An intestinal proton-dependent peptide transporter located on the lumenal surface of the enterocyte is responsible for the uptake of many orally absorbed beta-lactam antibiotics. Both cephalexin and loracarbef are transported by this mechanism into the human intestinal Caco-2 cell line. Forty-seven analogs of the carbacephalosporin loracarbef and the cephalosporin cephalexin were prepared to evaluate the structural features necessary for uptake by this transport carrier. Compounds were evaluated for their antibacterial activities and for their ability to inhibit 1 mM cephalexin uptake and, subsequently, uptake into Caco-2 cells. Three clinically evaluated orally absorbed carbacephems were taken up by Caco-2 cells, consistent with their excellent bioavailability in humans. Although the carrier preferred the L stereoisomer, these compounds lacked antibacterial activity and were hydrolyzed intracellularly in Caco-2 cells. Compounds modified at the 3 position of cephalexin and loracarbef with a cyclopropyl or a trifluoromethyl group inhibited cephalexin uptake. Analogs with lipophilic groups on the primary amine of the side chain inhibited cephalexin uptake, retained activity against gram-positive bacteria but lost activity against gram-negative bacteria. Substitution of the phenylglycl side chain with phenylacetyl side chains gave similar results. Compounds which lacked an aromatic ring in the side chain inhibited cephalexin uptake but lost all antibacterial activity. Thus, the phenylglycl side chain is not absolutely required for uptake. Different structural features are required for antibacterial activity and for being a substrate of the transporter. Competition studies with cephalexin indicate that human intestinal Caco-2 cells may be a useful model system for initially guiding structure-activity relationships for the rational design of new oral agents.

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A new cepham metabolite has been isolated from the filtered broth of Cephalosporium acremonium by high performance liquid chromatography (HPLC) and identified as 7ß-(5-Damino-adipamido)-3ß-hydroxy-3a-methyl-cepham-4a-carboxylic acid (I). Pure penicillin N was prepared using HPLC in the analytical mode. When I was added in place of penicillin N as substrate for the cell-free biosynthetic of cephalosporin, no formation of deacetoxycephalosporin C (II) was observed.A synthetic cepham derivative, 7ß-(5-D-aminoadipamido)-3-exomethylene-cepham-4a-carboxylic acid (III) was also tested in the cell-free system as a possible intermediate. The compound III was shown to be an inhibitor of the ring expansion enzyme that converts penicillin N to deacetoxycephalosporin C.We have shown earlier3) that using HPLC one can isolate metabolites directly from the fermentation broth.Modification of appropriate stationary, and liquid phases, employed in the course of that first investigation, led to the isolation1)of new tripeptides from the broth of P. chrysogenum (Fig. 1).When the same system was applied to the present investigation of the broth of C. acremonium (Fig. 2), a new cepham derivative (I) was obtained.Its structure was suggestive of a possible relationship to deacetoxycephalosporin C (DAC, deacetoxy ceph C) (II) and, therefore, we examined its role in biosynthesis of DAC in a cell-free system derived from C. acremonium.KOHSAKA and DEMAIN4) were first to describe a cell-free system from C. acremonium CW-19 that converted penicillin N into a penicillinase-resistant cephalosporinase-sensitive material. YOSHIDA et al.,5) have shown this compound to be II by paper electrophoresis and paper and TLC chromatography.More recently, in another cell-free system derived from C. acremonium mutant M-0198, this finding was further confirmed6) using the same HPLC system we introduced in the examination of the broth of C. acremonium.3) In all these experiments however impure penicillin N was used as the substrate for the cell-free synthesis of DAC. We are able to prepare essentially pure penicillin N for use in the cellfree experiment.The process of the ring expansion from penicillin N to DAC was followed by injection into the HPLC system of aliquots of 50 microliters of the reaction mixture. The UV absorbance profile at 254 nm of authentic DAC was compared to profiles obtained from aliquots of the reaction mixture observed at 0 time, then at 20-minute intervals after addition of penicillin N to a total of 60 minutes. At that

and eluted with 200 ml of dry benzene. Evaporation of the solvent gave 0.47 g of product.An analytical sample was obtained by rechromatographing the above material on acid-washed alumina (38.0 g). Elution with 100 ml of dry hexane provided pure 19: ir shows no absorptions above 3000 cm-1; nmr (CCh) 3.69 ppm (s,3, OCH3).

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