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Running title: Synthesis and antifungal activities of griseofulvin analogues
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Synthesis of Griseofulvin Analogues and Their Inhibitory
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Activities against Phytopathogenic Fungi
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YU XIAO-JIE,‡#, ZHU YU-JING,†#, LI ZHI-CONG,‡, PAN ZHI-ZHEN,†, CHEN QING-XI,‡, AND LIU BO†*
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† Agricultural
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350003, China
Bio-resource Institute, Fujian Academy of Agricultural Sciences, Fuzhou
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‡ Key
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361005, China
Laboratory for Chemical Biology of Fujian Province, Xiamen University, Xiamen
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# These authors contributed equally to this work.
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* Author for corresponding. Tel/fax: +86 591 87864601. E-mail address: liubofaas@163.com
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(B.L.).
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ABSTRACT
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In this study, six analogues of griseofulvin were synthesized and evaluated for inhibitory
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fungicidal activity against phytopathogenic fungi Fusarium moniliforme, Fusarium solani,
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Fusarium oxysporum and Colletotrichum truncatum. The results indicated that these
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compounds exhibited different antifungal activities. Especially, the activity of
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4′-hydroxylaminefriseofulvin against F. moniliforme and F. solani increased evidently, while
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griseofulvin was employed for comparison. Effect of the analogues on hyphal growth of
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fungi was also evaluated using microscope. The active analogues were found to be teratogenic
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against fungi just like griseofulvin. Also the solubility in different solvents of analogues and
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griseofulvin was roughly evaluated and compared. The variations in griseofulvin structure
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covered four positions, namely the 5, 2′, 3′and 4′ positions. Modification of the 5 position
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resulted in inactive compound. The alkoxy at the 2′ position was important and the 4′ position
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should keep its double bond form.
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Keywords: Synthesis; Griseofulvin; Antifungal Activity; Structure-activity Relationship;
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Comparison of Solubility
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INTRODUCTION
Griseofulvin (1, Fig. 1), first isolated from the mycelium of Penicillium griseofulvum
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Dierckx (1), was a classic antifungal agent against many pathogenic filamentous fungi (2-5).
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It had been used clinically for the treatment of dermatomycoses (6-8) for years, because it
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could treat fungus infections caused by tinea organisms of the hair, skin and nails. However, it
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was not found effective against bacterias and yeasts. Besides, griseofulvin was of great use in
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the treatment of cancer due to that it could block cell-cycle progression at the G2/M phase and
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induce apoptosis in human tumor cell lines (9). More recently griseofulvin has become the
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object of increased interest thanks to its anticancer potential (10, 11).
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The status of griseofulvin in crop protection introduced a desirable solution to the
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economics of its utilization which could contribute to the establishment of griseofulvin as a
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specific plant protection against numerous diseases induced by fungi (12, 13). Though
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griseofulvin was of importance in the development of agriculture, its use in plant protection
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was not popularized because much attention had been paid to its advantage and disadvantage
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when used as drug in the treatment of dermatophyte infections.
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As was known, water solubility and bioavailability of griseofulvin were extremely low,
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leading to the decrease of its effectivity. What’s worse, it also exhibited a number of
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undesirable side-effects, such as urticaria and fixed drug eruption (14), erythema multiforme
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(15) and so on. It had also been reported that griseofulvin was carcinogenioc and teratogenic
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in animal models (16). Hence, more research was needed on the structure of griseofulvin for
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enhancing its absorption, eliminating its defects and improving its activity.
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Nowadays more attention has been paid to related derivatives of possible physiological
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utilities with the sharp development of total synthesis of antifungal agent. Hundreds of papers
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describing griseofulvin derivative synthesis and structure-activity relationship studies related
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to antifungal activity have been published by now. In order to improve the use of griseofulvin
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in agriculture and the advancement of griseofulvin analogues as antibiotics, the authors
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synthesized six analogues of griseofulvin, tested and compared their fungicidal activity with
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griseofulvin against four phytopathogenic fungi including Fusarium moniliforme Sheld,
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Fusarium solani (M art.) Sacc, Fusarium oxysporum and Colletotrichum truncatum
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(Schw.) Andr et Moore. All analogues described in this paper have been synthesized from
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commercially available griseofulvin. Meanwhile, effect of analogues on hyphal growth of
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fungi was evaluated by using microscope. Also, the solubility of analogues were calculated
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roughly by the method of saturation and compared to griseofulvin. Finally, the correlation of
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structure and activity was studied. We were interested in determining the influence of the 2′,
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3′, 4′and 5 position on the antifungal activity. This study could help enhance the use of
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griseofulvin in agriculture and improve the development of biocides.
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MATERIALS AND METHODS
Fungi. All the fungi were pathogenic in agriculture and selected for testing from the
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laboratory collection in Fujian Academy of Agricultural Sciences including Fusarium
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moniliforme Sheld FJAT-165, Fusarium solani FJAT-176, Fusarium oxysporum FJAT-3701
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and Colletotrichum truncatum FJAT-9253. Fungi were grown on potato dextrose agar (PDA)
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in 9-cm petri dishes and incubated at 28±1 °C.
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Reagents. Griseofulvin (99.9% powder) was purchased from Shanghai Pharmaceuticals
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Holding Co., Ltd. (Shanghai, China). Dimethyl sulfoxide (DMSO) was the product of
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Sigma-Aldrich (St. Louis, MO, USA). The other reagents and solvents were the products of
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Sinopharm Chemical Reagent Co., Ltd. (Shanghai, China) and used without further
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purification. The water used was redistilled ion-free.
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Chemistry. The syntheses of some derivatives were according to previous reports (11, 17)
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and the methods were changed or improved more or less as showed in Scheme 1-3. The
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analogue 2 was synthesized by treatment of 1 with H2SO4 in acetic acid. Epoxidation of 1
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with H2O2 afforded the epoxy derivative 3. Nitration of 1 using nitric acid yielded 4.
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Modification of 4′-position of 1 gave 5, 6 and 7. The new compound hydrazine 5 and oxime 6
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could be synthesized by the similiar procedure: to the solution of 1 and thiosemicarbazide or
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hydroxylamine hydrochloride in ethanol and DMSO, added sodium methoxide and then
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refluxed. Besides, it could also yield 5 by treatment of 1 with thiosemicarbazide in methanol
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and acetic acid according to the special procedure. The analogue 7 derived from 1 by
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reduction with sodium hydride in tetrahydrofuran (THF) and H2O. The structures of these
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compounds were established by spectroscopic methods (MS and H1NMR). Additionally, the
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three active analogues (3, 5 and 6) were performed by high-resolution FT-ICR-MS (HRMS).
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2′-hydroxylgriseofulvin (2). Griseofulvin (10 mmol) was added to acetic acid (60 ml),
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and heated to 40 °C to dissolved, then added 2 mol/L sulfuric acid (15 ml) to the solution. The
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mixture was stirred at 60-70 °C and allowed to come to room temperature when the reaction
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was over. In the process of the reaction, there were white precipitates appeared. After being
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filtrated, the residue was washed with water and dried, affording the product 2.
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Epoxygriseofulvin (3). To a cooled suspension of griseofulvin (4 mmol) in methanol (40
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ml), added 30% H2O2 (5 ml) and KOH (7 mmol) and stirred, keeping the temperature at
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0-5 °C. Thirty minutes later, the reaction mixture was warmed to room temperature and stirred
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for another 30 min. Added ice water (50 ml) to the mixture and filtrated. The residue was
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purified by column chromatography to afford the product 3.
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5-nitrylgriseofulvin (4). To a cooled solution of griseofulvin (5 mmol) in acetic
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anhydride (Ac2O, 20 ml), added cautiously 65% nitric acid (3 ml). The mixture was allowed
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to come to room temperature and stirred for 48 h. Then washed with ice water (40 ml) and
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filtrated. The residue was purified by column chromatography to afford the product 4.
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4′-thiosemicarbazonegriseofulvin (5). Genaral procedure: To the solution of
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griseofulvin (10 mmol) in ethanol (40 ml) and DMSO (20 ml), added thiosemicarbazide (10
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mmol) and sodium acetate (NaOAc, 30 mmol). Then the solution was stirred and refluxed for
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72 h. After reaction, to the reaction solution added ice water (50 ml) and extracted with
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CH2Cl2 (3×50 ml). The organic phase was washed with water and saturated solution of NaCl
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and then dried and concentrated. The residue was purified by column chromatography to
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afford the product 5. Special procedure: Griseofulvin (10 mmol) and acetic acid (AcOH, 10
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ml) were added to a solution of thiosemicarbazide (10 mmol) in methanol (60 ml). The
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mixture was stirred and refluxed for 72 h and then cooled to room temperature. Then the
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reaction mixture was kept under 4 °C for night and filtrated. The residue was recrystallized in
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ethanol to afford product 5.
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4′-hydroxylaminegriseofulvin (6). To a solution of griseofulvin (5 mmol) in ethanol (25
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ml) and DMSO (20 ml), added hydroxylamine hydrochloride (15 mmol) and NaOAc (17
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mmol) and then stirred and refluxed for 24 h. After reaction, cooled to 0 °C and added ice
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water (50 ml). The combined aqueous phases were extracted with CH2Cl2 (3×50 ml). The
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combined organic phases were dried with NaSO4 and concentrated. The residue was purified
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by column chromatography to afford the product 6.
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4′-hydroxylgriseofulvin (7). A suspension of griseofulvin (10 mmol) in THF (60 ml) and
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H2O (2 ml) was heated to 40 °C to dissolve. NaBH4 (40 mmol) was slowly added to the
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solution. The reaction mixture was heated to 60 °C and refluxed for 2 h. The mixture was
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added saturated solution of sodium hydrogen carbonate and extracted with ethyl acetate (3×50
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ml), and then washed with water and saturated solution of sodium chloride and dried. The
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residue was purified by column chromatography to afford the product 7.
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Conidial suspension preparation. Conidia were harvested from 7-day-old cultures by
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flooding plates with 10 ml of sterile distilled water and dislodging conidia by softly brushing
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the colonies with aseptic glass spreader. Aqueous conidial suspensions were filtered through
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sterile gauze to remove hyphae. Conidial concentrations were determined by hemocytometer
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and adjusted with sterile water to the concentration of 107 conidial/ml.
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Mycelial growth assay. The mycelial growth assay was conducted on PDA medium
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according to the coating method (19). Prior to the fungal inoculation, 100 μl of different
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concentrations of the tested compounds were poured and spread on the surface of the agar
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medium. Dishes were then left opened to enable solvent to evaporate or penetrate. And then,
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the plates were inoculated in the middle with 50 μl fungal conidial suspension (~1×105
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CFU/ml). In parallel, control experiments only with DMSO were also tested. The Petri dishes
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were incubated at 28±1 °C and the fungal colony diameter was measured daily. The inhibition
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rates were calculated after 7 days of incubation when mycelium in the control experiments
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completely covered the dishes. It was expressed as an average diameter and calculated using
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the following equation:
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where Dc and Dt represent mycelia growth diameter in control and treated Petri plates,
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respectively.
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Effect of analogues on hyphal growth of fungi. Effect of analogues on hyphal growth of
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the four fungi was studied by using microscope. 10 ml of agar medium inoculated with fresh
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fungal culture (~1×105 CFU/ml) were poured into the surface of the agar medium in Petri
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dishes as the upper layer. After solidification, two wells of 8-mm diameter were created. Fifty
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μl of 4×10-6 mol/ml analogues solution and DMSO were deposited in wells. Petri dishes were
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then incubated at 28±1 °C for 72 h. The hyphae closely around the inhibition zones were
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collected and observed by Leica DMI 3000 M Inverted Microscope (Leica Corp.).
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Saturation method for determination of compound solubility. The same amounts (500
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mg) of griseofulvin and analogues were put into isovolumetric solvents (10 ml) respectively
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and stirred to resolve. Then filtrate the suspensions and collect the residues. The highness and
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slowness of the solubility of the compounds were preliminarily determined through the
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weight of the residues. If the weight of the residue of one analogue was larger than that of
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griseofulvin, then the solubility of the analogue is lower than griseofulvin and vice versa.
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Data Analysis. All the measurements were replicated three times for each treatment and
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the data were reported as mean. The statistical analysis was performed by one way analysis of
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variance (ANOVA). IC50 values of analogues on the tested fungi were analyzed using Probit
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Analysis. The statistical analyses were done using the China-DPS program (20).
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RESULTS AND DISCUSSION
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Chemical synthesis of analogues. Analogue 2 was purified as white powder. Yield: 3.17 g
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(93.8%). 1H NMR (DMSO-d6, 600 MHz): δ (ppm) 11.84 (2′-OH, H, s), 6.45 (bezene, H, s),
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5.32 (3′-CH, H, s), 4.03 (CH3O-bezene, 3H, s), 3.91 (CH3O-bezene, 3H, s), 3.42 (6′-CH, H, s),
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2.75, 2.50 (5′-CH2, 2H, m), 0.84 (CH3, 3H, d, J=6.2 Hz). ESI-MS: m/z 339.0 (M+H+).
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Analogue 3 was purified as yellow powder. Yield: 0.73 g (49.4%). 1H NMR (CDCl3, 600
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MHz): δ (ppm) 6.12 (bezene, 1H, s), 4.00 (CH3O-bezene, 6H, m), 3.49 (3′-CH, H, m),
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3.01(2′-OCH3, H, m), 2.82 (2′-OCH3, H, d, J=6.2Hz), 2.57 (2′-OCH3, H, m), 2.05 (6′-CH, H,
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s), 1.27 (5′-CH2, 2H, d), 0.95 (CH3, 3H, dd, J=6.7, 23.3 Hz). ESI-MS: m/z 369.2 (M+H+).
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HRMS calcd for [C17H17ClO7]+ 368.0663, found 369.0715 (M+H+).
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Analogue 4 was purified as yellow powder. Yield: 0.97 g (48.5%). 1H NMR (DMSO-d6,
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600 MHz): δ (ppm) 5.55 (3′-CH, H, s), 4.04 (CH3O-bezene, 3H, s), 3.98 (CH3O-bezene, 3H,
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s), 3.62 (2′-CH3O, 3H, s), 3.03 (5′-CH, H, m), 2.84 (5′-CH, H, m), 2.43 (6′-CH, H, dd, J=4.7,
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16.7 Hz), 0.96 (CH3, 3H, d, J=6.8 Hz). ESI-MS: m/z 398.3 (M+H+).
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Analogue 5 was purified or recrystallized as white solid. Yield: 3.47 g (81.6%) by
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general procedure; yield: 3.63 g (85.4%) by special procedure. 1H NMR (CDCl3, 600 MHz): δ
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(ppm) 7.25 (HN, 1H, s), 5.61 (CH, 2/3H, s), 5.72 (CH, 1/3H, s), 6.12 (bezene, H, s), 6.35,
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8.68 (NH2, 4/3 H, s), 6.32, 8.97 (NH2, 2/3 H, s), 3.98 (CH3O-bezene, 3H, s), 4.06
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(CH3O-bezene, 3H, s), 3.60 (2′-CH3O, 2H, s), 3.67(2′-CH3O, H, s), 2.72(CH, 2/3H, m), 3.10
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(CH, 1/3H, m), 1.26 (CH2, 2H, m), 0.96 (CH3, 3H, m). ESI-MS: m/z 426.2 (M+H+). HRMS
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calcd for [C18H20ClN3O5S]+ 425.0812, found 426.0892 (M+H+).
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Analogue 6 was purified as light yellow crystals. Yield: 1.56 g (84.6%). 1H NMR
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(DMSO-d6, 600 MHz): δ (ppm) 6.20 (bezene, H, s), 5.58 (3′-CH, H, s), 4.02 (CH3O-bezene,
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3H, s), 3.93 (CH3O-bezene, 3H, s), 3.54 (2′-CH3O, 3H, m), 2.84 (6′-CH, H, dt, J=14.6, 12.0
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Hz), 2.42 (5′-CH2, H, m), 1.06 (5′-CH2, H, t, J=6.6 Hz), 0.81 (CH3, 3H, s). ESI-MS: m/z
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368.3 (M+H+). HRMS calcd for [C17H18ClNO6]+ 367.0823, found 368.0903 (M+H+).
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Analogue 7 was purified as white powder. Yield: 2.94 g (83.0%). 1H NMR (CDCl3, 600
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MHz): δ (ppm) 5.34 (3′-CH, H, s), 6.15 (bezene, H, s), 3.97, 4.04 (CH3O-bezene, 6H, s), 3.42
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(5′-CH, H, s), 3.67 (2′-CH3O, 3H, s), 2.16 (CH2, 2H, m), 0.84 (CH3, 3H, d, J=6.8 Hz).
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ESI-MS: m/z 377.2 (M+Na+).
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Antifungal activity of griseofulvin and analogues in mycelial growth assay. The
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antifungal activities of synthesized analogues 2-7 were tested against FJAT-165, FJAT-176,
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FJAT-3701 and FJAT-9253. Griseofulvin was employed for comparison purposes as a
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standard in this test. Of the six synthesized compounds, three (2, 4 and 7) were totally
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inactive and the others (3, 5 and 6) performed differently against various fungi compared to
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griseofulvin. The fungal colony diameters treated with analogues were obviously
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smaller than that of control group treated with DMSO, such as analogue 3 against
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FJAT-3701 showed in Figure 2. The inhibition rates of them were measured and IC 50
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values were calculated. The results were presented in Table 1, and combining the data
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from Table 1 led to Table 2, the comparison of griseofulvin and analogues on activity.
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It was clear that the inhibition activities of 1, 3, 5 and 6 against four fungi, which could
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be indicated from IC 50 values, increased with their concentrations increasing as showed in
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Table 1. This meant they were dose-dependent. The antifungal activity of griseofulvin could
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be profoundly altered by changes in chemical structure and some synthesized analogues were
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even better than griseofulvin as we expected. The effects of 3 and 5 against FIAT-165 were
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similar to 1, while the effect of 6 was a little better than1; The antifungal activity of 6
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against FJAT-176 was dramatically increased compared to 1, though the activity of 3
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and 5 decreased; As to the activity against FJAT-3701, 3 was close to 1 while 5 and 6 were
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worse than 1; The fungicidal activity against FJAT-9253 of analogue 3, 5 and 6 were not so
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good as 1. However, the IC50 values of 5 and 6, 1.55 and 1.66 ×10-6 mol/ml respectively, were
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also very low which meant they could exhibit excellent inhibitory effects as well.
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The results definitely demonstrated that among the four fungi, analogue 1, 5 and 6
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performed best against FJAT-9253 compared to the other three fungi and 3 performed best
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against FJAT-3701. The activity of 1 against FJAT-9253 is fascinating, although 5 and 6
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also showed their best activities against FJAT-9253. The activity of 3 against FJAT-3701
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was the greatest among 3, 5 and 6, and it was similar to the activity of 1. Analogue 1, 3
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and 5 all performed worst against FJAT-176; however, 6 could do well against FJAT-176.
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Besides, the fungicidal activity of 6 was better than that of 1 against FJAT-165.
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Effect of analogues on hyphal morphology. The alternation of hyphae grown on PDA
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amended with analogues (3, 5 and 6) was studied under microscope. Degenerative changes
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were observed on the hyphal morphology in all analogue treatments such as the three
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analogues against four fungi showed in Figure 2. The hyphae of fungi treated with DMSO as
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control treatment were thick, elongated, even and smooth surfaced, whereas the growths of
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fungi were strongly inhibited, as indicated with mycelia sparsity, asymmetry, swelling, curling
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and twisting when treated with analogues. These indicated that the active analogues were also
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teratogenic in fungi just like griseofulvin.
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Comparison of solubility. The solubility of compounds were evaluated roughly and
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compared to that of griseofulvin by the method of saturation. The weights of residues of
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griseofulvin and analogues were compared. If the weight of the residue of one analogue was
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larger than that of griseofulvin, then the solubility of it is lower than griseofulvin and vice
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versa. The results were presented in Table 6. The solubility in methanol of all six analogues
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except 5 were higher than that of griseofulvin. As to the solubility in ethanol, 5 and 6 were
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lower than griseofulvin while the other four were higher. When it came to the solubility in
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solution of 10% NaOH, only 2 and 6 were increased. Unfortunately, their solubility in water
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were not improved evidently compared to griseofulvin. Combined these with the results of
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antifungal activities, both the activity and solubility of analogue 3, 5 and 6 were get improved
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in certain circumstances.
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Correlation of structure and activity. At ring A, the 5 position was altered. Introduction of
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a nitro (analogue 4) at the 5 position resulted in no activity, indicating that modification at this
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position was not tolerated. And this conformed to the previous report (22). The 2′ position and
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2′-3′ double bond of the ring C were altered in this study leading to the synthesis of 2 and 3,
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the activities of which were quite different even against the same fungus. The analogue 2
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showed no activity and this indicated that the alkoxy at the 2′ position played an important
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role in inhibiting fungi. However, the activity of 3 toward FJAT-165 and FJAT-3701 was
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comparable to griseofulvin. Besides, the analogue 3 was found to be less active t han
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griseofulvin toward FJAT-176 and FJAT-9253. From the modification of 4′ position, we
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got 3 compounds: analogue 5, 6 and 7. The analogue 5 appeared a slight increase in activity
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against FJAT-165 but an evident decrease in capability against FJAT-176, FJAT-3701 and
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FJAT-925. The activity of analogue 6 toward FJAT-165 and FJAT-176 increased
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considerably. Nevertheless, when it came to FJAT-3701 and FJAT-9253, the analogue 6
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showed a lower activity in comparison with griseofulvin. The analogue 7, available b y
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reduction of 1, did not show any activity at all. The results from the 4 ′ analogues
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indicated that this position was significant for the antifungal activity as removal of the ketone
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caused the compound 7 inactive. Combining with the activity of 5 and 6, it suggested that the
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4′ position should keep its double bond form.
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Modifications on the structure of griseofulvin could afford novel and effective biocides,
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analogue 3, 5 and 7 exhibited antifungal activity against phytopathogenic fungi such as
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Fusarium moniliforme Shel, Fusarium solani (M art.) Sacc, Fusarium oxysporum and
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Colletotrichum truncatum (Schw.) Andr et Moore. Their activity and solubility in various
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solvents got changed and even improved compared with griseofulvin. As the effect of active
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analogues on hyphal growth showed, they were teratogenic in fungi as well just like
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griseofulvin. All of these data showed that griseofulvin could be served as a leading
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compound for novel antifungal agent development. They also enriched the knowledge of
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biological control of phytopathogenic fungi.
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ABBREVIATIONS USED
DMSO, dimethyl sulfoxide; THF, tetrahydrofuran; Ac2O, acetic anhydride; NaOAc, sodium
acetate; AcOH, acetic acid; IC50, half maximal inhibitory concentration.
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ACKNOWLEDGEMENTS
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The present investigation was supported by the Special Fund for Agro-scientific Research in
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the Public Interest (200903049), the Fujian Natural Science Foundation (2007J0058), the
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Fujian Funds for Distinguished Young Scientists (2009J06010).
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16
341
Table 1. Compounds Included in the Article and Their IC 50 Values (×10-6 mol/ml) within a 95%
342
Confidence Interval
Compo-
Fungi
und
FJAT-165
FJAT-176
FJAT-3701
FJAT-9253
1
7.43±1.55
9.30±7.25
3.70±1.98
0.39±0.22
2
―
―
―
―
3
7.23±2.95
13.25±6.30
3.80±2.82
7.49±0.85
4
―
―
―
―
5
7.02±4.45
15.28±3.03
5.42±3.20
1.55±0.29
6
5.90±2.97
3.57±2.50
6.27±4.56
1.66±1.45
7
―
―
―
―
343
344
345
346
347
348
349
350
351
352
353
354
355
356
357
358
359
360
17
361
Table 2. Comparison of griseofulvin and analogues on solubility in different solvents.
analogues
methanol
ethanol
10%
water
NaOH
362
363
2
+
+
+
-
3
+
+
-
-
4
+
+
-
-
5
-
-
-
-
6
+
-
+
-
7
+
+
-
-
“+”: solubility was increased;
“-”: solubility was not increased or even decreased.
364
365
366
367
368
369
370
371
372
373
374
375
376
377
378
379
18
380
Legend for figures
381
Figure 1 Chemical structure of griseofulvin.
382
Figure 2 Reaction scheme. Figures of 1-7 denote griseofulvin and analogue 2-6
383
respectively.
384
Figure 3 Effects of griseofulvin on hyphal growth of FJAT-3701. Figures of A and B
385
denote the fungi colonies treated with analogue 3 and DMSO separately, while
386
figures of C, T1, T2 and T3 denote the control treatment, treatment with analogue 2,
387
treatment with analogue 3 and treatment with analogue 4 respectively. The
388
concentrations of analogues were 4×10-6 mol/ml.
389
390
391
392
393
394
395
396
397
398
399
400
401
19
402
Figure 1
403
MeO
4
5
A
O OMe
2'
B
O
MeO
404
Cl
405
406
407
408
409
410
411
412
413
414
415
416
417
418
419
420
421
422
20
C
3'
4'
O
423
Figure 2
Scheme 1
MeO
MeO
O OMe
O
CH3OH
H2O2, KOH
O
MeO
Cl
O
3
1
O OH
CH3COOH, H2SO4
60-70℃
O
O
MeO
Cl
2
Scheme 2
MeO
1
O OMe
O2N
Ac2O
O
HNO3
MeO
Cl
O
4
Scheme 3
MeO
O OMe
OH
O
7
MeO
Cl
THF
reflux H2O
NaBH4
MeO
MeO
O OMe
S
N
MeO
Cl
424
O
5
N
H
OH
N
general procedure CH3OH, DMSO
1
special procedure AcONa, reflux
NH2
MeO
Cl
O
6
general procedure: EtOH, DMSO, AcONa, reflux; special procedure: AcOH, CH3OH, reflux
425
426
427
428
429
430
431
432
O OMe
Figure 3
21
433
434
A
B
C
T1
T2
T3
435
436
437
438
439
440
441
22