A basis set of polyamine analogues was designed and synthesized. These compounds were used to initiate a systematic investigation of the role of chain length, terminal nitrogen alkyl group size, and symmetry of the methylene backbone in the antineoplastic properties of polyamine analogues. New synthetic methods predicated on our earlier polyamine fragment synthesis are described for accessing the tetraamines of interest. An unsymmetrically substituted diamine reagent, N-(tert-butoxycarbonyl)-N,N'-bis(mesitylenesulfonyl)-1,4-diaminobu tane, was developed for entry into unsymmetrical tetraamines. All of the tetraamines synthesized were first evaluated in a murine leukemia L1210 cell IC50 assay at 48 and 96 h. In an attempt to correlate this behavior with some aspect of polyamine metabolism, each compound was tested for its ability to compete with spermidine for the polyamine uptake apparatus, its impact on the polyamine biosynthetic enzymes ornithine decarboxylase (ODC) and S-adenosylmethionine decarboxylase (AdoMetDC), and its effect on the polyamine-catabolizing enzyme spermidine/spermine N1-acetyltransferase (SSAT) and on polyamine pools. While there was no obvious correlation between the 48 and 96 h IC50's and the impact of the analogues on polyamine metabolism, there were other structure-activity relationships. Correlations were observed to exist between chain length and IC50's and between terminal alkyl substituents and impact on Ki, ODC, and AdoMetDC. Also, preliminary studies suggest a relationship may exist between the 48 and 96 h IC50 activities and the analogue's chronic toxicity in vivo. Finally, when the overall length of the polyamine backbone was held constant, the symmetry of the methylene chains of the polyamine fragments was shown to be unimportant to the compound's activity.

Additional structure-activity studies of desferrithiocin analogues are carried out. The effects of stereochemistry at C-4 on the ligands' iron clearing efficiency are reviewed and assessed using the enantiomers 4,5-dihydro-2-(2, 4-dihydroxyphenyl)thiazole-4(R)-carboxylic acid and 4,5-dihydro-2-(2, 4-dihydroxyphenyl)thiazole-4(S)-carboxylic acid. The utility of 4'-hydroxylation as a method of reducing the toxicity of desazadesferrithiocin analogues is also examined further with the synthesis and in vivo comparison of 4, 5-dihydro-2-(2-hydroxyphenyl)-4-methylthiazole-4(S)-carboxylic acid, which is the natural product 4-methylaeruginoic acid, and 4, 5-dihydro-2-(2,4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylic acid. The stereochemistry at C-4 is shown to have a substantial effect on the iron clearing efficiency of desferrithiocin analogues, as does C-4'-hydroxylation on the toxicity profile. All of the compounds are evaluated in a bile-duct-cannulated rodent model to determine iron clearance efficiency and are carried forward to the iron-overloaded primate for iron clearing measurements. On the basis of the results of the present work, although 4,5-dihydro-2-(2, 4-dihydroxyphenyl)thiazole-4(S)-carboxylic acid is still the most promising candidate for clinical evaluation, 4,5-dihydro-2-(2, 4-dihydroxyphenyl)-4-methylthiazole-4(S)-carboxylic acid (4'-hydroxydesazadesferrithiocin) also merits further preclinical assessment.

A systematic investigation of the impact of spermidine analogues both in vitro and in vivo is described. The study characterizes the effects of these analogues on L1210 cell growth, polyamine pools, ornithine decarboxylase, S-adenosyl-L-methionine decarboxylase, spermidine/spermine N1-acetyltransferase, the maintenance of cellular charge, i.e., cationic equivalence associated with the polyamines and their analogues, and compares their ability to compete with spermidine for transport. The findings clearly demonstrate that the activity of the linear polyamine analogues is highly dependent on the length of the triamines and the size of the N(alpha),N(omega)-substituents. It appears that there is an optimum chain length for various activities and that the larger the N(alpha),N(omega)-alkyls, the less active the compound. Metabolic transformation including N-dealkylation of these compounds is also evaluated. While there is no monotonic relationship between chain length and the ability of the analogue to be metabolized, the dipropyl triamines are clearly more actively catabolized than the corresponding methyl and ethyl systems. A comparison of the triamines with the corresponding tetraamines is made throughout the text regarding both in vitro activity against L1210 cells and in vivo toxicity measurements, suggesting that several triamine analogues may offer therapeutic advantages over the corresponding tetraamines.

Further structure-activity studies of desferrithiocin analogues are carried out. (S)-Desazadesmethyldesferrithiocin, 2-(2-hydroxyphenyl)-Delta2-thiazoline-4(S)-carboxylic acid, serves as the principal framework in the current paper. Desazadesmethyldesferrithiocin can be structurally altered with facility, and data are already available on its iron-clearing properties and toxicity parameters. Four different kinds of structural modifications of this framework are undertaken: introduction of hydroxy, carboxy, or methoxy groups on the aromatic ring; alteration of the thiazoline ring; increasing the distance between the ligand donor atoms; and benz-fusion of the aromatic rings. The structural modifications described are shown to have a tremendous impact on both the iron clearance and toxicity profiles of the desazadesmethyldesferrithiocin molecule. All of the compounds are assessed in a bile-duct-cannulated rodent model to determine iron clearance efficiency. Ligands which demonstrate an efficiency of greater than 2% are carried forward to the iron-overloaded primate for iron-clearing measurements. Ligands with efficiencies greater than 3% in the primate are then evaluated in a formal toxicity study in rodents. On the basis of the results of the present work, 2-(2, 4-dihydroxyphenyl)-Delta2-thiazoline-4(S)-carboxylic acid is a promising candidate for clinical evaluation.

A series of analogues and homologues of N1,N12-diethylspermine (DESPM) was synthesized, and their biological properties were evaluated. These tetraamines include a simple linear analogue of DESPM, N1,N12-bis(2,2,2-trifluoroethyl)spermine (FDESPM), the cyclic analogues of DESPM, N,N'-bis(4-piperidinylmethyl)-1,4-diaminobutane [PIP(4,4,4)] and N,N'-bis[2-(4-piperidinyl)ethyl]-1,4-diaminobutane [PIP(5,4,5)], and their aromatic counterparts, N,N'-bis-(4-pyridylmethyl)-1,4-diaminobutane [PYR(4,4,4)] and N,N'-bis[2-(4-pyridyl)ethyl]-1,4-diaminobutane [PYR(5,4,5)]. The analogues FDESPM, PIP(4,4,4), and PYR(4,4,4) have distances between their nitrogen atoms almost identical to those of DESPM. The longer analogues PIP(5,4,5) and PYR(5,4,5) are very similar in the spacing of their amino groups. However, the pKa of the nitrogens in the groups differ; thus, the extent of protonation and the charge characteristics among the members of the groups differ. A comparison of the biological properties of these compounds clearly demonstrates that the tetraamines must be charged to be "recognized" by the cell. Analogues with low nitrogen pKa's such that the nitrogens are poorly protonated at physiological pH do not compete well with spermidine for uptake and, as expected, have high 96 h IC50 values and have little effect on S-adenosylmethionine decarboxylase, ornithine decarboxylase, and spermidine/spermine N1-acetyltransferase activities and on intracellular polyamine pools.

The impact of altering the octanol-water partition properties (log P) of analogues of desazadesferrithiocin, (S)-4,5-dihydro-2-(2-hydroxyphenyl)-4-methyl-4-thiazolecarboxylic acid, on the ligands' iron clearing properties is described. Increasing chelator lipophilicity can both substantially augment iron clearing efficiency in Cebus apella primates as well as alter the mode of iron excretion, favoring fecal over urinary output. The complications of iron overload are often associated with the metal's interaction with hydrogen peroxide, generating hydroxyl radicals (Fenton chemistry) and, ultimately, other related deleterious species. In fact, some iron chelators actually promote this chemistry. All of the compounds synthesized and tested in the current study are shown to be both inhibitors of the iron-mediated oxidation of ascorbate, thus removing the metal from the Fenton cycle, and effective radical scavengers.

The design, synthesis, and testing of a novel class of antidiarrheal drugs based on a tetraamine pharmacophore are reported. While N1,N14-diethylhomospermine (DEHSPM) (5 mg/kg) completely prevents diarrhea in rodents, tissue distribution studies demonstrated that the principal metabolite of DEHSPM, homospermine (HSPM), accumulates and persists in tissues for a protracted period of time. This accumulation accounts for a large part of the chronic toxicity of DEHSPM. Thus a major objective was to develop a metabolically labile analogue of DEHSPM which retained the desirable biological properties of the parent drug. Hydroxyl groups, sites vulnerable to further metabolic transformation, were introduced into the external aminobutyl segments providing N1,N14-diethyl-(3R),(12R)-dihydroxyhomospermine [(HO)2-DEHSPM]. The design concept was assisted by molecular modeling, which predicted that (HO)2DEHSPM would have a Ki for polyamine transport essentially identical with that of DEHSPM. The experimentally measured Ki and also the observed values of other biological properties of (HO)2DEHSPM were in fact identical with those of DEHSPM, including IC50 against L1210 cells, impact on the NMDA receptor, and impact on L1210 native polyamine pools. Most significantly, however, there was no accumulation of the dideethylated metabolite in tissues from mice treated chronically with (HO)2DEHSPM, and (HO)2DEHSPM was 3-fold less toxic than DEHSPM. Finally, (HO)2DEHSPM completely prevented diarrhea in the castor oil-treated rat model at a dose of 5 mg/kg, just as did DEHSPM.

Basic solutions of tetrapeptides derived from L-aspartic acid diketopiperazines are shown to form microcapsules when acidified to pH 2.4. An initial structure-activity study clearly demonstrates that a very delicate balance exists between the tetrapeptides' structure and their ability to self-assemble. Scanning electron micrographs confirm that microcapsules and not solid microspheres are formed.