Lacosamide isothiocyanate-based agents: Novel agents to target and identify lacosamide receptors

Article abstract of DOI:10.1021/jm9012054

(R)-Lacosamide ((R)-2, (R)-N-benzyl 2-acetamido-3-methoxypropionamide) has recently gained regulatory approval for the treatment of partial-onset seizures in adults.Whole animal pharmacological studies have documented that (R)-2 function is unique. A robust strategy is advanced for the discovery of interacting proteins associated with function and toxicity of (R)-2 through the use of (R)-2 analogues, 3, which contain "affinity bait (AB)" and "chemical reporter (CR)" functional groups. In 3, covalent modification of the interacting proteins proceeds at the AB moiety, and detection or isolation of the selectively captured protein occurs through the bioorthogonal CR group upon reaction with an appropriate probe. We report the synthesis, pharmacological evaluation, and interrogation of the mouse soluble brain proteome using 3 where the AB group is an isothiocyanate moiety. One compound, (R)-N-(4-isothiocyanato)benzyl 2-acetamido-3-(prop-2-ynyloxy) propionamide ((R)-9), exhibited excellent seizure protection in mice, and like (R)-2, anticonvulsant activity principally resided in the (R)-stereoisomer. Several proteins were preferentially labeled by (R)-9 compared with (S)-9, including collapsin response mediator protein 2. 2009 American Chemical Society.

Full text of DOI:10.1021/jm9012054

J. Med. Chem. 2009, 52, 6897–6911 6897
DOI: 10.1021/jm9012054
Lacosamide Isothiocyanate-Based Agents: Novel Agents To Target and Identify Lacosamide Receptors
†
†
†
‡
‡
ꢀ †
Ki Duk Park, Pierre Morieux, Christophe Salome, Steven W. Cotten, Onrapak Reamtong, Claire Eyers,
Simon J. Gaskell,‡ James P. Stables,§ Rihe Liu,*,†, and Harold Kohn*,†,^
†Division of Medicinal Chemistry and Natural Products, UNC Eshelman School of Pharmacy, University of North Carolina, Chapel Hill,
North Carolina 27599-7568, ‡Michael Barber Centre for Mass Spectrometry, Manchester Interdisciplinary Biocentre, University of Manchester,
131 Princess Street, Manchester, M1 7DN, U.K., §Anticonvulsant Screening Program, National Institute of Neurological Disorders and Stroke,
National Institutes of Health, 6001 Executive Boulevard, Suite 2106, Rockville, Maryland 20892-9523, Carolina Center for Genome Sciences,
University of North Carolina, Chapel Hill, North Carolina 27599-7264, and ^Department of Chemistry, University of North Carolina, Chapel Hill,
North Carolina 27599-3290
Received August 12, 2009
(R)-Lacosamide ((R)-2, (R)-N-benzyl 2-acetamido-3-methoxypropionamide) has recently gained
regulatory approval for the treatment of partial-onset seizures in adults. Whole animal pharmacological
studies have documented that (R)-2 function is unique. A robust strategy is advanced for the discovery
of interacting proteins associated with function and toxicity of (R)-2 through the use of (R)-2 analogues,
3, which contain “affinity bait (AB)” and “chemical reporter (CR)” functional groups. In 3, covalent
modification of the interacting proteins proceeds at the AB moiety, and detection or isolation of the
selectively captured protein occurs through the bioorthogonal CR group upon reaction with an
appropriate probe. We report the synthesis, pharmacological evaluation, and interrogation of the
mouse soluble brain proteome using 3 where the AB group is an isothiocyanate moiety. One compound,
(R)-N-(4-isothiocyanato)benzyl 2-acetamido-3-(prop-2-ynyloxy)propionamide ((R)-9), exhibited
excellent seizure protection in mice, and like (R)-2, anticonvulsant activity principally resided in the
(R)-stereoisomer. Several proteins were preferentially labeled by (R)-9 compared with (S)-9, including
collapsin response mediator protein 2.
Introduction
compound, (R)-lacosamide ((R)-2, (R)-N-benzyl 2-acetamido-
3-methoxypropionamide18), has recently gained regulatory
approval for adjunctive therapy for partial-onset seizures in
adults.19 Whole animal pharmacological studies have documen-
ted that (R)-2 function is unique and prevents seizure spread by
mechanisms different from those of current clinical agents.18,20
Radioligand displacement assays using more than 100 potential
binding sites, which include both ligand- and voltage-gated
receptors, failed to identify high-affinity binding targets,21
suggesting that either the target site(s) are novel or the binding
is weak or both. Only recently has (R)-2 function been reported
to be correlated to the modulation of both the slow inactivation
gate of sodium channels22 and collapsin response mediator
protein 2 (CRMP2).21 The full spectrum of pharmacological
pathways for (R)-2 remains elusive.
Epilepsy is a major neurological disorder that affects all
populations.1 Epilepsy describes the types of recurrent sei-
zures produced by paroxysmal excessive neuronal discharges
in the brain.2,3 The mainstay of treatment has been the
long-term and consistent administration of anticonvulsant
drugs.4-6 Unfortunately, current medications are ineffective
for approximately one-third of patients with epilepsy.7 Many
continue to have seizures, while others experience disturbing
sideeffects (e.g., drowsiness, dizziness, nausea, liver damage).8
The shortcomings of current regimens highlight the need for
new agents with novel mechanisms of action. The ability to
identify specific nervous system pathways and protein target
sites responsible for seizures would accelerate the develop-
ment of new and safer therapeutic agents.
In 1985, we discovered a novel class of anticonvulsant agents,
termed functionalized amino acids (FAA,a 1).9-18 The lead
*To whom correspondence should be addressed. For R.L.: telephone,
919-843-3635; fax, 919-966-0204; e-mail, rliu@email.unc.edu. For H.K.:
telephone, 919-843-8112; fax, 919-966-0204; e-mail, hkohn@email.unc.edu.
a Abbreviations: FAA, functionalized amino acids; AB, affinity bait;
CR, chemical reporter; CRMP2, collapsin response mediator protein 2;
NMDA, N-methyl D-aspartate; GABA, γ-aminobutyric acid; AMPA,
R-amino-3-hydroxyl-5-methyl-4-isoxazole propionate; SAR, structure-
-activity relationship; MES, maximal electroshock; IBCF, isobutyl
chloroformate; NMM, N-methylmorpholine; DMTMM, 4-(4,6-di-
methoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; DPT,
di(2-pyridyl) thionocarbonate; DITC, 1,10-thiocarbonyldiimidazole;
scMet, subcutaneous metrazol; TCEP, tris(2-carboxyethyl)phosphine;
iTRAQ, isobaric tag for relative and absolute quantitation; GST,
glutathione-S-transferase; CNS, central nervous system.
These findings have led us to suggest that (R)-2 may interact
with several targets and that (R)-2drug efficacy is a summation
of multiple beneficial interactions, similar to other neurologi-
cal agents.23 Using two antiepileptic agents as examples,24
carbamazepine interacts with sodium channels, reduces
sodium and calcium influx through N-methyl D-aspartate
r
2009 American Chemical Society
Published on Web 10/01/2009
pubs.acs.org/jmc

6898 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Park et al.
Scheme 1. “Affinity Bait” and “Chemical Reporter” (AB&CR) Strategy To Scan the Brain Proteome for (R)-Lacosamide Target(s)
(NMDA) activated channels at glutamate receptor sites,
blocks the reuptake of norepinephrine into presynaptic
terminals, and has adenosine receptor antagonistic activity.
Similarly, topiramate blocks sodium channels, enhances
γ-aminobutyric acid (GABA)A mediated chloride influx,
acts as an antagonist to R-amino-3-hydroxyl-5-methyl-4-isox-
azole propionate (AMPA) and kainate receptors, and inhibits
carbonic anhydrase.
A major objective of our laboratories is to identify and
characterize the target(s) and mechanism(s) of (R)-2 function
and to advance molecular tools useful to elucidate the path-
ways leading to seizure control. Here, we report three mod-
ified (R)-2 agents each with an appended isothiocyanate
moiety that have been designed to search the brain proteome
for site(s) of drug binding. These three compounds were used
to interrogate soluble mouse brain lysate. We show that
CRMP2 along with other potential lacosamide target(s) are
selectively captured.
(drugs) where binding does not occur at the enzyme
catalytic site. The captured (R)-2-bound target 8 in
the reaction mixture (e.g., cell lysate) is either detected by
in-gel fluorescence through the use of a reporter group
tethered to 7 or selectively isolated from the mixture using
affinity-based chromatographic methods (e.g., biotin
(strept)avidin).
b. Structural and Chemical Criteria for Lacosamide
AB&CR Probes ((R)-3). Six criteria have been established
for our AB&CR ((R)-3) agents. First, the compound
must conform to the structure-activity relationship (SAR)
observed for 1.9-18 In this case, we must limit the size of the
AB and the CR groups because of 1’s SAR.9-18 Second, the
reactive AB group must have documented success in pre-
vious target adduction studies.27 In this report, we describe
(R)-3 agents into which an electrophilic affinity bait has been
installed. For the electrophilic AB, we expect that this group
must be correctly aligned with the target-based nucleophile if
covalent modification is to take place.27 Third, the CR group
must be inert under the conditions necessary to bait the
target protein yet must be prone to react with probe
P. Fourth, syntheses of both enantiomeric forms of the
lacosamide AB&CR agents 3 are necessary. A distinguishing
feature of 1 is that its anticonvulsant activity resides princi-
pally in the D-configuration.11-13,15,18 Accordingly, prefer-
ential enantioselective irreversible inactivation with the
AB&CR agents serves as an important measure of target
site specificity.28,29 Fifth, the electrophilic (photoaffinity)
labels should be incorporated at different sites within the
(R)-2 structure to capture all its possible binding partners.
Sixth, the (R)-3 agents used to identify target sites should
ideally demonstrate significant anticonvulsant activity in in
vivo assays (e.g., maximal electroshock (MES) seizure
test30). Documentation of similar pharmacological activity
provides preliminary evidence that the mode of action of
(R)-3 is comparable with (R)-2. We recognize that promising
pharmacological data do not guarantee that these com-
pounds will serve as site-selective agents, since the ability
of the AB unit to efficiently modify the protein targets is a
composite of variables, some of which are unrelated to
function. Correspondingly, compounds inactive in vivo
may still function as in vitro affinity labels, since chemical
Results
a. The Proposed Method. Central to our efforts is the
expansion of traditional and contemporary methods of
identifying small-molecular-weight ligand protein target
sites where binding is modest and where moderate-to-exten-
sive ligand structural change abolishes target binding.
Accordingly, we have designed (R)-2 analogues that contain
an “affinity bait (AB)” and a “chemical reporter (CR)”25
group. These compounds are defined as lacosamide AB&CR
agents (R)-3. In Scheme 1, we diagram the proposed pathway
for (R)-3 function. We anticipate that (R)-3 reversibly binds
to the (R)-2 receptor(s) 4 to give 5, provided that the AB and
CR moieties do not perturb binding. This interaction is
followed by an irreversible, covalent modification wherein
receptor capture occurs through the AB moiety to give 6.
In most instances, the AB group is either an electrophilic
or a photoaffinity agent. Both groups have been extensively
utilized to capture biomolecular targets. Removal of the
selectively captured protein 6 occurs through the bioortho-
gonal25a CR group upon reaction with a probe (P, 7)
to provide 8. While similar target discovery approaches
have been reported,26 few, if any, have been designed
to identify receptors of small-molecular-weight ligands

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6899
Scheme 2. General Synthetic Pathway To Prepare Lacosamide AB&CR Agents 9-11
and cellular (e.g., transport, metabolic) processes may pre-
vent ligand-target site binding. Nonetheless, demonstrating
significant and stereospecific in vivo anticonvulsant activity
provides an initial stringent test for our agents.
with hydroxylic solvents (permitting its dissolution in
aqueous solutions) but does react with nucleophiles
(e.g., thiols (cysteines), amines (lysines)).27 Significantly,
isothiocyanate electrophilic units have proven effective
in selectively modifying target proteins (e.g., GABA,37
opioid31,34,35) within brain membrane fractions and have
been used for in vivo modification studies after intracerebro-
ventricular administration.31c Nonetheless, the isothio-
cyanate AB unit has limitations. First, only lysine and
cysteine residues are likely to be modified. This restriction
may prevent covalent adduction of some receptors. Fortu-
nately, the extended C(R) methylene chain within lysines
gives this nucleophilic amino acid considerable conforma-
tional flexibility to reach a bound AB&CR agent. Another
concern is that hydrophobic drug binding pockets may
not contain nearby lysine residues. Considerable support
exists for the selected CR moieties (i.e., alkyne, azide)
and for the notion that protein capture with P occurs
through a Cu(I)-mediated cycloaddition reaction (“click
chemistry”).25
c. Compound Selection. In this study, we selected (R)- and
(S)-9-11 as our initial AB&CR agents where AB is the
electrophilic isothiocyanate moiety.31-42 We have chosen
small AB and CR groups to minimize adverse binding of our
agents with possible receptor sites. In 9 and 11, we have
incorporated the isothiocyanate moiety at the 40 N-benzyl
position. Earlier studies showed that N-benzyl 40-substituted
1 compounds exhibited enhanced anticonvulsant activities
compared with their corresponding 30-isomers.43 In 9, the
CR group is an alkyne, while in 11 it is an azide. In compound
10, the sites of AB (isothiocyanate) and CR (alkyne) incor-
poration have been interchanged from that in 9.
d. Synthesis of Agents. The synthetic pathways used to
prepare 9-11 are provided in Scheme 2. Care was taken
to install the AB unit in the last step to avoid possible
isothiocyanate-related reactions during the synthesis and
reaction workup. In general, commercially available D- or
L-serine methyl ester hydrochloride (12) was converted to the
corresponding (R)- and (S)-methyl 1-acetylaziridine-2-
carboxylates (16), followed by stereospecific ring-opening
with the appropriate alcohol using catalytic amounts
The isothiocyanate AB group displays an excellent bal-
ance between stability and reactivity; it does not readily react
of BF3 Et2O to give the desired (R)- and (S)-methyl
3

6900 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Table 1. Pharmacological Evaluation of Lacosamide AB&CR Agents
Park et al.
mice (ip)a,c
rat (po)b,c
Tox,g TD50
(mg/kg)
>30
NDh
3.7 29 [0.5] (21-40) 7.9 [0.5] (4.7-11) >500
MES,d ED50
(mg/kg)
Tox,e TD50
(mg/kg)
6 Hz, ED50
MES, ED50
(mg/kg)
compd
(R)-9
(S)-9
R
X
PIf (mg/kg) (32 mA)
PIf
CH2CtCH
CH2CtCH
CH2CtCH
CH2CtCH
CH3
NCS
NCS
H
45 [1] (41-48)
>300
110 [0.25] (76-150) 2.4 53 [0.5] (42-68) >30
>300
59 [0.25] (55-66)
>300
NDh
NDh
(R)-28
(S)-28
(R)-31i
(S)-31
16 [0.25] (13-19)
>300
>63
>59
H
>30
2.0 NDh
NDh
>30
4.2 [4] (2.4-8.0)
>180
>30
>250
>30
NDh
NDh
NCS
NCS
24 [0.5] (21-27)
>100, <300
CtCH NDh
CtCH NDh
47 [0.25] (43-50)
>30, <100
NDh
CH3
(R)-10
(S)-10
CH2CH2NCS
CH2CH2NCS
CH3
NDh
NDh
NDh
NDh
NDh
(R)-29
(S)-29
CtCH >3, <10 [0.5]
CtCH >300 [0.5]
>10, <30 [0.5]
>300 [0.5]
>30, <100
>30, <100 [0.5]
>100, < 300 [0.5]
NDh
<30 [0.5 -4.0]
NDh
<30 [0.5-4.0]
NDh
CH3
NDh
(R)-27
(S)-27
CH2CH2NCS
CH2CH2NCS
CH2CH2N3
CH2CH2N3
CH2CH2N3
CH2CH2N3
CH3
H
>300 [0.5]
>300 [0.5]
>100, <300 [4.0]
NDh
<30 [4.0]
<30 [4.0]
NDh
NDh
>30 [4.0]
NDh
H
NDh
(R)-11
(S)-11
NCS
NCS
H
NDh
NDh
>100, <300 [0.25] >300
>100, <300 [4.0]
>300
NDh
NDh
>30
(R)-30
(S)-30
>100, <300 [4.0]
>300
44 [0.5] (32-65) >40 [0.25]
H
NDh
2.0 NDh
NDh
NDh
NDh
(R)-31
(S)-31
(R)-2j
phenytoink
phenobarbital
NCS
NCS
24 [0.5] (21-27)
>100, <300
47 [0.25] (43-50)
>30, <100
4.2 [4] (2.4-8.0)
>180
>250
>30
>59
CH3
4.5 [0.5] (3.7-5.5)
9.5 [2] (8.1-10)
22 [1] (15-23)
27 [0.25] (38-47)
66 [2] (53-72)
69 [0.5] (63-73)
6.0 10
6.9
3.2
3.9 [0.5] (2.9-5.5) >500 [0.5]
>130
>100
6.7
30 [4] (22-39)
9.1 [5] (7.6-12)
l
61 [0.5]
(44-96)
280 [0.5]
(190-350)
valproate
270 [0.25]
(250-340)
430 [0.25]
(370-450)
1.6
490 [0.5]
(350-730)
0.6
a The compounds were administered intraperitoneally. b The compounds were administered orally. c Numbers in parentheses are 95% confidence
intervals. The dose effect data was obtained at the “time of peak effect” (indicated in hours in the brackets). d MES = maximal electroshock seizure test.
e Tox = neurologic toxicity determined from rotorod test. f PI = protective index (TD50/ED50). g Tox = behavioral toxicity. h ND = not determined.
i LeTiran, A.; Stables, J. P.; Kohn, H. J. Med. Chem. 2002, 45, 4762-4773. j Choi, D.; Stables, J. P.; Kohn, H. J. Med. Chem. 1996, 39, 1907-1916. k Porter,
R. J.; Cereghino, J. J.; Gladding, G. D.; Hessie, B. J.; Kupferberg, H. J.; Scoville, B.; White, B. G. ClevelandClin. Q. 1984, 51, 293-305. l No ataxia observed up to
3000 mg/kg.
2-acetamido-3-(alkoxy)propanoates (17, 18).44,45 The esters
17 and 18 were converted to the free acids 19 and 20,
respectively, using stoichiometric amounts of LiOH at
0 ꢀC. Under these conditions, we detected little or no
racemization. The free acids were coupled with the appro-
priate substituted benzylamine using either the mixed anhy-
dride coupling method46a (i.e., isobutyl chloroformate
(IBCF), N-methyl morpholine (NMM)) or 4-(4,6-di-
methoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM)46b to give the lacosamide analogues 23-25. The
syntheses of 9-11 concluded with AB isothiocyanate moiety
installation. For 9 and 10, we used di-(2-pyridyl) thiono-
carbonate (DPT)47a and for 11, 1,10-thiocarbonyldiimidazole
(DITC).47b The spectral and analytical data for 9-11 and all
intermediates were in agreement with their proposed struc-
tures. We documented the enantiopurity of the final com-
pounds using the chiral resolving agent (R)-mandelic acid
and 1H NMR spectroscopy.48
anticonvulsant activity for the lacosamide isothiocyanate
(AB) probes (R)-31 and (S)-31.43a
e. Pharmacological Evaluation. Initial criteria we estab-
lished for our lacosamide AB&CR agents are that they prove
effective in preventing MES-induced seizures in animal
models30 and that the principal activity resides in the (R)-
stereoisomer (D-configuration). In the MES test, tonic hin-
dlimb seizures are induced by corneal electrical stimulation.
This assay is an established method to identify compounds
effective against generalized tonic-clonic seizures30 and was
the initial assay used to discover (R)-2. Table 1 lists the MES
anticonvulsant activities obtained for the lacosamide
In order to assess the individual effects of the AB
(isothiocyanate) and CR (alkyne, azide) units on anticon-
vulsant activities, the corresponding lacosamide analogues
that contained only the AB (27) or the CR (28-30) units
have also been prepared (Schemes S1-S3 in the Supporting
Information). We have previously reported the synthesis and

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6901
Figure 1. Chemical evaluation of the AB and the CR units in 3.
AB&CR compounds (9, 11), the analogues into which
only an AB (27, 31) or a CR (28-30) moiety was installed
in the lacosamide framework, (R)-2,18 and other clinical
antiepileptic agents. The compounds were also evaluated
in the subcutaneous metrazol (scMet) seizure model but
provided no protection (data not shown). The absence
of seizure protection in this assay is a hallmark of 1
activity.9-18
The findings for the lacosamide AB and the lacosamide
CR analogues guided our investigation of 9-11 where both
an AB and a CR moiety were installed in the molecule. Here,
we found that 9, containing a N-benzyl 40-isothiocyanate
moiety and an O-propargyl moiety, provided significant
seizure protection (MES ED50 = 45 mg/kg) and that activity
principally resided in the (R)-stereoisomer. For 11, we
observed that both stereoisomers gave only minimal seizure
protection, a finding that mirrored the anticonvulsant
activity for the corresponding CR-only analogues (R)-30
and (S)-30. Finally, we did not evaluate (R)-10 and (S)-10 in
animal models because the corresponding AB compounds
(R)-27 and (S)-27 were inactive at 300 mg/kg.
f. Chemical Evaluation of the AB and CR Units in 3. The AB
and CR moieties have been installed within 9-11 for specific
chemical functions. The isothiocyanate AB unit serves as an
electrophilic agent to trap either lysine or cysteine residue(s)
situated near the (R)-2 binding site, and either the alkyne or
azide CR group acts as a bioorthogonal unit that can be
selectively captured by the corresponding azide- or alkyne-
containing probe via a Cu(I)-mediated cycloaddition reac-
tion.50,51 Prior to conducting our studies we evaluated the
chemical reactivity of these groups using model reactions
(Figure 1). Treating AB (R)-31 with EtNH2 gave the
expected thiourea (R)-32 in high yield (94%).43 Substituting
the aliphatic isothiocyanate 33 for (R)-31 gave the expected
thiourea 34 (77% yield).43a Correspondingly, when (R)-9
was reacted with 1.0 equiv of the biotin-containing azide
probe 35,52 CuSO4, and sodium ascorbate,50 we isolated the
We found that we could incorporate a small moiety in the
40 position of the N-benzylamide unit of (R)-2 without
appreciable loss of MES anticonvulsant activity compared
with unmodified (R)-2 (MES ED50 = 4.5 mg/kg) in mice (ip).
For example, excellent seizure protection was observed
for CR (R)-29 (MES ED50 = 3-10 mg/kg) and AB (R)-31
(MES ED50 = 24 mg/kg).43 We also found that some
modifications of the C(2) side chain in (R)-2 led to excellent
activity retention in the MES seizure test while others did
not. The MES ED50 values for the CR O-propargyl ((R)-28),
the CR O-2-azidoethyl ((R)-30), and the AB O-2-isothio-
cyanatoethyl ((R)-27) lacosamide analogues were 16, 100-300,
and >300 mg/kg, respectively. We do not know if transport,
efflux, or metabolic factors are responsible for the
diminished activity for (R)-27 and (R)-30 in the MES test.
Interestingly, (R)-30 showed appreciable activity in the
psychomotor seizure test49 (6 Hz ED50 = 44 mg/kg). Con-
sistent with the pharmacological pattern observed for 218
and other FAAs (1),11-13,15 we found that for 28, 29, and 31,
the (R)-stereoisomer was consistently more potent than the
(S)-stereoisomer.

6902 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Park et al.
desired product, (R)-36, in 51% yield. No modification of the
isothiocyanate moiety was observed under these Cu(I)-
mediated cycloaddition conditions. Finally, we treated ali-
phatic azide (R)-30 with alkyne 37 under similar conditions
to obtain (R)-38 (96% yield).
g. Using Mouse Brain Soluble Lysates To Identify Lacosa-
mide Targets. The utility of the lacosamide AB&CR agents
9-11 to identify drug binding targets was first assessed using
mouse brain soluble lysates prepared from male (2 months)
ICR mice (Rockland Immunochemicals, Gilbertsville, PA).
The lysates were treated with (R)- and (S)-9-11 (5 μM) for
0.5 h at room temperature, and the lysate mixture reacted
with an appropriate rhodamine-containing probe (39,53 40)
under Cu(I)-mediated cycloaddition conditions (CuSO4,
tris(2-carboxyethyl)phosphine (TCEP)). The reaction
samples were separated by electrophoresis using a 10%
SDS-PAGE gel and then analyzed for proteins labeled by
the AB&CR agents by in-gel fluorescence using a typhoon
scanning laser (emission 532 nm, absorption 580 nm)
(Figure 2A). Gels were Coomassie-stained to visualize the
proteins (Figure 2C).
Figure 2. Proteome reactivity profiles of three different isothiocya-
nate (NCS) agents 9-11. (A) AB&CR agent-labeled proteins were
detected by in-gel fluorescence scanning after Cu(I)-mediated cy-
cloaddition to either a rhodamine-azide reporter probe 39 or a
rodamine-alkyne reporter probe 40. (B) CRMP2 protein was
detected by Western blot using anti-CRMP2. (C) All proteins in
lysate were visualized by Coomassie blue staining after in-gel
fluorescence scanning. All images are shown in gray scale.
Figure 2A showed that AB&CR 9 efficiently labeled many
proteins in the lysate while 10 did not. The in-gel fluorescence
pattern for 11 suggested the labeling of many of the proteins
seen with 9, along with additional background proteins.
Significantly, the rhodamine-based probe used for 9- and
10-modified reactions was different from that used for
11-modified reaction. The alkyne CR moieties in 9 and 10
required the use of the azide probe 39 for the Cu(I)-mediated
cycloaddition step, while the azide CR group in 11 required
the alkyne-based probe 40. Cravatt and co-workers have
previously reported higher levels of background protein
adduction with alkyne probes compared with their azide
counterparts.53 This observation was consistent with the gel
patterns observed for 11 versus 9 and 10 (Figure 2A) that
showed more proteins modified by the probe 40 compared
with probe 39 under Cu(I)-mediated cycloaddition condi-
tions. Similarly, we observed more labeled proteins with 40
compared with 39 in the absence of the AB&CR agent
(Figure 2A, no drug control lanes).
Analysis of the (R)-9- and (S)-9-modified lysate gel
patterns showed that some proteins were more extensively
modified by (R)-9 than (S)-9 (Figure 2A; see bands labeled by
arrow and asterisk). One of these bands appeared to be
∼62 kDa (arrow), the approximate molecular weight of
CRMP2.54 To confirm that the ∼62 kDa band contained
CRMP2, we performed a Western blot analysis using an
anti-CRMP2 antibody and observed prominent bands con-
sistent with full-length and truncated forms of CRMP254
(Figure 2B). In addition to the ∼62 kDa band, we observed
another protein band (Figure 2A) with stronger intensity for
the (R)-9 lane than for (S)-9 (Figure 2A; see band labeled by
asterisk). The identity of this protein(s) is currently under
investigation.
We recognized that both the increased fluorescence of the
∼62 kDa band for the (R)-9 treated lysate compared with the
(S)-9 reaction and the identification of CRMP2 by Western
blot analysis do not demonstrate, by themselves, the prefer-
ential modification of CRMP2 by (R)-9. We suspect that the
∼62 kDa band consists of several proteins, and thus, the
observed fluorescence pattern may be due to a protein(s)
other than CRMP2. Accordingly, additional studies were
conducted to verify the selective modification of CRMP2 by
(R)-9. First, the (R)-9 lysate adduction was repeated. The
modified lysate was treated with biotinyl CR probe 35 using
Cu(I)-mediated cycloaddition conditions. Substitution of
probe 35 for 39 permits the selective isolation of the (R)-9
captured proteins from the lysate. The biotinylated proteins
were captured with streptavidin beads, the beads stringently
washed to remove background proteins adsorbed on the
resin, and then the protein adducts released with SDS
treatment. SDS-PAGE electrophoresis showed bands at
∼62 kDa upon Coomassie blue staining. The 62 kDa band
was excised, trypsinized, and analyzed by mass spectrometry
(MS). Analysis of the tryptic fragments indicated the pre-
sence of several proteins (e.g., CRMP2, CRMP1, serum
albumin) whose molecular weights were consistent with their
electrophoretic mobility. The protein with the largest num-
ber of identified fragments in the (R)-9 treated reaction was

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6903
CRMP2 (17 peptides including 11 peptides unique to
CRMP2 and 6 peptides common to other CRMP proteins,
45% coverage), followed by CRMP1 (6 peptides including 4
peptides unique to CRMP1 and 2 peptides common to other
CRMP proteins, 17% coverage) and serum albumin
(4 peptides, 10% coverage), indicating that CRMP2 was a
likely major constituent in the ∼62 kDa band. Correspond-
ingly, CRMP2 and other proteins were not observed in
∼62 kDa band in the no drug control reaction. We made a
quantitative comparison of the 62 kDa trypsin digests using
differential stable isotope labeling (isobaric tag for relative
and absolute quantitation (iTRAQ)).55 The trypsin digests
from the (R)-9, (S)-9, the no drug control reaction, and
iTRAQ buffer alone were each separately treated with
distinct iTRAQ isotope labels, the samples combined, and
then analyzed by LC-MS/MS (Table S1 in the Supporting
Information). There was no difference in observed iTRAQ
ratio between fragments unique to CRMP2 and those com-
mon to other CRMP proteins. The peptide fragments
showed, on average, a ∼1.3:1 increase in the relative amounts
of each tryptic fragment from the (R)-9 treated sample
compared with (S)-9, after normalization to the tubulin
fragments, based on the assumption of no stereochemical
preference for lacosamide binding to tubulin. Minor
amounts (<6% compared with the (R)-9 sample) of CRMP2
fragments were observed in the no drug sample. Analysis of
the CRMP1 fragments also showed a 1.3:1 increase in the
(R)-9 reaction compared with the (S)-9 reaction. This differ-
ential amount in tryptic fragment abundances was in agree-
ment with the observed ratio for the fluorescence intensities
of the ∼62 kDa band in the (R)-9 reaction versus the (S)-9
experiment (Figure 2A), substantiating the identification of
CRMP1 and CRMP2 as the relevant components of the gel
bands from which they were derived. Finally, we over-
expressed CRMP2 as a glutathione-S-transferase (GST) fusion
protein.56 Attachment of the GST tag permitted protein
purification and increased the size of the CRMP2 protein
by ∼25 kDa. The purified recombinant GST-CRMP2 pro-
tein (10 μg) was included in the lysate (50 μL, 2 mg/mL), and
the AB&CR adduction reaction was repeated with (R)-9,
(S)-9, and no drug. The reaction products were treated with
rhodamine-azide 39 under Cu(I)-mediated cycloaddition
conditions and then separated by 10% SDS-PAGE
(Figure 3). The electrophoretic mobility of the GST-CRMP2
placed it in a region (∼82 kDa) that was relatively free of
other soluble lysate proteins. We observed an approximate
1.5-fold increase in fluorescent intensity for the (R)-9
modified GST-CRMP2 protein compared with the corres-
ponding (S)-9 modified recombinant protein (double
arrowhead, Figure 3A). By comparison, the (R)-9-modified
endogenous 62 kDa band was approximately 1.8-fold more
intense than the corresponding (S)-9-modified band (arrow,
Figure 3A). Western blot analysis of the ∼82 kDa band was
in agreement with the presence of CRMP2 and GST
(Figure 3B). Collectively, these studies show that CRMP2
is preferentially modified in the lysate by lacosamide
AB&CR agent (R)-9 and that (R)-9 leads to higher CRMP2
adduction levels than the corresponding (S)-9 derivative.
Figure 3. In vitro labeling of externally added GST-CRMP2 and
endogenous CRMP2 in mixture of mouse lysate and overexpressed
GST-CRMP2. (A) AB&CR agent-labeled proteins were detected by
in-gel fluorescence scanning after Cu(I)-mediated cycloaddition to
rhodamine-azide reporter probe 39. (B) CRMP2 and GST proteins
were detected by Western blot using anti-CRMP2 and anti-GST
antibodies. (C) All proteins in lysate were visualized by Coomassie
blue staining after in-gel fluorescence scanning. All images are
shown in gray scale.
could be general. As a test case, we have searched for the
central nervous system (CNS) binding partners for (R)-2, a
therapeutic agent recently approved for the treatment
of partial-onset seizures.19 Whole animal pharmacological
studies indicated that (R)-2 function is unique.20
Our strategy requires the installation of AB and CR units
within the drug molecule (Scheme 1). The AB group in 3
covalently modifies the drug target to give 6, rendering
permanently a reversible binding interaction. The CR moiety
in3 is a biologicallyinert group that specificallyreactswith the
bioorthogonal probe 7 designed to either detect or isolate the
modified protein adduct. Specific criteria have been estab-
lished for the designed AB&CR units. These requirements are
stringent and have been adopted to minimize false positive
results. In this study, we sought lacosamide AB&CR agents
that exhibited potent anticonvulsant activity in rodent models
for epilepsy and where the pharmacological activity princi-
pally resided in the enantiomer corresponding to D-serine,
similar to that seen for (R)-2. This stereochemical selectivity
Discussion
A robust strategy is advanced for the discovery of proteins
that interact (bind) with small-molecular-weight ligands
(drugs) thatexplicate function. Our target discovery approach

6904 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Park et al.
for (R)-2 function and related 1 is a distinguishing feature for
this class of compounds,11-13,15,18 and we now show that this
enantiospecificity may extend to receptor tagging as well.
An efficient synthesis was devised to permit the preparation
of enantiomerically pure samples of the AB&CR agents 9-11
and the corresponding AB 27 and 31 and CR 28-30 com-
pounds (Scheme 1 and Supporting Information Schemes
chose to interrogate the mouse brain lysates with all three
AB&CR agents (9-11) to gain further information about the
suitability of different AB and CR units for receptor identi-
fication, with the understanding that only animal pharmaco-
logical data supported the use of 9.
We first chose to examine the soluble proteome from
Charles River male ICR mouse brains. Comparison of
AB&CR agents (R)- and (S)-9-11 showed that (R)-9 and
(S)-9 provided the cleanest in-gel fluorescent patterns while
still showing appreciable protein labeling (Figure 2A). This is
particularly apparent when the lanes for 9 are compared with
that for 11. These sets of agents contain the same AB unit
(40-isothiocyanate N-benzyl moiety) but have different CR
moieties. Consistent with results reported in the literature, we
observed that the number of proteins modified by using
alkyne-based probe 40 were considerably higher than with
azide-based probe 39. We have attributed this, in part, to
nonspecific lysate modification by the rhodamine-alkyne 40
probe.53 This unwanted reaction complicates receptor identi-
fication. When the protein band pattern for lacosamide
AB&CR agent 10 was compared with that for 9, we observed
a marked decrease in the extent of protein modification.
Compounds 9 and 10 can be considered functional isomers
where the structural positions of the AB and CR units have
been reversed. Together, our findings indicate that AB&CR
agent9 maybe best suited for lacosamidetarget identification.
This study showed that CRMP2 was preferentially mod-
ified in the lysate by lacosamide AB&CR agent (R)-9 and that
(R)-9 leads to higher CRMP2 adduction levels than the
corresponding reaction with the (S)-9 derivative. Interroga-
tion of the mouse brain soluble lysate is complicated by the
complexity of the proteome. Thus, several findings were used
to validate CRMP2 labeling. First, Western blot analysis
verified the presence of CRMP2 in the ∼62 kDa band after
lysate modification by (R)-9 and treatment with 39
(Figure 2B). Second, MS analysis of the trypsin digest of the
modified proteins in the ∼62 kDa band indicated that
CRMP2 was among the most abundant proteins present in
this gel slice. Third, the enhanced adduction by (R)-9 com-
pared with (S)-9 was confirmed using iTRAQ analysis of the
tryptic fragments from the ∼62 kDa band. A modest increase
(1.3-fold) in the abundance of CRMP2 fragments was
observed in the (R)-9 adduction experiment compared to
(S)-9 reaction when compared to an internal protein
(tubulin R chain) (Table S1 in the Supporting Information).
The increased (R)-9 adduction levels are important because
whole animal pharmacological studies have demonstrated
that the principal anticonvulsant activity for 2 resided in the
(R)-stereoisomer. Finally, we overexpressed GST-CRMP2
and then added a small amount of the fusion protein to the
mouse lysate. Repetition of the 9 adduction experiments
followed by Cu(I)-mediated cycloaddition with probe 39
and SDS-PAGE showed increased fluorescent levels
(1.5- to 1.8-fold) for both the exogenous ∼82 kDa and the
endogenous ∼62 kDa bands in the (R)-9 reaction compared
with the (S)-9 experiment (Figure 3A).
S1-S3). Key to our route was the BF3 Et2O catalyzed ring-
3
opening of aziridine 16.44 We found this method versatile, and
it reliably gave optically pure 2-alkoxypropionamide deriva-
tives. The isothiocyanate moiety was chosen as the AB moiety
in 9-11 because of its relative stability in aqueous solutions
and its reactivity with amines and thiols.27 We installed this
group from the corresponding amine in the final step of the
synthesis to minimize product loss. For amines 23 and 26, we
used di-(2-pyridyl) thionocarbonate,47a and for amine 25 we
employed 1,10-thiocarbonyldiimidazole47b for this step. We
further tested the reactivity of the different AB and CR groups
to validate their use in our proteomic target search (Figure 1).
An important design component for our AB&CR agents is
the useof small ABand CRunits inordertominimize possible
adverse interactions that could either prevent or alter target
binding. In the case of the C(2) site in (R)-2, for example, we
demonstrated that progressive size increases of the oxygen
substituent led to a steady loss in seizure protection in the
MES test (mice, ip).57 A similar structure-activity relation-
ship has been found for the 40 benzyl position in (R)-2
derivatives.58 Thus, in (R)-9, we incorporated an isothio-
cyanate group at the 40 N-benzyl site for the AB moiety and
a propargyl unit at C(2) alkoxy site for the CR moiety. We
found that (R)-9 exhibited notable anticonvulsant activity in
mice (ip) (MES ED50 = 45 mg/kg) while the corresponding
(S)-9 did not (MES ED50 > 300 mg/kg). The anticonvulsant
activities of the corresponding AB (R)-31 (MES ED50
=
24 mg/kg)43a and CR (R)-28 (MES ED50 = 16 mg/kg)
derivatives exceeded that of the AB&CR agent (R)-9 (MES
ED50 = 45 mg/kg). The loss of activity observed for (R)-9 is
consistent with the cumulative adverse effects that may have
occurred when two groups were appended to the lacosamide
framework. We observed only modest MES seizure protection
for AB&CR agent (R)-11 (MES ED50 = 100-300 mg/kg).
Moreover, the animal behavioral studies did not differentiate
the activities of (R)-11 and its stereoisomer (S)-11. The 6 Hz
psychomotor seizure test49 for (R)-30 (mice, ip; 32 mA) pro-
vided evidence to support the use of the 2-azidoethoxy group in
this lacosamide AB&CR agent. The absence of detectable
anticonvulsant activity of (R)-27 in mice (>300 mg/kg) in the
MES seizure test (Table 1) discouraged our preparation and
evaluation of the structural isomer of (R)-11 where the posi-
tions of the isothiocyanate and azide moieties are reversed.
Collectively, the whole animal pharmacological data
showed that incorporating small AB and CR units at either
the 40 N-benzyl site or the C(2)-alkoxy position did not
markedly perturb anticonvulsant function and likely did not
impede binding to the drug’s cognate receptors. Our data
demonstrated that the seizure protection provided by (R)-9
and (S)-9 mirrored that of (R)-2 and (S)-2, respectively.
Correspondingly, the whole animal pharmacological data
for 10 and 11 provided neither support nor evidence against
their use for in vitro proteomic searches. Placement of either
an isothiocyanate or azido group at the C(2) aliphatic
site may diminish their activity, in part, presumably by
preventing these compounds from reaching the CNS due
to chemical, transport, efflux, and metabolic processes. We
The relative ratios of (R)-9 versus (S)-9 modification of
CRMP2 proteins (1.3- to 1.8-fold) were modest compared
with the observed MES ED50 values for (R)-2 and (S)-2 in this
seizure model in mice (>22-fold).18 Several factors likely
contributed to this finding. First, the MES ED50 values for
(R)-9 and (S)-9 in rodents represent a composite value
that encompasses numerous pharmacological factors that
include binding to target(s), metabolism, transport, efflux,

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6905
and nonspecific binding to abundant cellular proteins. There-
fore, the in vitro modification data are not likely to fully
recapitulate the in vivo pharmacological results. Second,
different (R)-2 targets likely display different stereochemical
preferences for the 9 enantiomers, and this stereochemical
preference may be further influenced by the AB and CR
groups that are incorporated within the lacosamide frame-
work to facilitate in vitro analysis. Finally, we expect that the
conditions of adduction such as drug and target concentra-
tion, temperature, and time will affect the levels of (R)-9 and
(S)-9 CRMP2 adduction. For our experiments, we used
relatively high amounts of9 and moderate reaction conditions
to provide substantial protein adduction levels.
Our results support a report by Beyreuther and co-workers
that CRMP2 may interact with (R)-2.21 In their study, (R)-2-
like derivatives (precise structures not disclosed) that con-
tained an appended isothiocyanate group and a biotin moiety
were prepared and then reacted with mouse brain lysates.
Subsequent treatment of the modified lysate with streptavidin
removed the biotinylated tagged proteins. Differentiating
Beyreuther and colleagues’ study from ours was their use of
significantly larger (R)-2-type agents and the absence of whole
animal pharmacological data to support their use. Knowing
these collective findings, detailed studies are warranted to
determine the site of (R)-3 CRMP2 adduction and to validate
this protein as an (R)-2 target.
(589 nm) using a 1 dm path length cell. NMR spectra were
obtained at 300 MHz (1H) and 75 MHz (13C) using TMS as an
internal standard. Chemical shifts (δ) are reported in parts per
million (ppm) from tetramethylsilane. Low-resolution mass
spectra were obtained with a BioToF-II-Bruker Daltonics spectro-
meter by Drs. Matt Crowe and S. Habibi at the Department of
Chemistry, University of North Carolina. The high-resolution
mass spectra were obtained on a Bruker Apex-Q 12 Telsa
FTICR spectrometer by Drs. Matt Crowe and S. Habibi.
Microanalyses were performed by Atlantic Microlab, Inc.
(Norcross, GA). Reactions were monitored by analytical thin-
layer chromatography (TLC) plates (Aldrich, catalog no.
Z12272-6) and analyzed with 254 nm light. The reaction
mixtures were purified by MPLC (CombiFlash Rf) with self-
packed columns (silica gel from Dynamic Adsorbents Inc.,
catalog no. 02826-25) or by flash column chromatography using
silica gel (Dynamic Adsorbents Inc., catalog no. 02826-25). All
chemicals and solvents were reagent grade and used as obtained
from commercial sources without further purification. THF was
distilled from blue sodium benzophenone ketyl. Yields reported
are for purified products and were not optimized. All com-
pounds were checked by TLC, 1H and 13C NMR, MS, and
elemental analyses. The analytical results are within (0.40% of
the theoretical values. The TLC, NMR, and the analytical data
confirmed that the purity of the products was g95%.
General Procedure for the Aziridine Ring-Opening of (R)/(S)-
16 with Alcohols. Method A. To a cooled CH2Cl2 solution (ice
bath) containing the N-acetylaziridine methyl ester (R)/(S)-16
([C] ∼ 0.5-1 M) and the appropriate alcohol (1-3 equiv) was
We observed other possible (R)-2 binding target(s)
(Figure 2A, asterisk band). The identity of this band is under
investigation. Only mouse soluble brain proteome was used in
this study. Future studies will examine the membrane-bound
fraction. In this regard, (R)-2 has been reported to selectively
enhance the slow inactivation of voltage-gated sodium chan-
nels.22 The combined use of our AB&CR (R)-3 agents,
enzymatic digestion, and MS of the protein fragments may
help reveal the site(s) of drug binding.
added BF3 Et2O (0.1-1 equiv) dropwise while stirring. After
3
addition, the mixture was warmed to room temperature and
stirred (90 min to 2 h), 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 product could be
detected (TLC analysis) and then all the organic layers were
combined, dried (Na2SO4), and concentrated in vacuo to yield a
residue that was purified by flash column chromatography.
General Procedure for the Ester Hydrolysis of (R)/(S)-17 or
(R)/(S)-18 with LiOH. Method B. To a THF solution of methyl
ester (2 volumes, [C] ∼ 0.1 M) was added an aqueous LiOH
(1 equiv) solution (1 volume). The mixture was stirred at room
temperature (90 min to 12 h), after which time the aqueous layer
was washed with Et2O (2 volumes). The aqueous layer was
acidified (pH ∼1) by the dropwise addition of aqueous concen-
trated HCl at 0 ꢀC, 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 when needed from EtOAc and
hexanes to provide an analytical sample.
Conclusions
We advance a strategy to identify molecular targets for
ligands (e.g., drugs) whose binding to their cognate receptors
is modest and where moderate-to-extensive structural change
abolishes target binding. Our method requires the installation
of AB and CR units within the ligand where the AB group
covalently modifies the target and the CR group is used to
either identify or isolate the ligand-modified receptor. Strict
guidelines for the use of the AB&CR strategy have been
established to increase the success of this method and to
minimize the isolation of false positive sites. Among these
guidelinesare thatthe ABandCRgroupsmustconformtothe
SAR for the ligand and that the pharmacological profile for
the AB&CR agent must mirror that of the ligand. Using this
approach we interrogated the mice brain soluble proteome for
(R)-2 binding sites. We identified several potential drug
targets including CRMP2, a protein previously suggested to
interact with (R)-2. Further studies will be needed to validate
these targets and to identify the sites of drug interaction.
General Procedure for the Mixed Anhydride Coupling Reac-
tion. Method C. To a cooled THF solution (-78 ꢀC, dry
ice/acetone bath) of acid (R)/(S)-19 ([C] ∼ 0.1 M) were succes-
sively added NMM (1.0-1.1 equiv), stirred for 2 min, IBCF
(1.0-1.2 equiv), stirred for 5 min, and then the desired benzyl-
amine (1.0-1.2 equiv). Upon addition the reaction mixture was
allowed to warm to room temperature and further stirred
(2-3 h). The salts were filtered and rinsed with THF, and the
filtrate was concentrated in vacuo. The residue obtained was
purified by flash chromatography, followed by recrystallization
from EtOAc and hexanes when necessary.
General Procedure for the DMTMM Amide Coupling
Reaction. Method D. To a THF solution of acid (R)/(S)-20
([C] ∼ 0.1 M) at room temperature was added the desired
benzylamine (1.2 equiv). The solution was stirred (5-10 min)
until the benzylammonium 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
Experimental Section
General Methods. Melting points were determined in open
capillary tubes using a Thomas-Hoover melting point apparatus
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 a Jasco P-1030 polarimeter at the sodium D line

6906 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Park et al.
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 recrystal-
lized from EtOAc and hexanes.
(R)-Methyl 2-Acetamido-3-(prop-2-ynyloxy)propionate ((R)-17).
Utilizing method A, (R)-methyl 1-acetylaziridine-2-carboxylate44
((R)-16, 1.50 g, 10.49 mmol), propargyl alcohol (0.92 mL,
(CDCl3) δ 2.06 (s, CH3C(O)NH), 3.23-3.42 (m, CH2N3),
3.31-3.64 (m, OCH2CH2N3, CHCHH0OCH2), 3.78 (s, OCH3),
3.96 (dd, J = 3.0, 9.1 Hz, CHCHH0OCH2), 4.76-4.81 (m,
CHCH2O), 6.30-6.41 (br d, CH3C(O)NH); 13C NMR
(CDCl3) δ 23.2 (CH3C(O)), 50.0 (OCH2CH2N3), 52.6 (OCH3
or CHCH2O), 52.8 (CHCH2O or OCH3), 70.9 (CHCH2O or
OCH2CH2N3), 71.1 (OCH2CH2N3 or CHCH2O), 170.1, 170.6
(C(O)OCH3, CH3C(O)NH); Mr (þESI) 253.0906 [M þ Na]þ
(calcd for C8H14N4O4Naþ 253.0913 [M þ Na]þ).
15.74 mmol), and BF3 Et2O (1.39 mL, 11.01 mmol) gave 1.43 g
3
(68%) of (R)-17 as a white solid after recrystallization with cold
hexanes: mp 58.5-59.5 ꢀC; [R]25D -64.6ꢀ (c 1.0, CHCl3); Rf = 0.35
(3/1 EtOAc/hexanes); IR (nujol mull) 3301, 2919, 2114, 1750, 1639,
1542, 1457 cm-1; 1H NMR (CDCl3) δ 2.06 (s, CH3C(O)), 2.46 (t,
J = 2.3 Hz, CH2CCH), 3.78 (s, OCH3), 3.79 (dd, J = 3.3, 9.6 Hz,
CHH0OCH2), 3.98 (dd, J = 2.9, 9.6 Hz, CHH0OCH2), 4.09-4.21
(m, OCH2C), 4.77-4.82 (m, CH), 6.33-6.35 (br d, J = 7.2 Hz,
NHCH); 13C NMR (CDCl3) δ 23.3 (CH3C(O)), 52.6 (CH), 52.8
(OCH3), 58.7 (CH2CCH), 69.6 (CH2OCH2), 75.3 (CH2CCH), 79.0
(CH2CCH), 170.1, 170.8 (2 C(O)); Mr (þESI) 200.0916 [M þ H]þ
(calcd for C9H13NO4Hþ 200.0923 [M þ H]þ). Anal. (C9H13NO4)
C, H, N.
(S)-Methyl 2-Acetamido-3-(2-azidoethoxy)propionate ((S)-18).
Utilizing the preceding procedure and using (S)-1644 (3.30 g,
23 mmol), 2-azidoethanol (6.3 mL, 92 mmol), and BF3 Et2O
3
(1.4 mL, 11.5 mmol) in CH2Cl2 (115 mL) gave 1.67 g (31%) of a
colorless residue: [R]25D -48.0ꢀ (c1.0; EtOAc); Rf =0.47(EtOAc);
IR (neat) 3302, 2948, 2107, 1746, 1667, 1534, 1443 cm-1; 1H NMR
(CDCl3) δ 2.06 (s, CH3C(O)NH), 3.23-3.42 (m, CH2N3),
3.31-3.64 (m, OCH2CH2N3, CHCHH0OCH2), 3.78 (s, OCH3),
3.96 (dd, J = 3.0, 9.1 Hz, CHCHH0OCH2), 4.76-4.81 (m,
CHCH2O), 6.30-6.41 (br d, CH3C(O)NH); 13C NMR (CDCl3)
δ
23.3 (CH3C(O)), 50.1 (OCH2CH2N3), 52.7 (OCH3 or
CHCH2O), 52.9 (CHCH2O or OCH3), 71.0 (CHCH2O or
OCH2CH2N3), 71.2 (OCH2CH2N3 or CHCH2O), 170.2, 170.7
(CH3C(O)NH and C(O)OCH3); Mr (þESI) 253.0906 [M þ Na]þ
(calcd for C8H14N4O4Naþ 253.0913 [M þ Na]þ).
(S)-Methyl 2-Acetamido-3-(prop-2-ynyloxy)propionate ((S)-17).
Utilizing the preceding procedure and using (S)-1644 (1.50 g, 10.49
mmol), propargyl alcohol (0.92 mL, 15.74 mmol), and BF3 Et2O
(1.39 mL, 11.01 mmol) gave 1.32 g (63%) of (S)-17 as a white solid:
3
(R)-2-Acetamido-3-(2-azidoethoxy)propionic Acid ((R)-20).
Utilizing method B and using compound (R)-18 (1.56 g,
6.78 mmol) in THF (60 mL) and LiOH (195 mg, 8.14 mmol)
in H2O (30 mL) gave 750 mg (51%) of (R)-20 as a white solid. An
analytical sample was obtained by recrystallization from EtOAc
and hexanes: mp 99-100 ꢀC; [R]25D -12.6ꢀ (c 1.8; MeOH); Rf =
0.21 (1/9 MeOH/CHCl3); IR (nujol mull) 3356, 2119, 1735,
mp 59.0-59.5 ꢀC; [R]25 þ65.3ꢀ (c 1.0, CHCl3); Rf = 0.35 (3/1
D
EtOAc/hexanes); IR (nujol mull) 3296, 2924, 2116, 1740, 1641,
1543, 1457 cm-1; 1H NMR (CDCl3) δ 2.06 (s, CH3C(O)), 2.46 (t,
J = 2.4 Hz, CH2CCH), 3.78 (s, OCH3), 3.79 (dd, J = 3.2, 9.4 Hz,
CHH0OCH2), 3.98 (dd, J = 3.0, 9.4 Hz, CHH0OCH2), 4.12
(1/2HH0q, J = 2.4, 16.2 Hz, OCHH0C), 4.18 (1/2HH0q, J = 2.4,
16.2 Hz, OCHH0C), 4.77-4.82 (m, CH), 6.32-6.34 (br d, J = 7.2,
NHCH); 13C NMR (CDCl3) δ 23.3 (CH3C(O)), 52.6 (CH), 52.8
(OCH3), 58.7 (CH2CCH), 69.6 (CH2OCH2), 75.3 (CH2CCH),
79.0 (CH2CCH), 170.1, 170.7 (2 C(O)); Mr (þESI) 200.0917 [M þ
H]þ (calcd for C9H13NO4Hþ 200.0923 [M þ H]þ). Anal.
(C9H13NO4) C, H, N.
1
1624, 1547 cm-1; H NMR (CDCl3) δ 2.07 (s, CH3C(O)NH),
3.23-3.42 (m, CH2N3), 3.60-3.80 (m, OCH2CH2N3,
CHCHH0O), 4.01 (dd, J = 3.0, 9.1 Hz, CHCHH0O), 4.76-
4.80 (m, CHCH2O), 6.00-7.00 (br, CO2H), 6.48 (d, J = 7.5 Hz,
CH3C(O)NH), no signal could be detected for the COOH
proton; 13C NMR (DMSO-d6) δ 22.3 (CH3C(O)), 49.9
(OCH2CH2N3), 52.2 (CHCH2O), 69.4 (CHCH2O or OCH2-
CH2N3), 70.1 (OCH2CH2N3 or CHCH2O), 169.4 (CH3-
C(O)NH or C(O)OH), 171.5 (C(O)OH or CH3C(O)NH); Mr
(þESI) 239.0750 [M þ Na]þ (calcd for C7H12N4O4Naþ
(R)-2-Acetamido-3-(prop-2-ynyloxy)propionic Acid ((R)-19).
Utilizing method B, (R)-17 (1.00 g, 5.03 mmol), and LiOH
(0.6 M, 8.4 mL, 5.03 mmol) gave 0.87 g of (R)-19 (94%) as a
yellow oil: Rf = 0.35 (15/5/1 EtOAc/hexanes/AcOH); IR (nujol
mull) 3264, 2924, 2120, 1726, 1642, 1548, 1459 cm-1; 1H NMR
(CD3OD) δ 2.01 (s, CH3C(O)), 2.88 (t, J = 2.6 Hz, CH2CCH),
3.77 (dd, J = 3.6, 9.7 Hz, CHH0OCH2), 3.92 (dd, J = 5.0, 9.7
Hz, CHH0OCH2), 4.18 (d, J = 2.6 Hz, OCH2C), 4.61-4.64 (m,
CH); 13C NMR (CD3OD) δ 22.5 (CH3C(O)), 54.1 (CH),
59.3 (CH2CCH), 70.4 (CH2OCH2), 76.5 (CH2CCH), 80.2
(CH2CCH), 173.1, 173.5 (2 C(O)); Mr (þESI) 186.0761 [M þ
H]þ (calcd for C8H11NO4Hþ 186.0766 [M þ H]þ). Anal.
239.0756 [M þ Na]þ). Anal. (C7H12N4O4 0.07EtOAc) C, H, N.
3
(S)-2-Acetamido-3-(2-azidoethoxy)propionic Acid ((S)-20).
Utilizing the preceding procedure and using (S)-18 (1.67 g,
7.26 mmol) in THF (60 mL) and LiOH (209 mg, 8.71 mmol)
in H2O (30 mL) gave 862 mg (55%) of (S)-20 as a white solid
upon workup and evaporation. An analytical sample was
obtained by recrystallization from EtOAc and hexanes: mp 99-
100 ꢀC; [R]25 þ12.4ꢀ (c 1.3; MeOH); Rf = 0.21 (1/9 MeOH/
D
(C8H11NO4 0.14H2O) C, H, N.
CHCl3); IR (nujol mull) 3356, 2119, 1735, 1624, 1547 cm-1; 1H
NMR (DMSO-d6) δ 1.86 (s, CH3C(O)NH), 3.30-3.45 (m,
CH2N3), 3.50-3.78 (m, OCH2CH2N3, CHCH2OCH2), 4.39-
4.48 (m, CHCH2O), 8.13 (d, J = 8.1 Hz, CH3C(O)NH), no
signal could be detected for the COOH proton; 13C NMR
(DMSO-d6) δ 22.3 (CH3C(O)), 49.9 (OCH2CH2N3), 52.2 (CH-
CH2O), 69.4 (CHCH2O or OCH2CH2N3), 70.1 (OCH2CH2N3
or CHCH2O), 169.4 (CH3C(O)NH or C(O)OH), 171.5
(C(O)OH or CH3C(O)NH); Mr (þESI) 239.0750 [M þ Na]þ
(calcd for C7H12N4O4Naþ 239.0756 [M þ Na]þ). Anal.
3
(S)-2-Acetamido-3-(prop-2-ynyloxy)propionic Acid ((S)-19).
Utilizing the preceding procedure and using (S)-17 (1.00 g,
5.03 mmol) and LiOH (0.6 M, 8.4 mL, 5.03 mmol) gave 0.86 g
(93%) of (S)-19 as a yellow oil: Rf = 0.35 (15/5/1 EtOAc/
hexanes/AcOH); IR (nujol mull) 3270, 2924, 2120, 1729, 1641,
1
1547, 1459 cm-1; H NMR (DMSO-d6) δ 1.87 (s, CH3C(O)),
3.48 (t, J = 2.4 Hz, CH2CCH), 3.62 (dd, J = 4.1, 9.6 Hz,
CHH0OCH2), 3.74 (dd, J = 2.4, 9.6 Hz, CHH0OCH2), 4.16 (d,
J = 2.4 Hz, OCH2C), 4.41-4.47 (m, CH), 8.21-8.23 (d, J = 8.4
Hz, NHCH); 13C NMR (DMSO-d6) δ 22.4 (CH3C(O)), 52.1
(CH), 57.8 (CH2CCH), 69.2 (CH2OCH2), 77.6 (CH2CCH), 79.9
(CH2CCH), 169.6, 171.5 (2 C(O)); Mr (þESI) 186.0760 [M þ
H]þ (calcd for C8H11NO4Hþ 186.0766 [M þ H]þ). Anal.
(C7H12N4O4 0.07EtOAc) C, H, N.
3
(R)-N-(4-Amino)benzyl
2-Acetamido-3-(prop-2-ynyloxy)-
propionamide ((R)-23). Utilizing method C, (R)-19 (0.25 g, 1.37
mmol), NMM (0.23 mL, 2.06 mmol), IBCF (0.23 mL, 1.73 mmol),
and 4-aminobenzylamine (22) (0.21 mL, 1.85 mmol) gave 0.24 g
(61%) of (R)-23 as a light-yellow solid: mp 97.5-98.5 ꢀC;
[R]25D þ4.2ꢀ (c 1.0, MeOH); Rf = 0.40 (1/9 MeOH/CHCl3); IR
(nujol mull) 3279, 2930, 2111, 1628, 1536, 1459 cm-1; 1H NMR
(CDCl3) δ 2.00 (s, CH3C(O)), 2.44 (t, J = 2.4 Hz, CH2CCH), 3.62
(dd, J = 6.9, 9.3 Hz, CHH0OCH2), 3.56-3.72 (br m, NH2),
3.88 (dd, J = 4.1, 9.3 Hz, CHH0OCH2), 4.12 (1/2HH0q, J = 2.4,
(C8H11NO4 0.25H2O) C, H, N.
3
(R)-Methyl 2-Acetamido-3-(2-azidoethoxy)propionate ((R)-
18). Using method A, compound (R)-1644 (2.35 g, 16.4 mmol),
2-azidoethanol (4.5 mL, 65.6 mmol) and BF3 Et2O (1 mL,
8.2 mmol) gave 1.56 g (41%) of (R)-18 after purification:
3
[R]25 þ49.1ꢀ (c 1.0; EtOAc); Rf = 0.47 (EtOAc); IR (neat)
D
3302, 2948, 2107, 1746, 1668, 1534, 1443 cm-1 1H NMR
;

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6907
15.9 Hz, OCHH0C), 4.19 (1/2HH0q, J = 2.4, 15.9 Hz, OCHH0C),
4.32 (d, J = 5.7 Hz, CH2Ar), 4.54-4.60 (m, CH), 6.55-6.58 (br d,
J = 7.2 Hz, NHCH), 6.60-6.65 (m, 2 ArH), 6.65-6.71 (br m,
NHCH2), 7.03-7.06 (m, 2 ArH); 13C NMR (CDCl3) δ 23.4
(CH3C(O)), 43.5 (CH2Ph), 52.7 (CH), 58.8 (CH2CCH), 69.4
(CH2OCH2), 75.5 (CH2CCH), 79.1 (CH2CCH), 115.4, 127.7,
129.1, 146.1 (ArC), 169.6, 170.5 (2 C(O)); Mr (þESI) 290.1500
[M þ H]þ (calcd for C15H19N3O3Hþ 290.1505 [M þ H]þ). Anal.
(C15H19N3O3) C, H, N.
NHCH2), 7.17-7.20 (m, 2 ArH), 7.25-7.27 (m, 2 ArH). Addition
of excess (R)-(-)-mandelic acid to a CDCl3 solution of (S)-9 gave
only a single signal for the acetyl methyl protons and the alkyne
proton. Addition of excess (R)-(-)-mandelic acid to a CDCl3
solution of (R)-9 and (S)-9 (1:2 ratio) gave two signals for the acetyl
methyl protons (δ2.019 (R) and 2.033 (S) (Δppm = 0.014)) and two
signals for the alkyne proton (δ 2.424 (S) and 2.462 (R) (Δppm =
0.038)). 13C NMR (CDCl3) δ 23.3 (CH3C(O)), 43.1 (CH2Ph), 52.7
(CH), 58.8 (CH2CCH), 69.3 (CH2OCH2), 75.6 (CH2CCH), 79.0
(CH2CCH), 126.1, 128.7, 130.5 (3 ArC), 135.7 (NCS), 137.5 (1
ArC), 170.0, 170.7 (2 C(O)); Mr (þESI) 332.1063 [M þ H]þ (calcd
for C16H17N3O3SHþ 332.1069 [M þ H]þ).Anal.(C16H17N3O3S) C,
H, N, S.
(S)-N-(4-Amino)benzyl 2-Acetamido-3-(prop-2-ynyloxy)pro-
pionamide ((S)-23). Utilizing the preceding procedure and using
(S)-19 (0.25 g, 1.37 mmol), NMM (0.20 mL, 1.78 mmol), IBCF
(0.19 mL, 1.44 mmol), and 22 (0.16 mL, 1.44 mmol) gave 0.30 g
(77%) of (S)-23 as a light-yellow solid: mp 97.0-98.0 ꢀC;
(R)-N-(4-Isothiocyanato)benzyl 2-Acetamido-3-(2-azidoethoxy)-
propionamide ((R)-11). Utilizing method D and using acid (R)-20
(1130 mg, 5.2 mmol), 4-aminobenzylamine (22) (760 mg,
6.3 mmol), and DMTMM (1730 mg, 6.3 mmol) in THF (100
mL) gave 715 mg (42%) of a dark-brown residue. The residue was
directly dissolved in dry acetonitrile (40 mL), and DITC (90%,
510 mg, 2.57 mmol) was added all at once. The reaction solution
was stirred at room temperature (1 h), the solvent was removed in
vacuo, and the residue was purified using flash chromatography
(5/95 MeOH/CHCl3) and then recrystallized from EtOAc to yield
502 mg (27%, 2 steps) of (R)-11 as an off-white solid: mp 142-
[R]25 -4.1ꢀ (c 1.0, MeOH); Rf = 0.40 (1/9 MeOH/CHCl3);
D
1
IR (nujol mull) 3282, 2924, 2112, 1630, 1537, 1458 cm-1; H
NMR (CDCl3) δ 2.03 (s, CH3C(O)), 2.45 (t, J = 2.4 Hz,
CH2CCH), 3.62 (dd, J = 7.1, 9.1 Hz, CHH0OCH2), 3.62-3.74
(br m, NH2), 3.92 (dd, J = 4.2, 9.1 Hz, CHH0OCH2), 4.14 (1/
2HH0q, J = 2.4, 15.9 Hz, OCHH0C), 4.22 (1/2HH0q, J = 2.4,
15.9 Hz, OCHH0C), 4.35 (d, J = 6.0, CH2Ar), 4.53-4.59 (m,
CH), 6.42-6.44 (br d, J = 6.3 Hz, NHCH), 6.48-6.56 (br m,
NHCH2), 6.62-6.67 (m, 2 ArH), 7.03-7.08 (m, 2 ArH); 13C
NMR (CDCl3) δ 23.6 (CH3C(O)), 43.8 (CH2Ph), 52.9 (CH),
59.0 (CH2CCH), 69.7 (CH2OCH2), 75.7 (CH2CCH), 79.4
(CH2CCH), 115.6, 128.0, 129.4, 146.3 (C6H4), 169.9, 170.7 (2 C(O));
Mr (þESI) 290.1499 [M þ H]þ (calcd for C15H19N3O3Hþ
290.1505 [M þ H]þ). Anal. (C15H19N3O3) C, H, N.
143 ꢀC; [R]25 -28.6ꢀ (c 0.81, CHCl3); Rf = 0.45 (1/9 acetone/
D
EtOAc); IR (nujol mull) 3276, 2181, 2114, 1631, 1547, 1459,
1
1374, 1285 cm-1; H NMR (CDCl3) δ 2.05 (s, CH3C(O)NH),
3.30-3.50 (m, CH2N3), 3.55 (dd,
J = 7.5, 9.3 Hz,
(R)-N-(4-Isothiocyanato)benzyl 2-Acetamido-3-(prop-2-yny-
loxy)propionamide ((R)-9). To a dry THF solution (20 mL) of
(R)-23 (0.17 g, 0.60 mmol) was added dropwise a solution of
DPT (0.21 mL, 0.89 mmol) in THF (8 mL). The reaction
solution was stirred under Ar at room temperature (2 h). The
solvent was removed in vacuo, and the product was purified by
silica gel column chromatography (1/9 acetone/EtOAc) to give
CHCHH0OCH2), 3.62-3.80 (m, OCH2CH2N3), 3.95 (dd, J =
4.0, 9.3 Hz, CHCHH0OCH2), 4.46 (d, J = 6.0 Hz, C(O)NH-
CH2Ph), 4.54-4.60 (m, CHCH2O), 6.45 (br d, J = 6.2 Hz,
CH3C(O)NH), 6.84-6.92 (br t, C(O)NHCH2Ph), 7.18-7.22
(m, 2 ArH), 7.23-7.29 (m, 2 ArH). Addition of excess (R)-(-)-
mandelic acid to a CDCl3 solution of (R)-11 gave only one signal
for the acetyl protons. Addition of excess (R)-(-)-mandelic acid
to a CDCl3 solution of (S)-11 and (R)-11 (1:2 ratio) gave two
signals for the acetyl methyl protons (δ 2.033 (S) and 2.020
(R) (Δppm = 0.013). 13C NMR (CDCl3) δ 23.2 (CH3C(O)),
41.0 (NHCH2Ph), 50.7 (CH2N3), 52.4 (CHCH2O), 70.0 (OCH2-
CH2N3 or CHCH2O), 70.3 (CHCH2O or OCH2CH2N3), 125.9,
128.6, 130.5 (3 ArC), 135.6 (NCS), 137.4 (1 ArC), 169.8, 169.2
(CH3C(O)NH, C(O)NHCH2); Mr (þESI) 363.1236 [M þ H]þ
(calcd for C15H18N6O3SHþ 363.1239 [M þ H]þ). Anal. (C15H18-
N6O3S) C, H, N, S.
0.15 g (77%) of (R)-9 as a white solid: mp 157-159 ꢀC; [R]25
D
-10.9ꢀ (c 1.5, CHCl3); Rf = 0.40 (1/9 acetone/EtOAc); IR
(nujol mull) 3270, 2924, 2084, 1634, 1553, 1459 cm-1 1H
;
NMR (CDCl3) δ 2.03 (s, CH3C(O)), 2.48 (t, J = 2.5 Hz,
CH2CCH), 3.65 (dd, J = 7.1, 9.3 Hz, CHH0OCH2), 3.92 (dd,
J = 4.2, 9.3 Hz, CHH0OCH2), 4.15 (1/2HH0q, J = 2.5, 15.9 Hz,
OCHH0C), 4.22 (1/2HH0q, J = 2.5, 15.9 Hz, OCHH0C), 4.40
(1/2HH0q, J = 5.9, 15.5 Hz, CHH0Ar), 4.49 (1/2HH0q, J = 5.9,
15.5 Hz, CHH0Ar), 4.60-4.66 (m, CH), 6.52 (br d, J = 6.9 Hz,
NHCH), 6.96 (br t, J = 5.9 Hz, NHCH2), 7.16-7.19 (m, 2 ArH),
7.24-7.27 (m, 2 ArH). Addition of excess (R)-(-)-mandelic acid
to a CDCl3 solution of (R)-9 gave only a single signal for the
acetyl methyl protons and the alkyne proton. Addition of excess
(R)-(-)-mandelic acid to a CDCl3 solution of (R)-9 and (S)-9
(1:2 ratio) gave two signals for the acetyl methyl protons (δ 2.019
(R) and 2.033 (S) (Δppm = 0.014)) and two signals for the
(S)-N-(4-Isothiocyanato)benzyl 2-Acetamido-3-(2-azidoethoxy)-
propionamide ((S)-11). Utilizing the preceding procedure and using
acid (S)-20 (1.61 g, 7.52 mmol), 22 (1.02 mL, 9.03 mmol), and
DMTMM (2.50 g, 9.03 mmol) in anhydrous THF (200 mL)
followed by DITC (90%, 1.58 g, 7.90 mmol) in acetonitrile
(20 mL) gave 706 mg (26%, two steps) of (S)-11 as an off-white
solid after flash chromatography and recrystallization: mp
alkyne proton (δ 2.424 (S) and 2.462 (R) (Δppm = 0.038)). 13
C
142-143 ꢀC; [R]25 þ28.5ꢀ (c 0.95; CHCl3); Rf = 0.45 (1/9
D
NMR (CDCl3) δ 23.4 (CH3C(O)), 43.1 (CH2Ph), 52.7 (CH),
58.9 (CH2CCH), 69.3 (CH2OCH2), 75.6 (CH2CCH), 79.0
(CH2CCH), 126.1, 128.8, 130.6 (3 ArC), 135.7 (NCS), 137.5 (1
ArC), 170.0, 170.6 (2 C(O)); Mr (þESI) 332.1065 [M þ H]þ
(calcd for C16H17N3O3SHþ 332.1069 [M þ H]þ). Anal.
(C16H17N3O3S) C, H, N, S.
(S)-N-(4-Isothiocyanato)benzyl 2-Acetamido-3-(prop-2-ynyloxy)-
propionamide ((S)-9). Utilizing the preceding procedure and using
(S)-23 (0.30 g, 1.02 mmol) and DPT (0.32 g, 1.38 mmol) gave 0.25 g
(75%) of pure (S)-9 as a white solid: mp 157-158 ꢀC; [R]25D þ10.6ꢀ
(c 1.5, CHCl3); Rf = 0.40 (1/9 MeOH/CHCl3); IR (nujol mull)
3272, 2924, 2084, 1633, 1553, 1459 cm-1; 1H NMR (CDCl3) δ 2.04
(s, CH3C(O)), 2.48 (t, J = 2.5 Hz, CH2CCH), 3.65 (dd, J = 7.1, 9.2
Hz, CHH0OCH2), 3.94 (dd, J = 4.1, 9.2 Hz, CHH0OCH2), 4.16
(1/2HH0q,J=2.5,16.0Hz,OCHH0C), 4.24 (1/2HH0q,J=2.5,16.0
Hz, OCHH0C), 4.42 (1/2HH0q, J = 6.3, 15.5 Hz, CHH0Ar), 4.50
(1/2HH0q, J = 6.3, 15.5 Hz, CHH0Ar), 4.57-4.63 (m, CH),
6.41-6.43 (br d, J = 6.6 Hz, NHCH), 6.81 (br t, J = 6.3 Hz,
acetone/EtOAc); IR (nujol mull) 3280, 2177, 2108, 1637, 1548,
1452, 1374, 1293 cm-1;1HNMR(CDCl3) δ2.01 (s, CH3C(O)NH),
3.28-3.48 (m, CH2N3), 3.55 (dd, J = 7.2, 9.3 Hz, CHCH-
H0OCH2), 3.62-3.78 (m, OCH2CH2N3), 3.91 (dd, J = 4.0, 9.3
Hz, CHCHH0OCH2), 4.43 (d, J = 6.0 Hz, C(O)NHCH2Ph),
4.56-4.62 (m, CHCH2O), 6.56 (br d, J = 6.2 Hz, CH3C(O)NH),
6.84-6.92 (br t, J = 6.0 Hz, C(O)NHCH2Ph), 7.14-7.20 (m,
2 ArH), 7.22-7.28 (m, 2 ArH). Addition of excess (R)-(-)-
mandelic acid to a CDCl3 solution of (S)-11 gave only one signal
for the acetyl protons. Addition of excess (R)-(-)-mandelic acid to
a CDCl3 solution of (S)-11 and (R)-11 (1:2 ratio) gave two signals
for the acetyl methyl protons (δ 2.033 (S) and 2.020 (R) (Δppm =
0.013). 13C NMR (CDCl3) δ 23.3 (CH3C(O)), 43.2 (NHCH2Ph),
50.9 (CH2N3), 52.7 (CHCH2O), 70.3 (OCH2CH2N3 or CHCH2O),
70.4(CHCH2OorOCH2CH2N3), 126.1, 128.8, 130.6 (3 ArC), 135.8
(NCS), 137.6 (1 ArC), 170.0, 170.7 (CH3C(O)NH, C(O)NHCH2);
Mr (þESI) 363.1241 [M þ H]þ (calcd for C15H18N6O3SHþ
363.1239 [M þ H]þ). Anal. (C15H18N6O3S) C, H, N, S.

6908 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Park et al.
(R)-N-(4-Ethynyl)benzyl 2-Acetamido-3-(2-azidoethoxy)propion-
amide ((R)-24). Using method C, (R)-2-acetamido-3-(2-azidoethoxy)-
propionic acid ((R)-20) (0.31 g, 1.4 mmol), NMM (189 μL,
1.7 mmol), IBCF (228 μL, 1.7 mmol), 4-ethynylbenzylamine (21)
(225 mg, 1.7 mmol), and THF (20 mL) gave 0.26 g (55%) of (R)-24 as
awhitesolid:Rf = 0.18 (EtOAc); mp 80-87 ꢀC (dec); [R]25D þ4.8ꢀ (c
0.5, DMSO); IR (nujol mull) 3282, 2103, 1636, 1548, 1458, 1375,
1303, 1258, 1124, 982, 821, 721 cm-1; 1H NMR (CDCl3) δ 2.03 (s,
CH3C(O)), 3.08 (s, HCtC), 3.27-3.46 (m, CH2N3), 3.56 (dd,
J = 7.2, 9.6 Hz, OCHH0), 3.62-3.77 (m, CH2CH2O), 3.92 (dd, J
= 3.6, 9.6 Hz, OCHH0), 4.46 (d, J = 5.7 Hz, CH2N), 4.56-4.62 (m,
CH), 6.53 (d, J = 6.9 Hz, NH), 6.88-7.01 (br t, CH2NH), 7.22 (d,
J=8.1Hz,2ArH),7.45(d,J=8.1Hz,2ArH);13CNMR(CDCl3) δ
23.1 (CH3CO), 43.3 (CH2N), 50.7 (CH2N3),52.5(CH), 70.1 (CH2O),
70.3 (CH2O), 77.4 (CtC), 83.3 (CtC), 121.3, 127.4, 132.4, 138.7 (4
ArC), 169.8, 170.5 (2 C(O));Mr (þESI) 330.1569 [M þ H]þ (calcdfor
C16H19N5O3Hþ 330.1566 [M þ H]þ).
(S)-N-(4-Ethynyl)benzyl) 2-Acetamido-3-(2-isothiocyanato-
ethoxy)propionamide ((S)-10). Utilizing the preceding proce-
dure, polymer bound triphenylphosphine (Fluka, catalog no.
93094) (1.6 mol/g, 3.3 mmol), H2O (205 μL, 11.4 mmol)/THF
(10 mL) solution, and (S)-24 (375 mg, 1.1 mmol) gave the free
amine (S)-26: 1H NMR (CDCl3) δ 2.01 (s, CH3C(O)), 2.77-2.91
(m, CH2NH2), 3.07 (s, HCtC), 3.49-3.57 (m, OCHH0, CH2O),
3.80 (dd, J = 4.5, 9.9 Hz, OCHH0), 4.38-4.51 (m, CH2N),
4.56-4.62 (m, CH), 6.88 (d, J = 6.9 Hz, CH3C(O)NH), 7.23 (d,
J = 8.7 Hz, 2 ArH), 7.45 (d, J = 8.7 Hz, 2 ArH), 7.88-8.01 (br t,
NHCH2); Mr (þESI) 304.1662 [M þ H]þ (calcd for C16H21-
N3O3Hþ 304.1661 [M þ H]þ).
With the preceding procedure, the free amine (S)-26 was
coupled with DPT (255 mg, 1.1 mmol) to give (S)-10 (125 mg,
32%): Rf = 0.45 (EtOAc); mp 127 ꢀC; [R]25 -9.6ꢀ (c 0.5,
D
DMSO); IR (nujol mull) 3277, 2730, 2208, 2107, 1635, 1549,
1458, 1375, 1307, 1130, 819, 726 cm-1; 1H NMR (CDCl3) δ 2.07
(s, CH3C(O)), 3.07 (s, HCtC), 3.53-3.77 (m, CH2NCS, OCHH0,
CH2CH2O), 4.00 (dd, J = 3.3, 9.3 Hz, OCHH0), 4.42-4.56 (m,
CH2N), 4.59-4.64 (m, CH), 6.55 (d, J = 6.9 Hz, CH3C(O)NH),
6.81-6.85 (br t, NHCH2), 7.24 (d, J = 8.4 Hz, 2 ArH), 7.46
(d, J = 8.4 Hz, 2 ArH); 13C NMR (CDCl3) δ 23.2 (CH3CO), 43.3
(CH2N), 45.4 (CH2NCS), 52.7 (CH), 69.3, 70.0 (2 CH2O),
77.3 (CtC), 83.3 (CtC), 121.3, 127.5, 132.4, 138.7 (4 ArC),
169.7, 170.6 (2 C(O)). The signal for the isothiocyanate
carbon resonance was not detected under the conditions used
for the acquisition of the NMR spectrum. Mr (þESI) 368.1046
[M þ Na]þ (calcd for C17H19N3O3SNaþ 368.1045 [M þ Na]þ).
(S)-N-(4-Ethynyl)benzyl 2-Acetamido-3-(2-azidoethoxy)propion-
amide ((S)-24). Utilizing the preceding procedure and using THF
(20mL),(S)-20 (0.31g,1.4mmol),NMM(189μL, 1.7 mmol), IBCF
(225 μL, 1.7 mmol), and 21 (225 mg, 1.7 mmol) gave 165 mg of a
white solid (35%): Rf = 0.18 (EtOAc); mp 107-109 ꢀC; [R]25
D
-6.0ꢀ (c 0.5, DMSO); IR (nujol mull) 3274, 2100, 1635, 1548, 1457,
1375, 1301, 1257, 1121, 980, 820, 719 cm-1;1HNMR(CDCl3) δ2.02
(s, CH3C(O)), 3.08 (s, HCtC), 3.27-3.46 (m, CH2N3), 3.56 (dd,
J = 7.5, 9.6 Hz, OCHH0), 3.61-3.76 (m, CH2CH2O), 3.91 (dd, J =
3.6, 9.6 Hz, OCHH0), 4.45 (d, J = 6.0 Hz, CH2N), 4.56-4.63 (m,
CH), 6.54 (d, J = 6.0 Hz, NH), 6.95-7.05 (br t, CH2NH), 7.22 (d,
J=7.9Hz, 2ArH), 7.45(d, J=7.9Hz, 2ArH);13CNMR(CDCl3)
δ 23.1 (CH3C(O)), 43.2 (CH2N), 50.6 (CH2N3), 52.4 (CH), 70.1,
70.2 (2 CH2O), 77.3 (CtC), 83.2 (CtC), 121.2, 127.4, 132.3, 138.7
(4 ArC), 169.8, 170.4 (2 C(O)); Mr (þESI) 352.1387 [M þ Na]þ
(calcd for C16H19N5O3Naþ 352.1386 [M þ Na]þ).
Anal. (C17H19N3O3S 0.16CH3OH) C, H, N, S.
3
Preparative Reaction of (R)-N-(4-Isothiocyanato)benzyl) 2-Acet-
amido-3-(prop-2-ynyloxy)propionamide ((R)-9) and N-2-(2-(2-(2-Azi-
doethoxy)ethoxy)ethoxy)ethyl Biotin Amide (35) To Give (R)-36.
(R)-9 (50 mg, 0.15 mmol) and 35 (81 mg, 0.18 mmol) were dissolved
in t-BuOH/H2O (1:2, 3 mL), and then a freshly prepared aqueous 1
M sodium ascorbate solution (15 μL, 15.0 μmol) was added, followed
(R)-N-(4-Ethynyl)benzyl) 2-Acetamido-3-(2-isothiocyanato-
ethoxy)propionamide ((R)-10). Polymer bound triphenylpho-
sphine (Fluka, catalog no. 93094) (1.6 mol/g, 3.0 mmol) was
added to a H2O (180 μL, 10.0 mmol)/THF (10 mL) solution
of (R)-24 (329 mg, 1.0 mmol). The mixture was shaken until
the starting material was no longer evident by TLC analysis
(18 h). The triphenylphosphine-based support was filtered and
washed with CH2Cl2, and the filtrate was concentrated in
vacuum to obtain the free amine (R)-26: 1H NMR (CDCl3)
δ 2.03 (s, CH3C(O)), 2.76-2.92 (m, CH2NH2), 3.07 (s, HCtC),
3.48-3.62 (m, OCHH0, CH2O), 3.84 (dd, J = 4.2, 10.2 Hz,
OCHH0), 4.39-4.52 (m, CH2N), 4.54-4.60 (m, CH), 6.71-6.79
(br d, CH3C(O)NH), 7.23 (d, J = 7.9 Hz, 2 ArH), 7.45 (d, J =
7.9 Hz, 2 ArH), 7.82-7.91 (br m, NHCH2); 13C NMR (CD3CN)
δ 23.1 (CH3CO), 42.5, 43.2 (2 CH2N), 54.7 (CH), 71.2, 74.2
(2 CH2O), 78.7 (CtC), 84.2 (CtC), 121.5, 128.3, 133.0, 141.7
(ArC), 171.2, 171.4 (2 C(O)).
by a freshly prepared aqueous 0.1 M CuSO4 5H2O solution (15 μL,
3
1.5 μmol). The reaction mixture was vigorously stirred at room
temperature (15 h), evaporated in vacuo, and then purified by
column chromatography (SiO2, 1/9 MeOH/CHCl3) to yield 60 mg
(51%) of (R)-36 as a white sticky foam: Rf = 0.30 (1/9 MeOH/
CHCl3); IR (nujol mull) 3284, 2925, 2108, 1657, 1544, 1459 cm-1
;
1H NMR (CDCl3) δ 1.37-1.44 (m, C(6)H2), 1.55-1.67 (m,
CH2CH2C(6)), 2.02 (s, CH3C(O)), 2.14 (t, J = 7.5 Hz, C(10)CH2),
2.70 (d, J = 12.6 Hz, C(5)HH0), 2.89 (dd, J = 4.8, 12.6 Hz,
C(5)HH0), 3.09-3.15 (m, C(2)H), 3.38-3.41 (m, OCH2CH2NHC-
(O)), 3.52-3.62 (m, 2 OCH2CH2O, triazole-CH2CH2O), 3.67
(dd, J = 6.5, 9.8 Hz, CHCHH0OCH2), 3.86-3.94 (m, OCH2CH2-
NHC(O), CHCHH0OCH2), 4.25-4.30 (m, C(3)H), 4.40-4.49 (m,
CH2Ar, C(4)H), 4.55 (t, J =5.0Hz, triazole-CH2CH2O), 4.62-4.68
(m, CHCH2OCH2-triazole, CHCH2OCH2-triazole), 5.57, 6.52 (s,
N(10)H, N(30)H), 6.84 (t, J = 5.4 Hz, OCH2CH2NHC(O)), 7.10-
7.15 (m, 2 ArH), 7.24 (d, J = 8.7 Hz, 2 ArH), 7.40 (d, J = 7.5 Hz,
CH3C(O)NHCH), 7.80 (s, CH triazole), 7.83 (t, J = 6.0 Hz,
NHCH2Ar); 13C NMR (CD3OD) δ 23.3 (CH3C(O)), 25.7 (C(6)),
28.3 (C(7)), 28.4(C(8)), 35.9(C(9)), 39.3(OCH2CH2NHC(O)), 40.8
(C(5)), 43.0 (CH2Ar), 50.4 (triazole-CH2CH2O), 53.2 (CHCH2-
OCH2-triazole), 55.8 (C(2)), 60.3 (C(3)), 62.1 (C(4)), 64.6 (CHCH2-
OCH2-triazole), 69.5 (CHCH2OCH2-triazole), 70.0, 70.1, 70.2, 70.4,
70.5, 70.6 (3 CH2OCH2 in PEG linker), 124.3 (triazole CH), 126.0,
128.7, 130.1, 138.1 (C6H4), 135.4 (NCS), 144.3 (triazole C), 164.1,
170.6, 171.2, 173.6 (4 C(O));HRMS(ESI) 776.3215[Mþ Hþ] (calcd
for C34H50N9O8S2 776.3224).
Anhydrous CH2Cl2 (5 mL) was added to the free amine (R)-26,
and DPT (232 mg, 1.0 mmol) was added. The yellow solution was
stirred at room temperature (18 h). The solvent was evaporated
under reduced pressure and the residue was purified by silica gel
column chromatography (EtOAc to 90/10 EtOAc/MeOH) to
obtain (R)-10 as a white solid (80 mg, 23%); Rf = 0.45 (EtOAc);
mp 124-126 ꢀC; [R]25 þ8.0ꢀ (c 0.5, DMSO); IR (nujol mull)
D
3266, 2571, 2201, 2103, 1631, 1538, 1458, 1375, 1304, 1120, 820, 725
cm-1; 1H NMR (CDCl3) δ 2.08 (s, CH3C(O)), 3.07 (s, HCtC),
3.55-3.77 (m, CH2NCS, OCHH0, CH2CH2O), 4.01 (dd, J = 3.3,
9.3 Hz, OCHH0), 4.43-4.57 (m, CH2N), 4.58-4.64 (m, CH), 6.54
(d, J = 6.3 Hz, CH3C(O)NH), 6.79-6.88 (br t, NHCH2), 7.25 (d,
J = 8.6 Hz, 2 ArH), 7.46 (d, J = 8.6 Hz, 2 ArH); 13C NMR
(CDCl3) δ 23.3 (CH3CO), 43.3 (CH2N), 45.4 (CH2NCS), 52.7
(CH), 69.3, 70.0 (2 CH2O), 77.4 (CtC), 83.3 (CtC), 121.3, 127.5,
132.4, 138.8 (4 ArC), 169.7, 170.6 (2 C(O)). The signal for the
isothiocyanate carbon resonance was not detected under the
conditions used for the acquisition of the NMR spectrum. Mr
(þESI) 368.1048 [M þ Na]þ (calcd for C17H19N3O3SNaþ
(R)-N-Benzyl
2-Acetamido-3-(2-(4-(methoxymethyl)-1H-
1,2,3-triazol-1-yl)ethoxy)propionamide ((R)-38). Compound
(R)-30 (400 mg, 1.3 mmol) was dissolved in a THF/H2O mixture
(1:1, 50 mL), and while the mixture was being stirred, propargyl
methyl ether (1 mL, 11.8 mmol), sodium ascorbate (25 mg,
0.13 mmol), and CuSO4 (3 mg, 0.01 mmol) were successively
added. The mixture was stirred at room temperature (24 h), and
saturated aqueous NaHCO3 (100 mL) was added. The aqueous
368.1045 [M þ Na]þ). Anal. (C17H19N3O3S 0.2H2O) C, H, N, S.
3

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6909
layer was extracted with CH2Cl2 (2 ꢀ 100 mL), and the
combined organic layers were washed with brine (100 mL) and
filtered through a Celite bed. Removal of solvents in vacuo gave
an off-white solid that was purified by flash chromatography
(10/90 MeOH/CH2Cl2) to yield (R)-38 (470 mg, 96%) as a
crystalline solid: mp 127-129 ꢀC; [R]25D þ1.8ꢀ (c 0.34; MeOH);
Rf = 0.49 (10/90 MeOH/CH2Cl2); IR (nujol mull) 3296, 2861,
Acknowledgment. The authors thank the NINDS and the
Anticonvulsant Screening Program (ASP) at the National
Institutes of Health for kindly performing the pharmacologi-
cal studies via the ASP’s contract site atthe University of Utah
with Drs. H. Wolfe, H. S. White, and K. Wilcox. Funds for
this study was generously provided by Grants R01NS054112
(H.K., R.L.) 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 responsibility of the
authors and does not necessarily represent the official views
of the National Institute of Neurological Disorders and
Stroke or the National Institutes of Health. Harold Kohn
has a royalty-stake position in (R)-2.
1
1641, 1545, 1457, 1377, 1143, 1095 cm-1; H NMR (CDCl3)
δ 2.00 (s, CH3C(O)), 3.38 (s, CH2OCH3), 3.50 (dd, J = 6.5, 9.6
Hz, CHH0OCH2CH2), 3.76-3.85 (m, OCH2CH2N(N)CH),
3.92 (dd, J = 4.2, 9.6 Hz, CHH0OCH2CH2), 4.34-4.51 (m, NH-
CH2Ph, OCH2CH2N(N)CH, CH2OCH3), 6.63 (d, J = 6.6 Hz,
NHCHC(O)), 7.00-7.10 (br t, NHCH2Ph), 7.18-7.35 (m,
C6H5), 7.52 (s, NCHC(N)). Addition of excess (R)-(-)-mandelic
acid to a CDCl3 solution of (R)-38 gave only one signal for
the acetyl protons. 13C NMR (CDCl3) δ 23.2 (CH3C(O)),
43.7 (NHCH2Ph), 50.0 (CH2N(N)CH), 52.9 (CHC(O)NH),
58.6 (CH2OCH3), 66.0 (OCH2CH2N), 69.4, 70.5 (CHCH2-
OCH2, CH2OCH3), 123.8 (NCHC(N)), 127.6, 127.7, 128.8,
138.3 (C6H5), 145.3 (NCHC(N)), 169.7, 170.8 (CH3C(O),
CHC(O)NH); Mr (þESI) 398.1806 [M þ Na]þ (calcd for
C18H25N5O4Naþ 398.1804 [M þ Na]þ). Anal. (C18H25N5O4)
C, H, N.
Preparation of Mouse Soluble Brain Proteomes. Mouse
brains (male, 2 months, ICR mice [Rockland Immunochem-
icals, Gilbertsville, PA]) were Dounce-homogenized in 50 mM
HEPES buffer (pH 7.4). The lysate was centrifuged at slow
speed (1200g for 12 min at 4 ꢀC) to remove debris. The super-
natant was then centrifuged at high speed (100000g for 1 h at
4 ꢀC). The resulting supernatant was collected and stored at
-80 ꢀC until use. The total protein concentration was deter-
mined by using the Bradford assay.
Three NCS Probe Labeling, Cycloaddition Reaction, and In-
Gel Fluorescence Scanning. Mouse brain lysate (1 mL, 50 mM
HEPES buffer (pH 7.4)) was passed through a NAP-10 column
(GE Healthcare) to exchange buffer to an aqueous 50 mM
HEPES buffer (pH 8.0). Lysate aliquots (50 μL of 2.0 mg/mL
protein in 50 mM HEPES buffer (pH 8.0)) were treated with
lacosamide AB (NCS) and CR (alkyne, N3) compounds (5 μM)
at room temperature (30 min). The modified lysates were
sequentially treated with rhodamine reporter tag (50 μM)
(either rhodamine-azide (Rho-N3, 39) or rhodamine-alkyne
(Rho-PEGt, 40)), TCEP (1 mM), TBTA (200 μM), and CuSO4
(1 mM). Samples were shaken and allowed to rotate using Roto-
shake (8 rpm, Scientific Industries Inc., model no. SI-1100,
Bohemia, NY) at room temperature (1 h). Proteins were sepa-
rated by SDS-PAGE after addition of 4ꢀ SDS-PAGE loading
buffer and visualized by in-gel fluorescence using a typhoon
9400 scanner (GE Healthcare/Amersham Bioscience) with
excitation at 532 nm and detection at 580 nm.
Probe Labeling of GST-CRMP2 in Mouse Lysate, Cycloaddi-
tion, and In-Gel Fluorescence Scanning. With the previous
method, lysate aliquots (150 μL, pH 8.0) were prepared after
passage through a NAP-10 column. Overexpressed GST-
CRMP2 (30 μg) was added to the lysate (150 μL) and divided
into three equal aliquots. The aliquots (50 μL) were treated with
(R)- and (S)-9 (5 μM) at room temperature (30 min). With the
previous method, the modified lysates were then treated with 39
under Cu(I)-mediated cycloaddition conditions, followed by
SDS-PAGE separation and in-gel fluorescence.
Supporting Information Available: Synthetic procedures for
the preparation of (R)- and (S)-13, 15, 16, 27-30, 42, 44-46,
52-55 and 21, 39, 40, 49, 50, 56-59, 61, 63-65; elemental
analysis results; 1H and 13C NMR spectra of compounds
reported in this study; MS table. This material is available free
of charge via the Internet at http://pubs.acs.org.
References
(1) Hauser, W. A.; Annegers, J. F.; Kurland, L. T. The prevalence of
epilepsy in Rochester, Minnesota, 1940-80. Epilepsia 1991, 32,
429–445.
(2) Evans, J. H. Post-traumatic epilepsy. Neurology 1962, 12, 665–674.
(3) Lindsay, J. M. Genetics and epilepsy. Epilepsia 1971, 12, 47–54.
(4) (a) Rogawski, M. A.; Porter, R. J. Antiepileptic drugs: pharmaco-
logical mechanisms and clinical efficacy with consideration of
promising development stage compounds. Pharmacol. Rev. 1997,
42, 223–286. (b) McNamara, J. O. In Goodman & Gilman’s The
Pharmacological Basis of Therapeutics, 10th ed.; Hardman, J. G.;
Limbird, L. E., Eds.; McGraw-Hill: New York, 2001; Chapter 21, pp
521-547. (c) Aiken, S. P.; Brown, W. M. Treatment of epilepsy:
existing therapies and future developments. Front. Biosci. 2000, 5,
124–152.
(5) Brodie, M. J.; Dichter, M. A. Antiepileptic drugs. N. Engl. J. Med.
1996, 334, 168–175.
(6) Dichter, M. A.; Brodie, M. J. New antiepileptic drugs. N. Engl.
J. Med. 1996, 334, 1583–1590.
(7) (a) McCorry, D.; Chadwick, D.; Marson, A. Current drug treat-
ment of epilepsy in adults. Lancet Neurol. 2004, 3, 729–735. (b)
Duncan, J. S. The promise of new antiepileptic drugs. Br. J. Clin.
Pharmacol. 2002, 53, 123–131. (c) Bauer, J.; Reuber, M. Medical
treatment of epilepsy. Expert Opin. Emerging Drugs 2003, 8, 457–
467. (d) Mattson, R. H.; Cramer, J. A.; Collins, J. F.; Smith, D. B.
Comparison of carbamazepine, phenobarbital, phenytoin, and primi-
done in partial and secondarily generalized tonic-clonic seizures. N.
Engl. J. Med. 1985, 313, 145–151.
(8) Pellock, J. M.; Willmore, L. J. A rational guide to monitoring in
patients receiving anticonvulsants. Neurology 1991, 41, 961–964.
(9) 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.
(10) Conley, J. D.; Kohn, H. Functionalized DL-amino acid deriva-
tives. Potent new agents for the treatment of epilepsy. J. Med.
Chem. 1987, 30, 567–574.
(11) Kohn, H.; Conley, J. D. New antiepileptic agents. Chem. Br. 1988,
24, 231–234.
(12) Kohn, H.; Conley, J. D.; Leander, J. D. Marked stereospecificity in
a new class of anticonvulsants. Brain Res. 1988, 457, 371–375.
(13) Kohn, H.; Sawhney, K. N.; LeGall, P.; Conley, J. D.; Robertson,
D. W.; Leander, J. D. Preparation and anticonvulsant activity of a
series of functionalized R-aromatic and R-heteroaromatic amino
acids. J. Med. Chem. 1990, 33, 919–926.
(14) 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.
Pharmacology. Compounds were screened under the auspices
of the National Institutes of Health’s Anticonvulsant Scree-
ning Program. Experiments were performed in male rodents
[albino Carworth Farms no. 1 mice (intraperitoneal route, ip),
albino Spague-Dawley rats (oral route, po)]. Housing, hand-
ling, and feeding were in accordance with recommendations
contained in the “Guide for the Care and Use of Laboratory
Animals”. Anticonvulsant activity was established using the
MES test59,60 and the scMet test,59 using previously reported
methods.43a
(15) Kohn, H.; Sawhney, K. N.; Bardel, P.; Robertson, D. W.; Leander,
J. D. Synthesis and anticonvulsant activities of R-heterocyclic
R-acetamido-N-benzylacetamide derivatives. J. Med. Chem. 1993,
36, 3350–3360.

6910 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21
Park et al.
(16) 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. Med. Chem. 1994, 37, 4567–4571.
(17) Kohn, H.; Sawhney, K. N.; Robertson, D. W.; Leander, J. D.
Anticonvulsant properties of N-substituted R,R-diamino acid
derivatives. J. Pharm. Sci. 1994, 83, 689–691.
(18) Choi, D.; Stables, J. P.; Kohn, H. Synthesis and anticonvulsant
activities of N-benzyl-2-acetamidopropionamide derivatives.
J. Med. Chem. 1996, 39, 1907–1916.
(31) (a) de Costa, B. R.; Rothman, R. B.; Bykov, V.; Jacobson, A. E.;
Rice, K. C. Selective and enantiospecific acylation of K opioid
receptors by (1S,2S)-trans-2-isothiocyanato-N-methyl-N-[2-(1-
pyrrolidinyl)cyclohexyl]-benzeneacetamide. Demonstration of K
receptor heterogeneity. J. Med. Chem. 1989, 32, 281–283. (b) Burke,
T. R., Jr.; Jacobson, A. E.; Rice, K. C.; Silverton, J. V.; Simonds, W. F.;
Streaty, R. A.; Klee, W. A. Probes for narcotic receptor mediated
phenomena. 12. cis-(þ)-3-Methylfentanyl isothiocyanate, a potent
site-directed acylating agent for δ opioid receptors. Synthesis, absolute
configuration, and receptor enantioselectivity. J. Med. Chem. 1986, 29,
1087–1093. (c) de Costa, B. D.; Rothman, R. B.; Bykov, V.; Band, L.;
Pert, A.; Jacobson, A. E.; Rice, K. C. Probes for narcotic receptor
mediated phenomena. 17. Synthesis and evaluation of a series of trans-
3,4-dichloro-N-methyl-N-[2-(1-pyrrolidinyl)cyclohexyl]benzeneacetamide
(U50,488) related isothiocyanate derivatives as opioid receptor affinity
ligands. J. Med. Chem. 1990, 33, 1171–1176. (d) de Costa, B. R.;
Rothman, R. B.; Bykov, V.; Jacobson, A. E.; Rice, K. C. Selective
and enantiospecific acylation of K opioid receptors by (1S,2S)-trans-
2-isothiocyanato-N-methyl-N-[2,(1-pyrrolidinyl)cyclohexyl]benzene-
acetamide. Demonstration of K receptor heterogeneity. J. Med. Chem.
1989, 32, 281–283.
(19) Perucca, E.; Yasothan, U.; Clincke, G.; Kirkpatrick, P. Lacosa-
mide. Nat. Rev. Drug Discovery 2008, 7, 973–974.
(20) Stoehr, T.; Kupferberg, H. J.; Stables, J. P.; Choi, D.; Harris, R. H.;
Kohn, H.; Walton, N.; White, H. S. Lacosamide, a novel antic-
onvulsant drug, shows efficacy with a wide safety margin in rodent
models for epilepsy. Epilepsy Res. 2007, 74, 147–154.
(21) Beyreuther, B.; Freitag, J.; Heers, C.; Krebsfaenger, N.;
Scharfenecker, U.; Stoehr, T. Lacosamide: a review of preclinical
properties. CNS Drug Rev. 2007, 13, 21–42.
(22) (a) Errington, A. C.; Stohr, T.; Heers, C.; Lees, G. The investiga-
tional anticonvulsant lacosamide selectively enhances slow inacti-
vation of voltage-gated sodium channels. Mol. Pharmacol. 2008,
73, 157–169. (b) Sheets, P. L.; Heers, C.; Stoehr, T.; Cummins, T. R.
Differential block of sensory neuronal voltage-gated sodium channels
by lacosamide, lidocaine and carbamazepine. J. Pharmacol. Exp.
Ther. 2008, 326, 89–99.
(32) deCosta, B. R.;Lewin, A. H.;Rice, K. C.;Skolnick, P.; Schoenheimer,
J. A. Novel site-directed affinity ligands for GABA-gated chloride
channels: synthesis, characterization, and molecular modeling of
1-(isothiocyanatophenyl)-4-tert-butyl-2,6,7-trioxabicyclo[2.2.2]octanes.
J. Med. Chem. 1991, 34, 1531–1538.
(23) (a) Roth, B. L.; Sheffler, D. J.; Kroeze, W. K. Magic shotguns
versus magic bullets: selectively non-selective drugs for mood
disorders and schizophrenia. Nat. Rev. Drug Discovery 2004, 3,
353–359. (b) Cavalli, A.; Bolognesi, M. L.; Minarini, A.; Rosini, M.;
Tumiatti, V.; Recanatini, M.; Melchiorre, C. Multi-target-directed
ligands to combat neurodegenerative diseases. J. Med. Chem. 2008,
51, 347–372.
(24) (a) Brunton, L. L.; Lazo, J. S.; Parker, K. L. Goodman & Gilman’s
The Pharmacological Basis of Therapeutics, 11th ed.; McGraw-Hill:
New York, 2006. (b) Errington, A. C.; Stoehr, T.; Lees, G. Voltage-gated
ion channels: targets for anticonvulsant drugs. Curr. Top. Med. Chem.
2005, 5, 15–30.
(33) Williams, E. F.; Rice, K. C.; Paul, S. M.; Skolnick, P. Hetero-
geneity of benzodiazepine receptors in the central nervous system
demonstrated with kenazepine, an alkylating benzodiazepine.
J. Neurochem. 1980, 35, 591–597.
(34) (a) Rice, K. C.; Jacobson, A. E.; Burke, T. R., Jr.; Bajwa, B. S.
Irreversible ligands with high selectivity toward μ or δ opiate
receptors. Science 1983, 220, 314–316. (b) Lessor, R. A.; Rice, K.
C.; Streaty, R. A.; Klee, W. A.; Jacobson, A. E. Probes for narcotic
receptor mediated phenomena. 10. Irreversibly ligands to opioid re-
ceptors based on biologically potent endoethenooripavines. Reversible
binding of fit to mu and delta opioid receptors. Neuropeptides 1984, 5,
229–232.
(25) (a) Saxon, E.; Bertozzi, C. R. Cell surface engineering by a modified
Staudinger reaction. Science 2000, 287, 2007–2010. (b) Speers, A. E.;
Cravatt, B. F. Chemical strategies for activity-based proteomics.
ChemBioChem. 2004, 5, 41–47. (c) Ovaa, H.; van Swieten, P. F.;
Kessler, B. M.; Leeuwenburgh, M. A.; Fiebiger, E.; van den Nieuwen-
dijk, A. M. C. H.; Galardy, P. J.; van der Marel, G. A.; Ploegh, H. L.;
Overkleeft, H. S. Chemistry in living cells: detection of active protea-
somes by a two-step labeling strategy. Angew. Chem., Int. Ed. 2003,
42, 3626–3629. (d) Kho, Y.; Kim, S. C.; Jiang, C.; Barma, D.; Kwon, S.
W.; Cheng, J.; Jaunbergs, J.; Weinbaum, C.; Tamanoi, F.; Falck, J.;
Zhao, Y. A tagging-via-substrate technology for detection and proteo-
mics of farnesylated proteins. Proc. Natl. Acad. Sci. U.S.A. 2004, 101,
12479–12484.
(26) (a) Evans, M. J.; Saghatelian, A.; Sorensen, E. J.; Cravatt, B. F.
Target discovery in small-molecule cell-based screens by in situ
proteome reactivity profiling. Nat. Biotechnol. 2005, 23, 1303–
1307. (b) Hosoya, T.; Hiramatsu, T.; Ikemoto, T.; Nakanishi, M.;
Aoyama, H.; Hosoya, A.; Iwata, T.; Maruyama, K.; Endo, M.; Suzuki,
M. Novel bifunctional probe for radioisotope-free photoaffinity label-
ing: compact structure comprised of photospecific ligand ligation and
detectable tag anchoring units. Org. Biomol. Chem. 2004, 2, 637–641.
(c) Ballell, L.; van Scherpenzeel, M.; Buchalova, K.; Liskamp, R. M. J.;
Pieters, R. J. A new chemical probe for the detection of the cancer-
linked galectin-3. Org. Biomol. Chem. 2006, 4, 4387–4394. (d)
Gubbens, J.; Ruijter, E.; De Fays, L. E. V.; Damen, J. M. A.; de Kruijff,
B.; Slijper, M.; Rijkers, D. T. S.; Liskamp, R. M. J.; de Kroon, A. I. P. M.
Photocrosslinking and click chemistry enable the specific detection of
proteins interacting with phospholipids at the membrane interface.
Chem. Biol. 2009, 16, 3–14. (e) Staub, I.; Sieber, S. A. β-Lactam
probes as selective chemical-proteomic tools for the identification and
functional characterization of resistance associated enzymes in MRSA.
J. Am. Chem. Soc. 2009, 131, 6271–6276.
(35) (a) Burke, T. R., Jr.; Bajwa, B. S.; Jacobson, A. E.; Rice, K. C.;
Streaty, R. A.; Klee, W. A. Probes for narcotic receptor mediated
phenomena. 7. Synthesis and pharmacological properties of
irreversible ligands specific for μ or δ opiate receptors. J. Med.
Chem. 1984, 27, 1570–1574. (b) Burke, T. R., Jr.; Jacobson, A. E.; Rice,
K. C.; Silverton, J. V.; Simonds, W. F.; Streaty, R. A.; Klee, W. A. Probes
for narcotic receptor mediated phenomena. 12. cis-(þ)-3-Methylfenta-
nyl isothiocyanate, a potent site-directed acylating agent for δ opioid
receptors. Synthesis, absolute configuration, and receptor enantioselec-
tivity. J. Med. Chem. 1986, 29, 1087–1093.
(36) Rice, K. C.; Brossi, A.; Tallman, J.; Paul, S. M.; Skolnick, P.
Irazepine, and noncompetitive, irreversible inhibitor of 3H-diaze-
pam binding to benzodiazepine receptors. Nature (London) 1979,
278, 854–855.
(37) Perret, P.; Sarda, X.; Wolff, M.; Wu, T.-T.; Bushey; Goeldner, M.
Interaction of non-competitive blockers within the γ-aminobutyric
acid type A chloride channel using chemical reactive probes as
chemical sensors for cysteine mutants. J. Biol. Chem. 1999, 274,
25350–25354.
(38) Shelton, R. L.; Langdon, R. G. Location of the maltose isothio-
cyanate binding site on the human erythrocyte glucose transporter.
Biochemistry 1985, 24, 2397–2400.
(39) Morse, K. L.; Fournier, D. J.; Li, X.; Grzybowska, J.; Makriyannis,
A. A novel electrophilic high affinity irreversible probe for the
cannabinoid receptor. Life Sci. 1995, 56, 1957–1962.
(40) Daphne, A.; Plotek, Y.; Miskin, I. An affinity label for
R2-adrenergic receptors in rat brain. Eur. J. Biochem. 1982, 126,
537–541.
(41) Soskic, V.; Maelicke, A.; Petrovic, G.; Ristic, B.; Petrovic, J.
Synthesis of some phenothiazine derivatives as potential affinity
ligands for the central dopamine receptors. J. Pharm. Pharmacol.
1991, 43, 27–31.
(27) (a) Takemori, A. E.; Portoghese, P. S. Affinity labels for opioid
receptors. Annu. Rev. Pharmacol. Toxicol. 1985, 25, 193–223.
(b) Robertson, J. G. Mechanistic basis of enzyme-targeted drugs.
Biochemistry 2005, 44, 5561–5571. (c) Drahl, C.; Cravatt, B. F.;
Sorensen, E. J. Protein-reactive natural products. Angew. Chem., Int.
Ed. 2005, 44, 5788–5809.
(42) Casalotti, S. O.; Kozikowski, A. P.; Fauq, A.; Tuckmantel, W.;
Krueger, K. E. Design of an irreversible affinity ligand for the
phencyclidine recognition site on N-methyl-D-aspartate-type
glutamate receptors. J. Pharmacol. Exp. Ther. 1992, 260, 21–28.
(43) (a) LeTiran, A.; Stables, J. P.; Kohn, H. Design and evaluation
of affinity labels of functionalized amino acid anticonvulsants.
J. Med. Chem. 2002, 45, 4762–4773. (b) Kohn, H.; Robertson,
D. W.; Leander, J. D. Unpublished results.
(44) Bucciarelli, M.; Forni, A.; Moretti, I.; Prati, F.; Torre, G. Regio-
selectivity of ring-opening reactions of optically active N-acetyl-
2-methoxycarbonylaziridine. Tetrahedron: Asymmetry 1995, 6,
2073–2080.
(28) Walle, T.; Walle, U. K. Pharmacokinetic parameters obtained with
racemates. TIPS 1986, 155–158.
€
(29) Ariens, E. J. Stereochemistry: a source of problems in medicinal
chemistry. Med. Res. Rev. 1986, 6, 451–466.
(30) Levy, R. H.; Mattson, R.; Meldrum, B. Antiepileptic Drugs, 4th ed.;
Raven Press: New York, 1995; pp 99-110.

Article
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 21 6911
(45) (a) Larsson, U.; Carlson, R. Synthesis of amino acids with modified
principal properties 2: amino acids with polar side chains. Acta
Chem. Scand. 1994, 48, 511–516. (b) Belohradsky, M.; Ridvan, L.;
Zavada, J. Synthesis of homochiral acyclic mono- and bis(alpha-amino
acids) with oligo(oxyethylene) chains. Collect. Czech. Chem. Com-
mun. 2003, 68, 1319–1328.
(46) (a) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. A
reinvestigation of the mixed carbonic anhydride method of peptide
synthesis. J. Am. Chem. Soc. 1967, 87, 5012–5017. (b) Kunishima,
M.; Kawachi, C.; Monta, J.; Terao, K.; Iwasaki, F.; Tani, S. 2-(4,6-
Dimethoxy-1,3,5-triazin-2-yl)-4-methyl-morpholinium chloride: an
efficient condensing agent leading to the formation of amides and
esters. Tetrahedron 1999, 55, 13159–13170.
(47) (a) Kim, S.; Yi, K. Y. Di-2-pyridyl thiocarbonate. A new reagent for
the preparation of isothiocyantes and carbodiimides. Tetrahedron
Lett. 1985, 26, 1661–1664. (b) Potashman, M. H.; Bready, J.; Coxon, A.;
DeMelfi, T. M.; DiPietro, L.; Doerr, N.; Elbaum, D.; Estrada, J.; Gallant,
P.; Germain, J.; Gu, Y.; Harmage, J.; Kaufman, S. A.; Kendall, R.; Kim,
J. L.; Kumar, G. N.; Long, A. M.; Neervannan, S.; Patel, V. F.; Polverino,
A.; Rose, P.; van der Plas, S.; Whittington, D.; Zanon, R.; Zhao, H.
Design, synthesis, and evaluation of orally active benzimidazoles and
benxoxazoles as vascular endothelial growth factor-2 receptor tyrosine
kinase inhibitors. J. Med. Chem. 2007, 50, 4351–4373.
(48) For comparable procedures for resolving stereoisomers, see the
following: (a) Weisman, G. R. In Asymmetric Synthesis-Analy-
tical Methods; Morrison, J. D., Ed.; Academic Press: New York, 1983;
Vol. 1, pp 153-171. (b) Parker, D.; Taylor, R. J. Direct 1H NMR assay
of the enantiomeric composition of amines and β-amino alcohols using
O-acetyl mandelic acid as a chiral solvating agent. Tetrahedron 1987,
43, 5431–5456.
(51) Tornoe, C. W.; Christensen, C.; Meldal, M. Peptidotriazoles on
solid phases [1,2,3]-triazoles by regiospecific copper(I)-catalyzed
1,3-dipolar cycloadditions of terminal alkynes to azides. J. Org.
Chem. 2002, 67, 3057–3064.
(52) (a) Park, K. D.; Liu, R.; Kohn, H. Useful tool for biomolecule
isolation, detection, and identification: acylhydrazone-based
cleavable linker. Chem. Biol. 2009, 16, 763–772. (b) Sun, X.-L.;
Stabler, C. L.; Cazalis, C. S.; Chaikof, E. L. Carbohydrate and
protein immobilization onto solid surfaces by sequential Diels-Alder
and azide-alkyne cycloadditions. Bioconjugate Chem. 2006, 17,
52–57.
(53) Speers, A. E.; Cravatt, B. F. Profiling enzyme activities in
vivo using click chemistry methods. Chem. Biol. 2004, 11, 535–
546.
(54) Zhang, Z.; Ottens, A. K.; Sadasivan, S.; Kobeissy, F. H.; Fang, T.;
Hayes, R. L.; Wang, K. K. W. Calpain-mediated collapsing
response mediator protein-1, -2, and -4 proteolysis after neurotoxic
and traumatic brain injury. J. Neurotrauma 2007, 24, 460–472.
(55) Ross, P. L.; Huang, U. N.; Marchese, J. N.; Williamson, B.; Parker,
K.; Hattan, S.; Khainovski, N.; Pillai, S.; Dey, S.; Daniels, S.;
Purkayastha, S.; Juhasz, P.; Martin, S.; Bartlet-Jones, M.; He, F.;
Jacobson, A.; Pappin, D. J. Multiplexed protein quantitation in
Saccharomyces cerevisiae using amine-reactive isobaric tagging
reagents. Mol. Cell. Proteomics 2004, 3, 1154–1169.
(56) Cotten, S.; Liu, R. Unpublished data.
(57) 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.
(58) Salome, C.; Salome-Grosjean, E.; Park, K. D.; Morieux, P.;
Swendiman, R. ; DeMarco, E.; Stables, J. P.; Kohn, H. Unpub-
lished data.
(59) Stables, J. P.; Kupferberg, H. J. In Molecular and Cellular Targets
for Antiepileptic Drugs; Avanzini, G., Tanganelli, P., Avoli, M., Eds.;
John Libbey: London, 1977; pp 191-198.
(60) 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, 393–408.
(49) Barton, M. E.; Klein, B. D.; Wolff, H. H.; White, H. S. Pharma-
cological characterization of the 6 Hz psychomotor seizure model
of partial epilepsy. Epilepsy Res. 2001, 47, 217–227.
(50) 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.