Spirocyclization reactions and antiproliferative activity of indole phytoalexins 1-methoxybrassinin and its 1-substituted derivatives

The effect of the reaction temperature and the solvent on the diastereoselectivity of the spirocyclization of 1-methoxybrassinin leading to 1-methoxyspirobrassinol methyl ether was studied. 1-Acyl derivatives of 1-methoxyspirobrassinol and 1-methoxyspirobrassinol methyl ether were prepared by the bromine-mediated spirocyclization reactions of derivatives of brassinin bearing an acyl group on the indole nitrogen with water or methanol as nucleophilic agents. The cyclization of 1-acyl derivatives of brassinin afforded the trans -diastereoisomer as the major product, whereas using 1-methoxybrassinin afforded the cis - and trans -isomers in a ratio near to 1:1. Bromospirocyclization of brassinin and 1-methylbrassinin in the presence of methanol resulted in the formation of spirobrassinin and 1-methylspirobrassinin. The newly synthesized analogues of indole phytoalexins exhibited more significant antiproliferative activity against human leukemia cell lines than the natural phytoalexins.


Introduction
In 1940 Müller first proposed the phytoalexin concept. 1 Phytoalexins play a significant role in the defence response of plants.These secondary metabolities, which are synthesized de novo in response to biotic or abiotic stress, are part of the plant chemical defense mechanism. 2Indole phytoalexins produced by crucifers were first reported in 1986 by Takasugi. 3To date, 44 indole phytoalexins have been isolated from economically and dietary important plants of the family Cruciferae (syn Brassicaceae), which are cultivated worldwide (e.g.cabbage, turnip, Chinese cabbage, Japanese radish, wasabi, broccoli, rapeseed and arabidopsis). 4The 44 cruciferous phytoalexins have been divided into six groups according to simple structural features. 4A unique group of these natural products are spiroindoline structures containing a spirocyclic ring in the C-3 position [(S)-()-spirobrassinin [()-1], 5 (R)-(+)-1-methoxyspirobrassinin [(+)-2], 6 1-methoxyspirobrassinol (3) 7

Results and Discussion
First the effect of the solvent (dichloromethane, diethyl ether, diisopropyl ether, tetrahydrofuran, 1,4-dioxane) was studied on the diastereoselectivity of the bromine-induced spirocyclization reaction of 1-methoxybrassinin (5a) at rt.A distinct change in diastereoselectivity was observed upon replacing 1,4-dioxane as a solvent with dichloromethane.Bromine was used instead of 1,4-dioxane dibromide as a convenient cyclization agent.The reaction mixture was stirred for 15 min at rt and then triethylamine was added to trap the hydrogen bromide liberated during the reaction.Under these conditions a mixture of isomers containing a small excess of the trans-diastereoisomer trans-(±)-4a was obtained (Table 1, entry 1).This result highlights the influence of the solvent on the diastereoselectivity of spirocyclization.When using 1,4-dioxane it is postulated that rather than direct addition of methanol on the methoxyiminium ion A, which is favourable from both sides, a solvent molecule attacks methoxyiminium ion A from the less hindered thiazoline CH2 side with the formation of an unstable oxonium ion B. 2 Subsequently methanol attacks the oxonium ion B 2 from the sulfur side, which results in the formation of the cis-diastereoisomer cis-(±)-4b (Scheme 2).
The designations trans-and cis-diastereoisomers are used for differentiation of diastereoisomers.The trans-diastereoisomer is regarded as the one with the sulfur of thiazoline ring and methoxy group at C-2 located on the opposite sides of indoline ring, whereas the cisdiastereoisomer has the sulfur and 2-methoxy group on the same side of indoline ring.Scheme 2. The mechanism of the spirocyclization of 1-methoxybrassinin (5a) in 1,4-dioxane.
The effect of triethylamine on the diastereoselectivity of spirocyclization of 1methoxybrassinin (5a) was also determined.Performing the spirocyclization with bromine in anhydrous dichloromethane and subsequent addition of a solution of Et3N in methanol, 1methoxyspirobrassinol methyl ethers were isolated in a 39:61 ratio in favor of cisdiastereoisomer cis-(±)-4b (Table 1, entry 8).It is postulated that in this case the Et3N preferably approaches the intermediate methoxyiminium ion A from the less hindered CH2 side of thiazoline ring with the formation of an unstable triethylammonium ion analogous to that produced from 1,4-dioxane.With the aim of influencing the diastereoselectivity of the spirocyclization of 5a, the effect of temperature on the reaction was examined.Performing experiments above and below rt confirmed that temperature has a distinct effect on the diastereoselectivity of the spirocyclization of 1-methoxybrasssinin (5a).Reactions performed at low temperature led predominantly to the trans-diastereoisomer trans-(±)-4a (Table 2), whereas at rt or at 40-60 °C in 1,4-dioxane a preference for the cis-diastereoisomer cis-(±)-4b was observed.The best ratio was achieved at -70 °C in THF as solvent (  Under these conditions a mixture of trans-diastereoisomer trans-(±)-4a and cisdiastereoisomer cis-(±)-4b was obtained in an 85:15 ratio.It is postulated that at low temperature, molecules of methanol form intermolecular hydrogen bonds with the solvent used as well as with each other to create bulky associates.Such associates reacts with the methoxyiminium ion A from the less hindered thiazoline CH2 side with the preferential formation of trans-diastereoisomer trans-(±)-4a.
The ratios of diastereoisomers (±)-26a-(±)-33b were determined by the 1 H NMR spectra of the crude products after dilution with dichloromethane, washing with brine, drying and evaporation of the solvent.The ratios of diastereoisomers (±)-26a-(±)-33b were determined by integration of well separated signals corresponding to the H-2, Ha and Hb protons.Chromatographic separation of the mixture of diastereoisomers of 1-methoxycarbonylspirobrassinol afforded pure trans-(±)-28a and pure cis-diastereoisomer cis-(±)-28b as crystalline substances.trans-and cis-Diastereoisomers 30a,30b-33a,33b were separated by column chromatography.In the case of 1-acetylspirobrassinol ( 26) and 1benzoylspirobrassinol ( 27), the trans-and cis-diastereoisomers were not separable owing to isomerization during the attempted separation on silica gel.This fact was confirmed by a simple experiment.Prepared products 26 or 27 were applied to a TLC plate and the plate was developed.After waiting for one hour, the plate was turned by 90° and developed again.Detection using UV showed that from each original spot there were now two spots for the two diastereoisomers (Figure 2).The products 26 and 27 were isolated as a mixture of trans-and cis-diastereoisomers by column chromatography.It is supposed that diastereoisomers (±)-26a-(±)-26b and (±)-27a-(±)-27b isomerize at C-2 atom like the diastereoisomers of 1methoxyspirobrassinol [trans-(±)-3a, cis-(±)-3b].In the case of 1-methoxyspirobrassinol (3), isomerization was explained by facile interconversion of hemi-aminal and aminoaldehyde.The structures of individual diastereoisomers were confirmed by NMR studies, including COSY, HSQC, HMBC and NOESY experiments.The cis-diastereoisomers 7b-11b and 26b-33b exhibited in their NOESY spectra a cross peak between H-2 and Hb protons confirming their cis-configuration.The NOESY specta of structures 7a-11a and 26a-33a did not show the interactions between Hb and OH or alkoxy group, which would have confirmed their transdiastereoisomeric structure.However, interactions between H-2 and Hb were also not observed thus the structures of trans-diastereoisomer was assigned to these products.
Inspection of the 1 H NMR spectra of 7-11 and 26-33 revealed a significant difference in the chemical shifts between the H-2 protons of the trans-and cis-diastereoisomers.In all cases the δ(H-2)trans appeared at lower field compared to δ(H-2)cis (Table 6).The higher shielding of H-2 in the cis-diastereoisomers is probably caused by anisotropic shielding by the C=N double bond of the thiazoline ring.This correlation is valid for trans-and cis-diastereoisomers of 1methoxyspirobrassinol (3), 7 1-methoxyspirobrassinol methyl ether (4) 7,11 and 1-Bocspirobrassinol (29), 12 in which the diastereoisomeric structures were confirmed by NOE experiments.This consistent chemical shift difference was observed in CDCl3 or DMSO-d6.
NT not tested

Experimental Section
General.Melting points were determined on a Koffler hot-stage apparatus and are uncorrected. 1H-and 13 C-NMR spectra were measured on a Varian Mercury Plus spectrometer (400 MHz for 1 H and 100 MHz for 13 C).Chemical shifts () are reported in ppm downfield from TMS as the internal standard and the coupling constants (J) are given in Hertz.Microanalyses were performed with a Perkin-Elmer, Model 2400 analyzer.The EI mass spectra were recorded on a GS-MS Trio 1000 (Fisons Instruments) spectrometer at an ionization energy of 70 eV.IR spectra were recorded on an IR-75 spectrometer (Zeiss Jena).Flash column chromatography was performed on the Kieselgel Merck Type 9385 at 230-400 mesh.The progress of chemical reactions was monitored by thin layer chromatography (TLC), using Macherey-Nagel plates Alugram Sil G/UV254.Preparative column chromatography was performed on Kieselgel 60 Merck Type 9385 (0.040-0.063).

General procedure for the preparation of 1-acyl derivatives of indole-3-ylmethyl amine 20-22.
To a solution of NiCl26H2O (1.05 g, 4.4 mmol) in MeOH (40 mL) was added oxime (17-19; 4.0 mmol) in MeOH (30 mL) followed by NaBH4 (1.51 g, 40.0 mmol) in one portion with stirring and cooling with flowing cold water.After 5 min, MeOH in the mixture was evaporated to ¼ of its original volume and mixture was poured into a saturated solution of NH4Cl (250 mL).After extraction with CH2Cl2 for compounds 20 and 21 or EtOAc for compound 22 (1 × 150 mL, 1 × 100 mL, 2 × 50 mL), drying the extract over Na2SO4 and evaporation of the solvent, the crude amine 20-22 was obtained.The crude amine 20-22 was employed in the next reaction without purification.
Stirring was continued for 5 min and the reaction mixture was diluted with CH2Cl2 (5 mL) and washed with brine (2 × 5 mL).The organic layer was dried over anhydrous Na2SO4.The residue obtained after evaporation of the solvent was subjected to chromatography on 8 g silica gel (nhexane/Me2CO 3:1) and diastereoisomers trans-(±)-29a and cis-(±)-29b were separated.General procedure for the spirocyclization of 1-acyl derivatives of brassinin 23-25 with bromine in the presence of methanol.To a stirred solution of 1-acyl derivatives of brassinin 23-25 (0.5 mmol) in a mixture of anhydrous CH2Cl2/MeOH (3.6 mL/0.4 mL) at rt was added freshly prepared solution of Br2 (1.26 mL, 0.55 mmol).The stock solution was obtained by dissolving of 0.04 mL of bromine in 1.76 mL of anhydrous CH2Cl2.The reaction mixture was stirred for 15 min, then Et3N (0.111 g, 0.15 mL, 1.1 mmol) was added.Stirring was continued for 5 min and the reaction mixture was diluted with CH2Cl2 (25 mL) and washed with brine (2 × 25 mL).The organic layer was dried over anhydrous Na2SO4 and the residue obtained after evaporation of the solvent subjected to chromatography.
The reaction mixture was stirred for 15 min, then Et3N (0.033 g, 0.046 mL, 0.33 mmol) was added.Stirring was continued for 5 min and the mixture poured mixture into water (10 mL), the product extracted with CH2Cl2 (2 × 10 mL), the extract washed with brine (2 × 10 mL).
To a stirred solution of brassinin (5c; 0.047 g, 0.15 mmol) in a mixture of 1,4-dioxane/MeOH (1.8 mL/0.2 mL) at rt was added freshly prepared solution of DDB (1.4 mL, 0.6 mmol).The stock solution was obtained by dissolving of 0.04 mL of bromine in 1.76 mL of anhydrous of 1,4-dioxane.The reaction mixture was stirred for 15 min, then Et3N (0.121 g, 0.167 mL, 1.2 mmol) was added.Stirring was continued for 5 min and the mixture poured mixture into water (10 mL), the product extracted with CH2Cl2 (2 × 10 mL), the extract washed with brine (2 × 10 mL).The organic layer was dried over anhydrous Na2SO4 and the residue obtained after evaporation of the solvent subjected to chromatography on 5 g of silica gel (n-hexane/EtOAc 2:1).

5-Bromospirobrassinin [(±)-43].
Yield: 0.031 g (64%), pale yellow oil, Rf 0.22 (nhexane/EtOAc 2:1).The spectral data were fully identical with those of previously described product (±)-43. 37The cells were routinely maintained in RPMI 1640 medium with L-glutamine and HEPES (Jurkat, HeLa and CCR-CEM) or Dulbecco's modified Eagle's medium with Glutamax-I (MCF-7, MDA-MB-231 and A-549) supplemented with 10% fetal calf serum, penicillin (100 IU x mL -1 ) and streptomycin (100 lg x mL -1 ) (all from Invitrogen, USA), in humidified air with 5% CO2 at 37 o C. Before each cytotoxicity assay, cell viability was determined by the trypan blue exclusion method and found to be greater than 95%.Cytotoxicity assay.The antiproliferative effects of compounds were studied using the colorimetric microculture assay with the MTT endpoint. 38Briefly, 5  10 3 cells were plated per well in 96-well polystyrene microplates (Sarstedt, Germany) in 100 μL of the culture medium containing tested chemicals at final concentrations of 10 -6 -10 -4 mol  L -1 .After 72 h incubation, 10 μL of MTT (5 mg  mL -1 , Sigma-Aldrich) was added into each well.After an additional 4 h at 37 °C, during which insoluble formazan was produced, 100 μL of 10% (m/m) sodium dodecylsulfate (SDS, Sigma-Aldrich) was added into each well and another 12 h were allowed for the dissolution of formazan.The absorbance was measured at 540 nm and 630 nm reference wavelenght by the automated uQuant TM Universal Microplate Spectrophotometer (Biotek Instruments Inc., Winooski, VT USA).The blank corrected absorbance of the control wells was taken as 100% and the results were expressed as a percentage of the control.
Jurkat (human T-cell acute lymphoblastic leukemia), HeLa (human cervical adenocarcinoma) and MCF-7 (human breast adenocarcinoma, estrogen receptor-positive) were obtained from the European Collection of Cell Cultures (United Kingdom), CCRF-CEM cell line (human T-cell acute lymphoblastic leukemia) from the German Collection of Microorganisms and Cell Cultures (Braunschweig, Germany).MDA-MB-231 (human breast adenocarcinoma, estrogen receptor-negative) and A-549 cell lines (human lung adenocarcinoma) were kindly provided by Dr. M. Hajdúch (Olomouc, Czech Republic).

Table 1 .
The effect of solvent on the diastereoselectivity of the spirocyclization of the 1methoxybrassinin (5a) at room temperature a The ratios of diastereoisomers trans-(±)-4a : cis-(±)-4b were determined by integration of separate signals corresponding to H-2, Ha and Hb protons in the 1 H NMR spectrum of the crude product mixture.b Crude product.

Table 2 .
The effect of temperature on the diastereoselectivity of the spirocyclization of 1methoxybrassinin (5a) b Crude product.

Table 3 .
Spirocyclization of the 1-methoxybrassinin (5a) in the presence of the complex MeONa/15-crown-5 ether at room temperature The ratios of diastereoisomers trans-(±)-4a : cis-(±)-4b were determined by integration of separate signals corresponding to H-2, Ha and Hb protons in the 1 H NMR spectrum of the crude product mixture.
b Crude product.

Table 4 .
The effect of the bulkiness of alcohols (ethanol, isopropyl alcohol, tert-butanol, b Crude product.

Table 5 .
Ratios b Crude product.