Multicomponent synthesis and anticonvulsant activity of monocyclic 2,6-diketopiperazine derivatives

Article abstract of DOI:10.1007/s00044-013-0800-4

In this study, a series of diastereomerically pure monocyclic 2,6-diketopiperazine (2,6-DKP) derivatives were synthesized. The key synthetic step involved a multicomponent Ugi five-center, four-component reaction which was used to generate the convertible tert -butylamidoesters with both good yields and high diastereoselectivity toward the desired bioactive (S ,S) absolute configuration. In subsequent steps, selective tertbutyl cleavage by use of BF₃·CH₃COOH and base-induced intramolecular cyclocondensation gave the final 2,6-DKP derivatives. The relative stereochemistry of the target molecules was confirmed by ¹H NMR experiments. The compounds obtained were submitted to in vivo screening in animal models of epilepsy. Some of them displayed good activity in maximal electroshock seizure and 6 Hz tests. The Author(s) 2013.

Full text of DOI:10.1007/s00044-013-0800-4

MEDICINAL
CHEMISTRY
RESEARCH
Med Chem Res (2014) 23:2007–2018
DOI 10.1007/s00044-013-0800-4
ORIGINAL RESEARCH
Multicomponent synthesis and anticonvulsant activity
of monocyclic 2,6-diketopiperazine derivatives
•
Maciej Dawidowski Jadwiga Turło
Received: 8 May 2013 / Accepted: 19 September 2013 / Published online: 1 October 2013
Ó The Author(s) 2013. This article is published with open access at Springerlink.com
Abstract In this study, a series of diastereomerically pure
monocyclic 2,6-diketopiperazine (2,6-DKP) derivatives
were synthesized. The key synthetic step involved a mul-
ticomponent Ugi five-center, four-component reaction
which was used to generate the convertible tert-butylami-
doesters with both good yields and high diastereoselectiv-
ity toward the desired bioactive (S,S) absolute
configuration. In subsequent steps, selective tertbutyl
cleavage by use of BF3ꢀCH3COOH and base-induced
intramolecular cyclocondensation gave the final 2,6-DKP
derivatives. The relative stereochemistry of the target
control in only 60 % of patients. Additionally, major
concerns of pharmacotherapy of epilepsy include high
incidence of severe side effects and drug–drug interactions
resulting from enzyme induction. Therefore, there is sub-
stantial need for new, more effective and safer AEDs
(Kwan and Brodie, 2000; Brodie, 2001). Another essential
challenge for epilepsy research is to develop therapeutics
that would not only symptomatically suppress seizures, but
would also inhibit or reverse progression of the sickness
(the so-called ‘‘disease modifying’’ drugs; Perucca et al.,
2007; Bialer and White, 2010).
1
molecules was confirmed by H NMR experiments. The
Presently, the compounds at different stages of devel-
opment belong to various chemical classes and display
diverse, often unknown mechanisms of action (Bialer et al.,
2013). Most of these agents have been identified initially
through in vivo screening in animal models of epilepsy
rather than by a mechanistic approach. Although the animal
models utilized for screening are associated with certain
endpoints, it is generally accepted that they offer a good
starting point in the early discovery of new AED candidates
compounds obtained were submitted to in vivo screening in
animal models of epilepsy. Some of them displayed good
activity in maximal electroshock seizure and 6 Hz tests.
Keywords Multicomponent reactions ꢀ Ugi reaction ꢀ
Anticonvulsant activity ꢀ 2,6-Diketopiperazine ꢀ
2,6-Piperazinedione
¨
(Loscher and Schmidt, 1994; Malawska, 2005; Rogawski,
Introduction
2006; Smith et al., 2007; Bialer and White, 2010; Banerjee
and Sharma, 2012; Mishra and Ganguly, 2012).
Epilepsy is a major neurological disorder characterized by
recurrent, spontaneous seizures. It affects approx. 50 mil-
lion people (*1 % of the world’s population). Currently,
the main treatment for epilepsy is the chronic administra-
tion of anticonvulsant drugs (AEDs). Although more than
30 AEDs are available, they provide satisfactory seizure
Recently, we have reported that chiral, bicyclic 2,6-
diketopiperazines (2,6-DKPs) derived from cyclic amino
acids display a broad anticonvulsant activity in various
animal models of epilepsy (Dawidowski et al., 2011,
2012a). Among the newly developed agents, compound
ADD408003 exhibited a broad spectrum of seizure-sup-
pressing activity. A preliminary structure–activity rela-
tionship (SAR) study of close analogs revealed that several
factors are responsible for the anticonvulsant activity
(Fig. 1): the (S,S) absolute configuration on the stereogenic
centers, the presence of imide moiety and the benzene ring
adjacent to 2,6-DKP core. Further, neither substitution of
M. Dawidowski (&) ꢀ J. Turło
Department of Drug Technology and Pharmaceutical
Biotechnology, Medical University of Warsaw, Banacha 1 Str.,
02-097 Warsaw, Poland
e-mail: maciej.dawidowski@wum.edu.pl
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Med Chem Res (2014) 23:2007–2018
the imide nitrogen of ADD408003 with different alkyl and
arylalkyl moieties nor expansion of the fused pyrrole chain
markedly influenced the antiseizure activity.
Results and discussion
Chemistry
These findings led us to ask whether the related mono-
cyclic 2,6-DKPs, derived from non-polar L-amino acids
other than L-proline or L-homoproline display comparable
anticonvulsant activity. The designed compounds fulfill all
requirements determined on the basis of the preliminary
SAR analysis, i.e., proper stereochemistry, the presence of
imide moiety and benzene ring attached to 2,6-DKP scaf-
fold. Further, due to the absence of the fused pyrrolidine or
piperidine rings, these agents are less sterically con-
strained, which might allow for a better fit to the putative
receptor(s).
The target enantiopure, monocyclic 2,6-DKP derivatives
3a–e were synthesized according to the reaction sequences
depicted in Scheme 1.
In the first step, the Ugi five-center four-component
reaction (U-5C-4CR; Demharter et al., 1996) of the
appropriate non-polar amino acid (L-valine, L-leucine,
L-isoleucine, L-phenylalanine, L-phenylglycine), benzalde-
hyde, tert-butyl isocyanide, and methanol in the presence
of a catalytic amount of iron(III) chloride gave the tert-
butylamidoesters 1a–e with chemical yields ranging from
Fig. 1 Preliminary SAR of anticonvulsant 2,6-DKPs and proposed structural modifications
Scheme 1 Synthesis of enantiopure 2,6-DKP derivatives 3a–e
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Med Chem Res (2014) 23:2007–2018
2009
41 to 75 %. The reaction proceeded with the formation of
the new stereocenter and in all cases, the major diaste-
1
reomer was (2S,1S)-1, as judged by the H NMR analyses
saturation of H-5, led to the enhancement of H-3 resonance
in (3S,5R)-3a (4.0 %), but not in (3S,5S)-3a.
The difference in the chemical shifts of the protons H-3
of the crude post-reaction mixtures. In general, the degree
of diastereoinduction depended on the steric bulkiness of
the side chain of the substrate amino acid. The highest
diastereomeric ratios were measured for L-valine and
L-isoleucine derivatives 1a (dr = 7.3/1) and 1c (dr = 9.0/1),
respectively, bearing branched alkyl chains directly adjacent
to the position C-2, located close to the newly formed ste-
reocenter. The U-5C-4CR adducts of L-leucine and L-phen-
ylalanine 1b and d, respectively, were formed with a slightly
lower diastereoinduction (dr & 5/1 for each). This could be
attributed to the lower steric hindrance of a methylene group
adjacent to the carbon C-2. A surprisingly small degree of
diastereoselectivity was found for the L-phenylglycine
derivative 1e (dr = 1.5/1), having a bulky phenyl substituent
in the position C-2. The possible explanation for this
unexpected observation is the stabilization of the six-mem-
bered cyclic Ugi intermediate (Demharter et al., 1996)
leading to (2S,1R)-1e by a pi–pi interaction of the two
phenyl rings occupying axial positions.
and H-5 adjacent to the stereogenic carbon atoms was
1
another H NMR spectroscopic feature useful for distin-
guishing between the respective diastereoisomers of 3.
With the exception of 3e, the signals corresponding to H-5
in (3S,5S)-3 were shifted downfield (Dd = 0.26–0.38 ppm
[parts per million]) compared to those of (3S,5R)-3. This
observation was in agreement with the pseudoequatorial
arrangement of these protons with respect to the 2,6-DKP
ring in (3S,5S)-3, and their pseudoaxial position in
(3S,5R)-3. On the contrary, resonances of the H-3 protons
in all (3S,5S)-3 isomers were shifted upfield (Dd = 0.14–
0.32 ppm). Although these protons in both respective
diastereoisomers occupy the same pseudoaxial positions,
the slightly stronger shielding in (3S,5R)-3 could be
attributed to the anisotropic effect of the phenyl ring
present in the spatial vicinity.
The racemic 2,6-DKPs 3f, g were synthesized from the
corresponding N-substituted glycines in a similar manner,
according to the reaction sequences depicted in Scheme 2.
Notably, the chemical yields of racemic U-5C-4CR products
1f and g (18 and 24 %, respectively) were significantly
lower than those observed for N-unsubstituted adducts 1a–e.
Attempts to quantitatively separate the diastereoisomers
of 1a–e by column chromatography or fractional recrys-
tallization failed. Therefore, the obtained diastereomeric
mixtures were used in the subsequent amide
N-detertbutylation. Reaction of (2S,1S)/(2S,1R)-1a–e with
BF3ꢀCH3COOH at 45–55 °C provided amidoesters 2a–e
with the yields range from 55 to 83 %. With the exception
of 2e, all diastereomeric mixtures could be efficiently
resolved by column chromatography.
Anticonvulsant screening
Compounds 3a–f were evaluated in the in vivo animal
models of epilepsy within the Anticonvulsant Screening
Program (ASP) of The National Institute of Neurological
Disorders and Stroke (NINDS), at The National Institutes
of Health according to well-established protocols described
in the ‘‘Experimental’’ section of this article. The com-
pounds were screened using maximal electroshock seizure
(MES) and subcutaneous metrazole (scMET) tests (White
et al., 2002). The first of these tests uses an electrical
stimulus to induce generalized tonic–clonic seizures and is
capable of identifying compounds that prevent the spread
of seizure. The latter employs chemically induced seizures
to recognize agents that raise the seizure threshold. The
most promising compounds were subjected to an evalua-
tion of anticonvulsant activity using a minimal clonic sei-
zure (6 Hz) test (Brown et al., 1953; Barton et al., 2001;
Kaminski et al., 2004), which is regarded as a preliminary
model of pharmacoresistant limbic seizures. Additionally,
a standardized rotorod test for neurological toxicity (TOX;
Dunham and Miya, 1957) was performed for each com-
pound. The results are summarized in Tables 1 and 2.
As shown in Table 1, compounds 3a, b, d–f exhibited
weak to good anticonvulsant activities in the MES model in
mice. (3S,5S)-3a and (3S,5S)-3e were most potent, dis-
playing a notable seizure protection in 2/3 and 2/4 of tested
In the last step, compounds (2S,1S)-2a–d were subjected
to base-induced intramolecular cyclization. The reaction
was accompanied by a notable degree of epimerization at
stereogenic centers C-5 of the products 3. Nevertheless, in
all cases, the unwanted (3S,5R) isomers could be separated
by means of column chromatography (compounds (3S,5R)-
3a, c, d) or recrystallization (compound (3S,5R)-3b).
Intramolecular cyclization of 1.4/1 diastereomeric mixture
of (2S,1S)/(2S,1R)-2e gave (3S,5S)-3e and (3S,5R)-3e (a
meso compound) in equal proportion. The isomers were
efficiently separated by column chromatography.
Relative stereochemistry of the respective diastereoiso-
mers of 2,6-DKPs 3 was confirmed with nuclear Overha-
user effect (nOe) 1H NMR experiments (Fig. 2) performed
for (3S,5S) and (3S,5R)-3a. Contrary to (3S,5R)-3a, the
interatomic distance between protons H-3 and H-5 in
(3S,5S)-3a should exclude any considerable nOe effect.
Indeed, the irradiation of the H-3 resonance in (3S,5R)-3a
resulted in a remarkable enhancement of the H-5 signal
(4.2 %), whereas no increase in intensity for the proton H-5
was observed in the analogous experiment performed for
(3S,5S)-3a. The reverse order of irradiation, consisting in
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Med Chem Res (2014) 23:2007–2018
Fig. 2 Selected nOe
correlations in (3S,5S)-3a and
(3S,5R)-3a
Scheme 2 Synthesis of racemic 2,6-DKP derivatives 3f, g
animals, respectively, at the dose of 100 mg/kg, after 0.5 h
postadministration. No neurotoxicity was detected at the
same dose. (3S,5S)-3e also proved effective also at the later
time points (1/4 and 4/4 at 100 and 300 mg/kg, respec-
tively, at 2.0 h). None of the compounds investigated were
significantly active in the scMET model.
Conclusions
We have synthesized a series of novel diastereomerically
pure, monocyclic 2,6-DKP derivatives by use of diastereo-
selective synthetic sequence using the U-5C-4CR multicom-
ponent reaction as the key step. The compounds displayed
weak to good anticonvulsant activities in the MES model,
while none of them were active in scMET screen. The most
promising compound (3S,5S)-3a exhibited notable action in
the 6 Hz test. Contrary to the recently reported activity of
bicyclic 2,6-DKPs, the activity of monocyclic derivatives
seemed to be less stereochemistry-dependent. We conclude
that this is due to increased conformational flexibility.
Although the seizure-suppressing potency of the newly syn-
thesized agents was generally weaker relative to bicyclic 2,6-
DKPs, they possess secondary amino groups that provide
additional points of diversification for further SAR studies.
According to our previous reports on bicyclic 2,6-DKP
derivatives, the (R,S) isomers were completely devoid of
any in vivo pharmacological activity (Dawidowski et al.,
2011, 2012a). In contrast, the monocyclic derivatives
(3R,5S)-3a and (3R,5S)-3e, displayed
a weak, yet
noticeable activity in the MES test (1/1 and 1/4 at
300 mg, respectively, at 0.5 h). This could mean that the
greater flexibility due to the lack of fused alkyl rings
allows for a better fit in the putative binding site within
the CNS. Nevertheless, the (S,S) isomers again proved
more active.
In general, the most significant levels of seizure pro-
tection were observed for derivatives bearing alkyl or aryl
substituents at carbon C-5. Among the compounds with
alkyl groups, the L-valine derivative with isopropyl side
chain (3S,5S)-3a was most potent in the MES test. High
levels of seizure protection was also observed for sym-
metrical (3S,5S)-3e having two benzene rings with a proper
stereochemistry with respect to the 2,6-DKP core. Impor-
tantly, the anticonvulsant activity of the investigated mol-
ecules was not dependant on their logP values.
Experimental
Chemistry
Melting points were determined on an Electrothermal 9100
apparatus in open capillary tubes and were uncorrected. The
IR spectra (thin film on KBr pellets) were recorded on a
Shimadzu FTIR-8300 instrument. The NMR spectra were
obtained on a Varian Inova 500 MHz spectrometer. Chem-
ical shifts (d) were expressed in ppm relative to tetrameth-
ylsilane or solvent used as the internal reference. The
following abbreviations were used to describe the peak
patterns: s (singlet), d (doublet), t (triplet), q (quartet), qt
(quintet), sp (septet), m (multiplet), p (pseudo-), and b
Derivatives (3S,5S)-3a and (3S,5S)-3e were further
assessed for their potential efficacy against pharmacore-
sistant epilepsy using the 6 Hz screen. The results are
summarized in Table 2. Notable activity was detected for
the first compound (2/4 and 1/4, at 0.25 and 0.5 h,
respectively, at 100 mg/kg), while the latter was inactive.
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Med Chem Res (2014) 23:2007–2018
2011
Table 1 Anticonvulsant activity and neurotoxicity of compounds in
the MES and scMET models following intraperitoneal (ip) adminis-
tration in mice
Table 2 Anticonvulsant activity and neurotoxicity of compounds in
the 6 Hz model following intraperitoneal (ip) administration in mice
Compounds
Testa
0.25 h
0.5 h
1.0 h
2.0 h
4.0 h
Compounds Dose
(mg/kg)
MESa
scMETb
TOXc
ClogPd
(3S,5S)-3a
6 Hzb
TOXc
6 Hz
2/4
0/4
–
1/4
0/4
0/4
0/8
0/4
0/4
–
0/4
0/4
0/4
0/8
0/4
0/4
–
0.5 h 4.0 0.5 h 4.0 h 0.5 h 4.0
he he
(3S,5S)-3e
(3S,5S)-3a
30
0/1 0/1 0/1 0/1 0/4 0/2 0.80
TOX
–
–
–
100
300
2/3 0/3 0/1 0/1 0/8 0/4
1/1 0/1 0/1 0/1 4/4f, 0/2
Ratios where at least one animal was protected or displayed neuro-
toxicity have been highlighted in bold to enhance data readability and
interpretation
g
a
(3S,5R)-3a
(3S,5S)-3b
(3S,5S)-3c
(3S,5S)-3d
(3S,5S)-3e
(3S,5R)-3e
rac-3f
30
100
300
30
0/1 0/1 0/1 0/1 0/4 0/2 0.80
0/3h 0/3 1/5i 0/1 0/8 0/4
1/1 0/1 0/1 0/1 2/4 0/2
0/1 0/1 0/1 0/1 0/4 0/2 1.19
0/3 0/3 0/1 0/1 0/8 0/4
1/1 0/1 0/1 0/1 3/4f 0/2
0/1 0/1 0/1 0/1 0/4 0/2 1.19
0/3 0/3 0/1 0/1 0/8 0/4
0/1 0/1 0/1 0/1 2/4 0/2
0/1 0/1 0/1 0/1 0/4 0/2 1.61
0/3 0/3 0/1 0/1 0/8 0/4
1/1 0/1 0/1 0/1 0/4 0/2
At dose 100 mg/kg
b
6 Hz test, 32 mA (number of animals protected/number of animals
tested)
c
Neurotoxicity test (number of animals exhibiting neurological
toxicity/number of animals tested)
100
300
30
(broad-). Coupling constants (J) were in hertz (Hz). The
electrospray ionization high-resolution mass spectra (ESI-
HRMS) were recorded on a LCT TOF (Micromass) instru-
ment. Optical rotations were measured with a Perkin-Elmer
241 polarimeter at 20 °C, using a sodium lamp (589 nm).
Thin-layer chromatography (TLC) was run on Merck Silica
gel-60 F254 plates. The spots were visualized by ultraviolet
light (254 nm) or iodine vapors. Flash column chromatog-
raphy (FC) was carried out on Merck Silica gel 60 (particle
size 0.040–0.063 mm). Solvents were dried and purified by
standard methods. Petroleum ether (PE) referred to the
fraction boiling at 40–60 °C. All reagents were purchased
from commercial sources and used as received. Unless
otherwise stated, the chemical yields were calculated for
pure (dr C95/5) compounds. Compound rac-3g was syn-
thesized as described previously (Dawidowski et al., 2012b).
100
300
30
100
300
30
0/4 0/4
2/4 1/4
4/4 4/4
0/4 0/4
0/4 0/4
1/4 0/4
0/4 0/4
0/4 0/4
0/4 3/4
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
–
0/8 0/8 2.12
0/8 0/8
100
300
30
2/8 1/8
0/8 0/8 2.12
0/8 0/8
100
300
30
0/8 0/8
0/8 0/8 2.29
0/8 0/8
100
300
30
0/8 0/8
rac-3g
0/1 0/1 0/1 0/1 0/4 0/2 2.12
0/3 0/3 0/1 0/1 0/8 0/4
0/1 0/1 0/1 0/1 0/4 0/2
100
300
Synthesis of compounds 1 by U-5C-4CR condensation
Ratios where at least one animal was protected or displayed neuro-
toxicity have been highlighted in bold to enhance data readability and
interpretation
Iron(III) chloride (5 mol.%) and tert-butyl isocyanide
(1.0 equiv.) were added to a stirred suspension of appro-
priate a-amino acid (1.2 equiv.) and benzaldehyde
(1.0 equiv.) in MeOH (100 mL). The mixture was stirred at
RT for 48 h and the volatiles were removed under reduced
pressure. The resulting crude products were purified FC.
a
Maximal electroshock test (number of animals protected/number of
animals tested)
b
Subcutaneous metrazole test (number of animals protected/number
of animals tested)
c
Neurotoxicity test (number of animals exhibiting neurological
toxicity/number of animals tested)
Methyl (2S,1S)- and (2S,1R)-2-(2-(tert-butylamino)-2-
oxo-1-phenylethylamino)-3-methylbutanoate (2S,1S)-1a
and (2S,1R)-1a
d
Theoretical logP value calculated by a logarithm included in Hy-
perChem 7.5 package
e
Compounds (3S,5S)-3e, (3S,5R)-3e and rac-3f were tested at 2.0 h
post administration
f
From L-valine (2.36 g, 20.16 mmol), benzaldehyde
(16.80 mmol, 1.71 mL) and tert-butyl isocyanide (2.00 mL,
16.80 mmol); FC (gradient: PE/AcOEt 6:1–3:1): yield
4.04 g (75 %) of chromatographically inseparable
Unable to grasp rotorod
g
Loss of righting reflex
h
Active also in 1/3 at 0.25 h post administration
i
Myoclonic jerks
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Med Chem Res (2014) 23:2007–2018
1
diastereomeric mixture (dr = 7.3/1, H NMR). Analytical
sample of (2S,1S)-1a was obtained by recrystallization from
PE/Et2O 10:1. (2S,1S)-1a: white wax; mp 37–38 °C;
[a]D = -97.2 (c 1, CHCl3); IR (KBr): 729, 764, 1200, 1454,
1516, 1678, 1736, 2874, 2962, 3333; TLC (PE/AcOEt 3:1):
Rf = 0.43; 1H NMR (CDCl3, 500 MHz): d 0.89 (d, 3J = 6.5,
3H, CH3), 0.93 (d, 3J = 6.5, 3H, CH30 ), 1.29 (s, 9H,
(C-30, C-50), 139.0 (C-10), 170.8 (CONH), 175.4 (COOCH3);
(2S,1R)-1b (minor isomer): d 22.2 (CH3), 23.2 (CH0 ), 24.9
3
(CH), 28.7 (C(CH3)3), 43.4 (CH2), 50.7 (C(CH3)3), 52.0
(OCH3), 59.0 (C-2), 66.9 (C-1), 127.2 (C-20, C-60), 128.1 (C-
40), 128.8 (C-30, C-50), 139.9 (C-10), 170.9 (CONH), 175.9
(COOCH3); HRMS (ESI) calcd for C18H28N2O3Na:
357.2154 (M?Na)? found 357.2171.
3
C(CH3)3), 1.96 (m, J = 6.5, 1H, CH), 2.34 (bs, 1H, NH),
3
2.87 (bpd, J = 5.0, 1H, H-2), 3.71 (s, 3H, OCH3), 4.08 (s,
Methyl (2S,1S,3S)- and (2S,1R,3S)-2-(2-(tert-butylamino)-
2-oxo-1-phenylethylamino)-3-methylpentanoate
(2S,1S,3S)-1c and (2S,1R,3S)-1c
1H, H-1), 6.37 (bs, 1H, CONH), 7.28–7.36 (m, 5H, H–Ar);
13C NMR (CDCl3, 125 MHz): d 18.4 (CH3), 19.2 (CH30 ),
28.6 (C(CH3)3), 31.4 (CH), 50.8 (C(CH3)3), 51.5 (OCH3),
64.6 (C-2), 66.6 (C-1), 127.9 (C-20, C-60), 128.2 (C-40), 128.8
(C-30, C-50), 138.8 (C-10), 170.9 (CONH), 174.7 (COOCH3);
HRMS (ESI) calcd for C18H28N2O3Na: 343.1998 (M?Na)?
found 343.1958. (2S,1R)-1a: 1H NMR (from diastereomeric
From L-isoleucine (2.64 g, 20.16 mmol), benzaldehyde
(16.80 mmol, 1.71 mL) and tert-butyl isocyanide
(2.00 mL, 16.80 mmol); FC (gradient: PE/AcOEt 9:1–4:1):
yield 3.97 g (71 %) of chromatographically inseparable
3
1
mixture, CDCl3, 500 MHz): 0.95 (d, J = 6.5, 3H, CH3),
diastereomeric mixture (dr = 9.0/1, H NMR). Colorless
1.06 (d, 3J = 6.5, 3H, CH30 ), 1.39 (s, 9H, C(CH3)3), 2.02 (m,
3J = 6.5, 1H, CH), 2.34 (bs, 1H, NH), 3.09 (m, 1H, H-2),
3.73 (s, 3H, OCH3), 3.92 (s, 1H, H-1), 6.37 (bs, 1H, CONH),
the remaining signals overlap with the signals of (2S,1S)-1a;
13C NMR (from diastereomeric mixture, CDCl3, 125 MHz):
oil; IR (KBr): 700, 741, 1148, 1200, 1225, 1265, 1454,
1516, 1678, 1736, 2876, 2930, 2964, 3329; TLC (PE/
AcOEt 3:1): Rf = 0.35; 1H NMR (from diastereomeric
mixture, CDCl3, 500 MHz): (2S,1S,3S)-1c (major isomer):
3
3
d 0.83 (t, J = 7.5, 3H, CH2CH3), 0.85 (d, J = 7.0, 3H,
CH3), 1.16 (m, 1H, CH2), 1.30 (s, 9H, C(CH3)3), 1.51 (m,
1H, CH20 ), 1.72 (m, 1H, CH), 2.35 (bs, 1H, NH), 2.94 (d,
3J = 6.0, 1H, H-2), 3.71 (s, 3H, OCH3), 4.07 (s, 1H, H-1),
6.37 (bs, 1H, CONH), 7.27–7.34 (m, 5H, H–Ar);
(2S,1R,3S)-1c (minor isomer): d 0.92 (t, 3J = 7.5, 3H,
CH2CH3), 1.00 (d, 3J = 7.0, 3H, CH3), 1.39 (s, 9H,
C(CH3)3), 3.17 (d, 3J = 5.5, 1H, H-2), 3.72 (s, 3H, OCH3),
3.93 (s, 1H, H-1), 5.30 (bs, 1H, CONH), the remaining
signals overlap with the signals of (2S,1S,3S)-1c; 13C NMR
(from diastereomeric mixture, CDCl3, 125 MHz):
d 18.0 (CH3), 19.6 (CH0 ), 28.8 (C(CH3)3), 31.5 (CH), 50.7
3
(C(CH3)3), 51.8 (OCH3), 66.3 (C-1), 67.0 (C-2), 127.3 (C-20,
C-60), 128.3 (C-40), 128.8 (C-30, C-50), 139.6 (C-10), 170.9
(CONH), 175.0 (COOCH3).
Methyl (2S,1S)- and (2S,1R)-2-(2-(tert-butylamino)-2-
oxo-1-phenylethylamino)-4-methylpentanoate (2S,1S)-1b
and (2S,1R)-1b
From L-leucine (2.64 g, 20.16 mmol), benzaldehyde
(16.80 mmol, 1.71 mL) and tert-butyl isocyanide (2.00 mL,
16.80 mmol); FC (gradient: PE/AcOEt 9:1–4:1): yield
(2S,1S,3S)-1c (major isomer): d 11.3, 15.6 (CH3, CH0 ),
3
25.3 (CH2), 28.6 (C(CH3)3), 38.0 (CH), 50.9 (C(CH3)3),
51.5 (OCH3), 63.5 (C-2), 66.6 (C-1), 127.9 (C-20, C-60),
128.2 (C-40), 128.8 (C-30, C-50), 138.8 (C-10), 170.9
(CONH), 174.7 (COOCH3); (2S,1R,3S)-1c (minor isomer):
1
3.58 g (64 %) of diastereomeric mixture (dr = 5.3/1, H
NMR). Colorless oil; IR (KBr): 700, 733, 1155, 1200, 1227,
1454, 1516, 1680, 1738, 2870, 2959, 3331; TLC (PE/AcOEt
3:1): Rf = 0.35 (major isomer) and 0.38 (minor isomer); 1H
NMR (from diastereomeric mixture, CDCl3, 500 MHz):
d 11.7, 16.4 (CH3, CH0 ), 25.0 (CH2), 28.8 (C(CH3)3), 38.5
3
(CH), 50.7 (C(CH3)3), 51.7 (OCH3), 65.3 (C-2), 67.1 (C-1),
127.2 (C-20, C-60), 128.0 (C-40), 128.8 (C-30, C-50), 139.6
(C-10), 171.0 (CONH), 174.7 (COOCH3); HRMS (ESI)
calcd for C18H28N2O3Na: 357.2154 (M?Na)? found
357.2148.
3
(2S,1S)-1b (major isomer): d 0.77 (d, J = 6.5, 3H, CH3),
0.87 (d, 3J = 6.5, 3H, CH30 ), 1.31 (s, 9H, C(CH3)3), 1.58 (m,
3
2H, CH2), 1.71 (m, J = 6.5, 1H, CH), 2.26 (bs, 1H, NH),
3.11 (pt, 3J = 7.5, 1H, H-2), 3.70 (s, 3H, OCH3), 4.11 (s, 1H,
H-1), 6.49 (bs, 1H, CONH), 7.28–7.37 (m, 5H, H–Ar);
3
(2S,1R)-1b (minor isomer): d 0.96 (d, J = 6.5, 3H, CH3),
Methyl (2S,1S)- and (2S,1R)-2-(2-(tert-butylamino)-2-
oxo-1-phenylethylamino)-3-phenylpropanoate (2S,1S)-1d
and (2S,1R)-1d
0.99 (d, 3J = 6.5, 3H, CH30 ), 1.38 (s, 9H, C(CH3)3), 1.86 (m,
3J = 6.5, 1H, CH), 3.32 (dd, 3J1 = 9.0, 3J2 = 5.0, 1H, H-2),
3.72 (s, 3H, OCH3), 3.95 (s, 1H, H-1), the remaining signals
overlap with the signals of (2S,1S)-1b; 13C NMR (from
diastereomeric mixture, CDCl3, 125 MHz): (2S,1S)-1b
From L-phenylalanine (3.33 g, 20.16 mmol), benzaldehyde
(16.80 mmol, 1.71 mL) and tert-butyl isocyanide
(2.00 mL, 16.80 mmol); FC (gradient: PE/AcOEt 9:1–2:1):
yield 3.23 g (52 %) of diastereomeric mixture (dr = 5.1/1,
1H NMR). Pale-yellow oil; IR (KBr): 700, 754, 1223, 1454,
(major isomer): d 22.0 (CH3), 22.8 (CH0 ), 24.8 (CH), 28.6
3
(C(CH3)3), 42.5 (CH2), 50.9 (C(CH3)3), 51.2 (OCH3), 57.5
(C-2), 66.4 (C-1), 127.8 (C-20, C-60), 128.2 (C-40), 128.9
123

Med Chem Res (2014) 23:2007–2018
2013
1516, 1680, 1738, 2872, 2966, 3326; TLC (PE/AcOEt 3:1):
Rf = 0.20 (major isomer) and 0.24 (minor isomer); 1H
NMR (from diastereomeric mixture, CDCl3, 500 MHz):
(2S,1S)-1d (major isomer): d 1.28 (s, 9H, C(CH3)3), 2.33
(C(CH3)3), 52.4 (OCH3), 64.1 (C-2), 66.9 (C-1), 127.3,
127.5 (C-20, C-60, C-200, C-600), 128.2, 128.4 (C-40, C-400),
128.9, 129.0 (C-30, C-50, C-300, C-500), 137.9, 139.0 (C-10,
C-100), 170.6 (CONH), 173.2 (COOCH3); HRMS (ESI?)
calcd for C21H26N2O3Na: 377.1841 (M?Na)? found
377.1843.
2
3
(bs, 1H, NH), 2.85 (dd, J = 13.5, J = 8.0, 1H, CH2),
3.03 (dd, 2J = 13.5, 3J = 6.0, 1H, CH20 ), 3.36 (dd,
3
3J = 8.0, J = 6.0, 1H, H-2), 3.68 (s, 3H, OCH3), 4.08 (s,
1H, H-1), 6.67 (bs, 1H, CONH), 7.06 (m, 2H, H–Ar), 7.10
(m, 2H, H–Ar), 7.21–7.37 (m, 6H, H–Ar); (2S,1R)-1d
(minor isomer): d 1.08 (s, 9H, C(CH3)3), 2.68 (dd,
2J = 13.5, 3J = 10.0, 1H, CH2), 3.47 (dd, 3J = 10.0,
3J = 4.0, 1H, H-2), 3.75 (s, 3H, OCH3), 3.96 (s, 1H, H-1),
6.78 (bs, 1H, CONH), the remaining signals overlap with
the signals of (2S,1S)-1d; 13C NMR (from diastereomeric
mixture, CDCl3, 125 MHz): (2S,1S)-1d (major isomer): d
28.6 (C(CH3)3), 39.4 (CH2), 50.8 (C(CH3)3), 51.9 (OCH3),
60.4 (C-2), 66.4 (C-1), 126.8 (C-400), 127.6 (C-20, C-60),
128.1 (C-40), 128.5 (C-200, C-600), 128.7 (C-30, C-50), 129.3
(C-300, C-500), 137.0 (C-100), 138.4 (C-10), 170.7 (CONH),
174.1 (COOCH3); (2S,1R)-1d (minor isomer): d 28.4
(C(CH3)3), 40.2 (CH2), 50.3 (C(CH3)3), 52.1 (OCH3), 62.4
(C-2), 66.8 (C-1),00127.0 (C-400), 127.2 (C-20, C-60), 128.1
(C-40), 128.7 (C-2 , C-600), 128.8 (C-30, C-50), 129.5 (C-300,
C-500), 137.6 (C-100), 139.5 (C-10), 170.5 (CONH), 174.8
(COOCH3); HRMS (ESI?) calcd for C22H28N2O3Na:
391.1998 (M?Na)? found 391.1995.
Methyl (?/-)-2-(2-benzyl-2-(tert-butylamino)-2-oxo-1-
phenylethylamino)-acetate rac-1f
From N-benzylglycine hydrochloride (4.06 g, 20.16 mmol),
triethylamine (2.81 mL, 20.16 mmol) benzaldehyde
(16.80 mmol, 1.71 mL) and tert-butyl isocyanide (2.00 mL,
16.80 mmol); FC (gradient: PE/AcOEt 10:1–3:1): yield
0.77 g (12 %). White powder; mp 87–89 °C; TLC (PE/
AcOEt 3:1): Rf = 0.40; IR (KBr): 700, 741, 1204, 1454,
1512, 1680, 1742, 2872, 2928, 2964, 3327; 1H NMR (CDCl3,
500 MHz): d 1.38 (s, 9H, C(CH3)3), 3.06 (d, 2J = 17.5, 1H,
PhCH2), 3.31 (d, 2J = 17.5, 1H, PhCH20 ), 3.59 (s, 3H,
2
2
OCH3), 3.67 (d, J = 13.5, 1H, CH2), 3.85 (d, J = 13.5,
1H, CH20 ), 4.43 (s, 1H, H-1), 7.26–7.39 (m, 10H, H–Ar), 7.60
(bs, 1H, CONH); 13C NMR (CDCl3, 125 MHz): d 28.7
(C(CH3)3), 50.9 (C(CH3)3), 51.5 (OCH3), 51.6 (PhCH2),
56.9 (CH2), 71.1 (C-1), 127.6, 128.1 (C-40, C-400), 128.5,
128.6 (C-20, C-60, C-200, C-600), 128.9, 129.6 (C-30, C-50, C-300,
C-500), 135.6, 137.8 (C-10, C-100), 170.5 (CONH), 172.1
(COOCH3); HRMS (ESI?) calcd for C22H28N2O3Na:
391.1998 (M?Na)? found 391.1985.
Methyl (2S,1S)- and (2S,1R)-2-(2-(tert-butylamino)-2-
oxo-1-phenylethylamino)-3-phenylacetate (2S,1S)-1e
and (2S,1R)-1e
Synthesis of compounds 2 by BF3ꢀ2CH3COOH
mediated N-detertbutylation
From L-phenylglycine (3.05 g, 20.16 mmol), benzaldehyde
(16.80 mmol, 1.71 mL) and tert-butyl isocyanide
(2.00 mL, 16.80 mmol); FC (gradient: PE/AcOEt 5:1–1:2):
yield 2.41 g (41 %) of chromatographically inseparable
diastereomeric mixture (dr = 1.5/1, 1H NMR). Pale-yellow
wax; mp 65–71 °C; IR (KBr): 700, 733, 1223, 1454, 1516,
1678, 1740, 2872, 2930, 2966, 3333; TLC (PE/AcOEt 3:1):
Rf = 0.28; 1H NMR (from diastereomeric mixture, CDCl3,
500 MHz): (2S,1S)-1e (major isomer): d 1.35 (s, 9H,
C(CH3)3), 2.85 (bs, 1H, NH), 3.69 (s, 3H, OCH3), 3.99 (s,
1H, H-1), 4.33 (s, 1H, H-2), 6.88 (bs, 1H, CONH),
7.23–7.38 (m, 10H, H–Ar); (2S,1R)-1e (minor isomer): d
1.27 (s, 9H, C(CH3)3), 2.78 (bs, 1H, NH), 3.69 (s, 3H,
OCH3), 4.05 (s, 1H, H-1), 4.29 (s, 1H, H-2), 6.97 (bs, 1H,
CONH); the remaining signals overlap with the signals of
(2S,1S)-1e; 13C NMR (from diastereomeric mixture,
CDCl3, 125 MHz): (2S,1S)-1e (major isomer): d 28.7
(C(CH3)3), 50.9 (C(CH3)3), 52.5 (OCH3), 63.6 (C-2), 65.1
(C-1), 127.5, 127.6 (C-20, C-60, C-200, C-600), 128.2, 128.5
(C-40, C-400), 128.9, 129.0 (C-30, C-50, C-300, C-500), 137.2,
139.1 (C-10, C-100), 170.5 (CONH), 172.6 (COOCH3);
(2S,1R)-1e (minor isomer): d 28.6 (C(CH3)3), 50.7
The appropriate Ugi product
1 was dissolved in
BF3ꢀ2CH3COOH (*36 % BF3 basis, 3 mL/1 mmol of
substrate), and stirred at 45–55 °C until full conversion of
the starting material is observed by TLC (typically for
4–6 h). The solution was cooled, poured onto excess of
crushed ice and made alkaline with 25 % aqueous solution
of ammonia. The resulting mixture was extracted with
DCM (3 9 50 mL). The combined organic phase was
washed with water (30 mL), brine (30 mL), dried over
anhydrous MgSO4, filtered and concentrated under reduced
pressure. The residue was purified by FC.
Methyl (2S,1S)- and (2S,1S)-2-(2-amino-2-oxo-1-
phenylethylamino)-3-methylbutanoate (2S,1S)-2a
and (2S,1R)-2a
From diastereomeric mixture of (2S,1S)-1a and (2S,1R)-1a
(3.98 g, 12.43 mmol) and BF3ꢀ2CH3COOH (37 mL); FC
(gradient: PE/AcOEt 2:1–0:1): yield 2.31 g (70 %): 1.95 g
123

2014
Med Chem Res (2014) 23:2007–2018
(59 %) of (2S,1S)-2a, 0.19 g (6 %) of (2S,1R)-2a and
0.17 g (5 %) of diastereomeric mixture. (2S,1S)-2a: col-
orless oil; [a]D = -133.5 (c 0.977, CHCl3); IR (KBr): 702,
759, 1152, 1205, 1456, 1682, 1732, 2874, 2960, 3196,
[a]D = -0.2 (c 1.030, CHCl3); IR (KBr): 702, 756, 1157,
1202, 1269, 1387, 1454, 1680, 1734, 2870, 2957, 3190,
1
3325, 3445; TLC (AcOEt): Rf = 0.63; H NMR (CDCl3,
500 MHz): d 0.95 (d, 3J = 6.5, 3H, CH3), 0.95 (d,
3J = 6.5, 3H, CH30 ), 1.49 (m, 2H, CH2), 1.83 (m, 3J = 6.5,
1H, CH), 2.25 (bs, 1H, NH), 3.40 (dd, 3J1 = 8.0, 3J1 = 6.0,
1H, H-2), 3.70 (s, 3H, OCH3), 4.10 (s, 1H, H-1), 6.08 (bs,
1H, CONH), 7.17 (bs, 1H, CONH0), 7.27–7.42 (m, 5H, H–
Ar); 13C NMR (CDCl3, 125 MHz): d 22.0 (CH3), 22.9
(CH03), 24.9 (CH), 43.1 (CH2), 51.9 (OCH3), 59.0 (C-2),
66.3 (C-1), 127.2 (C-20, C-60), 128.3 (C-40), 128.8 (C-30,
C-50), 138.7 (C-10), 175.0 (CONH), 175.7 (COOCH3);
HRMS (ESI) calcd for C15H22N2O3Na: 301.1528
(M?Na)? found 301.1534.
1
3332, 3445; TLC (AcOEt): Rf = 0.54; H NMR (CDCl3,
500 MHz): d 0.89 (d, 3J = 7.0, 3H, CH3), 0.93 (d,
0
3
3J = 7.0, 3H, CH3), 1.96 (m, J = 7.0, 1H, CH), 2.22 (bs,
1H, NH), 2.87 (bs, 1H, H-2), 3.72 (s, 3H, OCH3), 4.19 (s,
1H, H-1), 5.80 (bs, 1H, CONH), 6.23 (bs, 1H, CONH0),
7.30–7.40 (m, 5H, H–Ar); 13C NMR (CDCl3, 125 MHz): d
18.4 (CH3), 19.3 (CH03), 31.4 (CH), 52.6 (OCH3), 64.2 (C-
2), 65.6 (C-1), 128.1 (C-20, C-60), 128.5 (C-40), 128.9 (C-30,
C-50), 138.1 (C-10), 174.3 (CONH), 174.8 (COOCH3);
HRMS (ESI) calcd for C14H20N2O3Na: 287.1372
(M?Na)? found 287.1396. (2S,1R)-2a: white powder; mp
107–109 °C; [a]D = -5.2 (c 0.975, CHCl3); IR (KBr):
698, 758, 1150, 1202, 1456, 1685, 1733, 2874, 2960, 3196,
Methyl (2S,1R,3S)- and (2S,1S,3S)-2-(2-amino-2-oxo-1-
phenylethylamino)-3-methylpentanoate (2S,1R,3S)-2c
and (2S,1S,3S)-2c
1
3331, 3443; TLC (AcOEt): Rf = 0.58; H NMR (CDCl3,
500 MHz): d 0.96 (d, 3J = 7.0, 3H, CH3), 1.03 (d,
0
3
3J = 7.0, 3H, CH3), 2.02 (m, J = 7.0, 1H, CH), 2.18 (bs,
1H, NH), 3.17 (bs, 1H, H-2), 3.72 (s, 3H, OCH3), 4.06 (s,
1H, H-1), 5.93 (bs, 1H, CONH), 7.22 (bs, 1H, CONH0),
7.28–7.44 (m, 5H, H–Ar); 13C NMR (CDCl3, 125 MHz): d
18.2 (CH3), 19.6 (CH03), 31.6 (CH), 51.8 (OCH3), 66.2 (C-
1), 66.7 (C-2), 127.3 (C-20, C-60), 128.4 (C-40), 128.9 (C-30,
C-50), 138.8 (C-10), 174.8 (CONH), 174.9 (COOCH3);
HRMS (ESI) calcd for C14H20N2O3Na: 287.1372
(M?Na)? found 287.1359.
From diastereomeric mixture of (2S,1S,3S)-1c and
(2S,1R,3S)-1c (3.96 g, 11.85 mmol) and BF3ꢀ2CH3COOH
(35 mL); FC (gradient: PE/AcOEt 2:1–0:1): yield 2.75 g
(83 %): 1.92 g (58 %) of (2S,1S,3S)-2c, 0.05 g (1 %) of
(2S,1R,3S)-1c and 0.78 g (24 %) of diastereomeric mix-
ture. (2S,1S,3S)-2c: pale-yellow oil; [a]D = -124.1 (c
0.085, CHCl3); IR (KBr): 702, 758, 1151, 1202, 1384,
1456, 1682, 1734, 2878, 2964, 3190, 3325, 3447; TLC
1
(AcOEt): Rf = 0.55; H NMR (CDCl3, 500 MHz): d 0.83
3
3
Methyl (2S,1R)- and (2S,1S)-2-(2-amino-2-oxo-1-
phenylethylamino)-4-methylpentanoate (2S,1S)-2b
and (2S,1R)-2b
(t, J = 7.5, 3H, CH2CH3), 0.85 (d, J = 7.0, 3H, CH3),
1.17 (m, 1H, CH2), 1.52 (m, 1H, CH20 ), 1.71 (m, 1H, CH),
2.54 (bs, 1H, NH), 2.94 (d, 3J = 6.0, 1H, H-2), 3.71 (s, 3H,
OCH3), 4.19 (s, 1H, H-1), 5.73 (bs, 1H, CONH0), 6.23 (bs,
1H, CONH), 7.31–7.42 (m, 5H, H–Ar); 13C NMR (CDCl3,
From diastereomeric mixture of (2S,1S)-1b and (2S,1R)-1b
(3.11 g, 9.31 mmol) and BF3ꢀ2CH3COOH (28 mL); FC
(gradient: PE/AcOEt 2:1–0:1): yield 1.43 g (55 %): 1.03 g
(40 %) of (2S,1S)-2b, 0.08 g (3 %) of (2S,1R)-2b and
0.32 g (12 %) of diastereomeric mixture. (2S,1S)-2b: yel-
low wax; mp 65–72 °C; [a]D = -132.9 (c 1.107, CHCl3);
IR (KBr): 702, 739, 1155, 1202, 1271, 1367, 1454, 1676,
1732, 2872, 2957, 3192, 3329, 3441; TLC (AcOEt):
Rf = 0.51; 1H NMR (CDCl3, 500 MHz): d 0.73 (d,
125 MHz): d 11.3, 15.6 (CH3, CH0 ), 25.2 (CH2), 38.0
3
(CH), 51.6 (OCH3), 63.2 (C-2), 65.6 (C-1), 128.1 (C-20, C-
60), 128.5 (C-40), 128.9 (C-30, C-50), 138.1 (C-10), 174.3
(CONH), 174.8 (COOCH3); HRMS (ESI) calcd for
C15H22N2O3Na: 301.1528 (M?Na)? found 301.1516;
(2S,1R,3S)-2c: white wax; mp 86–89 °C; [a]D =?6.0 (c
0.833, CHCl3); IR (KBr): 700, 756, 1150, 1202, 1267,
1381, 1456, 1680, 1732, 2878, 2964, 3194, 3331, 3443;
0
3
1
3J = 6.5, 3H, CH3), 0.87 (d, J = 6.5, 3H, CH3), 1.47 (m,
TLC (AcOEt): Rf = 0.63; H NMR (CDCl3, 500 MHz): d
3
2H, CH2), 1.76 (m, J1 = 7.5, J1 = 6.5, 1H, CH), 2.44
3
3
3
0.91 (t, J = 7.5, 3H, CH2CH3), 0.97 (d, J = 7.0, 3H,
(bs, 1H, NH), 3.11 (dd, J1 = 8.5, J1 = 6.0, 1H, H-2),
3.70 (s, 3H, OCH3), 4.24 (s, 1H, H-1), 5.93 (bs, 1H,
CONH), 6.31 (bs, 1H, CONH0), 7.29–7.39 (m, 5H, H–A0r);
13C NMR (CDCl3, 125 MHz): d 21.8 (CH3), 22.9 (CH3),
24.7 (CH), 42.6 (CH2), 51.8 (OCH3), 57.0 (C-2), 65.3 (C-
1), 128.0 (C-20, C-60), 128.5 (C-40), 128.9 (C-30, C-50),
138.2 (C-10), 174.4 (CONH), 175.5 (COOCH3); HRMS
(ESI) calcd for C15H22N2O3Na: 301.1528 (M?Na)? found
301.1522; (2S,1R)-2b: pale-yellow powder; mp 88–95 °C;
CH3), 1.20 (m, 1H, CH2), 1.54 (m, 1H, CH20 ), 1.76 (m, 1H,
3
3
3
CH), 2.22 (bs, 1H, NH), 3.25 (d, J = 5.5, 1H, H-2), 3.71
(s, 3H, OCH3), 4.06 (s, 1H, H-1), 6.06 (bs, 1H, CONH0),
7.20 (bs, 1H, CONH), 7.28–7.42 (m, 5H, H–Ar); 13C NMR
(CDCl3, 125 MHz): d 11.6, 15.9 (CH3, CH0 ), 25.2 (CH2),
3
38.5 (CH), 51.7 (OCH3), 65.3 (C-2), 66.7 (C-1), 127.3 (C-
20, C-60), 128.3 (C-40), 128.9 (C-30, C-50), 138.8 (C-10),
174.8 (CONH), 175.0 (COOCH3); HRMS (ESI) calcd for
C15H22N2O3Na: 301.1528 (M?Na)? found 301.1501.
123

Med Chem Res (2014) 23:2007–2018
2015
Methyl (2S,1R)- and (2S,1S)-2-(2-amino-2-oxo-1-
phenylethylamino)-3-phenylpropanoate (2S,1R)-2d
and (2S,1S)-2d
(s, 3H, OCH3), 4.33 (s, 1H, H-2), 5.29 (bs, 1H, CONH),
6.80 (bs, 1H, CONH0); the remaining signals overlap with
the signals of (2S,1S)-2e; 13C NMR (from diastereomeric
mixture, CDCl3, 125 MHz): (2S,1S)-2e (major isomer): d
52.4 (OCH3), 63.3 (C-2), 64.3 (C-1), 127.6 (C-20, C-60,
C-200, C-600), 128.5, 128.6 (C-40, C-400), 129.0 (C-30, C-50,
C-300, C-500), 137.1, 138.3 (C-10, C-100), 172.6 (CONH),
174.3 (COOCH3); (2S,1R)-2e (minor isomer): d 52.5
(OCH3), 63.5 (C-2), 65.3 (C-1), 127.4, 127.7 (C-20, C-60,
C-200, C-600), 128.4, 128.5 (C-40, C-400), 128.9, 129.0 (C-30,
C-50, C-300, C-500), 137.3, 138.1 (C-10, C-100), 173.0
(CONH), 174.4 (COOCH3); HRMS (ESI?) calcd for
C17H18N2O3Na: 321.1215 (M?Na)? found 321.1227.
From diastereomeric mixture of (2S,1S)-1d and (2S,1R)-1d
(2.34 g, 6.36 mmol) and BF3ꢀ2CH3COOH (19 mL); FC
(gradient: PE/AcOEt 2:1–0:1): yield 1.32 g (67 %): 1.10 g
(55 %) of (2S,1S)-2d and 0.22 g (12 %) of (2S,1R)-2d.
(2S,1S)-2d: pale-yellow oil; [a]D = -72.3 (c 0.392,
CHCl3); IR (KBr): 702, 752, 1205, 1454, 1682, 1734, 2854,
2951, 3028, 3190, 3325, 3445; TLC (AcOEt): Rf = 0.46;
1H NMR (CDCl3, 500 MHz): d 2.40 (bs, 1H, NH), 2.85
2
3
2
(dd, J = 13.5, J = 8.0, 1H, CH2), 3.03 (dd, J = 13.5,
3J = 6.0, 1H, CH20 ), 3.38 (bpt, 3J = 6.0, 1H, H-2), 3.67 (s,
3H, OCH3), 4.22 (s, 1H, H-1), 5.60 (bs, 1H, CONH), 6.44
(bs, 1H, CONH0), 7.09 (m, 2H, H–Ar), 7.12 (m, 2H, H–Ar),
7.21–7.30 (m, 6H, H–Ar); 13C NMR (CDCl3, 125 MHz): d
39.4 (CH2), 51.9 (OCH3), 60.1 (C-2), 65.3 (C-1), 126.8 (C-
400), 127.7 (C-20, C-60), 128.3 (C-40), 128.4 (C-200, C-600),
128.8 (C-30, C-50), 129.2 (C-300, C-500), 136.9 (C-100), 137.7
(C-10), 174.1 (COOCH3), 174.2 (CONH); HRMS (ESI)
calcd for C18H20N2O3Na: 335.1372 (M?Na)? found
335.1363. (2S,1R)-2d: white powder; mp 124–127 °C;
[a]D = -37.8 (c 0.775, CHCl3); IR (KBr): 702, 739, 1209,
1452, 1693, 1734, 2951, 3030, 3188, 3335, 3429; TLC
Methyl (?/-)-2-(2-benzyl-2-amino-2-oxo-1-
phenylethylamino)-acetate rac-2f
From rac-1f (0.59 g, 1.60 mmol) and BF3ꢀ2CH3COOH
(5 mL); FC (gradient: PE/AcOEt 4:1–1:2): yield 0.40 g
(80 %) of rac-2f. White powder; mp 147–149 °C; TLC
(AcOEt): Rf = 0.63; IR (KBr): 700, 741, 1204, 1454, 1558,
1682, 1734, 2844, 2951, 3030, 3182, 3418; 1H NMR
(CDCl3, 500 MHz): d 3.07 (d, 2J = 17.5, 1H, PhCH2),
0
2
3.40 (d, J = 17.5, 1H, PhCH2), 3.61 (s, 3H, OCH3), 3.66
0
2
2
(d, J = 13.5, 1H, CH2), 3.85 (d, J = 13.5, 1H, CH2),
4.75 (s, 1H, H-1), 5.85 (bs, 1H, CONH), 7.26–7.42 (m,
10H, H–Ar), 7.63 (bs, 1H, CONH0); 13C NMR (CDCl3,
125 MHz): d 51.7 (OCH3), 51.8 (PhCH2), 56.8 (CH2), 69.9
(C-1), 127.7, 128.4 (C-40, C-400), 128.64, 128.65 (C-20,
C-60, C-200, C-600), 129.0, 129.6 (C-30, C-50, C-300, C-500),
134.7, 137.5 (C-10, C-100), 172.3 (CONH), 174.4
(COOCH3); HRMS (ESI?) calcd for C18H20N2O3Na:
335.1360 (M?Na)? found 335.1372.
1
(AcOEt): Rf = 0.58; H NMR (CDCl3, 500 MHz): d 2.21
2
3
(bs, 1H, NH), 2.68 (dd, J = 13.5, J = 10.0, 1H, CH2),
3.11 (dd, 2J = 13.5, 3J = 4.0, 1H, CH20 ), 3.47 (bps,
3J = 6.0, 1H, H-2), 3.76 (s, 3H, OCH3), 4.08 (s, 1H, H-1),
5.04 (bs, 1H, CONH), 6.32 (bs, 1H, CONH0), 7.23–7.42
(m, 10H, H–Ar); 13C NMR (CDCl3, 125 MHz): d 40.1
(CH2), 52.2 (OCH3), 62.3 (C-2), 66.4 (C-1), 127.0 (C-400),
127.3 (C-20, C-60), 128.4 (C-40), 128.6 (C-200, C-600), 128.9
(C-30, C-50), 129.6 (C-300, C-500), 137.7 (C-100), 138.6 (C-10),
174.5 (COOCH3), 174.6 (CONH); C18H20N2O3Na:
335.1372 (M?Na)? found 335.1366.
Synthesis of compounds 3 by base-induced
intramolecular cyclocondensation
Methyl (2S,1S)- and (2S,1R)-2-(2-amino-2-oxo-1-
phenylethylamino)-3-phenylacetate (2S,1S)-2e
and (2S,1R)-2e
To a stirred solution of appropriate amidoester 2 in abso-
lute EtOH (5 mL/1 mmol of amidoester), sodium hydrox-
ide (1 equiv.) was added at room temperature. After
dissolution of the hydroxide, the mixture was quenched
with saturated aqueous solution of ammonium chloride
(100 mL). The resulting cloudy solution was extracted with
CH2Cl2 (3 9 30 mL). The combined organic phase was
washed with water (20 mL), dried over anhydrous MgSO4,
filtered and concentrated under reduced pressure. The
residue was purified by FC.
From diastereomeric mixture of (2S,1S)-1e and (2S,1R)-1e
(2.26 g, 6.38 mmol) and BF3ꢀ2CH3COOH (19 mL); FC
(gradient: PE/AcOEt 4:1–1:2): yield 1.54 g (81 %) of
1
diastereomeric mixture (dr = 1.4/1, H NMR). Pale-yel-
low, sticky wax; mp 43–50 °C; IR (KBr): 700, 741, 1175,
1209, 1456, 1510, 1558, 1682, 1736, 2852, 2924, 3194,
3333, 3443; TLC (AcOEt): Rf = 0.51; 1H NMR (from
diastereomeric mixture, CDCl3, 500 MHz): (2S,1S)-2e
(major isomer): d 2.85 (bs, 1H, NH), 3.69 (s, 3H, OCH3),
4.13 (s, 1H, H-1), 4.39 (s, 1H, H-2), 5.29 (bs, 1H, CONH),
6.83 (bs, 1H, CONH0), 7.28–7.40 (m, 10H, H–Ar);
(2S,1R)-2e (minor isomer): d 2.98 (bs, 1H, NH), 3.68
(3S,5R)- and (3S,5S)-3-isopropyl-5-phenylpiperazine-2,6-
dione (3S,5S)-3a and (3S,5R)-3a
From (2S,1S)-2a (1.86 g, 7.04 mmol) and NaOH (0.28 g,
1 equiv.); FC (gradient: PE/EtOAc 6:1–1:1): yield 1.34 g
123

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Med Chem Res (2014) 23:2007–2018
(82 %): 0.72 g (44 %) of (3S,5S)-3a, 0.32 g (19 %) of
(3S,5R)-3a and 0.30 g (19 %) of diastereomeric mixture.
(3S,5S)-3a: white powder; mp 103–105 °C; [a]D = -152.1
(c 1, CHCl3); IR (KBr): 756, 1030, 1099, 1180, 1234, 1331,
1454, 1497, 1701, 2932, 1963, 3225; TLC (PE/AcOEt 3:1):
Rf = 0.35; 1H NMR (CDCl3, 500 MHz): d 0.99 (d,
(m, 1H, H-3), 4.57 (s, 1H, H-5), 8.18 (bs, 1H, CONHCO),
the remaining signals overlap with the signals of (3S,5S)-3b;
13C NMR (from diastereomeric mixture, CDCl3, 125 MHz):
d 21.3 (CH3), 23.4 (CH0 ), 24.5 (CH), 39.0 (CH2), 57.6 (C-3),
64.6 (C-5), 128.5 (C-203, C-60), 128.8 (C-30, C-50), 128.9 (C-
40), 136.3 (C-10), 171.8 (C-6), 173.3 (C-2).
0
3
3J = 7.0, 3H, CH3), 1.09 (d, J = 7.0, 3H, CH3), 2.18 (bs,
1H, NH), 2.49 (2 sp, 3J1 = 6.5, 3J2 = 5.0, 1H, CH), 3.26 (d,
3J = 4.5, 1H, H-3), 4.90 (s, 1H, H-5), 7.32–7.46 (m, 5H, H–
Ar), 8.34 (bs, 1H, CONHCO); 13C NMR (CDCl3,
125 MHz): d 17.1 (CH3), 19.3 (CH3), 27.7 (CH), 58.7 (C-
3), 59.8 (C-5), 127.1 (C-20, C-60), 128.5 (C-40), 129.0 (C-30,
C-50), 134.6 (C-10), 172.3 (C-6), 173.1 (C-2); HRMS
(ESI?) calcd for C13H16N2O2Na: 255.1109 (M?Na)?
found 255.1101. (3S,5R)-3a: white powder; mp
111–112 °C; [a]D = -117.5 (c 1, CHCl3); IR (KBr): 756,
1223, 1269, 1497, 1701, 2874, 2936, 3032, 3221; TLC (PE/
(3S,5R,1S)- and (3S,5S,1S)-3-sec-butyl-5-
phenylpiperazine-2,6-dione (3S,5S,1S)-3c
and (3S,5R,1S)-3c
From (2S,1S,3S)-2c (1.81 g, 6.51 mmol) and NaOH
(0.26 g, 1 equiv.); FC (gradient: PE/EtOAc 6:1–2:1): yield
1.44 g (90 %): 0.75 g (47 %) of (3S,5S,1S)-3c, 0.24 g
(15 %) of (3S,5R,1S)-3c and 0.45 g (28 %) of diastereo-
meric mixture. (3S,5S,1S)-3c: white powder; mp 66–67 °C;
[a]D = -118.2 (c 1, CHCl3); IR (KBr): 760, 1030, 1177,
1230, 1454, 1693, 2874, 2932, 2963, 3206; TLC (PE/AcOEt
1
AcOEt 3:1): Rf = 0.29; H NMR (CDCl3, 500 MHz):0 d
1.02 (d, J = 7.0, 3H, CH3), 1.09 (d, J = 7.0, 3H, CH3),
3
3
1
3:1): Rf = 0.38; H NMR (CDCl3, 500 MHz): d 0.94 (t,
3
1.76 (bs, 1H, NH), 2.60 (2 sp, J1 = 7.0, J2 = 2.5, 1H,
3
3J = 7.0, 3H, CH2CH3), 1.07 (d, 3J = 7.0, 3H, CH3), 1.26
(m, 1H, CH2), 1.47 (m, 1H, CH20 ), 2.20 (m, 2H, CH, NH),
3.30 (d, 3J = 4.5, 1H, H-3), 4.90 (s, 1H, H-5), 7.31–7.46 (m,
5H, H–Ar), 8.25 (bs, 1H, CONHCO); 13C NMR (CDCl3,
3
CH), 3.58 (d, J = 2.5, 1H, H-3), 4.54 (s, 1H, H-5), 7.36–
7.44 (m, 5H, H–Ar), 8.13 (bs, 1H, CONHCO); 13C NMR
(CDCl3, 125 MHz): d 16.7 (CH3), 19.3 (CH0 ), 28.8 (CH),
3
64.3 (C-3), 64.3 (C-5), 128.6 (C-20, C-60), 128.8 (C-30,
C-50), 128.9 (C-40), 136.4 (C-10), 171.6 (C-6), 172.3 (C-2);
HRMS (ESI?) calcd for C13H16N2O2Na: 255.1109
(M?Na)? found 255.1129.
125 MHz): d 12.0, 16.0 (CH3, CH0 ), 24.6 (CH2), 34.5 (CH),
3
58.5 (C-3), 59.8 (C-5), 127.0 (C-20, C-60), 128.5 (C-40),
129.0 (C-30, C-50), 134.5 (C-10), 172.2 (C-6), 173.2 (C-2);
HRMS (ESI?) calcd for C14H18N2O2Na: 269.1266
(M?Na)? found 269.1261; (3S,5R,1S)-3c: white powder;
mp 138–139 °C; [a]D = -94.5 (c 1, CHCl3); IR (KBr): 756,
1219, 1265, 1385, 1701, 2874, 2932, 2962, 3225; TLC (PE/
AcOEt 3:1): Rf = 0.30; 1H NMR (CDCl3, 500 MHz): d 0.94
(3S,5S)- and (3S,5R)-3-isobutyl-5-phenylpiperazine-2,6-
dione (3S,5S)-3b and (3S,5R)-3b
3
3
From (2S,1S)-2b (0.92 g, 3.31 mmol) and NaOH (0.13 g,
1 equiv.); FC (gradient: PE/EtOAc 5:1–2:1): yield 0.63 g
(77 %) of chromatographically inseparable diastereomeric
mixture (dr = 68/32, 1H NMR). Pure (3S,5S)-3b was
obtained by fractional recrystallization form PE/Et2O 1:1.
(3S,5S)-3b: white powder; mp 60–61 °C; [a]D = -30.3 (c
1, CHCl3); IR (KBr): 756, 1242, 1384, 1454, 1701, 2870,
2955, 3090, 3225, 3321; TLC (PE/AcOEt 3:1): Rf = 0.36;
1H NMR (CDCl3, 500 MHz): d 0.84 (d, 3J = 6.5, 3H, CH3),
0.97 (d, 3J = 6.5, 3H, CH30 ), 1.57 (m, 2J = 13.5, 3J1 = 9.5,
3J2 = 4.0, 1H, CH2), 1.85 (m, 1H, CH20 ), 1.89 (m, 1H, CH),
2.07 (bs, 1H, NH), 3.44 (pd, 3J = 9.5, 1H, H-3), 4.86 (s, 1H,
H-5), 7.30–7.47 (m, 5H, H–Ar), 8.34 (bs, 1H, CONHCO);
13C NMR (CDCl3, 125 MHz): d 21.1 (CH3), 23.3 (CH30 ),
24.4 (CH), 38.7 (CH2), 52.1 (C-3), 59.7 (C-5), 127.2 (C-20,
C-60), 128.5 (C-40), 128.9 (C-30, C-50), 134.7 (C-10),
172.3 (C-6), 174.3 (C-2); HRMS (ESI?) calcd for
C14H18N2O2Na: 269.1266 (M?Na)? found 269.1231;
(3S,5R)-3b: 1H NMR (from diastereomeric mixture, CDCl3,
500 MHz): d 0.95 (d, 3J = 6.5, 3H, CH3), 0.98 (d, 3J = 6.5,
3H, CH30 ), 1.61 (m, 1H, CH2), 1.87 (m, 2H, CH, NH), 2.02
(m, 2J = 14.0, 3J1 = 10.0, 3J2 = 4.0, 1H, CH20 ), 3.66
(t, J = 7.5, 3H, CH2CH3), 1.08 (d, J = 7.0, 3H, CH3),
1.39 (m, 1H, CH2), 1.53 (m, 1H, CH20 ), 1.76 (bs, 1H, NH),
2.29 (m, 1H, CH), 3.61 (bps, 1H, H-3), 4.52 (s, 1H, H-5),
7.36–7.42 (m, 5H, H–Ar), 8.11 (bs, 1H, CONHCO); 13C
NMR (CDCl3, 125 MHz): d 12.3, 16.2 (CH3, CH0 ), 24.7
3
(CH2), 35.8 (CH), 64.4 (C-3), 64.4 (C-5), 128.6 (C-20, C-60),
128.8 (C-30, C-50), 128.9 (C-40), 136.4 (C-10), 171.6 (C-6),
172.4 (C-2); HRMS (ESI?) calcd for C14H18N2O2Na:
269.1266 (M?Na)? found 269.1280.
(3S,5R)- and (3S,5S)-3-benzyl-5-phenylpiperazine-2,6-
dione (3S,5S)-3d and (3S,5R)-3d
From (2S,1S)-2d (1.02 g, 3.27 mmol) and NaOH (0.13 g,
1 equiv.); FC (gradient: PE/EtOAc 6:1–2:1): yield 0.71 g
(78 %): 0.44 g (48 %) of (3S,5S)-3d, 0.27 g (39 %) of
(3S,5R)-3d. (3S,5S)-3d: white powder; mp 114–115 °C;
TLC (PE/AcOEt 3:1): Rf = 0.34; [a]D = -88.2 (c 1,
CHCl3); IR (KBr): 764, 1261, 1342, 1450, 1497, 1701,
2812, 3028, 3159, 3263, 3287; 1H NMR (CDCl3,
500 MHz): d 2.12 (bs, 1H, NH), 3.16 (dd, 2J = 14.0,
3J = 8.0, 1H, CH2), 3.25 (dd, 2J = 14.0, 3J = 4.5, 1H,
123

Med Chem Res (2014) 23:2007–2018
2017
0
3
3
CH2), 3.72 (dd, J1 = 8.0, J2 = 4.5, 1H, H-3), 4.82 (s,
1H, H-5), 7.21–7.36 (m, 10H, H–Ar), 8.27 (bs, 1H,
CONHCO); 13C NMR (CDCl3, 125 MHz): d 35.5 (CH2),
54.7 (C-3), 59.8 (C-5), 127.1 (C-20, C-60), 127.3 (C-400),
128.5 (C-40), 128.9 (C-200, C-600), 128.9 (C-30, C-50), 129.4
(C-300, C-500), 134.4 (C-10), 136.3 (C-100), 171.7 (C-6), 172.7
(C-2); HRMS (ESI?) calcd for C17H16N2O2Na: 303.1109
(M?Na)? found 303.1132; (3S,5R)-3d: white powder; mp
98–99 °C; TLC (PE/AcOEt 3:1): Rf = 0.28; [a]D =
-184.2 (c 1, CHCl3); IR (KBr): 760, 1230, 1288, 1454,
(98 %): white powder; mp 156–169 °C; TLC (PE/AcOEt
3:1): Rf = 0.22; IR (KBr): 698, 744, 1246, 1454, 1699,
2814, 2852, 2924, 3030, 3209; 1H NMR (CDCl3,
500 MHz): d 3.30 (d, 2J = 17.5, 1H, PhCH2), 3.57 (d,
2J = 17.5, 1H, PhCH20 ), 3.63 (d, 2J = 13.5, 1H, H-3), 3.83
2
(d, J = 13.5, 1H, H0-3), 4.50 (s, 1H, H-5), 7.23–7.39 (m,
6H, H–Ar), 7.41 (m, 4H, H–Ar), 8.24 (bs, 1H, CONHCO);
13C NMR (CDCl3, 125 MHz): d 51.3 (PhCH2), 58.7 (C-3),
67.1 (C-5), 128.1, 128.8 (C-40, C-400), 128.1, 128.8 (C-20,
C-60, C-200, C-600), 128.9, 129.0 (C-30, C-50, C-300, C-500),
134.0, 136.2 (C-10, C-100), 170.1 (C-6), 171.0 (C-2); HRMS
(ESI-) calcd for C17H15N2O2 279.1133 (M-H)- found
279.1126.
1
1716, 2851, 3086, 3182; H NMR (CDCl3, 500 MHz): d
1.89 (t, 1H, NH), 2.93 (dd, J = 14.0, J = 9.5, 1H, H-7),
3.62 (dd, 2J = 14.0, J = 2.5, 1H, H-70), 3.86 (dd,
2
3
3
3J1 = 8.0, 3J2 = 2.5, 1H, H-3), 4.46 (s, 1H, H-5), 7.22–7.38
(m, 10H, H–Ar), 8.18 (bs, 1H, NH); 13C NMR (CDCl3,
125 MHz): d 36.5 (CH2), 60.5 (C-3), 64.5 (C-5), 127.2 (C-
400), 128.5 (C-20, C-60), 128.7 (C-30, C-50), 128.8 (C-40),
129.0 (C-200, C-600), 129.3 (C-300, C-500), 136.0 (C-10), 136.7
(C-100), 171.4 (C-6), 171.8 (C-2); HRMS (ESI?) calcd for
C17H16N2O2Na: 303.1109 (M?Na)? found 303.1115.
Pharmacological evaluation
The compounds obtained have been submitted for in vivo
evaluation in the ASP of NINDS, Bethesda, USA (White
et al., 2002). The experiments were performed in male
albino Carworth Farms No. 1 mice (weighing 18–25 g).
The animals had free access to feed and water except
during the 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.’’ The test compounds were dissolved or sus-
pended in 0.5 % (v/v) aqueous solution of methylcellulose.
(3S,5R)- and (3S,5S)-3,5-diphenylpiperazine-2,6-dione
(3S,5S)-3e and (3S,5R)-3e
From diastereomeric mixture of (2S,1S)-2e and (2S,1R)-2e
(1.43 g, 4.80 mmol) and NaOH (0.19 g, 1 equiv.); FC
(gradient: PE/EtOAc 6:1–2:1): yield 0.94 g (74 %): 0.52 g
(41 %) of (3S,5S)-3e, 0.42 g (33 %) of (3S,5R)-3e.
(3S,5S)-3e: white powder; mp 126–129 °C; TLC (PE/
AcOEt 3:1): Rf = 0.17; [a]D =?5.5 (c 0.887, CHCl3); IR
(KBr): 700, 744, 1240, 1454, 1695, 2855, 2922, 3070,
The MES test
In the MES test, an electrical stimulus of 0.2 s in duration
(50 mA in mice) was delivered via corneal electrodes
primed with an electrolyte solution containing an anes-
thetic agent. Mice were tested at least two different time
points (15 min, 30 min, 1 h or 4 h) following intraperito-
neal administration of 30, 100, and 300 mg/kg of test
compound. Abolition of the hindlimb tonic extensor com-
ponent indicated the test compound’s ability to inhibit
MES-induced seizure spread (White et al., 2002).
1
3204, 3312; H NMR (CDCl3, 500 MHz): d 2.48 (bs, 1H,
NH), 4.76 (s, 2H, H-3, H-5), 7.36–7.44 (m, 10H, H–Ar),
8.22 (bs, 1H, CONHCO); 13C NMR (CDCl3, 125 MHz): d
59.5 (C-3, C-5), 127.7 (C-20, C-60, C-200, C-600), 128.8 (C-40,
C-400), 129.1 (C-30, C-50, C-300, C-500), 135.2 (C-10, C-100),
171.5 (C-2, C-6); HRMS (ESI-) calcd for C16H13N2O2
265.0977 (M-H)- found 265.0982. (3S,5R)-3e: white
powder; mp 172–174 °C; TLC (PE/AcOEt 3:1): Rf = 0.10;
[a]D = 0 (c 0.733, CHCl3); IR (KBr): 698, 737, 1219,
1263, 1454, 1709, 3034, 3065, 3103, 3223, 3317; 1H NMR
(CDCl3, 500 MHz): d 2.22 (bs, 1H, NH), 4.75 (s, 2H, H-3,
H-5), 7.35–7.44 (m, 6H, H–Ar), 7.45–7.49 (m, 4H, H–Ar),
8.08 (bs, 1H, CONHCO); 13C NMR (CDCl3, 125 MHz): d
65.1 (C-3, C-5), 128.7 (C-20, C-60, C-200, C-600), 128.8 (C-30,
C-50, C-300, C-500), 129.0 (C-40, C-400), 135.9 (C-10, C-100),
171.2 (C-2, C-6); HRMS (ESI-) calcd for C16H13N2O2
265.0977 (M-H)- found 265.0976.
The scMET seizure test
The test utilized a dose of metrazole (pentylenetetrazole,
85 mg/kg in mice). This produced clonic seizures lasting for
a period of at least 5 s in 97 % (CD97) of animals tested. At
the anticipated time of testing, the convulsant was admin-
istered subcutaneously. The test compound was adminis-
tered intraperitoneally in mice and the animals were
observed over a 30 min period. Mice were tested at least
two different time points (15 min, 30 min, 1 h or 4 h) fol-
lowing intraperitoneal administration of 30, 100, and
300 mg/kg of test compound. The absence of clonic spasms
indicated a compound’s ability to abolish the effect of
pentylenetetrazol on seizure threshold (White et al., 2002).
(?/-)-4-Benzyl-3-phenylpiperazine-2,6-dione rac-3f
From rac-2f (0.32 g, 1.03 mmol) and NaOH (0.04 g,
1 equiv.); FC (gradient: PE/EtOAc 3:1–1:1): yield 0.28 g
123

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The acute neurological impairment (TOX)
Neurological toxicity induced by a compound was detected
in mice or rats using the standardized rotorod test (Dunham
and Miya, 1957). Mice were tested at a minimum of two
different time points (15 min, 30 min, 1 h or 4 h) follow-
ing intraperitoneal administration of 100 mg/kg of test
compound. Rats were tested at time intervals between 0.25
and 4 h following an oral or intraperitoneal dose of 30 mg/
kg. Neurological impairment was demonstrated by the
inability of animals to maintain equilibrium on a 6 rpm
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The minimal clonic seizure test (6 Hz)
The 6 Hz screen was carried out according to the protocol
originally described by Brown et al. (1953) and more
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It is an alternative electroshock paradigm that uses low-
frequency (6 Hz), long-duration (3 s) electrical stimulation.
Mice were tested at time intervals between 0.25 and 4 h
following intraperitoneal doses of 100 mg/kg of test com-
pound. Corneal stimulation (0.2 ms-duration monopolar
rectangular pulses at 6-Hz for 3 s) was delivered by a
constant-current device. During the stimulation, mice were
manually restrained and released into the observation cage
immediately after the current application. The seizures
manifested in ‘‘stunned’’ posture associated with rearing,
forelimb, automatic movements and clonus, twitching of
the vibrissae and Straub-tail. The duration of the seizure
activity ranged from 60 to 120 s in untreated animals. At the
end of the seizure, animals resumed their normal explor-
atory behavior. The experimental end point was protection
against the seizure. The animal was considered to be pro-
tected if it resumed its normal exploratory behavior within
10 s from the stimulation (Kaminski et al., 2004).
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Acknowledgments We would like to thank the Anticonvulsant
Screening Program (National Institute of Neurological Disorders and
Stroke, NINDS Rockville, MD, USA) staff for providing the phar-
macological data. We would also like to thank Carolyn Foster for her
assistance in preparation of the manuscript. Financial support was
provided by the Polish Ministry of Science and Higher Education
(Grant No. N N405 623138).
Perucca E, French J, Bialer
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antiepileptic drugs: challenges, incentives, and recent advances.
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the development pipeline. Epilepsy Res 69:273–294
Smith M, Wilcox KS, White HS (2007) Discovery of antiepileptic
drugs. Neurotherapeutics 4:12–17
Open Access This article is distributed under the terms of the
Creative Commons Attribution License which permits any use, dis-
tribution, and reproduction in any medium, provided the original
author(s) and the source are credited.
White HS, Woodhead JH, Wilcox KS, Stables JP, Kupferberg HJ,
Wolf HH (2002) General principles: discovery and preclinical
development of antiepileptic drugs. In: Levy RH, Mattson RH,
Meldrum BS, Perucca E (eds) Antiepileptic drugs, 5th edn.
Lippincott Williams and Wilkins Publishers, New York, pp 6–48
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