Synthesis of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate cancer cells

Article abstract of DOI:10.1080/14786419.2013.824440

The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3 and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives are reported. The synthesis of new compounds was first accomplished by the reaction of different indoles with trimethoxyacetophenone in the presence of catalytic amounts of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate [(CO ₂H)₂·2H₂O] and N-cetyl-N,N,N- trimethylammonium bromide in water was carried out and led to better yields. Compound 5b significantly reduced LNCaP prostate cancer cell viability in a dose-dependent manner, with an IC₅₀ of 0.64 ± 0.09 M. To determine whether the growth inhibition was associated with the induction of apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 M of 5b showed the morphological changes characteristic of apoptosis after 24 h of incubation.

Full text of DOI:10.1080/14786419.2013.824440

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Natural Product Research: Formerly
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Synthesis of a new bis(indolyl)methane
that inhibits growth and induces
apoptosis in human prostate cancer
cells
Mariangela Marrellia, Xavier Cachetb, Filomena Confortia, Rosa
Siriannia, Adele Chimentoa, Vincenzo Pezzia, Sylvie Michelb,
Giancarlo A. Stattia & Francesco Menichinia
a Department of Pharmacy, Health and Nutritional Sciences,
University of Calabria, via Pietro Bucci, I-87036, Rende (CS), Italy
b Laboratoire de Pharmacognosie, Faculté des Sciences
Pharmaceutiques et Biologiques, Université Paris Descartes, UMR
8638 CNRS, 4 Avenue de l'Observatoire, 75006, Paris, France
Published online: 20 Aug 2013.
To cite this article: Mariangela Marrelli, Xavier Cachet, Filomena Conforti, Rosa Sirianni, Adele
Chimento, Vincenzo Pezzi, Sylvie Michel, Giancarlo A. Statti & Francesco Menichini (2013) Synthesis
of a new bis(indolyl)methane that inhibits growth and induces apoptosis in human prostate
cancer cells, Natural Product Research: Formerly Natural Product Letters, 27:21, 2039-2045, DOI:
10.1080/14786419.2013.824440
To link to this article: http://dx.doi.org/10.1080/14786419.2013.824440
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Vol. 27, No. 21, 2039–2045, http://dx.doi.org/10.1080/14786419.2013.824440
Synthesis of a new bis(indolyl)methane that inhibits growth and induces
apoptosis in human prostate cancer cells
Mariangela Marrellia, Xavier Cachetb, Filomena Confortia, Rosa Siriannia, Adele Chimentoa,
Vincenzo Pezzia, Sylvie Michelb, Giancarlo A. Stattia* and Francesco Menichinia
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The synthesis and the antiproliferative activity against the human breast MCF-7, SkBr3
and the prostate LNCaP cancer cell lines of a series of bis(indolyl)methane derivatives
are reported. The synthesis of new compounds was first accomplished by the reaction
of different indoles with trimethoxyacetophenone in the presence of catalytic amounts
of hydrochloric acid. A second procedure involving the use of oxalic acid dihydrate
[(CO2H)2·2H2O] and N-cetyl-N ,N ,N-trimethylammonium bromide in water was carried
out and led to better yields. Compound 5b significantly reduced LNCaP prostate
cancer cell viability in a dose-dependent manner, with an IC50 of 0.64 ^ 0.09 mM. To
determine whether the growth inhibition was associated with the induction of
apoptosis, treated cells were stained using DAPI. LNCaP cells treated with 1 mM
of 5b showed the morphological changes characteristic of apoptosis after 24 h of
incubation.
Keywords: antiproliferative activity; apoptosis; bis(indolyl)methane; indole
1. Introduction
Breast and prostate cancers are frequently diagnosed malignancies in Western countries (Ferlay
et al. 2007). Although many anticancer drugs have been developed and used clinically for
controlling breast and prostate cancers, more specific and efficacious anticancer and/or
chemopreventive agents are still needed. Bis(indolyl)methanes and their derivatives constitute
an important class of heterocyclic compounds used in the pharmaceutical industry. These
molecules can be found in plants belonging to Cruciferae family and they are known to promote
oestrogen metabolism and to induce apoptosis in human tumour cells. Bis(3-indolyl)methane
(3,30-diindolylmethane, DIM, 1) is the principal compound derived from the metabolism of the
indole-3-carbinole (I3C, 2), an autolysis product of the glucosinolate glucobrassicin found in
vegetables such as broccoli, Brussels sprouts cabbage and kale (Figure 1). The diindolylmethane
is a promising antitumour agent (Hong et al. 2002). On the basis of the structure of previously
reported bis(indolyl)methanes, a novel series of derivatives were designed and synthesised as
anticancer agents. The cytotoxicity of the new compounds was determined towards different
cancer cell lines: MCF-7 and SkBr3 as models for breast cancer and LNCaP as a model for
prostate cancer.
*Corresponding author. Email: giancarlo.statti@unical.it
q 2013 Taylor & Francis

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Figure 1. Chemical structure of 3,30-diindolylmethane and its precursor, I3C.
2. Results and discussion
2.1 Synthesis of bis(indolyl)methanes
The synthesis of bis(indolyl)methanes is based on the indole reaction with aldehydes or ketones,
which leads to the formation of an intermediate that reacts with a second molecule of indole to
form bis(indol-3-yl)methanes (Ghorbani-Vaghei et al. 2010). Several methods, based on the use
of different promoters, were reported in previous years (Deb & Bhuyan 2005; Zhang & Du 2009;
Epifano et al. 2011). The new derivative 5a was obtained from 1H-indole 3a and 30,40,50-
trimethoxyacetophenone 4 in the presence of catalytic amounts of hydrochloric acid (Scheme 1).
This procedure provided a yield of 26%. We wanted to assess whether the procedure could also
be applied to the synthesis of substituted compounds. To this end, the 6-methoxy-indole was
used in place of 1H-indole. The substituted bis(indolyl)methane 5b was obtained with a yield of
9%. Our goal then was to improve the strategy of synthesis of compounds 5a and 5b in order to
increase the yield of reactions. The analogues were obtained using a more appropriate procedure
described by Ghorbani-Vaghei et al. (2010). The reaction is described in Scheme 1: the indole
reacts with trimethoxyacetophenone in the presence of oxalic acid dihydrate [(CO2H)2·2H2O]
and N -cetyl-N,N ,N-trimethylammonium bromide (CTAB) in water. This procedure gave
increased yields compared with previous attempts.
Compound 5a was obtained with a yield of 62%, whereas the substituted derivative 5b was
1
synthesised with a yield of 42%. The synthesised compounds were characterised by H NMR,
13C NMR and mass spectral data.
Scheme 1. Synthesis of bis(indolyl)methanes 5a and 5b. 3a: R ¼ ZH; 3b: R ¼ ZOCH3. Reagents and
conditions: (a) HCl, EtOH, 788C, 19 and 22 h for 5a and 5b, respectively. (b) (COOH)2·2H2O/CTAB, H2O,
room temperature, 7 and 10 h for 5a and 5b, respectively.

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2.2 Antiproliferative activity of new analogues
The effects of synthesised compounds on viability of human cancer cell lines (MCF-7, SkBR3
and LNCaP) were assessed using the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium
bromide (MTT) assay, a simple bioassay used for the primary screening of compounds. For each
cell line used in this study, there was a linear relationship between cell number and absorbance,
measured at 570 nm in both control and drug-treated wells. Cisplatin, doxorubicin and paclitaxel
were used as positive controls. The antiproliferative activity of synthesised compounds was
determined after 48 h of treatment. Effects on proliferation of MCF-7, SkBr3 and LNCaP cell
lines are shown in Table 1. Compound 5b significantly reduced LNCaP prostate cancer cell
viability in a dose-dependent manner compared with their respective vehicle (0.1% dimethyl
sulfoxide (DMSO))-treated control cells, with a 50% inhibition (IC50) value of 0.64 ^ 0.09 mM.
Between the other two cell lines, MCF-7 and SkBr3, the latter showed a better inhibition on its
proliferation rate in response to compound 5b, with IC50 values of 20.03 ^ 2.18 and
9.6 ^ 1.36 mM, respectively. On the contrary, compound 5a showed no activity.
Obtained data show that the presence of a methoxy group in position 6 of the indole leads to a
substantial improvement in cytotoxic activity. Some 6,60-methoxy-diindolylmethanes and
derivatives were already synthesised by Benabadji et al. (2004). These molecules showed good
antioxidant activity, which was related to the presence of the methoxy group, a strong electron-
donating group which increases the stability of the benzene ring and amplifies the radical
scavenging activity of compounds. Previous studies about a series of 3-formyl-2-phenylindoles
demonstrated as well that the presence of a methoxy group at p a r a position of the 2-phenyl ring
led to more active compounds against human breast cancer cells, a finding which was correlated
with a different binding mode/site (Gastpar et al. 1998). Here, the presence of a methoxy groups
clearly influenced the antiproliferative activity of tested compounds. Further studies will, of
course, be needed to confirm these observations and evaluate their significance.
2.3 Evaluation of the mechanism of action
Observation of LNCaP cells under a microscope revealed that cells treated for 48 h with vehicle
exhibited normal features with typical adherent, membrane intact morphology. On the contrary,
treatment with different concentrations of 5b (from 0.0156 to 100 mM) for 48 h revealed changes
in morphology for the majority of LNCaP cells (Figure 2, bright field). To assess if
morphological changes and growth inhibition were associated with an apoptotic mechanism,
nuclei of cell monolayers were stained with a fluorescent dye, DAPI. Cells were left untreated
(control) or treated for 24 h with compound 5b before being stained with DAPI (Sigma-Aldrich,
Milan, Italy). This method showed that LNCaP cells treated with compound 5b had brighter blue
nuclei compared with control, untreated cells (Figure 2, DAPI). This is consistent with more
condensed chromatin which is a feature of apoptotic cells.
Table 1. Antiproliferative activity of bis(indolyl)methanes.
IC50 (mM)
Cell line
LNCaP
SkBr3
MCF-7
5a
5b
ND
ND
ND
0.64 ^ 0.09a
2.01 ^ 0.12b
28.41 ^ 2.52f
–
9.60 ^ 1.36c
13.90 ^ 0.90dc
–
20.03 ^ 2.18e
15.46 ^ 1.08d
1.9 ^ 0.03b
–
Cisplatin
Doxorubicin
Paclitaxel
23.94 ^ 1.99e
Notes: Data are expressed as mean ^ SEM (n ¼ 6). Different letters indicate statistically significant differences at
P , 0.05 (Tukey’s test). ND: not detectable.

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Figure 2. LNCaP cells were treated with compound 5b (1 mM) for 24 h, fixed with paraformaldehyde, dyed
with DAPI and observed under a fluorescent microscope (400 £ ). Control, untreated LNCaP cells.
Brightness indicates condensed nuclei. Data presented is a representative from three separate experiments.
3. Experimental
3.1 Chemicals and instrumentation
Reagents were obtained from Sigma-Aldrich S.p.A. (Milan, Italy), Merck (Milan, Italy) and
Acros Organics (Milan, Italy) and used without further purification. Solvents were obtained from
VWR International (Milan, Italy) and purified and stored according to standard procedures.
Melting points were determined on a LEICA VM microscope equipped with a heating stage and
are uncorrected. NMR spectra were recorded at 300 MHz (1H) and at 75 MHz (13C) with an AC
300 Bruker spectrometer (Milan, Italy). Chemical shifts are given in parts per million (ppm, d)
relative to solvent peaks as internal standards (d: CDCl3: 7.27 ppm (1H), 77.0 ppm (13C);
acetone-d 6: 2.05 ppm (1H), 29.8 and 206.0 ppm (13C)); coupling constants are given in hertz (Hz,
J ). Mass spectra (MS) were measured with a Nermag R10-10C mass spectrometer (CI/NH3)
(Rueil-Malmaison, France) or with a ZQ 2000 Waters mass spectrometer (ESI) (Waters S.p.A.,
Milan, Italy). Flash column chromatography was carried out with silica gel (SDS 60 ACC 35–
70mM). The reactions were monitored by thin-layer chromatography using Merck Kieselgel 60
F254 silica gel; zones were detected visually under ultraviolet irradiation (254 and 366 nm) and
by spraying with sulphuric vanillin followed by heating.
3.2 Synthesis of bis(indolyl)methanes
3.2.1
3 - [ 1 - ( 1 H - I n d o l -3 -y l ) - 1 - ( , , - t r i m e t h o x y p h e n y l ) e t h y l ) - 1 H - i n d o le ( 5a)
3 0 4 0 5 0
(a) The indole 3a (100 mg, 0.854 mmol) and the 30,40,50-trimethoxyacetophenone 4
(89.8 mg, 0.427 mmol) were dissolved in absolute ethanol. Catalytic amounts (0 mL,

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0.0085 mmol) of hydrochloric acid were added. The reaction mixture was heated at
788C for 19 h. After cooling, the precipitate was recovered by vacuum filtration and
washed with absolute ethanol, resulting in 5a, obtained as a white solid (47.2 mg), with a
yield of 26%.
(b) A solution of indole 3a (100 mg, 0.854 mmol) and 30,40,50-trimethoxyacetophenone 4
(89.8 mg, 0.427 mmol) in the presence of CTAB (5 mol%) and oxalic acid dihydrate
[(CO2H)2·2H2O (50 mol%)] was placed under agitation at room temperature in water.
After about 7 h, the reaction was finished and the precipitated product was filtered and
obtained by recrystallisation from a solution of EtOH–H2O as a white solid (112.8 mg).
1
Yield ¼ 62%; m.p. ¼ 285–287 8C; H NMR (300 MHz, acetone-d 6): d 2.33 (s, 3H);
3.59 (s), 3.71 (s, 3H), 6.77 (s, 2H), 6.83 (ddd, 2H, J ¼ 8.1, 7.1, 1.1 Hz), 6.85 (s, 2H), 7.03
(ddd, 2H, J ¼ 8.1, 7.1, 1.1 Hz), 7.28 (d, 2H, J ¼ 8.1 Hz), 7.39 (d, 2H, J ¼ 8.1 Hz), 9.99
(br s, 2H, D2O exch, NH £ 2); 13C NMR (75 MHz, acetone-d 6): d34.0, 43.9, 55.4 ( £ 2),
59.6, 106.4 ( £ 2), 111.4 ( £ 2), 118.1 ( £ 2), 120.8 ( £ 2), 121.6 ( £ 2), 123.5 ( £ 4),
126.6 ( £ 2), 136.5, 137.6 ( £ 2), 144.2, 152,6 ( £ 2); MS (ZQ2000/ESI þ ): m/z 449
[M þ Na]þ, 465 [M þ K]þ.
3.2.2
i n d o l e (5b)
3 - [ 1 - ( 6 - M e t h o x y - 1 H - i n d o l - 3 - y l ) - 1 - ( , , - t r i m e t h o x y p h e n y l ) e t h y l ] - 6 - m e t h o x y - 1 H -
3 0 4 0 5 0
(a) The 6-methoxyindole 3b (100 mg, 0.68 mmol) and the 30,40,50-trimethoxyacetophenone
4 (71.4 mg, 0.34 mmol) were dissolved in absolute ethanol. Catalytic amounts of
hydrochloric acid were added. The reaction mixture was heated at 788C for 22 h. After
purification carried out using flash chromatography (eluent: CH2Cl2), compound 5b was
obtained as a yellow amorphous solid (14.84 mg, 9% yield).
(b) A solution of 6-methoxyindole 3b (50 mg, 0.34 mmol) and 30,40,50-trimethoxyaceto-
phenone 4 (35.7 mg, 0.17 mmol) in the presence of CTAB (5 mol%) and oxalic acid
dihydrate[(CO2H)2·2H2O (50 mol%)] was placed under agitation at room temperature in
water. After about 10 h, the reaction was finished. After purification carried out using
flash chromatography (eluent: CH2Cl2), compound 5b was obtained as a yellow
amorphous solid (34.7 mg, 42% yield). 1H NMR (300 MHz, CDCl3): d 1.62 (s, 3H), 3.78
(s, 9H), 3.79 (s, 6H), 6.19 (s, 4H), 6.66 (dd, J ¼ 8.6, 2.3 Hz, 2H), 6.81 (d, J ¼ 2.2 Hz,
2H), 7.24 (d, J ¼ 8.6 Hz, 2H), 7.85 (s, 2H, NH £ 2); 13C NMR (75 MHz, CDCl3): d
36.8, 37.5, 55.2 ( £ 2), 56.0, 55.5, 60.0, 104.5 ( £ 2), 109.0 ( £ 2), 119.1 ( £ 2), 119.5
( £ 2), 119.9 ( £ 2), 120.4 ( £ 4), 121.4 ( £ 2), 142.6, 152.2, 152.9 ( £ 2), 156.4 ( £ 2);
MS (ZQ2000/ESI þ ): m/z 509 [M þ Na]þ.
3.3 Assessment of cell proliferation
The MTT assay was conducted as previously reported (Conforti et al. 2010) to detect the cell
proliferation of the human cancer cell lines MCF-7, SkBr3 and LNCaP obtained from American
Type Culture Collection (LGC Standards, Teddington, Middlesex, UK) (Nos HTB-22, HTB-30
and CRL-1740, respectively). MCF-7 cells were cultured in DMEM medium (Sigma) while
SkBr3 and LNCaP cells were grown in RPMI-1640 medium, both supplemented with 10% fetal
bovine serum, 1% L-glutamine, 1% penicillin/streptomycin (complete medium). Cell
monolayers were subcultured onto 96-well culture plates (2 £ 104 cells/well) used for
experiments 24 h later.
Cells were then treated with serial concentrations of the synthesised compounds. A total of
100 mL/well of each concentration was added and used in triplicates to obtain final dilutions

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ranging from 0.0156 to 100 mM. Treatments never exceeded 0.5% of the solvent (DMSO), per
cent of solvent that was used to treat control wells. The culture plates were kept at 378C with 5%
(v/v) CO2. After 48 h of incubation, medium was removed from each well. Subsequently,
100 mL of 0.5% (w/v) MTT (Sigma), dissolved in phosphate-buffered saline (PBS), were added
to each well and allowed to incubate for an additional 4 h. MTT was then removed and 100 mL of
DMSO were added to each well to dissolve the formazan crystals. Absorbance values at 570 nm
were measured using a microplate reader (GDV DV 990 B/V, Roma, Italy). Cytotoxicity was
expressed as IC50 which is the concentration giving 50% inhibition compared with the control
(untreated cells).
3.4 Determination of nuclear morphological changes
Cells were cultured in complete medium for 24 h on microscope slides (1 £ 105 cells) and then
treated in serum-free medium for 24 h. Cells were washed with PBS and fixed in 4%
formaldehyde for 10 min. Fixed cells were washed and incubated with 2-(4-amidinophenyl)-6-
indolecarbamidine dihydrochloride (DAPI) (0.2 mg/mL) for 10 min in a humidified chamber,
protected from light, at 378C. Cells were then washed three times with cold PBS and one drop of
mounting solution was added. Cell nuclei were observed and imaged by an inverted fluorescence
microscope with excitation at 350 nm and emission at 460 nm. The number of apoptotic nuclei
was determined in at least six randomly selected areas from three coverslips of each
experimental group. The results were expressed as apoptotic index as a percentage of apoptotic
cells relative to the total number (1 £ 105) of cells.
3.5 Statistical analysis
All experiments were carried out in sestuplicate. Data were expressed as mean value ^ SEM.
The concentration giving 50% inhibition (IC50) was calculated by non-linear regression using
the GraphPad Prism Software. Statistical significance was assessed using a one-way analysis of
variance using the SigmaStat Software. The significant differences among the means were
analysed using Tukey’s multiple comparison test. Differences of P , 0.05 were considered
significant.
4. Conclusions
Dietary consumption of cruciferous vegetables may exert a protective effect against some
chronic diseases and specific cancers, because of the content of isothiocyanates and I3C,
considered as the major chemopreventive and chemotherapeutic phytochemicals of these plants.
I3C is highly unstable and gives a broad spectrum of products, but it is primarily converted into
the dimeric product DIM. Several i n v i v o studies demonstrated the anti-tumourigenic activity of
I3C. As a result of this discovery, a series of symmetrical DIM derivatives have been synthesised
during the last years and some of them showed anti-carcinogenic activity (Safe et al. 2008). To
further validate these previous observations, this study deals with the synthesis of some new
DIM analogues and with the evaluation of their biological activity. Analogue 5b showed the best
antiproliferative activity against human prostate cancer cells LNCaP (IC50 of 0.64 ^ 0.09 mM).
LNCaP cells incubated with 5b at a concentration of 1 mM showed morphological changes
characteristic of apoptosis after an incubation period of 24 h compared with the control
(untreated cells). Data indicating a more intense staining of nuclei of treated cells support the
idea of an apoptotic mechanism of action for this compound. In conclusion, compound 5b seems
to be a promising compound potentially useful as anticancer agent, and further modifications of
this molecule will be carried out in order to optimise the activity.

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Acknowledgement
The authors are thankful to the ‘Institut National du Cancer’ (INCa, Boulogne Billancourt, France) for
financial support.
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