α-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of α-cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

-cyano-substituted analogoues of decarboxylated S-adenosylmethionine as enzyme activated, irreversible inhibitors of S-adenosylmethionine decarboxylase

A pair of -cyano analogues of decarboxylated S-adenosylmethionine (2a and 2b) were synthesized as potential enzyme activated, irreversible inhibitors of the[pyruvoyl enzyme S-adenosylmethionine decarboxylase (AdoMet-DC). Each of these analogues acts as an irreversible inactivator for ADoMet-DC from Escherichia coli (IC₅₀ values of 9 and 50 μM, respectively). These analogues also inactivate human AdoMet-DC, with K_I values of 246.6 and 7.2 μM, and k_inact values of 0.29 and 0.03 min⁻¹, respectively.     

Carbocyclic Adenosine Analogues as S-Adenosylhomocysteine Hydrolase Inhibitors and Antiviral Agents: Recent Advances

Abstract

Various carbocyclic analogues of adenosine, including aristeromycin (carbocyclic adenosine), carbocyclic 3-deazaadenosine, neplanocin A, 3-deazaneplanocin A, the 5′-nor derivatives of aristeromycin, carbocylic 3-deazaadenosine, neplanocin A and 3-deazaneplanocin A, and the 2-halo (i.e., 2-fluoro) and 6′-R-alkyl (i.e., 6′-R-methyl) derivatives of neplanocin A have been recognized as potent inhibitors of S-adenosylhomocysteine (AdoHcy) hydrolase. This enzyme plays a key role in methylation reactions depending on S-adenosylmethionine (AdoMet) as methyl donor. AdoHcy hydrolase inhibitors have been shown to exert broad-spectrum antiviral activity against pox-, paramyxo-, rhabdo-, filo-, bunya-, arena-, and reoviruses. They also interfere with the replication of human immunodeficiency virus through inhibition of the Tat transactivation process.     

Irreversible Inhibition of Thymidylate Synthase by Pyridoxine (B6) Analogues

Abstract

Three vitamin B₆ analogues have been synthesized and tested as inhibitors of thymidylate synthase. The compounds are: 4′,5′-dichloro-, 4,5′-dibromo- and 4′, 5′-diiodo-pyridoxine. All three analogues inhibited the enzyme irreversibly. The kinetic data for the chloro- and bromo-analogues showed that a limiting rate of inhibition is approached as the inhibitor concentration is increased, which indicates that a reversible enzyme: inhibitor affinity complex is formed prior to the irreversible reaction. 4′,5′-Dibromo-pyridoxine exhibited a greater binding affinity (lower K_i) for thymidylate synthase than 4′,5′-dichloro-pyridoxine, and it also reacted faster to irreversibly inhibit the enzyme. The presence of the substrate dUMP (10μM) completely protected thymidylate synthase from inhibition. These data suggest that the halogenated vitamin B₆ analogues are active site-directed inhitors of thymidylate synthase, which first bind reversibly to the catalytic site and then react irreversibly with the enzyme.     

A New Series of S-Adenosyl-L-Methionine Synthetase Inhibitors

Abstract

A new series of epithio and epoxy amino acid analogues of L-methionine or L-methoxinine were examined as potential inhibitors of the enzyme S-adenosylmethionine (AdoMet) syn-thetase. The kinetic behaviour of these compounds was studied using recombinant rat liver S-adenosyl-L-methionine sythetase (α-isoform) fractionated from E. coli, transformed with the plasmid pSSRL-T7N. All the compounds tested were competitive inhibitors with respect to L-methionine and the (2S, 4S)-2-amino-4,5-epoxy pentanoic acid was found to be a very potent inhibitor of the enzyme compared to those already reported for AdoMet synthetase from other mammalian tissues.     

5′-Substituted 2′-Deoxycytidines as Non-Substrate Inhibitors of Human Deoxycytidine Kinase

Abstract

Several 5′-substituted analogues of 2′-deoxycytidine (dC), including 5′-amino2′,5′-dideoxycytidine (5′-amino-ddC), 5′-azido-2′,5′-dideoxycytidine (5′-azido-ddC) and 5′-O-ethyl-2′-deoxycytidine (5′-O-ethyl-dC), are non-substrate inhibitors of human dC kinase. Whereas 5′-amino-ddC inhibits phosphorylation of deoxyadenosine (dA) with K_i ～ 1 μ inhibition of phosphorylation of dC is bimodal and more effective at low inhibitor concentrations. In particular 5′-O-ethyl-dC inhibits phosphorylation of dA 10-fold more effectively (K_i ～ 3 μ) than that of dC (K_i ～ 30 μ). For 5′-azido-ddC inhibition was shown directly to be competitive with respect to substrate.     

Substrate/Inhibitor Properties of Human Deoxycytidine Kinase (dCK) and Thymidine Kinases (Tk1 And Tk2) Towards the Sugar Moiety of Nucleosides,Including O′-Alkyl Analogues

Abstract

Nucleoside analogues with modified sugar moieties have been examined for their substrate/inhibitor specificities towards highly purified deoxycytidine kinase (dCK) and thymidine kinases (tetrameric high-affinity form of TK1, and TK2) from human leukemic spleen. In particular, the analogues included the mono-and di-O′-methyl derivatives of dC, dU and dA, syntheses of which are described. In general, purine nucleosides with modified sugar rings were feebler substrates than the corresponding cytosine analogues. Sugar-modified analogues of dU were also relatively poor substrates of TK1 and TK2, but were reasonably good inhibitors, with generally lower K_i values vs TK2 than TK1. An excellent discriminator between TK1 and TK2 was 3′-hexanoylamino-2′,3′-dideoxythymidine, with a K_i of ～600 μM for TK1 and ～0.1 μM for TK2. 3′-OMe-dC was a superior inhibitor of dCK to its 5′-O-methyl congener, consistent with possible participation of the oxygen of the (3′)-OH or (3′)-OMe as proton acceptor in hydrogen bonding with the enzyme. Surprisingly α-dT was a good substrate of both TK1 and TK2, with K_i values of 120 and 30 μM for TK1 and TK2, respectively; and a 3′-branched α-L-deoxycytidine analogue proved to be as good a substrate as its α-D-counterpart. Several 5 ′-substituted analogues of dC were     

Phosphate Modified Analogues of 5′-O-Phosphorylated 2′,3′-Dideoxynucleosides: Synthesis and Anti-HIV Activity

Abstract

Phosphonate analogues of 5′-O-phosphoryl-2′,3′-dideoxyribofuranosyl adenine, cytosine, hypoxanthine and thymine were synthesized. The hypoxanthine and thymine analogues were inactive against HIV induced cyopathy in the CEM-4 T-cell lines. The 2′,3′-ddC and 2′,3′-ddA analogues were marginally active.     

Synthesis of Novel 2′-Fluoro-3′-Hydroxymethyl-5′-Deoxythreosyl Phosphonic Acid Nucleoside Analogues As Antiviral Agents

A series of purine 5′-deoxyphosphonate analogues were designed and synthesized to mimic naturally occurring purine monophosphate from 1,3-dihydroxyacetone as starting material. The discovery of threosyl phosphonate nucleoside (PMDTA, EC₅₀ = 2.53 μM) as a potent anti-HIV agent has led to the synthesis and biological evaluation of 2′,3′-modified 5′-deoxyversions of the threosyl phosphonate nucleosides. The synthesized 2′-fluoro-3′-hydroxymethyl 5′-deoxythreosyl phosphonic acid nucleoside analogues 14, 18, 23, and 27 were tested for anti-HIV activity as well as cytotoxicity. The adenine analogue 18 exhibits weak in vitro anti-HIV-1 activity (EC₅₀ = 19.2 μM).     

Efficient Electrophilic Fluorination for the Synthesis of Novel 2′-Fluoro-3′-Methyl-5′-Deoxyphosphonic Acid Apiosyl Nucleoside Analogues

Novel 5′-deoxyapiosyl purine phosphonic acid analogues with a 2′-electropositive moiety, such as, a fluorine atom were designed and synthesized from commercially available hydroxylacetone. Condensation of a glycosyl donor 10 with purines under Vorbruggen conditions and cross-metathesis give the desired nucleoside phosphonic acid analogues 14, 17, 21, and 24. The synthesized nucleoside analogues were subjected to antiviral screening against HIV-1, and the adenine analogue 17 exhibited weak in vitro anti-HIV-1 activity (EC₅₀ = 26.6 μM)     

Disaccharide Pyrimidine Nucleosides and Their Derivatives: A Novel Group of Cell-Penetrating Inhibitors of Poly(ADP-Ribose) Polymerase 1

Nearly 30 synthetic nucleosides were tested with human recombinant poly(ADP-ribose) polymerase 1 as potential inhibitors of this enzyme. The most active compounds were some disaccharide analogues of thymidine: 3′-O-β-D-ribofuranosyl-5-iodo-dUrd (2d; IC₅₀ = 45 μM), 3′-O-β-D-ribofuranosyl-2′-deoxythymidine (2e; IC₅₀ = 38 μM), and 3′-O-β-D-ribofuranosyl-2′-deoxythymidine oxidized (4; IC₅₀ = 25 μM). These compounds also reduced H₂O₂-induced synthesis of poly(ADP-ribose) in cultured human ovarian carcinoma (SKOV-3) cells in a dose-dependent manner. Furthermore, compounds 2d or 2e until a concentration of 1 mM did not affect growth of SKOV-3 cells, whereas dialdehyde compound 4, as well as thymidine, exhibited a significant cytotoxicity.     

Synthesis of Some 2′,3′-Dideoxy-2′-C-Methyl-Substituted Nucleosides

Abstract

Nucleoside analogues analogues1-(2′,3′-dideoxy-2′-C-hydroxymethyl-β-D-erythro-pentofuranos-yl)thymine (1), 2′,3′-dideoxy-2′-C-hydroxymethylcytidine (2), 2′,3′-dideoxy-2′-C-hydroxymethyladenosine (3), 1-(2′-C-azidomethyl-2′,3′-dideoxy-β-D-erythro-pento-furanosyl)thymine (4), 2′-C-azidomethyl-2′,3′-dideoxycytidine (5), and 2′3′-dideoxy-2′-C-methylcytidine (6) have been synthesized from (S)-4-hydroxymethyl-y-butyro-lactone (7)     

2-Bromoadenosine-Substituted 2′,5′-Oligoadenylates Modulate Binding and Activation Abilities of Human Recombinant RNase L

Abstract

2-Bromoadenosine-substituted analogues of 2–5A, p5′A2′p-5′A2′p5′(br²A), p5′(br²A)2′p5′A2′p5′A, and p5′(br²A)2′p5′(br²A)2′p-S′(br²A), were prepared via a modification of a lead ion-catalyzed ligation reaction and were subsequently converted into the corresponding 5′-triphosphates. Both binding and activation of human recombinant RNase L by various 2-bromoadenosine-substituted 2–5A analogues were examined. Among the 2-bromoadenosine-substituted 2–5A analogues, the analogue with 2-bromoadenosine residing in the 2′-terminal position, p5′A2′p5′A2′p-5′(br²A), showed the strongest binding affinity and was as effective as 2–5A itself as an activator of RNase L. The CD spectrum of p5′A2′p-5′A2′p5′(br²A) was superimposable on that of p5′A2′p5′A2′p5′A, indicative of an anti orientation about the base-glycoside bonds as in naturally occurring 2–5A.     

Synthesis and Antiherpetic Activities of Several Acyclic Analogues Of Guanosine

Abstract

Several acyclic analogues of guanosine, 2′-deoxy-2′, 3′-secoguanosine(3), 3′-deoxy-2′, 3′-secoguanosine (4), and 2′-, 3′-dideoxy-2′-, 3′-secoguanosine were synthesized from guanosine. In addition, the 3′-, 5′-cyclic phosphate (21) and 3′-, 5′-cyclic methylphosphonates (22a, b) of 3 were also prepared. At concentrations up to 300 μM none of these compounds had significant antiherpetic activity in antiviral assays in vitro.     

The Synthesis of Cyclonucleotides with Fixed Glycosidic Bond Linkages as Putative Agonists for P2-Purinergic Receptors

Abstract

Cyclonucleotides with fixed glycosidic bond linkages were investigated as possible ligands for purinoceptors in PC12 cells. P₂Y₂-purinoceptors were not activated by the ATP analogue, 8,2′-thioanhydroadenosine-5′-triphosphate (4) and only weakly by the UTP analogue, 2,2′-anhydrouridine-5′-triphosphate (6). However, both analogues were agonists for P2X₂-purinoceptors although the potencies were approximately 30-fold less than that of the parent nucleotides.     

Synthesis and Biological Activities of Sugar-Modified 2-(p-n-Butylanilino)-2′-deoxyadenosine Analogues

Abstract

Several sugar-modified 2-(p-n-butylanilino)-2′-deoxyadenosine analogues, including arabino and 2′(R)-azido-2′-deoxy analogues and their 5′-triphosphates were synthesized. These nucleosides thus obtained exhibited moderate cytotoxicity against P-388 leukemic cells in culture (IC₅₀ = 13–24 μ). In contrast to above results, the 5′-triphosphates have been shown to exert strong and selective inhibitory effects on mammalian DNA polymerase α (Ki= 0.02–0.04 μ).     

Synthesis and evaluation of 5′-modified thymidines and 5-hydroxymethyl-2′-deoxyuridines as Mycobacterium tuberculosis thymidylate kinase inhibitors

We report the synthesis of 5′-modified thymidines (16, 18, 21, 23) and 5,5′-bis-substituted 2′-deoxyuridine analogues (30, 47) as inhibitors of thymidine monophosphate kinase of Mycobacterium tuberculosis (TMPKmt). These analogues were evaluated for their capacity to inhibit TMPKmt and solely two 5′-modified thymidines were found to possess moderate inhibitory activity. In addition, a feasibility study of protecting groups for the 5-CH₂OH moiety of 2′-deoxyuridines is described that enables to introduce the desired 5′-modification.     

Sugar and Base-Modified 2′,3′-Dideoxynucleosides as Potential Anti-Aids Drugs

Abstract

Several 2′,3′-dideoxynucleosides have proven to be effective against HIV-1 in cell culture. The 2′,3-unsaturated analogues dideoxycytidinene (D4C) and dideoxythymidinene (D4T) are among the most potent and selective inhibitors of HIV-1 replication in vitro. However, 2′,3′-didehydro-2′,3′-dideoxynucleosides are relatively unstable. A chlorine substituent at the 2′-position increases the stability, and, therefore, some 2′-chloro-2′,3′-didehydro-2′,3′-dideoxynucleosides were synthesized and evaluated for anti-HIV-1 activity.     

Synthesis and Evaluation of Oligodeoxynucleotides Containing 3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine: Introduction of a Novel Class of Phosphodiester Internucleoside Linkages

Abstract

3′-O-Ethyl-4′-C-(hydroxymethyl)thymidine (5) was synthesized and converted into the phosphoramidite building block 8. Novel oligodeoxynucleotide analogues containing 4′-C-hydroxymethyl phosphodiester internucleoside linkages were synthesized on an automated DNA-synthesizer. The hybridization properties and enzymatic stability were studied on oligomers with one to four modifications. The 3′-end modified oligodeoxynucleotides were resistent towards 3′-exonuclease degradation and showed only moderate lowered affinity towards complementary DNA compared with oligodeoxynucleotides bearing modifications in the middle.