Synthesis and biological evaluation of a γ-cyclodextrin-based formulation of the anticancer agent 5,6,11,12,17,18,23,24- octahydrocyclododeca[1,2-b:4,5-b':7,8-b :10,11-b ']tetraindole (CTet)

Article abstract of DOI:10.3390/molecules15064085

5,6,11,12,17,18,23,24-octahydrocyclododeca[1,2-b:4,5-b':7,8-b :10, 11-b ']tetrai ndole (CTet), an indole-3-carbinol (I3C) metabolite endowed with anticancer properties, is poorly soluble in the solvents most frequently used in biological tests. This study indicates that the use of γ-cyclodextrin (γ-CD) avoids this problem. Formulated with γ-CD CTet is a potent inhibitor of DNA synthesis in both estrogen receptor positive (MCF-7) and estrogen receptor negative (MDA-MB-231) human breast cell lines (IC₅₀ = 1.20 ± 0.04 μM and 1.0 ± 0.1 μM, respectively).

Full text of DOI:10.3390/molecules15064085

Molecules 2010, 15, 4085-4093; doi:10.3390/molecules15064085
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis and Biological Evaluation of a γ-Cyclodextrin-based
Formulation of the Anticancer Agent 5,6,11,12,17,18,23,24-
Octahydrocyclododeca[1,2-b:4,5-b’:7,8-b’’:10,11-b’’’]tetraindole
(CTet)
Simone Lucarini 1, Mauro De Santi 2, Francesca Antonietti 1, Giorgio Brandi 2, Giuseppe
Diamantini 1, Alessandra Fraternale 2, Maria Filomena Paoletti 2, Andrea Tontini 1, Mauro
Magnani 2 and Andrea Duranti 1,*
1
Dipartimento di Scienze del Farmaco e della Salute, Università degli Studi di Urbino “Carlo Bo” I-
61029 Urbino, Piazza del Rinascimento 6, Italy; E-Mails: simone.lucarini@uniurb.it (S.L.);
francesca.antonietti@uniurb.it (F.A.); giuseppe.diamantini@uniurb.it (G.D.);
andrea.tontini@uniurb.it (A.T.)
2
Dipartimento di Scienze Biomolecolari, Università degli Studi di Urbino “Carlo Bo” I-61029
Urbino, Via Aurelio Saffi 2, Italy; E-Mails: mauro.desanti@uniurb.it (M.D.S.);
giorgio.brandi@uniurb.it (G.B.); alessandra.fraternale@uniurb.it (A.F.);
maria.paoletti@uniurb.it (M.F.P.); mauro.magnani@uniurb.it (M.M.)
* Author to whom the correspondence should be addressed; E-Mail: andrea.duranti@uniurb.it;
Tel.: +39 0722 303323; Fax: +39 0722 303313.
Received: 21 April 2010; in revised form: 27 May 2010 / Accepted: 1 June 2010 /
Published: 4 June 2010
Abstract: 5,6,11,12,17,18,23,24-octahydrocyclododeca[1,2-b:4,5-b’:7,8-b’’:10,11- b’’’]tetrai
ndole (CTet), an indole-3-carbinol (I3C) metabolite endowed with anticancer properties, is
poorly soluble in the solvents most frequently used in biological tests. This study indicates
that the use of γ-cyclodextrin (γ-CD) avoids this problem. Formulated with γ-CD CTet is a
potent inhibitor of DNA synthesis in both estrogen receptor positive (MCF-7) and estrogen
receptor negative (MDA-MB-231) human breast cell lines (IC50 = 1.20 ± 0.04 μM and
1.0 ± 0.1 μM, respectively).
Keywords: indole-3-carbinol; indole cyclic tetramer; γ-cyclodextrin; breast cancer

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1. Introduction
Edible cruciferous vegetables of the genus Brassica are endowed with chemopreventive and
chemotherapeutic properties [1–3]. These actions depend on an autolysis product of 3-indolylmethyl
glucosinolate (glucobrassicin), namely indole-3-carbinol (I3C, Figure 1) [4,5], and the resulting indole
oligomers produced in the acidic environment of the stomach: 3,3’-diindolylmethane (DIM) [4,5],
indolo[3,2-b]carbazole (ICZ) [6,7], the linear trimer LTr [6,8,9], the cyclic trimer CTr [6,8,10], and
the cyclic tetramer CTet [11,12] (Figure 1) [2]. The current interest of pharmacologists and medicinal
chemists in this topic has resulted in several reports which disclosed synthetic analogues of I3C
[13–15], DIM [16–31], and CTr [32] possessing anticancer properties.
Figure 1. Chemical structures of compounds I3C, DIM, ICZ, LTr, CTr, and CTet.
H
N
H
N
N
ICZ
H
HN
DIM
H
N
H
N
HN
OH
I3C
HN
LTr
NH
H
N
N
H
H
N
NH
HN
CTet
CTr
NH
In order to study the antitumor effects of CTet, we needed a reliable and gram-scale synthesis of
this compound. The methods reported in the literature for the preparation of CTet are three. The first
involves the one-pot reaction of indole and formaldehyde in acidic methanol, and is in principle
advantageous [33]. The second is in fact aimed at obtaining CTr and affords CTet as a by-product in
low yield after recrystallization with DMSO; this method, employing gramine as a source in a basic
environment of the presumed 3-methylene indolenine intermediate, is practical and efficient but not all
its reagents could be utilized on a large scale synthesis because of their toxicity [34]. The third
protocol utilizes I3C and acetic acid but the acidic conditions [8] and the purification by means of
silica gel column chromatography lead to the formation of polymers and degradation products, so it is
not possible to isolate pure CTet by recrystallization with methanol [12].

Molecules 2010, 15
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Unfortunately, CTet is poorly, if at all, soluble in the most common solvents, in particular those
usually employed in biological experiments (acetone: 0.04, pyridine: 0.22, 2-butanol: 0.11, DMSO:
0.1% w/v). Furthermore, in chloroform, ethanol, methanol, and toluene CTet solubility is less than
0.1% w/v and the compound is insoluble in water and physiological saline solutions. Several
procedures were therefore evaluated to increase CTet solubility in a pharmaceutically acceptable
formulation. We found the approach with γ-cyclodextrin (γ-CD) promising, therefore it was selected
for further investigation.
2. Results and Discussion
The synthesis of pure CTet was carried out by modifying Bergman et al.’s procedure [33]. When
we applied this protocol, we repeatedly obtained results not congruent with the reported ones. In
particular, the precipitate that separated from the mixture contained only a trace of the desired CTet,
being instead constituted of numerous side-products, probably formed through polymerization
processes. However, the filtrate of the reaction mixture did contain CTet, which was isolated by
chromatography and recrystallization. In addition, HPLC analysis of the chromatographic fractions
showing a single spot on TLC plates demonstrated that CTet was present together with CTr.
Bergman’s protocol was modified by prolonging reaction time, due to the presence of the starting
material in the mixture after one hour, and by purifying the crude by two rapid passages through short
aluminum oxide columns. CTet was finally obtained with a purity higher than 99% by recrystallization
from acetone, rather than pyridine [33] and DMSO [34], to facilitate solvent removal. The protocol
proved to be scalable, in that it was possible to run it using up to 150 mmol of indole (17.5 g); these
experiments gave yields and CTr/CTet ratios comparable with those reported on a lower scale
(amounts of reagents higher than those reported were not used) (Scheme 1).
Scheme 1. Synthesis of 5,6,11,12,17,18,23,24-octahydrocyclododeca[1,2-b:4,5-b’:7,8-
b’’:10,11-b’’’]tetraindole (CTet).
HN
H
N
H
N
N
H
1
NH
CTet
Reagents and conditions: 37% HC(O)H, MeOH, 96% H2SO4, reflux, 2 h.
With the aim of examining the antiproliferative activity of CTet, the drug was solubilized in
pyridine or suspended in ethanol or DMSO and tested on estrogen receptor positive (ER+) breast
cancer cell line MCF-7. It resulted that CTet in pyridine could not affect cell proliferation, whereas
CTet in DMSO did in a dose-dependent manner (IC50 = 11.3 ± 1.4 µM). Also, CTet suspended in

Molecules 2010, 15
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ethanol showed good antiproliferative activity in the same cell line (IC50 = 1.7 ± 0.1 μM) (Figure 2). A
pure ethanolic preparation, however, could not be used in clinical studies, thus we considered
important to investigate formulations of CTet in an aqueous system.
Several protocols such as Solvent Induced Activation (SIA) system with PVP-Cl
(polyvinylpyrrolidone-Cl) in different mediums, HP-55 (hydroxypropyl methyl cellulose phthalate),
and β- or γ-CD complexation, were investigated. Only γ-CD formulation gave encouraging results. So,
while the suspension obtained by diluting the CTet mixture in ethanol/water 1:10 showed a significant
loss of biological activity (IC50 = 7.9 ± 0.6 μM; P < 0.001) (Figure 2), when dilution of CTet was
carried out in a γ-CD EtOH/H2O (1:10) solution, the activity of CTet resulted superimposable to that
of CTet suspended in pure ethanol (IC50 = 1.20 ± 0.04 µM) (Figure 2).
Figure 2. Effect of CTet formulated in aqueous solutions on DNA synthesis of MCF-7
breast cancer cell line. Cells were treated with various concentrations of CTet suspended in
10% EtOH ({), 10% EtOH with 160 mM γ-CD (◊) or pure EtOH (▲); during the last 5 h
of treatment, cells were pulsed with [3H]thymidine, and the incorporation into DNA was
determined (1.5 μCi). Data are expressed as percentage of cells treated with vehicle only
and are means ± SEM of at least three experiments.
100
EtOH
EtOH 10%
IC50 = 1.7 ± 0.1 µM
IC50 = 7.9 ± 0.6 µM
80
60
40
20
0
EtOH 10% + γCD IC50 = 1.20 ± 0.04 µM
0
4
8
12
16
CTet (µM)
The antiproliferative activities of CTet both suspended in pure ethanol and formulated in γ-CD 10%
ethanol were also tested on an estrogen receptor negative (ER-) breast cancer cell line (MDA-MB-231);
the results were comparable with those obtained with MCF-7 cells (IC50 = 0.9 ± 0.1 and
1.0 ± 0.1 µM, respectively) (Figure 3). Notably, a 10% ethanolic solution of γ-CD did not have any
appreciable cytotoxicity in our tests.
Finally, we had ascertained by HPLC that these formulations were stable for many months at room
temperature in the dark; this observation is corroborated by the fact that antiproliferative tests in MCF-
7 cells were comparable with those reported above (data not shown).

Molecules 2010, 15
Figure 3. Effect of CTet formulated in aqueous solutions on DNA synthesis of MDA-MB-
4089
231 breast cancer cell line. Cells were treated with various concentrations of CTet
suspended in 10% EtOH with 160 mM γ-CD (◊) or pure EtOH (▲); during the last 5 h of
treatment cells were pulsed with [3H]thymidine, and the incorporation into DNA was
determined (1.5 μCi). Data are expressed as percentage of cells treated with vehicle only
and are means ± SEM of at least three experiments. A 10% ethanolic solution of γ-CD did
not have any appreciable cytotoxicity in our tests.
100
EtOH
IC50 = 0.9 ± 0.1 µM
80
60
40
20
0
EtOH 10% + γCD IC50 = 1.0 ± 0.1 µM
12
16
0
4
8
CTet (µM)
3. Experimental
3.1. General
All reagents were purchased from Sigma-Aldrich or Carlo Erba with the exception of PVP-Cl and
HP-55 which were furnished by Eurand, β-cyclodextrin (CAPTISOL®, CyDex), and γ-cyclodextrin
(CAVAMAX® W8, Wacker); they were in the highest quality commercially available. Solvents were
RP grade. Melting points were determined on a Büchi B-540 capillary melting point apparatus. The
1
structure of CTet was unambiguously assessed by MS, H-NMR, and 13C-NMR. MS (ESI) spectra
were recorded with a Waters Micromass ZQ spectrometer in a positive mode using a nebulizing
nitrogen gas at 400 L/min and a temperature of 250 ºC, cone flow 40 mL/min, capillary 3.5 Kvolts and
cone voltage 60 V; only molecular ion in positive ion mode [M+H]+ is given. Retention time (tR) value
was determined by direct HPLC analysis by Waters 2795 Separations Module, Alliance HT and
Waters 2996, Photodiode Array Detector spectrometers with
a
SupelcosilTM LC-18
(15 cm × 4 mm, 3 μM; Supelco) column using a combination of acetonitrile and aqueous solution
0.1% formic acid as eluent. 1H-NMR and 13C-NMR spectra were recorded on a Bruker AC 200 or 50,
instrument, respectively, and analyzed using the WIN-NMR software package. Chemical shifts were
measured by using the central peak of the solvent. Purification of the crude material was carried out by

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column chromatography on aluminum oxide (0.05–0.15 mm, Fluka). TLC analyses were performed on
precoated aluminum oxide on aluminum sheets (60 F254, neutral; Merck).
3.2. Synthesis of 5,6,11,12,17,18,23,24-octahydrocyclododeca[1,2-b:4,5-b’:7,8-b’’:10,11-
b’’’]tetraindole (CTet)
To a solution of indole (3.12 g, 26.7 mmol) and aqueous 37% HC(O)H (3.2 mL, 40 mmol) in
CH3OH (240 mL), 96% H2SO4 (1.74 mL) was added. The mixture was stirred at reflux in the dark for
1 h, then further HC(O)H (3.2 mL, 40 mmol) was added, the mixture was stirred in the same
conditions for 1 h, cooled to room temperature and concentrated in the dark. Purification of the solid
by two short, protected from light, and fast aluminum oxide column chromatographies
(cyclohexane/EtOAc 6:4, Rf = 0.82) and washing with hot CH3OH gave a white solid consisting
(HPLC/MS) in a 9:1 mixture of CTr and CTet [HPLC: SupelcosilTM LC-18; flow: 0.5 mL/min; λmax
:
284 nm; eluent: CH3CN/aqueous solution 0.1% HCOOH with a gradient 7:3 to 9:1 in 9 min; tR CTr:
4.95 min, tR CTr: 6.93 min]. Yield: 31% (1.08 g). Recrystallization [(CH3)2CO, 78 mL] afforded pure
CTet as a white solid. Mp: chars over 300 ºC. MS (ESI) m/z: 517.2 [M+H]+. 1H-NMR [(CD3) 2CO]: δ
3.88 (s, 8H, CH2), 6.85 (dd, 4H, ArH, J1 = 7.0 and J2 = 8.0 Hz), 6.99 (dd, 4H, ArH, J1 = 7.0 and
J2 = 8.0 Hz), 7.24 (d, 4H, ArH, J = 8.0 Hz), 7.33 (d, 4H, ArH, J = 8.0 Hz), 9.95 (s, 4H, NH); 13C-NMR
(pyridine-d5): δ 23.6, 109.1, 112.6, 119.4, 120.4, 122.0, 131.4, 137.7, 138.0.
3.3. CTet formulations
A suspension of CTet (0.0083 g, 0.016 mmol) in pure EtOH (1 mL) was magnetically stirred at
room temperature for different times (1 to 3 days, 1,000 rpm). The highest percentage of inhibition was
obtained when the suspension was stirred for at least 2 days. This time was routinely used in all further
experiments. The emulsion obtained was then diluted (volume ratio 1:10) by an aqueous solution of
γ-CD (177 mM); the resulting white emulsion had a final concentration of 1.6 mM. The
antiproliferative assays were performed with 10 μL of formulated product appropriately diluted in 1
mL of the cellular culture medium.
3.4. Cell cultures and antiproliferative assay
The human breast carcinoma ER+ (MCF-7) and ER- (MDA-MB-231) cell lines were cultured in
DMEM (Dulbecco’s Modified Eagle’s Medium) supplemented with 10% FCS (Fetal Calf Serum),
2 mM L-glutamine, 10 g/L NEAA (Non-Essential Amino Acid), 50 mg/L streptomycin, 1,000 U/L
penicillin, with (in the case of MCF-7) or without (in the case of MDA-MB-231) 10 mg/L insulin.
Cells (30,000/well in 24-well tissue culture plates) were treated with the several CTet formulations or
respective vehicles for 72 h, and during the last 4 h of treatment were pulsed with 1.5 μCi of
[3H]thymidine and processed [12].

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3.5. Statistical analyses
Data are means ± SEM of at least three separate experiments. Differences between means were
evaluated by Student t-test; differences were considered significant at P < 0.05 (Prism5, GraphPad
Software Inc., La Jolla, CA, USA).
4. Conclusions
A straightforward, reproducible, and scalable synthesis of CTet is reported, together with a
formulation of CTet that allows the molecule to exert its pharmacological potential as an inhibitor of
DNA synthesis in both ER+ and ER- human breast cancer cells. It is hypothesized that γ-CD is capable
to enhance the otherwise very low solubility of the drug in aqueous systems.
Acknowledgements
The authors thank Giorgio Tarzia for his helpful hints.
References
1. National Research Council. Diet, Nutrition and Cancer; Peter, F.M., Ed.; National Academy Press:
Washington, DC, USA, 1982; pp. 358–370.
2. Weng, J.-R.; Tsai, C.-H.; Kulp, S.K.; Chen, C.-S. Indole-3-carbinol as a chemopreventive and
anti-cancer agent. Cancer Lett. 2008, 269, 153–163.
3. Safe, S.; Papineni, S.; Chintharlapalli, S. Cancer chemotherapy with indole-3-carbinol, bis(3’-
indolyl)methane and synthetic analogs. Cancer Lett. 2008, 269, 326–338.
4. Virtanen, A.I. Studies on organic sulphur compounds and other labile substances in plants.
Pythochemistry 1965, 4, 207–228.
5. Wattenberg, L.W.; Loub, W.D. Inhibition of polycyclic aromatic hydrocarbon-induced neoplasia
by naturally occurring indoles. Cancer Res. 1978, 38, 1410–1413.
6. Bjeldanes, L.F.; Kim, J.-Y.; Grose, K.R.; Bartholomew, J.C.; Bradfield, C.A. Aromatic
hydrocarbon responsiveness-receptor agonists generated from indole-3-carbinol in vitro and in
vivo: Comparisons with 2,3,7,8-tetrachlorodibenzo-p-dioxin. Proc. Natl. Acad. Sci. USA 1991, 88,
9543–9547.
7. D'Argy, R.; Bergman, J.; Dencker, L. Effects of immunosuppressive chemicals on lymphoid
development in fetal thymus organ cultures. Pharmacol. Toxicol. 1989, 64, 33–38.
8. De Kruif, C.A.; Marsman, J.W.; Venekamp, J.C.; Falke, H.E.; Noordhoek, J.; Blaauboer, B.J.;
Wortelboer, H.M. Structure elucidation of acid reaction products of indole-3-carbinol: detection in
vivo and enzyme induction in vitro. Chem.-Biol. Interact. 1991, 80, 303–315.
9. Chang, Y.-C.; Riby, J.; Chang, G.H.-F.; Peng, B.C.; Firestone, G.; Bjeldanes, L.F. Cytostatic and
antiestrogenic effects of 2-(indol-3-ylmethyl)-3,3’-diindolylmethane, a major in vivo product of
dietary indole-3-carbinol. Biochem. Pharmacol. 1999, 58, 825–834.
10. Riby, J.E.; Feng, C.; Chang, Y.-C.; Schaldach, C.M.; Firestone, G.L.; Bjeldanes, L.F. The major
cyclic trimeric product of indole-3-carbinol is a strong agonist of the estrogen receptor signaling
pathway. Biochemistry 2000, 39, 910–918.

Molecules 2010, 15
4092
11. Grose, K.R.; Bjeldanes, L.F. Oligomerization of indole-3-carbinol in aqueous acid. Chem. Res.
Toxicol. 1992, 5, 188–193.
12. Brandi, G.; Paiardini, M.; Cervasi, B.; Fiorucci, C.; Filippone, P.; De Marco, C.; Zaffaroni, N.;
Magnani, M. A new indole-3-carbinol tetrameric derivative inhibits cyclin-dependent kinase 6
expression, and induces G1 cell cycle arrest in both estrogen-dependent and estrogen-independent
breast cancer cell lines. Cancer Res. 2003, 63, 4028–4036.
13. Weng, J.-R.; Tsai, C.-H.; Kulp, S.K.; Wang, D.; Lin, C.-H.; Yang, H.-C.; Ma, Y.; Sargeant, A.;
Chiu, C.-F.; Tsai, M.-H.; Chen, C.-S. A potent indole-3-carbinol–derived antitumor agent with
pleiotropic effects on multiple signaling patways in prostate cancer cells. Cancer Res. 2007, 67,
7815–7824.
14. Jump, S.M.; Kung, J.; Staub, R.; Kinset, M.A.; Cram, E.J.; Yudina, L.N.; Preobrazhenskaya, M.N.;
Bjeldanes, L.F.; Firestone, G.L. N-Alkoxy derivatization of indole-3-carbinol increases the
efficacy of the G1 cell cycle arrest and of I3C-specific regulation of cell cycle gene transcription
and activity in human breast cancer cells. Biochem. Pharmacol. 2008, 75, 713–724.
15. Guo, W.; Wu, S.; Liu, J.; Fang, B. Identification of a small molecule with synthetic lethality for
K-Ras and protein kinase C iota. Cancer Res. 2008, 68, 7403–7408.
16. Ramamoorthy, K.; Navo, M.; Gupta, M.S.; Safe, S.H. AhR-mediated antiestrogenicity of
diindolylmethane and analogs in vivo and in vitro. Organohalogen Compd. 1998, 37, 321–324.
17. Ramamoorthy, K.; McDougal, A.; Safe, S.H. Structure-Ah receptor agonist/binding activity
relationships of various chlorine-substituted diindolylmethane compounds. Organohalogen
Compd. 1999, 42, 363–367.
18. McDougal, A.; Gupta, M.S.; Ramamoorthy, K.; Sun, G.; Safe, S.H. Inhibition of carcinogen-
induced rat mammary tumor growth and other estrogen-dependent responses by symmetrical
dihalo-substituted analogs of diindolylmethane. Cancer Lett. 2000, 151, 169–179.
19. McDougal, A.; Gupta, M.S.; Morrow, D.; Ramamoorthy, K.; Lee, J.-E.; Safe, S.H. Methyl-
substituted diindolylmethanes as inhibitors of estrogen-induced growth of T47D cells and
mammary tumors in rats. Breast Cancer Res. Treat. 2001, 66, 147–157.
20. Benabadji, S.H.; Wen, R.; Zheng, J.-B.; Dong, X.-C.; Yuan, S.-G. Anticarcinogenic and
antioxidant activity of diindolylmethane derivatives. Acta Pharmacol. Sin. 2004, 25, 666–671.
21. Pisano, C.; Kollar, P.; Gianní, M.; Kalac, Y.; Giordano, V.; Ferrara, F.F.; Tancredi, R.; Devoto, A.;
Rinaldi, A.; Rambaldi, A.; Penco, S.; Marzi, M.; Moretti, G.; Vesci, L.; Tinti, O.; Carminati, P.;
Terao, M.; Garattini, E. Bis-indols: a novel class of molecules enhancing the cytodifferentiating
properties of retinoids in myeloid leukemia cells. Blood 2002, 100, 3719–3730.
22. Giannini, G.; Marzi, M.; Moretti, G.P.; Penco, S.; Tinti, M.O.; Pesci, S.; Lazzaro, F.; De Angelis,
F. Synthesis of cycloalkanoindoles by an unusual DAST-triggered rearrangement reaction. Eur. J.
Org. Chem. 2004, 2411–2420.
23. Maciejewska, D.; Rasztawicka, M.; Wolska, I.; Anuszewka, E.; Gruber, B. Novel 3,3’-
diindolylmethane derivatives: Synthesis and cytotoxicity, structural characterization in solid state.
Eur. J. Med. Chem. 2009, 44, 4136–4147.
24. Sanderson, J.T.; Slobbe, L.; Lansbergen, G.W.A.; Safe, S.; van der Berg, M. 2,3,7,8-
Tetrachlorodibenzo-p-dioxin and diindolylmethanes differentially induce cytochrome P450 1A1,
1B1, and 19 in H295R human adrenocortical carcinoma cells. Toxicol. Sci. 2001, 61, 40–48.

Molecules 2010, 15
4093
25. Qin, C.; Morrow, D.; Stewart, J.; Spencer, K.; Porter, W.; Smith, R., III; Phillips, T.; Abdelrahim,
M.; Samudio, I.; Safe, S. A new class of peroxisome proliferator-activated receptor γ (PPARγ)
agonists that inhibit growth of breast cancer cells: 1,1-Bis(3’-indolyl)-1-(p-substituted
phenyl)methanes. Mol. Cancer Ther. 2004, 3, 247–259.
26. Contractor, R.; Samudio, I.J.; Estrov, Z.; Harris, D.; McCubrey, J.A.; Safe, S.H.; Andreeff, M.;
Konopleva, M. A novel ring-substituted diindolylmethane, 1,1-bis[3’-(5-methoxyindolyl)]-1-(p-t-
butylphenyl) methane, inhibits extracellular signal-regulated kinase activation and induces
apoptosis in acute myelogenous leucemia. Cancer Res. 2005, 65, 2890–2898.
27. Chintharlapalli, S.; Burghardt, R.; Papineni, S.; Ramaiah, S.; Yoon, K.; Safe, S. Activation of
Nur77 by selected 1,1-bis(3’-indolyl)-1-(p-substitued phenyl)methanes induces apoptosis through
nuclear pathways. J. Biol. Chem. 2005, 280, 24903–24914.
28. Inamoto, T.; Papineni, S.; Chintharlapalli, S.; Cho S.-D.; Safe, S.; Kamat, A.M. 1,1-Bis(3’-
indolyl)-1-(p-chlorophenyl)methane activates the orphan nuclear receptor Nurr1 and inhibits
bladder cancer growth. Mol. Cancer Ther. 2008, 7, 3825–3833.
29. Noguchi-Yachide, T.; Tetsuhashi, M.; Aoyama, H.; Hashimoto, Y. Enhancement of chemically-
induced HL-60 cell differentiation by 3,3’-diindolylmethane derivatives. Chem. Pharm. Bull.
2009, 57, 536–540.
30. Chao, W.-R.; Yean, D.; Amin, K.; Green, C.; Jong, L. Computer-aided rational drug design: A
novel agent (SR13668) designed to mimic the unique anticancer mechanisms of dietary indole-3-
carbinol to block akt signaling. J. Med. Chem. 2007, 50, 3412–3415.
31. Vincent, E.; Shirani, H.; Bergman, J.; Rannug, U.; Janosik, T. Synthesis and biological evaluation
of fused thio- and selenopyrans as new indolocarbazole analogues with aryl hydrocarbon receptor
affinity. Bioorg. Med. Chem. 2009, 17, 1648–1653.
32. Xue, L.; Schaldach, C.M.; Janosik, T.; Bergman, J.; Bjeldanes, L.F. Effects of analogs of indole-
3-carbinol cyclic trimerization product in human breast cancer cells. Chem.-Biol. Interact. 2005,
152, 119–129.
33. Bergman, J.; Högberg, S.; Lindström, J.-O. Macrocyclic condensation products of indole and
simple aldehydes. Tetrahedron 1970, 26, 3347–3352.
34. Staub, R.E.; Bjeldanes, L.F. Convenient synthesis of 5,6,11,12,17,18-hexahydrocyclononal[1,2-
b:4,5-b’:7,8-b’’]triindole, a novel phytoestrogen. J. Org. Chem. 2003, 68, 167–169.
Sample Availability: A sample of the compound is available from the authors.
© 2010 by the authors; licensee MDPI, Basel, Switzerland. This article is an Open Access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/3.0/).