The structure?activity relationship of the 3-Oxy site in the anticonvulsant (R)- N -benzyl 2-acetamido-3-methoxypropionamide

Article abstract of DOI:10.1021/jm100508m

Lacosamide ((R)-N-benzyl 2-acetamido-3-methoxypropionamide, (R)-1) is a low molecular weight anticonvulsant recently introduced in the United States and Europe for adjuvant treatment of partial-onset seizures in adults. In this study, we define the structure?activity relationship (SAR) for the compound's 3-oxy site. Placement of small nonpolar, nonbulky substituents at the 3-oxy site provided compounds with pronounced seizure protection in the maximal electroshock (MES) seizure test with activities similar to (R)-1. The anticonvulsant activity loss that accompanied introduction of larger moieties at the 3-oxy site in (R)-1 was offset, in part, by including unsaturated groups at this position. Our findings were similar to a recently reported SAR study of the 4?-benzylamide site in (R)-1 (J. Med. Chem. 2010, 53, 1288 ?1305). Together, these results indicate that both the 3-oxy and 4?-benzylamide positions in (R)-1 can accommodate nonbulky, hydrophobic groups and still retain pronounced anticonvulsant activities in rodents in the MES seizure model.

Full text of DOI:10.1021/jm100508m

5716 J. Med. Chem. 2010, 53, 5716–5726
DOI: 10.1021/jm100508m
The Structure-Activity Relationship of the 3-Oxy Site in the Anticonvulsant (R)-N-Benzyl
2-Acetamido-3-methoxypropionamide
†
Pierre Morieux, Christophe Salome, Ki Duk Park, James P. Stables, and Harold Kohn*
†
‡
,†,§
ꢀ †
†Division of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina, Chapel Hill, North Carolina
27599-7568, ‡Epilepsy Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 6001 Executive Boulevard,
Suite 2106, Rockville, Maryland 20892, and §Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290
Received April 26, 2010
Lacosamide ((R)-N-benzyl 2-acetamido-3-methoxypropionamide, (R)-1) is a low molecular weight
anticonvulsant recently introduced in the United States and Europe for adjuvant treatment of partial-
onset seizures in adults. In this study, we define the structure-activity relationship (SAR) for the
compound’s 3-oxy site. Placement of small nonpolar, nonbulky substituents at the 3-oxy site provided
compounds with pronounced seizure protection in the maximal electroshock (MES) seizure test with
activities similar to (R)-1. The anticonvulsant activity loss that accompanied introduction of larger
moieties at the 3-oxy site in (R)-1 was offset, in part, by including unsaturated groups at this position.
Our findings were similar to a recently reported SAR study of the 40-benzylamide site in (R)-1 (J. Med.
Chem. 2010, 53, 1288-1305). Together, these results indicate that both the 3-oxy and 40-benzylamide
positions in (R)-1 can accommodate nonbulky, hydrophobic groups and still retain pronounced
anticonvulsant activities in rodents in the MES seizure model.
Epilepsy is a common neurological disorder that affects
0.5-1% of the world population.1 It is a broad term that
encompasses many different human seizure types.2,3 The
treatment mainstay for patients with epilepsy has been the
long-term and consistent administration of anticonvulsant
drugs, but these agents may be either poorly tolerated or
ineffective.4,5 Elucidation of new biological targets for seizure
control are expected to provide new treatment strategies and
lead to a better understanding of the pharmacological me-
chanisms underlying epileptic disorders.
and efforts to detect selective binding in rat brain homoge-
nates with radiolabeled (R)-1 were unsuccessful.11 These
findings suggest that either (R)-1 binds to its target(s) with
modest affinity or its binding partner(s’) levels are too low to
be detected or a combination of both factors. Electrophysiol-
ogy experiments in neuroblastoma cells demonstrated that
(R)-1 selectively enhanced sodium channel slow inactivation
in a time- and voltage-dependent manner without affecting
fast inactivation.12 Similarly, use of recombinant human
NaV1.3 and NaV1.7 channels and NaV1.8-type tetrodotoxin-
resistant (TTX-R) currents from dorsal root ganglion (DRG)
neurons demonstrated that (R)-1 selectively interacted with
the slow inactivation state in each of these sodium channel
subtypes.13 Use of (R)-1 analogues designed to irreversibly
modify (R)-1 targets preferentially modified collapsin re-
sponse mediator protein 2 (CRMP2) in brain lysates.9,14
While CRMP2 is a signaling protein involved in neuronal
differentiation and axonal guidance,15 its importance in (R)-1
function has not been determined. Thus, the pharmacological
actions of (R)-1 remain elusive.
Lacosamide6 ((R)-1) is a novel antiepileptic drug (AEDa)
that was recently introduced in the United States and Europe
for adjuvant treatment of partial-onset seizures in adults.7
Whole animal pharmacology studies (e.g., maximal electro-
shock seizure (MES), chemoconvulsant seizure tests, Frings
mouse model, hippocampal kindled rat test) have revealed a
distinctive profile for (R)-1 that differentiated it from all other
antiseizure drugs.8 Studies have appeared concerning the
mechanism of action of (R)-1. Radioligand displacement
studies using (R)-1 (10 μM) and more than 100 potential
pharmacological targets failed to show significant binding,9,10
*To whom correspondence should be addressed. Phone: 919-843-
8112. Fax: 919-966-0204. E-mail: harold_kohn@unc.edu.
a Abbreviations: AED, antiepileptic drug; MES, maximal electro-
shock; Nav,voltage-gated sodium channel; TTX, tetrodotoxin; DRG,
dorsal root ganglion; CRMP2, collapsin response mediator protein 2;
AB, affinity bait; CR, chemical reporter; AB&CR, affinity bait and
chemical reporter; SAR, structure-activity relationship; ASP, Antic-
onvulsant Screening Program; NINDS, National Institute of Neurolo-
gical Disorders and Stroke; DTPP, diethoxytriphenylphosphorane;
DTPP-F6, bis(2,2,2-trifluoroethoxy)triphenylphoshorane; DMTMM,
4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride;
HRMS, high resolution mass spectrometry; scMet, subcutaneous me-
trazol; IBCF, isobutylchloroformate; NMM, N-methylmorpholine.
In 2009, we advanced a strategy to identify molecular
drug targets within the proteome where binding is modest.14
Our approach required attaching an affinity bait (AB) and
a chemical reporter (CR) moiety to the drug framework
r
pubs.acs.org/jmc
Published on Web 07/08/2010
2010 American Chemical Society

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5717
Figure 1. Use of the AB&CR strategy to identify potential drug receptors.
(Figure 1, 2). The AB group is designed to irreversibly modify
the potential target site(s), and the CR unit permits either the
detection (e.g., by fluorescence) or isolation (e.g., by biotin
conjugation) of the covalently labeled receptor 3 after reaction
with a bioorthogonal probe. We outlined the critical require-
ments for the AB and CR units.14 Key to the success of this
strategy is that the AB and CR groups in 2 do not impede
binding to the cognate receptor(s) of the medicinal agent that
elicit drug function.
We have applied this strategy to search for molecular
targets in the rodent brain proteome by installing AB and
CR groups at select sites in (R)-1.14,16 Initial structure-activ-
ity relationship (SAR) studies6 showed that small structural
changes at the 3-oxy site in (R)-1 led to modest reductions in
anticonvulsant activity, indicating that including either an AB
or a CR moiety at this position was possible. Here, we report
an in-depth SAR of the 3-oxy site in (R)-4 and show the
importance of size, hydrophobic interactions, and polar mod-
ifications of this site on anticonvulsant activity in the MES
seizure test17 in rodents. We include in our SAR possible AB
and CR moieties. We demonstrate that placing nonpolar,
nonbulky substituents at the 3-oxy site provided compounds
with pronounced seizure protection in the MES seizure test.
This study complements a recently reported SAR study of the
40-benzylamide site in (R)-5.18 Together, these investigations
serve as the structural basis for the design and use of (R)-1
AB&CR agents that can be employed in target-based search
studies. Moreover, they provide important information about
the structural elements that can be incorporated at these
(R)-1 sites while still retaining significant protection in animal
tests.
for 4. The compounds were evaluated for seizure protection
at the Anticonvulsant Screening Program (ASP) of the
National Institute of Neurological Disorders and Stroke
(NINDS) at the National Institutes of Health. We examined
several potential interactions that might affect drug binding.
To test for the effect of steric size, we replaced the (R)-1
O-methyl unit with larger alkyl groups ((R)-6-(R)-9). We
introduced unsaturated aliphatic and (hetero)aromatic sys-
tems ((R)-10-(R)-16, (R)-18, (R)-19, (R)-26) to look for
potential hydrophobic, π-π, and cationic-π interactions
that might facilitate binding.22 Within this set, we varied the
length of the methylene spacer between several of these
groups and the 3-oxy site in (R)-4. Not to limit our study
to hydrophobic interactions, we prepared derivatives that
contained a polar side chain ((R)-24, (R)-27, (R)-28). These
compounds could accept and/or donate a hydrogen bond(s)
to a suitable amino acid residue within the putative drug
binding pocket(s). Finally, we evaluated (R)-4 oxy-substi-
tuted analogues that contained either an AB ((R)-20-(R)-
23) or a CR group ((R)-12, (R)-18, (R)-25). In those cases
where little or no anticonvulsant activity was observed for
the AB, we prepared the corresponding isostere (i.e., (R)-16
for (R)-21, (R)-17 for (R)-22, (R)-18 for (R)-23 and (R)-25) to
see if either metabolic factors or structural constraints con-
tributed to the observed activity loss.
Chemistry. We have reported a versatile method for the
introduction of 3-oxy substituents within the (R)-4 framework
(Scheme 1).23 The method was adopted from earlier reports
showing that N-substituted aziridines carboxylate esters serve as
valuable intermediates in the synthesis of O-substituted serine
amino acid derivatives.23,24-31 To expedite the synthesis of the
central aziridine intermediate (R)-32 from D-serine ((R)-29), we
adopted Evans’ and co-workers’ cyclodehydration protocol
using dialkoxytriphenylphosphoranes (PPh3(OR)2).32-36 Ac-
cordingly, serine methyl ester hydrochloride ((R)-30) was se-
quentially treated with Et3N and diethoxytriphenylphospho-
rane (PPh3(OCH2CH3)2, DTPP)37 to give aziridine (R)-31a and
(R)-31b as a mixture of methyl and ethyl esters,23 respectively.
The 24 h reaction provided good yields (50-70%) of (R)-31
after bulb-to-bulb distillation and was used to prepare multiple
grams of (R)-31 in a single experiment. We found that the
reaction only proceeded if (R)-30 was isolated as the free
amine.23 Acetylation of (R)-31a and (R)-31b with acetic anhy-
dride and catalytic amounts of DMAP afforded (R)-32a and
(R)-32b. Treatment of the (R)-32a and (R)-32b mixture with
alcohols under Lewis acid-catalyzed conditions produced the
desired 3-oxy substituted serine esters, (R)-33a and (R)-33b,
Results and Discussion
Choice of Compounds. Table 1 lists the (R)-4 3-oxy sub-
stituted compounds that constitute the SAR. Our previous
findings show that the anticonvulsant activity for (R)-16 and
similar analogues18-21 principally reside in the D-amino acid
derivative, and thus, we synthesized only the (R)-enantiomer

5718 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Table 1. Structure-Activity Relationship of the (R)-4a
Morieux et al.
a All compounds tested corresponded to the (R)-enantiomer except 11. The compounds were tested through the auspices of the NINDS ASP. b The
compounds were administered intraperitoneally. ED50 and TD50 values are in milligrams per kilogram. c The compounds were administred orally unless
otherwise indicated. ED50 and TD50 values are in mg/kg. d MES = maximal electroshock seizure test. e The 6 Hz test was carried out at 32 mA. f TD50
value determined from the rotorod test. g PI = protective index (TD50/ED50). h Tox = behavioral toxicity. i Ref 6. j Ref 23. k Ref 14. l Ref 51. m No ataxia
observed up to 3000 mg/kg.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5719
Scheme 1. Synthetic Pathway to Enantiopure (R)-4 Derivatives Using N-Acetyl Aziridine Ester (R)-32 Ring-Opening
Scheme 2. Alternative Synthetic Pathway to O-Substituted Derivatives (R)-4
with the same C(2) stereochemistry as its precursor.30 The (R)-
33a and (R)-33b esters were hydrolyzed with LiOH to the free
acid (R)-34 and then coupled with benzylamine using either the
mixed anhydride coupling method38 or the reagent 4-(4,6-
dimethoxy-1,3,5-triazine-2-yl)-4-methyl-morpholinium chlor-
ide (DMTMM).39 Using this protocol, we prepared (R)-6-
(R)-10, (R)-12-(R)-14, (R)-16, (R)-17, and (R)-20-(R)-28.
Although DTTP facilitated the production of aziridine
esters (R)-31, its preparation was tedious and required an
initial centrifugation step (8000 rpm, 10 min) to remove
finely divided NaBr, and then three or four evaporation/
filtration steps to precipitate PPh3(O), a byproduct of the
reaction.34 Accordingly, we examined the use of a hexafluoro
derivative of DTPP, PPh3(OCH2CF3)2 (DTPP-F6),40 to
generate aziridine (R)-31. DTPP-F6 formation proceeded
faster than DTPP and required only filtration and evapora-
tion to yield the cyclodehydration reagent.41 Unfortunately,
we found that compared with DTPP yields (50-70%),
DTPP-F6 provided lower yields (25-30%) of aziridine (R)-
31a after bulb-to-bulb transfer.
were obtained with DTPP (60-70%) over DTPP-F6 (30-
35%). Several factors, however, made this synthetic route
(Scheme 2) inconvenient. First, the low volatility of (R)-37
prevented its bulb-to-bulb distillation, thus requiring silica
gel flash chromatography for purification. Second, we ob-
served in the final step a byproduct that was not readily
separated from the desired product by column chromatog-
raphy. Accordingly, the pure 3-oxy-substituted (R)-4 deri-
vatives were obtained by recrystallization. We have tenta-
tively identified the additional product as the acid-catalyzed
rearranged31 2-oxazoline 39 on the basis of high resolution
mass spectrometry (HRMS) and the characteristic oxazoline
methyl resonance (δ 13.4 ppm) in the 13C NMR spectrum.43
Using this method, we prepared (R)-18 and (R)-19 on a suffi-
cient scale for the pharmacological studies.
Synthesis of the O-benzyl analogue (R)-15 was simpli-
fied by the commercially availability of (R)-O-benzylserine
((R)-40). Treatment of (R)-40 with acetic anhydride followed
by amide coupling with benzylamine and DMTMM gave
(R)-15 in 93% overall yield (Scheme 3).
We examined an alternative method to prepare the 3-oxy
substituted (R)-4 analogues, wherein the N-benzyl amide
group was installed at an early stage of the synthesis (Scheme 2).
(R)-N-Benzyl 2-amino-3-hydroxypropionamide42 ((R)-36)
was obtained by catalytic hydrogenation of (R)-3542 and
then reacted with either DTPP or DTPP-F6 to give (R)-N-
benzyl aziridine-2-carboxamide ((R)-37).14 Following acet-
ylation of (R)-37 to give (R)-38, the aziridine was ring-
opened with 3-butyn-1-ol and phenethyl alcohol to yield
(R)-18, and (R)-19, respectively. Paralleling the formation of
aziridine esters (R)-31a and (R)-31b, greater yields of (R)-37
For several of the 3-oxy-substituted (R)-4 derivatives, the
3-oxy group was modified in the last step of the synthesis.
Aldehyde (R)-21 was prepared from alcohol (R)-41 using
Swern oxidation conditions (Scheme 4).44 Attempts to pre-
pare the corresponding aldehyde AB derivative containing a
single methylene group between the 3-oxy site and the
aldehyde group were unsuccessful. Using the (S)-enantiomer
(S)-42, Swern oxidation gave the cyclized hemiaminal 44 as
a ∼9:1 mixture of diastereoisomers with no trace of the desired
product (S)-43 (Scheme 4). We have attributed the difference
in Swern oxidation products 44 and (R)-21 to the entropic

5720 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Scheme 3. Synthesis of (R)-1 Derivative (R)-15
Morieux et al.
Scheme 5. Synthesis of (R)-24 and (R)-26
Scheme 4. Synthesis of (R)-21 and 44
and the behavioral toxicity effects observed in rats, we
determined the median doses for 50% neurological impair-
ment (TD50). TD50 values were determined for compounds
that exhibited significant activity in the MES seizure test.
The protective index (PI=TD50/ED50) for these analogues
are also listed. Select compounds were evaluated in the psy-
chomotor 6 Hz (32 mA) seizure models (mice, ip).50 When
the 3-oxy (R)-4 derivatives were evaluated in the subcuta-
neous metrazol (scMet) seizure model,48 none provided
protection at 300 mg/kg doses at the times (0.5 and 4 h)
tested (data not shown), a finding similar to (R)-16 and
structurally related compounds.6,14,19-21,53-55
At the beginning of our studies, little was known about the
SAR of the 3-oxy site in (R)-4. Only the anticonvulsant
activities of the O-ethyl ((R,S)-6) and the O-allyl (R,S)-11) 4
analogues were reported.6 Both compounds exhibited sig-
nificant activity but were 2-8-fold less active then (R,S)-1 in
mice (MES ED50=8.3 mg/kg).6 Recently, a (R)-4 analogue
was prepared wherein the (R)-1 methyl moiety was replaced
by the difluoromethyl moiety to give (R)-45.56 This fluori-
nated derivative displayed excellent protection and pro-
longed duration of action (rat, po) in the MES seizure test
(ED50=3.0 mg/kg (0.5 h), 4.2 mg/kg (4 h)) when compared
with (R)-1 ((ED50 = 1.8 mg/kg (0.5 h), 8.3 mg/kg (4 h)) under
the same test conditions.56 Our development of a general,
versatile method to prepare enantiomerically pure 3-oxy-
substituted (R)-4 derivatives through N-acetyl aziridine (R)-
3223 permitted us to explore the SAR of the 3-oxy position.
ease in formation of a six-membered versus the correspond-
ing seven-membered ring system.45
We used azide (R)-25 to synthesize (R)-24 and (R)-26
(Scheme 5). Catalytic reduction (10% Pd/C, H2) of (R)-25
gave the terminal amine, which was treated directly with
acetic anhydride and base to give amide (R)-24. When azide
(R)-25 was treated with propargyl methyl ether under Cu(I)-
mediated cycloaddition conditions,46 we obtained the tria-
zolyl derivative (R)-26.14
The enantiopurity of (R)-6-(R)-10 and (R)-12-(R)-28
were assessed by the detection of a single acetyl methyl signal
in the 1H NMR spectrum for each compound when a
saturated solution of (R)-(-)-mandelic acid was added.47
We report in the Experimental Section the details (syn-
thetic procedure, characterization) of the final step for all
new compounds evaluated in the seizure models. In the
Supporting Information, we provide a synthetic scheme for
each new compound tested and the experimental procedures,
physical, and full spectroscopic properties for all new syn-
thetic compounds prepared in this study.
Pharmacological Activity. Compounds (R)-6-(R)-28 were
tested for anticonvulsant activity at the NINDS ASP using
the procedures described by Stables and Kupferberg.48
The pharmacological data from MES48,49 and 6 Hz50 seizure
tests are summarized in Table 1, along with clinical AEDs
phenytoin,51 valproic acid,51 and phenobarbital.51 All com-
pounds were administered intraperitoneally (ip) to mice and
orally (po) to rats, unless otherwise indicated. The table lists
the values determined to be protective in blocking hind limb
extension induced in the MES seizure model from the rodent
studies. For compounds that showed significant activity, we
report the 50% effective dose (ED50) obtained in quantita-
tive screening evaluations. Using the rotorod test52 in mice
In total, 23 O-substituted (R)-4 analogues were prepared
and evaluated for anticonvulsant activity at the NINDS ASP
(Table 1). We observed a steady increase in activity in mice
(ip) as the steric size of the (R)-4 R substituent decreased
from cyclohexyl ((R)-9, ED50 =100-300 mg/kg) and tert-
butyl ((R)-8, ED50 = 30-100 mg/kg) to isopropyl ((R)-7,
ED50 = 23 mg/kg), to ethyl ((R)-6, ED50=7.9 mg/kg), and to
methyl ((R)-1, ED50=4.5 mg/kg), indicating that nonbulky
3-oxy substituted groups in (R)-4 analogues provided the
highest anticonvulsant activity.23 Interestingly, we observed

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5721
a similar increase in activity as the size of the 3-oxy sub-
stituent decreased when the compounds were administered
orally to rats, but the range of activities was narrower. For
example, the O-cyclohexyl derivative (R)-9 exhibited an
ED50 of ∼30 mg/kg, while O-isopropyl (R)-7 (ED50 = 5.6
mg/kg), O-ethyl (R)-6 (ED50=5.2 mg/kg), and O-methyl (R)-
1 (ED50=3.9 mg/kg) all displayed excellent seizure protec-
tion. (R)-6 and (R)-7 showed no behavioral neurotoxicity in
the rat at the highest dose (500 mg/kg) tested, leading to high
PI values for both compounds ((R)-6:PI>89;(R)-7: PI> 58).
We concluded that including bulky alkyl substituents at the
3-oxy site in (R)-4 adversely affected seizure protection in the
MES seizure test.
Inserting one methylene (CH2) group between the oxygen
and the cyclohexyl ring in (R)-9 (ED50 = 100-300 mg/kg) to
give (R)-14 (ED50=100-300 mg/kg) did not improve activity
in mice (ip). However, we observed a pronounced increase in
potency in mice (ip) going from O-phenyl (R)-10 (ED50 =100-
300 mg/kg)23 to O-benzyl (R)-15 (ED50 = 64 mg/kg).
The effect of polar substituents at the C(2) site in (R)-4 was
assessed by incorporating either an ethylenoxy or an acetami-
doethoxy unit two methylene units removed from the 3-oxy site.
When we attached one ethylenoxy group to give methyl ether
(R)-27, we observed noticeable seizure protection (ED50 = 30-
100 mg/kg) in mice (ip). Adding a second ethylenoxy spacer to
(R)-27 to give (R)-28 produced no detectable activity under
comparable test conditions (ED50 = >300 mg/kg). Similarly,
the acetamidoethyl analogue (R)-24 (ED50=>300 mg/kg) was
inactive at the doses tested. The limited data do not allow us to
speculate on the factors (e.g., (R)-4 biodistribution in the brain,
metabolism, drug binding) responsible for the loss of antic-
onvulsant activity of (R)-24 and (R)-28. Nonetheless, our find-
ings indicated that introducing polar substituents at the 3-oxy
site in (R)-4 did not improve seizure protection and provided
valuable information for the installation of both AB and CR
units in the (R)-1 framework and for future drug design efforts.
We tested (R)-4 derivatives, (R)-21-(R)-23, that contained
AB groups at their 3-oxy sites. The electrophilic groups alde-
hyde (R)-21 (ED50=>300 mg/kg) and epoxide (R)-22 (ED50=
>300 mg/kg) exhibited no anticonvulsant activity and no
neurological toxicity at the doses tested in mice (ip). The lack
of neurotoxicity may suggest that the compounds did not
cross the blood-brain barrier. The aliphatic isothiocyanate
(R)-23 was inactive in mice (ED50 = >300 mg/kg), active in
We also found notable anticonvulsant activity for other
derivatives that contained an unsaturated substituent one
methylene group separated from the 3-oxy site in (R)-4. The
O-allyl6 ((R,S)-11, ED50 = 30-100 mg/kg), O-propargyl ((R)-
12, ED50=16 mg/kg),14 and O-but-2-ynyl ((R)-13, ED50 = 30-
100 mg/kg) (R)-1 analogues all provided pronounced seizure
protection in mice (ip). When the O-propargyl ((R)-12, ED50
=
rats (ED50 = <30 mg/kg), but neurologically toxic (TD50 =
7.9 mg/kg) and the O-but-2-ynyl ((R)-13, ED50 = 6.4 mg/kg)
(R)-4 derivatives were tested in the rat (po), we observed ex-
cellent seizure protection, which was comparable with pheny-
toin (ED50=30 mg/kg)51 and phenobarbital (ED50=9.1 mg/
kg),51 and no behavioral neurological toxicity at 500 mg/kg.
Significantly, the propargyl unit was shown to be a superior
CR unit in bioorthogonal Cu(I)-mediated cycloaddition reac-
tions.14,57 Our finding that (R)-12 and (R)-13 exhibited excellent
activity in the MES seizure model indicated that this CR unit in
(R)-1 AB&CR agents would not likely impact the unit’s binding
to its cognate receptor(s).
We next explored the effect of adding a second methylene
unit between the unsaturated unit and the (R)-4 3-oxy site to
determine if the location of the unsaturated site affected
anticonvulsant activity. When we added the second methy-
lene group to O-propargyl (R)-12 (ED50 = 16 mg/kg) and
O-benzyl (R)-15 (ED50 = 64 mg/kg) to give O-3-butynyl
(R)-18 (ED50=30-100 mg/kg) and O-2-phenylethyl (R)-19
(ED50 = 100-300 mg/kg) (R)-4 derivatives, we saw a de-
crease in anticonvulsant activity in mice (ip). Like the
O-phenylethyl derivative (R)-19 (ED50 =100-300 mg/kg),
the O-triazolylethyl (R)-26 compound (ED50=>300 mg/kg)
did not display activity in mice (ip) at the doses tested.
Nonetheless, when tested orally in rats, the O-3-butynyl
(R)-18 (ED50=11 mg/kg) provided excellent seizure protec-
tion. These results indicated that the site of unsaturation at
the 3-oxy site in (R)-4 may be important for anticonvulsant
activity. We observed one exception to this pattern, the
O-allyl ((R,S)-11, ED50 = 30-100 mg/kg) and the O-3-butenyl
((R)-16, ED50 = 30-100 mg/kg) (R)-4 derivatives showed
similar anticonvulsant activity in mice (ip). Like O-3-butynyl
(R)-18, the activity of the O-3-butenyl (R)-16 derivative
improved from mice (ip) (ED50=30-100 mg/kg) to rat (po)
(ED50 = 17 mg/kg). Collectively, these findings suggested
that a favorable interaction with the 3-oxy π system in (R)-4
at the target site(s) may foster binding, provided the group
was not large and the unsaturated system was correctly
positioned at the 3-oxy site.
30-100 mg/kg, mice (ip); TD50 =>30 mg/kg, rat (po)). We
suspect that several of these AB groups may have undergone
change or been metabolized under the test conditions. Indeed,
aldehydes and epoxides are known substrates for a variety of
enzymes (e.g., aldehyde reductases, epoxide hydrolases).58-63
Because we planned to use these AB groups for in vitro
experiments where metabolism would less likely be an issue,
we evaluated, where possible, the corresponding AB isostere to
determine if the steric size of the AB group precluded their use.
We were gratified to find that the O-but-3-enyl isostere (R)-16
(ED50=30-100 mg/kg, mice (ip); ED50 = 17 mg/kg, rat (po))
for aldehyde (R)-21 and the O-cyclopropyl isostere (R)-17
(ED50 = 46 mg/kg, mice (ip)) for epoxide (R)-22 displayed
pronounced-to-excellent anticonvulsant activity. Thus, we have
not attributed the absence of activity for aldehyde (R)-21 and
epoxide (R)-22 to steric factors. Similar to the O-cyclopropyl
(R)-17, the photolabile methyldiazirine AB derivative (R)-20
exhibited pronounced animal protection in the MES seizure test
(ED50 = 30-100 mg/kg, mice (ip); ED50 =44 mg/kg, rat (po)).
Several (R)-4 compounds were prepared that contained a
CR unit at the 3-oxy site ((R)-12, (R)-18, (R)-25). The alkyne
(R)-12 and azide (R)-25 CR compounds displayed excellent
anticonvulsant activity (ED50=<10 mg/kg) in the rat upon po
and ip administration, respectively. These findings supported
the use of these CR units in (R)-1 proteomic target searches. Of
all the (R)-4 3-oxy substituted analogues prepared, the whole
animal pharmacology of O-azidoethyl analogue (R)-25 was the
most intriguing. (R)-25 anticonvulsant activity was dependent
on the animal, the seizure test, and the route of administration.
While (R)-25 displayed poor MES seizure protection in mice
(ip) (ED50 = 100-300 mg/kg), it proved potent in the 6 Hz
(32 mA) test50 when also administered ip to mice (ED50=44
mg/kg). Furthermore, we observed that (R)-25 exhibited ex-
ceptional activity in the MES seizure test in the rat when
administered ip (ED50=5.7 mg/kg), but that activity dropped
with po administration (MES ED50=>40 mg/kg). When ad-
ministered ip to the rat, the TD50 for (R)-25 was 78 mg/kg,
providing a PI greater than 13.

5722 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Morieux et al.
Finally, we asked whether that a methyl-d3 analogue of
(R)-1, (R)-1-d3, would display enhanced anticonvulsant ac-
tivity over (R)-1. Recently, pharmaceutical companies have
prepared deuterated versions (“heavy drugs”) of marketed
medicinal agents.64 The greater strength of C-D bonds
compared with C-H bonds is predicted to confer increased
metabolic stability for the deuterated version of the drug.64
Significantly, the major metabolite for (R)-1 in humans is the
desmethyl analogue (R)-46.8 The metabolic conversion of
(R)-1 to (R)-46 has not been attributed to a specific metabolic
enzyme.65 Nonetheless, (R)-46 accounts for ∼30% of the
(R)-1 excreted in human urine,66 and it exhibits little antic-
onvulsant activity (ED50 = 100-300 mg/kg).6 Accordingly,
we reasoned that replacing the 3-oxy methyl group in (R)-1
with the perdeuterated methyl unit to give (R)-1-d3 might
reduce metabolic processes and thus lead to increased bioa-
vailability of the AED. We prepared (R)-1-d3 using a method
we reported for (R)-142 and substituting CD3I for CH3I. The
deuterated derivative (R)-1-d3 (ED50 = 5.2 mg/kg) showed
an anticonvulsant activity in the rat (po) comparable with
(R)-1 (ED50 = 3.9 mg/kg), without exceeding it, and exhibited
greater neurological toxicity ((R)-1-d3, TD50 = ∼200 mg/kg;
(R)-1, TD50 =>500 mg/kg).
Experimental Section
General Methods. The general methods used in this study
were identical to those previously reported14 and are summar-
ized in the Supporting Information. All compounds were
1
checked by TLC, H and 13C NMR, MS, and elemental ana-
lyses. The analytical results are within (40% of the theoretical
value. The TLC, NMR, and the analytical data confirmed the
purity of the products was g95%.
General Procedure for the Pd-Catalyzed Hydrogenation Reac-
tion. Method A. A MeOH suspension of the starting material
([C] ∼0.01-0.2 M) and 10% Pd/C (10% w/w) was vigorously
stirred under an atmosphere of H2 (balloon) at room tempera-
ture (16 h). The mixture was filtered through a bed of celite. The
bed was washed with MeOH and CH2Cl2, and the washings
were collected and evaporated in vacuo. The amine was used
without further purification.
General Procedure for the Ester Hydrolysis of (R)-33 with
LiOH. Method B. To a THF solution of ester (R)-33 (2 volumes,
[C] ∼0.1 M) was added an aqueous solution (1 volume) of LiOH
(1 equiv). The homogeneous solution was stirred at room
temperature (60 min), after which time Et2O (2 volumes) was
added. The aqueous layer was recovered and washed with Et2O.
The remaining aqueous layer was acidified (pH ∼1) by the
dropwise addition of aqueous concentrated HCl, saturated with
NaCl, and extracted with EtOAc until no further product was
detected (TLC analysis). The combined organic layers were
combined, dried (Na2SO4), and evaporated to an oily residue
that was used directly for the next step or recrystallized from
EtOAc and hexanes to provide an analytical sample.
General Procedure for the DMTMM Amide Coupling Reac-
tion.39 Method C. To a THF solution of acid (R)-34 ([C] ∼0.1 M)
at room temperature was added the desired benzylamine (1.2
equiv). The solution was stirred (5-10 min) until the benzylam-
monium carboxylate precipitated. With stirring, DMTMM (1.2
equiv) was added all at once, and the resulting suspension was
stirred at room temperature (3-12 h). In those cases where a salt
did not precipitate, DMTMM was added after 15 min to the
solution. The salts were removed by filtration and washed with
THF, and the solvent was removed in vacuo. The residue obtained
was purified by flash column chromatography to afford the
benzylamide and then recrystallized from EtOAc and hexanes.
General Procedure for the Aziridine Ring-Opening of (R)-32
and (R)-38 with Alcohols. Method D. To a cooled CH2Cl2
solution (ice bath) of either (R)-32 or (R)-38 ([C] ∼ 0.5-1 M)
Conclusions
This SAR study demonstrated that replacing the 3-oxy
methyl group in (R)-1 with small, nonbulky, hydrophobic
groups provided derivatives with pronounced anticonvulsant
activity in the MES seizure model. We showed that substitut-
ing the 3-oxy methyl group in (R)-1 with larger alkyl groups
diminished activity, but the loss could be offset, in part, by the
incorporating hydrophobic, unsaturated moieties. Our SAR
findings for the 3-oxy site in (R)-4 were similar to that
observed for the 40-benzylamide position in (R)-5, where
nonbulky, hydrophobic groups provided derivatives with
excellent anticonvulsant activities18 (Figure 2). Most impor-
tant, these findings supportouruse of (R)-1 AB&CR agents in
proteomic searches wherein either the AB or the CR unit is
installed at the 3-oxy site.
and the appropriate alcohol (1-5 equiv) was added BF3 Et2O
3
(1 equiv) dropwise while stirring. After addition, the mixture
was warmed to room temperature and stirred (30 min), and then
an equal volume of saturated aqueous NaHCO3 was added. The
reaction was vigorously stirred (15 min) and the organic layer
separated. The aqueous layer was extracted with CH2Cl2 until
no additional product could be detected (TLC analysis). All the
organic layers were then combined, dried (Na2SO4), and con-
centrated in vacuo to yield a residue that was either purified by
flash column chromatography, used directly for the next step or
recrystallized from EtOAc and hexanes.
(R)-N-Benzyl 2-Acetamido-3-(methoxy-d3)propionamide ((R)-
1-d3). Using method A, (R)-N-benzyl 2-N-(benzyloxycarbonyl)-
amino-3-(methoxy-d3)propionamide (1.5 g, 4.25 mmol) and
10% Pd/C (200 mg) in MeOH (50 mL) gave after evaporation
an oily residue that was directly dissolved in CH2Cl2 (50 mL).
While stirring, Et3N (590 μL, 4.25 mmol), DMAP (25 mg, 212
μmol), and Ac2O (400 μL, 4.25 mmol) were successively added and
the reaction was stirred at room temperature (1 h). The organic
layer was washed with aqueous 0.1 M H2SO4 (20 mL). The
aqueous layer was extracted with CH2Cl2 (4ꢀ 20 mL). The organic
layers were combined, washed with brine (50 mL), dried (Na2SO4),
and the solvents were removed under vacuum. The obtained solid
was recrystallized from EtOAc and hexanes to give (R)-1-d3
as a white solid (888 mg, 82% over 2 steps): mp 142-143 °C;
Figure 2. Structural sites in (R)-1, where incorporation of nonpolar
substituents lead to pronounced anticonvulsant activity in the MES
seizure model.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5723
[R]25D þ16.0° (c 0.7, MeOH); Rf = 0.31 (5/95 MeOH/CH2Cl2). 1H
NMR (CDCl3) δ 1.97 (s, 3H), 3.47 (dd, J = 6.6, 9.0 Hz, 1H), 3.84
(dd, J = 4.0, 9.0 Hz, 1H), 4.34-4.52 (m, 2H), 4.55-4.69 (m, 1H),
6.72 (d, J = 7.0 Hz, 1H), 7.05-7.16 (m, 1H), 7.20-7.38 (m, 5H).
Mr (þESI) 276.1 [M þ Na]þ (calcdforC13H15D3N2O3Naþ 276.1).
Anal. (C13H15D3N2O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(2-cyclohexyloxy)propionamide
((R)-9). Using method B, a ∼1:1 mixture of (R)-methyl 2-acet-
amido-3-(cyclohexyloxy)propionate and (R)-ethyl 2-acetami-
do-3-(cyclohexyloxy)propionate (2.10 g, 8.4 mmol) in THF
(80 mL) and LiOH (202 mg, 8.4 mmol) in H2O (40 mL) gave
upon workup the corresponding acid (1.62 g, 7.1 mmol, 84%) as
a yellow viscous oil (Mr (þESI) 252.1212 [M þ Na]þ (calcd for
C11H19NO4Naþ 252.1204)) that was directly dissolved in THF
(70 mL). Using method C, addition of benzylamine (930 μL, 8.5
mmol) and DMTMM (2.4 g, 8.5 mmol) gave (R)-9 as a white
solid (1.17 g, 65%) after purification by flash chromatography
(1:2 hexanes/EtOAc to 1/9 MeOH/CH2Cl2) followed by recrys-
for 2 steps) upon purification by flash chromatography (EtOAc
to 1/9 MeOH/CH2Cl2): mp 145-146 °C; [R]25 -28.7° (c 0.7;
D
CHCl3); Rf = 0.49 (EtOAc). 1H NMR (CDCl3) δ 2.01 (s, 3H),
3.51 (dd, J = 7.8, 9.0 Hz, 1H), 3.90 (dd, J = 4.2, 9.0 Hz, 1H),
4.38-4.64 (m, 5H), 6.40-6.52 (br d, 1H), 6.72-6.84 (br t, 1H),
7.18-7.38 (m, 10H). Mr (þESI) 349.2 [M þ Na]þ (calcd for
C19H22N2O3Naþ 349.2). Anal. (C19H22N2O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(but-3-enyloxy)propionamide
((R)-16). Using method C, (R)-2-acetamido-3-(but-3-en-1-yloxy)-
propionic acid (1.32 g, 6.57 mmol), benzylamine (860 μL, 7.88
mmol), and DMTMM (2.18 g, 7.88 mmol) gave (R)-16 (1.30 g,
67%) as a white solid after purification by flash chromatogra-
phy (EtOAc) and further recrystallization from EtOAc and
hexanes: mp 103-104 °C; [R]25 þ51.3° (c 0.6; MeOH); Rf =
D
1
0.37 (5/95 MeOH/CH2Cl2). H NMR (CDCl3) δ 2.03 (s, 3H),
2.22-2.36 (m, 2H), 3.40-3.62 (m, 3H), 3.85 (dd, J = 4.2, 9.0 Hz,
1H), 4.45 (d, J = 7.2 Hz, 2H), 4.48-4.57 (m, 1H), 4.92-5.06 (m,
2H), 5.62-5.74 (m, 1H), 6.42-6.56 (m, 1H), 6.84-6.96 (br t,
1H), 7.20-7.36 (m, 5H). Mr (þESI) 313.1528 [M þ Na]þ (calcd
for C16H22N2O3Naþ 313.1528). Anal. (C16H22N2O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(2-cyclopropylethoxy)propio-
namide ((R)-17). Using method B, a ∼1:1 mixture of (R)-methyl
2-acetamido-3-(2-cyclopropylethoxy)propionate and (R)-ethyl
2-acetamido-3-(2-cyclopropylethoxy)propionate (1.60 g, 6.6
mmol) in THF (60 mL) and LiOH (158 mg, 6.6 mmol) in H2O
(30 mL) gave upon workup the corresponding acid (1.27 g, 5.9
mmol, 90%) as a yellow oil (Mr (þESI) 238.1056 [M þ Na]þ
(calcd for C10H17NO4Naþ 238.1054)) that was directly dis-
solved in THF (60 mL). Using method C, addition of benzyl-
amine (740 μL, 7.13 mmol) and DMTMM (2.0 g, 7.13 mmol)
gave (R)-17 as a white solid (1.17 g, 65%) after purification by
flash chromatography (1:2 hexanes/EtOAc to 1/9 MeOH/
CH2Cl2) followed by recrystallization from EtOAc and hexanes:
mp 97-99 °C; [R]25D -32.1° (c 1.5; MeOH); Rf = 0.52 (EtOAc).
1H NMR (CDCl3) δ -0.08-0.01 (m, 2H), 0.30-0.38 (m, 2H),
0.50-0.64 (m, 1H), 1.32-1.48 (m, 2H), 2.02 (s, 3H), 3.40-3.62
(m, 3H), 3.84 (dd, J = 3.9, 9.0 Hz, 1H), 4.38-4.58 (m, 3H),
6.48-6.58 (br d, 1H), 6.90-7.00 (br t, 1H), 7.20-7.36 (m, 5H).
Mr (þESI) 327.2 [M þ Na]þ (calcd for C17H24N2O3Naþ 327.2).
Anal. (C17H24N2O3): C, H, N.
tallization from EtOAc and hexanes: mp 134-135 °C; [R]25
D
-33.5° (c 1.2; CHCl3); Rf = 0.52 (EtOAc). 1H NMR (CDCl3) δ
1.10-1.34, 1.42-1.88 (m, 10H), 2.03 (s, 3H), 3.24-3.36 (m, 1H),
3.43 (app t, J = 9.0 Hz, 1H), 3.86 (dd, J = 3.6, 9.0 Hz, 1H),
4.38-4.56 (m, 3H), 6.48-6.58 (br d, 1H), 6.92-7.02 (br t, 1H),
7.22-7.38 (m, 5H). Mr (þESI) 341.3 [M þ Na]þ (calcd for
C18H26N2O3Naþ 341.3). Anal. (C18H26N2O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(but-2-yn-1-yloxy)propionamide
((R)-13). To a cooled (-78 °C) solution of (R)-2-acetamido-
3-(but-2-ynyloxy)propionic acid (1.15 g, 5.78 mmol) in THF (50
mL) were successively added N-methylmorpholine (NMM)
(0.95 mL, 8.67 mmol), stirred (2 min), isobutylchloroformate
(IBCF) (0.95 mL, 7.28 mmol), stirred (15 min), and benzylamine
(0.75 mL, 6.94 mmol).38 The reaction was allowed to warm to
room temperature and further stirred (2 h), filtered, and con-
centrated in vacuo. Purification by flash chromatography (1/9
MeOH/CHCl3) gave 1.53 g (92%) of (R)-13 as a white solid: mp
149-151 °C; [R]25 -34.2° (c 0.5, CHCl3); Rf = 0.45 (1/9
D
MeOH/CHCl3). 1H NMR (CDCl3) δ 1.83 (t, J = 2.4 Hz, 3H),
2.03 (s, 3H), 3.61 (dd, J = 7.2, 9.3 Hz, 1H), 3.90 (dd, J = 4.2, 9.3
Hz, 1H), 4.08-4.21 (m, 2H), 4.41-4.54 (m, 2H), 4.57-4.63 (m,
1H), 6.48-6.51 (br d, J = 6.6 Hz, 1H), 6.77-6.84 (br m, 1H),
7.25-7.37 (m, 5H). Mr (þESI) 288.1 [M þ H]þ (calcd for
C16H20N2O3Hþ 288.1). Anal. (C16H20N2O3) C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(but-3-ynyloxy)propionamide
((R)-18). Using method D, compound (R)-38 (1.14 g, 5.22
mmol), homopropargyl alcohol (2.0 mL, 26.1 mmol), and
(R)-N-Benzyl 2-Acetamido-3-(2-cyclohexylmethoxy)propio-
namide ((R)-14). Using method B, a ∼1:4 mixture of (R)-methyl
2-acetamido-3-(cyclohexylmethoxy)propionate and (R)-ethyl
2-acetamido-3-(cyclohexylmethoxy)propionate (2.32 g, 8.6 mmol)
in THF (85 mL) and LiOH (207 mg, 8.6 mmol) in H2O (40 mL)
gave upon workup the corresponding acid (1.88 g, 7.7 mmol, 90%)
as a yellow viscous oil that was directly dissolved in THF (75 mL).
Using method C, addition of benzylamine (1.0 mL, 9.2 mmol) and
DMTMM (2.54 g, 9.2 mmol) gave (R)-14 as a white solid (1.50 g,
59%) after purification by flash chromatography (1:2 hexanes/
EtOAc to 1/9 MeOH/CH2Cl2) followed by recrystallization from
EtOAc and hexanes: mp 143-144 °C; [R]25D þ6.7° (c 1.6; MeOH);
Rf = 0.39 (5/95 MeOH/CH2Cl2). 1H NMR (CDCl3) δ 0.74-0.90,
1.00-1.26, 1.40-1.72 (m, 11H), 2.02 (s, 3H), 3.16-3.32 (m,
2H), 3.42 (app. t, J = 9.0 Hz, 1H), 3.79 (dd, J = 3.6, 9.0 Hz,
1H), 4.38-4.50 (m, 2H), 4.51-4.60 (m, 1H), 6.53 (d, J = 6.3 Hz,
1H), 6.88-7.00 (br t, 1H), 7.22-7.38 (m, 5H). Mr (þESI) 355.1998
[M þ Na]þ (calcd for C19H28N2O3Naþ 355.1997). Anal. (C19H28-
N2O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(benzyloxy)propionamide ((R)-
15). To a stirred suspension of O-benzyl-D-serine (Astatech Inc.)
(1.00 g, 5.13 mmol) in a 9:1 THF:H2O mixture (50 mL) was
added Ac2O (1.45 mL, 15.3 mmol) at room temperature, and the
reaction was stirred at room temperature (3 h). The solvents
were removed in vacuo to yield a viscous clear residue (1.22 g,
5.13 mmol, quant) that was directly dissolved in THF (60 mL).
Using method C, benzylamine (670 μL, 6.2 mmol) and DMT-
MM (1.71 g, 6.2 mmol) gave (R)-15 as a white solid (1.55 g, 93%
BF3 Et2O (500 μL, 3.97 mmol) in CH2Cl2 (30 mL) gave (R)-
3
18 as a white solid (450 mg, 30%) upon workup, purification by
flash chromatography (2/1 EtOAc/CH2Cl2), and subsequent
recrystallization from EtOAc and hexanes: mp 120-122 °C;
[R]25 -49.1° (c 0.8, CHCl3); Rf = 0.38 (2/1 EtOAc/CH2Cl2).
D
1H NMR (CDCl3) δ 1.80 (t, J = 2.7 Hz, 1H), 2.04 (s, 3H),
2.38-2.46 (m, 2H), 3.46-3.74 (m, 3H), 3.90 (dd, J = 3.9, 9.0 Hz,
1H), 4.38-4.58 (m, 3H), 6.48-6.56 (m, 1H), 6.92-7.06 (m, 1H),
7.20-7.38 (m, 5H).; Mr (þESI) 421.1 [M þ Cs]þ (calcd for
C16H20N2O3Csþ 421.1). Anal. (C16H20N2O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-phenethoxypropionamide ((R)-
19). Using method D, compound (R)-38 (2.00 g, 9.2 mmol),
phenethyl alcohol (6.0 mL, 49.2 mmol), and BF3 Et2O (1.0 mL,
3
8.0 mmol) in CH2Cl2 (30 mL) gave (R)-19 as a white solid (392
mg, 10%) upon workup and purification by flash chromatog-
raphy (2/1 EtOAc/CH2Cl2) and recrystallization from EtOAc
and hexanes: mp 90-92 °C; [R]25D -29.2° (c 0.5, CHCl3); Rf =
0.44 (3/1 EtOAc/CH2Cl2). 1H NMR (CDCl3) δ 1.97 (s, 3H), 2.84
(d, J = 6.3 Hz, 2H), 3.40 (app d, J = 8.1 Hz, 1H), 3.62-3.92 (m,
3H), 4.20-4.38 (m, 2H), 4.40-4.50 (m, 1H), 6.32-6.40 (m, 1H),
6.50-6.62 (m, 1H), 7.12-7.38 (m, 10H). Mr (þESI) 363.2 [M þ
Na]þ (calcd for C20H24N2O3Naþ 363.2). Anal. (C20H24N2O3):
C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(2-(3-methyl-3H-diazirin-3-yl)-
ethoxy)propionamide ((R)-20). Using method C, (R)-2-acetami-
do-3-(2-(3-methyl-3H-diazirin-3-yl)ethoxy)propionic acid (500 mg,
2.2 mmol), benzylamine (285 μL, 2.6 mmol), and DMTMM

5724 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Morieux et al.
(723 mg, 2.6 mmol) in THF (22 mL) gave (R)-20 as a white solid
(565 mg, 81%) after purification by flash chromatography (3/2
hexanes/EtOAc to EtOAc to 3/2 EtOAc/acetone): Rf = 0.43
THF (170 mL) and LiOH (415 mg, 17.3 mmol) in H2O (80 mL)
gave 1.10 g (31%, 5.36 mmol) of a crude viscous yellow oil upon
workup (Mr (þESI) 228.0848 [M þ Na]þ (calcd for C8H15NO5-
Naþ 228.0848)). Using method C, the oil was directly dissolved in
THF (60 mL), and then benzylamine (732 μL, 6.7 mmol) followed
by DMTMM (1.90 g, 6.7 mmol) were added. Purification by flash
chromatography (1/9 hexanes/EtOAc to 1/9 MeOH/CH2Cl2) fol-
lowed by recrystallization from EtOAc and hexanes afforded (R)-
27 (750 mg, 45%) as a white solid: mp 109-110 °C; [R]25D þ10.3° (c
0.5; MeOH); Rf = 0.43 (1/9 MeOH/CH2Cl2). 1H NMR (CDCl3) δ
2.02 (s, 3H), 3.18 (s, 3H), 3.43-3.53 (m, 3H), 3.62-3.78 (m, 2H),
3.85 (dd, J = 4.2, 9.6 Hz, 2H), 4.37-4.51 (m, 2H), 4.52-4.58 (m,
1H), 6.65 (d, J = 6.6 Hz, 1H), 7.21-7.38 (m, 6H). Mr (þESI)
317.1478 [M þ Na]þ (calcd for C15H22N2O4Naþ 317.1477). Anal.
(C15H22N2O4): C, H, N.
Pharmacology. Compounds were screened under the auspices
of the National Institutes of Health’s Anticonvulsant Screening
Program. Experiments were performed in male rodents (albino
Carworth Farms no. 1 mice (intraperitoneal route, ip), albino
Spague-Dawley rats (oral route, po)). Housing, handling, and
feeding were in accordance with recommendations contained in
the “Guide for the Care and Use of Laboratory Animals”. Antic-
onvulsant activity was established using the MES test,53,54 6 Hz,55
and the scPTZ test,53 according to previously reported methods.6
1
(EtOAc); mp 120-121 °C; [R]24.1 -33.4° (c 0.5, CHCl3). H
D
NMR (CDCl3) δ0.98 (s, 3H), 1.65 (t, J = 6.0 Hz, 2H), 2.07 (s, 3H),
3.14-3.22 (m, 1H), 3.33-3.44 (m, 2H), 3.84 (dd, J = 3.6, 9.0 Hz,
1H), 4.44-4.58 (m, 3H), 6.63 (d, J = 6.3 Hz, 1H), 6.97-7.05 (br t,
1H), 7.24-7.37 (m, 5H). Mr (þESI) 319.1769 [M þ H]þ (calcd
C16H22N4O3Hþ 319.1770). Anal. (C16H22N4O3): C, H, N.
(R)-N-Benzyl 2-Acetamido-3-(3-oxopropoxy)propionamide ((R)-
21). To a cooled solution (dry ice/acetone bath) of oxalyl chloride
(432 μL, 4.92 mmol) in CH2Cl2 (12 mL) was added dropwise a
solution of DMSO (720 μL, 9.84 mmol) in CH2Cl2 (24 mL). After
stirring at -78 °C (15 min), a solution of (R)-N-benzyl 2-acetamido-
3-(3-hydroxypropoxy)propionamide (1.20 g, 4.08 mmol) in CH2Cl2
(12 mL) was added dropwise at -78 °C and the reaction was stirred
at -78 °C (1 h). DIEA (3.55 mL, 14.4 mmol) was then added
dropwise, stirred (20 min), warmed to room temperature, and then
stirred (30 min). Aqueous 10% citric acid (50 mL) was added to the
solution, and the CH2Cl2 layer was separated. The aqueous layer
was extracted with CH2Cl2 (6 ꢀ 50 mL), the combined organic
layers were washed with brine (50 mL), dried (Na2SO4), and
evaporated to give an oily residue that was recrystallized from
EtOAc and hexanes to afford (R)-21 as white needles: mp 120-
121 °C; [R]25 -22.1° (c 0.9, CHCl3); Rf = 0.34 (5/95 MeOH/
D
CHCl3). 1H NMR (CDCl3) δ 2.04 (s, 3H), 2.60-2.80 (m, 2H), 3.48
(dd, J = 7.5, 9.3 Hz, 1H), 3.70-3.78 (m, 1H), 3.80-3.87 (m, 1H),
3.90 (dd, J = 3.6, 9.3 Hz, 1H), 4.46 (d, J = 6.0 Hz, 2H), 4.51-4.58
(m, 1H), 6.61 (d, J = 6.3 Hz, 1H), 6.85-7.04 (br t, 1H), 7.22-7.38
(m, 5H), 9.71 (t, J = 1.4 Hz, 1H). Mr (þESI) 315.1323 [M þ Na]þ
(calcdforC15H20N2O4Naþ 315.1321). Anal. (C15H20N2O4): C, H, N.
(2R)-N-Benzyl 2-Acetamido-3-(2-(oxiran-2-yl)ethoxy)propio-
namide ((R)-22) (Mixture of Diastereomers). A solution of
mCPBA (77 wt %, 420 mg, 1.88 mmol) in CH2Cl2 (3 mL) was
stirred in the presence of Na2SO4 (∼50 mg, 5 min), and (R)-16
(320 mg, 1.10 mmol) was added at once. The suspension was
stirred at room temperature (15 h), filtered, concentrated under
vacuum to give (R)-22 (265 mg, 72%) as a ∼1:1 mixture of
diastereoisomers after purification by flash chromatography
Acknowledgment. We thank the NINDS and the ASP at
the National Institutes of Health with Drs. Tracy Chen and
Jeffrey Jiang, for kindly performing the pharmacological stud-
ies via the ASP’s contract site at the University of Utah with
Drs. H. Wolfe, H. S. White, and K. Wilcox. The project was
supported by grants R01NS054112 (H.K.) and F31NS060358
(P.M.) from the National Institute of Neurological Disorders
and Stroke, and by the Korean Research Foundation Grant
funded by the Korean Government (MOEHRD) grant KRF-
2006-352-C00042 (K.D.P.). The content is solely the responsi-
bility of the authors and does not necessarily represent the
official views of the National Institute of Neurological Dis-
orders and Stroke or the National Institutes of Health. Harold
Kohn has a royalty-stake position in (R)-1.
(15/85 acetone/EtOAc): mp 104-110 °C; [R]25 -29.6° (c 0.3;
D
CHCl3); Rf = 0.31 (15/85 acetone/EtOAc). 1H NMR (CDCl3) δ
1.52-1.64 (m, 1H), 1.86-2.00 (m, 1H), 2.03, 2.04 (s, 3H),
2.41-2.46 (m, 1H), 2.66-2.71 (m, 1H), 2.82-2.96 (m, 1H),
3.44-3.52 (m, 1H), 3.54-3.76 (m, 2H), 3.84-3.93 (m, 1H),
4.39-4.52 (m, 2H), 4.50-4.58 (m, 1H), 6.56-6.68 (m, 1H),
6.88-6.98, 6.99-7.08 (m, 1H), 7.22-7.36 (m, 5H). Mr (þESI)
329.1478 [M þ Na]þ (calcd for C16H22N2O4Naþ 329.1477).
Anal. (C16H22N2O4): C, H, N.
Supporting Information Available: Synthetic procedures for
the intermediates leading to the preparation of (R)-1-d3, (R)-9,
1
(R)-13-(R)-22, (R)-24, (R)-27, and 44, elemental analyses, H
and 13C NMR spectra of compounds (R)-1-d3, (R)-9, (R)-13-
(R)-22, (R)-24, and (R)-27 evaluated in this study. This material
is available free of charge via the Internet at http://pubs.acs.org.
(R)-N-Benzyl 2-Acetamido-3-(2-acetamidoethoxy)propiona-
mide ((R)-24). Using method A, (R)-25 (1.14 g, 3.7 mmol) and
10% Pd/C (100 mg) in MeOH (25 mL) gave upon filtration and
evaporation a residue that was dissolved in CH2Cl2 (50 mL). Et3N
(570 μL, 4.1 mmol), DMAP (1 mg, catalytic), and Ac2O (390 μL,
4.1 mmol) were then successively added and the reaction was
stirred at room temperature (30 min), filtered, and the solvents
were removed under vacuum. The crude residue was purified by
flash chromatography (1/9 MeOH/CH2Cl2) to give an oily residue
that was dissolved in warm THF (25 mL). The white solid that
precipitated upon cooling was filtered to give (R)-24 (490 mg, 40%
overall yield for two steps): mp 166-168 °C; [R]25D þ11.7° (c 0.6,
MeOH); Rf = 0.30 (5/95 MeOH/CH2Cl2). 1H NMR (DMSO-d6)
δ 1.79, 1.88 (s, 6H), 3.10-3.24 (m, 2H), 3.36-3.46 (m, 2H),
3.52-3.64 (m, 2H), 4.29 (d, J=7.0 Hz, 2H), 4.42-4.52 (m, 1H),
7.20-7.36 (m, 5H), 7.80-7.88 (m, 1H), 8.07 (d, J = 7.0 Hz, 1H),
8.50 (t, J = 6.0 Hz, 1H). Mr (þESI) 344.2 [M þ Na]þ (calcd for
C16H23N3O4Naþ 344.2). Anal. (C16H23N3O4): C, H, N.
References
(1) Hauser, W. A.; Annegers, J. F.; Kurland, L. T. Prevalence of
epilepsy in Rochester, Minnesota: 1940-1980. Epilepsia 1991, 32,
429–445.
(2) Evans, J. H. Post-traumatic epilepsy. Neurology 1962, 12, 665–674.
(3) Lindsay, J. M. M. Genetics and epilepsy: a model from critical path
analysis. Epilepsia 1971, 12, 47–54.
(4) Willmore, L. J.; Triggs, W. J.; Pellock, J. M. Valproate toxicity:
risk-screening strategies. J. Child Neurol. 1991, 6, 3–6.
(5) Pellock, J. M.; Willmore, L. J. A rational guide to routine blood
monitoring in patients receiving antiepileptic drugs. Neurology
1991, 41, 961–964.
(6) Choi, D.; Stables, J. P.; Kohn, H. Synthesis and anticonvulsant
activities of N-benzyl-2-acetamidopropionamide derivatives.
J. Med. Chem. 1996, 39, 1907–1916.
(7) Perucca, E.; Yasothan, U.; Clincke, G.; Kirkpatrick, P. Lacosamide.
Nature Rev. Drug Discovery 2008, 7, 973–974.
€
(8) Stohr, T.; Kupferberg, H. J.; Stables, J. P.; Choi, D.; Harris, R. H.;
Kohn, H.; Walton, N.; White, H. S. Lacosamide, a novel anti-
convulsant drug, shows efficacy with a wide safety margin in rodent
models for epilepsy. Epilepsy Res. 2007, 74, 147–154.
(R)-N-Benzyl 2-Acetamido-3-(2-methoxyethoxy)propion-
amide ((R)-27). Using method B, a ∼5:95 mixture of (R)-methyl
2-acetamido-3-(2-methoxyethoxy)propionate and (R)-ethyl 2-
acetamido-3-(2-methoxyethoxy)propionate (4.00 g, 17.4 mmol) in
€
(9) Beyreuther, B. K.; Freitag, J.; Heers, C.; Krebsfanger, N.;
€
Scharfenecker, U.; Stohr, T. Lacosamide: a review of preclinical
properties. CNS Drug Rev. 2007, 13, 21–42.

Article
Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15 5725
€
(10) Errington, A. C.; Coyne, L.; Stohr, T.; Selve, N.; Lees, G. Seeking a
mechanism of action for the novel anticonvulsant lacosamide.
Neuropharmacology 2006, 50, 1016–1029.
aziridines with diethoxytriphenylphosphorane. J. Org. Chem.
1986, 51, 95–97.
(33) Kelly, J. W.; Evans, S. A. Regioselective phosphoranylation
and cyclodehydration of triols with diethoxytriphenylphosphor-
ane. J. Am. Chem. Soc. 1986, 108, 7681–7685.
(34) Mathieu-Pelta, I.; Evans, S. A. A new and improved preparation of
acyclic σ-dialkoxyphosphoranes. J. Org. Chem. 1994, 59, 2234–
2237.
(35) Murray, W. T.; Kelly, J. W.; Evans, S. A. Synthesis of substituted
1,4-oxathianes. Mechanistic details of diethoxytriphenylphosphor-
ane and triphenylphosphine/tetrachloromethane-promoted cyclo-
dehydrations and carbon-13 NMR spectroscopy. J. Org. Chem.
1987, 52, 525–529.
(36) Robinson, P. L.; Kelly, J. W.; Evans, S. A. The synthetic utility of
dioxyphosphoranes in organic synthesis. Phosphorus Sulfur 1986,
26, 15–24.
(37) Kuyl-Yeheskiely, E.; Dreef-Tromp, C.; van der Marel, G.; van
Boom, J. An expeditious in situ preparation of benzyl (S)-2-
aziridinecarboxylate. Recl. Trav. Chim. Pays-Bas 1989, 108, 314–
316.
(38) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. Reinvestiga-
tion of the mixed carbonic anhydride method of peptide synthesis.
J. Am. Chem. Soc. 1967, 89, 5012–5017.
(39) Kunishima, M.; Kawachi, C.; Monta, J.; Terao, K.; Iwasaki, F.;
Tani, S. 4-(4,6-Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholi-
nium chloride: an efficient condensing agent leading to the forma-
tion of amides and esters. Tetrahedron 1999, 55, 13159–13170.
(40) Kubota, T.; Miyashita, S.; Kitazume, T.; Ishikawa, N. Novel
synthetic reactions using bis(2,2,2-trifluoroethoxy)triphenylphos-
phorane. J. Org. Chem. 1980, 45, 5052–5057.
(41) Yang, Z. Bis(fluoroalkoxy)triphenylphosphorane: a new reagent
for the preparation of fluorinated ketals. J. Org. Chem. 1995, 60,
5696–5698.
(42) Andurkar, S. V.; Stables, J. P.; Kohn, H. Synthesis and antic-
onvulsant activities of (R)-(O)-methylserine derivatives. Tetrahe-
dron: Asymmetry 1998, 9, 3841–3854.
(43) Choi, D.; Kohn, H. Trimethylsilyl halides: effective reagents for the
synthesis of β-halo amino acid derivatives. Tetrahedron Lett. 1995,
36, 7011–7014.
(44) Mancuso, A. J.; Brownfain, D. S.; Swern, D. J. Structure of the
dimethyl sulfoxide-oxalyl chloride reaction product. Oxidation of
heteroaromatic and diverse alcohols to carbonyl compounds.
J. Org. Chem. 1979, 44, 4148–4150.
(45) Casadei, M. A.; Galli, C.; Mandolini, L. Ring-closure reactions. 22.
Kinetics of cyclization of diethyl (ω-bromoalkyl)malonates in the
range of 4- to 21-membered rings. Role of ring strain. J. Am. Chem.
Soc. 1984, 106, 1051–1056.
(46) Rostovtsev, V. V.; Green, L. G.; Fokin, V. V.; Sharpless, K. B. A
stepwise Huisgen cycloaddition process: copper(I)-catalyzed regio-
selective “ligation” of azides and terminal alkynes. Angew. Chem.,
Int. Ed. 2002, 41, 2596–2599.
(11) Standifer, K.; Kohn, H., see: LeTiran, A. Design and synthesis of
functionalized amino acids (FAAs), FAA peptidomimetics, and
FAA affinity labels. Studies on their mode of action for the control
of convulsive disorders, Ph.D. Dissertation, University of Houston,
2000, Chapter 3.
€
(12) Errington, A. C.; Stohr, T.; Heers, C.; Lees, G. The investigational
anticonvulsant lacosamide selectively enhances slow inactivation
of voltage-gated sodium channels. Mol. Pharmacol. 2008, 73,
157–169.
(13) Sheets, P. L.; Heers, C.; Stoehr, T.; Cummins, T. R. Differential
block of sensory neuronal voltage-gated sodium channels by
lacosamide [(2R)-2-(acetylamino)-N-benzyl-3-methoxypropana-
mide], lidocaine, and carbamazepine. J. Pharmacol. Exp. Ther.
2008, 326, 89–99.
ꢀ
(14) Park, K. D.; Morieux, P.; Salome, C.; Cotten, S. W.; Reamtong, O.;
Eyers, C.; Gaskell, S. J.; Stables, J. P.; Liu, R.; Kohn, H. Lacosa-
mide isothiocyanate-based agents: novel agents to target and
identify lacosamide receptors. J. Med. Chem. 2009, 52, 6897–6911.
(15) Deo, R. C.; Schmidt, E. F.; Elhabazi, A.; Togashi, H.; Burley, S. K.;
Strittmatter, S. M. Structural bases for CRMP function in plexin-
dependent semaphorin3A signaling. EMBO J. 2004, 23, 9–22.
(16) Park, K. D.; Stables, J. P.; Liu, R.; Kohn, H. Proteomic searches
comparing two (R)-lacosamide affinity baits: an electrophilic
arylisothiocyanate and a photoactivated arylazide group. Org.
Biomol. Chem. 2010, 8, 2803–2813.
(17) Levy, R. H.; Mattson, R. H.; Meldrum, B. Antiepileptic Drugs, 4th
ed.; Raven Press: New York, 1995, Chapter 6.
(18) Salome, C.; Salome-Grosjean, E.; Park, K. D.; Morieux, P.;
Swendiman, R.; DeMarco, E.; Stables, J. P.; Kohn, H. Synthesis
and anticonvulsant activities of (R)-N-(40-substituted)benzyl
2-acetamido-3-methoxypropionamides. J. Med. Chem. 2010, 53,
1288–1305.
(19) Conley, J. D.; Kohn, H. Functionalized DL-amino acid derivatives.
Potent new agents for the treatment of epilepsy. J. Med. Chem.
1987, 30, 567–574.
(20) Kohn, H.; Conley, J. D. New antiepileptic agents. Chem. Ber. 1988,
231–234.
(21) Kohn, H.; Conley, J. D.; Leander, J. D. Marked stereospecificity in
a new class of anticonvulsants. Brain Res. 1988, 457, 371–375.
(22) Gallivan, J. P.; Dougherty, D. A. Cation-pi interactions in
structural biology. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 9459–
9464.
(23) Morieux, P.; Stables, J. P.; Kohn, H. Synthesis and anticonvulsant
activities of N-benzyl (2R)-2-acetamido-3-oxysubstituted propio-
namide derivatives. Bioorg. Med. Chem. 2008, 16, 8968–8975.
(24) Axelsson, B. S.; O’Toole, K. J.; Spencer, P. A.; Young, D. W.
Versatile synthesis of stereospecifically labelled D-amino acids via
labelled aziridines-preparation of (2R,3S)-[3-2H1]- and (2R,3R)-
[2,3-2H2]-serine; (2S,20S,3S,30S)-[3,30-2H2]- and (2S,20S,3R,30R)-
[2,20,3,30-2H4]-cystine; and (2S,3S)-[3-2H1-] and (2S,3R)-[2,3-2H2]-
β-chloroalanine. J. Chem. Soc., Perkin Trans. 1 1994, 807–815.
(25) Baldwin, J. E.; Adlington, R. M.; Robinson, N. G. Nucleophilic
ring opening of aziridine-2-carboxylates with Wittig reagents; an
enantioefficient synthesis of unsaturated amino acids. J. Chem.
Soc., Chem. Commun. 1987, 153–155.
(26) Belohradsky, M.; Ridvan, L.; Zavada, J. Synthesis of homochiral
acyclic mono- and bis (alpha-amino acids) with oligo(oxyethylene)
ohains. Collect. Czech. Chem. Commun. 2003, 68, 1319–1328.
(27) Galonic, D. P.; van der Donk, W. A.; Gin, D. Y. Site-selective
conjugation of thiols with aziridine-2-carboxylic acid-containing
peptides. J. Am. Chem. Soc. 2004, 126, 12712–12713.
(28) Kato, S.; Harada, H.; Morie, T. Efficient synthesis of (6R)-6-
amino-1-methyl-4-(3-methylbenzyl)hexahydro-1H-1,4-diazepine
from methyl (2R)- and (2S)-1-benzyloxycarbonylaziridine-2-car-
boxylates. J. Chem. Soc., Perkin Trans. 1 1997, 3219–3226.
(29) Lee, T.; Bryan Jones, J. Probing enzyme stereospecificity. Evalua-
tion of β-alkoxy-R-amino acids with two stereocenters as inhibitors
of serine proteases. Tetrahedron 1995, 51, 7331–7346.
(30) Nakajima, K.; Oda, H.; Okawa, K. Studies on 2-aziridinecar-
boxylic acid. IX. convenient synthesis of optically active S-alkyl-
cysteine, threo-S-alkyl-β-methylcysteine, and lanthionine deriva-
tives via the ring-opening reaction of aziridine by several thiols.
Bull. Chem. Soc. Jpn. 1983, 56, 520–522.
ꢀ
ꢀ
(47) Weisman, G. R. In Asymmetric Synthesis-Analytical Methods;
Morrison, J.,Weisman, G., Ed.; Academic Press: New York, 1983; Vol. 1,
pp 153-171.
(48) Stables, J. P.; Kupferberg, H. G. In Molecular and Cellular Targets
for Antiepileptic Drugs; Avoli, M., Avanzini, G., Tanganelli, P., Eds.;
John Libbey: London, 1977; pp 191-196.
(49) Krall, R. L.; Penry, J. K.; Kupferberg, H. J.; Swinyard, E. A.
Antiepileptic Drug Development: I. History and a program for
progress. Epilepsia 1978, 19, 393–407.
ꢁ
ꢀ
ꢀ
(50) Barton, M. E.; Klein, B. D.; Wolf, H. H.; White, H. S. Pharmaco-
logical characterization of the 6 Hz psychomotor seizure model of
partial epilepsy. Epilepsy Res. 2001, 47, 217–227.
(51) Porter, R. J.; Cereghino, J. J.; Gladding, G. D.; Hessie, B. J.;
Kupferberg, H. J.; Scoville, B.; White, B. G. Antiepileptic drug
development program. Cleveland Clin. Q. 1984, 51, 293–305.
(52) Dunham, N. W.; Miya, T. S. A note on a simple apparatus for
detecting neurological deficit in rats and mice. J. Am. Pharm.
Assoc., Sci. Ed 1957, 46, 208–209.
(53) Kohn, H.; Sawhney, K. N.; LeGall, P.; Robertson, D. W.; Leander,
J. D. Preparation and anticonvulsant activity of a series of func-
tionalized R-heteroatom-substituted amino acids. J. Med. Chem.
1991, 34, 2444–2452.
(54) Bardel, P.; Bolanos, A.; Kohn, H. Synthesis and anticonvulsant
activities of R-acetamido-N-benzylacetamide derivatives contain-
ing an electron-deficient R-heteroaromatic substituent. J. Med.
Chem. 1994, 37, 4567–4571.
(31) Nakajima, K.; Neya, M.; Yamada, S.; Okawa, K. Studies on
2-aziridinecarboxylic acid. VI. Synthesis of β-alkoxy-R-amino
acids via ring-opening reaction of aziridine. Bull. Chem. Soc. Jpn.
1982, 55, 3049–3050.
(55) Cortes, S.; Liao, Z. K.; Watson, D.; Kohn, H. Effect of structural
modification of the hydantoin ring on anticonvulsant activity.
J. Med. Chem. 1985, 28, 601–606.
(32) Kelly, J. W.; Anderson, N. L.; Evans, S. A. Cyclodehydration of
(56) Higgins, G. A.; Isaac, M.; Slassi, A.; Xin, T. Fluorinated com-
N- and C-substituted β-amino alcohols to the corresponding
pounds. Patent WO/2007/076306, 2007.

5726 Journal of Medicinal Chemistry, 2010, Vol. 53, No. 15
Morieux et al.
(57) Speers, A. E.; Cravatt, B. F. Profiling enzyme activities in vivo
using click chemistry methods. Chem. Biol. 2004, 11, 535–546.
(58) Daly, A. K.; Mantle, T. J. The kinetic mechanism of the major form
of ox kidney aldehyde reductase with D-glucuronic acid. Biochem.
J. 1982, 205, 381–388.
(59) Whittle, S. R.; Turner, A. J. Effects of the anticonvulsant sodium
valproate on gamma-aminobutyrate and aldehyde metabolism in
ox brain. J. Neurochem. 1978, 31, 1453–1459.
(62) Morisseau, C.; Hammock, B. D. Epoxide hydrolases: mechanisms,
inhibitor designs, and biological roles. Annu. Rev. Pharmacol.
Toxicol. 2005, 45, 311–333.
(63) Greenfield, N. J.; Pietruszko, R. Two aldehyde dehydrogenases
from human liver. Isolation via affinity chromatography and
characterization of the isozymes. Biochim. Biophys. Acta 1977,
483, 35–45.
(64) Yarnell, A. T. Heavy-hydrogen drugs turn heads, again. Chem.
(60) Rane, A.; Peng, D. Phenytoin enhances epoxide metabolism
in human fetal liver cultures. Drug Metab. Dispos. 1985, 13, 382–
385.
(61) Marshall, A. D.; Caldwell, J. Influence of modulators of epoxide
metabolism on the cytotoxicity of trans-anethole in freshly isolated
rat hepatocytes. Food Chem. Toxicol. 1992, 30, 467–473.
Eng. News 2009, 87, 36–39.
(65) Assessment Report for Vimpat; European Medicines Agency, 2008.
(66) Bialer, M.; Johannessen, S. I.; Kupferberg, H. J.; Levy, R. H.;
Loiseau, P.; Perucca, E. Progress report on new antiepileptic drugs:
a summary of the Sixth Eilat Conference (EILATVI). Epilepsy Res.
2002, 51, 31–71.