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Sulfurization of protected [i.e., 2-cyanoethyl, methyl and 2-(4-nitrophenyl)ethyl] internucleosidic phosphite triesters can be conveniently executed with the easily accessible reagent, phenylacetyl disulfide (1). High-quality oligodeoxynucleotides containing predetermined combinations of natural and phosphorothioate linkages can be obtained via a solid-phase approach by using reagent 1 and 2-(4-nitrophenyl)ethyl-protected phosphoramidites.

Immobilized and fully protected oligodeoxynucleotides containing a 4-(1,2,4-triazolyl)-thymidine residue at a predetermined position were prepared according to a well-established phosphite triester methodology using 2-cyanoethyl phosphoramidites of a 4-(1,2,4-triazolyl)-substituted thymidine and standard protected nucleosides. Treatment of the immobilized oligomer with methanol, ethanol or n-propanol in the presence of DBU at 50°C gave the corresponding oligonucleotides containing 4-methoxy, 4-ethoxy- or 4-n-propoxythymidine residue.

High-quality oligodeoxynucleotides having an 6-O-alkyl-2′-deoxyguanosine (alkyl = methyl, ethyl, n-propyl or n-hexyl) residue at a predetermined position can be obtained via a solid-phase approach using the respective 2-cyanoethyl N,N-diisopropylphosphoramidites of 5′-O-(4.4′-dimethoxytrityl)-protected 6-O-alkyl-2′-deoxyguanosines having a free exocyclic amino group, and 5′-O-(4-4′-dimethoxytrityl) N-acyl-protected (i.e., benzoyl and isobutyryl for dC/dA and dG, respectively) 2′-deoxynucleosides.

A1 adenosine receptor agonists with reduced intrinsic activity may be therapeutically useful as result of an increased selectivity of action. In this study the tissue selectivity of three 8-alkylamino substituted analogues of N6-cyclopentyladenosine (CPA) was investigated for haemodynamic and anti-lipolytic effects using an integrated pharmacokinetic-pharmacodynamic approach. Chronically instrumented male Wistar rats received intravenous infusions of 4.0 mg kg−1 8-methylaminoCPA (8MCPA), 12.0 mg kg−1 8-ethylaminoCPA (8ECPA), 20.0 mg kg−1 8-butylaminoCPA (8BCPA) or vehicle during 15 min. During experimentation, serial arterial blood samples were drawn for the determination of agonist concentrations and plasma non-esterified fatty acid (NEFA) levels. Blood pressure and heart rate were monitored continuously. In addition to the CPA analogues, each rat received a rapid bolus infusion of CPA to determine the maximal effects of the full agonist. The concentration-time profiles of the CPA analogues could be described by a bi-exponential function. Values for clearance, volume of distribution at steady state and elimination half-life were 44±5, 48±6 and 39±2 ml min−1 kg−1, 0.97±0.09, 0.84±0.10 and 1.05±0.07 1 kg−1 and 25±2, 28±2 and 40±2 min for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8). Different models were used to derive the concentration-effect relationships for heart rate and NEFA, yielding estimates of potency (EC50) and instrinsic activity (Emax) for both effects of the compounds in vivo. On heart rate the compounds acted as partial agonists, with Emax values of −173±14, −131±11 and −71±6 beats min−1 for 8MCPA, 8ECPA and 8BCPA, respectively. These Emax values were significantly lower than the maximal effect of CPA (−208±8 beats min−1). With regard to the anti-lipolytic effect all three compounds were full agonists and lowered NEFA levels to the same extent as CPA (69%). The estimated Emax values were 63±5, 63±4 and 68±2%, respectively. Furthermore, the compounds were more potent in causing anti-lipolytic than cardiovascular effects. The EC50 values for the NEFA and heart rate lowering effects were 37±15, 68±22 and 659±108 ng ml−1 and 164±22, 341±76 and 975±190 ng ml−1 for 8MCPA, 8ECPA and 8BCPA, respectively (mean±s.e.mean, n=6–8). This study demonstrates that partial agonists for the A1 adenosine receptor have increased selectivity of action in vivo. The 8-alkylamino analogues of CPA may be useful anti-lipolytics with less pronounced haemodynamic side effects.

5'-(Alkylthio)-, 5'-(methylseleno)-, and 5'-(alkylamino)-substituted analogues of N6-cyclopen-tyladenosine (CPA) were synthesized in 30-50% overall yields. The affinities of these compounds for the adenosine A1 and A2A receptors were determined in rat brain membranes. The 5'-substituted CPA analogues proved selective for the adenosine A1 receptors, displaying affinities in the nanomolar range. The compounds were also evaluated for their ability to stimulate [35S]GTP gamma S binding, also in rat brain membranes. The Ki values in receptor binding studies corresponded well to the EC50 values thus obtained. Intrinsic activities of the compounds were tested in vitro by determining the GTP shift in receptor binding studies as well as the maximal binding of [35S]GTP gamma S. It appeared that the 5'-thio and 5'-seleno derivatives in particular behaved as partial agonists.

O4-Alkylthymines have been implicated as potential carcinogenic DNA lesions. We have studied the effects of O4-methylthymine, O4-ethylthymine, and O4-n-propylthymine in a model system in which a single lesion was located at a defined position on a SV40-based shuttle vector and have found large differences in the effects of these lesions in repair-proficient and nucleotide excision repair-deficient cells. In repair-competent human HeLa cells, normal fibroblasts, and XP-A (2OS) revertant cells, all 3 residues were highly mutagenic; a mutation frequency of approximately 20% was found for both O4-methylthymine and O4-ethylthymine, whereas that of O4-n-propylthymine was approximately 12%. These frequencies were independent of the activity of the O6-alkylguanine DNA alkyltransferase. All three O4-alkylthymines induced T-->C transitions exclusively. In nucleotide excision repair-deficient XP-A cells, however, these lesions were not mutagenic but strongly inhibited plasmid replication (> 90%). These results indicate that O4-alkylthymines are efficiently recognized by the nucleotide excision repair system and cause a complete cessation of plasmid replication if this system is deficient. Nevertheless, proficiency in the nucleotide excision repair pathway correlates with a high frequency of mutation induction by these lesions.

One-pot coupling of 2′,3′-di-O-benzoyl-uridine and 4,5,6,8-tetra-O-acetyl-3,7-anhydro-2-deoxy-D-glycero-D-ido-octitol with appropriate bifunctional phosphoro-, phosphono- and thiophosphonylation reagents gave, after removal of the base-labile protecting groups, analogues of UDP-Glc containing charged phosphate, neutral alkyl- and arylphosphonate and phosphonothioate linkages. In an analogous manner, GDP-Man analogues, in which guanosine and octitol units are similarly linked via phosphate, alkyl phosphate or phosphotriester bonds, were prepared.

Immobilized and fully protected oligodeoxynucleotides containing a 4-(1,2,4-triazolyl)-thymidine residue at a predetermined position were prepared according to a well-established phosphite triester methodology using 2-cyanoethyl phosphoramidites of a 4-(1,2,4-triazolyl)-substituted thymidine and standard protected nucleosides. Treatment of the immobilized oligomer with methanol, ethanol or n-propanol in the presence of DBU at 50°C gave the corresponding oligonucleotides containing 4-methoxy, 4-ethoxy- or 4-n-propoxythymidine residue.

We described earlier the effect of tris-gal-chol (a triantennary galactose structure coupled to cholesterol) on the fate of low density lipoprotein (LDL) and high density lipoprotein (HDL). Tris-gal-chol-loaded LDL and HDL are both efficiently cleared from blood by hepatic galactose-specific receptors. Thus, tris-gal-chol combines a beneficial LDL-reducing effect with an equally effective but undesirable HDL-lowering effect. We recently synthesized a cholesterol derivative with a single terminal galactose residue, denoted mono-gal-chol. In the present study we show that this compound, which incorporates readily into both LDL and HDL, induces rapid association of LDL and HDL to the liver. The mono-gal-chol-stimulated hepatic association of HDL, however, was about fivefold lower than that of LDL. In the liver, Kupffer cells were mainly (90%) responsible for the liver uptake of mono-gal-chol-loaded LDL, whereas the complex of mono-gal-chol with HDL was predominantly (95%) taken up by parenchymal cells. Uptake by both cell types proceeded via galactose-specific receptors and was followed by degradation of the apolipoproteins in the lysosomes. Thus, compared with tris-gal-chol, mono-gal-chol is equally effective in the induction of galactose-specific uptake of LDL by Kupffer cells. However, the galactose-specific receptor on parenchymal cells recognizes mono-gal-chol-loaded HDL less efficiently than tris-gal-chol-containing HDL. These results indicate that mono-gal-chol might be used to specifically lower LDL levels in patients with a high LDL cholesterol level.

Monophosphonylation of 2′-protected ribonucleosides (i.e. 2′-O-THP-uridine and 2′-O-THP-N 6-levulinoyl-adenosine) with the bifunctional reagents bis[(6-trifluoromethyl)benzotriazol-1-yl] methyl(phenyl)phosphonates or the analogous phosphonothioates, and subsequent addition of N-methylimidazole, gave the chirally pure 3′,5′-cyclic methyl(phenyl)phosphonate or phosphonothioate derivatives, respectively. Deblocking of the fully protected compounds yielded, as evidenced by X-ray analysis, the corresponding pure Sp-diastereoisomers.

The in vivo mutagenicity of 7-hydro-8-oxo-2'-deoxyguanosine (8-oxodG) and N-(guanin-8-yl)-N-acetyl-2-aminofluorene (8-AAFdG) in human cells was determined by transfecting various cell lines with plasmids that carried a single adduct at a defined site. 8-OxodG is one of the many DNA modifications formed by oxygen radicals, and was found to be highly miscoding during replication with purified DNA polymerases in vitro. Here we show that the frequency of mutations induced by 8-oxodG during replication in vivo is at most only 2% above background. The most predominant mutation found was a single G----T transversion. The frequency of this transversion was found to be 3 to 5-fold increased in excision repair deficient XP-A cells. Interestingly, also the replication of 8-oxodG containing plasmids was significantly impaired (approximately 4-fold) in the XP-A cells, but not in HeLa cells, normal fibroblasts or XP-A revertant cells. When 8-AAFdG containing plasmids were used, the mutation frequencies did not exceed background levels (less than 2%) with any of the cell lines tested. The presence of 8-AAFdG almost completely inhibited plasmid replication (more than 50-fold) in XP-A cells. Apparently, both 8-AAFdG and 8-oxodG are not or poorly repaired in these cells, causing a block of DNA replication. This suggests that both lesions are substrates for excision repair, although to a varying extent.

The synthesis of several monogalactoside-terminated phosphorothiolated cholesteryl derivatives is described. Monogalactosyl derivatives are coupled by phosphorothiolation to cholesterol by using ethylene glycol units as hydrophilic spacer moieties. The resulting compounds are easily soluble in water. Upon addition of such solutions to human serum (to 2 mM final concentration) the compounds are readily incorporated into lipoproteins. Isolated low-density lipoprotein (LDL) and high-density lipoprotein (HDL), preloaded with the compounds, are rapidly cleared from the circulation by the liver. The hepatic association is blocked by N-acetylgalactosamine, which indicates that galactose-specific recognition sites are responsible for the increased liver uptake. The plasma clearance and hepatic uptake of LDL loaded with the compounds is substantially higher (about 2-fold) than clearance and uptake of HDL containing the compounds. The selectivity of the effects of monogalactoside-terminated phosphorothiolated cholesteryl derivatives on the in vivo behavior of LDL as compared to that of HDL indicates that these compounds might be used to lower specifically LDL levels in patients with a high LDL-cholesterol level.

The myo -inositol phosphorothioates 6 , 9 , 12 , and 17 were readily accessible from properly protected precursors by phosphitylation with N,N -diisopropyl dibenzyl phosphoramidite ( 1 ), subsequent in situ sulfurization of the intermediate phosphite triesters with phenacetyl disulfide ( 3 ), and removal of all benzyl protecting groups.

One-pot coupling of 2′,3′-di-O-benzoyl-uridine and 4,5,6,8-tetra-O-acetyl-3,7-anhydro-2-deoxy-D-glycero-D-ido-octitol with appropriate bifunctional phosphoro-, phosphono- and thiophosphonylation reagents gave, after removal of the base-labile protecting groups, analogues of UDP-Glc containing charged phosphate, neutral alkyl- and arylphosphonate and phosphonothioate linkages. In an analogous manner, GDP-Man analogues, in which guanosine and octitol units are similarly linked via phosphate, alkyl phosphate or phosphotriester bonds, were prepared.

Publisher Summary This chapter discusses the partial agonists for adenosine receptors. Adenosine is generally considered as a “local hormone” with profound physiological activity. It is thought to mediate a large variety of effects, as diverse as vasodilation in the cardiovascular system, inhibition of lipolysis in fat cells, and depression of neuronal activity in the central nervous system (CNS). Most of its effects are mediated by membrane-bound receptors, called P i -purinoceptors, of which currently three subclasses are defined: A l , A 2 and A 3 . All three classes have been cloned, and are coupled to the enzyme adenylate cyclase, A 1 and A 3 adenosine receptors in an inhibitory, and A 2 receptors, of which two further subtypes A 2a and A 2b exist, in a stimulatory fashion. The chapter discusses some methods for screening partial agonists as well as application of these screening methods to adenosine receptors. There is also a description of how partial agonists for adenosine receptors were obtained for the purpose of this chapter. In view of the overwhelming number of therapeutic strategies already in clinical practice, the hypotensive effects of adenosine receptor agonists are probably of limited use. On the contrary, the potentially beneficial metabolic, antiarrhythmic, and CNS effects elicited by these compounds are confounded by the cardiovascular actions.

A series of 8-substituted adenosine and theophylline-7-riboside analogues (28 and 9 compounds, respectively) was tested on adenosine A1 and A2A receptors as an extensive exploration of the adenosine C8-region. Alkylamino substituents at the 8-position cause an affinity decrease for adenosine analogues, but an affinity increase for theophylline-7-riboside derivatives. The affinity decrease is probably due to a direct steric hindrance between the C8-substituent and the binding site as well as to electronic effects, not to a steric influence on the ribose moiety to adopt the anti conformation. The 8-substituents increase the affinity of theophylline-7-riboside analogues probably by binding to a lipophilic binding site. The intrinsic activity was tested in vitro for some 8-substituted adenosine analogues, by determining the GTP shift in receptor binding studies and the inhibition of adenylate cyclase in a culture of rat thyroid FRTL-5 cells, and in vivo in the rat cardiovascular system for 8-butylaminoadenosine. Thus, it was shown that 8-ethyl-, 8-butyl-, and 8-pentylamino substituted analogues of adenosine may be partial agonists in vitro, and that 8-butylaminoadenosine is a partial agonist for the rat cardiovascular A1 receptor in vivo.

Easily accessible phenacetyl or benzoyl disulfide proved to be very convenient reagents for a rapid P-sulfurization of phosphite-triesters and H-phosphonate diesters, respectively.

The reagent obtained in situ by treating methylphosphonothioic dichloride with 1-hydroxy-6-trifluoromethylbenzotriazole could be used for the introduction of methylphosphonothioate linkages. The individual diastereomers of the protected dimer d-Tp(S,Me)A were applied in the synthesis of the chiral pure (R or S) hexamers d-[CpCpTp(S,Me)ApGpG]. The reagent showed also to be very effective for the preparation of the 3',5'-cyclic methylphosphonothioate of uridine.

It will be demonstrated that 5'-O-DMT-N-acyl-deoxyribonucleosides, 5'-O-Lev-2'-O-MTHP-N-acyl-ribonucleosides and, also, 2'-O-MTHP-N-acyl-ara-cytidine can be coupled, via the hydroxybenzotriazole phosphotriester approach, to afford two types of DNA-RNA hybrids as well as ara-C containing DNA-fragments. The final removal of acid-labile DMT and MTHP groups could be effected by 1 h treatment with 80% acetic acid of the otherwise unprotected DNA-RNA hybrids. The same acidic hydrolysis did not result in complete removal of the 2'-O-MTHP group from the ara-C unit. Complete deblocking was accomplished after an additional 2 h aqueous HC1 (0.01 M; pH 2.00) treatment.

A 1H-NMR investigation was carried out on the tetranucleotides U-m6(2)A-U-m6(2)A and m6(2)A-m6(2)A-U-m6(2)A (m6(2) = N6-dimethyladenosine) as well as on the hybrid trinucleotide dA-r(U-A). An extensive comparison with m6(2)A-U-m6(2)A and other relevant compounds is made. Previous proton NMR studies on trinucleotides have shown that purine-pyrimidine-purine sequences prefer to adopt a mixture of states which have as a common feature that the interior pyrimidine residue bulges out, whereas the flanking purine residues stack upon each other. A stacking interaction on the 3' side of the bulge is known to have no measurable effect on the bulge population. Chemical-shift data, ribose ring conformational analysis and information from NOE experiments now show unambiguously that the moderate U(1)-m6(2)A(2) stack in U-m6(2)A-U-m6(2)A diminishes the population of bulged-out structures in favour of a regular stack. This tendency towards conformational transmission in the downstream 5'----3' direction is fully confirmed by the fact that the strong m6(2)A(1)-m6(2)A(2) stack in the tetranucleotide m6(2)A-m6(2)A-U-m6(2)A virtually precludes the formation of bulged-out structures. The conformational characteristics of dA-r(U-A) appear comparable with those of m6(2)A-U-m6(2)A, which indicates that the presence of a 2'-hydroxyl group in the first purine residue is not a necessary prerequisite for the formation of a bulge.

Acetalisation of 3′,5′-di-O-acetyluridine in DMF with 4,4-dimethoxytetrahydro-4H-pyran and 4,4-dimethoxytetrahydro-4H-1-thiopyran in the presence of trimethylsilyl chloride (TMSiCl) afforded, after removal of the acetyl groups, 2′-O-(4-methoxytetrahydropyranyl)-uridine and its sulfur analogue, respectively. It was also established that a 3′,5′-O-(tetraisopropyldisiloxane-1,3-diyl) protected ribonucleoside was rapidly isomerised into the corresponding 2′,3′-isomer using the TMSiCl promoted acetalisation method.