Total Syntheses of (±)-Gusanlung A, (±)-Gusanlung D and 8-Oxyberberrubine and the Uncertainty Concerning the Structures of (-)-Gusanlung A, (-)-Gusanlung D and 8-Oxyberberrubine

(±)-Gusanlung A, 8-oxyberberrubine and (±)-gusanlung D have been synthesized by radical cyclisation of the corresponding 2-aroyl-1-methylenetetra- hydroisoquinolines. The 1H and 13C spectra of (-)-gusanlung D were found to be different from those of synthetic (±)-gusanlung D. Careful analyses of the 13C spectra of (–)-gusanlung A and natural 8-oxyberberrubine also cast doubt on the correctness of the structures previously assigned to these two compounds. (±)-Gusanlung A and (±)-gusanlung D were inactive against Staphylococcus aureus ATCC25932, Escherichia coli ATCC10536 and Candida albicans ATCC90028.

The 1 H-NMR data of synthetic (±)-gusanlung A (2) were in reasonably good agreement with those reported for natural (-)-gusanlung A (2). However, a number of carbons in the 13 C-NMR spectrum of natural (-)-gusanlung A (2) were found to have quite different chemical shifts from the corresponding carbons in the spectrum of (±)-gusanlung A (2). We therefore carried out 1 H-1 H-COSY, HMQC and HMBC experiments to allow complete assignments of chemical shifts of (±)-gusanlung A (2). Details of the HMBC correlations are shown in Figure 2 and Table 4. The 1 H-NMR spectral data of natural 8oxyberberrubine (3) were found to be in good agreement with those of synthetic 8-oxyberberrubine (3). However, from HMBC correlation experiment, it was possible to establish that the chemical shifts of H-1 and H-13 previously assigned should be interchanged. On the other hand, the 13 C spectrum of natural 8-oxyberberrubine (3) had a number of features which were quite different from those of synthetic 8-oxyberberrubine (3). These differences were highlighted and the HMBC correlations were shown in Figure 2 and Table 5. In summary, it can be concluded that while the 1 H-NMR analysis lent good support to the structures proposed for (-)-gusanlung A (2) and 8-oxyberberrubine (3), in view of the discrepancies in a number of carbon chemical shifts in the 13 C-NMR spectra of (-)-gusanlung A (2) versus those of (±)-gusanlung A (2) on the one hand, and natural 8-oxyberberrubine (3) versus synthetic 8-oxyberberrubine (3) on the other, no definite conclusions can be drawn at this time concerning the correctness of the structures previously assigned to (-)-gusanlung A (2) and 8oxyberberrubine (3). Synthesis of (±)-gusanlung D The synthesis of (±)-gusanlung D (1) was uneventful. Thus, 2-iodobenzoyl chloride (4d) was reacted with 5 [8] in the presence of triethylamine to give the highly unstable 2-(2′-iodobenzoyl)-1methylene-6,7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (6b). Treatment of 6b with tributyltin hydride in presence of a catalytic amount of 2,2′-azobis(isobutyronitrile) gave a 39.0% yield of a mixture of 1 and 8b in a ratio of 87:23 from 1 H-NMR analysis. Treatment of the mixture with hydrazine and palladium/charcoal gave (±)-gusanlung D (1), whose 1 H-and 13 C-NMR data were in good agreement with those of (±)-gusanlung D (1) and (-)-gusanlung D obtained from previous syntheses [2,3,4] but differed significantly from those of natural (-)-gusanlung D [1]. The structure previously assigned to (-)-gusanlung D [1] therefore remains uncertain.       Antimicrobial activity (±)-Gusanlung D (1) and (±)-gusanlung A (2) at the concentration value 256 μg/mL were inactive against Staphylococcus aureus ATCC25932, Escherichia coli ATCC10536 and Candida albicans ATCC90028.

Conclusions
Based on spectral analysis, there were significant discrepancies between the spectral data of natural (-)-gusanlung D and synthetic (±)-gusanlung D. Hence, the structure previously proposed for (-)gusanlung D remains doubtful. While the 1 H spectral data of natural (-)-gusanlung A and 8oxyberberrubine were in reasonably good agreement with those of synthetic (±)-gusanlung A and 8oxyberberrubine, the 13 C spectral data of natural (-)-gusanlung A and 8-oxyberberrubine were not entirely in good agreement with those of synthetic (±)-gusanlung A and 8-oxyberberrubine. The structures previously proposed for natural (-)-gusanlung A and 8-oxyberberrubine must therefore be treated with caution.

General
Melting points were determined on a SMP 2 Stuart Scientific melting point apparatus and are uncorrected. Infrared spectra were recorded on CH 2 Cl 2 -films with a Perkin Elmer Spectrum GX FT-IR spectrophotometer. Ultraviolet spectra were recorded on methanol solutions with a Perkin Elmer Lambda 35 UV-VIS spectrophotometer. 1 H-and 13 C-NMR spectra were recorded on (D) chloroform solutions at 300 MHz for 1 H and 75 MHz for 13 C with a Bruker AVANCE 300 spectrometer. Tetramethylsilane was used as the internal standard. MS spectra were recorded on a POLARIS Q mass spectrometer. Benzyloxy-6-bromo-3-methoxybenzoic acid (4b). A solution of sodium chlorite (0.36 g, 3.6 mmol) in H 2 O (5 mL) was added to a solution of 2-benzyloxy-6-bromo-3-methoxybenzaldehyde (4a) [7] (1.0 g, 3.1 mmol) and sulfamic acid (0.5 g) in tert-butanol (10 mL) and H 2 O (3 mL). The solution was stirred for 1 h. The mixture was shaken with ethyl acetate (20 mL) and the ethyl acetate layer was extracted with 5% sodium carbonate (3 × 20 mL). The aqueous layer was then acidified with concentrated hydrochloric acid and extracted with chloroform (3 × 20 mL). The chloroform layer was dried over anhydrous sodium sulfate. Removal of the solvent under vacuum gave a solid which was recrystallized from benzene-hexane to give 4b as pale white crystals (0.
A solution of iodine (4.6 g, 18.3 mmol) in dioxane (100 mL) was added dropwise over 30 min. to a refluxing solution of the mixture of 7a and 8a (1.3 g, 3.0 mmol) and sodium acetate (1.5 g) in dioxane (50 mL), then the mixture was refluxed for 6 h. On cooling, the sodium acetate was filtered off and the precipitate was washed with chloroform (100 mL). The chloroform layer was washed with 5% NaHSO 3 (100 mL), dilute NH 3 (30 mL), H 2 O (100 mL) then dried over anh. Na 2 SO 4 . Removal of the solvent under vacuum gave a red solid which was recrystallized with ethanol to give 9-benzyl-8oxyberberrubine (8a) as red crystals (0.  (3). A solution of 8a (100.0 mg, 0.2 mmol) in ethanol (30 mL) and conc. HCl (30 mL) was refluxed for 3 h. On cooling, the solution was extracted with chloroform (50 mL). The extract was washed with water (50 mL), then dried over anh. Na 2 SO 4 . Removal of the solvent under vacuum gave a yellow solid which was recrystallized with ethanol to give 8-oxyberberrubine (3) Table 5.

Minimum inhibitory concentration (MIC)
MIC of (±)-gusanlung A (2) and (±)-gusanlung D (1) were determined by NCCLS microbroth dilution methods [9]. (±)-Gusanlung A (2) and (±)-gusanlung D (1) were weighed and dissolved in DMSO to make a solution of concentration 2.56 mg/mL. From this stock solution two-fold serial dilution has been carried out to give a series of solutions from 256 μg/mL to 0.50 μg/mL with culture medium in 96-well microplates (100 μL of total volume). Three different microorganisms were selected viz. Staphytolcoccus aureus ATCC25932, Escherichia coli ATCC10536 and Candida albicans ATCC90028. They were subcultured on nutrient broth supplemented with 10% glucose (NBG) (for bacteria) or Sabouraud glucose broth (for yeast) and incubated at 37 °C for 24 h. A final concentration of 1 x 10 5 cfu/mL of test bacteria or yeast was added to each dilution. The plates were incubated at 37 °C for 48 h. MIC was defined as the lowest concentration of test agent that inhibited bacterial or yeast growth, as indicated by the absence of turbidity. Test agent-free broth containing 5% DMSO was incubated as growth control.