Defining the Structural Parameters that Confer Anticonvulsant Activity by the Site-by-Site Modification of (R)-N′-Benzyl 2- Amino-3-methylbutanamide

Abstract

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

INTRODUCTION

Epilepsy is a heterogeneous mixture of disorders characterized by neuronal hyperexcitability and hypersynchronous neuronal firing, and it affects up to 1% of the world’s population.¹ There are over 40 antiepileptic drugs (AEDs) currently in clinical use,² yet, some 30% of patients are pharmacoresistant and do not respond to at least two of the first-line AEDs.³ Furthermore, compliance is often limited by adverse side effects (e.g., drowsiness, dizziness, nausea), experienced by nearly 40% of patients.⁴ Novel compounds with increased efficacy and decreased toxicity are needed to improve the quality of life of those suffering from epilepsy.

We recently reported that Primary Amino Acid Derivatives (PAADs^a, 1) exhibit potent anticonvulsant activities in the maximal electroshock seizure (MES) test⁵ in rodents,⁶ with activities approaching that of many clinical AEDs.⁷ Significantly, the structure-activity relationship (SAR) for PAADs and their N-acetylated analogs, the Functionalized Amino Acids (FAAs, 2), differed in several ways. First, PAAD anticonvulsant activity, unlike the FAAs, did not improve when a substituted heteroatom was included one atom removed from the C(2) chiral center.⁶ Second, PAAD activity was sensitive to the electronic properties of para-substituents on the N′-benzylamide ring, where activity was retained with an electron-withdrawing group but lost upon inclusion of an electron-releasing group.⁸ By comparison, FAAs containing either electron-donating or electron-withdrawing groups at the para-position provided compounds with excellent anticonvulsant activities.⁹ Third, the maximal activity for C(2)-hydrocarbon PAADs resided in the D-configuration, similar to the FAAs, but the differences in the ratio of the active stereoisomer to the less-active stereoisomer (eudismic ratio)¹⁰ differed widely. For (R)-3 and (S)- 3, the eudismic ratio in mice exceeded 20, while for (R)-4 and (S)-4 it was 3. For FAAs, the eudismic ratio was consistently above 10.^{9, 11, 12} The stark contrast between PAAD and FAA activity was most apparent for C(2)-hydrocarbon derivatives, where the C(2)-hydrocarbon was a branched alkyl moiety. We found that the C(2)-isopropyl PAAD (R)-3 (MES ED₅₀ = 15 mg/kg) and C(2)-tert-butyl PAAD (R)-4 (MES ED₅₀ = 14 mg/kg) were potent anticonvulsants, while their FAA counterparts (R)-5 (MES ED₅₀ = >100, <300 mg/kg) and (R)-6 (MES ED₅₀ = > 300 mg/kg) were not.⁶ Collectively, these results suggest that the C(2)-hydrocarbon PAADs represent a novel class of anticonvulsants.

The differences in the PAAD and FAA SAR have led us to question the optimal chemical framework for PAADs. At the outset,⁶ we assumed that PAADs and FAAs would require similar structural features for maximal activity. Among these were the diamine backbone containing a carbonyl unit and a N′-terminal benzylamide. In the present study, we systematically modified six sites in the core structure of PAAD 3. Significantly, we found that several key structural motifs associated with maximal FAA seizure protection were not necessary for potent PAAD activity. These findings provided additional evidence that C(2)-hydrocarbon PAADs are a distinct, new class of potent anticonvulsants. The structural simplicity of these PAADs, and the excellent water solubility of their corresponding salts, provide opportunities for further development.

RESULTS AND DISCUSSION

Choice of Compounds

C(2)-Hydrocarbon PAADs possess significant anticonvulsant activities and our studies indicated that many of the FAA structural hallmarks do not apply to PAADs.^{6, 8} Therefore, we investigated the originally proposed PAAD structural framework. For most of our studies, we systematically modified (R)-C(2)-isopropyl PAAD 3, which was among the most potent PAADs discovered. Six structural features common to both PAADs and FAAs were examined by preparing 7–24 (Figure 1): the carbonyl unit (Site A); the amide bond (Site B); the amide methylene linker length (Site C); the regiosubstitution of the N′-benzylamide (Site D); the need for a N′-benzylamide (Site E); and the C(2)-amino functionality (Site F). All C(2)- isopropyl PAAD analogs (7–18, 23, 24) were synthesized in the (R)-configuration. For N′-benzyl 2-aminobutanamide 19 and N-benzyl 2-hydroxybutanamide 22, we synthesized the (R)- configuration, for 21, we prepared the (R,S) and (S)-configurations, and N-benzyl butanamide (20) does not have a chiral center.

Figure 1.

C(2)-Isopropyl PAAD (R)-3 Analogs (Sites A-F)

C(2)-Isopropyl PAAD analogs (Sites A–C)

The importance of the carbonyl unit for FAA anticonvulsant activity has been demonstrated by an isoelectronic substitution of the amide carbonyl with a thiocarbonyl group,¹² and we conducted a similar investigation using (R)-7 (Site A). We further examined the value of the carbonyl unit by replacing it with a methylene group ((R)-8).

We have reported that functionalized amido ketones (FAKs) exhibit significant anticonvulsant activities that were comparable to FAAs; however, an increase in neurological toxicity was also observed (Site B).¹³ In a similar manner, we replaced the nitrogen of the amide bond in (R)-3 with a methylene group ((R)-9) or oxygen ((R)-10) to create either a primary amino ketone or primary amino ester, respectively.

The distance between the amide bond and the aromatic ring was briefly investigated for FAAs, and maximal activity was seen for the N′-benzylamide moiety (n = 1). Therefore, we examined the anticonvulsant activities of PAADs that contain 0–4 methylene units between the amide bond and the aromatic ring ((R)-3, (R)-11–14) (Site C).

Regiosubstitution in N′-Benzylamide PAADs (Site D)

Recently, we systematically evaluated the effect of a fluoro and trifluoromethoxy group at the 2′, 3′, and 4′ postions in the N′-benzyl moiety of FAAs and determined that the 4′ derivatives displayed the highest level of anticonvulsant activity,⁹ consistent with previous studies.^{11, 14} Accordingly, we evaluated the effect of a trifluoromethoxy group at the 2′, 3′, and 4′ positions of PAAD (R)-3 by preparing (R)-15, (R)-16, and (R)-17,⁸ respectively.

N′-Substituted C(2)-isopropyl PAADs (Site E)

We evaluated several N′-substituted FAAs, including a N′-methylamide, N′-benzhydrylamide, and N′-benzylamide, and found that the N′-benzylamide unit is necessary for the excellent anticonvulsant activity of FAAs.¹⁵ We substituted the aromatic ring in (R)-3 for a cyclohexyl ring ((R)-18) to examine the influence of the planar ring system on anticonvulsant activity.

α-Substituted N-benzylbutanamides (Site F) and N-Substituted C(2)-Isopropyl PAADs (Site F′)

In our final analysis we examined the importance of the amino group and amine substitution. The limited availability of 2-substituted 3-methylbutanoic acid reagents prompted the investigation of N-benzylbutanamide derivatives ((R)-19, 20, (R,S)-21, (S)-21, and (R)-22) instead of N′-benzyl 3-methylbutanamide derivatives (Site F). Previously, the C(2)-acetamido of (R,S)-N′-benzyl 2-acetamido-2-phenylacetamide ((R,S)-25, ED₅₀ = 20 mg/kg) was systematically replaced with a hydrogen, methyl, hydroxy, methoxy, or halogen group. The hydroxy and methoxy groups provided moderate MES activities (ED₅₀ = >30, <100 mg/kg) but were appreciably less active than the parent FAA.¹⁶ In a similar study, the C(2)-acetamido group of (R,S)-N′-benzyl 2-acetamido-3-methoxypropionamide ((R,S)-26, ED₅₀ = 8.3 mg/kg) was replaced with a methyl, hydroxy, and amino group. The methyl and amino groups also resulted in moderate MES activities (ED₅₀ = >30, <100 mg/kg) but, again, the activities were lower than the corresponding FAA.¹⁷ Accordingly, both studies concluded that the C(2)-acetamido group showed superior anticonvulsant activity. We conducted a similar investigation in which we replaced the amino group in (R)-19 with a hydrogen (20), methyl ((R,S)-21), or hydroxy group ((R)-22). The moderate activity of (R,S)-21 prompted our efforts to prepare (R)-21 and (S)-21 to determine if anticonvulsant activity resided in the (R)-stereoisomer. We successfully synthesized (S)-21 but were unable to prepare (R)-21.

Finally, to assess the importance of N-terminal substitution (Site F′), we prepared the Secondary Amino Acid Derivative (SAAD) (R)-23 and the Tertiary Amino Acid Derivative (TAAD) (R)-24 in the C(2)-isopropyl series. Earlier studies did not reveal a general pattern to correlate anticonvulsant activity with amine substitution.^18–20 We observed that N-amine substitution improved anticonvulsant activity in several cases, but in other compounds, we saw a loss of activity.

Chemistry

Thioamide (R)-7 was prepared by treating (R)-27⁶ with excess Lawesson’s reagent at reflux,¹² followed by trifluoroacetic acid (TFA) deprotection of the t-Boc group (Scheme 1). We were mindful of the potential for thiation of the carbamate carbonyl in (R)-27, but the ¹³C NMR spectrum contained a signal at ~155 ppm, the typical shift for a carbamate carbon, rather than ~190 ppm for the thiocarbamate.²¹ In agreement with the proposed structure of (R)-27, we observed the thioamide carbon signal in the ¹³C NMR at 205 ppm.

Scheme 1.

Synthesis of (R)-N′-benzyl 2-amino-3-methylthiobutanamide ((R)-7)

Diamine (R)-8¹⁴ was synthesized by directly reducing (R)-3⁶ with borane in THF (Scheme 2). We followed a previously reported procedure¹⁴ but changed the workup.

Scheme 2.

Synthesis of (R)-1-N-benzylamino-2-amino-3-methylbutane ((R)-8)

We synthesized PAAD (R)-9 by coupling (R)-29⁶ with N,O-dimethylhydroxylamine hydrochloride (HCl) in the presence of 2-chloro-4,6-dimethoxy-1,3,5-triazine (CMDT) and base²² to give Weinreb amide (R)-30 (Scheme 3). Weinreb amides readily react with Grignard reagents and are widely known as useful precursors to ketones.^22–24 Accordingly, (R)-30 was reacted with phenethylmagnesium bromide to give (R)-31, followed by HCl deprotection to give PAAD (R)-9 as a HCl salt.

Scheme 3.

Synthesis of (R)-4-amino-2-methyl-6-phenyl-4-hexanone hydrochloride ((R)- 9)

We synthesized (R)-32 from (R)-29⁶ using a mild esterification method ²⁵ that employed benzylchloroformate in the presence of base and catalytic amounts of 4-dimethylaminopyridine (DMAP) (Scheme 4). Subsequent TFA deprotection of (R)-32 gave PAAD (R)-10.

Scheme 4.

Synthesis of (R)-O-benzyl 2-amino-3-methylbutanoate ((R)-10)

The C(2)-isopropyl PAAD analogs (R)-11–16 and (R)-18 were synthesized by coupling (R)-29⁶ with commercially available amines (33–39) and using the standard mixed anhydride coupling (MAC) procedure,²⁶ to give amides (R)-40–46, followed by TFA deprotection to the corresponding PAADs (Scheme 5).

Scheme 5.

Synthesis of C(2)-isopropyl PAADs (R)-11–16 and (R)-18

PAADs 20, (R,S)-21, (S)-21, and (R)-22 were synthesized from commercially available carboxylic acids 47, (R,S)-48, (S)-48, and (R)-49 (Scheme 6). For (R)-20, (R,S)-21, and (S)-21, we used the MAC procedure with benzylamine. We attempted to prepare (R)-21 to compare its pharmacological activity with (S)-21 but were unsuccessful.²⁷ PAAD (R)-22 was prepared by treating (R)-49 with benzylamine in the presence of the commercially available catalyst, 3,5- bis(trifluoromethyl)benzeneboronic acid²⁸ (50) (Scheme 6).

Scheme 6.

Synthesis of C(2)-isopropyl analogs: α-Substituted N-benzylbutanamides 20, (R,S)-21, (S)-21, and (R)-22

SAAD (R)-23 was prepared in three steps, beginning with the t-Boc protection of commercially available acid (R)-51 to give (R)-52 (Scheme 7). (R)-52 was coupled with benzylamine using the MAC procedure to give amide (R)-53 and then HCl deprotected to give SAAD (R)-23 as the HCl salt.

Scheme 7.

Synthesis of (R)-N′-benzyl N,N-dimethylamino-3-methylbutanamide ((R)-23)

Synthesis of TAAD (R)-24 began with reductive condensation of D-valine ((R)-54) with formaldehyde using 10% Pd-C in the presence of H₂ to give (R)-55 (Scheme 8).^29–31 (R)-55 was coupled with benzylamine using the condensing reagent 4-(4,6-dimethoxy-1,3,5-triazin-2-yl)-4- methylmorpholinium chloride (DMTMM)³² to give TAAD (R)-24.

Scheme 8.

Synthesis of (R)-N′-benzyl N,N-dimethylamino-3-methylbutanamide ((R)-24)

In the experimental section, we detail the final step (synthetic procedure and characterization) for all compounds evaluated in the animal models. In the Supporting Information, we provide our experimental procedures for all compounds prepared and their physical and spectroscopic properties.

Pharmacological Activity

PAADs 7–22 and 24 were evaluated for anticonvulsant activity using the MES test at the National Institute of Neurological Disorders and Stroke Anticonvulsant Screening Program (NINDS ASP), following the procedures described by Stables and Kupferberg,⁵ and described in the preceeding paper.⁸ PAAD 23 was evaluated for anticonvulsant activity at UCB Pharma, following the procedures described by Klitgaard,³³ as previously reported.⁸ The pharmacological data from the MES tests are summarized in Tables 1 and 2. PAADs tested at the NINDS ASP were evaluated in the subcutaneous Metrazol^® (scMet) seizure model⁵ and displayed little to no protection.²⁷

Table 1.

Pharmacological activities of C(2)-isopropyl PAAD amide analogs (Sites A–E) in mice (mg/kg)^a and rats (mg/kg)^b

						Mice (ip)a			Rat (po)b
Cmpd No	Site(s)	X	Y	n	R	MES,c ED50	Tox,d TD50	PIe	MES,c ED50	Tox,f TD50	PIe
(R)-3g		C=O	NH	1	C6H5	15 [0.25] (13–18)	70 [0.25] (63–80)	4.8	11 [0.25] (9.1–13)	>500	>45
(R)-7	A	C=S	NH	1	C6H5	>30, <100 [0.5]	>30, <100 [0.5]		<30 [0.5–2.0]	>30 [0.25–4.0]
(R)-8	A	CH2	NH	1	C6H5	>30, <100 [0.5]	>100, <300 [0.5]		>30 [0.25–4.0]	>30 [0.25–4.0]
(R)-9	B	C=O	CH2	1	C6H5	>30, <100	>100, <300		NDh	NDh
(R)-10	B	C=O	O	1	C6H5	>300 [0.5]	>300 [0.5]		>30 [0.25]	>30 [0.25–4.0]
(R)-11	C	C=O	NH	0	C6H5	>30, <100 [0.5]	>30, <100 [0.5]		NDh	NDh
(R)-12	C	C=O	NH	2	C6H5	10 [0.25] (8.3–14)	50 [0.25] (42–80)	5.0	<30 [0.25–2.0]	>30 [0.24–4.0]
(R)-13	C	C=O	NH	3	C6H5	16 [0.25] 13–17	43 [0.25] (38–47)	2.7	<30 [0.25–0.5]	>30 [0.25–4.0]
(R)-14i	C	C=O	NH	4	C6H5	11 [0.25] (9.9–13)	38 [0.25] (34–42)	3.5	>30 [0.25–1.0]	>30 [0.25–4.0]
(R)-15j,k	D	C=O	NH	1	C6H4(2′-OCF3)	9.2 [0.25] (7.7–11)	51 [0.25] (38–65)	5.5	33 [0.5] (27–44)	>500	>15
(R)-16l	D	C=O	NH	1	C6H4(3′-OCF3)	7.1 [0.25] (6.3–8.1)	30 [0.25] (27–32)	4.2	10 [1.0] (7.5–14)	43 [1.0] (35–57)	4.3
(R)-17m,n,o	D	C=O	NH	1	C6H4(4′-OCF3)	16 [0.25] (14–20)	84 [0.25] (67–109)	5.3	18 [1.0] (12–28)	>500	>27
(R)-18	E	C=O	NH	1	C6H11	>30, <100 [0.5]	>100, <300 [0.5]		~15 [0.25–1.0]	>30 [0.25–4.0]
(R)-56	C+D	C=O	NH	2	C6H4(2′-F)	27 [0.25] (22–32)	79 [0.25] (71–84)	2.9	20 [4.0] (11–31)	>500	>25
(R)-57	C+D	C=O	NH	2	C6H4(3′-F)	20 [0.25] (17–22)	67 [0.25] (61–75)	3.4	NDh	NDh
(R)-58	C+D	C=O	NH	2	C6H4(4′-F)	>10, <30	>30, <100		NDh	NDh
(R)-59m	D	C=O	NH	1	C6H4(4′-F)	32 [0.25] (28–39)	97 [0.25] (88–105)	3.0	21 [0.5] (13–31)	>500	>24
(R)-26p						4.5 [0.5] (3.7–5.5)	27 [0.25] (26–28)	6.0	3.9 [2.0] (2.9–6.2)	>500	>120
phenytoinq						9.5 [2.0] (8.1–10)	27 [0.25] (26–28)	2.8	30 [4.0] (22–39)	>3000	>100
phenobarbitalq						22 [1.0] (15–23)	66 [0.5] (63–73)	3.0	9.1 [5.0] (7.6–12)	61 [0.5] (44–96)	6.7
valproateq						270 [0.5] (250–340)	430 [0.25] (370–450)	1.6	490 [0.5] (350–730)	280 [0.5] (190–350)	0.6

^aThe compounds were administered intraperitoneally to adult male albino CF-1 mice under the auspices of the NINDS ASP. ED₅₀ and TD₅₀ values are in mg/kg. A dose-response curve was generated for all compounds that displayed sufficient activity (4–6 doses tested, n = 8 per dose) and the dose-effect 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.

^bThe compounds were administered orally to adult male albino Sprague Dawley rats under the auspices of the NINDS ASP. ED₅₀ and TD₅₀ values are in mg/kg.

^cMES = maximal electroshock seizure test.

^dTD₅₀ value determined from the rotorod test.

^ePI = protective index (TD₅₀/ED₅₀).

^fTox = behavioral toxicity.

^gRef 6.

^hND = not determined.

ⁱ6 Hz (32 mA) ED₅₀ = 24 mg/kg [0.25 h] (17–29).

^j6 Hz (32 mA) ED₅₀ = < 100 mg/kg;

^kFormalin ED₅₀ = 9.2 mg/kg [0.25 h].

^l6 Hz (32 mA) ED₅₀ = 18 mg/kg [0.5 h] (14–24).

^mRef 8.

ⁿ6 Hz (32 mA) ED₅₀ = 28 mg/kg [0.5 h] (18–40).

^oFormalin ED₅₀ = 16 mg/kg [0.25 h].

^pRef 11.

^qRef 7.

Table 2.

Pharmacological activities of C(2)-substituted N-benzyl butanamides (Site F) in mice (mg/kg)^a and rats (mg/kg)^b and primary (PAAD), secondary (SAAD), and tertiary (TAAD) amino acid derivatives of (R)-N-benzyl 2-amino-3-methylbutanamide (Site F′) in mice (mg/kg)^a

				Mice (ip)a			Rat (po)b
Cmpd No.	Site	R	X	MES,c ED50	Tox,d TD50	PIe	MES,c ED50	Tox,f TD50	PIe
(R)-19g	F	CH2CH3	NH2	18 [0.25] (10–25)	80 [0.25] (65–95)	4.4	11 [2.0] (7.8–16)	>500	>45
20	F	CH2CH3	H	>30, <100 [0.5]	>100, <300 [0.5]		>30 [0.25–4.0]	>30 [0.25–4.0]
(R,S)-21h	F	CH2CH3	CH3	56 [0.25] (45–69)	165 [0.25] (148–180)	2.9	51 [0.5] (35–71)	>500	>9.8
(S)-21	F	CH2CH3	CH3	>100, <300 [0.5]	~300 [0.5]		>30 [0.25–4.0]	>30 [0.25–4.0]
(R)-22	F	CH2CH3	OH	>30, <100 [0.5]	>100, <300 [0.5]		>30 [0.25–4.0]	>30 [0.25–4.0]
(R)-3g	F′	CH(CH3)2	NH2	15 [0.25] (13–18)	70 [0.25] (63–80)	4.7	11 [0.25] (9.1–13)	>500	>45
(R)-23i	F′	CH(CH3)2	N(H)CH3	25	NDj		NDj	NDj
(R)-24	F′	CH(CH3)2	N(CH3)2	>30, <100 [0.5]	>100, <300 [0.5]		NDj	NDj

^aThe compounds were administered intraperitoneally to adult male albino CF-1 mice under the auspices of the NINDS ASP. ED₅₀ and TD₅₀ values are in mg/kg. A dose-response curve was generated for all compounds that displayed sufficient activity (4–6 doses tested, n = 8 per dose) and the dose-effect 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.

^bThe compounds were administered orally to adult male albino Sprague Dawley rats under the auspices of the NINDS ASP. ED₅₀ and TD₅₀ values are in mg/kg.

^cMES = maximal electroshock seizure test.

^dTD₅₀ value determined from the rotorod test.

^ePI = protective index (TD₅₀/ED₅₀).

^fTox = behavioral toxicity.

^gRef 6.

^h6 Hz (32 mA) = 63 mg/kg.

ⁱThe compounds were administered intraperitoneally to adult male NMRI mice under the auspices of UCB. ED₅₀ and TD₅₀ values are in mg/kg and were determined 30 min after ip administration.

^jND = not determined.

Compound (R)-3 emerged as a potent anticonvulsant (MES ED₅₀ = 15 mg/kg) that possessed pain attenuating properties (formalin ED₅₀ = 15 mg/kg) from the SAR investigation at the C(2)-carbon.⁶ This excellent activity came as a surprise since it did not follow the trends observed in the FAA series. Therefore, we have questioned several aspects of the original PAAD structural framework to determine if the basic tenets of the FAA blueprint applied to this C(2)- hydrocarbon PAAD. We investigated the importance of six properties (Sites A–F) that were common in the FAAs and report their anticonvulsant activity and neurological toxicity in Tables 1 and 2.

First, we determined the effect of the carbonyl unit (Site A) on anticonvulsant activity (Table 1). Replacement of the carbonyl unit in (R)-3 with a thiocarbonyl unit to give (R)-7 resulted in decreased MES activity in mice (ED₅₀ (mg/kg): (R)-3, 15; (R)-7, >30, <100), but notable activity was observed in rats (ED₅₀ = <30 mg/kg). Reducing the carbonyl unit in (R)-3 to a methylene group to provide (R)-8 led to decreased activity in both mice (ED₅₀ = >30, <100 mg/kg) and rats (ED₅₀ = >30 mg/kg).

Next, we determined the effect of the amide bond (Site B) on anticonvulsant activity (Table 1). Converting the amide (R)-3 (Y = NH) to ketone (R)-9 (Y = CH₂) decreased the MES activity (ED₅₀ (mg/kg): (R)-3, 15; (R)-9, >30, <100) and conversion to the ester (R)-10 (Y = O) abolished activity (ED₅₀ = >300 mg/kg).

Then, at Site C we looked at the optimal methylene linker length between the amide bond and the aromatic ring (Table 1). Direct linkage of the aromatic ring to the amide bond to give (R)-11 (n = 0) resulted in a significant drop in activity from the parent compound (R)-3 (n = 1) (ED₅₀ (mg/kg): (R)-3, 15; (R)-11, >30, <100). However, extending the linkage of the parent compound (R)-3 (n = 1) by one methylene unit to provide (R)-12 (n = 2) increased anticonvulsant activity by ~30% (ED₅₀ = 10 mg/kg). The activity increase was associated with a neurotoxicity increase over the parent compound (TD₅₀ (mg/kg): (R)-3, 70; (R)-12, 50); however, the PI of (R)-3 (4.8) and (R)-12 (5.0) were nearly equal. Extending the linker by another carbon to give (R)-13 (n = 3) led to comparable activity with (R)-3 (ED₅₀ (mg/kg): (R)-3, 15; (R)-13, 16), but the increased toxicity for (R)-13 (TD₅₀ = 43 mg/kg) reduced the PI to 2.7. A final extension to n = 4 to give (R)-14 resulted in comparable activity in mice (ip) with (R)-3 and (R)- 13 (ED₅₀ (mg/kg): (R)-14, 11; (R)-3, 15; (R)-13, 16), with a PI of 3.5. The anticonvulsant activities of (R)-12–14 were surprising since in the FAA series, an increase in the methylene linkage led to a sharp drop in activity.³⁴

Site D compared the effect of substitution on the N′-benzylamide ring, where we systematically placed a trifluoromethoxy group at the 2′ ((R)-15), 3′ ((R)-16), and 4′-positions ((R)-17) (Table 1). All regiosubstitutions displayed excellent MES activities in mice (ED₅₀ (mg/kg): (R)-15, 9.2; (R)-16, 7.1; (R)-17, 16). The ED₅₀ value for (R)-16 was 2-fold more active than the parent compound (R)-3 (ED₅₀ = 15 mg/kg) but the increase in activity was associated with an increase in neurotoxicity (ED₅₀ (mg/kg): (R)-3, 70; (R)-16, 30). Comparison of the MES activity in rats (Table 1) showed ~3-fold drop in activity going from (R)-3 to (R)-15 (ED₅₀ (mg/kg): (R)-3, 11; (R)-15, 33). The MES activity of (R)-16 in the rat (ED₅₀ = 10 mg/kg) was comparable with (R)-3 (ED₅₀ = 11 mg/kg) but there was a sharp increase in behavioral toxicity (TD₅₀ (mg/kg): (R)-3, >500; (R)-16, 43). The potent anticonvulsant activity observed for (R)-15 led us to evaluate this compound in the formalin neuropathic pain model³⁵ where we observed excellent activity in the inflammatory phase (ED₅₀ = 9.2 mg/kg, data not shown).³⁶

Next, we examined the need for a benzyl moiety (Site E) (Table 1). We replaced the aromatic ring ((R)-3) with a cyclohexyl ring ((R)-18) and observed a decrease in anticonvulsant activity (ED₅₀ (mg/kg): (R)-3, 15; (R)-18, >30, <100). When (R)-18 was evaluated in the rat, we found this PAAD to have excellent activity (ED₅₀ = ~15 mg/kg). While our data indicates that the N′-benzyl substituent is preferred over the saturated N′-cyclohexylmethyl unit, we did not anticipate the anticonvulsant activity of (R)-18.

Finally, we investigated the importance of the C(2)-amino functionality (Site F) by comparing the unsubstituted N-benzyl butanamide 20 (X = H), the methyl-substituted N-benzyl butanamide (R,S)-21 and (S)-21 (X = CH₃), and the hydroxy-substituted N-benzyl butanamide (R)-22 (X = OH) with the parent PAAD (R)-19 (X = NH₂) (Table 2). Compounds 20 and (R)-22 gave similar, modest activity (ED₅₀ = >30, <100 mg/kg) in mice and no appreciable activity in rats (ED₅₀ = >30 mg/kg). (R,S)-21 also displayed modest protection in mice (ED₅₀ = 56 mg/kg) with minimal neurotoxicity (TD₅₀ = 165 mg/kg), and moderate activity in rats (ED₅₀ = 51 mg/kg) without any detectable behavioral toxicity (TD₅₀ = >500 mg/kg). We were surprised by the activity of (R,S)-21 since the methyl group lacks the hydrogen bonding capabilities afforded by an amino or a hydroxy group. Evaluation of (S)-21 showed a decrease in anticonvulsant activity (ED₅₀ = >100, <300 mg/kg), suggesting that activity resided predominantly in the (R)-isomer. Unfortunately, we were unable to complete the synthesis of (R)-21 to confirm this suggestion. The activities of the primary (PAAD), secondary (SAAD), and tertiary (TAAD) amino acid analogs of (R)-N′-benzyl 2-amino-3-methylbutanamide ((R)-3) are summarized in Table 2 (Site F′). Comparison of the SAAD (R)-23 with the PAAD (R)-3 showed decreased activity in the MES test (ED₅₀ (mg/kg): (R)-3, 15; (R)-23, 25). Furthermore, comparison of the TAAD (R)-24 with the SAAD (R)-23 showed an additional decrease in activity in the MES test (ED₅₀ (mg/kg): (R)-23, 25; (R)-24, >30, <100). Therefore, the MES data for PAAD (R)-3, SAAD (R)-23, and TAAD (R)-24 revealed a linear decrease in anticonvulsant activity as we successively incorporated methyl groups at the C(2)-amine site. This trend differed from the reported pattern for racemic C(2)-methyl and C(2)-CH₂OCH₃ PAADs, in which successive N-methylation led to non-linear trends in MES activity.¹⁸

Our findings indicated that PAAD anticonvulsant activity increased when a N-phenethylamide moiety (Table 1, (R)-12) was used and when an electron-withdrawing substituent (OCF₃) was incorporated on the aromatic ring (Table 1, (R)-15–17). Accordingly, we prepared three mono-fluoro regioisomers of (R)-N-phenethyl 2-amino-3-methylbutanamide, ((R)- 56–58) using the same general procedure outlined in Scheme 5. We observed that anticonvulsant activity remained high in the MES test (mice, ip) for all three regioisomers (ED₅₀ (mg/kg): (R)- 56, 27; (R)-57, 20; (R)-58, >10, <30), but there was no apparent benefit over the parent, unsubstituted PAAD (R)-12 (MES ED₅₀ = 10 mg/kg). However, we observed an increase in activity and a decrease in toxicity in the MES test for (R)-56 going from mice (ip) to rats (po). (R)-58 exhibited only slightly better activity than the corresponding benzyl analog (R)-59⁸ (ED₅₀ = 32 mg/kg), in terms of potency in the MES test and PI values (Table 1).

CONCLUSIONS

Examination of PAAD Sites A–F revealed that the amide bond (Sites A and B) was important for effective seizure protection and altering this unit affected the hydrogen bonding properties of the compounds. Assessment of the methylene linker (Site C) showed that at least one carbon between the amide bond and the aromatic rings was necessary for anticonvulsant activity but incorporation of up to three additional methylene units provided excellent activity. When considering activity, toxicity, and the protective index for these C(2)-isopropyl PAADs, the optimal linker length appeared to be when n = 2, instead the n = 1, as seen in the FAAs. Similarly, we found that replacement of the benzylamide group by a cyclohexylmethylamide (Site E) led to lower anticonvulsant activity, but the drop was modest. Thus, there seems to be structural latitude at Sites C and E, where modifications retain excellent activities. Comparison of trifluoromethoxy regioisomers of (R)-N′-benzyl 2-amino-3-methylbutanamide (Site D) revealed that superb seizure protection was associated with all positions. The activity increase was associated with a neurotoxicity increase but the PI values were comparable with the parent, unsubstituted PAAD (R)-3. Lastly, we evaluated our choice of the C(2)-amino group (Site F) since the earlier SAR studies were centered around primary amino acid derivatives.^{6, 8} Of the C(2)-groups investigated, the amino group was most active but was not mandatory for activity. The activities of the primary (PAAD), secondary (SAAD), and tertiary (TAAD) amino acid analogs of (R)-N′-benzyl 2-amino-3-methylbutanamide ((R)-3) showed a steady loss of anticonvulsant activity with N-methylation. Collectively, the findings of this study, along with the earlier observations that the anticonvulsant activity of C(2)-hydrocarbon PAADs do not improve when a substituted heteroatom is included one atom removed from the C(2)-center⁶ and that PAAD activity is sensitive to the electronic effects of N′-benzylamide substituents,⁸ indicated that the C(2)-hydrocarbon PAADs have an unique SAR and. Future work will investigate PAAD mechanism of action to determine if the difference in observed efficacy of PAADs and FAAs is a consequence of varying pharmacokinetics or if PAADs interact with a novel molecular target.

EXPERIMENTAL SECTION

General methods

The general methods used in this study were identical to those previously reported⁶ and are summarized in the Supporting Information. All compounds were checked by TLC, ¹H and ¹³C NMR, MS, and elemental analyses. The analytical results were within ±0.40% of the theoretical value. The TLC, NMR, and the analytical data confirmed the purity of the products was ≥95%.

General Procedure for PAAD Preparation Using TFA Deprotection (Method A)

TFA (15 equiv) was added to an anhydrous CH₂Cl₂ solution of the tBoc-protected N′-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 (3x). The combined aqueous layers were washed with CH₂Cl₂ (2x), basified (pH 10–12) with aqueous 4 M NaOH, and extracted with CH₂Cl₂ (3x). The combined organic layers were washed with brine (2x), dried (Na₂SO₄), evaporated in vacuo, and purified by column chromatography (SiO₂). Basic: The crude product was diluted with CH₂Cl₂ and washed with aqueous 1 M Na₂CO₃ (3x). The aqueous layers were combined and washed with CH₂Cl₂ (2x). All of the CH₂Cl₂ layers were combined and successively washed with H₂O (2x) and brine (2x), dried (Na₂SO₄), evaporated in vacuo, and purified by column chromatography (SiO₂).

General Procedure for the Preparation of N′-Benzylamide Amino Acid Derivatives Using the Mixed Anhydride Coupling (MAC) Method (Method B)

An anhydrous THF solution of carboxylic acid (0.5–2.0 M) was cooled to −78 °C in a dry ice/acetone bath under an inert atmosphere (Ar or N₂), and 4-methylmorpholine (NMM) (1.3–1.5 equiv) was added. After the mixture was stirred (2–10 min), isobutyl chloroformate (IBCF) (1.1–1.5 equiv) was added leading to the precipitation of a white solid. The reaction was allowed to proceed for an additional 15–25 min, and then benzylamine (1.05–1.36 equiv) was added at −78 °C. The reaction mixture was allowed to stir at room temperature (1.5 h), and then the insoluble salts were filtered. The organic layer was concentrated in vacuo, and the product was purified by column chromatography (SiO₂).

(R)-N′-Benzyl 2-Amino-3-methylbutanethioamide ((R)-7)

Utilizing Method A with (R)-28 (1.72 g, 5.34 mmol), TFA (5.95 mL, 80.1 mmol), and CH₂Cl₂ (18 mL) gave the crude product after acidic workup and further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (0.89 g, 76%) as a yellow oil: [α]^28.5_D +43.7° (c 1.0, CHCl₃); R_f 0.50 (1:1 EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 0.72 (d, J = 6.8 Hz, 3H), 1.07 (d, J = 6.8 Hz, 3H), 1.36 (s, 2H), 2.75–2.87 (m, 1H), 3.78 (d, J = 2.8 Hz, 1H), 4.84 (dd, J = 4.6, 15.0 Hz, 1H), 4.91 (dd, J = 5.0, 15.0 Hz, 1H), 7.29–7.38 (m, 5H), 9.75–9.95 (br s, 1H); LRMS (ESI) 223.13 [M + H⁺] (calcd for C₁₂H₁₈N₂SH⁺ 223.14); Anal. (C₁₂H₁₈N₂S): C, H, N.

(R)-1-N′-Benzylamino-2-amino-3-methylbutane ((R)-8).³⁷

(R)-3⁶ (3.38 g, 16.4 mmol) was dissolved in anhydrous THF (65 mL) and cooled to 0 °C. BH₃•THF (1 M, 49.19 mL, 49.19 mmol) was added dropwise and the mixture was heated to reflux (18 h). The mixture was cooled to 0 °C and aqueous 0.5 M HCl (100 mL) was slowly added. The THF was evaporated in vacuo and Et₂O (100 mL) was added to the acidic aqueous solution. The aqueous layer was separated and the organic layer was extracted with aqueous 0.5 M HCl (3 × 50 mL). All of the aqueous layers were combined and washed with Et₂O (2 × 100 mL). The aqueous layer was basified to pH 10–12 using aqueous 4 M NaOH and extracted with CH₂Cl₂ (3 × 100 mL). The CH₂Cl₂ layers were combined and were successively washed with a 1:1 mixture of EtOH/H₂O (2 × 100 mL) and saturated aqueous brine (2 × 100 mL), dried (Na₂SO₄), evaporated in vacuo, and purified three times by flash column chromatography (SiO₂; 1:100 MeOH/CH₂Cl₂) to give the desired product (0.53 g, 17%) as a pale yellow oil: [α]²⁸_D −31.4° (c 0.51, CH₂Cl₂) (lit.³⁷ [α]²⁰_D − 30.4° (c 0.55, CH₂Cl₂)), [α]²⁸_D −31.4° (c 1.9, CHCl₃) (lit.³⁸ (S): [α]^25.4_D +33.4° (c 1.80, CHCl₃)); R_f 0.53 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.90 (d, J = 6.8 Hz, 6H), 1.58–1.69 (m, 1H), 2.42–2.48 (m, 4H), 2.64–2.69 (m, 1H), 2.74 (dd, J = 3.2, 12.0 Hz, 1H), 3.78 (1/2 AB_q, J = 13.4 Hz, 1H), 3.84 (1/2 AB_q, J = 13.4 Hz, 1H), 7.22–7.35 (m, 5H); HRMS (ESI) 193.1705 [M + H⁺] (calcd for C₁₂H₂₀N₂H⁺ 193.1695); Anal. (C₁₂H₂₀N₂•H₂O): C, H, N.

(R)-4-Amino-2-methyl-6-phenyl-4-hexanone Hydrochloride ((R)-9)

(R)-31^{39, 40} (3.04 g, 9.96 mmol) was dissolved in MeOH (50 mL) and aqueous concentrated HCl (4.9 mL, 59 mmol) was added. The reaction was heated at reflux (1 h) and then cooled to room temperature before evaporating the solvent in vacuo to give a crude oil. The crude product was triturated with hexanes (3x) to give the desired product (0.60 g, 25%) as a white solid: mp 123–124 °C; [α]²⁸_D − 59.7° (c 1.1, CHCl₃); R_f 0.49 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.97 (d, J = 7.0 Hz, 3H), 1.17 (d, J = 7.0 Hz, 3H), 2.34–2.44 (br m, 1H), 2.56–2.98 (m, 4H), 4.18–4.26 (br d, 1H), 7.16–7.24 (m, 5H), 8.57 (br s, 3H); LRMS (ESI) 206.13 [M – Cl⁻] (calcd for C₁₃H₂₀NO 206.13); Anal. (C₁₃H₂₀ClNO): C, H, Cl, N.

(R)-O-Benzyl 2-Amino-3-methylbutanoate ((R)-10).⁴¹

1 M HCl in Et₂O (165 mL) was added to a Et₂O solution (10 mL) of (R)-32^{25, 42} (2.02 g, 6.58 mmol) at 0 °C. The reaction was stirred at room temperature (18 h) to give a cloudy white solution. The solution was filtered but no substantial filtrate was collected. The solvent was evaporated in vacuo to give a crude oil that was then redissolved in CH₂Cl₂ (10 mL). The organic layer was extracted with aqueous 1 M HCl (3 × 10 mL). The aqueous layers were combined and washed with CH₂Cl₂ (2 × 30 mL). The aqueous layer was basified to pH 10–12 with aqueous 4 M NaOH and extracted with CH₂Cl₂ (3 × 50 mL). The second set of organic layers were combined and washed with saturated aqueous brine (2 × 150 mL), dried (Na₂SO₄), and evaporated in vacuo. The crude product was purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired product (0.99 g, 72%) as a pale yellow oil: [α]^28.5_D −10.2° (c 1.1, CHCl₃); R_f 0.39 (1:1 EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 0.88 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 1.44 (s, 2H), 1.99–2.10 (m, 1H), 3.34 (d, J = Hz, 1H), 5.14 (1/2 AB_q, J = 12.2 Hz, 1H), 5.18 (1/2 AB_q, J = 12.2 Hz, 1H), 7.30–7.39 (m, 5H); LRMS (ESI) 208.15 [M + H⁺] (calcd for C₁₂H₁₇NO₂H⁺ 208.15); Anal. (C₁₂H₁₇NO₂): C, H, N.

(R)-N′-Phenyl 2-Amino-3-methylbutanamide ((R)-11).⁴³

Utilizing Method A with (R)-40⁴⁴ (1.75 g, 5.99 mmol), TFA (6.67 mL, 89.8 mmol), and CH₂Cl₂ (20 mL) gave the crude product after acidic workup and was further purified by flash column chromatography (SiO₂; 1:10–1:1 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (0.94 g, 82%) as a pale orange oil: [α]²⁵_D +82.5° (c 1.6, CH₂Cl₂); R_f 0.34 (1:1 EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 0.87 (d, J = 7.2 Hz, 3H), 1.04 (d, J = 7.2 Hz, 3H), 1.46 (s, 2H), 2.40–2.48 (m, 1H), 3.37 (d, J = 3.6 Hz, 1H), 7.09 (t, J = 7.2 Hz, 1H), 7.32 (t, J = 8.2 Hz, 2H), 7.60 (d, J = 8.2 Hz, 2H), 9.46–9.56 (br s, 1H); HRMS (ESI) 193.1343 [M + H⁺] (calcd for C₁₁H₁₆N₂OH⁺ 193.1341); Anal. (C₁₁H₁₆N₂O•0.01H₂O): C, H, N.

(R)-N′-Phenethyl 2-Amino-3-methylbutanamide ((R)-12)

Utilizing Method A with (R)-41 (1.37 g, 4.28 mmol), TFA (4.77 mL, 64.2 mmol), and CH₂Cl₂ (15 mL) gave the crude product after acidic workup and further purified by flash column chromatography (SiO₂; 1:10–1:1 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (873 mg, 93%) as a pale yellow solid: mp 36–37 °C; [α]²⁵_D +32.9° (c 1.0, CH₂Cl₂); R_f 0.19 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.77 (d, J = 7.2 Hz, 3H), 0.96 (d, J = 7.2 Hz, 3H), 1.20–1.26 (s, 2H), 2.22–2.33 (m, 1H), 2.77–2.88 (m, 2H), 3.19 (d, J = 4.0 Hz, 1H), 3.46– 3.62 (m, 2H), 7.20–7.32 (m, 5H); HRMS (ESI) 221.1643 [M + H⁺] (calcd for C₁₃H₂₀N₂OH⁺ 221.1654); Anal. (C₁₃H₂₀N₂O): C, H, N.

(R)-N′-Phenylpropyl 2-Amino-3-methylbutanamide ((R)-13)

Utilizing Method A with (R)-42 (5.00 g, 15.0 mmol), TFA (16.7 mL, 225 mmol), and CH₂Cl₂ (50 mL) gave the crude product after acidic workup and further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (3.02 g, 86%) as a pale yellow oil: [α]^28.5_D +30.1° (c 1.2, CHCl₃); R_f 0.21 (1:1 EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 0.81 (d, J = 7.2 Hz, 3H), 0.98 (d, J = 7.2 Hz, 3H), 1.30 (s, 2H), 1.79–1.90 (m, 2H), 2.23–2.34 (m, 1H), 2.65 (t, J = 8.4 Hz, 2H), 3.19 (d, J = 4.0 Hz, 1H), 3.22–3.37 (m, 2H), 7.16–7.20 (m, 3H), 7.26–7.35 (m, 3H); HRMS (ESI) 235.1818 [M + H⁺] (calcd for C₁₄H₂₂N₂OH⁺ 235.1810); Anal. (C₁₄H₂₂N₂O•0.16H₂O): C, H, N.

(R)-N′-Phenylbutyl 2-Amino-3-methylbutanamide ((R)-14)

Utilizing Method A with (R)-43 (3.18 g, 9.13 mmol), TFA (10.2 mL, 137 mmol), and CH₂Cl₂ (30 mL) gave the crude product after basic workup that was further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (2.16 g, 96%) as a pale yellow oil: [α]^28.5_D +29.9° (c 1.0, CHCl₃); R_f 0.53 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.81 (d, J = 6.8 Hz, 3H), 0.97 (d, J = 6.8 Hz, 3H), 1.51–1.59 (m, 4H), 1.62–1.70 (m, 2H), 2.22–2.34 (m, 1H), 2.63 (t, J = 8.0 Hz, 2H), 3.20 (d, J = 4.0 Hz, 1H), 3.22–3.44 (m, 2H), 7.15–7.19 (m, 3H), 7.25–7.36 (m, 3H); HRMS (ESI) 249.1954 [M + H⁺] (calcd for C₁₅H₂₄N₂OH⁺ 249.1967); Anal. (C₁₅H₂₄N₂O•0.0.6CH₂Cl₂): C, H, N.

(R)-2′-N′-(Trifluoromethoxy)benzyl 2-Amino-3-methylbutanamide ((R)-15)

Utilizing Method A with (R)-44 (6.00 g, 15.4 mmol), TFA (17.1 mL, 231 mmol), and CH₂Cl₂ (50 mL) gave the crude product after acidic workup that was further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (3.70 g, 83%) as a pale yellow solid: mp 54–55 °C; [α]^28.5_D +28.0° (c 1.0, CHCl₃); R_f 0.59 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.81 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 1.34 (s, 2H), 2.28–2.39 (m, 1H), 3.27 (d, J = 4.0 Hz, 1H), 4.48–4.57 (m, 2H), 7.22–7.33 (m, 3H), 7.41–7.43 (m, 1H), 7.71–7.79 (br t, 1H); LRMS (ESI) 291.12 [M + H⁺] (calcd for C₁₃H₁₇F₃N₂O₂H⁺ 291.12); Anal. (C₁₃H₁₇F₃N₂O₂): C, H, F, N.

(R)-3′-N′-(Trifluoromethoxy)benzyl 2-Amino-3-methylbutanamide ((R)-16)

Utilizing Method A with (R)-45 (3.00 g, 7.69 mmol), TFA (8.57 mL, 115 mmol), and CH₂Cl₂ (25 mL) gave the crude product after acidic workup that was further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (1.81 g, 81%) as a pale yellow oil: [α]^28.5_D +20.6° (c 1.3, CHCl₃); R_f 0.47 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.83 (d, J = 7.0 Hz, 3H), 1.00 (d, J = 7.0 Hz, 3H), 1.38 (s, 2H), 2.31–2.40 (m, 1H), 3.30 (d, J = 4.0 Hz, 1H), 4.43 (dd, J = 6.2, 15.0 Hz, 1H), 4.51 (dd, J = 6.6, 15.0 Hz, 1H), 7.10–7.13 (m, 2H), 7.21–7.22 (m, 1H), 7.34 (t, J = 8.0 Hz, 1H), 7.78–7.86 (br t, 1H); LRMS (ESI) 291.11 [M + H⁺] (calcd for C₁₃H₁₇F₃N₂O₂H⁺ 291.11); Anal. (C₁₃H₁₇F₃N₂O₄): C, H, F, N.

(R)-N′-Cyclohexylmethyl 2-Amino-3-methylbutanamide ((R)-18)

Utilizing Method A with (R)-46 (3.60 g, 11.5 mmol), TFA (12.8 mL, 173 mmol), and CH₂Cl₂ (38 mL) gave the crude product after acidic workup that was further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (2.34 g, 96%) as a white solid: mp 85–86 °C; [α]²⁵_D +38.1° (c 1.1, CHCl₃); R_f 0.59 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.82 (d, J = 7.0 Hz, 3H), 0.89–0.99 (m, 2H), 0.99 (d, J = 7.0 Hz, 3H), 1.10–1.28 (m, 3H), 1.36 (s, 2H), 1.41–1.52 (m, 1H), 1.65–1.74 (m, 5H), 2.26–2.37 (m, 1H), 3.03–3.18 (m, 2H), 3.23 (d, J = 3.6 Hz, 1H), 7.30–7.40 (br t, 1H); LRMS (ESI) 213.18 [M + H⁺] (calcd for C₁₂H₂₄N₂OH⁺ 213.18); Anal. (C₁₂H₂₄N₂O): C, H, N.

N-Benzyl Butanamide (20).⁴⁵

Utilizing Method B with 47 (2.00 mL, 21.9 mmol), NMM (3.13 mL, 28.5 mmol), IBCF (3.10 mL, 24.1 mmol), and benzylamine (2.51 mL, 23.0 mmol) gave the crude product that was recrystallized from hot EtOAc/hexanes to give the desired compound (1.18 g, 30%) as a white solid: mp 54–55 °C (lit.⁴⁵ mp 36.9–38 °C); R_f 0.67 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.96 (t, J = 7.6 Hz, 3H), 1.65–1.74 (m, 2H), 2.20 (t, J = 8.0 Hz, 2H), 4.45 (d, J = 5.6 Hz, 2H), 5.66–5.78 (br s, 1H), 7.26–7.35 (m, 5H); HRMS (ESI) 178.1238 [M + H⁺] (calcd for C₁₁H₁₅NOH⁺ 178.1232); Anal. (C₁₁H₁₅NO•0.06H₂O): C, H, N.

(R,S)-N-Benzyl 2-Methylbutanamide ((R,S)-21).⁴⁶

Utilizing Method B with (R,S)-48 (2.00 mL, 18.3 mmol), NMM (2.62 mL, 23.8 mmol), IBCF (2.60 mL, 20.2 mmol), and benzylamine (2.10 mL, 19.3 mmol) gave the crude product that was purified twice by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes) to give the desired compound (2.32 g, 66%) as a white solid: mp 54–55 °C (lit.⁴⁶ mp 47.5–48.5 °C); R_f 0.80 (1:1 EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 0.91 (t, J = 7.2 Hz, 3H), 1.15 (d, J = 6.2 Hz, 3H), 1.39–1.50 (m, 1H), 1.64–1.75 (m, 1H), 2.09– 2.17 (m, 1H), 4.39–4.49 (m, 2H), 5.79–5.89 (br s, 1H), 7.26–7.35 (m, 5H); HRMS (ESI) 214.1199 [M + Na⁺] (calcd for C₁₂H₁₇NOH⁺ 214.1208); Anal. (C₁₂H₁₇NO): C, H, N.

(S)-N-Benzyl 2-Methylbutanamide ((S)-21).⁴⁷

Utilizing Method B with (S)-48 (0.90 mL, 8.27 mmol), NMM (1.18 mL, 10.8 mmol), IBCF (1.17 mL, 9.10 mmol), and benzylamine (0.95 mL, 8.6 mmol) gave the crude product that was purified twice by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes) to give the desired compound (1.01 g, 64%) as a white solid: mp 55–56 °C; [α]²⁸_D +15.5° (c 1.0, acetone) (lit.⁴⁷ [α]_D +16.96° (c 1.0, acetone)); R_f 0.80 (1:1 EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃) δ 0.91 (t, J = 7.6 Hz, 3H), 1.16 (d, J = 7.6 Hz, 3H), 1.40–1.50 (m, 1H), 1.65–1.75 (m, 1H), 2.09–2.18 (m, 1H), 4.39–4.49 (m, 2H), 5.80–5.88 (br s, 1H), 7.25–7.35 (m, 5H); LRMS (ESI) 193.16 [M + H⁺] (calcd for C₁₂H₁₇NOH⁺ 193.16); Anal. (C₁₂H₁₇NO): C, H, N.

(R)-N-Benzyl 2-Hydroxybutanamide ((R)-22)

To anhydrous toluene (80 mL) was added (R)- 49 (1.70 g, 16.3 mmol), benzylamine (1.78 mL, 16.3 mmol), and 3,5-bis(trifluoromethyl)benzene boronic acid (50) (0.42 g, 1.6 mmol). A pressure equalizing dropping funnel containing a cotton plug was filled 1/3 of the way with 3 Å molecular sieves that were oven dried (120 °C) and a condenser was placed above the dropping funnel. The mixture was heated at reflux (18 h) before cooling to room temperature and then the solvent was evaporated in vacuo. The crude product was purified by flash column chromatography (SiO₂; 1:20–1:1 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) followed by recrystallization from hot EtOAc/hexanes to give the desired compound (1.05 g, 33%) as a white solid: mp 63–64 °C; [α]^28.5_D +28.3° (c 2.2, CHCl₃); R_f 0.56 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.96 (t, J = 7.6 Hz, 3H), 1.63–1.74 (m, 1H), 1.82–1.92 (m, 1H), 3.32 (d, J = 4.8 Hz, 1H), 4.07–4.11 (m, 1H), 4.38–4.48 (m, 2H), 6.95– 7.20 (br t, 1H), 7.24–7.34 (m, 5H); LRMS (ESI) 216.12 [M + Na⁺] (calcd for C₁₁H₁₅NO₂Na⁺ 216.12); Anal. (C₁₁H₁₅NO₂): C, H, N.

(R)-N′-Benzyl N-(Methyl)amino-2-methylbutanamide Hydrochloride ((R)-23)

HCl in dioxane (4 mL, 4 M) was added at 0 °C to an Et₂O (1 mL) solution of (R)-53 (620 mg, 4.7 mmol). The reaction was stirred at room temperature (16 h) and then concentrated in vacuo. The residue was triturated with Et₂O to give the desired compound (450 mg, 90%) as a white solid: mp 120–125 °C; [α]²⁷_D +93.8°(c 0.5, CH₃OH); ¹H NMR (400 MHz, CD₃OD) δ 1.02 (d, J = 7.2 Hz, 3H), 1.05 (d, J = 6.9 Hz, 3H), 2.18–2.24 (m, 1H), 2.65 (s, 3H), 3.62 (d, J = 5.1 Hz, 1H), 4.40 (1/2 AB_q, J = 15.0 Hz, 1H), 4.50 (1/2 AB_q, J = 15.0 Hz, 1H), 7.28–7.34 (m, 5H); HRMS (ESI) 221.1654 [M+H⁺] (calcd. for C₁₃H₂₀N₂OH⁺ 221.1653); Anal. (C₁₃H₂₀N₂O): C, H, Cl, N.

(R)-N′-Benzyl N,N-Dimethylamino-3-methylbutanamide ((R)-24)

(R)-55 (2.28 g, 15.7 mmol) and benzylamine (2.06 mL, 18.9 mmol) were added to anhydrous THF (160 mL) at room temperature. The mixture stirred (15 min) and then DMTMM (5.22 g, 18.9 mmol) was added in one portion. The reaction continued at room temperature overnight (18 h) and then the insoluble salts were filtered, evaporated in vacuo, and purified by flash column chromatography (SiO₂; 2:1 EtOAc/CH₂Cl₂ followed by 1:10 MeOH/CH₂Cl₂) to give the desired product (0.38 g, 10%) as a white solid: mp 78–79 °C; [α]²⁸_D −11.2° (c 0.5, CHCl₃); R_f 0.55 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.89 (d, J = 6.6 Hz, 3H), 1.03 (d, J = 6.6 Hz, 3H), 2.04–2.17 (m, 1H), 2.25 (s, 6H), 2.48 (d, J = 6.0 Hz, 1H), 4.42–4.51 (m, 2H), 6.58–6.65 (br t, 1H), 7.25–7.35 (m, 5H); LRMS (ESI) 235.17 [M + H⁺] (calcd for C₁₄H₂₂N₂OH⁺ 235.17); Anal. (C₁₄H₂₂N₂O): C, H, N.

(R)-2′-N′-(Fluoro)phenethyl 2-Amino-3-methylbutanamide ((R)-56)

Utilizing Method A with (R)-2′-N′-(fluoro)phenethyl 2-N-(t-butoxycarbonyl)amino-3-methylbutanamide (9.00 g, 26.6 mmol), TFA (30 mL, 0.40 mol), and CH₂Cl₂ (90 mL) gave the crude product after acidic workup and further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (5.76 g, 91%) as a white solid: mp 51–52 °C; [α]²⁸_D +32.6° (c 1.5, CHCl₃); R_f 0.26 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.77 (d, J = 7.0 Hz, 3H), 0.95 (d, J = 7.0 Hz, 3H), 1.25 (s, 2H), 2.24–2.32 (m, 1H), 2.82–2.93 (m, 2H), 3.19 (d, J = 3.6 Hz, 1H), 3.46–3.61 (m, 2H), 6.99–7.09 (m, 2H), 7.17–7.23 (m, 2H), 7.34–7.42 (br t, 1H); LRMS (ESI) 239.13 [M + H⁺] (calcd for C₁₃H₁₉FN₂OH⁺ 239.13); Anal. (C₁₃H₁₉FN₂O): C, H, F, N.

(R)-3′-N′-(Fluoro)phenethyl 2-Amino-3-methylbutanamide ((R)-57)

Utilizing Method A with (R)-3′-N-(fluoro)phenethyl 2-N-(t-butoxycarbonyl)amino-3-methylbutanamide (8.50 g, 25.1 mmol), TFA (28 mL, 0.38 mol), and CH₂Cl₂ (85 mL) gave the crude product after acidic workup and further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (5.08 g, 85%) as a pale yellow oil: [α]²⁸_D +31.4° (c 1.1, CHCl₃); R_f 0.26 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.77 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 1.23 (s, 2H), 2.23–2.34 (m, 1H), 2.77–2.88 (m, 2H), 3.20 (d, J = 4.0 Hz, 1H), 3.45–3.61 (m, 2H), 6.89–6.93 (m, 2H), 6.97–6.99 (m, 1H), 7.23–7.28 (m, 1H), 7.34–7.44 (br t, 1H); HRMS (ESI) 239.1553 [M + H⁺] (calcd for C₁₃H₁₉FN₂OH⁺ 239.1560); Anal. (C₁₃H₁₉FN₂O•0.01H₂O): C, H, F, N.

(R)-4′-N′-(Fluoro)phenethyl 2-Amino-3-methylbutanamide ((R)-58)

Utilizing Method A with (R)-4′-N′-(fluoro)phenethyl 2-N-(t-butoxycarbonyl)amino-3-methylbutanamide (9.00 g, 26.6 mmol), TFA (30 mL, 0.40 mol), and CH₂Cl₂ (90 mL) gave the crude product after acidic workup and further purified by flash column chromatography (SiO₂; 1:20 EtOAc/hexanes followed by 1:10 MeOH/CH₂Cl₂) to give the desired compound (5.82 g, 92%) as a pale yellow oil: [α]²⁸_D +31.8° (c 1.5, CHCl₃); R_f 0.19 (1:20 MeOH/CH₂Cl₂); ¹H NMR (400 MHz, CDCl₃) δ 0.77 (d, J = 7.0 Hz, 3H), 0.96 (d, J = 7.0 Hz, 3H), 1.23 (s, 2H), 2.22–2.33 (m, 1H), 2.74–2.85 (m, 2H), 3.19 (d, J = 3.6 Hz, 1H), 3.42–3.59 (m, 2H), 6.95–7.00 (m, 2H), 7.13–7.18 (m, 2H), 7.34– 7.42 (br t, 1H); LRMS (ESI) 239.13 [M + H⁺] (calcd for C₁₃H₁₉FN₂OH⁺ 239.13); Anal. (C₁₃H₁₉FN₂O): C, H, F, 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.”⁴⁸ Pharmacological evaluation by UCB Pharma consisted of the MES test³³ to assess anticonvulsant activity and the rotorod test³³ to assess neurological toxicity. Pharmacological evaluation by the NINDS ASP utilized male albino Carworth Farms No. 1 mice (ip) or male albino Sprague-Dawley rats (po) and consisted of the MES test (mice and rats) and the subcutaneous pentylenetetrazol (Metrazol^®) (scMet) seizure threshold test₅ to assess anticonvulsant activity (mice), the rotorod test to assess neurological toxicity (mice), and the positional sense test or gait and stance test to assess behavioral toxicity (rats).⁵