New 6- and 7-heterocyclyl-1H-indole derivatives as potent tubulin assembly and cancer cell growth inhibitors

Article abstract of DOI:10.1016/j.ejmech.2018.04.042

We designed new 3-arylthio- and 3-aroyl-1H-indole derivatives 3–22 bearing a heterocyclic ring at position 5, 6 or 7 of the indole nucleus. The 6- and 7-heterocyclyl-1H-indoles showed potent inhibition of tubulin polymerization, binding of colchicine to tubulin and growth of MCF-7 cancer cells. Compounds 13 and 19 inhibited a panel of cancer cells and the NCI/ADR-RES multidrug resistant cell line at low nanomolar concentrations. Compound 13 at 50 nM induced 77% G2/M in HeLa cells, and at 20 nM caused 50% stable arrest of mitosis. As an inhibitor of HepG2 cells (IC₅₀ = 20 nM), 13 was 4-fold superior to 19. Compound 13 was a potent inhibitor of the human U87MG glioblastoma cells at nanomolar concentrations, being nearly one order of magnitude superior to previously reported arylthioindoles. The present results highlight 13 as a robust scaffold for the design of new anticancer agents.

Full text of DOI:10.1016/j.ejmech.2018.04.042

Accepted Manuscript
New 6- and 7-heterocyclyl-1H-indole derivatives as potent tubulin assembly and
cancer cell growth inhibitors
Giuseppe La Regina, Ruoli Bai, Antonio Coluccia, Valentina Naccarato, Valeria
Famiglini, Marianna Nalli, Domiziana Masci, Annalisa Verrico, Paola Rovella, Carmela
Mazzoccoli, Eleonora Da Pozzo, Chiara Cavallini, Claudia Martini, Stefania Vultaggio,
Giulio Dondio, Mario Varasi, Ciro Mercurio, Ernest Hamel, Patrizia Lavia, Romano
Silvestri
PII:
S0223-5234(18)30376-3
10.1016/j.ejmech.2018.04.042
EJMECH 10391
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 20 February 2018
Revised Date: 19 April 2018
Accepted Date: 21 April 2018
Please cite this article as: G. La Regina, R. Bai, A. Coluccia, V. Naccarato, V. Famiglini, M. Nalli, D.
Masci, A. Verrico, P. Rovella, C. Mazzoccoli, E. Da Pozzo, C. Cavallini, C. Martini, S. Vultaggio, G.
Dondio, M. Varasi, C. Mercurio, E. Hamel, P. Lavia, R. Silvestri, New 6- and 7-heterocyclyl-1H-indole
derivatives as potent tubulin assembly and cancer cell growth inhibitors, European Journal of Medicinal
Chemistry (2018), doi: 10.1016/j.ejmech.2018.04.042.
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to
our customers we are providing this early version of the manuscript. The manuscript will undergo
copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please
note that during the production process errors may be discovered which could affect the content, and all
legal disclaimers that apply to the journal pertain.

ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
New 6- and 7-heterocyclyl-1H-indole derivatives as potent tubulin
assembly and cancer cell growth inhibitors
a
b
a
a
a
Giuseppe La Regina, Ruoli Bai, Antonio Coluccia, Valentina Naccarato, Valeria Famiglini,
a
a
c
c
d
g
Marianna Nalli, Domiziana Masci, Annalisa Verrico, Paola Rovella, Carmela Mazzoccoli,
e
e
e
f
Eleonora Da Pozzo, Chiara Cavallini, Claudia Martini, Stefania Vultaggio, Giulio Dondio,
f
f
b
c
a,*
Mario Varasi, Ciro Mercurio, Ernest Hamel, Patrizia Lavia, and Romano Silvestri
a
Department of Drug Chemistry and Technologies, Sapienza University of Rome, Laboratory
affiliated to Istituto Pasteur Italia – Fondazione Cenci Bolognetti, Piazzale Aldo Moro 5, I-
00185 Roma, Italy
b
Screening Technologies Branch, Developmental Therapeutics Program, Division of Cancer
Treatment and Diagnosis, Frederick National Laboratory for Cancer Research, National Cancer
Institute, National Institutes of Health, Frederick, Maryland 21702, United States
c
Institute of Molecular Biology and Pathology (IBPM), CNR Consiglio Nazionale delle
Ricerche, c/o Sapienza University of Rome, Via degli Apuli 4, I-00185 Roma, Italy
d
Laboratorio di Ricerca Pre-Clinica e Traslazionale, Istituto di Ricovero e Cura a Carattere
Scientifico (IRCCS), Centro di Riferimento Oncologico della Basilicata, Via Padre Pio 1, I-
85028 Rionero in Vulture, Italy
e
Department of Pharmacy, University of Pisa, Via Bonanno Pisano 6, I-56126 Pisa, Italy
f
Experimental Therapeutics IFOM-the FIRC Institute of Molecular Oncology Foundation, Via
Adamello 16, 20139 Milan, Italy
g
APHAD, Via della Resistenza 65, 20090 Buccinasco (MI), Italy
* Corresponding author
e-mail address: romano.silvestri@uniroma1.it; phone: +39 06 4991 3800

ACCEPTED MANUSCRIPT
Keywords:
Microtubule
Tubulin
Cancer cell
Inhibitor
Indole
ABSTRACT
We designed new 3-arylthio- and 3-aroyl-1H-indole derivatives 3-22 bearing a heterocyclic
ring at position 5, 6 or 7 of the indole nucleus. The 6- and 7-heterocyclyl-1H-indoles showed
potent inhibition of tubulin polymerization, binding of colchicine to tubulin and growth of MCF-
7 cancer cells. Compounds 13 and 19 inhibited a panel of cancer cells and the NCI/ADR-RES
multidrug resistant cell line at low nanomolar concentrations. Compound 13 at 50 nM induced
77% G2/M in HeLa cells, and at 20 nM caused 50% stable arrest of mitosis. As an inhibitor of
HepG2 cells (IC50 = 20 nM), 13 was 4-fold superior to 19. Compound 13 was a potent inhibitor
of the human U87MG glioblastoma cells at nanomolar concentrations, being nearly one order of
magnitude superior to previously reported arylthioindoles. The present results highlight 13 as a
robust scaffold for the design of new anticancer agents.

ACCEPTED MANUSCRIPT
1. Introduction
Microtubules (MTs) are hollow, cylindrical, filamentous structures made of dimerized α- and
β-tubulin subunits and are characterized by a highly dynamic equilibrium involving
polymerization, where tubulin dimers bind non-covalently to a MT, and depolymerization to
tubulin dimers [1]. MTs regulate key cellular functions, such as cell growth and division,
intracellular trafficking, preservation of the architecture of the cell, and motility. Due to these
key roles, disrupting the dynamic equilibrium of MTs at either the tubulin assembly or MT
disassembly level, results in a fatal cellular event. Cancer cells are characterized by a high rate of
cell division: hence, the strategy of inducing cell death through an interference with the
dynamics of MTs has proved successful for the design of effective antitumor drugs [2-6].
MT binding agents fall into two main groups: (i) inhibitors of tubulin polymerization,
including colchicine [7,8], combretastatin A-4 (CSA4) [9] (Chart 1), vincristine (VCR),
vinorelbine (VRB) and vinblastine (VBL); and (ii) MT stabilizers, including taxoids and
epothilones. The stabilizers stimulate MT polymerization and stabilization at high
concentrations, whereas at lower concentrations paclitaxel (PTX) inhibits MT dynamics with
little effect on the proportion of tubulin in polymer [10]. Taxoids and epothilones bind at a
luminal site on the β-subunit [11,12] following entry into the MT through pores in its wall [13]
that are shaped by various tubulin subunits on the MT surface. Some evidence indicates a
transient binding of MT stabilizers at a specific pore site.
Despite considerable clinical successes [14,15], the anticancer therapies based on tubulin
binding agents still have limitations perhaps due to multi-drug resistance (MDR), toxicity and
unwanted side effects [16,17]. Therefore, there is a quest for new effective MT inhibitors with
fewer side effects to become components of improved anticancer treatments [18,19].

ACCEPTED MANUSCRIPT
Chart 1. Structures of compounds 1−22.
Currently, there are no US Food and Drug Administration approved anticancer drugs on the
market binding at the colchicine site [20]. Compared to other tubulin agents, these compounds (i)
have shown less transporter mediated drug resistance in preclinical studies [21], (ii) are
minimally affected by the overexpression of βIII-tubulin [22], and (iii) have generally better
water solubility [9].
Improvements of activity of colchicine site binding agents by replacement or introduction of
heterocyclic ring(s) to the parent scaffold have been reported [16,23,24]. Accordingly, as tubulin
polymerization inhibitors, 2-heterocyclyl-3-arylthio-1H-indole (2-HATI) derivatives were more
effective than the corresponding 2-aryl-3-arylthio-1H-indole counterparts in both the sulfur and

ACCEPTED MANUSCRIPT
aroyl series [25]. For example, 2-(thiophen-2-yl)-3-[(3′,4′,5′-trimethoxyphenyl)thio]-1H-indole
(1) inhibited tubulin assembly with an IC50 of 0.74 µM and MCF-7 cancer cell growth with an
IC50 of 39 nM [26]. Compound 1 was superior to VRB, VBL, and PTX as an inhibitor of the P-
glycoprotein (Pgp) overexpressing NCI/adriamycin-resistant (ADR-RES) MDR cell line and
showed satisfactory metabolic stability. Recently, introduction of halogen or methoxy
substituent(s) at positions 4−7 of 3-arylthio-1H-indoles (ATIs) bearing the phenyl at position 2
of the indole provided potent tubulin polymerization inhibitors (for example 2) [27].
Herein we report the synthesis of new indole derivatives 3-22 bearing a five-membered
heterocyclic ring at position 5, 6 or 7 of the indole ring (Chart 1 and Table 1). Our findings
demonstrate that the new derivatives are potent anticancer agents, superior to the corresponding
reference derivative 1 [26]. Compounds 13 and 19 inhibited the HT29, HCT116, HepG2, T98G,
U87MG, U343G and NCI/ADR-RES cell lines with IC50 values in the nanomolar range.
Compound 13 was superior to the previously reported compounds [26] as an inhibitor of HepG2
and glioblastoma cells.
2. Chemistry
2.1. Synthetic procedures
Arylthioindoles 3-7, 9, 11, 13, 15, 19 and 37 were prepared according to our previously
reported venting-while-heating microwave (MW)-assisted procedure [28] by treating the
appropriate indole with bis(3,4,5-trimethoxyphenyl)disulfide [29] in the presence of sodium
hydride in anhydrous N,N-dimethylformamide (DMF) at 130 °C for 2 min (120 W) (Scheme 1).
Reaction of the indole with 3,4,5-trimethoxybenzoyl chloride in the presence of diethylaluminum
chloride in dichloromethane at -10 °C for 2 h furnished methanones 8, 10, 12 and 14. Derivatives
16-18, 20-22, 30-32, 35 and 36 were synthesized by a coupling reaction of the appropriate indole

ACCEPTED MANUSCRIPT
with the boronic derivative in the presence of tris(dibenzylideneacetone)dipalladium(0)
(Pd2dba3), 2-dicyclohexylphosphino-2′,6′-dimethoxybiphenyl (SPhos) and potassium phosphate
tribasic in 1-butanol at 100 °C for 15 h. A palladium(II) acetate (Pd(OAc)2)-catalyzed reaction of
5-iodo-1H-indole (24) with 2-furanboronic or 2-thienylboronic acid in the presence of tri(o-
tolyl)phosphine (P(o-tol)3) and potassium phosphate tribasic in ethanol/toluene at 80 °C for 2 h
gave the corresponding heteroaryl indoles 27 and 29, respectively. MW-assisted treatment of
indole 24 with 3-furanboronic acid pinacol ester in the presence of Pd(OAc)2 and potassium
carbonate in methylpyrrolidone/water at 110 °C for 15 min (200 W) furnished 5-(furan-2-yl)-1H-
indole (28). Compound 38 was prepared by MW-assisted Friedel-Crafts reaction of 7-iodo-1H-
indole (26) with 3,4,5-trimethoxybenzoyl chloride in the presence of anhydrous aluminum
chloride in 1,2-dichloroethane at 110 °C for 2 min (150 W).

ACCEPTED MANUSCRIPT
Scheme 1. Synthesis of Compounds 3-22 and 27-37. Reagents and reaction conditions: (a) (27, 29)
boronic derivate, Pd(OAc)2, P(o-tol)3, K3PO4, EtOH/PhMe, 80 °C, 2 h, 73-87%; (b) (28) 3-furanboronic
acid pinacol ester, Pd(OAc)2, K2CO3, methylpyrrolidone/water, closed vessel, 200 W, 110 °C, 15 min,
45%; (c) (3-7, 9, 11, 13, 15, 19 and 37) bis(3,4,5-trimethoxyphenyl)disulfide, NaH, anhydrous DMF,
closed vessel, 120 W, 130 °C, 2 min, 5-50%; (d) (8, 10, 12, 14) (i) diethylaluminum chloride, CH2Cl2, -78
°C, Ar stream; (ii) 3,4,5-trimethoxybenzoyl chloride, -10 °C
→ 25 °C within 2 h, 14-69%; (e) (16-18, 20-
22, 30-32, 35 and 36) boronic derivative, Pd2dba3, SPhos, K3PO4, BuOH, 100 °C, 15 h, 5-75%; (f) (38)
3,4,5-trimethoxybenzoyl chloride, CH2Cl2, anhydrous AlCl3, closed vessel, 150 W, 110 °C, 2 min, 51%.

ACCEPTED MANUSCRIPT
Table 1
Inhibition of tubulin polymerization, growth of MCF-7 human breast carcinoma cells, and colchicine
binding by compounds 3-22.a
Compd
R
X
IC50 ± SD (nM)
MCF-7c
(% ± SD)
IC50 ± SD (µM)
Tubulinb
Inh. Colch. Bind.d
5-
5-
5-
5-
6-
6-
6-
6-
6-
6-
6-
6-
7-
S
S
0.87 ± 0.1
1.8 ± 0.1
70 ± 10
200 ± 100
150 ± 70
200 ± 0
10 ± 3
81 ± 1
63 ± 1
3
4
S
1.3 ± 0.09
1.7 ± 0.03
0.59 ± 1
76 ± 2
5
S
72 ± 3
6
S
97 ± 1
7
C=O
S
0.76 ± 0.04
2.3 ± 0.04
0.61 ± 0.06
0.47 ± 0.05
0.38 ± 0.1
0.58 ± 0.06
0.60 ± 0.04
0.64 ± 0.06
4.7 ± 0.6
10 ± 0
96 ± 0.1
90 ± 2
8
9
C=O
S
4.3 ± 0.6
6.0 ± 1
96 ± 0.1
95 ± 1
10
11
12
13
14
15
C=O
S
9.6 ± 0.6
4.5 ± 1
96 ± 0.8
97 ± 0.3
94 ± 0.6
95 ± 0.6
C=O
S
18 ± 4
15 ± 4

ACCEPTED MANUSCRIPT
16
17
18
19
20
21
22
7-
7-
7-
7-
7-
7-
7-
C=O
S
1.9 ± 0.03
1.3 ± 0.1
3.0 ± 0.3
0.57 ± 0.03
2.6 ± 0.04
1.3 ± 0.01
6.2 ± 1
320 ± 10
24 ± 2
70 ± 1
86 ± 1
29 ± 0.6
89 ± 1
48 ± 0.5
77 ± 0.1
Nde
C=O
S
1300 ± 0
29 ± 1
C=O
S
550 ± 70
38 ± 10
C=O
1400 ± 100
—
—
—
—
—
—
0.74 ± 0.05
3.2 ± 0.4
1.0 ± 0.1
39 ± 10
5 ± 1
88 ± 2
—
1
Colch.
CSA4
13 ± 3
98 ± 0.6
a Experiments were performed in duplicate or triplicate.
b Inhibition of tubulin polymerization. Tubulin was at 10
µM in the assembly assay.
c Inhibition of growth of MCF-7 human breast carcinoma cells.
d Inhibition of [3H]colchicine binding: tubulin, [3H]colchicine, inhibitor at 1:5:5 µM.
e Nd, not done.
f CSA4 yielded IC50 of 0.65 ± 0.03
µM in the assay with 5-7, 10, 12, 15 and 17, and IC50 of 0.64 ± 0.01
µ
M in the assay with 9 and 11, in which different tubulin preparations were used.
3. Results and discussion
3.1. Inhibition of tubulin polymerization
We synthesized arylthioindole and aroylindole derivatives 3-22 to explore the effects of five-
membered heterocyclic rings at position 5, 6 or 7 of the indole nucleus (Table 1). Ten new
derivatives (3, 7, 8, 10-15, and 19) inhibited tubulin polymerization with IC50 values at
submicromolar concentrations, six compounds (4-6, 16, 17 and 21) were in the 1.0−2.0 µM
range, as compared with colchicine (IC50 = 3.2 µM) and CSA4 (IC50 = 1.0 µM). With the

ACCEPTED MANUSCRIPT
exception of 9, the most potent tubulin polymerization inhibitors (7, 8 and 10-14) were
characterized by the presence of the heterocyclic ring at position 6 of the indole; among others,
compound 3, with the heterocyclic ring at position 5 of the indole, and 16, 17 and 21, with the
heterocyclic ring at position 7, showed IC50 values <1.0 µM. As tubulin assembly inhibitors, the
compounds bearing the furanyl-2-yl or the thiophen-2-yl ring were generally superior to the
corresponding furan-3yl-/thiophen-3-yl derivatives (compare 3 with 4, 5 with 6, 7 with 9, 15
with 17, and 19 with 21). Replacement of the sulfur bridging atom with the carbonyl group also
provided potent tubulin assembly inhibitors. The presence of the heterocycle at position 7 of the
indole conferred significant differences of inhibition of tubulin polymerization among
arylylthio/aroyl derivatives (compare 15 with 16, 17 with 18, 19 with 20, and 21 with 22), while
compounds 7-14 with the heterocycle at position 6 were almost equipotent, except 9 and 10, with
12 (IC50 = 0.38 µM) being the most potent tubulin polymerization inhibitor among the new
compounds.
3.2. MCF-7 breast cancer cell growth
Several new indole derivatives inhibited the growth of human MCF-7 nonmetastatic breast
cancer epithelial cells with IC50 values at nanomolar concentrations (Table 1). Structure-activity
relationship (SAR) analysis show three distinct groups: (i) introduction of the heterocyclic ring at
position 5 of the indole had a relatively weak effect on inhibition of MCF-7 cell growth, with
IC50 values ranging from 70 (3) to 200 nM (4, 6); (ii) compounds 7-14, bearing the heterocyclic
ring at position 6 of the indole, all were highly potent MCF-7 cell growth inhibitors, with IC50
values ranging from 4.3 (10) to 18 (14) nM, with five compounds (8 and 10-13) having single
digit nanomolar IC50 values; (iii) the indoles with the heterocycle at position 7 showed different
behavior, depending on the bridging group: the 7-heterocyclyl-3-arylthio-1H-indoles 15, 17, 19

ACCEPTED MANUSCRIPT
and 21 (IC50s ranging from 15 to 38 nM) were more than one order of magnitude superior to the
corresponding aroyl derivatives 16, 18, 20 and 22.
A SAR summary of tubulin polymerization inhibition and inhibition of MCF-7 cell growth of
compounds 3-22 is depicted in Chart 2. It should be noted that the SARs differ from those of the
2-phenyl-1H-indoles [27]. Compounds 3-6 with the heterocyclic ring at position 5 showed
moderate inhibition of MCF-7 cell growth; compounds 7-14, bearing the heterocycle at position
6 of the indole, all were potent MCF-7 cell growth inhibitors and showed comparable IC50
values; derivatives 15, 17, 19 and 22, with the heterocyclic ring at position 7 and the sulfur atom
bridging the two aromatic ring systems, were substantially superior to the corresponding aroyl
compounds (16, 18, 20 and 22).
3.3. Inhibition of the binding of [3H]colchicine to tubulin
Compounds 3-22 were also examined for potential inhibition of the binding of [3H]colchicine
to tubulin (Table 1). Thirteen compounds, 3, 5, 7-17, and 19 yielded >75% inhibition of the
binding reaction. Interestingly, all the indoles with the heterocycle at position 7 were strong
inhibitors of [3H]colchicine binding, with 7 and 13 (97% inhibition) nearly as potent as CSA4
(98% inhibition). In general, good correlation between [3H]colchicine binding and MCF-7 cell
growth inhibition was observed: compounds which inhibited [3H]colchicine binding >90%,
inhibited the growth of the MCF-7 cancer cell with IC50s ≤15 nM; compounds with 80-90%
inhibition of [3H]colchicine binding inhibited the growth of the MCF-7 cancer cell line with
IC50s in the range of 24-70 nM; except for 21, compounds with 70-80% inhibition of
[3H]colchicine binding inhibited the growth of the MCF-7 cancer cells with IC50s in the range of
150-350 nM.

ACCEPTED MANUSCRIPT
Chart 2. SAR summary for tubulin polymerization
inhibition (TPI) and MCF-7 cell growth inhibition
(CGI) by compounds 3-22.
3.4. Molecular modeling studies
Compounds 6, 13 and 19, representative members of the heterocyclic substitutions at position
5-, 6- or 7- of the indole nucleus, were docked into the tubulin-DAMA-colchicine complex (PDB
code 1SA0) [8] by following our previously reported procedure [30]. The docking studies were
also performed for five newly available tubulin crystal structures [31]. The docking results in the
different tubulin structures revealed a consistent binding mode for compounds bearing the
heterocyclic ring at position 5-, 6- or 7- of the indole: (i) the 3,4,5-trimethoxyphenyl moiety
formed an H-bond with the Cys241β side chain and hydrophobic contacts with Leu248β and
Leu255β; (ii) the indole established hydrophobic contacts with Asn258β and Met259β; (iii) the
heterocyclic ring of 6, 13 and 19 at position 5, 6 or 7 of the indole, respectively, arranged
hydrophobic interactions with Met259β, Lys353β, Ala180α and Val181 in the same cleft of the
colchicine site (Figs. 1 and 2).

ACCEPTED MANUSCRIPT
Fig. 1. Plants proposed binding mode for derivatives 6 (yellow), 19 (pink) and 21 (green) into the 1SA0
tubulin structure. Residues involved in interactions are shown as stick diagrams. The tubulin polypeptide
chains are shown as ribbon cartoons.
Fig. 2. Plants proposed binding mode for derivatives 13 (left panel) and 19 (right panel) for the studied tubulin
structures: 1SA0 (green), 3HKC (cyan), 4O2B (magenta), 5CA0 (purple), 5CB4 (sky blue), 5LYJ (pink).
Residues involved in interactions are shown as stick diagrams. The tubulin polypeptide chains are shown as
ribbon cartoons. Residues and cartoon are from 1SA0.

ACCEPTED MANUSCRIPT
3.5. Inhibition of growth of glioblastoma T98G, U87MG and U343MG cell lines
Treatments of T98G (human glioblastoma), U87MG (human glioblastoma-astrocytoma) and
U343MG (human glioblastoma-astrocytoma) cells with increasing concentrations of 13 and 19
significantly inhibited cell growth in a dose-dependent manner (Table 2 and Figs. 1SD-3SD,
supplementary data). The IC50 values were calculated taking into account the relative cellular
doubling times [32,33] of 48 h for the T98G and U87MG cells and of 72 h for the U343MG
cells. Compound 13 potently inhibited the glioblastoma cells at nanomolar concentrations.
Arylthioindoles with the imidazol-1-yl or pyridin-4-yl heterocyclic ring at position 2 of the
indole were weaker inhibitors of glioblastoma U87MG cells than were 13 and 19, even though
such compounds exhibited potent inhibition of the MCF-7 cancer cell growth [26].
3.6. Inhibition of HT29, HCT116 and HepG2 cancer cell growth
Compounds 13 and 19 were evaluated as growth inhibitors of the human cell lines HT29
(colon adenocarcinoma), HCT116 (colon carcinoma), and HepG2 (hepatocellular carcinoma)
using PTX as reference compound (Table 2). Compounds 13 and 19 showed strong inhibition of
the HT29 and HCT116 cell lines. As inhibitors of the HepG2 cells, 13 and 19 were more
effective than PTX (IC50 = 2660 nM).
3.7. Inhibition of MV4-11, THP-1, A-549 and PC3 cell lines
Compounds 13 and 19 were evaluated as inhibitors of MV4-11 (leukemia, acute myeloid),
THP1 (leukemia, acute monocytic), A549 (lung carcinoma) and PC3 (prostate carcinoma) cancer
cells. Compound 13 inhibited MV4-11, THP-1, A-549 and PC3 cells at single digit nanomolar
concentration, while 19 was consistently less active (Table 2).

ACCEPTED MANUSCRIPT
Table 2
Growth Inhibition of HT29, HCT116, HepG2, T98G, U87MG, U343G, MV4-11, THP-1, A-549 and PC3 cells by 13 and 19.a,b
Compd
IC50 ± SD (nM)
HT29
HCT116
21 ± 1.5
70 ± 1.7
HepG2
20 ± 1.1
80 ± 1.2
T98G
U87MG
U343G
31 ± 4.3
154 ± 6.9
MV4-11
6 ± 2.0
THP-1
2 ± 1
A-549
2 ± 0.1
58 ± 1
PC3
10 ± 1.4
69 ± 1.4
26 ± 3.6
211 ± 20
16 ± 2.0
96 ± 14
3.5 ± 2
39 ± 4
13
19
70 ± 16
20 ± 2
a Cytotoxic concentrations for the indicated cell lines; cytostatic concentrations for A-549 and PC3 cells.
b Incubation time was 48 h; for U343G, MV4-11 and PC3 cells, incubation was 72 h.
3.8. MDR cell lines
Compounds 13 and 19, representative of the 6- and 7-heterocyclyl series, respectively, were
compared with CSA4, VRB, VLB and PTX in the ovarian carcinoma cell lines OVCAR-8 and
its cognate Pgp overexpressing line NCI/ADR-RES (Table 3). In contrast to CSA4, the standard
agents VRB, VLB and PTX showed weak inhibition of the MDR line NCI/ADR-RES. The IC50
values of 13 and 19 for the MCF-7 cells, repeated in this study, resembled those obtained in the
studies summarized in Table 1.
Table 3
Growth Inhibition of the OVCAR-8 and NCI/ADR-RES cells by
compounds 13 and 19 and reference compounds CSA4, VRB,
VLB and PTX.a
Compd
IC50 ± SD (nM)
NCI/ADR_RES
7.5 ± 2
OVCAR-8
9.3 ± 2
58 ± 8
MCF-7
6.7 ± 2
55 ± 7
5.0 ± 1
5.5 ± 0.7
Ndc
13
19
34 ± 8
CSA4
PTX
VRB
VBL
4.0 ± 0b
4.0 ± 1
300 ± 0
15 ± 7
3.5 ± 0.7
3100 ± 600
5000 ± 1000
200 ± 0
Nd
a Inhibition of growth of the indicated cell lines.
b Same value obtained in all experiments. cNd, not done.

ACCEPTED MANUSCRIPT
3.9. Compounds 13 and 19 induce cell cycle arrest in the G2/M phase in HeLa cells
We compared cell cycle progression in HeLa cells exposed to 20, 50 and 100 nM 13 and 19,
using 20 nM VBL as a reference compound (a concentration that effectively arrests cells in
mitosis [27]) and DMSO as a control. Cells were treated for 24 h to cover the average duration of
an entire cell cycle, then harvested, incubated with propidium iodide (PI) and analyzed for their
genomic content by FACS analysis. Representative cell cycle profiles are shown in Fig. 3A, left
panel; data from three experiments are quantified in the graph in Fig. 3A, right panel. Both
molecules inhibited cell cycle progression in a dose-dependent manner but with different
effectiveness: treatment with 100 nM 19 yielded a substantial fraction (over 71%) of the cell
population in the G2/M phase, whereas lower doses had no significant effect; compound 13 was
already partly effective at 20 nM and induced substantial G2/M arrest at 50 nM (77% arrest).
Immunofluorescence (IF) analysis of 13 and 19-treated HeLa cell cultures confirmed that
treatment with 20 nM 13 (24 h) induced arrest in mitosis in over 50% of all cells, similar to the
effect of VBL. The mitotic arrest was stable, and only 15% of the cell population included
multinucleated cells, resulting from mitotic “slippage” and interphase re-entry with
unsegregated, or randomly segregating, chromosomes. Compound 19 induced mitotic arrest with
lower effectiveness than 13: (i) 100 nM 19 arrested 50% of the cell population in mitosis,
compared with 20 nM 13 or VBL; (ii) the induction of mitotic arrest by 19 was not fully
sustained. The higher accumulation of cells in mitosis observed with 100 nM 19 was
accompanied by induction of a relevant fraction (around 32%) of multinucleated cells: this
suggests that cells that reached mitosis arrested only transiently, and eventually resumed mitotic
progression with an inefficient mitotic apparatus, generating multinucleated cellular offspring
(Fig. 3B).

ACCEPTED MANUSCRIPT
Fig. 3. FACS analysis of the cell cycle and cell death in HeLa cell populations treated with 13 or 19.
Panel A left: distribution of cells after a 24 h treatment with 13 or 19 or VBL (DMSO as control). The
DNA content indicates the cell cycle phase (2C: G1; 4C: G2+M; intermediate values between 2C and 4C:
S phase). The panel shows a representative analysis of the cell cycle phase distribution, arranged
according to the compound concentration (on the z axis). In the lower two diagrams, 20 nM, green curves;
50 nM, red curves; 100 nM; blue curves. The profiles illustrate that 19 only induces 4C cell accumulation
when used at 100 nM, whereas 13 is fully effective at 50 nM. Panel A right: the histograms show the
mean % values
± SD of 4C cells (G2+M phases) counted in 3 independent experiments. Panel B: the
graph represents the frequency of mitotic figures (grey bars) and multinucleated cells (blue bars) in cell
populations treated for 24 h under the indicated conditions, processed for immunofluorescence and scored
by microscopy. Between 300 and 650 cells were counted for each condition in 2 independent
experiments. Panel C: FACS analysis of cell populations treated for 24 h under the indicated conditions
and stained with annexin V. Mean
± SD values were calculated from three experiments.

ACCEPTED MANUSCRIPT
We wondered whether cell cycle arrest by 13 and 19 was paralleled by cell death induction, as
is the case with classical MT-targeting drugs. Cultures treated for 24 h were incubated with
annexin V to identify cells with damaged plasma membranes committed to death. Both
compounds activated cell death parallel to arresting cell cycle progression; again, 19 was
effective when used at 100 nM, whereas 20 nM 13 was already partly effective and reached the
same death-inducing capacity as VBL at the 50 nM dose (Fig. 3C).
The tubulin inhibitory effects of 13 and 19 at the cytological level were evaluated by IF
analysis (Fig. 4). These assays provided a visual demonstration of the differential effectiveness
of the two compunds. Compund 19 at 20 or 50 nM induced a mixture of apparently normal, or
fragmented or multipolar spindles in variable proportions (examples are shown in Fig. 4A,
phenotypes are quantified in Fig. 4C); 100 nM 19 largely induced small formations of radially
arranged short microtubules that failed to elongate (labeled as “MT asters”, Fig. 4A, central
row). Compound 13 had a more pronounced effect at all tested concentrations, and no cell
showed a normal or only weakly affected spindle. Even with 13 at 20 nM, most mitotic cells
displayed multipolar (Fig. 4B, top row) or fragmented spindles (Fig. 4B, central row); at higher
concentration, 50 nM or above, 13 inhibited MT formation altogether, with sparse tubulin foci in
virtually all treated cells (Fig. 4B, bottom row). Representative phenotypes are shown in Fig.
4A,B and quantified in Fig. 4C.

ACCEPTED MANUSCRIPT
Fig. 4. IF analysis of mitotic cells in HeLa cultures treated with 13 or 19. Panel A: representative
phenotypes induced by 19: all cells were arrested in prometaphase (compare the configuration of
chromosomes in DAPI panels); the green channel shows the extent of inhibition of tubulin polymerization
at increasing compound concentrations (indicated at the right of the panels in nM). Panel B:
representative phenotypes induced by 13: the DAPI panels show the prometaphase configuration of
chromosomes; the green channel shows the extent of inhibition of tubulin polymerization at the indicated
concentrations. Panel C: Distribution of tubulin cytological phenotypes under the indicated conditions.
The histograms represent the frequency of the indicated phenotypes (n, 100 to 250 prometaphase-arrested
cells per condition).
In summary, both 13 and 19 arrested cells in mitosis, with formation of a defective mitotic
apparatus, yet the two compounds showed differential effectiveness. Compound 13 induced the

ACCEPTED MANUSCRIPT
inhibition of MT polymerization to a greater extent than 19, and it showed a full inhibitory effect
on tubulin polymerization in cells at 50 nM, comparable to the effect of VBL. Compound 19
induced milder inhibition: it essentially affected the mitotic spindle structural organization but
did not fully inhibit tubulin polymerization in cells below 100 nM. The parallel observation that
13 was a more effective inducer of cell death than 19 correlated with cellular data on microtubule
disruption and with inhibition of [3H]colchicine binding.
3.10. Effects of 13 or 19 on viability of HepG2 cells
HepG2 cells were treated with different concentrations of 13 or 19 and analyzed by the MTT
assay. Treatment with compound 19 for 48 h caused a dose-dependent decrease of cell viability:
at 80 nM nearly 30% of cells displayed reduced viability as compared to untreated cells (Fig.
4SD, panel A, supplementary data). The dose-dependent decrease of cell viability on treatment
with 13 became significant at 20 nM, nearly 40% of cells were affected (Fig. 4SD, panel B,
supplementary data). These experiments indicated 80 nM 19 and 20 nM 13 are optimal
concentrations to induce significant modifications in cell viability but, at the same time,
preserving a sufficient proportion of the cell population to permit further analysis. At such
concentrations, 13 and 19 caused a marked impairment of HepG2 cell growth after a 48 h
treatment. The cell-cycle blocker p21Cip1/Waf1 was significantly up-regulated in HepG2 treated
cells at these doses. After 80 nM 19 (Fig. 5SD, panel A, supplementary data) or 20 nM 13 (Fig.
5SD, panel B, supplementary data) treatments, the transcript levels of p21Cip1/Waf1, assessed by
qRT-PCR, were increased by 1.5- and 3.9-fold, respectively, as compared with untreated cells.
3.11. Evaluation of the drug-like properties of 13 and 19

ACCEPTED MANUSCRIPT
Drug like properties of compounds 13 and 19 were predicted through the most common
descriptors of drug-likeness (Table 4). Oral absorption according to Lipinski’s rule of five [34]
and Veber’s rule [35] was estimated by FAF drug server [36]. We referred to the 3/75 rule (logP
> 3 and topological PSA < 75 Å2) [37] to estimate compound toxicity. Derivatives 13 and 19
properly fitted with both Lipinsky and Veber’s rules, suggesting a potential good absorption after
oral administration. Furthermore, a low likelihood of in vivo toxicological outcome was inferred
by the 3/75 rule (Fig. 5).
Table 4
Physico-chemical profiles of compounds 13 and 19
LogPa
5.34
MWb
397.51
397.51
tPSAd
97.02
97.02
Compd
13
LogSwc
-5.58
HBA-HBDe RoF violf Veberg
3/75h
5
5
1
1
good
good
warning
warning
5.37
-5.60
19
a Logarithm of the partition coefficient between n-octanol and water computed by XLOGP3 method [38].
b Molecular weight.
c LogSw represents the logarithm of compound water solubility computed by the ESOL method [39].
d Molecular polar surface area, this parameter has been shown to correlate with human intestinal
absorption (<140) [40].
e Number H-bond acceptors and H-bond donors.
f Violation of the rule of five [34].
g Veber’s rule matching [35].
h 3/75 rule matching [36].

ACCEPTED MANUSCRIPT
Fig. 5. Physico-chemical properties. Panel A. Compounds 13 and 19 (blue lines) are compared with the
optimal light green area defined by Lipinski’s rule-of-five and Veber’s rule. The computations includes
logP, molecular weight (MW), topological polar surface area (tPSA), rotatable bonds (RotB), H-bond
acceptors and donors (HBA, HBD. The red area indicates low oral bioavailability. Estimated physico-
chemical profiles of compounds 13 and 19 fall within the light green area. Panel B. Likelihood of in vivo
toxicity according 3/75 rule [37]. tPSA is expressed as a function of logP. Compounds located in the red
square are likely to cause toxicity. Estimated toxicities of compounds 13 and 19 (blues point) fall within
the light green area.
4. Conclusions
We designed new 3-arylthio- and 3-aroyl-1H-indole derivatives 3-22 bearing a heterocyclic
ring at position 5, 6 or 7 of the indole nucleus. Ten new derivatives inhibited polymerization of

ACCEPTED MANUSCRIPT
purified tubulin with IC50 values at submicromolar concentrations. Seven of the new indole
derivatives inhibited the growth of MCF-7 cells with IC50 values ≤10 nM. Inhibition of cell
growth showed good correlation with inhibition of [3H]colchicine binding to tubulin. Two
representative highly potent members of the 6- and 7-heterocyclyl-1H-indoles, 13 and 19,
inhibited an extensive panel of cancer cell lines, including the Pgp overexpressing NCI/ADR-
RES cell line, with nanomolar IC50s. In cell cycle analysis, compound 13 became effective at 20
nM and induced 77% G2/M arrest at 50 nM, whereas 19 accumulated 71% cells in the G2/M
phase at 100 nM. Compound 13 extensively inhibited cellular tubulin polymerization at 50 nM,
and its activity was comparable to that of VBL. Compound 19 at 100 nM affected mitotic spindle
structural organization but did not fully inhibit cellular tubulin polymerization. As inhibitors of
HepG2 cells, 13 and 19 (IC50 values of 20 nM and 80 nM, respectively) were distinctly superior
to PTX. Compound 13 was also a potent inhibitor of the U87MG glioblastoma cell line at
nanomolar concentrations [41] being almost one order of magnitude more active than the
previously reported arylthioindoles with the imidazol-1-yl or pyridin-4-yl ring at position 2 of
the indole nucleus [26]. Analysis of the gene expression levels of p21Cip1/Waf1, the cell cycle
blocker, indicated that at sub-cytotoxic concentrations 13 caused a decrease in cell growth with
strong up-regulation of p21Cip1/Waf1 [42].
In conclusion, these new indoles are potent inhibitors of tubulin polymerization and cancer
cell growth, including human liver carcinoma and glioblastoma cells. The introduction of the
heterocyclic ring at position 5, 6 or 7 of the 3-(3’,4’,5’-trimethoxyarylthio)-1H-indole and 3-
(3’,4’,5’-trimethoxyaroyl)-1H-indole scaffold was a productive strategy for the design of new
effective anticancer agents, with compound 13 as a robust lead agent. Based on SAR findings,

ACCEPTED MANUSCRIPT
the synthesis of new analogues is in progress in our laboratory, and the results will be reported in
due course.
5. Experimental protocols
5.1. Chemistry
All reagents and solvents were handled according to the material safety data sheet of the
supplier and were used as purchased without further purification. 5-Chloro-1H-indole (23), 5-
iodo-1H-indole (24), 6-bromo-1H-indole (25), 7-iodo-1H-indole (26), 6-(thiophen-2-yl)-1H-
indole (33) and 6-(thiophen-3-yl)-1H-indole (34) were commercially available. MW-assisted
reactions were performed on a CEM Discover SP single-mode reactor equipped with an Explorer
72 autosampler, controlling the instrument settings by PC-running CEM Synergy 1.60 software.
Closed vessel experiments were carried out in capped MW-dedicated vials (10 mL) with a
cylindrical stirring bar (length 8 mm, diameter 3 mm). Stirring, temperature, irradiation power,
maximum pressure (Pmax), pressure set point, times at set point, delta pressure, PowerMAX
(simultaneous cooling-while-heating), ActiVent (simultaneous venting-while-heating), and ramp
and hold times were set as indicated. Reaction temperature was monitored by an external CEM
fiber optic temperature sensor. After completion of the reaction, the mixture was cooled to 25 °C
via air-jet cooling. Organic solutions were dried over anhydrous sodium sulfate. Evaporation of
solvents was carried out on a Büchi Rotavapor R-210 equipped with a Büchi V-850 vacuum
controller and a Büchi V-700 vacuum pump. Column chromatography was performed on
columns packed with silica gel from Macherey-Nagel (70−230 mesh). Silica gel thin layer
chromatography (TLC) cards from Macherey-Nagel (silica gel precoated aluminum cards with
fluorescent indicator visualizable at 254 nm) were used for TLC. Developed plates were
visualized with a Spectroline ENF 260C/FE UV apparatus. Melting points (mp) were determined

ACCEPTED MANUSCRIPT
on a Stuart Scientific SMP1 apparatus and are uncorrected. Infrared (IR) spectra were recorded
on a PerkinElmer Spectrum 100 FT-IR spectrophotometer equipped with a universal attenuated
total reflectance accessory and IR data acquired and processed by PerkinElmer Spectrum
10.03.00.0069 software. Band position and absorption ranges are given in cm−1. Proton nuclear
magnetic resonance (1H NMR) spectra were recorded with a Varian Mercury (300 MHz) or a
Bruker Avance (400 MHz) spectrometer in the indicated solvent, and the corresponding fid files
were processed by MestreLab Research SL MestreReNova 6.2.1-769 software. Chemical shifts
are expressed in δ units (ppm) from tetramethylsilane. Mass spectra were recorded on a Bruker
Daltonics MicroTOF LC/MS mass spectrometer equipped with a positive ion ESI source.
Compound purity was checked by high pressure liquid chromatography (HPLC). Purity of tested
compounds was found to be >95%. The HPLC system used (Thermo Fisher Scientific Inc.
Dionex UltiMate 3000) consisted of an SR-3000 solvent rack, a LPG-3400SD quaternary
analytical pump, a TCC-3000SD column compartment, a DAD-3000 diode array detector, and
an analytical manual injection valve with a 20 µL loop. Samples were dissolved in acetonitrile (1
mg/mL). HPLC analysis was performed by using a Thermo Fisher Scientific Inc. Acclaim 120
C18 column (5 µm, 4.6 mm × 250 mm) at 25 ± 1 °C with an appropriate solvent gradient
(acetonitrile/water), flow rate of 1.0 mL/min and signal detector at 206, 230, 254 and 365 nm.
Chromatographic data were acquired and processed by Thermo Fisher Scientific Inc.
Chromeleon 6.80 SR15 Build 4656 software.
5.1.1. General procedure for the synthesis of derivatives 3-7, 9, 11, 13, 15, 19 and 37. Example.
5-(Furan-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (3)
A mixture of indole 27 (183 mg, 1 mmol), bis(3,4,5-trimethoxyphenyl)disulfide [29] (438 mg,
1.1 mmol), and sodium hydride (88 mg, 2.2 mmol; 60% in mineral oil) in anhydrous DMF (3

ACCEPTED MANUSCRIPT
mL) was placed into the MW cavity (closed vessel mode, Pmax = 250 psi). Starting MW
irradiation of 120 W was used, the temperature being ramped from 25 to 130 °C, while rapidly
stirring and venting (pressure set point: 100 psi; times at set point: 100; delta pressure: 20 psi).
Once 130 °C was reached, taking about 1 min, the reaction mixture was held at this temperature
for 2 min. The mixture was diluted with water and extracted with ethyl acetate. The organic layer
was washed with brine and dried. Removal of the solvent gave a residue that was purified by
column chromatography (silica gel, n-hexane:ethyl acetate = 3:2 as eluent) to give 3 (122 mg,
yield 32%), mp 118-120 °C (from ethanol). 1H NMR (DMSO-d6, 400 MHz): δ 3.98 (s, 9H), 6.82
(s, 2H), 6.95 (s, 1H), 7.22 (d, J = 1.7 Hz, 1H), 7.93-7.96 (m, 2H), 8.07 (s, 1H), 8.17 (s, 1H), 8.22
(s, 1H), 12.20 ppm (br s, disappeared after treatment with D2O, 1H). IR: ν 3448 cm-1. MS (ESI):
382.4 (MH+). C21H19NO4S (381.45).
5.1.2. 5-(Furan-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (4)
1
Synthesized as 3 starting from 28. Yield 20%, mp 125-130 °C (from ethanol). H NMR
(DMSO-d6, 400 MHz): δ 3.57 (s, 3H), 3.59 (s, 6H), 6.44 (s, 2H), 6.89-6.91 (m, 1H), 7.45-7.47
(m, 2H), 7.61-7.65 (m, 1H), 7.70 (d, J = 1.7 Hz, 1H), 7.77 (s, 1H), 8.05-8.10 (m, 1H), 11.70 ppm
(br s, disappeared after treatment with D2O, 1H). IR: ν 3425 cm-1. MS (ESI): 382.2 (MH+).
C21H19NO4S (381.45).
5.1.3. 5-(Thiophen-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (5)
1
Synthesized as 3 starting from 29. Yield 31%, mp 115-120 °C (from ethanol). H NMR
(CDCl3, 400 MHz): δ 3.69 (s, 6H), 3.78 (s, 3H), 6.44 (s, 2H), 7.00-7.07 (m, 1H), 7.24 (dd, J =
2.9 and 7.1 Hz, 1H), 7.28 (s, 1H), 7.44 (d, J = 8.5 Hz, 1H), 7.52 (d, J = 2.6 Hz, 1H), 7.55-7.60
(m, 1H), 7.89 (s, 1H), 8.49 ppm (br s, disappeared after treatment with D2O, 1H). IR: ν 3439 cm-
1. MS (ESI): 398.4 (MH+). C21H19NO3S2 (397.51).

ACCEPTED MANUSCRIPT
5.1.4. 5-(Thiophen-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (6)
1
Synthesized as 3 starting from 30. Yield 40%, mp 116-120 °C (from ethanol). H NMR
(DMSO-d6, 400 MHz): δ 3.57 (s, 3H), 3.58 (s, 6H), 6.44 (s, 2H), 7.49-7.51 (m, 1H), 7.53 (s, 1H),
7.55 (d, J = 1.3 Hz, 1H), 7.59-7.62 (m, 1H), 7.72-7.74 (m, 2H), 7.79 (d, J = 2.4 Hz, 1H), 11.71
ppm (br s, disappeared after treatment with D2O, 1H). IR: ν 3448 cm-1. MS (ESI): 398.5 (MH+).
C21H19NO3S2 (397.51).
5.1.5. 6-(Furan-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (7)
1
Synthesized as 3 starting from 31. Yield 44%, mp 164-169 °C (from ethanol). H NMR
(DMSO-d6, 300 MHz): δ 3.56 (s, 9H), 6.37 (s, 2H), 6.56-6.60 (m, 1H), 6.85 (d, J = 3.2 Hz, 1H),
7.45 (s, 2H), 7.70-7.74 (m, 3H), 11.74 ppm (br s, disappeared after treatment with D2O, 1H). IR:
ν 3179 cm-1. MS (ESI): 382.2 (MH+). C21H19NO4S (381.45).
5.1.6. 6-(Furan-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (9)
1
Synthesized as 3 starting from 32. Yield 4%, slurry. H NMR (DMSO-d6, 400 MHz): δ 3.57
(s, 9H), 6.39 (s, 2H), 6.90-6.95 (m, 1H), 7.34-7.36 (m, 1H), 7.40-7.43 (m, 1H), 7.63 (s, 1H),
7.70-7.73 (m, 2H), 8.14 (s, 1H), 11.66 ppm (br s, disappeared after treatment with D2O, 1H). IR:
ν 3401 cm-1. MS (ESI): 382.2 (MH+). C21H19NO4S (381.45).
5.1.7. 6-(Thiophen-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (11)
1
Synthesized as 3 starting from 33. Yield 50%, mp 158-161 °C (from ethanol). H NMR
(DMSO-d6, 400 MHz): δ 3.58 (s, 3H), 3.59 (s, 6H), 6.41 (s, 2H), 7.10-7.13 (m, 1H), 7.41-7.43
(m, 1H), 7.44-7.48 (m, 3H), 7.70-7.72 (m, 1H), 7.82 (s, 1H), 11.74 ppm (br s, disappeared after
treatment with D2O, 1H). IR: ν 3372 cm-1. MS (ESI): 398.3 (MH+). C21H19NO3S2 (397.51).
5.1.8. 6-(Thiophen-3-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (13)

ACCEPTED MANUSCRIPT
1
Synthesized as 3 starting from 39. Yield 30%, mp 188-190 °C (from ethanol). H NMR
(DMSO-d6, 400 MHz): δ 3.58 (s, 3H), 3.59 (s, 6H), 6.40 (s, 2H), 7-45-7.47 (m, 2H), 7.52-7.57
(m, 1H), 7.60-7.64 (m, 1H), 7.76 (s, 1H), 7.79 (s, 1H), 7.77-7.81 (m, 1H), 11.72 ppm (br s,
disappeared after treatment with D2O, 1H). IR: ν 3376 cm-1. MS (ESI): 398.5 (MH+).
C21H19NO3S2 (397.51).
5.1.9. 7-(Furan-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (15)
Synthesized as 3 starting from 35. Yield 15%, mp 80-83 °C (from ethanol). 1H NMR (DMSO-
d6, 400 MHz): δ 3.58 (s, 9H), 6.42 (s, 2H), 6.71-6.75 (m, 1H), 7.16-7.21 (m, 2H), 7.44-7.50 (m,
1H), 7.59-7.63 (m, 1H), 7.80-7.83 (m, 2H), 11.59 ppm (br s, disappeared after treatment with
D2O, 1H). IR: ν 3351 cm-1. MS (ESI): 382.2 (MH+). C21H19NO4S (381.45).
5.1.10. 7-(Thiophen-2-yl)-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (19)
1
Synthesized as 3 starting from 36. Yield 29%, mp 153-155 °C (from ethanol). H NMR
(DMSO-d6, 300 MHz): δ 3.56 (s, 3H), 3.58 (s, 6H), 6.43 (s, 2H), 7.14 (t, J = 5.8 Hz, 1H), 7.24 (t,
J = 3.8 Hz, 1H), 7.32 (d, J = 5.5 Hz, 1H), 7.46 (d, J = 5.9 Hz, 1H), 7.56 (d, J = 2.7 Hz, 1H), 7.64
(d, J = 3.8 Hz, 1H), 7.76 (s, 1H), 11.57 ppm (br s, disappeared after treatment with D2O, 1H). IR:
ν 3322 cm-1. MS (ESI): 398.4 (MH+). C21H19NO3S2 (397.51).
5.1.11. 7-Iodo-3-((3,4,5-trimethoxyphenyl)thio)-1H-indole (37)
1
Synthesized as 3 starting from 26. Yield 27%, mp 100-103 °C (from ethanol). H NMR
(DMSO-d6, 400 MHz): δ 3.56 (s, 3H), 3.57 (s, 6H), 6.40 (s, 2H), 6.90 (t, J = 7.6 Hz, 1H), 7.46
(d, J = 7.8 Hz, 1H), 7.58 (d, J = 7.4 Hz, 1H), 7.77 (s, 1H), 11.61 ppm (br s, disappeared after
treatment with D2O, 1H). IR: ν 3270 cm-1.