A Facile Synthesis of Deaza-Analogues of the Bisindole Marine Alkaloid Topsentin

A series of substituted ethyl 1-[(tert-butoxycarbonyl)amino]-2-methyl-5-(1-methyl-1H-indol-3-yl)-4-[(1-methyl-1H-indol-3-yl)carbonyl]-1H-pyrrole-3-carboxylates were prepared in excellent yields (82-98%) by one-pot reactions between β-dicarbonyl compounds 12a–e and 1,2-diaza-1,3-diene (DD) 13. Derivatives 10a,c–e, deazaanalogues of the bis-indole alkaloid topsentin, screened by the National Cancer Institute (Bethesda, MD, USA) in the in vitro one dose primary anticancer assay against a panel of about 60 human tumor cell lines, showed no significant activity, with the exception of compound 9e, which showed moderate activity against the HOP-92 cell line of the non small cell lung cancer sub-panel and the SNB-75 cell line of the CNS sub-panel.

We have recently reported the synthesis and the antitumor activity of bis-indolyl thiophene [20] 5, pyrazoles [21] 6, furans [22] 7 and isoxazoles [22] 8 ( Figure 1) that showed inhibitory activity against a wide range of human tumor cell lines, generally in the micro-and submicromolar range. Even more recently bis-indolyl pyrroles [23] 9 have exhibited concentration-dependent antitumor activity towards a panel of 42 human tumor cell lines, with mean IC 50 values of 1.54 µM and 0.67 µM, respectively. Moreover, investigating human tumor xenografts in an ex vivo clonogenic assay revealed selective antitumor activity.
Four pyrrole derivatives 10a,c-e were selected for a single dose concentration, in vitro disease-oriented antitumor screenings against the full NCI panel of about 60 human tumor cell lines that have grouped in disease sub-panels including leukaemia, non-small lung, colon, central nervous system, melanoma, ovarian, renal, prostate, and breast tumors cell lines [26]. The results obtained take into consideration the percent growth of the treated cells. The results revealed that compound 10e, tested at the concentration of 10 −4 M, showed a growth of −27.31% against the SNB-75 cell line of the CNS cancer sub-panel and 4.73% against the HOP-92 of the non-small cell lung cancer sub-panel. The other tested compounds showed no significant activity.

General Procedures
All the commercially available reagents and solvents were used without further purification. 1,2-Diaza-1,3-diene (DD, 13) was synthesized as a mixture of E/Z isomers as previously reported [27][28][29]. Column chromatography was performed with Merck silica gel 230-400 mesh ASTM or with a Büchi Sepacor chromatography module (a prepacked cartridge system). TLC analysis was performed on pre-loaded (0.25 mm) glass-supported silica gel plates (Kieselgel 60); compounds were visualized by exposure to UV light and by dipping the plates in 1% Ce(SO 4 )·4H 2 O, 2.5% (NH 4 ) 6 Mo 7 O 24 ·4H 2 O in 10% sulfuric acid followed by heating on a hot plate. 1 H-NMR and 13 C-NMR spectra were recorded in DMSO-d 6 or CDCl 3 solution on 200 (Bruker AC) or 400 (Varian Mercury) MHz instruments. Proton and carbon spectra were referenced internally to solvent signals, using values of δ = 2.49 ppm for proton (middle peak) and δ = 39.50 ppm for carbon (middle peak) in DMSO-d 6 and δ = 7.26 ppm for proton and δ = 77.00 ppm for carbon (middle peak) in CDCl 3 . The following abbreviations are used to describe peak patterns where appropriate: s = singlet, d = doublet, t = triplet q = quartet and m = multiplet. All coupling constants (J) are given in Hz. All melting points were taken on a Büchi-Tottoli capillary apparatus and are uncorrected; IR spectra were determined in bromoform or nujol with a Jasco FT/IR 5300 spectrophotometer. Mass spectra were recorded in the EI mode (70eV) on a Shimadzu GC-MS QP5050A spectrometer. Elemental analyses (C, H, N) were within ±0.4% of the theoretical values.
3.1.1.1. General Procedure for the Preparation of 1,3-bis(Indol-3-yl)propane-1,3-diones 12a-e A solution of malonyl dichloride (1 mL, 10 mmol) in DCM (10 mL) was added dropwise to a cold solution of the appropriate N-methylindole 11a-e (20 mmol) in DCM (20 mL). The reaction mixture was stirred for 2 h at room temperature. The mixture was then added to 5% aqueous sodium carbonate and shaken for 2 min. This was extracted with DCM, dried and evaporated. The residue was purified by chromatography eluting with DCM/Ethyl acetate 9/1 to give derivatives 12a-e. Analytical and spectroscopic data are reported elsewhere [12].

Methodology of the in Vitro Cancer Screen
The human tumor cell lines of the cancer screening panel are grown in RPMI 1640 medium containing 5% fetal bovine serum and 2 mM L-glutamine. For a typical screening experiment, cells are inoculated into 96 well microtiter plates in 100 µL at plating densities ranging from 5,000 to 40,000 cells/well depending on the doubling time of individual cell lines. After cell inoculation, the microtiter plates are incubated at 37 °C, 5% CO 2 , 95% air and 100% relative humidity for 24 h prior to addition of experimental drugs. After 24 h, two plates of each cell line are fixed in situ with TCA, to represent a measurement of the cell population for each cell line at the time of drug addition (Tz). Experimental drugs are solubilized in dimethyl sulfoxide at 400-fold the desired final maximum test concentration and stored frozen prior to use. At the time of drug addition, an aliquot of frozen concentrate is thawed and diluted to twice the desired final maximum test concentration with complete medium containing 50 µg/mL gentamicin. Additional four, 10-fold or ½ log serial dilutions are made to provide a total of five drug concentrations plus control. Aliquots of 100 µL of these different drug dilutions are added to the appropriate microtiter wells already containing 100 µL of medium, resulting in the required final drug concentrations. Following drug addition, the plates are incubated for an additional 48 h at 37 °C, 5% CO 2 , 95% air, and 100% relative humidity. For adherent cells, the assay is terminated by the addition of cold TCA. Cells are fixed in situ by the gentle addition of 50 µL of cold 50% (w/v) TCA (final concentration, 10% TCA) and incubated for 60 min at 4 °C. The supernatant is discarded, and the plates are washed five times with tap water and air dried. Sulforhodamine B (SRB) solution (100 µL) at 0.4% (w/v) in 1% acetic acid is added to each well, and plates are incubated for 10 min at room temperature. After staining, unbound dye is removed by washing five times with 1% acetic acid and the plates are air dried. Bound stain is subsequently solubilized with 10 mM trizma base, and the absorbance is read on an automated plate reader at a wavelength of 515 nm. For suspension cells, the methodology is the same except that the assay is terminated by fixing settled cells at the bottom of the wells by gently adding 50 µL of 80% TCA (final concentration, 16% TCA). Using the seven absorbance measurements [time zero, (Tz), control growth, (C), and test growth in the presence of drug at the five concentration levels (Ti)], the percentage growth is calculated at each of the drug concentrations levels. Percentage growth inhibition is calculated as: For further information to see NCI website [30].