A number of new parenteral cephalosporin and cephamycin derivatives with improved use potential over the available marketed products are currently under development. These compounds are reported to have a broader spectrum of antibacterial activity and good stability to a large number of f-lactamases produced by bacterial isolates (2,7,9,11,12,14,15). One such compound is SK&F 75073 [7-D-mandelamido-3-(1-sulfomethyltetrazole-5-ylthiomethyl) -3-cephem-4-carboxylic acid, disodium salt] a parenteral cephalosporin with broad antibacterial activity and high and extended serum levels (Fig. 1) Efficacy tests: (i) in vitro activity. The minimum inhibitory concentrations (MICs) were determined by the agar dilution method as previously reported (1).The surface of the agar was inoculated with the aid of a Steers apparatus (13). After overnight incubation at 37°C, the MICs were read as the lowest concentration of antibiotic completely inhibiting growth. For determination of effect of serum on antibiotic activity, pooled human serum was added in a final concentration of 50% to Muelier-Hinton broth. The MIC determinations were carried out using microtiter methodology (6). The MICs for the inoculum size experiments were also carried out in Mueller-Hinton broth using the microtiter apparatus.The test medium employed for the staphylococci and the gram-negative bacterial species was Mueller-

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The synthesis of a series of related broad-spectrum 7-phenylglycyl cephalosporins with 3-heterocyclicthiomethyl substituents is described. The effects of benzene-ring hydroxylation and 3-substituent variation on the in vitro antibacterial activity, height and duration of mouse serum levels, and effectiveness in protecting against bacterial infection in the mouse are examined. Included for comparison are cephalexin, cephaloglycin and their ortho-, meta-and para-hydroxy derivatives. The biological properties examined were influenced by the position of the hydroxyl group and by the nature of the 3-substituent. The 7-(p-hydroxyphenylglycyl)-3-heterocyclicthiomethyl analogs were found to produce significantly higher serum levels on oral administration to mice than their unhydroxylated counterparts. This effect was not observed with the 7-(m-hydroxyphenylglycyl)-3-heterocyclicthiomethyl cephalosporins, nor with the p-hydroxyphenylglycyl analog of cephalexin. While m-and p-hydroxylation had little effect on in vitro activity the o-hydroxyphenylglycyl cephalosporins tested had very low antibacterial activities and were not examined further. One derivative, 7-[R-2-amino-2-(4-hydroxyphenyl)acetamido]-3-(1H-1, 2, 3-triazole-4(5)-ylthiomethyl)-3-cephem-4-carboxylic acid (SK&F 60771) was found to have outstanding in vitro and in vivo activities along with oral and subcutaneous serum levels in the mouse that were significantly higher than those obtained with cephalexin. This derivative which has been given the generic name cefatrizine was selected for extensive additional biological evaluation.The criteria used in selecting a cephalosporin antibiotic as a candidate for clinical use require that it exhibit simultaneously a number of desirable chemotherapeutic and pharmacokinetic characteristics. Three of the characteristics examined in our laboratory for evaluating the potential utility of a cephalosporin are the in vitro activity (MIC) against gram-positive and gram-negative bacteria, the effectiveness in protecting small laboratory animals against bacterial infection (PD5O, mouse) and the serum concentrations attained when the cephalosporin is administered orally or by injection. Frequently these characteristics, and others of importance to the successful use of the cephalosporin, can be altered by modifying the chemical structure; but the alterations in biological properties do not necessarily proceed in a parallel direction. For example, recent studies',') demonstrate that in certain cases in vitro activity can be increased against a number of bacteria by substituting an appropriate heterocyclicthiomethyl group for the methyl or acetoxymethyl group at the 3-position of the cephalosporin nucleus. The efficacy of this structural change is illustrated by cefazolin and cefamandole, two injectable cephalosporins with 3-heterocyclicthiomethyl substituents. Both

The 2-aminothiazol-4-yl-2-alkoxyiminoacetamido substituent-containing i3-lactam antibiotics (cephalosporins and monobactams) develop a stable, concentration-dependent purple or cherry-red color after reaction with sodium nitrite in acidic condition. The color-formation is highly specific; it requires certain defined structural features such as the simultaneous presence of the intact aminothiazole-ring and an alkoxyimino substituent in the syn configuration. Other substituents on the (3-lactam nucleus have effect only on the intensity of the color. This simple and fast colorimetric procedure was found to be useful not only for the detection of this class of (3-lactam antibiotics but also for their quantitative spectrophotometric determination (2me .g 500 nm). A linear relationship exists between the intensity of the color plotted on a logarithmic scale and the concentration (12.5-200 i g/ml) of the compounds on an arithmetic scale. The j3-lactams studied in this class with definitely positive purple-red color reaction are; cefotaxime, ceftizoxime, ceftazidime, ceftriaxone, cefmenoxime, cefodizime, ceftiolene, cefpirome, aztreonam, HR 109, FK 027 (cefixime), FR 19346, SK&F 88070, FR 13300, carumonam, YM 13115, BMY 28142, DN 9550, deacetylcefotaxime, deacetoxycefotaxime and deacetylcefotaxime lactone.Most of the new semisynthetic cephalosporins and the monobactam, aztreonam, belong to the class of compounds containing the 2-aminothiazoleacetamido group. Those which contain a 2-alkoxyimino substituent at the syn-configuration are of considerable therapeutic interest. They have broad antibacterial spectra, high levels of activity especially against Gram-negative bacteria and excellent (3-lactamase stability1). These antibiotics are currently assayed using biological methods.A simple colorimetric procedure was developed based on the structural similarity between these 43-lactams and the sulfonamides, procaine (novocain) and other p-aminobenzoic acid derivatives. It was reasoned that the aromatic amino group of the aminothiazole ring of the (3-lactams similar to that of the sulfanilamides and procaine should form a colored compound after diazotization with sodium nitrite followed by coupling with N-1-naphthylethylenediamine.However, when this reaction was carried out as described in the literature2-7), there was apparently no coupling reaction, as evidenced by the lack of characteristic color. A concentration-dependent purple color did develop, however, when the sodium nitrite-treated solutions of the alkoxyiminoaminothiazolyl cephalosporins were acidified. This observation served as a basis for the development of the colorimetric detection and the spectrophotometric determination of these ,8-lactam antibiotics, as described herewith. Part of these data were previously reported8). Materials and Methods Antibiotics and Other CompoundsThe aminothiazolyl 19-lactam antibiotics and the aminothiazole-containing non-antibiotic control

isolates from patients later treated with cefazolin. Upon receipt, the isolates were classified and tested for response to 12 commercial antibiotics. A single passage of the organism was made, and samples were frozen at -70 C for additional trials.Antibiotic disks. Cephalothin 30-,ug disks were purchased from BBL. The 30-iAg cefazolin disks were prepared in the BBL laboratories under controlled conditions conforming to their commercial production of susceptibility disks. The disks were assayed for stability over a 1-year period in both BBL and our laboratories and found to have no significant loss in potency when stored at 4 C. The assays were performed by the FDA performance plate test method described in the Federal Register for cephalosporin disks (2).Disk susceptibility tests. The procedure employed was essentially that of Bauer and Kirby (1) and later was detailed in the Federal Register (3). Clinical isolates first were grown in Trypticase soy broth, adjusted to a standard optical density, and swabbed on the surface of a petri dish containing Mueller-Hinton agar (BBL). Duplicate disks were placed on the agar surface with sterile forceps. All plates were incubated at 37 C for 18 h.

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The synthesis and in vitro and in vivo activities of a series of cephalosporins having side chains derived from 2-[(2,2,2-trifluoroethyl)thio]acetic acid or 2-(cyanomethylthio)acetic acid and with acetoxymethyl or 3-heterocyclic thiomethyl substituents at the 3 position are described. In both series, increasing the oxidation state of the side-chain sulfur atom from sulfide to sulfoxide/sulfone decreased the in vitro gram-positive activity, but the effect on gram-negative activity was variable and less pronounced. The protective effectiveness in mice infected with Escherichia coli increased as the oxidation level of the side-chain sulfur was raised from sulfied to sulfoxide/sulfone. Replacement of the 3-acetoxymethyl by a 3-heterocyclic thiomethyl group resulted in overall improvement of activity both in vitro and in vivo for all oxidation states.