4.2. Pharmacology
NaOH was added and left overnight in cold. To a resulting white
oil CH2Cl2 was added, mixed and layers were then separated.
Organic layer was dried over sodium sulphate, filtered and
evaporated. Resulting product was used for further reactions after
CC purification.
4.2.1. [3 H]Nα -Methylhistamine hH3 R displacement
assay
Competition
binding
data
were
analyzed
using
GraphPadPrism (V6.01, San Diego, CA, USA) software, using
non-linear least squares/regression fit. Ki values were calculated
from IC50 values according to Cheng-Prusoff equation [29].
Affinity values (Ki) were expressed as mean (with 95%
confidence interval) from at least three experiments, each
performed at least in duplicates.
4.1.1.1. 1-(4-(3-Bromopropoxy)phenyl)ethan-1-one (1a)
Synthesis from 1-(4-hydroxyphenyl)ethan-1-one (13.62 g, 0.1
mol), 1,3-dibromopropane (40.38 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 18.51 g of raw product was
obtained, purified by CC (I) with yield of 72%.
The displacement binding assay was carried out as described
by Kottke et al. with slight modifications [20]. Frozen crude
membrane preparations of HEK-293 cells stably expressing the
recombinant hH3R in full length were thawed, homogenized,
incubated for 90 min at RT (continuous shaking) with [3H]Nα-
methylhistamine (2 nM) and different concentrations of the test
compounds (five to eleven appropriate concentrations between
0.01 nM and 10 µM were used) in a final assay volume of 200 µl
per well. Non-specific binding was determined in the presence of
Pitolisant (10 µM). The bound radioligand was separated from
free radioligand by filtration through GF/B filters (pretreated
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
water. Scintillation cocktail was added and the liquid scintillation
counting was performed with a Perkin Elmer TriluxBeta counter
(Perkin Elmer, Germany).
4.1.1.2. 1-(4-(4-Bromobutoxy)phenyl)ethan-1-one (1b)
Synthesis from 1-(4-hydroxyphenyl)ethan-1-one (13.62 g, 0.1
mol), 1,4-dibromobutane (43.18 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 19.25 g of raw product was
obtained, purified by CC (I) with yield 71%.
4.1.1.3. 1-(4-(3-Bromopropoxy)phenyl)propan-1-one (1c)
Synthesis from 1-(4-hydroxyphenyl)propan-1-one (15.02 g,
0.1 mol), 1,3-dibromopropane (40.38 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 18.43 g of raw product was
obtained, purified by CC (I) with yield 68%.
4.1.1.4. 1-(4-(4-Bromobutoxy)phenyl)propan-1-one (1d)
Synthesis from 1-(4-hydroxyphenyl)propan-1-one (15.02 g,
0.1 mol), 1,4-dibromobutane (43.18 g, 0.2 mol) in sodium
propanolate (0.1 mol, 100 ml). 17.68 g of raw product was
obtained, purified by CC (I) with yield 62%.
4.2.2. In vivo studies
4.2.2.1. Drugs and compounds
4.1.2. General synthetic procedure for compounds 2–25
To a suspension of potassium carbonate and catalytic amount
of potassium iodide in water, a mixture of proper cyclic amine
and compound 1a–1d in ethanol was added. Mixture was then
refluxed for 8–20 h (TLC controlled). After cooling down to
room temperature, reaction mixture was filtrated, evaporated and
purified. To a resulting oil, 100 ml of CH2Cl2 was added and
washed with: 0.5% HCl solution, 0.5% NaOH solution and
water. After drying over anhydrous Na2SO4 and evaporation of
organic layer product was further purified using CC (II).
Heparin was delivered by Polfa Warszawa S.A. (Warsaw,
Poland), while thiopental sodium was from Sandoz International
(France).
4.2.2.2. Animals
The experiments were carried out on male CD-1 mice, body
weight 20 - 22 g (locomotor activity) or Wistar rats, initial body
weight 160
- 180 g (body weight, biochemical assays,
spontaneous activity). Animals were housed in pairs in plastic
cages in constant temperature facilities exposed to light-dark
cycle, water and food available ad libitum. Control and
experimental groups consisted of six to eight animals each. All
experiments were conducted according to the Guidelines of the
Animal Use and Care Committee of the Jagiellonian University.
Detailed synthetic procedure and analytical data for
compounds 2–25 could be found in Supplementary Data.
4.1.2.1. 1-(4-(3-(4-(Pyridin-2-yl)piperazin-1-
yl)propoxy)phenyl)ethan-1-one hydrogen oxalate
(2)
4.2.2.3. The influence of test compounds on locomotor activity
Synthesis from 1-(pyridin-2-yl)piperazine (0.82 g, 5 mmol),
and compound 1a (1.29 g, 5 mmol) in ethanol (50 ml) in the
presence of K2CO3 (1.17 g, 8.5 mmol) and catalytic amount KI in
water (10 ml). Reaction was refluxed for 20 h and then purified.
Obtained 1.44 g of oil with yield of 72%. Raw product was
transformed into oxalic acid salt yielding 0.88 g of final
The locomotor activity was recorded with an Opto M3
multichannel activity monitor (MultiDevice Software v1.3,
Columbus Instruments, USA). Locomotor activity was evaluated
as the distance travelled by the animals while attempting to climb
upward [30]. Mice were inserted into the parameter-counting
cage immediately after the intraperitoneal administration of the
test compound, although the activity measurement started 30
minutes after administration of the test compound over a 20
minutes period.
1
compound. Mp: 182–183 °C. H NMR (300 MHz, DMSO-d6) δ
ppm: 8.13 (dd, J=4.69, 1.17 Hz, 1H, 2Pyr-6H), 7.91 (m, J=8.79
Hz, 2H, Ph-3,5H), 7.57 (ddd, J=8.79, 7.03, 1.76 Hz, 1H, 2Pyr-
4H), 7.03 (m, J=8.79 Hz, 2H, Ph-2,6H), 6.90 (d, J=8.21 Hz, 1H,
2Pyr-3H), 6.70 (dd, J=6.74, 4.98 Hz, 1H, 2Pyr-5H), 4.13 (t,
J=5.86 Hz, 2H, CH2-O), 3.70 (br. s., 4H, Pip-3,5H), 3.04-3.15
(m, 6H, Pip-2,6H + NCH2), 2.50 (s, 3H, CO-CH3), 2.07-2.18 (m,
2H, CH2-CH2-O). 13C NMR (DMSO-d6): 196.74, 164.52, 162.63,
158.69, 148.03, 138.26, 130.93, 130.45, 114.73, 114.30, 108.00,
66.00, 53.73, 51.47, 42.98, 26.87, 24.36. LC–MS: purity 100% tR
= 2.72, (ESI) m/z [M+H]+ 341.33. Anal. Calcd for C20H25N3O2 x
C2H2O4: C, 61.53, N, 9.78, H, 6.34%. Found: C, 60.95, N, 9.82,
H, 6.28.
4.2.2.4. The influence of test compounds on body weight and food
and water intake in rats fed with palatable diet (Western-style
diet)
In order to determine the anorectic activity of compounds, its
effect on calories and water intake in the model of excessive
eating was assessed [31]. Four groups composed of six rats were
fed during two weeks with a diet consisting of milk chocolate
with nuts, cheese, salted peanuts and 7% condensed milk.