Primary amino acid derivatives: Compounds with anticonvulsant and neuropathic pain protection activities

ARTICLE

pubs.acs.org/jmc

Primary Amino Acid Derivatives: Compounds with Anticonvulsant

and Neuropathic Pain Protection Activities

Amber M. King,^†Christophe Salomꢀe,^†Jason Dinsmore,^†Elise Salomꢀe-Grosjean,^†Marc De Ryck,^‡

Rafal Kaminski,^‡Anne Valade,^‡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

^‡UCB Pharma SA, CNS Research, Chemin du Foriest, B-1420 Braine-l’Alleud, Belgium

^§Department of Chemistry, University of North Carolina, Chapel Hill, North Carolina 27599-3290, United States

S Supporting Information

b

ABSTRACT: Pharmacological management remains the primary method

to treat epilepsy and neuropathic pain. We have advanced a novel class of

anticonvulsants termed functionalized amino acids (FAAs). In this study,

we examine FAA derivatives from which the terminal acetyl moiety was

removed and termed these compounds primary amino acid derivatives

(PAADs). Twenty-seven PAADs were prepared; the central C(2) R-

substituent was varied, including C(2) stereochemistry, and the compounds

were tested in rodent models of seizures and neuropathic pain. C(2)-

Hydrocarbon N-benzylamide PAADs were potent anticonvulsants and

excellent anticonvulsant activity (mice, ip; rat, po) was observed for C(2)

R-substituted PAADs in which the R group was ethyl, isopropyl, or tert-

butyl, and the C(2) stereochemistry conformed to the ^D-amino acid

conﬁguration ((R)-stereoisomer). These values surpassed the activities

of several clinical antiepileptic drugs. The C(2) (R)-ethyl and C(2) (R)-isopropyl PAADs also displayed excellent activities in the

mouse (ip) formalin neuropathic pain model. Signiﬁcantly, unlike the FAA structureꢀactivity relationship, PAAD anticonvulsant

activity increased upon substitution of a methylene unit for a heteroatom in the R-substituent that was one atom removed from the

C(2) site, suggesting that these PAADs function by a diﬀerent pathway than FAAs.

’ INTRODUCTION

the C(2) carbon and whose activity principally resided in the

(R)-stereoisomer.^4,5,10ꢀ12

Epilepsy is a serious neurological disorder aﬀecting up to 1% of

the world’s population, including more than 2 million Americans.^1,2

Currently, its estimated annual medical cost exceeds $15 billion,

and approximately 140,000 new cases were diagnosed in the US

in 2010.¹Epilepsy is characterized by recurring, unprovoked

seizures that result from neuronal hyperexcitability and hyper-

synchronous neuronal ﬁring²and is commonly, but incorrectly,

deﬁned as a singular disease. More appropriately, epilepsy is a

heterogeneous mixture of disorders with the commonality of

neuronal dysregulations that result from varying external, brain

developmental, or genetic causes.³

We have advanced a novel class of anticonvulsants termed

functionalized amino acids (FAAs, 1).^4ꢀ15The structureꢀactivity

relationship (SAR) for FAAs provided distinctive trends that

permitted our discovery of the lead FAA lacosamide ((R)-2),⁵

which is marketed in the US and Europe for the adjuvant treat-

ment of partial-onset seizures in adults.¹⁶We found that FAA 3,

wherein R¹was methyl and R³was benzyl, provided excellent

anticonvulsant activity in the maximal electroshock seizure (MES)

model. Most important, activity improved when we incorporated

a substituted heteroatom in R²that was one atom removed from

Neuropathic pain aﬀects up to 8% of the world’s population,

and results from dysfunctioning neuronal pathways within the

peripheral nervous system (PNS), the central nervous system

(CNS), or both.^17ꢀ19Thus, it is not surprising that several anti-

epileptic drugs (AEDs) are used to manage neuropathic pain.^20,21

Received: April 11, 2011

Published: June 03, 2011

r

2011 American Chemical Society

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Figure 1. C(2)-(CH₂)_nX(R⁰)_yPAADs.

N-benzylamide substituents. A preliminary SAR study of 4ꢀ6

examined the importance of the C(2) R²-group and the termi-

nal amine substituents. While representative 4ꢀ6 displayed ex-

cellent activity in the MES model, the limited data did not

provide clear trends to warrant further development.³³In this

investigation, we restricted our compound set to 4s that had an

unsubstituted N-benzylamide unit and focused on the central

C(2) R²-substituent, including C(2) stereochemistry. We de-

monstrated that select PAADs exhibited outstanding activities in

established animal models for seizures and neuropathic pain.

Signiﬁcantly, PAADs and FAAs did not share similar SARs;

PAADs that contained a branched hydrocarbon at the C(2)

position displayed excellent activities while the corresponding

FAAs were largely inactive. The implications of these ﬁndings are

discussed.

Figure 2. C(2)-Hydrocarbon PAADs.

(R)-2 has shown signiﬁcant protection in neuropathic models for

inﬂammatory pain, osteoarthritis pain, diabetic neuropathy, bone

cancer pain, chemotherapy-induced pain, and spinal-cord nerve

injury.^22ꢀ28

Pharmacological management remains the primary method to

treat epilepsy and neuropathic pain disorders. Although newer

generation AEDs have improved patient care, it is estimated that

30% of epilepsy patients fail at least two ﬁrst-line AED treat-

ments, so their epilepsy is deemed to be pharmacoresistant. Addi-

tionally, 40% of the patients who do respond to AEDs experience

adverse side eﬀects.^29,30Similarly, neuropathic pain medications

provide 30ꢀ50% pain reduction in approximately 50% of patients.³¹

There remains, therefore, a need for potent new neurological

agents with improved eﬃcacy, decreased toxicity, and favorable

physiochemical and pharmacokinetic properties to better treat

these disorders.

We, and others, have brieﬂy examined FAA derivatives from

which the terminal acetyl moiety was removed.^32ꢀ38We named

these compounds primary amino acid derivatives (PAADs, 4),

secondary amino acid derivatives (SAADs, 5), or tertiary amino

acid derivatives (TAADs, 6), depending on the terminal amine

substitution pattern. We saw potential therapeutic beneﬁts in

these amine derivatives. Chief among these was the protonation

of the terminal amine at physiological pH values to give the cor-

responding ammonium species. We anticipated amine proton-

ation would allow these compounds to exhibit improved water

solubility while permitting greater diversity for the C(2) R²and

’ RESULTS

Choice of Compounds. We used PAAD 4, which contained

an unsubstituted N-benzylamide moiety, as our structural tem-

plate and systematically modified the C(2) R²-position according

to two categories: heteroatom-containing substituents (PAADs

7ꢀ19) (Figure 1) or hydrocarbon substituents (PAADs 20ꢀ28)

(Figure 2). Heteroatom-containing substituents correspond to

the general formula (CH₂)_nX(R⁰)_y, where n equals the number of

methylene carbons, X is an oxygen, nitrogen, or sulfur, R⁰is an

alkyl group, and y is the number of R⁰groups. For select com-

pounds in this series, we compared the (R)-, (S)-, and (R,S)-

stereoisomers.

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Scheme 1. Synthesis of C(3)-Hydroxy PAAD 7 and C(3)-Alkoxy PAADs 8ꢀ10

Scheme 2. Synthesis of Unsaturated C(3)-Alkoxy PAADs (R,S)-11 and (R,S)-12

The heteroatom-containing PAADs were further classiﬁed

into three subcategories (Figure 1): C(3)-alkoxy PAADs (7ꢀ12),

C(3)-amino PAADs (13ꢀ16), and C(4)-substituted PAADs

(17ꢀ19). In the C(3)-alkoxy series, R⁰was successively replaced

with a methyl, ethyl, propyl, allyl, and propargyl group. All C(3)-

alkoxy PAADs were synthesized in the (R,S)-conﬁguration, with

the exception of the C(3)-hydroxy PAAD 7 and the C(3)-

methoxy PAAD 8, which were synthesized in the (R)-, (S)-,

and (R,S)-conﬁgurations. In the C(3)-amino series, we prepared

the parent C(3)-amino PAAD 13, the N⁰,N⁰-dimethylamino

PAAD 15 (y = 2), and morpholino 16. Eﬀorts to prepare the

N⁰-monomethyl PAAD 14 were unsuccessful. All C(3)-amino

PAADs were synthesized in the (R,S)-conﬁguration. In the C(4)-

substituted series, X is oxygen or sulfur, and R⁰is either a hydro-

gen or methyl group (17ꢀ19). All C(4)-substituted PAADs were

synthesized in the (R)-conﬁguration. Eﬀorts to prepare (R)-18

on a scale suﬃcient for testing were unsuccessful.

In the C(2)-hydrocarbon PAADs 20ꢀ28, R²is a linear alkyl

group (20ꢀ23; methyl, ethyl, propyl, and n-butyl), a branched

alkyl group (24ꢀ26; isopropyl, tert-butyl, and 2-methylpropyl),

or a cyclic group (27ꢀ28; cyclohexyl and benzyl) (Figure 2).

Because the C(2)-(CH₂)_nX(R⁰)_yPAADs revealed a preference

for stereochemistry in the ^D-amino acid conﬁguration, all hydro-

carbon PAADs were synthesized as the (R)-stereoisomer, except

PAAD 26, which contained two chiral centers and was a mixture

of four diastereomers. The excellent anticonvulsant activity of

PAADs (R)-24 and (R)-25 prompted the synthesis of the cor-

responding (S)-stereoisomers to determine if pharmacological

activity paralleled the C(2)-(CH₂)_nX(R⁰)_yPAADs, where activ-

ity preferentially resided in the C(2) ^D-amino acid conﬁguration.

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Scheme 3. Pathway for the Synthesis of N⁰,N⁰-Disubstituted C(3)-Amino PAADs (R,S)-15 and (R,S)-16 Utilizing Cbz-Protected

Dehydroalanine Methyl Ester 51^a

^aMixture = mixture of (R,S)-57 and (R,S)-N-methyl 2-N⁰⁰-(benzyloxycarbonyl)amino-3-(N⁰⁰-methylamino)propionamide.

Scheme 4. Synthesis of (R,S)-N-Benzyl 2,3-Diaminopropionamide ((R,S)-13)

Chemistry. C(3)-Hydroxy and C(3)-alkoxy PAADs 7ꢀ10

were synthesized in 3ꢀ4 steps using commercially available reagents

and established synthetic procedures (Scheme 1). Treatment of

(R)-, (S)-, and (R,S)-serine (29) with benzyl chloroformate

under basic conditions gave (R)-, (S)-, and (R,S)-30,³⁹which

were then converted to the amides (R)-, (S)-, and (R,S)-31 using

the mixed anhydride coupling (MAC) procedure,⁴⁰followed by

subsequent hydrogenolysis to obtain the corresponding PAADs

(R)-, (S)-, and (R,S)-7. Alkylation of (R)-, (S)-, and (R,S)-31

using methyl iodide (32) and Ag₂O gave (R)-, (S)-, and (R,S)-

35³⁹before hydrogenolysis to the corresponding PAADs (R)-,

(S)-, and (R,S)-8.^32,33Similarly, treatment of (R,S)-31 with either

ethyl iodide (33) or propyl iodide (34) and Ag₂O gave (R,S)-36

and (R,S)-37, followed by hydrogenolysis to the PAADs (R,S)-9

and (R,S)-10. We observed an increase in reaction time and tem-

perature, as well as a decrease in yield, as the alkyl iodide went

from methyl to ethyl to propyl. A similar finding was observed for

the corresponding FAAs.⁵

We attempted to synthesize (R,S)-11 and (R,S)-12 using allyl

iodide and propargyl iodide under the alkylation conditions from

Scheme 1, but no reaction was observed. Therefore, we prepared

PAADs (R,S)-11 and (R,S)-12 via an aziridine intermediate

(Scheme 2). First, we executed a 3-step procedure beginning

with the N-tritylation of commercially available (R,S)-38, fol-

lowed by cyclodehydration to give (R,S)-40.⁴¹Completion of the

remaining steps required consideration of the sensitivity of the

N-protecting group to BF₃Et₂O and of the sensitivity of the

3

unsaturated alkenyl or alkynyl group to the ﬁnal deprotection

conditions. Aziridines exhibit modest reactivity toward ring-

opening by oxygen nucleophiles, so we incorporated an elec-

tron-withdrawing group at the nitrogen site to enhance ring-

opening.⁴²Thus, deprotection of (R,S)-40 under acidic condi-

tions was followed by reprotection with Boc₂O under basic

conditions to give aziridine (R,S)-41.⁴³Ring-opening with allyl

alcohol (42) or propargyl alcohol (43) in the presence of BF₃Et₂O

3

gave (R,S)-44 and (R,S)-45,⁴⁴which were hydrolyzed to give

(R,S)-46 and (R,S)-47 and then directly used for the MAC

procedure to give amides (R,S)-48 and (R,S)-49. Finally, amides

(R,S)-48 and (R,S)-49 were triﬂuoroacetic acid (TFA) depro-

tected to the corresponding PAADs (R,S)-11 and (R,S)-12.

We prepared the N⁰,N⁰-disubstituted C(3)-amino PAADs (R,

S)-15 and (R,S)-16 using a procedure similar to our earlier

synthesis of racemic 2,3-diaminopropionic acid derivatives.⁴⁵

Accordingly, we generated dehydroalanine 51⁴⁶via the mesyla-

tion and elimination of (R,S)-50 using Et₃N (Scheme 3). Michael

addition of dimethylamine (54) or morpholine (55) to 51 gave

(R,S)-58 and (R,S)-59, which were subsequently hydrolyzed and

directly used in the MAC procedure to give the corresponding

amides (R,S)-60 and (R,S)-61. Deprotection of amides (R,S)-60

and (R,S)-61 by hydrogenolysis gave PAADs (R,S)-15 and (R,S)-

16. Use of ammonia (52) in this protocol was unsuccessful. We

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observed a competing side reaction when methylamine (53) was

employed and could not isolate suﬃcient quantities of (R,S)-57.⁴⁷

To prepare (R,S)-13, we treated commercially available di-

amine (R,S)-62 with Boc₂O under basic conditions to give (R,S)-

63 (Scheme 4).⁴⁸Using the MAC procedure, (R,S)-63 was

coupled with benzylamine to give amide (R,S)-64, followed by

TFA deprotection to give PAAD (R,S)-13.

Evaluation of (R)-, (S)-, and (R,S)-8 indicated that the

pharmacological activity resided largely in the ^D-amino acid con-

ﬁguration (see Pharmacological Activity). Therefore, the C(4)-

substituted PAADs were synthesized as the (R)-stereoisomer.

Synthesis of (R)-19 was achieved in 3 steps, starting with the

N-tBoc protection of commercially available ^D-methionine ((R)-

65),⁴⁹followed by coupling with benzylamine using the MAC

procedure to provide (R)-67, and ﬁnally, TFA deprotected to

give (R)-19 (Scheme 5).

To synthesize the oxygen equivalent of (R)-19 (S f O, (R)-18),

we attempted to follow the synthetic route depicted in Scheme 1

using commercially available ^D-homoserine ((R)-68) to obtain

the C(4)-hydroxy PAAD (R)-17 and the C(4)-methoxy PAAD

(R)-18. However, lactonization during N-Cbz protection of (R)-

68 gave (R)-69, so we modiﬁed our route to take advantage of

(R)-69 (Scheme 6).⁵⁰Treatment of (R)-69 under basic condi-

tions with benzylamine gave amide (R)-70, followed by hydro-

genolysis to give PAAD (R)-17. Several attempts were made

to obtain (R)-18, but large scale eﬀorts to methylate (R)-70 were

unsuccessful.

C(2)-Hydrocarbon PAADs were prepared using a 3-step

procedure beginning with commercially available amino acids

(Scheme 7). (R)-71ꢀ74, (S)-73ꢀ74, (R,S)-75, and (R)-76 were

N-tBoc protected following standard procedures, coupled with

benzylamine using the MAC method, and then TFA deprotected

to give PAADs (R)-21, (R)-23ꢀ25, (S)-24ꢀ25, (R,S)-26, and

(R)-27. PAADs (R)-, (S)-, (R,S)-22 and (R)-28 were prepared

using a N-Cbz protecting group similar to that used to synthesize

(R)-, (S)-, and (R,S)-7 (Scheme 1). Accordingly, (R)-, (S)-, and

(R,S)-norvaline (89) were used to prepare the corresponding

N-Cbz intermediates (R)-, (S)-, and (R,S)-91 and (R)-, (S)-, and

(R,S)-93. Similarly, (R)-phenylglycine ((R)-90) was used to

prepare the corresponding N-Cbz intermediates (R)-92 and

(R)-94 (Scheme 8).

We detail in the Experimental Section 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 7ꢀ13, 15ꢀ17, and

19ꢀ28 were evaluated for anticonvulsant activity using the

MES test at UCB Pharma, following the procedures reported

by Klitgaard,⁵¹or at the National Institute of Neurological Disorders

and Stroke Anticonvulsant Screening Program (NINDS ASP),

following the procedures reported by Stables and Kupferberg,⁵²

or at both. Anticonvulsant activity using the 6 Hz test was per-

formed at UCB Pharma, following the procedures reported by

Kaminski and co-workers (44 mA),⁵³or at the NINDS ASP,

following the procedures reported by Stables and Kupferberg

(32 mA),⁵²or at both. Several PAADs evaluated at UCB Pharma

were tested in the formalin model of neuropathic pain.^54,55

Recently, Visser and co-workers demonstrated a good correla-

tion between findings in the second phase of the formalin test

and results for cold allodynia in the chronic constriction injury

(CCI) model for both rats (r = 0.72) and gerbils (r = 0.68) using

drugs with pain-attenuating effects in humans.⁵⁶Therefore, the

formalin model has an advantage over other well-characterized

models of neuropathic pain (e.g., CCI) due to its ease of

administration and standardization, and is an effective tool to

prescreen compounds for neuropathic pain protection. All

compounds were administered intraperitoneally (ip) to mice at

UCB Pharma or ip to mice and orally (po) to rats at the NINDS

ASP. For compounds that exhibited significant activity, we report

the effective dose (50%, ED₅₀) values obtained in quantitative

screening evaluations. Also provided are the median doses for

neurological impairment (50%, TD₅₀) in mice using the rotorod

test⁵⁷and the behavioral toxicity effects observed in rats. TD₅₀

values were determined for compounds that exhibited significant

activity in the MES test. The pharmacological data from the

MES, 6 Hz, and formalin tests are summarized in Tables 1ꢀ3.

The MES activities of PAADs are compared with those of their

corresponding FAAs in Tables 4, 5, and Supporting Informa-

tion Table S1. The pharmacological data from PAADs synthe-

sized as individual isomers ((R), (S), and (R,S)) are reported in

Tables 1ꢀ3 and then summarized in Supporting Information

Table S2. Several compounds were evaluated at both UCB Pha-

rma and the NINDS ASP and displayed comparable activities

(Supporting Information Table S3). The protective indices (PI =

TD₅₀/ED₅₀) are provided, when applicable. PAADs tested at the

Scheme 5. Synthesis of (R)-N-Benzyl 2-Amino-

4-(methylthio)butanamide ((R)-19)

Scheme 6. Synthesis of (R)-N-Benzyl 2-Amino-4-hydroxybutanamide ((R)-17)

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Scheme 7. Synthesis of C(2)-Hydrocarbon PAADs (R)-21, (R)-23ꢀ25, (S)-24ꢀ25, (R,S)-26, and (R)-27

Scheme 8. Synthesis of (R)-, (S)-, (R,S)-N-Benzyl 2-Aminopentanamide ((R)-, (S)-, (R,S)-22) and (R)-N-Benzyl 2-Amino-3-

phenylpropionamide ((R)-28)

NINDS ASP were evaluated in the subcutaneous Metrazol

(scMet) seizure model, but little or no protection was observed

at the doses (30, 100, 300 mg/kg) and times (0.5 and 4 h) tested

(data not shown).

approximate 2-fold preference, in regard to MES activity, for

the (R)-isomer. This selectivity was considerably lower than that

exerted for (R)-2, where we saw a >22-fold diﬀerence in activity

between the (R) and (S) isomers in mice.⁵Overall, comparing

the MES activities of PAADs 7ꢀ9, 11, and 12 with their

corresponding FAAs (Table 4) revealed a consistent drop in

activity (3ꢀ10-fold) as we went from the FAA to the PAAD. In

all instances, the MES activity was greater than the 6 Hz activity

(44 and 32 mA), and no signiﬁcant pain attenuation was

observed in the formalin assay.

Table 1 also lists the pharmacological activities for the C(3)-

amino N-benzylamide PAADs. Here, we evaluated the unsub-

stituted C(3)-amino PAAD ((R,S)-13), the corresponding sub-

stituted C(3)-amino PAAD with two methyl groups ((R,S)-15),

and the PAAD in which the C(3)-amino was embedded within a

morpholino moiety ((R,S)-16). No signiﬁcant neurological acti-

vity was observed in either anticonvulsant models or the pain

model, and toxicity was not evaluated because there was no activity.

A similar ﬁnding was observed for the C(3)-amino substituted

FAAs (Table 4).

Table 1 lists the neurological activities for the ﬁrst category of

C(2)-substituted PAADs, which included the C(3)-alkoxy,

C(3)-amino, and C(4)-substituted derivatives. Beginning with

the C(3)-oxy N-benzylamide PAADs (R)-, (S)-, and (R,S)-7,

(R)- and (S)-8, and (R,S)-9ꢀ12 in mice, we systematically

evaluated the eﬀect of a hydrogen, methyl, ethyl, propyl, allyl,

and propargyl group placed at the C(3)-oxy terminus (R⁰) on

anticonvulsant activity and pain attenuation. The clinical AEDs

(R)-2,⁵phenytoin,⁵⁸phenobarbital,⁵⁸and valproate⁵⁸served as

the reference compounds. The MES activities of the C(3)-alkoxy

PAADs (R)-, (S)-, and (R,S)-8, and (R,S)-9ꢀ12 remained rela-

tively constant throughout the series, but we observed a slight in-

crease in activity as we went from a saturated alkyl group ((R,S)-

10, ED₅₀= 69 mg/kg) to unsaturated hydrocarbon groups (ED₅₀

(mg/kg): (R,S)-11, 45; (R,S)-12, 46). (R)-8 displayed the high-

est anticonvulsant activity in this series (ED₅₀= 34 mg/kg), but it

was an approximate 10-fold drop in activity from the correspond-

ing FAA (R)-2 (ED₅₀= 3.3 mg/kg).⁵PAAD 8 showed an

The next compounds listed in Table 1 were the C(4)-

substituted N-benzylamide PAADs (R)-17 and (R)-19. PAADs

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Table 1. Pharmacological Activities of Primary Amino Acid Derivatives (PAADs) in Mice (mg/kg) at UCB and the NINDS ASP

mice (ip)^a

compd no.

test site

R⁰

MES,^bED₅₀

6 Hz,^cED₅₀

formalin, ED₅₀

Tox,^dTD₅₀

PI,^eMES PI,^eform

(R)-7

(S)-7

UCB

NINDS

UCB

OH

ND^f

>62

>110

(R,S)-7

OH

>62

>110

>1.8

(R)-8

OCH₃

34

>67

ND^f

>67

ND^f

>120

>3.5

1.0

>1.8

(R)-8

OCH₃

48 [0.25] (40ꢀ61)

>30, <100 [0.25]

(S)-8

OCH₃

64

>70

120

63

ND^f

0.5

(R,S)-9

OCH₂CH₃

OCH₂CH₂CH₃

OCH₂CHdCH₂

OCH₂CꢁCH

NH₂

73

120

71^g(19%)

>130

75^g(12%)

74^g(27%)

ND^f

(R,S)-10

(R,S)-11

(R,S)-12

(R,S)-13

(R,S)-15

(R,S)-16

(R)-17

69

>130

45

130 (MAD)^h

ND^f

46

47

ND^f

1.0

>68

N(CH₃)₂

N(CH₂CH₂)₂O

CH₂OH

94

>110

83^g(14%)

89

>84

69

>160

>120

>67

(R)-19

CH₂SCH₃

CH₂CH₃

(CH₂)₂CH₃

75

21

130 (MAD)^h

66 (MAD)^h

93

>76

(R)-22

35

57

ND^f

2.8

1.6

1.3

(R)-23

23

>71

lacosamide ((R)-2)

3.3

10

15

19

5.8

6.0

lacosamideⁱ((R)-2) NINDS

phenytoin^j

phenobarbital^j

4.5 [0.5] (3.7ꢀ5.5)

9.5 [2.0] (8.1ꢀ10)

22 [1.0] (15ꢀ23)

270 [0.25] (250ꢀ340)

10

ND^f

27 [0.25] (26ꢀ28)

66 [0.5] (53ꢀ72)

69 [0.5] (63ꢀ73)

430 [0.25] (370ꢀ450)

valproate^j

^aThe compounds were administered either 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. ED₅₀and TD₅₀values are in mg/kg and were determined 30 min after ip

administration (UCB) or a doseꢀresponse curve was generated for all compounds that displayed suﬃcient activity 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. ^bMES = maximal electroshock seizure test. ^c6 Hz test = psychomotor seizure model (44 mA, UCB; 32 mA, NINDS ASP). ^dTox = neurological

toxicity. TD₅₀value determined from the rotorod test. ^ePI = protective index (TD₅₀/ED₅₀). ^fND = not determined. ^gSingle dose experiments where the

mg/kg used is followed by the percentage protected in parentheses. ^hMAD = minimal active dose. ⁱReference 5. ^jReference 58.

containing an oxygen ((R)-17) or a sulfur ((R)-19) two atoms

from the C(2) carbon did not display appreciable anticonvulsant

activity in the MES test (ED₅₀(mg/kg): (R)-17, >160; (R)-

19, 75). (R)-17 and (R)-19 were also inactive in the 6 Hz and

formalin tests (ED₅₀= >60 mg/kg), therefore, the inactivity did

not justify toxicity studies.

Because the substituted heteroatom one atom removed from

the C(2) carbon in R²led to increased FAA anticonvulsant

activity, we compared the activities of (R)-8 and (R,S)-9 with

their R²-hydrocarbon equivalents (R)-22 and (R)-23 (Table 1).

Signiﬁcantly, methylene substitution of the C(3)-oxy moiety

provided PAADs with appreciable anticonvulsant activity (ED₅₀

(mg/kg): (R)-22, 21; (R)-23, 23) that surpassed their hetero-

atom counterparts (ED₅₀(mg/kg): (R)-8, 48; (R,S)-9, 73). (R)-

counterpartsdistinctly diﬀerentiates the PAAD SAR from the cor-

responding FAA SAR.

The signiﬁcant activities of (R)-22 and (R)-23 prompted us to

expand our study of C(2)-hydrocarbon N-benzylamide PAADs.

Table 2 lists the neurological activities in mice (ip) for C(2)-

hydrocarbon N-benzylamide PAADs (R)-, (S)-, and (R,S)-20,

(R)-21, (R)-, (S)-, and (R,S)-22, (R)-23, (R)- and (S)-24, (R)-

and (S)-25, (R,S)-26, (R)-27, and (R)-28. The C(2)-hydrocar-

bon PAADs displayed signiﬁcant anticonvulsant activity in the

MES test and, to a lesser degree, in the 6 Hz test. MES activity

decreased slightly as the length of the alkyl chain increased (ED₅₀

(mg/kg): ethyl ((R)-21, 16) > propyl ((R)-22, 21) > n-butyl

((R)-23, 23)), but the MES activity improved slightly with an

increase in branching (ED₅₀(mg/kg):ethyl((R)-21, 16) < isopropyl

((R)-24, 15) < tert-butyl ((R)-25, 13)). Substituting the terminal

methyl group of (R)-21 (ED₅₀= 16 mg/kg) with a phenyl group

((R)-28, ED₅₀= 40 mg/kg) resulted in a 2.5-fold decrease in

MES activity. The same trends were observed for anticonvulsant

activity in the 6 Hz test, although 3ꢀ5-fold less sensitive. The

C(2)-hydrocarbon PAADs (R)-20,³³(R)-21, (R)-24, and (R)-25

22 also displayed signiﬁcant activity in the formalin test (ED₅₀

=

35 mg/kg). We concluded that C(2)-acyclic PAADs containing a

heteroatom one and two atoms removed from the C(2) center in

R²did not exhibit notable seizure protection and that including a

substituted heteroatom in R²did not increase activity. The dimi-

nished activity of the C(3)-alkoxy PAADs versus their hydrocarbon

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ARTICLE

Table 2. Pharmacological Activities of C(2)-Hydrocarbon N-Benzylamide PAADs in Mice (mg/kg) at UCB and the NINDS ASP

mice (ip)^a

compd no. test site

R²

MES,^bED₅₀

6 Hz,^cED₅₀

formalin, ED₅₀

Tox,^dTD₅₀

PI,^eMES PI,^eform

(R)-20^f

(S)-20^f

(R,S)-20^fNINDS

NINDS

CH₃

>10, <30

ND^g

69

ND^g

>100, <300

CH₃

>300

CH₃

>100, <300

>300

(R)-21

(R)-22

(S)-22

(R,S)-22

(R)-23

(R)-24

(S)-24

(R)-25

(S)-25

(R,S)-26

(R)-27

(R)-28

UCB

NINDS

UCB

CH₂CH₃

(CH₂)₂CH₃

(CH₂)₃CH₃

CH(CH₃)₂

C(CH₃)₃

16

62 (MAD)^h

∼50 [0.5]

66 (MAD)^h

>210

22

ND^g

18 [0.25] (10ꢀ25)

80 [0.25] (65ꢀ95)

4.4

21

35

57

ND^g

2.8

1.6

2.4

UCB

>37

39

100

68ⁱ(17%)

UCB

120 (MAD)^h

UCB

23

93

>71

UCB

16 (MAD)^h

15 [0.25] (13ꢀ18)

>300 [0.5]

13

74

20

47

70 [0.25] (63ꢀ80)

>300 [0.5]

ND^g

NINDS

UCB

<100 [0.25ꢀ1.0]

ND^g

15 [0.25]

ND^g

4.7

>71

>22

ND^g

NINDS

14 [0.25] (11ꢀ17)

42 [0.25] (37ꢀ46)

∼30 [0.25ꢀ0.5]

ND^g

66 [0.25] (58ꢀ73)

100 [0.25] (100ꢀ110)

ND^g

NINDS

UCB CH(CH₃)CH₂CH₃46 (MAD)^h46ⁱ(100%)

>120

>22

25 (inactive) 79ⁱ(95%)

>37

UCB

C₆H₁₁

28

140 (MAD)^h

81 (MAD)^h

ND^g

UCB

CH₂C₆H₅

40

^aThe 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. ED₅₀and TD₅₀values are in mg/kg and were determined 30 min after ip administration

(UCB) or a doseꢀresponse curve was generated for all compounds that displayed suﬃcient activity 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. ^bMES =

maximal electroshock seizure test. ^c6 Hz test = psychomotor seizure model (44 mA, UCB; 32 mA, NINDS ASP). ^dTox = neurological toxicity. TD₅₀

value determined from the rotorod test. ^ePI = protective index (TD₅₀/ED₅₀). ^fReference 33. ^gND = not determined. ^hMAD = minimal active dose.

ⁱSingle dose experiments where the mg/kg used is followed by the percentage protected in parentheses.

all exhibited excellent activity in the rat (po) (MES ED₅₀= 11ꢀ

19 mg/kg) (Table 3) and the values exceeded those of phenytoin

(MES ED₅₀= 30 mg/kg).⁵⁸The MES ED₅₀values for (R)-21,

(R)-24, and (R)-25 were the same (11 mg/kg), but interestingly,

the time of peak eﬃciency increased as the size of the C(2)-

hydrocarbon moiety decreased (0.25ꢀ2.0 h). None of the three

compounds exhibited neurotoxic eﬀects, resulting in high PI

values (>45).

All of the C(2)-hydrocarbon N-benzylamide PAADs that were

tested as individual enantiomers (20, 22, 24, and 25) revealed a

>1.8ꢀ20-fold increase in anticonvulsant activity in the MES test

for the (R)-isomer compared with the (S)-isomer (Table 2). The

isopropyl group at the C(2) carbon displayed the largest (R)-

versus (S)-selectivity (ED₅₀(mg/kg): (R)-24, 15; (S)-24, >300),

but the stereoselectivity was diminished to 3-fold using the tert-

butyl group (ED₅₀(mg/kg): (R)-25, 14; (S)-25, 42). The stereo-

selectivity of (R)-24 resembles that of (R)-2, wherein (R)-2 is

>22-fold more potent than its (S)-stereoisomer.⁵The large

variation in (R)- versus (S)-selectivity with respect to MES

activity in the C(2)-hydrocarbon series was unexpected. In the

FAAs, the eudismic ratio⁵⁹(the ratio of the aﬃnity between the

more tightly bound isomer [eutomer] and the less tightly bound

isomer [distomer]) ranged between 10 and 22. We are uncer-

tain why this ratio varied within the PAAD series but suspect

that multiple factors (e.g., site selectivity, metabolism, transport,

eﬄux) aﬀected the observed anticonvulsant activities for the

individual stereoisomers. The PI values in the MES test in mice of

(R)-24 (PI = 4.7) and (R)-25 (PI = 4.7) are similar to (R)-2

(PI = 6.0).⁵When (R)-24 was tested in the rat, no apparent

behavioral toxicity (TD₅₀= >500 mg/kg) was observed, provid-

ing a PI >45 (Table 3). Remarkably, the anticonvulsant activities

in the MES test (mice, ip) of PAADs (R)-24 (ED₅₀= 15 mg/kg)

and (R)-25 (ED₅₀= 14 mg/kg) greatly surpassed those of their

FAA counterparts, (R)-105 (ED₅₀= >100, <300 mg/kg) and

(R)-106 (ED₅₀= >300 mg/kg) (Table 5), and exceeded the

MES activity of the traditional antiepileptic phenobarbital (ED₅₀=

22 mg/kg).⁵⁸The observed increase in anticonvulsant activity in

proceeding from FAA (R)-105 and (R)-106 to PAAD (R)-24 and

(R)-25, respectively, exceeded 6.7-fold, and the trend was opposite

to the pattern observed for the C(3)-alkoxy PAADs compared

with their corresponding FAA counterparts (Table 4).

PAADs (R)-21, (R)-22, and (R)-24 displayed excellent activ-

ity in the formalin test (ED₅₀(mg/kg): (R)-21, 22; (R)-22,

35; (R)-24, 20) and approached the formalin activity of (R)-2

(ED₅₀= 15 mg/kg) (Table 2). We did not observe a correlation

between the C(2)-alkyl length and formalin activity, as seen

in the MES test. There was an initial 3-fold increase in activity

from methyl to ethyl (ED₅₀(mg/kg): (R)-20, 69; (R)-21, 22),

but then a steady decrease in activity was observed as the C(2)-

alkyl length increased (ED₅₀(mg/kg): ethyl ((R)-21, 22) > propyl

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ARTICLE

Table 3. Pharmacological Activities of C(2)-Hydrocarbon N-Benzylamide PAADs in Rats (mg/kg) at the NINDS ASP

rat (po)^a

compd no.

R²

MES,^bED₅₀

Tox,^cTD₅₀

PI^d

(R)-20^e

(S)-20^e

CH₃

19 [2.0] (13ꢀ25)

>80

>30

>80

>1.5

(R,S)-20^e

CH₃

14 [1.0] (7ꢀ22)

11 [2.0] (7.8ꢀ16)

11 [0.25] (9.1ꢀ13)

11 [0.25] (8.7ꢀ15)

>30 [0.25ꢀ4.0]

3.9 [0.5] (2.6ꢀ6.2)

30 [4.0] (22ꢀ39)

9.1 [5.0] (7.6ꢀ12)

490 [0.5] (350ꢀ730)

>500

>36

>45

(R)-21

CH₂CH₃

CH(CH₃)₂

C(CH₃)₃

>500

(R)-24

>500

(R)-25

>500

(S)-25

>30 [0.25ꢀ4.0]

>500 [0.5]

>3000

lacosamide^f((R)-2)

phenytoin^g

phenobarbital^g

valproate^g

>130

>100

6.7

61 [0.5](44ꢀ96)

280 [0.5] (190ꢀ350)

0.6

^aThe compounds were administered orally to adult male albino SpragueꢀDawley rats. ED₅₀and TD₅₀values are in mg/kg. A doseꢀresponse curve was

generated for all compounds that displayed suﬃcient activity 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. ^bMES = maximal electroshock seizure test. ^cTox = behavioral

toxicity. ^dPI = protective index (TD₅₀/ED₅₀). ^eReference 33. ^fReference 5. ^gReference 58.

((R)-22, 35) > n-butyl ((R)-23, >71). We observed an activity

increase in the formalin test upon branching. A three carbon

C(2) substituent displayed greater activity when it was in the

isopropyl conﬁguration ((R)-24, ED₅₀= 20 mg/kg) compared

with the n-propyl conﬁguration ((R)-22, ED₅₀= 35 mg/kg).

corresponded to each other and there was a modest variance

among the neurological toxicities, resulting in diﬀerent PI values.

The anticonvulsant activities observed for the PAADs sug-

gested that there were reasonable CNS concentrations for many

of these compounds. Nonetheless, we determined the relative

plasma and brain concentration levels for two hydrophilic PAADs,

(R)-7 and (R)-8, 30 min after ip administration (data not shown).

Appreciable brain-to-plasma (B:P) ratios were observed in mice

((R)-7, 1.2:1; (R)-8, 1.2:1). These ﬁndings indicated that the

PAADs may exert their function by targeting sites in the PNS, or

the CNS, or both.

We found that the C(2)-hydrocarbon N-benzylamide PAADs

were the most potent anticonvulsants (Table 2). Hydrocar-

bon PAADs (R)-21, (R)-22, (R)-24, and (R)-25 exhibited ex-

cellent anticonvulsant activity in the MES test (mice, ip) (ED₅₀

(mg/kg): (R)-21, 16; (R)-22, 21; (R)-24, 15; and (R)-25, 13)

that surpassed the activity observed for the traditional AED

phenobarbital (ED₅₀= 22 mg/kg)⁵⁸(Table 2). Similarly, superb

activities in the MES test were observed for the C(2)-hydro-

carbon PAADs when tested in the rat (po) (Table 3). The MES

ED₅₀value for (R)-21, (R)-24, and (R)-25 were all 11 mg/kg.

Surprisingly, we observed that activity increased upon substitu-

tion of a methylene unit for a heteroatom in R²that was one atom

removed from the C(2) site (ED₅₀(mg/kg): (R)-22, 21; (R)-8,

34). This ﬁnding was unexpected because in the FAA series

activity increased with the incorporation of a substituted

heteroatom at this site,^{4ꢀ6,10ꢀ12,39}and this structural deter-

minant proved critical in our optimization of this class of com-

pounds⁵(Figure 3). For example, FAA anticonvulsant activity in

the MES test (mice, ip) increased going from (R,S)-104³⁹to

(R,S)-2⁵(ED₅₀(mg/kg): (R,S)-104, 38; (R,S)-2, 8.3). Similarly,

incorporation of a nitrogen in the C(2) aromatic ring of (R,S)-

107⁶to give (R,S)-108⁴led to an improvement in anticonvulsant

activity (ED₅₀(mg/kg): (R)-107, 20; (R)-108, 11). Finally,

we showed that for C(2)-thiophene FAAs,¹¹the 2-thienyl FAA

(R,S)-109 was more active than the isomeric 3-thienyl FAA

’ DISCUSSION

In this study, we asked whether C(2)-substituted PAADs

exhibit potent anticonvulsant activities and neuropathic pain

protection in animal models. We assumed that the close struc-

tural correspondence of FAAs and PAADs would facilitate our

identiﬁcation of active PAAD agents and prepared 27 PAADs

containing C(2) R²-substituents (i.e., CH₂OR⁰, CH₂N(R⁰)R⁰,

CH₂CH₂XR⁰, hydrocarbon) that tested the hallmarks of the FAA

SAR^4ꢀ13with respect to anticonvulsant activity. Accordingly, we

investigated the importance for activity of a heteroatom in the R²

group that was one atom removed from the C(2) center, the need

for heteroatom substitution (R⁰), and the stereochemical pre-

ference at the C(2) position.

For many PAADs, anticonvulsant activity was determined in

both the MES and the 6 Hz psychomotor seizure assays. Each

model has shown diﬀerent sensitivities to distinct classes of anti-

epileptic agents. A prime example is the eﬃcacy of levetiracetam

in the 6 Hz test but not in the MES test.⁵¹Conversely, traditional

antiepileptic agents are active in the MES test but are largely

inactive in the 6 Hz test.⁶⁰Including the MES test in our studies

proved beneﬁcial in determining the anticonvulsant activity of

PAADs because many were largely insensitive to the 6 Hz test.

We employed the formalin model for neuropathic pain and report

values for the late (chronic) inﬂammation phase. Because the

collective animal studies were conducted at two testing facilities,

we determined the pharmacological activities of several active

PAADs ((R)-8, (R)-21, (R)-24, (R)-25) at both sites (Support-

ing Information Table S3). We found that the MES activities

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Journal of Medicinal Chemistry

ARTICLE

Table 4. Comparison of the Pharmacological Activities of C(3)-Alkoxy and C(3)-Amino N-Benzylamide FAAs and Their PAAD

Counterparts in Mice (mg/kg)

FAA

PAAD

mice (ip)^a

FAA

MES,^bED₅₀

FAA

Tox,^cTD₅₀

PAAD

MES,^bED₅₀

PAAD

Tox,^cTD₅₀

R⁰

compd no. Test Site

compd no. test site

OH

(R)-95^d

(R,S)-95^d

(R)-2^d

NINDS

53 [2.0] (38ꢀ67)

>500 [2.0]

<300

(R)-7

(R,S)-7

(R)-8

UCB

>62

>110

>100, <300

OCH₃

NINDS 4.5 [0.5] (3.7ꢀ5.5) 27 [0.25] (26ꢀ28)

NINDS >100, <300 >300

NINDS 8.3 [0.5] (7.9ꢀ9.8) 43 [0.25] (38ꢀ47)

NINDS

NINDS 48 [0.25] (40ꢀ61)

UCB 64

>30, <100 [0.25]

63

OCH₃

(S)-2^d

(S)-8

OCH₃

(R,S)-2^d

(R,S)-96^d

(R,S)-97^d

(R,S)-98^f

(R,S)-99^h

(R,S)-100^hNINDS

(R,S)-101^hNINDS

(R,S)-8^e

(R,S)-9

(R,S)-11

(R,S)-12

(R,S)-13

(R,S)-15

(R,S)-16

NINDS 84 [0.25] (65ꢀ97) 290 [0.25] (240ꢀ320)

OCH₂CH₃

OCH₂CHdCH₂

OCH₂CꢁCH

NH₂

17 [0.25] (15ꢀ19) 78 [0.25] (64ꢀ90)

>30, <100 >30, <100

UCB

73

45

ND^g

47

ND^g

16 [0.25] (13ꢀ19) 59 [0.25] (55ꢀ66)

46

>100

>100, <300

300

>68

94

N(CH₃)₂

>30, <100

>100, <300

morpholino

89

^aThe compounds were administered either 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. ED₅₀and TD₅₀values are in mg/kg and were determined 30 min after ip

administration (UCB) or a doseꢀresponse curve was generated for all compounds that displayed suﬃcient activity 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. ^bMES = maximal electroshock seizure test. ^cTox = neurological toxicity. TD₅₀value determined from the rotorod test. ^dReference 5.

^eReference 33. ^fReference 41. ^gND = not determined. ^hChoi, D. et al., unpublished results.

(R,S)-110 (ED₅₀(mg/kg): (R)-109, 45; (R)-110, 88). This

diﬀerence in the PAAD and FAA SARs led us to compare the

eﬀect of the structural size of the C(2)-hydrocarbon substituent

on anticonvulsant activity for both series of compounds. For

FAAs, we found that anticonvulsant activity in the MES test

(mice, ip) was higher for R²nonbulky alkyl substituents (ED₅₀

(mg/kg): (R)-102,⁹55; (R)-104,³⁹38) compared with the more

sterically bulky R²moieties (ED₅₀(mg/kg): (R)-105, 100ꢀ300;

(R)-106, >300). The opposite was true for the PAADs, where the

more bulky R²alkyl substituents exhibited slightly improved

anticonvulsant activities (ED₅₀(mg/kg): (R)-24, 15; (R)-25, 14)

compared with PAADs with less sterically bulky R²groups (ED₅₀

(mg/kg): (R)-20, 10ꢀ30; (R)-21, 18). Together these two

structural features (i.e., eﬀect of heteroatom substitution, steric

size of the R²-substituent) diﬀerentiated C(2)-hydrocarbon

PAADs from all previously reported FAAs.

Evaluation of the individual stereoisomers of C(2)-hydrocar-

bon PAADs 24 and 25 demonstrated that the (R)-stereoisomer

consistently exhibited greater anticonvulsant activity than the

(S)-stereoisomer (Supporting Information Table 2). A high

eudismic ratio was observed for 24 (>20), but for 25, this value

decreased to 3. (R)-24 displayed signiﬁcant activity in the

formalin test (ED₅₀= 20 mg/kg); however, the lack of data

from the corresponding (S)-stereoisomer prevented any general-

ized conclusions concerning the importance of C(2) stereo-

chemistry for activity in this animal model.

(R)-21 and (R)-22 displayed signiﬁcant activity (ED₅₀(mg/kg):

(R)-21, 22; (R)-22, 35). For 22, both stereoisomers were evaluated

in the formalin test with the (R)-stereoisomer being 2.8-fold

more active (ED₅₀(mg/kg): (R)-22, 35; (S)-22, 100).

Results for the C(3)-alkoxy and the C(4)-substituted

N-benzylamide PAADs (Table 1) were diﬀerent. The C(3)-alkoxy

PAADs exhibited moderate anticonvulsant activity in the MES

model. While we observed a similar trend in the MES activities

for the C(3)-alkoxy PAADs and the corresponding FAAs

(Table 4), PAAD activity decreased 3ꢀ10-fold compared with

the FAAs. Moreover, the degree of separation between (R)- and

(S)-speciﬁcity for the C(3)-alkoxy PAADs was typically 1.9-fold,

compared with the 10 to >22-fold diﬀerence in the FAAs.^5,8,9,11

The C(3)-alkoxy PAADs provided little or no appreciable

protection in the formalin assay (ED₅₀(mg/kg): (R)-8, >67;

(S)-8, 120; (R,S)-9, >71; (R,S)-10, >120; (R,S)-11, >75; (R,S)-

12, >74). The poor activity of the C(3)-methoxy PAAD (R)-8

(ED₅₀= >67 mg/kg) in the formalin test contrasted with the

activity of its FAA counterpart (R)-2 (ED₅₀= 15 mg/kg). The

C(3)-amino and the C(4)-substituted PAADs were largely

inactive in the anticonvulsant models, with (R)-19 (MES ED₅₀

=

75 mg/kg) displaying the greatest activity in this class of PAADs.

Similarly, we observed little or no protection in the formalin assay

for the C(3)-amino PAADs (ED₅₀(mg/kg): (R,S)-15, >83;

(R,S)-16, 69) and the C(4)-substituted PAADs (ED₅₀(mg/kg):

(R)-17, >67; (R)-19, >76).

Where possible, we evaluated other C(2)-hydrocarbon PAADs

for neuropathic pain protection using the formalin test. PAADs

Does the FAA SAR correlate with the corresponding PAAD

bioactivity? Implicit in this question is whether FAAs and PAADs

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Journal of Medicinal Chemistry

Table 5. Comparison of the Pharmacological Activities of C(2)-Hydrocarbon FAAs and Their PAAD Counterparts in Mice (mg/kg)

ARTICLE

FAA

PAAD

mice (ip)^a

PAAD Tox,^c

FAA

FAA MES,^b

ED₅₀

FAA Tox,^c

TD₅₀

PAAD

PAAD MES,^b

ED₅₀

R²

compd no. Test Site

TD₅₀

CH₃

(R)-102^d

(S)-102^d

(R,S)-102^dNINDS

(R)-103

ND^f

(R,S)-103^gNINDS

NINDS

55 [0.5] (50ꢀ60)

210 [0.5] (150ꢀ260)

(R)-20^e

(S)-20^e

(R,S)-20^e

(R)-21

NINDS

>10, <30

>300

>100, <300

>300

NINDS 550 [0.5] (460ꢀ740) 840 [0.5] (690ꢀ950)

CH₃

76 [0.5] (67ꢀ89)

450 [0.5] (420ꢀ500)

>100, <300

>300

CH₂CH₃

ND^f

NINDS 18 [0.25] (10ꢀ25) 80 [0.25] (65ꢀ95)

>100, <300

38 [0.25] (35ꢀ45)

>100, <300

>300

160 [0.25] (150ꢀ170)

>300

(R,S)-21

(R,S)-22

(R)-24

ND^f

39

ND^f

CH₂CH₂CH₃(R,S)-104^hNINDS

UCB

CH(CH₃)₂

(R)-105

NINDS

NINDS 15 [0.25] (13ꢀ18) 70 [0.25] (63ꢀ80)

NINDS 14 [0.25] (11ꢀ17) 66 [0.25] (58ꢀ73)

C(CH₃)₃

(R)-106

NINDS

>300

(R)-25

^aThe 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. ED₅₀and TD₅₀values are in mg/kg and were determined 30 min after ip administration

(UCB) or a doseꢀresponse curve was generated for all compounds that displayed suﬃcient activity 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. ^bMES =

maximal electroshock seizure test. ^cTox = neurological toxicity. TD₅₀value determined from the rotorod test. ^dReference 9. ^eReference 33. ^fND = not

determined. ^gReference 65. ^hReference 39.

minimally active. These results suggest that these PAADs and

other C(2)-hydrocarbon PAADs possibly operate by diﬀerent

mechanism(s) than FAAs. The excellent activities of the C(2)-

hydrocarbon PAADs (R)-21, (R)-24, and (R)-25, and the water

solubility of their corresponding salts, makes these new com-

pounds worthy of further study.

’ CONCLUSIONS

Our data indicated that small hydrocarbon substituents at the

C(2) center of PAADs aﬀorded excellent anticonvulsant activity

in the MES test (mice, ip; rats, po) and that activity principally

resided in the (R)-stereoisomer. Additionally, C(2)-hydrocarbon

PAADs signiﬁcantly attenuated pain in the formalin model (mice, ip)

Figure 3. The eﬀect of heteroatom substitution on anticonvulsant activity.

of neuropathic pain. Most notably, incorporation of a hetero-

function by similar pathways. We can only compare the anti-

atom that was one atom removed from the C(2) center was

convulsant activity for these two series in the MES test because

detrimental to anticonvulsant activity. The dramatic improve-

we have limited data in the formalin test. For the C(3)-alkoxy

ment in MES protection for PAADs (R)-24 and (R)-25, com-

N-benzylamide PAADs 7ꢀ9, 11, and 12, we saw a 3ꢀ10-fold

pared with their FAA counterparts (R)-105 and (R)-106,

reduction in activity from the FAAs to the PAADs; for the C(3)-

suggested that C(2)-hydrocarbon PAADs may function, in part,

amino PAADs 13, 15, and 16, both the FAAs and PAADs

by diﬀerent pharmacological pathways than other PAADs and

displayed modest activity; and for the C(2)-linear hydrocarbon

the FAA class. PAADs (R)-21, (R)-24, and (R)-25 emerged as

PAADs 20 and 22, both FAAs and PAADs showed comparable

the lead compounds in this series.

activity (Tables 4 and 5). These diﬀerences do not allow us to

speculate if most PAADs exert their anticonvulsant activities by

pathway(s) similar to FAAs or if the observed diﬀerences may be

attributed to changes in bioavailability, metabolism, and physio-

’ EXPERIMENTAL SECTION

chemical properties. Only for the C(2)-branched hydrocarbon

PAADs 24 and 25 (ED₅₀(mg/kg): (R)-24, 15; (R)-25, 14 mg/kg)

did we see a dramatic diﬀerence compared with their FAA

counterparts (R)-105 and (R)-106 (ED₅₀(mg/kg): (R)-105,

>100, <300; (R)-106, >300) (Table 5), where the PAADs

exhibited excellent activity and the corresponding FAAs were

General Methods. The general methods used in this study were

identical to those previously reported¹⁴and are summarized in the

Supporting Information. All compounds were checked by TLC, ¹H and

¹³C NMR, MS, and elemental analyses. The analytical results are within

(0.40% of the theoretical value. The TLC, NMR, and the analytical data

confirmed the purity of the products was g95%.

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dx.doi.org/10.1021/jm2004305 |J. Med. Chem. 2011, 54, 4815–4830

Journal of Medicinal Chemistry

ARTICLE

General Procedure for the Conversion of Cbz-Pro-

tected PAADs to PAADs Using Pd-Catalyzed Hydrogenation

(Method A). A MeOH solution of Cbz-protected PAAD (0.05ꢀ0.1

M) was hydrogenated (1 atm) in the presence of 10% PdꢀC at room

temperature (3 hꢀ7 d). The mixture was filtered through a bed of Celite,

the filtrate was evaporated in vacuo, and the product was purified by

column chromatography (SiO₂).

CHCl₃). ¹H NMR (300 MHz, DMSO-d₆) δ 1.84 (s, 2H), 3.25 (s, 3H),

3.35ꢀ3.44 (m, 3H), 4.23ꢀ4.36 (m, 2H), 7.20ꢀ7.34 (m, 5H),

8.36ꢀ8.45 (br t, 1H). Anal. (C₁₁H₁₆N₂O₂0.15H₂O): C, H, N.

3

(R,S)-N-Benzyl 2-Amino-3-methoxypropionamide ((R,S)-8).

The previous procedure was repeated using (R,S)-35 (2.00 g, 5.85 mmol),

10% PdꢀC (0.2 g), and MeOH (100 mL) to give the desired product

1

(0.42 g, 32%) as a pale-yellow oil: R_f0.37 (1:20 MeOH/CHCl₃). H

NMR (300 MHz, DMSO-d₆) δ 1.82 (br s, 2H), 3.25 (s, 3H), 3.37ꢀ3.44

(m, 3H), 4.23ꢀ4.36 (m, 2H), 7.19ꢀ7.34 (m, 5H), 8.36ꢀ8.44 (br t, 1H).

Anal. (C₁₁H₁₆N₂O₂): C, H, N.

General Procedure for the Conversion of tBoc-Protected

PAADs to PAADs Using TFA Deprotection (Method B). TFA

(15 equiv) was added to an anhydrous CH₂Cl₂solution of the N-t-

butoxycarbonyl N⁰-benzylamide (0.3 M) at room temperature. The

solution was stirred (1 h), and then the solvent was evaporated in vacuo.

The crude product was diluted with CH₂Cl₂and extracted with aqueous

1 M HCl (3ꢂ). The combined aqueous layers were washed with

CH₂Cl₂(2ꢂ), basified (pH 10ꢀ12) with aqueous 4 M NaOH, and

extracted with CH₂Cl₂(3ꢂ). The combined organic layers were washed

with brine (2ꢂ), dried (Na₂SO₄), evaporated in vacuo, and purified by

column chromatography (SiO₂).

(R,S)-N-Benzyl 2-Amino-3-ethoxypropionamide ((R,S)-9).

Utilizing method A and (R,S)-36 (1.00 g, 2.81 mmol), 10% PdꢀC (0.1 g),

and MeOH (30 mL) (18 h) gave the crude product that was purified by

flash column chromatography (SiO₂; 1:10 MeOH/CH₂Cl₂). The

resulting oil was dissolved in CH₂Cl₂(10 mL) and was extracted with

aqueous 0.1 N HCl (3 ꢂ 10 mL). The aqueous layers were combined

and washed with CH₂Cl₂(2 ꢂ 30 mL). The aqueous layer was basified

to pH 10ꢀ12 with aqueous 0.1 N NaOH and then extracted with

CH₂Cl₂(3 ꢂ 60 mL). The CH₂Cl₂layers were combined, dried (MgSO₄),

and concentrated in vacuo to give the desired product (0.42 g, 68%) as a

(R)-N-Benzyl 2-Amino-3-hydroxypropionamide ((R)-7). Uti-

lizing method A with (R)-31 (1.82 g, 5.53 mmol), 10% PdꢀC (180 mg),

and MeOH (200 mL) (8 h) gave the crude product that was purified

by medium pressure liquid chromatography to give the desired product

1

pale-yellow oil: R_f0.33 (1:1 EtOAc/hexanes). H NMR (300 MHz,

DMSO-d₆) δ 1.10 (t, J = 7.2 Hz, 3H), 1.82 (s, 2H), 3.29ꢀ3.49 (m, 5H),

4.22ꢀ4.37 (m, 2H), 7.20ꢀ7.33 (m, 5H), 8.36ꢀ8.45 (1H). HRMS (ESI)

223.1450 [M þ H^þ] (calcd for C₁₂H₁₈N₂O₂H^þ223.1447). Anal.

(0.56 g, 53%) as a white solid: mp 95ꢀ96 °C; [R]²⁵ꢀ0.48° (c 1.5,

D

MeOH). ¹H NMR (300 MHz, DMSO-d₆) δ 1.84 (br s, 2H), 3.25 (dd, J =

4.8, 4.8 Hz, 1H), 3.42ꢀ3.56 (m, 2H), 4.29 (d, J = 6.0 Hz, 2H), 4.72ꢀ4.86

(br s, 1H), 7.20ꢀ7.33 (m, 5H), 8.35 (t, J = 6.0 Hz, 1H). HRMS (ESI)

217.0953 [M þ Na^þ] (calcd for C₁₀H₁₄N₂O₂Na^þ217.0953). Anal.

(C₁₀H₁₄N₂O₂): C, H, N.

(C₁₂H₁₈N₂O₂0.06CH₂Cl₂): C, H, N.

3

(R,S)-N-Benzyl 2-Amino-3-propoxypropionamide ((R,S)-

10). Utilizing method A and (R,S)-37 (1.42 g, 3.85 mmol), 10% PdꢀC

(0.15 g), and MeOH (50 mL) (6 h) gave the crude product that was

purified by flash column chromatography (SiO₂; 1:100 MeOH/CH₂Cl₂).

The resulting oil was dissolved in CH₂Cl₂(10 mL) and extracted with

aqueous 0.1 N HCl (3 ꢂ 10 mL). The aqueous layers were combined

and washed with CH₂Cl₂(2 ꢂ 30 mL). The aqueous layer was basified

to pH 10ꢀ12 with aqueous 0.1 N NaOH and then extracted with

CH₂Cl₂(3 ꢂ 60 mL). The second set of CH₂Cl₂layers were combined,

dried (NaSO₄), and concentrated in vacuo to give the desired product

(0.66 g, 72%) as a pale-orange oil: R_f0.52 (1:100 MeOH/CH₂Cl₂). ¹H

NMR (300 MHz, DMSO-d₆) δ 0.85 (t, J = 7.8 Hz, 3H), 1.44ꢀ1.56

(m, 2H), 1.82 (br s, 2H), 3.31ꢀ3.46 (m, 5H), 4.23ꢀ4.37 (m, 2H),

7.19ꢀ7.32 (m, 5H), 8.36ꢀ8.45 (br t, 1H). Anal. (C₁₃H₂₀N₂O₂): C, H, N.

(R,S)-N-Benzyl 2-Amino-3-allyloxypropionamide ((R,S)-11).

Utilizing method B with (R,S)-48 (1.17 g, 3.50 mmol), TFA (3.90 mL,

52.52 mol), and CH₂Cl₂(12 mL) gave the crude product after workup that

was further purified by flash column chromatography (SiO₂; 1:100ꢀ1:10

MeOH/CH₂Cl₂) to give the desired product (285 mg, 35%) as a pale-

yellow oil: R_f0.44 (1:20 MeOH/CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃) δ

1.61ꢀ1.73 (br s, 2H), 3.62 (t, J = 5.6 Hz, 1H), 3.66ꢀ3.74 (m, 2H), 4.00

(app t, J = 1.2 Hz, 1H), 4.02 (app t, J = 1.6 Hz, 1H), 4.41ꢀ4.51 (m, 2H),

5.17ꢀ5.29 (m, 2H), 5.84ꢀ5.93 (m, 1H), 7.24ꢀ7.35 (m, 5H), 7.73ꢀ7.81

(br t, 1H). HRMS (ESI) 235.1452 [M þ H^þ] (calcd for C₁₃H₁₈N₂O₂H^þ

(S)-N-Benzyl 2-Amino-3-hydroxypropionamide ((S)-7). The

previous procedure was repeated using (S)-31 (1.46 g, 4.44 mmol), 10%

PdꢀC (140 mg), and MeOH (200 mL) (8 h) to give the desired product

(0.54 g, 63%) as a white solid: mp 91ꢀ92 °C; [R]²⁵þ4.88° (c 1.5,

D

MeOH). ¹H NMR (300 MHz, DMSO-d₆) δ 3.27 (dd, J = 5.1, 5.1 Hz, 1H),

3.42ꢀ3.57 (m, 2H), 4.30 (d, J = 6.2 Hz, 2H), 7.19ꢀ7.34 (m, 5H), 8.37

(t, J = 6.2 Hz, 1H). HRMS (ESI) 217.0953 [M þ Na^þ] (calcd for

C₁₀H₁₄N₂O₂Na^þ217.0953). Anal. (C₁₀H₁₄N₂O₂): C, H, N.

(R,S)-N-Benzyl 2-Amino-3-hydroxypropionamide ((R,S)-7).

The previous procedure was repeated using (R,S)-31 (1.33 g, 4.07 mmol),

10% PdꢀC (130 mg), and MeOH (200 mL) (8 h) to give the desired

product (0.52 g, 66%) as a white solid: mp 90ꢀ91 °C. ¹H NMR (300 MHz,

DMSO-d₆) δ 2.14 (br s, 2H), 3.27 (dd, J = 4.5, 4.8 Hz, 1H), 3.42ꢀ3.57

(m, 2H), 4.30 (d, J = 6.5 Hz, 2H), 4.62ꢀ4.88 (br s, 1H), 7.19ꢀ7.34

(m, 5H), 8.37 (t, J = 6.5 Hz, 1H). HRMS (ESI) 217.0953 [M þ Na^þ]

(calcd for C₁₀H₁₄N₂O₂Na^þ217.0953). Anal. (C₁₀H₁₄N₂O₂): C, H, N.

(R)-N-Benzyl 2-Amino-3-methoxypropionamide ((R)-8).³²

Utilizing method A with (R)-35 (2.00 g, 5.85 mmol), 10% PdꢀC (0.2 g),

and MeOH (100 mL) (5 h) gave the crude product that was purified by

flash column chromatography (SiO₂; 1:10 MeOH/CHCl₃). The result-

ing oil was dissolved in CH₂Cl₂(20 mL) and extracted with aqueous 0.1

N HCl (3 ꢂ 20 mL). The aqueous layers were combined and washed

with CH₂Cl₂(2 ꢂ 60 mL). The aqueous layer was basified to pH 10ꢀ12

with aqueous 0.1 N NaOH and then extracted with CH₂Cl₂(3 ꢂ

100 mL). The CH₂Cl₂layers were combined, dried (MgSO₄), and

concentrated in vacuo to give the desired product (0.89 g, 73%) as a

235.1447). Anal. (C₁₃H₁₈N₂O₂0.20H₂O): C, H, N.

3

(R,S)-N-Benzyl 2-Amino-3-(prop-2-ynyloxy)propionamide

((R,S)-12). Utilizing method B and using (R,S)-49 (973 mg, 2.93 mmol),

TFA (3.26 mL, 43.94 mol), and CH₂Cl₂(10 mL) gave the crude product

after workup that was further purified by flash column chromatography

(SiO₂; 1:100ꢀ1:10 MeOH/CH₂Cl₂) to give the desired product (512 mg,

75%) as a pale-yellow oil: R_f0.29 (1:100 MeOH/CH₂Cl₂). ¹H NMR (100

MHz, CDCl₃) δ1.64ꢀ1.72 (br s, 2H), 2.45 (t, J= 2.4 Hz, 1H), 3.62 (dd, J=

4.0, 6.2 Hz, 1H), 3.76 (dd, J = 6.2, 9.2 Hz, 1H), 3.84 (dd, J = 4.0, 9.2 Hz,

1H), 4.13ꢀ4.24 (m, 2H), 4.41ꢀ4.51 (m, 2H), 7.24ꢀ7.35 (m, 5H),

7.73ꢀ7.81 (br t, 1H). HRMS (ESI) 233.1294 [M þ H^þ] (calcd for

waxy solid: mp 39ꢀ40 °C; [R]²⁵_Dꢀ1.5° (c 1.6, MeOH) (lit.³²[R]²³

D

ꢀ2.0° (c 1.5, MeOH)); R_f0.26 (1:20 MeOH/CHCl₃). ¹H NMR (300

MHz, DMSO-d₆) δ 1.82 (s, 2H), 3.25 (s, 3H), 3.35ꢀ3.44 (m, 3H),

4.23ꢀ4.36 (m, 2H), 7.20ꢀ7.34 (m, 5H), 8.36ꢀ8.44 (br t, 1H). Anal.

(C₁₁H₁₆N₂O₂0.18H₂O): C, H, N.

3

(S)-N-Benzyl 2-Amino-3-methoxypropionamide ((S)-8).³⁹

The previous procedure was repeated using (S)-35 (2.00 g, 5.85 mmol),

10% PdꢀC (0.2 g), and MeOH (100 mL) to give the desired product

C₁₃H₁₆N₂O₂H^þ233.1290). Anal. (C₁₃H₁₆N₂O₂0.09CH₂Cl₂): C, H, N.

3

(R,S)-N-Benzyl 2,3-Diaminopropionamide ((R,S)-13). Utiliz-

ing method B and using (R,S)-64 (1.71 g, 4.35 mmol), TFA (4.86 mL,

65.23 mmol), and CH₂Cl₂(15 mL) gave the crude product after workup

(1.12 g, 92%) as a waxy solid: mp 39ꢀ40 °C; [R]²⁵þ1.7° (c 1.5,

D

MeOH) (lit.³⁹[R]²³þ1.8° (c 0.8, MeOH)); R_f0.39 (1:20 MeOH/

D

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Journal of Medicinal Chemistry

ARTICLE

that was further purified by recrystallization from hot EtOAc/hexanes to

give the desired product (427 mg, 51%) as a white solid: mp 119ꢀ

120 °C; R_f0.17 (1:10 MeOH/CH₂Cl₂). ¹H NMR (300 MHz, CD₃OD)

δ 2.70ꢀ2.95 (m, 2H), 3.35ꢀ3.42 (m, 1H), 4.40 (s, 2H), 7.21ꢀ7.35

(m, 5H). HRMS (ESI) 216.1107 [M þ Na^þ] (calcd for C₁₀H₁₅N₃ONa^þ

with CH₂Cl₂(2 ꢂ 30 mL). The aqueous layer was basified to pH 10ꢀ12

with aqueous 1 M NaOH, and then extracted with CH₂Cl₂(3 ꢂ 60 mL).

The CH₂Cl₂layers were combined, dried (NaSO₄), and concentrated in

vacuo to give the desired product (0.95 g, 79%) as a pale-yellow oil:

[R]²⁵_Dꢀ9.4° (c 1.0, MeOH); R_f0.64 (1:10 MeOH/CH₂Cl₂). ¹H NMR

(300 MHz, DMSO-d₆) δ 0.86 (t, J = 7.4 Hz, 3H), 1.19ꢀ1.42 (m, 3H),

1.47ꢀ1.61 (m, 1H), 1.78 (br s, 2H), 3.15ꢀ3.20 (app t, 1H), 4.28 (d, J =

5.7 Hz, 2H), 7.20ꢀ7.34 (m, 5H), 8.34 (t, J = 5.7 Hz, 1H). HRMS (ESI)

229.1317 [M þ Na^þ] (calcd for C₁₂H₁₈N₂ONa^þ229.1317). Anal.

(C₁₂H₁₈N₂O): C, H, N.

216.1113). Anal. (C₁₀H₁₅N₃O H₂O): C, H, N.

3

(R,S)-N-Benzyl 2-Amino-3-(N⁰,N⁰-dimethyl)aminopropiona-

mide ((R,S)-15). Utilizing method A and using (R,S)-60 (1.08 g,

3.03 mmol), 10% PdꢀC (0.1 g), and MeOH (30 mL) gave a crude oil

that was purified by flash column chromatography (SiO₂; 1:100ꢀ1:20

MeOH/CH₂Cl₂) to give the desired product (0.40 g, 60%) as a pale-orange

oil: R_f0.26 (1:10 MeOH/CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ 1.86

(s, 2H), 2.26 (s, 6H), 2.42 (dd, J = 8.8, 12.3 Hz, 1H), 2.60 (dd, J = 6.0, 12.3

Hz, 1H), 3.50 (dd, J = 6.0, 8.8 Hz, 1H), 4.38ꢀ4.52 (m, 2H), 7.24ꢀ7.36 (m,

5H), 8.20ꢀ8.34 (br t, 1H). HRMS (ESI) 244.1434 [M þ Na^þ] (calcd for

(S)-N-Benzyl 2-Aminopentanamide ((S)-22). The previous

procedure was repeated using (S)-93 (3.00 g, 8.82 mmol), 10% PdꢀC

(0.3 g), and MeOH (100 mL) to give the desired product (1.76 g, 97%)

as a pale-yellow oil: [R]²⁵_Dþ9.4° (c 1.5, MeOH); R_f0.65 (1:10 MeOH/

CH₂Cl₂). ¹H NMR (300 MHz, DMSO-d₆) δ 0.86 (t, J = 6.6 Hz, 3H),

1.22ꢀ1.42 (m, 3H), 1.48ꢀ1.61 (m, 1H), 1.78 (br s, 2H), 3.17 (t, J = 6.9

Hz, 1H), 4.28 (d, J = 6.0 Hz, 2H), 7.20ꢀ7.34 (m, 5H), 8.34 (t, J = 4.8 Hz,

1H). LRMS (ESI) 207.12 [M þ H^þ] (calcd for C₁₂H₁₈N₂OH^þ

C₁₂H₁₉N₃ONa^þ244.1426). Anal. (C₁₂H₁₉N₃O₂0.33H₂O): C, H, N.

3

(R,S)-N-Benzyl 2-Amino-3-morpholinopropionamide ((R,S)-

16). Utilizing method A with (R,S)-61 (2.50 g, 6.29 mmol), 10% PdꢀC

(0.25 g), and MeOH (60 mL) gave the crude product that was further

purified by flash column chromatography (SiO₂; 1:100 MeOH/CH₂Cl₂)

to give the desired compound (0.94 g, 57%) as a white solid: mp 84ꢀ

85 °C; R_f0.24 (1:20 MeOH/CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ

1.83 (s, 2H), 2.37ꢀ2.55 (m, 5H), 2.67 (dd, J = 5.7, 12.3 Hz, 1H),

3.53ꢀ3.69 (m, 5H), 4.43 (d, J = 5.7 Hz, 2H), 7.24ꢀ7.36 (m, 5H),

8.06ꢀ8.18 (br t, 1H). HRMS (ESI) 286.1533 [M þ Na^þ] (calcd for

C₁₄H₂₁N₃O₂Na^þ286.1532). Anal. (C₁₄H₂₁N₃O₂): C, H, N.

207.12). Anal. (C₁₂H₁₈N₂O 0.32H₂O): C, H, N.

3

(R,S)-N-Benzyl 2-Aminopentanamide ((R,S)-22). The pre-

vious procedure was repeated using (R,S)-93 (2.00 g, 11.76 mmol),

10% PdꢀC (0.2 g), and MeOH (60 mL) to give the desired product

(0.88 g, 72%) as a pale-yellow oil: R_f0.65 (1:10 MeOH/CH₂Cl₂). ¹H

NMR (300 MHz, DMSO-d₆) δ 0.86 (t, J = 6.6 Hz, 3H), 1.24ꢀ1.42 (m,

3H), 1.48ꢀ1.61 (m, 1H), 1.74 (br s, 2H), 3.15ꢀ3.18 (app t, 1H), 4.28

(d, J = 5.9 Hz, 2H), 7.20ꢀ7.34 (m, 5H), 8.36 (t, J = 5.9 Hz, 1H). HRMS

(ESI) 229.1320 [M þ Na^þ] (calcd for C₁₂H₁₈N₂ONa^þ229.1317).

(R)-N-Benzyl 2-Amino-4-hydroxybutanamide ((R)-17). Uti-

lizing method A and using (R)-70 (1.50 g, 4.38 mmol), 10% PdꢀC

(0.15 g), and MeOH (45 mL) gave the crude product that was further

purified by flash column chromatography (SiO₂; 1:100ꢀ1:10 MeOH/

CH₂Cl₂) to give the desired product (0.53 g, 57%) as a white solid: mp

Anal. (C₁₂H₁₈N₂O 0.18H₂O): C, H, N.

3

(R)-N-Benzyl 2-Aminohexanamide ((R)-23). Utilizing method

B and using (R)-84 (3.33 g, 9.78 mmol), TFA (10.9 mL), and CH₂Cl₂

(33 mL) gave crude product that was further purified by flash column

chromatography (1:10 MeOH/CH₂Cl₂) to give the desired product

88ꢀ89 °C; R_f0.40 (1:10 MeOH/CH₂Cl₂); [R]²⁵þ4.8° (c 1.1,

D

CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ 1.79ꢀ1.90 (m, 1H), 1.93ꢀ

2.19 (m, 3H), 3.61 (t, J = 6.6 Hz, 1H), 3.76ꢀ3.88 (2H), 4.46 (d, J = 6.3

Hz, 2H), 7.25ꢀ7.38 (m, 5H), 7.56ꢀ7.64 (br t, 1H). HRMS (ESI)

209.1288 [M þ H^þ] (calcd for C₁₁H₁₆N₂O₂H^þ209.1290). Anal.

(C₁₁H₁₆N₂O₂): C, H, N.

(1.83 g, 80%) as a clear oil: R_f0.55 (1:10 MeOH/CH₂Cl₂); [R]²⁵

D

1

ꢀ3.5° (c 2.6, EtOH). H NMR (CDCl₃) δ 0.87ꢀ0.92 (br t, 3H),

1.31ꢀ1.60 (m, 7H), 1.80ꢀ1.95 (br m, 1H), 3.31ꢀ3.43 (m, 1H), 4.42

(d, J = 6.0 Hz, 2H), 7.25ꢀ7.32 (m, 5H), 7.59ꢀ7.78 (br s, 1H). HRMS

(ESI) 221.1655 [M þ H] ^þ(calcd for C₁₃H₂₁N₂O^þ221.1654). Anal.

(R)-N-Benzyl 2-Amino-4-(methylthio)butanamide ((R)-19).

Utilizing method B and using (R)-67 (1.64 g, 4.85 mmol), TFA (5.40 mL,

72.74 mol), and CH₂Cl₂(15 mL) gave the crude product that was further

purified by flash column chromatography (SiO₂; 1:100ꢀ1:10 MeOH/

CH₂Cl₂) to give the desired product (0.87 g, 76%) as a pale-yellow oil: R_f

0.58 (1:20 MeOH/CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ 1.48 (s, 2H),

1.73ꢀ1.87 (m, 1H), 2.10 (s, 3H), 2.16ꢀ2.27 (m, 1H), 2.62 (t, J = 7.4 Hz,

2H), 3.55 (dd, J = 4.4, 8.3 Hz, 1H), 4.45 (d, J = 6.0 Hz, 2H), 7.25ꢀ7.36 (m,

5H), 7.57ꢀ7.64 (br t, 1H). HRMS (ESI) 239.1224 [M þ H^þ] (calcd for

C₁₂H₁₈N₂OSH^þ239.1218). Anal. (C₁₂H₁₈N₂OS): C, H, N, S.

(C₁₃H₂₀N₂O 0.15H₂O): C, H, N.

3

(R)-N-Benzyl 2-Amino-3-methylbutanamide ((R)-24). Uti-

lizing method B and using (R)-85 (4.00 g, 13.06 mmol), TFA (14.56 mL,

0.20 mol), and CH₂Cl₂(45 mL) gave the crude product after work-

up that was further purified by flash column chromatography (SiO₂;

1:100ꢀ1:10 MeOH/CH₂Cl₂) to give the desired compound (2.25 g,

83%) as a pale-yellow oil: R_f0.70 (1:20 MeOH/CH₂Cl₂). ¹H NMR (300

MHz, CDCl₃) δ 0.82 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 1.31

(s, 2H), 2.24ꢀ2.35 (m, 1H), 3.23 (d, J = 3.9 Hz, 1H), 4.35ꢀ4.49

(m, 2H), 7.21ꢀ7.33 (m, 5H), 7.72ꢀ7.81 (br t, 1H). HRMS (ESI)

207.1501 [M þ H^þ] (calcd for C₁₂H₁₈N₂OH^þ207.1497). Anal.

(R)-N-Benzyl 2-Aminobutanamide ((R)-21). Utilizing method

B and using (R)-83 (4.87 g, 16.67 mmol), TFA (18.57 mL, 0.25 mol),

and CH₂Cl₂(55 mL) gave the crude product after workup that was

further purified by flash column chromatography (SiO₂; 1:100ꢀ1:10

MeOH/CH₂Cl₂) to give the desired compound (1.86 g, 58%) as a pale-

yellow oil: R_f0.53 (1:20 MeOH/CH₂Cl₂). ¹H NMR (300 MHz,

CDCl₃) δ 0.97 (t, J = 7.2 Hz, 3H), 1.38ꢀ1.46 (br s, 2H), 1.51ꢀ1.66

(m, 1H), 1.83ꢀ1.97 (m, 1H), 3.35 (dd, J = 4.5, 7.8 Hz, 1H), 4.44 (d, J =

5.7 Hz, 2H), 7.26ꢀ7.35 (m, 5H), 7.61ꢀ7.73 (br t, 1H). HRMS (ESI)

193.1348 [M þ H^þ] (calcd for C₁₁H₁₆N₂OH^þ193.1341). Anal.

(C₁₂H₁₈N₂O 0.04CH₂Cl₂): C, H, N.

3

(S)-N-Benzyl 2-Amino-3-methylbutanamide ((S)-24).⁶¹The

previous procedure was repeated using (S)-85 (2.57 g, 8.39 mmol), TFA

(9.35 mL, 0.13 mol), and CH₂Cl₂(28 mL) to give the crude product after

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 (1.65 g, 96%) as a white solid: mp 52ꢀ53 °C; [R]^28.5_Dꢀ27.1°

(c 0.6, CH₂Cl₂) (lit.⁶¹[R]²⁰ꢀ27.2° (c 0.5, CH₂Cl₂)); R_f0.47 (1:20

D

MeOH/CH₂Cl₂). ¹H NMR (400 MHz, CDCl₃) δ 0.84 (d, J = 6.8 Hz, 3H),

0.99(d, J= 6.8 Hz, 3H), 1.43 (s, 2H), 2.28ꢀ2.40 (m, 1H), 3.27 (d, J=4.0Hz,

1H), 4.42 (dd, J= 6.2, 14.8 Hz, 1H), 4.48 (dd, J= 6.0, 14.8 Hz, 1H), 7.24ꢀ7.34

(5H), 7.62ꢀ7.70 (br t, 1H). HRMS (ESI) 207.1502 [M þ H^þ] (calcd for

(C₁₁H₁₆N₂O 0.06CH₂Cl₂): C, H, N.

3

(R)-N-Benzyl 2-Aminopentanamide ((R)-22). Utilizing meth-

od A and using (R)-93 (2.00 g, 5.88 mmol), 10% PdꢀC (0.2 g), and

MeOH (60 mL) gave the crude product that was further purified by flash

column chromatography (SiO₂; 1:100 MeOH/CH₂Cl₂). The resulting

oil was dissolved in CH₂Cl₂(10 mL) and was extracted with aqueous

1 M HCl (3 ꢂ 10 mL). The aqueous layers were combined and extracted

C₁₂H₁₈N₂OH^þ207.1497). Anal. (C₁₂H₁₈N₂O 0.30H₂O): C, H, N.

3

(R)-N-Benzyl 2-Amino-3,3-dimethylbutanamide ((R)-25).⁶²

Utilizing method B and using (R)-86 (3.80 g, 11.87 mmol), TFA

(13.22 mL, 0.18 mol), and CH₂Cl₂(40 mL) to give the crude product

4827

dx.doi.org/10.1021/jm2004305 |J. Med. Chem. 2011, 54, 4815–4830

Journal of Medicinal Chemistry

ARTICLE

after 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 (2.17 g, 83%) as a pale-yellow solid: mp 65ꢀ66 °C

(s, 3H), 2.04ꢀ2.11 (m, 1H), 4.25 (app t, J = 8.2 Hz, 1H), 4.37 (1/2 AB_q, J=

5.4, 14.8 Hz, 1H), 4.47 (1/2 AB_q, J = 5.8, 14.8 Hz, 1H), 6.18ꢀ6.21 (br d,

1H), 6.42ꢀ6.50 (br t, 1H), 7.24ꢀ7.34 (m, 5H), addition of excess

(R)-(ꢀ)-mandelic acid to a CDCl₃solution of (R)-105 gave only one

signal for the acetyl methyl protons. LRMS (ESI) 271.16 [M þ Na^þ]

(calcd for C₁₄H₂₀N₂O₂Na^þ271.14). Anal. (C₁₄H₂₀N₂O₂): C, H, N.

(R)-N-Benzyl 2-Acetamido-3,3-dimethylbutanamide ((R)-

106). (R)-N-Benzyl 2-amino-3,3-dimethylbutanamide hydrochloride

(0.64 g, 2.5 mmol) was dissolved in CH₂Cl₂(50 mL), and then Et₃N

(1.0 mL, 7.50 mmol) and acetyl chloride (0.2 mL, 3.00 mmol) were

carefully added at 0 °C and the resulting solution was stirred at room

temperature (2 h). Aqueous 10% citric acid (50 mL) was added, the

layers separated, and the organic layer was washed with a saturated

aqueous NaHCO₃solution (50 mL). The aqueous layer was extracted

with CH₂Cl₂(3 ꢂ 50 mL). All the organic layers were combined, dried

(Na₂SO₄), and concentrated in vacuo. The residue was purified by

chromatography (SiO₂; 30:70ꢀ70:30 EtOAc/hexanes) to give crude

product that was recrystallized with EtOAc to give the desired product as

a white solid: mp 199ꢀ200 °C; [R]²⁵_Dꢀ6.1° (c 0.5, CHCl₃); R_f0.19

(30:70 EtOAc/hexanes). ¹H NMR (400 MHz, CDCl₃) δ 1.00 (s, 9H),

1.86 (s, 3H), 4.29 (1/2 AB_q, J = 5.5, 14.8 Hz, 1H), 4.43 (1/2 AB_q, J = 5.8,

14.8 Hz, 1H), 4.45 (d, J = 9.4 Hz, 1H), 6.45 (d, J = 9.4 Hz, 1H), 7.18 (br t,

J = 5.4 Hz, 1H), 7.23ꢀ7.32 (m, 5H), addition of excess (R)-(ꢀ)-

mandelic acid to a CDCl₃solution of (R)-106 gave only one signal for

the acetyl methyl protons. LRMS (ESI) 263.20 [M þ H^þ] (calcd for

C₁₅H₂₂N₂O₂H^þ263.18). Anal. (C₁₅H₂₂N₂O₂): C, H, N.

Pharmacology. Compounds were screened under the auspices of

UCB Pharma (Braine L’Alleud, 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.⁶⁴Pharmacological evaluation by UCB Pharma

consisted of four assays using male NMRI mice (ip): the 6 Hz test⁵³

and the MES test⁵¹to assess anticonvulsant activity, the formalin

test^54,55to assess neuropathic pain attenuation, and the rotorod test⁵¹

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 test (mice) to assess anticonvulsant activity, the rotorod test

(mice) to assess neurological toxicity, and the positional sense test or

gait and stance test (rats) to assess behavioral toxicity.⁵²

(lit.⁶²mp 53ꢀ54 °C); [R]^28.5þ21.8° (c 0.5, CH₂Cl₂); R_f0.48 (1:20

D

1

MeOH/CH₂Cl₂). H NMR (400 MHz, CDCl₃) δ 1.00 (s, 9H), 1.45

(s, 2H), 3.12 (s, 1H), 4.42 (dd, J = 4.0, 12.8 Hz, 1H), 4.46 (dd, J = 4.0,

12.8 Hz, 1H), 7.05ꢀ7.13 (br m, 1H), 7.24ꢀ7.34 (m, 5H). HRMS

(þESI) 221.1654 [M þ H]^þ(calcd for C₁₃H₂₀N₂OH^þ221.1653). Anal.

(C₁₃H₂₀N₂O): C, H, N.

(S)-N-Benzyl 2-Amino-3,3-dimethylbutanamide ((S)-25).^61,63

The previous procedure was repeated using (S)-86 (2.50 g, 7.81 mmol),

TFA (8.70 mL, 0.12 mol), and CH₂Cl₂(26 mL) to give the crude product

after workup that was further purified by flash column chromatography

(SiO₂; 1:100ꢀ1:10 MeOH/CH₂Cl₂) to give the desired compound (1.58 g,

92%) as a white solid: mp 65ꢀ66 °C (lit.⁶³mp 53ꢀ54 °C); [R]²⁵_Dꢀ15.2°

(c 0.51, CH₂Cl₂) (lit.⁶¹[R]²⁰_Dꢀ17.5° (c 0.56, CH₂Cl₂); R_f0.19 (100%

EtOAc). ¹H NMR (300 MHz, CDCl₃) δ 1.00 (s, 9H), 1.47 (s, 2H), 3.14 (s,

1H), 4.44 (d, J = 5.7 Hz, 2H), 7.04ꢀ7.12 (br t, 1H), 7.24ꢀ7.36 (m, 5H).

HRMS (ESI) 221.1652 [M þ H^þ] (calcd for C₁₃H₂₀N₂OH^þ221.1654).

Anal. (C₁₃H₂₀N₂O): C, H, N.

(R,S)-N-Benzyl 2-Amino-3-methylpentanamide ((R,S)-26).

Utilizing method B and using (R,S)-87 (4.00 g, 12.49 mmol), TFA

(13.92 mL, 0.19 mol), and CH₂Cl₂(40 mL) gave the crude product after

workup that was further purified by flash column chromatography (SiO₂;

1:100ꢀ1:10 MeOH/CH₂Cl₂) to give the desired compound (2.24 g,

81%) as a pale-yellow oil and as a 1:1 mixture of diastereomers: R_f0.73

(1:20 MeOH/CH₂Cl₂). ¹H NMR (300 MHz, CDCl₃) δ 0.77ꢀ0.96 (m,

6H), 1.00ꢀ1.46 (m, 4H), 1.92ꢀ2.17 (m, 1H), 3.26 (d, J = 3.6 Hz, 0.5H),

3.36 (d, J = 3.6 Hz, 0.5H), 4.36ꢀ4.49 (m, 2H), 7.21ꢀ7.33 (m, 5H),

7.74ꢀ7.82, 7.83ꢀ7.91 (1H). HRMS (ESI) 221.1663 [Mþ H^þ] (calcd for

C₁₃H₂₀N₂OH^þ221.1654). Anal. (C₁₃H₂₀N₂O 0.03CH₂Cl₂): C, H, N.

3

(R)-N-Benzyl 2-Amino-2-cyclohexylacetamide ((R)-27). Utiliz-

ing method B and using (R)-88 (1.10 g, 3.2 mmol), TFA (2.40 mL,

31.8 mmol), and CH₂Cl₂(50 mL) gave the crude product after workup that

was further purified by flash column chromatography (SiO₂; 0:95:5ꢀ5:90:5

MeOH/EtOAc/Et₃N) to give the desired product (530 mg, 68%) as a

yellow solid: mp 94ꢀ96 °C; [R]²⁷_Dþ24.0° (c 0.5, CHCl₃); R_f0.55 (95:5

1

EtOAc/Et₃N). H NMR (300 MHz, CDCl₃) δ 0.92ꢀ1.34 (m, 1H),

1.45ꢀ1.71 (m, 5H), 1.85ꢀ1.94 (m, 5H), 3.20 (d, J = 3.9 Hz, 1H), 4.39

(d, J = 5.7 Hz, 2H), 7.17ꢀ7.30 (m, 5H), 7.49ꢀ7.62 (br m, 1H). HRMS

(ESI) 247.1810 [M þ H^þ] (calcd for C₁₅H₂₂N₂OH^þ247.1810). Anal.

(C₁₅H₂₂N₂O 0.08H₂O): C, H, N.

3

’ ASSOCIATED CONTENT

(R)-N-Benzyl 2-Amino-3-phenylpropionamide ((R)-28).

Utilizing method A and using (R)-94 (3.50 g, 9.02 mmol), 10% PdꢀC

(0.35 g), and MeOH (90 mL) gave the crude product that was purified by

flash column chromatography (SiO₂; 1:10 MeOH/CH₂Cl₂) to give the

S

Supporting Information. Synthetic procedures and spec-

b

tral properties (IR, H and ¹³C NMR, MS) for the synthetic

1

desired compound (2.22 g, 97%) as a pale-yellow solid: mp 66ꢀ67 °C; [R]²⁵

intermediates, the PAADs, (R)-105, and (R)-106; table of ele-

mental analyses and table of MS spectra. This material is available

free of charge via the Internet at http://pubs.acs.org.

D

1

þ63.3° (c 1.4, CH₂Cl₂); R_f0.43 (1:20 MeOH/CH₂Cl₂). H NMR (300

MHz, CDCl₃) δ 0.98ꢀ1.62 (br s, 2H), 2.75 (dd, J = 9.0, 13.7 Hz, 1H), 3.29

(dd, J = 4.2, 13.7 Hz, 1H), 3.65 (dd, J = 4.2, 9.0 Hz, 1H), 4.37ꢀ4.50 (m, 2H),

7.20ꢀ7.35 (m, 10H), 7.56ꢀ7.64 (br t, 1H). HRMS (ESI) 277.1321 [M þ

Na^þ] (calcd for C₁₆H₁₈N₂ONa^þ277.1317). Anal. (C₁₆H₁₈N₂O): C, H, N.

(R)-N-Benzyl 2-Acetamido-3-methylbutanamide ((R)-105).

(R)-N-Benzyl 2-amino-3-methylbutanamide hydrochloride (0.79 g,

3.26 mmol) was dissolved in CH₂Cl₂(60 mL), and then Et₃N (1.4 mL,

9.78 mmol) and acetyl chloride (0.28 mL, 3.91 mmol) were carefully added

at 0 °C. The resulting solution was stirred at room temperature (2 h). Aque-

ous 10% citric acid (60 mL) was added, the layers separated, and the organic

layer was washed with a saturated aqueous NaHCO₃solution (60 mL). The

aqueous layer was extracted with CH₂Cl₂(3 ꢂ 60 mL). All of the organic

layers were combined, dried (Na₂SO₄), and concentrated in vacuo. The

residue was recrystallized with EtOAc to give the desired compound as a

’ AUTHOR INFORMATION

Corresponding Author

*Phone: 919-843-8112. Fax: 919-966-0204. E-mail: hkohn@

email.unc.edu.

’ ACKNOWLEDGMENT

The project was supported by UCB Pharma (Braine-l’Alleud,

Belgium) with the assistance of Dr. Robert Kramer. We thank

the NINDS and the ASP at the National Institutes of Health

with James P. Stables, and 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 solid: mp 223ꢀ224 °C; [R]²⁶þ32.8° (c 0.5, CHCl₃); R_f0.53

D

(100% EtOAc). ¹H NMR (400 MHz, CDCl₃) δ 0.94ꢀ0.97 (m, 6H), 1.98

4828

dx.doi.org/10.1021/jm2004305 |J. Med. Chem. 2011, 54, 4815–4830

Journal of Medicinal Chemistry

ARTICLE

White, and K. Wilcox. Harold Kohn has a royalty-stake position

in (R)-2.

(16) Perucca, E.; Yasothan, U.; Clincke, G.; Kirkpatrick, P. Lacosamide.

Nature Rev. Drug Discovery 2008, 7, 973–974.

(17) Baron, R.; Binder, A.; Wasner, G. Neuropathic pain: diagnosis,

pathophysiological mechanisms, and treatment. Lancet Neurol. 2010,

9, 807–819.

’ ABBREVIATIONS USED

(18) Costigan, M.; Scholz, J.; Woolf, C. J. Neuropathic pain: a mala-

daptive response of the nervous system to damage. Annu. Rev. Neurosci.

2009, 32, 1–32.

(19) O’Connor, A. B. Neuropathic pain: quality-of-life impact, costs

and cost eﬀectiveness of therapy. Pharmacoeconomics 2009, 27, 95–112.

(20) Dickenson, A. H.; Matthews, E. A.; Suzuki, R. Neurobiology of

neuropathic pain: mode of action of anticonvulsants. Eur. J. Pain 2002,

6, 51–60.

(21) McQuay, H.; Carroll, D.; Jadad, A. R.; Wiﬀen, P.; Moore, A.

Anticonvulsant drugs for management of pain: a systematic review. BMJ

[Br. Med. J.] 1995, 311, 1047–1052.

(22) Bee, L. A.; Dickenson, A. H. Eﬀects of lacosamide, a novel

sodium channel modulator, on dorsal horn neuronal responses in a rat

model of neuropathy. Neuropharmacology 2009, 57, 472–479.

(23) Beyreuther, B.; Callizot, N.; St€ohr, T. Antinociceptive eﬃcacy

of lacosamide in a rat model for painful diabetic neuropathy. Eur.

J. Pharmacol. 2006, 539, 64–70.

(24) Beyreuther, B.; Callizot, N.; St€ohr, T. Antinociceptive eﬃcacy

of lacosamide in the monosodium iodoacetate rat model for osteoar-

thritis pain. Arthritis Res. Ther. 2007, 9, R14.

(25) Beyreuther, B. K.; Callizot, N.; Brot, M. D.; Feldman, R.; Bain,

S. C.; St€ohr, T. Antinociceptive eﬃcacy of lacosamide in rat models for

tumor- and chemotherapy-induced cancer pain. Eur. J. Pharmacol. 2007,

565, 98–104.

(26) Beyreuther, B. K.; Geis, C.; St€ohr, T.; Sommer, C. Antihyper-

algesic eﬃcacy of lacosamide in a rat model for muscle pain induced by

TNF. Neuropharmacology 2007, 52, 1312–1317.

AED, antiepileptic drug; ASP, Anticonvulsant Screening Pro-

gram; B:P, brain-to-plasma; CCI, chronic constriction injury; CNS,

central nervous system; ED₅₀, eﬀective dose (50%); FAA, func-

tionalized amino acid; ip, intraperitoneally; MAC, mixed anhy-

dride coupling; MES, maximal electroshock seizure; NINDS,

National Institute of Neurological Disorders and Stroke; PAAD,

primary amino acid derivative; PI, protective index; po, orally; PNS,

peripheral nervous system;SAAD, secondary amino acid deriva-

tive; SAR, structureꢀactivity relationship; scMet, subcutaneous

Metrazol; TAAD, tertiary amino acid derivative; TD₅₀, neurolo-

gical impairment (toxicity, 50%); TFA, triﬂuoroacetic acid

’ REFERENCES

(1) CDC Epilepsy; http://www.cdc.gov/epilepsy/.

(2) Stafstrom, C. E. Epilepsy: a review of selected clinical syndromes

and advances in basic science. J. Cereb. Blood Flow Metab. 2006,

26, 983–1004.

(3) Fisher, R. S.; Boas, W. v. E.; Blume, W.; Elger, C.; Genton, P.;

Lee, P.; J., E., Jr. Epileptic seizures and epilepsy: deﬁnitions proposed by

the International League Against Epilepsy (ILAE) and the International

Bureau for Epilepsy (IBE). Epilepsia 2005, 46, 470–472.

(4) Bardel, P.; Bolanos, A.; Kohn, H. Synthesis and anticonvulsant

activities of R-acetamido-N-benzylacetamide derivatives containing an

electron-deﬁcient R-heteroaromatic substituent. J. Med. Chem. 1994, 37,

4567–4571.

(27) Hao, J.-X.; St€ohr, T.; Selve, N.; Wiesenfeld-Hallin, Z.; Xu, X.-J.

Lacosamide, a new anti-epileptic, alleviates neuropathic pain-like beha-

viors in rat models of spinal cord or trigeminal nerve injury. Eur.

J. Pharmacol. 2006, 553, 135–140.

(28) St€ohr, T.; Krause, E.; Selve, N. Lacosamide displays potent

antinociceptive eﬀects in animal models for inﬂammatory pain. Eur.

J. Pain 2006, 10, 241–249.

(29) McCorry, D.; Chadwick, D.; Marson, A. Current drug treat-

ment of epilepsy in adults. Lancet Neurol. 2004, 3, 729–735.

(30) Picot, M. C.; Baldy-Moulinier, M.; Daurs, J. P.; Dujols, P.;

Crespel, A. The prevalence of epilepsy and pharmacoresistant epilepsy

in adults: a population-based study in a western European country.

Epilepsia 2008, 49, 1230–1238.

(31) Butera, J. A. Current and emerging targets to treat neuropathic

pain. J. Med. Chem. 2007, 50, 2543–2546.

(32) Andurkar, S. V.; Stables, J. P.; Kohn, H. Synthesis and anti-

convulsant activities of (R)-(O)-methylserine derivatives. Tetrahedron:

Asymmetry 1998, 9, 3841–3854.

(33) Bꢀeguin, C.; LeTiran, A.; Stables, J. P.; Voyksner, R. D.; Kohn, H.

N-Substituted amino acid N⁰-benzylamides: synthesis, anticonvulsant,

and metabolic activities. Bioorg. Med. Chem. 2004, 12, 3079–3096.

(34) Paruszewski, R.; Rostaﬁnska-Suchar, G.; Strupinska, M.; Jaworski,

P.; Stables, J. P. Synthesis and anticonvulsant activity of some amino acid

derivatives. Part 1: Alanine derivatives. Pharmazie 1996, 51, 145–148.

(35) Paruszewski, R.; Rostaﬁnska-Suchar, G.; Strupinska, M.; Jaworski,

P.; Winiecka, I.; Stables, J. P. Synthesis and anticonvulsant activity of some

amino acid derivatives. Part 2: Derivatives of Gly, Ala, Leu, Pro, Trp, Phe

(4 Cl), and Ala(R-Me). Pharmazie 1996, 51, 212–215.

(5) Choi, D.; Stables, J. P.; Kohn, H. Synthesis and anticonvulsant

activities of N-benzyl-2-acetamidopropionamide derivatives. J. Med.

Chem. 1996, 39, 1907–1916.

(6) Conley, J. D.; Kohn, H. Functionalized ^DL-amino acid deriva-

tives. Potent new agents for the treatment of epilepsy. J. Med. Chem.

1987, 30, 567–574.

(7) Cortes, S.; Liao, Z. K.; Watson, D.; Kohn, H. Eﬀect of structural

modiﬁcation of the hydantoin ring on anticonvulsant activity. J. Med.

Chem. 1985, 28, 601–606.

(8) Kohn, H.; Conley, J. D. New antiepileptic agents. Chem. Br.

1988, 24 (231ꢀ232), 234.

(9) Kohn, H.; Conley, J. D.; Leander, J. D. Marked stereospeciﬁcity

in a new class of anticonvulsants. Brain Res. 1988, 457, 371–375.

(10) Kohn, H.; Sawhney, K. N.; Bardel, P.; Robertson, D. W.; Leander,

J. D. Synthesis and anticonvulsant activities of R-heterocyclic R-acetamido-

N-benzylacetamide derivatives. J. Med. Chem. 1993, 36, 3350–3360.

(11) Kohn, H.; Sawhney, K. N.; LeGall, P.; Conley, J. D.; Robertson,

D. W.; Leander, J. D. Preparation and anticonvulsant activity of a series

of functionalized R-aromatic and R-heteroaromatic amino acids. J. Med.

Chem. 1990, 33, 919–926.

(12) Kohn, H.; Sawhney, K. N.; LeGall, P.; Robertson, D. W.;

Leander, J. D. Preparation and anticonvulsant activity of a series of

functionalized R-heteroatom-substituted amino acids. J. Med. Chem.

1991, 34, 2444–2452.

(13) Kohn, H.; Sawhney, K. N.; Robertson, D. W.; Leander, J. D.

Anticonvulsant properties of N-substituted R,R-diamino acid deriva-

tives. J. Pharm. Sci. 1994, 83, 689–691.

(14) Morieux, P.; Salomꢀe, C.; Park, K. D.; Stables, J. P.; Kohn, H.

The structureꢀactivity relationship of the 3-oxy site in the anticonvul-

sant (R)-N-benzyl 2-acetamido-3-methoxypropionamide. J. Med. Chem.

2010, 53, 5716–5726.

(15) Salomꢀe, C.; Salomꢀe-Grosjean, E.; Park, K. D.; Morieux, P.;

Swendiman, R.; DeMarco, E.; Stables, J. P.; Kohn, H. Synthesis and

anticonvulsant activities of (R)-N-(4⁰-substituted)benzyl 2-acetamido-

3-methoxypropionamides. J. Med. Chem. 2010, 53, 1288–1305.

(36) Paruszewski, R.; Rostaﬁnska-Suchar, G.; Strupinska, M.; Winiecka,

I.; Stables, J. P. Synthesis and anticonvulsant activity of some amino acid

derivatives. Part 3: Derivatives of Ala, Arg, Tzl, Gly, and χAbu. Pharmazie

2000, 55, 27–30.

(37) Paruszewski, R.; Strupinska, M.; Rostaﬁnska-Suchar, G.;

Stables, J. P. Anticonvulsant activity of benzylamines of some amino

acids and heterocyclic acids. Protein Pept. Lett. 2003, 10, 475–482.

4829

dx.doi.org/10.1021/jm2004305 |J. Med. Chem. 2011, 54, 4815–4830

Journal of Medicinal Chemistry

ARTICLE

(38) Paruszewski, R.; Strupinska, M.; Stables, J. P.; Swiader, M.;

Czuczwar, S.; Kleinrok, Z.; Turski, W. Amino acid derivatives with

anticonvulsant activity. Chem. Pharm. Bull. 2001, 49, 629–631.

(39) LeTiran, A.; Stables, J. P.; Kohn, H. Design and evaluation of

aﬃnity labels of functionalized amino acid anticonvulsants. J. Med. Chem.

2002, 45, 4762–4773.

(58) Porter, R. J.; Cereghino, J. J.; Gladding, G. D.; Hessie, B. J.;

Kupferberg, H. J.; Scoville, B.; White, B. G. Antiepileptic Drug Devel-

opment Program. Cleveland Clin. Q. 1984, 51, 293–305.

(59) Lehmann, P. A. Quantifying stereoselectivity or how to choose

a pair of shoes when you have two left feet. Trends Pharmacol. Sci. 1982,

3, 103–106.

(40) Anderson, G. W.; Zimmerman, J. E.; Callahan, F. M. Reinves-

tigation of the mixed carbonic anhydride method of peptide synthesis.

J. Am. Chem. Soc. 1967, 89, 5012–5017.

(41) Park, K. D.; Morieux, P.; Salomꢀe, C.; Cotten, S. W.; Reamtong,

O.; Eyers, C.; Gaskell, S. J.; Stables, J. P.; Liu, R.; Kohn, H. Lacosamide

isothiocyanate-based agents: novel agents to target and identify lacosa-

mide receptors. J. Med. Chem. 2009, 52, 6897–6911.

(60) Barton, M. E.; Klein, B. D.; Wolf, H. H.; Steve White, H.

Pharmacological characterization of the 6 Hz psychomotor seizure

model of partial epilepsy. Epilepsy Res. 2001, 47, 217–227.

(61) Ramalingam, B.; Neuburger, M.; Pfaltz, A. Synthesis of chiral

C₂-symmetric methylene- and boron-bridged bis(imidazolines). Synth-

esis 2007, 4, 572–582.

(62) Vachal, P.; Jacobsen, E. N. Enantioselective catalytic addition of

HCN to ketoimines. Catalytic synthesis of quaternary amino acids. Org.

Lett. 2000, 2, 867–870.

(42) Hu, X. E. Nucleophilic ring opening of aziridines. Tetrahedron

2004, 60, 2701–2743.

(43) Whitehouse, D.; Hu, S.; Van Zandt, M. C.; Parker, G. Sub-

stituted amino carboxylic acids. U.S. Patent 7,498,356, March 3, 2009.

(44) Wei, Z.-L.; Petukhov, P. A.; Bizik, F.; Teixeira, J. C.; Mercola,

M.; Volpe, E. A.; Glazer, R. I.; Willson, T. M.; Kozikowski, A. P.

Isoxazolyl-serine-based agonists of peroxisome proliferator-activated

receptor: design, synthesis, and eﬀects on cardiomyocyte diﬀerentiation.

J. Am. Chem. Soc. 2004, 126, 16714–16715.

(63) Vachal, P.; Jacobsen, E. N. Structure-based analysis and opti-

mization of a highly enantioselective catalyst for the Strecker reaction.

J. Am. Chem. Soc. 2002, 124, 10012–10014.

(64) National Resource Council, Institute of Laboratory Animal

Resources. Guide for the Care and Use of Laboratory Animals; National

Academy Press: Washington, DC, 1996.

(65) Shen, M.; LeTiran, A.; Xiao, Y.; Golbraikh, A.; Kohn, H.;

Tropsha, A. Quantitative StructureꢀActivity Relationship Analysis of

Functionalized Amino Acid Anticonvulsant Agents Using k Nearest

Neighbor and Simulated Annealing PLS Methods. J. Med. Chem. 2002,

45, 2811–2823.

(45) Choi, D.; Kohn, H. Expedient syntheses of racemic 2,3-diamino-

propanoic acid derivatives. Tetrahedron Lett. 1995, 36, 7371–7374.

(46) Cernak, T. A.; Gleason, J. L. Synthesis of 5-chloromethylene

hydantoins and thiohydantoins. Heterocycles 2007, 71, 117–134.

(47) King, A. M. Synthesis and pharmacological evaluation of

primary amino acid derivatives (PAADs): novel neurological agents

for the treatment of epilepsy and neuropathic pain. Ph.D. Dissertation.

University of North Carolina at Chapel Hill, Chapel Hill, NC, 2010.

(48) Efange, S. M. N.; Kung, H. F.; Billings, J. J.; Blau, M. Synthesis

and biodistribution of 99mTc-labeled piperidinyl bis(aminoethanethiol)

complexes: potential brain perfusion imaging agents for single photon

emission computed tomography. J. Med. Chem. 1988, 31, 1043–1047.

(49) Macdonald, S. J. F.; Clarke, G. D. E.; Dowle, M. D.; Harrison,

L. A.; Hodgson, S. T.; Inglis, G. G. A.; Johnson, M. R.; Shah, P.; Upton,

R. J.; Walls, S. B. A ﬂexible, practical, and stereoselective synthesis of

enantiomerically pure trans-5-oxohexahydropyrrolo[3,2-β]pyrroles

(pyrrolidine-trans-lactams), a new class of serine protease inhibitors,

using acyliminium methodology. J. Org. Chem. 1999, 64, 5166–5175.

(50) Lall, M. S.; Karvellas, C.; Vederas, J. C. β-Lactones as a new class

of cysteine proteinase inhibitors: inhibition of hepatitis A virus 3C

proteinase by N-Cbz-serine β-lactone. Org. Lett. 1999, 1, 803–806.

(51) Klitgaard, H.; Matagne, A.; Gobert, J.; W€ulfert, E. Evidence for a

unique proﬁle of levetiracetam in rodent models of seizures and epilepsy.

Eur. J. Pharmacol. 1998, 353, 191–206.

(52) Stables, J. P.; Kupferberg, H. J. The NIH anticonvulsant drug

development (ADD) program: preclinical anticonvulsant screening

project. In Molecular and Cellular Targets for Anti-Epileptic Drugs;

Avanzini, G., Regesta, G., Tanganelli, O., Avoli, M., Eds.; John Libby

& Company Ltd: London, 1997; pp 191ꢀ198.

(53) Kaminski, R. M.; Livingood, M. R.; Rogawski, M. A. Allopreg-

nanolone analogs that positively modulate GABA_Areceptors protect

against partial seizures induced by 6-Hz electrical stimulation in mice.

Epilepsia 2004, 45, 864–867.

(54) Lambeng, N.; Lebon, F.; Christophe, B.; Burton, M.; De Ryck,

M.; Quꢀerꢀe, L. Arylsulfonamides as a new class of cannabinoid CB1

receptor ligands: identiﬁcation of a lead and initial SAR studies. Bioorg.

Med. Chem. Lett. 2007, 17, 272–277.

(55) Tjølsen, A.; Berge, O.-G.; Hunskaar, S.; Rosland, J. H.; Hole, K.

The formalin test: an evaluation of the method. Pain 1992, 51, 5–17.

(56) Vissers, K. C. P.; Geenen, F.; Biermans, R.; Meert, T. F.

Pharmacological correlation between the formalin test and the neuro-

pathic pain behavior in diﬀerent species with chronic constriction injury.

Pharmacol. Biochem. Behav. 2006, 84, 479–486.

(57) Dunham, N. W.; Miya, T. S. A note on a simple apparatus for

detecting neurological deﬁcit in rats and mice. J. Am. Pharm. Assoc. 1957,

46, 208–209.

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dx.doi.org/10.1021/jm2004305 |J. Med. Chem. 2011, 54, 4815–4830

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Primary amino acid derivatives: Compounds with anticonvulsant and neuropathic pain protection activities

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