Design, Synthesis And Antibacterial Evaluation Of Descladinosylazithromycin Derivatives Against Resistant Bacteria

The macrolide antibiotics are the most important drug for the treatment of bacterial infections, the activity of which stems from the large lactone ring. Azithromcin and clarithromycin are great examples of the second generation macrolides, which have better pharmacokinetic profiles and are more stable than the first generation macrolide erythromycin. Nevertheless, the emergence of bacterial drug-resistance makes it an urgent task to develop novel macrolide candidates with better antibacterial activity againt resistant bacteria.In the 23S rRNA of bacterial 50S subunit, there are three binding sites from peptidyl transferase center to the peptide releasing tunnel. Macrolides can inhibit protein synthesis by selectively binding to the 50S subunit of bacterial ribosome and blocking up the peptidyl release tunnel. It is reported that the 2'-OH of erythromycin can bind to A2058 in domain V of the 23S rRNA, which is the first binding site. The C-11,12 carbamate chain of ketolides may bind with A752 in domain II, which is the second binding site. The strong affinity for resistant ribosome will result in good activity against bacteria. The 4"-O-arylalkyl group of CP-544372 may interact with nucleotides in A and P sites of 50S subunit, which is regarded as the third binding site.In recent years, the increasing drug resistance makes it an urgent need to discover novel macrolide antibotics. The macrolide resistances include erm-expressing methylation and mef-expressing efflux pumps. The ribosomal methylase encoded by the erm gene can methylate the key nucleotide A2058, which should be responsible for high-level macrolide resistance. The efflux pump, which is encoded by the mef (A) gene, pumps the drug out of intracellular environment and results in intermediate resistance. Hence, it is an urgent need to develop new macrolide antibiotics that can overcome the pneumoniae resistance while still being active against other resistant and sensitive bacteria. The new structures are supposed to conserve the fine pharmacokinetic properties and be stable to acid.According to the above information, targeting G2505, C2610, U2586 and other nucleotides in the peptidyl transferase center (PTC) region, a series of 3-O-arylalkylcarbamoyl-descladinosylazithromycin 11,12-carbonates were designed and evaluated for their antibacterial actitiy in vitro. We hoped that the 3-O-arylalkylcarbamoyl side chain can bind with G2505, U2506, C2452 or 2451 in the PTC region via hydrogen bonding, ?-stacking and electrostatic interactions. Besides, a series of 3-O-arylalkylcarbamoyl-11-O-arylalkylcarbamoyl-descladinosyl azithromycin, targeting peptidyl transferase center (PTC) region and A752 in domain ?, were also designed, synthesized and evaluated in vitro. The 11-O-arylalkylcarbamoyl groups were hoped to interact with A752 or other nucleotides and produce additional affinity for the ribosome.In addition, we designed and synthesized 2'-O-arylalkylcarbamoyl-descladinosyl azithromycin which targeted ermB-mediating dimethylated ribosome. In general, these synthesized compounds belonged to the three new subclasses C-E. The 2'-O-arylalkylcarbamoyl groups could interact with A2058 (A2041 in Deinococcus radiodurans) and other nucleotides through hydrophobic interaction, hydrophobic interaction and aromatic stacking effects. We prepared 71 novel azilides and confirmed their structures by MS,13C NMR and 1H NMR. In vitro antibacterial activity of these target compounds were presented as minimum inhibitory concentrations (MICs) by using the the broth microdilution method.The results were summarized as follows: 1. Antibacterial activity against susceptible strains of Gram-negative bacteria: compound C6, D1, and D2 exhibited the most potent activity against Escherichia. coli (32 ?g/mL). The MIC value of the best compound against Paeruginosa was 4?g/mL, which was remarkblely improved compared with that of the refereces.2. Antibacterial activity against susceptible strains of Gram-positive bacteria: compound B7 showed the best antibacterial activity against susceptible Staphylococcus aureus (1 ?g/mL) and Streptococcus pyogenes (0.03 ?g/mL). Encouragingly, compound B8 had the most improved activity against Bacillus subtilis with an MIC value as low as 0.5?g/mL, showing 2-fold higher activity than azithromycin (1?g/mL).3. Antibacterial activity against resistant strains of Gram-positive bacteria: Compound B8 showed the best activity against S. epidermidis (0.125 ?g/mL), which was 2-fold better activity than azithromycin (0.25 ?g/mL) and clarithromycin (0.25 ?g/mL).Compound A1 had potent activity against erythromycin resistant S. pyogenes with an MIC value of 8 ?g/mL, which was 32-fold higher than that of controlls (256 ?g/mL). In addition, compound E3 exhibited 16-fold higher activity against resistant Streptococcus pyogenes than controlled drugs with the MIC values of 16 ?g/mL. Compounds A2, A3, A8, A15, B4, B6, B8, C13, D8, E2, E4, E8 and E9 also showed moderate activity against resistant S. pyogens (32 ?g/mL).A majority of the target compounds showed improved activity against the ermB type resistant S. pneumoniae than the references. Amazingly, compound B8 (1 ?g/mL) displayed favorable activity against resistant S. pneumoniae expressing the ermB gene, which were 256-fold better than the references (256 ?g/mL).As for mefA type resistant S. pneumoniae, compounds B6, B7, A7, and A8 showed the MIC values of 0.5 ?g/mL,0.25 ?g/mL,1 ?g/mL, and 1 ?g/mL, repectively, which were better than the controlled drugs (4 ?g/mL).Compounds A3 (1 ?g/mL), B8 (1 ?g/mL), B2 (2 ?g/mL), B7 (2 ?g/mL) and C15 (2 ?g/mL) demonstrated potent activity against ermB+mefA type resistant S. pneumoniae, which were 256,256,128,128, and 128-fold better than azithromycin (256 ?g/mL), respectively.Structure-activity relationships (SARs) based on the MIC value analysis of compound A-E were summarized as below:1. Compound B8 showed the best antibacterial activity against susceptible B. subtilis (0.5 ?g/mL), S. epidermidis (0.125 ?g/mL), ermB resistant S. pneumoniae (1 ?g/mL) and ermB+mefA type resistant S. pneumoniae (1 ?g/mL). We speculated that its 11-O-arylalkylcarbamoyl group could interact with A752, and its 3-O-arylalkylcarbamoyl group could interact with nucleotide binding sites near the PTC region. The interactions, such as hydrogen bonding, electrostatic force and ?-stacking, increased affinity for the bacteria ribosome, thus providing a remarkable improvement in activity against the susceptible and resistant strains, especially the ermB and ermB +mefA resistant S. pneumoniae.2. For susceptible S. aureus, B. subtilis, S. epidermidis, ermB+mefA type resistant S. pneumoniae and resistant S. pyogenes,3-O-hexylcarbamoyl-descladinosyl azithromycin 11,12-carbonate D8 had better antibacterial activity than 3-O-pentylcarbamoyl-descladinosyl azithromycin 11,12-carbonate D7. And compound B8 with the 3-O-hexylcarbamoyl side chain also exhibited excellent activity against the above five strains. So the terminal hexyl group of C-3 side chain was favorable for better activity against the five strains. This indicated that the 3-O-hexylcarbamoyl side chain had suitble length and steric configuration to interact with nucleotides in the PTC region.3. The antibacterial activity evaluation indicated that compounds C3, C6-C7, C11-C13, D7-D8, E2-E4 and E7-E9 showed the MIC values of 16-64 ?g/mL, which were 4-16 fold better than that of azithromycin. We speculated that 2'-OH may interact with A2058 (A2041) or the nucleotides nearby through hydrophobic interaction and hydrophobic interaction.In conclusion, we designed and sythesized 71 of descladinosylazithromycin carbamate derivatives and evaluated their in vitro activity against susceptible and resistant strains. Based on this, we summarized their SAR.