J. Med. Chem. 1996, 39, 1907-1916
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Synthesis and Anticonvulsant Activities of N-Benzyl-2-acetamidopropionamide Derivatives Daeock Choi,† James P. Stables,‡ and Harold Kohn*,† Department of Chemistry, University of Houston, Houston, Texas 77204-5641, and Epilepsy Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Federal Building, Room 114, Bethesda, Maryland 20892-9020 Received November 29, 1995X
Studies have demonstrated that 2-substituted N-benzyl-2-acetamidoacetamides (2) are potent anticonvulsants. A recent investigation has led to the hypothesis that an important structural feature in 2 for maximal anticonvulsant activity is the placement of a small, substituted heteroatom moiety one atom from the C(2) site. This paper validates this hypothesis. Twelve derivatives of N-benzyl-2-acetamidopropionamide have been prepared in which six different heteroatom substituents (chloro, bromo, iodo, oxygen, nitrogen, and sulfur) were incorporated at the C(3) site. Highly potent activities were observed for the two oxygen-substituted derivatives, N-benzyl-2-acetamido-3-methoxypropionamide (18) and N-benzyl-2-acetamido-3ethoxypropionamide (19). The ED50 values in mice following intraperitoneal (ip) dosing for the maximal electroschock-induced seizure test for 18 and 19 were 8.3 and 17.3 mg/kg, respectively. These values compared favorably to the ED50 value found for phenytoin (ED50 ) 6.5 mg/kg). Comparable activities were observed for 18 and 19 upon oral (po) administration to rats (18, ED50 ) 3.9 mg/kg; 19, ED50 ) 19 mg/kg; phenytoin, ED50 ) 23 mg/kg). Evaluation of the individual stereoisomers for 18 demonstrated that the principal anticonvulsant activity resided in the (R)-stereoisomer. The ED50 value for (R)-18 was 4.5 mg/kg, and the ED50 for (S)-18 exceeded 100 mg/kg. This difference in activity for the two stereochemical isomers surpassed comparable values for other members within this class of compounds. The protective indices (PI ) TD50/ED50) (where TD50 represents a neurotoxic dose impairing rotorod performance) for (R)-18 in mice (ip) and in rats (po) were 6.0 and >130, respectively. Introduction Over the past 10 years we have reported on the anticonvulsant properties of functionalized amino acid derivatives that conform to the general structure 1.1-9
Systematic evaluation of over 100 derivatives has led to our proposal that stringent steric and electronic requirements exist for this novel class of compounds for protection against maximal electroshock (MES)-induced seizure. Our structure-activity studies indicated two important trends. First, in cases where an aromatic moiety was positioned at the C(2) site improved activity was noted for those compounds that contained a small, electron-rich R2 substituent.5,7 Second, enhanced protection against MES-induced seizures was observed for C(2) cyclic and acyclic R2 groups that contained a substituted heteroatom one atom from the C(2) site.6,7,9 Consistent with these contentions were the relative activities (mice, ip) of 3 (ED50 ) 10 mg/kg) versus the 2-tetrahydrofuran-2-yl diastereomers (7) (ED50 ) 5290 mg/kg),7 the relative potencies of 2-furan-2-yl (3) (ED50 ) 10 mg/kg), 2-pyrrol-2-yl (4) (ED50 ) 16 mg/kg), and 2-thien-2-yl (5) (ED50 ) 45 mg/kg) N-benzyl-2† ‡ X
University of Houston. NIH. Abstract published in Advance ACS Abstracts, April 1, 1996.
0022-2623/96/1839-1907$12.00/0
acetamidoacetamide derivatives,5 the decrease in activity of the 2-thien-3-yl (6) (ED50 ) 88 mg/kg) adduct versus the isomeric 2-thien-2-yl (5) (ED50 ) 45 mg/kg) compound, and the dramatic increase in activity for the O-methylhydroxylamine (9) (ED50 ) 6.2 mg/kg) and O,N-dimethylhydroxylamine (10) (ED50 ) 6.7 mg/kg) derivatives versus the C(2) hydroxylamine compound (8) (ED50 ≈ 100 mg/kg).6 Another critical aspect of the structure-activity profile observed for N-benzyl-2-acetamidoacetamide derivatives (2) was the discovery that in three different cases the (R)-stereoisomer was approximately 10 times more potent in the control of MES seizures than the (S)-enantiomer.1,4,5,7 These differences in activities were the greatest eudismic ratio10 reported to date for MES-selective anticonvulsant agents.
Recently, we found that the three C(2) electrondeficient heteroaromatic compounds (11-13) possessed © 1996 American Chemical Society
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Journal of Medicinal Chemistry, 1996, Vol. 39, No. 9
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Table 1. Physical and Pharmacological Data in Mice for N-Benzyl-2-acetamidopropionamidesa
R2
no.
mpb
14f
CH3
138-139
15 16 17 18
CH2Cl CH2Br CH2I CH2OCH3
143-144 123-125 169-170 121-122
19
CH2OCH2CH3
113-114
20 21 22 23 24 25 26 27 8 9
CH2OCH2CHdCH2 CH2NH2 CH2N(H)CH3 CH2N(H)CH2CH3 CH2N(CH3)2 CH2-morpholino CH2SCH3 CH2OH N(H)OH N(H)OCH3
76-77 119-120 107-108 90-91 125-126 147-148 142-143 201-203 144-146 dec 95-97
10
N(CH3)OCH3
165-167
phenytoini phenobarbitalj valproatei
MESc ED50
TD50d
PIe
77 [1] (67-89) ∼100 >30, 30, 30, 100 >100, 100 >30, 100, 30, 100, 100, 100, 30, 100 >100, 300 >300 >100, 26 >22 6.7 2.2
a ED 50 and TD50 values are in mg/kg. Numbers in parentheses are 95% confidence intervals. The dose effect data was obtained at the “time of peak effect” (indicated in hours in the brackets). b MES ) maximal electroshock seizure test. c Tox ) neurologic toxicity (the rotorod test). d PI ) protective index (TD50/MES ED50). e No ataxia observed up to 1000 mg/kg. f No attaxia observed up to 500 mg/kg. g Reference 11a. h Reference 11b.
(15) was also briefly examined for the synthesis of N-benzyl-3-substituted-2-acetamidopropionamide derivatives. Treatment of 15 with KOH or NaH provided the elimination product 34, while addition of sodium methanethiolate to 15 in MeOH gave the substitution product 26 in 89% yield. Information concerning the mechanism of this transformation was obtained by rerunning this reaction in CD3OD. Under these conditions the C(2) methine site was fully deuterated (NMR analysis, data not shown). This finding coupled with the observation that only 15% C(2)H f C(2)D exchange occurred upon dissolution of 26 in basic CH3OD indicated that conversion of 15 to 26 proceeded through 34, rather than by direct SN2 displacement of the chloride group in 15.
of the hydrochloride salts of the optically pure serine methyl esters 36 with benzylamine gave benzylamide 37, which was then acylated with acetic anhydride to provide 27. Three criteria were used to assess the enantiopurity of 27. These were melting point, optical rotation, and the detection of only a single acetyl methyl signal in the 1H NMR spectrum of 27 upon addition of a saturated solution of (R)-(-)-mandelic acid.20 NMR analysis indicated that conversion of 36 to 27 proceeded to give an approximate 2:1 mixture of the enantiomers. Accordingly, 27 was repeatedly recrystallized until optically pure and then alkylated with MeI and Ag2O to give the desired ether 18 without racemization. The pronounced anticonvulsant activity observed for (R)-18 led us to devise an alternative, more expeditious synthetic route for this compound (Scheme 3). Beginning with D-serine ((R)-35), treatment with acetic anhydride in acetic acid gave the N-acylated derivative 38, which was converted to N-benzylamide (R)-27 using the mixed anhydride coupling procedure.21 NMR and optical measurements indicated that both steps proceeded without racemization. Alkylation of (R)-27 with MeI and Ag2O gave (R)-N-benzyl-2-acetamido-3-methoxypropionamide ((R)-18) in 30% overall yield. The success of this protocol permitted the rapid synthesis of derivatives of (R)-18 in which the substitution pattern on the benzylamide group was altered (Table 3). Accordingly, treatment of (R)-38 with 3-fluorobenzylamine and 4-fluorobenzylamine provided (R)-39 and (R)-40, respectively, which were then converted to the methyl ethers (R)-41 and (R)-42, respectively, with MeI and Ag2O. Pharmacological Evaluation
The pharmacological activity observed for N-benzyl2-acetamido-3-methoxypropionamide (18) warranted our preparation of the individual (R)- and (S)-enantiomers (Table 2). The original preparative route developed took advantage of the commercial availability of both D- and L-serine derivatives (Scheme 2). Treatment
The anticonvulsant activities for 2-acetamido-N-benzylpropionamide derivatives 15-26 were determined using the procedure described by Kupferberg,22 and these results compared to 8-10,6 14,2 27, and the clinically proven antiepileptic agents phenytoin,11a phenobarbital,11b and valproate.11a All compounds were administered intraperitoneally (ip) to mice. Table 1 lists the results obtained from the initial mouse identification and quantitation screening studies. They include the ED50 values for racemates 15-26 that are protective
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Journal of Medicinal Chemistry, 1996, Vol. 39, No. 9
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Scheme 2. Preparation of Enantiopure 18
Scheme 3. Improved Procedure for the Preparation of Enantiopure (R)-18 and Related Compounds
in blocking hind limb extension induced in the MES test. Also contained in Table 1 are the median doses for neurological impairment (TD50) using either the rotorod23 or the horizontal screen24 (HS) test. In most cases, the TD50s were only determined for those compounds that had good activity in the MES test. The protective index (PI ) TD50/ED50) for these adducts, where appropriate, is also shown in Table 1. Inspection of the composite data in Table 1 provided strong support for our current structure-activity relationship for this class of anticonvulsants. Highlighted were the beneficial value accrued by the placement of a heteroatom one atom from the C(2) site, the need for the heteroatom to be substituted, and the stringent steric requirements that limit the size of the C(2) substituent. Six different heteroatoms (O, N, S, Cl, Br, and I) were incorporated in N-benzyl-2-acetamidopropionamide (14). Of these, the oxygen-substituted derivatives 18-20 proved to be the most effective for the control of MES-induced seizures. The anticonvulsant activities of both 18 (ED50 ) 8.3 mg/kg) and 19 (ED50 ) 17 mg/kg) exceeded that of 14 (ED50 ) 77 mg/kg). Moreover, the MES ED50 values and protective indices of racemic 18 and 19 were comparable to those reported for phenytoin11a (ED50 ) 6.5 mg/kg) and phenobarbital11b (ED50 ) 22 mg/kg), respectively. A similar trend with
respect to heteroatom substituent was observed for the C(2) aromatic compounds 3 (ED50 ) 10 mg/kg), 4 (ED50 ) 16 mg/kg), and 5 (ED50 ) 45 mg/kg).5 The comparatively low activity observed for amines 21-25 has been tentatively attributed, in part, to the basicity of this substituent. This difference in activity was reminiscent of the decreased activity observed for the C(2) imidazole derivative 43 (ED50 >100 mg/kg) versus the less basic, isomeric C(2) pyrazole adduct 44 (ED50 ) 17 mg/kg).7
Support for our suggestion that increased anticonvulsant protection accompanied substitution of the heteroatom moiety was provided by the comparison of the ED50 values for methyl ether 18 (ED50 ) 8.3 mg/ kg) and ethyl ether 19 (ED50 ) 17 mg/kg) versus alcohol 27 (ED50 >100, 100, 300 mg/kg5). The pharmacological activities of racemic 18 and 19 warranted their further evaluation. Table 2 lists the MES ED50, TD50, and PI value for these compounds upon oral administration (po) to rats with similar data
N-Benzyl-2-acetamidopropionamide Derivatives
Journal of Medicinal Chemistry, 1996, Vol. 39, No. 9 1911
Table 3. Selected Physical and Pharmacological Data for Functionalized N-Benzyl-2-acetamidopropionamide Stereoisomers mice (ip)b R2
no.
Ar
mpa
(R,S)-14
CH3
Ph
139-141
(R)-14
CH3
Ph
139-141
(S)-14
CH3
Ph
139-142
(R,S)-18
CH2OCH3
Ph
121-122
(R)-18
CH2OCH3
Ph
143-144
(S)-18 (R,S)-27 (R)-27
CH2OCH3 CH2OH CH2OH
Ph Ph Ph
143-144 201-203 148-149
(R)-41
CH2OCH3
Ph(m-F)
150-151
(R)-42
CH2OCH3
Ph(p-F)
144-145
MES,c
ED50
77 [1]g (67-89) 55 [0.5]i (50-60) 550 [0.5] (460-740) 8.3 [0.5] (7.9-9.8) 4.5 [0.5] (3.7-5.5) >100, 100, 300 >300 >500 [2] 46 [0.25] (40-55) 28 [0.25] (22-34)
PIe 5.9 3.9 1.5 5.2 6.0
>9.4 6.7 6.7
MES,c
ED50
48 [1] (32-72) 28 [4] (22-35) -j 3.8 [2] (2.9-5.5) 3.9 [0.5] (2.6-6.2) >30 -j -j 6.9 [0.5] (4.3-9.9) 2.6 [2] (1.9-3.6)
Tox,d TD50
PIe
-h
>21
-h
>36
-j 390 [1] (320-510) >500 [0.5]
100 >130
>30 -j -j >400 [0.5]
>58
>125, 22 and surpassed comparable values found for 3, 14, and 46. Similarly, we noted a significant improvement in anticonvulsant activity of (R)-27 (ED50 ) 53 mg/kg) versus the corresponding racemate (R,S)-27 (ED50 >100,
