Prenylated Coumarins , Chalcone and New Cinnamic Acid and Dihydrocinnamic Acid Derivatives from Brosimum gaudichaudii

Três novos derivados naturais dos ácidos cinâmico e diidrocinâmico foram isolados das raízes de Brosimum gaudichaudii, além de mais quatorze substâncias naturais conhecidas (dez cumarinas, uma chalcona, os dois esteróides -sitosterol e 3 -O-D-glicopiranosil-sitosterol e o triterpeno amirina). As estruturas destas substâncias foram estabelecidas com base na análise de dados espectrais, inclusive experiências de RMN 2D e espectros de massas. O ácido 4-hidroxi-3-prenilcinâmico pode ser postulado como precursor para todas substancias aromáticas. As novas substâncias naturais são derivadas do precursor das cumarinas que perdeu a possibilidade de formar o anel lactônico devido a O-metilação.

The genus Brosimum, which is traditionally employed in the photochemotherapy of psoriasis, 4,5 is known for the production of prenylated coumarins, [6][7][8] including furanocoumarins.These linear furanocoumarins, also known as psoralens (e.g. 4 and 5), are widely distributed in plants and have also been used internally and externally to promote skin pigmentation and skin tanning.Methoxysalen (xanthotoxin=8-methoxypsoralen) is used in medicine to facilitate skin repigmentation in patients affected by severe vitiligo. 9
The fact that all isolated aromatic compounds bear a prenyl-unit or prenyl-derived substituent at the carbon corresponding to the 3-position in cinnamic acid, led to the conclusion that 4-hydroxy-3-prenylcinnamic acid should be the common precursor, implicating that the prenylation should precede the formation of the lactone ring.This contrasts with some previous studies on the biosynthesis of furanocoumarins, in which the prenylation was considered to occur only after the coumarin lactone ring was formed. 19,20In the most recent study on this subject it was shown that umbelliferone in Apium graveolens is prenylated and that this prenylation is achieved via the novel mevalonate independent pathway. 19It remains to be investigated if in Brosimum the biosynthesis is different, as was suggested by the results presented here.

Experimental Section
General NMR spectra in CDCl 3 or CD 3 SOCD 3 solvents were recorded at 300, 400, 500 and 600 MHz for 1 H and 75, 100, 125 and 150 MHz for 13 C on a Varian Unit Plus 300, Bruker Avance DPX 400, Avance 500 or Avance 600 spectrometers, respectively, using TMS as int.standard or by reference to solvent signals CHCl 3 at d H 7.26 or CD 2 HSOCD 3 at d H 2.49 and 13 CDCl 3 at d C 77.00 or 13 CD 3 SO 13 CD 3 at d C 39.50; LRMS was obtained on a GS/MS-QP 5000 or HP 5989 mass spectrometer.The 13 C multiplicity was deduced by comparative analysis of the HBBD-and DEPT-13 C NMR spectra.Homonuclear 1 H connectivity was determined by 1 H-1 H-COSY spectra.Heteronuclear 1 H and 13 C connectivity was deduced by 13 C-1 H-COSY-1 J CH [spin-spin coupling of carbon and hydrogen via one bond ( 1 J CH 138.0 Hz) and 13 C-1 H-COSY-n J CH [n = 2 and 3, spin-spin interaction of carbon and hydrogen via two ( 2 J CH ) and three ( 3 J CH ) bonds, optimized for n J CH of 8.0 Hz].IR spectra with KBr plates were obtained on a FT-IR Perkin Elmer 1600/1605 spectrometer.Silica gel 60 (70-230 mesh, Merck) and neutral alumine oxide (BDH Laboratory Supplies) were used for column chromatography and silica gel 60 F 254 plates (Merck) for TLC.

Plant material
The roots of a specimen of Brosimum gaudichaudii Trécul., Moraceae family were collected in Araguari, Minas Gerais State, Brazil, in July, 1994, and compared with voucher specimen deposited at the Reserva Florestal da Companhia Vale do Rio Doce (CVRD), Espírito Santo State, Brazil.

Extraction and isolation
Air-dried and powdered root bark (1.89 kg) was successively extracted at room temperature with CH 2 Cl 2 followed by MeOH.

Peracetyl derivative 13
Natural product 13 (80.0mg) was treated with Ac 2 O (9.0 mL) and dry pyridine (1.0 mL) at room temperature.After the usual workup, the crude peracetyl derivative was chromatographed on a silica gel column eluting with increasing polarity of CH

3b-O-b-D-glucopyranosylsitosterol
1 H and 13 C NMR spectral data, including of the peracetyl derivative, in agreement with literature data. 16The natural product 3b-O-b-D-glucopyranosylsitosterol (16.0 mg) was treated with Ac 2 O (9.0 mL) and dry pyridine (1.0 mL) at room temperature.After the usual workup, the crude peracetyl derivative was chromatographed on a silica gel column eluting with increasing polarity gradient of CH 2 Cl 2 /EtOAc/MeOH to furnish the peracetyl derivative (19.3 mg), amorphous powder, mp 160-164 °C.

Table 1 )
. On the basis of the relative intensities of the singlet signals corresponding to H-5 (7/8: d H 7.13/6.74)and H-4 (9 d H 7.33) in the 1 H NMR spectrum (500 MHz) the percentages of 7 (43.9%), 8 (13.8 %) and 9 (42.3 %) in the mixture were calculated.The composition of the mixture was confirmed by GC/EIMS analysis: 7 yielded a peak at