A New Biphenyl from Clusia melchiorii and a New Tocotrienol from C . obdeltifolia

O extrato diclorometânico do tronco de Clusia melchiorii, após fracionamento e purificação por cromatografia em gel de sílica, produziu um novo bifenilo, 2,2-dimetil-5-hidroxi-7fenilcromeno, ao lado de outros compostos conhecidos, 2,2-dimetil-5,10-diidro-2Hbenzo[g]cromeno-5,10-diona (xiloidona), ácido betulínico, friedelina, friedelinol, eufol, sitostenona, estigmastenona e uma mistura de β-sitosterol e estigmasterol. O extrato hexânico do tronco de Clusia obdeltifolia depois de fracionado em gel de sílica produziu um novo álcool δ-tocotrienílico, 2Z, 6E, 10E-13-(6-hidroxi-2,8-dimetil-3,4-diidro-2H,2-cromenil)-2,6,10trimetil-2,6,10-tridecatrien-1-ol, ao lado dos ácidos 2E e 2Z-δ-tocotrienolóico, betulínico e betulônico, betulinaldeído, glutinol, friedelina, sitostenona e uma mistura de β-sitosterol e estigmasterol. As estruturas dos compostos isolados foram determinadas através de seus dados espectroscópicos.


Introduction
The genus Clusia belongs to Clusiaceae and comprises about 250 species that occur in tropical and subtropical regions of South and Central America.Clusia species have been used as purgative, antirheumatic, for stomach problems and to heal wounds.The literature indicated that species of this genus produce polyprenylated benzophenones, 1,2 triterpenes and sterols, 3,4 flavonoid glycosides 5 and not so often tocotrienols, 6 biphenyls 7,8 and quinones. 9iphenyls in Clusiaceae are rare and the literature related the isolation of only eight of them in this family, two from Pentaphalangium solomonse, 10 five from C. paralicola 7,8 and one from Kielmeyera coriacea. 11The presence of these compounds in plants was associated to a phytoalexin response. 12copherols and tocotrienols are lipid soluble molecules that differ in the degree of saturation of prenyl side chain.They are compounds of vitamin E group and are essential to maintain membrane integrity.These compounds protect the plant from oxygen toxicity.Tocopherols, especially α-tocopherol, are found in fruits, roots, tubers, cotyledons, stems, leaves, and flowers of higher plants, while tocotrienols are found principally in seeds and specialized cells like latex tuber. 13From our knowledge, the presence of tocotrienols in Clusiaceae is described just to Kielmeyera lathrophyton, 14 K. reticulata, 15 Garcinia kola, 16 Tovomitopsis psychotriifolia 17 and Clusia grandiflora. 6

Results and Discussion
The dichloromethane extract of C. melchiorii yielded, after repeated silica gel column chromatography, one new biphenyl, 2,2-dimethyl-5-hydroxi-7-phenylchromene, 1.In addition were identified one naphtoquinone, four triterpenes and four sterols.The structure of 1 was determined by NMR and MS experiments.Its molecular formula was deduced to be C 17 H 16 O 2 based in the M + ion peak at m/z 252 in the EIMS spectrum and in the 1 H and 13 C NMR data (Table 1).The 1 H NMR presented two multiplets at δ 7.53 and δ 7.36 integrating to 2H and 3H, respectively.These results combined with the presence of the ion m/z 77 in the EIMS spectrum indicated the presence of a phenyl group.Two broad singlets at δ 6.56 and δ 6.68 suggested the presence of two meta-coupled aromatic protons.Two doublets at δ 6.65 and δ 5.61 (J 9.9 Hz) integrating to 1H beside the singlet at δ 1.45 integrating to 6H, indicated the presence of a dimethylpyrane moiety.The 13 C NMR data confirmed the presence of a phenyl and a dimethylpyrane groups in the molecule and also indicated the presence of other six aromatic carbons being two oxygenated and two methine.COLOC cross peaks indicated a 1,3-relation between the methine carbons.Data from COLOC and NOEDIF experiments were not conclusive about the position of the dimethylpyrane ring.With these data two structures were proposed: a biphenyl with the 2,2-dimethylpyrane ring at C-3 and C-4 and one hydroxyl group at C-5 and another with the 2,2-dimethylpyrane ring at C-2 and C-3.In order to deduce the correct location of the dimethylpyrane ring, compound 1 was submitted to methylation with diazomethane yielding 2. The irradiation of methoxyl hydrogens of 2 enhanced the signal of H-4' allowing to locate unambiguously the dimethylpyrane ring at C-3 and C-4 and not at C-2 and C-3 (Figure 1).Compound 2 had been previously isolated from Mourera fluviatilis 18 along with its isomer with the dimethylpyrane ring at C-2 and C-3.The authors differentiated the two isomers using an argument based only in the analysis of 1 H chemical shifts differences for the meta-coupled aromatic hydrogens.However, the comparison of the NMR data of 2 with those of compounds obtained from M. fluviatilis demonstrated that the data of the two isomers were changed.Recently we have reported the isolation of five polyprenylated benzophenones from the trunk of C. obdeltifolia. 1 A re-examination of the hexane extract of the trunk from same plant provides, after repeated silica gel column chromatography, one new δ-tocotrienilic alcohol, 2Z, 6E, 10E-13-(6-hydroxy-2,8dimethyl-3,4-dihydro-2H,2-chromenyl)-2,6,10trimethyl-2,6,10-tridecatrien-1-ol, 3, along with the known 2Z and 2E-δ-tocotrienoloic acids, five triterpenes and three sterols.
The molecular formula of compound 3, C 27 H 40 O 3 , was determined by EIMS and by 1 H and 13 C NMR data.The IR data were consistent with a phenol moiety.In the 1 H NMR spectrum, a pair of doublets at δ 6.48 and δ 6.38 (J 2.8 Hz) of meta-coupled aromatic protons, defined a tetrasubstitution pattern for the aromatic ring.A singlet integrating to 3H at δ 2.12 suggested the presence of a benzylic methyl group.The presence of a prenyl side chain was deduced from the observation of three triplets of olefinic protons at δ 5.08, δ 5.12 and δ 5.27 and three singlets of allylic methyl groups at δ 1.57, δ 1.58 and δ 1.78.Another singlet at δ 4.11 integrating to 2H suggested the presence of a primary allylic alcohol.The analysis of hydrogen broadband decoupled (HBBD) and DEPT 135 o 13 C NMR spectra indicated the presence of five methyl groups, nine methylene carbons, five methine carbons and eight nonhydrogenated carbons.The 13 C chemical shifts were coherent with a benzopyrane moiety containing one hydroxyl and one methyl in a 1,3-substitution pattern and a prenyl side chain with three trisubstituted double bonds.These results were corroborated by fragments at m/z 177 [C 11 H 13 O 2 ] + , 58%, 3a, originated from cleavage of bond between C-2 and C-1', at m/z 137 [C 8 H 9 O 2 ] + , 99%, 3b, produced by retro Diels-Alder rearrangement involving a hydrogen transfer to the oxygen, which likely rearranges to the more stable tropylium ion and at m/z 136 [C 8 H 9 O 2 ] +• , 22%, 3c, originated from a retro Diels-Alder rearrangement of benzopyrane moiety without hydrogen transfer (Figure 1).The structural formula of 3 (Figure 1) was confirmed by comparison of its spectroscopic data with spectroscopic data of the product obtained by reduction of 2Ztocotrienoloic acid with LiAlH 4 in ethyl ether and with data of the 2E isomer.

Experimental
General procedures IR spectra were obtained with a JASCO FT-IR spectrophotometer.A VARIAN Gemini 300 spectrometer, operating at 300 MHz for 1 H and 75 MHz for 13 C in CDCl 3 with TMS as internal standard were used to obtain NMR data.EIMS was obtained with direct probe insertion at 70 eV in an HP MSD 5973 apparatus.

Plant material
Clusia melchiorii Gleason was collected in the vicinity of Mucugê at Parque Nacional da Chapada Diamantina, Bahia, Brazil, in October 1999.Clusia obdeltifolia Bittrich was collected in an area near Palmeiras at Parque Nacional da Chapada Diamantina, Bahia, Brazil, in April 1996.Voucher specimens number ALCB-038358 and ALCB-035997, respectively, have been deposited at Alexandre Leal Costa Herbarium, Instituto de Biologia, Universidade Federal da Bahia, Salvador, Brazil.
Compound 1 (8.4 mg) was further methylated with ethereal diazomethane prepared from Diazald (Aldrich Chemical Co.) in the usual procedure 26 to yield 2 (8.9 mg).The dried powdered trunk (4400 g) of C. obdeltifolia was extracted with hexane.Evaporation of solvent under reduced pressure yielded 39.2 g of extract.This extract was submitted to chromatography on silica gel column using hexane-EtOAc (0-100%) in order to give 19 fractions.