Psychopharmacology
acetic acid (step 2). The mixture was neutralized with saturat-
ed aqueous sodium hydrogen carbonate, separated and re-
duced under vacuum. The crude product was recovered in
quantitative yield and used without purification in the next
step. Then, α-bromoketone was dissolved in dichloromethane
and pyrrolidine was added (step 3). The reaction mixture was
stirred for 16 h at room temperature and diluted with CH2Cl2,
washed with water, dried over sodium sulfate, filtered and
concentrated at reduced pressure. The crude product was dis-
solved in 2-propanol and, while stirring, hydrogen chloride
solution in ethyl ether was added to this solution, producing
a white precipitate. The solid was rinsed with cold 2-propanol
and dried to give the hydrochloride salt of MDPV as a white
solid. The identification of MDPV hydrochloride was
assessed by 1H NMR (250 MHz, DMSO) and mass spectrom-
etry yielding the following results: δ 7.74 (d, 1H, J = 1.6 Hz);
7.55 (dd, 1H, J = 1.6 Hz, J = 8.2 Hz); 7.16 (d, 1H, J = 8.0 Hz);
6.2 (s, 2H); 5.35 (dd, 1H, J = 6.8, J = 12.2 Hz); 3.59 (m, 1H);
3.42 (m, 1H); 3.17 (m, 1H); 2.97 (m, 1H); 1.85–2.07 (m, 6H);
1.11 (m, 2H); 0.79 (t, 3H, J = 7.20 Hz); MS (ESI) m/z 276.2
[M + 1]+.
demonstrate the onset of additive effects if both drugs are
administered in combination. Drugs were dissolved in H2O
and administered in volumes of 1 mL/kg of body weight by
gavage. Thereafter, locomotor activity (non-catheterized rats;
N = 11 rats/group) and pharmacokinetics (catheterized rats;
N = 5 rats/group) were studied in two distinct series of rats.
Locomotor activity measurement
Locomotor activity was assessed in an enclosed Plexiglas
chamber open at the top, divided into four equal-sized areas
(45 × 50 × 50 cm) under low illumination (10 lx). Twenty-four
hours prior to the experiment, rats received one habituation
session during 15 min. At the experimentation time, the rat
was placed in each area, and locomotor activity recorded dur-
ing each 10-min interval starting immediately after the drug
administration until 4-h post-administration. The floor of the
chamber was fitted with an infrared floor connected to a min-
iature overhead infrared video connected to a PC that used an
automated video tracking software (ViewPoint®, Videotrack,
Lyon, France) to determine the horizontal locomotor activity
(distance traveled).
4-MMC was prepared in two steps as the hydrochloride
salt. To a solution of 4-methylpropiophenone in CH2Cl2 was
added one drop of hydrobromic acid (48% aqueous solution)
and one drop of bromine. The mixture was stirred at room
temperature until the bromine color was discharged and addi-
tional bromine was introduced dropwise with stirring. The
mixture was stirred for 1 h and then concentrated in vacuo
to reveal an orange oil which solidified on standing (step 1).
The crude product was recrystallized from diethyl ether to
give 4-methyl-2-bromopropiophenone. Then, α-
bromoketone was dissolved in CH3CN and methylamine
(2M) in THF was added. The reaction mixture was stirred
for 1–2 h at room temperature and concentrated at reduced
pressure (step 2). The crude product was dissolved in ethyl
acetate and hydrogen chloride solution in ethyl ether added to
this solution while stirring, producing a white precipitate. The
solid was rinsed with ethyl ether and dried to give the hydro-
chloride salt as a white solid. The identification of 4-MMC
hydrochloride was assessed by 1H NMR (250 MHz, DMSO)
and mass spectrometry yielding the following results: δ 9.35
(br s, 2H); 7.96 (d, 2H, J = 8.0 Hz); 7.48 (d, 2H, J = 8.0 Hz);
5.10 (q, 1H, J = 7.2 Hz); 2.60 (s, 3H); 2.43 (s, 3H); 1.47 (d,
3H, J = 7.2 Hz); MS (ESI) m/z 178.1 [M + 1]+.
Jugular vein catheterization
The day before the pharmacokinetic study, the jugular vein
was catheterized using a 20-cm silastic tubing with external
and internal diameters of 0.94 and 0.51 mm, respectively
(Dow Corning Co., Midland, MI, US). The jugular catheter
was tunneled subcutaneously and fixed at the back of the
neck. Heparinized saline was injected into the catheter to
avoid thrombosis and catheter obstruction. Rats were then
returned to their individual cages for a minimum recovery
period of 24 h, to allow complete anesthesia washout. On
the day of experimentation, rats were placed in horizontal
Plexiglas cylinders (6.5-cm internal diameter, up to 20-cm
adjustable length) (Harvard Apparatus, Inc., Holliston, MA,
US), modified by the addition of several holes at the cephalic
end to avoid CO2 rebreathing. Before the drug administration,
the catheter was exteriorized, purged, and its permeability
checked.
Measurement of the plasma concentrations of MDPV
and 4-MMC
Study design
The plasma concentrations of MDPVand 4-MMC were mea-
sured using an Agilent 1100 liquid chromatography (LC)/
mass selective detector (MSD) G1946D (Agilent, Les Ulis,
France) equipped with a column Nucleodur C18 Pyramid
125 mm × 2 mm; 3 μm (Macherey Nagel, Hoerdt, France).
Three hundred microliters of blood samples were obtained
from the venous catheter at T10, 20, 30, 60, 90, 120, 180,
240, 480, and 1440 min after drug administration. After
Rats were randomized in four groups to receive H2O (control
group), MDPV (3 mg/kg), 4-MMC (30 mg/kg), or MDPV
(3 mg/kg)/4-MMC (30 mg/kg) combination. We chose the
doses of MDPV and 4-MMC able to induce significant but
not peak locomotor effects, as previously assessed (Aarde et
al. 2013; Martínez-Clemente et al. 2013), thus allowing us to