(+)-N-(2-Hydroxypropyl)lindcarpine: A New Cytotoxic Aporphine Isolated from Actinodaphne pruinosa Nees

One new alkaloid; (+)-N-(2-hydroxypropyl)lindcarpine (1), together with four known aporphine alkaloids, (+)-boldine (2) (+)-norboldine (3), (+)-lindcarpine (4) and (+)-methyllindcarpine (5) were isolated from the stem bark of Actinodaphne pruinosa Nees (Lauraceae). (+)-N-(2-Hydroxypropyl)lindcarpine (1) exhibited cytotoxic activity against P-388 murine leukemia cells with an IC50 value of 3.9 μg/mL. Structural elucidation of all the compounds were performed by spectral methods such as 1D- and 2D- NMR, IR, UV, and HRESIMS.


Results and Discussion
(+)-N-(2-Hydroxypropyl)lindcarpine (1; see Figure 1) exhibited a molecular formula of C 21 H 26 NO 5 based on the HRESIMS spectrum (positive mode) which showed a pseudomolecular ion at m/z 372.1797 [M+H] + (calcd. 372.1811, -1.4 mmu). The IR spectrum revealed an absorption band at 3,180 cm -1 due to the OH stretching vibration. The overall physical properties and NMR spectral profile revealed its identity as a member of the aporphine group of isoquinoline, a characteristic and distinguishable chemical marker of Actinodaphne plants [3,4].
In the 1 H-NMR spectrum ( Table 1) (Table 1) and DEPT spectra, showed a total of 21 carbon signals; three methyls, four methylenes, one methine bearing hydroxyl group, four methines, and nine quaternary carbons in which four are aromatic oxygenated carbon signals. The complete 1 H-and 13 C-NMR ( Figure 2) spectral assignment of 1 was accomplished by thorough analysis of DEPT, COSY (Figure 3), HMQC (Figure 4), and HMBC data. The 1 H-1 H COSY, combined with the HMQC spectrum revealed that 1 has the following partial structure: -CH 2 CH 2 -(C4 and C5); -CHCH 2 -(C6a-C7); =CHCH= (C8-C9); -CH 2 CH-(C12-C13); and -CHCH 3 -(C13-C14). All of these segments were compatible for rings B, C, and D of a 1,2,9,10-tetrasubstituted aporphine type linked to -CH 2 CH(OH)CH 3 unit. The HMBC spectrum of 1 provided conclusive evidence for the presence of the 2-hydroxypropyl chain unit. The HMBC spectrum showed cross peaks of H-3 with C1, C2, C3a and C4; H-12 with C5, C6a and C14; H-8 with C7, C11a and C-10; and H-9 with the C7a and C11.   The NOE differential measurements, showed enhancement of H-4 ( 2.67) upon radiation of H-3 ( 6.79). In addition the irradiation of H-9 showed enhancement of 10-OMe protons and H-8, suggesting that the methoxyl groups are placed at C-1 and C-10, respectively. The absolute configuration of the asymmetric carbon at C-13 was not determined due to the limited amount of compound available, so alkaloid 1 can be considered a racemic mixture.
To our knowledge, there has been no report on the phytochemical study and medicinal value of Actinodaphne pruinosa. This is the first report on the occurrence of N-(2-hydroxypropyl)aporphine type of alkaloid; (+)-N-(2-hydroxypropyl)lindcarpine (1) which exhibited significant cytotoxicity against P-388 murine leukemia cells.

Conclusions
In summary, we have observed that Actinodaphne pruinosa produces alkaloids closely related to those found in A. nitida, A. acutivena, A. abovata and A. sesquipedalis which were studied previously [4,9,10]. These species yielded noraporphine or N-methylaporphine types. The presence of N-(2hydroxypropyl)aporphine type which significantly exhibited a potent cytotoxicity against P-388 [11] murine leukemia cells with IC 50 3.9 μg/mL, suggesting its potential for further investigation as anticancer agent.

General
The optical rotations were recorded on s Jasco (Japan) P1010 instrument equipped with a tungsten lamp. HRESIMS 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 recorded 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. Chemical shifts are reported in ppm on  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 of Actinodaphne pruinosa (2.0 kg) was ground and extracted exhaustively for 12 hours by Soxhlet extraction with hexane, followed by CH 2 Cl 2 . Extraction of alkaloids was carried out in the usual manner, which has been described in detail [12,13] and gave 43.0 g of crude alkaloid. The crude alkaloid was submitted to exhaustive column chromatography over silica gel using CH 2 Cl 2 gradually enriched with methanol to yield 26 fractions. Fractions were combined on the basis of TLC behavior.  Table 1; Principle NOE's (%) in CDCl 3 : OCH 3 -10 to H-9 (2.6%).