Molecular design, synthesis, and biological evaluation of bisamide derivatives as cyclophilin A inhibitors for HCV treatment

Article abstract of DOI:10.1016/j.ejmech.2019.112031

Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. Direct-acting antivirals (DAAs), including inhibitors of nonstructural proteins (NS3/4A protease, NS5A, and NS5B polymerase), represent key components of anti-HCV treatment. However, so

Full text of DOI:10.1016/j.ejmech.2019.112031

Journal Pre-proof
Molecular design, synthesis, and biological evaluation of bisamide derivatives as
cyclophilin A inhibitors for HCV treatment
Jinhe Han, Hye Won Lee, Yifeng Jin, Daulat B. Khadka, Suhui Yang, Xiaoli Li,
Meehyein Kim, Won-Jea Cho
PII:
S0223-5234(19)31189-4
DOI:
https://doi.org/10.1016/j.ejmech.2019.112031
EJMECH 112031
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 September 2019
Revised Date: 20 December 2019
Accepted Date: 31 December 2019
Please cite this article as: J. Han, H.W. Lee, Y. Jin, D.B. Khadka, S. Yang, X. Li, M. Kim, W.-J. Cho,
Molecular design, synthesis, and biological evaluation of bisamide derivatives as cyclophilin A inhibitors
for HCV treatment, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2019.112031.
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Graphical abstract
1

Molecular design, synthesis, and biological evaluation of bisamide
derivatives as cyclophilin A inhibitors for HCV treatment
Jinhe Han a,1, Hye Won Lee b,1,†, Yifeng Jin a, Daulat B. Khadka a, Suhui Yang a, Xiaoli Li a,
Meehyein Kim b,**, Won-Jea Cho a,*
a College of Pharmacy, Chonnam National University, Gwangju, 61186, Republic of Korea.
b
Virus Research Group, Therapeutics and Biotechnology Division, Korea Research
Institute of Chemical Technology, Daejeon, 34114, Republic of Korea
* Corresponding author.
** Corresponding author.
E-mail address: wjcho@jnu.ac.kr (W.-J. Cho), mkim@krict.re.kr (M. Kim)
1 These authors contributed equally to this work.
†
Present address: Center for Infectious Disease Research, Korea National Institute of
Health, Republic of Korea
1

ABSTRACT
Hepatitis C virus (HCV) is a major cause of end-stage liver diseases. Direct-acting
antivirals (DAAs), including inhibitors of nonstructural proteins (NS3/4A protease, NS5A,
and NS5B polymerase), represent key components of anti-HCV treatment. However, some
DAAs are associated with increased drug resistance and undesired side effects. Previous
reports have shown that bisamides could be a novel class of cyclophilin A (CypA)
inhibitors for treating HCV as a member of combinational therapies. To fully elucidate
structure-activity relationships of bisamide derivatives and find a better hit compound with
diverse binding modes, 16 biamides were designed with the help of docking program. They
were then synthesized using one-pot four-component Ugi reaction. 7e with selectivity index
of more than 18.9 (50% effective concentration of 5.3 µM, but no cytotoxicity at 100 µM)
and unique binding mode that could be dived into gatekeeper pocket was selected as a new
hit compound. Surface plasmon resonance experiments revealed that 7e is able to bind to
CypA with a KD of 3.66 µM. Taken together, these results suggest that 7e as a CypA
inhibitor could be used as an alternative anti-HCV agent in combinational therapy in the
future.
Keywords:
Hepatitis C Virus, Cyclophilin A inhibitor, Bisamide, Ugi reaction, Molecular Modeling
2

Graphical abstract
3

Highlights
ò A new group of bisamide derivatives, totally 16 compounds were designed and
aimed to switch the binding mode against CypA.
ò The EC50 and CC50 values of the newly synthesized compounds were helped to
enrich the structure-activity relationships.
ò Docking studies were proofed 7e located into gatekeeper pocket with
selectivity index of more than 18.9 (50% effective concentration of 5.3 µM, but no
cytotoxicity at 100 µM).
ò SPR results revealed that 7e is able to bind to CypA with a KD of 3.66 µM.
4

1.
Introduction
Despite recent advances in treatment, hepatitis C virus (HCV) infection continues
to be a burden of disease. Around 3% of the population are affected by HCV infection
which leads to 300,000 deaths per year. Approximately 80% of infected patients develop
chronic symptoms that can result in cirrhosis (27%) and hepatocellular carcinoma (25%)
[1-3].
There are multiple strains of at least seven HCV genotypes (GTs) identified.
Among them, GT 1a and GT 1b are well characterized clinically and molecular
virologically. Different types of HCV infection need different treatment strategies [4, 5].
Before 2011, the standard treatment for HCV infection was mainly through a combination
of pegylated interferon (pegIFN) alpha and ribavirin (RBV) for 24 to 48 weeks depending
on HCV genotype [6]. This combination therapy can produce a sustained virologic
response (SVR) rate of 40% to 50%. However, both regimens in this combination therapy
have unwanted and intolerable side effects [7-9].
To improve the problem of side effects caused by the double combination therapy,
telaprevir and boceprevir (NS3/4A protease inhibitors), the first two direct-acting antivirals
(DAAs), were developed. When used in therapeutic combination, they could dramatically
increase viral clearance rate by around 70% compared to the effect of double combination
of pegIFN and ribavirin [10, 11]. However, these newly developed DAAs still possess
several drawbacks including cumbersome dosing regimens, strict dietary requirements, and
unfavorable adverse effect profiles. With the discovery of additional DAAs, therapy
without using pegIFN became possible for curing all HCV genotypes while prominently
improving unwanted side effects [12, 13].
5

In the next few years, several new DAAs were additionally developed and
approved by the FDA as alternative regimens to supplement current combination therapies,
including glecaprevir, grazoprevir (NS3/4A inhibitors), daclatasvir, and elbasvir (NS5A
inhibitors) [14]. Combined DAAs therapy has higher efficacy than their monotherapy.
Meanwhile, several recommended combination therapies such like sofosbuvir (an NS5B
polymerase inhibitor) and simeprevir (an NS3 protease inhibitor) approved by FDA already
put DAAs into the arsenal as new regimens to treat HCV [14]. However, ensuing drug-drug
interaction has become an issue to be solved. For example, sofosbuvir plus pegIFN or RBV
have been reported to cause the most common drug-drug interaction that leads patients to
have fatigue, headache, nausea, insomnia, and anemia [15, 16]. To avoid flaws of current
combination therapies including simultaneous viral mutations, drug-drug interactions,
and/or drug resistance, a new class of anti-HCV agents with potential to replace existing
regimen should be developed. In the present, we suggest that cyclophilin A (CypA)
inhibitors could be one of acceptable options.
CypA plays critical roles in many biological signal networks. As a candidate of
cellular peptidyl-prolyl cis-trans isomerase (PPIase), it is involved in cellular cycles such as
protein folding and trafficking [17]. The first CypA inhibitors that showed antiviral
activities were cyclosporine A (CsA) (Fig. 1) and sanglifehrin A, both of which were
isolated from natural products [18]. Up to date, most of existing Cyp inhibitors are derived
from these two natural products, sharing the same scaffold. Blockage of
dephosphorylation-driven nuclear translocation of nuclear factor of activated T cells
(NFAT) by CsA-CypA complex suggests that CsA can be an immunosuppressant, but not
an anti-HCV agent [19]. Many non-immunosuppressive CsA analogs such as CPI-431-32
6

[20] and SCY-635 [21] have been developed. Due to disadvantages of such large CsA-like
structures, including high risk
Fig. 1. Chemical structures of CsA 1 and bisamide hit 2.
of having clinical safety profiles, complex synthetic route, and high cost [22], small
synthetic molecules as new anti-HCV agents have been developed [20, 23-25].
Our previous research has reported that bisamide analogs as CypA inhibitors
possess potent antiviral activity against HCV without cytotoxicity at a maximum
concentration tested [26]. Bisamide hit 2 (Fig. 1) inhibited HCV replication but not
mediated by immunosuppressive effects. The objective of the present study was to further
investigate the structure-activity relationship of the bisamide analogs. New derivatives
were synthesized and their anti-HCV properties were tested using a JFH1-derived
infectious clone.
2.
Results and Discussion
2.1.
Design of bisamides
7

Our previous report has revealed that bis-amides can be novel CypA inhibitors by
possessing selectivity without showing immunosuppressive effects. In addition, they are
structurally highly different from current CsA-based inhibitors. Particularly, hit compound
2 had strong interactions with Arg55, Trp121, and Lys 125 of CypA through H-bonds by
oxygen atoms of trimethoxyphenyl group as shown in our published report [26]. By
investigating the binding mode of other bisamides reported in the previous studies, we
found that the cyclohexyl moiety all occupied in the catalytic pocket. Meanwhile,
hydrophobic interaction generated by the indolylmethyl moiety also seemed to be crucial
for the binding affinity of bisamides derivatives to CypA.
Since our aim was to optimize bisamide structure by altering substituents so that a
promising target compound could located in both of the two hydrophobic regions (the
catalytic pocket and the gatekeeper pocket), the main strategy was to reserve the indole
moiety and the cyclohexyl moiety while changing the rest of the compound. In this report,
we mainly focused on bis-amides with di- or tri- methoxy groups as substitutions for R2
moiety.
2.2.
Chemistry
In the present study, bisamide derivatives were synthesized by the four-component Ugi
reaction according to the first published report [27]. The possible mechanism is that the
reaction starts from the formation of an imine between amine and aldehyde with a loss of
one equivalent of water. The formed imine intermediate was protonated by the carboxylic
acid to form iminium ion. Then nucleophilic addition between the iminium ion and
isocyanide gives the nitrilium ion which is then reacted with carboxylic acid by another
8

nucleophilic addition. The final rearrangement called a Mumm rearrangement takes place
with the transfer of acyl group from oxygen to nitrogen. Rearrangement is the only non-
reversible reaction among all steps. It is considered a critical step that drives all reaction
sequences. Because of the one-pot reaction property, the Ugi reaction gives the possibility
to synthesize a large number of bisamide derivatives just by using various aldehydes,
amines, isocyanides, and carboxylic acids. The reaction also has an easy workup procedure
with high efficiency. Sixteen bis-amides were prepared by the Ugi reaction using methanol
or 2,2,2-trifluoroethanol at room temperature (RT) or 55 °C (Schemes 1 and 2).
8h, 8m
7a-p
R1
R2
3a
3b
4a
4d
4c
4b
R3
R4
OMe
OMe
*
NH
6a
*
5a
5b
5c
5d
PMB
N
6b
*
NH
5e
5f
5g
5h
5i
6c
*
Scheme 1. Ugi multi-component reaction. (a) MeOH or 2,2,2-trifluoroethanol, room
temperature or 55 °C.
9

Scheme 2. Synthesis of imines 8h and 8m. (a) EtOH or MeOH or 2,2,2-trifluoroethanol,
room temperature.
2.3.
Evaluation of antiviral activity and cytotoxicity
To test cytotoxicity, Huh7.5 cells were treated with increasing concentrations of
compounds and their half maximal cytotoxic concentrations (CC50) were determined by the
standard 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. In
parallel, half-maximal effective concentrations (EC50) were calculated by Renilla luciferase
assay from Huh7.5 cells which were infected with the JFH1-Luc virus and treated with
different concentrations of bisamides derivatives [28]. 7a (Table 1) with a PMB substitution
on the amino group of the indole moiety at R4 showed no antiviral activity under its
subtoxic concentrations (CC50 = 24.4 ± 7.2 µM) (Table 1). Similarly, all three bisamide
derivatives with 2,3,4-trimethoxyphenyl at R2 position showed cytotoxicities but no
antiviral activity (Table 1). Some bisamide derivatives with 3,4-dimethoxyphenyl at R2
position were less toxic compared to those with 2,3,4-trimethoxyphenyl at R2 position
(Table 1). Notably, 7j and 7m displayed selectivity index of 10.5 and 8.6, respectively.
Given that the hit compound 2 had EC50 of 5.2 ± 0.7 µM and CC50 > 100 µM, it could be
suggested that both compounds, 7j (EC50 = 5.3 ± 0.6 µM; CC50 = 55.3 ± 2.4 µM) and 7m
(EC50 = 3.8 ± 0.7 µM; CC50 = 32.7 ± 5.9 µM), maintained comparable anti-HCV activity.
10

Finally, among the bisamide derivatives with 3,4,5-trimethoxyphenyl at R2 position, five
compounds (7b, 7d, 7e, 7o, 7p) showed selectivity indices over 5.0 (Table 1). It is
noteworthy that 7e (EC50, 5.3 ± 0.7 µM; CC50, >100 µM), showed promising antiviral
activity and no cytotoxicity at a maximum concentration tested. However, by only
switching the cyclohexyl group to tert-butyl at R1 position of the compound, 7n (EC50, 6.3
± 3.0 µM; CC50, 13.9 ± 0.1 µM) exhibited lower selectivity index compared with 7e,
mainly due to increase in cytotoxicity rather than enhancement of its antiviral effect (Table
1). After changing the indolylmethyl group of 7n to indolyl group, the resulting 7o partially
alleviated the cytotoxicity problem, without affecting anti-HCV activity.
Table 1. Anti-HCV activity and cytotoxicity of bisamides.
a
b
EC50
CC50
Compd
7a
R1
R2
R3
R4
SIc
n.d.d
9.0
(µM)
(µM)
>24.4
24.4 ± 7.2
5.0 ± 2.4 44.8 ± 4.0
9.4 ± 4.6 33.2 ± 1.6
10.2 ± 2.9 56.7 ± 1.4
7b
3.4
7c
5.5
7d
11

5.3 ± 0.7
>18.8
> 100.0
>18.9
n.d.
7e
7f
18.8 ± 0.4
7g
7h
>7.2
7.2 ± 0.2
n.d.
n.d.
>5.9
5.9 ± 0.1
6.9 ± 0.1
2.0 ± 0.6
3.5
10.5
n.d.
n.d.
8.6
7i
7j
5.3 ± 1.0 55.3 ± 2.4
>14.6
>54.5
14.6 ± 0.7
54.5 ± 4.6
7k
7l
3.8 ± 0.7 32.7 ± 5.9
7m
12

*
6.3 ± 3.0 13.9 ± 0.1
5.9 ± 3.3 36.7 ± 4.7
2.3
6.2
7n
7o
*
2.0 ± 1.4 19.4 ± 3.0
9.7
7p
5.2 ± 0.7
>100.0
>100.0
> 100.0
>19.2
>6.1
2
Ribavirin
-
-
-
-
-
-
-
-
16.3 ± 3.0
INF-αe
0.31 ± 0.1 >322.6
a
b
c
The half-maximal effective concentration. The half-maximal cytotoxic concentration;
d
Selectivity index, the ratio of CC50 to EC50. Not determined. All experiments were
performed in triplicate. e Treatment concentration unit is ng/mL.
2.4.
Structure and activity relationship
To identify preferential elements of the bis-amide compound as an anti-HCV agent,
structure and activity relationship study in cells was investigated from its newly
synthesized derivatives and structural priority in each group was summarized (Fig. 2).
13

According to our previous report [26], the indole moiety in the R4 of the hit compound 2
can generate a π- π interaction with Trp121 based on docking study. When a para-
methoxybenzyl (PMB) group was introduced to the nitrogen of the indolylmethyl group, 7a
showed cytotoxicity against the tested cell line. As for R4 position, the carbon number
between the indole moiety and adjacent amide also showed a significant impact on both
cytotoxicity and antiviral activity. When the indolylmethyl group of the compound 2 was
switched to the indolyl group to generate 7p, it led to a 2.5-fold increase in anti-HCV
activity (EC50 values of 5.2 and 2.0 µM, respectively). Meanwhile, its cytotoxicity was also
over 5-fold enhanced (CC50 values of >100 and 19.4 µM, respectively). Innovation with the
cyclohexyl moiety at the R1 of 7p to the t-butyl group, resulting in 7o caused selectivity
index to decrease over 1.5-fold (from >9.7 to 6.2), indicating that the cyclohexyl group at
this position is more favorable than the t-butyl group.
Antiviral effects and cytotoxicity seemed to vary primarily depending on the
substituent groups of the phenyl ring at the R2 position. The introduction of one more
methoxy group at ortho-position of R2 resulted in loss of anti-HCV activity and showed
strong cytotoxicity (Table 1). Among all newly synthesized bis-amides, 7i (EC50, 2.0 µM)
showed best anti-HCV activity. At the same time, strong cytotoxicity was observed by 7i
(CC50, 6.9 µM). It was related to the deletion of one of methoxy groups in meta-position.
The antiviral activity seems to be less influenced by the substitution groups of the
phenyl ring at the R3 position. The three compounds, 7b (with a 4-methoxy substituent), 7c
(with a naphthalene moiety) and 7d (with 3,4-dimethoxy substituents) showed similar
antiviral activity ranging from 5.0 to 10.2 µM as well as comparable cytotoxicity ranging
from 33.2 to 56.7 µM. Intriguingly, 7e with more distance by inserting two carbons
14

Fig. 2. Structure-activity relationship (SAR) of newly synthesized bisamides.
between the phenyl ring and the nitrogen dramatically reduced cytotoxicity (over 100 µM),
subsequently enhancing its selectivity index over 18.9, as observed in the previous hit 2. It
might be related to the obviously computational binding mode change compared to that of
bisamide with a substituted phenyl group at the R3 position because of the massive
structure difference. Accordingly, among the five hit compounds, 7b, 7e, 7j, 7m, and 7p,
7e was selected as a new hit of CypA inhibitor.
2.5.
Docking study
15

To figure out the potential of computational binding mode of the five potential
active bisamide derivatives including 7b, 7e, 7j, 7m, and 7p and the rest of the bisamide
compounds, the binding mode with a reference to a tertiary structure of the CsA-CypA
complex (PDB code: 1CWA) or the hit compound 2-CypA complex were revealed in silico
by utilizing Surflex-Dock. As a representative, binding modes of 7e as well as CsA and hit
2 were visualized by using Flare docking program (Fig. 3).
Fig. 3. A) Overlay of CsA (green), hit 2 (pink), and 7e (cyan) bound to human
CypA without protein structure. B) Overlay of hit 2 (pink), and 7e (cyan) bound to human
CypA, showing its active site with the hydrophobic pocket.
By analyzing the binding pattern of the newly synthesized analogs, surprisingly, a
total of five compounds (7c, 7e, 7j, 7k, and 7l) showed more or less flip for the mode of the
gatekeeper pocket (Fig.4 and Fig. 5A). And the rest 11 bisamides remained the similar
binding mode compared with the hit 2 which had no interaction with the other hydrophobic
pocket (gatekeeper pocket) (Fig. 5B).
16

Fig. 4. Docking mode of biamides and CsA. A) hit 2. B) 7e. C) CsA. D) 7l. E) 7j. F) 7c. G)
7k.
A
B
Fig. 5. Overlaid Docking modes of bisamide derivatives. A) the compounds failed to flip
into gatekeeper pocket. B) the compounds succeeded to flip into gatekeeper pocket.
17

Among the docked compounds (Fig. 4.), 7e was the only compound that showed
antiviral activity (EC50, 5.3 µM) and no cytotoxicity at the concentration of 100 µM. The
docking results showed that binding modes of 7e and hit compound 2 were obviously
distinct, even though they share the catalytic pocket at the binding site (Fig. 3). The
overlapping area was the cyclohexyl moiety that could have hydrophobic interaction with
Phe113 of CypA. The extension of the carbon chain between the nitrogen and the phenyl
ring at the R3 position totally flipped the binding mode of 7e from the catalytic site to the
adjacent gatekeeper pocket. The hydrogen bond generated between the nitrogen of the
indolylmethyl moiety and His54 of CypA increased the binding affinity (Fig. 6). The
electrostatic charge of the dimethoxyphenylethyl moiety located in the gatekeeper can
highly conform to its charge. The previous report [26] has revealed that this region is a
highly attractive target pocket that can be used to develop isoform-specific CypA inhibitor
because of residue distinction at position 103 in CyP isoforms. Ala103 participating in the
formation of the gatekeeper pocket of CypA may contribute to the hydrophobic interaction
with 7e. It is surprising that trimethylphenyl moiety can also establish hydrophobic
interaction with the vicinity amino acid Ala103 of CypA. This unique binding mode of 7e
potentially could make it an anti-HCV compound by blocking CypA.
18

Fig. 6. Stereotype view of 7e (yellow) bind to human CypA. Active site residues
participating in the catalytic site and gatekeeper pocket are shown as ball and stick mode
according to atom type (red: oxygen, blue: nitrogen, grey: carbon).
2.6.
Surface Plasmon Resonance
The binding affinity of 7e was evaluated by performing surface plasmon resonance
(SPR). By testing different running concentration of 7e (0.3125, 0.625, 1.25, 2.5, 5, 10, 20
µM), the kinetics parameters were obtained (Fig. 7). 7e showed slow dissociation rate and
large association rate (Ka = 7.2 × 102 M-1S-1 Kd = 2.6 × 10-3 S-1). The dissociation constant
,
(KD) was calculated from Ka and Kd (KD [M] = Kd [s-1] / Ka [M-1s-1]) that indicated that 7e
is able to bind to CypA with a KD of 3.66 µM.
19

Fig. 7. Relative response in resonance units showing interactions of 7e with CypA at the
different concentrations. The KD value (mean ± S.D.) was calculated from three
independent experiments.
3.
Conclusion
By switching each of starting components in Ugi reaction, a new group of
bisamides were synthesized with easy workup and promising yield for the purpose of
finding analogs that could have interaction with the gatekeeper pocket of CypA protein.
Meanwhile, the structure-activity relationship was further investigated after screening their
cytotoxicities and anti-HCV activities. To identify new hit compounds among the newly
synthesized compounds, docking study was carried out in parallel. As a result, five
bisamides showed different binding modes that slightly shifted to the direction of the
gatekeeper pocket. However, only 7e generated hydrophobic interaction with the
gatekeeper pocket. It also had better potential as specific CypA inhibitor than other
20

isoforms of Cyps. Thus, 7e was selected as a new hit compound for further biological tests
or pharmacokinetic studies prior to in vivo experiments. SPR binding assay confirmed that
7e is able to bind to CypA with a KD of 3.66 µM. As all data displayed, 7e as a CypA
inhibitor could be used as an alternative anti-HCV agent in combinational therapy in the
future.
4.
Experimental section
4.1.
Chemistry
Melting points were determined by the capillary method with a MEL-TEMP
1
capillary melting point apparatus. They were uncorrected. H NMR was recorded with
Varian Unity Plus 300 MHz at the Korea Basic Science Institute. Chemical shifts are
reported in ppm downfield to tetramethylsilane (TMS) (δ = 0). Coupling constant (J) is
shown in Hz. Data are reported in the following order: chemical shift, multiplicity (s,
singlet; bs, broad singlet; b, broad; d, doublet; t, triplet; q, quartet; m, multiplet; dd, double
doublet), coupling constant, and a number of protons. 13C NMR was recorded with FT-
NMR Spectrometer 400MHz in Chonnam Center for Research Facilities (CCRF). Mass
spectra were obtained on a Shimadzu LCMS-2010 EV liquid chromatograph mass
spectrometer using electron spray ionization (ESI) method. High-resolution mass
spectrometry (HRMS) results were obtained on a Waters Synapt G2 high definition mass
spectrometer. Elemental analyses were performed using
a
vario MICRO
cube/Elementar/Germany elemental analyzer. Values obtained were within ± 0.3% of
theoretical values. Column chromatography (gravity) was performed with a Merck silica
gel 60 (70−230 mesh). Thin layer chromatography (TLC) was performed using plates
21

coated with silica gel 60 F254 (Merck). All chemical reagents were purchased from Sigma-
Aldrich or Tokyo Chemical Industry Co., Ltd., Japan. They were used without further
purification. Solvents were distilled prior to use. All reactions were conducted in oven-
dried glassware with magnetic stirring.
Syntheses of bis-amides are outlined in Scheme 1. Bis-amides were prepared by
employing an Ugi multi-component reaction. Conventional Ugi four-component
condensation involves in situ formation of imine from an aldehyde 4 and an amine 5 which
further reacts with isocyanide 3 and carboxylic acid 6 to an intermediate which in turn
rearranges via an acyl transfer to yield desired bis-amides. Ugi reaction can also be carried
out with preformed imine 8, isocyanide 3, and carboxylic acid 6 (Schemes 1 and 2).
4.1.1 N-Cyclohexyl-2-({2-[1-(4-methoxy-benzyl)-1H-indol-3-yl]-acetyl}[12]-phenyl-
amino)-2-phenyl-acetamide (7a)
Procedure A: A solution of benzaldehyde 4a (50 mg, 0.47 mmol) (70 mg, 0.35
mmol) and aniline 5a (47 mg, 0.50 mmol) in methanol was stirred at RT for 6 h. To this
solution, cyclohexyl isocyanide 3a (51 mg, 0.47 mmol) and [1-(4-Methoxy-benzyl)-1H-
indol-3-yl]-acetic acid 6b (149 mg, 0.50 mmol) were added. The reaction mixture was
heated at 55 °C overnight, evaporated to dryness, washed with saturated NaHCO3 aqueous
solution, and extracted with EtOAc. The organic phase was dried over anhydrous sodium
sulfate and evaporated in vacuo. The resulting residue was purified to obtain bis-amide 7a
as a white solid (33 mg, 11%) after purification by column chromatography (n-
1
hexane:EtOAc=3:1). Mp: 187.3-191.9 °C. H NMR (300 MHz, CDCl3) δ: 7.41 (d, J = 7.5
Hz, 2H), 7.23 – 7.00 (m, 14H), 6.82 – 6.79 (m, 3H), 6.06 (s, 1H), 5.73 (d, J = 7.2 Hz, 1H),
22

5.15 (s, 2H), 3.82 – 3.72 (m, 1H), 3.77 (s, 3H), 3.58 (s, 2H), 1.87 – 1.77 (m, 2H), 1.62 –
1.51 (m, 3H), 1.35 – 1.21 (m, 2H), 1.10 – 0.85 (m, 3H). 13C NMR (100 MHz, DMSO-d6): δ
170.7, 169.2, 159.0, 140.46, 136.1, 131.5, 130.6, 130.5, 129.1, 128.6, 128.2, 128.0, 127.8,
121.5, 119.4, 119.0, 114.3, 110.4, 108.6, 64.1, 55.5, 48.9, 48.4, 32.6, 32.5, 31.8, 25.1, 25.0.
MS (ESI) m/z = 608 (M+Na)+, 584 (M-H)-.
4.1.2 N-Cyclohexyl-2-[(2-1H-indol-3-yl-acetyl)-(4-methoxy-phenyl)-amino]-2-(3,4,5-
trimethoxy-phenyl)-acetamide (7b)
Procedure A was used with cyclohexyl isocyanide 3a (56 mg, 0.51 mmol), 3,4,5-
trimethoxybenzaldehyde 4b (100 mg, 0.51 mmol), p-anisidine 5b (77 mg, 0.63 mmol), 3-
indoleacetic acid 6a (186 mg, 1.06 mmol), and methanol to obtain bis-amide 7b as an ivory
solid (217 mg, 72%) after purification by column chromatography (n-hexane:EtOAc=3:1).
Mp: 130.5-134.7 °C. 1H NMR (300 MHz, CDCl3) δ: 8.03 (s, 1H), 7.42 (d, J = 7.8 Hz, 2H),
7.31 (d, J = 8.1 Hz, 1H), 7.14 (t, J = 7.8 Hz, 1H), 7.04 (t, J = 8.1 Hz, 1H), 6.98 (s, 1H), 6.79
– 6.60 (m, 3H), 6.31 (s, 2H), 6.07 (s, 1H), 5.74 (d, J = 8.7 Hz, 1H), 3.77 (s, 4H), 3.74 (s,
3H), 3.62 – 3.54 (m, 8H), 1.19 – 1.80 (m, 2H), 1.66 – 1.53 (m, 3H), 1.36 – 1.23 (m, 2H),
13
1.10 – 0.99 (m, 3H). C NMR (100 MHz, DMSO-d6): 171.2, 169.3, 158.7, 152.5, 137.4,
136.4, 133.3, 132.5, 131.6, 127.8, 124.2, 121.3, 119.1, 118.6, 113.7, 111.7, 108.9, 108.4,
64.1, 60.4, 56.2, 55.7, 48.4, 32.7, 32.5, 31.8, 25.7, 25.1, 25.0. MS (ESI) m/z = 608
(M+Na)+, 584 (M-H)-. Anal. Calcd for C34H39N3O6·0.1H2O: C, 69.51; H, 6.73; N, 7.51.
Found: C, 69.57; H, 6.83; N, 7.23.
4.1.3 N-Cyclohexyl-2-[(2-1H-indol-3-yl-acetyl)-naphthalen-1-yl-amino]-2-(3,4,5-
23

trimethoxy-phenyl)-acetamide (7c)
Procedure A was used with cyclohexyl isocyanide 3a (56 mg, 0.51 mmol), 3,4,5-
trimethoxybenzaldehyde 4b (100 mg, 0.51 mmol), 1-aminonapthalene 5c (95 mg, 0.66
mmol), 3-indoleacetic acid 4a (116 mg, 0.66 mmol), and methanol at RT to obtain bis-
amide 7c as a violet solid (62 mg, 20%) after purification by column chromatography (n-
1
hexane:EtOAc=3:1). Mp: 206.5-215.1 °C (dec). H NMR (300 MHz, CDCl3) δ: 7.99 (s,
1H), 7.87 (d, J = 8.4 Hz, 1H), 7.76 (t, J = 8.7 Hz, 2H), 7.62 (d, J = 6.6 Hz, 1H), 7.40 – 7.28
(m, 5H), 7.13 – 7.08 (m, 1H), 7.04 – 6.96 (m, 1H), 6.85 (d, J = 2.1 Hz, 1H), 6.29 (s, 2H),
5.96 (s, 1H), 5.77 (d, J = 8.1 Hz, 1H), 3.85 – 3.79 (m, 1H), 3.73 (s, 3H), 3.49 (s, 2H), 3.47
(s, 6H), 1.91 – 1.86 (m, 2H), 1.65 – 1.56 (m, 3H), 1.40 – 1.23 (m, 2H), 1.14 – 1.01 (m, 3H).
13C NMR (100 MHz, DMSO-d6): 171.4, 169.5, 151.6, 137.1, 136.8, 136.3, 134.0, 132.1,
130.0, 129.8, 128.9, 128.2, 127.6, 127.1, 126.7, 126.4, 125.6, 124.3, 124.0, 121.3, 119.0,
119.0, 118.6, 111.6, 109.0, 108.7, 108.0, 65.0, 60.3, 60.2, 56.0, 55.7, 48.5, 32.5, 32.4, 31.7,
25.6, 25.1, 25.0. MS (ESI) m/z = 628 (M+Na)+, 604 (M-H)-.
4.1.4 N-Cyclohexyl-2-[(3,4-dimethoxy-phenyl)-(2-1H-indol-3-yl-acetyl)-amino]-2-
(3,4,5-trimethoxy-phenyl)-acetamide (7d)
Procedure A was used with cyclohexyl isocyanide 3a (55 mg, 0.50 mmol), 3,4,5-
trimethoxybenzaldehyde 4b (100 mg, 0.51 mmol), 4-aminoveratrole 5d (97 mg, 0.63
mmol), 3-indoleacetic acid 6a (133 mg, 0.76 mmol), and 2,2,2-trifluoroethanol at RT to
obtain bis-amide 7d as a light orange solid (239 mg, 76%) after purification by column
chromatography (n-hexane:EtOAc=3:1). Mp: 113.5-115.6 °C. 1H NMR (300 MHz, CDCl3)
δ: 7.99 (s, 1H), 7.39 – 7.37 (m, 1H), 7.31 (d, J = 7.8 Hz, 4H), 7.17 – 7.12 (m, 1H), 7.06 –
24

7.01 (m, 2H), 6.34 (s, 2H), 6.05 (s, 1H), 5.70 (d, J = 8.7 Hz, 1H), 3.82 (s, 4H), 3.77 (s, 4H),
3.63 – 3.61 (m, 9H), 3.40 – 3.31 (m, 1H), 1.92 – 1.81 (m, 2H), 1.68 – 1.60 (m, 3H), 1.35 –
13
1.23 (m, 2H), 1.18 – 1.03 (m, 3H). C NMR (100 MHz, DMSO-d6): 171.2, 169.4, 152.5,
148.3, 136.4, 131.7, 127.7, 124.2, 123.8, 121.3, 119.0, 118.6, 115.4, 111.7, 111.2, 109.0,
108.5, 60.4, 56.2, 56.0, 55.5, 48.4, 39.5, 32.7, 32.5, 31.9, 25.7, 25.1, 25.0. MS (ESI) m/z
= 638 (M+Na)+, 614 (M-H)-. Anal. Calcd for C35H41N3O7·0.65H2O: C, 67.00; H, 6.79; N,
6.70. Found: C, 67.12; H, 6.96; N, 6.76.
4.1.5 N-Cyclohexyl-2-[[2-(3,4-dimethoxy-phenyl)-ethyl]-(2-1H-indol-3-yl-acetyl)-
amino]-2-(3,4,5-trimethoxy-phenyl)-acetamide (7e)
Procedure A was used with cyclohexyl isocyanide 3a (55 mg, 0.50 mmol), 3,4,5-
trimethoxybenzaldehyde 4a (100 mg, 0.51 mmol), 3,4-dimethoxyphenethylamine 5e (144
mg, 0.63 mmol), 3-indoleacetic acid 6a (133 mg, 0.76 mmol), and 2,2,2-trifluoroethanol at
RT to obtain bis-amide 7e as a light yellow solid (18 mg, 5%) after purification by column
chromatography (n-hexane:EtOAc=3:1). Mp: 177.7-179.4 °C. 1H NMR (300 MHz, CDCl3)
δ: 8.11 (s, 1H), 7.60 (d, J = 8.1 Hz, 2H), 7.36 (d, J = 8.1 Hz, 2H), 7.22 – 7.08 (m, 3H), 6.70
(s, 2H), 6.42 – 6.39 (m, 1H), 5.93 (s, 1H), 5.75 (d, J = 8.4 Hz, 1H), 3.85 – 3.72 (m, 18H),
3.58 – 3.50 (m, 2H), 2.76 – 2.63 (m, 2H), 1.94 – 1.82 (m, 2H), 1.68 – 1.56 (m, 3H), 1.40 –
13
1.23 (m, 2H), 1.12 – 1.03 (m, 3H). C NMR (100 MHz, DMSO-d6): 172.1, 169.0, 153.3,
153.1, 149.1, 147.7, 137.8, 136.6, 132.7, 131.6, 127.5, 124.6, 123.8, 121.6, 120.7, 119.0,
118.9, 112.5, 112.4, 111.9, 108.7, 107.1, 106.5, 61.2, 60.6, 56.3, 56.1, 56.0, 55.7, 48.3, 32.6,
32.4, 31.6, 25.6, 25.0. MS (ESI) m/z = 666 (M+Na)+.
25

4.1.6 N-Cyclohexyl-2-[(2-1H-indol-3-yl-acetyl)-phenyl-amino]-2-(2,3,4-trimethoxy-
phenyl)-acetamide (7f)
Procedure A was used with cyclohexyl isocyanide 3a (55 mg, 0.50 mmol), 2,3,4-
trimethoxybenzaldehyde 4c (100 mg, 0.51 mmol), aniline 5a (59 mg, 0.63 mmol), 3-
indoleacetic acid 6a (88 mg, 0.50 mmol), and 2,2,2-trifluoroethanol at RT to obtain bis-
amide 7f as a light yellow solid (75 mg, 26%) after purification by column chromatography
(EtOAc). Mp: 108.5-112.3 °C. 1H NMR (300 MHz, CDCl3) δ: 8.04 (s, 1H), 7.38 (d, J = 7.8
Hz, 2H), 7.30 (d, J = 8.1 Hz, 1H), 7.15 – 7.10 (m, 4H), 7.05 – 6.98 (m, 3H), 6.67 (d, J = 8.7
Hz, 1H), 6.39 (s, 1H), 6.32 (d, J = 9.0 Hz, 1H), 5.64 (d, J = 8.1 Hz, 1H), 3.83 – 3.79 (m,
1H), 3.81 (s, 3H), 3.76 (s, 3H), 3.75 (s, 3H), 3.57 (s, 2H), 1.89 – 1.80 (m, 2H), 1.70 – 1.50
(m, 3H), 1.35 – 1.23 (m, 2H), 1.12 – 0.89 (m, 3H). 13C NMR (100 MHz, DMSO-d6): 170.6,
169.6, 153.6, 152.1, 141.4, 136.4, 128.6, 128.0, 124.9, 124.1, 121.9, 121.3, 119.0, 118.6,
111.7, 109.0, 107.2, 61.1, 60.4, 58.8, 56.1, 48.6, 39.8, 32.8, 32.6, 31.8, 25.7, 25.3, 25.1. MS
(ESI) m/z = 578 (M+Na)+, 554 (M-H)-.
4.1.7 2-[(4-Bromo-phenyl)-(2-1H-indol-3-yl-acetyl)-amino]-N-cyclohexyl-2-(2,3,4-
trimethoxy-phenyl)-acetamide (7g)
Procedure A was used with cyclohexyl isocyanide 3a (55 mg, 0.50 mmol), 2,3,4-
trimethoxybenzaldehyde 4c (100 mg, 0.51 mmol), 4-bromoaniline 5f (108 mg, 0.63 mmol),
3-indoleacetic acid 6a (88 mg, 0.50 mmol), and 2,2,2-trifluoroethanol at RT to obtain bis-
1
amide 7g as a white solid (163 mg, 50%) after filtration. Mp: 143.2-146.5 °C. H NMR
(300 MHz, CDCl3) δ: 8.00 (s, 1H), 7.37 – 7.29 (m, 4H), 7.24 – 7.19 (m, 2H), 7.16 – 7.11
(m, 1H), 7.07 – 7.00 (m, 2H), 6.63 (d, J = 8.7 Hz, 1H), 6.37 – 6.34 (m, 2H), 5.51 (d, J = 8.4
26

Hz, 1H), 3.83 – 3.75 (m, 1H), 3.82 (s, 3H), 3.77 (s, 3H), 3.76 (s, 3H), 3.56 (s, 2H), 1.92 –
1.82 (m, 2H), 1.68 – 1.54 (m, 3H), 1.38 – 1.23 (m, 2H), 1.14 – 0.91 (m, 3H). 13C NMR
(100 MHz, DMSO-d6): 170.4, 169.5, 152.1, 140.1, 136.4, 131.5, 127.6, 124.8, 124.1, 121.7,
121.3, 121.0, 119.0, 118.6, 111.7, 109.8, 107.4, 61.1, 60.4, 58.7, 56.2, 48.6, 32.8, 32.6, 32.0,
25.7, 25.3, 25.1. MS (ESI) m/z = 656 (M+Na)+. Anal. Calcd for C33H36BrN3O5·0.2H2O: C,
62.11; H, 5.75; N, 6.58. Found: C, 62.22; H, 5.87; N, 6.58.
4.1.8 N-Cyclohexyl-2-[(2-1H-indol-3-yl-acetyl)-(4-isopropyl-phenyl)-amino]-2-(2,3,4-
trimethoxy-phenyl)-acetamide (7h)
Procedure B: A solution of 2,3,4-trimethoxybenzaldehyde 4c (200 mg, 1.02 mmol)
and 4-isopropylaniline 5g (172 mg, 1.27 mmol) in ethanol was stirred at RT. The solid thus
formed was filtered off to obtain imine. The imine (160 mg, 0.51 mmol) was then added to
a solution of cyclohexyl isocyanide 3a (56 mg, 0.51 mmol) and 3-indole acetic acid 6a (112
mg, 0.64 mmol) in methanol and the reaction mixture was heated at 55 °C overnight. The
mixture was evaporated to dryness, washed with saturated NaHCO3 aqueous solution, and
extracted with EtOAc. The organic phase was dried over anhydrous sodium sulfate and
evaporated in vacuo. The resulting residue was purified by column chromatography using
n-hexane:EtOAc (3:1) to obtain bis-amide 7h as a light yellow solid (187 mg, 61%). Mp:
1
171.3-175.5 °C. H NMR (300 MHz, CDCl3) δ: 7.99 (s, 1H), 7.37 – 7.29 (m, 4H), 7.15 –
7.10 (m, 1H), 7.04 – 6.91 (m, 4H), 6.68 (d, J = 8.7 Hz, 1H), 6.35 – 6.31 (m, 2H), 5.67 (d, J
= 8.1 Hz, 1H), 3.79 – 3.74 (m, 1H), 3.78 (s, 3H), 3.76 (s, 3H), 3.75 (s, 3H), 3.58 (s, 2H),
2.81 (heptet, J = 6.9 Hz, 1H), 1.88 – 1.80 (m, 2H), 1.66 – 1.52 (m, 3H), 1.26 – 1.19 (m,
2H), 1.16 (d, J = 6.9 Hz, 6H), 1.08 – 0.91 (m, 3H). 13C NMR (100 MHz, DMSO-d6): 170.7,
27

169.5, 153.6, 148.0, 141.5, 138.4, 136.4, 130.5, 127.7, 126.3, 125.0, 124.1, 121.3, 119.0,
118.6, 111.6, 109.1, 107.2, 61.0, 60.3, 58.9, 56.2, 48.6, 33.4, 32.8, 32.6, 31.8, 25.7, 25.3,
25.1, 24.2, 24.1. MS (ESI) m/z = 620 (M+Na)+, 596 (M-H)-. Anal. Calcd for
C36H43N3O5·1.4H2O: C, 69.41; H, 7.41; N, 6.75. Found: C, 69.12; H, 7.78; N, 6.73.
4.1.9 N-Cyclohexyl-2-(3,4-dimethoxy-phenyl)-2-[(2-1H-indol-3-yl-acetyl)-(4-isopropyl-
phenyl)-amino]-acetamide (7i)
Procedure A was used with cyclohexyl isocyanide 3a (66 mg, 0.60 mmol), 3,4-
dimethoxybenzaldehyde 4d (100 mg, 0.60 mmol), 4-isopropylaniline 5g (101 mg, 0.75
mmol), 3-indoleacetic acid 6a (210 mg, 1.20 mmol), and methanol at RT to obtain bis-
amide 7i as a white solid (176 mg, 51%) after purification by column chromatography (n-
1
hexane:EtOAc=5:1). Mp: 163.7-168.4 °C. H NMR (300 MHz, CDCl3) δ: 8.01 (s, 1H),
7.37 – 7.29 (m, 3H), 7.16 – 7.10 (m, 2H), 7.05 – 6.98 (m, 4H), 6.76 (dd, J = 8.4, 1.8 Hz,
1H), 6.68 (d, J = 8.4 Hz, 1H), 6.47 (d, J = 2.1 Hz, 1H), 6.08 (s, 1H), 5.75 (d, J = 8.1 Hz,
1H), 3.82 (s, 3H), 3.80 – 3.74 (m, 1H), 3.58 (s, 2H), 3.50 (s, 3H), 2.84 (heptet, J = 6.9 Hz,
1H), 1.87 – 1.76 (m, 2H), 1.55 – 1.52 (m, 3H), 1.40 – 1.23 (m, 2H), 1.19 (d, J = 6.9 Hz,
13
6H), 1.10 – 0.87 (m, 3H). C NMR (100 MHz, DMSO-d6): 170.1, 169.5, 148.7, 148.2,
148.1, 138.3, 136.4, 131.3, 128.4, 127.7, 126.4, 124.1, 123.4, 121.3, 119.0, 118.6, 114.3,
111.7, 111.4, 108.9, 63.7, 55.8, 55.7, 48.3, 39.7, 33.4, 32.6, 32.5, 31.9, 25.7, 25.1, 25.0,
24.3, 24.1. MS (ESI) m/z = 590 (M+Na)+, 566 (M-H)-.
4.1.10 N-Cyclohexyl-2-(3,4-dimethoxy-phenyl)-2-[(3,4-dimethoxy-phenyl)-(2-1H-indol-
3-yl-acetyl)-amino]-acetamide (7j)
28