The DNA alkylation and sequence specificity of a group of natural and synthetic pyrrolo- [1,4]benzodiazepines [P(1,4)Bs] were evaluated by using an exonuclease I11 stop assay, and the results were compared with in vitro and in vivo biological potency and antitumor activity. The P(1,4)B antibiotics are potent antitumor agents produced by various Actinomycetes, which are believed to mediate their cytotoxic effects by covalent bonding through N-2 of guanine in the minor groove of DNA. In this article we describe the results of a sensitive DNA alkylation assay using exonuclease I11 which permits both estimation of the extent of DNA modification as well as location of the precise guanines to which the drugs are covalently bound. Using this assay, we have evaluated a series of natural and synthetic compounds of the P(1,4)B class for their ability to bind to DNA and also determined their DNA sequence preference. The compounds included in this study are P(1,4)Bs carrying different substituents in the aromatic ring, having varying degrees of saturation in the five-membered ring, or differing in the stereochemistry a t C-lla. These same compounds were evaluated for in vitro cytotoxic activity against B16 melanoma cells, for potency in vivo in B6D2F1 mice (LDm), and for antitumor activity (ILS,) against P388 leukemia cells. A good correlation was found between extent of DNA alkylation and in vitro and in vivo potency. Furthermore, on the basis of electronic and steric considerations, it was possible to rationalize why those compounds that showed negligible biological activity were unable to bond covalently to DNA. Last, we have determined that the degree of saturation in the five-membered ring of the P(1,4)Bs has a significant effect on the DNA bonding reactivity and biological activity of this class of compounds.

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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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