Design, synthesis, and characterization of the antitumor activity of novel ceramide analogues.

A deficiency in apoptosis is one of the key events in the proliferation and resistance of malignant cells to antitumor agents; for these reasons, the search for apoptosis-inducing drugs represents a valuable approach for the development of novel anticancer therapies. In this study we report the first example of conformationally restrained analogues of ceramide (compounds 1-4), where the polar portion of the molecule has been replaced by a thiouracil (1, 3) or uracil (2, 4) ring. The evaluation of their biologic activity on CCRF-CEM human leukemia cells demonstrated that the most active was compound 1 followed by compound 2 (mean 50% inhibition of cell proliferation [IC(50)] 1.7 and 7.9 microM, respectively), while compounds 3 and 4 were inactive, as were uracil, thiouracil, and 5,6-dimethyluracil, the pyrimidine moieties of compounds 1-4. For comparison, the IC(50) of the reference substance, the cell-permeable C2-ceramide, was 31.6 microM. Compounds 1 and 2 and C2-ceramide were able to trigger apoptosis, as shown by the occurrence of DNA and nuclear fragmentation, and to release cytochrome c from treated cells. The treatment of female CD-1 nu/nu athymic mice bearing a WiDr human colon xenograft with the most active compound 1 at 2, 10, 50, and 200 mg/kg ip daily for 10 days resulted in an antitumor effect that was equivalent at 50 mg/kg or superior (200 mg/kg) to that of cyclophosphamide, 20 mg/kg ip daily, delivered on the same schedule, with markedly lower systemic toxicity. In conclusion, the present study demonstrates that the new ceramide analogues 1 and 2 are characterized by in vitro and in vivo antitumor activity and low toxicity.

Purification, enzymatic characterization, and inhibition of the Z-farnesyl diphosphate synthase from Mycobacterium tuberculosis.

We have recently shown that open reading frame Rv1086 of the Mycobacterium tuberculosis H37Rv genome sequence encodes a unique isoprenyl diphosphate synthase. The product of this enzyme, omega,E,Z-farnesyl diphosphate, is an intermediate for the synthesis of decaprenyl phosphate, which has a central role in the biosynthesis of most features of the mycobacterial cell wall, including peptidoglycan, arabinan, linker unit galactan, and lipoarabinomannan. We have now purified Z-farnesyl diphosphate synthase to near homogeneity using a novel mycobacterial expression system. Z-Farnesyl diphosphate synthase catalyzed the addition of isopentenyl diphosphate to omega,E-geranyl diphosphate or omega,Z-neryl diphosphate yielding omega,E,Z-farnesyl diphosphate and omega,Z,Z-farnesyl diphosphate, respectively. The enzyme has an absolute requirement for a divalent cation, an optimal pH range of 7-8, and K(m) values of 124 micrometer for isopentenyl diphosphate, 38 micrometer for geranyl diphosphate, and 16 micrometer for neryl diphosphate. Inhibitors of the Z-farnesyl diphosphate synthase were designed and chemically synthesized as stable analogs of omega,E-geranyl diphosphate in which the labile diphosphate moiety was replaced with stable moieties. Studies with these compounds revealed that the active site of Z-farnesyl diphosphate synthase differs substantially from E-farnesyl diphosphate synthase from pig brain (Sus scrofa).

Polyprenyl phosphate biosynthesis in Mycobacterium tuberculosis and Mycobacterium smegmatis.

Mycobacterium smegmatis has been shown to contain two forms of polyprenyl phosphate (Pol-P), while Mycobacterium tuberculosis contains only one. Utilizing subcellular fractions from M. smegmatis and M. tuberculosis, we show that Pol-P synthesis is different in these species. The specific activities of the prenyl diphosphate synthases in M. tuberculosis are 10- to 100-fold lower than those in M. smegmatis. In M. smegmatis decaprenyl diphosphate and heptaprenyl diphosphate were the main products synthesized in vitro, whereas in M. tuberculosis only decaprenyl diphosphate was synthesized. The data from both organisms suggest that geranyl diphosphate is the allylic substrate for two distinct prenyl diphosphate synthases, one located in the cell membrane that synthesizes omega,E,Z-farnesyl diphosphate and the other present in the cytosol that synthesizes omega,E,E,E-geranylgeranyl diphosphate. In M. smegmatis, the omega,E, Z-farnesyl diphosphate is utilized by a membrane-associated prenyl diphosphate synthase activity to generate decaprenyl diphosphate, and the omega,E,E,E-geranylgeranyl diphosphate is utilized by a membrane-associated activity for the synthesis of the heptaprenyl diphosphate. In M. tuberculosis, however, omega,E,E,E-geranylgeranyl diphosphate is not utilized for the synthesis of heptaprenyl diphosphate. Thus, the difference in the compositions of the Pol-P of M. smegmatis and M. tuberculosis can be attributed to distinct enzymatic differences between these two organisms.

Synthesis and HIV-1 inhibitory properties of new tetrahydrobenzoquinazolinedione and tetrahydrobenzocycloheptenuracil derivatives and of their thioxo analogues.

Some new tetrahydrobenzoquinazolinediones 2a-4a, tetrahydrobenzocycloheptenuracils 5a, 6a and their thioxo analogues 2b-6b were synthesized within a project aimed at obtaining new HIV-1 tricyclic inhibitors whose scaffold includes a pyrimidine and a phenyl ring, which are present in various HIV-1 non-nucleoside inhibitors. Among the tetrahydrobenzoquinazolinediones 2a-4a, compounds 3a and 4a, in which the tricyclic system is respectively in an angular or linear arrangement, proved to possess a HIV-1 inhibitory activity which was in the micromolar range, while compound 2a, in which the tricyclic system is in the angular arrangement opposite to that of 3a, was found to be completely inactive. As regards the tetrahydrobenzocycloheptenuracil derivatives (5a and 6a), only 5a showed an inhibitory activity similar to that of 3a and 4a. Furthermore, all thioxo analogues 2b-6b were found to be devoid of any activity.

New N-substituted 7-aminocephalosporanic acid derivatives as potential agents against Streptococcus pneumoniae. Synthesis and in vitro activity.

The synthesis and the antimicrobial properties of a new series of cephalosporinic beta-lactam antibiotics is described. The data reported in the present paper show the potential of this type of substituted cephalosporins as new anti Gram-positive antibiotic drugs. In fact, all compounds tested showed a good in vitro antibacterial activity against the most relevant Gram-positive pathogens including resistant species that currently represent unmet medical need. On the contrary, the new synthesized compounds were found to be completely devoid of any activity on Gram-negative bacteria up to a concentration of the single agent of 128 microg/ml.

Synthesis and aldose reductase inhibitory activity of new N-(benzyloxy) glycine derivatives.

This paper reports the synthesis and aldose reductase (AR) inhibitory properties of some N-(benzyloxy) glycine derivatives (compounds 2-6), structurally related to the previously described N-(aroyl)-N-(arylmethyloxy) glycines A which had proved to possess an appreciable AR inhibitory activity. In compounds 2-5, spacers of different lengths and degrees of rigidity were inserted between the phenyl ring and the carbonyl group of type A derivatives; compound 6 differs from the most active type A derivative (compound 1) in the replacement of the methoxy moiety in the para position of the benzoyl side-chain with a group with different electronic characteristics, such as the trifluoromethyl moiety. Biological results indicated that among compounds 2-5 only derivative 3, which presents a CH2CH2 spacer between the phenyl and the carbonyl moiety, proved the possess AR inhibitory properties analogous to those of 1, while all the other compounds proved to be devoid of any significant activity. Furthermore, compound 6 showed an inhibitory activity about 3 times lower than that of 1.

Fluorescent probes for adenosine receptors: synthesis and biology of N6-dansylaminoalkyl-substituted NECA derivatives.

New fluorescent ligands for adenosine receptors are described; these compounds were obtained by the insertion, in the N6 position of NECA (a potent adenosine agonist), of dansylaminoalkyl moieties with alkyl spacers of increasing carbon chain length (from 3 to 12). Among them, the compound with a C6 alkyl spacer proved to be the most interesting one, showing a marked selectivity for the A1 receptor subtype; furthermore, in fluorescence microscopy assays it proved to be able to visualize and localize this receptor subtype at the level of the molecular layer of the rate cerebellar cortex.

Toward the rational development of peptidomimetic analogs of the C-terminal endothelin hexapeptide: development of a theoretical model.

In an early report on the structure-activity relationship of endothelin (ET) peptides, it was reported that the C-terminal hexapeptide ET(16-21), His-Leu-Asp-Ile-Ile-Trp, is the minimum ET fragment which maintains biological activity in some, but not all the tissues responding to ETs. Subsequently, other authors described a series of analogs of this peptide, in which the His 16 residue was replaced by non-natural amino acids, characterized by bulky aromatic side chains. Among them, two well-characterized non-selective ETA/ETB antagonists were PD 142893 and PD 145065; interest in these potent ET antagonists was, however, reduced by their peptidic structure which was likely to lead to undesirable properties such as poor bioavailability and short duration of action. On the basis of these premises, our previous studies led to the development of a peptidomimetic ligand of ET receptors (compound 3), based on the replacement of the His 16 residue of ET(16-21) with an (E)-N-(benzyloxy)iminoacyl moiety; compound 3 proved to possess a certain affinity for ET receptors, albeit lower than that shown by PD 142893 and PD 145065. We report here on ETA/ETB binding affinity of compounds 4-12, designed as a new series of ET(16-21) analogs. Compounds 4 and 5 were practically devoid of any affinity; derivatives 6-12 exhibited appreciable affinity indices for ETB receptors higher than that shown by 3, even if still lower than that obtained for PD 145065. This paper also describes the development of a pharmacophoric model able to explain the ET receptor binding properties of our hexapeptide analogs compared with those of PD 142893 and PD 145065 and IRL2500, recently reported as a potent ETB selective endothelin antagonist.