Synthesis of new perhydropyrrolo[1,2-A]pyrazine derivatives and their evaluation in animal models of epilepsy

Article abstract of DOI:10.3390/molecules191015955

A series of novel stereochemically pure derivatives of the investigative broad-spectrum anticonvulsant ADD408003 was designed and synthesized. Five-center four-component (U-5C-4CR) and four-center three-component (U-4C-3CR) variants of Ugi reaction were used in the key step of the synthetic pathways. The compounds obtained were evaluated for the anticonvulsant activitiy in the maximal electroshock seizure (MES), subcutaneous Metrazole (scMET) and minimal clonic seizure (6 Hz) animal models of epilepsy. The efficacies of most derivatives in the 6 Hz model of pharmacoresistant partial seizures were markedly higher than in the 'classical' MES and scMET models. The most active compounds, (4R,8aR)-3a, and (4S,8aS)-6 displayed median effective doses (ED50) of 47.90 and 126.19 mg/kg, respectively, for the 6 Hz test.

Full text of DOI:10.3390/molecules191015955

Molecules 2014, 19, 15955-15981; doi:10.3390/molecules191015955
OPEN ACCESS
molecules
ISSN 1420-3049
www.mdpi.com/journal/molecules
Article
Synthesis of New Perhydropyrrolo[1,2-a]pyrazine Derivatives
and Their Evaluation in Animal Models of Epilepsy
Maciej Dawidowski 1,*, Wojciech Lewandowski 2 and Jadwiga Turło 1
1
Department of Drug Technology and Pharmaceutical Biotechnology, Medical University of
Warsaw, Banacha 1 Str., 02-097 Warszawa, Poland; E-Mail: jadwiga.turlo@wum.edu.pl
2
Faculty of Pharmacy, Medical University of Warsaw, Banacha 1 Str., 02-097 Warszawa, Poland;
E-Mail: lewandowski.w@o2.pl
* Author to whom correspondence should be addressed; E-Mail: maciej.dawidowski@wum.edu.pl;
Tel.: +48-22-572-0646; Fax: +48-22-572-0643.
External Editor: Derek J. McPhee
Received: 20 August 2014; in revised form: 24 September 2014 / Accepted: 26 September 2014 /
Published: 7 October 2014
Abstract: A series of novel stereochemically pure derivatives of the investigative
broad-spectrum anticonvulsant ADD408003 was designed and synthesized. Five-center
four-component (U-5C-4CR) and four-center three-component (U-4C-3CR) variants of
Ugi reaction were used in the key step of the synthetic pathways. The compounds obtained
were evaluated for the anticonvulsant activitiy in the maximal electroshock seizure (MES),
subcutaneous Metrazole (scMET) and minimal clonic seizure (6 Hz) animal models of
epilepsy. The efficacies of most derivatives in the 6 Hz model of pharmacoresistant partial
seizures were markedly higher than in the ‘classical’ MES and scMET models. The most
active compounds, (4R,8aR)-3a, and (4S,8aS)-6 displayed median effective doses (ED50)
of 47.90 and 126.19 mg/kg, respectively, for the 6 Hz test.
Keywords: 2,6-diketopiperazine; pyrrolo[1,2-a]pyrazine; multicomponent reaction; Ugi
reaction; U-5C-4CR; U-4C-3CR; anticonvulsant agent; antiseizure agent; antiepileptic agent

Molecules 2014, 19
15956
1. Introduction
According to epidemiological studies, epilepsy affects approximately 1% of the world’s population [1].
Although significant advances have been achieved in pharmacotherapy of this disorder, currently
available anticonvulsant drugs (AEDs) produce satisfactory seizure control only in 60%–70% of
patients. Moreover, their usage is often associated with disturbing side-effects which seriously limit
quality of patients’ life. Thus, there is a substantial need for further development of novel, safer and
more efficient AEDs [2–6].
In our recent reports, we described a structurally novel class of investigative anticonvulsants,
derivatives of perhydropyrrolo[1,2-a]pyrazines [7]. Among them, compound ADD408003 (Figure 1)
revealed high and broad activity in various preclinical animal models of epilepsy, including the models
of pharmacoresistant epilepsy. A preliminary structure–activity relationship (SAR) study revealed that
several structural patterns are necessary for the high anticonvulsant activity: the (S,S) absolute
configuration on the stereogenic centers, the presence of the annulated pyrrolidine ring, the presence of
imide moiety and the benzene ring in C-4 position of the pyrrolo[1,2-a]pyrazine core.
Figure 1. Structure of ADD408003.
f used aliphatic ring
ADD408003
H
8a
O
N
ED50 (6 Hz)= 9.93 mg/kg
ED50 (MES) = 39.7 mg/kg
4
NH
aromatic site
H
O
imide moiety
To complement our previous extensive SAR investigations in this group of active compounds [7–10],
we synthesized and pharmalogically evaluated a new series of ADD408003 derivatives. Since the
mechanism of action of the parent molecule remains unknown, the new compounds have been
designed according to diverse classical medicinal chemistry methods. First, we decided to examine if
increasing the distance of the benzene ring from the imide moiety by methylene insertion would
influence the anticonvulsant activity of ADD408003. Next, we asked whether replacing the hydrogen
atom at C-4 of the parent compound with a non-epimerizable substituent would prevent the putative
metabolic conversion to the inactive (4R,8aS) isomer, thus prolonging the anticonvulsant activity.
Further, we incorporated alkyl residues in the C-4 position. Finally, based on the common structural
motifs and the stereochemistry of ADD408003 and the potent broad-spectrum AED Levetiracetam, we
have designed a hybrid of both molecules (Figure 2).
Since our previous studies on the stereochemistry-activity relationship of structurally related
pyrido[1,2-a]pyrazines revealed interesting activity of (R,R) stereoisomers, we now focused on
synthesizing both (R,R) and (S,S) enantiomers of the new ADD408003 derivatives [8].

Molecules 2014, 19
15957
Figure 2. Hybrid compound of Levetiracetam and ADD408003.
H
S
O
O
N
S
Levetiracetam
N
S
NH2
NH
Ph
H
H
O
O
Me
S
O
O
N
S
NH
Ph
H
O
2. Results and Discussion
2.1. Chemistry
Target compounds 3a–g of the desired absolute configurations were obtained using the Ugi
five-center four-component reaction (U-5C-4CR [11]) → amide N-detertbutylation → intramolecular
cyclocondensation sequence [12], as shown on Scheme 1. For clarity, only the routes to (4S,8aS)-3
and (8aS)-3 isomers are shown and discussed. The respective opposite mirror images (4R,8aR)-3 and
(8aR)-3 were synthesized by the same sequence, using (R)-amino acids as starting materials.
The appropriate (S)-amino acids were condensed with aldehydes or aliphatic ketones, tert-butyl
isocyanide and methanol in the presence of catalytic amount of FeCl3 or TiCl4 to give Ugi adducts 1a–g
with chemical yields ranging from 17% to 64% (Table 1). When aldehydes or unsymmetrical ketones
were employed as the starting materials, the reactions proceeded with the formation of the new
stereocenters at carbons C-1 of the products 1a–d. In cases of adducts 1a–c the diastereoinduction was
in favor of the (2S,1S) configurations, whereas equal amounts of (2S,1S)-1d and (2S,1R)-1d isomers
were obtained for benzylmethyl ketone as the carbonyl component. In all cases the isomeric mixtures
could be efficiently resolved by flash column chromatography on silica.
In the subsequent step, (2S,1S)-1a–g were subjected to N-detertbutylation mediated by
BF3•2CH3COOH complex. Amido esters (2S,1S)-2a–c were obtained in good yields (43%–62%) and
without loss of the stereochemical purity. Interestingly, in the reaction of Ugi adducts derived from
ketones (2S,1S)-1d and (2S)-1e–g, the respective cyclized products (4S,8aS)-3d, (8aS)-3f and (8aS)-3g
were formed in considerable (24%–70%) amounts. The amido ester (2S)-2e could not be isolated from
the postreaction mixture due to the complete acid mediated deprotection/cyclisation of (2S)-1e to (8aS)-3e.
In the last step of the synthesis, amido esters 2 were converted to their cyclic derivatives 3 upon
treatment with 1 eq. of NaOH in EtOH, with chemical yields ranging from 55% to 95%. The
cyclocondensation of (2S,1S)-2a–c was accompanied by a slight degree of epimerization at carbon
C-4. Pure diastereomers (4S,8aS)-3a–c were obtained by recrystallization.

Molecules 2014, 19
15958
Scheme 1. Synthesis of compounds 1–3a–g.
n
n
n
H
H
H
8a
n
i
O
ii
H
2
2
N
4
N
1
N
1
COOMe
COOMe
+
N
COOH
NH
H
R1
R1
R1
CONHtBu
R2
CONH2
R2
R2
O
(2 ,1 )-2a-g
(2S,1S)-1a-g
S
S
(4S,8aS)-3a-g
iii
H
H
H
H
H
8a
8a
9a
8a
O
O
O
O
O
N
4
N
N
N
N
4
4
4
NH
NH
Ph
NH
Ph
NH
Ph
NH
Ph
Ph
H
H
H
H
Me
O
O
O
O
O
(4S,8aS)-3b
(4S,9aS)-3c
(4S,8aS)-3d
(4R,8aR)-3a
ADD408003
H
8a
H
8a
H
8a
O
O
O
N
N
N
Ph
NH
NH
NH
Me
Me
O
O
Ph
O
(8aS)-3f
(8aS)-3g
(8aS)-3e
Reagents and conditions: i. (a) carbonyl component, tBuNC, MeOH, FeCl3(cat.) (or TiCl4(cat.) for 1d–
g), MeOH, rt, 24 h (or 72 h for 1d–g); (b) Chromatographic separation (1a–d); ii. BF3•2CH3COOH,
40–50 °C, 4–12 h; iii. NaOH, EtOH, rt, 10 min.
Table 1. Synthesis of compounds 1–3a–g.
U-5C-4CR
N-de-tert-butylation
Cyclocondensation
Ugi adduct Amino acid Carbonyl component Yield Dr a Yield 1→2 Yield 1→3 Yield 1→3
c
(2S,2S)-1a L-Proline
(2S,2S)-1b L-Proline
Benzaldehyde
Phenylacetaldehyde 54% b 4:1 61%
64% b 4:1 62%
-
65% d
69% d
55% d
88% d
-
-
(2S,2S)-1c L-Pipecoline Phenylacetaldehyde 60% b 4:1 43%
(2S,2S)-1d L-Proline Phenylacetone
38% b 1:1 46%
1,3-Diphenylacetone 17%
-
24%
46%
36%
70%
(2S)-1e
(2S)-1f
L-Proline
L-Proline
L-Proline
-
-
-
-
Acetone
61%
60%
33%
26%
95%
-
(2S)-1g
Cyclopentanone
a
1
Dr of (2S,1S)-1/(2S,1R)-1 astimated by H analyses of the crude postreaction mixtures (1a–c) or LC/MS
(1d); b Sum of (2S,1S)-1 and (2S,1R)-1 diastereoisomers; c Not applicable; d Yield of pure diastereoisomers.
It is well known that the U-5C-4CR reaction is highly versatile with regard to the substrate
scope [11–17]. In this work and in our previous investigations, we have successfully coupled cyclic
amino acids with aliphatic ketones [13]. However, to the best of our knowledge, aromatic ketones have
not been employed as carbonyl components in this process. We were particularly interested in
L-Proline and acetophenone adduct, as it could lead to the C-4 methylated ADD408003 derivative
(4S,8aS)-3h. Propitiously, we observed by LC/MS that the U-5C-4CR product 1h was formed after

Molecules 2014, 19
15959
1 day, at room temperature, with the use of a TiCl4 catalyst. The diastereoinduction slightly in favored
(2S,1S)-1h (dr = 2:1, LC/MS). Unfortunately, attempts to optimize this particular reaction led to only a
13% yield after 7 days of stirring at room temperature.
Nevertheless, enough (4S,8aS)-3h was obtained for initial evaluation in animal models of epilepsy,
following the synthetic route described for 3a–g (Scheme 2 and Figure 3). Similar to what had been
observed for 1d–g cyclic imides 3h were formed upon treatment of the respective diastereomers of 1h
with BF3•2CH3COOH complex. Interestingly, cyclocondensation of (2S,1S)-1h diastereomer to
(4S,8aS)-3h was more facile than in the case of its epimer (2S,1R)-1h.
Scheme 2. Synthesis of compounds 1–3h.
H
H
H
8a
O
ii
2
2
N
4
N
N
COOMe
COOMe
+
1
1
NH
Ph
Ph
Ph
CONHtBu
CONH2
Me
Me
Me
O
i
H
(2S,1R)-2h, 60%
(4R,8aS)-3h, 20%
(2S,1R)-1h, 4%
N
COOH
H
H
H
H
O
ii
N
N
N
COOMe
CONH2
+
NH
Ph
Ph
Ph
CONHtBu
CONH2
Me
Me
Me
O
(2 ,1 )-1h
, 8%
(2S,1S)-2h, 26%
(4S,8aS)-3h, 60%
S
S
iii
90%
Reagents and conditions: i. (a) PhCOCH3, tBuNC, MeOH, TiCl4(cat.), MeOH, rt, 7 days; (b)
Chromatographic separation; ii. BF3•2CH3COOH, 40–50 °C, 4–12 h; iii. NaOH, EtOH, rt, 10 min.
Figure 3. Selected nuclear Overhauser effect (nOe) correlations in (4R,8aS)-3h (left) and
(4S,8aS)-3h (middle, right).
5.2%
4.6%
1.7%
H
H
H
O
O
O
N
N
N
6.5%
3.8%
5.2%
NH
NH
NH
Ph
Ph
Ph
Me
Me
Me
O
O
O
3.2%
Encouraged by the results of U-5C-4CR of acetophenone, L-proline, tert-butyl isocyanide and
MeOH, we tested if the biphenyl analog of ADD408003 could be obtained in the same manner as 3h
(Scheme 3). However, the more bulky benzophenone failed to react and no traces of Ugi adduct were
detected by LC/MS of the crude postcondensation mixture. On the other hand, when tert-butyl
isocyanide was replaced by the linear n-butyl isocyanide, we observed formation of the product (2S)-1i
in a modest 2% yield. This was in agreement with our previous observations that steric factors played
the most important role for the outcome of U-5C-4CR of ketones [13]. As a further example of this

Molecules 2014, 19
15960
relationship, the yield of acetophenone adduct 1j incresed from 13% to 32%, when less bulky
n-butylisocyanide was used in lieu of the branched tert-butyl isomer. A loss of stereoselectivity was
observed at the same time.
Scheme 3. U-5C-4CR of L-proline aromatic ketones.
H
H
i
H
8a
O
2
N
COOMe
N
N
H
COOH
1
NH
Ph
Ph
CONHtBu
Ph
Ph
O
H
ii
H
N
2
COOMe
N
H
COOH
1
(2S)-1i, 2%
Ph
CONHnBu
Ph
iii
H
H
H
+
N
1
2
N
COOMe
N
H
COOMe
COOH
Ph
Ph
CONHnBu
CONHnBu
Me
Me
(2S,1S)-1j, 17%
(2S,1R)-1j, 15%
Reagents and conditions: i. Ph2CO, tBuNC, MeOH, TiCl4(cat.), MeOH, rt, 7 days; ii. Ph2CO, nBuNC,
MeOH, TiCl4(cat.), MeOH, rt, 7 days; iii. (a) PhCOCH3, nBuNC, MeOH, TiCl4(cat.), MeOH, rt, 7 days;
(b) Chromatographic separation.
The desired (4R,8aR) and (4S,8aS) diastereomers of 6, bearing oxygen atom at carbon C-6, were
synthesized using the intramolecular Ugi five-center four-component reaction (U-4C-3CR) of methyl
(S)-phenylglycinate, levulinic acid, tert-butyl isocyanide and MeOH as a key step of the synthetic
sequence depicted in Scheme 4 [18–20]. The uncatalyzed reaction proceeded with a high yield and the
resulting equimolar mixture of (2R,αS)-4 and (2S,αS)-4 was quantitatively separated by column
chromatography on silica. The subsequent treatment of the respective diastereomers of 4 with
BF3•2CH3COOH gave the dealkylated amido-esters 5 and cyclic imides 6. Similar to what had been
observed for dealkylation reactions of 1h, acid-mediated cyclocondensation was more facile in case of
the (2S,αS)-4 diastereomer. Finally, base-mediated cyclization of (2R,αS)-5 proceeded with a high
degree of epimerization on C-4 of the products, resulting in the chromatographically separable mixture
of (4S,8aR)-6 and the desired (4R,8aR)-6. No epimerization took place in the analogous
cyclocondensation reaction of (2S,αS)-5.

Molecules 2014, 19
15961
Scheme 4. Synthesis of compounds 6.
Me
Me
Me
8a
O
O
O
ii
O
2
2
N
N
4
N
CONHtBu
CONH2
+
α
α
NH
Ph
Ph
Ph
COOMe
COOMe
H
H
H
O
i
NH2 x HCl
(2R, S)-5
(4S,8aR)-6
(2R,αS)-4, 43%
α
, 58%
, 10%
Ph
COOMe
H
Me
Me
Me
O
O
O
Ph
O
ii
N
H
CONHtBu
N
H
CONH2
N
+
NH
Ph
Ph
COOMe
COOMe
H
O
(2S,αS)-5, 36%
(4S,8aS)-6, 55%
(2S,αS)-4, 47%
iii
87%
Me
Me
O
Me
O
O
Ph
O
iii
O
N
H
N
H
N
CONH2
+
NH
NH
Ph
Ph
COOMe
H
O
O
(4R,8aR)-6
, 52%
(2R,αS)-5
(4S,8aR)-6
, 35%
Reagents and conditions: i. (a) CH3COCH2CH2COOH, tBuNC, MeOH, MeOH, rt, 24 h; (b)
Chromatographic separation; ii. BF3•2CH3COOH, 40–50 °C, 4–12 h; iii. NaOH, EtOH, rt, 10 min.
The selected NOESY correlations have been shown for (4S,8aR)-6 and (4R,8aR)-6.
2.2. Anticonvulsant Evaluation
Compounds 3 and 6 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 the established decision schemes
and using protocols described in the Experimental Section of this article [21]. Primary anticonvulsant
studies involved two tests: maximal electroshock seizure (MES) and subcutaneous Metrazol (scMET),
in mice [22]. It is emphasized that nearly all clinically significant AEDs are protective in at least one
of these two models, thus making them very useful tools for initial high througput screening of
candidate anticonvulsants. The MES test employs an electrical stimulus to induce generalized
tonic-clonic seizures and identifies which compounds prevent the spread of seizures. The scMET
model utilizes chemically induced myoclonic seizures and recognizes the agents that act by raising the
seizure threshold. Selected compounds were subjected to evaluation of anticonvulsant activity in the
minimal clonic seizure (6 Hz) model of pharmacoresistant partial seizures [23–25]. Recently, it was
found that Levetiracetam, a novel broad-spectrum AED, is ineffective in most of the classic tests (e.g.,
MES and scMET) and that it suppresses seizures in the 6 Hz model at the same time. Therefore, an
expanded approach employed by the ASP is to utilize this test as a screening procedure to examine the
compounds that are inactive in conventional models, but which can possess novel activity spectra. In

Molecules 2014, 19
15962
addition to the primary anticonvulsant evaluation in the MES, scMET and 6Hz models, the acute
neurological impairment (TOX) was assessed in rotorod test [26]. The results are summarized in
Tables 2 and 3.
Table 2. Anticonvulsant activity and neurotoxicity of compounds in the MES and scMET
models following intraperitoneal (ip.) administration in mice.
Compound Dose
(mg/kg)
MES a
0.5 h
0/3
1/1
0/3
1/1
0/3
1/1
1/3
1/1
1/3
1/1
0/4
-
scMET b
0.5 h
0/1
TOX c
0.5 h
0/8
4/4
0/8
0/4
0/8
0/4
0/8
04
4.0 h
0/3
0/1
0/3
0/1
0/3
0/1
0/3
0/1
0/3
0/1
0/4
-
4.0 h
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
0/1
-
4.0 h
0/4
0/2
0/4
0/2
0/4
0/2
0/4
0/2
0/4
2/2 d
0/4
-
(4R,8aR)-3a 100
300
(4S,8aS)-3b 100
300
1/1
0/1
0/1
(4R,8aR)-3b 100
300
0/1
0/1
(4S,9aS)-3c 100
300
0/1
5/5
(4R,9aR)-3c 100
300
0/1
0/8
4/4 d,e
0/4
-
0/1
f
(4S,8aS)-3d 100
300
-
-
-
(4R,8aR)-3d 100
300
0/4
-
0/4
-
-
-
0/4
-
0/4
-
-
-
(8aS)-3e
(8aR)-3e
(8aS)-3f
(8aS)-3g
100
300
100
300
100
300
100
300
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
0/4
-
1/4 d
0/4
0/4
-
0/1
0/3
0/4
-
3/4 h
-
0/1
0/2
0/4
-
0/1
0/1
0/4
-
0/1
0/1
0/4
-
3/8
4/4 g
0/4
-
(4S,8aS)-3h 100
0/4
-
0/4
-
0/4
-
0/8
-
0/8
-
300
(4S,8aS)-6 100
300
3/4
-
3/4
-
0/4
-
0/8
-
0/8
-
0/8
-
(4R,8aR)-6 100
300
3/4
-
0/4
-
0/4
-
0/8
-
0/8
-
0/8
-
a
b
Maximal electroshock test (number of animals protected/number of animals tested); Subcutaneous
c
Metrazole test (number of animals protected/number of animals tested); Neurotoxicity test (number of
d
e
animals exhibiting neurological toxicity/number of animals tested); One animal died; Unable to grasp
rotorod; f Not determined; g Four animals died; h Active also in 1/4 at 0.25 h post administration.

Molecules 2014, 19
15963
Table 3. Anticonvulsant activity and neurotoxicity of compounds in the 6 Hz model
following intraperitoneal (ip.) administration in mice.
Compound Test a
0.25 h 0.5 h 1.0 h 2.0 h 4.0 h
(4R,8aR)-3a 6 Hz b 4/4
4/4
-
1/4
-
1/4
-
0/4
-
TOX c
-
(4S,9aS)-3c 6 Hz
1/4
0/4
3/4
0/4
1/4
0/4
3/4
0/4
0/4
0/4
0/4
0/4
-
2/4
0/4
1/4
0/4
1/4
0/4
2/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
1/4 e
0/4
0/4
2/2
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
-
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
3/3
1/4 f
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
-
TOX
(4R,9aR)-3c 6 Hz
TOX
(4S,8aS)-3d 6 Hz
TOX
(4R,8aR)-3d 6 Hz
TOX
(8aS)-3e
(8aR)-3e
(8aS)-3f
(8aR)-3f
(8aS)-3g
(8aR)-3g
6 Hz
TOX
6 Hz
TOX
6 Hz
TOX
-
-
-
6 Hz d 0/4
1/3
1/4 f
0/4
0/4
2/2
2/4 g
0/4
0/4
2/4
0/4
1/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
0/4
TOX d 2/4 e
6 Hz
TOX
0/4
0/4
6 Hz d 1/2
TOX d 4/4 e,g,h,i 2/4 g
(4S,8aS)-3h 6 Hz j
TOX j 0/4
1/4
3/4
0/4
2/4
0/4
(4S,8aS)-6
6 Hz
TOX
3/4
0/4
a
b
At dose 100 mg/kg; 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); d At dose
e
f
g
h
50 mg/kg;
Clonic seizures; One animal died;
Two animals died;
Unable to grasp rotorod;
i Tremors; j At dose 75 mg/kg.
In contrast to the parent compound ADD408003, its opposite enantiomer (4R,8aR)-3a displayed
only a weak activity in the ‘classical’ MES model (1/1 at 300 mg/kg, at 0.5 h). A considerable level of
neurotoxicity was observed at the same dose and timepoint. An increase in the distance of the benzene
ring from the imide moiety of ADD408003 and (4R,8aR)-3a by a methylene unit resulted in (4S,8aS)-3b
and (4R,8aR)-3b, respectively. This structural modification did not improve the activity in MES model,
when compared to parent molecules, although no signs of neurotoxicity were observed in the TOX test.
The piperidine homologs of (4S,8aS)-3b and (4R,8aR)-3b, (4S,9aS)-3c and (4R,9aR)-3c, respectively,
showed somewhat greater potency in the MES test (1/1 and 1/3 at 300 and 100 mg/kg, respectively, at
0.5 h). On the other hand, (4R,9aR)-3c isomer showed pronounced unwanted neurotoxic effect at
300 mg/kg. Replacing the hydrogen atom at C-4 with methyl group in (4S,8aS)-3b and (4R,8aR)-3b
resulted in inactive (4S,8aS)-3d and (4R,8aR)-3d, respectively. Dibenzyl analogs (8aS)-3e and (8aR)-

Molecules 2014, 19
15964
3e displayed no activity in MES model and no neurotoxicity in TOX test at a dose of 100 mg/kg. The
alkyl derivatives (8aS)-3f and (8aS)-3g were devoid of desired anticonvulsant activity and the former
produced severe neurotoxicity. The C-4 methylated analog of ADD408003, (4S,8aS)-3h, showed high
levels of seizure protection in MES model (1/4 and 3/4 at 0.25 and 0.5 h, respectively, at 100 mg/kg)
and lack of neurotoxic effects. However, the compound was not competitive when compared to the
parent molecule, and due to the low synthetic accessibility it was not scheduled for secondary
anticonvulsant evaluations. A comparable activity was observed for the oxo- derivatives (4S,8aS)-6
and (4R,8aR)-6.
The anticonvulsant activity of investigated compounds in the scMET model in mice was markedly
lower than in the MES screen. Only (4R,8aR)-3a and (4S,9aS)-3c showed considerable levels of
seizure protection (1/1 and 5/5, respectively, at 0.5 h) at dose of 300 mg/kg.
To conclude the above results, the investigated compounds showed only moderate anticonvulsant
activity in the ‘classical’ MES and scMET screening. Encouragingly, the levels of seizure protection in
the 6 Hz model of pharmacoresistant epilepsy were markedly higher (Table 3).
Interestingly, the C-4 phenylmethyl derivatives (4R,8aR)-3c and (4R,8aR)-3d showed a slightly
greater potency than their respective (4S,8aS) enantiomers in this test. Dibenzyl analogs (8aR)-3e and
(8aS)-3e were devoid of anticonvulsant activity and neurotoxicity, whereas the alkyl derivatives
(8aR)-3f and (8aS)-3f displayed severe motor impairment and proconvulsant effects.
Compounds (4R,8aR)-3a and (4S,8aS)-6 were most potent in the primary 6 Hz screening, at both
early and late time points. Therefore they were further subjected to in vivo quantification in this model
(Table 4). They displayed median effective dose (ED50) values of 47.90 mg/kg and 126.19 mg/kg,
respectively, at 0.25 h. Although the compounds proved less competitive than the parent ADD408003,
their potencies and protective indices were comparable to those of standard AEDs.
Table 4. Quantification studies of (4R,8aR)-3a and (4S,8aS)-6 in the 6 Hz and
neurotoxicity tests following intraperitoneal (ip.) administration in mice.
Compound
(4R,8aR)-3a
(4S,8aS)-6
1 d
ED50 6 Hz (mg/kg)
TD50 (mg/kg) a
PI b 6Hz TPE c (h)
47.90 (35.6–71.08)
277.09 (245.78–350.13)
5.8
2.1
12.2
-
0.25
0.25
0.25
-
126.19 (105.87–145.68) 262.22 (242.15–290.77)
9.93 (5.97–14.55)
121.5 (104.9–142.8)
41.0 (39.4–43.0)
69.0 (62.8–72.9)
341 (290–384)
>500
Phenytoin e
>60
Phenobarbital e 14.8 (8.9–23.9)
Ethosuximide e
167 (114–223)
Levetiracetam e 19.4 (9.90–36.0)
4.7
2.0
>25.8
0.5
0.25
1.0
a
b
Values in parentheses are 95% confidence intervals determined by probit analysis; Neurotoxicity;
c
d
e
Protective index (TD50/ED50 6 Hz); Time to peak effect for the 6Hz test; Data from Ref. [8]; Data from
Ref. [25]
3. Experimental Section
3.1. Chemistry
The NMR spectra were obtained on a Varian VNMRS 300 MHz (Agilent Technologies, Santa
Clara, CA, USA) or Varian Inova 500 MHz (Agilent Technologies, Santa Clara, CA, USA)

Molecules 2014, 19
15965
spectrometer at room temperature. Chemical shifts (δ) were expressed in parts per million (ppm)
relative to tetramethylsilane (TMS) or residual solvent peaks used as the internal references. The
following abbreviations were used to describe the signal patterns: s (singlet), d (doublet), t (triplet), q
(quartet), m (multiplet), p (pseudo-) and b (broad-). Coupling constants (J) were in hertz (Hz). The
FT-IR spectra (thin film on KBr pellets) were recorded on a FT-IR8300 instrument (Shimadzu
Corporation, Kyoto, Japan). High resolution mass spectra (HRMS) were obtained using a LCT-TOF
(Micromass) spectrometer (Waters Corporation, Milford, MA, USA) with electrospray ionization
(ESI). Optical rotations were measured with a Perkin-Elmer 241 polarimeter (Perkin-Elmer, Waltham,
MA, USA) using a sodium lamp (589 nm). Melting points were determined on an Electrothermal 9100
apparatus (Bibby-Scientific, Stone, UK) in open capillary tubes and were uncorrected. LC/MS
analyses were performed using a Shimadzu Nexera UHPLC system (Shimadzu Corporation, Kyoto,
Japan) with LCMS-2020 single quadrupole spectrometer (Shimadzu Corporation, Kyoto, Japan)
equipped with an ESI ion source. Supelcosil LC-18-DB column (length: 25 mm, internal diameter: 4.6
mm, particle size: 5 µm, (Supelco, Bellefonte, PA, USA) was used. The analyte concentration was
approximately 50 ng/mL, injection: 5 µL, flow rate 0.4 mL/min, temperature: 60 °C, mobile phase:
acetonitrile/water 60:40 (v/v). Thin-layer chromatography (TLC) was run on Merck silica gel (60-F254)
plates (Merck, Darmstadt, Germany). The spots were visualized by ultraviolet light (254 nm) or iodine
vapors. Flash column chromatography (FC) was carried out on silica gel 60 (particle size: 0.040-0.063
mm, Merck, Darmstadt, Germany). 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. Compounds (2S,1S)-1d-g and (2S,1R)-1d were synthesized as
previously described [13].
3.1.1. Synthesis of Compounds 1 by U-5C-4CR Condensation
FeCl3 (for 1a–c) or TiCl4 (for 1d–g) (5 mol%) and isocyanide (1.0 eq.) were added to a stirred
solution of appropriate α-amino acid (1.2 eq.) and carbonyl component (1.0 eq.) in MeOH (100 mL).
The mixture was stirred at rt for 24 h (72 h for 1d–g) and the volatiles were removed under reduced
pressure. The resulting crude products were purified by FC.
Methyl (2R,1R)- and (2R,1S)-1-(1-(tert-butylcarbamoyl)-1-phenylmethyl)-pyrrolidine-2-carboxylate
(2R,1R)-1a and (2R,1S)-1a. From D-proline (2.32 g, 20.16 mmol), benzaldehyde (1.78 mL, 16.80 mmol)
and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 8:1 to 2:1): yield 3.43 g (64%): 2.26 g
(42%) of (2R,1R)-1a, 0.45 g (8%) of (2R,1S)-1a and 0.72 g (14%) of diastereomeric mixture. (2R,1R)-1a:
Yellow oil; TLC (PE/AcOEt 3:1): Rf = 0.15; [α]D = +3.6 (c 0.546, CHCl3); IR (KBr): 759, 1171, 1215,
1
1362, 1454, 1520, 1673, 1736, 2842, 2967, 3295; H-NMR (CDCl3, 500 MHz): δ 1.41 (s, 9H,
2
3
3
C(CH3)3), 1.78–1.92 (m, 3H, H-3, H-4, H′-4), 2.02 (m, 1H, H′-3), 2.70 (pq, J = J1 = J2 =7.5, 1H,
3
3
H-5), 3.22 (m, 1H, H′-5), 3.30 (dd, J1 = 9.0, J2 = 4.0, 1H, H-2), 3.49 (s, 3H, OCH3), 4.09 (s, 1H,
13
H-α), 7.23–7.32 (m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′), 7.70 (bs, 1H, CONH); C-NMR (CDCl3, 125
MHz): δ 24.3 (C-4), 28.8 (C(CH3)3), 30.8 (C-3), 50.7 (C(CH3)3), 51.6 (OCH3), 54.4 (C-5), 61.6 (C-2),
74.0 (C-α), 128.2 (C-4′), 128.5 (C-2′, C-6′), 129.2 (C-3′, C-5′), 137.3 (C-1′), 171.0 (CONH), 175.9
(COOCH3); HRMS (ESI+) calcd for C18H26N2O3Na 341.1841 (M+Na)+ found 341.1845. (2R,1S)-1a:

Molecules 2014, 19
15966
Yellow oil; TLC (PE/AcOEt 3:1): Rf = 0.26; [α]D = +81.8 (c 0.672, CHCl3); IR (KBr): 758, 1164,
1
1203, 1363, 1453, 1513, 1678, 1739, 2856, 2964, 3317; H-NMR (CDCl3, 500 MHz): δ 1.35 (s, 9H,
C(CH3)3), 1.77 (m, 1H, H-4), 1.83 (m, 1H, H′-4), 1.91 (m, 1H, H-3), 2.10 (m, 1H, H′-3), 2.53 (m,
2J = 9.0, 3J1=3J2 = 7.5, 1H, H-5), 2.92 (4d, 2J = 9.0, 3J1 = 7.5, 3J2 = 4.5, 1H, H′-5), 3.55 (dd, 3J1 = 9.4,
3J2 = 3.0, 1H, H-2), 3.69 (s, 3H, OCH3), 4.20 (s, 1H, H-α), 7.26-7.35 (m, 6H, H-2′, H-3′, H-4′, H-5′,
H-6′, CONH); 13C-NMR (CDCl3, 125 MHz): δ 23.9 (C-4), 28.6 (C(CH3)3), 29.8 (C-3), 50.7 (C(CH3)3),
50.9 (C-5), 51.8 (OCH3), 63.7 (C-2), 73.2 (C-α), 128.0 (C-4′), 128.3 (C-2′, C-6′), 129.0
(C-3′, C-5′), 136.9 (C-1′), 170.6 (CONH), 175.3 (COOCH3); HRMS (ESI+) calcd for C18H26N2O3Na
341.1841 (M+Na)+ found 341.1850.
Methyl (2S,1S)- and (2S,1R)-1-(1-(tert-butylcarbamoyl)-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate
(2S,1S)-1b and (2S,1R)-1b. From L-proline (2.32 g, 20.16 mmol), phenylacetaldehyde (2.10 mL,
16.80 mmol) and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 7:1 to 2:1): yield 3.02 g
(54%): 2.01 g (36%) of (2S,1S)-1b, 0.39 g (7%) of (2S,1R)-1b and 0.62 (11%) of diastereomeric
mixture. (2S,1S)-1b: Yellow oil; TLC (PE/AcOEt 3:1): Rf = 0.25; [α]D = −26.2 (c 1, CHCl3); IR (KBr):
745, 1092, 1173, 1204, 1362, 1454, 1520, 1670, 1736, 2874, 2966, 3310; 1H-NMR (CDCl3, 500 MHz):
δ 1.26 (s, 9H, C(CH3)3), 1.76 (m, 1H, H-4), 1.81 (m, 1H, H′-4), 1.89 (m, 1H, H-3), 1.98 (m, 1H, H′-3),
2
3
2.88 (m, J = 14.0, J = 6.5, 1H, CH2), 2.91 (m, 1H, H-5), 3.18 (m, 1H, H′-5), 3.23 (dd,
2J = 14.0, 3J = 8.0, 1H, CH2), 3.48 (t, 3J = 7.5, 1H, H-2), 3.66 (m, 1H, H-1), 3.70 (s, 3H, OCH3), 6.52
(bs, 1H, CONH), 7.16–7.30 (m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′); 13C-NMR (CDCl3, 125 MHz): δ 23.6
(C-4), 28.6 (C(CH3)3), 30.7 (C-3), 36.5 (CH2), 50.7 (C-5), 51.9 (OCH3), 51.9 (C(CH3)3), 60.5 (C-2),
67.3 (C-1), 126.3 (C-4′), 128.4 (C-3′, C-5′), 129.0 (C-2′, C-6′), 138.8 (C-1′), 171.5 (CONH), 175.9
(COOCH3); HRMS (ESI+) calcd for C19H28N2O3Na: 355.1998 (M+Na)+ found 355.2004. (2S,1R)-1b:
White wax; M.p. 75–77 °C; TLC (PE/AcOEt 3:1): Rf = 0.31; [α]D = +9.0 (c 1, CHCl3); IR (KBr): 748,
1
1207, 1454, 1520, 1678, 1732, 2874, 2962, 3317; H-NMR (CDCl3, 500 MHz): δ 1.33 (s, 9H,
2
3
C(CH3)3), 1.72–1.81 (m, 4H, H-3, H′-3, H-4, H′-4), 2.89 (dd, J = 14.5, J = 9.5, 1H, CH2), 2.91 (m,
2
3
1H, H-5), 2.96 (m, 1H, H′-5), 3.25 (m, 1H, H-2), 3.38 (dd, J = 14.5, J = 5.0, 1H, CH2), 3.62 (dd,
3J1 = 9.5, 3J2 = 5.0, 1H, H-1), 3.66 (s, 3H, OCH3), 7.16-7.29 (m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′), 7.58
(bs, 1H, CONH); HRMS (ESI+) calcd for C19H28N2O3Na: 355.1998 (M+Na)+ found 355.1995.
Methyl (2R,1R)- and (2R,1S)-1-(1-(tert-butylcarbamoyl)-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate
(2R,1R)-1b and (2R,1S)-1b. From D-proline (2.32 g, 20.16 mmol), phenylacetaldehyde (2.10 mL,
16.80 mmol) and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 7:1 to 2:1): yield 2.53 g
(45%): 1.25 g (22%) of (2R,1R)-1b, 0.87 g (16%) of (2R,1S)-1b and 0.42 (7%) of diastereomeric
mixture. (2R,1R)-1b: Yellow oil; [α]D = +33.1 (c 1.3, CHCl3); HRMS (ESI+) calcd for C19H28N2O3Na:
355.1998 (M+Na)+ found 355.2008. (2R,1S)-1b: White wax; M.p. 65–70 °C; [α]D = −6.9 (c 1, CHCl3);
HRMS (ESI+) calcd for C19H28N2O3Na: 355.1998 (M+Na)+ found 355.1999.
Methyl (2S,1S)- and (2S,1R)-1-(1-(tert-butylcarbamoyl)-3-phenyl-1-ethyl)-piperidine-2-carboxylate
(2S,1S)-1c and (2S,1R)-1c. From L-pipecolinic acid (2.60 g, 20.16 mmol), phenylacetaldehyde
(2.10 mL, 16.80 mmol) and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 9:1 to 2:1):
yield 3.49 g (60%): 2.32 g (40%) of (2S,1S)-1c, 0.83 (14%) of (2S,1R)-1c and 0.34 g (6%) of

Molecules 2014, 19
15967
diastereomeric mixture. (2S,1S)-1c: Colorless oil; TLC (PE/AcOEt 3:1): Rf = 0.40; [α]D = −33.6 (c 1,
1
CHCl3); IR (KBr): 748, 1161, 1366, 1454, 1512, 1678, 1732, 2858, 2936; H-NMR (CDCl3,
500 MHz): δ 1.25 (s, 9H, C(CH3)3), 1.43 (m, 1H, H-4), 1.58 (m, 3H, H′-4, H-5, H′-5), 1.76 (m, 1H,
H-3), 1.83 (m, 1H, H′-3), 2.61 (m, 2J = 12.5, 3J = 5.0, 1H, H-6), 2.88 (dd, 2J = 13.0, 3J = 4.0, 1H, H-2),
3.13–3.22 (m, 2H, H′-6, CH2), 3.24 (dd, 2J = 8.5, 3J = 4.0, 1H, CH2′), 3.44 (dd, 3J1 = 7.5, 3J2 = 4.0, 1H,
H-1), 3.68 (s, 3H, OCH3), 5.95 (bs, 1H, CONH), 7.15–7.25 (m, 5H, H-2′, H-3′, H-4′, H-5′, H-6′); 13C-
NMR (CDCl3, 125 MHz): δ 22.2 (C-4), 25.8 (C-3), 28.6 (C(CH3)3), 29.8 (C-5), 36.2 (CH2), 45.9 (C-6),
51.0 (C(CH3)3), 51.7 (OCH3), 62.7 (C-2), 69.1 (C-1), 126.1 (C-4′), 128.2 (C-2′, C-6′), 129.3 (C-3′,
C-5′), 139.3 (C-1′), 169.9 (CONH), 174.3 (COOCH3); HRMS (ESI+) calcd for C20H30N2O3Na
369.2154 (M+Na)+ found 369.2158. (2S,1R)-1c: Colorless oil; TLC (PE/AcOEt 3:1): Rf = 0.50;
[α]D = −0.6 (c 1, CHCl3); IR (KBr): 740, 799, 1196, 1225, 1454, 1516, 1682, 1744, 2858, 2963, 3344;
1H-NMR (CDCl3, 500 MHz): δ 1.34 (s, 9H, C(CH3)3), 1.44 (m, 2H, H-4, H-5), 1.58 (m, 1H, H′-5),
1.70 (m, 2H, H′-4, H-6), 2.53 (m, 2J = 11.5, 3J1 = 10.0, 3J2 = 2.5, 1H, H′-3), 2.76 (m, 1H, H-CH2), 2.79
3
3
2
3
(m, 1H, H′-6), 3.14 (dd, J1 = 9.5, J2 = 4.0, 1H, H-2), 3.28 (dd, 1H, J = 14.5, J2 = 6.0, CH2′), 3.48
(dd, 3J1 = 12.0, 3J2 = 6.0, 1H, H-1), 3.63 (s, 3H, OCH3), 7.18 (tt, 3J = 7.0, 4J = 2.0, 1H, H-4′), 7.23 (tt,
3J = 7.0, J = 2.0, 2H, H-3′, H-5′), 7.27 (d, J = 7.0, 2H, H-2′, H-6′), 7.40 (bs, 1H, CONH); HRMS
(ESI+) calcd for C20H30N2O3Na 369.2154 (M+Na)+ found 369.2161.
4
3
Methyl (2R,1R)- and (2R,1S)-1-(1-(tert-butylcarbamoyl)-3-phenyl-1-ethyl)-piperidine-2-carboxylate
(2R,1R)-1c and (2R,1S)-1c. From D-pipecolinic acid (2.60 g, 20.16 mmol), phenylacetaldehyde
(2.10 mL, 16.80 mmol) and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 9:1 to 2:1):
yield 3.20 g (55%): 2.11 g (36%) of (2R,1R)-1c, and 1.09 (19%) of (2R,1S)-1c. (2R,1R)-1c: Colorless
oil; [α]D = +31.2 (c 0.720, CHCl3); HRMS (ESI+) calcd for C20H30N2O3Na 369.2154 (M+Na)+ found
369.2155. (2S,1R)-1c: Colorless oil; [α]D = +1.9 (c 0.880, CHCl3); HRMS (ESI+) calcd for
C20H30N2O3Na 369.2154 (M+Na)+ found 369.2159.
Methyl (2R,1S)- and (2R,1R)-1-(1-(tert-butylcarbamoyl)-1-methyl-3-phenyl-1-ethyl)-pyrrolidine-2-
carboxylate (2R,1S)-1d and (2R,1R)-1d. From D-proline (2.32 g, 20.16 mmol), phenyl-2-propanone
(2.24 mL, 16.80 mmol) and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 12:1 to 7:1):
yield 1.80 g (31%): 0.76 g (13%) of (2R,1S)-1d, 0.58 g (10%) of (2R,1R)-1d and 0.46 g (8%) of
diastereomeric mixture. (2R,1S)-1d: Pale-yellow wax; M.p. 61–68 °C; TLC (PE/AcOEt 5:1):
Rf = 0.37; [α]D = +30.1 (c 0.811, CHCl3); LC/MS: 347 [M+H]+, retention time: 17.2 min; HRMS
(ESI+) calcd for C20H30N2O3Na: 369.2154 (M+Na)+ found 369.2157. (2R,1R)-1d: Pale-yellow wax;
M.p. 35-42 °C; TLC (PE/AcOEt 5:1): Rf = 0.26; [α]D = +9.1 (c 0.920, CHCl3); LC/MS: 347 [M+H]+,
retention time: 20.0 min; HRMS (ESI+) calcd for C20H30N2O3Na: 369.2154 (M+Na)+ found 369.2162.
Methyl (2R)-1-(1-(tert-butylcarbamoyl)-1-benzyl-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate (2R)-1e.
From L-proline (2.32 g, 20.16 mmol), diphenyl-2-propanone (3.54 g, 16.80 mmol) and tert-butyl
isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 12:1 to 8:1): yield 1.63 g (23%). Yellow oil; TLC
(PE/AcOEt 3:1): Rf = 0.35; [α]D = +38.0 (c 1, CHCl3); HRMS (ESI+) calcd for C26H34N2O3Na:
445.2467 (M+Na)+ found 445.2474.

Molecules 2014, 19
15968
Methyl (2R)-1-(1-(tert-butylcarbamoyl)-1-methyl-1-ethyl)-pyrrolidine-2-carboxylate (2R)-1f. From
L-proline (2.32 g, 20.16 mmol), 2-propanone (1.24 mL, 16.80 mmol) and tert-butyl isocyanide
(2.00 mL, 16.80 mmol); FC (PE/AcOEt 9:1 to 3:1): yield 2.04 g (45%). White wax; M.p. 40–43 °C;
TLC (PE/AcOEt 3:1): Rf = 0.35; [α]D = +32.5 (c 0.950, CHCl3); HRMS (ESI+) calcd for
C14H26N2O3Na: 293.1841 (M+Na)+ found 293.1848.
Methyl (2R)-1-(1-(tert-butylcarbamoyl)-cyclopentyl)-pyrrolidine-2-carboxylate (2R)-1g. From L-proline
(2.32 g, 20.16 mmol), cyclopentanone (1.48 mL, 16.80 mmol) and tert-butyl isocyanide (2.00 mL,
16.80 mmol); FC (gradient: PE/AcOEt 10:1 to 3:1): yield 2.74 g (55%). Colorless oil; TLC (PE/AcOEt
5:1): Rf = 0.41; [α]D = +15.2 (c 0.513, CHCl3); HRMS (ESI+) calcd for C16H28N2O3Na: 319.1998
(M+Na)+ found 319.2004.
Methyl (2S,1S)- and (2S,1R)-1-(1-(tert-butylcarbamoyl)-1-methyl-1-phenylmethyl)-pyrrolidine-2-
carboxylate (2S,1S)-1h and (2S,1R)-1h. From L-proline (2.32 g, 20.16 mmol), acetophenone
(2.02 mL, 16.80 mmol) and tert-butyl isocyanide (2.00 mL, 16.80 mmol); FC (PE/AcOEt 10:1 to 5:1):
yield 0.72 g (14%): 0.40 g (8%) of (2S,1S)-1h, 0.20 (4%) of (2S,1R)-1h and 0.12 g (2%) of
diastereomeric mixture. (2S,1S)-1h: Pale-yellow powder; M.p. 109–111 °C; TLC (PE/EA 5:1):
Rf = 0.23; [α]D = −43.3 (c 0.800; CHCl3); IR (KBr): 760, 1169, 1203, 1447, 1502, 1679, 1738, 2872,
1
2963, 3300; H-NMR (CDCl3, 500 MHz): δ 1.40 (s, 9H, C(CH3)3), 1.68 (s, 3H, CH3), 1.71 (m, 1H,
2
3
3
H-4), 1.77 (m, 1H, H′-4), 1.87 (m, 2H, H-3, H′-3), 2.93 (pq, J = J1 = J2 =8.5, 1H, H-5), 3.11 (m,
3
3
3
3
2J = 9.5, J1 = 6.0, J2 = 4.0, 1H, H′-5), 3.35 (dd, J1 = 8.5, J2 = 3.0, 1H, H-2), 3.43 (s, 3H, OCH3),
7.22 (t, 3J = 7.5, 1H, H-4′), 7.28 (t, 3J = 7.5, 2H, H-3′, H-5′), 7.43 (d, 3J = 7.5, 2H, H-2′, H-6′), 7.91 (bs,
1H, CONH); 13C-NMR (CDCl3, 125 MHz): δ 18.1 (CH3), 25.4 (C-4), 28.9 (C(CH3)3), 31.9 (C-3), 49.6
(C-5), 50.9 (C(CH3)3), 51.7 (OCH3), 61.2 (C-2), 69.1 (C-1), 127.7 (C-2′, C-6′), 127.8 (C-4′), 128.3 (C-
3′, C-5′), 141.5 (C-1′), 173.4 (CONH), 177.0 (COOCH3); LC/MS: 333 [M+H]+, retention time: 14.0
min; HRMS (ESI+) calcd for C19H28N2O3Na: 355.1998 (M+Na)+ found 355.1996. (2S,1R)-1h: White
powder; M.p. 112–113 °C; TLC (PE/EA 5:1): Rf = 0.32; [α]D = −14.7 (c 0.600; CHCl3); IR (KBr): 758,
1
1167, 1203, 1447, 1503, 1679, 1741, 2871, 2964, 3351; H-NMR (CDCl3, 500 MHz): δ 1.21 (s, 9H,
C(CH3)3), 1.62 (s, 3H, CH3), 1.64–1.78 (m, 2H, H-4, H′-4), 1.89–2.00 (m, 2H, H-3, H′-3), 2.37 (m, 2J
= 3J1 = 9.5, 3J2 = 7.0, 1H, H-5), 2.99 (m, 2J = 9.5, 3J1 = 7.0, 3J2 = 3.0, 1H, H′-5), 3.73 (s, 3H, OCH3),
3
3
3
3
3.79 (dd, J1 = 9.0, J2 = 2.5, 1H, H-2), 7.22 (t, J = 7.5, 1H, H-4′), 7.31 (t, J = 7.5, 2H,
3
13
H-3′, H-5′), 7.42 (bs, 1H, CONH), 7.55 (d, J = 7.5, 2H, H-2′, H-6′); C-NMR (CDCl3, 125 MHz): δ
16.4 (CH3), 24.9 (C-4), 28.6 (C(CH3)3), 32.2 (C-3), 50.0 (C-5), 50.8 (C(CH3)3), 52.2 (OCH3), 61.7
(C-2), 70.5 (C-1), 127.3 (C-4′), 127.5 (C-2′, C-6′), 128.2 (C-3′, C-5′), 142.2 (C-1′), 173.4 (CONH),
177.2 (COOCH3); LC/MS: 333 [M+H]+, retention time: 17.0 min; HRMS (ESI+) calcd for
C19H28N2O3Na: 355.1998 (M+Na)+ found 355.1985.

Molecules 2014, 19
15969
3.1.2. Synthesis of Compounds 2 and 5 by BF3•2CH3COOH Mediated N-detertbutylation
The appropriate Ugi product 1 or 4 was dissolved in BF3•2CH3COOH (~36% BF3 basis, 3 mL per
1 mmol of substrate), and stirred at 40–50 °C until total consumption of the starting material (TLC),
typically for 4–12 h. The resulting solution was cooled, poured onto excess of crushed ice and made
alkaline with 25% aqueous solution of ammonia. The mixture was extracted with DCM (3 × 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 (2R,1R)-1-(1-carbamoyl-1-phenylmethyl)-pyrrolidine-2-carboxylate (2R,1R)-2a. From (2R,αR)-1a
(2.13 g, 6.70 mmol); FC (PE/AcOEt 1:1 to 1:2); Yield 1.21 g (69%). White powder; M.p. 155–157 °C;
TLC (AcOEt): Rf = 0.28; [α]D = −4.0 (c 0.612, CHCl3); HRMS (ESI+) calcd for C18H26N2O3Na
258.1210 (M+Na)+ found 258.1208.
Methyl (2S,1S)-1-(1-carbamoyl-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate (2S,1S)-2b. From (2S,αS)-1b
(1.91 g, 5.75 mmol); FC (PE/AcOEt 2:1 to 0:1, then EA/MeOH 95:5); Yield 1.02 g (61%). Yellow oil;
TLC (AcOEt): Rf = 0.36; [α]D = −35.5 (c 0.827, CHCl3); IR (KBr): 758, 1173, 1213, 1456, 1678, 1735,
1
2854, 2925, 2951, 3025, 3192; H-NMR (CDCl3, 500 MHz): δ 1.66–1.86 (m, 1H, H-3, H-4,
2
3
H′-4), 1.93 (m, 1H, H′-3), 2.84 (m, 1H, H-5), 2.93 (dd, J = 14.5, J = 7.5, 1H, CH2), 3.23 (m, 2H,
H′-5, CH2), 3.66 (t, 3J = 7.5, 1H, H-2), 3.70–3.72 (m, 4H, H-1, OCH3), 5.40 (bs, 1H, CONH), 6.97 (bs,
1H, CONH′), 7.20 (t, 3J = 7.5, 1H, H-4′), 7.23 (d, 3J = 7.5, 2H, H-2′, H-6′), 7.28 (t, 3J = 7.5, 2H, H-3′,
13
H-5′); C-NMR (CDCl3, 125 MHz): δ 24.0 (C-4), 30.9 (C-3), 36.9 (CH2), 52.3 (OCH3), 52.7 (C-5),
60.8 (C-2), 66.6 (C-1), 126.7 (C-4′), 128.8 (C-3′, C-5′), 129.1 (C-2′, C-6′), 138.7 (C-1′), 175.8 (CONH),
176.4 (COOCH3); HRMS (ESI+) calcd for C15H20N2O3Na: 299.1372 (M+Na)+ found 299.1401.
Methyl (2R,1R)-1-(1-carbamoyl-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate (2R,1R)-2b. From (2R,1R)-1b
(1.20 g, 3.61 mmol); FC (PE/AcOEt 2:1 to 0:1, then EA/MeOH 95:5); Yield 0.51 g (52%). Yellow oil;
[α]D = +40.8 (c 1.1; CHCl3); HRMS (ESI+) calcd for C15H20N2O3Na: 299.1372 (M+Na)+ found 299.1380.
Methyl (2S,1S)-1-(1-carbamoyl-3-phenyl-1-ethyl)-piperidine-2-carboxylate (2S,1S)-2c. From (2S,1S)-1c
(2.79 g, 8.06 mmol); FC (PE/AcOEt 3:1 to 1:1); Yield 1.00 g (43%). White wax; M.p. 80–84 °C; TLC
(AcOEt): Rf = 0.55; [α]D = −47.1 (c 0.670, CHCl3); IR (KBr): 698, 748, 1173, 1200, 1439, 1686, 1736,
1
2854, 2936, 3209, 3445; H-NMR (CDCl3, 500 MHz): δ 1.42 (m, 1H, H-4), 1.59 (m, 3H, H′-4, H-5,
2
3
H′-5), 1.76 (m, 1H, H-3), 1.84 (m, 1H, H-6), 2.55 (m, 1H, H′-3), 2.94 (dd, J = 13.5, J = 5.5, 1H,
2
3
3
3
H-β), 3.16 (m, 1H, H′-6), 3.19 (dd, J = 13.5, J = 8.5, 1H, H′-β), 3.41 (dd, J1 = 8.5, J2 = 5.5, 1H,
H-α), 3.48 (dd, 3J1 = 7.0, 3J2 = 4.0, 1H, H-2), 3.68 (s, 3H, OCH3), 5.77 (bs, 1H, CONH), 6.24 (bs, 1H,
CONH′), 7.18 (tt, 3J = 7.0, 4J = 1.5, 1H, H-4′), 7.23 (tt, 3J = 6.0, 4J = 1.5, 2H, H-3′, H-5′), 7.26 (tt, 3J = 6.0,
13
4J = 1.5, 2H, H-2′, H-6′); C-NMR (CDCl3, 125 MHz): δ 22.0 (C-4), 25.7 (C-3), 29.6 (C-5), 35.8
(C- β), 46.1 (C-6), 51.8 (OCH3), 62.7 (C-2), 68.2 (C-α), 126.2 (C-4′), 128.3 (C-3′, C-5′), 129.1 (C-2′,
C-6′), 173.4 (COOCH3), 174.2 (CONH2); HRMS (ESI+) calcd for C16H22N2O3Na 313.1528 (M+Na)+
found 313.1522.

Molecules 2014, 19
15970
Methyl (2R,1R)-1-(1-carbamoyl-3-phenyl-1-ethyl)-piperidine-2-carboxylate (2R,1R)-2c. From (2R,1R)-1c
(1.63 g, 4.71 mmol); FC (PE/AcOEt 3:1 to 1:1); Yield 0.74 g (54%). Pale-yellow wax; M.p.
79–81 °C; [α]D = −45.5 (c 0.540, CHCl3); HRMS (ESI+) calcd for C16H22N2O3Na 313.1528 (M+Na)+
found 313.1531.
Methyl (2S,1S)-1-(1-carbamoyl-1-methyl-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate (2S,1S)-2d and
(4S,8aS)-4-Methyl-4-benzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4S,8aS)-3d. From (2S,1S)-1d
(1.54 g, 4.45 mmol); FC (PE/AcOEt 4:1 to 0:1): Yield 70%: 0.60 g (46%) of (2S,1S)-2d and 0.28 g
(24%) of (4S,8aS)-3d. (2S,1S)-2d: White powder, M.p. 121–123 °C; TLC (AcOEt): Rf = 0.54;
[α]D = −12.3 (c 0.790, CHCl3); IR (KBr): 702, 1171, 1202, 1456, 1506, 1684, 1736, 2843, 2870, 2950,
1
3445; H-NMR (CDCl3, 500 MHz): δ 1.12 (s, 3H, CH3), 1.76 (m, 1H, H-4), 1.81–1.95 (m, 2H, H-3,
2
3
H′-4), 2.12 (m, 1H, H′-3), 2.70 (d, 2J = 12.5, 1H, CH2), 2.79 (m, J = J1 = 9.0, 3J2 = 6.0, 1H, H-5),
3
2.99-3.08 (m, 2H, H′-5, CH2), 3.72 (s, 3H, OCH3), 3.84 (dd, J1 = 9.5, 3J2 = 3.5, 1H, H-2), 7.16–7.28
13
(m, 5H, H-Ar), 7.34 (bs, 1H, CONH); C-NMR (CDCl3, 75 MHz): δ 14.3 (CH3), 24.7 (C-4), 32.0
(C-3), 45.0 (CH2), 48.9 (C-5), 52.1 (OCH3), 58.9 (C-2), 66.5 (C-1), 126.8 (C-4′), 128.0 (C-2′, C-6′),
130.2 (C-3′, C-5′), 136.4 (C-1′), 177.2 (CONH), 177.5 (COOCH3); HRMS (ESI+) calcd for
C16H22N2O3Na 313.1528 (M+Na)+ found 313.1525. (4S,8aS)-3d: White powder; M.p. 151–153 °C;
TLC (PE/AcOEt 3:1): Rf = 0.20; [α]D = −20.6 (c 0.775; CHCl3); IR (KBr): 702, 770, 1194, 1265, 1508,
1701, 2819, 2953, 2988, 3196; 1H-NMR (CDCl3, 500 MHz): δ 1.36 (s, 1H, CH3), 1.86 (m, 2H, H-7, H-
8), 2.36 (m, 2H, H′-7, H′-8), 2.58 (q, 2J = 3J1 = 3J2 = 8.5, 1H, H-6), 3.01 (d, 2J = 13.5, 1H, CH2), 3.11
(m, 1H, H′-6), 3.17 (d, 2J = 13.5, 1H, CH2’), 4.20 (dd, 3J1 = 8.5, 3J2 = 3.5, 1H, H-8a), 7.20 (m, 2H, H-
13
Ar), 7.33 (m, 3H, H-Ar), 8.00 (bs, 1H, NH); C-NMR (CDCl3, 125 MHz): δ 20.9 (CH3), 22.2 (C-7),
27.5 (C-8), 43.0 (CH2), 47.7 (C-6), 59.1 (C-8a), 63.0 (C-4), 127.1 (C-4′), 128.2 (C-2′,
C-6′), 130.4 (C-3′, C-5′), 135.3 (C-1′), 174.5 (C-3), 175.0 (C-1); HRMS (ESI+) calcd for
C15H18N2O2Na: 281.1266 (M+Na)+ found 281.1272.
Methyl (2R,1R)-1-(1-carbamoyl-1-methyl-3-phenyl-1-ethyl)-pyrrolidine-2-carboxylate (2R,1R)-2d and
(4R,8aR)-4-Methyl-4-benzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4R,8aR)-3d. From (2R,1R)-1d
(1.31 g, 3.79 mmol); FC (PE/AcOEt 4:1 to 0:1); Yield 65%: 0.48 g (44%) of (2R,1R)-2d and 0.21 g
(21%) of (4R,8aR)-3d. (2R,1R)-2d: Pale yellow, M.p. 118–120 °C; [α]D = +11.7 (c 0.880, CHCl3);
HRMS (ESI+) calcd for C16H22N2O3Na 313.1528 (M+Na)+ found 313.1524. (4R,8aR)-3d: Pale-yellow
powder; M.p. 150–152 °C; [α]D = +20.9 (c 0.942; CHCl3); HRMS (ESI+) calcd for C15H18N2O2Na:
281.1266 (M+Na)+ found 281.1267.
(8aS)-4,4-Dibenzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (8aS)-3e. From (2S)-1e (1.12 g,
2.65 mmol); FC (PE/AcOEt 10:1 to 3:1), then recrystallization from PE/Et2O; Yield 0.41 g (46%).
White powder, M.p. 121–122 °C; TLC (PE/AcOEt 3:1): Rf = 0.36; [α]D = −8.3 (c 0.775, CHCl3); IR
1
(KBr): 700, 748, 1218, 1267, 1354, 1452, 1504, 1697, 2831, 2926, 3028, 3207; H-NMR (500 MHz,
2
CDCl3): δ 1.76-1.92 (m, 3H, H-7, H′-7, H-8), 1.99 (m, 1H, H-8, H′-8), 3.11 (d, J = 15.0, 1H, CH2),
3
2
3.16–3.27 (m, 4H, H-6, H′-6, CH2, CH2′), 3.30 (t, J = 7.0, 1H, H-8a), 3.35 (d, J = 14.0, 1H, CH2′),
7.12–7.31 (m, 10H, H-Ar), 7.77 (bs, 1H, NH), 13C-NMR (CDCl3, 125 MHz): δ 21.7 (C-7), 26.3 (C-8),
41.0, 41.2 (CH2, CH2′), 45.2 (C-6), 58.8 (C-8a), 68.3 (C-4), 126.6, 127.3, 128.1, 128.6, 130.06, 130.11,

Molecules 2014, 19
15971
136.0, 136.5 (C-Ar), 172.1 (C-3), 174.9 (C-1); HRMS (ESI+) calcd for C21H22N2O2Na: 357.1579
(M+Na)+ found 357.1577.
(8aR)-4,4-Dibenzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (8aR)-3e. From (2R)-1e (1.30 g, 3.08 mmol);
FC (PE/AcOEt 10:1 to 3:1), then recrystallization from PE/Et2O; Yield: 0.35 g (34%). White powder,
M.p. 116–118 °C; [α]D = +7.7 (c 0.803, CHCl3); HRMS (ESI+) calcd for C21H22N2O2Na: 357.1579
(M+Na)+ found 357.1589.
Methyl (2S)-1-(1-carbamoyl-1-methyl-1-ethyl)-pyrrolidine-2-carboxylate (2S)-2f and (8aS)-4,4-
Dimethylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (8aS)-3f. From (2S)-1f (1.27 g, 4.70 mmol); FC
(PE/AcOEt 5:1 to 0:1, then AcOEt/MeOH 95/5); Yield 69%: 0.33 g (33%) of (2S)-2f and 0.31 g (36%)
of (8aS)-3f. (2S)-2f: White powder; M.p. 56–58 °C; TLC (AcOEt): Rf = 0.37; [α]D = −38.4
(c 0.906, CHCl3); IR (KBr): 1203, 1435, 1578, 1681, 1736, 2874, 2951, 3206; 1H-NMR (CDCl3,
500 MHz): δ 1.24 (s, 3H, CH3), 1.26 (s, 3H, CH3’), 1.74–1.92 (m, 3H, H-3, H-4, H′-4), 1.89 (m, 1H,
2
3
3
2
3
H′-3), 2.04 (m, 1H, H′-3), 2.72 (td, J = J1 = 9.0, J2 = 6.0, 1H, H-5), 3.02 (m, J = 8.5, J1 = 6.5,
3J2 = 3.0, 1H, H′-5), 3.61 (dd, 3J1 = 9.5, 3J2 = 3.0, 1H, H-2), 3.71 (s, 3H, OCH3), 5.39 (bs, 1H, CONH),
13
7.44 (bs, 1H, CONH′); C-NMR (CDCl3, 125 MHz): δ 20.0 (CH3), 23.5 (CH3’), 24.8 (C-4), 31.7
(C-3), 48.4 (C-5), 52.0 (OCH3), 59.4 (C-2), 62.2 (C-α), 177.4 (COOCH3), 179.8 (CONH2); HRMS
(ESI+) calcd for C10H18N2O3Na: 237.1215 (M+Na)+ found 237.1218. (8aS)-3f: White powder, M.p.
105–107 °C; TLC (PE/AcOEt 3:1): Rf = 0.10; [α]D = +15.1 (c 0.933, CHCl3); IR (KBr): 1157, 1261,
1315, 1454, 1701, 2820, 2920, 2982, 3163; 1H-NMR (500 MHz, CDCl3): δ 1.43 (s, 3H, CH3), 1.46 (s,
2
3
3
3H, CH3’), 1.86 (m, 2H, H-7, H′-7), 2.26 (m, 2H, H-8, H′-8), 2.56 (pq, J = J1 = 7.5, J2 = 8.5, 1H,
H-6), 3.01 (m, 2J = 8.5, 3J1 = 7.0, 3J2 = 5.5, 1H, H′-6), 3.89 (dd, 3J1 = 8.0, 3J2 = 4.0, 1H, H-8a), 7.78 (bs,
1H, NH), 13C-NMR (CDCl3, 125 MHz): δ 22.2, 23.8, 24.0 (C-7, 2×CH3), 27.5 (C-8), 47.7 (C-6), 59.6
(C-4), 60.0 (C-8a), 174.3 (C-3), 176.9 (C-1).
Methyl (2R)-1-(1-carbamoyl-1-methyl-1-ethyl)-pyrrolidine-2-carboxylate (2R)-1f and (8aR)-4,4-
Dimethylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (8aR)-3f. From (2R)-1f (2.66 g, 9.85 mmol); FC
(PE/AcOEt 5:1 to 0:1, then AcOEt/MeOH 95/5); Yield 75%: 0.31 g (14%) of (2R)-2f and 1.13 g (61%)
of (8aR)-3f. (2R)-1f: Pale-yellow powder; M.p. 53–56 °C; [α]D = +36.1 (c 1.085, CHCl3); HRMS
(ESI+) calcd for C10H18N2O3Na: 237.1215 (M+Na)+ found 237.1231. (8aR)-3f: White powder, M.p.
110–112 °C; [α]D = +14.2 (c 0.847, CHCl3).
Methyl
(2S)-1-(1-carbamoyl-1-cyclopentyl)-pyrrolidine-2-carboxylate
(2S)-2g
and
(8a’S)-
perhydrospiro[cyclopentane-1,4'-pyrrolo[1,2-a]pyrazine]-1′,3′-dione (8aS)-3g. From (2S)-1g (0.74 g,
2.50 mmol); FC (PE/AcOEt 3:1 to 0:1, then AcOEt/MeOH 95/5); Yield 96%: 0.16 g (26%) of (2S)-2g
and 0.36 g (70%) of (8aS)-3g. (2S)-2g: Brown oil; TLC (AcOEt): Rf = 0.43; [α]D = +3.8 (c 0.987,
CHCl3); IR (KBr): 1200, 1454, 1569, 1680, 1732, 2872, 2953, 3441; 1H-NMR (CDCl3, 500 MHz): δ
1.56–1.67 (m, 4H), 1.68–2.11 (m, 8H), 2.24 (m, 1H, H′-3), 2.67 (m, 2J = 3J1 = 9.0, 3J2 = 6.5, 1H, H-5),
2
3
3
3
3.05 (m, J = 8.5, J1 = 6.0, J2 = 2.5, 1H, H′-5), 3.65 (dd, J1 = 9.0, 3J2 = 3.0, 1H, H-2), 3.72 (s, 3H,
OCH3), 5.40 (bs, 1H, CONH), 7.56 (bs, 1H, CONH′); 13C-NMR (CDCl3, 125 MHz): δ 24.7, 25.0, 25.2,
31.3, 31.7, 35.9, 49.3 (C-5), 52.0 (OCH3), 60.0 (C-2), 72.9 (C-1), 177.5 (COOCH3), 179.9 (CONH2);
HRMS (ESI+) calcd for C12H20N2O3Na: 263.1372 (M+Na)+ found 263.1386. (8aS)-3g: White powder,

Molecules 2014, 19
15972
M.p. 84–85 °C; TLC (PE/AcOEt 3:1): Rf = 0.11; [α]D = +41.0 (c 0.550, CHCl3); IR (KBr): 1176, 1246,
1327, 1456, 1697, 2814, 2874, 2959, 3234; 1H-NMR (500 MHz, CDCl3):
1.69–197 (m, 8H, H-cPent), 2.05 (m, 1H, H-7′), 2.22 (m, 1H, H-8′), 2.34 (m, 2H, H′-7′, H′-8′), 2.45 (q,
δ
3
3
2
3
3
2J = J1 = J2 = 8.5, 1H, H-6′), 3.07 (m, J = 8.5 = J1 = 8.5, J2 = 4.0, 1H, H′-6′), 3.89 (dd,
3J1 = 8.5, J2 = 2.5, 1H, H-8a′), 7.90 (bs, 1H, NH), C-NMR (CDCl3, 125 MHz): δ 22.2 (C-7′), 24.6,
25.1 (2×C-cPent), 27.4 (C-8′), 33.8, 37.4 (2×C-cPent), 48.2 (C-6′), 60.6 (C-8a′), 69.6 (C-4′), 174.1
(C-3′), 176.3 (C-1′); HRMS (ESI-) calcd for C11H15N2O2: 207.1134 (M−H)− found 207.1130.
3
13
Methyl (2R)-1-(1-carbamoyl-1-cyclopentyl)-pyrrolidine-2-carboxylate
(2R)-2g
and
(8a’S)-
perhydrospiro[cyclopentane-1,4′-pyrrolo[1,2-a]pyrazine]-1′,3′-dione (8aR)-3g. From (2R)-1g (1.02 g,
3.45 mmol); FC (PE/AcOEt 3:1 to 0:1, then AcOEt/MeOH 95/5); Yield 81%: 0.25 g (30%) of (2R)-2g
and 0.37 g (51%) of (8aR)-3g. (2R)-2g: Brown oil; [α]D = −4.0 (c 0.833, CHCl3); HRMS (ESI+) calcd
for C12H20N2O3Na: 263.1372 (M+Na)+ found 263.1371. (8aR)-3g: Pale-yellow powder, M.p. 85–87 °C;
[α]D = −43.8 (c 0.807, CHCl3); HRMS (ESI-) calcd for C11H15N2O2: 207.1134 (M−H)− found 207.1127.
Methyl (2S,1S)-1-(1-carbamoyl-1-methyl-1-phenylmethyl)-pyrrolidine-2-carboxylate (2S,1S)-2h and
(4S,8aS)-4-Methyl-4-phenyl-perhydropyrrolo[1,2-a]pyrazine-1,3-dione (4S,8aS)-3h. From (2S,1S)-1h
(200 mg, 0.60 mmol); FC (PE/AcOEt 6:1 to 0:1); Yield 86%: 43 mg (26%) of (2S,1S)-2h and 88 mg
(60%) of (4S,8aS)-3h. (2S,1S)-2h: White powder; M.p. 152–154 °C; TLC (AcOEt): Rf = 0.35;
[α]D = +50.1 (c 0.600; CHCl3); IR (KBr): 695, 754, 1169, 1205, 1376, 1681, 1737, 2842, 2923, 3179,
1
3413; H-NMR (CDCl3, 500 MHz): δ 1.72 (s, 3H, CH3), 1.78 (m, 2H, H-4, H′-4), 1.87 (m, 2H, H-3,
2
3
3
3
H′-3), 2.97 (m, 1H, H-5), 3.20 (m, J = 9.5, J1 = 6.5, J2 = 4.0, 1H, H′-5), 3.39 (dd, J1 = 8.5,
3J2 = 3.5, 1H, H-2), 3.44 (s, 3H, OCH3), 5.71 (bs, 1H, CONH), 7.24 (t, J = 7.5, 1H, H-4′), 7.30 (t,
3
3J = 7.5, 2H, H-3′, H-5′), 7.49 (d, 3J = 7.5, 2H, H-2′, H-6′), 7.72 (bs, 1H, CONH′); 13C-NMR (CDCl3,
125 MHz): δ 18.5 (CH3), 25.4 (C-4), 31.8 (C-3), 49.8 (C-5), 51.9 (OCH3), 61.4 (C-2), 69.2 (C-α),
127.8 (C-2′,C-6′), 128.1 (C-4′), 128.4 (C-3′, C-5′), 141.5 (C-1′), 177.2 (CONH) 177.4 (COOCH3);
HRMS (ESI+) calcd for C15H20N2O3Na: 299.1372 (M+Na)+ found 299.1362. (4S,8aS)-3h: White
powder; M.p. 72–73 °C; TLC (PE/AcOEt 3:1): Rf = 0.38; [α]D = −166.1 (c 0.800; CHCl3); IR (KBr):
702, 764, 1249, 1693, 2814, 2987, 3221; 1H-NMR (CDCl3, 500 MHz): δ 1.65 (s, 3H, CH3), 1.84–2.00
(m, 2H, H-7, H′-7), 2.13–2.24 (m, 1H, H-8), 2.24–2.32 (m, 1H, H′-8), 2.62 (q, 2J = 3J1 = 3J2 = 8.5, 1H,
2
3
3
3
3
H-6), 3.30 (td, J = J1 = 8.5, J2 = 3.0, 1H, H′-6), 3.65 (dd, J1 = 8.0, J2 = 2.5, 1H, H-8a), 7.29 (t,
3J = 7.0, 1H, H-4′), 7.35 (t, J = 7.0, 2H, H-3′, H-5′), 7.46 (d, J = 7.0, 2H, H-2′, H-6′), 8.31 (bs, 1H,
NH); 13C-NMR (CDCl3, 125 MHz): δ 22.8 (CH3), 26.6 (C-7), 27.6 (C-8), 47.4 (C-6), 60.2 (C-8a), 66.5
(C-4), 125.7 (C-2′, C-6′), 128.4 (C-4′), 129.2 (C-3′, C-5′), 141.3 (C-1′), 174.5 (C-3), 175.0 (C-1);
HRMS (ESI+) calcd for C14H16N2O2Na: 267.1110 (M+Na)+ found 267.1118.
3
3
Methyl (2S,1R)-1-(1-carbamoyl-1-methyl-1-phenylmethyl)-pyrrolidine-2-carboxylate (2S,1R)-2h and
(4R,8aS)-4-Methyl-4-phenyl-perhydropyrrolo[1,2-a]pyrazine-1,3-dione (4R,8aS)-3h. From (2S,1R)-
1h (200 mg, 0.60 mmol); FC (PE/AcOEt 6:1 to 0:1); Yield 80%: 98 mg (60%) of (2S,1R)-2h and 28
mg (20%) of (4R,8aS)-3h. (2S,1R)-2h: White powder; M.p. 124–125 °C; TLC (AcOEt): Rf = 0.55;
1
[α]D = −43.6 (c 0.800; CHCl3); IR (KBr): 700, 1169, 1208, 1446, 1683, 1734, 2872, 2950, 3421; H-
NMR (CDCl3, 500 MHz): δ 1.62–1.68 (m, 5H, H-4, H′-4, CH3), 1.96 (m, 2H, H-3, H′-3), 2.38 (m,

Molecules 2014, 19
15973
2J = 3J1 = 9.5, 3J2 = 6.5, 1H, H-5), 3.02 (m, 2J = 9.5, 3J1 = 7.0, 3J2 = 3.0, 1H, H′-5), 3.71 (s, 3H, OCH3),
3
3
3
3.84 (dd, J1 = 8.5, J2 = 3.5, 1H, H-2), 5.41 (bs, 1H, CONH), 7.25 (t, J = 7.5, 1H, H-4′), 7.33 (t,
3J = 7.5, 2H, H-3′, H-5′), 7.51 (bs, 1H, CONH′), 7.56 (d, 3J = 7.5, 2H, H-2′, H-6′); 13C-NMR (CDCl3,
125 MHz): δ 18.4 (CH3), 24.9 (C-4), 32.0 (C-3), 50.0 (C-5), 52.4 (OCH3), 61.3 (C-2), 69.8 (C-α),
127.6 (C-4′), 127.8 (C-2′, C-6′), 128.4 (C-3′, C-5′), 141.2 (C-1′), 177.3 (CONH), 177.4 (COOCH3);
HRMS (ESI+) calcd for C15H20N2O3Na: 299.1372 (M+Na)+ found 299.1360. (4R,8aS)-3h: White
powder; M.p. 126–128 °C; TLC (PE/AcOEt 3:1): Rf = 0.22; [α]D = −80.0 (c 0.700; CHCl3); IR (KBr):
701, 758, 1234, 1705, 2830, 2977, 3217; 1H-NMR (CDCl3, 500 MHz): δ 1.70-1.90 (m, 5H, H-7, CH3),
2.22 (m, 2H, H-8), 2.40 (q, 2J = 3J1 =3J2 = 8.0, 1H, H-6), 2.49 (td, 2J = 3J1 = 8.0, 3J2 = 4.0, 1H, H′-6),
3
3
3
3.67 (t, J = 8.0, 1H, H-8a), 7.31 (t, J = 7.5, 1H, H-4′), 7.37 (t, J = 7.5, 2H, H-3′, H-5), 7.56 (d,
3J = 7.5, 2H, H-2′, H-6′), 7.96 (bs, 1H, NH); C-NMR (CDCl3, 125 MHz): δ 14.2 (CH3), 21.4 (C-7),
13
25.8 (C-8), 46.2 (C-6), 58.7 (C-8a), 65.7 (C-4), 127.4 (C-2′, C-6′), 128.3 (C-4′), 128.7 (C-3′, C-5′),
141.1 (C-1′), 172.5 (C-3), 176.1 (C-1); HRMS (ESI+) calcd for C14H16N2O2Na: 267.1110 (M+Na)+
found 267.1106.
Methyl (2S,αS)-α-(2-carbamoyl-2-methyl-5-oxopyrrolidin-1-yl)-α-phenylacetate (2S,αS)-5 and
(4S,8aS)-4-phenyl-8a-methylperhydropyrrolo[1,2-a]pyrazine-1,3,6-trione (4S,8aS)-6. From (2S,αS)-4
(2.65 g, 7.66 mmol); FC (PE/AcOEt 3:1 to 0:1, then AcOEt/MeOH 95/5); Yield 91%: 0.81 g (36%) of
(2S,αS)-5 and 1.08 g (55%) of (4S,8aS)-6. (2S,αS)-5: White powder; M.p. 174–175 °C; TLC (AcOEt):
Rf = 0.29; [α]D = +18.5 (c 0.700; CHCl3); IR (KBr): 723, 757, 1209, 1399, 1605, 1679, 1742, 3007,
1
3193, 3351; H-NMR (CDCl3, 500 MHz): δ 1.14 (s, 3H, CH3), 2.06 (m, 1H, H-3), 2.37-2.40 (m, 2H,
H′-3, H-4), 2.55 (1H, H′-4), 3.75 (s, 3H, OCH3), 5.20 (s, 1H, H-α), 5.39 (bs, 1H, CONH), 7.24 (bs, 1H,
13
CONH′), 7.36-7.40 (m, 3H, H-3′, H-4′, H-5′), 7.42-7.46 (m, 2H, H-2′, H-6′); C-NMR (CDCl3, 125
MHz): δ 23.3 (CH3), 28.6 (C-4), 35.4 (C-3), 53.2 (OCH3), 61.5 (C-2), 68.0 (C-α), 125.2 (C-2′,
C-6′), 129.4 (C-4′), 133.0 (C-1′), 171.5 (C-5), 176.4 (CONH2), 176.5 (COOCH3); HRMS (ESI+) calcd
for C15H18N2O4Na: 313.1164 (M+Na)+ found 313.1172. (4S,8aS)-6: White powder; M.p.
164–166 °C; TLC (AcOEt): Rf = 0.60; [α]D = +50.4 (c 0.800; CHCl3); IR (KBr): 758, 1188, 1290,
1
1341, 1710, 2839, 3104, 3193; H-NMR (CDCl3, 500 MHz): δ 1.16 (s, 3H, CH3), 2.17 (m, 1H, H-8),
2.48-2.58 (m, 2H, H-7, H′-8), 2.66 (m, 1H, H′-7), 6.29 (s, 1H. H-4), 7.32-7.40 (m, 5H, H-Ar), 8.54 (bs,
1H, NH); 13C-NMR (CDCl3, 125 MHz): δ 25.1 (CH3), 28.8 (C-8), 30.6 (C-7), 55.5 (C-8a), 62.4 (C-4),
127.0 (C-2′, C-6′), 128.6 (C-4′), 129.1 (C-3′, C-5′), 135.2 (C-1′), 167.8 (C-6), 173.8 (C-1), 174.1 (C-3);
HRMS (ESI-) calcd for C14H13N2O3: 257.0926 (M−H)− found 257.0916.
Methyl (2R,αS)-α-(2-carbamoyl-2-methyl-5-oxopyrrolidin-1-yl)-α-phenylacetate (2R,αS)-5 and
(4S,8aS)-4-phenyl-8a-methylperhydropyrrolo[1,2-a]pyrazine-1,3,6-trione (4S,8aR)-6. From (2R,αS)-4
(1.46 g, 4.22 mmol); FC (PE/AcOEt 3:1 to 0:1, then AcOEt/MeOH 95/5); Yield 65%: 0.71 g (58%) of
(2R,αS)-5 and 0.10 g (10%) of (4S,8aR)-6. (2R,αS)-5: White powder; M.p. 174–175 °C; TLC
(AcOEt): Rf = 0.25; [α]D = –124.5 (c 0.6; CHCl3); IR (KBr): 721, 754, 1268, 1401, 1605, 1692, 3007,
1
3193, 3369; H-NMR (CDCl3, 500 MHz): δ 1.33 (s, 3H, CH3), 2.00 (m, 1H, H-3), 2.42-2.61 (m, 2H,
H′-3, H-4, H′-4), 2.55 (1H, H′-4), 3.79 (s, 3H, OCH3), 4.77 (s, 1H, H-α), 5.72 (bs, 1H, CONH),
13
7.32–7.40 (m, 5H, H-Ar), 8.48 (bs, 1H, CONH′); C-NMR (CDCl3, 125 MHz): δ 24.0 (CH3), 29.2
C-4), 34.3 (C-3), 53.7 (OCH3), 60.6 (C-2), 68.3 (C-α), 128.4 (C-4′), 128.5 (C-2′, C-6′), 128.6 (C-3′, C-

Molecules 2014, 19
15974
5′), 136.2 (C-1′), 171.5 (C-5), 175.5 (CONH2), 176.5 (COOCH3); HRMS (ESI+) calcd for
C15H18N2O4Na 313.1164 (M+Na)+ found 313.1165. (4S,8aR)-6: White powder; M.p. 188–189 °C;
TLC (EA): Rf = 0.51; [α]D = +87.6 (c 0.600; CHCl3/MeOH 10:3); IR (KBr): 727, 764, 1187, 1262,
1
1306, 1692, 1713, 2828, 3065; H-NMR (CDCl3, 500 MHz): δ 1.60 (s, 3H, CH3), 2.27 (m, 1H, H-8),
2.46–2.57 (m, 3H, H-7, H′-7, H′-8), 5.20 (s, 1H. H-4), 7.31–7.40 (m, 5H, H-Ar), 8.01 (bs, 1H, NH);
13C-NMR (CDCl3, 125 MHz): δ 23.5 (CH3), 28.0 (C-8), 29.2 (C-7), 60.1 (C-8a), 63.0 (C-4), 126.9
(C-2′, C-6′), 128.9 (C-4′), 129.0 (C-3′, C-5′), 135.1 (C-1′), 169.3 (C-6), 171.8 (C-1), 172.3 (C-3);
HRMS (ESI-) calcd for C14H13N2O3: 257.0926 (M−H)− found 257.0917.
3.1.3. Synthesis of Compounds 3 and 6 by Base Induced Intramolecular Cyclocondensation
To a stirred solution of appropriate amidoester 2 or 5 in absolute EtOH (5 mL per 1 mmol of
amidoester), sodium hydroxide (1 eq.) 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 × 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.
(4R,8aR)-4-Phenylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4R,8aR)-3a. From (2R,1R)-2a (1.09 g,
4.16 mmol) and NaOH (0.17, 4.16 mmol); Recrystallization from H2O/MeOH; Yield 0.62 g (65%) of
(4R,8aR)-3a. White powder; M.p 115–117 °C; [α]D = +135.3 (c 0.823, CHCl3); HRMS (ESI-) calcd
for C13H13N2O2: 229.0971 (M−H)− found 229.0978.
(4S,8aS)-4-Benzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4S,8aS)-3b. From (2S,αS)-2b (1.02 g,
3.70 mmol) and NaOH (0.15 g, 3.70 mmol); FC (PE/AcOEt 4:1 to 2:1), then recrystallization form
H2O/MeOH; Yield 0.62 g (69%). White powder; M.p. 188–190 °C; TLC (PE/AcOEt 3:1): Rf = 0.20;
[α]D = −46.7 (c 0.689, CHCl3); IR (KBr): 700, 746, 1115, 1264, 1305, 1695, 2694, 2786, 2969;
1H-NMR (500 MHz, CDCl3): δ 1.78 (m, 2H, H-7, H-7′), 2.23 (m, 1H, H-8), 2.32 (m, 1H, H′-8), 2.55 (q,
2J = 3J1 = 3J2 = 8.5, 1H, H-6), 3.01 (m, 1H, H′-6), 3.07 (m, 2H, CH2), 3.84 (m, 2H, H-4, H-8a), 7.23 (d,
3
3J = 8.0, 2H, H-2′, H-6′), 7.26 (t, 3J = 7.5, 1H, H-4′), 7.31 (t, J = 7.5, 2H, H-3′, H-5′); 7.86 (bs, 1H,
NH); 13C-NMR (CDCl3, 125 MHz): δ 22.5 (C-7), 27.9 (C-8), 38.1 (CH2), 52.9 (C-6), 57.2 (C-8a), 63.0
(C-4), 127.2 (C-4′), 128.8 (C-3′, C-5′), 129.3 (C-2′, C-6′), 137.4 (C-1′), 173.4 (C-3), 174.2
(C-1); HRMS (ESI+) calcd for C14H16N2O2Na: 267.1109 (M+Na)+ found 267.1108.
(4R,8aR)-4-Benzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4R,8aR)-3b. From (2R,1R)-2b (0.51 g,
1.85 mmol) and NaOH (74 mg, 1.85 mmol); FC (PE/AcOEt 4:1 to 2:1), then recrystallization form
H2O/MeOH; Yield 0.29 g (62%). White powder; M.p. 189–191 °C; [α]D = +47.3 (c 0.713; CHCl3);
HRMS (ESI+) calcd for C14H16N2O2Na: 267.1109 (M+Na)+ found 267.1115.
(4S,9aS)-4-Benzylperhydropyrido[1,2-a]pyrazine-1,3-dione (4S,9aS)-3c. From (2S,1S)-2c (1.00 g,
3.45 mmol) and NaOH (0.14 mg, 3.45 mmol); FC (PE/AcOEt 4:1 to 2:1), then recrystallization form
H2O/MeOH; Yield 0.49 g (55%). White powder; M.p. 133 °C; TLC (PE/AcOEt 3:1): Rf = 0.29;
[α]D = −23.7 (c 0.667; CHCl3); IR (KBr): 698, 1111, 1246, 1698, 2806, 2947, 3429; 1H-NMR (CDCl3,

Molecules 2014, 19
15975
500 MHz): δ 1.45 (m, 1H, H-8), 1.52 (m, 1H, H′-8), 1.56 (m, 2H, H-7, H′-7), 1.83 (m, 1H, H-9), 2.11
(m, 1H, H′-9), 2.59 (m, 1H, H-6), 2.64 (m, 1H, H′-6), 2.98 (dd, 2J = 14.0, 3J = 6.5, 1H, H-α), 3.14 (dd,
3
3
3
3
2J = 14.0, J = 8.0, 1H, H′-α), 3.62 (dd, J1 = 8.0, J2 = 6.5, 1H, H-4), 3.72 (pt, J = 5.0, 1H, H-9a),
1
7.20-7.34 (m, 5H, H-Ar), 7.93 (bs, 1H, NH); H-NMR ((CD3)2CO), 500 MHz): δ 1.41 (m, 1H, H-8),
1.45-1.58 (m, 3H, H-7, H′-7, H′-8), 1.75 (m, 1H, H-9), 2.01 (m, 1H, H′-9), 2.61 (m, 1H, H-6), 2.67 (m,
1H, H′-6), 2.97 (dd, 2J = 14.0, 3J = 6.5, 1H, CH2), 3.26 (dd, 2J = 14.0, 3J = 8.0, 1H, CH2’), 3.58 (dd, 3J1
3
3
= 8.0, J2 = 6.5, 1H, H-4), 3.86 (pt, J = 5.0, 1H, H-9a), 7.18-7.34 (m, 5H, H-Ar), 9.67 (bs, 1H, NH);
13C-NMR ((CD3)2CO, 125 MHz): δ 22.6 (C-8), 26.2 (C-7), 26.4 (C-9), 34.4 (C-α), 52.0 (C-6) 55.4
(C-8a), 68.7 (C-4), 127.2 (C-4′), 129.1 (C-3′, C-5′), 130.2 (C-2′, C-6′), 139.5 (C-1′), 173.3
(C-3), 173.1 (C-1); HRMS (ESI+) calcd for C15H18N2O2Na: 281.1266 (M+Na)+ found 281.1269.
(4R,9aR)-4-Benzylperhydropyrido[1,2-a]pyrazine-1,3-dione (4R,9aR)-3c. From (2R,1R)-2c (0.50 g,
1.72 mmol) and NaOH (69 mg, 1.85 mmol); FC (PE/AcOEt 4:1 to 2:1), then recrystallization form
H2O/MeOH; Yield 0.28 g (64%). White powder; M.p. 130–131 °C; [α]D = +27.1 (c 0.493; CHCl3);
HRMS (ESI+) calcd for C15H18N2O2Na: 281.1266 (M+Na)+ found 281.1269.
(4S,8aS)-4-Methyl-4-Benzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4S,8aS)-3d. From (2S,1S)-2d
(0.45 g, 1.55 mmol) and NaOH (62 mg, 1.55 mmol); FC (PE/AcOEt 4:1 to 2:1); Yield 0.35 g (88%).
The [α]D value matches this recorded for (4S,8aS)-3d via acid-mediated cyclocondensation.
(4R,8aR)-4-Methyl-4-Benzylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (4R,8aR)-3d. From (2R,1R)-2d
(0.42 g, 1.45 mmol) and NaOH (58 mg, 1.45 mmol); FC (PE/AcOEt 4:1 to 2:1); Yield 0.35 g (93%).
The [α]D value matches this recorded for (4R,8aR)-3d via acid-mediated cyclocondensation.
(8aS)-4,4-Dimethylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (8aS)-3f. From (2S)-2f (0.25 g, 1.17 mmol)
and NaOH (47 mg, 1.17 mmol); FC (PE/AcOEt 1:1 to 0:1); Yield 0.20 g (95%). The [α]D value
matches this recorded for (8aS)-3f via acid-mediated cyclocondensation.
(8aR)-4,4-Dimethylperhydropyrrolo[1,2-a]pyrazine-1,3-dione (8aR)-3f. From (2R)-2f (0.29 g, 1.36 mmol)
and NaOH (54 mg, 1.36 mmol); FC (PE/AcOEt 1:1 to 0:1); Yield 0.21 g (85%). The [α]D value
matches this recorded for (8aR)-3f via acid-mediated cyclocondensation.
(4R,8aR)-4-Phenyl-8a-methylperhydropyrrolo[1,2-a]pyrazine-1,3,6-trione (4R,8aR)-6. From (2R,αS)-5
(1.28 g, 4.42 mmol) and NaOH (0.18 g, 4.42 mmol); FC (PE/AcOEt 3:1 to 0:1); Yield 1.00 g (87%):
0.59 (52%) of (4R,8aR)-6 and 0.40 g (35%) of (4S,8aR)-6. (4S,8aR)-6: White powder; M.p.
164–167 °C; [α]D = −50.7 (c 0.600; CHCl3); HRMS (ESI-) calcd for C14H13N2O3: 257.0926 (M−H)−
found 257.0923.
Methyl (2S)-1-(1-(n-butylcarbamoyl)-1,1-diphenylmethyl)-pyrrolidine-2-carboxylate (2S)-1i. From
L-proline (0.64 g, 5.57 mmol), benzophenone (0.85 g, 4.64 mmol) and n-butyl isocyanide (0.50 mL,
4.64 mmol); FC (gradient: PE/AcOEt 10:1 to 3:1): yield 0.04 g (2%). Colorless oil; TLC (PE/AcOEt
5:1): Rf = 0.26; [α]D = −39.6 (c 0.833, CHCl3); IR (KBr): 706, 779, 1168, 1205, 1445, 1508, 1680,
1
3
1736, 2851, 2920, 2955, 3358; H-NMR (CDCl3, 300 MHz): δ 0.83 (t, J = 7.0, 1H, CH3), 0.86–1.04
(m, 2H), 1.09–1.40 (m, H), 1.41-1.73 (m, 4H), 2.87 (dt, J =3J1 = 11.0, J1 = 7.5, 1H, H-5), 3.13 (m,
2
3

Molecules 2014, 19
15976
2
3
3
3
2H), 3.34 (m, J = 11.0, J1 = 8.0, J1 = 5.0, 1H, H′-5), 3.72 (s, 3H, OCH3), 3.92 (dd, J1 = 9.0,
3J2 = 2.0, 1H, H-2), 7.24–7.36 (m, 6H, H-Ar), 7.37–7.44 (m, 2H, H-Ar), 7.60 (m, 2H, H-Ar), 7.76 (pt,
3J = 5.0, 1H, CONH); 13C-NMR (CDCl3, 75 MHz): 13.7, 19.9, 24.2, 30.7, 31.5, 39.2, 50.0 (C-5), 52.1
(OCH3), 63.2 (C-2), 79.3 (C-1), 127.17, 127.24 (C-4′, C-4″), 127.7 (C-2′, C-2″, C-6′, C-6″), 129.7,
129.9 (C-3′, C-3″, C-5′, C-5″), 139.1, 141.1 (C-1′, C-1″), 172.3 (CONH), 176.9 (COOCH3); HRMS
(ESI+) calcd for C24H30N2O3Na: 417.2154 (M+Na)+ found 417.2148.
Methyl (2S,1S)- and (2S,1R)-1-(1-(n-butylcarbamoyl)-1-methyl-1-phenylmethyl)-pyrrolidine-2-carboxylate
(2S,1S)-1j and (2S,1R)-1j. From L-proline (0.64 g, 5.57 mmol), acetophenone (0.54 mL, 4.64 mmol)
and n-butyl isocyanide (0.50 mL, 4.64 mmol); FC (PE/AcOEt 8:1 to 3:1): yield 0.49 g (32%): 0.24 g
(15%) of (2S,1R)-1j, 0.25 (17%) of (2S,1S)-1j. (2S,1S)-1j: Colorless oil; TLC (PE/AcOEt 5:1):
Rf = 0.16; [α]D = −29.5 (c 1.190, CHCl3); IR (KBr): 700, 1167, 1205, 1441, 1522, 1682, 1736, 2872,
2957, 3334; 1H-NMR (CDCl3, 300 MHz): δ 0.94 (t, 3J = 7.5, 3H, CH2CH2CH2CH3), 1.30–1.44 (m, 2H,
CH2), 1.49-1.61 (m, 2H, CH2), 1.71 (s, 3H, CH3), 1.71-1.98 (m, 4H, H-4, H′-4, H-3, H′-3), 2.93 (pq, 2J
= 3J1 = 9.5, 3J2 = 7.5, 1H, H-5), 3.11 (m, 2J = 9.5, 3J1 = 6.5, 3J2 =4.0, 1H, H′-5), 3.21–3.40 (m, 3H, H-2,
CH2′′), 3.43 (s, 3H, OCH3), 7.17–7.33 (m, 3H, H-4′, H-3′, H-5′), 7.41 (d, 3J = 7.0, 2H, H-2′, H-6′), 7.91
(pt, 3J1 = 5.0, 1H, CONH); 13C-NMR (CDCl3, 75 MHz): δ 13.8, 17.9, 20.2, 25.2, 31.6, 31.7, 39.2, 49.5
(C-5), 51.6 (OCH3), 61.1 (C-2), 68.9 (C-1), 127.5 (C-2′, C-6′), 127.6 (C-4′), 128.1 (C-3′, C-5′), 141.2
(C-1′), 174.1 (CONH), 177.0 (COOCH3); LC/MS: 333 [M+H]+, retention time: 12.6 min; HRMS
(ESI+) calcd for C19H28N2O3Na: 355.1998 (M+Na)+ found 355.1981. (2S,1R)-1j: Colorless oil; TLC
(PE/AcOEt 5:1): Rf = 0.24; [α]D = −34.4 (c 0.833, CHCl3); IR (KBr): 700, 764, 1167, 1209, 1443,
1
3
1522, 1682, 1736, 2870, 2945, 3369; H-NMR (CDCl3, 300 MHz): δ 0.87 (t, J = 7.5, 3H,
CH2CH2CH2CH3), 1.18–1.32 (m, 2H, CH2), 1.33–1.46 (m, 2H, CH2′), 1.62–1.82 (m, 5H, H-4, H′-4,
CH3), 1.89–2.05 (m, 2H, H-3, H′-3), 2.36 (m, 2J = 3J1 = 9.5, 3J2 = 7.0, 1H, H-5), 2.98 (m, 2J = 9.5, 3J1
= 7.5, 3J2 = 3.0, 1H, H′-5), 3.11 (m, 1H, CH′2′′), 3.18 (m, 1H, CH′2′′), 3.72 (s, 3H, OCH3), 3.81 (dd, 3J1
3
3
3
3
= 8.0, J2 = 3.5, 1H, H-2), 7.23 (t, J = 7.5, 1H, H-4′), 7.32 (t, J = 7.5, 2H, H-3′, H-5′), 7.55 (d, J =
3
13
7.5, 2H, H-2′, H-6′), 7.72 (pt, J1 = 5.0, 1H, CONH); C-NMR (CDCl3, 75 MHz): δ 13.9 (br), 17.4
(br), 20.2 (br), 24.8 (br), 31.7 (br), 32.0 (br), 39.3, 49.9, (C-5), 52.2 (OCH3), 61.4 (C-2), 69.9 (C-1),
127.3 (C-4′), 127.6 (C-2′, C-6′), 128.2 (C-3′, C-5′), 141.8 (C-1′), 174.2 (CONH), 177.3 (COOCH3);
LC/MS: 333 [M+H]+, retention time: 15.6 min; HRMS (ESI+) calcd for C19H28N2O3Na: 355.1998
(M+Na)+ found 355.1990.
3.1.4. Synthesis of Compounds 4 by Intramolecular U-4C-3CR Condensation
Triethylamine (1.0 eq.) was added to a cold, stirred solution of methyl (S)-phenylglycinate (1.0 eq.)
and levulinic acid (1.0 eq.) in MeOH (100 mL), followed by tert-butyl isocyanide (1.0 eq.) The
mixture was stirred at rt for 24 h and the volatiles were removed under reduced pressure. The residue
was dissolved in DCM (100 mL) and washed with water (3 × 20 mL), brine (20 mL), dried and
concentrated under reduced pressure. The residue was purified by FC.
Methyl (2R,αS)- and (2S,αS)-α-(2-(tert-butylcarbamoyl)-2-methyl-5-oxopyrrolidin-1-yl)-α-phenylacetate
(2R,αS)-4 and (2S,αS)-4. From methyl (2S)-2-phenylglycinate (3.39 g, 16.80 mmol), triethylamine
(2.34 mL, 16.80 mmol), levulinic acid (1.95 g, 16.8 mmol) and tert-butyl isocyanide (2.0 mL,

Molecules 2014, 19
15977
16.80 mmol); FC (PE/AcOEt 6:1 to 1:1); Yield 5.23 g (90%): 2.70 g (47%) of (2S,αS)-4 and 2.53 g
(43%) of (2R,αS)-4. (2R,αS)-4: White powder; M.p. 160–163 °C; TLC (AcOEt): Rf = 0.70;
[α]D = –90.2 (c 0.7; CHCl3); IR (KBr): 704, 1001, 1266, 1395, 1532, 1666, 1694, 1724, 2972, 3223,
1
3390; H-NMR (CDCl3, 500 MHz): δ 1.26 (s, 3H, CH3), 1.41 (s, 9H, C(CH3)3), 1.90–2.00 (m, 1H,
H-3), 2.38–2.54 (m, 2H, H′-3, H-4), 3.78 (s, 3H, OCH3), 4.64 (s, 1H, H-α), 7.30–7.37 (m, 6H, H-Ar),
13
7.91 (bs, 1H, CONH); C-NMR (CDCl3, 125 MHz): δ 24.0 (CH3), 28.6 (C(CH3)3), 29.3 (C-4), 34.4
(C-3), 51.7 (OCH3), 53.5 (C(CH3)3), 60.6 (C-2), 68.8 (C-α), 128.4 (C-4′), 128.6 (C-2′, C-6′), 128.6 (C-
3′, C-5′), 136.5 (C-1′), 170.7 (C-5), 172.4 (CONH), 175.7 (COOCH3); HRMS (ESI+) calcd for
C19H26N2O4Na: 369.1790 (M+Na)+ found 369.1803. (2S,αS)-4: White powder; M.p. 162–164 °C; TLC
(AcOEt): Rf = 0.55; [α]D = −105.4 (c 0.7; CHCl3); IR (KBr): 715, 1005, 1229, 1393, 1513, 1673, 1699,
1745, 2970, 3333, 3412; TLC (EA): Rf = 0.55; 1H-NMR (CDCl3, 500 MHz): δ 1.02 (s, 9H, C(CH3)3),
2
3
3
2
3
1.41 (s, 3H, CH3), 2.11 (m, J = 16.0, J1 = 9.0, J2 = 7.5, 1H, H-3), 2.28 (m, J = 16.0, J1 = 9.5,
3J2 = 7.0, 1H, H′-3), 2.48 (m, 2H, H-4), 3.73 (s, 3H, OCH3), 4.84 (s, 1H, H-α), 7.26 (bs, 1H, CONH),
7.38 (m, 3H, H-3′, H-4′, H-5′), 7.49 (dd, J = 7.5, J = 3.5, 2H, H-2′, H-6′); 13C-NMR (CDCl3,
125 MHz): δ 21.9 (CH3), 27.9 (C(CH3)3), 28.9 (C-4), 34.8 (C-3), 51.0 (OCH3), 53.0 (C(CH3)3), 60.9
(C-2), 69.0 (C-α), 129.1 (C-4′), 129.2 (C-2′, C-6′), 129.8 (C-3′, C-5′), 135.5 (C-1′), 170.1 (C-5), 172.4
(CONH), 177.0 (COOCH3); HRMS (ESI+) calcd for C19H26N2O4Na: 369.1790 (M+Na)+
found 369.1800.
3
4
3.2. Pharmacological Evaluation
The compounds obtained have been submitted for in vivo evaluation in the Anticonvulsant
Screening Program (ASP) of The National Institute of Neurological Disorders and Stroke (NINDS),
Bethesda, USA [21,22]. 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 suspended
in 0.5% (v/v) aqueous solution of methylcellulose.
3.2.1. The Maximal Electroshock Seizure Test (MES) [22]
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 anesthetic agent. Mice were tested
at at least two different time points (15 min, 30 min, 1 h or 4 h) following intraperitioneal
administration of 100 and 300 mg/kg of test compound. Abolition of the hindlimb tonic extensor
component indicated the test compound’s ability to inhibit MES-induced seizure spread.
3.2.2. The subcutaneous Metrazol seizure test (scMET) [22]
The test utilized a dose of Metrazol (pentylenetetrazole, 85 mg/kg in mice). This produced clonic
seizures lasting for a period of at least five seconds in 97% (CD97) of animals tested. At the anticipated
time of testing the convulsant was administered subcutaneously. The test compound was administered
intraperitoneally in mice and the animals were observed over a 30 min period. Mice were tested at at

Molecules 2014, 19
15978
least two different time points (15 min, 30 min, 1 h or 4 h) following intraperitioneal administration of
100 and 300 mg/kg of test compound. Absence of clonic spasms indicated a compound’s ability to
abolish the effect of pentylenetetrazol on seizure threshold.
3.3.3. The Acute Neurological Impairment (TOX) [26]
Neurological toxicity induced by a compound was detected in mice or rats using the standardized
rotorod test. Mice were tested at a minimum of two different time points (15 min, 30 min, 1 h or 4 h)
following intraperitioneal administration of 100 mg/kg of test compound. Neurological impairment
was demonstrated by the inability of animals to maintain equilibrium on a 6 r.p.m. rotation rod for a
given time.
3.3.4. The minimal clonic seizure test (6 Hz) [23–25]
The 6 Hz screen 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 compound. 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
exploratory behavior. The experimental end point was protection against the seizure. The animal was
considered to be protected if it resumed its normal exploratory behavior within 10 s from the
stimulation. The quantitative determination of the median effective (ED50) and toxic doses (TD50) was
conducted at previously calculated time of peak effect (TPE) using the intraperitoneal route in mice.
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 in 50% of the animals in each test,
and 95% confidence interval were calculated by a computer program based on methods described by
Finney [27].
4. Conclusions
We have synthesized a series of novel stereochemically pure pyrrolo[1,2-a]pyrazine derivatives by
use of pathways based on the U-5C-4CR and the U-4C-3CR multicomponent reactions. The
compounds displayed weak to good anticonvulsant activities in the MES model, while only few of
them were active in the scMET screen. The efficacy of most of the new derivatives in the 6 Hz model
of pharmacoresistant partial seizures was significantly higher than in the ‘classical’ models. As a
supplement to our previous extensive SAR studies within this group of compounds, we learned that:
(1) increasing the distance of the benzene ring from the imide moiety of ADD408003 lowered its
potency in MES, scMET and 6 Hz models, (2) introduction of non-epimerizable residue in lieu of
hydrogen atom at C-4 did not enhance the pharmacological properties of the parent compound,

Molecules 2014, 19
15979
(3) introduction of alkyl residues at C-4 produced either inactive or strongly neurotoxic and
proconvulsant compounds, (4) for most of the newly synthesized derivatives, the differences in
pharmacological activities between (R,R) and (S,S) were only slight.
The opposite enantiomer of ADD408003 (4R,8aR)-3a was most active from the series with an ED50
value of 47.90 mg/kg and a protective index (PI) of 5.8 in the 6 Hz model of pharmacoresistant
epilepsy. The ADD408003-Levetiracetam hybrid (4S,8aS)-6 was markedly less potent in this test, with
ED50 value of 126.19 mg/kg and PI of 2.1. However, the overall synthetic feasibility of this compound
was very high. Thus, manipulating the amino acid and keto acid component in the initial
intramolecular U-4C-3CR condensation opens the access to chemical diversity of potentially
interesting (4S,8aS)-6 analogs.
Acknowledgements
The authors would like to thank the Anticonvulsant Screening Program (National Institute of
Neurological Disorders and Stroke, NINDS Rockville, MD, USA) staff for providing the
pharmacological data. Financial support was provided by the Polish Ministry of Science and Higher
Education (Grant No. N N405 623138). The autors would like to thank Sławomir Sobczak, Krzysztof
Kubica and Magdalena Wyszomierska for technical support during the synthesis.
Author Contributions
M.D. designed the compounds, planned synthetic pathways, performed chemical synthesis,
analyzed the results of biological testing and wrote the manuscript. W.L. performed part of chemical
synthesis. J.T. performed LC/MS analyses. All authors commented on the manuscript.
Conflicts of Interest
The authors declare no conflict of interest.
References and Notes
1. Sander, J.W.; Shorvon, S.D. Epidemiology of the epilepsies. J. Neurol. Neurosurg. Psychiat.
1996, 61, 433–443.
2. Kwan, P.; Schachter, S.C.; Brodie, M.J. Drug-resistant epilepsy. N. Engl. J. Med. 2011, 365,
919–926.
3. Brodie, M.J. Do we need any more new antiepileptic drugs? Epilepsy Res. 2001, 45, 3–6.
4. French, J.A. Refractory epilepsy: Clinical overview. Epilepsia 2007, 48, 3–7.
5. Gerlach, A.C.; Krajewski, J.L. Antiepileptic drug discovery and development: What have we
learned and where are we going? Pharmaceuticals 2010, 3, 2884–2899.
6. Zaccara, G.; Gangemi, P.F.; Cincotta, M. Central nervous system adverse effects of new
antiepileptic drugs: A meta-analysis of placebo-controlled studies. Seizure 2008, 17, 405–421.
7. Dawidowski, M.; Herold, F.; Chodkowski, A.; Kleps, J.; Szulczyk, P.; Wilczek, M. Synthesis and
anticonvulsant activity of novel 2,6-diketopiperazine derivatives. Part 1: Perhydropyrrole[1,2-a]
pyrazines. Eur. J. Med. Chem. 2011, 46, 4859–4569.

Molecules 2014, 19
15980
8. Dawidowski, M.; Herold, F.; Chodkowski, A.; Kleps, J. Synthesis and anticonvulsant activity of
novel 2,6-diketopiperazine derivatives. Part 2: Perhydropyrido[1,2-a]pyrazines. Eur. J. Med. Chem.
2012, 48, 347–353.
9. Dawidowski, M.; Wilczek, M.; Kubica, K.; Skolmowski, M.; Turło, J. Structure–activity
relationships of the aromatic site in novel anticonvulsant pyrrolo[1,2-a]pyrazine derivatives.
Bioorg. Med. Chem. Lett. 2013, 23, 6106–6110.
10. Dawidowski, M.; Turło, J. Multicomponent synthesis and anticonvulsant activity of monocyclic
2,6-diketopiperazine derivatives. Med. Chem. Res. 2014, 23, 2007–2018.
11. Demharter, A.; Hörl, W.; Herdtweck, E.; Ugi, I. Synthesis of chiral 1,1′-iminodicarboxylic acid
derivatives from α-amino acids, aldehydes, isocyanides, and alcohols by the diastereoselective
five-center–four-component reaction. Angew. Chem. Int. Ed. 1996, 35, 173–175.
12. Dawidowski, M.; Herold, F.; Wilczek, M.; Turło, J.; Chodkowski, A.; Gomółka, A.; Kleps, J.
Synthesis of bicyclic 2,6-diketopiperazines via a three-step sequence involving an Ugi five-center,
four-component reaction. Tetrahedron 2012, 68, 8222–8230.
13. Dawidowski, M.; Sobczak, S.; Wilczek, M.; Kulesza, A.; Turło, J. Expanding the substrate scope
of ugi five-center, four-component reaction (U-5C-4CR): Ketones as coupling partners for
secondary amino acids. Mol. Divers. 2014, 18, 61–77.
14. Ugi, I.; Hörl, W.; Hanusch-Kompa, C.; Schmid, T.; Herdtweck, E. MCR 6: Chiral
2,6-piperazinediones via Ugi reactions with α-amino acids, carbonyl compounds, isocyanides and
alcohols. Heterocycles 1998, 47, 965–975.
15. Mandai, H.; Irie, S.; Mitsudo, K.; Suga, S. Studies on the synthesis of DMAP derivatives by
diastereoselective Ugi reactions. Molecules 2011, 16, 8815–8832.
16. Sung, K.; Chen, F.-L.; Chung, M.-J.; Application of MCR: Facile one-pot diastereoselective
syntheses of novel chiral alpha, alpha′-iminodiacetic acid analogues. Mol. Divers. 2003, 6,
213–221.
17. Szakonyi, Z.; Sillanpää, R.; Fülöp, F. Synthesis of conformationally constrained tricyclic β-lactam
enantiomers through Ugi four-center three-component reactions of a monoterpene-based β-amino
acid. Mol. Divers. 2010, 14, 59–65.
18. Short, K.M.; Mjalli, A.M.M. A solid-phase combinatorial method for the synthesis of novel 5-and
6-membered ring lactams. Tetrahedron Lett. 1997, 38, 359–362.
19. Harriman, G.C.B. Synthesis of small and medium sized 2,2-disubstituted lactams via the
“intramolecular” three component Ugi reaction. Tetrahedron Lett. 1997, 38, 5591–5594.
20. Hanusch-Kompa, C.; Ugi, I. Multi-component reactions 13: Synthesis of γ-lactams as part of a
multiring system via Ugi-4-centre-3-component reaction. Tetrahedron Lett. 1998, 39, 2725–2728.
21. Anticonvulsant screening program. Available online: http://www.ninds.nih.gov/research/asp/
(accessed on 11 June 2014).
22. White, H.S.; Woodhead, J.H.; Wilcox, K.S.; Stables, J.P.; Kupferberg, H.J.; Wolf, H.H. General
principles: Discovery and preclinical development of antiepileptic drugs. In Antiepileptic Drugs,
5th ed.; Levy, R.H., Mattson, B.S., Meldrum, E., Perucca, E., Eds.; Lippincott Williams &
Wilkins Publishers: New York, NY, USA, 2002; pp. 36–48.

Molecules 2014, 19
15981
23. Brown, W.C.; Schiffman, D.O.; Swinyard, E.A.; Goodman, L.S. Comparative assay of
antiepileptic drugs by psychomotor seizure test and minimal electroshock threshold test.
J. Pharmacol. Exp. Ther. 1953, 107, 273–283.
24. Kaminski, R.F.; Livingood, M.R.; Rogawski, M.A. Allopregnanolone analogs that positively
modulate GABA receptors protect against seizures induced by 6-Hz electrical stimulation in mice.
Epilepsia 2004, 45, 864–867.
25. Barton, M.E.; Klein, B.D.; Wolf, H.H.; White, H.S. Pharmacological characterization of the 6 Hz
psychomotor seizure model of partial epilepsy. Epilepsy Res. 2001, 47, 217–227.
26. Dunham, M.S.; Miya, T.A. A note on a simple apparatus for detecting neurological deficit in rats
and mice. J. Am. Pharmac. Assoc. Sci. Edit. 1957, 46, 208–209.
27. Finney, D.J. Probit Analysis, 3rd ed.; Cambridge University Press: London, UK, 1971; pp. 1–333.
Sample Availability: Samples of the compounds are not available from the authors.
© 2014 by the authors; licensee MDPI, Basel, Switzerland. This article is an open access article
distributed under the terms and conditions of the Creative Commons Attribution license
(http://creativecommons.org/licenses/by/4.0/).