1

Pharmacy 754, Spring-2007Medicinal Chemistry/Pharmacology

STRUCTURAL CLASSES OF ANTIMICROBIAL AGENTS

I: β-Lactams

Reading assignment: W&G 11th Ed., pp 301 – 334Recommended reading: G&G 10th Ed., pp 1189-1218

β-Lactams: Introduction• Largest group of compound used to treat bacterial infections

– >100,000 known– includes: penicillins, cephalosporins, monobactams and others

• Most bacterial species are sensitive to at least one β-lactam

History/Background• 1929 – Alexander Fleming (Oxford) discovers a substance from a

fungus inhibits the growth of a culture a Stapholococcus sp.– Fleming goes on holiday – leaves lab window open– Spores from a strain of Penicillium notatum in another lab enter Fleming’s

window and land on agar plates of staph left uncovered on bench– Weather turns cool – favors fungal growth on the plates

and slows bacterial growth– Temperature warms up, allows bacteria

not killed by fungus to grow rapidly– Result was a clear zone (lysed bacteria) surrounding

the fungus

fungus

bacteria

2

Discovery of Penicillin• Remarkable series of coincidences

– very few Penicillium species secrete penicillin

– most Penicillium species produce very low amounts of penicillin

– weather and timing

• Fleming recognized the significance of the clear zone but was unable to isolatethe responsible substance

– looked at effect of broth extract on bacteria grown in presence of blood but nevertested extract in a pathogen-infected animal

– did not foresee the clinical applications in humans

• 1938 – Florey and Chain (Oxford)– recognized potential of penicillin project –responsible for getting it into clinic

– responsible for scale-up production and isolation of pure, active substance

– whole university department converted to “factory “

• 1940 – First in vivo tests in mice are successful– an English Bobby – dying from mixed Staph/Strep infection was nearly saved by

penicillin before supplies ran out

– even isolated unmetabolized drug from the patient’s urine

• Promising results and WWII prompted need formassive scale-up

– used deep fermentors in the US developed forvitamin production

• 1945 – Fleming, Ernst Chain and Howard Floreyshare Nobel Prize in Medicine

• Working with Abraham (chemist) structure of thepenicillin produced in the US is solved

Discovery of Penicillin

N

S

HHN

CO2H

CH3

CH3OO

3

Development of PenicillinsEarly 50’s• Directed biosynthesis – Discovered that adding excess amounts of aromatic carboxylic

acids to growth medium led to new penicillins containing these acids as side chains

Late 50’s• Major breakthrough – Strain of P. chrysogenum isolated that accumulated (6-APA)

6-aminopenicillanic acid when side chain precursors are omitted from medium– Could isolate this core structure with all critical stereochemistry intact and add various side

chains in the lab– Nearly all penicillins are now semisynthetic– 6-APA is produced in ton quantities– > 20,000 different penicillins now reported in literature

• 1957 – Penicillins shown to inhibit bacterial cell wall biosynthesis The first synthesis of benzylpenicillin

• 1965 – Penicillins found to block the crosslinking of the peptidoglycan strands• Final step in cell wall assembly

N

S

HHHN

H

CO2H

CH3

CH3

O

OO

O CO2H

Phenoxyacetic acid

Penicillin V

add to

Penicillium cultures

e.g.,

N

S

H

H2N

CO2H

CH3

CH3

O

1

2

3

4

56

7

H

6-APA

General Features of β-Lactams• Common structural feature

– Chemistry and biological activity of these antibiotics isdominated by this strained amide

– Amides are normally very stable to hydrolysis– 4-membered ring creates severe strain

• Common β-lactam-containing ring systems

Ring strain plays a major role in …• Antibacterial mechanism of action• Deactivation by enzymes found in resistant organisms• Instability in acidic media (pH <5)

– Most amide are quite stable under these conditions– Leads to poor oral availability of many β-lactams

N

O

!-lactam

R

N

S

O

penam

N

O

carbapenam

N

O

O

oxapenam

N

O

S

cephem

N

O R

monobactam

4

β-Lactams: Mechanism of Action• OVERALL: They inhibit bacterial cell wall biosynthesis• SPECIFICALLY: The block the crosslinking (transpeptidation) step of

peptidoglycan biosynthesis– The enzymes that form the crosslinks are PBPs - for Penicillin Binding Proteins

Structure of bacterial cell walls

lipopolysaccharides

Gram-(-)

bacterium

cytoplasmic

membraneperiplasm

peptidoglycan

layerouter membrane

porin channels

PBP !-lactamase

(if present)

cytoplasmic

membrane

peptidoglycan

layer

Gram-(+)bacterium

!-lactamase

(if present)PBP

Peptidoglycan structure and biosynthesis• Peptidoglycan (PG) – net-like polymer

– Like a chainlink fence – really strong, but porous• main structural component of bacterial cell wall

– maintains integrity and shape of cell– prevents lysis under normal hypotonic conditions (osmotic pressure ~ 30 atm)

• PG is made of long polymeric strands of alternating β-1,4-linked N-Acetylglucosamine (GlcNAc or NAG) and its 3-O-D-lactyl ether derivative N-Acetylmuramic acid (MurNAc or NAM)

• The NAM residues are substituted with a pentapeptide (5 amino acids)

L-Ala–D-!-Glu–Dap–D-Ala–D-Ala

CH3

O

O

O O

O

HO

O

HO

NHAc

NHAc

OHO

HN

O

NH

O

CO2H

CH3

NH

HN

CH3

CO2H

NH2

CO2H

O

CH3

HN

O

NAG

NAG

NAM

NAM

5

Peptidoglycan structure and biosynthesis• Peptidoglycan gets its rigidity and insolubility from crosslinks between

the pentapeptides– peptide (amide) bonds are formed between the penultimate D-Ala

of one PG strand and an ε-NH2 groups of a Lysine or Diaminopimelate(Dap) on a second PG strand

– Usually L-Ala-γ-D-Glu-Dap-D-Ala-D-Ala orL-Ala-γ-D-Glu-L-Lys-D-Ala-D-Ala in Gram-(-)

• Many Gram-(+) bacteria have a short peptide (usually 5 Glycines)attached to the lysine. The NH2 on the terminal Gly forms the crosslinkwith the penultimate D-Ala of a second PG strand

NAGNAG

NAMNAM

L-Ala–D-!-Glu–Lys–D-Ala–D-AlaCH3

O

O

O O

O

HO

O

HO

NHAc

NHAc

OHO

HN

O

NH

O

CO2H

CH3

NH

HN

CH3

CO2H

HN

O

CH3

HN

O

O

HN

NH

O

HN

O

NH

O

NH2

O

Gly5

OP

PPOD-Ala–D-Ala–Dap–D-!-Glu–L-Ala

PPOD-Ala–D-Ala–Dap–D-!-Glu–L-Ala

PO

UDP L-Ala–D-!-Glu–Dap

UDP L-Ala–D-!-Glu–Dap–D-Ala–D-Ala

OPP

L-Ala

D-Ala

D-Ala–D-Ala

Cytoplasm Cell SurfaceCytoplasmicMembrane

D-Ala

Overview of Peptidoglycan Biosynthesis

UDP-GlcNAc

UDP-MurNAc

Stage I

Stage II Stage III

UDP-GlcNAc

OPP L-Ala–D-Glu–Dap–D-Ala–D-Ala

L-Ala–D-Glu–Dap–D-Ala

L-Ala–D-Glu–Dap–D-Ala–D-Ala

L-Ala–D-Glu–Dap–D-Ala–D-Ala

OPP L-Ala–D-Glu–Dap–D-Ala–D-Ala

D-Ala–D-Ala–Dap–D-Glu–L-Ala

D-Ala–Dap–D-Glu–L-Ala

L-Ala–D-Glu–Dap–D-Ala–D-Ala

D-Ala-Dap–D-Glu–L-Ala

L-Ala–D-Glu–Dap–D-Ala–D-Ala

D-Ala-Dap–D-Glu–L-Ala

6

Stage III steps of PG biosynthesis: Transglycosylation

Lengthening the PG monomer chain

CH3

O

O

OO

O

OH

HO

AcHNAcHN

OH

HN

O

NH

O

CO2

H3C

NH

HN

H3C

CO2

H2N

CO2

O

CH3

NH

O

P

O

P

O

CH3

O

O

OO

O

OH

OH

AcHNAcHN

OH

HN

O

NH

O

CO2

H3C

NH

HN

H3C

CO2

H2N

CO2

O

CH3

NH

O

O

O O

O

O

O

P

O

P

O

O O

O

O

O

C45

H3C

O

O

OO

O

OH

OH

OH

AcHN

AcHN

OH

NH

O

HN

O

O2C

CH3

HN

NH

CH3

O2C

NH2

O2C

O

H3C

HN

O

C45

transglycosylasecell surface

CH3

O

O

OO

O

OH

HO

AcHNAcHN

OH

HN

O

NH

O

CO2

H3C

NH

HN

H3C

CO2

H2N

CO2

O

CH3

NH

O

CH3

O

O

OO

O

OH

OH

AcHNAcHN

OH

HN

O

NH

O

CO2

H3C

NH

HN

H3C

CO2

H2N

CO2

O

CH3

NH

O

O

P

O

P

O

O O

O

O

O

C45

H3C

O

O

OO

O

OH

O

OH

AcHN

AcHN

OH

NH

O

HN

O

O2C

CH3

HN

NH

CH3

O2C

NH2

O2C

O

H3C

HN

O

transglycosylase

P

O

P

O

O O

O

O

O

C45

Transglycosylase (a glycosyltransferase)

Stage III steps of PG biosynthesis: Transpeptidation

“Crosslinking”

Transpeptidase (TPase)H3C

O

O

OOO

OHO

OH

AcHNAcHN

OH

NH

O

HN

O

O2C

CH3

HN

NH

CH3

O2C

O

H3C

HN

O

H3C

O

O

OOO

OHO

OH

AcHN

AcHN

OH

L-Ala

D-!-Glu

Dap

D-Ala

D-Ala

TPase

OH

H3C

O

O

OOO

OHO

OH

AcHN

AcHN

OH

HN

O

NH

O

CO2H3C

NH

NH

CH3

O2C

H2N

CO2

O

CH3

HN

O

NH2

O2C

H3C

O

O

OOO

OHO

OH

AcHNAcHN

OH

NH

O

O

HN

NH

CH3

O2C

O

H3C

HN

O

H3C

O

O

OOO

OHO

OH

AcHN

AcHN

OH

L-Ala

D-!-Glu

Dap

D-Ala

D-Ala

TPase

O

D-Ala O2C

NH2

H3C

O

O

OO

O

OH

O

OH

AcHNAcHN

OH

NH

O

O

HN

NH

CH3

O2C

O

H3C

HN

O

O

H3C

O

O

OO

O

OH

O

OH

AcHN

AcHN

OH

HN

O

NH

O

CO2

H3C

NH

NH

CH3

O2C

HN

CO2

O

CH3

HN

O

O2C

NH2

TPaseOH

7

β-lactams inactivate the transpeptidase• Transpeptidase is one of the family of PBPs: Penicillin Binding Proteins

– Located along the exterior of the cytoplasmic membrane (Gram-(+)) or inthe periplasmic space (Gram-(-))

– PBPs have varied roles in PG synthesis and cell wall maintenance– Several types of PBPs known, some are bifunctional

Overall structure of PBP2.

Lovering et al., Science 315,1402 -1405 (2007)

Inactivation of the PBPs• β-lactams mimic the D-Ala-D-Ala end of the pentapeptide and are bound

by the Transpeptidase/PBPs

• The active site serine of TPase attacks the β-lactam– A stable acyl-enzyme complex is formed– The PBP/PCN adduct is not attacked by a Lys or Dap on another PG strand– The PBP/PCN adduct is only slowly hydrolyzed by H2O

• Selectivity for Gram-(+) organisms by many β-lactams is a result of targetaccess (refer back to the diagram of cell wall types)

R

OHN

NS

H

O

O2C

O

B-H

Ser

B:

R

O

HN

HN

S

H

O

CO2

O

B-H

Ser

B:

TP-ase TP-ase

H

8

Resistance to the β-Lactams• Resistance to β-lactams is primarily through acquisition of

genes for enzymes that hydrolyze the β-lactam ring• β-lactamases

– ~ 600 β-lactamase variants have been identified• Evidence for evolution from PBPs

– Different mechanisms suggest they independently evolved 4 separate times– > 130 variants of TEM-1 β-lactamase have developed since it was

discovered in 1963• Do not bind peptidoglycan or retain transpeptidase activity

– Two general types of β-lactamases: grouped into four classes• Zinc-requiring β-lactamases (metallo-β-lactamases): Class B

RHN

N

S

H

OCO2

Zn2+

Zn2+

OH

Zinc !-lactamase

RHN

HN

S

H

OCO2

O

Resistance to the β-Lactams• The Serine β-Lactamases: Classes A, C & D

– Destroy the β-lactams antibiotics by a mechanism that is similar to how theβ-lactams inactivate the PBP targets

– BUT, the acyl-enzyme complex is unstable - easily hydrolyzed by H2O– Some Class A & C β-lactamase have achieved ‘catalytic perfection’

• They function at diffusion controlled limit

RHN

N

S

H

OCO2H

RHN

HN

S

H

OCO2H

O

H2O

!-lactamase

RHN

HN

S

H

OCO2H

HOSer

RHN

HN

S

H

OCO2H

O

Ser

unstable acyl-enzyme intermediate

PBP

!-lactamase

stable acyl-enzyme

Transpeptidase(PBP)

!-lactamase

OH

Ser

Enz

9

β-Lactamases• In Gram-(+) bacteria that produce β-lactamases the enzymes are

secreted from the cell or found along the exterior of the cytoplasmicmembrane– often produced in very large quantities– the cells need to be surrounded by a ‘sea of β-lactamases’

• β-lactams are hydrolyzed before they can reach the cell and its porous cell wall

• In Gram-(-) species the enzymes are found in the periplasmic space orbound to the exterior of the cytoplasmic membrane.– only need to act on β-lactams that make it through the outer membrane

and into the periplasmic space– 2nd line of defense, after the outer membrane– typically produce lower amounts of β-lactamases than Gram-(+)

Evolution of β-Lactams Resistance

• Evidence that β-lactamases evolved along with the biosynthesis of thecompounds

– First β-lactamase was identified in 1940• Clinical use accelerated the selection process

– β-lactams still make up ~55% of all ABX used

• Penicillin-resistant organisms emerged within 2-3 years after clinical use began– esp. Staph. aureus

• by mid 1950’s 80% of all S. aureus isolates from some hospitals were resistantto Pen G and Pen V

– driving force to discover new/better β-lactams– led to development of agents like methicillin (1960)– methicillin-resistant Staph. aureus (MRSA) appeared in 1961

10

CLASSES OF β-LACTAMSPenicillins: General SAR

• 6-APA nucleus

– Must have a free carboxyl on thiazolidine ring

– Must have substituted amide at position 6

– R varies widely• All clinically important PCNs have an aromatic ring in the R group

– R group has pronounced effect on many properties of the penicillins• Varying the R group on the amide at C-6 can…

– Modulate the reactivity of the ring– Alter solubility– Protect from β-lactamases– Affect spectrum of activity– Bioavailability/protein binding

N

S

HHN

CO2R'

CH3

CH3

R

OO

1

2

3

4

56

7

Penicillin SAR:Effect of R group on Acid Stability• β-lactam strain results in poor amide resonance

– The result is an amide Nitrogen that is fairly basic• Decomposition in acid is assisted by lone pair of electrons on sidechain

carbonyl oxygen

– t1/2 of Pen G at pH 2 and 37 °C is 5 min (R = benzyl)

• Remedy: place electron withdrawing group (EWG) adjacent to amide carbonyl.Decreases rate of acid hydrolysis and increase oral bioavailability.

R

O

HN

N

S

H

OCO2H

R

O

HN

N

S

H

OCO2H

N

S

CO2H

N

HO2C

R

Penillic Acid

H3O+

H

N

S

HHHN

H

CO2H

CH3

CH3

O

OO

N

S

HHN

CO2H

CH3

CH3OO

NH3

N

S

HHN

CO2H

CH3

CH3

EWG

OO

Pen V ampicillin

t1/2 at pH 1 & 37 °C = 5 hr Stable in acid

11

Penicillin SAR:Effect of R group on β-lactamase sensitivity

• R groups with steric bulk adjacent to the sidechain carbonyl preventdestruction by β-lactamases

– Avoidance tactic– The β-lactamase active site Serine can’t get close enough to attack– The trade-off: the steric bulk also decrease access to the PBP targets of the PCN

• All the β-lactamase resistant PCNs are less potent than the natural PCNs

• NOTE: These modifications do not provide protection from most Class B β-lactamases

Methicillin Nafcillin

O

HN

N

S

H

OCO2H

OCH3

OCH3

O

HN

N

S

H

OCO2H

OEt

Penicillin SAR:Effect of R group on Spectrum of Activity

• Activity of natural PCNs is limited to Gram-(+) bacteria• Adding polar or charged R groups can extend spectrum to include

Pseudomonas aeruginosa and other Gram-(-) pathogens

• The increased polarity helps the compounds pass through the porin channels

Carbenicillin

N

S

HHN

CO2Na

CH3

CH3OO

CO2Na

N

S

HHN

CO2H

OO

NHO

NO

EtNO

Pipericillin

12

Classes of PenicillinsFour Classes:• Natural Penicillins

– Produced by fermentation– ~30 known, only Pen G and Pen V are used clinically

• Only advantage of Pen V is oral availability– Most potent of all PCNs versus susceptible bacteria– Readily hydrolyzed by β-lactamases

• β-Lactamase Resistant Penicillins– All are semisynthetic– Stable to hydrolysis by most staphylococcal β-lactamases

• NOTE: these avoid destruction by β-lactamases, they are not inhibitors of the enzymes

– Methicillin (Staphcillin®) was first• Not orally active• Dimethoxyphenyl sidechain is electron rich - more acid labile than Pen G

– Nafcillin (Unipen®)• Most active of the class - based on mass-to-mass comparison of potency• Orally active

Classes of Penicillins• β-Lactamase Resistant Penicillins (cont’d)

– Isoxazoyl Penicillins• Oxacillin (Prostaphlin®)• Cloxacillin (Tegopen®)• Dicloxacillin (Dynapen®, Pathocil®)

– All are orally active– Very good resistance to β-lactamases

• Aminopenicillins

– All are semisynthetic– Sidechains all have an amine on the carbon adjacent to amide carbonyl– Easily cleaved by β-lactamases– Spectrum of susceptible bacteria extended to include many Gram-(-)

• Protonated amine at physiological pH helps diffuse through the porins

NO

O

6-APA

CH3X2

X1

Oxacillin: X1 = X2 = H

Cloxacillin: X1 = Cl; X2 = H

Dicloxacillin: X1 = X2 = Cl

R

O

HN

N

S

H

OCO2H

NH2

α

13

Classes of Penicillins• Aminopenicillins (cont’d)

– Ampicillin (Penbritin®, Polycillin®)• The NH2 creates a new stereocenter

– (R)-isomer has better Gram-(-) activity• Ampicillin is available as the prodrug Bacampicillin

– Amoxicillin (Amoxil®)• p-hydroxyampicillin• Same features as ampicillin but ~2x better absorbed with less GI upset• Often combined with the β-lactamases inhibitor clavulanic acid to further extend the

spectrum of activity

O

HN

N

S

H

O

NH2

CH3

O

O

O

Et

O

O

O

HN

N

SH

OCO2H

NH2

ampicillin

amoxicillin

O

HN

N

S

H

OCO2H

NH2

HO

Classes of Penicillins• Extended Spectrum Penicillins

– All are semisynthetic– Most active PCNs towards Gram-(-) bacteria including Ps. aeruginosa– Most are β-lactamase sensitive– Most are acid labile

Two Subclasses1) α-Carboxypenicillins– Carbenicillin (Geopen®)

• The diacid is very water soluble– Usually used as the di-sodium salt

• α-Carboxyl group introduces new stereocenter: no significant difference between isomer• No significant difference in activity between isomers• Decarboxylate in acid to give Pen G• Some prodrug forms are available that increase oral availability• Carbenicillin has been shown to affect platelet function and increase bleeding times

– Ticarcillin (Ticar®)– Thiophene analog of carbenicillin

• 2-4x more potent than carbenicillin vs. Ps. aeruginosa• Can require very high doses

• These are often used with a β-lactamase inhibitor or aminoglycoside to extend the spectrum of activity

N

S

HHN

CO2Na

CH3

CH3OO

CO2Na

α

O

HN

N

S

H

OCO2H

S

CO2H

14

Classes of Penicillins• Extended Spectrum Penicillins (cont’d)

2) α-Acylureidopenicillins (aka, acylaminopenicillins)All derivatives of ampicillin– Pipericillin (Piperacil®)

• Contains a 2,3-diketopiperazine– Fairly common in sidechains of PCNs and some

cephalosporins• Can require high doses• Good activity vs. variety of Gram-(-) and retains good Gram-(+) activity• Acid labile• β-lactamase sensitive

– Appears ‘bulky’ but the bulk is not close enough to the sidechain carbonyl to effectivelyblock attack

– Azlocillin (Azlin®) and Mezlocillin (Mezlin®) R = H R = SO2CH3

• Both are more potent tha carbenicillin towards Ps. aeruginosa• Both are acid labile• Both are readily inactivated by β-lactamase

• Pipericillin can also be combined with a β-lactamase inhibitor to extend the spectrum of activity

N

S

HHN

CO2H

OO

NHO

NO

EtNO

NH

N

O

O

N

R

O N

S

HHN

CO2HO

Penicillin Side Effects• Among the safest drugs available• Hypersensitivity is most common adverse reaction

– Estimated at 8-9% of US population– Ranges from rash to anaphylaxis and death– All β-lactams have potential to cause hypersensitivity

• Most common with Pen G and ampicillin– Caused by covalent modification of a host protein(s) with the PCN or a

PCN degradation product• Makes the protein appear foreign

– Ampicillin can polymerize to form a potent antigen

O

HN

N

S

H

OCO2

NH2

O

HN

N

S

H

OCO2

NH2

O

NH

NS

H

O

O2C

NH

ONH

HN

S

H

O

CO2

NH2

O

NH

NS

H

O

O2C

NH

ONH

HN

S

H

O

CO2

NH

ONH

HN

S

H

O

CO2

NH2

etc.

ampicillin

15

Cephalosporins• History/Background

– 1945: the fungus Cephalosporium acremonium is isolated from asewer outlet on Sardinia

• Crude extract killed Staph. spp. and could cure typhoid fever– 1948: E.P. Abraham (Oxford) isolated two compounds with good

antibacterial activity and determined the structures

– Ceph C was resistant to hydrolysis by some β-lactamases• Little initial interest

– Interest increased with: 1) growing resistance to PCNs, and2) enzymatic method to remove the aminoadipoyl sidechain

O

HN

NH2

HO2C

N

S

H

OCO2H

O

HN

N

H

O

S

CO2H O

O CH3NH2

HO2C

penicillin N cephalosporin C

Cephalosporin SAR• All cephalosporins are semisynthetic

– Derived from 7-Amino Cephalosporanic Acid (7-ACA)

General SAR

– Must have free carboxyl at C-4• Can be derivatized as an ester prodrug

– Alterations at R1 affect spectrum and acid stability– R2 affects reactivity and acid stability

• If the “Y” group above is a good leaving group, this increases reactivity butdecreases stability – a balance must be reached

H2N

N

H

O

S

CO2H O

O CH3

7-ACA

R1

HN

N

H

O

S

R2

CO2H

1

2

3

4

5

67

8

R1

HN

N

H

O

S

CH2

CO2

Y

R1 NH

HN

H

O

S

CH2

CO2

O

R1 NH

HN

H

O

S

CH2

CO2

O Y

H

Y

TPase

Ser-OH

Ser

TPase

Ser

TPase

16

Cephalosporins and β-Lactamases• Cephalosporins are inherently less sensitive to hydrolysis by

β-lactamases than all but the β-lactamase-resistant PCNs– Resistance to β-lactamases can be increased, BUT the sidechains that

make PCNs β-lactamase stable lead to inactive Cephalosporins

To increase β-lactamase resistance in the cephalosporins– Add aromatic alkoximino group on sidechain

• Contributes important bulk and electronic effects

– Replace H-7 with OCH3creates Cephamycin skeleton

O

Ar

NOCH3

HN

N

H

O

S

R2

CO2H

The syn alkoximinoisomers interfer withβ-lactamase attackbetter than the antiisomers

R1

NH

N

H

O

S

R2

CO2H

7

H3CO

Classes of CephalosporinsGrouped by “generations”• Based on TIME of discovery and SPECTRUM of activity

– See tables on pp. 1207 & 1208 in G&G 10th ed. and pp. 320-321 in W&G 11th ed.

• Representative Examples:First Generation– Good activity vs. most Gram-(+) except methicillin resistant S.

aureusand S. epidermidis

– Relatively modest activity vs. most Gram-(-)

– Cephalothin (Keflin®)• Not orally active• Spectrum of activity similiar to ampicillin• Most β-lactamase resistant of the First Gen Cephs

O

HN

N

H

O

S

CO2H O

O CH3S

cephalothin

17

First Generation Cephalosporins (cont’d)

• Cefazolin (Ancef®, Kefzol®)

– Given parenterally

– Active towards many gram-(-)

– longest t1/2 of First Gen Cephs

• Cephalexin (Keflex®, Keflet®)

– Same side chain as ampicillin

– orally active – acid stability results from amino group and the methyl at C-3

– dominated market for years

– Not metabolized – 90% excreted in urine

O

HN

N

H

O

S

CO2H

S

NN

S

NN

N

N

CH3

O

HN

N

H

O

S

CH3

CO2H

NH2

Second Generation Cephalosporins• Usually active against same organisms susceptible to First Generation

Cephs PLUS active against most strains of H. influenzae• Generally, more active towards Gram-(-) than First Gen Cephs

• Cefuroxime (Zinacef®, Ceftin Axetil® (oral))– First e.g. of an oxime ether

• note syn configuration– has good β-lactamase resistance

• anti methyloxime is hydrolyzedby β-lactamases

– good penetration into CSF• some use to Tx H. influenzae meningitis

– Axetil prodrug, 1-(acetyloxy)ethyl ester

O

HN

N

H

O

S

CO2R O

O NH2O

N

OCH3

CH3

O

O

CH3

cefuroxime R = Hcefuroxime axetil R =

1-(acetyloxy)ethyl ester

18

Second Generation Cephs (cont’d)• Cefoxitin (Mefoxin®)

– Example of a Cephamycin– Grouped with 2nd Gen Cephs based on spectrum– Semisynthetic derivative of cephamycin C

– Not as potent towards many Gram-(+) as some2nd Gen Cephs, but has activity versus someanaerobes and Gram-(-) bacilli that are notsensitive to cephalosporins infections

– The C-7 OCH3 group confers good β-lactamase resistance• Cefoxitin is actuall a competitive inhibitor of many β-lactamases

O

HN

N

H

O

S

CO2H O

O NH2S

OCH3

Third Generation Cephalosporins• Generally less active than 1st Generation Cephs against Gram-(+)• Broader Gram-(-) activity than 1st or 2nd Gen Cephs• Some exhibit antipseudomonal activity

• Cefotaxime (Claforan®)– The first 3rd Gen Ceph released in the US– syn-oxime ether is most potent AND

most β-lactamase resistant– Aminothiazole increases activity

• Particularly towards Enterobacteriaceae– Includes Yersinia, Salmonella

– Given parenterally (acetoxymethyl at C-3)

• Ceftriaxone (Rocephin®)– Most widely used injectable Ceph– Very long t1/2 – once/day injection– Broad spectrum– Resistant to most β-lactamase– Increased use to Tx gonorrhea because cefixime is not longer available

O

HN

N

H

O

S

CO2H O

O CH3

S

N

H2N

N

OCH3

cefotaxime

O

HN

N

H

O

S

CO2 N

S N

S

N

H2N

N

OCH3

N

O

O

CH3

Na+

Na+

19

Fourth Generation Cephalosporins• Broad spectrum antibiotics

– Gram-(+) activity similar to 1st Gen Cephs– Gram-(-) activity similar to 3rd Gen Cephs

• Broad resistance to many β-lactamases

• Cefepime (Maxipime®)– The first ‘true’ 4th Gen Ceph released in the US– Given parenterally

• Quaternary amine (methyl pyrrolidinium)– Very good leaving group– Charge helps access Gram-(-)

– 100% excretion in urine– Good access and high concentration in CSF

• Effective for bacterial meningitis

• Cefpirome (Cefrom®)– Spectrum of activity similar to cefipime– Given parenterally

• Quaternary amine (pyridinium)

O

HN

N

H

O

S

CO2H

N

S

N

H2N

N

OCH3

CH3

cefepime

O

HN

N

H

O

S

CO2H

N

S

N

H2N

N

OCH3

cefpirome

Monobactams:Sulfonated β-lactam is not fused with another ring• First monobactam discovered was sulfazecin

– produced by a soil microbe Ps. acidophila• Highly resistant to β-lactamase hydrolysis

– but weak antibacterial activityInitial SAR work showed:• Adding a methyl group at C-4 of ring increased β-lactamase resistance

– Also increased Gram-(-) activity while lowering activity toward Gram-(+)• Adding two methyl groups at C-4 increased oral availability

– Also a slight increase in potency

– Aztreonam (Azactam®)• Only monobactam on the US market• Produced by total synthesis• Narrow spectrum

– includes many aerobic Gram-(-) no activity towards anaerobes and Gram-(+)• Injected only

O

HN

N

O SO3H

OCH3

NH

HO2C

CH3O

NH2H

Sulfazecin

O

HN

N

H3C

O

H

S

N

H2N

N

OC

SO3Na

H3C CH3

CO2Na

Aztreonam

20

β-lactamase inhibitorsTwo methods of dealing with these enzymes• Avoidance – alter the structure of the penicillin so it is no

longer a substrate for the β-lactamase– Successful – but all examples had decrease antibacterial activity

• e.g., methicillin, nafcillin

• Neutralization – inhibit the β-lactamase whilesimultaneously administering a penicillin that wouldotherwise be hydrolyzed– Early attempts to develop reversible inhibitors failed

• Focus was on competitive inhibitors– Low affinity for β-lactamases meant large doses were required

• 1976 – two natural products discovered that wereirreversible inhibitors (inactivators) of serine β-lactamases

β-lactamase inhibitors• Clavulanic Acid

– Prototype Class I β-lactamase inhibitor– Example of an oxapenam

• Complex mechanism of inactivation– Can cause reversible or irreversible inhibition

• Weak broad spectrum antibacterial action• Inhibits β-lactamases from wide variety of bacteria• Often see pronounced synergistic effect when coadministered

with some β-lactams• The clavulanate extends the life of the PCN so the antibiotic can

reach the target PBPs.

– Amoxicillin + Clavulanate (Augmentin®) Oral– Ticarcillin + Clavulanate (Timentin®) Injected

N

O

H

OCO2H

OH

21

β-lactamase inhibitors• Other Class I β-lactamase inhibitors

– Sulbactam• Synthetic• Weak antibiotic activity• Potentiates the activity of ampicillin and carbenicillin

– Not against all species– Ampicillin + Sulbactam (Unasyn®) parenteral

– Tazobactam• synthetic• Inhibits β-lactamases from many Gram-(+)

and Gram-(-) species

– Pipericillin + Tazobactam (Zosyn®) parenteral

N

S

H

O

CO2H

O

O

N

S

H

O

CO2H

O

O

N

N N

β-lactamase inhibitors• Class II β-lactamase inhibitors• Thienamycin served as design prototype

– other natural product isolated in 1976• Example of a carbapenem

– The double bond in the 5-membered ringincreases the strain and reactivity of the β-lactam

– Good intrinsic antibacterial activity– Unstable in solution

• Rapidly polymerizes

– Easily inactivated by host renal dehydropeptidase-1

N

H

O

CO2H

SH3C

HO

NH2

HH

thienamycin

N

H

O

CO2H

SH3C

HO

NH2

HH

N

H

O

CO2H

SH3C

HO

NH2

HH

N

H

O

CO2H

SH3C

HO

NH

HH

HN

H

O

CO2H

S

H3C

HO

NH2

HH

etc.

N

H

O

CO2H

SH3C

HO

NH2

HH

DHP-1

HO2C HN

H

CO2H

SH3C

HO

NH2

HH

H2O

22

Carbapenem antibiotics• Imepenem

– Formimide derivative of thienamycin– Extremely broad spectrum– Stable to hydrolysis by most serine β-lactamases and inhibits many

Gram-(-) β-lactamases– Used for antibacterial activity and β-lactamase resistance – not as aβ-lactamase inhibitor

– Can cause seizures in ~2% of patients– Co-administered with Cilastatin

• Reversible competitive inhibitor of renal DHP-1• Not an antibiotic

• Inhibition of DHP-1 extends the t1/2 of imepenem– Imepenem + Cilastatin = Primaxin® injected

N

H

O

CO2H

SH3C

HO

NH

H

NH

HH

imepenem

HN S

O CO2H

CO2H

H NH2H

cilastatin

Newer Carbapenems• Meropenem Merrem®

– Extremely broad spectrum• not quite as potent as imepenem vs Gram-(+)

– Greater in vitro antipseudomonal activity than imepenem– Resistant to most β-lactamases, except metallo-β-lactamases

• No activity versus MRSA– Does not require coadministration with cilastatin

• Only slowly hydrolyzed by DHP-1– Good penetration of CSF, heart valves, lungs

• Fewer reports of seizures

• Ertapenem (Invanz®)– Resistant to DHP-1 inactivation– Resistant to most β-lactamases, except metallo-β-lactamases– Not active vs Ps. Aeruginosa or Acinetobacter sp.– 1g once/day dosing

N

H

OCO2H

SH3C

HO

NH

N CH3

O

CH3H H3C

N

H

OCO2H

SH3C

HO

NH

HN

O

CH3H

CO2H

23

Carbapenems (cont’d)• Biapenem Omegacin®

– Same broad spectrum as otherthienamycin derivatives

– Resistant to DHP-1 inactivation• not quite as potent as imepenem vs Gram-(+)

– Better antipseudomonal activity than other carbapenems– Resistant to most β-lactamases, except metallo-β-lactamases– Twice/day dosing

N

H

OCO2

SH3C

HO

N

N

CH3H N