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Enzymes involved in tetrahydrofolate metabolism are of particular pharmaceutical interest, as their function is crucial for amino acid and DNA biosynthesis. The crystal structure of the human cytosolic methylenetetrahydrofolate dehydrogenase/cyclohydrolase (DC301) domain of a trifunctional enzyme has been determined previously with a bound NADP cofactor. While the substrate binding site was identified to be localized in a deep and rather hydrophobic cleft at the interface between two protein domains, the unambiguous assignment of catalytic residues was not possible. We succeeded in determining the crystal structures of three ternary DC301/NADP/inhibitor complexes. Investigation of these structures followed by site-directed mutagenesis studies allowed identification of the amino acids involved in catalysis by both enzyme activities. The inhibitors bind close to Lys56 and Tyr52, residues of a strictly conserved motif for active sites in dehydrogenases. While Lys56 is in a good position for chemical interaction with the substrate analogue, Tyr52 was found stacking against the inhibitors' aromatic rings and hence seems to be more important for proper positioning of the ligand than for catalysis. Also, Ser49 and/or Cys147 were found to possibly act as an activator for water in the cyclohydrolase step. These and the other residues (Gln100 and Asp125), with which contacts are made, are strictly conserved in THF dehydrogenases. On the basis of structural and mutagenesis data, we propose a reaction mechanism for both activities, the dehydrogenase and the cyclohydrolase.

Analogs of the antitumor agents cryptophycins 1 and 8 with dialkyl substitution at C-6 (fragment C) were synthesized and evaluated for in vitro cytotoxicity against human leukemia cells (CCRF-CEM). The activity of these analogs decreased as the size of the substituents at C-6 increased. The C-6 spirocylopropyl compound (2g) was highly potent in vitro and showed excellent antitumor activity in animal models.

5,6,7,8-Tetrahydro-N5,N10-carbonylfolic acid (LY354899) has been demonstrated to inhibit the dehydrogenase activity of C1-tetrahydrofolate synthase. This compound was only moderately antiproliferative toward CCRF-CEM lymphocytic leukemia cells in culture, but induced apoptosis after long incubation times. Slightly greater potency was observed in CEM cells adapted to grow in low folate media. Cell cycle alterations induced by LY354899 were unique relative to antifolates that inhibit either the purine or thymidine de novo biosynthetic pathways. Based on the observed changes in DNA content, we hypothesized that inhibition of the dehydrogenase resulted in two temporally distinct events: the first was a purineless-like effect and the second was a thymineless-like effect that resulted in apoptosis. To test this hypothesis, we combined LY354899 with the purine salvage metabolite, hypoxanthine. This combination resulted in an earlier and more dramatic apoptotic response, indicating that the thymineless effect had been potentiated. Biochemical analysis of ribo- and deoxyribonucleoside triphosphates confirmed that inhibition of the dehydrogenase activity initially resulted in decreased pools of deoxypurines and deoxypyrimidines, followed 16 hr later by an increase in deoxyadenosine triphosphate (dATP) and a further decrease in deoxythymidine triphosphate (dTTP). These studies demonstrate that the inhibition of the dehydrogenase activity of C1-tetrahydrofolate synthase may represent a viable target for the development of novel antifolates. The results are discussed in terms of deoxypurine and deoxypyrimidine biosynthesis.

The disposition and metabolism of LY295501 was studied in mice, rats, and monkeys. This novel diaryl sulfonylurea oncolytic agent is structurally related to sulofenur and shows excellent activity in a broad range of mouse antitumor models. The compound is well absorbed, giving plasma concentrations greater than 200 micrograms/ml after oral doses of 30-100 mg/kg, where it appears to be completely bound (> 99.9%) to plasma proteins. The high degree of protein binding may be a factor in its relatively long half-life, which ranges from about 8 hr in rats and 15 hr in mice to 50 hr in monkeys. While more material was excreted in feces than in urine from mice and rats given single oral doses of [14C]LY295501, urine was the major route of elimination in monkeys. Three major metabolites-all formed via oxidation of the saturated part of the benzodihydrofuran moiety-were characterized in the urine of mice, rats, and monkeys. It is interesting that two of these metabolites are derived from opening of this saturated ring, an unusual metabolic process which represents a significant part of the metabolism of LY295501. As with sulofenur, metabolites of 3,4-dichloroaniline formed after metabolic cleavage of the sulfonylurea linkage were also found in urine. Unlike sulofenur, these do not seem to have major toxicological significance, but their formation does explain the minor methemoglobinemia observed in toxicology studies of LY295501. Even though only trace amounts of LY295501 were found in urine, LY295501 is the predominant drug-related material in plasma, along with small amounts of other, relatively nonpolar, metabolites.

Several diarylsulfonylureas (DSU), including Sulofenur (LY186641) and LY181984, have been described that exhibit wide spectrum and high therapeutic activity against murine solid tumors and human tumor xenografts. The mechanism for antitumor activity is poorly understood. Moreover, in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity for DSU. Since DSU are extensively bound to serum albumin (> 99%), we sought to determine the effect of albumin on tumor cytotoxicity. We adapted human CCRF-CEM leukemia and GC3 colon carcinoma cells for growth in UltraCHO serum- and albumin-free medium. In comparisons between normal growth medium (RPMI-1640 with 10% fetal bovine serum) and UltraCHO medium, the unbound fraction of drug correlated better with cytotoxic activity than did the total drug. Tumor cytotoxicity by DSU required > 24 h and was markedly enhanced in UltraCHO medium. For example, LY181984 and Sulofenur had IC50 values of 7.4 and 12.1 micrograms/ml against CCRF-CEM in normal growth medium and 0.6 and 0.2 microgram/ml in UltraCHO. Moreover, DSU with the lowest IC-50s in albumin-free medium displayed the most potent in vivo antitumor activity in the 6C3HED lymphosarcoma. A Sulofenur-resistant CCRF-CEM cell line was developed by culturing the cells for > 20 passages in UltraCHO medium containing LY186641 at 2 micrograms/ml (10X IC-50). This line showed approximately 18-fold resistance to LY186641, but did not show cross-resistance to vinblastine, actinomycin D, or doxorubicin. The albumin-free conditions may be useful for further mechanistic studies on the antitumor action by DSU. Further studies are underway to determine whether DSU structural requirements for cytotoxicity an albumin binding are intrinsically linked.

Human pancreatic carcinoma xenograft models were developed from established MIA PaCa-2 and PANC-1 cell lines (ATCC, Rockville, MD). Tumors were maintained by serial trocar implantation in CD1 nu/nu mice, and attempts were made to test all drugs under optimal schedules at maximum tolerated doses. In both models, adriamycin, cisplatin, and 5-fluorouracil were inactive (< 60% inhibition of tumor weight), whereas gemcitabine (LY188011] produced modest activity (69% inhibition in MIA PaCa-2 and 76% inhibition in PANC-1. Major differences in tumor sensitivity were noted with diarylsulfonylureas (DSU) and taxol. The DSU (Sulofenur [LY186641] and LY295501) produced complete inhibition in the MIA PaCa-2 xenograft, but were inactive in PANC-1. Conversely, taxol produced 80% inhibition of PANC-1 tumor growth, but was inactive against MIA PaCa-2. In general, in vivo antitumor activity roughly correlated with in vitro tumor cytotoxicity with the exception of DSU. We have previously shown that DSU are extensively bound to albumin and that in vitro cytotoxic activity in serum-containing medium is not predictive of in vivo antitumor activity. The MIA PaCa-2 and PANC-1 xenograft models may be useful for selecting potential candidates for therapy of human pancreatic cancer.

Diarylsulfonylureas, such as N-(4-chlorophenyl)aminocarbonyl-2,3-dihydro-1-indene-5-sulfonamide (LY186641, Sulofenur) and N-(4-chlorophenyl)aminocarbonyl-4-methylbenzene sulfonamide (LY181984), have been shown to be effective antitumor agents in a variety of in vivo and in vitro animal models. Their mechanism of action is unknown but does not appear to be the result of nonselective destruction of actively dividing cell populations. Mitochondria have been shown to accumulate Sulofenur and therefore may be targets of drug action. The purpose of these investigations was to examine the effects of a variety of diarylsulfonylureas in mitochondria and attempt to determine the relevance of these changes to antitumor activity. Many of the diarylsulfonylureas which were effective antitumor agents in animal models were also uncouplers of mitochondrial oxidative phosphorylation. They increased state 4 respiration and dissipated the mitochondrial membrane potential in a concentration-related fashion. The mechanism of uncoupling appeared to be related to a dissociable hydrogen ion as these molecules had pKa values that ranged from 6.0 to 6.2 and were highly lipophilic. Thus, the uncoupling action appears to be the result of hydrogen ion translocation. The mechanism of antitumor activity does not appear to be the result of uncoupling as no correlation was evident between inhibition of cell growth and uncoupling action of a variety of active and inactive diarylsulfonylureas. In vitro, Sulofenur is cytotoxic at high concentrations and inhibits cell growth at lower concentrations in the absence of any overt cell kill. The inhibition of cell growth also did not appear to be related to the uncoupling action of these drugs. In contrast, uncoupling may have played a partial role in the early, high exposure cell kill that can occur with these compounds.

The metabolic formation of p-chloroaniline from the oncolytic agent sulofenur [N-(5-indanesulfonyl)-N'-(4-chlorophenyl)urea, LY186641,] and from similar diaryl-substituted sulfonylureas, and its possible relevance to the compound's toxicity, was studied. In previous studies it was found that significant amounts of metabolites such as 2-amino-5-chlorophenyl sulfate (II), which is also a metabolite of p-chloroaniline, are formed from sulofenur in mice, rats, monkeys, and humans. The metabolism of N-(4-tolyl)-N'-(2-hydroxy-4-chlorophenyl)-urea (V) was studied, and V was not found to be an intermediate in the metabolic formation of II from the sulfonylurea N-(4-tolyl)-N'-(4-chlorophenyl)urea (LY181984, III). The amounts of this p-chloroaniline metabolite (II) formed in C3H mice from a series of diarylsulfonylureas were found to correlate with the compound's propensities to form methemoglobin, one notable toxicity of p-chloroaniline. This metabolism was also found to correlate with the structure of the arylsulfonyl moiety of the sulfonylurea. Other evidence supports the hypothesis that p-chloroaniline is directly formed by metabolism of sulfofenur and similar diarylsulfonylureas as well. Metabolic formation of p-chloroaniline thus appears to be a plausible explanation for the methemoglobinemia and anemia found to be dose-limiting toxicities of sulofenur in Phase I trials.

Forty-five aminoglycoside antibiotics and related compounds were compared for their ability to induce the accumulation of dihydrostreptomycin in Escherichia coli K12. The common aminoglycosides and a streptothricin antibiotic all induced enhanced uptake within a relatively narrow concentration range. These concentrations were lethal to the bacteria. Comparison of aminoacyl derivatives of tobramycin and apramycin, the latter synthesized utilizing transition-metal cations to selectively control the site of substitution, revealed that 1-N-aminoacyl modifications resulted in an increased ability to induce enhanced uptake. 2'-N-Aminoacyl modifications were also effective at inducing enhanced uptake, albeit without noticeable improvement over parent. The findings from this structure-activity comparison support the proposition that aminoglycosides share a common critical target (most likely the ribosome), which, when acted upon, results in both drug accumulation and killing.

A rigid dopamine congener, 2,3-dihydroxy-9-amino-9,10-dihydrophenanthrene, was synthesized and tested for the ability to dilate the renal artery in dogs. The compound was found to be inactive in this assay at molar doses 1000 times greater than required for dopamine. This result is similar to isoapomorphine. The title compound was also examined for its ability to stimulate rat striatal adenylate cyclase. In concentrations of 10-100 micrometer no effect was observed. The phenanthrene also showed no ability to block dopamine-induced stimulation of adenylate cyclase.

Modification of the aldehyde group in tylosin and related macrolide antibiotics dramatically enhanced the oral efficacy of the derivatives against experimental infections caused by susceptible bacteria in laboratory animals. A large number and wide variety of aldehyde-modified macrolide derivatives were prepared, utilizing the Mitsunobu reaction and other chemical transformations. Evaluation of in vitro and in vivo antimicrobial activity indicated that derivatives of demycarosyltylosin (desmycosin) combined the broadest spectrum of antimicrobial activity with the best efficacy and bioavailability after oral administration.