去氫環肽素衍生物的合成研究
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2022
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天然物Cyclopenin及Viridicatin是在1960年代在圓弧青黴菌(P. cyclopium)及鮮綠青黴菌(P. viridicatum)的代謝物中被分離出來。Viridicatin為結構中含有quinolinone骨架的生物鹼，被發現具有抑制人類免疫缺陷病毒(HIV)複製的Sodium methoxide活性。而結構中含有苯并二氮呯(benzodiazepine)骨架的Cyclopenin家族，則於2021年被發現對於SARS-CoV-2 Mpro具有良好的抑制活性潛力。Luckner團隊在進行Cyclopenin的結構鑑定過程中發現，當Cyclopenin被置於稀釋的酸性溶液中，會失去其光學活性，並得到Viridicatin，隨後White團隊在1970年以化學合成的方式確立兩者天然物在生物體內的關係。而目前在生物合成上發現Cyclopenin會在AsqI酵素催化下進行縮環反應，推測會經由離去異氰酸甲酯進行重排得到Viridicatin，不過確切的反應機制仍尚未確立。由於根據White團隊的方法，Cyclopenin縮環形成Viridicatin的步驟在不需經由酵素催化便可進行，由於本實驗室對於此步驟的反應機制深感興趣，所以想先透過化學合成的方式來合成Dehydrocyclopeptin及衍生物，作為合成Cyclopenin及其衍生物的前驅物，以利後續探討反應機制。我們使用鄰硝基苯甲酸(o-nitrobenzoic acid)作為起始物，透過乙二醯氯(oxalyl chloride)進行醯氯化後，與甘胺酸(glycine)進行縮合反應得到o-nitro-hippuric acid。接著與4-甲基苯甲醛(4-methylbenzaldehyde)進行Erlenmeyer反應，建立具有立體選擇性的 (Z)-4-(4-methylbenzylidene)-2-(2-nitrophenyl)oxazol-5-one的單一產物。隨後使用甲醇鈉對其碳氧雙鍵進行加成後開環，再使用氫化鈉對醯胺基團的氮去質子化，與甲基碘進行取代反應，得到methyl-2-(N-methyl-2-nitrobenzamido)-3-(p-tolyl)acrylate的Z : E = 1 : 0.8的混合物後直接進行還原反應。為了避免benzylidene取代的雙鍵也被還原，我們使用鐵粉及硫酸鐵作為溫和的還原條件，選擇性對硝基進行還原，原先預期會得到以Z組態為主的產物methyl (Z)-2-(2-amino-N-methylbenzamido)-3-(p-tolyl)acrylate，卻在經由NMR鑑定之後發現E組態才是主產物，不過在後續使用MeOH : Piperidine = 1:1的迴流條件下，可成功進行分子內合環建立benzodiazepine骨架，得到雙鍵為Z組態的4’-Methyldehydrocyclopeptin。
In 1960s, Cyclopenin and Viridicatin were isolated from metabolites of Penicillium cyclopium Westling and Penicillium viridicatum Westling. Viridicatin, belonged to quinolone alkaloids, has showed the biological activity to inhibit virus production by blocking TNF-α activation of the HIV long terminal repeat (LTR). Cyclopenin, one of the benzodiazepine alkaloids, has been identified to have a good activity against SARS-CoV-2 Mpro.Luckner et al. found that Cyclopenin loses its optical activity in diluted acid solution with concomitant appearance in high yield of Viridicatin in the process of the structural characterization of cyclopenin. White et al. described a chemical synthesis which confirmed that Cyclopenin can be chemically converted to Viridicatin. In biosynthesis, it was observed that AsqI can catalyze the ring contraction of Cyclopenin to form Viridicatin. The possible mechanism involves the leaving of methyl isocyanate and rearrangement but has not been confirmed. Therefore, we would like to investigate the synthesis of Dehydrocyclopeptin derivatives, as precursors of Cyclopenin derivatives, in order to explore the ring-contraction mechanism.Starting with the preparation of o-nitrobenzoic chloride from commercially available o-nitrobenzoic acid, followed by the nucleophilic acyl substitution with glycine in one pot, (Z)-4-(4-methylbenzylidene)-2-(2-nitrophenyl)oxazol-5-one was efficiently prepared through Erlenmeyer reaction of o-nitrohippuric acid and 4-methylbenzaldehyde. Basic methanolysis of the (Z)-oxazolone resulted in the formation of (Z)-2-(4-methylbenzylidene)-o-nitrohippurate with retention of stereochemistry in the 4-methylbenzylidene. Subsequently, methylation at the N-position of carboxamide afforded inseparable (Z/E)-isomers, Z:E=1:0.8. Reduction of the nitro group with Fe/FeSO4 formed a primary amineand avoided the reduction of 4-methylbenzylidene group. Although we obtained methyl (E)-2-(2-amino-N-methylbenzamido)-3-(p-tolyl)acrylate, the undesired stereoisomer, subsequent intramolecular cyclization still took place to afford 4’-Methyldehydrocyclopeptin with the desired (Z) -configuration under refluxing MeOH and piperidine condition.
In 1960s, Cyclopenin and Viridicatin were isolated from metabolites of Penicillium cyclopium Westling and Penicillium viridicatum Westling. Viridicatin, belonged to quinolone alkaloids, has showed the biological activity to inhibit virus production by blocking TNF-α activation of the HIV long terminal repeat (LTR). Cyclopenin, one of the benzodiazepine alkaloids, has been identified to have a good activity against SARS-CoV-2 Mpro.Luckner et al. found that Cyclopenin loses its optical activity in diluted acid solution with concomitant appearance in high yield of Viridicatin in the process of the structural characterization of cyclopenin. White et al. described a chemical synthesis which confirmed that Cyclopenin can be chemically converted to Viridicatin. In biosynthesis, it was observed that AsqI can catalyze the ring contraction of Cyclopenin to form Viridicatin. The possible mechanism involves the leaving of methyl isocyanate and rearrangement but has not been confirmed. Therefore, we would like to investigate the synthesis of Dehydrocyclopeptin derivatives, as precursors of Cyclopenin derivatives, in order to explore the ring-contraction mechanism.Starting with the preparation of o-nitrobenzoic chloride from commercially available o-nitrobenzoic acid, followed by the nucleophilic acyl substitution with glycine in one pot, (Z)-4-(4-methylbenzylidene)-2-(2-nitrophenyl)oxazol-5-one was efficiently prepared through Erlenmeyer reaction of o-nitrohippuric acid and 4-methylbenzaldehyde. Basic methanolysis of the (Z)-oxazolone resulted in the formation of (Z)-2-(4-methylbenzylidene)-o-nitrohippurate with retention of stereochemistry in the 4-methylbenzylidene. Subsequently, methylation at the N-position of carboxamide afforded inseparable (Z/E)-isomers, Z:E=1:0.8. Reduction of the nitro group with Fe/FeSO4 formed a primary amineand avoided the reduction of 4-methylbenzylidene group. Although we obtained methyl (E)-2-(2-amino-N-methylbenzamido)-3-(p-tolyl)acrylate, the undesired stereoisomer, subsequent intramolecular cyclization still took place to afford 4’-Methyldehydrocyclopeptin with the desired (Z) -configuration under refluxing MeOH and piperidine condition.
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