10
K.J. Kuder et al. / Bioorganic & Medicinal Chemistry xxx (2017) xxx–xxx
After drying over anhydrous Na2SO4 and evaporation of organic
layer product was further purified using CC (CH2Cl2:MeOH).
Procedure B: To a resulting oil 100 ml of CH2Cl2 was added and
washed with: 0.5% HCl solution. After drying over anhydrous
Na2SO4 and evaporation of organic layer, product was suspended
in 50 ml of 3% HCl solution and washed with diethyl ether. Organic
layer was then alkalized and extracted to CH2Cl2.
the deposition number CCDC 1485473. Copies of the data can be
obtained free of charge on application to CCDC, 12 Union Road,
CambridgeCB2 1EW, UK (Fax: Int code +(1223)336-033; E-mail:
deposit@ccdc.cam.ac.uk).
4.4. Docking studies
Resulting oils were transformed into hydrogen oxalates, using
10% excess of oxalic acid ethanol solution in room temperature,
and then precipitated by addition of ethyl ether.
Detailed synthetic procedure and analytical data for compounds
2–21 could be found in Appendix A: Supplementary Data.
In silico visualization of binding to histamine H3R model
described by Levoin et al., was held using Glide module of
Schrödinger MacroModel 10.5.43–46 All of the tested compounds
were used in their N-protonated form, and generated using
Schrödinger suite, despite compound 18 where crystallographic
structure was used for docking. For the tested compounds energet-
ically optimal conformers were found using ConfGen (MMFFs
forcefield, PRCG, convergence threshold = 0.05, 20 steps per rotat-
able bond, max. ring conformations = 16). For all of the structures
5 energetically best conformations were used for docking.
Receptor grid was generated by GlideGrid module and validated
with previously described BP.294 (Pitolisant). Docking studies
were performed via GlideDock module (OPLS 2001 forcefield, sol-
vent = water, standard precision, post-docking minimization).
Results were interpreted by the means of Docking Score functions.
Receptor-ligand interactions were visualized using PyMOLver
1.4.1.47
4.1.2.1. 1-(4-(4-(tert-Pentyl)phenoxy)butyl)piperidine hydrogen oxa-
late (2). Synthesis from piperidine (0.85 g, 10 mmol), and com-
pound 1a (1.5 g, 5 mmol) in ethanol (105 ml) in the presence of
K2CO3 (2.07 g, 15 mmol) and catalytic amount KI in water
(20 ml). Refluxed for 20 h. Purified by Procedure A, CC eluent:
CH2Cl2:MeOH (9:1). Obtained 0.75 g of oil. Yield 35%. Raw product
was transformed into oxalic acid salt yielding 0.63 g of final
compound. Mp: 133–135 °C. Rf = 0.28. 1H NMR (DMSO-d6) d:
7.22–7.17 (d, J = 8.7 Hz, 2H, Ph-3,5-H), 6.85–6.80(d, J = 8.7 Hz, 2H,
Ph-2,6-H), 3.95–3.92 (t, J = 6.2 Hz, 2H, CH2-O), 3.03–2.98(m, 6H,
Pip-2,6-CH2 + N-CH2), 1.80–1.60 (m, 8H, 3ꢁ CH2 + t-p-CH2), 1.59–
1.51(m, 4H, 2ꢁ CH2), 1,18 (s, 6H, 2ꢁ CH3), 0.61–0.56(t, J = 7.3 Hz,
3H, CH2-CH3). 13C NMR (DMSO-d6)TM: 165.11, 156.61, 141.27,
127.13, 114.35, 67.14, 56.11, 52.43, 37.36, 36.72, 28.92, 26.52,
4.5. Pharmacology
23.00, 21.98, 20.78, 9.51;UV–VIS k [nm] (lg
e
): 216 (3.89), 223
4.5.1. General remarks
(3.89), 276 (3.78), 282 (3.71). IR (cmꢀ1): 3429.78 (N(CH2–)4),
3022.87 (aromatic CH@), 2961.16 (het. CH2–), 2875.34, 2679.60,
2537.86, 2365.26, 1719.23, 1700.91, 1608.34, 1579.41, 1491.67,
1475.28, 1384.64, 1362.46, 1294.97 (O–CH aromatic), 1186.01,
1115.62, 1034.62, 993.16, 953.63, 831.17, 779.10, 704.85 (alif.
CH2–), 662.43, 521.65. LC–MS: purity 100% tR = 5.90, (ESI) m/z [M
+H]+ 304.24. Anal. Calcd for C20H33NO ꢁ C2H2O4: C, 67.14, N, 3.56,
H, 8.96%. Found: C, 67.22, N, 3.61, H, 9.25.
Competition binding data were analyzed by the GraphPad-
PrismTM (200, version 3.02, San Diego, CA, USA) software, using
non-linear least squares fit. Affinity values (Ki) were expressed as
mean from at least two experiments in triplicates with SEM. Ki val-
ues were calculated from IC50 values according to Cheng-Prusoff
equation.48
4.5.2. [125I]Iodoproxyfan hH3R displacement assay
The displacement binding assay was carried out as described by
Ligneau et al.23 In brief, membrane preparations of CHO-K1 cells
expressing the recombinant hH3R gene were homogenized, incu-
bated for 90 min at 25 °C and shook at 250 rpm with [125I]
Iodoproxyfan (16–50 pM) and different concentrations of the test
compound. Non-specific binding was determined in the presence
of imetit (1 mM). In saturation binding experiments Bmax was found
to be 0.6 pmol/mg and the Kd value was 0.044 pM. The bound radi-
oligand was separated from free radioligand by filtration through
GF/B filters (pre-treated with 0.3% (m/v) polyethyleneimine using
an Inotech cell harvester Dottikin, Switzerland). Unbound radioli-
gand was removed by four washing steps with 5 ml/well of binding
buffer.
4.2. Lipophilicity
Lipophilicity, by means of RM0 values, was estimated using RP-
TLC planar method. Methanol:acetic acid:water (with constant,
10% acid concentration) solvent mixture was used as a mobile
phase. Organic solvent concentration varied from 85% to 55%, by
5% each step. Mobile phase composition as well as concentrations
were chosen experimentally. For each concentration, 10 ml of
compounds methanol solution in 1 mg/ml concentration was used.
Planar chromatographic chambers (Chromdes) were saturated
with proper mixture for 45 min. followed by 15 min. saturation
together with the plate. Glass plates (Merck RP-18, FP254s) were
then evaluated on the distance of 90 mm, dried and spots
were visualized using iod fumes. For each of the compounds, on
the base of Rf values, RM values were determined. RM0 values were
read from RM/methanol concentration charts, after extrapolation to
zero methanol concentration using in-house script.
a
4.5.3. [3H]N -Methylhistamine hH3R displacement assay
The displacement binding assay was carried out as described by
Kottke et al.24 Frozen crude membrane preparations of HEK-293
cells stably expressing the recombinant hH3R in full length were
thawed, homogenized, incubated for 90 min at 25 °C and shook
a
4.3. X-ray structure analysis
at 250 rpm with [3H]N -methylhistamine (2 nM) and different
concentrations of the test compounds (seven appropriate concen-
trations between 0.01 nM and 10 mM were used) in a final assay
volume of 200 ml per well. Non-specific binding was determined
in the presence of Pitolisant (10 mM). In saturation binding experi-
ments Bmax was found to be 0.89 pmol/mg and the Kd value of [3H]
Crystal data for 18: C24 H42 N O, C2 H2 O4, M = 449.61, triclinic,
space group P1, a = 8.4543(2) Å, b = 11.1330(3) Å, c = 14.7601
(11) Å, h = 89.363(1) (°), Ò = 84.8700(9) (°), Ó = 73.511(6) (°),
V = 1326.63(12) Ǻ3,
Z = 2,
Dx = 1.126 g cmꢀ3
,
T = 296 K,
m = 0.612 mmꢀ1, k = 0.71073 Ǻ, data/parameters = 3703/559; final
R1 = 0.06.
Crystallographic data (excluding structural factors) for the
structure reported in this paper has been deposited at
the Cambridge Crystallographic Data Centre and allocated with
N -methylhistamine was 2.98 nM. The bound radioligand was sep-
a
arated from free radioligand by filtration through GF/B filters (pre-
treated with 0.3% (m/v) polyethyleneimine) using an Inotech cell
harvester (Dottikin, Switzerland). Unbound radioligand was
removed by three washing steps with 0.3 ml/well of ice-cold