Phenylethylpyranone and Aristolochic Acid Derivatives from Aristolochia urupaensis

A new pyranone, (S)-2-(4-hydroxyphenylethyl)-6-methyl-2,3-dihydro-4H-pyran-4-one, with unusual carbon skeleton, and three new aristolochic acid derivatives (7-O-methylaristolochic acid F, sodium 7-O-methylaristolochate F and sodium aristolochate F) were isolated from Aristolochia urupaensis (Aristolochiaceae) stems together with 31 known compounds. The structures of the compounds were determined by spectroscopic analyses, including Fourier transform infrared (FTIR) and 1D and 2D nuclear magnetic resonance (NMR) techniques, and high-resolution mass spectrometry (HRMS).


Introduction
Aristolochia is the largest genus of the family Aristolochiaceae with about 500 species worldwide and 92 native species to Brazil. 1,2The interest in phytochemical studies on Aristolochia is due to the extensive use of its species in traditional and homeopathic medicine. 3ccording to Heirinch et al., 4 99 species of Aristolochia have been reported for medicinal uses, including for treatment of sexually transmitted diseases (STDs), gastrointestinal complaints, snakebites and poisoning, eczema and fungal skin diseases, as well as abortifacient.Many studies on Aristolochia species are linked to aristolochic acid nephropathy (AAN), a disease associated with kidney failure and upper urothelial carcinoma (UUC).Although aristolochic acids I and II (AAs) are considered to be responsible for these nephrotoxic and carcinogenic effects, 5 other aristolochic acids and aristolactams, also present in these species, may be considered as nephrotoxic agents. 6,7n continuation to previous chemical studies on plants belonging to the Aristolochiaceae family, we report the isolation and structural elucidation of 35 compounds (Figure S1 in the Supplementary Information (SI) section) from the stems of Aristolochia urupaensis Hoehne.Among them, a pyranone, an aristolochic acid and two sodium aristolochates were isolated and identified for the first time.The known compounds were identified by comparing their physical and spectroscopic data with those of authentic samples and/or data reported in the literature.

Plant material
The plant materials (stems and leaves) were collected in the city of Porto Nacional (Tocantins State, Brazil) in December 2014, and identified as Aristolochia urupaensis Hoehne by Dr Vinicius Castro Souza and MSc Joelcio Freitas.A voucher specimen (MBML 50517, 28/07/2016) was deposited at the herbarium of Museu de Biologia Prof Mello Leitão (MBML) in the city of Santa Teresa (Espírito Santo State, Brazil).The materials were separated according to the plant parts and dried (ca.45 °C).

Extraction and isolation
The stems (194.7 g) were ground and exhaustively extracted by maceration at room temperature with hexanes, acetone and ethanol (3 × ca.200 mL, 48 h, and shaken manually every 12 h for 2 min for each extraction), successively.Then, the residue was extracted with ethanol in a Soxhlet apparatus and extracts were individually concentrated.
Phenylethylpyranone and Aristolochic Acid Derivatives from Aristolochia urupaensis J. Braz.Chem.Soc.2278   342.0613).The FTIR spectrum of this compound showed characteristic absorption bands to carboxylic acid at 1699 and 2800-3500 cm -1 , and to nitro group at 1340 and 1521 cm -1 .The 1 H and 13 C NMR, HMBC and HSQC spectra (Table 1) of a mixture comprising compounds 5 + 4 (2:1) showed signals for aristolochic acid I (4) which were identified with those of authentic sample.In addition, these spectra showed signal for 14 aromatic carbons, one acyl (d C 168.5), one methylenedioxyl (d H 6.46 s, 2H, d C 103.2) and one methoxyl (d H 3.94 s, 3H, d C 56.0) groups.The 1 H NMR spectrum of 5 showed, in the aromatic region, a trisubstituted system with three mutual coupled ABX pattern signals at d 8.99 (d, 1H, J 9.0 Hz), 7.51 (dd, 1H, J 9.0, 2.4 Hz), and 7.79 (d, 1H, J 2.4 Hz), which were assigned to H-5, H-6 and H-8, respectively.In addition, two aromatic CH were observed in these spectra (d H-2 7.72 s, d C-2 111.5 and d H-9 8.47 br s, d C-9 125.5).These data and the UV absorption at 264, 308 and 380 nm are in accordance with a nitrophenanthrene structure, such as shown by aristolochic acids. 33The substituent positions on the AA structure were assigned with the help of HMBC experiments (Figure 1).These experiments showed correlations between H-2 (d H 7.72) and C-11 (d C 168.5) confirming the acyl group position in the structure, between H-9 (d H 8.47) and C-8 (d C 111.6), as well as H-5 (d H 8.99) and OCH 3 (d H 3.94) and C-7 (d C 159.3).These latter correlations are also in accordance with a methoxyl group linked to C-7 on the C ring.Thus, this new compound was determined as 7-O-methylaristolochic acid F.
The 1 H and 13 C NMR spectra of compounds 8 and 9 are very similar to those of 5.However, compounds 8 and 9 showed in their FTIR spectra absorption bands at ca. 1590 cm -1 , characteristic of carboxylate instead of carboxylic acid of AAs (1660-1710 cm -1 ). 34The spectra revealed also absorption bands that indicated the presence of NO 2 group (ca.1350 and 1540 cm -1 ), and for 9 showed absorption band for hydroxyl group at 3240 cm -1 .The HRMS spectra of compounds 8 and 9 showed peaks for protonated molecules at m/z 364.0428 and 350.0268, in accordance with the molecular formulae C 17 H 10 NO 7 Na and C 16 H 8 NO 7 Na, respectively, and suggest 8 had a methoxyl substituent, whereas 9 a hydroxyl in the nitrophenanthrene structure.In addition, the protonated molecule of compound 8 is 22 Da higher than 5. Thus, a sodium aristolochate derivative could be proposed for 8 and 9.The correlations observed by HMBC experiments between the oxygenated carbons C-4, C-11 and H-2, C-7 and H-5, as well as C-8 and H-9 corroborate with this suggestion (Figure 1).Comparison of 1 H NMR data of 5, 8 and 9 confirmed the lower values for d H-9 observed for sodium aristolochates than aristolochic acids (5: d H-9 8.47, 8: d H-9 8.24, 9: d H-9 8.37). 34Thus, compounds 8 and 9 were determined as 7-methoxy and 7-hydroxy sodium aristolochates, respectively.Moreover, an analogous acid of 9, which is known as aristolochic acid F, 33 showed NMR data considerably different from those observed for 9. (−)-(S)-2-(4-Hydroxyphenylethyl)-6-methyl-2,3-dihydro-4H-pyran-4-one(1)