Accumulation of 5-Ethyl-2′-deoxyuridine and its 5,6-Dihydro Prodrugs in Murine Lung and its Potential Clinical Application

The accumulation of 5-ethyl-2′-deoxyuridine (EDU), (–)-trans-(5S, 6S)-5-bromo-5-ethyl-6-methoxy-5,6-dihydro-2′-deoxyuridine [(5S, 6S)-BMEDU], (+)-trans-(5R, 6R)-5-bromo-5-ethyl-6-methoxy-5,6-dihydro-2′-deoxyuridine [(5R, 6R)-BMEDU], (+)-trans-(5.R, 6R)-5-bromo-5-ethyl-6-ethoxy-5, 6-dihydro-2′-deoxyuridine (BEEDU), (+)-trans-(5R, 6R)-5-bromo-5-ethyl-6-ethoxy-5,6-dihydro-5′-O-valeryl-2′- deoxyuridine (VBEEDU) and (+)-trans-(5R, 6R)-5-bromo-5-ethyl-6-ethoxy-5,6-dihydro-3′-5′-di-O-valeryl-2′-deoxyuridine (DVBEEDU) in lung and other tissues was investigated in male Balb-C mice following intravenous injection of the corresponding 4-¹⁴C-labelled compounds. EDU showed a rapid distribution into liver and lung immediately after injection, and the overall levels of radioactivity in blood, liver and lung were similar. The distribution of radioactivity in lung after injection of [4-¹⁴C](5S, 6S)-BMEDU and [4–14C](5R, 6R)-BMEDU were substantially different from one another and also from that of [4-¹⁴C]EDU. The radioactivity level present in lung samples after injection of both [4-¹⁴C](5S, 6S)-BMEDU and [4-¹⁴C](5R, 6R)-BMEDU was substantially higher than that in blood samples. Radioactivity levels present in lung samples taken at 18 min after injection of [4-¹⁴C]BEEDU were significantly higher (P < 0·05) than those for liver and blood samples. Although the radioactivity present in lung samples after injection of [4-¹⁴C]VBEEDU was significantly higher (P < 0·05) than those of liver and blood samples, [4-¹⁴C]VBEEDU did not provide a higher radioactivity level in lung samples than did [4-¹⁴C]BEEDU. The level of radioactivity in lung samples following injection of [4-¹⁴C]DVBEEDU was also higher than that of blood samples. EDU undergoes glycosidic bond cleavage to form EU in lung following intravenous injection into Balb-C mice. The concentrations of EDU and 5-ethyluracil (EU) following intravenous injection of EDU, and its prodrugs BEEDU and VBEEDU, were quantitated in lung and blood samples. In contrast to lung samples, EU was not detected in blood samples at 120 min post injection of EDU. The concentrations of both EDU and EU in lung tissues after injection of BEEDU and VBEEDU were substantially higher than those in blood samples.     

Synthesis of [1,2,4]triazolo[3′,4′:3,4][1,2,4]triazino[5,6-b]indole,tetrazolo[5′,1′:3,4][1,2,4]triazino[5,6-b]indole and their Derivatives

Reactions of 5-ethyl 5H-1,2,4-triazino|5,6-b|indol-3-thione ( 1 ) with various reagents have been studied. 1 and hydrazine hydrate in anhydrous ethanol gave 5-ethyl-3-hydrazino-5H-1,2,4-triazino|5,6-b|indole ( 2 ). This compound was condensed with formic acid and acetic acid to give 10-ethyl-10H-|1,2,4|triazolo|3′,4′:3,4||1,2,4|triazino|5,6-b|indole ( 4 , R=H) and 10-ethyl-1 -methyl-10H-|1,2,4|triazolo-|3′,4′:3,4‖ 1,2,4|triazino|5,6-b|indole ( 4 , R = CH₃), respectively. Treatment of 2 with nitrous acid gave 10-ethyl-10H-tetrazolo|5′,1′:3,4‖ 1,2,4|triazino|5,6-b|indole ( 7 ). Interaction of 2 with acetylacetone in alcoholic KOH gave the pyrazole 8 , whereas reaction of 2 with ethyl acetoacetate in anhydrous ethanol led to the expected hydrazone 9 , which on reflux with alcoholic KOH gives 1-(5-ethyl-5H-1,2,4-triazino| 5,6-b|indol-3-yl)-3-methyl-2-pyrazolin-5-one 10 . Treatment of 2 with ethyl chloroformate gives 11 , which can be converted to 10-ethyl-2,10-dihydro-1H-|1,2,4|triazolo|3′,4′:3,4|triazino|5,6-b|-indol-1-one 12 by fusion.     

Prodrugs of 5-ethyl-2'-deoxyuridine, I. Syntheses and antiviral activities of some 5'-O-acyl derivatives

A series of 5′-O- acyl derivatives (2a-e) of 5-ethyl-2′-deoxyuridine (EDU, EtUdR) were prepared by direct acylation of the parent nucleoside 1 in pyridine/N,N-dimethylformamide (DMF). These compounds, designed as prodrugs of 1 , offer a wider range of solubilities and lipophilicities than 1. The antiviral activities (against herpes simplex virus types 1 and 2) of all compounds were determined in primary rabbit kidney (PRK) cell cultures.     

Synthesis of [1,2,4]triazolo[3′,4′: 3,4] [1,2,4]triazino[5,6-b]indole derivatives and 3-substituted 5-ethyl-H-1,2,4-triazino[5,6-b] Indole

5-Ethyl-3-hydrazino-5H-1,2,4-triazino|5,6-b|indole ( 2 ) reacts with aromatic aldehydes in anhydrous ethanol to yield substituted arylidene-(5-ethyl-5H-1,2,4-triazino|5,6-b|indol-3-yl)hydrazones 3 . Treatment of 3 with excess SOCl₂ gave 10-ethyl-1-aryl-10H-| 1,2,4]triazolo|3′,4′:3,4| 1,2,4|triazino|5,6-b|indoles 4 , while the reaction of 2 with CS₂ in methanolic KOH afforded 10-ethyl-2,10-dihydro-1 H-| 1,2,4|triazolo|3′,4′3,4‖ 1,2,4|triazino|5,6-b|indol-1-thione ( 5 ). Fusion of 1 with aromatic amines leads to 3-(arylamino)-5-ethyl-5H-1,2,4-triazino|5,6-b|indoles 6 , whereas the reaction of 1 with alkyl or aralkyl halides in anhydrous acetone and in the presence of anhydrous K₂CO₃ results in the alkylation of the thiol group to give the alkylthio or aralkylthio derivatives 7.     

Synthesis and antiviral activity of acyclic derivatives of 5-ethyl-2'-deoxyuridine

Four acyclic pyrimidine derivatives of 5-ethyl-2′-deoxyuridine (1) (EDU) have been synthesized and tested for their activities against vesicular stomatitis virus, vaccinia virus and various strains of herpes simplex virus type 1 (HSV-1) and herpes simplex virus type 2 (HSV-2) in primary rabbit kidney (PRK) cell cultures. The acyclic nucleoside analogues of EDU exhibited no activity against any of the viruses tested.     

Prodrugs of 5-ethyl-2'-deoxyuridine, II. Syntheses and antiviral activities of 5'- and 3'-ester derivatives

With the aim of obtaining derivatives of the well established antiherpes compound 5-ethyl-2′-deoxyuridine ( 1 ) (Aedurid©, EtUdR, EDU), which are more lipophilic and therapeutically superior, 5′- and 3′-ester derivatives of 1 were synthesized. Tested in primary rabbit kidney cell cultures against various strains of herpes simplex type 1 (HSV-1) and type 2 (HSV-2), all EtUdR esters, with the exception of compounds 8 and 13 , proved almost as active as EtUdR itself, suggesting that they were readily hydrolized.     

Bequeme Synthese von Bis-3′-0-(5-ethyl-2′-desoxyuridin)-sulfoxid (KK-Ro 258)

Convenient Synthesis of Bis-3′-0-(5-ethyl-2′-deoxyuridine) Sulfoxide (KK-Ro 258) The title compound 4 was prepared by treating 5′-0-trityl-5-ethyl-2′-deoxyuridine with thionyl chloride in a mixture of dichloromethane and pyridine. The trityl group was removed by standard procedures.     

Antiherpes activity of [E]-5-(1-propenyl)-2′-deoxyuridine and 5-(1-propenyl)-1-β-d-arabinofuranosyluracil

5-(1-Propenyl)-1-β-d-arabinofuranosyluracil has been synthesized, and this compound and [E]-5-(1-propenyl)-2′-deoxyuridine have been tested for inhibition of herpes virus multiplication. Only [E]-5-(1-propenyl)-2′-deoxyuridine was found to be an active inhibitor reducing by 50% the plaque formation of herpes simplex virus type 1 (HSV-1) at about 1 μM. A comparison to the bromovinyl derivatives showed the following order of decreasing activity; $\text{[}\text{E}\text{]-5-(2-}\text{bromovinyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(2-}\text{bromovinyl}\text{)-1-β-}\text{d}\text{-}\text{arabinofuranosyluracil}\text{≥ [}\text{E}\text{]-5-(1-}\text{propenyl}\text{)-2′-}\text{deoxyuridine}\text{> 5-(1-}\text{propenyl}\text{)-1-β-}\text{arabinofuranosyluracil}$. HSV-1 mutants lacking thymidine kinase or resistant against acycloguanosine were resistant against [E]-5-(1-propenyl)-2′-deoxyuridine. All compounds seemed to be phosphorylated by HSV-1 thymidine kinase in a cell-free assay. The compounds were phosphorylated to a lower extent by cellular or HSV-2 thymidine kinase, and the HSV-2 strains tested were inhibited by less than 50% at 100 μM in plaque assays. A selective inhibition of HSV-1 DNA synthesis by [E]-5-(1-propenyl)-2′-deoxyuridine was observed in infected cells indicating an effect on viral DNA polymerase. [E]-5-(1-Propenyl)-2′-deoxyuridine had a low cellular toxicity and a therapeutic effect when applied topically to HSV-1-infected guinea pig skin.     

Studies on the antiviral activity of 5′-amino-2′,5′-dideoxy-5-iodouridine (AIU) against herpes viruses in vivo and in vitro

5′-Amino-2′,5′-dideoxy-5-iodouridine (AIU) is known to have antiviral activity against herpes simplex virus type 1 (HSV-1) in cell cultures but is less potent than the parent compound 5-iodo-2′-deoxyuridine (IDU). Studies on its activity in vivo are limited. AIU showed antiviral effects in BHK cells against wild-type HSV-1 but not a thymidine kinase-negative (TK^?) mutant, indicating the importance of phosphorylation of the compound by HSV-1-induced thymidine kinase for antiviral effects. When cells were coinfected with the TK^? mutant and a wild-type TK⁺ strain, both strains were inhibited by AIU, suggesting that the failure to phosphorylate AIU accounts for the resistance of the TK^? strain alone. AIU failed to limit death after systemic HSV-1 infection of mice when the drug was administered parenterally or orally, although IDU in similar treatment regimens was effective. Tested for efficacy in a local HSV-1 skin infection of mice, topical AIU in a petrolatum ointment or dimethyl sulphoxide (DMSO) did not reduce titres of virus in the skin or modify the clinical course of infection, whereas topical application of IDU in DMSO caused a significant reduction in the titres of virus in the skin and reduced both the occurrence and the severity of lesions. When administered subcutaneously or orally AIU had a slight antiviral effect against HSV-1 infection in the skin of mice. Moreover, intraperitoneally (i.p.) administered AIU limited HSV-1 replication in peritoneal cells of i.p. infected mice, indicating that AIU is inherently antiviral in vivo. The poor antiviral activity of AIU in vivo compared with IDU is attributed to its lower antiviral potency as judged by its activity in cell cultures and its inability to enter neural tissue.     

5-Ethyl-2′-deoxyuridine-5′-monophosphate inhibition of the thymidylate synthetase from escherichia coli

The synthesis of 5-ethyl-2′-deoxyuridine-5-monophosphate (EtdUrdMP) and an improved method for the purification of deoxythymidylate synthetase (TdRMP synthetase) from E. coli are described. TdRMP synthetase was inhibited competitively by EtdUrdMP (K₁ = 2·2 × 10^?5 M). Under similar conditions the K_i for deoxythymidine monophosphate (TdRMP) was 2·0 × 10^?5 M. The possible role of EtdUrdMP in the virostatic activity of the nucleoside 5-ethyl-2′-deoxyuridine (EtdUrd) is considered.     

Nucleosides and Nucleotides,Part 144 Synthesis and Antiviral Activity of 5-Substituted (2′S)-2′-Deoxy-2′-C-Methylcytidines and -uridines[1]

Synthesis of several 5-substituted (2′S)-2′-deoxy-2′-C-methylcytidines ( 8 ) and -uridines ( 6, 11 ) has been accomplished using radical deoxygenation of the 2′-tert-alcohols via their methyl oxalyl esters as a key reaction. Anti-herpes simplex virus type-1 and -2, and anti-varicella-zoster virus activities of the newly synthesized nucleosides were evaluated. Among them, the 5-iodouracil derivative 6e showed the most potent activity against herpes simplex virus type-1, with an EC₅₀ of 0.14 μg/mL without showing cytotoxicity up to 100 μg/mL, but had a weak activity against herpes simplex virus type-2 and no activity against varicella-zoster virus up to 50 μg/mL in vitro. Although the 5-fluorocytosine derivative 8b had a potent anti-herpes simplex virus type-1 activity (EC₅₀ = 0.22 μg/mL), it was rather cytotoxic to the CCRF-HSB-2 human T-cell line (IC₅₀ ≥ 1.0 μg/mL).     

Synthesis and antiviral activity of novel 5-(1-cyanamido-2-haloethyl) and 5-(1-hydroxy(or methoxy)-2-azidoethyl) analogues of uracil nucleosides

A new class of 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) and arabinouridines (7, 8) were synthesized by the regiospecific addition of halogenocyanamides (X-NHCN) to the 5-vinyl substituent of the respective 5-vinyl-2'-deoxyuridine (2) and 2'-arabinouridine (3). Reaction of 2 with sodium azide, ceric ammonium nitrate, and acetonitrile-methanol or water afforded the 5-(1-hydroxy-2-azidoethyl)-(10) and 5-(1-methoxy-2-azidoethyl)-2'-deoxyuridines (11). In vitro antiviral activities against HSV-1-TK(+) (KOS and E-377), HSV-1-TK(-), HSV-2, VZV, HCMV, and DHBV were determined. Of the newly synthesized compounds, 5-(1-cyanamido-2-iodoethyl)-2'-deoxyuridine (6) exhibited the most potent anti-HSV-1 activity, which was equipotent to acyclovir and superior to 5-ethyl-2'-deoxyuridine (EDU). In addition, it was significantly inhibitory for thymidine kinase deficient strain of HSV-1 (EC(50) = 2.3-15.3 microM). The 5-(1-cyanamido-2-haloethyl)-2'-deoxyuridines (4-6) all were approximately equipotent against HSV-2 and were approximately 1.5- and 15-fold less inhibitory for HSV-2 than EDU and acyclovir, respectively. Compounds 4-6 were all inactive against HCMV but exhibited appreciable antiviral activity against VZV. Their anti-VZV activity was similar or higher to that of EDU and approximately 5-12-fold lower than that of acyclovir. The 5-(1-cyanamido-2-haloethyl)-(7,8) analogues of arabinouridine were moderately inhibitory for VZV and HSV-1 (strain KOS), whereas compounds 10 and 11 were inactive against herpes viruses. Compounds 5 and 6 also demonstrated modest anti-hepatitis B virus activity against DHBV (EC(50) = 19.9-23.6 microM). Interestingly, the related 5-(1-azido-2-bromoethyl)-2'-deoxyuridine (1n) analogue proved to be markedly inhibitory to DHBV replication (EC(50) = 2.6-6.6 microM). All compounds investigated exhibited low host cell toxicity to several stationary and proliferating host cell lines as well as mitogen-stimulated proliferating human T lymphocytes.     

Synthesis of [1,3, NH2‐15N3] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine

To facilitate NMR studies and low‐level detection in biological samples by mass spectrometry, [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was synthesized from imidazole‐4,5‐dicarboxylic acid in 21 steps. The three ¹⁵N isotopes were introduced during the chemo‐enzymatic preparation of [1,3, NH₂‐¹⁵N₃]‐2′‐deoxyguanosine using an established procedure. The ¹⁵N‐labeled 2′‐deoxyguanosine was converted to a 5′‐phenylthio derivative, which allowed the 8‐5′ covalent bond formation via photochemical homolytic cleavage of the C–SPh bond. SeO₂ oxidation of C‐5′ followed by sodium borohydride reduction and deprotection gave the desired product in good yield. The isotopic purity of the [1,3, NH₂‐¹⁵N₃] (5′S)‐8,5′‐cyclo‐2′‐deoxyguanosine was in excess of 99.94 atom% based on liquid chromatography–mass spectrometry measurements. Copyright © 2013 John Wiley & Sons, Ltd.     

Efficacy of (E)-5-(2-bromovinyl)-2′-deoxyuridine in the treatment of experimental herpes simplex virus encephalitis in mice

Systemic treatment of mice with (E)-5-(2-bromovinyl)-2′-deoxyuridine (BVDU) showed a significant therapeutic efficacy against herpes simplex type 1 virus (HSV-1) encephalitis. With treatment initiated 12 h after viral inoculation and continued for 10 consecutive days, BVDU administered intraperitoneally in daily doses of 100–500 mg/kg increased the 21-day survival rates from 30 to 100% and reduced brain virus titers by 3–4 log₁₀ on day 6 post-infection. Furthermore, at doses of 300–500 mg/kg per day BVDU prevented the establishment of latent virus infection in the trigeminal ganglia following intracerebral HSV-1 inoculation.     

Antiviral activity of 5-ethyl pyrimidine deoxynucleosides.

The antiviral activity of the α- and β-anomers of 5-ethyl-2′-deoxyuridine (EtUdR) and 5-ethyl-2′-deoxycytidine (EtCdR) has been assessed in primary rabbit kidney (PRK) cell cultures. Cytosine arabinoside (ara-C) and 5-iodo-2′-deoxyuridine (IUdR) were included as reference materials. When inhibition of vaccinia virus multiplication was measured the following order of (decreasing) activity was established: ara-C > IUdR > (β-)EtUdR > (β-)EtCdR. The α-anomers of EtUdR and EtCdR were completely inactive.In marked contrast with ara-C and IUdR which were found to inhibit PRK cell growth and thymidine incorporation into cell DNA at relatively low doses (0.08–0.4 μg/ml and 8–40 μg/ml respectively), EtUdR did not inhibit cell growth or thymidine incorporation into DNA unless very high doses were used (200–500 μg/ml). At 0.4–200 μg/ml EtUdR had a stimulatory effect on the subsequent incorporation of thymidine and deoxycytidine into DNA, most probably due to a starvation of the cells for these precursors during their contact period with EtUdR. EtCdR did not markedly alter thymidine incorporation into DNA.Antiviral indices were calculated for EtUdR, IUdR and ara-C. These were defined as the ratios of the minimum toxic doses (inhibiting cell growth or thymidine incorporation by 50 per cent) to the minimum effective doses (inhibiting vaccinia virus induced cytopathogenicity by 50 per cent). The antiviral indices of EtUdR and IUdR were almost identical but considerably greater than that of ara-C.     

10-Carbethoxymethyl-3-phenyl-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indole and Derivatives at its 10-Position

Reaction of 3-phenyl-10H-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indole ( 4 ) with ethyl chloroacetate gave 10-carbethoxymethyl-3-phenyl-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indole ( 3 ). Condensation of 3 with hydrazine hydrate gave (3-phenyl-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indol-10-yl)acetylhydrazine ( 5 ). Reactions of 5 with a number of aromatic aldehydes, acetophenone, cyclohexanone and D-galactose gave the corresponding hydrazones 6 - 12 . Condensation of 5 with acetylacetone gave the pyrazole 15 . Cyclization of 5 with CS₂ gave (3-phenyl-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indol-10-yl)(2-thiol-1,3,4-oxadiazol-5-yl)methane ( 16 ). Reaction of 16 with ethyl chloroacetate gave the carbethoxy alkylated derivative (3-phenyl-1,2,4-triazolo[4′,3′:2,3][1,2,4]triazino[5,6-b]indol-10-yl)[2-(thiocarbethoxymethyl)1,3,4-oxadiazol-5-yl]methane ( 17 ).     

Synthesis of tritium‐labelled 3′‐azido‐3′‐deoxythymidine

New approaches to the synthesis of 3′‐azido‐3′‐deoxythymidine labelled with tritium in the heterocyclic base have been developed. With this aim, enzymatic transribosylation with [³H]thymine using the enzyme preparation from rat liver and a three‐step chemical synthesis with use of the tritium labelled precursor were studies. The enzyme preparation did not catalyse the transfer of the 3′‐azido‐3′‐deoxyribosyl fragment to the [³H]thymine residue. 5′‐O‐Benzoyl‐2,3′‐anhydrothymidine was taken as a precursor for the tritium labelling by the chemical methods. The resulting [³H]3′‐azido‐3′‐deoxythymidine was obtained with a specific radioactivity of 18.3 Ci/mmol, the tritium is located in the C‐6 position of the thymine residue. Copyright © 2003 John Wiley & Sons, Ltd.     

Rac‐ and R‐(+)‐[4,4′,5,5′‐2H4]‐2‐(1′‐[2′′,6′′‐dichlorophenoxy]‐ethyl)‐Δ2‐imidazoline (lofexidine)

The synthesis of the d₄‐forms of rac‐ and R‐lofexidine was accomplished. Two methods are described; one method is a two‐step synthesis of rac‐d₄‐lofexidine from 2‐chloropropionitrile, the second method is a three‐step preparation of R‐d₄‐lofexidine in absolute enantiomeric purity from S‐methyl lactate. The commercial availability of R‐methyl lactate makes this latter enantioselective synthesis applicable also to the synthesis of S‐d₄‐ lofexidine. These procedures also conserve the utilization of the relatively expensive [1,1′,2,2′‐²H₄]ethylene diamine precursor. The availability of S‐ and R‐d₄‐lofexidines will enable pharmacokinetic studies to be carried out to determine if differential in vivo metabolism of the two enantiomers of lofexidine occurs. Copyright © 2009 John Wiley & Sons, Ltd.     

5-alkynyl analogs of arabinouridine and 2'-deoxyuridine: cytostatic activity against herpes simplex virus and varicella-zoster thymidine kinase gene-transfected cells

A group of arabinouridines (TMSEAU, EAU, IEAU-TA) and 2'-deoxyuridines (TMSEDU, EDU, IEDU) having a variety of substituents at the uracil C-5 position (trimethylsilylethynyl, TMSE; ethynyl, E; or iodoethynyl, IE), and the sugar C-2' position (2'-arabino OH in arabinouridine, AU; or 2'-deoxyribo H in 2'-deoxyuridine, DU) were prepared to acquire antiviral structure-activity relationships. A broad-spectrum viral panel screen showed that these 5-alkynylarabino/deoxy-uridines exhibit moderate anti-HSV-1 activity, with no difference in potency between arabinouridines and 2'-deoxyuridines. The 2'-deoxyuridines TMSEDU, EDU, and IEDU, unlike the arabinouridines, exhibited potent antiviral activity against cytomegalovirus, but they were also highly cytostatic. The abilities of the 5-alkynylarabino/deoxy-uridines to inhibit nontransfected (wild-type or thymidine kinase-deficient, tk-) and viral gene transfected (HSV-1, HSV-2, or VZV thymidine kinase-positive, tk+) FM3A and OST (osteosarcoma) cells were determined. This group of 5-alkynylarabino/deoxy-uridines showed an enhanced ability to inhibit cells transfected with a viral thymidine kinase gene (HSV-1tk+, HSV-2tk+, VZVtk+) relative to wild-type or thymidine kinase-deficient (tk-) cells.