JBCS

INTRODUCTION

The genus Rauvolfia, family Apocynaceae, continues to be fascinating as it produces a number of indole alkaloids with novel skeletons, which are interesting from the biosynthetic point of view as well as for their medicinal aspect and spectroscopic analysis.^1,2 Species of this genus have several biological activities, such as, cholinesterase inhibitors,³ and antimicrobial,⁴ anticonvulsant,⁵ anxiolytic,⁶ antimalarial, antipyretic,⁷ and antipsychotic effects,⁸ in addition to sedative activity.⁹

Rauvolfia capixabae I. Koch & Kinoshita-Gouveia,¹⁰ commonly known as "Grão-de-Gato" in Atlantic forest in the North of Espírito Santo State, appears as a tree of 6-12 m. This species has not been reported in studies on its chemical composition described in the literature.

As part of the research program for the Natural Products Chemistry Group of the North Fluminense State University (UENF) on the identification of alkaloids in species of the Rauvolfia genus,^4,11,12 a phytochemical investigation of the stem bark extracts of R. capixabae is described. In the present paper, we describe the isolation and characterization of a novel sarpagine-type alkaloid, N_a-methylrauflorine (1), along with five known alkaloids. The known and new alkaloid structures were established on the basis of spectral data, mainly ¹H and ¹³C (1D and 2D) NMR spectra, mass spectrometry and by comparison with literature data.

EXPERIMENTAL

General procedures

Optical rotation measurements were obtained on a Perkin Elmer 343 digital polarimeter. Melting points were obtained on a Microquímica MQRPF and are uncorrected. FTIR spectra were recorded on a FTIR-8300 Shimadzu spectrometer using KBr disk. EI-MS (low resolution) mass spectra were obtained on Shimadzu QP5050A mass spectrometer. Column chromatographic purifications were carried out over silica gel (70-230 mesh). Silica gel 60F₂₅₄ was used in thin layer chromatography analysis.

¹H and ¹³C NMR spectra were measured on a Jeol Eclipse 400 spectrometer, operating at 400 (¹H) and 100 (¹³C) MHz. CDCl₃ was used as solvent and TMS as internal reference. Chemical shifts are given in the δ scale (ppm) and coupling constants J in Hz. 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 spectra by using a multinuclear inverse detection 5 mm probe with field gradient.

Plant materials

The stem bark of Rauvolfia capixabae I. Koch & Kinoshita-Gouveia were collected in November 2004 at Cia Vale, Linhares City, Espírito Santo State, Brazil. The specimen was identified by taxonomist Ingrid Koch. A voucher specimen (CVRD 338) is deposited at the Cia Vale herbarium, Linhares, Espírito Santo State.

Extraction and isolation

The stem bark (3.08 kg) was extracted with CH₂Cl₂ from R. capixabae I. Koch & Kinoshita-Gouveia at room temperature, furnishing 125 g of crude dichloromethane extract after solvent evaporation.

A portion of the dichloromethane extract (10.1 g) was chromatographed over silica gel column with a polarity gradient of CH₂Cl₂/MeOH to afford fourteen fractions. Fraction 1 (58.4 mg) yielding isoreserpiline (2). The fraction 7 (1.35 g) was rechromatographed over a silica gel column with a polarity gradient of CH₂Cl₂/MeOH yielding thirteen fractions. Fraction 7.2 (6.8 mg) presented as an amorphous yellow solid identified as Compound (1). Fraction 7.13 (384.8 mg) was rechromatographed over a silica gel column with a polarity gradient of CH₂Cl₂/MeOH yielding the compounds isoreserpiline (2, 7.1 mg) and perakine (5, 166 mg).

Fraction 8 (0.8 g) was rechromatographed over silica gel column with a polarity gradient of CH₂Cl₂/MeOH supplying eight fractions. Fraction 8.1(92.0) yielding ajmalicine (4, 92.0 mg). Fraction 8.8 (301.2) was rechromatographed over silica gel column with a polarity gradient of CH₂Cl₂/MeOH yielding compound 6 (199 mg).

Fraction 11 (1.2 g) was rechromatographed over silica gel column with a polarity gradient of CH₂Cl₂/MeOH supplying seven fractions. Fraction 11.7 (217.5 mg) was rechromatographed over silica gel column with a polarity gradient of CH₂Cl₂/MeOH yielding compound N_b-oxide isoreserpiline (3, 13.0 mg).

N_a-methylrauflorine (1), yellow amorphous solid, mp 166-168 ºC; [α]_D²³ = + 8.06º (CHCl₃, c 0.062); LREI-MS (rel. int.) 336 [M^+. (100%,)] (Scheme 1); ¹H and ¹³C NMR: see Table 1.

Scheme 1. Proposed fragmentation mechanisms to justify principal peaks observed in the mass spectrum (LREIMS, 70 eV) of 1

Figure 1. Compounds Isolated from R. Capixabae

Figure 2. Select dipolar-dipolar interactions revealing spatial correlations (NOE) and relative stereochemistry for Alkaloid 1. Arrows denote NOE spatial correlations principals

RESULTS AND DISCUSSION

The CH₂Cl₂ extracts of R. capixabae stem bark were subjected to classical chromatographic methods to yield the a new sarpagine-type alkaloid, N_a-methylrauflorine (1), in addition to the known alkaloids, isoreserpiline (2),¹¹ N_b-oxide-isoreserpiline (3),¹² ajmalicine (4),¹³ perakine (5),¹⁴ and vinorine (6),¹⁵ that were identified in the analysis of ¹H and APT-¹³C NMR spectra data, including 2D ¹H-¹H-COSY, ¹H-¹H-NOESY, HSQC and HMBC NMR experiments,^16,17 which were also used to complete unambiguous ¹H and ¹³C chemical shift assignments of the new alkaloid 1.

The new monoterpenic indole alkaloid (1) [a]_D²³ = + 8.6, (CHCl₃, c 0.062), was obtained as a yellow amorphous solid. Comparative analysis of {¹H}- and APT-¹³C NMR spectra (Table 1), involving the corroboration of ¹H NMR spectra (1D ¹H NMR and 2D ¹H-¹H-COSY), allowed to recognize the presence of 21 signals corresponding to six nonhydrogenated [(C)₆: one sp³, five sp² (including one carbonyl group at δ_C 213.6 (C-17) and one sp² olefinic at δ_C 137.5 (C-20)], nine methine [(CH)₉: three sp³ (all linked to a nitrogen atom: CH-2, CH-3 and CH-5) and four sp² including one olefinic at δ_C 115.5 (CH-19)], three methylene [(CH₂)₃, all sp³, including one linked to a nitrogen atom at δ_C 55.7 (CH2-21)] and three methyl [(CH₃)₃: including one linked to a nitrogen atom at δ_C 36.9/δ_H 3.06, one linked to an sp² olefinic carbon atom at δ_C 12.9/δ_H 1.65 and methoxyl group (δ_C 56.0/δ_H 3.80,s)] carbon atoms, allowing to deduce the expanded molecular formula (C)₅(C=O)(CH)₉(CH₂)₃N(CH₃)(NCH₃)(OCH₃) = C₂₁H₂₄N₂O₂ for 1.

The LREI-MS (70 eV) spectrum of 1 (Scheme 1) showed of molecular peak [M^+.] at m/z 336 Daltons, allowing in conjugation with the ¹³C NMR spectral data to deduce the molecular formula C₂₁H₂₄N₂O₂ (1), containing eleven degrees of unsaturation and consistent with the presence of one carbonyl group and one double bond in a methoxylated pentacyclic alkaloid, compatible with the alkaloidic structure sustaining a carbonyl group at carbon atom C-17 (δ_C 213.6) in rauflorine skeleton (1a).⁴ In fact, heteronuclear long-range couplings (³J_HC) of this carbon atom C-17 (δ_C 213.6) with H-2 (δ_H 2.64), H-5 (δ_H 3.18-3.24) and H-6a (δ_H 2.38) was used to confirm the presence of a function carbonyl linked at atom carbon C-17, as shown in Table 1 together with additional heteronuclear long range couplings ¹H-¹³C-COSY-ⁿJ_HC (n=2, HMQC or HSQC and HMBC, n=3) that were also used to complete unambiguous ¹H and ¹³C chemical shift assignments.

¹H NMR spectrum of 1 showed three signals corresponding to the hydrogens linked to aromatic carbons at δ_H 6.86 (br s, H-9), 6.84 (d, J = 9.2 Hz, H-12) and 6.80 (dd, J = 9.2 and 3.0 Hz, H-1) typical of a 1,2,4-trisubstituted benzene ring (ring A),^18,19 which was confirmed by direct heteronuclear correlations (¹J_HC) between CH-9 (δ_C 121.3)/δ_H 6.86), CH-11 (δ_C 112.7/δ_H 6.80), C-12 (δ_C 116.0/δ_H 6.84) observed in the HMQC spectrum. The presence of a single signal at δ_H 3.80 was attributed to three hydrogens of a methoxyl group (¹J_HC δ_C 56.0/δ_H 3.80), in agreement with a methoxyl group in the indole nucleus.^18,19 The single signal at δ_H 3.06 (three hydrogen atoms) suggested the presence of a N-Me group that was confirmed in the HMQC by the correlation ¹J_HC Me-N_a (δ_C 37.0/δ_H 3.06) and compatible with a substituted indole nucleus and N-methylated.²⁰

The location of the methoxyl and Me-N_a groups in the indole moiety was confirmed by heteronuclear long-range couplings observed in the HMBC spectrum by correlations ³J_HC between by C-8 (δ_C 131.1) and H-12 (δ_H 6.84), C-10 (δ_C 147.5), H-12 (δ_H 6.84) and MeO-10 (δ_H 3.80), C-13 (δ_C 141.9), H-9 (δ_H 6.86), H-11 (δ_H 6.80) and Me-N_a (δ_H 3.06), CH-11 (δ_C 112.7) and H-9 (δ_H 6.86), and CH-2 (δ_C 79.5) and Me-N_a (δ_H 3.06), shown in Table 1.

The presence of a methyl group bonded to a sp² methine carbon was recognized by a double signal at δ_H 1.65 (d, J = 7.0 Hz, 3H-18) and the multiplet at δ_H 5.29-5.32 (m, H-19), characterizing a C=CH-CH₃ moiety corresponding to CH-19 and CH₃-18, respectively, present in several alkaloids. This moiety was further confirmed by long-range correlations revealed by the HMBC spectrum through correlations observed between the CH₃-18 (δ_C 12.9) and H-19 (δ_H 5.29-5.32, ²J_HC), CH-19 (δ_C 115.5), 3H-18 (δ_H 1.65, ²J_HC) and C-20 (δ_C 137.5) and 3H-18 (δ_H 1.65, ³J_HC).^18,19,21

The presence of a carbonyl carbon at C-17 was confirmed by the heteronuclear correlations observed in the HMBC spectrum, which revealed long-range correlations between C-17 (δ_C 213.6) and H-16 (δ_H 2.58, ³J_HC), H-2 (δ_H 2.64, ³J_HC), H-5 (δ_H 3.22, ³J_HC) and H-6a (δ_H 1.66, ³J_HC).^18,19,21 The other correlations are shown in Table 1.

All these data allowed the definition of a sapargine-type skeleton^18,19,21 and propose the Structure 1 for the alkaloid isolated. The comparison of the ¹H and ¹³C NMR spectral data of 1 with values described in the literature for the rauflorine alkaloid (1a)¹⁸ allowed to verify that the significant difference between these two alkaloids can be justified by presence of the methyl group linked to N-atom of the alkaloid 1.

The relative stereochemistry proposed for alkaloid 1 was based on biogenic argument,²² by comparison with data described in the literature for sapargine-type alkaloids skeleton^18-21 and results revealed by dipolar-dipolar interactions observed in a ¹H-¹H-NOESY experiment (Table 1).

CONCLUSION

From dichloromethane extract of R. capixabae stem bark a new sarpagine-type alkaloid was isolated, N_a-methylrauflorine (1), in addition to known alkaloids, isoreserpiline (2), N_b-oxide-isoreserpiline (3), ajmalicine (4), perakine (5) and vinorine (6).

SUPPLEMENTARY MATERIAL

The LREIMS, ¹H NMR, ¹³C NMR -APT, ¹H-¹H COSY NMR, HMQC, HMBC and NOESY spectra for compound 1 are freely available at http://quimicanova.sbq.org.br in PDF file.

ACKNOWLEDGEMENTS

The authors are grateful to Fundação Carlos Chagas Filho de Amparo à Pesquisa do Rio de Janeiro (FAPERJ), Consenho Nacional de Desenvolvimento Científico e Tecnológico (CNPq) and Coordenação de Aperfeiçoamento de Pessoal de Nível Superior (CAPES) for scholarships and financial support.

REFERENCES

1. Roberts, M. F.; Wink, M.; Alkaloids: Biochemistry, Ecology and Medicinal Applications; Plenum Press: New York, 1998.

2. Bruneton, J.; Pharmacognosy, Phytochemistry, Medicinal Plants, Lavoisier: Londres, 1995.

3. Tappayuthpijarn, P.; Jansom, C.; Jansom, V.; Ingkaninun, K.; Planta Med. 2010, 76, 1209.

4. Carlos, L. A.; Silva, A. K. A.; Vieira, I. J. C.; Mathias, L.; Braz-Filho, R.; Samarão, S. S.; Vieira-da-Motta, O.; Braz. J. Microbiol. 2010, 41, 612. DOI: http://dx.doi.org/10.1590/S1517-83822010000300011

5. Quintans-Junior, L. J.; Siqueira, J. S.; Melo, M. S.; Silva D. A.; Morais, L. C. S. L.; Souza, M. F. V.; Almeida, R. N.; Rev. Bras. Farmacogn. 2010, 20, 54. DOI: http://dx.doi.org/10.1590/S0102-695X2010000100012

6. Netto, S. M.; Warela R. W. B.; Fechine, M. F.; Queiroga, M. N.; Quintans-Júnior, L.; J. Rev. Bras. Farmacogn. 2009, 19, 888. DOI: http://dx.doi.org/10.1590/S0102-695X2009000600017

7. Amole, O. O.; Onabanjo, A. O.; Agbaje, E. O.; Arch. Pharmacol. 1998, 358, 497.

8. Obembe, A.; Sokomba, E. N.; Sijuwola, O. A.; Olorunfemi, P. O.; Alemika, T. O. E.; Isichei, C. O.; Ogunkeye, O. O.; Bangudu, A. B.; Phytother. Res. 1994, 8, 218. DOI: http://dx.doi.org/10.1002/ptr.2650080406

9. Madawala, P. G.; Arambewela, L. S. R.; Premakumara, G. A. S.; Ratnasooria, W. D.; J. Ethnopharmacol. 1994, 42, 63. DOI: http://dx.doi.org/10.1016/0378-8741(94)90024-8 PMID: 8046945

10. Koch, I.; Kinoshita, L. S.; Bittrich, V.; Novon 2007, 17, 462. DOI: http://dx.doi.org/10.3417/1055-3177(2007)17[462:TNIRAR]2.0.CO;2

11. Cancelieri, N. M.; Vieira, I. J. C.; Mathias, L.; Schripsema, J.; Braz-Filho, R.; Tetrahedron Lett. 2002, 43, 1783. DOI: http://dx.doi.org/10.1016/S0040-4039(02)00151-X

12. Cancelieri, N. M.; Vieira, I. J. C.; Mathias, L.; Braz-Filho, R.; Magn. Reson. Chem. 2003, 41, 287. DOI: http://dx.doi.org/10.1002/mrc.1168

13. Lounasmaa, M.; Tolvanem, A.; Heterocycles 1986, 24, 3229. DOI: http://dx.doi.org/10.3987/R-1986-11-3229

14. Rahman, A.-U.; Sultana, A.; Nighat, F.; Bhatti, K.; Kuruku, S.; Kartal, M.; Phytochemistry 1995, 38, 1057. DOI: http://dx.doi.org/10.1016/0031-9422(94)00638-A

15. Kato, L.; Braga, R. M.; Kock, I.; Kinoshita, L. S.; Phytochemistry, 2002, 60, 315. DOI: http://dx.doi.org/10.1016/S0031-9422(02)00122-X PMID: 12031452

16. Claridge, T. D. W.; High-Resolution NMR Techniques in Organic Chemistry, Pergamon: Amsterdam, 1999.

17. Friebolin, H.; Basic One and Two-Dimensional NMR Spectroscopy, 2^nd ed., VCH: New York, 1993.

18. Jokela, R.; Lounasmaa, M. A.; Heterocycles 1996, 43, 1015. DOI: http://dx.doi.org/10.3987/COM-96-7401

19. Chatterjee, A.; Ghosh, A. K.; Hagaman, E. W.; J. Org. Chem. 1982, 47, 1732. DOI: http://dx.doi.org/10.1021/jo00348a026

20. Azoug, M.; Loucaci, A.; Richard, B.; Nuzillard, J.; Moreti, C.; Hanrot, Z.; Men-Oliver, L.; Phytochemistry 1995, 39, 1223. DOI: http://dx.doi.org/10.1016/0031-9422(95)00050-H

21. Braga, R. M.; Reis, F. A. M.; Phytochemistry 1987, 26, 833. DOI: http://dx.doi.org/10.1016/S0031-9422(00)84797-4

22. Danieli, B.; Palmisano, G.; In The Alkaloids; Brossi, A., ed.; Academic Press: New York, 1986.