast and the initial SAR study for NS2B-NS3 protease inhibition.
In addition, computational docking analysis identified a possible binding site for zafirlukast on
the NS3 protein.
2. Results and Discussion
2.1
Inhibitor validation and Enzyme inhibitor kinetic study

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The NS2B-NS3 protease activity was measured by a fluorescence resonance energy transfer
(FRET) enzymatic assay. The assay involves a synthetic reporter peptide that contains both
fluorophore and quencher molecules attached to amino acids located across the peptide cleave
site [24]. As the WNV NS2B-NS3 cleaves the synthetic peptide an increase in fluorescence
from the fluorophore is measured and is proportional to enzymatic activity [24]. The assay was
used to identify the inhibitor, validate inhibition, determine the IC50 (inhibitor concentration that
lower enzymatic activity by 50 %)
value and mode of inhibition. The
IC50 value of zafirlukast was
determined to be 32 µM for
NS2B-NS3 protease. Zafirlukast
was validated by monitoring
inhibition in the presence of
detergents at various
concentrations. The inhibitor
activity of zarfirlukast was
consistent with a range of
Figure 2: Zafirlukast inhibition of NS2B-NS3
activity in the presence of various
concentrations of CHAPS and Brij35
detergents. The inhibitor activity was
consistent in the presence of detergents at
varying concentrations.
concentrations (0-0.2 %) in both
CHAPS and Brij35 detergents (Figure
2). This experiment confirms that the
observed inhibitory effects of zafirlukast were specific to the NS2B-NS3 protease and not caused
by non-specific aggregation.

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In addition, an enzyme inhibitor kinetic study was performed varying both the inhibitor and
substrate concentrations to generate a Lineweaver-burke plot (Figure 3). Based on the plot,
zafirlukast inhibitors the protease through a mixed inhibition mechanism. This mechanism is
further supported by a Michaelis-Menten plot (see supplementary data) at the various inhibitor
concentrations, which indicates a
decrease in the appVmax and
increase in the appKm of the
reaction as the concentration of
zafirlukast is increased. This type
of mechanism would indicate that
zafirulkast is inhibiting the protease
by binding at an allosteric site on
the protease. This is consistent with
previously reported small molecule
inhibitors that act via uncompetitive
or mixed inhibition modes [13, 25,
26]. Collectively, these results
suggest that inhibitors including
Figure 3. Lineweaver-burk plot of WNV NS2B-NS3
protease with concentrations of zafirlukast at 0 (•), 10
zafirlukast bind to an allosteric site
(¢), 20 (p), 30 (Ç), 40 (∑) µM. Substrate
concentrations of 1, 2, 3, 6 and 10 µM for each
on the NS3 protein and either alter
inhibitor concentration.
the conformation of the NS3 active

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site or disrupts binding of the cofactor NS2B, to render the NS2B-NS3 protease catalytically
inactive.
2.2
Structure activity relationship study
The binding of zafirlukast to the NS2B-NS3 protease was studied through a structure activity
relationship (SAR) study via chemical synthesis and enzymatic assays of the analogs.
Zafirlukast analogs were synthesized following literature procedures [27, 28] and all compounds
were examined by the FRET based enzymatic assay to determine the percent of NS2B-NS3
protease inhbition (Table 1) [24]. Zafirlukast contains a toluic acid-N-aryl sulfonamide domain
connected at the C-2 of the indole. Complete removal of the N-aryl sulfonamide (1) significantly
decreased inhibition. In addition, substituting the sulfonamide for an amide linker (2) resulted in
similar poor inhibition. These results indicate both the aromatic ring and the sulfonamide group
are essential for inhibition. However, the methyl group attached to the benzene of the N-aryl
sulfonamide was shown to have no significant effects on inhibitor activity, as an analog without
the methyl group (3) displayed comparable inhibition to zafirlukast. The sulfonamide can aid in
binding through hydrogen bonding and orientating groups in a favorable position that cannot be
achieved with the amide. It is not clear if the hydrogen bonding or directing ability of the
sulfonamide is the element responsible for inhibition.
The toluic acid contains a methoxy group at the meta position. Removal of the methoxy (4)
lowers inhibitor activity, but not greatly. This would indicate that either the methoxy is not in
close hydrogen bond distance to residues of the protein or the methoxy is donating electron
density to the aromatic ring, thereby increasing the strength of π-stacking interactions between
the phenyl and nearby aromatic residues. Removal of the methoxy group would not result in

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complete loss of the phenyl-residue interaction, but would weaken it, which is observed
experimentally.
Cmpd
R1
R2
R3
% Inhibitiona
Table 1. SAR of
(1-8).
zafirlukast analogs
1
OMe
OMe
12
2
OMe
5
3
4
OMe
H
81
64
5
6
H
OMe
OMe
14
12
7
8
OMe
OMe
33
92

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a at an inhibitor concentration of 60 µM
Zafirlukast also contains a cyclopentyl carbamate connected at the C-5 of the indole. Removal
of the cylcopentyl carbamate (5) resulted in poor inhibition and replacement of the cylcopentyl to
an ethyl (6) gave similar poor inhibition as 5. Substitution of the carbamate for an amide (7) also
lowered inhibition. These results indicate the cyclopentyl group is occupying a hydrophobic
binding site. This particular binding site is able to accommodate the cyclopentyl group, which
suggests the site is relatively large. To further probe the size and hydrophobic nature of the
binding site the cyclopentyl was replaced with a phenyl (8). This analog displayed better
inhibition than zafirlukast with an IC50 of 22 µM. The phenyl group is larger than cyclopentyl,
however it is planer and can participate in pi-stacking, which would increase interactions with
aromatic residues leading to improved binding and inhibition. The carbamate functional group
was also revealed to be important for binding, as removal of this group (7) lowered inhibition.
The carbamate can participate in hydrogen bonding between the carbonyl of the carbamate and
polar residues in the active site. The amide analog could also participate in the same interaction,
but at a lesser degree than the carbamate resulting in weakened interactions leading to the lower
inhibition, which is observed experimentally.
The overall shape of zafirlukast was investigated for inhibitor activity (Table 2.). The toluic
acid-N-aryl sulfonamide moiety of Zafirlukast is connected at the C-2 of the indole. The moiety
can also be connected at the N-1 of the indole to generate analogs with a different overall shape
but does not restrict molecular rotation. Similar to the parent, when the toluic acid-N-aryl
sulfonamide moiety or the N-aryl sulfonamide are removed (9 and 10 respectively), there is
effectively no inhibition. Interestingly when the toluic acid-N-aryl sulfonamide domain is

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attached to N-1 (11) the inhibitor activity is comparable to zafirlukast. This would suggest that
the binding site occupied by zafirlukast is large and can accommodate the cyclopentyl carbamate
and the toluic acid-N aryl sulfonamide domains in different molecular shapes. This evidence
also supports the role of the indole core as a linker for the two domains. This theory was
explored by synthesizing analogs that contained a benzene linking the two domains. Two
analogs were
Table 2. SAR of zafirlukast analogs (9-11)
Cmpd
R
% Inhibition
6
9
CH3
10
11
5
79
a at an inhibitor concentration of 60 µM
synthesized and examined for NS2B-NS3 protease inhibitor activity. Both analogs substituted
the indole for a benzene with the cyclopentyl carbamate directly attached to the ring (Figure 4).
One analog did not contain the toluic acid moiety
(12) resulting in minimal inhibition, which is
consistent with previous experimental data.
Compound 13 contained the toluic acid moiety
linked to the benzene at the meta position, with
Figure 4. Analogs of zafirlukast (12 and
13), replacing the indole core with a
benzene. Percent inhibition at a
concentration of 60 µM.

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moderate inhibition. It is concluded that 13 was better able to inhibit the NS2B-NS3 protease
due to the spatial orientation of the two domains, with the benzene connecting the two groups at
the meta position, resulting in an overall molecular shape similar to zafirlukast. However, in the
case of 12, the benzene connects the two domains at the para position adopting a linear
molecular shape and also lacked the toluic acid moiety, vastly different from zafirlukast. The
experimental data indicates that the indole may not be essential for inhibition, however the
overall molecular shape is vital for binding.
2.3 Molecular docking
To better understand how zafirlukast inhibits the viral protease, computational docking studies
[29] of zafirlukst and 8 binding to the NS3 protein (PDB #2IJO) were conducted via Autodock
Vina. Previously, researchers have proposed small molecules interacting with the NS3 protease
at allosteric sites that would dislocate the NS2B co-factor from binding to the NS3 protein
resulting in protease inhibition [13, 25, 26]. Disruption of the NS2B-NS3 complex has been
experimentally proven to abate protease activity [8, 30]. In addition, mutation of NS2B residues
vital for binding to the NS3 protease also inactivated catalytic activity of the protease [31].
Therefore, the focus of our docking studies was to explore these allosteric sites on the NS3
protein to determine if they are viable binding sites for zafirlukast and 8. There are three
positions on the NS3 protein that have been identified as NS2B binding sites. One region is at
the catalytic active site, another interface is located in proximity to Gln96 and the final binding
site is adjacent to Phe116 of the NS3 protein [8, 32]. The initial docking simulations were
performed with the ligand binding site defined
as the entire molecular surface of the NS3
protein to identify all favorable binding
Figure 5. Lowest energy binding
mode between zafirlukast and NS3
protein via Autodock Vina.

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locations for zafirlukast. The results from this docking simulation revealed the lowest energy
complex is zafirulkast binding to the NS3 protein in proximity to Phe116 as determined by
AutoDock Vina scoring function (Figure 5). A visual inspection of this lowest energy binding
mode revealed zafirlukast interacting with two binding pockets. One pocket is formed by
residues Phe116, Val154 Gly153 and Thr118 interacting with the phenyl sulfonamide (Figure
6A). In this pocket there is pi-stacking observed between the phenyl sulfonamide and Phe116.
In addition, there are other contacts between the phenyl sulfonamide and hydrophobic residues in
this pocket. The second pocket interacts with the cyclopentyl carbamate and is composed of
Trp89, Ile147, Ile165, lys88 and Gln167 (Figure 6B). This binding site is hydrophobic with
many contacts between the cyclopentyl and hydrophobic residues including the carbon side chain
of lys88. Based on a visual inspection, both binding pockets could accommodate larger
hydrophobic groups, particularly aromatic systems, which may improve binding and overall
inhibition. The docking model also revealed Asn152 as a possible hydrogen bonding pattern for
the methoxy on the toluic acid. There is no hydrogen bonding observed in the docking complex,
however rotation of the toluic acid could induce hydrogen bonding formation, which would
explain the important of the methoxy group in the SAR study. The indole core has hydrophobic

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contacts with Ile123 and the overall shape of zafirlukast formed by the connection of toluic N-
aryl-sulfonamide and cylcopentyl carbamate domains at the indole C-2 and C-5 respectively,
positions the domains into favorable binding sites.
Figure 6. Zafirlukast docked to the NS3 protein. A: NS3 protein binding pocket interacting
with the phenyl sulfonamide and B: additional binding pocket interacting with cyclopentyl
carbamate of zafirlukast.
Figure 7. Lowest energy docking complex for zafirlukast (teal) and 8 (green) superimposed
B
A
on the NS3 protein.

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Docking of 8 to the NS3 protein resulted in a similar binding model as zafirlukast, which is
observed by superimposing the docked complex of zafirlukast and 8 on the NS3 protein (Figure
7). Based on docking scores and supported by experimental inhibitor data, 8 is a better binder
than zafirlukast to the NS3 protein. The planar nature of the phenyl carbamate is better able to
bind in the pocket, which has also improved the interactions of toluic acid and phenyl
sulfonamide as both are interacting with Phe116, which is not observed in the zafirlukast
docking complexes. These increased interactions improved binding and also resulted in greater
disruption of NS2b binding. The docking models of zafirlukast and 8 are consistent with
experimental data, indicating both domains are important for inhibitor activity. In both docking
models the phenyl sulfonamide would be in position to disrupt NS2B binding resulting in NS2B-
NS3 protease inhibition (Figure 8). This supports experimental data implying that zafirlukast is
inhibiting through a mixed mode inhibition due to binding at an allosteric site.
Figure 8. Docking image of zafirlukast interfering with NS2 binding to the NS3.
3. Chemistry

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The initial syntheses centered on creating zafirlukast analogs with variations of the alkyl
carbamate and N-aryl sulfonamide domains (Scheme 1). The indole nitrogen was methylated
and underwent alkylation in the presence of silver oxide to give 17a-c [27]. The nitro group
was reduced to the amine and acylated to give the carbamate or amide precursors (1, 20a-d).
The methyl ester was hydrolyzed to the corresponding carboxylic acid and reacted with either
the o-toluenesulfonamide, benzenesulfonamide or benzylamine to give the target compounds
2-4 and 6-8. In addition, 5 and 9 were synthesized following similar reactions described in
scheme 1.
Scheme 1. Synthesis of 1-9: a) MeI, acetone 40ο C; b) H2,10 % Pd/C, THF, 24-48 hr; c) cyclopentyl
chloroformate, NaHCO3, THF/H2O; d) Ag2O, dioxane, 60ο C, 20 hr; g) cyclopentylacetic acid,
DMAP, EDC, DCM, 16 hr; g) ethyl chloroformate, NaHCO3, THF/H2O; i) 2M NaOH aq.,
MeOH/THF, 60ο C; j) benzylamine or o-toluene sulfonamide, DMAP, EDC, DCM, 16 h;
Table 3. Substitution pattern of compounds 14-21.

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Analogs probing the molecular shape of zafirlukast were synthesized according to scheme 2.
Briefly, 14a was alkylated at the N-1 of the indole with methyl-4-(bromomethyl)-3-
methoxybenzoate followed by catalytic hydrogenation of the nitro to the corresponding amine
(23) [28]. The amine was acylated with cyclopentyl chloroformate (10) and the toluate was
hydrolyzed to toluic acid and coupled with o-toluenesulfonamide under EDC/DMAP
conditions to give 11.

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Scheme 2. Syntheses of 10 and 11. a) methyl-4-(bromomethyl)-3-methoxybenzoate, NaH,
DMF, RT, overnight; b) H2,10 % Pd/C, THF, 24-48 h; c) cyclopentyl chloroformate, NaHCO3,
THF/H2O; d) 2M NaOH aq., MeOH/THF, 60ο C; e) o-toluene sulfonamide, DMAP, EDC,
DCM, 16 h.
The final compounds substituted the indole for a phenyl as depicted in schemes 3 and 4.
Compound 12 was synthesized beginning with a Suzuki coupling [33] between 3-
nitrophenylboronic acid and methyl-4-(bromomethyl)-3-methoxybenzoate followed by
reduction of the nitro to an amine (26). The amine was acylated with cyclopentyl
chloroformate and the ester hydrolyzed and coupled with o-toluene sulfonamide to give 12.
As indicated in scheme 4, methyl 4-aminobenzoate was acylated with cyclopentyl
chloroformate under basic conditions to give the cyclopentyl carbamate (30). The ester was
hydrolyzed to the carboxylic acid and then coupled with o-toluene sulfonamide to yield 13.

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Scheme 3. Synthesis of 12. Reagents and conditions: a) methyl-4-(bromomethyl)-3-
methoxybenzoate, CsCO3, Pd(PPh3)4, H2O/DME, 90-100ο C, 24 h; b) H2,10 % Pd/C, THF,
24-48 h; c) cyclopentyl chloroformate, NaHCO3, THF/H2O; d) 2M NaOH aq., MeOH/THF,
60ο C e) o-toluenesulfonamide, DMAP, EDC, DCM, 16 h.
Scheme 4. Synthesis of 13. Reagents and conditions: a) cyclopentyl chloroformate, NaHCO3,
THF/H2O; b) 2M NaOH aq., MeOH/THF, 60ο C; c) o-toluene sulfonamide, DMAP, EDC,
DCM, 16 h
4.
Conclusion
In summary, we report the discovery of zafirlukast as an inhibitor for the NS2B-NS3 protease.
Zafirlukast was determined to inhibit the protease via a mixed mode mechanism with an IC50
value of 32 µM. The SAR study revealed the alkyl carbamate and N- toluic acid-N-aryl
sulfonamide domains to be required for inhibition. Substituting the cyclopentyl with a phenyl
improved inhibition with an IC50 value of 22 µM. An in-silico model of zafirlukast and 8 docked

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to the NS3 protein indicated binding at an allosteric site that disrupts formation of the NS2B-
NS3 protease complex, thereby inhibiting catalytic activity, which is supported by experimental
results. Future studies will continue to investigate the indole core and substitution of the indole
with other privileged scaffolds to better orientate the carbamate and toluic acid-N-aryl
sulfonamide domains for improved NS2B-NS3 protease inhibition.
5.
Experimental section
5.1 Chemistry
5.1.1 Materials
All chemicals and reagents were obtained from Fisher Scientific and used without further
purification. An authentic sample of zafirlukast was purchased from Fisher Scientific. Column
chromatography was performed on a Teledyne Isco CombiFlash Rf+ with an EtOAc/Hex
gradient utilizing RediSep Rf silica columns. NMR spectra was recorded utilizing a Bruker 400
MHz NMR operating at 400 MHz for proton NMR and 100 MHz for carbon NMR in either
CDCl3 or d6-DMSO solvent as indicated. High resolution mass spectra (HRMS) data was
collected by a Q Exactive Focus mass spectrometer.
5.1.2 Synthetic procedures
All compounds were prepared following schemes 1-4 based on literature procedures for the
synthesis of zafirlukast and analogs.
1-methyl-5-nitro-1H-indole (15a): KOH (5 eq.) was added to a solution of indole (1 eq.)
dissolved in acetone (5 mL/mmol of indole) at 0°C. Iodomethane (2 eq.) was added. Solution

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was stirred at 40°C until disappearance of indole by TLC. Reaction was then cooled to room
temperature. DCM (33 mL/mmol of indole) was added to reaction and stirred for 30 minutes.
The solid was then filtered out and filtrate was washed 2 x H2O, 1 x 1M HCl, and 2 x H2O.
Organic layer was dried with Mg2SO4 and rotovapped. Crude product was recrystallized. Yellow
solid; 65-100%; 1H NMR (CDCl3):
δ8.61 (m, 1H), 8.16 (dd, 1H), 7.36 (dt, 1H), 7.23 (d, 1H),
6.70 (dd, 1H), 3.89 (s, 3H); 13C NMR (CDCl3):
103.86, 99.99, 33.32
δ139.43, 132.00, 127.63, 118.18, 117.27, 109.08,
Compounds 15b was synthesized following the procedure describe for 17a.
1-methyl-1H-indole (15b): Yellow oil; 50-100%; 1H NMR (DMSO):
7.39 (m, 1H), 7.31 (d, J=3.2 Hz, 1H), 7.21 (ddd, J=8.2, 6.9, 1.2 Hz, 1H), 7.16-7.02 (m, 1H),
δ
7.66-7.56 (m, 1H), 7.47-
6.51-6.41 (m, 1H), 3.77 (d, J=0.9 Hz, 3H); 13C NMR (DMSO):
120.81, 119.38, 110.07, 100.76, 32.83;
δ136.89, 129.94, 128.58, 121.48,
Methyl 4-[(1-methyl-5-nitroindol-3-yl)methyl]benzoate (17a): Silver (I) oxide (1 eq.) was added
to a stirred solution of indole (1 eq.) and benzyl bromide (1 eq.) in dioxane (1 mL/mmol of
indole) in a microwave tube under nitrogen gas. The reaction was sealed and stirred at 60°C for
20 hours. EtOAc (2 mL/mmol of indole) was added to the reaction. The reaction was then
filtered through celite and the filtrate was rotovapped. Product was purified by column
chromatography to give a yellow solid; 25-45%; 1H NMR (DMSO):
δ8.42 (d, J=2.3 Hz, 1H),
8.02 (dd, J=9.1, 2.3 Hz, 1H), 7.93-7.84 (m, 2H), 7.60 (d, J=9.1 Hz, 1H), 7.44 (d, J=8.3 Hz, 3H),

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4.21 (s, 2H), 3.83 (d, J=4.1 Hz, 6H); 13C NMR (DMSO):
δ166.59, 147.18, 140.80, 140.05,
132.12, 129.82, 129.25, 127.93, 126.82, 116.99, 116.29, 116.03, 110.86, 52.45, 33.30, 30.65
Compounds 17b-c were synthesized following the procedure described for 17a.
Methyl 3-Methoxy-4-[(1-methyl-5-nitroindol-3-yl)methyl]benzoate. (17b): Yellow solid; 20-
50%; 1H NMR (DMSO):
7.29 (d, 1H), 4.12 (s, 2H), 3.86 (m, 9H); 13C NMR (d6-DMSO):
δ8.51 (d, 1H), 8.02 (dd, 1H), 7.59 (m, 1H), 7.49 (d, 2H), 7.38 (s, 1H),
δ
166.56, 157.16, 140.76, 139.90,
135.10, 132.08, 130.38, 126.93, 122.07, 116.41, 115.56, 111.21, 110.81, 56.02, 52.57, 33.30,
24.94
Methyl 3-methoxy-4-[(1-methyl-1H-indol-3-yl)methyl]benzoate (17c): Green oil; 40%; 1H NMR
(DMSO):
δ7.57-7.31 (m, 4H), 7.24-7.04 (m, 3H), 6.98 (ddd, J=8.0, 7.0, 1.0 Hz, 1H), 4.04 (s,
2H), 3.95-3.81 (m, 6H), 3.73 (s, 3H); 13C NMR (DMSO):
δ
166.64, 157.22, 137.14, 135.81,
130.17, 129.07, 128.31, 127.83, 121.92, 121.53, 119.89, 119.07, 118.91, 111.91, 118.91, 111.91,
111.04, 110.05, 56.01, 52.51, 32.70, 25.17
1-methyl-1H-indol-5-amine (16): 10% Pd/C (.18 g/g of starting material) was added to a solution
of starting material dissolved in THF (54 mL/g of starting material). Reaction flask was sealed
and vacuum was used to remove air. A balloon filled with hydrogen gas was then attached to the
flask. The reaction was stirred at room temperature for 24 to 48 hours or until disappearance of
starting material was observed by TLC. The reaction was then filtered through celite. Filtrate was
rotovapped to give a brown solid; 100%; 1H NMR (DMSO):
δ7.15-7.05 (m, 2H), 6.71 (dd,

ACCEPTED MANUSCRIPT
J=3.0, 0.9 Hz, 1H), 6.57 (dd, J=8.5, 2.1 Hz, 1H), 6.12 (dd, J=3.0, 0.9 Hz, 1H), 3.67 (s, 3H); 13C
NMR (DMSO): δ141.30, 131.12, 129.49, 129.44, 129.41, 112.31, 110.11, 104.21, 99.13, 32.86
Compounds 19a,19b, 23, 26 were synthesized following the procedure described for 16.
Methyl 4-[(5-amino-1-methyl-1H-indol-3-yl)methyl]benzoate (19a): brown solid; 50-70%; 1H
NMR (DMSO):
δ7.90-7.82 (m, 2H), 7.41-7.33 (m, 2H), 7.11-7.03 (m, 1H), 6.96 (s, 1H), 6.56-
6.48 (m, 2H), 4.47 (s, 2H), 3.99 (s, 2H), 3.82 (s, 3H), 3.63 (s, 3H); 13C NMR (DMSO):
δ166.68,
148.24, 141.51, 131.41, 129.63, 129.09, 128.55, 127.94, 127.56, 112.32, 110.69, 110.25, 102.27,
52.42, 32.69, 31.47
4-[(5-amino-1-methyl-1H-indol-3-yl)methyl]-3-methoxy-N-(2-
methylbenzenesulfonyl)benzamide (19b): A grey solid; 75%; 1H NMR (DMSO):
δ7.88 (dd,
J=7.7, 1.6 Hz, 1H), 7.50 (d, J=1.5 Hz, 1H), 7.39 (dd, J=7.7, 1.5 Hz, 1H), 7.34-7.13 (m, 4H), 7.09
(dd, J=8.5, 0.6 Hz, 1H), 6.95 (d, J=7.7 Hz, 1H), 6.86 (s, 1H), 6.65-6.52 (m, 2H), 5.60 (s, 3H),
3.84 (d, J=4.0 Hz, 7H), 3.62 (s, 3H); 13C NMR (DMSO):
δ169.54, 156.54, 144.59, 139.82,
136.27, 131.75, 131.44, 130.39, 128.89, 128.12, 125.29, 120.93, 112.50, 110.84, 103.40, 55.68,
32.68, 25.10, 20.57
Methyl 4-[(5-amino-1H-indol-1-yl)methyl]-3-methoxybenzoate (23). Brown oil; 70%; 1H NMR
(CDCl3): δ7.59 (d, J=1.5 Hz, 1H), 7.49 (dd, J=7.8, 1.6 Hz, 1H), 7.15-7.03 (m, 2H), 7.01-6.94 (m,
1H), 6.71-6.59 (m, 2H), 6.41 (dd, J=3.1, 0.9 Hz, 1H), 5.31 (s, 2H), 3.95 (d, J=23.7 Hz, 6H), 3.43
(s, 2H); 13C NMR (CDCl3):
δ
166.83, 156.41, 139.52, 131.56, 131.38, 130.46, 129.54, 128.95,
127.35, 122.18, 112.70, 110.79, 110.24, 105.79, 100.48, 55.61, 52.18, 45.16
Methyl 4-[(3-aminophenyl)methyl]-3-methoxybenzoate (26): Yellow oil; 55%; 1H NMR
(DMSO): δ7.55-7.45 (m, 2H), 7.20 (d, J=7.7Hz, 1H), 6.90 (td, J=7.2, 1.6 Hz, 1H), 6.41-6.31 (m,

ACCEPTED MANUSCRIPT
3H), 4.94 (s, 2H), 3.88-3.77 (m, 8H); 13C NMR (DMSO):
δ166.62, 157.30, 149.11, 140.76,
135.73, 130.72, 129.26, 121.96, 116.86, 114.73, 112.27, 111.16, 56.03, 52.55, 35.66
Methyl 4-[(5-[27]-1-methyl-1H-indol-3-yl)methyl]-3-methoxybenzoate (1): NaHCO3 (3.1 eq.)
and chloroformate (1.1 eq.) were added to a solution of amine (1 eq.) in THF (3.1 mL/mmol of
amine) and H2O (3.1 mL/mmol of amine). The reaction was stirred at room temperature until
disappearance of amine was observed by TLC. The reaction was washed 3 x EtOAc. Combined
organic layer was dried with Mg2SO4 and rotovapped. White oil; 60-90%; 1H NMR (DMSO):
δ
9.21 (s, 1H), 7.60 (s, 1H), 7.52-7.42 (m, 2H), 7.27 (d, J=8.6 Hz, 1H), 7.14 (d, J=7.8 Hz, 2H),
7.05 (s, 1H), 5.06 (td, J=6.0, 2.9 Hz, 1H), 3.94 (d, J=16.8 Hz, 5H), 3.83 (s, 3H), 3.69 (s, 3H),
1.85 (d, J=6.9 Hz, 1H), 1.67 (s, 4H), 1.57 (s, 3H); 13C NMR (DMSO):
δ166.65, 157.19, 154.14,
135.72, 133.73, 129.95, 129.02, 128.93, 127.70, 121.90, 111.42, 110.93, 109.95, 76.68, 56.00,
52.51, 35.47, 32.82, 32.77, 25.14, 23.72, 23.40; HRMS: C25H28N2O5 Calculated [M+H]:
437.2071 Observed: 437.2066
Compounds 9, 10, 20a, 20b, 20d, 27 and 29 were synthesized following the procedure described
for 1.
Cyclopentyl-N-(1-methyl-1H-indol-5-yl)carbamate (9): Brown solid; 85%; 1H NMR (DMSO):
δ
9.26 (s, 1H), 7.67 (s, 1H), 7.34-7.16 (m, 3H), 6.33 (dd, J=3.0, 0.8 Hz, 1H), 5.09 (tt, J=5.9, 2.8
Hz, 1H), 3.33 (s, 3H), 1.96-1.77 (m, 2H), 1.78-1.50 (m, 6H); 13C NMR (DMSO):
δ
154.20,

ACCEPTED MANUSCRIPT
133.40, 131.83, 130.44, 128.38, 114.72, 109.87, 100.44, 76.69, 32.94, 32.84, 23.75; HRMS:
C15H18N2O2 Calculated [M+H]: 259.1441 Observed: 259.1442.
Methyl 4-[(5-{[(cyclopentloxy)carbonyl]amino}-1H-indol-1-yl)methyl]-3-methoxybenzoate
(10): Brown solid; 74%; 1H NMR (CDCl3):
δ7.73 (s, 1H), 7.58 (d, J=1.5 Hz, 1H), 7.48 (dd,
J=7.9, 1.5 Hz, 1H), 7.22-7.08 (m, 3H), 6.64 (d, J=7.9 Hz, 1H), 6.57-6.49 (m, 2H), 5.34 (s, 2H),
5.24 (tt, J=6.1, 2.7 Hz, 1H), 3.97 (s, 3H), 3.91 (s, 3H), 1.99-1.56 (m, 8H); 13C NMR (CDCl3):
δ
166.77, 156.44, 133.43, 131.16, 130.61, 130.57, 129.32, 128.83, 127.36, 122.17, 110.86,
109.81, 101.70, 99.99, 77.74, 55.62, 52.18, 45.21, 32.81, 23.70; HRMS: C24H26N2O5 Calculated
[M+H]: 423.1914 Observed: 423.1907
Methyl 4-[(5-{[(cyclopentyloxy)carbonyl]amino}-1-methyl-1H-indol-3-yl)methyl]benzoate
(20a): Purple solid; 90%; 1H NMR (DMSO):
δ9.21 (s, 1H), 7.91-7.83 (m, 2H), 7.57 (s, 1H),
7.42-7.34 (m, 2H), 7.31-7.24 (m, 1H), 7.14 (d, J=28.3 Hz, 2H), 5.05 (tt, J=5.3, 2.5 Hz, 1H), 4.05
(s, 2H), 3.82 (s, 3H), 3.70 (s, 3H), 1.88-1.79 (m, 2H), 1.69-1.53 (m, 6H); 13C NMR (DMSO):
δ
166.66, 154.14, 147.87, 133.84, 129.69, 129.10, 128.85, 127.66, 127.50, 112.17, 110.01, 76.67,
52.43, 32.83, 32.79, 31.28, 23.73
Methyl 4-({5-[(ethoxycarbonyl)amino]-1-methyl-1H-indol-3-yl}methyl)-3-methoxybenzoate
(20b): Brown solid; 64%; 1H NMR (CDCl3):
δ7.61-7.49 (m, 2H), 7.22-7.16 (s, 3H), 6.78 (s, 1H),
6.66 (s, 1H), 4.23 (q, J=7.1 Hz, 2H), 4.09 (s, 2H), 3.93 (d, J=11.0 Hz, 6H), 3.71 (s, 3H), 1.32 (t,
J=7.1 Hz, 3H); 13C NMR (CDCl3):
δ
167.23, 157.11, 154.36, 135.49, 134.35, 129.88, 129.65,
129.03, 128.22, 128.06, 122.02, 115.37, 112.54, 110.87, 110.41, 109.36, 60.92, 55.58, 52.03,
32.72, 25.24, 14.65

ACCEPTED MANUSCRIPT
Methyl 3-methoxy-4-({1-methyl-5-[(phenoxycarbonyl)amino]-1H-indol-3-yl}methyl)benzoate
(20d): White solid; 46%; 1H NMR (CDCl3):
δ7.69 (s, 1H), 7.55 (d, J=7.7 Hz, 2H), 7.45-7.35 (m,
2H), 7.31-7.13 (m, 5H), 6.97 (s, 1H), 6.81 (s, 1H), 4.09 (s, 2H), 3.93 (d, J=9.6 Hz, 6H), 3.74 (s,
3H); 13C NMR (CDCl3):
δ
167.22, 157.10, 150.88, 135.39, 129.60, 129.32, 129.06, 128.41,
128.10, 125.43, 122.02, 121.72, 112.67, 110.87, 109.50, 99.99, 55.59, 52.03, 32.76, 25.22
Methyl 4-[(3-{[(cyclopentyloxy)carbonyl]amino}phenyl]-3-methoxybenzoate (27): Yellow oil;
100%; 1H NMR (DMSO):
δ9.46-9.41 (m, 1H), 7.55-7.44 (m, 2H), 7.34-7.10 (m, 4H), 6.85-6.78
(m, 1H), 5.05 (tt, J=5.3, 2.4 Hz, 1H), 3.93-3.82 (m, 8H), 1.91-1.78 (m, 2H), 1.74-1.51 (m, 6H);
13C NMR (DMSO):
δ
166.58, 157.34, 153.76, 140.83, 139.81, 135.23, 130.74, 129.48, 129.05,
123.16, 122.03, 118.94, 116.42, 111.23, 77.03, 56.05, 52.57, 35.76, 32.76, 23.70
Methyl 4-{[(cyclopentyloxy)carbonyl]amino}benzoate (29): White solid; 80%; 1H NMR
(DMSO): δ9.98 (s, 1H), 7.92-7.81 (m, 2H), 7.64-7.55 (m, 2H), 5.11 (tt, J=5.9, 2.8 Hz, 1H), 3.81
(s, 3H), 1.94-1.80 (m, 2H), 1.77-1.54 (m, 6H); 13C NMR (DMSO):
130.76, 123.45, 117.82, 77.65, 52.27, 32.73, 23.71.
δ166.34, 153.61, 144.39,
Methyl 4-{[5-(2-cyclopentylacetamido)-1-methyl-1H-indol-3-yl]methyl}-3-methoxybenzoate
(20c): Carboxylic acid (1 eq.), DMAP (1.5 eq.), EDC (1.2 eq.), and amine (1.2 eq.) were added
to DCM (10 mL/mmol of carboxylic acid) in that order. The reaction was stirred at room
temperature for 16 hours. 2M HCl was added to the reaction. Organic layer was extracted 3 x
EtOAc. Combined organic layers were washed with brine. Organic layer was dried with Mg2SO4
and rotovapped. Product was purified by column chromatographyPink solid; 60%; 1H NMR
(DMSO):
δ9.61 (s, 1H), 7.78-7.72 (m, 1H), 7.52-7.42 (m, 2H), 7.33-7.22 (m, 2H), 7.13 (d, J=7.8
Hz, 1H), 7.06 (s, 1H), 3.98 (s, 2H), 3.93 (s, 3H), 3.83 (s, 3H), 3.70 (s, 3H), 2.19-2.03 (m, 1H),

ACCEPTED MANUSCRIPT
1.81-1.68 (m, 3H), 1.68-1.42 (m, 4H), 1.26-1.04 (m, 3H); 13C NMR (DMSO):
δ174.50, 170.64,
166.64, 157.20, 135.72, 134.00, 131.76, 129.93, 129.04, 128.97, 127.57, 121.90, 115.43, 111.54,
110.93, 109.82, 109.70, 55.99, 52.51, 43.00, 37.23, 36.47, 32.78, 32.40, 25.15, 24.99
3-methoxy-4-[(1-methyl-1H-indol-3-yl)methyl]benzoic acid (18): 2M NaOH (11.1 mL/mmol of
ester) was added to a solution of ester (1 eq.) in MeOH (11.1 mL/mmol of ester) and THF (5.55
ML/mmol of ester). The reaction was stirred at 60°C until disappearance of ester was observed
by TLC. 2M HCl (11.1 mL/mmol of ester) was added to the reaction. The reaction was washed 3
x EtOAc. The combined organic layer was washed with brine. The organic layer was then dried
with Mg2SO4 and rotovapped. White solid; 70%; 1H NMR (DMSO):
δ12.86 (s, 1H), 7.58-7.31
(m, 4H), 7.22-7.03 (m, 3H), 6.98 (ddd, J=9.0, 6.9, 1.0 Hz, 1H), 4.01 (s, 2H), 3.90 (s, 3H), 3.73 (s,
3H); 13C NMR (DMSO)
δ
167.73, 157.12, 137.12, 135.26, 130.23, 130.07, 128.27, 127.83,
122.04, 121.52, 119.09, 118.90, 112.07, 111.25, 110.04, 55.93, 32.70, 25.15
Compounds 21a-21e, 24 28 and 30 were synthesized following the procedure described for 18
4-[(5-[27]-1-methyl-1H-indol-3-yl)methyl]-3-methoxybenzoic acid (21a) White solid; 40-75%;
1H NMR (DMSO):
δ12.03 (s, 1H), 9.22 (s, 1H), 7.61 (s, 1H), 7.55 (d, J=2.8 Hz, 1H), 7.51-7.41
(m, 1H), 7.31-7.23 (m, 1H), 7.13 (d, J=8.4 Hz, 2H), 7.04 (d, J=2.5 Hz, 1H), 5.05 (d, J=5.0 Hz,
1H), 3.98-3.88 (m, 5H), 3.69 (t, J=2.1 Hz, 3H), 1.84 (d, J=13.4 Hz, 2H), 1.66 (d, J=12.8 Hz, 4H),
1.57 (s, 2H); 13C NMR (CDCl3):
δ171.50, 157.13, 136.36, 134.32, 129.98, 129.69, 128.39,

ACCEPTED MANUSCRIPT
128.23, 128.05, 122.77, 112.38, 111.21, 109.34, 99.99, 74.16, 55.58, 35.49, 32.82, 32.71, 25.29,
23.67, 23.29.
4-[(5-{[(cyclopentyloxy)carbonyl]amino}-1-methyl-1H-indol-3-yl)methyl]benzoic acid (21b):
Purple solid; 40%; 1H NMR (DMSO):
δ12.73 (s, 1H), 9.21 (s, 1H), 7.89-7.80 (m, 2H), 7.58 (s,
1H), 7.39-7.32 (m, 2H), 7.28 (d, J=8.7 Hz, 1H), 7.11 (s, 1H), 5.06 (tt, J=4.9, 2.3 Hz, 1H), 4.04 (s,
2H), 3.70 (s, 3H), 1.84 (dd, J=12.1, 5.7 Hz, 2H), 1.70-1.53 (m, 6H); 13C NMR (DMSO):
δ
167.74, 154.14, 147.33, 133.83, 129.84, 128.90, 128.80, 127.52, 112.31, 109.99, 90.16, 76.68,
32.83, 32.78, 31.27, 23.73
4-({5-[(ethoxycarbonyl)amino]-1-methyl-1H-indol-3-yl}methyl)-3-methoxybenzoic acid (21c):
Brown solid; 84%; 1H NMR (CDCl3):
δ7.74-7.56 (m, 2H), 7.32 (s, 1H), 7.32-7.17 (m, 2H), 7.04
(d, J=9.2 Hz, 1H), 6.84 (s, 1H), 4.12 (s, 2H), 3.94 (s, 3H), 3.79-3.60 (m, 5H), 1.69-1.54 (m, 3H);
13C NMR (CDCl3):
δ170.93, 157.12, 156.66, 135.86, 135.75, 129.77, 128.80, 128.34, 128.02,
122.61, 121.61, 118.11, 112.92, 111.29, 109.51, 62.40, 55.58, 32.76, 25.37, 14.66
4-{[5-(2-cyclopentylacetamido}-1-methyl-1H-indol-3-yl]methyl}-3-methoxybenzoic acid (21d):
70%; 1H NMR (DMSO):
δ12.80 (s, 1H), 9.62 (s, 1H), 7.79-7.73 (m, 1H), 7.51-7.40 (m, 2H),
7.36-7.23 (m, 2H), 7.14-7.03 (m, 2H), 3.97 (s, 2H), 3.91 (s, 3H), 3.70 (s, 3H), 2.30-2.17 (m, 4H),
1.74 (dd, 11.6, 5.5 Hz, 2H), 1.67-1.45 (m, 4H), 1.20 (dt, J=12.3, 7.1 Hz, 1H); 13C NMR
(DMSO): δ170.67, 167.76, 157.11, 135.18, 134.02, 131.76, 130.24, 129.76, 128.91, 127.61,
122.04, 115.47, 111.74, 111.16, 109.78, 60.21, 55.89, 43.02, 37.25, 36.48, 32.74, 32.42, 25.15,
25.01
3-methoxy-4-({1-methyl-5-[(phenoxycarbonyl)amino]-1H-indol-3-yl}methyl)benzoic acid
(21e): Brown solid; 33%; 1H NMR (CDCl3):
δ7.66-7.55 (m, 2H), 7.43 (s, 1H), 7.34-7.26 (m,
2H), 7.25-7.08 (m, 2H), 7.03 (s, 1H), 6.86 (d, J=13.7 Hz, 1H), 6.71 (s, 3H), 4.15 (s, 2H), 3.94 (s,

ACCEPTED MANUSCRIPT
3H), 3.74 (s, 3H); 13C NMR (CDCl3):
δ157.15, 154.80, 151.54, 135.95, 132.99, 129.77, 129.06,
128.54, 128.08, 125.09, 122.68, 121.68, 121.40, 118.21, 112.95, 111.32, 109.70, 55.60, 32.79,
25.41
4-[(5-{[(cyclopentyloxy)carbonyl]amino}-1H-indol-1-yl)methyl]-3-methoxybenzoic acid (24):
Purple solid; 65%; 1H NMR (CDCl3):
δ7.72 (s, 1H), 7.64-7.52 (m, 2H), 7.21-7.08 (m, 3H), 6.66
(d, J=7.8 Hz, 1H), 6.57-6.50 (m, 2H), 5.36 (s, 2H), 5.27-5.20 (m, 1H), 3.98 (s, 3H), 1.99-1.59
(m, 8H); 13C NMR (CDCl3):
δ
170.81, 156.46, 133.45, 132.14, 130.54, 129.67, 129.33, 128.84,
127.40. 122.94, 128.84, 127.40, 122.94, 111.17, 109.78, 101.80, 99.99, 55.63, 45.25, 32.81,
23.69
4-[(3-{[(cyclopentyloxy)carbonyl]amino)phenyl)methyl]-3-methoxybenzoic acid (28): White
solid; 40%; 1H NMR (DMSO):
δ12.88 (s, 1H), 9.44 (s, 1H), 7.53-7.45 (m, 2H), 7.33-7.25 (m,
2H), 7.22-7.11 (m, 2H), 6.82 (dt, J=7.6, 1.4 Hz, 1H), 5.06 (ddd, J=6.7, 5.1, 1.9 Hz, 1H), 3.87 (d,
J=19.3 Hz, 5H), 1.84 (dp, J=11.8, 8.3, 6.6 Hz, 3H), 1.74-1.51 (m, 5H); 13C NMR (DMSO):
δ
157.24, 149.27, 140.99, 134.31, 130.58, 129.25, 123.17, 122.15, 118.71, 116.11, 111.45,
107.54, 98.06, 77.03, 55.97, 35.40, 32.76, 23.71
4-{[(cyclopentyloxy)carbonyl]amino}benzoic acid (30): Pink solid; 60%; 1H NMR (DMSO):
δ
12.59 (s, 1H), 9.92 (s, 1H), 7.91-7.81 (m, 2H), 7.63-7.53 (m, 2H), 5.11 (td, J=5.9, 2.9 Hz, 1H),
1.96-1.80 (m, 2H), 1.77-1.52 (m, 6H); 13C NMR (DMSO):
124.62, 117.70, 77.58, 32.74, 23.7
δ167.43, 153.63, 144.00, 130.87,
Cyclopentyl N-[1-methyl-3-({4-[(2-methylbenzenesulfonyl)carbamoyl]phenyl}methyl)-1H-
indol-5-yl]carbamate (2): Carboxylic acid (1 eq.), DMAP (1.5 eq.), EDC (1.2 eq.), and

ACCEPTED MANUSCRIPT
amine/sulfonamide (1.2 eq.) were added to DCM (10 mL/mmol of carboxylic acid) in that order.
The reaction was stirred at room temperature for 16 hours. 2M HCl was added to the reaction.
Organic layer was extracted 3 x EtOAc. Combined organic layers were washed with brine.
Organic layer was dried with Mg2SO4 and rotovapped. Product was purified by column
chromatography. Grey solid; 69%; 1H NMR (DMSO):
δ12.53 (s, 1H), 9.20 (s, 1H), 8.02 (d,
J=7.9 Hz, 1H), 7.80 (d, J=8.0 Hz, 2H), 7.57 (t, J=7.3 Hz, 2H), 7.48-7.23 (m, 5H), 7.13 (d, J=27.9
Hz, 2H), 5.10-5.01 (m, 1H), 4.03 (s, 2H), 3.69 (s, 3H), 2.60 (s, 3H), 1.88-1.82 (m, 2H), 1.66 (s,
4H), 1.56 (s, 2H); 13C NMR (DMSO):
δ165.65, 154.14, 147.78, 137.30, 133.81, 132.76, 131.57,
130.82, 128.93, 128.88, 127.46, 126.61, 112.19, 110.01, 76.68, 32.83, 32.81, 23.73, 20.06;
HRMS: C30H31N3O6S Calculated [M+H]: 562.2006 Observed: 562.2007
Compounds 3-8 and 11-13 were synthesized following the procedure described for 2.
Cyclopentyl N-[3-({4-[(benzenesulfonyl)carbamoyl]-2-methoxyphenyl}methyl)-1-methyl-1H-
indol-5-yl]carbamate (3): Grey solid; 42%; 1H NMR (DMSO):
δ12.46 (s, 1H), 9.20 (s, 1H),
8.05-7.95 (m, 2H), 7.77-7.56 (m, 4H), 7.48 (d, J=1.7 Hz, 1H), 7.38 (dd, J=7.8, 1.7 Hz, 1H), 7.32-
7.22 (m, 1H), 7.12 (dd, J=18.4, 8.1 Hz, 2H), 7.02 (s, 1H), 5.05 (tt, J=5.2, 2.5 Hz, 1H), 3.92 (s,
3H), 3.68 (s, 3H), 1.84-1.57 (m, 8H); 13C NMR (DMSO):
δ165.46, 157.11, 154.14, 139.98,
135.80, 134.10, 133.72, 130.58, 129.81, 129.57, 128.90, 128.12, 127.66, 121.19, 111.39, 110.53,
109.94, 76.68, 56.15, 32.82, 32.76, 25.13, 23.72; HRMS: C31H33N3O4 Calculated [M+H]:
512.2544 Observed: 512.2545