In Silico, In Vitro and In Vivo Pharmacodynamic Characterization of Novel Analgesic Drug Candidate Somatostatin SST4 Receptor Agonists

Background: Somatostatin released from the capsaicin-sensitive sensory nerves mediates analgesic and anti-inflammatory effects via its receptor subtype 4 (SST4) without influencing endocrine functions. Therefore, SST4 is considered to be a novel target for drug development in pain, especially chronic neuropathy which is a great unmet medical need. Purpose and Experimental Approach: Here, we examined the in silico binding, SST4-linked G protein activation and β-arrestin activation on stable SST4 expressing cells and the effects of our novel pyrrolo-pyrimidine molecules (20, 100, 500, 1,000, 2,000 µg·kg−1) on partial sciatic nerve ligation-induced traumatic mononeuropathic pain model in mice. Key Results: The novel compounds bind to the high affinity binding site of SST4 the receptor and activate the G protein. However, unlike the reference SST4 agonists NNC 26-9100 and J-2156, they do not induce β-arrestin activation responsible for receptor desensitization and internalization upon chronic use. They exert 65–80% maximal anti-hyperalgesic effects in the neuropathy model 1 h after a single oral administration of 100–500 µg·kg−1 doses. Conclusion and Implications: The novel orally active compounds show potent and effective SST4 receptor agonism in vitro and in vivo. All four novel ligands proved to be full agonists based on G protein activation, but failed to recruit β-arrestin. Based on their potent antinociceptive effect in the neuropathic pain model following a single oral administration, they are promising candidates for drug development.


Supplementary Material
Novel pyrrolo-pyrimidine compounds  hydride (60 % dispersion in mineral oil) was added in small amounts. After the addition the reaction mixture was stirred for 30 min at room temperature, then 1.83 g (11 mmol) 3bromopropyl)dimethylamine was added, and the reaction mixture was stirred overnight. After the starting chlorine compound disappeared by TLC (eluent: chloroform/methanol 10/1) the mixture was diluted with 100 ml ice-cold water. The pH was set to 8-9 with saturated NaHCO3 solution, and the product was extracted with 3x40 ml ethyl acetate. The organic layers were separated, combined, and dried over Na2SO4. The solvent was removed under vacuum, the remaining oil was treated with diisopropyl-ether to obtain the solid product which was used for the next step without further purification.
Yield: 2.05 g (77 %) yellow material. (obtained from the previous step), and 1.556 g (10 mmol) 2-(2-Chlorophenyl)ethylamine were solved in 6 ml dimethylsulfoxide, and the mixture was stirred at 100 o C for 12 hours. The reaction mixture was cooled down to room temperature, then it was diluted with 120 ml saturated NaHCO3 solution. The product was extracted with 3x40 ml ethyl acetate. The organic layers were separated, combined, and dried over Na2SO4. The solvent was removed under vacuum, the crude product was purified by column chromatography (eluent: chloroform/methanol 10/1, with 1% NH3.aq). The yielded oil was dissolved in 30 ml diethyl ether, and 5 ml saturated HCl/ethyl acetate was added.
The formed HCl salt was filtered out, and dried.
Yield: 1.33 g (63 %) off-white crystals.  N,N-dimethylformamide. The solution was cooled down to 0 o C, and 385 mg (9.6 mmol) sodium hydride (60 % dispersion in mineral oil) was added in small amounts. After the addition the reaction mixture was stirred for 30 min at room temperature, then 1.88 g (11 mmol) benzyl bromide was added and stirred overnight. After the starting chlorine compound disappeared by TLC (eluent: chloroform/methanol 10/1) the mixture was diluted with 100 ml ice-cold water. The pH was set to 8-9 with saturated NaHCO3 solution, and the product was extracted with 3x40 ml ethyl acetate.
The organic layers were separated, combined, and dried over Na2SO4. The solvent was removed under vacuum, the remaining oil was treated with diisopropyl-ether to obtain the solid product which was used for the next step without further purification.
Yield: 1.76 g (81 %) off-white material. N,N-dimethylformamide. The solution was cooled down to 0 o C, and 385 mg (9.6 mmol) sodium hydride (60 % dispersion in mineral oil) was added in small amounts. After the addition the mixture was stirred for 30 min at room temperature, then 1.77 g (11 mmol) 2-(bromomethyl)furan was added, and the mixture was stirred for 24 hours. After the starting chlorine compound disappeared by TLC (eluent: chloroform/methanol 10/1) the mixture was diluted with 100 ml ice-cold water.
The pH was set to 8-9 with saturated NaHCO3 solution, and the product was extracted with 3x40 ml ethyl acetate. The organic layers were separated, combined, and dried over Na2SO4. The solvent was removed under vacuum, the remaining oil was treated with diisopropyl-ether to obtain the solid product which was used for the next step without further purification. Changes of the mechanonociceptive thresholds. Columns represent the mechanonociceptive thresholds before the operation (white) and on the 7 th postoperative day before (black) and 60 min after the treatment with Compounds 1-4 (striped). Each column represents the mean+S.E.M. of n.