New ester and furocoumarins from the roots of Pituranthos tortuosus

Abstracts

Sete compostos foram isolados, a partir das frações solúveis em CHCl3, 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-beta-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.

ARTICLE

New ester and furocoumarins from the roots of Pituranthos tortuosus

Maged S. Abdel-Kader

Department of Pharmacognosy, College of Pharmacy, University of Alexandria, Alexandria, Egypt 21215

^{Address to correspondence}

RESUMO

Sete compostos foram isolados, a partir das frações solúveis em CHCl₃, 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-b-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.

ABSTRACT

Seven compounds were isolated from the CHCl₃ soluble fraction of the roots of Pituranthos tortuosus; in addition, mannitol was crystallized out of the total alcoholic extract. Using different spectroscopic techniques, the isolated compounds were identified as bergapten, graveolone, xanthotoxin, isopimpinellin, aesculetin dimethyl ether, stigmasterol glucoside, in addition to the new ester 4-methoxyphenylumbellate. The structure of the new ester was confirmed by total synthesis and it was found to be inactive in antimicrobial and cytotoxicity assays.

Keywords: Pituranthos tortuosus, Apiaceae, 4-methoxyphenylumbellate, furanocoumarins, mannitol, stigmasterol glucoside, synthesis

Introduction

Members of the family Apiaceae (Umbelliferae) are well known producers of furanocoumarins. In this regard, Apiaceae is in the first place followed by Rutaceae and Moraceae.¹ Furanocoumarins have several interesting biological activities, such as analgesic, antiinflammatory, antibacterial, antiviral, anticoagulant, in addition to their well known photosensitizing effect.^2-8 In Egypt, the genus Pituranthos (Deverra) is represented by two species.⁹ While five furanocoumarins were isolated from P. triradiatus, the aerial parts of P. tortuosus was found to be free from these compounds.¹⁰ This 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 ¹³C-NMR data with those in the literature¹¹.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.¹² That ion in 3 (xanthotoxin) was of very low intensity indicating C-8 oxygenation.

Compounds 2, 4 and 5 were identified as graveolone,^{13- 15} isopimpinellin¹⁶ and aesculetin dimethyl ether,¹⁷ respectively, by comparison of their data with those of the literature. However, the ¹³C-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}

Compound 6 gives a positive reaction with FeCl₃ indicating the presence of free phenolic OH group(s). In the ¹H-NMR (Table 1), theABX 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. The chemical shifts of the protons and carbons supported a 1, 2, 4-trisubstitution with two oxygenations at 2, 4 rather than 1, 3, 4-trisubstitutions.^18-21 Two other doublets (J 16.0 Hz) each integrated for 1 proton at d 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 ¹³C-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 C₁₆H₁₄O₅. Fourteen carbon signals were observed in the ¹³C-NMR spectrum (Table 1). However, two of these signals (114.18 and 122.41 ppm) correlated by an HMQC experiment to two doublets at d 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 OCH₃ (d 3.77, 54.85 ppm in ¹H- and ¹³C-NMR respectively) at C-4' was determined by a GOESY experiment where irradiation of the OCH₃ signal resulted in an enhancement in the doublet at d 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 4-methoxyphenol.

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.²² The 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.

Experimental

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 ¹H and 100.578 MHz for ¹³C. 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.

Extraction and isolation

1.2 kg of the dried roots was extracted with MeOH (6L). The methanolic extract was concentrated to 200 mL. At that point 400 mg of colourless crystals of mannitol were separated out of the solution. The concentrated methanolic solution was diluted with water to 300 mL and extracted exhaustively with hexane (700 mL), CHCl₃ (700mL) and EtOAc (500 mL). The CHCl₃ soluble fraction (3 g) was fractionated over silica gel column (200 g, 3 cm) eluting with 1% MeOH in CHCl₃, with gradual increasing of the MeOH contents and 200 mL fractions were collected. Fractions 2-4 (1% MeOH, 1.0 g) were rechromatographed over silica gel column (100 g, 2.5 cm) eluting with 25% hexane in CHCl₃, CHCl₃/ hexane and then CHCl₃/MeOH mixtures. Fraction 2 (50% hexane in CHCl₃, 150 mg) was subjected to repeated prep TLC using hexane/CHCl₃ (2: 1) as developing system (triple run) to afford 1 (8 mg), 2 (5 mg), 3 (14 mg), 4 (6 mg) and 5 (4 mg).

Fraction 5 (2% MeOH, 100 mg) was further purified over flash silica gel column (30 g, 2.5 cm) eluting with 1% MeOH in CHCl₃. Fraction 3 (7 mg) was subjected to prep TLC over RP18 and MeOH/ H₂O (7: 3) as developing system to afford 2 mg of 6.

Fraction 9 afforded 50 mg of stigmasterol glucoside after crystallization from MeOH.

Bergapten (5-Methoxypsoralen) (1). C₁₂H₈O₄, mp 191-192 °C. EIMS m/z (rel. Int.): 216 (90, M⁺). Negative CIMS m/z (rel. Int.): 217 (7, M⁺+1), 216 (10, M⁺), 201 (100, M⁺-CH₃).

Graveolone (2). C₁₄H₁₂O₄, mp 176-178 ⁰C. UV l_max/ nm (MeOH) 253, 303, 308, 329, 344. IR (film): n_max/cm^-1: 1733 (lactone C=O), 1688 (C=O), 1620, 1589, 1542, 821. ¹H-NMR (ppm,CDCl₃): d 1.49(6H, s, 2XCH₃), 2.77 (2H, s, H-2'), 6.30 (1H, d, J 9.7 Hz, H-3), 6.83 (1H, s, H-8), 7.66 (1H, d, J 9.7 Hz, H-4), 8.03 (1H, s, H-5). ¹³C-NMR (ppm, CDCl₃): d 26.93 (C-4', C-5'), 48.86 (C-2'), 80.98 (C-3'), 105.83 (C-8), 113.53 (C-6), 114.82 (C-3), 117.85 (C-10), 127.60 (C-5), 143.56 (C-4), 159.49 (C-9), 160.15 (C-7), 162.61 (C-2), 191.07 (C-1'). EIMS m/z (rel. Int.): 244 (43, M⁺), 229 (100, M⁺-CH₃), 216 (8, M⁺-CO), 201 (7, M⁺-CH₃-CO), 189 (81, M⁺-C₄H₇), 188 (40, M⁺-C₄H₈), 160 (28), 132 (12), 104 (15), 76 (37).

Xanthotoxin (8-Methoxypsoralen) (3). C₁₂H₈O₄, mp 150-151 °C. EIMS m/z (rel. Int.): 216 (100, M⁺). Negative CIMS m/z (rel. Int.):217 (16, M⁺+1), 216 (100, M⁺), 201 (6, M⁺-CH₃).

Isopimpinellin (4). C₁₃H₁₀O₅, mp 115-117 °C. EIMS m/z (rel. Int.): 246 (100, M⁺), 231 (100, M⁺-CH₃), 216 (15), 203 (17, M⁺-CH₃-CO).

Aesculetin dimethyl ether (5). C₁₁H₁₀O₄, mp 145-146 °C. EIMS m/z (rel. Int.): 206 (100, M⁺), 191 (36, M⁺-CH₃), 178 (21, M⁺-CO), 163 (44, M⁺-CH₃-CO), 135 (42), 107 (43), 79 (52), 69 (74).

4-Methoxyphenylumbellate (6). C₁₆H₁₄O₅, mp 169 °C. UV l_max/ nm (MeOH) 221, 244, 297, 337. ¹H- and ¹³C-NMR (Table 1). EIMS m/z (rel. Int.): 286 (50, M⁺), 255 (92, M⁺-OCH₃), 223 (56), 211 (55), 195 (42), 194 (57), 179 (31, M⁺-C₇H₇O), 177 (68), 171 (37), 165 (58), 163 (52, M⁺-C₇H₇O₂ ), 161 (100). CIMS m/z (rel. Int.): 287 (47, M⁺+1), 286 (21, M⁺), 269 (24), 255 (19), 213 (23), 179(27), 177 (45), 163 (100). HRCIMS m/z: 286.281 (M⁺), calculated for C₁₆H₁₄O₅ 286.282.

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 CHCl₃ and CHCl₃/MeOH mixtures. 455 mg of umbelliferone were recovered in the early fractions. Fractions 5-11 eluted with 2% MeOH in CHCl₃ afforded 516 mg of umbellic acid: UV l_max/nm (MeOH) 218, 239, 287, 323. ¹H- and ¹³C-NMR Table 1. Found: C, 60.12; H, 4.61. Calc. for C₉H₈O₄ (180.1): C, 60.00; H, 4.48.

Protection of umbellic acid to (7a). To a solution of 7 (410 mg, 2.3 mmol) in 0.75 mL DMF, imidazol (938 mg, 13.8 mmol) and tert-butyldimethylsilylchloride (TBSCl) (810 mg, 5.4 mmol) were added. The reaction mixture was stirred for 15 min under argon, quenched with NaHCO₃, and stirring was continued for 5 min. The resulted solution was extracted with 300 mL EtOAc. The organic layer was washed twice with H₂O, brine solution, then H₂O again and finally dried over Na₂SO₄. Part of the residue left after evaporation of the EtOAc (7a) was checked by ¹H-NMR and the rest was dried for the next step. UV l_max/nm (CHCl₃) 228, 241, 293, 325. ¹H-NMR Table 1. Found: C, 61.94; H, 8.79; Si, 13.91. Calc. for C₂₁H₃₆O₄Si₂ (408.6): C, 61.72; H, 8.88; Si, 13.74.

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 CHCl₃ to afford 97 mg of (6a). UV l_max/nm (MeOH) 241, 287, 296, 330. ¹H-¹³C-NMR Table 1. Found: C, 65.40; H, 8.36; Si, 10.72. Calc. for C₂₈H₄₂O₅Si₂ (514.8): C, 65.32; H, 8.22; Si, 10.91.

Deprotection of (6a) to4-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% NaHCO₃, then H₂O. 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 C₁₆H₁₄O₅ 286.282.

Received: October 8, 2001

Published on the web: November 14, 2002

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Publication Dates

• Publication in this collection
  07 Apr 2003
• Date of issue
  Jan 2003

History

• Received
  08 Oct 2001