New Ester and Furocoumarins from the Roots of Pituranthos tortuosus

Sete compostos foram isolados, a partir das frações solúveis em CHCl 3 , de raízes de Pituranthos tortuosos; adicionalmente manitol foi cristalizado a partir do extrato alcoólico. Usando diferentes técnicas espectroscópicas os compostos isolados foram identificados como bergaptana, graveolana, xanthotoxinona, isopimpinela, dimetoxi aesculetina, 3-O-β-glicopiranosil estigmasterol e também o novo éster umbelato de 4-metoxifenila. A estrutura do novo éster foi confirmada pela síntese completa e se mostrou inativo a testes antimicrobiais e de citotoxicidade.


Introduction
Members of the family Apiaceae (Umbelliferae) are well known producers of furanocoumarins.3][4][5][6][7][8] In Egypt, the genus Pituranthos (Deverra) is represented by two species. 9While five furanocoumarins were isolated from P. triradiatus, the aerial parts of P. tortuosus was found to be free from these compounds. 10This result intiated the study of the plant roots for the presence of furanocoumarins since they are taxonomic markers for the family.

Results and Discussions
Spectral data indicated that compounds 1 and 3 are furanocoumarins with a methoxyl group at either C-5 or C-8.The position of the methoxyl was assigned based on comparison of the 13 C-NMR data with those in the literature 11 .The negative CIMS data of 1 (bergapten) showed a base peak at m/z 201 resulting from the fission of the aromatic ether bond indicating a 1, 3, 5 oxygenation of the aromatic ring which stabilizes the resulting phenoxide anion. 12That ion in 3 (xanthotoxin) was of very low intensity indicating C-8 oxygenation.
Compounds 2, 4 and 5 were identified as graveolone, 13-15 isopimpinellin 16 and aesculetin dimethyl ether, 17 respectively, by comparison of their data with those of the literature.However, the 13 C-NMR data for the linear dihydrobenzodipyrandione; graveolone (2) is reported here for the first time.Graveolone is a compound of very limited occurrence and has only been isolated from dill and parsley. 13,16 mpound 6 gives a positive reaction with FeCl 3 indicating the presence of free phenolic OH group(s).In the 1 H-NMR (Table 1), the ABX system (6.33,dd, J 2.2, 9.0 Hz; 6.31, d, J 2.2 Hz; 7.38, d, J 9.0 Hz) was assigned for a trisubstituted aromatic system.9][20][21] Two other doublets (J 16.0 Hz) each integrated for 1 proton at δ 6.58 and 8.03 with their correlated carbons (Table 1) were assigned to trans oriented conjugated vinyl protons.The substituted aromatic system along with the vinyl protons as well as the carbonyl signal at 167.91 ppm in the 13 C-NMR (Table 1) were assigned for a 2, 4-dioxygenated cinnamate moiety.
Both EIMS (M + at m/z 286) and CIMS (M + +1 at m/z 287) (see experimental) were consistent with the molecular formula C 16 H 14 O 5 .Fourteen carbon signals were observed in the 13 C-NMR spectrum (Table 1).However, two of these signals (114.18 and 122.41 ppm) correlated by an HMQC experiment to two doublets at δ 6.92 (2H, J 8.8 Hz) and 7.03 (2H, d, J 8.8 Hz) were assigned for a p-dioxygenated benzene ring.The position of the OCH 3 (δ 3.77, 54.85 ppm in 1 H-and 13 C-NMR respectively) at C-4' was determined by a GOESY experiment where irradiation of the OCH 3 signal resulted in an enhancement in the doublet at δ 6.92 (2H, J 8.8 Hz).
The above discussion indicated that 6 is an ester of umbellic acid (2, 4-dihydroxy cinnamic acid) with 4methoxyphenol.
As final proof, 6 was obtained by total synthesis (Figure 1).The commercially available umbelliferone (Aldrich) was treated with 5% alcoholic solution of NaOH to open the lactone ring.This reaction resulted in the formation of umbellic acid (2, 4-dihydroxy trans-cinnamic acid) 7 which was protected by TBDMSCl to give 7a.Protected umbellic acid 7a was then coupled with 4-methoxyphenol to produce 6a.Deprotection of 6a gave 6. Umbellic acid is a possible precursor for umbelliferone which is the key compound in the biosynthesis of furanocoumarins. 22The isolation of compounds 1-6 from the roots of P. tortuosus indicated that its biosynthetic pathway is consistent with those in other members of the family Apiaceae.
Compound 6 was inactive when tested against A2780 (Human Ovarian cancer cell), Escherichia coli, Staphylococcus albus and Candida albicans.

General procedure
Melting points were determined using Kofler , s hot stage instrument and are uncorrected.UV spectra were determined using UV-1201 Shimadzu spectrometer.NMR spectra were recorded on a Varian Unity 400 NMR instrument at 399.951 MHz for 1 H and 100.578MHz for 13 C. MS were taken on a VG 7070 E-HF.All chemicals used in the synthesis of 6 were obtained from Aldrich Chemicals Company.

Plant material
Roots of Pituranthos tortuosus (Desf.)Benth.were collected on April 3, 2000 from Borg El-Arab, Alexandria, Egypt.The plant was identified by Dr Sanyia Kamal, Department of Botany, Faculty of Science, University of Alexandria.A voucher specimen MS-7 is deposited in the Pharmacognosy Department, Faculty of Pharmacy, University of Alexandria, Egypt.
Fraction 9 afforded 50 mg of stigmasterol glucoside after crystallization from MeOH.

Synthesis of 4-Methoxyphenylumbellate (6)
Preparation of Umbellic acid (7). 1 gm of umbelliferone (Aldrich) was dissolved in 100mL MeOH and stirred with an equal volume of 5% alcoholic KOH for 4 h.The reaction mixture was neutralized with diluted HCl and then extracted with EtOAc (500 mL).The residue left after evaporation of the solvent was purified over silica gel column (100 g, 3 cm) eluting with CHCl 3 and CHCl 3 /MeOH mixtures.455 mg of umbelliferone were recovered in the early fractions.Esterification of (7a) to (6a).To a solution of 7a (165 mg, 0.49 mmol) in dry toluene, ECDI (141 mg, 0.74 mmol) and DMAP (90 mg, 0.74 mmol) were added.After stirring for 10 min, a solution of 92 mg 4-methoxyphenol (0.74 mmol) in dry toluene was injected into the reaction solution and kept overnight at 55 °C with stirring.The product of the reaction was purified by silica gel column (30 g, 2 cm) eluted with CHCl 3 to afford 97 mg of (6a).UV λ max /nm (MeOH) 241, 287, 296, 330. 1  Deprotection of (6a) to 4-Methoxyphenylumbellate (6).The protected ester (6a)(100 mg) was dissolved in dry THF, 2 mL of THF/pyridine were added and the solution was stirred at room temperature for 10 min.The reaction mixture was extracted with 5% NaHCO 3 , then H 2 O.The organic layer after evaporation and prep TLC on silica gel afforded 45 mg (6), which was identical with the isolated natural compound.HRCIMS m/z: 286.280 (M + ), calculated for C 16 H 14 O 5 286.282.

Table 1 .
1H-a and 13 C-NMR data for compounds 6

, 6a, 7 and 7a
a J values in Hz; b Spectra were measured in CDCl 3 ; c Spectra were measured in CD 3 OD.Abdel-Kader J. Braz.Chem.Soc.