Pro-nucleotide inhibitors of adenylyl cyclases in intact cells.

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 beta-l-2',5'-dd-Ado, protected 5'-phosphoryl derivatives of beta-l-2',3'-dd-Ado, 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) approximately 2 microm) 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) approximately 30 nm) and suppressed opening of cAMP-dependent Cl(-) channels in cardiac myocytes (IC(50) approximately 800 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.


9-(2,5-Dideoxy-$ $ $
$-L-erythro-pentofuranosyl)adenine ($ $ $ $-L-2',5'-dd-Ado) -By use of the procedure previously described in the D-series by Désaubry et al. [40], $-L-2'-deoxyadenosine (0.7g, 2.8 mmol) was dissolved in pyridine (10 ml) and treated with diphenyldisulfide (0.76 g, 3.5 mmol) and tributylphosphine (0.87 ml, 3.5 mmol) (Scheme 2). After 24 h, 10 ml of methanol was added, the solution was concentrated to dryness and co-evaporated with toluene, then was co-evaporated with methanol. The resulting material was subjected to column chromatography on silica gel with a stepwise gradient of 0 to 12% methanol in   After further stirring at   room temperature for 30 min, the mixture was diluted with dichloromethane and was washed successively with 10% aqueous sodium sulfite solution and then with water. The organic layer was dried over sodium sulfate, filtered, and evaporated to dryness under reduced pressure. Purification of the residue by column chromatography on silica gel with a stepwise gradient of 0 to 4% methanol in dichloromethane afforded Figure 1; 65 mg, 56%) as a colorless syrup. Figure 1) -    Figure 1) and $-L-2',3'- (Ph-SATE); compound  12 1,2,4-triazole (193 mg, 0.65 mmol, 5.0 eq) was added and the reaction mixture was stirred for 3 days at room temperature. The reaction was stopped by the addition of 20 ml of saturated aqueous sodium bicarbonate and the resulting mixture was extracted with 30 ml of methylene chloride. The organic phase was dried by filtration through MgSO 4 and the filtrate was evaporated to dryness. The residue was purified by flash chromatography (20 g silica gel) with a step gradient of 0 to 4% methanol in methylene chloride. The appropriate fractions were combined and evaporated to dryness. The residue was dissolved in 5 ml of acetic acid:water:methanol = 8:1:1 (v:v:v). The reaction mixture was stirred for 24 h at ambient temperature. The solvent was evaporated in vacuo and the residue was co-evaporated twice with 5 ml of toluene, twice with 5 ml of methanol, and then with 5 ml of methylene chloride. The residue was purified by flash chromatography (15 g silica gel) with a step gradient of 0 to 20% methanol in methylene chloride yielding (R)-bis(S-pivaloyl-2-thioethyl)-9-(2-phosphono-methoxypropyl)adenine (t-Bu-SATE-PMPA; compound 11 in  mm, 5 :m) with linear gradients of 0 to 100% acetonitrile in water over a 40 min period at a flow rate of 1 ml/min. Solvents, pyridine, tetrahydrofuran, and dimethylformamide, were of analytical quality absolute from Fluka. 1H-tetrazole and 1-(mesitylenesulfonyl)-3-nitro-1H-1,2,4-triazole were from Aldrich.
Human macrophages were prepared from leukocyte packs obtained from the Long Island Blood Service as described by Cheng et al. [46]. Briefly, cells were diluted with phosphate-buffered saline and then were

[ 3 H]cAMP formation in cells prelabeled with [ 3 H]adenine -This procedure followed suggestions of
Salomon [47]. Macrophages or OB1771 cells were grown in 35 mm culture dishes, growth medium was  [48]. A portion of the eluate from the alumina column was removed and used to quantify the recovery of cAMP and the remainder was used to quantify the formation of [ 3 H]cAMP by scintillation spectrometry.
Adenylyl Cyclase Preparation and Assay -Adenylyl cyclase from rat brain was prepared as a detergentdispersed extract as previously described [24,48]. Membranes from OB1771 or F442A cells were prepared as described above under "Cell homogenates and membrane preparations". Activity of adenylyl cyclase from rat brain was determined with a reaction mixture containing 50 mM triethanolamineCHCl, pH 7.5, Inhibition kinetics were evaluated as previously described [49] with variable concentrations of MnC5'-ATP and free cation held fixed in excess of the 5'-ATP concentration.

Cardiac ventricular myocytes and Clcurrent measurements.
Adult male guinea pigs, weighing 300-500 g, were sacrificed by peritoneal injection of sodium pentobarbitone solution (1 ml of 390 mg/ml). Hearts were removed and cardiac myocytes were enzymatically isolated as described by Gao   . These two agents also inhibit adenylyl cyclase in vitro [7]. Compounds 16 and 17 are enantiomeric derivatives of 2'-phosphoryl-9-(cyclopentyl)adenine, for which the corresponding 2'-triphosphate forms are also competitive inhibitors of adenylyl cyclase. Phosphate groups were protected by either cyclo-saligenyl-derivatives (compounds 1 and 2) [29,[34][35][36] or S-acyl-2-thioethyl-(SATE-) derivatives [33,39] (compounds 3 -17). The comparison of these several ligands allows a distinction to be made in the efficacy of pre-and post-transition-state inhibitors of adenylyl cyclases for use in intact cell systems and a distinction to be made between pro-nucleotides that are spontaneously deprotected (cyclo-saligenyl-derivatives) and those that require deprotection by cellular enzymes (SATE-derivatives).

Inhibition of adenylyl cyclase and of [ 3 H]cAMP formation in preadipocytes -
The adenylyl cyclase in membranes isolated from OB1771 preadipocytes was inhibited by adenine nucleosides with a rank-order of potency similar to that previously reported for inhibition of the enzyme isolated from rat brain ( Table 1).
Inhibitory potency increased significantly as the number of 3'-phosphates was increased, but potency was somewhat less in the preadipocytes. These data suggest a probable range within which pro-nucleotide potency should fall. Potency of inhibition of cAMP formation in intact cells will depend upon the efficacy of pro-nucleotide uptake by cells and the extent of its subsequent deprotection and phosphorylation within cells.
To evaluate effects of agents to inhibit cAMP formation in intact cells, responses were tested on cells  Table 1). The data imply that intact cells are required for inhibition of cAMP formation by the protected nucleotide and suggest that inhibition occurs as a consequence of the nucleotide's being taken up and metabolized to a ligand more potent than the precursor nucleoside 3'-monophosphate.  Table 1). The H-Sal-derivative (1; IC 50 ~1.2 µM) was slightly more potent than the Me-Salderivative (2; IC 50 ~2.0 µM), consistent with its more rapid hydrolysis [29,34]. In fact, the general lack of efficacy of these ligands likely lies with their relative stability; they undergo spontaneous deprotection in aqueous solutions with relatively long half-lives. For related compounds the half-lives were: 2',3'-dd-5'-  [29,35].

Pro-nucleotide comparison -
Consequently, the saligenyl-derivatives of 2',5'-dd-3'-AMP (compounds 1 and 2) were substantially less effective inhibitors of [ 3 H]cAMP formation in intact cells than was 2',5'-dd-3'-AMP-bis(t-Bu-SATE) (4;  (Table 1).  Table 2). The acyl group clearly influenced potency. And second, these compounds exhibited slightly lower potencies from those seen with the OB1771 cells (cf. Table 2). Differences in efficacy of the pro-nucleotides were more pronounced when human macrophages were tested (Table 2). Although potency of the bis(t-Bu-SATE)-protected 2',5'-dd-3'-AMP (IC 50 76 nM) was between those observed with OB1771 (IC 50 ~51 nM) and F442A (IC 50 ~146 nM) cells and the bis(Ph-SATE)-pro-nucleotide also exhibited a potency in this range (IC 50 ~95 nM), the bis(Me-SATE)-pronucleotide, rather than being the more potent was actually somewhat less potent (IC 50 ~247 nM). In each set of experiments cells were exposed to pro-nucleotide for 15 min before the challenge with forskolin, which again was for 15 min. These observations suggest that uptake and processing of pro-nucleotides differ between cell types and that this may be dependent on the protecting group. By accepted models, processing of these pro-nucleotides requires uptake, deprotection, and subsequent poly-phosphorylation. Therefore, variations in pro-nucleotide potency could be due to differences at any stage. For example, some differences in potency among the bis(S-acyl-2-thioethyl)-phosphate derivatives would be expected to be due to differences in their processing rates. The half-lives for similar phosphotriester derivatives of 3'-azido-5'-TMP 22

Comparisons of other pro-nucleotides -
A similar conundrum occurred with S-acyl-2-thioethyl-derivatives of β-L-2',3'-dd-5'-AMP. None of these pro-nucleotides inhibited the forskolin-stimulated formation of [ 3 H]cAMP in isolated cells, yet β-L-2',3'-dd-5'-ATP is one of the most potent known inhibitors of adenylyl cyclases (IC 50 ~24 nM; Table 1). That is, the expectation was that pro-nucleotide forms of either 2',5'-dd-3'-AMP or β-L-2',3'-dd-5'-AMP would have behaved similarly because the triphosphate forms of these nucleotides were comparably potent inhibitors of adenylyl cyclases. However, only protected derivatives of 2',5'-dd-3'-AMP yielded effective inhibitors of or that this was with minced tissue, into which agents might diffuse poorly, or because of which the preparation contained a lot of damaged tissue. To circumvent these problems and to establish that the efficacy of these pro-nucleotides was not, in fact, a consequence of the tritium-labeling assay method, a different approach was used with guinea-pig cardiac ventricular myocytes. These cells express a cAMPdependent Cl --channel; Cl --current is increased with increasing cellular levels of cAMP [52,53]. When isolated cells were treated for 15 min with varying concentrations of 2',5'-dd-3'-AMP-bis(t-Bu-SATE), isoproterenol-activated Cl --current was suppressed with an IC 50 ~800 nM (Figure 7). This concentration was in the range of that noted above for inhibition of forskolin-stimulated formation of [ 3 H]cAMP in atrial tissue, suggesting that the less potent inhibition by this pro-nucleotide was a characteristic of cardiac tissue and was not a consequence of the assay method. When compared with other cell types, sensitivity to inhibition by 2',5'-dd-3'-AMP-bis(t-Bu-SATE) followed the order macrophages / OB1771 cells > 3T3 F442A > cardiomyocytes (Figure 7). Because the bis(SATE)-pro-nucleotides inhibited either cAMP formation or a cAMP-mediated process in four different cell types as measured with two different end-points, the differences in sensitivity likely lie with tissue-dependent characteristics of pro-nucleotide processing, with differences in sensitivities of the adenylyl cyclase isozymes present in these several cell types, or with 24 differences imposed by the techniques and conditions we used.

Pro-nucleotide metabolism and the effects of metabolites -Deprotection of bis(SATE)-protected
nucleotides is thought to occur through a carboxyesterase catalyzed elimination of one SATE-group, followed by spontaneous intramolecular decomposition and elimination of episulfide, yielding the nucleotide-SATE-diester (Scheme 3) [33]. With whole homogenates of OB1771 preadipocytes, a time-and tissue-dependent appearance of a major product (retention time ~110 min) was noted ( Figure 8, lower panel). It was also evident upon exposure of 2',5'-dd-3'-AMP-bis(t-Bu-SATE) to minced rat atrial tissue (not shown). As expected, formation of thẽ 25 of interest to determine if this also occurred with intact cells. Incubation of preadipocytes or macrophages with the pro-nucleotide resulted in its substantial breakdown (Figure 9). This was more apparent with macrophages ( Figure 9, middle panel) than with the preadipocytes (Figure 9,
The data are fully consistent with the concept that the protected ligand must be taken up, be deprotected, and be polyphosphorylated to be active (cf. Scheme 3). The overall elimination of the bis(SATE)-protective groups would be expected to follow the order Me-SATE-> t-Bu-SATE-> Ph-SATE-[cf. 33], and this was reflected in the potencies noted here ( Table 2). The differences in rates and mechanism of deprotection appeared not to hinder the efficacy of 2',5'-dd-3'-AMP protected with bis(Me-SATE)-and possibly bis(t-Bu-SATE)-groups, but could account for the reduced apparent potency of the bis(Ph-SATE)-protected ligand, resulting in the rank-order of potency of the 2',5',-dd-3'-AMP-bis(SATE) derivatives tested ( Figure 6 and Table 2). That is, longer exposure of cells to bis(t-Bu-SATE)-and bis(Ph-SATE)-protected ligand might have resulted in potencies similar to that noted with the bis(Me-SATE)-nucleotide.
Attempts were made to verify the sequence of uptake and deprotection events, but the HPLC-UV isolation and detection techniques available to us were not sufficiently sensitive for the purpose. One 28 predominant metabolic product was noted. On this chromatography system 3 it exhibited a slightly shorter retention time (~110 min peak) than the initial pro-nucleotide (~118 min peak) and it conformed spectrally to the mono-(t-Bu-SATE)-diester of 2',5'-dd-3'-AMP. The possibility was further examined that this compound was, in fact, the active agent, but it neither inhibited adenylyl cyclase per se (Figure 8) nor blocked the formation of [ 3 H]cAMP in prelabeled cells (Figure 9). This suggests that the active agent for inhibiting To be clear, it remains a slight possibility that this pro-nucleotide need not be taken up by cells to exert its effects and that it might act indirectly, for example, through a cell-surface receptor-mediated mechanism.
There is no evidence to support this alternative possibility. Although these compounds are adenine nucleotides, they do not fit the structure profile for purinergic receptor agonists. Taken together, the data fit best the model of prodrugs that are deprotected within cells to agents that are then further metabolized to active inhibitors of adenylyl cyclases. Once deprotected, 2',5'-dd-3'-AMP presumably undergoes sequential phosphorylation to the intracellularly active and most potent form, 2',5'-dd-3'-ATP (Scheme 3). Nucleoside kinases are numerous and are relatively promiscuous and the intracellular phosphorylation of 2',5'-dd-3'-AMP may be expected. Although phosphorylation of 2',5'-dd-3'-AMP to the corresponding diphosphate has not been described, the subsequent phosphorylation to 2',5'-dd-3'-ATP can be catalyzed by several common enzymes, for example, creatine kinase or 3-phosphoglycerate kinase, but not pyruvate kinase [59].
The efficacy of pro-nucleotides to block cAMP-mediated events in intact cells will depend not only on their structures, but also on the mechanisms of uptake, deprotection, phosphorylation, and subsequent breakdown.
Uptake and deprotection can be influenced if not controlled by the nature of the protective group. Simple changes in the structures of the pro-nucleotide protecting groups can have significant effects on the rates of deprotection. For example, the addition of a hydroxyl group to the t-Bu-SATE-group, increases the half-life of a protected 3'-azido-5'-TMP in CEM-SS cell extracts from 1.2 to 7.5 hr [54]. Behavior of pro-nucleotides can be modified significantly by the use of mixed phosphotriester derivatives, e.g. with one SATE-group and a second group susceptible to hydrolysis by an enzyme other than carboxyesterases. Examples include thioesterases for dithioethyl-groups, or phosphoramidases for amidate moieties (glycine, [54]; tyrosine, [56]) or alkylamines (isopropyl-amine [54]). These substituents also influence solubility, effecting a balance between aqueous solubility and lipophilicity. But perhaps more important, because the uptake mechanisms and the deprotecting enzymes expressed in different cell types will differ, it may be possible that Although the considerations regarding variations in processing efficacy or mechanism could explain differences in responsiveness of the several cell types to the pro-nucleotides, the differences might also 30 reflect in part the different structures and hence susceptibilities of the adenylyl cyclase isozymes being expressed. For example, the SATE-protected forms of 2',5'-dd-3'-AMP reduced [ 3 H]cAMP formation in OB1771 cells in the low nanomolar range but a comparable response in cardiac ventricular myocytes required high nanomolar concentrations (Table 2). If these pro-nucleotide were effectively converted to the suggested 2',5'-dd-3'-ATP, then the number, amounts, and sensitivity of the different isozymes expressed in the respective tissues could influence the response seen. From past experience, however, the differences in selectivity for adenine nucleoside 3'-polyphosphates among the isozymes we have tested (I, II, VI, VII, and VIII) was not great, with IC 50 s varying from 90 nM (type VII) to 280 nM (type II) (5). The other isozymes have not yet been tested nor have we explored which isozymes are expressed in the cells studied here.
And lastly, the mechanisms of inhibition of adenylyl cyclase differ considerably for the agents tested here. β-L-2',3'-dd-5'-ATP is a competitive inhibitor of adenylyl cyclase [6], inhibition by PMEApp or PMPApp is mixed 5 [7], whereas 2',5'-dd-3'-ATP elicits non-competitive or un-competitive inhibition, depending on reaction conditions [2]. This and the observation that 2'-d-3'-AMP is competitive with cAMP in inhibiting the reverse reaction of a chimeric construct of adenylyl cyclase [60] suggest that 2',5'-dd-3'-ATP is a post-transition-state inhibitor, binding to adenylyl cyclase only after products cAMP and metalpyrophosphate have left the catalytic cleft. Consequently, this class of pro-nucleotide would not be susceptible to the competitive pressures characteristic of the mechanism for β-L-2',3'-dd-5'-ATP and that pronucleotides leading to the formation of 2',5'-dd-3'-ATP should be very specific inhibitors of adenylyl cyclases in intact cells.
The expectation is that the number and characteristics of prodrug inhibitors of adenylyl cyclases can be expanded and that tissue and possibly isozyme selective ligands will result. Much can be learned about cell and tissue functions through the inhibition of adenylyl cyclases by ligands such as these.  [61], can the inhibition of adenylyl cyclase during G2 provide information regarding changes in gene expression necessary for mitosis per se? If the inhibition of adenylyl cyclase causes an increased rate of differentiation of preadipocytes [18], are the changes in gene expression similar to those induced by insulin and triiodothyronine or by bromo-palmitate? That is, is the cassette of genes whose expression is altered by reduced cAMP similar to or different from those associated with differentiation per se? By use of selective adenylyl cyclase inhibitors, one can gain insights into the roles of the adenylyl cyclase-cAMP signaling cascade in cell and tissue function that are not otherwise possible. Moreover, this concept may well be expanded easily to guanylyl cyclases and to other enzymes in signaling cascades with the attendant additional insights that can be gained from blockade of the respective pathways.  b) Values are from Shoshani et al. [6].