P -Lactam Antibiotics

By 1938, Fleming had moved on to other research, and Howard Florey and Ernst Chain of the School of Pathology at Oxford were just beginning a program aimed at developing antibacterial agents from natural sources. A candidate that especially appealed to them was Fleming s penicillin. 

Their most daunting initial problem was making enough penicillin. Enter Norman Heatley, of whom it has been said .. . without Heatley, no penicillin. Heatley, an inventive and careful experimentalist, devised procedures to make and isolate penicillin on a scale sufficient to begin testing. By 1941, Florey, Chain, and Heatley had a drug that was both effective and safe. 

England was at war, and the United States soon would be the need for large amounts of penicillin was obvious. Working with U.S. Department of Agriculture, Heatley and Andrew J. Moyer of the USDA laboratories in Peoria, Illinois, found better penicillium sources and developed novel fermentation methods to produce ever-increasing amounts of penicillin. Treatment of wounded soldiers with penicillin became possible early in 1943 and was widely practiced before the war ended in August 1945. Four 

Penicillin G originated in a mold Penicillium chrysogenum) found on a cantaloupe in Peoria, Illinois. 

Chapter 19 Carboxylic Acid Derivatives Nucleophilic Acyl Substitution 

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Another example is the purification of a P-lactam antibiotic, where process-scale reversed-phase separations began to be used around 1983 when suitable, high pressure process-scale equipment became available. A reversed-phase microparticulate (55—105 p.m particle size) C g siUca column, with a mobile phase of aqueous methanol having 0.1 Af ammonium phosphate at pH 5.3, was able to fractionate out impurities not readily removed by hquid—hquid extraction (37). Optimization of the separation resulted in recovery of product at 93% purity and 95% yield. This type of separation differs markedly from protein purification in feed concentration ( i 50 200 g/L for cefonicid vs 1 to 10 g/L for protein), molecular weight of impurities (<5000 compared to 10,000—100,000 for proteins), and throughputs ( i l-2 mg/(g stationary phasemin) compared to 0.01—0.1 mg/(gmin) for proteins). 

Unit sales prices of from 800 to 900 fine chemicals are fisted weekly in the Chemical Marketing Reporter. This number reflects those fine chemicals produced and sold in industrial quantities. Some market studies on fine chemicals, fisting important product families, such as side chains for P-lactam antibiotics (qv). A/- and A-heterocycfic compounds, fluoroaromatics, etc, do exist (14,15). 

The number of naturally occurring antibiotics increased from about 30 known in 1945, to 150 in 1949, 450 in 1953, 1200 in 1960, and to 10,000 by 1990 (1,9). Table 1 Hsts the years of historical importance to the development of antibiotics used for treatment in humans. Most of the antibiotics introduced since the 1970s have been derived from synthetic modifications of the P-lactam antibiotics (qv). 

C QHyN O SNa, as a potentially useful P-lactamase inhibitor capable of potentiating the activity of a number of clinically important P-lactam antibiotics against resistant strains (153). 

P-lactam antibiotics, exert thek antibacterial effect by interfering with the synthesis of the bacterial cell wall. These antibiotics tend to be "kreversible" inhibitors of cell wall biosynthesis and they are usually bactericidal at concentrations close to thek bacteriostatic levels. Cephalospotins are widely used for treating bacterial infections. They are highly effective antibiotics and have low toxicity. 

Bacteria produce chromosomady and R-plasmid (resistance factor) mediated P-lactamases. The plasmid-mediated enzymes can cross interspecific and intergeneric boundaries. This transfer of resistance via plasmid transfer between strains and even species has enhanced the problems of P-lactam antibiotic resistance. Many species previously controded by P-lactam antibiotics are now resistant. The chromosomal P-lactamases are species specific, but can be broadly classified by substrate profile, sensitivity to inhibitors, analytical isoelectric focusing, immunological studies, and molecular weight deterrnination. Individual enzymes may inactivate primarily penicillins, cephalosporins, or both, and the substrate specificity predeterrnines the antibiotic resistance of the producing strain. Some P-lactamases are produced only in the presence of the P-lactam antibiotic (inducible) and others are produced continuously (constitutive). 

Fermentation. The commercial P-lactam antibiotics which act as starting material for all of the cephalosporins ate produced by submerged fermentation. The organisms used for the commercial production of the penicillins and cephalosporins ate mutants of PenicU/in chTysogenum and Cephalosporium acremonium respectively (3,153,154). Both ate tme fungi (eucaryotes). In contrast, the cephamycins ate produced by certain species of procaryotic Streptomyces including Streptomyces clavuligerus and Streptomyces lipmanii (21,103). 

The P-lactam antibiotics ate produced by secondary metaboHc reactions that differ from those responsible for the growth and reproduction of the microorganism. In order to enhance antibiotic synthesis, nutrients must be diverted from the primary pathways to the antibiotic biosynthetic sequences. Although most media for the production of penicillins and cephalosporins are similar, they ate individually designed for the specific requkements of the high yielding strains and the fermentation equipment used. 

Isolation. Isolation procedures rely primarily on solubiHty, adsorption, and ionic characteristics of the P-lactam antibiotic to separate it from the large number of other components present in the fermentation mixture. The penicillins ate monobasic catboxyHc acids which lend themselves to solvent extraction techniques (154). Pencillin V, because of its improved acid stabiHty over other penicillins, can be precipitated dkecdy from broth filtrates by addition of dilute sulfuric acid (154,156). The separation process for cephalosporin C is more complex because the amphoteric nature of cephalosporin C precludes dkect extraction into organic solvents. This antibiotic is isolated through the use of a combination of ion-exchange and precipitation procedures (157). The use of neutral, macroporous resins such as XAD-2 or XAD-4, allows for a more rapid elimination of impurities in the initial steps of the isolation (158). The isolation procedure for cephamycin C also involves a series of ion exchange treatments (103). 

The antibacterial effectiveness of penicillins cephalospotins and other P-lactam antibiotics depends upon selective acylation and consequentiy, iaactivation, of transpeptidases involved ia bacterial ceU wall synthesis. This acylating ability is a result of the reactivity of the P-lactam ring (1). Bacteria that are resistant to P-lactam antibiotics often produce enzymes called P-lactamases that inactivate the antibiotics by cataly2ing the hydrolytic opening of the P-lactam ring to give products (2) devoid of antibacterial activity. 

One approach to combating antibiotic resistance caused by P-lactamase is to inhibit the enzyme (see Enzyme inhibition). Effective combinations of enzyme inhibitors with P-lactam antibiotics such as penicillins or cephalosporins, result in a synergistic response, lowering the minimal inhibitory concentration (MIC) by a factor of four or more for each component. However, inhibition of P-lactamases alone is not sufficient. Pharmacokinetics, stability, ability to penetrate bacteria, cost, and other factors are also important in determining whether an inhibitor is suitable for therapeutic use. Almost any class of P-lactam is capable of producing P-lactamase inhibitors. Several reviews have been pubUshed on P-lactamase inhibitors, detection, and properties (8—15). 

Sulbactam (25, R = R = H, R" = R " = dl ) is produced by Pfizer. The oral version of sulbactam in combination with ampicillin is called Unasyn Oral which is the mutual prodmg sultamicillin. Two sulbactam parenteral products are sold, a combination product with ampicillin called Unasyn and a combination with cefoperazone [62893-19-0] called Sulperazon. In addition, sulbactam is sold alone for parenteral use with any P-lactam antibiotic as Betamaze. In 1990 worldwide sales of sulbactam containing products were over 280 million. 

P-Lactam antibiotics exert their antibacterial effects via acylation of a serine residue at the active site of the bacterial transpeptidases. Critical to this mechanism of action is a reactive P-lactam ring having a proximate anionic charge that is necessary for positioning the ring within the substrate binding cleft (24). 

The cost of 6-APA, ampicillin, and amoxicillin is invariably linked to that of penicillin G. 6-APA remains one of the cheapest raw starting materials available for the preparation of semisynthetic penicillins and other related P-lactam antibiotics. 

The total U.S. antibiotic market for 1990 was about 4.73 biUion, 233 million of that was tetracyclines. The development of the semisynthetic P-lactam antibiotics (see Antibiotics, P-LACTAMs) and emergence of resistance to the tetracyclines has steadily diminished the clinical usefulness of tetracyclines. 

In the relatively few years since the preparation of the previous volume in this series, the explosion of synthetic and clinical experimentation on the semi and totally synthetic antibacterial p-lactam antibiotics has continued, providing a rich body of literature from which to assemble this chapter. The search for utopiasporin, the perfect cephalosporin, continues. The improvements in. spectrum and clinical properties achieved to date, however, are largely incremental and have been achieved at the price of substantially higher costs to the patient. Nonetheless, these newer compounds are truly remarkable when compared with the properties of the fermentation-derived substances from which they have sprung. 

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