Synthesis of sulfur containing dihydro-pyrrolo derivatives and their biological evaluation as antioxidants

Article abstract of DOI:10.1016/j.bmc.2012.07.014

The 1,3-dipolar cycloaddition to N-phenylmaleimide of azomethine ylides, generated in situ from sulfanyl-substituted imines of glycine esters, yields 5H-dihydro-pyrrolo products with syn diastereoselectivity. The syn (major) and anti (minor) products were

Full text of DOI:10.1016/j.bmc.2012.07.014

Bioorganic & Medicinal Chemistry 20 (2012) 5103–5109
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of sulfur containing dihydro-pyrrolo derivatives and their
biological evaluation as antioxidants
Dimitra Georgiou a, , Vasilios Toutountzoglou a, Kenneth W. Muir b, Dimitra Hadjipavlou-Litina c,
,
⇑
Yiannis Elemes a,
⇑
a Department of Chemistry, University of Ioannina, 45110 Ioannina, Greece
b Chemistry Department, University of Glasgow, Glasgow G12 8QQ, Scotland, UK
c Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
a r t i c l e i n f o
a b s t r a c t
Article history:
The 1,3-dipolar cycloaddition to N-phenylmaleimide of azomethine ylides, generated in situ from sulfa-
nyl-substituted imines of glycine esters, yields 5H-dihydro-pyrrolo products with syn diastereoselectiv-
ity. The syn (major) and anti (minor) products were isolated chromatographically and fully characterized
by spectroscopic methods and in two cases also by X-ray analysis. The diastereomeric cycloadducts were
tested for their antioxidant activity with good results.
Received 31 May 2012
Revised 29 June 2012
Accepted 6 July 2012
Available online 15 July 2012
Ó 2012 Elsevier Ltd. All rights reserved.
Keywords:
Azomethine ylides
Imines
Aminoacids
1,3-Dipolar cycloadditions
Dihydro-pyrrole
Sulfides
Syn Diastereoselectivity
X-ray
Antioxidant activity
1. Introduction
in situ); numerous reviews testify to the importance of this topic.4
Sulfur-containing imines are important intermediate substrates for
Cycloaddition reactions are among the most important and
powerful tools for ring construction in organic synthesis.1 In parti-
cular, 1,3-dipolar cycloadditions have been a fertile source of ring
systems with extended applications in medicine, industry and
the laboratory.2 Azomethine ylides are reactive 1,3-dipoles and,
with electrophiles, give rise to a variety of five-membered ring ni-
trogen heterocyclic compounds which have synthetic applications
in heterocyclic and natural product chemistry.3 Heterocyclic
compounds are significant because of their biological activity and
their applications in diverse fields. Heterocyclic moieties are
important in naturally occurring compounds, as in alkaloids,
vitamins, hormones, and antibiotics. They are also widely present
in pharmaceutical products, herbicides, and dyes.
amino acid synthesis: with a chiral auxiliary they produce excel-
lent precursors for the highly diastereoselective synthesis of
optically active amino acid derivatives,5 which has found applica-
tions on the synthesis of labeled amino acid derivatives.5e,f
Our recent interest in 1,3-dipolar cycloadditions of sulfur-
substituted azomethine ylides to C60,6 led us to a systematic inves-
tigation of the fore mentioned reaction between sulfur-containing
imines of glycine esters with electrophile such as N-phenylmalei-
mide (NPM). Sulfur-containing nitrogen heterocycles might be of
biological interest and may play an antioxidative or a biological
role in general due to the sulfur functionality.7 We decided to
explore the applicability of this reaction in terms of different sul-
fide- and alkoxy-substituents in the imines. We report here on
the isolation of syn and anti 5H-dihydro-pyrrolo derivatives, from
the cycloaddition of a series of N,N-[bis-methylsulfanyl]-imines
of glycine esters to NPM, in good yields under neutral conditions
at elevated temperatures, and their full spectroscopic characteriza-
tion. In addition we tested most of the cycloadducts for their pos-
sible antioxidant activity. In the literature there was only one
example of such a reaction (with imine 1a and NPM) in strongly
acidic solution (stoichiometric, to the reactants, quantity of AcOH)
A major route to azomethine ylides involves imines of amino
acids or their derivatives (either previously synthesized or formed
⇑
Corresponding authors. Tel.: +30 26510 08432; fax: +30 26510 08799.
E-mail addresses: hadjipav@pharm.auth.gr (D. Hadjipavlou-Litina), yelemes@c-
c.uoi.gr (Y. Elemes).
Present address: Department of Chemistry, Faculty of Pure and Applied Sciences,
University of Cyprus, 1678 Nicosia, Cyprus.
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.07.014

5104
D. Georgiou et al. / Bioorg. Med. Chem. 20 (2012) 5103–5109
reported to give only the 2a-syn adduct,9 which was isolated and
O
H
H
O
Ph
N
briefly characterized by 1H NMR.
N
H
N
OR
R1S
R1S
OR
O
O
5
5
O
3
4
3 4
R1S
R1S
HO
O
H
N
H
+
2. Results and discussion
OR
O
O
N
Ph
PhMe, ~185oC,
24h,
N
Ph
anti
The following methyl 2-[bis (alkylsulfanyl or aralkylsulfanyl)
methyleneamino] acetates, 1a–f, were prepared by known proce-
dures,5a Figure 1.
1
2
-R1SH
syn
Scheme 1. Mixture of syn and anti products 2, from the 1,3-dipolar cycloaddition of
imines 1, to N-phenylmaleimide.
In general, the cycloaddition reaction was performed by placing
in a screw-capped test-tube flushed with Ar the desired quantity of
the imine of methyl glycinate, 1, dissolved in dry toluene. To this
solution solid NPM (10% excess with respect to the imine) was
added in one portion. The tube was closed and heated to ꢀ185–
190 °C (below this temperature little or no reaction took place as
detected by TLC). The reaction was followed by TLC and stopped
after 24 h. Volatile compounds were removed with a rotary eva-
porator and high vacuum pump. Column chromatography on
SiO2, EtOAc/n-hexanes, ꢀ1/2 v/v) afforded syn and anti cycloaddi-
tion products 2, Scheme 1.
These were the only products detected by TLC during the reac-
tion. An aliquot taken periodically from the reaction mixture
showed the formation of the syn product already within 20 min-
utes after the beginning of the reaction. Throughout of this work
the expected cycloaddition products (with two –SR substituents)
never detected nor isolated. In addition the elimination of a mer-
captane molecule during the reaction course was indirectly de-
tected with the isolation and characterization of the Thia Michael
addition of MeSH to NPM (see footnote b in Table 2).
Table 1
Yields of syn and anti diastereomers isolated from the reaction of imines 1 to NPMa,b
Adduct
R
syn (%)
anti (%)
Total (%)
d.r.
6.6
2.8
1.3
1.3
19.7
3.3
2a
2b
2c
2d
2e
2f
Me
Et
n-Pr
i-Pr
n-Bu
Bn
66
10
76
94
78
62
62
39
69 (60)c
44
25 (0)c
34
27
3
35
59
30
9
a
b
c
10% excess of NPM was used in every run, unless otherwise specified.
Yields % of isolated adducts after chromatographic separation.
Under strong acidic conditions, see Ref. 9.
Table 2
Isolated yields of syn and anti cycloadducts and diastereomeric ratios after
chromatographic purification, for the cycloaddition of imines 1g–i with NMPa
The two diastereomers isolated from the reaction of each imine,
1, were fully characterized by spectrometric means and by elemen-
tal analysis. In general, 13C NMR reveals four resonances for the
three carbonyls and the imine carbon nuclei. In the 1H NMR the
stereogenic carbon center imposes multiplicity to the diastereoto-
pic methylene hydrogens of the sulfide substituents. Furthermore
in the syn adducts, there is a large coupling constant between H4
and H5 (JH4,H5 ꢀ9.50 Hz), whereas the corresponding coupling con-
stant is smaller in the anti isomers (JH4,H5 ꢀ2.20 Hz), testifying to
their relative stereochemistry (i.e., cis and trans, respectively).
Smaller differences were observed for the JH3,H4 coupling constants
(i.e., ꢀ9.20 Hz for the syn and ꢀ8.40 Hz for the anti isomer. The
above coupling constants are in close relation with the reported
values for 2a stereoisomers in Ref. 9: JH4,H5 = 9.9 Hz and
JH3,H4 = 8.8 Hz for the syn and JH4,H5 = 2.5 Hz and JH3,H4 = 8.45 Hz
for the anti isomer.
Ester
syn (%)
anti (%)
Total (%)
d.r.
–OMe
–OEt
66.0
37.8
38.0
32.2
10.0
10.2
8.2b
5.7
76.0
48.0
46.2
37.9
6.6
3.7
4.6
5.6
–OBn
–OtBu
a
10% excess of NPM was used in every run, unless otherwise specified.
Characterized as a mixture with the MeSH Michael addition product to NPM.8
b
t-butyl- esters of the –SMe substituted glycine imines 1g–i,
Scheme 2. Isolated yields of the two diastereomers and the ob-
tained d.r. are listed in Table 2.
At this point we should comment on the absence of any clear
correlation between the d.r. and the size of either the sulfide or
the alkoxy substituents used in this work.
According to an earlier report, that only closed adducts were
isolated in the reaction of a cyclic thioiminoether (cyclic monosub-
stituted imine) with NPM,9 and the results of the present work we
have to conclude that the reaction follows the azomethine ylide
path. More work though is needed to a definite answer for a more
detailed mechanism of this process.
It is interesting to note that in all cases the anti adduct invari-
ably eluted first from the column, revealing its smaller polarity
with regard to the syn diastereomer. Yields and molar ratios of
the isolated products, together with diastereomeric ratios (d.r.)
are shown in Table 1.
In the case of adducts 2a, crystals of adequate quality for XRD
were obtained from CH2Cl2/n-hexanes, 2/1, v/v. From the resulting
diffraction analyses, Figure 2, the anti- or syn-relation of the ester
group and the maleimide ring and also between the ring hydrogens
is evident.
3. Biological studies
We tested the new compounds, from Table 1, with regard to
their antioxidant ability and in comparison to well known antiox-
idant agents, for example, nordihydroguaiaretic acid (NDGA) and
trolox.
Finally, the cycloaddition was performed under the same, as
above, experimental conditions with the ethyl-, benzyl-, and
O
RS
RS
O
S
K2CO3/Acetone
RI
1) CS2/Et3N/CHCl3/Δ
2) RI
H
N
N
HCl.Gly-OMe
OMe
OMe
RS
1
R-: Me-, Et-, n-Pro-, i-Pro-, n-Bu-, Bn-
1a 1b 1c 1d 1e 1f
Figure 1. General synthetic procedure for methyl 2-[bis (alkylsulfanyl or aralkylsulfanyl) methyleneamino] acetate 1, used as substrates for the 1,3-dipolar cycloaddition to
NPM.

D. Georgiou et al. / Bioorg. Med. Chem. 20 (2012) 5103–5109
5105
ability in an iron-free system, was measured at 50
lM (equimolar
to the DPPH concentration) for 20/60 min). In the DPPH assay, the
dominant chemical reaction involved is the reduction of the DPPH
radical by an ET from the antioxidant. Particularly effective such
antioxidants are the phenoxide anions from phenolic compounds
like catechol and derivatives, such as NDGA. Under our experimen-
tal conditions very low interaction values were observed.
The use of the free radical reactions initiator 2,2-azobis(2-ami-
dinopropane) dihydrochloride (AAPH) is recommended as more
appropriate for measuring radical-scavenging activity in vitro, be-
cause the activity of the peroxyl radicals produced by the action of
AAPH shows a greater similarity to cellular activities such as lipid
peroxidation.11 Oxidation of sodium linoleate by a thermal free ra-
dical initiator (AAPH) is followed by UV spectrophotometry in a
highly diluted sample.12 In the AAPH assay, the highly reactive al-
kylperoxyl radicals are intercepted mainly by a hydrogen atom
transfer (HAT) from the antioxidant.13 Therefore, particularly effec-
tive HAT agents are compounds with high hydrogen atom donating
ability, that is, compounds with low heteroatom-H bond dissocia-
tion energies and/or compounds from which hydrogen abstraction
leads to sterically hindered radicals as well as compounds from
which abstraction of hydrogen leads to C-centered radicals stabi-
lized by resonance. All the present compounds caused strong inhi-
bition of lipid peroxidation (72–98%) and higher than trolox (63%)
(Table 3, third row). Not much difference is observed between syn
and anti isomers. Since these compounds lack the easily oxidizable
phenol unit present in trolox, their inhibitory potency should be at-
tributed to other factors. The antioxidant effect of 5H-dihydro-pyr-
rolo products is possible to be supported by the following
mechanism of C centered radical stabilization by resonance, Figure
3.
Figure 2. Views of the molecules of the 2a-anti (left) and 2a-syn (right) isomers.
The samples were racemic. In the 2a-anti molecule shown all three chiral centers
have S configurations whereas in the 2a-syn molecule the configurations of the
chiral carbon atoms are 3-S, 4-S, 6-R.
O
H
H
O
Ph
N
N
H
N
OR
MeS
MeS
OR
O
O
5
5
O
3
4
3 4
MeS
MeS
HO
O
H
N
H
+
OR
O
O
N
Ph
PhMe, ~185oC,
24h,
N
Ph
anti
1
t
R-: Et, Bn, Bu
1g, 1h, 1i
2
-R1SH
syn
Scheme 2. 1,3-Dipolar cycloadditions of imines 1g–i with NPM.
Eicosanoids are oxygenated metabolites of arachidonic acid
with a broad implication in a diversity of diseases. Upon appropri-
ate stimulation of neutrophils, arachidonic acid (AA) is cleaved
from membrane phospholipids and can be converted into leuko-
trienes (LTs). The lipoxygenase (LOX) catalyzes the first two steps
in the metabolism of arachidonic acid to leukotrienes. LTB4 gen-
eration is considered to be important in the pathogenesis of neu-
trophil-mediated inflammatory diseases14 with a marked relation
to the severity of cardiovascular diseases, asthma and cancer. In
this context, we decided to further evaluate the synthesized com-
pounds for their ability to inhibit soybean LOX by the UV absor-
bance based enzyme assay.15 Most of the LOX inhibitors are
antioxidants or free radical scavengers. NDGA, a known inhibitor
of soybean LOX, has been used as a reference compound (64%)
and as a positive control. All the compounds inhibit soybean
LOX, with the exception of Me-syn isomer (Table 3, first column,
second row). No big differences are observed between the % inhibi-
tion values of Pr-syn/anti isomers and iPr-syn/anti isomers. In two
cases, the anti-isomers of Bu- and Bn-substituted adducts, are more
potent than their respective syn. This might be attributed to a bet-
ter fit of the former isomer in the active space of the enzyme due to
steric reasons compared to the latter.
Antioxidants are defined as substances that, even at low con-
centration, significantly delay or prevent oxidation of easily oxidiz-
able substrates. The formation of Reactive Oxygen Species (ROS) is
characteristic of aerobic organisms, as an unavoidable conse-
quence of cell metabolism. Their involvement in inflammation,
cancer, myocardial and central nervous system (CNS) ischemia is
under intensive study. For the estimation of the antioxidative po-
tential of chemical components, different experimental approaches
were used. The most popular screening assays have been devel-
oped to be fast and easy. Most of them require a spectrophoto-
metric measurement and a certain reaction time in order to
obtain reproducible results.10 However, all the used assays do
not measure the same chemistry. In this way, factors such as solu-
bility or steric hindrance, which may be of overriding importance
in one environment but not in another, can be varied and the
antioxidant ability of a compound in a variety of milieus may be
evaluated.
The interaction/reducing activity (RA) of the examined com-
pounds with the stable free radical DPPH is shown in Table 3, first
row. This interaction, which indicates their radical scavenging
Table 3
Biological evaluation of cycloadducts 2a–f as antioxidants
Method/adduct
DPPH%@50
M 20mina
%LOX Inh. @100
Mb
AAPH%@100
Mc
Me syn
Me anti
Et syn
Et anti
Pr syn
Pr anti
i-Pr syn
i-Pr anti
Bu syn
Bu anti
Bn syn
Bn anti
NDGA
Trolox
63
l
8
dece
84
26
51
93
nad
26
89
11
81
91
9
39
86
7
43
86
13
44
72
12
49
89
7
56
89
11
91
82
11
73
76
15
83
98
81
64
l
l
a
b
c
% Interaction with DPPH (RA%).
In vitro % inhibition of soybean lipoxygenase (LOX).
% Inhibition of lipid peroxidation (AAPH).
No activity under the experimental conditions.
Not stable under the experimental conditions.
d
e

5106
D. Georgiou et al. / Bioorg. Med. Chem. 20 (2012) 5103–5109
O
NMR peak assignments were made by 2D NMR spectroscopy
(COSY, HSQC, and HMBC experiments).
O
H
O
N
N
N
MeS
OR
MeS
OR MeS
-H
OR
H
H
O
H
H
H
H
O
5.2.1. Methyl 3-(methylthio)-4,6-dioxo-5-phenyl-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2a
O
O
N
Ph
O
N
O
N
Ph
Ph
5.2.1.1. Compound 2a-syn.
White needles. Mp: 163.5–164 °C.
Rf (EtOAc/n-hexanes 1:2, v/v) 0.15. 1H NMR (250 MHz, CDCl3): dH
7.49–7.17 (m, 5H, aromatics), 5.20 (dd, 1H5, JH5,H4 = 9.51 Hz,
JH5,H3 = 0.93 Hz), 4.27 (dd, 1H3, JH3,H4 = 9.19 Hz, JH3,H5 = 0.93 Hz),
4.00 (dd, 1H4, JH4,H3 = 9.19 Hz, JH4,H5 = 9.51 Hz), 3.77 (s, 3H, OMe),
2.53 (s, 3H, –SCH3). 13C NMR (62.9 MHz, CDCl3): dC 174.2 (C@O),
170.9 (C@N), 170.5 (C@O), 169.9 [C(@O)OR], 131.3, 129.0, 128.8,
126.4 (aromatics), 75.7 (C5), 59.4 (C3), 52.6 (OMe), 47.1 (C4), 14.3
(–SMe). FT-IR (KBr pellet) 3484, 3066, 3045, 3002, 2950, 2936,
2847, 1785(C@O), 1715 (C@O),1575 (C@N), 1494, 1454, 1436,
1379, 1350, 1290, 1200, 1092, 1041, 993, 942, 916, 888, 871, 844,
823, 796, 770, 757, 736, 691, 656, 635, 618, 573, 544, 500, 475,
434, 413 cmꢂ1. UV–vis (CHCl3): kmax 244 nm. Anal. Calcd for
C15H14N2SO4: N, 8.80; C, 56.59; H, 4.43; S, 10.07. Found: N, 8.84;
C, 56.67; H, 4.45; S, 10.03. MS (FAB+) m/z: 319, [M+H]+. MS (ESI+)
m/z: 319.21 [M+H]+, [M]: 318.35. HRMS Calcd for C15H15N2SO4, m/
z: 319.0747, [M+H]+. Found: 319.0740.
Figure 3. Radical stabilization by resonance.
4. Conclusions
In summary, we show here that syn diastereoselectivity prevails
when a series of N,N-bis[sulfanyl]-substituted imines of glycinates
undergo 1,3-dipolar cycloaddition to N-phenylmaleimide, and the
two diastereomeric dihydro-pyrrolo adducts bearing a vinylic sul-
fide substituent were isolated and characterized in each reaction.
Furthermore, it was found that the size of the sulfanyl and alkoxy
substituents in the starting imine plays no definite role on the rate
or the diastereoselectivity of the reaction. Qualitatively, and also in
the light of a previous study the observed behavior is consonant
with a cycloaddition through an azomethine ylide dipole, although
more mechanistic studies are needed to settle more accurate the
mechanism. These cycloadducts present interesting biological
activities as antioxidant agents. Especially the anti- Bu and Bn sub-
stituted adducts with the higher lipophilicity values16 (clogP 3.31
and 3.38, respectively) highly inhibit lipid peroxidation and soy-
bean LOX and they might serve as lead compounds. Finally the
isolated diastereomeric cycloadducts showed a hyperoxidation
activity with three major standard methods.
5.2.1.2. Compound 2a-anti.
White needles. Mp: 170.5–
171 °C. Rf (EtOAc/n-hexanes 1:2, v/v) 0.28. 1H NMR (250 MHz,
CDCl3): dH 7.50–7.25 (m, 5H, aromatics), 5.21 (dd, 1H5,
JH5,H4 = 2.27 Hz, JH5,H3 = 2.04 Hz), 4.34 (dd, 1H3, JH3,H4 = 8.40 Hz,
JH3,H5 = 2.04 Hz), 4.13 (dd, 1H4, JH4,H3 = 8.40 Hz, JH4,H5 = 2.27 Hz),
3.84 (s, 3H, OMe), 2.53 (s, 3H, –SCH3). 13C NMR (62.9 MHz, CDCl3):
dC 175.1 (C@O), 171.0 (C@N), 170.9 (C@O), 170.1 [C(@O)OR],
131.1, 129.1, 128.9, 126.2 (aromatics), 76.7 (C5), 58.9 (C3), 53.0
(OMe), 48.1 (C4), 14.3 (–SMe). FT-IR (KBr pellet): 3478, 3006, 2976,
2955, 2853, 1782 (C@O), 1742 (C@O), 1713 (C@O), 1575 (C@N),
1498, 1442, 1387, 1351, 1321, 1278,,1209, 1188, 1186, 1097,
1032, 1013, 975, 956, 945, 915, 804, 788, 757, 733, 693, 662, 622,
571, 526, 494, 459, 381 cmꢂ1. UV–vis (CHCl3): kmax 243 nm. Anal.
Calcd for C15H14N2SO4: N, 8.80; C, 56.59; H, 4.43; S, 10.07. Found:
N, 8.75; C, 56.70; H, 4.45; S, 10.11. MS (FAB+) m/z: 319, [M+H]+.
MS (ESI+) m/z: 319.27 [M+H]+, [M]: 318.35. HRMS Calcd for
5. Experimental
5.1. General
All NMR spectra were taken in CDCl3 98% D, on a Bruker-Spec-
trospin, Avance spectrometer. ESI MS spectra were taken on a Agi-
lent Technologies 1100 Series LC/MSD-Trap-SL spectrometer. FAB
MS were taken on a MICROMASS ZABSPEC spectrometer (FAB
modus, with 3-nitrobenzylalcohol as matrix). HRMS were taken
on an Orbitrap LTQ/XL instrument. FT IR spectra were taken on a
Perkin Elmer Spectrum GX, FT IR System, spectrometer. UV–vis
spectra were recorded on a Hitachi U-2001 spectrophotometer.
All reagents and solvents were obtained from commercial suppli-
ers and used without further purification. Dry quality solvents
were obtain0 ed according to literature procedures,17 and stored
over MS 4ÅA under Ar atmosphere. Thus, PhMe distilled from Na
with benzophenone as an indicator. Compounds 1, were prepared
by known literature methods.5a
C
15H15N2SO4, m/z: 319.0747, [M+H]+. Found: 319.0745.
5.2.2. Methyl 3-(ethylthio)-4,6-dioxo-5-phenyl-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2b
5.2.2.1. Compound 2b-syn.
White powder. Mp: 169.5–170 °C.
Rf (EtOAc/n-hexanes 1:2, v/v) 0.25. 1H NMR (250 MHz, CDCl3): dH
7.46–7.24 (m, 5H, aromatics), 5.21 (dd, 1H5, JH5,H4 = 9.48 Hz,
JH5,H3 = 0.89 Hz), 4.26 (dd, 1H3, JH3,H4 = 9.20 Hz, JH3,H5 = 0.89 Hz),
3.98 (dd, 1H4, JH4,H3 = 9.20 Hz, JH4,H5 = 9.48 Hz), 3.77 (s, 3H, OMe),
3.20–3.06 (m, 2H, –SCH2–, not well resolved), 1.36 (t, 3H, –CH3,
J = 7.39 Hz); 13C NMR (62.9 MHz, CDCl3): dC 174.2 (C@O), 170.6
(C@N), 170.3 (C@O), 169.9 [C(@O)OR], 131.4, 129.2, 128.9, 126.5
(aromatics), 76.1 (C5), 59.8 (C3), 52.7 (-OMe), 47.0 (C4), 26.2 (–
SCH2–), 13.7 (Me); FT-IR (KBr pellet): 3488, 3063, 2945, 1720
(C@O),1573 (C@N), 1495, 1454, 1437, 1383, 1348, 1291, 1248,
1200, 1176, 1090, 1044, 943, 870, 826, 797, 732, 692, 661, 623,
545, 476, 425 cmꢂ1. UV–vis (CHCl3): kmax 240 nm. Anal. Calcd for
5.2. General procedure for 1,3-dipolar cycloaddditions; isolation
of adducts 2
A screw-capped 10 mL tube flashed with Ar. In it were placed
5.17 ꢁ 10ꢂ4 mol of glycine ester imine 1, and 5.69 ꢁ 10ꢂ4 mol of
N-Phenylmaleimide (NMP) in 3.5 mL of dry toluene. This solution
was stirred at ꢀ185 °C for 24 h. The tube was left to reach room
temperature. Volatiles were removed in a rotary evaporator and
the remaining material was then chromatographed on a silica gel
column (SiO2, 12 cm height), starting with a mixture of EtOAc/
n-hexanes, 1/8, v/v, as eluant, in order to remove safely the unreac-
tive starting materials without contaminating the desired
products. The solvent polarity was increased to elute the products
2. A mixture of EtOAc/n-hexanes, 1/3, v/v, was normally used as
eluant for efficient separation. Yields are listed in the text. Products
usually solidified by treatment with mixtures of CHCl3/n-hexanes.
C
16H16N2SO4: N, 8.43; C, 57.82; H, 4.85; S, 9.65. Found: N, 8.40; C,
57.94; H, 4.83; S, 9.62. MS (FAB+) m/z: 333, [M+H]+. MS (ESI+) m/z:
333.34 [M+H]+, [M]: 332.37. HRMS calcd for C16H17N2SO4, m/z:
333.0904, [M+H]+. Found: 333.0898.
5.2.2.2. Compound 2b-anti.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.47. 1H NMR (250 MHz, CDCl3): dH 7.46–7.17 (m, 5H, aromatics),
5.21 (dd, 1H5, JH5,H4 = 2.14 Hz, JH5,H3 = 2.02 Hz), 4.32 (dd, 1H3,
JH3,H4 = 8.39 Hz, JH3,H5 = 2.27 Hz), 4.02 (dd, 1H4, JH4,H3 = 8.39 Hz,
JH4,H5 = 2.27 Hz), 3.83 (s, 3H, OMe), 3.24 (qd, –SCH–, J1=13.02 Hz,

D. Georgiou et al. / Bioorg. Med. Chem. 20 (2012) 5103–5109
5107
J2 = 7.47 Hz), 3.13 (qd, –SCH–, J1 = 13.02 Hz, J2 = 7.47 Hz), 1.40 (t,
3H, –CH3, J1 = 7.47 Hz). 13C NMR (62.9 MHz, CDCl3): dC 175.2
(C@O), 170.9 (C@N), 170.4 (C@O), 170.3 [C(@O)OR], 131.2, 129.2,
128.9, 126.4, 126.2 (aromatics), 76.8 (C5), 59.1 (C3), 53.1 (OMe),
47.9 (C4), 26.1 (–SCH2–), 13.7 (Me). FT-IR (KBr pellet): 3488,
3063, 2945, 1720 (C@O), 1573 (C@N), 1495, 1454, 1437, 1383,
1348, 1291, 1248, 1200, 1176, 1090, 1044, 943, 870, 826, 797,
732, 692, 661, 623, 545, 476, 425 cmꢂ1. UV–vis (CHCl3): kmax
238 nm. Anal. Calcd for C16H16N2SO4: N, 8.43; C, 57.82; H, 4.85;
S, 9.65. Found: N, 8.36; C, 58.07; H, 4.81; S, 9.60. MS (FAB+) m/z:
333, [M+H]+. MS (ESI+) m/z: 333.34 [M+H]+, [M]: 332.37. HRMS
calcd for C16H17N2SO4, m/z: 333.0904, [M+H]+. Found: 333.0902.
C
17H18N2SO4: N, 8.09; C, 58.94; H, 5.24; S, 9.26. Found: N, 7.92;
C, 58.64; H, 5.27; S, 9.13. MS (FAB+) m/z: 347 [M+H]+.
5.2.4.2. Compound 2d-anti.
White powder. Mp: 136–137 °C.
Rf (EtOAc/n-hexanes 1:2, v/v) 0.46. 1H NMR (250 MHz, CDCl3): dH
7.46–7.26 (m, 5H, aromatics), 5.23 (dd, 1H5, JH5,H4 = 2.21 Hz,
JH5,H3 = 2.02 Hz), 4.29 (dd, 1H3, JH3,H4 = 8.32 Hz, JH3,H5 = 2.02 Hz),
4.05 (dd, 1H4, JH4,H3 = 8.32 Hz, JH4,H5 = 2.21 Hz), 3.90 (septet, par-
tially overlapped, –SCH<, J = 6.85 Hz), 3.83 (s, 3H, OMe), 1.41 (d,
3H, CH3, J = 6.85 Hz), 1.38 (d, 3H, CH3, J = 6.85 Hz). 13C NMR
(62.9 MHz, CDCl3): dC 175.2 (C@O), 171.0 (C@N), 170.4 (C@O),
170.1 [C(@O)OR], 131.2, 129.1, 128.8, 126.2 (aromatics), 76.9 (C5),
59.3 (C3), 53.0 (OMe), 47.7 (C4), 37.2 (–SCH<), 22.7 (Me), 22.5
(Me). FT-IR (KBr pellet): 3479, 2954, 2929, 2868, 1742 (C@O),
1718 (C@O), 1595, 1564 (C@N), 1496, 1439, 1387, 1320, 1276,
1239, 1205, 1185, 1086, 1031, 1012, 980, 957, 911, 806, 787, 738,
696, 665, 647, 625, 540, 494, 459 cmꢂ1. UV–vis (CHCl3): kmax
244 nm. Anal. Calcd for C17H18N2SO4: N, 8.09; C, 58.94; H, 5.24; S,
9.26. Found: N, 7.92; C, 58.84; H, 5.27; S, 9.13. MS (FAB+) m/z:
347 [M+H]+. HRMS calcd for C17H19N2SO4, m/z: 347.1060, [M+H]+.
Found: 347.1056.
5.2.3. Methyl 4,6-dioxo-5-phenyl-3-(propylthio)-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2c
5.2.3.1. Compound 2c-syn.
White powder. Mp: 145–146 °C.
Rf (EtOAc/n-hexanes 1:2, v/v) 0.35. 1H NMR (250 MHz, CDCl3): dH
7.49–7.23 (m, 5H, aromatics), 5.17 (dd, 1H5, JH5,H4 = 9.50 Hz,
JH5,H3 = 0.89 Hz), 4.23 (dd, 1H3, JH3,H4 = 9.16 Hz, JH3,H5 = 0.89 Hz),
3.95 (dd, 1H4, JH4,H3 = 9.16 Hz, JH4,H5 = 9.50 Hz), 3.76 (s, 3H, OMe),
3.13 (td, 1H, –SCH–, J1 = 12.76 Hz, J2=7.31 Hz), 3.06 (td, 1H, –SCH–
,J1 = 12.76 Hz, J2=7.31 Hz), 1.73 (sextet, 2H, –CH2–, J = 7.31 Hz),
1.01 (t, 3H, Me, J = 7.34 Hz). 13C NMR (62.9 MHz, CDCl3): dC 174.2
(C@O), 170.6 (C@N), 170.4 (C@O), 169.9 [C(@O)OR], 131.4, 129.1,
128.8, 126.5 (aromatics), 76.0 (C5), 59.7 (C3), 52.7 (OMe), 47.0
(C4), 33.6 (–S–CH2–), 21.9 (–CH2–), 13.4 (Me). FT-IR (KBr pellet):
3487, 2946, 2875, 1721 (C@O), 1575 (C@N), 1496, 1439, 1381,
1352, 1291, 1199, 1088, 1040, 944, 827, 768, 734, 691, 636, 544,
5.2.5. Methyl 3-(butylthio)-4,6-dioxo-5-phenyl-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2e
5.2.5.1. Compound 2e-syn.
White powder. Mp: 102–103 °C.
Rf (EtOAc/n-hexanes 1:2, v/v) 0.41. 1H NMR (250 MHz, CDCl3): dH
7.50–7.23 (m, 5H, aromatics), 5.17 (dd, 1H5, JH5,H4=9.51 Hz,
JH5,H3 = 0.86 Hz), 4.22 (dd, 1H3, JH3,H4 = 9.20 Hz, JH3,H5 = 0.86 Hz),
3.95 (dd, 1H4, JH4,H3 = 9.20 Hz, JH4,H5 = 9.51 Hz), 3.76 (s, 3H, OMe),
3.16 (td, 1H, –SCH–, J1 = 12.54 Hz, J2 = 7.35 Hz), 3.09 (td, 1H, –
SCH–, J1 = 12.54 Hz, J2=7.35 Hz), 1.68 (quintet, 2H, –CH2–,
J = 7.35 Hz), 1.43 (sextet, 2H, –CH2–, J = 7.31 Hz), 0.93 (t, 3H, Me,
J = 7.31 Hz). 13C NMR (62.9 MHz, CDCl3): dC 174.2 (C@O), 170.6
(C@N), 170.4 (C@O), 169.9 [C(@O)OR], 131.3, 129.1, 128.8, 126.4
(aromatics), 75.9 (C5), 59.7 (C3), 52.6 (OMe), 46.9 (C4), 31.4 (–S–
CH2–), 30.3 (–CH2–), 21.8 (–CH2–), 13.5 (Me). FT-IR (KBr pellet):
3490, 2937, 2841, 1784, 1722 (C@O), 1578 (C@N), 1438, 1384,
1352, 1291, 1244, 1198, 1085, 1038, 945, 889, 825, 797, 748,
734, 691, 638, 619, 545, 481, 442, 414 cmꢂ1. UV–vis (CHCl3): kmax
242 nm. Anal. Calcd for C18H20N2SO4: N, 7.77; C, 59.98; H, 5.59;
S, 8.90. Found: N, 7.81; C, 59.73; H, 5.57; S, 8.93. MS (FAB+) m/z:
361, [M+H]+. MS (ESI+) m/z: 361.41 [M+H]+, [M]: 360.43.
478 cmꢂ1
.
UV–vis (CHCl3): kmax 241 nm. Anal. Calcd for
C
17H18N2SO4: N, 8.09; C, 58.94; H, 5.24; S, 9.26. Found: N, 7.99; C,
58.75; H, 5.26; S, 9.22. MS (FAB+) m/z: 347 [M+H]+. HRMS calcd
for C17H19N2SO4, m/z: 347.1060, [M+H]+. Found: 347.1056.
5.2.3.2. Compound 2c-anti.
White powder. Mp: 151–152 °C.
Rf (EtOAc/n-hexanes 1:2, v/v) 0.47. 1H NMR (250 MHz, CDCl3): dH
7.50–7.25 (m, 5H, aromatics), 5.20 (dd, 1H5, JH5,H4=2.21 Hz,
JH5,H3 = 2.02 Hz), 4.32 (dd, 1H3, JH3,H4 = 8.38 Hz, JH3,H5 = 2.02 Hz),
4.08 (dd, 1H4, JH4,H3 = 8.38 Hz, JH4,H5 = 2.21 Hz), 3.83 (s, 3H, OMe),
3.17 (td, 1H, –SCH-, J1 = 12.80 Hz, J2 = 7.27 Hz), 3.05 (td, 1H, –SCH–
, J1=12.80 Hz, J2 = 7.27 Hz), 1.72 (sextet, 2H, –CH2–, J = 7.27 Hz)
1.01 (t, 3H, Me, J = 7.27 Hz). 13C NMR (62.9 MHz, CDCl3): dC 175.2
(C@O), 171.0 (C@N), 170.6 (C@O), 170.4 [C(@O)OR], 147.1, 129.2,
128.9, 126.2 (aromatics), 76.8 (C5), 59.2 (C3), 53.1 (OMe), 48.0 (C4),
33.7 (–S–CH2–), 21.9 (–CH2–), 13.4 (Me). FT-IR (KBr pellet): 3478,
3059, 2963, 2934, 2873, 1968, 1748, 1712 (C@O), 1578 (C@N),
1502, 1455, 1436, 1390, 1342, 1291, 1264, 1241, 1196, 1087,
1044, 1007, 990, 957, 941, 914, 807, 784, 756, 733, 694, 665, 618,
577, 541, 506, 480, 432 cmꢂ1. UV–vis (CHCl3): kmax 243 nm. Anal.
Calcd for C17H18N2SO4: N, 8.09; C, 58.94; H, 5.24; S, 9.26. Found:
N, 7.97; C, 58.846; H, 5.26; S, 9.15. MS (FAB+) m/z: 347 [M+H]+. HRMS
calcd for C17H19N2SO4, m/z: 347.1060, [M+H]+. Found: 347.1059.
5.2.5.2. Compound 2e-anti.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.53. 1H NMR (250 MHz, CDCl3): dH 7.50–7.24 (m, 5H, aromatics),
5.20 (dd, 1H5, JH5,H4 = 2.14 Hz, JH5,H3 = 1.99 Hz), 4.32 (dd, 1H3,
JH3,H4 = 8.34 Hz, JH3,H5 = 1.99 Hz), 4.09 (dd, 1H4, JH4,H3 = 8.34 Hz,
JH4,H5 = 2.14 Hz), 3.83 (s, 3H, OMe), 3.18 (td, 1H, –SCH–,
J1 = 12.72 Hz, J2 = 7.28 Hz), 3.08 (td, 1H, –SCH–, J1 = 12.72 Hz,
J2 = 7.28 Hz), 1.67 (quintent, 2H, –CH2–, J = 7.28 Hz), 1.42 (sextet,
2H, –CH2–, J = 7.30 Hz), 0.92 (t, 3H, Me, J = 7.30 Hz). 13C NMR
(62.9 MHz, CDCl3): dC 175.2 (C@O), 171.0 (C@N), 170.7 (C@O),
170.4 [C(@O)OR], 131.2, 129.2, 128.9, 126.3 (aromatics), 76.8
(C5), 59.2 (C3), 53.1 (OMe), 48.0 (C4), 31.5 (–S–CH2–), 30.4 (–CH2–
), 21.9 (–CH2–), 13.6 (Me). FT-IR (KBr pellet): 3437, 2954, 2915,
2856, 1742, 1721 (C@O), 1571 (C@N), 1491, 1386, 1207, 1191,
1086, 794, 735, 694, 464 cmꢂ1. UV–vis (CHCl3): kmax 244 nm. Anal.
Calcd for C18H20N2SO4: N, 7.77; C, 59.98; H, 5.59; S, 8.90. Found: N,
7.80; C, 59.81; H, 5.57; S, 8.96. MS (FAB+) m/z: 361, [M+H]+. MS
(ESI+) m/z: 361.40 [M+H]+, [M]: 360.43.
5.2.4. Methyl 3-(isopropylthio)-4,6-dioxo-5-phenyl-
1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2d
5.2.4.1. Compound 2d-syn.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.34. 1H NMR (250 MHz, CDCl3): dH 7.44–7.24 (m, 5H, aromatics),
5.21 (dd, 1H5, JH5,H4 = 9.53 Hz, JH5,H3 = 0.98 Hz), 4.23 (dd, 1H3,
JH3,H4 = 9.20 Hz, JH3,H5 = 0.98 Hz), 3.94 (dd, 1H4, JH4,H3 = 9.20 Hz,
JH4,H5 = 9.53 Hz), 3.86 (septet, 1H, –SCH<, J = 6.81 Hz), 3.77 (s, 3H,
OMe), 1.39 (d, 6H, 2-CH3, J = 6.81 Hz). 13C NMR (62.9 MHz, CDCl3):
dC 174.2 (C@O), 170.6 (C@N), 170.0 (C@O), 169.9 [C(@O)OR], 131.4,
129.1, 128.9, 126.5 (aromatics), 76.3 (C5), 60.0 (C3), 52.7 (OMe),
46.7 (C4), 37.3 (–SCH<), 22.7 (Me), 22.6 (Me). FT-IR (KBr pellet):
3340, 2959, 1718 (C@O), 1581 (C@N), 1498, 1383, 1187, 740,
5.2.6. Methyl 3-(benzylthio)-4,6-dioxo-5-phenyl-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2f
5.2.6.1. Compound 2f-syn.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.33. 1H NMR (250 MHz, CDCl3): dH 7.46–7.24 (m, 10H, aromatics),
5.21 (dd, 1H5, JH5,H4 = 9.51 Hz, JH5,H3 = 0.98 Hz), 4.42 (d, 1H,
692 cmꢂ1
.
UV–vis (CHCl3): kmax 242 nm. Anal. Calcd for

5108
D. Georgiou et al. / Bioorg. Med. Chem. 20 (2012) 5103–5109
benzylic, J = 13.14 Hz), 4.30 (d, 1H, benzylic, J = 13.14 Hz), 4.24 (dd,
1H3, JH3,H4 = 9.20 Hz, JH3,H5 = 0.98 Hz), 3.97 (dd, 1H4, JH4,H3 = 9.20 Hz,
JH4,H5 = 9.51 Hz), 3.78 (s, 3H, OMe). 13C NMR (62.9 MHz, CDCl3): dC
174.1 (C@O), 170.4 (C@N), 170.0 (C@O), 169.9 [C(@O)OR], 136.1,
131.4, 129.2, 129.1, 128.9, 128.6, 127.6, 126.5 (aromatics), 76.1
(C5), 59.6 (C3), 52.7 (OMe), 47.1 (C4), 36.1 (benzylic). FT-IR (KBr pel-
let): 3480, 3063, 3029, 2952, 1716 (C@O), 1581 (C@N), 1497, 1454,
1435, 1383, 1347, 1286, 1177, 1086, 1030, 944, 913, 873, 819, 766,
734, 693, 641, 618 cmꢂ1. UV–vis (CHCl3): kmax 242 nm. Anal. Calcd
for C21H18N2SO4: N, 7.10; C, 63.94; H, 4.60; S, 8.13. Found: N, 7.12;
C, 63.74; H, 4.57; S, 8.15. MS (FAB+) m/z: 395 [M+H]+, [M]: 394.44.
(C4), 14.4 (SMe). Anal. Calcd for C21H18N2SO4: N, 7.10; C, 63.94;
H, 4.60; S, 8.13. Found: N, 7.06; C, 64.21; H, 4.63; S, 8.10. MS
(FAB+) m/z: 417.0 [M+Na]+, 393.1 [MꢂH]ꢂ, [M]: 394.44.
5.2.8.2. Compound 2h-anti.
Oil. Rf (EtOAc/n-hexanes 1:2, v/
v) 0.28. 1H NMR (250 MHz, CDCl3): dH 7.47–7.23 (m, 5H, aro-
matics), 5.24 (br s, 3H, 2H benzylic+1H5), 4.30 (dd, 1H3,
JH3,H4 = 9.19 Hz, JH3,H5 = 1.87 Hz), 4.07 (dd, 1H4, JH4,H3 = 9.19 Hz,
JH4,H5 = 2.25 Hz), 2.50 (s, 3H, SMe). 13C NMR (62.9 MHz, CDCl3): dC
175.0 (C@O), 171.1 (C@N), 170.9 (C@O), 169.5 (C@O), 135.0,
131.0, 129.1, 128.8, 128.5, 128.4, 128,1, 126.3, 126.1 (aromatics),
76.7 (C5), 67.6 (benzylic), 58.9 (C3), 48.1 (C4), 14.3 (SMe). MS
(FAB+) m/z: 417.0 [M+Na]+, [M]: 394.44.
5.2.6.2. Compound 2f-anti.
130 °C; Rf (EtOAc/n-hexanes 1:2, v/v) 0.39; 1H NMR (250 MHz,
CDCl3): dH 7.49–7.23 (m, 10H, aromatics), 5.24 (dd, 1H5, JH5,H4
White powder, m.p. 129.5–
=
5.2.9. Michael adduct of MeSH to NPM
2.23 Hz, JH5,H3 = 2.04 Hz), 4.44 (d, 1H, benzylic, J = 13.08 Hz), 4.33
(dd, 1H3, JH3,H4 = 8.37 Hz, JH3,H5 = 2.04 Hz), 4.28 (d, 1H, benzylic,
J = 13.08 Hz), 4.12 (dd, 1H4, JH4,H3 = 8.37 Hz, JH4,H5 = 2.23 Hz), 3.84
(s, 3H, OMe). 13C NMR (62.9 MHz, CDCl3): dC 175.1 (C@O), 170.8
(C@N), 170.2 (C@O), 170.1 [C(@O)OR], 135.9, 131.1, 129.2, 129.1,
128.9, 128.6, 127.6, 126.2 (aromatics), 76.9 (C5), 59.1 (C3), 53.1
(OMe), 48.0 (C4), 36.1 (benzylic). FT-IR (KBr pellet): 3447, 3030,
2958, 2927, 1716 (C@O), 1574 (C@N), 1497, 1454, 1382, 1260,
1181, 1089, 1028, 911, 803, 747, 693, 618 cmꢂ1. UV–vis (CHCl3):
kmax 244 nm. Anal. Calcd for C21H18N2SO4: N, 7.10; C, 63.94; H,
4.60; S, 8.13. Found: N, 7.13; C, 63.78; H, 4.58; S, 8.16. MS (FAB+)
m/z: 395 [M+H]+, [M]: 394.44. HRMS calcd for C21H19N2SO4, m/z:
395.1060, [M+H]+. Found: 395.1052.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v) 0.33. 1H NMR (250 MHz) dH
(ppm): 7.47–7.23 (m, 5H, aromatics), 3.77 (dd, 1H3, J1 = 9.00 Hz,
J2 = 3.57 Hz), 3.28 (dd, 1H4a, J1 = 18.82 Hz, J2 = 9.00 Hz), 2.66 (dd,
1H4b, J1 = 18.82 Hz, J2 = 3.57 Hz), 2.36 (s, 3H, SMe). 13C NMR
(62.9 MHz, CDCl3): dC 175.2 (C@O), 173.6 (C@O), 134.1, 131.5,
128.7, 127.9, 126.0 (aromatics), 40.6 (C3), 35.8 (C4), 14.8 (SMe).
MS (FAB+) m/z: 244.0 [M+Na]+, [M]: 221.28.
5.2.10. tert-Butyl 3-(methylthio)-4,6-dioxo-5-phenyl-
1,3a,4,5,6,6a-hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2i
5.2.10.1. Compound 2i-syn.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.17. 1H NMR (250 MHz, CDCl3): dH 7.49–7.26 (m, 5H, aromatics),
5.01 (d, 1H5, JH5,H4 = 9.61 Hz), 4.23 (d, 1H3, JH3,H4 = 9.36 Hz), 3.95
(dd, 1H4, JH4,H5 = 9.61 Hz, JH4,H3 = 9.36 Hz), 2.52 (s, 3H, SMe), 1.48
(s, 9H, –OBu-t). 13C NMR (62.9 MHz, CDCl3): dC 174.0 (C@O),
170.8 (C@N), 170.4 (C@O), 168.8 (C@O), 131.5, 129.1, 128.8,
126.6 (aromatics), 83.1 (OBu-t), 76.9 (C5), 59.6 (C3), 47.3 (C4),
28.0 (CH3), 14.4 (SMe). Anal. Calcd for C18H20N2SO4: N, 7.77; C,
59.98; H, 5.59; S, 8.90. Found: N, 7.80; C, 59.71; H, 5.58; S, 8.94.
MS (FAB+) m/z: 383.0 [M+Na]+, 359.3 [MꢂH]ꢂ, [M]: 360.42.
5.2.7. Ethyl 3-(methylthio)-4,6-dioxo-5-phenyl-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2g
5.2.7.1. Compound 2g-syn.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.11. 1H NMR (250 MHz, CDCl3): dH 7.50–7.24 (m, 5H, aromatics),
5.18 (dd, 1H5, JH5,H4 = 9.53 Hz, JH5,H3 = 0.74 Hz), 4.26 (dd, 1H3,
JH3,H4 = 9.39 Hz, JH3,H5 = 0.74 Hz), 4.28–4.16 (m, 3H, –OCH2–,+H3),
3.99 (dd, 1H4, JH4,H3 = 9.39 Hz, JH4,H5 = 9.53 Hz), 2.52 (s, 3H, –SMe),
1.28 (t, 3H, Me, J = 7.22 Hz). 13C NMR (62.9 MHz, CDCl3): dC 174.1
(C@O), 170.9 (C@N), 170.6 (C@O), 169.6 [C(@O)OR], 131.4, 129.2,
128.9, 126.5 (aromatics), 76.1 (C5), 62.1 (–OCH2–), 59.6 (C3), 47.3
(C4), 14.5 (SMe), 14.0 (Me). Anal. Calcd for C16H16N2SO4: N, 8.43;
C, 57.82; H, 4.85; S, 9.65. Found: N, 8.46; C, 57.59; H, 4.88; S,
9.63. MS (FAB+) m/z: 355.0 [M+Na]+, [M]: 332.57.
5.2.10.2. Compound 2i-anti.
Oil. Rf (EtOAc/n-hexanes 1:2, v/
v) 0.39. 1H NMR (250 MHz, CDCl3): dH 7.50–7.24 (m, 5H, aro-
matics), 5.09 (dd, 1H5, JH5,H4 = 2.17 Hz, JH5,H3 = 1.99 Hz), 4.32 (dd,
1H3, JH3,H4 = 9.00 Hz, JH3,H5 = 1.99 Hz), 4.01 (dd, 1H4, JH4,H3 = 9.00 Hz,
JH4,H5 = 2.17 Hz), 2.52 (s, 3H, SMe) 1.51 (s, 9H, –OBu-t). 13C NMR
(62.9 MHz, CDCl3): dC 175.3 (C@O), 171.2 (C@N), 170.7 (C@O),
168.9 (C@O), 131.2, 129.2, 128.9, 126.3 (aromatics), 82.9 (OBu-t),
77.7 (C5), 58.9 (C3), 48.4 (C4), 27.9 (CH3), 14.6 (SMe). Anal. Calcd
for C18H20N2SO4: N, 7.77; C, 59.98; H, 5.59; S, 8.90. Found: N,
7.79; C, 59.79; H, 5.61; S, 8.94. MS (FAB+) m/z: 361.0 [M+H]+,
383.0 [M+Na]+, 359.3 [MꢂH]ꢂ, [M]: 360.42.
5.2.7.2. Compound 2g-anti.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.25. 1H NMR (250 MHz, CDCl3): dH 7.50–7.25 (m, 5H, aromatics),
5.19 (dd, 1H5, JH5,H4 = 2.19 Hz, JH5,H3 = 2.00 Hz), 4.34 (dd, 1H3,
JH3,H4 = 8.32 Hz, JH3,H5 = 2.00 Hz), 4.30 (dq, 1H, -OCH-, J1 = 7.13 Hz,
J2 = 3.22 Hz), 4.28 (dq, 1H, -OCH-, J1 = 7.13 Hz, J2 = 3.22 Hz), 4.11
(dd, 1H4, JH4,H3 = 8.32 Hz, JH4,H5 = 2.19 Hz), 2.53 (s, 3H, –SMe), 1.35
(t, 3H, Me, J = 7.13 Hz). 13C NMR (62.9 MHz, CDCl3): dC 175.2
(C@O), 171.0 (C@N), 169.8 (C@O), 131.1, 129.2, 128.9, 126.2 (aro-
matics), 76.9 (C5), 62.3 (–OCH2–), 58.9 (C3), 48.2 (C4), 14.4 (SMe),
14.1 (Me). Anal. Calcd for C16H16N2SO4: N, 8.43; C, 57.82; H,
4.85; S, 9.65. Found: N, 8.47; C, 58.07; H, 4.90; S, 9.58. MS (FAB+)
m/z: 355.0 [M+Na]+, [M]: 332.57.
5.3. Biological evaluation
All the reagents used were commercially available by Merck,
1,1-diphenyl-2-picrylhydrazyl (DPPH), nordihydroguaiaretic acid
(NDGA) were purchased from the Aldrich Chemical Co. Milwaukee,
WI, (USA). Soybean Lipoxygenase, linoleic acid sodium salt, indo-
methacin were obtained from Sigma Chemical, Co. (St. Louis, MO,
USA).
5.2.8. Benzyl 3-(methylthio)-4,6-dioxo-5-phenyl-1,3a,4,5,6,6a-
hexahydropyrrolo[3,4-c]pyrrole-1-carboxylate, 2h
5.3.1. Determination of the reducing activity of the stable
radical 1,1-diphenyl-picrylhydrazyl (DPPH)15
To a solution of DPPH (final concentration 50 lM) in absolute
ethanol was added an equal volume of the compounds dissolved
in dimethylsulfoxide. As control solution ethanol was used. The fi-
nal concentration of the test compounds was 50 lM. After 20 min
at room temperature, the absorbance was recorded at 517 nm
(Table 3)
5.2.8.1. Compound 2h-syn.
Oil. Rf (EtOAc/n-hexanes 1:2, v/v)
0.14. 1H NMR (250 MHz, CDCl3): dH 7.47–7.20 (m, 5H, aromatics),
5.18–5.20 (d+s, partially overlaped, 3H, 2H benzylic+H5), 5.18 (s,
2H, benzylic), 4.22 (d, 1H3, JH3,H4 = 8.94 Hz), 3.97 (dd, 1H4,
JH4,H3 = 8.94 Hz, JH4,H5 = 9.38 Hz), 2.52 (s, 3H, SMe). 13C NMR
(62.9 MHz, CDCl3): dC 174.1 (C@O), 171.0 (C@N), 170.6 (C@O),
169.5 (C@O), 134.9, 131.3, 129.1, 128.8, 128.6, 128.5, 128.4,
128,1, 126.5 (aromatics), 75.9 (C5), 67.8 (benzylic), 59.5 (C3), 47.2

D. Georgiou et al. / Bioorg. Med. Chem. 20 (2012) 5103–5109
5109
5.3.2. Soybean lipoxygenase inhibition study in vitro15
The in vitro study was evaluated as reported previously. The
test compounds dissolved in ethanol were incubated at room tem-
Soc. 2007, 129, 10646; (e) Ess, D. H.; Houk, K. N. J. Am. Chem. Soc. 2008, 130,
10187.
3. (a) Harwood, L. M.; Vickers, R. J. In Synthetic Applications of 1,3-Dipolar
Cycloaddition Chemistry Toward Heterocycles and Natural Products; Padwa, A.,
Pearson, W. H., Eds.; Wiley: New York, 2002. Chapter 3; (b) Tsuge, O.;
Kanemasa, S. Adv. Heterocycl. Chem. 1989, 45, 231; (c) Lown, J. W. In 1,3-Dipolar
Cycloaddition Chemistry; Padwa, A., Ed.; Wiley: New York, 1984. Chapter 6; (d)
Tsuge, O.; Kanemasa, S.; Ohe, M.; Yorozu, K.; Takenaka, S.; Ueno, K. Bull. Chem.
Soc. Jpn. 1987, 60, 4067; (e) Prassana, P.; Palamurugan, K.; Perumal, S.;
Vogeeswari, P.; Sriram, D. Eur. J. Med. Chem. 2010, 45, 5653; (f) Dandia, A.; Jain,
A. K.; Bhati, D. S. Tetrahedron Lett. 2011, 52, 5333; (g) Maheswari, S. U.;
Perumal, S.; Almansur, A. I. Tetrahedron Lett. 2012, 53, 349; (h) Maheswari, S.
U.; Balamurugan, K.; Perumal, S.; Vogeeswari, P.; Sriram, D. Bioorg. Med. Chem.
Lett. 2010, 20, 7278; (i) Li, Q.-H.; Xue, Z.-Y.; Tao, H.-Y.; Wang, C.-J. Tetrahedron
Lett. 2012, 53, 3650; (j) Wo, H.; Wang, B.; Liu, H.; Wang, L. Tetrahedron 2012, 67,
1210; (k) Martin-Rodriguez, M.; Najera, C.; Sansano, J. M.; Wu, F-L. Tetrahedron:
Asymmetry 2010, 21, 1184.
4. (a) Nájera, C.; Sansano, J. M. Curr. Org. Chem. 2003, 7, 1105; (b) Gothelf, K.;
Jørgensen, K. A. Chem. Rev. 1998, 98, 863; (c) Gothelf, K. V. In Cycloaddition
Reactions in Organic Synthesis; Kobayashi, S., Jørgensen, K. A., Eds.; Wiley-VCH:
Weinheim, 2002; pp 211–245; (d) Kanemasa, S. Synlett 2002, 1371; (e)
Huisgen, R.; Scheer, W.; Huber, H. J. Am. Chem. Soc. 1967, 89, 1753; (f) Grigg, R.;
Donegan, G.; Gunaratne, H. Q. N. Tetrahedron 1989, 45, 1723; (g) Grigg, R.;
Montgomery, J.; Somasunderam, A. Tetrahedron 1992, 48, 10431; (h) Copper, D.
M.; Grigg, R.; Hargreaves, S.; Kennewell, P.; Redpath, J. Tetrahedron 1995, 51,
7791; (i) Kanemasa, S.; Hayashi, T.; Tanaka, J.; Yamamoto, H.; Sakurai, T. J. Org.
Chem. 1991, 56, 4473; (j) Allway, P.; Grigg, R. Tetrahedron Lett. 1991, 32, 5817;
(k) Grigg, R.; Kilner, C.; Sarker, M. B. M.; Orgaz de la Cierva, C.; Dondas, H. A.
Tetrahedron 2008, 64, 8974; (l) Garner, P.; Kaniskan, H. Ü. J. Org. Chem. 2005, 70,
10868; (m) Stanley, L. M.; Sibi, M. P. Chem. Rev. 2008, 108, 2887; (n) Naodovic,
M.; Yamamoto, H. Chem. Rev. 2008, 108, 3132; (o) Álvarez-Corral, M.; Muñoz-
Dorado, M.; Rodriguez-García, I. Chem. Rev. 2008, 108, 3174; (p) Patil, N. T.;
Yamamoto, Y. Chem. Rev. 2008, 108, 3395; (q) Pellissier, H. Tetrahedron 2007,
63, 3235; Awata, A.; Arai, T. Chem. Eur. J. 2012, 18, 8278; (r) Tong, M.-C.; Li, J.;
Tao, H.-Y.; Li, Y.-X.; Wang, C-J. Chem. Eur. J. 2011, 17, 12922; (s) Rodriguez-
Martin, M.; Nacera, C.; Mansano, J. M.; de Gozar, A.; Cosio, F. P. Chem. Eur. J.
2011, 17, 14224; (t) Najera, C.; Sansano, J. Org. Biobol. Chem. 2009, 7, 4567; (u)
Huck, L.; Conzalez, J. F.; de la Guesta, E.; Menendez, J. C.; Avandano, C. Org.
Biomol. Chem. 2011, 9, 4567; (v) Yu, J.; Shi, F.; Gong, L-Z. Acc. Chem. Res. 2011,
44, 1156.
perature with sodium linoleate (100 lM) and 0.2 mL of enzyme so-
lution (1/9 ꢁ 10ꢂ4 w/v in saline). The conversion of sodium
linoleate to 13-hydroperoxylinoleic acid at 234 nm was recorded
and compared with the appropriate standard inhibitor (caffeic acid
600 lM) (Table 3)
5.3.3. Inhibition of linoleic acid peroxidation15
Production of conjugated diene hydroperoxide by oxidation of
linoleic acid in an aqueous dispersion was monitored at 234 nm.
2,20-Azobis(2-amidinopropane) dihydrochloride (AAPH) is used as
a free radical initiator. This assay can be used to follow oxidative
changes and to determine the inhibition of linoleic acid peroxida-
tion induced by each tested compound. Ten microliters of the
16 mM linoleic acid dispersion were added to the UV cuvette con-
taining 0.93 mL of 0.05 M phosphate buffer, pH 7.4, thermostatted
at 37 °C. The oxidation reaction was initiated at 37 °C under air by
the addition of 50
lL of 40 mM AAPH solution. Oxidation was car-
ried out in the presence of aliquots (10
l
L) of the tested com-
pounds. In the assay without antioxidant, lipid oxidation was
measured in the presence of the same level of DMSO. The rate of
oxidation at 37 °C was monitored by recording the increase in ab-
sorption at 234 nm caused by conjugated diene hydroperoxide for-
mation (Table 3).
Acknowledgments
This work supported by the Research Committee of the Univer-
sity of Ioannina, Program No. 667 and by Glasgow University. We
thank the MS and NMR centers at the University of Ioannina, and
especially Dr. Vasiliki Exarxou for 2D NMR experiments. The Insti-
tute of Organic Chemistry, University of Erlangen-Nürnberg, Ger-
many is gratefully acknowledged for NMR spectra, and FAB MS
measurements. Y.E. thanks Prof. Dr. Andreas Hirsch for the hospi-
tality, encouragement and helpful discussions during a sabbatical
leave at the Insitute of Organic Chemistry, University of Erlan-
gen-Nürnberg. We thank Prof. L. Hadjiarapoglou for comments
and discussions. We thank Biobyte and Dr. C. Hansch and A. Leo
for the free access to the C-QSAR program.
5. (a) Hoppe, D.; Beckmann, L. Liebigs Ann. Chem. 1979, 2066; (b) Lankiewicz, L.;
Nyasse, B.; Fransson, B.; Grehn, L.; Ragnarsson, U. J. Chem. Soc., Perkin Trans. 1
1994, 2503; (c) Oppolzer, W.; Moretti, R.; Thomi, S. Tetrahedron Lett. 1989, 30,
6009; (d) Oppolzer, W.; Moretti, R.; Zhou, C. Helv. Chim. Acta 1994, 77, 2363; (e)
Elemes, Y.; Ragnarsson, U. J. Chem. Soc., Perkin Trans. 1 1996, 537; (f) Elemes, Y.;
Ragnarsson, U. Chem. Commun. 1996, 935.
6. (a) Naxakis, G.; Sofou, P.; Elemes, Y. Fullerenes Nanot. Carbon Nanostruct. 2004,
12, 781; (b) Sofou, P.; Elemes, Y.; Panou-Pomonis, E.; Stavrakoudis, A.; Tsikaris,
V.; Sakarellos, C.; Sakarellos-Daitsiotis, M.; Maggini, M.; Formaggio, F.; Toniolo,
C. Tetrahedron 2004, 60, 2823; (c) Ioannou, E.; Hirsch, A.; Elemes, Y. Tetrahedron
2007, 63, 7070.
7. Damani, L. A. Sulphur containing drugs and related compounds: Chemistry,
Biochemistry and Toxicology In Part A: Metabolism of Sulfur Functional Groups;
Ellis Horwood Limited, 1989; Vol. 1,.
8. Hoveyda, H. R.; Hall, D. G. Org. Lett. 2001, 3, 3491.
9. Grigg, R.; Basanagoudar, D. A.; Kennedy, D. A.; Malone, J. F.; Thianpatanagul, S.
Tetrahedron Lett. 1982, 23, 2803.
Supplementary data
10. Prior, R. L.; Wu, X.; Schaich, K. J. Agric. Food Chem. 2005, 53, 4290.
11. Niki, E. Chem. Phys. Lipids 1987, 44, 227.
12. Liegois, C.; Lermusieau, G.; Colin, S. J. Agric. Food Chem. 2000, 48, 1129.
13. Huang, D.; Ou, B.; Prior, R. L. J. Agric. Food Chem. 2005, 53, 1841. and references
cited therein.
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.07.014.
14. Jovanovic, D. V.; Fermandes, J. C.; Martel-Pelletier, J.; Jolicoeur, F. C.; Reboul, P.;
Laufer, S.; Tries, S.; Pelletier, J. P. Arthritis Rheum. 2001, 2001, 2320.
15. Hadjipavlou-Litina, D.; Garnelis, T.; Athanassopoulos, C. M.; Papaioannou, D. J.
Enzyme Inhib. Med. Chem. 2009, 24, 1188.
16. Biobyte Corp., C-QSAR Database 201 West 4th Str., Suite 204, Claremont CA,
California 91711, USA.
References and notes
1. (a) Kobayashi, S.; Jorgensen, K. A. Cycloaddition Reaction in Organic Synthesis;
Wiley: New York, 2001; (b) Carruthers, W. Cycloaddition Reactions in Organic
Synthesis; Pergamon: Oxford, 1990.
2. (a)1,3-Dipolar Cycloaddition in Chemistry; Padwa, A., Ed.; Wiley: New York,
1984; (b) Gothelf, K. V.; Jorgensen, K. A. Chem. Rev. 1998, 98, 863; (c) Coldham,
I.; Hufton, R. Chem. Rev. 2005, 105, 2765; (d) Ess, D. H.; Houk, K. N. J. Am. Chem.
17. Armarego, W. L. F.; Perrin, D. D. Purification of Laboratory Chemicals; Reed
Educational and Profesional Publishing: Oxford, 1996.