, Following procedure A with compound 13 (95 mg, 0.25 mmol), 67% isolated yield). 1 H NMR (MeOD, 400 MHz, 298 K) ? 7.70 (s, 1H), 7.13 (m, 1H), 6.81-6.77 (m, 2H), 6.69 (m, 1H), vol.6

, HRMS m/z calcd. for C 21 H 26 N 2 O 4 Cl, MS, vol.3439, p.697, 1229.

, We obtained compound 6, as a brown powder (97 mg, 51% isolated yield). 1 H NMR (MeOD, 400 MHz) ? 7.79 (s, 1H, H 6 ), 7.14 (td, 1H, J = 7.5, 1.2 Hz), 6.79 (m, 1H), 6.78 (m, 1H), 6.71 (m, 1H), 6.50 (s, 1H), 3.91 (s, 3H), 3.53 (s, 2H), 3.28 (d, 2H

. M-+-h]-+-404, 64-406.64; HRMS (m/z) calcd. for C 21 H 27 N 3 NaO 3 [M + H] + 404.1741, found 404.1736; m.p. 92-105 ? C; IR (neat, cm ?1 ) ? max 3391, MS, vol.3204, p.770, 1148.

, mL/mmol). The resulting mixture was stirred at room temperature for 1h. Removal of the solvent under vacuum afforded the crude product, which was directly engaged in the next step. The residue obtained (1.0 equiv) was dissolved in DMF (10 mL/mmol) and 12 (1.3 equiv) and K 2 CO 3 (10.0 equiv) were added. The resulting mixture was stirred at 110 ? C for 1 h, then concentrated in vacuo. Ethyl acetate was added, the organic layer was washed several times with brine, dried over MgSO 4 and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel column, Procedure B. To a stirred solution of tert-butyl piperidine-1-carboxylate derivative (1.0 equiv.) in DCM (20 mL/mmol) was added TFA

J. 2h, 03 (s, 1H), 6.97 (dd, 1H, J = 7.8, 2.4 Hz), 6.51 (s, 1H), 5.84 (s, 2H), 3.81 (s, 3H), 3.45 (s, 2H), 3.39 (q, We obtained compound 7, as a brown oil (80 mg, 50% isolated yield). 1 H NMR (DMSO, 400 MHz, 363 K) ? 7.51 (s, 1H), 7.29 (t, 1H, J = 7.8 Hz), 7.12 (d, 1H, J = 7.8 Hz), vol.7, p.400, 1167.

, 05 (t, 1H, J = 2.2 Hz), 6.98 (dd, 1H, J = 7.8, 2.2 Hz), 6.50 (s, 1H), MHz, 363 K) ? 7.57 (s, 1H), 7.30 (t, 1H, J = 7.8 Hz), 7.13 (d, 1H, J = 7.8 Hz), vol.7, p.100

. Mhz,

, HRMS (m/z) calcd. for C 25 H 33 ClN 3 O 5 [M + H] + 490.2109, found 490.2106; IR (neat, cm ?1 ) ? max 3470, vol.3352, p.694, 1167.

, Eagle medium (DMEM) supplemented with 10% dialyzed fetal calf serum (dFCS) and antibiotics. Cells were transiently transfected by electroporation with plasmids encoding HA-tagged 5-HT 4 R (100 ng/10 6 cells), then seeded in 96-well plates (16,000 cells/well). Twenty-four hr after transfection, cells were exposed to the indicated concentrations of 5-HT 4 R ligands in the presence of 0.1 mM of the phosphodiesterase inhibitor RO-20-1724, GF/C filters (Alpha Biotech) pre-of cAMP Production COS-7 cells were grown in Dulbecco's modified

. Bsa), After 10 min, cells were then lysed by the addition of the same volume of Triton-X100 (0.1%). Quantification of cAMP production was performed by HTRF ® using the cAMP Dynamic kit

, USA) according to the protocol provided by the manufacturer. Briefly, the donecopride fumarate solution (20 mM in DMSO) was diluted in Prisma HT buffer (pH 7.4; pION) to 100 µM; 200 µL this solution (n = 6) was added to the donor plate (P/N 110,243). Five microliters BBB-1 Lipid (P/N 110,672) was used to coat the membrane filter of the acceptor plate (P/N 110,243). Two hundred microliters Brain Sink Buffer (P/N 110,674) was added to each well of the acceptor plate. The PAMPA sandwich was assembled and allowed to incubate at room temperature for 4 h without stirring. The sandwich was then separated, and the UV-visible spectra were measured for both the donor and receiver wells with the microplate reader, Parallel Artificial Membrane Permeability Assay The parallel artificial membrane permeability assay (PAMPA) blood-brain barrier (BBB) experiments were conducted using the Pampa Explorer Kit

, A reversed phase column C18 (Zorbax1 Eclipse Plus RRHD, 1.8 µm, 2.1 × 50 mm, Agilent) has been used for all components. A binary pump was used and the mobile phase was always composed of a mixture of (A) water (0.1% formic acid) and (B) acetonitrile (0.1% formic acid). Solutions of compound 19 and 20 were prepared in a PBS buffer, The HPLC analyses were performed using an Agilent pump 1290, an autosampler 1290 and a diode array UV detector 1260 (Agilent technologies

, Adult male NMRI mice (3 months old, weighing 35-40 g) from Janvier labs (Le Genest-Saint-Isle, France) were used to perform experiments. Mice were housed by ten in standard polycarbonate cages in standard controlled conditions (22 ± 2 ? C, 55 ± 10% humidity) with a reversed Molecules 2019, 24, 2786 the cycle and were in agreement with the European Directives and French law on animal experimentation, In Vivo Biological Studies Animals

, Behavioral and neurological changes induced by graded doses (1, 10, 100 mg/kg) of the tested derivatives were evaluated in mice, in groups of four, by a standardized observation technique at different times (30 min, 3 and 24 h) after intraperitoneal administration, CNS-activity and acute toxicity test, vol.25

, The approximate DL 50 of the compounds were also calculated through the quantification of mortality after 24 h. Amphetamine (2 mg/kg) and chlorpromazine (10 mg/kg) were used as the stimulant and depressive references, respectively. Locomotor activity. The locomotion of mice was measured using an actimeter (Imetronic ® , Pessac, France) through infrared detection. Eight individual removable polycarbonate cages (21 cm length, 7 cm wide and 12 cm high), Major changes of behavioral data (for example, hypo-or hyperactivity, ataxia, tremors, convulsion, etc.) were noted in comparison to the control group

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, This article is an open access article distributed under the terms and conditions of the Creative Commons Attribution, Sample Availability: Samples of all the synthesized compounds are available from the authors. © 2019 by the authors. Licensee MDPI