Functionalized amino acid anticonvulsants: Synthesis and pharmacological evaluation of conformationally restricted analogues

Article abstract of DOI:10.1016/S0968-0896(01)00204-8

Proven conformationally restricted analogues of anticonvulsant functionalized amino acids (FAAs) were prepared using short-range cyclizations and evaluated in pharmacological assays providing new information concerning the structural requirements for FAA function.

Full text of DOI:10.1016/S0968-0896(01)00204-8

Bioorganic & Medicinal Chemistry 9 (2001) 2693–2708
Functionalized Amino Acid Anticonvulsants: Synthesis and
Pharmacological Evaluation of Conformationally Restricted
Analogues
Arnaud LeTiran,a James P. Stablesb and Harold Kohnc,*
aDepartment of Chemistry, University of Houston, Houston, TX 77204-5641, USA
bEpilepsy Branch, National Institute of Neurological Disorders and Stroke, National Institutes of Health, Federal Building,
Room 114, Bethesda, MD 20892-9020, USA
cDivision of Medicinal Chemistry and Natural Products, School of Pharmacy, University of North Carolina at Chapel Hill,
Chapel Hill, NC 27599-7360, USA
Received 14 March 2001; accepted 24 May 2001
Abstract—Proven conformationally restricted analogues of anticonvulsant functionalized amino acids (FAAs) were prepared using
short-range cyclizations and evaluated in pharmacological assays providing new information concerning the structural require-
ments for FAA function. # 2001 Elsevier Science Ltd. All rights reserved.
Introduction
the use of conformationally constrained (semirigid)
analogues.3 It is proposed that appropriate structural
constraints will restrict a residue or group of residues to
a sufficiently small region of conformational space
thereby permitting the drug to bind, with high affinity,
to its designated receptor.3 An expectation of this
approach is that the semirigid analogue will have
improved biological activity through the elimination of
bioactive conformers that give unwanted biological
responses.4 This strategy is limited because it is impos-
sible to search all accessible conformations for most
biomolecules. We tried to determine in this study whe-
ther we could learn the preferred bioactive FAA con-
former(s) through the use of a select set of proven,
constrained peptidomimetics.
We have described a new class of anticonvulsant agents,
termed functionalized amino acids (FAAs, 1).1 N-Ben-
zyl-2-acetamidopropionamide (2) is the parent com-
pound in this series, and (R)-N-benzyl-2-acetamido-3-
methoxypropionamide1i (3) is the lead antiepileptic
agent, currently in phase II clinical trials. Structure–
activity relationships studies have determined the cor-
rect stereochemical orientation of the C(2) substituent
and the key units within 1 that are necessary for activity.
However, we have neither identified the site of action of
FAAs nor have we learned the bioactive conformation
for this class of compounds.
Results and Discussion
Selection of compounds
There has been considerable effort in recent years to
determine the relationship of three-dimensional, con-
formational structure to biological activity for small
molecules.2 Understanding the preferred structure pro-
vides new leads for therapeutic agents with improved
pharmacological properties. One approach to this is to
minimize the flexibility of the lead compound through
Introduction. The interconnecting network of single
bonds within FAAs leads to a spectrum of possible
conformations. Using 2 as the prototype the four
torsion (dihedral) angles, o1 [CH3–C(1)–N(1)–C(2)], f
[C(1)–N(1)–C(2)–C(3), c (N(1)–C(2)–C(3)–N(2)] and o2
[C(2)–C(3)–N(2)–CH2], can be used to define the com-
pound’s structural backbone (Fig. 1). Resonance theory
predicts that o1 and o2 will be either cis (0o) or trans
(180o). Greater conformational freedom exists for the f
*Corresponding author. Tel.: +1-919-966-2680; fax: +1-919-843-
7835; e-mail: harold_kohn@unc.edu
0968-0896/01/$ - see front matter # 2001 Elsevier Science Ltd. All rights reserved.
PII: S0968-0896(01)00204-8

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A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
and c angles. Nonetheless, studies have shown that
preferred angles exist for f and c, and two dimensional
probability distribution functions have been reported
for these dihedral angles (Ramachadran map) in vacuo
and in solvent.5,6
Incorporation of an l-proline unit within the peptide
framework constrains the two central torsion angles, f
and c. Studies document that f is restricted to
ꢁ65ꢂ15ꢀ,7,9c and the peptide adopts a conformation
that affords
c
angles of either +150ꢂ15ꢀ or
ꢁ40ꢂ15ꢀ.13 Finally, the proline tertiary amide bond
(o1) exists in both the trans-(180ꢀ) and the cis-(0ꢀ) con-
formations, with the trans-conformer being pre-
dominant. Using this conformational constraint, we
synthesized 7 and the two acyclic reference compounds,
8 and 9; the N(1) amide moiety was further modified.
Figure 1. The backbone dihedral angles f, c, o1, and o2 within 2.
In our study, four conformationally restricted classes of
peptidomimetic compounds of the parent FAA 2 were
evaluated. Conformational constraints were enforced
using short-range cyclizations.7 The first analogue
selectively limited the o2 dihedral angle while the second
and third set of compounds constrained the c and o2,
and the f and c angles, respectively. The final class of
agents restricted the o1, f, and c angles. For these four
sets of peptidomimetics, we elected to prepare the race-
mates and to compare their biological activities with the
corresponding acyclic racemates.
Simultaneous restriction of the three adjoining f, c,
and o2 torsion angles can be achieved by bridging the
N(1), N(2) sites in dipeptides14,15 with a carbonyl unit to
give substituted hydantoins. X-ray diffraction studies of
5,5-disubstituted hydantoins showed the ring to be
nearly planar with c and o2 torsion angles at 0ꢀ and
180ꢀ, respectively.16,17 We prepared hydantoin 10 and
the corresponding thiohydantoin 11, to study the effect
of restriction of the f, c and o2 dihedral angles on
anticonvulsant activity. This investigation was expan-
ded to the 5-phenyl 12 and the 5-methoxymethylene 13
derivatives.
Selection of compounds. The preferred dihedral angles
for o1 and o2 are cis (0ꢀ) and trans (180ꢀ). Of these two,
the trans arrangement in peptides is the energetically
more stable,8 unless the peptide is incorporated in a
cyclic array or unusual steric and/or hydrogen-bonding
interactions exist that favor the cis conformer.9 While
we expected the FAA o2 dihedral angle to be 180ꢀ in the
absence of the receptor, we were anxious to learn if a
cis-amide-type bond (o2=0ꢀ) would show equal or
improved bioactivity. The 1,5-disubstituted tetrazole
(É[CN4]) ring system has been employed as a peptido-
mimetic unit for the cis-amide bond that exhibited bond
lengths and bond angles close to those observed for the
cis-peptide conformer.10 Accordingly, we prepared the
1,5-disubstituted tetrazole 4.
Synthesis and structural evaluation
1,5-Disubstituted tetrazole 4. We prepared tetrazole 4
using the von Braun18 procedure (Scheme 1). Beginning
with 14, treatment with benzylamine gave N-benzyl-a-
Freidinger11 and others12 have demonstrated that bridg-
ing the C(2) and N(2) terminal amide units through the
use of a g-lactam constrains the c torsion angle and
restricts o2 to 180ꢀ. Conformational energy minimiza-
tion studies and X-ray structure analyses for 3-amino-2-
pyrrolidones indicated that the most stable conforma-
tions have c angles in a restricted region around
ꢁ130ꢂ15ꢀ for the (S)-enantiomer and +130ꢂ15ꢀ for
the corresponding (R)-enantiomer.12b,c We adopted this
short-range cyclization strategy and prepared 5. This
approach converts one of the two secondary amide
groups [N(2)] in 2 to a tertiary amide unit. Removal of
the N(H) moiety may affect the binding of the drug
candidate to the putative receptor and so we selected 6
as the reference compound for 5.
Scheme 1. Synthesis of tetrazole analogue 4. Reagents: (a)
PhCH2NH2, TEA, THF, 0 ꢀC to rt, 3 h, 73%; (b) PCl5, benzene, rt, 12
h; (c) HN3, benzene, 65 ꢀC, 5 h, 80%; (d) potassium phthalimide, KI,
DMF, 110 ꢀC, 3 h, 74%; (e) N2H4, DMF, rt, 2h; (f) 1 N NaOH, rt,
99%; (g) Ac2O, TEA, THF, rt, 45 min, 84%.

A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
2695
chloro amide 15,19 which was sequentially treated with
PCl5 and hydrazoic acid20 to give 5-a-chloroalkyl-
tetrazole 16. Substitution of the a-chloro group in 16
with potassium phthalimide and KI yielded 17, which
upon hydrazinolysis gave the free amine 18. Acetylation
[Ac2O, TEA, DMAP (cat)] of the amine afforded 4
(36% overall yield from 14).
Proline 7 and N(1)-alkyl FAA analogues 8 and 9. Pro-
line 7 was prepared from commercially available N-ace-
tyl-dl-proline 25 by mixed anhydride coupling with
benzylamine (64% yield) (Scheme 4).
The two reference compounds, 8 and 9, were synthe-
sized according to the procedure of Olsen24 using Cbz-
protected amino acids (Scheme 5). Commercially avail-
able Cbz-dl-Ala (26) was treated with EtI and Ag2O in
DMF to produce the N-ethylamino ethyl ester 27 as an
oil. Similarly, using readily available Cbz-dl-Abu25 (31)
and MeI, we produced 32 in 70% yield. Saponification
of the esters in aqueous alcoholic 2N KOH solution
followed by acid workup gave the desired carboxylic
acids, 28 and 33, which were then converted to their
N-benzylamides 29 and 34, respectively, using the mixed
anhydride coupling procedure. Hydrogenolysis (H2, Pd/
C in MeOH)23b,26 of the Cbz protecting groups pro-
duced the amines 30 and 35, which were then acylated
[Ac2O, TEA, DMAP (cat)] to give the reference N(1)-
alkyl FAA analogues 8 and 9 in 62and 44% overall
yields, respectively.
X-ray crystallographic analyses of 4 confirmed the
restriction of the o2 dihedral angle to near 0ꢀ [(R)-iso-
mer: o2=ꢁ1.9(6)ꢀ; (S)-isomer: o2=ꢁ0.1(6)ꢀ] (data not
shown).21
Lactam 5 and N-methyl-N-benzylamide 6. The Frei-
dinger lactam 5 required the cyclization of the N(2)
amide group with the C(2) side chain. Commercially
available tBoc-dl-methionine (19) was treated with
benzylamine to provide 20, using the standard, mixed
anhydride coupling methodology (Scheme 2).1i,22
Treatment of 20 with MeI gave sulfonium salt 21 and
then we cyclized it to the g-lactam 22 with NaH11 in
DMF. Acid deprotection23 of the tBoc group in 22
afforded the corresponding hydrochloride salt of 23,
which was then acylated to provide 5 in excellent yield
(60% overall yield from 19).
1
The H and 13C NMR spectra of 7 showed two sets of
peaks corresponding to the expected cis-(o1=0ꢀ) and
trans-(o1=180ꢀ) isomers. Our 13C NMR assignment of
isomer geometry was made on the basis of chemical
shift values for the pyrrolidinone carbons in CDCl3.27
We measured the trans- to cis-amide ratio to be 3:1 by
1H NMR integration of the N(1)H proton signals
(trans: d 8.35; cis: d 8.66–8.68) in DMSO-d6; a value
substantiated by previous data for N(1)-acetylprolyl
amides.28 The 1H and 13C NMR data for reference
compounds 8 and 9 and their synthetic precursors (27–
29, 32–34) also showed the presence of two major con-
formations in CDCl3, and we have arbitrarily assigned
the major conformer as the o1-trans isomer, based on
those findings.28
The reference compound 6 was prepared using com-
mercially available N-acetyl-dl-alanine (24) (83% yield)
(Scheme 3).
The g-lactam ring in 5 restricted the o2 torsional angle
to trans (o2=180ꢀ) and reduced the conformational
freedom (c) for this molecule. Only one conformer was
observed by 1H NMR for 5 and synthetic precursors 22
and 23, while two conformers (65:35 ratio) were detec-
ted for 6. This finding suggested that a single o1-con-
formation existed for
6
and for pyrrolidinone
derivatives 5, 22, and 23.
Scheme 4. Synthesis of 7. Reagents: (a) NMM, IBCF, benzylamine,
THF, ꢁ78 ꢀC to rt, 1 h, 64%.
Scheme 2. Synthesis of lactam 5. Reagents: (a) NMM, IBCF, benzyl-
amine, THF, ꢁ78 ꢀC to rt, 1 h, 86%; (b) CH3I, rt, 12h, 100%; (c)
NaH, DMF, 0 ꢀC to rt, 2h, 74%; (d) 3 N HCl, EtOAc, rt, 2days,
99%; (e) Ac2O, TEA, DMAP (cat), THF, rt, 10 h, 96%.
Scheme 5. Synthesis of the N-alkyl FAAs 8 and 9. Reagents: (a)
Ag2O, RI (R=Et or Me), DMF, 50 ꢀC (R=Et) or 35 ꢀC (R=Me), 1–2
days, 88 and 70% (R=Et and Me, respectively); (b) 2N aq KOH,
ROH (R=Et or Me), 35 ꢀC, 1–3 h, 99%; (c) NMM, IBCF, benzyl-
amine, THF, ꢁ78 ꢀC to rt, 1 h, 83 and 90% (29 and 34, respectively);
(d) H2 (1 atm), Pd/C, MeOH, rt, 12h, 96 and 99% ( 30 and 35,
respectively); (e) Ac2O, TEA, DMAP (cat), THF, rt, 16–24 h, 89 and
76% (8 and 9, respectively).
Scheme 3. Synthesis of 6. Reagents: (a) EDCI, NMM, HOBt, N-
methylbenzylamine, THF, ꢁ78 ꢀC to rt, 1 h, 83%.

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A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
(Thio)hydantoins 12–15. Using established procedures
we prepared 37, 39, and 41 in moderate-to-good yield
(55–82%) by treating 36, 38, and 40, respectively, with
either benzyl isocyanate or benzyl isothiocyanate and
triethylamine (Scheme 6). Cyclization of the inter-
mediate phenylglycine urea derivative 41 to the hydan-
toin 42 required an additional equivalent of
triethylamine (40 ꢀC, 4 h). Acetylation [Ac2O, TEA and
DMAP (cat)] of the hydantoin N(1) ring nitrogen pro-
vided (thio)hydantoins (10, 11, and 12) (41–81% overall
yield).
activity using the procedures described by Stables and
Kupferberg,30 and these results were compared with the
findings previously reported for FAAs 2 and 3,1b,i and
appropriate anticonvulsant agents31,32 (Tables 1–3). All
compounds were administrated intraperitoneally (ip) to
mice and, when appropriate, orally (po) to rats. For
specific compounds, we report the ED50 values required
to prevent tonic extension of the hind limbs in mice and
in rats in the MES-induced test and the neurological
toxicity (TD50) values using the rotorod test.33
Tetrazole 4. Tetrazole 4 exhibited diminished anti-
convulsant activity (mice: ED50 >100, <300 mg/kg)
compared with its parent analogue 2 (mice: ED50=77
mg/kg). This finding suggests that constraining o2 to a
cis conformation (o2=0ꢀ) does not enhance drug func-
tion. Several factors prohibit further conclusions con-
cerning this conformational constraint. First, only one
compound was tested. Second, the cis-amide bond offers
a unique arrangement of an adjacent hydrogen-bond
donor and acceptor interactions that does not exist in
tetrazole 4. Finally, the tetrazole unit is sterically larger
than the cis-amide bond and this structural feature may
adversely impact anticonvulsant activity.
The final thiohydantoin 13 was prepared beginning with
N-benzyl-2-N-(Cbz)amino-3-hydroxypropionamide29 (43)
(Scheme 7). Methylation (CH3I, Ag2O) followed by
Cbz-deprotection (H2, Pd/C) and treatment with di-2-
pyridylthiocarbonate (DPT) afforded thiohydantoin 46.
N(1)-Acetylation (Ac2O, TEA, DMAP) gave 13 (51%
overall yield from 43).
Freidinger lactams 5. The pharmacological data for
g-lactam 5 (mice: ED50 >300 mg/kg; rats: ED50 >30
mg/kg) and the acyclic N(2)-methyl reference com-
pound 6 (mice: ED50 >100, <300 mg/kg; rats: ED50
>30 mg/kg) showed that both compounds exhibited
weak in vivo anticonvulsant activity. Comparison of
these findings with the MES activity for parent FAA 2
(mice: ED50=77 mg/kg; rats: ED50=48 mg/kg) indi-
cated that N(2)-alkylation of the N-benzylamide moiety
caused a significant decrease in anticonvulsant activity.
To verify this trend, we synthesized 47 and compared
the pharmacological activity of 47 with 3.1i Compound
47 was prepared in four steps beginning with Cbz-serine
(48) (Scheme 8). Conversion of 48 to the N-benzyl-N-
methylamide 49 followed by methylation (CH3I, Ag2O)
and Cbz-deprotection (H2, Pd/C) permitted acetylation
(Ac2O, TEA) of the terminal amine to give 47.
Scheme 6. Synthesis of (thio)hydantoins 10, 11, and 12. Reagents: (a)
aq KOH, PhCH2NCO, 65 ꢀC, 20 min; (b) 6 N HCl, 75 ꢀC, 12h, 55%;
(c) Ac2O, TEA, DMAP (cat), THF, rt, 1.5 days, 84%; (d) PhCH2NCS,
TEA, CH2Cl2, reflux, 30 min, 82%; (e) Ac2O, TEA, DMAP (cat),
THF, rt, 1 h, 99%; (f) PhCH2NCO, TEA, THF, 60 ꢀC, 10 min, 72%;
(g) TEA, THF, 40 ꢀC, 4 h, 84%; (h) Ac2O, TEA, DMAP (cat), THF,
rt, 16 h, 67%.
Scheme 8. Synthesis of 47. Reagents: (a) EDCI, NMM, HOBt, N-
methylbenzylamine, THF, ꢁ78 ꢀC to rt, 1 h, 49%; (b) Ag2O, CH3I,
CH3CN, rt, 3 days, 97%; (c) H2 (1 atm), Pd/C, MeOH, rt, 12h, 91%;
(d) Ac2O, TEA, DMAP (cat), THF, rt, 12h, 96%.
Scheme 7. Synthesis of thiohydantoin 13. Reagents: (a) CH3I, Ag2O,
CH3CN, rt, 3 days, 93%; (b) H2 (1 atm), Pd/C, MeOH, rt, 4 h, 99%;
(c) DPT, THF, rt, 24 h; (d) Ac2O, TEA, DMAP (cat), THF, rt, 6 h,
55% (2steps).
Once again, we saw a large drop in activity. The MES
ED50 values for 47 and 3 in mice were >100, <300 mg/
kg and 8.3 mg/kg,1i respectively. These results may
indicate that the secondary benzylamide unit serves as a
key hydrogen bond donor upon binding to the putative
receptor, and this hydrogen bond is lost upon N(2)-
Pharmacological evaluation
Introduction. All new compounds and appropriate
reference compounds were tested for anticonvulsant

A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
2697
alkylation.34 Alternatively, the incorporation of a g-lac-
tam ring (5) within the FAA backbone and/or N(2)
methylation (6) may prevent adoption of the conforma-
tion needed for maximal anticonvulsant activity.
Finally, we note that the steric size of the terminal
amine unit for lactam 5 and for the N(2)-methyl com-
pound 6 is larger than FAAs 2 and 3. This factor may
prevent binding to the putative receptor and may be
responsible for the observed drop of anticonvulsant
activity.
Proline 7 and N-alkyl amino acid derivatives 8 and 9.
The pharmacological data for proline 7 (mice: ED50
>100, <300 mg/kg; rats: ED50>30 mg/kg) and the two
standard compounds 8 (mice: ED50=85 mg/kg; rats:
ED50=69 mg/kg) and 9 (mice: ED50 >30, <100 mg/kg;
rats: ED50 ꢃ30 mg/kg) showed that incorporation of a
proline unit in the FAA backbone leads to appreciable loss
of anticonvulsant activity. Significantly, both reference
compounds 8 and 9 exhibited anticonvulsant activity
comparable with the parent FAA 2 (mice: ED50=77
mg/kg; rats: ED50=48 mg/kg). The finding that 7 was
less active than its reference compounds 8 and 9 sug-
gested that the proline-induced f1 and/or c1 dihedral
angles constraints7,9 impacted the anticonvulsant function
of these FAA peptidomimetics. Recently, Paruszewski
Table 1. Pharmacological data for the conformationally constrained derivatives and their reference compounds
Compd
Mice (ip)a
Tox,d TD50
Rats (po)b
Tox,d TD50
MES,c ED50
PIe
5.9
5.2
>1.0
>1.0
—
>1.0
2.1
>3.0
6.9
MES,c ED50
PIe
f
2
3
4
5
6
7
8
9
77 [1] (67–89)
8.3 [0.5] (7.9–9.8)
>100, <300
>300
>100, <300
>100, <300
85 [0.25] (74–99)
>30, <100
450 [0.5] (420–500)
43 [0.25] (38–47)
>300
48 [1] (32–72)
3.8 [2] (2.9–5.5)
ꢃ60g
—
390 [1] (320–520)
>20
101
—
—
—
—
>3.5
—
>30
>30
>30
>30
>240
>30
>300
>30
>30
>30
ꢃ300
>300
180 [0.25] (160–200)
>300
66 [2] (53–72)
69 [6] (35–290)
ꢃ30g,h
Phenytoini
9.5 [2] (8.1–10)
30 [4] (22–39)
—
>100
j
aThe compounds were administered intraperitoneally. ED50 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 square brackets).
bThe compounds were administered orally.
cMES=maximal electroshock seizure test.
dTox=neurologic toxicity determined from rotorod test.
ePI=protective index (TD50/ED50).
fNo ataxia observed up to 1000 mg/kg.
g2/4 animals were protected at this dose.
hED50 <30 mg/kg by ip administration in rats (4/4 animals were protected at 30 mg/kg after 0.25 h).
iRef 31.
jNo ataxia observed up to 3000 mg/kg.
Table 2. Pharmacological data for (thio)hydantoin derivatives and their acyclic FAA analogues
Compd
Mice (ip)a
Tox,d TD50
Rats (po)b
Tox,d TD50
MES,c ED50
PIe
MES,c ED50
PIe
10
11
12
13
2
52
3
>30, <100 [0.5]
100 [0.5] (89–120)
>300
>300f
120 [0.5] (96–150)
>300
—
1.2
27 [0.5] (17–41)
ꢄ40g
>500
>50
>30
>18
—
—
—
>20
—
100
>100
6.7
—
<1.0
5.9
4.8
5.1
>30
h
h
>300
>100, <300
—
—
—
i
77 [1] (67–89)
20 [0.5] (17–25)
8.3 [0.5] (7.9–9.8)
9.5 [2] (8.1–10)
22 [1] (15–23)
450 [0.5] (420–500)
97 [0.5] (80–120)
43 [0.25] (38–47)
66 [2] (53–72)
69 [0.5] (63–73)
48 [1] (32–72)
48 [4] (31–68)
3.8 [2] (2.9–5.5)
30 [4] (22–39)
9.1 [5] (7.6–12)
i
—
390 [1] (320–520)
Phenytoinj
Phenobarbitall
6.9
3.2
—
61 [0.5] (44–96)
k
aThe compounds were administered intraperitoneally. ED50 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 square brackets).
bThe compounds were administered orally.
cMES=maximal electroshock seizure test.
dTox=neurologic toxicity determined from rotorod test.
ePI=protective index (TD50/ED50).
f2/4 animals were protected at this dose.
gNo linear dose response was obtained in rats by po for this compound. ED50=12[0.5] (7.9–17) mg/kg by ip administration in rats.
hNot determined.
iNo ataxia observed up to 1000 mg/kg.
jRef 31.
kNo ataxia observed up to 3000 mg/kg.
lRef 32.

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A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
Table 3. Subcutaneous Metrazol1 (scMet) seizure threshold test for selected (thio)hydantoin derivatives and their acyclic FAA analogues
Compd
Mice (ip)a
Tox,d TD50
Rats (po)b
Tox,d TD50
scMet,c ED50
PIe
scMet,c ED50
PIe
10
11
12
13
2
52
3
>100, <300
46 [0.5] (36–60)
>300
ꢃ300f
>1.0
2.5
—
<1.0
<0.6
<0.8
<0.2
—
>250
>50
>500
>50
>30
—
—
—
—
—
—
<1.5
—
120 [0.5] (96–150)
>300
g
—
—
g
g
>100, <300
>100, <300
—
—
>800f
450 [0.5] (420–500)
97 [0.5] (79–120)
43 [0.25] (38–47)
66 [2] (53–72)
69 [0.5] (63–73)
>1000
>1000
>260
h
h
>120
—
390 [1] (320–520)
>300
Phenytoini
Phenobarbitall
—
13 [1] (5.9–16)
—
—
j
j
k
5.62112[0.5] (44–96)
5.3
aThe compounds were administered intraperitoneally. ED50 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 square brackets).
bThe compounds were administered orally.
cscMet=subcutaneous Metrazol1 seizure threshold test.
dTox=neurologic toxicity determined from rotorod test.
ePI=protective index (TD50/ED50).
f2/4 animals were protected at this dose.
gNot determined.
hNo ataxia observed up to 1000 mg/kg.
iRef 31.
jNot effective.
kNo ataxia observed up to 3000 mg/kg.
lRef 32.
and co-workers reported the anticonvulsant activity for
optically pure (R)-7 (ED50=67 mg/kg) in mice (ip).35
The increased potency for (R)-7, compared with 7, was
in agreement with the C(2) stereochemical requirements
of FAAs.1cꢁe,g,i Finally, we note that our finding that
N(1)-alkylation did not affect activity contrasted with
the discovery that N(2)-alkylation leads to loss of
anticonvulsant activity (e.g., 6; Table 1).
aromatic moiety at C(2) (i.e., 52 vs 2). This trend was
not maintained in our (thio)hydantoins. We found that
replacement of the methyl group in 10 (mice: ED50
>30, <100 mg/kg) by a phenyl moiety to give 12 (mice:
ED50 >300 mg/kg) led to a significant drop in anti-
convulsant activity. Similarly, the C(5) methyl thiohy-
dantoin
11
(mice:
ED50=100
mg/kg)
was
considerably more potent than the C(5) methoxy-
methylene analogue 13 (mice: ED50 >300 mg/kg).
Third, hydantoin 10 and thiohydantoins 11 and 13
possessed moderate-to-good activity in the scMet-seizure
test30 in mice (Table 3). The most active compound was
11 (ED50=46 mg/kg). This finding contrasted with the
lack of scMet-activity exhibited by FAA analogues1
2, 3, and 52 and phenytoin.31 Previous SAR studies
have shown that hydantoin N(3) alkylation confers
significant sc-Met activity without appreciable loss in
MES-activity.36,37
3.5(Thio)hydantoins 10–13. The in vivo anticonvulsant
activities for (thio)hydantoins 10–13, the corresponding
FAAs 2, 3, and 52,1b,i and the potent antiepileptic
agents, phenytoin,31 and phenobarbital,32 are summarized
in Tables 2and 3.
We concluded that FAAs and their monosubstituted
hydantoin analogues exhibited different anticonvulsant
profiles. To gain additional insight into the possible
mechanism of action of these hydantoins, we examined
the activities of 10, 2, and (R)-3 in the voltage-sensitive
Na+ channel test using patch-clamp electrophysiology
techniques.38 Table 4 documents that hydantoin 10
exhibits a large voltage-dependant blockage of Na+
channels (35% blockage at ꢁ60 mV), while the two
FAAs [2 and (R)-3] had no significant effect on peak
current at 100 mM. For comparison, two prototypical
antiepileptic Na+ channel blockers, phenytoin and
lamotrigine, provide 48 and 53% blockage at ꢁ60 mV,
respectively.39 These findings indicate that 10 expresses
its anticonvulsant activity, in part, by acting at the Na+
channel. Together, the divergent SAR and the different
electrophysiological findings for (thio)hydantoins and
FAAs provide evidence that these two classes of com-
pounds function by different mechanisms.
These results provided several important findings. First,
no significant differences in anticonvulsant activities
were observed for hydantoin 10 and thiohydantoin 11.
Both compounds provided protection in the MES-
induced seizure test in mice and rats. The ED50 values
for compound 10 in rats (po) was 27 mg/kg, while
compound 11 protected half of the rats tested at 40 mg.
We observed that 11 did not give a linear dose-response
(4/8 rats were protected at 40 mg/kg but only 5/16 at 80
mg/kg). Accordingly, we tested ip administration of 11
in rats to eliminate potential absorption problems. The
ED50 (ip) was impressive at 12mg/kg in the MES-seiz-
ure test. Second, the SAR patterns for FAAs1 were not
useful for predicting the activities of newly prepared
(thio)hydantoins. For FAAs, improved anticonvulsant
activities in the MES-seizure assay were observed by
either incorporating a heteroatom one atom removed
from the C(2) site (i.e., 3 vs 2) or by placing a small

A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
2699
Table 4. Effect of 2, (R)-3 and 10 on voltage sodium channels in NI
E-115 neuroblastoma cells at 100 mM
General procedure for the preparation of N-benzylamide
amino acids derivatives using the mixed anhydride
coupling (MAC) (method A)1i,22
Compd
No. of cells
% Control (ꢂSEM)a
ꢁ90 mV
ꢁ60 mV
83ꢂ4
A dry THF solution (ꢃ0.5–2mmol carboxylic acid/mL
of THF) containing the carboxylic acid was cooled to
ꢁ78 ꢀC under Ar, and 4-methylmorpholine (1.1–2.2
equiv) was added. After stirring (2min), isobutyl chlor-
oformate (1.1–1.25 equiv) was added leading to the
precipitation of a white solid. The reaction was allowed
to proceed for an additional 2min, and then benzyla-
mine (1.1–1.25 equiv) was added at ꢁ78 ꢀC. The reac-
tion mixture was allowed to stir at room temperature
(30 min–3 h), and then the insoluble salts filtered. The
organic layer was concentrated in vacuo and the pro-
duct purified by column chromatography on SiO2 gel.
2
(R)-3
10
Phenytoinc
Lamotriginec
5, 5
6
6, 9
5, 5
7, 9
100ꢂ1
102ꢂ5
93ꢂ3
b
—
65ꢂ3
ꢂ4
78ꢂ252
67ꢂ5
47ꢂ7
aThe results from several individual cells were averaged, the SEM cal-
culated and statistical significance was determined using the Student’s
t-test. Significance was taken to be in the range of p values: p<0.05.
bNot determined.
cRef 39.
Conclusions
General procedure for the preparation of N-alkyl-
benzylamide-substituted amino acids (method B)
We have synthesized and evaluated a series of proven,
conformationally constrained analogues and compared
their anticonvulsant activities with their corresponding
reference FAAs. We designed these compounds to learn
the preferred bioactive FAA conformer(s). No
improvement in pharmacological activity was observed
upon conformational constraint thus preventing our
identifying new lead compounds. Important new
information on the SAR of FAAs was obtained. We
showed that FAA N(1)-alkylation did not reduce anti-
convulsant activity while N(3)-alkylation led to appreci-
able activity loss. We also learned that structurally
similar (thio)hydantoins function by pathway(s) distinct
from FAAs. This finding is important since the mode of
action of FAAs has not been determined. Finally, we
observed that hydantoin 10 and thiohydantoin 11, upon
po and ip administration, respectively, displayed activ-
ities comparable with phenytoin in the MES-seizure test
in rats and that both compounds displayed significant
protection against sc-Met-induced seizures.
To a dry DMF or THF solution (ꢃ0.1–0.5 mmol car-
boxylic acid/mL of solvent) of the carboxylic acid were
successively added under Ar EDCI (1.1–1.8 equiv),
HOBt (1.1–1.8 equiv), and 4-methylmorpholine (1.1–1.8
equiv). After stirring (5 min), the amine (0.8–1.2equiv)
was added dropwise and the reaction was stirred at
room temperature (12–24 h). The mixture was diluted
with 1 M HCl and extracted with EtOAc. The organic
layers were combined, washed with brine, concentrated
in vacuo, and the product purified by column chromato-
graphy on SiO2 gel.
General procedure for the preparation of N(1)-acetyl-
substituted amino acids (method C)
To a dry THF solution of amine or ammonium hydro-
chloride salt (ꢃ0.1–1 mmol amine/mL of THF) were
successively added TEA (1–2equiv), Ac O (1–2equiv),
2
and a catalytic amount of DMAP. The reaction was
allowed to stir at room temperature (1 h–1 day). The
organic layer was concentrated in vacuo and the pro-
duct purified by column chromatography on SiO2 gel or
by recrystallization.
Experimental
General methods
Nuclear magnetic resonance spectra were measured at
300 MHz for 1H NMR and 75 MHz for 13C NMR.
Chemical shifts (d) are reported in parts per million
(ppm) downfield from tetramethylsilane and coupling
constants (J values) are in Hertz. In cases where con-
formational isomers were observed the number in par-
enthesis in the NMR assignments correspond to the
minor conformer and signals not detected are believed
to overlap with those of the major conformer. Yields
reported are for purified products and were not opti-
mized. Reactions involving water- or air-sensitive com-
pounds were conducted in dried glassware and carried
out under a positive pressure of Ar. Mass spectral
studies were conducted by Dr. M. Moini (Unversity of
Texas, Austin, TX, USA) and the X-ray crystallo-
graphic determination of 4 was performed by Dr. James
Korp (University of Houston, Houston, TX, USA).
Microanalyses were provided by Atlantic Microlab, Inc.
(Northcross, GA, USA).
1-Benzyl-5-(1-chloroethyl)-1H-tetrazole (16). Phospho-
rous pentachloride (6.79 g, 32.6 mmol) was added with
stirring and cooling (5–10 ꢀC) to a suspension of 1519
(5.85 g, 29.7 mmol) in dry benzene (45 mL). The reac-
tion was exothermic and was accompanied by the evo-
lution of HCl. The reaction was stirred overnight at
room temperature and then concentrated in vacuo by
approximately one half. A benzene solution (92mL) of
hydrazoic acid20 (CAUTION) (ꢃ4 M) was then added
in portions with vigorous stirring at 5–10 ꢀC. The mix-
ture was allowed to warm to room temperature and
stirred at 65 ꢀC (5 h). The solvent was evaporated in
vacuo and iced cooled H2O (100 mL) was added. The
product was extracted from the cooled suspension with
benzene (3ꢅ60 mL). The organic layers were combined,
dried (Na2SO4), and the solvent evaporated in vacuo.
The residue was purified by column chromatography
(SiO2; 1:99, EtOAc/hexanes to 1:9, EtOAc/hexanes) to
yield 5.29 g (80%) of pure 16 as a clear oil: Rf 0.43 (3:1,

2700
A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
hexanes/EtOAc); IR (neat) 3067, 3035, 2990, 1498
1
N-Benzyl-2- N-(tert-butoxycarbonyl)amino-4-(thiome-
thyl)-butanamide (20). Utilizing method A and N-tBoc-
dl-methionine (19) (2.00 g, 8.0 mmol), THF (80 mL), 4-
methylmorpholine (1.1 mL, 9.63 mmol), isobutyl
chloroformate (1.3 mL, 9.63 mmol) and benzylamine
(1.1 mL, 9.63 mmol) gave 2.32 g (86%) of pure 20 as a
white solid after purification by column chromato-
graphy (SiO2; 2:1, hexanes/EtOAc): mp 95–96ꢀC; Rf
0.30 (2:1, hexanes/EtOAc); IR (KBr) 3336, 3305, 2970,
2924, 1682, 1655, 1570, 1523 cmꢁ1; 1H NMR (CDCl3) d
1.41 (s, (CH3)3), 1.89–1.98 (m, CHH0CH), 2.05–2.15 (m,
CHH0CH), 2.06 (s overlapping with CHH0CH, SCH3),
2.49–2.60 (m, CH2SCH3), 4.25–4.33 (m, CH), 4.37–4.50
(m, CH2Ph), 5.35 (d, J=8.4 Hz, NHCH), 6.81–6.85
(m, NHCH2), 7.20–7.36 (m, 5 PhH); 13C NMR (CDCl3)
d 15.5, 28.5, 30.4, 31.9, 43.7, 53.7, 80.0, 127.7, 127.8,
128.9, 138.1, 155.8, 171.6; MS (+CI) (rel intensity)
339 (M++1, 27), 283 (100), 239 (42); Mr (+CI)
339.175 12[M ++1] (calcd for C17H27N2O3S 339.174
24). Anal. calcd for C17H26N2O3S: C, 60.33%; H,
7.74%; N, 8.28%. Found: C, 60.19%; H, 7.79%; N,
8.33%.
cmꢁ1; H NMR (CDCl3) d 1.98 (d, J=6.8 Hz, CH3),
4.97 (q, J=6.8 Hz, CH), 5.55 (d, J=15.3 Hz, CHH0Ph),
5.83 (d, J=15.3 Hz, CHH0Ph), 7.26–7.29 (m, 2 PhH),
7.35–7.41 (m, 3 PhH); 13C NMR (CDCl3) d 22.4,
44.4, 51.7, 127.9, 129.4, 129.5, 132.9, 154.7; MS
(+CI) (rel intensity) 225 (33), 223 (M++1, 100); Mr
(+CI) 223.075 67 [M++1] (calcd for C10H1235ClN4
223.075 05). Anal. calcd for C10H11ClN4: C, 53.94%; H,
4.98%; N, 25.16%. Found: C, 54.18%; H, 5.07%; N,
24.96%.
2-[1-(1-Benzyl-1H-tetrazole-5-yl)-ethyl]-isoindole-1,3-
dione (17).20a To a DMF solution (60 mL) of 16 (3.92
g, 17.6 mmol) were added potassium phthalimide (3.92
g, 21.2 mmol) and KI (0.35 g, 1.8 mmol). The mixture
was stirred at 110 ꢀC (3 h), and the solvent was evapo-
rated in vacuo to give crude 17 as a pale-yellow solid.
The residue was recrystallized from toluene (2ꢅ) to
yield 4.33 g (74%) of pure 17 as a white solid: mp 148–
149 ꢀC (lit.20a mp 146–147 ꢀC); Rf 0.35 (3:1, hexanes/
1
EtOAc); H NMR (DMSO-d6) d 1.83 (d, J=7.1 Hz,
CH3), 5.33 (d, J=16.7 Hz, CHH0Ph), 5.79 (d, J=16.7
Hz, CHH0Ph), 5.89 (q, J=7.1 Hz, CH), 6.71–6.80 (m, 3
PhH), 6.91 (t, J=7.5 Hz, 2Ph H), 7.60 (dd, J=3.0, 6.0
Hz, 2Pht H), 7.72(dd, J=3.0, 6.0 Hz, 2Pht H).
N-Benzyl-2-N-(tert-butoxycarbonyl)amino-4-(S,S-dime-
thyl sulfonium)-butanamide iodide (21) and 1-benzyl-3-
(tert-butoxycarbonyl)amino-pyrrolidin-2-one (22). Com-
pound 20 (1.49 g, 4.4 mmol) was dissolved in CH3I (10
mL, 160 mmol) and stirred at room temperature (12h).
Evaporation of the solvent in vacuo gave 2.10 g (100%)
of 21 as a pale-yellow solid which was used without
further purification: mp 103–110 ꢀC; Rf 0.26 (1:9,
MeOH/CHCl3); IR (KBr) 3328, 3270, 2976, 1704, 1674,
1-(1-Benzyl-1H-tetrazole-5-yl)-ethylamine (18)20a and N-
acetyl-[1-(1-benzyl-1H-tetrazole-5-yl)ethyl]-ethylamine
(4). To a DMF solution (30 mL) of 17 (3.00 g, 9.0
mmol) was added hydrazine (570 mL, 18.0 mmol) and
the solution was stirred at room temperature (2h). The
solvent was evaporated in vacuo and the residue was
redissolved in a binary mixture of an aqueous 1 N
NaOH solution (60 mL) and CH2Cl2 (100 mL) and then
stirred (30 min). The organic layer was separated and
then extracted with an aq 1 N HCl solution (6ꢅ50 mL).
The aqueous layers were combined, adjusted to pH 13
with an aq 5 N NaOH solution and extracted with
CH2Cl2 (6ꢅ60 mL). The organic layers were combined,
dried (Na2SO4), evaporated in vacuo to yield 1.85 g
(99%) of 18 as an oil: Rf 0.29 (1:19, MeOH/CHCl3); 1H
NMR (CDCl3) d 1.45 (d, J=6.8 Hz, CH3), 1.65 (s,
NH2), 4.26 (q, J=6.8 Hz, CH), 5.72(s, C H2Ph), 7.23–
7.26 (m, 2 PhH), 7.33–7.39 (m, 3 PhH).
1
1563 cmꢁ1; H NMR (DMSO-d6) d 1.40 (s, (CH3)3),
1.95–2.16 (m, CH2CH), 2.90 (s, S+CH3), 2.91 (s,
S+CH3), 3.27–3.35 (m, CH2S(CH3)2), 4.03–4.12(m,
CH), 4.27 (dd, J=5.7, 15.2Hz, C HH0Ph), 4.36 (dd,
J=5.9, 15.2Hz, CH H0Ph), 7.17–7.34 (m, 5 PhH and
NHCH2), 8.47 (t, J=5.7 Hz, NH).
The crude sulfonium salt 21 (5.14 g, 10.7 mmol) was
dissolved in DMF (50 mL) under Ar and cooled (0 ꢀC)
and then sodium hydride (428 mg (60% dispersion in
mineral oil), 10.7 mmol) was added all at once. The
mixture was stirred at 0 ꢀC (10 min) and then allowed to
warm to room temperature (2h). The reaction mixture
was quenched with H2O (50 mL) at 0 ꢀC. The solution
was concentrated in vacuo and the residue purified by
column chromatography (SiO2; 1:1, hexanes/EtOAc) to
give 2.30 g (74%) of pure 22 as a white solid: mp 126–
128 ꢀC; Rf 0.48 (1:1, hexanes/EtOAc); IR (KBr) 3294,
Utilizing method C and using crude 18 (187 mg, 0.92
mmol), THF (10 mL), TEA (150 mL, 1.10 mmol) and
Ac2O (100 mL, 1.10 mmol) gave crude 4 as an oil after
45 min. The product was purified by column chromato-
graphy (SiO2; 1:19, MeOH/CHCl3) to give 250 mg
(84%) of pure 4 as a white solid: mp 110–112 ꢀC; Rf 0.50
(1:19, MeOH/CHCl3); IR (KBr) 3242, 3066, 2994, 1639,
1
2978, 1712, 1666, 1512 cmꢁ1; H NMR (CDCl3) d 1.38
(s, (CH3)3), 1.68–1.82(m, C HH0CH), 2.48–2.56 (2.73–
2.78) (m, CHH0CH (CH2CH)), 3.11–3.16 (m,
CH2CH2N), 4.06–4.20 (m, CH), 4.34–4.50 (s, CH2Ph),
1
1554, 1511 cmꢁ1; H NMR (CDCl3) d 1.42(d, J=6.9
1
Hz, CH3), 1.94 (s, CH3C(O)), 5.36–5.41 (m, CH), 5.75
(s, CH2Ph), 7.25–7.27 (m, 2 PhH), 7.35–7.39 (m, 3
PhH), 7.57 (d, J=7.8 Hz, NH); 13C NMR (CDCl3)
19.2, 22.8, 38.7, 51.1, 127.7, 129.0, 129.3, 133.9, 156.7,
170.4; MS (+CI) (rel intensity) 247 (11), 246 (M++1,
100); Mr (+CI) 246.135 68 [M++1] (calcd for
C12H16N5O 246.135 49). Anal. calcd for C12H15N5O: C,
58.76%; H, 6.16%; N, 28.55%. Found: C, 58.50%; H,
6.15%; N, 28.41%.
5.15–5.24 (br s, NHCH), 7.13–7.28 (m, 5 PhH), the H
NMR structural assignments were in agreement with
the 1H–1H COSY experiment; 13C NMR (CDCl3) d
28.3, 28.4 (28.5), 43.5 (43.1), 47.2 (47.3), 52.7, 79.9,
127.9, 128.2, 128.9, 136.0, 156.0, 172.3, the 13C NMR
structural assignments were in agreement with the
HETCOR experiment; MS (+CI) (rel intensity) 291
(M++1, 22), 235 (100); Mr (+CI) 291.170 00 [M++1]
(calcd for C16H23N2O3 291.170 87). Anal. calcd for

A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
2701
C16H22N2O3: C, 66.18%; H, 7.64%; N, 9.65%. Found:
C, 66.23%; H, 7.64%; N, 9.64%.
lamine (950 mL, 8.77 mmol) gave crude 7. The product
was purified by column chromatography (SiO2; 1:19,
MeOH/CHCl3) to obtain 1.10 g (64%) of pure 7 as a
clear oil, which crystallized upon standing (24 h, in
3-Amino-1-benzyl-pyrrolidin-2-one hydrochloride (23)
and 3-acetylamino-1-benzyl-pyrrolidin-2-one (5). Com-
pound 22 (2.28 g, 7.86 mmol) was dissolved in 3 M aq
HCl/EtOAc (20/60 mL) and stirred at room tempera-
ture. After 2days, the solution was concentrated in
vacuo to yield a green hygroscopic oil. The crude
hydrochloride salt was redissolved in water (50 mL) and
extracted with EtOAc (2ꢅ25 mL). The aqueous layer
was concentrated in vacuo to give 1.77 g (99%) of crude
23 as a thick colorless, highly hygroscopic oil, which
was used without further purification: 1H NMR
(DMSO-d6+1 drop D2O) d 1.87–1.97 (m, CHH0CH),
2.37–2.41 (m, CHH0CH), 3.26–3.30 (m, CHH0CH2N),
4.06 (t, J=9.3 Hz, CH), 4.38 (d, J=15.0 Hz, CHH0Ph),
4.47 (d, J=15.0 Hz, CHH0Ph), 7.24–7.38 (m, 5 PhH),
the structural assignments were in agreement with the
1H–1H COSY experiment; 13C NMR (DMSO-d6+1
drop D2O) d 23.9, 43.7, 46.3, 50.2, 127.8, 127.9, 129.0,
136.1, 169.4.
vacuo): mp 96–97 ꢀC; Rf 0.61 (1:9, MeOH/CHCl3); H
1
NMR (DMSO-d6) d 1.86–2.24 (m, CH3C(O), C(3)HH0
and C(4)H2), 3.42–3.55 (m, C(5)HH0), 3.58–3.66 (m,
C(5)HH0), 4.31–4.40 (m, CH2Ph and CH), 7.25–7.40 (m,
1
5 PhH), 8.35 (8.66–8.68) (t (m), J=6.0 Hz, NH), H
NMR analysis indicated the major and minor con-
formational isomers existed in a 75:25 ratio in CDCl3;
13C NMR (CDCl3) d 22.1, 24.5 (22.8), 28.2 (31.8), 42.8,
47.9 (46.3), 59.4 (61.6), 126.7, 127.0, 127.2, 128.1, 138.3,
138.4, 170.2(169.9), 171.5 (171.8); MS (+CI) (rel
intensity) 248 (16), 247 (M++1, 100); Mr (+CI)
247.144 32 [M++1] (calcd for C14H19N2O2 247.144 65).
Anal. calcd for C14H18N2O2: C, 68.27%; H, 7.37%; N,
11.37%. Found: C, 68.40%; H, 7.36%; N, 11.51%.
2-(N-Benzyloxycarbonyl-N-ethyl)aminopropionic
acid
ethyl ester (27). To a DMF (80 mL) solution of 26
(5.34 g, 23.9 mmol) was successively added Ag2O (22.16
g, 95.7 mmol) and EtI (15.3 mL, 107 mmol) at room
temperature. The reaction mixture was stirred at 50 ꢀC
(2days), filtered and the filtrate evaporated in vacuo. The
oily residue was purified by column chromatography
(SiO2; 4:1, hexanes/EtOAc) to give 5.86 g (88%) of pure
27 as a clear oil: Rf 0.25 (4:1, hexanes/EtOAc); IR (neat)
2983, 2941, 1741, 1702 cmꢁ1; 1H NMR (CDCl3) d 1.10–
1.25 (m, NCH2CH3 and OCH2CH3), 1.46 (d, J=7.2Hz,
CHCH3), 3.42–3.50 (3.12–3.20) (m, NCH2CH3), 4.14–
4.17 (3.94–4.06) (m, OCH2CH3), 4.53–4.63 (4.25–4.33)
(m, CH), 5.12(5.13) (s, OC H2Ph), 7.27–7.35 (m, 5
PhH), 1H NMR analysis indicated the major and minor
conformational isomers existed in a 55:45 ratio in
CDCl3, the structural assignments were in agreement
Utilizing method C and using crude 23 (2.00 g, 8.85
mmol), THF (100 mL), TEA (2.5 mL, 17.7 mmol),
Ac2O (920 mL, 9.73 mmol) and DMAP (ꢃ200 mg) gave
crude 5 after 10 h. The product was purified by column
chromatography (SiO2; 1:19, MeOH/CHCl3) to obtain
1.97 g (96%) of pure 5 as a white solid: mp 130–132 ꢀC;
Rf 0.34 (1:19, MeOH: CHCl3); IR (KBr) 3267, 3082,
2993, 2939, 1697, 1639, 1558 cmꢁ1; 1H NMR (CDCl3) d
1.76–1.84 (m, CHH0CH), 2.05 (s, CH3C(O)), 2.65–2.72
(m, CHH0CH), 3.21–3.26 (m, CHH0CH2N), 4.32–4.50
(m, CH), 4.44 (d, J=14.7 Hz, CHH0Ph), 4.53 (d,
J=14.7 Hz, CHH0Ph), 6.69 (d, J=4.5 Hz, NHCH),
7.21–7.37 (m, 5 PhH), the structural assignments were in
agreement with the 1H–1H COSY experiment; 13C
NMR (CDCl3) d 23.2, 28.1, 43.9, 47.4, 52.1, 128.0,
128.2, 129.0, 135.8, 171.1, 172.6; MS (+CI) (rel intensity)
233 (M++1, 27), 215 (10), 191 (13); Mr (+CI) 233.128
61 [M++1] (calcd for C13H17N2O2 233.129 00). Anal.
1
with the H–1H COSY experiment; 13C NMR (CDCl3)
d 14.2, 15.2, 16.1 (17.0), 40.6 (41.8), 55.2, 61.1, 67.2,
127.8, 127.9, 128.2, 128.5, 136.7, 136.8, 156.2 (155.4),
172.2; MS (+CI) (rel intensity) 281 (14), 280 (M++1,
100), 236 (16); Mr (+CI) 280.155 17 [M++1] (calcd for
C15H22NO4 280.154 88). Anal. calcd for C15H21NO4: C,
64.50%; H, 7.58%; N, 5.01%. Found: C, 64.59%; H,
7.64%; N, 5.09%.
.
calcd for C13H16N2O2 0.1H2O: C, 66.70%; H, 6.98%;
N, 11.97%. Found: C, 66.56%; H, 6.86%; N, 11.86%.
N-Benzyl-N-methyl-2-acetamidopropionamide (6).40 Uti-
lizing method B and using 24 (1.43 g, 10.9 mmol), THF
(150 mL), EDCI (2.51 g, 13.09 mmol), 4-methylmor-
pholine (1.4 mL, 13.09 mmol), HOBt (1.77 g, 13.09
mmol) and N-methylbenzylamine (1.1 mL, 8.73 mmol)
gave crude 6 after 24 h. The product was purified by
column chromatography (SiO2; 1:33, MeOH/CHCl3) to
give 1.70 g (83%) of pure 6 as a clear oil: Rf 0.57 (1:19,
MeOH/CHCl3); 1H NMR (CDCl3) d 1.37 (1.32) (d,
J=6.9 (4.5) Hz, CHCH3), 2.01 (1.97) (s, CH3C(O)), 2.99
(2.92) (s, NCH3), 4.50–4.69 (m, CH2Ph), 4.94–5.05 (m,
CH), 7.00 (d, J=5.4 Hz, NH), 7.18–7.35 (m, 5 PhH), 1H
NMR analysis indicated the major and minor
conformational isomers existed in a 65:35 ratio in CDCl3.
2-(N-Benzyloxycarbonyl-N-ethyl)aminopropionic
acid
(28). Compound 27 (4.57 g, 16.39 mmol) was saponi-
fied with 2M KOH/EtOH solution (20 /40 mL) at 35 ꢀC
(3 h). The solution was partially concentrated in vacuo
and the remaining aqueous solution was washed with
Et2O (20 mL), cooled (0 ꢀC), and brought to pH 2with
aqueous 4 N HCl. The mixture was extracted with
EtOAc (3ꢅ50 mL), and then the combined extracts were
dried (Na2SO4) and evaporated in vacuo to give 4.10 g
(99%) of pure 28 as a clear oil: Rf 0.57 (1:9, MeOH/
CHCl3); IR (neat) 3381–2900 (br), 1699 (br) cmꢁ1; H
1
NMR (CDCl3) d 1.03–1.07 (m, NCH2CH3), 1.36 (d,
J=6.9 Hz, CHCH3), 3.01–3.17 (3.33–3.40) (m,
NCH2CH3), 4.48 (4.24) (q, J=6.9 (6.9) Hz, CH), 5.05
(5.02) (s, OCH2Ph), 7.13–7.23 (m, 5 PhH), 10.20 (s,
N-Benzyl-1-acetyl-2-pyrrolidinecarboxamide (7).41 Uti-
lizing method A and using 25 (1.10 g, 7.02mmol), THF
(90 mL), 4-methylmorpholine (960 mL, 8.77 mmol), iso-
butyl chloroformate (1.10 mL, 8.77 mmol), and benzy-
1
C(O)OH), H NMR analysis indicated the major and
minor conformational isomers existed in a 55:45 ratio in
CDCl3; 13C NMR (CDCl3) d 15.0 (14.1), 15.2(15.8),

2702
A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
40.9 (41.9), 55.1 (55.0), 67.5, 127.7, 128.0, 128.1, 128.4,
136.2, 136.4, 156.4 (155.6), 176.9; MS (+CI) (rel inten-
sity) 252 (M++1, 78), 208 (100); Mr (+CI) 252.123 75
[M++1] (calcd for C13H18NO4 252.123 58).
2.07 (2.03) (s, CH3C(O)), 3.34 (q, J=7.2Hz,
NCH2CH3), 4.37 (d, J=5.7 Hz, CH2Ph), 5.03 (q, J=7.2
Hz, CH), 7.10–7.14 (br s, C(O)NH), 7.20–7.31 (m, 5
PhH), 1H NMR analysis indicated the major and minor
conformational isomers existed in a 90:10 ratio in
CDCl3; 13C NMR (CDCl3) d 14.4, 15.5, 21.7, 40.0, 43.3,
53.3, 127.2, 127.5, 128.5, 138.5, 171.7, 171.8; MS (+CI)
(rel intensity) 249 (M++1, 21), 142 (100); Mr (+CI)
249.160 54 [M++1] (calcd for C14H21N2O2 249.160 30).
Anal. calcd for C14H20N2O2: C, 67.71%; H, 8.12%; N,
11.28%. Found: C, 67.83%; H, 8.16%; N, 11.31%.
N-Benzyl-2-(N-benzyloxycarbonyl-N-ethylamino)propion-
amide (29). Utilizing method A and using 28 (3.80 g,
15.1 mmol), THF (80 mL), 4-methylmorpholine (2.0
mL, 18.2mmol), isobutyl chloroformate (2.4 mL, 18.2
mmol), and benzylamine (2.0 mL, 18.2 mmol) gave
crude 29. The product was purified by column chroma-
tography (SiO2; 2:1, hexanes/EtOAc) to obtain 4.27 g
(83%) of pure 29 as a white solid: mp 88–89 ꢀC; Rf 0.39
(2:1, hexanes/EtOAc); IR (KBr) 3304, 3067, 3031, 2980,
2-(N-Benzyloxycarbonyl-N-methyl)aminobutyric
acid
methyl ester (32). Utilizing a procedure comparable for
the synthesis of 27 and using 3133 (86 mg, 0.36 mmol),
DMF (2.5 mL), Ag2O (336 mg, 1.45 mmol), and MeI
(0.2mL, 2.90 mmol) at 35 ꢀC (1 d) gave pure 32 (67 mg,
70%) as a clear oil after purification by column chro-
matography (SiO2; 2:1, hexanes/EtOAc): Rf 0.57 (2:1,
hexanes/EtOAc); IR (neat) 2970, 2955, 1744, 1703
1
1683, 1649, 1531 cmꢁ1; H NMR (CDCl3) d 1.11 (t,
J=6.8 Hz, NCH2CH3), 1.41 (d, J=7.2Hz, CHC H3),
3.21–3.39 (m, NCH2CH3), 4.36 (d, J=4.2Hz, C H2Ph),
4.62–4.67 (m, CH), 5.11 (5.15) (s, OCH2Ph), 7.17–7.32
(m, 10 PhH), the structural assignments were in agree-
ment with the 1H–1H COSY experiment; 13C NMR
(CDCl3) d 15.1 (14.8), 15.2(15.3), 39.3 (39.4), 43.6, 55.1
(55.3), 67.6, 127.4, 127.9, 128.3, 128.7, 136.5, 138.3,
157.0, 171.7; MS (+CI) (rel intensity) 342 (22), 341
(M++1, 100), 233 (21); Mr (+CI) 341.186 01 [M++1]
(calcd for C20H25N2O3 341.186 52). Anal. calcd for
C20H24N2O3: C, 70.57%; H, 7.11%; N, 8.23%. Found:
C, 70.64%; H, 7.08%; N, 8.34%.
1
cmꢁ1; H NMR (CDCl3) d 0.93 (0.91) (t, J=7.2Hz,
CH2CH3 (m, CH2CH3 minor conformer, the signal
overlaps the major conformer peak)), 1.66–1.78 (m,
CHH0CH3), 1.94–2.06 (m, CHH0CH3), 2.88 (2.89) (s,
NCH3), 3.70 (3.65) (s, C(O)OCH3), 4.75 (4.53) (dd,
J=5.1 (5.0), 10.5 (10.7) Hz, CH), 5.17 (5.15) (s,
OCH2Ph), 7.26–7.47 (m, 5 PhH), 1H NMR analysis
indicated the major and minor conformational isomers
existed in a 60:40 ratio in CDCl3, the structural assign-
ments were in agreement with the 1H–1H COSY
experiment; 13C NMR (CDCl3) d 10.8, 22.1 (22.4), 30.4
(30.9), 52.1, 60.0 (60.3), 67.5, 127.8, 128.1, 128.6, 136.8,
157.2(156.5), 172.2(172.0); MS (+CI) (rel intensity)
267 (15), 266 (M++1, 100); Mr (+CI) 266.139 29
[M++1] (calcd for C14H20NO4 266.139 23). Anal. calcd
for C14H19NO4: C, 63.38%; H, 7.22%; N, 5.28%.
Found: C, 63.29%; H, 7.23%; N, 5.38%.
N-Benzyl-2-ethylaminopropionamide (30). A methanolic
solution of 29 (2.09 g, 6.15 mmol) was hydrogenated (1
atm) in the presence of 10% Pd/C (ꢃ200 mg) at room
temperature (12h). The mixture was carefully filtered
through a bed of Celite1 521 and the clear filtrate was
evaporated in vacuo to obtain a pale-yellow oil. Puri-
fication of the product by column chromatography
(SiO2; 1:9, MeOH/CHCl3) gave 30 (1.22 g, 96%) as a
clear oil: Rf 0.54 (1:9, MeOH/CHCl3); IR (neat) 3303
1
(br), 3064, 2969, 1656, 1525 cmꢁ1; H NMR (CDCl3) d
1.03 (dt, J=2.0, 7.2 Hz, NHCH2CH3), 1.19–1.25 (br s,
NHCH2CH3), 1.32(d, J=7.1 Hz, CHCH3), 2.50–2.68
(m, NHCH2CH3), 3.21 (q, J=7.1 Hz, CH), 4.44 (d,
J=5.7 Hz, CH2Ph), 7.24–7.35 (m, 5 PhH), 7.60–7.63 (br
s, C(O)NH); 13C NMR (CDCl3) d 15.6, 20.1, 43.1, 43.2,
58.5, 127.5, 127.7, 128.8, 138.9, 175.4; MS (+CI) (rel
intensity) 208 (12), 207 (M++1, 100); Mr (+CI)
207.149 72 [M++1] (calcd for C12H19N2O 207.149 74).
2-(N-Benzyloxycarbonyl-N-methylamino)butyric
acid
(33). Utilizing a procedure comparable for the syn-
thesis of 28 and using 32 (3.22 g, 12.1 mmol) and aq 2 M
KOH/MeOH solution (20/40 mL) at 35 ꢀC (1 h) gave
3.06 g (99%) of pure 33 as a clear oil: Rf 0.59 (1:9,
MeOH/CHCl3); IR (neat) 3112 (br), 2972, 2939, 1741,
1
1705, 1680 cmꢁ1; H NMR (CDCl3) d 0.93 (0.90) (t,
J=7.2Hz, CH 2CH3 (m, CH2CH3 minor conformer, the
signal overlaps the major conformer peak)), 1.69–1.79
(m, CHH0CH3), 1.97–2.10 (m, CHH0CH3), 2.89 (2.90)
(s, NCH3), 4.76 (4.57) (dd, J=5.2(4.8), 11.0 (10.8) Hz,
CH), 5.17 (s, OCH2Ph), 7.26–7.47 (m, 5 PhH), 10.60 (s,
.
Anal. calcd for C12H18N2O 0.4H2O: C, 67.51%; H,
8.88%; N, 13.12%. Found: C, 67.52%; H, 8.81%; N,
13.17%.
1
N-Benzyl-2-(N-acetyl-N-ethyl)aminopropionamide
(8).
C(O)OH), H NMR analysis indicated the major and
Compound 8 was prepared utilizing method C and
using 30 (1.60 g, 7.76 mmol), THF (100 mL), TEA (1.2
mL, 8.54 mmol), Ac2O (800 mL, 8.54 mmol), and a cat-
alytic amount of DMAP (ꢃ100 mg). The reaction was
stirred at room temperature (16 h), and then the solvent
was evaporated in vacuo to provide a pale-yellow resi-
due. The product was purified by column chromato-
graphy (SiO2; 1:33, MeOH/CHCl3) to give 1.71 g (89%)
of pure 8 as a white solid: mp 60–63 ꢀC; Rf 0.42(1:19,
MeOH/CHCl3); IR (KBr) 3309, 2977, 2917, 1673, 1626,
minor conformational isomers existed in a 55:45 ratio
in CDCl3; 13C NMR (CDCl3) d 10.9, 21.9 (22.2), 30.4
(30.8), 60.1, 67.8, 127.9, 128.1, 128.6, 136.5, 157.6
(156.8), 176.9 (176.7); MS (+CI) (rel intensity) 253 (14),
252 (M++1, 100), 208 (61); Mr (+CI) 252.123 55
[M++1] (calcd for C13H18NO4 252.123 58). Anal. calcd
.
for C13H17NO4 0.2H2O: C, 61.48%; H, 6.87%; N,
5.51%. Found: C, 61.48%; H, 6.83%; N, 5.47%.
N-Benzyl-2-(N-benzyloxycarbonyl-N-methyl)aminobutan-
amide (34). Utilizing method A and using 33 (3.38 g,
13.4 mmol), THF (100 mL), 4-methylmorpholine
;
1526 cmꢁ1 1H NMR (CDCl3) d 1.29 (t, J=7.2Hz,
NCH2CH3), 1.37 (1.49) (d, J=7.2(6.0) Hz, CHC H3),

A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
2703
(1.8 mL, 16.2mmol), isobutyl chloroformate (2.1 mL,
16.2mmol), and benzylamine (1.8 mL, 16.2mmol)
gave crude 34. The product was purified by column
chromatography (SiO2; 3:1, hexanes/EtOAc) to obtain
4.12g (90%) of pure 34 as a clear oil: Rf 0.40 (2:1,
hexanes/EtOAc); IR (KBr) 3327, 3064, 3032, 2968,
21.0, 22.1, 31.4, 43.3, 57.6, 127.4, 127.6, 128.7, 138.5,
170.6, 172.2; MS (+CI) (rel intensity) 250 (13), 249
(M++1, 100), 142(97); Mr (+CI) 249.159 93 [M++1]
(calcd for C14H21N2O2 249.160 30). Anal. calcd for
C14H20N2O2: C, 67.71%; H, 8.12%; N, 11.28%. Found:
C, 67.82%; H, 8.11%; N, 11.17%.
2936, 1694, 1674, 1531 cmꢁ1 1H NMR (CDCl3) d
;
0.97 (t, J=7.2Hz, CH 2CH3), 1.74–1.84 (m, CHH0CH3),
2.02–2.18 (m, CHH0CH3), 2.95 (s, NCH3), 4.46 (br d,
J=4.8 Hz, NHCH2Ph), 4.62–4.70 (m, CH), 5.17 (s,
OCH2Ph), 6.82–6.94 (6.56–6.60) (br s, NH), 7.27–7.39
(m, 10 PhH), 1H NMR analysis indicated the major
and minor conformational isomers existed in a 75:25
ratio in CDCl3, the structural assignments were in
agreement with the 1H–1H COSY experiment; 13C
NMR (CDCl3) d 10.5, 21.2 (20.8), 29.6 (29.7), 43.3, 60.3
(60.4), 67.5, 127.3, 127.5, 127.6, 128.1, 128.5, 128.6,
136.4, 138.4, 157.4 (156.4), 170.6, the structural
3-Benzyl-5-methyl-imidazolidin-2,4-dione (37).36,42 dl-
Alanine (36) (1.52g, 17.06 mmol) was dissolved in an
aqueous KOH solution (1.15 g, 20.5 mmol, 31 mL H2O)
at 0 ꢀC and then benzyl isocyanate (2.5 mL, 20.5 mmol)
was added over a 20 min period. The reaction was
warmed to 65 ꢀC for 20 min and then the precipitate was
filtered, and the filtrate acidified to pH 2with aqueous
3 N HCl leading to the precipitation of a white solid.
The solid was collected by filtration and then suspended
in an aqueous 6 N HCl solution (14 mL) and the reac-
tion mixture heated at 75 ꢀC (12h). The resulting solu-
tion was allowed to return to room temperature leading
to a precipitation of a solid that was filtered, and
recrystallized (EtOH) to give 1.88 g (55%) of pure 37 as
a white solid: mp 112–113 ꢀC (lit.42 mp 112–114 ꢀC); Rf
1
assignments for the H and 13C NMR were in agree-
ment with the HETCOR and DEPT experiments; MS
(+CI) (rel intensity) 342(17), 341 (M ++1, 100); Mr
(+CI) 341.187 12[M ++1] (calcd for C20H25N2O3
341.186 52). Anal. calcd for C20H24N2O3: C, 70.56%;
H, 7.11%; N, 8.23%. Found: C, 70.41%; H, 7.13%; N,
8.25%.
1
0.60 (EtOAc); H NMR (CDCl3) d 1.43 (d, J=6.9 Hz,
CHCH3), 4.08 (q, J=6.9 Hz, CH), 4.65 (s, CH2Ph), 6.30
(s, NH), 7.26–7.39 (m, 5 PhH).
N-Benzyl-2-N-methylaminobutanamide (35). Utilizing
the procedure for 30 and using 34 (3.70 g, 10.8 mmol),
MeOH (50 mL), 10% Pd/C (ꢃ300 mg) and H2 (1 atm)
gave after 1 day pure 35 (2.30 g, 99%) as a clear oil: Rf
0.53 (1:9, MeOH/CHCl3); IR (neat) 3301 (br), 2966,
1-Acetyl-3-benzyl-5-methyl-imidazolidin-2,4-dione (10).
Utilizing method C and using 37 (1.58 g, 7.74 mmol),
THF (30 mL), TEA (2.6 mL, 18.58 mmol), Ac2O (880
mL), and DMAP (ꢃ150 mg) gave crude 10 after 1.5
days. The product was purified by column chromato-
graphy (SiO2; 1:1, hexanes/EtOAc) to give 1.60 g (84%)
of pure 10 as a white solid: mp 64–65 ꢀC; Rf 0.64 (1:1,
hexanes/EtOAc); IR (KBr) 3066, 2935, 1793, 1705 (br)
cmꢁ1; 1H NMR (CDCl3) d 1.57 (d, J=6.9 Hz, CHCH3),
2.56 (s, CH3C(O)), 4.53 (q, J=6.9 Hz, CH), 4.69 (s,
CH2Ph), 7.31–7.42(m, 5 Ph H); 13C NMR (CDCl3) d
16.6, 25.3, 42.9, 55.3, 128.5, 128.9, 129.1, 135.3, 153.5,
169.2, 171.8; MS (+CI) (rel intensity) 248 (12), 247
(M++1, 100); Mr (+CI) 247.108 71 [M++1] (calcd for
C13H15N2O3 247.108 27). Anal. calcd for C13H14N2O3:
C, 63.40%; H, 5.73%; N, 11.38%. Found: C, 63.36%;
H, 5.74%; N, 11.29%.
1
2934, 1650, 1525 cmꢁ1; H NMR (CDCl3) d 0.96 (t,
J=7.5 Hz, CH2CH3), 1.32(br s, N HCH3), 1.58–1.68
(m, CHH0CH3), 1.75–1.86 (m, CHH0CH3), 2.37 (s,
NCH3), 2.95 (dt, J=1.5, 7.2Hz, C H), 4.47 (d, J=5.7
Hz, CH2Ph), 7.26–7.33 (m, 5 PhH), 7.49 (br s, NH); 13
C
NMR (CDCl3) d 10.4, 26.8, 35.8, 43.2, 66.7, 127.5,
127.9, 128.9, 138.9, 174.2; MS (+CI) (rel intensity) 208
(13), 207 (M++1, 100); Mr (+CI) 207.149 37 [M++1]
(calcd for C12H19N2O 207.149 74). Anal. calcd for
.
C12H18N2O 0.4H2O: C, 67.51%; H, 8.88%; N, 13.12%.
Found: C, 67.56%; H, 8.73%; N, 13.08%.
N-Benzyl-2-(N-acetyl-N-methyl)aminobutanamide
(9).
Compound 9 was prepared utilizing method C and
using 35 (2.15 g, 10.4 mmol), THF (120 mL), TEA (1.6
mL, 11.5 mmol), Ac2O (1.1 mL, 11.5 mmol), and a cat-
alytic amount of DMAP (ꢃ200 mg). The reaction was
stirred at room temperature (1 day) and then the solvent
was evaporated in vacuo to provide a pale-yellow resi-
due. The product was purified by column chromato-
graphy (SiO2; 1:33, MeOH/CHCl3) to give 1.94 g (76%)
of pure 9 as a thick clear oil: Rf 0.58 (1:19, MeOH/
CHCl3); IR (neat) 3315 (br), 2927, 1683, 1647, 1526
3-Benzyl-5-methyl-2-thioxo-imidazolidin-4-one (39). To a
solution of 38 (3.13 g, 22.4 mmol) in CH2Cl2 (70 mL)
was added TEA (3.1 mL, 22.4 mmol) and benzyl iso-
thiocyanate (3.0 mL, 22.4 mmol). The solution was
stirred at room temperature (30 min) and then heated to
reflux (30 min). The solvent was evaporated in vacuo,
the residue redissolved in CH2Cl2 (50 mL), and washed
with H2O (20 mL). The organic phase was concentrated
in vacuo to yield crude 39. The solid was recrystallized
(EtOH) to obtain 4.00 g (82%) of pure 39 as a white
solid: mp 149–150 ꢀC; Rf 0.52(1:1, hexanes/EtOAc); IR
(KBr) 3170, 3012, 1743, 1535 cmꢁ1; 1H NMR (CDCl3) d
1.46 (d, J=7.2Hz, CHC H3), 4.15 (q, J=7.2Hz, C H),
4.95 (d, J=14.6 Hz, CHH0Ph), 5.02(d, J=14.6 Hz,
CHH0Ph), 7.26–7.45 (m, 5 PhH), 7.80 (s, NH); 13C
NMR (CDCl3) d 17.0, 44.7, 55.2, 128.1, 128.7, 128.9,
135.8, 174.8, 183.7; MS (+CI) (rel intensity) 221
(M++1, 100); Mr (+CI) 221.075 25 [M++1] (calcd for
C11H13N2OS 221.074 86). Anal. calcd for C11H12N2O2:
cmꢁ1
;
1H NMR (CDCl3) d 0.88 (t, J=7.5 Hz,
CH2CH3), 1.60–1.72(m, C HH0CH3), 1.91–1.99 (m,
CHH0CH3), 2.07 (2.17) (s, CH3C(O)), 2.91 (2.75) (s,
NCH3), 4.37–4.41 (m, NHCH2Ph), 4.95 (dd, J=7.2, 8.4
Hz, CH), 6.83 (7.01) (br s, NH), 7.20–7.33 (m, 5 PhH),
1H NMR analysis indicated the major and minor con-
formational isomers existed in a 90:10 ratio in CDCl3,
the structural assignments were in agreement with the
1H–1H COSY experiment; 13C NMR (CDCl3) d 10.6,

2704
A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
C, 59.97%; H, 5.49%; N, 12.72%. Found: C, 59.96%;
H, 5.48%; N, 12.72%.
N-Benzyl-2-N-(benzyloxycarbonyl)amino-3-methoxy-pro-
pionamide (44). To a CH3CN (250 mL) solution of 4329
(6.72g, 20.4 mmol) was successively added Ag 2O (23.70
g, 0.15 mol) and CH3I (12.75 mL, 0.30 mol) at room
temperature. The reaction was maintained at room
temperature (3 days), filtered and the solvent evapo-
rated in vacuo. The residue was purified by column
chromatorgraphy (SiO2; 1:9, MeOH/CHCl3) to give
6.50 g (93%) of 44 as a white solid: mp 140–141 ꢀC;
Rf 0.31 (1:1, hexanes/EtOAc); IR (KBr) 3304, 3062,
1-Acetyl-3-benzyl-5-methyl-2-thioxo-imidazolidin-4-one
(11). Utilizing method C and using 39 (1.50 g, 6.82
mmol), THF (60 mL), TEA (1.1 mL, 8.18 mmol), Ac2O
(770 mL, 8.18 mmol) and DMAP (ꢃ100 mg) gave crude
11 after 1 h. The product was recrystallized (EtOH) to
give 1.78 g (99%) of pure 11 as a white solid: mp 76–
77 ꢀC; Rf 0.69 (1:1, hexanes/EtOAc); IR (KBr) 3034,
1
1
2928, 1759, 1701 cmꢁ1; H NMR (CDCl3) d 1.59 (d,
2954, 1690, 1546 cmꢁ1; H NMR (CDCl3) d 3.36 (s,
J=6.9 Hz, CHCH3), 2.80 (s, CH3C(O)), 4.68 (q, J=6.9
Hz, CH), 5.00 (d, J=14.4 Hz, CHH0Ph), 5.08 (d,
J=14.4 Hz, CHH0Ph), 7.28–7.42 (m, 5 PhH); 13C NMR
(CDCl3) d 17.0, 28.2, 45.1, 57.4, 128.2, 128.7, 128.9,
135.1, 170.4, 172.8, 180.6; MS (+CI) (rel intensity) 264
(12), 263 (M++1, 100); Mr (+CI) 263.085 17 [M++1]
(calcd for C13H15N2O2S 263.085 43). Anal. calcd for
C13H14N2O2S: C, 59.52%; H, 5.38%; N, 10.68%.
Found: C, 59.64%; H, 5.33%; N, 10.61%.
OCH3), 3.50 (dd, J=6.6, 9.2Hz, C HH0OCH3), 3.85
(dd, J=3.9, 9.2Hz, CH H0OCH3), 4.30–4.36 (m, CH),
4.47 (d, J=5.7 Hz, CH2NH), 5.11 (s, CH2OC(O)), 5.60–
5.70 (m, NH), 6.65–6.72(m, N H), 7.26–7.36 (m, 10 PhH);
13C NMR (CDCl3) d 43.8, 54.6, 59.3, 67.5, 72.2, 127.7,
128.4, 128.5, 128.8, 128.9, 136.3, 138.1, 156.3, 170.1; MS
(+CI) (rel intensity) 344 (22), 343 (M++1, 100); Mr
(+CI) 343.165 81 [M++1] (calcd for C19H23N2O4
343.165 78). Anal. calcd for C19H22N2O4: C, 66.65%; H,
6.48%; N, 8.18%. Found: C, 66.93%; H, 6.55%; N,
8.15%.
2-(3-Benzylureido)-2-phenylacetic acid methyl ester
(41).43 Utilizing a similar procedure for 39 and using 40
(1.50 g, 7.43 mmol), THF (70 mL), TEA (2.1 mL, 14.87
mmol), and benzyl isocyanate (1.80 mL, 14.8 mmol)
gave crude 41 after 10 min at 60 ꢀC. The residue was
recrystallized (EtOH) to give 3.17 g (72%) of pure 41 as
a white solid: mp 105–107 ꢀC; Rf 0.43 (1:1, hexanes/
N-Benzyl-2-amino-3-methoxypropionamide (45). Utiliz-
ing the procedure for 30 and using 44 (4.00 g, 11.7
mmol) and 10% Pd/C (ꢃ400 mg) gave 2.43 g (99%) of
45 after purification by flash column chromatography
(SiO2; 1:9, MeOH/CHCl3) as a pale-yellow oil: Rf 0.33
(1:9, MeOH/CHCl3); IR (liquid film) 3357, 3312, 3063,
2927, 2896, 2826, 1655, 1527 cmꢁ1; 1H NMR (CDCl3) d
1.74 (br s, NH2), 3.34 (s, OCH3), 3.53–3.61 (m,
CHCH2), 4.36–4.48 (m, CH2NH), 7.22–7.33 (m, 5
PhH), 7.75–7.86 (m, NH); 13C NMR (CDCl3) d 43.1,
54.9, 58.8, 74.6, 127.3, 127.6, 128.6, 138.4, 172.8; MS
(+CI) (rel intensity) 210 (12), 209 (M++1, 100); Mr
(+CI) 209.129 15 [M++1] (calcd for C11H17N2O2
1
EtOAc); H NMR (DMSO-d6) d 3.62(s, OC H3), 4.22
(d, J=5.9 Hz, CH2Ph), 5.31 (d, J=7.8 Hz, CH), 6.54 (t,
J=5.9 Hz, NHCH2), 7.05 (d, J=7.8 Hz, NHCH), 7.24–
7.42(m, 10 Ph H).
3-Benzyl-5-phenylimidazolidin-2,4-dione (42).36 To
a
methanolic solution (40 mL) of 41 (3.00 g, 10.07 mmol)
was added TEA (1.4 mL, 10.07 mmol), and the reaction
solution was stirred at 40 ꢀC (4 h). The solvent was
evaporated in vacuo to yield crude 42. The residue was
recrystallized (iPrOH–H2O) to give 2.25 g (84%) of pure
42 as a white solid: mp 171–172 ꢀC (lit.36a mp 171–
.
209.129 00). Anal. calcd for C11H16N2O2 0.2H2O: C,
62.36%; H, 7.80%; N, 13.22%. Found: C, 62.43%; H,
7.78%; N, 13.11%.
173 ꢀC); H NMR (DMSO-d6) d 4.54 (d, J=15.6 Hz,
1-Acetyl-3-benzyl-5-methoxymethyl-2-thioxo-imidazolidin-
4-one (13). To a THF solution (100 mL) of 45 (2.50 g,
12.0 mmol), was added di-2-pyridyl thionocarbonate
(2.66 g, 11.4 mmol) and then the solution was stirred at
room temperature (24 h). The reaction was con-
centrated in vacuo and the residue redissolved in
MeOH, stirred at room temperature (2h), and then the
solvent was evaporated in vacuo to yield a dark orange
oil. The crude was used without further purification and
was dissolved in THF (125 mL) and then TEA (1.9 mL,
13.6 mmol), Ac2O (1.3 mL, 13.6 mmol), and DMAP
(ꢃ200 mg) were added. The reaction was stirred at
room temperature (6 h), and the solvent evaporated in
vacuo to give crude 13. The product was purified by
column chromatography (SiO2; 1:1, hexanes/EtOAc) to
yield 1.94 g (55%) of pure 13 as a pale-orange solid: mp
98–99 ꢀC; Rf 0.41 (4:1, hexanes/EtOAc); IR (KBr) 2931,
1
CHH0Ph), 4.59 (d, J=15.6 Hz, CHH0Ph), 5.31 (s, CH),
7.24–7.44 (m, 10 PhH), 8.79 (s, NH).
1-Acetyl-3-benzyl-5-phenylimidazolidin-2,4-dione
(12).
Utilizing method C and using 42 (1.42g, 5.30 mmol),
THF (30 mL), TEA (890 mL, 6.40 mmol), Ac2O (600
mL, 6.40 mmol), and DMAP (ꢃ150 mg) gave after 16 h
crude 12. The product was recrystallized (EtOH) to give
1.10 g (67%) of pure 12 as a white solid: mp 112–113 ꢀC;
Rf 0.67 (1:1, hexanes/EtOAc); IR (KBr) 3035, 2966,
1
1782, 1716 (br) cmꢁ1; H NMR (CDCl3/DMSO-d6) d
2.50 (s, CH3C(O)), 4.64 (d, J=15.2Hz, C HH0Ph), 4.70
(d, J=15.2Hz, CH H0Ph), 5.58 (s, CH), 7.26–7.35 (m, 10
PhH); 13C NMR (CDCl3) d 24.9, 43.1, 62.3, 126.5, 128.4,
128.8, 128.9, 129.1, 133.5, 135.2, 153.7, 168.2, 169.2, one
of the aromatic carbon peaks was not detected and is
believed to overlap with the observed peaks; MS (+CI)
(rel intensity) 310 (19), 309 (M++1, 100), 121 (46); Mr
(+CI) 309.123 16 [M++1] (calcd for C18H17N2O3
1
2827, 1759, 1697 cmꢁ1; H NMR (CDCl3) d 2.84 (s,
CH3C(O)), 3.25 (OCH3), 3.86 (dd, J=1.2, 9.9 Hz,
CHH0OCH3), 4.10 (dd, J=2.1, 9.9 Hz, CHH0OCH3),
4.71–4.74 (br s, CH), 5.03 (d, J=14.9 Hz, CHH0Ph),
5.12(d, J=14.9 Hz, CHH0Ph), 7.26–7.39 (m, 5 PhH);
13C NMR (CDCl3) d 28.1, 45.2, 59.6, 62.2, 69.3, 128.0,
.
309.123 92). Anal. calcd for C18H16N2O3 0.45H2O: C,
68.32%; H, 5.38%; N, 8.85%. Found: C, 68.23%; H,
5.22%; N, 8.80%.

A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
2705
128.4, 128.6, 134.9, 170.7, 170.8, 181.4; MS (+CI)
(rel intensity) 294 (14), 293 (M++1, 100); Mr (+CI)
293.095 60 [M++1] (calcd for C14H17N2O3S 293.095
99). Anal. calcd for C14H16N2O3S: C, 57.52%; H,
5.52%; N, 9.58%. Found: C, 57.61%; H, 5.45%; N,
9.54%.
N-Benzyl-N-methyl-2-amino-3-methoxypropionamide (51).
Utilizing the procedure for 30 and employing 50 (3.66
g, 10.2mmol) and 5% Pd/C ( ꢃ350 mg) gave 2.08 g
(91%) of 51 as a pale-yellow oil after purification by
flash column chromatography (SiO2; 1:9, MeOH/
CHCl3): Rf 0.45 (1:9, MeOH/CHCl3); IR (liquid film)
3474, 3368, 3299, 2980, 2929, 2894, 1640 cmꢁ1 1H
;
N-Benzyl-N-methyl-2-(benzyloxycarbonyl)amino-3-hy-
droxypropionamide (49). Utilizing method B and using
carbobenzyloxy-dl-serine (48) (12.60 g, 52.7 mmol),
DMF (100 mL), EDCI (16.10 g, 84.3 mmol), 4-methyl-
morpholine (9.3 mL, 84.3 mmol), HOBt (10.60 g, 79
mmol) and N-methylbenzylamine (8.2mL, 63.2mmol)
gave after 16 h crude 49 as a yellow oil. The product
was purified by column chromatography (SiO2; 1:1,
hexanes/EtOAc) to give 8.76 g (49%) of pure 49 as a
white solid: mp 89–91 C; Rf 0.53 (1:19, MeOH/CHCl3);
IR (liquid film) 3433, 3309, 3032, 2931, 1712, 1628
NMR (CDCl3) d 1.72(br s, N H2), 2.98 (2.95) (s,
NCH3), 3.36 (3.31) (s, OCH3), 3.47–3.55 (3.40–3.46) (m,
CH2OCH3), 4.00 (3.92) (t, J=6.6 (6.6) Hz, CH), 4.51 (d,
J=14.6 Hz, CHH0Ph), 4.73 (d, J=14.6 Hz, CHH0Ph),
1
7.18–7.37 (m, 5 PhH and NH), H NMR analysis indi-
cated the major and minor conformational isomers
existed in a 60:40 ratio in CDCl3; 13C NMR (CDCl3) d
34.5 (34.1), 51.1 (52.8), 51.2, 59.0, 76.1 (76.2), 126.4,
127.2, 127.5, 127.8, 128.5, 128.8, 136.6, 136.9, 173.4
1
(173.9), the H and 13C NMR assignments for the ali-
phatic carbons signals were in agreement with the
HETCOR and DEPT experiments; MS (+CI) (rel
intensity) 224 (13), 223 (M++1, 100), 122 (11); Mr
(+CI) 223.144 45 [M++1] (calcd for C12H19N2O2
1
cmꢁ1; H NMR (CDCl3) d 3.01 (2.91) (s, NCH3), 3.42
(br s, OH), 3.70–3.86 (m, CH2OH), 4.50–4.70 (m,
CH2Ph), 4.74–4.84 (m, CH), 5.10 (5.03–5.05) (s (m),
OCH2Ph), 6.05–6.14 (m, NH), 7.18–7.34 (m, 10 PhH),
1H NMR analysis indicated the major and minor con-
formational isomers existed in a 60:40 ratio in CDCl3;
13C NMR (CDCl3) d 35.0 (34.2), 51.4 (53.4), 52.7 (51.9),
64.1, 67.3, 126.2, 127.7, 128.0, 128.1, 128.3, 128.6, 128.9,
129.0, 136.1, 136.3, 136.4, 156.5, 170.9 (171.6), the 13C
NMR assignments for the aliphatic carbons signals were
in agreement with the DEPT experiment; MS (+CI) (rel
intensity) 343 (M++1, 100), 299 (22), 235 (18); Mr
(+CI) 343.165 70 [M++1] (calcd for C19H23N2O4
.
223.144 65). Anal. calcd for C12H18N2O2 0.32H2O: C,
63.20%; H, 8.16%; N, 12.28%. Found: C, 63.29%; H,
8.15%; N, 12.13%.
N-Benzyl-N-methyl-2-acetamido-3-methoxypropionamide
(47). Utilizing method C and employing 51 (360 mg,
1.62mmol), THF (10 mL), TEA (250 mL, 1.78 mmol),
Ac2O (170 mL, 1.78 mmol) and a catalytic amount of
DMAP gave crude 47 after 3 h at room temperature.
The product was purified by column chromatography
(SiO2; 1:19, MeOH/CHCl3) to obtain 412mg (96%) of
pure 47 as a colorless oil: Rf 0.44 (1:19, MeOH/CHCl3);
.
343.165 78). Anal. calcd for C19H22N2O4 0.7H2O: C,
64.28%; H, 6.64%; N, 7.89%. Found: C, 64.17%; H,
6.33%; N, 7.89%.
1
IR (neat) 3302 (br), 3062, 2931, 1766, 1635 cmꢁ1; H
NMR (CDCl3) d 1.99 (1.96) (s, CH3C(O)), 3.00 (2.90)
N-Benzyl-N-methyl-2-(benzyloxycarbonyl)amino-3-meth-
oxypropionamide (50). To a CH3CN (250 mL) solution
of 49 (8.70 g, 25.4 mmol) was successively added Ag2O
(29.00 g, 0.13 mol) and MeI (16.0 mL, 0.25 mmol) at
room temperature. The reaction was maintained at
room temperature (3 days), filtered and the solvent
evaporated in vacuo. The residue was purified by col-
umn chromatography (SiO2; 1:1, hexanes/EtOAc) to
give 8.41 g (97%) of pure 50 as a pale-yellow 0:1: Rf
0.29 (1:1, hexanes/EtOAc); IR (liquid film) 3292, 3032,
(s, NCH3), 3.32(35.2) (s, OC
H3), 3.43–3.63 (m,
CH2OCH3), 4.44 (4.56) (d, J=14.9 (17.0) Hz, CHH0Ph),
4.57 (4.71) (d, J=14.9 (17.0) Hz, CHH0Ph), 5.13–5.20
(m, CH), 6.52–6.62 (m, NH), 7.18–7.37 (m, 5 PhH), H
1
NMR analysis indicated the major and minor con-
formational isomers existed in a 65:35 ratio in CDCl3;
13C NMR (CDCl3) d 23.4, 35.2 (34.2), 49.3, 51.6 (53.4),
58.4, 73.2, 127.0, 127.7, 127.9, 128.9, 129.1, 136.2, 136.7,
169.9, 170.8, the 13C NMR assignments were in agree-
ment with the DEPT experiment; MS (+CI) (rel inten-
sity) 265 (M++1, 100), 122 (53); Mr (+CI) 265.154 95
[M++1] (calcd for C14H21N2O3 265.155 22). Anal.
1
2931, 1712, 1643 cmꢁ1; H NMR (CDCl3) d 3.00 (2.90)
(s, NCH3), 3.33 (3.26) (s, OCH3), 3.48–3.65 (m,
CH2OCH3), 4.44 (4.57) (d, J=14.7 (16.8) Hz, CHH0Ph),
4.77 (4.71) (d, J=14.7 (16.8) Hz, CHH0Ph), 4.91–4.98
(br q, J=6.7 Hz, CH), 5.10 (5.07) (s, OCH2Ph), 5.94 (m,
.
calcd for C14H20N2O3 0.2H2O: C, 62.76%; H, 7.67%;
N, 10.46%. Found: C, 62.75%; H, 7.76%; N, 10.32%.
1
NH), 7.19–7.33 (m, 10 PhH), H NMR analysis indi-
Pharmacology
cated the major and minor conformational isomers
existed in a 65:35 ratio in CDCl3; 13C NMR (CDCl3) d
35.0 (34.0), 50.7, 51.4 (53.3), 59.3, 66.9, 73.4, 126.9,
127.5, 127.9, 128.0, 128.1, 128.5, 128.7, 128.8, 136.2,
Compounds were screened under the auspices of the
National Institutes of Health’s Anticonvulsant Screening
Project. Experiments were performed in male rodents
[albino Carworth Farms No. 1 mice (intraperitoneal
route, ip), albino Sprague–Dawley rats (oral route, po)].
The mice weighed between 18 and 25 g while rats were
between 100 and 150 g. All animals had free access to
feed and water except during actual testing period.
Housing, handling and feeding were all in accordance
with recommendations contained in the ‘Guide for the
Care and Use of Laboratory Animals’. All of the test
1
136.5, 136.7, 156.0, 170.6, the H NMR and 13C NMR
assignments for the aliphatic carbons signals were in
agreement with the HETCOR and DEPT experiments;
MS (+CI) (rel intensity) 358 (19), 357 (M++1, 100),
249 (15); Mr (+CI) 357.181 69 [M++1] (calcd for
C20H25N2O4 357.181 43). Anal. calcd for C20H24N2O4:
C, 67.40%; H, 6.79%; N, 7.86%. Found: C, 67.24%; H,
6.88%; N, 7.80%.

2706
A. LeTiran et al. / Bioorg. Med. Chem. 9 (2001) 2693–2708
compounds were administrated in suspensions of 0.5%
(w/v) of methylcellulose in water. The volumes admi-
nistered were 0.01 mL/g of body weight for mice and 0.2
mL/10 g for rats. Anticonvulsant activity was estab-
lished using the maximal electroshock (MES) test.30,44
For the MES test, a drop of electrolyte solution with an
anesthetic (0.5% butacaine hemisulfate in 0.9% sodium
chloride) was placed in the eyes of the animals prior to
positioning the corneal electrodes and delivery of the
non-lethal current. A 60-cycle alternating current was
administered for 0.2s in both species, utilizing 50 mA in
mice and 150 mA in rats. Protection endpoints were
defined as the abolition of the hind limb tonic extensor
component of the induced seizure.45 The subcutaneous
pentylenetetrazole (Metrazol1) seizure threshold test
(scMet) entailed administration of either 85 mg/kg of
pentylenetetrazole in mice (Carworth Farms No. 1) or
70 mg/kg in rats (Sprague–Dawley) as a 0.5% solution
subcutaneously in the posterior midline. This amount of
pentylenetetrazole produces clonic seizures in 97%
(CD97) of animals tested. The animal is observed for 30
min. Protection is defined as the failure to observe even
a threshold seizure (a single episode of clonic spasms of
at least 5-s duration). In mice, effects of compounds on
forced spontaneous motor activity were determined
using the rotorod test. The inability of experimental
mice to maintain their balance for 1 min on a 1-in dia-
meter knurled rod rotating at 6 rpm in three successive
trials was interpreted as a demonstration of motor
impairment. Under these conditions, mice can normally
maintain their balance indefinitely. Motor impairment
in rats was assessed by observing the overt evidence of
ataxia, abnormal gait and stance, and/or loss of placing
response and muscle tone. In the mouse identification
screens, all compounds were administered at three dose
levels (30, 100, 300 mg/kg) and two time periods (0.5
and 4 h). Typically, in the MES seizure test one animal
was used at 30 and 300 mg/kg, and three animals at 100
mg/kg. In the rotorod toxicity test four animals were
used at 30 and 300 mg/kg, and eight animals at 100 mg/
kg (Tables 1–3). Oral rat identification screening was
performed using four animals at a fixed dose of 30 mg/
kg for both the MES and the rotorod toxicity tests over
five time periods ranging from one quarter to 4 h post
drug administration. The quantitative determination of
the median effective (ED50) and toxic doses (TD50) were
conducted at previously calculated time of peak effect
using ip route in mice and oral route in rats. Groups of
at least eight animals were tested using different doses of
test compound until at least two points were determined
between 100 and 0% protection and minimal motor
impairment. The dose of the candidate substance
required to produce the desired endpoint (abolition of
hindlimb tonic extensor component) in 50% of the
animals in each test, and 95% confidence interval were
calculated by a computer program based on methods
described by Finney.46
blastoma cells and whole-cell voltage-clamp recording
techniques. The N1 E-115 neuroblastoma cell line was
maintained at 35 ꢀC in Dulbecco’s modified Eagles
Medium supplemented with 5% fetal calf serum, 20
mM HEPES, 80 mg/mL gentamicin and 4 mM gluta-
mine. Prior to electrophysiological studies, cells were
plated and incubated for 3–5 days in a differentiation
medium similar to above with reduced (2.5%) fetal calf
serum and 2% DMSO. Recordings were carried out at
room temperature in a bathing solution containing 130
mM NaCl, 5 mM KCl, 1.5 mM CaCl2, 5 mM glucose, 5
mM HEPES. The buffer also contained 0.1 mM CdCl2
and 25 mM tetraethylammonium chloride to block vol-
tage-gated Ca2+ and K+ channels, respectively. Whole
cell recordings were obtained using patch electrodes (1–
2 Mꢀ) filled with the intracellular solution described
above. The currents were filtered at 10 KHz and
acquired on computer using PClamp 6 (Axon Instru-
ments). Series resistance and capacitive currents were
compensated using the internal clamp circuitry. The
series resistance was 3–5 Mꢀ, and 75–85% of the series
resistance was compensated. Cells were initially voltage
clamped at ꢁ60 mV and in separate experiments the test
compound was applied using the perfusion system
noted above. To activate voltage-gated sodium chan-
nels, cells were hyperpolarized to ꢁ90 mV for 90 ms and
then depolarized to 0 mV for five trials in control solu-
tion then again in the solution containing test com-
pound. The data from the multiple trials were averaged.
The process was then repeated but this time hyperpo-
larizing at ꢁ60 mV. Drug was applied using a gravity
fed perfusion system coupled to a piezoelectric stepper
controlled electronically.
Acknowledgements
The authors thank Dr. Harvey J. Kupfergberg and the
Anticonvulsant Screening Project (ASP) at the National
Institutes of Health, for kindly performing the pharma-
cological studies via the ASP’s contract site at the Uni-
versity of Utah with Drs. H. Wolf, S. White, and Karen
Wilcox, and Dr. James Korp (University of Houston)
for conducting the X-ray crystallographic analysis of 4.
Funds for this project were provided, in part, by the
University of Houston and the University of North
Carolina—Chapel Hill.
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