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    1

    Patrick

    An Introduction to Medicinal Chemistry3/e

    Chapter 16

    ANTIBACTERIAL AGENTS

    Part 1: Penicillins

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    1

    PENICILLINS

    N

    S Me

    Me

    HN

    CO2H

    O

    C H H

    O

    R

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    1

    INTRODUCTION

    Antibacterial agents which inhibit bacterial cell wall synthesis Discovered by Fleming from a fungal colony (1928)

    Shown to be non toxic and antibacterial

    Isolated and purified by Florey and Chain (1938)

    First successful clinical trial (1941)

    Produced by large scale fermentation (1944)

    Structure established by X-Ray crystallography (1945)

    Full synthesis developed by Sheehan (1957)

    Isolation of 6-APA by Beechams (1958-60)

    - development of semi-synthetic penicillins Discovery of clavulanic acid and b-lactamase inhibitors

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    Acyl side

    chain

    6-Aminopenicillanic acid

    (6-APA)

    STRUCTURE

    Side chain varies depending on carboxylic acid present in fermentation medium

    b-Lactamring

    Thiazolidine

    ring

    present in corn steep liquor

    Penicillin GCH2 CO2H

    Benzyl penicillin (Pen G)

    R =

    Phenoxymethyl penicillin (Pen V)

    R =

    CH2

    O CH2

    N

    S Me

    Me

    HN H H

    CO2HO

    C

    O

    R

    Penicillin V

    (first orally active penicillin)

    OCH2 CO2H

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    Shape of Penicillin G

    Folded envelope shape

    H

    NO

    NHC

    O

    R

    H

    S

    CO2

    H

    H

    Me

    Me

    ..

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    1CYS VAL

    Biosynthesis of Penicillins

    S

    N

    Me

    MeO

    HN

    CO2H

    C

    R

    O

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    Properties of Penicillin G

    Active vs. Gram +ve bacilli and some Gram -ve cocci

    Non toxic Limited range of activity

    Not orally active - must be injected

    Sensitive to b-lactamases(enzymes which hydrolyse the b-lactam ring)

    Some patients are allergic

    Inactive vs.Staphylococci

    Drug DevelopmentAims

    To increase chemical stability for oral administration

    To increase resistance to b-lactamases

    To increase the range of activity

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    SAR

    Conclusions

    Amide and carboxylic acid are involved in binding

    Carboxylic acid binds as the carboxylate ion

    Mechanism of action involves the b-lactam ring Activity related to b-lactam ring strain

    (subject to stability factors)

    Bicyclic system increases b-lactam ring strain

    Not much variation in structure is possible Variations are limited to the side chain (R)

    Bicyclic system essential

    N

    S Me

    Me

    HN

    CO2H

    O

    C H H

    O

    R

    Carboxylic acid essentia

    Cis Stereochemistry essential

    bLactam essential

    Amide essentia

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    Penicillins inhibit a bacterial enzyme called the transpeptidase

    enzyme which is involved in the synthesis of the bacterial cellwall

    The b-lactam ring is involved in the mechanism of inhibition Penicillin becomes covalently linked to the enzymes active site

    leading to irreversible inhibition

    Covalent bond formed

    to transpeptidase enzyme

    Irreversible inhibition

    N

    S Me

    Me

    HN

    H H

    CO2HO

    C

    O

    R

    Nu

    Enz

    CHN

    C

    CO2H

    HH

    Me

    MeS

    HN

    O

    R

    O

    Nu-Enz-H N

    S Me

    Me

    HN

    H H

    CO2HO

    C

    H

    Enz-Nu

    O

    R

    Mechanism of action

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    L-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-LysL-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-Lys

    L-Ala

    D-Glu

    L-Lys

    Mechanism of action - bacterial cell wall synthesis

    NAM NAM NAMNAGNAG

    NAM NAM NAMNAGNAG

    NAM NAM NAMNAGNAG

    Bond formationinhibited by

    penicillin

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    1

    D-AlanineTRANSPEPTIDASE

    PENICILLIN

    SUGARBACKBONE

    NAM

    L-Ala

    NAG

    D-Glu

    L-Lys

    D-Ala

    D-Ala

    Gly Gly Gly Gly Gly Gly GlyGlyGlyGlyL-Lys

    NAG

    D-Ala

    D-Ala

    D-Glu

    L-Ala

    NAM

    SUGAR

    BACKBONE

    L-Lys Gly Gly

    D-Ala

    NAM

    L-Ala

    NAG

    D-Glu

    L-L ys Gly Gly Gly Gly GlyGlyGlyGly

    NAG

    D-Ala

    D-Glu

    L-Ala

    NAM

    Cross linking

    Mechanism of action - bacterial cell wall synthesis

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    Penicillin inhibits final crosslinking stage of cell wall synthesis

    It reacts with the transpeptidase enzyme to form an

    irreversible covalent bond

    Inhibition of transpeptidase leads to a weakened cell wall

    Cells swell due to water entering the cell, then burst (lysis) Penicillin possibly acts as an analogue of the L-Ala-g-D-Glu

    portion of the pentapeptide chain. However, the carboxylate

    group that is essential to penicillin activity is not present in

    this portion

    Mechanism of action - bacterial cell wall synthesis

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    Alternative theory- Pencillin mimics D-Ala-D-Ala.

    Normal Mechanism

    PeptideChain

    D-Ala D-Ala CO2H

    OH

    Peptide

    Chain

    Gly

    HOH

    Peptide

    Chain

    Peptide

    Chain

    D-Ala GlyPeptide

    Chain

    O

    D-Ala

    Mechanism of action - bacterial cell wall synthesis

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    Alternative theory- Pencillin mimics D-Ala-D-Ala.

    Mechanism inhibited by penicillin

    OH

    Peptide

    Chain

    Gly

    H

    Blocked H2O

    Blocked

    Blocked Irreversibly blocke

    HCR

    O

    CO2H

    NH

    OMe

    Me

    N

    S

    S

    HN

    O

    O

    Me

    Me

    NH

    CO2H

    C

    O

    R HS

    HN

    O

    O

    Me

    Me

    NH

    CO2H

    C

    O

    R H

    Mechanism of action - bacterial cell wall synthesis

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    Penicillin can be seen to mimic acyl-D-Ala-D-Ala

    But 6-methylpenicillin is inactive

    despite being a closer analogue

    H

    S

    N

    Me

    MeO

    HN

    CO2

    H

    MeC

    R

    O

    Penicillin Acyl-D-Ala-D-Ala

    HH

    CO2H

    HN

    OMe

    Me

    N

    SC

    R

    O

    Me

    CO2H

    HN

    O CH3

    HN

    HH

    C

    R

    O

    Mechanism of action - bacterial cell wall synthesis

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    H

    CO2H

    HN

    O

    Me

    Me

    N

    SC

    R

    O

    Penicillin may act as an umbrella inhibitor

    Mechanism of action - bacterial cell wall synthesis

    HO

    OH

    Active Site

    D-Ala-D -Ala

    Blocked

    H

    Acylation

    OH

    O

    H

    CO2H

    HN

    Me

    Me

    HN

    SC

    R

    O

    O

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    1

    Resistance to Penicillins

    Penicillins have to cross the bacterial cell wall in order toreach their target enzyme

    But cell walls are porous and are not a barrier

    The cell walls of Gram +ve bacteria are thicker than Gram -ve cell walls, but the former are more susceptible to

    penicillins

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    Resistance to Penicillins

    Cell

    Cell membrane

    Thick porous cell wall

    Gram +ve bacteria

    Thick cell wall

    No outer membrane

    More susceptible to penicillins

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    Resistance to Penicillins

    L

    CellCell

    membrane

    Thin cell wall

    Lactamaseenzymes

    Outer

    membrane

    Hydrophobic barrier

    Periplasmic

    space

    Porin

    L

    L

    L

    Gram -ve bacteria

    Thin cell wall Hydrophobic outer membrane

    More resistant to penicillins

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    Resistance to Penicillins

    Factors

    Gram -ve bacteria have a lipopolysaccharide outer

    membrane preventing access to the cell wall

    Penicillins can only cross via porins in the outer membrane

    Porins only allow small hydrophilic molecules that can exist

    as zwitterions to cross

    High levels of transpeptidase enzyme may be present The transpeptidase enzyme may have a low affinity for

    penicillins (e.g. PBP 2a forS. aureus)

    Presence ofb-lactamases

    Concentration ofb-lactamases in periplasmic space Mutations

    Transfer ofb-lactamases between strains Efflux mechanisms pumping penicillin out of periplasmic

    space

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    Penicillin Analogues - Preparation

    1) By fermentation vary the carboxylic acid in the fermentation medium

    limited to unbranched acids at the a-position i.e. RCH2CO2H tedious and slow

    2) By total synthesis

    only 1% overall yield (impractical)

    3) By semi-synthetic procedures

    Use a naturally occurring structure as the starting material foranalogue synthesis

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    1

    Penicillin Analogues - Preparation

    Fermentation

    Penicillin acylase

    or chemical hydrolysis

    O

    C

    ClR

    Penicillin

    HC

    HN

    CO2H

    OMe

    Me

    N

    S

    O

    CH2

    6-APA

    OCO2H

    HH

    H2N

    Me

    MeS

    N

    Semi-synthetic penicillinsN

    S Me

    Me

    HN

    H H

    CO2HO

    C

    O

    R

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    1

    Penicillin Analogues - Preparation

    Problem - How does one hydrolyse the side chain by chemical

    means in presence of a labileb-lactam ring?Answer - Activate the side chain first to make it more reactive

    Note - Reaction with PCl5 requires involvement of nitrogens

    lone pair of electrons. Not possible for the b-lactam nitrogen.

    N

    SNH

    OCO2H

    C

    O

    PhCH2PCl5

    PENNC

    Cl

    PhCH2

    ROH

    PENNC

    OR

    PhCH2H2O

    6-APA

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    1

    Problems with Penicillin G

    It is sensitive to stomach acids

    It is sensitive to b-lactamases - enzymes which hydrolyse theb-lactam ring it has a limited range of activity

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    Problem 1 - Acid Sensitivity

    Reasons for sensitivity

    1) Ring Strain

    H2O

    H

    N

    S Me

    Me

    HN

    H H

    CO2H

    O

    C

    O

    R

    Relieves ring strain

    CHN

    HO2C

    CO2H

    HH

    Me

    MeS

    HN

    O

    R

    Acid or

    enzyme

    N

    S Me

    Me

    HN

    H H

    CO2H

    O

    C

    HO

    O

    R

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    1

    Problem 1 - Acid Sensitivity

    2) Reactive b-lactam carbonyl groupDoes not behave like a tertiary amide

    Interaction ofnitrogens lone pair with the carbonyl group is not possible

    Results in a reactive carbonyl group

    Tertiary amide

    C

    O

    R

    NR2 Unreactive

    R

    C

    R

    N

    O

    R

    b-Lactam

    Folded ring

    system

    N

    S

    OH

    Me

    Me

    CO2H

    Impossibly

    strained

    N

    SMe

    Me

    CO2H

    O

    Reasons for sensitivity

    X

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    Problem 1 - Acid Sensitivity

    3) Acyl Side Chain

    - neighbouring group participation in the hydrolysis mechanism

    H

    Further

    reactions

    -

    H

    N

    S

    O

    N

    O

    R R

    O

    N

    O

    S

    HN

    C N

    O

    S

    N

    H

    R

    O

    H

    Reasons for sensitivity

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    1

    C

    E.W.G.

    O

    HN

    O

    S

    N

    H

    Problem 1 - Acid Sensitivity

    Conclusions

    The b-lactam ring is essential for activity and must be retained Therefore, cannot tackle factors 1 and 2

    Can only tackle factor 3

    Strategy

    Vary the acyl side group (R) to make it electron withdrawing to

    decrease the nucleophilicity of the carbonyl oxygen

    Decreases

    nucleophilicity

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    1

    N

    S

    HN

    O

    C

    O

    CH2PhOH

    Penicillin V(orally active)

    Problem 1 - Acid Sensitivity

    Examples

    electronegative

    oxygen

    Very successful semi-

    synthetic penicillins

    e.g. ampicillin, oxacillin

    Better acid stability and orally active

    But sensitive to b-lactamases Slightly less active than Penicillin G

    Allergy problems with some patients

    X = NH2, Cl, PhOCONH,Heterocycles, CO2H

    C

    HC

    O

    HN

    O

    S

    N

    X

    R

    H

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    Problem 2 - Sensitivity to b-Lactamases

    Notes on b-Lactamases Enzymes that inactivate penicillins by opening b-lactam rings Allow bacteria to be resistant to penicillin Transferable between bacterial strains (i.e. bacteria can

    acquire resistance)

    Important w.r.t.Staphylococcus aureus infections in hospitals

    80% Staph. infections in hospitals were resistant to penicillinand other antibacterial agents by 1960

    Mechanism of action for lactamases is identical to the

    mechanism of inhibition for the target enzyme

    But product is removed efficiently from the lactamase active

    site

    b-LactamaseN

    S Me

    Me

    HN

    CO2HO

    CH

    O

    R

    CHN

    CO2H

    Me

    MeS

    HN

    H

    HO2C

    O

    R

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    Bulky

    group

    Problem 2 - Sensitivity to b-Lactamases

    Strategy

    Enzyme

    C

    R

    HN

    O

    CO2H

    Me

    MeS

    N

    H H

    O

    Block access of penicillin to active site of enzyme byintroducing bulky groups to the side chain to act as steric

    shields

    Size of shield is crucial to inhibit reaction of penicillins with b-lactamases but not with the target enzyme (transpeptidase)

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    ortho groupsimportant

    Problem 2 - Sensitivity to b-Lactamases

    Examples - Methicillin (Beechams - 1960)

    Methoxy groups block access to b-lactamases but not to transpeptidases Active against some penicillin G resistant strains (e.g.Staphylococcus)

    Acid sensitive (no e-withdrawing group) and must be injected

    Lower activity w.r.t. Pen G vs. Pen G sensitive bacteria (reduced access

    to transpeptidase)

    Poorer range of activity

    Poor activity vs. some streptococci

    Inactive vs. Gram -ve bacteria

    N

    S Me

    Me

    HN

    CO2H

    O

    MeO

    OMe

    H HC

    O

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    Problem 2 - Sensitivity to b-Lactamases

    Examples - Oxacillin

    Orally active and acid resistant

    Resistant to b-lactamases

    Active vs.Staphylococcus aureus Less active than other penicillins

    Inactive vs. Gram -ve bacteria

    Nature of R & R influences absorption and plasma protein binding

    Cloxacillin better absorbed than oxacillin

    Flucloxacillin less bound to plasma protein, leading to higher

    levels of free drug

    Bulky ande- withdrawing

    Oxacillin R = R' = H

    Cloxacillin R = Cl, R' = H

    Flucloxacillin R = Cl, R' = FN

    O

    C

    O

    HN

    OCO2H

    Me

    MeS

    N

    R'

    R

    Me

    H H

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    Problem 3 - Range of Activity

    Factors

    1. Cell wall may have a coat preventing access to the cell

    2. Excess transpeptidase enzyme may be present

    3. Resistant transpeptidase enzyme (modified structure)

    4. Presence ofb-lactamases5. Transfer ofb-lactamases between strains6. Efflux mechanisms

    Strategy

    The number of factors involved make a single strategy

    impossible

    Use trial and error by varying R groups on the side chain

    Successful in producing broad spectrum antibiotics

    Results demonstrate general rules for broad spectrum activity.

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    Problem 3 - Range of Activity

    1. R= hydrophobic results in high activity vs. Gram +ve bacteria

    and poor activity vs. Gram -ve bacteria

    2. Increasing hydrophobicity has little effect on Gram +ve activity

    but lowers Gram -ve activity

    3. Increasing hydrophilic character has little effect on Gram+ve activity but increases Gram -ve activity

    4. Hydrophilic groups at the a-position (e.g. NH2, OH, CO2H)increase activity vs Gram -ve bacteria

    Results of varying R in Pen G

    P bl 3 R f A i i

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    Problem 3 - Range of Activity

    Examples of Broad Spectrum Penicillins

    Class 1 - NH2 at the a-positionAmpicillin and Amoxycillin (Beechams, 1964)

    Ampicillin (Penbritin)2nd most used penicillin

    Amoxycillin (Amoxil)

    H

    C

    H

    O

    HNC

    NH2

    HO

    O

    C

    NH2

    CHN

    O

    H

    H

    O

    P bl 3 R f A i i

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    Problem 3 - Range of Activity

    Active vs Gram +ve bacteria and Gram -ve bacteria which

    do not produce b-lactamases Acid resistant and orally active

    Non toxic

    Sensitive to b-lactamases Increased polarity due to extra amino group

    Poor absorption through the gut wall

    Disruption of gut flora leading to diarrhoea

    Inactive vs.Pseudomonas aeruginosa

    Examples of Broad Spectrum Penicillins

    Properties

    P bl 3 R f A ti it

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    1

    O

    N

    HNC

    C

    NH2

    S Me

    Me

    CO2R

    H H

    H

    O

    Problem 3 - Range of Activity

    Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969)

    Properties

    Increased cell membrane permeability

    Polar carboxylic acid group is masked by the ester

    Ester is metabolised in the body by esterases to give the free

    drug

    PIVAMPICILLINR = CH2OC

    O

    CMe3

    TALAMPICILLINR = O

    O

    BACAMPICILLIN

    R = CH

    Me

    OC

    O

    O CH2Me

    P bl 3 R f A ti it

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    Problem 3 - Range of Activity

    Mechanism

    Ester is less shielded by penicillin nucleus

    Hydrolysed product is chemically unstable and degrades

    Methyl ester of ampicillin is not hydrolysed in the

    body - bulky penicillin nucleus acts as a steric shield

    ENZYME

    PEN

    C O CH2

    O

    OH

    HH

    PEN

    C O CH2

    O

    O

    C

    O

    CMe3

    Formaldehyde

    PEN

    C OH

    O

    P bl 3 R f A ti it

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    Problem 3 - Range of Activity

    Examples of Broad Spectrum Penicillins

    Class 2 - CO2H at the a-position (carboxypenicillins)

    Examples

    Carfecillin = prodrug for carbenicillin

    Active over a wider range of Gram -ve bacteria than ampicillin

    Active vs.Pseudomonas aeruginosa Resistant to most b-lactamases Less active vs Gram +ve bacteria (note the hydrophilic group)

    Acid sensitive and must be injected

    Stereochemistry at the a-position is important

    CO2H at the a-position is ionised at blood pH

    R = H CARBENICILLIN

    R = Ph CARFECILLINH H

    O

    N

    HNC

    CH

    CO2R

    S Me

    Me

    CO2H

    O

    P bl 3 R f A ti it

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    Problem 3 - Range of Activity

    Examples of Broad Spectrum Penicillins

    Class 2 - CO2H at a-position (carboxypenicillins)

    Examples

    Administered by injection

    Identical antibacterial spectrum to carbenicillin

    Smaller doses required compared to carbenicillin More effective againstP. aeruginosa

    Fewer side effects

    Can be administered with clavulanic acid

    TICARCILLINS

    N

    Me

    MeO

    HH

    CO2H

    HN

    O

    CO2H

    S

    P bl 3 R f A ti it

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    Problem 3 - Range of Activity

    Examples of Broad Spectrum Penicillins

    Class 3 - Urea group at the a-position (ureidopenicillins)Examples

    Administered by injection

    Generally more active than carboxypenicillins vs. streptococci and

    Haemophilus species Generally have similar activity vs Gram -ve aerobic rods

    Generally more active vs other Gram -ve bacteria

    Azlocillin is effective vsP. aeruginosa

    Piperacillin can be administered alongside tazobactam

    Azlocillin

    Mezlocillin

    Piperacillin

    S

    N

    Me

    MeO

    HH

    CO2H

    HN

    O

    NH

    O

    R2N

    HNN

    O

    NN

    OMeO2S

    N N

    OO

    Et