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Medicinal

Chemistry of Beta -
Lactam Antibiotics

- ;
This section focuses on the medicinal chemistry of beta lactam antibiotics the second part of our series on the medicinal chemistry

antibacterial compounds .

, , .
Penicillin derivatives cephalosporins monobactams and carbapenems all belong to this popular class of drugs A four membered -
,
lactam ring known as a β-lactam ring, is a common structural feature of this class (see below). To this day, the pharmacology of be
.
lactam antibiotics has clearly bore out an excellent safety and efficacy profile Most of these medicines work by interfering with bact

; ,
cell wall synthesis the cell wall being an optimum drug target because it is something that bacterial cells possess but not human ce

Penicillin and its Derivatives

Penicillin consists of a fused a β-lactam ring and a thiazolidine ring; part of the heterocyclic bicyclic system is the β-lactam ring. The
bicyclic system confers greater ring strain on the β-lactam ring, an aspect important for activity. An amide and a carboxylic acid gro
.
are also present The carboxylic acid group is a possible site of modification to make prodrugs Also . – note the stereochemistry of th
-
acylamino side chain with respect to the 4 membered ring and the cis stereochemistry for the hydrogen atoms highlighted in green T .
key structural features of penicillins can be summarised as follows :

Fused β-lactam and thiazolidine ring forming a bicyclic system (Penam)

Free carboxylic acid

 Acylamino side chain

Cis stereochemistry for the hydrogen

; ,
Texts describing penicillins may appear to have conflicting numbering systems as there are two different widely used numbering

. . ,
systems The USP assigns the nitrogen atom at number 1 and the sulfur atom at number 4 In contrast the Chemical Abstracts

.
assigns sulfur as number 1 and the nitrogen as number 4 Keep these differing numbering systems in mind when reviewing texts on b

lactam medicinal chemistry .

Chemical Properties & Reactions

’ - . , ,
Penicillin s overall shape is similar to a half open book As we talked about earlier the bicyclic ring system has large torsional strain  
. , -
angle strain Unlike typical tertiary amides the carbonyl group of the strained four membered ring is very reactive and susceptible to

.
nucleophilic attack Think about amide resonance from introductory organic chemistry and its effect on amide reactivity In the case .
the β-lactam ring, amide resonance is diminished. For steric reasons the bonds to the nitrogen cannot be planar; the opening of the f
membered ring relieves strain .

. -
Penicillins can react with amines to form inactive amides This has implications on co administration and formulation .

Activity – Can you draw a reaction mechanism for the scheme shown below?

.
Penicillins are also generally susceptible to hydrolysis under alkaline conditions Alkaline hydrolysis can be catalysed by the presenc

metal ions such as Cu

+.The
2
;
resulting hydrolysis products do not possess antibacterial activity this is valuable knowledge for the

, ,
storage analysis and processing of these medicinal chemistry compounds .
Penicillins also tend to be sensitive to acids (see reaction scheme below). Penillic acid is the major product of acid degradation. In th
, .
stomach where conditions are acidic the drug breaks down Acidic conditions must also be avoided during production and analysis .
,
Penicillins such as phenoxymethylpenicillin (Penicillin V), have enhanced acid stability as they have electron withdrawing R groups.

, - . 
Like many other drugs penicillins face enzyme catalysed degradation in vivo Amidases catalyse the conversion of the C6 amide to a

.
amine Amidases are useful in industry for the production of 6-Aminopenicillanic acid (6-APA). This compound is used a precursor f
many semisynthetic penicillins .

Drug resistance is also a growing problem .β-lactamases (beta lactamases) are mainly responsible for this.

β-lactamases are serine protease enzymes that act against the β-lactam drugs through a similar mechanism as the transpeptidase
; .
enzyme the bacterial enzyme targeted by penicillin The mechanism of action will be shown later .

β-lactamase inhibitors such as clavulanic acid are given to patients in combination with penicillins such as amoxicillin. The use of
. ,
clavulanic acid in penicillin formulations allows for a reduction in dosage Furthermore the spectrum of activity is also improved .
that there is no single β-lactamase enzyme. Clavulanic acid does not inhibit all β-lactamase enzymes.
 Augmentin
®: Amoxicillin + clavulanic acid

Timentin
®: Ticarcillin + clavulanic acid

Tazobactam and sulbactam are examples of β-lactamase inhibitors that contain the β-lactam ring. Avibactam is an example of a β-
lactamase inhibitor that does not have the β-lactam ring in its structure.

Now lets turn our attention to the synthesis of semisynthetic derivatives .

Synthesis of Semisynthetic Derivatives

We briefly mentioned 6-APA earlier. 6-APA is acquired through enzymatic hydrolysis of Penicillin G or Penicillin V, or through tradition
.
organic chemistry Reaction with an acid chloride at the C6 primary amine allows the synthesis of many semisynthetic penicillin

derivatives (try to draw the reaction mechanism as practice!). The free carboxylic acid is, though, a possible site of modification. Este
prodrugs such as pivampicillin were developed to improve the pharmacokinetic properties of their parent drug Pivampicillin is a.
pivaloyloxymethyl ester prodrug of ampicillin .

Spectroscopy

- - .
We emphasized the cis stereochemistry of the H5 H6 protons at the start of this beta lactam review Generally speaking the H ,
coupling constant ( H NMR) is in the range of 4-5 Hz.
1 13
C NMR studies and DEPT experiments would help distinguish between CH CH ,
. ,
and CH3 When studying the IR spectra of penicillins one must be on the lookout for the carbonyl stretches – particularly for the
characteristic β-lactam carbonyl stretch at around 1770-1790 -1
cm .

,
Depending on which penicillin is being studied the side chain would give characteristic NMR and IR signals The predicted . 1
H NMR

. .
spectrum of ampicillin is shown below Note that the spectrum below is merely an estimate Can you explain why the benzylic proton

appear shifted downfield than expected ?

Mechanism of Action

. . -
Recall the structure of bacteria from microbiology The bacterial cell wall is needed by most bacteria in order to survive Gram positiv

. - ,
bacteria possess a thick peptidoglycan layer in the cell wall and an inner cell membrane Gram negative bacteria on the other hand ,
. -
possess an outer membrane and an inner cell membrane Porins are present in the outer membrane of Gram negative bacteria The .
- .
much thinner peptidoglycan layer of Gram negative bacteria is found between these two membranes The significance of this outer

-
membrane in drug design of broad spectrum penicillins is assessed later .

Peptidoglycan (or murein) consists of sugar and amino acid units. This mesh-like polymeric layer outside the inner cell membrane fo
. -
the cell wall of bacteria The sugars N acetylglucosamine (GlcNAc or NAG) and N-acetylmuramic acid (MurNAc or NAM) alternate,
are connected through a β-(1,4)-glycosidic bond. An amino acid chain is found in each NAM. D-amino acids are also found in these
. - ; -
amino acid chains The sugars are cross linked via these peptides cross linking adding structural integrity to the bacterial cell wall .

.
Transpeptidase enzymes are bacterial enzymes responsible for the formation of these crosslinks Penicillins act by interfering with th

- . ,
cross linking of peptidoglycan by inhibiting the transpeptidase enzyme Without an intact cell wall bacteria are generally unable to

. ,
survive Thus penicillins are bactericidal in effect .

-
Structure Activity Relationships

,
Through the synthesis and studies of many semisynthetic penicillins the following conclusions were reached .

Cis stereochemistry of H 5 and H6 essential

 The bicyclic ring is very important

The free carboxylate is essential

The acylamino side chain is necessary

, , ,
Variation is mostly limited to the R group of the amide and as mentioned earlier prodrugs have been developed by modifying the

. , ’
carboxylate group So far we ve reviewed the following structural modifications :

-
Enhancing acid stability by using electron withdrawing R groups

Converting the carboxylate functional group to an ester to give a prodrug

We will now consider several other structural modifications .

Other Structural modifications

-
Gram negative bacteria possess an outer lipopolysaccharide membrane which surrounds the thinner cell wall The outer membrane .
.
serves as a protective layer against compounds that may pose harm to the bacteria This partly explains why Gram negative bacteri -
. ,
are generally resistant to antibacterial compounds As examined earlier porins are proteins present in the outer membrane Water an .
.
essential nutrients can pass through these proteins Small drugs can also pass through porins The ability of drugs to pass through .
,
porins is dependent on their size structure and charge .

, ,
Generally speaking molecules that are large anionic and hydrophobic are unable to pass through proteins On the other hand . ,
, .
molecules that are small zwitterionic and hydrophilic tend to pass through easily During the search for broad spectrum penicillins -
,
through variation of the acylamino side chain the following conclusions were reached :

Hydrophobic groups tend to enhance ability against Gram positive bacteria -

Hydrophilic groups generally increase ability against Gram negative bacteria -

Attachment of a hydrophilic group at Cα appear to improve activity against Gram negative bacteria -

-
Broad spectrum antibiotics such as amoxicillin and ampicillin both have -NH2 groups attached to Cα (as shown below); both
. , -
compounds being orally active As well as this the presence of the electron withdrawing amino group increases acid stability Ampic - .
.
is poorly absorbed by the gut due to ionisation of both the amino and carboxylic groups Oral absorption of amoxicillin is in contras ,
much higher .

, ,
Modifications such as changing the carboxylic acid to esters were made at the carboxylic group to alleviate this problem Pivampici .
.
and bacampicillin are examples of ester prodrugs of ampicillin The prodrugs undergo metabolism in the body to give ampicillin .
remember the names of the enzymes involved in the ester hydrolysis of prodrugs ?
 - ,
The ureidopenicillins are broad spectrum penicillins typically used parenterally and are active against Pseudomonas aeruginosa

, . .
name suggests a urea group is present in the molecule The urea group is situated at Cα Azlocillin and piperacillin are examples of

ureidopenicillins .

β-lactamases gave bacteria resistance to the traditional penicillins, driving the need for β-lactamase-resistant penicillins. Steric shield
.
may also be used This strategy involves designing penicillins to resist β-lactamases. By placing a bulky group on the acylamino sid
,
chain degradation of the drug by β-lactamases is minimized. However, if a steric shield is too bulky, the penicillin is not able to bind t
transpeptidase .

. -
Methicillin is an example of a penicillin with a bulky group This semi synthetic penicillin possesses a dimethoxybenzene R group Bo .
.
the methoxy groups of the benzene are at the ortho position Nafcillin possesses a naphthalene ring in its acylamino side chain whic

. -
acts as a steric shield Flucloxacillin contains a bulky and electron withdrawing heterocyclic acylamino side chain Thus flucloxacillin . ,
- ,
an acid resistant narrow spectrum - , β-lactamase-resistant penicillin.

Summary

-
Penicillins are bactericidal beta lactam antibiotics
 ’
Penicillin s core structure consists of a fused β-lactam ring and a thiazolidine ring

The bicyclic system is highly strained

Modifications can also be made at the acylamino side chain

Cis stereochemistry of H5 and H6 is essential

6-Aminopenicillanic acid (6-APA) is mainly used as a precursor for semisynthetic penicillin drugs

Structural modifications :

The carboxylic acid group can be modified to give ester prodrugs

-
Attach electron withdrawing groups at the amide to enhance acid stability

Hydrophilic groups at Cα at the acylamino side chain improves spectrum of activity

Steric shields at the acylamino side chain generally improves resistance to β-lactamase enzymes

Cephalosporins

Cephalosporin C was the first cephalosporin discovered from a fungus obtained from Sardinian sewer waters during the mid 1 - 940s. J
,
like penicillins Cephalosporin C has a bicyclic system made up of a β-lactam ring fused with a sulfur heterocycle, which, in this case
. .
the dihydrothiazine ring The side chain is referred to as the aminoadipic side chain The acetoxy group of Cephalosporin C is a key

;
feature a feature examined in more detail later .

,
Cephalosporin C is less potent compared to penicillins but this compound has many advantageous properties Cephalosporin C is m .
- , . ,
acid resistant and has a better spectrum of activity for example Moreover the likelihood of causing allergic reactions is considerabl

. ,
less As a result cephalosporin C became a useful lead compound for the development of better more clinically robust antibiotics , .

Chemical Properties & Reactivity

,
Compared to the penicillins the ring system strain is not as great but like the , , β-lactam carbonyl of penicillins, the β-lactam carbony
.
group of Cephalosporin C is also reactive for similar reasons Diminished amide resonance and ring strain confers reactivity The fou .
.
membered ring is also susceptible to nucleophilic attack Cephalosporin C inhibits the transpeptidase enzyme through the mechanism

. . ,
shown below A serine residue is involved As shown below the acetoxy group serves as a leaving group .

Synthesis of Semisynthetic Derivatives

7-aminocephalosporinic acid (7-ACA) is used as the precursor of many cephalosporins. Unlike 6-APA which can be acquired from
enzymatic hydrolysis of certain penicillins , 7-ACA cannot be acquired by enzymatic hydrolysis of Cephalosporin C. 7-ACA is produce
.
chemical hydrolysis of Cephalosporin C Due to the presence of the reactive β-lactam ring, a special method of chemical hydrolysis
.
devised Can you draw a mechanism for the formation of the imino chloride ?
Cephalosporin analogues may be formed by reacting 7-ACA with acid chlorides.

-
Structure Activity Relationships

The β-lactam ring is crucial for activity

Bicyclic ring system important in increasing ring strain

-
The cis stereochemistry at the positions highlighted in green is important

.
Other groups may be substituted for the acetoxy group which may or may not serve as good leaving groups The nature of the leav

.
group is important for activity Better leaving groups tend to give cephalosporin C analogues with better activity .

The acylamino side chain may be altered

.
Sites of possible modifications are highlighted in red boxes

First Generation Cephalosporins

 ,
Cefalexin cephalothin (cefalotin), cephaloglycin, and cephaloridine are examples of first-generation cephalosporins. The methyl gro
, .
in cephalexin is a poor leaving group which is bad for activity However the use of a methyl group appears to improve absorption , .
-
Cefalexin may be synthesized through an acid catalysed ring expansion of a penicillin Cephalothin has an acetoxy as a leaving gro .
and a 1 -(thiophen-2-yl)propan-2-one in its acylamino side chain. Despite being a good leaving group, the acetoxy moiety is suscept
- . ’
to enzyme catalysed hydrolysis Cephaloglycin s acylamino side chain is the same as that for ampicillin Cephaloridine has pyridiniu .
, .
as a leaving group giving pyridine Unlike the acetoxy group the pyridinium is stable to metabolism , .

-
First generation cephalosporins share the following features :

,
In general their activity is comparably lower than penicillins but possess a broader spectrum of activity

Apart from the methyl substituted cephalosporins gut wall absorption is poor ,

-
Most of the first generation cephalosporins are administered by injection

-
Activity against Gram positive bacteria is greater than Gram negative bacteria -

Second Generation Cephalosporins

, ,
Cefamandole cefaclor and cefuroxime are examples of second generation cephalosporins The second generation have increased - . -
-
activity against Gram negative species of bacteria such as Neisseria gonorrhoeæ while some have decreased Gram positive activity , -
-
Many cephalosporins that fall under the second generation group are also able to cross the blood brain barrier Cefuroxime is also a - .
.
example of an oximinocephalosporin The presence of the iminomethoxy group appears to increase stability against certain β-
lactamases .

Third Generation Cephalosporins

-
Cefdinir and ceftriaxone belong to the third generation of cephalosporins During 200 . 8, cefdinir was one of the highest-selling
.
cephalosporins Ceftriaxone is marketed by Hoffmann La Roche under the trade name Rocephin - ® (known for its painful administrati
, -
Overall third generation cephalosporins are more stable to β-lactamase degradation, and have even greater anti-Gram-negative act
, -
Like the previous generation the third generation are able to cross the blood brain barrier making them useful against meningococc ,

Fourth Generation Cephalosporins

- . -
The fourth generation compounds exist are zwitterionic The fourth generation cephalosporins are not only better at traversing the ou

- ,
membrane of Gram negative bacteria but these compounds also have similar activity against Gram positive bacteria as first genera - -
cephalosporins Moreover . , β-lactamase resistance is also greater. Like the 2 nd
and 3
rd
,
generations many of the 4
th
generation can c

.
the blood brain barrier They are also used against Pseudomonas aeruginosa .
Fifth Generation Cephalosporins

,
Currently members of the scientific community have not reached agreement with regards to the use of the term fifth generation ‘ -
cephalosporins ’. Fifth-generation compounds have demonstrable activity against MRSA. Ceftobiprole is often described as a fifth-
. - .
generation cephalosporin This compound possesses good anti Pseudomonal activity Ceftaroline fosamil is also another example o

cephalosporin described as fifth generation - .

Cephamycins

.
Recall that one of the hydrogens emphasised earlier is a possible site of modification The methoxy substituted versions are referred

.
cephamycins A compound called cephamycin C can be isolated from Streptomyces clavuligerus A urethane group is present instea .
,
the acetoxy group enhancing the compounds metabolic stability Can you explain why . ?

Derivatives may be synthesised from the methoxy substituted analogue of - 7-ACA, or through reactions with cephalosporins. Note th
.
some refer to cephamycins as a separate class of antibacterial compounds altogether The cephamycins appear to have greater

resistance against β-lactamases. Cephalosporins and cephamycins are sometimes collectively referred to as cephems.

Summary

Fused β-lactam and dihydrothiazine ring form a bicyclic system.

7-aminocephalosporinic acid (7-ACA) is used as precursor for many semisynthetic cephalosporins

-
Structure activity relationships are similar to penicillins

Nature of leaving group is important to activity

Generations of cephalosporins :

: .
1st Activity is comparably lower than penicillins but possess a broader spectrum of activity Greater activity for Gram positive orga -
-
than Gram negative organisms .

: -
2nd Have increased activity against Gram negative bacteria but some have a concomitant reduction of Gram positive activity Ma - .
-
cross the blood brain barrier .

: - .
3rd Even better activity against Gram negative bacteria Some compounds have the same problem of decreased Gram positive ac -
.
with the previous generation They are also associated with improved β-lactamase resistance and many can also cross the blood-b
barrier .

: -
4th Better Gram negative bacteria activity , β-lactamase resistance, and many can also cross the blood-brain barrier. Gram-positiv
 activity similar to the 1
st
generation .

5th: Currently no universal agreement on its definition. Some drugs classified as ‘fourth-generation’ are classified as ‘fifth-generatio
. , .
vice versa Examples often include ceftobiprole ceftaroline and ceftolozane Ceftobiprole has potent anti Pseudomonal activity and -
. -
confers little resistance Fifth generation drugs also show activity against MRSA .

Monobactams

,
As the name suggests the monobactams such as aztreonam are , , β-lactam compounds that are not fused to another ring.
-
Monobactams exhibit moderate activity against certain Gram negative bacteria in vitro including Neisseria and Pseudomonas ,

Carbapenems

The carbapenem class of β-Lactam antibiotics have broad-spectrum activity and exhibit resistance to many β-lactamases.
Carbapenems are used as antibiotics of last resort for infections of bacteria such as Escherichia coli and Klebsiella pneumoniae

Thienemycin is a carbapenem first discovered and isolated from Streptomyces cattleya in 1 976. Thienamycin exhibits excellent activit
- -
against Gram positive and Gram negative bacteria and displays resistance to many β-lactamases. Meropenem and doripenem
.
analogues of thienemycin and are examples of carbapenems currently in clinical use Both compounds have been described as ultra

-
broad spectrum .

,
From the structure of the compounds shown it is easy to see that the carbapenems have some structural features that penicillins do

- - .
The double bond on the five membered ring leads to high ring strain A sulfur atom is also missing in the five membered ring The - .
.
acylamino side chain is absent Also note the trans stereochemistry of the hydrogens .

- ;
This concludes our review of the medicinal chemistry of beta lactam antibiotics a subject often featured on pharmacy pharmaceutic ,
- . -
chemistry and other medicinal related courses It informs the reader of structure activity relationships the development of synthetic ,
,
derivatives and how medicinal chemistry relates to broader concepts such as formulation and dosage The growing problem of .
- ;
resistance is an ever present challenge for medicinal chemists a biological mechanism that spurs on the engine of research in this f

Further Reading

Total synthesis of thienemycin :

. .
J Org Chem ., 1990, 55 (10), pp 3098–3103
 : ,
Antibacterial resistance worldwide causes challenges and responses

Nature Medicine ., 2004, 10, S122 – S129

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