Synthesis and anti-HIV activity of 4'-azido- and 4'-methoxynucleosides.

A series of nucleosides were synthesized in which the 4'-hydrogen was substituted with either an azido or a methoxy group. The key steps in the syntheses of the 4'-azido analogues were the stereo- and regioselective addition of iodine azide to a 4'-unsaturated nucleoside precursor followed by an oxidatively assisted displacement of the 5'-iodo group. The 4'-methoxynucleosides were made via epoxidation of 4'-unsaturated nucleosides with in suit epoxide opening by methanol. Reaction-mechanism considerations, empirical conformation rules, NMR-based conformational calculations, and NOE experiments suggest that the 4'-azidonucleosides prefer a 3'-endo (N-type) conformation of the furanose moiety. When evaluated for their inhibitory effect on HIV in A3.01 cell culture, all the 4'-azido-2'-deoxy-beta-D-nucleosides exhibited potent activity. IC50's ranged from 0.80 microM for 4'-azido-2'-deoxyuridine (6c) to 0.003 microM for 4'-azido-2'-deoxyguanosine (6e). Cytotoxicity was detected at 50-1500 times the IC50's in this series. The 4'-methoxy-2'-deoxy-beta-D-nucleosides were 2-3 orders of magnitude less active and less toxic than their azido counterparts. Modifications at the 2'- or 3'-position of the 4'-substituted-2'-deoxynucleosides tended to diminish activity. Further evaluation of 4'-azidothymidine (6a) in H9, PBL, and MT-2 cells infected with HIV demonstrated a similar inhibitory profile to that of AZT. However, 4'-azidothymidine (6a) retained its activity against HIV mutants which were resistant to AZT.

Evolution of therapy for cytomegalovirus infection.

The first antiherpes agents discovered in the early 1960s were 2'-deoxy-5-iodouridine (idoxuridine; IUDR), beta-D-arabinofuranosylcytosine (cytarabine; ara-C), and 9-beta-D-arabinofuranosyladenine (vidarabine; ara-A), 2'-deoxy-5-fluorouridine (floxuridine; FUDR), and 2'-deoxy-5-trifluoromethyluridine (trifluridine; TFT). All of these drugs showed potency against herpes simplex virus (HSV) and cytomegalovirus (CMV) in vitro but had a narrow therapeutic margin. Although ribavirin, a nucleoside analogue synthesized in 1972, was much less toxic than earlier drugs, it proved ineffective against CMV. Phosphonoformic acid (PFA), a pyrophosphate analogue, has recently shown encouraging results for CMV infections in bone marrow and renal transplant recipients. Several interferons, alone or in combination with other antiviral agents, have proved clinically ineffective against CMV infections. Antiviral nucleoside analogues synthesized in the late 1970s generated much hope and enthusiasm, and acyclovir (9-[(2-hydroxyethoxy)methyl]guanine; ACV) has emerged as a safe and efficacious anti-HSV agent and has shown some promise in the treatment of CMV infection in transplant recipients. The most recent anti-CMV agent, ganciclovir (9-[(1,3-dihydroxy-2-propoxy)methyl]guanine; DHPG), is an analogue of 2'-deoxyguanosine. It was reported independently in 1982 by four laboratories. DHPG is a potent agent against all five human herpesviruses. Its toxicity, unfortunately, limits its clinical use at present to life- and sight-threatening CMV infections.

Synthesis and antiviral activity of various esters of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

The synthesis and in vivo biological activity of a series of mono-O-, di-O-, and N2-acyl derivatives of 9-[(1,3-dihydro-2-propoxy)-methyl]guanine (DHPG) are described.

Synthesis and anti-herpes-virus activity of acyclic 2'-deoxyguanosine analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

Several "sugar" modified acyclic nucleoside analogues related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 2) were synthesized and evaluated for antiviral activity. The preparation generally involved the condensation of the acetoxymethyl ether of alcohols 6c-g and 10-12a with diacetylguanine to give adducts 7c-g and 14-16, which were then deprotected to afford analogues 9c-g and 17-19. Alternatively, alcohols 12a and 13a were converted to iodides via their tosylates 12b and 13b and then reacted with the sodium salt of guanine to afford, after deprotection, analogues 22 and 23. A crossed aldol-Cannizzaro reaction on aldehyde 27 readily afforded 28, which was deprotected to give analogue 29. An in vitro assay against HSV-1 showed that all compounds tested were less active than DHPG, though several were good substrates for the viral thymidine kinase. The more promising acyclic nucleosides 9c, 19, and 29 were evaluated in a mouse encephalitis model and proved ineffective at preventing death at a dose of 20 mg/kg.

Synthesis and antiherpes virus activity of phosphate and phosphonate derivatives of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine.

A series of phosphate esters of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) were synthesized and evaluated for antiherpes virus activity. The cyclic phosphate esters were made by a new, efficient method utilizing stannic chloride as a solubilizing agent. Monophosphate 2 and bisphosphate 4 showed comparable activity to DHPG and probably acted as prodrugs of DHPG. On the other hand, the cyclic phosphate of DHPG 3 was taken up by cells and bypassed the virus-specified thymidine kinase. As a result, 3 was active against DHPG-resistant HSV mutants that lacked the viral-specified thymidine kinase and was more toxic than DHPG to uninfected cells. The phosphonate 5, the least toxic of the derivatives tested, was only marginally active against HSV but showed substantial activity against human cytomegalovirus in vitro.

Digitoxigenin 3-O-beta-D-furanosides.

The syntheses of the title compounds were accomplished by Koenig-Knorr condensation of acylated furanoses with digitoxigenin followed by basic hydrolysis of protecting groups. In this manner the riboside, 5-amino-5-deoxyriboside, 3,6-anhydroglucoside, and 3,6-dideoxy-3,6-iminoglucoside of digitoxigenin were prepared. These compounds as well as several of the synthetic intermediates showed weak to moderate cardiotonic activity.

Synthesis and antiherpes simplex virus activity of 9-[(1,3-dihydroxy-2-propylthio)methyl]guanine.

The synthesis of the thio analogue (thio-DHPG, 2) of 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) is described. The synthesis of 2 proceeded via the condensation of acetoxymethyl sulfide 9 with diacetylguanine 10 to give the protected nucleoside analogue 11. Although catalytic hydrogenolysis failed, the benzyl ether functionalities of 11 were successfully cleaved by an acetolysis reaction to furnish 14. Ammonolysis of 14 gave 2, which was also transformed to sulfoxide 15 and sulfone 16. Preliminary in vitro screening indicated that 2 exhibited comparable activity to DHPG against herpes simplex virus type 1 (HSV-1) but was less active against the type 2 virus (HSV-2) and human cytomegalovirus (HCMV). In a mouse encephalitis model (HSV-2), subcutaneous treatment with 2 led to a 53% reduction in mortality at a dose of 100 mg/kg per day.

Acyclic analogues of 2'-deoxynucleosides related to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine as potential antiviral agents.

A series of acyclic analogues of 2'-deoxynucleosides related in structure to 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1) have been synthesized and evaluated for antiviral activity against herpes simplex virus type 1 (F strain). Additionally, the ability of these analogues to function as substrates for the virus-specified thymidine kinase was examined. Phosphorylation by this kinase is essential for antiviral activity. Although the acyclic 4-oxopyrimidine nucleosides were substrates for the kinase, they were devoid of antiviral activity. In the purine series, most analogues similar in structure to DHPG did exhibit significantly lower antiviral activity, indicating that even small modifications in the purine substituents substantially reduce the antiviral potency. The most active agent, 2,6-diaminopurine 27, was only poorly phosphorylated by the viral kinase; therefore, its activity was most likely due to a prior enzymatic deamination to give DHPG. Evaluation of 27 in a mouse encephalitis model has shown it to be nearly as potent as DHPG (1).

9-[(1,3-Dihydroxy-2-propoxy)methyl]guanine: a new potent and selective antiherpes agent.

The synthesis of a new acyclic analogue of deoxyguanosine, 9-[(1,3-dihydroxy-2-propoxy)methyl]guanine (DHPG, 1), is described starting from epichlorohydrin via condensation of 2-O-(acetoxymethyl)-1,3-di-O-benzylglycerol (5) with N2,9-diacetylguanine (6). In vitro studies indicate that DHPG is a potent and broad-acting (herpes simplex virus types 1 and 2, cytomegalovirus, and Epstein-Barr virus) antiherpetic agent. In vivo studies indicate its lack of toxicity [LD50 (mice) = 1-2 g/kg, ip] and its superiority over acyclovir [oral ED50 = 7 (mg/kg)/day vs. 550 (mg/kg)/day in HSV-2 infected mice].

Anti-herpesvirus activity of the acyclic nucleoside 9-(1,3-dihydroxy-2-propoxymethyl)guanine.

The antiherpetic effects of a novel purine acyclic nucleoside, 9-(1,3-dihydroxy-2-propoxymethyl)guanine (DHPG), were compared with those of acyclovir in cell cultures and in mice. The modes of action of DHPG and acyclovir were similar in that herpes thymidine kinase phosphorylated each compound, and both agents selectively inhibited viral over host cell DNA synthesis. In 50% plaque reduction assays in Vero cells, the drugs inhibited herpes simplex virus types 1 and 2 thymidine kinase-positive strains at 0.2 to 2.4 microM. DHPG was markedly more active than acyclovir against human cytomegalovirus (50% inhibitory doses were 7 and 95 microM, respectively). Each nucleoside inhibited uninfected cell macromolecule synthesis and cell proliferation at concentrations far above those required to inhibit herpes simplex virus replication. Although the two compounds had many similarities in their behavior in vitro, the important difference was the superior performance of DHPG against herpesvirus-induced encephalitis and vaginitis in vivo. Thus, mortality in mice infected with herpesvirus type 2 was reduced 50% by daily doses of 7 to 10 mg of DHPG/kg, whereas an equally effective daily dose of acyclovir was approximately 500 mg/kg. DHPG at a daily dose of 50 mg/kg was also superior to acyclovir at 100 mg/kg per day in its inhibition of herpetic vaginal lesions in mice.

Analogs of (A2'p)nA. Correlation of structure of analogs of ppp(A2'p)2A and (A2'p)2A with stability and biological activity.

Analogs of (A2'p)2A core and ppp(A2'p)2A were chemically synthesized and their susceptibility to phosphodiesterase degradation and ability to either activate an endonuclease or to inhibit cell growth were determined. The absence of the internal 3'-OH groups ((3'dA2'p)2A) resulted in a 5-fold increase in stability, but also in a 10-fold decrease in activity, as measured by (a) activation of an endonuclease in cell-free extracts and inhibition of protein synthesis in intact cells by the 5'-triphosphate species and (b) inhibition of DNA synthesis in synchronized cells by the core analogs. An uncharged derivative of this analog containing two methylphosphotriesters, although significantly more stable, was even less active. Additional deletion of the terminal 3'-OH ((3'd A2'p)23'dA) resulted in a further 6-fold increase in stability (30-fold overall increase in stability), as well as approximately a 2-fold increase in ability to inhibit cell growth, as compared to the natural 2'5' A core. The analog lacking a terminal 2'-OH as well as lacking the internal 3'-OH group ((3'dA2'p)22'dA) showed an overall 15-fold increased stability, yet showed very little activity in inhibiting cell growth. The most stable (120-fold increased overall stability) as well as most active analog was a xyloadenosine analog of 2'5' A core, (xyloA2'p)2xyloA. These results show that modification of the 3'-terminal OH appears to be most important in increasing 2'-5' A core stability as well as biological activity. However, the mechanism of cell growth inhibition by these 2'-5' A core analogs may involve pathways different from those utilized by the 2'-5' A-dependent endonuclease.

Reactions of 2-acyloxyisobutyryl halides with nucleosides. 6. Synthesis and biological evaluation of some 3'-acyl derivatives of 2,2'-anhydro-1-(beta-D-arabinofuranosyl)cytosine hydrochloride.

The reactions of cytidine with 22 different 2-O-acyloxyisobutyril chlorides lead to the isolation of the corresponding 2,2'-anhydro-1-(3'-O-acyl-beta-d-arabinofuranosyl)cytosine hydrochlorides 9. These compounds, which all show cytotoxicity against HeLa cells in tissue culture, have been examined for antiviral and antileukemic activity. Activity against DNA viruses (vaccinia and Herpes) in tissue culture is maximal in compounds containing acyl groups with 8--12 carbon atoms. Activity against L1210 leukemia in mice varies markedly according to the length of the acyl groups, and high activities were observed in the case of long-chain (C16--C22) esters. The reaction between cytidine and O-acetylsalicyloyl chloride provides an alternate route for the synthesis of 3'-O-Ac cycloC hydrochloride.

Reactions of 2-acyloxyisobutyryl halides with nucleosides. Synthesis and biological evaluation of some 3"-acyl and 3',5'-diacyl derivatives of 1-beta-D-arabinofuranosylcytosine.

Previous papers in this series have described efficient syntheses of 3'-O-acyl and 3',5'-di-O-acyl and 3',5'-di-O-acyl derivatives of 2,2'-anhydro-1-(beta-D-arabinofuranosyl)cytosine hydrochloride (1,3). It has now been shown that the 2,2'-anhydro linkage in 1 and 3 can be selectively and efficiently cleaved by treatment with a mixture of pyridine and methanol giving the corresponding 3'-O-acyl derivatives of 1-beta-D-arabinofuranosylcytosine (2,4). The selective hydrolysis of the more soluble derivatives can also be achieved using either aqueous pyridine or a mixture of sodium carbonate and sodium bicarbonate in aqueous dioxane. Using the above procedures 3'-O-acyl araCs and 3',5'-di-O-acyl araCs with saturated or unsaturated ester groups containg from 2 to 22 carbon atoms have been prepared, and these substances have been evaluated for cytotoxicity and antiviral activity in tissue culture and for antitumor activity these substances have been evaluated for cytotoxicity and antiviral activity in tissue culture and for antitumor activity against L1210 leukemia in mice. Many of the compounds show high anti-L1210 activity relative to araC itself.

Reactions of 2-acyloxyisobutyryl halides with nucleosides. 7. Synthesis and biological evaluation of some 2,2'-anhydro-1(3',5'-di-O-acyl-beta-D-arabinofuranosyl)cytosine hydrochlorides.

The direct acylation of 2,2'-anhydro-1(beta-D-arabinofuranosyl)cytosine hydrochloride (cycloC) with a homologous series of saturated and unsaturated acyl chlorides in dimethylacetamide has been investigated. Such acylation reactions have made available a considerable number of 3',5'-diesters of cycloC that have been examined for biological activities. The compounds all show cytotoxicity against HeLa cells in tissue culture, and with the exception of the highly insoluble long-chain diesters (C16--C22), show pronounced activity against vaccinia and Herpes simplex viruses. Against L1210 leukemia in mice the compounds show varied activities, the C12--C14 saturated diesters and the C18--C22 unsaturated diesters being highly effective. Other diesters, varying by only a few methylene groups, show dramatically different results.

Synthesis of certain 4'-substituted nucleosides.

We have developed general methods for the synthesis of 4'-fluoro- and 4'-methoxynucleosides by addition of iodinemonofluoride or iodine and methanol across the double bond of suitably protected 4',5'-unsaturated pyrimidine and purine nucleosides. The structures of these adducts have been determined by a combination of chemical, spectroscopic, and electrophoretic methods. The 4'-methoxy- and the uridine analogs of nucleocidin have been synthesized from the corresponding 4'-fluorouridine and 4'-methoxyadenosine derivatives.