Classical And Non-Classical Beta-Lactam Antibiotics PDF

Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics Antibacterial agents Antibacterial agent is a synthetic or naturally occurring agent which can kill (Bactericidal) or inhibit the growth (Bacteriostatic) o...

Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics Antibacterial agents Antibacterial agent is a synthetic or naturally occurring agent which can kill (Bactericidal) or inhibit the growth (Bacteriostatic) of bacterial cells. Antibiotic agent is an antibacterial agent derived from a natural source. Antibiotics: are classified into ◼ β-Lactam Antibiotics ✓ Classical β-Lactam Antibiotics : Penicillins and Cephalosporins. ✓ Non-classical β-Lactam Antibiotics : ✓ Carbapenems, Monobactams and β-Lactamase inhibitors. ◼ Non β -lactam Antibiotics: Tetracyclins, Marcolides, Aminoglycosides, Chloramphenicol,etc ◼ Synthetic chemotherapeutic Agents. ✓ Sulfonamides ✓ Antimycobactrial drugs ✓ Quinolones ✓ Antiparasitic Drugs ✓ Antifungal Agents ✓ Antiviral Agents ✓ Anticancer Agents The success of antibacterial agents results from their selective action against bacterial cells rather than animal cells due to the difference in their structure and biosynthetic pathways. Animal cell Bacterial cell ✓ has a defined nucleus ✓ does not have a defined nucleus ✓ contain a variety of structures called ✓ relatively simple organelles (e.g. mitochondria, etc.), ✓ It can acquire intact essential vitamins ✓ Synthesize essential vitamins, so having from food enzymes catalyzing these reactions. ✓ has only cell membrane ✓ has a cell membrane and a cell wall β-lactam antibiotics β-lactam antibiotics are antibiotics which contain the β-lactam ring (4-membered cyclic ring, azetidinone: 1-azacyclobutan-4-one). β-lactam antibiotics are classified into: ✓ Classical types: Penicillins and Cephalosporins. ✓ Non-classical β-Lactams: Carbapenams, Monobactams and other related antibiotics. Page 1  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics 1- Penicillins ❑ Penicillin contains highly unstable bicyclic ring system consisting of: ✓ 4-membered β-lactam ring ✓ Fused to a 5-membered thiazolidine ring. The skeleton of the molecule suggests that it is derived from the amino acids: ✓ Cysteine ✓ Valine The overall shape of the molecule is like a half-open book. Nomenclature of Penicillin ◼ Chemical abstract system: - As bicyclic system ◼ Penam derivatives ✓ It is the name given to unsubstituted bicyclic lactam. Page 2  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics ◼ Penicillanic acid derivatives ◼ Penicillin derivatives: Mechanism of action of Penicillin Animal cells do not have a cell wall, making it the perfect target for antibacterial agents such as penicillins. The bacterial cell wall is a peptidoglycan structure: it is made up of peptide and sugar units. The structure of the wall consists of a parallel series of sugar backbones containing two types of sugar ✓ N -acetylmuramic acid (NAM) ✓ N - acetylglucosamine (NAG) Peptide chains* are bound to the NAM sugars. The peptide chains are linked together (by the displacement of D-alanine from one chain by glycine in another) by transpeptidase enzyme (Penicillin binding proteins; PBPs). Page 3  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics In human biochemistry, there are only L-amino acids, whereas bacteria have racemase enzymes that can convert L-amino acids to D-amino acids. The enzyme responsible for the cross-linking reaction is known as the transpeptidase enzyme. Penicillin inhibits final crosslinking stage of cell wall synthesis via reacting with the transpeptidase enzyme (PBPs) to form an irreversible covalent bond. Inhibition of transpeptidase leads to a weakened cell wall. Cells swell due to water entering the cell, then burst (lysis). The penicillins and the other β-lactam antibiotics have a structure that closely resembles that of acylated D-alanyl-D-alanine. The enzyme mistakenly accepts the penicillin as though it were its normal substrate. Once bound, penicillin is subjected to nucleophilic attack by serine moiety of the transpeptidase enzyme (PBPs). Normal Mechanism Page 4  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics Mechanism inhibited by penicillin Sources of Penicillins 1- Natural Penicillins ❑ Fermentation: varying the fermentation conditions ✓ Adding different carboxylic acids* to the fermentation medium resulted in penicillins with different acyl side chains 1. Benzylpenicillin (penicillin G) 2. Phenoxymethylpenicillin (Penicillin V) There was a limitation to the sort of carboxylic acid which was accepted by the biosynthetic route (i.e. only acids of general formula RCH2COOH). This, in turn, restricted the variety of analogues which could be obtained. The other major disadvantage was the tedious and time- consuming nature of the method. 2- Semi-Synthetic Penicillins Acylation of 6-Aminopenicillanic acid (6-APA) The isolated biosynthetic intermediate 6-APA from Penicillium chrysogenum grown in a fermentation medium was treated with a range of acid chlorides. Page 5  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics SAR of penicillins ✓ The strained β-lactam ring and the bicyclic system is essential. ✓ This confers further strain on the β-lactam ring: - The greater the strain, the greater the activity, but the greater the instability of the molecule ✓ The free carboxylic acid is essential. ✓ This is usually ionized and penicillins are administered as sodium or potassium salts. ✓ The carboxylate ion binds to the charged nitrogen of a lysine residue in the binding site. ✓ The acylamino side chain is essential. ✓ The stereochemistry of the bicyclic ring with respect to the acylamino side chain is important. ✓ Sulphur is usual but not essential. Any variations are restricted to the acylamino side chain (R) Resistance to penicillin Bacterial strains vary in their susceptibility to penicillin. There are several reasons for this varied susceptibility. 1. Physical barriers. 2. Presence of β-lactamase enzymes. 3. High levels of transpeptidase enzyme (PBPs) produced. 4. Affinity of transpeptidase enzyme to penicillin. 5. Transport back across the outer membrane of Gram-negative bacteria (Efflux pumps). 6. PBP Mutations and genetic transfers. Page 6  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics 1- Physical barriers ✓ Penicillin has to pass through the cell walls of both Gram-positive and Gram-negative bacteria to reach the outer surface of the bacterial cell membrane to inhibit the transpeptidase enzyme. ✓ The cell wall is highly porous, so small molecules as penicillin can move easily through it. ✓ In Gram-positive bacteria, there is no barrier preventing penicillin reaching the cell membrane so penicillin G has good activity against these organisms. Thick porous cell wall Cell membrane Cell However, Gram-negative bacteria have an outer lipopolysaccharide membrane surrounding the cell wall which is impervious to water and polar molecules, such as penicillin. So, Gram-negative bacteria are generally resistant to penicillin. ✓ Why some Gram-negative bacteria are susceptible and some are not? ✓ The outer membrane has a protein structures called porins which act as pores through which water and essential nutrients can pass to reach the cell. Small drugs can also pass through porins, but whether they do or not depends on the characteristics of the penicillin (i.e. its size, structure, and charge), HOW? ✓ Large, negatively charged, and hydrophobic (lipophilic) drugs have less chance of passing through the porins. ✓ Small, hydrophilic drug that can exist as a zwitterion can pass through porins. 2- Presence of β-lactamase enzymes β-lactamases are enzymes which have mutated from transpeptidases. They hydrolyze (open up) the β-lactam ring of benzylpenicillin. Some Gram-positive bacterial strains are resistant to penicillin as they can release β- lactamase into the surrounding environment so penicillin is inactivated before reaching the cell membrane. The enzyme eventually dissipates through the cell wall and is lost, so the bacterium has to keep generating the enzyme to maintain its protection. All Gram-negative bacteria produce β-lactamases which makes them more resistant to penicillins. The released β-lactamase cannot pass through the outer membrane so are trapped in the periplasmic space between the cell membrane and outer membrane. As a Page 7  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics result, any penicillin penetrates the outer membrane encounters a higher concentration of β-lactamase than it would with Gram-positive bacteria. Properties & drawbacks of Penicillin G Benzylpenicillin (penicillin G; Pen G) is Active against a range of bacterial infections. Non-toxic, penicillins are amongst the safest drugs. Lacks serious side effects for most patients. However, there are various drawbacks. Short duration. Causes allergic reactions in some individuals. Acid sensitivity (cannot be taken orally because it is broken down by stomach acids). β-Lactamase sensitivity. Limited or a narrow spectrum of activity (mainly against Gram-positive bacteria). Penicillins with long duration The problem of short duration of penicillin could be solved by reacting penicillin G with high molecular weight amine such as procaine or benzathine, the resulted salt is less water soluble than the alkali metal salts and possesses a long duration. Penicillin G procaine (procaine penicillin): ✓ It is a combination of penicillin G and procaine HCl. ✓ It is slowly absorbed into the circulation after IM injection and hydrolyzed to benzylpenicillin. ✓ It is used where prolonged low concentrations of benzylpenicillin are required (????). ✓ At high doses procaine penicillin can cause seizures and CNS abnormalities due to procaine present in it. Penicillin G Benzathine: Page 8  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics ✓ It is very water insoluble, so injected IM, where benzathine slowly releases the penicillin making the combination long acting (2-4 weeks after single IM dose). ✓ It is also used to prevent rheumatic fever. Allergic Reactions Penicillins cause allergic reactions in some individuals, varying from rash to immediate anaphylactic shock, Why?? Penicillins molecules can react with nucleophilic groups on body proteins, the β-lactam ring is opened and the penicillin is covalently linked to the protein. Acid sensitivity of penicillins. There are three reasons for the acid sensitivity of penicillin G. 1- Ring strain: ✓ The bicyclic system in penicillin consists of a four-membered ring fused to a five-membered ring. As a result, penicillin suffers large angle and torsional strains. ✓ Acid-catalyzed ring-opening relieves these strains by breaking open the more highly strained β-lactam ring. 2- A highly reactive β-lactam carbonyl group ✓ The carbonyl group in the β-lactam ring is highly susceptible to nucleophiles and does not behave like a normal tertiary amide!? ✓ 3ry amide is resistant to nucleophilic attack because the carbonyl group is resonance stabilized by the neighboring nitrogen atom. ✓ The nitrogen can feed its lone pair of electrons into the carbonyl group to form a dipolar structure with bond angles of 120°. Page 9  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics ✓ This resonance stabilization is impossible for the β-lactam ring because of the increase in angle strain that would result in having a double bond within a four-membered β-lactam ring. ✓ The preferred bond angles for a double bond are 120° but the bond angles of the β-lactam ring are constrained to 90°. ✓ As a result, the lone pair is localized on the nitrogen atom and the carbonyl group is more electrophilic. 3- Influence of the acyl side chain (neighboring group participation): Penicillin G has a self-destruct mechanism built into its structure. Acid-Resistant (Stable) Penicillins ✓ Nothing can be done about the first two factors, as the β-lactam ring is vital for antibacterial activity. ✓ Inserting an electron-withdrawing group (EWG) in the acyl side will decreases the electron density on the side-chain carbonyl and reduce its tendency to act as a nucleophile thus protects penicillins from acid degradation. Page 10  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics β-Lactamase-resistant penicillins ✓ The strategy of steric shields was used successfully to block penicillin from accessing the penicillinase or β-lactamase active site by placing a bulky group on the side chain. ✓ In general, increasing the steric hindrance at the -carbon of the acyl group increased resistance to -lactamase. The -acyl carbon could be part of an aromatic (e.g. phenyl or naphthyl) or heteroaromatic (e.g., 4-isoxazoyl) system.  Methicillin: ✓ Substitutions at the ortho positions of ring system increase the steric hindrance of the acyl group and confer more -lactamase resistance. ✓ In methicillin, Steric shields are two O–OCH3 on the aromatic ring. Movement of one OCH3 groups to the p-position or replacing one with H-atom → β- lactamases-sensitive agent. Inserting a methylene between the aromatic ring and 6-APA → a β-lactamase-sensitive agent. ✓ With no EWG on the side chain, so must be injected*. ✓ It has narrow spectrum of activity*. Page 11  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics  Nafcillin ✓ Substitution at the 2-position of a 1-naphthyl system by ethoxy group increases the steric hindrance of the acyl group and confer more -lactamase resistance. ✓ With no EWG on the side chain, so must be injected.  Steric factors that confer -lactamase resistance, however, do not necessarily also confer acid stability.  The -lactamase–resistant penicillins tend to be lipophilic molecules that do not penetrate well into Gram-negative bacteria.  The isoxazolyl penicillins ✓ The 4-isoxazoyl penicillins require both 3-aryl & 5-methyl substituents for effectiveness against -lactamase–producing S. aureus*. ✓ The isoxazoyl penicillins, particularly those with an electronegative substituent in the 3- phenyl group are also resistant to acid-catalyzed hydrolysis of the -lactam (i.e. orally active). The isoxazolyl ring acts as steric shield (giving the structure β-lactamase stability) and electron with drawing (giving the structure acid stability). ✓ Because they are highly serum protein bound, they are not good choices for treatment of septicemia.  Temocillin ✓ Temocillin is a β-lactamase-resistant carboxypenicillin with 6-OCH3 group. ✓ It is not active against Gram-positive bacteria (Why??), or bacteria with altered penicillin binding proteins (PBPs). ✓ It is reserved for the treatment of infections caused by β-lactamase producing strains of Gram-negative bacteria, including those resistant to third generation cephalosporins. Page 12  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics ✓ It is used by injection for the treatment of septicemia, UT-infections, and lower respiratory tract infections caused by susceptible Gram-negative bacteria. Broad-spectrum penicillins The spectrum of activity shown by any penicillin depends on: - ✓ Its structure and Its ability to cross the cell membrane of Gram-negative bacteria. ✓ Its susceptibility to β-lactamases. ✓ Its affinity for the transpeptidase target enzyme. ✓ The rate at which it is pumped back out of cells by Gram-negative organisms. The variations in the side chain and gave the following results: Hydrophobic (lipophilic) groups on the side chain favor activity against Gram-positive bacteria, but result in poor activity against Gram-negative bacteria. If the hydrophobic character is increased, there is little effect on Gram-positive activity, but activity drops even further against Gram-negative bacteria. Hydrophilic groups on the side chain have little effect on Gram-positive activity (e.g. penicillin T) or cause a reduction of activity (e.g. penicillin N), 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*, COOH) is attached to the carbon that is α to the carbonyl group on the side chain, why? ✓ Hydrophilic group aids the passage of these penicillins through the porins of the Gram- negative bacterial outer membrane. ✓ Those penicillins having useful activity against both Gram-positive and Gram-negative bacteria are known as broad-spectrum antibiotics. ✓ The introduction of an ionized or polar group into the -position of the side chain benzyl carbon atom of penicillin G confers activity against Gram-negative bacilli. ✓ There are three classes of broad-spectrum antibiotics, all of which have an α-hydrophilic group: -  The aminopenicillins.  The carboxypenicillins.  The Ureidopenicillins. A- The -aminopenicillins Page 13  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics ✓ Ampicillin is acid stable (orally active): the electron-withdrawing character of the protonated –NH2 group reducing participation in hydrolysis of the β-lactam bond. ✓ Sensitive to β-lactamase enzyme (no steric shields present). ✓ Only 40% of the oral dose was absorbed, Why? ✓ The problem of poor absorption through the gut wall is due to the dipolar nature of the molecule since it has both free NH2 and COOH groups which are amphoteric and forms Zwitterr ions (difficult solubility in both acid and alkaline media). This problem can be solved by: 1- Shifting the isoelectric point to the acid side by introducing para-phenolic OH group into the side-chain phenyl → Amoxicillin. ✓ Amoxicillin: 80% of the dose is absorbed after oral administration. ✓ Better oral absorption leads to less disturbance of the normal GI flora and, therefore, less drug-induced diarrhea. 2- 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. ✓ Simple methyl esters are not used, Why? ✓ The bulky penicillin skeleton is so close to the ester that it acts as a steric shield and prevents the esterase enzymes from accepting the penicillin ester as a substrate (i.e. not metabolized in humans) ✓ Thus we ‘extended or double’ esters (Acyloxymethyl esters) which contain a second ester group further away from the penicillin nucleus, so is more exposed to attack by esterases. ✓ Double ester of formaldehyde hydrate in which one of the OH groups has been esterified with ampicillin and the other with ….... ✓ The “double ester,” would extend the terminal ester carbonyl away from the thiazolidine ring and eliminate the inherent steric hindrance with the enzyme. ✓ In contrast to ampicillin (which is amphoteric), pivampicillin and bacampicillin are weak bases and are very well absorbed in the duodenum. ✓ Hydrolysis of the terminal ester gives an unstable hydroxymethyl ester, which spontaneously decomposes to ampicillin. Page 14  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics B- The -carboxypenicillins ✓ Carbenicillin* shows a broad spectrum of activity due to the hydrophilic COOH group (ionized at pH 7) on the side chain. ✓ The drug is sensitive to β-lactamases, Why? ✓ It is acid unstable and thus must be given by injection. ✓ Carbenicillin is readily decarboxylated producing benzyl penicillin, which has no activity against the organisms for which carbenicillin is indicated. Overcome: Esterification of the -carboxyl group partially protects it from acid-catalyzed destruction and provides an orally active derivative that is hydrolyzed to carbenicillin in the plasma. ✓ Carfecillin is prodrug for carbenicillin and show an improved absorption through the gut wall. ✓ Aryl esters are better than alkyl esters as the former are more chemically susceptible to hydrolysis, because of the electron-withdrawing inductive effect of the aryl ring. ✓ An extended ester is not required as the aryl ester is further from the β-lactam ring and is not shielded. Page 15  Prof. Dr Mohammed Abubakr Classical and Non-classical -lactam antibiotics Ticarcillin ✓ Ticarcillin is a sulfur-based bioisostere of carbenicillin that cannot decarboxylate as carbenicillin does. ✓ It is similar in antibacterial spectrum to carbenicillin (mainly against Gran-negative infections). ✓ Like carbenicillin, is unstable in acid and, therefore, must be administered parenterally. ✓ It is currently administered with clavulanic acid to broaden its spectrum of activity! ✓ It has fewer side effects. C- The ureidopenicillins ✓ They have a urea functional group at the α-position (i.e. are acylated derivatives of ampicillin). ✓ They have better properties and activity than carboxypenicillins. ✓ They are generally more active against Gram-negative bacteria*. ✓ β-lactamase sensitive. ✓ They are unstable under acidic conditions; therefore, they are not available for oral administration (They have to be injected). ✓ Tazobactam is often co-administered with piperacillin because of tazobactam’s ability to inhibit β-lactamases. Page 16