Lecture 2 Penicillin LMS PDF

New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Department of Medicinal Chemistry Program: : Pharm D Program Course Title : Medicinal Chemistry-I...

New Mansoura University Faculty of Pharmacy Pharm D Program ___________________________________________________________________________ Department of Medicinal Chemistry Program: : Pharm D Program Course Title : Medicinal Chemistry-I Course Code: PMC-306  Chemotherapy (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:  β-Lactamase inhibitors, Carbapenems and Monobactams.  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  Relatively simple. called organelles (e.g. mitochondria, etc.),  It can acquire intact essential  Synthesize essential vitamins, so vitamins from food. having enzymes catalyzing these reactions.  Has only cell membrane.  Has a cell membrane and a cell wall.  There are five main mechanisms by which antibacterial agents act: 1. Inhibition of bacterial cell wall synthesis 2. Interactions with the plasma membrane (so affect membrane permeability which fatal results for the cell) 3. Disruption (inhibition) of protein synthesis (so essential proteins and enzymes can no longer be made) 4. Inhibition of cell metabolism 5. Inhibition of nucleic acid transcription and replication  The name “Lactam” is given to cyclic amides and is analogous to the name “Lactone” which is given to cyclic esters.  The name “Lactam” is given to cyclic amides and is analogous to the name “Lactone” which is given to cyclic esters.  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 β-Lactams: Penicillins and Cephalosporins.  Non-classical β-Lactams: Carbapenams, Monobactams and β-Lactamase inhibitors. 1- Penicillins  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. CYS VAL  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. CYS VAL  Penicillin contains highly unstable bicyclic ring system consisting of:  4-membered β-lactam ring.  Fused to a 5-membered thiazolidine ring. Acyl side Chain West End 6-aminopenicillanic acid (6-APA) b-Lactam ring Thiazolidine ring  It is the name given to unsubstituted bicyclic lactam  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).  NAM NAG NAM NAG NAM L-Ala NAM L-Ala NAG NAM L-Ala NAG NAM D-Glu D-Glu D-Glu L-Ala L-Ala L-Ala NAM NAG NAM NAG NAM L-Lys L-Lys L-Lys D-Glu D-Glu D-Glu L-Ala L-Ala L-Ala L-Lys L-Lys L-Lys D-Glu D-Glu D-Glu L-Lys L-Lys L-Lys  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). NAM NAG NAM NAG SUGAR BACKBONE L-Ala L-Ala D-Glu D-Glu L-Lys Gly Gly Gly Gly Gly L-Lys Gly Gly Gly Gly Gly D-Ala D-Ala D-Ala D-Ala NAM NAG NAM NAG SUGAR BACKBONE L-Ala L-Ala D-Glu D-Glu L-Lys Gly Gly Gly Gly Gly L-Lys Gly Gly Gly Gly Gly D-Ala D-Ala Cross linking  The penicillins and the other β-lactam antibiotics have a structure that closely resembles that of acylated D-alanyl-D-alanine.  The transpeptidase enzyme (PBPs) 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).  Mode of action of the β-lactam antibiotics is a selective and irreversible inhibition of transpeptidase enzyme (PBPs).  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)  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.  cis Stereochemistry No substitution allowed 6-OCH3 →β-lactamase resistance is essential Sulphur is usual but not essential Amide side chain is essential It is responsible for Presence of one -CH3  Acid stability. and one b-CH3 is  b-lactamase essential for activity resistance  Broad spectrum of Free “COOH” is essential:- activity  Usually ionized and administered as Na or K salt. Strained b-Lactam  Carboxylate ion binds to the The carbonyl group is is essential charged N- of lysine residue in highly electrophilic the binding site. for nucleophilic  Activity diminished when attack (Why?). Bicyclic system is essential modified to alcohol or ester  Confers further strain on the β-lactam ring.  The greater the strain, the greater the activity, but the greater the instability of the molecule to other factors.  The strained β-lactam ring is essential.  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)  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.  Thick porous cell wall Cell membrane Cell  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, i.e. small molecules like 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.  Outer membrane Hydrophobic barrierPorin Periplasmic Lactamase L L space enzymes L L Thin 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.  Outer membrane Hydrophobic barrierPorin Periplasmic Lactamase L L space enzymes L L Thin cell wall Cell membrane 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.   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.  Outer membrane Hydrophobic barrierPorin Periplasmic Lactamase L L space enzymes L L Thin cell wall Cell membrane  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 result, any penicillin penetrates the outer membrane encounters a higher concentration of β-lactamase than it would with Gram-positive bacteria.  In some Gram-negative bacteria, excess quantities of transpeptidase are produced and penicillin is incapable of inactivating all the enzyme molecules present.  Several forms of the transpeptidase enzyme are present within any bacterial cell and has different affinity for the different β-lactams.  Penicillin-resistant strains of S. aureus acquired a transpeptidase enzyme called PBP2a which has a much lower affinity to penicillins.  Some Gram negative bacteria have proteins in the outer membrane which capable of pumping penicillin out (efflux) of the periplasmic space, thus lowering its concentration and effectiveness.  The extent to which this happens varies from species to species and also depends on the structure of the penicillin.  Mutations can occur which will affect any or all of the above mechanisms such that they are more effective in resisting the effects of β-lactams.  Small portions of DNA carrying the genes required for resistance can also be transferred from one cell to another by means of genetic vehicles called plasmids.  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 (can not 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).  The problem of short duration of penicillin could be 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.  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.  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.  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.  There are three reasons for the acid sensitivity of penicillin G.  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.  There are three reasons for the acid sensitivity of penicillin G.  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.  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°.  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.  Inhibit cell wall synthesis Irreversible inhibition of Nature Penicillin transpeptidase enzyme (PBPs) (Cross linking) (bactericidal) Acid β-Lactamase Short Allergy sensitive sensitive Duration Narrow spectrum  To avoid Short To be acid duration stable To be β-Lactamase resistant 43 Choose the correct answer: Which of the following drugs has long duration of action? a. Penicillin V b. Penicillin G c. Penicillin G procaine d. All of them Choose the correct answer: The addition of EWG as shown below is responsible for? a. Acid stability b. Acid sensitivity c. Broad spectrum d. Short duration 47