Synthesis and antiproliferative effects of novel 5'-fluorinated analogues of 5'-deoxy-5'-(methylthio)adenosine

5'-Deoxy-5'-[(monofluoromethyl)thio]adenosine (9) and 5'-deoxy-5'-fluoro-5'-(methylthio)adenosine (10), two novel analogues of 5'-deoxy-5'-(methylthio)adenosine (MTA), have been synthesized and evaluated for their substrate and inhibitory activities toward MTA phosphorylase and for their biological effects in L1210 (MTA phosphorylase deficient) and L5178Y (MTA phosphorylase containing) murine leukemia cell lines. Compound 9 was a potent competitive inhibitor of MTA phosphorylase with a Ki value of 3.3 microM and was also a substrate, with activity approximately 53% that of MTA. Compound 10 was significantly less inhibitory toward the phosphorylase with a Ki value of 141 microM; its lack of substrate activity was attributed to rapid nonenzymatic degradation. The 50% growth inhibitory concentrations (48 h) of 9 were 300 and 200 microM in L1210 and L5178Y cells, respectively; for 10, these respective values were 2 and 0.7 microM. The initial characterization of 9 in these systems reveals that it differs from MTA by not acting as a product regulator of the polyamine biosynthetic pathway.     

5'-Deoxy-5'-methylthioadenosine phosphorylase--III. Role of the enzyme in the metabolism and action of 5'-halogenated adenosine analogs

5'-Deoxy-5'-halogenated adenosines are alternative substrates for 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAPase), an enzyme responsible for the metabolism of 5'-deoxy-5'-methylthioadenosine (MTA), a by-product of polyamine biosynthesis. The relative reactivity of these nucleosides with MTAPase from HL-60 human promyelocytic leukemia cells is MTA greater than 5'-deoxy-5'-fluoroadenosine (5'-FlAdo) greater than 5'-chloro-5'-deoxyadenosine (5'-ClAdo) greter than 5'-bromo-5'-deoxyadenosine (5'-BrAdo) greater than 5'-deoxy-5'-iodoadenosine (5'-IAdo). In MTAPase-containing cells, the adenine released from the 5'-halogenated adenosine was incorporated into adenine nucleotide pools; cleavage by (MTAPase appeared to be the rate-limiting step in this process. 5'-BrAdo and 5'-IAdo were growth inhibitors (EC50 values less than 10 microM) of MTAPase-containing cell lines (HL-60 human promyelocytic leukemia and the L5178Y murine lymphoblastic leukemia) but were much less active (EC50 values greater than 65 microM) against MTAPase-deficient cell lines (the CCRF-CEM human T cell leukemia and the L1210 murine leukemia). The full cytotoxicity of these compounds, therefore, appeared to be related to their phosphorolysis by MTAPase. Indirect evidence suggests that 5-halogenated ribose-1-phosphate derivatives of 5'-BrAdo or 5'-IAdo produced by the MTAPase reaction were the active metabolites of these 5'-halogenated adenosines.     

5'-deoxy-5'-methylthioadenosine phosphorylase--IV. Biological activity of 2-fluoroadenine-substituted 5'-deoxy-5'-methylthioadenosine analogs

5'-Deoxy-5'-methylthioadenosine phosphorylase (MTAPase) phosphorolyzes 5'-deoxy-5'-methylthioadenosine (MTA) generated during polyamine biosynthesis to adenine and 5-methylthioribose-1-phosphate. Two doubly-substituted, 2-fluoroadenine-containing analogs of MTA, 5'-deoxy-2-fluoroadenosine (5'-dFAdo) and 5'-deoxy-5'-iodo-2-fluoroadenosine (5'-IFAdo), were synthesized and studied as substrates of MTAPase: their reaction with this enzyme resulted in the liberation of the cytotoxic base, 2-fluoroadenine, as well as potentially cytotoxic analogs of 5-methylribose-1-phosphate. The activities of these MTA analogs were compared to that of the singly-substituted analog, 5'-deoxy-5'-methylthio-2-fluoroadenosine (5'-MTFAdo). The cytotoxic action of these MTA analogs depended primarily on their conversion to 2-fluoroadenine-containing nucleotides, as a cell line that contains both MTAPase and adenine phosphoribosyltransferase (APRT) activity (HL-60 human promyelocytic leukemia) readily converted these MTA analogs to 2-fluoroadenine-containing nucleotides (especially 2-fluoroadenosine triphosphate) and was highly sensitive to the growth-inhibitory effects of all three compounds (IC50 values in the 10(-8) M range), whereas cell lines lacking MTAPase (CCRF-CEM human T-cell leukemia) or APRT (HL-60/aprt1 cells) did not form analog nucleotides and were relatively insensitive to these compounds (IC50 values in the 10(-5) M range). The doubly-substituted analogs were not more growth inhibitory than 5'-MTFAdo in wild type HL-60 cells as the potent effects of 2-fluoroadenine may mask the activity of the 5-methylthioribose-1-phosphate analogs generated in the reaction of these compounds with MTAPase. 5'-dFAdo and 5'-IFAdo also were irreversible inhibitors of S-adenosylhomocysteine hydrolase, which may explain in part the weak but observable growth inhibitory action of these compounds against MTAPase-deficient cell lines.     

Antitrypanosomal activity of 5'-deoxy-5'-(iodomethylene)adenosine and related 6-N-cyclopropyladenosine analogues

Treatment of the 6-N-cyclopropyl-2',3'-di-O-isopropylideneadenosine 5'-aldehyde with sulfone-stabilized phosphonate or fluorophosphonate reagents followed by stannyldesulfonylations and subsequent iodo- or protiodestannylation gave 6-N-cyclopropyl-5'-deoxy-5'-(iodomethylene)adenosine 8b or its 5'-fluoromethylene analogue 11. Treatment of the 5'-aldehyde with hydroxylamine or dibromomethylene- or cyanomethylene-stabilized Wittig reagents and deprotections gave the oxime 4b, 5'-cyanomethylene 5b, and 5'-dibromomethylene 13b analogues. Dehydrobromination of 13b gave acetylenic compound 14b. From the tested 6-N-cyclopropyladenosine analogues modified at the 5' carbon, the 5'-iodomethylene 8b had the most potent activity against Trypanosoma brucei in vitro with an IC50 of 12 microg/mL. The IC50 value was 19 microg/mL for both the 5'-fluoromethylene 11 and the 5'-cyanomethylene 5b compounds. The (E)-5'-deoxy-5'-(iodomethylene)adenosine 2a, a known inhibitor of AdoHcy hydrolase not modified with a cyclopropyl ring at 6-amino group, also inhibited T. brucei with an IC50 of 9 microg/mL. In contrast to some other adenosine analogues modified at C5', the 6-N-cyclopropyladenosine analogues described here do not exhibit an inhibitory effect on AdoHcy hydrolase and displayed only marginal antiviral activity.     

Inhibition of muscarinic receptor binding and acetylcholine-induced contraction of guinea pig ileum by analogues of 5'-(isobutylthio)adenosine

(Isobutylthio)adenosine (SIBA, 1) and its derivatives have been shown to produce a variety of biological effects on the basis of the hypothesis that such agents act directly as inhibitors of transmethylation reactions, as inhibitors of S-adenosylhomocysteine hydrolase, or as inhibitors of polyamine biosynthesis. We report here the ability of selected analogues of SIBA to inhibit the binding of the muscarinic antagonist quinuclidinyl benzilate (QNB) to cultured N4TG1 neuroblastoma cells and to antagonize the acetylcholine-induced contraction of guinea pig ileum. The most potent inhibitors were 5'-deoxy-5'-(isobutylthio)-1-deazaadenosine (1-deaza-SIBA, 5) and 5'-deoxy-5'-(isobutylthio)-3-deazaadenosine (3-deaza SIBA, 3), while the parent nucleoside SIBA and the carbocyclic derivative 5'-(isobutylthio)-3-deazaaristeromycin were less active. The same agents had no effect on the nicotinic receptors of NG108-15 neuroblastoma X glioma hybrid cells. The acyclic derivative 9-[[2-(isobutylthio)ethoxy]methyl]adenine, 3-deazaadenosine, 5'-(isobutylthio)tubercidin, and 5'-(isobutylamino)adenosine were inactive at the 1-mM level. These results suggest that SIBA and 3-deaza-SIBA may have profound effect on membrane-mediated phenomenon, including inhibition of muscarinic receptor binding.     

5'-deoxy-5'-methylthioadenosine phosphorylase--II. Role of the enzyme in the metabolism and antineoplastic action of adenine-substituted analogs of 5'-deoxy-5'-methylthioadenosine

The biological activities of several previously synthesized [J. A. Montgomery et al., J. med. Chem. 17, 1197 (1974)] adenine-substituted analogs of 5'-deoxy-5'-methylthio- or 5'-deoxy-5'-ethyl-thioadenosine, including the 2-fluoroadenine, 2-chloroadenine, 2,6-diaminopurine, 8-azaadenine, and 4-aminopyrazolo [3,4-d]pyrimidine-containing derivatives, have been reexamined. It is demonstrated that many of these analogs are cleaved to their respective free base analogs by 5'-deoxy-5'-methyl-thioadenosine phosphorylase (MTAPase), an enzyme associated with polyamine biosynthesis, and that this reaction is necessary for the cytotoxic action of these MTA analogs to be fully expressed. Evidence to support this includes: (1) the growth of two MTAPase-containing human colon carcinoma cell lines (the HCT-15 and DLD-1 lines) was inhibited by these analogs, whereas an MTAPase-deficient cell line, the CCRF-CEM human T-cell leukemia, was relatively insensitive to their cytotoxic action; (2) extracts of the MTAPase-containing colon carcinoma cell lines were able to cleave these analogs to their respective free base analogs; in contrast, extracts of MTAPase-deficient CCRF-CEM cells were unable to cleave these analogs; (3) intact colon carcinoma cells converted these MTA analogs to their corresponding 5'-phosphorylated analog nucleotides, whereas CCRF-CEM cells did not, at least to detectable levels; and (4) the MTA analog, 5'-deoxy-5'-ethylthio-4-aminopyrazolo [3,4-d]pyrimidine ribonucleoside, which is not a substrate of MTAPase, did not form analog nucleotides and was essentially noncytotoxic to all cell lines tested, whereas the corresponding adenine analog, 4-aminopyrazolo [3,4-d]pyrimidine, readily formed analog nucleotides and was highly cytotoxic to all the lines. It is postulated that the corresponding adenine analog 5'-phosphorylated nucleotides are the primary active metabolites of these MTA analogs, having been formed by the cleavage of these nucleosides to free adenine analogs by MTAPase, followed by the conversion of these base analogs to analog nucleotides by adenine phosphoribosyltransferase and the enzymes of adenine nucleotide phosphorylation. This pathway represents a novel drug-activation system for the synthesis of analog nucleotides and has the potential to be exploited chemotherapeutically.     

5'-deoxy-5'-methylthioadenosine phosphorylase--V. Acycloadenosine derivatives as inhibitors of the enzyme

Various adenosine acyclonucleoside derivatives were tested as inhibitors of 5'-deoxy-5'-methylthioadenosine (MeSAdo) phosphorylase, an enzyme involved in the salvage of adenine and methionine from MeSAdo. The 2-halogenated derivatives of acyloadenosine [9-(2-hydroxyethoxy-methyl)adenine], including the chloro-, bromo- and iodo-congeners, all inhibited murine Sarcoma 180 (S180) MeSAdo phosphorylase, with Ki values in the range of 10(-6) to 10(-5) M. Halogenated derivatives of 9-(1,3-dihydroxy-2-propoxymethyl)adenine, which more closely resemble the natural substrate, were substantially more potent inhibitors of the enzyme, with Ki values in the range of 2-7 x 10(-7) M. 5'-Methylthio and 5'-halogenated analogs of 2'-deoxy-1',2'-seco-adenosine were weak inhibitors, with Ki values of 10(-4) M or greater. 9-[(1-Hydroxy-3-iodo-2-proxy)methyl]adenine. (HIPA), the derivative with the lowest Ki values among these analogs, was a competitive inhibitor of S180 MeSAdo phosphorylase. In preliminary studies, HIPA inhibited MeSAdo phosphorylase in intact HL-60 human promyelocytic leukemia cells, as it limited the incorporation of [8-14C]MeSAdo into cellular adenine nucleotide pools. In addition, 9-(phosphonoalkyl)adenines, representing potential multisubstrate inhibitors of MeSADo phosphorylase, were synthesized. Of these the heptyl derivative was the most potent inhibitor, with a Ki of 1.5 x 10(-5) M at low (3.5 mM) phosphate concentrations. The inhibitory effects of these analogs could be ablated at high phosphate concentrations (50 mM), suggesting that they interact with the phosphate binding site on the enzyme. Some of these novel MeSAdo phosphorylase inhibitors may have a role in cancer chemotherapy as potentiators of agents that block purine de novo synthesis, e.g. antifolates and 6-methylmercaptopurine ribonucleoside.     

Synthesis and biological properties of purine and pyrimidine 5'-deoxy-5'-(dihydroxyphosphinyl)-beta-D-ribofuranosyl analogues of AMP, GMP, IMP, and CMP

Methyl 2,3-O-isopropylidene-D-ribofuranoside (1) was converted to 1-O-acetyl-5-bromo-5-deoxy-2,3-di-O-benzoyl-D-ribofuranose (6) in five steps with good yield. The Arbuzov condensation of compound 6 with triethyl phosphite resulted in the synthesis of 1-O-acetyl-2,3-di-O-benzoyl-5-deoxy-5-(diethoxyphosphinyl)-D-ribofuranos e (7). Compound 7 was used for direct glycosylation of both purine and pyrimidine bases. The glycosylation was accomplished with the dry silylated heterocyclic base in the presence of trimethylsilyl triflate. Deblocking of the glycosylation products gave exclusively the beta anomer of the 5'-phosphonate analogues of 9-[5'-deoxy-5'-(dihydroxyphosphinyl)-beta-D-ribofuranosyl]adenine (13), 9-[5'-deoxy-5'-(dihydroxyphosphinyl)-beta-D-ribofuranosyl]guanosin e (16), 9-[5'-deoxy-5'-(dihydroxyphosphinyl)-beta-D-ribofuranosyl]hypoxant hine (17), and 9-[5'-deoxy-5'-(dihydroxyphosphinyl)-beta-D-ribofuranosyl]cytosine (15), described here for the first time. The target compounds as well as their intermediates showed no in vitro antiviral or antitumor activity, although phosphorylation of 15 and 16 to di- and triphosphate analogues was demonstrated with use of isolated cellular enzymes.     

Synthesis of a potent transition-state inhibitor of 5'-deoxy-5'-methylthioadenosine phosphorylase

Human 5'-deoxy-5'-methylthioadenosine phosphorylase (MTAP) participates in the purine salvage pathway to generate adenine and methylthioribose-1-phosphate, which in turn is converted into adenine nucleotides and methionine. Hence, inhibition of MTA phosphorylase may be an effective target in the design of potential antiproliferative agents. Presented herein is the synthesis of 2-(4-amino-5H-pyrrolo[3,2-d]pyrimidin-7-yl)-5-methylsulfanylmethylpyrrolidin-3,4-diol (1), a potent inhibitor of MTAP.     

Characterization of the effects of adenosine 5'-[beta-thio]-diphosphate in rat liver.

1. In rat liver cells micromolar concentrations of adenosine 5'-[beta-thio]diphosphate (ADP beta S), activate glycogen phosphorylase by an adenosine 3':5'-cyclic monophosphate (cyclic AMP)- independent mechanism. 2. As with adenosine 5'-triphosphate (ATP), ADP beta S also inhibits the rise in cyclic AMP after glucagon. 3. Cytosolic Ca2+ measured in single cells is rapidly increased with a pattern similar for ADP beta S and for ATP. 4. At variance with ATP, ADP beta S hardly increases inositol 1,4,5-trisphosphate (IP3) levels. 5. Phorbol myristic acetate, which inhibits only slightly the glycogenolytic effect of ATP, almost completely abolishes this effect of ADP beta S. 6. With adenosine 5'-[beta-[35S]thio]diphosphate (ADP beta[35S]) as radioligand, we detected specific purinoceptors on rat liver plasma membranes. Binding consists of a major binding component with KD = 0.7 microM and Bmax = 51 pmol mg-1 of protein, probably mediating the activation of glycogen phosphorylase, and a minor high affinity, low capacity binding component with no obvious function. 7. It is concluded that the differences in biological effects between ATP and ADP beta S may involve different receptors and/or different transduction mechanisms and that ADP beta[35S] can be used to detect the specific binding sites for ADP beta S.     

Antineoplastic activity of 3'-(chloroethyl)nitrosourea analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine

The (chloroethyl)nitrosourea analogues of 2'-deoxyuridine and 2'-deoxy-5-fluorouridine, 3'-[3-(2-chloroethyl)-3-nitrosoureido]-2',3'-dideoxyuridine (3'-CdUNU, 7) and 3'-[3-(2-chloroethyl)-3-nitrosoureido]-2,3'-dideoxy-5-fluorouridine (3'-CFdUNU, 8), have been synthesized by treatment of the corresponding 3'-amino nucleosides with chloroethyl isocyanate, followed by nitrosation of the resulting ureas. Nucleoside nitrosoureas 7 and 8 exhibited marked anticancer activity against L1210 leukemia in tumor-bearing mice. At an optimum dosage level of 40 mg/kg, 7 and 8 produced 90% and 60% "cures" (greater than 60-day survivors), respectively. The structure-activity relationships are discussed.     

Irreversible inhibition of S-adenosylmethionine decarboxylase of Trypanosoma brucei brucei by S-adenosylmethionine analogues.

S-Adenosylmethionine analogues designed as active-site directed inhibitors were tested in vitro for their effects on S-adenosylmethionine decarboxylase (AdoMetDC) of Trypanosoma brucei brucei. These analogues contained a tertiary nitrogen atom in place of the sulfonium and had a side chain of variable length ending in a reactive group (hydrazino-, aminooxy-, hydrazido- or a methylnitrosourea). The hydrazino- derivatives were the most potent inhibitors with IC50 values in the range of 40-100 nM. The most active compound (IC50 of 0.04 microM) was 5'-deoxy-5'-[(2-hydrazinoethyl)-methylamino]adenosine (MHZEA). Addition of MHZEA produced a time-dependent inactivation with an apparent Ki of 0.4 microM, and the enzyme half-life at a saturating concentration of MHZEA was 0.4 min. Increasing the length of the side chain or changing the methyl group attached to the nitrogen to an ethyl group reduced the potency. Replacement of the hydrazino moiety with an aminooxy group resulted in about a 30- to 35-fold decrease in inhibition potency. However, the relative order of activities of these aminooxy analogues was similar to that found in the hydrazino series with 5'-deoxy-5'-[(2-aminooxyethyl)methylamino]adenosine (MAOEA), which had an IC50 of 1.3 microM, being the most active. The hydrazido analogs were even less effective with 5'-deoxy-5'-[(3-hydrazino-3-oxopropyl)-methylamino]adenosine, the best inhibitor, having an IC50 value of 8.7 microM. The methylnitrosourea derivatives were inactive. The inactivation of trypanosomal AdoMetDC with MHZEA or MAOEA was irreversible and was greatly stimulated by putrescine, a known activator of the enzyme, indicating that the compounds bind to the active site and form a covalent bond with the enzyme. These inhibitors may have considerable potential as chemotherapeutic agents against trypanosomiasis and other protozoal infections and may also be useful in studying the role of AdoMetDC in the regulation of polyamine levels in these organisms.     

5'-thioadenosine derivatives as potent and selective inhibitors of histamine N-methyltransferase

Several new analogues of adenosine bearing a lipophilic side chain at the 5'-position have been synthesized and investigated for their ability to inhibit histamine N-methyltransferase (HNMT). The 5'-deoxy-5'-[4-(3-indolyl)but-1-yl]thio]adenosine (2e), exhibited a pI50 of 5.00 against guinea pig brain HNMT. Interestingly, the polar methyl sulphonium analogue (1c) was a more potent inhibitor of this enzyme (pI50 = 5.26). Both compounds were relatively ineffective inhibitors of rabbit adrenal phenylethanolamine N-methyltransferase (PNMT), rabbit lung indoleamine N-methyltransferase (INMT), and rat brain catechol O-methyltransferase (COMT). 5'-[N(4-phenylbutyl)]-amino-5'deoxyadenosine (2a) and 5'-[N-methyl,N-(4-phenylbutyl]-amino-5'deoxyadenosine (2b) also exhibited potent and selective inhibition against guinea pig brain HNMT. Results from kinetic studies indicate that the above compounds are inhibitors that compete for both the histamine and the S-adenosylmethionine (SAM) binding sites of HNMT. Compound 1c is one of the most potent adenosine analogue inhibitors of HNMT known.     

Adenosine analogues as selective inhibitors of glyceraldehyde-3-phosphate dehydrogenase of Trypanosomatidae via structure-based drug design

In our continuation of the structure-based design of anti-trypanosomatid drugs, parasite-selective adenosine analogues were identified as low micromolar inhibitors of glyceraldehyde-3-phosphate dehydrogenase (GAPDH). Crystal structures of Trypanosoma brucei, Trypanosoma cruzi, Leishmania mexicana, and human GAPDH's provided details of how the adenosyl moiety of NAD(+) interacts with the proteins, and this facilitated the understanding of the relative affinities of a series of adenosine analogues for the various GAPDH's. From exploration of modifications of the naphthalenemethyl and benzamide substituents of a lead compound, N(6)-(1-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (6e), N(6)-(substituted-naphthalenemethyl)-2'-deoxy-2'-(substituted-benzamido)adenosine analogues were investigated. N(6)-(1-Naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (6m), N(6)-[1-(3-hydroxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (7m), N(6)-[1-(3-methoxynaphthalene)methyl]-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (9m), N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3-methoxybenzamido)adenosine (11e), and N(6)-(2-naphthalenemethyl)-2'-deoxy-2'-(3,5-dimethoxybenzamido)adenosine (11m) demonstrated a 2- to 3-fold improvement over 6e and a 7100- to 25000-fold improvement over the adenosine template. IC(50)'s of these compounds were in the range 2-12 microM for T. brucei, T. cruzi, and L. mexicana GAPDH's, and these compounds did not inhibit mammalian GAPDH when tested at their solubility limit. To explore more thoroughly the structure-activity relationships of this class of compounds, a library of 240 N(6)-(substituted)-2'-deoxy-2'-(amido)adenosine analogues was generated using parallel solution-phase synthesis with N(6) and C2' substituents chosen on the basis of computational docking scores. This resulted in the identification of 40 additional compounds that inhibit parasite GAPDH's in the low micromolar range. We also explored adenosine analogues containing 5'-amido substituents and found that 2',5'-dideoxy-2'-(3,5-dimethoxybenzamido)-5'-(diphenylacetamido)adenosine (49) displays an IC(50) of 60-100 microM against the three parasite GAPDH's.     

Second generation transition state analogue inhibitors of human 5'-methylthioadenosine phosphorylase

The polyamine biosynthetic pathway is a therapeutic target for proliferative diseases because cellular proliferation requires elevated levels of polyamines. A byproduct of the synthesis of spermidine and spermine is 5'-methylthioadenosine (MTA). In humans MTA is processed by 5'-methylthioadenosine phosphorylase (MTAP) so that significant amounts of MTA do not accumulate. Products of the MTAP reaction (adenine and 5-methylthio-alpha-D-ribose-1-phosphate) are recycled to S-adenosylmethionine, the precursor for polyamine synthesis. Potent inhibitors of MTAP might allow the build-up of sufficient levels of MTA to generate feedback inhibition of polyamine biosynthesis and/or reduce S-adenosylmethionine levels. We recently reported the design and synthesis of a family of potent transition state analogue inhibitors of MTAP. We now report the synthesis of a second generation of stable transition state analogues with increased distance between the ribooxocarbenium ion and purine mimics. These compounds are potent inhibitors with equilibrium dissociation constants as low as 10 pM. The first and second generation inhibitors represent synthetic approaches to mimic early and late features of a dissociative transition state.     

5'-Deoxy-5'-methylthioadenosine: a nucleoside which differentiates between adenosine receptor types

The activities of an endogenous nucleoside, 5'-deoxy-5'-methylthioadenosine (MTA), on adenosine sensitive sites such as adenosine A1 and A2 receptors and the P-site, as well as on purine nucleoside transport, have been studied. This nucleoside competitively antagonized the A2 receptor-mediated stimulation of neuroblastoma adenylate cyclase, produced a GTP-dependent and 8-p-sulfophenyltheophylline-sensitive inhibition of adenylate cyclase activity in rat cerebellar membranes, and decreased the spontaneous contractile activity of isolated segments of rabbit jejunum. MTA was neither active at the P-site nor did it diminish the binding of [3H]nitrobenzylthioinosine, a nucleoside transport inhibitor. We conclude that (a) MTA is an agonist at the adenosine A1 receptor but an antagonist at the A2 receptor, and (b) the adenosine receptor which causes relaxation of rabbit jejunum is not a neuroblastoma-type A2 receptor which activates adenylate cyclase.     

Effects of adenosine and adenosine analogues on glycogen metabolism in isolated rat hepatocytes

Adenosine and adenosine analogues were incubated with isolated rat hepatocytes. Adenosine and 5'-deoxy-5'-chloroadenosine stimulated glucose release, glycogen loss, and the conversion of glycogen phosphorylase b to a. The effect was of short duration for adenosine, but of long duration for 5'-deoxy-5'-chloroadenosine. The effects on glucose release and phosphorylase were blocked by theophylline, an R-receptor blocking agent, but not by nitrobenzylthioinosine or dipyridamol which are nucleoside transport inhibitors. A dose-dependent rise in cyclic AMP concentration was observed in hepatocytes 1 min after adding adenosine. It is concluded that adenosine exerts these effects in liver by activating adenylcyclase. Adenosine may be involved in the short-term regulation of hepatic glycogen phosphorylase.     

N6,5'-Disubstituted adenosine derivatives as partial agonists for the human adenosine A3 receptor

5'-(Alkylthio)-substituted analogues of N6-benzyl- and N6-(3-iodobenzyl)adenosine were synthesized in 37-61% overall yields. The affinities of these compounds for the adenosine A1, A2A, and A3 receptors were determined using rat brain cortex, rat brain striata, and stably transfected human A3 receptors in HEK 293 cells, respectively. The compounds proved to be selective for the adenosine A3 receptor and displayed affinities in the nanomolar range. Compounds 8, 10, and 11 had the highest affinities for the A3 receptor with Ki values ranging from 8.8 to 27.7 nM. In the N6-benzyl series, compound 4 (LUF 5403), with a 5'-methylthio group, maintained a reasonable affinity and had the highest selectivity for the A3 receptor. Compound 12 (LUF 5411), with an N6-(3-iodobenzyl) group and a 5'-(n-propylthio) substituent, had the highest A3 selectivity of all of the compounds and also displayed high affinity for this receptor (Ki = 44.3 nM). The compounds were also evaluated for their ability to stimulate [35S]GTPgamma[S] binding in cell membranes expressing the human adenosine A3 receptor. It appeared that the N6,5'-disubstituted adenosine derivatives behaved as partial agonists. Compounds 2, 4, 8, and 10 had the highest intrinsic activities. Additionally, when tested in a cAMP assay, these compounds also behaved as partial agonists.     

Extracellular adenosine deprivation induces epithelial differentiation of HT29 cells: evidence for a concomitant adenosine A(1)/A(2) receptor balance regulation

HT29 cells display an undifferentiated phenotype in culture. However, numerous treatments are able to induce both epithelial differentiation and cell growth inhibition. We have previously demonstrated that adenosine and its analogues act through specific adenosine receptors to modulate cell proliferation in HT29 and other human colon adenocarcinoma cell lines. Among the treatments tested, the most potent inhibition of HT29 cell growth was induced by deprivation of extracellular adenosine using adenosine deaminase. Here, we investigated the capacity of adenosine deaminase to initiate epithelial differentiation. After 1 month of daily addition of 10 U/ml adenosine deaminase to the culture medium, HT29 cells were cloned by limited dilution. Among the clones obtained, we focused our attention on clone 13. Microscopic visualization and proliferation studies indicated that cells from this clone grew very slowly and in a pseudo-monolayer, in marked contrast with the situation observed in the mother HT29 cell line. In addition, clone 13 cells displayed epithelial features that mimic the enterocytic differentiation of Caco-2 cells. These modifications were accompanied by dramatic changes in the activity of adenosine receptors, as demonstrated by pharmacological studies. In contrast to the original HT29 cells, clone 13 as well as Caco-2 cells displayed (i) a very low number of adenosine A(1) receptors, and (ii) increases in intracellular cAMP levels when challenged with adenosine analogues. It is hypothesized that a loss of adenosine A(1) receptors, with no change or a concomitant increase in adenosine A(2) receptors, results in the emergence of adenosine A(2) receptor-mediated differentiation and inhibition of proliferation, through a cAMP-dependent pathway.     

Synthesis and anticancer and antiviral activities of various 2'- and 3'-methylidene-substituted nucleoside analogues and crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride.

Various 2'- and 3'-methylidene-substituted nucleoside analogues have been synthesized and evaluated as potential anticancer and/or antiviral agents. Among these compounds, 2'-deoxy-2'-methylidene-5-fluorocytidine (22) and 2'-deoxy-2'-methylidenecytidine (23) not only demonstrated potent anticancer activity in culture against murine L1210 and P388 leukemias, Sarcoma 180, and human CCRF-CEM lymphoblastic leukemia, producing ED50 values of 1.2 and 0.3 microM, 0.6 and 0.4 microM, 1.5 and 1.5 microM, and 0.05 and 0.03 microM, respectively, but also were active in mice against murine L1210 leukemia. Of all the tested drug dosage levels (25, 50, and 75 mg/kg, respectively) compound 23 had no toxic deaths and compound 22 yielded only one toxic death at the highest dosage level. On the contrary, in the same study, 1-beta-D-arabinofuranosylcytosine (ara-C) resulted in 2/5, 5/5, and 5/5 toxic deaths, respectively. Both compounds 22 and 23 have shown better anticancer activity than ara-C, yielding higher T/C x 100 values and some long-term survivors (greater than 60 days). In addition, compounds 22 and 23 were found to have, respectively, approximately 130 and 40 times lower binding affinity for cytidine/deoxycytidine deaminase derived from human KB cells compared to ara-C, suggesting that the two 2'-methylidene-substituted analogues may be more resistant to deamination. Cytoplasmic deoxycytidine kinase (dCK) was required for compounds 22 and 23 action. Furthermore, compounds 14, 22, 23, and 24 also have antiherpes simplex virus type 1 (HSV-1) and type 2 (HSV-2) activity in cell culture. In addition, the crystal structure of 2'-deoxy-2'-methylidenecytidine hydrochloride (23-HCl) was determined by X-ray crystallography.