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M. Dawidowski et al. / European Journal of Medicinal Chemistry 48 (2012) 347e353
b), 40.6 (C-
a), 50.8 (C-6), 56.0 (C-9a), 69.6 (C-4), 126.7, 128.3, 128.6,
4.1.3. Synthesis of (9aS)- and (9aR)-perhydropyrido[1,2-a]
129.0, 129.3, 133.3, 138.4 (C-Ar), 171.0 (C-3), 172.5 (C-1); Hydro-
chloride: white powder; mp 196e199 ꢁC; Elemental analysis:
calculated for C22H24N2O2 ꢄ HCl: C 68.65%, H 6.55%, N 7.28%; found:
C 69.09%, H 6.31%, N 7.40%.
pyrazine-1,3-dione (9aS)-9 and (9aR)-9
General method: To a stirred solution of appropriate amidoester
(2S)-8 or (2R)-8 (1 equivalent) in absolute ethanol (10 mL per
1 mmol of amidoester), sodium hydroxide (1 equivalent) pellet was
added at room temperature. After dissolution of the hydroxide, the
mixture was quenched with saturated aqueous solution of
ammonium chloride (100 mL). The resulting cloudy solution was
extracted with methylene chloride (2 ꢄ 30 mL) and subsequently
4.1.1.6. (4R,9aR)-2-(2-Phenylethyl)-4-phenyl-perhydropyrido[1,2-a]
pyrazine-1,3-dione
(4R,9aR)-7b. From
(4R,9aR)-6
(0.50 g,
2.05 mmol) and 2-bromoethylobenzene (2.0 mL, 14.34 mmol); FC
(gradient: petroleum ether/ethyl acetate 9:1e7:1): yield 0.53 g
(75%).
with
a mixture of dichloromethane/methanol 70:30 (v/v)
(2 ꢄ 30 mL). The combined organic extracts were dried with
magnesium sulphate, filtered, and the solvent was evaporated in
vacuo. The residue was purified by FC (petroleum ether/ethyl
acetate 1:3).
Colourless crystals; mp 121e122 ꢁC; [
a
]
¼ þ96.3 (c 1, CHCl3);
D
Hydrochloride: white powder; mp 201e203 ꢁC; Elemental anal-
ysis: calculated for C22H24N2O2 ꢄ HCl: C 68.65%, H 6.55%, N 7.28%;
found: C 68.39%, H 6.50%, N 7.03%.
4.1.3.1. (9aS)-Perhydropyrido[1,2-a]pyrazine-1,3-dione (9aS)-9. From
(2S)-8 (1.10 g, 5.50 mmol); yield 0.75 g (81%).
4.1.1.7. Ethyl
pyrazin-2-yl)-acetate
2.13 mmol) and ethyl bromoacetate (1.7 mL, 14.91 mmol); FC
(4S,9aS)-
a
-(1,3-dioxo-4-phenyl-perhydropyrido[1,2-a]
(4S,9aS)-7c. From
(4S,9aS)-6 (0.52 g,
White powder; mp 96e98 ꢁC; [
a
]D ¼ þ14.6 (c 1, CHCl3); IR: 837,
1119, 1284, 1331, 1732, 2858, 2939, 3221; TLC (ethyl acetate):
(gradient: petroleum ether/ethyl acetate 9:1e4:1): yield 0.64 g (91%).
Rf ¼ 0.62; 1H NMR (500 MHz, CDCl3):
d 1.38 (m, 1H, H-8), 1.60 (m,
White powder; mp 82e84 ꢁC; [
a]
¼ ꢀ68.9 (c 1.1, CHCl3); IR:
2H, H-7, H-9), 1.69 (m, 1H, H0-7), 1.83 (m, 1H, H0-8), 2.23 (m, 2H, H0-
9, H-6), 2.82 (dd, 3J1 ¼10.0, 3J2 ¼ 2.5, 1H, H-9a), 2.91 (dt, 2J ¼ 11.5,
1H, H0-6), 3.14 (d, 2J ¼ 17.0, 1H, H-4), 3.57 (d, 2J ¼ 17.0, 1H, H0-4), 8.33
D
707, 746, 1210, 1333, 1377, 1405, 1451, 1690, 1738, 2821, 2859, 2928,
2939; TLC (petroleum ether/ethyl acetate 3:1): Rf ¼ 0.45; 1H NMR
(CDCl3, 500 MHz):
d
1.30 (t, 3J ¼ 7.0, 3H, OCH2CH3), 1.47 (m, 2H, H-8,
(bs, 1H, NH); 13C NMR (CDCl3, 125 MHz):
d 23.2 (C-8), 25.0 (C-7),
H0-8), 1.66 (m, 2H, H0-7, H-7), 1.86 (m, 1H, H-9), 2.02 (m, 1H, H0-9),
26.5 (C-9), 54.6 (C-6), 58.3 (C-9a), 62.8 (C-4), 170.0 (C-3), 172.3 (C-
1); Elemental analysis: calculated for C8H12N2O2: C 57.13%, H 7.19%,
N 16.66%; found: C 56.92%, H 7.33%, N 16.34%
2.63 (m, 1H, H-6), 2.89 (m, 1H, H0-6), 3.50 (t, 3J ¼ 5.0, 1H, H-9a), 4.23
(m, 2H, OCH2CH3), 4.59 (d, 2J ¼ 16.5, 1H, H-
a
), 4.65 (d, 2J ¼ 16.5, 1H,
H0- ), 4.72 (s, 1H, H-4), 7.32e7.44 (m, 5H, H-Ar); 13C NMR (CDCl3,
a
125 MHz):
d
14.4 (OCH2CH3), 21.9 (C-8), 25.3 (C-7), 26.5 (C-9), 40.5
4.1.3.2. (9aR)-Perhydropyrido[1,2-a]pyrazine-1,3-dione (9aR)-9. From
(2R)-8 (1.23 g, 6.15 mmol); yield 0.76 g (74%).
(C- ), 50.9 (C-6), 55.6 (OCH2CH3), 61.9 (C-9a), 69.8 (C-4), 128.6,
a
128.8, 129.0, 133.2 (C-Ar), 168.1 (COOCH2CH3), 171.0 (C-3), 172.3 (C-
1); Elemental analysis: calculated for C18H22N2O4: C 65.44%, H
6.71%, N 8.48%; found: C 65.21%, H 6.89%, N 8.55%.
White powder; mp 102e104 ꢁC; [
a
]D ¼ ꢀ17.2 (c 1, CHCl3);
Elemental analysis: calculated for C8H12N2O2: C 57.13%, H 7.19%, N
16.66%; found: C 57.02%, H 7.40%, N 16.82%.
4.1.2. Synthesis of (2S)- and (2R)-
a-(2-carbamoylpiperidinyl)-
4.2. Pharmacology
acetic acid ethyl ester (2S)-8 and (2R)-8
General method: To a stirred, cooled (0 ꢁC, ice-salt bath) mixture
of (S)- or (R)-2-piperidinecarboxylic acid amide (S)-4 or (R)-4
(1.53 g, 11.94 mmol), N,N-diisopropyl-N-ethylamine (2.08 mL,
11.94 mmol) and acetonitrile (70 mL), ethyl bromoacetate (1.1 mL,
9.95 mmol) was added over 10 min. The mixture was then allowed
to warm to room temperature, stirred (20 h) and the solvent was
evaporated in vacuo. The residue was purified by FC (gradient:
petroleum ether/ethyl acetate 1:1 to ethyl acetate, then ethyl
acetate/methanol 9.5:0.5).
The obtained compounds have been submitted for in vivo
evaluation in the Anticonvulsant Screening Program (ASP) of
National Institute of Neurological Disorders and Stroke (NINDS),
Bethesda, USA [6]. The experiments were performed in male albino
Carworth Farms No. 1 mice (weighing 18e25 g) or albino Sprague-
Dawley rats (weighing 100e150 g). The animals had free access to
feed and water except during actual testing period. Housing,
handling and feeding were all in accordance with recommenda-
tions contained in the ‘Guide for the Care and Use of Laboratory
Animals’. The test compounds were dissolved or suspended in 0.5%
(v/v) aqueous solution of methylcellulose.
4.1.2.1. (2S)-
a
-(2-Carbamoylpiperidinyl)-acetic acid ethyl ester (2S)-
8. Yield 1.45 g (73%). White powder; mp 169e170 ꢁC; [
a]D ¼ ꢀ70.2
(c 1, CHCl3); IR: 1184, 1450, 1647, 1728, 2866, 2959, 2194, 3414; TLC
4.2.1. The maximal electroshock seizure test (MES)
(ethyl acetate): Rf ¼ 0.30; 1H NMR (500 MHz, CDCl3):
d
1.27 (t,
In the MES test, an electrical stimulus of 0.2 s in duration (50 mA
in mice and 150 mA in rat at 60 Hz) was delivered via corneal
electrodes primed with an electrolyte solution containing an
anaesthetic agent (0.5% butacaine hemisulfate in 0.9% sodium
chloride). Mice were tested at 30 min and 4 h following intraperi-
toneal administration of 30, 100 and 300 mg/kg of test compound
while rats were tested at time intervals between 0.25 and 4 h
following a standard oral dose of 30 mg/kg. Abolition of the hin-
dlimb tonic extensor component indicated the test compound’s
ability to inhibit MES-induced seizure spread [6].
3J ¼ 7.0, 3H, CH2CH3), 1.32 (m, 1H, H-4), 1.51e1.67 (m, 3H, H-3, H-5,
H0-5), 1.75 (m, 1H, H0-4), 1.99 (m, 1H, H0-3), 2.26 (td, 2J ¼ 11.5,
3J1 ¼4.5, 3J2 ¼ 3.5, 1H, H-6), 2.95 (dd, 3J1 ¼10.5, 3J2 ¼ 3.5, 1H, H-2),
2.99 (m, 2J ¼ 11.5, 3J ¼ 4.0, 4J ¼ 1.0, 1H, H0-6), 3.08 (d, 2J ¼ 17.0, 1H, H-
a
), 3.38 (d, 2J ¼ 17.0, 1H, H0-
a
), 4.18 (q, 3J ¼ 7.0, 2H, CH2CH3), 5.34 (bs,
1H, CONH), 6.97 (bs, 1H, CONH’); 13C NMR (CDCl3, 125 MHz):
d 14.4
(OCH2CH3), 23.2 (C-5), 24.9 (C-4), 30.1 (C-3), 52.9 (C-6), 58.2 (C-2),
61.0 (OCH2CH3), 66.6 (C-a), 171.3 (CONH), 176.9 (COOCH2CH3);
HRMS (ESI) calculated for C10H18N2O3Na: 237.1215 (M þ Na)þ found
237.1237.
4.2.2. The subcutaneous Metrazol seizure test (scMET)
4.1.2.2. (2R)-
a
-(2-Carbamoylpiperidinyl)-acetic acid ethyl ester (2R)-
This test utilised a dose of Metrazol (pentylenetetrazol, 85 mg/
kg in mice and 70 mg/kg in rats). This produced clonic seizures
lasting for a period of at least 5 s in 97% (CD97) of animals tested. At
the anticipated time of testing the convulsant was administered
8. Yield 1.23 g (62%). White powder; mp 168e170 ꢁC; [
a]
¼ þ73.4
D
(c 1, CHCl3); HRMS (ESI) calculated for C10H18N2O3Na: 237.1215
(M þ Na)þ found 237.1181.