X-Ray Crystallographic and Kinetic Analysis of Human Purine Nucleoside Phosphorylase Complexes with 1-β-D-Ribofuranosyl-1,2,4-triazole-3-carobxamide and 1-β-D-Ribofuranosyl-1,2,4-triazole-3-carboxamidine

Abstract

The three-dimensional structures of the complexes between human erythrocytic purine nucleoside phosphorylase (PNP) and both 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide (ribavirin) and 1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamidine (TCNR) have been determined using X-ray crystallographic techniques. The structures have been refined at 2.9 Å resolution using simulated annealing and conjugate-gradient minimization techniques to an R value of 21.8% for ribavirin and 20.8% for TCNR. Ribavirin and TCNR are truncated nucleosides corresponding to adenosine and inosine, respectively, and are of potential interest as PNP inhibitors. Kinetic parameters have been determined for recombinant wild-type PNP and for a mutant PNP in which Asn 243 is converted to Asp. The K_i value for ribavirin is 4.9 mM with wild-type PNP and 4.7 mM with the Asn243Asp mutant, while the K_i values for TCNR are 17.6 μM and 3.8 μM with wild-type and mutant, respectively. X-ray crystallographic studies showed that the binding geometry for both of these substrate analogues was similar to that seen for natural substrates. The glycosidic torsion angles (χ) were ?34° for ribavirin and ?39° for TCNR which are in good agreement with values seen for other studied nucleoside complexes with PNP, but which are unusual when compared to those seen for free nucleic acid derivatives. Based upon the three-dimensional structure, interactions of Asn 243 and Glu 201 with a protonated carboxamidine of TCNR explain the stronger inhibition of PNP observed for TCNR over ribavirin.     

The broad spectrum antiviral agent ribavirin inhibits capping of mRNA.

Ribavirin (1-β-D-ribofuranosyl-1,2,4-triazole-3-carboxamide) is a broad spectrum antiviral substance active against a wide range of both DNA and RNA viruses. It is, however, virtually inactive against polio virus. Its pharmacological mechanism of action was obscure. A possible common target for a chemotherapeutic agent in both DNA and RNA viruses is the “capping” reaction of mRNAs which $\text{inter}\text{alia}$ involves the formation of a guanine pyrophosphate structure at the 5′ terminus by mRNA guanylyl transferase. We have observed that Ribavirin triphosphate is a potent competitive inhibitor of the capping guanylation of viral mRNA. This finding could account for the antiviral potency of the drug against both DNA and RNA viruses and its ineffectiveness against a virus in which the mRNAs derived from them are not capped.     

Chemical ribosylation of 5-substituted 1,2,4-triazole-3-carboxylates

The chemical ribosylation pathways of 5-substituted-1,2,4-triazole-3-carboxylates are discussed. For the products of the chemical synthesis of the 3(5)-alkyl- or 3(5)-aryl-substituted ribavirin analogues the anomer configuration and isomer composition were determined.     

Synthesis and properties of acyclic analogs of ribavirin

Acyclic analogues of ribavirine, viz. 1-(1-hydroxy-4-oxahex-3-yl)-, 1-(1-chloro-4-oxahex-3-yl)-, 1-(1,2-dihydroxy-4-oxahex-3-yl)-, 1-(1,6-dihydroxy-4-oxahex-3-yl)-, 1-(1-chloro-6-hydroxy-4-oxahex-3-yl)-, and 1-(1,2,6-trihydroxy-4-oxahex-3-yl)-1,2,4-triazole-3-carboxamide, with the C3'-C4' bond of the furanose ring cleaved, have been prepared by condensation of trimethylsilyl derivatives of 3-ethoxycarbonyl-1,2,4-triazole with alkylating agents in the presence of SnCl4 followed by treatment with methanolic ammonia. Convenient methods for synthesis of the alkylating agents were elaborated.     

Compounds, similar to acyclovir V. Synthesis and antiviral activity of carbethoxyalkoxymethyl derivatives of nucleic bases

Ethyl esters and amides of carboxymethoxy-, alpha- and beta-carboxyethoxy- and gamma-carboxypropoxymethyl derivatives of adenine, guanine, cytosine, uracil, thymine, 1,2,4-triazole-3- and -5-carboxamide were synthesized and their antiviral activity was studied.     

Investigation of potent inhibitors of cholinesterase based on thiourea and pyrazoline derivatives: Synthesis,inhibition assay and molecular modeling studies

Owing to the desperate need of new drugs development to treat Alzheimer's ailment the synthesis of 1-aroyl-3-(5-(4-chlorophenyl)-1,2,4-triazole-3-thioneaminylthioureas (2–6) starting from (4-amino-5-(4-chlorophenyl)-4H-1,2,4-triazole-3-thiol) (1) and synthesis of 1-(3-(4-aminophenyl)-5-phenyl-4,5-dihydro-1H-pyrazol-1-yl)-2-(4-isobutylphenyl)propan-1-one (7–9) starting from 2-(4-isobutylphenyl)propanehydrazide (a) with the cyclization with substituted chalcones (c-e) was carried out. To check the biological potential of the synthesized compounds, all were subjected to acetylcholinesterase (AChE) and butrylcholinesterase (BChE) inhibition assays. The most potent and selective inhibitor for the acetylcholinesterase was compound 7 having an inhibitory concentration of 123 ± 51 nM, whereas, compound 6 was found as selective inhibitor of butyrylcholinesterase (BChE) with an IC₅₀ value of 201 ± 80 nM. However, the compounds 1 and 2 were found as dual inhibitors i.e. active against both acetylcholinesterase as well as butyrylcholinesterase.     

Synthesis of some new hybride molecules containing several azole moieties and investigation of their biological activities

1,2,4-Triazole-3-one prepared from tryptamine was converted to the corresponding carbothioamides by several steps. Their treatment with ethyl bromoacetate or 4-chlorophenacyl bromide produced the corresponding 5-oxo-1,3-thiazolidine or 3-(4-chlorophenyl)-1,3-thiazole derivatives. Acetohydrazide derivative that was obtained starting from tryptamine, was converted to the corresponding Schiff basis and sulfonamide by the treatment with suitable aldehydes and benzensulphonyl chloride, respectively. 2-[(4-Amino-5-thioxo-4,5-dihydro-1H-1,2,4-triazole-3-yl)methyl]-4-[2-(1H-indole-3-yl)ethyl]-5-methyl-2,4-dihydro-3H-1,2,4-triazole-3-one was synthesized starting from hydrazide via the formation of the corresponding 1,3,4-oxadiazole compound, while the other bitriazole compounds were obtained by intramolecular cyclisation of carbothioamides in basic media. The treatment of 1,2,4-triazole or 1,3,4-oxadiazole compound with several amines generated the corresponding Mannich bases. Ethyl (2-amino-1,3-thiazole-4-yl)acetate was converted to the corresponding 1,3,4-oxadiazole derivative, arylidenehydrazides, 1,2,4-triazole-3-one and 5-oxo-1,3-oxazolidine derivatives by several steps. The structural assignments of new compounds were based on their elemental analysis and spectral (FT IR, ¹H NMR, ¹³C NMR and LC-MS) data. The antimicrobial, antilipase and antiurease activity studies revealed that some of the synthesized compounds showed antimicrobial, antilipase and/or antiurease activity.     

Synthesis of 2′,3′-Dideoxyribavirin

Abstract

A synthesis of 1-(2,3-dideoxy-β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (2′,3′-dideoxyribavirin, ddR) is described. Glycosylation of the sodium salt of 1,2,4-triazole-3-carbonitrile (5) with 1-chloro-2-deoxy-3,5-di-0-p-toluoyl-α-D-erythro-pentofuranose (1) gave exclusively the corresponding N-1 glycosyl derivative with β-anomeric configuration (6), which on ammonolysis provided a convenient synthesis of 2′-deoxyribavirin (7). Similar glycosylation of the sodium salt of methyl 1,2,4-triazole-3-carboxylate (2) with 1 gave a mixture of corresponding N-1 and N-2 glycosyl derivatives (3) and (4), respectively. Ammonolysis of 3 furnished yet another route to 7. A four-step deoxygenation procedure using imidazolylthiocarbonylation of the 3′-hydroxy group of 5′-0-toluoyl derivative (9a) gave ddR (11). The structure of 11 was proven by single crystal X-ray studies. In a preliminary in vitro study ddR was found to be inactive against HIV retrovirus.     

Synthesis of acyclic analogs of ribavirin

A number of ribavirin analogues were prepared in which the ribose moiety was replaced with acyclic substituents imitating some fragments of the ribose ring: 2,3-dihydroxy-prop-1-yl, 3-hydroxymethyl-4-hydroxy-2-oxabut-1-yl, 4,5-dihydroxy-2-oxapent-1-yl and 1,5-dihydroxy-3-oxapent-2-yl. These analogues were synthesized by direct alkylation of ethyl 1,2,4-triazole-3-carboxylate with suitable agents followed by ammonolysis. New convenient methods for preparing the alkylating agents were developed.     

Nucleosides. II. Synthesis and Properties of 3,4-Diaryl-4,5-dihydro-1-(β-D-ribofuranosyl)-1,2,4-triazole-5-thiones

Abstract

3,4-Diaryl-4,5-dihydro-1,2,4-triazole-5-thiones (1a-c) were silylated to give compounds (2a-c) which were condensed with 1-O-acetyl-2,3,5-tri-O-benzoyl-β-D-ribofuranose (3) in the presence of trimethylsilyl trifluoromethane sulfonate to afford the corresponding nucleosides 4a-c. Treatment of 4a-c with sodium methoxide in methanol at room temperature afforded the debenzoylated nucleosides 5a-c. The reaction of 5a with acetone in the presence of p-toluenesulfonic acid gave the 2′, 3′-isopropylidene derivative (6a). Phosphorylation of 6a with phosphoryl chloride and triethylphosphate followed by treatment with barium hydroxide afforded barium 3,4-diphenyl-4,5-dihydro(β-D-ribofuranosyl)-1,2,4-triazole-5-thione-5′- monophosphate, which gave after lyophilization the free acid (7a)     

Microbiological synthesis of virazole by immobilized cells]

Based on the isotherms of adsorption of the cells of Xanthomonas campestris B-610 to glass and polyvinyl fibers, immobilized systems were produced, in which the cell content was sufficient for enzymatic synthesis of Virazole (1-[beta-D-ribofuranosyl]-1,2,4-triazole-3-carboxamide) using adenosine as a donor of ribose (50-60% conversion of 1,2,4-triazole-3-carboxamide). The immobilized cells thus obtained retain their enzymatic activity for six months and can be used repeatedly.     

Synthesis of 3'-deoxy-3'-C-methyl nucleoside derivatives

2',3'-Dideoxy-3'-C-methyl nucleosides bearing the five naturally occurring nucleic acid bases were synthesized. Additionally, the 3'-deoxy-3'-C-methyl nucleoside analogues bearing 5-aminoimidazole-4-carboxamide as well as 1,2,4-triazole-3-carboxamide moieties were prepared. The synthesis of the corresponding 2',3'-dideoxy-3'-C-methyl triazole derivative was also accomplished. The dideoxynucleoside derivatives were prepared by radical deoxygenation from their 3'-deoxy-3'-C-methyl parent ribonucleosides. When evaluated for their antiviral activity in cell culture experiments, none of these compounds showed any significant antiviral activity.     

Ribavirin cures cells of a persistent infection with foot-and-mouth disease virus in vitro.

Ribavirin (1-beta-D-ribofuranosyl-1H-1,2,4-triazole-3-carboxamide) eliminates foot-and-mouth disease virus from persistently infected cell cultures. The latter are 10-fold more sensitive to ribavirin than lytically infected cells. In treated cells no viral RNA or proteins could be detected by dot-blot hybridization to cDNA probes, virus and RNA infectivity assays, or immunofluorescence. A potential application of the drug for the treatment of animals carrying the virus is suggested.     

The antiviral drug ribavirin is a selective inhibitor of S-adenosyl-L-homocysteine hydrolase from Trypanosoma cruzi

Ribavirin (1,2,4-triazole-3-carboxamide riboside) is a well-known antiviral drug. Ribavirin has also been reported to inhibit human S-adenosyl-L-homocysteine hydrolase (Hs-SAHH), which catalyzes the conversion of S-adenosyl-L-homocysteine to adenosine and homocysteine. We now report that ribavirin, which is structurally similar to adenosine, produces time-dependent inactivation of Hs-SAHH and Trypanosoma cruzi SAHH (Tc-SAHH). Ribavirin binds to the adenosine-binding site of the two SAHHs and reduces the NAD(+) cofactor to NADH. The reversible binding step of ribavirin to Hs-SAHH and Tc-SAHH has similar K(I) values (266 and 194 microM), but the slow inactivation step is 5-fold faster with Tc-SAHH. Ribavirin may provide a structural lead for design of more selective inhibitors of Tc-SAHH as potential anti-parasitic drugs.     

Some 3-thioxo/alkylthio-1,2,4-triazoles with a substituted thiourea moiety as possible antimycobacterials.

A series of novel N-alkyl/aryl-N'-[4-(4-alkyl/aryl-2,4-dihydro-3H-1,2,4-triazole-3-thione-5-yl)phenyl]thioureas 1-19 and three S-alkylated representatives of the former, N-alkyl/aryl-N'-[4-(3-aralkylthio-4-alkyl/aryl-4H-1,2,4-triazole-5-yl)phenyl]thioureas 20-22, were synthesized and tested for antimycobacterial activity against Mycobacterium tuberculosis H37Rv as well as Mycobacterium fortuitum ATCC 6841 which is a rapid growing opportunistic pathogen. Compounds 4 and 9-11 were found to possess the same MIC value with that of Tobramycin against M. fortuitum ATCC 6841 whereas 1-3 and 21 had positive response against M. tuberculosis H37Rv at varying degrees. Compound 21 was identified as the most potent derivative of the 1-22 series by an MIC value of 6.25 microg/mL and selectivity index of 1.6.     

Synthesis and antiviral activity of amino acid esters of ribavirin

The synthesis and antiviral activity of amino acid esters of 1-(β-D-ribofuranosyl)-1,2,4-triazole-3-carboxamide (ribavirin,1) is described.     

Synthesis and evaluation of arylpiperazines derivatives of 3,5-dioxo-(2H,4H)-1,2,4-triazine as 5-HT1AR ligands

5-HT_1AR agonist or partial agonists are established drug candidates for psychiatric and neurological disorders. We have reported the synthesis and evaluation of a series of high affinity 5-HT_1AR partial agonist PET imaging agents with greater selectivity over α-1AR. The characteristic of these molecules are 3,5-dioxo-(2H,4H)-1,2,4-triazine skeleton tethered to an arylpiperazine unit through an alkyl side chain. The most potent 5-HT_1AR agonistic properties were found to be associated with the molecules bearing C-4 alkyl group as the linker. Therefore development of 3,5-dioxo-(2H,4H)-1,2,4-triazine bearing arylpiperazine derivatives may provide high affinity selective 5-HT_1AR ligands. Herein we describe the synthesis and evaluation of the binding properties of a series of arylpiperazine analogues of 3,5-dioxo-(2H,4H)-1,2,4-triazine.     

The effect of 1-β-d-ribofuranosyl- 1,2,4-triazole-3-carboxamide on bud and shoot formation in tobacco petiole cultures

Ribavirin (1-β- d -ribofuranosyl- 1,2,4-triazole-3-carboxamide) increases starch accumulation in explants of Nicotiana tabacum L. ev. Xanthi nc and increases the number of meristematic zones; adventitious bud and shoot formation is significatntly increased. It overcomes auxin inhibition of bud and shoot formation in this system but studies on interaction with exogenous auxin and triiodobenzoic acid do not support the hypothesis that ribavirin acts directly on auxin metabolism. The results are discussed in relation to the induction and maintenance of permissive or receptive cell states in tobacco explant culture.     

Synthesis and structure–activity relationships of C-glycosylated oxadiazoles as inhibitors of glycogen phosphorylase

A series of per-O-benzoylated 5-β-d-glucopyranosyl-2-substituted-1,3,4-oxadiazoles was prepared by acylation of the corresponding 5-(β-d-glucopyranosyl)tetrazole. As an alternative, oxidation of 2,6-anhydro-aldose benzoylhydrazones by iodobenzene I,I-diacetate afforded the same oxadiazoles. 1,3-Dipolar cycloaddition of nitrile oxides to per-O-benzoylated β-d-glucopyranosyl cyanide gave the corresponding 5-β-d-glucopyranosyl-3-substituted-1,2,4-oxadiazoles. The O-benzoyl protecting groups were removed by base-catalyzed transesterification. The 1,3,4-oxadiazoles were practically inefficient as inhibitors of rabbit muscle glycogen phosphorylase b while the 1,2,4-oxadiazoles displayed inhibitory activities in the micromolar range. The best inhibitors were the 5-β-d-glucopyranosyl-3-(4-methylphenyl- and -2-naphthyl)-1,2,4-oxadiazoles (K_i = 8.8 and 11.6 μM, respectively). A detailed analysis of the structure–activity relationships is presented.     

The synthesis of cholestane and furostan saponin analogues and the determination of sapogenin's absolute configuration at C-22

A facile and efficient way for the synthesis of cholestane and furostan saponin analogues was established and adopted for the first time. Following this strategy, starting from diosgenin, three novel cholestane saponin analogues: (22S,25R)-3β,22,26-trihydroxy-cholest-5-ene-16-one 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 11, (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 14 and (25R)-3β,16β,26-trihydroxy-cholest-5-ene-22-one 16-O-[O-α-l-rhamnopyranosyl-(1 → 2)-β-d-glucopyranoside] 17, three novel furostan saponin analogues: (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 23, (22R,25R)-furost-5-ene-3β,22,26-triol 22-O-(α-d-glucopyranoside) 24 and (22S,25R)-furost-5-ene-3β,22,26-triol 22-O-[O-α-l-rhamnopyranosyl-(1 → 2)-α-d-glucopyranoside] 26, were synthesized ultimately. The structures of all the synthesized analogues were confirmed by spectroscopic methods. The S-chirality at C-22 of cholestane was confirmed by Mosher's method. The absolute configuration at C-22 of furostan saponin analogues was distinguished by conformational analysis combined with the NMR spectroscopy. The cytotoxicities of the synthetic analogues toward four types of tumor cells were shown also.