2-MeS-beta,gamma-CCl2-ATP is a potent agent for reducing intraocular pressure

Abstract

Extracellular nucleotides can modify the production or drainage of the aqueous humor via activation of P2 receptors and therefore affect the intraocular pressure (IOP). We have synthesized slowly hydrolyzable nucleoside di- and triphosphate analogues, 1, and 8-14. Analogues 8-14 were completely resistant to hydrolysis by alkaline phosphatase over 30 min at 37 degrees C. In human blood serum, analogues 8-14 exhibited high stability, e.g., analogues 9 and 10-14 were only 15% and 0% degraded after 24 h, respectively. Moreover, analogues 8-14 were highly stable at pH 1.4 (t(1/2) 1 h-30 days). Analogues 8-14 were agonists of the P2Y(1) receptor (EC(50) 0.57-9.54 muM). Ocular administration of most analogues into rabbits reduced IOP, e.g., analogue 9 reduced IOP by 32% (EC(50) 95.5 nM). Analogue 9 was more effective at reducing IOP than several common glaucoma drugs and represents a promising alternative to timolol maleate, which cannot be used for the treatment of patients suffering from asthma or cardiac problems.

Figure 1

Structures of previously tested nucleotides.

Figure 2

Structures of nucleotides tested in this study.

Figure 3

Rates of hydrolysis of analogues 9 and 10B at pH 1.4 at 37 °C, as monitored by HPLC. Hydrolysis of 7.5 mM analogue 9 or 10B in KCl/HCl buffer at pH 1.4 and 37 °C was recorded for 5 days at 7–17 h intervals. (A) HPLC chromatograms of analogue 9 and hydrolysates at t = 6, 52, 216 h. (B) Kinetics of acidic hydrolysis of analogue 9 (t_1/2 = 65 h) and time-dependent formation of degradation products. (C) Kinetics of acidic hydrolysis of analogue 10B (t_1/2 = 8 h).

Figure 4

Hydrolysis of ATP and analogues 1, 8, and 9 in human blood serum (180 μL) and RPMI-1640 medium (540 μL) over 24 h at 37 °C, as monitored by HPLC. (A) Hydrolysis of 0.25 mM ATP and production of ADP and AMP. (B) Degradation of analogues 1, 8, and 9 in human blood serum.

Figure 5

(A) Effect of analogues 1, 8–9, and 11–14 (100 μM) on rabbit IOP measured over 8 h vs control: (1) an equal volume of saline was administered to the contralateral eye; (2) an equal volume of saline was administered to other animals. IOP was measured twice before the application of any drug and was nearly identical in all animals. (B) Time course for the effects of analogues 1, 9, 13, and 14 (100 μM) on rabbit IOP over 8 h vs control. Values are the means ± SEM of results from 10 independent experiments. (C) Time-course for the effects of analogues 11B, 12A, and 12B (100 μM) on rabbit IOP over 8 h. Values are the means ± SEM of results from eight independent experiments. (D)Dose–response curves for the maximal effectsof analogues 1, 9, 13, and 14 (100 μM) on rabbit IOP. Values are the means ± SEM of results from eight independent experiments. (E) Maximal effects of analogues 1 and 9 (100 μM, 10 μL) on rabbit IOP, as compared to latanoprost (0.005%), dorzolamide hydrochloride (2%), and timolol maleate (0.5%) applied in 40 μL, as compared to control. (F) Duration of the effects of analogues 1 and 9 (100 μM, 10 μL), as compared to latanoprost (0.005%), dorzolamide hydrochloride (2%), and timolol maleate (0.5%) applied in 40 μL, as compared to control. The duration of the effect, termed mean-time effect, was calculated as the time between 50% maximal decrease in IOP after drug administration and a 50% recovery of the initial IOP value.

Scheme 1^a

^aReaction conditions for analogue 8: (a) trimethylphosphate, POCl₃, proton sponge, 0 °C, 3 h; (b) 0.5 M bis(tributylammonium)difluoromethylene diphosphonate in dry DMF, Bu₃N, 0 °C, 1.5 h; (c) 0.5 M TEAB, pH 7, RT, 1 h; (d) (1) 18% HCl, pH 2.3, RT, 3 h, (2) 24% NH₄OH, pH 9, RT, 45 min. Reaction conditions for analogue 9: (a) trimethylphosphate, POCl₃, proton sponge, 0 °C, 1 h; (b) 1 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu₃N, 0 °C, 40 min, and then RT, 5 min; (c) 0.5 M TEAB, pH 7, RT, 1 h; (d) as described for analogue 8.

Scheme 2^a

^aReaction conditions for analogue 10: (a) trimethylphosphate, PCl₃, proton sponge, 0 °C, 30 min; (b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu₃N, 0 °C, 1 h; (c) 2 M BH₃·SMe in THF, 0 °C, 5 min, and then RT, 60 min; (d) 0.5 M TEAB, pH 7, RT, 1 h; (e) (1) 18% HCl, pH 2.3, RT, 3 h, (2) 24% NH₄OH, pH 9, RT, 45 min. Reaction conditions for analogue 11: (a) trimethylphosphate, PCl₃, proton sponge, 0 °C, 30 min; (b) 0.5 M bis(tributylammonium)dichloromethylene diphosphonate in dry DMF, Bu₃N, 0 °C, 25 min; (c) 2 M BH₃·SMe₂ in THF, 0 °C, 5 min and then RT, 25 min; (d) 0.5 M TEAB, pH 7, RT, 45 min; (e) as described for analogue 10. Reaction conditions for analogue 12: (a) trimethylphosphate, PCl₃, proton sponge, 0 °C, 45 min; (b) 0.5 M bis(tributylammonium)difluoromethylene diphosphonate in dry DMF, Bu₃N, 0 °C, 50 min; (c) 2 M BH₃·SMe in THF, 0 °C, 5 min then RT, 60 min; (d) and (e) as described for analogue 10.

Scheme 3^a

^aReaction conditions for analogue 15: (a) CH₂Cl₂, DMAP, TsCl, RT, 12 h; (b) tetra-(n-butylammonium)difluoromethylene diphosphonate in dry DMF, RT, 72 h; (c) (1) 18% HCl, pH 2.3, RT, 3 h; (2) 24% NH₄OH, pH 9, RT, 45 min. Reaction conditions for analogue 33: (a) CH₂Cl₂, DMAP, TsCl, RT, 12 h; (b) tetra-(n-butylammonium) dichloromethylene diphosphonate in dry DMF, RT, 72 h; (c) TFA, argon bubbling, RT, 10 min.