Synthesis and anticonvulsant activity of a class of 2-amino 3-hydroxypropanamide and 2-aminoacetamide derivatives

Article abstract of DOI:10.1016/j.bmc.2005.12.064

Several studies have demonstrated that N-substituted aminoacid derivatives exhibit weak anticonvulsant activities in vivo. In the present study, a series of amides of aminoacids structurally related to aminoacetamide have been synthesised and investigated

Full text of DOI:10.1016/j.bmc.2005.12.064

Bioorganic & Medicinal Chemistry 14 (2006) 3263–3274
Synthesis and anticonvulsant activity of a class of 2-amino
3-hydroxypropanamide and 2-aminoacetamide derivatives
Eleonora Ghidini,a,* Maurizio Delcanale,a Renato De Fanti,a Andrea Rizzi,a
Manuela Mazzuferi,b Donata Rodi,b Michele Simonato,b Milco Lipreri,a Franco Bassani,a
Loredana Battipaglia,a Marco Bergamaschia and Gino Villettia
aR&D Department, Chiesi Farmaceutici S.p.a., Via Palermo 26/A, 43100 Parma, Italy
bDepartment of Clinical and Experimental Medicine, Section of Pharmacology and Neuroscience Center,
Via Fossato di Mortara 17-19, University of Ferrara, 44100 Ferrara, Italy
Received 12 January 2005; accepted 16 December 2005
Available online 3 February 2006
Abstract—Several studies have demonstrated that N-substituted aminoacid derivatives exhibit weak anticonvulsant activities in vivo.
In the present study, a series of amides of aminoacids structurally related to aminoacetamide have been synthesised and investigated
for anticonvulsant activity. Among the molecules investigated, those containing a bicyclic (tetralinyl, indanyl) group linked to the
aminoacetamide chain (40, 47 and 59) were among the most active as anticonvulsants (ED50 > 10, <100 mg/kg after oral adminis-
tration) against tonic seizures in the mouse maximal electroshock, bicuculline and picrotoxin tests at doses devoid of neurotoxic
activity. Altogether, these results suggest the described compounds as a class of orally available anticonvulsants. The ability of these
compounds to partially block veratridine-induced aspartate efflux from rat cortical synaptosomes suggests that their anticonvulsant
activity may be only partly the consequence of an interaction with neuronal voltage-dependent sodium channels. Some of the most
potent compounds appear worthy of a further investigation aimed at assessing their anticonvulsant activity in other models and at
elucidating the underlying mechanism of action.
ꢀ 2006 Elsevier Ltd. All rights reserved.
1. Introduction
explore the potential of the a-aminoacetamide deriva-
tives as anticonvulsant agents.
The conditions grouped under the term epilepsy consti-
tute an area of continuous medical need. Various litera-
ture reports estimate that approximately 25–30% of all
epileptic patients do not respond to current therapeutic
treatments,1 and therefore new, less toxic and more
effective drugs are required.2 Even some of the most
recently introduced drugs retain significant adverse ef-
fects.3 Therefore, first-generation drugs, such as valproic
acid and carbamazepine, are still widely used.
Milacemide (n-pentylaminoacetamide, 1, Fig. 1) was
reported some years ago4 as a promising antiepileptic
agent, but its full development was not pursued due to
marginal effects in clinical trials. However, further stud-
ies5,6 led to the development of safinamide (NW 1015, 2,
Fig. 1) currently in Phase I trials.3
In this work, the anticonvulsant activity of a new series
of compounds, most of which contain either the 2-ami-
no-3-hydroxypropanamide (serinamide) or 2-acetamide
(glycinamide) group, was evaluated.
The strategy of investigating compounds structurally
related to first generation of antiepileptic drugs seems
appropriate to better understand the mechanism dis-
played by these old drugs and possibly improve them.
The work reported in the present paper aimed to further
2. Chemistry
The synthesis of most compounds (Tables 1–3) was
accomplished by reductive amination of an aldehyde
or ketone with the appropriate a-aminoacid ester using
a literature protocol7 (Scheme 1). The ester derivative
was purified by simple trituration with a solvent and
Keywords: Milacemide; a-Aminoamides; Anticonvulsant activity;
MES.
*
Corresponding author. E-mail: E.Ghidini@chiesigroup.com
0968-0896/$ - see front matter ꢀ 2006 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2005.12.064

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E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3. Pharmacology
O
H
N
H3C
NH2
CH3
Compounds were tested and selected for an extensive
pharmacological evaluation on the basis of a widely
accepted screening programme for the identification of
anticonvulsant activity.8
1
NH2
N
H
Initial screening was carried out by the maximal electro-
shock test (MES) in mice (see Tables 1–3 for results).
The horizontal screen test was employed to quantify
motor impairment induced by the most promising com-
pounds (see Table 3).
O
F
O
2
NH2
N
A subset of three compounds that were found to have a
significant potency and a good protective index were
then evaluated in a series of secondary tests. These tests,
whose methods have been described in the literature,9
included the following: clonic convulsions induced by
subcutaneous injection of pentylentetrazole (scPTZ)
and tonic convulsions induced by subcutaneous injec-
tion of bicuculline (scBIC) and picrotoxin (scPIC).
H
O
64
Figure 1.
dissolved in an ammonia–methanol solution to convert
the ester into amide. The reductive amination of alde-
hydes occurred with moderate to good yields using Pd
on carbon in methanol as a catalyst. With ketones
(Scheme 2) it was necessary to use sodium cyanoborohy-
dride in a EtOH/MeOH mixture as a reducing agent to
obtain good yields. Previously unreported aldehydes or
ketones were synthesised by literature methods.
The new compounds appeared to antagonise specifically
the tonic seizures induced by both supramaximal electri-
cal and chemical stimuli. For these reasons, the bio-
chemical tests performed to elucidate their mechanism/
s of action included in vitro N-methyl-D-aspartate
(NMDA),
a-amino-3-hydroxy-5-methyl-4-isoxazole
Table 1. Structural and physical data and MES activity (ED50, mg/kg) for compounds 3–24 (Scheme 1)
R1
H
N
R
N
H
H
O
Configurationa
[a]D
ED50
b
Compound
R
R1
Milacemide
Pentyl
Heptyl
H
—
R
—
—
698 (402–1212)
206 (109–389)
421 (303–587)
>300
3
4
Phenyl
Benzyl
Methyl
Heptyl
Heptyl
rac
rac
rac
R
—
—
5
6
Heptyl
Pentyl
2-Thienyl
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
—
ꢀ500
7
—
>1000
>1000
>500
8
Heptyl
iso-Propyl
R
+3.75c
+17.0d
+12.6d
À0.3d
+11.5d
—
9
R
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
Cyclohexylmethyl
1-Methylhexyl
2-Phenylethyl
Octyl
R
427 (337–542)
101 (62–164)
65 (46–93)
138 (71–268)
161 (64–402)
>500
R
R
rac
rac
rac
rac
R (R,S)
R (S)
R
Nonyl
—
—
1-Ethylheptyl
1-Methylnonyl
1-Methyl-3-phenylpropyl
1-Methyl-2-phenylethyl
3-Phenylbutyl
1-Methylbenzyl
1,1-Diphenylmethyl
4-Phenylbutyl
2-Phenoxyethyl
3-Phenylpropyl
—
—
295 (203–430)
>500
À28.8d
ꢀ300
—
—
172 (115–257)
144 (82–254)
41 (33–50)
159 (69–364)
217 (156–302)
47(29–76)
R
R
+12.1d
R
—
—
R
R
—
a The configuration between brackets is referred to the chiral centre in the R chain; when not indicated, it is a racemic mixture.
b Values in brackets are 95% confidence limits.
c 0, 4% in CHCl3/CH3OH.
d 1% in methanol.

E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3265
Table 2. Structural and physical data and MES activity (ED50, mg/kg) for compounds 25–38 (Scheme 1)
R1
R2
N
R3
O
X
a
Compound
R1
R2
R3
X
Configuration
ED50
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OCOCH3
CH2OH
C6H5
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NH2
NCH3
H
H
H
H
H
H
H
H
H
H
H
H
H
CH3
H
H
R
47 (29–76)
51 (33–80)
>300
H
S
p-OCH3
o-OCH3
m-OCH3
p-CH3
p-F
RS
RS
RS
RS
RS
RS
R
ꢀ300
>300
>300
>300
>300
p-Cl
p-CF3
H
291 (159–532)
79 (48–131)
>100
R
N(CH3)2
OH
NH2
H
RS
R
H
>300
ꢀ300
H
S
NH2
NH2
H
RS
RS
71 (45–113)
>100
H
a Values in brackets are 95% confidence limits.
propionate (AMPA), kainic acid (KA), c-aminobutyric
acid (GABA) and voltage-dependent sodium channel
receptor binding. Furthermore, effects on veratridine-in-
duced [3H]D-aspartate release in rat synaptosomes were
investigated, in order to analyse the compounds ability
to functionally block neuronal voltage-dependent sodi-
um channels.
The good activity of the R-3-phenylpropylderivative
(24) prompted us to investigate the effect of the amino-
acid configuration and of the substitution on the aro-
matic ring (Table 2). Whereas the S-enantiomer 25
showed substantially the same activity of 24, no positive
effect was obtained by substituting the aromatic ring of
24 with a series of either electron-donating or electron-
withdrawing groups in the various positions (26–32).
Methylation of the amide (33) and of the amino nitrogen
(37), respectively, maintained the activity, although a
second methyl group (34) on the former was
detrimental.
On the basis of the collected data, several tested com-
pounds have been investigated not only in epilepsy,10
but also in hypoxic-ischaemic brain injury, inflammato-
ry and neuropathic pain and degenerative disorders.11,12
The best results were obtained by constraining the pro-
pyl chain of 24 into a ring. In the case of the 2-tetralinyl
derivatives (Table 3, 39–42), all the four stereoisomers
were prepared and tested. The most active compounds
are those with opposite configuration at the two centres
(40 and 42) and this finding holds also for the N-meth-
ylamino derivative 47 versus 46. The activity was main-
tained when the CH2OH substituent was reduced to
CH3 (43, 44), or in the case of compounds with a naph-
thylmethyl (45) or a 1-tetralinyl (48) ring. The best series
appeared to be that of the 2-indanyl derivatives. Here
again it was confirmed that the configuration of the ami-
noacid carbon has no relevance (compare 49 with 50, 51
with 52 and 53 with 54) for the anticonvulsant activity.
It was also confirmed that reduction of the CH2OH sub-
stituent to CH3 (55–58) and even its deletion (59, 60)
was well tolerated, compounds 53 and 59 being the most
active in the whole series. A similar conclusion holds for
the N-methylation either at the amino (51, 52, 57 and
58) or at the amide (53, 54 and 60) nitrogen. Again
the introduction of a bulky substituent, such as a cyclo-
alkyl (61, 62) or phenyl (63), was detrimental. Shifting
the chain onto the 1-position of indane to give the Teva
compound, 2-[(1-indanyl)-amino]acetamide (64), allows
to maintain a good activity.14 The overall result is that
4. Discussion
With the aim to identify compounds more potent and
with a wider range of anticonvulsant spectra than
milacemide, different substitution first introduced in po-
sition 2 on the basis that glycinamide analogues bearing
substitutions in the carbon at that position have been
shown13 to be resistant to the monoamino oxidase
activity.
In a first group of compounds, the effect of alkyl or aryl
substitution at C2 of milacemide was assessed (Table 1).
The four compounds 3–6 showed only a weak activity,
so that further investigation of this kind of compounds
was not pursued.
Instead the introduction of a CH2OH substituent in-
creased the activity when the alkyl group linked to the
amino nitrogen reached a sufficient length (13, 14 and
16). A further improvement was obtained, up to two-
digit activity, when a phenyl group was tethered to the
amino nitrogen by a short (C1–C2, C1–C3) alkyl chain
(12, 24 vs 22 and 23). Alkyl (17–19) but not phenyl
(21) substitution on the tether chain was deleterious.

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E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
Table 3. Structural and physical data, MES activity (ED50, mg/kg) and neurologic toxicity (TD50, mg/kg) for carbocyclic derivatives 39–63
(Scheme 2)
R1
R
NHR2
N
R3
O
a
b
Compound
R
R1
R2
R3
Tetralinyl
configuration
Aminoacid
configuration
[a]D
ED50
TD50
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
61
62
63
6411
2-Tetralinyl
2-Tetralinyl
2-Tetralinyl
2-Tetralinyl
2-Tetralinyl
2-Tetralinyl
2-Naphthylmethyl
2-Tetralinyl
2-Tetralinyl
1-Tetralinyl
2-Indanyl
CH2OH
CH2OH
CH2OH
CH2OH
CH3
H
H
R
R
À105.1c
+92.0c
À94.2c
+110.0c
—
143 (81–251)
43 (24–74)
ꢀ150
—
H
H
S
R
1300
—
H
H
S
S
H
H
R
S
44 (32–62)
76 (46–118)
100 (62–160)
113 (76–170)
10% at 100e
70% at 100e
85 (49–149)
36 (22–61)
34 (21–55)
40% at 100e
40% at 100e
35 (25–48)
70% at 100e
22 (11–42)
20% at 100e
60% at 100e
80% at 100e
21 (14–31)
70% at 100e
40% at 100e
30% at 100e
10% at 100e
38 (27–55)
1099
—
H
H
rac
rac
—
R
S
CH3
H
H
R
—
—
—
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH2OH
CH3
H
H
rac
R
—
CH3
CH3
H
H
+48.5d
À93.0d
—
—
—
H
S
R
H
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
rac
S
—
H
H
+15.8c
+26.2c
+2.0c
À2.0c
À15.6d
+19.8d
+22.6d
À24.9d
À10.6d
+10.1d
—
1172
>1500
—
2-Indanyl
2-Indanyl
H
H
R
CH3
CH3
H
H
R
2-Indanyl
2-Indanyl
H
S
—
CH3
CH3
H
R
689
—
2-Indanyl
2-Indanyl
H
S
H
S
251
—
2-Indanyl
2-Indanyl
CH3
CH3
H
H
R
CH3
CH3
H
H
R
—
—
2-Indanyl
2-Indanyl
CH3
H
H
S
H
—
—
rac
rac
rac
—
274
—
2-Indanyl
2-Indanyl
H
CH3
H
H
—
—
Cyclopropyl
Cyclopentyl
C6H5
H
—
—
2-Indanyl
2-Indanyl
1-Indanyl
H
H
—
—
À1.4d
H
H
—
—
H
H
H
—
a See Experimental section.
b Values in brackets are 95% confidence limits.
c 1% in methanol.
d % in DMSO.
e At mg/kg.
OH
OH
O
OH
NH2
NH3/MeOH
Pd/C
R
O
O
+
R
R
H2N
CH3
N
CH3
N
H
MeOH
H
H
O
O
O
Scheme 1.
Indanyl (n=1),tetralinyl (n=2)
OH
OH
OH
NH2
NaBH3CN
(
)
NH3/MeOH
CH3
(
)
n
O
n
+
O
O
(
)
H2N
CH3
N
N
n
MeOH/EtOH
H
H
O
O
O
Scheme 2.
a bulky, cyclic, lipophilic group linked to the nitrogen of
the aminoacetamide chain is determinant for activity in
this series, but without too stringent spatial requisites.
A lead optimisation exploration was performed to im-
prove the noncompetitive antagonism of 59
(Ki = 8.8 lM) in the phencyclidine site of NMDA

E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3267
120
100
80
receptor, as in parallel research 59 (CHF 3381) was
found to be a NMDA functional antagonist.12 However,
data (not reported) for the displacement or [3H]TCP
from the phencyclidine site of NMDA receptor showed
clearly that modifications in the structure of 59 did not
improve the potency of the analogues. Some of the com-
pounds which exhibited an excellent activity in the MES
test (ED50 < 40 mg/kg) and a low neurotoxic potential in
the horizontal screen test (TD50 > 250 mg/kg), namely
40, 49 and 59, were subjected to other
in vitro and in vivo tests to better characterise the anti-
convulsant profile of this new series.
Control
Lamotrigine
*
**
*
*
**
**
**
60
40
25 40 41 50 55 59
Compound
Figure 2. Effect of compounds 25, 40, 41, 50, 55 and 59 on veratridine-
induced [3H]D-aspartate release in rat cortical synaptosomes. Results
(n = 3–5) are expressed as the mean percentage ( SEM) of the release
In in vivo studies, compounds 40, 49 and 59 antagonised
BIC- and PIC-induced tonic convulsions in mice (Table
4), whereas they appeared inactive against pentylenetet-
razole-induced clonic convulsions in mice (data not
shown), thus appearing similar to the commonly used
antiepileptic drugs carbamazepine, lamotrigine and phe-
nytoin in their behaviour.
observed in the control and statistical analysis ( p < 0.05; p < 0.01)
* **
was determined by Student’s t test for unpaired data.
effect, being the reduction of [3H]D-aspartate efflux equal
to 51%.
Compound 59 has demonstrated also to be a very prom-
ising drug candidate for the treatment of chronic pain.
Clinical trials are ongoing to investigate the therapeutic
potential of the compound.
In in vitro studies with molecular targets which may be
linked to the anticonvulsant effect, none of them (at
10À5 M concentration), but compound 59, showed a sig-
nificant interaction with molecular targets which may be
linked to the anticonvulsant effect. Indeed, compound
59 was found to be a low-affinity noncompetitive N-
methyl-D-aspartate receptor antagonist (Ki = 8.8 lM).
5. Conclusions
In conclusion, we have identified a new series of bicy-
cloalkyl-substituted aminoacetamides that are active
against tonic convulsions induced by electrical and
chemical stimuli in mice. Preliminary data suggest that
this class of compounds only partially exerts its anticon-
vulsant activity by functionally blocking neuronal volt-
age-sensitive sodium channels. Some of the most
potent compounds appear worthy of a further investiga-
tion aimed at assessing their anticonvulsant activity in
other models and at elucidating the underlying mecha-
nism of action.
Even if the binding of the radioligand [3H]batrachotoxin
in rat brain membrane preparations was not displaced
by our compounds, we sought to investigate their ability
to functionally inhibit veratridine-dependent release of
[3H]D-aspartate from rat cortical synaptosomes, a model
to investigate pharmacological blockade of neuronal
voltage-sensitive sodium channels.15 At a concentration
of 300 lM, compounds 40 and 59 significantly reduced
veratridine-induced [3H]D-aspartate release to 87% and
77% of controls, respectively, indicating that part of
their effects may depend upon sodium channel blockade
(for results, see Fig. 2). A similar activity, ranging from
74% to 80% of control, was shown by compounds 25,
41, 50 and 55. However, the well-established sodium
channel blocker lamotrigine showed a slightly superior
6. Experimental
6.1. Chemistry
Melting points were determined in open glass capillaries
with a Buchi melting point apparatus and are uncorrect-
¨
Table 4. Anticonvulsant activity of selected compounds (ED50, po)
versus reference and antiepileptic drugs (mice, po)a
ed. The IR spectra were determined on a Perkin-Elmer
1310 spectrophotometer. H NMR spectra were record-
1
c
c
Compound
sc BICb ED50
(mg/kg)
sc PICb ED50
(mg/kg)
ed at 200.13 MHz on a Bruker ACF 200 spectrometer;
chemical shifts are in d (ppm), with tetramethylsilane
as internal standard. Several spectra do not show N–H
and O–H proton signals because they are in rapid ex-
change with the protons of the water present in the sol-
vent (DMSO). Mass spectra were measured with a
Fisons-VG Trio 2000 single quadrupole spectrometer
equipped with a dual EI/CI source. Optical rotations
were measured on a Perkin-Elmer 241 and 241-MC
polarimeter. Sodium sulfate was employed as a drying
agent for diethyl ether extracts. The petroleum ether
used throughout this work had a boiling point of 40–
70 ꢁC. TLC on silica gel plates (Merck, 60F 254) was
used to check product purity and the spots were detected
Na-Valproate
Phenytoin
Lamotrigine
Vigabatrin
Safinamide (2)
49
214 (189-242)
108 (68–170)
1.14 (0.55–2.36)
1.4 (1–2.4)
18d
4d
N.A. up to 1500
20 (11–38)
65 (48–87)
128d
N.A. up to 1000
15 (10–22)
14 (6–31)
59
40
13 (7–22)
53 (37–74)
68 (55–83)
a Values are as ED50 (mg/kg po) with 95% c.l. at the time of the peak
effect in antagonising the tonic convulsion.
b BIC, bicuculline test; PIC, picrotoxin test; N.A., not active.
c Values in brackets are 95% confidence limits.
d Indicative value calculated from regression analysis.

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E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
with ninhydrin or visualized by iodine vapour. Prepara-
tive chromatography was carried out on silica gel ICN
(32–63 lm). The structures of all compounds were con-
sistent with their analytical and spectroscopic data.
NH), 1.1–1.6 (br, 10H, CH3–(CH2)5–CH2–NH), 2.3–
2.5 (m, 2H, CH3–(CH2)5–CH2–NH, overlay with sol-
vent), 4.3 (s, 1H, NH–CH–CO), 6.3–7.0 (m, 2H,
C@H), 7.3–7.4 (dd, 1H, C = H), 7.1 and 7.5 (2br, 2H,
CO–NH2).
6.1.1. General reductive amination of a-aminoacid deriv-
atives (Scheme 1). To the appropriate a-aminoacid ester
(0.9 mol) in dry methanol (350 ml) at room temperature
was added the corresponding aldehyde (0.9 mol) and the
mixture was hydrogenated under 45 psi in the presence
of 10% Pd/C, until the hydrogen absorption ceased.
The composition of the crude product was determined
by t.l.c. and infrared spectroscopy. When the conversion
was complete, the catalyst was filtered off and the filtrate
was evaporated to dryness under vacuum. The resulting
oil was taken up with methylene chloride (500 ml) and
extracted in acidic aqueous phase by means of a 0.1 N
HCl solution. The separated aqueous phase was basified
with sodium bicarbonate and the product was extracted
again with methylene chloride (2· 250 ml), the com-
bined organic solutions were washed with water and
dried. The solvent was removed in vacuo at 35 ꢁC. The
recovered pale yellow oil was used without further puri-
fication, only in a few cases it was purified by crystalli-
zation or by flash chromatography. The residue was
dissolved in dry methanol (150 ml). Ammonia was bub-
bled through the solution, cooled at À5 ꢁC, to a ꢀ15 M
concentration. The hermetically sealed system was react-
ed for 5 days at room temperature and then evaporated
to dryness under vacuum. The product was recovered
generally as hydrochloride by dissolution in ethanol
(40 ml), acidification with diethyl ether saturated with
HCl and precipitation with diethyl ether (400 ml). The
following compounds were prepared according to this
procedure; the reported yields calculated from the corre-
sponding starting aminoacid ester:
6.1.1.5. (2R)-3-Hydroxy-2-[pentylamino]propanamide
1
hydrochloride (7). Mp 58–63 ꢁC; (48% yield); H NMR
(300 MHz, DMSO-d6/CHCl3-d3 1:1, TMS) d 0.95 (t,
J = 6.0 Hz, 3H, CH3–(CH2)4ÀNH), 1.2–1.5 (m, 4H,
CH3–(CH2)2–(CH2)2ÀNH),
2.8–3.0
(m,
2H,
CH3À(CH2)2–CH2–CH2–NH), 3.7–4.0 (m, 3H, HN–
CH1–CH2–OH), 5.3–5.6 (br, 1H, OH), 7.5–7.9 (br, 2H,
CO–CH2), 8.1–8.6 (br, 2H, N+–H2); MS/EI+ 175
[MH]+, 130 [MÀ(CONH2)]+.
6.1.1.6. (2R)-3-Hydroxy-2-[heptylamino]propanamide
1
hydrochloride (8). Mp 62–65 ꢁC; (53% yield); H NMR
(200 MHz, DMSO-d6) d 0.6–1.0 (t, J = 6.5 Hz, 3H,
CH3–(CH2)6–NH), 1.1–1.7 (br, 10H, CH3–(CH2)5–
CH2–NH), 2.3–2.7 (br, 2H, CH3–(CH2)5–CH2–NH),
2.8–3.1 (t, J = 6 Hz, 1H, HN–CH1–CH2–OH), 3.4–3.7
(t, J = 8 Hz, 2H, HN–CH–CH2–OH), 4.5–5.1 (br, 1H,
OH), 7.0–7.4 (br, 2H, CO–NH2).
6.1.1.7. (2R)-3-Hydroxy-2-[isopropylamino]propana-
mide (9). Mp 65–69 ꢁC; (38% yield); 1H NMR
(200 MHz, DMSO-d6) d 0.9–1.0 (d, J = 6.5 Hz, 6H,
(CH3)2–CH–NH), 2.6–2.8 (m, 1H, CH–(CH3)2), 3.0–
3.12 (t, J = 6.0 Hz, 1H, NH–CH–CH2OH), 3.3–3.53
(m, 2H, NH–CH–CH2–OH), 6.93–7.4 (br, 2H, CO–
NH2); MS/EI+ 147 [MH]+, 115 [MÀ(CH2–OH)]+.
6.1.1.8.
(2R)-3-Hydroxy-2-[cyclohexylmethylami-
1
no]propanamide (10). Mp 106–109 ꢁC; (37% yield); H
NMR (200 MHz, DMSO-d6) d 0.7–2.0 (m, 11H, cyclo-
hexyl), 2.2–2.4 (d, J = 6.0 Hz, 2H, cyclohexyl–CH2–
NH), 2.8–3.0 (t, J = 6.0 Hz, 1H, NH–CH–CH2–OH),
3.3–3.7 (m, 2H, NH–CH–CH2–OH), 4.5–4.8 (br, 1H,
OH), 6.9–7.3 (br, 2H, CO–NH2); MS/EI+ 201 [MH]+,
169 [MÀ(CH2–OH)]+.
6.1.1.1. (2R)-2-Phenyl-2-(heptylamino)acetamide (3).
Mp 113–116 ꢁC; (45% yield); 1H NMR (200 MHz,
DMSO-d6) d 0.8–0.9 (t, J = 5.3 Hz, 3H, CH3–(CH2)6–
NH), 1.1–1.6 (br, 10H, CH3–(CH2)5–CH2–NH), 2.2–
2.4 (br, m, 3H, N–H, CH3–(CH2)5–CH2–NH), 4.1 (s,
1H, NH–CH–CO), 6.9–7.5 (br, m, 7H, Ar-H e CO–
NH2); MS/EI+ 249 [MH]+ 205 [MÀ(CH3–(CH2)2)]+.
6.1.1.9. (2R)-3-Hydroxy-2-[1-methylhexylamino]pro-
panamide hydrochloride (11). Mp 88–92 ꢁC; (34%
yield); 1H NMR (200 MHz, DMSO-d6) d 0.9–1.0 (t,
J = 6.2 Hz, 3H, CH3–(CH2)5–CH–NH), 1.1–1.9 (m,
13H, CH3–(CH2)5–CH–CH3), 3.3–3.25 (br, 1H, CH3–
CH–NH), 3.7–3.8 (br, 2H, CH2–OH), 3.81–4.1 (br,
1H, CO–CH–NH), 4.9–5.1 (br, 1H, OH), 7.5–8.2 (2br,
2H, CO–NH2), 8.3–9.4 (2br, 2H, N+–H2); MS/EI+ 217
[MH]+, 172 [MÀ(CH2OH)]+.
6.1.1.2. (2R,S)-3-Phenyl-2-(heptylamino)propanamide
(4). Mp 89–93 ꢁC; (40% yield); 1H NMR (200 MHz,
DMSO-d6) d 0.7–0.8 (t, J = 5.8 Hz, 3H, CH3–(CH2)6–
NH), 1.1–1.4 (br, 10H, CH3–(CH2)5–CH2–NH), 2.2–
2.4 (m, 2H, CH3–(CH2)5–CH2–NH), 2.6–2.9 (m, m,
3H, NH–CH–CH2-Ar), 6.8–7.4 (br, m, 7H, Ar-H e
CO–NH2).
6.1.1.10. (2R)-3-Hydroxy-2-[2-phenylethylamino]pro-
panamide hydrochloride (12). Mp 94–99 ꢁC; (51%
yield); 1H NMR (200 MHz, DMSO-d6 and CF3COOH-
d1) d 3.4–3.7 (m, 4H, Ar-CH2–CH2–NH), 4.1–4.6 (m,
3H, NH–CH–CH2–OH), 7.7–8.5 (m, br, 7H, Ar-H,
CO–NH2); MS/EI+ 209 [MH+], 164 [MÀ(CO–NH2)]+.
6.1.1.3. (2R,S)-2-(Heptylamino)propanamide (5). Mp
68–72 ꢁC; (39% yield); H NMR (200 MHz, CHCl3-d1)
d 0.7–1.0 (t, J = 5.8 Hz, 3H, CH3–(CH2)6–NH), 1.1–1.6
(br, 13H, CH3À(CH2)5-CH2–NH and CH3–CH–NH),
2.4–2.7 (m, 3H, CH3–(CH2)5–CH2–NH and CH3–CH–
NH), 5.9 and 7.1 (2br, 2H, CO–NH2).
1
6.1.1.11. (2R,S)-3-Hydroxy-2-[octylamino]propana-
mide (13). Mp 71–72 ꢁC; (59% yield); 1H NMR
(200 MHz, DMSO-d6) d 0.7–1.0 (t, J = 6.0 Hz, 3H,
CH3–(CH2)7–NH), 1.1–1.6 (m, 12H, CH3–(CH2)6–
6.1.1.4. (2R,S)-[2-Thienyl]-2-[heptylamino]acetamide
(6). Mp 74–76 ꢁC; (34% yield); 1H NMR (200 MHz,
DMSO-d6) d 0.7–0.9 (t, J = 5.9 Hz, 3H, CH3–(CH2)6–

E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3269
CH2–NH), 2.3–2.6 (m, 2H, CH3–(CH2)6–CH2–NH),
2.9–3.0 (t, J = 6.3 Hz, 1H, NH–CH–CH2–OH), 3.3–3.6
(m, 2H, NH–CH–CH2–OH), 4.2–4.8 (br, 1H, OH), 7.0
and 7.3 (2br, 2H, CO–NH2).
6.1.1.18.
(2R)-3-Hydroxy-2–[1-methylbenzylami-
no]propanamide hydrochloride (20). Mp 180–183 ꢁC;
(45% yield); 1H NMR (200 MHz, CH3OH-d4) d 1.6–
1.7 (d, J = 6.3 Hz, 3H, NH–CH–CH3), 3.4–4.1 (m, 3H,
NH–CH–CH2–OH), 4.4–4.6 (m, 1H, Ar-CH–NH),
7.4–7.6 (m, 5H, Ar-H); MS/EI+ 209 [MH]+ 164
[MÀ(CO–NH2)]+.
6.1.1.12.
(2R,S)-3-Hydroxy-2-[nonylamino]propan-
amide (14). Mp 63–65 ꢁC; (51% yield); 1H NMR
(200 MHz, DMSO-d6) d 0.7–0.9 (t, J = 6.0 Hz 3H,
CH3–(CH2)8–NH), 1.1–1.5 (br, 14H, CH3–(CH2)7–
CH2–NH), 2.3–2.6 (m, 2H, CH3–(CH2)7–CH2–NH),
2.8–3.0 (t, J = 6.4 Hz, 1H, NH–CH–CH2–OH), 3.3–3.6
(m, 2H, NH–CH–CH2–OH), 4.2–5.0 (br, 1H, OH), 7.0
and 7.3 (2br, 2H, CO–NH2).
6.1.1.19. (2R)-3-Hydroxy-2–[1,1-diphenylmethylami-
no]propanamide (21). Mp 134–137 ꢁC; (36% yield); H
1
NMR (200 MHz, DMSO-d6) d 2.6–2.7 (br, 1H, NH–
CH–CH2–OH), 2.8–3.0 (br, 1H, NH–CH–(Ar)2), 3.4–
3.6 (t, J = 4.5 Hz, 2H, NH–CH–CH2–OH), 4.6–4.7 (t,
J = 5.5 Hz, 1H, OH), 4.8–4.9 (br, 1H, NH), 6.9–7.6
(br, m, 12H, Ar-H, CO–NH2); MS/EI+ 226 [MÀ(CO–
NH2)]+.
6.1.1.13. (2R,S)-3-Hydroxy-2-[1-ethylheptylamino]-
1
propanamide (15). Mp 60–62 ꢁC; (32% yield); H NMR
(200 MHz, CHCl3-d3) d 0.7–1.0 (m, 6H, CH3–CH2–
CH–(CH2)5–CH3), 1.1–1.6 (m, 12H, CH3–CH2–CH–
(CH2)5–CH3), 2.3–2.6 (t, J = 6.2 Hz, 1H, CH3–CH2–
CH–NH), 3.1–3.3 (t, J = 6.5 Hz, 1H, NH–CHCH2–
OH), 3.6–3.9 (m, 2H, NH–CH–CH2–OH), 6.6 and 7.4
(2br, 2H, CO–NH2).
6.1.1.20. (2R)-3-Hydroxy-2-[4-phenylbutylamino]pro-
panamide (22). Mp 159–161 ꢁC; (42% yield); H NMR
1
(200 MHz, DMSO-d6) d 1.3–1.7 (m, 4H, NH–CH2–
CH2–CH2), 2.4–2.7 (m, 4H, Ar-CH2–(CH2)2–CH2–NH
overlay with solvent), 2.9–3.1 (t, J = 5.8 Hz, 1H, NH–
CH–CH2–OH), 3.3–3.6 (m, 2H, NH–CH–CH2–OH),
4.4–5.0 (br, 1H, NH–CH–CH2–OH), 6.9– 7.4 (br, m,
7H, Ar-H, CO–NH2); MS/EI+ 237 [MH]+ 192
[MÀ(CONH2)]+.
6.1.1.14. (2R,S)-3-Hydroxy-2–[1-methylnonylamino]-
1
propanamide (16). Mp 71–73 ꢁC; (34% yield); H NMR
(200 MHz, CHCl3-d3) d 0.8–0.9 (t, J = 6.2 Hz, 3H,
CH3–(CH2)7–CH–NH), 1.0–1.1 (dd, J = 7 Hz, 3H,
CH3–CH–NH), 1.2–1.5 (br, 14H, CH3–(CH2)7–CH–
NH), 2.5–2.7 (m, 1H, CH3–CH–NH), 3.1–3.3 (m, 1H,
NH–CH–CH2–OH), 3.6–3.9 (m, 2H, NH–CH–CH2–
OH), 6.0 e 7.4 (2br, 2H, CO–NH2).
6.1.1.21. (2R)-3-Hydroxy-2-[2-Phenoxyethylamino]-
propanamide hydrochloride (23). Mp 59–61 ꢁC; (42%
yield); 1H NMR (200 MHz, DMSO-d6 and
CF3COOH-d1) d 3.1–3.6 (t, J = 5.2 Hz, 2H, NH–CH2–
CH2–O), 3.7–4.1 (m, 3H, NH–CH–CH2–OH), 4.2–4.4
(t, J = 5.2 Hz, 2H, NH–CH2), 5.0–6.0 (br, 1H, OH),
6.9–7.1 (m, 3H, Ar-H), 7.2–7.4 (m, 2H, Ar-H), 7.6 and
8.0 (2br, 2H, CO–NH2), 8.7–9.5 (2br, 2H, N+–H2);
MS/EI+ 225 [MH]+ 193 [MÀ(CH2OH)]+.
6.1.1.15.
(2R)-3-Hydroxy-2–[1-methyl-3-phenylpro-
pylamino]propanamide (17). (34% yield); 1H NMR
(200 MHz, CHCl3-d1) d 0.9–1.1 (dd, J = 6.1 Hz, 3H,
CH3–CH–NH), 1.5–1.9 (m, 2H, Ar-CH2–CH2–CH–
NH), 2.5–2.7 (m, 3H, Ar-CH2–CH2–CH–NH), 2.8–3.1
(br, 1H, OH), 3.2–3.3 (m, 1H, NH–CH–CH2–OH),
3.6–3.9 (m, 2H, NH–CH–CH2–OH), 6.4 and 7.5 (br,
2H, CO–NH2), 7.0–7.3 (m, 5H, Ar-H).
6.1.1.22.
(2R)-3-Hydroxy-2-[3-phenylpropylamino]-
propanamide hydrochloride (24). Mp 70–72 ꢁC; (53%
1
yield); H NMR (200 MHz, DMSO-d6) d 1.8–2.1 (m,
2H, Ar-CH2–CH2–CH2–NH), 2.6–2.7 (t, J = 6.4 Hz
2H, Ar-CH2–CH2–CH2ÀNH), 2.8–3.0 (m, 2H, CH2–
NH), 3.8–4.0 (br, 3H, NH–CH–CH2–OH), 5.4–5.7 (br,
1H, OH), 7.1–7.3 (m, 5H, Ar-H), 7.6 and 8.1 (2br, 2H,
CO–NH2), 8.6–9.4 (2br, 2H, N+–H2); MS/EI+ 223
[MH+], 178 [MÀ(CO–NH2)]+.
6.1.1.16. (2R)-3-Hydroxy-2–[1-methyl-2-phenylethyl-
amino]propanamide hydrochloride (18). Mp 105–
107 ꢁC; (37% yield); 1H NMR (200 MHz, DMSO-d6)
d 1.0–1.2 (d, J = 6.2 Hz, 3H, CH3–CH–NH), 2.6–2.8
(m, 1H, CH3–CH–NH), 3.2–3.4 (m, 2H, Ar-CH2–
CH–NH), 3.8–3.9 (br, 2H, NH–CH–CH2–OH), 4.1–
4.2 (br, 1H, NH–CH–CH2–OH), 5.5–5.8 (br, 1H,
OH), 7.1–7.4 (m, 5H, Ar-H), 7.7 and 8.2 (2br, 2H,
CO–NH2), 8.7–9.5 (2br, 2H, N+–H2). Anal Calcd.
for C13H20N2O 2ÆHCl: C, 57.24; H, 7.76; Cl, 13.00;
N, 10.27; O, 11.73. Found: C, 55.82; H, 7.74; Cl,
13.82; N, 13.91.
6.1.1.23. (2S)-3-Hydroxy-2-[3-phenylpropylamino]pro-
panamide (25). Mp 70–72 ꢁC; (53% yield); 1H NMR
(200 MHz, DMSO-d6) d 1.8–2.1 (m, 2H, Ar-CH2–
CH2–CH2–NH), 2.6–2.7 (t, J = 6.4 Hz 2H, Ar-CH2–
CH2–CH2ÀNH), 2.8–3.0 (m, 2H, CH2–NH), 3.8–4.0
(br, 3H, NH–CH–CH2–OH), 5.4–5.7 (br, 1H, OH),
7.1–7.3 (m, 5H, Ar-H), 7.6 and 8.1 (2br, 2H, CO–
NH2), 8.6–9.4 (2br, 2H, N+–H2); MS/EI+ 223 [MH+],
178 [MÀ(CO–NH2)]+.
6.1.1.17. (2R)-3-Hydroxy-2-[-3-phenybutylamino]pro-
panamide hydrochloride (19). Mp 135–139 ꢁC; (33%
yield); 1H NMR (200 MHz, DMSO-d6) d 1.1–1.2 (d,
J = 6.0 Hz, 3H, Ar-CH–CH3), 1.8–2.1 (m, 2H, NH–
CH2–CH2), 2.6–3.0 (m, 3H, Ar-CH–CH2–CH2–NH),
3.7–3.9 (br, 3H, NH–CH–CH2–OH), 5.3–5.6 (br, 1H,
OH), 7.1–7.4 (m, 5H, Ar-H), 7.1 and 8.1 (2br, 2H,
CO–NH2), 8.6–9.5 (2br, 2H, N+–H2); MS/EI+ 237
[MH]+ 192 [MÀ(CO–NH2)]+.
6.1.1.24. (2R)-3-Hydroxy-2-[3-(4-methoxyphenyl)pro-
pylamino]propanamide hydrochloride (26). Mp 168–
1
170 ꢁC; (33% yield); H NMR (200 MHz, CH3OH-d4)
d 1.9–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–2.8 (t,
J = 6.2 Hz, 2H, NH–CH2–CH2–CH2), 2.9–3.2 (m, 2H,
NH–CH2–CH2–CH2), 3.7 (s, 3H, O–CH3), 3.8–4.1 (m,

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E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3H, NH–CH–CH2–OH), 6.8–6.9 (d, J = 8.2 Hz, 2H, Ar-
H), 7.1–7.2 (d, J = 8.2 Hz, 2H, Ar-H); MS/EI+ 253
[MH]+ 208 [MÀ(CO–NH2)]+.
CO–NH2), 8.0 (br, 1H, CO–NH2), 8.5–9.4 (2br, 2H,
N+–H2); MS/EI+ 291 [MH]+ 259 [MÀ(CH2–OH)]+.
6.1.1.31. (2R)-3-Hydroxy-2-[3-phenyl-propylamino]N-
methyl propanamide (33). Mp 76–78 ꢁC; (41% yield);
41%; 1H NMR (200 MHz, DMSO-d6) d 1.6–1.8 (m,
2H, NH–CH2–CH2–CH2), 1.8–2.0 (br,1H, N–H–
(CH2)3), 2.3–2.5 (t, J = 6.3 Hz, 2H, NH–CH2–CH2–
CH2 overlay with solvent), 2.6–2.7 (t, J = 6.3 Hz, d,
J = 6.0 Hz, 5H, NH–CH2–CH2–CH2, N–CH3), 2.9–
3.1 (t, J = 5.8 Hz, 1H, NH–CH–CH2–OH), 3.4–3.6
(m, 2H, NH–CH–CH2–OH), 4.6–4.8 (t, J = 6.0 Hz,
1H, O–H), 7.0–7.4 (m, 5H, Ar-H), 7.6–7.8 (br, 1H,
CO–NH–CH3); MS/EI+ 237 [MH]+ 205 [MÀ(CH2–
OH)]+.
6.1.1.25.
(2R,S)-3-Hydroxy-2-[3-(2-methoxyphe-
nyl)propylamino]propanamide hydrochloride (27). Mp
154–156 ꢁC; (30% yield); 1H NMR (200 MHz,
CH3OH-d4) d 1.9–2.2 (m, 2H, NH–CH2–CH2–CH2),
2.6–2.8 (t, J = 6.4 Hz, 2H, NH–CH2–CH2–CH2), 2.9–
3.2 (m, 2H, NH–CH2–CH2–CH2), 3.8 (S, 3H, O–CH3),
3.8–4.1 (m, 3H, NH–CH–CH2–OH), 6.8–7.0 (m, 2H,
Ar-H), 7.1–7.3 (m, 2H, Ar-H); MS/EI+ 253 [MH]+ 208
[MÀ(CO–NH2)]+.
6.1.1.26. (2R,S)-3-Hydroxy-2-[3-(3-methoxyphenyl-)-
propylamino]propanamide hydrochloride (28). Mp 142–
1
145 ꢁC; (33% yield); H NMR (200 MHz, DMSO-d6) d
6.1.1.32. (2R,S)-3-Hydroxy-2-[3-phenylpropylami-
no]N,N-dimethyl propanamide hydrochloride (34). Mp
1.8–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–2.7 (t,
J = 6.1 Hz, 2H, NH–CH2–CH2–CH2), 2.7–3.0 (br, 2H,
NH–CH2–CH2–CH2), 3.7 (s, 3H, O–CH3), 5.0–5.7 (br,
1H, O–H), 6.7–6.9 (m, 3H, Ar-H), 7.1–7.3 (m, 1H, Ar-
H), 7.6 and 8.0 (2br, 2H, CO–NH2), 8.1–9.3 (2br, 2H,
N+–H2); MS/EI+ 253 [MH]+ 208 [MÀ(CO–NH2)]+.
1
125–129 ꢁC; (41% yield); H NMR (200 MHz, DMSO-
d6) d 1.8–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–2.7 (t,
J = 6.1 Hz, 8H, NH–CH2–CH2–CH2 and N(CH3)2),
2.8–3.3 (m, 4H, NH–CH2–CH2–CH2), 3.7–3.8 (d,
J = 5.2 Hz, 2H, NH–CH–CH2–OH), 4.3–4.4 (t,
J = 5.2 Hz, 1H, NH–CH–CH2–OH), 4.8–5.8 (br, 1H, -
OH), 7.1–7.4 (m, 5H, Ar-H), 8.4–9.8 (br, 2H, N+–H2);
MS/EI+ 251 [MH]+ 219 [MÀ(CH2–OH)]+.
6.1.1.27. (2R,S)-3-Hydroxy-2-[3-(4-methylphenyl)-
propylamino]propanamide hydrochloride (29). Mp 183–
1
186 ꢁC; (35% yield); H NMR (200 MHz, DMSO-d6) d
1.8–2.1 (br, 2H, NH–CH2–CH2–CH2), 2.2 (s, 3H, Ar-
CH3), 2.6–2.7 (t, J = 6.2 Hz, 2H, NH–CH2–CH2–
CH2), 2.8–3.0 (br, 2H, NH–CH2–CH2–CH2), 3.8–4.0
(br, 3H, NH–CH–CH2–OH), 5.3–5.7 (br, 1H, O–H),
7.1 (s, 4H, Ar-H), 7.6 and 8.1 (2br, 2H, CO–NH2),
8.6–9.4 (2br, 2H, N+–H2); MS/EI+ 237 [MH]+ 192
[MÀ(CO–NH2)]+.
6.1.1.33.
(2R)-3-Hydroxy-2-[3-phenylpropylami-
no]propionic acid hydrochloride (35). Mp 165–168 ꢁC;
1
(41% yield); H NMR (200 MHz, DMSO-d6) d 1.8–2.1
(m, 2H, NH–CH2–CH2–CH2), 2.6–2.7 (t, J = 6.2 Hz,
2H, NH–CH2–CH2–CH2), 2.9–3.1 (m, 2H, NH–CH2–
CH2–CH2), 3.8–4.0 (m, 3H, NH–CH–CH2–OH), 7.1–
7.4 (m, 5H, Ar-H), 8.0–10.0 (br, N-H, COOH, chemical
exchange); MS/EI+ 223 M+ 179 [MÀ(CO–NH2)]+.
6.1.1.28. (2R,S)-3-Hydroxy-2-[3-(4-fluorophenyl)-pro-
pylamino]propanamide hydrochloride (30). Mp 188–
6.1.1.34.
(2S)-3-(Acetoxy)-2-[3-phenylpropylami-
1
191 ꢁC; (37% yield); H NMR (200 MHz, DMSO-d6) d
no]propanamide hydrochloride (36). Mp 165–167 ꢁC;
1
1.8–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–2.7 (t,
J = 6.2 Hz, 2H, NH–CH2–CH2–CH2), 2.8–3.0 (br, 2H,
NH–CH2–CH2–CH2), 3.7–3.9 (br, 3H, NH–CH–CH2–
OH), 5.3–5.7 (br, 1H, O–H), 6.9–7.2 (2m, 4H, Ar-H),
7.6 and 8.1 (2br, 2H, CO–NH2), 8.6–9.5 (2br, 2H,
N+–H2); MS/EI+ 241 [MH]+ 196 [MÀ(CO–NH2)]+.
(41% yield); H NMR (200 MHz, DMSO-d6) d 1.9–2.1
(m, 5H, CO–CH3, NH–CH2–CH2–CH2), 2.6–2.7 (t,
J = 6.4 Hz, 2H, NH–CH2–CH2–CH2), 2.8–3.0 (br, 2H,
NH–CH2–CH2–CH2), 4.0–4.2 (br, 1H, NH–CH–CH2),
4.3–4.6 (m, 2H, NH–CH–CH2), 7.0–7.3 (m, 5H, Ar-
H), 7.6 and 8.3 (2br, 2H, CO–NH2), 9.0 and 9.8 (br,
2H, N+–H2); MS/DEI+ 265 [MH]+ 220 [MÀ(CO–
NH2)]+.
6.1.1.29. (2R,S)-3-Hydroxy-2-[3-(4-chlorophenyl)-pro-
pylamino]propanamide hydrochloride (31). Mp 198–
1
200 ꢁC; (41% yield); H NMR (200 MHz, DMSO-d6) d
6.1.1.35. (2R,S)-3-Hydroxy-2-[3-phenyl-propyl-(N-
methyl)-amino]propanamide hydrochloride (37). Mp
1.7–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–2.7 (t,
J = 6.1 Hz, 2H, NH–CH2–CH2–CH2), 2.8–3.0 (br, 2H,
NH–CH2–CH2–CH2), 3.7–3.9 (br, 3H, NH–CH–CH2–
OH), 5.3–5.7 (br, 1H, O–H), 7.1–7.4 (2m, 4H, Ar-H),
7.6 and 8.0 (2br, 2H, CO–NH2), 8.6–9.4 (2br, 2H,
N+–H2); MS/EI+ 256 M+ 212 [MÀ(CO–NH2)]+.
1
111–113 ꢁC; (41% yield); H NMR (200 MHz, DMSO-
d6) d 1.7–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–2.7 (t,
J = 6.3 Hz, 2H, NH–CH2–CH2–CH2), 2.9 (s, 3H, N–
CH3), 3.8–4.1 (br, 3H, NH–CH–CH2–OH), 5.5–5.8
(br, 1H, O–H), 7.1–7.4 (m, 5H, Ar-H), 7.7 and 8.3 (br,
d, 2H, CO–NH2), 9.7 and 10.3 (2br, 2H, N+–H2); MS/
EI+ 236 M+ 192 [MÀ(CO–NH2)]+.
6.1.1.30. (2R)-3-Hydroxy-2-[3-(4-trifluoromethylphe-
nyl)-propylamino]propanamide hydrochloride (32). Mp
1
222–224 ꢁC; (42% yield); H NMR (200 MHz, DMSO-
6.1.1.36.
(2R,S)-2-Phenyl-2-[3-phenylpropyl-ami-
d6) d 1.8–2.1 (m, 2H, NH–CH2–CH2–CH2), 2.6–3.7 (t,
J = 6.2 Hz, 2H, NH–CH2–CH2–CH2), 2.8–3.1 (br, 2H,
NH–CH2–CH2–CH2), 3.7–3.9 (br, 3H, NH–CH–CH2–
OH), 5.4–5.6 (br, 1H, O–H), 7.3–7.5 (d, J = 8.1 Hz,
2H, Ar-H), 7.5–7.7 (d, br, J = 8.1 Hz, Ar-H,
no]acetamide hydrochloride (38). Mp 230–231 ꢁC; (41%
1
yield); H NMR (200 MHz, DMSO-d6) d 1.8–2.1 (m,
2H, NH–CH2–CH2–CH2), 2.6–2.9 (t, J = 6.3 Hz, m,
4H, NH–CH2–CH2–CH2), 5.0 (br, 1H, Ar-CH–CO),
7.1–7.7 (3m, 12H, Ar-H, CO–NH2), 8.1 (br, 2H,

E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3271
CO–NH2), 9.3–9.7 (br, 2H, N+–H2); MS/EI+ 269 [MH]+
224 [MÀ(CO–NH2)]+.
6.1.2.6. (2R)-2-[(1,2,3,4-Tetrahydro-2-(R,S)-napht-
halenyl)amino]propanamide hydrochloride (44). Mp
244–246 ꢁC; (37% yield); H NMR (200 MHz, DMSO-
1
6.1.2. General reductive amination of a-aminoacid deriv-
atives (Scheme 2). The appropriate aminoacid ester
(13 mmol) was dissolved in a dry mixture of 10:1 etha-
nol/methanol (110 ml), added with corresponding ke-
tone (13 mmol) and, after half an hour, with sodium
cyanoborohydride in 3 portions at room temperature.
The suspension was left overnight. The solvent was
evaporated in vacuo and the residue was treated in the
same way as described in Section 6.1.1. The final prod-
ucts were tested as either the free base or its hydrochlo-
ride salt. Following the procedure above these
compounds were synthesised:
d6) d 1.4–1.6 (d, J = 8.2 Hz, 3H, CH3), 1.7–2.0 and
2.1–2.4 (2m, 2H, Ar-CH2–CH2–CH–NH), 2.7–3.2 (m,
4H, Ar-(CH2)2), 3.3–3.5 (br, 1H, Ar-CH2–CH–NH),
4.0–4.2 (br, 1H, CH–CH3), 7.0–7.2 (m, 4H, Ar-H), 7.6
and 8.1 (2br, 2H, CO–NH2), 8.9–9.6 (2br, 2H, N+–
H2); MS/EI+ 219 [MH]+, 174 [MÀCO–NH2]+.
6.1.2.7.
3-Hydroxy-(2R,S)-2-[(2-naphthyl)-methyl-
amino]propanamide hydrochloride (45). Mp 240–241 ꢁC;
1
(39% yield); H NMR (200 MHz, DMSO-d6) d 3.7–4.0
(2m, 3H, NH–CH–CH2–OH), 4.3–4.4 (br, 2H, Ar-
CH2–NH), 7.5–7.7 and 7.8–8.2 (2br, 9H, Ar-H and
CO–NH2), 9.0–9.8 (2br, 2H, N+–H2); MS/EI+ 244
[M]+, 200 [MÀCO–NH2]+.
6.1.2.1. 3-Hydroxy-(2R)-2-[(1,2,3,4-tetrahydro-2-(R)-
naphthalenyl)amino]propanamide hydrochloride (39). mp
226–231 ꢁC; (41% yield); 1H NMR (200 MHz,
CH3OH-d4) d 1.7–2.0 and 2.3–2.5 (2m, 2H, Ar-CH2–
CH2–CH), 2.8–3.1 and 3.2–3.3 (2m, 4H, Ar-CH2–CH
and Ar-CH2–CH–NH), 3.4–3.7 (m, 1H, Ar-CH2–CH–
NH), 3.7–4.1 (m, 2H NH–CH–CH2–OH), 4.2–4.3 (m,
1H, NH–CH–CH2–OH), 7.1–7.2 (m, 4H, Ar-H); MS/
EI+ 235 [MH]+ 190 [MÀCO–NH2]+.
6.1.2.8. 3-Hydroxy-(2R)-2-[(1,2,3,4-tetrahydro-2-(R)-
naphthalenyl)-amino]N-methyl propanamide hydrochlo-
ride (46). Mp 179–182 ꢁC; (43% yield); 1H NMR
(200 MHz, CH3OH-d4) d 1.8–2.1 and 2.3–2.4 (2m, 2H,
Ar-CH2–CH2–CH), 2.8 (s, 3H, CO–NH–CH3), 2.9–3.1
and 3.2–3.4 (2m, 4H, Ar-[CH2]2), 3.5–3.7 (m, 1H, Ar-
CH2–CH–NH), 3.7–4.1 (m, 2H, CH–CH2–OH), 4.2–
4.3 (m, 1H, NH–CH–CH2–OH), 7.1 (s, 4H, Ar-H);
MS/EI+ 217 [MÀCH2OH]+.
6.1.2.2. 3-Hydroxy-(2R)-2-[(1,2,3,4-tetrahydro-2-(S)-
naphthalenyl)amino]propanamide hydrochloride (40).
Mp 143–146 ꢁC; (40% yield); 1H NMR (200 MHz,
CH3OH-d4) d 1.5–1.7 and 1.9–2.1 (2m, 2H, Ar-CH2–
CH2–CH), 2.5–3.1 (2m, 5H, Ar-CH2–CH2–CH–NH
and Ar-CH2–CH–NH), 3.4–3.5 (t, J = 5.8 Hz, 1H,
NH–CH–CH2–OH), 3.6–3.8 (m, 2H, NH–CH–CH2–
OH), 6.9–7.1 (m, 4H, Ar-H); MS/EI+ 235 [MH]+ 190
[MÀCO–NH2]+.
6.1.2.9. 3-Hydroxy-(2R)-2-[(1,2,3,4-tetrahydro-2-(S)-
naphthalenyl)amino]N-methyl propanamide hydrochloride
(47). Mp 163–167 ꢁC; (45% yield);1H NMR (200 MHz,
CH3OH-d4) d 1.8–2.1 and 2.3–2.4 (2m, 2H, Ar-CH2–
CH2–CH), 2.8 (s, 3H, CO–NH–CH3), 2.9–3.1 and 3.2–
3.4 (2m, 4H, Ar-[CH2]2), 3.5–3.7 (m, 1H, Ar-CH2–
CH–NH), 3.7–4.1 (m, 2H, CH–CH2–OH), 4.2–4.3 (m,
1H, NH–CH–CH2–OH), 7.1 (s, 4H, Ar-H); MS/EI+
217 [MÀCH2OH]+.
6.1.2.3. 3-Hydroxy-(2S)-2-[(1,2,3,4-tetrahydro-2-(S)-
naphthalenyl)amino]propanamide hydrochloride (41).
Mp 232–236 ꢁC; (41% yield); 1H NMR (200 MHz,
CH3OH-d4) d 1.7–2.0 and 2.3–2.5 (2m, 2H, Ar-CH2–
CH2–CH), 2.8–3.1 and 3.2–3.3 (2m, 4H, Ar- CH2–CH
and Ar-CH2–CH–NH), 3.4–3.7 (m, 1H, Ar-CH2–CH–
NH), 3.8–4.1 (m, 2H, NH–CH–CH2–OH), 4.2–4.3
(m,1H, NH–CH–CH2–OH), 7.0–7.2 (m, 4H, Ar-H).
6.1.2.10. 3-Hydroxy-(2R,S)-2-[(1,2,3,4-tetrahydro-1-
(R,S)-naphthalenyl)amino]propanamide (48). Mp 115–
1
122 ꢁC; (45% yield); H NMR (200 MHz, DMSO-d6) d
1.5–2.0 (m, 5H, Ar-CH2–CH2–CH2–CH–NH), 2.9–2.9
(br, 4H. Ar-CH2–CH2 and NH–CH–CH2–OH), 3.6–
3.7 (q, J = 3.4 Hz, 1H, Ar-CH–NH), 3.8–4.0 (m, 1H,
NH–CH–CO), 5.2–5.5 (br, 1H, OH), 7.0–7.4 (m, 6H,
Ar-H, CO–NH2).
6.1.2.4. 3-Hydroxy-(2S)-2-[(1,2,3,4-tetrahydro-2-(R)-
naphthalenyl)amino]propanamide (42). Mp 143–148 ꢁC;
(41% yield); 1H NMR (200 MHz, CH3OH-d4) d 1.5–
1.7 and 1.9–2.1 (2m, 2H, Ar-CH2–CH2–CH), 2.5–3.1
(2m, 5H, Ar-CH2–CH2–CH–NH and Ar-CH2–CH–
NH), 3.4–3.5 (t, J = 5.8 Hz, 1H, NH–CH–CH2–OH),
3.6–3.8 (m, 2H, NH–CH–CH2–OH), 6.9–7.1 (m, 4H,
Ar-H).
6.1.2.11. 3-Hydroxy-(2S)-2-[(2-indanyl)amino]propan-
amide hydrochloride (49). Mp 190–192 ꢁC; (44% yield);
1H NMR (200 MHz, DMSO-d6) d 3.1–3.5 (m, 4H, Ar-
(CH2)2), 3.8–4.1 (m, 4H, CH–NH–CH–CH2–OH), 5.4–
5.7 (br, 1H, OH), 7.1–7.3 (m, 4H, Ar-H), 7.7 and 8.1
(2br, 2H, CO–NH2), 9.1–9.9 (2br, 2H, N+–H2). Anal.
Calcd. for C12H16N2O 2ÆHCl: C, 56.14; H, 6.67; Cl,
13.81; N, 10.91; O, 12.46. Found: C, 56.27; H, 6.52;
Cl, 13.92; N, 10.78.
6.1.2.5. (2S)-2-[(1,2,3,4-Tetrahydro-2-(R,S)-napht-
halenyl)amino]propanamide hydrochloride (43). Mp
1
247–249 ꢁC; (35% yield); H NMR (200 MHz, DMSO-
d6) d 1.4–1.6 (d, J = 8.2 Hz, 3H, CH3), 1.7–2.0 and
2.1–2.4 (2m, 2H, Ar-CH2–CH2–CH–NH), 2.7–3.2 (m,
4H, Ar-(CH2)2), 3.3–3.5 (br, 1H, Ar-CH2–CH–NH),
4.0–4.2 (br, 1H, CH–CH3), 7.0–7.2 (m, 4H, Ar-H), 7.6
and 8.1 (2br, 2H, CO–NH2), 8.9–9.6 (2br, 2H, N+–
H2); MS/EI+ 219 [MH]+, 174 [MÀCO–NH2]+.
6.1.2.12. 3-Hydroxy-(2R)-2-[(2-indanyl)amino]propan-
1
amide hydrochloride (50). Mp 94–96 ꢁC; (40% yield); H
NMR (200 MHz, DMSO-d6) d 2.1–2.4 (br, 1H, NH),
2.6–2.8 (m, 2H, Ar-(CH2)2 Hax), 2.9–3.1 (m, 3H, Ar-
(CH2)2 Heq and Ar-(CH2)2–CH–NH), 3.4–3.7 (m, 3H,
NH–CH–CH2–OH), 4.6–4.8 (br, 1H, OH), 6.9–7.5

3272
E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
(m,br, 6H, Ar-H and CO–NH2); MS/EI+ 221 [MH]+,
189 [MÀCH2OH]+. Anal Calcd. for C12H16N2O2ÆHCl:
C, 56.14; H, 6.67; Cl, 13.81; N, 10.91; O, 12.46. Found:
C, 56.08; H, 6.76; Cl, 13.62; N, 10.82.
NMR (200 MHz, DMSO-d6) d 1.3–1.6 (d, J = 5.8 Hz,
3H, CH3–CH–CO), 2.6–2.7 (d, J = 4.9 Hz, 3H, CH3–
NH), 3.1–3.3 (m, 4H, Ar-[CH2]2), 3.7–4.1 (m, 2H,
CH–N–CH–CH3), 7.1–7.3 (m, 4H, Ar-H), 8.7–8.9 (q,
1H, CO–NH), 9.2–10.2 (2br, 2H, N+–H2); MS/EI+ 160
[MÀCH3–NH–CO]+.
6.1.2.13. 3-Hydroxy-(2R)-2-[(2-indanyl)-amino]N-
methyl-propanamide hydrochloride (51). Mp 189–
1
190 ꢁC; (41% yield); H NMR (200 MHz, DMSO-d6) d
6.1.2.20. (2S)-2-[(2-Indanyl)amino]N-methyl propana-
mide hydrochloride (58). Mp 194–196 ꢁC; (41% yield);1H
NMR (200 MHz, DMSO-d6) d 1.3–1.6 (d, J = 5.8 Hz,
3H, CH3–CH–CO), 2.6–2.7 (d, J = 4.9 Hz, 3H, CH3–
NH), 3.1–3.3 (m, 4H, Ar-[CH2]2), 3.7–4.1 (m, 2H,
CH–N–CH–CH3), 7.1–7.3 (m, 4H, Ar-H), 8.7–8.9 (q,
1H, CO–NH), 9.2–10.2 (2br, 2H, N+–H2); MS/EI+ 219
[MH]+, 160 [MÀCH3–NH–CO]+.
2.6–2.8 (d, J = 6.0 Hz, 3H, CH3–NH–CO), 3.0–3.4 (m,
4H, Ar-[CH2]2–CH), 3.7–4.1 (m, 4H, CH–NH–CH–
CH2–OH), 5.4–5.7 (br, 1H, OH), 7.1–7.3 (m, 4H, Ar-
H), 8.6–8.8 (q, 1H, CO–NH), 9.1–9.8 (2br, 2H, N+–
H2); MS/EI+ 235 [MH]+, 203 [MÀCH2–OH]+.
6.1.2.14. 3-Hydroxy-(2S)-2-[(2-indanyl)-amino]N-
methyl-propanamide hydrochloride (52). Mp 190–
1
192 ꢁC; (41% yield); H NMR (200 MHz, DMSO-d6) d
6.1.2.21. 2-[(2-Indanyl)amino]l-acetamide hydrochlo-
ride (59). Mp 207–209 ꢁC; (48% yield);1H NMR
(200 MHz, DMSO-d6) 3.0–3.2 (m, 4H, Ar-(CH2)2–CH)
3.7 (s, 2H, CO–CH2–N+) 3.9–4.1 (m, 1H, Ar-(CH2)–
CH) 7.1–7.3 (m, 4H, Ar-H) 7.5–7.6 and 7.9–8.1 (2br,
2H, CO–NH2) 9.5–9.7 (br, 2H, N+–H2); MS/DEI+ 191
[MH]+ 146 [MÀCO–NH2]+. Anal Calcd. for
C11H14N2OÆHCl: C, 58.28; H, 6.67; Cl, 15.64; N,
12.36; O, 7.06. Found: C, 58.36; H, 6.73; Cl, 15.75; N,
12.49.
2.6–2.8 (d, J = 6.0 Hz, 3H, CH3–NH–CO), 3.0–3.4 (m,
4H, Ar-[CH2]2–CH), 3.7–4.1 (m, 4H, CH–NH–CH–
CH2–OH), 5.4–5.7 (br, 1H, OH), 7.1–7.3 (m, 4H, Ar-
H), 8.6–8.8 (q, 1H, CO–NH), 9.1–9.8 (2br, 2H, N+–
H2); MS/EI+ 235 [MH]+, 203 [MÀCH2–OH]+.
6.1.2.15.
3-Hydroxy-(2R)-2-[(2-indanyl),-(N-meth-
yl)amino]propanamide hydrochloride (53). Mp 208–
210 ꢁC; (41% yield);1H NMR (200 MHz, DMSO-
d6+CF3COOD) d 2.7–2.9 (s, 3H, N–CH3), 2.9–3.5 (m,
4H, Ar-[CH2]2–CH), 3.9–4.1 (2br, 3H, N–CH–CH2–
OH), 4.2–4.4 (br, 1H, Ar-[CH2]2–CH), 7.0–7.3 (4H,
Ar-H); MS/ES+ 235 [MH]+.
6.1.2.22.
2-[(2-Indanyl)amino]N-methylacetamide
hydrochloride (60). Mp 219–220 ꢁC; (41% yield); 1H
NMR (200 MHz, DMSO-d6) 2.6–2.7 (d, J = 4.7 Hz,
3H, N–CH3) 3.03–3.3 (m, 4H, Ar-(CH2)2–CH) 3.7 (s,
2H, CO–CH2–N+) 3.9–4.1 (m, 1H, AR-(CH2)2–CH)
7.0–7.3 (m, 4H, Ar-H) 8.5–8.7 (m, 1H, CO–NH)
9.4–9.8 (br, 2H, N+–H2) ; MS/ES+ 205 [MH]+.
6.1.2.16. 3-Hydroxy-(2S)-2-[(2-indanyl)-(N-methyl)-
amino]propanamide hydrochloride (54). Mp 209–211 ꢁC;
(41% yield);1H NMR (200 MHz, CH3OH-d4) d 2.7–3.0
(br, 3H, N–CH3), 3.2–3.5 (m, 4H, Ar-[CH2]2), 3.9–4.2
(m, 3H, CO–CH–CH2–OH), 4.3–4.5 (m, 1H, N–CH3–
CH–[CH2]2), 4.7–5.6 (br, 1H, OH), 7.1–7.3 (m, 4H,
Ar-H), 7.6–7.8 and 8.1–8.4 (2br, 2H, CO–NH2), 10.1–
10.4 and 11.1–11.4 (2br, 1H, N+–H).
6.1.2.23. 2-[(2-Indanyl)amino]-2-cyclopropylacetamide
hydrochloride (61). Mp 195–198 ꢁC; (41% yield); 1H
NMR (200 MHz, DMSO-d6) 1.4–1.7 (2m, 4H, CO–C–
(CH2)2) 3.1–3.4 (m, 4H, Ar-(CH2)2–CH) 4.1–4.3 (m,
1H, Ar-(CH2)2–CH) 7.1–7.3 (m, 4H, Ar-H) 7.4–7.7
(br, 2H, CO–NH2) 9.8–10.2 (br, 2H, N+–H2); MS/
DCI+ 217 [MH]+.
6.1.2.17.
hydrochloride (55). Mp 217–219 ꢁC; (41% yield); 1H
NMR (200 MHz, DMSO-d6) 1.2–1.4 (d, 3H,
(2S)-2-[(2-Indanyl)amino]propanamide
d
J = 6.1 Hz, CH3–CH), 2.9–3.3 (m, 4H, Ar-[CH2]2–
CH), 3.7–3.9 (m, 2H, CH–N–CH–CH3), 6.9–7.2 (m,
4H, Ar-H), 7.5 and 8.1 (2br, 2H, CO–NH2), 8.9–10.0
(2br, 2H, N+-H2); MS/DEI+ 205 [MH]+, 160 [MÀCO–
NH2]. Anal Calcd. for C12H16N2OÆHCl: C, 59.87; H,
7.12; Cl, 14.73; N, 11.64; O, 6.65. Found: C, 59.95; H,
7.24; Cl, 14.86; N, 11.57.
6.1.2.24. 2-[(2-Indanyl)amino]-2-cyclopentylacetamide
hydrochloride (62). Mp 235–239 ꢁC; (41% yield); 1H
NMR (200 MHz, DMSO-d6) 1.6–1.8 (m, 4H, 2CH2)
2.0–2.3 (2m, 4H, 2CH2) 3.1–3.4 (m, 4H, Ar-(CH2)2–
CH) 3.8–4.0 (m, 1H, Ar-(CH2)2–CH) 7.1–7.3 (m, 4H,
Ar-H) 7.7–7.9 (br, 2H, CO–NH2) 9.5–9.8 (br, 2H,
N+–H2); MS/DEI+ 245 [MH]+ 200 [MÀCO–NH2].
6.1.2.18.
hydrochloride (56). Mp 217–219 ꢁC; (41% yield); 1H
NMR (200 MHz, DMSO-d6) 1.2–1.4 (d, 3H,
J = 6.1 Hz, CH3–CH), 2.9–3.3 (m, 4H, Ar-[CH2]2–
CH), 3.7–3.9 (m, 2H, CH–N–CH–CH3), 6.9–7.2 (m,
4H, Ar-H), 7.5 and 8.1 (2br, 2H, CO–NH2), 8.9–10.0
(2br, 2H, N+–H2). Anal Calcd. for C12H16N2OÆHCl:
C, 59.87; H, 7.12; Cl, 14.73; N, 11.64; O, 6.65. Found:
C, 59.74; H, 7.06; Cl, 14.66; N, 11.76.
(2R)-2-[(2-Indanyl)amino]propanamide
6.1.2.25. 2-[(2-Indanyl)amino]phenylacetamide hydro-
chloride (63). Mp > 250 ꢁC; (41% yield); 1H NMR
(200 MHz, DMSO-d6) 3.1–3.3 (m, 4H, Ar-(CH2)2–CH)
3.6–3.8 (m, 1H, Ar-(CH2)2–CH) 5.0–5.2 (br, 1H,
N+–CH–CO) 7.1–7.2 (m, 4H, Ar-H) 7.4–7.6 (m, 3H,
Ar-H) 7.7–7.8 (m, 3H, Ar-H, CO–NH2) 8.1–8.3 (br, 1H,
CO–NH2) 9.7–10.3 (br, 2H, N+–H2).
d
6.2. Pharmacology
6.1.2.19. (2R)-2-[(2-Indanyl)amino]N-methyl propana-
mide hydrochloride (57). Mp 194–196 ꢁC; (41% yield); 1H
6.2.1. Maximal electroshock seizure test in mice. Male
CD-1 mice (20–30 g, Charles River Laboratories, Italy,

E. Ghidini et al. / Bioorg. Med. Chem. 14 (2006) 3263–3274
3273
Calco) were used in this study. An electrical stimulus
(50 mA, 60 Hz; 0.2 s ECT unit model 7801, Ugo Basile,
Varese, Italy) was delivered through corneal electrodes
after the application of a local anaesthetic (lidocaine in
0.9% sodium chloride solution). This stimulus was suffi-
cient to produce a hind limb tonic extension in 100% of
control animals. The anticonvulsant activity of orally
administered test compounds was initially evaluated at
different time points (15, 30, 60, 120, 180 and 240 min)
from treatment to establish the time of peak effect. At
least three doses of test compounds were then adminis-
tered to groups of 10–20 mice at time of peak effect in
a dose volume of 10 ml/kg to calculate the respective
doses at which 50% of the animals were protected from
seizures (ED50). All controls received the identical vol-
ume of corresponding vehicle. The complete suppression
of the hind limb tonic component of the seizures was
taken as the end-point for this test. The method of
Litchfield–Wilcoxon (1949) was used to calculate the
ED50 values with 95% confidence interval.16
ual plexiglas cages and observed continuously for the
presence or absence of hind limb tonic extension. Obser-
vation period was of 30 min for BIC and of 45 min for
PIC.18 Dose–response curves consisted of at least three
dose levels for each compound, with 12–24 animals used
for dose.
6.2.4. Modulation of veratridine-induced [3H]D-aspartate
release in cortical synaptosomes. Synaptosomes were pre-
pared following previously described protocols.19,20
Adult male Sprague–Dawley rats (200–250 g) were sacri-
ficed by decapitation. Cortices (200–300 mg) was quick-
ly dissected and homogenized in ice cold 0.32 M Tris-
buffered sucrose (pH 7.4). The homogenate was centri-
fuged at 900 g for 5 min at 4 ꢁC. The supernatant was
stratified on a Percoll discontinuous gradient (20%,
10% and 6%) and then centrifuged at 36,000g for
5 min. The synaptosomal fraction (interface between
Percoll 20% and 10%) was extracted, added with 5 ml
cold, oxygenated (95% O2, 5% CO2) Krebs solution
(mM NaCl 125; KCl 3; CaCl2 1.2; MgSO4 1.2; NaH2-
PO4 1; NaHCO3 22; glucose 10) and centrifuged again
at 39,000g for 15 min. Finally, the resulting pellet was
resuspended in 1 ml of oxygenated Krebs solution at
room temperature (as above).
6.2.2. Horizontal screen test in mice. In this study, neural
impairment was determined by failure of mice (male
CD-1 mice of 20–30 g, Charles River Laboratories, Ita-
ly, Calco) to perform successfully the horizontal screen
test.17 The apparatus consisted of a 13 · 14 cm square
wire screens which was mounted horizontally on a steel
rod. The rod was supported at both ends and could be
inverted through an arc of 180ꢁ. Untrained mice were
placed individually on the top of the screen and the
rod was then rotated (mice unable to climb to an upright
position within 1 min were rated as failures). The neuro-
toxic activity of orally administered test compounds was
initially evaluated at different time points from treat-
ment to establish the time of peak effect. At least three
doses of test compounds were then administered to
groups of 10–20 mice at time of peak effect in a dose vol-
ume of 10 ml/kg to calculate the respective doses at
which 50% All controls received the identical volume
of corresponding vehicle. After drug treatment, two val-
ues were recorded: (1) the number of mice that fall from
the screen and (2) the number of mice that fail to climb
the top of the screen (i.e., the sum of those that remain
clinging to the bottom of the screens and those that fall
from the screen). The method of Litchfield–Wilcoxon
(1949) was used to calculate the respective doses at
which 50% of the animals failed the test (TD50).16
Synaptosomes were incubated in oxygenated Krebs solu-
tion containing 150 nM [3H]D-aspartate (16. Ci/mmol)
for 15 min at 37 ꢁC. At the end of the incubation time they
were transferred into superfusion chambers according to
Simonato et al..21 The preparation was stimulated by
pulses (60 s duration) of 10 lM veratridine. Treatments
were applied 6 min before stimulation. The perfusate
was collected at 3 min intervals, and [3H]D-aspartate con-
tent was determined using a scintillation b-spectrometer
(LS1800 Beckman). Protein content of the preparation
was determined by the method of Lowry.22
Veratridine-evoked overflow of [3H]D-aspartate was cal-
culated by subtracting the presumed basal outflow from
the total tritium found in the sample at stimulus applica-
tion and in the following sample. The presumed basal
outflow was obtained by interpolation between the prec-
edent and the following sample. Data have been ex-
pressed as % of the overflow obtained in the control
(vehicle-treated) filters.
6.2.3. Chemically induced tonic seizures in mice. Testing
for efficacy against chemically induced tonic convulsions
occurred at the time of the maximal anticonvulsant ef-
fect of the compounds obtained in previous studies or
from published data showing anticonvulsant activity in
mice (male CD-1 mice of 20–30 g, Charles River Labo-
ratories, Italy, Calco). All the anticonvulsants were
administered orally. Control animals received the vehi-
cle in the same way. Chemical convulsants were admin-
istered sc at doses that caused convulsions in 97% of the
animals (CD97 values), 2.2 mg/kg for bicuculline (BIC)
and 13.4 mg/kg for picrotoxin (PIC). For po and sc
administration a volume of 10 ml/kg was used. Follow-
ing injections of chemical convulsants in a fold of skin in
the midline of the neck, animals were placed in individ-
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