The synthesis of four hydroxylated polyamine analogues, (2R, 10R)-N(1),N(11)-diethyl-2,10-dihydroxynorspermine, (2S,10S)-N(1), N(11)-diethyl-2,10-dihydroxynorspermine, (3S,12S)-N(1), N(14)-diethyl-3,12-dihydroxyhomospermine, and (3R,12R)-N(1), N(14)-diethyl-3,12-dihydroxyhomospermine, is described along with their impact on the growth and polyamine metabolism of L1210 murine leukemia cells. Four different synthetic approaches are set forth, two each for the hydroxylated norspermines and for the hydroxylated homospermines. The key step in the assembly of the norspermines was the coupling of either N-[(2R)-2,3-epoxypropyl]-N-ethyl p-toluenesulfonamide or N-[(2S)-2,3-epoxypropyl]-N-ethyl trifluoromethanesulfonamide to N,N'-dibenzyl-1,3-diaminopropane. The key step with homospermines employed alkylation of putrescine with (3S)-N-(benzyloxycarbonyl)-N-ethyl-3,4-epoxybutylamine or of N, N'-bis(mesitylenesulfonyl)-1,4-butanediamine with (2R)-2-benzyloxy-4-[N-(mesitylenesulfonyl)ethylamino]-O-tosyl-1-++ +butan ol. All of the hydroxylated analogues were active against L1210 cells with 96-h IC(50) values of =2 microM, and they also effectively reduced putrescine and spermidine, although the effect on spermine pools ranged from moderate to insignificant. Interestingly, the impact of the hydroxylated analogues on ornithine decarboxylase (ODC) was significantly less than that of unhydroxylated parent drug (e.g., N(1),N(11)-diethylnorspermine [DENSPM]) at 1 microM; however, S-adenosylmethionine decarboxylase (AdoMetDC) depletion was nearly identical to what was observed in cells treated with parent drug. The most notable difference between the parent and hydroxylated analogues was seen with spermidine/spermine N(1)-acetyltransferase (SSAT) upregulation in the DENSPM series. The hydroxylated analogues, especially (R, R)-(HO)(2)DENSPM, were much less effective at upregulation than the parent DENSPM. Finally, a comparison of the toxicity of (R, R)-(HO)(2)DENSPM with that of DENSPM at subchronic doses revealed that the neurological effects seen with DENSPM were now absent.
A new means of accessing N(1)-cyclopropylmethyl-N(11)-ethylnorspermine (CPMENSPM) and the first synthesis of (2R,10S)-N(1)-cyclopropylmethyl-2,10-dihydroxy-N(11)-ethylnorspermine [(2R,10S)-(HO)(2)CPMENSPM] are described. Both of these polyamine analogues are shown to be more active against L1210 murine leukemia cell growth than either N(1),N(11)-diethylnorspermine (DENSPM) or (2R,10R)-N(1),N(11)-diethyl-2,10-dihydroxynorspermine [(2R,10R)-(HO)(2)DENSPM] after 96 h of treatment; the activity was comparable to that of (2S,10S)-N(1),N(11)-diethyl-2,10-dihydroxynorspermine [(2S,10S)-(HO)(2)DENSPM] at 96 h. Both cyclopropyl compounds reduced putrescine and spermidine pools, but less effectively than did DENSPM and its derivatives. Only CPMENSPM, and not (2R,10S)-(HO)(2)CPMENSPM, lowered spermine pools. As with DENSPM and (2R,10R)-(HO)(2)DENSPM, both cyclopropyl analogues diminished ornithine decarboxylase and S-adenosylmethionine decarboxylase activity. Unlike the hydroxylated DENSPM compounds, both cyclopropyl norspermines substantially upregulated spermidine/spermine N(1)-acetyltransferase. The most interesting effect of hydroxylating CPMENSPM is the profound reduction in toxicity compared with that of the parent drug. The same phenomenon had been observed for the DENSPM/(2R,10R)-(HO)(2)DENSPM pair. Thus, hydroxylation of norspermine analogues appears to be a way to maintain the compounds' antiproliferative activity while reducing their toxicity.
The synthesis of a reagent that enables the incorporation of the unusual amino acid (2S,9R)-hypusine (Hpu) into peptide sequences is described. The reagent, (2S,9R)-11-[(benzyloxycarbonyl)amino]-7-(carbobenzyloxy)-2-[(9-fluorenylmethoxycarbonyl)amino]-9-(tetrahydropyran-2-yloxy)-7-azaundecanoic acid, is utilized in the synthesis of a hexapeptide containing the primary pentapeptide sequence of the eukaryotic initiation factor eIF-5A, L-Cys-L-Thr-Gly-Hpu-L-His-Gly. The reagent is shown to be effective for both solution phase and Merrifield resin synthesis.
Two new synthetic methods which allow access to (2S)-deoxyhypusine, natural (2S,9R)-hypusine, (2S,9S)-hypusine, and deoxyhypusine- and hypusine-containing peptides are described. The methods involve both the construction of a deoxyhypusine reagent in which the alpha-nitrogen protecting group is orthogonal to the N-7 and N-12 protecting groups and an alternate synthesis of our previous hypusine reagent, a synthesis which provides for better stereochemical control at C-9. Synthetic hypusine and deoxyhypusine can be generated from these reagents. The hypusine-containing hexapeptide (Cys-Thr-Gly-Hpu-His-Gly) is conjugated to ovalbumin (OVA), keyhole limpet hemocyanin (KLH), and a bis-maleimide; KLH conjugates are also made with the deoxyhypusine- and lysine-containing hexapeptides. Monoclonal antibodies are generated to the hypusine-containing hexapeptide-OVA conjugate in mice. These are isolated and screened against the hypusine-containing hexapeptide-KLH and hypusine-containing hexapeptide-bis-maleimide conjugates, as well as against the deoxyhypusine-containing and lysine-containing hexapeptide-KLH conjugates. These antibodies may be useful in localizing intracellular hypusine-containing peptides as well as peptides containing hypusine analogues.
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