Synthesis and activity of pyrimidinylpropenamide antibiotics: the alkyl analogues of sparsomycin.

Facile syntheses of sparsomycin (3) and its four analogues (4-7) based on diastereoselective oxidation of sulfide, sulfenylation, and coupling of 6-methyluracylacryllic acid with monooxodithioacetal amine, are described. Studies on the biological activity of morphological reversion on src(ts)-NRK cells were also carried out.

Synthesis and morphological reversion activity on srctsNRK cells of pyrimidinylpropanamide antibiotics, sparsomycin, sparoxomycin A1, A2, and their analogues.

Three pyrimidinylpropanamide antibiotics sparsomycin (1), sparoxomycins A1, A2 (2, 3), and also six analogues (4-9) have been synthesized by employing asymmetric sulfide oxidation conditions as a key step. Sparsomycin (1) and its alkyl analogues (5-7) showed higher morphological reversion activities on srctsNRK cells than 2 and 3.

The chiral synthesis and biochemical properties of electron rich phenolic sulfoxide analogs of sparsomycin.

A novel route to activated phenolic sulfoxide analogs of sparsomycin has been developed. These analogs display an enhanced "preincubation effect" as inhibitors of peptide-bond formation. This time-dependent component of inhibition, which is postulated to result from an enzyme-mediated Pummerer rearrangement, is the dominant route to inhibition in these activated analogs.

Lipophilic analogues of sparsomycin as strong inhibitors of protein synthesis and tumor growth: a structure-activity relationship study.

Fourteen derivatives of sparsomycin (1) were synthesized. Six of them were prepared following a novel synthetic route starting from the L-amino acid alanine. Some physicochemical properties, viz. lipophilicity and water solubility, of selected derivatives were measured. The biological activity was tested in vitro in cell-free protein synthesis inhibition assays, in bacterial and tumor cell growth inhibition assays, and in the L1210 leukemia in vivo model in mice. Also for selected drugs the acute toxicity in mice was determined. Ribosomes from both an eukaryotic and a prokaryotic organism were used in the protein synthesis inhibition systems. A linear correlation between the lipophilicity parameters measured was observed. Water solubility and drug toxicity in mice were found to be linearly correlated with lipophilicity. All the derivatives studied are more lipophilic than 1. The deshydroxysparsomycin analogues (30-33) showed an interesting phenomenon: increase in hydrophobicity was accompanied by a considerable increase in water solubility. We found that an increase in hydrophobicity of the drug as a result of replacing the SMe group of 1 with larger alkylthio groups causes an increase in the biological activity of the drug. However, not only the hydrophobicity but also shape and size of the substituent are important; in the homologous series 1-9-10-11-12, 21-22-23-24, and 30-31-32-33, highest protein synthesis inhibitory and in vitro cytostatic activity is found with compounds 11, 23, and 32, respectively, and in comparison with the highly active n-butyl compound 10, the isomeric tert-butyl compound 13 is rather inactive. Polar substituents replacing the SMe group, i.e. Cl in 17 and 35, also render the molecule inactive. Substituting the bivalent sulfur atom for a methylene group decreases the drug's activity. This effect can be compensated for by increasing the length of the alkylsulfinyl side chain. The agreement between the results derived from cell-free and "in vivo" tests is good. The assays using ribosomes of bacterial and eukaryotic organisms give similar results although the latter seem to be more sensitive to changes in hydrophobicity of the drug. Our results confirm the presence of a hydrophobic region at the peptidyl transferase center of the ribosome; the interaction of sparsomycin with this region is more pronounced in the eukaryotic particles. The sparsomycin analogues 11, 23, and 30 show the highest antitumor activity against L1210 leukemia in mice, their median T/C values are 386, 330, and 216%, respectively.(ABSTRACT TRUNCATED AT 400 WORDS)

Sparsomycin analogs. VI. Synthesis and antitumor activity of octylsparsomycin analogs.

Five sparsomycin analogs (9-13) were prepared and examined for their ability to inhibit deoxyribonucleic acid (DNA) synthesis in L5178Y lymphoma cells. All of the compounds showed significant activity in the DNA synthesis assay. The compounds having Rc configuration exhibited almost the same activities independently of the configuration at the sulfoxide sulfur atom. Among the Sc isomers, the Rs configuration was advantageous for the appearance of activity.

The development of sparsomycin as an anti-tumour drug.

The synthesis of sparsomycin and eight lipophilic analogues is discussed. Sparsomycin, a bacterial metabolite, is a potent inhibitor of protein biosynthesis; in addition it has anti-tumour properties. Structure-activity relationship studies indicate that the anti-tumour activity is a direct consequence of the inhibition of protein synthesis. Two of the analogues show a higher anti-tumour activity in vitro as well as in vivo, and are less toxic to mice than sparsomycin. Finally, some properties of sparsomycin are discussed: the drug potentiates the cytotoxicity of cisplatin and is selectively active on tumour cells without affecting human bone-marrow.

Structure-activity relationships of sparsomycin and its analogues. Inhibition of peptide bond formation in cell-free systems and of L1210 and bacterial cell growth.

The biological activity of 14 analogues of sparsomycin (1) was studied in cell-free systems of Escherichia coli, Saccharomyces cerevisiae, and Sulfolobus solfataricus by measuring the inhibition of protein synthesis. The inhibition of L1210 colony formation in soft agar and bacterial cell growth in solid as well as in liquid medium was also examined. Each analogue possesses not more than two structural modifications of the sparsomycin molecule. This enabled us to determine unambiguously several structural and stereochemical features that are required for an optimal biological activity in these assays. Sparsomycin, having the SCRS chirality, is the most potent of the four possible stereoisomers. The results obtained with compounds 5-7 indicate that the presence of an oxygen atom on the S (alpha) atom is essential. Substitution of the bivalent sulfur atom by a CH2 group (10) or of the SCH3 moiety by a Cl atom (12) affects the activity of the molecule partially. Compound 12 is surprisingly active against intact cells. Substitution of the C(6)-CH3 group by a H(14) reduces the activity of the molecule. Isomerization of the trans double bond into the cis double bond yields cis-sparsomycin (15), which is inactive. The hydrophobic derivatives 8, 9, and 11 are considerably more active than sparsomycin; thus the ribosomal binding site for sparsomycin may have a hydrophobic character.

Synthesis and biological evaluation of sparsomycin analogues.

Three series of sparsomycin analogues were prepared and examined for their ability to inhibit DNA or protein synthesis in bone marrow, P388 lymphocytic leukemia, and P815 mastocytoma cells. The compounds of series I and II, distinguished by the inclusion or exclusion of a hydroxymethyl functional group, were designed to elucidate the effect on activity of replacing the oxodithioacetal side chain of sparsomycin with 4-substituted benzyl groups. The series III analogues, which excluded the hydroxymethyl group and replaced the oxodithioacetal moiety of sparsomycin with a benzyl amide group, were designed to investigate the potential interaction of an amide oxygen in contrast to the sulfoxide oxygen of sparsomycin. Overall, the bromobenzyl-substituted analogues imparted the greatest inhibitory activity in the protein synthesis assay, while the methoxybenzyl-substituted analogues displayed the least. The methylbenzyl and the unsubstituted benzyl compounds were intermediate in inhibitory potential. The activity in the protein synthesis assay may correspond to the lipophilic and electronic characteristics of the substituents on the benzyl moiety of the analogues. All of the compounds were inactive in the DNA synthesis assay.

Necessity of the sulfoxide moiety for the biochemical and biological properties of an analog of sparsomycin.

An analog of the peptidyl transferase inhibitor sparsomycin was a competitive inhibitor (Ki = 1.8 microM) of peptidyl-puromycin synthesis on E. coli polysomes. Preincubation of polysomes with the compound enhanced the degree of inhibition of peptide bond formation. A model for the involvement of a histidine residue in peptidyl transferase activity is presented as a result of our observations which include direct association of [3H] labelled analog with 70S ribosomes. The correct oxidation state of sulfur in the compound was necessary for the "preincubation effect" and entry of the compound into bacterial cells.

Effect of sparsomycin analogues on the puromycin-peptidyl transferase reaction on ribosomes.

Sparsomycin analogues in which the unique -S(O)CH2SCH3 moiety was replaced by a variety of more easily accessible side chains were evaluated as inhibitors of the peptidyl transferase reaction with bacterial ribosomes. Competitive inhibition of acetyl[14C]phenylalanylpuromycin formation revealed that the sulfur-containing side chain of sparsomycin could be replaced with hydrophobic moieties, whereas complete removal of the -S(O)CH2SCH3 side chain eliminated the ribosomal binding affinity of sparsomycin. The specificity for the D isomer of S-deoxo-S-propylsparsomycin has established that the chiral carbon of sparsomycin analogues must be identical with the chirality of D-cysteinol for ribosomal binding.

Pyrimidinylpropenamides as antitumor agents. Analogues of the antibiotic sparsomycin.

A series of pyrimidinylpropenamides 9 and their oxidation products 10 was prepared, as analogues of sparsomycin (1), for antitumor evaluation. Syntheses involved condensation of the appropriate amino alcohol 5 with acid 8. The resulting sulfides 9 were then oxidized with NaIO4 or H2O2 to sulfoxides 10. Activity was studied in lymphocytic leukemia P-338 and KB cell culture. With the exception of the n-decyl analogue, all of the deoxygenated compounds 9 were inactive regardless of the stereochemical form. In the sulfoxide series 10, those compounds prepared with an L configuration at the asymmetric carbon were also inactive. The completely racemic sulfoxides, on the other hand, displayed substantial antitumor activity (ILS = 37-61% in P-388; ED50 = 1.2-2.4 mug/ml in KB) suggesting that both the presence of a sulfoxide moiety and a D configuration at the chiral carbon atom were structural requirements for a positive antitumor response. There appeared to be a large tolerance for the group substituted at the sulfoxide moiety, however.