(6,7-Dimethoxy-4-methylisoquinolinyl)-(4’-methoxyphenyl)-methanone, a New Benzylisoquinoline Alkaloid from Beilschmiedia brevipes

The leaves of Beilschmiedia brevipes provided a new benzylisoquinoline alkaloid: (6,7-dimethoxy-4-methylisoquinolinyl)-(4’-methoxyphenyl)-methanone (1) and O,O-dimethylannocherin A (2), a new natural compound which has been synthesized before. Complete 1H- and 13C-NMR data of both compounds were reported. The structures were established through various spectroscopic methods notably 1D- and 2D-NMR, UV, IR and HRESIMS.


Figure 2. Selected HMBC correlation of alkaloids
, was obtained as a brownish amorphous solid. The HRESI mass spectrum showed a molecular ion peak at m/z 326.1396 [M+H] + , thus giving a molecular formula of C 19 H 20 NO 4 (calc. 326.1392 Δ 0.4 mmu). One fragmentation peak was observed at m/z 308.1457, [M-H 2 O] + which can be attributed to the loss of a water molecule thus suggesting the presence of an aliphatic hydroxyl group. The IR spectrum revealed absorption bands at 3387 and 1597 cm −1 for -OH and C=N stretching vibrations [17,18].
The 1 H-NMR spectrum showed a singlet at δ 7.05 ascribable to H-3. A singlet representing C-8 proton appeared at δ 7.11 thus, suggesting that ring A was tetrasubstituted. A pair of doublets with a coupling constant of 5.6 Hz appeared at δ 8.38 and 7.52 which belong to H-3 and H-4 respectively [14]. In addition, an imine carbon signal (C-1) appeared at δ 156.6. HMBC spectrum showed correlation between H-α at δ 6.23 and C-1 (δ 156.6) thus, indicating that ring B is fully unsaturated. Two doublets at δ 7.24 and 6.78 (J = 8.4 Hz, each two protons) are also observed for a disubstituted ring C.
The 13 C-NMR spectrum of (2) showed the presence of 19 carbons, which was in agreement with the molecular formula. The C-α hydroxyl peak was observed at δ 72.1. The complete 1 H-and 13 C-NMR spectral assignment of (2) was accomplished by thorough analysis of DEPT, COSY, HMQC, and HMBC data. The absolute configuration at C-α is determined as S due to the positive sign of the specific rotation for alkaloid (2) [α] 25 D = +91° (c = 0.22, MeOH) [18,19].

Biological Activity
The six alkaloids isolated in this study were tested for cytotoxic activity against P-388 murine leukemia cell lines. Only O,O-dimethylcoclaurine [12] exhibited any significant cytotoxicity, with an IC 50 of 6.5 μg/mL (Table 2). Table 2. In vitro cytotoxic activity against P388 cell lines.

General
The optical rotations were recorded on a Jasco (Japan) P1010 instrument equipped with a tungsten lamp. HRMS were obtained on a Thermo Finnigan Automass Multi. The ultraviolet spectra were obtained in MeOH on a Shimadzu UV-310 ultraviolet-visible spectrometer. The Fourier Transform Infrared (FTIR) spectra were obtained with CHCl 3 (NaCl window technique) on a Perkin Elmer 2000 instrument. The 1 H-NMR and 13 C-NMR spectra were recorded in deuterated chloroform on a JEOL 400 MHz (unless stated otherwise) instrument; 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) was used for column chromatography. Mayer's reagent was used for alkaloid screening. TLC aluminium sheets (20 × 20 cm Silica gel 60 F 254 ) were used in the TLC analysis. The TLC spots were visualized under UV light (254 and 366 nm) followed by spraying with Dragendorff's reagent for an alkaloid detection.

Plant Materials
Beilschmiedia brevipes (Lauraceae), collected from Lenggor, Kluang, Johor, Malaysia was identified by Mr Teo Leong Eng. A voucher specimen (KL4978) is deposited at the Herbarium of the Department of Chemistry, University of Malaya, Kuala Lumpur, Malaysia and at the Herbarium of the Forest Research Institute, Kepong, Malaysia.

Cytotoxic Assays
The cytotoxicity of all the alkaloids was tested against P-388 murine leukemia cells using the MTTmicroculture tetrazolim assay [21].

Conclusions
To the knowledge of the authors, alkaloid 1 is the first benzylisoquinoline alkaloid bearing a methyl group at C-4 reported in the family of Lauraceae and this is the first communication on alkaloids from the Beilschmiedia brevipes. Alkaloid 2, which was identified in this study belongs to the rare C-α hydroxybenzylisoquinoline class. Only two examples of this type of alkaloid have been previously reported from Ocotea pulchella (Lauraceae) and Annona cherimola (Annonaceae), respectively [18,20]. According to Botega et al. [20], this type of alkaloids tends to oxidize easily to the corresponding ketones in air therefore alkaloid 3 is most probably formed through oxidation of alkaloid 2.