Grandine A, a New Proaporphine Alkaloid from the Bark of Phoebe grandis

The stem bark of Phoebe grandis afforded one new oxoproaporphine; (–)-grandine A (1), along with six known isoquinoline alkaloids: (–)-8,9-dihydrolinearisine (2), boldine, norboldine, lauformine, scortechiniine A and scortechiniine B. In addition to that of the new compound, complete 1H- and 13C-NMR data of the tetrahydroproaporphine (–)-8,9-dihydrolinearisine (2) is also reported. The alkaloids’ structures were elucidated primarily by means of high field 1D- and 2D-NMR and HRMS spectral data.


Results and Discussion
The alkaloid grandine A (1) The IR spectrum revealed absorption bands at 3,376 and 1,729 cm -1 due to the OH and the C=O stretching vibrations respectively. The 1 H-NMR spectrum (Table 1) showed a pair of doublets (J = 5.4 Hz) typically found in oxoaporphines [9]. These signals are attributable to H-4 (δ 7.60) and H-5 (δ 8.68), respectively. A singlet ascribable to H-3 appeared at δ 7.19. Two broad singlets representing the methylenedioxy protons were apparent at δ 6.16 and 6.12, respectively. The respective protons in scortechiniine B 3, which lacks the C-8,9 double bond, gave a singlet at δ 6.13 corresponding to both protons ( Table 1). The deshielded aliphatic proton, H-10, resonated as a broad singlet at δ 4.45. The olefinic protons, H-8 and H-9, resonated at δ 5.43 and 6.14 as a doublet and a doublet of doublets, similar to the resonances of the same protons in prooxocryptochine (4) (Figure 1, Table 1). However, H-8 appeared as a doublet in the former as compared to doublet of doublets in the latter. The 13 C-NMR spectrum of grandine A (1) showed the presence of 17 carbons, which is in agreement with the molecular formula. The resonance of the quaternary spiro carbon, C-7a, at δ 52.7 implied the proaporphinic nature of compound 1. The C-7 carbonyl peak was observed at δ 204.4. The carbon bearing the hydroxyl group, C-10, resonated at δ 65.1. Thorough analysis of the COSY, HMQC, HMBC and NOESY spectrums allowed the complete assignments of all protons and carbons of grandine A 1 (Table 1).
The 1 H-NMR spectrum ( Table 2) displayed one methoxyl singlet at δ 3.76. In addition, one proton singlet was observed at δ 6.41, which may be ascribed to H-3. This observation also indicated that C-2 is substituted. The N-methyl group resonated at δ 2.38 and the aliphatic protons gave a multiplet between δ 1.82 to δ 3.27. H-6a resonated at δ 3.23 (dd, J, J' = 11.0, 6.3 Hz) while H-7α appeared as a doublet of doublets at δ 2.72. The 13 C-NMR spectrum of 2 showed the presence of eighteen carbon atoms; two methyls, seven methylenes, two methines, one carbonyl carbon and six quarternary carbons. The quaternary C-7a resonated at δ 47.3, while the C-1 methoxyl group peak appeared at δ 61.0. The NOE differential experiment showed signal enhancements of H-7α and H-8eq upon irradiation of H-6a, therefore indicating that H-6a is syn to H-7α and H-8eq [11]. Irradiation of H-3 resulted in the enhancement of H-4 (δ 2.55), thus suggesting that the methoxyl group is attached to C-1. (-)-8,9-Dihydrolinearisine (2) adopts an S configuration at C-6a, based on its negative optical rotation value [α] 23 D -50.0 o [12]. Alkaloid 2 is actually the enantiomer of (+)-N-methyltetrahydrocrotonosine, which occurred in Croton species [14].

Conclusions
In summary, we have observed that Phoebe grandis produces alkaloids similar to those of Phoebe scortechinii [2][3]. Both plants yielded proaporphines, aporphines and proaporphine-tryptamines. Earlier work on the barks of Phoebe grandis, collected from the northern part of Peninsular Malaysia, had indicated only aporphine alkaloids [1] while another study on the same plant species collected from Pahang, on the east coast of Peninsular Malaysia, has shown the presence of aporphines and proaporphines. This could be due to either seasonal variations or a difference in the soil types (the former was collected from a lowland area while the latter was collected from a highland area). The occurrence of the new proaporphine in Phoebe grandis is of special interest, in view of the fact that this type of alkaloid is a precursor of aporphines [15,16] and proaporhine-tryptamines, found in Phoebe species. To the knowledge of the authors, only two oxoproaporphines have been previously reported; scortechiniine B (3) [11] and prooxocryptochine (4) [17]. Grandine A (1) and scortechiniine B (3) occurred in the Phoebe species, while prooxocryptochine (4) was isolated from the wood of Cryptocarya chinensis [17]. Incidently, both Phoebe and Cryptocarya belong to the family Lauraceae.

General
The optical rotations were recorded on s Jasco (Japan) P1010 instrument equipped with a tungsten lamp. HRMS was obtained on a Thermo Finnigan Automass Multi. The ultraviolet spectra were obtained in MeOH on a Shimadzu UV-160A ultraviolet-visible spectrometer. The infrared spectra were taken on a Perkin Elmer 1600 Double-Beam recording spectrometer, using chloroform as solvent. The 1 H-NMR and 13 C-NMR spectra were recorded in deuterated chloroform on a JEOL 400 MHz (unless stated otherwise); chemical shifts are reported in ppm on δ scale, and the coupling constants are given in Hz. Silica gel 60, 70-230 mesh ASTM (Merck 7734) and silica gel 60, 230-400 Mesh ASTM (Merck 9385) were used for column and flash chromatography, respectively. Mayer's reagent was used for alkaloid screening.

Extraction and isolation of the alkaloids
The dried stem bark (1.0 kg) of Phoebe grandis was ground and extracted exhaustively with hexane followed by CH 2 Cl 2 by Soxhlet extractor for 17 hours. Extraction of alkaloids was carried out in the usual manner, which has been described in detail [1,2] and gave 4.02 g of crude alkaloid. CH 2 Cl 2extracted crude alkaloid (1.0 g) was subjected to column chromatography. The isolation and purification of compound 1 and 2 were carried out by chromatography on a small column and preparative TLC (Silica gel 60F 254 ) yielding 4.2 mg of grandine A (1) (CH 2 Cl 2 : MeOH, 98:2) and 7.4 mg of (-)-8,9-dihydrolinearisine (2) (CH 2 Cl 2 : MeOH, 95:5).  Table 1.  Table 2.