Antitumour activity and retinotoxicity of ethyldeshydroxy-sparsomycin in mice.

The colony formation in agar of human tumour xenografts was used as a test system to study the cytostatic activity of ethyldeshydroxy-sparsomycin (EdSm) at the cellular level. EdSm was additionally studied in vivo in human tumour xenografts and murine tumour models. EdSm showed a clear dose-response effect in vitro. At continuous exposure with 0.01 micrograms/ml, 2 out of 11 of the tumours responded (a gastric and a small cell lung carcinoma). At 0.1 mu/ml EdSm, the tumour response was 5/11 tumours and at 1 microgram/ml the compound was active in all tumours. The maximal tolerable doses of EdSm in vivo have been determined in non-tumour bearing CDF1 mice. In the intraperitoneally (i.p.) given multiple dose schedules the respective LD10 doses indicated that the tolerable cumulative dose increases when lower doses are given more frequently. This also enhances the antitumour activity in L1210 leukaemia to 172% T/C. On the other hand, continuous infusion strongly diminished the tolerable dose as well as the antitumour activity. EdSm was also active against i.p. inoculated P388 leukaemia (150% T/C), B16 melanoma (156% T/C), and RC carcinoma (197% T/C), and the subcutaneously (s.c.) inoculated L1210 (139% T/C) and RC (138% T/C). Absence of tumour responses was found in the following s.c. implanted murine tumours: M5076 sarcoma, osteosarcomas C22LR and CP369, and the LL carcinoma, as well as in the human tumour xenografts: LXFG 529, a non-small cell lung carcinoma; GXF 251, a gastric carcinoma; and FMa, an ovary carcinoma. Possible long-range retinotoxic effects of EdSm were investigated in tumour-bearing mice, cured after surviving treatment with LD50 doses of EdSm, by assaying the protein biosynthetic capacity of the retinal by assaying the ocular rhodopsin and opsin levels as parameters. In none of these cases could a significant reduction in either opsin or rhodopsin levels be measured and no changes were seen histologically.

Chemical, biochemical and genetic endeavours characterizing the interaction of sparsomycin with the ribosome.

Sparsomycin interaction with the ribosome and characteristics of the drug binding site in the particle were studied using chemical modification of the drug, affinity labeling methods and isolation of drug resistant mutants. The structure-function relationship studies, performed with a large number of drug derivatives, indicate that the drug interacts with the ribosome by its western and eastern moieties. The uracil ring, in the western end of the drug molecule, probably forms hydrogen bonds with the rRNA, while the apolar CH3-S-CH3 group in the eastern end interacts with a hydrophobic ribosomal domain that affinity labeling results seem to indicate is formed by protein. An increase in lipophilicity in this part of the antibiotic results in a dramatic increase in the inhibitory activity of the drug. The sparsomycin binding site is not accessible in free ribosomes, but the presence of an N-blocked amino acyl-tRNA at the P-site turns the particles capable of reversible interaction with the drug. After failure using Escherichia coli, a sparsomycin-resistant mutant was obtained by direct mutagenesis on Halobacterium halobium, a species with a unique copy of rRNA genes, stressing the role of rRNA on the drug interaction site.

Structure-activity relationships of sparsomycin: modification at the hydroxyl group.

Ten analogues of the antibiotic sparsomycin were prepared and evaluated in several in vitro tests. Nine of them carry a modification at the hydroxymethylene group of the molecule, two have a disulfide bond instead of the S(O)-CH2-S moiety at the sulfur-containing side chain of the molecule. While the presence of the S-S group decreases the activity of the analogues in all the tests performed, the modification at the OH group has no deleterious effects on the activity when a polyphenylalanine synthesis assay is used in an Escherichia coli extract. The same modifications, however, diminish drastically the activity of the analogues when tested in a similar Saccharomyces cerevisiae extract. A polymerization system in the archaebacterium Halobacterium halobium extract behaves like the eukaryotic preparations. A discrepancy is also found between the results of the polymerization tests and those of the 'puromycin reaction' which is also less sensitive to the modified sparsomycin analogues. The results of cell growth inhibition tests in bacteria as well as in eukaryotic organisms agree only partially with the in vitro data.

Structure-activity relationships of sparsomycin and its analogues. Octylsparsomycin: the first analogue more active than sparsomycin.

Nine analogues of sparsomycin were synthesized, and their cytostatic activity was studied in an in vitro clonogenic L1210 assay by measuring the inhibition of colony formation. The activity of an analogue, expressed as an ID50 value, was compared to that of sparsomycin. Each possesses not more than two structural modifications of the sparsomycin molecule 1. Comparison of the activity of with that of the stereomers, having RCSS, SCSS, and RCRS chirality, respectively, shows that the S configuration of the chiral carbon atom is essential for an optimal activity, whereas the R chirality of the sulfoxide sulfur atom of sparsomycin is of importance. Study of the ID50 values of the S-deoxo analogues, as well as the compounds having the beta-sulfoxide function, indicate that the presence of an oxygen atom on the alpha-sulfur atom is essential. Isomerization of the trans double bond into the cis double bond yields isosparsomycin, (Scheme II), which has a low activity. The cytostatic activity of sparsomycin seems to be related to its lipophilicity: octylsparsomycin was shown to be three times as effective as sparsomycin.

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.

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)

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.

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.

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.

Potentiation of cisplatin antitumor activity on L1210 leukemia s.c. by sparsomycin and three of its analogues.

Sparsomycin (Sm) is a known antibiotic derived from Streptomyces. Its potential antitumor activity stimulated the search for a synthetic production method and the development of new derivatives. In a recent screening investigation, three Sm analogues appeared to be more active and considerably less toxic than the parent drug. Sparsomycins became especially interesting when it was shown that Sm potentiates the antitumor activity of cisplatin. In the present study Sm and its three promising analogues: deshydroxy-Sm (dSm), ethyl-deshydroxy-Sm (EdSm) and n-pentyl-Sm (PSm) were studied for their cisplatin potentiating effect. The experiments were performed on CD2F1 mice inoculated with 10(6) L1210 cells s.c. Sparsomycins were administered i.p. 3 h before cisplatin on days 1, 5 and 9. Three of the drugs, Sm, dSm and PSm, showed no potentiating effect in this tumor model. At a dose of 10 mg/kg, EdSm potentiated cisplatin antitumor activity 2.8 times (P less than 0.01) without an increase in weight loss. These results warrant further investigation.

Inhibition of Trypanosoma brucei brucei peptidyl transferase activity by sparsomycin analogs and effects on trypanosome protein synthesis and proliferation.

Peptidyl transferase activity of Trypanosoma brucei brucei polyribosomes was competitively inhibited by analogs of sparsomycin (Ki = 1-100 microM). The analogs were also potent inhibitors of [3H]-leucine and [3H]mannose incorporation into the proteins of intact trypanosomes with little or no effect on overall respiratory rate, suggesting a specific site of action for these analogs on protein synthesis. The peptidyl transferase inhibitors were effective at low concentrations at limiting the proliferation of trypanosomes both in vitro and in vivo. The potency of the compounds as inhibitors of cell proliferation was positively correlated with their efficacy as inhibitors of peptidyl transferase activity. One compound, MDL 20828 (1 mg/kg), increased the survival time of T. b. brucei-infected mice 4-fold in the absence of any overt drug toxicity to the hosts.

Correlation of the in vitro cytotoxicity of ethyldeshydroxysparsomycin and cisplatin with the in vivo antitumour activity in murine L1210 leukaemia and two resistant L1210 subclones.

The cultured murine leukaemia L1210 cell populations used in the present study were derived from L1210 cells that had been grown in vivo. Subclones resistant to sparsomycin (L1210/Sm) or cisplatin (L1210/CDDP) were also developed in vivo. The doubling times of the cultured cell populations were identical. Fractions surviving after drug treatment in vitro were determined by colony formation in soft agar. The results, based on the differential sensitivity of the cell populations to ethyldeshydroxysparsomycin (EdSm) and CDDP, indicated that after a short exposure, cultured L1210/CDDP cells were cross-resistant to EdSm. L1210/Sm cells, however, were not cross-resistant to CDDP. The results obtained in cultured cell populations were confirmed in vivo. CD2f1 mice bearing i.p. implants of 1 x 10(5) tumour cells were given EdSm or CDDP and a combination of the two agents. Drugs were given once daily every 4 days for 3 doses starting at 24 h after tumour implantation. Treatment of mice bearing L1210/wt leukaemia with combined EdSm and CDDP caused strongly synergistic antitumour activity. In animals bearing the two resistant subclones, however, combined drug treatment did not improve the antitumour activity. The corresponding median survival of mice receiving combined drug treatment was 60 days in each group containing 6 mice bearing L1210/wt, with 4-6 cures being noted; 19 days in animals harbouring L1210/Sm, with 2 cures being recorded among 6 mice; and 11 days in mice bearing L1210/CDDP, with no cure being obtained. The results of this study indicate that the synergism resulting from combined treatment with CDDP and EdSm is a function of the cellular properties of the target tumour-cell populations and is independent of host factors.

Preclinical pharmacokinetics of the antitumor antibiotic deshydroxy-sparsomycin in beagle dogs.

Sparsomycin (Sm) is a well known inhibitor of protein synthesis with anticancer potential. In order to minimize toxicity of this drug and increase its activity, several analogues were synthesized. Deshydroxy-Sm (dSm) appeared to be a good candidate for further investigations because of its lower toxicity and significantly higher antitumor activity in several ascitic tumors in mice. Pharmacokinetic evaluation in beagle dogs was performed using either single iv bolus or continuous infusion administrations. The drug was eliminated with a terminal t1/2 beta of 0.8 +/- 0.08 hours (48 +/- 5 minutes). The mean volume of distribution was 0.4 +/- 0.06 l.kg-1. The mean total body clearance was 6.4 +/- 0.8 ml.min-1.kg-1. The drug is eliminated mainly by the kidneys (54%). Active tubular secretion and active tubular reabsorption of the drug were observed. The pharmacokinetics was linear until the lethal dose. The results of this study provided additional data useful in selection of potentially useful analogues for further preclinical studies.

Potentiation of cisplatin antitumour activity by Ethyldeshydroxy-Sparsomycin in L1210 leukemia.

The combination of Ethyldeshydroxy-Sparsomycin (EdSm) with cisdiamminedichloroplatinum(II) (CDDP) caused significant antitumour activity against murine L1210 leukemia. Although single drug treatment by cisplatin generated some cures, all schedules of combined treatment, using nontoxic doses of EdSm (5mg/kg) and cisplatin (3 mg/kg), resulted in the cure of 4 to 6 mice in each group consisting of 6 mice. No differences in antitumour activity were observed between pretreatment, simultaneous treatment or posttreatment of cisplatin with EdSm. Increasing the number of tumour cells implanted i.p. diminished the antitumour effect of both EdSm as well as CDDP, but not for the drug combination. Changing the route of administration from i.p. to i.v. for one of the drugs out of the combination resulted in loss of antitumour activity.

Synthesis of sparsomycin analogs as potential antitumor agents.

No information is available on the structural requirements for the antitumor activity of sparsomycin, an antibiotic obtained from the fermentation broth of Streptomyces sparsogenes. Its high in vivo and in vitro activity, novel structure, and uncommon mode of action have, therefore, suggested the synthesis of analogs. This report describes the preparation and screening of a series of N-substituted 3-aryl acrylamides which are closely related to sparsomycin. Three compounds exhibited some tumor inhibition but insufficient to warrant further testing.

Absolute binding free energy calculations of sparsomycin analogs to the bacterial ribosome.

The interactions of the 50S subunit of bacterial ribosome with antibiotic sparsomycin (SPS) and five analogs (AN) are investigated through the calculation of the standard (absolute) binding free energy and the characterization of conformational dynamics. The standard binding free energies of the complexes are computed using free energy perturbation molecular dynamics (FEP/MD) simulations with explicit solvent. Restraining potentials are applied and then released during the simulation to efficiently sample the changes in translational, orientational, and conformational freedom of the ligand and receptor upon binding. The biasing effects of the restraining potentials are rigorously removed. The loss of conformational freedom of the ligand upon binding is determined by introducing a potential of mean force (PMF) as a function of the root-mean-square deviation (rmsd) of the ligand relative to its conformation in the bound state. To reduce the size of the simulated system, the binding pocket of the ribosome is simulated in the framework of the generalized solvent boundary potential (GSBP). The number of solvent molecules in the buried binding site is treated via grand canonical Monte Carlo (GCMC) during the FEP/MD simulations. The correlation coefficient between the calculated and measured binding free energies is 0.96, and the experimentally observed ranking order for the binding affinities of the six ligands is reproduced. However, while the calculated affinities of the strong binders agree well with the experimental values, those for the weak binders are underestimated.

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.

Pharmacokinetics of the antitumor antibiotic n-pentyl-sparsomycin in beagle dogs.

N-pentyl-sparsomycin (PSm) is a lipophilic analogue of sparsomycin (Sm), which is a well known inhibitor of protein synthesis. This compound was selected for preclinical pharmacokinetic studies because of its high in vitro and in vivo antitumor activity. In this study in which the drug was evaluated in beagle dogs under anaesthesia, the drug concentrations in plasma, urine and bile samples were determined using high performance liquid chromatography (HPLC). Plasma protein binding was approximately 54%. The mean t1/2 beta was 0.2 hours (12 minutes) and t1/2 tau was 0.75 +/- 0.1 hours (45 +/- 6 minutes). During continuous infusions up to 5.25 hours, the steady state was reached in 3 out of 6 experiments, suggesting that in some cases the real t1/2 tau was longer than measured. PSm was actively reabsorbed from the renal tubuli. This process was saturable at the higher doses. Tubular reabsorption played only a minor role in pharmacokinetics as most of the drug (67%) was eliminated by the non-renal clearance. The non-renal clearance was saturable at higher doses of PSm and was the reason for non-linearity of pharmacokinetics.

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.