Acyclic analogues of adenosine bisphosphates as P2Y receptor antagonists: phosphate substitution leads to multiple pathways of inhibition of platelet aggregation

Abstract

Activation by ADP of both P2Y(1) and P2Y(12) receptors in platelets contributes to platelet aggregation, and antagonists at these receptor subtypes have antithrombotic properties. In an earlier publication, we have characterized the SAR as P2Y(1) receptor antagonists of acyclic analogues of adenine nucleotides, containing two phosphate groups on a symmetrically branched aliphatic chain, attached at the 9-position of adenine. In this study, we have focused on antiaggregatory effects of P2Y antagonists related to a 2-chloro-N(6)-methyladenine-9-(2-methylpropyl) scaffold, containing uncharged substitutions of the phosphate groups. For the known nucleotide (cyclic and acyclic) bisphosphate antagonists of P2Y(1) receptors, there was a significant correlation between inhibition of aggregation induced by 3.3 microM ADP in rat platelets and inhibition of P2Y(1) receptor-induced phospholipase C (PLC) activity previously determined in turkey erythrocytes. Substitution of the phosphate groups with nonhydrolyzable phosphonate groups preserved platelet antiaggregatory activity. Substitution of one of the phosphate groups with O-acyl greatly reduced the inhibitory potency, which tended to increase upon replacement of both phosphate moieties of the acyclic derivatives with uncharged (e.g., ester) groups. In the series of nonsymmetrically substituted monophosphates, the optimal antagonist potency occurred with the phenylcarbamate group. Among symmetrical diester derivatives, the optimal antagonist potency occurred with the di(phenylacetyl) group. A dipivaloyl derivative, a representative uncharged diester, inhibited ADP-induced aggregation in both rat (K(I) 3.6 microM) and human platelets. It antagonized the ADP-induced inhibition of the cyclic AMP pathway in rat platelets (IC(50) 7 microM) but did not affect hP2Y(1) receptor-induced PLC activity measured in transfected astrocytoma cells. We propose that the uncharged derivatives are acting as antagonists of a parallel pro-aggregatory receptor present on platelets, that is, the P2Y(12) receptor. Thus, different substitution of the same nucleoside scaffold can target either of two P2Y receptors in platelets.

Figure 1.

Aggregometry traces for ADP-induced rat platelet aggregation in the presence or absence of various inhibitors: A, riboside 2a; B, acyclic monophosphate monopivaloyl derivative 21; C, acyclic bisphosphate 14; D, acyclic dipivaloyl derivative 26. In each case 3.3 μM ADP was present.

Figure 2.

Concentration-response curves for ADP-induced rat platelet aggregation in the presence or absence of various inhibitors: A, riboside 2b; B, acyclic phenylurethane monophosphate derivative 24; C, acyclic bisphosphate 14; D, diphenylacetate derivative 31. In each case 3.3 μM ADP was present.

Figure 3.

Correlation of inhibition of phospholipase C activity in turkey erythrocytes expressing a native P2Y₁ receptor with the potency in the inhibition of rat platelet aggregation of previously reported antagonists 2a, 2b, 3, and 12–17. Values for the PLC inhibition are taken from refs , , and . Dashed curves represent the 95% confidence range (r² = 0.842).

Figure 4.

Concentration-response curves for inhibition of P2Y receptor-induced second messengers, either (□) cyclic AMP levels in rat platelets (in the presence of 3.3 μM ADP) or (●) phospholipase C activity in 1321N1 astrocytoma cells expressing the hP2Y₁ receptor. Measurements were made in the presence of increasing concentrations of the following inhibitors: A, acyclic bisphosphate 14 and B, acyclic dipivaloyl derivative 26.

Figure 5.

Concentration-response curves for inhibition of ADP-induced aggregation of human platelets by the acyclic dipivaloyl derivative 26 (■) and the di(phenylacetyl) derivative 31 (▼), expressed as percent (mean ± SD) of ADP (5 μM)-induced platelet aggregation in the presence of 1% DMSO (control samples).

Scheme 1.

Synthesis of Adenine 9-Riboside Derivatives Similar to 2b, in Which the 3′-Phosphate Group Has Been Replaced with a Urethane Group^{a a} Reagents: (a) bis-2-cyanoethyl-N,N-diisopropylphosporamidite, tetrazole, THF; then t-BuOOH; (b) R-NCO, CuBr, Py; (c) bistrimethylsilylacrylamide, DBU, Py. B = 2-chloro-6-methylaminopurin-9-yl.

Scheme 2.

Synthesis of Adenine 9-(N)-Methanocarba Derivatives Similar to 3, in Which the 3′- or 5′-Phosphate Ester Groups Have Been Removed or Replaced with Acetyl Groups^{a a} Reagents: (a) Ac₂O, DMAP, Py, CH₂Cl₂; (b) Et₂NP(OBu^−t)₂, 1H-tetrazole, m-CPBA, THF/CH₂Cl₂; (c) 10% TFA in CH₂Cl₂; (d) NH₃/H₂O; (e) DHP, TsOH–H₂O; (f) KOH/MeOH; (g) Ac₂O, DMAP, Et₃N, CH₂Cl₂. B = 2-chloro-6-methylaminopurin-9-yl.

Scheme 3.

Synthesis of 2-Chloro-N⁶-methyladenine-9-(2-methylpropyl) Derivatives Similar to 14, in Which Either or Both Phosphate Ester Groups Have Been Removed or Replaced with an Acetyl Group^{a a} Reagents: (a) Me₂C(OMe)₂, TsOH-H₂O, THF; (b) MsCl, Et₃N, CH₂Cl₂; (c) 2-chloro-6-methylaminopurine, K₂CO₃, DMF, 60 °C; (d) AcOH/H₂O/THF (35:65:10), 70 °C; (e) ⁱPr₂NP(OBn)₂ or Et₂NP-(OBu^−t)₂, 1H-tetrazole, m-CPBA, THF/CH₂Cl₂; (f) K₂CO₃, CH₃OH; (g) 80% HOAc; (h) Ac₂O, DMAP, Py, CH₂Cl₂. B = 2-chloro-6-methylaminopurin-9-yl.

Scheme 4.

Synthesis of 2-Chloro-N⁶-methyladenine-9-(2-methylpropyl) Derivatives Similar to 14, in Which One of the Phosphate Ester Groups Has Been Removed or Replaced with an Acyl Group^{a a} Reagents: (a) pivalic anhydride, pyridine, CH₂Cl₂; (b) (PhCO)₂O, pyridine, DMAP, CH₂Cl₂; (c) ClCOOEt, Et₃N, DMAP, CH₂Cl₂; (d) PhNCO, Et₃N, CH₂Cl₂; (e) (i) TMSBr, CH₂Cl₂; (ii) triethylammonium bicarbonate buffer. B = 2-chloro-6-methylaminopurin-9-yl.

Scheme 5.

Synthesis of 2-Chloro-N⁶-methyladenine-9-(2-methylpropyl) Derivatives Similar to 14, in Which Both of the Phosphate Ester Groups Has Been Replaced Nonsymmetrically with Acyl Groups^{a a} Reagents: (a) for 25, (CH₃CH₂CO)₂O, DMAP, pyridine, CH₂Cl₂; for 26, (Me₃CCO)₂O, DMAP, pyridine, CH₂Cl₂; for 27, crotonic anhydride, DMAP, pyridine, CH₂Cl₂; for 28, (PhCO)₂O, pyridine, DMAP, CH₂Cl₂; for 29, (2-F-PhCO)₂O, pyridine, DMAP, CH₂Cl₂; for 30, (2,6-di-F-PhCO)₂O, pyridine, DMAP, CH₂Cl₂; for 31, (PhCH₂CO)₂O, DMAP, pyridine, CH₂Cl₂; for 32, PhNCO, Et₃N, CH₂Cl₂; for 33, ClCOOPh, Et₃N, DMAP, CH₂Cl₂; for 34, succinic anhydride, pyridine; for 35, CS₂, NaOH, PhCH₂Br, DMSO. (b) For 36, (PhCO)₂O, DMAP, pyridine, CH₂Cl₂; for 37, (PhCH₂CO)₂O, DMAP, pyridine, CH₂Cl₂. B = 2-chloro-6-methylaminopurin-9-yl.

Scheme 6.

Synthesis of 2-Chloro-N⁶-methyladenine-9-(2-methylpropyl) Bisphosphonate Derivatives Similar to 14^{a a} Reagents: (a) LiAlH₄, THF; (b) NaH, THF; (c) n-Bu₄NI, BnBr; (d) 9-BBN, THF; (e) H₂O₂, NaOH; (f) Br₂, Ph₃P, DMF; (g) P(OEt)₃, 155°; (h) H₂ , 5% Pd/carbon, MeOH; (i) P(OEt)₃, 155°; (j) 9-BBN, THF, reflux; (k) 2,6-dichloropurine, Ph₃P, DEAD, THF; (l) MeNH₂, CH₃CN; (m) Me₃SiI, CH₃CN; (n) Dowex-Na.