Synthesis and anticancer activity of 4-amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine.

4-Amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (4) was recently synthesized and evaluated in our laboratories for anticancer activities. This compound showed potent in vitro inhibitory effects on the growth of HTB-81 prostate cancer cells and Daudi-lymphoma. In vivo studies showed that the compound could inhibit HTB-81 tumor growth in syngeneic mice by 93% at a daily dose of 8.5 mg/kg for 10 days.

Synthesis and cytotoxicity of 4'-C- and 5'-C-substituted toyocamycins.

Toyocamycin and some analogues have shown potent antitumor activities; however, none of them could be used clinically primarily owing to their cytotoxicity to normal human cells. In order to overcome the weakness of these nucleoside analogues, substitution of a variety of modified sugars for the ribofuranose was explored in our laboratories with expectation that certain sugar-modified toyocamycin analogues may be selectively cytotoxic to cancer cells. In this article, we report synthesis and cytotoxicity of 4'-C- and 5'-C-substituted toyocamycins, which were prepared via the condensations of 4-C- and 5-C-substituted ribofuranose derivatives 11, 12, 13, 20, 21, and 26 with the silylated form of 4-amino-6-bromo-5-cyanopyrrolo[2,3-]pyrimidine (27) and subsequent debromination and debenzoylation. When compared to the parent toyocamycin, all these analogues showed much lower cytotoxicity to human prostate cancer cells (HTB-81), mouse melanoma cancer cells (B16) as well as normal human fibroblasts. Compound 1e showed a significant cytotoxicity to the prostate cancer cells and a moderate selectivity. The results suggested that sugar modifications, especially those that may affect phosphorylation of nucleosides, could alter cytotoxicity profile significantly.

Synthesis and cytotoxicity of 4-amino-5-oxopyrido[2,3-d]pyrimidine nucleosides.

A number of nucleoside analogues have been either used clinically as anticancer drugs or evaluated in clinical studies, while new nucleoside analogues continue to show promise. In this article, we report synthesis and cytotoxicity of a series of new pyrido[2, 3-d]pyrimidine nucleosides. 2-Amino-3-cyano-4-methoxypyridine was converted, in two steps, to 4-amino-5-oxopyrido[2,3-d]pyrimidine. A variety of 1-O-acetylated pentose sugar derivatives were condensed with silylated 4-amino-5-oxopyrido[2,3-d]pyrimidine, followed by protection, to afford a series of 4-amino-5-oxopyrido[2, 3-d]pyrimidine nucleosides. Further derivatizations provided an additional group of pyrido[2,3-d]pyrimidine nucleosides. These nucleosides were evaluated for in vitro cytotoxicity to human prostate cancer (HTB-81) and mouse melanoma (B16) cells as well as normal human fibroblasts (NHF). A number of compounds (1a,b, 2a-c,f, 3f+4d) showed significant cytotoxicity to cancer cells, with 4-amino-5-oxo-8-(beta-D-ribofuranosyl)pyrido[2,3-d]pyrimidine (1b) being the most potent proliferation inhibitor (EC(50): 0.06-0.08 microM) to all types of cells tested. However, a selective inhibition to the cancer cells was observed for 4-amino-5-oxo-8-(beta-D-xylofuranosyl)pyrido[2,3-d]pyrimidine (2b), which is a potent inhibitor of HTB-81 (EC(50): 0.73 microM) and has a favorable in vitro selectivity index (28).

Synthesis and in vitro antiviral activity of some symmetrical phosphoramidate dimers of AZT.

The synthesis of some symmetrical phosphoramidate dimers of AZT is presented. The synthetic scheme includes the formation of the symmetrical H-phosphonate diester of AZT, followed by its conversion to several dinucleoside phosphoramidate analogues. The compounds were evaluated for their anti-retroviral activity.

Conformationally locked nucleosides. Synthesis and hybridization properties of oligodeoxynucleotides containing 2',4'-C-bridged 2'-deoxynucleosides.

A conformationally locked, 2',4'-C-bridged 2'-deoxyribofuranoside2 was condensed with silylated pyrimidines to give 2',4'-C-bridged bicyclonucleosides, which were converted to the phosphoramidites and incorporated into oligodeoxynucleotides (ODNs). The hybridization data of the modified ODNs to DNA and RNA are presented.

The S-acyl-2-thioethyl pronucleotide approach applied to acyclovir: part I. Synthesis and in vitro anti-hepatitis B virus activity of bis(S-acyl-2-thioethyl)phosphotriester derivatives of acyclovir.

The synthesis and in vitro anti-hepatitis B virus (HBV) activity of two mononucleoside phosphotriester derivatives of acyclovir incorporating S-acyl-2-thioethyl (SATE) groups are reported. In contrast to the parent nucleoside, the described phosphotriesters emerged as potent and selective inhibitors of HBV replication in HepG2.2.15 cells. This result can be attributed to the unique cellular metabolism of the SATE pronucleotides giving rise to the delivery to acyclovir 5'-monophosphate inside the infected cells. Moreover, the in vitro anti-HBV activities of one of these bis(SATE)phosphotriesters and of (-)-beta-L-2',3'-dideoxy-3'-thiacytidine (lamivudine, 3TC) were compared alone and in combination. Analysis of the combination data indicates that 3TC and the studied SATE pronucleotide of acyclovir exhibited strong synergistic interactions. The present study provides an example where the use of a pronucleotide approach extends the antiviral spectrum of a nucleoside analogue. Given the potency of SATE pronucleotides of acyclovir against HBV in HepG2.2.15 cells, further studies including animal experiments seem warranted to evaluate the potential of these compounds as anti-HBV agents.

Synthesis and anti-retroviral activity of O,O'-bis(3'-azido-2',3'-dideoxythymidin-5'-yl) phosphoramidate derivatives.

A simple and efficient protocol for the preparation of various symmetrical dinucleoside phosphoramidates derived from AZT, is presented. It consists of the phosphonylation of AZT with phosphonic acid in the presence of DCC to produce the symmetrical H-phosphonate diester, followed by its oxidative conversion to various phosphoramidate analogues. The synthesized compounds were evaluated for their anti-HIV activity in different cell cultures.

Design, synthesis, and antiviral activity of alpha-nucleosides: D- and L-isomers of lyxofuranosyl- and (5-deoxylyxofuranosyl)benzimidazoles.

Several 2-substituted alpha-D- and alpha-L-lyxofuranosyl and 5-deoxylyxofuranosyl derivatives of 5,6-dicholro-2-(isopropylamino)-1-(beta-L-ribofuranosyl) benzimidazole (1263W94) and 2,5,6-trichloro-1(beta-D-ribofuranosyl)benzimidazole (TCRB) were synthesized and evaluated for activity against two herpesviruses (HSV-1 and HCMV) and for their cytotoxicity against HFF and KB cells. Condensation of 1,2,3,5-tetra-O-acetyl-L-lyxofuranose (2a) with 2,5,6-trichlorobenzimidazole (1) yielded the alpha-nucleoside 3a. The 2-bromo derivative and 2-methylamino derivative were prepared by treatment of 3a with HBr followed by deprotection or from methylamine, respectively. Compound 3a was deprotected and the resultant nucleoside used to prepare the 2-cyclopropylamino and 2-isopropylamino derivatives. The 2-alkylthio nucleosides were prepared by condensing 2a with 5,6-dichlorobenzimidazole-2-thione followed by deprotection. Alkylation of this adduct gave the 2-methylthio and 2-benzylthio derivatives. Condensation of 5-deoxy-1,2,3-tri-O-acetyl-L-lyxofuranosyl, prepared from L-lyxose, with 1 or 2-bromo-5,6-dichlorobenzimidazole (15), followed by deprotection, gave the 2-chloro or 2-bromo-5'-deoxylyxo-furanosyl derivative, respectively. The cyclopropylamino derivative was prepared from the 2-chloro derivative. All D-isomers were prepared in an analogous fashion from D-lyxose. Either compounds were inactive against HSV-1 or weak activity was poorly separated from cytotoxicity. In contrast, the 2-halogen derivatives in both the alpha-lyxose and 5-deoxy-alpha-lyxose series were active against the Towne strain of HCMV. The 5-deoxy alpha-L analogues were the most active, IC50's = 0.2-0.4 microM, plaque assay; IC90's = 0.2-2 microM, yield reduction assay. All of the 2-isopropylamino or 2-cyclopropylamino derivatives were less active (IC50's = 60-100 microM, plaque assay; IC90's = 17-100 microM, yield reduction assay) and were not cytotoxic. The methylamino, thio, and methylthio derivatives were neither active nor cytotoxic. The benzylthio derivatives were weakly active, but this activity was poorly separated from cytotoxicity. The alpha-lyxose L-isomers were more active in a plaque assay against the AD169 strain of HCMV compared to the Towne strain, thereby providing additional evidence of antiviral specificity.

Decomposition pathways and in vitro HIV inhibitory effects of isoddA pronucleotides: toward a rational approach for intracellular delivery of nucleoside 5'-monophosphates.

The decomposition pathways and kinetics in various biological media and the in vitro anti-HIV-1 and anti-HIV-2 activities of four derivatives of the 5'-mononucleotide of isoddA incorporating carboxylate esterase-labile transient phosphate protecting groups are reported and compared: namely, two mononucleoside aryl phosphoramidate derivatives 1a,b and two mononucleoside phosphotriester derivatives incorporating two S-acyl-2-thioethyl groups 2a,b. All four compounds show better antiviral activity, compared to the parent nucleoside analog isoddA. The results highlight that both types of compounds act as pronucleotides, i.e. they exert their antiviral effect via intracellular delivery of the 5'-mononucleotide of isoddA. The results may give insights for the design of new more efficient pronucleotides.

Anti-human immunodeficiency virus type 1 activities of dideoxynucleoside phosphotriester derivatives in primary monocytes/macrophages.

Mononucleoside phosphotriester derivatives of dideoxynucleosides, following intracellular enzymatic activation, are likely to liberate their parent 5'-nucleoside monophosphate. Prodrugs of 3'-azido-2',3'-dideoxythymidine (AZT), 2',3'-didehydro-2',3'- dideoxythymidine (d4T), 2',3'-dideoxyinosine (ddl) and 2',3'-dideoxyadenosine (ddA) were evaluated for their anti-HIV1 activities in monocyte-derived macrophages, cells which are known to have low levels of nucleoside kinases. Prodrugs were found to be much more active, or just as active, as the corresponding dideoxynucleoside. Furthermore, their selectivity index was enhanced by a factor of 2 to 200.

New insights regarding the potential of the pronucleotide approach in antiviral chemotherapy.

The rationale for a pronucleotide approach based on the use of phosphotriesters which incorporate enzyme-mediated bio-labile protection is discussed in detail. Among the studied bio-labile phosphate protecting groups, the S-acyl-2-thioethyl (SATE) groups appeared the most promising as exemplified in cell culture systems in the case of the pronucleotides of 3'-azido-3'-deoxythymidine, 2',3'-didehydro-3'-deoxythymidine, 2',3'-dideoxyadenosine and acyclovir In vivo implementations of such bis(SATE) pronucleotides have been planned for future animal studies.

Mononucleoside phosphotriester derivatives with S-acyl-2-thioethyl bioreversible phosphate-protecting groups: intracellular delivery of 3'-azido-2',3'-dideoxythymidine 5'-monophosphate.

The synthesis, in vitro anti-HIV-1 activity, and decomposition pathways of several mononucleoside phosphotriester derivatives of 3'-azido-2',3'-dideoxythymidine (AZT) incorporating a new kind of carboxylate esterase-labile transient phosphate-protecting group, namely, S-acyl-2-thioethyl, are reported. All the described compounds showed marked antiviral activity in thymidine kinase-deficient CEM cells in which AZT was virtually inactive. The results strongly support the hypothesis that such pronucleotides exert their biological effects via intracellular delivery of the 5'-mononucleotide of AZT. This point was corroborated by decomposition studies in cell extracts and culture medium.

Equal inhibition of the replication of human immunodeficiency virus in human T-cell culture by ddA bis(SATE)phosphotriester and 3'-azido-2',3'-dideoxythymidine.

It is shown that ddA bis(SATE)phosphotriester is one of the most potent anti-HIV agents in cell culture. Compared with the parent nucleoside, ddA, an increase of 3 orders of magnitude was observed in the EC50, which makes this compound as active as AZT. This can be tentatively explained if one considers that direct ddAMP intracellular delivery shunts the well established ddA/ddI metabolism pathway.

Localization of a putative magnesium-binding site within the cytoplasmic domain of the sarcoplasmic reticulum Ca(2+)-ATPase.

The amino acid sequences of several P-type ATPases share regions of high similarity. The functions of some of these regions, although several proposals have been made, have not yet been absolutely identified. In particular, one of these domains, located within the cytoplasmic loop in the area known as the 'hinge' domain, exhibits the highest degree of conservation. In the sarcoplasmic reticulum Ca(2+)-ATPase (SERCA-1), this region is located at residues 700-712. Comparison of the sequence in this domain with calcium-binding proteins reveals similarities with the center of the helix-loop-helix EF-hand structure that is known to form divalent-cation-binding sites. A 38-residue polypeptide, corresponding to the domain 682-719 of the Ca(2+)-ATPase was synthesized and tested for its ability to bind divalent cations. Circular-dichroism, intrinsic-fluorescence and fluorescence-energy-transfer studies performed on this polypeptide in solution support the hypothesis that this domain has, in the protein, the ability to bind a divalent cation, presumably Mg2+, with an affinity of 10-15 mM. This property is observed for the isolated polypeptide in aqueous solvent and in the presence of low concentrations of the alpha-helix promoter 2,2,2-trifluoroethanol. Substitution of either one or two critical amino acids in the sequence induces a significant reduction of the binding properties. It is proposed that this sequence is involved in the co-ordination of a Mg2+ in the nucleotide-binding site and/or in the phosphorylation site of P-type ATPases.

Characterisation of ATP binding inhibition to the sarcoplasmic reticulum Ca(2+)-ATPase by thapsigargin.

The inhibition of Ca(2+)-ATPase of sarcoplasmic reticulum by thapsigargin has been reported to be associated with a suppression of calcium binding to the high affinity transport sites. We report here that thapsigargin also acts as an inhibitor of ATP binding by reducing its apparent affinity by about two orders of magnitude. This inhibition is non-competitive indicating that thapsigargin does not bind to the ATP binding site. This is confirmed by the fact that thapsigargin binding to the Ca(2+)-ATPase does not affect the binding of 2',3'-O-(2,4,6-trinitrocyclohexadienylidene)-ATP (TNP-ATP).

Fluoroaluminate complexes are bifunctional analogues of phosphate in sarcoplasmic reticulum Ca(2+)-ATPase.

The mechanism of inhibition of the sarcoplamc reticulum (SR) Ca(2+)-ATPase by the fluoroaluminate complexes was investigated. First, AlF4- was shown to bind to the Ca(2+)-free conformation of the enzyme by a slow quasi-irreversible process. The rate constants of the reaction are k+ = 16 x 10(3) M-1 s-1 and k- < 1.5 10(-3) s-1. We directly measured a stoichiometry of about 4.8 nmol of AlF4- bound/mg of protein. Mg2+ was a necessary cofactor for the reaction with a dissociation constant of 3 mM. It was demonstrated (Dupont, Y., and Pougeois, R. (1983) FEBS Lett. 156, 93-98) that phosphorylation by P(i) induced a dehydration of the catalytic site. The same process has been shown here to occur upon AlF4- binding either by the use of Me2SO or by demonstration of an increase of bound 2',3'-O-(2,4,6-trinitrocyclohexadienyldene)adenosine triphosphate fluorescence. Phosphorylation by P(i) is inhibited by the binding of AlF4-. Second, a fluoroaluminate complex, presumably AlF4-, was also shown to bind to the Ca(2+)-bound conformation of the Ca(2+)-ATPase in the presence of ADP and stabilize a E1.Ca2.ADP.AlFx complex. The dissociation constant of the nucleotidic site for ADP was shifted to the micromolar range. The Ca2+ ions bound on the external high affinity sites became occluded upon binding of (ADP + AlFx). We propose that AlF4- mimics P(i) binding to the Ca(2+)-free conformation of the ATPase and stabilizes an intermediate similar to the acyl-phosphate derivative; it also acts as an analogue of the gamma-phosphate of ATP and stabilizes an E1.[Ca2].ADP.AlF4 complex where the Ca2+ ions are occluded.

A reexamination of the stopped-flow measured hydrogen-deuterium exchange rates in nucleic acid double helices.

Using the deuteration labelling method in conjunction with an improved stopped-flow instrument, we have reexamined the proton exchange process in poly(dA-dT).poly(dA-dT). A single proton exchange class is found with a rate constant at 20 degrees C of 0.4 S-1. This exchange rate is independent of buffer concentration. From the comparison of these results with those obtained in a former study where two rates were measured, it is concluded that the present deuteration signal corresponds to the exchange of the amino protons, whereas the imino proton exchange is not detected.

Ellipticity changes of the sarcoplasmic reticulum Ca(2+)-ATPase induced by cation binding and phosphorylation.

The sarcoplasmic reticulum (SR) Ca(2+)-ATPase is a member of the 'P-type' class of cation transport ATPases which form a covalent phosphorylated intermediate. It has been proposed that during ion transport, these proteins cyclically adopt two major enzymatic states E1 and E2, that are related to two essential conformations of the protein. By the use of especially sensitive circular dichroism (CD) instrumentation it is shown here that Ca2+ addition induces 5% or 2.5% increases in Ca(2+)-ATPase ellipticity at 225 nm in the absence or in the presence of Mg2+, respectively. Furthermore, a 2% change in the same direction was observed when the enzyme was phosphorylated with Pi in the absence of Ca2+. These results suggest that the E1----E2 transition and the E2-P formation are associated with structural changes of the polypeptide backbone structure of the calcium pump protein.

Fluoride, beryllium and ADP combine as a ternary complex in aqueous solution as revealed by a multinuclear NMR study.

Different kinds of nucleotide binding enzymes are sensitive to fluoroberyllate complexes (BeFx) and fluoroaluminate complexes (AlFy). It has been hypothesized that the effects of these fluorometals are related to the generation at a nucleotide binding site of a pseudo nucleoside triphosphate, consisting of a fluorometal moiety bound to the beta phosphate group of a molecule of nucleoside diphosphate (Bigay et al. 1985; Lunardi et al. 1985). In order to establish whether ternary complexes comprising ADP, beryllium and fluoride can exist in slightly alkaline solution in the absence of enzyme, we have carried out a multinuclear (31P, 9Be and 19F) NMR study. In preliminary experiments, pyrophosphate (PPi) was substituted for ADP and taken as a simpler analog of nucleoside diphosphate. In the absence of fluoride, three types of PPi-Be complexes were generated: two of these were bidentate chelates with either one or two pyrophosphate molecules bound per beryllium; the third one was a monodentate complex. It is probable that the same types of combination exist between the polyphosphate chain of ADP and Be. In the presence of fluoride, both ADP and PPi combined with beryllium to form ternary complexes. These complexes consisted of monofluoroberyllate(-BeF) or difluoroberyllate(-BeF2) bound to the two phosphates of one molecule of ADP or PPi as a bidentate chelate. We failed to observe the formation of complexes between ADP and trifluoroberyllate(-BeF3). The relevance of this study to the biological effects of fluoride and beryllium on various enzymic reactions is discussed.

Evidence of a calcium-induced structural change in the ATP-binding site of the sarcoplasmic-reticulum Ca2+-ATPase using terbium formycin triphosphate as an analogue of Mg-ATP.

Terbium ions and terbium formycin triphosphate have been used to investigate the interactions between the cation and nucleotide binding sites of the sarcoplasmic reticulum Ca2+-ATPase. Three classes of Tb3+-binding sites have been found: a first class of low-affinity (Kd = 10 microM) corresponds to magnesium binding sites, located near a tryptophan residue of the protein; a second class of much higher affinity (less than 0.1 microM) corresponds to the calcium transport sites, their occupancy by terbium induces the E1 to E2 conformational change of the Ca2+-ATPase; a third class of sites is revealed by following the fluorescence transfer from formycin triphosphate (FTP) to terbium, evidencing that terbium ions can also bind into the nucleotide binding site at the same time as FTP. Substitution of H2O by D2O shows that Tb-FTP binding to the enzyme nucleotide site is associated with an important dehydration of the terbium ions associated with FTP. Two terbium ions, at least, bind to the Ca2+-ATPase in the close vicinity of FTP when this nucleotide is bound to the ATPase nucleotide site. Addition of calcium quenches the fluorescence signal of the terbium-FTP complex bound to the enzyme. Calcium concentration dependence shows that this effect is associated with the replacement of terbium by calcium in the transport sites, inducing the E2----E1 transconformation when calcium is bound. One interpretation of this fluorescence quenching is that the E1----E2 transition induces an important structural change in the nucleotide site. Another interpretation is that the high-affinity calcium sites are located very close to the Tb-FTP complex bound to the nucleotide site.