Neurotox Res
VION in HDMSe mode, in the range 50–1000 m/z, with
N2 as the drift gas. Two independent scans with different
collision energies were acquired during the run: a colli-
sion energy of 6 eV for low energy (LE) and a ramp of
In Vivo Studies
Animals
2
8–56 eV for high energy (HE). The LE and HE functions
Male CD-1 mice (Charles River, France), fed with standard
laboratory chow (4RF21 LPG, Mucedola Srl, Milan, Italy)
and water ad libitum, were kept at an average temperature of
21 ± 1 °C with 12-h light/dark cycle. Animal experiments
were carried out in accordance with the relevant European
Community and National rules on the protection of animals
used for experimental and other scientific purposes (Directive
2010/63/EU; Decreto-Lei 113/2013) and were approved by
the animal Ethics Committee of the Faculty of Pharmacy,
Universidade de Lisboa and by the Portuguese National
Authority (Directorate-General of Food and Veterinary
Medicine). According to the 3R’s principle, every effort was
made to minimize the number and suffering of animals.
settings were for both a scan time of 0.3 s. Nitrogen was
used as collision-induced dissociation (CID) gas. All data
was examined using an accurate mass screening workflow
within UNIFI informatics platform from Waters
Corporation.
General Procedure for Phenone Brominations Bromine
(
(
0.16 mL, 3.0 mmol) was added to a solution of phenone
3.0 mmol) in dichloromethane (5 mL). After stirring for
2
h, dichloromethane was added (20 mL), and the mixture
washed with sat. Na CO (20 mL), sat. Na S O (20 mL)
2
3
2 2 3
and pumped to dryness, affording the respective bromi-
nated phenone. When necessary, products were purified
by flash chromatography, using n-hexane/AcOEt mixtures
as eluent.
Study Procedure
Single- and repeated-dose regimens were used to study the
in vivo effects of the selected SC—Buph and NEH. Mice were
handled daily prior to each dosing study in order to acclima-
tize them to routine contact. Behaviour tests took place during
the illuminated part of the cycle (between 8:00 and 16:00) in a
room with low noise. For single-dose studies, mice were ran-
domly divided in groups of 3 mice/dose. Dose increments
were based on a double scale increase from 1 up to 128 mg/kg.
Animal behaviour and clinical signs (morbidity and mortality)
were monitored for 3 h and then 24 h (D2) and 7 days after
dosing according to the behaviour Irwin test. For repeated-
dose studies, mice were randomly assigned to groups of 7
mice/dose each. Animals were daily administered with 4 and
16 mg/kg of each tested synthetic cathinone, or vehicle, for 4
consecutive days. On post-dose day 1 (D5) conditioned place
preference (CPP) and marble burying behaviour were
assessed, the latest occurring 2 h after the CPP test. On post-
dose day 3 (D7) the splash test was conducted under reduced
illumination before sacrifice. Mentioned behaviour tests are
described below. Individual body weights were monitored
during both the single- and the repeated-dose studies. Body-
weight changes were calculated with, final body weights re-
corded prior to animal sacrifice, and a gross necropsy was
conducted. Figure 2 depicts a diagram of both single- and
repeated-dose protocol.
General Procedure for Amination of Brominated Phenones
Amine aqueous solution (12 mmol) was added to brominated
products (3 mmol). After stirring overnight at r.t., the mixture
was pumped to dryness, affording the respective aminated
phenone. When necessary, products were purified by flash
chromatography, using CH Cl /MeOH mixtures as eluent.
2
2
HCl was bubbled for 2 min in ethanol solutions of the pure
cathinones. Solvent was removed under reduced pressure and
products were washed with ethyl ether, affording the respec-
tive hydrochlorides.
1
N-Ethylhexedrone hydrochloride: white solid; η = 74%. H
+
2
NMR (400 MHz, DMSO-d6) δ 9.51 (br. s, 1H, NH Et), 9.08,
+
2
(
br. s, 1H, NH Et), 8.10 (d, 2H, J = 7.6 Hz, H Ph), 7.76 (t,
2,6
1
1
1
H, J = 7.6 Hz, H Ph), 7.61 (t, 2H, J = 7.6, H Ph), 5.32 (s,
4 3,5
H, H ), 3.03 (s, 1H, NCH CH ), 2.92 (s, 1H, NCH CH ),
2
2
3
2
3
.85–2.00 (m, 2H, H ), 1.02–1.29 (comp., 7H, H , H +
3
5
4
1
3
NCH CH ), 0.74 (t, 3H, J = 7.2 Hz, 3H ); C NMR
2
3
6
(
(
(
(
[
100 MHz, DMSO-d6) δ 196.4 (C ), 135.4 (C Ph), 134.4
1 4
C Ph), 129.7 (C3,5Ph), 129.2 (C2,6Ph), 61.0 (C ), 41.7
1
2
NCH CH ), 29.8 (C3), 26.1 (C ), 22.3 (C ), 14.0
2
3
4
5
NCH CH ), 11.6 (C ). ESI-HRMS (+) m/z: 220.16989
2
3
6
+
M+H] ; exact mass calcd. for C H N O : 220.17014.
Buphedrone hydrochloride: white solid; η = 80%. H NMR
2
0 34 7 6
1
(
300 MHz, DMSO-d6) δ 9.82 (br. s, 1H, NH ), 9.34 (br. s, 1H,
2
+
NH ), 8.04 (d, 2H, J = 7.6 Hz, H2,6Ph), 7.74 (t, 1H, J =
2
7
.6 Hz, H Ph), 7.60 (t, 2H, J = 7.6 Hz, H Ph), 5.27 (s, 1H,
Primary Observation (Irwin) Test
4
3,5
H ), 2.56 (s, 3H, NCH ), 2.06–2.14 (m, 1H, H ), 1.85–2.04
2
3
3
1
3
(
m, 1H, H ), 0.76 (t, 3H, J = 7.0 Hz, H ); C NMR (75 MHz,
Assessment of mice behaviour was done simultaneously by
two observers, blind to the treatment, using the procedure
previously described (Roux et al. 2005). Briefly, for each
studied dose, three mice were placed in a transparent container
(26.8 × 21.5 × 14.1 cm) 24 h before administration in order to
3
4
DMSO-d6) δ 196.5 (C ), 135.2 (C Ph), 134.4 (C Ph), 129.6
(
8
1
4
1
C Ph), 129.2 (C Ph), 63.2 (C ), 31.7 (NCH ), 23.1 (C ),
3,5 2,6 2 3 3
.6 (C ). ESI-HRMS (+) m/z: 178.12242 [M+H] ; exact mass
4
+
calcd. for C H N O : 178.12319.
2
0 34 7 6