Patrick: An Introduction

to Medicinal Chemistry 5e
Chapter 19

PENICILLINS
O
H
C N H H
S Me
R

N Me
O
CO2H

© Oxford University Press, 2013

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 b-lactamase inhibitors

© Oxford University Press, 2013

 STRUCTURE
R=
O
CH2 H H H
C N
S Me
Benzyl penicillin (Pen G) R 6-Aminopenicillanic acid
N (6-APA)
R= Acyl side Me
chain O
O CH2 CO2H Thiazolidine
ring
Phenoxymethyl penicillin (Pen V) b-Lactam
ring

Side chain varies depending on carboxylic acid present in fermentation medium

CH2 CO2H Penicillin G

present in corn steep liquor

OCH2 CO2H Penicillin V

(first orally active penicillin)
© Oxford University Press, 2013
Shape of Penicillin G

O
Me
C H
R NH
S

Me

O N CO2H
H H
..

Folded ‘envelope’ shape

© Oxford University Press, 2013

Biosynthesis of Penicillins

O
H
C N
S Me
R

N
Me
O
CO2H

CYS VAL

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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 b-lactamases
(enzymes which hydrolyse the b-lactam ring)
•Some patients are allergic
•Inactive vs. Staphylococci

Drug Development
Aims
•To increase chemical stability for oral administration
•To increase resistance to b-lactamases
•To increase the range of activity
© Oxford University Press, 2013
 SAR
Amide essential
Cis S tereo ch em istry essen tia l
O
H
C N H H
S Me
R

N Me
O
Ca r b o x y l i c aci d e s s e nt i
b-Lactam essential
CO2H

Bicyclic system essential

Conclusions
•Amide and carboxylic acid are involved in binding
•Carboxylic acid binds as the carboxylate ion
•Mechanism of action involves the b-lactam ring
•Activity related to b-lactam ring strain (subject to stability factors)
•Bicyclic system increases b-lactam ring strain
•Not much variation in structure is possible
•Variations are limited to the side chain (R) © Oxford University Press, 2013
 Mechanism of action
•Penicillins inhibit a bacterial enzyme called the transpeptidase
enzyme which is involved in the synthesis of the bacterial cell wall
•The b-lactam ring is involved in the mechanism of inhibition
•Penicillin becomes covalently linked to the enzyme’s active site by
means of an ester link to a serine residue
•Penicillin is not split in two and acts as a steric shield to prevent
access of substrate or water to the active site
• Results in 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 -H+ R R
Enz-Nu
N N O C HN
Nu Me Me Me
O O Nu-Enz
Enz CO2H H CO2H CO2H

•Covalent bond formed

to transpeptidase enzyme
•Irreversible inhibition
© Oxford University Press, 2013
 Mechanism of action - bacterial cell wall synthesis

NAM NAG NAM NAG NAM

L-Ala NAM L-Ala

NAG NAM L-Ala
NAG 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

L-Lys L-Lys L-Lys

Bond formation
inhibited by
penicillin

© Oxford University Press, 2013

Mechanism of action - bacterial cell wall synthesis
NAM NAG
SUGAR BACKBONE
L-Ala NAM NAG
SUGAR BACKBONE
D-Glu L-Ala

L-Lys Gly Gly Gly Gly Gly D-Glu

D-Ala L-Lys Gly Gly Gly Gly Gly

D-Ala D-Ala

D-Ala

TRANSPEPTIDASE PENICILLIN
D-Alanine

NAM NAG
SUGAR BACKBONE
L-Ala NAM NAG
SUGAR BACKBONE
D-Glu L-Ala

L-Lys Gly Gly Gly Gly Gly D-Glu

D-Ala L-Lys Gly Gly Gly Gly Gly

Cross linking D-Ala

© Oxford University Press, 2013

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 ester
linkage with a serine residue
• The ring-opened penicillin acts as a steric shield
• Neither substrate nor water is capable of reaching the ester link
• Results in irreversible inhibition
•Inhibition of transpeptidase leads to a weakened cell wall
•Cells swell due to water entering the cell, then burst (lysis)
•Penicillin thought to mimic D-Ala-D-Ala

© Oxford University Press, 2013

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

Pe ptide
Pe ptide Chain
Chain
Peptide
Peptide Chain
Peptide D-Ala Gly
Chain
Chain

Gly
D-Ala D-Ala CO 2H
D-Ala
OH
OH O H

© Oxford University Press, 2013

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

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

CO2H O CO2H
OH O

Blocked Irreversibly blo cked

© Oxford University Press, 2013

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 N
Me
O O CH3
CO2H CO2H

Penicillin Acyl-D-Ala-D-Ala

© Oxford University Press, 2013

Gram +ve and Gram -ve Cell Walls

•Penicillins have to cross peptidoglycan layers in order to reach

their target enzyme

•Peptidoglycan layers are porous and are not a barrier

•The peptidoglycan layers of Gram +ve bacteria are thicker

than Gram -ve cell walls, but the former are more susceptible to
penicillins

© Oxford University Press, 2013

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

Thick porous cell wall

Cell membrane

Cell

•Thick porous peptidoglycan cell wall

•No outer membrane
•Penicillins cross cell wall easily
•Gram +ve more susceptible to penicillins
© Oxford University Press, 2013
Gram +ve and Gram -ve Cell Walls
Gram -ve bacteria

Hydrophobic barrier
Outer
membrane Porin

Lactamase
L enzymes L
L
Periplasmic Thin peptidoglycan layer
space
L

Cell
Cell
membrane

•Thin peptidoglycan layer

•Hydrophobic outer membrane
- acts as a barrier to penicillins
•Gram –ve more resistant to penicillins
© Oxford University Press, 2013
Resistance to Penicillins
Factors
•Gram -ve bacteria have a lipopolysaccharide outer membrane
preventing access to the periplasmic space
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 and concentration of b-lactamases in the periplasmic
space
•Efflux mechanisms pumping penicillins out of the periplasmic
space
•Transfer of b-lactamases between strains
•Mutations

© Oxford University Press, 2013

Penicillin Analogues - Preparation

1) By fermentation
•Vary the carboxylic acid in the fermentation medium
•Limited to unbranched acids at the a-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

© Oxford University Press, 2013

Penicillin Analogues - Preparation
O
H H
C N
S Me
CH2 Penicillin G
N
Me
O
CO2H
Penicillin acylase
or chemical hydrolysis
H H
H2N
S Me
Fermentation
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
© Oxford University Press, 2013
Penicillin Analogues - Preparation
Problem - How does one hydrolyse the side chain by chemical
means in presence of a labile b-lactam ring?

Answer - Activate the side chain first to make it more reactive

O
PhCH2 C NH Cl OR
S PCl5 ROH H2O
PhCH2 C N PhCH2 C N 6- APA
N PEN PEN
O
CO2H

Note - Reaction with PCl5 requires the involvement of a lone pair of electrons
from nitrogen. Not possible for the b-lactam nitrogen.

© Oxford University Press, 2013

Problems with Penicillin G
•It is sensitive to stomach acids

•It is sensitive to b-lactamases - enzymes which hydrolyse the b-

lactam ring

• It has a limited range of activity

© Oxford University Press, 2013

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

O O
O
H H H Acid or H
N
H H
C
H
N
H H
C
C N
S Me enzyme S Me
R
S Me
R R
HO HO2C
N Me HN Me
H2O N Me
O O
CO2H CO2H
CO2H H

Relieves ring strain

© Oxford University Press, 2013

 Problem 1 - Acid Sensitivity
Reasons for sensitivity
2) Reactive b-lactam carbonyl group
Does not behave like a tertiary amide

Tertiary amide
R R
R

C NR2 C N Unreactive
O O R

b-Lactam Me

S S
Me
Me

O
N
H
CO2H X O
N Me

Folded ring CO2H

Impossibly
system strained

•Interaction of nitrogen’s lone pair with the carbonyl group is not possible
•Results in a reactive carbonyl group
© Oxford University Press, 2013
Problem 1 - Acid Sensitivity
Reasons for sensitivity
3) Acyl side chain
Neighbouring group participation in the hydrolysis mechanism

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

Further
reactions

© Oxford University Press, 2013

Problem 1 - Acid Sensitivity
Conclusions
•The b-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

H H
E.W.G. N
S
C

O N
O
Decreases
nucleophilicity

© Oxford University Press, 2013

 Problem 1 - Acid Sensitivity

Examples
X
H H
PhO CH2 N HC
a H H
S N
C S
R C

O N N
electronegative O
O
O
oxygen
Penicillin V X= NH2, Cl, PhOCONH,
(orally active) heterocycles. CO2H

•Better acid stability and orally active Very successful semi-synthetic

•But sensitive to b-lactamases penicillins
•Slightly less active than penicillin G e.g. ampicillin, oxacillin
•Allergy problems with some patients

© Oxford University Press, 2013

Problem 2 - Sensitivity to b-Lactamases
b-Lactamases
•Enzymes that inactivate penicillins by opening b-lactam rings
•Allow bacteria to be resistant to penicillin
•Transferable between bacterial strains (i.e. bacteria can acquire
resistance)
•Important w.r.t. 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
O O
H H H H
C N C N
S Me S Me
R R

N Me HO2C HN Me
O b-Lactamase
CO2H CO2H
© Oxford University Press, 2013
Problem 2 - Sensitivity to b-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 b-
lactamases, but not with the target transpeptidase enzyme
O
Bulky H H H
C N
group S Me
R

N Me
Enzyme O
CO2H

© Oxford University Press, 2013

Problem 2 - Sensitivity to b-Lactamases
Examples - Methicillin (Beechams - 1960)
O
ortho groups H
H H
important MeO
C N
S Me

N Me
OMe
O
CO2H

•Methoxy groups block access to b-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
•Poorer 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 © Oxford University Press, 2013
 Problem 2 - Sensitivity to b-Lactamases
Examples

R'
O
H H H Oxacillin R = R' = H
C N
S Me Cloxacillin R = Cl, R' = H
R
N
Flucloxacillin R = Cl, R' = F
N Me
O Me
O
Bulky and CO2H
electron withdrawing

•Orally active and acid resistant

•Resistant to b-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
© Oxford University Press, 2013
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 b-lactamases
5) Transfer of b-lactamases between strains
6) Efflux mechanisms

Strategy
•The number of factors involved make a single strategy impossible
•Use of trial and error to vary R groups on the side chain
•Successful in producing broad spectrum antibiotics
•Results demonstrate general rules for broad spectrum activity.

© Oxford University Press, 2013

 Problem 3 - Range of Activity

Results of varying R in Pen G

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 a-position (e.g. NH2, OH, CO2H)
increase activity vs Gram -ve bacteria

© Oxford University Press, 2013

Problem 3 - Range of Activity
Examples of Broad Spectrum Penicillins

Class 1 - NH2 at the a-position

Ampicillin and amoxicillin (Beechams, 1964)

H NH2 H NH2

C HO C
H H
C N H C N H

O O

O O

Ampicillin (Penbritin) Amoxicillin (Amoxil)

2nd most used penicillin

© Oxford University Press, 2013

Problem 3 - Range of Activity
Examples of Broad Spectrum Penicillins

Properties
•Active vs Gram +ve bacteria and Gram -ve bacteria which do
not produce b-lactamases
•Acid resistant and orally active
•Non toxic
•Sensitive to b-lactamases
•Increased polarity due to extra amino group
•Poor absorption through the gut wall
•Disruption of gut flora leading to diarrhoea
•Inactive vs. Pseudomonas aeruginosa

© Oxford University Press, 2013

Problem 3 - Range of Activity
Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969)
O
C
H NH2 R= CH2O CMe3 PIVAMPICILLIN

C
H O
C N H H
S R= O TALAMPICILLIN
O Me

N Me
O O

CO2R C
R= CH O O CH2Me

Me
Properties BACAM PICILLIN

•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

© Oxford University Press, 2013

 Problem 3 - Range of Activity

Mechanism of prodrug activation

O
H PEN H PEN
PEN
C H
C O CH2 O C OH
C O CH2 O CMe3
O O
O
EN
ZYME
Formaldehyde

•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
© Oxford University Press, 2013
Problem 3 - Range of Activity
Examples of broad spectrum penicillins
Class 2 - CO2H at the a-position (carboxypenicillins)
CO2R
Examples CH
H R=H Carbenicillin
C N H H
S
R = Ph Carfecillin
O Me

N Me
O
CO2H

•Carfecillin = prodrug for carbenicillin

•Active over a wider range of Gram -ve bacteria than ampicillin
•Active vs. Pseudomonas aeruginosa
•Resistant to most b-lactamases
•Less active vs Gram +ve bacteria (note the hydrophilic group)
•Acid sensitive and must be injected
•Stereochemistry at the a-position is important
•CO2H at the a-position is ionised at blood pH
© Oxford University Press, 2013
Problem 3 - Range of Activity
Examples of broad spectrum penicillins
Class 2 - CO2H at the a-position (carboxypenicillins)
Examples
CO2H
H H H
N S
Me
TICARCILLIN
S O N Me
O
CO2H

•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

© Oxford University Press, 2013

Problem 3 - Range of Activity
Examples of broad spectrum penicillins
Class 3 - Urea group at the a-position (ureidopenicillins)
O
Examples Azlocillin HN
N
O

O R2N NH
MeO2S H H H
Mezlocillin N
N
N S
Me
O N
Et N N
Me
Piperacillin O
O O CO2H

•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 Pseudomonas aeruginosa
•Piperacillin can be administered alongside tazobactam
© Oxford University Press, 2013