Complete 1 H and 13 C NMR Assignments of Isojuripidine from Solanum asterophorum Mart .

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 H, RMN C{1H}, RMNC-DEPT135; 2D: COSY, H-H-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. 1The 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. 2 Solanum asterophorum is known as jurubeba-de-fogo and its roots are popularly used in the treatment of liver diseases. 2s part of our chemical and pharmacological studies of Brazilian Solanum, [4][5][6][7][8][9][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 1 H and 13 C NMR data of isojuripidine 1 and derivatives 1a and 1b are reported for the first time and involved a combination of homo-(1D 1 H NMR, 13 C NMR-HBBD and 13 C NMR-DEPT) and heteronuclear 2D NMR techniques ( 1 H-1 H-COSY, 1 H-1 H-NOESY, HSQC, HMBC), and HREIMS.Isojuripidine was isolated previously only from Solanum paniculatum 11 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 (ν 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 1 H NMR 13,14 and 13 C NMR. 15,16 As expected the 1 H 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 β-amino and a carbinolic hydrogen, indicated an amino and hydroxyl group, respectively.The amino group would most probably be at C-3.
The 13 C 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 C 27 H 45 NO 3 (Calc.431.33994), expected for 1. Full assignment of 1 H and 13 C chemical shifts of 1 (Table 1) was achieved with the aid of 1 H-1 H COSY, HSQC, HMBC and NOESY experiments, discussed below.
Only a few characteristic assignments can be made immediately from 1 H NMR, e.g.H-3, H-16, H-26 eq and 26 ax at δ H 2.82, 4.23, 3.33 and 3.16, respectively.Comparative analysis of the 13   4 =C 27 H 42 NO 3 was deduced, which after considering the presence of one hydroxyl and one amino group was established as C 27 H 45 NO 3 in accordance with the HREIMS.However, the complete assignments, especially in the most congested regions ( 1 H from δ H 0.6 to δ H 2.0, 13 C from δ 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 CH 3 -18 and H-6 were used on the COSY.The hydrogens on methyl-18 were used because steroidal compounds show a characteristic coupling between CH 3 -18 (δ H 0.65) and CH 2 -12 (δ 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 (δ H 3.14) was correlated with H-5 (δ H 0.89) and H-5 with H-4 (δ H 1.18).The assignment on H-4 leads to the assignment of ring A. The connection of H-6 to H-7 (δ 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 δ H 3.33 and 3.16 and were correlated to the other hydrogens on ring F and to methyl CH 3 -27 (δ H 0.70).
The 1 H- 13 C 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 1 H- 13 C correlations in a straightforward manner.
The 3-aminospirostane alkaloid (jurubidine-type) form a relatively small group of Solanum alkaloids. 18Only few reports of this type of compound are described on the literature 19 juripidine 20 itself and isojuripidine, 11 besides soladunalinidine 21 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 1 H and 125 MHz for 13 C) in DMSO-d 6 .The compounds 1a and 1b were taken on a

Table 1 .
13 (500 MHz) and13C (125 MHz) spectral data for isojuripidine 1 obtained by heteronuclear 2D shift-correlated HSQC and HMBC spectra, in DMSO-d 6 .Chemical shifts (δ, ppm) and coupling constants (J in Hz, in parenthesis) a .40 b  H eq ; 1.18 b , H ax 12 39.3 1.63 b , H eq ; 1.07 b , H ax 15 31.41.85 b , H α ; 1.11 b , H β 23 30.9 1.57 b , H eq ; 1.47 b , H ax 2D homonuclear 1 H-1 H-COSY and heteronuclear HMBC spectra were also used for these assignments.Chemical shifts of hydrogen atoms obtained from 1D 1 H NMR spectra.Carbon signals corresponding to C, CH, CH 2 and CH 3 deduced by comparative analysis of HBBD-and DEPT-13 C NMR spectra.Superimposed 1 H signals are described without multiplicity and chemical shifts deduced by HSQC, HMBC and 1 H-1 H-COSY; a b Superimposed by other signals.

Table 2 .
13C (200 MHz) and1H NMR (50.3 MHz) chemical shifts* of the derivatives of isojuripidine from S. asterophorum Spectra were recorded in CDCl 3 and the chemical shifts are expressed on the δ scale with TMS as internal standard (values in parenthesis are coupling constants in Hz).Bruker AC 200 (200 MHz for 1 H and 50.3 MHz for 13 C) in CDCl 3 .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. *