Synthesis, anticancer and antioxidant properties of new indole and pyranoindole derivatives

Article abstract of DOI:10.1016/j.bioorg.2020.104440

The indole scaffold has been recognized, over the years, as a model for the synthesis of compounds with anticancer activity by dint of its substantiated ability to act via multiple mechanisms, which also involves the inhibition of enzymes engaged in DNA replication. In this regard, a new series of indole and pyranoindole derivatives have been prepared, some of which showed good antitumor activity and proved their inhibitory effects on the tubulin target. The anticancer activity of the newly synthesized compounds has been evaluated on breast cancer cell lines, as MCF-7 and MDA-MB231, cervical cancer cells line HeLa and Ishikawa endometrial cancer cell line. Among the compounds under study, 7 exhibited a good antitumor activity on HeLa cell line (IC₅₀ = 3.6 ± 0.5), leading to cell death by apoptosis due to the inhibition of tubulin polymerization, which demonstrated that the compound can explicate its function in a similar way to Vinblastine, a well-known inhibitor of tubulin polymerization. The data were also confirmed by in silico assays. No cytotoxicity against normal cells has been detected. Furthermore, in order to investigate the antioxidant properties, DPPH and ABTS tests were performed, together with fluorescence assays on 3T3-L1 cells. All our findings taken together led us to consider compound 7 a favourable candidate for the battle against cancer.

Full text of DOI:10.1016/j.bioorg.2020.104440

Journal Pre-proofs
Synthesis, anticancer and antioxidant properties of new indole and pyranoin-
dole derivatives
Domenico Iacopetta, Alessia Catalano, Jessica Ceramella, Alexia Barbarossa,
Alessia Carocci, Alessia Fazio, Chiara La Torre, Anna Caruso, Marco
Ponassi, Camillo Rosano, Carlo Franchini, Maria Stefania Sinicropi
PII:
S0045-2068(20)31738-7
DOI:
https://doi.org/10.1016/j.bioorg.2020.104440
YBIOO 104440
Reference:
To appear in:
Bioorganic Chemistry
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Revised Date:
Accepted Date:
20 August 2020
30 September 2020
24 October 2020
Please cite this article as: D. Iacopetta, A. Catalano, J. Ceramella, A. Barbarossa, A. Carocci, A. Fazio, C. La
Torre, A. Caruso, M. Ponassi, C. Rosano, C. Franchini, M. Stefania Sinicropi, Synthesis, anticancer and
antioxidant properties of new indole and pyranoindole derivatives, Bioorganic Chemistry (2020), doi: https://
doi.org/10.1016/j.bioorg.2020.104440
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Synthesis, anticancer and antioxidant properties of new indole and pyranoindole derivatives
Domenico Iacopettaa,1, Alessia Catalanob,1, Jessica Ceramellaa, Alexia Barbarossab, Alessia
Caroccib,*, Alessia Fazioa, Chiara La Torrea, Anna Carusoa, Marco Ponassic, Camillo Rosanoc, Carlo
Franchinib, Maria Stefania Sinicropia
aDepartment of Pharmacy, Health and Nutritional Sciences, University of Calabria, 87036,
Arcavacata di Rende (Italy)
bDepartment of Pharmacy-Drug Sciences, University of Bari “Aldo Moro”, 70126, Bari (Italy)
cBiopolymers
Largo R. Benzi 10, 16132 Genova, Italy
and
Proteomics
IRCCS,
Ospedale
Policlinico
San
Martino–IST,
* Corresponding author: e-mail address: alessia.carocci@uniba.it
1 These authors equally contributed to this work.
Abstract
The indole scaffold has been recognized, over the years, as a model for the synthesis of compounds
with anticancer activity by dint of its substantiated ability to act via multiple mechanisms, which also
involves the inhibition of enzymes engaged in DNA replication. In this regard, a new series of indole
and pyranoindole derivatives have been prepared, some of which showed good antitumor activity and
proved their inhibitory effects on the tubulin target. The anticancer activity of the newly synthesized
compounds has been evaluated on breast cancer cell lines, as MCF-7 and MDA-MB231, cervical
cancer cells line HeLa and Ishikawa endometrial cancer cell line. Among the compounds under study,
7 exhibited a good antitumor activity on HeLa cell line (IC50 = 3.6±0.5), leading to cell death by
apoptosis due to the inhibition of tubulin polymerization, which demonstrated that the compound can
explicate its function in a similar way to Vinblastine, a well-known inhibitor of tubulin
polymerization. The data were also confirmed by in silico assays. No cytotoxicity against normal
cells has been detected. Furthermore, in order to suggest a possible relationship between anticancer
effects and to investigate the antioxidant properties, DPPH and ABTS tests were performed, together
with fluorescence assays on 3T3-L1 cells. All our findings taken together led us to consider
compound 7 a favourable candidate for the battle against cancer.
Key words: indole derivatives, tubulin, molecular docking studies, anticancers, antioxidants

Introduction
Despite the technological and social development, cancer is one of the most common diseases of
concern and a leading cause of human suffering. It was estimated that deaths from cancer worldwide
would rise over 13.1 million in 2030 (WHO report, Globocan 2012, IARC). Therefore, the design of
newer and potent anticancer agents with higher selectivity on neoplastic cells and few side effects,
able to overcome problems like severe toxicity as well as resistance with the existing drugs, may be
envisaged. An ongoing source of debate is the possibility of some molecules with antioxidant
properties to explicate chemopreventive action. Indeed, many prior studies corroborate that the
antioxidants can enhance the known chemotherapy protocols by ameliorating the toxic side effects
without affecting treatment efficacy [1]. Besides, other in vitro investigations suggest the relevant
role of this kind of molecules in inducing the apoptosis in cancer cells [2]. In the last decades, research
efforts resulted in the development of numerous antineoplastic agents with a variety of structures.
Among them, the indole nucleus, due to its biodiversity and versatility, has been a highly privileged
motif for the target-based design and development of anticancer agents [3]. Furthermore, FDA
approved the use of some naturally occurring indole alkaloids for anti-tumor activity, including
vinblastine (1961, breast cancer, choriocarcinoma, Hodgkin lymphoma, Kaposi sarcoma, mycosis
fungoides, non-Hodgkin lymphoma, testicular cancer), vincristine (1963, leukemia), vinorelbine
(1994, non-small cell lung cancer) and vindesine (1994, melanoma, lung cancers and uterine cancer)
(Figure 1). Vinca alkaloids (vinblastine and vincristine) isolated from Catharanthus roseus
(Apocynaceae) were the first plant alkaloids investigated for their use as anticancer agents [4]. They
cause disruption of microtubules and arrest of cells in the metaphase, ultimately causing cell death
by apoptosis. A number of semisynthetic analogues of vinblastine and vincristine, including
vinorelbine and vindesine that are comparatively more effective, have been then developed [5].
Taking inspiration from these natural compounds, several synthetic analogues were synthesized,
some of which showing good anti-tumor activity [6]. Recently, indole derivatives have been studied
as inhibitors of enzymes involved in DNA replication as topoisomerase, telomerase, tubulin [7-9].

O
CH3
N
MeOOC
HO
H
MeOOC
HO
H
N
Me
OMe
O
Me
OMe
O
COOMe
Et
COOMe
Et
O
H
N
H
O
H
N
H
H
N
H
N
Et
HO
Et
HO
N
N
vincristine
vinblastine
O
CH3
CH3
N
H2N
MeOOC
HO
H
H
N
HO
HO
Et
Me
OMe
O
OMe
COOMe
Et
COOMe
O
H
N
H
H
N
H
H
H
N
N
Et
HO
N
N
Et
vinorelbine
vindesine
Figure 1. Structure of Vinca alkaloids
Due to the multiple functions of microtubules in cell mitosis, tubulin has become a highly
attractive target for new anticancer drugs discovery [10]. Therefore, tubulin/microtubule-interacting
drugs are used successfully for treatment of a wide variety of human cancers [11]. In the last decade,
the synthesis and biological evaluation of some indole and corresponding pyranoindole derivatives
(I‒IV, Figure 2) was reported [12]. The compounds were evaluated for their inhibition of NO
production, antioxidant activity and for their ability to inhibit in vitro the growth of four human tumor
cell lines (large lung carcinoma COR-L23, alveolar basal epithelial carcinoma A549, amelanotic
melanoma C32 and melanoma A375) [12]. Two indole derivatives (I, II) showed to be active against
amelanotic melanoma cell lines, while inhibiting NO production in murine monocytic macrophage.
Moreover, several researches highlighted the ability of indole derivatives to suppress the proliferation
of a large panel of tumor cells, including breast and uterine cancer cells. In particular, it was reported
that indole-3-carbinol and its metabolite exerted a potent effect in cervical cancer and in human
Ishikawa endometrial cancer cells [13, 14]. In addition, the same compounds caused a selective cell

growth inhibition and induction of apoptosis in several breast tumor cells (MDA-MB-468, MCF-7
and MDA-MB-231) [15] [16]. More recently, Prasad et al also demonstrated the capability of three
2-amino-2-(1H-indole-2-yl) derivatives to suppress the proliferation of breast cancer cell lines, MCF-
7 and MDA-MB-231 [17].
O
O
I, III
: R = H
O
O
N
R
II, IV
: R = Me
N
R
I, II
III, IV
Figure 2. Structure of indoles (I, II) and pyranoindoles (III, IV) described in the literature
Based on these evidences, new indole and pyranoindole derivatives (5‒13, Figure 3) have been
synthesized in order to enlarge studies on this class of compounds towards the breast and uterine
cancer cells, representing the leading cause of death in women [18]. Thus, the anticancer activity on
breast cancer (MCF-7 and MDA-MB-231) and human uterine carcinoma cell lines (HeLa and
Ishikawa) of these new compounds is reported herein, along with the evaluation of their ability to
inhibit tubulin microtubule polymerization. Furthermore, in order to delve into the possible
relationship between the antioxidant properties and anticancer mechanisms, the antioxidant activity
of the synthesized compounds have been evaluated by means of both in vitro and cellular assay.
Finally, molecular docking studies have also been conducted to support the results obtained.

O
R
O
O
R
O
N
N
H
H
5:
6:
7:
R = CH2CH3
R = CH2CH2CH3
R = Ph
11:
12:
R = CH2CH3
R = CH2CH2CH3
O
R
O
O
O
R
N
N
8:
9:
R = CH2CH3
13:
R = CH2CH3
R = CH2CH2CH3
10:
R = Ph
Figure 3. Structure of indoles (5‒10) and pyranoindoles (11‒13) under study.
2. Results and Discussion
2.1. Chemistry
The synthesis of 5-hydroxy-1-methylindole 3 is depicted in Scheme 1. 5-Methoxyindole 1 was
treated with NaH to obtain the N-anion followed by reaction with methyl iodide to give 5-methoxy-
1-methylindole 2. Selective demethylation of the methoxy group by heating compound 2 with
anhydrous pyridine hydrochloride gave the desired 3 [12].
O
O
HO
i
ii
N
H
N
N
1
2
3
Scheme 1. Reagents and conditions: (i) NaH 60% oil dispersion, CH3I, DMF, 0 °C→rt, 1 h (81%);
(ii) pyridine hydrochloride, Δ, 3 h (46%).

The synthesis of the indole and pyranoindole derivatives is depicted in Scheme 2. Commercial 5-
hydroxyindole 4 was submitted to esterification with pent-2-ynoic acid, es-2-ynoic acid or
phenylpropiolic acid to give the corresponding esters 5, 6, 7, respectively. The same reactions, run
on 5-hydroxy-1-methylindole 3 gave esters 8, 9, 10. When esters 5, 6, 8 were heated under reflux in
dioxane-dichloroethane in the presence of PtCl4 as a catalyst, an intramolecular cyclization occurred
giving pyranoindoles 11,12,13, respectively, as confirmed by NMR analyses.
O
R
O
O
HO
i
ii
R
O
N
R
N
N
R1
R1
3,4
5 10

13

Scheme 2. Reagents and conditions: (i) pent-2-ynoic acid (for compounds 5 and 8, 48% and 40%,
respectively) or es-2-ynoic acid (for compounds 6 and 9, 42% and 11%, respectively) or
phenylpropiolic acid (for compounds 7 and 10, 40% and 34%, respectively), DCC, DMAP,
CH2Cl2/DMF (10:1), rt, 4 h; (ii) PtCl4, 1,4-dioxane/1,2-dicloroethane (1:1), reflux, 5 h (55%, 16%
and 62%, for 10, 11 and 13, respectively).
2.1.1 Thermal stability test
Prior research has thoroughly investigated the possibility of several organic compounds to decompose
by heat action[19]. Compound 7, the most bioactive molecule of the series, presents in its structure a
triple bond, a possible source of molecular instability. For this reason, through the modification of
already known protocols aimed at verifying heat stability, we performed a “stress test assay” on 7 by
heating it for 6 hours at 60 °C. At the end of the process, we compared the NMR spectra of 7 in the
usual conditions and after the heat treatment. The match of the two spectra surmises that 7 is heat
resistant.
2.2. Biological Results
2.2.1 Cell viability assay

The cytotoxic activities of synthesized compounds were evaluated against four human cancer cell
lines, including two breast cancer cells (MCF-7 and MDA-MB-231) and two human uterine cancer
cells (HeLa and Ishikawa), using MTT assay. The calculated IC50 values of studied compounds in
comparison with the reference drug, Vinblastine, are listed in Table 1. Among them, the indole
bearing the phenylpropiolic group, compound 7, exhibited the best anticancer activity, particularly
toward the human cervical cancer cells HeLa and the breast cancer cells MCF-7, with the IC50 values
amounting to 3.6±0.5 and 3.8±0.7 μM, respectively (dose response curve is shown in Figure 11 of
supplementary materials). A moderate anticancer activity toward all the cell lines used was also
shown by compounds 5 and 6, mostly toward HeLa cells (IC50= 9.8±0.3 and 14.5±0.6 μM,
respectively). Compounds 5 and 6 are structurally indole analogues of compound 7, bearing the pent-
2-ynoyl and es-2-ynoyl groups, respectively. The methylindole derivatives 8, 9 and 10 possessed a
lower activity than the indole analogues, except for methylindole 8 that showed an interesting
anticancer activity, in particular against the uterine cancer cells Ishikawa and HeLa (IC50= 13.1±0.3
and 14.0±0.7 μM, respectively). Thus, the addition of a methyl group on the nitrogen indole ring
appears to cause a decrease in antiproliferative activity. Moreover, the pyranoindole compounds 11,
12 and 13 exhibited a lower o no antiproliferative activity on the cell lines tested, highlighting the
importance of the indole group in the moiety of these compounds. The known microtubule-binding
agent Vinblastine showed a stronger anticancer activity than our synthesized compounds on all cancer
cell lines, with IC50 values between 2.3x10-2±0.6 μM and 1.6x10-1±0.3 μM. However, Vinblastine
exhibited also a dramatic cytotoxic effect on normal cells used in this assay (MCF-10A, Hek-293 and
3T3-L1), unlike our compounds that did not interfere with the normal cells viability, at least until the
concentration of 200 μM and under the conditions used for this assay.

Table 1. IC50 values of compounds 5-13 and Vinblastine expressed in µMa.
Compounds
MCF-7
MDA-MB-231
HeLa
Ishikawa MCF-10A Hek-293 3T3-L1
5
6
13.1±0.3
30.2±0.2
3.8±0.7
23.0±0.4
>200
10.7±0.2
15.9±0.5
9.9±0.3
9.8±0.3
14.5±0.6
3.6±0.5
14.0±0.7
>200
12.1±0.6
30.5±0.5
10.1±0.7
13.1±0.3
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
>200
7
8
23.5±0.4
>200
9
10
11
12
13
37.6±0.5
86.0±0.3
>200
40.7±0.8
44.4±0.3
172.4±0.6
80.2±0.5
49.5±0.9
21.1±0.7
141.4±0.7
>200
77.7±0.4
84.0±0.5
>200
131.5±0.3
>200
Vinblastine 4.5x10-2±0.6 1.6x10-1±0.3 6.7x10-2±0.5 2.5x10-2±0,7 2.3x10-2±0.6 80.8±0.4 1.9x10-4±0.8
aValues are the mean of at least three determinations performed in triplicate.
2.2.2 Effect of compound 7 on microtubule dynamics and cell death by apoptosis
Numerous studies have shown that the alteration of microtubule dynamics induced by different
compounds causes mitochondrial membrane potential activation, cell cycle and mitotic process arrest
[11, 20-22]. To demonstrate the specific action of the synthesized compounds on tubulin network, we
chose compound 7 and performed an immunofluorescence test using the anti-β-tubulin antibody on
HeLa cells, on which compound 7 displayed the best antiproliferative activity. In Figure 4, the
vehicle-treated cells (panel A, CTRL) showed a normal distribution of the microtubule network, with
intact tubulin polymers regularly distributed throughout the cytoplasmic region (panel B, CTRL).
After only 24 hours of compound 7 treatment (used at its IC50 value), tubulin microfilaments appeared
as distinct punctiform structures and accumulates unfairly throughout the cell cytoplasm and nucleus
(Panel B, compound 7, see white arrows). A similar behavior can be observed in Figure 3, Panel B,
Vinblastine, where microtubules became irregular and the arrangement and organization of the
tubulin network showed a severe disruption with a stronger formation of dot-like structures.

A
C
B
CTRL
Vinblastine
7
Figure 4. Immunofluorescence studies. HeLa cells were treated with the compound 7 or with
Vinblastine (both used at their IC50 values) or with a vehicle (CTRL) for 24 h. After treatment, the
cells were incubated with antibodies and observed under the inverted fluorescence microscope at 63×
magnification, as in depth described in Materials and Methods. Vehicle-treated HeLa cells (CTRL)
exhibited a normal arrangement and organization of the cytoskeleton. Instead, HeLa cells treated with
compound 7 and Vinblastine showed an irregular arrangement and organization of tubulin. Panels A:
DAPI (λex = 350 nm/λem = 460 nm); Panels B: β-tubulin (Alexa Fluor® 488; λex = 490 nm/λem = 515
nm); Panels C: show the overlay channels. Representative fields are shown. DAPI (λex = 350 nm/λem
= 460 nm); Panels B: β-tubulin (Alexa Fluor® 488; λex = 490 nm/λem = 515 nm); Panels C: show the
overlay channels. Representative fields are shown.
To confirm these data, we examined the effect of compound 7 on the tubulin polymerization using a
specific in vitro assay and measuring the turbidity variation every 30 seconds at 350 nm for 90
minutes. We used two well-known compounds as reference molecules, a tubulin polymerization
inhibitor, Vinblastine, and a stabilizing agent, Paclitaxel. In the only vehicle (CTRL) reaction, tubulin
heterodimers rapidly self-assembled to form linear tubulin polymers, in a time-dependent manner,
with a final optical density (OD350) of above 0.44, as showed in Figure 5. The microtubule-stabilizing
agent Paclitaxel caused an increased rate of tubulin heterodimers assembly, whereas using the

microubule-destabilizing agent such Vinblastine, the polymerization reaction was hampered (final
OD350 of 0.19). Compound 7 interfered with the tubulin polymerization with a similar effectiveness
to Vinblastine, indeed the polymerization curve reached the steady state at an OD350 1.5-fold lesser
than the control reaction (OD350 = 0.48 and 0.3 for CTRL and compound 7, respectively), with a final
OD350 value of about 0.24. Thus, compound 7 resulted an inhibitor of tubulin polymerization.
CTRL
Paclitaxel
Vinblastine
7
0.6
0.5
0.4
0.3
0.2
0.1
0
900
1800
2700
3600
0
Time (sec)
Figure 5. Tubulin-polymerization assay. The effect of compound 7 on in vitro tubulin polymerization
was evaluated by measuring the turbidity variation at 350 nm for 3600 s, as described in the
experimental section. Paclitaxel and Vinblastine were used, at a concentration of 10 μM, as tubulin-
stabilizing and tubulin-destabilizing agents, respectively. DMSO was used as a negative control.
The alteration of microtubules network causes a blockage of the mitotic process progression with the
induction, in the last phase, of the apoptotic process [23, 24]. For this reason, we decided to perform
a TUNEL assay with the aim to demonstrate the ability of compound 7 to induce cell death by
apoptosis on the HeLa tumor cell line. The induction of apoptosis promotes morphological
characteristic changes such as the cellular narrowing (the cytoplasm is dense and the organelles are
narrower), the picnosis, which is the result of the condensation of the chromatin, and the
fragmentation of the DNA[25]. The experiment was conducted by treating HeLa with compound 7,

used at its IC50 value, or with the vehicle (DMSO) for 24 h, and then performing the TUNEL assay,
as described in the materials and methods section. The images obtained (Figure 6) clearly show the
development of a nuclear green fluorescence in the cells treated with compound 7, due to DNA
damage related to the apoptotic process. This fluorescence, however, is absent in the cells treated
with the vehicle only (Panel B, CTRL) used as control.
CTRL
7
A
B
C
Figure 6. Tunel assay. HeLa cells were treated with compound 7 at its IC50 value or with vehicle
(CTRL) for 24 h. After treatment, cells were incubated with rTdT enzyme reaction and then observed
and imaged under an inverted fluorescence microscope at 20x magnification (see materials and
methods). The green nuclear fluorescence, visible in cells treated with compound 7, and absent in the
CTRL cells, indicates the apoptotic death. Panels A: DAPI (λex = 350 nm/λem = 460 nm); Panels B:
CFTM488A (λex = 490 nm/λem = 515 nm) Panels C: show the overlay. Fields are representative of
three separate experiments.

It is known that reactive oxygen species (ROS), at low doses, are essential for regulation of
normal physiological functions of the cells. However, high cellular levels of ROS cause serious
damages to proteins, nucleic acids, lipids, membranes and others organelles, which can lead to trigger
cell death pathway such as apoptosis [26]. Considering the main role of ROS in the regulation of the
apoptosis process, we investigated whether the most active compound 7 could act as ROS scavenger
in a non-tumoral cell model, the mouse fibroblasts 3T3-L1, by using fluorescence assays. In
particular, we treated 3T3-L1 with compound 7 at a concentration of 10 μM for 24 h and then we
induced ROS production treating with menadione (Men) for 1 h at 40 μM. At the end of the treatment
cells were incubated with 2’,7’-dichlorofluorescein diacetate (DCFH-DA) for 2 h. DCFH-DA is
hydrolyzed by cellular esterases to 2’,7’-dichlorodihydrofluorescein and, then, oxidized to the green
fluorescent 2’,7’- dichlorofluorescein (DCF) by intracellular H2O2. The green fluorescence was then
observed and imaged under a fluorescence microscope. The obtained results, showed in Figure 7,
highlighted that the pre-treatment with compound 7 significantly reduced the oxidative stress induced
by menadione (Figure 7a, panel B, 7+Men), if compared with the cells treated only with menadione
(Figure 6a, panel B, Men). The quantification of the green fluorescence (Figure 7b) demonstrated, in
fact, that compound 7 was able to cause a three-fold decrease of ROS generation in the 3T3-L1 cells,
acting as a good scavenging of ROS.

A
B
C
a
CTRL
Men
7 + Men
b
ROS Scavenging
25000
20000
15000
10000
5000
0
*
CTRL
Men
7 + Men
Figure 7. ROS scavenging activity. (a) Evaluation of ROS scavenging activity using DCF-DA
(CFTM488A), in 3T3-L1 cells treated with compound 7 and Men (used as ROS inducer). Images were
acquired at 20x magnification. The pre-treatment with compound 7 causes a 3-fold reduction of ROS
production induced by Men (Figure 6a, panels B, 7 + Men). Panels A: 2-(4-amidinophenyl)-6-
indolecarbamidine dihydrochloride (CTRL, Men, 7 + Men) λex/em= 350 nm/460 nm. Panels B:
CFTM488A (CTRL, Men, 7 + Men,) λex/em= 490 nm/515 nm. Panels C (CTRL, Men, 7 + Men) show
the overlay channel. (b) Fluorescence quantification; * p < 0.001, 7 + Men versus Men.

2.3 In vitro antioxidant activity
The radical scavenging activity of the synthesized compounds against the two most commonly used
stable radicals (DPPH• and ABTS•+) was evaluated by in vitro studies.
2.3.1 DPPH assay
The free radical scavenging activity against DPPH was determined for all compounds under study
(5-13). The DPPH assay, which is based on the reduction of the purple DPPH to 1, 1-diphenyl-2-
picryl hydrazine (DPPH-H) via hydrogen transfer (HT) mechanisms, measures the ability of
antioxidant compounds for trapping free radicals by donating hydrogen atoms, producing in
consequence the bleaching of the colored radical solutions.
The results obtained as a function of concentration (8.33, 3.33, 1.67, 0.33 μg mL−1) were reported in
Table 2 (Figure 8 in supporting information). The results showed that the scavenging effects of the
compounds on DPPH radical increased with the concentration. The data indicated that, among the
synthetic compounds, 6 with the inhibition percentage of 90.45% at the highest concentration (8.33
μg mL−1), displayed higher antioxidant activity than the other compounds. Compound 6 exhibited a
high value of antioxidant ability even at lower concentration (78.2% at 1.67 μg mL−1). Compounds 9
and 10 showed moderate antioxidant activities (79.04 and 73.40 %, respectively, at the highest
concentration).
Compounds 7 and 12 showed a DPPH inhibition percentage of 43% and 50% at the highest
concentration. The lowest antioxidant activity with a DPPH inhibition value of 10% at 8.33 μg mL−1
belongs to 8. Compound 11 was the only compound that did not exhibit any antioxidant activity at
the lowest concentration tested (0.33 μg mL−1).
The antioxidant activity does not appear to rely on the presence or absence of a methyl group onto
the indole nitrogen atom, but it seems to depend mostly on the alkyl or aryl group in the side chain of
the tested compounds. From our results it seems that the presence of a n-propyl group is crucial for
antioxidant activity both in the series of indoles and pyranoindoles (see compounds 6, 9 and 12),

followed by the phenyl one (compounds 7 and 10). On the contrary, the methyl group is detrimental
for this activity (see compounds 5, 8, 11 and 13).
Table 2. %IDPPH of compounds 5‒13 ± standard deviation
Concentration (µg mL-1)
8.33
3.33
1.67
0.33
Compound
Trolox
99.0±0
93.33±0.51
32.02±2.85
92.68±0.53
30.13±0.69
92.68±0.53
17.75±0.53
5
36.02±0.78
6
7
90.45±0.52
43.25±1.36
10.44±1.61
79.04±2.69
73.40±0.30
35.60±2.21
50.53±0.25
14.39±1.66
87.96±1.06
31.42±3.48
6.93±1.22
78.18±0.54
26.58±3.52
4.81±0.76
24.90±2.98
5.96±1.35
2.02±0.76
11.83±3.65
11.80±0.45
0
8
9
49.11±1.68
42.96±3.08
26.83±1.47
37.72±3.27
15.25±0.90
35.00±2.14
24.76±4.69
11.23±0.41
28.16±1.38
13.16±0.25
10
11
12
13
21.48±0.40
9.55±2.79
Antioxidant capacities were also expressed as Trolox equivalent antioxidant capacity (TEAC). The
results reported in Table 3 highlighted that 6 was the compound with the highest antioxidant activity
at all concentrations (from 37.90±8.25 at 0.33 µg mL-1 to 219.56±1.43 at 8.33 µg mL-1), while lowest
TEAC value results from 8 (2.90±0.02 at 8.33 µg mL-1 ).

Table 3. DPPH radical scavenging of compounds 5‒13 expressed as µg of TE/g DW
Concentration (µg mL-1)
8.33
3.33
1.67
0.33
Compound
5
70.05±2.17
59.08±7.87
53.86±1.89
19.81±1.49
6
7
219.56±1.43
89.56±3.78
2.90±0.02
212.67±2.96
43.60±9.74
0
185.52±1.48
56.94±9.58
0
37.90±8.25
0
8
0
9
189.09±7.28
173.62±0.81
71.23±5.98
111.71±0.70
13.62±4.54
107.91±4.58
90.75±8.36
47.43±3.96
76.94±8.86
16.00±2.46
69.56±5.85
41.22±12.77
5.05±1.08
51.00±0.14
10.29±0.72
6.70±0.89
6.00±1.24
0
10
11
12
13
32.90±1.08
0.51±0.02
In order to furtherly assess which extracts have the greatest antioxidant activity, EC50 values were
determined (Table 4). Low EC50 values reflect a strong ability of the molecule to act as a DPPH
scavenger whereas a high EC50 value indicates low scavenging activity of the scavengers, as more
amount of the scavengers was required to achieve 50 % scavenging reaction. Consequently, the
scavengers are less effective at scavenging DPPH radicals. Data obtained showed that the order of
reactivity found was 6 > 9 > 10 > 12 > 7 > 5 >11 > 13 > 8. Considering these findings, it is possible
to confirm that the compound 6 is the compound with the highest DPPH radical scavenging capacity
while the compound 8 is a poor DPPH radical scavenger.

Table 4. EC50 values of compounds 5‒13
expressed in µg mL-1 ± standard deviation
EC50± SD
Compound
Trolox
5
0.04±0.01
8.67 ± 1.21
6
7
0.66 ± 0.2
8.30 ± 2.43
61.76 ± 2.36
2.96 ± 0.54
4.00 ± 0.12
12.79 ± 1.17
5.76 ± 1.32
32.55 ± 1.41
8
9
10
11
12
13
2.3.2 ABTS assay
The free radical scavenging activity against ABTS (2,2’-azino-bis(3-ethylbenzothiazoline-6-
sulfonate)) was determined at four different concentrations (2.5, 1.0, 0.5 and 0.1 mg mL−1 in DMSO)
and it is shown in Table 5 (Figure 9 in supporting information). All the derivatives exhibited a lower
capacity to inhibit the cationic radical ABTS•+ than the DPPH radical. Similarly to DPPH assay, the
scavenging effects of the compounds on cationic radical ABTS•+ increased with the compound
concentration. An opposite trend was observed for the ABTS assay, where 9 and 10 had higher
scavenging activity (30.34% and 21.56, respectively) than 6 (14.96%). The antioxidant ability
determined by in vitro assays can significantly differ[27] but the combination of the results obtained
from these two assays is considered of higher significance for a reliable evaluation of compounds
radical scavenging capacity than the use of only one method. The ABTS assay is based on the in situ
generation of a blue/green radical (ABTS•+) that can be reduced by antioxidants by either single

electron transfer (SET) or hydrogen transfer (HT) mechanisms, whereas the DPPH assay is based on
its reduction to 1,1-diphenyl-2-picryl hydrazine (DPPH-H) via HT mechanism.
6 at the highest concentration did not exceed 15 % of inhibitory activity, while recording the zero at
the lowest concentration. For all the other tested molecules no important inhibitory activity was
highlighted and 11, 8, 13 were confirmed as the least active compounds also against the radical cation
ABTS•+.
Table 5. %IABTS of compounds 5‒13 ± standard deviation
Concentration (µg mL-1)
25
10
5
1
Compound
Trolox
99.00±0
99.04±0.41
3.12±0.51
51.52±1.33
2.36±0.28
18.81±0.65
1.18±0.69
5
4.82±0.48
14.96±0.33
11.59±0.16
3.52±1.56
30.34±1.35
21.56±1.48
1.26±0.24
6
7
3.43±0.33
5.89±0.70
3.13±1.17
15.98±1.96
8.86±1.38
0
0
2.36±0.07
4.62±0.33
2±0.73
6.86±0.57
3.81±0.28
0
0.58±0.02
8
0
9
2.85±2.01
1.67±0.51
0
10
11
12
13
4.12±0.66
3.22±0.14
0
0.59±0.38
0
5.25±0.27
2.51±0.5
0.51±0.03
Antioxidant capacities were also expressed as Trolox equivalent antioxidant capacity (TEAC) (Table
6). The results highlighted that the values were very low even for the 9 which showed the highest
ABTS inhibition percentage among all the compounds (2.07% at 8.33 µg mL-1).

Table 6. ABTS radical scavenging of compounds 5‒13 expressed as µg of TE/g DW
Concentration (µg mL-1)
25
10
5
1
Compounds
5
0.34±0.04
0.21±0.04
0.16±0.02
0.07±0.04
6
7
1.10±0.02
0.63±0.01
0.31±0.12
2.07±0.11
1.67±0.11
0.07±0.02
0.07±0.02
0.22±0.18
0.23±0.02
0.17±0.06
0.28±0.09
0.94±0.16
0.71±0.11
0
0.15±0.01
0.07±0.03
0.18±0.06
0.22±0.03
0.33±0.01
0
0
0.58±0.02
8
0
0
9
10
11
12
13
0.17±0.10
0
4.12±0.66
0.07±0.08
3.22±0.14
0.03±0
0.59±0.38
0.03±0
The EC50 values for the ABTS inhibition percentage are shown in Table 7. Data highlighted that the
order of compound reactivity was 9 >10 > 6 > 12 > 7> 9 >8> >13 > 11, thus 9 and 11 are the
compounds with the highest and lowest ABTS radical scavenging, respectively.

Table 7. EC50 values expressed in µg mL-1 ±
standard deviations
EC50± DS
Molecules
Trolox
5
2.20±0.12
435.5 ± 0.5
6
7
156.6 ± 0.7
178.5 ± 1.0
560.5 ± 0.9
54.96 ± 1.48
92.33 ± 1.16
2494 ± 1
8
9
10
11
12
13
377 ± 1
1436 ± 1
2.4 Docking Studies
To select among our compounds the best lead candidates and to identify their possible binding modes
to human tubulin, we performed different docking simulations using as a target the atomic structures
of the polymeric complex formed between Tubulin  Tubulin , Stathmin4 and the Tubulin Tyrosine
Ligase[28] [PDB code 5J2T]. We compared the binding modes or our moieties to the ones of the
known ligands Colchicine and Vinblastine (Figure 10). For all our molecules, we performed a “blind
docking”: i.e. the docking of small molecules to their targets was done without a priori knowledge of
the binding mode of the ligand in the active site of the protein. Our procedure was firstly validated
by correctly positioning Vinblastine in its correct binding site, with a RMSD of less than 0.2 Å with
respect to the one determined by X-ray crystallography.

The binding site of our moieties is placed on the interface between the alfa and beta subunit of the
quaternary assembly of tubulin, in a cleft placed about 7.5Å far from the Vinblastine binding site.
Table 8 reports the residues involved in ligand binding and the binding energies of all the complexes
formed by tubulin and our compounds.

Table 8. Binding energies of the complexes formed by tubulin and compounds 5-13 and residues
involved in ligand binding.
Binding
Calculated
Compound
Tubulin residues involved in ligand binding §
Energy
Ki (M) *
2.0
(Kcal/mol)
Ala 314, Cys  Pro 348, Pro
Asp 179, Gln 394
5
6
-7.77
-8.17
-9.48
-7.13
-8.80
-7.25
-6.18
173, Pro 175, Val 181, Pro 184
Ala 314, Cys  Pro 348, Pro
Asp 179, Gln 394
1.02
173, Pro 175, Val 181, Pro 184
Ala 314, Cys  Pro 348, Pro
Asp 179, Gln 394
7
0.113
5.99
173, Pro 175, Val 181, Pro 184
Ala 314, Cys  Pro 348, Pro
Asp 179
8
173, Pro 175, Val 181, Pro 184
Ala 314, Cys  Pro 348, Pro
Asp 179
9
0.357
4.86
173, Pro 175, Val 181, Pro 184
Ala 314, Cys  Pro 348, Pro
Asp 179
10
11
173, Pro 175, Val 181, Pro 184
Pro 348, Pro 173, Pro 175, Pro
Asp 179, Gln 394
184
29.57
Pro 348, Pro 173, Pro 175, Pro
12
-6.33
-6.04
23.03
37.52
Asp 179, Gln 394
184
13 +
Gln 11, Gln 15
Leu 209, Leu 227
* Ki values as calculated by Autodock algorithm: Ki = exp(G/(R*T)
§ Residues involved in Hydrogen bond are listed in bold. Hydrophobic contacts in Italic.
+ Binds in a different cleft, on the surface of the beta subunit, out from the dimeric interface

Based on the geometry, the binding energy and the clusterization of the results, we selected as best
candidates, molecules 7, 6 and 9 in ranked order.
A
B
C
D
Figure 10. Panel A) A cartoon representation of a Tubulin tetramer (2alpha-2beta subunits).
Two different binding sites for the compounds tested are indicated by arrows. Vinblastine and
compounds 5-13 binding site are separated by about 11Å. Panel B) a more detailed view of
compound 5 (violet sticks) binding site. Panel C) reports the binding mode of compound 6
(drawn in purple) and Panel D) illustrate the pose of compound 7 within the binding site cleft.
Residues involved in the binding of the three molecules to tubulin quaternary structure are
evidenced and properly labelled.
3. Conclusions
Over the last years, the indole-based compounds have occupied an important position as powerful
and versatile pharmacological weapons in the battle against life-threatening diseases as, for instance,
cancer. Indeed, several indole derivatives have been employed pre-clinically and clinically and their

multi-target features offered better and more promising therapies, overcoming the drug resistance
mechanism and diminishing the undesirable side effects. The assessment of the basic importance of
cancer microenvironment have enlarged the number of potential targets hit by indole derivatives and
drugs that can control the microtubule assembly by hampering tubulin polymerization or, as well, by
obstructing microtubule disassembly, and it could represent pivotal leads to block cancer
metastasization. In this paper, we reported the design, the synthesis and the biological evaluation of
molecules bearing the indole scaffold. Structure-activity relationships were made, based on the
anticancer activities obtained against four cancer cell models (MCF-7, MDA-MB-231, HeLa and
Ishikawa). We selected a good lead compound with no effects on the viability of three different
normal cells. We proved that compound 7 is able to hamper cancer cells viability, used in these
experiments, by blocking the tubulin polymerization reaction, as demonstrated by a direct enzymatic
assay and immunostaining studies on the native tubulin. Finally, in silico studies corroborated the
observed tubulin inhibition. Targeting tubulin, compound 7 lead to cancer cells death by inducing
apoptosis. Moreover, we studied the antioxidant properties of this series, finding out that compound
7 can reduce the menadione-induced ROS production in 3T3-L1 cells and that compound 6, 9 and 10
showed the greatest scavenging activity in DPPH and ABTS assays. We are confident that these
outcomes will be exploitable for a better understanding of the great potential of indole derivatives as
anticancer drugs.
4. Experimental Protocols
4.1. Chemistry
Chemicals were purchased from Sigma-Aldrich or Lancaster. Yields refer to purified products and
were not optimized. Compound structures were confirmed by routine spectrometric and spectroscopic
analyses. Melting points were determined on a Gallenkamp melting point apparatus in open glass
capillary tubes and are uncorrected. The infrared spectra were recorded on a Perkin-Elmer (Norwalk,
CT) Spectrum One FT spectrophotometer and band positions are given in reciprocal centimetres (cm–

1). 1H NMR and 13C NMR spectra were recorded on a Varian Mercury-VX spectrometer operating at
300 and 75 MHz for 1H and 13C, respectively using CDCl3 or DMSO-d6 as solvents. Chemical shifts
are reported in parts per million (ppm) relative to solvent resonance: δ 7.26 (1H NMR) and δ 77.3
(13C NMR); DMSO-d6, δ 2.48 (1H NMR) and δ 40.3 (13C NMR). J values are given in Hz. EI mass
spectra were recorded on a Hewlett-Packard 6890-5973 MSD gas chromatograph/mass spectrometer
at low resolution. Elemental analyses (C, H, N) were used to confirm the purity of all new compounds
and were performed on a Eurovector Euro EA 3000 analyser (results within ± 0.4 of the theoretical
values). Chromatographic separations were performed on silica gel columns by flash chromatography
(Kieselgel 60, 0.040–0.063 mm, Merck, Darmstadt, Germany). TLC analyses were performed on
precoated silica gel on aluminium sheets (Kieselgel 60 F254, Merck).
Synthesis of 5-methoxy-1-methylindole (2). To a stirred cold solution (0 °C) of 5-methoxyindole (1)
(2.0 g, 13.6 mmol) in dry DMF (60 mL), NaH 60% oil dispersion (0.54 g, 20.4 mmol) was added.
After 15 min, iodomethane (2.54 mL, 40.8 mmol) was added and the mixture was further stirred at
room temperature for 1 h. Water (200 mL) was then added to the reaction mixture and the solid
product obtained was filtered, washed with water and dried to give 2.7 g of pure 2 as a white solid.
Yield: 81%; mp: 114–115 °C; GC/MS (70 eV) m/z (%): 161 (M+, 100). Spectroscopic data were in
agreement with the literature[12].
Synthesis of 5-hydroxy-1-methylindole (3). A mixture of 5-methoxy-1-methylindole (2, 1.0 g, 6.21
mmol) and anhydrous pyridine hydrochloride (17.9 g, 156 mmol) was heated to melting temperature
for 3 h. The reaction mixture was left to cool to room temperature, then ice water was added. The
product was extracted with Et2O. The organic layers were washed with a solution of 2N HCl, dried
(Na2SO4) and concentrated to give 0.37 g of 3 as a yellow solid. Yield: 46%; mp: 130–132 °C; lit.120
156 °C; GC/MS (70 eV) m/z (%): 147 (M+, 100). Spectroscopic data were in agreement with the
literature [12].
Synthesis of 1H-indol-5-yl pent-2-ynoate (5). To a stirred solution of 5-hydroxyindole (4, 0.30 g, 2.3
mmol) in a mixture of CH2Cl2 and DMF (10+1 ml), pent-2-ynoic acid (0.24 g, 2.5 mmol),

dicyclohexylcarbodiimide (0.47 g, 2.3 mmol) and dimethyl aminopyridine (0.024 g, 0.2 mmol) were
added. The reaction mixture was stirred at room temperature for 4 h, and then concentrated under
reduced pressure. The solid residue was suspended in ethyl acetate, filtered and the filtrate was
washed with brine. The organic layer was dried over anhydrous Na2SO4 and evaporated under
reduced pressure. The solid residue was purified by silica gel chromatography (ethyl acetate/hexane
2:8) to give 0.24 g (48%) of a white solid: mp 116–117 °C; IR (KBr): 3386 (NH), 2231 (C≡C), 1706
(C=O) cm–1; 1H NMR (500 MHz, CDCl3): δ 1.25 (t, J = 7.3 Hz, 3H, CH3), 2.41 (q, J = 7.3 Hz, 2H,
CH2), 6.52 (s, 1H, Ar HC), 6.93 (dd, J = 8.8, 2.4 Hz, 1H, Ar HC), 7.18 (t, J = 2.4, 8.8 Hz, 1H, Ar
HC), 7.28 (d, J = 8.8 Hz, 1H, Ar HC), 7.37 (d, J = 1.9 Hz, 1H, Ar HC), 8.28 (br s, 1H, NH); 13C NMR
(125 MHz, CDCl3): δ 12.5 (1C, CH2), 12,6 (1C, CH3), 77.0 (1C, C-C=O), 92.9 (1C, C-CH2), 102.8
(1C, Ar-C), 111.5 (1C, Ar-C), 112.5 (1C, Ar-C), 115.7 (1C, Ar-C), 125.8 (1C, Ar-C), 128.0 (1C, Ar-
C), 133.9 (1C, Ar-C), 143.7 (1C, C=O), 153.3 (1C, Ar-C); MS (70 eV) m/z (%) 213 (M+, 39), 133
.
(100). Anal. Calcd. for C13H11NO2 0,25H2O: C 71.71; H 5.32; N 6.43. Found: C 71.80; H 5.15; N
6.52.
Synthesis of 1H-indol-5-yl hex-2-ynoate (6). Prepared as reported for 5 starting from 5-
hydroxyindole (4) and es-2-ynoic acid. Light brown solid. Yield: 42%; mp 82–83 °C; IR (KBr): 3394
(NH), 2228 (C≡C), 1708 (C=O) cm–1; 1H NMR (500 MHz, CDCl3): δ 1.04 (t, J = 7.3 Hz, 3H, CH3),
1.61–1.68 (m, 2H, CH2- CH3), 2.37 (t, J = 7.3 Hz, 2H, CH2-C), 6.52 (s, 1H, Ar HC-3), 6.93 (dd, J =
8.8, 2.4 Hz, 1H, Ar HC), 7.19 (t, J = 2.9 Hz, 1H, Ar HC), 7.29 (d, J = 8.8 Hz, 1H, Ar HC), 7.37 (d, J
13
= 1.9 Hz, 1H, Ar HC), 8.27 (br s, 1H, NH); C NMR (125 MHz, CDCl3): δ 13.5 (1C, CH3), 20.8
(1C, CH2-C), 21.0 (1C, CH2-CH3), 73.2 (1C, C-C=O), 91.8 (1C, C-CH2), 102.9 (1C, Ar-C), 111.5
(1C, Ar-C), 112.6 (1C, Ar-C), 115.7 (1C, Ar-C), 125.7 (1C, Ar-C), 128.1 (1C, Ar-C), 133.9 (1C, Ar-
C), 143.8 (1C, C=O), 153.3 (1C, Ar-C); MS (70 eV) m/z (%) 227 (M+, 81), 133 (100). Anal. Calcd.
for C14H13NO2: C 73.99; H 5.77; N 6.16. Found: C 73.84; H 5.84; N 6.29.

Synthesis of 9-ethylpyrano[3,2-e]indol-7(3H)-one (11). To a solution of 5 (0.25 g, 1.17 mmol) in a
mixture of 1,4-dioxane and 1,2-dichloroethane (6+6 ml), PtCl4 (0.02 g, 0.06 mmol) was added. The
reaction mixture was heated under reflux for 4 h then concentrated under reduced pressure. The
residue was dissolved in ethyl acetate and washed with water and brine. The organic layer was then
dried (Na2SO4) and concentrated under reduced pressure to give a solid residue. The solid product
was purified by silica gel chromatography (ethyl acetate/hexane 1:1) to give 40 mg (55%) of a ochre-
yellow solid: mp 242–243 °C; IR (KBr): 3253 (NH), 1671 (C=O) cm–1; 1H NMR (500 MHz, DMSO-
d6): δ 1.3 (t, J = 7.3 Hz, 3H, CH3), 3.08 (q, J = 7.3 Hz, 2H, CH2), 6.24 (s, 1H, CH), 6.83 (s, 1H, Ar
HC), 7.14 (d, J = 8.3 Hz, 1H, Ar HC), 7.59 (t, J = 2.9 Hz, 1H, Ar HC), 7.68 (d, J = 8.3 Hz, 1H, Ar
HC), 11.7 (br s, 1H, NH); 13C NMR (125 MHz, DMSO-d6): δ 12.7 (1C, CH3), 31.1 (1C, CH2), 103.0
(1C, Ar-C), 111.2 (1C, Ar-C), 111.3 (1C, CH),116.7 (1C, Ar-C), 122.2 (1C, Ar-C), 128.2 (2C, Ar-
C), 133.2 (1C, Ar-C), 150.0 (1C, C-O), 160.3 (1C, C-CH2), 161.1 (1C, C=O); MS (70 eV) m/z (%)
213 (M+, 87), 170 (100). Anal. Calcd. for C13H11NO2·0.34 H2O: C 71.20; H 5.36; N 6.39. Found: C
71.44; H 5.50; N 6.41.
Synthesis of 9-propylpyrano[3,2-e]indol-7(3H)-one (12). Prepared as reported for 11 starting from
6. Ochre-yellow solid. Yield: 16%; mp 234–235 °C; IR (KBr): 3188 (NH), 1667 (C=O) cm–1; 1H
NMR (500 MHz, DMSO-d6): δ 1.03 (t, J = 7.3 Hz, 3H, CH3), 1.66–1.74 (m, 2H, CH2), 2.98 (t, J =
7.3 Hz, 2H, CH2C), 6.23 (s, 1H, CH), 6.75 (s, 1H, Ar HC), 7.13 (d, J = 8.8 Hz, 1H, Ar HC), 7.59 (t,
J = 2.9 Hz, 1H, Ar HC), 7.68 (d, J = 8.8 Hz, 1H, Ar HC), 11.7 (br s, 1H, NH); 13C NMR (125 MHz,
DMSO-d6): δ 14.1 (1C, CH3), 21.4 (1C, CH2-CH3), 36.6 (1C, CH2-C), 102.7 (1C, Ar-C), 111.2 (1C,
Ar-C), 111.3 (1C, Ar-C), 112.4 (1C, CH), 116.8 (1C, Ar-C), 122.2 (1C, Ar-C), 128.2 (1C, Ar-C),
133.3 (1C, Ar-C), 150.2 (1C, C-O), 158.6 (1C, C-CH2), 161.0 (1C, C=O); MS (70 eV) m/z (%) 227
(M+, 100). Anal. Calcd. for C14H13NO2·0.34 H2O: C 72.09; H 5.91; N 6.00; N 6.39. Found: C 71.83;
H 5.87; N 5.86.

Synthesis of 1H-indol-5-yl 3-phenylprop-2-ynoate (7). Prepared as reported for 5 starting from 5-
hydroxyindole (4) and phenylpropiolic acid. Light beige solid. Yield: 40%; mp 142–143 °C; IR
(KBr): 3409 (NH), 2221 (C≡C), 1705 (C=O) cm–1; 1H NMR (500 MHz, CDCl3): δ 6.56 (s, 1H, Ar
HC), 7.0 (dd, J = 8.8, 1.9 Hz, 1H, Ar HC), 7.22–7.28 (m, 1H, Ar HC), 7.36–7.50 (m, 6H, Ar HC),
7.62 (d, J = 7.8 Hz, 1H, Ar HC), 8.26 (br s, 1H, NH); 13C NMR (125 MHz, CDCl3): δ 80.6 (1C, C-
C=O), 88.4 (1C, C-CH2), 103.1 (1C, Ar-C), 111.5 (1C, Ar-C), 112.7 (1C, Ar-C), 115.8 (1C, Ar-C),
119.5 (1C, Ar-C), 125.7 (C, Ar-C), 128.2 (1C, Ar-C), 128.6 (2C, Ar-C), 130.9 (1C, Ar-C), 133.2 (2C,
Ar-C), 133.9 (1C, Ar-C), 143.9 (1C, C=O), 153.5 (1C, Ar-C); MS (70 eV) m/z (%) 261 (M+, 23), 129
.
(100). Anal. Calcd. for C17H11NO2 0.17 H2O: C 77.41; H 4.50; N 5.21; N 6.39. Found: C 77.70; H
4.37; N 5.47.
Synthesis of 1-methyl-1H-indol-5-yl pent-2-ynoate (8). Prepared as reported for 5 starting from 5-
methoxyindole (3) and pent-2-ynoic acid. Tan solid. Yield: 40%; mp 47–48 °C; IR (KBr): 2221
(C≡C), 1723 (C=O) cm–1; 1H NMR (300 MHz, CDCl3): δ 1.25 (t, J = 1.1 Hz, 3H, CH2CH3), 2.40 (q,
J = 6.4 Hz, 2H, CH2), 3.80 (s, J = 1.1 Hz, 3H, NCH3), 6.46 (d, J = 2.9 Hz, 1H, Ar HC), 6.97 (dd, J =
8.7, 2.3 Hz, 1H, Ar HC), 7.08 (d, J = 2.9 Hz, 1H, Ar HC), 7.25–7.35 (m, 2H, Ar HC); 13C NMR (75
MHz, CDCl3): δ 12.5 (1C, CH2-C), 12.6 (1C, CH3-CH2), 33.0 (1C, CH3-N), 72.5 (1C, C=CO), 92.6
(1C, C-CH2), 101.3 (1C, Ar-C), 109.6 (1C, Ar-C), 112.8 (1C, Ar-C), 115.3 (1C, Ar-C), 128.6 (2C,
Ar-C), 130.2 (1C, Ar-C), 134.8 (1C, C=O), 143.6 (1C, C-O); MS (70 eV) m/z (%) 227 (M+, 41); 147
.
(100). Anal. Calcd. for C14H13NO2 0.20 H2O: C 72.84; H 5.85; N 6.07. Found: C 72.87; H 5.77; N
6.22.
Synthesis of 9-ethyl-3-methylpyrano[3,2-e]indol-7(3H)-one (13). Prepared as reported for 11
starting from 8. Tan solid. Yield: 62%; mp 174–175 °C; IR (KBr): 1630 (C=O) cm–1; 1H NMR (300
MHz, CDCl3): δ 1.41 (t, J = 7.0 Hz, 3H, CH2CH3), 3.13 (q, J = 5.8 Hz, 2H, CH2), 3.87 (s, 3H, NCH3),
6.32 (s, 1H, CH), 6.82 (d, J = 4.1 Hz, 1H, Ar HC), 7.23–7.28 (m, 2H, Ar HC), 7.5 (d, J = 8.7 Hz, 1H,

13
Ar HC); C NMR (75 MHz, CDCl3): δ 12.1 (1C, CH3-CH2), 28.0 (1C, CH3-N), 30.8 (1C, CH2),
100.4 (1C), 102.2 (1C, Ar-C ), 111.8 (1C, Ar-C), 111.9 (1C, Ar-C), 113.4 (1C, Ar-C), 119.3 (2C, Ar-
C), 130.4 (2C, C-O + CH), 159.5 (1C, C-CH2), 163.4 (1C, C=O); MS (70 eV) m/z (%) 227 (M+, 100).
Anal. Calcd. for C14H13NO2: C 73.99; H 5.77; N 6.16. Found: C 73.79; H 5.85; N 6.13.
Synthesis of 1-methyl-1H-indol-5-yl hex-2-ynoate (9). Prepared as reported for 5 starting from 5-
1
methoxyindole (3) and es-2-ynoic acid. Slightly yellowish oil. Yield: 11%; H NMR (300 MHz,
CDCl3): δ 1.04 (t, J = 7.0 Hz, 3H, CH2CH3), 1.65 (q, J = 7.0 Hz, 2H, CH2CH3), 2.37 (t, J = 7.0 Hz,
2H, CCH2), 3.77 (s, 3H, NCH3), 6.46 (d, J = 2.9 Hz, 1H, Ar HC), 6.97 (dd, J = 8.8, 2.3 Hz, 1H, Ar
HC), 7.08 (d, J = 3.5 Hz, 1H, Ar HC), 7.29 (d, J = 8.8 Hz, 1H, Ar HC), 7.35 (d, J = 2.3 Hz, 1H, Ar
13
HC); C NMR (75 MHz, CDCl3): δ 13.4 (1C, CH3-CH2), 20.7 (1C, CH2-C), 21.0 (1C, CH2-CH3),
33.0 (1C, CH3-N), 73.1 (1C, C=CO), 91.5 (1C, C-CH2), 101.3 (1C, Ar-C), 109.6 (1C, Ar-C), 112.8
(1C, Ar-C), 115.3 (1C, Ar-C), 128.6 (1C, Ar-C), 130.1 (1C, Ar-C), 134.8 (1C, Ar-C), 143.7 (1C,
C=O), 153.1 (1C, C-O); MS (70 eV) m/z (%) 241 (M+, 33), 147 (100).
Synthesis of 1-methyl-1H-indol-5-yl 3-phenylprop-2-ynoate (10). Prepared as reported for 5 starting
from 5-methoxyindole (3) and phenylpropiolic acid. Brown oil. Yield: 34%; IR (NaCl): 2222 (C≡C),
1723 (C=O) cm–1; 1H NMR (500 MHz, CDCl3): δ 3.79 (s, 3H, CH3), 6.50 (s, 1H, HC), 7.06 (d, J =
8.8 Hz, 1H, Ar HC), 7.10 (s, 1H, Ar HC), 7.32 (d, J = 8.8 Hz, 1H, Ar HC), 7.38–7.52 (m, 5H, Ar
13
HC), 7.64 (d, J = 7.3 Hz, 2H, Ar HC); C NMR (125 MHz, CDCl3): δ 33.1 (1C, CH3), 80.7 (1C,
C=CO), 88.2 (1C, C-CH2), 101.3 (1C, Ar-C), 109.7 (1C, Ar-C), 112.8 (1C, Ar-C), 115.3 (1C, Ar-C),
119.5 (1C, Ar-C), 128.6 (1C, Ar-C), 128.7 (2C, Ar-C), 130.3 (1C, Ar-C), 130.9 (1C, Ar-C), 133.2
(2C, Ar-C), 134.9 (1C, Ar-C), 143.7 (1C, C=O), 153.5 (1C, C-O); MS (70 eV) m/z (%) 275 (M+, 38),
129 (100).
4.1.2 Thermal stability test
20 mg of 7 were heated in a reaction flask in oil bath for 6 hours at 60 °C.