Antibacterial agents which inhibit cell wall synthesis -lactam antibiotics (penicillins and cephalosporins)

Introduction
The name lactam is given to cyclic amides and is anologues to lactone (cyclic esters)

The second carbon atom in an aliphatic caroxylic acid was designated as The third carbon was designated as and so on The current name for this ring system is azetidinone (-lactam) This ring is the principal component of the pharmacophore.
Posesses both medicinal and chemical significance

 The penicillin subclass of -lactam antibiotics is characterized by the

presence of a substituted 5-membered thiazoldine ring fused to the lactam ring. This fusion and the chirality of the -lactam ring results in the molecule roughly possessing a V -shape This considerably interferes with the planarity of the lactam bond and

inhibits resonance of the lactam nitrogen with its carbonyl group.

Consequently, the -lactam ring is much more reactive and, therefore, more sensitive to nucleophilic attack when compared with normal planar

amides.

 -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. -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 -Lactam ring structure.
4

I. Penicillins
HIstory
1928- Alexander Fleming discovers a mold (Penicillium notatum (P.

chrysogenum) which inhibits the growth of staphylococcus bacteria

1940- penicillin is isolated and tested on mice by researchers at Oxford (Chain et al.) 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 -lactams/ -lactamase inhibitors are discovered

and modified from the natural products of bacteria

Structure of penicillin
Penicillin contains a highly unstable-looking bicyclic system consisting of a four membered -lactam ring fused to a five-membered thiazolidine ring. The skeleton of the molecule suggests that it is derived from the amino acids cysteine and valine
O OH H2N SH
HO O NH 2

Cysteine
H R C O N CH3 H N H S CH3

Valine

6-Aminopenicillanic acid

Acyl side chain

COOH

-lactam ring

Thiazoline ring

Fig. Structure of Penicillin

H R C O H N

H S CH3 CH3

Cysteine

O COOH

Valine

The acyl side-chain (R) varies, depending on the make up of the fermentation media

 Types of Penicillins
1.
R1OCHN S CH3 CH3

N O CO2H

Fernentation Derived Penicilins 6-Aminopenicillanic acid (6-PAS) Benzylpenicillin (Penicillin G) Phenoxymethylpenicillin (Penicillin V)

R1 H C6H5-CH2 C6H5-OCH2

II. Semisynthetic Penicillinase resistant

R1OCHN S CH3 CH3 N O CO2H

Semisynthetic-Penicilinase Resistant Parental Penicillins

R1
CH3

Methicillin

CH3

Nafcillin

C2H5O

O R" N

CH3

Semisynthetic-Penicilinase Resistant Oral Penicillins

R'

R' Oxacillin Cloxacillin Dichloxacillin H H Cl

R" H Cl Cl

IIIa. Semi-synthetic penicillinase sensitive, Broad spectrum, Parentral

R1OCHN S CH3 CH3 N O CO2H

Semisynthetic-Penicilinase Sensitive, Broad Spectrum, Parental Penicillins Carbenicillin

R1
CO2H

CO2

Carbenicillin Indanyl
CO2H

Ticarcillin
S O

Mezlocillin

O N N H

C6H5

H3SO2

N O

O N N H

Piperacillin

O C2H5 N

C6H5

10

IIIB. Semi-synthetic penicillinase sensitive, Broad spectrum, Oral

R1OCHN S CH3 CH3

N O CO2H

Semisynthetic-Penicilinase Sensitive, Broad Spectrum, Oral Penicillins Ampicillin

R1
NH2

H NH2

Amoxacillin
HO

11

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 -lactams disrupt the synthesis of the bacterial cell

wall by interfering with the transpeptidase which

catalyzes the cross linking process.
12

Peptidoglycan

Peptidoglycan is a carbohydrate composed of alternating units of N-acetylmuramic acid (NAMA) and N-

acetylglucosamine (NAGA) linked (1,4)- (Fig Below)

. NAM = N-Acetyl muramic acid

OH O OH NAM O HO O O NHAc NHAc NAG O

NAM = N-Acetyl glucosamine

13

 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

14

Mechanism

15

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-AlaD-Ala sequence of the NAMA peptide side chain by the PBP Interfering with this recognition disrupts the cell wall synthesis -lactams mimic the structure of the D-Ala-D-Ala link and

bind to the active site of PBPs, disrupting the cross-linking

process (Pic. Next slide)
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17

Mechanism of -Lactam Drugs

The amide of the -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 -Lactams acylate the hydroxyl group on the serine residue of Penicillin binding protein (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.

18

 The hydroxyl attacks the amide and forms a tetrahedral intermediate.

19

 The tetrahedral intermediate collapses, the amide bond is

broken, and the nitrogen is reduced.

20

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

21

SAR
A large number of penicillin analogues have been synthesized and studied. The results of these studies led to the following conclusions
The strained -lactam ring is essential. The free carboxylic acid is essential. The bicyclic system is important (confers strain on the -lactam ringthe greater the strain, the greater the activity, but the greater the instability of the molecule to other factors). The acylamino side-chain is essential (exceptional for thienamycin) Sulfur is usual but not essential.

The stereochemistry of the bicyclic ring with respect to the acylamino sidechain is important.

22

Amide E
O H C H N H

Cis Steriochemistry E

5 4
N

1 2 3

CH3

CH3

Free Acid E
O COOH

-lactam E Bicyclic ring system E Fig. Structure activity relationship of Penicillin (E=Essential)

23

 The chemical substituents attached to the penicillin nucleus can greatly influence the stability of the penicillins as well as the spectrum of activity. It is important to recognize whether the structural changes

Affect drug stability on the shelf or in the GI tract (in vivo)

Improve stability toward bacterial metabolism, Enlarge the spectrum of activity The substitution of a side-chain R group on the primary amine with an electron-withdrawing group decreases the electron density on the side-

chain carbonyl and protects these penicillins from acid degradation.

24

 This property has clinical implications since these compounds survive passage through the stomach better and many can be given orally for systemic purposes (table below)

25

 In addition, in vitro degradation reactions of penicillins can be

retarded by keeping the pH of solutions between 6.0 and 6.8 and by refrigerating them.

Metal ions, such as mercury, zinc, and copper, catalyze the

degradation of penicillins, so they should be kept from contact with penicillin solutions. The lids of containers used today are routinely made of inert plastics, in part, to minimize such problems. The more lipophilic the side chain of a penicillin, the more serum protein bound is the antibiotic

26

Table. Protein binding of Pencillin

This has some advantages in terms of protection from degradation, but it does reduce measurably the effective bactericidal concentration of the drug in whole blood.

27

 Stability of the penicillins toward -lactamase is influenced by the bulk in the acyl group attached to the primary amine. -Lactamases are much less tolerant to the presence of steric hindrance near the side-chain amide bond than are the penicillin binding proteins. When the aromatic ring is attached directly to the side-chain carbonyl and both ortho positions are substituted by methoxy groups, -lactamase stability results (Fig. Below)
O OCH3 C H N H H S CH3 CH3 OCH3 O O C H N H H S CH3 CH3 COOH

N OCH3 O

COOH

-lactamase resistant

28

 Movement of one of the methoxy groups to the para position, or replacing one of them by a hydrogen, resulted in an analogue sensitive to lactamases. Putting in a methylene between the aromatic ring and 6-APA produced a -lactamasesensitive agent
O H C N H3CO H H S N CH3 CH3 OCH3 COOH O COOH O H C N H H S N CH3 CH3

OCH3O

H H C N O OCH3

H S N CH3 CH3 COOH

-lactamase Senstitive

29

 These findings provide strong support for the hypothesis that

its resistance to enzyme degradation is based on differential steric hindrance.

Examples of Penicillin with this effect are seen in the drugs

Methicillin,

Nafcillin
oxacillin, cloxicillin, and Dicloxicillin
30

Bacterial Resistance
Bacteria have many methods with which to combat the effects of -lactam type drugs
Intrinsic defenses such as efflux pumps can remove the lactams from the cell

-Lactamases are enzymes which hydrolyze the amide bond

of the -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 -lactam

31

Penicillin sensitivity to -lactamases

-Lactamases are enzymes produced by penicillin-resistant bacteria

A solution to the problem was the design of penicillinase-resistant penicillins The strategy is to block the penicillin from reaching the penicillinase active site. One way of doing that is to place a bulky group on the side-chain. This bulky group can then act as a 'shield' to ward off the penicillinase and therefore prevent binding

32

 Fortunately, 'shields' were found which could make that discrimination. Methicillin was the first semisynthetic penicillin unaffected by penicillinase and was developed just in time to treat the Staph. aureus problem The principle of the steric shield can be seen by the presence of two ortho-methoxy groups on the aromatic ring. Both of these are important in shielding the lactam ring.

33

Range of Activity
-Lactams can easily penetrate Gram (+) bacteria, but the outer cell membrane of Gram (-) bacteria prevents diffusion

of the drug
-Lactams can be modified to make use of import porins in the cell membrane.

-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 -lactams.
34

 Most penicillins show a poor activity against Gram-negative bacteria. There are several reasons for this resistance Permeability barrier High levels of transpeptidase enzyme produced Modification of the transpeptidase enzyme Presence of -lactamase Transfer of the -lactamase enzyme Tackling the problem of narrow activity spectrum The search for broad-spectrum antibiotics has been one of trial and error

which involved making a huge variety of analogues.

These changes were again confined to variations in the side chain and gave the following results:
35

Hydrophobic groups on the side-chain (e.g. penicillin G) favour

activity

against Gram-positive bacteria, but result in poor activity against Gramnegative bacteria If the hydrophobic character is increased, there is little effect on the Grampositive activity, but what activity there is against Gram-negative bacteria drops even more. Hydrophilic groups on the side-chain have either little effect on Gram-positive activity (e.g. penicillin T) or cause a reduction of activity (e.g. penicillin N)

36

 However, they lead to an increase in activity against Gram-negative

bacteria. Enhancement of Gram-negative activity is found to be greatest if

the hydrophilic group (e.g. NH2, OH, CO2H) is attached to the

carbon, alpha to the carbonyl group on the side-chain. Those penicillins having useful activity against both Gram-positive and Gram negative bacteria are known as broad-spectrum antibiotics. There are two classes of broad-spectrum antibiotics. Both have an alpha-hydrophilic group

37

 However, in one class the hydrophilic group is an amino function as in ampicillin or amoxycillin (Fig Below),

Whereas in the other the hydrophilic group is an acid group as in carbenicillin (Fig. below)

38

Modification of -Lactams
-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 -lactamases Acylate the PBPs of resistant species (there are many different PBPs)

The penicillins usually are discussed under various groups based on spectrum of activity and sensitivity or resistance toward -lactamase
39

Class I broad-spectrum antibiotics (ampicillin and amoxycillin)

Ampicillin is the second most used penicillin in medical practice Amoxycillin differs merely in having a phenolic group It has similar properties, but is better absorbed through the gut wall

40

Properties
Active versus Gram-positive bacteria and against those Gramnegative bacteria which do not produce penicillinase.

Acid-resistant due to the NH2 group, and is therefore orally active

Non-toxic Sensitive to penicillinase (no 'shield') Inactive against Pseudomonas aeruginosa (a particularly resistant species).

Can cause diarrhoea due to poor absorption through the gut wall
leading to disruption of gut flora.

41

The last problem of poor absorption through the gut wall is due to the dipolar nature of the molecule since it has both a free amino group and a free carboxylic acid function.

This problem can be alleviated by using a prodrug where one of the polar groups is

masked with a protecting group.

This group is removed metabolically once the prodrug has been absorbed through the gut wall.

42

 These three compounds are all prodrugs of ampicillin

In all three examples, the esters used to mask the carboxylic acid group seem rather elaborate and one may ask why a simple methyl ester is not used methyl esters of penicillins are not metabolized

in man
Perhaps the bulkiness of the penicillin skeleton being so close to the ester functional group prevents the esterases from

binding the penicillin

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Class II broad-spectrum antibioticscarbenicillin

Carbenicillin has an activity against a wider range of Gramnegative bacteria than ampicillin

It is resistant to most penicillinases and is also active against

the stubborn Pseudomonas aeruginosa This particular organism is known as an 'opportunist' pathogen since it strikes patients when they are in a weakened condition
The organism is usually present in the body, but is kept under control by the body's own defence
mechanisms

44

 There are drawbacks to carbenicillin It shows a marked reduction in activity against Gram-positive bacteria (note the hydrophilic acid group) It is also acid sensitive and has to be injected

45

 In general, carbenicillin is used against penicillin-resistant Gramnegative bacteria The broad activity against Gram-negative bacteria is due to the hydrophilic acid group (ionized at pH 7) on the side-chain It is particularly interesting to note that the stereochemistry of this group is important.

The alpha-carbon is chiral and only one of the two enantiomers is

active This implies that the acid group is involved in some sort of binding

interaction with the target enzyme.

Carfecillin is the prodrug for carbenicillin and shows an improved absorption through the gut wall
46

Penicillins- Natural (Bezylpenicillin Group)

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. A. Penicillin G

not orally active

47

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

48

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 possible.

49

Production
All commercially available -lactams are initially produced through the fermentation of bacteria.

Bacteria assemble the penicillin molecule from L-AAA, Lvaline, 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.

50

Penicillin Biosynthetic Pathway

51

52

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.

53

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

54

 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)

55

 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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Toxicity
-Lactams target PBPs exclusively, and because human cell membranes do
not have this type of protein -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 -lactams A previous history of allergy to penicillins is a contraindicating factor to their use Topical wheal and flare tests are available when there is doubt

When an allergic reaction develops, the drug must be discontinued, and,

because cross-sensitivity is common, other -lactam drugs should generally be avoided.
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Clavulanic Acid and Sulbactam

Clavulanic acid is a mold product with weak intrinsic antibacterial activity, It is irreversible inhibitor of most -lactamases. It is believed to acylate the active site serine by mimicking the normal substrate. Hydrolysis occurs with some -lactamases, but in many cases, subsequent reactions occur that inhibit the enzyme irreversibly This leads to its classification as a mechanism-based inhibitor (or so-called suicide substrate). The precise chemistry is not well understood but when clavulanic acid is added to ampicillin and amoxicillin preparations, the potency against lactamaseproducing strains is markedly enhanced.
58

H O CH2OH

N O CO2H

Clavulanic Acid

-lactamases
H OH N O CO2H O CH2OH

-lactamases
O O O N Co2H O

H+

-lactamases
O

H N

-lactamases
O

n:

CO2H

O O HN Co2H

59

 Sulbactam is another -lactamasedisabling agent Sulbactam is prepared by partial chemical synthesis from penicillins The oxidation of the sulfur atom to a sulfone greatly enhances the potency of sulbactam
H S CH3 N O CH3

Sulbactam
H S CH3 N O CH3

CO2H

-lactamases
O

H N SO2H

Sulbactam

CO2H

CO2H

Instead of a -lactamase, ampicillin resistance is caused by a penetration barrier, clavulanic acid and sulbactam are not able to overcome this, because the underlying mechanism is different.
60

II. Cephalosporins
The second major group of -lactam antibiotics Cephalosporins were discovered shortly after penicillin entered into

widespread product, but not developed till the 1960s.

The first cephalosporin was cephalosporin Cisolated from a fungus obtained from sewer water on the island of Sardinia

Cephalosporins are similar to penicillins structurally, but have a 6

member dihydrothiazine ring instead of a 5 member thiazolidine ring

This larger ring relieves the strain in the bicyclic system to

some extent, but it is still a reactive system.
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 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. A study of the cephalosporin skeleton reveals that cephalosporins can be derived from the same biosynthetic precursors as penicillin, i.e. cysteine and valine

62

Cephalosporin C

Properties of cephalosporin C
Difficult to isolate and purify due to a highly polar side-chain. Low potency (one-thousandth of penicillin G). Not absorbed orally Non-toxic

63

 Low risk of allergenic reactions Relatively stable to acid hydrolysis compared to penicillin G More stable than penicillin G to penicillinase (equivalent to oxacillin) Good ratio of activity against Gram-negative bacteria and Gram-positive bacteria

Cephalosporin C has few clinical uses However, its importance lies in its potential as a lead

Compound to something better.

This potential resides in the last property mentioned above.
64

 Cephalosporin C may have low activity, but the antibacterial activity which it does have is more evenly directed against Gram-negative and Grampositive bacteria than is the case with penicillins. By modifying Cephalosporin C it might be able to increase the potency whilst retaining the breadth of activity against both Gram positive and Gram-negative bacteria Another in-built advantage of Cephalosporin C over penicillin is that it is already resistant to acid hydrolysis and to penicillinase enzymes. Cephalosporin C has been used in the treatment of urinary tract infections since it is found to concentrate in the urine and survive the body's hydrolytic enzymes.

65

Structure-activity relationships of Cephalosporin C

Many analogues of Cephalosporin C have been made and SAR conclusions are as follows. The -lactam ring is essential. A free carboxyl group is needed at position 4. The bicyclic system is essential.

The stereochemistry of the side-groups and the rings is

important. These results tally closely with those obtained for the penicillins and once

again there are only a limited number of places where modifications can
be made

66

 the 7-acylamino side-chain; the 3-acetoxymethyl side-chain;

substitution at carbon 7.

67

 Access to analogues with varied side-chains at the 7-position initially

posed a problem. Unlike penicillins, it proved impossible to obtain cephalosporin analogues by fermentation. likewise, it was not possible to obtain the 7-ACA (7-aminocephalosporinic acid) skeleton (Fig. Next slide ) either by fermentation or by enzymic hydrolysis of cephalosporin, thus preventing the semisynthetic approach analogous to the preparation of penicillins from 6-APA. Therefore, a way had to be found of obtaining 7-ACA from cephalosporin C by chemical hydrolysis. After all, a secondary amide has to be hydrolysed in the presence of a highly reactive -lactam ring
68

Normal hydrolytic procedures are not suitable and so a special method had to be worked out (Fig. Below)

Fig. Synthesis of 7-ACA and cephalosporin analogues

69

 The strategy used takes advantage of the fact that the -lactam nitrogen is
unable to share its lone pair of electrons with its neighbouring carbonyl group The first step of the procedure requires the formation of a double bond between the nitrogen on the side-chain and its neighbouring carbonyl group This is only possible for the secondary amide group since ring constraints prevent the -lactam nitrogen forming a double bond with the -lactam ring A chlorine atom is now introduced to form an imino chloride which can then be reacted with an alcohol to give an imino ether.

70

 This product is now more susceptible to hydrolysis than the -lactam ring and so treatment with aqueous acid successfully gives the desired 7-AC A which can then be acylated to give a range of analogues The most commonly used of these cephalosporin analogues is cephalothin

71

Properties of cephalothin
Less active than penicillin G versus cocci and Gram-positive bacilli. More active than penicillin G versus some Gram-negative bacilli (Staph.

aureus and E. coli).

Resistant to penicillinase from Staph. aureus infections. Not active against Pseudomonas aeruginosa.

Poorly absorbed in the gastrointestinal tract and has to be injected.

Metabolized in man by deacetylation to give a free 3-hydroxymethyl group which has reduced activity Less chance of allergic reactions and can be used for patients with allergies to penicillin.
72

 The study of several analogues has demonstrated the

following SAR results relevant to the 7-acylamino side-chain. Best activity is obtained if the alpha-carbon is monosubstituted (i.e. RCH2CO-7 AC A). Further substitution leads to a drop in Gram-

positive activity.
Lipophilic substituents on the aromatic or

heteroaromatic ring increase the Gram positive

activity and decrease the Gram-negative activity.

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Analogues of cephalosporin C by variation of the 3-acetoxymethyl side-chain

The first observation which can be made about this area of the molecule is that losing the 3-acetyl group releases the free alcohol group and results

in a drop of activity.
This hydrolysis occurs metabolically and therefore it would be useful if this process was blocked to prolong the activity of cephalosporins

An example is cephaloridine (Fig. below) which contains a pyridinium

group in place of the acetoxy group.

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Properties of cephaloridine
Stable to metabolism Soluble in water because of the positive charge. Low serum protein binding leads to good levels of free drug in the circulation Excellent activity against Gram-positive bacteria. Same activity as cephalothin against Gram-negative bacteria. Slightly lower resistance than cephalothin to penicillinase. Some kidney toxicity at high doses. Poorly absorbed through gut wall and has to be injected. A second example is cephalexin (Fig. next slide) which has no substitution at position 3. This is one of the few cephalosporins which is absorbed through the gut wall and can be taken orally.

75

Fig. Cephalexin This better absorption appears to be related to the presence of the 3methyl group. Usually, the presence of such a group lowers the activity of cephalosporins, but if the correct 7-acylamino group is present as in cephalexin, then activity can be retained.

76

Synthesis of 3-methylated cephalosporins

The synthesis of 3-methylated cephalosporins from cephalosporins is very difficult and it is easier to start from the penicillin nucleus

77

Summary of properties of cephalosporins

Injectable cephalosporins of clinical use have a high activity against a large number of Gram-positive and Gram-negative organisms including the penicillin-resistant staphylococci. Most cephalosporins are poorly absorbed through the gut wall.

Cephalosporins have lower activity than comparable penicillins, but a better range. This implies that the enzyme which is attacked by penicillin and cephalosporin has a binding site which fits the penam skeleton better than the cephem skeleton. The ease of oral absorption appears to be related to an alpha-amino group on the 7- acyl substituent, plus an uncharged group at position 3.
78

 The cephalosporins mentioned so far are all useful agents

Resistant organisms has posed a problem Gram-negative organisms appear to have a -lactamase which can

degrade even those cephalosporins which are resistant to -lactamase

enzymes in Gram-positive species Attempts to introduce some protection against these lactamases by

means of steric shields were successful, but led to inactive compounds

The introduction of such groups in cephalosporins not only prevents access to the - lactamase enzyme, but also to the target transpeptidase

enzyme
The next advance came when it was discovered that cephalosporins substituted at the 7-position were active.
79

Analogues of cephalosporin C by substitution at position 7

The only substitution which has been useful at position 7 has been the introduction of the 7-alpha-methoxy group to give a class of compounds

known as the cephamycins

The parent compound cephamycin C was isolated from a culture of Streptomyces clavuligerus
80

 Modification of the side-chain gave cefoxitin (Fig. below) which showed a

broader spectrum of activity than most cephalosporins, due to greater resistance to penicillinase enzymes. This increased resistance is thought to be due to the steric hindrance provided by the extra methoxy group.

However, it is interesting to note that introduction of the methoxy group at the corresponding 6-alpha-position of penicillins results in loss of activity.
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Properties of cefoxitin
Stable to -lactamases. Stable to mammalian hydrolytic enzymes (due to NH2 in place

of CH3 )
Broader spectrum of activity than previous cephalosporins. Poor absorption through the gut wall and therefore administered by injection. Painful at injection site and therefore administered with a

local anaesthetic
Poor activity against Pseudomonas aeruginosa.
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Classification of Cephalosporins
Most cephalosporins have generic names beginning with cef- or ceph-. The cephalosporins are classified by a inconsequential nomenclature system loosely derived from the chronology of their introduction but more closely related to their antimicrobial spectrum. The first-generation cephalosporins primarily are active in vitro against Gram-positive cocci (penicillinase-positve and -negative Staphylococcus aureus and S. epidermis) , group A -hemolytic streptococci (Streptococcus pyogenes), group B streptococci (Streptococcus agal actiae), and Streptococcus pneumoniae.

83

 They are not effective against MRSA.

They are not significantly active against Gram-negative bacteria, although some strains of Escherichia coli , Klebsiella pneumoniae, Proteus mirabi li s, and Shigella sp. may be sensitive. The second-generation cephalosporins generally retain the antiGram-positive activity of the first generation agents but include Haemophilus influenzae as well and add to this better anti -Gramnegative activity so that some strains of Acinetobacter, Citrobacter, Enterobacter, Escherichia coli , Klebsiella, Neisseria, Proteus, Providencia, and Serratia also are sensitive

84

 The third generation cephalosporins are less active against staphylococci

than the first -generation agents but are much more active against Gramnegative bacteria than either the first - or the second-generation drugs. They frequently are useful against nosocomial multidrug-resistant hospital-acquired strains. One also adds Morganella sp. and Pseudomonas aeruginosa to the list of species that often are sensitive The fourth-generation cephalosporins have an antibacterial spectrum like the third-generation drugs but add some enterobacteria that are resistant to the third-generation cephalosporins. They also are more active against some Gram-positive organisms

85

Table. First generation Cephalosporins

O R CHN S N O CO2H X

Name

Salt

Parentral agents Na

Cephapirin

SCH2

OAc

Cefazolin Oral Agents Cephalexin

N N

N N

CH2

N N S S CH3

Na

H
NH2

HCl

Cephadroxil

HO

H
NH2

Oral and parental agent

Cepharadine
NH2

86

 Good activity against gram (+) and modest against gram (-) microbes

Penetration of the cerebrospinal fluid (CSF) is inadequate

UTI, minor staph lesions, minor polymicrobial infections cellulitis, soft

tissue abscess

Not used in serious systemic infections

87

Table. Second generation Cephalosporins

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 Better activity against anaerobes and gram (-)

aerobes Only Cefuroxime can produce sufficient Sinusitis, otitis, LRTI, mixed anaerobic such as peritonitis/diverticulitis CSF level infections

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Table. Third generation Cephalosporins

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 Generally less active than the first generation against gram (+) cocci but most active against gram (-) including B-lactamase-producing strains Cefoperazone, Ceftazidime (more active) against Pseudomona Cefoperazone, Cefotaxime Active against anaerobes
Ceftizoxime, B. fragilis Cefoperazone & Ceftriaxone -excreted primarily in the bile; Probenecid does not affect renal excretion Cross BBB except Cefoperazone, Cefixime, Ceftibuten and Cefpodoxime proxetil

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Fourth Generation
Cefepime
It is a semisynthtihc agent containing a Z-methoxyimine moeity and an aminothiazolyl group at C-7 This broadened spectrum of activity as well as resistance to -lactamases Also its antistaphylococcal activity is increased
H2N N S N O CH3 N O CH3 COO O H H S

Cefepime

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 More resistant to hydrolysis by chromosomal -lactamases (eg. Those produced by enterobacter) Good activity against P. aeruginosa, enterobacteriaceae, S. aureus, S. pneumoniae Highly active against Haemophilus & Neisseria Penetrates well into CSF

Cleared by kidneys
T1/2 2 hrs Good activity against most penicillin resistant strains of streptococci Useful in the treatment of enterobacter infections

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III. Carbapenems A. Thienamycin

The first of the carbapenems, was isolated from Streptomyces cattleya.

Because of its

extremely intense and broad-spectrum antimicrobial

activity as well as its ability to inactivate -lactamases, it combines in one molecule the functional features of the best of the -lactam antibiotics as

well as the -lactamase inhibitors.

It differs structurally in several important respects from the penicillins and cephalosporins.

The sulfur atom is not part of the 5-membered ring but, rather, has been
replaced by a methylene moiety at that position.
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OH H

CH3H NH2 S N O Co2H

Thienamycin

Carbon is roughly half the molecular size of sulfur Consequently, the carbapenem ring system is highly strained and very susceptible to reactions cleaving the -lactam bond. The sulfur atom is now attached to C-3 as part of a functionalized sidiechain.
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 The endocyclic olefinic linkage also enhances the reactivity of the -lactam
ring. Both make thienamycin unstable, which caused great difficulties in the original isolation studies. The terminal amino group in the side chain attached to C-3 is nucleophilic and attacks the -lactam bond of a nearby molecule through an intermolecular reaction destroying activity (fig. below)
OH H CH3 H N S N O Co2H CO2H S O HN HO CH3 NH2

Thienamycin

Fig. Intermolecular instability reaction of thienamycin

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 Ultimately, this problem was overcome by changing the amino group to a

less nucleophilic N-formiminoyl moiety by a semisynthetic process to produce imipenem.
OH H S N O Co2H CH3 NH HN

Imipenem

At C-6, there is a 2-hydroxyethyl group attached with -stereochemistry. Thus, the absolute stereochemistry of the molecule is 5R,6S,8S. With these striking differences from the penicillins and cephalosporins, it is not surprising that thienamycin analogues bind differently to the PBPs (especially strongly to PBP-2), but it is satisfying that the result is very potent broad-spectrum activity.
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B. Imipenem
Imipenem, penetrates very well through porins and is very stable, even inhibitory, to many - lactamases. Imipenem is not, however, orally active. When used to treat urinary tract infections, renal dehydropeptidase-1 hydrolyzes imipenem through hydrolysis of the -lactam and deactivates it. An inhibitor for this enzyme, cilastatin, is coadministered with imipenem to protect it.
H3C NH2 HN O CO2 Na S H C COOH CH3

Cilastatin Sodium

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 Inhibition of human dehydropeptidase does not seem to have

deleterious consequences to the patient This make the combination highly efficacious against urinary tract

infections
The combination of imipenem and cilastatin is approximately 25% serum protein bound.

On injection, it penetrates well into most tissues, but not cerebrospinal

fluid, and is subsequently excreted in the urine. It is broader in its spectrum than any other antibiotic presently available

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 This very potent combination is especially useful for treatment of serious

infect ions by aerobic Gram-negative bacilli, anaerobes, and Staphylococcus aureus. It is used clinically for severe infections of the gut in adults as well as of

the genitourinary tract , bone, skin, and endocardia,

Unfortunately, imipenem also has the property of being a good lactamase inducer.

Because of these features, imipenemcilastatin is rarely a drug of first

choice but, rather , is reserved for use in special circumstances.

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IV. Monobactams Aztreonam

Fermentation of unusual microorganisms led to the discovery of a class of monocyclic -lactam antibiotics, named monobactams None of these natural molecules have proven to be important , but the group served as the inspiration for the synthesis of aztreonam.
S O HN N H3C N O N C CH3 O CO2Na SO3Na H CH3 H

H2N

Aztreonam Disodium

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 Aztreonam is a totally synthetic parenteral antibiotic, the antimicrobial spectrum of which is devoted almost exclusively to Gram-negative microorganisms Also it is capable of inactivating some -lactamases. Its molecular mode of action is closely similar to that of the penicillins, cephalosporins, and carbapenems, the action being characterized by strong affinity for PBP-3, Whereas the principal side chain closely resembles that of ceftazidime, the sulfamic acid moiety attached to the -lactam ring was extraordinary
S O HN N H3C N O N C CH3 O CO2Na CO2H N H H S

H2N

Ceftazidime

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 Remembering the comparatively large size of sulfur atoms, this

assembly may sufficiently spatially resemble the corresponding C-2 carboxyl group of the precedent -lactam antibiotics. This confuse the penicillin binding protons.

The strongly electron-withdrawing character of the sulfamic acid group probably also makes the -lactam bond more vulnerable to hydrolysis.

In any case, the monobactams demonstrate that a fused ring is not

essential for antibiotic activity. The -oriented methyl group at C-2 is associated with the stability of aztreonam towards -lactamases.

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 Aztreonam is given by injection and is primarily excreted in

the urine. The primary clinical use of aztreonam is against severe infect ions caused by Gram-negative microorganisms, especially those acquired in the hospital .

These are mainly urinary tract , upper respiratory tract, bone,

cartilage, abdominal , obstetric and gynecologic infections, and septicemias.

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Summary
-Lactam antibiotics have dominated the clinical market since their introduction in the 1940s and today consist of nearly 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 -lactams has led to the development of resistant

strains, new modifications are necessary in order for -lactams to remain viable.

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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. -Lactamase Inhibitors from Laboratory to Clinic. Clinical Microbiology Reviews, Jan. 1988, p. 109-123. Web. Elander, R.P. Industrial production of -Lactam antibiotics. Journal of Applied Microbiology and Biotechnology (2003) 61:385392. 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 -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 Cephalosprins? (0.75 EACH) 2. How were the natural penicillins modified to be orally available? (WITH PLAUSABLE STRUCTURAL

CLASSIFICATION) (2 MARKS)
3. What are two ways that the -lactam can be

protected from -lactamases? (0.75 EACH)

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