Solvent-Free addition of indole to aldehydes: Unexpected synthesis of novel 1-[1-(1h-indol-3-yl) alkyl]-1h-indoles and preliminary evaluation of their cytotoxicity in hepatocarcinoma cells

Article abstract of DOI:10.3390/molecules22101747

New 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles, surprisingly, have been obtained from the addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles. In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction, are discussed in detail. The effect of some selected 3,3'- and 1,3'-diindolyl methane derivatives on cell proliferation of the hepatoma cell line FaO was also evaluated.

Full text of DOI:10.3390/molecules22101747

molecules
Article
Solvent-Free Addition of Indole to Aldehydes:
Unexpected Synthesis of Novel 1-[1-(1H-Indol-3-yl)
Alkyl]-1H-Indoles and Preliminary Evaluation of
Their Cytotoxicity in Hepatocarcinoma Cells
Graziella Tocco 1,
*
ID , Gloria Zedda 2, Mariano Casu 3, Gabriella Simbula 4,†
ID
and Michela Begala 1,†
1
Department of Life and Environmental Sciences, Unit of Drug Sciences, University of Cagliari,
via Ospedale 72, 09124 Cagliari, Italy; gsimbula@unica.it
Merck Millipore, 39 Route Industrielle de la Hardt, 67120 Molsheim, France; gloria.zedda@merckgroup.com
Department of Physics, University of Cagliari, 09042 Monserrato CA, Italy; mcasu@unica.it
Department of Biomedical Science, Oncology and Molecular Pathology Unit, University of Cagliari,
2
3
4
09124 Cagliari, Italy; gsimbula@unica.it
*
†
Correspondence: toccog@unica.it; Tel.: +39-070-675-8711; Fax: +39-070-675-8553
These authors equally contributed to the work.
Received: 14 September 2017; Accepted: 13 October 2017; Published: 17 October 2017
Abstract: New 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles, surprisingly, have been obtained from the
addition of indole to a variety of aldehydes under neat conditions. CaO, present in excess, was
fundamental for carrying out the reaction with paraformaldehyde. Under the same reaction
conditions, aromatic and heteroaromatic aldehydes afforded only classical bis (indolyl) aryl indoles.
In this paper, the role of CaO, together with the regiochemistry and the mechanism of the reaction,
are discussed in detail. The effect of some selected 3,30- and 1,30-diindolyl methane derivatives on
cell proliferation of the hepatoma cell line FaO was also evaluated.
Keywords: solvent-free reaction; 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles; hepatocarcinoma; cytotoxicity
1. Introduction
Indole is one of the most versatile heterocyclic nuclei, identified as a pharmacophore in a large
number of natural and synthetic biologically active molecules [1]. Due to its electron-rich character,
indole promptly reacts with hard and soft electrophiles; thus, it is fair to consider it a privileged
structure, quaintly referred to as the ‘lord of the rings‘ [2].
Today, considerable attention is focused on a number of 3,30-diindolyl methanes (or bis (indolyl)
methanes) (3,30-BIMs), structurally dimers of the dietary component indole-3-carbinol (I3C), with
whom they share the same ability to suppress proliferation and induce apoptosis in various cancer
cells [
3
–
5
]. In view of their importance, many methods have been reported for the synthesis of
0
3,3 -BIM compounds, using a multitude of catalysts such as protic or Lewis acids, I2, zeolite, K-10 clay,
ZnO, water, gold(I)-complexes, oxalic acid dehydrate, cobalt nanocatalyst, nanoparticles, graphene
oxide [6–17] and various reaction conditions (e.g., ionic liquids, flow chemistry, etc.) [18,19]. However,
to the best of our knowledge, reports on the preparation of 1,30-diindolyl methane derivatives are
rare [20–23].
We report herein the first example of the synthesis of racemic 1-[1-(1H-indol-3-yl) alkyl]-1H-
indoles through a direct solvent-free Mannich-type addition reaction of indole and aliphatic aldehydes
(Scheme 1).
Molecules 2017, 22, 1747; doi:10.3390/molecules22101747
www.mdpi.com/journal/molecules

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Scheme 1. Reaction between indole 1 and aliphatic aldehydes 2.
Interestingly, several natural [24] and synthetic 3,30-BIMs have shown important anti-cancer
properties and demonstrated the capacity to sensitize cancer cells to apoptosis by signaling various
proapoptotic genes and proteins [25–27]. In this regard, we present a preliminarily evaluation of the
effect of these novel 3,30- and 1,30-BIM derivatives on the growth of the hepatoma cell line FaO.
2. Results and Discussion
In the quest to develop a simple and eco-friendly protocol to 3,30-BIM derivatives, we
unexpectedly observed, from the very beginning of our study, that, when the reaction was carried
out with formaldehyde or aliphatic aldehydes, both 3,30- and 1,30-bisindolyl methane (3,30- and
1,30-BIM) derivatives were obtained. Conversely, aromatic aldehydes gave only classical 3,30-BIMs.
To understand these unusual results, the reaction between indole
from paraformaldehyde, was first investigated (Scheme 2).
1, and formaldehyde 2a, derived
Scheme 2. Reaction between indole 1 and formaldehyde 2a.
Surprisingly, apart from the expected 3,30-diindolyl methane 3a, we observed, as shown in
Scheme 2, the formation of indole-1-carbinol
5 [28] and 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a,
which was fully characterized via mass spectrometry (MS) and nuclear magnetic resonance (NMR)
spectroscopy experiments (spectra available in Supplementary Materials). The best results were
achieved at 100 ◦C, and also observed an improvement in the yields when an excess of calcium oxide
(CaO) was used. The reaction did not take place at all at room temperature, both with or without CaO.
Similarly, when the operative temperature was fixed at 60 ◦C, traces of both isomers were detectable.
Thus, it was speculated that CaO, which has recently been shown to catalyse some Mannich reactions
for the preparation of lariat ethers [29], might affect our experiments. Hence, the role of CaO was
explored, noticing that, when it was used in stoichiometric amounts or in large excess, the reaction
seemed to proceed smoothly. Use of CaO in a catalytic amount (10 mol %), did not seem to produce
any effect. Reasonably, the temperature and CaO might have a synergic effect in the reaction activation.
We can plausibly assume that, in these conditions, the paraformaldehyde is rapidly converted to
free formaldehyde and the traces of formic acid produced during prolonged heating are rapidly
neutralized by CaO. Following the experiment by means of gas chromatography–mass spectrometry
(GC–MS) analysis (Figure 1), we also observed, from the very beginning of the reaction and only in the
presence of CaO, the formation of the indole-1-carbinol
condition [30,31].
5, which is usually obtained in a strong basic

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Figure 1. Gas chromatogram (GC) and mass spectra of compounds 3a, 4a and 5.
We postulate that, in our experimental conditions, CaO induces a partial N–H proton abstraction
and, despite the modest ionic character of the N–Ca bond, formaldehyde is so reactive as to undergo
a nucleophilic attack, generating indole-1-carbinol 5 [28]. As a matter of fact, when DMSO was used
as polar aprotic solvent, the reaction afforded 1-[1-(1H-indol-3-yl) methyl]-1H-indole 4a as a major
product in a high regioselective manner.
In addition, an interesting yield improvement, especially for isomer 4a, was noticed when KOH
was used instead of CaO. A possible explanation, apart from the stronger electropositivity of K+, might
be that indole-1-carbinol
5, when heated in the presence of KOH, decomposes to regenerate indole and
HCHO [30] that react again to afford both 3,30- and 1,30-BIM isomers (Table 1).
◦
Table 1. Base effect on reaction between indole 1 and formaldehyde 2a at 100 C.
Yields (%)
Entry
Base
Base/(1)
Time (h)
3a
4a
1
2
3
4
5
6
-
-
6
6
6
3
3
N.I. a
N.I. a
50
N.I. a
N.I. a
8
25
62
CaO
CaO
CaO
CaO b
KOH
0.1/1
1/1
17/1
17/1
1/1
65
N.I. a
61
1.5
37
a
Not Isolated. GC yields; b DMSO was used as solvent.
To gain a better comprehension of the reaction mechanism, we examined the possible
role of compound It did not demonstrate being a real intermediate in the formation of
1-[1-(1H-indol-3-yl)methyl]-1H-indole 4a. In fact, when could react with indole and CaO, only
5.
5
1
traces of the two isomers were detected (Scheme 3a). Interestingly, the classical bis (indolyl) methane
3a, could derive from a thermal-induced isomerisation of 4a (Scheme 3b) [20].
Scheme 3. Investigation of the role of indole-1-carbinol 5 in the formation of compounds 3a and 4a.

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With the perception that the reaction, if successful with formaldehyde, would allow similar
1,3 -diindolyl isomers when different carbonyl substrates were used, some other exploratory reactions
with diverse ketones and aldehydes were carried out (Table 2).
0
a
Table 2. Reaction of indole 1 with aliphatic and aromatic aldehydes.
Products
Yields (%) [ref.]
Entry
Base
Time (h)
Substrate
3
4
3
4
CH3CH2CHO
N.I. b
1
2
CaO
-
5
5
2b
N
CH3CH2CHO
3.5
57 [32]
43 [32]
38
34
NH
HN
NH
2b
4
CH3(CH2)4CHO
N
4
3
4
-
-
3.5
3.5
2c
NH
HN
NH
5
CH3(CH2)5CHO
N
45 [33]
10
22
5
2d
5
NH
HN
NH
10
CH3(CH2)10CHO
N
5
6
-
-
3.5
3.5
2e
NH
10
HN
NH
O
-
70 [32]
-
2
2f
NH
HN
O
O
7
8
-
3.5
5
-
-
50 [9]
N.I. b
-
-
2g
2
O
O
O
CaO
N
H
N
H
2
2g
O
O
9
-
-
3.5
3.5
-
-
10 [13]
5 [32]
-
-
O
2
2h
HN
NH
O
O
10
O
2
2j
HN
NH
O
NO2
NO2
11
-
3.5
-
70 [34]
-
2k
N
H
N
H
NO2
O
N.I. b [34]
N.I.b [32]
12
13
-
-
3.5
3.5
-
-
-
-
NO2
2l
HN
NH
NO2
O
NO2
HN
NH
2m
O
O
O
14
-
3.5
-
5 [8]
-
O2
O
2n
HN
NH

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Table 2. Cont.
Products
Yields (%) [ref.]
Entry
Base
Time (h)
Substrate
3
4
3
4
O
S
S
15
-
3.5
-
73 [34]
-
2
2o
HN
NH
O
O
O
16
a
-
3.5
-
88 [32]
-
2
2p
HN
NH
Operative temperature: 100 ◦C. All the reactions did not occur at room temperature; b Not Isolated. GC yields.
It was immediately evident that the reaction was highly chemoselective for aldehydes;
in fact, ketones such as 2-octanone, 2-hexanone, 40-methylacetophenone and 1-(p-methoxyphenyl)-2-
propanone did not react at all. Besides, only aliphatic aldehydes afforded both 3,30- and 1,30-isomers,
while aromatic and heteroaromatic ones produced only bis (indolyl) aryl methanes. Interestingly,
the reactions efficiently proceeded under neat conditions [35] and the yields seemed to be positively
affected by the absence of CaO, which demonstrated its importance mainly for the paraformaldehyde
activation. Remarkably, the reactions almost did not occur or took place very slowly, with or without
CaO, when a solvent (toluene, CH3CN, CHCl3, THF, DMF) was employed [35].
Mechanistically, we presume (Scheme 4) that two reaction pathways can be conceived for the
formation of 3,30- and 1,30-BIMs isomers. Both routes share the same well-known 2-azafulvene
intermediate [20–23,36,37] which may add to nucleophilic species. Specifically, if the path a could
be merely assumed as a Friedel–Crafts alkylation, the path b is presumably a Mannich-type
N-aminoalkylation. In this respect, the different reactivity of aliphatic and aromatic aldehydes seems
plausible given their intrinsically diverse steric and electronic features, and associated with the modest
nucleophilicity of the indole nitrogen.
Scheme 4. Proposed reaction mechanism.
Electron-impact (EI) mass spectra allowed us to characterize and differentiate both isomers easily
(Figure 2).

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N
NH
Figure 2. Electron-impact mass spectrometry (EI-MS) spectra of compounds 4b and 3b.
In fact, in the mass spectra of all 1,30-diindolyl alkane isomers, the formation of the [M-116]+
ion, presumably due to N–C bond cleavage, is the most favored fragmentation process (RA % 100),
while in the case of 3,30-isomers, the formation of [M-116]+ ion (RA % 5), related to the rupture
of the more stable C–C bond, is suppressed in favor of m/z 245 ion (RA % 100), generated by the
radical loss of R substituent. MS/MS experiments are now in progress to investigate the most
characteristic fragmentation pathways. NMR analysis included 1H, 13C, COSY, gHSQC, gHMQC and
ROESY (spectra available in Supplementary Materials), and confirmed the structure of 1,30-diindolyl
alkane isomers.
Recent studies have reported on the pleiotropic protective properties on the chronic liver injuries
steatohepatitis [38] and hepatocarcinoma [39
,
40], and on the multiple anti-tumour activities, including
0
the apoptotic, anti-proliferative and anti-angiogenetic effects [24], of 3,3 -bisindolylmethane, the parent
0
compound of 3,3 -BIMs and one of the most abundant dietary compounds derived from Brassica-genus
0
0
vegetables. Hence, we decided to evaluate the effect of our novel 1,3 and 3,3 -BIMs on cell proliferation
of the rat hepatoma cell line FaO by comparing their effects to those induced by 3,3 -bisindolylmethane
3a and indole-3-carbinol (I3C), the natural active precursor of 3,3 -bisindolylmethane. In experiments,
0
0
carried out on a selection of compounds, FaO cells were treated with increasing concentrations of
3b
,
4a and 4b as well as 400
µM of I3C for 24 h. As shown in Figure 3, the hepatoma cells were
highly susceptible to the anti-proliferative effect of these BIMs. In particular, compounds 4a
,
3b and
4b exhibited a concentration-dependent growth inhibitory effect in FaO cells similar to that observed
after treatment with the well-characterized BIM 3a [38,39].
Figure 3. Effect of 400
µM I3C and increasing concentrations of the indole derivatives (A) 3a, 4a and
(
B
)
3b, 4b on FaO cell viability, determined by neutral red uptake (NRU) assay after 24 h of treatment.
Data are expressed as percentage of viable cells assessed by neutral red.
More importantly, our novel derivatives, as well as 3a, were 20- to 4-fold more potent than I3C
in suppressing the viability of FaO cells with an IC50 value ranging from 50–100
µM after treatment
with 4a and from 20–100 M and 25–100 M after treatment with 3b and 4b, respectively. It was
µ
µ
noteworthy that all the tested BIMs induced a significant inhibition of growth of hepatoma cells at
lower concentrations than I3C. Furthermore, 3b was more effective in inducing loss of viability at very
low doses.

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3. Materials and Methods
3.1. Chemistry
All starting materials were purchased from commercial sources (Sigma-Aldrich, Milan, Italy) and
AlfaAesar Thermo Fisher Scientific, Karlsruhe, Germany with purity 98%) and used without further
≥
purification. Reaction progress was monitored by thin layer chromatography (TLC) using Aldrich
silica gel 60 F254 (0.25 mm) with detection by ultra-violet (UV) light. Chromatography was performed
using Aldrich silica gel (60–120) mesh size with freshly distilled solvents.
1H- and 13C-NMR were recorded by Varian 400 and 500 MHz (Varian Medical Systems, Palo Alto,
CA, USA) using tetramethylsilane as the internal standard. Microanalysis for carbon, nitrogen and
hydrogen (CHN) were performed by a Carlo Erba 1106 Elemental Analyzer (Milan, Italy).
GC–MS: Low resolution mass spectra were carried out on a Saturn 2000 ion-trap coupled with
a Varian 3800 gas chromatograph (Varian, Walnut Creek, CA, USA) operating under EI conditions
(electron energy 70 eV, emission current 20
µ
A, ion-trap temperature 200 ◦C, manifold temperature
◦
80 C, automatic gain control (AGC) target 21,000) with the ion trap operating in scan mode (scan range
−5
from m/z 40–400 at a scan rate of 1 scan
have been introduced into the gas chromatographer inlet. A Varian factor four low-bleed/MS capillary
·
s
−1). Aliquot of 1
µL of solutions 1.0
×
10 M in chloroform
column (VF-5ms 30 m, 0.25 mm i.d., 0.25
µ
m film thickness) (Varian) was used. The oven temperature
◦
◦
◦
was programmed from 150 C (held for 2 min) to 310 C at 30 C/min (held for 2 min). The temperature
◦
◦
◦
was then ramped up to 350 C at 20 C/min. The transfer line was maintained at 250 C and the
injector port 30/1 split) at 270 ◦C.
High-resolution mass spectra were obtained from the Department of Environmental Health
Sciences Mass Spectrometry Laboratory of Mario Negri Institute for Pharmacological Research, Milan.
High-resolution mass spectrometry (HRMS) experiments were carried out on a Finnigan LTQ hybrid
ion trap/orbitrap.
3.2. Experimental Procedures
Synthesis of 1-[1-(1H-indol-3-yl) methyl]-1H-indole (4a)
Indole
1 (8.54 mmol), paraformaldehyde (10.25 mmol) and CaO (145.18 mmol) were blended
with a magnetic stirrer for the period indicated (TLC) at 100 ◦C. After reaction, the crude mixture was
extracted with CH2Cl2 and the obtained extract was passed through celite. The concentrated filtrate
was flash chromatographed (hexane/ethyl acetate 3/1) on silica gel, obtaining the desired product.
Typical procedure for 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles (4b–e)
A mixture of indole
1 (8.54 mmol) and the appropriate aldehyde (10.25 mmol) was stirred for
the period indicated (TLC) at 100 ◦C. After reaction, the crude mixture was flash chromatographed
(hexane/ethyl acetate 10/1, 5/1 or 3/1) on silica gel, obtaining the desired product.
Synthesis of 3-(1-(1H-indol-3-yl) dodecyl)-1H-indole (3e)
A mixture of indole
1 (8.54 mmol) and dodecanal 2e (10.25 mmol) was stirred for the period
indicated (TLC) at 100 ◦C. After reaction, the crude mixture was washed with CH2Cl2 and filtered
through celite. The concentrated filtrate was flash chromatografied (hexane/ethyl acetate 10/1) on
silica gel, obtaining the desired product.
3.3. Biology
Cell Line and Culture
Rat FaO cell line was supplied by the Interlab Cell Line Collection (Servizio Biotecnologie, IST,
Genova, Italy). FaO cells were maintained in Dulbecco’s Modified Eagle’s Medium (DMEM plus

Molecules 2017, 22, 1747
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Glutamax I, Invitrogen S.r.l., Milano, Italy) supplemented with penicillin, streptomycin and 10%
heat-inactivated fetal calf-serum (FCS) (Invitrogen) in a humidified atmosphere of 5% CO2/95% air,
at 37 ◦C. Indole 3-carbinol (I3C) and compound 3a
, 3b, 4a and 4b were dissolved in DMSO and were
added to the culture media to the final concentrations specified in the text. Control cells were treated
with an equivalent amount of the solvent alone.
Cell Viability
Cell viability was determined by the uptake of neutral red by lysosome of viable cells.
Determination of viability of adherent cells by NRU assay was performed according to Borefreund and
Puerner [40]. The value obtained for treated cells was expressed as percentage of the value obtained in
control cells. All experiments were performed in triplicate.
Statistical Analysis
Instant software (GraphpAd Prism 5, GraphPad Software Inc., San Diego, CA, USA) was used to
analyse data. One-way analysis of variance (ANOVA) with post hoc analysis using Tukey’s multiple
comparison test was used for parametric data. The results of multiple observations were presented as
the means ± S.D. of three experiments. A p value of <0.05 was considered statistically significant.
4. Conclusions
We succeeded in the synthesis of new the 1-[1-(1H-indol-3-yl) alkyl]-1H-indoles by a simple and
eco-friendly method, also assuming that a 2-azafulvene intermediate
reaction mechanism. Moreover, CaO was demonstrated to be a valid alternative to organolithium
reagents for the formation of indole-1-carbinol , a potential building-block for biologically active
6 could represent the key to the
5
compounds. Work continues on the improvement of the yields and on the development of new
0
regioselective and stereoselective approaches to 1,3 -BIM isomers. Besides, we demonstrated that BIM
derivatives 3b, 4a and 4b show a behaviour similar to 3a and are more potent than I3C in inducing
loss of viability in hepatoma cells FaO, suggesting the possible employment of these compounds
for therapeutic purposes, including liver injury and the selective elimination of tumoral liver cells.
However, further studies are needed to elucidate the possible underlying mechanisms in action and
their effects on the expression of some proteins involved in both Akt pathway-mediated oncogenic
signaling, possibly leading to changes in the functional status of diverse effects or involved in cell
cycle progression and apoptosis.
Supplementary Materials: Compound characterization and NMR spectra associated with this article are
available online.
Acknowledgments: We thank Enrico Davoli (Mario Negri Institute for Pharmacological Research, Milan) and
Silvana A.M. Urru (CRS4–Division of Biomedicine, Pula, Cagliari) for the HRMS.
Author Contributions: G.T. conceived and designed the experiments; G.T., G.Z., M.C., G.S. and M.B. performed
the experiments, analyzed the data and contributed to reagents/materials/analysis tools; G.T. wrote the paper.
Conflicts of Interest: The authors declare no conflict of interest.
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Sample Availability: Samples of the compounds are not available from the authors.
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