Glial Cell Line–Derived Neurotrophic Factor Receptor Rearranged During Transfection Agonist Supports Dopamine Neurons In Vitro and Enhances Dopamine Release In Vivo

Abstract Background Motor symptoms of Parkinson's disease (PD) are caused by degeneration and progressive loss of nigrostriatal dopamine neurons. Currently, no cure for this disease is available. Existing drugs alleviate PD symptoms but fail to halt neurodegeneration. Glial cell line–derived neurotrophic factor (GDNF) is able to protect and repair dopamine neurons in vitro and in animal models of PD, but the clinical use of GDNF is complicated by its pharmacokinetic properties. The present study aimed to evaluate the neuronal effects of a blood‐brain‐barrier penetrating small molecule GDNF receptor Rearranged in Transfection agonist, BT13, in the dopamine system. Methods We characterized the ability of BT13 to activate RET in immortalized cells, to support the survival of cultured dopamine neurons, to protect cultured dopamine neurons against neurotoxin‐induced cell death, to activate intracellular signaling pathways both in vitro and in vivo , and to regulate dopamine release in the mouse striatum as well as BT13's distribution in the brain. Results BT13 potently activates RET and downstream signaling cascades such as Extracellular Signal Regulated Kinase and AKT in immortalized cells. It supports the survival of cultured dopamine neurons from wild‐type but not from RET‐knockout mice. BT13 protects cultured dopamine neurons from 6‐Hydroxydopamine (6‐OHDA) and 1‐methyl‐4‐phenylpyridinium (MPP+)–induced cell death only if they express RET. In addition, BT13 is absorbed in the brain, activates intracellular signaling cascades in dopamine neurons both in vitro and in vivo, and also stimulates the release of dopamine in the mouse striatum. Conclusion The GDNF receptor RET agonist BT13 demonstrates the potential for further development of novel disease‐modifying treatments against PD. © 2019 The Authors. Movement Disorders published by Wiley Periodicals LLC. on behalf of International Parkinson and Movement Disorder Society.

The individual steps of the synthesis were carried out as follows.  To a 2-L round-bottom flask equipped with thermometer, mechanical stirrer, dropping funnel 54 and drying tube chlorosulfonic acid (4) (1.14 kg, 9.78 mol, 0.65 L) was charged. With well stirring 55 and cooling to keep the temperature below 5 °C the solution of the piperazine derivative (3) (129.0 56 4 g; 384 mmol) in dry dichloromethane (500 ml) was added dropwise. The reaction mixture was let to 57 warm up to room temperature and the stirring was continued for an additional hour. 58 To a five liter beaker, equipped with thermometer, mechanical stirrer and immersed into a 59 saltice cooling mixture ca. 1 kilogram of ice was placed, and then the reaction mixture was slowly 60 poured into it such a rate, that the temperature kept below 5 °C. The phases were separated and the dropwise. After the addition had taken place the reaction mixture was let to warm up to room 74 temperature and the stirring was continued for additional 30 min, by that time the reaction has been 75 completed. 76 The reaction mixture was diluted with water and the phases were separated. The organic phase 77 was washed twice with water and once with brine, dried over MgSO4 and evaporated to dryness. The 78 crystalline residue was suspended in diisopropyl ether, filtered off and washed with the same solvent.   To a suspension of sulfonamide (7) (31.30 g, 66.2 mmol) in tetrahydrofuran (300 ml) was 87 added the aqueous solution of potassium hydroxide (9.30 g, 166 mmol, 2.50 equiv., in 40 mL water) 88 in one portion and the mixture was stirred at 40-50 °C for four hours. The same amount of potassium 89 hydroxide (9.30 g, 166 mmol, 2.50 equiv) was dissolved in water (10 mL) and it was also added to 90 the reaction mixture and the stirring was continued at 50 °C for another four hours then at room 91 temperature overnight.

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After completion of the reaction the solvent was evaporated, the aqueous residue was diluted 94 with water and extracted with dichloromethane three times. The targeted piperazine derivative (8) 95 was extracted from the organic phase three times with 3% HCl solution. The combined aqueous 96 solution was cooled off, basified with 20% aqueous NaOH and extracted three times with  To a suspension of 4-fluoro-2-(trifluoromethyl)benzoic acid (10) (9.53 g, 46 mmol) in 110 tetrahydrofuran (400 ml) was added 1,1'carbonyldiimidazole (8.17 g, 50 mmol, 1.10 equiv.) and the 111 reaction mixture was stirred at room temperature for 2 hours. The solution of the piperazine derivative 112 (9) (15.00 g, 46 mmol) in a minimal amount of tetrahydrofuran was added in one portion and the 113 reaction mixture was stirred at 50 °C for 20 hours.

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After the reaction had completed the solvent was evaporated. The residue was dissolved in 116 dichloromethane and was washed with 3% aqueous HCl solution, water, saturated aqueous NaHCO3 117 solution and brine, dried over MgSO4, filtered and finally evaporated to dryness. This crude product 118 was recrystallized from hot, 50% aqueous ethanol (500 ml) using norit. Yield: 12.91 g of 11 (54%)       Plates were prepared as described previously (7)  Imaging System (Molecular Devices) 10×magnification. Images were analyzed using CellProfiler 316 image analysis software (9).

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The dopamine, HVA and DOPAC concentrations in midbrain were measured 30 minutes post-493 intravenous administration. The measurement time point was was selected based on pharmacokinetics 494 data for BT13. BT13 significantly increased the level of HVA by 2.4-fold (P=0.0488, unpaired t-test).

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We also observed trends to increase in DA and DOPAC concentrations. The DOPAC/DA ratio 496 remained unchanged, but HVA/DA ratio was slightly higher in the brain of animals treated with BT13