New pemetrexed-peptide conjugates: synthesis, characterization and in vitro cytostatic effect on non-small cell lung carcinoma (NCI-H358) and human leukemia (HL-60) cells.

Pemetrexed (Pem) is a novel antimetabolite type of anticancer drug that demonstrated promising clinical activity in a wide variety of solid tumors, including non-small cell lung carcinoma and malignant pleural mesothelioma. It inhibits enzymes involved in the folate pathway, for which the presence of its free carboxylic groups is necessary. The heteroaromatic ring system of Pem has a modifiable amino group, which opens a possibility to apply a new strategy to conjugate Pem to carrier molecules. Considering this as well as the necessity of untouched carboxylic groups of Pem in the new conjugates, we developed a new synthesis strategy. Here, we describe the synthesis and the characterization of new Pem-peptide conjugates in which cell-penetrating octaarginine or/and lung-targeting H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH(2) peptide is attached to the drug by thioether bond. The conjugates characterized by RP-HPLC and MS exhibited cytostatic effect in vitro on non-small cell lung carcinoma as well as on human leukemia cell lines. The IC(50) values of the conjugates were similar, but the conjugates with H-Ile-Glu-Leu-Leu-Gln-Ala-Arg-NH(2) sequence were slightly more effective. Our data show that the in vitro cytostatic effect of the free Pem was essentially maintained after conjugation with cell-penetrating or cell-targeting peptides. Thus, the conjugation strategy reported could lead to the development of a new generation of active Pem conjugates.

New daunomycin-oligoarginine conjugates: synthesis, characterization, and effect on human leukemia and human hepatoma cells.

In this article, the synthesis, a novel chromatographic procedure and characteristics of a new class of daunomycin (Dau)-oligoarginine conjugates are described. In these compounds oligoarginine with 6 or 8 residues (Arg(n), n = 6, 8) is attached to Dau by different covalent bond: squaric amide (Dau- square-Arg(n)), oxime (Dau=N-O-CH2-CO-Arg(n)), or hydrazone (H-Glu(Arg(n))-NH-N=Dau). Conjugates were characterized by RP-HPLC and mass spectrometry. We report also on our findings concerning chemical and biological properties of Dau-conjugates as a function of covalent linkage, site of conjugation and length of the oligoarginine moiety. Stability, fluorescent properties as well as cytostatic effect and cellular uptake of these compounds were studied. Dau-conjugates with squaric amide or oxime linkage were stable, but continuous release of free Dau was observed from the hydrazone conjugate in solution. We found that some spectral characteristics (e.g., the amplitude of the emission spectrum) of conjugates could be sensitive for the site of coupling (amino vs. oxo function). Cytostasis and cellular uptake of conjugates were investigated both on human leukemia (HL-60) and human hepatoma (HepG2) cell lines by MTT assay and flow cytometry We found that cytostatic effect and uptake properties of Dau-conjugates were dependent on the acid stability of the linkage (hydrazone vs. oxime/amide) applied and more markedly on the cell line studied.

New ferrocene containing peptide conjugates: synthesis and effect on human leukemia (HL-60) cells.

Data reported in this article describe the synthesis of Arg-rich oligopeptide conjugates of ferrocenecarboxylic acid on solid support with two different strategies and for the first time, the successful preparation of peptide conjugates of ferrocenylacrylic acid in solution. The antitumor effect of conjugates was analyzed by MTT assay in vitro. We demonstrated that ferrocenylacrylic acid possessing an enone (--CH==CH--CO--) moiety exhibited remarkable antiproliferative effect against human leukemia cells (HL-60) in vitro, but its effect was not improved by conjugation with hexa- or octaarginines. However, we observed highly increased water-solubility. In contrast, the results provide evidence that conjugation of ferrocenecarboxylic acid to Arg(n) (n = 6, 8) improved not only its water-solubility, but also antitumor effect on human leukemia cells in vitro.

A new class of semisynthetic anthracycline glycoside antibiotics incorporating a squaric acid moiety.

Treatment of the squaric acid amide esters (7, 9) of anthracycline glycoside antibiotics with aliphatic and aromatic primary and secondary amines, amino acids, peptides and aminodeoxy sugars furnished the new asymmetric diamides 16-19, 25-30, 32, 34 and 38-40 in stereoselective reactions which do not require protecting group-manipulations. The IC50 = 0.12 microM value measured for daunorubicin (1) on human leukemia (HL-60) cells is comparable to those obtained for the daunomycin-L-leucyl squaric acid diamide (30, IC50 = 0.18 microM) and the corresponding D-galactosamine derivative (40, IC50=0.22 microM).