Synthesis and evaluation of bioactive naphthoquinones from the Brazilian medicinal plant, Tabebuia avellanedae����� ����

Bioorganic & Medicinal ChemistryVol.17 / No.17 (2009)

バイオオーガニック アンド メディシナル ケミストリー

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In our precedent paper,9 we reported a concise and stereoselective synthesis of 1 and its antiproliferative effect toward several tumor cell lines and in vitro cancer preventive activity. Through our preliminary results, we clearly recognized that there were significant differences between 1 and (R)-1 in their biological activities mentioned above. This finding promoted us to synthesize both 2 and its enantiomer (R)-2 and to evaluate their biological activities since it has been reported by Wagner et al. that compound 2 was isolated in the state of a racemic mixture.5c In fact, we also could obtain 2 only as a racemate through repeated HPLC of a CHCl3 extract of T. avellanedae. On the other hand, Fujimoto’s group, who

two-stage carcinogenesis tests. Therefore, compound 1 is expected to be a potential cancer chemopreventer.8

Ⅰ. Introduction

Tabebuia avellanedae LORENTZ ex GRISEB1 (Bignoniaceae) (syn. T. impetiginosa), which is widespread in South America throughout Brazil to north Argentina, has been well known as a traditional medicine since the Incan Era.2 The stem bark of Tabebuia avellanedae has been used as a diuretic and as an astringent. In addition, the stem bark of this plant shows a wide array of biological activities such as antitumor, antibacterial, antifungal and anti-inflammatory activity.3 In particular, the discovery of its antitumor activity has made T. avellanedae an important medicinal resource.4 A series of naphthoquinones and anthraquinones, a number of simple benzoic acid derivatives and iridoid glycosides has been isolated as secondary metabolites of this plant.5 Among these constituents, naphtho[2,3–b]furan–4, 9–dione analogues such as (–)–5–hydroxy–2–(1'–hydoxyethyl)naphtho[2,3–b] furan–4,9–dione (1) and its positional isomer, (–)–8–hydroxy–2–(1'–hydoxyethyl)naphtho[2,3–b] furan–4,9–dione (2) are, in particular, worthy of attention because of their potent antiproliferative activity against various tumor cells as well as lapachone (3) and β-lapachol (4) that are congeners of 1 in this plant (Fig.1).6 In addition, compound 1 strongly inhibits Epstein-Barr virus early antigen (EBV-EA) activation induced by the tumor promoter, 12-O-tetradecanoylphorbol-13-acetate (TPA) in Raji cell7 and TPA-induced tumor promotion on mouse skin initiated with 7,12-dimethylbenz[a]anthracene (DMBA) in

Abstract: A series of naphthoquinones based on the naphtho[2,3–b]furan–4,9–dione skeleton such as (–)–5–hydroxy–2–(1'–hydoxyethyl)

naphtho[2,3–b]furan–4,9–dione (1) and its positional isomer, (–)–8–hydroxy–2–(1'–hydoxyethyl)naphtho [2,3–b]furan–4,9–dione (2), which are secondary metabolites found in the inner bark of Tabebuia avellanedae, were stereoselectively synthesized and their biological activities were evaluated in conjunction with those of their corresponding enantiomers. Compound 1 exhibited potent antiproliferative effect against several human tumor cell lines, but its effect against some human normal cell lines was much lower than that of mitomycin. On the other hand, its enantiomer (R)-1 was less active toward the above tumor cell lines than 1. The antiproliferative effect of 2 against all tumor cell lines was significantly reduced. These results indicated the presence of the phenolic hydroxy group at C-5 is of great important for increasing antiproliferative effect. In addition, 1 also showed higher cancer chemopreventive activity than 2, while there were no significant differences between 1 and 2 in antimicrobial activity. Both compounds displayed modest antifungal and antibacterial activity (Gram-positive bacteria), whereas they were inactive against Gram-negative bacteria.

Akira Iida (Faculty of Agriculture, Kinki University), Mitsuaki Yamashita and Masafumi Kaneko (Faculty of Pharmacy,

Takasaki University of Health and Welfare), Harukuni Tokuda (Department of Biochemistry, Kyoto Prefectural University of

Medicine), Katsumi Nishimura (Organic Chemistry, Kobe Pharmaceutical University) others

Figure 1.

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first obtained 1 and (R)-1 together with 2 and (R)-2 by optical resolution of the synthetic racemates 1 and 2, isolated 2 from the same plant as a mixture in which the ratio of the (R)- and (S)-enantiomer was 3 : 1. Furthermore, they reported that there were no differences between the two pairs of enantiomers in their antiproliferative activity toward L1210 cells.6c These results also encouraged us to advance our research program. Herein we report the stereoselective synthesis of two pairs of enantiomers described above via Noyori reduction as a key step and their antiproliferative effect against some human tumor cell lines and their corresponding human normal cell lines, in vitro cancer chemopreventive activity and antimicrobial activity.10 In addition, in vivo cancer chemopreventive activity of 1 and (R)-1 is also described.

Ⅱ. Results and Discussion

2.1 Chemistry The gram-scale synthesis of 1 and 2 were accomplished

similarly according to our preliminary synthesis of 1.9 The synthetic way to 1 and 2 is shown in Scheme 1. The synthesis of juglone (6) was started from commercially available 1,5-hydroxynaphthalene, which was oxidized with air in the presence of CuCl in the dark to give 6 in 47% yield.11 Chemical transformations of 6 to 8 was carried out according to the reported methods12 with some modifications. Oxidative amination of 6 with dimethylamine (2.0 M solution in THF) in toluene at –40 °C gave 2-dimethylaminojuglone (7a) and 3-dimethylaminojuglone (7b) in 42% and 11% yields, respectively. As reported in the precedent paper,9 a THF solution of dimethylamine is suitable reagent for practical use, and moderate chemical yield and regioselectivity were observed.

Deamination of 2-dimethylaminojuglone (7a) with 10% aqueous HCl gave 2,5-dihydroxy-1,4-naphthoquinone (8a) in quantitative chemical yield.12b The naphtho[2,3–b]furan– 4,9–dione skeleton was constructed by the reaction of 8a with 3,4-dibromobutan-2-one, which was synthesized from commercially available but-3-en-2-one and bromine, in the presence of DBU in THF according to the method reported by Hagiwara and others13 to give naphthodihydrofuran 9a. It should be noted that the gram-scale synthesis afforded only 9a without the simultaneous formation of 10a differently from the case of the milligram-scale synthesis.9a After the crude product was washed with MeOH, naphthodihydrofuran 9a was treated with MnO2 in chloroform to provide the desired naphthofuran 10a in 50% yield in two steps. Compound 10b was synthesized from 8b via 9b in the same manner as that described above. Subsequent Noyori reduction completed the stereoselective synthesis of 1 and 2 (Scheme 2).14 Asymmetric transfer hydrogenation of naphthofurans 10a and 10b, which was catalyzed by a commercially available chiral Ru(II) complex, Ru [(S,S)-Tsdpen] (p-cymene)15 in a formic acid-triethylamine mixture and CH2Cl2, successfully afforded the corresponding secondary alcohols 1 and 2 in high chemical yield and enantiomeric excess (89-97% yield, 95-97% ee). The physico-chemical properties of all the compounds synthesized in this study were identical in all respects to those reported previously.13 The corresponding enantiomers (R)-1 and (R)-2 were similarly prepared using Ru [(R,R)-Tsdpen] (p-cymene) as a chiral catalyst.16

2.2. Biology As reported so far, naphthoquinones isolated from the

Tabebuia plants are recognized as antiproliferative compounds.3b,6,7 Thus, the two pairs of enantiomers synthesized were also evaluated for their ability to suppress the growth of human tumor cell lines including PC-3 (prostate), A549 (lung) and MCF-7 (breast) (Table 1). Compound 1 exhibited rather potent antiproliferative activity against all the three cell lines (0.14-0.78 μM), which was almost equal to that of mitomycin (0.14-0.96 μM) and much higher than that of β-lapachone (1.13-9.96 μM). However, its antiproliferative effect against human normal cell lines including Hs888Lu (lung) and SVCT-M12 (breast) was noticeably lower than that of mitomycin (1: 3.36-54.5 μM, mitomycin: 0.56-1.46 μM) (Table 2).

Scheme 1. Synthesis of 10. Reagents and conditions: (a) CuCl, CH3CN, air, rt, 47%; (b) Me2NH, toluene, THF, –40°C, 42% and 11% for 7a and 7b, respectively; (c) 10% HCl, dioxane, reflux, 97%; (d) 3,4-dibromobutan-2-one, DBU, THF, room temperature; (e) MnO2, CHCl3, reflux, 50-60% in two steps.

Scheme 2. Chiral Ru catalyst mediated asymmetric hydrogenation of naphthofuran 10

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Unexpectedly, ketone 10a exhibited potent antiproliferative effect toward the tumor cell lines (Table 1). In particular, it strongly suppressed the growth of MCF-7 cells, which was comparable to that of 1. On the other hand, its antiproliferative effect against Hs888Lu and SVCT-M12 was not as high as that of 1 (Table 2). Therefore, 10a is also eminently suitable for the development of anticancer drugs in addition to 1.

The mechanism of action of compound 1 for antiproliferative effect is still unknown and now being under investigation. However, our preliminary results indicated that compound 1 affects E2F1, a multifunctional transcription factor. On the other hand, β-lapachone, which is a congener of 1, has been shown to induce apoptosis in a variety of cancer cell lines and to be involved in transcription processes. Thus, we currently deduce that compound 1 also has a similar mechanism to that of β-lapachone. 6d

Compound 1 has already been known to act as a cancer chemopreventive agent and showed potent inhibition on EBV-EA activation without cytotoxicity against Raji cells dose-dependently.8 In order to compare in vitro cancer chemopreventive activity of (R)-1, 2 and (R)-2 with that of 1, they were evaluated for their ability to inhibit EBV-EA activation induced by TPA in Raji cells as a primary screening test for antitumor promoters along with β-lapachone and lapachol (Table 3). Among them, both 1 and (R)-1 were found to be potent inhibitors with IC50 values (mol ratio/32 pmol TPA) of 33.2 and 38.9, respectively. In particular, compound 1 showed 10-fold higher inhibition than the positive control substance curcumin. Although (R)-2, which was unambiguously more potent than 2, exhibited modest inhibitory effect that was comparable with that of β-lapachone, it was much less potent than 1. These results strongly suggest that the hydroxy group at C-5 plays an important role in increasing the inhibitory effect as well as the antiproliferative effect against the tumor cell lines described above. Moreover, the values of cell viability at 1000 mol ratio in Table 3 indicate that the naphtho[2,3–b] furan–4,9–dione analogues are less cytotoxic than β-lapachone and lapachol.

On the other hand, the antiproliferative activity of its enantiomer (R)-1 was discernibly reduced toward the tumor cell lines, in particular against MCF-7 cells, compared with that of 1. These results suggested that the orientation of the hydroxy group at C-1’ is crucial. Interestingly, ketone 10a was between 1 and (R)-1 in antiproliferative activity. This finding suggests the superiority of the α-oriented hydroxy group of 1. Regarding the four human normal cell lines tested (Table 2), the antiproliferative activity of (R)-1 was somewhat higher than 1 only against IE cells.

The comparison between 1 and 2 showed that the position of a hydroxy group attached to the aromatic ring also affects their ability to suppress the growth of the tumor cell lines. As shown in Table 1, the antiproliferative effect of 2 that possesses a hydroxy function at C-8 was considerably lower than that of 1 (1.57-4.31 μM) although it was comparable with that of β-lapachone. Thus, the presence of a phenolic hydroxy group at C-5 seems to play an important role in increasing antiproliferative effect. A similar effect was also observed between 10a and 10b, supporting its role described above.

Table 1. Antiproliferative effects of 10a, 10b, 1, (R)-1, racemate 1, 2, β-lapachone, lapachol and mitomycin against human tumor cell linesa

a human tumor cell lines: PC-3 = prostate, A549 = lung, MCF-7 = breast.

Table 2. Antiproliferative effects of 10a, 10b, 1, (R)-1, racemate 1, 2 and mitomycin against human normal cell linesa

a human normal cell lines: Fb = skin, Hc = liver, MPC-5 = lung, IE = colon, Hs888Lu = lung, SVCT-M12 = breast. b NT, not tested.

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In addition to in vitro cancer chemopreventive activity mentioned above, it has already been known that compound 1 exhibits in vivo cancer chemopreventive activity.8a These findings inevitably led to the idea that naphtho[2,3–b]furan–4,9–dione analogues are likely to prove extremely useful for the development of cancer chemopreventive agents. Therefore, we reexamined inhibitory effect of 1 that was the most active in vitro and compared with that of (R)-1 on mouse skin tumor promotion in two-stage carcinogenesis experiments in which DMBA is used as an initiator and TPA as a promoter. Figure 2 a shows the incidence (%) of papilloma bearing mice during 20 weeks of promotion. In group 1, in which only TPA was applied to mouse skin initiated by DMBA (positive control), the first tumors were observed at 6 weeks with about 7% incidence. The rate of papilloma-bearing mice reached 100% in additional 4 weeks. On the other hand, those in groups II-IV which were treated with 1, (R)-1, racemate 1 before application of TPA was approximately 7-20% at 10 weeks, 47-53% at 15 weeks and 67-80% even at 20 weeks, respectively. Twenty weeks of promotion allowed group I to form 8.6 papillomas per mouse, while groups II-IV possessed only 3.9-4.7 papillomas per mouse. The inhibitory activities of 1, (R)-1 and racemate 1 were also observed as the average numbers of papillomas per mouse during 20 weeks of promotion (Fig. 2b). These results clearly demonstrate the potential of naphtho[2,3–b]furan–4,9–dione analogues as cancer chemopreventive agents. It is noteworthy that (R)-1 was somewhat more potent than 1 in this in vivo experiment model.

Antimicrobial activity of naphtho[2,3–b]furan–4,9– dione analogues synthesized was also examined because some constituents isolated from the Tabebuia plants including 3 and 4 are known to be active toward bacteria and fungi (Table 4). All compounds including a synthetic precursor 10a, which is also one of constituents of T. avellanedae, were found to have modest or potent antibacterial activity against several Gram-positive bacteria (0.78-6.25 μg/mL) and antifungal activity (1.56-100 μg/mL) in comparison with penicillin G and amphotericin B. On the other hand, all compounds were not active against common Gram-negative bacteria at concentrations up to 100μg/mL. From these results, it is likely that the presence of the hydroxy group at C-5 makes a slight contribution toward increasing antimicrobial activity, while the other hydroxy group is not so important although detailed structural requirements cannot be discussed.

Figure 2. Inhibitory effect of 1 on mouse skin carcinogenesis induced by DMBA and TPA: X: control DMBA (390 nmol) and TPA (1.7 nmol)(group I); ○: DMBA (390 nmol), TPA (1.7 nmol) and 1 (85 nmol)(group II), ●: DMBA (390 nmol), TPA (1.7 nmol) and (R)-1 (85 nmol)(group III) , □: DMBA (390 nmol), TPA (1.7 nmol) and racemate 1 (85 nmol)(group IV). (a) Percentage of mice with papillomas. (b) Average number of papillomas per mouse. At 20 weeks of promotion, group II-IV were significantly different from group I (p<0.05) on papillomas per mouse.

Table 3. Inhibitory effect of 1, (R)-1, racemate 1, 2, (R)-2, racemate 2, β-lapachone and lapachol on TPA-induced EBV-EA activation

a Values represent relative percentage to the positive control value (100%). b Values in parentheses represent viability percentages of Raji cells measured through trypan blue staining; unless otherwise stated, the viability percentage of Raji cells were more than 80%. c Positive control substance.

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Ⅲ. Conclusions In conclusion, the concise stereoselective synthesis of 1

and 2 starting from 5 was accomplished by using Noyori reduction as a key step, and the total yields of products 1 and 2 were 8.5% and 2.9%, respectively. Naphtho[2,3–b] furan–4,9–dione 1 synthesized in this study showed potent antiproliferative effect and in vitro and in vivo cancer chemopreventive activity. In addition, it displayed relatively strong antimicrobial activity against several Gram-positive bacteria and fungi. On the other hand, its positional isomer 2 was less active in antiproliferative effect and in vitro cancer chemopreventive activity, but exhibited similar antimicrobial activity to that of 1. Furthermore, significant differences between 2 and (R)-2 were recognized at least in the in vitro cancer chemopreventive activity. Interestingly, (R)-1 showed somewhat higher cancer chemopreventive activity in vivo. Further modification toward structure-activity relationship studies is now in progress to better understand structural requirements for increasing their biological activities.

Ⅴ. Chemistry 5.1. 5-Hydroxy-1,4-naphthalenedione (6)11

This compound was prepared according to the reported procedure in 47% yield as yellow needles of mp 163-165 °C. 1H-NMR: 6.96 (1H, s), 7.29 (1H, dd, J = 2.0, 8.0 Hz), 7.62-7.67 (2H, m), 11.9 (1H, s). 13C NMR: 114.9, 119.2, 124.5, 131.7, 136.6, 138.6, 139.6, 161.4, 184.3, 190.3.

5.2. 2-(Dimethylamino)-5-hydroxy-1,4-naphthalenedione (7a) and 2-(dimethylamino)-8-hydroxy-1,4-naphthalenedione (7b)12a

These compounds were prepared according to the reported procedure12 with some modifications. To the solution of 6 (4.28 g, 24.5 mmol) in toluene (125 mL) was added dimethyl amine (18.4 mL, 2.0M solution in THF, 36.8 mmol) at –40 °C. After the mixture was stirred for 12 h, the solvent was removed under reduced pressure. The residue was purified by column chromatography (hexane/EtOAc = 4/1) gave 7a (2.23 g, 42% yield) as yellow needles of mp 145-146 °C and 7b (0.58 g, 11% yield) as yellow needles of mp 155-157 °C.

5.3. 2,5-Dihydroxy-1,4-naphthalenedione (8a)12b

This compound was prepared from 7a according to the reported procedure in 97% yield as yellow needles of mp >210 °C (dec.). 1H NMR: 6.31 (1H, s),7.35 (1H, dd, J = 1.2, 8.5 Hz), 7.44 (1H, s), 7.59 (1H, t, J = 8.5 Hz), 7.69 (1H, dd, J = 1.2, 8.5 Hz), 12.33 (1H, s). 13C-NMR: 110.3, 114.5, 119.8, 126.7, 129.3, 135.2, 156.9, 161.4, 181.2, 191.2.

5.4. 2,8-Dihydroxy-1,4-naphthalenedione (8b)12b

Compound 8b was prepared from 7b according to the reported procedure in 97% yield as yellow needles of mp >215 °C (dec.). 1H NMR: 6.31 (1H, s), 7.35 (1H, dd, J = 1.2, 8.5 Hz), 7.44 (1H, s), 7.59 (1H, t, J = 8.5 Hz), 7.69 (1H, dd, J = 1.2, 8.5 Hz), 12.33 (1H, s). 13C NMR: 111.6, 113.1, 119.6, 123.4, 132.7, 138.2, 156.1, 161.6, 184.1, 185.1.

Ⅳ. Experimental

1H and 13C NMR spectra were taken in CDC13 unless otherwise noted. Chemical shift values are expressed in ppm relative to internal tetramethylsilane. Abbreviations are as follows: s, singlet; d, doublet; t, triplet; m, multiplet. IR was expressed in cm－1. The extract was dried over Na2SO4 unless otherwise noted. Purification was carried out using silica gel column chromatography. All reactions were carried out under an argon atmosphere unless otherwise stated.

Table 4. Antimicrobial activitya of 1, (R)-1, 2, (R)-2 and 10a

a Values represent minimum growth inhibitory concentration, MIC (μg/mL). b NT, not tested. c Positive control substance.

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5.9. 5-Hydroxy-2-[(1S )-1-hydroxyethyl]-naphtho [2,3-b]furan-4,9-dione (1)5c

To a flask was added ketone 10a (128 mg, 0.5 mmol), Noyori asymmetric transfer hydrogenation catalyst Ru [(S,S)-Tsdpen](p-cymene) (15 mg, 0.025 mmol, 5 mol %), CH2Cl2 (5.0 mL), formic acid/Et3N (5:2, 1.3 ml). The resulting solution was stirred at room temperature for 24 h. The reaction mixture was diluted by addition of H2O and 10% HCl aq, and extracted with CHCl3. The organic extracts were washed with brine, and then dried over Na2SO4. Concentration and column chromatography (hexane/EtOAc = 2/1) gave 1 (115 mg, 89% yield, 96% ee) as yellow needles of mp 171-172 °C. [α]24

D –22.7 (c 0.58, CH3OH). 96% ee (HPLC, Daicel Chiralpak AD-H; hexane/i-PrOH = 9/1, 1.0 mL/min; 254 nm, minor 37.9 min and major 40.9 min). 1H NMR: 1.66 (3H, d, J = 6.8 Hz), 2.31 (1H, br s), 5.05 (1H, m), 6.84 (1H, s), 7.27 (1H, dd, J = 1.0, 8.3 Hz), 7.62 (1H, dd, J = 8.0, 8.3 Hz), 7.75 (1H, dd, J = 0.9, 8.0 Hz), 12.18 (1H, s). 13C NMR: 21.5, 63.8, 103.4, 115.2, 120.0, 125.3, 131.0, 132.6, 136.3, 152.0, 162.3, 165.4, 172.7, 186.5.

5.10. 5-Hydroxy-2-[(1R )-1-hydroxyethyl]-naphtho [2,3-

b]furan-4,9-dione ((R )-1) [α]24

D ＋22.5 (c 0.42, CH3OH). 5.11. 8-Hydroxy-2-[(1S )-1-hydroxyethyl]-naphtho [2,3-

b]furan-4,9-dione (2)5c Compound 2 was prepared from 10b in a manner similar

to that described for compound 10a. Concentration and column chromatography (hexane/EtOAc = 2/1) gave 2 (97% yield, 97% ee) as yellow needles of mp 181-182 °C. [α]24

D –22.3 (c 0.16, CH3OH). 97% ee (HPLC, Daicel Chiralpak AD-H; hexane/i-PrOH = 9/1, 1.0 mL/min; 254 nm, minor 21.1 min and major 24.1 min).1H NMR: 1.66 (3H, d, J = 6.5 Hz), 2.61 (1H, br s), 5.05 (1H, q, J = 6.5 Hz), 6.85 (1H, s), 7.26 (1H, dd, J = 1.5, 8.0 Hz), 7.59 (1H, dd, J = 7.5, 8.0 Hz), 7.70 (1H, dd, J = 1.5, 7.5 Hz), 12.00 (1H, s). 13C-NMR: 21.5, 63.8, 104.7, 114.7, 120.1, 125.3, 131.9, 133.2, 136.3, 151.1, 162.6, 165.8, 178.5, 179.7.

5.12. 8-Hydroxy-2-[(1R )-1-hydroxyethyl]-naphtho [2,3-

b]furan-4,9-dione ((R )-2) [α]24

D ＋21.7 (c 0.16, CH3OH). 5.13. Antiproliferative effect assay

The antiproliferative effects of naphthoquinones were examined in cancer cell lines and normal cell lines derived from human origin. These cells were maintained in usual 10% fetal serum Dulbecco’s minimum essential medium (DMEM) through experiments and exposed to four dose concentrations of naphthoquinones in a humidified atmosphere (37 °C, 5% CO2) for 72 h. After reaction, cells were further incubated with 0.25% trypan blue dye for 20 min and counted for viable cells under light microscopic apparatus. IC50 values were calculated from separate experiments performed in triplicate.

5.5. 2-Acetyl-2,3-dihydro-5-hydroxy-naphtho[2,3-b]furan-4,9-dione (9a)

This compound was prepared by the method of Hagiwara. To a solution of methyl vinyl ketone (215 g, 3.07 mol) in pentane (700 mL) was slowly added a solution of bromine (500g, 3.13 mol) in pentane (600 mL) at –15 °C. After stirred for 10 min, concentration under reduced pressure gave 3,4-dibromobutan-2-one, which was immediately added to the solution of 8a (97.3 g, 512 mmol) in THF (2.5 mL). To the solution was slowly added DBU (551 mL, 3.69 mol) at 0 °C, and the mixture was stirred for overnight at room temperature. The mixture was quenched with 10% HCl at 0 °C. The mixture was extracted with CHCl3. The organic extracts were washed with brine, and then dried. Concentration and crystallization (CHCl3/benzene = 2/1), then washing with MeOH gave 9a (92 g, 70% yield) as yellow needles of mp 186-188 °C. 1H NMR: 2.67 (3H, s), 7.32 (1H, dd, J = 1.0, 8.5 Hz), 7.59 (1H, s), 7.67 (1H, dd, J = 7.0, 8.5 Hz), 7.77 (1H, dd, J = 1.0, 7.0 Hz), 7.77 (1H, dd, J = 1.0, 7.0 Hz), 11.90 (1H, s). 13C NMR: 26.6, 29.5, 87.4, 114.6, 119.7, 123.5, 126.0, 131.6, 135.4, 159.8, 161.3, 176.4, 187.5, 204.3. IR (KBr): 3400, 1717, 1678, 1647, 1616, 1450, 1234, 760. HRMS (ESI) m/z: [M–H]－ calcd for [C14H9O5]－, 257.0450; found, 257.0452.

5.6. 2-Acetyl-2,3-dihydro-8-hydroxy-naphtho[2,3-

b]furan-4,9-dione (9b) Compound 9b was prepared from 8b using the same

procedure as described for compound 9a. Concentration and column chromatography (chloroform only) gave 9b (72% yield) as yellow needles of mp 131-133 °C. 1H-NMR: 2.40 (3H, s), 3.40 (1H, d, J = 9.0 Hz), 3.40 (1H, d, J = 11.0 Hz), 5.29 (1H, dd, J = 9.0, 11.0 Hz), 7.23 (1H, dd, J = 3.5, 6.0 Hz), 7.63-7.61 (2H, m), 11.60 (1H, s). 13C NMR: 26.4, 30.0, 87.2, 114.5, 119.4, 124.4, 124.6, 132.9, 137.1, 158.9, 162.1, 180.7, 181.8, 204.2. IR (KBr): 1736, 1650, 1620, 1439, 1246, 1177, 957, 756, 732. HRMS (ESI) m/z: [M–H]－ calcd for [C14H9O5]－, 257.0450; found, 257.0465.

5.7. 2-Acetyl-5-hydroxy-naphtho[2,3-b]furan-4,9-dione (10a)5c

Under Ar atmosphere, a solution of compound 64 (20 g, 78 mmol) in chloroform (1.0 L) was heated with MnO2 (90 g, <5μm, activated, 1.04 mol) to reflux for 1 d. The mixture was passed through a silica gel column chromatography. Concentration gave 10a (13.6 g, 71% yield) as yellow needles of mp >247 °C (dec.). 1H NMR: 2.67 (3H, s), 7.32 (1H, dd, J = 1.0, 8.5 Hz), 7.60 (1H, s), 7.67 (1H, dd, J = 7.0, 8.5 Hz), 7.82 (1H, dd, J = 1.0, 7.0 Hz), 12.13 (1H, s). 13C NMR: 26.8, 111.9, 115.3, 120.5, 125.9, 130.5, 132.7, 136.7, 152.9, 155.7, 162.7, 173.2, 185.6, 187.5.

5.8. 2-Acetyl-8-hydroxy-naphtho[2,3-b]furan-4,9-dione (10b)5c

Compound 10b was prepared from 9b using the same procedure as described for compound 10a. Concentration gave 10b (84% yield) as yellow needles of mp 220-221 °C. 1H NMR: 2.67 (3H, s), 7.32 (1H, dd, J = 1.0, 8.5 Hz), 7.59 (1H, s), 7.67 (1H, dd, J = 7.0, 8.5 Hz), 7.77 (1H, dd, J = 1.0, 7.0 Hz), 11.90 (1H, s). 13C NMR: 26.7, 112.4, 115.3, 120.5, 125.5, 131.3, 133.2, 137.0, 152.3, 155.8, 163.0, 178.8, 178.8, 187.2.

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5.14. In vitro EBV-EA activation assay The inhibition of EBV-EA activation was assayed

according to the reported method.17 The cells were incubated for 48 h at 37 °C in a medium containing n-butyric acid (4 mM), TPA (32 pmol) and various amounts of the test compounds. Smears were made from the cell suspension and the EBV-EA inducing cells were stained by means of an indirect immunofluorescence technique.18 In each assay, at least 500 cells were counted and the number of stained cells (positive cells) among them was recorded. Triplicate assays were performed for each data point. The EBV-EA inhibitory activity of the test compound was compared with that of the control experiment (100%) with n-butyric acid plus TPA. In the experiments, the EBV-EA activities were ordinarily abound 40% and these values were taken as the positive control (100%). The viability of cells was assayed against treated cells by the trypan-blue staining method. For the determination of cytotoxicity, the cell viability was required to be more than 60 %.19 Student’s t-test was used for all statistical analyses.

5.15. In vitro antibacterial activity

MICs of (1, (R)-1, 2, (R)-2 and 10a) were determined by slightly modified NCCLS M27-P broth dilution method.20 A 50 μL aliquot of 0.85% sodium chloride solution containing 106/mL bacterial cells was added to 1 mL of Muller-Hinton broth (OXOID) containing (1, (R)-1, 2, (R)-2 and 10a) at a concentration range of 0.1-100 μg/mL, followed by incubation for 24 h at 37 °C. Similarly, fungal conidia suspended in 50 μL of 0.05% Tween 80 solution (105/mL conidia concentration) were added to 1mL of potato dextrose broth (DIFCO) containing (1, (R)-1, 2, (R)-2 and 10a) at a concentration range of 0.1-100 μg/mL and incubated for 3 to 5 days at 28 °C.

5.16. In vivo two-stage carcinogenesis test on mouse

skin papillomas promoted by TPA Mice were divided into groups, with 15 females in each

group and housed in a controlled environment. All mice were shaved with electric clippers 1 day before application of the agents to backs of the mice. 7,12-Dimethylbenz[a]anthracene (DMBA, 390 nmol) in 0.1 mL acetone were applied topically with pipet. After one week, the same skin portion was painted with 1, (R)-1, racemate 1 (85 nmol) in acetone or acetone alone, 60 min before TPA (1.7 nmol). Results of tumor response are present as the average number of mice with papillomas and the percentage of mice with papillomas.

Acknowledgments

The authors are grateful to Taheebo Japan Co., Ltd. for generously providing the powdered inner bark of Tabebuia avellanedae and SANSHIN METAL WORKING CO., LTD for generous financial support of this project.

References and notes 1 ) Gentry, A. H. Ann. Missouri Bot. Garden. 1973, 60,

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4 ) (a) Ueda, S.; Tokuda H. Planta Med. 1990, 56, 669; (b) Zani, C L.; Oliveira, A. B.; O1iveira, G. G. Phytochemistry 1991, 30, 2379.

5 ) For quinones and benzene derivatives: (a) Burnett, A. R.; Thomson, R. H. J. Chem. Soc. C 1967, 1261; (b) Burnett, A. R.; Thomson, R. H. J. Chem. Soc. C 1967, 2100; (c) Wagner, H.; Kreher, B.; Lotter H.; Hamburger, M. O.; Cordell, G. A. Helv. Chim. Acta 1989, 72, 659; For iridoids: (d) Nakano, K.; Maruyama, K.; Murakami, K.; Takaishi, Y.; Tomimatsu, T. Phytochemistry 1993, 32, 37l; (e) Warasina, T.;Nagatani, Y.; Noro, T. Phytochemistry, 2004, 65, 2003. (f) Warashina, T.;Nagatani, Y.; Noro, T. Phytochemistry 2005, 66, 589.

6 ) (a) Hussain, H.; Krohn, K.; Ahmad, V. U.; Miana, G. A.; Green, I. R. Arkivoc 2007, 145; (b) Pardee, A. B.; Li, Y. Z.; Li, C. J. Curr. Cancer Drug Targets 2002, 2, 227; (c) Fujimoto, Y.; Eguchi, T.; Murasaki, C.; Ohashi, Y.; Kakinuma, K.; Takagi, H.; Abe, M.; Inazawa, K.; Yamazaki, K.; Ikekawa, N.; Yoshikawa, O.; Ikekawa, T. J. Chem. Soc., Perkin Trans. 1 1991, 2323; (d) Cragg, G. M.; Grothaus, P. G.; Newman, D. J. Chem . Rev. 2009 109, 3012.

7 ) Ueda, S.; Umemura, T.; Dohguchi, K.; Matsuzaki, T.; Tokuda, H.; Nishino, H.; Iwashima, A. Phytochemistry 1994, 36, 323.

8 ) Ueda, S. U.S. Patent 5663197, 2005; Chem. Abstr. 1994, 121, 50088.

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10) (a) Guiraud, P.; Steiman, R.; Campos-Takaki, G. M.; Seigle-Murandi, F.; Simeon de Buochberg, M. Planta Medica 1994, 60, 373; (b) Nagata, K.; Hirai, K.; Koyama, J.; Wada, Y.; Tamura, T. Ahtmicrob. Agents Chemother. 1998, 42, 700-702; (c) Eyong, K. O.; Folefoc, G. N.; Kuete, V.; Beng, V. P.; Krohn, K.; Hussain, H.; Nkengfack, A. E.; Saeftel, M.; Salem Ramadan Sarite, S. R.; Hoerauf, A. Phytochemistry 2006, 67, 605.

1-114

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13) Hagiwara, H.; Sato, K.; Nishino, D.; Hoshi, T.; Suzuki, T.; Ando, M. J. Chem. Soc, Perkin Trans. I 2001, 2946.

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15) Tsdpen = N-(p-toluenesulfonyl)-1,2-diphenylethanediamine. 16) The purity of synthesized 1 and (R)-1, 2, (R)-2 used for

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M.; Imanaka, H. Cancer Lett. 1981, 13, 29. 18) Henle, G.; Henle, W. J. Bacteriol. 1966, 91, 1248. 19) Ohigashi, H.; Takamura, H.; Koshimizu, K.; Tokuda,

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ブラジル産薬用植物Tabebuia avellanedaeから得られた 生物活性ナフトキノン類の合成と評価

バイオオーガニック アンド メディシナル ケミストリー 第17巻 No.17 (2009)

近畿大学 農学部 飯田　彰

飯田　彰（近畿大学農学部）、山下光明・金子雅文（高崎健康福祉大学薬学部）、 徳田春邦（京都府立医科大学分子医科学教室）、西村克己（神戸薬科大学）、他

（要旨） (–)–5–hydroxy–2–(1'–hydoxyethyl)naphtho[2,3–b]furan–4,9–dione (1) やその位置異性体(–)–8–hydroxy–2–(1'–hydoxyethyl)

naphtho[2,3–b]furan–4,9–dione (2)、それらはいずれもTabebuia avellanedaeの内皮から見つかった二次代謝産物であるが、そのようなnaphtho[2,3–b]furan–4,9–dione骨格を基盤とする一連のナフトキノン類が、それらの鏡像体とともに立体選択的に合成されるとともに、それらの生物活性が評価された。化合物１は数種のヒト由来腫瘍細胞に対して強力な細胞増殖抑制活性を示したが、そのヒト由来正常細胞に対する効果はマイトマイシンのそれよりずっと低かった。一方、その鏡像体である(R)-1は、上記腫瘍細胞株に対して1より活性は低かった。2の腫瘍細胞に対する増殖抑制効果は著しく減少した。これらの結果は、５位の炭素のフェノール性水酸基の存在が細胞増殖抑制活性の増強に非常に重要であることを示している。さらに1は2より高い発がん予防効果も示したが、抗菌活性に関しては1と2の間で顕著な差は認められなかった。両化合物とも、真菌やグラム陽性菌に対しては適度な抗菌活性を示したが、グラム陰性菌に対しては活性を示さなかった。

われわれは直前の論文で、1の簡便で立体選択的

合成ならびに数種の腫瘍細胞株に対する増殖抑制

活性とin vitroでのがん予防活性を報告した。その予備

的結果によって、われわれは1と(R)-1の間においては、

先に述べた生物活性において顕著な差があることを

明瞭に確認した。この知見により、われわれは2とその

鏡像体(R)-2を合成し、それらの生物活性を評価する

こととなった。なぜならば、化合物2はラセミ混合物の状態

抗原の活性化、ならびに二段階発がん試験において

DMBAを用いて初期化されたマウス皮膚上で TPAに

よって誘導される発がん促進効果を強力に阻害する。

それゆえ、化合物1は潜在的ながん化学予防剤である

ことが期待される。

1. 緒　　　　　言

Tabebuia avellanedae LORENTZ ex GRISEB

(ノウゼンカズラ科) (異名 T. impetiginosa)、それらは

ブラジルから北アルゼンチンに至る南米に広く分布して

いるが、インカの時代から伝統薬物としてよく知られて

いる。その樹皮は利尿薬や収斂剤として利用されてきた。

さらに、この樹皮は、抗腫瘍、抗菌、抗真菌、抗炎症など

幅広い生物活性を示す。特に、その抗腫瘍活性の

発見は、本植物を重要な薬用資源に位置づけた。また、

一連のナフトキノン類やアントラキノン類、多くの安息

香酸類、イリドイド配糖体が本植物の二次代謝産物と

して単離されている。

これら成分の中で、(–)–5–hydroxy–2–(1'–hydoxyethyl)

naphtho[2,3–b]furan–4,9–dione (1)やその位置異性体

(–)–8–hydroxy–2–(1'–hydoxyethyl)naphtho[2,3–b]

furan–4,9–dione (2)のようなnaphtho[2,3–b]furan–4,

9–dione 類緑体が、1とともに本植物に含まれるβ-ラパコン

(3)やラパコール(4)と同様、さまざまな腫瘍細胞に対して

強力な増殖抑制活性を示すことは特筆に値する(図1)。

さらに、化合物1はRaji細胞において、発がんプロモーター

TPAによって誘導されるEpstein-Barrウイルス初期

図1.

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で単離されることがWagnerによって報告されていたか

らである。事実、われわれもまた、T. avellanedaeのクロ

ロフォルムエキスをHPLCで繰り返し精製してもラセミ体

の2しか得られなかった。一方、藤本のグループ、彼ら

は合成したラセミ体1と2を光学分割し、最初に四つの

鏡像体を得たのだが、彼らは同じ植物から2を(R)と(S)

の比率が 3 : 1の混合物として単離した。さらに彼らは、

両鏡像体間でL1210細胞に対する増殖抑制活性に

差がないことを報告した。これらの結果もまた、われわれ

の研究プログラムを推し進めるきっかけとなった。ここに、

われわれは先に述べた鏡像体2組の野依還元を重要

段階とする立体選択的合成とそれらのヒト由来の腫瘍

細胞と正常細胞に対する増殖抑制活性、in vitroでの

がん化学予防活性ならびに抗菌活性を報告する。さらに、

1 と (R)-1のin vivoでのがん化学予防効果についても

述べる。

2. 結果と考察

2.1. 化学

1と2のグラムスケールでの合成は、われわれの1の

予備合成に従って同様に行なわれた。1と2の合成経

路をスキーム1に示す。ユグロン(6)の合成は、購入可能

な1,5-hydroxynaphthaleneから開始して、それを

塩化銅の存在下、空気酸化を遮光下で行い、47%収率

で6を得た。6 から 8への化学変換は、少し改良を加え、

報告の方法に従って行われた。–40°Cでトルエン中、

ジメチルアミンを用いた6の酸化的アミノ化により、7aと

7bをそれぞれ42と11%の収率で得た。前報で述べたと

おり、ジメチルアミンのTHF溶液は実用上、ふさわしい

試薬であり、適度な化学収率と位置選択性を示した。

7aを10%塩酸で脱アミノ化すると8aを定量的に与えた。

naphtho[2,3–b]furan–4,9–dione骨格9aの構築は、

Hagiwaraらの方法に従い、8aを購入可能なbut-3-en-2-

oneとブロミンから生成される3,4-dibromobutan-2-oneと

DBU存在下、THF中で反応させることによって行った。

ミリグラムスケールでの合成とは異なり、10aの生成は

伴わず9aのみを与えたことは注意すべき点である。

9aをMeOHで洗浄後、クロロホルム中、二酸化マン

ガンで処理し、望ましい10aを2工程、50%で得た。続く

野依還元により、1と2の立体選択的合成が完成した

(スキーム2)。10aと10bの不斉水素化移動は、蟻酸―

トリエチルアミン混合物とジクロロメタン中、購入可能な

キラルなRu(II)錯体、Ru [(S,S)-Tsdpen] (p-cymene)

により触媒されるが、高い化学収率と鏡像体過剰率で

対応する2級アルコール1と2を与えることに成功した。

この研究で合成されたすべての化合物の物理化学

的特性は以前報告されたそれらと完全に一致した。

対応する鏡像体(R)-1と(R)-2は、キラル触媒としてRu

[(R,R)-Tsdpen] (p-cymene)を用いることにより、

同様に合成された。

2.2. 生物学

既に報告されているとおり、タベブイア植物から単離

されたナフトキノン類は、細胞増殖抑制化合物として

認識されている。そこで、合成された2組の鏡像体も、

ヒト由来腫瘍細胞PC-3 (前立腺), A549 (肺) and

MCF-7 (胸)の成長を抑制する能力で評価された(表1)。

化合物1は、3つの細胞株に対してマイトマイシンに匹敵

するほどの、またβ-ラパコンのそれよりはずっと強力な

増殖抑制活性を示した。しかし、ヒト由来正常細胞

Hs888Lu (肺)やSVCT-M12 (胸)に対する増殖抑制

活性は、マイトマイシンのそれよりもかなり低かった(表2)。

スキーム 1. Synthesis of 10. Reagents and conditions: (a) CuCl, CH3CN, air, rt, 47%; (b) Me2NH, toluene, THF, –40°C, 42% and 11% for 7a and 7b, respectively; (c) 10% HCl, dioxane, reflux, 97%; (d) 3,4-dibromobutan-2-one, DBU, THF, room temperature; (e) MnO2, CHCl3, reflux, 50-60% in two steps.

スキーム 2. Chiral Ru catalyst mediated asymmetric hydrogenation of naphthofuran 10

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予想以上に、ケトン10aは腫瘍細胞に対して強力な

増殖抑制活性を示した(表1)。特に、それはMCF-7

細胞の成長を強力に抑制し、その活性は1のそれと

同等であった。一方、そのHs888LuやSVCT-M12に

対する増殖抑制活性は1のそれほど高くなかった(表2)。

それゆえ、1同様10aもまた、抗がん剤開発に向けて

非常に有望である。

1の増殖抑制活性の作用機構はまだ解明されて

おらず、現在、研究途上である。しかし、われわれの

予備的結果は、化合物1は多機能転写因子E2F1に

影響していることを示した。一方、1の類縁体であるβ-

ラパコンはさまざまながん細胞株においてアポトーシスを

誘導し、転写プロセスに関与していることが示されている。

したがって、われわれは現在、1もまた、β-ラパコンのそれ

と同様の機構を持っているものと推定している。

化合物1はすでにがんの化学予防剤として作用する

ことが知られており、用量依存的にRajiに対して毒性を

示すことなく、EBウイルス初期抗原の活性化を強力に

阻害した。in vitroでの(R)-1, 2 および (R)-2のがん

化学予防活性を1のそれと比較するために、抗発がん

プロモーター探索の一次スクリーニングとして、β-ラパコン

やラパコールとともに、それらがRaji細胞中でTPAに

よって誘導されるEBウイルス初期抗原の活性化を

阻害する能力を評価した(表3)。

それらの中で、1と(R)-1の両方はともにIC50 値33.2

and 38.9を示し、強力な阻害剤であることが分かった。

特に化合物1は、陽性対照であるクルクミンより10倍

高い活性を示した。2より明らかにより強力ではある

(R)-2はβ-ラパコンのそれと同程度の適度な阻害効果

を示したが、1よりはずっと活性は低かった。これらの

結果は、5位炭素上の水酸基が上述した腫瘍細胞株

に対する増殖抑制活性と同様、阻害効果増強におい

て重要な役割を担っていることを強く示唆している。

さらに、表3で示された1000モル比における細胞生存率

の値は、naphtho[2,3–b]furan–4,9–dione類縁体の

細胞毒性が、β-ラパコンやラパコールのそれよりも

ずっと低いことを示している。

一方、その鏡像体(R)-1の腫瘍細胞株、特にMCF-7

細胞対する増殖抑制活性は、1のそれと比較してかな

り低下した。これらの結果は、1’位炭素上の水酸基の

配向が重要であることを示唆している。興味深いことに、

ケトン10aの増殖抑制活性は、1と(R)-1の中間であった。

この知見は、1の水酸基のα-配向性の優位を示唆して

いる。試験されたヒト由来の4種の正常細胞に関して、

(R)-1の増殖抑制活性はIE細胞に対してのみ1より

やや高かった(表2)。

1と2の比較から、芳香環に結合した水酸基の位置も

また、腫瘍細胞株の成長抑制に影響を与えることが

示された。表2に示されるように、8位炭素に水酸基を有

する2の増殖抑制活性はβ-ラパコンとほぼ同じであるが、

1のそれよりかなり低い。それゆえ、増殖抑制活性増強

には、5位炭素上のフェノール性水酸基の存在が重要

な役割を担っているように思える。同様の効果は10aと

10bの間でも観察され、上述を支持した。

表 1. Antiproliferative effects of 10a, 10b, 1, (R)-1, racemate 1, 2, β-lapachone, lapachol and mitomycin against human tumor cell linesa

a human tumor cell lines: PC-3 = prostate, A549 = lung, MCF-7 = breast.

表 2. Antiproliferative effects of 10a, 10b, 1, (R)-1, racemate 1, 2 and mitomycin against human normal cell linesa

a human normal cell lines: Fb = skin, Hc = liver, MPC-5 = lung, IE = colon, Hs888Lu = lung, SVCT-M12 = breast. b NT, not tested.

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上で述べたin vitroでのがん化学予防活性に加えて、

化合物1がin vivoでのがん化学予防活性を示すことは

既に知られている。これらの知見により、naphtho

[2,3–b]furan–4,9–dione類縁体ががんの化学予防剤

開発にきわめて有用であるということを証明してい

そうだという発想に必然的にいたった。それゆえ、われ

われはin vitroで最も活性の高い1のDMBAをイニ

シエーター、TPAをプロモーターとする二段階発がん

実験におけるマウス皮膚発がん促進に関する阻害

効果を再度試験し、(R)-1のそれと比較した。

図2 (a)は、20週のプロモーション期間にパピローマを

もったマウスの発生率を示している。グループI、DMBA

によってイニシエーションを受けたマウス皮膚にTPA

のみを塗布している(陽性対照)グループでは、最初の

がんは6週間、約7％の発生率で観察された。パピ

ローマをもったマウスの比率は、さらに10週で100％に

達した。一方、TPA塗布まえに1, (R)-1, ラセミ体1を

処理したグループII-IVにおいては、それぞれ、10週で

7-10％、15週で47-53％、20週でも67-80%であった。

20週のプロモーションで、グループIではマウス当たり

8.6個のパピローマが形成された。一方、グループII-IV

ではマウス当たり3.9-4.7個のパピローマが形成された

だけであった。1, (R)-1, ラセミ体 1の阻害活性は、20週

のプロモーション期間にマウス当たりのパピローマの

平均個数としても観察された（図2(b)）。これらの結果は、

naphtho[2,3–b]furan–4,9–dione類縁体のがん化学

予防剤としての潜在力を明確に示している。このin vivo

実験系では、(R)-1が1よりやや強力であったことは注目

に値する。

3や4を含むTabebuia属植物から単離された成分の

いくつかはバクテリアやカビに対して活性であることが

知られているので、合成したnaphtho[2,3–b]furan–4,

9–dione類縁体の抗微生物活性についても調べた。

合成前駆体10a（これもまたT. avellanedaeの成分の

一つである）を含むすべての化合物が、ペニシリンGや

アンフォテリシンBと比較しても、グラム陽性菌（0.78-

6.25μg/mL）やカビに対して適度なあるいは強力な

抗菌活性をもつことが分かった。一方、すべての化合物

は、100μg/mLの濃度で、グラム陰性菌に対しては抗菌

活性を示さなかった。これらの結果から、5位炭素上の

水酸基の存在することが、抗微生物活性増強にわずか

ではあるが重要であるが、もう一方の水酸基はそれほど

重要ではないようである。ただし、詳細な構造要因は

議論されてはいない。

表 3. Inhibitory effect of 1, (R)-1, racemate 1, 2, (R)-2, racemate 2, β-lapachone and lapachol on TPA-induced EBV-EA activation

a Values represent relative percentage to the positive control value (100%). b Values in parentheses represent viability percentages of Raji cells measured through trypan blue staining; unless otherwise stated, the viability percentage of Raji cells were more than 80%. c Positive control substance.

図 2. Inhibitory effect of 1 on mouse skin carcinogenesis induced by DMBA and TPA: X: control DMBA (390 nmol) and TPA (1.7 nmol)(group I); ○: DMBA (390 nmol), TPA (1.7 nmol) and 1 (85 nmol)(group II), ●: DMBA (390 nmol), TPA (1.7 nmol) and (R)-1 (85 nmol)(group III) , □: DMBA (390 nmol), TPA (1.7 nmol) and racemate 1 (85 nmol)(group IV). (a) Percentage of mice with papillomas. (b) Average number of papillomas per mouse. At 20 weeks of promotion, group II-IV were significantly different from group I (p<0.05) on papillomas per mouse.

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3. 結論

結論として、5から出発して1と2の簡便で立体選択的

な合成が、野依還元を鍵反応として完成した。1と2の

全収率は、それぞれ8.5%と2.9%であった。今回合成された

naphtho[2,3–b]furan–4,9–dione 1は、強力な細胞増殖

抑制活性とin vitroとin vivoでのがん化学予防活性を

示した。さらに、それは、グラム陽性菌やカビに相対的に

強い抗菌活性を示した。一方、その位置異性体2の

細胞増殖抑制活性とin vitroでのがん化学予防活性

は低下したが、抗菌性に関しては1とほぼ同等であった。

さらに、in vitroでのがん化学予防活性においては、

少なくとも2と(R)-2の間には有意な差が認められた。

興味深いことに、(R)-1はin vivoでやや高いがん化学

予防効果を示した。現在、それらの生物活性を増強

するための構造要因をよりよく理解するために、構造

活性相関研究に向けたさらなる構造修飾を行っている。

表 4. Antimicrobial activitya of 1, (R)-1, 2, (R)-2 and 10a

a Values represent minimum growth inhibitory concentration, MIC (μg/mL). b NT, not tested. c Positive control substance.

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