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Patrick
An Introduction to Medicinal Chemistry3/e
Chapter 16
ANTIBACTERIAL AGENTS
Part 1: Penicillins
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PENICILLINS
N
S Me
Me
HN
CO2H
O
C H H
O
R
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INTRODUCTION
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
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Acyl side
chain
6-Aminopenicillanic acid
(6-APA)
STRUCTURE
Side chain varies depending on carboxylic acid present in fermentation medium
b-Lactamring
Thiazolidine
ring
present in corn steep liquor
Penicillin GCH2 CO2H
Benzyl penicillin (Pen G)
R =
Phenoxymethyl penicillin (Pen V)
R =
CH2
O CH2
N
S Me
Me
HN H H
CO2HO
C
O
R
Penicillin V
(first orally active penicillin)
OCH2 CO2H
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Shape of Penicillin G
Folded envelope shape
H
NO
NHC
O
R
H
S
CO2
H
H
Me
Me
..
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1CYS VAL
Biosynthesis of Penicillins
S
N
Me
MeO
HN
CO2H
C
R
O
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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 DevelopmentAims
To increase chemical stability for oral administration
To increase resistance to b-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 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)
Bicyclic system essential
N
S Me
Me
HN
CO2H
O
C H H
O
R
Carboxylic acid essentia
Cis Stereochemistry essential
bLactam essential
Amide essentia
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Penicillins inhibit a bacterial enzyme called the transpeptidase
enzyme which is involved in the synthesis of the bacterial cellwall
The b-lactam ring is involved in the mechanism of inhibition Penicillin becomes covalently linked to the enzymes active site
leading to irreversible inhibition
Covalent bond formed
to transpeptidase enzyme
Irreversible inhibition
N
S Me
Me
HN
H H
CO2HO
C
O
R
Nu
Enz
CHN
C
CO2H
HH
Me
MeS
HN
O
R
O
Nu-Enz-H N
S Me
Me
HN
H H
CO2HO
C
H
Enz-Nu
O
R
Mechanism of action
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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-LysL-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 NAM NAMNAGNAG
NAM NAM NAMNAGNAG
NAM NAM NAMNAGNAG
Bond formationinhibited by
penicillin
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D-AlanineTRANSPEPTIDASE
PENICILLIN
SUGARBACKBONE
NAM
L-Ala
NAG
D-Glu
L-Lys
D-Ala
D-Ala
Gly Gly Gly Gly Gly Gly GlyGlyGlyGlyL-Lys
NAG
D-Ala
D-Ala
D-Glu
L-Ala
NAM
SUGAR
BACKBONE
L-Lys Gly Gly
D-Ala
NAM
L-Ala
NAG
D-Glu
L-L ys Gly Gly Gly Gly GlyGlyGlyGly
NAG
D-Ala
D-Glu
L-Ala
NAM
Cross linking
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-g-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.
Normal Mechanism
PeptideChain
D-Ala D-Ala CO2H
OH
Peptide
Chain
Gly
HOH
Peptide
Chain
Peptide
Chain
D-Ala GlyPeptide
Chain
O
D-Ala
Mechanism of action - bacterial cell wall synthesis
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Alternative theory- Pencillin mimics D-Ala-D-Ala.
Mechanism inhibited by penicillin
OH
Peptide
Chain
Gly
H
Blocked H2O
Blocked
Blocked Irreversibly blocke
HCR
O
CO2H
NH
OMe
Me
N
S
S
HN
O
O
Me
Me
NH
CO2H
C
O
R HS
HN
O
O
Me
Me
NH
CO2H
C
O
R H
Mechanism of action - bacterial cell wall synthesis
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Penicillin can be seen to mimic acyl-D-Ala-D-Ala
But 6-methylpenicillin is inactive
despite being a closer analogue
H
S
N
Me
MeO
HN
CO2
H
MeC
R
O
Penicillin Acyl-D-Ala-D-Ala
HH
CO2H
HN
OMe
Me
N
SC
R
O
Me
CO2H
HN
O CH3
HN
HH
C
R
O
Mechanism of action - bacterial cell wall synthesis
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H
CO2H
HN
O
Me
Me
N
SC
R
O
Penicillin may act as an umbrella inhibitor
Mechanism of action - bacterial cell wall synthesis
HO
OH
Active Site
D-Ala-D -Ala
Blocked
H
Acylation
OH
O
H
CO2H
HN
Me
Me
HN
SC
R
O
O
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Resistance to Penicillins
Penicillins have to cross the bacterial cell wall in order toreach their target enzyme
But 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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Resistance to Penicillins
Cell
Cell membrane
Thick porous cell wall
Gram +ve bacteria
Thick cell wall
No outer membrane
More susceptible to penicillins
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Resistance to Penicillins
L
CellCell
membrane
Thin cell wall
Lactamaseenzymes
Outer
membrane
Hydrophobic barrier
Periplasmic
space
Porin
L
L
L
Gram -ve bacteria
Thin cell wall Hydrophobic outer membrane
More resistant to penicillins
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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 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 (e.g. PBP 2a forS. aureus)
Presence ofb-lactamases
Concentration ofb-lactamases in periplasmic space Mutations
Transfer ofb-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 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 foranalogue synthesis
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Penicillin Analogues - Preparation
Fermentation
Penicillin acylase
or chemical hydrolysis
O
C
ClR
Penicillin
HC
HN
CO2H
OMe
Me
N
S
O
CH2
6-APA
OCO2H
HH
H2N
Me
MeS
N
Semi-synthetic penicillinsN
S Me
Me
HN
H H
CO2HO
C
O
R
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Penicillin Analogues - Preparation
Problem - How does one hydrolyse the side chain by chemical
means in presence of a labileb-lactam ring?Answer - Activate the side chain first to make it more reactive
Note - Reaction with PCl5 requires involvement of nitrogens
lone pair of electrons. Not possible for the b-lactam nitrogen.
N
SNH
OCO2H
C
O
PhCH2PCl5
PENNC
Cl
PhCH2
ROH
PENNC
OR
PhCH2H2O
6-APA
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Problems with Penicillin G
It is sensitive to stomach acids
It is sensitive to b-lactamases - enzymes which hydrolyse theb-lactam ring it has a limited range of activity
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Problem 1 - Acid Sensitivity
Reasons for sensitivity
1) Ring Strain
H2O
H
N
S Me
Me
HN
H H
CO2H
O
C
O
R
Relieves ring strain
CHN
HO2C
CO2H
HH
Me
MeS
HN
O
R
Acid or
enzyme
N
S Me
Me
HN
H H
CO2H
O
C
HO
O
R
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Problem 1 - Acid Sensitivity
2) Reactive b-lactam carbonyl groupDoes not behave like a tertiary amide
Interaction ofnitrogens lone pair with the carbonyl group is not possible
Results in a reactive carbonyl group
Tertiary amide
C
O
R
NR2 Unreactive
R
C
R
N
O
R
b-Lactam
Folded ring
system
N
S
OH
Me
Me
CO2H
Impossibly
strained
N
SMe
Me
CO2H
O
Reasons for sensitivity
X
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Problem 1 - Acid Sensitivity
3) Acyl Side Chain
- neighbouring group participation in the hydrolysis mechanism
H
Further
reactions
-
H
N
S
O
N
O
R R
O
N
O
S
HN
C N
O
S
N
H
R
O
H
Reasons for sensitivity
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C
E.W.G.
O
HN
O
S
N
H
Problem 1 - Acid Sensitivity
Conclusions
The b-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 oxygen
Decreases
nucleophilicity
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N
S
HN
O
C
O
CH2PhOH
Penicillin V(orally active)
Problem 1 - Acid Sensitivity
Examples
electronegative
oxygen
Very successful semi-
synthetic penicillins
e.g. ampicillin, oxacillin
Better acid stability and orally active
But sensitive to b-lactamases Slightly less active than Penicillin G
Allergy problems with some patients
X = NH2, Cl, PhOCONH,Heterocycles, CO2H
C
HC
O
HN
O
S
N
X
R
H
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Problem 2 - Sensitivity to b-Lactamases
Notes on 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 penicillinand 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
b-LactamaseN
S Me
Me
HN
CO2HO
CH
O
R
CHN
CO2H
Me
MeS
HN
H
HO2C
O
R
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Bulky
group
Problem 2 - Sensitivity to b-Lactamases
Strategy
Enzyme
C
R
HN
O
CO2H
Me
MeS
N
H H
O
Block access of penicillin to active site of enzyme byintroducing bulky groups to the side chain to act as steric
shields
Size of shield is crucial to inhibit reaction of penicillins with b-lactamases but not with the target enzyme (transpeptidase)
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ortho groupsimportant
Problem 2 - Sensitivity to b-Lactamases
Examples - Methicillin (Beechams - 1960)
Methoxy groups block access to b-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 -ve bacteria
N
S Me
Me
HN
CO2H
O
MeO
OMe
H HC
O
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Problem 2 - Sensitivity to b-Lactamases
Examples - Oxacillin
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
Bulky ande- withdrawing
Oxacillin R = R' = H
Cloxacillin R = Cl, R' = H
Flucloxacillin R = Cl, R' = FN
O
C
O
HN
OCO2H
Me
MeS
N
R'
R
Me
H H
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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 ofb-lactamases5. Transfer ofb-lactamases between strains6. 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. 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 a-position (e.g. NH2, OH, CO2H)increase activity vs Gram -ve bacteria
Results of varying R in Pen G
P bl 3 R f A i i
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Problem 3 - Range of Activity
Examples of Broad Spectrum Penicillins
Class 1 - NH2 at the a-positionAmpicillin and Amoxycillin (Beechams, 1964)
Ampicillin (Penbritin)2nd most used penicillin
Amoxycillin (Amoxil)
H
C
H
O
HNC
NH2
HO
O
C
NH2
CHN
O
H
H
O
P bl 3 R f A i i
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Problem 3 - Range of Activity
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
Examples of Broad Spectrum Penicillins
Properties
P bl 3 R f A ti it
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O
N
HNC
C
NH2
S Me
Me
CO2R
H H
H
O
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
PIVAMPICILLINR = CH2OC
O
CMe3
TALAMPICILLINR = O
O
BACAMPICILLIN
R = CH
Me
OC
O
O CH2Me
P bl 3 R f A ti it
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Problem 3 - Range of Activity
Mechanism
Ester is less shielded by 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
ENZYME
PEN
C O CH2
O
OH
HH
PEN
C O CH2
O
O
C
O
CMe3
Formaldehyde
PEN
C OH
O
P bl 3 R f A ti it
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Problem 3 - Range of Activity
Examples of Broad Spectrum Penicillins
Class 2 - CO2H at the a-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 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
R = H CARBENICILLIN
R = Ph CARFECILLINH H
O
N
HNC
CH
CO2R
S Me
Me
CO2H
O
P bl 3 R f A ti it
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Problem 3 - Range of Activity
Examples of Broad Spectrum Penicillins
Class 2 - CO2H at a-position (carboxypenicillins)
Examples
Administered by injection
Identical antibacterial spectrum to carbenicillin
Smaller doses required compared to carbenicillin More effective againstP. aeruginosa
Fewer side effects
Can be administered with clavulanic acid
TICARCILLINS
N
Me
MeO
HH
CO2H
HN
O
CO2H
S
P bl 3 R f A ti it
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Problem 3 - Range of Activity
Examples of Broad Spectrum Penicillins
Class 3 - Urea group at the a-position (ureidopenicillins)Examples
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 vsP. aeruginosa
Piperacillin can be administered alongside tazobactam
Azlocillin
Mezlocillin
Piperacillin
S
N
Me
MeO
HH
CO2H
HN
O
NH
O
R2N
HNN
O
NN
OMeO2S
N N
OO
Et