NMR structural analysis of <A HREF="#back2"></A>braznitidumine: a new indole alkaloid with 1,2,9-triazabicyclo[7.2.1] system, isolated from Aspidosperma nitidum (Apocynaceae)

Abstracts

The phytochemical study of the stem bark of Aspidosperma nitidum led to the isolation of a new type of indole alkaloid with a 1,2,9-triazabicyclo[7.2.1] system, which has been called braznitidumine 1. The characterization of its chemical structure was carried out by IR, UV, ESIMS, and ¹H, 13C, and 15N NMR by using 1D and 2D (¹H ¹H COSY, ¹H ¹H NOESY, ¹H 13C HSQC and ¹H 13C HMBC) experiments. ¹H ¹H NOESY results showed that 1 presents a folded conformation with the approximation of the indole and the imidazolidine di-hydropyran groups. This configuration was investigated by theoretical calculations involving geometry optimization (DFT/BLYP/6-31G*) for the conformational analysis of this alkaloid. It confirmed the distance between the two groups in agreement with the NOESY experimental data.

Apocynaceae; Aspidosperma nitidum; indole alkaloid; 1,2,9-triazabicyclo[7.2.1] system

O estudo fitoquímico das cascas do cerne de Aspidosperma nitidum permitiu o isolamento de um novo tipo de alcalóide indólico, contendo um sistema 1,2,9-triazabiciclo[7.2.1], que foi denominado braznitidumina 1. A caracterização de sua estrutura química foi realizada pela análises dos dados de IV, UV, ESI-EM e RMN de ¹H, 13C e 15N, empregando experimentos 1D e 2D (¹H ¹H COSY, ¹H ¹H NOESY, ¹H 13C HSQC e ¹H 13C HMBC). Pela análise do experimento ¹H ¹H NOESY, verificou-se que 1 apresenta uma conformação dobrada com aproximação entre os grupos indólico e imidazolidino-di-hidropirano. Essa configuração foi investigada por cálculos teóricos envolvendo otimizações de geometria (DFT/BLYP/6-31G*) para análise conformacional desse alcalóide, pela qual a distância entre os dois grupos mostrou-se compatível com as informações obtidas pelo experimento NOESY.

ARTICLE

NMR structural analysis of ^#braznitidumine: a new indole alkaloid with 1,2,9-triazabicyclo[7.2.1] system, isolated from Aspidosperma nitidum (Apocynaceae)

Maria M. Pereira^{I, II}; Antônio Flávio de C. Alcântara^{II, III}; Dorila Piló-Veloso^{*, II}; Délio S. Raslan^II

^IDepartamento de Medicamentos e Alimentos, Faculdade de Farmácia, Universidade Federal do Amazonas, 69010-300 Manaus-AM, Brazil

^IIDepartamento de Química, ICEx, Universidade Federal de Minas Gerais, 31270-901 Belo Horizonte-MG, Brazil

^IIIDepartamento de Química, ICE, Universidade Federal do Amazonas, 69077-000 Manaus-AM, Brazil

ABSTRACT

The phytochemical study of the stem bark of Aspidosperma nitidum led to the isolation of a new type of indole alkaloid with a 1,2,9-triazabicyclo[7.2.1] system, which has been called braznitidumine 1. The characterization of its chemical structure was carried out by IR, UV, ESIMS, and ¹H, ¹³C, and ¹⁵N NMR by using 1D and 2D (¹H ¹H COSY, ¹H ¹H NOESY, ¹H ¹³C HSQC and ¹H ¹³C HMBC) experiments. ¹H ¹H NOESY results showed that 1 presents a folded conformation with the approximation of the indole and the imidazolidine di-hydropyran groups. This configuration was investigated by theoretical calculations involving geometry optimization (DFT/BLYP/6-31G*) for the conformational analysis of this alkaloid. It confirmed the distance between the two groups in agreement with the NOESY experimental data.

Keywords: Apocynaceae, Aspidosperma nitidum, indole alkaloid, 1,2,9-triazabicyclo[7.2.1] system

RESUMO

O estudo fitoquímico das cascas do cerne de Aspidosperma nitidum permitiu o isolamento de um novo tipo de alcalóide indólico, contendo um sistema 1,2,9-triazabiciclo[7.2.1], que foi denominado braznitidumina 1. A caracterização de sua estrutura química foi realizada pela análises dos dados de IV, UV, ESI-EM e RMN de ¹H, ¹³C e ¹⁵N, empregando experimentos 1D e 2D (¹H ¹H COSY, ¹H ¹H NOESY, ¹H ¹³C HSQC e ¹H ¹³C HMBC). Pela análise do experimento ¹H ¹H NOESY, verificou-se que 1 apresenta uma conformação dobrada com aproximação entre os grupos indólico e imidazolidino-di-hidropirano. Essa configuração foi investigada por cálculos teóricos envolvendo otimizações de geometria (DFT/BLYP/6-31G*) para análise conformacional desse alcalóide, pela qual a distância entre os dois grupos mostrou-se compatível com as informações obtidas pelo experimento NOESY.

Introduction

The Aspidosperma Mart. (Apocinaceae) genus is native to Americas and is found from Mexico to Argentina.¹ The literature reports the presence of alkaloids in species of this genus, mainly indole ones.²Aspidosperma nitidum, popularly known as carapanaúba, is widely distributed in the Amazonas State, Brazil. The stem barks of this species are largely employed in folk medicine as a contraceptive, antimalarial, antiinflammatory (uterus and ovary), anticarcinogenic, antidiabetic, antileprosy, and for stomach upsets.³ Nevertheless, just one report has been found in literature on its chemical components.⁴ In that work the only reported alkaloid from this species was 10-methoxydihydrocorynantheol.

In this paper, we report the results of the phytochemical study of the Aspidosperma nitidum species. From the ethanolic extract was isolated the indole alkaloid (1) named braznitidumine, which has a very interesting bridged chemical structure containing a 1,2,9-triazabicyclo[7.2.1] system. The structure was determined by means of spectroscopic techniques, mainly ESIMS, IR and 1D and 2D NMR spectra.

Results and Discussion

Purification of the stem bark ethanol extract of Aspidosperma nitidum by silica gel column chromatography resulted in the isolation of the new indole alkaloid (1). Its molecular formula, C₂₄H₃₂N₄O₆ , was deduced from the precisely determined mass of the molecular ion peak [M⁺] at m/z 472.682 in the positive mode ESIMS. This molecular formula has 11 degrees of unsaturations, which is in agreement with ¹H (1D and 2D COSY), ¹³C (1D and 2D HSQC, HMBC) NMR spectral data. 1D ¹H and ¹³C ({¹H} and DEPT) and 2D (¹H ¹³C HSQC, ¹H ¹⁵N HSQC, ¹H ¹³C HMBC, ¹H ¹⁵N HMBC) NMR experiments led to the elucidation of a very interesting new indole alkaloid, name braznitidumine, as yet unreported in the literature.

A NH and a conjugated carbonyl group were identified by absorption bands at 3430 and 1697 cm^-1, respectively, observed in the IR spectrum of 1.⁵ The UV spectral data of 1 in methanol, l_max 208.80, 217.37, 272.20, and 305.50 nm, were characteristic of indole systems.⁶

Tables 1 and 2 show ¹H, ¹³C, and ¹⁵N NMR data of compound 1. The ¹H NMR spectrum showed most of the hydrogen resonance as unresolved broad signals (290 to 300 K). This spectrum revealed the presence of an indole group by singlets at d_H 7.01 (H-9) and 6.90 (H-6), which show correlations in the ¹H ¹³C HSQC with the ¹³C signals at d_C 101.1 (C-9) and 95.4 (C-6), respectively. It also displayed long range correlations in the ¹H ¹³C HMBC of the signal at d_H 7.01 with the ¹³C signals at d_C 95.4 (C-6), 102.1 (C-9b), 130.8 (C-5a), and 146.9 (C-7); of the signal at d_H 6.90 with the ¹³C signals at d_C 101.1 (C-9), 118.2 (C-9a), and 144.6 (C-8); of the signal at d_H 3.76, relative to six hydrogens of methoxyl groups (MeO-7 and MeO-8), with the ¹³C signals at d_C 95.4 (C-6), 101.1 (C-9), 144.6 (C-8), and 146.9 (C-7) (Figure 1). These last correlations indicate the phenyl moiety of an indole group, with two orto methoxyl substituents. The ¹H NMR spectrum also shows a singlet at d_H 11.13 (H-5), which is not correlated to any signal either by ¹H ¹H COSY or ¹H ¹³C HSQC, but that is correlated with the ¹⁵N signal at d_N 127.4 (N-5) by ¹H ¹⁵N HSQC. Through these last data, it was identified an NH by the indole group. Finally, a correlation was observed of the ¹H signal at d_H 6.90 (H-6) with the ¹⁵N signal at d_N 127.4 (N-5) by ¹H ¹⁵N HMBC.

The connectivity of the indole group with the rest of the molecule was deduced from the following ¹H ¹³C HMBC correlations: the signal at d_H 2.98 (H-10) with the ¹³C signals at d_C 102.1 (C-9b) via ²J, 127.3 (C-4a) via ³J, and 118.2 (C-9a) via ³J; the signal at d_H 3.84 (H-11) with the ¹³C signal at d_C 102.1 (C-9b) via ³J; the signal at d_H 4.90 (H-4) with the ¹³C signals at d_C 102.1 (C-9b) via ³J and 127.3 (C-4a) via ²J.

The homonuclear spin-spin couplings of H-10 with H-11 and of H-4 with both hydrogens having signals at d_H 2.00 (H-2) and 2.64 (H-3) were revealed by 2D ¹H ¹H COSY and so was the coupling between H-2 and H-3. Other ¹H ¹³C HSQC correlations were observed: H-3 with the ¹³C signal at d_C 32.3 (C-3); H-10 with the ¹³C signal at d_C 17.8 (C-10); H-11 with the ¹³C signal at d_C 54.3 (C-11); H-4 with the ¹³C signal at d_C 64.0 (C-4). No HSQC correlation was observed to the H-2 signal. In addition, ¹H ¹³C HMBC long range ²J and ³J correlations were observed for the H-11 signal and the MeO-4 signal at d_H 3.26 with the ¹³C signals at d_C 17.8 (C-10) and 64.0 (C-4), respectively. ¹H ¹⁵N HSQC shows correlation of the ¹H signal at d_H 2.00 (H-2) with the ¹⁵N signal at d_N 55.0 (N-2), which shows long range correlations with the signals at d_H 2.64 (H-3), 3.00 (H-16a), and 3.26 (MeO-4) in ¹H ¹⁵N HMBC.

Some broad and relatively very small signals characteristic of nitrogen-bonded aliphatic carbons⁷ were observed in the ¹³C NMR spectrum at d_C 27.6 (CH-16a), 32.3 (CH₂-3), 35.7 (CH-12a), 54.3 (CH₂-11), and 61.4 (CH₂-17). The ¹H ¹³C HMQC and ¹H ¹³C HSQC experiments confirmed these signals were due to hydrogenated carbons.

The substituted indole fragment shown in Figure 1 was inferred from all the data discussed above. In this fragment, the proximity of the NH indole and the oxygen of MeO-4 may lead to the formation of a hydrogen bond and thus explain the deshielding at d_H 11.13 observed for H-5.

In addition, the combination of ¹H and ¹³C NMR data with the IR spectrum revealed a molecular fragment of dihydropyran methyl ester (Figure 2). All the three signals of both non-hydrogenated ¹³C at d_C 107.6 (C-16) and 166.4 (C-18) as well as the one of ¹³C at d_C 155.0 (C-15), show correlations by ¹H ¹³C HSQC with the ¹H signal at d_H 7.53 (H-15), and are therefore compatible with the conjugated carbonyl system deduced from the IR spectrum. The ¹H signals at d_H 3.66 (MeO-18) and at d_H 3.00 (H-16a) are both correlated with the carbonyl ¹³C signal at d_C 166.4 (C-18) in ¹H ¹³C HMBC. On the other hand, H-16a is correlated with the nitrogenated ¹³C signal at d_C 27.6 (C-16a) in ¹H ¹³C HSQC and with the ¹³C signal at d_C 71.3 (C-13) in ¹H ¹³C HMBC. H-16a is coupled with a hydrogen with a signal at d_H 2.26 (H-12a) in ¹H ¹H COSY (Table 2). The latter shows spin-spin coupling with hydrogens presenting signals at d_H 4.55 (H-13) and 4.03 (H-17) in ¹H ¹H COSY and it correlates with the nitrogenated ¹³C signal at d_C 35.7 (C-12a) in ¹H ¹³C HSQC. The spin-spin coupling between H-12a and H-17 is weak and is only detected by ¹H ¹H COSY, possibly due to a W ⁴J coupling.

The methyl signal at d_H 1.44 (Me-13), which is correlated with the ¹³C signal at d_C 17.9 (Me-13) in ¹H ¹³C HSQC, is correlated with the signal at d_H 4.55 (H-13) in ¹H ¹H COSY and with the ¹³C signal at d_C 35.7 (C-12a) in ¹H ¹³C HMBC. Finally, in ¹H ¹³C HSQC, the ¹³C signal at d_C 61.4 (C-17) is correlated with the signal at d_H 4.03 and so must be the remaining of the cited aliphatic nitrogenated CH₂ detected in the DEPT experiment. As its attached hydrogen shows weak coupling with H-12a in the ¹H ¹H COSY and also because of its chemical shift, this CH₂ group is thought to be linked to two nitrogen atoms in a fused 5-6 ring system, i.e. a substituted fused imidazolidine-pyran system fragment of 1, as shown in Figure 2. The literature reports some other indole alkaloids isolated from Aspidosperma species as isomitrafiliane, 3-isoajamalicine, mitrafiline, and isomitrafilinic acid with a substituted pyran fragment, but none with the fused imidazolidine-pyran system shown in Figure 2.⁸ Taking into account the molecular formula and the deduced structure of both the indole (Figure 1) and the imidazolidine-pyran (Figure 2) fragments, one of the aliphatic nitrogen atoms of each of these fragments must be shared to connect them. Hence, the structure containing the bridgehead-nitrogen bridged-bicyclic system shown in Figure 3 is proposed for braznitidumine 1. In accord with this, ¹H ¹⁵N HMBC shows long range correlation of the signal at d_N 55.0 (N-2) with the signal at d_H 2.64 (H-3, via ²J), with the signal at d_H 3.00 (H-16a, via ³J), and with the signal at d_H 3.26, showing weak coupling (MeO-4, via ⁵J). No other correlations were observed in the NMR experiments performed (¹H ¹H COSY, ¹H ¹³C HMBC, ¹H ¹⁵N HMBC) via scalar homonuclear or heteronuclear spin-spin coupling of imidazolidine-pyran system atoms with indole fragment atoms. However, the proposed structure 1 was confirmed by dipolar interaction due to spatial proximity observed as correlations in ¹H ¹H NOESY experiment (Table 2). Figure 3 shows the ¹H ¹H NOESY correlations detected for braznitidumine 1.

No correlation of the signal at d_H 2.26 (H-12a) with the signal at d_H 3.00 (H-16a) in ¹H ¹H NOESY was observed. The correlations of H-12a with both Me-13 and H-17 show that they are on the same side of the molecule. The latter correlates also with OMe-4. Thus, the relative configuration of H-12a is established relatively to those groups in the molecule. However, nothing may be deduced for H-16a configuration, because the only correlation it presents is with the NH-2 (Figure 3). Therefore, H-16a may be cis or trans relatively to H-12a.

The correlations of MeO-18 with H-4, H-5, and H-6, as well as the correlation of Me-13 with H-11 indicates their spatial proximity, which could be explained if braznitidumine show a folded conformation. This conformation may be accounted for if the imidazolidine-pyran ring fusion is trans or cis as may be seen by the Dreiding model as well by the DFT/BLYP/6-31G* optimized geometry (Figures 4a and 4b, respectively).

Figure 4 shows the DFT/BLYP/6-31G* optimized geometry of 1 considering the solvent (DMSO) effect by PCM method. By single bond rotations of this geometry, the folding could be verified from spatial proximity of Me-13 with H-11, as well as of MeO-18 with H-4, H-6, and H-5. In the folded trans configuration (Figure 4a), the hydrogen interatomic distances are: CH₃O-18 – H-5 = 2.470 Å; CH₃O-18 – H-6 = 4.326 Å; CH₃O-18 – H-4 = 1.210 Å; CH₃O-4 – H-17 = 3.140 Å; CH₃-13 – H-17 = 4.320 Å. In the folded cis configuration (Figure 4b), the hydrogen interatomic distances are: CH₃O-18 – H-5 = 2.241 Å; CH₃O-18 – H-6 = 2.594 Å; CH₃O-18 – H-4 = 1.192 Å; CH₃O-4 – H-17 = 2.792 Å; CH₃-13 – H-17 = 2.464 Å.

Experimental

General Procedures

Melting point was obtained on a Mettler FP82 HT and was uncorrected. FTIR spectrum was determined in KBr disk on an FTIR Perkin Elmer Spectrum 200 spectrometer. ESI-MS spectrum was obtained in positive mode in a Q-TOF Micro^TM MICROMASS spectrometer. UV spectrum was obtained in 1% methanol solution in a Perkin Elmer 202 spectrometer. Chromatographic purification was carried out on silica gel (70-230 mesh). In the thin layer chromatography analysis was used silica gel 60F254 and 60G mixture (1:3).

¹H and ¹³C NMR spectra were measured on a Bruker DRX400 – AVANCE spectrometer, with inverse probes and field gradient, operating at 400.129 and 100.613 MHz, respectively. DMSO-d₆ was used as a solvent (the sample was dissolved in 0.75 mL of solvent and transferred to a 5 mm NMR tube), with TMS as an internal reference (d = 0). ¹⁵N spectra were measured on a Bruker DRX400 AVANCE spectrometer with inverse probe and field gradient, operating at 40.549 MHz, at Centro Nacional de Ressonância Magnética Nuclear (CNRMN), UFRJ. DMSO-d₆ was used as a solvent with urea as external reference (78 ppm in DMSO-d₆). Chemical shifts are given in the d-scale (ppm) and coupling constants J in Hz. Experiments were carried out using pulse sequence and programs provided by the manufacturer. 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 (2D) homonuclear and heteronuclear shift correlation spectra by using a multinuclear inverse detection 5 mm probe with field gradient at z axis. For ¹H ¹³C HMBC, three delays for evolution of long range coupling [1/(ⁿJ_C-H)] 65, 125, and 130 ms] were used. For the 2D ¹H ¹H NOESY experiment two mixing times (350 and 700 ms) were pre-optimized by a specific Bruker computer program.

Theoretical calculations

Theoretical studies were carried out using the software package GAUSSIAN03.⁹ Spatial arrangements determined through NOESY experiments were used as initial models for geometry optimization calculations by the semi-empirical PM3 method in the gaseous state.¹⁰ Geometries obtained by PM3 method were optimized again by the Density Functional Theory (DTF)¹¹ method with BLYP functionals¹² with a 6-31G*¹³ basis set (DFT/BLYP/6-31G*). All structures obtained by theoretical calculations were characterized as true energy minima in PES through frequency calculations (when the frequencies are real, they correspond to a true minimal energy structure). Calculations of solvent effects were performed for optimized geometries in the DFT/BLYP/6-31G* level by using the Polarizable Continuum Model (PCM) at the same calculation level.¹⁴

Material and isolation of 1

Stem of A. nitidum was collected near Manaus City, Amazon State in June 2000. A voucher specimen (181832) is deposited in the Herbarium of Instituto Nacional de Pesquisas da Amazônia (INPA). The dried and powered stem barks (1.5 kg) of A. nitidum were extracted with EtOH and yielded 173 g of crude ethanol extract after the removal of the solvent. This extract was chromatographed on silica gel column and eluted with methylene chloride, acetyl acetate, and ethanol. The ethanol solution evaporated under vacuum yielded 40.20 g of a residue, which was rechromatographed on silica gel with ethyl acetate:methanol 9:1 as an eluent. Compound 1 (120.0 mg) was obtained as a yellow solid; mp 272.9-273.1 °C; Dragendorff positive test for alkaloids (orange-yellow color); IR (KBr) n_max/cm^-1: 3430, 2949, 1697, 1485, 1442, 1308, 1285. MS (ESI+) m/z: 472.682 (M⁺; 0.3), 427.214 ([M–C₂H₆O]⁺; 1). For NMR data see Tables 1 and 2.

Conclusions

The structure of alkaloid braznitidumine (1) could be elucidated through the analysis of its 1D and 2D ¹H, ¹³C, and ¹⁵N NMR spectra. It showed a new and interesting framework including a 1,2,9-triazabicyclo[7.2.1] system in a folded molecule. The new compound may show either a trans or a cis fused imidazolidine-pyran system fragment in its structure. Geometry optimization calculations (DFT/BLYP/6-31G*) are compatible with nOe data and corroborate ¹H ¹H NOESY results.

Acknowledgments

The authors thank CNPq, FAPEAM, and FAPEMIG the financial support and Prof. Fábio C. L. Almeida from Centro Nacional de Ressonância Magnética Nuclear (CNRMN), UFRJ, the ¹⁵N NMR spectra. M. M. Pereira thanks CAPES for PICDT grant.

Supplementary Information

Supplementary data are available at free of charge at http://jbcs.sbq.org.br, as PDF file.

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12. Becke, A. D.; Phys. Rev. A 1988, 38, 3098; Lee, C.; Yang, W.; Parr, R. G.; Phys. Rev. B 1993, 37, 785.

13. Ditchfield, R.; Hehre, W. J.; Pople, J. A.; J. Chem. Phys. 1971, 54, 724; Hehre, W. J.; Ditchfield, R.; Pople, J. A.; J. Chem. Phys. 1972, 56, 2257; Hariharan, P. C.; Pople, J. A.; Theor. Chim. Acta 1973, 28, 213; Hariharan, P. C.; Pople, J. A.; Mol. Phys. 1974, 27, 209; Gordon, M. S.; Chem. Phys. Lett. 1980, 76, 163.

14. Ditchfield, R.; Hehre, W. J.; Pople, J. A.; J. Chem. Phys. 1971, 54, 724; Hehre, W. J.; Ditchfield, R.; Pople, J. A.; J. Chem. Phys. 1972, 56, 2257; Cossi, M.; Barone, V.; Camimi, R.; Tomasi, J.; Chem. Phys. Lett. 1996, 255, 327; Barone, V.; Cossi, M.; Tomasi, J.; J. Chem. Phys. 1997, 107, 3210.

Received: April 27, 2006

Published on the web: August 22, 2006

Supplementary Information

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#

This name is an homage to Professor Raimundo Braz-Filho. This article was submitted to the special issue dedicated to Professor Raimundo Braz-Filho on the occasion of his 70

^th birthday.

Publication Dates

• Publication in this collection
  29 Jan 2007
• Date of issue
  Dec 2006

History

• Accepted
  22 Aug 2006
• Received
  27 Apr 2006