Third International Electronic Conference on Synthetic Organic Chemistry (ECSOC-3), www.reprints.net/ecsoc-3.htm, September 1-30, 1999

[c0007]

Steroidal Tetraoxanes - New Class of Antitumor Compounds

Dejan Opsenica^a, Gabriela Pocsfalvi^b, Zorica Juranic^c, Bogdan Solaja^d*

^aInstitute of Chemistry, Technology and Metallurgy, Belgrade

^bCentro Internazionale di Servizi di Spettrometria di Massa del C.N.R, Naples

^cInstitute for Oncology and Radiology of Serbia, Belgrade

^dFaculty of Chemistry, University of Belgrade, Studentski trg 16, P.O. Box 158, YU-11001 Belgrade, Yugoslavia

E-mail: [email protected]

Received: 29 July 1997 / Uploaded: 4 August 1997

Abstract: Cholic acid derived steroidal tetraoxanes possessing methyl ester, carboxylic acid, primary and secondary amide termini were synthesised and their in vitro antitumor activity against Fem-X and HeLa cell cultures was determined. IC₅₀ range between 3.1 and 166 M. Five out of seven tested compounds exhibited cell rounding and / or apoptic activity. Their liquid secondary ionization (LSI) and electronspray ionization (ESI) spectra confirmed the tetraoxane structure.

Keywords: Steroids, cholic acid, tetraoxanes, LSIMS, ESI, antitumor activity.

• Introduction
  
• Scheme
  
• Results and Discussion
  
• Figures
  
• Tables
  
• References

Introduction

1,2,4,5-Tetraoxanes are a class of compounds usually synthesised for ring-enlargement purposes [1], and more recently, for their established antimalarial activity [2]. Activity of 1,2,4,5-tetraoxanes against Plasmodium falciparum D6 and W2 clones is very similar to that of trioxanes such as naturally occurring artemisinin, its semi-synthetic derivatives [3] and related compounds [4]. Cholestane based steroidal tetraoxanes were prepared [5] in order to explore the influence of bulky substituent(s) on 1,2,4,5-tetraoxane ring on their activity, as well as the possible influence of natural carrier. So far no report on tetraoxane antitumor activity has been published.

Here, we present the results of our further studies on steroidal tetraoxanes, the synthesis, (preliminary) structure determination and in vitro antitumor activity of some of cholic acid derived tetraoxanes [6].

Scheme 1

Results and Discussion

Tetraoxanes 2a (15%, 250-251 ^oC) and 3a (13%, mp 167-171 ^oC) were synthesised from parent ketone 1a (X = OCH₃) using procedure given in [5], Scheme 1. The acids 2d (56%, mp 206-210 ^oC) and 3d (47 %, mp 189-192 ^oC) were obtained by alkali hydrolysis of corresponding esters. Tetraoxanes 2b (26%, mp 211-217 ^oC), 3b (24%, mp 196-199 ^oC) and 3c (20%, mp 171-174 ^oC) were prepared using (TMSO)₂ / TMSOTf method of Jefford et al. [7]. In each case (except hydrolysis reaction) the complex mixture of products was obtained, from which the given tetraoxanes were obtained by chromatography on SiO₂ column. Two isomeric methyl esters (2a and 3a, hence two acids 2d and 3d) were isolated, as well as a pair of primary amides (2b and 3b). Propyl amide 1c afforded only tetraoxane 3c. The stereochemistry of steroid substituents at 1,2,4,5-tetraoxane ring is not determined as yet (lowest energy conformations of the pair of stereoisomers is shown in Figure 1 (X = OCH₃)). The ¹H and ¹³C NMR spectra (vide infra) of each pair are almost identical (at the level of 200 MHz spectrometer), but clear distinction between tetraoxanes within each pair can be made based on the appearance of corresponding acetate methyl groups (compare ¹H NMR spectra, Figure 2). In one series, the acetate methyls appear as broad singlet (at ca. 2.10 ppm), in the another, as broad singlet (at ca. 2.12 ppm) followed by another smaller broad singlet at 2.07 ppm. Tetraoxane 3c belongs to the second group. Liquid secondary ionization and electrospray ionization mass spectra of the above compounds resulted in molecular ion peaks in all of the cases. These experiments along with the LSIMS high resolution probe accurate mass measurements confirmed that we are dealing with tetraoxanes and not with hexaoxanes [8], Table 1.

The effect of tetraoxanes 2a-3d on Fem-x and HeLa cell survival, 72 h after the agent's action is given in Table 2. Investigated compounds expressed the dose dependent antiproliferative action toward investigated cell lines. In order to compare the extent of the antiproliferative action between the members of this group, IC₅₀ were determined under exactly the same conditions. They were similar for Fem-X and HeLa cells with exemption for 3c. This compound inhibited selectively more melanoma then HeLa cell growth. Morphological examination of target cells on inverted microscope showed that cytotoxic action of 3a, 2b, 3b, 2d and 3d resulted in target cell rounding and / or cell fragmentation to apoptic bodies, typical for action of known cytostatic cis-diamminedichloroplatinum(II), an apoptosis inducing agent. Tetraoxane 3c inhibited HeLa cell growth without the signs of direct cell cytotoxicity up to 66 M.

Figure 1. Computer generated structures of two possible diastereomeric tetraoxanes (X = OCH₃)

Figure 2. ¹H and ¹³C NMR spectra of 2a and 3a, respectively.

2a 1H NMR	2a 13C NMR	3a 1H NMR	3a 13C NMR

Table 2. IC50 (M) for the antiproliferative action of tetraoxanes 2a-3d on Fem-X and HeLa cells, determined 72 h after continuous agents' action, by MTT test
	IC50 (M) 72h
Compound	Fem-X	HeLa
2a	>29	>29
3a	19.5	20.5
2b	3.1	3.7
3b	6.2	6.0
3c	94	166
2d	4.2	5.2
3d	7.6	9.2

References and Notes

[1] a) P. R. Story, D. D. Denson, C. E. Bishop, B. C. Clark Jr., J-C. Farine, J. Am. Chem. Soc., 1968, 90, 817. b) J. R. Sanderson, P. R. Story, K. Paul, J. Org. Chem., 1975, 40, 691.
[2] J. L. Vennerstrom, H-N. Fu, W. Y. Ellis, A. L. Ager Jr., J. K. Wood, S. L. Andersen, L. Gerena,W. K. Milhous, J. Med. Chem., 1992, 35, 3023. b) Y. Dong, H. Matile, J. Chollet, R. Kaminsky, J. K. Wood, J. L. Vennerstrom, J. Med. Chem., 1999, 42, 1477. c) K. Tsuchiya, Y. Hamada, A. Masuyama, M. Nojima, Tetrahedron Lett., 1999, 40, 4077..
[3] Selected references: a) J. N. Cumming, D. Wang, S. B. Park, T. A. Shapiro, G. H. Posner, J. Med. Chem., 1998, 41, 952. b) P. M. O'Neill, N. L. Searie, K. J. Raynes, J. L. Maggs, S. A. Ward, R. C. Storr, B. K. Park, G. H. Posner, Tetrahedron Lett., 1998, 39, 6065. c) T. T. Thanh Nga, C. Menage, J-P. Beque, D. Bonnet-Delpon, J-C. Gantier, B. Pradines, J-C. Doury, T. Dinh Thac, J. Med. Chem., 1998, 41, 4101. d) G. H. Posner, M. H. Parker, J. Northorp, J. S. Elias, P. Ploypradith, S. Xie, T. A. Shapiro, J .Med. Chem., 1999, 42, 300.
[4] a) Y. Takaya, K-i. Kurumada, Y. Takeuji, H-S. Kim, Y. Shibata, N. Ikemoto, Y. Wataya, Y. Oshima, Tetrahedron Lett., 1998, 39, 1361. b) G. H. Posner, H. O' Dowd, P. di Ploypradith, J. N. Cumming, S. Xie,T. A. Shapiro, J. Med. Chem., 1998, 41, 2164. c) M. D' Ambrosio, A. Guerriero, E. Deharo, C. Debitus, V. Munoz, F. Pietra, Helv. Chim. Acta, 1998, 81, 1285.
[5] N.M. Todorovic, M. Stefanovic, B. Tinant, J-P. Declercq, M.T. Makler, B.A. Solaja, Steroids, 1996, 61, 688-696.
[6] The antimalarial activity of cholic acid-derived tetraoxanes will be published soon.
[7] C. W. Jefford, A. Jaber, J. Boukouvalas, Synthesis, 1998, 391.
[8] For problems arising with mass determination of tetraoxanes and corresponding hexaoxanes see Y. Dong, J. L. Vennerstrom, J. Org. Chem., 1998, 63, 8582.

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