Primary amino acid derivatives: Substitution of the 4′-N′- benzylamide site in (R)-N′-benzyl 2-amino-3-methylbutanamide, (R)-N′-benzyl 2-amino-3,3-dimethylbutanamide, and (R)-N′-benzyl 2-amino-3-methoxypropionamide provides potent anticonvulsants with pain-attenuating properties

Article abstract of DOI:10.1021/jm200759t

Recently, we reported that select N′-benzyl 2-substituted 2-amino acetamides (primary amino acid derivatives (PAADs)) exhibited pronounced activities in established whole animal anticonvulsant (i.e., maximal electroshock seizure (MES)) and neuropathic pain (i.e., formalin) models. The anticonvulsant activities of C(2)-hydrocarbon N′-benzyl 2-amino acetamides (MES ED₅₀ = 13-21 mg/kg) exceeded those of phenobarbital (ED ₅₀ = 22 mg/kg). Two additional studies defining the structure-activity relationship of PAADs are presented in this issue of the journal. In this study, we demonstrated that the anticonvulsant activities of (R)-N′-benzyl 2-amino-3-methylbutanamide and (R)-N′-benzyl 2-amino-3,3-dimethylbutanamide were sensitive to substituents at the 4′-N′-benzylamide site; electron-withdrawing groups retained activity, electron-donating groups led to a loss of activity, and incorporating either a 3-fluorobenzyloxy or 3-fluorophenoxymethyl group using a rationally designed multiple ligand approach improved activity. Additionally, we showed that substituents at the 4′-N′-benzylamide site of (R)-N′-benzyl 2-amino-3-methoxypropionamide also improved anticonvulsant activity, with the 3-fluorophenoxymethyl group providing the largest (～4-fold) increase in activity (ED₅₀ = 8.9 mg/kg), a value that surpassed phenytoin (ED₅₀ = 9.5 mg/kg). Collectively, the pharmacological findings provided new information that C(2)-hydrocarbon PAADs represent a novel class of anticonvulsants.

Full text of DOI:10.1021/jm200759t

ARTICLE
pubs.acs.org/jmc
Primary Amino Acid Derivatives: Substitution of the 40-N0-Benzylamide
Site in (R)-N0-Benzyl 2-Amino-3-methylbutanamide, (R)-N0-Benzyl
2-Amino-3,3-dimethylbutanamide, and (R)-N0-Benzyl 2-Amino-
3-methoxypropionamide Provides Potent Anticonvulsants with
Pain-Attenuating Properties
Amber M. King,† Christophe Salomꢀe,† Elise Salomꢀe-Grosjean,† 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-l0Alleud, 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: Recently, we reported that select N0-benzyl
2-substituted 2-amino acetamides (primary amino acid deriva-
tives (PAADs)) exhibited pronounced activities in established
whole animal anticonvulsant (i.e., maximal electroshock seizure
(MES)) and neuropathic pain (i.e., formalin) models. The
anticonvulsant activities of C(2)-hydrocarbon N0-benzyl 2-ami-
no acetamides (MES ED50 = 13ꢀ21 mg/kg) exceeded those of
phenobarbital (ED50 = 22 mg/kg). Two additional studies
defining the structureꢀactivity relationship of PAADs are
presented in this issue of the journal. In this study, we demon-
strated that the anticonvulsant activities of (R)-N0-benzyl 2-amino-3-methylbutanamide and (R)-N0-benzyl 2-amino-3,3-dimethyl-
butanamide were sensitive to substituents at the 40-N0-benzylamide site; electron-withdrawing groups retained activity, electron-
donating groups led to a loss of activity, and incorporating either a 3-fluorobenzyloxy or 3-fluorophenoxymethyl group using a
rationally designed multiple ligand approach improved activity. Additionally, we showed that substituents at the 40-N0-benzylamide
site of (R)-N0-benzyl 2-amino-3-methoxypropionamide also improved anticonvulsant activity, with the 3-fluorophenoxymethyl
group providing the largest (∼4-fold) increase in activity (ED50 = 8.9 mg/kg), a value that surpassed phenytoin (ED50 = 9.5 mg/kg).
Collectively, the pharmacological findings provided new information that C(2)-hydrocarbon PAADs represent a novel class of
anticonvulsants.
’ INTRODUCTION
We recently reported7 that select primary amino acid deri-
vatives (PAADs, 1) displayed potent activities in established
animal models of seizures (maximal electroshock seizure
(MES)8)9 and neuropathic pain (formalin).10 Our investigation
of PAAD activity was prompted by the excellent pharmacological
properties observed for their N-acetylated counterparts, the
functionalized amino acids (FAAs, 2),11ꢀ22 from which lacosa-
mide (LCM, (R)-3)12 emerged as a first-in-class AED that is
currently marketed in the US and Europe for the adjuvant
treatment of partial-onset seizures in adults.23 Several patch-clamp
electrophysiology studies have shown that (R)-3 enhances slow
Epilepsy and neuropathic pain are chronic neurological dis-
orders that arise from dysregulations in neuronal function,
including neuronal hyperexcitability and hypersynchronous neu-
ronal firing.1,2 Pharmacological management remains the pri-
mary treatment option,3 and the similar pathophysiology of these
disorders result in the clinical application of antiepileptic drugs
(AEDs) to treat neuropathic pain.4 Although newer generation
AEDs have made improvements in patient care, pharmacoresis-
tance (failure to respond to two first-line AEDs)5 and adverse
side effects (e.g., drowsiness, dizziness, nausea)6 limit their thera-
peutic usefulness. The development of new compounds with a
novel mechanism of action may circumvent the shortcomings of
current AEDs.
Received: June 13, 2011
Published: August 23, 2011
r
2011 American Chemical Society
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inactivation of voltage-gated sodium channels without affecting
fast inactivation, a pharmacological process that helps to regulate
the firing frequency of hyperexcitable neurons.24ꢀ26 We found
that the structureꢀactivity relationship (SAR) for 1 and 2 differed,
suggesting that their underlying mechanism(s) of action might
differ as well. The C(2)-hydrocarbon PAADs (4) were the most
active compounds in this study, and they surpassed the activities
of the corresponding C(3)-O-alkoxy PAADs (5). This trend was
unexpected because anticonvulsant activity improved in the FAA
series when a substituted heteroatom was incorporated one atom
removed from the chiral C(2) center.11,12,17ꢀ19
Figure 1. 40-N0-Benzylamide-substituted C(2)-hydrocarbon PAADs
(series 1).
Figure 2. 40-N0-Benzylamide-substituted C(3)-O-methoxy PAADs
(series 2).
In this study, we ask whether 40-substitution of the N0-
benzylamide moiety in C(2)-hydrocarbon PAADs 4 and C(3)-
O-alkoxy PAADs 5, to give PAADs 6 and 7, would lead to
improved pharmacological activity. We assessed anticonvulsant
activity using the MES and 6 Hz psychomotor assays and, when
possible, evaluated pain attenuation in the late (chronic) phase of
the formalin neuropathic pain model. The 40-substituents (R0)
tested included small electron-donating (ED) or electron-with-
drawing (EW) groups, as well as more complex groups using a
rationally designed multiple ligand (DML) approach.27 The
combination of two ligands within a single agent to produce
dual or chimeric agents has been used in a number of drug design
programs28ꢀ33 and has been shown to affectively modulate
multiple targets at the same time (polypharmacology). In our
study, we overlapped the commonalities of PAADs (1) and α-
aminoamides (AAAs, 8), a class of amino acid-based anti-
convulsants that exhibit excellent activity in various animal
seizure models,34,35 to create chimeric PAADs. The archetypal
AAA, safinamide ((S)-9), was initially discovered because of its anti-
convulsant activity, and it has been advanced for the treatment of
Parkinson0s disease.34,36ꢀ38
in which EW groups largely retained excellent anticonvulsant
activity and ED groups were inactive. We noted that while none
of the 40-EW substituents for 6 increased the anticonvulsant
activity compared with the parent, unsubstituted PAADs, activity
was enhanced through the DML strategy for PAADs 6 and 7. The
activity of the chimeric PAADs rivaled that found for most
clinical AEDs. Moreover, where testing permitted, we showed
that 40-N0-benzylamide-substituted PAADs also exhibited potent
activity in the formalin model of neuropathic pain.
’ RESULTS AND DISCUSSION
Choice of Compounds. The 40-unsubstituted N0-benzyl-
amide PAADs (R)-10ꢀ12 served as the parent compounds in
this study (Figures 1 and 2). We systematically modified the
40-N0-benzylamide position according to four categories: 40-N0-
benzylamide-substituted C(2)-hydrocarbon PAADs ((R)-13ꢀ23)
(series 1, Figure 1); 40-N0-benzylamide-substituted C(3)-O-
methoxy PAADs ((R)-24ꢀ27) (series 2, Figure 2); 40-chimeric
C(2)-hydrocarbon PAADs ((R)-28ꢀ32) (series 3, Figure 3);
40-chimeric C(3)-O-methoxy PAADs ((R)-33 and (R)-34)
(series 4, Figure 3). Our initial study documented that the
C(2) stereochemical preference corresponded to the D-amino
acid ((R)-configuration).7 Accordingly, we prepared the
40-N0-benzylamide-substituted PAADs in the (R)-configuration.
Within the 40-N0-benzylamide-substituted C(2)-hydrocarbon
PAADs (series 1), we focused on C(2)-isopropyl and C(2)-tert-
butyl derivatives because the parent, unsubstituted PAADs
(R)-10 (ED50 = 15 mg/kg) and (R)-11 (ED50 = 14 mg/kg)
exhibited excellent activities in the MES test (mice, ip).7 For
C(2)-isopropyl PAADs, the 40-N0-benzylamide position (R0) of
(R)-10 was systematically modified with a fluoro, chloro, methyl,
trifluoromethyl, methoxy, trifluoromethoxy, and phenyl group
We demonstrated that PAAD anticonvulsant activity (MES
test) for the C(2)-hydrocarbon PAADs 6 was markedly affected
by the electronic properties of the 40-N0-benzylamide substituent,
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activity of chimeric PAADs resided predominantly in the (R)-
stereoisomer.
Chemistry. The 40-N0-benzylamide-substituted C(2)-hydro-
carbon PAADs (R)-13ꢀ23 were synthesized by coupling either
(R)-357 or (R)-367 with commercially available 40-substituted
benzylamines (37ꢀ43) using the standard mixed anhydride
coupling (MAC) procedure,39 followed by trifluoroacetic acid
(TFA) deprotection (Scheme 1).
The 40-N0-benzylamide-substituted C(3)-O-methoxy PAADs
(R)-2422 and (R)-2722 were prepared by TFA deprotection of
the tBoc-protected amides (R)-5522 and (R)-58,22 while catalytic
removal (10% PdꢀC, H2) ofthe2-N-(benzyloxycarbonyl) group in
Cbz-protected amides (R)-5622 and (R)-5722 gave PAADs (R)-25
and (R)-26.
Figure 3. 40-Chimeric PAADs (series 3 and 4).
The 40-chimeric C(2)-hydrocarbon PAADs (R)-28, (R)-29,
(R)-30, (S)-30, (R)-31,40 and (R)-32 were synthesized from
either (R)-59, (R)-35, (S)-35, or (R)-36 following standard
MAC procedures and using the appropriate 40-modified benzyl-
amines 6140 or 6240 (Scheme 2). PAADs (R)-33, (R)-34,
and (S)-34 were synthesized in the same manner beginning with
(R)- and (S)-60, except alkylation of (R)-68,40 (R)-69,40 and
(S)-69 using methyl iodide and Ag2O gave (R)-70,40 (R)-71,40
and (S)-71, respectively, before TFA deprotection to the corre-
sponding PAADs.
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 prepared compounds and
their physical and spectroscopic properties.
Pharmacological Activity. PAADs 13ꢀ34 were evaluated for
anticonvulsant activity using the MES test at either UCB Pharma,
following the procedures described by Klitgaard,41 or at the
National Institute of Neurological Disorders and Stroke Anti-
convulsant Screening Program (NINDS ASP), following the
procedures described by Stables and Kupferberg,8 or both.
Anticonvulsant activity using the 6 Hz test was performed either
at UCB Pharma, following the procedures described by Kaminski
and co-workers (44 mA),42 or at the NINDS ASP, following the
procedures described by Stables and Kupferberg (32 mA),8 or
both. Several PAADs evaluated at UCB Pharma and the NINDS
ASP were also tested in the formalin model of neuropathic
pain.10,43 The formalin model of neuropathic pain is an effective
tool to prescreen compounds for analgesic activity. Using drugs
with pain-attenuating effects in humans, Vissers and co-workers
demonstrated a good correlation between the findings in the second
phase of the formalin test and the results for cold allodynia in the
chronic constriction injury (CCI) model for both rats (r = 0.72)
and gerbils (r = 0.68).44 Therefore, the formalin model is
((R)-13ꢀ19) (Figure 1). Similarly, in the C(2)-tert-butyl PAAD
set, the 40-N0-benzylamide position of (R)-11 was modified with
a chloro, trifluoromethyl, trifluoromethoxy, and phenyl group
((R)-20ꢀ23). This series permitted us to assess the importance
of size, electronics, and hydrophobic interactions of the 40-
substituents on anticonvulsant activity.
A smaller series of 40-N0-benzylamide-substituted C(3)-O-
methoxy PAADs (series 2) was prepared because we had
observed a lower level of anticonvulsant activity in the MES test
(mice, ip) for (R)-12 (ED50 = 34 mg/kg) than for either (R)-10
(ED50 = 15 mg/kg) or (R)-11 (ED50 = 14 mg/kg).7 Accordingly,
we systematically modified the 40-N0-benzylamide position (R0)
of (R)-12 with a bromo, trifluoromethyl, trifluoromethoxy, and
phenyl group ((R)-24ꢀ27) (Figure 2). The compounds were
purified and tested as their hydrochloride (HCl) salts.
Our pharmacological data for (R)-13ꢀ27 indictated that small
substituents at the 40-N0-benzylamide position of C(2)-hydro-
carbon and C(3)-O-methoxy PAADs affected the anticonvulsant
activity. In the third and fourth series of 40-N0-benzylamide-
substituted PAADs, we evaluated the effect of a substantially
larger substituent utilizing a DML approach in an effort to
improve anticonvulsant activity and neuropathic pain attenua-
tion. We overlaid the N-benzylamino unit in PAADs (1) and
AAAs (8) (see structural unit in box in 1 and (S)-9 in Figure 3) to
create chimeric PAADs (R)-28ꢀ34. Each chimeric PAAD was
defined by the R and R0 moieties, where R consisted of the PAAD
functionality and R0 consisted of the AAA functionality. In series 3,
R was a hydrocarbon (CH3, CH(CH3)2, or C(CH3)3), and in
series 4, R was CH2OCH3. In both series, R0 was either a 3-fluor-
obenzyloxy group or a 3-fluorophenoxymethyl group. All chi-
meric PAADs were synthesized in the (R)-configuration. The
excellent activities of (R)-30 and (R)-34 prompted the synthesis
of the (S)-stereoisomers to determine if the anticonvulsant
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Scheme 1. Synthesis of 40-N0-Benzylamide-Substituted Hydrocarbon PAADs (R)-13ꢀ23
comparable with other well-characterized models of neuropathic
pain (e.g., CCI) and is advantageous due to the ease of admin-
istration and standardization. All compounds were administered
intraperitoneally (ip) to mice at UCB Pharma and the NINDS
ASP or orally (po) to rats at the NINDS ASP. The pharma-
cological data from the MES, 6 Hz, and formalin tests are
summarized in Tables 1ꢀ4. The tables list the results obtained
from either qualitative (dose range) or quantitative (ED50)
testing in mice (ip) and rats (po). We also included qualitative
(dose range) or median neurological impairing dose (TD50)
values using the rotorod tes in mice (ip) and the positional
sense test or gait and stance test in rats (po). ED50 and TD50
values were calculated from doseꢀresponse curves containing
4ꢀ6 dose points (n = 8ꢀ10 per dose). The protective indices
(PI = TD50/ED50) were provided, when applicable. PAADs
tested at the NINDS ASP were evaluated in the subcutaneous
metrazol (scMet) seizure model8 and displayed little to no
protection.45
when possible. We gauged the importance of electronic effects,
hydrophobic interactions, and size of the 40-substituent in C(2)-
hydrocarbon PAADs (R)-13ꢀ23 (series 1) on anticonvulsant
activity in mice (Table 1). The MES activity for the 40-chloro-
substituted C(2)-isopropyl ((R)-14) and C(2)-tert-butyl ((R)-20)
PAADs were similar (ED50 (mg/kg): (R)-14, 22; (R)-20, 25)
but both were slightly lower than their respective parent com-
pounds (R)-10 and (R)-11 (ED50 (mg/kg):(R)-10,15;(R)-11, 14).
The 40-fluoro derivative (R)-13 was evaluated in the isopropyl
series and displayed slightly lower activity than the 40-chloro
derivative (R)-14 (ED50 (mg/kg): (R)-13, 32; (R)-14, 22). There-
fore, the EW effects of halogens (F and Cl) slightly decreased
anticonvulsant activity compared with the unsubstituted parent
compounds but, nonetheless, provided substantial anticonvul-
sant activity. Halogenated PAADs (R)-13, (R)-14, and (R)-20
displayed significant activity in the formalin test (ED50 (mg/kg):
(R)-13, 32; (R)-14, 22; (R)-20, 25) (Table 1). In rats (Table 1),
the 40-fluoro-substituted isopropyl PAAD (R)-13 resulted in a
2-fold decrease in seizure protection from the parent PAAD (R)-
10 (ED50 (mg/kg): (R)-10, 11; (R)-13, 21). Next, we compared
the 40-methyl-substitued C(2)-isopropyl PAAD (R)-15 and the
40-trifluoromethyl analogue (R)-16 in mice. The ED 40-methyl
moiety resulted in the complete loss of anticonvulsant activity in
the MES test (ED50 = >300 mg/kg), while the EW 40-trifluoro-
methyl moiety displayed excellent activity (ED50 = 14 mg/kg)
(Table 1). Similar results were obtained when we evaluated
the 40-methoxy ((R)-17) and 40-trifluoromethoxy ((R)-18)
The C(2)-hydrocarbon PAADs (R)-10 (ED50 = 15 mg/kg)7
and (R)-11 (ED50 = 14 mg/kg)7 displayed excellent activity in
the MES test that surpassed the activity of the traditional
antiepileptic phenobarbital (ED50 = 22 mg/kg).46 Additionally,
(R)-10 displayed significant activity (ED50 = 15 mg/kg) in the
late (chronic) phase of the formalin model of neuropathic pain.
Therefore, we chose to expand the SAR of (R)-10 and (R)-11 to
include functionalization of the N0-benzylamide moiety in an attempt
to optimize anticonvulsant activity and assess pain attenuation,
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Scheme 2. Synthesis of 40-Chimeric PAADs (R)-28ꢀ34, (S)-30, and (S)-34
derivatives in the C(2)-isopropyl series (ED50 (mg/kg): (R)-17,
>300; (R)-18, 16). The 40-trifluoromethyl ((R)-21) and 40-
trifluoromethoxy ((R)-22) compounds also displayed significant
anticonvulsant activities in the C(2)-tert-butyl series (ED50 (mg/
kg): (R)-21, 24; (R)-22, 28), although to a lesser degree than
found in the isopropyl series, and were less active than the parent
compound (R)-11 (ED50 = 14 mg/kg). (R)-16, (R)-18, (R)-21,
and (R)-22 also showed substantial MES activity in the rat (ED50
(mg/kg): (R)-16, 13; (R)-18, 18; (R)-21, <30; (R)-22, 23)
(Table 1). Additionally, the 40-trifluoromethoxy PAAD (R)-18
showed excellent activity in the formalin test (ED50 = 16 mg/kg)
(Table 1). Finally, the 40-phenyl-substituted derivatives (R)-19
and (R)-23 displayed moderate anticonvulsant activity in mice
(ED50 = >30, <100 mg/kg).
incorporating fluorine substituents in CNS agents has been
previously noted.48 The greatest activity was observed in
C(2)-isopropyl PAADs containing a CF3 ((R)-16) or OCF3
((R)-18) group at the 40-N-benzyl position. When the corre-
sponding nonfluorinated moieties CH3 ((R)-15) and OCH3
((R)-17) were incorporated at this position, the compounds
were inactive, suggesting that electronic factors and, to a lesser
extent, hydrophobic interactions (defined by their π-value)47
(Supporting Information Table S1) may also have contributed
to their anticonvulsant activity. However, there was not a
direct correlation between hydrophobicity and pharmacolo-
gical activity.
The correlation between the pharmacological activities of
C(2)-hydrocarbon PAADs and the electronic properties of the
40-substitutent was unexpected. In a related study, we showed
that for 40-substituted derivatives of the FAA (R)-3, anticonvul-
sant activity did not correlate with the electronic properties of the
40-group, and that both ED and EW substituents displayed
excellent protection in the MES model.22 The sensitivity
of C(2)-hydrocarbon PAADs to the electronic properties of
the 40-N-benzylamide substituent differentiates the SAR of our
series from that reported for the FAA (R)-3.
Our data indicated that the electronic properties of 40-N0-
benzylamide substituents impacted pharmacological activity to a
greater extent than the hydrophobic properties. EW 40-substit-
uents (defined as a positive Hammett σp value)47 (Supporting
Information Table S1) maintained seizure protection while ED
groups (defined as a negative Hammett σp value) at this site
dramatically decreased activity. Many, but not all, of the EW
moieties contained fluorine substituents, and the benefit of
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Table 2. Pharmacological Activities of 40-N0-Benzylamide-Substituted C(3)-O-Methoxy PAADs (Series 2) in Mice (mg/kg)a
ARTICLE
compd
R0
MES,b ED50
6 Hz,c ED50
Formalin, ED50
Tox,d TD50
PI,e MES
PI,e Formalin
1.6
(R)-12
(R)-24
H
34
31
>67
>180
55
NDf
59 (MAD)g
>67
70
>120
115
>3.5
3.7
Br
(R)-25
CF3
OCF3
C6H5
19
>31
NDf
>33
15
48
2.5
(R)-26
>10, <30
15
>30, <100
NDf
(R)-27
LCMh ((R)-3i)
3.3
10
19
5.8
1.3
a The compounds were administered intraperitoneally to adult male NMRI mice under the auspices of UCB. ED50 and TD50 values are in mg/kg (4ꢀ6
doses tested, n = 10 per dose) and were determined 30 min after ip administration. b MES = maximal electroshock seizure test. c 6 Hz test = psychomotor
seizure model (44 mA). d Tox = neurological toxicity. TD50 value determined from the rotorod test. e PI = protective index (TD50/ED50). f ND = not
determined. g MAD = minimal active dose. h LCM = lacosamide. i Reference 7.
The neurological activities for 40-N0-benzylamide-substituted
C(3)-O-methoxy PAADs (R)-24ꢀ27 (series 2) in mice are listed
in Table 2. We systematically evaluated the effect of a bromo,
trifluoromethyl, trifluoromethoxy, and phenyl group placed at
the 40-N0-benzylamide position on anticonvulsant activity and
pain attenuation. The MES activities of all 40-N0-benzylamide-
substituted C(3)-O-methoxy PAADs increased over the parent,
unsubstituted PAAD (R)-12 (ED50 = 34 mg/kg). The
40-phenyl derivative (R)-27 (ED50 = 15 mg/kg) and the
40-trifluoromethyl derivative (R)-25 (ED50 = 19 mg/kg) dis-
played the highest MES activities, a ∼2-fold improvement
40-N0-benzylamide position (R0) in C(2)-methyl, C(2)-isopro-
pyl, C(2)-tert-butyl (series 3), or C(3)-O-methoxy (series 4)
PAADs affected pharmacological activity. When R0 was 3-fluor-
obenzyloxy, we observed that most R moieties provided com-
pounds with excellent anticonvulsant activity in the MES test,
and the C(2)-tert-butyl displayed diminished activity (i.e., C(2)-
methyl (R)-28, ED50 = 17 mg/kg; C(2)-isopropyl (R)-29,
ED50 = 12 mg/kg; C(2)-tert-butyl (R)-31, ED50 = >30, <100
mg/kg; C(2)-CH2OCH3 (R)-33, ED50 = 15 mg/kg) (Table 3).
Similarly, when R0 was 3-fluorophenoxymethyl, we observed
decreased MES activity for the C(2)-tert-butyl (R)-32 (ED50 = >30,
from (R)-12. However, the 40-bromo derivative (R)-24 (ED50
=
<100 mg/kg), while the C(2)-isopropyl (R)-30 (ED50 =
31 mg/kg) showed marginally improved anticonvulsant activity.
While PAADs (R)-24ꢀ27 displayed significant activity in the
MES test, they were 2ꢀ6-fold less sensitive to the 6 Hz test. This
result is not surprising because a similar decrease in sensitivity
(∼3-fold) was observed for (R)-3 (MES ED50 = 3.3 mg/kg, 6 Hz
ED50 = 10 mg/kg),49 and a >2-fold decrease in sensitivity was
observed for (R)-12 (MES ED50 = 34 mg/kg, 6 Hz ED50 >67
mg/kg).7 None of the 40-N0-benzylamide-substituted C(3)-O-
methoxy PAADs displayed appreciable activity in the formalin
test at the tested doses. Comparing MES activities of (R)-24ꢀ27
with their corresponding FAAs22 revealed a consistent drop in
activity (2ꢀ10-fold) as we went from FAA to PAAD (Supporting
Information Table S2). This drop was similar to the activity drop
observed for the unsubstituted N0-benzylamide C(3)-alkoxy
PAADs and FAAs.7 Therefore, we concluded that placement of
a small substituent at the 40-N0-benzylamide position of C(3)-O-
methoxy PAADs moderately improved the anticonvulsant activ-
ity of PAADs in the MES test but does not provide an advantage
over C(2)-hydrocarbon PAADs or FAAs.
18 mg/kg) and C(2)-CH2OCH3 (R)-34 (ED50 = ∼10 mg/kg)
exhibited potent anticonvulsant activity. This general pattern for
the R group was observed when the compounds were tested in
the rat (po) (Table 4). The C(2)-tert-butyl derivatives (R)-31
and (R)-32 exhibited no or modest MES activity in rats at the
doses tested (ED50 (mg/kg): (R)-31, >30; (R)-32, ∼30), while
for a C(2)-CH2OCH3 group we saw excellent activity, irrespec-
tive of the ether orientation in the R0 unit (ED50 (mg/kg): (R)-33,
13; (R)-34, 12).
The near equivalence in anticonvulsant activity of the C(2)-
isopropyl ((R)-29, (R)-30) and C(3)-O-methoxy ((R)-33, (R)-
34) groups is unique to chimeric PAADs. In the unsubstituted
PAADs (R)-10 and (R)-12, we observed that C(2)-isopropyl
(R)-10 was 2.3-fold more active than C(3)-O-methoxy (R)-12
(ED50 (mg/kg): (R)-10, 15; (R)-12, 34) (Tables 1 and 2). This
activity difference was even greater in the corresponding FAA
chimeric series, wherein the terminal amine in the PAAD had
been acetylated (Table 5). Here, the C(2)-isopropyl deriva-
tive (R)-74 was inactive, while the C(3)-O-methoxy com-
pound (R)-76 displayed excellent anticonvulsant activity (ED50
(mg/kg): (R)-74, >300; (R)-76, 13). The comparable activity
of the C(2)-isopropyl ((R)-29, (R)-30) and C(3)-O-methoxy
((R)-33, (R)-34) chimeric PAADs may be due to a DML
effect, where adding either a 3-fluorobenzyloxy or a 3-fluor-
ophenoxymethyl pharmacophore at the 40-N-benzylamide
position on the (R)-12 PAAD backbone significantly improved
the PAAD anticonvulsant activity. A similar but smaller boost
was observed for C(2)-isopropyl PAAD (R)-10 when these
pharmacophores were included to give PAADs (R)-29 and
(R)-30 (Table 3).
Next, we evaluated the neurological activities of 40-N0-benzyl-
amide chimeric PAADs (R)-28ꢀ34 (series 3 and 4) in mice
(Table 3) and rats (Table 4). The data was first analyzed from the
standpoint of R, allowing us to identify the optimal R substituent,
irrespective of the R0 unit. Then, we examined the effect of R0 on
activity; we kept R the same, permitting us to see if there was a
preference in the ether linkage orientation of R0 (-OCH2- versus -
CH2O-). Finally, we evaluated the stereochemical preference for
40-N0-benzylamide chimeric PAADs 30 and 34.
Using this approach, we determined how introducing either
a 3-fluorobenzyloxy or a 3-fluorophenoxymethyl moiety at the
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Table 3. Pharmacological Activity of 40-N0-Benzylamide Chimeric PAADs (Series 3 and 4) in Mice (mg/kg)a
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Table 3. Continued
a The compounds were administered intraperitoneally to adult male NMRI mice under the auspices of UCB or administered intraperitoneally to adult
male albino CF-1 mice under the auspices of the NINDS ASP. ED50 and TD50 values are in mg/kg (4ꢀ6 doses tested, n = 10 per dose) and were
determined 30 min after ip administration (UCB) or 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)
(NINDS ASP). Numbers in parentheses are 95% confidence intervals. b MES = maximal electroshock seizure test. c 6 Hz test = psychomotor seizure
e
model (44 mA, UCB; 32 mA, NINDS ASP). d Tox = neurological toxicity. TD50 value determined from the rotorod test. PI = protective index
(TD50/ED50). f ND = not determined. g MAD = minimal active dose. h Single dose experiments where the mg/kg used is followed by the percentage
protected in parentheses.
Lastly, we examined the C(2)-isopropyl, C(2)-tert-butyl, and
C(3)-O-methoxy PAADs that contained either a 3-fluorobenzy-
loxy or a 3-fluorophenoxymethyl moiety (R0) to determine if
there was a preferred ether linker orientation between the two
aromatic rings. Each R set (C(2)-isopropyl, C(2)-tert-butyl,
C(2)-CH2OCH3) displayed similar MES activities in mice for
the two ether linkages (-OCH2- ED50 (mg/kg): (R)-29, 12;
(R)-31, >30, <100; (R)-33, 15; and -CH2O- ED50 (mg/kg): (R)-
30, 12; (R)-32, >30, <100; (R)-34, 8.9) (Table 3). The formalin
activity was evaluated for the C(2)-CH2OCH3 PAAD (R)-34
(37 mg/kg (94% reduction)), but the lack of data for (R)-31,
(R)-32, and (R)-33 prevented any generalized statements. There-
fore, on the basis of the available data, both 3-fluorobenzyloxy
and 3-fluorophenoxymethyl moieties at the 40-N0-benzylamide
position of C(2)-isopropyl ((R)-29 and (R)-30) and C(2)-
CH2OCH3 ((R)-33 and (R)-34) PAADs showed increased MES
activity over the corresponding, unsubstituted parent compounds
((R)-10 and (R)-12).
Comparison of the MES activities of (R)-30 with (S)-30 and
(R)-34 with (S)-34 revealed that the higher activity was asso-
ciated with the (R)-isomer (ED50 (mg/kg): (R)-30, 18; (S)-30,
>30, <100; (R)-34, ∼10; (S)-34, >30, <100). This was not
surprising because similar trends were observed in the unsub-
stituted PAADs (ED50 (mg/kg): (R)-72, >10, <30; (S)-72, >300;
(R)-12, 34; (S)-12, 64; (R)-10, 15; (S)-10, >300; (R)-11, 14;
(S)-11, 42)7 and in the FAA series (ED50 (mg/kg): (R)-76, 13;
(S)-76, >300).22
and electronic properties of the 40-N0-benzylamide substituent
further distinguished PAADs from FAA (R)-3, where activity was
independent of electronic factors.22
Incorporation of the AAA pharmacophore at the 40-N0-ben-
zylamide position of PAADs resulted in potent anticonvulsants,
with both the 40-chimeric C(2)-isopropyl PAAD (R)-30 and
C(3)-O-methoxy PAAD (R)-34 displaying superb anticonvul-
sant activity in the MES model (ED50 = e12 mg/kg). This SAR
pattern was unique to PAADs because in the corresponding
N-acetylated series, the C(2)-hydrocarbon 40-chimeric deriva-
tives (R)-74 and (R)-75 were devoid of anticonvulsant activity
(ED50 = >300 mg/kg), while the C(3)-O-methoxy 40-chimeric
compounds (R)-76 and (R)-77 exhibited excellent activity
(ED50 = e13 mg/kg).40 When considering seizure protection,
toxicity, the protective indices, and the amount of compound
administered in μmol/kg, chimeric PAAD (R)-34 displayed
superior anticonvulsant activity (MES ED50 = 8.9 mg/kg; UCB
value), which approached the therapeutic capabilities of the
clinical AED (R)-3 (MES ED50 = 3.3 mg/kg). Using the
conventional mg/kg dose, it was 2.7-fold less active than (R)-3,
but it exhibited nearly the same PI value as (R)-3 (PI: (R)-34, 5.4;
(R)-3, 5.8). When the ED50 values are converted to μmol/kg, the
activity difference between (R)-34 and (R)-3 reduced to 2.0-fold
(∼25% increase).
The sensitivity of the C(2)-hydrocarbon PAADs to the
electronic properties of the 40-N0-benzylamide substituent pro-
vided an important difference in the SAR of this series of
compounds compared with the FAAs. Additionally, the excellent
anticonvulsant activity observed for both the C(2)-hydrocarbon
and C(3)-O-methoxy chimeric PAADs also differientates this
SAR from that of the FAAs. We recognize that the observed
differences in the PAAD and FAA SAR may be attributed
to changes in pharmacokinetic parameters (e.g., bioavailabil-
ity, metabolism), but these findings, along with the earlier
observation7 that the anticonvulsant activity for hydrocarbon
PAADs did not improve when a substituted heteroatom was
included one atom removed from the C(2)-center, suggests
that the C(2)-hydrocarbon PAADs have a unique SAR and
possibly a mechanism(s) of action that differs from FAAs and
other PAADs.
’ CONCLUSIONS
We evaluated 24 C(2)-hydrocarbon and C(3)-O-methoxy
PAADs that included a 40-N0-benzylamide substituent in whole
animal seizure models (MES, 6 Hz psychomotor) and, in some
cases, the formalin model of neuropathic pain. The 40-N0-benzyl-
amide substituents included either EW or ED moieties, or struc-
tural units found in AAAs (8). We discovered that the pharma-
cological activity of C(2)-hydrocarbon PAADs was highly
dependent upon the electronic properties of the 40-substituent,
where 40-EW groups provided compounds with excellent activity
(ED50 = <30 mg/kg) and 40-ED groups provided compounds
that were inactive. The correlation between anticonvulsant activity
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Table 4. Pharmacological Activity of 40-N0-Benzylamide Chimeric PAADs (Series 3 and 4) in Rats (mg/kg)a
a 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. 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 MES = maximal electroshock seizure test. c Tox = behavioral toxicity. d PI = protective index (TD50/ED50).
’ EXPERIMENTAL SECTION
(15.5 mL, 209 mmol), and CH2Cl2 (46 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 (2.73 g, 87%) as a white solid: mp 86ꢀ87 ꢀC;
[α]25D +32.9ꢀ (c 1.1, CHCl3); Rf 0.47 (1:20 MeOH/CH2Cl2); 1H NMR
(400 MHz, CDCl3) δ0.82 (d, J=6.8Hz, 3H), 0.99(d, J= 6.8 Hz, 3H), 1.35
(s, 2H), 2.29ꢀ2.41 (m, 1H), 3.27 (d, J = 3.2 Hz, 1H), 4.39 (dd, J = 6.0, 15.0
Hz, 1H), 4.45 (dd, J = 6.0, 14.8 Hz, 1H), 6.97ꢀ7.03 (m, 2H), 7.23ꢀ7.27
(m, 2H), 7.64ꢀ7.72 (br t, 1H); LRMS (ESI) 225.13 [M + H+] (calcd for
C12H17FN2OH+ 225.13); Anal. (C12H17FN2O): C, H, F, N.
General Methods. The general methods used in this study were
identical to those previously reported7 and are summarized in the
1
Supporting Information. All compounds were checked by TLC, H
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 the purity of the products was g95%.
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 chromatography
(SiO2). Basic: The crude product was diluted with CH2Cl2 andwashedwith
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 chromatography (SiO2).
(R)-N0-40-Fluorobenzyl 2-Amino-3-methylbutanamide
((R)-13). Utilizing Method A with (R)-44 (4.50 g, 13.9 mmol), TFA
(R)-N0-40-Chlorobenzyl 2-Amino-3-methylbutanamide
((R)-14). Utilizing Method A with (R)-45 (2.86 g, 8.41 mmol), TFA
(6.09 mL, 82.0 mmol), and CH2Cl2 (18 mL) gave the crude product
after acidic workup that was further purified by flash column chroma-
tography (SiO2; 1:20ꢀ1:1 EtOAc/hexanes followed by 1:10 MeOH/
CH2Cl2) to give the desired compound (1.52 g, 76%) as a white solid:
mp 72ꢀ73 ꢀC; [α]28.5D +26.7ꢀ (c 1.1, CHCl3); Rf 0.26 (1:20 MeOH/
CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 0.82 (d, J = 7.2 Hz, 3H), 0.99
(d, J = 6.4 Hz, 3H), 1.30 (s, 2H), 2.28ꢀ2.37 (m, 1H), 3.27 (d, J =
3.6 Hz, 1H), 4.37 (dd, J = 6.0, 14.8 Hz, 1H), 4.43 (dd, J = 6.0, 14.8 Hz,
1H), 7.21 (d, J = 8.2 Hz, 2H), 7.28 (d, J = 8.2 Hz, 2H), 7.72ꢀ7.80 (br t,
1H); LRMS (ESI) 241.12 [M + H+] (calcd for C12H17ClN2OH+
241.12); Anal. (C12H17ClN2O): C, H, Cl, N.
(R)-N0-40-Methylbenzyl 2-Amino-3-methylbutanamide
((R)-15). Utilizing Method A with (R)-46 (3.50 g, 10.9 mmol),
TFA (12.2 mL, 164 mmol), and CH2Cl2 (36 mL) gave the crude
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Table 5. Comparison of the Pharmacological Activities of 40-N0-Benzylamide Chimeric FAAs and Their PAAD Counterparts in
Mice (mg/kg)a
R
X
Y
FAA compd no.
FAA MES,b ED50
FAA Tox,c TD50
PAAD compd no. PAAD MES,b ED50
PAAD Tox,c TD50
CH3
O
O
O
O
O
CH2
CH2
CH2
CH2
CH2
O
(R)-73d,e
(R)-74d,e
(R)-75d,e
(R)-76d,e
(S)-76d,e
(R)-77d,e
(S)-77d,e
>30, <100
>300
>300
>300
(R)-28f
(R)-29f
(R)-31e
(R)-33e
(S)-33e
(R)-34f
(S)-34e
17
12
NDg
NDg
CH(CH3)2
C(CH3)3
CH2OCH3
CH2OCH3
>300
>300
>30, <100 [0.5]
15 [0.5] (13ꢀ17)
NDg
∼300 [0.5]
58 [0.25] (53ꢀ62)
NDg
13 [0.25] (11ꢀ16)
>300
26 [0.5] (21ꢀ34)
>300
CH2OCH3 CH2
5.9 [0.25] (4.3ꢀ7.3) 10 [0.25] (9.1ꢀ13)
8.9
46
CH2OCH3 CH2
O
NDg NDg
>30, <100
>100, <300
a The compounds were administered intraperitoneally to adult male NMRI mice under the auspices of UCB or administered intraperitoneally to adult
male albino CF-1 mice under the auspices of the NINDS ASP. ED50 and TD50 values are in mg/kg (4ꢀ6 doses tested, n = 10 per dose) and were
determined 30 min after ip administration (UCB) or 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)
(NINDS ASP). Numbers in parentheses are 95% confidence intervals. b MES = maximal electroshock seizure test. c Tox = neurological toxicity. TD50
value determined from the rotorod test. d Reference 40. e The compounds were administered intraperitoneally to adult male albino CF-1 mice under the
auspices of the NINDS ASP. f The compounds were administered intraperitoneally to adult male NMRI mice under the auspices of UCB. g ND = not
determined.
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 (1.70 g, 71%) as a
white solid: mp 67ꢀ68 ꢀC; [α]28.5D +26.7ꢀ (c 1.1, CHCl3); Rf 0.53
(1:10 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 0.83 (d, J =
7.0 Hz, 3H), 0.99 (d, J = 7.0 Hz, 3H), 1.33 (s, 2H), 2.29ꢀ2.40 (m, 1H),
2.33 (s, 3H), 3.27 (d, J = 4.0 Hz, 1H), 4.38 (dd, J = 6.0, 14.8 Hz, 1H), 4.44
(dd, J = 6.0, 14.8 Hz, 1H), 7.13 (d, J = 8.4 Hz, 2H), 7.17 (d, J = 8.4 Hz,
2H), 7.54ꢀ7.62 (br t, 1H); HRMS (ESI) 221.1664 [M + H+] (calcd for
C13H20N2OH+ 221.1654); Anal. (C13H20N2O•0.05H2O): C, H, N.
(R)-N0-40-(Trifluoromethyl)benzyl 2-Amino-3-methylbuta-
namide ((R)-16). Utilizing Method A with (R)-47 (4.00 g, 10.7 mmol),
TFA (11.9 mL, 161 mmol), and CH2Cl2 (35 mL) gave the crude product
after acidic workup that was further purified by flash column chromatog-
raphy (SiO2; 1:10ꢀ1:1 EtOAc/hexanes followed by 1:10 MeOH/
CH2Cl2) to give the desired compound (2.18 g, 75%) as a white solid:
mp 86ꢀ87 ꢀC; [α]28.5D +26.0ꢀ (c 1.0, CHCl3); Rf 0.37 (1:20 MeOH/
CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 0.83 (d, J = 7.2 Hz, 3H), 1.00
(d, J = 7.2 Hz, 3H), 1.32 (s, 2H), 2.30ꢀ2.41 (m, 1H), 3.30 (d, J = 4.0 Hz,
1H), 4.47 (dd, J = 6.0, 15.4 Hz, 1H), 4.53 (dd, J = 6.0, 15.4 Hz, 1H), 7.39
(d, J = 7.6 Hz, 2H), 7.57 (d, J = 7.6 Hz, 2H), 7.84ꢀ7.92 (br t, 1H); LRMS
(ESI) 275.14 [M + H+] (calcd for C13H17F3N2OH+ 275.14); Anal.
(C13H17F3N2O): C, H, F, N.
LRMS (ESI) 237.17 [M + H+] (calcd for C13H20N2O2H+ 237.17);
Anal. (C13H20N2O2): C, H, N.
(R)-N0-40-(Trifluoromethoxy)benzyl 2-Amino-3-methylbu-
tanamide ((R)-18). Utilizing Method A with (R)-49 (3.20 g, 8.20
mmol), TFA (9.14 mL, 123 mmol), and CH2Cl2 (27 mL) gave the crude
product after acidic workup that was further purified by flash column
chromatography (SiO2; 1:20ꢀ1:1 EtOAc/hexanes followed by 1:10
MeOH/CH2Cl2) to give the desired compound (1.33 g, 56%) as a pale
yellow solid: mp 63ꢀ64 ꢀC; [α]28.5 +26.6ꢀ (c 1.0, CHCl3); Rf 0.47
D
(1:20 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 0.83 (d, J = 7.2
Hz, 3H), 1.00 (d, J = 7.2 Hz, 3H), 1.36 (s, 2H), 2.33ꢀ2.41 (m, 1H), 3.30
(d, J = 3.2 Hz, 1H), 4.40ꢀ4.51 (m, 2H), 7.17 (d, J = 8.4 Hz, 2H), 7.31 (d,
J = 8.4 Hz, 2H), 7.70ꢀ7.80 (br t, 1H); LRMS (ESI) 291.15 [M + H+]
(calcd for C13H17F3N2O2H+ 291.15); Anal. (C13H17F3N2O2): C, H, F, N.
(R)-N0-(Biphenyl-40-yl)methyl 2-Amino-3-methylbutana-
mide ((R)-19).50 Utilizing Method A with (R)-50 (2.51 g, 6.57 mmol),
TFA (7.32 mL, 98.5 mmol), and CH2Cl2 (22 mL) gave the crude
product after basic workup that was further purified by flash column
chromatography (SiO2; 1:10ꢀ1:1 EtOAc/hexanes) to give the desired
compound (1.81 g, 98%) as a white solid: mp 96ꢀ97 ꢀC; [α]25D +16.5ꢀ
(c 1.0, CH2Cl2); Rf 0.28 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 0.85 (d, J = 7.0 Hz, 3H), 1.01 (d, J = 7.0 Hz, 3H), 1.45 (s, 2H),
2.33ꢀ2.42 (m, 1H), 3.32 (d, J = 3.2 Hz, 1H), 4.45ꢀ4.55 (m, 2H),
7.32ꢀ7.37 (m, 3H), 7.43 (m, 2H), 7.56 (m, 4H), 7.62ꢀ7.68 (br t, 1H);
HRMS (ESI) 283.1800 [M + H+] (calcd for C18H22N2OH+ 283.1810);
Anal. (C18H22N2O•0.07CH2Cl2): C, H, N.
(R)-N0-40-Methoxybenzyl 2-Amino-3-methylbutanamide
((R)-17). Utilizing Method A with (R)-48 (4.50 g, 13.4 mmol), TFA
(14.9 mL, 201 mmol), and CH2Cl2 (45 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
(R)-N0-40-Chlorobenzyl 2-Amino-3,3-dimethylbutanamide
((R)-20). Utilizing Method A with (R)-51 (2.50 g, 7.06 mmol), TFA
(7.86 mL, 106 mmol), and CH2Cl2 (23 mL) gave the crude product after
acidic workup that was further purified by flash column chromatography
(SiO2; 1:100ꢀ1:10 MeOH/CH2Cl2) to give the desired compound
the desired compound (3.04 g, 96%) as a white solid: mp 81ꢀ82 ꢀC;
1
[α]28.5 +25.5ꢀ (c 1.1, CHCl3); Rf 0.42 (1:20 MeOH/CH2Cl2); H
D
NMR (400 MHz, CDCl3) δ 0.83 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.0 Hz,
3H), 1.33 (s, 2H), 2.28ꢀ2.40 (m, 1H), 3.26 (d, J = 3.6 Hz, 1H), 3.79 (s,
3H), 4.36 (dd, J = 5.6, 14.6 Hz, 1H), 4.41 (dd, J = 6.0, 14.6 Hz, 1H), 6.86
(d, J = 8.6 Hz, 2H), 7.21 (d, J = 8.6 Hz, 2H), 7.52ꢀ7.58 (br t, 1H);
(1.31 g, 73%) as a white solid: mp 78ꢀ79 ꢀC; [α]25 +14.2ꢀ (c 1.0,
D
CH2Cl2); Rf 0.23 (1:20 MeOH/CH2Cl2); 1H NMR (300 MHz, CDCl3)
δ 1.00 (s, 9H), 1.47 (s, 2H), 3.14 (s, 1H), 4.36 (1/2 ABq, J = 17.6 Hz, 1H),
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4.43 (1/2 ABq, J = 17.6 Hz, 1H), 7.22 (d, J = 8.6 Hz, 2H), 7.29 (d, J =
8.6 Hz, 2H); HRMS (ESI) 255.1256 [M + H+] (calcd for C13H19ClN2-
OH+ 255.1264); Anal. (C13H19ClN2O): C, H, Cl, N.
was evaporated in vacuo to obtain a yellow solid. The solid was dissolved
in Et2O (10 mL) and HCl (1.0 M in Et2O, 5 mL) was added dropwise.
The resulting mixture was stirred (5 min), and the white precipitate was
filtered and dried to give the desired compound as a white solid: mp
169ꢀ170 ꢀC; [α]25D ꢀ4.7ꢀ (c 1, MeOH); Rf 0.08 (EtOAc); 1H NMR
(DMSO-d6) δ 3.31 (s, 3H), 3.72ꢀ3.74 (m, 2H), 4.01ꢀ4.12 (br s, 1H),
4.31ꢀ4.44 (m, 2H), 7.34 (d, J = 8.1 Hz, 2H), 7.40 (d, J = 8.1 Hz, 2H),
8.26ꢀ8.44 (br s, 3H), 9.19ꢀ9.23 (app. t, 1H); HRMS (ESI) 293.1105
[M + H+] (calcd for C12H15F3N2O3H+ 293.1108); Anal. (C12H16-
ClF3N2O3): C, H, Cl, F, N.
(R)-N0-(Biphenyl-40-yl)methyl 2-Amino-3-methoxypropion-
amide Hydrochloride ((R)-27). A saturated HCl solution in dioxane
(1 mmol/2 mL, 11.5 mL) was added to (R)-5822 (2.20 g, 5.73 mmol) at
0 ꢀC, and the solution was stirred at room temperature (12 h). A second
saturated HCl solution in dioxane (1 mmol/2 mL, 11.5 mL) was added,
and the solution was stirred at room temperature (6 h). The white solid
(R)-N0-40-(Trifluoromethyl)benzyl 2-Amino-3,3-dimethyl-
butanamide ((R)-21). Utilizing Method A with (R)-52 (2.50 g,
6.44 mmol), TFA (7.18 mL, 96.6 mmol), and CH2Cl2 (21 mL) gave
the crude product after acidic workup that was further purified by
flash column chromatography (SiO2; 1:10ꢀ1:1 EtOAc/hexanes) to give
the desired compound (0.68 g, 37%) as a white solid: mp 90ꢀ91 ꢀC;
[α]25D +17.2ꢀ (c 1.0, CH2Cl2); Rf 0.14 (1:1 EtOAc/hexanes); 1H NMR
(400 MHz, CDCl3) δ 1.01 (s, 9H), 1.49 (s, 2H), 3.17 (s, 1H), 4.44ꢀ4.54
(m, 2H), 7.29ꢀ7.37 (br t, 1H), 7.40 (d, J = 7.6 Hz, 2H), 7.58 (d, J = 7.6
Hz, 2H); HRMS (+ESI) 289.1515 [M + H+] (calcd. for C14H19F3-
N2OH+ 289.1528); Anal. (C14H19F3N2O): C, H, F, N.
(R)-N0-40-(Trifluoromethoxy)benzyl 2-Amino-3,3-dimethyl-
butanamide ((R)-22). Utilizing Method A with (R)-53 (2.00 g,
4.95 mmol), TFA (5.51 mL, 74.2 mmol), and CH2Cl2 (16 mL) gave
the crude product after acidic workup that was further purified by
flash column chromatography (SiO2; 1:10ꢀ1:1 EtOAc/hexanes) to
give the desired compound (0.93 g, 62%) as a white solid: mp
was filtered to give the desired compound (1.25 g, 70%): mp
1
145ꢀ148 ꢀC; [α]27.0 +33.2ꢀ (c 0.5, H2O); H NMR (DMSO-d6) δ
D
3.32 (s, 3H), 3.75 (d, J = 4.6 Hz, 2H), 4.09 (t, J = 4.6 Hz, 1H), 4.36ꢀ4.44
(m, 2H), 7.34ꢀ7.41 (s, 3H), 7.43ꢀ7.90 (m, 2H), 7.61ꢀ7.68 (m, 4H),
8.31ꢀ8.44 (br s, 3H), 9.22 (t, J = 5.7 Hz, 1H); Anal. (C17H21ClN2O2):
C, H, Cl, N.
68ꢀ69 ꢀC; [α]25 +14.7ꢀ (c 1.1, CH2Cl2); Rf 0.29 (1:1 EtOAc/
D
hexanes); 1H NMR (400 MHz, CDCl3) δ 1.00 (s, 9H), 1.56 (s, 2H),
3.14 (s, 1H), 4.43 (d, J = 6.0 Hz, 2H), 7.17 (d, J = 8.6 Hz, 2H),
7.23ꢀ7.29 (br s, 1H), 7.31 (d, J = 8.6 Hz, 2H); HRMS (ESI)
305.1463 [M + H+] (calcd for C14H19F3N2O2H+ 305.1477); Anal.
(C14H19F3N2O2): C, H, F, N.
(R)-N0-40-(300-Fluorobenzyloxy)benzyl 2-Aminopropiona-
mide Hydrochloride ((R)-28). HCl in dioxane (4.0 M, 2.4 mL,
4.7 mmol) was added to (R)-63 (1.90 g, 4.72 mmol) at room tem-
perature (2 h). The precipitate was filtered to give the desired compound
(1.55 g, 100%) as a white solid: mp 105ꢀ107 ꢀC; [α]26D ꢀ11.4ꢀ (c 0.5,
DMSO); Rf 0.00 (EtOAc); 1H NMR (DMSO-d6) δ 1.37 (s, J = 6.6 Hz,
3H), 3.80ꢀ3.87 (br m, 1H), 5.12 (s, 2H), 6.97 (d, J = 8.1 Hz, 2H),
7.10ꢀ7.30 (m, 5H), 7.40ꢀ7.45 (m, 1H), 8.17ꢀ8.25 (br d, 3H), 8.86ꢀ
8.92 (br s, 1H); HRMS (ESI) 303.1518 [M + H+] (calcd for C17H19-
(R)-N0-(Biphenyl-40-yl)methyl 2-Amino-3,3-dimethylbut-
anamide ((R)-23). Utilizing Method A with (R)-54 (1.53 g, 3.86 mmol),
TFA (4.30 mL, 57.9 mmol), and CH2Cl2 (13 mL) gave the crude product
after basic workup that was further purified by flash column chromatography
(SiO2; 1:10ꢀ1:1 EtOAc/hexanes) to give the desired compound (0.76 g,
67%) as a white solid: mp 75ꢀ76 ꢀC; [α]28.5D +14.4ꢀ (c 1.0, CH2Cl2); Rf
0.50 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 1.03 (s, 9H),
1.50 (s, 2H), 3.16 (s, 1H), 4.44ꢀ4.54 (m, 2H), 7.06ꢀ7.12 (br s, 1H),
7.32ꢀ7.37 (m, 3H), 7.42ꢀ7.45 (m, 2H), 7.55ꢀ7.59 (m, 4H); LRMS (ESI)
297.20 [M + H+] (calcd for C19H24N2OH+ 297.20); Anal. (C19H24N2O):
C, H, N.
(R)-N0-40-Bromobenzyl 2-Amino-3-methoxypropionamide
Hydrochloride ((R)-24). HCl (4.0 M in dioxane, 16 mL) was added
at 0 ꢀC to (R)-5522 (2.24 g, 5.78 mmol), and the solution was stirred at
room temperature overnight. The reaction was concentrated in vacuo and
trituratedwith Et2O to give the desired compound (1.18 g, 63%) as a white
solid: mp 165ꢀ167 ꢀC; [α]25D +1.2ꢀ (c 1, MeOH); Rf 0.13 (EtOAc); 1H
NMR (DMSO-d6) δ 3.30 (s, 3H), 3.67ꢀ3.77 (m, 2H), 4.07 (t, J = 4.8 Hz,
1H), 4.25ꢀ4.39 (m, 2H), 7.25 (d, J = 8.4 Hz, 2H), 7.53 (d, J = 8.4 Hz, 2H),
8.26ꢀ8.42 (br s, 3H), 9.20 (t, J = 5.7 Hz, 1H); LRMS (ESI) 287.0
[M + H+] (100%), 289.1 [M + 2 + H+] (100%) (calcd for C11H1579Br-
N2O2H+ 287.0); Anal. (C11H16ClBrN2O2): C, H, Br, Cl, N.
(R)-N0-40-(Trifluoromethyl)benzyl 2-Amino-3-methoxy-
propionamide Hydrochloride ((R)-25). HCl (4.0 M in dioxane,
16 mL) was added at 0 ꢀC to (R)-5622 (3.00 g, 7.98 mmol), and the
solution was stirred at room temperature overnight. The reaction was
concentrated in vacuo and triturated with Et2O to give the desired
compound (2.28 g, 91%) as a white solid: mp 181 ꢀC; [α]24.5D ꢀ5.14ꢀ
(c 1, MeOH); Rf 0.08 (EtOAc); 1H NMR (DMSO-d6) δ 3.32 (s, 3H),
3.73ꢀ3.75 (m, 2H), 4.08ꢀ4.11 (m, 1H), 4.37ꢀ4.51 (m, 2H), 7.51
(d, J = 8.1 Hz, 2H), 7.71 (d, J = 8.1 Hz, 2H), 8.32 (br s, 3H), 9.28 (br s,
1H); HRMS (ESI) 277.1164 [M + H+] (calcd for C12H15F3N2O2H+
277.1158); Anal. (C12H16ClF3N2O2): C, H, Cl, F, N.
(R)-N0-40-(Trifluoromethoxy)benzyl 2-Amino-3-methoxy-
propionamide Hydrochloride ((R)-26). An EtOH solution
(100 mL) of (R)-5722 (1.00 g, 2.65 mmol) was treated with H2
(1 atm) in presence of 10% PdꢀC (100 mg) at roomtemperature (16 h).
The mixture was carefully filtered through a bed of Celite, and the filtrate
FN2O2H+ 303.1503); Anal. (C17H20ClFN2O2 0.10H20): C, H.
3
(R)-N0-40-(300-Fluorobenzyloxy)benzyl 2-Amino-3-methyl-
butanamide ((R)-29). Utilizing Method A with (R)-64 (2.00 g,
4.65 mmol), TFA (5.18 mL, 69.7 mmol), and CH2Cl2 (15 mL) gave
the crude product after basic workup that was further purified by flash
column chromatography (SiO2; 1:100ꢀ1:10 MeOH/CH2Cl2) to give
the desired compound (481 mg, 32%) as a white solid: mp 77ꢀ78 ꢀC;
1
[α]25 +16.8ꢀ (c 1.0, CH2Cl2); Rf 0.57 (1:20 MeOH/CH2Cl2); H
D
NMR (400 MHz, CDCl3) δ 0.83 (d, J = 6.8 Hz, 3H), 0.99 (d, J = 7.2 Hz,
3H), 1.31ꢀ1.58 (br s, 2H), 2.31ꢀ2.38 (m, 1H), 3.26 (d, J = 2.8 Hz, 1H),
4.33ꢀ4.44 (m, 2H), 5.05 (s, 2H), 6.91 (d, J = 7.2 Hz, 2H), 7.00 (t, J = 8.4
Hz, 1H), 7.13ꢀ7.26 (m, 4H), 7.31ꢀ7.39 (m, 1H), 7.51ꢀ7.59 (br t, 1H);
HRMS (ESI) 331.1827 [M + H+] (calcd for C19H23FN2O2H+
331.1822); Anal. (C19H23FN2O2): C, H, F, N.
(R)-N0-40-(300-Fluorophenoxymethyl)benzyl 2-Amino-3-
methylbutanamide ((R)-30). Utilizing Method A with (R)-65
(3.00 g, 6.97 mmol), TFA (7.77 mL, 105 mmol), and CH2Cl2 (23 mL)
gave the crude product after basic workup that was further purified by
flash column chromatography (SiO2; 1:100ꢀ1:10 MeOH/CH2Cl2) to
give the desired compound (0.92 g, 40%) as a white solid: mp 78ꢀ
79 ꢀC; [α]25D +17.5ꢀ (c 1.1, CH2Cl2); Rf 0.50 (1:20 MeOH/CH2Cl2);
1H NMR (400 MHz, CDCl3) δ 0.84 (d, J = 6.8 Hz, 3H), 1.00 (d, J =
7.2 Hz, 3H), 1.26ꢀ1.58 (br s, 2H), 2.33ꢀ2.41 (m, 1H), 3.29 (d, J =
3.6 Hz, 1H), 4.41ꢀ4.52 (m, 2H), 5.03 (s, 2H), 6.64ꢀ6.69 (m, 2H),
6.73ꢀ6.76 (m, 1H), 7.12ꢀ7.39 (m, 5H), 7.62ꢀ7.84 (br t, 1H); HRMS
(ESI) 331.1828 [M + H+] (calcd for C19H23FN2O2H+ 331.1822); Anal.
(C19H23FN2O2): C, H, F, N.
(S)-N0-40-(300-Fluorophenoxymethyl)benzyl 2-Amino-3-
methylbutanamide ((S)-30). The previous procedure was repeated
using (S)-65 (4.00 g, 9.30 mmol), TFA (10.4 mL, 139 mmol), and
CH2Cl2 (30 mL) to give 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
6428
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Journal of Medicinal Chemistry
ARTICLE
compound (2.67 g, 87%) as a white solid: mp 75ꢀ76 ꢀC; [α]28D ꢀ18.2ꢀ
(c 1.2, CH2Cl2); Rf 0.45 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz,
CDCl3) δ 0.84 (d, J = 7.0 Hz, 3H), 0.99 (d, J = 7.0 Hz, 3H), 1.20ꢀ1.52
(br s, 2H), 2.34ꢀ2.40 (m, 1H), 3.24ꢀ3.34 (br d, 1H), 4.41ꢀ4.52 (m,
2H), 5.02 (s, 2H), 6.64ꢀ6.70 (m, 2H), 6.73ꢀ6.76 (m, 1H), 7.12ꢀ7.25
(m, 1H), 7.30 (d, J = 8.2 Hz, 2H), 7.38 (d, J = 8.2 Hz, 2H), 7.64ꢀ7.70 (br
t, 1H); LRMS (ESI) 331.14 [M + H+] (calcd for C19H23FN2O2H+
331.14); Anal. (C19H23FN2O2): C, H, F, N.
Pharmacology. Compounds were screened under the auspices of
UCB Pharma (Braine L0Alleud, Belgium) and the NINDS ASP
(Rockville, MD). Housing, handling, and feeding were in full accordance
with recommendations contained in the Guide for the Care and Use of
Laboratory Animals.51 Pharmacological evaluation by UCB Pharma con-
sisted of four assays using male NMRI mice (ip): the 6 Hz test42 and the
MES test41 to assess anticonvulsant activity, the formalin test10,43 to
assess neuropathic pain attenuation, and the rotorod test41 to assess
neurological toxicity. Pharmacological evaluation by the NINDS ASP
utilized male albino Carworth Farms No. 1 mice (ip) or male albino
SpragueꢀDawley rats (po) and consisted of the MES test (mice and rats)
and the subcutaneous pentylenetetrazol (metrazol) (scMet) seizure
threshold test8 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).8
(R)-N0-40-(300-Fluorophenoxymethyl)benzyl 2-Amino-3,
3-dimethylbutanamide ((R)-32). Utilizing Method A with (R)-
67 (1.82 g, 4.10 mmol), TFA (4.56 mL, 61.5 mmol), and CH2Cl2
(14 mL) gave the crude product after basic workup that was further
purified by flash column chromatography (SiO2; 1:10ꢀ1:1 EtOAc/
hexanes) to give the desired compound (1.03 g, 73%) as a white solid:
mp 59ꢀ60 ꢀC; [α]25 +9.3ꢀ (c 1.1, CH2Cl2); Rf 0.30 (1:20 MeOH/
D
CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 1.01 (s, 9H), 1.50 (s, 2H),
3.14 (s, 1H), 4.45 (d, J = 4.2 Hz, 2H), 5.03 (s, 2H), 6.65ꢀ6.69 (m, 2H),
6.75 (d, J = 6.0 Hz, 1H), 7.02ꢀ7.14 (br t, 1H), 7.19ꢀ7.25 (m, 1H), 7.31
(d, J = 5.3 Hz, 2H), 7.39 (d, J = 5.3 Hz, 2H); HRMS (ESI) 345.1977
[M + H+] (calcd for C20H25FN2O2H+ 345.1978); Anal. (C20H25-
FN2O2): C, H, F, N.
’ ASSOCIATED CONTENT
S
Supporting Information. Synthetic procedures and spec-
b
tral properties (IR, H and 13C NMR, MS) for the synthetic
1
intermediates and the PAADs, and tables of elemental analyses
(Table S3) and MS spectra (Table S4). This material is available
free of charge via the Internet at http://pubs.acs.org.
(R)-N0-40-(300-Fluorobenzyloxy)benzyl 2-Amino-3-methoxy-
propionamide ((R)-33). Utilizing Method A with (R)-70 (3.00 g,
6.94 mmol), TFA (7.73 mL, 104 mmol), and CH2Cl2 (23 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 product (2.05 g, 89%) as a pale
yellow solid: mp 76ꢀ77 ꢀC; [α]28.5D +7.9ꢀ (c 1.1, CHCl3); Rf 0.74 (1:20
’ AUTHOR INFORMATION
Corresponding Author
*E-mail: hkohn@email.unc.edu, tel: 919-843-8112, fax: 919-
966-0204.
1
MeOH/CH2Cl2); H NMR (400 MHz, CDCl3) δ 1.67 (s, 2H), 3.36
(s, 3H), 3.56ꢀ3.64 (m, 3H), 4.33ꢀ4.44 (m, 2H), 5.04 (s, 2H), 6.91 (d,
J = 8.4 Hz, 2H), 6.98ꢀ7.02 (m, 1H), 7.13ꢀ7.21 (m, 4H), 7.34 (q, J = 8.0
Hz, 1H), 7.68ꢀ7.72 (br t, 1H); LRMS (ESI) 333.17 [M + H+] (calcd for
C18H21FN2O3H+ 333.17); Anal. (C18H21FN2O3): C, H, F, N.
’ ACKNOWLEDGMENT
The project was supported by UCB Pharma (Braine-l0Alleud,
Belgium) with the assistance of Dr. Robert Kramer. The authors
thank the NINDS and the ASP at the National Institutes of
Health with Drs. Tracy Chen and Jeffrey Jiang, for kindly per-
forming the pharmacological studies via the ASP0s 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)-3.
(R)-N0-40-(300-Fluorophenoxymethyl)benzyl 2-Amino-3-
methoxypropionamide ((R)-34). Utilizing Method A with (R)-
71 (2.45 g, 5.67 mmol), TFA (6.32 mL, 85.1 mmol), and CH2Cl2
(19 mL) gave the crude product after basic workup that was further
purified twice by flash column chromatography (SiO2; 1:20ꢀ1:1
EtOAc/hexanes followed by 1:10 MeOH/CH2Cl2) followed by recrys-
tallization from hot EtOAc/hexanes to give the desired product (1.30 g,
69%) as a pale yellow solid: mp 60ꢀ61 ꢀC; [α]25D +6.1ꢀ (c 1.1, CHCl3);
Rf 0.76 (1:20 MeOH/CH2Cl2); 1H NMR (400 MHz, CDCl3) δ 1.63 (s,
2H), 3.38 (s, 3H), 3.59ꢀ3.65 (m, 3H), 4.42ꢀ4.52 (m, 2H), 5.03 (s, 2H),
6.64ꢀ6.69 (m, 2H), 6.75 (d, J = 9.6 Hz, 1H), 7.22 (q, J = 8.4 Hz, 1H),
7.30 (d, J = 7.8 Hz, 2H), 7.38 (d, J = 7.8 Hz, 2H), 7.74ꢀ7.83 (br t, 1H);
LRMS (ESI) 333.17 [M + H+] (calcd for C18H21FN2O3H+ 333.17);
Anal. (C18H21FN2O3): C, H, F, N.
’ ABBREVIATIONS
AAA, α-aminoamide; AED, antiepileptic drug; ASP, Anticonvul-
sant Screening Program; CCI, chronic constriction injury; CNS,
central nervous system; DMLs, designed multiple ligands; ED,
electron-donating; ED50, effective dose (50%); EW, electron-
withdrawing; FAA, functionalized amino acid; HCl, hydrochlor-
ide; 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; SAR, structureꢀactiv-
ity relationship; scMet, subcutaneous metrazol; TD50, neurolo-
gical impairment (toxicity, 50%); TFA, trifluoroacetic acid
(S)-N0-40-(300-Fluorophenoxymethyl)benzyl 2-Amino-3-
methoxypropionamide ((S)-34). The previous procedure
was repeated using (S)-71 (2.71 g, 6.27 mmol), TFA (6.99 mL,
94.1 mmol), and CH2Cl2 (21 mL) to give 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 a mixture of desired product and impurity as an orange oil. The
crude oil obtained after the acid workup was then further purified by
flash column chromatography (SiO2; 1:100ꢀ1:10 MeOH/CH2Cl2)
to give the desired compound (0.27 g, 19%) as a pale yellow solid: mp
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