Class I inhibitors: β-lactamse inhibitors

H CH2OH
Clavulanic acid O

N
O COOH

• Isolated from Streptomyces clavuligerus

• Weak antibacterial activity
• Powerful irreversible inhibitor of β-lactamases (suicide inhibitor)
• Orally active, used as a guard drug for amoxicillin (Augmentin)

• decreases the dose levels of amoxicillin and increases the spectrum of activity
• It acylates the active site of β-lactamases (serine) by mimicking the normal substrate
• effective against most (not all) types of β-lactamases
• When Clavulanic acid is added to amoxicillin/ampicillin preparations, the potency
against β-lactamase producing strains is markedly enhanced
 Class I inhibitors: β-lactamse inhibitors

Clavulanic acid

• Unusual structure:
• Fused to an oxazolidine ring rather than sulphur-containing ring
• have no acylamino side chain

• Essential requirements for β-lactamases inhibition:

• A strained β-lactam ring
• The enol ether
• The Z configuration for the double bond of the enol ether (activity is reduced but not
eliminated if the double bond is E)
• No substitution at C-6
• (R) stereochemistry at positions 2 and 5
• The COOH group
 β-Lactamase Inhibitors
Clavulanic acid - mechanism of action

1 2

NH 2 NH2

O
CH2OH
O CH2OH
N
O HN
O H Base
H
CO2H
OH O CO2H

3 4 5
O CH2OH

H2N

NH NH NH
CO2H

H CH
O CH2OH HC

O HN O O
H
O CO2H O O

Irreversibly blocked
 β-Lactamase Inhibitors

Suggested mechanism of irreversible inactivation of β-lactamase by clavulanic acid

and sulbactam
 β-lactamse inhibitors

H O
Sulbactam S OCH
3
N CH3
O
COOH

• Prepared by partial chemical synthesis from Penicillin

• The oxidation of the sulfur atom to a sulfone greatly enhances the potency of the
sulbactam, but is less potent than clavulanic acid
• Suicide substrates for β -lactamase enzymes
• Sulbactam has a broader spectrum of activity vs β-lactamases than clavulanic acid,
but is less potent
 β-lactamse inhibitors

H O
Tazobactam S O
N N
N N
O
COOH

• Tazobactam (more hydrophilic) has a broader spectrum of activity vs β-lactamases

than clavulanic acid, and has similar potency

• Not all β-lactamases are sensitive to the presence of these inhibitors

•Other mechanism of resistance?? Physical barrier, high level of trans peptidase

enzyme and lower affinity to Penicillins

• If penicillin resistant is caused by a penetration barrier or by decreased affinity for

PBPs, the β-lactamase inhibitors are not able to overcome this, because the
underlying mechanism is different
 Carbapenems
• Inhibitors that do not have heteroatom at position 4 4
6 5
H3C H H 3
Thienamycin HO N
NH2 7 1
H N
S O 2

O
COOH

• Potent and excellent broad-spectrum antimicrobial activity (G + and -)

• highly resistant to β-lactamases
• The endocyclic olefinic double bond enhances the reactivity of the
β-lactam ring and creates ring strain making Thienomycin unstable
• The Carbapenem ring is highly strained and very sensitive to reaction cleaving
the β-lactam bond
double bond is highly reactive
• Poor metabolic and chemical stability, and is not absorbed from the GIT
• Missing sulfur and acylamino side chain (both were thought to be essential to activity

• Penetrate very well through porins

 Thienamycin

• C-6: Hydroxyethyl side chain is responsible for the resistant to β-lactamases, it also
has stereoselectivity of antibacterial action (5R: 6S: 8S)
• C-3: The terminal amino group in the side chain is nucleophilic and attacks the
β-lactam bond of a nearby molecule through an intermolecular reaction
destroying activity (not stable in acidic and basic solutions due to the strained
nature of the fused ring having endocyclic double bond).

• Changing amino moiety to a less nucleophilic N-formiminoyl moiety by a

semisynthetic process to produce imipenem

• With all these differences, Carbapenems bind differently than Penicillins, they bind
to PBP2
 Imipenem
H3C HN
H H
HO
NH
H S
N
O
COOH
• Good penetration through porins (zwitterions)
• Inhibit many β-lactamases with very broad spectrum
• Orally inactive
• Renal dehydropeptidase-1 hydrolyzes Imipenem (β-lactam hydrolysis), resulting
in Imipenem deactivation
Coadministration of Cilastatin (inhibitor of human dehydropeptidase 1)

H3C

H3C NH2
NH COOH
O S
H
CO2 Na
 Meropenem

O CH3
N
CH3
H3C H H CH3
HO NH
H S
N
O
COOH

• C-3: bulky side chain

• C-4: introduction of methyl group

effective resistant (stablility) to hydrolysis by dehydropeptidase 1

can be used as a single agent

 Other carbapenems

Ertapenem

HO H H3C
HN
H3C S
N O COOH
O NH
COOH

Doripenem

NH2
O
CH3 CH3 S
H H O
HO H H NH
H
S H
N NH
O
COOH
 Monobactams
Nocardicins

N OH

HO2C O C
D H H
HC CH2 CH2 C N
OH

H2N O

N
Nocardicin A
C
O

CO2H
H

• Monocyclic b-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
 Monobactams
Clinically useful monobactam

Me
Me CO2H

O
N
H Me
N N
H2N Aztreonam
S O 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
•The strongly electron-withdrawing character of the sulfamic acid group probably also makes
the β-lactam bond more vulnerable to hydrolysis
Drugs Acting On Bacterial Cell
Wall Biosynthesis
 Cell Wall Biosynthesis
Growing cell wall

.. ... ... ... ... ... ...

Vancomycin
b-Lactams

.
Cross-linking

... ... ... ... ... ... ... ... ...

Transglycosidation
.Gly NAG

Carrier
Bacitracin
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala- D-Ala

Amino acid
Cycloserine

•Building block partially constructed in cytoplasm

•Constructed from 2 sugars (NAM, NAG) and a peptide chain
•Transported across cell membrane and completed
•Linked to growing cell wall by enzyme (transglycosidation)
•Final cross-linking reaction catalysed by transpeptidase enzymes
 D-Cycloserine
Growing cell wall

.. ... ... ... ... ... ...

Vancomycin
b-Lactams

.
Cross-linking

... ... ... ... ... ... ... ... ...

Transglycosidation
.Gly NAG

Carrier
Bacitracin
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala- D-Ala

Amino acid
Cycloserine

•Natural product produced by Streptomyces garyphalus

•Inhibits L-alanine racemase and D-Ala-D-Ala ligase
•Blocks biosynthesis of D-Ala-D-Ala
•Mimics the structure of D-Ala
 D-Cycloserine

H HO
N
O O
O Me
H
H
NH2
NH2
D-Cycloserine D-Alanine

• Simple molecule
• Broad spectrum activity
• Acts within the cytoplasm to prevent the formation of D-Ala-D-Ala
• Has similar structure to D-Ala and inhibit:
 enzyme (L-alanine racemase): responsible for racemizing L-Ala to D-Ala
 enzyme (D-Ala-D-Ala ligase): responsible for linking the two D-Ala units together
 Bacitracin
Growing cell wall

.. ... ... ... ... ... ...

Vancomycin
b-Lactams

.
Cross-linking

... ... ... ... ... ... ... ... ...

Transglycosidation
.Gly NAG

Carrier
Bacitracin
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala- D-Ala

Amino acid
Cycloserine

•Polypeptide produced by Bacillus subtilis

•Binds to the carrier lipid
•Prevents the carrier lipid from transporting the NAM
-pentapeptide building block across the cell membrane
 Vancomycin and vancomycin analogues
Growing cell wall

.. ... ... ... ... ... ...

Vancomycin
b-Lactams

.
Cross-linking

... ... ... ... ... ... ... ... ...

Transglycosidation
.Gly NAG

Carrier
Bacitracin
Cell lipid
membrane

Cytoplasm
L-Ala
D-Glu
L-Lys

NAM

L-Ala D-Ala D-Ala- D-Ala

Amino acid
Cycloserine

•Narrow spectrum bactericidal glycopeptide

•Produced by Streptomyces orientalis
•Blocks transglycosidation
 Vancomycin and vancomycin analogues

HO CH2OH
H3N Me
HO O
HO

O O
O
Cl
Vancomycin
Me O O

HO OH H3C CH3
Cl
O O
H H H H O
O N O
N N N
H H H Peptide chain
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH

•Important antibacterial agent

•Caps the building block used in the synthesis of the bacterial cell wall
•Contains a peptide chain which forms hydrogen bonds to the target
•Vancomycin acts as a binding site for the building block
 Vancomycin and vancomycin analogues
Biosynthesis of vancomycin
Glycosidations

Chlorination Chlorination

H OH H
Hydroxylation
O O Hydroxylation
Tyr Tyr

O O O
H H H
HO2C N N N NHMe
N N N
H H H
O O O
H2NOC
Asn Val

HO OH
OH

Oxidative couplings

•Derived from a flexible hexapeptide

•Cyclisations result in a rigid structure
•Peptide backbone is held in a fixed conformation
 Vancomycin and vancomycin analogues
Mechanism of inhibition
Vancomycin

Building
block

Cell membrane

Notes
•Vancomycin provides binding pocket for tail of biosynthetic building block
•Vancomycin binds to the tail of the building block’s peptide chain
•Caps the building block
•Disguises the building block from the transglycosidation enzyme
Vancomycin and vancomycin analogues

... Vancomycin

.. .. dimer

Growing cell wall

. . Building
block

•Dimerization occurs
•Dimer is highly stable
•Large vancomycin molecule acts as steric shield
 Vancomycin and vancomycin analogues
HO CH2OH
H3N Me
HO O
HO

O
O O Cl
Me O O
C D E
HO OH H3C CH3
Cl
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
B CONH2
A
HO OH
OH
H-bonding interactions between the
peptide backbone of vancomycin
O H O and the biosynthetic building block
H Me
N
N O
Cell wall building block H
O Me H
L-Lys-D-Ala-D-Ala 'tail'
 Vancomycin and vancomycin analogues
Binding interactions
H Me O
in dimer H Cell wall building block D-Ala-D-AlaL-Lys- tail
O N
N
Me H
O H O

R7
R3 H
H
NH2Me O2C N
N H O R5
H
Heptapeptide
backbone N N N
R1 O N O
O H
O R4 H O
R2 R6

R6 R2
O H R4 O
H
O
O N O R1
N N N Heptapeptide
backbone
R5 O H N H
N CO2 NH2Me
R3 H
H
R7

O H O
H Me
N
Cell N O L-Lys-D-Ala-D-Ala tail
Cellwall
wallbuilding
buildingblock
block H
O Me H
 Vancomycin and vancomycin analogues
Drug resistance
•Vancomycin-resistant Staphylococcus aureus (VRSA) (1996)
•Vancomycin-resistant enterococci (VRE) (1989)
•Resistance due to mutation in pentapeptide chain of cell wall building block
•Terminal D-alanine replaced by D-lactate

O O O O
H Me H Me
H Mutation
N O
N O Cell wall building block N O
Cell wall building block H H
O Me H O Me H

L-Lys-D-Ala-D-Ala tail L-Lys-D-Ala-D-Lactate tail

•Peptide link replaced by ester link

•Loss of NH (HBD)
•Weakens binding affinity of vancomycin with ‘tail’
•Lactate acts as a leaving group in cell wall synthesis
 Vancomycin and vancomycin analogues
Drug resistance
•Vancomycin-resistant Staphylococcus aureus (VRSA) (1996)
•Vancomycin-resistant enterococci (VRE) (1989)
•Resistance due to mutation in pentapeptide chain of cell wall building block
•Terminal D-alanine replaced by D-lactate

O O O O
H Me H Me
H Mutation
N O
N O Cell wall building block N O
Cell wall building block H H
O Me H O Me H

L-Lys-D-Ala-D-Ala tail L-Lys-D-Ala-D-Lactate tail

•Peptide link replaced by ester link

•Loss of NH (HBD)
•Weakens binding affinity of vancomycin with ‘tail’
•Lactate acts as a leaving group in cell wall synthesis
 Vancomycin and vancomycin analogues

Teicoplanin HO CH2OH
Alkyl anchor O
HO O

OH N O
H Cl
HO NHAc O O
C D E
HO O Cl OH
O
O O
H H H H O
O N O NH3
N N N H
Heptapeptide backbone H H
O H N H
N CO2
H
H
B
HO OH
A
O
HO OH
O
HO
O
OH
HO
OH
 Vancomycin and vancomycin analogues

Teicoplanin
•Isolated from a soil micro-organism
•Does not dimerise
•Alkyl chain anchors the antibiotic to the outer surface of the cell membrane
•Less toxic than vancomycin

Teicoplanin

Building
block

Alkyl chain anchor

Cell membrane
 Vancomycin and vancomycin analogues
Eremomycin - naturally occurring glycopeptide

H3N Me
HO HO CH2OH

Me O HO O

O
H3N Me O Cl
HO O O

O OH H3C CH3
H
Me O
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH
 Vancomycin and vancomycin analogues
LY 333 328 - analogue of eremomycin

Biphenyl hydrophobic tail

Me CH2 NH2 Me
HO HO CH2OH

Me O HO O

O
H3N Me O Cl
HO O O

O OH H3C CH3
Cl
Me O
O O
H H H H O
O N O
N N N
H H H
O H N H
N CO2 NH2Me
H
H
CONH2

HO OH
OH

1000 x more active than vancomycin

 Vancomycin and vancomycin analogues
Simplification
•Simplified structures capable of binding D-Ala-D-Ala or D-Ala-D-Lac
•Lead compounds for further development

OH
O

H3C CH3
O
H H O
N O
AA1-AA2-AA3 N N
H H
O H N H
NH2Me
H
CONH2
 Bacitracin A

S
H2N
N
L-His-D-Asp-L-Asn O
D-Phe  L-Leu
L-Ile-D-Orn-L-Lys-L-Ile-D-Glu


• A polypeptide complex (natural)

• Binds to the lipid carrier responsible for transporting the NAM/pentapeptide unit across the cell membrane

preventing the lipid carrier from carrying its role

Inhibitors of protein synthesis

• Aminoglycosides

• Tetracyclines

• Chloramphenicol

• Macrolides
https://www.ncbi.nlm.nih.gov/books/NBK22022/
 Aminoglycosides-cidal-

• These compounds inhibit protein synthesis by binding to ribosomes

• Inhibit different stages of the translation process (prevents repair, cellular growth and reproduction)

• Their selective toxicity is due to:

1. Different diffusion rates through the cell barriers of bacteria vs mammalian cells
2. Difference between the ribosomal target structures [prokaryotic ribosomes (70S), eukaryotic ribosomes (80S)]

• Sufficient difference in ribosomes structures

• Possible for drugs to distinguish between them

(lower affinity for ribosomes in human cells, which explains their selectivity)

• At normal doses, they do not bind or interfere with the function of eukaryotic ribosomes (80S)
 Aminoglycosides and aminocyclitols
Pharmacophore is 1,3-diaminoinositol moiety consisting of either:
H2NC=NHHN
H3CHN OH H2N
HO HO HO
HO OH
HO HO
OH NHC=NHNH2 NHCH3 NH2
OH OH
Streptidine Spectinamine 2-Deoxystreptamine
(Streptamine)
• A carbohydrate structure which includes basic amine group and form acid salts
• Highly water soluble (polar), have to be injected (if given orally, their action is limited to the GIT)
• Active against G-ve bacteria omly
• At pH 7.4, they have a positive charge that is beneficial to activity

Enhance absorption through the outer membrane of G-ve

An ionic interaction takes place with many –ve charged groups on the outer surface of the cell membrane
Displaces Mg2+ and Ca2+ ions (these ions act as bridges between lipopolysaccharides)
Their displacement results in rearrangement of cell membrane components to produce pores through
which an aminoglycoside can pass
Crosses the cell membrane, trapped inside the cell and accumulates in high concentrations
Binding to bacterial ribosomes to inhibit protein synthesis
• Binding specifically to the 30S ribosomal subunit (through formation of hydrogen bond)
• Prevents the movement of the ribosome along mRNA (the triplet code on mRNA can no longer be read)
• In some cases, protein synthesis is terminated and the shortened proteins end up in the cell membrane
leads to a further increase in cell permeability
Greater uptake of the drug

• Aminoglycosides are bactericidal

• Their activity may be due to their effects both on the ribosomes and the outer cell membrane
 Mechanism of action

Aminoglycosides bind 30S ribosomal sub-particle

• Inhibit the initiation of protein synthesis

• Misreading mutations of the genetic code conformational changes in the site
of the ribosome on binding to aminogylcoside mistranslation of RNA templates
and the selection of wrong amino acid formation of nonsense proteins
 H2NH2C
HO
O
HO H2NH2C
HO
R O
HO
H2N
O HO
2-deoxystreptamin ring HO NH2 O
H2N
O
HO NHCOCHOHCH2CH2NH2
O
HOH2C O OH
HO HOH2C O OH
H2N
HO
H2N
Kanamycin A, R= OH
Kanamycin B, R= NH2

Kanamycin Amikacin

H2NH2C H2N(CH3)CH
HO
O O
H
H2N H2N
H2N H2N
O O
HO HO NH2
NH2 O
O

O O OH
HOH2C OH
H3C
HO NHCH3
H2N
OH

Tobramycin Gentamicin C-2

 HO
NHCH3 HO
HO H3CHN OH
O H2N O
NH O
HO O HN O
O
NHCH3
HO HO OH NH OH
-Streptose O O
OHC HO NH NH2 CH3
CH3
Streptobiosamine Streptidine

Spectinomycin
Streptomycin
• The diaminoinositol unit (spectinamine)
• It was introduced in 1943 primarily for the treatment of contains two mono-N-methyl groups, and
tuberculosis. the hydroxyl between them has a
• Streptomycin differs from the typical aminoglycosides with a stereochemistry opposite to that in
modified pharmacophore in that the diaminoinositol unit is streptomycin.
• streptamine • The glycosidically attached sugar is also
• Two highly basic guanido groups at C-l and C-3 in place of unusual and is fused by two adjacent
the primary amine moieties of 2-deoxystreptamine linkages to spectinamine to produce an
unusual fused three ring structure.
• Spectinomycin is bacteriostatic.
• It is used in a single bolus injection
intramuscularly against Neisseria gonorrhea
Drug-drug incompatibility between β-lactams and aminoglycosides

• The two drugs react with each other, resulting in inactivation of both antibiotics

• Should not be mixed in the same solution and should be administered into different
tissue compartments (one in each arm) to prevent this
because NH group will bind to beta lactam (interaction btw nucleophy and elecrophy
 Macrolides-static-

• Macrolide Large lactone (Macrocyclic lactone) Cyclic ester

• Have two or more characteristic sugars (cladinsoe and desosamine-amino sugar)

attached to the ring macro-ring (14 membered ring)
 Characteristics

1. Large lactone ring (12, 14, 16 membered ring)

2. Amino sugar at position 5 is essential for activity

3. Hydroxyl grope at position 6 is essential for activity

4. Contain at least one ketone moiety

5. Weak bases (N-dimethyl group present in the amino sugar) form salts with acids

6. They bind to the 50S subunit of bacterial ribosomes

7. Prevents the growing peptide from becoming longer

8. Resulting in the dissociation of peptidyl tRNA (inhibit translocation)

 Erythromycin

N(CH3)2
CH3 HO
O

H3C CH3
HO O O CH3
HO
OH
H3C CH3
C2H5 O O CH3
O
CH3
O OH
H3C OCH3

• Free, salts, pro-drug

• Chemical modifications to improve oral absorption and increase acid stability

 Acid instability (ketal formation)

Acid sensitivity is due to the presence of a ketone and 2 alcohol groups

(acid catalyzed intramolecular formation of a ketal)

Chemically unstable because of rapid acid catalyzed internal cyclic ketal formation
leading to inactive compounds, which occurs in the GIT and thus associated with
GI cramping
How to improve acid instability (ketal formation)
 Semisynthetic compounds
N(CH3)2 N(CH3)2
CH3 HO CH3 HO
O 8
9
H3C CH3 H3C 9aNCH3 CH3
O CH3 N(CH3)2
HO H3CO O O CH3 CH3
HO HO O HO
OH NH
CH3 OH CH3OCH2CH2O
H3C CH3
C2H5 O O CH3 H3C H3C CH3
O C2H5 O O CH3 O O O CH3
CH3 O HO
CH3 OH
O OH CH3
OH H3C
H3C OCH3 O C2H5 O O CH3
H3C OCH3 O
CH3
O OH
H3C OCH3
Clarithromycin Azithromycin

• Prevent ketal formation by
protecting 6-OH
• C-6 hydroxyl converted
Semi-synthetically to methoxy
Dirithromycin
N-methyl inserted between
C-9 and C-10 (ring expansion)
And absent of the carbonyl moiety

Lipophilic, prevents ketal formation

Prevents inactivation, less GI cramp No ketal formation
More stable to acid
Improved oral absorption
 Tetracyclines –static-
7 6 5 4 7 6 5 4
6a 5a 4a 5a 4a
8 3 3
Partial reduction 8

9
D C B A
2 A, B, C 9 2
10a 11a 12a 11a 12a
10 11 12 1 10 11 12 1

Naphthacene 1,2,3,4,4a,5,5a,6,11,11a,12,12a-
dodecahydronaphthacene
HO CH3H H N(CH3)2
OH
H
NH2
OH
OH O OH O O
Tetracycline

• Four linearly fused-6-membered rings

• Amphoteric compounds forms salts with acids or bases

C-4 dimethylamino (ring A) Basic

C-1 to C-3 conjugated trione Acidic
 Tetracyclines

•C-4 dimethylamino Basic

C-1 to C-3 conjugated trione Acidic

• C-4 dimethylamino must be α-oriented

• 6-demethyl-deoxytetracycline(1,2,3,4,10,11,11a,12,12a) represents the hydrophilic

parts of the molecule and can not be changed.
Binding interaction of tetracycline with RNA of bacterial ribosomes
 Chelation

H3C CH3
H3C OH N
OH

NH2
OH
OH O O O O
M

• The acidic functions of the tetracyclines are capable of forming salts (complex)
through chelation with metal ions. So we cant give it with milk
• Tetracyclines are incompatible with multivalent ion rich antacids, milk……..
• α,β-unsaturated carbonyl with acidic center at β-position.

• make chelation Stable Complexes at acidic and physiological conditions,

insoluble in water and reduce tetracyclines absorption

• Chelation occurs at C-11, C-12.

 Chelation

• Bones of which teeth are the most visible are calcium rich

• Tetracyclines accumulates when bones and teeth are being formed

• Because tetracyclines are yellow this leads to permanent discoloration

(in advanced cases, the teeth are even brown-photochemical process-)

• Cosmetically unattractive

• To avoid this, tetracyclines are not given to children

 Epimerization CH3
H3C
N H
OH
CH3 CH3 H3C CH3 NH2
H3C N H H3C N H
N
OH OH enolization OH
H H O O
NH2 Tetracycline
NH2 NH2
CH3
O O O O H3C
OH O N H
OH
Tetracycline Enol NH2

O O
4-Epitetracycline
• The α-stereo orientation of the C-4 dimethylamino moiety is essential for the activity
• The presence of tricarbonyl system of ring A allows enolization alcohol with double bond loss of C-4 hydrogen
• Reprotonation takes place either from:
top of the enol tetracycline 50%
bottom of the enol 4-epitetracycline 50% (inactive)
• At equilibrium, the mixture consists of equal amounts of both diastereoisomers

Old preparation can lose 50 % of their potency

• The epimerization process is most rapid at pH 4, and is slower in the solid-state.
 Dehydration
H3C CH3 H3C CH3
H3C OH N H3C OH2 N
OH OH
H H H
NH2 NH2
OH OH
OH O OH O O OH O OH O O

H2O

H3C CH3 H3C CH3

CH3 N CH3 N
OH OH
H
H
NH2 enolization NH2
OH OH
OH OH O O O OH O O O O
H
5,6-anhydrotetracycline
(inactive)

• Most of the natural tetracyclines have 3° and benzylic OH group at C-6

• Ideal geometry for acid catalysed dehydration (-H2O) including the C-5aα-oriented hydrogen (trans with respect to each other

• Resulting in naphthalene derivative (energetic reasons for the reaction proceeding in that direction)

• C-5a,6-Anhydrotetracycline is inactive and much deeper in color than tetracyclines

• Discolored old tetracylines are suspect, and should be discarded
 Fanconi-like syndrome

• Epimerization and dehydration to produce 4-epianhydrotetracycline

• Toxic to the kidneys and leads to Fanconi-like syndrome, in certain cases is fatal

• Tetracyclines lacking C-6 hydroxyl, can not undergo dehydration free from this toxicity
 Tetracyclines in basic medium

H3C CH3 H3C CH3

H3C OH N N
H3C O
OH OH
OH
NH2 NH2
OH OH
OH O OH O O OH O OH O O

H3C CH3
CH3 N
OH
O
NH2
OH
OH O O O O

Isotetracycline
(inactive)

• In alkaline solutions at or above pH 8.5

• Cleavage of ring C (involving C-6 OH)

• Resulting in lactone product (isotetracycline) which is inactive

 Phototoxicity

Cl HO H H N(CH3)2
H
OH
H
NH2
OH
OH O OH O O

C-7 chlorine atom is associated with phototoxicity

Absorbs light in the visible region

Leading to free radical generation

Causes severe erythema (redness of skin) upon exposure to sunlight

Sunburns
Cl HO H N(CH3)2 H3C CH3
H H N H N(CH3)2
H H
OH H
OH
H
H
NH2
OH NH2
OH O OH O OH
O OH O OH O O
Demeclocycline Minocycline

H H H H N(CH3)2
OH HO CH3OHH N(CH3)2
OH
H
H
NH2
OH NH2
OH O OH O O OH
OH O OH O O
Sancycline Oxytetracycline

CH2 OH N(CH3)2
H CH3 OH N(CH3)2
OH H H
OH
H
H
NH2 NH2
OH OH
OH O OH O O OH O OH O O
Methacycline Doxycycline
 Chloramphenicol

H CH2OH
Cl2CHCON H
O
H OH
OH
palmitic acid

NO2
O
O
• Two chiral centers OH
OH
hemisuccinate
• (1R,2R) is the only active one

• Binds to 50S subunit, inhibit the movement of ribosomes along mRNA

• Prodrug: C-3 palmitate to mask the bitter taste

C-3 hemisuccinate to improve water solubility

• The nitro group and both alcohols are involved in binding interactions

• The nitro group is thought to be responsible for chloramphenicol toxicity

 Bacterial drug targets
Inhibitors of cell wall
synthesis
Inhibitors of folic acid
synthesis

Pteridine

Tetrahydrofolic Folic acid

acid

Ribosomes
Purines Pyrimidines
50S & 30S

mRNA
DNA
Inhibitors of protein
synthesis Inhibitors of DNA
synthesis
 Lead compound

SO2NH2
NH2 SO2NH2
N Liver [H]
N
H2N
H2N

Prontosil Rubrum Sulfanilamide

• Prontosil - red dye

• Antibacterial activity in vivo (1935)

• Inactive in-vitro
• Acts as pro-drug

• Metabolized to active sulfonamide

• Sulfanilamide; the first synthetic antibacterial agent

 Quiolones

First-generation Quinolones:

O O O
COOH O COOH O COOH

N
H3C N N O N O N

Nalidixic acid Cinoxacin Oxolinic acid

 SAR and mechanism of action

• Inhibit DNA gyrase and topoisomerase IV inhibition of DNA synthesis

SAR
2nd, 3rd and 4th generation of flouroquiolones
 SAR

Carboxy-4-pyridone

• The pharmacophore, essential for activity, must be fused with aromatic ring
• The nucleus for all quiolones, responsible for affinity
• Any modification on the pharmacophore loss of activity

Position 1

• Lipophilic substituents are essential (alkyl substitution, methyl, ethyl, cyclopropyl)

• Excessive lipophilic (2,4-fluorobenzene) decrease activity
• Influence pharmacokinetics and control potency, cyclopropyl is optimal

Position 2 =3=4

• Must be free, it is the position where the enzyme acts

• Any substitution loss of activity
 SAR
Position 6

• Almost all clinically useful quinolones bear a fluorine atom at this position
• Fluoro substituent is optimal increased antibacterial activity
increased lipophilicity
Position 8

• Substituents need to be small

• Fluoro substituent improves absorption and half life, and increase incidence
of photosensitivity

• Methoxy substituent decrease the incidence of photosensitivity

Position 5
• Small hydrogen bond donor or acceptor
• Increase activity against gram + bacteria
• amino moiety decrease incidence of photosensitivity among fluoroquinolones
 SAR

Position 7
• Heterocyclic substitution improves the spectrum of activity against gram -bacteria

• Piperazinyl and pyrrolidinyl greatest antimicrobial improvements

• Bind to GABA receptor, responsible for CNS side effects

• Adding methyl group at position 3' and/or 5' decreases the binding to the GABA
 Drugs Acting On DNA
Topoisomerase poisons - non-intercalating
Quiolones and FluoroQuinolons

O O
COOH F COOH

N N N N
HN

Nalidixic acid Ciprofloxacin

• Synthetic agents used as antibacterial agents (bactericidal)

• [inhibit DNA gyrase and Topo IV, key bacterial enzymes that command the conformation of DNA]

• Binding site for agents revealed once DNA strands are ‘nicked’

• Inhibit the replication and transcription of bacterial DNA by stabilizing the complex between bacterial DNA and
topoisomerases

• G +ve: the stabilized complexes are between DNA and Topo IV, drugs showing 1000 fold selectivity for the bacterial
enzyme over the corresponding enzyme in human cells
• G -ve: the stabilized complexes are between DNA and Topo II (DNA gyrase), has the same role as Topo IV
 Drugs Acting On DNA
Topoisomerase poisons - non-intercalating
Quiolones and FluoroQuinolons

Topoisomerase
enzyme
Region binding to DNA
R5 O O
Region
R6 binding
Region O
binding to enzyme
to enzyme
R7 X8 N
R1
Stacking domain

Fluoroquinolones

• Four drug molecules are stacked in the bound complex

• Bound to DNA and enzyme by hydrogen and ionic bonds
Supercoiling of circular DNA catalyzed by DNA gyrase

(+) (-)
Step 1 Step 2 Step 3

(-) (-) (-)

A. View from the top: Step 1. Stabilize positive node. Step 2.

Break both strands of the back segment. Step 3. Pass
unbroken segment through the break and reseal on the front side.

Upper (crossing)
DNA segment
Lower DNA
segment

Step 1
Step 2
Step 3

Step 4

B. View from the side: Step 1. Staggered cuts in each strand.

Step 2. Gate opens. Step 3. Transverse segment passed through
the break. Step 4. Reseal cut segment.
https://www.youtube.com/watch?v=EYGrElVyHnU
 Drugs Acting On DNA

• Different Fluoroquinolones inhibit these essential enzymes to different extents, which explains some of the
differences in the spectrum of activity of Fluoroquinolones

• Humans shape their DNA with Topo II, an analog enzyme to bacterial DNA gyrase.

• Human Topo II does not bind Fluoroquinolones at normally achievable doses

• Commercial Fluoroquinolones do not kill host cells (human calls)

 Quiolones and FluoroQuinolons
Early quinolones
O O
5 4 COOH F COOH
6
3 From bedside to bench
7 2
N N N N N
8 1
By adding F at C-6 greatly HN
Increased biological activity Enoxacin
Nalidixic acid

• 1st marketed quinolone (discontinued) • Improved broad spectrum activity due to:

• Classified as 1st generation quinolone
(based on spectrum and pharmacokinetic properties)
• Limited to small number of G-ve, rapid resistant developed
(little clinical significance)
 F at C-6: increased activity and cellular uptake
 A basic substituent at C-7 (piperazine) enhanced
pharmacokinetic properties by forming zwitterion
with COOH at C-3

• Various analogs synthesized but no great advantage • Due to the great impact from adding Fluorine, the
chemical class have been named Fluoroquinolones
• Quinolones went to bedside
 Quiolones and FluoroQuinolons
Fluoroquinolone optimization
O
O O
F COOH
F COOH F COOH
Bio-isoster Intense research N N
N N N N N
HN
HN Replacing N HN Resulted in thousands
by C at 8 of analogs
Enoxacin Norfloxacin Ciprofloxacin

• 1st generation • 2nd generation • 2nd generation

• Replacing N at 8 with C: • Introducing cyclopropyl at N-1:

 Reduced side effects  increased broad spectrum
 Broad spectrum (increased activity against G-ve)
(increased activity against G-ve)+
• Optimum substituent at N-1
• Equivalent potency to
many natural antibiotics
 Quiolones and FluoroQuinolons
Fluoroquinolone optimization
O O
F COOH F COOH

N-methylation
N N N N
HN N
H3C

Ciprofloxacin

• Piperazine binds to GABA receptors (CNS) • Alkyl substitution on the Piperazine nitrogen decreases
binding to GABA receptors
• Accounts for CNS side effects
• Resulting in less CNS side effects
 Quiolones and FluoroQuinolons
Synthesis of large number of Fluoroquinolone O O O
F COOH F COOH F COOH

O O N N N N N N
 HN
F COOH F COOH N O O HN O
H
N N N N Ofloxacin (racemic)
Moxifloxacin Gatifloxacin
HN HN Levofloxacin (S)

Norfloxacin Ciprofloxacin O O
F COOH F COOH
2 generation
nd
O
N N N N N
N
Cl
O O
H2N
F NH2
OH Besifloxacin
Pharmacophore Gemifloxacin

N N
HN R
3rd and 4th generation

• All have similar bicyclic ring system that includes a pyridone ring and COOH at C-3

• 1st and 2nd generation show moderate activity against S. aureus followed by resistance and only slight activity against
aerobic bacteria

• 3rd and 4th generation solved this problem and have broad spectrum
 SAR
R5 O O
Cell wall penetration R6 OH Pharmacophore
R7 N
R8 R
Spectrum of activity
Potency

• Pharmacophore: carboxy-4-pyridine
• COOH + Ketone: involved in binding to the DNA and DNA gyrase enzyme
• Reduction of the C 2,3-double bond or the 4-keto: inactive compounds
• Substitution at C-2 interferes with the enzyme-drug complexation
• F at C-6: greatly improves antimicrobial activity by:
1. Increasing lipophilicity: improves penetration through the bacterial cell wall
2. Increasing DNA gyrase and Topoisomerase IV inhibitory action (through binding)
• F or Cl at C-8: further improves drug absorption and half-life, but also increase drug-induced sensitivity
• OCH3 at C-8 reduces the photosensitivity
 SAR
R5 O O
Cell wall penetration R6 OH Pharmacophore
R7 N
R8 R
Spectrum of activity
Potency

• C-7 heterocycles improves the spectrum of activity especially against G-ve

(piperazine and pyrrolidine) show most significant antimicrobial improvement
• They also increases binding to GABA receptors in the CNS (which accounts for CNS side effects)
• Alkyl substitution on the piperazine N decreases binding to GABA (less CNS side effects)
• Cyclopropyl at C-1: broaden spectrum to include atypical bacteria
• Substitution of a 2,4-difluorophenyl at N-1 improves antimicrobial potency, but produces serious side effects
(withdrawn from the market: trovafloxacin and temafloxacin)
• Introducing a 3rd ring to the nucleus of the quinolone enhances potency
 SAR NH
N N
M O
F
O O
O O
F C O
Mg
N N
O O
R
F
O

N N
HN

• Chemical incompatibility is common to all the quinolones, they are able to chelate polyvalent metal ions
(Ca2+, Mg 2+, Zn 2+, Fe 2+, Al 3+)

Resulting in decreased solubility and absorption of the drug

• Chelation occurs between the metal and the 3-carboxylic acid and 4-keto groups (α,β-unsaturated keto acid)

Substances containing polyvalent metals should be given separately from the quinolones
 Pharmacokinetics

• 1st generation Fluoroquinolones were rapidly excreted into the urine

Their therapeutic application was limited to UTIs

• 2nd, 3rd and 4th generations are distributed to alveolar macrophages, bronchial mucosa and epithelial lining fluid

Improving their use in various systemic infections