Antimicrobials Medicinal Chemistry Fall 2005 Antimicrobial

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Antimicrobials

• Medicinal Chemistry
• Fall 2005

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Antimicrobial Agents

• Antibiotics natural substances produced by
  microorganisms
• Semi-synthetic antibiotics chemically modified
  natural products
• Synthetic antibiotics chemically synthesized
  natural substances
• Chemotherapeutic agents chemically synthesized
  agents

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Microbes in History
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Historical Perspective of Antibiotics

• Ancient remedies and observations
• 1500 BC Ancient Chinese recognized the
  therapeutic properties of moldy soybean curd on
  boils and similar infections
• 1871 Joseph Lister noted that urine samples
  contaminated with mold did not allow the growth
  of bacteria and tried to identify the agent
  antibacterial agent in the mold.
• 1874 William Roberts observed that cultures of
  the mold Penicillium glaucum did not exhibit
  bacterial contamination
• 1877 Pasteur and Joubert noted that anthrax
  bacilli were inhibited when the cultures were
  contaminated with mold
• 1897 Ernest Duchesne reported in his
  dissertation the discovery, partial refinement
  and successful testing of a substance with
  antibiotic properties
• Modern Era of antimicrobial therapeutics
• 1928 Flemmings discovery of penicillin
• 1935 Domagks discovery of sulfonamides
• 1939 Ernst Chain, Howard Florey, Edward Abraham
  purified and stabilized a form of penicillin
• 1940s WWII Production of penicillin
• Isolation of Streptomycin
• Isolation of Chloramphenicol
• Isolation of Tetracycline
• 1950S Antibiotics in clinical usage

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Antimicrobial Agents

• Antibiotic therapy is prescribed for 30 of all
  hospitalized patients.
• Antibiotic therapy has grown to be one of the
  most misused by physicians.
• Widespread use has allowed for the emergence of
  antibiotic resistant pathogens.
• Selection of an antimicrobial agent is a complex
  procedure microbiological factors and
  pharmacological considerations.

Ultimate Goal a drug that is selective against
the infecting organism and has the least
potential to cause harm to the host patient.
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Antimicrobial Agents

• Effect on microbes
• Cidal (killing) effect
• Static (inhibitory) effect
• Spectrum of action
• Broad Spectrum effective against procaryotes
  which kill or inhibit a wide range of Gram and
  Gram- bacteria
• Narrow spectrum effective against mainly Gram
  or Gram- bacteria
• Limited spectrum effective against a single
  organism or disease

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Antimicrobial Agents
Characteristics of a clinically-useful antibiotic

• Wide spectrum of activity
• Nontoxic to the host and without undesirable side
  effects
• Non-allergenic to the host
• Not eliminate the normal flora of the host
• Be able to reach the part of the body where the
  infection is occurring
• Inexpensive and easy to produce
• Chemically stable (long shelf life)
• Unlikely to develop microbial resistance

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Your Basic Bacteria
How we think of bacteria
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Antibiotic Targets

• Inhibition of bacterial cell wall synthesis
• Interactions with the cell membrane
• Disruption of protein synthesis
• Inhibition of DNA and RNA synthesis
• Inhibition of cell metabolism-Folate synthesis

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Antibiotic Targets
www.ratsteachmicro.com
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Antimicrobial Drugs Mode of Action

• ?-lactams
• Penicillin G, Cephalothin
• Semisynthetic penicillin
• Ampicillin, Amoxycillin
• Glycopeptides
• Vancomycin
• Clavulanic Acid
• Clavamox
• (clavulanic acid amoxycillin)
• Sulfonamides
• Sulfa drugs

Inhibit steps in cell wall (peptidoglycan)
synthesis and murein assembly
suicide inhibitor of beta-lactamases
Inhibit cell metabolism Folate synthesis
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Antimicrobial Drugs Modes of Action

• Aminoglycosides
• Streptomycin
• Macrolides
• Erythromycin
• Tetracyclines
• Tetracycline
• Quinolones
• Ciprofloxacin
• Rifamycins
• Rifampicin
• Polypeptides
• Bacitracin

Inhibits translation (protein synthesis)
Inhibits nucleic acid synthesis
Damages cytoplasmic membranes
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Types of Antimicrobial Resistance

• Intrinsic Resistance
• Natural or built in resistance based on the
  characteristics of a particular strain or
  species.
• Acquired Resistance
• Acquisition of new genetic information or
  mutation of the existing genome that protects the
  bug from the effects of an antibiotic.

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Mechanisms of Resistance

• Impermeability
• Some bacteria are naturally resistant to
  antibiotics because their cell envelope is
  impermeable to a particular class of antibiotics.
• Antibiotic Modification
• Enzyme inactivation
• Organism spontaneously produces an enzyme that
  degrades the antibiotic.
• Staphylococcus arueus produces an extracellular
  enzyme
• ?-lactamase.
• Enzyme addition
• Bacteria may express enzymes that add a chemical
  group to the antibiotic, inhibiting its activity.
• Addition of an amino, acetyl or adenosine group
  to aminoglycosides.

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Mechanisms of Resistance

• Efflux mechanisms
• Acquisition of an inner membrane protein which
  actively pumps the antibiotic out of the cell.
• E. coli acquires resistance to tetracyclines
• Alternative pathway
• Bacteria acquire a gene to code an alternative
  penicillin binding protein which is not
  inhibited.
• Alteration of the target site
• Point mutations, insertions or deletions alters
  the site of inhibition thus conferring resistance.

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Transmission of Antimicrobial Resistance

• Transformation
• bacteria takes up naked DNA and incorporate it
  into their genome.
• Conjugation
• Plasmids (circular portions of DNA found in the
  cytoplasm) are passed from one bacterium to
  another.
• Transposons
• Moveable genetic elements able to encode
  transposition. Can move between the chromosome
  and the plasmids and between bacteria.

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Its an uphill battle with the bugs
Antibiotic (e.g. penicillin)
Antibiotic (e.g. streptomycin)
Enzymes that degrade antibiotics (e.g.
beta-lactamases)
Enzymes that alter antibiotics addition of
amino, acetyl or adenosine group to
aminoglycosides
Plasmid with resistance genes.
Antibiotic (e.g. tetracycline,
fluoroquinolone)
Chromosome Changes to an antibiotics target
Pumps that transport antibiotics out
of the cell.
(e.g. a protein involved in cell wall synthesis
prevents inhibition.)
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Factors That Accelerate Microbial Resistance

• Inadequate levels of antibiotics at the site of
  infection.
• Duration of treatment too short
• Overwhelming numbers of organisms
• Overuse/misuse of antibiotics

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Mechanisms to Reduce Antibiotic Resistance

• Control, reduce or cycle usage
• Improve hygiene personal and in hospitals
• Discover or develop new antibiotics
• Modify existing antibiotics chemically to produce
  compounds inert to known mechanisms of resistance
• Develop inhibitors of antibiotic-modifying
  enzymes
• Define agents that would cure resistance
  plasmids

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Cell Wall Structure of Gram() and Gram(-)
Bacterium
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Bacterial Cell Wall Synthesis Stage I

• Formation of starting materials takes place in
  the cytoplasm
• N-Acetylglucosamine 1-Phosphate and uridine
  triphosphate (UTP) are converted to
  uridinediphosphoN-acetylglucosamine (UDPNAG)
• Condensation with elimination of pyrophosphate
• UDPNAG reaction with phosphoenolpyruvic acid
  (PEP) with transferase gives the enolic ether.
• Reduction of the double bond by NADPH utilizing
  reductase enzyme gives N-acetylmuramic acid (as
  the uridine derivative)
• Three amino acids are added to the muramyl
  peptide to give the tripeptide using ATP and
  enzymes specific for the aa.
• Two more aa are added D-alanine-D-alanine
  added after 2 D-alanines were synthesized via
  D-ala-Dalasynthetase. D-ala is from racemization
  of L-ala by racemase enzyme.
• UDPNAM-pentapeptide

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Inhibitors of Bacterial Cell Wall Synthesis
Stage I

• Fosfomycin
• inhibits the enol-pyruvyl transferase by direct
  nucleophilic attack on the enzyme.
• Note Mammalian enzymes are not inhibited,
  thus no effect
• on the host.

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Inhibitors of Bacterial Cell Wall Synthesis
Stage I

• Cycloserine
• inhibits both alanine racemase and
  D-alaninyl-D-alanine synthetase
• Note the similarity in cycloserine and
  D-alanine.
• Cycloserine actually binds to the enzymes
  better than the D-alanine

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Bacterial Cell Wall Synthesis Stage II

• Peptidoglycan Synthesis Reactions take place at
  and are catalyzed by membrane bound enzymes
• The pentapeptide is linked to a phospholipid
  membrane- bound carrier bactoprenol (55C
  isoprenoid alcohol esterified with phosphoric
  acid).
• It is now anchored and the subsequent events
  occur in the interior of the cell membrane.
• A second sugar moiety is added by glycosidation
  and the UDP released are rephosphorylated to UTP
  and recycled to stage 1.
• 5 glycines are added (S. aureus) in sequence to
  the lycine residue each carried by the specific
  glycyl-t-RNA.
• The disaccharide-decapeptide monomer unit, which
  upon movement through the membrane is transferred
  following pyrophosphatase cleavage to an acceptor
  not yet identified.
• Separation from the membrane bound anchor leaves
  undecaprenyl phosphate which regenerate the
  original phosphate alcohol ester on hydrolysis by
  phosphotase and repeat the cycle.

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Inhibitors of Bacterial Cell Wall Synthesis
Stage II
binds to the membrane bound bactoprene
phosphate (membrane anchor) thus inhibiting
cleavage from the anchor to allow for transport
of the monomer unit to the outside of the cell.
- and of lesser significance, inhibits lycine
inclusion (stage I) into the murein structure.
Bacitracin
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Inhibitors of Bacterial Cell Wall Synthesis
Stage II
Vancomycin
interaction with the D-alanyl-D-alanine
portion of the forming mucopeptide involving
strong, but not covalent, bonding with the
hydroxylated phenyl glycine residues of the
antibiotic. Separation of the murein component
to the outside of the membrane is thus impaired
and cell wall synthesis is inhibited.
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Bacterial Cell Wall Synthesis Stage III

• Peptidoglycan Cross-Link outside the cell wall
• Polymerization of the subunits transfer of the
  new peptidoglycan chain from its carrier in the
  membrane to the cell wall.
• The terminal amine function of the pentaglycine
  side chain forms a new peptide bond at the
  expense of the terminal d-alanyl-D-alanine
  linkage of a neighboring peptidoglycan chain ?
  transpeptidation
• Transpeptidase enzyme cleaves the peptide bond
  between two D-alanine residues in the
  pentapeptide and become acylated via the carbonyl
  group of the penultimate D-alanine residue.

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Bacterial Cell Wall Synthesis Stage III
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Inhibitors of Bacterial Cell Wall Synthesis
Stage III
Cefalosporin C
Ceftriaxone
?-lactams inhibit the enzyme transpeptidase
responsible for
crosslinking peptidoglycans that comprise the
cell wall.
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?-Lactam General Mode of Action
A residue of the transpeptidase opens the
B-lactam ring thus acylating the enzyme. The
acylated enzyme is now too sterically crowded to
allow the cross-linking reaction to occur.
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?-Lactam Antibiotics
Penicillin
Cephalosporin
Clavulanic Acid
Thienamycin
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Cell Wall Synthesis Key Antibiotic Targets
Cleavage of monomer unit from cell membrane
anchor to allow for transport to exterior of cell.
Synthetase
D-Ala
L-Ala--------------?D-Ala
Racemase
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?-Lacatams
Penicillins
Cephalosporins

• Absolute Requirement
• ?-Lacatam ring
• Sulfur can be replaced
• Sulfur can be omitted
• Second ring is not necessary
• Carboxyl group can be replaced
• Amide side chain unnecessary

Moxalactam
Norcardicins
Tetrazolyl Penam
Thienamycin
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Its an uphill battle with the bugs
Antibiotic (e.g. penicillin)
Antibiotic (e.g. streptomycin)
Enzymes that degrade antibiotics (e.g.
beta-lactamases)
Enzymes that alter antibiotics addition of
amino, acetyl or adenosine group to
aminoglycosides
Plasmid with resistance genes.
Antibiotic (e.g. tetracycline,
fluoroquinolone)
Chromosome Changes to an antibiotics target
Pumps that transport antibiotics out
of the cell.
(e.g. a protein involved in cell wall synthesis
prevents inhibition.)
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Bacteria Fight Back

• Strategy I
• Decrease the penetration of the antibiotic to its
  target
• Strategy II
• Alter the target of the antibiotic
• Strategy III
• Inactivate the antibiotic with an enzyme

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Bacteria Fight Back
Abraham and Chain (1940) During purification of
penicillin they discovered bacteria that
inactivated antibiotics. Enzymes ?
penicillinases General class ? ?-Lactamases
Hydrolytic enzymes
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Cell Wall Structure of Gram() and Gram(-)
Bacterium
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Antibiotic Pathway
Penetrate outer membrane
Antibiotic
Arrive at cell wall and associate with penicillin
binding proteins (transpeptidases)
Avoid ?-Lactamase enzymes in the periplasmic space
Penicillins inactivate these by acylation of the
active sites
suicide substrate
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?-Lactamases
?-Lactamases cleave the N-carbonyl bond of the
?-Lactam thus inactivating the molecule.

• Plasmid-mediated
• Chromosome-mediated
• Physical properties
• Substrate specificity
• 5. Inhibition profiles

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Cell Wall Structure of Gram() and Gram(-)
Bacterium
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Hydrolysis by ?-Lactamase
?-Lactamase
Penicillin
?-Lactamase
Cephalosporin
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Degradation of Penicillin
?-Lactamase
Degradation H2O
Penicilloic Acid
Penicillamine
Penaldic acid
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One Strategy to Combat Penicillinases
Penicillin G
Methicillin
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Plan B not planned
Soil samples from various parts of the world were
tested
Streptomyces olivaceus
Potent ?-Lactamase inhibitor
Metabolites isolated olivanic acids
Streptomyces clavuligerus
Potent inhibitor
clavulanic acid
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Clauvlanic Acid Characterization
1 HPLC isolation 2 1HNMR 3 X-ray
(z)-(2R, 5R)-3-(?-hydroxyethylidene)-7-oxo-4-oxa-1
-azabicyclo3.2.0heptane-2-carboxylic acid
First example of a non-traditional ?-Lactam from
a natural source.
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Bioassay
?-Lactamase producing organism
Organism Grows
Penicillin G
Sample to be screened
Organism doesnt grow
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Inhibition Study
Table 14.3
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Irreversible Inhibitor of ?-Lactamases
NH2
NH2
NH
Clavulanic Acid
NH
NH
NH
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Proposed Biosynthetic Pathway
Liras Rodriquez-Garcia, 2000
oxidative deamination
Other clavams
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Synthesis of (?)Methyl clavulanate
See scheme 14.3 for synthesis
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Bacterial Protein Synthesis
http//www.cat.cc.md.us/courses/bio141/lecguide/un
it4/genetics/protsyn/translation/translation.html
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Inhibition Sites of Bacterial Protein Synthesis

• Binding of aminoacyl-t RNA to acceptor site
• Peptidyl transfer from the peptidyl t RNA to the
  newly bound aminoacyl t RNA on the acceptor site
• Translocation of the synthesized peptidyl t RNA
  from the acceptor site to the donor site

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Antibiotics that Inhibit Protein Synthesis

• Aminoacyl - tRNA formation use of imposter amino
  acids

N-ethylglycine
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Antibiotics that Inhibit Protein Synthesis

• Inhibitors of initiation complex formation and
  tRNA-ribosome interactions

Tetracyclines Aminoglycosides
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Inhibitors of peptide bond formation
translocation
Antibiotics that Inhibit Protein Synthesis
Erythromycin A
Chloramphenicol
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Tetracyclines

• Discovered in 1947
• Bacteriostatic (almost always)
• Enter via porins (G-) and by their lipophilicity
  in (G).
• Low toxicity, broad spectrum for both Gram- and
  Gram bacteria
• Selectivity results from transfer into bacterial
  cells but not mammalian cells
• Primary binding site is 30s ribosomal subunit.
  Prevents the attachment of amino acyl-tRNA to the
  ribosome and protein synthesis is stopped
• Resistance associated with ability of compound to
  permeate membranes and alteration of the target
  of the antibiotic by the microbe

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Tetracyclines SAR

• Carbon Modification
  Effect
• 1 any No activity
• 2 Slight activity
• 3 any No activity
• 4 must have ?-N(CH3)2
• 5 Active
• 5a lose H Inactive

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Carbon Modification
Effect 6 loss of OH or CH3
Active more stable 7 Cl, Br, NO3, N(CH3)2
Active 8 ----
---- 9 Cl and
CH3 Less active 10
Cannot change 11 Cannot
change Loss of activity 11a Cannot
change 12 Cannot change 12a
Change OH stereochemistry Decreases
activity
or remove
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Amphoteric Compound
pKa 9.1-9.7
Cannot be modified
Strong Chromophore pKa 2.8 3.3
Strong Chromophore pKa 7.2 7.8
Zwitterion exists at ph 4 7 (ph of duodenum)
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Degradation/Instability of Tetracyclines
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Aminoglycosides
Streptomycin

• represents some of the oldest antibiotics
• bactericiocidal
• works against Gm and Gm- bacteria
• binds to the S12 protein on the 30s ribosome to
  block normal
• activation of the initiation complex
• can alter membrane permeability increase
  membrane leakage
• can alter elongation of the peptide chain

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Macrolide Antibiotics
Erythromycin A

• active against Gm bacteria
• can be bacteriostatic or bacteriocidal
  depending upon the organism
• binds with high affinity to bacterial 50s
  ribosomes and interacts
• with the 23s ribosomal RNA
• protein synthesis is inhibited by the blockage
  of chain elongation

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Mode of Action of Erythromycin A

• Penetrates the periplasmic area by diffusing
  through porin lined aqueous channels.
• Enters bacterial cycoplasm by using energy
  dependent electron transport associated with
  oxidative phosphorylation.
• Drug interacts with ribosomes to prevent protein
  synthesis
• Drug binds the ribosome and causes a
  conformational change
• Ribosomes break down and associate with mRNA
  causing inhibition of normal synthesis

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Chloramphenicol

• active against Gm and Gm
• bacteriostatic
• mode of action involves reversible binding to
  the 50s
• ribosomal subunit
• binds aminoacyl-tRNA and prevents translocation
  of the
• peptide chain

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Bacterial DNA Synthesis

• For DNA replication to occur, the two strands
  must be separated.
• Separation results in excessive positive
  supercoiling (overwinding).

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Bacterial DNA Synthesis

• To avoid this, DNA gyrase is responsible for
  continually introducing negative supercoils into
  DNA.
• Both strands of the DNA are cut during this
  process thus allowing passage of a portion of the
  DNA through the break which is then resealed.

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Synthetic Antibiotics - Quinolones
Quinoline

• Inhibit DNA synthesis
• Interfere with the activity of DNA gyrase
• Bacteriocidal
• Newer agents are called fluoroquinolones
• Broad spectrum of activity

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Mode of Action

• Quinolones selectively inhibit bacterial DNA
  synthesis.
• Target DNA-gyrase (topoisomerase II enzyme
  found in procaryotic cells)
• 4-Quinolones inhibit ATP-dependent DNA
  supercoiling by binding to subunit A of
  DNA-gyrase.

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Bacterial Resistance to Quinolones

• Alteration of the target of the antibiotic
• in G resistance is due to a change in the DNA
  gyrase (mutation)
• Decrease penetration of the antibiotic
• in G- resistance is primarily due to a change
  in the porin proteins (can also be due to a
  change in the DNA gyrase)
• decreased uptake of the antibiotic by increased
  efflux of antibiotic from the cell.

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QSAR of Quinolones
Looked at ?, ?, and Es factors 78 Compounds

• R2 not a good correlation but
  proved to be the best group at the R2 site.
• For R4 Et, the following R1 groups were
  examined
• H, F, Cl, NO2, Br, CH3, OCH3, I
• Steric effects seemed most important optimal
  size F Cl
• R3 groups also exhibited steric effects with Cl
  and CH3 being optimal.
• R4 groups showed increased activity as steric
  bulk increased.

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Naldixic Acid

• 1st Generation Quinolone
• Used to treat urinary tract infections
• Polar nature of this compound allows for
  significant concentrations in the urinary tract
• Acts on DNA Gyrase
• Resistance acquired by a single mutation.

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Ciprofloxacin

• Introduced in 1991
• 2nd Generation Quinolon
• Broad Spectrum
• Typical MIC is .5 ?g/ml

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Rosaxacin

• Another 2nd generation 4-quinolone
• Pyridinyl substitution at position 7
• Not yet introduced in the us

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Combination pro-drug Approach
Cleaved by ?-Lactamase also

• ?-Lactam and 4-Quinolone
• Compound is broad spectrum antibiotic and
  exhibits 2 modes of action

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Where do we stand?Brown and Wright, Chemical
Reviews, 2005, 105, 759-774

• The existing arsenal of antimicrobials is
  insufficient.
• This is due to the drive of evolution that leads
  to antimicrobial resistance.
• Were unable to predict the nature of new
  emerging infections (HIV, SARS, avian flu).
• Drug companies are putting their resources into
  chronic diseases that promise long term profits.
• Humbling, isnt it.
• BUT Were better equipped to discover new
  targets and pathways for drug development.

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Nitro Compounds

• Nitrofurazone
• Broad spectrum antiparasitic compound

Mode of Action
bacterial nitroreductase
attacks DNA strands
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Nitroimidazoles

• Most effective compound

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Nitroimidazoles
Miconazole

• Imadazole crucial for activity

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Evolution of Discovery
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Mode of Action
Inhibition of Ergosterol Synthesis
Inhibited by imidazole

• Necessary for fungal membranes

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(No Transcript)
82

• Compounds selective for fungal demethylase vs.
  mammalian enzyme
• Increased levels of methylated sterols and
  decreased availability of ergosterol have
  negative effects on fungal membranes ? become
  more permeable and lose components.
• Imidazole portion of the molecule interacts with
  Fe atom in cytochrome P-450 which causes
  demethylation of sterol

SAR

• Need both N in imidazole

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Early Discovery of Activity

• Active in vitro against a broad range of fungal
  pathogens.

Chlortrimazole
Mode of Action

• Inhibition of fungal C-14 demethylase
  (cytochrome p-450 containing enzyme which is
  necessary for the fungal sterol, ergosterol

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• Initial Compounds
• Good topical activity
• Poor activity in animal models when taken orally
  or intravenously
• Rationale
• Imidazoles were susceptable to metabolic
  inactivation.
• Too lipophillic and were bound to plasma
  proteins.
• Result
• Low bioavailability

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Ketoconazole

• More stable to metabolism
• Less lipophillic
• Better bioavailability
• Still binds to plasma proteins

86
SAR
Initial Analogs

• Compounds compared well to ketoconazole but were
  still metabolized to a significant extent.

Metabolism site
87
Most active Compound
Fluconazole (Pfizer)
88
(No Transcript)
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Sulphonamides inhibitors of folate synthesis
p-aminobenzoic acid pteridine
dihydropteroate synthase
dihydropteroic acid
dihydrofolic acid
tetrahydrofolic acid