Spruceanumines A and B, novel plumeran indole alkaloids from Aspidosperma spruceanum(Apocynaceae)

Abstracts

Two novel indole alkaloids with plumeran skeleton, spruceanumines A (1) and B (2), and eight known indole alkaloids, aspidospermidine (3), demethoxypalosine (4), aspidocarpine (5), aspidolimine (6), fendlerine (7), aspidolimidine (8), obscurinervidine (9) and obscurinervine (10) were isolated from stem bark and seeds methanolic extracts of Aspidosperma spruceanum. Compounds structures were elucidated on the basis of spectroscopic data, mainly those obtained by ¹H and 13C NMR (1D and 2D) and mass spectrometry.

Aspidosperma spruceanum; Apocynaceae; plumeran indole alkaloids

Dois novos alcalóides indólicos com esqueleto plumerano, spruceanuminas A (1) e B (2), e oito alcalóides indólicos conhecidos, aspidospermidina (3), desmetoxipalosina (4), aspidocarpina (5), aspidolimina (6), fendlerina (7), aspidolimidina (8), obscurinervidina (9) e obscurinervina (10), foram isolados do extrato metanólico das cascas do caule e sementes de Aspidosperma spruceanum. As estruturas dos compostos foram elucidadas com base na análise de dados espectroscópicos, principalmente os obtidos por espectros de RMN ¹H e 13C (1D e 2D) e por espectrometria de massas.

ARTICLE

Spruceanumines A and B, novel plumeran indole alkaloids from Aspidosperma spruceanum (Apocynaceae)

Vilma B. Oliveira^I; Ivo J. Curcino Vieira^I; R. Braz-FilhoII, ^*; Leda Mathias^I; Norberto P. Lopes^III; Antonio E. M. Crotti^IV; Daniel E. de A. Uchôa^V

^ILaboratório de Ciências Químicas (LCQUI)-CCT, Universidade Estadual do Norte Fluminense Darcy Ribeiro (UENF), 28013-602 Campos dos Goytacazes-RJ, Brazil

^IIPesquisador Visitante Emérito-FAPERJ, Laboratório de Ciências Químicas(LCQUI)-CCT-UENF/PPGQO-DEQUIMUFRRJ, 28013-602 Campos dos Goytacazes-RJ, Brazil

^IIIFaculdade de Ciências Farmacêuticas de Ribeirão Preto-USP, 14040-903 Ribeirão Preto-SP, Brazil

^IVNúcleo de Pesquisas em Ciências Exatas e Tecnológicas, Universidade de Franca, 14404-600 Franca-SP, Brazil

^VCentro Nordestino de Aplicação e Uso da Ressonância Magnética Nuclear, Departamento de Química Orgânica e Inorgânica, Universidade Federal do Ceará, 60021-97 Fortaleza-CE, Brazil

ABSTRACT

Two novel indole alkaloids with plumeran skeleton, spruceanumines A (1) and B (2), and eight known indole alkaloids, aspidospermidine (3), demethoxypalosine (4), aspidocarpine (5), aspidolimine (6), fendlerine (7), aspidolimidine (8), obscurinervidine (9) and obscurinervine (10) were isolated from stem bark and seeds methanolic extracts of Aspidosperma spruceanum. Compounds structures were elucidated on the basis of spectroscopic data, mainly those obtained by ¹H and ¹³C NMR (1D and 2D) and mass spectrometry.

Keywords:Aspidosperma spruceanum, Apocynaceae, plumeran indole alkaloids

RESUMO

Dois novos alcalóides indólicos com esqueleto plumerano, spruceanuminas A (1) e B (2), e oito alcalóides indólicos conhecidos, aspidospermidina (3), desmetoxipalosina (4), aspidocarpina (5), aspidolimina (6), fendlerina (7), aspidolimidina (8), obscurinervidina (9) e obscurinervina (10), foram isolados do extrato metanólico das cascas do caule e sementes de Aspidosperma spruceanum. As estruturas dos compostos foram elucidadas com base na análise de dados espectroscópicos, principalmente os obtidos por espectros de RMN ¹H e ¹³C (1D e 2D) e por espectrometria de massas.

Introduction

The Aspidosperma(Apocynaceae) genus is endemic to Americas and is found mainly in regions between Mexico and Argentina.¹ Aspidospermagenus continues to be fascinating as an expressive source of indole alkaloids with novel skeletons, which are interesting from a biosynthetic perspective and reported biological properties. Several species of Aspidospermaare broadly used in popular medicine as potential antimalarial agents, leishmaniose treatment, uterus and ovary inflammation, as contraceptive, in diabetes, in stomach problems, against cancer, fever and rheumatism.²

Aspidosperma spruceanum (A. spruceanum ), commonly known as "Paratudo-Branco" inAtlantic forest in the North of Espírito Santo State, appears as a tree of 5-20 m. The isolation and structure elucidation of two alkaloids from stem bark of A. spruceanumcollected in Rio de Janeiro State, Brazil, were reported.³

In the present paper, we describe the isolation and characterization of two novel plumeran indole alkaloids named as spruceanumines A (1) and B (2), along with known indole alkaloids: aspidospermidine (3),^4-7 demethoxypalosine (4),^7-9 aspidocarpine (5),^8,10,14 aspidolimine (6),^8,14 fendlerine (7),^15,16 aspidolimidine (8),^8,13,15 obscurinervidine (9)^14,17 and obscurinervine (10).^14,17 Their structures were established by spectrometric techniques, mainly one-and two-dimensional nuclear magnetic resonance (NMR), as well as high resolution electron spray ionization mass spectra (HRESIMS).

Results and Discussion

Elaboration of stem bark and seeds methanol extract of A. spruceanumby classical chromatographic methods resulted in the isolation of ten plumeran indole alkaloids (1-10), whose structures are shown in Figure 1. The well-known plumeran indole alkaloids, aspidospermidine (3), demethoxypalosine (4), aspidocarpine (5), aspidolimine (6), fendlerine (7), aspidolimidine (8), obscurinervidine (9) and obscurinervine (10) were identified on the basis of ¹H and ¹³C NMR spectral data, including ¹H-¹H correlation spectroscopy (COSY), ¹H-¹H nuclear overhauser effect spectroscopy (NOESY), heteronuclear single quantum coherence (HSQC) and heteronuclear multiple bond correlation (HMBC) NMR experiments,¹⁸ which were also used to complete unambiguous ¹H and ¹³C chemical shift assignments of 1 and 2.

Spruceanumines A (1) and B (2), were obtained as a mixture of amorphous form, = -101.7 (CHCl₃, c0.61). Infrared (IR) spectrum showed bands at V_max 3100-2890 (C-H stretching), V_max 1755 (stretching of the γ-lactone carbonyl group) in addition to other bands at V_max 1624, 1606 and 1497 (C=C stretching of the benzene ring), and 887 and 739 cm^-1 (C-H bending of substituted benzene ring).¹⁹

Comparative analysis of the {¹H}- and distortionless enhancement by polarization transfer (DEPT) 135º- ¹³C NMR spectra (Table 1) revealed signals corresponding to 24 (1) or 25 (2) carbon atoms, allowing to recognize the presence of signals corresponding to nine nonhydrogenated [(C)₉: three sp³ (including one bounded to nitrogen and oxygen atoms at δ_C 106.79), six sp² (including one carbonyl group at δ_C 175.10 and five sp² attributed to aromatic ring], five methine [(CH)₅: two sp³ linked to nitrogen atom (δ_C 68.91/δ_H 3.50 and δ_c44.73/δ_H 3.27 correlated in the HSQC pectrum with ¹H chemical shifts at δ_H 3.50 and 3.27, respectively, as indicated also in the direct subsequent correlations, ¹J_CH) and three sp² (one aromatic at δ_C 101.78/ δ_H 6.63 (s) and two olefinic at δ_c123.31/δ_H 5.81 (ddd) and 130.79/δ_H 5.37 (brd)], seven (1) and eight (2) sp³ methylene [(CH)or (CH), including one linked to oxygen atom at δ_C 72.26 (1) and 70.20 (2, revealing γ-effect of the methyl group CH-4')] and three methyl [(CH): δ_c15.10/δ_H 1.12 (d, J= 6.2 Hz), 1; δ_C 9.39/δ_H 0.98 (t, J= 7.5 Hz), 2; and (MeO)₂ represented by signals at δ_c 56.49/δ_H 3.70 (s) and 61.18/δ_H 3.81 (s), 1; δ_c 56.97/δ_H 3.74 (s) and 61.18/δ_H 3.86 (s), 2] carbon atoms, allowing to deduce the expanded molecular formulae (C)₇(C=O)(N-C-O)(CH)₅(O-CH₂)(CH₂)₆ (CH₃)(MeO)₂and (C)₇(C=O)(N-C-O)(CH)₅(O-CH₂) (CH₂)₇(CH₃)(MeO)₂ for 1and 2, respectively. This later contains additional metylene group CH₂ (δ_C 22.56/δ_H 1.69 (m) and 1.46 (m) coupled to the hydrogens of an adjacent methyl group (δ_C 9.39/δ_H 0.98 (t, J= 7.5 Hz).

The high resolution electro-spray ionization mass spectrum (ESI-MS) of 1and 2showed peaks corresponding to the protonated molecules [M+H]⁺ at m/z425.2170 of 1(C₂₄H₂₉N₂O₅= m/z425.2076, Δ_m/z 0.0094) and 439.2332 of 2 (C₂₅H₃₁N₂O₅ = m/z439.2233, Δ_m/z 0.0099) Daltons, which together with the NMR ¹³C spectrum enable to propose molecular formulas C₂₄H₂₈N₂O₅ (1) and C₂₄H₂₈N₂O₅ (2), respectively, containing twelve degrees of unsaturation (C₂₄H₂₈N₂O₅ - C₂₄H₂₈ N₂O₅ = H₂₄ or C₂₅H₅₄N₂O₅ -C₂₄H₃₀N₂O₅ = H₂₄), which is consistent with the structure of alkaloids containing the nucleus of 21-oxo-aspidoalbidine²⁰ (11, aspidospermidin-18,21-olide, using actual numeration) as basic structure (eleven degrees of unsaturation = four corresponding to aromatic ring, two to carbonyl lactone group and additional pentacyclic moiety), which after the location of one 1,2-disubstituted double bond between the carbon atoms CH-14 and CH-15 and of one heterocyclic involving the N-substituent and the oxygen atom sustained by carbon atom C-12, justifying the presence of OCH₂ (1: δ_c 72.26/δ_H 4.27 and 3.90; 2: δ_H 70.20/δ_H 4.35 and 4.00, revealing shielding induced by γ-effect of the methyl 3H-4'), methyl group represented by a doublet signal (J= 6.2 Hz) at δ_H 1.12 (3H-3' correlated in the HSQC spectrum with ¹³C chemical shift at δ_C 15.10) coupled hydrogen linked to nitrogenated carbon atom (δ_H 3.27 , m, H-2' correlated with ¹³C signal at δ_C 44.73, CH-2') in the alkaloid 1and by a triplet signal (J= 7.5 Hz) at 0.98 (3H-4') coupled to hydrogen atoms of the additional methylene of 2 (δ_H 1.69 and 1.46 correlated in the HSQC with ¹³C chemical shift at δ_C 22.56). The lower field ¹³C chemical shift CH-2' (δ_C 50.43) in compound 2when to that of 1 (δ_C 44.73) is indicative of a β-effect induced by the methyl group CH₃-4', as shown in Table 1.

The identity of the six-membered heterocyclic ring containing and oxygen, was supported by ³J_CH HMBC correlations between C-12 [(δ_C 136.24, 1and 2)] and 2H-1' [δ_H 4.27 and 3.90 (1); δ_H 4.35 and 4.00 (2) (Table 1), as well as by ¹H-¹H-COSY cross-peaks displayed by H-1'b (δ_H 4.27 in 1; 4.35 in 2), H-1'a (δ_H 3.90 in 1; 4.00 in 2), H-2' (δ_H 3.27 in 1; 3.13 in 2).

The ¹H-¹H-COSY spectrum (Table 1) showed coupling of methylenic hydrogens at δ_H 4.27 [(dd, J= 10.7 and 2.7 Hz, H-1'b (1)] and δ_H 3.90 [(dd, J= 10.7 and 8.8 Hz, H-1'a (1)] with the methinic hydrogen at δ_H 3.27 (m, H-2', 1) and at δ_H 4.35 [(dd, J= 10.8 and 2.6 Hz, H-1'b (2)] and δ_H 4.00 [(dd, J= 10.8 and 8.6 Hz, H-1'a (2)] correlated with the signal at δ_H 3.13 [(m, H-2', 2)], in agreement with the presence six-membered ring formation.

The assignment of a methyl group at C-2' was confirmed by its ¹H-¹H-COSY and ³J_CH HMBC correlations with H-2' (δ_H 3.27) and 2H-1' (δ_H 4.35 and 4.00), respectively.

In spruceanumine B (2), the presence of an ethyl group at C-2 was confirmed by the coupling of the methylenic hydrogens CH₂-3' (δ_H 1.69 and 1.46) with the vicinal methyl group (δ_H 0.98) and H-2' (δ_H 3.13).

The ¹H NMR spectrum of mixture showed signals at δ_H 3.70 (1), 3.74 (2) and δ_H 3.81 (1), 3.86 (2), which are characteristics of methoxyl groups linked to the benzene ring.¹⁹ These signals showed heteronuclear interaction via one bond (¹J_CH) with the signals at δ_C 56.49 (1), 56.97 (2) and 61.18 (1e 2) observed in the HSQC spectrum, suggesting the presence of two methoxyl groups linked to the ring A. This, was confirmed by long range heteronuclear coupling (ⁿJ_CH, n= 2 and 3) observed in the HMBC spectrum, as summarized in Table 1. The signal at δ_C 61.18 (Table 1) observed in the ¹³C NMR of 1and 2is a typical value corresponding to signal of methoxyl groups located at forbidden position (MeO-11), as also observed in the aromatic ring of 11(MeO-11). These data allowed to and postulate the same substitution for 1and 2, as indicated in Figure 1.

The ¹³C NMR spectrum (Table 1) revealed the presence of a γ-lactone covering the carbon atoms C-20 e CH-21 by the signal at δ_C 175.10 (C-18), consistent with carbonyl carbon lactone of five members,^20-21 that was also confirmed by long-range coupling of C-18 (δ_C 175.10) with both hydrogen atoms 2H-19 represented by the signals at δ_H 2.50 (H-19b) and δ_H 2.12 (H-19a). Additional heteronuclear long-range couplings are summarized in Table 1.

The main ions fragments observed in the ESI-MS/MS spectrum (low resolution) of 1and 2are summarized in Scheme 1. These fragmentation pattern are compatible with that of plumeran alkaloids, as 21-oxo-aspidoalbidine (18-oxo by actual numeration utilized in the literature), previously isolated from Aspidosperma exalatum²⁰, and they are also in agreement with the presence of 18, 21-olide function in 1and 2, as suggested by signals at δ_C 175.10 (C-18) and 106.79 (C-21).

The location of a double bond at CH-14, CH-15 was deduced from the HMBC correlations of carbons resonating at δ_C 123.51 (CH-14, 1and 2), 130.79 (CH-15, 1) and 130.66 (CH-15, 2), with olefinic hydrogens at δ_H 5.81 (H-14), and δ_H 5.37 (H-15). The vicinal coupling between these hydrogen atoms was confirmed in the ¹H-¹H-COSY spectrum.^18,21

The relative stereochemistry of spruceanumine A (1) and B (2) was suggested from the nuclear overhauser effect (nOe) interactions displayed in the NOE spectrum, as summarized in Figure 2.

¹H-¹H-NOESY correlations of H-2 and H-2' of 1 and 2 indicated both a-orientations; of H-2 with one hydrogen H-6 of the methylene group CH₂-6 of 1 and 2 was also used to establish the relative configuration 7(S); of H-2 with both H-2' and 2H-3 of the methylene group CH₂-3' of 2 revealed a-orientation of H-2; of H-16β with methyl group CH₃-3' of 1 and with methylene group CH₂-3' of 2 are consistent with β orientation of this hydrogen atom H-16; spatial interaction of the of the H-15 with both H-19 and H-17 indicated to these hydrogen atoms a and β-orientation, respectively, as shown in Figure 2.

The relative intensity of ¹H NMR signals from the methyl groups CH₃-3' (1, δ_H 1.12) and CH₃-4' (2, δ_H 0.98) was used to deduce the approximated percentage of the 32.9% and 67.1% to spruceanumine A (1) and, spruceanumine B (2) in the mixture, respectively.

Experimental

General Procedures

Measures of optic rotation were obtained on a Perkin Elmer 343 digital polarimeter. Melting points were obtained on a Microquímica MQRPF and were uncorrected. Fourier transform infrared spectroscopy (FTIR) spectra were recorded on a FTIR-8300 Shimadzu spectrometer using KBr disk. ESI-MS (high resolution) and ESI-MS/ MS (low resolution) mass spectra were obtained on a MICROMASSUltrOTOF-Q (Brüker Daltonics, Billerica, MA) mass spectrometer, using the positive ion mode of analysis. Chromatographic purifications were carried out over silica gel (70-230 mesh). Silica gel 60F₂₅₄ was used in thin layer chromatography analysis.

¹H and ¹³C NMR spectra were measured on a Brüker DRX500 spectrometer, equipped with inverse probes and field gradient, operating at 500 (¹H) and 125 (¹³C) MHz. CDCl₃ was used as solvent and tetramethylsilane (TMS) as internal reference. Chemical shifts are given in the δ scale (ppm) and coupling constants Jin Hz. One dimensional (1D) ¹H and ¹³C NMR spectra were acquired under standard conditions by using a direct detection 5 mm ¹H/¹³C dual probe. Standard pulse sequences were used for two dimensional spectra by using a multinuclear inverse detection 5 mm probe with field gradient.

Plant materials

The stem bark and seeds of A. spruceanumBenth ex. Mull. Arg. were collected in November 2004 at Reserva Florestal de Linhares, Linhares, Espírito Santo State, Brazil. A voucher specimen (CVRD-273) is deposited at the Reserva Florestal herbarium, Cia. Vale do Rio Doce, Linhares, Espírito Santo State.

Extraction and isolation

Dried and powdered stem bark (3.09 kg) and seeds (530.1 g) from A. spruceanumBenth ex. Mull Arg were extracted with methanol at room temperature, furnishing, after solvent evaporation, 63.7 g and 18.5 g of crude methanol extracts, respectively.

The methanol extract (63.7 g) from stem bark was successively partitioned with CH₂Cl₂/H₂O. The CH₂Cl₂ fraction (7.7 g) was chromatographed over silica gel column with a gradient of hexane/ethyl acetate to afford ten fractions. Fraction 8 (475.8 mg) was rechromatographed over a silica gel column with a gradient of MeOH in CH₂Cl₂ yielding aspidolimine (6, 15.9 mg) and demethoxypalosine (4, 34.7 mg). Fraction 10 (364.5 mg) was rechromatographed over a silica gel column with a gradient of MeOH in CH₂Cl₂ supplying aspidocarpine (5, 97.9 mg) and aspidospermidine (3, 19.1 mg) alkaloids.

The methanol extract (18.5 g) from seeds was partitioned with CH₂Cl₂/H₂O. CH₂Cl₂ fraction (7.4 g) was chromatographed over silica gel column with a gradient of CH₂Cl₂/methanol supplying six fractions. Fraction 3 (3.9 g) was rechromatographed over a silica gel column with a gradient of MeOH in CH₂Cl₂ supplying four fractions. Fraction 3.1 (74.6 mg) provided the spruceanumines A-B (1-2) alkaloids mixture. Fraction 3.2 (103.2 mg) was rechromatographed over a silica gel column with a gradient of MeOH in CH₂Cl₂ supplying five fractions. Fraction 3.2.2 (20.6 mg) yielded the fendlerine (7) and aspidolimidine (8) alkaloids mixture, and fraction 3.2.4 (68.2 mg) afforded a mixture of obscurinervidine (9) and obscurinervine (10).

Spruceanumine A(1)

Amorphous solid, mp 195ºC;

= -101.7º (CHCl₃, c0.61); IR (KBr disk)V _max/cm^-1: 3100-2890 (C-H stretching), 1755 (C=O;) 1624, 1606, 1479 (benzene ring), 887, 739 (benzene ring). HRESI-MS ([M+H]⁺) Found: m/z425.2170. Calc. for C₂₄H₂₉N₂O₅⁺: 425.2071 (see Scheme 1); ¹H and ¹³C NMR: see Table 1.

Spruceanumine B (2)

Amorphous solid, mp 195ºC; = -101.7º(CHCl₃, c0.61); IR (KBr disk) V_max/cm^-1: 3100-2890 (C-H stretching), 1755 (C=O;) 1624, 1606, 1479 (benzene ring), 887, 739 (benzene ring). HRESI-MS ([M+H]⁺) Found: m/z439.2233. Calc. for C₂₅H₃₁N₂O₅⁺: 439.2227 (see Scheme 1); ¹H and ¹³C NMR: see Table 1.

Acknowledgements

The authors thank to Fundação de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ-RJ-Brazil) for a visitant research fellowship and grants, to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq-Brazil) for a research fellowship and grants and to Centro Nordestino de Aplicação e Uso da Ressonância Magnética Nuclear (CENAUREMN), Universidade Federal do Ceará (UFC), Fortaleza, Ceará, for the facilities in the obtention of the NMR (1D and 2D) spectra.

Supplementary Information

Available free of charge at http://jbcs.org.br, as PDF file.

Received: November 17, 2008

Web Release Date: April 30, 2009

FAPESP helped in meeting the publication costs of this article.

Supplementary Information

IR, ESI-MS, ESI-MS/MS, ¹H NMR, {¹H)-¹³C NMR, DEPT 135º¹³C NMR, ¹H-¹H-COSY, ¹H-¹H-NOESY, HSQC (¹J_CH) and HMBC (ⁿJ_CH, n=2 and 3) spectra of 1 and 2 are available free of charge at http://jbcs.sbq.org.br, as PDF file.

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Publication Dates

• Publication in this collection
  10 June 2009
• Date of issue
  2009

History

• Received
  17 Nov 2008
• Accepted
  30 Apr 2009