Antibacterial antibiotics

Reference Wilson and gisvold textbook of Organic Medical And Pharmaceutical chemistry

Antibiotics (literally “against life”) as the biological concept of survival of the fittest, in which one organism destroys another to preserve itself

The word antibiotic was derived from this root.

An antibiotic or antibiotic substance is a substance

produced by microorganisms, which has the capacity of inhibiting the growth and even of destroying other microorganisms.”

Later proposals have sought both to expand and to restrict the definition to include any substance produced by a living organism that is capable of inhibiting the growth or survival of one or more species of microorganisms in low concentrations

The advances made by medicinal chemists to modify naturally occurring antibiotics and to prepare synthetic analogs necessitated the inclusion of semisynthetic and synthetic derivatives in the definition.

Therefore, a substance is classified as an antibiotic if the following conditions are met:

1. It is a product of metabolism (although it may be duplicated or even have been anticipated by chemical synthesis).

2. It is a synthetic product produced as a structural analog of a naturally occurring antibiotic.

3. It antagonizes the growth or survival of one or more species of microorganisms.

4. It is effective in low concentrations.

CHEMICAL CLASSIFICATION

The chemistry of antibiotics is so varied that a chemical classification is of limited value

Some similarities can be found, however, indicating that some antibiotics may be the products of similar mechanisms in different organisms and that these structurally similar products may exert their activities in a similar manner

For example, several important antibiotics have in common a macrolide structure (i.e., a large lactone ring) e.g. erythromycin and oleandomycin

The tetracycline family comprises a group of compounds very closely related chemically.

Several compounds contain closely related amino sugar moieties, such as those found in streptomycins , kanamycins, neomycins, paromomycins, and gentamicins.

The bacitracins, tyrothricin, and polymyxin are among a large group of polypeptides that exhibit antibiotic action.

The penicillins and cephalosporins are -lactam ring–containing antibiotics derived from amino acids.

β-Lactam Antibiotics

Introduction

β-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.

β-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 β-Lactam ring structure.

History

1928- Alexander Fleming discovers a mold which inhibits the growth of staphylococcus bacteria

1940- penicillin is isolated and tested on mice by researchers at Oxford

1941- penicillin mass produced by fermentation for use by US soldiers in WWII

1950’s- 6-APA is discovered and semi-synthetic penicillins are developed.

1960’s to today- novel β-lactams/ β-lactamase inhibitors are discovered and modified from the natural products of bacteria

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 β-lactams disrupt the synthesis of the bacterial cell wall by interfering with the transpeptidase which catalyzes the cross linking process.

Peptidoglycan

Peptidoglycan is a carbohydrate composed of alternating units of NAMA and NAGA.

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.

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-Ala-D-Ala sequence of the NAMA peptide side chain by the PBP. Interfering with this recognition disrupts the cell wall synthesis.

β-lactams mimic the structure of the D-Ala-D-Ala link and bind to the active site of PBPs, disrupting the cross-linking process.

Mechanism

=.

Bacterial cell wall of gram-positive bacteria.

NAM = N-acetylmuramic acid; NAG N-

acetylglucosamine; PEP = cross-linking peptide

Mechanism of β-Lactams activity

The amide of the β-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.

β-Lactams acylate the hydroxyl group on the serine residue of 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.

Mechanism of β-Lactams activity

The hydroxyl attacks the amide and forms a tetrahedral intermediate.

Mechanism of β-Lactams activity

The tetrahedral intermediate collapses, the amide bond is broken, and the nitrogen is reduced.

Mechanism of β-Lactams activity

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

Nomenclature

The nomenclature of penicillins is somewhat complex and very cumbersome.

Two numbering systems for the fused bicyclic heterocyclic system exist.

The Chemical Abstracts system initiates the numbering with the sulfur atom and assigns the ring nitrogen the 4-position

The numbering system adopted by the USP is the reverse of the Chemical Abstracts procedure, assigning number 1 to the nitrogen atom and number 4 to the sulfur atom.

Stereochemistry

The penicillin molecule contains three chiral carbon atoms(C-3, C-5, and C-6). All naturally occurring and microbiologically active synthetic and semisynthetic penicillins have

The carbon atom bearing the acylamino group (C-6) has the L configuration, whereas the carbon to which the carboxyl group is attached has the D configuration

Bacterial Resistance

Bacteria have many methods with which to combat the effects of β-lactam type drugs.

Intrinsic defenses such as efflux pumps can remove the β-lactams from the cell. β-Lactamases are enzymes which hydrolyze the amide bond of the β-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 β-lactam .

Range of Activity

β-Lactams can easily penetrate Gram (+) bacteria, but the outer cell membrane of Gram (-) bacteria prevents diffusion of the drug. β-Lactams can be modified to make use of import porins in the cell membrane.

β-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 β-lactams.

Toxicity

β-Lactams target PBPs exclusively, and because human cell membranes do not have this type of protein β-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 β-lactams.

Classes of β-Lactams

The classes of β-lactams are distinguished by the variation in the ring adjoining the β-lactam ring and the side chain at the α position.

Penicillin

Modification of β-Lactams

β-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 β-lactamases

-acylate the PBPs of resistant species (there are many different PBPs)

Chemical Degradation

The early commercial penicillin was a yellow-to-brown amorphous powder . Improved purification procedures provided the white crystalline material in use today

Crystalline penicillin must be protected from moisture, but when kept dry, the salts will remain stable for years without refrigeration

The solubility and other physicochemical properties of the penicillins are affected by the nature of the acyl side chain and by the cations used to make salts of the acid

The sodium and potassium salts of most penicillins, are soluble in water and readily absorbed orally or parenterally

Salts of penicillins with organic bases, such as benzathine, procaine, and hydrabamine, have limited water solubility and are, therefore, useful as depot forms to provide effective blood levels over a long period in the treatment of chronic infections

Penicillins- Natural

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.

Penicillin G

not orally active

Penicillin G in Acidic Conditions

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

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 viable.

Production

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

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.

Penicillin Biosynthetic Pathway

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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.

Penicillins- Antistaphylococcal

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

Penicillins- Antistaphylococcal

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)

Penicillins- Antistaphylococcal

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.

Penicillins- Aminopenicillins

In order to increase the range of activity, the penicillin has been modified to have more hydrophilic groups, allowing the drug to penetrate into Gram (-) bacteria via the porins.

Ampicillin R=Ph

Amoxicillin R= Ph-OH

Penicillins- Aminopenicillins

These penicillins have a wider range of activity than natural or antistaphylococcal drugs, but without the bulky side groups are once again susceptible to attack by β-lactamases

The additional hydrophilic groups make penetration of the gut wall difficult, and can lead to infections of the intestinal tract by H. pylori

Penicillins- Aminopenicillins

Due to the effectiveness of the aminopenicillins, a second modification is made to the drug at the carboxyl group.

Changing the carboxyl group to an ester allows the drug to penetrate the gut wall where it is later hydrolyzed into the more polar active form by esterase enzymes.

This has greatly expanded the oral availability of the aminopenicillin class.

Penicillins- Extended Spectrum

Extended spectrum penicillins are similar to the aminopenicillins in structure but have either a carboxyl group or urea group instead of the amine

Penicillins- Extended Spectrum

Like the aminopenicillins the extended spectrum drugs have an increased activity against Gram (-) bacteria by way of the import porins.

These drugs also have difficulty penetrating the gut wall and must be administered intravenously if not available as a prodrug.

These are more effective than the aminopenicillins and not as susceptible to β-lactamases

Cephalosporins

Cephalosporins were discovered shortly after penicillin entered into widespread product, but not developed till the 1960’s.

Cephalosporins are similar to penicillins but have a 6 member dihydrothiazine ring instead of a 5 member thiazolidine ring.

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.

Cephalosporins

Unlike penicillin, cephalosporins have two side chains which can be easily modified. Cephalosporins are also more difficult for β-lactamases to hydrolyze.

Mechanism of Cephalosporins

The acetoxy group (or other R group) will leave when the drug acylates the PBP.

Cephalosporins- Classification

Cephalosporins are classified into four generations based on their activity.

Later generations generally become more effective against Gram (-) bacteria due to an increasing number of polar groups (also become zwitterions.)

Ceftazidime (3rd gen) in particular can cross blood brain barrier and is used to treat meningitis.

Later generations are often the broadest spectrum and are reserved against penicillin resistant infections to prevent the spread of cephalosporin resistant bacteria.

Carbapenems

Carbapenems are a potent class of β-lactams which attack a wide range of PBPs, have low toxicity, and are much more resistant to β-lactamases than the penicillins or cephalosporins.

Carbapenems

Thienamycin, discovered by Merck in the late 1970’s, is one of the most broad spectrum antibiotics ever discovered.

It uses import porins unavailable to other β-lactams to enter Gram (-) bacteria.

Due to its highly unstable nature this drug and its derivatives are created through synthesis, not bacterial fermentation.

Carbapenems

Thienamycin was slightly modified and marked as Imipenem. Due to its rapid degradation by renal peptidase it is administered with an inhibitor called cilastatin under the name Primaxin. Imipenem may cause seizures or sever allergic reactions.

Other modifications of Thienamycin have produced superior carbapenems called Meropenem and Ertapenem, which are not as easily degraded by renal peptidase and do not have the side effects of Imipenem.

Monobactams

The only clinically useful monobactam is aztreonam. While it resembles the other β-lactam antibiotics and targets the PBP of bacteria, its mechanism of action is significantly different.

It is highly effective in treating Gram (-) bacteria and is resistant to many β-lactamases

β-Lactamases

β-Lactamases were first discovered in 1940 and originally named penicillinases.

These enzymes hydrolyze the β-lactam ring, deactivating the drug, but are not covalently bound to the drug as PBPs are.

Especially prevalent in Gram (-) bacteria.

Three classes (A,C,D) catalyze the reaction using a serine residue, the B class of metallo- β-lactamases catalyze the reaction using zinc.

β-Lactamase Inhibitors

There are currently three clinically available β-lactamase inhibitors which are combined with β-lactams; all are produced through fermentation.

These molecules bind irreversibly to β-lactamases but do not have good activity against PBPs. The rings are modified to break open after acylating the enzyme.

β-Lactam/Inhibitor combinations

Aminopenicillins:

ampicillin-sulbactam = Unasyn

amoxicillin-clavulante = Augmentin

Extended-Spectrum Penicillins

piperacillin-tazobactam = Zosyn

ticarcillin-clavulanate = Timentin

Summary

β-Lactam antibiotics have dominated the clinical market since their introduction in the 1940’s and today consist of nearly ¾ 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 β-lactams has led to the development of resistant strains, new modifications are necessary in order for β-lactams to remain viable.

Assigned reading:

Patrick, Graham L. An Introduction to Medicinal Chemistry 4th Edition. New York: Oxford University Press, 2009. 388-414. Print.

Optional References/ Reading

Brunton, Laurence L. et al. Goodman and Gillman’s Pharmaceutical Basis of Therapeutics 11th Edition. McGraw-Hill, 2006 1134- 52. Print.

Bush, Karen. β-Lactamase Inhibitors from Laboratory to Clinic. Clinical Microbiology Reviews, Jan. 1988, p. 109-123. Web.

Elander, R.P. Industrial production of β-Lactam antibiotics. Journal of Applied Microbiology and Biotechnology (2003) 61:385–392. 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 β-lactam research. Journal of Antimicrobial Chemotherapy (1998) 41, 589–603. Web.

Questions

1. What are two ways by which a bacteria could become resistant to carbapenems?

2. How were the natural penicillins modified to be orally available?

3. How are extended spectrum penicillins modified to be orally available?

4. What are two ways that the β-lactam can be protected from β-lactamases?