Phenylethylpyranone and Aristolochic Acid Derivatives from Aristolochia urupaensis

Abstract

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).

Keywords:
Aristolochia urupaensis; Aristolochiaceae; sodium aristolochate; phenylethylpyranone

Introduction

Aristolochia is the largest genus of the family Aristolochiaceae with about 500 species worldwide and 92 native species to Brazil.^1,2 The interest in phytochemical studies on Aristolochia is due to the extensive use of its species in traditional and homeopathic medicine.³ According 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.^6,7

In 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.

Experimental

General experimental procedures

One-dimensional (¹H, ¹³C and TOCSY (total correlation spectroscopy)) and two-dimensional (¹H-¹H COSY (homonuclear correlation), HSQC (heteronuclear single-quantum correlation) and HMBC (heteronuclear multiple bond correlation) NMR (nuclear magnetic resonance) experiments were performed on a Bruker Avance III 600 spectrometer (14.1 T) at 600 MHz (¹H) and 151 MHz (¹³C), using deuterated solvents (CDCl₃ and DMSO-d₆) (99.98% D) as internal standards for ¹³C NMR chemical shifts and residual solvent as an internal standard for ¹H NMR. δ values are reported relative to TMS (tetramethylsilane). High-resolution mass spectra (HRMS) were obtained on a Q-TOF Bruker MaXis Impact^TM mass spectrometer. Fourier transform infrared (FTIR) spectra were obtained on a Bruker VERTEX 70 FTIR spectrometer using ATR (attenuated total reflectance). Optical rotations were measured on a PerkinElmer 341-LC polarimeter. Ultraviolet (UV) absorptions were measured on a PerkinElmer UV-Vis Lambda 1050. Circular dichroism (CD) spectra were recorded on a JASCO J-815 spectrometer, using 1.0 mm cell. High performance liquid chromatography (HPLC) analyses were performed using a Jasco LC-NetII/ADC, equipped with photodiode array (MD-2018 Plus) and CD (2095 Plus) detectors. A Zorbax RX C18 (5 µm, 250 × 9.4 mm, Agilent) and Microsorb 100 Å Phenyl (5 µm, 250 × 4.6 mm, Agilent) columns were used for semi-preparative analysis. Solvents were HPLC grade from Mallinckrodt. Ultrapure water was obtained from Direct-Q3 UV System from Millipore.

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.

The crude ethanol extract (3.5 g) was washed with methanol. The insoluble fraction gave 33 (530.0 mg). The soluble fraction was concentrated (3.0 g) and subjected to the column chromatography (CC) (C18, 18.7 × 3.0 cm, H₂O-MeOH gradient, 9:1 to 100% MeOH) to give 11 fractions (ca. 100 mL each; Fr1-Fr11). Fr6 gave 2 (21.7 mg). Fr2, Fr4, Fr5, Fr9 and Fr10 were subjected to C18 prep-HPLC by using different H₂O-MeOH gradients for further separation. Fr2 gave 25 + 34 (2.0 mg), 26 (0.9 mg) and 34 (1.0 mg); Fr4 gave 10 (0.3 mg), 21 (0.5 mg), 27+30 (0.5 mg) and 28 + 31 (0.4 mg); Fr5 gave 18 + 20 (0.4 mg), 19 (3.9 mg), 22 (0.7 mg) and 23 (0.5 mg); Fr9 gave 3 (1.4 mg), 4 + 5 (1.2 mg), 7 (0.7 mg), 8 (0.8 mg), 9 (1.1 mg) and 15 (2.5 mg); and Fr10 gave 3 (0.3 mg) and 11 (0.2 mg). The mixture 4 + 5 was subjected to HPLC by using phenyl column and eluted with H₂O-ACN 11:9 to give 5 (0.4 mg).

The crude ethanolic Soxhlet extract (7.7 g) was fractioned on Amberlite XAD-16 column eluting with H₂O (600 mL), MeOH (300 mL) and EtOAc (200 mL), successively. The MeOH portion was concentrated (1.5 g) and subjected to CC (C18, 13.0 × 1.0 cm, H₂O-MeOH gradient, 9:1 to 100% MeOH) to give 12 fractions (ca. 100 mL each; Fr1-Fr12). Fr1 and Fr9 gave 33(26.3 mg) and 3 (87.2 mg), respectively. Fr7, Fr8 and Fr10 were subjected to C18 prep-HPLC by using different H₂O-MeOH gradients for further separation. Fr7 gave 1 (1.0 mg), 2(1.0 mg), 6 (1.1 mg), 6+9 (1.0 mg), 12 + 13 (1.0 mg), 16 (0.7 mg), 24 (1.2 mg) and 29 + 32 (0.3 mg). Fr8 gave 2 (1.0 mg), 9 (0.7 mg), 14 (0.2 mg) and 16 (0.5 mg), and Fr10 gave 3 (1.0 mg), 11 (0.6 mg), 17 (0.2 mg) and 35 (0.9 mg).

(−)-(S)-2-(4-Hydroxyphenylethyl)-6-methyl-2,3-dihydro-4H-pyran-4-one (1)

Yellow amorphous powder; [α]_D²⁸ –12.9 (c 0.001, MeOH); CD (c 0.0107, MeOH) [Θ]₂₇₀ −131826, [Θ]₃₁₈−202997; UV (MeOH) λ / nm 268, 324; ATR-FTIR ν / cm^-1 3386, 1647; ¹H NMR (600 MHz, DMSO-d₆) δ 1.85 (dddd, 1H, J13.1, 9.0, 5.6, 4.4 Hz, H-1’a), 1.97 (dddd, 1H, J13.1, 9.6, 8.1, 5.9 Hz, H-1’b), 1.98 (br s, 3H, H-7), 2.31 (ddd, 1H, J16.8, 3.7, 0.7 Hz, H-3a), 2.40 (dd, 1H, J16.8, 13.2 Hz, H-3b), 2.58 (ddd, 1H, J13.1, 9.0, 5.9 Hz, H-2’a), 2.64 (ddd, 1H, J13.1, 9.6, 4.4 Hz, H-2’b), 4.32 (dddd, 1H, J13.2, 8.1, 5.6, 3.7 Hz, H-2), 5.26 (br s, 1H, H-5), 6.67 (d, 2H, J8.4, H-3’’/5’’), 7.00 (d, 2H, J8.4 Hz, H-2’’/6’’); ¹³C NMR (151 MHz, DMSO-d₆) δ 20.8 (C-7), 29.7 (C-2’), 35.8 (C-1’), 40.3 (C-3), 78.4 (C-2), 104.4 (C-5), 115.3 (C-3’’/5’’), 129.4 (C-2’’/6’’), 131.3 (C-1’’), 155.6 (C-4’’), 174.2 (C-6), 192.1 (C-4); HRMS (ESI QTOF, positive mode) m/z (rel. int.): 233.1175 [M + H]⁺ (100) (calcd. for C₁₄H₁₇O₃, 233.1178).

7-O-Methylaristolochic acid F (5)

Yellow oil; UV (MeOH) λ / nm 264, 308, 380; ATR-FTIR ν / cm^-1 1340, 1521, 1699, 3145; ¹H and ¹³C NMR data, see Table 1; HRMS (ESI QTOF, positive mode) m/z (rel. int.): 342.0610 [M + H]⁺ (45), 298.0707 [M + H − CO₂]⁺ (100) (calcd. for C₁₇H₁₂NO₇, 342.0613).

Table 1
NMR spectroscopic data for compounds 5, 8 and 9 (14.1 T, DMSO-d₆)

Position	5		8		9
	δC	δH (J in Hz)	δC	δH (J in Hz)	δC	δH (J in Hz)
1	125.7		a		125.3
2	111.5	7.72, s	113.9	7.61, s	111.1	7.67, s
3	145.9		145.7		147.4
4	145.3		143.4		147.5
4a	117.9		117.6		118.1
4b	123.2		122.3		122.0
5	128.6	8.99, d (9.0)	128.5	8.97, d (9.0)	128.8	8.92, d (9.0)
6	120.5	7.51, dd (9.0, 2.4)	119.5	7.44, dd (9.0, 3.0)	120.8	7.37, dd (9.0, 1.8)
7	159.3		158.0		157.8
8	111.6	7.79, d (2.4)	111.0	7.69, br s	114.0	7.50, d (1.8)
8a	131.1		130.5		131,2
9	125.5	8.47, br s	123.8	8.24, br s	125.2	8.37, br s
10	146.3		146.6		146.1
10a	116.1		117.3		115.9
11	168.5		168.0		168.7
OCH2O	103.2	6.46, s	101.6	6.36, s	103.0	6.44, s
OCH3	56.0	3.94, s	55.8	3.91, s

• ^a
  Signal not observed.

Sodium 7-O-methylaristolochate F (8)

Yellow amorphous powder; UV (MeOH) λ / nm 260, 308, 376; ATR-FTIR ν / cm^-1 1350, 1590; ¹H and ¹³C NMR data, see Table 1; HRMS (ESI QTOF, positive mode) m/z (rel. int.): 364.0428 [M + H]⁺ (20), 342.0608 [M + H − Na]⁺ (35) (calcd. for C₁₇H₁₁NO₇Na, 364.0428).

Sodium aristolochate F (9)

Yellow amorphous powder; UV (MeOH) λ / nm 268, 308, 376; ATR-FTIR ν / cm^-1 1360, 1542, 1591, 3240; ¹H and ¹³C NMR data, see Table 1; HRMS (ESI QTOF, positive mode) m/z (rel. int.): 350.0268 [M + H]⁺ (40) (calcd. for C₁₆H₉NO₇Na, 350.0271); HRMS (ESI QTOF, negative mode) m/z (rel. int.): 326.0315 [M − Na]⁻ (100) (calcd. for C₁₆H₈NO₇, 326.0300).

Results and Discussion

Compounds 1-35 (Figure S1, in the SI section) were isolated from the ethanolic and ethanolic Soxhlet extracts of the stems by column chromatography followed by semipreparative HPLC. The structures of the known compounds were determined by comparison of their physical and spectroscopic data with those of authentic samples and/or data reported in the literature. The known compounds were identified as aristolochic acid IIIa (2), aristolochic acid II (3), aristolochic acid I (4),⁸ sodium aristolochate IIIa (6), sodium aristolochate II (7), aristolactam IIIa N-β-glucoside (10),⁹ aristolactam II (11),¹⁰ cepharanone A N-β-glucoside (12),¹¹ aristolactam IIIa (13),¹² aristolactam AII (14),¹³ cepharadione A (15),¹⁴ tuberosinone (16),¹⁵ magnoflorine (17), trans-N-feruloyltyramine (18), trans-N-feruloyl-3-O-methyldopamine (19), cis-N-feruloyltyramine (20),¹⁶ quercetin-3-O-β-glucopyranosyl-(1→6)-β-glucopyranoside (21),¹⁷ quercetin-3-O-β-glucopyranoside (22),¹⁸ kaempferol-3-O-β-glucopyranosyl-(1→6)-β-glucopyranoside (23),¹⁹ tiliroside (24),²⁰ icariside D₂ (25),²¹ tyrosol-1-O-β-xylopyranosyl-(1→6)-O-β-glucopyranoside (26),²²trans-ferulic acid (27),²³trans-6-O-(p-coumaroyl)-glucopyranoside (28),²⁴ (E)-ethyl p-coumarate (29),²⁵cis-ferulic acid (30),²³cis-6-O-(p-coumaroyl)-glucopyranoside (31),²⁴ (Z)-ethyl p-coumarate (32),²⁵ (R)-allantoin (33),⁹ adenosine (34)²⁶ and (−)-9,9’-di-[O-(E)-feruloyl]secoisolariciresinol (35).²⁷ The flavonoids 21-23, the glycosidic phenylpropanoid 26 and the lignan 35 are being reported for the first time in the Aristolochiaceae family.

Compound 1 showed UV absorption bands at 268 and 324 nm, and IR absorption bands at 1647 and 3386 cm^-1 characteristics of α,β-unsaturated ketone and hydroxyl group, respectively. The HRMS spectrum of 1 showed peak at m/z 233.1175 [M + H]⁺ for protonated molecule, indicating the molecular formula C₁₄H₁₆O₃ (calcd. for C₁₄H₁₇O₃, 233.1178). The ¹H NMR and HSQC spectra showed signals for an aromatic ring 1,4-substituted (δ_H7.00 d, J8.4 Hz, 2H, δ_C 129.4 and δ_H 6.67 d, J8.4 Hz, 2H, δ_C 115.3), one olephinic CH (δ_H 5.26 br s; δ_C 104.4), one carbinolic CH (δ_H 4.32 dddd, J13.2, 8.1, 5.6, 3.7 Hz; δ_C 78.4) and one methyl (δ_H 1.98 br s; δ_C 20.8) groups. In addition, three non-equivalent methylenes were observed (d_H 2.40 dd, J16.8, 13.2 Hz and δ_H 2.31 ddd, J16.8, 3.7, 0.7 Hz, δ_C 40.3, CH₂-3; δ_H 1.97 dddd, J13.1, 9.6, 8.1, 5.9 Hz and δ_H 1.85 dddd, J13.1, 9.0, 5.6, 4.4 Hz, δ_C 35.8, CH₂-1’; and δ_H 2.64 ddd, J13.1, 9.6, 4.4 Hz and d_H 2.58 ddd, J13.1, 9.0, 5.9 Hz, d_C 29.7, CH₂-2’). The multiplicities of the methylene hydrogens were determined with the help of spectral simulations using the WINDNMR-Pro program²⁸ (Figure S3, in the SI section). ¹H-¹H COSY experiment showed correlations between H-3 and H-2, as well as between H-1’ and H-2’ and H-2 (Figure 1). The correlations observed by HMBC experiment between C-2’ (δ_C 29.7) and H-2’’,6’’ (δ_H 7.00); C-3 (δ_C 40.3) and H-5 (δ_H 5.26); C-5 (δ_C 104.4) and H-7 (δ_H 1.98), as well as the molecular formula determined for this compound led to establishing of a 2-(4-hydroxyphenylethyl)-6-methyl-2,3-dihydro-4H-pyran-4-one structure for 1 (Figure 1), which was confirmed by correlations between H-2 and 2H-3, 2H-1’, and 2H-2’ observed by 1D-TOCSY experiments.

Figure 1.
Select HMBC (→) correlations and ¹H-¹H COSY (↔) interactions for 1, 5, 8 and 9.

Based on the magnitude of the coupling constant between H-2 and H-3 (J13.2 Hz), a pseudo-axial conformation was assigned to H-2. Similar synthetic (S)-2-(phenylethyl)-2,3-dihydro-4H-pyran-4-ones showed negative optical rotation,^29-31 while the (R)-2-ethyl-6-methyl-2,3-dihydro-4H-pyran-4-one hepialone showed [α]_D²⁰ +106.4 (c 1.09, EtOH) and positive Cotton effects at 261 and 312 nm in its CD curve.³² Since compound 1 showed [α]_D²⁸ –12.9 and negative Cotton effects at 270 and 318 nm, the structure could be established as (S)-2-(4-hydroxyphenylethyl)-6-methyl-2,3-dihydro-4H-pyran-4-one. This is the first time that a pyranone, isolated from a natural source, with this carbon skeleton is being described in the literature.

A molecular formula C₁₇H₁₁NO₇ was determined for 5 based on the HRMS spectra, which showed peak at m/z342.0610 [M + H]⁺ (calcd. for C₁₇H₁₂NO₇, 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 ¹H and ¹³C 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 (δ_C 168.5), one methylenedioxyl (δ_H 6.46 s, 2H, δ_C 103.2) and one methoxyl (δ_H 3.94 s, 3H, δ_C 56.0) groups. The ¹H NMR spectrum of 5 showed, in the aromatic region, a trisubstituted system with three mutual coupled ABX pattern signals at δ 8.99 (d, 1H, J9.0 Hz), 7.51 (dd, 1H, J9.0, 2.4 Hz), and 7.79 (d, 1H, J2.4 Hz), which were assigned to H-5, H-6 and H-8, respectively. In addition, two aromatic CH were observed in these spectra (δ_H-2 7.72 s, δ_C-2 111.5 and δ_H-9 8.47 br s, δ_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.³³ The substituent positions on the AA structure were assigned with the help of HMBC experiments (Figure 1). These experiments showed correlations between H-2 (δ_H 7.72) and C-11 (δ_C 168.5) confirming the acyl group position in the structure, between H-9 (δ_H 8.47) and C-8 (δ_C 111.6), as well as H-5 (δ_H 8.99) and OCH₃ (δ_H 3.94) and C-7 (δ_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 ¹H and ¹³C NMR spectra of compounds 8 and9 are very similar to those of 5. However, compounds8and9 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).³⁴ The spectra revealed also absorption bands that indicated the presence of NO₂ 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 C₁₇H₁₀NO₇Na and C₁₆H₈NO₇Na, respectively, and suggest8 had a methoxyl substituent, whereas 9 a hydroxyl in the nitrophenanthrene structure. In addition, the protonated molecule of compound8 is 22 Da higher than 5. Thus, a sodium aristolochate derivative could be proposed for8and9. 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 ¹H NMR data of 5,8 and 9 confirmed the lower values for δ_H-9 observed for sodium aristolochates than aristolochic acids (5:δ_H-98.47, 8: δ_H-9 8.24, 9: δ_H-9 8.37).³⁴ Thus, 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.

Conclusions

To date, 35 different compounds have been isolated from A. urupaensis, including the new 2,3-dihydro-4H-pyran-4-one (1). Compounds with this pyranone carbon skeleton have not been isolated from natural sources yet. The new compounds 5, 8 and 9 are aristolochic acid derivatives with unusual C-7 oxygenated substituents, and the compounds 21-23, 26 and 35 are being reported for the first time in the Aristolochiaceae family.

Acknowledgments

The authors thank Dr Vinicius C. Souza and MSc Joelcio Freitas for plant identification, and FAPESP, CAPES and CNPq for financial support and fellowships to L. M. X. L. and J. C. H.

Supplementary Information

Supplementary information (1D and 2D NMR, MS and FTIR spectroscopic data of compounds 1, 5, 8 and 9) is available free of charge at http://jbcs.sbq.org.br as PDF file.

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Publication Dates

• Publication in this collection
  Nov 2017

History

• Received
  21 Dec 2016
• Accepted
  27 Mar 2017