Aristolactams and Alkamides of Aristolochia gigantea

A new aristolactam, aristolactam 9-O-β-D-glucopyranosyl-(1→2)-β-D-glucoside, and two alkamides, N-cis- and N-trans-p-coumaroyl-3-O-methyldopamine, were isolated from stems of Aristolochia gigantea, together with the known compounds allantoin, E-nerolidol, β-sitosterol, (+)-kobusin, (+)-eudesmin, trans-N-feruloyltyramine, trans-N-coumaroyltyramine, trans-N-feruloyl-3-O-methyldopamine, aristolactam Ia-N-β-D-glucoside, aristolactam Ia 8-β-D-glucoside, aristolactam IIIa, and magnoflorine. Their structures were determined by spectroscopic analyses.


Introduction
The Aristolochiaceae family consists of 450 to 600 species, among which more than 200 have been at least partially studied [1]. Most of these studies have focused on a characteristic group of phenanthrenic compounds, which includes the aristolochic acids (AAs) and the aristolactams (ALs), the former of which occur mainly in species of the genus Aristolochia.
In some European countries and until recently in Brazil, Aristolochia herbs have been used in weight-loss regimens. The clinical application of aristolochic acid (AA) has been limited due to its severe nephrotoxic activity. Recent studies have revealed that AA-I can cause direct damage to renal tubular cells, and its carcinigenicity is associated with the formation of promutagenic AA-DNA adducts [2,3]. The cytotoxic potency of AL-I is higher than that of AA-I, and the cytotoxic effects of these molecules are mediated through the induction of apoptosis in a caspase 3-dependent pathway [3]. Consequently many countries have now banned the use of herbs containing AAs and ALs and the US

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Food and Drug Administration has banned the sale of all products that contain AAs and ALs in their formulations [4].
Recently, the aristolactams have received much attention due to an interesting array of biological properties, including anti-inflammatory, antiplatelet, antimycobacterial, and neuro-protective activities [5]. Naturally occurring aristolactams and several synthetic aristolactam derivatives have been shown to have potent antitumor activities against a broad array of human cancer cell lines. Several aristolactams which may possess postcoital antifertility activity have been isolated from Aristolochia indica. In addition, neurological disorders, especially Parkinson's disease, have been treated by administration of the aristolactam taliscanine [6]. Brazilian Aristolochia species, including Aristolochia gigantea, have been used in traditional medicine as abortifacients and in the treatment of wounds and skin diseases [7].
Aristolochia gigantea develops a strong system of subterranean stems and roots (tuberous or rhizomatous roots). -Phellandrene (60.9%) and linalool (16.6%) are the major constituents of the essential oil obtained from these plant parts [8], whereas germacrene D and -elemene are the most abundant compounds in the leaf oils. trans-Nerolidol and geraniol are the major constituents in the stem and flower oils, respectively [9]. Previous studies on the leaves of this plant have also led to the isolation of allantoin and sitosterol [7], which are also found in significant quantities in other Aristolochiaceae species. In addition, salsolinol, higenamine, and pinitol have been isolated together with several bisbenzylisoquinolinic and 8-benzylberberinic alkaloids from A. gigantea. These latter compounds have an unusual carbon skeleton [7,10,11]. As part of our continuing studies on the Aristolochiaceae family, we report here the isolation and structural elucidation of aristolactams and alkamides, among other compounds, from aerial and ground (rhizomes) stems of A. gigantea.
A molecular formula of C 29 H 31 O 15 N was determined for compound 10 based on its HRMS spectra, which showed quasi-molecular ions at m/z 632.1614 [M -H]  . The IR spectrum of compound 10 showed characteristic absorption bands of a lactam group at 1,654 cm −1 and hydroxyl groups at 3,442 and 1,088 cm −1 . The DEPT and 13 C-NMR spectra of 10 (Table 1)       The 1 H-and 13 C-NMR spectra of 14 + 15 (Table 2) were very similar to those of 13, except for the absence of a methoxyl group at C-3' in 13, and suggested that it consisted of cis-and trans-alkamides with p-disubstituted and trisubstituted aromatic rings. The molecular formula (C 18 H 19 O 4 N) deduced from the HRMS spectra was also consistent with the lack of an OCH 3 substituent. Based on the integration of the signals corresponding to the olefinic hydrogens [cis:  H-2 5.75 (d, J = 13.0) and  H-3 6.48 (d, J = 13.0); trans:  H-2 6.38 (d, J = 15.5) and  H-3 7.28 (d, J = 15.5)] it was possible to determine that the isolated mixture was in a 1:2 cis/trans proportion. Although cis and trans isomers can isomerize under UV light, both alkamide isomers may be natural compounds [26,27]. To assign with confidence all of the chemical shifts for carbons and hydrogens in the structures, this mixture was exposed to daylight for four hours. The subsequent 1 H-NMR spectrum revealed that the cis/trans proportion had changed to 2:1. The linkage of the methoxyl group to C-3''' was established based on the observation of a correlation between this carbon and the methoxyl hydrogens by gHMBC experiments, as well as by the spatial interactions of methoxyl hydrogens with H-2''', as observed by 1D-NOESY experiments (Figure 3). Correlations observed by gHMBC experiments also assisted to determine the carbon skeleton. These alkamides 14 and 15 were named N-cis-and N-trans-pcoumaroyl-3-O-methyldopamine, respectively.  Allantoin (1) is a product of purine metabolism and is widely distributed in biological systems. It has been isolated from marine sponges, animals, and numerous plants, including Aristolochia species. It is used as an anti-inflammatory, antipsoriatic (disputed), and topical vulnerary agent [28]. Allantoin is one of the best-known wound-healing agents, and exerts keratolytic and astringent effects and stimulates new tissue formation [29]. Other well-known compounds that were isolated from A. gigantea include E-nerolidol, which has been shown to possess larvicidal activity against Aedes aegypti [30] and antifungal activity against Microsporum gypseum [31], and magnoflorine, which exhibits insecticidal activity against Spodoptera frugiperda [32], among others activities [33].

Plant material
The plant material was collected in Araraquara, SP, Brazil, in February, 2004, and identified as Aristolochia gigantea Mart. (Aristolochiaceae) by Dr. Lindolpho Capellari Júnior (Escola Superior de Agricultura "Luiz de Queiroz" (ESALQ), Piracicaba, SP, Brazil). A voucher specimen (ESA 88281) was deposited at the herbarium of the ESALQ, Piracicaba, SP, Brazil. The material was separated according to the plant parts and dried (ca. 45 °C). The stems were further separated into aerial stems and rhizomes.

Extraction and isolation of the chemical constituents
The rhizomes (433.6 g) and aerial stems (379.4 g) were ground and exhaustively extracted successively at room temperature with hexane, acetone, and ethanol. The residues were extracted with ethanol in a Soxhlet apparatus and the extracts were individually concentrated. A portion of the crude ethanol extract of rhizomes (8.10 g) was washed with CH 3 OH. Compound 1 (43.0 mg) was isolated from the insoluble fraction. The methanol-soluble fraction was subjected to CC (6.0 by 40.0 cm, silica gel 60H, 127.3 g, hexane/CH 3 OH gradient, 19:1 to 100% CH 3 OH) to give 25 fractions (ca. 125 mL each).

Conclusions
Extracts from different parts of A. gigantea showed a diverse chemical composition. As previously observed, the characteristic chemical constituents of the leaves of this species are bisbenzylisoquinolinic and 8-benzylberberinic alkaloids, whereas stems contain lignans at high concentrations, alkamides, and aristolactams. Among the compounds that were isolated from stems, two alkamides and an aristolactam are described here for the first time.