Design, synthesis and antiproliferative activity of a novel class of indole-2-carboxylate derivatives

Article abstract of DOI:10.1016/j.ejmech.2014.05.043

Based on the chemical structure of Pyrroloquinoline quinone (PQQ), a novel class of indole-2-carboxylate derivatives was designed, synthesized and assayed for antiproliferative activity in cancer cells in vitro. The biological results showed that some der

Full text of DOI:10.1016/j.ejmech.2014.05.043

Accepted Manuscript
Design, synthesis and antiproliferative activity of a novel class of indole-2-carboxylate
derivatives
Xing-yue Ji , Si-tu Xue , Yue-chen Zhan , Jia-jia Shen , Lin-tao Wu , Jie Jin , Zhen
Wang , Zhuo-rong Li
PII:
S0223-5234(14)00459-0
10.1016/j.ejmech.2014.05.043
EJMECH 7001
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 November 2013
Revised Date: 17 April 2014
Accepted Date: 14 May 2014
Please cite this article as: X.-y. Ji, S.-t. Xue, Y.-c. Zhan, J.-j. Shen, L.-t. Wu, J. Jin, Z. Wang, Z.-r. Li,
Design, synthesis and antiproliferative activity of a novel class of indole-2-carboxylate derivatives,
European Journal of Medicinal Chemistry (2014), doi: 10.1016/j.ejmech.2014.05.043.
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ACCEPTED MANUSCRIPT
Compounds 6e and 9l were identified as potent antiproliferative agents inducing ROS generation.

ACCEPTED MANUSCRIPT
Design, synthesis and antiproliferative activity of a novel class of
indole-2-carboxylate derivatives
Xing-yue Jia, Si-tu Xuea, Yue-chen Zhan, Jia-jia Shen, Lin-tao Wu, Jie Jin, Zhen
Wang**, and Zhuo-rong Li*
Institute of Medicinal Biotechnology, Chinese Academy of Medical Science and
Peking Union Medical College, Beijing 100050, China
*Corresponding author: Tel: +86-10-63027185; Fax: +86-10-63017302; E-mail:
lizhuorong@ imb.pumc.edu.cn.
**Corresponding
wangzhen@imb.pumc.edu.cn.
author.
Tel
&
Fax:
+86-10-63165289;
E-mail:
aThese authors made equal contributions to this work.
Abstract: Based on the chemical structure of Pyrroloquinoline quinone (PQQ), a
novel class of indole-2-carboxylate derivatives was designed, synthesized and assayed
for antiproliferative activity in cancer cells in vitro. The biological results showed that
some derivatives exhibited significant antiproliferative activity against HepG2, A549
and
MCF7
cells.
Notably,
the
novel
compounds,
(6e) and
methyl
methyl
6-amino-4-cyclohexylmethoxy-1H-indole-2-carboxylate
4-isopropoxy-6-methoxy-1H-indole-2-carboxylate (9l) exhibited more potent
antiproliferative activity than the reference drugs PQQ and etoposide in vitro, with
IC50 values ranging from 3.78 ± 0.58 to 24.08 ± 1.76 ꢀM. Further biological assay
showed that both compounds 6e and 9l increased ROS generation dose-dependently,
and induced PARP cleavage in A549 cells. Consequently, 6e and 9l appeared as
promising anticancer lead compounds for further optimization.
Keywords: PQQ; antiproliferative activities; indole-2-carboxylate derivatives; ROS
generation
1. Introduction
As a leading cause of death in humans, cancer is a major public health problem
worldwide [1]. Although a variety of anticancer drugs are available in clinic, their
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therapeutic effects are always impaired either by severe toxic side effects or by the
emergence of drug resistance, especially multidrug resistance (MDR) [2, 3]. Therefore,
the development of novel anticancer agents to circumvent these obstacles is still
desirable and encouraged.
Pyrroloquinoline quinone (PQQ), also known as methoxatin, was first identified
in methylotrophic bacteria as a coenzyme for methanol dehydrogenase in 1979 [4]. It
is widely distributed in a variety of food and other sources [5, 6], and is considered to
be a redox active nutrient that can produce or scavenge superoxide depending on
different cellular context [7]. In recent years, PQQ has garnered much attention due to
its versatile roles in biological processes, including antioxidant function, cognitive
promotion, neuroprotection and cardioprotection [8-10]. Also, PQQ is reported to
have anticancer activity by inducing apoptosis in human Jurkat cells and
promonocytic leukemia U937 cells, and this event is associated with generation of
reactive oxygen species (ROS) [7, 11].
Similar to other quinone compounds, PQQ serves as a Michael addition acceptor
to form covalent bonds with macromolecules (i.e., protein, DNA). In addition, the C-5
position in PPQ is also highly reactive to nucleophiles (Figure 1) [12]. Undoubtedly,
those features could boost the anticancer activity of PPQ; however, its high affinity
for nucleophiles could also induce nonselective cytotoxicity to normal cells [13].
Additionally, there are three carboxyl groups in the structure of PQQ, which probably
make it undruggable due to potentially inferior PK profiles (Low permeability).
Consequently, it is desirable to optimize its structure to retain its anticancer activity
and improve its druggability.
In this work, the structural simplification was made to PQQ aiming to retain its
antiproliferative activity and improve its druggability. As shown in Figure 2, the
designed target compounds shared some common fragments with PQQ; hence,
compounds harboring this scaffold would potentially possess antiproliferative
activities as well. Additionally, the Michael addition receptor fragment, the highly
reactive electrophilic center and the redundant carboxyl groups on ring C were all
eliminated from our designed structure. Last but not least, as indole scaffold
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represents a privileged structure widely distributed among successful drugs [14], and
furthermore nowadays, new potential and very promising antitumor agents bearing an
indole nucleus are reported [15-18]. A series of substituted indole-2-carboxylate
derivatives were consequently synthesized and assayed for their antiproliferative
activity against several cancer cell lines in vitro, and some preliminary
structure-activity relationships (SAR) were concluded. Our results showed that
compounds 6e and 9l exhibited potent antiproliferative activity against all of the
cancer cells tested. Additionally, the preliminary mechanism of action for compounds
6e and 9l was investigated in comparison with PQQ.
2. Results and discussion
2.1 Design
Based on the chemical structure of PQQ, the structure modifications in this work
were primarily made to the 4, 5 and 6-positions of the indole ring. As depicted in
Figure 3, compounds 6a-e with substituents at the 4 and 6-positions of indole ring
were synthesized and screened for their antiproliferative activity. Additionally,
compound 10 without alkoxyl substituents at the 4-position of indole scaffold, and
compounds 9c-h without amino group at the 6-position of indole scaffold were also
designed to probe the importance of alkoxyl group and amino group for the
antiproliferative activity, respectively. Meanwhile, compounds 9i-l with alkoxyl
substituents at both 4 and 6-positions, and compounds 9m-o with alkoxyl substituents
at both 4 and 5-positions were also designed to gain an insight into the SAR at those
positions.
2.2 Chemistry
As shown in Scheme 1, compounds 6a-6e were synthesized according to a
reported method with some modifications [19]. The commercially available methyl
p-acetamidosalicylate was used as the starting material to react with a variety of alkyl
halides affording compounds 1a-e. When substituted benzyl bromides or allyl
bromides were used (1a-c), a mild base (anhydrous K2CO3) was potent enough to
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drive the reaction to completion. However, in the case of saturated alkyl bromides
(1d and 1e), a strong base (NaH) was employed for a reasonable yield (60-70%).
Reduction of compounds 1a-e by lithium aluminum hydride (LiAlH4) in
tetrahydrofuran (THF) yielded corresponding alcohol derivatives 2a-e, which were
further oxidized to afford corresponding aldehydes in the presence of a mild oxidation
reagent (BaMnO4). However, usage of BaMnO4 suffered from moderate yield and
prolonged reaction time (24 h). Using an alternative oxidant pyridinium dichromate
(PDC) [20], the reaction time was reduced (2 h), and compounds 3a-e were obtained
in high yield (85-90%). During the aldehydes (3a-e) condensing step with methyl
azidoacetate [21-24], an intermediate compound with high polarity (i.e., small Rf
values of TLC) was observed, which was subsequently transformed into the target
compounds 4a-e as the reaction proceeded.. Thermolysis of compounds 4a-e in
boiling xylene afforded the expected indole compounds 5a-5e in 60-70% yield over
two steps. The acid cleavage of acetamide in a boiling hydrogen chloride solution of
methanol produced compounds 6a-e, among which compound 6a was
hydrogenolyzed to generate compound 7 in the presence of 5% Pd/C and under 1
atmosphere hydrogen pressure.
Compounds 9a-o with various alkoxyl substituents at 4, 5 and 6 positions of the
indole scaffold were obtained using a similar method to that employed to synthesize
compounds 5a-e, and compound 9a was further deacetylated to afford compound 10
(Scheme 2).
2.3 Antiproliferative activity and structure-activity relationships
Compounds 5d, 5e, 6a-e, 7, 9b-o and 10 were assayed for antiproliferative activity
against human hepatocellular (HepG2), lung adenocarcinoma (A549) and breast
adenocarcinoma cells (MCF7) using MTT assay. PQQ and two conventional
anticancer drugs etoposide (topoisomerase inhibitor) and paclitaxel (mitotic inhibitor),
were used as reference drugs, and the results were summarized in Table 1.
As shown in Table 1, among all of the tested compounds, derivatives 6d, 6e, and
9l showed definitive antiproliferative activity against HepG2, A549 and MCF7 cells.
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Compounds 9d and 9i-k also showed some inhibitory activity against A549 and
MCF7 cells. Notably, compounds 6e and 9l, with IC50 ranging from 3.78 ± 0.58 to
24.08 ± 1.76 ꢀM, exhibited more potent antiproliferative activity against the entire
cancer cell lines tested, and their antiproliferative activity was superior to that of
positive control drugs etoposide and PQQ, but both were less potent compared to
paclitaxel. Additionally, compounds 6d, 6e, 9d, 9e, 9g, 9i-m and 9o exhibited superior
antiproliferative activity against A549 cells compared to reference drugs PQQ and
etoposide.
In terms of the structure-activity relationships, substitution of the benzyloxy or
allyloxy (6a-c: IC50 > 200 ꢀM) by the alkyloxy moieties (6d: IC50 11.38 ± 2.12 -
97.44 ± 11.32 ꢀM and 6e: IC50 3.78 ± 0.58 - 14.50 ± 1.18 ꢀM) at the 4-position of the
indole ring decreased the antiproliferative activity. Given that compounds 7 (IC50 >
200 ꢀM) and 10 (IC50 > 200 ꢀM) did not show any antiproliferative activity, we
concluded that the alkyloxy at the 4-position of indole ring was crucial to the
antiproliferative activities. When the amino group at the 6-position of the indole ring
(6d: IC50 11.38 ± 2.12 - 97.44 ± 11.32 ꢀM and 6e IC50 3.78 ± 0.58 - 14.50 ± 1.18 ꢀM )
was replaced by a hydrogen atom (9d: IC50 31.07 ± 8.34 - > 200 ꢀM and 9e: IC50
51.36 ± 11.36 - > 200 ꢀM), a dramatic decrease in the antiproliferative activity was
observed. These results indicated that the amino group at the 6-indole ring is
indispensable to the antiproliferative activities. Additionally, acetyl substituent at the
amino group (5d: IC50 > 200 ꢀM and 5e: IC50 > 200 ꢀM) also disfavored the
antiproliferative activities.
Substitution of the methoxy group (9i-l: IC50 3.80 ± 0.61 - > 200 ꢀM) by a
hydrogen atom at 6-position of the indole ring (9d-h: IC50 31.07 ± 8.34 - > 200 ꢀM)
decreased the antiproliferative activity with the exception of compound 9d (IC50:
31.07 ± 8.34 ꢀM against A549), which showed increased activity against A549 cells
compared to compound 9i (IC50: 107.4 ± 21.51 ꢀM against A549). Based on the
activity of compounds 9i-l (IC50 3.80 ± 0.61 - > 200 ꢀM), it can be concluded that the
optimal alkoxyl substituent at 4-position is isopropoxy group. Substitution ethoxy
(9m-o: IC50 63.49 ± 13.54 - >200 ꢀM) for hydrogen atom at 5-position of the indole
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ring (9d: IC50 31.07 ± 8.34 - >200 ꢀM, 9f: IC50 > 200 ꢀM and 9g: IC50 87.14 ± 9.61 - >
200 ꢀM) has little influence on the antiproliferative activity.
2.4 Generation of intracellular ROS
ROS is one of major factors to mediate the cytotoxicity of many anticancer drugs
[25, 26]. PQQ was reported to induce apoptosis in Jurkat and U937 cells accompanied
by cellular ROS induction [7, 11]. Given that the chemical structures of compounds
6e, 9l and PQQ have some common fragments, we wonder whether the synthesized
compounds may increase ROS as well. As depicted in Figure 4, both compounds 6e
and 9l dose-dependently triggered the increase of cellular ROS in A549 cells after 48
h treatment. As expected, PQQ also induced ROS generation in A549 cells at a
concentration of 200 ꢀM. This suggests that the anti-cancer activity of compounds 6e
and 9l may, like PQQ, be associated with ROS production.
2.5 Cell cycle analysis
Cytotoxicity of conventional anti-cancer drugs normally results from either cell
cycle arrest or apoptosis [27]. We then determined whether both compounds 6e and 9l
were able to affect the cell cycle distribution in A549 cells. The cells were treated
with compound 6e or 9l for 48 h and then subjected to flow cytometry analysis. As
shown in Figure 5, compounds 6e or 9l arrested cells at G1 or G2/M phase in a dose
dependent manner, respectively. The difference in cell cycle arrest caused by both
agents indicates that compounds 6e and 9l may differentially pose effects on G1 and
G2/M cell cycle regulators, respectively. As ROS was reported to induce inhibition of
proliferation in G1, S and G2 phases of the cell cycle [28], the cell cycle inhibition
may be relevant to ROS production.
2.6 PARP cleavage
We lastly determined whether both compounds 6e and 9l may induce apoptosis
in A549 cells. Poly ADP ribose polymerase (PARP) is a zinc-finger DNA-binding
protein which catalyzes the synthesis of poly(ADP-ribose) from its substrate
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beta-NAD. In addition, this enzyme is implicated in the maintenance of genomic
stability and DNA damage-triggered signaling cascade. It is widely accepted that
PARP cleavage, which separates the carboxy-terminal catalytic domain (89 kDa) from
the full-length protein (116 kDa), is a hallmark of cell apoptosis [29]. In this work,
compounds 6e and 9l were subjected to western blot analysis of PARP cleavage in
A549 cells. As shown in Figure 6, both compounds 6e and 9l dose-dependently
induced PARP cleavage in A549 cells, indicating that apoptosis was indeed induced.
Meanwhile, β-actin, as an internal loading control, did not demonstrate any change.
Given the close correlation between ROS and apoptosis in cancer treatment [25, 26],
this induction of apoptosis by both agents may also be associated with ROS
generation.
3. Conclusion
In summary, a series of indole-2-carboxylate derivatives was synthesized based
on the chemical structure of PQQ. All the target compounds were assayed for their
antiproliferative activities in HepG2, A549 and MCF7 cells using MTT assay. Among
the tested compounds, 6e and 9l with highly simplified chemical structure compared
to PQQ showed potent cytotoxicity to the cancer cells, especially A549, tested at low
micromolar concentrations, and the potentially undruggable fragments in the structure
of PQQ were all eliminated from the structure of 6e and 9l. Additionally, compound
6e and 9l have a molecular weight and AlogP values of 302.37, 3.70 and 263.29, 2.96,
respectively (Data obtained from Discovery studio 3.0), and possess an indole
scaffold structure, a privileged structure widely distributed among successful drugs
[14]. Consequently, compounds 6e and 9l represent promising lead compounds for the
development of novel anticancer drugs and show potential for optimization to
improve their antiproliferative activities without sacrificing their druggability.
In terms of mechanism of action, our results showed that both compound 6e and
9l could trigger the generation of ROS in A549 cells, just like PQQ did. Also, both
compounds are able to arrest cell cycle and induce apoptosis in the cells, and these
effects may be associated with the increase of ROS.
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4. Experimental section
4.1 Synthesis and characterisation
1H NMR and 13C NMR spectra were recorded using TMS as the internal
standard in DMSO-d6 or CDCl3 with a Bruker BioSpin GmbH spectrometer at 400,
500 and 600 MHz, respectively. The mass spectra (MS) were recorded on a Thermo
Scientific LTQ ORBITRAP instrument with an ESI mass selective detector. Melting
points (m.p.) were determined using an SRS-OptiMelt automated melting point
instrument, without correction. Flash column chromatography was performed with
silica gel (200-300 mesh) purchased from Qingdao Haiyang Chemical Co. Ltd.
4.1.1 General procedure A for the preparation of 1a-e
Methyl 4-acetamido-2-hydroxybenzoate (100 mmol) was dissolved in anhydrous
DMF (100 mL), followed by the addition of anhydrous K2CO3 (150 mmol) or NaH
(150 mmol) and alkyl bromides (150 mmol). The mixture was heated to 60 °C for
4-6 h, and the reaction mixture was cooled to room temperature and filtrated. The
filtrate was concentrated in vacuo, and the resulting residue was recrystallized from
ethyl acetate and petroleum ether to afford the target compounds.
4.1.1.1 Methyl 4-acetamido-2-(benzyloxy)benzoate (1a)
Methyl 4-acetamido-2-hydroxybenzoate and benzyl bromide was treated with K2CO3
in DMF according to general procedure A to afford compound 1a as a white solid.
1
Yield, 90%; m.p. 107-108 °C (113-115°C, lit. 19). H NMR (400 MHz, CDCl3) δ
(ppm): 2.18 (s, 3H), 3.88 (s, 3H), 5.18 (s, 2H), 6.82 (d, J = 8.4 Hz, 1H), 7.30 (t, J =
7.6 Hz, 1H), 7.38 (t, J = 7.6 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H), 7.69 (s, 1H), 7.83 (d, J
= 8.4 Hz, 1H), 10.21 (s, 1H).
4.1.1.2 Methyl 4-acetamido-2-(4-fluorobenzyloxy)benzoate (1b)
Methyl 4-acetamido-2-hydroxybenzoate and 4-fluorobenzyl bromide was treated with
K2CO3 in DMF according to general procedure A to afford compound 1b as a white
1
solid. Yield, 85%; m.p. 132-133 °C. H NMR (400 MHz, CDCl3) δ (ppm): 2.20 (s,
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3H), 3.88 (s, 3H), 5.15 (s, 2H), 6.76 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.04-7.07 (m,
2H), 7.48-7.51 (m, 2H), 7.76 (s, 1H), 7.83 (d, J = 8.4 Hz, 1H). 13C NMR (126 MHz,
DMSO-d6) δ 24.7, 52.0, 69.3, 104.3, 111.0, 114.4, 115.6, 129.5, 132.7, 133.4, 144.8,
158.9, 163.0, 165.7, 169.8. HRMS (ESI+): found 318.11341 (Calcd for C17H17O4NF
[M+H]+: 318.11361)
4.1.1.3 Methyl 4-acetamido-2-(allyloxy)benzoate (1c)
Methyl 4-acetamido-2-hydroxybenzoate and allyl bromide was treated with K2CO3 in
DMF according to general procedure A to afford compound 1c as a white solid. Yield,
90%; m.p. 114-115 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.06 (s, 3H), 3.74 (s,
3H), 4.54 (d, J = 6.4 Hz, 2H), 5.25 (d, J = 6.4 Hz, 1H), 5.51 (d, J = 16.0 Hz, 1H),
6.01-6.04 (m, 1H), 7.16 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H), 7.47 (d, J = 1.6 Hz, 1H),
7.66 (d, J = 8.4 Hz, 1H), 10.18 (s, 1H).
4.1.1.4 Methyl 4-acetamido-2-isobutoxybenzoate (1d)
Methyl 4-acetamido-2-hydroxybenzoate and isobutyl bromide was treated with NaH
in DMF according to general procedure A to afford compound 1d as a white solid.
1
Yield, 70%; m.p. 112-113 °C. H NMR (400 MHz, DMSO-d6) δ (ppm): 1.00 (d, J =
6.8 Hz, 6H), 2.01 (s, 3H), 2.03-2.05 (m, 1H), 3.72 (d, J = 7.2 Hz, 2H), 3.74 (s, 3H),
7.15 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.44 (d, J = 2.0 Hz, 1H), 7.64 (d, J = 8.4 Hz,
1H), 10.16 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 19.3, 24.6, 28.2, 51.8, 74.6,
103.6, 110.4, 114.1, 132.5, 144.7, 159.5, 166.0, 169.4. HRMS (ESI+): found
266.13845 (Calcd for C14H20O4N [M+H]+: 266.13868).
4.1.1.5 Methyl 4-acetamido-2-(cyclohexylmethoxy)benzoate (1e)
Methyl 4-acetamido-2-hydroxybenzoate and cyclohexylmethyl bromide was treated
with NaH in DMF according to general procedure A to afford compound 1e as a white
1
solid. Yield, 65%; m.p. 132-133 °C. H NMR (400 MHz, CDCl3) δ (ppm): 1.05-1.32
(m, 5H), 1.68-1.90 (m, 6H), 2.23 (s, 3H), 3.83 (d, J = 6.0 Hz, 2H), 3.87 (s, 3H), 6.79
13
(d, J = 8.4 Hz, 1H), 7.29 (s, 1H), 7.53 (s, 1H), 7.80 (d, J = 8.4 Hz, 1H). C NMR
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(126 MHz, DMSO-d6) δ 24.69, 25.82, 26.54, 29.54, 37.59, 51.88, 73.73, 103.60,
110.43, 114.11, 132.59, 144.78, 159.61, 165.90, 169.41. HRMS (ESI+): found
306.17002 (Calcd for C17H24O4N [M+H]+: 306.16998).
4.1.2 General procedure B for the preparation of compounds 2a-e
A suspension of LiAlH4 (25 mmol) in anhydrous THF (100 mL) was added dropwise
a solution of compounds 1a-e (20 mmol) in anhydrous THF (50 mL), maintaining the
reaction temperature below 0 °C. After completion of the addition, the reaction
mixture was warmed up to room temperature for another 2 h, and was cooled again
with ice bath. Na2SO4•7H2O was added to quench the reaction, and the resulting
mixture was stirred for additional 2 h at room temperature. After that, the mixture was
filtered, and the filtrate was concentrated in vacuo to give a white solid, which was
triturated with diethyl ether to afford the target compounds.
4.1.2.1 N-(3-(benzyloxy)-4-(hydroxymethyl)phenyl)acetamide (2a)
Compound 1a was treated with LiAlH4 in anhydrous THF according to procedure B
to afford compound 2a as a white solid. Yield, 85%; m.p. 115-116 °C (115-116 °C,
lit.19); 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.02 (s, 3H), 4.48 (d, J = 5.6 Hz, 2H),
4.89 (t, J = 5.6 Hz, 1H), 5.05 (s, 2H), 7.12 (dd, J1 = 1.6 Hz, J2 =8.0 Hz, 1H), 7.26 (d, J
= 8.0 Hz, 1H), 7.32-7.46 (m, 6H), 9.87 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ
24.5, 58.2, 69.5, 103.5, 111.3, 125.8, 127.7, 127.9, 128.2, 128.8, 137.6, 139.5, 155.5,
168.6. HRMS (ESI+): found 272.12446 (Calcd for C16H18O3N [M+H]+: 272.12812).
4.1.2.2 N-(3-(4-fluorobenzyloxy)-4-(hydroxymethyl)phenyl)acetamide (2b)
Compound 1b was treated with LiAlH4 in anhydrous THF according to procedure B
to afford compound 2b as a white solid. Yield, 80%; m.p. 124-125 °C. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 2.01 (s, 3H), 4.46 (d, J = 5.6 Hz, 2H), 4.87 (t, J = 5.6 Hz,
1H), 5.02 (s, 2H), 7.09 (d, J = 7.6 Hz, 1H), 7.21-7.24 (m, 3H), 7.37 (s, 1H), 7.49 (dd,
J1 = 8.0 Hz, J2 = 5.6 Hz, 2H), 9.86 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 24.5,
58.2, 68.8, 103.6, 111.3, 115.7, 126.7, 129.9, 130.0, 132.6, 135.6, 139.5, 155.4, 168.6.
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HRMS (ESI+): found 290.11830 (Calcd for C16H17O3NF [M+H]+: 290.11870).
4.1.2.3 N-(3-(allyloxy)-4-(hydroxymethyl)phenyl)acetamide (2c)
Compound 1c was treated with LiAlH4 in anhydrous THF according to procedure B
to afford compound 2c as a white solid. Yield, 75%; m.p. 129-131 °C. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 2.01 (s, 3H), 4.44 (d, J = 6.4 Hz, 2H), 4.49 (d, J = 5.6 Hz,
2H), 4.86 (t, J = 5.6 Hz, 1H), 5.25 (d, J = 6.4 Hz, 1H), 5.40 (d, J = 17.2 Hz, 1H),
6.01-6.07 (m, 1H), 7.08 (dd, J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H),
13
7.28 (d, J = 1.6 Hz, 1H), 9.85 (s, 1H). C NMR (126 MHz, DMSO-d6) δ 24.5, 58.1,
68.5, 103.3, 111.2, 117.5, 125.6, 127.8, 134.1, 139.5, 155.4, 168.6. HRMS (ESI+):
found 222.11237 (Calcd for C12H16O3N [M+H]+: 222.11247).
4.1.2.4 N-(4-(hydroxymethyl)-3-isobutoxyphenyl)acetamide (2d)
Compound 1d was treated with LiAlH4 in anhydrous THF according to procedure B
to afford compound 2d as a white solid. Yield, 85%; m.p. 120-121 °C. 1H NMR (400
MHz, DMSO-d6) δ (ppm): 0.97 (d, J = 6.8 Hz, 6H), 2.00 (s, 3H), 2.02-2.03 (m, 1H),
3.65 (d, J = 6.4 Hz, 2H), 4.44 (d, J = 5.6 Hz, 2H), 4.84 (t, J = 5.6 Hz, 1H), 7.06 (dd,
J1 = 8.4 Hz, J2 = 2.0 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 7.26 (d, J = 2.0 Hz, 1H), 9.83
13
(s, 1H). C NMR (126 MHz, DMSO-d6) δ 19.5, 24.5, 28.2, 58.0, 74.0, 102.9, 110.8,
125.6, 127.6, 139.4, 155.8, 168.5. HRMS (ESI+): found 238.14290 (Calcd for
C13H20O3N [M+H]+: 238.14377).
4.1.2.5 N-(3-(cyclohexylmethoxy)-4-(hydroxymethyl)phenyl)acetamide (2e)
Compound 1e was treated with LiAlH4 in anhydrous THF according to procedure B
1
to afford compound 2e as a white solid. Yield, 79%; m.p. 112-114 °C. H NMR (400
MHz, DMSO-d6) δ (ppm): 1.03-1.29 (m, 5H), 1.68-1.80 (m, 6H), 2.00 (s, 3H), 3.68 (d,
J = 6.0 Hz, 2H), 4.42 (d, J = 5.2 Hz, 2H), 4.83 (t, J = 5.2 Hz, 1H), 7.07 (dd, J1 = 8.4
Hz, J2 = 1.6 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 7.24 (d, J = 1.6 Hz, 1H), 9.82 (s, 1H).
11

ACCEPTED MANUSCRIPT
13C NMR (126 MHz, DMSO-d6) δ 24.5, 25.8, 26.5, 29.7, 37.5, 58.0, 73.1, 102.9,
110.8, 125.6, 127.6, 139.4, 155.9, 168.5. HRMS (ESI+): found 278.17588 (Calcd for
C16H24O3N [M+H]+: 278.17507).
4.1.3 General procedure C for the preparation of compounds 3a-3e
Compounds 2a-e (20 mmol) were dissolved in CH2Cl2 (100 mL), and PDC (30 mmol)
was added. The resulting mixture was heated under reflux for 2 h. The reaction
mixture was cooled and filtered through a thin layer of silica gel, and the filtrate was
concentrated in vacuo. The residue was recrystallized from ethyl acetate and
petroleum ether to afford the target compounds as white solid.
4.1.3.1 N-(3-(benzyloxy)-4-formylphenyl)acetamide (3a)
Compound 2a was treated with PDC according to general procedure C to afford
1
compound 3a as a white solid. Yield, 90%; m.p. 126-128 °C (l56-158 °C, lit. 19). H
NMR (400 MHz, DMSO-d6) δ (ppm): 2.06 (s, 3H), 5.21 (s, 2H), 7.21 (d, J = 8.4 Hz,
1H), 7.35 (t, J = 7.6 Hz, 1H), 7.42 (t, J = 7.6 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H), 7.65
13
(s, 1H), 7.68 (d, J = 8.4 Hz, 1H), 10.25 (s, 1H), 10.34 (s,1H). C NMR (126 MHz,
DMSO-d6) δ 24.7, 70.2, 103.2, 111.6, 120.6, 128.0, 128.5, 129.0, 129.4, 136.7, 146.9,
161.9, 169.7, 187.8. HRMS (ESI+): found 270.11276 (Calcd for C16H16O3N [M+H]+:
270.11247).
4.1.3.2 N-(3-(4-fluorobenzyloxy)-4-formylphenyl)acetamide (3b)
Compound 2b was treated with PDC according to general procedure C to afford
1
compound 3b as a white solid. Yield, 87%; m.p. 158-160 °C. H NMR (400 MHz,
CDCl3) δ (ppm): 2.22 (s, 3H), 5.16 (s, 2H), 6.69 (d, J = 8.0 Hz, 1H), 7.06-7.10 (m,
2H), 7.35 (s, 1H), 7.43 (dd, J1 = 8.4 Hz, J2 = 5.6 Hz, 2H), 7.79 (d, J = 8.0 Hz, 1H),
7.93 (s, 1H), 10.34 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 24.7, 69.5, 103.4, 111.7,
115.8, 120.2, 129.5, 130.3, 130.3, 133.0, 146.9, 161.8, 169.7, 187.8. HRMS (ESI+):
found 288.10329 (Calcd for C16H15O3NF [M+H]+: 288.10305).
12

ACCEPTED MANUSCRIPT
4.1.3.3 N-(3-(allyloxy)-4-formylphenyl)acetamide (3c)
Compound 2c was treated with PDC according to general procedure C to afford
1
compound 3c as a white solid. Yield, 90%; m.p. 121-122 °C. H NMR (400 MHz,
DMSO-d6) δ (ppm): 2.08 (s, 3H), 4.65 (d, J = 6.8 Hz, 2H), 5.30 (d, J = 6.4 Hz, 1H),
5.48 (d, J = 16.0 Hz, 1H), 6.05-6.12 (m, 1H), 7.17 (dd, J1 = 8.4 Hz, J2 = 1.6 Hz, 1H),
13
7.58 (d, J = 1.6 Hz, 1H), 7.64 (d, J = 8.4 Hz, 1H), 10.24 (s, 1H), 10.32 (s, 1H). C
NMR (126 MHz, DMSO-d6) δ 24.7, 69.1, 103.2, 111.5, 118.3, 120.1, 129.43, 133.2,
146.9, 161.8, 169.7, 187.8. HRMS (ESI+): found 220.09723 (Calcd for C12H14O3N
[M+H]+: 220.09682).
4.1.3.4 N-(4-formyl-3-isobutoxyphenyl)acetamide (3d)
Compound 2d was treated with PDC according to general procedure C to afford
1
compound 3d as a white solid. Yield, 84%, m.p. 133-134 °C. H NMR (400 MHz,
DMSO-d6) δ (ppm): 1.00 (d, J = 6.8 Hz, 6H), 2.07 (s, 3H), 2.10-2.11 (m, 1H), 3.82 (d,
J = 6.4 Hz, 2H), 7.15 (d, J = 8.4 Hz, 1H), 7.56 (s, 1H), 7.63 (d, J = 8.4 Hz, 1H),
10.24 (s, 1H), 10.31 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 19.4, 24.7, 28.1, 74.6,
102.8, 111.2, 120.0, 129.2, 147.0, 162.5, 169.6, 187.7. HRMS (ESI+): found
236.12852 (Calcd for C13H18O3N [M+H]+: 236.12812).
4.1.3.5 N-(3-(cyclohexylmethoxy)-4-formylphenyl)acetamide (3e)
Compound 2e was treated with PDC according to general procedure C to afford
1
compound 3e as a white solid. Yield, 90%, m.p. 156-157 °C. H NMR (400 MHz,
DMSO-d6) δ (ppm): 1.06-1.27 (m, 5H), 1.70-1.83 (m, 6H), 2.07 (s, 3H), 3.85 (d, J =
6.0 Hz, 2H), 7.17 (d, J = 8.4 Hz, 1H), 7.53 (s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 10.22 (s,
1H), 10.30 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 24.7, 25.7, 26.4, 29.6, 37.4,
73.7, 102.8, 111.2, 120.0, 129.2, 147.0, 162.5, 169.6, 187.5. HRMS (ESI+): found
276.15988 (Calcd for C16H22O3N [M+H]+: 276.15942).
13

ACCEPTED MANUSCRIPT
4.1.4 General procedure D for the preparation of compounds 5a-5e, 9a-9o
To a solution of NaOMe (30 mmol) in MeOH (80 mL) were added compounds 3a-e
(20 mmol) and methyl 2-azidoacetate (200 mmol) in anhydrous THF (80 mL) at
-15 °C, and the mixture was stirred for another 4 h. After that, the reaction was
quenched by the addition of saturated ammonium chloride solution, and extracted
with ethyl acetate (3 × 100 mL). The combined organic layer was washed with brine,
and dried over anhydrous MgSO4, filtered and concentrated. The residue was
triturated with MeOH to give the intermediate compounds 4a-4e (8a-8o) as a yellow
solid. The crude compounds 4a-4e (8a-8o) were dissolved in xylene, and was heated
under reflux for 2 h. After cooling, a white solid was crystallized. Filtration afforded
the target compounds.
4.1.4.1 Methyl 6-acetamido-4-(benzyloxy)-1H-indole-2-carboxylate (5a)
Compounds 3a and methyl 2-azidoacetate were treated with NaOMe in MeOH
according to the general procedure D to afford compound 5a as a white solid. Yield,
60%; m.p. 264-265 °C (263-265 °C, lit. 19). 1H NMR (400 MHz, DMSO-d6) δ (ppm):
2.04 (s, 3H), 3.83 (s, 3H), 5.18 (s, 2H), 6.78 (s, 1H), 7.07 (s, 1H), 7.36 (t, J = 7.6 Hz,
1H), 7.42 (t, J =7.6 Hz, 2H), 7.51 (d, J = 7.6 Hz, 2H), 7.62 (s, 1H), 9.92 (s, 1H),
11.79 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 24.6, 52.0, 69.5, 95.8, 105.8,
106.5, 115.0, 125.8, 127.8, 128.3, 128.9, 137.4, 138.1, 139.4, 152.8, 161.9, 168.6.
HRMS (ESI+): found 339.13379, (Calcd for C19H19O4N2 [M+H]+: 339.13393)
4.1.4.2 Methyl 6-acetamido-4-(4-fluorobenzyloxy)-1H-indole-2-carboxylate (5b)
Compounds 3b and methyl 2-azidoacetate was treated with NaOMe in MeOH
according to the general procedure D to afford compound 5b as a white solid. Yield,
60%; m.p. 273-273 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.03 (s, 3H), 3.82 (s,
3H), 5.15 (s, 2H), 6.78 (s, 1H), 7.05 (s, 1H), 7.23 (t, J = 8.4 Hz, 2H), 7.55 (dd, J1 =
5.6 Hz, J2 = 8.4 Hz, 2H), 7.60 (s, 1H), 9.92 (s, 1H), 11.79 (s, 1H). 13C NMR (126
MHz, DMSO-d6) δ 24.6, 52.0, 68.8, 95.8, 105.7, 115.0, 115.6, 115.8, 125.8, 130.0,
130.1, 133.7, 138.1, 139.3, 152.7, 161.9, 168.6. HRMS (ESI+): found 357.12429
14

ACCEPTED MANUSCRIPT
(Calcd for C19H18O4N2F [M+H]+: 357.12451).
4.1.4.3 Methyl 6-acetamido-4-(allyloxy)-1H-indole-2-carboxylate (5c)
Compounds 3c and methyl 2-azidoacetate was treated with NaOMe in MeOH
according to the general procedure D to afford compound 5c as a pale yellow solid.
Yield, 62%; m.p. 200-201 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.01 (s, 3H),
3.82 (s, 3H), 4.61 (d, J = 5.2 Hz, 2H), 5.28 (d, J = 6.4 Hz, 1H), 5.45 (d, J = 17.2 Hz,
1H), 6.06-6.14 (m, 1H), 6.68 (s, 1H), 7.03 (s, 1H), 7.59 (s, 1H), 9.90 (s, 1H), 11.77 (s,
1H). HRMS (ESI+): found 289.11805 (Calcd for C15H17O4N2 [M+H]+: 289.11828).
4.1.4.4 Methyl 6-acetamido-4-isobutoxy-1H-indole-2-carboxylate (5d)
Compounds 3d and methyl 2-azidoacetate was treated with NaOMe in MeOH
according to the general procedure D to afford compound 5d as a white solid. Yield,
66%; m.p. 107-108 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.01 (d, J = 6.8 Hz,
6H), 2.03 (s, 3H), 2.06-2.11 (m, 1H), 3.78 (d, J = 6.4 Hz, 2H), 3.82 (s, 3H), 6.65 (s,
1H), 7.01 (s, 1H), 7.57 (s, 1H), 9.88 (s, 1H), 11.74 (s, 1H). 13C NMR (150 MHz,
DMSO-d6) δ (ppm): 19.5, 24.6, 28.2, 52.0, 74.1, 95.2, 95.3, 105.7, 114.9, 125.7, 138.2,
139.3, 153.3, 161.9, 168.6. HRMS (ESI+): found 305.14938 (Calcd for C16H21O4N2
[M+H]+: 305.14958).
4.1.4.5 Methyl 6-acetamido-4-(cyclohexylmethoxy)-1H-indole-2-carboxylate (5e)
Compounds 3e and methyl 2-azidoacetate was treated with NaOMe in MeOH
according to the general procedure D to afford compound 5e as a pale yellow solid.
Yield, 63%; m.p. 253-255 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.04-1.30 (m,
5H), 1.65-1.75 (m, 6H), 2.02 (s, 3H), 3.81 (s, 3H), 3.82 (d, J = 6.0 Hz, 2H), 6.63 (s,
1H), 7.01 (s, 1H), 7.57 (s, 1H), 9.87 (s, 1H), 11.74 (s, 1H). 13C NMR (126 MHz,
DMSO-d6) δ 24.6, 25.7, 26.5, 29.7, 37.5, 52.0, 73.1, 95.1, 95.2, 105.7, 115.0, 125.6,
138.2, 139.3, 153.3, 161.9, 168.5. HRMS (ESI+): found 345.18094 (Calcd for
C19H25O4N2 [M+H]+: 345.18088).
15

ACCEPTED MANUSCRIPT
4.1.4.6 Methyl 6-acetamido-1H-indole-2-carboxylate (9a)
4-Acetamidophenzaldehyde and methyl 2-azidoacetate was treated with NaOMe in
MeOH according to the general procedure D to afford compound 9a as a white solid.
Yield, 70%; m.p. 235-237 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.05 (s, 3H),
3.83 (s, 3H), 7.07 (s, 1H), 7.10 (d, J = 8.8 Hz, 1H), 7.52 (d, J = 8.8 Hz, 1H), 8.01 (s,
1H), 9.95 (s, 1H), 11.75 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ 24.5, 52.1, 102.4,
108.4, 114.4, 122.5, 123.2, 127.1, 137.1, 138.2, 162.1, 168.6. HRMS (ESI+): found
233.09207 (Calcd for C12H13O3N2 [M+H]+: 233.09188).
4.1.4.7 Methyl 6-(dimethylamino)-1H-indole-2-carboxylate (9b)
4-Dimethylaminobenzaldehyde and methyl 2-azidoacetate was treated with NaOMe
in MeOH according to the general procedure D to afford compound 9b as a brown
solid. Yield, 67%; m.p. 177-178 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 2.91 (s,
6H), 3.80 (s, 3H), 6.56 (s, 1H), 6.74 (dd, J1 = 8.8 Hz, J2 = 2.0 Hz, 1H), 6.99 (s, 1H),
13
7.43 (d, J = 8.8 Hz, 1H), 11.34 (s, 1H). C NMR (126 MHz, DMSO-d6) δ (ppm):
41.2, 51.8, 93.6, 108.9, 111.0, 119.1, 122.6, 124.9, 139.9, 149.6, 161.9. HRMS (ESI+):
found 219.11261 (Calcd for C12H15O2N2 [M+H]+: 219.11280).
4.1.4.8 Methyl 4-methoxy-1H-indole-2-carboxylate (9c)
2-Methoxybenzaldehyde and methyl 2-azidoacetate was treated with NaOMe in
MeOH according to the general procedure D to afford compound 9c as a white solid.
1
Yield, 65%; m.p. 143-145 °C (143-145°C, lit. 30). H NMR (400 MHz, DMSO-d6) δ
(ppm): 3.86 (s, 3H), 3.88 (s, 3H), 6.53 (d, J = 7.6 Hz, 1H), 7.02 (d, J = 7.6 Hz, 1H),
7.09 (s, 1H), 7.18 (t, J = 7.6 Hz, 1H), 11.92 (s, 1H). 13C NMR (126 MHz, DMSO-d6)
δ (ppm): 52.4, 55.3, 100.0, 105.3, 105.9, 118.3, 126.2, 126.3, 139.2, 154.3, 162.0.
HRMS (ESI+): found 206.08092 (Calcd for C11H12O3N [M+H]+: 206.08117).
4.1.4.9 Methyl 4-(cyclohexylmethoxy)-1H-indole-2-carboxylate (9d)
2-Cyclohexylmethoxybenzaldehyde and methyl 2-azidoacetate was treated with
NaOMe in MeOH according to the general procedure D to afford compound 9d as
pale yellow solid. Yield, 67%; m.p. 183-185 °C. 1H NMR (500 MHz, CDCl3) δ (ppm):
16

ACCEPTED MANUSCRIPT
1.16-1.37 (m, 5H), 1.71-1.94 (m, 6H), 3.90 (d, J = 6.0 Hz, 2H), 3.94 (s, 3H), 6.48 (d,
J = 8.0 Hz, 1H), 6.98 (d, J = 8.0 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.36 (s, 1H), 8.79
13
(s, 1H). C NMR (126 MHz, DMSO-d6) δ (ppm): 25.8, 26.5, 29.8, 37.6, 52.1, 73.0,
100.6, 105.5, 105.7, 118.7, 126.2, 126.4, 139.1, 153.7, 161.9. HRMS (ESI+): found
288.15940 (Calcd for C17H22O3N [M+H]+: 288.15942).
4.1.4.10 Methyl 4-isobutoxy-1H-indole-2-carboxylate (9e)
2-Isobutoxybenzaldehyde and methyl 2-azidoacetate was treated with NaOMe in
MeOH according to the general procedure D to afford compound 9e as a white solid.
1
Yield, 70%; m.p. 166-167 °C. H NMR (500 MHz, CDCl3) δ (ppm): 1.08 (d, J = 7.0
Hz, 6H), 2.16-2.22 (m, 1H), 3.87 (d, J = 6.5 Hz, 2H), 3.94 (s, 3H), 6.48 (d, J = 8.0 Hz,
1H), 6.98 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H),7.37 (s, 1H), 8.78 (s, 1H). 13C
NMR (150 MHz, DMSO-d6) δ (ppm): 19.5, 28.2, 52.2, 73.8, 100.8, 105.5, 105.8,
118.7, 126.2, 126.4, 139.1, 153.5, 162.2. HRMS (ESI+): found 248.12806 (Calcd for
C14H18O3N [M+H]+: 248.12812).
4.1.4.11 Methyl 4-propoxy-1H-indole-2-carboxylate (9f)
2-Propoxybenzaldehyde and methyl 2-azidoacetate was treated with NaOMe in
MeOH according to the general procedure D to afford compound 9f as a white solid.
1
Yield, 68%; m.p. 140-141 °C (69 °C, lit. 31). H NMR (500 MHz, CDCl3) δ (ppm):
1.09 (t, J = 7.5 Hz, 3H), 1.86-1.93 (m, 2H), 3.93 (s, 3H), 4.07 (t, J = 6.5 Hz, 2H),
6.49 (d, J = 8.0 Hz, 1H), 6.99 (d, J =8.0 Hz, 1H), 7.21 (t, J = 8.0 Hz, 1H), 7.36 (s,
13
1H), 8.80 (s, 1H). C NMR (126 MHz, DMSO-d6) δ (ppm): 11.0, 22.8, 52.2, 69.3,
100.6, 105.5, 105.8, 118.7, 126.2, 126.4, 139.1, 153.5, 161.9. HRMS (ESI+): found
234.11255 (Calcd for C13H16O3N [M+H]+: 234.11247).
4.1.4.12 Methyl 4-butoxy-1H-indole-2-carboxylate (9g)
2-Butoxybenzaldehyde and methyl 2-azidoacetate was treated with NaOMe in MeOH
according to the general procedure D to afford compound 9g as a white solid. Yield,
1
65%; m.p. 153-154 °C. H NMR (500 MHz, CDCl3) δ (ppm): 1.00 (t, J = 7.5 Hz,
17

ACCEPTED MANUSCRIPT
3H), 1.53-1.58 (m, 2H), 1.83-1.88 (m, 2H), 3.93 (s, 3H), 4.11 (t, J = 6.5 Hz, 2H), 6.49
(d, J = 7.5 Hz, 1H), 6.99 (d, J = 8.0 Hz, 1H), 7.22 (t, J = 8.0 Hz, 1H), 7.36 (s, 1H),
8.78 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ (ppm): 13.8, 19.1, 31.3, 51.9, 67.5,
100.4, 105.5, 105.8, 118.7, 126.2, 126.3, 139.4, 153.5, 162.2. HRMS (ESI+): found
248.12822 (Calcd for C14H18O3N [M+H]+: 248.12822).
4.1.4.13 Methyl 4-isopropoxy-1H-indole-2-carboxylate (9h)
2-Isopropoxybenzaldehyde and methyl 2-azidoacetate was treated with NaOMe in
MeOH according to the general procedure D to afford compound 9h as a white solid.
Yield, 68%; m.p. 207-208 °C. 1H NMR (500 MHz, CDCl3) δ (ppm): 1.41 (d, J = 6.0
Hz, 6H), 3.93 (s, 3H), 4.69-4.74 (m, 1H), 6.52 (d, J = 8.0 Hz, 1H), 6.98 (d, J = 8.0 Hz,
1H), 7.21 (t, J = 8.0 Hz, 1H), 7.35 (s, 1H), 8.76 (s, 1H). 13C NMR (126 MHz,
DMSO-d6) δ (ppm): 22.3, 52.2, 69.8, 102.1, 105.6, 105.8, 119.3, 126.1, 126.3, 139.3,
152.4, 161.8. HRMS (ESI+): found 234.11255 (Calcd for C13H16O3N [M+H]+:
234.11247).
4.1.4.14 Methyl 4-(cyclohexylmethoxy)-6-methoxy-1H-indole-2-carboxylate (9i)
2-Cyclohexylmethoxy-4-methoxybenzaldehyde and methyl 2-azidoacetate was
treated with NaOMe in MeOH according to the general procedure D to afford
compound 9i as a pale yellow solid. Yield, 70%; m.p. 186-188 °C. 1H NMR (500
MHz, CDCl3) δ (ppm): 1.07-1.40 (m, 5H), 1.70-1.91 (m, 6H), 3.83-3.85 (m, 5H), 3.91
(s, 3H), 6.16 (s, 1H), 6.40 (s, 1H), 7.28 (s, 1H), 8.67 (s, 1H). 13C NMR (151 MHz,
DMSO-d6) δ (ppm): 25.6, 26.4, 29.6, 37.5, 51.8, 55.6, 73.0, 86.9, 93.2, 105.9, 113.5,
124.6, 139.6, 153.9, 159.7, 161.7. HRMS (ESI+): found 318.16990 (Calcd for
C18H24O4N [M+H]+: 318.16998).
4.1.4.15 Methyl 6-methoxy-4-propoxy-1H-indole-2-carboxylate (9j)
2-Propoxy-4-methoxybenzaldehyde and methyl 2-azidoacetate was treated with
NaOMe in MeOH according to the general procedure D to afford compound 9j as a
white solid. Yield, 67%; m.p. 178-179 °C. 1H NMR (500 MHz, CDCl3) δ (ppm): 1.08
18

ACCEPTED MANUSCRIPT
(t, J = 7.0 Hz, 3H), 1.86-1.90 (m, 2H), 3.84 (s, 3H), 3.90 (s, 3H), 4.02 (t, J = 6.0 Hz,
2H), 6.17 (s, 1H), 6.41 (s, 1H), 7.28 (s, 1H), 8.66 (s, 1H). 13C NMR (151 MHz,
DMSO-d6) δ (ppm): 10.9, 22.3, 51.9, 55.6, 69.4, 86.9, 93.4, 106.0, 113.2, 124.8, 139.4,
153.9, 159.8, 161.8. HRMS (ESI+): found 264.12304 (Calcd for C14H18O4N [M+H]+:
264.12303).
4.1.4.16 Methyl 4-butoxy-6-methoxy -1H-indole-2-carboxylate (9k)
2-Butoxy-4-methoxybenzaldehyde and methyl 2-azidoacetate was treated with
NaOMe in MeOH according to the general procedure D to afford compound 9k as a
white solid. Yield, 68%; m.p. 141-143 °C. 1H NMR (500 MHz, CDCl3) δ (ppm): 0.99
(t, J = 7.5 Hz, 3H), 1.52-1.58 (m, 2H), 1.80-1.86 (m, 2H), 3.83 (s, 3H), 3.90 (s, 3H),
13
4.06 (t, J = 6.0 Hz, 2H), 6.17 (s, 1H), 6.40 (s, 1H), 7.27 (s, 1H), 8.66 (s, 1H). C
NMR (151 MHz, DMSO-d6) δ (ppm): 9.3, 14.4, 26.3, 47.1, 50.7, 62.9, 80.8, 88.4,
102.4, 109.2, 119.8, 133.5, 149.5, 155.6, 157.4. HRMS (ESI+): found 278.13870
(Calcd for C15H20O4N [M+H]+: 278.13868).
4.1.4.17 Methyl 4-isopropoxy -6-methoxy -1H-indole-2-carboxylate (9l)
2-Isopropoxy-4-methoxybenzaldehyde and methyl 2-azidoacetate was treated with
NaOMe in MeOH according to the general procedure D to afford compound 9l as a
1
white solid. Yield, 69%; m.p. 158-159 °C. H NMR (500 MHz, DMSO-d6) δ (ppm):
1.32 (d, J = 6.0 Hz, 6H), 3.76 (s, 3H), 3.83 (s, 3H), 4.67-4.72 (m, 1H), 6.19 (s, 1H),
13
6.45 (s, 1H), 7.01 (s, 1H), 11.69 (s, 1H). C NMR (151 MHz, DMSO-d6) δ (ppm):
22.4, 51.8, 55.5, 70.2, 86.9, 94.6, 106.0, 114.2, 124.6, 139.7, 152.7, 159.9, 161.9.
HRMS (ESI+): found 264.12304 (Calcd for C14H18O4N [M+H]+: 264.12303).
4.1.4.18 Methyl 4-(cyclohexylmethoxy)-5-ethoxy-1H-indole-2-carboxylate (9m)
2-Cyclohexylmethoxy-3-ethoxybenzaldehyde and methyl 2-azidoacetate was treated
with NaOMe in MeOH according to the general procedure D to afford compound 9m
as a white solid. Yield, 70%; m.p. 132-134 °C. 1H NMR (500 MHz, CDCl3) δ (ppm):
1.07-1.35 (m, 5H), 1.40 (t, J = 6.5 Hz, 3H), 1.69-1.97 (m, 6H), 3.94 (s, 3H), 4.02 (d, J
19

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= 6.0 Hz, 2H), 4.08 (q, J = 6.5 Hz, 2H), 7.01 (d, J = 8.0 Hz, 1H), 7.05 (d, J = 8.0 Hz,
1H), 7.27 (s, 1H), 8.70 (s, 1H). 13C NMR (151 MHz, DMSO-d6) δ (ppm): 15.7, 25.8,
26.5, 29.8, 38.5, 52.2, 67.1, 78.4, 104.9, 107.6, 118.3, 122.4, 127.5, 135.2, 142.3,
143.4, 161.9. HRMS (ESI+): found 332.18546 (Calcd for C19H26O4N [M+H]+:
332.18563).
4.1.4.19 Methyl 5-ethoxy -4-propoxy -1H-indole-2-carboxylate (9n)
2-Propoxy-3-ethoxybenzaldehyde and methyl 2-azidoacetate was treated with
NaOMe in MeOH according to the general procedure D to afford compound 9n as a
1
white solid. Yield, 70%; m.p. 99-100 °C. H NMR (500 MHz, CDCl3) δ (ppm): 1.08
(t, J = 7.5 Hz, 3H),1.41 (t, J = 7.5 Hz, 3H), 1.79-1.86 (m, 2H), 3.93(s, 3H), 4.09 (q, J
= 7.5 Hz, 2H), 4.19 (t, J =7.5 Hz, 2H), 7.02 (d, J = 8.0 Hz, 1H), 7.06 (d, J = 8.0 Hz,
13
1H), 7.29 (s, 1H), 8.70 (s, 1H). C NMR (151 MHz, DMSO-d6) δ (ppm): 10.9, 15.6,
23.5, 52.3, 66.9, 74.4, 105.0, 107.6, 118.2, 122.4, 127.6, 134.9, 142.2, 143.4, 161.9.
HRMS (ESI+): found 278.13880 (Calcd for C15H20O4N [M+H]+: 278.13868).
4.1.4.20 Methyl 4-butoxy-5-ethoxy-1H-indole-2-carboxylate (9o)
2-Butoxy-3-ethoxybenzaldehyde and methyl 2-azidoacetate was treated with NaOMe
in MeOH according to the general procedure D to afford compound 9o as a white
solid. Yield, 65%; m.p. 87-88 °C. 1H NMR (500 MHz, CDCl3) δ (ppm): 1.00 (t, J =
7.0 Hz, 3H), 1.41 (t, J = 7.0 Hz, 3H), 1.51-1.60 (m, 2H), 1.75-1.82 (m, 2H), 3.94 (s,
3H), 4.09 (q, J = 7.0 Hz, 2H), 4.23 (t, J = 6.5 Hz, 2H), 7.02 (d, J = 8.0 Hz, 1H), 7.06
13
(d, J = 8.0 Hz, 1H), 7.27 (s, 1H), 8.70 (s, 1H). C NMR (151 MHz, DMSO-d6) δ
(ppm): 13.9, 15.7, 19.2, 32.2, 52.2, 66.7, 72.5, 104.9, 107.7, 118.2, 122.3, 127.4,
135.1, 142.1, 143.4, 161.9. HRMS (ESI+): found 292.15446 (Calcd for C16H22O4N
[M+H]+: 292.15433).
4.1.5 General procedure E for the preparation of compounds 6a-6e/10
A solution of compounds 5a-e/9a (10 mmol) in MeOH (50 mL) presaturated with
hydrogen chloride was heated under reflux for 2 h. After cooling, the solution was
20

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poured into saturated sodium hydrogen carbonate (200 mL), and extracted with ethyl
acetate (3 × 80 mL). The combined organic layer was dried over MgSO4. Filtered and
concentrated, the residue was recrystallized from anhydrous EtOH to afford the target
compounds.
4.1.5.1 Methyl 6-amino-4-(benzyloxy)-1H-indole-2-carboxylate (6a)
Compounds 5a was treated with a solution of hydrogen chloride in MeOH according
to general procedure E to afford the title compound 6a as a pale yellow solid. Yield,
1
75%; m.p. 222-223 °C (217-220°C, lit.19). H NMR (400 MHz, DMSO-d6) δ (ppm):
3.78 (s, 3H), 5.12 (s, 2H), 5.16 (s, 2H), 6.06 (s, 1H), 6.14 (s, 1H), 6.95 (s, 1H), 7.36 (t,
J = 7.6 Hz, 1H),7.41 (t, J = 7.6 Hz, 2H), 7.49 (d, J = 7.6 Hz, 2H), 11.19 (s, 1H). 13C
NMR (126 MHz, DMSO-d6) δ 51.5, 71.2, 89.8, 94.2, 108.4, 110.9, 125.1, 127.1,
127.5, 129.0, 141.2, 142.1, 142.8, 152.9, 159.9
for C17H16O3N2 [M]+: 296.1161).
.
HRMS (EI+): found 296.1146 (Calcd
4.1.5.2 Methyl 6-amino-4-(4-fluorobenzyloxy)-1H-indole-2-carboxylate (6b)
Compounds 5b was treated with a solution of hydrogen chloride in MeOH according
to general procedure E to afford the title compound 6b as a pale yellow solid. Yield,
70%; m.p. 254-256 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.84 (s, 3H), 5.21 (s,
2H), 6.49 (s, 1H), 6.94 (s, 1H), 7.12 (s, 1H), 7.24 (t, J = 8.4 Hz, 2H), 7.56 (dd, J1 =
5.6 Hz, J2 = 8.4 Hz, 2H), 9.54 (br, 2H), 12.04 (s, 1H). 13C NMR (126 MHz, DMSO-d6)
δ (ppm): 52.2, 69.0, 97.4, 105.6, 115.7, 115.9, 127.1, 130.2, 130.2, 133.0, 138.4,
153.2, 153.6, 161.3, 161.7, 163.3. HRMS (ESI+): found 315.11399 (Calcd for
C17H16O3N2F [M+H]+: 315.11395).
4.1.5.3 Methyl 4-(allyloxy)-6-amino-1H-indole-2-carboxylate (6c)
Compounds 5c was treated with a solution of hydrogen chloride in MeOH according
to general procedure E to afford the title compound 6c as a pale yellow solid. Yield,
65%; m.p. 230-231 °C. 1H NMR(400 MHz, DMSO-d6) δ (ppm): 3.85 (s, 3H), 4.68 (d,
21

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J = 5.2 Hz, 2H), 5.30 (d, J = 6.4 Hz, 1H), 5.48 (d, J = 17.2 Hz, 1H), 6.06-6.16 (m,
13
1H), 6.46 (s, 1H), 6.99 (s, 1H), 7.11 (s, 1H), 9.75 (br, 2H), 12.08 (s, 1H). C NMR
(126 MHz, DMSO-d6) δ (ppm): 52.1, 69.0, 97.1, 100.2, 105.6, 117.1, 118.3, 127.2,
131.1, 133.6, 138.4, 153.6, 161.7. HRMS (ESI+ ): found 247.10758 (Calcd for
C13H15O3N2 [M+H]+: 247.10772).
4.1.5.4 Methyl 6-amino-4-isobutoxy-1H-indole-2-carboxylate (6d)
Compounds 5d was treated with a solution of hydrogen chloride in MeOH according
to general procedure E to afford the title compound 6d as a pale yellow solid. Yield,
68%; m.p. 230-233 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 1.02 (d, J = 6.8 Hz,
6H), 2.08-2.14 (m, 1H), 3.84 (d, J = 6.4 Hz, 2H), 3.85 (s, 3H), 6.42 (s, 1H), 6.95 (s,
1H), 7.10 (s, 1H), 9.62 (br, 2H), 12.04 (s, 1H). 13C NMR (126 MHz, DMSO-d6) δ
(ppm): 19.3, 28.2, 52.4, 74.5, 96.5, 105.4, 126.7, 138.4, 149.1, 149.3, 153.9, 161.5.
HRMS (ESI+): found 263.13892, (Calcd for C14H19O3N2 [M+H]+: 263.13902).
4.1.5.5 Methyl 6-amino-4-(cyclohexylmethoxy)-1H-indole-2-carboxylate (6e)
Compounds 5e was treated with a solution of hydrogen chloride in MeOH according
to general procedure E to afford the title compound 6e as a pale yellow solid. Yield,
73%; m.p. 240-242 °C.1H NMR (400 MHz, CDCl3) δ (ppm): 1.08-1.31 (m, 5H),
1.65-1.85 (m, 6H), 3.83 (s, 3H), 3.87 (d, J = 5.6 Hz, 2H), 6.34 (s, 1H), 6.82 (s, 1H),
13
7.06 (s, 1H), 9.18 (br, 2H), 11.92 (s, 1H). C NMR (126 MHz, DMSO-d6) δ (ppm):
25.8, 26.5, 29.5, 37.1, 52.4, 73.4, 85.8, 96.5, 105.6, 115.0, 116.7, 126.9, 138.4, 154.1,
161.8. HRMS (ESI+): found 303.17031, (Calcd for C17H23O3N2 [M+H]+: 303.17032).
4.1.5.6 Methyl 6-amino-1H-indole-2-carboxylate (10)
Compounds 9a was treated with a solution of hydrogen chloride in MeOH according
to general procedure E to afford the title compound 10 as a pale yellow solid. Yield,
65%; m.p. 240-242 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.87 (s, 3H), 7.01 (s,
1H), 7.19 (s, 1H), 7.40 (s, 1H), 7.72 (d, J = 8.4 Hz, 1H), 9.79 (br, 2H), 12.01 (s, 1H).
13C NMR (151 MHz, DMSO-d6) δ (ppm): 52.3, 107.2, 107.9, 115.7, 123.3, 126.2,
22

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128.6, 129.8, 137.4, 161.9. HRMS (ESI+ ): found 191.08137 (Calcd for C10H11O2N2
[M+H]+: 191.08150).
4.1.6 Methyl 6-amino-4-hydroxy-1H-indole-2-carboxylate (7)
To a solution of compounds 6a (1.0 g, 3.4 mmol) in EtOH (30 mL) and AcOH (5 mL)
was added Pd/C (50 mg, 10%), and the resulting mixture was stirred at room
temperature under 1 atm H2 for 4 h. After that, the mixture was filtered, and the
filtrate was concentrated in vacuo to give the title compound as a pale brown solid.
Yield, 90%; m.p. 207-208 °C. 1H NMR (400 MHz, DMSO-d6) δ (ppm): 3.85 (s, 3H),
6.38 (s, 1H), 6.84 (s, 1H), 7.18 (s, 1H), 9.80 (br, 2H), 10.37 (s, 1H), 11.96 (s, 1H). 13C
NMR (126 MHz, DMSO-d6) δ (ppm): 52.2, 98.6, 99.8, 106.1, 117.4, 126.9, 130.2,
138.8, 153.1, 161.9. HRMS (ESI+): found 207.07626 (Calcd for C10H11O3N2 [M+H]+:
207.07642).
4.2 Biological experiment
A549, HepG2 and MCF7 cell lines were purchased from the American Type
Culture Collection. The positive control drug PQQ and Etoposide was purchased from
J&K Chemical. The antibodies against cleaved/total PARP were from Cell Signaling
Technology, Inc. (Danvers, Massachusetts, USA). All the tested compounds were
dissolved in DMSO to 100 mM stock solutions, and they were further diluted with
culture medium prior to use.
4.2.1 MTT assay
The cells were seeded into a 96-well plate at a density of 5000 (100 ꢀL) per well
for 24 h, followed by drug treatment (100 ꢀL) for 48 h. Next, 20 ꢀL of 5 mg/ml MTT
was added to the medium, and the cells were incubated for 4 h at 37 °C and 5% CO2.
After removing the culture medium, 150 ꢀL of dimethyl sulfoxide was added. The
plates were read using an enzyme-linked immunosorbent assay plate reader at 570 nm.
The viability of untreated cells was set as 100%, and the viability in the other groups
was calculated by comparing the optical density reading with the control. The IC50
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values were calculated using nonlinear regression analysis. The assays were repeated
three times.
4.2.2 Measurement of reactive oxygen species
A549 cells were treated with various concentrations of the indicated compounds
(The concentration for PQQ was 200 ꢀM) for 24 h. Next, the cells were stained with 5
mmol/L H2DFFDA, ROS specific dye, for 1 h at 37 °C. The cells were harvested and
then immediately underwent flow cytometric analysis with excitation and emission
settings of 488 nm and 530 nm, respectively. For each sample, 10,000 cells were
counted.
4.2.3 Western Blotting
A549 cells were treated with various concentrations of the indicated compound
for 48 h. Next, the cells were harvested by centrifugation at 1000 g for 5 min. The cell
pellets were washed with PBS, resuspended in lysis buffer (150 mM NaCl, 50 mM
Tris (pH 8.0), 0.02% NaN3, 0.01% PMSF, 0.2% Aprotinin, and 1% TritonX-100,
supplemented with protease inhibitor cocktail (Thermo Scientific)), and centrifuged at
12000 g for 10 min. The total protein concentration was determined using the
Bio-Rad protein assay. 50 µg protein of each sample was loaded and resolved by
SDS-PAGE and transferred to a polyvinylidene fluoride membrane. After blocking
with 5% non-fat milk in blocking buffer (PBS containing 0.1% Tween 20, pH 7.5),
the membrane was incubated with the indicated primary antibody for 2 h at room
temperature and then incubated with the appropriate peroxidase-conjugated secondary
antibody. The immunoreactive bands were visualised using the ECL Plus Western
Blotting Detection System (Piscataway, New Jersey, USA). Beta-actin was used as a
loading control.
4.2.3 Cell Cycle Analysis
A549 cells were seeded at 2×105 cells/well in 6-well plates and cultured for 48 h.
Next, the cells were incubated with the test compounds for 48 h. The cells were then
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treated with cold PBS. After harvest, the cells were fixed in 70% ice-cold ethanol
overnight. Subsequently, the cells were centrifuged (1200 rpm for 5 min), the
supernatant was discarded and the pellet was treated with RNase A (100 ꢀg/mL) for
30 min at room temperature. The cells were stained using propidium iodide at a final
concentration of 50 ꢀg/mL. The stained cells were then analysed for cell cycle
distribution using flow cytometry (BECKMAN), and the changes in the cell cycle
profiles was analysed using CELL QUEST PRO.
Acknowledgement
This work was supported by the International Science & Technology Cooperation
Program of China (2012DFA30530) and the grants from the National Science
Foundation of China (81373438).
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