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)

5,6,11,12,17,18,23,24-octahydrocyclododeca[1,2-b:4,5-b’:7,8-b’’:10,11-b’’’]tetraindole (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).

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.

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). 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 (IC 50 = 11.3 ± 1.4 µM). Also, CTet suspended in ethanol showed good antiproliferative activity in the same cell line (IC 50 = 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.
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).

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 structure of CTet was unambiguously assessed by MS, 1 H-NMR, and 13 C-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 (t R ) value was determined by direct HPLC analysis by Waters 2795 Separations Module, Alliance HT and Waters 2996, Photodiode Array Detector spectrometers with a Supelcosil TM LC-18 (15 cm × 4 mm, 3 μM; Supelco) column using a combination of acetonitrile and aqueous solution 0.1% formic acid as eluent. 1 H-NMR and 13 C-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 EtOH IC 50 = 0.9 ± 0.1 µM EtOH 10% + γCD IC 50 = 1.0 ± 0.1 µM column chromatography on aluminum oxide (0.05-0.15 mm, Fluka). TLC analyses were performed on precoated aluminum oxide on aluminum sheets (60 F 254 , neutral; Merck). 5,6,11,12,17,18,23,24-octahydrocyclododeca [1,2-b:4,5-b':7,8-

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.

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 [ 3 H]thymidine and processed [12].

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).

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.