Floral Resin of Tovomitopsis saldanhae (Guttiferae) and 7-Epi-nemorosone: Structural Revision Volker Bittrichb, Maria do Carmo E. Amaralb, Samı´sia M. F. Machadoa,c, and Anita J. Marsaiolia* a Universidade Estadual de Campinas, UNICAMP, Instituto de Quı´mica, CP 6154, CEP 13083-970 Campinas, SP, Brazil. E-mail: [email protected] b Universidade Estadual de Campinas, UNICAMP, Instituto de Biologia, CEP 13083-970 Campinas, SP, Brazil c Universidade Federal de Sergipe, UFS, Departamento de Quı´mica, CEP 49100-000, S. Cristo´ va˜o, Sergipe, Brazil * Author for correspondence and reprint requests Z. Naturforsch. 58c, 643Ð648 (2003); received January 27/April 2, 2003 The floral resin of Tovomitopsis saldanhae (Guttiferae) is composed of poliisoprenylated benzophenone and the major constituent is 7-epi-nemorosone which has now been revised. Key words: Clusia, Tovomitopsis, Guttiferae, Resin Introduction tribe Clusieae, one of the two major monophyletic groups of the subfamily Clusioideae (Gustafsson The flowers of angiosperms offer various re- et al., 2002). Clusiella, the other genus well known wards for their pollinators, the most common be- for producing flora resins, is only very distantly ing nectar and pollen. Floral resins are very un- related to the Clusieae, belonging to the subfamily common rewards, known to occur up to now only Kielmeyeroideae. In this case there is no doubt in two angiosperm families and a few genera: Eu- that its floral resins have evolved independently phorbiaceae (species of the genus Dalechampia L., from Clusia. The presence of floral resin in To- Armbruster, 1984) and Guttiferae (species of the vomitopsis, however, suggests two possibilities: genera Clusia L., Chrysochlamys Poeppig, and either the resin evolved independently in both Clusiella Planch. & Triana; Bittrich and Amaral, genera, or the flowers of the last common ancestor 1997; Hammel, 1999). One has to be aware that of Clusia and Tovomitopsis already produced flo- the physical aspect of the reward (resin) cannot be ral resin and rewards like nectar and pollen taken as a clue of the chemical composition. Thus evolved more recently in Clusia. If the first were triterpenoids are the main constituents of the Da- true, the chemical composition of the floral resin lechampia floral resin (Armbruster, 1984) and po- in both genera might be different, if the latter is lyisoprenylated benzophenones are major compo- true, the chemical composition should be rather nents of Clusia floral resins (de Oliveira et al., similar. To test these hypotheses, we proposed to 1996, 1999; Porto et al., 2000; Cuesta-Rubio et al., analyze the floral resin of Tovomitopsis. Tovomi- 2001). Not all Clusia species offer a resinous re- topsis is a genus very little investigated from a ward, alternatively some offer pollen and others chemical point of view. nectar. Thus the evolution of floral rewards within A review of previous investigations showed the genus prompted an investigation. We have that an antibacterial vitamin E derivative was looked for floral resinous rewards in other closely isolated from Tovomitopsis psychotriifolia Oerst., related genera. Recently we found a floral resin, Planch. & Triana leaves (ethanolic extract) (Setzer secreted by the stamen filaments, in Tovomitopsis et al., 1995). This species from Costa Rica belongs saldanhae Engl., a rare species from southeastern probably rather into the genus Chrysochlamys Brazil. Tovomitopsis is a small genus closely re- (Hammel, 1999) and not into Tovomitopsis. The lated to Clusia. Together with the genera Chryso- delimitation of both genera is controversial and chlamys, Dystovomita and Tovomita it forms the needs further studies. 0939Ð5075/2003/0900Ð0643 $ 06.00 ” 2003 Verlag der Zeitschrift für Naturforschung, Tübingen · http://www.znaturforsch.com · D 644 V. Bittrich et al. · Floral Resin of Tovomitopsis saldanhae (Guttiferae) and 7-Epi-nemorosone Results and Discussion 17 A flowering Tomovitopsis saldanhae specimen O O O O was located in the Atlantic rain forest in the north- 3 3 1 7 1 7 10 eastern part of Minas Gerais, Brazil. The resin 5 5 27 O from the stamens of 5 flowers were collected with O O 22 O glass rods and then put into vials containing di- 4 ethyl acetate. Solvent evaporation produced 70 mg 3 (Cuesta-Rubio, 2001) of a resinous residue. Evaluation of the composi- (de Oliveira, 1996) tion complexity and polarity by silica gel thin layer CH2N2 chromatography revealed that the mixture had one major component that was too polar to moni- tor by GC/MS. The crude mixture was therefore MeO O O OMe evaluated by 1H NMR revealing signals that were + characteristic of polyisoprenylated benzophenones O O possessing the bicyclo[3,3,1]nonanetrione moiety. O O Following the methodology previously established 4a 4b in our group (de Oliveira et al., 1996), the crude (de Oliveira, 1999) resin was methylated with diazomethane and the Fig. 2. Nemorosone methyl derivatives obtained with di- resulting methylated mixture was submitted to azomethane, 4a (revised by Cuesta-Rubio, 2001) and 4b preparative TLC. Isolation (18 mg) of the major (de Oliveira, 1999, reconfirmed). component as its methyl derivative (~ 25% of the mixture) and spectral analysis (1H and 13C NMR, MS, IR) revealed that this component was iden- in fruits (Fuller et al., 1999; Alves et al., 1999; tical to one of 7-epi-nemorosone methyl deriva- Henry et al., 1999, 1995; Delle Monache et al., tives (1a; Fig. 1), previously isolated from Clusia 1991, 1988; Rama Rao et al., 1978; Venkatswamy nemorosa (male flowers) (de Oliveira et al., 1999), et al., 1975; Krishnamurthy et al., 1981; Santos C. insignis (male) (Porto et al., 2000) and C. et al., 1998; Karanjgoakar et al., 1973), latex (Lok- renggerioides. This class of secondary metabolites vam et al., 2000) and other plant parts (Gustafson (polyisoprenylated benzophenones) is characteris- et al., 1992; Fuller et al., 1999) of several Guttiferae tic of Clusia and now of Tovomitopsis floral resin- genera (Clusia, Garcinia, Symphonia, Rheedia (= ous rewards. Its occurrence is relatively common, Garcinia s.lat.), Vismia, Hypericum and Allan- blackia). 7-Epi-nemorosone structural revision O 3 OMe O 3 O Revising the literature for the occurrence of po- 1 1 CH2N2 lyisoprenylated benzophenones we became aware 7 7 5 5 (de Oliveira, 1999) that structure 3 suggested for nemorosone by de O O O O Oliveira et al. (1996) was recently revised to struc- 1a ture 4 by Cuesta-Rubio (2001) (Fig. 2), who also 1 suggested that an analogous structural misassign- ment might have occurred in the 7-epi-nemoro- sone methyl derivative (1) but due to the lack of MeO O O O an authentic sample their suggestion remained as CH N A 2 2 a hypothesis. This prompted us to reinvestigate O O O O nemorosone and 7-epi-nemorosone methyl deriva- tives. Methylation of Clusia rosea flower resin with 2 2a diazomethane allowed the isolation of the two Fig. 1. 7-epi-nemorosone and 7-epi-nemorosone methyl nemorosone methyl derivatives I (4a) and II (4b), derivative structural revisions. respectively (Fig. 2). V. Bittrich et al. · Floral Resin of Tovomitopsis saldanhae (Guttiferae) and 7-Epi-nemorosone 645 20 21 With these data in hands the spectroscopic rein- vestigation of 7-epi-nemorosone was a must. As 1 13 31 mentioned above the H and C NMR spectra of 17 MeO the O-methyl derivative of 7-epi-nemorosone iso- O 12 2 27 11 4 32 lated from Tovomitopsis were identical to the pre- 16 10 8 30 1 7 viously reported ones. 5 O The main questions were not related to proton 9 22 33 6 O and carbon assignments which relied on C,H con- 25 nectivities using 2D NMR and the HSQC and g- HMBC pulse sequences (Fig. 4), but about the 26 substituents on carbon 1 and 5 and the relative configuration at C-7 which led us to focus on No. C H No. C H nOe experiments. 1 73.0 18 120.9 4.95 Using a non-degassed sample, NOESY 1D pulse 2 170.9 19 134.8 sequence and setting the selective pulse onto one 3 122,1 20 17.9 1.64 δ 4 193.5 21 25.7 1.66 of the geminal methyl group ( 1.48) provoked sig- 5 63.4 22 30.1 2.50 e 2.60 nal increments at the methyl group (δ 1.38, 6 41.9 2.12 e 2.18 23 119.5 5.05 1.17%), O-methyl group (δ 3.51, 0.26%), H-27 7 48.5 1.44 24 133.1 δ 8 49.5 25 18.1 1.55 ( 2.28, 2.54%) thus linking the O-methyl enol 9 209.4 26 25.6 1.67 ether moiety to the gem-dimethyl group. Setting 10 197.8 27 30.0 1.92 e 2.28 the selective pulse onto the aromatic hydrogen 11 137.0 28 4.87 δ 12 128.6 7.61 29 132.6 ( 7.61) provoked a 0.40% enhancement of the 13 127.9 7.29 30 17.8 1.55 methoxy group signal at δ 3.51 which set the ben- 14 132.1 7.42 31 26.0 1.64 zoyl moiety on carbon 1 thus sharing with the me- 15 127.9 7.29 32 27.2 1.48 16 128.6 7.61 33 23.8 1.38 thoxy and the gem-dimethyl groups the same 17 23.5 3.16 e 3.30 34 61.3 3.51 hemisphere of 2a (Fig. 1 and Fig. 4). Finally shap- ing the soft pulse in order to excite both hydrogens 1 13 Fig. 3. H and C NMR chemical shift assignments for H-22 (δ 2.50 and δ 2.60) produced 2.57% en- 2a.