N-(benzyloxycarbonyl)glycine esters and amides as new anticonvulsants

Article abstract of DOI:10.1021/jm970086f

Glycine is a small neutral amino acid exhibiting weak anticonvulsant activities in vivo. Recently, studies have demonstrated that N- (benzyloxycarbonyl)glycine (1) antagonized seizures superior to glycine in addition to activity in the maximal electroshock (MES) test, a convulsive model where glycine is inactive. In the present study a series of ester and amide derivatives of 1 as well as esters of N-(3-phenylpropanoyl)glycine (5) have been prepared. The compounds were evaluated in the MES test as well as in several chemically induced seizure models. Among the derivatives investigated, N-(benzyloxycarbonyl)glycine benzylamide (16) was the most potent compound exhibiting an anticonvulsant activity in the MES test comparable to the drug phenytoin. Median effective doses (ED₅₀) of 4.8 and 11.6 mg/kg were determined at 30 min and 3 h after ip administration, respectively. Compound 16 also effectively suppressed tonic seizures in different chemically induced models such as the strychnine, 3- mercaptopropionic acid, and pentylenetetrazole tests. Moreover, the compound studied here did not show acute neurotoxicity in the rotorod test up to a dose of 150 mg/kg. It is concluded that N-(benzyloxycarbonyl)glycine amides, especially 16, are potent anticonvulsant agents.

Full text of DOI:10.1021/jm970086f

24
J . Med. Chem. 1998, 41, 24-30
Articles
N-(Ben zyloxyca r bon yl)glycin e Ester s a n d Am id es a s New An ticon vu lsa n ts
Muriel Geurts,†,§ J acques H. Poupaert,† Gerhard K. E. Scriba,‡ and Didier M. Lambert*,†
Laboratory of Pharmaceutical Chemistry and Radiopharmacy, Department of Pharmaceutical Sciences, School of Pharmacy,
Faculty of Medicine, Catholic University of Louvain, 73, Avenue E. Mounier, UCL-CMFA 73.40, B-1200 Brussels, Belgium, and
Department of Pharmaceutical Chemistry, University of Mu¨nster, Hittorfstrasse 58-62, 48149 Mu¨nster, Germany
Received February 10, 1997X
Glycine is a small neutral amino acid exhibiting weak anticonvulsant activities in vivo.
Recently, studies have demonstrated that N-(benzyloxycarbonyl)glycine (1) antagonized seizures
superior to glycine in addition to activity in the maximal electroshock (MES) test, a convulsive
model where glycine is inactive. In the present study a series of ester and amide derivatives
of 1 as well as esters of N-(3-phenylpropanoyl)glycine (5) have been prepared. The compounds
were evaluated in the MES test as well as in several chemically induced seizure models. Among
the derivatives investigated, N-(benzyloxycarbonyl)glycine benzylamide (16) was the most potent
compound exhibiting an anticonvulsant activity in the MES test comparable to the drug
phenytoin. Median effective doses (ED50) of 4.8 and 11.6 mg/kg were determined at 30 min
and 3 h after ip administration, respectively. Compound 16 also effectively suppressed tonic
seizures in different chemically induced models such as the strychnine, 3-mercaptopropionic
acid, and pentylenetetrazole tests. Moreover, the compound studied here did not show acute
neurotoxicity in the rotorod test up to a dose of 150 mg/kg. It is concluded that N-
(benzyloxycarbonyl)glycine amides, especially 16, are potent anticonvulsant agents.
In tr od u ction
tion or systemic administration of large doses of glycine
(10-40 mmol/kg) are necessary for a significant increase
in CNS glycine levels.5
In the central nervous system (CNS) glycine exhibits
unique properties as one of the major inhibitory neu-
rotransmitters1 and simultaneously as a coagonist of the
excitatory neurotransmitter glutamate.2 The inhibitory
activity is mediated via the strychnine-sensitive glycine
receptor, a ligand-gated ion channel, localized mainly
in the brain stem and the spinal cord.3 Interaction with
the strychnine-insensitive glycine binding site of the
N-methyl-D-aspartate (NMDA) receptor complex poten-
tiates the excitatory action of the neurotransmitter
glutamate.4
In vivo, glycine displays weak anticonvulsant activity
in several seizure models including the 3-mercaptopro-
pionic acid test and the strychnine test, while it is
inactive in the maximal electroshock (MES) test and the
pentylenetetrazole (ScMet) test.5-11 Moreover, glycine
exhibits analgesic12 and neuroprotective13 effects. As
other agonists of the two glycine receptors, such as
L-alanine, L-serine, â-alanine, and taurine for the strych-
nine-sensitive receptor and D-serine and D-alanine for
the strychnine-insensitive glycine receptor, glycine does
not easily cross the blood-brain barrier (BBB)14-16 due
to the zwitterionic character and the absence of active
transport mechanisms at the luminal side of the endo-
thelial cells. Thus, intracerebroventricular administra-
Recently, we have shown that N-(benzyloxycarbonyl)-
glycine17 (1) exhibits a higher anticonvulsant activity
than glycine after ip administration especially in the
MES test.18 Carbamate derivatives such as N-(benzyl-
oxycarbonyl)valine, N-(benzyloxycarbonyl)phenylala-
nine, and N-(benzyloxycarbonyl)ethanolamine were de-
void of anticonvulsant activity indicating that the
N-benzyloxycarbonyl group is not responsible for the
activity.19 A log P ) 1.47 of 119 is in agreement with
values known to allow sufficient penetration of the BBB.
Besides parameters such as the molecular size, hydro-
gen bonding, and protein binding, an optimal lipophi-
licity for brain penetration appears to exist at about log
P ) 2.20 Intravenous administration of N-(benzyloxy-
carbonyl)[14C]glycine displayed a 13-fold higher brain
uptake index compared to glycine.21 N-(Benzyloxycar-
bonyl)glycine is hydrolyzed in vitro by brain homogenate
suggesting a prodrug mechanism.21,22 Moreover, 1 did
not bind to either of the two glycine receptors.19
To increase the brain uptake ester- and amide-type
lipid conjugates of glycine and N-(benzyloxycarbonyl)-
glycine (1) have been developed.23 Some of these lipids
antagonized MES-induced seizures as well as 3-mer-
captopropionic acid- and strychnine-induced seizures.
Amide lipids were generally more active than the
corresponding ester lipids.
* To whom correspondence should be addressed. Phone: + 32-2-
764-73 47. Fax: + 32-2-764-73 63. E-mail: lambert@cmfa.ucl.ac.be.
† Catholic University of Louvain.
‡ University of Mu¨nster.
The present study was conducted in order to further
evaluate the potential of N-acylglycine derivatives as
anticonvulsant agents. A series of ester and amide
§ Present address: Laboratory of Experimental Pharmacology,
Catholic University of Louvain, Brussels, Belgium.
X Abstract published in Advance ACS Abstracts, December 15, 1997.
S0022-2623(97)00086-1 CCC: $15.00 © 1998 American Chemical Society
Published on Web 01/01/1998

N-(Benzyloxycarbonyl)glycines as New Anticonvulsants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 25
Sch em e 1
Sch em e 2
derivatives of N-(benzyloxycarbonyl)glycine and N-(3-
phenylpropanoyl)glycine24 were prepared. The anticon-
vulsant activity of the compounds was tested in several
seizure models.
at very high doses.5 In addition, the MES test and the
ScMet test were selected in which glycine itself is
inactive,18,22 while the acute neurotoxicity was assayed
by the rotorod test. None of the compounds exhibited
any acute neurotoxicity up to doses of 150 mg/kg. The
results of the anticonvulsant testing are summarized
in Tables 1 and 2. The N-benzyloxycarbonyl derivatives
1 and 2 displayed anticonvulsant activity in the MES
test as well as in the strychnine- and 3-mercaptopro-
pionic acid-induced seizures models. The N-3-phenyl-
propanoyl derivatives 5 and 6 were essentially inactive
in the chemically induced seizure models while display-
ing moderate activity in the MES test. None of the
compounds showed activity in the ScMet test. The
higher anticonvulsant activity of 2 compared to 1 can
be explained by an increased BBB penetration of 2 due
to its higher lipophilicity (log P ) 1.9019) compared to
the parent compound 1.
Due to the superiority of the carbamates 1 and 2
compared to the amides 5 and 6, additional benzyl and
n-decyl esters of 1 were prepared. The benzyl ester 327
was chosen because this derivatization greatly enhanced
the CNS activity of the peptide-derived drug S-acetyl-
thiorphan,28-30 while the n-decyl ester 4 was designed
to further facilitate membrane penetration due to a
resemblance to fatty acid ester. However, both esters
3 and 4 displayed only poor activity in the MES test
(Table 1).
Resu lts a n d Discu ssion
Ch em istr y. N-(Benzyloxycarbonyl)glycine17 (1), N-
(benzyloxycarbonyl)glycine methyl ester (2), N-(3-phen-
ylpropanoyl)glycine24 (5), and N-(3-phenylpropanoyl)-
glycine methyl ester (6) were prepared as outlined in
Scheme 1. The N-(benzyloxycarbonyl)glycine esters 3
and 4 as well as amides 8-17 were readily obtained by
treatment of N-(benzyloxycarbonyl)glycine N′-hydroxy-
succinimidyl ester with the respective alcohols or amines
(Scheme 2). The lipophilicity of the compounds (Table
1) was estimated by calculation using the ClogP pro-
gram25 and measured by the determination of isocratic
capacity factors, log ko, using a RP-HPLC assay slightly
modified from Bechalany et al.26 The HPLC method
generally gave higher values than the computational
method. Between the calculated values and the log ko
values, a good correlation (r2 ) 0.98) was obtained
(ClogP ) 1.08 × log ko - 1.37).
P h a r m a cology. Preliminary evaluation of N-(benz-
yloxycarbonyl)glycine (1), N-(3-phenylpropanoyl)glycine
(5), and their respective methyl esters 2 and 6 in
comparison to glycine was performed in four different
seizure models. The strychnine and the 3-mercapto-
propionic acid tests were selected, because glycine
showed anticonvulsant effects in these tests though only
To increase the metabolic stability of the derivatives,
several aliphatic and aromatic amides of 1 were pre-

26 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Geurts et al.
Ta ble 3. Anticonvulsant Activity of 16 in the Strychnine,
Ta ble 1. Median Effective Dose (ED50) and Lipophilicity
Values (log ko and ClogP) of Compounds 1-17, Glycine, and
Standard Anticonvulsant Drugs in the MES Test after ip
Administration to Mice
3-Mercaptopropionic Acid, and Pentylenetetrazole Tests after ip
Administration of 119 mg/kg to Micea
test
clonic
tonic
seizures
(%)
ED50 (mg/kg)a or animals
protected/animals tested
time seizures
(h)
lethality
(%)
compd
(%)
compd
glycine
1
2
3
4
5
6
7
8
9
10
11
12
13
14
log ko ClogP
30 min
3 h
pentylenetetrazole test
0/8 at 750 mg/kgb
2/8 at 84 mg/kgc
0/8 at 750 mg/kgb
56.5 (40.8-78.1)
16.5 (8.3-32.6)
(85 mg/kg)
controls
16
100 ( 8
67 ( 7
67 ( 7
67 ( 9
2.64 1.54 2/8 at 89 mg/kgc
0.5
3
0 ( 2*** 22 ( 4**
4.35 3.25 4/10 at 120 mg/kgd 6/9 at 120 mg/kgd
80 ( 11 70 ( 15
60 ( 16
6.30 1/8 at 150 mg/kgc
3/9 at 166 mg/kgc
5/10 at 150 mg/kgc
6/9 at 166 mg/kgc
4/10 at 89 mg/kgc
8.3 (4.0-17.7)
pentylenetetrazole test
>5
(120 mg/kg)
controls
16
3-mercaptopropionic acid test
controls
2.37 1.16 3/8 at 89 mg/kgc
1.52 0.34 15.2 (8.5-27.7)
2.03 0.68 13.6 (6.9-26.4)
2.65 1.52 14.0 (6.8-28.5)
3.25 1.92 39.4 (20.9-74.0)
3.70 2.72 33.4 (13.9-80.4)
100 ( 7 100 ( 11
83 ( 9
100 ( 8
0.5
0 ( 1*** 50 ( 6*
7.8 (2.7-21.8)
9.5 (3.5-25.0)
100 ( 9
67 ( 8
67 ( 14
0 ( 2**
30 ( 12
4/8 at 106 mg/kgc
3/8 at 116 mg/kgc
16
0.5
3
83 ( 10 17 ( 5*
100 ( 10
0 ( 2**
>5
5.45 2/10 at 150 mg/kgc 3/8 at 150 mg/kgc
strychnine test
controls
16
3.49 2.54 3/10 at 114 mg/kgc 4/8 at 114 mg/kgc
100 ( 7
33 ( 8**
80 ( 8
100 ( 6
17 ( 4**
80 ( 7
3.49 2.54 1/8 at 120 mg/kgc 74.3 (10.4-130)
0.5
3
15
16
17
3.99 3.04 0/8 at 120 mg/kgc
27.4 (6.3-121)
11.6 (3.0-33.4)
16.6 (5.3-50.6)
30.2 (10.1-41.8)
11.4 (5.7-21.5)
80.6 (50.0-128)
3.5
2.40 4.8 (2.7-8.6)
a
3.88 2.63 8.1 (4.1-16.6)
Pearson chi square test: *p < 0.1, **p < 0.05, ***p < 0.01.
milacemide
valpromide
sodium
valproate
phenytoin
2/10 at 58 mg/kgc
14.9 (11.7-17.2)
44.5 (22.1-86.4)
(compounds 1032 and 1133) led to a decreased activity
as did a phenyl ring or substituted phenyl groups
(compounds 13,34 14,35 and 1531). However, separation
of the amide bond and the aromatic ring by one or two
methylene groups, compounds 1631 and 17,36 respec-
tively, resulted in highly MES active derivatives. N-
(Benzyloxycarbonyl)glycine benzylamide (16) was the
most active derivative of the present series. On a molar
basis, the MES activity of 16 can be compared to the
MES activity of the drug phenytoin (Table 1).
3.5 (2.5-4.9)
3.0 (1.8-5.3)
a
The data were calculated from 6-7 doses (n ) 8-10 animals/
dose). The values are expressed in mg/kg; 95% confidence intervals
are given in parentheses. Corresponds to 10 mmol/kg. c Corre-
b
d
sponds to 0.4 mmol/kg. Corresponds to 0.8 mmol/kg.
Ta ble 2. Evaluation of Glycine and Compounds 1, 2, 5, and 6
in the Strychnine and 3-Mercaptopropionic Acid Tests after ip
Administration to Mice
Because of the high MES activity, compound 16 was
selected for further pharmacological evaluations in
chemically induced seizure models. The benzylamide
16 antagonized tonic seizures in the strychnine, 3-mer-
captopropionic acid, and pentylenetetrazole tests (Table
3). Compared to the parent compound N-(benzyloxy-
carbonyl)glycine (1), 16 was by far superior even at
lower doses. Moreover, 16 also reduced the lethality
consecutive to these tests.
3-mercaptopropionic
strychnine testa
acid testb
dose
compd (mg/kg)
30 min
3 h
30 min
3 h
vehicle
1
100 ( 10
100 ( 7
95 ( 7
90 ( 8
45 (7*
209
418
220
207
220
150
300
150 ( 27** 142 ( 12*** 55 ( 6*
nd
nd
33 ( 4** 27 ( 4**
2
5
6
154 ( 10** 139 ( 15** nd
nd
99 ( 8
107 ( 7
101 ( 7
96 ( 14
92 ( 12
80 ( 9
100 ( 5
100 ( 8
100 (7
53 ( 7*
nd
glycine
131 ( 10 * 60 (15
121 ( 7** 136 ( 10** nd
The time curves of anticonvulsant activities in the
MES test of N-(benzyloxycarbonyl)glycine (1), N-(benz-
yloxycarbonyl)glycine methyl ester (2), and the benzyl-
amide 16 are summarized in Figure 1. While 1 and 2
exhibit essentially the same behavior with a peak of the
activity at 3 h, 16 is highly active 15 min to 1 h after
the application with a gradual decrease thereafter.
Besides a pharmacokinetic difference, this might be
explained by a different mechanism. Neither the amides
nor the esters studied displayed significant binding to
the strychnine-sensitive glycine receptor or to the gly-
cine binding site of the NMDA receptor. Compounds
1-16 failed to displace either [3H]strychnine or [3H]-
5,7-dichlorokynurenic acid at concentrations of up to 100
mM. However, while a prodrug mechanism can be
assumed for 1,21,22 inhibition of esterases by treatment
of the animals with bis(p-nitrophenyl phosphate), an
acylamidase and carboxylesterase inhibitor, did not
affect the anticonvulsant potency of compound 16 in the
MES test (data not shown). The MES activity of the
amides 7-17 did not correlate with their lipophilicity
as estimated by ClogP or log ko. Comparable lipophi-
licities were calculated for 13, 14, and 16, but only 16
displayed high pharmacological activity. The less lipo-
a
The data are expressed as percent of the average latency of
the onset of the seizures compared to controls (mean ( SD, n )
8-10 animals). The data are expressed as percent of the tonic
seizures of the controls. Statistical evaluation was performed using
the t-test for unpaired observations (strychnine test) or Pearson
chi square test (3-mercaptopropionic test): *p < 0.1,** p < 0.05,***
p < 0.01.
b
pared. The results of the MES test are summarized in
Table 1. Compared to the respective esters, the amides
exhibited a generally higher anticonvulsant activity
except for the n-decylamide 12. This may be explained
by the poor solubility of the compound even in the
vehicle dimethyl sulfoxide which might also result in
insufficient absorption from the injection site.
N-(Benzyloxycarbonyl)glycine amide (7) and the short-
chain alkylamides 831 and 9 displayed high anticonvul-
sant activity in the MES test (Table 1). All three
compounds are highly active 30 min and 3 h after ip
administration. Based on a molar ratio the median
effective doses (ED50) are superior to those of the
anticonvulsant drugs sodium valproate, milacemide,
and valpromide (Table 1). Increase of the steric hin-
drance by introduction of a bulky aliphatic substituent

N-(Benzyloxycarbonyl)glycines as New Anticonvulsants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 27
solution of 1 (6 g, 28.7 mmol) until the mixture became slightly
yellowish, and it was stirred at room temperature for 4 h. After
removal of the solvents under reduced pressure, the residue
was dissolved in 75 mL of ethyl acetate and washed three
times with saturated NaHCO3. The organic layer was dried
(MgSO4) and evaporated under reduced pressure to give a
white solid: yield 5.1 g, 80%; mp 22-25 °C; TLC Rf 0.72
(methylene chloride/acetone, 1:1); 13C NMR (CDCl3) 42.7 (CH2),
52.3 (COOCH3), 67.1 (C6H5-CH2), 128.1, 128.2, 128.5 (CH
arom), 136.3 (C arom), 156.4 (O-CO-NH), 171.5 (COOCH3); MS
m/z 224 (MH+); IR 1740-1735 (COOCH3). Anal. (C11H13NO4)
C,H,N.
N-(3-P h en ylp r op a n oyl)glycin e (5). Glycine (3.7 g 50
mmol) was dissolved in 15 mL of 1 M NaOH; 16.8 g of
3-phenylpropanoyl chloride (100 mmol) dissolved in 25 mL of
ethyl acetate was added under vigorous stirring and ice
cooling. The mixture was stirred at 0-4 °C for 1 h and allowed
to warm to room temperature. Excess 3-phenylpropanoyl
chloride was removed by extraction with diethyl ether. The
aqueous layer was acidified to pH 1 by addition of 6 M HCl
and extracted with CHCl3. The organic layer was dried (Na2-
SO4) and evaporated to yield a yellowish solid. Washing with
diisopropyl ether gave analytically pure 5: yield 5.2 g, 51%;
mp 113-114 °C (lit.24 mp 110-111 °C); TLC Rf 0.1 (methylene
chloride/acetone, 1:1); 13C NMR (CDCl3) 30.9 (C6H5CH2), 36.8
(CH2-CH2-CO), 40.9 (NH-CH2-CO), 126.3, 128.2, 128.5 (CH
arom), 140.4 (C arom), 173.3 (CONH), 176.1 (COOH); MS m/z
207 (M+); IR 1640 (CO-NH). Anal. (C11H13NO3) C,H,N.
N-(3-P h en ylp r op a n oyl)glycin e Meth yl Ester (6). Gly-
cine methyl ester (3.3 g, 26 mmol) and 6.6 g of triethylamine
(65.7 mmol) were dissolved in 350 mL of ethyl acetate; 6.7 g
of phenylpropanoyl chloride (40 mmol) dissolved in 25 mL of
ethyl acetate was added dropwise to the mixture at room
temperature, and it stirred for 20 h. Following removal of
triethylamine hydrochloride by filtration, the ethyl acetate
solution was washed successively with 10% citric acid, water,
and brine. The organic layer was dried and removed under
reduced pressure. Crude 6 was purified by column chroma-
tography using acetone/methylene chloride (1:1) and recrystal-
lized from diethyl ether: yield 2.6 g, 44%; mp 71-72 °C; TLC
Rf 0.4 (hexane/propan-2-ol, 4:1); 13C NMR (CDCl3) 31.4
(C6H5CH2), 38.0 (CH2-CH2-CO), 41.2 (NH-CH2-CO), 52.4
(COOCH3), 126.3, 128.3, 128.6 (CH arom), 140.3 (C arom),
170.4 (COOCH3), 173.9 (CO-NH); MS m/z 222 (MH+); IR
1740-1735 (COOCH3), 1635 (CO-NH). Anal. (C12H15NO3)
C,H,N.
F igu r e 1. Time curves of anticonvulsant activity in the MES
test of compounds (0) 16 (24 mg/kg, 0.08 mmol/kg), (×) 1 (84
mg/kg, 0.4 mmol/kg), and (b) 2 (89 mg/kg, 0.4 mmol/kg) after
ip administration to mice (n ) 6-8 aminals per time point
and compound).
philic derivatives 7-9 were also active in the test.
Thus, the exact mechanism of action of the N-(benzy-
loxycarbonyl)glycine amides remains to be elucidated.
However, the structural similarity to the 2-substituted
N-acyl amino acid benzylamides developed by Kohn and
co-workers37 suggests that especially 16 might be an
effective anticonvulsant agent due to its broad activity
in various seizure models.
In conclusion, this paper reports the anticonvulsant
activity of esters and amides of N-(benzyloxycarbonyl)-
glycine and N-(3-phenylpropanoyl)glycine. From this
series, N′-benzyl-N-(benzyloxycarbonyl)glycine amide
(16) displayed high anticonvulsant activity in the MES
test comparable to the drug phenytoin. Moreover, 16
also effectively antagonized seizures in several chemi-
cally induced models.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined in
open capillary tubes with an electrothermal melting point
apparatus and are uncorrected. Elemental analyses were
performed on a Carlo Erba EA 1108 Analyzer (Carlo Erba,
Milano, Italy) and are within (0.4% of the theoretical values.
13C NMR spectra were recorded at ambient temperature on a
AC-300 Bruker spectrometer. The chemical shifts are reported
as δ (ppm) values relative to tetramethylsilane. Mass spectra
were obtained on a Kratos MS-80 (FAB mode) or LKB GC-
MS 9000S instrument. IR spectra were run on a Perkin-Elmer
457 spectrophotometer. Absorption values are expressed as
wavenumbers (cm-1). Column chromatography was carried
out on silica gel 60 (230-400 mesh; Merck-Belgolabo, Overijse,
Belgium). All TLC data were determined using aluminum-
backed sheets with silica gel 60 F254 (Merck-Belgolabo).
Phenytoin was obtained from Sigma (Boornem, Belgium), bis-
(p-nitrophenyl phosphate) from Aldrich (Boornem, Belgium),
and milacemide from Continental Pharma (Mont-Saint Guib-
ert, Belgium). Sodium valproate and valpromide were pur-
chased from Sanofi (Brussels, Belgium); N-(benzyloxycarbonyl)-
glycine N′-hydroxysuccinimidyl ester and N-(benzyloxy-
carbonyl)glycine amide (7) were from Fluka (Boornem, Bel-
gium). N-(Benzyloxycarbonyl)glycine (1) was synthesized as
described.17 Ethyl acetate and methylene chloride were
distilled over P2O5; triethylamine was distilled over KOH.
N-(Ben zyloxyca r bon yl)glycin e Meth yl Ester (2). Dia-
zomethane in diethyl ether was prepared by reaction of KOH
and [(p-tolylsulfonyl)methyl]nitrosamide (Diazald Kit, Aldrich).
Diazomethane was slowly added at 0-4 °C to an ethanolic
Gen er a l P r oced u r e for th e P r ep a r a tion of Ester s 3′
a n d 4 a n d Am id es 8-17. To a solution of N-(benzyloxycar-
bonyl)glycine N′-hydroxysuccinimidyl ester (3 g, 9.8 mmol) and
freshly distilled triethylamine (1 g, 10 mmol) in methylene
chloride (200 mL) was added 10 mmol of the amine or the
alcohol, and the mixture was stirred overnight at room
temperature. The mixture was successively washed with
water and 10% aqueous citric acid. The organic layer was
dried (MgSO4) and evaporated under reduced pressure. The
compounds were recrystallized from water/ethanol.
N-(Ben zyloxyca r bon yl)glycin e Ben zyl Ester (3). This
compound was synthesized according to the procedure de-
scribed above. After evaporation of the organic layer, a
yellowish oil was obtained and purified by chromatography
(silica gel, 70-230 mesh; eluent methylene chloride/acetone,
9:1): yield 2.1 g, 70%; mp 71-73 °C (lit.27 mp 70.5-72 °C);
TLC Rf 0.76 (methylene chloride/acetone, 1:1); 13C NMR
(CDCl3) 42.8 (CH2), 67.1 (C6H5-CH2-O), 67.2 (C6H5-CH2-O),
127.8, 128.1, 128.2, 128.4, 128.5, 128.6 (CH arom), 135.2, 136.2
(C arom), 156.3 (O-CO-NH), 169.9 (CH2-CO-NH); MS m/z 300
(MH+); IR 3340 (CH arom), 1720 (COO), 1700 (OCONH).
Anal. (C17H17NO4) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e d ecyl ester (4): yield 2.7
g, 73%; mp 55-57 °C; TLC Rf 0.73 (hexane/2-propanol, 8:2);
13C NMR (CDCl3) 13.8 (CH3), 22.4, 25.5, 28.2, 28.9, 29.0, 29.2,
29.2, 29.3, 31.6 (CH2), 42.5 (NH-CH2-CO), 65.4 ((CH2)10-CH2-
O), 66. 8 (C6H5-CH2-O), 127.8, 127.9, 128.2 (CH arom), 135.9
(C arom), 156.0 (O-CO-NH), 169.7 (CH2-CO-NH); MS m/z 377

28 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1
Geurts et al.
(MH+); IR 3350 (CH arom), 2920-2850 (CH alkyl), 1735
(COO), 1690 (OCONH). Anal. (C22H35NO4) C,H,N.
35.6 (CH2), 40.6 (CH2), 44.7 (NH-CH2-CO), 67.3 (C6H5-CH2-
O), 126.6, 128.1, 128.3, 128.6, 128.7, 128.7 (CH arom), 136.1,
138.6 (C arom), 156.5 (O-CO-NH), 168.8 (CH2-CO-NH); MS
m/z 313 (MH+); IR 3320-3300 (CH arom), 1700 (OCONH),
1650 (NHCO). Anal. (C18H20N2O3) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e m eth yla m id e (8): yield
0.6 g, 16%; mp 105-107 °C (lit.31 mp 106-107 °C); TLC Rf
0.46 (methylene chloride/acetone, 1:1); 13C NMR (CDCl3) 26.2
(CH3), 44.6 (NH-CH2), 67.2 (C6H5-CH2), 128.1, 128.3, 128.6 (CH
arom), 136.1 (C arom), 156.8 (O-CO-NH), 169.8 (CH2-CO-NH);
MS m/z 223 (MH+); IR 3320-3040 (CH arom), 1710 (OCO-
NH), 1660 (CO-NH). Anal. (C11H14N2O3‚0.33H2O) C,H,N.
N-(Ben zyloxycar bon yl)glycin e isopr opylam ide (9): yield
3 g, 74%; mp 96-97 °C; TLC Rf 0.76 (methylene chloride/
acetone, 1:1); 13C NMR (CDCl3) 22.2 (CH3), 41.3 (NH-CH), 44.4
(NH-CH2), 66.6 (C6H5-CH2), 128.0, 128.1, 128.2 (CH arom),
135.9 (C arom), 156.4 (O-CO-NH), 167.7 (CH2-CO-NH); MS
m/z 251 (MH+); IR 3280 (CH arom), 1690 (OCONH), 1540
(NHCO). Anal. (C13H18N2O3) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e ter t-bu tyla m id e (10):
yield 1.3 g, 48%; mp 68-70 °C (lit.32 mp 68.5-71 °C); TLC Rf
0.87 (methylene chloride/acetone, 1:1); 13C NMR (CDCl3) 28.7
(CH3), 45.1 (NH-CH2), 51.5 (NH-C), 67.1 (C6H5-CH2), 128.2,
128.3, 128.5 (CH arom), 136.3 (C arom), 156.7 (O-CO-NH),
168.1 (CH2-CO-NH); MS m/z 265 (MH+); IR 3310-3250 (CH
arom), 1715 (OCONH), 1650 (NHCO). Anal. (C14H20N2O3‚
0.5H2O) C,H,N.
Lip op h ilicity. 1. log ko Deter m in a tion s: The log ko
values were measured by RP-HPLC according to the method
of Bechalany26 with slight modifications. The HPLC apparatus
consisted of a Perkin-Elmer series 10 liquid chromatograph
injector, a UV detector Perkin-Elmer LC-85B variable wave-
length spectrophotometer (set at 254 nm), and a Perkin-Elmer
LCI-100 laboratory computing integrator. The lipophilic
stationary phase was a Bio-Sil C18 HL 90-5 S column, 250 ×
4.6 mm, particle size 5 µm (Bio-Rad, Nazareth, Belgium), and
the flow rate was set to 1.0 mL/min. The mobile phases were
prepared volumetrically from a combination of 10-50% water
and 50-90% aqueous phosphate buffer (0.01 M, pH 7.4). All
solutions were purified by filtration using a Whatman filtra-
tion unit. The logarithms of isocratic capacity factors, log ko,
were defined as log ko ) log(tr - to)/to, where tr is the retention
time of the solute and to is the column dead time (methanol
was used as the nonretained compound). Assays were made
in triplicates with each eluent. log ko values were extrapolated
from at least four determinations using different eluents.
2. log P Ca lcu la tion s: The log P calculated values were
calculated on a Macintosh Power PC using the ClogP 2.0
program25 (Biobyte Corp., Claremont, CA).
P h ar m acology. 1. Electr ically In du ced Seizu r es (MES)
Test: Male OF1 mice, 15-25 g, were obtained from Iffa-Credo
(Les Oncins, France) and housed in colony cages in a 12-h
light-dark cycle with free access to commercial rodent chow
and water. During the experiments the animals were only
allowed free access to water. Maximal electroshock seizures
were induced by delivering an electrical stimulus of 50 mA
for 0.2 s via corneal electrodes. Blockade of the tonic extension
of the hind limbs was considered as protection against
seizures.38 The compounds were dissolved in dimethyl sul-
foxide. A volume of 2 mL/kg of the freshly made solutions was
injected intraperitoneally. Typically, 8-10 animals were used
per compound, dose, and time point. All compounds were
screened at an initial dose corresponding to 0.4 mmol/kg. ED50
values were determined when the compounds exhibited more
than 50% protection at the initial dose of 0.4 mmol/kg and were
calculated according to Lietchfield and Wilcoxon.39 For es-
terase inhibition experiments, 200 mg/kg bis(p-nitrophenyl
phosphate) dissolved in 10% Tween 80 saline solution was
injected subcutaneously 30 min prior to the MES test or
simultaneously with compound 16.
2. Ch em ica lly In d u ced Seizu r es: Male NMRI mice (bred
at the animal facilities at UCL), weighing 18-25 g, were
housed as described above. The compounds were dissolved in
dimethyl sulfoxide and injected ip in a constant volume of 2
mL/kg.
3. Str ych n in e-In d u ced Seizu r es: At 30 min or 3 h after
the administration of the compounds, the animals received a
sc injection of a solution of strychnine chlorhydrate in saline
(1.2 mg/mL, 1 mL/kg). The mice were placed in individual
cages and observed for 30 min. The time of onset of the
seizure,6 the number of tonic seizures,38 and the lethality were
recorded.
4. P en tylen etetr a zole-In d u ced Seizu r es: At 30 min and
3 h after the administration of the compounds, 85 or 120 mg/
kg pentylenetetrazole dissolved in saline was administered sc.
The animals were placed in individual cages and observed for
30 min. The number of clonic and tonic seizures as well as
the number of deaths was noted.
5. 3-Mer ca p top r op ion ic Acid -In d u ced Seizu r es: At 30
min and 3 h after the administration of the compounds, 40
mg/kg 3-mercaptopropionic acid in saline solution were in-
jected ip. The animals were placed in individual cages and
observed for 30 min. The number of clonic and tonic seizures
as well as the number of deaths was recorded.
N-(Ben zyloxyca r bon yl)glycin e cycloh exyla m id e (11):
yield 1 g, 41%; mp 108-110 °C (lit.33 mp 108-110 °C); TLC Rf
0.28 (methylene chloride/acetone, 9:1); 13C NMR (CDCl3) 24.8,
25.5, 33.0 (NH-C6H11), 44.8 (NH-CH2), 48.4 (NH-C6H11), 67.2
(C6H5-CH2), 128.1, 128.3, 128.6 (CH arom), 136.2 (C arom),
156.7 (O-CO-NH), 168.0 (CH2-CO-NH); MS m/z 291 (MH+); IR
3320-3260 (CH arom), 2900 (CH alkyl), 1710 (OCONH),
1650-1640 (NHCO). Anal. (C16H22N2O3) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e d ecyla m id e (12): yield
1.3 g, 64%; mp 115-116 °C; TLC Rf 0.85 (methylene chloride/
acetone, 1:1); 13C NMR (CDCl3) 14.1 (CH3), 22.7, 26.9, 29.3,
29.4, 29.5, 29.6, 29.6, 29.67, 31.9, 39.7 (CH2), 44.8 (NH-CH2),
67.3 (C6H5-CH2), 128.1, 128.3, 128.7 (CH arom), 136.2 (C
arom), 156.6 (O-CO-NH), 168.7 (CH2-CO-NH); MS m/z 377
(MH+); IR 3340 (CH arom), 2920-2860 (CH alkyl), 1690
(OCONH), 1640 (NHCO). Anal. (C22H36N2O3) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e a n ilid e (13): yield 2 g,
44%; mp 148-149 °C (lit.34 mp 145-149 °C); TLC Rf 0.90
(methylene chloride/acetone, 1:1); 13C NMR (CDCl3) 45.7 (NH-
CH2), 67.5 (C6H5-CH2), 120.0, 124.7, 128.1, 128.4, 128.6, 129.0
(CH arom), 135.9, 137.3 (C arom), 157.0 (O-CO-NH), 167.1
(CH2-CO-NH); MS m/z 285 (MH+); IR 3340-3280 (CH arom),
1700 (OCONH), 1610 (NHCO). Anal. (C16H16N2O3) C,H,N.
N-(Ben zyloxyca r b on yl)glycin e o-t olu id id e (14): yield
2.4 g, 64%; mp 122-124 °C (lit.35 mp 114-116°C); TLC Rf 0.82
(hexane/2-propanol, 1:1); 13C NMR (CDCl3) 17.6 (CH3), 45.8
(NH-CH2), 67.5 (C6H5-CH2), 123.0, 125.4, 126.8, 128.2, 128.4,
128.6, 129.2, 130.5 (CH arom), 135.2, 136.0 (C arom), 157.0
(O-CO-NH), 167.4 (CH2-CO-NH); MS m/z 299 (MH+); IR 3340-
3270 (CH arom), 1690 (OCONH), 1660 (NHCO). Anal.
(C17H18N2O3) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e p-tolu id id e (15): yield 2
g, 72%; mp 160-162 °C (lit.31 mp 158-160 °C); TLC Rf 0.9
(methylene chloride/acetone, 1:1); 13C NMR (CDCl3) 20.9 (CH3),
45.6 (NH-CH2), 67.5 (C6H5-CH2), 120.1, 128.2, 128.4, 128.7,
129.6, 134.4 (CH arom), 134.8, 136.0 (C arom), 156.9 (O-CO-
NH), 167.0 (CH2-CO-NH); MS m/z 299 (MH+); IR 3320 (CH
arom), 1690 (OCONH), 1610 (NHCO). Anal. (C17H18N2O3)
C,H,N.
N-(Ben zyloxyca r bon yl)glycin e ben zyla m id e (16): yield
5 g, 92%; mp 119-120 °C (lit.31 mp 116-118 °C); TLC Rf 0.76
(methylene chloride/acetone, 1:1); 13C NMR (CDCl3) 43.6 (CH2-
C6H5), 44.8 (NH-CH2-CO), 67.3 (C6H5-CH2-O), 127.7, 127.8,
128.1, 128.3, 128.6, 128.8 (CH arom), 136.0, 137.7 (C arom),
156.6 (O-CO-NH), 166.7 (CH2-CO-NH); MS m/z 299 (MH+); IR
3320-3300 (CH arom), 1690 (OCONH), 1640 (NHCO). Anal.
(C17H18N2O3) C,H,N.
N-(Ben zyloxyca r bon yl)glycin e p h en eth yla m id e (17):
yield 2.3 g, 46%; mp 109-110 °C (lit.31 mp 104-107 °C); TLC
Rf 0.82 (methylene chloride/acetone, 1:1); 13C NMR (CDCl3)
6. Acu te Neu r otoxicity: At the time of the peak effect of
the MES test, the animal was subjected to the rotorod test.38

N-(Benzyloxycarbonyl)glycines as New Anticonvulsants
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 1 29
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to maintain equilibrium on a rod rotating at 6 rpm for 1 min
in each of three trials.
7. Ra d ioliga n d Bin d in g Assa ys: Male Wistar rats (bred
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and spinal cord were carefully removed and stored at -80 °C
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tions (0.3 mg of protein/mL, final volume 1 mL) were incubated
for 15 min at 4 °C in sodium phosphate buffer (50 mM, pH
7.4) containing [3H]strychnine (New England Nuclear; specific
activity 906.5 GBq/mmol, 24.5 Ci/mmol, final concentration 2
nM). Nonspecific binding was determined with 10 mM
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