Synthetic Studies with Pinus elliottiis ́ Rosin Derivatives . Oxidation of Maleopimaric Anhydride Methyl Ester and Trimethyl Fumaropimarate

Sonia C. Hess , Maria I. S. Farah, Silvia Y. Eguchib, and Paulo M. Imamura Departamento de Morfofisiologia/CCBS, Universidade Federal de Mato Grosso do Sul, CP 549, CEP 79070-900, Campo Grande MS, Brazil Fundação Tropical de Pesquisas e Tecnologia André Tosello, R. Latino Coelho, 1301, CEP 13087-010, Campinas SP, Brazil Instituto de Química, Universidade Estadual de Campinas, CP 6154, CEP 13083-970, Campinas SP, Brazil


Introduction
In the search for biologically active substances, we have envisioned that maleopimaric anhydride methyl ester (1) and fumaropimaric monomethyl ester (2), easily prepared through Diels-Alder reaction of abietane acids present in Pinus elliottiis' rosin 1 with maleic anhydride and fumaric acid, respectively, would be potential starting materials mainly for the synthesis of some C-17 oxygenated naturally occurring polycyclic systems such as 4, 5 and 6 2,3 (Figure 1), if a way could be found to cleave the ∆ 13 double bond in C-ring.Oxidation of 1 and 2 with KMnO 4 4,5 , RuO 4 and O 3 [6][7][8] has been already studied during the establishment of the correct stereochemistry of the Diels-Alder products.As part of our research program on the use of Pinus elliottiis' rosin as chiral synthons 9 , we prepared 1 and 2 in order to cleave the ∆ 13 double bond using different conditions.In the present paper, we describe the results of our work on the oxidative transformations of 1 and trimethyl fumaropimarate (3), from which two new compounds were isolated and characterized by spectroscopic data.Some products obtained during our investigation were submitted to biological assays and shown to be active against Staphylococcus aureus, Bacillus subtilis and Micrococcus luteus.

Results and Discussion
It is well known that the product of the ozonolysis of olefins depends on the solvent medium, i.e., protic or aprotic 10 .Zalkow 6,7 and Halbrook 8 reported that ozonolysis of 1 and 2 carried out in acetic acid leads to a mixture of many products.
Thus we decided to follow the literature suggestion 11 to carry out the ozonolysis of 1 at 0 o C in CH 2 Cl 2 solution in the presence of tetracyanoethylene (TCNE) in attempt to minimize the formation of the undesired products obtained previously by Zalkow 4,6,7 and Halbrook 8 .Although in this case the mechanism is not clear, the catalytic action of TCNE on the alcoholysis of epoxides is well known due to its π-acid and one-electron acceptor properties 12 .Carrying out the reaction under these conditions we isolated, after purification, a known epoxide 7 7 in 20% yield and the ozonide 8 in 7% yield (Scheme 1).This ozonide proved to be stable at low temperature 13 and has not been observed before; this is not surprising since there are reports in the literature concerning the isolation of many stable ozonides 14,15 .The ozonide was characterized by careful analysis of 1 H and 13 C NMR data.The hydrogen H-14 appeared at δ 3.19 as a singlet and the carbons C-13 and C-14 of ozonide appeared, respectively, at δ 112.5 and 106.6.These chemical shifts are in good agreement with those observed for the ozonide of methyl abietate (9) previously prepared in our laboratory 9 .
The desired keto-acid 13 was obtained in only moderate and the present result showed that ozonation seems to be the best way to oxidize the hindered ∆ 13 double bond of 3. The easiest ozonolysis of 3, in comparison with 1, is probably due to the less hindered α-orientation of the carbomethoxyl group at C-24.
The compounds obtained from the oxidation of 1 and 3 were evaluated for antibacterial activity by means of bioautographic tests, following the methodology previously described [23][24] using chloramphenicol as standard.Compounds 1, 2, 12 and 14 proved to be active against grampositive bacteria (Bacillus subtilis, Micrococcus luteus, Staphylococcus aureus) and were submitted to tests for the determination of the Minimal Inhibitory Concentration (MIC) following the cylinder-cup method 25 .MIC values are presented in Table 1 and as can be seen, compound 12 was the most active against the gram-positive bacteria.In contrast, Salmonella chokerauesuis, a gram-negative bacteria, was resistant to every substance tested.
Further investigation of the synthesis of some C-17 oxygenated polycyclic systems are underway in our laboratory.

Experimental
All melting points were determined on Kofler block and are uncorrected.TLC was performed on silica gel with fluorescent indicator on glass plates (Silica gel GF 254 , Merck).Column chromatography was carried out on silica gel (Silica gel 60, 0.06-0.2mm, Merck).NMR spectra were measured on Varian Gemini -300 and Bruker ACP -300 ( 1 H NMR at 300 MHz, 13 C at 75.6 MHz) instruments, in deuterated chloroform with tetramethylsilane as internal standard.IR spectra were measured on FT IR Perkin-Elmer 16 PC.Optical On the other hand, epoxide 7 was the only product isolated from the ozonolysis of 1, in the absence of tetracyanoethylene (-78 o C; CH 2 Cl 2 , 20% yield), after treatment with Me 2 S.An intractable mixture was obtained when the reaction mixture was treated with NaBH 4 or Zn/HOAc.The reaction of 1 with RuO 4 (25 o C; CH 2 Cl 2 ; NaIO 4 / RuCl 3 ) 16,17 also gave the epoxide 7 in 10% yield and did not cleave the ∆ 13 double bond to the corresponding keto-acid.
Although Zalkow 4,5 reported the oxidation of 3 with KMnO 4 in basic medium leading to a mixture of products, our protocol was carried out using recent and improved conditions described in the literature: a) 19 KMnO 4 with dibenzo- Maleopimaric anhydride methyl ester (1).Pinus elliottiis' rosin was esterified with dimethylsulfate (NaOH, Na 2 CO 3 /H 2 O, Me 2 SO 4 ).After purification by column chromatography with hexane-ethyl acetate mixture (95:5), the mixture of methyl esters was reacted with maleic anhydride according to the literature 4