Abstract: P2 receptors are widely distributed in animal and human organisms; however, selective antagonists of these receptors are still lacking. One of the well-known and mostly used P2 receptor antagonist at present is pyridoxal-phosphate 6-azophenil 2’, 4’-disulphonic acid (PPDAS) which has no selectivity to either P2X or P2Y receptors. In this study, a series of new PPADS analogues for their potential antagonistic effects on P2 receptors was tested. The compounds (lab codes 2a-2g) were tested at a concentration of 10 mM in vitro for antagonistic activity towards P2X and P2Y receptors using isolated rat smooth muscle preparations of urinary bladder, vas deferens and duodenum. Contractions of urinary bladder and vas deferens preparations were evoked by either agonist of P2X-receptors, α,β-methylene-ATP (0.1-3 mM), or by electrical field stimulation (EFS, 0.5 msec, 100 V, 1-32 Hz) in the presence of M-cholinoblocker atropine (10 μM) and alpha-adrenoblocker phentolamine (10 μM). Relaxant responses of carbachol-precontracted duodenum preparations were induced by either ATP (1 μM-1 mM) or by EFS (0.5 msec, 100 V) with frequencies of 0.5, 1, 2 and 4 Hz. Effects of tested compounds were compared with that of PPADS (10 μM). It was found that the majority of the tested compounds showed some degree of antagonism against EFS-evoked contractions of rat isolated urinary bladder and vas deferens mediated via P2X receptors, however only compound 2c produced antagonism comparable with that of parent antagonist, PPADS. Further, compound 2c, unlike PPADS, did not antagonize P2Y-receptor-mediated relaxation in rat isolated duodenum preparations. It is concluded from this study that compound 2c is an effective antagonists of P2X but not P2Y receptors and it’s selectivity towards subtypes of P2 receptors needs to be proved in further experiments.
INTRODUCTION
P2 receptors, where ATP is a main endogenous agonist, are present in all tissues of any biological organism. Up to date 15 subtypes of P2 receptors have been identified which belong to two major classes, namely P2X and P2Y. Wide distribution and diversity of P2 receptors make them promising targets for pharmacological intervention. Antagonists of P2Y12 receptors (clopidogrel, ticlopidine etc.) are effective antiplatelet drugs and are widely used as alternatives to aspirin for prevention of myocardial infarction and stroke. Several other agonists and antagonists of P2 receptors are currently studied in clinical trials for treatment of different pathological conditions (Ziganshin and Ziganshina, 2009).
Development of P2 receptor antagonists is a promising area of modern medicinal chemistry and pharmacology. Despite considerable numbers of synthesized and studied P2 receptor antagonists with different chemical structures, each of them has certain disadvantages, in particular the lack of selectivity or efficiency of their antagonism or significant effect on the activity of ecto-ATPase (Ziganshin et al., 1994a). One of the well-known P2 receptors antagonists is pyridoxal-phosphate 6-azophenil 2’, 4’-disulphonic acid (PPDAS)-an azosulfophenyl derivative of pyridoxal phosphate (Ziganshin et al., 1993; McLaren et al., 1994). Various structural modifications of PPADS have been made through functional group substitution on the sulfophenyl ring (Connolly, 1995; Ziganshin et al., 1998). However, the influence of substituents at the pyridoxal moiety on the P2 receptor antagonism was less studied. It was shown that a phosphate linkage is not required for P2 receptor antagonism through the inclusion of phosphonates (Kim et al., 1998) and substitution of phosphate moiety by carboxylic acid groups (Jung et al., 2013; Cho et al., 2013). This study presents the biological activity of novel PPADS analogs based on azosulfophenyl derivatives of pyridoxine with acetal protection of hydroxymethyl groups. Such structure provides opportunities to vary the lipophilicity of resulting compounds which has significant impact on biological activity of pyridoxine derivatives.
MATERIALS AND METHODS
Tested compounds: Compounds with laboratory codes of 2a-2g were synthesized in the Centre for Research and Education in Pharmaceutics, Kazan (Volga Region) Federal University, Kazan, Russia (Pugachev et al., 2013a, b). All compounds are close analogues of PPADS with modifications in pyridoxal moiety. Compounds were diluted in distilled water to a concentration of 10-2 M; this was used as a stock solution for all experiments (Fig. 1).
General procedures: Antagonistic activity of the synthesized compounds towards P2 receptors was evaluated in vitro on isolated smooth muscle preparations of male Wistar rats weighing 150-200 g (nursery "Puschino", Moscow region). All experiments were performed in accordance with the workplace policies on the use of experimental animals. Method for studying the mechanical activity of isolated tissues was applied (Ziganshin et al., 1993). All new compounds as well as PPADS were tested at a concentration of 10-5 M in triple or quadruple experiments on tissues of at least three different animals.
Antagonistic activity against P2X receptors: The smooth muscle preparations of rat urinary bladder and vas deferens were placed in a 10 mL thermostatic bath with Krebs solution of the following composition (mM): NaCl 144.3, KCl 4.7, NaHCO3 5.0, MgCl2 0.6, NaH2PO4 1.35, glucose 7.8 and CaCl2 2.5. The pH of the solution was 7.3-7.4, temperature 37.0±0.5°C.
| Fig. 1: | Chemical structure of the tested compounds |
One end of the preparation was fixed while another end by a silk thread was attached to the transducer of mechanical activity. Mechanical activity of the muscle preparations was detected by a FSG-01 force-displacement transducer (Linton, Great Britain) and recorded by the MP100WSW Data Acquisition System (Biopack, Great Britain) and displayed on a computer screen. The preparations were allowed to equilibrate under the initial tension of 1 g for at least 60 min in the bath media while the Krebs solution was replaced every 15 min.
Contractions induced by electrical field stimulation: Electrical Field Stimulation (EFS) was applied using a Digitimer MultiStim D330 stimulator (Digitimer Ltd, UK) via two platinum wire rings 2.5 mm in diameter, 15 mm apart, through which the muscles were threaded. Contractile activity of the tissue was elicited by applying rectangular impulses at frequencies of 0.5-32.0 Hz, 0.5 msec in length and 100 V in voltage. After initial contractions induced by EFS being recorded, EFS and recording were conducted again in the presence of either atropine sulfate (10 mM, urinary bladder) or phentolamine (10 mM, vas deferens) to exclude the effects of M-cholinergic or α-adrenergic receptors on electrical stimulation. The following EFS and recording were produced after 20 min incubation of muscle preparations with a given tested compound at a concentration of 10-5 M.
Contractions induced by α,β-methylene ATP (α,β-meATP): A non-selective agonist of P2X receptors, α,β-meATP (0.1-3 mM), produced concentration-dependent contractions of isolated rat urinary bladder and vas deferens. Such concentration-contraction relationship for α,β-meATP was obtained before and after 20 min incubation of muscle preparations with tested compounds or PPADS at a concentration of 10-5 M.
Antagonistic activity against P2Y-receptors: Longitudinal smooth muscle layer of rat duodenum was gently removed by damp cotton ball and placed vertically in the 10 mL thermostatic bath with Krebs solution as written above. Smooth muscle preparations were allowed to equilibrate under the initial tension of 500 mg for at least 60 min in the bath media while the Krebs solution was replaced every 15 min. In preliminary experiments the concentration-response relationship for carbachol-induced contractions of the longitudinal smooth muscles of duodenum was assessed. It was found that carbachol at a concentration of 0.3 μM causes persistent contractile responses; this concentration was used for further experiments to raise tissue tone. Relaxation responses were evaluated as a percentage of the maximal relaxation possible.
Relaxation induced by EFS: The EFS (0.5 msec, 100 V) with frequencies of 0.5, 1, 2 and 4 Hz induced frequency-dependent relaxation of carbachol-precontracted duodenum preparations. A frequency-relaxation relationship for EFS was constructed before and after 20 min incubation of the duodenum preparations with tested compounds or PPADS at a concentration of 10-5 M.
Relaxation induced by ATP: A non-selective agonist of P2 receptors, ATP (1 μM-1 mM), elicited a concentration-dependent relaxation of carbachol-precontracted duodenum preparation. A concentration-relaxation relationship for ATP was constructed before and after 20 min incubation of the duodenum preparations with tested compounds or PPADS at a concentration of 10-5 M.
Drugs: All agents for these experiments were purchased from Sigma-Aldrich.
Statistics: Data was expressed as Mean±Standard errors. The significances of differences were evaluated using the Student’s t-test. The p value being not less than 0.05 were deemed significant. The n value reported in the text refer to the number of individual experiments.
RESULTS AND DISCUSSION
Antagonistic activity towards P2X receptors: PPADS at a concentration of 10 mM significantly down-shifted the frequency-response relationship curve for EFS-evoked contractions in the rat isolated urinary bladder and vas deferens in the presence of atropine and phentolamine. The reduction of contractile responses in the presence of PPADS was up to 50% in vas deferens and up to 30% in urinary bladder comparing to corresponding control contractions.
It was found that all tested compounds showed some degree of antagonism against EFS-evoked contractions of rat isolated urinary bladder and vas deferens, although none of the compound was significantly better in their antagonism than that shown by PPADS. However, one of the tested compound (2c) produced antagonism towards P2X-mediated contractions of rat urinary bladder and vas deferens comparable with that of parent antagonist (PPADS).
To keep the paper short, further we present results only for compound 2c with comparison for that of PPADS. Compound 2c was tested in experiments where contractions of rat isolated urinary bladder and vas deferens preparations were induced by a non-selective agonist of P2X receptors, α,β-meATP (0.1-3 μM). Pre-incubation of smooth muscle tissues with compound 2c (10 μM) caused downshift of the concentration-response curve for a,b-meATP, although this effect was less prominent than that of PPADS (Fig. 2).
Antagonistic activity towards P2Y receptors: To test potential antagonistic effects of compound 2c on P2Y receptor-mediated relaxation, we evaluated its activity on rat isolated longitudinal smooth muscle preparations of duodenum. It was found that compound 2c (10 μM) dZiganshinid not significantly affect P2Y receptor-mediated relaxation of duodenum induced by either EFS (0.5-8 Hz) or ATP (1-30 μM). In similar experiments, PPADS caused small but significant decrease of relaxant effects mediated by P2Y receptors (Fig. 3).
PPADS initially was introduced as a selective P2X receptor antagonist (Ziganshin et al., 1993, 1994b), however soon its antagonistic activity against P2Y receptors was observed by Windscheif et al. (1995). Since then many analogues of PPADS were tested for their possible selective antagonistic activity towards different P2 receptors (Kim et al., 1998; Ziganshin et al., 1998), however not much progress was achieved.
| Fig. 2(a-b): | Contractile responses of rat isolated vas deferens evoked by agonist of P2X receptors, α,β-meATP (10-7-3×10-6 М), before and after 20 min incubation of tissues with (a) Compound 2c and (b) PPADS at a concentration of 10-5 М. Results presented as Mean±SD, n = 3-4 |
| Fig. 3(a-b): | Relaxant responses of carbachol-precontracted rat isolated longitudinal smooth muscle preparations of duodenum evoked by ATP (10-6-3×10-5 М) before and after 20 min incubation of tissues with (a) Compound 2c and (b) PPADS at a concentration of 10-5 М. Results presented as Mean±SD, n = 3-4 |
In this study, 7 new analogues of PPADS were tested with substitutions at the pyridoxal moiety for their antagonistic activity against P2X and P2Y receptor-mediated responses in rat isolated smooth muscle tissues. Although most of the tested compounds did not appear to be effective antagonists, one of compounds (2c) showed a notable antagonism against P2X receptor-mediated contractions of urinary bladder and vas deferens, both in experiments with EFS and with α,β-meATP. Although the antagonistic effect of compound 2c towards P2X receptors was not greater than that of PPADS, unlike PPADS, compound 2c did not significantly affect the P2Y receptor-mediated relaxation. Thus compound 2c might be more selective to some P2 receptor subtypes than PPADS. This fact confirms that phosphate moiety is not necessary for antagonistic activity (Kim et al., 1998) and offers new opportunities for synthesis of novel purinoceptor antagonists. It is interesting to mention that unlike other biological activity of studied substances (Pugachev et al., 2013a, b), in the present in vitro experiments, antagonism against P2 receptors seems to be not dependent on the lipophilicity of the compounds.
CONCLUSION
In conclusion, we have shown in this study that azophenilsulfonic acid derivatives demonstrated activity towards certain types of purinoceptors and in particular compound 2c is an effective antagonists of P2X but not P2Y receptors and it’s selectivity towards subtypes of P2 receptors needs to be proved in further experiments.
ACKNOWLEDGMENTS
The study was supported by the Russian Foundation for Basic Research 13-04-00188. This study was partly funded by the subsidy allocated to Kazan Federal University for the state assignment in the sphere of scientific activities, Russia.