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Anais da Academia Brasileira de Ciências

Print version ISSN 0001-3765

An. Acad. Bras. Ciênc. vol.78 no.1 Rio de Janeiro Mar. 2006

http://dx.doi.org/10.1590/S0001-37652006000100003 

CHEMICAL SCIENCES

 

Pimarane Diterpenes and a Sesquiterpene from Salzmmania nitida

 

 

Mário G. de CarvalhoI; Josinete S. AlvesII; Emidio V. Leitão da-CunhaII, III; José M. Barbosa-FilhoII; Marcelo S. da SilvaII

IDepartamento de Química, ICE, Universidade Federal Rural do Rio de Janeiro, BR 465, Km. 7, 23850-480 Seropédica, RJ, Brasil
IILaboratório de Tecnologia Farmacêutica, Universidade Federal da Paraíba, C.P. 5009, 58051-970 João Pessoa, PB, Brasil
IIIDepartamento de Farmácia e Biologia, Universidade Estadual da Paraíba, 58100-000 Campina Grande, PB, Brasil

 

 


ABSTRACT

Two new terpenoids, (+)-3-oxo-thermarol and 11-acetoxyeudesman-4a-methyl-5a-ol along with the (+)- thermarol were isolated from the aerial parts of Salzmmania nitida. The structures and unambiguous 1H and 13C chemical shift assignments were established by spectroscopic means, including homo and heteronuclear techniques.

Key words: Salzmmania nitida, Rubiaceae, diterpenes, eudesmane.


RESUMO

O estudo fitoquímico de Salzmmania nitida D.C. (Rubiaceae) conduziu ao isolamento e identificação de dois novos terpenoides, (+)-3-oxo-thermarol e 11-acetoxi-4alfa-metil-5alfa-eudesmanol além do (+)-termarol. As estruturas foram estabelecidas com base na análise de espectros de IV, Massas e RMN de 1H e 13C (1D e 2D).

Palavras-chave: Salzmmania nitida, Rubiaceae, diterpenos, eudesmano.


 

 

INTRODUCTION

The Rubiaceae family has been shown to be one of much interest in phytochemical investigation due to the presence of species produced biologically active compounds such as alkaloids, flavonoids, antraquinones, saponins and triterpenes. Chinchona, Coffea, Psychotria and Rubia species are genera of this family known due to biosynthesize interesting naphthoquinones and alkaloids such as quinine, caffeine, emetine (Bruneton 1995, Evans 1991). Continuing our phytochemical investigation of plants from the northeastern region of Brazil, we recently reported the first study of Salzmmania nitida D.C. (Rubiaceae), describing the structures of triterpenes isolated from this plant (Alves et al. 2000). This species is a monotypic plant and common in the "restinga" (a kind of sand bank covered with vegetation) of the Northeast. This work describes the isolation and structural determination of two pimarane diterpenes besides 5a-hydroxy-4a-methyl-11-O-acethyl-eudesmane obtained from Salzmania nitida (Rubiaceae).

 

MATERIALS AND METHODS

GENERAL EXPERIMENTAL PROCEDURE

Mp's are uncorrected. NMR spectra were recorded on Bruker AC-200 (1H: 200 MHz, 13C: 50,3 MHz) and AMX 400 (1H: 400 MHz, 13C: 100 MHz) spectrometers using approximately 10-15 mg of sample dissolved in 0,5 ml of CDCl3 in 5 mm NMR tubes. Residual CHCl3 (dH 7.24) and 13CDCl3 (dC 77.00) signals were used as references. Homonuclear 2D (1H-1H-COSY and NOESY) and heteronuclear 2D {1H-13C-COSY- nJCH [n=1, HMQC (modulated with JCH 130 Hz); and n=2 and 3, HMBC (modulated with JCH = 9.0 Hz)]} spectra were obtained with standard pulse sequences. FT-IR spectra were recorded using KBr disks or NaCl film on a Perkin-Elmer 1600 spectrometer. Mass spectra were obtained using a VG Auto Spec-300 spectrometer. Chromatography was performed using Aldrich silica gel with a suitable granulation for column and preparative TLC. The visualization of spots was done by UV (254 and 366 nm) and exposure to iodine vapor.

PLANT MATERIAL

Salzmania nitida D.C. (Rubiaceae) was collected in January 1998 in the surroundings of Santa Rita, State of Paraíba, Brazil, and identified by botanist Dr. Maria de Fátima Agra of the Universidade Federal da Paraíba. A Voucher specimen (Agra 2986) is deposited at the Herbarium Prof. Lauro Pires Xavier (JPB), Universidade Federal da Paraíba.

ISOLATION OF CONSTITUENTS

The dried and ground aerial parts of S. nitida (3.40 Kg) were extracted in a Soxhlet apparatus with 95% EtOH. The solvent was removed under vacuum to yield a residue (180.0 g). This residue was dissolved in MeOH-water(9:1) and subjected to partition with CHCl3 and AcOEt. The residue from the AcOEt fraction (80.3 g) was submitted to a C.C. on silica gel using hexane-CHCl3 (1:1) as eluent. 76 fractions of 50 mL each were obtained. Fractions 10-30 and 35-55 yielded 1 (0.10 g), and 2 (0.15 g), respectively, after recrystallization from methanol. Fractions 57-76 were submitted to preparative TLC using hexane:AcOEt (8:2) to yield the sesquiterpene 3 (0.010 g, oil).

(+)-8b, 19-dihydroxy-3-oxopimar-15b-ene, (+)-3-oxo-thermarol (1): M.P. 158 ºC, [a]D = + 22.60 (CHCl3, c 0.045). 1H and 13C NMR data in Table I.

 

 

(+)-8b, 19-dihydroxy-pimar-15b-ene, (+)-thermarol (2): M.P. 148 ºC, [a]D = +8.28 (CHCl3, c 0.052). 1H and 13C NMR data in Table I.

11-acetoxyeudesman-4a-methyl-5a-ol (3): oil; dH(CDCl3, 500 MHz): 2.2 (H-7, m), 1.93 (H-4, H-1, m), 1.70 (H-2, 6, 9, m), 1,45 (H-2, 3, 8, m), 1.30 (H-1, 3, 9, m), 1.2 ( H-6, 8, m), 1.95 (s, H3CCO), 1.42 and 1.49 (3H, s, H-12 and 13), 0.84 (3H, s, H-14), 0.77 (3H, d, 7.0 Hz, H-15); dC (CDCl3, 125 MHz): 170.5 (H3C CO), 85.2 (C-11), 73.2 (C-5), 41.1 (C-10), 39.4 (C-7), 36.5 (C-9), 32.9 (C-4), 32.0 (C-1), 30.0 (C-6), 29.7 (C-3), 23.4 and 23.2 (C-12 and C-13), 22.9 (C-2), 22.4 (C-8), 22.1 (H3CCO), 15.2 (C-14), 15.9 (C-15).

 

RESULTS AND DISCUSSION

The analysis of 13C NMR [HBBD and DEPT (q: 135 and 90º)] spectra of 1 allowed to identify three methyl, nine methylene, three methyne and five quaternary carbons. The IR spectra of this compound showed bands at nmax 1715 cm-1 (nC = O), 3470 cm-1 (nOH) and 3080, 1630, 975 and 910 cm-1 characteristic of a vinyl group. These observations together with a peak at m/z = 320(M) in the mass spectrum are in agreement with the molecular formula C20H32O3 for 1. These data are in accordance with an oxo-pimarane diterpene. The 1H NMR spectrum shows singlets at dH 0.92, 0.98 and 1.28 for three methyl groups, two signals at dH 3.45 (d, 11.0 Hz, 1H) and 4.05 (d, 11.0 Hz, 1H) for a hydroxymethylene group and three double doublets at dH 5.13 (9.0 Hz and 2.0 Hz, 1H), 5.19 (16.0 Hz and 2.0 Hz, 1H) and 5.98 (16.0 Hz, 9.0 Hz, 1H) of the identified vinyl group. These groups were also confirmed by cross-peaks in the 2D (1H-13C-COSY, 1JCH) NMR spectrum between those signals and carbon-13 chemical shifts at 16.5, 22.3, 32.5 (CH3), 66.0 (CH2), 112.8 (CH2) and 147.9 (CH) relative to connections for methyl groups, hydroxymethylene and vinyl hydrogen, respectively. The HMBC analysis showed 2,3JCH cross-peak correlation as described in Table I. The NOE observed between H3C-20, H-1 (eq) and H2C-19 in 1H-1H-NOESY experiment, along with the considerable shielding effect on C-18 (22.3) and deshielding on C-4 (51.1), were used to confirm the presence of a carbonyl at C-3 of ring A. The location of the hydroxyl group at C-8 was confirmed by the chemical shift at 72.0 ppm besides the signals of long-range coupling with H-6, H-9 and 2H-14 in the HMBC experiment. Additional signals showing correlation between 3H-20 and C-1, C-5 and C-9, 3H-17 with C-12, C-14 and C-15 together with comparison of (-)-thermarol 13C NMR chemical shifts (Matsuo et al. 1976, Ramos et al. 1984) allowed to assign hydrogen and carbon chemical shifts as shown in Table I. The peaks in the mass spectrum of 1 have the same m/z values described in the literature for a thermarol derivative (Takaishi et al. 1997). The difference between the C-17 chemical shift in 1 (dCH3 32.5) and that described in the literature (dCH3 24.3) for 8b,19-dihydroxy-3-oxopimar-15a-ene (Takaishi et al. 1997) along with the NOE cross peak between H-17 and H-14a in the 1H-1H-NOESY spectrum led us to established an equatorial position for the C-17 methyl group, such as in the representation for (-)-thermarol (Matsuo et al. 1976). Finally, the [a]D = + 22.60 (CHCl3, c 0.045) allowed to define the structure of 1 as (+)-8b,19-dihydroxy-3-oxopimar-15b-ene represented for the new diterpene named (+)-3-oxo-thermarol (Fig. 1).

 

 

Spectral analysis for 2 led us to identify 20 carbon signals including three methyl, ten methylene, three methyne and four quaternary carbons including two oxigenated carbons (H2C-O and C-O). The molecular formula of 2, C20H34O2, was compatible with M+. 306 observed in the mass spectrum. The 13C NMR spectra data besides the peaks observed in the mass spectrum of 2 were identical to those of (-)-thermarol (Matsuo et al. 1976, Ramos et al. 1984). The cross peaks of NOE between H-9/H-12, H-1/H-20, H-12/H-9, H-17, H-14/H-17, H-19(dH 3.48)/H-1,H-20 and H-17/H-14a, H-15 observed in the NOESY spectra along with information from homonuclear 1Hx1H-COSY were used to confirm the structure for 2. Furthermore, this analysis allowed us to make the first detailed assignment of all hydrogen chemical shifts of thermarol, Table I. The optical rotation of 2, [a]D = +8.28 (CHCl3, c 0.052), has the same sign of (+)-thermarol prepared by reduction of 8b-Hydroxypimar-15-en-19-oic acid, isolated from Taxodium mucronatum (Ramos et al. 1984) (Fig. 1). So, the optical rotation permitted to identify 2 as being the natural enantiomer compound (+)-thermarol.

The acetyl derivative of eudesmanediol 3 was identified by IR and NMR spectral analysis besides comparison with data for 5,11-dihydroxy-eudesmane and 11-Acetoxyeudesman-4-ol isolated from Cryptomeria japonica (Su et al. 1995) and from Ursinia species (Jakupovic et al. 1992). The HMQC and HMBC experiments were useful to assign the carbon and hydrogen chemical shift of 3 (see experimental). The analysis of carbon-13 chemical shifts confirmed the location of the tertiary hydroxyl and the acetyl groups in the carbon with chemical shifts at d73.5 (C-5) and 85,2 (C-11), respectively, together with comparison with values for 3a described in literature (Su et al. 1995). The shielding g-effect of an axial HO-5 on C-15 (dCH3 15.9, Dd = 6.6 ppm) and on C-7 (dCH 39.4, Dd = 5.8 ppm) besides the expected deshielding effect on C-10 (dC 41.1, Dd = 3.5 ppm) were used to justify the a-orientation for HO-5 and H3C-15. The chemical shift of C-11 (dC 85.2) and of 12/13 methyl groups (dCH3 23.4/23.2) are in agreement with an acetoxy group (dC = O 170.5 and dCH3 22.1) at C-11. On the basis of these data, the new sesquiterpene (3) was identified as 11-acetoxyeudesman-4a-methyl-5a-ol (Fig. 1).

 

ACKNOWLEDGMENTS

The authors are grateful to Conselho Nacional de Desenvolvimento Científico e Tecnológico (CNPq), Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES), Financiadora de Estudos e Projetos (FINEP) and Fundação Carlos Chagas Filho de Amparo à Pesquisa do Estado do Rio de Janeiro (FAPERJ) for grants and fellowships. We also thank Dra. M. de F. Agra (Universidade Federal da Paraíba, João Pessoa, Brasil) for botanical identification and Prof. Alexander I. Gray (Phytochemistry Research Laboratories, Department of Pharmaceutical Sciences, University of Strathclyde, Glasgow, UK) for recording the 400 MHz NMR spectra (Bruker AMX 400).

 

REFERENCES

ALVES JS, CASTRO JCM DE, FREIRE MO, DACUNHA EVL, BARBOSA-FILHO JM AND SILVA MS DA. 2000. Complete Assignment of the 1H and 13C NMR Spectra of four Triterpenes of the Ursane, Artane, Lupane and Fridelane groups. Magn Reson Chem 38: 201-206.         [ Links ]

BRUNETON J. 1995. Pharmacognosy, Phytochemistry, Medicinal Plants, 2nd ed., Lavoisier, Adover, p. 330-887.         [ Links ]

EVANS WC. 1991. Farmacognosia, 13ª ed. - Interamericana, M. Graw Hill, p. 224-229.         [ Links ]

JAKUPOVIC J, GANZER U, PRITSCHOW P, LEHMANN L, BOHLMANN F AND KING RM. 1992. Sesquiterpene Lactones and other Constituents from Ursinia species. Phytochemistry 31: 863-880.         [ Links ]

MATSUO A, UTO S, NAKAYAMA N, HAYASHI S, YAMASAKI K, KASAI R AND TANAKA O. 1976. (-)-Thermarol, A New Ent-pimarane-Class Diterpene Diol From Jungermannia thermarum (Liverwort). Tetrahedron Lett 2: 2451-2454.         [ Links ]

RAMOS AR, ESCAMILIA EM, CALDERÓN J AND RODRÍGUES B. 1984. 8?-hydroxypimar-15-en-19-oic acid from Taxodium mucronatum. Phytochemistry 23: 1329-1330.         [ Links ]

SU W, FANG J AND CHENG Y. 1995. Sesquiterpenes from Leaves of Cryptomeria japonica. Phytochemistry 39: 603-607.         [ Links ]

TAKAISHI Y, MIYAGI K, KAWAZOE K, NAKANO K, LI K AND DUAN H. 1997. Terpenoids From Tripterygium wilfordii Var. Regelii. Phytochemistry 45: 975-978.         [ Links ]

 

 

Manuscript received on June 3, 2004; accepted for publication on August 27, 2005; presented by FERNANDO GALEMBECK

 

 

Correspondence to: Dr. Mário Geraldo de Carvalho
E-mail: mgeraldo@ufrrj.br