Muscicapines , a New Class of Guaiane-Type Sesquiterpene Alkaloids from Croton muscicapa

Vicente T. de Araújo-Júnior, Marcelo S. da Silva, Emídio V. Leitão da-Cunha, Maria de Fátima Agra, Petrônio F. de Athayde-Filho, Ivo J. Curcino Vieira, Raimundo Braz-Filho, and José M. Barbosa-Filho* Laboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, CP 5009, 58051-970 João Pessoa PB, Brazil Departamento de Farmácia e Biologia, Universidade Estadual da Paraíba, 58100-000 Campina Grande PB, Brazil


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). 2n 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 Spreng 3 and diterpenes and alkaloids from Croton moritibensis Baill. 4n 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. 5The 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
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 CH 3 -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 1 H NMR (500 MHz) and 13 C 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 CDCl 3 , and the solvent signals (7.27 and 77.0 ppm) were used as references.The chemical shifts (δ) 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 [α] value was obtained on a Perkin Elmer model 343 Digital Polarimeter using CHCl 3 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 PF 254 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.

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 ( 1 H and 13 C NMR) are given in Table 1.