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 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
C

H
N

H
S

R
N

C
Me
Me

O
CO2H

H
N

H
S

-Lactamase

HO2C

HN

Me
Me

CO2H

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

O

Bulky

group

H
N

H
S

Me

R
N

Enzyme

Me

O
CO2H

Problem 2 - Sensitivity to -Lactamases

Examples - Methicillin (Beechams - 1960)

ortho groups

important
MeO

O
C

H
N

H
S
N

OMe

Me
Me

O
CO2H

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

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

Problem 2 - Sensitivity to -Lactamases

Examples

R'
O
C
R

N
O

H
N

H
S
N

Me

Bulky and
electron withdrawing

Me

Oxacillin R = R' = H
Cloxacillin R = Cl, R' = H
Flucloxacillin R = Cl, R' = F

Me

O
CO2H

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

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 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.

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

Problem 3 - Range of Activity

Examples of Broad Spectrum Penicillins

Class 1 - NH2 at the -position

Ampicillin and amoxicillin (Beechams, 1964)

H

NH2

HO

C
C

H
N

NH2
C
C

H
N

Ampicillin (Penbritin)

2nd most used penicillin

Amoxicillin (Amoxil)

Problem 3 - Range of Activity

Examples of Broad Spectrum Penicillins


Properties

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 diarrhoea

Inactive vs. Pseudomonas aeruginosa

Problem 3 - Range of Activity

Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969)

O

R=

NH2

C
CH2O

CMe3

PIVAMPICILLIN

C
C

H
N

H
S

Me

TA LAMPICILLIN

R=

Me

O
CO2R

R=

CH

CH2Me

Me
BACAMPICILLIN
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

Problem 3 - Range of Activity

Mechanism of prodrug activation

PEN

C O CH
2 O

O
C

PEN
CM
e 3

ENZYME

PEN

C O CH
2 O

H
O

C OH

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

Problem 3 - Range of Activity

Examples of broad spectrum penicillins

Class 2 - CO2H at the -position (carboxypenicillins)

Examples

CO2R
CH
C

H
N

R = H Carbenicillin
R = Ph Carfecillin

H
S

Me
Me

N
O
CO2H

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

Problem 3 - Range of Activity

Examples of broad spectrum penicillins

Class 2 - CO2H at the -position (carboxypenicillins)

Examples

CO2H

H H
N

N
O

Me

TICARCILLIN

Me
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

Problem 3 - Range of Activity

Examples of broad spectrum penicillins

Class 3 - Urea group at the -position (ureidopenicillins)

O

Examples

Azlocillin

Mezlocillin

HN

MeO2S

O
N
O

R2N

NH

H H
N

Piperacillin

Et N

N
O

Me
Me

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

CEPHALOSPORINS

1. Introduction

Antibacterial agents which inhibit bacterial cell wall synthesis

Discovered from a fungal colony in Sardinian sewer water (1948)

Cephalosporin C identified in 1961

2. Structure of Cephalosporin C

7-Aminoadipic side chain

H H
N

H2N
H

CO2H

S
1

2
3

Dihydrothiazine

ring

-Lactam

ring

Me

CO2H

H2N

O
CO2H

Me

7-Aminocephalosporinic acid (7-ACA)

H H
N

H2N

3. Properties of Cephalosporin C

CO2H

S
1

2
3

O
CO2H

Me

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 -lactamases

Ratio of activity vs Gram -ve and Gram +ve bacteria is better

Conclusion

Useful as a lead compound

4. Biosynthesis of Cephalosporins

H2N
R

OH
O

H Cys SH

HO

CO2H Me Val
Me
H2N
CO2H

C
O

Me

H H
N

O
CO2H

Me

5. SAR of Cephalosporins

H H
N

R
O

S
1

2
3

Me

O
CO2H


Similar to penicillins

The -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-Acetoxymethylene side chain

Substitution at C-7

6. Mechanism of Action

H H
N

O
CO2H

En zyme

N
O

O
Ser

OH
Ser

Me

-CH3CO2-

H H
N

CO2H

Enzyme

Note

The acetoxy group acts as a good leaving group and aids the
mechanism

7. Variation of the 7-Acylamino Side Chain

Not possible to generate analogues by fermentation

Not possible to generate analogues by a full synthesis

Restricted to semi-synthetic procedure

H2N

RCOCl
N

O
CO2H

H H
N

Me

O
CO2H

Me

7-ACA

7-ACA not available by fermentation

7-ACA not available by enzymatic hydrolysis of cephalosporin C

Generated by a chemical hydrolysis

7. Variation of the 7-Acylamino Side Chain

Generation of 7-ACA

Need to hydrolyse a relatively unreactive secondary amide in the
presence of a labile -lactam ring



H
N

R1

PCl5

7
O

N
4

R1

R1

ROH

CO2SiMe3

OR

Cl

OAc

H2O

-R1CO2H

Imino ether

Imino chloride

Protecting group

H2N

H
N

R2

R2COCl

N

OAc

O
CO2H

7-ACA

OAc

O
CO2H

Range of cephalosporins

8. First Generation Cephalosporins

Cephalothin

H H
N

7
S

OAc

O
CO2H

More active than penicillin G vs. some Gram -ve bacteria

Less likely to cause allergic reactions

Useful vs. penicillinase producing strains of S. aureus

Not active vs. Pseudonomas aeruginosa

Poorly absorbed from GIT

Administered by injection

Metabolised to give a free 3-hydroxymethylene group
(deacetylation)

Metabolite is less active

8. First-generation Cephalosporins

Cephalothin - drug metabolism



H H
N

H H
N

7
S

O
CO2H

OAc

Metabolism

OH

O
CO2H

Less active

OH is a poorer leaving group

Strategy

Replace the acetoxy group with a metabolically stable leaving
group

8. First-generation Cephalosporins

Cephaloridine

H H
N

7
S

O
CO2

The pyridine ring is stable to metabolism

The pyridine ring is a good leaving group (neutralisation of
charge)

Cephaloridine exists as a zwitterion and is soluble in water

Poorly absorbed through the gut wall

Administered by injection

8. First-generation Cephalosporins

Cefalexin

H2N

H H
N

Me

O
CO2H

The 3-methyl group is a poor leaving group

Methyl group is bad for activity but aids oral absorption

Can be administered orally

A hydrophilic amino group at the -carbon of the side chain
helps to compensate for the loss of activity due to the methyl group

8. First Generation Cephalosporins

Synthesis of cephalosporins with a 3-methyl substituent

H
N

H
S

OH

Me

H2O2

2
N

Me

S
N

Me

Toluene/
PTSA

S
N

Me

OH

CH2
N

Me

O
CO2Me

S
N

OH
CH3

CO2Me

CO2Me

CO2Me

CO2Me

-H

H
OH

CH3
CO2Me

CH3

S
3
CH3
CO2Me

8. 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 -lactamases)

9. Second-generation Cephalosporins

9.1 Cephamycins

H OMe H
N

HO2C
H2N

O
CO2H

NH2

Cephamycin C

Isolated from a culture of Streptomyces clavuligerus

First -lactam to be isolated from a bacterial source

Contains a 7-methoxy group

Modifications carried out on the 7-acylamino side chain

9. Second-generation Cephalosporins

9.1 Cephamycins

H OMe H
N

7
S

O
CO2H

NH2

Cefoxitin

Broader spectrum of activity than most first-generation

cephalosporins

Greater resistance to -lactamase enzymes

The 7-methoxy group may act as a steric shield

The urethane group is stable to metabolism

9. Second-generation Cephalosporins

9.2 Oximinocephalosporins

Me
O

N
C

H H
N

O
CO2H

NH2

Cefuroxime

Much greater stability against some -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 Pseudomonas aeruginosa

Used clinically against respiratory infections

10. Third-generation Cephalosporins

Oximinocephalosporins

R
Me

Aminothiazole

ring

O
N

H2N
S

Cefotaxime
Ceftizoxime

CH2OCOMe
H

N
C

H H
N

Me
CH2S

N
N

Ceftriaxone

OH
O

CO2H

Aminothiazole ring enhances penetration of cephalosporins

across the outer membrane of Gram -ve bacteria

May also increase affinity for the transpeptidase enzyme

Good activity against Gram -ve bacteria

Variable activity against Gram +ve cocci

Variable activity vs. Pseudomonas aeruginosa

Lack activity vs MRSA

Generally reserved for troublesome infections

10. Third-generation Cephalosporins

Oximinocephalosporins

Me

Me
O

N
C

N
H2N

CO2H

H H
N

Ceftazidime

O
CO2

Injectable cephalosporin

Excellent activity vs. P. aeruginosa and other Gram -ve bacteria

Can cross the blood brain barrier

Used to treat meningitis

11. Fourth-generation Cephalosporins

Oximinocephalosporins

Me
O
N
H2N
S

R
N
C

H H
N

Me
CH2

CH2 N

Cefipime

Cefpirome

CO2

Zwitterionic compounds

Enhanced ability to cross the outer membrane of Gram negative
bacteria

Good affinity for the transpeptidase enzyme

Low affinity for some -lactamases

Active vs. Gram +ve cocci and a broad array of Gram -ve
bacteria

Active vs. Pseudomonas aeruginosa

THIENAMYCIN

MONOBACTAMS

-LACTAMASE INHIBITORS

1. Thienamycin

Acylamino side

chain absent

OH

Plays a role

in -lactamase

resistance

Opposite
stereochemistry
to penicillins

Merck 1976

Carbon

H
H
H3C

NH3
S
N
O
CO2

Double bond leading to

high ring strain and an increase
in -lactam ring reactivity

Carbapenam nucleus

Isolated from Streptomyces cattleya

Potent and wide range of activity vs Gram +ve and Gram -ve
bacteria

Active vs. Pseudomonas aeruginosa

Low toxicity

High resistance to -lactamases

Poor stability in solution (ten times less stable than Pen G)

1. Thienamycin

Thienamycin analogues used in the clinic

H OH
Me

H OH
Me

H OH
Me

NH
HN

Imipenem

N
CO2

H
N

O
C

Me

Meropenem

Me
S

N
CO2

H
N

Me

O
C

Ertapenem(2002)

N
CO2

CO2

2. Monobactams

Nocardicins (Fujisawa 1975)

HO2C

D
HC

H2N

O
CH2

OH

H
N

CH2

H
OH

Nocardicin A

N
C

O
H

CO2H

Monocyclic -lactam ring

Moderately active in vitro vs narrow group of Gram -ve bacteria

Active vs. Pseusomonas aeruginosa

Inactive vs. Gram +ve bacteria

Different spectrum of activity from penicillins

Thought to operate by a different mechanism from penicillins

Low toxicity

2. Monobactams

Clinically useful monobactam

Me
N

Me

CO2H

O
H
N

Me

Aztreonam

H2N
S

N
O

SO3-

Administered by intravenous injection

Can be used for patients with allergies to penicillins and
cephalosporins

No activity vs. Gram +ve or anaerobic bacteria

Active vs. Gram -ve aerobic bacteria

3. -Lactamase Inhibitors

Clavulanic acid (Beechams 1976)

Sulphur replaced by O
No acylamino

side chain

6
7

OH

3
2
H

CO2H

-Lactam

Oxazolidine ring

Isolated from Streptomyces clavuligerus

Weak, unimportant antibacterial activity

Powerful irreversible inhibitor of -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 = ticarcillin + clavulanic acid

3. -Lactamase Inhibitors

Clavulanic acid - mechanism of action

1

NH

NH

CH2OH
O

CH2OH

N
O

HN

Base

CO2H
O

OH

CO2H

CH2OH

H2N

NH

NH

O
HN

NH

CO2H

CH

CH2OH

HC
O

CO2H

Irreversibly blocked

3. -Lactamase Inhibitors

Penicillanic acid sulfone derivatives

O
S

Me

2
3

Me
CO2 Na

Sulbactam

Me
N
N

CO2

Tazobactam

Suicide substrates for -lactamase enzymes

Sulbactam has a broader spectrum of activity vs -lactamases than clavulanic acid, but is less
potent

Unasyn = ampicillin + sulbactam

Tazobactam has a broader spectrum of activity vs -lactamases than clavulanic acid, and has
similar potency

Tazocin or Zosyn = piperacillin + tazobactam

Next-genera8on inhibitors

CYCLOSERINE

BACITRACIN

VANCOMYCIN

VANCOMYCIN ANALOGUES