Immobilized artificial membranes (IAMs) are chromatographic surfaces prepared by covalently immobilizing cell membrane phospholipids. IAM surfaces mimic fluid cell membranes. Solute capacity factors (k'IAM) measured on IAM columns correlate very well with solute equilibrium partition coefficients (Km') measured in fluid liposome systems. For 23 structurally unrelated compounds, log-(k'IAM) correlates with log(Km') with a linear correlation coefficient r = 0.907. This indicates that solute partitioning between the IAM bonded phase and the aqueous mobile phase is similar to the solute partitioning between liposomes and the aqueous phase. Although both IAM chromatography and liposome partitioning can be used as in vitro methods to predict solute partitioning into cell membranes, IAM chromatography is experimentally convenient compared to liposome systems. To study the effect of lipid structure on drug binding to IAMs, IAMs were prepared from three different phosphatidylcholine ligands: (i) a diacylated phosphatidylcholine ligand, (ii) a single chain ether phosphatidylcholine ligand, and (iii) a single chain phosphatidylcholine ligand that lacks a glycerol backbone. Solute retention data were identical for all of these IAMs, and consequently, predictions of solute binding to fluid membranes were also identical. This indicates that the structure of the phosphatidylcholine ligand that is immobilized is not critical for the binding of solutes. Since the structure is not important, the binding of solutes to membranes is a bulk phase property, i.e., it is the interface created by the ligands that determines the solute binding properties, not the ligands themselves. Solute partitioning using octanol/water systems does not correlate with k'IAM unless a homologous series of hydrophobic solutes is being evaluated.

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The chemical synthesis of certain 4-substituted pyrazolo[3,4-d]pyrimidine nucleosides is described. Using 1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidin-4-one (1) as the starting material, the reactive intermediate 4-chloro-1-(2,3,5-tri-O-acetyl-beta-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (2) was prepared in excellent yield. Compound 2 served as a versatile precursor for the synthesis of a number of 4-substituted pyrazolo[3,4-d]pyrimidine nucleosides. In antitumor studies of these nucleosides, in vitro and in vivo, it was found that any alteration of the 4-amino substituent of 4-amino-1-beta-D-ribofuranosylpyrazolo[3,4-d]pyrimidine (3) was accompanied by a significant decrease or loss of antitumor activity. On the other hand, introduction of certain substituents at the 3 position of 3 (synthesis reported previously) led to a dramatic increase in antitumor activity in comparison to the parent compound.

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The synthesis of certain tricyclic nucleosides with a dihydroimidazole, imidazole, triazole, or tetrazole ring fused to the pyrazolo[3,4-d]pyrimidine ring system in an angular position (C-4 and N-5) has been accomplished. The 4aziridinyl derivative (2) was prepared by a nucleophilic displacement of the chlorine atom of 4-chloro-l -(2,3,5-tri-O-acetyl-~-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine (1) with ethylenimine. The nucleoside (2) was then treated with sodium iodide to furnish 7-(~-D-ribofuranosyl)-2,3-dihydroimidazo[l,2-c]pyrazolo[4,3-e] pyrimidine (3). The reaction of (1) with lithium azide gave 7-(2,3,5-tri-0-acetyl-~-D-ribofuranosyl)pyrazolo[4,3-e]tetrazolo[l,5-c] pyrimidine (5) and cyclization of 4-amino-1 -(P-D-ribofuranosyl)pyrazolo[3,4-d]pyrimidine with chloroacetaldehyde provided the tricyclic nucleoside 7-(~-~-ribofuranosyl)imidazo[l,2-c]pyrazolo[4,3-e]pyrimidine (7). 2.4-Din i trophenoxami n e was treated with 4 -amino-1 -( p -Dri bof uranosyl) pyrazolo [3,4-d] pyrimidine (6) to give an intermediate (8) which on cyclization with diethoxymethyl acetate gave 7-(2,3-0-methoxymethylenep-D-ribofuranosyl) pyrazolo [4,3-e] -1,2,4-triazolo [1,5-c] pyrimidine (9). Acid-catalysed deblocking of (9) provided the desired tricyclic nucleoside (1 0). The reaction of trimethyl orthoformate with 4-hydrazino-1 -(P-Dribofuranosyl)pyrazolo[3,4-d]pyrimidine under different experimental conditions resulted in the formation of a mixture of diastereoisomers due to the 2',3'~O-methoxymethylene group. Treatment of (1 0) with alkali gave a ring-opened intermediate which on treatment with sodium nitrite and acetic acid cyclized to give an aza-derivative (16) of (10). R = COMe