Design, Synthesis And Activity Evaluation Of Novel Broad-spectrum β-Lactamase Inhibitors

Β-lactam antibiotics play an important role in antimicrobial drugs by acting on penicillin-binding protein(PBPs)and impeding the synthesis of peptidoglycan of bacterial cell wall. The bacteria perform abnormal morphology due to the uncompleted cell wall and the action of osmotic pressure, ultimately, the bacteria is ruptured to achieve the purpose of sterilization. However, since the clinical use of penicillin, bacteria began to generate resistance to antibiotics under the pressure to survive. The most important mechanism of resistance is the generation of β-lactamase enzymes that can hydrolyze the β-lactam antibiotics. With the increase of β-lactam antibiotics, the variety of β-lactamase is generating and the related hydrolyzing enzymes emerge soon after the new kind antibiotics appear on the market.According to Ambler classification, β-lactamases can be divided into four classes: A, B, C and D. Ambler class A, C and D have the same active-site serine residue, whereas class B consists of enzymes with a metal ion(one or two Zn2+)on the active site. At present, the multiple resistant bacteria(MDR) with multiple resistance mechanisms and expression in a variety of β-lactamases have been one of the most clinically threatening factors.To overcome the problem of bacterial resistance, people invented the use of β-lactamase inhibitors to collaborate with β-lactam antibiotics, in which the β-lactamase inhibitors are capable of inhibiting β-lactamase and improving the activity of antibiotics. When β-lactamase inhibitors and β- lactamase act together, a Michaelis complex is formed in the beginning, and then serine residue at the active site makes an nucleophilic attack at the carbonyl of β-lactam ring, resulting in a lactam ring amide bond rupture and the formation of an enzyme-substrate covalent compounds to inhibit the activity of the enzyme. Currently, the β-lactamase inhibitors on the market mainly involve clavulanic acid, sulbactam and tazobactam, all of which have a β-lactam ring structure that is similar to the β-lactam antibiotics.However, clavulanic acid, sulbactam and tazobactam only act against class A serine enzyme, and there is no inhibitory effect on classes B, C, D enzyme. Faced with the growing of broad-spectrum enzyme and the increasing of multiple resistant bacteria, existing β-lactamase inhibitors seem to be not enough to meet the clinical needs.Avibactam belongs to the diazabicyclooctane compounds. In February 2015, Avycaz, which was developed by Actavis and AstraZeneca, was approved by FDA for the treatment of adult patients with a complicated intra-abdominal infection(cIAI) or a complicated urinary tract infection(cUTI), as well as the application of kidney infected patients with limited or no alternative treatment.Compared with the classic "suicide inhibitors", Avibactam has its unique advantage in mechanism : the serine enzyme nucleophilic attack on the carbonyl group of Avibactam, leading to the amide bond of Avibactam rupture and the formation of enzyme-inhibitor covalent compounds in which the enzyme is inhibited, and the covalent compounds can’t behydrolyzed, then the β-lactam ring recovered and reform Avibactam by cyclization.The open-loop rate is greater than that of forming the rings,so the enzyme was basically kept in the state of inhibition. Avibactam has a long-term nature due to the restored structure. Avibactam was breakthrough of the new β- lactamase inhibitors in the past 30 years. Avibactam has opened new way for the further development of broad-spectrum β-lactamase inhibitors according to the inhibitory effect of class A, C and some kinds of D enzyme. But so far there’s still no β-lactamase inhibitors specific acting on metallo-β- lactamase enzymes of class B and most enzymes of class D.This thesis aims to take Avibactam as lead compound to design two series of new compounds with the structure of diazabicyclooctane, according to the structure-activity relationship of Avibactam, the crystal of enzyme-inhibitor complex spatial conformation and the mechanism of action analysis. We expect the novel β-lactamase inhibitors have a broader spectrum and better inhibitory activity.In this thesis, the synthesis of Avibactam process was modified and optimized. With(S)-1-(benzyloxycarbonyl)-5-oxo-pyrrolidine-2 – carboxylic acid as a starting material, we synthesized the Avibactam sodium salt via 13 steps of reaction. We shorten the reaction reported in the literature from 15 to 13 steps; The reaction product in the literature was separated and purified by silica gel column chromatography, but we use the recrystallization instead in this thesis. The reaction parameters are optimized and determined, making the reaction with the controllability and repeatability; Optical purity and chemical purity of Avibactam sodium were detected through HPLC. The sodium content was measured through Agilent ICP-MS. This thesis also established an effective method to detect the chemical purity and optical purity of Avibactam sodium. The improved process with high yield and less steps is easy to operate, which has laid an important foundation for further research and development production process of Avibactam.Referring to the optimized synthesis route of Avibactam, We synthesized the(2S, 5R)-6-(benzyloxy)-7-oxo-1,6-diazabicyclo [3.2.1] octane-2-carboxylic acid, which is the intermediate of target compound. On the basis of key intermediates, two types of target compounds were designed and synthesized to obtain 19 target compounds. All the structures of the new compounds and intermediates were confirmed by ESI-MS and 1H-NMR.At present, 20 compounds, including Avibactam, were preliminary evaluated for their biological activity. We first performed the enzyme titration experiments, and then the target compounds were detected with 12 concentration gradients, which react with class A TEM-1, class C AmpC and class D OXA-23 enzyme respectively. The nitrocefin, as a substrate, was added to a mixture of the target compounds and β-lactamase enzyme. The content of the substrate is determined by measuring the absorbance of the mixture, thereby the inhibition rate of the target compound to the enzyme is obtained, and the IC50 value of the target compound is used to indicate the activity of the inhibitor.According to the results of biological activity, the IC50 value of the first class I target compounds substituted by benzyl oxygen group was higher(>1000nM) than that of R2 group. The second class of compounds that retained sulfonate and transformation of Avibactam on the R1 groups have a lower IC50 with outstanding enzyme inhibition, especially the R1 groups with saturated fatty hydrocarbons substitution. The IC50 values of compounds L13, L14, L15 and L19 are less than 10 nm for both TEM-1 and AmpC enzyme, which is lower than the positive compound. These compounds have thepotentiality of further research.According to the literature and the activity of the compounds, we discussed the activity effects of R1 and R2 groups. When R1 is substituted by saturated aliphatic chain with 4 to 5 carbon, and R2 is replaced by sulfonic acid ester, the compound performs better inhibition activity to β-lactamase enzyme of class A and C. However, if R2 is replaced by benzyl oxygen, the activity is poor. The analysis above will provide reference for the further design of new broad-spectrum β- lactamase inhibitors.