Complete ¹H and 13C NMR assignments of isojuripidine from Solanum asterophorum Mart.

Abstracts

Isojuripidine was isolated from the aerial parts of Solanum astherophorum Mart. Its structure was determined using a combination of homo- (1D ¹H NMR, 13C NMR-HBBD and 13C NMR-DEPT) and heteronuclear 2D NMR techniques (¹H-¹H-COSY, ¹H-¹H-NOESY, HSQC, HMBC), and HREIMS. The unambiguous assignments of ¹H and 13C NMR data of derivatives 3-N,6-O-diacetyl-isojuripidine and 3-N-cinnamoyl-isojuripidine are described.

Solanaceae; Solanum asterophorum; isojuripidine

Isojuripidina, um alcalóide 3-aminoespirosolano foi isolado das partes aéreas de Solanum asterophorum Mart. A estrutura foi determinada usando uma combinação de técnicas de RMN homo (1D: RMN ¹H, RMN 13C{1H}, RMN13C-DEPT135; 2D: COSY, ¹H-¹H-NOESY) e heteronuclear 2D (HSQC e HMBC) e espectro de massas de alta resolução. Foi feita também a atribuição inequívoca dos deslocamentos químicos dos átomos de carbono e hidrogênio dos derivados 3-N,6-O-diacetil-isojuripidina e 3-N-cinamoil-isojuripidina.

SHORT REPORT

Complete ¹H and ¹³C NMR assignments of isojuripidine from Solanum asterophorum Mart.

Tania M. S. Silva^*; Rodrigo A. Costa; Eduardo J. Oliveira; José M. Barbosa-Filho; Maria F. Agra; Celso A. Camara

Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, Campus I, Cidade Universitária, s/n, CP 5009, 58051-970 João Pessoa - PB, Brazil

ABSTRACT

Isojuripidine was isolated from the aerial parts of Solanum astherophorum Mart. Its structure was determined using a combination of homo- (1D ¹H NMR, ¹³C NMR-HBBD and ¹³C NMR-DEPT) and heteronuclear 2D NMR techniques (¹H-¹H-COSY, ¹H-¹H-NOESY, HSQC, HMBC), and HREIMS. The unambiguous assignments of ¹H and ¹³C NMR data of derivatives 3-N,6-O-diacetyl-isojuripidine and 3-N-cinnamoyl-isojuripidine are described.

Keywords: Solanaceae, Solanum asterophorum, isojuripidine

RESUMO

Isojuripidina, um alcalóide 3-aminoespirosolano foi isolado das partes aéreas de Solanum asterophorum Mart. A estrutura foi determinada usando uma combinação de técnicas de RMN homo (1D: RMN ¹H, RMN ¹³C{1H}, RMN¹³C-DEPT135; 2D: COSY, ¹H-¹H-NOESY) e heteronuclear 2D (HSQC e HMBC) e espectro de massas de alta resolução. Foi feita também a atribuição inequívoca dos deslocamentos químicos dos átomos de carbono e hidrogênio dos derivados 3-N,6-O-diacetil-isojuripidina e 3-N-cinamoil-isojuripidina.

Introduction

The genus Solanum is considered to be one of the largest and most complex among the Angiosperms. It is comprised of about 1000 species and approximately 3000 epithets are described.¹ The genus is well represented in Brazil and is widely distributed from north to south in diverse phytogeographic regions. Many of the species are endemic to the country. In the Northeast of Brazil, many Solanum species are widely used in popular medicine and are commonly known as jurubeba, the word originating from the Tupi-guarani, yu'beba, which refers to the presence of prickles on some of them.²Solanum asterophorum is known as jurubeba-de-fogo and its roots are popularly used in the treatment of liver diseases.²

As part of our chemical and pharmacological studies of Brazilian Solanum,^4-10 we report the first chemical investigation on S. asterophorum. From the methanol extract of S. asterophorum the steroidal alkaloid isojuripidine 1 was isolated. The spectroscopic data of 1 and of its derivative 3-N,6-O-diacetyl-isojuripidine 1a and 3-N-cinnamoyl-isojuripidine 1b (Figure 1) are described. The compound 1b is a new derivative. The unambiguous assignments of ¹H and ¹³C NMR data of isojuripidine 1 and derivatives 1a and 1b are reported for the first time and involved a combination of homo- (1D ¹H NMR, ¹³C NMR-HBBD and ¹³C NMR-DEPT) and heteronuclear 2D NMR techniques (¹H-¹H-COSY, ¹H-¹H-NOESY, HSQC, HMBC), and HREIMS. Isojuripidine was isolated previously only from Solanum paniculatum¹¹ and no NMR data was published.

Results and Discussion

The steroidal alkaloid isojuripidine 1 was isolated from the methanol extract of the aerial parts of Solanum asterophorum (Solanaceae). Compound 1 was obtained as an amorphous powder and gave a positive Dragendorff reagent test. The spectral data were in agreement of a (25R)-spirostan steroidal type skeleton, with the IR spectrum (KBr) showing absorptions at 3409 cm^-1 (n_max N-H and O-H), with a 25R-spirostan structure (supported by peaks at 982, 956, 920 and 899 cm^-1, intensity 920<899,)¹² and confirmed by ¹H NMR^13,14 and ¹³C NMR.^15,16 As expected the ¹H NMR spectrum (Table 1) showed the signals of two methyl singlets, two methyl doublets, and a multiplet due to an oxymethine hydrogen. The presence of a b-amino and a carbinolic hydrogen, indicated an amino and hydroxyl group, respectively. The amino group would most probably be at C-3.

The ¹³C NMR spectrum of 1 showed 27 signals. From these, the signals of C-16, C-22 and C-26 and one additional signal for a carbon-bearing nitrogen additionally supports the proposed aminospirosolane structure for 1. EIMS fragments at m/z 115 and 139 corroborated this structure, while the peaks at m/z 56 and m/z 98 provided confirmation of the amino group at C-3,¹⁷ and hydroxyl at C-6, respectively. HREIMS (IE, 70 eV) gave [M]⁺ at 431.33979 corresponding to the molecular formula C₂₇H₄₅NO₃ (Calc. 431.33994), expected for 1. Full assignment of ¹H and ¹³C chemical shifts of 1 (Table 1) was achieved with the aid of ¹H-¹H COSY, HSQC, HMBC and NOESY experiments, discussed below.

Only a few characteristic assignments can be made immediately from ¹H NMR, e.g. H-3, H-16, H-26_eq and 26_ax at d_H 2.82, 4.23, 3.33 and 3.16, respectively. Comparative analysis of the ¹³C NMR-HBBD and ¹³C NMR-DEPT spectra was used to identify the number of signals attributed to three quaternary [(C)₃: all sp³, ten methine [(CH)₁₀: all sp³, including two oxygenated and one with amino group = (CH)₇(O-CH)₂(HN-CH)], ten methylene [(CH₂)₁₀: all sp³ including one oxygenated = (CH₂)₉(CH₂-O)], and four methyl [(CH₃)₄] carbon atoms. Consequently, the expanded formula (C)₃(CH)₇(O-CH)₂(N-CH)(CH₂) ₉(CH₂-O)(CH₃ )₄=C₂₇H₄₂NO₃ was deduced, which after considering the presence of one hydroxyl and one amino group was established as C₂₇H₄₅NO₃ in accordance with the HREIMS. However, the complete assignments, especially in the most congested regions (¹H from d_H 0.6 to d_H 2.0, ¹³C from d_C 25 to 35) had to be made from 2D data.

The hydrogen assignment was divided into two spin systems. The first system includes the steroidal nucleus and ring E. The second system is the six-membered heterocyclic moiety at position 22 (Figure 1). For spin system 1, two starting points CH₃-18 and H-6 were used on the COSY. The hydrogens on methyl-18 were used because steroidal compounds show a characteristic coupling between CH₃-18 (d_H 0.65) and CH₂-12 (d_H 1.63). This helps to penetrate into the heart of the convoluted region of the spectrum and to verify assignments made by the use of H-6 as the starting point. The carbinolic hydrogen H-6 (d_H 3.14) was correlated with H-5 (d_H 0.89) and H-5 with H-4 (d_H 1.18). The assignment on H-4 leads to the assignment of ring A. The connection of H-6 to H-7 (d_H 0.75) leads to continuous correlations which result in the assignment of rings B, C, D and E. The second spin system was assigned by using the two geminal hydrogens H-26 as the starting point. The H-26 hydrogens absorb at d_H 3.33 and 3.16 and were correlated to the other hydrogens on ring F and to methyl CH₃-27 (d_H 0.70).

The ¹H-¹³C correlation was used interactively with COSY to differentiate the correlations between geminal and vicinal hydrogens. Subsequent to the completion of the hydrogen assignment, the assignments of the hydrogenated carbons were made from the one-bond ¹H-¹³C correlations in a straightforward manner.

The quaternary carbons were assigned by using long-range ¹H-¹³C correlations obtained from the HMBC experiment, which primarily reveals connectivity between hydrogens and carbons separated by two and three bonds. The angular methyl hydrogen correlations are particularly useful in rendering this information. The quaternary carbons C-10 (d_C 35.7), C-13 (d_C 40.1), and C-22 (d_C 108.4) showed correlations to methyl hydrogen CH₃-19 (d_H 0.75), CH₃-18 (d_H 0.65), and CH₃-21 (d_H 1.87), respectively. In addition C-22 exhibited three bond correlations to H-16 (d_H 4.23), H-17 (d_H 1.61), and two bond correlation to H-20 (d_H 1.75). This signal (H-20) also showed a three bond correlation to C-13.

The NOESY spectrum of 1 was used to confirm the trans junctions of the A/B and B/C rings and their corresponding conformations. The a-axial orientation of H-5 was confirmed by a strong NOE observed between H-5 (d_H 0.89) and the hydrogens assigned to H-2_eq (d_H 1.69), H-3_ax (d_H 2.82), H-4_ax (d_H 2.12), H-4_eq (d_H 1.18), and H-9 (d_H 0.60). The axial orientation of H-6 was confirmed by the strong correlation with H-7_eq (d_H 1.85), H-8 (d_H 1.52), H-11_ax (d_H 1.18), and CH₃-19 (d_H 0.75). The dipolar interaction was observed between CH₃-27_eq (d_H 0.70), with H-23_ax (d_H 1.47), and 26_ax (d_H 3.16), indicating spatial proximity of these hydrogen atoms. These data were used to confirm the configuration 25R-3b-amino-5-22a-O-spirostan-6a-ol. This proposal is in agreement with the specific rotation [a]_D=-50 (literature [a]_D=-47).¹¹

The acetylation with acetic anhydride and acylation with cinnamoyl chloride furnished diacetylisojuripidine 1a and the new N-cinnamoylisojuripidine 1b respectively. The last compound was chemoselectively formed. The structures were confirmed by ¹H and ¹³C NMR assignments (Tables 2). As expected, H-6 was shifted from d_H 3.14 to 4.50 in 1a. This is the first report of ¹³ C NMR data assignments for the isojuripidine 1 (25R-3b-amino-5-22a-O-spirostan-6a-ol) and the derivatives 1a (25R-3b-acetanamide-5a-22a-O-spirostan-6 a-acetate) and 1b (25R-3b-N-cinnamoyl-5a-22a-O-spirostan-6 a-ol).

The 3-aminospirostane alkaloid (jurubidine-type) form a relatively small group of Solanum alkaloids.¹⁸ Only few reports of this type of compound are described on the literature¹⁹ juripidine²⁰ itself and isojuripidine,¹¹ besides soladunalinidine²¹ and few others aza-ketal isomers are solely representative members.

Experimental

The NMR spectra of compound 1 were run on a Bruker Advance 500 (500 MHz for ¹H and 125 MHz for ¹³C) in DMSO-d₆. The compounds 1a and 1b were taken on a Bruker AC 200 (200 MHz for ¹H and 50.3 MHz for ¹³C) in CDCl₃. FT-IR spectra were obtained on a Bomen-Michelson spectrophotometer using KBr. High-resolution mass spectra were obtained by electron impact on a VG Autospec spectrometer.

Plant material

The aerial parts of Solanum asterophorum were colleted in Brazil, Paraiba, municipality of Areia, in 2003. Voucher specimens (Agra 1744) are deposited at the Herbarium Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraiba. The powdered aerial parts of S. asterophorum (396.0 g) were extracted with MeOH in a Soxhlet apparatus. The extract was concentrated under vacuum in a rotaevaporator. The crude residue (37.5 g), after standing in the refrigerator, furnished a white precipitate that was separated from the extract and recristallyzed from methanol to yield 205.0 mg of 1, mp 200 ºC (Lit.¹¹ 204-5 ºC); [a]²⁰_D -50.0 (Lit.¹¹ -47.0); IR (KBr) n_max/cm^-1: 3409, 2951, 2929, 2873, 2850, 1617, 1449, 1385, 1242, 1176, 1154, 1055, 982, 956, 920, 899 and 866; HREIMS Calc. for C₂₇H₄₅NO₃ 431.33994; Found 431.33979.

Synthesis of 1a.

A mixture of 90.0 mg of isojuripidine 1 with 1 mL of pyridine and 1 mL acetic anhydride was stirred at room temperature for 24 h. After TLC inspection, the reaction was added to crushed ice with the aid of ethanol. The solids were collected on a Buchner and recrystallized from methanol, yielding 75.1 mg (70 %) of 1b as white crystals. IR (KBr) n_max/cm^-1: 3416, 2950, 2935, 2872, 1753, 1639, 1452, 1239, 1042, 982, 900 and 865.

Synthesis of 1b²²

To a mixture of 1 (23.0 mg) in 10 mL of dichloromethane with 1 mL of triethylamine at room temperature was added a solution of cinnamoyl chloride (10.0 mg, 1.12 mmol) in 1 mL of dichloromethane. The resulting mixture was stirred for 24 hours at room temperature, the solvents removed by vacuum and the product crystallized in methanol, giving the N-cinnamoyl derivative 1a as colourless crystals (14.7 mg, 52 % yield), with mp 327-330 ºC; IR (KBr) n_max/cm^-1: 3409, 2949, 2927, 2851, 1651, 1620, 1451, 1240, 1176, 1052, 900 and 788; MS (70 eV) 544 (4%, M⁺ - OH), 415 (2%), 387 (2%), 256 (13%), 131 (7%), 77 (5%) and 69 (100%).

Supplementary Information

All spectra obtained for the assignments are given as PDF file for consulting, free of charge, at http://jbcs.sbq.org.br next to the article PDF link.

Acknowledgments

The authors thanks IMSEAR-CNPq, CAPES and PIBIC-UFPB for financial support. TMSS thanks to Prof. Edilberto R. Silveira and Daniel E. Uchoa (CENAUREM – Centro Nordestino de Aplicação e Uso de RMN) for kindly recording the NMR data.

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Received: June 14, 2005

Published on the web: December 5, 2005

Supplementary Infotmation

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Publication Dates

• Publication in this collection
  20 Jan 2006
• Date of issue
  Nov 2005

History

• Received
  14 June 2005