• β-lactam antibiotics – penicillins
and properties of penicillin
and activity relationships of penicillins
of penicillins – due to acid, β-lactamase
spectrum of activity of penicillins
(broad) spectrum Penicillins
degradation of penicillins – various pathways
of individual penicillins including structures and specific uses
• β-lactamase inhibitors –
of action of β-lactamase
of individual compounds used as β-lactamase
end of this lecture, student will be able to
Discuss the structure and properties of Penicillins
Explain the SAR of penicillins
Explain the sensitivity of penicillins towards acid and
Describe the causes for narrow spectrum of activity of
Discuss the structural modifications to design extended
spectrum of penicillins
Discuss the chemical degradation of Penicillin
Discuss the structures, specific uses and side effects of
Explain the mode of action of β-lactamase inhibitors
Discuss the structures, specific uses β-lactamase inhibitors
spectrum of antibacterial action. The unequaled importance of β-lactam antibiotics in chemotherapy is due to
lethal bactericidal action in the growth phase
frequency of toxic and other adverse effects
lethal antibacterial action is due to the selective imbibition of bacterial
cell wall synthesis. Specifically it inhibits the biosynthesis of peptidoglycan
which provides strength and rigidity to the cell wall.
and cephalosporins acylate specific bacterial transpeptidases (Penicillin
binding proteins) and make them inactive
1a &1b – transpeptidases involved in peptidoglycansynthesis associated with
cell elongation. Inhibition causes lysis
2- transpeptidase involved in maintaining the rod shape in bacilli. Inhibition
causes ovoid or round forms which undergo lysis
3- transpeptidase required for septum formation in cell division. Inhibition
results in formation of filamentous forms thast cannot separate
4- carboxypeptidase responsible for the hydrolysis of the terminal peptide
bonds of crosslinking peptides. This cleavage of bond is required before
peptide crosslinkage. But inhibition of theses enzymes are not lethal
various β-lactam antibiotics
differ in their affinities for the PBPs
β-lactam Antibiotics – Pencillins
of Penicillin was established in 1945 by “ DOROTHY HODGKINS” by the use of X-ray
contains a bicyclic system consisting of a four-membered β-lactam ring fused to
a five membered thiazolidine ring
skeleton of the molecule suggests that it is derived from the amino acids
‘CYSTEINE’ and ‘VALINE’
over-all shape of the molecule is like a HALF OPEN BOOK.
It is a product of metabolism
side chain (R) varies depending on the make-up of the fermentation media.
example, Corn steep liquor as the medium give penicillin G (R= Benzyl). This
was due to the high levels of Phenyl acetic acid (PhCH2COOH) present
in the medium.
method of varying the side chain is to add different carboxylic acids to the
example, Addition of phenoxy acetic acid (PhOCH2COOH) gives
acids of general formula ‘RCH2COOH’ can be added to the fermentation
medium, which restricts the variety of analogues.
disadvantages – Tedious -Time-consuming process.
Properties of Penicillin G:-
against Gram +ve bacilli (Staphylococci, meningitis and gonorrhea) and many
(but not all) Gram –ve Cocci.
– The penicillins are amongst the safest drugs known to medicine.
active over a wide range (or spectrum) of bacteria.
when taken orally since it breaks down in the acid condition of the stomach
G can only be administered by injection.
to all known β-lactamases. These are enzymes produced by penicillin-resistant
bacteria which catalyzes the degradation of penicillins.
reactions are suffered by some individuals
There are several
problems associated with the use of Penicillin G; most serious ones
spectrum of activity
purpose of making semi-synthetic penicillin analogues is to find compounds
which do not suffer from these disadvantages.
study of ‘SAR’ and finding out the features important to its activity is vital
for making new effective analogues of ‘Penicillin G’.
STRUCTURE –ACTIVITY RELATIONSHIPS OF PENICILLINS
β-lactam and thiazolidine ring forming a bicyclic system (Penam). Bicyclic
system confers strain on the β-lactam ring
carboxylic acid group is essential.
strained β-lactam ring is essential (increase strain, increase in activity,
amino side chain is essential except for thienamycin
is usual but not essential
of the bicyclic ring with respect to the acyl amino side chain is important –
Cis stereochemistry for the hydrogens
little variation is tolerated by penicillin nucleus. Also, any variation which
can be made is restricted to the acyl amino side chain
Acid sensitivity of Penicillins:-
There are THREE
reasons for the acid sensitivity of ‘Penicillin G’
1) Ring Strain:
The bicyclic system in penicillin consists of a four-membered ring and a
five-membered ring. As a result, Penicillin suffers ‘LARGE ANGLE STRAIN and
catalyzed ring openings relieves these strains by breaking open the more highly
stained four-membered lactam ring.
2) A highly
reactive β-lactam carbonyl group:
carbonyl group in the β-lactam ring is highly susceptible to nucleophiles and
does not behave like a normal tertiary amide which is usually quite resistant
to Nucleophilic attack.
difference in reactivity is due mainly to the fact that stabilization of the
carbonyl is possible in the tertiary amide, but is not possible in the β-lactam
β-lactam nitrogen is unable to feed its lone pair of electrons into the
carbonyl group since this would require the bi-cyclic rings to adopt an
impossibly strained flat system.
result the lone pair of electrons is localized on the nitrogen and the carbonyl
group is far more electrophilic than one would expect for a tertiary amide.
normal tertiary amide is far less susceptible to nucleophiles since the
resonance structures reduce the electrophilic character of the carbonyl group
3) Influence of
the acyl side chain [Neighboring group participation]
neighboring acyl group can actively participate in a mechanism to open up the
lactam ring. Thus penicillin G has self – destructive mechanism built into its
problems of acid sensitivity:-
can be done about the first two factors since the β-lactam ring is vital for
only the third factor can be tackled i.e. reducing the effect or participation
of the neighboring group, by introducing a good electron withdrawing group
attached to the carbonyl group. This
electron-withdrawing group will draw the electrons away from the carbonyl
oxygen by inductive effect and reduce its tendency to act as a nucleophile
of the acyl side chain [Neighboring group participation]
Penicillin V, has an electronegative oxygen on the acyl side chain, so the
molecule has better acid stability than Penicillin G and is stable enough to
survive the acid in the stomach. Thus, it can be given orally
Penicillin V is still sensitive to penicillinases and is slightly less active
than penicilln G
Penicillin sensitivity to β-lactamases:
are enzymes produced by penicillin-resistant bacteria, which can catalyse the
reaction, in which the same ring opening and deactivation of penicillin which
occurred with acid hydrolysis.
deactivation of penicillin
design of penicillinase-resistant penicillins involves the blocking of the
penicillin from reaching the penicillinase active site.
to do this is to “place a bulky group on the side chain”. Thus bulky group act
as a ‘shield’ to prevent binding with the enzyme, penicillinase”.
the shield is too bulky, then the steric shield also prevents the penicillin
from attacking the enzyme responsible for bacterial cell wall synthesis.
Ideal ‘Shield’ will be that which would be large enough to ward off the
lactamase enzyme, but would be small enough to allow the penicillin to act on
the enzyme responsible for cell wall synthesis.
Methicillin was the first semi-synthetic penicillin unaffected by penicillinase
and was developed to treat S.aureus infections, which was due to
virulent penicillin – resistant strains. Both the methoxy groups (ortho) on the
aromatic ring are important in shielding the lactam ring.
is not an ideal drug, since there are no electron-withdrawing groups on the
side chain, it is acid sensitive, and so has to be injected.
It has ⅕
of the activity of Penicillin G against organisms sensitive to Penicillin G, it
shows poor activity against some streptococci, and it is inactive
against Gram –ve bacteria. This problem of acid sensitivity was solved by incorporating
into the side chain a five-membered heterocycle which was designated to act as
a steric shield and also to be electron-withdrawing.
compounds (Oxacillin, Cloxacillin and Flucloxacillin) are resistant to acid and
penicillinase and are useful against S. aureus infections.
difference between the above three compounds is the type of halogen
substitution on the aromatic ring.
influence of these groups is found to be pharmacokinetic i.e. they influence
such factors as absorption of the drug and plasma protein binding.
Cloxacillin is better absorbed through the gut wall than Oxacillin, whereas
flucloxacillin is less bound to plasma protein, resulting in higher levels of
the free drug in the blood.
also are inactive against Gram –ve bacteria.
acid resistant penicillin would be the choice of the drug against an infection.
if the bacteria proved resistance because of penicillinase enzyme, then the
therapy would be changed to penicillinase – resistance penicillin.
Narrow – spectrum of activity:-
penicillins show a poor activity against Gram –ve bacteria.
There are several
reasons for this resistance.
difficult for penicillins to invade a Gram –ve bacterial cell because of the
make of the cell wall.
bacteria have a coating on the outside of their cell wall which consists of a mixture of fats, sugars
coating can act as barrier in various ways.
The outer surface may have an overall –ve and +ve charge depending on its
excess of phosphatidyl Glycerol would result in an overall anionic charge
whereas an excess of lysyl phosphatidyl
Glycerol would result in an overall cationic charge.
has a free carboxylic acid which if ionized, would be repelled by the former
type of cell coat.
the fatty portion of the coating may act as a barrier to the polar hydrophilic
way in which penicillin can pass such a barrier is through protein channels in
the outer coating-but most of these are usually closed.
levels of transpeptidase enzyme produced:-
transpeptidase enzyme is the enzyme attacked by penicillin. In some Gram –ve
bacteria, a lot of transpeptidase enzyme is produced and the penicillin is
incapable of inactivitating all the enzyme molecules present.
Modifications of the transpeptidase enzyme:-
mutation may occur which allows the bacterium to produce a transpeptidase
enzyme which is not antagonized by penicillin.
Presence of β-lactamase:-
are enzymes which degrade penicillin. They are situated between the cell wall
and its outer coating.
Transfer of the β-lactamase enzyme:-
can transfer small portion of DNA from one cell to another through structures
called Plasmids. These are small pieces of circular bacterial DNA. If the
transferred DNA contains the code for the β-lactamase enzyme, then the
recipient cell acquires immunity.
Extended (Broad) Spectrum Penicillins
to tackle the problem of narrow spectrum activity all the above factors has to
be considered. But the changes are confined only to the variations in the side
of hydrophilic groups on the side chain (eg., Penicillin G) favour activity
against Gram +ve bacteria, but has poor activity against Gram –ve bacteria.
hydrophilic groups on the side chain have either little effect (eg., Penicillin
T) or cause a reduction of Gram +ve activity (eg., Penicillin N). But they lead
to an increase in activity against Gram –ve bacteria.
of Gram –ve activity is found to be greatest if the hydrophilic groups (e.g.,
NH2, OH, COOH) is attached to the carbon that is ‘Alpha’ to the carbonyl group
on the side chain.
which are active against, both Gram +ve and Gram –ve bacteria are known as
There are two
classes of broad-spectrum antibiotics
classes have an ‘alpha’ hydrophilic group
broad spectrum antibiotics: Ampicillin and Amoxycillin
hydrophilic group is –NH2 group:
broad spectrum antibiotics: Carbenicillin
hydrophilic group is acid group,
Degradation of Penicillins:-
main cause of deterioration of penicillins is the reactivity of the strained
β-lactam ring, particularly by hydrolysis.
course of the hydrolysis and the nature of the degradation products are
influenced by the pH of the solution.
β-lactam carbonyl group of penicillin readily undergoes Nucleophilic attack by
water or hydroxide ion to form inactive ‘Penicilloic acid’ which is reasonably
stable in neutral to alkaline solutions but readily undergoes de-carboxylation
and further hydrolytic reactions in acidic solutions.
Nucleophiles as hydroxyl amines, alkyl amines and alcohols – open the β-lactam
ring to form the corresponding hydroxamic acids, amides and esters.
strongly acidic solution (pH < 3), penicillin undergoes a complex series of
reactions forming a variety of inactive degradation products. The first step in
the rearrangement to the penicillanic acid. This process is initiated by
protonation of the β-lactam nitrogen,
followed by Nucleophilic attack of the acyl oxygen atom on the β-lactam
opening of the β-lactam ring destabilizes the thiazolidine ring, suffers
acid-catalyzed ring opening to form penicillanic acid.
acid is very unstable and undergoes TWO major degradation pathways.
Path’ is hydrolysis of oxozolone ring to form unstable penamaldic acid, an
enamine easily undergoes hydrolysis to penicillamine (a major degradation
product) and penaldic acid.
Path’ involves a complex rearrangement of penicillanic acid to penillic acid.
acid (an imidazoline -2-carboxylic acid) readily decarboxylates and on
hydrolytic ring opening to form major end product penilloic acid.
acid (cannot be detected as intermediate), the major product formed. Weakly
acidic (neutral?) to alkaline hydrolytic conditions (also enzymatic
conditions), exists in equilibrium with penamaldic acid, undergoes
decarboxylation to give penilloic acid.
third major product of the degradation is penicilloaldehyde formed by
decarboxylation of penaldic acid (a derivative of malonaldehyde) .
Penicillin G: [Benzyl
of choice for the treatment of different kinds of bacterial infections than any
orally. But by combining antacids as calcium carbonate, aluminium hydroxide and
magnesium trisilicate or a strong buffer as sodium citrate and by giving large
doses, as it is poorly absorbed from intestinal tract, will increase the oral
activity of the drug.
soluble potassium or sodium salts are used orally and parenterally to achieve
high plasma concentration of pencillin G rapidly.
All pencillins should be administered with caution due to
allergic side effects
Penicillin V [Phenoxy
is resistant to hydrolysis by gastric acid and it produces uniform
concentration in blood, (when administered orally).
parenteral solution, potassium salt is usually used.
cloxacillin and dicloxacillin are highly resistant to inactivation by
penicillinase. The steric effects of 3-phenyl and 5-phenyl groups prevent
binding to the B- lactamase active site.
of Cl in ortho position causes increase in activity due to increase in oral
absorption. It attains high plasma levels
• [6-(2-ethoxy-1-naphthyl) penicillin sodium]
group and second ring of naphthalene group increase the stability against
to acid so it can be given by oral route
in infection caused solely by penicillin G-resistant staphylococci or
effective against pneumococci & group a – β-hemolytic streptococci.
be administered with care, due to its allergic side effects.
6-[D-α-aminophenyl acetamido] Penicillanic acid or D-α-amino benzyl penicillin
has an anti-bacterial spectrum broader than that of Penicillin G.
against Gram –Ve organism that are susceptible to other penicillins
active against Gram –ve bacteria and enterococci than other penicillins
resistant to penicillinases
• Use:- Particularly useful for the treatment of
acute urinary tract infections caused by E.Coli or Proteus mirailis.
is the agent of choice against Haemophilic influenzae infections
in combination with Probenicid for the treatment of Gonorrhea.
in treating Salmonellosis and Shigellosis.
6-[D-α-amino-p-hydroxy phenyl acetamido] Penicillanic acid (semi synthetic
antibacterial spectrum is same as that of ampicillin (resistant to acid,
susceptible to alkaline and β-lactamase hydrolysis).
complete gastro-intestinal absorption to give higher plasma and urine levels.
• Little or no effect of food on absorption.
β-lactamase inhibitors (Suicidal substrates)
β-lactamase inhibitors, such as sulbactam and tazobactam and natural occurring
β-lactams, such as the thienamycins, inhibit both β-lactamases and interact
with penicillin binding protein (PBP) present in the bacterial cell wall.
inhibitors are given along with β-lactamase sensitive penicillin, so that they
competitively bind to the enzyme and protect the penicillin from destruction.
inhibitors are of 2 classes.
• Class. I – inhibitors:- have a heteroatom at
Clavulanic acid & sulbactam.
• Class. II – inhibitors:- do not have a heteroatom at position 1
Eg. Carbapenams – as Thienamycin
action of β-lactamase inhibitors:-
of β-lactamases is done by mechanism-based inhibitors, which act by reacting
with the enzyme in the same way as that of the substrate.
acyl enzyme intermediate is formed by the reaction of the β-lactam with an
active-site serine hydroxyl group of the enzyme.
normal substrates (Penicillins), the acyl enzyme intermediate readily undergoes
hydrolysis, destroying the substrate and freeing the enzyme to attack more
mechanism based-inhibitor, the acyl enzyme intermediate formed is diverted by
tautomerism to a more stable imine that undergo hydrolysis more slowly to
eventually free the enzyme (transient inhibitors).
these inhibitors are also substrates for the enzymes that they inactivate, they
are sometimes called as ‘Suicidal substrates’
Mechanism based inhibition of β-lactamase
Compounds of β-lactamase inhibitor:-
acid is isolated from Streptomyces Clavuligeris
6-acylamino side chain
possess 2-hydroxyethylidene moiety at C-2.
has very weak anti-bacterial activity
is a potent inhibitor of S.aureus β-lactamase and plasmid-mediated
β-lactamases produced by Gram-ve bacilli.
with Amoxacillin and potassium salt of Clavulanic acid (Augmentin) is intended
for the treatment of skin, respiratory,
ear and urinary tract infections caused by β-lactamase producing bacterial
strains- the oral bioavailability of both are the same
is effective against strains which are
resistant to Amoxacillin alone
is stable to acid
of potassium clavulanate and ticarcillin sodium (extended–spectrum penicillin)
is recommended for Septicemia, lower respiratory tract infections and urinary
tract infections, bone and joint infections, skin & structure infections
caused by β-lactamase producing strains of S.aureus, Klebsiella, E.coli,
P.aeruginosa and other pseudomonas spp. Citrobacter spp. Enterobacter
spp. Serratia Marcescens, etc
Ticarcillin disodium ‘α-carboxy-3-thienyl penicillin’
Class II inhibitors – Carbepenams
of highly effective antibiotic agents commonly used for the treatment of severe
or high-risk bacterial infections. This class of antibiotics is usually
reserved for known or suspected multidrug-resistant (MDR) bacterial infections.
coverage of antibacterial activity
Gm+, and Gm- (especially drug resistant species), anaerobic coverage -Cover MSSA,
Enterococcus, streptococcus spp.
of choice for ESBL infections (ESBL-producing bacteria can’t be killed by many
of the antibiotics )
isolated from of Streptomyces Cattleya.
two structural features of thienamycins are shared with the penicillins and
fused bicyclic ring system containing a β-lactam.
equivalently attached 3-carboxyl group.
presence of double bond between C-2 and C-3 in the bicyclic structure creates a
considerable ring strain and increases the reactivity of the β-lactam to ring
has a 1-hydroxylethyl gp at 6th position (not the acyl amino side
chain) & this is oriented to the α ring
has broad spectrum antibacterial properties-active against most Gram+ &
gram- bacteria and resistant to activation by most β-lactamases (could be
because of hydroxyl ethyl side chain)
is more susceptible to acid & alkaline hydrolysis coz the strain nature of
the fused ring system. Stable at pH between 6-7
stable to most β-lactamases. It is an inhibitor of β-lactamases from certain
Gram+ve & Gram-ve bacteria resistant to other β-lactamam antibiotics
is used for the treatment of a wide variety of bacterial infections of the skin
& tissues, lower respiratory tract, bones & joints and genitourinary
are also used for septicemia & endocarditis caused by β-lactamases