Synthesis and antiproliferative activity of 1-methoxy-, 1-( α-D-ribofuranosyl )-and 1-( β-D-ribofuranosyl ) brassenin B

Syntheses of indole phytoalexin 1-methoxybrassenin B and a linear synthesis of its nucleoside analogs 1-(α-D-ribofuranosyl)brassenin B and 1-(β-D-ribofuranosyl)brassenin B are reported from the corresponding 1-substituted indole-3-carboxaldehydes and carboxylic acids as key intermediates. Examination of the antiproliferative activity of synthesized compounds on human tumor cell lines Jurkat, CEM, CEM-VCR, MCF-7 and HeLa revealed the highest activity for 1methoxybrassenin B, whereas activity of the nucleoside analogs decreased and appeared to be dependent on lipophilicity.


Introduction
Indole phytoalexins are inducible phytochemicals produced by Brassica plants in response to several forms of stress, including microbial infection. 1However, their isolation from plants requires complicated procedures and does not afford sufficient quantities for biological studies.With respect to the well known anticarcinogenicity of Brassica vegetables, 1c,2 there is a strong need to investigate the synthetic approaches to 1-methoxyindole phytoalexins and their analogs, including N-glycosylated ones.Indole nucleosides represent a rare type of natural products with interesting biological properties.Among them, nucleoside antibiotic rebeccamycin (1) 3a and its analogs have been identified as attractive cancer chemotherapy agents.3b Glycosylation of the natural indolocarbazole BE 13793C (2) 4a and its further modification lead to the improvement of its biological properties and yielded a potent anticancer drug J-107,088 (3a), active against human stomach cancer cells MKN-45 implanted to mice.4b,c From the series of sugar analogues of J-107,088 (3a), the β-D-ribofuranoside J-107,534 (3b) was found to be 6 times more potent than 3a at inhibiting topoisomerase I, an attractive target for cancer chemotherapy.4d A series of recently described 2,3-substituted-5,6-dichloro-1-(β-D-ribofuranosyl)indole compounds exhibit activity against human cytomegalovirus and herpes simplex virus type I, but this activity was not well separated from cytotoxicity. 5thin our continuing investigation of the synthesis of cruciferous phytoalexins and their nucleoside analogs, 6 we have focused our effort also towards the synthesis and biological activity of 1-methoxybrassenin B (4a), isolated in 1991 from cabbage (Brassica oleracea var.capitata) inoculated with Pseudomonas cichorii, 7 and its ribofuranosyl analogs 6a and 6b.
of the α-anomer is not known, it was suggested that DDQ eliminated a hydride ion from the anomeric center to form a carbocation, which can be stabilized by conjugation with the unshared electron pair of the neighboring nitrogen and oxygen atoms.A proton is then eliminated and a hydrogen transferred from the pyrrole ring to the anomeric carbon of the planar carbonium ion from either face to produce a mixture of β-and α-anomers. 11The unexpected formation of αribofuranosylindole 11a opened the way to synthesis of 6a.Indole 11a was subjected to a Vilsmeier reaction and aldehyde 12 was obtained in 86% yield.Because of the low yield of β-anomeric indole 11b, we have been searching for another way to obtain suitably protected 1-(β-D-ribofuranosyl)indole-3-carboxaldehyde.Consequently, we decided to verify the information that the reaction of peracetylated ribofuranose 13 with indoline (7) produces only 1-acetylindoline. 11It was found that condensation of protected ribose 13 with 3 equivalents of indoline under reflux during 6 hours afforded the desired 1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)indoline (14) in 63% yield after column chromatography in addition to the 1acetylindoline (Scheme 2).We presented this result in 2003 on the 10 th Blue Danube Symposium on Heterocyclic Chemistry in Vienna.6e In the same year, an analogous preparation of 14 in 55% yield after preparative TLC was published using 2 equivalents of indoline under reflux for 27 hours. 13Oxidation of 14 with DDQ afforded 1-(2,3,5-tri-O-acetyl-β-D-ribofuranosyl)indole (15)  of very high purity in 98% yield without chromatography.Vilsmeier formylation of 15 gave the required intermediate aldehyde 16 in 61% yield.In analogy to the linear synthesis of target nucleoside analogs 6a and 6b, we decided to use 1-methoxyindole-3-carboxaldehyde (17) 14 as the key synthetic intermediate in the synthesis of 1methoxybrassenin B (4a).Aldehyde 17 was prepared by Vilsmeier-Haack formylation of 1methoxyindole that was obtained by Somei's method, based on Na 2 WO 4 catalyzed oxidation of indoline to 1-hydroxyindole with H 2 O 2 and subsequent O-methylation with dimethyl sulphate. 15ith the main intermediates in hand, we continued in the synthesis of target compounds by oxidation of aldehydes 17, 12 and 16 to carboxylic acids 18-20 (Scheme 3).First we tried KMnO 4 as in the synthesis of glucopyranosylindole-3-carboxylic acid.6a In this way, 1methoxyindole-3-carboxylic acid 18 was prepared in 50% yield.However, oxidation with NaClO 2 in a mixture of t-BuOH and 2-methylbut-2-ene over 39 hours returned an excellent 95% yield in accordance with the literature. 16Application of a NaClO 2 oxidation to α-and βribofuranosylindol-3-carbaldehydes 12 and 16 afforded acids 19 and 20 in 91% and 81% yield with shorter reaction times of 8.5 and 5.5 hours respectively.Owing to the low solubility of aldehydes 12 and 16 in t-BuOH and 2-methylbut-2-ene, dioxane had to be added to achieve complete dissolution of starting compounds.In the next step, treatment of acids 18-20 with PCl 3 afforded the unstable acid chlorides 21-23, which were used in subsequent reactions as crude products.In the synthesis of 1-methoxybrassenin B (4a), we examined an approach via the acyl isothiocyanate used in the preparation of its demethoxyanalog 4b. 8 Transformation of acid chloride 21 to acyl isothiocyanate 24 (Scheme 3) proceeded smoothly and the product could be isolated by column chromatography and crystallization or preferably used in the next step as a crude product.Reaction of isothiocyanate 24 with NaSH and methyl iodide afforded dithiocarbamate 25 (23% yield from acid 18) and thioester 26 (25% yield from acid 18) as a side product.Methylation of 25 afforded 1-methoxybrassenin B (4a, 81%).The efficiency of this method was low, because the important intermediate 25 was formed only in 23% yield.Finally, the target phytoalexin 4a was advantageously prepared in 47% yield by the acylation of dimethyl carbonimidodithioate hydroiodide 17 with acid chloride 21 in pyridine.This reaction was previously used in the case of pyridine-4-carbonyl chloride 18 and a protected derivative of glucopyranosylindol-3-carbonyl chloride 6a .Application of this method to ribofuranosyl acid chlorides 22 and 23 afforded the α-and β-anomers of protected ribofuranosyl derivatives of 27 and 29 in 46 and 39% yield from the acids 19 and 20, respectively.The target nucleosides derived from 1-methoxybrassenin B were obtained by final deprotection.The removal of the isopropylidene group of 27 with trifluoroacetic acid and subsequent treatment of 5'-O-acetyl derivative 28 with sodium methoxide afforded 1-(α-D-ribofuranosyl)brassenin B (6a).The deprotection of peracetylated compound 29 with catalytic amount of sodium methoxide in methanol smoothly afforded target compound 6b.The structures of all nucleoside analogs were confirmed by spectral methods.In their 1 H and 13 C NMR spectra, the signals were assigned on the basis of 2D COSY and hetero-correlated HSQC spectra.The structures of α-and β-ribofuranosylindole 11a and 11b were determined by NOE experiments depicted on Figure 1 and 2. Irradiation of H-1´ signal enhanced the signal of H-2´ (8.7%) for 11a and signal H-4´for 11b (2.0%), in agreement with their respective α-and βconfigurations.The observed NOE between H-1´and H-2´, H-3´ (Figure 3) in the 1-(5-O-acetylα-D-ribofuranosylbrassenin B (28) confirmed its α-anomeric structure.The β-anomeric configuration of 1-(β-D-ribofuranosyl)brassenin B (6b) was confirmed by interaction between H-1´ and H-4´ (Figure 4).The conformation of the aglycone relative to the sugar moiety was suggested on the basis of strong interaction between H-1´ and H-7 as well as H-1´ and H-2 signals (Figure 1-4).It was our final goal to obtain information about antiproliferative activities of the synthesized compounds and compare them with phytoalexin aglycons as well as previously prepared 1-(β-Dglucopyranosyl) derivatives 5a and 5b.The antiproliferative activities were examined towards Jurkat cells (acute T-lymphoblastic leukemia), CEM (acute T-lymphoblastic leukemia), CEM-VCR (acute T-lymphoblastic leukemia, VCR-resistant), MCF-7 (mammary gland adenocarcinoma, estrogen receptor expressed), HeLa (cervix carcinoma) by an MTT (thiazolyl blue) test 19 in a culture medium containing the tested chemicals at a concentration of 10 -4 mol×L - 1 after 72 h incubation.The determined activity of compounds is given in Table 1 as the percent of living cells compared to solvent control (100%).The highest activity was found with indole phytoalexin 1-methoxybrassenin B (4a), whereas its demethoxy analog 4b was less active in all tested cell lines.Replacement of the methoxy group by a protected glycosyl moiety resulted in a small decrease of activity, with ribofuranosyl derivatives 27 and 29 being generally more active compared to glucopyranosyl derivative 5a.Nucleoside analogs 5b, 6a and 6b obtained after removal of the protecting groups almost completely lost antiproliferative activity, probably because of decreased lipophilicity.Compounds with a marked activity at 10 -4 mol×L -1 were examined at the concentrations 10 -5 -10 -9 mol×L -1 .A weak activity was found only in Jurkat cells, where compounds 4a, 4b, 5a and 29 at a concentration of 10 -6 mol×L -1 inhibited proliferation to 80-90%.Antiproliferative activity is given as the percent of living cells compared to solvent control (100%) at a concentration 10 -4 mol×L -1 after 72 h incubation.

Experimental Section
General Procedures.Melting points were determined on a Kofler micro melting point apparatus and are uncorrected.IR spectra were recorded on an IR-75 spectrometer (Zeiss Jena). 1 H and 13 C NMR spectra were measured on a Varian Gemini 2000 spectrometer using TMS as an internal standard.All new compounds were characterized by 1 H, 13 C NMR as well as 1 H-1 H and 1 H- 13 C correlation experiments.Specific optical rotations were measured on a digital polarimeter P3002 (Kruess) in a 1 dm cell and are given in 10 -1 deg×cm 2 ×g -1 ; concentration is given in g/100 ml.
Microanalyses were performed with a Perkin-Elmer, Model 2400 analyzer.The EI mass spectra were recorded on a Finigan SSQ 700 spectrometer at ionization energy of 70 eV, whereas MALDI-TOF mass spectra were measured on a MALDI IV (Shimadzu, Kratos Analytical).The samples were ionized with a N 2 -laser (λ = 337 nm).The progress of chemical reactions was monitored by thin layer chromatography, using Macherey-Nagel plates Alugram ® Sil G/UV254.Preparative column chromatography was performed on Kieselgel Merck Typ 9385 (40-63 µm).

General procedure for the preparation of acid chlorides 21-23
To a suspension of acid (0.5 mmol of 18, or 1 mmol of 19 or 20) in dry toluene (2 mL, or 8 mL) and dry acetonitrile (0.3 mL, or 1.2 mL) was added phosphorus trichloride (0.5 mmol for 18, 1 mmol for 19 and 2 mmol for 20) and the mixture was stirred at room temperature for 1 h (18 and 19) or 2.5 h (20).The resulting solution was decanted from phosphorus acid deposited on the flask walls, the flask washed with dry toluene (3 ml) and obtained solution concentrated to approximately ¼ of its original volume to remove the excess of phosphorus trichloride.The obtained solution of unstable crude product was immediately used in the next reaction.

General procedure for preparation of 1-methoxybrassenin B (4a) and its analogs 27, 29
To a stirred solution of crude acid chloride 21, 22 or 23 freshly prepared from 0.5 mmol of acid 18, or 1 mmol of 19 or 20, was added a solution of dimethyl carbonimidothioate hydroiodide (0.5 mmol for 4a and 1 mmol for 27 or 29) in pyridine (2 mL or 8 mL) and reaction mixture was stirred at room temperature for 30 min (4a), 1 h (27) and 1.5 h (29).After pouring into water (50 mL or 150 mL), the product was extracted with diethyl ether (3×20 mL or 3×75 mL), the extract washed with saturated solution NaHCO 3 (3×60 mL or 3×70 mL), dried over Na 2 SO 4 , solvent evaporated and the residue chromatographed on silica gel.ARKAT USA, Inc.

Assessment of antiproliferative activity by MTT assay
The antiproliferative activity of the synthesized compounds was examined by MTT (thiazolyl blue) test 19 using the selected human cancer cell lines.Briefly, 1×10 4 cells were plated per well in 96-well polystyrene microplates (Sarstedt, Germany) in the culture medium containing the tested chemicals at final concentrations 10 -4 -10 -9 mol×L -1 .After 72 h incubation, 10 µL of MTT (5 mg×mL -1 ) were added in each well.After additional 4 h, during which insoluble formazan was produced, 100 µl of 10% sodium dodecylsulphate were added in each well and another 12 h were allowed the formazan to be dissolved.The absorbance was measured at 540 nm using the automated MRX microplate reader (Dynatech laboratories UK).Absorbance of control wells was taken as 100%, and the results were expressed as a percent of control.

Table 1 .
Antiproliferative activity a of 1-methoxybrassenin B and its analogs