An approach to the total synthesis of sinefungin.

Condensation of N6-benzoyl-2',3'-O-isopropylideneadenosine-5'-aldehyde with nitromethane followed by acid catalyzed acetylation and borohydride reduction leads to N6-benzoyl-9-(5,6-dideoxy-2,3-O-isopropylidene-6-nitro-beta-D-ribo-hexofuranosyl)adenine (4). A second nitroaldol condensation between 4 and N-benzyloxycarbonly-L-aspartic acid-beta-semialdehyde alpha-benzyl ester (5) followed by acetylation and borohydride reduction leads to a fully protected 6'-nitro modification of sinefungin and its C6'-epimer (7). Hydrolysis of the acetonide followed by sequential reduction of the benzyl derived protecting groups and the nitro group and debenzoylation leads to a modest yield of a 3:1 mixture of sinefungin (1) and 6'-episinefungin which can only be separated by analytical ion exchange chromatography.

Regression of tumors in guinea pigs after treatment with synthetic muramyl dipeptides and trehalose dimycolate.

A high incidence of tumor regression was observed in guinea pigs bearing transplantable, line-10 hepatocellular carcinomas when synthetic muramyl dipeptides combined with trehalose dimycolate in oil-in-water emulsions were injected directly into the tumors. These compounds are promising candidates to replace viable bacillus Calmette-Guérin in cancer immunotherapy in humans and animals.

A radioimmunoassay for 1-beta-D-arabinofuranosyluracil with reference to cross-reactivity of 1-beta-D-arabinofuranosylcytosine with an antibody.

Antibodies directed against 1-beta-D-arabinofuranosyluracil have been produced in rabbits by immunization with a conjugate of 1-(5-O-succinyl-beta-D-arabinofuranosyl)uracil with human serum albumin. Two of four antibodies so obtained showed high specificity for 1-beta-D-arabinofuranosyluracil and allowed the development of a sensitive and reliable radioimmunoassay for this substrate. On the other hand, one antibody had a high affinity for 1-beta-D-arabinofuranosylcytosine. The binding of 1-beta-D-arabinofuranosylcytosine to this antibody was practically constant between pH 5.2 and 9.0, whereas 1-beta-D-arabinofuranosyluracil binding was affected drastically by pH. The pH-binding profile for 1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosyluracil was reminiscent of the specificity of ara-C-specific antibodies, which we previously obtained after immunization of rabbits with 1-(5-O-succinyl-beta-D-arabinofuranosyl)cytosine as a hapten.

A radioimmunoassay method for 1-beta-D-arabinofuranosyluracil using antibodies directed against 1-beta-D-arabinofuranosylcytosine.

Above pH 7.0 1-beta-D-arabinofuranosyluracil (ara-U) shows marked pH-dependent cross-reactivity with antibodies directed towards 1-beta-D-arabinofuranosylcytosine. Since this peculiar phenomenon has not been observed with other nucleosides and nucleotides thus far tested, it is probably the result of base-catalyzed tautomerism of ara-U to its enolic form which renders it more structurally similar to 1-beta-D-arabinofuranosylcytosine. By performing the radioimmunoassay at both pH 6.2 and 8.6 we could determine 1-beta-D-arabinofuranosylcytosine and ara-U simultaneously. This method for ara-U assay is simple, fairly reliable, and applicable to blood level studies.

Comparative study of the antiviral activity of acyl derivatives of 2,2'-anhydro-1-beta-D-arabinofuranosylcytosine and other nucleosides against encephalitis in mice.

Anti-deoxyribonucleic acid virus activities of 3'-O-acyl derivatives of 2,2'-anhydro-1-beta-d-arabinofuranosyl cytosine (cyclo-C) and 1-beta-d-arabinofuranosylcytosine (Ara-C) were evaluated by using an in vivo test system in mice. Among the derivatives tested, 3'-O-decanoyl cyclo-C hydrochloride was the most effective against herpes simplex virus-induced encephalitis in mice when the drug was administered directly into infected brains of mice (target-organ treatment). A comparative study of the treatment of herpetic encephalitis in mice with 3'O-decanoyl cyclo-C and other nucleosides, including Ara-C, 9-beta-d-arabinofuranosyladenine (Ara-A), and 5-iodo-2'-deoxyrudine (IUdR), proved Ara-A to be more efficacious than the other nucleosides, followed by 3'-O-decanoyl cyclo-C, which was more active than Ara-C and IUdR. Administration of 3'-O-acyl cyclo-C's by intraperitoneal injection, however, failed to demonstrate activity against herpetic encephalitis in mice. The antivaccinial activity of 3'-O-decanoyl cyclo-C was also compared with that of other compounds against encephalitis and dermal tail lesions in mice caused by vaccinia virus infection. Interaperitoneally administered 3'-O-decanoyl cyclo-C and Ara-C also showed no significant activity against the diseases. Under these test conditions, N-methylisatin-beta-thiosemicarbazone (Marboran) was the most active compound, followed by Ara-A.

A radioimmunoassay for 1-beta-D-arabinofuranosylcytosine.

A rapid and reliable radioimmunoassay method for 1-beta-D-arabinofuranosylcytosine (ara-C) has been developed using antibody induced in rabbits, [3H]ara-C, and a Millipore filtration technique. The sensitivity of this assay was such that ara-C, 0.02 mug/ml, in plasma could be detected, and the assay was practically free from interference by deoxycytidine, cytidine, 1-beta-D-arabinofuranosyluracil, and other nucleosides, as well as from various antibiotics. Blood levels of ara-C in C57BL X DBA/2F1 mice were determined after injection of 1-(3-O-octanoyl-beta-D-arabinofuranosyl) cytosine. Relatively high ara-C levels could be maintained for a fairly long period. Plasmas of mouse, rat, and rabbit contained high esterase activity which hydrolyzed the 3'-octanoyl group in 1-(3-O-octanoyl-beta-D-arabinofuranosyl)cytosine, whereas this activity was relatively low in dog and human plasmas.

Antiviral activities of acyl derivatives of 2,2'-anhydro-1-beta-D-arabinofuranosylcytosine and 1-beta-D-arabinofuranosylcytosine in cell culture.

Antiviral activities of acyl derivatives (3'-O-octanoyl and 3'-O-decanoyl) of 2,2'-anhydro-1-beta-d-arabinofuranosylcytosine (cyclo-C) and 1-beta-d-arabinofuranosylcytosine (Ara-C) were compared with other antiviral nucleosides, and some biological characteristics of the antiviral activity were investigated. Among those synthesized acyl derivatives, 3'-O-decanoyl ara-C was the most active against deoxyribonucleic acid viruses, with an activity comparable to that of Ara-C. Acyl derivatives of cyclo-C were somewhat less active than their Ara-C counterparts. In the value of therapeutic index, 1-beta-d-arabinofuranosyladenine was superior to the others, followed by 5-iodo-2'-deoxyuridine. In comparing the sensitivity of two serotypes of herpes simplex virus it was found that Ara-C and its ester, as well as its cyclo-C counterpart, were more active against the type 2 than the type 1 strain. The activity of 3'-O-decanoyl Ara-C, like that of its parent, was diminished by treatment with cytidine deaminase from mouse kidney, but 3'-O-decanoyl cyclo-C was resistant to this treatment. In comparative studies of 3'- and 5'-O-acyl Ara-C's, antivaccinia virus activity of 3'-O-palmitoyl Ara-C was significantly superior to its 5'-counterpart. The inhibitory activity of 5'-O-decanoyl Ara-C was markedly reduced by the presence of a threefold molar excess of eserine sulfate, a choline esterase inhibitor, whereas the 3'-acyl Ara-C was not affected by the inhibitor in any combination. This result indicates that enzymatic hydrolysis of the 3'-ester to Ara-C, which is inhibited by eserine sulfate, did not occur in this cell culture.

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.