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Home , Bacampicillin, Cephaloridine, Cephalosporin, Cephalosporin C, Cephamycin, Flucloxacillin

Topic 8-1 Antibacterial Agents

β-Lactam -Chapter 16 Patrick PENICILLINSPENICILLINS

O H C N H H S Me R

N Me O

CO2H INTRODUCTION

• Antibacterial agents which inhibit bacterial 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 • Discovery of and β-lactamase inhibitors STRUCTURE

R = O CH H H H 2 C N S Me Benzyl (Pen G) R 6-Aminopenicillanic acid N (6-APA) R = Acyl side Me chain O O CH 2 CO2H ring Phenoxymethyl penicillin (Pen V) β-Lactam ring

Side chain varies depending on present in fermentation medium

CH2 CO2H Penicillin G

present in corn steep liquor

OCH2 CO2H Penicillin V (first orally active penicillin) Shape of Penicillin G

O Me C R NH H S

Me

O N H H CO2H ..

Folded ‘envelope’ shape Biosynthesis of Penicillins

O H C N S Me R

N Me O

CO2H CYS VAL Properties of Penicillin G

• Active vs. Gram + bacilli and some Gram - cocci • Non toxic • Limited range of activity • Not orally active - must be injected • Sensitive to β-lactamases ( which hydrolyse the β-lactam ring) • Some patients are allergic • Inactive vs. Staphylococci

Drug Development Aims • To increase chemical stability for oral administration • To increase resistance to β-lactamases • To increase the range of activity SAR essential Cis Stereochemistry essential O H C N H H S Me R

N Me O Carboxylic acid essential !"Lactam essential CO2H

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

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

O O O H H H H H H H H H C N C N C N S Me S Me S Me R R R Enz-Nu O C HN Nu N Me -H N Me Me O O Nu-Enz CO H Enz CO2H H CO2H 2 Covalent bond formed OH to transpeptidase enzyme Irreversible inhibition Szer Mechanism of action - bacterial cell wall synthesis

NAM NAG NAM NAG NAM

L-Ala NAM NALG-Ala NAM NALG-Ala NAM

D-Glu D-Glu D-Glu L-Ala L-Ala L-Ala NAM NAG NAM NAG NAM L-Lys L-Lys L-Lys D-Glu D-Glu D-Glu L-Ala L-Ala L-Ala L-Lys L-Lys L-Lys D-Glu D-Glu D-Glu

Bond formation L-Lys L-Lys L-Lys inhibited by penicillin Mechanism of action - bacterial cell wall synthesis

NAM NAG NAM NAG SUGAR BACKBONE L-Ala L-Ala

D-Glu D-Glu

L-Lys Gly Gly Gly Gly Gly L-Lys Gly Gly Gly Gly Gly

D-Ala D-Ala

D-Ala D-Ala

PENICILLIN TRANSPEPTIDASE D-Alanine

NAM NAG NAM NAG SUGAR BACKBONE L-Ala L-Ala

D-Glu D-Glu

L-Lys Gly Gly Gly Gly Gly L-Lys Gly Gly Gly Gly Gly

D-Ala D-Ala Cross linking Mechanism of action - bacterial cell wall synthesis

• 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-γ-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 Alternative theory- Pencillin mimics D-Ala-D-Ala.

Normal Mechanism

Peptide Peptide Chain Chain Peptide Peptide Chain Peptide D-Ala Gly Chain Chain

Gly D-Ala D-Ala CO H 2 D-Ala OH OH O H Mechanism of action - bacterial cell wall synthesis Alternative theory- Pencillin mimics D-Ala-D-Ala.

Mechanism inhibited by penicillin

Peptide Chain Blocked Blocked H2O O O R C NH H O Gly S Me R C NH H R C NH H S S Me Me N Me O O O HN HN H Me CO2H Me O CO2H OH O CO2H

Blocked Irreversibly blocked Mechanism of action - bacterial cell wall synthesis Penicillin can be seen to mimic acyl-D-Ala-D-Ala R R H H C N H H C N Me S Me O O H H H N Me N O O CH3 CO2H CO2H Penicillin Acyl-D-Ala-D-Ala Resistance to Penicillins

• Penicillins have to cross the bacterial cell wall in order to reach their target enzyme

• But cell walls are porous and are not a barrier

• The cell walls of Gram + are thicker than Gram - cell walls, but the former are more susceptible to penicillins Resistance to Penicillins

Gram + bacteria

• Thick cell wall • No outer membrane • More susceptible to penicillins

Thick porous cell wall

Cell membrane

Cell Resistance to Penicillins

Gram - bacteria • Thin cell wall • Hydrophobic outer membrane • More resistant to penicillins

Hydrophobic barrier Outer membrane Porin

Lactamase L enzymes L L

Periplasmic Thin cell wall space L

Cell Cell membrane Resistance to Penicillins

Factors • Gram - bacteria have a lipopolysaccharide outer membrane preventing access to the cell wall • Penicillins can only cross via porins in the outer membrane • Porins only allow small hydrophilic molecules that can exist as zwitterions to cross • High levels of transpeptidase enzyme may be present • The transpeptidase enzyme may have a low affinity for penicillins • Presence of β-lactamases • Concentration of β-lactamases in periplasmic space • Transfer of β-lactamases between strains • Efflux mechanisms pumping penicillin out of periplasmic space Penicillin Analogues - Preparation

1) By fermentation • vary the carboxylic acid in the fermentation medium • limited to unbranched acids at the α-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 Penicillin Analogues - Preparation O H H C N S Me CH2 Penicillin G N Me O CO2H Penicillin acylase or chemical H H H N 2 S Fermentation Me

N Me O 6-APA CO2H O C R Cl O H H H C N S Me R Semi-synthetic penicillins N Me O CO2H Problems with Penicillin G

• It is sensitive to stomach acids • It is sensitive to β-lactamases - enzymes which hydrolyse the β- lactam ring • it has a limited range of activity

O H H C N S Me CH2 Penicillin G N Me O CO2H Problem 1 - Acid Sensitivity Reasons for sensitivity Factor 1) Ring Strain

O O O H H H H H Acid or H H H H C N C N C N S S S enzyme Me Me Me R R R HO HO2C N Me HN Me H2O N Me O O CO2H CO2H CO2H H Relieves ring strain Problem 1 - Acid Sensitivity Reasons for sensitivity Factor 2) Reactive β-lactam Does not behave like a tertiary amide

Tertiary amide R R R

C NR2 C N Unreactive

O O R

β-Lactam Me

S S Me Me

N N O CO2H X Me H O

Folded ring CO2H Impossibly system strained • Interaction of ’s lone pair with the carbonyl group is not possible • Results in a reactive carbonyl group Problem 1 - Acid Sensitivity Reasons for sensitivity Factor 3) Acyl Side Chain - neighbouring group participation in the hydrolysis mechanism

R H N N C N H R S R S S - O H O N O HN N O O O H

Further reactions Problem 1 - Acid Sensitivity Conclusions • The β-lactam ring is essential for activity and must be retained • Therefore, 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

H H E.W.G. N S C

O N Decreases O nucleophilicity Problem 1 - Acid Sensitivity

Examples X

H H ! H H PhO CH2 N HC N S S C R C

N O O N electronegative O O oxygen

Penicillin V X = NH2, Cl, PhOCONH, (orally active) Heterocycles, CO2H • Better acid stability and orally active • Very successful semi- • But sensitive to β-lactamases synthetic penicillins • Slightly less active than Penicillin G e.g. , • problems with some patients Problem 2 - Sensitivity to b-Lactamases Notes on β-Lactamases • Enzymes that inactivate penicillins by opening β-lactam rings • Allow bacteria to be resistant to penicillin • Transferable between bacterial strains (i.e. bacteria can acquire resistance) • Important to aureus 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

O O H H H H C N C N S Me S Me R R

HO C N Me 2 HN Me O β-Lactamase CO2H CO2H Problem 2 - Sensitivity to b-Lactamases

Strategy • Block 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 β- lactamases but not with the target enzyme (transpeptidase)

O Bulky H H H C N group S Me R

N Me Enzyme O CO2H Problem 2 - Sensitivity to b-Lactamases Examples - (Beechams - 1960)

O ortho groups H C N H H important MeO S Me

N OMe Me O

CO2H

• Methoxy groups block access to β-lactamases but not to transpeptidases • Active against some penicillin G resistant strains (e.g. Staphylococcus) • Acid sensitive (no e-withdrawing group) and must be injected • Lower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced access to transpeptidase) • Poorer range of activity • Poor activity vs. some streptococci • Inactive vs. Gram - bacteria Problem 2 - Sensitivity to β-Lactamases Examples - Oxacillin

R' O H H H Oxacillin R = R' = H C N S Me R = Cl, R' = H R R = Cl, R' = F N N Me O Me O Bulky and CO2H e- withdrawing

• Orally active and acid resistant • Resistant to β-lactamases • Active vs. • 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 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 β-lactamases 5. Transfer of β-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 antibiotics • Results demonstrate general rules for broad spectrum activity. Problem 3 - Range of Activity

Results of varying R in Pen G

1. R= hydrophobic results in high activity vs. Gram + bacteria and poor activity vs. Gram -ve bacteria 2. Increasing hydrophobicity has little effect on Gram + activity but lowers Gram -ve activity 3. Increasing hydrophilic character has little effect on Gram + activity but increases Gram - activity 4. Hydrophilic groups at the α-position (e.g. NH2, OH, CO2H) increase activity vs Gram - bacteria Problem 3 - Range of Activity

Examples of Broad Spectrum Penicillins

Class 1 - NH2 at the α-position Ampicillin and Amoxycillin (Beechams, 1964)

H NH2 H NH2

C HO C H H C N H C N H

O O

O O

Ampicillin (Penbritin) Amoxycillin (Amoxil) 2nd most used penicillin Problem 3 - Range of Activity

Examples of Broad Spectrum Penicillins

Properties • Active vs Gram + bacteria and Gram - bacteria which do not produce β-lactamases • Acid resistant and orally active • Non toxic • Still sensitive to β-lactamases • Increased polarity due to extra amino group • Poor absorption through the gut wall • Disruption of gut flora leading to • Inactive vs. aeruginosa Problem 3 - Range of Activity Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969)

O C H NH2 R = CH2O CMe3 C H O C N H H S R = O O Me

N Me O O

CO2R C R = CH O O CH2Me Me Properties • Increased cell membrane permeability • Polar carboxylic acid group is masked by the • Ester is metabolised in the body by esterases to give the free drug Problem 3 - Range of Activity Examples of Broad Spectrum Penicillins

Class 2 - CO2H at the α-position ()

CO R Examples 2 CH H R = H C N H H R = Ph CARFECILLIN O S Me

N Me O

CO2H

• Carfecillin = prodrug for carbenicillin • Active over a wider range of Gram -ve bacteria than ampicillin • Active vs. • Resistant to most β-lactamases • Less active vs Gram + bacteria (note the hydrophilic group) • Acid sensitive and must be injected • Stereochemistry at the α-position is important

• CO2H at the α-position is ionized at blood pH CEPHALOSPORINSCEPHALOSPORINS

O H H H R C N S

N OAc O

CO2H 1. Introduction

• Antibacterial agents which inhibit bacterial cell wall synthesis • Discovered from a fungal colony in Sardinian sewer water (1948) • C identified in 1961 2. Structure of

7-Aminoadipic side chain H H H H N N S 2 1 7 6 2 H CO H O 8 5 3 2 N 4 O Me O C O CO2H β-Lactam Dihydrothiazine ring ring

H H S H2N

N O Me O C O CO2H

7-Aminocephalosporinic acid (7-ACA) H H H H N N S 2 1 7 6 2 H CO H O 8 5 3 3. Properties of Cephalosporin C 2 N 4 O Me O C O CO2H

Disadvantages • Polar due to the side chain - difficult to isolate and purify • Low potency - limited to the treatment of urinary tract infections where it is concentrated in the urine • Not absorbed orally Advantages • Non toxic • Lower risk of allergic reactions compared to penicillins • More stable to acid conditions • More stable to β-lactamases • Ratio of activity vs Gram - and Gram + bacteria is better Conclusion • Useful as a lead compound 4. SAR of

H H H R N S 1 7 6 2 O 8 5 3 N 4 O Me O C O CO2H Similar to penicillins • The β-lactam ring is crucial to the mechanism • The carboxylic acid at position 4 is important to binding • The bicyclic system is important in increasing ring strain • Stereochemistry is important • The acetoxy substituent is important to the mechanism

Possible modifications • 7-Acylamino side chain • 3-Acetoxymethyl side chain • Substitution at C-7 5. First Generation Cephalosporins Cephalothin H H H N S 7 S O N 3 OAc O

CO2H • First generation cephalosporin • More active than penicillin G vs. some Gram - bacteria • Less likely to cause allergic reactions • Useful vs. penicillinase producing strains of S. aureus • Not active vs. Pseudonomas aeruginosa • Poorly absorbed from gut • Administered by injection • Metabolised to give a free 3-hydroxymethyl group (deacetylation) • Metabolite is less active 5. First Generation Cephalosporins Cephalothin - drug

H H H H H H N S N S 7 S O S O N 3 OAc N OH O Metabolism O

CO2H CO2H Less active OH is a poorer leaving group

Strategy • Replace the acetoxy group with a metabolically stable leaving group 5. First Generation Cephalosporins

H H H N S 7 S O N 3 N O

CO2 • The pyridine ring is stable to metabolism • The pyridine ring is a good leaving group (neutralisation of charge) • Exists as a zwitterion and is soluble in water • Poorly absorbed through the gut wall • Administered by injection 5. First Generation Cephalosporins H N H 2 H H H N S 7 O N 3 O Me

CO2H

• The methyl group at position 3 is not a good leaving group • The methyl group is bad for activity but aids oral absorption - mechanism unknown • Cefalexin can be administered orally • A hydrophilic amino group at the α- of the side chain helps to compensate for the loss of activity due to the methyl group 6. First Generation Cephalosporins Summary

• Generally lower activity than comparable penicillins • Better range of activity than comparable penicillins • Best activity is against Gram-positive cocci • Useful against some Gram negative infections • Useful against S. aureus and streptococcal infections when penicillins have to be avoided • Poorly absorbed across the gut wall (except for 3-methyl substituted cephalosporins) • Most are administered by injection • Resistance has appeared amongst Gram negative bacteria (presence of more effective β-lactamases) 6. Second Generation Cephalosporins

H OMe H S HO2C N

H N H 2 O C N O NH2 O C O CO2H • Isolated from a culture of Streptomyces clavuligerus • First β-lactam to be isolated from a bacterial source • Modifications carried out on the 7-acylamino side chain 6. Second Generation Cephalosporins 6 Cephamycins

H OMe H N S 7 S O 3 N O NH2 O C O CO2H

• Broader spectrum of activity than most first generation cephalosporins • Greater resistance to β-lactamase enzymes • The 7-methoxy group may act as a steric shield • The urethane group is stable to metabolism compared to the ester • Introducing a methoxy group to the equivalent position of penicillins (position 6) eliminates activity. 6. Second Generation Cephalosporins Oximinocephalosporins

Me O N H H H N S C O O N O NH2 O C O CO2H • Much greater stability against some β-lactamases • Resistant to esterases due to the urethane group • Wide spectrum of activity • Useful against organisms that have gained resistance to penicillin • Not active against P. aeruginosa • Used clinically against respiratory infections Newer β-Lactam Antibiotics (Merck 1976)(from Streptomyces cattleya) Acylamino side Opposite chain absent stereochemistry OH to penicillins Plays a role H Carbon in ß-lactamase H resistance H3C NH3 S N

O Double bond leading to CO2 high ring strain and an increase in !-lactam ring reactivity Carbapenam nucleus • Potent and wide range of activity vs Gram + and Gram - bacteria • Active vs. Pseudomonas aeruginosa • Low toxicity • High resistance to β-lactamases • Poor stability in solution (ten times less stable than Pen G) Newer β-Lactam Antibiotics Clinically useful

Me Me CO2H

O N H N N Me H2N S O N - O SO3

• Administered by intravenous injection • Can be used for patients with to penicillins and cephalosporins • No activity vs. Gram + or anaerobic bacteria • Active vs. Gram - aerobic bacteria β-Lactamase Inhibitors Clavulanic acid (Beechams 1976)(from Streptomyces clavuligerus)

Sulphur replaced by O No acylamino side chain H O 9 4 OH 6 5 3 7 1 N 2 H O H CO2H !-Lactam Oxazolidine ring • Weak, unimportant antibacterial activity • Powerful irreversible inhibitor of β-lactamases - suicide substrate • Used as a sentry drug for ampicillin • Augmentin = ampicillin + clavulanic acid • Allows less ampicillin per dose and an increased activity spectrum • Timentin = + clavulanic acid β-Lactamase Inhibitors Penicillanic acid sulfone derivatives

O O O O S S 1 Me Me 6 5 6 2 7 N N 3 Me N 3 N O O N CO2 Na CO2

• Suicide substrates for β-lactamase enzymes • Sulbactam has a broader spectrum of activity vs β-lactamases than clavulanic acid, but is less potent • Unasyn = ampicillin + sulbactam • Tazobactam has a broader spectrum of activity vs β-lactamases than clavulanic acid, and has similar potency • Tazocin or Zosyn = + tazobactam