_Sulfa drugs and beta-lactam antibiotics part 1.pdf

Sulfa drugs and lactam antibiotics CHMI3427EL Winter 2024 Dr. S. Montaut 1 Introduction Antibiotics are microbial metabolites or synthetic analogues that, in small doses, inhibit the growth and survival of microorganisms without serious toxicity to the host. Selective toxicity is the key concept. S...

Sulfa drugs and lactam antibiotics CHMI3427EL Winter 2024 Dr. S. Montaut 1 Introduction Antibiotics are microbial metabolites or synthetic analogues that, in small doses, inhibit the growth and survival of microorganisms without serious toxicity to the host. Selective toxicity is the key concept. Synthetic substances that are unrelated to natural products but still inhibit or kill microorganisms are referred to as antimicrobial agents. 2 History 1676: discovery of bacteria by van Leeuwenhoek. 19th century: Robert Koch isolation of microorganisms from infected patients, growth on culture media and administration to healthy individuals so as to reproduce in healthy individuals all of the classic symptoms of the same disease. 1877: Louis Pasteur reported in 1877 that when what he termed “common bacteria” were introduced into a pure culture of anthrax bacilli, the bacilli died, and that an injection of deadly anthrax bacillus into a laboratory animal was harmless if “common bacteria” were injected along with it. This did not always work but led to the appreciation of antibiosis, wherein two or more microorganisms competed with one another for survival. 3 1929: observation of a clear zone of inhibition (lysis) in a bacterial colony surrounding a colony of contaminating airborne Penicillium mold by Alexander Fleming in England. 1936: discovery of the sulfonamides in France and German. late 1930s and early 1940s: purification of penicillin by Florey, Chain, Abraham, and Heatley. 1939: discovery of tyrothricin 1941: first successful clinical trial of crude penicillin. 1943: discovery of streptomycin. 1947: discovery of chloramphenicol. 1948: discovery of chlortetracycline. 1949: discovery of neomycin. 1952: discovery of erythromycin. 4 There is an increasing impact of bacterial resistance. Intrinsic resistance to antimicrobial agents (resistance present before exposure to antibiotics) was recognized from the beginning. Some bacteria are immune to treatment from the outset because they do not take up the antibiotic or lack a susceptible target. Starting in the 1940s, however, and encountered with increasing frequency to this day, bacteria that were previously expected to respond were found to be resistant, many bacteria became resistant during the course of chemotherapy, and others were simultaneously resistant to several different antibiotics. The organisms were found to be capable of passing this trait on to other bacteria, even to those belonging to different genera. The spread of this phenomenon is aided by microorganisms’ short generation time (sometimes measured in fractions of an hour) and genetic versatility, as well as by poor antibiotic prescribing and utilization practices. 5 6 General Therapeutic Approach Drug Nomenclature Penicillins are derived from fungi and have names ending in the suffix -cillin, as in the term ampicillin. The cephalosporins are likewise fungal products, although their names mostly begin with the prefix cef- (or sometimes, following the English practice, spelled ceph-). The synthetic fluoroquinolones mostly end in the suffix -floxacin. Most of the remaining antibiotics are produced by fermentation of soil microorganisms belonging to various Streptomyces species. By convention, these have names ending in the suffix -mycin, as in streptomycin. 7 Some prominent antibiotics are produced by fermentation of various soil microbes known as Micromonospora sp. These antibiotics have names ending in -micin (e.g., gentamicin). In earlier times, the terms “broad spectrum” and “narrow spectrum” had specific clinical meaning. The widespread emergence of microbes resistant to single agents and multiple agents has made these terms less meaningful. It is, nonetheless, still valuable to remember that some antimicrobial families have the potential of inhibiting a wide range of bacterial genera belonging to both gram-positive and gram-negative cultures and so are called broad spectrum (such as the tetracyclines). Others inhibit only a few bacterial genera and are termed narrow spectrum (such as the glycopeptides, typified by vancomycin, which are used almost exclusively for a few gram-positive and anaerobic microorganisms). 8 Gram Stain - method for staining bacteria so that they are more readily visible under the microscope Gram-positive microorganisms are stained purple by contact with a methyl violet-iodine process. This is largely a consequence of their lack of an outer membrane and the nature of the thick cell wall surrounding them. Gram-negative microorganisms do not retain the methyl violetiodine stain when washed with alcohol but rather are colored pink when subsequently treated with the red dye safranin. The lipopolysaccharides on their outer membrane apparently are responsible for the staining behavior of gram-negative cells. 9 Since Gram stain is dependent on the outer layers of bacterial cells and this also strongly influences the ability of antimicrobial agents to reach their cellular targets, knowing the Gram staining behavior of infectious bacteria helps one decide which antimicrobial might be effective in therapy. Not all bacteria can be stained by the Gram procedure. These often require special staining processes for visualization. Among the more prominent of these for our purposes are the mycobacteria (the causative agents of tuberculosis, for example). These very waxy cells are called acid-fast and are stained by a carbol fuchsin mixture. 10 The Importance of Identification of the Pathogen Experimentally Based Therapy. The selection of an appropriate antibiotic involves sampling infectious material from a patient before instituting anti-infective chemotherapy, culturing the microorganism on suitable growth media, and identifying its genus and species. The bacterium in question is then grown in the presence of a variety of antibiotics to see which of them will inhibit its growth or survival and what concentrations will be needed to achieve this result. This is expressed in minimum inhibitory concentration (MIC) units. The term MIC refers to the concentration that will inhibit 99% or more of the microbe in question and represents the minimum quantity that must reach the site of the infection in order to be useful (see figure next slide). 11 In the top tubes (viewed from the top), a serially decreasing amount of antimicrobial agent is added to a suitable growth medium inoculated with a microorganism. Following incubation, microbial growth is detected by turbidity. The last concentration that produces no visible growth is scored as the minimum inhibitory concentration (m.i.c.) (1/8). Next a loopful is taken from each tube and placed in fresh medium (bottom row). In tubes where the organisms were killed by the drug there is no resumption of growth. Where the organisms were inhibited but not killed, removal of drug allows for resumption of growth. The last concentration that produces no visible growth under these conditions 12 is scored as the minimum bactericidal concentration (m.b.c.) (1/2). One of the most convenient experimental procedures is that of Kirby and Bauer. With this technique, sterile filter paper disks impregnated with fixed doses of commercially available antibiotics are placed on the seeded Petri dish. The dish is then incubated for a period of time. If the antibiotic is active against the particular strain of bacterium isolated from the patient, a clear zone of inhibition will be seen around the disk. If a given antimicrobial agent is ineffective, the bacterium may even grow right up to the edge of the disk. The diameter of the inhibition zone is directly proportional to the degree of sensitivity of the bacterial strain and the concentration of the antibiotic in question. 13 Currently, a given zone size in millimeters is dictated above which the bacterium is sensitive and below which it is resistant. When the zone size obtained is near this break point (the break point represents the maximum clinically achievable concentration of an anti-infective agent), the drug is regarded as intermediate in sensitivity, and clinical failure can occur. This powerful methodology gives the clinician a choice of possible antibiotics to use. This method is illustrated next slide. The widespread occurrence of resistance of certain strains of bacteria to given antibiotics reinforces the need to perform susceptibility testing. In outpatient practice, the choice of antimicrobial agents is more commonly made empirically. 14 Looking down upon a Petri dish containing solidified nutrient agar to which had been added a suspension of a bacterial species. Next, six filter paper discs containing six different antimicrobials were added followed by overnight incubation. The antimicrobials in discs 1, 4, and 5 were inactive. Of the active agents in discs 2, 3, and 6, antibiotic 2 was much more active, as the microorganism was not able to grow as near this impregnated disc. 15 Bactericidal Versus Bacteriostatic. Almost all antibiotics have the capacity to be bactericidal in vitro; that is, they will kill bacteria if the concentration or dose is sufficiently high. In the laboratory, it is almost always possible to use such doses. Subsequent inoculation of fresh, antibiotic-free media with a culture that has been so treated will not produce growth of the culture because the cells are dead. When such doses are achievable in live patients, such drugs are clinically bactericidal. At somewhat lower concentrations, bacterial multiplication is prevented even though the microorganism remains viable (bacteriostatic action). The smallest concentration that will kill a bacterial colony is the minimum bactericidal concentration. 16 With gentamicin, doubling or quadrupling the dose changes the effect on bacteria from bacteriostatic to bactericidal. Such doses are usually achievable in the clinic, so gentamicin is termed bactericidal. The difference between bactericidal and bacteriostatic doses with tetracycline is approximately 40-fold, and it is not possible to achieve such doses safely in patients, so tetracycline is referred to as bacteriostatic. If a bacteriostatic antibiotic is withdrawn prematurely from a patient, the microorganism can resume growth, and the infection can reestablish itself because the organism is still viable. 17 When a patient is immunocompetent or the infection is not severe, a bacteriostatic concentration will break the fulminating stage of the infection (when bacterial cell numbers are increasing at a logarithmic rate). In immunocompromised patients who are unable to contribute natural defenses to fight their own disease, having the drug kill the bacteria is more important for recovery. Thus, although it is preferred that an antibiotic be bactericidal, bacteriostatic antibiotics are widely used and are usually satisfactory. 18 Microbial Susceptibility Resistance. Resistance is the failure of microorganisms to be killed or inhibited by antimicrobial treatment. Resistance can either be intrinsic (be present before exposure to drug) or acquired (develop subsequent to exposure to a drug). Resistance of bacteria to the toxic effects of antimicrobial agents and to antibiotics develops fairly easily both in the laboratory and in the clinic and is an ever-increasing public health hazard. In the laboratory, resistance is almost always found to be due to an alteration in the biochemistry of the colony so that the molecular target of the antibiotic has become less sensitive, or it can be due to decreased uptake of antibiotic into the cells. This is genomically preserved and passes to the next generation. 19 The altered progeny may be weaker than the wild strain so that they die out if the antibiotic is not present to give them a competitive advantage. In some cases, additional compensatory mutations can occur that restore the vigor of the resistant organisms. Resistance of this type is usually expressed toward other antibiotics with the same mode of action and thus is a familial characteristic. Most tetracyclines show extensive crossresistance with other agents in the tetracycline family. In the clinic, resistance more commonly takes place by resistance (R) factor mechanisms. In this case, enzymes are elaborated that attack the antibiotic and inactivate it. Mutations leading to resistance occur by many mechanisms. 20 They can result from point mutations, insertions, deletions, inversions, duplications, and transpositions of segments of genes or by acquisition of foreign DNA from plasmids, bacteriophages, and transposable genetic elements. The genetic material coding for this form of resistance is often carried on extrachromosomal elements consisting of small circular DNA molecules known as plasmids. A bacterial cell may have many plasmids or none. The plasmid may carry DNA for several different enzymes capable of destroying structurally dissimilar antibiotics. Such plasmid DNA may migrate within the cell from plasmid to plasmid or from plasmid to chromosome by a process known as transposition. 21 Such plasmids may migrate from cell to cell by conjugation (passage through a sexual pilus), transduction (carriage by a virus vector), or transformation (uptake of exogenous DNA from the environment). These mechanisms can convert an antibiotic-sensitive cell to an antibiotic-resistant cell. This can take place many times in a bacterium’s already short generation time. The positive selecting pressure of inadequate levels of an antibiotic favors explosive spread of R-factor resistance. This provides a rationale for conservative but aggressive application of appropriate antimicrobial chemotherapy. 22 Bacterial resistance is generally mediated through one of three mechanisms: - failure of the drug to penetrate into or stay in the cell, - destruction of the drug by defensive enzymes, or - alterations in the cellular target of the drug. In many cases, a resistant microorganism can still be controlled by achievable, although higher, doses than are required to control sensitive populations. 23 Persistence Sensitive bacteria may not all be killed. Survivors are thought to have been resting (not metabolizing) during the drug treatment time and are still viable. These bacteria are still sensitive to the drug even though they survived an otherwise toxic dose. Some bacteria also can aggregate in films. A poorly penetrating antibiotic may not reach the cells lying deep within such a film. Such cells, although intrinsically sensitive, may survive antibiotic treatment. Bacteria living in host cells, living in cysts, or existing as an abscess are also harder to reach by drugs and thus are more difficult to control. 24 Combination Therapy A common example is the use of a β-lactam antibiotic and an aminoglycoside for empiric therapy of overwhelming sepsis of unknown etiology. Both of the antibiotic families applied in this example are bactericidal in readily achievable parenteral doses. The β-lactams inhibit bacterial cell wall formation, and the aminoglycosides interfere with protein biosynthesis and membrane function. Their modes of action are supplementary. One may also often successfully combine two bacteriostatic antibiotics for special purposes, for example, a macrolide and a sulfonamide. 25 This combination is occasionally used for the treatment of an upper respiratory tract infection caused by Haemophilus influenzae because the combination of a protein biosynthesis inhibitor and an inhibitor of DNA biosynthesis results in fewer relapses than the use of either agent alone. 26 Serum Protein Binding It is considered in most instances that the percentage of antibiotic that is protein bound is not available at that moment for the treatment of infections so must be subtracted from the total blood level in order to get the effective blood level. The tightness of the binding is also a consideration. A highly bound but readily released antibiotic will have a comparatively short half-life (the time it takes for the amount of a drug's active substance in your body to reduce by half) and work well for systemic infections. An antibiotic that is not significantly protein bound will normally be rapidly excreted and have a short half-life. Thus, some protein binding of poorly water-soluble agents is normally regarded as helpful. 27 Agricultural Use of Antibiotics Their use for treatment of infections of plants and animals is not to be discouraged so long as drug residues from the treatment do not contaminate foods. In contamination, problems such as penicillin allergy or subsequent infection higher up the food chain by drug-resistant microbes can occur. Animals demonstrably grow more rapidly to marketable size when antibiotics are added to their feed even though the animals have no apparent infection. This is believed to be due in large part to suppression of subclinical infections that would consequently divert protein biosynthesis from muscle and tissue growth into proteins needed to combat the infection. 28 Under appropriate conditions, antibiotic feed supplementation is partly responsible for the comparative wholesomeness and cheapness of our food supplies. This practice has the potential, however, to contaminate the food we consume or to provide reservoirs of drug-resistant enteric microorganisms, so it is imperative that antibiotics used for agriculture be utilized appropriately. 29 Sulfonamides The antibacterial properties of the sulfonamides were discovered in the mid-1930s. Prontosil rubrum, a red dye, was one of a series of dyes examined by Gerhard Domagk of Bayer of Germany in the belief that it might be taken up selectively by certain pathogenic bacteria and not by human cells, in a manner analogous to the way that the Gram stain works, and thus serve as a selective poison to kill these cells. The dye, indeed, proved active in vivo against streptococcal infections in mice. Curiously, it was not active in vitro. Trefouel and others soon showed that the urine of Prontosil rubrum–treated animals was bioactive in vitro. 30 Fractionation led to identification of the active substance as paminobenzenesulfonic acid amide (sulfanilamide), a colorless cleavage product formed by reductive liver metabolism of the administered dye. Today, we would call prontosil rubrum a prodrug. 1 4 The discovery of sulfanilamide’s in vivo antibacterial properties ushered in the modern anti-infective era, and Domagk was awarded a Nobel Prize for Medicine in 1939. 31 Mechanism of Action The sulfonamides are bacteriostatic when administered to humans in achievable doses. They inhibit the enzyme dihydropteroate synthase, an important enzyme needed for the biosynthesis of folic acid derivatives and, ultimately, the thymidine required for DNA. They do this by competing at the active site with paminobenzoic acid (PABA), a normal structural component of folic acid derivatives. PABA is otherwise incorporated into the developing tetrahydrofolic acid molecule by enzyme-catalyzed condensation with 6-hydroxymethyl-7,8-dihydropterin- pyrophosphate to form 7,8-dihydropteroate and pyrophosphate. Thus, sulfonamides may also be classified as antimetabolites (see next slide). 32 Microbial biosynthetic pathway leading to tetrahydrofolic acid synthesis and major site of action () of sulfonamides as well as site of action seen in some bacteria (), resulting in incorporation of sulfanamide as a false metabolite. 33 The antimicrobial efficacy of sulfonamides can be reversed by adding significant quantities of p-aminobenzoic acid into the diet (in some multivitamin preparations and as metabolites of certain local anesthetics) or into the culture medium. Most susceptible bacteria are unable to take up preformed folic acid from their environment and convert it to a tetrahydrofolic acid but, instead, synthesize their own folates de novo. Folates are essential intermediates for the biosynthesis of thymidine without which bacteria cannot multiply. Thus, inhibition of the dihydropteroate synthase is bacteriostatic. Humans are unable to synthesize folates from component parts, lacking the necessary enzymes (including dihydropteroate synthase), and folic acid is supplied to humans in our diet. 34 Sulfonamides consequently have no similarly lethal effect on human cell growth, and the basis for the selective toxicity of sulfonamides is clear. In a few strains of bacteria, sulfonamides are attached to the dihydropteroate diphosphate in place of the p-aminobenzoic acid. The resulting unnatural product is not capable of undergoing the next necessary reaction, condensation with glutamic acid. This false metabolite is also an enzyme inhibitor, and the net result is inability of the bacteria to multiply when the folic acid in their cells is used up, and further nucleic acid biosynthesis becomes impossible. The net result is the same, but the molecular basis of the effect is somewhat different in these strains. Bacteria that are able to take up preformed folic acid into their cells are intrinsically resistant to sulfonamides. 35 Structure-Activity Relationships The basis of the structural resemblance of sulfonamides to with paminobenzoic acid (PABA) is clear. The functional group that differs in the two molecules is the carboxyl of PABA and the sulfonamide moiety of sulfanilamide. The strongly electron-withdrawing character of the aromatic SO2 group makes the N atom to which it is directly attached partially electropositive. This, in turn, increases the acidity of the H atoms attached to the N so that this functional group is slightly acidic (pKa = 10.4). The pKa of the carboxyl group of PABA is approximately 4.9. 36 It was soon found that replacement of one of the NH2 hydrogens by an electron-withdrawing heteroaromatic ring enhanced the acidity of the remaining H and dramatically enhanced potency. With suitable groups in place, the pKa is reduced to the same range as that of with p-aminobenzoic acid itself. Not only did this markedly increase the antibacterial potency of the product, but it also dramatically increased the water solubility under physiologic conditions. 37 The pKa of sulfisoxazole, one of the sulfonamides in present use, is approximately 5.0. The poor water solubility of the earliest sulfonamides led to occasional crystallization in the urine (crystalluria) and resulted in kidney damage because the molecules were un-ionized at urine pH values. 38 It is still recommended to drink increased quantities of water to avoid crystalluria when taking certain sulfonamides, but this form of toxicity is now comparatively uncommon with the more important agents used today because they form sodium salts that are at least partly ionized and hence reasonably water soluble at urinary pH values. They are poorly tolerated on injection, however, because these salts are corrosive to tissues. Structural variation among the clinically useful sulfonamides is restricted primarily to installation of various heterocyclic aromatic substituents on the sulfonamide N. 39 Pharmacokinetics. The orally administered sulfonamides are well absorbed from the gastrointestinal tract, distributed fairly widely, and excreted by the kidney. The drugs are bound to plasma protein (sulfisoxazole 30%-70%, sulfamethoxazole 70%) and, as such, may displace other protein-bound drugs as well as bilirubin. The latter phenomenon disqualifies them for use in late-term pregnancy because they can cause neonatal jaundice. Sulfonamides are partly deactivated by acetylation at N-4 and glucuronidation at N-1 in the liver. Plasmid-mediated resistance development is common, particularly among gram-negative microorganisms and usually takes the form of decreased sensitivity of dihydropteroate synthase or increased production of p-aminobenzoic acid. 40 Therapeutic Applications. Of the thousands of sulfonamides that have been evaluated, only a few are still available and are often used in combination with other agents. The surviving sulfonamides (Table 29.1 next slide) include sulfisoxazole, which is used in combination with erythromycin. It has a comparatively broad antimicrobial spectrum in vitro, especially against gramnegative organisms, but clinical use is generally restricted due to the development of bacterial resistance. Susceptible organisms may include enterobacteriaceae (E. coli, Klebsiella, and Proteus) and Streptococcus pyogenes, Streptococcus pneumoniae, and Haemophilus. Sulfamethoxazole in combination with trimethoprim (antimicrobial agent) is more commonly seen. 41 42 Adverse Effects. - Allergic reactions are the most common (rash, photosensitivity, and drug fever). - Less common problems are kidney and liver damage, hemolytic anemia, and other blood problems. - The most serious adverse effect is the Stevens-Johnson syndrome characterized by sometimes fatal erythema multiforme and ulceration of mucous membranes of the eye, mouth, and urethra. These effects are comparatively rare. 43 Antibiotics: Inhibitors of Bacterial Cell Wall Biosynthesis The Bacterial Cell Wall Bacterial cells are enclosed within a complex and largely rigid cell wall. The main functions of the bacterial cell wall are: (1) to provide a semipermeable barrier interfacing with the environment through which only desirable substances may pass; (2) to provide a sufficiently strong barrier so that the bacterial cell is protected from changes in the osmotic pressure of its environment; and (3) to prevent digestion by host enzymes. 44 The initial units of the cell wall are constructed within the cell, but soon the growing and increasingly complex structure must be extruded. Final assembly takes place outside of the inner membrane. This circumstance makes the enzymes involved in late steps more vulnerable to inhibition because they are at or near the cell surface. 45 Gram-Positive Bacteria. Schematic of some features of the gram-positive bacterial cell wall. 46 On the very outside of the cell is a set of characteristic carbohydrates and proteins that together make up the antigenic determinants that differ from species to species and that also cause adherence to particular target cells. There may also be a lipid-rich capsule surrounding the cell (not shown in the diagram). The next barrier that the wall presents is the peptidoglycan layer. This is a spongy, gel-forming layer consisting of a series of alternating sugars (N-acetylglucosamine and N-acetylmuramic acid) linked (1,4)-β in a long chain (see next slide). To the lactic acid carboxyl moieties of the N-acetylmuramic acid units is attached, through an amide linkage, a series of amino acids of which l-alanyl-d-glutamyl-l-lysyl-d-alanine is typical of Staphylococcus aureus. One notes the d-stereochemistry of the glutamate and the terminal alanine. This feature is presumably important in protecting the peptidoglycan from hydrolysis by host peptidases, particularly in the gastrointestinal tract. 47 Schematic of cell wall cross-linking. Pentaglycyl group replaces terminal D-alanine. 48 The peptidoglycan layer is traversed by complex glycophospholipids called teichoic and teichuronic acids. These are largely responsible for the acid mantle of gram-positive bacteria. Beneath the peptidoglycan layer is the lipoidal cytoplasmic cell membrane in which a number of important protein molecules float in a lipid bilayer. Among these proteins are the βlactam targets, the penicillin binding proteins. These are enzymes that are important in cell wall formation and remodeling. In gram-positive bacteria, the outer layers are relatively ineffective in keeping antibiotics out. 49 The inner membrane and its protein components provide the principal barrier to uptake of antibiotics. The penicillin binding proteins are important in construction and repair of the cell wall. βLactam antibiotics bind to these proteins and kill bacteria by preventing the biosynthesis of a functional cell wall. Various β-lactam antibiotics display different patterns of binding to the penicillin binding proteins. These proteins must alternate in a controlled and systematic way between their active and inert states so that bacterial cells can grow and multiply in an orderly manner. Selective interference by β-lactam antibiotics with their functioning prevents normal growth and repair and creates serious problems for bacteria, particularly young cells needing to grow and mature cells needing to repair damage or to divide. 50 Gram-Negative Bacteria. Schematic of some features of the gram-negative bacterial cell wall. 51 These cells usually contain an additional outer lipid membrane that differs considerably from the inner membrane. The outer layer contains complex lipopolysaccharides that encode antigenic responses, cause septic shock, provide the serotype, and influence morphology. This exterior layer also contains a number of enzymes and exclusionary proteins. Important among these are the porins. These are transmembranal supermolecules made up of two or three monomeric proteins. The center of this array is a transmembranal pore of various dimensions. Some allow many kinds of small molecules to pass, and others contain specific receptors that allow only certain molecules to come in. The size, shape, and lipophilicity of drugs are important considerations controlling porin passage. 52 Antibiotics have greater difficulty in penetrating into gram-negative bacterial cells as a consequence. Next comes a periplasmic space containing a somewhat less impressive and thinner, as compared to gram-positive organisms, layer of peptidoglycan. Also present is a phospholipid-rich cytoplasmic membrane in which floats a series of characteristic proteins with various functions. The β-lactam targets (penicillin binding proteins) are found here. Other inner membrane proteins are involved in transport, energy, and biosynthesis. In many such cells, there are proteins that actively pump out antibiotics and other substances at the expense of energy and that may require the simultaneous entrance of oppositely charged materials to maintain an electrostatic balance. 53 β-Lactam Antibiotics A β-lactam is a cyclic amide with four atoms in its ring. The contemporary name for this ring system is azetidinone. This ring proved to be the main component of the pharmacophore, so the term possesses medicinal as well as chemical significance. The penicillin subclass of β-lactam antibiotics is characterized by the presence of a substituted five-membered thiazolidine ring fused to the β-lactam ring. 54 This fusion and the chirality of the β-lactam ring result in the molecule roughly possessing a “V” shape. This drastically interferes with the planarity of the lactam bond and inhibits resonance of the lactam nitrogen with its carbonyl group. Consequently, the β-lactam ring is much more reactive and thus more sensitive to nucleophilic attack when compared with normal planar amides. The earliest penicillins were produced by fungi from media constituents. 55 The cephalosporins were discovered as secondary metabolites of a different fungal species. Because it was stable to many activity-destroying β-lactamases, its core nucleus, 7- aminocephalosporanic acid was substituted with a wide variety of unnatural side chains, and three generations of clinically useful analogues have resulted. Later work produced the carbapenems, monobactams, and β-lactamase inhibitors. Many thousands of these compounds have been prepared by partial or total chemical synthesis, and a significant number of these remain on the market many years after their discovery. 56 Penicillins. There are 3 asymmetric centers. This absolute stereochemistry must be preserved for useful antibiotic activity. 57 Preparation of Penicillins. The original fermentation-derived penicillins were produced by growth of the fungus Penicillium chrysogenum on complex solid media with the result that they were mixtures differing from one another in the identity of the side chain moiety. When a sufficient supply of phenylacetic acid is present in liquid media, this is preferentially incorporated into the molecule to produce mainly benzylpenicillin (penicillin G in the old nomenclature). Use of phenoxyacetic acid instead leads to phenoxymethylpenicillin (penicillin V). More than two dozen different penicillins have been made in this way, but these two are the only ones that remain in clinical use. 58 The sodium and potassium salts of penicillins are crystalline, hydroscopic, and water soluble. They can be employed orally or parentally. When dry, they are stable for long periods, but they hydrolyze rapidly when in solution. Their best stability is noted at pH values between 5.5 and 8, especially at pH 6.0-7.2. The procaine and benzathine salts of benzylpenicillin, on the other hand, are water insoluble. Because they dissolve slowly, they are used for repository purposes following injection when long-term blood levels are required. 59 Nomenclature. Ring and numbering systems of clinically available β-lactam antibiotic types. 60 Chemical Instabilities. The most unstable bond in the penicillin molecule is the highly strained and reactive β-lactam amide bond. This bond cleaves moderately slowly in water unless heated, but breaks down much more rapidly in alkaline solutions to produce penicilloic acid, which readily decarboxylates to produce penilloic acid (see next slide). Penicilloic acid has a negligible tendency to reclose to the corresponding penicillin, so this reaction is essentially irreversible under physiologic conditions. Because the β-lactam ring is an essential portion of the pharmacophore, its hydrolysis deactivates the antibiotic. A fairly significant degree of hydrolysis also takes place in the liver. The bacterial enzyme, βlactamase, catalyzes this reaction also and is a principal cause of bacterial resistance in the clinic. 61 Instability of β-lactams to nucleophiles. 62 Alcohols and amines bring about the same cleavage reaction, but the products are the corresponding esters and amides. These products are inactive. A reaction with a specific primary amino group of aminoglycoside antibiotics is of clinical relevance as it inactivates penicillins and cephalosporins. When proteins serve as the nucleophiles in this reaction, the antigenic conjugates that cause many penicillin allergies are produced. Small molecules that are not inherently antigenic but react with proteins to produce antigens in this manner are called haptens. 63 Commercially available penicillin salts may be contaminated with small amounts of these antigenic penicilloyl proteins derived from reaction with proteins encountered in their fermentative production or by high-molecular-weight self-condensation–derived polymers resulting when penicillins are concentrated and react with themselves. Both of these classes of impurities are antigenic and may sensitize some patients. In acidic solutions, the hydrolysis of penicillins is complex. The main end products of the acidic degradation are penicillamine, penilloic acid, and penilloaldehyde (see next slide). 64 Instability of penicillins in acid. Hydrolysis involves the C-6 side chain. 65 The intermediate penicillenic acid is highly unstable and undergoes subsequent hydrolysis to the corresponding penicilloic acid. An alternate pathway involves sulfur ejection to a product that in turn fragments to liberate penicilloic acid also. Penicilloic acid readily decarboxylates to penilloic acid. The latter hydrolyzes to produce penilloaldehyde and penicillamine. Several related fragmentations to a variety of other products take place. None of these products has antibacterial activity. At gastric pH (∼2.0) and a temperature of 37°C, benzylpenicillin has a half-life measured in minutes. The less water-soluble amine salts are more stable. 66 Structure-Activity Relationship. The substitution of a side chain R group on the primary amine of 6-aminopenicillanic acid with an electron-withdrawing group decreases the electron density on the side chain carbonyl and protects these penicillins in part from acid degradation. This property has clinical implications because these compounds survive passage through the stomach better and many can be given orally for systemic purposes. The survival of passage and degree of absorption under fasting conditions are shown in next slide. 67 68 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 commonly made from inert materials in part to minimize such problems. 69