ANTIBACTERIAL ANTIBIOTICS

1
 ANTIBIOTICS
 The word "antibiotics" comes from the Greek
anti ("against") and bios ("life"). Antibiotics are
drugs that either destroy bacteria or prevent their
reproduction. Antibiotics that kill bacteria are called
"bactericidal" and the ones that stop the growth of
bacteria are called "bacteriostatic".

 Some antibiotics are 'bactericidal', meaning that

they work by killing bacteria. Other antibiotics are
'bacteriostatic', meaning that they work by stopping
bacteria multiplying. 2
 Each different type of antibiotic affects different
bacteria in different ways. For example, an
antibiotic might inhibit a bacterium's ability to turn
glucose into energy, or its ability to construct its
cell wall. When this happens, the bacterium dies
instead of reproducing.

 Some antibiotics can be used to treat a wide

range of infections and are known as 'broad-
spectrum' antibiotics. Others are only effective
against a few types of bacteria and are called
'narrow-spectrum' antibiotics.

3
 Targets for Antibiotics

Fig 24.9
4
pg. 741
 Antibiotics That Inhibit Cell Wall
Synthesis
Penicillin and Cephalosporins

1. One major class of antibiotics inhibit the

synthesis of peptidoglycan.

2. Once cell wall synthesis (involving penicillin

binding proteins) is inhibited, enzymatic autolysis
of the cell wall can occur.

5
 Antibiotic resistance
 Antibiotics are extremely important in medicine, but
unfortunately bacteria are capable of developing resistance to
them. Antibiotic-resistant bacteria are germs that are not killed
by commonly used antibiotics. When bacteria are exposed to the
same antibiotics over and over, the bacteria can change and are
no longer affected by the drug.

 Bacteria have number of ways how they become antibiotic-

resistant. For example, they possess an internal mechanism of
changing their structure so the antibiotic no longer works, they
develop ways to inactivate or neutralize the antibiotic. Also
bacteria can transfer the genes coding for antibiotic resistance
between them, making it possible for bacteria never exposed to
an antibiotic to acquire resistance from those which have.
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 ANTIBIOTICS CLASSIFICATION

Based on
Based on chemical pharmacological
structure activity

•β-lactum antibiotics
•Aminoglycoside • Antifungal antibiotics
•Tetracyclines • Anticancer antibiotics
•Macrolide antibiotics • Anti typhoid antibiotics
•Lincomycins • Anti diarrheal antibiotics
•Polypeptide antibiotics • Antituberculor antibiotics
•miscellaneous

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 Β-LACTAMS

β-lactam ring

8
 HOW DO THEY WORK?

1. The β-lactam binds to Penicillin Binding

Protein (PBP)
2. PBP is unable to crosslink peptidoglycan
chains
3. The bacteria is unable to synthesize a stable
cell wall
4. The bacteria is lysed

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 Mechanism of action of β-lactam antibiotics.

Normally, a new subunit of N-acetylmuramic acid (NAMA) and N-

acetylglucosamine (NAGA) disaccharide with an attached peptide
side chain is linked to an existing peptidoglycan polymer. This may
occur by covalent attachment of a glycine () bridge from one peptide
side chain to another through the enzymatic action of a penicillin-
binding protein (PBP).

In the presence of a β-lactam antibiotic, this process is disrupted. The

β-lactam antibiotic binds the PBP and prevents it from cross-linking
the glycine bridge to the peptide side chain, thus blocking
incorporation of the disaccharide subunit into the existing
peptidoglycan polymer.
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 Mechanism of penicillin-binding protein
(PBP)inhibition by β-lactam antibiotics.
PBPs recognize and catalyze the peptide bond between
two alanine subunits of the peptidoglycan peptide side
chain. The β-lactam ring mimics this peptide bond.
Thus, the PBPs attempt to catalyze the β-lactam ring,
resulting in inactivation of the PBPs.

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Peptidoglycan Layer

12
13
 The beta lactam antibiotics include penicillins
(e.g. ampicillin), cephalosporins and monobactams.

They bind to and inhibit enzymes (penicillin binding

proteins) involved in the transpeptidation (cross-linking) of
peptidoglycan. These antibiotics have in common the four
membered lactam ring.

Attached to the lactam, penicillins have an additional five

membered ring and cephalosporins a six membered ring.

Monobactams consist of the lactam ring alone and display

antibiotic activity.
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 penicillins
Penicillin is the oldest known antibiotic in the whole world and
what is really surprising that it was discovered by total
coincidence.
It was discovered by Alexander Fleming in the late nineteenth
century when he was trying to multiply the penicillum fungi in the
lab.
Penicillin is still used till now for the treatment of many bacterial
and fungal infections and of course it was developed immensely
since that time
.
There are now different categories of penicillin but the core of
these categories is the natural penicillin or what is known the
penicillin G. natural penicillin is effective against most of the gram
positive microorganisms like streptococcus and staphylococcus. It
is also effective against some gram negative bacteria and it is
mainly used for treatment of oral cavity infections. 15
The second category of penicillin antibiotic is penicillinase resistant
antibiotics. Oxacillin and cloxacillin are prominent examples for this
group. This group does not target a wide range of microorganisms like
the natural penicillin but it targets a specific group of bacteria which
produces the beta lactamase enzyme. The lactamase producing
bacteria are resistant to natural penicillin.

The third category of penicillin antibiotics is the aminopenicillins.

Amoxicillin is the best example for this group and it is widely used
today. This is group is very potent against gram negative bacteria like E-
coli and Hemophelus influenza. The best feature of this group of
penicillin antibiotics is that they can tolerate the gastric acid. This
means that they could be administered orally without being degraded
through the acid medium of the stomach.

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The fourth group is called the extended penicillin antibiotics,
although this group is effective against many gram - negative
microorganisms but they are still inactive against beta- lacatam
producing bacteria like the aminopinicillins
.
The four categories of penicillin are still used till now to combat
different types of microorganisms and every category is used to
perform its specific role.

All penicillin drugs act in the same way by affecting the bacterial
cell wall. Suppression the formation of the bacterial cell wall will
cause the bacteria to die and omit the effect of the microorganism
on the body, the best feature of penicillin is that it develops the
least side effects on the patient.

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 Penicillin
• Prevents crosslinking of peptidoglycan, gram +/some -
• Beta-lactam nucleus
– Resistance through beta-lactamases
• In low concentrations,kills gram + cells, gram (-) in higher
doses
• Allergic reactions

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 History of penicillins
In 1875 Pasteur and Joubert discovered that certain moulds could produce
toxic substances which killed bacteria. Unfortunately, these substances
were also toxic to humans and of no clinical value. However, they did
demonstrate that moulds could be a potential source of antibacterial
agents.

In 1928, Fleming noted that a bacterial culture which had been left several
weeks open to the air had become infected by a fungal colony. Of more
interest was the fact that there was an area surrounding the fungal colony
where the bacterial colonies were dying. He correctly concluded that the
fungal colony was producing an antibacterial agent which was spreading
into the surrounding area. Recognizing the significance of this, he set out to
culture and identify the fungus and showed it to be a relatively rare species
of Penicillium. It has since been suggested that the Penicillium spore
responsible for the fungal colony originated from another laboratory in the
building and that the spore was carried by air currents and eventually
blown through the window of Fleming's laboratory.
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Fleming spent several years investigating the novel antibacterial
substance and showed it to have significant antibacterial properties
and to be remarkably non-toxic to humans. Unfortunately, the
substance was also unstable and Fleming was unable to isolate and
purify the compound. He therefore came to the conclusion that
penicillin was too unstable to be used clinically.

The problem of isolating penicillin was eventually solved in 1938 by

Florey and Chain by using a process known as freeze-drying which
allowed isolation of the antibiotic under much milder conditions than
had previously been available. By 1941, Florey and Chain were able to
carry out the first clinical trials on crude extracts of penicillin and
achieved spectacular success. Further developments aimed at
producing the new agent in large quantities were developed in the
United States such that by 1944, there was enough penicillin for
casualties arising from the D-Day landings.
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Penicillins were used widely and often carelessly, so that the
evolution of penicillinresistant bacteria became more and more of
a problem. The fight against these penicillin-resistant bacteria was
promoted greatly when, in 1976, Beechams discovered a natural
product called clavulanic acid which has proved highly effective in
protecting penicillins from the bacterial enzymes which attack
penicillin.

STRUCTURE OF PENICILLIN

Penicillin contains a highly unstable-looking bicyclic system

consisting of a fourmembered lactam ring fused to a five-
membered thiazolidine ring. The skeleton of the molecule suggests
that it is derived from the amino acids cysteine and valine (Fig.),
and this has been established.

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STRUCTURE OF PENICILLIN
O
S
CH3

C NH CH CH C

CH3

O C N CH COOH

Site of penicillinase action.

Breakage of the β lactam ring.

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

O CH2 Thiazolidine
b-Lactam ring
Phenoxymethyl penicillin (Pen V) ring

Side chain varies depending on carboxylic acid present in fermentation

medium .For example, corn steep liquor was used as the medium when
penicillin was first mass-produced in the United States and this gave penicillin
G (R=benzyl). This was due to high levels of phenylacetic acid (PhCH2CO2H)
present in the medium.
CH2 CO2H Penicillin G

present in corn steep liquor

OCH2 CO2H Penicillin V

(first orally active penicillin)
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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
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 CONSTITUTION OF PENICILLIN
Molecular formula of penicillin is C9H11N2O4SR

 presence of carboxylic group:

They form mono salts indicating the presence of a carboxylic group
Penicillins are not found to posses a free amino group , thiol group

 Hydrolysis:
penicillins are hydrolysed by hot dilute inorganic acids , they lose one carbon atom
in the Form of carbondioxide and they are also degraded to the equimolar
amounts of an amine, PENICILLAMINE, and an aldehyde PENILLOALDEHYDES. All
the penicillins are degraded to the Same amine but different aldehydes because
the fragement comes in the aldehyde portion.

C9H11N2O4SR + 2H2O CO2 + C5H11NO2S + C3H4NO2R

PENICILLAMINE PENILLOALDEHYDE
Now since the fragment R comes in the aldehyde portion, all the penicillins
give the same amine but different aldehydes.

 Structure of D-Penicillamine :
C5H11NO2S.this ha been elucidated on the basisof following facts:

Molecular formula of Penicillamine is C5H11NO2S.

As Penicillamine respond to the indigo colour reaction with ferric chloride solution
and Also gives colour reaction with sodium nitroprusside , this shows that D-
Penicillamine is probably a substituted cysteine.

From electrometric titrations ,it is revealed that there are three pka values 1.8, 7.9,and
10.5 corresponding to the carboxylic , α-amino and thiol groups respectively. The
presence Of these groups also reveals that D-penicillamine is probably substituted
cysteine.
Moreover, the chemical reactions are parrell to those of cysteine . For example
like cysteine D-penicillamine also reacts with acetone to yield an isopropylidene
derivative. The latter Compound does not contain a free amino or thiol group
and is reconverted into pencillamine On hydrolysis. These reactions reveal that
these groups,i.e, amino and thiol group , are Attached to adjacent carbon
atoms.further oxidation of penicillamine with brominewater Yields a sulphonic
acid(this reaction is characterstic of a thiol)

When kuhn-roth method for the determination of methy side chain is applied to
D-PENICILLAMINE It gives a very low value (~ 0.2molecules): this low value
reveals that D-PENICILLAMINE contains An isopropyl end-group and not a
methyl end-group.

so D-PENICILLAMINE is β,β-dimethylcysteine which has been confirmed by its

synthesis.
 NaOH
(CH3)2CHCHCO2H ClCH2COCL (CH3)2CHCHCO2H
-HCL
NH2 NHCOCH2Cl
DL-valine
(CH3CO)2O

(H3C)2C C C
(H3C)2C C CHCO2H
1.H2S N O
(CH3)2C CHCO2H H2S S N
2.-H + H
C
BOIL C
3.+H+
NHCOCH3 SH
SH
CH3
CH3
Azalactone
HCL(boil) 2,5-trimethyl-2-
thiazoline-4-carboxylic acid
pyridine

(CH3)2C CHCO2H

SH
NH2

DL-penicillamine
 From the above synthesis, the recemic miture DL is obtained.for
resolution of this racemic mixture , this is treated with formic acid to
yield the formyl derivative which is then resolved by means of brucine .
The formyl group is removed by hydrolysis.

HCO2H
(CH3)2C CHCO2H CH3)2C CHCHO2H

SH NH2 SH NHCHO
DL-form
DL-formyl derivative

1.resolution by brucine
2.HCL
3.pyridine

(CH3)2C CHCO2H

NH2
SH

D-penicillamine
The D- penicillamine odtained from the resoltuion of the recemic mixture has
been found to be identical with the natural penicillamine.

When the penicillamine is treated with diazomethane , it is converted into its

methyl ester.
 The lattes compound when treated with aqueous solution of mercuric chloride
yields the methyl ester of penicillamine .these reactions reveal that the
carboxylic group in penicillin itself .

Structure of penilloaldehyde:
This has been elucidated on the basis of the following facts.:

The general formula of penilloaldehyde has been found to be C3H4NO2R .

When hydrolysed vigorously , all penilloaldehydes yield a substitued acetic acid

and Amino acetaldehyde . This reaction reveals that penilloaldehyde are
acyl derivatives
RCONHCH2CHO of aminoacetaldehyde.
(or C3H4NO2R) + H2O RCOOH + NH2CH2CHO
substitued aminoacetaldehyde
penilloaldehydes acetic acid
The above structure of penilloaldehyde has been confirmes by its synthesis from the
Corresponding acid chloride and amino acetal.

RCOCl + NH2CH2CH(OC2H5)2 RCONHCH2CH(OC2H5)2

HCL

RCONHCH2CHO

penilloaldehydes
In this point it was stated that acid hydrolysis of penicillinyields
penicillamine,penilloaldehyde And corbondioxide. The formation of co2 molecule is
indicative of the fact that some unstable acid is formed as an intermediate which
undergoes ready decarboxylation to yield carbon dioxide .such an acid forme as an
intermediate in the hydrolysis of penicillin.

RCONHCHCHO CO2 + RCONHCH2CHO

penilloaldehyde
COOH
penaldic acid
Mode of lincking of penicillamine and penilloaldehyde.: now the next question arises
How are the two fragments ,penicillamine and penilloaldehyde, linked in penicillin …
So that it may explain tha formation of panaldic acid that is to explain the formation
of co2 ?

Tha mode of linkage of these fragments has been established on the basis of the
following facts;

When penicillin is hydrolysed with dil alkali or with the enzyme penicillinase it It
yields penicilloic acid which is a dicarboxylic acid and readily eliminates a molecule of
co2 to yield a monocarboxylic acid, penilloic acid . this suggest that in penicilloic acid
one of the carboxylic groups is in the β-position with respect to anelectron-attractin g
group.

The structure of penillonic acid has been proved on the basis of the fact that it,when
hydrolysed with aqueous mercuric chloride,yields pwnicillamine and penilloaldehyde.
This type of hydrolysis is characteristic of compounds having a thiazolidine ring. The
presence of thiazolidine type of nucleus in penicillin has been proved by the fact that
it possesses neither free amino group nor a free thiol group .thus penilloic acid could
be represented as
•Because this structure would yield the desired products

SH
S
ROCHNH2CHC C(CH3)2
CH(CH3)2
H2O + RCONHCH2CHO
HN HgCl2 H2N CHCO2H
CHCO2H

Penilloic acid (A ) penicillamine penilloaldehyde

If A is the structure of penilloic acid , then the structure of penicilloic acid would
be represented as

S
R.CO.NH.CH CH C(CH3)2
CO2 + (A)
HOOC HN CHCOOH

PENICILLOIC ACID
The structure B of penicilloic acid has been confirmed by the fact that
penicillin when treated with methanol yields methyl penicilloate which on
hydrolysis with aq. Mercuric chloride yields methyl penaldate

S
R.CO.NH.CH CH C(CH3)2 H2O
CH3OH CH(CH3)2
PENICILLIN CO2H3C HN CHCO2H HgCl2
HO2CHCNH2
Methl penicilloate
penicillamine
+

RCONHCHCHO

COOCH3
methl penaldate
 Structure of penicillin:
From the above evidences , two structures C , D are possible for penicillin . But
penicillin on treatment with dil acid undergoes molecular rearrangement readly
to yield penillic acid . Therefore , the chemical evidence could not decide about
the correct structure C (or) D of penicillin. However , the physical methods could
decide about the correct structure of penicillin.

S
N N ROCHNHC HC C(CH3)
H3CR CH HC CH(CH3)
CO N CHCO2H
O CO HN CHCO2H

beta lactum structure (D)

oxazolone structure (C)
 Definite evidence of the existing of the β-lactum ring in penicillin was
obtained by desulphurisation of the benzylpenicillin with raney nickel to
yield desthiobenzylpenicillin (E) Which on hydrolysis by acid yields
desthiopenicilloic acid (F) or on boiling with benzylamine in Dioxane
solution yields benzylaminde of desthiobenzyopenicilloic acid(G).
S

C6H5H2COC.HN HC CH3 C.(CH3) RANEY Ni

B A
OC N H2C COOH

BENZYLPENICILLIN C6H5CH2CO.NH.CH CH2 CH(CH3)2

OC N CH.COOH
desthiobenzylpenicilln(E)

C6H5CH2NH2
C6H5CH2CO.NH.CH CH2 CH(CH3)2

COOH HN CH.COOH

desthiobenzylpenicilloic acid (G)

(H3C)2HC
C6H5CH2CO.NH.CH CH2

NH CH.COOH
CO.NH.CH2.C6H3

benzylamide of desthiobenzylpenicilloic acid (H)

The compounds G , H can be obtained by the desulphation of benzyl penicilloic acid and
Its benzyl amide respectively.

However it was not possible to decide between th two possible structure of penicillami
On chemical evidenca alone , because penicillin readly undergoes moleclar rearrangem

Eg; on treatment with dil acid , penicillin rearranges to yield penillic acid

S
H
HCL CHOO C C C(CH3)2
penicillin

N N CH.COOH
C

R
PENILLIC ACID
CLASSIFICATION OF PENICILLINS

NATURAL PENICILLINS

BENZYL PENICILLIN(PENICILLIN-G)

PENTYL PENICILLIN(PENICILLIN-F)

3-PENTYL PENICILLIN(PENICILLIN-K)

PHENOXY METHYL PENICILLIN(PENICILLIN-V)

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Semi-synthetic Penicillin

• Ampicillin
• Amoxicillin
• Carbenicillin
• Methicillin

40
Penicillins

41
Figure 20.6
Cephalosporins

42
 Cephalosporins
• Semisynthetic B-lactams derived
from chemical side chains added to
7-aminocephalosporanic acid.
• Generally more resistant to B-
lactamases.

43
Other Inhibitors of Cell Wall Synthesis
• Cephalosporins
– 2nd, 3rd, and 4th
generations more
effective against
gram-negatives

44
Figure 20.9
Other Inhibitors of Cell Wall Synthesis
• Polypeptide antibiotics
– Bacitracin
• Topical application
• Against gram-positives
– Vancomycin
• Glycopeptide
• Important "last line" against antibiotic resistant S. aureus

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 The Cephalosporins (generalized)
*Not effective vs. Enterococcus or Listeria

1st Generation Gram (+)

Decreasing Gram (+) and

2nd Generation
Increasing Gram (-)

3rd Generation Gram (-), but also some GPC

4th Generation Gram (+) and Gram (-)

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Discovery and structure of cephalosporin C

The second major group of β-lactam antibiotics to be discovered were

the cephalosporins. The first cephalosporin was cephalosporin C—
isolated in 1948 from a fungus obtained from sewer waters on the
island of Sardinia. Although its antibacterial properties were
recognized at the time, it was not until 1961 that the structure was
established.

It is perhaps hard for modern chemists to appreciate how difficult and

painstaking structure determination could be, even in the post-war
period. The advent of NMR spectroscopy in the sixties and seventies
has revolutionized the field so that if a new fungal metabolite is
discovered today, its structure can be worked out in a matter of
days rather than a matter of years.

. 47
The structure of cephalosporin C (Fig. 10.41) has similarities to that of
penicillin in that it has a bicyclic system containing a four-membered (β-
lactam ring. However, this time the (3-lactam ring is fused with a six-
membered dihydrothiazine ring. This larger ring relieves the strain in the
bicyclic system to some extent, but it is still a reactive system

A study of the cephalosporin skeleton reveals that cephalosporins can be derived

from the same biosynthetic precursors as penicillin, i.e. cysteine and valine (Fig.
10.42).

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Properties of cephalosporin C

The properties of cephalosporin C can be summarized as follows.

• Difficult to isolate and purify due to a highly polar side-chain.
• Low potency (one-thousandth of penicillin G).
• Not absorbed orally.
• Non-toxic.
• Low risk of allergenic reactions.
• Relatively stable to acid hydrolysis compared to penicillin G.
• More stable than penicillin G to penicillinase (equivalent to
oxacillin).
• Good ratio of activity against Gram-negative bacteria and Gram-
positive bacteria.

49
Cephalosporin C has few clinical uses, is not particularly potent and at
first sight seems rather uninteresting. However, its importance lies in
its potential as a lead compound to something better. This potential
resides in the last property mentioned above. Cephalosporin C may
have low activity, but the antibacterial activity which it does have is
more evenly directed against Gram-negative and Gram-positive
bacteria than is the case with penicillins.

By modifying Cephalosporin C we might be able to increase the

potency whilst retaining the breadth of activity against both
Grampositive and Gram-negative bacteria. Another in-built advantage
of Cephalosporin C over penicillin is that it is already resistant to acid
hydrolysis and to penicillinase enzymes. Cephalosporin C has been
used in the treatment of urinary tract infections since it is found to
concentrate in the urine and survive the body's hydrolytic enzymes.

50
 Monobactams
• Monobactams: Aztreonam
• Spectrum: ONLY  Gram negative aerobic bacteria
• Lack of Coverage:
– Some resistant P. aeruginosa, E. cloacae, and C. freundii
– Acinetobacter sp., Stenotrophomonas sp.
• Pharmacokinetics:
– Well distributed into tissues, esp. inflamed tissues
– Excretion: renal clearance
• Adverse reactions:
– Skin rash
– Very low cross-reactivity with Beta-Lactam class – highest risk
in patient allergic to ceftazidime.

51