Acyclic derivatives of adenine, known as highly effective nucleotide analogs with broad spectrum antiviral activity, were evaluated for potential cross-reactivity with adenylyl cyclases, a family of membrane-bound enzymes that share putative topologies at their catalytic sites with oligonucleotide polymerases and reverse transcriptases. A series of derivatives of 9-(2-phosphonylmethoxyethyl)adenine (PMEA) inhibited a preparation of adenylyl cyclase derived from rat brain with IC 50 values that ranged from 66 M (PMEA) to 175 nM for its diphosphate derivative (PMEApp) and mimics of it. PMEApp mimics included PMEAp(NH)p, PMEAp(CH 2 )p, PMEAp(CX 2 )p (X ‫؍‬ fluorine, chlorine, or bromine), PMEAp(CHX)pp, and PMEAp(C(OH)CH 3 pp. The data suggest that inhibition of adenylyl cyclases may contribute to the therapeutic action of some of these or similar compounds or constitute part of their side effects in therapeutic settings.Adenylyl cyclases are a family of membrane-bound enzymes central to one of the most important signal transduction systems and influence regulation of cell function in virtually all cells. The putative membrane topology of adenylyl cyclases conforms to a repeated sequence of a six-membrane spanning region followed by a cytosolic domain (1). The two cytosolic domains (C 1 and C 2 ) are homologous and contain regions that are highly conserved among adenylyl cyclase isozymes (1). The cleft formed by interaction of C 1 and C 2 contains the enzyme catalytic site (2, 3), the topology of which resembles aspects of the palm domain of DNA polymerases and human immunodeficiency virus (HIV) 1 reverse transcriptase (4 -7). Moreover, aspects of the catalytic mechanisms of adenylyl cyclases and enzymes involved in polymerization of oligonucleotides are similar as well. Each involves nucleoside triphosphate as substrate and divalent cation-dependent catalysis that includes attack involving the substrate 3Ј-OH group, to catalyze either chain elongation of a primer oligonucleotide or formation of the 3Ј:5Ј-cyclic phosphate, with pyrophosphate as a leaving group. Adenine nucleoside 3Ј-polyphosphates are among the most potent inhibitors of adenylyl cyclases (8 -11) and of these 2Ј-d-

9-Substituted adenine derivatives with protected phosphoryl groups were synthesized and tested as inhibitors of adenylyl cyclase in isolated enzyme and intact cell systems. Protected 3-phosphoryl derivatives of 2,5-dideoxyadenosine (2,5-dd-Ado) and ␤-L-2,5-ddAdo, protected 5-phosphoryl derivatives of ␤-L-2,3-ddAdo, and protected phosphoryl derivatives of two 9-(2-phosphonomethoxy-acyl)-adenines were synthesized. Protection was afforded by two cyclosaligenyl-or three S-acyl-2-thioethyl-substituents. These pro-nucleotides were tested for their capacity to block forskolin-induced increases in [ 3 H]cAMP in OB1771 and F442A preadipocytes and human macrophages prelabeled with [ 3 H]adenine. A striking selectivity for 2,5-dd-Ado-3-phosphoryl derivatives was observed. Cyclosaligenyl-derivatives (IC 50 ϳ2 M) were much less potent than S-acyl-2-thioethyl-derivatives. Best studied of these was 2,5-dd- Ado-3-O-bis(S-pivaloyl-2-thioethyl)-phosphate, which blocked [3 H]cAMP formation in preadipocytes (IC 50 ϳ30 nM) and suppressed opening of cAMP-dependent Cl ؊ channels in cardiac myocytes (IC 50 ϳ800 nM). None of the pro-nucleotides inhibited adenylyl cyclase per se, whether isolated from rat brain or OB1771 cells. These compounds exhibit the hallmarks of prodrugs. Data suggest they are taken up, are deprotected, and are converted to a potent inhibitory form to inhibit adenylyl cyclase, but only by intact cells. The availability and characteristics of these prodrugs should make them useful for blocking cAMP-mediated pathways in intact cell systems, in biochemical, pharmacological, and potentially therapeutic contexts.

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The enantioselective synthesis of a-hydroxyarylmethylphosphonates 2a-p by the oxazaborolidine-catalyzed reduction of a-keto phosphonates la-p using different boranes 4a-c was studied in detail. Moderate to good enantioselectivities are found (up to 80% ee) by using the borane-dimethyl sulfide complex 4a as reducing reagent and the 2-n-butyl deri-vative of the (S)-5,5-diphenyl-3,4-trimethylene-l,3,2-oxazaborolidine catalyst 3b. In contrast, excellent enantiomeric excesses (up a-Hydroxy phosphonates are biologically active and have been shown to inhibit the enzymes renin"], EPSP synthetaseL2], and HIV proteaser3]. The absolute configuration in the a-position of substituted phosphonic acids has been shown to be important for biological acti~ity[~.~]. In addition, chiral, nonracemic a-hydroxy phosphonates are useful precursors of the extensively studied a-amino phosphonic acid esters (P-amino a c i d~) [~,~] .The latter compounds are used as analogues of a-amino acids. In contrast to P-amino acids, there is only a limited number of synthetic approaches to optically active a-hydroxy phosphonates with one stereogenic center. Chirality was introduced by i) the addition of chiral aldehydes to phosphorus nucleophilesL' 1, ii) the enantioselective addition of chiral phosphites to aldehydes ['], iii) the use of the Pudovik reaction [9] in the presence of chiral base catalysts[l01, iv) two different approaches of stereoselective reductions of a-["] and p-keto phosphonates[I2], or v) by enzymatic resolution of racemic a-hydroxy pho~phonates['~~'~1. As part of an ongoing program investigating the use of chiral nucleoside a-hydroxy phosphonate diesters as pr~drugs['~I or prooligonucleowe developed a method for the stereoselective synthesis of a-hydroxyarylmethyl-and a-hydroxyalkylphosphonate diesters involving the enantioselective reduction of prochiral a-keto phosphonates by oxazaborolidine catalysis and borane reducing agents ['7J']. In this paper we report on the enantioselective reduction of a-ketoarylphosphonates as well as the different parameters influencing the obtained enantiomeric excess and on an application of the optimized reaction condition to the enantioselective synthesis of a-hydroxyalkylphosphonates. Additionally we describe the enantioselective synthesis of p-and y-hydroxy phosphonates by using the same methodology. Results and DiscussionAs starting materials different aromatic a-keto phosphonates la-p were used. In order to elucidate the effect of different substitution pattern of the aryl ring various acceptor and donor groups were introduced into 1. Additionally, the effect of the ester residues in the phosphoryl moiety of 1 was studied by changing these from methyl to ethyl, isopropyl, and tert-butyl groups. These compounds were subsequently stereoselectively reduced to the corresponding chiral, nonracemic dialkyl a-hydroxyarylmethylphosphonates 2a-p (Scheme 1).The dimethyl (la), diethyl (lp), and the diisopropyl (ly) a-keto phosphonates were obtained by an Arbuzov reaction of trialkyl phosphites wit...