Beta-Lactam Antibiotics

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Beta-Lactam Antibiotics

• Clinically Important ß-Lactam Antibiotics
• Medicinal Chemistry Presentation
• David McLeod
• Southern Methodist University

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Introduction

• ß-Lactam antibiotics are the most widely produced
  and used antibacterial drugs in the world, and
  have been ever since their initial clinical
  trials in 1941.
• ß-Lactams are divided into several classes based
  on their structure and function and are often
  named by their origin, but all classes have a
  common ß-Lactam ring structure.

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History

• 1928- Alexander Fleming discovers a mold which
  inhibits the growth of staphylococcus bacteria
• 1940- penicillin is isolated and tested on mice
  by researchers at Oxford
• 1941- penicillin mass produced by fermentation
  for use by US soldiers in WWII
• 1950s- 6-APA is discovered and semi-synthetic
  penicillins are developed.
• 1960s to today- novel ß-lactams/ ß-lactamase
  inhibitors are discovered and modified from the
  natural products of bacteria

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Target- Cell Wall Synthesis

• The bacterial cell wall is a cross linked polymer
  called peptidoglycan which allows a bacteria to
  maintain its shape despite the internal turgor
  pressure caused by osmotic pressure differences.
• If the peptidoglycan fails to crosslink the cell
  wall will lose its strength which results in cell
  lysis.
• All ß-lactams disrupt the synthesis of the
  bacterial cell wall by interfering with the
  transpeptidase which catalyzes the cross linking
  process.

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Peptidoglycan

• Peptidoglycan is a carbohydrate composed of
  alternating units of NAMA and NAGA.
• The NAMA units have a peptide side chain which
  can be cross linked from the L-Lys residue to the
  terminal D-Ala-D-Ala link on a neighboring NAMA
  unit.
• This is done directly in Gram (-) bacteria and
  via a pentaglycine bridge on the L-lysine residue
  in Gram () bacteria.

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Mechanism
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Transpeptidase- PBP

• The cross linking reaction is catalyzed by a
  class of transpeptidases known as penicillin
  binding proteins
• A critical part of the process is the recognition
  of the D-Ala-D-Ala sequence of the NAMA peptide
  side chain by the PBP. Interfering with this
  recognition disrupts the cell wall synthesis.
• ß-lactams mimic the structure of the D-Ala-D-Ala
  link and bind to the active site of PBPs,
  disrupting the cross-linking process.

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Mechanism of ß-Lactam Drugs

• The amide of the ß-lactam ring is unusually
  reactive due to ring strain and a conformational
  arrangement which does not allow the lone pair of
  the nitrogen to interact with the double bond of
  the carbonyl.
• ß-Lactams acylate the hydroxyl group on the
  serine residue of PBP active site in an
  irreversible manner.
• This reaction is further aided by the oxyanion
  hole, which stabilizes the tetrahedral
  intermediate and thereby reduces the transition
  state energy.

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Mechanism of ß-Lactam Drugs

• The hydroxyl attacks the amide and forms a
  tetrahedral intermediate.

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Mechanism of ß-Lactam Drugs

• The tetrahedral intermediate collapses, the amide
  bond is broken, and the nitrogen is reduced.

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Mechanism of ß-Lactam Drugs

• The PBP is now covalently bound by the drug and
  cannot perform the cross linking action.

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Bacterial Resistance

• Bacteria have many methods with which to combat
  the effects of ß-lactam type drugs.
• Intrinsic defenses such as efflux pumps can
  remove the ß-lactams from the cell. ß-Lactamases
  are enzymes which hydrolyze the amide bond of the
  ß-lactam ring, rendering the drug useless.
• Bacteria may acquire resistance through mutation
  at the genes which control production of PBPs,
  altering the active site and binding affinity for
  the ß-lactam .

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Range of Activity

• ß-Lactams can easily penetrate Gram () bacteria,
  but the outer cell membrane of Gram (-) bacteria
  prevents diffusion of the drug. ß-Lactams can be
  modified to make use of import porins in the cell
  membrane.
• ß-Lactams also have difficulty penetrating human
  cell membranes, making them ineffective against
  atypical bacteria which inhabit human cells.
• Any bacteria which lack peptidoglycan in their
  cell wall will not be affected by ß-lactams.

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Toxicity

• ß-Lactams target PBPs exclusively, and because
  human cell membranes do not have this type of
  protein ß-lactams are relatively non toxic
  compared to other drugs which target common
  structures such as ribosomes.
• About 10 of the population is allergic
  (sometimes severely) to some penicillin type
  ß-lactams.

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Classes of ß-Lactams

• The classes of ß-lactams are distinguished by the
  variation in the ring adjoining the ß-lactam ring
  and the side chain at the a position.
• Penicillin

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Modification of ß-Lactams

• ß-Lactam type antibiotics can be modified at
  various positions to improve their ability to
• -be administered orally (survive acidic
  conditions)
• -be tolerated by the patient (allergies)
• -penetrate the outer membrane of Gram (-)
  bacteria
• -prevent hydrolysis by ß-lactamases
• -acylate the PBPs of resistant species (there are
  many different PBPs)

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Penicillins- Natural

• Natural penicillins are those which can be
  obtained directly from the penicillium mold and
  do not require further modification. Many species
  of bacteria are now resistant to these
  penicillins.
• Penicillin G
• not orally active

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Penicillin G in Acidic Conditions

• Penicillin G could not be administered orally due
  to the acidic conditions of the stomach.

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Penicillin V

• Penicillin V is produced when phenoxyacetic acid
  rather than phenylacetic acid is introduced to
  the penicillium culture. Adding the oxygen
  decreases the nucleophilicity of the carbonyl
  group, making penicillin V acid stable and orally
  viable.

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Production

• All commercially available ß-lactams are
  initially produced through the fermentation of
  bacteria.
• Bacteria assemble the penicillin molecule from
  L-AAA, L-valine, and L-cysteine in three steps
  using ACV synthase, IPN synthase, and
  acyltransferase.
• Modern recombinant genetic techniques have
  allowed the over expression of the genes which
  code for these three enzymes, allowing much
  greater yields of penicillin than in the past.

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Penicillin Biosynthetic Pathway
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Semi-Synthetic Penicillins

• The acyl side chain of the penicillin molecule
  can be cleaved using enzyme or chemical methods
  to produce 6-APA, which can further be used to
  produce semi-synthetic penicillins or
  cephalosporins
• 75 of the penicillin produced is modified in
  this manner.

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Penicillins- Antistaphylococcal

• Penicillins which have bulky side groups can
  block the ß-Lactamases which hydrolyze the lactam
  ring.

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Penicillins- Antistaphylococcal

• These lactamases are prevalent in S. aureus and
  S. epidermidis, and render them resistant to
  Penicillin G and V. This necessitated the
  development of semi-synthetic penicillins through
  rational drug design.
• Methicillin was the first penicillin developed
  with this type of modification, and since then
  all bacteria which are resistant to any type of
  penicillin are designated as methicillin
  resistant. (MRSA- methicillin-resistant S.
  aureus)

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Penicillins- Antistaphylococcal

• Methicillin is acid sensitive and has been
  improved upon by adding electron withdrawing
  groups, as was done in penicillin V, resulting in
  drugs such as oxacillin and nafcillin.
• Due to the bulky side group, all of the
  antistaphylococcal drugs have difficulty
  penetrating the cell membrane and are less
  effective than other penicillins.

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Penicillins- Aminopenicillins

• In order to increase the range of activity, the
  penicillin has been modified to have more
  hydrophilic groups, allowing the drug to
  penetrate into Gram (-) bacteria via the porins.
• Ampicillin RPh
• Amoxicillin R Ph-OH

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Penicillins- Aminopenicillins

• These penicillins have a wider range of activity
  than natural or antistaphylococcal drugs, but
  without the bulky side groups are once again
  susceptible to attack by ß-lactamases
• The additional hydrophilic groups make
  penetration of the gut wall difficult, and can
  lead to infections of the intestinal tract by H.
  pylori

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Penicillins- Aminopenicillins

• Due to the effectiveness of the aminopenicillins,
  a second modification is made to the drug at the
  carboxyl group.
• Changing the carboxyl group to an ester allows
  the drug to penetrate the gut wall where it is
  later hydrolyzed into the more polar active form
  by esterase enzymes.
• This has greatly expanded the oral availability
  of the aminopenicillin class.

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Penicillins- Extended Spectrum

• Extended spectrum penicillins are similar to the
  aminopenicillins in structure but have either a
  carboxyl group or urea group instead of the amine

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Penicillins- Extended Spectrum

• Like the aminopenicillins the extended spectrum
  drugs have an increased activity against Gram (-)
  bacteria by way of the import porins.
• These drugs also have difficulty penetrating the
  gut wall and must be administered intravenously
  if not available as a prodrug.
• These are more effective than the
  aminopenicillins and not as susceptible to
  ß-lactamases

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Cephalosporins

• Cephalosporins were discovered shortly after
  penicillin entered into widespread product, but
  not developed till the 1960s.
• Cephalosporins are similar to penicillins but
  have a 6 member dihydrothiazine ring instead of a
  5 member thiazolidine ring.
• 7-aminocephalosporanic acid (7-ACA) can be
  obtained from bacteria, but it is easier to
  expand the ring system of 7-APA because it is so
  widely produced.

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Cephalosporins

• Unlike penicillin, cephalosporins have two side
  chains which can be easily modified.
  Cephalosporins are also more difficult for
  ß-lactamases to hydrolyze.

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Mechanism of Cephalosporins

• The acetoxy group (or other R group) will leave
  when the drug acylates the PBP.

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Cephalosporins- Classification

• Cephalosporins are classified into four
  generations based on their activity.
• Later generations generally become more effective
  against Gram (-) bacteria due to an increasing
  number of polar groups (also become zwitterions.)
• Ceftazidime (3rd gen) in particular can cross
  blood brain barrier and is used to treat
  meningitis.
• Later generations are often the broadest spectrum
  and are reserved against penicillin resistant
  infections to prevent the spread of cephalosporin
  resistant bacteria.

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Carbapenems

• Carbapenems are a potent class of ß-lactams which
  attack a wide range of PBPs, have low toxicity,
  and are much more resistant to ß-lactamases than
  the penicillins or cephalosporins.

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Carbapenems

• Thienamycin, discovered by Merck in the late
  1970s, is one of the most broad spectrum
  antibiotics ever discovered.
• It uses import porins unavailable to other
  ß-lactams to enter Gram (-) bacteria.
• Due to its highly unstable nature this drug and
  its derivatives are created through synthesis,
  not bacterial fermentation.

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Carbapenems

• Thienamycin was slightly modified and marked as
  Imipenem. Due to its rapid degradation by renal
  peptidase it is administered with an inhibitor
  called cilastatin under the name Primaxin.
  Imipenem may cause seizures or sever allergic
  reactions.
• Other modifications of Thienamycin have produced
  superior carbapenems called Meropenem and
  Ertapenem, which are not as easily degraded by
  renal peptidase and do not have the side effects
  of Imipenem.

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Monobactams

• The only clinically useful monobactam is
  aztreonam. While it resembles the other ß-lactam
  antibiotics and targets the PBP of bacteria, its
  mechanism of action is significantly different.
• It is highly effective in treating Gram (-)
  bacteria and is resistant to many ß-lactamases

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ß-Lactamases

• ß-Lactamases were first discovered in 1940 and
  originally named penicillinases.
• These enzymes hydrolyze the ß-lactam ring,
  deactivating the drug, but are not covalently
  bound to the drug as PBPs are.
• Especially prevalent in Gram (-) bacteria.
• Three classes (A,C,D) catalyze the reaction using
  a serine residue, the B class of metallo-
  ß-lactamases catalyze the reaction using zinc.

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ß-Lactamase Inhibitors

• There are currently three clinically available
  ß-lactamase inhibitors which are combined with
  ß-lactams all are produced through fermentation.
• These molecules bind irreversibly to ß-lactamases
  but do not have good activity against PBPs. The
  rings are modified to break open after acylating
  the enzyme.

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ß-Lactam/Inhibitor combinations

• Aminopenicillins
• ampicillin-sulbactam Unasyn
• amoxicillin-clavulante Augmentin
• Extended-Spectrum Penicillins
• piperacillin-tazobactam Zosyn
• ticarcillin-clavulanate Timentin

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Summary

• ß-Lactam antibiotics have dominated the clinical
  market since their introduction in the 1940s and
  today consist of nearly ¾ of the market.
• Development of natural products such as
  penicillin G into more potent forms through
  rational modification has increased the range of
  activity of these drugs, although this has led to
  some toxicity problems.
• Widespread use of ß-lactams has led to the
  development of resistant strains, new
  modifications are necessary in order for
  ß-lactams to remain viable.

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Assigned reading Patrick, Graham L. An
Introduction to Medicinal Chemistry 4th Edition.
New York Oxford University Press, 2009. 388-414.
Print.
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Optional References/ Reading

• Brunton, Laurence L. et al. Goodman and Gillmans
  Pharmaceutical Basis of Therapeutics 11th
  Edition. McGraw-Hill, 2006 1134- 52. Print.
• Bush, Karen. ß-Lactamase Inhibitors from
  Laboratory to Clinic. Clinical Microbiology
  Reviews, Jan. 1988, p. 109-123. Web.
• Elander, R.P. Industrial production of ß-Lactam
  antibiotics. Journal of Applied Microbiology and
  Biotechnology (2003) 61385392. Web.
• Hauser, Alan R. Antibiotic Basics for Clinicians
  Choosing the Right Antibacterial Agent.
  Philadelphia Lippincott, 2007. 18-46. Print.
• Patrick, Graham L. An Introduction to Medicinal
  Chemistry 4th Edition. New York Oxford
  University Press, 2009. 388-420. Print.
• Rolinson, George N. Forty years of ß-lactam
  research. Journal of Antimicrobial Chemotherapy
  (1998) 41, 589603. Web.

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Questions

• 1. What are two ways by which a bacteria could
  become resistant to carbapenems?
• 2. How were the natural penicillins modified to
  be orally available?
• 3. How are extended spectrum penicillins modified
  to be orally available?
• 4. What are two ways that the ß-lactam can be
  protected from ß-lactamases?