Defining the structural parameters that confer anticonvulsant activity by the site-by-site modification of (R)-N′-benzyl 2-amino-3-methylbutanamide

Article abstract of DOI:10.1021/jm200760a

Primary amino acid derivatives (PAADs) (N′-benzyl 2-substituted 2-amino acetamides) are structurally related to functionalized amino acids (FAAs) (N′-benzyl 2-substituted 2-acetamido acetamides) but differ by the absence of the terminal N-acetyl group. Both classes exhibit potent anticonvulsant activities in the maximal electroshock seizure animal model, and the reported structure-activity relationships (SARs) of PAADs and FAAs differ in significant ways. Recently, we documented that PAAD efficacy was associated with a hydrocarbon moiety at the C(2)-carbon, while in the FAAs, a substituted heteroatom one atom removed from the C(2)-center was optimal. Previously in this issue, we showed that PAAD activity was dependent upon the electronic properties of the 4′-N′-benzylamide substituent, while FAA activity was insensitive to electronic changes at this site. In this study, we prepared analogues of (R)-N′-benzyl 2-amino-3-methylbutanamide to identify the structural components for maximal anticonvulsant activity. We demonstrated that the SAR of PAADs and FAAs diverged at the terminal amide site and that PAADs had considerably more structural latitude in the types of units that could be incorporated at this position, suggesting that these compounds function according to different mechanism(s).

Full text of DOI:10.1021/jm200760a

ARTICLE
pubs.acs.org/jmc
Defining the Structural Parameters That Confer Anticonvulsant
Activity by the Site-by-Site Modification of (R)-N0-Benzyl
2-Amino-3-methylbutanamide
Amber M. King,† Marc De Ryck,‡ Rafal Kaminski,‡ Anne Valade,‡ James P. Stables,§ 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, United States
‡CNS Research, UCB Pharma S.A., Chemin du Foriest, B-1420 Braine-l’Alleud, Belgium
§Anticonvulsant Screening Program, National Institute of Neurological Disorders and Stroke, National Institutes of Health, 6001
Executive Boulevard, Suite 2106, Rockville, Maryland 20892, United States
^Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, United States
S Supporting Information
b
ABSTRACT: Primary amino acid derivatives (PAADs) (N0-benzyl 2-sub-
stituted 2-amino acetamides) are structurally related to functionalized
amino acids (FAAs) (N0-benzyl 2-substituted 2-acetamido acetamides)
but differ by the absence of the terminal N-acetyl group. Both classes
exhibit potent anticonvulsant activities in the maximal electroshock seizure
animal model, and the reported structureꢀactivity relationships (SARs) of
PAADs and FAAs differ in significant ways. Recently, we documented that PAAD efficacy was associated with a hydrocarbon moiety
at the C(2)-carbon, while in the FAAs, a substituted heteroatom one atom removed from the C(2)-center was optimal. Previously in
this issue, we showed that PAAD activity was dependent upon the electronic properties of the 40-N0-benzylamide substituent, while
FAA activity was insensitive to electronic changes at this site. In this study, we prepared analogues of (R)-N0-benzyl 2-amino-3-
methylbutanamide to identify the structural components for maximal anticonvulsant activity. We demonstrated that the SAR of
PAADs and FAAs diverged at the terminal amide site and that PAADs had considerably more structural latitude in the types of units
that could be incorporated at this position, suggesting that these compounds function according to different mechanism(s).
’ INTRODUCTION
N0-benzylamide ring, where activity was retained with an
electron-withdrawing group but lost upon inclusion of an
electron-releasing group.8 By comparison, FAAs containing
either electron-donating or electron-withdrawing groups at
the para-position provided compounds with excellent anti-
convulsant activities.9 Third, the maximal activity for C(2)-
hydrocarbon PAADs resided in the D-configuration, similar
to the FAAs, but the differences in the ratio of the active ste-
reoisomer to the less-active stereoisomer (eudismic ratio)10
differed widely. For (R)-3 and (S)-3, the eudismic ratio in
mice exceeded 20, while for (R)-4, and (S)-4 it was 3. For
FAAs, the eudismic ratio was consistently above 10.9,11,12
The stark contrast between PAAD and FAA activity was most
apparent for C(2)-hydrocarbon derivatives, where the C(2)-
hydrocarbon was a branched alkyl moiety. We found that the
C(2)-isopropyl PAAD (R)-3 (MES ED50 = 15 mg/kg) and
C(2)-tert-butyl PAAD (R)-4 (MES ED50 = 14 mg/kg) were
potent anticonvulsants, while their FAA counterparts (R)-5
Epilepsy is a heterogeneous mixture of disorders characterized
by neuronal hyperexcitability and hypersynchronous neuronal
firing, and it affects up to 1% of the world’s population.1 There
are over 40 antiepileptic drugs (AEDs) currently in clinical use,2
yet, some 30% of patients are pharmacoresistant and do not
respond to at least two of the first-line AEDs.3 Furthermore,
compliance is often limited by adverse side effects (e.g., drowsi-
ness, dizziness, nausea), experienced by nearly 40% of patients.4
Novel compounds with increased efficacy and decreased toxicity
are needed to improve the quality of life of those suffering from
epilepsy.
We recently reported that primary amino acid derivatives
(PAADs, 1) exhibit potent anticonvulsant activities in the
maximal electroshock seizure (MES) test5 in rodents,6 with
activities approaching that of many clinical AEDs.7 Signifi-
cantly, the structureꢀactivity relationship (SAR) for PAADs
and their N-acetylated analogues, the functionalized amino
acids (FAAs, 2), differed in several ways. First, PAAD anti-
convulsant activity, unlike the FAAs, did not improve when a
substituted heteroatom was included one atom removed from
the C(2) chiral center.6 Second, PAAD activity was sensi-
tive to the electronic properties of para-substituents on the
(MES ED50 = >100, <300 mg/kg) and (R)-6 (MES ED50
=
>300 mg/kg) were not.6 Collectively, these results suggest
Received: June 13, 2011
Published: August 23, 2011
r
2011 American Chemical Society
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Figure 1. C(2)-Isopropyl PAAD (R)-3 analogues (sites AꢀF).
that the C(2)-hydrocarbon PAADs represent a novel class of
anticonvulsants.
’ RESULTS AND DISCUSSION
Choice of Compounds. C(2)-Hydrocarbon PAADs possess
significant anticonvulsant activities and our studies indicated that
many of the FAA structural hallmarks do not apply to PAADs.6,8
Therefore, we investigated the originally proposed PAAD struc-
tural framework. For most of our studies, we systematically
modified (R)-C(2)-isopropyl PAAD 3, which was among the
most potent PAADs discovered. Six structural features common
to both PAADs and FAAs were examined by preparing 7ꢀ24
(Figure 1): the carbonyl unit (site A); the amide bond (site B);
the amide methylene linker length (site C); the regiosubstitution
of the N0-benzylamide (site D); the need for a N0-benzylamide
(site E); the C(2)-amino functionality (site F). All C(2)-iso-
propyl PAAD analogues (7ꢀ18, 23, 24) were synthesized in the
(R)-configuration. For N0-benzyl 2-aminobutanamide 19 and N-
benzyl 2-hydroxybutanamide 22, we synthesized the (R)-config-
uration, for 21, we prepared the (R,S) and (S)-configurations,
and N-benzyl butanamide (20) does not have a chiral center.
C(2)-Isopropyl PAAD Analogues (Sites AꢀC). The impor-
tance of the carbonyl unit for FAA anticonvulsant activity has
been demonstrated by an isoelectronic substitution of the
amide carbonyl with a thiocarbonyl group,12 and we con-
ducted a similar investigation using (R)-7 (site A). We further
examined the value of the carbonyl unit by replacing it with a
methylene group ((R)-8).
We have reported that functionalized amido ketones (FAKs)
exhibit significant anticonvulsant activities that were comparable
to FAAs; however, an increase in neurological toxicity was also
observed (site B).13 In a similar manner, we replaced the nitrogen
of the amide bond in (R)-3 with a methylene group ((R)-9) or
oxygen ((R)-10) to create either a primary amino ketone or
primary amino ester, respectively.
The distance between the amide bond and the aromatic ring
was briefly investigated for FAAs, and maximal activity was seen
for the N0-benzylamide moiety (n = 1). Therefore, we examined
The differences in the PAAD and FAA SAR have led us to
question the optimal chemical framework for PAADs. At the
outset,6 we assumed that PAADs and FAAs would require similar
structural features for maximal activity. Among these were the
diamine backbone containing a carbonyl unit and a N0-terminal
benzylamide. In the present study, we systematically modified six
sites in the core structure of PAAD 3. Significantly, we found that
several key structural motifs associated with maximal FAA seizure
protection were not necessary for potent PAAD activity. These
findings provided additional evidence that C(2)-hydrocarbon
PAADs are a distinct, new class of potent anticonvulsants. The
structural simplicity of these PAADs, and the excellent water
solubility of their corresponding salts, provide opportunities for
further development.
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the anticonvulsant activities of PAADs that contain 0ꢀ4 methy-
lene units between the amide bond and the aromatic ring ((R)-3,
(R)-11ꢀ14) (site C).
Scheme 1. Synthesis of (R)-N0-Benzyl 2-Amino-3-
methylthiobutanamide ((R)-7)
Regiosubstitution in N0-Benzylamide PAADs (Site D).
Recently, we systematically evaluated the effect of a fluoro and
trifluoromethoxy group at the 20, 30, and 40 postions in the N0-
benzyl moiety of FAAs and determined that the 40 derivatives
displayed the highest level of anticonvulsant activity,9 consistent
with previous studies.11,14 Accordingly, we evaluated the effect of
a trifluoromethoxy group at the 20, 30, and 40 positions of PAAD
(R)-3 by preparing (R)-15, (R)-16, and (R)-17,8 respectively.
N0-Substituted C(2)-Isopropyl PAADs (Site E). We evaluated
several N0-substituted FAAs, including a N0-methylamide, N0-
benzhydrylamide, and N0-benzylamide, and found that the N0-
benzylamide unit is necessary for the excellent anticonvulsant
activity of FAAs.15 We substituted the aromatic ring in (R)-3 for a
cyclohexyl ring ((R)-18) to examine the influence of the planar
ring system on anticonvulsant activity.
Scheme 2. Synthesis of (R)-1-N-Benzylamino-2-amino-3-
methylbutane ((R)-8)
α-Substituted N-Benzylbutanamides (Site F) and N-Sub-
stituted C(2)-Isopropyl PAADs (Site F0). In our final analysis we
examined the importance of the amino group and amine
substitution. The limited availability of 2-substituted 3-methyl-
butanoic acid reagents prompted the investigation of N-benzyl-
butanamide derivatives ((R)-19, 20, (R,S)-21, (S)-21, and
(R)-22) instead of N0-benzyl 3-methylbutanamide derivatives
(site F). Previously, the C(2)-acetamido of (R,S)-N0-benzyl
2-acetamido-2-phenylacetamide ((R,S)-25, ED50 = 20 mg/kg)
was systematically replaced with a hydrogen, methyl, hydroxy,
methoxy, or halogen group. The hydroxy and methoxy groups
provided moderate MES activities (ED50 = >30, <100 mg/kg)
but were appreciably less active than the parent FAA.16 In a similar
study, the C(2)-acetamido group of (R,S)-N0-benzyl 2-acetamido-
3-methoxypropionamide ((R,S)-26, ED50 = 8.3 mg/kg) was
replaced with a methyl, hydroxy, and amino group. The methyl
and amino groups also resulted in moderate MES activities
(ED50 = >30, <100 mg/kg), but again the activities were lower
than the corresponding FAA.17 Accordingly, both studies con-
cluded that the C(2)-acetamido group showed superior antic-
onvulsant activity. We conducted a similar investigation in which
we replaced the amino group in (R)-19 with a hydrogen (20),
methyl ((R,S)-21), or hydroxy group ((R)-22). The moderate
activity of (R,S)-21 prompted our efforts to prepare (R)-21 and
(S)-21 to determine if anticonvulsant activity resided in the (R)-
stereoisomer. We successfully synthesized (S)-21 but were
unable to prepare (R)-21.
trifluoroacetic acid (TFA) deprotection of the t-Boc group
(Scheme 1). We were mindful of the potential for thiation of
the carbamate carbonyl in (R)-27, but the 13C NMR spectrum
contained a signal at ∼155 ppm, the typical shift for a carbamate
carbon, rather than ∼190 ppm for the thiocarbamate.21 In
agreement with the proposed structure of (R)-27, we observed
the thioamide carbon signal in the 13C NMR at 205 ppm.
Diamine (R)-814 was synthesized by directly reducing (R)-36
with borane in THF (Scheme 2). We followed a previously
reported procedure14 but changed the workup.
We synthesized PAAD (R)-9 by coupling (R)-296 with N,O-
dimethylhydroxylamine hydrochloride (HCl) in the presence
of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CMDT) and base22
to give Weinreb amide (R)-30 (Scheme 3). Weinreb amides re-
adily react with Grignard reagents and are widely known as useful
precursors to ketones.22ꢀ24 Accordingly, (R)-30 was reacted with
phenethylmagnesium bromide to give (R)-31, followed by HCl
deprotection to give PAAD (R)-9 as a HCl salt.
We synthesized (R)-32 from (R)-296 using a mild ester-
ification method25 that employed benzyl chloroformate in
the presence of base and catalytic amounts of 4-dimethyla-
minopyridine (DMAP) (Scheme 4). Subsequent TFA de-
protection of (R)-32 gave PAAD (R)-10.
The C(2)-isopropyl PAAD analogues (R)-11ꢀ16 and (R)-18
were synthesized by coupling (R)-296 with commercially available
amines (33ꢀ39) and using the standard mixed anhydride coupling
(MAC) procedure,26 to give amides (R)-40ꢀ46, followed by TFA
deprotection to the corresponding PAADs (Scheme 5).
PAADs 20, (R,S)-21, (S)-21, and (R)-22 were synthesized
from commercially available carboxylic acids 47, (R,S)-48,
(S)-48, and (R)-49 (Scheme 6). For (R)-20, (R,S)-21, and
(S)-21, we used the MAC procedure with benzylamine. We
attempted to prepare (R)-21 to compare its pharmacological
activity with (S)-21 but were unsuccessful.27 PAAD (R)-22
was prepared by treating (R)-49 with benzylamine in the
presence of the commercially available catalyst, 3,5-bis-
(trifluoromethyl)benzeneboronic acid28 (50) (Scheme 6).
Finally, to assess the importance of N-terminal substitution
(site F0), we prepared the secondary amino acid derivative
(SAAD) (R)-23 and the tertiary amino acid derivative
(TAAD) (R)-24 in the C(2)-isopropyl series. Earlier studies
did not reveal a general pattern to correlate anticonvulsant
activity with amine substitution.18ꢀ20 We observed that N-amine
substitution improved anticonvulsant activity in several cases, but
in other compounds, we saw a loss of activity.
Chemistry. Thioamide (R)-7 was prepared by treating (R)-
276 with excess Lawesson’s reagent at reflux,12 followed by
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Scheme 3. Synthesis of (R)-4-Amino-2-methyl-6-phenyl-4-hexanone Hydrochloride ((R)-9)
investigated the importance of six properties (sites AꢀF) that
were common in the FAAs and report their anticonvulsant
activity and neurological toxicity in Tables 1 and 2.
Scheme 4. Synthesis of (R)-O-Benzyl 2-Amino-3-methylbu-
tanoate ((R)-10)
First, we determined the effect of the carbonyl unit (site A) on
anticonvulsant activity (Table 1). Replacement of the carbonyl
unit in (R)-3 with a thiocarbonyl unit to give (R)-7 resulted in
decreased MES activity in mice (ED50 (mg/kg): (R)-3, 15; (R)-
7, >30, <100), but notable activity was observed in rats (ED50
=
<30 mg/kg). Reducing the carbonyl unit in (R)-3 to a methylene
group to provide (R)-8 led to decreased activity in both mice
(ED50 = >30, <100 mg/kg) and rats (ED50 =>30 mg/kg).
Next, we determined the effect of the amide bond (site B)
on anticonvulsant activity (Table 1). Converting the amide
(R)-3 (Y = NH) to ketone (R)-9 (Y = CH2) decreased the
MES activity (ED50 (mg/kg): (R)-3, 15; (R)-9, >30, <100)
and conversion to the ester (R)-10 (Y = O) abolished activity
(ED50 = >300 mg/kg).
Then, at site C, we looked at the optimal methylene linker
length between the amide bond and the aromatic ring
(Table 1). Direct linkage of the aromatic ring to the amide
bond to give (R)-11 (n = 0) resulted in a significant drop
in activity from the parent compound (R)-3 (n = 1) (ED50
(mg/kg): (R)-3, 15; (R)-11, >30, <100). However, extending
the linkage of the parent compound (R)-3 (n = 1) by one
methylene unit to provide (R)-12 (n = 2) increased antic-
onvulsant activity by ∼30% (ED50 = 10 mg/kg). The activity
increase was associated with a neurotoxicity increase over the
parent compound (TD50 (mg/kg): (R)-3, 70; (R)-12, 50);
however, the PI of (R)-3 (4.8) and (R)-12 (5.0) were nearly
equal. Extending the linker by another carbon to give (R)-13
(n = 3) led to comparable activity with (R)-3 (ED50 (mg/kg):
(R)-3, 15; (R)-13, 16), but the increased toxicity for (R)-13
(TD50 = 43 mg/kg) reduced the PI to 2.7. A final extension to
n = 4 to give (R)-14 resulted in comparable activity in mice
(ip) with (R)-3 and (R)-13 (ED50 (mg/kg): (R)-14, 11; (R)-3,
15; (R)-13, 16), with a PI of 3.5. The anticonvulsant activities of
(R)-12ꢀ14 were surprising because in the FAA series, an increase
in the methylene linkage led to a sharp drop in activity.34
SAAD (R)-23 was prepared in three steps, beginning with the
t-Boc protection of commercially available acid (R)-51 to give
(R)-52 (Scheme 7). (R)-52 was coupled with benzylamine using
the MAC procedure to give amide (R)-53 and then HCl
deprotected to give SAAD (R)-23 as the HCl salt.
Synthesis of TAAD (R)-24 began with reductive condensation
of D-valine ((R)-54) with formaldehyde using 10% PdꢀC in the
presence of H2 to give (R)-55 (Scheme 8).29ꢀ31 (R)-55 was
coupled with benzylamine using the condensing reagent 4-(4,6-
dimethoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride
(DMTMM)32 to give TAAD (R)-24.
In the Experimental Section, we detail the final step (synthetic
procedure and characterization) for all compounds evaluated in
the animal models. In the Supporting Information, we provide
our experimental procedures for all compounds prepared and
their physical and spectroscopic properties.
Pharmacological Activity. PAADs 7ꢀ22 and 24 were eval-
uated for anticonvulsant activity using the MES test at the
National Institute of Neurological Disorders and Stroke Anti-
convulsant Screening Program (NINDS ASP), following the
procedures described by Stables and Kupferberg5 and described
in the preceding paper.8 PAAD 23 was evaluated for anti-
convulsant activity at UCB Pharma, following the procedures
described by Klitgaard,33 as previously reported.8 The pharma-
cological data from the MES tests are summarized in Tables 1
and 2. PAADs tested at the NINDS ASP were evaluated in the
subcutaneous metrazol (scMet) seizure model5 and displayed
little to no protection.27
Site D compared the effect of substitution on the N0-
benzylamide ring, where we systematically placed a trifluoro-
methoxy group at the 20 ((R)-15), 30 ((R)-16), and 40-positions
((R)-17) (Table 1). All regiosubstitutions displayed excellent
MES activities in mice (ED50 (mg/kg): (R)-15, 9.2; (R)-16,
7.1; (R)-17, 16). The ED50 value for (R)-16 was 2-fold more
active than the parent compound (R)-3 (ED50 = 15 mg/kg),
but the increase in activity was associated with an increase in
neurotoxicity (ED50 (mg/kg): (R)-3, 70; (R)-16, 30). Com-
parison of the MES activity in rats (Table 1) showed a ∼3-fold
drop in activity going from (R)-3 to (R)-15 (ED50 (mg/kg):
(R)-3, 11; (R)-15, 33). The MES activity of (R)-16 in the rat
Compound (R)-3 emerged as a potent anticonvulsant (MES
ED50 = 15 mg/kg) that possessed pain attenuating properties
(formalin ED50 = 15 mg/kg) from the SAR investigation at the
C(2)-carbon.6 This excellent activity came as a surprise because it
did not follow the trends observed in the FAA series. Therefore,
we have questioned several aspects of the original PAAD
structural framework to determine if the basic tenets of the
FAA blueprint applied to this C(2)-hydrocarbon PAAD. We
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Scheme 5. Synthesis of C(2)-Isopropyl PAADs (R)-11ꢀ16 and (R)-18
Scheme 6. Synthesis of C(2)-Isopropyl Analogues: α-Substituted N-Benzylbutanamides 20, (R,S)-21, (S)-21, and (R)-22
Scheme 7. Synthesis of (R)-N0-Benzyl N,N-Dimethylamino-
3-methylbutanamide ((R)-23)
Scheme 8. Synthesis of (R)-N0-Benzyl N,N-Dimethylamino-
3-methylbutanamide ((R)-24)
(ED50 = 10 mg/kg) was comparable with (R)-3 (ED50 = 11
mg/kg), but there was a sharp increase in behavioral toxicity
(TD50 (mg/kg): (R)-3, >500; (R)-16, 43). The potent anti-
convulsant activity observed for (R)-15 led us to evaluate this
compound in the formalin neuropathic pain model35 where we
protection in mice (ED50 = 56 mg/kg) with minimal neurotoxicity
(TD50 = 165 mg/kg) and moderate activity in rats (ED50 = 51
mg/kg) without any detectable behavioral toxicity (TD50 =
observed excellent activity in the inflammatory phase (ED50
=
9.2 mg/kg, data not shown).36
Next, we examined the need for a benzyl moiety (site E)
(Table 1). We replaced the aromatic ring ((R)-3) with a
cyclohexyl ring ((R)-18) and observed a decrease in anti-
convulsant activity (ED50 (mg/kg): (R)-3, 15; (R)-18, >30,
<100). When (R)-18 was evaluated in the rat, we found this
PAAD to have excellent activity (ED50 = ∼15 mg/kg). While
our data indicates that the N0-benzyl substituent is preferred
over the saturated N0-cyclohexylmethyl unit, we did not
anticipate the anticonvulsant activity of (R)-18.
Finally, we investigated the importance of the C(2)-amino
functionality (site F) by comparing the unsubstituted N-
benzyl butanamide 20 (X = H), the methyl-substituted N-
benzyl butanamide (R,S)-21 and (S)-21 (X = CH3), and
the hydroxy-substituted N-benzyl butanamide (R)-22 (X =
OH) with the parent PAAD (R)-19 (X = NH2) (Table 2).
Compounds 20 and (R)-22 gave similar, modest activity
(ED50 =>30, <100 mg/kg) in mice and no appreciable activity
in rats (ED50 = >30 mg/kg). (R,S)-21 also displayed modest
>500 mg/kg). We were surprised by the activity of (R,S)-21
because the methyl group lacks the hydrogen bonding cap-
abilities afforded by an amino or a hydroxy group. Evaluation
of (S)-21 showed a decrease in anticonvulsant activity (ED50
=
>100, <300 mg/kg), suggesting that activity resided predo-
minantly in the (R)-isomer. Unfortunately, we were unable to
complete the synthesis of (R)-21 to confirm this suggestion.
The activities of the primary (PAAD), secondary (SAAD), and
tertiary (TAAD) amino acid analogues of (R)-N0-benzyl
2-amino-3-methylbutanamide ((R)-3) are summarized in Ta-
ble 2 (site F0). Comparison of the SAAD (R)-23 with the
PAAD (R)-3 showed decreased activity in the MES test (ED50
(mg/kg): (R)-3, 15; (R)-23, 25). Furthermore, comparison of
the TAAD (R)-24 with the SAAD (R)-23 showed an addi-
tional decrease in activity in the MES test (ED50 (mg/kg):
(R)-23, 25; (R)-24, >30, <100). Therefore, the MES data for
PAAD (R)-3, SAAD (R)-23, and TAAD (R)-24 revealed a
linear decrease in anticonvulsant activity as we successively
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Table 2. Pharmacological Activities of C(2)-Substituted N-Benzyl Butanamides (site F) in Mice (mg/kg)a and Rats (mg/kg)b and
Primary (PAAD), Secondary (SAAD), and Tertiary (TAAD) Amino Acid Derivatives of (R)-N-Benzyl 2-Amino-3-methylbuta-
namide (site F0) in Mice (mg/kg)a
mice (ip)a
Tox,d TD50
rat (po)b
Tox,f TD50
compd no.
site
R
X
MES,c ED50
PIe
4.4
MES,c ED50
PIe
(R)-19g
20
F
F
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH2CH3
CH(CH3)2
CH(CH3)2
CH(CH3)2
NH2
H
18 [0.25] (10ꢀ25)
>30, <100 [0.5]
56 [0.25] (45ꢀ69)
>100, <300 [0.5]
>30, <100 [0.5]
15 [0.25] (13ꢀ18)
25
80 [0.25] (65ꢀ95)
>100, <300 [0.5]
165 [0.25] (148ꢀ180)
∼300 [0.5]
>100, <300 [0.5]
70 [0.25] (63ꢀ80)
NDj
11 [2.0] (7.8ꢀ16)
>30 [0.25ꢀ4.0]
51 [0.5] (35ꢀ71)
>30 [0.25ꢀ4.0]
>30 [0.25ꢀ4.0]
11 [0.25] (9.1ꢀ13)
NDj
>500
>30 [0.25ꢀ4.0]
>500
>45
(R,S)-21h
(S)-21
(R)-22
(R)-3g
(R)-23i
(R)-24
F
CH3
2.9
4.7
>9.8
>45
F
CH3
>30 [0.25ꢀ4.0]
>30 [0.25ꢀ4.0]
>500
F
OH
F0
F0
F0
NH2
N(H)CH3
N(CH3)2
NDj
NDj
>30, <100 [0.5]
>100, <300 [0.5]
NDj
a The compounds were administered intraperitoneally to adult male albino CF-1 mice under the auspices of the NINDS ASP. ED50 and TD50 values are
in mg/kg. A doseꢀresponse curve was generated for all compounds that displayed sufficient activity (4ꢀ6 doses tested, n = 8 per dose) and the
doseꢀeffect data for these compounds was obtained at the “time of peak effect” (indicated in hours in the brackets). Numbers in parentheses are 95%
confidence intervals. b The compounds were administered orally to adult male albino SpragueꢀDawley rats under the auspices of the NINDS ASP. ED50
and TD50 values are in mg/kg. c MES = maximal electroshock seizure test. d TD50 value determined from the rotorod test. e PI = protective index (TD50/
ED50). f Tox = behavioral toxicity. g Reference 6. h 6 Hz (32 mA) = 63 mg/kg. i The compounds were administered intraperitoneally to adult male NMRI
mice under the auspices of UCB. ED50 and TD50 values are in mg/kg and were determined 30 min after ip administration. j ND = not determined.
incorporated methyl groups at the C(2)-amine site. This trend
differed from the reported pattern for racemic C(2)-methyl
and C(2)-CH2OCH3 PAADs, in which successive N-methyla-
tion led to nonlinear trends in MES activity.18
protection and that altering this unit affected the hydrogen
bonding properties of the compounds. Assessment of the
methylene linker (site C) showed that at least one carbon
between the amide bond and the aromatic ring was necessary
for anticonvulsant activity but incorporation of up to three
additional methylene units provided excellent activity. When
considering activity, toxicity, and the protective index for
these C(2)-isopropyl PAADs, the optimal linker length ap-
peared to be when n = 2, instead the n = 1, as seen in the FAAs.
Similarly, we found that replacement of the benzylamide
group by a cyclohexylmethylamide (site E) led to lower
anticonvulsant activity, but the drop was modest. Thus, there
seems to be structural latitude at sites C and E, where
modifications retained excellent activities. Comparison of
trifluoromethoxy regioisomers of (R)-N0-benzyl 2-amino-3-
methylbutanamide (site D) revealed that superb seizure
protection was associated with all positions. The activity
increase was associated with a neurotoxicity increase, but
the PI values were comparable with the parent, unsubstituted
PAAD (R)-3. Lastly, we evaluated our choice of the C(2)-amino
group (site F) because the earlier SAR studies were centered
around primary amino acid derivatives.6,8 Of the C(2)-groups
investigated, the amino group was most active but was not man-
datory for activity. The activities of the primary (PAAD), second-
ary (SAAD), and tertiary (TAAD) amino acid analogues of (R)-
N0-benzyl 2-amino-3-methylbutanamide ((R)-3) showed a steady
loss of anticonvulsant activity with N-methylation. Collectively,
the findings of this study, along with the earlier observations that
the anticonvulsant activity of C(2)-hydrocarbon PAADs do not
improve when a substituted heteroatom is included one atom
removed from the C(2)-center6 and that PAAD activity is sensitive
to the electronic effects of N0-benzylamide substituents,8 indicated
that the C(2)-hydrocarbon PAADs have a unique SAR. Future
work will investigate PAAD mechanism of action to determine if
Our findings indicated that PAAD anticonvulsant activity
increased when a N-phenethylamide moiety (Table 1, (R)-
12) was used and when an electron-withdrawing substituent
(OCF3) was incorporated on the aromatic ring (Table 1, (R)-
15ꢀ17). Accordingly, we prepared three monofluoro regio-
isomers of (R)-N0-phenethyl 2-amino-3-methylbutanamide,
((R)-56ꢀ58) using the same general procedure outlined in
Scheme 5. We observed that anticonvulsant activity re-
mained high in the MES test (mice, ip) for all three
regioisomers (ED50 (mg/kg): (R)-56, 27; (R)-57, 20; (R)-
58, >10, <30), but there was no apparent benefit over the
parent, unsubstituted PAAD (R)-12 (MES ED50 = 10 mg/kg).
However, we observed an increase in activity and a decrease in
toxicity in the MES test for (R)-56 going from mice (ip) to rats
(po). (R)-58 exhibited only slightly better activity than the
corresponding benzyl analogue (R)-598 (ED50 = 32 mg/kg), in
terms of potency in the MES test and PI values (Table 1).
’ CONCLUSIONS
Examination of PAAD sites AꢀF revealed that the amide
bond (sites A and B) was important for effective seizure
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Journal of Medicinal Chemistry
ARTICLE
the difference in observed efficacy of PAADs and FAAs is a
consequence of varying pharmacokinetics or if PAADs interact
with a novel molecular target.
with Et2O (2 ꢁ 100 mL). The aqueous layer was basified to pH 10ꢀ12
using aqueous 4 M NaOH and extracted with CH2Cl2 (3 ꢁ 100 mL).
The CH2Cl2 layers were combined and successively washed with a 1:1
mixture of EtOH/H2O (2 ꢁ 100 mL) and saturated aqueous brine
(2 ꢁ 100 mL), dried (Na2SO4), evaporated in vacuo, and purified
three times by flash column chromatography (SiO2; 1:100 MeOH/
CH2Cl2) to give the desired product (0.53 g, 17%) as a pale yellow
oil: [α]28 ꢀ31.4ꢀ (c 0.51, CH2Cl2) (lit.37 [α]20 ꢀ30.4ꢀ (c 0.55,
’ EXPERIMENTAL SECTION
General Methods. The general methods used in this study
were identical to those previously reported6 and are summarized in
the Supporting Information. All compounds were checked by TLC,
1H and 13C NMR, MS, and elemental analyses. The analytical
results were within (0.40% of the theoretical value. The TLC, NMR,
and the analytical data confirmed that the purity of the products was
g95%.
D
D
CH2Cl2)), [α]28D ꢀ31.4ꢀ (c 1.9, CHCl3) (lit.38 (S): [α]25.4D +33.4ꢀ
(c 1.80, CHCl3)); Rf 0.53 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 0.90 (d, J = 6.8 Hz, 6H), 1.58ꢀ1.69 (m, 1H), 2.42ꢀ2.48
(m, 4H), 2.64ꢀ2.69 (m, 1H), 2.74 (dd, J = 3.2, 12.0 Hz, 1H), 3.78 (1/2
ABq, J = 13.4 Hz, 1H), 3.84 (1/2 ABq, J = 13.4 Hz, 1H), 7.22ꢀ7.35
(m, 5H); HRMS (ESI) 193.1705 [M + H+] (calcd for C12H20N2H+
General Procedure for PAAD Preparation Using TFA
Deprotection (Method A). TFA (15 equiv) was added to an
anhydrous CH2Cl2 solution of the tBoc-protected N0-benzylamide
PAAD (0.3 M) at room temperature. The solution was stirred (1 h),
and then the solvent was evaporated in vacuo. The crude product was
subjected to acidic workup or basic workup. Acidic: The crude product
was diluted with CH2Cl2 and extracted with aqueous 1 M HCl (3ꢁ).
The combined aqueous layers were washed with CH2Cl2 (2ꢁ), basified
(pH 10ꢀ12) with aqueous 4 M NaOH, and extracted with CH2Cl2
(3ꢁ). The combined organic layers were washed with brine (2ꢁ), dried
(Na2SO4), evaporated in vacuo, and purified by column chromatogra-
phy (SiO2). Basic: The crude product was diluted with CH2Cl2 and
washed with aqueous 1 M Na2CO3 (3ꢁ). The aqueous layers were
combined and washed with CH2Cl2 (2ꢁ). All of the CH2Cl2 layers were
combined and successively washed with H2O (2ꢁ) and brine (2ꢁ),
dried (Na2SO4), evaporated in vacuo, and purified by column chroma-
tography (SiO2).
193.1695); Anal. (C12H20N2 H2O): C, H, N.
3
(R)-4-Amino-2-methyl-6-phenyl-4-hexanone Hydrochlor-
ide ((R)-9). (R)-3139,40 (3.04 g, 9.96 mmol) was dissolved in MeOH
(50 mL), and aqueous concentrated HCl (4.9 mL, 59 mmol) was added.
The reaction was heated at reflux (1 h) and then cooled to room
temperature before evaporating the solvent in vacuo to give a crude oil.
The crude product was triturated with hexanes (3ꢁ) to give the desired
product (0.60 g, 25%) as a white solid: mp 123ꢀ124 ꢀC; [α]28D ꢀ59.7ꢀ
(c 1.1, CHCl3); Rf 0.49 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 0.97 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 2.34ꢀ2.44
(br m, 1H), 2.56ꢀ2.98 (m, 4H), 4.18ꢀ4.26 (br d, 1H), 7.16ꢀ7.24 (m,
5H), 8.57 (br s, 3H); LRMS (ESI) 206.13 [M - Clꢀ] (calcd for
C13H20NO 206.13); Anal. (C13H20ClNO): C, H, Cl, N.
(R)-O-Benzyl 2-Amino-3-methylbutanoate ((R)-10).41 HCl
(1 M) in Et2O (165 mL) was added to an Et2O solution (10 mL) of (R)-
3225,42 (2.02 g, 6.58 mmol) at 0 ꢀC. The reaction was stirred at room
temperature (18 h) to give a cloudy white solution. The solution was
filtered but no substantial filtrate was collected. The solvent was
evaporated in vacuo to give a crude oil that was then redissolved in
CH2Cl2 (10 mL). The organic layer was extracted with aqueous 1 M
HCl (3 ꢁ 10 mL). The aqueous layers were combined and washed with
CH2Cl2 (2 ꢁ 30 mL). The aqueous layer was basified to pH 10ꢀ12 with
aqueous 4 M NaOH and extracted with CH2Cl2 (3 ꢁ 50 mL). The
second set of organic layers were combined and washed with saturated
aqueous brine (2 ꢁ 150 mL), dried (Na2SO4), and evaporated in vacuo.
The crude product was purified by flash column chromatography (SiO2;
1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give the
desired product (0.99 g, 72%) as a pale yellow oil: [α]28.5D ꢀ10.2ꢀ (c 1.1,
CHCl3); Rf 0.39 (1:1 EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) δ
0.88 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 1.44 (s, 2H), 1.99ꢀ2.10
(m, 1H), 3.34 (d, J = Hz, 1H), 5.14 (1/2 ABq, J = 12.2 Hz, 1H), 5.18 (1/2
ABq, J = 12.2 Hz, 1H), 7.30ꢀ7.39 (m, 5H); LRMS (ESI) 208.15
[M+H+] (calcd forC12H17NO2H+ 208.15); Anal. (C12H17NO2):C, H, N.
(R)-N0-Phenyl 2-Amino-3-methylbutanamide ((R)-11).43
Utilizing method A with (R)-4044 (1.75 g, 5.99 mmol), TFA (6.67 mL,
89.8 mmol), and CH2Cl2 (20 mL) gave the crude product after acidic
workup and was further purified by flash column chromatography (SiO2;
1:10ꢀ1:1 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give the
desired compound (0.94 g, 82%) as a pale orange oil: [α]25D +82.5ꢀ (c 1.6,
CH2Cl2); Rf 0.34 (1:1 EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) δ
0.87 (d, J = 7.2 Hz, 3H), 1.04 (d, J = 7.2 Hz, 3H), 1.46 (s, 2H), 2.40ꢀ2.48
(m, 1H), 3.37(d, J= 3.6 Hz, 1H), 7.09 (t, J= 7.2 Hz, 1H), 7.32 (t, J=8.2Hz,
2H), 7.60 (d, J= 8.2 Hz, 2H), 9.46ꢀ9.56 (br s, 1H); HRMS (ESI) 193.1343
General Procedure for the Preparation of N0-Benzylamide
Amino Acid Derivatives Using the Mixed Anhydride Cou-
pling (MAC) Method (Method B). An anhydrous THF solution of
carboxylic acid (0.5ꢀ2.0 M) was cooled to ꢀ78 ꢀC in a dry ice/acetone
bath under an inert atmosphere (Ar or N2), and 4-methylmorpholine
(NMM) (1.3ꢀ1.5 equiv) was added. After the mixture was stirred
(2ꢀ10 min), isobutyl chloroformate (IBCF) (1.1ꢀ1.5 equiv) was
added, leading to the precipitation of a white solid. The reaction was
allowed to proceed for an additional 15ꢀ25 min, and then benzylamine
(1.05ꢀ1.36 equiv) was added at ꢀ78 ꢀC. The reaction mixture was
allowed to stir at room temperature (1.5 h), and then the insoluble salts
were filtered. The organic layer was concentrated in vacuo, and the
product was purified by column chromatography (SiO2).
(R)-N0-Benzyl 2-Amino-3-methylbutanethioamide ((R)-7).
Utilizing method A with (R)-28 (1.72 g, 5.34 mmol), TFA (5.95 mL,
80.1 mmol), and CH2Cl2 (18 mL) gave the crude product after acidic
workup and was further purified by flash column chromatography
(SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give
the desired compound (0.89 g, 76%) as a yellow oil: [α]28.5D +43.7ꢀ (c
1.0, CHCl3); Rf 0.50 (1:1 EtOAc/hexanes); 1H NMR (400 MHz,
CDCl3) δ 0.72 (d, J = 6.8 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 1.36 (s,
2H), 2.75ꢀ2.87 (m, 1H), 3.78 (d, J = 2.8 Hz, 1H), 4.84 (dd, J = 4.6, 15.0
Hz, 1H), 4.91 (dd, J = 5.0, 15.0 Hz, 1H), 7.29ꢀ7.38 (m, 5H), 9.75ꢀ9.95
(br s, 1H); LRMS (ESI) 223.13 [M + H+] (calcd for C12H18N2SH+
223.14); Anal. (C12H18N2S): C, H, N.
(R)-1-N0-Benzylamino-2-amino-3-methylbutane ((R)-8).37
(R)-36 (3.38 g, 16.4 mmol) was dissolved in anhydrous THF
[M + H+] (calcd for C11H16N2OH+ 193.1341); Anal. (C11H16N2O
0.01H2O): C, H, N.
(R)-N0-Phenethyl 2-Amino-3-methylbutanamide ((R)-12).
Utilizing method A with (R)-41 (1.37 g, 4.28 mmol), TFA (4.77 mL,
64.2 mmol), and CH2Cl2 (15 mL) gave the crude product after acidic
workup that was further purified by flash column chromatography
(SiO2; 1:10ꢀ1:1 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2)
(65 mL) and cooled to 0 ꢀC. BH3 THF (1 M, 49.19 mL, 49.19 mmol)
3
3
was added dropwise, and the mixture was heated to reflux (18 h). The
mixture was cooled to 0 ꢀC, and aqueous 0.5 M HCl (100 mL) was
slowly added. The THF was evaporated in vacuo, and Et2O (100 mL)
was added to the acidic aqueous solution. The aqueous layer was
separated, and the organic layer was extracted with aqueous 0.5 M HCl
(3 ꢁ 50 mL). All of the aqueous layers were combined and washed
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Journal of Medicinal Chemistry
ARTICLE
to give the desired compound (873 mg, 93%) as a pale yellow solid: mp
(d, J = 7.0 Hz, 3H), 1.10ꢀ1.28 (m, 3H), 1.36 (s, 2H), 1.41ꢀ1.52 (m,
1H), 1.65ꢀ1.74 (m, 5H), 2.26ꢀ2.37 (m, 1H), 3.03ꢀ3.18 (m, 2H), 3.23
(d, J = 3.6 Hz, 1H), 7.30ꢀ7.40 (br t, 1H); LRMS (ESI) 213.18 [M + H+]
(calcd for C12H24N2OH+ 213.18); Anal. (C12H24N2O): C, H, N.
N-Benzyl Butanamide (20).45 Utilizing method B with 47
(2.00 mL, 21.9 mmol), NMM (3.13 mL, 28.5 mmol), IBCF (3.10 mL,
24.1 mmol), and benzylamine (2.51 mL, 23.0 mmol) gave the crude
product that was recrystallized from hot EtOAc/hexanes to give the desired
compound (1.18 g, 30%) as a white solid: mp 54ꢀ55 ꢀC (lit.45 mp
36ꢀ37 ꢀC; [α]25 +32.9ꢀ (c 1.0, CH2Cl2); Rf 0.19 (1:20 MeOH/
D
CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 0.77 (d, J = 7.2 Hz, 3H), 0.96
(d, J = 7.2 Hz, 3H), 1.20ꢀ1.26 (s, 2H), 2.22ꢀ2.33 (m, 1H), 2.77ꢀ2.88
(m, 2H), 3.19 (d, J = 4.0 Hz, 1H), 3.46ꢀ3.62 (m, 2H), 7.20ꢀ7.32 (m,
5H); HRMS (ESI) 221.1643 [M + H+] (calcd for C13H20N2OH+
221.1654); Anal. (C13H20N2O): C, H, N.
(R)-N0-Phenylpropyl 2-Amino-3-methylbutanamide ((R)-13).
Utilizing method A with (R)-42 (5.00 g, 15.0 mmol), TFA (16.7 mL,
225 mmol), and CH2Cl2 (50 mL) gave the crude product after acidic
workup that was further purified by flash column chromatography (SiO2;
1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give the desired
1
36.9ꢀ38 ꢀC); Rf 0.67 (1:20 MeOH/CH2Cl2); H NMR (400 MHz,
CDCl3) δ 0.96 (t, J = 7.6 Hz, 3H), 1.65ꢀ1.74 (m, 2H), 2.20 (t, J = 8.0 Hz,
2H), 4.45 (d, J = 5.6 Hz, 2H), 5.66ꢀ5.78 (br s, 1H), 7.26ꢀ7.35 (m, 5H);
HRMS (ESI) 178.1238 [M + H+] (calcd for C11H15NOH+ 178.1232);
compound (3.02 g, 86%) as a pale yellow oil: [α]28.5 +30.1ꢀ (c 1.2,
D
CHCl3); Rf 0.21 (1:1 EtOAc/hexanes); 1H NMR (400 MHz, CDCl3) δ
0.81 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 7.2 Hz, 3H), 1.30 (s, 2H), 1.79ꢀ1.90
(m, 2H), 2.23ꢀ2.34 (m, 1H), 2.65 (t, J = 8.4 Hz, 2H), 3.19 (d, J = 4.0 Hz,
1H), 3.22ꢀ3.37 (m, 2H), 7.16ꢀ7.20 (m, 3H), 7.26ꢀ7.35 (m, 3H); HRMS
(ESI) 235.1818 [M + H+] (calcd for C14H22N2OH+ 235.1810); Anal.
Anal. (C11H15NO 0.06H2O): C, H, N.
3
(R,S)-N-Benzyl 2-Methylbutanamide ((R,S)-21).46 Utilizing
method B with (R,S)-48 (2.00 mL, 18.3 mmol), NMM (2.62 mL,
23.8 mmol), IBCF (2.60 mL, 20.2 mmol), and benzylamine (2.10 mL, 19.3
mmol) gave the crude product that was purified twice by flash column
chromatography (SiO2; 1:20 EtOAc/hexanes) to give the desired com-
pound (2.32 g, 66%) as a white solid: mp 54ꢀ55 ꢀC (lit.46 mp
(C14H22N2O 0.16H2O): C, H, N.
3
(R)-N0-Phenylbutyl 2-Amino-3-methylbutanamide ((R)-14).
Utilizing method A with (R)-43 (3.18 g, 9.13 mmol), TFA (10.2 mL, 137
mmol), and CH2Cl2 (30 mL) gave the crude product after basic workup
that was further purified by flash column chromatography (SiO2; 1:20
EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give the desired
1
47.5ꢀ48.5 ꢀC); Rf 0.80 (1:1 EtOAc/hexanes); H NMR (400 MHz,
CDCl3) δ 0.91 (t, J = 7.2 Hz, 3H), 1.15 (d, J = 6.2 Hz, 3H), 1.39ꢀ1.50 (m,
1H), 1.64ꢀ1.75 (m, 1H), 2.09ꢀ2.17 (m, 1H), 4.39ꢀ4.49 (m, 2H),
5.79ꢀ5.89 (br s, 1H), 7.26ꢀ7.35 (m, 5H); HRMS (ESI) 214.1199
[M+Na+] (calcdforC12H17NOH+ 214.1208); Anal. (C12H17NO): C, H, N.
(S)-N-Benzyl 2-Methylbutanamide ((S)-21).47 Utilizing meth-
od B with (S)-48 (0.90 mL, 8.27 mmol), NMM (1.18 mL, 10.8 mmol),
IBCF (1.17 mL, 9.10 mmol), and benzylamine (0.95 mL, 8.6 mmol) gave
the crude product that was purified twice by flash column chromato-
graphy (SiO2; 1:20 EtOAc/hexanes) to give the desired compound (1.01 g,
64%) as a white solid: mp 55ꢀ56 ꢀC; [α]28D +15.5ꢀ (c 1.0, acetone) (lit.47
[α]D +16.96ꢀ (c 1.0, acetone)); Rf 0.80 (1:1 EtOAc/hexanes); 1H
NMR (400 MHz, CDCl3) δ 0.91 (t, J = 7.6 Hz, 3H), 1.16 (d, J =
7.6 Hz, 3H), 1.40ꢀ1.50 (m, 1H), 1.65ꢀ1.75 (m, 1H), 2.09ꢀ2.18 (m, 1H),
4.39ꢀ4.49 (m, 2H), 5.80ꢀ5.88 (br s, 1H), 7.25ꢀ7.35 (m, 5H);
LRMS (ESI) 193.16 [M + H+] (calcd for C12H17NOH+ 193.16); Anal.
(C12H17NO): C, H, N.
compound (2.16 g, 96%) as a pale yellow oil: [α]28.5 +29.9ꢀ (c 1.0,
D
CHCl3); Rf 0.53 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ
0.81 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 1.51ꢀ1.59 (m, 4H),
1.62ꢀ1.70 (m, 2H), 2.22ꢀ2.34 (m, 1H), 2.63 (t, J = 8.0 Hz, 2H), 3.20 (d,
J = 4.0 Hz, 1H), 3.22ꢀ3.44 (m, 2H), 7.15ꢀ7.19 (m, 3H), 7.25ꢀ7.36 (m,
3H); HRMS (ESI) 249.1954 [M + H+] (calcd for C15H24N2OH+
249.1967); Anal. (C15H24N2O 0.6CH2Cl2): C, H, N.
3
(R)-20-N0-(Trifluoromethoxy)benzyl 2-Amino-3-methylbu-
tanamide ((R)-15). Utilizing method A with (R)-44 (6.00 g, 15.4
mmol), TFA (17.1 mL, 231 mmol), and CH2Cl2 (50 mL) gave the crude
product after acidic workup that was further purified by flash column
chromatography (SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/
CH2Cl2) to give the desired compound (3.70 g, 83%) as a pale yellow
solid: mp 54ꢀ55 ꢀC; [α]28.5 +28.0ꢀ (c 1.0, CHCl3); Rf 0.59 (1:20
(R)-N-Benzyl 2-Hydroxybutanamide ((R)-22). To anhydrous
toluene (80 mL) were added (R)-49 (1.70 g, 16.3 mmol), benzylamine
(1.78 mL, 16.3 mmol), and 3,5-bis(trifluoromethyl)benzene boronic
acid (50) (0.42 g, 1.6 mmol). A pressure equalizing dropping funnel
containing a cotton plug was filled 1/3 of the way with 3 Å molecular
sieves that were oven-dried (120 ꢀC) and a condenser was placed above
the dropping funnel. The mixture was heated at reflux (18 h) before
cooling to room temperature, and then the solvent was evaporated in
vacuo. The crude product was purified by flash column chromatography
(SiO2; 1:20ꢀ1:1 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2)
followed by recrystallization from hot EtOAc/hexanes to give the
D
MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 0.81 (d, J = 6.8 Hz,
3H), 0.98 (d, J = 6.8 Hz, 3H), 1.34 (s, 2H), 2.28ꢀ2.39 (m, 1H), 3.27 (d,
J = 4.0 Hz, 1H), 4.48ꢀ4.57 (m, 2H), 7.22ꢀ7.33 (m, 3H), 7.41ꢀ7.43 (m,
1H), 7.71ꢀ7.79 (br t, 1H); LRMS (ESI) 291.12 [M + H+] (calcd for
C13H17F3N2O2H+ 291.12); Anal. (C13H17F3N2O2): C, H, F, N.
(R)-30-N0-(Trifluoromethoxy)benzyl 2-Amino-3-methylbuta-
namide ((R)-16). Utilizing method A with (R)-45 (3.00 g, 7.69 mmol),
TFA (8.57 mL, 115 mmol), and CH2Cl2 (25 mL) gave the crude product
after acidic workup that was further purified by flash column chro-
matography (SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/
CH2Cl2) to give the desired compound (1.81 g, 81%) as a pale yellow
desired compound (1.05 g, 33%) as a white solid: mp 63ꢀ64 ꢀC;
1
[α]28.5 +28.3ꢀ (c 2.2, CHCl3); Rf 0.56 (1:20 MeOH/CH2Cl2); H
oil: [α]28.5 +20.6ꢀ (c 1.3, CHCl3); Rf 0.47 (1:20 MeOH/CH2Cl2);
D
D
1H NMR (400 MHz, CDCl3) δ 0.83 (d, J = 7.0 Hz, 3H), 1.00 (d, J = 7.0
Hz, 3H), 1.38 (s, 2H), 2.31ꢀ2.40 (m, 1H), 3.30 (d, J = 4.0 Hz, 1H),
4.43 (dd, J = 6.2, 15.0 Hz, 1H), 4.51 (dd, J = 6.6, 15.0 Hz, 1H),
7.10ꢀ7.13 (m, 2H), 7.21ꢀ7.22 (m, 1H), 7.34 (t, J = 8.0 Hz, 1H),
7.78ꢀ7.86 (br t, 1H); LRMS (ESI) 291.11 [M + H+] (calcd for
C13H17F3N2O2H+ 291.11); Anal. (C13H17F3N2O4): C, H, F, N.
(R)-N0-Cyclohexylmethyl 2-Amino-3-methylbutanamide
((R)-18). Utilizing method A with (R)-46 (3.60 g, 11.5 mmol), TFA
(12.8 mL, 173 mmol), and CH2Cl2 (38 mL) gave the crude product after
acidic workup that was further purified by flash column chromatography
(SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give
the desired compound (2.34 g, 96%) as a white solid: mp 85ꢀ86 ꢀC;
[α]25D +38.1ꢀ (c 1.1, CHCl3); Rf 0.59 (1:20 MeOH/CH2Cl2); 1H NMR
(400 MHz, CDCl3) δ 0.82 (d, J = 7.0 Hz, 3H), 0.89ꢀ0.99 (m, 2H), 0.99
NMR (400 MHz, CDCl3) δ 0.96 (t, J = 7.6 Hz, 3H), 1.63ꢀ1.74 (m,
1H), 1.82ꢀ1.92 (m, 1H), 3.32 (d, J = 4.8 Hz, 1H), 4.07ꢀ4.11 (m, 1H),
4.38ꢀ4.48 (m, 2H), 6.95ꢀ7.20 (br t, 1H), 7.24ꢀ7.34 (m, 5H); LRMS
(ESI) 216.12 [M + Na+] (calcd for C11H15NO2Na+ 216.12); Anal.
(C11H15NO2): C, H, N.
(R)-N0-Benzyl N-(Methyl)amino-2-methylbutanamide Hy-
drochloride ((R)-23). HCl in dioxane (4 mL, 4 M) was added at 0 ꢀC
to an Et2O (1 mL) solution of (R)-53 (620 mg, 4.7 mmol). The reaction
was stirred at room temperature (16 h) and then concentrated in vacuo.
The residue was triturated with Et2O to give the desired compound
(450 mg, 90%) as a white solid: mp 120ꢀ125 ꢀC; [α]27D +93.8ꢀ (c 0.5,
CH3OH); 1H NMR (400 MHz, CD3OD) δ 1.02 (d, J = 7.2 Hz, 3H),
1.05 (d, J = 6.9 Hz, 3H), 2.18ꢀ2.24 (m, 1H), 2.65 (s, 3H), 3.62 (d, J = 5.1
Hz, 1H), 4.40 (1/2 ABq, J = 15.0 Hz, 1H), 4.50 (1/2 ABq, J = 15.0 Hz,
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Journal of Medicinal Chemistry
ARTICLE
1H), 7.28ꢀ7.34 (m, 5H); HRMS (ESI) 221.1654 [M+H+] (calcd for
C13H20N2OH+ 221.1653); Anal. (C13H20N2O): C, H, Cl, N.
or male albino SpragueꢀDawley rats (po) and consisted of the MES test
(mice and rats) and the subcutaneous pentylenetetrazol (metrazol)
(scMet) seizure threshold test5 to assess anticonvulsant activity (mice),
the rotorod test to assess neurological toxicity (mice), and the positional
sense test or gait and stance test to assess behavioral toxicity (rats).5
(R)-N0-Benzyl N,N-Dimethylamino-3-methylbutanamide
((R)-24). (R)-55 (2.28 g, 15.7 mmol) and benzylamine (2.06 mL,
18.9 mmol) were added to anhydrous THF (160 mL) at room
temperature. The mixture was stirred (15 min), and then DMTMM
(5.22 g, 18.9 mmol) was added in one portion. The reaction continued at
room temperature overnight (18 h), and then the insoluble salts were
filtered, evaporated in vacuo, and purified by flash column chromatog-
raphy (SiO2; 2:1 EtOAc/CH2Cl2 followed by 1:10 MeOH/CH2Cl2) to
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures and spec-
b
tral properties (IR, H and 13C NMR, MS) for the synthetic
1
give the desired product (0.38 g, 10%) as a white solid: mp 78ꢀ79 ꢀC;
intermediates and the PAADs, table of elemental analyses
(Supporting Information Table S1) and MS spectra (Supporting
Information Table S2). This material is available free of charge via
the Internet at http://pubs.acs.org.
1
[α]28 ꢀ11.2ꢀ (c 0.5, CHCl3); Rf 0.55 (1:20 MeOH/CH2Cl2); H
D
NMR (400 MHz, CDCl3) δ 0.89 (d, J = 6.6 Hz, 3H), 1.03 (d, J = 6.6 Hz,
3H), 2.04ꢀ2.17 (m, 1H), 2.25 (s, 6H), 2.48 (d, J = 6.0 Hz, 1H),
4.42ꢀ4.51 (m, 2H), 6.58ꢀ6.65 (br t, 1H), 7.25ꢀ7.35 (m, 5H); LRMS
(ESI) 235.17 [M + H+] (calcd for C14H22N2OH+ 235.17); Anal.
(C14H22N2O): C, H, N.
’ AUTHOR INFORMATION
(R)-20-N0-(Fluoro)phenethyl 2-Amino-3-methylbutanamide
((R)-56). Utilizing method A with (R)-20-N0-(fluoro)phenethyl 2-N-(tert-
butoxycarbonyl)amino-3-methylbutanamide (9.00 g, 26.6 mmol), TFA
(30 mL, 0.40 mol), and CH2Cl2 (90 mL) gave the crude product after
acidic workup that was further purified by flash column chromatography
(SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give
the desired compound (5.76 g, 91%) as a white solid: mp 51ꢀ52 ꢀC;
[α]28D +32.6ꢀ (c 1.5, CHCl3); Rf 0.26 (1:20 MeOH/CH2Cl2); 1H NMR
(400 MHz, CDCl3) δ0.77 (d, J= 7.0 Hz, 3H), 0.95 (d, J= 7.0 Hz, 3H), 1.25
(s, 2H), 2.24ꢀ2.32 (m, 1H), 2.82ꢀ2.93 (m, 2H), 3.19 (d, J = 3.6 Hz, 1H),
3.46ꢀ3.61 (m, 2H), 6.99ꢀ7.09 (m, 2H), 7.17ꢀ7.23 (m, 2H), 7.34ꢀ7.42
(br t, 1H); LRMS (ESI) 239.13 [M + H+] (calcd for C13H19FN2OH+
239.13); Anal. (C13H19FN2O): C, H, F, N.
(R)-30-N0-(Fluoro)phenethyl 2-Amino-3-methylbutanamide
((R)-57). Utilizing method A with (R)-30-N-(fluoro)phenethyl 2-N-(tert-
butoxycarbonyl)amino-3-methylbutanamide (8.50 g, 25.1 mmol), TFA
(28 mL, 0.38 mol), and CH2Cl2 (85 mL) gave the crude product after
acidic workup that was further purified by flash column chromatography
(SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give the
desired compound (5.08 g, 85%) as a pale yellow oil: [α]28D +31.4ꢀ (c 1.1,
CHCl3); Rf 0.26 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ
0.77 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 1.23 (s, 2H), 2.23ꢀ2.34
(m, 1H), 2.77ꢀ2.88 (m, 2H), 3.20 (d, J = 4.0 Hz, 1H), 3.45ꢀ3.61 (m, 2H),
6.89ꢀ6.93 (m, 2H), 6.97ꢀ6.99 (m, 1H), 7.23ꢀ7.28 (m, 1H), 7.34ꢀ7.44
(br t, 1H); HRMS (ESI) 239.1553 [M + H+] (calcd for C13H19FN2OH+
Corresponding Author
*E-mail: hkohn@email.unc.edu, tel: 919-843-8112, fax: 919-966-
0204.
’ ACKNOWLEDGMENT
The project was supported by UCB Pharma (Braine-l’Alleud,
Belgium) with the assistance of Dr. Robert Kramer. We thank
Dr. Christophe Salomꢀe for preparing (R)-23. The authors thank
the NINDS and the ASP at the National Institutes of Health with
Drs. Tracy Chen and Jeffrey Jiang, for kindly performing the
pharmacological studies via the ASP’s contract site at the
University of Utah with Drs. H. Wolfe, H. S. White, and K.
Wilcox. Harold Kohn has a royalty-stake position in (R)-26.
’ ABBREVIATIONS
AED, antiepileptic drug; ASP, Anticonvulsant Screening Program;
CMDT, 2-chloro-4,6-dimethoxy-1,3,5-triazine; CNS, central nervous
system; DMAP, 4-dimethylaminopyridine; DMTMM, 4-(4,6-di-
methoxy-1,3,5-triazin-2-yl)-4-methylmorpholinium chloride; ED50,
effective dose (50%); FAA, functionalized amino acid; FAK,
functionalized amido ketone; ip, intraperitoneally; MAC, mixed
anhydride coupling; MES, maximal electroshock seizure; NINDS,
National Institute of Neurological Disorders and Stroke; PAAD,
primary amino acid derivative; PI, protective index; po, orally;
SAAD, secondary amino acid derivative; SAR, structureꢀactivity
relationship; scMet, subcutaneous metrazol; TD50, neurological
impairment (toxicity, 50%); TAAD, tertiary amino acid deriva-
tive; TFA, trifluoroacetic acid
239.1560); Anal. (C13H19FN2O 0.01H2O): C, H, F, N.
3
(R)-40-N0-(Fluoro)phenethyl 2-Amino-3-methylbutanamide
((R)-58). Utilizing method A with (R)-40-N0-(fluoro)phenethyl 2-N-(tert-
butoxycarbonyl)amino-3-methylbutanamide (9.00 g, 26.6 mmol), TFA
(30 mL, 0.40 mol), and CH2Cl2 (90 mL) gave the crude product after
acidic workup that was further purified by flash column chromatography
(SiO2; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) to give the
desired compound (5.82 g, 92%) as a pale yellow oil: [α]28D +31.8ꢀ (c 1.5,
CHCl3); Rf 0.19 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ
0.77 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 1.23 (s, 2H), 2.22ꢀ2.33
(m, 1H), 2.74ꢀ2.85 (m, 2H), 3.19 (d, J = 3.6 Hz, 1H), 3.42ꢀ3.59 (m, 2H),
6.95ꢀ7.00 (m, 2H), 7.13ꢀ7.18 (m, 2H), 7.34ꢀ7.42 (br t, 1H); LRMS
(ESI) 239.13 [M + H+] (calcd for C13H19FN2OH+ 239.13); Anal.
(C13H19FN2O): C, H, F, N.
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