Quinoline alkaloids and friedelane-type triterpenes isolated from leaves and wood of Esenbeckia alata kunt (Rutaceae)

Abstract

This work describes the phytochemical exploration of the ethanol extract from leaves and wood of Esenbeckia alata, leading to the isolation and identification of quinoline alkaloids 4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one, N-methylflindersine, dictamine, kokusaginine, Γ-fagarine, flindersiamine, as well as the fridelane-type triterpenes, frideline, fridelanol and its acetate derivative. Identification of these compounds was based on full analyses of spectroscopic data (¹H, 13C, 1D, 2D, IR, MS) and comparison with data reported in literature. Compound 4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one is reported for the first time for the genus Esenbeckia.

Esenbeckia alata; quinoline alkaloids

ARTIGO

Quinoline alkaloids and friedelane-type triterpenes isolated from leaves and wood of Esenbeckia alata kunt (Rutaceae)

Luis Enrique Cuca-Suarez^I; Ericsson David Coy Barrera^I, ^#; Juan Manuel Alvarez CaballeroII, ^*

^IDepartamento de Química, Facultad de Ciencias, Universidad Nacional de Colombia, Kr 30 45-03, Ciudad Universitaria, Bogotá D.C., Colombia

^IIFacultad de Ciencias Básicas, Universidad del Magdalena, Kr 32 22-08 Santa Marta D.T.C.H., Colombia

ABSTRACT

This work describes the phytochemical exploration of the ethanol extract from leaves and wood of Esenbeckia alata, leading to the isolation and identification of quinoline alkaloids 4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one, N-methylflindersine, dictamine, kokusaginine, Γ-fagarine, flindersiamine, as well as the fridelane-type triterpenes, frideline, fridelanol and its acetate derivative. Identification of these compounds was based on full analyses of spectroscopic data (¹H, ¹³C, 1D, 2D, IR, MS) and comparison with data reported in literature. Compound 4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one is reported for the first time for the genus Esenbeckia.

Keywords:Esenbeckia alata; quinoline alkaloids.

INTRODUCTION

Rutaceae family is gathered in 140 genera including ca 1600 species, which are distributed in temperate and tropical zones on both hemispheres¹ involving biological forms such as trees, shrubs and herbs.² One of the most abundant taxa for Rutaceae family is the Esenbeckia genus involving a number of 30 species of the family.³ There are several reports to this taxon indicating their uses in traditional medicine and its biological activity. In Mexico, leaves and roots of E. yaxhoob are used by local people for the treatment of gastrointestinal diseases, epilepsy, headaches and as antidiuretic agent.^4,5 Metabolites isolated from E. leiocarpa exhibited antifeedant activity against worm Pectinophora gossypiella.⁶ In addition, a geranylcoumarin has been isolated from E. febrifuga, which significantly inhibited the growth of tropical parasite Leishmania major.⁷ Phytochemical studies on this genus has allowed the isolation of several secondary metabolites such as flavonoids from E. yaxhoob,⁴E. grandiflora subsp. brevipetiolata, E. almawillia, and E. berlandieri ssp. Acapulcensis;⁸ terpenoids from E. conspecta, E. ovata, E. stephani, E. yaaxhokob, E. almawillia, and E. nesiotica;⁹ limonoids from E. litoralis, E. flava and E. berlandieri;¹⁰ cinnamic acid derivatives from E. almawillia;¹¹ alkaloids from E. pentaphylla,¹²E. grandiflora, E. litoralis,¹³E. almawillia,^11,14 and E. belizencis;¹⁵ coumarins from E. grandiflora, E. litoralis,¹³E. febrífuga,⁷ and E. pentaphylla.¹² From the former group of metabolites, quinoline alkaloids are considered as taxonomic markers for Esenbeckia genus and they have been identified in various species of the genus such as E. belizencis,¹⁵E. pentaphylla,¹²E. flava,¹⁰E. grandiflora and E. litoralis.¹³

E. alata is a medicinal shrub whose ecology is diverse being identified in different colombian areas. On the Atlantic coast of Colombia, its aerial parts are used as febrifuge and insecticide.¹⁶ This fact has prompted that many studies had been particularly focused on this plant. In a previous work, phytochemical examination of the ethanol extract of the bark from E. alata led to the isolation of four metabolites which were identified as 5-hydroxy-2-methylchromanone, the lignan (-)-episesamin, the amide pellitonin and sitosterol. In that study it was evaluated the antimicrobial activity of the obtained lignan, showing significant results against the microorganisms Bacillus subtilis, Klebsiella pneumoniae and Pseudomonas aeruginosa.¹⁶ On the other hand, from the ethanol extract of the leaves of this species were isolated furanocoumarins, pyranocoumarins, lignans and furoquinoline alkaloids.¹⁷ The present work aims to contribute to the chemotaxonomic hoard of genus through chemical study of the ethanol extract of both leaves and wood of E. alata, consisting the first phytochemical report for the ethanol extract from wood of E. alata, herein described therefore is the isolation and identification of quinoline alkaloids and friedelane-type triterpenes.

EXPERIMENTAL

General procedures

Silica gel 60 (0.063-0.200 mm, 70-230 mesh ASTM) (Merck) was used for column chromatography (CC) and silica gel 60 F₂₅₄ chromatoplates Merck, (20 x 20 and 0.30 mm thickness) for thin layer chromatography (TLC). Preparative TLC was held on plate coated with Merck silica gel 60G F₂₅₄ (1.0 and 2.0 mm thickness). TLC was revealed in UV lamp (254 nm), iodine vapor and ceric ammonium sulfate solution in sulfuric acid with subsequent heating at 100 ºC. Vacuum column chromatography (VCC) was developed with silica gel 60 G, Merck. ¹H NMR (400 MHz) and ¹³C NMR (100 MHz) spectra were recorded on a Bruker Avance 400 using TMS as internal standard, in deuterated chloroform (CDCl₃) as solvent. High Resolution Mass spectra (HRMS) were determined on a Shimadzu IT-ToF spectrometer (with an ESI source and in the positive ion mode), and electron impact mass spectra (IEMS) were recorded in a Jeol JMS-SX102A spectrometer. Infrared spectra (IR) were taken on film on a KBr window, in a Perkin Elmer 500 series FTIR Panagon 1000. Optical rotations were measured on a polartronic-E Schmidt-Hänsch polarimeter in CHCl₃ at 20 °C. Acetylation was carried out by conventional procedure, by refluxing with pyridine and acetic anhydride for 2 h.

Plant material

Plant sample corresponding to the wood and leaves of Esenbeckia alata was collected in Los Montes de María (9°39'58.77"N and 75°20'3.45"O), Department of Bolívar, Colombia, on October 2005. Specimen was identified by the botanist E. Carbonó and a voucher was deposited in the Herbarium of the University of Magdalena with the collection number 001(UTMC).

Extraction and fractionation

Isolation of metabolites from wood

Wood of E. alata was air dried at room temperature for 8 days. A total of 1200 g sample were extracted by percolation using 96% EtOH (15 L) for 10 days. Resulting ethanol extract (called EaM) was concentrated under reduced pressure to obtain 16.3 g crude extract. EaM was subjected to CC (80 x 5 cm) on silica gel using toluene/iPrOAc 9:1 to 1:1 (14 L) as elution system, collecting 138 fractions (100 mL each one), combined into 16 fractions by their CCD profiles (EaM1-EaM16). EaM6 fraction (1088 mg) was subjected to successive washing with methanol (4 x 3 mL) to obtain a liquid subfraction (EaM6L) (647 mg) and a solid (EaM6S) (421 mg). EaM6L and EaM6S subfractions were separately purified by CC (40 x 3.5 and 50 x 2.5 cm, respectively) on silica gel eluting with petroleum ether/EtOAc 7:3 (0.6 L) affording 1 (30 mg), and 3 (8 mg), respectively. EaM7 fraction (793 mg) was subjected to CC (40 x 3.5 cm) on silica gel eluting with toluene/EtOAc 7:3 (0.8 L) obtaining 2 (33 mg), and 4 (3 mg). EaM9 fraction (1682 mg) was purified by CC (60 x 3.5 cm) on silica gel using toluene/EtOAc 8:2 (3 L) as eluting system yielding 5 (15 mg).

Isolation of metabolites from leaves

Dried and powdered leaves (2875 g) of the specimen were extracted by percolation using 96%EtOH (25 L) for 10 days. Resulting ethanol extract (called EaH) was concentrated under reduced pressure to obtain 113.8 g crude extract. A sample of this extract (65 g) was fractionated by VCC (70 x 7 cm) on silica gel using hexane/acetone (18 L) as mobile phase by increasing polarity producing 23 fractions (EaH1-EaH23). EaH5 fraction (3220 mg) was subjected to VCC (28 x 5 cm) on silica gel using dichloromethane/acetone (2.5 L) as eluent by increasing polarity to collect ten subfractions (EaH5.1-EaH5.10). EaH5.3 subfraction (987 mg) was purified by VCC (20 x 2.5 cm) eluting with dichloromethane/acetone 9:1 (2 L) to obtain 6 (102 mg). A similar procedure was carried out on EaH6 fraction (2330 mg), which was subjected to VCC (28 x 5 cm) on silica gel with hexane/acetone (4L) as mobile phase obtaining EaH6.4 subfraction (410 mg), which was then purified by VCC (20 x 1.5 cm) eluted with dichloromethane/acetone 9:1 (0.7 L) to yield 4 (76 mg). EaH10 fraction (1367 mg) was subjected to VCC (20 x 2.5 cm) on silica gel using hexane/EtOAc (2.5 L) by increasing polarity as eluent, collecting 25 subfractions (EaH10.1-EaH10.25). From EaH10.15 (92 mg) and EaH10.21 (146 mg) subfractions resulted into two solids, which were recrystallized from chloroform/methanol 2:8 (35 mL), affording 7 (76 mg) and 8 (130 mg), respectively. In order to increase the solubility in chloroform and thus facilitate spectra recording, 8 was acetylated to yield 9 (56 mg), which was recrystallized from chloroform-methanol 9:1.

4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one (1): Needles, mp 199-200 ºC; HRESIMS [M+H]⁺m/z 258.1472, calcd for C₁₆H₂₀NO_2, 258.1494; IR (KBr, cm^-1) 3272, 1731, 1633, 1467, 756; ¹H RMN (CDCl₃, 400 MHz): δ 7.5 (ddd, J =1.5, 7.2, 8.6 Hz, H-7), 7.8 (dd, J =1.5, 7.2 Hz, H-5), 7.3 (d, J =8.6 Hz, H-8), 7.22-7.28 (m, H-6), 5.2 (m, H-2'), 3.4 (d, J =6.8Hz, H-1'), 1.8 (s, H-4'), 1.6 (s, H-5'), 3.7 (s, N-Me), 3.9 (s, O-Me); ¹³C RMN (CDCl₃,100 MHz) δ 17.9 (C-4'), 24.3 (C-1'), 25.7 (C-5'), 29.7 (N-Me), 61.7 (O-Me), 114 (C-8), 117.8 (C-4a), 121.5 (C-2'), 121.8 (C-6), 122.6 (C-3), 123.4 (C-5), 130 (C-7), 132.5 (C-3'), 139 (C-8a), 160.1(C-4), 163.9 (C-2).

RESULTS AND DISCUSSION

Ethanol extract of leaves and wood of E. alata was fractionated and purified by conventional chromatographic methods in order to isolate eight compounds corresponding to quinolone-type alkaloid 4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one (1), pyranoquinolone alkaloid N-methylflindersine (2), furoquinoline alkaloids dictamine (3), kokusaginine (4), Γ-fagarine (5), flindersiamine (6), as well as the friedelane-type triterpenes friedeline (7) and fridelanol (8). Metabolites were identified by spectroscopic techniques ¹H and ¹³C NMR and by comparison with published data in the literature (Figure 1).

Compound 1 is a crystalline solid (needles, mp 199-200 °C, MeOH), possessing a molecular formula C₁₆H₁₉NO₂, as deduced from HRESIMS analysis ([M+H]⁺m/z 258.1472, calcd for C₁₆H₂₀NO_2, 258.1494), whose IR spectrum showed tension bands at 1633 and 1467 cm^-1 characteristic of C=C stretching of aromatic ring, as well as flexion bands in 756 and 1363 cm^-1 for the aromatic group =CH and -CH₃, respectively. Other bands were also identified at 1102 and 1730 cm^-1 characteristic of C-O and C=O stretchings, respectively.¹⁸ In ¹H NMR spectrum were observed signals of four aromatic hydrogens at δ_H 7.5 (1H, ddd, J = 1.5, 7.2, 8.6 Hz), δ_H 7.8 (1H, dd, J = 1.5, 7.2 Hz), δ_H 7.3 (1H, d, J = 8.6 Hz), and δ_H 7.22-7.28 (1H, m), whose chemical shift, multiplicity and coupling constants indicated the presence of a disubstituted aromatic ring.¹⁹ There were also signals at δ_H 5.2 (1H, m), 3.4 (2H, d, J = 6.8Hz), 1.89 (s, 3H) and 1.69 (s, 3H) corresponding to an isoprenyl moiety.²⁰ Same spectrum exhibited two singlets at δ_H 3.7 (3H) and δ_H 3.9 (3H), whose assignment was defined to be N-methyl and O-methyl groups.⁶¹³C NMR spectrum and DEPT experiments showed a signal at δ_C 163.9 for a quaternary carbon, corresponding to a carbonyl group,²¹ and it confirms the presence of N-methyl and O-methyl groups whose carbon signals were observed at δ_C 29.7 and δ_C 61.7, respectively.²¹ Above-mentioned spectral data allowed identifing signals of quaternary and methylene carbons for the isoprenyl moiety at δ_C 121.4 and δ_C 132.5, respectively.²⁰ HMBC and HMQC spectra confirmed the location of the isoprenyl and carbonyl groups. Information provided by ¹H and ¹³C NMR spectra led to determine the presence of a quinoline alkaloid, named as 4-methoxy-3-(3'-methyl-but-2'-enyl)-N-methyl-quinolin-2(1 H)-one.^19-21 Compound 1 is reported for the first time for the genus Esenbeckia. Similarly, full-analyses of ¹H and ¹³C NMR (one- and two-dimensional) spectra of compound 2 having a condensed formula C₁₅H₁₅NO₂ [HRESIMS analysis ([M+H]⁺m/z 242,1171, calcd for C₁₅H₁₆NO_2, 242,1181) allowed identifying it as another pyranoquinolone-type alkaloid, N-methylflindersine (2). This metabolite was previously isolated from species belonging to the genus Esenbeckia.¹²

Compounds 3-6 have molecular formulas assigned by HRESIMS analyses as C₁₂H₉NO₂ ([M+H]⁺m/z 200.0707, calcd for C₁₂H₁₀NO_2, 200.0712), C₁₄H₁₃NO₄ ([M+H]⁺m/z 260.0912, calcd for C₁₄H₁₄NO_4, 260.0923), C₁₃H₁₁NO₃ ([M+H]⁺m/z 230.0808, calcd for C₁₃H₁₂NO_3, 230.0817), and C₁₄H₁₁NO₅ ([M+H]⁺m/z 274.0715, calcd for C₁₄H₁₂NO_5, 274.0715), respectively. ¹H NMR spectra of those compounds showed similar profiles including signals at δ_Hca 7.5 (d, J ≈ 2.5 Hz, 1H) and δ_Hca 6.9 (d, J ≈ 2.5 Hz, 1H), for vinyl protons at furan ring.²²¹³C NMR spectra of 3-6 revealed signals for oxygenated quaternary carbons at δ_Cca 165-150 range, and nitrogen atom-bonded carbon at δ_Cca 145. Differences among them were stablished through NMR spectra according to the presence of a methoxy groups signals, whose location was defined by HMBC experiments. According to the above-mentioned information obtained from ¹H and ¹³C NMR (one- and two-dimensional), on comparing spectroscopic data with the literature,²³ compounds 3-6 were identified as furoquinoline alkaloids dictamine, kokusaginine, Γ-fagarine, and flindersiamine, respectively, which had been previously isolated from Rutaceae specimens such as Boronia pinnata, Dictamnus angustifolius,Teclea ouabanguiensis, Haplophyllum vulcanicum, Melicope lunu-ankenda, Acronychia laurifolia, Melicope ptelefolia,²³ and particularly from genus Esenbeckia from the species E. pentaphylla,¹²E. grandiflora, E. litoralis,¹³E. almawillia,¹⁵E. belizencis,¹⁶ and E. febrífuga.²⁴

Compounds 7, 8 and 9 were characterized by analyses of ¹H and ¹³C NMR and MS spectroscopic data and optical rotation values, thereby identifying them as friedelane-type triterpenes, friedeline (7) ([α]_D²⁵ -75.2, c 0.1, CHCl₃; EIMS M⁺m/z 426), friedelanol ([α]_D²⁵ 16.2, c 0.1, CHCl₃; EIMS M⁺m/z 428) (8), and its acetate derivative (friedelanyl acetate) ([α]_D²⁵ -12.5, c 0.1, CHCl₃; EIMS M⁺m/z 470) (9), whose analyses of both NMR and optical rotation data, in comparison with reported data in literature,¹⁴ allowed establishing the configuration showed in Figure 1 for 7-9. Compounds 7 and 8 have been previously identified in E. litoralis.¹⁴

ACKNOWLEDGEMENTS

Authors thank Universidad Nacional de Colombia for the support to the development of this project. Our gratitude is extended to Nuclear Magnetic Resonance and LCMS laboratories at Universidad Nacional de Colombia by NMR and HRESIMS records, respectively.

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Recebido em 8/9/10; aceito em 10/1/11; publicado na web em 29/3/11

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Present address: Facultad de Ciencias Básicas, Universidad Militar Nueva Granada, Campus Universitario Rio Grande, km 3 Vía Cajicá-Zipaquirá, Cundinamarca, Colombia

Publication Dates

• Publication in this collection
  01 Aug 2011
• Date of issue
  2011

History

• Accepted
  10 Jan 2011
• Received
  08 Sept 2010