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 (1)H and (13)C spectra of (-)-gusanlung D were found to be different from those of synthetic (+/-)-gusanlung D. Careful analyses of the (13)C 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.

Introduction

(–)-Gusanlung D, isolated from Acangelisia gusanlung H. S. Lo (Menispermaceae), is the first natural 8-oxotetrahydroprotoberberine alkaloid with an unoxygenated ring D [1]. Based on spectral data analysis, structure 1 was proposed for (–)-gusanlung D. Prior to the isolation of (–)-gusanlung D, Kessar et al. synthesized in 1992 a compound which is essentially (±)-gusanlung D [2]. However, a close comparison of the 1H-NMR data of (±)-gusanlung D with those reported for (–)-gusanlung D revealed significant differences. In 2003 Reimann, Grasberger and Polborn reported another synthesis of (±)-gusanlung D [3]; in this case the 13C-NMR spectral data were found to show significant differences to those reported for (–)-gusanlung D. Subsequently, an unsymmetric synthesis of (–)-gusanlung D was achieved by Chrzanowska, Dreas and Razwadowska in 2004 [4]. Comparison of the 1H- and 13C-NMR data of synthetic (–)-gusanlung D with those of natural (–)-gusanlung D also showed significant differences. Finally, Chang and Chang reported a total synthesis of (±)-gusanlung D [5], whose spectral data were said to agree with those in references [1,2,3,4]. This last conclusion added further confusion to the matter since, if the spectral data of (±)-gusanlung D [5] are in good agreement with those reported for (±)-gusanlung D [2,3] and synthetic (–)-gusanlung D [4], they cannot also be consistent with those reported for natural (–)-gusanlung D [1]. In view of these discrepancies in the 1H- and 13C-NMR data of natural (–)-gusanlung D [1] and the synthetic alkaloids, it was therefore highly desirable to perform another independent synthesis of (±)-gusanlung D to shed further light on the possible structure of (–)-gusanlung D.

Structures of (–)-gusanlung D (1), (–)-gusanlung A (2) and 8-oxyberberrubine (3).

Furthermore, two new related alkaloids: (–)-gusalung A [6] and 8-oxyberberrubine [1], for which structures 2 and 3 were proposed based on spectral analysis, were isolated from Acangelisia gusanlung H. S. Lo. In view of the uncertainty regarding the correct structure of (–)-gusanlung D (1), it was therefore highly desirable to also confirm the correctness of the structures proposed for (–)-gusanlung A (2) and 8-oxyberberrubine (3) by total syntheses.

Results and Discussion

Syntheses of (±)-gusanlung A (2) and 8-oxyberberrubine (3)

The synthesis of (±)-gusanlung A (2) was based on the radical-initiated cyclization of 2-(2'-benzyloxy-6'-bromo-3'-methoxybenzoyl)-1-methylene-6,7-methylenedioxy-1,2,3,4-tetrahydroisoqui- noline (6a), as outlined in Scheme 1, with subsequent catalytic hydrogenolysis of the benzyl protecting group.

Scheme 1

Synthetic routes to (±)-gusanlung A (1), (±)-gusanlung D (2), and 8-oxyberberrubine (3).

Reagents and Conditions A) NaClO2, sulphamic acid/ tert-butanol-H2O; B) SOCl2/ benzene; C) Et3N/ dry benzene; D) Bu3SnH, AIBN/ dry benzene; E) H2, Pd/C/ ethanol; F) hydrazine, Pd/C/ ethyl acetate-ethanol; G) I2/ dioxane; H) conc. HCl/ ethanol.

Thus, oxidation of 2-benzyloxy-6-bromo-3-methoxybenzaldehyde (4a) [7] with sodium chlorite gave 2-benzyloxy-6-bromo-3-methoxybenzoic acid (4b), whose acid chloride (4c) was then reacted with 6,7-methylenedioxy-1-methyl-3,4-dihydroisoquinoline (5) [8] in the presence of triethylamine to give thee moderately stable compound 6a. Treatment of 6a with tributyltin hydride in the presence of a catalytic amount of 2,2'-azobis(isobutyronitrile) gave a 31.3% yield of a mixture of (±)-9-benzylgusanlung A (7a) and 9-benzyl-8-oxyberberrubine (8a) in a ratio of 78:22 according to 1H-NMR analysis. Catalytic hydrogenolysis of the mixture of 7a and 8a to remove the benzyl protecting group also resulted in the concurrent hydrogenation of the C-C double bond to give pure (±)-gusanlung A (2). On the other hand, oxidation of the mixture of 7a and 8a with iodine gave 9-benzyl-8-oxyberberrubine (8a), whose benzyl protecting group was removed by acid treatment to give 8-oxy-berberrubine (3).

The 1H-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 13C-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 1H-1H-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 1H-NMR spectral data of natural 8-oxyberberrubine (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 13C 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 1H-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 13C-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 8-oxyberberrubine (3).

Figure 2

HMBC correlations of (±)-gusanlung A (1) and 8-oxyberberrubine (3).

Table 4

Comparison of 13C-NMR spectral data between natural (-)-gusanlung A [6] and synthetic (-)-gusanlung A [this work] and HMBC correlations of (±)-gusanlung A [this work].

(position)	(-)-gusanlung A(DMSO-d6) [6]m.p. 260-262 °C	(±)-gusanlung A(DMSO-d6) [this work]m.p. 188-189 °C	(±)-gusanlung A(CDCl3) [this work]m.p. 188-189 °C	(±)-gusanlung A(DMSO-d6) [this work]
				HMBC
	13C	13C	13C	2J	3J
1	106.1	106.6	105.8	C-2	C-3, 4a, 14
2	145.9a	146.7c	146.8*	-	-
3	147.7a	146.5c	146.7*	-	-
4	107.8	108.7	108.6	C-3	C-2, 5, 14a
4a	129.1b	128.3	128.1	-	-
5	29.0	28.9	29.4	C-4a, 6	C-4, 14a
6	37.8	38.5	38.4	C-5	C-4a, 8, 14
8	161.4	168.4	168.6	-	-
8a	122.3b	111.4	111.4	-	-
9	149.7a	151.4	151.8	-	-
10	145.7a	147.2	147.5	-	-
11	118.9	116.7	115.4	C-10	C-9, 12a
12	122.1	116.9	116.1	C-11	C-8a, 10, 13
12a	128.2b	129.6	128.7	-	-
13	37.7	35.9	37.1	C-12a, 14	C-8a, 12, 14a
14	54.4	55.4	55.7	C-13, 14a	-
14a	129.3b	129.1	128.4	-	-
C10-OCH3	60.5	56.3	56.3	-	C-10
OCH2O	100.5	101.3	101.2	-	C-2, 3
OH				C-9	C-8a, 10

a, b, c,* assignments may be interchangeable.

Table 5

Comparison of 1H- and 13C-NMR spectral data between natural 8-oxyberberubine (3) [1], synthetic 8-oxyberberubine (3) [this work] and HMBC correlations of 8-oxyberberrubine [this work].

(position)	natural 8-oxy-berberrubine (3)CDCl3 [1]m.p. 240-241 °C	synthetic 8-oxy-berberrubine (3)CDCl3 [this work]m.p. 238-239 °C	natural 8-oxy-berberrubine (3)CDCl3 [1]m.p. 240-241 °C	synthetic 8-oxy-berberrubine (3)CDCl3 [this work]m.p. 238-239 °C	synthetic 8-oxyberberrubine (3)(CDCl3) [this work]
					HMBC
	1H	1H	13C	13C	2J	3J
1	6.83 (s)	7.21 (s)	104.0	104.8	C-2	C-3, 4a, 14
2			141.6	147.5*	-	-
3			146.4	148.6*	-	-
4	6.72 (s)	6.71 (s)	107.1	108.0	C-3	C-2, 5, 14a
4a			109.6	129.5	-	-
5	2.91 (t, 7.2)	2.92 (t, 6.1)	28.4	28.4	C-4a, 6	C-4, 14a
6	4.27 (t, 7.2)	4.27 (t, 6.1)	39.1	39.1	C-5	C-4a, 8, 14
8			164.0	165.4	-	-
8a			129.9	111.0	-	-
9			149.0	150.3	-	-
10			147.5	144.9	-	-
11	7.30 (AB q, 8.0)	7.28 (d, 8.5)	114.9	119.1	C-10	C-9, 12a
12	7.00 (AB q, 8.0)	6.99 (d, 8.5)	120.0	115.3	C-11	C-8a, 10, 13
12a			128.9	130.5	-	-
13	7.21 (s)	6.83 (s)	103.6	103.6	C-14	C-8a, 12, 14a
14			133.6	134.6	-	-
14a			122.1	123.5	-	-
C10-OCH3	3.96 (s)	3.97 (s)	56.7	56.7	-	C-10
OCH2O	6.02 (s)	6.02 (s)	100.6	101.5	-	C-2, 3
OH	-	13.14	-	-	-	-

*assignments may be interchangeable.

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)-1-methylene-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 1H-NMR analysis. Treatment of the mixture with hydrazine and palladium/charcoal gave (±)-gusanlung D (1), whose 1H- and 13C-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 1H spectral data of natural (-)-gusanlung A and 8-oxyberberrubine were in reasonably good agreement with those of synthetic (±)-gusanlung A and 8-oxyberberrubine, the 13C 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.

Experimental

General

Melting points were determined on a SMP 2 Stuart Scientific melting point apparatus and are uncorrected. Infrared spectra were recorded on CH2Cl2-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. 1H- and 13C-NMR spectra were recorded on (D) chloroform solutions at 300 MHz for 1H and 75 MHz for 13C with a Bruker AVANCE 300 spectrometer. Tetramethylsilane was used as the internal standard. MS spectra were recorded on a POLARIS Q mass spectrometer.

2-Benzyloxy-6-bromo-3-methoxybenzoic acid (4b). A solution of sodium chlorite (0.36 g, 3.6 mmol) in H2O (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 H2O (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 (0.8 g, 76.2%), m.p. 112-115 °C; 1H-NMR: δ 7.47-7.42 (2H, m, Ph-H); 7.38-7.25 (4H, m, Ph-H × 3 and Ar-H); 6.88, (1H, d, J = 8.9 Hz, Ar-H); 5.10 (2H, s, CH2Ph); 3.89 (3H, s, OCH3). 13C-NMR: δ 171.0 (C), 152.2 (C), 145.9 (C), 136.7 (C), 130.6 (C), 128.4 (CH), 128.3 (CH), 128.2 (CH), 114.9 (CH), 108.7 (C), 76.0 (CH2), 56.2 (OCH3).

2-(2'-Benzyloxy-6'-bromo-3'-methoxybenzoyl)-1-methylene-6,7-methylenedioxy-1,2,3,4-tetrahydro-isoquinoline (6a). A solution of acid 4b (3.6 g, 10.0 mmol) and thionyl chloride (3.9 g, 32.8 mmol) in benzene (20 mL) was refluxed for 1 h. The solvent and excess thionyl chloride were removed under vacuum to give acid chloride 4c as a yellow oil (3.7 g, 94.9%) which was used in the next step without further purification. A solution of acid chloride 4c (1.9 g, 5.3 mmol) in dry benzene (20 mL) was added dropwise over 10 min. to a solution of isoquinoline 5 [8] (1.0 g, 5.3 mmol) and triethylamine (1.0 g) in dry benzene (20 mL), then the mixture was refluxed for 2 h. On cooling, the precipitated triethylamine hydrochloride was filtered off. The filtrate was evaporated under vacuum to give enamide 6a as a yellow oil (2.6 g, 84.4%) which was unstable and decomposed on standing. It was immediately used in the next step without further purification. 1H-NMR: δ 7.38-7.23 (5H, m, Ph-H), 7.18(1H, d, J = 8.8 Hz, H-5'), 6.89 (1H, s, H-8), 6.75 (1H, d, J = 8.8 Hz, H-4'), 6.41 (1H, s, H-5), 5.90 (2H, AB q, J = 1.3 Hz, OCH2O), 5.14 (1H, d, J = 1.3 Hz, =CH2), 5.00 (2H, AB q, J = 10.8 Hz, CH2Ph), 4.81 (1H, d, J = 1.3 Hz, =CH2), 4.13-4.02, 3.57-3.50 (2H, 2 m, CH2-3), 3.80 (3H, s, OCH3), 2.90-2.59 (2H, m, CH2-4); 13C-NMR: δ 165.0 (C), 152.1 (C), 147.8 (C), 146.5 (C), 145.3 (C), 141.4 (C), 137.4 (C), 134.3 (C), 129.0 (C), 128.4 (CH), 128.1 (CH), 127.9 (CH), 127.7 (CH), 125.1 (C), 113.4 (CH), 110.0 (C), 108.4 (CH), 104.4 (CH2), 103.8 (CH), 101.1 (CH2), 75.4 (CH2), 55.9 (OCH3), 41.6 (CH2), 28.8 (CH2).

(±)-Gusanlung A (1) and 9-benzyl-8-oxyberberrubine (8a). A solution of enamide 6a (2.7 g, 5.3 mmol), tributyltin hydride (3.4 g, 11.7 mmol) and 2,2'-azobis(isobutyronitrile) (0.2 g, 0.7 mmol) in dry benzene (50 mL) was refluxed with stirring for 3 h., then the solvent was removed under vacuum. The residue was washed with hexane (4 × 15 mL) and dissolved in chloroform (30 mL). The chloroform layer was washed with brine, then dried over anhydrous sodium sulfate. The solvent was removed under vacuum to give a yellow solid which was recrystallized from ethanol to give a 31.3% yield of a mixture of (±)-9-benzylgusanlung A (7a) and 9-benzyl-8-oxyberberrubine (8a) in a ratio of 78:22 from 1H-NMR analysis.

A solution of the mixture of 8a and 7a (303.7 mg, 0.7 mmol) in ethanol (50 mL) was hydrogenated over 10% Pd/C (30.4 mg) at atmospheric pressure for 48 h. The catalyst was fittered off and the solvent was removed under vacuum to give a crude yellow solid. Recrystallization of the crude solid from ethanol gave (±)-gusanlung A (2) as a pale yellow soild (82.4 mg, 34.3%), m.p. 188-189 °C; UV (MeOH) λmax nm (log ε): 219 (4.54), 271sh (3.87), 281 (3.98), 308 (4.16), 319 (4.15); IR νmax (film): 3737, 3650, 3585, 2919, 2852, 1748, 1634, 1615, 1581, 1542, 1506, 1488, 1456, 1386, 1356, 1336, 1315, 1262, 1239, 1154, 1084, 1069, 1037, 1001, 933, 858, 804, 792, 728 cm-1; MS (EI) m/z (%): 339 (M+, 55), 176 (100). 1H-NMR see Table 3, 13C-NMR and HMBC see Table 4.

Table 3

Comparison of 1H-NMR spectral data between natural (-)-gusanlung A [1] and synthetic (±)-gusanlung A [this work].

(position)	(-)-gusanlung A (DMSO-d6) [6]m.p. 260-262 °C	(±)-gusanlung A(DMSO-d6) [this work]m.p. 188-189 °C	(±)-gusanlung A(CDCl3) [this work]m.p. 188-189 °C
	1H	1H	1H
1	6.96 (s)	7.00 (s)	6.71 (s)
4	6.80 (s)	6.79 (s)	6.66 (s)
5	2.73-2.81 (m)	2.75-2.89 (m)	2.72-2.84 (m)
6α	2.73-2.81 (m)	2.89-3.01 (m)	2.94-3.40 (m)
6β	4.71 (m)	4.69-4.59(m)	4.80-4.87 (m)
11	6.99 (d, 8.1)	7.09 (d, 8.1)	6.94 (d, 8.1)
12	6.86 (d, 8.1)	6.71 (d, 8.1)	6.63 (d, 8.1)
13α	3.13 (dd, 15.3, 3.1)	3.36 (dd, 15.2, 3.6)	3.14 (dd, 15.2, 3.8)
13β	2.62 (dd, 15.3, 13.3)	2.66-2.75 (m)	2.80-2.94 (m)
14	4.68 (dd, 13.3, 3.1)	4.84 (dd, 13.3, 3.4)	4.80 (dd, 13.6, 3.5)
C10-OCH3	3.76 (s)	3.78 (s)	3.90 (s)
OCH2O	5.98, 5.99 (s)	5.98, 6.00 (s)	5.96 (s)
OH	-	12.88 (s)	12.83 (s)

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% NaHSO3 (100 mL), dilute NH3 (30 mL), H2O (100 mL) then dried over anh. Na2SO4. Removal of the solvent under vacuum gave a red solid which was recrystallized with ethanol to give 9-benzyl-8-oxyberberrubine (8a) as red crystals (0.6 g, 50.0%), m.p. 190-192 °C. UV (MeOH) λmax nm (log ε): 206sh (4.62), 224 (6.31), 255sh (5.78), 312 (5.76), 342 (6.03), 369 (5.86), 387 (5.71); IR νmax (film): 2938, 2898, 2841, 1651, 1619, 1599, 1494, 1484, 1386, 1372, 1317, 1277, 1225, 1176, 1100, 1083, 939, 871, 834, 777, 734 cm-1; 1H-NMR: δ 7.73-7.68 (2H, m, Ph-H); 7.43-7.32 (3H, m, Ph-H); 7.32-7.28 (2H, m, H-11 and H-12); 7.22 (1H, s, H-1), 6.72 (1H, s, H-13); 6.70 (1H, s, H-4); 6.00 (2H, s, OCH2O); 5.16 (2H, s, CH2Ph); 4.31 (2H, t, J = 6.1 Hz, CH2-6); 3.88 (3H, s, OCH3); 2.88 (2H, t, J = 6.1 Hz, CH2-5); 13C-NMR: δ 160.2 (C), 151.7 (C), 148.4 (C), 148.2 (C), 147.3 (C), 138.1 (C), 135.6 (C), 132.4 (C), 130.1 (C), 128.7 (CH), 128.2 (CH), 127.7 (CH), 123.8 (C) , 122.5 (CH), 119.8 (C), 119.1 (CH), 107.9 (CH), 104.7 (CH), 101.4 (CH2), 101.3 (CH), 75.7(CH2), 56.9 (OCH3), 39.5 (CH2), 28.7 (CH2).

8-Oxyberberrubine (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. Na2SO4. Removal of the solvent under vacuum gave a yellow solid which was recrystallized with ethanol to give 8-oxyberberrubine (3) as pale yellow crystals (42.2 mg, 53.5%), m.p. 238-239 °C (Lit. [2] m.p. 240-241 °C); UV (MeOH) λmax nm (log ε): 225 (4.44), 258sh (3.99), 270 (3.87), 288 (3.69), 345 (4.16), 369 (4.13); IR νmax (film): 3011, 2893, 2836, 1645, 1594, 1490, 1393, 1320, 1267, 1228, 1181, 1087, 1033, 932, 826, 665 cm-1. 1H-NMR, 13C-NMR and HMBC see Table 5.

2-(2'-Iodobenzoyl)-1-methylene-6, 7-methylenedioxy-1,2,3,4-tetrahydroisoquinoline (6b). A solution of 2-iodobenzoyl chloride 4d (1.4 g, 5.4 mmol) in dry benzene (20 mL) was added dropwise over 10 min. to a solution of isoquinoline 5 [8] (1.0 g, 5.3 mmol) and triethylamine (1.0 g) in dry benzene (20 mL), then the mixture was refluxed for 2 h. On cooling, the precipitated triethylamine hydrochloride was filtered off and the filtrate was evaporated under vacuum to give enamide 6b as a yellow oil (2.2 g, 99.1%) which was unstable and decomposed on standing, so it was immediately used in the next step without further purification. 1H-NMR: δ 8.07-6.84 (5H, m, Ar-H); 6.58 (1H, s, Ar-H); 5.92 (2H, s, OCH2O); 5.18 (1H, br s, =CH2); 4.50 (1H, br s, =CH2); 4.12( 2H, br s, CH2); 2.95 (2H, br s, CH2); 13C-NMR: δ 169.0 (C), 161.2 (C), 148.2 (C), 146.8 (C), 142.6 (C), 142.2 (CH), 139.3 (CH), 135.9 (C), 132.5 (C), 129.9 (CH), 128.3 (CH), 125.0 (C), 108.4 (CH), 106.2 (CH2), 103.9 (CH), 101.2 (CH2), 41.8 (CH2), 29.0 (CH2).

(±)-Gusanlung D (1) and 13,14-didehydrogusanlung D (8b). A solution of enamide 6b (2.9 g, 10.0 mmol) tributyltin hydride (11.7 g, 40.0 mmol) and 2,2'-azobis(isobutyronitrile) (1.6 g, 10.0 mmol) in dry benzene (50 mL) was refluxed with stirring for 3 h., then the solvent was removed under vacuum. The residue was washed with hexane (4 ×15 mL) and dissolved in chloroform (30 mL). The chloroform layer was washed with brine, then dried over anhydrous sodium sulfate. The solvent was removed under vacuum to give a solid which was recrystallized from ethanol to give a 39.0% yield of a mixture of 1 and 8b in a ratio of 23:87 from 1H-NMR analysis.

A mixture of 1 and 8b (200.0 mg, 0.7 mmol), Pd/C (300.0 mg), hydrazine (50 mL), ethanol (50 mL) and ethyl acetate (50 mL) was refluxed for 48 h. The Pd/C was filtered and the filtrate extracted with chloroform (80 mL). The extract was washed with 10% HCl (2 × 50 mL), water (50 mL) then dried over anh. Na2SO4. Removal of the solvent under vacuum gave a yellow solid which was recrystallized with ethanol to give pure (±)-gusanlung D (1) as pale yellow crystals (99.4 mg, 49.4%), m.p. 175-176 °C (lit. [5] m.p. 175-177 °C). UV (MeOH) λmax nm (log ε): 206 (6.27), 230 (5.78), 253sh (5.42), 290 (5.42), 335 (5.02), 365 (4.77); IR νmax (film): 2922, 1646, 1602, 1576, 1487, 1412, 1362, 1333, 1285, 1241, 1218, 1178, 1141, 1038, 936, 906, 853, 743, 636, 505 cm-1. 1H-NMR and 13C-NMR see Table 1 and Table 2.

Table 1

Comparison of 1H-NMR spectral data between natural (-)-gusanlung D [1], synthetic (-)-gusanlung D [4] and synthetic (±)-gusanlung D [2] and [this work].

(position)	(–)-gusanlung D CDCl3 [1]m.p. 250-251 °C	(–)-gusanlung DCDCl3 [4]m.p. 195-197 °C	(±)-gusanlung DCDCl3 [2] m.p. 175-177 °C	(±)-gusanlung D CDCl3 [this work]m.p. 175-176 °C
	1H	1H	1H	1H
1	7.35 (s)	6.71 (s)	6.76 (d)	6.72 (s)
4	6.80 (s)	6.67 (s)	6.76 (d)	6.67 (s)
5α	2.70-3.40 (m)	2.7-2.8 (m)	2.83-3.35 (m)	2.70-2.82 (m)
5β	2.70-3.40 (m)	2.82-3.02 (m)	2.83-3.35 (m)	2.87-3.07 (m)
6α	2.70-3.40 (m)	2.82-3.02 (m)	2.83-3.35 (m)	2.87-3.07 (m)
6β	4.8 (m)	4.93-4.99 (m)	4.7-5.1 (m)	4.88-4.99 (m)
9	8.07 (d, 8.0)	8.13 (d, 7.4)	8.1-8.37 (m)	8.13 (dd, 7.6, 1.4)
10	7.29-7.41 (m)	7.34-7.40 (m)	7.25-7.65 (m)	7.39 (br t, 7.4)
11	7.29-7.41 (m)	7.41-7.49 (m)	7.25-7.65 (m)	7.46 (dt, 7.4, 1.5)
12	7.29-7.41 (m)	7.24 (d, 7.4)	7.25-7.65 (m)	7.22-7.29 (m)
13α	2.70-3.40 (m)	2.82-3.02 (m)	2.83-3.35 (m)	2.87-3.07 (m)
13β	2.70-3.40 (m)	3.18 (dd, 15.3, 3.7)	2.83-3.35 (m)	3.18 (dd, 15.7, 3.7)
14	3.95 (m)	4.83 (dd, 13.3, 3.7)	4.7-5.1 (m)	4.84 (dd, 13.3, 3.7)
OCH2O	6.20, 6.06 (s)	5.96 (s)	5.93 (s)	5.96 (s)

Table 2

Comparison of 13C-NMR spectral data between natural (-)-gusanlung D [1], synthetic (-)-gusanlung D [4] and synthetic (±)-gusanlung D [3] and [this work].

(position)	(–)-gusanlung DCDCl3 [1]m.p. 250-251 °C	(–)-gusanlung DCDCl3 [4]m.p. 195-197 °C	(±)-gusanlung DCDCl3 [3]m.p. 175-177 °C	(±)-gusanlung DCDCl3 [this work]m.p. 175-176 °C
	13C	13C	13C	13C
1	107.3	105.8	105.97	105.9
2	135.0	146.5 b	146.57	146.6c
3	147.0	146.7b	146.77	146.8c
4	107.5	108.6	108.81	108.7
4a	126.5	128.8	128.85	128.9
5	29.7	29.6	29.61	29.7
6	42.0	38.7	38.49	38.8
8	162.0	164.5	158.67	164.6
8a	117.3	137.2	137.24	137.3
9	128.7a	128.6	128.60	128.6
10	127.9a	127.3	127.37	127.4*
11	127.1a	131.8	132.33	131.9*
12	126.8a	126.8	126.87	126.9
12a	124.6	129.0	131.81	129.1
13	33.5	38.1	37.78	38.1
14	49.4	55.2	55.18	55.3
14a	126.5	128.5	128.55	128.6
OCH2O	100.9	101.1	101.00	101.1

a, b, c,* assignments may be interchangeable.

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 105 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.