medicinal chemistry penecilins

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INTRODUCTION TO PENICILLINS Antibacterial agents which inhibit bacterial cell wall synthesis Discovered by Fleming from a fungal colony (1928) Shown to be non toxic and antibacterial Isolated and purified by Florey and Chain (1938) First successful clinical trial (1941) Produced by large scale fermentation (1944) Structure established by X-ray crystallography (1945) Full synthesis developed by Sheehan (1957) Isolation of 6-APA by Beechams (1958-60) - development of semi-synthetic penicillins Discovery of clavulanic acid and -lactamase inhibitors

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STRUCTURE Side chain varies depending on carboxylic acid present in fermentation medium

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Shape of Penicillin G Folded ‘envelope’ shape

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Biosynthesis of Penicillins

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Properties of Penicillin G Active vs. Gram +ve bacilli and some Gram -ve cocci Non toxic Limited range of activity Not orally active - must be injected Sensitive to -lactamases (enzymes which hydrolyse the -lactam ring) Some patients are allergic Inactive vs. Staphylococci Drug Development Aims To increase chemical stability for oral administration To increase resistance to -lactamases To increase the range of activity

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SAR Conclusions Amide and carboxylic acid are involved in binding Carboxylic acid binds as the carboxylate ion Mechanism of action involves the-lactam ring Activity related to -lactam ring strain (subject to stability factors) Bicyclic system increases -lactam ring strain Not much variation in structure is possible Variations are limited to the side chain (R)

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Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in the synthesis of the bacterial cell wall The -lactam ring is involved in the mechanism of inhibition Penicillin becomes covalently linked to the enzyme’s active site leading to irreversible inhibition Mechanism of action

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Mechanism of action - bacterial cell wall synthesis

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Mechanism of action - bacterial cell wall synthesis

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Penicillin inhibits final crosslinking stage of cell wall synthesis It reacts with the transpeptidase enzyme to form an irreversible covalent bond Inhibition of transpeptidase leads to a weakened cell wall Cells swell due to water entering the cell, then burst (lysis) Penicillin possibly acts as an analogue of the L-Ala--D-Glu portion of the pentapeptide chain. However, the carboxylate group that is essential to penicillin activity is not present in this portion Mechanism of action - bacterial cell wall synthesis

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Alternative theory- Pencillin mimics D-Ala-D-Ala. Mechanism of action - bacterial cell wall synthesis

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Alternative theory- Pencillin mimics D-Ala-D-Ala. Mechanism of action - bacterial cell wall synthesis

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Penicillin can be seen to mimic acyl-D-Ala-D-Ala Mechanism of action - bacterial cell wall synthesis

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Penicillin may act as an ‘umbrella’ inhibitor Mechanism of action - bacterial cell wall synthesis

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Gram +ve and Gram -ve Cell Walls Penicillins have to cross the bacterial cell wall in order to reach their target enzyme Cell walls are porous and are not a barrier The cell walls of Gram +ve bacteria are thicker than Gram -ve cell walls, but the former are more susceptible to penicillins

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Gram +ve bacteria Gram +ve and Gram -ve Cell Walls

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Gram -ve bacteria Gram +ve and Gram -ve Cell Walls

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Resistance to Penicillins Factors Gram -ve bacteria have a lipopolysaccharide outer membrane preventing access to the cell wall Penicillins can only cross via porins in the outer membrane Porins allow small hydrophilic molecules such as zwitterions to cross High levels of transpeptidase enzyme may be present The transpeptidase enzyme may have a low affinity for penicillins (e.g. PBP 2a for S. aureus) Presence of -lactamases Concentration of -lactamases in periplasmic space Mutations Transfer of -lactamases between strains Efflux mechanisms pumping penicillin out of periplasmic space

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Penicillin Analogues - Preparation 1) By fermentation Vary the carboxylic acid in the fermentation medium Limited to unbranched acids at the -position i.e. RCH2CO2H Tedious and slow 2) By total synthesis Only 1% overall yield Impractical 3) By semi-synthetic procedures Use a naturally occurring structure as the starting material for analogue synthesis

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Penicillin Analogues - Preparation

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Penicillin Analogues - Preparation Problem - How does one hydrolyse the side chain by chemical means in presence of a labile -lactam ring? Answer - Activate the side chain first to make it more reactive Note - Reaction with PCl5 requires the involvement of a lone pair of electrons from nitrogen. Not possible for the -lactam nitrogen.

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Problems with Penicillin G It is sensitive to stomach acids It is sensitive to -lactamases - enzymes which hydrolyse the -lactam ring It has a limited range of activity

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Problem 1 - Acid Sensitivity Reasons for sensitivity 1) Ring strain

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Problem 1 - Sensitivity 2) Reactive -lactam carbonyl group Does not behave like a tertiary amide Interaction of nitrogen’s lone pair with the carbonyl group is not possible Results in a reactive carbonyl group Reasons for sensitivity X

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Problem 1 - Sensitivity Acyl side chain Neighboring group participation in the hydrolysis mechanism Reasons for sensitivity

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Problem 1 - Sensitivity Conclusions The -lactam ring is essential for activity and must be retained Cannot tackle factors 1 and 2 Can only tackle factor 3 Strategy Vary the acyl side group (R) to make it electron-withdrawing to decrease the nucleophilicity of the carbonyl oxygen

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Problem 1 - Sensitivity Examples

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Problem 2 - Sensitivity to -Lactamases -Lactamases Enzymes that inactivate penicillins by opening -lactam rings Allow bacteria to be resistant to penicillin Transferable between bacterial strains (i.e. bacteria can acquire resistance) Important with respect to Staphylococcus aureus infections in hospitals 80% Staph. infections in hospitals were resistant to penicillin and other antibacterial agents by 1960 Mechanism of action for lactamases is identical to the mechanism of inhibition for the target enzyme But product is removed efficiently from the lactamase active site

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Problem 2 - Sensitivity to -Lactamases Strategy Use of steric shields Block access of penicillin to the active site of the enzyme by introducing bulky groups to the side chain Size of shield is crucial to inhibit reaction of penicillins with-lactamases, but not with the target transpeptidase enzyme

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Problem 2 - Sensitivity to -Lactamases Examples - Methicillin (Beechams - 1960) Methoxy groups block access to -lactamases but not to transpeptidases Binds less readily to transpeptidases compared to penicillin G Lower activity compared to Pen G against Pen G sensitive bacteria Poor activity vs. some streptococci Inactive vs. Gram -ve bacteria Poor range of activity Active against some penicillin G resistant strains (e.g. Staphylococcus) Acid sensitive since there is no electron-withdrawing group Orally inactive and must be injected

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Problem 2 - Sensitivity to -Lactamases Examples - Oxacillin Orally active and acid resistant Resistant to -lactamases Active vs. Staphylococcus aureus Less active than other penicillins Inactive vs. Gram -ve bacteria Nature of R & R’ influences absorption and plasma protein binding Cloxacillin better absorbed than oxacillin Flucloxacillin less bound to plasma protein, leading to higher levels of free drug Oxacillin R = R' = H Cloxacillin R = Cl, R' = H Flucloxacillin R = Cl, R' = F

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Problem 3 - Range of Activity Factors 1) Cell wall may have a coat preventing access to the cell 2) Excess transpeptidase enzyme may be present 3) Resistant transpeptidase enzyme (modified structure) 4) Presence of -lactamases 5) Transfer of -lactamases between strains 6) Efflux mechanisms Strategy The number of factors involved make a single strategy impossible Use trial and error by varying R groups on the side chain Successful in producing broad spectrum antibiotics Results demonstrate general rules for broad spectrum activity.

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Problem 3 - Range of Activity 1) Hydrophobic side chains result in high activity vs. Gram +ve bacteria and poor activity vs. Gram -ve bacteria 2) Increasing hydrophobicity has little effect on Gram +ve activity but lowers Gram -ve activity 3) Increasing hydrophilic character has little effect on Gram +ve activity but increases Gram -ve activity 4) Hydrophilic groups at the -position (e.g. NH2, OH, CO2H) increase activity vs Gram -ve bacteria Results of varying R in Pen G

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Problem 3 - Range of Activity Examples of Broad Spectrum Penicillins Class 1 - NH2 at the -position Ampicillin and amoxicillin (Beechams, 1964) Ampicillin (Penbritin) 2nd most used penicillin Amoxicillin (Amoxil)

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Problem 3 - Range of Activity Active vs Gram +ve bacteria and Gram -ve bacteria which do not produce -lactamases Acid resistant and orally active Non toxic Sensitive to -lactamases Increased polarity due to extra amino group Poor absorption through the gut wall Disruption of gut flora leading to diarrhea Inactive vs. Pseudomonas aeruginosa Examples of Broad Spectrum Penicillins Properties

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Problem 3 - Range of Activity Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969) Properties Increased cell membrane permeability Polar carboxylic acid group is masked by the ester Ester is metabolised in the body by esterases to give the free drug

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Problem 3 - Range of Activity Mechanism of prodrug activation Extended ester is less shielded by the penicillin nucleus Hydrolysed product is chemically unstable and degrades Methyl ester of ampicillin is not hydrolysed in the body Bulky penicillin nucleus acts as a steric shield for methyl ester

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Problem 3 - Range of Activity Examples of broad spectrum penicillins Class 2 - CO2H at the -position (carboxypenicillins) Examples Carfecillin = prodrug for carbenicillin Active over a wider range of Gram -ve bacteria than ampicillin Active vs. Pseudomonas aeruginosa Resistant to most -lactamases Less active vs Gram +ve bacteria (note the hydrophilic group) Acid sensitive and must be injected Stereochemistry at the -position is important CO2H at the -position is ionised at blood pH R = H Carbenicillin R = Ph Carfecillin

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Problem 3 - Range of Activity Class 2 - CO2H at the -position (carboxypenicillins) Examples Administered by injection Identical antibacterial spectrum to carbenicillin Smaller doses required compared to carbenicillin More effective against P. aeruginosa Fewer side effects Can be administered with clavulanic acid Examples of broad spectrum penicillins

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Problem 3 - Range of Activity Administered by injection Generally more active than carboxypenicillins vs. streptococci and Haemophilus species Generally have similar activity vs Gram -ve aerobic rods Generally more active vs other Gram -ve bacteria Azlocillin is effective vs P. aeruginosa Piperacillin can be administered alongside tazobactam Examples of broad spectrum penicillins

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