Discovery, synthesis, and optimization of an N-alkoxy indolylacetamide against HIV-1 carrying NNRTI-resistant mutations from the Isatis indigotica root

Article abstract of DOI:10.1016/j.ejmech.2020.112071

From an aqueous decoction of the traditional Chinese medicine “ban lan gen” (the Isatis indigotica root), an antiviral natural product CI - 39 was isolated as an NNRTI (non-nucleoside reverse transcriptase inhibitor) (EC₅₀ = 3.40 μM). Its novel structure was determined as methyl (1-methoxy-1H-indol-3-yl)acetamidobenzoate by spectroscopic data and confirmed by single crystal X-ray diffraction. Through synthesis and structure-activity relationship (SAR) investigation of CI - 39 and 57 new derivatives (24 with EC₅₀ values of 0.06–8.55 μM), two optimized derivatives 10f and 10i (EC₅₀: 0.06 μM and 0.06 μM) having activity comparable to that of NVP (EC₅₀ = 0.03 μM) were obtained. Further evaluation verified that 10f and 10i were RT DNA polymerase inhibitors and exhibited better activities and drug resistance folds compared to NVP against seven NNRTI-resistant strains carrying different mutations. Especially, 10i (EC₅₀ = 0.43 μM) was more active to the L100I/K103N double-mutant strain as compared to both NVP (EC₅₀ = 0.76 μM) and EFV (EC₅₀ = 1.08 μM). The molecular docking demonstrated a possible binding pattern between 10i and RT and revealed activity mechanism of 10i against the NNRTI-resistant strains.

Full text of DOI:10.1016/j.ejmech.2020.112071

Journal Pre-proof
Discovery, synthesis, and optimization of an N-alkoxy indolylacetamide against HIV-1
carrying NNRTI-resistant mutations from the Isatis indigotica root
Chengbo Xu, Yijing Xin, Minghua Chen, Mingyu Ba, Qinglan Guo, Chenggen Zhu,
Ying Guo, Jiangong Shi
PII:
S0223-5234(20)30038-6
DOI:
https://doi.org/10.1016/j.ejmech.2020.112071
EJMECH 112071
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 November 2019
Revised Date: 4 January 2020
Accepted Date: 13 January 2020
Please cite this article as: C. Xu, Y. Xin, M. Chen, M. Ba, Q. Guo, C. Zhu, Y. Guo, J. Shi, Discovery,
synthesis, and optimization of an N-alkoxy indolylacetamide against HIV-1 carrying NNRTI-resistant
mutations from the Isatis indigotica root, European Journal of Medicinal Chemistry (2020), doi: https://
doi.org/10.1016/j.ejmech.2020.112071.
This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition
of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of
record. This version will undergo additional copyediting, typesetting and review before it is published
in its final form, but we are providing this version to give early visibility of the article. 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.
©
2020 Published by Elsevier Masson SAS.

1

Discovery, synthesis, and optimization of an N-alkoxy
indolylacetamide against HIV-1 carrying NNRTI-resistant
mutations from the Isatis indigotica root
a,†
a,†
a,b
a
a
a
Chengbo Xu , Yijing Xin , Minghua Chen , Mingyu Ba , Qinglan Guo , Chenggen Zhu , Ying
a,
a,
Guo *, Jiangong Shi *
a
State Key Laboratory of Bioactive Substance and Function of Natural Medicines, Institute of Materia
Medica, Chinese Academy of Medical Sciences and Peking Union Medical College, Beijing 100050,
China
b
Institute of Medicinal Biotechnology, Chinese Academy of Medical Sciences and Peking Union
Medical College, Beijing 100050, China
�
Corresponding authors.
E-mail addresses: yingguo6@imm.ac.cn (Y. Guo), shijg@imm.ac.cn (J. Shi).
†
These authors contributed equally.
1

Abstract
From an aqueous decoction of the traditional Chinese medicine “ban lan gen” (the Isatis indigotica
root), an antiviral natural product CI-39 was isolated as an NNRTI (non-nucleoside reverse transcriptase
inhibitor) (EC50
=
3.40 µM). Its novel structure was determined as methyl
(1-methoxy-1H-indol-3-yl)acetamidobenzoate by spectroscopic data and confirmed by single crystal
X-ray diffraction. Through synthesis and structure-activity relationship (SAR) investigation of CI-39
and 57 new derivatives (24 with EC values of 0.06 – 8.55 µM), two optimized derivatives 10f and 10i
50
(
EC : 0.06 µM and 0.06 µM) having activity comparable to that of NVP (EC = 0.03 µM) were
5
0
5
0
obtained. Further evaluation verified that 10f and 10i were RT DNA polymerase inhibitors and
exhibited better activities and drug resistance folds compared to NVP against seven NNRTI-resistant
strains carrying different mutations. Especially, 10i (EC50 = 0.43 µM) was more active to the
L100I/K103N double-mutant strain as compared to both NVP (EC50 = 0.76 µM) and EFV (EC50 = 1.08
µM). The molecular docking demonstrated a possible binding pattern between 10i and RT and revealed
activity mechanism of 10i against the NNRTI-resistant strains.
KEY WORDS
Isatis indigotica; N-Alkyloxy indole derivative; HIV-1 inhibitor; Derivative synthesis, Structure-activity
relationship; NNRTI
2

1
. Introduction
Acquired immunodeficiency syndrome (AIDS) is caused by the human immunodeficiency virus
HIV) infection, which destroys the body's immune system by infecting human immune cells and
causing patients to eventually die from complications such as severe infections or secondary tumors
1−2]. In 2018, there were approximately 37.9 million people worldwide living with HIV/AIDS,
(
[
including 1.7 million children [3]. Moreover, 1.7 million individuals became newly infected in 2018. As
there is no AIDS vaccine available, highly active antiretroviral therapy (HAART) is still the main
clinical treatment of HIV/AIDS. Many HAART comprises two or three antiretroviral drugs acting on
different targets, such as reverse transcriptase (RT), protease (PR), and integrase (IN) [4−6]. However,
long-term widespread use of HAART can lead to drug-resistant viruses and treatment failure [7]. At
least 50% of patients have drug-resistant viruses. The non-nucleoside reverse transcriptase inhibitors
(NNRTIs) nevirapine (NVP) and efavirenz (EFV) have been used for more than 10 years in clinical
practice, and stable drug-resistant strains have emerged [8]. Clinical studies have found that after 48
weeks of etravirine and rilpivirine administration, the body produces a virus resistant to both the drugs.
Several new forms of NNRTIs have been reported in recent years [9−16]. In consideration of drug
resistance continues to increase, the development of new NNRTIs is still worth the challenge.
“Ban lan gen” (Isatis indigotica root) is one of the most important traditional Chinese medicines
used for the treatment of influenza and various infectious diseases [17] as well as anti-severe acute
respiratory syndrome (SARS) [18]. Previous studies demonstrated that indirubin, one of the bioactive
indole alkaloids of the herbal drug, had the most significant cytotoxicity on IL-60 cells and inhibitory
effect on pesudorabies virus (PrV) replication, while extracts from roots and leaves of I. indigotica also
showed cytopathic effect (CPE) reduction either before or after infection of PrV on porcine kidney
(PK-15) cells [18]. Moreover, from the ethanol extract of “da qing ye” (I. indigotica leaves) an isatisine
A-derived artificial acetonide was reported to have a moderate anti-HIV-1 activity (EC50 = 37.8 µM)
3

[19]. However as far as we know, was neither extract or purified compound from “ban lan gen” reported
to inhibit HIV virus replication. Because the “ban lan gen” decoction is practically used in clinic,
whereas the ethanolic extracts were mainly focused in the previous studies, we systematically studied
the chemical constituents and biological activities of an aqueous extract of the I. indigotica root,
resulted in characterization of more than 100 chemical constituents including around 50 alkaloids and
some with antiviral (influenza virus A/Hanfang/359/95, herpes simplex virus 1, and/or Coxsackie virus
B3) and cell-damage protective activities [20−34]. A continuation of the work led to characterization of
a novel anti-HIV compound methyl (1-methoxy-1H-indol-3-yl)acetamidobenzoate (CI-39) (Fig. 1A)
with an EC value of 3.40 µM from a remaining subfraction. CI-39 represents the first anti-HIV-1
50
indolylacetamidobenzoate derivative from I. indigotica, though from other plants a variety of natural
products and extracts with inhibition of HIV-1 replication were reported [35−40].
In view of the structure of CI-39 and its inhibitory effect on the HIV-1 virus, along with the recent
reports on indole derivatives with anti-HIV activity [41−55], we determined that the optimization of
activity and physicochemical properties by synthesis to obtain optimized lead compounds or drug
candidates was worthy of further study. Because of limitation of the sample amount from the plant
extract, CI-39 was firstly synthesized with confirmation of the anti-HIV-1 replication. For the structural
optimization, the effect of the following structural changes on the anti-HIV-1 activity were investigated
in this study: (i) the shift of methoxyformyl on the phenylamine moiety with replacement or
introduction of other functional groups; (ii) the replacement of the phenyl ring with an aromatic
heterocyclic ring; (iii) the substitution of the N-methoxy group on the indole nucleus by other N-alkoxy
groups; (iv) the extension or shortening of the acetamide linker between the indole and phenyl ring
units. The synthetic lead compound CI-39 and most potent derivatives were further tested for their
activity against the HIV-1 strains carrying some of the most clinically relevant mutations that confer
resistance to NNRTIs. Herein, reported are isolation, structural determination, and synthesis of the lead
4

compound CI-39, along with synthesis, anti-HIV-1 activity, and structure-activity relationship of 57
new derivatives as well as activity of three optimized compounds 6d, 10f, and 10i as novel NNRTIs
against seven NNRTI-resistant HIV-1 strains carrying different RT-mutations. Molecular docking of the
lead compound CI-39 and the most potent compound 10i is also discussed.
2
2
2
. Results and discussion
.1. Chemistry
.1.1. Isolation, structure elucidation, and antiviral assay of CI-39
The aqueous decoction of the air-dried and pulvarized I. indigotica root was concentrated under
reduced pressure and separated by column chromatography (CC) over macroporous adsorbent resin
HPD-110), eluting successively with H O, 50% EtOH, and 95% EtOH, to yield three corresponding
(
2
fractions A, B and C. Fraction C was subjected to CC over silica gel, with elution using a gradient of
increasing acetone concentration (0–100%) in petroleum ether, to afford fractions C1-C11 [20]. Fraction
C9 was separated successively by CC over Sephadex LH-20, reversed phase silica gel (C18), and silica
gel to give a mixture, from which CI-39 was isolated by HPLC using a semipreparative C18 column
(see Experimental section 4.1.1).
Compound CI-39 was obtained as colorless prisms in acetone. Its IR spectrum showed the
-1
-1
presence of amino (3247 cm ), ester and amide carbonyl (1701 and 1677 cm ), and aromatic ring (1585
-1
and 1511 cm ) functionalities in the molecule. Combinatory analysis of HRMS(ESI+) and NMR
spectroscopic data led to determination of the molecular formula as C19 . In the molecular, the
18 2 4
H N O
occurrence of a 3-substituted indole nucleus, an ortho-substituted benzene ring, two methoxy groups, an
isolated methylene, and two ester and amide carbonyl carbons was deduced from analysis of the NMR
spectroscopic data (Table 1). The connection of these structural units was further established by the 2D
NMR experimental data as illustrated in Fig. 1B. Briefly a linkage between the indole nucleus and one
5

carbonyl carbon via the methylene unit to form a 2-(1H-indol-3-yl)acetyl moiety was unambiguously
1
1
established by cross-peaks H-4/H-5/H-6/H-7 in the H− H COSY spectrum as well as the two- and
three-bond heteronuclear correlations from H-2 to C-3, C-3a, C-7a, and C-8 and from H -8 to C-2, C-3,
C-3a, and C-9 in the HMBC spectrum, along with the chemical shifts of these proton and carbon signals
Fig. 1B). Location of an methoxyformyl at the ortho-substituted benzene ring to construct an methyl
2
(
1
1
ortho-substituted benzoate moiety was deduced from the H− H COSY coupling correlations of
H-3′/H-4′/H-5′/H-6′ and the long-range HMBC correlations from H-3′ to C-1′ and C-5′ and from H-6′ to
C-2′, C-4, and C-7′ as well as from OCH to C-7′. To satisfy the requirement of the molecular
3
composition, the remaining methoxy unit must be located at the N atom of the 2-(1H-indol-3-yl)acetyl
moiety, in turn which must link via an amide bond to the only opened position of the methyl
ortho-substituted benzoate moiety. The deduction was finally proved by X-ray diffraction analysis of a
suitable single crystal crystallized in the acetone solution of CI-39, an ORTEP drawn of the crystal
structure shown in Fig. 1C. Thus, the structure of CI-39 was determined as methyl
(1-methoxy-1H-indol-3-yl)acetamidobenzoate.
According to clinic application of “Ban lan gen” (I. indigotica root) in traditional Chinese medicine
17], anti-viral activity of CI-39 was assayed using cell-based protocols as reported in our previous
[
publications [20−34, 56]. This compound showed significant effects on H3N2 (influenza virus
A/Hanfang/359/95, EC = 2.59 µM), HSV-1 (herpes simplex virus 1, EC = 0.95 µM), Coxsackie virus
5
0
50
B3 (EC50 = 3.70 µM), and HIV-1 (EC50 = 3.40 µM).
2
.1.2. Synthesis of CI-39 and its inhibitory activity against RT RNA-dependent DNA polymerase
The synthesis of CI-39 is shown in Scheme 1. Methyl 2-(2-(1H-indol-3-yl)acetamido)benzoate
(CI-a) was obtained through an amidation reaction of indole-3-carboxylic acid with 2-aminobenzoate.
Methyl 2-(2-(indolin-3-yl)acetamido)benzoate (CI-b) was prepared through reduction using
6

trifluoroacetic acid and triethylsilane [57]. The oxidation of CI-b was carried out by sodium tungstate
and hydrogen peroxide to produce methyl 2-(2-(1-hydroxy-1H-indol-3-yl)acetamido)benzoate (CI-c)
[58]. Then, the methylation of CI-c with trimethylsilyldiazomethane produced CI-39.
With the synthesized CI-39, inhibitory activities against RT RNA-dependent DNA polymerase
59] and ribonuclease H [60] were detected, along with an activity test against VSVG/HIV-1 replication
56]. The result (Table 3) indicated that CI-39 was an inhibitor of the RT RNA-dependent DNA
[
[
polymerase with the IC50 value of 7.20 µM, but inactive to ribonuclease H (IC50 > 30 µM).
2
.1.3. Structural modification of CI-39
The synthesis of N-alkoxy indole derivatives is shown in Scheme 2. The first two steps to obtain
the intermediates 2, 3, 7 and 8 are as same as the method of preparing CI-a, CI-b. The oxidation of 3
was carried out by m-CPBA to produce an indole N-hydroxyl derivative (4) [61]. The methylation of 4
with trimethylsilyldiazomethane produced the target product (5), and 6 was synthesized from 4 in the
presence of potassium carbonate and a halogenated alkane. The intermediates 8 were transformed into
the indole N-hydroxyl derivative (9) through an oxidation reaction using sodium tungstate and hydrogen
peroxide. Then, alkylation of 9 under the basic condition provided 10 (Scheme 3).
The synthesis of 14 and 15 is depicted in Scheme 4. The intermediate (13) was prepared
sequentially by oxidation of 11 and alkylation of the resulting product 12. A reaction of 13 with oxalyl
chloride and 3-chloro-4-aminopyridine yielded 14, which was further transformed into 15 by reduction
with sodium borohydride. The reaction of indole (16) and pyridinecarboxaldehyde under the basic
condition [62] afforded 17, which was sequentially reduced and oxidized to yield the intermediates 19
via 18. Then, propylation of 19 synthesized 20 (Scheme 5).
2
.1.4. Structure-activity relationships
7

All the synthesized CI-39 and 58 derivatives were tested against replication of the wild-type HIV-1
virus and their cytotoxicities on HEK 293T cell as well. As shown in Table 2, replacement of either the
N-OMe in CI-39 by N-H (CI-a) and N-OH (CI-c) or the indole by an indoline (CI-b) resulted in loss of
activity. This indicates that the N-substituted indole moiety is one of the key pharmacophores. Among
the phenylamine ring substituted derivatives (5), only those with ortho-halogen atoms Cl, Br, and I (5j,
5
m, and 5n) retained activity with the EC50 values of 3.30, 3.20, and 4.40 µM, respectively, whereas all
the others (5a−5i, 5k, 5l, and 5o−5u) as well as those with the acetyl linker replaced by propionyl and
butyryl (5v and 5w) resulted in loss of activity. Accordingly, ortho-substitution at the phenylamine
moiety by the electron-withdrawing groups (COOMe, Cl, Br, and I) and retention of the acetyl linker
are essential to maintain activity.
For the N-alkoxy-substituted derivatives of CI-39, activity was decreased by prolonging the linear
alkyl chain (6a and 6b). However, for the halogenated analogues of 5j, activity was enhanced by
extending of the linear alkyl chain from one to five carbon atoms, e.g. methoxy (5j: EC = 3.30 µM),
5
0
ethoxy (6c: EC = 0.86 µM), propoxy (6d: EC = 0.29 µM), n-butoxy (6e: EC = 0.64 µM), and
5
0
50
50
n-pentyloxy (6f: EC = 0.63 µM). When the number of alkyl linear carbon atoms exceeded to six, the
50
HIV-1 virus replication inhibitions were gradually decreased (6g: EC = 1.02 µM; 6h: EC = 1.95 µM;
5
0
50
6
i: EC = 4.40 µM), and completely lost when the carbon atom number reached to 9 (6j). In addition,
50
introduction of the bulky units at the N-atom of the indole moiety also weakened activity, e.g.
benzyloxy (6l: EC50 = 8.55 µM), phenethyloxy (6m: EC50 = 5.50 µM), and p-methoxybenzyloxy (6n).
Complete loss of activity was observed also when electron-withdrawing acetyloxy or benzyloxy
substituted at the N-atom of the indole moiety (6o and 6p). Interestingly, as compared with 6d (EC =
5
0
0
.29 µM), the potency was decreased by ortho-substitution of the electron-withdrawing group CF on
3
the phenylamine moiety (6q: EC = 4.28 µM), which differed from 5j, 5m, and 5n. The aforementioned
5
0
results indicate that the N-alkoxy in the CI-39 derivatives is required for activity against the HIV-1 virus
8

replication, and the best activity is gained when the number of the linear alkyl carbon atoms is three (6d:
EC = 0.29 µM).
5
0
With replacement of the phenylamine moiety by pyridinamines (10a-10c), activity was retained
only for the derivative of pyridine-4-amine (10c: EC = 4.95 µM). Simultaneous introduction of Cl or
50
Br at the ortho-position of the amino group in the pyridine-4-amine moiety led to a remarkable gain of
activity by almost an order of magnitude (10d: EC50 = 0.53 µM and 10e: EC50 = 0.66 µM). Further
substitution of N-methoxy by N-propoxy (10f: EC50 = 0.06 µM) or N-butoxy (10i: EC50 = 0.06 µM)
increased activity by one more order of magnitude, while relatively less increase of activity was
observed by substitution of N-methoxy with N-pentyloxy (10j: EC50 = 0.13 µM). In addition, as
compared with 10f, activity was reduced by an ortho-fluorine substituent on the pyridine-4-amine
moiety (10g: EC = 0.33 µM) or by hydroxylation of the acetyl linker (15: EC = 2.01 µM). Moreover,
50
50
comparison of 6d (EC50 = 0.29 µM) and 10h (EC50 = 3.30 µM) indicated that replacement of
-chloroaniline by pyrimidin-4-amine reduced activity by an order of magnitude. Further modification
2
of the acetyl linker through oxidation (14) and replacement with a methylene unit (20) resulted in the
disappearance of activity. Therefore, in the synthesized derivatives the 3-chloropyridin-4-amine moiety
is optimal to replace the methyl 2-aminobenzoate moiety in the natural product CI-39. The three potent
compounds 6d, 10f, and 10i were selected for following investigations. Notably, we discovered that the
V617F
intermediate 4o (ZT55, inactive to HIV-1) was a highly-selective tyrosine kinase inhibitor of JAK2
against myeloproliferative neoplasms [63] while other derivatives were inactive at 10 µM. As shown in
Table 2, all compounds cytotoxicity was tested, and the cell viabilities were influenced somehow by
treating with the compounds at the maximum concentration of 30 µM; nevertheless, the CC50 of all 59
compounds was higher than 10 µM, which was the highest concentration on anti-HIV replication assay.
2
.2 Biology
9

The time-of-drug addition (TOA) assay was conducted to identify the stage at which the active
compounds had the inhibitory effects on HIV-1. The reverse transcriptase inhibitors zidovudine (AZT)
and EFV, and the integrase inhibitor raltegravir (RAL) were used as reference drugs. The assay showed
that the activity lost curves of 6d, 10f, and 10i were close to those of AZT and EFV. They had a range
of time of 50% failure (FT ) from 8.2 to 9.1 h comparable to AZT at 8.5 h and EFV at 9.4 h, which
5
0
were far ahead of the integrase inhibitor RAL with a FT50 of 12.2 h (Figure 2), indicating that
compounds 6d, 10f and 10i inhibit HIV-1 replication at the reverse transcription step.
Reverse transcription is a process of generating double-stranded proviral DNA with an HIV-1
RNA genome as the template. This complex process is preceded by RT catalysis with DNA polymerase
and Ribonuclease H activities, during which (−)DNA strand is generated by DNA polymerase activity
based on the template RNA and the template RNA is hydrolyzed by Ribonuclease H [64−65]. Since the
lead compound CI-39 was proved as a RT DNA polymerase inhibitor, we also tested the effects of
compounds 6d, 10f and 10i on both HIV-1 RT DNA polymerase and Ribonuclease H activities. As
shown in Table 3, all three compounds displayed inhibitory activity on RT RNA-dependent DNA
polymerase, but not on Ribonuclease H, which indicated that compounds 6d, 10f, and 10i were RT
DNA polymerase inhibitors.
HIV-1 RT comprises two subunits p66 and p51. The p66 is the catalytic subunit designated as
fingers, palm, thumb, connection, and RNase H domains [66]. All members of approved NNRTIs are
RT-DNA polymerase inhibitors by binding to a hydrophobic pocket located between the base of the
thumb and the β-sheets of the palm domain, about 10 Å under the catalytic active center. Due to the low
fidelity of HIV-1 replication and over two decades of NNRTIs use in clinic, the NNRTI-resistant
mutants have developed. Among these, K103N, Y181C or Y188L, carried by RT, are the most prevalent
mutants [67]. Lys103 locates on the outer rim of the NNRTI-binding pocket and in the vicinity of the
entrance to the pocket; Tyr181 and Tyr188 locates at NNRTI-binding pocket. When Lys103 changes to
1
0

asparagine, the RT catalytic pocket is preferred in its closed form, resulting in less electrostatic forces
within the NNRTIs [68]. Both Tyr181 and Tyr188 exist at the catalytic pocket, which the NNRTIs act
on through the π-π interaction of their aromatic rings. As the mutations of Y181C or Y188L occur, the
forces between NNRTIs and RT by π-π interaction disappear [69]. For novel NNRTIs, the activity
against existing NNRTI-resistant HIV-1 is a pivotal characteristic.
In this study, we evaluated the activities of the natural product CI-39 and the potent derivatives 6d,
1
0f, and 10i against replication of the seven NNRTI-resistant HIV-1 strains. Overall, 10f and 10i
exhibited better activities and drug resistance folds compared to NVP against six of the seven resistant
strains (Table 4). Particularly, 10i (EC = 0.43 µM) was more notably more active against the double
5
0
mutant L100I/K103N comparing to both NVP (EC = 0.76 µM) and EFV (EC = 1.08 µM). It is worth
50
50
noting that the natural product CI-39 is the only one fully active on all the viral mutants though it did
not exhibit the best activity among the four tested compounds.
2
.3. Molecular Modeling
In order to better understand the possible binding mode of the active compounds, the potent 10i
and CI-39 as well as the positive controls NVP and EFV were docked into the HIV-1 WT RT (PDB
code: 1TL1 ) [69] and the HIV-1 RT mutants RT-Y181C (PDB code: 1JLB) [70], RT-K103N/Y181C
(PDB code: 5VQY) [71], and RT-L100I/K103N (PDB code: 2ZE2) [72], respectively. The docking
results of 10i into WT RT (Figure 3) showed: (i) π-alkyl interactions between the indole ring with
Lys103, Leu100, Val179, and Val106; (ii) alkyl interactions between the end of the butoxy chain with
Val106, Leu234, Phe227, and Pro225, respectively, in addition to an H-bond between the
oxygen-germinal hydrogen on the N-alkoxy chain and the Lys101 carbonyl oxygen; (iii) π-π interactions
between the pyridine ring with Tyr181, Tyr188, and Trp229, respectively; and (iv) interactions between
chlorine with the Tyr181, Pro95, and Leu100 residues. The docking into the Y181C mutated RT (1JLB)
(
Figure 4a) indicated that the interaction between chlorine and Tyr181 in WT RT was replaced by a
1
1

π-alkyl interaction between the pyridine ring and Cys181, while Tyr188 retained the π-π interaction with
the pyridine ring. In addition, the pyridine ring lost its acting force with Leu100, in turn which had a
π-sigma interaction with the indole ring. Differently the docking result of CI-39 into 1JLB showed that
the terminal methyl group of methoxyformyl on the phenylamine moiety shared the π-alkyl interactions
with Trp229, Phe227, and Tyr188 and the alkyl interaction with the Leu234 (Figure S187b in
Supporting Information).
Compound 10i docking into the K103N/Y181C double-mutated RT (5VQY) (Figure 4b) showed
2
that an H-bond was formed between the Asn103 amide NH group and the N-alkoxy oxygen atom,
replacing the π-alkyl interaction of the indole ring with Lys103 in WT RT. Meanwhile, the π-π
interaction between the pyridine ring and Tyr181 was substituted by a π-sulfur interaction between the
indole nucleus and -SH group. Additionally, the pyridine N atom hydrogen-bonded to the phenolic
hydroxy proton of Tyr188 and the terminal -NH of Lys223, while the pyridine ring maintained the π-π
2
interaction with Tyr188. However, the carbonyl oxygen of methoxyformyl on the phenylamine moiety
2
of CI-39 hydrogen-bonded to the terminal -NH of Lys223 and the phenolic hydroxy proton of Tyr188,
while Cys181 maintained the same mode of action as 10i (Figure S187c in Supporting Information). For
the positive control NVP, which is resisted by K103N/Y181C double-mutated RT, the docking
simulation (Figure S185c in Supporting Information) showed no interaction of NVP with Asn103. This
suggests that the H-bond between the N-alkoxy oxygen atom and the Asn103 amide NH group may
2
play an important role in the effect of 10i, which is supported by the experimental data (Table 4).
In the case of the L100I/K103N double-mutated RT (Figure 6c), the docking exhibited a position
exchange between the indole and pyridine ring moieties in the interacting model, and the hydroxy group
from isomerization of the acetamide unit in 10i was hydrogen-bonded to the terminal carbamide oxygen
of the mutated Asn103. The indole ring began to have a π-π interaction with Tyr188, and the pyridine
ring maintained the π-π effect on Tyr181 as well as the acting force with Ile100, in turn which the
1
2

alkyl-π interaction with the indole nucleus was replaced by an alkyl interaction with the N-butoxy chain.
Similarly the docking of CI-39 displayed the terminal carbamide oxygen of Asn103 hydrogen-bonded
to the hydroxy group from isomerization of the acetamide unit (Figure S187d in Supporting
Information). However, the mutated Ile100 exhibited the alkyl interactions with both the methyl group
of methoxyformyl and the indole nucleus of CI-39. As the L100I/K103N double-mutated RT resisted
EFV, the docking (Figure S186d in Supporting Information) showed the absence of interaction between
the mutated Ile100 and EFV. This suggests that the interactions between 10i/CI-39 and the mutated
Ile100 are effective on their activities compared to EFV (Table 4).
3
. Conclusions
A novel antiviral lead compound CI-39 was characterized from an aqueous decoction of the
traditional Chinese medicine “ban lan gen” (the I. indigotica root), which should have a contribution to
the clinical effect of the herbal medicine. Based on the unique structure with potent activity against
HIV-1 replication, 57 new derivatives were designed and synthesized, of which 24 were active with
EC values of 0.06 – 8.55 µM. Structure-activity relationship of these derivatives was clarified. The
5
0
biological assays demonstrated that CI-39 and its derivatives displayed their activities by inhibiting RT
DNA polymerase, but no acting on RNase H. The synthetic representatives 10f and 10i exhibited much
better inhibitory effects on the six of seven tested resistant strains compared to NVP. Moreover, the two
candidates strongly suppressed NNRTI-resistant HIV-1 carrying the RT-K103N/Y181C mutation.
Especially 10i was superior to both NVP and EFV against the HIV-1RT-L100I/K103N, which suggested that
it has the potential for use in developing a new NNRTI. Additionally, the computational docking results
also hinted at possible binding mechanisms of the potent 10i. In summary, this class of derivatives has a
good antiviral prospect and deserves further investigation.
4
. Experimental
1
3

4
4
.1. Chemistry
.1.1 Isolation and structural determination of CI-39
General experimental procedures, plant material, extraction, and preliminary fractionation of the
extract, see reference [20]. Fraction C9 (2.8 g) was subjected to CC over Sephadex LH-20
(
CHCl /MeOH, 1:1) to give C9-1−C9-3, of which C9-2 (1.2 g) was fractionated by RP flash CC
3
(
30–70% MeOH in H O) to give fractions C9-2-1−C9-2-3. Subfraction C-9-2-1 (500 mg) was separated
2
by CC over silica gel eluting with a gradient of increasing MeOH in CHCl (1 − 5%) afforded C-9-2-1-1
3
－
C-9-2-1-5, of which C-9-2-1-4 (15.4 mg) was isolated by reversed phase HPLC using a
semipreparative C18 column and the mobile phase of 33% MeOH in H O to yield CI-39 (2.1 mg).
2
Methyl (1-methoxy-1H-indol-3-yl)acetamidobenzoate (CI-39). Yield 0.0000042%. M.p. 95−96 ℃;
UV (MeOH) λmax (logε) 222(4.81), 252 (4.25), 260 (4.19), 290 (3.99), 300 (3.99) nm; IR (KBr) νmax
-1 1
3
247, 2948, 2842, 1701, 1677, 1585, 1511, 1452, 1410, 1294, 1264, 1092, 1031, 956, 762, 741 cm ; H
1
3
NMR (acetone-d
6
, 500 MHz) data see Table 1; C NMR (acetone-d
6
, 125 MHz) data, see Table 1.
, 339.1339).
+
+
ESIMS m/z 361 [M + Na] ; HR-ESIMS m/z 339.1320 [M + H] (Calcd for C19
X-Ray Crystallography of CI-39. Molecular formula C19 , monoclinic, P2
19 2 4
H N O
H
18
N
2
O
4
1
/c, a = 7.449 (3)
3
Å, b = 23.336 (12) Å, c = 10.026 (4) Å,
α
=
γ
= 90°,
β
= 93.84 (8)°, V = 1738.9 (13) Å , Z = 4, D
=
=
calcd
3
2
2
1
0
.292 g/cm , 3391 reflections independent, 2069 reflections observed (|F| ≥ 2σ|F| ), R
1
= 0.0521, wR
2
.1246, S = 1.004.
The data were collected on a Rigaku MicroMax 002+ diffractometer with CuKα radiation by using
the ω and κ scan technique to a maximum 2 value of 144.60°. The crystal structure was solved by
θ
direct methods by using SHELXS-97, and all non-hydrogen atoms were refined anisotropically using
the least-squares method. All hydrogen atoms were positioned by geometric calculations.
Crystallographic data have been deposited with the Cambridge Crystallographic Data Center (CCDC
1
4

8
66444).
Copies
of
these
data
can
be
obtained
free
of
charge
via
www.ccdc.cam.ac.uk/conts/retrieving.html (or from the Cambridge Crystallographic Data Centre, 12
Union Road, Cambridge CB21EZ, UK; fax: (+44) 1223-336-033; or deposit@ccdc.cam.ac.uk).
4
.1.2 Synthesis
All the synthetic starting materials and reagents were purchased from commercial suppliers and
used directly without further purification. Column chromatography was performed using silica gel
200–300 mesh) purchased from Qingdao Haiyang Chemical Co., Ltd. Thin-layer chromatography was
(
performed on precoated silica gel F-254 plates and was visualized under UV light. 1H NMR and 13C
NMR spectra were recorded on Varian Mercury 300 MHz, 400 MHz or 500 MHz with TMS or solvent
peaks as references. Chemical shifts (
δ) are reported in parts per million (ppm) and coupling constants
(J) are reported in hertz (Hz). The splitting patterns are indicated as: s = singlet, d = doublet, t = triplet,
q = quartet, dd = doublet of doublet, m = multiplet. HR-ESIMS data were obtained on an Agilent 6520
Accurate-Mass Q-TOF LCMS spectrometers (Agilent Technologies, Ltd., Santa Clara, CA, USA).
Analysis of sample purity was performed on an Agilent HPLC system consisting of 1100 controller,
1
100 pump, and 1100 dual λ absorbance detector with an CAPCELL PAK AQ-C18 column (5µm, 250
4.6 mm). HPLC conditions: solvent A: H O; solvent B: CH OH; flow rate 1.0 mL/min; compounds
×
2
3
were eluted with a gradient of 30% CH OH in H O with to 100% CH OH for 20 min. All tested
3
2
3
compounds have a purity ≥95.0%.
.1.2.1 Synthesis of CI-39
To a solution of indole-3-acetic acid (1.05 g, 1 equiv.) in dry CH Cl (100 mL) was added EDCI
4
2
2
(
1.27 g, 1.1 equiv.). The reaction mixture was stirred at room temperature for 1 h, then methyl
-aminobenzoate (1.0 g, 1.1 equiv.) and DMAP (0.07 g, 0.1 equiv.) were added. The mixture was stirred
at room temperature for 3 h; 2 M hydrochloric acid (100 mL) was added and stirred strongly for 10 min.
2
1
5

The organic phase was separated and the water phase was extracted with CH Cl (50 mL) twice. The
2
2
combined organic phase was washed with brine and dried with Na SO . Filtration and removal of the
2
4
solvent at 40 � under reduced pressure yielded methyl 2-(2-(1H-indol-3-yl)acetamido)benzoate (CI-a,
.03 g).
To a solution of CI-a (1.03 g, 1 equiv.) in CF
1
3
3
COOH (30 mL) was added Et SiH (1.2 g, 3 equiv.),
stirred at 60 � for 1 h, followed by recovery of the solvent under reduced pressure to yield a residue.
The residue was dissolved in ethyl acetate and washed by saturated Na CO and brine in order,
2
3
subsequent
removal
of
the
solvent
under
reduced
pressure
afforded
methyl
2
-(2-(indolin-3-yl)acetamido)benzoate (CI-b, 0.98 g). To a solution of CI-b (0.98 g, 1 equiv.) in MeOH
50 mL) was added sodium tungstate dehydrate (0.3 g, 0.3 equiv.) at 0 °C, followed by dropwise
addition of hydrogen peroxide (30%, 3.5 mL, 10 equiv.) then stirred at 10−15 °C for 1 h. The reaction
mixture was partitioned between CH Cl and water, and the organic phase was washed with brine and
(
2
2
evaporated, the resulting residue was purified by column chromatography (silica gel, petroleum
ether/ethyl acetate 2:1) to furnish methyl 2-(2-(1-hydroxy-1H-indol-3-yl)acetamido)benzoate (CI-c,
0
.69 g). Selectively the CH Cl2 solution of CI-c (0.69 g) was added a solution of
2
trimethylsilyldiazomethane in n-hexane (6 M, 3.5mL, 10 equiv), stirred at room temperature overnight,
followed by recovery of the organic solvent to give a residue. The residue was purified by silica gel
column chromatography (petroleum ether/ethyl acetate 2:1) to yield CI-39 (0.49 g, four steps yield
2
4.2%).
Methyl 2-(2-(1H-indol-3-yl)acetamido)benzoate (CI-a) [73]. Yield 55.5%. H NMR (500 MHz,
DMSO-d ): δ 11.07 (s, 1H, CONH), 10.59 (s, 1H, NH), 8.41 (d, J = 8.5 Hz, 1H, ArH), 7.84 (dd, J = 2.0,
1
6
8
.0 Hz, 1H, ArH), 7.58 (t, J = 7.5 Hz, 1H, ArH), 7.51 (d, J = 7.5 Hz, 1H, ArH), 7.38 (m, 2H, ArH), 7.10
(
m, 2H, ArH), 6.97 (t, J = 7.5 Hz, 1H, ArH), 3.83 (s, 2H, CH ), 3.64 (s, 3H, CH ). ESIMS m/z 331 [M +
2
3
+
-
Na] , 344 [M + Cl] .
1
6

1
Methyl 2-(2-(indolin-3-yl)acetamido)benzoate (CI-b). Yield 95.0%. H NMR (500 MHz,
acetone-d ): δ 11.00 (s, 1H, CONH), 8.74 (d, J = 8.5 Hz, 1H, ArH), 8.03 (dd, J = 2.0, 8.5 Hz, 1H, ArH),
6
7
4
1
1
.60 (t, J = 8.0 Hz, 1H, ArH), 7.13 (m, 2H, ArH), 6.94 (t, J = 7.5 Hz, 1H, ArH), 6.57 (m, 2H, ArH),
.89 (s, 1H, NH), 3.90 (s, 3H, CH ), 3.75 (m, 2H, CH ), 3.30 (m, 1H, CH), 2.87 (m, 1H, CH ), 2.67 (m,
3
2
2
13
H, CH ); C NMR (125 MHz, acetone-d ): δ 170.9, 169.1, 153.1, 142.3, 135.2, 132.1, 131.6, 128.4,
2
6
+
24.5, 123.2, 120.9, 118.3, 116.0, 109.8, 53.9, 52.8, 43.5, 39.5; HR-ESIMS m/z 311.1398 [M + H]
Calcd for C18 , 311.1390).
Methyl 2-(2-(1-hydroxy-1H-indol-3-yl)acetamido)benzoate (CI-c). Yield 67.5%. H NMR (500
(
19 2 3
H N O
1
MHz, acetone-d ): δ 10.87 (1H, s, NH), 10.21 (1H, s, OH), 8.71 (1H, d, J = 8.5 Hz, ArH), 7.93 (1H, dd,
6
J = 1.5, 8.0 Hz, ArH), 7.56 (2H, m, ArH), 7.49 (1H, s, ArH), 7.42 (1H, d, J = 8.0 Hz, ArH), 7.17 (1H, t,
J = 7.5 Hz, ArH), 7.09 (1H, t, J = 7.5 Hz, ArH), 7.02 (1H, t, J = 7.5 Hz, ArH), 3.86 (2H, s, CH
2
), 3.74
): δ 170.8, 168.2, 142.4, 135.0 (2C), 131.5, 125.8,
24.7, 123.1, 122.8, 120.7, 120.0, 119.6, 116.4, 109.2, 104.2, 52.6, 35.8; HR-ESIMS m/z 325.119 [M +
1
3
(
3 6
3H, s, COOCH ); C NMR (150 MHz, acetone-d
1
+
H] (Calcd for C H N O , 325.1183).
1
8
17
2
4
4
.1.2.2 General procedure for the synthesis of 5, 6 and 10
The first two steps of general procedure for the synthesis of intermediates 2, 3, 7 and 8 are same as
that of preparing CI-a, CI-b. To a solution of 3 (1 equiv.) in MeOH was added m-CPBA (2 equiv.),
stirred at room temperature for 2 h, then concentrated under reduced pressure to yield a residue, which
was dissolved in CH Cl and washed successively by saturated Na CO and brine. The CH Cl solution
2
2
2
3
2
2
was evaporated and the resulting residue was purified by column chromatography (silica gel, petroleum
ether/ethyl acetate 2:1) to furnish 4. Similarly, 9 was obtained as same as the procedure of prepare CI-c.
2 2
Selectively the CH Cl solution of 4 or 9 was added to a solution of trimethylsilyldiazomethane in
n-hexane (6 M, 10 equiv) or the alkyl halide (1.5 equiv.), stirred at room temperature overnight or for 2
h in DMF with K CO (2 equiv.), followed by partition between CH Cl and water, then dry with
2
3
2
2
1
7

Na SO and recovery of the organic solvent or to give a residue. The residue was purified by silica gel
2
4
column chromatography (petroleum ether/ethyl acetate 2:1) to yield 5, 6, and 10.
1
2
-(1-Methoxy-1H-indol-3-yl)-N-phenylacetamide (5a). Four steps yield 29.6%. H NMR (400
6
MHz, acetone-d ): δ 9.16 (s, 1H, CONH), 7.64 (m, 3H, ArH), 7.46 (s, 1H, ArH), 7.42 (d, J = 8.4 Hz,
13
1
3 2
H, ArH), 7.23 (m, 3H, ArH), 7.04 (m, 2H, ArH), 4.05 (s, 3H, OCH ), 3.79 (s, 2H, CH ); C NMR
(
100 MHz, acetone-d ): δ 169.9, 140.4, 133.4, 129.4 (2C), 124.9, 124.1, 123.4, 123.2, 120.4, 120.2 (2C),
6
+
1
20.1, 109.0, 106.6, 66.1, 34.6; HR-ESIMS m/z 281.1287 [M + H] (Calcd for C H N O , 281.1285).
1
7
17
2
2
1
2
-(1-Methoxy-1H-indol-3-yl)-N-(o-tolyl)acetamide (5b). Four steps yield 20.0%. H NMR (400
MHz, acetone-d ): δ 8.33 (s, 1H, CONH), 7.72 (d, J = 8.0 Hz, 1H, ArH), 7.68 (d, J = 8.0 Hz, 1H, ArH),
.54 (s, 1H, ArH), 7.45 (d, J = 8.4 Hz, 1H, ArH), 7.24 (t, J = 7.6 Hz, 1H, ArH), 7.10 (m, 3H, ArH), 6.99
6
7
13
(
t, J = 7.6 Hz, 1H, ArH), 4.10 (s, 3H, OCH ), 3.84 (s, 2H, CH ), 2.01 (s, 3H, CH ); C NMR (100 MHz,
3 2 3
acetone-d ): δ 169.7, 137.6, 133.4, 131.0, 130.7, 126.9, 125.4, 124.8, 124.1, 123.6, 123.3, 120.6, 120.1,
6
+
1
09.0, 106.6, 66.2, 34.3, 17.7; HR- ESIMS m/z 295.1447 [M + H] (Calcd for C H N O , 295.1441).
1
8
19
2
2
1
2
-(1-Methoxy-1H-indol-3-yl)-N-(p-tolyl)acetamide (5c). Four steps yield 27.8%. H NMR (400
MHz, acetone-d ): δ 9.11 (s, 1H, CONH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.51 (d, J = 8.4 Hz, 2H, ArH),
.44 (s, 1H, ArH), 7.41 (d, J = 8.4 Hz, 1H, ArH), 7.21 (t, J = 8.0 Hz, 1H, ArH), 7.06 (t, 3H, ArH), 4.06
6
7
1
3
(
s, 3H, OCH ), 3.77 (s, 2H, CH ), 2.24 (s, 3H, CH ); C NMR (100 MHz, acetone-d ): δ 169.7, 137.8,
3 2 3 6
1
2
33.3, 129.8 (2C), 124.9, 123.4, 123.1, 120.4, 120.2, 108.9, 106.7, 66.1, 34.6, 20.7; HR-ESIMS m/z
+
95.145 [M + H] (Calcd for C H N O , 295.1441).
1
8
19
2
2
1
N-(2-Ethylphenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5d). Four steps yield 27.8%. H NMR
(
400 MHz, acetone-d ): δ 8.20 (s, 1H, CONH), 7.76 (d, J = 8.0 Hz, 1H, ArH), 7.68 (d, J = 8.0 Hz, 1H,
6
ArH), 7.57 (s, 1H, ArH), 7.46 (d, J = 7.6 Hz, 1H, ArH), 7.25 (t, J = 7.6 Hz, 1H, ArH), 7.11 (m, 3H,
ArH), 7.03 (t, J = 6.8 Hz, 1H, ArH), 4.12 (s, 3H, OCH ), 3.84 (s, 2H, CH ), 2.33 (q, J = 7.6, 14.8 Hz,
3
2
1
8

1
3
2
1
H, CH ), 0.85 (t, 3H, J = 7.6 Hz, CH ); C NMR (100 MHz, acetone-d ): δ 169.8, 136.8, 136.4, 133.4,
2 3 6
29.4, 126.9, 125.7, 124.7, 124.4, 123.7, 123.5, 120.7, 120.1, 109.1, 106.5, 66.3, 34.4, 24.7, 14.3;
+
HR-ESIMS m/z 309.1601 [M + H] (Calcd for C H N O , 309.1598).
1
9
21
2
2
1
Methyl 3-[2-(1-methoxy-1H-indol-3-yl)acetamido]benzoate (5e). Four steps yield 21.4%. H NMR
400 MHz, acetone-d ): δ 9.37 (s, 1H, CONH), 8.29 (s, 1H, ArH), 7.89 (d, J = 8.0 Hz, 1H, ArH), 7.67
d, J = 7.6 Hz, 2H, ArH), 7.48 (s, 1H, ArH), 7.40 (m, 2H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (t, J
(
6
(
1
3
=
7.6 Hz, 1H, ArH), 4.09 (s, 3H, OCH ), 3.84 (s, 3H, COOCH ), 3.83 (s, 2H, CH ); C NMR (100
3 3 2
MHz, acetone-d ): δ 170.2, 167.0, 140.6, 133.4, 131.6, 129.7, 124.9, 124.86, 124.4, 123.5, 123.2, 120.9,
6
+
1
3
20.5, 120.1, 109.0, 106.3, 66.2, 52.3, 34.6; HR-ESIMS m/z 339.1347 [M + H] (Calcd for C H N O ,
1
9
19
2
4
39.1339).
Methyl 4-[2-(1-methoxy-1H-indol-3-yl)acetamido]benzoate (5f). Four steps yield 24.7%. H NMR
400 MHz, acetone-d ): δ 9.49 (s, 1H, CONH), 7.92 (d, 2H, J = 8.4 Hz, ArH), 7.76 (d, J = 8.4 Hz, 2H,
1
(
6
ArH), 7.65 (d, J = 8.0 Hz, 1H, ArH), 7.46 (s, 1H, ArH), 7.41 (d, J = 8.0 Hz, 1H, ArH), 7.21 (t, J = 7.6
Hz, 1H, ArH), 7.07 (t, J = 7.6, 1H, ArH), 4.10 (s, 3H, OCH ), 3.84 (s, 2H, CH ), 3.83 (s, 3H,
3
2
13
COOCH ); C NMR (100 MHz, acetone-d ): δ 170.5, 166.7, 144.5, 133.3, 131.1 (2C), 125.6, 124.8,
3
6
1
23.5, 123.2, 120.5, 120.1, 119.3 (2C), 109.0, 106.1, 66.1, 52.0, 34.7; HR-ESIMS m/z 339.1344 [M +
+
H] (Calcd for C19
H
19
N O
2 4
, 339.1339).
1
Ethyl 2-[2-(1-methoxy-1H-indol-3-yl)acetamido]benzoate (5g). Four steps yield 20.0%. H NMR
(
400 MHz, acetone-d ): δ 11.00 (s, 1H, CONH), 8.76 (d, J = 8.4 Hz, 1H, ArH), 7.92 (d, J = 8.0 Hz, 1H,
6
ArH), 7.60 (s, 1H, ArH), 7.59 (d, J = 9.2Hz, 1H, ArH), 7.53 (t, J = 8.0 Hz, 1H, ArH), 7.46 (d, J = 8.0
Hz, 1H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (m, 2H, ArH), 4.180 (m, 5H, CH , OCH ), 3.87 (s,
): δ 170.7, 168.2, 142.4, 134.9,
2
3
1
3
2
1
2 3 6
H, CH ), 1.25 (t, J = 6.8 Hz, 3H, CH ); C NMR (100 MHz, acetone-d
33.3, 131.5, 124.8, 124.2, 123.3, 123.0, 120.6, 120.5, 119.8, 116.2, 109.0, 105.1, 66.4, 61.9, 35.7, 14.3;
+
HR-ESIMS m/z 353.1504 [M + H] (Calcd for C H N O , 353.1496).
2
0
21
2
4
1
9

1
2
-(1-Methoxy-1H-indol-3-yl)-N-(4-nitrophenyl)acetamide (5h). Four steps yield 25.9%. H NMR
(
400 MHz, acetone-d ): δ 9.76 (s, 1H, CONH), 8.17 (d, J = 9.2 Hz, 2H, ArH), 7.88 (d, J = 8.8 Hz, 2H,
6
ArH), 7.64 (d, J = 7.6 Hz, 1H, ArH), 7.49 (s, 1H, ArH), 7.42 (d, J = 7.6 Hz, 1H, ArH), 7.21 (t, J = 7.6
1
3
Hz, 1H, ArH), 7.07 (t, J = 7.2 Hz, 1H, ArH), 4.09 (s, 3H, OCH ), 3.88 (s, 2H, CH ); C NMR (100
3
2
MHz, acetone-d ): δ 170.8, 146.3, 143.8, 133.3, 125.5 (2C), 124.8, 123.6, 123.3, 120.5, 120.1, 119.7
6
+
(
2C), 109.0, 105.8, 66.2, 34.7; HR-ESIMS m/z 326.1144 [M + H] (Calcd for C17
H
16
N
3
O
4
, 326.1135).
1
N-(2-Fluorophenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5i). Four steps yield 18.5%. H NMR
(
400 MHz, acetone-d ): δ 8.83 (s, 1H, CONH), 8.22 (t, J = 8.0 Hz, 1H, ArH), 7.68 (d, J = 8.0 Hz, 1H,
6
ArH), 7.52 (s, 1H, ArH), 7.44 (d, J = 8.0 Hz, 1H, ArH), 7.22 (t, J = 7.6 Hz, 1H, ArH), 7.09 (m, 4H,
1
3
ArH), 4.09 (s, 3H, OCH ), 3.91 (s, 2H, CH ); C NMR (100 MHz, acetone-d ): δ 170.1, 133.4, 125.2,
3
2
6
1
25.14, 125.09, 125.06, 124.8, 123.5, 123.4, 123.3, 120.5, 120.1, 115.8, 115.6, 109.0, 106.3, 66.2, 34.3;
+
HR-ESIMS m/z 299.1193 [M + H] (Calcd for C17
H
16
N
2
O
2
F, 299.1190).
1
N-(2-Chlorophenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5j). Four steps yield 22.0%. H NMR
(
400 MHz, acetone-d ): δ 8.46 (s, 1H, CONH), 8.29 (d, J = 8.0 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H,
6
ArH), 7.61 (s, 1H, ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.10 (t, J = 7.6 Hz, 1H, ArH), 7.05 (t, J = 8.0 Hz, 1H, ArH), 4.12 (s, 3H, OCH ), 3.925 (s, 2H,
3
1
3
CH
2
); C NMR (100 MHz, acetone-d
6
): δ 169.9, 136.1, 133.4, 129.9, 128.3, 125.5, 124.7, 123.9, 123.5,
+
1
22.9 (2C), 120.8, 120.0, 109.1, 105.8, 66.3, 34.5; HR-ESIMS m/z 315.0905 [M + H] (Calcd for
C H N O Cl, 315.0895).
17
16
2
2
1
N-(3-Chlorophenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5k). Four steps yield 23.1%. H NMR
400 MHz, acetone-d ): δ 9.33 (s, 1H, CONH), 7.89 (s, 1H, ArH), 7.64 (d, J = 8.0 Hz, 1H, ArH), 7.44
m, 3H, ArH), 7.26 (t, J = 8.0 Hz, 1H, ArH), 7.21 (t, J = 8.0 Hz, 1H, ArH), 7.06 (m, 2H, ArH), 4.09 (s,
(
6
(
1
3
3
3 2 6
H, OCH ), 3.80 (s, 2H, CH ); C NMR (100 MHz, acetone-d ): δ 170.3, 141.7, 134.6, 133.3, 130.9,
2
0

1
3
24.8, 123.9, 123.5, 123.2, 120.5, 120.1, 119.9, 118.3, 109.0, 106.2, 66.2, 34.6; HR-ESIMS m/z
+
15.0897 [M + H] (Calcd for C H N O Cl, 315.0895).
1
7
16
2
2
1
N-(4-Chlorophenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5l). Four steps yield 20.4%. H NMR
400 MHz, acetone-d ): δ 9.30 (s, 1H, CONH), 7.65 (m, 3H, ArH), 7.46 (s, 1H, ArH), 7.41 (d, J = 8.4
Hz, 1H, ArH), 7.27 (d, J = 8.4 Hz, 2H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (t, J = 7.6 Hz, 1H,
(
6
1
3
ArH), 4.08 (s, 3H, OCH ), 3.79 (s, 2H, CH ); C NMR (100 MHz, acetone-d ): δ 170.1, 139.2, 133.3,
3
2
6
1
29.3 (2C), 128.4, 124.8, 123.5, 123.2, 121.6 (2C), 120.5, 120.1, 109.0, 106.3, 66.2, 34.6; HR-ESIMS
+
m/z 315.0905 [M + H] (Calcd for C H N O Cl, 315.0895).
1
7
16
2
2
1
N-(2-Bromophenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5m). Four steps yield 11.1%. H NMR
(
400 MHz, acetone-d ): δ 8.32 (s, 1H, CONH), 8.27 (d, J = 8.0 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz, 1H,
6
ArH), 7.64 (s, 1H, ArH), 7.49 (d, J = 7.6 Hz, 1H, ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.32 (t, J = 8.0
Hz, 1H, ArH), 7.25 (t, J = 7.6 Hz, 1H, ArH), 7.10 (t, J = 7.6 Hz, 1H, ArH), 6.99 (t, J = 8.0 Hz, 1H,
1
3
ArH), 4.13 (s, 3H, OCH ), 3.91 (s, 2H, CH ); C NMR (100 MHz, acetone-d ): δ 169.9, 137.2, 133.4,
3
2
6
1
33.2, 129.0, 126.0, 124.7, 124.0, 123.6, 122.9, 120.9, 120.0, 114.2, 109.1, 105.6, 66.4, 34.6;
+
HR-ESIMS m/z 359.0403 [M + H] (Calcd for C H N O Br, 359.0390).
1
7
16
2
2
1
N-(2-Iodophenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5n). Four steps yield 10.3%. H NMR
(
400 MHz, acetone-d ): δ 8.17 (d, J = 8.4 Hz, 1H, ArH), 8.13 (s, 1H, CONH), 7.72 (d, J = 7.6 Hz, 1H,
6
ArH), 7.65 (m, 2H, ArH), 7.47 (d, J = 8.4 Hz, 1H, ArH), 7.33 (t, J = 8.0 Hz, 1H, ArH), 7.25 (t, J = 8.0
Hz, 1H, ArH), 7.10 (t, J = 7.6 Hz, 1H, ArH), 6.83 (t, J = 7.6 Hz, 1H, ArH), 4.14 (s, 3H, OCH ), 3.89 (s,
3
1
3
2
1
H, CH ); C NMR (100 MHz, acetone-d ): δ 169.9, 139.8 (2C), 133.5, 129.7, 126.7, 124.7, 124.2,
2 6
+
23.6, 122.7 (2C), 120.9, 120.1, 109.2, 105.4, 66.5, 34.6; HR-ESIMS m/z 407.0269 [M + H] (Calcd for
17 2 2
C H16IN O
, 407.0251).
2
1

1
2
-(1-Methoxy-1H-indol-3-yl)-N-(2-methoxyphenyl)acetamide (5o). Four steps yield 35.2%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.33 (d, J = 7.6 Hz, 1H, ArH), 7.67 (d, J = 7.6 Hz,
6
1
7
H, ArH), 7.56 (s, 1H, ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.24 (t, J = 7.6 Hz, 1H, ArH), 7.10 (t, J =
.6 Hz, 1H, ArH), 6.96 (t, J = 8.0 Hz, 1H, ArH), 6.87 (m, 2H, ArH), 4.13 (s, 3H, OCH ), 3.86 (s, 2H,
3
1
3
CH ), 3.64 (s, 3H, OCH ); C NMR (100 MHz, acetone-d ): δ 169.5, 149.1, 133.4, 129.1, 124.7, 124.2,
2
3
6
1
3
23.7, 123.4, 121.3, 120.7, 120.13, 120.09, 111.2, 109.1, 106.4, 66.3, 56.0, 34.8; HR-ESIMS m/z
+
11.1397 [M + H] (Calcd for C18
H
19
N
2
O
3
, 311.1390).
1
2
-(1-Methoxy-1H-indol-3-yl)-N-(3-methoxyphenyl)acetamide (5p). Four steps yield 34.6%. H
NMR (400 MHz, acetone-d ): δ 9.30 (s, 1H, CONH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.46 (s, 1H, ArH),
6
7
.41 (d, J = 6.8Hz, 2H, ArH), 7.20 (t, J = 7.6 Hz, 1H, ArH), 7.13 (m, 2H, ArH), 7.06 (t, J = 7.6 Hz, 1H,
13
3 2 3
ArH), 6.59 (d, J = 6.8Hz, 1H, ArH), 4.08 (s, 3H, OCH ), 3.78 (s, 2H, CH ), 3.72 (s, 3H, OCH ); C
NMR (100 MHz, acetone-d
6
): δ 169.9, 160.9, 141.6, 133.3, 130.1, 124.9, 123.4, 123.1, 120.4, 120.2,
+
1
12.3, 109.5, 108.9, 106.6, 105.9, 66.1, 55.4, 34.6; HR-ESIMS m/z 311.1399 [M + H] (Calcd for
C H N O , 311.1390).
18
19
2
3
1
2
-(1-Methoxy-1H-indol-3-yl)-N-(4-methoxyphenyl)acetamide (5q). Four steps yield 32.9%. H
NMR (400 MHz, acetone-d
6
): δ 9.02 (s, 1H, CONH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.52 (d, J = 8.8 Hz,
H, ArH), 7.45 (s, 1H, ArH), 7.41 (d, J = 8.0 Hz, 1H, ArH), 7.20 (t, J = 7.6 Hz, 1H, ArH), 7.06 (t, J =
.6 Hz, 1H, ArH), 6.82 (d, J = 8.8 Hz, 2H, ArH), 4.08 (s, 3H, OCH ), 3.75 (s, 2H, CH ), 3.73 (s, 3H,
2
7
3
2
1
3
OCH ); C NMR (100 MHz, acetone-d ): δ 169.5, 156.7, 133.5, 133.4, 124.9, 123.4, 123.2, 121.7 (2C),
3
6
+
1
20.4, 120.2, 114.5 (2C), 108.9, 106.7, 66.1, 55.6, 34.5; HR-ESIMS m/z 311.1398 [M + H] (Calcd for
C H N O , 311.1390).
18
19
2
3
1
N-(3,4-Dimethylphenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5r). Four steps yield 26.9%. H
NMR (400 MHz, acetone-d ): δ 8.99 (s, 1H, CONH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.45 (s, 1H, ArH),
.41 (d, J = 8.0 Hz, 1H, ArH), 7.38 (s, 1H, ArH), 7.35 (d, J = 8.0 Hz, 1H, ArH), 7.20 (t, J = 7.6 Hz, 1H,
6
7
2
2

ArH), 7.06 (t, J = 7.6 Hz, 1H, ArH), 6.99 (d, J = 8.0 Hz, 1H, ArH), 4.08 (s, 3H, OCH ), 3.76 (s, 2H,
3
1
3
CH ), 2.16 (m, 6H, CH ×2); C NMR (100 MHz, acetone-d ): δ 169.6, 138.1, 137.3, 133.4, 132.0,
2
3
6
1
30.4, 124.9, 123.4, 123.2, 121.4, 120.4, 120.2, 117.7, 108.9, 106.7, 66.1, 34.6, 19.9, 19.1; HR-ESIMS
+
m/z 309.1601 [M + H] (Calcd for C H N O , 309.1598).
1
9
21
2
2
1
N-(3,5-Dimethylphenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5s). Four steps yield 29.9%. H
NMR (400 MHz, acetone-d ): δ 9.00 (s, 1H, CONH), 7.66 (d, J = 8.0 Hz, 1H, ArH), 7.46 (s, 1H, ArH),
.41 (d, J = 8.0 Hz, 1H, ArH), 7.25 (s, 2H, ArH), 7.20 (t, J = 7.6 Hz, 1H, ArH), 7.06 (t, J = 7.6 Hz, 1H,
6
7
1
3
ArH), 6.66 (s, 1H, ArH), 4.08 (s, 3H, OCH ), 3.77 (s, 2H, CH ), 2.20 (s, 6H, CH ×2); C NMR (100
3
2
3
MHz, acetone-d ): δ 169.7, 140.2, 138.8 (2C), 133.4, 125.7, 124.9, 123.4, 123.2, 120.4, 120.2, 117.9
6
+
(
2C), 108.9, 106.7, 66.1, 34.7, 21.4 (2C); HR-ESIMS m/z 309.1608 [M + H] (Calcd for C H N O ,
1
9
21
2
2
3
09.1598).
N-(3,5-Dimethoxyphenyl)-2-(1-methoxy-1H-indol-3-yl)acetamide (5t). Four steps yield 25.4%. H
NMR (400 MHz, acetone-d ): δ 9.19 (s, 1H, CONH), 7.65 (d, J = 8.0 Hz, 1H, ArH), 7.44 (s, 1H, ArH),
.41 (d, J = 8.0 Hz, 1H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (t, J = 7.6 Hz, 1H, ArH), 6.92 (s, 2H,
1
6
7
1
3
ArH), 6.20 (s, 1H, ArH), 4.07 (s, 3H, OCH ), 3.78 (s, 2H, CH ), 3.70 (s, 6H, OCH ×2); C NMR (100
3
2
3
MHz, acetone-d ): δ 170.0, 161.9 (2C), 141.9, 133.3, 124.8, 123.4, 123.2, 120.4, 120.2, 108.9, 106.5,
6
+
9
3
8.4 (2C), 96.2, 66.1, 55.4 (2C), 34.7; HR-ESIMS m/z 341.1506 [M + H] (Calcd for C H N O ,
1
9
21
2
4
41.1496).
-(1-Methoxy-1H-indol-3-yl)-N-(3,4,5-trimethoxyphenyl)acetamide (5u). Four steps yield 23.9%.
H NMR (400 MHz, acetone-d ): δ 9.12 (s, 1H, CONH), 7.65 (d, J = 8.0 Hz, 1H, ArH), 7.45 (s, 1H,
2
1
6
ArH), 7.42 (d, J = 8.0 Hz, 1H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (t, J = 7.6 Hz, 1H, ArH), 7.03
1
3
(
s, 2H, ArH), 4.09 (s, 3H, OCH ), 3.78 (s, 2H, CH ), 3.72 (s, 6H, OCH ×2), 3.65 (s, 3H, OCH ); C
3 2 3 3
NMR (100 MHz, acetone-d ): δ 169.8, 154.2 (2C), 136.4, 133.4, 124.9, 123.4, 123.2, 120.4, 120.2,
6
2
3

+
1
09.0, 106.6, 98.0 (2C), 66.2, 60.5, 56.2 (2C), 34.7; HR-ESIMS m/z 371.1613 [M + H] (Calcd for
C H N O , 371.1601).
20
23
2
5
1
Methyl 2-[3-(1-methoxy-1H-indol-3-yl)propanamido]benzoate (5v). Four steps yield 11.7%. H
NMR (400 MHz, acetone-d ): δ 10.95 (s, 1H, CONH), 8.72 (d, J = 8.8 Hz, 1H, ArH), 7.99 (d, J = 8.0
6
Hz, 1H, ArH), 7.64 (d, J = 7.6 Hz, 1H, ArH), 7.58 (t, J = 7.6 Hz, 1H, ArH), 7.38 (d, J = 8.4 Hz, 1H,
ArH), 7.35 (s, 1H, ArH), 7.19 (t, J = 7.6 Hz, 1H, ArH), 7.11 (t, J = 7.6 Hz, 1H, ArH), 7.05 (t, J = 7.6
Hz, 1H, ArH), 4.03 (s, 3H, OCH ), 3.89 (s, 3H, COOCH ), 3.15 (t, J = 7.6 Hz, 2H, CH ), 2.83 (t, J =
3
3
2
1
3
7
1
3
.6 Hz, 2H, CH ); C NMR (100 MHz, acetone-d ): δ 171.6, 169.1, 142.5, 135.2, 133.6, 131.6, 124.7,
2 6
23.1 (2C), 122.1, 120.9, 120.2, 119.8, 115.9, 111.9, 109.0, 65.9, 52.8, 39.5, 21.4; HR-ESIMS m/z
+
53.1508 [M + H] (Calcd for C H N O , 353.1496).
2
0
21
2
4
1
Methyl 2-(4-(1-methoxy-1H-indol-3-yl)butanamido)benzoate (5w). Four steps yield 9.2%. H NMR
(
500 MHz, acetone-d ): δ 10.96 (s, 1H, CONH), 8.72 (d, J = 8.5Hz, 1H, ArH), 8.01 (dd, J = 1.5, 8.0 Hz,
6
1
H, ArH), 7.59 (m, 2H, ArH), 7.39 (d, J = 8.0 Hz, 1H, ArH), 7.32 (s, 1H, ArH), 7.18 (t, J = 7.5Hz, 1H,
ArH), 7.12 (t, J = 7.5Hz, 1H, ArH), 7.04 (t, J = 7.5Hz, 1H, ArH), 4.06 (s, 3H, OCH ), 3.91 (s, 3H,
3
1
3
COOCH ), 2.83 (t, J = 7.5 Hz, 2H, CH ), 2.52 (t, J = 7.0 Hz, 2H, CH ), 2.11 (m, 2H, CH ); C NMR
3
2
2
2
(
125 MHz, acetone-d
6
): δ 172.0, 169.2, 142.6, 135.2, 133.8, 131.6, 124.9, 123.0 (2C), 122.1, 120.8,
+
1
20.1, 119.9, 115.8, 112.6, 109.0, 65.8, 52.8, 38.3, 26.6, 24.9; HR-ESIMS m/z 367.166 [M + H] (Calcd
23 2 4
for C21H N O , 367.1652).
1
Methyl 2-(2-(1-ethoxy-1H-indol-3-yl)acetamido)benzoate (6a). Four steps yield 24.1%. H NMR
(
400 MHz, acetone-d ): δ 10.93 (s, 1H, CONH), 8.75 (d, J = 8.4 Hz, 1H, ArH), 7.90 (d, J = 8.4 Hz, 1H,
6
ArH), 7.56 (m, 3H, ArH), 7.46 (d, J = 8.4 Hz, 1H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (m, 2H,
1
3
ArH), 4.43 (m, 2H, OCH
2
), 3.87 (s, 2H, CH
2 3 3
), 3.71 (s, 3H, COOCH ), 1.43 (t, J = 6.8 Hz, 3H, CH ); C
NMR (100 MHz, acetone-d
6
): δ 170.2, 168.0, 141.7, 134.4, 133.5, 130.9, 124.4, 124.1, 122.6, 122.5,
2
4

+
1
20.0, 119.9, 119.1, 115.4, 108.7, 104.2, 74.3, 51.9, 35.1, 13.6; HR-ESIMS m/z 353.1502 [M + H]
(
Calcd for C H N O , 353.1496).
2
0
21
2
4
1
Methyl 2-(2-(1-propoxy-1H-indol-3-yl)acetamido)benzoate (6b). Four steps yield 23.8%. H NMR
(
400 MHz, acetone-d ): δ 10.92 (s, 1H, CONH), 8.75 (d, J = 8.4 Hz, 1H, ArH), 7.90 (d, J = 8.4 Hz, 1H,
6
ArH), 7.56 (m, 3H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.21 (t, J = 7.6 Hz, 1H, ArH), 7.06 (m, 2H,
ArH), 4.34 (t, J = 6.4Hz, 2H, OCH ), 3.87 (s, 2H, CH ), 3.72 (s, 3H, COOCH ), 1.86 (m, 2H, CH ),
2
2
3
2
1
3
1
1
.10 (t, J = 7.6 Hz, 3H, CH ); C NMR (100 MHz, acetone-d ): δ 170.8, 168.6, 142.3, 135.0, 133.9,
3 6
31.5, 124.9, 124.7, 123.2, 123.1, 120.6, 120.5, 119.7, 116.0, 109.2, 104.9, 80.8, 52.5, 35.7, 22.3, 10.6;
+
HR-ESIMS m/z 367.1664 [M + H] (Calcd for C H N O , 367.1652).
2
1
23
2
4
1
N-(2-Chlorophenyl)-2-(1-ethoxy-1H-indol-3-yl)acetamide (6c). Four steps yield 25.3%. H NMR
(
400 MHz, acetone-d ): δ 8.44 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H,
6
ArH), 7.60 (s, 1H, ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.32 (d, J = 8.0 Hz, 1H, ArH), 7.25 (m, 2H,
ArH), 7.09 (t, J = 7.6 Hz, 1H, ArH), 7.04 (t, J = 7.6 Hz, 1H, ArH), 4.35 (m, 2H, ArH), 3.93 (s, 2H,
1
3
CH ), 1.38 (t, J = 7.2 Hz, 3H, CH ); C NMR (100 MHz, acetone-d ): δ 169.9, 136.0, 134.0, 129.8,
2
3
6
1
25.5, 124.7, 124.5, 123.4 (2C), 122.8 (2C), 120.7, 119.9, 109.3, 105.4, 74.7, 34.5, 14.1; HR-ESIMS
+
m/z 329.106 [M + H] (Calcd for C18
H
18
N
2
2
O Cl, 329.1051).
1
N-(2-Chlorophenyl)-2-(1-propoxy-1H-indol-3-yl)acetamide (6d). Four steps yield 24.9%. H NMR
(
400 MHz, acetone-d ): δ 8.44 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.67 (d, J = 8.0 Hz, 1H,
6
ArH), 7.61 (s, 1H, ArH), 7.47 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.09 (t, J = 7.6 Hz, 1H, ArH), 7.04 (t, J = 7.6 Hz, 1H, ArH), 4.26 (t, J = 6.8 Hz, 2H, OCH ), 3.92
2
1
3
(
s, 2H, CH
2
), 1.81 (m, 2H, CH
2 3 6
), 1.07 (t, J = 7.6 Hz, 3H, CH ); C NMR (100 MHz, acetone-d ): δ
1
8
69.9, 136.1, 133.9, 129.9, 128.3, 125.5 (2C), 124.6, 123.5 (2C), 122.8, 120.7, 119.9, 109.3, 105.5,
+
20 2 2
0.7, 34.6, 22.3, 10.5 ; HR-ESIMS m/z 343.1221 [M + H] (Calcd for C19H N O Cl, 343.1208).
2
5

1
2
-(1-Butoxy-1H-indol-3-yl)-N-(2-chlorophenyl)acetamide (6e). Four steps yield 26.2%. H NMR
(
400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz, 1H,
6
ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.09 (t, J = 7.6 Hz, 1H, ArH), 7.04 (t, J = 7.6 Hz, 1H, ArH), 4.30 (t, J = 6.4 Hz, 2H, OCH ), 3.92
2
1
3
(
s, 2H, CH ), 1.78 (m, 2H, CH ), 1.56 (m, 2H, CH ), 0.98 (t, J = 7.2Hz, 3H, CH ); C NMR (100 MHz,
2 2 2 3
acetone-d
6
): δ 169.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C), 122.8, 120.7, 120.0,
+
1
3
09.2, 105.5, 79.0, 34.6, 31.0, 19.7, 14.1; HR-ESIMS m/z 357.1373 [M + H] (Calcd for C20
H
22
N O
2 2
Cl,
57.1364).
1
N-(2-Chlorophenyl)-2-(1-(pentyloxy)-1H-indol-3-yl)acetamide (6f). Four steps yield 24.7%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz,
6
1
H, ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.07 (m, 2H, ArH), 4.30 (t, J = 6.4 Hz, 2H, OCH ), 3.92 (s, 2H, CH ), 1.80 (m, 2H, CH ), 1.51
); C NMR (100 MHz, acetone-d ): δ
2
2
2
1
3
(m, 2H, CH
2
), 1.41 (m, 2H, CH
2
), 0.93 (t, J = 7.2 Hz, 3H, CH
3
6
1
7
3
69.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C), 122.8, 120.7, 120.0, 109.3, 105.5,
+
9.3, 34.6, 28.70, 28.68, 23.1, 14,2; HR-ESIMS m/z 371.1528 [M + H] (Calcd for C H N O Cl,
2
1
24
2
2
71.1521).
N-(2-Chlorophenyl)-2-(1-(hexyloxy)-1H-indol-3-yl)acetamide (6g). Four steps yield 23.0%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz,
H, ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.09 (t, J = 7.6 Hz, 1H, ArH), 7.04 (t, J = 7.6 Hz, 1H, ArH), 4.30 (t, J = 6.4 Hz, 2H, OCH ), 3.92
1
6
1
2
(
s, 2H, CH ), 1.80 (m, 2H, CH ), 1.53 (m, 2H, CH ), 1.35 (m, 4H, CH ), 0.90 (t, J = 6.4 Hz, 3H, CH );
2 2 2 2 3
1
3
C NMR (100 MHz, acetone-d ): δ 169.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C),
6
1
22.8, 120.7, 120.0, 109.3, 105.5, 79.3, 34.6, 32.3, 29.0, 26.2, 23.2, 14.2; HR-ESIMS m/z 385.1678 [M
+
+
H] (Calcd for C22
H N
26 2
O
2
Cl, 385.1677).
2
6

1
N-(2-Chlorophenyl)-2-(1-(heptyloxy)-1H-indol-3-yl)acetamide (6h). Four steps yield 24.3%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz,
6
1
H, ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.07 (m, 2H, ArH), 4.30 (t, J = 6.4 Hz, 2H, OCH ), 3.92 (s, 2H, CH ), 1.81 (m, 2H, CH ), 1.53
2
2
2
1
3
(
m, 2H, CH ), 1.34 (m, 6H, CH ), 0.88 (t, J = 6.0Hz, 3H, CH ); C NMR (100 MHz, acetone-d ): δ
2 2 3 6
1
7
69.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C), 122.8, 120.7, 120.0, 109.3, 105.5,
+
9.3, 34.6, 32.4, 29.8, 29.0, 26.5, 23.2, 14.3; HR-ESIMS m/z 399.184 [M + H] (Calcd for
C
23
H
28
N
2
O
2
Cl, 399.1834).
N-(2-Chlorophenyl)-2-(1-(octyloxy)-1H-indol-3-yl)acetamide (6i). Four steps yield 24.4%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz,
1
6
1
H, ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.07 (m, 2H, ArH), 4.31 (t, J = 6.4 Hz, 2H, OCH ), 3.92 (s, 2H, CH ), 1.81 (m, 2H, CH ), 1.53
); C NMR (100 MHz, acetone-d ): δ
2
2
2
1
3
(m, 2H, CH
2
), 1.32 (m, 8H, CH
2
), 0.88 (t, J = 6.4 Hz, 3H, CH
3
6
1
7
69.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C), 122.8, 120.7, 120.0, 109.3, 105.5,
+
9.3, 34.6, 32.5, 30.1, 29.9, 29.0, 26.5, 23.3, 14.3; HR-ESIMS m/z 413.1996 [M + H] (Calcd for
C H N O Cl, 413.1990).
24
30
2
2
1
N-(2-Chlorophenyl)-2-(1-(nonyloxy)-1H-indol-3-yl)acetamide (6j). Four steps yield 25.8%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz,
H, ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.09 (t, J = 7.6 Hz, 1H, ArH), 7.05 (t, J = 7.6 Hz, 1H, ArH), 4.30 (t, J = 6.4 Hz, 2H, OCH ), 3.92
6
1
2
(
s, 2H, CH ), 1.80 (m, 2H, CH ), 1.53 (m, 2H, CH ), 1.33 (m, 10H, CH ), 0.87 (t, J = 6.4 Hz, 3H, CH );
2 2 2 2 3
1
3
C NMR (100 MHz, acetone-d ): δ 169.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C),
6
1
22.7, 120.7, 120.0, 109.3, 105.5, 79.3, 34.6, 32.5, 30.2, 30.1, 29.9, 29.0, 26.5, 23.3, 14.3; HR-ESIMS
+
m/z 427.2158 [M + H] (Calcd for C25
H
32
N
2
O
2
Cl, 427.2147).
2
7

1
N-(2-Chlorophenyl)-2-(1-(decyloxy)-1H-indol-3-yl)acetamide (6k). Four steps yield 26.1%. H
NMR (400 MHz, acetone-d ): δ 8.42 (s, 1H, CONH), 8.30 (d, J = 8.4 Hz, 1H, ArH), 7.66 (d, J = 8.0 Hz,
6
1
H, ArH), 7.61 (s, 1H, ArH), 7.46 (d, J = 8.0 Hz, 1H, ArH), 7.33 (d, J = 8.0 Hz, 1H, ArH), 7.26 (m, 2H,
ArH), 7.07 (m, 2H, ArH), 4.30 (t, J = 6.4 Hz, 2H, OCH ), 3.92 (s, 2H, CH ), 1.81 (m, 2H, CH ), 1.53
2
2
2
1
3
(
m, 2H, CH ), 1.32 (m, 12H, CH ), 0.87 (t, J = 6.4 Hz, 3H, CH ); C NMR (100 MHz, acetone-d ): δ
2 2 3 6
1
7
69.9, 136.1, 133.9, 129.9, 128.4, 125.5 (2C), 124.6, 123.5 (2C), 122.7, 120.7, 120.0, 109.3, 105.5,
+
9.3, 34.6, 32.6, 30.2 (2C), 30.1, 30.0, 29.0, 26.5, 23.3, 14.3; HR-ESIMS m/z 441.2309 [M + H] (Calcd
for C26
H
34
N
2
O
2
Cl, 441.2303).
-(1-(Benzyloxy)-1H-indol-3-yl)-N-(2-chlorophenyl)acetamide (6l). Four steps yield 22.9%. H
NMR (300 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.31 (dd, J = 1.2, 8.4 Hz, 1H, ArH), 7.35 (m, 6H,
1
2
6
13
ArH), 7.07 (m, 2H, ArH), 5.28 (2H, s, CH
2
), 3.90 (s, 2H, CH
2 6
); C NMR (75 MHz, acetone-d ): δ
1
1
69.9, 136.0, 135.8, 134.0, 130.5 (2C), 129.81, 129.78, 129.7, 129.3 (2C), 128.3, 125.5, 124.8, 124.5,
+
23.4, 122.9, 120.7, 119.9, 109.4, 105.5, 80.9, 34.5; HR-ESIMS m/z 391.1215 [M + H] (Calcd for
C H N O Cl, 391.1208).
23
20
2
2
1
N-(2-Chlorophenyl)-2-(1-phenethoxy-1H-indol-3-yl)acetamide (6m). Four steps yield 22.6%. H
NMR (400 MHz, acetone-d ): δ 8.43 (s, 1H, CONH), 8.28 (d, J = 8.4 Hz, 1H, ArH), 7.65 (d, J = 8.0 Hz,
H, ArH), 7.57 (s, 1H, ArH), 7.31 (m, 8H, ArH), 7.18 (t, 1H, ArH), 7.06 (m, 2H, ArH), 4.54 (t, J = 6.8
6
1
13
2 2 2 6
Hz, 2H, CH ), 3.91 (s, 2H, CH ), 3.14 (t, J = 6.8 Hz, 2H, CH ); C NMR (100 MHz, acetone-d ): δ
1
1
69.9, 138.8, 136.1, 133.9, 129.9 (2C), 129.8, 129.3 (2C), 128.4, 127.4, 125.5, 124.61, 124.58, 123.5,
+
22.9 (2C), 120.8, 119.9, 109.3, 105.7, 79.7, 35.3, 34.5; HR-ESIMS m/z 405.1345 [M + H] (Calcd for
C H N O Cl, 405.1364).
24
22
2
2
N-(2-Chlorophenyl)-2-{1-[(4-methoxybenzyl)oxy]-1H-indol-3-yl}acetamide (6n). Four steps yield
1
2
8.9%. H NMR (400 MHz, acetone-d
6
): δ 8.39 (s, 1H, CONH), 8.28 (d, J = 8.0 Hz, 1H, ArH), 7.65 (d,
J = 8.0 Hz, 1H, ArH), 7.42 (m, 4H, ArH), 7.34 (d, J = 8.0 Hz, 1H, ArH), 7.28 (t, J = 8.0 Hz, 1H, ArH),
2
8