Isomerization of Cytidine 2′,3′-Thionocarbonates

Abstract

N⁴,5′-Profecied cytidine 2′,3′-thionocarbonaies isomerize at elevated temperatures to 2′-deozy-2″-thiocylidine 2′,3′ -carbonates.     

Improved Synthesis of 2′,3′-Dideoxycytidine (d2C) and Its Correlated Nucleoside Analogues

Abstract

2′,3′-dideoxy- and 2′,3′-dideoxy-2′,3′-didehydrocy-tidine (d2C and d4C) have been synthesized in good yields from 2′-deoxyuridine via dichlorinated derivatives 7a-b. The same synthetic strategy was used in the synthesis of d2C^Me and d4C^Me from thymidine. Following this method the evaluable 3′-chloro-2′-deoxycytidine derivatives 9-12 can easily be obtained.     

Enzymatic Preparation of 32P-Labeled β-L-2′, 3′-dd-5′ ATP and Its Use as a High-Affinity,Conformation-Specific Ligand for Labeling Adenylyl Cyclases

Abstract

An enzymatic method was developed for the preparation of unlabeled and [β-³²P]-labeled β-L-2′,3′-dd-5′ATP from the monophosphate with near quantitative yields. β-L-2′,3′-dd-5′ATP was a competitive and potent inhibitor of adenylyl cyclases (IC₅ ～ 30 nM). Upon uvirradiation β-L-2′,3′-dd-[β-³²P]-5′ATP directly crosslinked to a chimeric construct of this enzyme. Data suggest that this is a pre-transition state inhibitor and contrasts with the equipotent 2′,5′-dd-3′ATP, a post-transition state, noncompetitive inhibitor.     

An Improved Synthesis of 1-β-D-Arabinofuranosylcytosine 5′-Phosphate-L-1,2-diacylglycerols

Abstract

5′-O-MMTr-cytosine arabinoside was prepared on a large scale from 5′-O-MMTr-cytidine with diphenyl carbonate via 5′-protected cytidine-2′,3′-carbonate-aracytidine-2′,2-anhydro derivative at a 67 % yield. The synthesis of 1,2-L-dipalmitoyl-snglycerol, 1,2-L-distearoyl-sn-glycerol and 1,2-L-dioleoyl-sn-glycerol described here using 9-fluorenylmethoxycarbonyl (FMOC) group for protection of 3-position of glycerol which can be selectively removed by Et₃N treatment on the overall 60–70 % yield based on 1.2,-isopropilidene-sn-glycerol. These glycerols were phosphorylated first with 2-chlorophenyl-phosphoro-bis-triazolide quantitatively¹ in order to avoid acyl migration, then the glycerophosphate intermediates were condensed with 2′,3′,N⁴-trileulinyl-l-β-D-arabinofuranosylcytosine in the presence of 2-mesytilenesulphonyl chloride (MsCl) and 1 -methylimidazole (Melm)-which was used in the coupling of nucleotides²-^? in an 85–95 % yield compared with the low yielding diester method of Ryu³. Deblocking was carried out in two steps with tetrabutylammonium fluoride (TBAF) and hydrazine hydrate, producing target compouns (14a, 14b, 14c) at a 50 % yield.     

Nucleosides and Nucleotides. 104. Radical and Palladium-Catalyzed Deoxygenation of the Allylic Alcohol Systems in the Sugar Moiety of Pyrimidine Nucleosides§,1

Abstract

New methods for the synthesis of 2′,3′-didehydro-2′,3′-dideoxy-2′ (and 3′)-methyl-5-methyluridines and 2′,3′-dideoxy-2′ (and 3′)-methylidene pyrimidine nucleosides have been developed from the corresponding 2′ (and 3′)-deoxy-2′ (and 3′)-methylidene pyrimidine nucleosides. Treatment of a 3′-deoxy-3′-methylidene-5-methyluridine derivative 8 with 1,1′-thiocarbonyldiimidazole gave the allylic rearranged 2′,3′-didehydro-2′,3′-dideoxy-3′-[(imidazol-1-yl)carbonylthiomethyl] derivative 24. On the other hand, reaction of 8 with methyloxalyl chloride afforded 2′-O-methyloxalyl ester 25. Radical deoxygenation of both 24 and 25 gave 26 exclusively. Palladium-catalyzed reduction of 2′,5′-di-O-acetyl-3′-deoxy-3′-methylidene-5-methyluridine (32) with triethylammonium formate as a hydride donor regioselectively afforded the 2′,3′-dideoxy-3′-methylidene derivative 35 and 2′,3′-didehydro-2′,3′-dideoxy-3′-methyl derivative 34 in a ratio of 95:5 in 78% yield. These reactions were used on the corresponding 2′-deoxy-2′-methylidene derivatives. An alternative synthesis of 2′,3′-dideoxy-2′-methylidene pyrimidine nucleosides (43, 52, and 54) was achieved from the corresponding 1-(3-deoxy-β-D-thero-pentofuranosyl)pyrimidines (44 and 45). The cytotoxicity against L1210 and KB cells and inhibitory activity of the pathogenicity of HIV-1 are also described     

Chemical-Enzymatic Synthesis of 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of Natural Heterocyclic Bases and Their 5′-Monophosphates

Abstract

Treatment of O², 3′-anhydro-5′-O-trityl derivatives of thymidine (1) and 2′-deoxyuridine (2) with lithium azide in dimethylformamide at 150 °C resulted in the formation of the corresponding isomeric 3′-azido-2′, 3′-dideoxy-5′-O-trityl-β-D-ribofuranosyl N¹- (the major products) and N³-nucleosides (3/4 and 5/6). 3′-Amino-2′, 3′-dideoxy-β-D-ribofuranosides of thymidine [Thd(3′NH₂)], uridine [dUrd(3′NH₂)], and cytidine [dCyd(3′NH₂)] were synthesized from the corresponding 3′-azido derivatives. The Thd(3′NH₂) and dUrd(3′NH₂) were used as donors of carbohydrate moiety in the reaction of enzymatic transglycosylation of adenine and guanine to afford dAdo(3′NH₂) and dGuo(3′NH₂). The substrate activity of dN(3′NH₂) vs. nucleoside phosphotransferase of the whole cells of Erwinia herbicola was studied.     

Variation in isomeric products of a phosphodiesterase from the chloroplasts of Phaseolus vulgaris in response to cations

ABSTRACT

Fast-atom bombardment mass spectrometry (FABMS), and collisionally-induced dissociation and mass-analyzed ion kinetic energy spectrum scanning (CID/MIKES) have been used to examine cation effects on a Phaseolus chloroplast complex phosphodiesterase activity. The kinetic parameters of the activity, and the effects of Li⁺, Na⁺, K⁺, Mg²⁺, Mn²⁺ and Fe³⁺ upon them, were determined with 3′,5′-cyclic AMP, -GMP and -CMP, and 2′,3′-cyclic AMP, -GMP and -CMP as substrates. Irrespective of the presence of cations and of the complex nucleotidase, the preferred substrate is a 3′,5′-cyclic nucleotide, not a 2′,3′-cyclic nucleotide. In the presence of the nucleotidase 3′,5′-cyclic AMP and 3′,5′-cyclic GMP are the best substrates, unless Fe³⁺ ions are present. Mg²⁺ and Mn²⁺ stimulate hydrolysis of 3′,5′-cyclic AMP and 3′,5′-cyclic GMP by the complex. However, Fe³⁺ inhibits these activities but stimulates the hydrolysis of 3′,5′-cyclic CMP. Kinetic data indicate that each of these six substrates is hydrolyzed at a single, common, catalytic site. Differentiation of the phosphodiesterase isomeric mononucleotide products by FABMS CID/MIKES analysis indicates that in the absence of ions and after removal of the nucleotidase, the 3′-ester linkage of the 3′,5′-cyclic substrates was hydrolyzed exclusively. Addition of monovalent and divalent ions results in hydrolysis of both the 5′- and 3′-ester linkages.     

Synthesis of 2′,3′-Dideoxy-2′-methylene Pyrimidine Nucleosides as Potential Anti-Human Immunodeficiency Virus (HIV) Agents

Abstract

Several 2′,3′-dideoxy-2′-methylene pyrimidine nucleosides, 2′,3′-dideoxy-2′-methylenecytidine hydrochloride (20), 2′,3′-dideoxy-2′-methyleneuridine (21), and 2′,3′-dideoxy-2′-methylene-5-methyluridine (22), have been synthesized via a multi-step synthesis from uridine and 5-methyluridine, respectively. These compounds were tested for their cytotoxicity against L1210, S-180, CCRF-CEM, and P388 cells in culture and their antiviral activity is under investigation.     

Intracellular Metabolism of β-L-ddAMP-bis(tbutylSATE), a Potent Inhibitor of Hepatitis B Virus Replication

Abstract

β-L-ddAMP-bis(tbutylSATE) is a potent inhibitor of HBV replication with an EC₅₀ = 0.1 μM. Following a 0-to72-hrs exposure of human hepatocytes to a 10 μM [2′,3′^?3H] β-L-ddAMP-bis(tbutylSATE), the pharmacologically active β-L-ddATP was the predominant metabolite attaining a concentration of 268.53 ± 107.97 pmoles/10⁶ cells at 2 hrs. In Hep-G2 cell, β-L-ddATP accounted for 146.8 ± 29.8 pmoles/10⁶ cells at 2 hrs with an half life of approximately 5.4 hrs. This study reveals that extensive intracellular concentrations of β-L-ddATP after incubation of cells to the parent drug is accounting for its potent antiviral activity.     

Conformation Analysis of Two Anti-HIV Nucleoside Analogues 2′,3′-Dideoxy-3′-fluorocytidine and Its N4-Dimethylaminomethylene Prodrug Derivative

Abstract

Crystal structure analysis of 2′,3′-dideoxy-3′-fluorocytidine (1) and its prodrug derivative, N⁴-dimethylaminomethylene-2′,3′-dideoxy-3′-fluorocytidine (2), active anti-HIV nucleoside analogues, reveals that both structures adopt an anti conformation about the glycosyl bond. The furanose ring is C2′-endo for (2) and C2′-endo/C1′-exo and C2′-endo/C3′-exo for the two independent molecules of (1), respectively.     

2′,5′-Adenylate and Cordycepin Trimer Cores: Metabolic Stability and Evidence for Antimitogenesis without 5′-Rephosphorylation

Abstract

The effect of the 2′,5′-adenylate and cordycepin trimer cores on DNA and protein synthesis in human umbilical cord lymphocytes, lymphoblasts, peripheral blood lymphocytes and Epstein-Barr virus infected lymphocytes and their metabolism in tissue culture medium have been studied. [³²P]Adenylate and [³²P]- and [³H]cordycepin trimer cores were synthesized either enzymatically or chemically and added to cells in culture. The half-lives of the 2′,5′-A₃ core and 2′,5′-3′dA₃ core in tissue culture were 3 and 17 hr, respectively. Chromatographic analysis of the TCA-soluble extracts of the lymphocytes and lymphoblasts treated with 2′,5′-[³H]A₃ showed that 0.25% of the ³²P was identified as AMP, ADP, ATP and inorganic phosphate. By the more sensitive 2′,5′-p₃A₄[³²P]pCp radiobinding assay, 2′,5′-A₃ was detected in the TCA supernatants; however, there was no 5′-rephosphorylation. With the [³H]- and [³²P]cordycepin trimer core, 0.55% and 1.3% of the radioactivity was in the TCA soluble extracts. Although there was no 5′-rephosphorylation as determined by radiobinding assay, the intact cordycepin trimer core was detected by tlc, radiobinding assay, and HPLC.

Furthermore, in two experiments, the concentration of the cordycepin trimer core bound to or taken up by the lymphocytes was three-fold greater than the concentration in the medium. 2′,5′-A₃ and 2′,5′-3′dA₃ cores were both antimitogenic, but did not inhibit protein synthesis.     

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.     

Study on Disulfur-Backboned Nucleic Acids: Part 3. Efficient Synthesis of 3′,5′-Dithio-2′-Deoxyuridine and Deoxycytidine

A general method is described for synthesizing 3′,5′-dithio-2′-deoxypyrimidine nucleosides 6 and 13 from normal 2′-deoxynucleosides. 2,3′-Anhydronucleosides 2 and 9 are applied as intermediates in the process to reverse the conformation of 3′-position on sugar rings. The intramolecular rings of 2,3′-anhydrothymidine and uridine are opened by thioacetic acid directly to produce 3′-S-acetyl-3′-thio-2′-deoxynucleosides 3 or 5. To cytidine, OH^? ion exchange resin was used to open the ring and 2′-deoxycytidine 10 was abtained in which 3′-OH group is in threo-conformation. The 3′-OH is activated by MsCl, and then substituted by potassium thioacetate to form the S,S′-diacetyl-3′,5′-dithio-2′-deoxycytidine 12. The acetyl groups in 3′,5′ position are removed rapidly by EtSNa in EtSH solution to afford the target molecules 6 and 13. The differences of synthetic routes between uridine and cytidine are also discusssed.     

Potent and selective activity of a new carbocyclic nucleoside analog (carbovir: NSC 614846) against human immunodeficiency virus in vitro

Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir: NSC 614846), a novel nucleoside analog, emerged as a potent and selective anti-HIV agent from a large screening program conducted by the National Cancer Institute and its contractors. Its hydrolytic stability and its ability to inhibit the infectivity and replication of HIV in T-cells at concentrations of approximately 200- to 400-fold below toxic concentrations make carbovir a top-priority candidate for development as a potential antiretroviral agent in the treatment of AIDS patients.     

Cyclic nucleotide esterification: relationship to phosphate ring geometry and growth regulation in synchronized human lymphocytes.

Infrared spectra of neutral aqueous solutions of nucleoside 3′,5′-cyclic monophosphates indicate an increase in the antisymmetric phosphoryl stretching frequency to 1236 cm^?1 from 1215 cm^?1 in trimethylene cyclic phosphates. A further increase to 1242 cm^?1 accompanies esterification of the 2′-ribose hydroxyl. The O²′-esterified and 2′-deoxy cyclic nucleotides examined display both reduced kinase binding and altered phosphoryl stretching frequencies, suggesting that modification of the phosphate ring represents a common feature in decreased kinase activation. Reversible inhibition of mitosis in thymidine-synchronized human lymphocytes by 2 mmN⁶,O²′-dibutyryladenosine 3′,5′-cyclic monophosphate and N⁶-monobutyryladenosine 3′,5′-cyclic monophosphate was observed. However, adenosine 3′,5′-cyclic monophosphate, O²′-monobutyryladenosine 3′,5′-cyclic monophosphate, butyric acid, and ethyl butyrate had no effect on mitosis when present at 2 mm concentrations during S and G2. These results are consistent with hydrolysis of O²′-monobutyryladenosine 3′,5′-cyclic monophosphate and adenosine 3′,5′-cyclic monophosphate by esterase and phosphodiesterase enzymes and suggest that modification of the N⁶ amino group is necessary for the antimitotic activity of N⁶,O²′-dibutyryladenosine 3′, 5′-cyclic monophosphate.     

Assignment of 13C Chemical Shifts of α-D-Ribonucleoside Sugar Carbons by 1J(CH) Coupling Constants

Abstract

The ¹J(CH) coupling constant of C-1 in nucleosides is increased compared to those of the other carbons of the sugar moiety. Applying this to several D-ribonucleosides the signals C-4′/C-1′of these a-anomers are reversed to those of the 8-counterparts (C-1′/C-4′). This phenomenon and the broadening of the C-3′ signal compared to that of C-2′ establishes the seauence C-4′,1′,2′,3′,5′ (increasing field) for a number of α-D-ribonucleosides.     

Synthesis,Solution Conformation and Biological properties of 2′,3′-Dideoxy-3′-fluoro-D-erythro-pentofuranosides of 2-Thiouracil and 2-Thiothymine

Abstract

The synthesis of the α- and β-anomers of 2′,3′-dideoxy-3′-fluoro-2-thiouridine and 2′,3′-dideoxy-3′-fluoro-2-thiothymidine via Lewis acid catalysed nucleoside condensation is described. High resolution ¹H NMR data, solution conformations and biological properties are also presented.     

Resolution of racemic carbovir and selective inhibition of human immunodeficiency virus by the (-) enantiomer

(+-) Carbocyclic 2',3'-didehydro-2',3'-dideoxyguanosine (Carbovir; NSC 614846) is an antiretroviral agent which is undergoing preclinical evaluation for the treatment of AIDS. Racemic carbovir was separated into its D and L enantiomers by the action of adenosine deaminase on the 2,6-diaminopurine precursor. Subsequent evaluation of the enantiomers against human immunodeficiency virus type 1 revealed that the antiviral activity of carbovir resides in the (-) isomer that is analogous to the nucleoside, beta-D-2',3'-didehydro-2',3'-dideoxyguanosine.     

Stannylation Approach to the Synthesis of 2′- and 3′-Substituted Analogues of 2′,3′-Didehydro-2′,3′-dideoxynucleosides

Abstract

Three methods are described for the introduction of a tributylstannyl group to the sp²-carbon of 2′,3′-didehydro-2′,3′-dideoxy nucleosides (d44Ns). The resulting stannylated products serve as versatile intermediates for the synthesis of d4Ns having various types of carbon-substituent.