Alkaloids and Flavone Acyl Glycosides from Acanthus arboreus

O estudo fitoquímico de Acanthus arboreus resultou no isolamento de 3 novos alcalóides: 6hidróxi-2-benzoxazolinona, 4-hidróxi-acantamina (3,4-di-hidróxi-1,4-benzoxazino-2-ona) e acantaminosídeo (3-O-glicopiranosídeo-1,4-benzoxazino-2-ona). Além destes alcalóides foi também isolado o novo flavonóide 7-O-β-D(6”-trans-p-cumaroil)3”-O-acetilglicopiranosídeo apigenina e os seguintes compostos de estruturas já conhecidas: apigenina, 7-O-β-D(6”-trans-p-cumaroil)3”-Oglicopiranosídeo apigenina, ácido vanílico, lupeol, estigmasterol e 3-β-glicopiranosídeo sitosterol. As estruturas dos compostos foram determinadas por métodos espectroscópicos e transformações químicas.


Introduction
3][4][5] A. ilicifolius is used as anticonvulsant, hypnotic and skeletal muscle relaxant due to the presence of benzoxazolinone; an alkaloid with CNS depressant activity. 6,7enzoxazolinone also exhibited antiprotozoal activity against Leishmania donovani in vitro; while its ribose derivatives were active as anticancer and antiviral agents. 5,8

Results and Discussion
The HRCI-MS of 1 showed an M + +1 at m/z 579.151 for the molecular formula C 30 H 26 O 12 .Physical and spectral data of 1 were identical with those reported for apigenin O-β-D-(6"-trans-p-coumaroyl)-glucoside isolated from Pogostemon cablin. 9However, Singh et al in 1986 reported the same compound as a novel product from Echinops echinatus under the name echinacin. 10Based on COSY and HMQC experiments, complete assignments for the sugar protons and all carbons were achieved.Our assignments were in complete agreement with first publication. 9However, some of the 13 C-NMR assignments in the latter publication 10 must be revised.
The HRCI-MS (M + +1 at m/z 621.161 for the molecular formula C 32 H 28 O 13 ) and other spectral data of 4 (see experimental) indicated an additional acetyl group compared with 1. Complete assignments of the sugar protons could be achieved by a combination of COSY and HMQC experiments.A major difference was observed in the chemical shift of H-3″ (δ 4.97, t) compared to that of 1 (δ 3.30, m), indicating that the acetyl group is located at C-3″.Further evidence for the position of the acetyl group was obtained from the 13 C-NMR, where a substantial downfield shift ∆ δC-3" = 1.20 ppm and the upfield shift of C-2″ and C-4″ (∆ δC-2" =1.66 ppm and (∆ δC-4" = 1.67 ppm) relative to those of 1 were observed. 11A literature search revealed that 4 (apigenin 7-O-β-D(6″-transp-coumaroyl)3″-O-acetylglucopyranoside) is a previously unreported natural compound.
The new alkaloid 2 gave positive reactions with FeCl 3 , Lassaigne and ninhydrin reagents indicating its phenolic nitrogenous nature.HREI-MS (experimental) showed an M + at m/z 151.027 for the molecular formula C 7 H 5 O 3 N.It also showed characteristic mass fragments at m/z 105, 95, 91 and 77, suggesting the presence of 2-benzoxazolinone. 12n the 13 C-NMR spectrum (Table 1) the seven carbons were resolved as three aromatic methines (δ 99.1, 111.0 and 111.6) assigned to C-7, C-5 and C-4 respectively, three quanternary aromatic carbons (δ 129.9, 146.1 and 154.8) attributed to C-9, C-8 and C-6 respectively, and a fourth quaternary carbon at δ 157.7 assigned to the lactone carbonyl.The 1 H-NMR spectrum (Table 1) showed an ABX system at δ 6.68 (1H, d, J 2.3 Hz), 6.85 (1H, d, J 8.5 Hz) and 6.60 (1H, dd, J 2.3, 8.5 Hz) indicating a trisubstituted aromatic system.Compound 2 was identified as the demethyl derivative of 6-methoxy-benzoxazolinone i.e. 6-hydroxy-benzoxazolinone.Even the known 6-methoxybenzoxazolinone is a rare natural product isolated only once from Corn plants. 13hemical reactions and solubility in NaOH, followed by recovery after acidification, suggested that 3 is a nitrogenous compound with a lactone function.This was confirmed by the IR absorption bands for carbonyl (1670 cm -1 ), hydroxy and/or NH groups (3325 cm -1 ).The HRCI-MS showed an M + +1 at m/z 182, and an M + at m/z 181.144 for the molecular formula C 8 H 7 O 4 N.The 13 C-NMR spectrum (Table 1) showed five methine signals and three quaternary carbons.In the 1 H-NMR spectrum the coupling pattern of the 4 aromatic protons suggested the presence of an O-disubstituted benzene ring.In the 1 H-NMR spectrum the singlet at δ 5.68, diagnostic for a proton flanked by two electronegative atoms, was assigned for H-3.This was supported by a 13 C-NMR signal which appeared at δ 93.7 and was attributed to the oxymethine carbon.The remaining three quaternary carbon absorptions at δ 129.7, 142.5 and 160.1 were assigned to C-10, C-9 and the carbonyl group respectively.The violet colour with FeCl 3 14 as well as the M + of the diacetate derivative 3a at m/z 265 indicated the presence of a hydroxylamine group.The exact positions of the carbonyl and hydroxyl groups were established from alkaline hydrolysis and by the inability of 3 to give O-aminophenol after fusion with KOH.This clearly distinct the new alkaloid 3 (3,4-dihydroxy-1,4-benzoxazine-2-one) from blepharigenin (2-hydroxy-1,4-benzoxazine-3-one) a compound with very close 1 H-NMR data. 15he HRCI-MS of the third new alkaloid 5 showed M + at m/z 327.095 for the molecular formula C 14 H 17 O 8 N. The 14 carbon signals were clear in the 13 C-NMR spectrum (Table 1), of which 8 signals were accounted for the aglycone part, while the remaining 6 carbon signals were assigned for the sugar moiety.The 1,4-benzoxazine-2-one skeleton was assigned to 5 rather than the 1,4-benzoxazine-3-one as indicated from its reaction with KOH solution and fusion test.15 The spectral data of 5 aglycone (Table 1 and Experimental) showed a close similarity to 3.However, the CI-MS and the negative reaction with FeCl 3 suggested the absence of a hydroxyl group attached to nitrogen atom.Consequently, the only possible site for glycosylation is the C-3 hydroxyl.The identity of the sugar moiety was established as glucose by spectral evidences and by TLC comparison after acid hydrolysis.On the basis of the chemical shift and coupling constant of the anomeric proton (J 1',2' 7.8 Hz), the glucosidic linkage should have the β-orientation.The identity of 5 was therefore established as 1,4-benzoxazine-2-one-3-O-glucoside.The known compounds were identified by direct comparison with reference materials (Aldrich).
Compounds 1 and 3-5 were subjected to antimicrobial testing using 10 microorganisms.Only compounds 1 and 4 were active against Bacillus subtilis with an MIC 64 and 128 µg/mL respectively.

General procedure
The CI-MS of 3a was measured on a Finnigan SSQ7000 mass spectrometer.NMR spectra were recorded on a JEOL 500 NMR instrument at 500 MHz for 1 H and 125 MHz for 13 C.Other experimental conditions were as previously described. 16

Plant material
The whole plants of Acanthus arboreus Forssk.growing wild in Wadi Dhar, Sana'a, Yemen was collected during the flowering stage in August 1998 and was identified by Prof. Nabil El-Hadidy, Department of Plant Taxonomy, Faculty of Science, Cairo University.A voucher sample (YA1) is preserved in the Department of Pharmacognosy, Faculty of Pharmacy, University of Alexandria, Egypt.
Alkaline hydrolysis of 3. A sample (5 mg) of 3 was dissolved in MeOH/3N NaOH (1:1) and heated for fifteen minutes on water bath.The resulting solution was first extracted with EtOAc, acidified with dil.HCl and extracted with EtOAc.The EtOAc fraction, after acidification, showed a TLC spot with same R f value of material 3.
Potassium hydroxide fusion test of 3. A mixture of 3 (3 mg) and KOH (15 mg) was fused in an oil bath for 30 min.The reaction mixture was then allowed to cool, diluted with water and filtered.The filtrate was neutralized with dil.HCl and extracted with EtOAc.TLC revealed that the EtOAc extract resulting from the KOH fusion test was devoid of an ortho-aminophenol spot.
Acid hydrolysis of 5.A sample (6 mg) of 5 was dissolved in methanol (10 mL)/2N HCl (1 mL) and heated under reflux for 1 hour.After cooling, the aqueous solution was extracted with EtOAc (3x5 mL) and neutralized with 5% Na 2 CO 3 .TLC identified the sugar in the aqueous layer as glucose using CHCl 3 /MeOH (6:4) as developing system and thymol/H 2 SO 4 as spray reagent.

Table 1 .
13-and13C-NMR spectral data of compounds 2, 3 and 5 (coupling constants in Hz) a a Assignments are based on HMQC experiments; b Spectra were measured in CD 3 OD; c Spectra were measured in CD 3 OD/DMSO mixture Amer et al.J. Braz.Chem.Soc.