Improvement in the Anticancer Activity of 6-Mercaptopurine via Combination with Bismuth(III).

6-Mercaptopurine (6-MP) is a clinically important antitumor drug and its commercially available form is provided as monohydrate, belonging to biopharmaceuticals classification system (BCS) class II category. The combination of bismuth(III) (Bi(III)) with 6-MP was proved to significantly improve the anticancer activity of 6-MP, leading to the discovery of a new amorphous complex ([Bi(MP)3(NO3)2]NO3). The prepared [Bi(MP)3(NO3)2]NO3 was characterized by the matrix assisted laser desorption-ionization time-of-flight (MALDI-TOF)-MS, etc. Noticeably, according to the in vitro evaluations of cytotoxicity, cellular apoptotic, colony formation as well as cell migration, the anticancer activity of amorphous [Bi(MP)3(NO3)2]NO3 was found to be of high therapeutic effect over 6-MP.

Fabrication of biodegradable micelles with reduction-triggered release of 6-mercaptopurine profile based on disulfide-linked graft copolymer conjugate.

This research is aimed to develop a biodegradable micelle delivery system with sheddable poly (ethylene glycol) shell to achieve the reduction-triggered intracellular sustained release of 6-mercaptopurine (6-MP) and decreased toxicity. Firstly, the amino-disulfide linked poly (ethylene glycol) monomethyl ether (mPEG-SS-NH(2)) was synthesized by the amidation reaction between cystamine and active ester of mPEG and p-nitrophenyl chloroformate (p-NPC) (mPEG-NPC). And then, the five-member rings in poly (l-succinimide) (PSI) were successively opened by mPEG-SS-NH(2) and 2-(pyridyldithio)-ethylamine (PDA) to produce the graft copolymer of mPEG-SS-NH-graft-PAsp-PDA. To avoid the drug initial burst, 6-MP was covalently conjugated with mPEG-SS-NH-graft-PAsp-PDA by thoil-disulfide exchange reaction to give the resultant product mPEG-SS-NH-graft-PAsp-MP. The product was found to form spherical micelles in aqueous media because of its amphiphilic nature with average particle size of 160 nm measured by dynamic light scattering (DLS). It was found that the mPEG-SS-NH-graft-PAsp-MP micelles, though stable in phosphate buffer solution (PBS), were prone to aggregation in the presence of dithiothreitol (DTT). The in vitro drug release studies revealed the release of 6-MP were distinct from the conventional micelles whose drugs loaded by physical encapsulation. Sustained release profile of 6-MP over 85 h was found in the presence of DTT (40 mM) simulating the intracellular condition while minimal drug release was observed within 24h at the level of DTT corresponding to extracellular environment. Remarkably, the cell viability results showed there was essential decrease of cytotoxicity to HL-60 cell line compared to free 6-MP.

6-Mercaptopurine complexes with silver and gold ions: anti-tuberculosis and anti-cancer activities.

Synthesis, characterization and biological studies of silver and gold complexes with 6-mercaptopurine (H2MP) are described. The Ag(I) and Au(I) complexes with HMP-, AgHMP and AuIHMP, were obtained by mixing an acidified H2MP aqueous solution with an equimolar aqueous solution of AgNO3 or Au(CN)2. The Au(III) complex with HMP-, AuIIIHMP, was obtained by adding an aqueous solution of K(AuCl4) to an acidified H2MP aqueous solution (1:1 molar ratio) and the final solution was acidified with HCl to pH=1.0. The Au(III)MP complex, KAu(MP)2, was obtained by adding an aqueous solution of K(AuCl4) to a basic H2MP solution (M:L - 1:2 molar ratio). Formulas for the complexes are: (Ag[C5H3N4S])*½H2O for AgHMP, (Au[C5H3N4S]) for AuIHMP, (Au[C5H3N4S][Cl]2)*2H2O for AuIIIHMP and K(Au[C5H2N4S]2)·2H2O for KAu(MP)2. The AuIHMP and KAu(MP)2 complexes decreased cell viability of HeLa cancer cells in vitro. The IC50 values for AuIHMP and KAu(MP)2 are 3.0 and 30.0 µM, respectively. Anti-M.tuberculosis assays showed a MIC value of 2.24 µM for AuIHMP and 5.12 µM for free MP while AgHMP is active at the concentration 93.2 µM.

Thiopurine derivatives containing triazole and steroid: synthesis, antimalarial and antileishmanial activities.

A series of novel 6-thiopurine derivates containing 1,2,3-triazole were synthesized and their in vivo antimalarial activity and in vitro antileishmanial activity were examined. The compounds 10, 11, 12 and 14 presented higher values of inhibition of parasite multiplication than chloroquine. For antileishmanial activity, the compound 14 showed activity against the three species of Leishmania tested. None of compounds showed cytotoxicity against mammalian cells.

Design and in vitro evaluation of branched peptide conjugates: turning nonspecific cytotoxic drugs into tumor-selective agents.

The use of peptide receptors as targets for tumor-selective therapies was envisaged years ago with the findings that receptors for different endogenous regulatory peptides are overexpressed in several primary and metastatic human tumors, and can be used as tumor antigens. Branched peptides can retain or even increase, through multivalent binding, the biological activity of a peptide and are very resistant to proteolysis, thus having a markedly higher in vivo activity compared with the corresponding monomeric peptides. Oligo-branched peptides, containing the human regulatory peptide neurotensin (NT) sequence, have been used as tumor-specific targeting agents. These peptides are able to selectively and specifically deliver effector units, for cell imaging or killing, to tumor cells that overexpress NT receptors. Results obtained with branched NT conjugated to different functional units for tumor imaging and therapy indicate that branched peptides are promising novel multifunctional targeting molecules. This study is focused on the role of the releasing pattern of drug-conjugated branched NT peptides. We present results obtained with oligo-branched neurotensin peptides conjugated to 6-mercaptopurin (6-MP), combretastain A-4 (CA4) and monastrol (MON). Drugs were conjugated to oligo-branched neurotensin through different linkers, and the mode-of-release, together with cytotoxicity, was studied in different human cancer cell lines. The results show that branched peptides are very promising pharmacodelivery options. Among our drug-armed branched peptides, NT4-CA4 was identified as a candidate for further development and evaluation in preclinical pharmacokinetic and pharmacodynamic studies. This peptide-drug exhibits significant activity against pancreas and prostate human cancer cells. Consequently, this derivative is of considerable interest due to the high mortality rates of pancreas neuroendocrine tumors and the high incidence of prostate cancer.

Novel derivatives of 6-mercaptopurine: synthesis, characterization and antiproliferative activities of S-allylthio-mercaptopurines.

Biologically active S-allylthio derivatives of 6-mercaptopurine (6-MP) and 6-mercaptopurine riboside (6-MPR) were synthesized. The products, S-allylthio-6-mercaptopurine (SA-6MP) and S-allylthio-6-mercaptopurine riboside (SA-6MPR) were characterized. The antiproliferative activity of the new prodrugs was tested on human leukemia and monolayer cell lines, and compared to that of their parent reactants. The new prodrugs acted by a concentration-dependent mechanism. They inhibited cell proliferation and induced-apoptosis more efficiently than the parent molecules. Leukemia cell lines were more sensitive to the new prodrugs than monolayer cell lines. Higher hydrophobicity of the derivatives improves their penetration into cells, where upon reaction with glutathione, S-allylthioglutathione (GSSA) is formed, and 6-MP or 6-MPR is released for further processing.

Synthesis and cytotoxic activity of imidazo[1,2-a]-1,3,5-triazine analogues of 6-mercaptopurine.

A series of 2-substituted imidazo[1,2-a]-1,3,5-triazines with various aliphatic and aromatic amines has been prepared and characterized by IR,1H-NMR, and elemental analysis. The initial in-vitro cytotoxicity studies with five human cancer cell lines showed that all but one of the compounds are without cytotoxic activity. The one active compound, 2-(indolin-1-yl)-7,8-dihydroimidazo[1,2-a]-1,3,5-triazine-4(6H)-thione 12, was tested on 12 human cancer cell lines. Of these, two lines, RT-4 and MCF-7, were the most sensitive to the compound, with IC50 values of 6.98 microM and 8.43 microM, respectively. When compared with the reference anticancer drug 6-mercaptopurine, only the RT-4 urinary bladder and KYSE-70 oesophagus cancer cell lines were more sensitive to the new compound. The antimetabolite thioguanine was more cytotoxic than 12 for all common cell lines tested.

A simple method for the preparation of PEG-6-mercaptopurine for oral administration.

A new and efficient method for the synthesis of PEG-6-mercaptopurine is described. The key feature of the proposed approach is the protection of the thiol group against metabolic inactivation. Preliminary in vivo and in vitro evaluations of the macromolecular prodrug have been carried out.

Water-soluble antitumor agents. I. Synthesis and biological activity of 6-S-aminoacyloxymethyl mercaptopurine derivatives.

In an attempt to improve the effectiveness and bioavailability of 6-mercaptopurine, various kinds of water-soluble analogues, such as 6-S-aminoacyloxymethyl mercaptopurine derivatives (3a--m) and 6-S,9-disubstituted derivates (7a,b and 9a,b), were synthesized. These compounds were evaluated for activity to augment antitumor immunity by using a double grated tumor system. Antitumor activities against solid tumors (sarcoma 180 and colon 26) were also evaluated. Many compounds exhibited potent activities in both test systems. In particular, the aminopropionate derivative (3a) and the L-glutamate derivative (3f) showed significant enhancement of antitumor immunity together with potent antitumor activities.

Purines. LXX. An extension of the "phenacylamine route" to the syntheses of the 7-N-oxides of 6-mercaptopurine and 6-methylthiopurine, and antileukemic activity of some purine N-oxides.

A full account is given of the first syntheses of 6-mercaptopurine 7-N-oxide (4) and 6-methylthiopurine 7-N-oxide (5). The synthesis of 4 followed a "phenacylamine route", which started from condensation of 4,6-dichloro-5-nitropyrimidine (15) with N-(4-methoxybenzyl)phenacylamine to form the phenacylaminopyrimidine derivative (11) and proceeded through conversion into the mercapto derivative, intramolecular cyclization between the NO2 nitrogen atom and the phenacyl carbanion to give 6-mercapto-9-(4-methoxybenzyl)purine 7-N-oxide (12), and removal of the 4-methoxybenzyl group. S-Methylation of 12 and removal of the 4-methoxybenzyl group afforded 5. The location of the oxygen function in 4,5, and 12 was confirmed by X-ray crystallographic analysis of 5.H2O, which was shown to exist in the N(7)-OH form (19). A UV spectroscopic approach suggested that the neutral species of 4 exists in H2O as the N(7)-OH tautomer (21), whereas that of 5 exists as an equilibrated mixture of the N(7)-oxide (5) and the N(7)-OH (19) tautomers. In the in vitro bioassay of antileukemic activity against murine L5178Y cells, the N-oxides 4 and 12 were found to be weakly cytotoxic.

Synthesis and SAR of 6-substituted purine derivatives as novel selective positive inotropes.

A series of purine derivatives was prepared and examined for selective inotropic activity in vitro and in vivo. Thioether-linked derivatives were superior to their oxygen and nitrogen isosteres. Substitution of electron-withdrawing groups on the benzhydryl moiety of these agents increased potency. The best compound of the study, 17 (carsatrin), was examined further and demonstrated selective oral activity as a positive inotrope. These compounds are presumed to act by affecting the kinetics of the cardiac sodium channel by analogy to the prototypic agent DPI 201106 (1). Their high selectivity for increasing contractile force and dP/dt without affecting blood pressure or heart rate is consistent with this mechanism. Carsatrin (17) was selected as a potential development candidate.

Prodrugs of 6-thiopurines: enhanced delivery through the skin.

Soft-alkylated derivatives of 6-mercaptopurine, its riboside, and 2-amino-6-mercaptopurine riboside have been prepared and evaluated to improve the delivery of the thiopurines through the skin. The soft-alkylated derivatives were prepared by the alkylation of the thiopurines with acylheteroalkyl halides under neutral or basic conditions. The penetration of the derivatives through hairless mouse skin was measured using diffusion cells. All of the derivatives underwent extensive degradation during their diffusion through skin so that the parent thiopurine, even in the case of the ribosides, was the major product observed in the receptor phase. The pivaloyloxymethyl derivatives showed the greatest potential for enhancing the penetration of the thiopurines through the skin. Among the 6-mercaptopurine derivatives, VII and XI were the most effective; they delivered 5 and 13 times, respectively, more 6-mercaptopurine than 6-mercaptopurine itself.

Probes of eukaryotic DNA-dependent RNA polymerase II-I. Binding of 9-beta-D-arabinofuranosyl-6-mercaptopurine to the elongation subsite.

9-beta-D-Arabinofuranosyl-6-mercaptopurine (ara-6-MP) was used to affinity-label wheat germ DNA-dependent RNA polymerase II (or B) (nucleosidetriphosphate:RNA nucleotidyltransferase, EC 2.7.7.6). This nucleoside analogue was found to be a competitive inhibitor with respect to [3H]UMP incorporation. Natural substrates protected the enzyme from inactivation by ara-6-MP when the enzyme was preincubated with excess concentrations of substrates, suggesting that the inhibitor binds at the elongation subsite. The inhibitor bound the catalytic center of the enzyme with a stoichiometry of 0.6:1. The sulfhydryl reagent, dithiothreitol, reversed the inhibition by ara-6-MP, suggesting that the 6-thiol group of the inhibitor was interacting closely with an essential cysteine residue in the catalytic center of the enzyme. Chromatographic analysis of the pronase-digestion products of the RNA polymerase II-ara-6-MP complex also showed that ara-6-MP had bound a cysteine residue. Sodium dodecyl sulfate-polyacrylamide gel electrophoresis of the denatured [6-35S]ara-6-MP-labeled RNA polymerase II revealed that over 80% of the radioactivity was associated with the IIb subunit of the enzyme.