Design and evaluation of affinity labels of functionalized amino acid anticonvulsants

Article abstract of DOI:10.1021/jm020225f

Studies have shown that functionalized amino acids (FAA) exhibit outstanding activity in the maximal electroshock-induced seizure (MES) test in rodents. Affinity labels patterned in part after the potent antiepileptic (R)-N-benzyl-2-acetamido-3-methoxypropionamide ((R)-2) have been prepared as mechanistic probes to learn the pharmacological basis for FAA function. The chemical reactivity of the affinity labels with nucleophiles was assessed, and the labels were evaluated in in vitro radioligand assays and in the MES tests in rodents. The affinity labels did not bind to receptors known to effect seizure spread. Three affinity labels, (R,S)-N-benzyl-2-acetamido-6-isothiocyanatohexanamide ((R,S)-5), (R)-N-(4-isothiocyanatobenzyl)-2-acetamido-3-methoxypropionamide ((R)-6), and (R)-N-(3-isothiocyanatobenzyl)-2-acetamido-3-methoxy-propionamide ((R)-7), possessed excellent in vivo anticonvulsant activity and exhibited maximal activity at later time periods than typically observed for FAA. The anticonvulsant activity of 6 and 7 resided primarily in the (R)-enantiomer and the activity of (R)-6 and (R)-7 in rats (po) exceeded that of phenytoin. The chemical properties, pharmacological profile, and marked stereospecificity associated with 6 and 7 anticonvulsant activity make these compounds useful pharmacological tools for the study of the mode of action of FAA.

Full text of DOI:10.1021/jm020225f

4762
J . Med. Chem. 2002, 45, 4762-4773
Design a n d Eva lu a tion of Affin ity La bels of F u n ction a lized Am in o Acid
An ticon vu lsa n ts
Arnaud LeTiran,† J ames P. Stables,‡ and Harold Kohn*,§
Department of Chemistry, University of Houston, Houston, Texas 77204-5641, Epilepsy Branch,
National Institute of Neurological Disorders and Stroke, National Institutes of Health, Federal Building, Room 114,
Bethesda, Maryland 20892-9020, and Division of Medicinal Chemistry and Natural Products, School of Pharmacy,
University of North Carolina, Chapel Hill, North Carolina 27599-7360
Received May 24, 2002
Studies have shown that functionalized amino acids (FAA) exhibit outstanding activity in the
maximal electroshock-induced seizure (MES) test in rodents. Affinity labels patterned in part
after the potent antiepileptic (R)-N-benzyl-2-acetamido-3-methoxypropionamide ((R)-2) have
been prepared as mechanistic probes to learn the pharmacological basis for FAA function. The
chemical reactivity of the affinity labels with nucleophiles was assessed, and the labels were
evaluated in in vitro radioligand assays and in the MES tests in rodents. The affinity labels
did not bind to receptors known to effect seizure spread. Three affinity labels, (R,S)-N-benzyl-
2-acetamido-6-isothiocyanatohexanamide ((R,S)-5), (R)-N-(4-isothiocyanatobenzyl)-2-acetamido-
3-methoxypropionamide ((R)-6), and (R)-N-(3-isothiocyanatobenzyl)-2-acetamido-3-methoxy-
propionamide ((R)-7), possessed excellent in vivo anticonvulsant activity and exhibited maximal
activity at later time periods than typically observed for FAA. The anticonvulsant activity of
6 and 7 resided primarily in the (R)-enantiomer and the activity of (R)-6 and (R)-7 in rats (po)
exceeded that of phenytoin. The chemical properties, pharmacological profile, and marked
stereospecificity associated with 6 and 7 anticonvulsant activity make these compounds useful
pharmacological tools for the study of the mode of action of FAA.
Functionalized amino acids (FAA, 1) are a new class
Affinity labeling has emerged as a powerful technique
useful for elucidating the structural site(s) for drug
action.8 An affinity label consists of two functionally
different groups: an affinity group, which structurally
resembles the biological substrate or drug, and a reac-
tive group, which covalently modifies amino acid resi-
dues at the drug binding site. An essential premise in
this concept is that the close structural correspondence
of the drug and the affinity group permit the affinity
label to target the correct binding site.
In this study, we describe the synthesis of a family of
FAA-based electrophilic affinity labels designed to
identify the site(s) of FAA function. We report on the
reactivity of the FAA affinity labels with nucleophiles
and summarize their activities in in vivo and in vitro
pharmacological assays. The biological properties docu-
mented that the placement of electrophilic tags at select
sites within the FAA does not lead to noticeable losses
in anticonvulsant activity, and they provided evidence
that these compounds may aid future mechanistic
studies.
of potent anticonvulsants.1-3 More than 250 compounds
have been prepared and tested in animal model
systems.1-3 Representative FAA have been shown to
have activity comparable with or exceeding that of
phenytoin (dilantin) in the maximal electroshock (MES)-
induced seizure test in mice.1e-h,j The MES test is a
model of generalized tonic-clonic seizures and identifies
compounds that prevent seizure spread,4,5 and pheny-
toin is considered the prototypical agent in this model.6
(R)-N-Benzyl-2-acetamido-3-methoxypropionamide1j ((R)-
2) has emerged as the lead FAA and has entered phase
II clinical trials for the treatment of epilepsy and
neuropathic pain under Schwarz Pharma sponsorship.
Little is known about the mechanism of action of FAA.
The preclinical pharmacological profile for 2 and 1
differed from established agents such as phenytoin,
carbamazepine, lamotrigine, gabapentin, felbamate,
valproic acid, clonazepam, and ethosuximide.7 Informa-
tion concerning function is vital for maximizing the
therapeutic potential of these agents.
Resu lts a n d Discu ssion
1. Ch oices of Su bstr a tes. Four criteria were estab-
lished in our design of FAA affinity labels. First, the
compounds needed to conform to the SAR observed for
1.1 When possible, we fashioned the affinity label after
2. Second, the reactive unit within the affinity label
must have documented success in previous receptor site
inactivation studies.8 Accordingly, we selected the isothio-
cyanate,9 the R-bromoacetamide,10 and the acrylamide11
electrophilic tags. These functional groups display an
* To whom correspondence should be addressed. Phone: (919) 966-
2680. Fax: (919) 843-7835. E-mail: harold_kohn@unc.edu.
† University of Houston.
‡ National Institutes of Health.
§ University of North Carolina.
10.1021/jm020225f CCC: $22.00 © 2002 American Chemical Society
Published on Web 09/06/2002

Amino Acid Anticonvulsants
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4763
Sch em e 1. Group 1 Affinity Label Candidates:
Preparation of (R)-, (S)-, and (R,S)-10
pharmacological changes compared with the corre-
sponding unsubstituted R3 benzyl FAA.1b,e,g,j Finally,
compound 5 was structurally similar to (R,S)-81j (MES
ED50 ) 17 mg/kg (mice, ip)) and (R,S)-917 (MES ED50
)
38 mg/kg (mice, ip)). The excellent anticonvulsant
F igu r e 1. Design of functionalized amino acid affinity labels.
excellent balance between reactivity and stability; they
react with nucleophiles (e.g., thiols, amines) but do not
readily react with hydroxylic solvents, thus allowing
their dissolution in aqueous buffer solutions.8 Third, we
required syntheses that permitted preparation of
both enantiomeric forms of the affinity label. A distin-
guishing feature of 1 is the pronounced anticonvulsant
activity of the (R)-stereoisomer compared with the (S)-
isomer.1c-e,g,j Since enantioselective irreversible inacti-
vation is an important measure of receptor site speci-
ficity,12 we have prepared and tested both isomers in
select cases. Fourth, the type and placement of the
reactive group in the FAA was varied. We incorporated
this criterion to satisfy the possible structural require-
ments for affinity label-receptor site adduction8 and to
maximize the chances of identifying multiple sites of
FAA function.13
activities for 8 and 9 suggested that 5 may serve as an
effective affinity label to target the site(s) of FAA
function.
2. Syn th eses. The synthetic schemes adopted for
affinity labels 3-7 considered the reactivity of the
electrophilic tags. The isothiocyanate and R-bromoac-
etamide moieties were not expected to survive the amide
coupling and functional group deprotection reactions.
Accordingly, we introduced the electrophilic unit at a
late stage in the synthesis.
2.1. Gr ou p 1 Affin ity La bel Ca n d id a tes. Affinity
labels 3 and 417 were prepared from the common
intermediate 10 (Scheme 1). The procedure previously
employed for (R)-1018 and (R,S)-1015 was also used to
synthesize (S)-10. Synthesis of (R)-, (S)-, and (R,S)-10
began with readily available Cbz-D-, Cbz-L-, and Cbz-
DL-serine ((R)-, (S)-, and (R,S)-11), respectively.19 Com-
pound 11 was converted to 12 using benzylamine and
the mixed-anhydride coupling (MAC) procedure.20 Me-
thylation of 12 (MeI, Ag2O) followed by hydrogenolysis
of 13 afforded (R)-, (S), and (R,S)-10, which were the
precursors for group 1 affinity label candidates. Treat-
ment of either (R)-10 or (S)-10 with R-bromoacetyl
bromide afforded (R)-3 and (S)-3, respectively (Scheme
2). The overall yield for affinity labels (R)-3, (S)-3, and
(R,S)-4 was 44-71% beginning with (R)-11, (S)-11, or
(R,S)-11.
Five different FAA affinity labels (3-7) were evalu-
ated (Figure 1). For 3, 6, and 7, we prepared both the
(R)- and (S)-enantiomers for a total of eight test
compounds. The five affinity labels were divided by the
site of the electrophilic tag. In group 1 (compounds 3
and 4), the label was placed at the N terminus of the
FAA.14 In group 2 (compound 5) we incorporated the
tag at the C(2) position, and in group 3 (compounds 6
and 7) the electrophilic unit was positioned at the FAA
C terminus.
Including electrophilic tags in the core structure
affected the size and electronic properties of the FAA.
Our earlier structure-activity relationship (SAR) stud-
ies showed that in several cases, increasing the R1 group
in 1 from methyl to higher alkyl gave compounds with
significant anticonvulsant activities, suggesting the use
of either an R-bromoacetamide unit (3) or an acrylamide
moiety (4) as an electrophilic tag.1b The SAR for 1
further predicted that inclusion of an isothiocyanate
unit within the R3 benzyl unit would lead to small
Three methods were used to assess the enantiopurity
of (R)-13, (S)-13, (R)-3, and (S)-3. These were the use
of NMR and a chiral resolving agent, optical rotation,
1
and melting point. In particular, we detected in the H
NMR only a single O-methyl ether signal for (R)-13 and
(S)-13 and a single signal for the O-methyl ether and

4764 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
LeTiran et al.
Sch em e 4. Group 3 Affinity Label Candidates:
Sch em e 2. Group 1 Affinity Label Candidates:
Preparation of (R)-3 and (S)-3
Preparation of (R)-6, (S)-6, (R)-7, and (S)-7
Sch em e 3. Group 2 Affinity Label Candidates:
Preparation of (R,S)-5
bromoacetyl methylene peaks for (R)-3 and (S)-3 when
the chiral resolving agent, (R)-(-)-mandelic acid,21 was
added to the CDCl3 solutions of these compounds.1j We
also verified the enantiopurity of (R)-10, (R)-12, and (R)-
13 by comparison with reported data.18
the formation of N,O-diacetyl serine (27) in the first
step. The binary mixtures (19 + 20, 23 + 24) formed in
2.2. Gr ou p 2 Affin ity La bel Ca n d id a tes. Com-
mercially available Nꢀ-Cbz-DL-lysine (Novabiochem)
((R,S)-14) served as the starting material for the
synthesis of the affinity label (R,S)-5 (Scheme 3). Using
a previously reported procedure,22 we acetylated (R,S)-
14 to get (R,S)-15 in near-quantitative yield. Acid (R,S)-
15 was coupled with benzylamine using 1-[3-(dimeth-
yla m in o)pr opyl]-3-et h ylca r bodiim ide
(E DCI)/1-
each of these reactions were separated by column
chromatography. Furthermore, 20 and 24 were con-
verted (91-99% yield) to 19 and 23, respectively,
without racemization upon treatment with basic MeOH.
Methylation of 19 and 23 with MeI and Ag2O provided
21 and 25, respectively. Catalytic reduction (H2, PtO2)
of the aromatic nitro groups in 21 and 25 gave amines
22 and 26, respectively, which served as immediate
precursors for the affinity labels. Treatment of THF
solutions containing (R)-22, (S)-22, (R)-26, or (S)-26 with
DPT24 efficiently produced (R)-6, (S)-6, (R)-7, or (S)-7,
respectively. The overall yield for group 3 affinity labels
was 21-29% beginning with either D- or L-serine (18)
(five steps). Spectroscopic evidence for the isothiocyan-
ate unit in 6 or 7 was obtained both by detecting the
NCS carbon resonance (135.6-135.8 ppm) in the 13C
NMR25 and by observing the NCS absorption band
(2125-2183 cm-1) in the FT-IR.26 We documented the
enantiopurity of group 3 affinity labels (R)- and (S)-6
and 7 and their synthetic precursors ((R)- and (S)-19-
26) by determining their NMR chemical shift values in
the presence of mandelic acid (e.g., N-acetyl methyl
group, O-methyl group), optical activities, and melting
points.
hydroxybenzotriazole (HOBt) to give (R,S)-16 (89%
yield). Hydrogenolysis (H2, Pd/C in MeOH)18,23 of the
Cbz protecting group provided lysine derivative (R,S)-
17 in a near-quantitative yield. Conversion of the amine
(R,S)-17 to the affinity label (R,S)-5 was accomplished
with di-2-pyridyl thionocarbonate24 (DPT) (64% overall
yield from (R,S)-14). In agreement with our structural
assignment for isothiocyanate (R,S)-5, we observed a
signal at 130.2 ppm in the 13C NMR spectrum25 and two
absorption bands at 2184 and 2110 cm-1 in the FT-IR26
spectrum for this moiety.
2.3. Gr ou p 3 Affin ity La bel Ca n d id a tes. The
preparation of the group 3 affinity label candidates (R)-
6, (S)-6, (R)-7, and (S)-7 is outlined in Scheme 4. Our
protocol is similar to that used by Choi and Kohn1j for
the synthesis of (R)-2 except that p- or m-nitrobenz-
ylamine hydrochloride (Aldrich) was substituted for
benzylamine. Accordingly, beginning with enantiopure
serine ((R)-18, (S)-18) acetylation and then treatment
with either p- or m-nitrobenzylamine hydrochloride
using MAC20 methodology gave binary mixtures of
amides 19 and 20 or amides 23 and 24, respectively.
The diacetylated products (20, 24) likely resulted from

Amino Acid Anticonvulsants
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4765
Ta ble 1. Selected Physical and Pharmacological Data for FAA Affinity Labels and Their Reference Compounds
mice (ip)a rat (po)b
Tox,e TD50
f
f
compd
(R,S)-2
mpc
MES,d ED50
PI
MES,d ED50
Tox,e TD50
PI
121-122
8.3 [0.5]
(7.9-9.8)
4.5 [0.5]
(3.7-5.5)
>100, <300
38 [0.25]
(35-45)
43 [0.25]
(38-47)
5.2
6.0
3.8 [2]
(2.9-5.5)
3.9 [0.5]
(2.6-6.2)
>30
37 [1]
(320-520)
>500
101
(R)-2
143-144
27 [0.25]
(26-28)
>128
(S)-2
(R,S)-9
143-144
138-139
>300
>30
>30
160 [0.25]
(150-170)
>30, <100
>30, <100
>300
4.2
∼30
(R)-3
(S)-3
(R,S)-4
(R,S)-5
(R)-6
169-171 (dec)
169-171 (dec)
138-139
98-99 (dec)
172-173
>300
g
g
>30
>30
4.2 [4]
(2.4-8.0)
>180
28 [4]
(19-40)
>150
30 [4]
(22-39)
9.1 [5]
(7.6-12)
490 [0.5]
(350-730)
g
g
>30, <100
>100, <300
>30, <100
24 [0.5]
(21-27)
>100, <300
74 [1]
(61-85)
>100, <300
9.5 [2]
(8.1-10)
22 [1]
>30
>30
>250
>30, <100
47 [0.25]
(43-50)
>30, <100
120 [0.25]
(93-170)
>100, <300
66 [2]
(53-72)
69 [0.5]
(63-73)
430 [0.25]
(370-450)
2.0
1.6
>59
(S)-6
(R)-7
171-172
161-163
>30
>250
>9.0
(S)-7
162-163
>150
i
phenytoinh
6.9
3.2
1.6
>100
6.7
phenobarbitalh
valproateh
61 [0.5]
(15-23)
270 [0.25]
(250-340)
(44-96)
280 [0.5]
(190-350)
0.6
a
The compounds were administered intraperitoneally. ED50 and TD50 values are in mg/kg. Numbers in parentheses are 95% confidence
b
intervals. The dose effect data was obtained at the “time of peak effect” (indicated in hours in the brackets). The compounds were
administered orally. c Melting points (°C) are uncorrected. MES ) maximal electroshock seizure test. e Tox ) neurologic toxicity
d
determined from rotorod test. f PI ) protective index (TD50/ED50). Not determined. Reference 31. No ataxia observed up to 3000 mg/
g
h
i
kg.
3. Ch em ica l Rea ctivity. A key criterion for affinity
labels 3-7 is that they readily, efficiently, and selec-
tively react with biological nucleophiles. Accordingly, we
have treated each compound with surrogate nucleo-
philes to test whether the reactive label functioned
as designed and to predict the reaction conditions
necessary for irreversible inactivation of the receptor
site(s). We chose to use ethanethiol, ethylamine, and
imidazole as mimics for cysteine-, lysine-, and histidine-
containing peptides, respectively. The affinity labels
were treated with these nucleophiles at room temper-
ature (0.5-20 h) in aqueous buffer solutions (0.1 M KH2-
PO4/NaOH, pH 7.4), and the reaction was monitored by
TLC. For those compounds (3, 6, 7) for which the two
enantiomers had been individually prepared, we chose
one of the stereoisomers for evaluation. Probes 3, 6, and
7 reacted with ethanethiol and ethylamine. For 5, we
used only ethylamine. In each case, the products (28-
34) were isolated and identified (see Supporting Infor-
mation). For 4, we observed no reaction with any of the
test nucleophiles, and we did not detect any product
formation when 3 and 5-7 were treated with imidazole.
We found that 3-7 were moderately stable in hydroxylic
solvents (aqueous KH2PO4/KOH buffer, pH 7.4) with
approximate half-lives of 2 h for 5 and >1 day for 3, 4,
6, and 7.
4. P h a r m a cologica l Eva lu a tion . All the affinity
labels (Table 1) were tested for anticonvulsant activity
using the procedure described by Stables and Kupfer-
berg.4 We wished to learn if their in vivo pharmacologi-
cal profile followed the general profile previously ob-
served for FAAs (1).1 Moreover, we anticipated that
whole animal pharmacological studies would provide
preliminary information concerning their utility as
affinity labels. First, we expected that useful agents
would likely display significant anticonvulsant activities
in the MES-induced seizure test upon administration
to mice (ip) and rats (po). Second, we hypothesized that
if the affinity label irreversibly modified the receptor
site(s), then the duration of action of the affinity label
may exceed that of the reference FAA.8 We understood
that both of these experimental predictions hinged upon
the assumption that the affinity labels survived admin-
istration and passage to the receptor site. The results
were compared with findings previously reported for 2,1j
9,17 and the proven antiepileptic agents phenytoin,
phenobarbital, and valproate.27
The FAA affinity labels were administrated intrap-
eritoneally (ip) to mice and orally (po) to rats. Table 1
lists the results obtained from qualitative testing in mice
(ip) along with quantitative mice (ip) and rat (po)
evaluations. We include in this table the MES ED50
values required to prevent tonic extension of the limbs
and the median neurologically impairing dose (TD50
)
values using the rotorod test.28 Finally, the protective

4766 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
LeTiran et al.
index (PI ) TD50/(MES ED50)) for these adducts, when
provided by this compound, since testing was ended
after 4 h). When the dose was 20 mg/kg, 25% of the
animals were still protected after 6 h. These results
demonstrated that maximal anticonvulsant activity for
(R)-6 and (R)-7 was achieved at a later time period than
our lead compound (R)-2 and that seizure protection was
likely maintained for longer time periods. At least four
hypotheses can be offered to account for these findings.
First, (R)-6 and (R)-7 acted as affinity labels and
covalently modified the putative receptor providing
prolonged seizure protection. Second, the site and func-
tion of affinity label candidates (R)-6 and (R)-7 differed
from those of (R)-2 and other FAA. Third, (R)-6 and
(R)-7 underwent oral bioactivation, metabolism, or both
to a species that exhibits increased biological activity,
and the TPE reflects this change. Finally, (R)-6 and
(R)-7 have different absorption properties compared
with (R)-2. Our data do not permit us to distinguish
among these possibilities, but this aspect of the phar-
macological data warrants future attention.
appropriate, is shown.
4.1. Gr ou p I Affin ity La bel Ca n d id a tes. Inspection
of the composite data in Table 1 revealed that group 1
(3, 4) affinity label candidates displayed moderate or
poor anticonvulsant activity in the MES-induced seizure
test. The estimated ED50 value for these compounds was
>30 mg/kg in mice (ip) and rats (po). The low activity
observed for 3 and 4 was in contrast to the potent
activity reported for 2.1j Even more surprising was the
difference in the anticonvulsant activities (mice, ip) for
(R)-3 (ED50 > 300 mg/kg) and (S)-3 (30 mg/kg < ED50
< 100 mg/kg). This finding is opposite the stereochem-
ical pattern learned in our SAR studies for FAAs.1c-e,g,j
4.2. Gr ou p II Affin ity La bel Ca n d id a tes. Com-
pound (R,S)-5 displayed moderate protection against
MES-induced seizures in mice (ip) (30 mg/kg < ED50
<
100 mg/kg). Significantly, (R,S)-5 showed only modest
loss of anticonvulsant activity compared with the refer-
ence anticonvulsant FAA (R,S)-9 (ED50 ) 38 mg/kg).
Further, (R,S)-5 showed significant inhibitory activity
at 4 h (mice, ip). By comparison, the “time of peak effect”
(TPE) for (R,S)-9 was 0.25 h. Additional whole-animal
pharmacological testing was not conducted because of
the high neurological toxicity observed in mice (30 mg/
kg < TD50 < 100 mg/kg) and the death of both test
animals at the 300 mg/kg dose.
FAA constitute a novel class of anticovulsant agents
whose mode of action remains unknown. Prior to this
study, we reported the anticonvulsant activities of four
pair of stereoisomers.1c-e,g,j In each case, the (R)-
enantiomer was more potent (10× to 22×) than the (S)-
isomer. This finding led to our suggestion that FAA
function, in part, was associated with stereoselective
binding to a receptor or enzyme.1d Other factors may
account for these differences, and so we tested all the
FAA affinity labels and (R)- and (S)-2 against receptors
and channels known to be involved in seizure processes.
We chose GABAA (agonist), GABAA (bzp), NMDA (PCP),
NMDA (MK-801), Na+ (type 2), and Ca2+ (type L) sites.
The tests were conducted through the auspices of the
National Institute of Mental Health (NIMH) Psychoac-
tive Drug Screening Program. None of the affinity labels
or (R)- and (S)-2 displaced the radioligand in these
receptor assays when tested at 10 µM concentrations.
These findings indicate that the receptors are probably
not involved in the mechanism of FAA action.
4.3. Gr ou p III Affin ity La bel Ca n d id a tes. Evalu-
ation of the group 3 affinity labels 6 and 7 showed that
placement of an isothiocyanate moiety at either the 4′-
or the 3′-phenyl sites, respectively, within the benzyl
unit provided compounds effective for the control of
MES-induced seizures in mice (ip). We found, of note,
that the (R)-stereoisomer in both series was more potent
than the (S)-isomer and that the activity of (R)-6 (ED50
) 24 mg/kg) matched that of phenobarbital (ED50 ) 22
mg/kg).
The pharmacological activities of (R)-6 and (R)-7
warranted further evaluation. Table 1 lists the MES
ED50, TD50, and PI values for these compounds as a
result of oral administration (po) to rats. Significantly,
the ED50 values for (R)-6 (ED50 ) 4.2 mg/kg) and (R)-7
(ED50 ) 28 mg/kg) compared favorably with phenytoin
(ED50 ) 30 mg/kg). The excellent activity of (R)-6
coupled with its low neurological toxicity (>250 mg/kg)
led to a PI value greater than 59. A distinguishing
pharmacological feature for FAAs is their stereochem-
ical differences in anticonvulsant activities.1c-e,g,j For 2,
the eudismic ratio29 was >7.6 in rats. We observed
similar high eudismic ratios for 6 and 7 with values
exceeding 43 and 5, respectively.
Con clu sion s
We have prepared three FAA affinity labels ((R,S)-5,
(R)-6, and (R)-7) that exhibit significant anticonvulsant
activities in animal models of MES-induced seizures.
The three compounds readily reacted with either sulfur-
or nitrogen-containing nucleophiles and underwent a
slow change in hydroxylic solvents. For 6 and 7, we
demonstrated that the principal anticonvulsant activity
resided in the (R)-stereoisomer. The chiral specificity
for 5 anticonvulsant activity has not been determined,
but both enantiomers should be accessible from com-
mercial materials using the methodology outlined in
Scheme 3. The FAA and (R)- and (S)-2 were tested at
receptors previously identified to be important for
controlling seizure spread using in vitro radioligand
binding assays. No significant binding was observed for
any of the test compounds, a finding in line with the
preclinical pharmacological profile that showed 1 dif-
fered from established antiepileptic agents.7 Mechanistic
studies concerning FAA function are underway in which
FAA and affinity labels 4-7 serve as important test
substrates.
The in vivo pharmacological data documented that
(R)-6 and (R)-7 were the most effective anticonvulsants
within our family of affinity labels. Significantly, we
observed that the TPE for (R)-6 and (R)-7 was 4 h (rats,
po), which was 8 times longer than that for (R)-2 (0.5
h). Protective activity for (R)-2 was highest at 30 min
and then gradually diminished. Correspondingly, (R)-6
showed maximal activity after 4 h with three out of four
animals protected after 2 and 6 h at 13 mg/kg.30
Similarly, we observed that (R)-7 afforded full protection
after 4 h at 30 mg/kg with 25% of the animals protected
after 0.5 h (we do not have the length of full protection

Amino Acid Anticonvulsants
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4767
(CDCl3) δ 3.34 (s, OCH3), 3.49 (dd, J ) 6.6, 9.2 Hz, CHH′OCH3),
3.84 (dd, J ) 3.9, 9.2 Hz, CHH′OCH3), 4.31-4.36 (m, CH),
4.46 (d, J ) 5.7 Hz, CH2NH), 5.10 (s, CH2OC(O)), 5.65-5.75
(m, NH), 6.68-6.75 (m, NH), 7.22-7.33 (m, 10 PhH). Addition
of excess (R)-(-)-mandelic acid to a CDCl3 solution of (S)-13
gave only one signal for the ether methyl protons. 13C NMR
(CDCl3) δ 43.7 (CH2Ph), 54.6 (CH), 59.2 (OCH3), 67.4 (CH2-
OC(O)), 72.2 (CH2OCH3), 127.5, 128.2, 128.3, 128.6, 128.8,
136.1, 138.0 (2 C6H5), 156.1 (C(O)O), 169.9 (C(O)NH); MS
(+CI) m/z (rel intensity) 344 (20), 343 (M+ + 1, 100), 299 (23);
Mr (+CI) 343.166 41 [M+ + 1] (calcd for C19H23N2O4 343.165 78).
Anal. (C19H22N2O4‚0.2H2O) C, H, N.
Exp er im en ta l Section
Gen er a l Meth od s. Melting points were determined in open
capillary tubes using a Thomas-Hoover melting point ap-
paratus and are uncorrected. Infrared spectra (IR) were run
on an ATI Mattson Genesis FT-IR spectrometer. Absorption
values are expressed in wavenumbers (cm-1). Optical rotations
were obtained on Perkin-Elmer 241 MC and J asco P-1030
polarimeters at the sodium D line (589 nm) using a 1 dm path
length cell. Nuclear magnetic resonance spectra were mea-
sured at 300 MHz for 1H NMR and at 75 MHz for 13C NMR
either on General Electric QE-300 NMR or Varian Gemini
2000 spectrometers. Chemical shifts (δ) are reported in parts
per million (ppm) downfield from tetramethylsilane. Low-
resolution mass spectra (CI+) were obtained with a Varian
MAT CH-5 spectrometer by Dr. M. Moini at the University of
TexassAustin. The high-resolution chemical ionization mass
spectrum was performed on a Finnigan MAT TSQ-70 by Dr.
M. Moini at the University of TexassAustin. Microanalyses
were provided by Atlantic Microlab, Inc. (Norcross, GA).
Analytical thin-layer chromatography (TLC) and preparative
TLC (PTLC) were performed on precoated silica gel slides (20
cm × 20 cm; Sigma Z12272-6). All column chromatography
separations were performed on Merck silica gel (SiO2) (grade
9385, 230-400 mesh, 60 Å). CH2Cl2 was distilled from CaH2,
and THF was distilled from blue sodium benzophenone ketyl.
Yields reported are for purified products and were not opti-
mized.
Gen er a l P r oced u r e for th e P r ep a r a tion N-Ben zyla -
m id e Am in o Acid s Der iva tives Usin g Mixed -An h yd r id e
Cou p lin g (MAC) (Meth od A).1j,20 A dry THF solution of the
carboxylic acid (∼0.5-2.0 M) was cooled to -78 °C under Ar,
and 4-methylmorpholine (NMM) (1.1-2.2 equiv) was added.
After the solution was stirred (2 min), isobutyl chloroformate
(IBCF) (1.1-1.25 equiv) was added, leading to the precipitation
of a white solid. The reaction was allowed to proceed for an
additional 2 min, and then benzylamine (1.1-1.25 equiv) was
added at -78 °C. The reaction mixture was allowed to stir at
room temperature (30 min to 3 h), and then the insoluble salts
were filtered. The organic layer was concentrated in vacuo,
and the product was purified by column chromatography on
SiO2 gel.
(S)-N-Ben zyl-2-a m in o-3-m et h oxyp r op ion a m id e ((S)-
10). A methanolic solution (100 mL) of (S)-13 (3.85 g, 11.2
mmol) was hydrogenated (1 atm) in the presence of 10% Pd/C
(0.60 g) at room temperature (4 h). The mixture was filtered
through a bed of Celite, and the clear filtrate was evaporated
in vacuo. Purification of the product by column chromatogra-
phy (SiO2; 1:9 MeOH/CHCl3) gave (S)-10 (2.15 g, 91%) as a
pale-yellow oil: [R]23 +1.8° (c 0.8, MeOH); Rf ) 0.33 (1:9
D
MeOH/CHCl3); IR (liquid film) 3312, 3063, 2926, 2894, 2825,
1660, 1524 cm-1; 1H NMR (CDCl3) δ 1.69 (br s, NH2), 3.38 (s,
OCH3), 3.59-3.69 (m, CH and CH2), 4.43 (dd, J ) 6.0, 14.7
Hz, CHH′NH), 4.49 (dd, J ) 6.0, 14.7 Hz, CHH′NH), 7.25-
7.36 (m, 5 PhH), 7.75-7.85 (m, NH); 13C NMR (CDCl3) δ 43.1
(CH2NH), 54.9 (CH), 58.8 (OCH3), 74.6 (CH2OCH3), 127.3,
127.5, 128.6, 138.4 (C6H5), 172.8 (C(O)NH); MS (+CI) m/z (rel
intensity) 210 (13), 209 (M+ + 1, 100); Mr (+CI) 209.129 34
[M+ + 1] (calcd for C11H17N2O2 209.129 00). Anal. (C11H16N2O2‚
0.22H2O) C, H, N.
(R)-N-Ben zyl-2-(2-br om o)a ceta m id o-3-m eth oxyp r op i-
on a m id e ((R)-3). A mixture of (R)-10 (1.33 g, 6.39 mmol) in
CH2Cl2 (70 mL), aqueous saturated NaHCO3 (70 mL), and
aqueous 2 N Na2CO3 (15 mL) was cooled to 5 °C and treated
with R-bromoacetyl bromide (0.85 mL, 9.76 mmol). The reac-
tion mixture was allowed to warm to room temperature, stirred
(10 min), and then poured into H2O (200 mL). The organic
layer was removed, and the aqueous layer was washed with
CH2Cl2 (2 × 50 mL). The combined CH2Cl2 extracts were
washed with aqueous saturated NaHCO3 (50 mL) and brine
(50 mL). The solvent was evaporated in vacuo, and the residue
recrystallized (acetone) to give 1.64 g (78%) of (R)-3 as a white
(S)-N-Ben zyl-2-N-(ben zyloxycar bon yl)am in o-3-h ydr ox-
yp r op ion a m id e ((S)-12). Utilizing method A, (S)-1119b,c (2.00
g, 8.4 mmol), NMM (1.4 mL, 12.7 mmol), IBCF (1.4 mL, 10.8
mmol), and benzylamine (1.1 mL, 11.6 mmol) gave crude (S)-
12. The product was purified by column chromatography (SiO2;
1:9 MeOH/CHCl3) to obtain 1.97 g (72%) of pure (S)-12 as a
solid: mp 169-171 °C (dec); [R]23 +3.5° (c 1.5, DMSO); Rf )
D
0.55 (EtOAc); IR (KBr) 3284, 3075, 2953, 1634, 1551 cm-1; 1H
NMR (CDCl3) δ 3.40 (s, OCH3), 3.46 (dd, J ) 8.1, 9.0 Hz,
CHH′OCH3), 3.83 (dd, J ) 4.1, 9.0 Hz, CHH′OCH3), 3.88 (s,
CH2Br), 4.43-4.57 (m, CH and CH2NH), 6.62-6.72 (m, NH),
7.25-7.37 (m, 5 PhH and NH). Addition of excess (R)-(-)-
mandelic acid to a CDCl3 solution of (R)-3 gave only one signal
for the bromoacetyl methylene protons and one signal for the
ether methyl protons. 13C NMR (CDCl3) δ 28.8 (CH2Br), 43.9
(CH2NH), 53.1 (CH), 59.4 (OCH3), 71.5 (CH2OCH3), 127.7,
127.8, 128.9, 137.9 (C6H5), 166.1 (C(O)CH2Br), 169.4 (CHC(O));
MS (+CI) m/z (rel intensity) 332 (16), 331 (86), 330 (21), 329
(M+ + 1, 100), 251 (11); Mr (+CI) 329.048 54 [M+ + 1] (calcd
for C13H1879BrN2O3 329.050 08). Anal. (C13H17BrN2O3) C, H,
N.
white solid: mp 148-149.5 °C; [R]23 -5.4° (c 1.04, MeOH);
D
Rf ) 0.51 (1:9 MeOH/CHCl3); IR (KBr) 3294, 1689, 1645, 1535
cm-1 1H NMR (DMSO-d6) δ 3.53-3.63 (m, CH2OH), 4.03-
;
4.10 (m, CH), 4.27 (d, J ) 5.7 Hz, CH2NH), 4.88 (t, J ) 5.1
Hz, OH), 5.02 (s, CH2OC(O)), 7.17-7.35 (m, 10 PhH and NHC-
(O)O), 8.41 (t, J ) 5.7 Hz, NHC(O)); 13C NMR (DMSO-d6) δ
42.0 (CH2NH), 57.3 (CH), 61.8 (CH2OH), 65.5 (OCH2Ph),
126.6, 127.0, 127.7, 128.1, 128.3, 136.9, 139.3 (2 C6H5), 155.9
(C(O)O), 170.1 (C(O)NH); MS (+CI) m/z (rel intensity) 330 (19),
329 (M+ + 1, 87), 285 (19), 221 (100), 203 (13), 191 (18); Mr
(+CI) 329.149 18 [M+ + 1] (calcd for C18H21N2O4 329.150 13).
Anal. (C18H20N2O4) C, H, N.
(S)-N-Ben zyl-2-(2-br om o)a ceta m id o-3-m eth oxyp r op i-
on a m id e ((S)-3). Using (S)-10 (1.85 g, 8.89 mmol), R-bro-
moacetyl bromide (1.3 mL, 14.8 mmol), and the preceding
procedure gave after recrystallization (acetone) 2.75 g (94%)
Gen er a l P r oced u r e for th e P r ep a r a tion of 3-Meth oxy-
2-a m id op r op ion a m id e Der iva tives (Meth od B). To a CH3-
CN solution of alcohol (∼0.05-0.1 M) was successively added
Ag2O (5 equiv) and MeI (10 equiv) at room temperature. The
reaction mixture was maintained at room temperature (1-3
days) and filtered, and the solvent was evaporated in vacuo.
The residue was purified by column chromatography on SiO2.
(S)-N-Ben zyl-2-N-(b en zyloxyca r bon yl)a m in o-3-m et h -
oxyp r op ion a m id e ((S)-13). Utilizing method B, (S)-12 (3.96
g, 12.1 mmol), Ag2O (14.0 g, 60 mmol), and MeI (7.5 mL, 0.12
mol) gave crude (S)-13 after 3 days. The product was purified
by column chromatography (SiO2; 1:9 MeOH/CHCl3) to obtain
of (S)-3 as a white solid: mp 169-171 °C (dec); [R]23 -2.9° (c
D
2.04, DMSO); Rf ) 0.55 (EtOAc); IR (KBr) 3287, 3074, 2953,
1633, 1550 cm-1; 1H NMR (CDCl3) δ 3.39 (s, OCH3), 3.47 (dd,
J
) 8.8, 8.8 Hz, CHH′OCH3), 3.82 (dd, J ) 4.1, 8.8 Hz,
CHH′OCH3), 3.87 (s, CH2Br), 4.42-4.56 (m, CH and CH2NH),
6.65-6.78 (m, NH), 7.25-7.37 (m, 5 PhH and NH). Addition
of excess (R)-(-)-mandelic acid to a CDCl3 solution of (S)-3 gave
only one signal for the bromoacetyl methylene protons and one
signal for the ether methyl protons. Addition of excess (R)-
(-)-mandelic acid to a CDCl3 solution of (R)-3 and (S)-3 (1:1
ratio) gave two signals for the bromoacetyl methylene protons
(δ 3.85 and 3.86) and two signals for the ether methyl protons
(δ 3.36 and 3.37). 13C NMR (DMSO-d6) δ 29.4 (CH2Br), 42.0
3.86 g (94%) of (S)-13 as a white solid: mp 130-132 °C; [R]24
D
-3.3° (c 1.1, MeOH); Rf ) 0.31 (1:1 hexanes/EtOAc); IR (KBr)
3295, 3059, 3030, 2880, 1688, 1641, 1541 cm-1 1H NMR
;

4768 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
LeTiran et al.
(CH2NH), 53.0 (CH), 58.2 (OCH3), 71.9 (CH2OCH3), 126.6,
126.9, 128.1, 139.0 (C6H5), 166.0 (C(O)CH2Br), 168.9 (CHC(O)).
The structural assignments were in agreement with the
HETCOR experiment. MS (+CI) m/z (rel intensity) 332 (12),
331 (74), 330 (17), 329 (M+ + 1, 100); Mr (+CI) 329.049 24 [M+
128.1, 128.3, 129.4, 139.9 (C6H5), 173.0, 174.3 (2 C(O)); MS
(+CI) m/z (rel intensity) 279 (15), 278 (M+ + 1, 100); Mr (+CI)
278.186 30 [M+ + 1] (calcd for C15H24N3O2 278.186 85). Anal.
(C15H23N3O2‚H2O) C, H, N.
(R,S)-N-Ben zyl-2-a ceta m id o-6-isoth iocya n a toh exa n a -
m id e ((R,S)-5). A dry THF/DMF (2:1, 60 mL) solution of (R,S)-
17 (1.37 g, 4.94 mmol) was added (10 min) to a THF solution
(25 mL) of DPT (1.15 g, 4.94 mmol). The reaction solution was
stirred at room temperature (1 h), and then the solvent was
evaporated in vacuo. The product was purified by column
chromatography (SiO2; 4:1 EtOAc/hexanes) to give 1.15 g (73%)
of pure (R,S)-5 as a white solid: mp 98-99 °C (dec); Rf ) 0.36
(4:1 EtOAc/hexanes); IR (KBr) 3274, 3084, 2935, 2184, 2110,
+ 1] (calcd for C13H1879BrN2O3 329.050 08). Anal. (C13H17
BrN2O3‚0.2C3H6O)) C, H, N.
-
(R,S)-2-Aceta m id o-6-N-(ben zyloxyca r bon yl)a m in oh ex-
a n oic Acid ((R,S)-15).22b (R,S)-6-N-(Benzyloxycarbonyl)-DL-
lysine ((R,S)-14) (3.17 g, 11.3 mmol) was dissolved in aqueous
0.5 N NaOH (28 mL) and cooled in an ice bath, and Ac2O (1.52
mL, 15.8 mmol) and TEA (3.2 mL, 32.2 mmol) were added in
two portions over 30 min. The solution was stirred at 0 °C (1
h) and then at room temperature (1 h). The reaction solution
was washed with Et2O (15 mL), and the layers were separated.
The pH of the aqueous layer was adjusted to 2 with an aqueous
3 N HCl solution, and then the layer was extracted with EtOAc
(100 mL). The organic layer was washed with brine (20 mL),
dried (Na2SO4), and evaporated in vacuo to give 3.65 g (99%)
of (R,S)-15 as a white solid: mp 119-120 °C (lit.22b 115-116
°C); Rf ) 0.22 (95:4:1 CHCl3/MeOH/AcOH); 1H NMR (CD3OD)
δ 1.38-1.46 (m, C(4)H2), 1.48-1.55 (m, C(5)H2), 1.63-1.76 (m,
C(3)HH′), 1.79-1.91 (m, C(3)HH′), 1.98 (s, CH3C(O)), 3.12 (t,
J ) 6.9 Hz, C(6)H2), 4.34 (dd, J ) 4.9, 8.8 Hz, CH), 5.06 (s,
CH2OC(O)), 7.27-7.35 (m, 5 PhH); 13C NMR (CD3OD) δ 22.4
(CΗ3C(Ã)), 24.1 (C(4)H2), 30.5 (C(5)H2), 31.3 (C(3)H2), 41.6
(C(6)H2), 53.7 (CH), 67.4 (OCH2Ph), 128.8, 129.0, 129.5, 138.5
(C6H5), 159.0 (C(O)O), 173.4, 175.5 (2 C(O)).
1
1638, 1554 cm-1; H NMR (CDCl3) δ 1.27-1.45 (m, C(4)H2),
1.54-1.80 (m, C(3)H2 and C(5)H2), 1.89 (s, CH3C(O)), 3.39 (t,
J ) 6.3 Hz, C(6)H2), 4.28 (dd, J ) 5.7, 14.8 Hz, CHH′Ph), 4.37
(dd, J ) 5.5, 14.8 Hz, CHH′Ph), 4.59-4.67 (m, CH), 7.19-
7.28 (m, 5 PhH), 7.45 (d, J ) 8.4 Hz, NHCH), 8.01 (app br t,
J ) 5.7 Hz, NHCH2); 13C NMR (CDCl3) δ 22.6 (C(4)H2), 22.8
(CΗ3C(Ã)), 29.5 (C(5)H2), 31.9 (C(3)H2), 43.3 (CH2Ph), 44.8
(C(6)H2), 52.7 (CH), 127.3 (C4′), 127.5, 128.6 (C2′ and C3′),
130.2 (NCS), 138.1 (C1′), 170.7, 172.1 (2 C(O)). The 1H NMR
and 13C NMR structural assignments were in agreement
with the HETCOR and DEPT experiments. MS (+CI) m/z
(rel intensity) 321 (21), 320 (M+ + 1, 100); Mr (+CI) 320.142 00
[M+
+ 1] (calcd for C16H22N3O2S 320.143 27). Anal.
(C16H21N3O2S) C, H, N.
(R)-N-(4-Nitr oben zyl)-2-acetam ido-3-h ydr oxypr opion a-
m id e ((R)-19) a n d (R)-N-(4-Nitr oben zyl)-2-a ceta m id o-3-
a cetoxyp r op ion a m id e ((R)-20). Compounds (R)-19 and (R)-
20 were prepared utilizing a procedure comparable to the
synthesis of (R)-3 and using D-serine ((R)-18) (10.00 g, 95
mmol), Ac2O (9.9 mL, 0.11 mol), NMM (20.9 mL, 0.19 mol),
IBCF (24.6 mL, 0.19 mol), and 4-nitrobenzylamine hydrochlo-
ride (25.00 g, 0.13 mol). The 4-nitrobenzylamine hydrochloride
was solubilized in DMF (50 mL) upon addition of NMM (14.3
mL, 0.13 mol), and then the generated free amine was added
dropwise by cannulation to the mixed anhydride at -78 °C.
After evaporation of the solvent, the crude product was purified
by column chromatography (SiO2; EtOAc) to yield 9.12 g (34%)
of (R)-19 and 2.64 g (9%) of (R)-20 as white solids.
(R,S)-N-Ben zyl-2-a cet a m id o-6-(b en zyloxyca r b on yl)-
a m in oh exa n a m id e ((R,S)-16). To a dry DMF solution (30
mL) of (R,S)-15 (2.06 g, 6.39 mmol) were successively added
under Ar EDCI (1.47 g, 7.7 mmol), HOBt (951 mg, 7.0 mmol),
and NMM (700 µL, 7.7 mmol). After the mixture was stirred
(5 min), benzylamine (840 µL, 7.7 mmol) was added dropwise
and the reaction mixture was stirred at room temperature (12
h). The mixture was diluted with 1 M HCl and extracted with
EtOAc (2 × 50 mL). The organic layers were combined, washed
with brine (50 mL), and concentrated in vacuo. The product
was purified by column chromatography (SiO2; 1:19 MeOH/
CHCl3) and recrystallized (EtOH) to obtain 2.35 g (89%) of pure
(R,S)-16 as a white solid: mp 157-158 °C; Rf ) 0.31 (1:19
MeOH/CHCl3); IR (KBr) 3287, 3063, 3032, 2923, 1688, 1632,
1553 cm-1; 1H NMR (CD3OD) δ 1.33-1.43 (m, C(4)H2), 1.46-
1.52 (m, C(5)H2), 1.60-1.70 (m, C(3)HH′), 1.73-1.85 (m, C(3)-
HH′), 1.98 (s, CH3C(O)), 3.10 (t, J ) 6.9 Hz, C(6)H2), 4.30 (dd,
J ) 5.7, 8.4 Hz, CH), 4.37 (s, CH2Ph), 5.06 (s, CH2OC(O)),
7.20-7.36 (m, 10 PhH). The structural assignments were in
(R)-19: mp 164-166 °C; [R]27 +8.14° (c 1.03, DMSO); Rf )
D
0.15 (1:9 MeOH/CHCl3); IR (KBr) 3370, 3352, 3281, 2954, 1657,
1638, 1546, 1520 cm-1; 1H NMR (CDCl3) δ 2.08 (s, CH3C(O)),
3.21 (dd, J ) 3.4, 9.7 Hz, OH), 3.58-3.68 (m, CHH′OH), 4.23
(dt, J ) 3.4, 11.4 Hz, CHH′OH), 4.22-4.46 (m, CH), 4.53 (d, J
) 6.3 Hz, NHCH2), 6.66 (d, J ) 6.6 Hz, NHCH), 7.42 (d, J )
8.9 Hz, 2 C2′H), 7.45-7.51 (m, CH2NH), 8.20 (d, J ) 8.9 Hz, 2
C3′H). Addition of excess (R)-(-)-mandelic acid to a CDCl3
solution of (R)-19 gave only one signal for the acetyl methyl
protons. 13C NMR (DMSO-d6) δ 22.7 (CH3C(O)), 41.7 (CH2-
NH), 55.4 (CH), 61.6 (CH2OH), 123.3 (2C3′), 128.0 (2C2′), 146.4,
147.7 (C1′ and C4′), 169.5, 170.7 (2 C(O)); MS (+CI) m/z (rel
intensity) 283 (12), 282 (M+ + 1, 100), 264 (11), 252 (10), 130
(29), 119 (11), 132 (32); Mr (+CI) 282.108 41 [M+ + 1] (calcd
for C12H16N3O5 282.109 00). Anal. (C12H15N3O5) C, H, N.
1
agreement with the H-1H COSY experiment. 13C NMR (CD3-
OD) δ 22.4 (CΗ3C(Ã)), 24.1 (C(4)H2), 30.5 (C(5)H2), 32.8 (C(3)-
H2), 41.5 (C(6)H2), 44.0 (NHCH2Ph), 55.0 (CH), 67.3 (OCH2Ph),
128.2, 128.5, 128.8, 128.9, 129.4, 129.5, 138.4, 139.9 (2 C6H5),
159.0 (C(O)O), 173.4, 174.4 (2 C(O)). The 13C NMR structural
assignments were in agreement with the DEPT experiment.
MS (+CI) m/z (rel intensity) 413 (24), 412 (M+ + 1, 100), 343
(22), 305 (29), 304 (15); Mr (+CI) 412.223 01 [M+ + 1] (calcd
for C23H30N3O4 412.223 63). Anal. (C23H29N3O4) C, H, N.
(R)-20: mp 177-180 °C (dec); [R]26 +13.5° (c 1.0, DMSO);
D
(R,S)-N-Ben zyl-2-acetam ido-6-am in oh exan am ide ((R,S)-
17). Utilizing a similar procedure employed for the synthesis
of (S)-10 and using (R,S)-16 (2.10 g, 5.10 mmol) and a catalytic
amount of 5% Pd/C gave, after filtration over a bed of Celite,
(R,S)-17 (1.41 g, 99%) as a pale-yellow oil: Rf ) 0.01 (2:1
EtOAc/hexanes); IR (liquid film) 3987, 3064, 3033, 2934, 2962,
Rf ) 0.30 (1:9 MeOH/CHCl3); IR (KBr) 3285, 3114, 1733, 1647,
1556, 1515 cm-1 1H NMR (CDCl3) δ 2.02, 2.06 (s, 2 CH3C-
;
(O)), 4.29 (dd, J ) 5.0, 11.6 Hz, CHH′OAc), 4.46 (dd, J ) 5.9,
11.6 Hz, CHH′OAc), 4.50 (dd, J ) 5.5, 15.2 Hz, CHH′NH), 4.57
(dd, J ) 5.8, 15.2 Hz, CHH′NH), 4.76-4.80 (m, CH), 6.56 (d,
J ) 7.5 Hz, NHCH), 7.32-7.37 (t, J ) 5.3 Hz, NHCH2), 7.42
(d, J ) 8.7 Hz, 2 C2′H), 8.17 (d, J ) 8.7 Hz, 2 C3′H). Addition
of excess (R)-(-)-mandelic acid to a CDCl3 solution of (R)-20
gave only one signal for each of the two acetyl methyl protons.
13C NMR (DMSO-d6) δ 20.6, 22.5 (2 CH3C(O)), 41.7 (CH2NH),
51.7 (CH), 63.5 (CH2OAc), 123.4, 127.9, 128.0, 146.4, 147.4
(C6H4), 169.3, 169.6, 170.0 (3 C(O)); MS (+CI) m/z (rel
intensity) 325 (18), 324 (M+ + 1, 100); Mr (+CI) 324.119 75
[M+ + 1] (calcd for C14H18N3O6 324.119 56). Anal. (C14H17N3O6)
C, H, N.
1
1650, 1553 cm-1; H NMR (CD3OD) δ 1.29-1.55 (m, C(4)H2
and C(5)H2), 1.60-1.71 (m, C(3)HH′), 1.72-1.86 (m, C(3)HH′),
1.99 (s, CH3C(O)), 2.63 (3.04) (t, J ) 7.1 (6.3) Hz, C(6)H2), 4.33
(dd, J ) 5.9, 8.6 Hz, CH), 4.38 (s, CH2Ph), 7.20-7.33 (m, 5
PhH). 1H NMR analysis indicated the major and minor
conformational isomers existed in a 90:10 ratio in CD3OD, the
number in parentheses corresponds to the value observed for
the minor conformer; all other signals for the minor conformer
are believed to overlap with those for the major conformer.
13C NMR (CD3OD) δ 22.7 (CΗ3C(Ã)), 24.1 (C(4)H2), 32.5, 32.9
(C(3)H2 and C(5)H2), 41.9 (C(6)H2), 43.8 (CH2Ph), 54.9 (CH),
(S)-N-(4-Nitr oben zyl)-2-acetam ido-3-h ydr oxypr opion a-
m id e ((S)-19) a n d (S)-N-(4-Nitr oben zyl)-2-a ceta m id o-3-

Amino Acid Anticonvulsants
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4769
a cetoxyp r op ion a m id e ((S)-20). Compound (S)-19 and (S)-
20 were prepared utilizing the preceding procedure and
L-serine ((S)-18) (4.50 g, 42.8 mmol), Ac2O (4.6 mL, 48 mmol),
NMM (5.9 mL, 53.5 mmol), IBCF (6.90 mL, 53.5 mmol), and
4-nitrobenzylamine hydrochloride (6.04 g, 54.9 mmol). The
4-nitrobenzylamine hydrochloride was solubilized in DMF (30
mL) upon addition of NMM (6.0 mL, 54.9 mmol). After
evaporation of the solvent, the crude product was purified by
column chromatography (SiO2; EtOAc) and then recrystallized
(EtOH) to yield 5.48 g (46%) of (S)-19 and 1.19 g (9%) of (S)-
20 as white solids.
0.32 (1:9 MeOH/CHCl3); IR (KBr) 3278, 3082, 2935, 1639, 1547,
1516 cm-1 1H NMR (CDCl3) δ 2.05 (s, CH3C(O)), 3.41 (s,
;
OCH3), 3.47 (dd, J ) 7.1, 9.3 Hz, CHH′OCH3), 3.84 (dd, J )
3.9, 9.3 Hz, CHH′OCH3), 4.50-4.62 (m, CH2NH and CH),
6.35-6.37 (m, NHCH), 6.96 (br s, NHCH2), 7.42 (d, J ) 8.7
Hz, 2 C2′H), 8.19 (d, J ) 8.7 Hz, 2 C3′H). Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (S)-21 gave only
one signal for the acetyl methyl and one signal for the ether
methyl protons. Addition of excess (R)-(-)-mandelic acid to a
CDCl3 solution of (R)-21 and (S)-21 (1:1 ratio) gave two signals
for the acetyl methyl protons (δ 2.02 and 2.03) and two signals
for the ether methyl protons (δ 3.34 and 3.38). 13C NMR
(CDCl3) δ 23.4 (CH3C(O)), 43.0 (CH2NH), 52.8 (CH), 59.4
(OCH3), 71.6 (CH2OCH3), 124.1 (2C3′), 128.1 (2C2′), 145.7,
147.6 (C1′ and C4′), 170.6, 170.7 (2 C(O)); MS (+CI) m/z (rel
intensity) 297 (16), 296 (M+ + 1, 100), 254 (22); Mr (+CI)
296.124 97 [M+ + 1] (calcd for C13H18N3O5 296.124 65). Anal.
(C13H17N3O5) C, H, N.
(S)-19: mp 162-164 °C; [R]27 -8.0° (c 1.07, DMSO); Rf )
D
0.15 (1:9 MeOH/CHCl3); IR (KBr) 3371, 3352, 2955, 2939, 1662,
1638, 1547, 1519 cm-1; 1H NMR (CDCl3) δ 2.08 (s, CH3C(O)),
3.16 (dd, J ) 3.5, 9.6 Hz, OH), 3.59-3.68 (m, CHH′OH), 4.23
(dt, J ) 3.5, 11.4 Hz, CHH′OH), 4.14-4.46 (m, CH), 4.53 (d, J
) 6.0 Hz, CH2NH), 6.65 (d, J ) 6.9 Hz, NHCH), 7.41 (d, J )
8.7 Hz, 2 C2′H), 7.45-7.50 (m, CH2NH), 8.20 (d, J ) 8.7 Hz, 2
C3′H). Addition of excess (R)-(-)-mandelic acid to a CDCl3
solution of (S)-19 gave only one signal for the acetyl methyl
proton. Addition of excess (R)-(-)-mandelic acid to a CDCl3
solution of (R)-19 and (S)-19 (1:1 ratio) gave two signals for
the acetyl methyl protons (δ 2.07 and 2.08). 13C NMR (DMSO-
d6) δ 22.6 (CH3C(O)), 41.7 (CH2NH), 55.3 (CH), 61.6 (CH2-
OH), 123.3 (2C3′), 128.0 (2C2′), 146.3, 147.6 (C1′ and C4′), 169.5,
170.6 (2 C(O)); MS (+CI) m/z (rel intensity) 283 (11), 282 (M+
+ 1, 100), 264 (33), 246 (22); Mr (+CI) 282.109 60 [M+ + 1]
(calcd for C12H16N3O5 282.109 00). Anal. (C21H15N3O5‚0.46H2O)
C, H, N.
(R)-N-(4-Am in oben zyl)-2-a ceta m id o-3-m eth oxyp r op i-
on a m id e ((R)-22). A methanolic solution (200 mL) of (R)-21
(2.50 g, 8.47 mmol) was hydrogenated (1 atm) in the presence
of a catalytic amount of PtO2 (200 mg) at room temperature
(1.5 h). The catalyst was filtered over a bed of Celite, and the
solvent was evaporated in vacuo to give 1.97 g (87%) of pure
(R)-22 as a white solid: mp >176 °C (dec); [R]25D -4.1° (c 1.21,
DMSO); Rf ) 0.39 (1:9 MeOH/CHCl3); IR (KBr) 3435, 3356,
1
3236, 3071, 2926, 1678, 1642, 1519 cm-1; H NMR (CDCl3) δ
2.03 (s, CH3C(O)), 3.36 (s, OCH3), 3.41 (dd, J ) 7.2, 9.1 Hz,
CHH′OCH3), 3.65 (br s, NH2), 3.79 (dd, J ) 4.1, 9.1 Hz,
CHH′OCH3), 4.35 (d, J ) 5.7 Hz, CH2NH), 4.47-4.53 (m, CH),
6.43 (d, J ) 6.0 Hz, NHCH), 6.59 (br s, NHCH2), 6.65 (d, J )
8.4 Hz, 2 C3′H), 7.05 (d, J ) 8.4 Hz, 2 C2′H). Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (R)-22 gave only
one signal for the acetyl methyl protons and one signal for the
ether methyl protons. 13C NMR (CDCl3) δ 23.4 (CH3C(O)), 43.4
(CH2NH), 52.6 (CH), 59.2 (OCH3), 71.9 (CH2OCH3), 115.4
(2C3′), 127.8 (C1′), 129.0 (2C2′), 146.0 (C4′), 169.9, 170.4 (2 C(O));
MS (+CI) m/z (rel intensity) 266 (M+ + 1, 37), 106 (100); Mr
(+CI) 265.141 46 [M+] (calcd for C13H19N3O3 265.142 64). Anal.
(C13H19N3O3) C, H, N.
(S)-20: mp 179-180 °C (dec); [R]26D -13.2° (c 1.03, DMSO);
Rf ) 0.30 (1:9 MeOH/CHCl3); IR (KBr) 3286, 3113, 2954, 1736,
1643, 1558, 1516 cm-1 1H NMR (CDCl3) δ 2.02, 2.05 (s, 2
;
CH3C(O)), 4.29 (dd, J ) 5.1, 11.4 Hz, CHH′OAc), 4.46 (dd, J
) 6.0, 11.4 Hz, CHH′OAc), 4.49 (dd, J ) 6.1, 16.1 Hz,
CHH′NH), 4.55 (dd, J ) 6.4, 16.1 Hz, CHH′NH), 4.76-4.83
(m, CH), 6.56 (d, J ) 7.2 Hz, NHCH), 7.34-7.38 (m, NHCH2),
7.42 (d, J ) 8.7 Hz, 2 C2′H), 8.17 (d, J ) 8.7 Hz, 2 C3′H).
Addition of excess (R)-(-)-mandelic acid to a CDCl3 solution
of (S)-20 gave only one signal for each of the two acetyl methyl
protons. Addition of excess (R)-(-)-mandelic acid to a CDCl3
solution of (R)-20 and (S)-20 (1:1 ratio) gave two signals for
each acetyl methyl proton (δ 1.94, 1.98, 2.00, and 2.01). 13C
NMR (DMSO-d6) δ 20.6, 22.5 (2 CH3C(O)), 41.7 (CH2NH), 51.7
(CH), 63.5 (CH2OAc), 123.4 (2C3′), 128.0 (2C2′), 146.4, 147.4
(C1′ and C4′), 169.3, 169.7, 170.1 (3 C(O)); MS (+CI) m/z (rel
intensity) 324 (M+ + 1, 100), 282 (11), 264 (49), 172 (12), 106
(20); Mr (+CI) 324.120 00 [M+ + 1] (calcd for C14H18N3O6
324.119 56). Anal. (C14H17N3O6) C, H, N.
(S)-N-(4-Am in ob en zyl)-2-a cet a m id o-3-m et h oxyp r op i-
on a m id e ((S)-22). Using the preceding procedure and using
(S)-21 (2.45 g, 8.30 mmol) and PtO2 (∼200 mg) gave 1.82 g
(83%) of pure (S)-22 as a pale-yellow solid after purification
by column chromatography (SiO2; 1:17 MeOH/CHCl3): mp
>176 °C (dec); [R]25 +4.3° (c 1.30, DMSO); Rf ) 0.39 (1:9
D
MeOH/CHCl3); IR (KBr) 3437, 3352, 3244, 3066, 2927, 1678,
1520 cm-1 1H NMR (CDCl3) δ 2.02 (s, CH3C(O)), 3.37 (s,
;
OCH3), 3.41 (dd, J ) 7.2, 9.2 Hz, CHH′OCH3), 3.67 (br s, NH2),
3.79 (dd, J ) 3.9, 9.2 Hz, CHH′OCH3), 4.34 (d, J ) 5.4 Hz.
NHCH2), 4.47-4.54 (m, CH ), 6.44 (d, J ) 6.3 Hz, NH CH),
6.60-6.67 (m, NHCH2 and 2 C3′H), 7.05 (d, J ) 8.7 Hz, 2 C2′H).
Addition of excess (R)-(-)-mandelic acid to a CDCl3 solution
of (S)-22 gave only one signal for the acetyl methyl protons
and one signal for the ether methyl protons. Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (R)-22 and (S)-22
(1:1 ratio) gave two signals for the acetyl methyl protons (δ
1.93 and 1.94) and two signals for the ether methyl protons (δ
3.24 and 3.28). 13C NMR (CDCl3) δ 23.5 (CH3C(O)), 43.5 (CH2-
NH), 52.6 (CH), 59.3 (OCH3), 71.9 (CH2OCH3), 115.4 (2C3′),
127.8 (C1′), 129.1 (2C2′), 146.1 (C4′), 169.9, 170.5 (2 C(O)); MS
(+CI) m/z (rel intensity) 266 (M+ + 1, 28), 106 (100); Mr (+CI)
266.149 54 [M+ + 1] (calcd for C13H20N3O3 266.150 47). Anal.
(C13H19N3O3‚0.18H2O) C, H, N.
(R)-N-(4-Nit r ob en zyl)-2-a cet a m id o-3-m et h oxyp r op i-
on a m id e ((R)-21). Utilizing method B, (R)-19 (2.65 g, 9.40
mmol), Ag2O (10.90 g, 47.0 mmol), and MeI (5.85 mL, 94.0
mmol) gave 2.55 g (92%) of pure (R)-21 as a white solid after
purification by column chromatography (SiO2; EtOAc): mp
163-164 °C; [R]27D +9.6° (c 1.35, MeOH); Rf ) 0.32 (1:9 MeOH/
CHCl3); IR (KBr) 3275, 3096, 2939, 1639, 1544, 1517 cm-1; 1H
NMR (CDCl3) δ 2.04 (s, CH3C(O)), 3.40 (s, OCH3), 3.49 (dd, J
) 7.1, 9.2 Hz, CHH′OCH3), 3.82 (dd, J ) 4.1, 9.2 Hz,
CHH′OCH3), 4.48-4.58 (m, CH2NH), 4.59-4.66 (m, CH), 6.55
(d, J ) 6.6 Hz, NHCH), 7.24 (t, J ) 5.4 Hz, NHCH2), 7.42 (d,
J ) 8.7 Hz, 2 C2′H), 8.17 (d, J ) 8.7 Hz, 2 C3′H). Addition of
excess (R)-(-)-mandelic acid to a CDCl3 solution of (R)-21 gave
only one signal for the acetyl methyl and one signal for the
ether methyl protons. 13C NMR (CDCl3) δ 23.3 (CH3C(O)), 42.9
(CH2NH), 52.8 (CH), 59.3 (OCH3), 71.7 (CH2OCH3), 124.0
(2C3′), 128.0 (2C2′), 145.8, 147.3 (C1′ and C4′), 170.6, 170.7 (2
C(O)); MS (+CI) m/z (rel intensity) 297 (16), 296 (M+ + 1, 100);
Mr (+CI) 296.124 34 [M+ + 1] (calcd for C13H18N3O5 296.124 65).
Anal. (C13H17N3O5) C, H, N.
(R)-N-(4-Isot h iocya n a t ob en zyl)-2-a cet a m id o-3-m et h -
oxyp r op ion a m id e ((R)-6). To a dry THF solution (50 mL)
of (R)-22 (1.30 g, 4.9 mmol) was added dropwise a solution of
DPT (1.73 g, 7.43 mmol) in THF (20 mL). The reaction mixture
was stirred under Ar at room temperature (2 h). The solvent
was removed in vacuo, and the product was purified by column
chromatography (SiO2; EtOAc) to give 1.14 g (76%) of pure
(S)-N-(4-Nit r ob en zyl)-2-a cet a m id o-3-m et h oxyp r op i-
on a m id e ((S)-21). Utilizing method B, (S)-19 (2.50 g, 8.89
mmol), Ag2O (10.30 g, 44.4 mmol), and MeI (5.50 mL, 89.0
mmol) gave 2.46 g (94%) of pure (S)-21 as a white solid after
purification by column chromatography (SiO2; 1:17 MeOH/
(R)-6 as a white solid: mp 172-173 °C; [R]23 +0.55° (c 2.56,
D
acetone); Rf ) 0.24 (1:9 acetone/EtOAc); IR (KBr) 3276, 3075,
1
CHCl3): mp 163-164 °C; [R]27 -10.0° (c 1.35, MeOH); Rf )
2929, 2181, 2130, 1638, 1543 cm-1; H NMR (CDCl3) δ 2.05
D

4770 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
LeTiran et al.
(s, CH3C(O)), 3.40 (s, OCH3), 3.46 (dd, J ) 7.7, 9.2 Hz,
CHH′OCH3), 3.82 (dd, J ) 4.1, 9.2 Hz, CHH′OCH3), 4.38-
4.55 (m, CH2NH), 4.56-4.60 (m, CH), 6.50 (d, J ) 5.4 Hz,
NHCH), 6.95 (br s, NHCH2), 7.19 (d, J ) 8.6 Hz, 2 C2′H or 2
C3′H),), 7.26 (d, J ) 8.6 Hz, 2 C2′H or 2 C3′H). Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (R)-6 gave only
one signal for the acetyl methyl protons and one signal for the
ether methyl protons. 13C NMR (CDCl3) δ 23.4 (CH3C(O)), 43.1
(CH2NH), 52.7 (CH), 59.3 (OCH3), 71.6 (CH2OCH3), 126.2
(2C3′), 128.7 (C2′), 130.6 (C4′), 135.7 (NCS), 137.5 (C1′), 170.3,
170.8 (2 C(O)); MS (+CI) m/z (rel intensity) 336 (14),
309 (16), 308 (M+ + 1, 100), 148 (11), 144 (11); Mr (+CI)
308.106 03 [M+ + 1] (calcd for C14H18N3O3S 308.106 89). Anal.
(C14H17N3O3S) C, H, N.
7.59-7.67 (m, NH and C6′H), 8.10-8.12 (m, C2′H and C4′H).
Addition of excess (R)-(-)-mandelic acid to a CDCl3 solution
of (R)-24 gave only one signal for each of the two acetyl methyl
protons. 13C NMR (DMSO-d6) δ 20.5, 22.6 (2 CH3C(O)), 41.5
(CH2NH), 51.6 (CH), 63.4 (CH2OAc), 121.5, 121.7 (C2′ and C4′),
129.7 (C5′), 133.7 (C6′), 141.7 (C1′), 147.7 (C3′), 169.2, 169.5,
170.0 (3 C(O)); MS (+CI) m/z (rel intensity) 324 (M+ + 1, 100),
264 (24); Mr (+CI) 324.119 85 [M+ + 1] (calcd for C14H18N3O6
324.119 56). Anal. (C14H17N3O6) C, H, N.
(S)-N-(3-Nitr oben zyl)-2-acetam ido-3-h ydr oxypr opion a-
m id e ((S)-23) a n d (S)-N-(3-Nitr oben zyl)-2-a ceta m id o-3-
a cetoxyp r op ion a m id e ((S)-24). Compounds (S)-23 and (S)-
24 were prepared utilizing the preceding procedure, L-serine
((S)-18) (5.00 g, 47.6 mmol), Ac2O (5.20 mL, 54.7 mmol), NMM
(6.5 mL, 59.5 mmol), IBCF (7.7 mL, 59.5 mmol), and 3-ni-
trobenzylamine hydrochloride (11.22 g, 59.5 mmol). The 3-ni-
trobenzylamine hydrochloride was solubilized in DMF (30 mL)
upon the addition of NMM (6.5 mL, 59.5 mmol). After
evaporation of the solvent, the crude product was purified by
column chromatography (SiO2; 1:25 MeOH/CHCl3) to yield a
mixture of 4.54 g (34%) of (S)-23 and 1.38 g (9%) of (S)-24 as
white solids.
(S)-N-(4-Isoth iocya n a toben zyl)-2-a ceta m id o-3-m eth ox-
yp r op ion a m id e ((S)-6). Using the preceding procedure, (S)-
22 (1.46 g, 5.50 mmol), and DPT (1.28 g, 5.50 mmol) gave 1.23
g (73%) of pure (S)-6 as a white solid after purification by
column chromatography (SiO2; 3:17 acetone/EtOAc): mp 171-
172 °C; [R]24 -0.76° (c 2.17, acetone); Rf ) 0.24 (1:9 acetone/
D
EtOAc); IR (KBr) 3274, 3070, 2927, 2183, 2125 (br), 1639, 1543,
1504 cm-1 1H NMR (CDCl3) δ 2.04 (s, CH3C(O)), 3.39 (s,
;
(S)-23: mp 136-138 °C; [R]27D -12.5° (c 0.50, DMSO); Rf )
0.20 (1:9 MeOH/CHCl3); IR (KBr) 3391, 3359, 3310, 3065, 1642,
1551, 1531 cm-1; 1H NMR (CDCl3) δ 2.10 (s, CH3C(O)), 3.24-
3.30 (m, OH ), 3.59-3.68 (m, CH H′OH), 4.20-4.25 (m,
CHH′OH), 4.45-4.50 (m, CH), 4.49 (dd, J ) 6.4, 15.8 Hz,
CHH′Ph), 4.59 (dd, J ) 6.1, 15.8 Hz, CHH′Ph), 6.67-6.75 (m,
NHCH), 7.48-7.61 (m, C5′H, C6′H, and NHCH2), 8.09-8.15
(m, C2′H and C4′H). Addition of excess (R)-(-)-mandelic acid
to a CDCl3 solution of (S)-23 gave only one signal for the acetyl
methyl protons. Addition of excess (R)-(-)-mandelic acid to a
CDCl3 solution of (R)-23 and (S)-23 (1:1 ratio) gave only one
signal for the acetyl methyl protons. 13C NMR (DMSO-d6) δ
22.6 (CH3C(O)), 41.4 (CH2NH), 55.3 (CH), 61.6 (CH2OH), 121.6
(C2′ and C4′), 129.6 (C5′), 133.7 (C6′), 142.0 (C1′), 147.8 (C3′),
169.5, 170.6 (2 C(O)); MS (+CI) m/z (rel intensity) 283 (13),
282 (M+ + 1, 100); Mr (+CI) 282.108 69 [M+ + 1] (calcd for
C12H16N3O5 282.109 00). Anal. (C12H15N3O5) C, H, N.
OCH3), 3.44 (dd, J ) 7.4, 9.2 Hz, CHH′OCH3), 3.81 (dd, J )
4.1, 9.2 Hz, CHH′OCH3), 4.42 (dd, J ) 5.6, 15.2 Hz, CHH′Ar),
4.48 (dd, J ) 6.1, 15.2 Hz, CHH′Ar), 4.51-4.57 (m, CH), 6.41
(d, J ) 6.0 Hz, NHCH), 6.84 (br s, NHCH2), 7.18 (d, J ) 8.6
Hz, 2 C2′H or 2 C3′H), 7.24 (d, J ) 8.6 Hz, 2 C2′H or 2 C3′H).
Addition of excess (R)-(-)-mandelic acid to a CDCl3 solution
of (S)-6 gave only one signal for the acetyl methyl protons and
one signal for the ether methyl protons. Addition of excess (R)-
(-)-mandelic acid to a CDCl3 solution of (R)-6 and (S)-6 (1:1
ratio) gave two signals for the acetyl methyl protons (δ 1.99
and 2.00) and two signals for the ether methyl protons (δ 3.32
and 3.34). 13C NMR (CDCl3) δ 23.4 (CH3C(O)), 43.1 (CH2NH),
52.7 (CH), 59.3 (OCH3), 71.7 (CH2OCH3), 126.2 (2C3′), 128.7
(2C2′), 130.6 (C4′), 135.6 (NCS), 137.6 (C1′), 170.3, 170.6 (2
C(O)); MS (+CI) m/z (rel intensity) 309 (17), 308 (M+ + 1, 100),
247 (20); Mr (+CI) 308.106 82 [M+ + 1] (calcd for C14H18N3O3S
308.106 89). Anal. (C14H17N3O3S) C, H, N.
(S)-24: mp 151-154 °C; [R]26 -14.4° (c 1.00, DMSO); Rf )
(R)-N-(3-Nitr oben zyl)-2-acetam ido-3-h ydr oxypr opion a-
m id e ((R)-23) a n d (R)-N-(3-Nitr oben zyl)-2-a ceta m id o-3-
a cetoxyp r op ion a m id e ((R)-24). Compounds (R)-23 and (R)-
24 were prepared utilizing the procedure for the synthesis of
(R)-19 and (R)-20, respectively, and using D-serine ((R)-18)
(17.80 g, 0.169 mol), Ac2O (19.8 mL, 0.203 mol), NMM (23.2
mL, 0.21 mol), IBCF (27.4 mL, 0.21 mol), and 3-nitroben-
zylamine hydrochloride (35.00 g, 0.19 mol). The 3-nitroben-
zylamine hydrochloride was solubilized in DMF (100 mL) upon
addition of NMM (20.4 mL, 0.19 mol). After evaporation of the
solvent, the crude product was purified by column chroma-
tography (SiO2; EtOAc) to yield a mixture of 12.95 g (27%) of
(R)-23 and 4.36 g (8%) of (R)-24 as white solids.
D
0.39 (1:9 MeOH/CHCl3); IR (KBr) 3282 (br), 3097, 1743, 1639,
1562, 1523 cm-1 1H NMR (CDCl3) δ 2.00, 2.04 (s, 2 CH3C-
;
(O)), 4.28 (dd, J ) 5.1, 11.4 Hz, CHH′OAc), 4.43 (dd, J ) 6.0,
11.4 Hz, CHH′OAc), 4.52 (d, J ) 5.7 Hz, CH2NH), 4.79-4.86
(m, CH), 6.72 (d, J ) 7.5 Hz, NHCH), 7.46-7.53 (m, C5′H),
7.59-7.66 (m, NHCH2 and C6′H), 8.10-8.12 (m, C2′H and
C4′H). Addition of excess (R)-(-)-mandelic acid to a CDCl3
solution of (S)-24 gave only one signal for each of the two acetyl
methyl protons. Addition of excess (R)-(-)-mandelic acid to a
CDCl3 solution of (R)-24 and (S)-24 (1:1 ratio) gave only one
signal for each acetyl methyl proton. 13C NMR (DMSO-d6) δ
20.5, 22.5 (2 CH3), 41.5 (CH2NH), 51.6 (CH), 63.4 (CH2OAc),
121.5, 121.7 (C2′ and C4′), 129.7 (C5′), 133.7 (C6′), 141.7 (C1′),
147.7 (C3′), 169.2, 169.6, 170.0 (3 C(O)); MS (+CI) m/z (rel
intensity) 324 (M+ + 1, 100), 264 (26), 233 (47), 172 (21); Mr
(+CI) 324.119 78 [M+ + 1] (calcd for C14H18N3O6 324.119 56).
Anal. (C14H17N3O6) C, H, N.
(R)-23: mp 137-140 °C; [R]27 +12.7° (c 1.45, DMSO); Rf )
D
0.20 (1:9 MeOH/CHCl3); IR (KBr) 3312, 3271, 3079, 1646, 1635,
1
1553, 1528 cm-1; H NMR (CDCl3) δ 2.10 (s, CH3C(O)), 3.26
(dd, J ) 3.3, 9.6 Hz, OH), 3.60-3.69 (m, CHH′OH), 4.23 (br
d, J ) 11.4 Hz, CHH′OH), 4.41-4.47 (m, CH), 4.49 (dd, J )
6.3, 15.5 Hz, CHH′Ph), 4.60 (dd, J ) 6.6, 15.5 Hz, CHH′Ph),
6.68-6.71 (m, NHCH), 7.49-7.61 (m, C5′H, C6′H, and NHCH2),
8.09-8.15 (m, C2′H and C4′H). Addition of excess (R)-(-)-
mandelic acid to a CDCl3 solution of (R)-23 gave only one signal
for the acetyl methyl protons. 13C NMR (DMSO-d6) δ 22.6
(CH3C(O)), 41.4 (CH2NH), 55.3 (CH), 61.6 (CH2OH), 121.5,
121.7 (C2′ and C4′), 129.7 (C5′), 133.7 (C6′), 142.0 (C1′), 147.8
(C3′), 169.5, 170.6 (2 C(O)); MS (+CI) m/z (rel intensity) 282
(R)-N-(3-Nit r ob en zyl)-2-a cet a m id o-3-m et h oxyp r op i-
on a m id e ((R)-25). Utilizing method B, (R)-23 (3.00 g, 10.40
mmol), Ag2O (12.41 g, 53.6 mmol), and MeI (6.7 mL, 0.11 mol)
gave 2.81 g (89%) of pure (R)-25 as a white solid after
purification by column chromatography (SiO2; EtOAc): mp
152-153 °C; [R]26 +18.3° (c 1.31, MeOH); Rf ) 0.40 (1:9
D
MeOH/CHCl3); IR (KBr) 3308, 3093, 2941, 2839, 1633, 1558,
1530 cm-1 1H NMR (CDCl3) δ 2.05 (s, CH3C(O)), 3.42 (s,
;
(M+ + 1, 100), 264 (13), 130 (64); Mr (+CI) 282.108 37 [M+
+
OCH3), 3.49 (dd, J ) 7.4, 9.0 Hz, CHH′OCH3), 3.82 (dd, J )
4.1, 9.0 Hz, CHH′OCH3), 4.56-4.64 (m, CH and CH2NH), 6.49
(d, J ) 6.3 Hz, NHCH), 7.14 (br s, NHCH2), 7.48-7.52 (m,
C5′H), 7.61 (d, J ) 7.5 Hz, C6′H), 8.05-8.15 (m, C2′H and C4′H).
Addition of excess (R)-(-)-mandelic acid to a CDCl3 solution
of (R)-25 gave only one signal for the acetyl methyl protons
and one signal for the ether methyl protons. 13C NMR (CDCl3)
δ 23.4 (CH3C(O)), 42.8 (CH2NH), 52.8 (CH), 59.4 (OCH3), 71.7
(CH2OCH3), 122.1, 122.6 (C2′ and C4′), 129.7 (C5′), 133.6 (C6′),
1] (calcd for C12H16N3O5 282.109 00). Anal. (C12H15N3O5) C, H,
N.
(R)-24: mp 152-154 °C; [R]26 +14.6° (c 1.0, DMSO); Rf )
D
0.39 (1:9 MeOH/CHCl3); IR (KBr) 3282, 3097, 2970, 1743, 1639,
1562, 1523 cm-1 1H NMR (CDCl3) δ 2.00, 2.04 (s, 2 CH3C-
;
(O)), 4.28 (dd, J ) 5.3, 11.3 Hz, CHH′OAc), 4.44 (dd, J ) 5.8,
11.3 Hz, CHH′OAc), 4.52 (d, J ) 5.8 Hz, CH2NH), 4.80-4.85
(m, CH), 6.71 (d, J ) 7.5 Hz, NHCH), 7.42 (t, J ) 8.2 Hz, C5′H),

Amino Acid Anticonvulsants
J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21 4771
140.5 (C1′), 148.7 (C3′), 170.6, 170.7 (2 C(O)); MS (+CI) m/z
(rel intensity) 296 (M+ + 1, 100), 144 (69), 116 (15); Mr (+CI)
296.123 82 [M+ + 1] (calcd for C13H18N3O5 296.124 65). Anal.
(C13H17N3O5) C, H, N.
(+CI) 266.150 72 [M+ + 1] (calcd for C13H20N3O3 266.150 47).
Anal. (C13H19N3O3‚0.12H2O) C, H, N.
(R)-N-(3-Isot h iocya n a t ob en zyl)-2-a cet a m id o-3-m et h -
oxyp r op ion a m id e ((R)-7). Utilizing the procedure for (R)-6
and using (R)-26 (431 mg, 1.62 mmol), CH2Cl2 (40 mL), and
DPT (378 mg, 1.62 mmol) gave 400 mg (80%) of pure (R)-7 as
a white solid after purification by column chromatography
(S)-N-(3-Nit r ob en zyl)-2-a cet a m id o-3-m et h oxyp r op i-
on a m id e ((S)-25). Utilizing method B, (S)-23 (2.50 g, 8.89
mmol), Ag2O (10.30 g, 44.4 mmol), and MeI (5.5 mL, 89.0
mmol) gave 2.60 g (99%) of pure (S)-25 as a white solid after
purification by column chromatography (SiO2; 1:17 MeOH/
(SiO2; EtOAc): mp 161-163 °C; [R]27 +5.7° (c 1.77, acetone);
D
Rf ) 0.46 (1:9 MeOH/CHCl3); IR (KBr) 3281, 3120, 2929, 2165,
2139, 1633, 1543 cm-1; 1H NMR (CDCl3) δ 2.05 (s, CH3C(O)),
3.44 (s, OCH3), 3.53 (dd, J ) 7.1, 9.2 Hz, CHH′OCH3), 3.83
(dd, J ) 4.4, 9.2 Hz, CHH′OCH3), 4.43 (dd, J ) 6.0, 15.5 Hz,
CHH′Ar), 4.51 (dd, J ) 6.2, 15.5 Hz, CHH′Ar), 4.54-4.59 (m,
CH), 6.90 (br d, J ) 6.0 Hz, NHCH), 7.14-7.36 (m, 4 ArH
and NHCH2). Addition of excess (R)-(-)-mandelic acid to a
CDCl3 solution of (R)-7 gave only one signal for the acetyl
methyl protons and one signal for the ether methyl protons.
13C NMR (CDCl3) δ 23.4 (CH3C(O)), 43.0 (CH2NH), 52.7 (CH),
59.4 (OCH3), 71.8 (CH2OCH3), 124.7, 124.8 (C2′ and C4′), 126.4
(C6′), 130.0, 131.8 (C3′ and C5′), 135.8 (NCS), 140.2 (C1′), 170.4,
170.6 (2 C(O)); MS (+CI) m/z (rel intensity) 336 (M+ + 1, 15),
309 (14), 308 (100), 144 (12); Mr (+CI) 308.105 68 [M+ + 1]
(calcd for C14H18N3O3S 308.106 89). Anal. (C14H17N3O3‚0.45H2O)
C, H, N.
CHCl3): mp 152-153 °C; [R]26 -18.4° (c 1.20, MeOH); Rf )
D
0.40 (1:9 MeOH/CHCl3); IR (KBr) 3310, 3089, 2941, 2837, 1639,
1531 cm-1 1H NMR (CDCl3) δ 2.05 (s, CH3C(O)), 3.42 (s,
;
OCH3), 3.49 (dd, J ) 7.4, 8.9 Hz, CHH′OCH3), 3.84 (dd, J )
3.9, 8.9 Hz, CHH′OCH3), 4.54-4.64 (m, CH and CH2NH), 6.51
(d, J ) 6.2 Hz, NHCH), 7.16 (br s, NHCH2), 7.49-7.53 (m,
C5′H), 7.61 (d, J ) 7.2 Hz, C6′H), 8.10-8.13 (m, C2′H and C4′H).
Addition of excess (R)-(-)-mandelic acid to a CDCl3 solution
of (S)-25 gave only one signal for the acetyl methyl protons
and one signal for the ether methyl protons. Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (R)-25 and (S)-25
(1:1 ratio) gave two signals for the acetyl methyl protons (δ
1.99 and 2.00) and two signals for the ether methyl protons (δ
3.33 and 3.36). 13C NMR (CDCl3) δ 23.3 (CH3C(O)), 42.7 (CH2-
NH), 52.9 (CH), 59.4 (OCH3), 71.9 (CH2OCH3), 122.0, 122.5
(C2′ and C4′), 129.7 (C5′), 133.6 (C6′), 140.6 (C1′), 148.6 (C3′),
170.6, 170.7 (2 C(O)); MS (+CI) m/z (rel intensity) 297 (15),
296 (M+ + 1, 100), 254 (41), 144 (14); Mr (+CI) 296.124 78 [M+
+ 1] (calcd for C13H18N3O5 296.124 65). Anal. (C13H17N3O5) C,
H, N.
(S)-N-(3-Isoth iocyan atoben zyl)-2-acetam ido-3-m eth oxy-
p r op ion a m id e ((S)-7). Utilizing the preceding procedure, (S)-
26 (2.50 mg, 9.43 mmol), THF (110 mL), and DPT (2.20 g, 9.43
mmol) gave 1.77 g (61%) of pure (S)-7 as a white solid after
purification by column chromatography (SiO2; 1:9 acetone/
(R)-N-(3-Am in oben zyl)-2-a ceta m id o-3-m eth oxyp r op i-
on a m id e ((R)-26). Utilizing the procedure for (R)-22 and
using a methanolic solution (80 mL) of (R)-25 (1.49 g, 5.05
mmol), PtO2 (∼115 mg), and H2 (1 atm) gave, after 45 min,
pure (R)-26 (1.34 g, 99%) as a clear oil, which solidified upon
standing: mp 183-184 °C (dec); [R]25D +3.59° (c 1.33, DMSO);
Rf ) 0.28 (1:9 MeOH/CHCl3); IR (KBr) 3356, 3308, 3290, 2922,
1650, 1622, 1560, 1544 cm-1; 1H NMR (CDCl3) δ 2.04 (s, CH3C-
(O)), 3.38 (s, OCH3), 3.43 (dd, J ) 7.5, 9.1 Hz, CHH′OCH3),
3.64-3.69 (br s, NH2), 3.81 (dd, J ) 4.1, 9.1 Hz, CHH′OCH3),
4.38 (d, J ) 5.7 Hz, CH2NH), 4.51-4.57 (m, CH), 6.43 (d, J )
5.4 Hz, NHCH), 6.58 (s, C2′H), 6.60-6.69 (m, C4′H, C6′H, and
NHCH2), 7.11 (br t, J ) 8.1 Hz, C5′H). Addition of excess (R)-
(-)-mandelic acid to a CDCl3 solution of (R)-26 gave only one
signal for the acetyl methyl protons and one signal for the
ether methyl protons. 13C NMR (DMSO-d6) δ 22.6 (CH3C(O)),
42.3 (CH2NH), 52.5 (CH), 58.2 (OCH3), 72.2 (CH2OCH3), 112.5,
112.7 (C2′ and C4′), 114.6 (C6′), 128.7 (C5′), 139.7 (C1′), 148.5
(C3′), 169.3, 169.5 (2 C(O)); MS (+CI) m/z (rel intensity) 267
(13), 266 (M+ + 1, 100), 123 (21), 119 (13); Mr (+CI) 266.149 70
[M+ + 1] (calcd for C13H20N3O3 266.150 47). Anal. (C13H19N3O3)
C, H, N.
EtOAc): mp 162-163 °C; [R]26 -5.8° (c 1.77, acetone); Rf )
D
0.46 (1:9 MeOH/CHCl3); IR (KBr) 3281, 3099, 2928, 2138 (br),
1
1637, 1545 cm-1; H NMR (CDCl3) δ 2.05 (s, CH3C(O)), 3.41
(s, OCH3), 3.46 (dd, J ) 7.5, 9.0 Hz, CHH′OCH3), 3.83 (dd, J
) 3.9, 9.0 Hz, CHH′OCH3), 4.46 (J ) 6.0 Hz, CH2Ar), 4.54-
4.60 (m, CH), 6.45 (d, J ) 5.7 Hz, NHCH), 6.88-6.91 (m,
NHCH2), 7.13-7.33 (m, 4 ArH). Addition of excess (R)-(-)-
mandelic acid to a CDCl3 solution of (S)-7 gave only one signal
for the acetyl methyl protons and one signal for the ether
methyl protons. Addition of excess (R)-(-)-mandelic acid to a
CDCl3 solution of (R)-7 and (S)-7 gave two signals for the acetyl
methyl protons (δ 2.02 and 2.03) and two signals for the ether
methyl protons (δ 3.36 and 3.38). 13C NMR (CDCl3) δ 23.4
(CH3C(O)), 43.0 (CH2NH), 52.7 (CH), 59.4 (OCH3), 71.8 (CH2-
OCH3), 124.7, 124.8 (C2′ and C4′), 126.4 (C6′), 130.0, 131.8 (C3′
and C5′), 135.8 (NCS), 140.2 (C1′), 170.4, 170.6 (2 C(O)); MS
(+CI) m/z (rel intensity) 336 (M+ + 1, 24), 309 (22), 308 (100),
165 (12); Mr (+CI) 308.105 88 [M+ + 1] (calcd for C14H18N3O3S
308.106 89). Anal. (C14H17N3O3S) C, H, N.
P h a r m a cology. 1. In Vivo Stu d ies. Compounds were
screened under the auspices of the National Institutes of
Health’s Anticonvulsant Screening Project. Experiments were
performed in male rodents (albino Carworth Farms No. 1 mice
(intraperitoneal route, ip), albino Sprague-Dawley rats (oral
route, po)). The mice weighed between 18 and 25 g, while rats
were between 100 and 150 g. All animals had free access to
feed and water except during the actual testing period.
Housing, handling, and feeding were all in accordance with
recommendations contained in the “Guide for the Care and
Use of Laboratory Animals”. All of the test compounds were
administrated in suspensions of 0.5% (w/v) of methylcellulose
in water. The volumes administered were 0.01 mL/g of body
weight for mice and 0.2 mL/10 g for rats. Anticonvulsant
activity was established using the maximal electroshock (MES)
test.4,31 For the MES test, a drop of electrolyte solution with
an anesthetic (0.5% butacaine hemisulfate in 0.9% sodium
chloride) was placed in the eyes of the animals prior to
positioning the corneal electrodes and delivery of a nonlethal
current. A 60-cycle alternating current was administered for
0.2 s in both species, utilizing 50 mA in mice and 150 mA in
rats. Protection endpoints were defined as the abolition of the
hind limb tonic extensor component of the induced seizure.5
In mice, effects of compounds on forced spontaneous motor
activity were determined using the rotorod test.28,32 The
inability of experimental mice to maintain their balance for 1
(S)-N-(3-Am in ob en zyl)-2-a cet a m id o-3-m et h oxyp r op i-
on a m id e ((S)-26). Utilizing the preceding procedure and
using a methanolic solution (80 mL) of (S)-25 (1.89 g, 6.40
mmol), MeOH (180 mL), PtO2 (∼180 mg), and H2 (1 atm) gave
1.59 g (94%) of pure (S)-26 as a clear oil, which solidified upon
standing: mp 184-185 °C (dec); [R]25D -3.71° (c 1.30, DMSO);
Rf ) 0.28 (1:9 MeOH/CHCl3); IR (KBr) 3356, 3308, 3091, 2884,
1650 cm-1 1H NMR (CDCl3) δ 2.04 (s, CH3C(O)), 3.38 (s,
;
OCH3), 3.45 (dd, J ) 7.7, 9.0 Hz, CHH′OCH3), 3.60-3.70 (br
s, NH2), 3.81 (dd, J ) 4.1, 9.0 Hz, CHH′OCH3), 4.38 (d, J )
5.7 Hz, CH2NH), 4.52-4.57 (m, CH), 6.44 (br d, J ) 6.6 Hz,
NHCH), 6.58 (s, C2′H), 6.60-6.65 (m, C4′H, C6′H), 6.65-6.71
(m, NHCH2), 7.11 (br t, J ) 8.1 Hz, C5′H). Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (S)-26 gave only
one signal for the acetyl methyl protons and one signal for the
ether methyl protons. Addition of excess (R)-(-)-mandelic acid
to a CDCl3 solution of (R)-26 and (S)-26 (1:1 ratio) gave two
signals for the acetyl methyl proton (δ 1.99 and 2.00) and two
signals for the ether methyl protons (δ 3.33 and 3.36). 13C NMR
(DMSO-d6) δ 22.5 (CH3C(O)), 42.2 (CH2NH), 52.4 (CH), 58.1
(OCH3), 72.1 (CH2OCH3), 112.4, 112.6 (C2′ and C4′), 114.6 (C6′),
128.6 (C5′), 139.6 (C1′), 148.5 (C3′), 169.3, 169.4 (2 C(O)); MS
(+CI) m/z (rel intensity) 267 (14), 266 (M+ + 1, 100); Mr

4772 J ournal of Medicinal Chemistry, 2002, Vol. 45, No. 21
LeTiran et al.
tionalized R-Heteroatom-Substituted Amino Acids. J . Med.
Chem. 1991, 34, 2444-2452. (g) Kohn, H.; Sawhney, K. N.;
Bardel, P.; Robertson, D. W.; Leander, J . D. Synthesis and
Anticonvulsant Activities of R-Heterocyclic R-Acetamido-N-ben-
zylamide Derivatives. J . Med. Chem. 1993, 36, 3350-3360. (h)
Bardel, P.; Bolanos, A.; Kohn, H. Synthesis and Anticonvulsant
Activities of R-Acetamido-N-benzylacetamide Derivatives Con-
taining an Electron-Deficient R-Heteroaromatic Substituent. J .
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N.; Robertson, D. W.; Leander, J . D. Anticonvulsant Properties
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1994, 83, 689-691. (j) Choi, D.; Stables, J . P.; Kohn, H. Synthesis
and Anticonvulsant Activities of N-Benzyl-2-acetamidopropi-
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(2) (a) Paruszewski, R.; Rostafinska-Suchar, G.; Strupinska, M.;
J aworski, P.; Stables, J . P. Synthesis and Anticonvulsant
Activity of Some Amino Acid Derivatives. Pharmazie 1996, 3,
145-148. (b) Paruszewski, R.; Rostafinska-Suchar, G.; Strupin-
ska, M.; J aworski, P.; Winiecka, I.; Stables, J . P. Synthesis and
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Suchar, G.; Strupinska, M.; Winiecka, I.; Stables, J . P. Synthesis
and Anticonvulsant Activity of Some Amino Acid Derivatives.
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min on a 1 in. diameter knurled rod rotating at 6 rpm in three
successive trials was interpreted as a demonstration of motor
impairment. Under these conditions, mice can normally main-
tain their balance indefinitely. Motor impairment in rats was
assessed by observing the overt evidence of ataxia, abnormal
gait and stance, and/or loss of placing response and muscle
tone. In the mouse identification screens, all compounds were
administered at three dose levels (30, 100, 300 mg/kg) and in
two time periods (0.5 and 4 h). Typically, in the MES seizure
test, one animal was used at 30 and 300 mg/kg and three
animals were used at 100 mg/kg. In the rotorod toxicity test,
four animals were used at 30 and 300 mg/kg and eight animals
were used at 100 mg/kg (Table 1). Oral rat identification
screening was performed using four animals at a fixed dose of
30 mg/kg for both the MES and the rotorod toxicity tests over
1
5 time periods ranging from /4 to 4 h postdrug administration.
The quantitative determinations of the median effective (ED50)
and toxic doses (TD50) were conducted at previously calculated
time of peak effect using the ip route in mice and the oral route
in rats. Groups of at least eight animals were tested using
different doses of test compound until at least two points were
determined between 100% and 0% protection and minimal
motor impairment. The dose of the candidate substance
required to produce the desired endpoint (abolition of hindlimb
tonic extensor component) in 50% of the animals in each test
and the 95% confidence interval were calculated by a computer
program based on methods described by Finney.33
(3) Ho, B.; Venkatarangan, P. M.; Cruse, S. F., Hinko, C. N.;
Andersen, P. H.; Crider, A. M.; Adloo, A. A.; Roane, D. S.;
Stables, J . P. Synthesis of 2-Piperidinecarboxylic Acid Deriva-
tives as Potential Anticonvulsants. Eur. J . Med. Chem. 1998,
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2. Recep tor Scr een . Assays34 for the following receptors
were performed by the NIMH Psychoactive Drug Screening
Program: (1) GABAA, agonist ([3H] muscimol), (2) GABAA, bzp
([3H] flunitrazepam], (3) NMDA, PCP ([3H] TCP), (4) NMDA,
MK-801 ([3H] MK-801), (5) Na+, type 2 ([3H]batrachotoxin),
and (6) Ca2+, type L ([3H]nifedipine). Detailed on-line protocols
for the binding assays are described at http://meds20785.
cwru.edu./myweb/protocol.htm. For screening purposes, 10 µM
of each compound (dissolved in 10% DMSO) was incubated
with the appropriate receptor preparation and the percent
inhibition was determined for duplicate determinations each
performed in duplicate.
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Raven Press: New York, 1995; pp 99-110.
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Evaluation of Anticonvulsant Drugs: The Profile of Anticonvul-
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Prototype Antiepileptic Drugs in Mice and Rats; Technical
Report, Contract No. N01-NS-4-2361; Epilepsy Branch, Neuro-
logical Disorders Program, National Institute of Neurological
and Communicative Disorders and Stroke: Bethesda, MD,
October 29, 1996.
(8) Takemori, A. E.; Portoghese, P. S. Affinity Labels for Opioid
Receptors. Annu. Rev. Pharmacol. Toxicol. 1985, 25, 193-223.
(9) For recent examples of aromatic isothiocyanate as affinity labels,
see the following. (a) Yamada, K.; Rice, K. C.; Flippen-Anderson,
J . L.; Eissenstat, M. A.; Ward, S. J .; J ohnson, M. R.; Howlett,
A. C. (Aminoalkyl)indole Isothiocyanates as Potential Electro-
philic Affinity Ligands for the Brain Cannabinoid Receptor. J .
Med. Chem. 1996, 39, 1967-1974. (b) Chang, A.-C.; Takemori,
A. E.; Ojala, W. H.; Gleason, W. B.; Portoghese, P. S. κ Opioid
Receptor Selective Affinity Labels: Electrophilic Benzeneaceta-
mides as κ-Selective Opioid Antagonists. J . Med. Chem. 1994,
37, 4490-4498. (c) For recent examples of aliphatic isothiocy-
anate as affinity labels, see the following. Husbands, S. M.;
Izenwasser, S.; Loeloff, R. J .; Katz, J . L.; Bowen, W. D.; Vilner,
B. J .; Newman, A. A. Isothiocyanate Derivatives of 9-[3(cis-3,5-
Dimethyl-1-piperazinyl)propyl]-carbazole (Rimcazole): Irrevers-
ible Ligands for the Dopamine Transporter. J . Med. Chem. 1997,
40, 4340-4346.
(10) For recent examples of R-bromoacetamide as affinity labels, see
the following. (a) Aliau, S.; Delettre, G.; Mattras, H.; El Garrouj,
D.; Nique, F.; Teutsch, G.; Borgna, J .-L. Stereoidal Affinity
Labels of the Estrogen Receptor R. 4. Electrophilic 11â-Aryl
Derivatives of Estradiol. J . Med. Chem. 2000, 43, 613-628. (b)
Maeda, D. Y.; Ishmael, J . E.; Murray, T. F.; Aldrich, J . V.
Synthesis and Evaluation of N,N-Dialkyl Enkephalin-Based
Affinity Label for δ Opioid Receptors. J . Med. Chem. 2000, 43,
3941-3948.
(11) For recent examples of acrylamide as affinity labels, see the
following. Smaill, J . B.; Rewcastle, G. W.; Loo, J . A.; Greis, K.
D.; Chan, O. H.; Reyner, E. L.; Lipka, E.; Showalter, H. D. H.;
Vincent, P. W.; Elliott, W. L.; Denny, W. A. Tyrosine Kinase
Inhibitors. 17. Irreversible Inhibitors of the Epidermal Growth
Factor: 4-(Phenylamino)quinazoline- and 4-(Phenylamino)py-
rido[3,2-d]pyrimidine-6-acrylamides Bearing Additional Solubi-
lizing Functions. J . Med. Chem. 2000, 43, 1380-1397.
(12) (a) Walle, T.; Walle, U. K. Pharmacokinetic Parameters Obtained
with Racemates. Trends Pharmacol. Sci. 1986, 7, 155-156. (b)
Arie¨ns, E. J . Stereochemistry: A Source of Problems in Medici-
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Ack n ow led gm en t. The authors thank the NINDS
and the Anticonvulsant Screening Project (ASP) at the
National Institutes of Health, for kindly performing the
pharmacological studies via the ASP’s contract site at
the University of Utah with Drs. H. Wolf, S. White, and
Karen Wilcox. Funds for this project were provided, in
part, by the University of Houston. We thank Dr. Bryan
L. Roth (BLR) and Mr. J on Evans at the National
Institutes of Mental Health (NIMH) Psychoactive Drug
Screening Project for performing the in vitro receptor
binding studies. This work was supported by NIMH
Psychoactive Drug Screening Program Contract to BLR;
BLR was supported by K02MH01366.
Su p p or tin g In for m a tion Ava ila ble: Experimental de-
tails on the saponification of (R)-20, (S)-20, (R)-24, and (S)-24
and the chemical reactivity of (S)-3, (R,S,)-5, (R,S)-6, and (R)-
7. This material is available free of charge via the Internet at
http://pubs.acs.org.
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