1. 1 © Patrick An Introduction to Medicinal Chemistry 3/e Chapter 16 ANTIBACTERIAL AGENTS Part 1: Penicillins

2. 1 © PENICILLINS

3. 1 © INTRODUCTION Antibacterial agents which inhibit bacterial cell wall synthesisAntibacterial agents which inhibit bacterial cell wall synthesis Discovered by Fleming from a fungal colony (1928)Discovered by Fleming from a fungal colony (1928) Shown to be non toxic and antibacterialShown to be non toxic and antibacterial Isolated and purified by Florey and Chain (1938)Isolated and purified by Florey and Chain (1938) First successful clinical trial (1941)First successful clinical trial (1941) Produced by large scale fermentation (1944)Produced by large scale fermentation (1944) Structure established by X-Ray crystallography (1945)Structure established by X-Ray crystallography (1945) Full synthesis developed by Sheehan (1957)Full synthesis developed by Sheehan (1957) Isolation of 6-APA by Beechams (1958-60) - development of semi-synthetic penicillinsIsolation of 6-APA by Beechams (1958-60) - development of semi-synthetic penicillins Discovery of clavulanic acid and  -lactamase inhibitorsDiscovery of clavulanic acid and  -lactamase inhibitors

4. 1 © Acyl side chain 6-Aminopenicillanic acid (6-APA) STRUCTURE Side chain varies depending on carboxylic acid present in fermentation medium  -Lactam ring Thiazolidinering present in corn steep liquor Penicillin G Benzyl penicillin (Pen G) R = Phenoxymethyl penicillin (Pen V) R = Penicillin V (first orally active penicillin)

5. 1 © Shape of Penicillin G Folded ‘envelope’ shape

6. 1 © CYS VAL Biosynthesis of Penicillins

7. 1 © Properties of Penicillin G Active vs. Gram +ve bacilli and some Gram -ve cocciActive vs. Gram +ve bacilli and some Gram -ve cocci Non toxicNon toxic Limited range of activityLimited range of activity Not orally active - must be injectedNot orally active - must be injected Sensitive to  -lactamases (enzymes which hydrolyse the  -lactam ring)Sensitive to  -lactamases (enzymes which hydrolyse the  -lactam ring) Some patients are allergicSome patients are allergic Inactive vs. StaphylococciInactive vs. Staphylococci Drug Development Aims To increase chemical stability for oral administrationTo increase chemical stability for oral administration To increase resistance to  -lactamasesTo increase resistance to  -lactamases To increase the range of activityTo increase the range of activity

8. 1 © SAR Conclusions Amide and carboxylic acid are involved in bindingAmide and carboxylic acid are involved in binding Carboxylic acid binds as the carboxylate ionCarboxylic acid binds as the carboxylate ion Mechanism of action involves the  -lactam ringMechanism of action involves the  -lactam ring Activity related to  -lactam ring strainActivity related to  -lactam ring strain (subject to stability factors) Bicyclic system increases  -lactam ring strainBicyclic system increases  -lactam ring strain Not much variation in structure is possibleNot much variation in structure is possible Variations are limited to the side chain (R)Variations are limited to the side chain (R)

9. 1 © Penicillins inhibit a bacterial enzyme called the transpeptidase enzyme which is involved in the synthesis of the bacterial cell wallPenicillins 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 inhibitionThe  -lactam ring is involved in the mechanism of inhibition Penicillin becomes covalently linked to the enzyme’s active site leading to irreversible inhibitionPenicillin becomes covalently linked to the enzyme’s active site leading to irreversible inhibition Covalent bond formed to transpeptidase enzyme Irreversible inhibition Mechanism of action

10. 1 © L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys L-Ala D-Glu L-Lys Mechanism of action - bacterial cell wall synthesis NAM NAG NAM NAG NAM NAG Bond formation inhibited by penicillin

11. 1 © Cross linking Mechanism of action - bacterial cell wall synthesis

12. 1 © Penicillin inhibits final crosslinking stage of cell wall synthesisPenicillin inhibits final crosslinking stage of cell wall synthesis It reacts with the transpeptidase enzyme to form an irreversible covalent bondIt reacts with the transpeptidase enzyme to form an irreversible covalent bond Inhibition of transpeptidase leads to a weakened cell wallInhibition of transpeptidase leads to a weakened cell wall Cells swell due to water entering the cell, then burst (lysis)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 portionPenicillin 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

13. 1 © Alternative theory- Pencillin mimics D-Ala-D-Ala. Normal Mechanism Mechanism of action - bacterial cell wall synthesis

14. 1 © Alternative theory- Pencillin mimics D-Ala-D-Ala. Mechanism inhibited by penicillin Mechanism of action - bacterial cell wall synthesis

15. 1 © Penicillin can be seen to mimic acyl-D-Ala-D-Ala But 6-methylpenicillin is inactive despite being a closer analogue PenicillinAcyl-D-Ala-D-Ala Mechanism of action - bacterial cell wall synthesis

16. 1 © Penicillin may act as an ‘umbrella’ inhibitor Mechanism of action - bacterial cell wall synthesis HO OH Active Site D-Ala-D-Ala Blocked Acylation OH O

17. 1 © Resistance to Penicillins Penicillins have to cross the bacterial cell wall in order to reach their target enzymePenicillins have to cross the bacterial cell wall in order to reach their target enzyme But cell walls are porous and are not a barrierBut 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 penicillinsThe cell walls of Gram +ve bacteria are thicker than Gram - ve cell walls, but the former are more susceptible to penicillins

18. 1 © Resistance to Penicillins Cell Cell membrane Thick porous cell wall Gram +ve bacteria Thick cell wallThick cell wall No outer membraneNo outer membrane More susceptible to penicillinsMore susceptible to penicillins

19. 1 © Resistance to Penicillins L Cell membrane Thin cell wall Lactamase enzymes Outer membrane Hydrophobic barrier Periplasmic space Porin L L L Gram -ve bacteria Thin cell wallThin cell wall Hydrophobic outer membraneHydrophobic outer membrane More resistant to penicillinsMore resistant to penicillins

20. 1 © Resistance to Penicillins Factors Gram -ve bacteria have a lipopolysaccharide outer membrane preventing access to the cell wallGram -ve bacteria have a lipopolysaccharide outer membrane preventing access to the cell wall Penicillins can only cross via porins in the outer membranePenicillins can only cross via porins in the outer membrane Porins only allow small hydrophilic molecules that can exist as zwitterions to crossPorins only allow small hydrophilic molecules that can exist as zwitterions to cross High levels of transpeptidase enzyme may be presentHigh levels of transpeptidase enzyme may be present The transpeptidase enzyme may have a low affinity for penicillins (e.g. PBP 2a for S. aureus)The transpeptidase enzyme may have a low affinity for penicillins (e.g. PBP 2a for S. aureus) Presence of  -lactamasesPresence of  -lactamases Concentration of  -lactamases in periplasmic spaceConcentration of  -lactamases in periplasmic space MutationsMutations Transfer of  -lactamases between strainsTransfer of  -lactamases between strains Efflux mechanisms pumping penicillin out of periplasmic spaceEfflux mechanisms pumping penicillin out of periplasmic space

21. 1 © Penicillin Analogues - Preparation 1) By fermentation vary the carboxylic acid in the fermentation mediumvary the carboxylic acid in the fermentation medium limited to unbranched acids at the  -position i.e. RCH 2 CO 2 Hlimited to unbranched acids at the  -position i.e. RCH 2 CO 2 H tedious and slowtedious and slow 2) By total synthesis only 1% overall yield (impractical)only 1% overall yield (impractical) 3) By semi-synthetic procedures Use a naturally occurring structure as the starting material for analogue synthesisUse a naturally occurring structure as the starting material for analogue synthesis

22. 1 © Penicillin Analogues - Preparation Fermentation Penicillin acylase or chemical hydrolysis Semi-synthetic penicillins

23. 1 © 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 PCl 5 requires involvement of nitrogen’s lone pair of electrons. Not possible for the  -lactam nitrogen.

24. 1 © Problems with Penicillin G It is sensitive to stomach acidsIt is sensitive to stomach acids It is sensitive to  -lactamases - enzymes which hydrolyse the  -lactam ringIt is sensitive to  -lactamases - enzymes which hydrolyse the  -lactam ring it has a limited range of activity it has a limited range of activity

25. 1 © Problem 1 - Acid Sensitivity Reasons for sensitivity 1) Ring Strain Relieves ring strain Acid or enzyme

26. 1 © Problem 1 - Acid 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 possibleInteraction of nitrogen’s lone pair with the carbonyl group is not possible Results in a reactive carbonyl groupResults in a reactive carbonyl group Tertiary amide Unreactive  -Lactam Folded ring system Impossibly strained Reasons for sensitivity X

27. 1 © Problem 1 - Acid Sensitivity 3) Acyl Side Chain - neighbouring group participation in the hydrolysis mechanism Further reactions Reasons for sensitivity

28. 1 © Problem 1 - Acid Sensitivity Conclusions The  -lactam ring is essential for activity and must be retainedThe  -lactam ring is essential for activity and must be retained Therefore, cannot tackle factors 1 and 2Therefore, cannot tackle factors 1 and 2 Can only tackle factor 3Can only tackle factor 3 Strategy Vary the acyl side group (R) to make it electron withdrawing to decrease the nucleophilicity of the carbonyl oxygen Decreasesnucleophilicity

29. 1 © Penicillin V (orally active) Problem 1 - Acid Sensitivity Examples electronegativeoxygen Very successful semi- synthetic penicillinsVery successful semi- synthetic penicillins e.g. ampicillin, oxacillin Better acid stability and orally activeBetter acid stability and orally active But sensitive to  -lactamasesBut sensitive to  -lactamases Slightly less active than Penicillin GSlightly less active than Penicillin G Allergy problems with some patientsAllergy problems with some patients

30. 1 © Problem 2 - Sensitivity to b-Lactamases Notes on  -Lactamases Enzymes that inactivate penicillins by opening  -lactam ringsEnzymes that inactivate penicillins by opening  -lactam rings Allow bacteria to be resistant to penicillinAllow bacteria to be resistant to penicillin Transferable between bacterial strains (i.e. bacteria can acquire resistance)Transferable between bacterial strains (i.e. bacteria can acquire resistance) Important w.r.t. Staphylococcus aureus infections in hospitalsImportant w.r.t. Staphylococcus aureus infections in hospitals 80% Staph. infections in hospitals were resistant to penicillin and other antibacterial agents by 196080% 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 enzymeMechanism 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 But product is removed efficiently from the lactamase active site  -Lactamase

31. 1 ©Bulkygroup Problem 2 - Sensitivity to b-Lactamases Strategy Enzyme Block access of penicillin to active site of enzyme by introducing bulky groups to the side chain to act as steric shieldsBlock access of penicillin to active site of enzyme by introducing bulky groups to the side chain to act as steric shields Size of shield is crucial to inhibit reaction of penicillins with  - lactamases but not with the target enzyme (transpeptidase)Size of shield is crucial to inhibit reaction of penicillins with  - lactamases but not with the target enzyme (transpeptidase)

32. 1 © ortho groups important Problem 2 - Sensitivity to b-Lactamases Examples - Methicillin (Beechams - 1960) Methoxy groups block access to  -lactamases but not to transpeptidasesMethoxy groups block access to  -lactamases but not to transpeptidases Active against some penicillin G resistant strains (e.g. Staphylococcus)Active against some penicillin G resistant strains (e.g. Staphylococcus) Acid sensitive (no e-withdrawing group) and must be injectedAcid sensitive (no e-withdrawing group) and must be injected Lower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced accessLower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced access to transpeptidase) Poorer range of activityPoorer range of activity Poor activity vs. some streptococciPoor activity vs. some streptococci Inactive vs. Gram -ve bacteriaInactive vs. Gram -ve bacteria

33. 1 © Problem 2 - Sensitivity to b-Lactamases Examples - Oxacillin Orally active and acid resistantOrally active and acid resistant Resistant to  -lactamasesResistant to  -lactamases Active vs. Staphylococcus aureusActive vs. Staphylococcus aureus Less active than other penicillinsLess active than other penicillins Inactive vs. Gram -ve bacteriaInactive vs. Gram -ve bacteria Nature of R & R’ influences absorption and plasma protein bindingNature of R & R’ influences absorption and plasma protein binding Cloxacillin better absorbed than oxacillinCloxacillin better absorbed than oxacillin Flucloxacillin less bound to plasma protein, leading to higherFlucloxacillin 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

34. 1 © 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 strategyThe number of factors involved make a single strategyimpossible Use trial and error by varying R groups on the side chainUse trial and error by varying R groups on the side chain Successful in producing broad spectrum antibioticsSuccessful in producing broad spectrum antibiotics Results demonstrate general rules for broad spectrum activity.Results demonstrate general rules for broad spectrum activity.

35. 1 © Problem 3 - Range of Activity 1.R= hydrophobic results 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. NH 2, OH, CO 2 H) increase activity vs Gram -ve bacteria Results of varying R in Pen G

36. 1 © Problem 3 - Range of Activity Examples of Broad Spectrum Penicillins Class 1 - NH 2 at the  -position Ampicillin and Amoxycillin (Beechams, 1964) Ampicillin (Penbritin) 2nd most used penicillin Amoxycillin (Amoxil)

37. 1 © Problem 3 - Range of Activity Active vs Gram +ve bacteria and Gram -ve bacteria which do not produce  -lactamasesActive vs Gram +ve bacteria and Gram -ve bacteria which do not produce  -lactamases Acid resistant and orally activeAcid resistant and orally active Non toxicNon toxic Sensitive to  -lactamasesSensitive to  -lactamases Increased polarity due to extra amino groupIncreased polarity due to extra amino group Poor absorption through the gut wallPoor absorption through the gut wall Disruption of gut flora leading to diarrhoeaDisruption of gut flora leading to diarrhoea Inactive vs. Pseudomonas aeruginosaInactive vs. Pseudomonas aeruginosa Examples of Broad Spectrum Penicillins Properties

38. 1 © Problem 3 - Range of Activity Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969) Properties Increased cell membrane permeabilityIncreased cell membrane permeability Polar carboxylic acid group is masked by the esterPolar carboxylic acid group is masked by the ester Ester is metabolised in the body by esterases to give the free drugEster is metabolised in the body by esterases to give the free drug

39. 1 © Problem 3 - Range of Activity Mechanism Ester is less shielded by penicillin nucleusEster is less shielded by penicillin nucleus Hydrolysed product is chemically unstable and degradesHydrolysed product is chemically unstable and degrades Methyl ester of ampicillin is not hydrolysed in theMethyl ester of ampicillin is not hydrolysed in the body - bulky penicillin nucleus acts as a steric shield PEN COCH 2 O O H COCH 2 O O C O CMe 3 Formaldehyde COH O

40. 1 © Problem 3 - Range of Activity Examples of Broad Spectrum Penicillins Class 2 - CO 2 H at the  -position (carboxypenicillins) Examples Carfecillin = prodrug for carbenicillinCarfecillin = prodrug for carbenicillin Active over a wider range of Gram -ve bacteria than ampicillinActive over a wider range of Gram -ve bacteria than ampicillin Active vs. Pseudomonas aeruginosaActive vs. Pseudomonas aeruginosa Resistant to most  -lactamasesResistant to most  -lactamases Less active vs Gram +ve bacteria (note the hydrophilic group)Less active vs Gram +ve bacteria (note the hydrophilic group) Acid sensitive and must be injectedAcid sensitive and must be injected Stereochemistry at the  -position is importantStereochemistry at the  -position is important CO 2 H at the  -position is ionised at blood pHCO 2 H at the  -position is ionised at blood pH R = H CARBENICILLIN R = Ph CARFECILLIN

41. 1 © Problem 3 - Range of Activity Examples of Broad Spectrum Penicillins Class 2 - CO 2 H at  -position (carboxypenicillins) Examples Administered by injectionAdministered by injection Identical antibacterial spectrum to carbenicillinIdentical antibacterial spectrum to carbenicillin Smaller doses required compared to carbenicillinSmaller doses required compared to carbenicillin More effective against P. aeruginosaMore effective against P. aeruginosa Fewer side effectsFewer side effects Can be administered with clavulanic acidCan be administered with clavulanic acid TICARCILLIN

42. 1 © Problem 3 - Range of Activity Examples of Broad Spectrum Penicillins Class 3 - Urea group at the  -position (ureidopenicillins) Examples Administered by injectionAdministered by injection Generally more active than carboxypenicillins vs. streptococci and Haemophilus speciesGenerally more active than carboxypenicillins vs. streptococci and Haemophilus species Generally have similar activity vs Gram -ve aerobic rodsGenerally have similar activity vs Gram -ve aerobic rods Generally more active vs other Gram -ve bacteriaGenerally more active vs other Gram -ve bacteria Azlocillin is effective vs P. aeruginosaAzlocillin is effective vs P. aeruginosa Piperacillin can be administered alongside tazobactamPiperacillin can be administered alongside tazobactam Azlocillin Mezlocillin Piperacillin