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Cytotoxic Activity of Silyl- and Germyl-Substituted 4,4-Dioxo-3a,6a-Dihydrothieno[2,3−d]isoxazolines-2
Abstract
The [2+3] dipolar cycloaddition of nitrile oxides to the double C = C bonds of thiophene-1, 1-dioxides leads to formation of the fused isoxazolines-2 (1, 2). Tumor growth inhibition of these compounds strongly depends on the nature of group IV A element increasing from slightly active tert-butyl derivatives to silicon and germanium containing analogues. The products of benzonitrile oxide cycloaddition have greater cytotoxic effect than the compounds obtained from the cycloaddition reaction of 2, 5-disubstituted thiophene-1, 1-dioxides with acetonitrile oxide. Fused silyl substituted isoxazolines-2 are stronger NO-inducers than their germyl and tert-butyl analogues.
CYTOTOXIC ACTIVITY OF SILYL- AND GERMYL-SUBSTITUTED 4,4-DIOXO-3a,6a-DIHYDROTHIENO [2,3-d] ISOXAZOLINES-2 

 E. Lukevics*, P. Arsenyan, I. Shestakova, O. Zharkova, I. Kanepe, R. Mezapuke, and O. Pudova Latvian Institute of Organic Synthesis, Aizkraukles 21, Riga, LV-1006, Latvia 

 ABSTRACT The [2+3] dipolar cycloaddition of nitrile oxides to the double (3=(3 bonds of thiophene-l,l-dioides leads to formation of the fused isoxazolines-2 (1, 2). Tumor growth inhibition of these compounds strongly depends on the nature of group IV A element increasing from slightly active ert-butyl derivatives to silicon and germanium containing analogues. The products of benzonitdle oxide cycloaddition have greater cytotoic effect than the compounds obtained from he cycloaddition reaction of 2,5-disubstituted thiophene-l,l-dioxides with acetonitrile oxide. Fused silyl substituted isoxazolines-2 are stronger NO-inducers than their germyl and tert-butyl analogues. 

 INTRODUCTION The interest in silyl substituted thiophene-l,l-dioxides stems from the fact that they are useful synthetic intermediates for the preparation of various types of organic compounds by Diels-Alder cycloaddition [1], amine induced ring-opening reaction [2], or coupling of bromothiophene-l,l-dioxides with thienyl stannanes in the presence of a palladium (0) catalyst [3]. It has been shown that unsubstituted thiophene-l,l-dioxide prepared in situ is a quite reactive dipolarophile in the [2+3] cycloaddition reactions with N,o-diphenylnitrone [4], benzonitrile [4, 5] and mesitonitrile [4, 5] oxides yielding mono- and diisoxazolines-2 and N-substituted isoxazolidines. Moreover, our recent studies indicate that silyl- and germylcontaining isoxazolines have gained a great deal of attention as compounds possessing a wide spectrum of the biological properties. The vasodilating, anticoagulant and cardioprotective activity of 5-Si-(Ge)substituted isoxazolines-2 has been studied in vitro and in vivo [6, 7]. The most active isoxazoline -3-(5"-triethylgermyl-3"-isoxazolino)pyridine hydrochloride protected the heart from rhythm disturbances and lethality during ischemiareperfusion [7]. It has been shown that silylisoxazolines-2 are more potent in protection against hypoxia and corazole convulsions than germanium analogues. However, germylisoxazolines-2 are stronger tumor growth inhibitors and NO-inducers than their silicon analogue [8]. This work presents the results of cytotoxic activity for fused isoxazolines-2 bearing a group 14 element as substituent (1, 2) in function of the nature of the group 14 element. 

 M 

 e3M.CH3 N 

 -0\ R 

 O\ M 

 e3M S 

 N 

 02 

 02 2 

 1 

 MATERIALS AND METHODS CHEMISTRY Seven tert-butyl-, trimethylsilyl-, and trimethylgermyl-substituted 4,4-dioxo-3a,6adihydrothieno[2,3-d]isoxazoline-2 1 and 2 (Table 1)were prepared by the [2+3] dipolar 63 

 Vol. 7, No. 2, 2000 

 Cytotoxic Activity ofSilyl-and Germyl-Substituted 4, 4-dioxo-3a ,6a-Dihydrothieno[2, 3-d]Isoxazolines-2 

 cycloaddition of aceto- and benzonitrile oxides to 2,5-disubstituted thiophene-l,l-dioxides. Their synthesis and characterization are given in ref. [9]. 

 _,,/0\N Me3M M'Me3 

 Me3M 

 02 M, M'=C, Si, Ge; R=Me, Ph; R'=H, Me3Ge Table 1. Me3C, MeSi, Investigated dihydrothieno[2,3-d]isoxazolines-2 

 02 Me3Ge substituted 

 %, \R 4,4-dioxo-3a,6a

 Compound 

 'Type la 

 M 

 R H Me3Ge Me3Ge 

 Yield 

 4,4-dioxo-3 methyl"5-tert-butyl-3a,6adihydrothieno[2,3-d]isoxazoline-2 

 (3' C Si 

 (%) 80 

 4,4-dioxo-3-methyl-3a-trimethylgermyl-5-tert-butyl3a, 6a-d ihyd rothieno[2,3-d]isoxazoli ne-2 4,4-dioxo-3-methyl-3a-trimethyigermyl-5tri methylsilyl-3a, 6a-d hyd roth ien o[2,3d]isoxazoline-2 4,4-d io x o-3- meth y I-3a, 5- b s tri m ethy Ig e rmy I)3a, 6a-d hyd rothieno[2,3-d] isoxazo ne-2 4,4-d ioxo-3-ph en y I-5-tert-b uty I-3a, 6adihydrothieno[2,3-d]isoxazoline-2 4,4-d ioxo-3-p h e ny I-5-tri meth y si ly I-3a, 6adihydrothieno[2,3-d]isoxazoline-2 4,4-d ioxo-3-p h e n y I-5-tri methy Ig e rmy I-3a, 6adi,hydr0thieno[2,3, d]isoxazoline-2 

 lb lc 

 58 45 67 84 

 ld 2a 2b 

 Ge 

 Me3Ge 

 C Si 

 77 85 

 2c 

 Ge 

 IN VITRO CYTOTOXITY ASSAY Monolayer cells lines were cultivated for 72 h in DMEM standard medium without an indicator and antibiotics. After the ampoule was defreezed not more than four passa4ges were performed. The control cells and cells with tested substances in the range of 2-5 10 cell/mL concentration (depending on line nature)were placed on a separate 96 wells plates. Solutions containing test compounds were diluted and added in wells to give the final concentrations of 50, 25, 12.5, and 6.25 #g/mL Control cells were treated in the same manner only in the absence of test compounds. Plates were cultivated for 72 h. A quantity of survived cells was determined using crystal violet (CV) or 3-(4,5-dimethylthiazol-2-yl)-2,5diphenyltetrazolinium bromide (MTT) coloration which was assayed by multiscan spectrofotometer. The quantity of alive cells on control plate was taken in calculations for 100% [10, 11]. Concentration of NO was determined according to [10]. RESULTS AND DISCUSSION Potential cytotoxic activity of synthesized fused isoxazolines 1 and 2 was tested in vitro on four monolayer tumor cell lines: MG-22A (mouse hepatoma), HT-1080 (human fibroblastoma), B16 (mouse melanoma), Neuro 2A (mouse neiroblastoma). Concentrations providing 50% of tumor death effect were determined according to the known procedure [12] using 96 well plates. The experimental evaluation of cytotoxicity properties is presented in Table 2. A preliminary analysis of the structure-activity relationship for the cytotoxic action clearly indicates the strong influence of the MeM (M=C, Si, Ge) group in position 5 of fused isoxazolines 1 and 2. Derivatives bearing tert-butyl substituent (la,b and 2a) have a slight cytotoxic effect (> 10 #g/mL). The substitution of the tert-butyl group by trimethylsilyl or 64 

 E. Lukevics et al. 

 Metal-Based Drugs 

 A 

 oO 

 {D 

 0 

 0 

 65 

 Vol. 7, No. 2, 2000 

 Cytotoxic Activity ofSilyl-and Germyl-Substituted 4, 4-dioxo-3a ,6a-Dihydrothieno[2, 3-d]Isoxazolines-2 

 trimethylgermyl ones leads to considerable increase of cytotoxicity. It must be noted that the activity of silicon- and germanium-containing compounds (1 and l d) depends on the tumor type. 5-Trimethylsilyl-substituted fused isoxazoline le is more active than the germanium analogue l d in tests on HP-1080 and MG-22A cell lines. However, the germanium compound ld has greater cytotoxic effect on Neuro 2A and B16 cell lines than the silicon derivative lc. Comparison of the tumor growth inhibition for derivatives 1 and 2 shows a higher activity of the condensed isoxazolines 2 containing a phenyl group in position 3 with respect to 4,4-dioxo-3-methyl-3a-trimethylgermyl-5-MeM-3a,6a-dihyd rothieno[2,3d]isoxazolines l b-d. Silyl- and germyl-substituted fused isoxazolines have a medium NOinduction ability, 4,4-dioxo-3-phenyl-5-trimethylsilyl-3a,6a-dihydrothieno[2,3-d]isoxazoline-2 (2b) being the most active (250% in the MG-22A test). 

 ACKNOWLEGMENT We are grateful to Latvian Taiho Foundation for financial support. REFERENCES 1. A. R. M. Donovan, M. K. Shepherd, Tetrahedron Lett., 35 (1994)4425. 2. S. Gronowitz, A.-B. H0rnfeldt, E. Lukevics, O. Pudova, Synthesis, (1994)40. 3. G. Barbarella, L. Favaretto, G. Sotgiu, M. Zambianchi, L. Antolini, O. Pudova, A. Bongini, J. Org. Chem., 63 (1998) 5497. 4. A. Bened, R. Durand, D. Pioch, P. Geneste, J. P. Declerq, G. Germain, J. Rambaud, R. Roques, J. Org. Chem., 46 (1981) 3502. 5. F. M. Albini, P. Ceva, A. Mascherpa, E. Albini, P. Caramella, Tetrahedron, 38 (1982) 

 6. 7. 8. 

 9. 10. 11. 12. 

 3629. E. Lukevics, M. Veveris, V. Dirnens, Appl. Organomet. Chem., 11 (1997) 805. E. Lukevics, P. Arsenyan, M. Veveris, Metal Based Drugs, 5 (1998) 251. E. Lukevics, P. Arsenyan, S. Germane, I. Shestakova, Applied Organomet. Chem., 13 (1999) 795. E. Lukevics, P. Arsenyan, S. Belyakov, J. Popelis, O. Pudova, Organometallics, 18 (1999) 3187. D.J. Fast, R.C. Lynch, R.W. Leu, J. Leuckocyt. Biol., 52 (1992) 255. P.J. Freshney, Culture of Animal Cells (A Manual of Basic Technique), Wiley-Liss, New York, 1994, pp. 296-297. R. J. Riddell, R. H. Clothier, M. Fd. Balls, Chem. Toxicol., 24 (1986) 469. 

 Received: January 21, 2000 Accepted: February 1, 2000 Received in revised camera-ready format" February 2, 2000 

 66
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