Beta-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. Clinically useful families of beta-lactam compounds include the penicillins, cephalosporins, monobactams carbapenems

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 MOA 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 transpeptidases which catalyzes the cross linking process.

Peptidoglycan Peptidoglycan is a carbohydrate composed of alternating units of NAMA (N-acetyl muramic acid) and NAGA (N-acetyl Gluc0samine). 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.

Mechanism

Mechanism of action - bacterial cell wall synthesis Cross linking

Transpeptidase- PBP The cross linking reaction is catalyzed by a class of transpeptidases known as penicillin binding proteins(PBPs) 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 of action - bacterial cell wall synthesis Normal Mechanism

Mechanism of β-Lactam Drugs 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 action - bacterial cell wall synthesis Alternative theory- Pencillin mimics D-Ala-D-Ala. Mechanism inhibited by penicillin

Mechanism of β-Lactam Drugs The hydroxyl attacks the amide and forms a tetrahedral intermediate.

Mechanism of β-Lactam Drugs The tetrahedral intermediate collapses, the amide bond is broken, and the nitrogen is reduced.

Mechanism of β-Lactam Drugs The PBP is now covalently bound by the drug and cannot perform the cross linking action.

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. Thus Inactivation of the antibiotic by beta lactamase Imparied penetration of drug to target PBPs 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.

PENICILLIN Classification 3. Synthetic Phenethicillin Naturally 1. Ocurring 2. Semisynthetic 3. Synthetic Acid Resistant Penicillins Eg. Peniciliin V Penicillinase resistant P. Eg. Methicillin, Nafcillin, Oxacillin, Cloxacillin Extended spectrum Penicillins Amino penicillins - Eg. Ampicillin, Amoxicillin, Carboxy Penicillins- Carbenicillin Penicillin G Phenethicillin

Nomenclature Numbering of fused bicyclic ring initiates the at N atom and assigns the ring S the 4-position. Thus, penicillins are named as 4-thia-l-azabicyclo[3.2.0]heptanes, according to this system

Three simplified forms of penicillin nomenclature have been adopted for general use. The first uses the name “penam” for the unsubstituted bicyclic system, including the amide carbonyl group, with numbering systems as just described Thus, penicillins generally are designated according to this system as 6- acylamino- 3,3-dimethylpenam-2-carboxylic acids.

Three simplified forms of penicillin nomenclature have been adopted for general use. The second, seen more frequently in the medical literature, uses the name “penicillanic acid” to describe the ring system with substituents that are generally present (i.e., 3,3-dimethyl and 2-carboxyl acid)

Three simplified forms of penicillin nomenclature have been adopted for general use. A third form, uses trivial nomenclature to name the entire 6-carbonylaminopenicillanic acid portion of the molecule as “penicillin” and then distinguishes compounds on the basis of the R group of the acyl portion of the molecule

Thus, penicillin G is named benzylpenicillin and penicillin V is phenoxymethylpenicillin

Degradation of Penicillin

Penicillin -SAR Penicillin

1.Penicillin 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)

Penicillins- Natural 2. 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 It is not orally active

Penicillin G in Acidic Conditions Penicillin G could not be administered orally due to the its inactivation in acidic conditions of the stomach.

Penicillin V 3. 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.

So Next focus was on resistance to β-lactamase Penicillin V (orally active) electronegative oxygen Better acid stability and orally active But sensitive to b-lactamases Slightly less active than penicillin G Allergy problems with some patients So Next focus was on resistance to β-lactamase

Semi-Synthetic Penicillins 4.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. 6-APA

Penicillins- Antistaphylococcal 5. In general, it was observed that, increasing the steric hindrance at the α- carbon of the acyl group increased resistance to staphylococcal β-lactamase . Thus Penicillins which have bulky side groups can block the β-Lactamases (of bacteria)which hydrolyze the lactam ring. This leads to development of penicillinase (β-Lactamases) resistant penicillins.

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. 7. 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)

Methoxy groups block access to b-lactamases but not to transpeptidases Examples – Methicillin ortho groups important Methoxy groups block access to b-lactamases but not to transpeptidases Binds less readily to transpeptidases compared to penicillin G Lower activity compared to Pen G against Pen G sensitive bacteria Poor activity vs. some streptococci Inactive vs. Gram -ve bacteria Poor range of activity Active against some penicillin G resistant strains (e.g. Staphylococcus) Acid sensitive since there is no electron-withdrawing group Orally inactive and must be injected

Penicillins- Antistaphylococcal the -acyl carbon could be part of an aromatic (e.g.,phenyl or naphthyl) or heteroaromatic (e.g., 4-isoxazoyl)system,which increases antibacterial potency by making them acid stable. Substitutions at the ortho positions of a phenyl ring (e.g., 2,6- dimethoxy [methicillin]) or the 2-position of a 1-naphthyl system (e.g., 2-ethoxyl [nafcillin]) increase the steric hindrance of the acyl group and confer more β-lactamase resistance than shown by the unsubstituted compounds

Penicillins- Antistaphylococcal 8. Methicillin is acid sensitive hence orally inactive and has been improved upon by adding electron withdrawing groups, as was done in penicillin V, resulting in drugs such as oxacillin and nafcillin.

Among five-membered–ring heterocyclic derivatives bulkier substituents are required to confer effective β-lactamase resistance. Thus, members of the 4-isoxazoyl penicillin family (e.g., oxacillin, cloxacillin, and dicloxacillin) require both the 3- aryl and 5-methyl (3-methyl and 5-aryl) substituents for effectiveness against β - lactamase–producing S. aureus.

Dicloxacillin R = Cl, R' = Cl Flucloxacillin R = Cl, R' = F Examples - Oxacillin Oxacillin R = R' = H Cloxacillin R = Cl, R' = H Dicloxacillin R = Cl, R' = Cl Flucloxacillin R = Cl, R' = F Orally active and acid resistant Resistant to b-lactamases Active vs. Staphylococcus aureus Less active than other penicillins Inactive vs. Gram -ve bacteria Nature of R & R’ influences absorption and plasma protein binding Cloxacillin better absorbed than oxacillin Flucloxacillin less bound to plasma protein, leading to higher levels of free drug

9. Due to the bulky side group, all of the antistaphylococcal drugs have difficulty penetrating the cell membrane and are less effective than other penicillins. All of the clinically available penicillinase-resistant penicillins are significantly less active than either penicillin G or penicillin V against most non–β-lactamase-producing bacteria normally sensitive to the penicillins.

Penicillins- Aminopenicillins 10.In order to increase the range of activity(efficacy), the penicillin has been modified to have more hydrophilic groups, allowing the drug to penetrate into Gram (-) bacteria via the porin channels. Introduction of an ionized or polar group into the α-position of the side chain benzyl carbon atom of penicillin G confers activity against Gram-negative bacilli Ampicillin R=Ph Amoxicillin R= Ph-(p)OH

Penicillins- Aminopenicillins 11. derivatives with an ionized -amino group, such as ampicillin and amoxicillin, are generally effective against such Gram-negative genera as Escherichia, Klebsiella, Haemophilus, Salmonella, Shigella, and non–indole-producing Proteus. Furthermore, activity against penicillin G–sensitive, Gram-positive species is largely retained. 12. 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 12.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 13.Due to the effectiveness of the aminopenicillins, a second modification is made to the drug at the carboxyl group to produce prodrug eg. BACAMPICILLIN prodrug of ampicillin. 14.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.

Prodrugs of Ampicillin (Leo Pharmaceuticals - 1969) Properties Increased cell membrane permeability Polar carboxylic acid group is masked by the ester Ester is metabolised in the body by esterases to give the free drug

Penicillins- Extended Spectrum 15.Extended spectrum penicillins are similar to the aminopenicillins in structure but have either a carboxyl group or urea group instead of the amine. Thus Incorporation of an acidic substituent at the α-benzyl carbon atom of penicillin G also imparts clinical effectiveness against Gram-negative bacilli and, furthermore, extends the spectrum of activity to include organisms resistant to ampicillin

Penicillins- Extended Spectrum 16.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

Eg. Carbenicillin Carbenicillin has a broad range of antimicrobial activity, broader than any other known penicillin, a property attributed to the unique carboxyl group. It has been proposed that the carboxyl group improves penetration of the molecule through cell wall barriers of Gram-negative bacilli, compared with other penicillins.

Eg. Piperacillin Piperacillin (Pipracil) is the most generally useful of the extended- spectrum acylureidopenicillins. Piperacillin is destroyed rapidly by stomach acid; therefore, it is active only by intramuscular or intravenous administration

Therapeutic Uses Pneumococcal Infections Pneumococcal Meningitis Pneumococcal Pneumonia Streptococcal Infections Streptococcal Pharyngitis (including Scarlet Fever) Streptococcal Pneumonia, Arthritis, Meningitis, and Endocarditis Staphylococcal Infections Meningococcal Infections Gonococcal Infections Syphilis Actinomycosis Diphtheria Anthrax Clostridial Infections Fusospirochetal Infections Rat-Bite Fever Listeria Infections Lyme Disease Erysipeloid Surgical Procedures in Patients with Valvular Heart Disease

IUPAC names of penicillins Penicillin G 6-Benzyl penicillin 6-(phenylacetamido)penicillanic acid

IUPAC names of penicillins Methicillin 2,6-dimethoxyphenylpenicillin

IUPAC names of penicillins Oxacillin (5-methyl-3-phenyl-4-isoxazolyl)penicillin

IUPAC names of penicillins Cloxacillin [3-(o-chlorophenyl)-5-methyl-4-isoxazolyl] penicillin

IUPAC names of penicillins Ampicillin D-α-aminobenzylpenicillin 6-[D-α-aminophenylacetamido]penicillanic acid

IUPAC names of penicillins Amoxicillin 6-[D- α -amino-p- hydroxyphenylacetamido]penicillanic acid

IUPAC names of penicillins Carbenicillin α -carboxybenzylpenicillin 6-[D-α-carboxyphenylacetamido]penicillanic acid

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 6-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.

Cephalosporins penicillins cephalorsporins -lactam ring

Cephalosporins Mechanism of Action: Cephalosporins are composed of a dihydrothiazine ring and a b-lactam ring. The mechanism of action is identical to penicillins. Mechanism of Resistance: Same as penicillins. Cephalosporins are less susceptible to Staphylococcus beta- lactamase; therefore have a broader spectrum of activity; however they are not the drug of choice. Other bacteria are resistant, because they produce distinct beta-lactamases. Methicillin-resistant Staphylococcus is resistant to most cephalosporins.

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

Inhibition of Beta lactamase by Cephalosporines

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.

First generation cephalosporins: cephalothin, cefazolin, cefalexin. These drugs have good activity against most Gram positive cocci (Streptococcus, pneumococcus but not or methicillin-resistant Staphylococcus). They are more active against Gram negative organisms (Escherichia co1i Kiebsiella pneumoniae, and the indole negative Proteus mirabilis) than are the natural penicillins. They are effective against some anaerobic cocci (Peptococcus and Peptosteptococcus, but NOT Bacteroides fragilis). They are ineffective against Pseudomonas aeruginosa, Enterobacter, and indole-positive Proteus species. These drugs do not cross the blood-brain barrier.

Second generation cephalosporins: cefuroxime, cefamandole, cefoxitin, cefaclor. The spectrum is extended to more Gram negative bacteria Enterobacter species, Klebsiella species, and indole-positive Proteus species. Also, Haemophilus influenza is covered by cefuroxime, cefamandole, cefaclor; Bacteroides fragilis by cefoxitin. These drugs do not achieve adequate levels in the CSF.

Third generation cephalosporins: moxalactam, cefaperazone, ceftazidirne, ceftriaxone. These drugs demonstrate extended Gram negative coverage, are more resistant to non-Staphylococcus b- lactamase, and readily cross the blood-brain barrier. The spectrum is extended to include: Enterobacter, Pseudomonas (ceftazidime and cefaperazone only), Serratia, b-lactamase producing Haemophillus influenza and Neisseria species. Only cetizoxime and moxalactam retain good activity against Bacteroides fragilis.

Fourth generation forth generation of cephalosporins (e.g. cefepime) are available, these are comparable to third-generation but more resistant to some beta­lactamases.

DEGRADATION OF CEPHALOSPORIN Pharmacokinetics Some cephalosporins may be given orally but most are given parenterally (IM or IV). They are widely distributed in the body like penicillins. Some such as cefoperazone, cefotaxime, cefuroxime, ceftriaxone, and ceftazidime (third generation) also cross the blood-brain barrier and are drugs of choice for meningitis due.

Uses: A cephalosporin with or without an aminoglycoside is first-line treatment of Klebsiella. First generation cephalosporins are used for surgical prophylaxis of wound infection. Third generation cephalosporins are used to treat meningitis due to pneumococci, meningococci, and Haemophillus influenza. Ceftriaxone is the drug of choice for treating beta- lactamase producing Neisseria gonorrhea.

THANK YOU -PHARMA STREET