Optical Control of Adenosine-Mediated Pain Modulation

Adenosine receptors (ARs) play many important roles in physiology and have been recognized as potential targets for pain relief. Here, we introduce three photoswitchable adenosine derivatives that function as light-dependent agonists for ARs and confer optical control to these G protein-coupled receptors. One of our compounds, AzoAdenosine-3, was evaluated in the classical formalin model of pain. The molecule, active in the dark, was not metabolized by adenosine deaminase and effectively reduced pain perception in a light-dependent manner. These antinociceptive effects suggested a major role for A1R and A3R in peripheral-mediated pain sensitization, whereas an average adenosine-mediated antinociceptive effect will be facilitated by A2AR and A2BR. Our results demonstrate that a photoswitchable adenosine derivative can be used to map the contribution of ARs mediating analgesia in vivo.


Drug Synthesis
Unless stated otherwise indicated all reactions were carried out with magnetic stirring using oven-dried glassware (160°C) under inert gas atmosphere (nitrogen or argon). Syringes used to transfer reagents and solvents were purged with nitrogen prior to use. Low temperature reactions were carried out in a Dewar vessel filled with the appropriate cooling agent e.g.
H2O/ice (0°C). Heating was conducted using a heated oil bath. Yields refer to spectroscopically pure compounds unless otherwise stated.
Reaction solvents were purchased from Acros Organics as 'extra dry' over molecular sieves and handled under inert gas atmosphere. Tetrahydrofuran (THF) was distilled from Na/benzophenone prior to use. Dichloromethane (DCM), triethylamine (TEA) and diisopropylethylamine (DIPEA) were distilled from calcium hydride. Ethanol and acetic acid were purchased from commercial suppliers and used as received. Solvents for extraction and flash column chromatography were purchased in technical grade purity and distilled under reduced pressure on a rotary evaporator prior to use. All other reagents and solvents were purchased from commercial suppliers and used as received.
Reactions and chromatography fractions were monitored by qualitative thin-layer monochromator, or a Prizmatix ultra high-power LED (460 nm), connected to a fiber-optic cable through which the sample in the spectrophotometer was irradiated from the top.
After turning homogenous at approximately 70°C, the mixture was stirred at 110°C for 3 h.
The reaction mixture was allowed to cool to rt, dissolved in methanol and purified using silica  max (DMSO): 292 nm, 328 nm ( → *).
Oxone (689 mg, 1.12 mmol, 4.5 eq.) in H2O (5 mL      formalin/0.92% formaldehyde; Sigma-Aldrich) was intraplantarly (i.pl.) injected in the mid-S11 plantar surface of the right hind paw of the mouse, as previously described. 8 The formalininduced nociceptive behaviour in light or mock manipulated (dark) conditions was quantified as the time spent licking or biting the injected paw during the 30 min after the injection of formalin. The initial acute phase (0-5 min; phase I), reflecting the acute peripheral pain, was followed by a relatively short quiescent period (10 min), which was then followed by a Absorbance @ 265 nm (% Veh) Figure S3. AA-3 induced locomotor activity. Locomotor activity was assessed by quantifying the total distance travelled of mice (n=5) systemically (i.p.) administered with vehicle (saline), adenosine (ADO, 10 mg/kg) or AzoAdenosine-3 (AA-3, 10 mg/kg) during 10 min. *P<0.05 and ***P < 0.001, one-way ANOVA with Dunnett's multiple comparison test using Veh as a control.