Synthesis and in vitro antiproliferative effect of isomeric analogs of cyclobrassinin phytoalexin possessing the 1,3-thiazino[5,6-b ]indole-4-one skeleton

Isomeric analogs of cyclobrassinin phytoalexin possessing the 1,3-thiazino[5,6-b ]indole-4-one skeleton have been prepared. Starting from 1 H -indole-2-carbonyl isothiocyanate, N -aryl- N’ -(indole-2-carbonyl)-substituted thiourea or the corresponding S -aryl- N -(indole-2-carbonyl)dithiocarbamate intermediates were prepared and then transformed by ring-closure via Hugerschoff reaction with phenyltrimethylammonium tribromide. Attempted synthesis of N ’ -(indole-2-carbonyl)- N -(3,4,5-trimethoxyphenyl)thiourea unexpectedly delivered the 2-(indole-2-carbonylamino)-5,6,7-trimethoxy-benzothiazole ring system. The structures of the new ring systems were determined by means of IR and NMR spectroscopy. The new derivatives synthesized exert moderate in vitro antiproliferative effects on a panel of adherent human cell lines (HeLa, A2780, A431 and MCF7).


Introduction
Among the possible isomers of 1,3-thiazinoindoles condensed at bond b of the indole skeleton, the most interesting derivatives are 1,3-thiazino [6,5-b]indoles.Compounds possessing this ring system, such as cyclobrassinin and their biosynthetic or synthetic precursors (brassinin), belong to the family of natural phytoalexins and were isolated from cruciferous plants, first from Chinese cabbage (Figure 1). 1 Phytoalexins are antimicrobial substances of low molecular weight produced by plants in response to infection or stress, which form part of their active defense mechanisms, probably as a result of the de novo synthesis of enzymes, as a part of the immune system of plants. 2 In addition to their antimicrobial activities, brassinin and cyclobrassinin have various other pharmacological effects.They inhibit the formation of preneoplastic mammary lesions in culture. 3They also exerts in vitro antiproliferative effect on a number of different human cell lines. 4,5As for the pharmacodynamic mechanism, brassinin and its derivatives are inhibitors of indolamine 2,3-dioxygenase, a new cancer immunosuppression target. 6Izutani et al. revealed, that brassinin induces G1 phase arrest through increase of p21 and p27 by inhibition of the phosphatidylinositol 3-kinase signalling pathway in human colon cancer cells. 7Kello et al. investegated ROSdependent antiproliferative effect of homobrassinin, a brassinin derivative, in human colorectal cancer caco2 cells. 8Lee et al. showed that brassinin inhibits STAT3 signaling pathway through modulation of PIAS-3 and SOCS-3 expression and sensitizes human lung cancer xenograft in nude mice to paclitaxel. 9Extensive investigations have been focused on the natural members of S,N-phytoalexins from antiproliferative point of view.In contrast, relatively few isomeric bioisoster indole compounds of this type were sythesized and investigated from pharmacological aspects.A series of 2-alkyl-or 2-arylimino-1,3thiazino [5,4-b]indol-4-one derivatives 10 inhibits human leukocyte elastase and α-chymotrypsin.The 4-oxo-1,3thiazino [6,5-b]indole derivatives of cyclobrassinone were prepared Kutschy et al.It turned out later, however, that the original structure of this phytoalexin was most probably misinterpreted. 11n our previous work we prepared isobrassinin (1), 2-methylthio-1,3-thiazino [5,6-b]indole (isocyclobrassinin, 2, X = S, R = Me, Figure 1) and its 2-benzylthio-analog 2 (X = S, R = Bn, Figure 1) and found them to exert good in vitro antiproliferative effects on cervical adenocarcinoma (HeLa), breast adenocarcinoma (MCF7) and squamous skin carcinoma (A431) cell lines. 5For the investigation of structure-activity relationships, further 2-aryl-1,3-thiazino [5,6-b]indole analogs were synthesized.The highest cytotoxic effect was displayed by 2-phenylimino-1,3-thiazino [5,6-b]indole 2 (X = N, R = Ph, Figure 1), which demonstrated inhibitory activity on the above three cell lines comparable to that of cisplatin. 5n order to improve the possible efficacy our attention next turned to the incorporation of indol-2carboxamide moiety (3, 4; Figure 1) into isobrassinin and isocyclobrassinin analogon compounds (1, 2; Figure 1).Motifs containing the indole-2-carboxamide moiety have the increased ability to bind to different receptors and enzymes.Compounds containing this pharmacophore, therefore, can be found in varied biologically active compounds.Certain derivatives, which target allosteric modulation of cannabnoid receptor 1 (CB1), 12 glycogene phosphorylase inhibitors, 13 neurotensin (NT) (8-13) mimetics, 14 monoamine oxidase inhibitors, 15 agonists of nociceptin/orphanin FQ (N/OFQ receptors, 16 dopa D3 receptor, 17 selective antagonists of NR2B subunit containing N-methyl-D-aspartate (NMDA) receptor, 18 can be useful in different mental and CNS disorders.Furthermore, other indole-2-carboxamide counterparts may act as antioxidants, 19 coagulation factor Xa inhibitors 20 and can have useful in different pathological conditions, such as heart failure conditions, 21 diabetes, 22 osteoporosis, 23 Chron's dishease, 24 and certain infections. 25These are also inhibitors of different enzymes (Rho kinase, 26 endothelin-converting enzyme, 27 vascular endothelial growth factor FR2 VEGFR2 tyrosine kinase, 28 topoisomerase I, 29 type 5 17β-hydroxysteroid dehydrogenase, 30 tubulin polimerization 31 ), may act as apoptosis inducers 32 and potential DNA-intercalating compounds 33 (like duocarmycin A).In addition, they exert promising and remarkable in vitro antiproliferative effects.
The key intermediate N,N'-disubstituted thioureas 9a-c were synthesized form acyl isothiocyanate 7 by its reaction with substituted aniline derivatives 8a-c in acetone in good yields (Scheme 1).According to our previous results for the preparation of our target 1,3-thiazino [5,6-b]indole-4-one derivatives, the Hugerschoff reaction was applied.The oxidative cyclization of (4-chlorophenyl)thiourea derivative 9a with phenyltrimethylammonium tribromide in dichloromethane yielded thiazinoindole 10.It turned out, however, that this compound exists as an equilibrium mixture of tautomers 10-endo and 10-exo in hexadeuterodimethyl sulfoxide under NMR measuring conditions.

Scheme 2
The structures of the synthesized compounds were verified by IR, 1 H and 13 C NMR spectroscopy including 2D-HMQC, DEPT, 1 H, 13 C and 13 C, 15 N-HMBC measurments.Spectral data are given in Tables 1 and 2.
➢ The benzene ring bearing the three methoxy substituents is penta-substituted (only a single aromatic carbon is protonated).➢ The six benzene carbons have carbon lines with different chemical shifts.➢ The substitution is asymmetric and the three methoxy groups have different 1 H and 13 C NMR signals.➢ The C-2 atom in the indole moiety is unsubstituted (the appropriate 1 H NMR singlet appears at 7.74 ppm).➢ The ring-chain tautomerism and the N-inversion around the exo sp 2 N atom result in slow motion of the electron distribution in molecules 10 and 11.Consequently, the carbon lines of S-C= and lactam C=O groups have broadened and thus in the 13 C NMR spectrum cannot be assigned in every cases.
Similarly, the H-2',6' signals in the 1 H NMR are broadened.Such broadenings are not observed in the spectra of 12.It is to be noted that in compounds 9a-c a chelate-type hydrogen bonding can form within the CO-NH-CS moiety.(A six-membered ring is formed by H-bonding between the carbonyl oxygen and the SH-hydrogen of the CO-N=C-SH tautomeric form).This assumption is supported by the following observations.
➢ The νNH IR band of this group is extremely broadened (around 2000 cm -1 ) and thus hardly observable.Such a diffuse absorption band is characteristic of chelates. 38➢ The C=S line in 13 C NMR appears at 179.5-179.7 ppm for 9a-c, outside the usual interval (173-176 ppm 39 ).➢ The electron-withdrawing or electron-releasing character of the substituents in the NH-aryl group does not have any influence on the IR frequencies and the NMR shifts of the CO-NH-CS group.This makes highly probable that these two parts of the molecule (NHAr and CONHCS) are isolated from one another.All these features are in good agreement with the presence of the stable chelate ring.Concerning the ring-chain tautomerism possible in 10-12, the difference in the shape and shift of NH signal between 10-11 and 12, respectively, is striking.In 12 the preferred structure of the aromatic benzothiazole part excludes the tautomerism and the amide NH signal is sharp and has higher shift at 12.0 ppm.In contrast, the tautomeric equilibrium of 10 and 11 results in a broad NH signal and a lower shift (11.1 ppm).The in vitro antiproliferative activities of the compounds prepared were examined on human tumor cell lines 40 HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma), A2780 (ovarian carcinoma) and A431 (squamous carcinoma) by means of MTT assay.The results are summarized in Table 3.

Experimental Section
General.Melting points were determined on a Kofler micro melting apparatus and are uncorrected.Elemental analyses were performed with a Perkin-Elmer 2400 CHNS elemental analyser.Merck Kieselgel 60F254 plates were used for TLC, and Merck Silica gel 60 (0.063-0.100) for column chromatography.The 1 H and 13 C NMR spectra were recorded in CDCl3 solution in 5 mm tubes at room temperature, on a Bruker DRX 500 spectrometer at 500 ( 1 H) and 126 ( 13 C) MHz, with the deuterium signal of the solvent as the lock and TMS as internal standard.DEPT spectra were run in a standard manner, using only the  = 135 0 pulse to separate CH/CH3 and CH2 lines phased "up" and "down", respectively.The 2D-HSQC and -HMBC spectra were obtained by using the standard Bruker pulse programs.Indole-2-carboxylic acid was purchased from Fluka.Indole-2carbonyl chloride was prepared by an earlier method. 35he antiproliferative properties of the prepared analogs were determined in vitro against a panel of human adherent cancer cell lines 40 including HeLa (cervix adenocarcinoma), MCF7 (breast adenocarcinoma), A2780 (ovarian carcinoma) and A431 (squamous carcinoma).All cell lines were purchased from the European Collection of Cell Cultures (ECCAC, Salisbury, UK).The cells were maintained in minimal essential medium (Lonza Ltd, Basel, Switzerland) supplemented with 10% foetal bovine serum, 1% non-essential amino acids and an antibiotic-antimycotic mixture.Near-confluent cancer cells were seeded onto a 96-well microplate at the density of 5000 cells/well and, after overnight standing, new medium (200 μL) containing the tested compound at 10 and 30 µM was added.After incubation for 72 h at 37 ºC in humidified air containing 5% CO2, the viability of the cells was determined by the addition of 5 mg/mL 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolium bromide (MTT) solution.During a 4-h contact period, the MTT was converted by intact mitochondrial reductase and the precipitated formazan crystals were dissolved in 100 μL DMSO.Finally, the reduced MTT was assayed at 545 nm, using a microplate reader; wells with untreated cells were utilized as controls.All in vitro experiments were carried out on two microplates with at least five parallel wells.Stock solutions of the tested substances (10 mM) were prepared in DMSO.The highest DMSO content of the medium (0.3%) did not have any substantial effect on the cell proliferation.Cisplatin (Ebewe Pharma GmbH, Unterach, Austria) was used as the reference agent.

General procedure for the preparation of N-aryl-N'-(1H-indole-2-carbonyl)thioureas (9a-c).
To a stirred solution of potassium thiocyanate (2.0 g, 20.6 mmol) in dry acetone (30 mL) a solution of freshly prepared indole-2-carbonyl chloride (3.4 g, 20,0 mmol) in acetone (10 ml) was added in one portion at room temperature.The reaction mixture was stirred for 1 h at room temperature and filtered through a sintered glass.After the evaporation of the solvent, the crystalline product was taken up in diethyl ether (2 x 10 mL) and filtered.Isothiocyanate (7) was used in the next step without any further purification.To the stirred solution of 1H-indole-2-carbonyl isothiocyanate 7 (0.5 g, 2,4 mmol) in acetone (10 mL) the appropriately substituted aniline 8a-c derivative was added portionwise.The reaction mixture was stirred at room temperature for 1 h (9c precipitated as white crystalline product) and then concentrated.The crystalline residues were triturated with acetone (9a-c), filtered off and purified as indicated.General procedure for the preparation of 4,5-dihydro-1,3-thiazino[5,6-b]indole-4-one derivatives (10, 11) and benzothiazole (12).To an intensively stirred suspension of thiourea derivatives 9a-c (0.7 mmol) in dichloromethane (20 mL), phenyltrimethylammonium tribromide (0.26 g, 0.69 mmol) was added in one portion at room temperature.After stirring for 5 min, triethylamine (0.39 mL, 2. 8 mmol) was added in one portion to the clear solution.The solvent was evaporated (water bath <50 °C) and the residue was purified by column chromatography, using n-hexane/ethyl acetate 3:2 as an eluent, to give 10-12 as crystalline powders.(15).To a stirred solution of acyl isothiocyanate 7 (0.5 g, 2,4 mmol) in acetone (10 mL) thiophenol 13 derivative was added portionwise.The reaction mixture was stirred at room temperature for 1 h and then concentrated.

Table 1 .
Characteristic IR frequencies a and 1 H NMR data b of compounds