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Muscicapines, a new class of guaiane-type sesquiterpene alkaloids from Croton muscicapa

Abstracts

Three new guaiane-type sesquiterpene alkaloids, muscicapine A (1), muscicapine B (2), and muscicapine C (3) were isolated from the roots of Croton muscicapa. The structures were established by analysis of spectroscopic data, mainly 1D and 2D NMR and MS. This is the first report of a new class of guaiane-type sesquiterpene alkaloids.

Croton muscicapa; Euphorbiaceae; guaiane sesquiterpene alkaloid; muscicapines


Três novos alcalóides sesquiterpênicos do tipo guaiano, muscicapina A (1), muscicapina B (2) e muscicapina C (3) foram isolados das raízes de Croton muscicapa. As estruturas foram elucidadas através de análises espectroscópicas, principalmente RMN de 1D e 2D e espectrometria de massas. Este é o primeiro relato na literatura de uma nova classe de alcalóides sesquiterpênicos com esqueleto guaiano.


ARTICLE

Muscicapines, a new class of guaiane-type sesquiterpene alkaloids from Croton muscicapa

Vicente T. de Araújo-JúniorI; Marcelo S. da SilvaI; Emídio V. Leitão da-CunhaI,II; Maria de Fátima AgraI; Petrônio F. de Athayde-FilhoI; Ivo J. Curcino VieiraIII; Raimundo Braz-FilhoIII; and José M. Barbosa-FilhoI,* * e-mail: jbarbosa@ltf.ufpb.br

ILaboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CP 5009, 58051-970 João Pessoa - PB, Brazil

IIDepartamento de Farmácia e Biologia, Universidade Estadual da Paraíba, 58100-000 Campina Grande - PB, Brazil

IIISetor de Química de Produtos Naturais-LCQUI, Universidade Estadual do Norte Fluminense, 28015-620 Campos - RJ, Brazil

ABSTRACT

Three new guaiane-type sesquiterpene alkaloids, muscicapine A (1), muscicapine B (2), and muscicapine C (3) were isolated from the roots of Croton muscicapa. The structures were established by analysis of spectroscopic data, mainly 1D and 2D NMR and MS. This is the first report of a new class of guaiane-type sesquiterpene alkaloids.

Keywords:Croton muscicapa, Euphorbiaceae, guaiane sesquiterpene alkaloid, muscicapines

RESUMO

Três novos alcalóides sesquiterpênicos do tipo guaiano, muscicapina A (1), muscicapina B (2) e muscicapina C (3) foram isolados das raízes de Croton muscicapa. As estruturas foram elucidadas através de análises espectroscópicas, principalmente RMN de 1D e 2D e espectrometria de massas. Este é o primeiro relato na literatura de uma nova classe de alcalóides sesquiterpênicos com esqueleto guaiano.

Introduction

Plants of the genus Croton (Euphorbiaceae), widely distributed throughout tropical areas, are used in South America as sources of traditional medicines for the treatment of wounds, inflammation, and cancer.1 Their species are known to be rich in terpenoids (essential oils and diterpenes) and alkaloids (indole and mainly isoquinoline derivatives).2

In a search for new drugs from plants of the genus Croton, a previous work described the isolation of clerodane and labdane diterpenes from Croton polyandrus Spreng3 and diterpenes and alkaloids from Croton moritibensis Baill.4 In this work, the chemical investigation of Croton muscicapa Müll. Arg., a native shrub to Northeastern Brazil, popularly known as "velame de cheiro", is reported. From the ethanolic extract of the roots, four alkaloids (1-4) were isolated through a series of partitions followed by chromatographic procedures.5 The alkaloids isolated were the known nicotine derivative anabasine (4) and three new guaiane-type sesquiterpene alkaloids 1, 2 and 3 which are described here for the first time (Figure 1).


The literature on components of essential oil refers to an enormous number of lower terpenes, but no mono- and sesquiterpene alkaloids. However, alkaloids derived from diterpenes and steroids are widespread in plants. To the best of our knowledge the alkaloid with the carbon skeleton closest to the guaiane-type sesquiterpene alkaloids is epiguaipyridine (5) isolated from the essential oil of Pogostemon pachouli.6

Results and Discussion

The known alkaloid anabasine (4) was identified by spectral data, mainly 1H and 13C NMR spectra, and comparison with literature values.7

Alkaloid 1 was obtained as an amorphous yellow solid and showed [a]2 0D – 44º (CHCl3, c 0.05). The IR spectrum revealed bands at nmax 3366 (N-H), 1650 (conjugated carbonyl group stretching) and 2932-2833 cm-1 (C-H stretching).8 The EIMS of 1 (Scheme 1) showed a molecular ion peak at m/z 245 daltons ([M]•+) which, together with 1H and 13C (HBBD and APT) NMR spectral data (Tables 1 and 2), were in agreement with the molecular formula C15H19NO2, compatible with a sesquiterpenoid skeleton with an additional nitrogen atom.6


The 1H and 13C NMR spectral data of 1, including 1H-13C-COSY-nJ CH (n=1, HMQC; n=2 and 3, HMBC) (Table 2), are in agreement with a guaiane sesquiterpene skeleton oxidized with the formation of an enamine group between C-10 (dC 110.30) and C-14 (dC 159.27). The location of the enamine group at C-10 (dC 110.30) was unequivocally deduced by HMBC correlations between H-14 (dH 7.89) and carbons C-10 (dC 110.30, 2JCH) and C-9 (dC 188.32, 3JCH). The presence of a primary enamine was suggested by the correlations of H-14 (dH 7.89, dd, J 8.2 and 13.9 Hz) and amino group NH2 (dH 11.71) in the 1H-1H-COSY spectrum. The 2-ene-1,4-dione system was confirmed by HMBC correlations between the carbonyl carbon C-9 (dC 188.32,) and hydrogens H-14 (dH 7.89, 3JCH) and H-8 (dH 6.76, 2JCH) (Table 2). The isopropyl group, was characterized by 1H NMR spectra (1D and 2D 1H-1H-COSY) by two doublets (J 6.7 Hz) at dH 1.15 and 1.14 (H3-12 and H3-13) and a multiplet at dH 3.38 (H-11). This together with HMQC (CH3-12 and CH3-13: dH/dC 1.15/22.84 and 1.14/22.41; CH-11: dH/dC 3.38/30.42) and HMBC correlations (Table 2) of C-7 with both methyl groups H3-12 (dH 1.15, 3JCH) and H3-13 (dH 1.14, 3JCH), H-8 (dH 6.76, 2JCH) and H-11 (dH 3.38, 2JCH) confirm the presence of the isopropyl group at C-7. The presence of the methyl group CH3-15 at position C-4, was determined by the doublet signal at dH 1.17 (J 6.9 Hz) observed in the 1H (1D and 2D 1H-1H-COSY) NMR spectra (Table 1) and by the HMBC correlations with the quaternary sp2 carbon C-5 (dC 139.73) (Table 2). Additional heteronuclear long-range correlations observed in the HMBC are summarized in Table 2.

The alkaloids 2, [a]20D – 6º (CHCl3, c 0.02), and 3, [a]20D – 9º (CHCl3, c 0.05), were separated by recycling HPLC using reverse phase silica gel. Analysis of the spectral data of 1, 2, and 3 allowed their identification as guaiane sesquiterpene alkaloids containing the common basic skeleton. The EIMS of isomers 2 and 3 (Scheme 1) showed molecular ion peaks at m/z 315 daltons ([M]•+), which, together with 1H and 13C (HBBD and APT) NMR spectral data (Table 2), suggested the deduction of the molecular formula C20H29NO2. Comparison of this molecular formula with that of 1 (C15H19NO2) suggested the presence of an additional reduced isoprene moiety C5H10 (C20H29NO2 - C15H19NO2 = 70 daltons) in 2 and 3.

The isopentyl group bonded to the nitrogen atom of 2 was characterized as 3-methylbutyl by the doublet signal at dH 0.96 (J 6.6 Hz, H3-4' and H3-5') and multiplet signals at dH 1.74 (H-3'), 1.60 (H2-2') and 3.50 (H2-1') in the 1H (1D and 2D 1H-1H-COSY) NMR, which showed heteronuclear correlations with 13C signals at dC 22.28 (CH3-4' and CH3-5'), 25.64 (CH-3'), 39.47 (CH2-2'), and 49.13 (CH2-1') in the HMQC spectrum (Table 1). The location of this group at the nitrogen atom [N-(3-methylbutyl) derivative of 1] was suggested by the 1H and 13C chemical shifts of the methylene group CH2-1' (dH 3.50 and dC 49.13) and confirmed by HMBC correlations between CH-14 (dC 160.40) and 2H-1' (dH 3.50, 3JCH) (Table 2). Additional HMBC correlations are indicated in Table 2.

The presence of a 2-methylbutyl group in 3 was deduced by the signals at dH 1.01 (d, J 6.9 Hz, H3-5'), 0.95 (t, J 7.7, H3-4'), 1.27 (m, H2-3'), 1.48 (m, H-2'), and 3.38/3.31 (m, H2-1') in the 1H (1D and 2D 1H-1H-COSY) NMR spectra, which was confirmed by the HMQC and HMBC spectra (Tables 1 and 2). HMBC correlations between CH-14 (dC 160.87) and H2-1' (dH 3.38 and 3.31), together with the 13C chemical shift of the methylene group CH2-1' (dC 56.91) (Table 2) were used to locate the 2-methylbutyl group at the nitrogen atom [N-(2-methylbutyl) derivative of 1]. Additional heteronuclear long-range couplings are summarized in Table 2. The alkaloids were named muscicapine A, muscicapine B, and muscicapine C, respectively.

To our knowledge, the muscicapines represent the first members of a new class of guaiane-type sesquiterpene alkaloids in plants. Biogenetically, their precursor should be the guaiadiene 6, which had the CH3-14 oxidized to the corresponding aldehyde, which, after reductive amination, incorporated the amino groups. The introduction of a double bond between C-10 and C-14 by NADP/FAD and oxidation of C-6 and C-9 furnishes muscicapine A (1). The other two muscicapines B (2) and C (3) probably originate by condensation of the aldehyde with the corresponding 3-methyl and 2-methyl butylamines probably originated by descarboxylation of leucine and isoleucine, respectively.

Experimental

General procedures

1H NMR (500 MHz) and 13C NMR (125 MHz) were recorded at room temperature with a Bruker NMR spectrometer (DRX 500) with an inverse multinuclear 5 mm probe head equipped with a shielded gradient coil. The spectra were recorded in CDCl3, and the solvent signals (7.27 and 77.0 ppm) were used as references. The chemical shifts (d) are given in ppm, and the coupling constant (J) in Hz. All programs used for performing the 2D NMR experiments are part of the Bruker library. EIMS data were determined on a JEOL mass spectrometer using direct insertion probe at 70 eV. IR spectra were obtained using KBr pellets in a Shimadzu Infrared Spectrometer model FTIR-8300. The optical rotation [a] value was obtained on a Perkin Elmer model 343 Digital Polarimeter using CHCl3 as solvent. CC was carried out over alumina (activity II-III, 70-230 mesh ASTM) using different mixtures of chloroform and methanol gradient. The alkaloids were purified by repeated PTLC (1 mm thick, 20 x 20 cm Si gel PF254 plates) and recycling HPLC using Shimadzu Chromatograph in reverse phase (ODS, Shim-Pack (H), 5 µm, MeOH, 3.0 mL min-1).

Plant material

Croton muscicapa Muell. Arg. was collected in the "caatinga" vegetation near the municipality of Caicó, State of Rio Grande do Norte, Brazil in April 2002. A voucher specimen (AGRA 5995) is deposited in the Herbarium Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraíba, Brazil.

Extraction and isolation

The dried and powdered roots (2 kg) of C. muscicapa were extracted at room temperature using EtOH 95%, furnishing, after solvent evaporation under vacuum, 73.5 g of extract. This extract was dissolved in 3% HCl, filtered over celite and extracted with CHCl3. The aqueous phase was alkalinized with NH4OH and extracted again with CHCl3 furnishing 3.2 g of a residue. The residue was chromatographed on a neutral aluminum oxide column with a gradient of MeOH in CHCl3 yielding seventy fractions. Alkaloid 4 (583 mg) was purified from fractions 5-8 (1.0 g) after rechromatography on silica gel PTLC using CHCl3:MeOH (99.5:0.5). Fractions 42-49 (15.0 mg) were purified by recycling HPLC using reverse phase with MeOH as solvent to give alkaloid 2 (2.1 mg) and alkaloid 3 (1.7 mg). Alkaloid 1 (5.8 mg) was purified from the fractions 67-70 (15.0 mg) using a silica gel column and a gradient of MeOH in CHCl3 as eluent. Alkaloid 1 was acetylated with acetic anhydride in the presence of pyridine (1:2) overnight at room temperature to furnish the monoacetate 1a (2.3 mg).

Muscicapine A (1)

C15H19NO2; red oil; [a]20D – 44º (CHCl3, c 0.05); IR (KBr) nmax/cm-1 3366, 2932, 2833 and 1650); EIMS (70 eV) m/z (rel. int.): 245 (73, M+•), 230 (29), 202 (100), 174 (14), 91 (12), 79 (11), 77 (16), 53 (13); NMR data (1H and 13C NMR, 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC) are given in Tables 1 and 2.

Muscicapine A acetate (1a)

Acetylation of 1 (4 mg) with acetic anhydride (0.3 mL) in pyridine (0.5 mL) and work-up in the usual way afforded 1a (3.1 mg) as a yellow oil. NMR data (1H and 13C NMR) are given in Table 1.

Muscicapine B (2)

C20H29NO2; yellow oil; [a]20D – 6º (CHCl3, c 0.02); EIMS (70 eV) m/z (rel. int.): 315 (87, M+•), 300 (45), 287 (15), 273 (18), 272 (100), 258 (38), 244 (42), 230 (20), 216 (13), 202 (27), 187 (13), 55 (13), 43 (69), 41 (43); NMR data (1H and 13C NMR, 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC) are given in Tables 1 and 2.

Muscicapine C (3)

C20H29NO2; yellow oil; [a]20D – 9º (CHCl3, c 0.05); EIMS (70 eV) m/z (rel. int.): 315 (79, M+•), 300 (41), 273 (18), 272 (100), 258 (8), 245 (18), 244 (94), 230 (16), 228 (12), 216 (13), 202 (18), 187 (20), 91 (13), 77 (12), 55 (15), 43 (850, 41 (49); NMR data (1H and 13C NMR, 1H-1H COSY, 1H-13C HMQC and 1H-13C HMBC) are given in Tables 1 and 2.

Acknowledgments

The authors are grateful to the Programa de Apoio ao Desenvolvimento Científico e Tecnológico (PADCT)/Financiadora de Estudos e Projetos (FINEP), Instituto do Milênio do Semi-Árido (IMSEAR), CNPq and FAPERJ for grants, and to CNPq for research fellowships.

Received: July 8, 2003

Published on the web: March 9, 2005

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  • Publication Dates

    • Publication in this collection
      09 Aug 2005
    • Date of issue
      June 2005

    History

    • Accepted
      09 Mar 2005
    • Received
      08 July 2003
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