Download Chemistry of Beta Lactam Antibiotics

Chemistry of Beta Lactam Antibiotics
Historical Perspectives
Humans have been plagued with the threat of bacterial infection since before the dawn of recorded
history, and have thus always had a keen interest in finding a suitable treatment for these maladies. In the
early part of the 19th century, colonial Americans were fearful of death by bacterial infections that are
now considered routine, and usually inquired about one another's health in their letters (indeed, the only
casualty during the Lewis and Clark expedition in 1803 was due to a bacterial infection!). Folk medicine
is replete with accounts of the use of various materials for the treatment of bacterial infection (moldy
bread, moldy cheese, plant and animal preparations), which we now assume were effective due to the
presence of some unknown antibacterial agent. The science of microbiology was significantly advanced
by Hans Christian Gram, a danish biologist who developed a stain that allowed the facile visualization of
some bacteria (now called Gram positive). The ability of certain bacteria to retain this stain lies in the
biochemistry of their cell wall. Early researchers such as Koch, Pasteur and Joubert, and Ehrlich
demonstrated fundamental properties of bacterial infection, such as the reliable isolation of a single type
of organism from a given infection, the absence of such bacteria in normal tissue, and the ability to kill
these bacteria with other more common bacteria or organometallics. The modern era of chemotherapy
began in 1936 with the discovery of the antibacterial effects of the sulfonamides. Prior to this time,
Fleming showed that a contamination of Penicillium mold in a bacterial culture produced a clear zone of
inhibition in the culture, and bacteria were killed therein by lysis. Penicillin was not isolated and
identified from this source until the early 1940's, and was first employed as an antibiotic agent in 1941.
The subsequent discovery of streptomycin from the soil organism Streptomyces griseus (Waksman,
1943), chloramphenicol (1947), chlortetracycline (1948), neomycin (1949) and erythromycin (1951)
ushered in the era of the miracle drug. Broad screening programs were then undertaken which identified
many of the antibiotic agents in use today. Currently, soil microbes remain the richest source of new
antibiotic agents. In 1990, the world consumed literally tons of antibiotics valued in excess of 7 billion
dollars; more than half of these antibiotics were of the beta lactam type.
Cell Wall Biochemistry
The biochemical makeup of the bacterial cell wall differs significantly from that of the mammalian lipid
bilayer, and as such provides multiple targets for the development of specific bacteriocidal agents. This
cell wall structure serves three purposes: 1. to provide a semipermeable barrier through which only
desired substances may pass; 2. to provide a barrier against osmotic stress; 3. to prevent digestion by host
enzymes. The gram positive cell wall is comprised of a characteristic set of carbohydrates and proteins
located on the outside aspect of the cell wall, which are the antigenic determinants that differ between
bacterial species and generate an immune response. Inside this layer is a layer comprised of
peptidoglycan made up of repeating units of N-acetylglucosamine (NAG) and N-acetylmuramic acid
(NAM) in polymeric chain form. Each NAM is linked to an oligopeptide chain as shown in the figure
below (L-ala-D-glu-L-lys-D-ala-D-ala is typical). A pentaglycyl oligomer is bonded to the central lysine
as shown. Of particular note is the presence of D-amino acids (D-ala and D-glu) which are produced from
host amino acids by specific bacterial racemases. The D-amino acids are included by the bacteria
presumably to reduce degradation of the cell wall by host proteases. The final step in cell wall
biosynthesis is a transamidation reaction, catalyzed by cell wall transamidase (CWT). This step
crosslinks the peptidoglycan strands as shown, lending greater stability to the barrier. Note that CWT
transiently binds to strand 1 during this process through a serine on the surface of the enzyme. Attack at
this bond by the free glycine amino terminus of a second strand then produces the crosslink and
regenerates CWT.
In addition to the structures described above, the gram positive cell wall contains specialized
transmembrane proteins called porins, which serves as a conduit to import various substances. Amino
acids in the outer protion of the porins are hydrophobic, and interact with the lipid-based membrane of the
bacteria, while amino acids on the inside of the pore are hydrophilic. This creates an environment through
which a variety of water soluble substances can pass through the cell wall.
The crosslinking process shown above is extremely sensitive to beta lactam antibiotics, as will be
discussed below, and in fact, CWT is also known as penicillin binding protein 1 (PBP-1). In addition to
CWT, there are also thought to be at least six other PBP's whose functions are not well understood.
Various penicillins bind differently to different PBP's, producing a variety of effects. Binding to PBP-1 (a
transpeptidase or transamidase) produces cell lysis, while binding to PBP-2 (also a transpeptidase) leads
to oval cells which cannot divide. Binding to PBP-4-6, which are carboxypeptidases, has no lethal effects.
The gram negative cell wall is more complex than the gram positive, and usually contains an outer
membrane and a periplasm. The outer layer contains a complex system of lipopolysaccharides that can
induce septic shock in the host. The peptidoglycan layer of gram negative bacteria is not as extensive as
gram positive, but is also sensitive to beta lactam antibiotics due to the presence of PBP's.
Beta Lactams - Mechanism of Action
The bacteriocidal action of beta lactam antibiotics is directly attributable to their ability to react with
PBP's. The beta lactam ring system is a highly strained and highly reactive chemical entity, and as such is
able to react with the serine hydroxyl group of PBP-1. Recall that in the final step of peptidoglycan
synthesis, the serine hydroxyl of PBP-1 attacks the amide linkage of the D-ala-D-ala segment of the
amino acid sidechain, as shown below. This process forms the enzyme-bound intermediate that is
eventually displaced by the glycine amino terminus of a second strand. The spatial arrangement of the
beta lactam ring system closely resembles the conformation of the D-ala-D-ala segment of the
peptidoglycan strand, and as such the PBP recognizes it as the natural substrate. Reaction of the
peptidoglycan with the beta lactam thus results in an irreversible inhibition of the PBP, and results in lysis
and death of the bacterial cell.
Penicillins
The beta lactam antibiotics that are currently available all feature the reactive beta lactam ring system, a
highly strained and reactive cyclic amide. There are five relevant ring systems which appear in active beta
lactams, including the penam, penem, carbapenem, cefem and monobactam ring structures (shown
below).
The reactive nature of the beta lactam ring system makes penicillins and related compounds susceptible to
a variety of degradative processes. As shown below, the beta lactam ring in these analogues can react with
hydroxide ion, which opens up the beta lactam ring to produce an inactive penicilloic acid. Spontaneous
loss of carbon dioxide then affords the corresponding penilloic acid. Beta lactams are also acid sensitive,
and degrade at low pH by a more complex mechanism. Degradation reactions can be retarded clinically
by buffering solution of penicillins between pH 6.0 and 6.8. These solutions should not be exposed to
sources of metal ions, since metals like zinc and copper accelerate the chemical degradation process.
Another result of the reactivity of beta lactams is the formation of allergenic haptens in vivo.
Nucleophilic OH or SH groups on certain proteins can react with the beta lactam ring system, creating a
covalent PCN-protein conjugate that can induce an allergic response, thus accounting for the inherent
allergenicity of these antibiotics. About 6 to 8% of the population is sensitive to beta lactam antibiotics
In addition to chemical means of degradation, many bacteria produce a group of enzymes specifically
designed to degrade and inactivate beta lactam antibiotics. These enzymes are collectively known as
penicillinases. By far the most prevelant type of penicillinase is the beta lactamase, which directly
attacks and opens the beta lactam bond, inactivating the antibiotic. There are also a variety of acylases
that have been isolated from some bacteria, and these enzymes cleave the acylamino sidechain of the
antibiotic, a modification which also inactivates the molecule. Today, there are compounds available,
such as clavulanic acid and the sulbactams, which irreversibly inactivate beta lactamases, and are thus
given in combination with beta-lactamase sensitive penicillins. The mechanism for this inactivation by
clavulanic acid is shown below, and a similar mechanism can be drawn for the sulbactams.
Resistance to beta lactam antibiotics is becoming increasingly common, and is a significant problem.
Resistance can be mediated through several cellular mechanisms, including production of beta lactamases,
mutation of PBP's to a form with lower affinity for the antibiotic, or decreased permeability of the cell
wall to beta lactams.
The original penicillins were produced by fermentation, and were often mixtures of various beta lactams
such as penicillins G and V, shown below. It should be noted that the penicillin pharmacophore contains
three critical chiral centers. The availability of 6-aminopenicillanic acid (6-APA) has allowed the
creation of hundreds of synthetic and semisynthetic penicillins, a few of which appear below. Whereas
penicillin G and V are still clinically useful drugs, they do suffer from deficiencies that have been
addressed in some of the later generation penicillins. For example, the inclusion of a side chain R group
that is electron withdrawing decreases the electron density of the side chain carbonyl and protects these
penicillins from acid degradation. For this reason, penicillin V is more acid resistant than penicillin G,
since it contains an electronegative oxygen in the sidechain. In order for a penicillin to be orally active, it
must be at least partially acid resistant; otherwise it will only be useful by injection.
Beta lactamases are not particularly tolerant to steric hinderance when it occurs at the sidechain amide,
even though they act to cleave the beta lactam amide bond. Thus the drug methacillin is a beta-lactamase
resistant antibiotic. However, these modifications cause a general decrease in the effectiveness of beta
lactams, and almost all clinically available beta-lactamase resistant penicillins are less potent than the
parent molecules. Methacillin is also more acid sensitive that penicillin G.
The antibiotics oxacillin, cloxacillin and dicloxacillin, in which a substituted isoxazoyl ring system is
used as a bioisosteric replacement for the benzyl group in penicillin G, are less potent against bacteria that
do not produce beta lactamase, but are effective against those that do. They are sufficiently acid stable to
be taken orally.
Ampicillin and amoxicillin belong to a group of penicillinase-sensitive, orally active antibiotics in which
one of the hydrogen atoms of the sidechain phenylacetic acid moiety is replaced by a phenylglycine in the
D-configuration. These antibiotics are orally active, and have a broader spectrum than penicillin G, but
are quite susceptible to beta lactamase, they are often given with clavulanic acid to avoid enzymatic
degradation.
Carbenicillin and ticarcillin differ from fermented penicillins in that they contain a carboxylate moiety
in the sidechain portion of the molecule. These molecules are more effective against gram negative
bacteria, presumably because they penetrate the cell wall more extensively that fermented penicillins.
Ticarcillin is a simple bioisostere of carbenicillin.
Cephalosporins
Unlike the penicillins, where the initial agent in the series was marketed with little SAR development, the
cephalosporin structure required a great deal of refinement before a clinically useful agent was
discovered. The original Cephalosporium acremonium culture was discovered in a sewer outlet in
Sardinia, and researchers Abraham and Newton isolated cephalosporin C, a weakly antibiotic compound
that showed some activity against penicillin-resistant cultures. Chemical removal of the cephalosporin C
sidechain gave 7-aminocephalosporanic acid (7-ACA), which like its cogener 6-APA was used as a
synthetic starting point for most of the cephalosporins available today. It is now more economically
feasible to produce 7-ACA from penicillin G in a seven step synthesis, rather than to incur the cost of
large scale fermentation of cephalosporin C.
Because the beta lactam structure in cephalosporins is appended to a 6 rather than a 5 membered ring, the
system is less strained and hence less reactive. However, much of this loss of strain is made up for by the
olefinic linkage in the 6-membered ring (which adds strain) and the acetoxy group appended to the
molecule, which provides a suitable leaving group. When the beta lactam ring is attacked by a
nucleophile, the electrons can flow to the acetoxy moiety, thus providing an electron sink and increasing
the reactivity of the beta lactam ring. The presence of this acetoxy functional group also provides a handle
for metabolism of these agents to the corresponding free alcohols, which are less active, and which
cyclize to the corresponding lactone derivative, which is inactive. The other modes of metabolism of the
cephalosporins are analogous to those described for penicillin. In terms of their chemical mechanism,
cephalosporins act in a very similar fashion to the penicillins, forming a covalent bond with PBPs and
causing cell lysis. Susceptible cephalosporins can be hydrolyzed by beta lactamases, and in fact some beta
lactamases are more efficient at hydrolyzing cephalosporins than penicillin itself. Allergic reactions are
not as common in this chemical class as in the penicillin class.
Cephalosporins are currently classified by belonging to either the first, second or third generation
series. They differ from one another in antimicrobial spectrum, beta lactamase stability, absorption from
the gut, metabolism, stability and side effects. The structure-activity features responsible for various
prooerties in the penicillins (oral activity, beta lactamase stability, etc.) are similar with the
cephalosporins.
First Generation Cephalosporins - Representative Agents
Second Generation Cephalosporins - Representative Agents
Third Generation Cephalosporins - Representative Agents
Carbapenems and Monobactams
The beta lactam antibiotic thienamycin was isolated from the mold Streptomyces cattlea. It is a very
interesting molecule, since it is an extremely potent antibiotic, and is also an effective inhibitor of beta
lactamase. Note that the sulfur atom is not part of the cyclic structure, but is exocyclic to the five
membered ring. Thienamycin was never marketed, because in concentrated form the primary amine
functionality attacks the beta lactam structure, thus inactivating the molecule. This problem was overcome
by changing this amine to a less nucleophilic imino functionality, producing imipenem.
Imipenem penetrates bacterial porins well, and is stable to and inhibitory for many beta lactamases. It is
not orally active, however. Imipenem can be used for urinary tract infections, but must be co-administered
with cilastin sodium, an inhibitor of renal dehydropeptidase 1, which attacks and inactivates imipenem.
Fermentation of unusual microorganisms resulted in the isolation of certain monocyclic beta lactams
called monobactams, of which aztreonam is the only important example. It is a totally synthetic
parenteral antibiotic which is active almost entirely against gram negative bacteria. Its mechanism is
similar to the penicillins, and it has a particularly strong affinity for PBP-3.