1,1-Dioxo-6-(substituted)penicillanic acids의 합성 및 β-lactamase저해 효과에 관한 연구

Abstract

β-Lactamase는 β-lactam계 항생제를 불활성화 시키며, 현재 가장 널리 쓰이고 있는 β-lactam계 항생제에 대한 균주 내성의 주 원인이 된다. β-Lactam계 항생제와 함께 사용하여 항생제를 보호할 목적으로 β-lactamase의 mechanism-based-inhibitors로서 6번위치에 다양한 tricycle을 가진 치환기를 도입한 1,1-dioxo-6-(substituted)penicillanic acids를 합성하였다. 즉, β-lactamase와 친화력을 가지면서 β-lactamase의 70번 serine-OH와 acyl-enzyme intermediate를 형성한 후 안정화되어 β-lactamase를 불활성화 시킬 수 있도록 3-thiazolo[3,2,a]benzimidazolyl, 6-methyl-3-thiazolo[3,2,a]-benzimidazolyl, 2-fluorenyl, 9-ethyl-3-carbazolyl, 9-anthracenyl, 10-methyl-9-anthracenyl, 10-chloro-9-anthracenyl, phenanthrenyl기를 도입한 1,1-dioxo-6-bromo-6-(tricyclic)hydroxymethyl penicillanic acids, 1,1-dioxo-6-bromo-6-(tricyclic)acetoxymethyl penicillanic acids, 1,1-dioxo-6-(tricyclic)methylene penicillanic acids 등을 합성하고 이들의 4가지 type의 β-lactamase에 대한 저해 효과와 항생제와 병용시 여러 균주에 대한 상승 효과를 알아보았다. 합성한 화합물들은 tazobactam이나 sulbactam, clavulanic acid와 마찬가지로 모두 assay에 사용할 type Ⅰ enzyme에 대해서는 효과가 없었지만 type Ⅲ와 type Ⅳ enzyme에 대해서는 상당히 효과가 있는 화합물도 있었다. 1,1-Dioxo-6-(tricyclic)methylene penicillanic acids는 type Ⅱ, type Ⅳ enzyme에 대해 비교적 특이적인 저해 효과를 보였는데, 이는 methylene기가 효소의 active site 내의 nucleophile의 공격을 받아 acyl-enzyme intermediate를 안정화 시키기 때문인 것으로 추정하였다. 1,1-Dioxo-6-bromo-6-(tricyclic)hydroxymethyl penicillanic acids는 type Ⅲ, type Ⅳ enzyme에 대해 상당히 좋은 저해 효과를 보였는데, 이는 6-bromo기가 떨어져나가면서 acyl-enzyme intermediate를 안정화 시키고 hydroxymethyl기가 효소와의 친화력을 크게하기 때문인 것으로 추정하였다. 특히 1,1-dioxo-6-(2-fluorenyl) methylene penicillanic acid의 경우 type Ⅱ, type Ⅲ, type Ⅳ enzyme에 대해 모두 양호한 효과가 있었으며 1,1-dioxo-6-bromo-6-(2-fluorenyl)hydroxymethyl penicillanic acid나 1,1-dioxo-6-bromo-6-(2-fluorenyl)acetoxymethyl penicillanic acid의 경우 type Ⅲ, type Ⅳ enzyme에 대해 tazobactam보다 상당히 좋은 효과가 있었는데 이는 fluorenyl기의 크기나 모양 전자밀도 등이 효소와의 친화력을 증가시키는데 적당하였기 때문인 것으로 추정하였다.;The increasing threat of conventional chemotherapy has posed by bacteria which had developed resistance to β-lactam antibiotics via β-lactamase. β-Lactamase inhibitors which have no antibacterial activity can be administered in conjunction with conventional β-lactam antibiotics to protect β-lactam antibiotics from β-lactamase. In our studies, we synthesized three classes of β-lactamase inhibiors ; 1,1-dioxo-6-(tricyclic)methylene penicillanic acid, 1,1-dioxo-6-bromo-6-(tricyclic)hydroxymethyl penicillanic acid and 1,1-dioxo-6-bromo-6-(tricyclic)acetnxymethyl penicillanic acid with variety of tricyclic substituents and tested β-lactamase inhibitory activity against four types of β-lactamase and synergistic effect with piperacillin against β-lactam antibiotics resistant bacteria. 1,1-Dioxo-6-(tricyclic)methylene penicillanic acid showed specific inhibitory activity against typeⅡ or type Ⅳ enzymes, we believe that acylation of an active site serine by the β-lactam followed by a nucleophilic attack on the methylene C(8) position of the β-lactam by another active site residue. 1,1-Dioxo-6-bromo-6-(tricyclic)hydroxymethyl penicillanic acid and 1,1-dioxo-6-bromo-6-(tricyclic)acetoxymethyl penicillanic acid showed good inhibitory activity against type Ⅲ and type Ⅳ enzymes. we believe that 6-bromo substituent stabilized acyl-enzyme intermediate by acting as a leaving group and hydroxymethyl substituent increased enzyme affinity. In particular, compound 29, 32, which have fluorenyl moiety showed most notable improvement in inhibitory activity against type Ⅲ and type Ⅳ β- lactamases compared to tazobactam and clavulanic acid. We believe that fluorenyl moiety must participate in some favorable interaction within the active site hydrophobic pocket of the β-lactamase.