Design, Synthesis And Antibacterial Evaluation Of Novel 15-Membered Azalide Carbamate Derivatives Against Resistant Bacteria

Macrolides are weak basic and lipophilic compounds produced by streptomycetes, which are characterized by the macrolide skeleton and saccharide groups. The first and second generation macrolides, such as erythromycin A, clarithromycin and azithromycin, demonstrate high inhibitory activity against Gram-positive bacteria, mycoplasma and chlamydia. And they have been widely used for treatment of respiratory, gastrointestinal and genitourinary infections. In recent years, more and more inappropriate use of macrolides leads to increasing drug resistance, which severely threatens therapeutic effectiveness of the macrolides and limites their clinical use. Currently, a wide variety of structural modifications are investigated to develop novel macrolides with significantly enhanced anti-resistant bacterial activity.The 23S rRNA of 50S subunit in bacterial ribosome can be divided into five domains, and peptidyl transferase center (PTC) is located in domain V. It has been reported that there are three binding sites from PTC to the contraction of peptidyl release tunnel. The first is A2058 or A2059 in domain V, which situates in the entrance of peptide releasing tunnel. The second site is U2609 in domain V or A752 in domain II, which is located in another side of entrance of peptidyl release tunnel. The third is the binding site of amphemycin or clindamycin, which is located A and P sites of 50S subunit. Two of the commonest mechanisms of macrolides resistance are erm-expressing methylation and mef-expressing efflux pumps. The methylase encoded by the erm gene can modify the key nucleotide A2058 in domain V through methylation, which leads to the MLSB resistance. The efflux pumps encoded by the mef gene can limit the steady-state accumulation of drugs by transporting antibiotics out of cytoplasm.In order to fight against the increasing resistance to macrolides, many analogs were obtained by introducing some side chains with appropriate length and terminal groups to C-11, C-6 or C-4" position of existing macrolides, such as ketolides, acylides and 4"-carbamates. These analogs are on the target of the binding sites of macrolides, such as A2058, A2059, A752, U2609 and the nucleotide residues between A and P sites of 50S subunit. On the basis of the consideration above, three series of 4"-O-carbamoylerythromycin A 6,9-imino ether 11,12-cyclic carbonate derivatives,4"-carbamoylazithromycin 11,12-cyclic carbonate analogs and 4",11-bicarbamoylazithromycin derivatives were designed, synthesized and evaluated in this thesis. The structures of the target compounds have been confirmed by MS, IR and 1H NMR.In vitro antibacterial activity of these target compounds was determined using the the broth microdilution method. The results were as follows:(1) Antibacterial activity against susceptible streptococcus pneumoniae:series A showed excellent activity similar to the control drugs (MIC=0.03 p.g/mL). The antibacterial activity of series C was slightly better than series B. Among series B, compounds B20, B29 and B30 were the most potent (MIC=0.12μg/mL). In addition, all analogs of series C possessed the similar MICs (0.03～0.12μg/mL). (2) Antibacterial activity against resistant S. pneumoniae strains:series B and C showed improved activity against all of the resistant S. pneumoniae strains, particularly, the activity of compounds against S. pneumoniae A22072 expressing the mef gene was the best. Series C possessed better activity against resistant strains than the other two series. Among series C, compounds C14 and C17～C19 showed the most improved activity against MLSB-resistant bacteria B1 (MIC=0.25μg/mL), showing 512-,512- and 256-fold more potent activity than EMA, AZM and CAM, respectively. Compounds C17～C19 were the most potent against S. pneumoniae A22072 (MIC=0.03 ug/mL), showing 256-,128- and 128-fold improved activity in comparison with the control drugs EMA, AZM and CAM, respectively. Additionally, compounds C17～C19 exhibited 128-fold, 128-fold and 64-fold higher activity than EMA, AZM and CAM, respectively, against resistant bacteria AB11. Series A showed moderate activity against resistant strains, and compound A6 exhibited the most potent activity against resistant strains B1 and AB11, showing 16- and 32-fold better activity than EMA and AZM, respectively.Structural modification and structure-activity relationships of these azalide analogs were summarized as follows:(1) Macrolide skeletons are essential for restoring the activity against susceptible bacterial strains. And the modifications of the lactonic ring or tethering side chains to the skeleton may reduce the activity against susceptible bacterial. (2) The introduction of side chains to the C-4" position can improve the activity against resistant bacteria. Compounds containing electron-withdrawing groups on the terminal aromatic ring of their C-4" side chains, demonstrate the most potent activity. The appropriate distance from the 4"-oxygen atom to the terminal benzene ring is 6 atoms. (3) The introduction of C-4"and C-11 aryl carbamate side chains to the skeleton simultaneously can significantly improve the activity against resistant bacteria strains. And the length of the side chains and the terminal aromatic ring can affect the activity. Particularly, derivatives with electron-withdrawing groups on the terminal aromatic ring of their C-4" side chains show more potent antibacterial activity.In summary, azithromycin is a parent nucleus for its beneficial antibacterial activity and excellent pharmacokinetic profiles. And the macrolide skeletons are essential for restoring the activity against susceptible bacterial strains. C-4", C-11 and C-12 positions are promising sites for structural modifications. It's hopeful that the introduced 4"-O-carbamate and C-11-O-carbamate side chains with appropriate length and terminal aromatic ring can interact with the binding sites in 23s rRNA and produce additional affinity for the resistant ribosome, resulting in potent activity against the resistant bacteria.