Synthesis and biological evaluation of (2,5-dihydro-1H-pyrrol-1-yl)-2H- chromen-2-ones as free radical scavengers

Article abstract of DOI:10.1016/j.ejmech.2011.09.053

The allylation of aminocoumarins in the presence of excess of anhydrous K₂CO₃ and allyl bromide to diallylaminocoumarins is described. The Ring Closing Metathesis reaction of the later with the Grubbs' 1rst generation catalyst under reflux or MW irradiation has resulted mainly to (2,5-dihydro-1H-pyrrol-1-yl)coumarins and (1H-pyrrol-1-yl)coumarins. The new compounds were tested in vitro for their ability: (i) to interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, (ii) to inhibit lipid peroxidation, (iii) to scavenge the superoxide anion, (iv) to inhibit the activity of soybean lipoxygenase LO and (v) to scavenge hydroxyl radicals. Most of them were found to be potent lipid peroxidation inhibitors in vitro. The majority of the compounds showed significant hydroxyl radical scavenging activity. Compounds 11a and 12c presenting higher LO inhibitory activity as well as compound 17 were found to present a promising antioxidant and LO inhibitory profile.

Full text of DOI:10.1016/j.ejmech.2011.09.053

European Journal of Medicinal Chemistry 46 (2011) 5894e5901
Contents lists available at SciVerse ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Synthesis and biological evaluation of (2,5-dihydro-1H-pyrrol-1-yl)-2H-chromen-
q
2
-ones as free radical scavengers
Anna Balabani , Dimitra J. Hadjipavlou-Litina , Konstantinos E. Litinas *, Maria Mainou a,
a
b
a,
a
a
Crystal-Catherine Tsironi , Anastasia Vronteli
Laboratory of Organic Chemistry, Department of Chemistry, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
Department of Pharmaceutical Chemistry, School of Pharmacy, Aristotle University of Thessaloniki, Thessaloniki 54124, Greece
a
b
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 22 February 2011
Received in revised form
The allylation of aminocoumarins in the presence of excess of anhydrous K CO and allyl bromide to
2
3
diallylaminocoumarins is described. The Ring Closing Metathesis reaction of the later with the Grubbs’
rst generation catalyst under reflux or MW irradiation has resulted mainly to (2,5-dihydro-1H-pyrrol-1-
yl)coumarins and (1H-pyrrol-1-yl)coumarins. The new compounds were tested in vitro for their ability:
i) to interact with 1,1-diphenyl-2-picryl-hydrazyl (DPPH) stable free radical, (ii) to inhibit lipid perox-
idation, (iii) to scavenge the superoxide anion, (iv) to inhibit the activity of soybean lipoxygenase LO and
v) to scavenge hydroxyl radicals. Most of them were found to be potent lipid peroxidation inhibitors
1
26 September 2011
Accepted 29 September 2011
Available online 6 October 2011
(
(
Keywords:
in vitro. The majority of the compounds showed significant hydroxyl radical scavenging activity.
Compounds 11a and 12c presenting higher LO inhibitory activity as well as compound 17 were found to
present a promising antioxidant and LO inhibitory profile.
Ring Closing Metathesis
Grubbs’ catalysts
Allylation
(
(
2,5-Dihydro-1H-pyrrol-1-yl)coumarins
1H-Pyrrol-1-yl)coumarins
Ó 2011 Elsevier Masson SAS. All rights reserved.
Free radicals scavengers
1. Introduction
Anti-Inflammatory Drugs (NSAIDs) which simultaneously possess
radical scavenging activity [4].
Reactive oxygen species (ROS) play an essential role in the control
of cell functions. Many components of the vascular system, such as
leukocytes, monocytes and endothelial cells are able to release ROS
upon the appropriate stimulation. In vivo molecular oxygen is easily
converted to reactive free radicals, which contain the superoxide
Coumarin derivatives are an interesting class of heterocyclic
systems, since coumarin ring consist an essential core moiety for
a variety of natural and synthetic biologically active compounds
[5e8]. The biological activities include anticoagulation, antibiotic,
antifungal, antipsoriasis, antitumor, anti-HIV, anti-inflammatory,
etc. Functionalized pyrrolines are an important class of bioactive
compounds with diverse biological activities [9e12]. A variety of
synthetic strategies has been developed for the synthesis of those
compounds [10e20]. Two of them apply the Ring Closing Metath-
esis (RCM) [10,13,18], a powerful synthetic method [21] for the
construction of different ring systems. Moreover, since the combi-
nation of two pharmacophores on the same scaffold is a well-
ꢀ
ꢁ
ꢁ
anion (O ), hydroxyl radical (HO ) and they are highly reactive
2
substances that react with lipids, proteins and DNA, provoking irre-
versible changes of their biomolecular structure [1]. Tissues with
high oxygen consumption rate are more easily susceptible to oxida-
tive damage, due to the presence of excitatory amino acids, elevated
iron stores, cell membranes rich in polyunsaturated fatty acids and
low levels of the natural antioxidant glutathione in neurons [2].
Furthermore, blood brain barrier reduces the permeability and the
protective efficacy of most antioxidants [3]. Therefore it is evident
that the treatment of various diseases could benefit from the use of
drugs that present free radical scavenging activity. This has already
been proven for a number of commercially available Non Steroidal
established approach to the synthesis of more potent drugs
3
[22e27], we decided to incorporate the
D
-pyrroline ring moiety in
the coumarin derivatives and to examine the influence of this
modification on their activity as free radical scavengers.
2. Chemistry
q
Preliminary communication presented at 1st Hellenic Symposium Organic
Synthesis, November 4e6, 2004, Athens, Greece, Book of Abstracts, p. 84.
The reactions studied and the title new compounds received are
depicted in Schemes 1e3. 6-Aminocoumarin (1a) [prepared [28]
*
Corresponding author. Tel.: þ30 2310997864; fax: þ30 2310997679.
E-mail address: klitinas@chem.auth.gr (K.E. Litinas).
0223-5234/$ e see front matter Ó 2011 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2011.09.053

A. Balabani et al. / European Journal of Medicinal Chemistry 46 (2011) 5894e5901
5895
R
O
R
R
i
H
N
H2N
N
+
O
O
O
O
O
4
a,b
1a,b
3a,b
ii
1
,3,4,6,7a: 6-amino, R=H
b: 7-amino, R=CH3
R
O
R
O
(
PCy3)2Cl2Ru=CH-Ph
+
N
N
O
O
5
6a,b
7a,b
Scheme 1. Reagents and conditions: (i) Allyl bromide (2), K
2
3
CO , acetone, reflux, 5 days. (ii) Grubbs’ catalyst, first generation (5), DCM, reflux, 3 days (r.t., 5 days for 4b) or MW,
ꢂ
100 C, 1 h.
from coumarin quantitavely by nitration with HNO
through 6-nitrocoumarin followed by reduction (in 78% yield) of the
later with Et N/HCO H in the presence of 10% Pd-C] allylated by allyl
bromide (2) (2 equiv.) in the presence of K CO (4 equiv.) in refluxing
dry acetone for 5 days to give 6-allylaminocoumarin (3a) (18%) and
-diallylaminocoumarin (4a) (64%) (Scheme 1). Analogous allylation
of the commercially available 7-amino-4-methylcoumarin (1b)
resulted to 7-allylamino-4-methylcoumarin (3b) (41%) and 7-dially-
lamino-4-methylcoumarin (4b) (56%).
3
and H
2
SO
4
derivative 4b performed with the catalyst 5 (17 mol %) at r.t. for 5
days to give the dihydropyrrolocoumarin 6b (70%) and the pyrro-
locoumarin 7b (6%), while 9% of the starting material remained
3
2
1
2
3
unchanged. For the elucidation of compounds 6a and 7a, in the H-
NMR spectra there are two singlets at 4.13 (4H) and 5.98 (2H) for 3-
pyrroline 6a and two triplets at 6.38 (2H, J ¼ 2.1 Hz) and 7.06 (2H,
J ¼ 2.1 Hz) for pyrrole 7a. The formation of pyrrole derivative 7a
could be explained by the oxidation of dihydropyrrolocoumarin 6a
under the reaction conditions, although the reactions were per-
formed under Argon atmosphere.
The allylation of 6-amino-7-methylcoumarin (8a) [28] resulted
to 6-allylamino-7-methylcoumarin (9a) (32%) and 6-diallylamino-
7-methylcoumarin (10a) (52%) (Scheme 2). The products 9a and
10a received in 54% and 25% yield respectively, when the reaction
6
The Ring Closing Metathesis (RCM) of compound 4a in the
presence of the Grubbs’ catalyst first generation 5 (11 mol %, added
in 7 portions every 9e10 h) in refluxing dichloromethane (DCM) for
3
days led to the dihydropyrrolocoumarin derivative 6a in 66% yield
and the pyrrolocoumarin derivative 7a (27%). When the same
reaction with the catalyst 5 (6 mol %) is performed at r.t. for 3 days
the yields for compounds 6a and 7a are 12% and 2% respectively,
while 78% of the starting material 4a recovered. By application of
MW irradiation with the same catalyst 5 (3.5 mol %, added in 2
2 3
repeated with equimolar amount of anhydrous K CO , The RCM
reaction of the later with the first generation’s Grubbs catalyst
[11,12] 5 (8 moles %) in DCM solution under argon atmosphere at
room temperature for 4 days gave the 6-(2,5-dihydro-1H-pyrrol-1-
yl)-7-methyl-2H-chromen-2-one (11a) in 49% yield and 7-methyl-
6-(1H-pyrrol-1-yl)-2H-chromen-2-one (12a) (45%). The same
reaction under reflux for 2 days with the catalyst 5 (7% mol %) led to
ꢂ
portions after 30 min) in DCM at 100 C for 1 h derivative 6a is
isolated in 81% yield accompanied from 7a (6%) and unreacted
compound 4a (9%). The analogous RCM reaction of coumarin
H2N
H3C
N
N
H
i
+
O
O
O
O
O
O
H3C
3
H C
8
a-c
9a-c
10a-c
8
-12 a: 6-N-substituent, 7-methyl
b: 6-methyl, 7-N-substituent
c: 5-N-substituent, 6-methyl
ii
N
N
Mes-N
N-Mes
Ph
+
Cl
Cl
Ru
PCy3
O
O
O
O
H3C
H3C
1
1a-c
12a-c
13
Scheme 2. Reagents and conditions: (i) Allyl bromide (2), K
2 3
CO , acetone, reflux, 5 days. (ii) Grubbs’ catalyst, first generation (5), DCM, r.t., 3 days (reflux, 2 days for 10a or MW,
ꢂ
100 C, 1 h for 10a,c).

5896
A. Balabani et al. / European Journal of Medicinal Chemistry 46 (2011) 5894e5901
H
H
N
O
N
N
i
+
ii
O
O
O
O
O
N
O
14
16
15
O
17
2 3
Scheme 3. Reagents and conditions: (i) Allyl bromide (2), K CO , acetone, reflux, 12 days. (ii) Grubbs’ catalyst, first generation (5), DCM, r.t., 3 days.
compounds 11a (83%) and 12a (7%), while under MW irradiation
dihydropyrrolocoumarins and pyrrolocoumarins in moderate to very
goodyields atr.t. orunder reflux. Bythe application ofMWirradiation
the amounts of pyrrolocoumarins are diminished. The pyrrolocou-
marins (oxidation products) are favored, by prolonged reaction time
at r.t.
ꢂ
(
(
catalyst 5 3.5 mol % at 100 C for 1 h) resulted to derivatives 11a
75%) and 12a (9%) (12% of the starting material recovered).
The 6-methyl-7-nitrocoumarin (starting material for the
synthesis of aminocoumarin 8b) is received by the nitration of 6-
methylcoumarin with a mixture of HNO and H SO in 22% yield
3
2
4
after the separation by column chromatography from the main
nitration product 6-methyl-5-nitrocoumarin [28] (49%). This
nitrocoumarin by reduction resulted to aminocoumarin 8b (56%),
which by allylation gave 7-allylamino-6-methylcoumarin (9b)
3. Biology
Taking the multifactorial character of oxidative stress into
account, we decided to evaluate the in vitro antioxidant activity of
the synthesized molecules using four different antioxidant assays.
Coumarins are referred to affect the formation and scavenging of
ROS, exhibiting tissues protective antioxidant properties, which
may include numerous different molecular mechanisms and are
probably related to their structural analogy with flavonoids and
benzophenones [31]. Thus, they can bind Fe (III) and through this
reaction to inhibit hydroxyl radical and hydrogen peroxide forma-
tion produced by Fenton’s reactions.
Several methods are used for the estimation of efficiency of
synthetic/natural antioxidants, like the 2,2 -azobis(2-amidino-
propane) dihydrochloride (AAPH)/linoleic acid assay [32], 1,1-
dipheny l-1-picrylhydrazyl (DPPH) assay [33]. DPPH assay is one
of the best-known, frequently employed and accurate methods.
(
2
48%) and 7-diallylamino-6-methylcoumarin (10b) (51%) (Scheme
). The RCM reaction of the later at r.t. for 4 days gave the 7-(2,5-
dihydro-1H-pyrrol-1-yl)-6-methyl-2H-chromen-2-one (11b) in
4% yield and 6-methyl-7-(1H-pyrrol-1-yl)-2H-chromen-2-one
12b) (21%) (14% of the starting material 10b recovered) after the
6
(
separation by PTLC.
The 5-allylamino-6-methylcoumarin (9c) (80%) and 5-diallyla-
mino-6-methylcoumarin (10c) (7%) (Scheme 2) are synthesized by
the allylation of aminocoumarin 8c [29] with equimolar amounts of
0
anhydrous K
llylcoumarin 10c at r.t. for 4 days in the presence of the catalyst 5
12 mol %) resulted to 5-(2,5-dihydro-1H-pyrrol-1-yl)-6-methyl-2H-
CO
2 3
and allyl bromide (2). The RCM reaction of dia-
(
chromen-2-one (11c) (56%), 6-methyl-5-(1H-pyrrol-1-yl)-2H-chro-
men-2-one (12c) (21%) and unreacted starting material 10c (19%)
after the separation of the reaction mixture by PTLC. The same
reaction for 15 days gave 11c (37%), 12c (44%) and 10c (18%). This
reaction was performed under MW irradiation (catalyst 5 3.5 mol %
Reduction of DPPH stable free radical (50
compounds at 50 and 100 M [34], was studied after 20 and 60 min
(Table 1). The results indicate their radical scavenging ability in an
iron-free system. No result was observed at 50 M. The results for
the 100 M are time independent with the exception of compounds
mM) by the examined
m
m
ꢂ
at 100 C for 1 h) and led to pyrrolino derivative 11c (65%), pyrrolo
m
derivative 12c (10%) and unreacted compound 10c (19%). When we
use the second generation Grubbs’ catalyst 13 (12 mol %) under
stirring at r.t. for 2 days we received again the compounds 11c (46%),
6a, 6b and 17. We tested the new derivatives in comparison to
a well-known antioxidant agent, for example, nordihydroguaiaretic
acid (NDGA). The presented % values are low compared to NDGA
with the exception of compound 6a (Table 1). No significant
differences are observed among (2,5-dihydro-1H-pyrrol-1-yl)-2H-
chromen-2-ones. Free coumarin as well as 7-methylcoumarin
12c (20%) and the deallylation products 9c (20%) and 8c (13%). The
tertiary amine’s deallylation has been referred earlier in the litera-
ture by using the catalyst 5 as a method of choice [30].
The allylation reaction of 4-allylaminocoumarin (14) [29] in the
presented very low reducing abilities. The results are indepen-
3
presence of K
acetone for 12 days resulted to 3-allyl-4-allylaminocoumarin (15)
31%) and 4-diallylaminocoumarin (16) (54%), while 14% of the
2
CO
3
with excess of allyl bromide (2) under reflux in
dent of the position of methyl- and/or of the
D
-pyrroline ring.
Hydroxyl radicals are among the most reactive oxygen species
and are considered to be responsible for some of the tissue damage
occurring in inflammation. It has been claimed that hydroxyl radical
scavengers could serve as protectors. The competition of compounds
(
starting material was recovered. Compound 15 is formed most
probably after Claisen rearrangement of 4-allylaminocoumarin (14)
followed by allylation of the intermediate 4-aminocoumarin
derivative. The RCM reaction of coumarin 16 with the catalyst 5
9 mol %) at r.t. for 3 days led to 4-(2,5-dihydro-1H-pyrrol-1-yl)-2H-
chromen-2-one (17) (75%), while 23% of the starting compound
was remained unchanged.
ꢁ
with dimethyl sulfoxide (DMSO) for OH radicals generated by the
3þ
Fe /ascorbic acid system, expressed as the inhibition of formalde-
hyde production [35], was used for the evaluation of their hydroxyl
radical scavenging activity. All the tested derivatives highly compete
(
with DMSO (33 mM) at 100 mM in comparison to trolox (Table 1).
From the above reactions we can firstly conclude that the allyla-
Perusal of the % results shows that derivatives 6a, 6b, 11a, 12b, 12c
and 17 are more potent in comparison to trolox used as a standard.
Compound 11b presents lower activity (41%). The hydroxyl radical
2 3
tionwith excess of anhydrousK CO (4 equivalents)and allyl bromide
(
2 equivalents) leads mainly to diallylaminocoumarins, while with
equimolar amounts of K
allylaminocoumarins are prepared. The 1rst generation Grubbs’
catalyst is efficient in the RCM reactions for the preparation of
2
CO
3
and allyl bromide the mono-
scavenging activity is similar for 6a and 17, regardless the position of
3
D
-pyrroline ring (4 or 7). No result is given by compound 11c,
whereas the activity for 7b is very low. Lipophilicity, as theoretically

A. Balabani et al. / European Journal of Medicinal Chemistry 46 (2011) 5894e5901
5897
Table 1
ꢁ
Interaction % with 1,1-diphenyl-2-picrylhydrazyl (DPPH) at 0.1 mM; Competition % with DMSO for hydroxyl radical (HO %) at 100
mM; Inhibition of lipid peroxidation at
100
m
M (LP %); In vitro inhibition of soybean lipoxygenase (LO) % at 100
mM.
ꢁ
Clog P36
No
DPPH % 20 min DPPH % 60 min
HO (%)
LP % inh.
LO % inh.
at 100
m
M
at 100
m
M
at 100
m
M
at 100
m
M
at 100 mM
6
6
7
7
1
1
1
1
1
1
1
a
b
a
b
1a
1b
1c
2a
2b
2c
7
49
5
18
14
9
5
5
14
13
7
71
20
19
14
10
7
90
85
No
9
77
41
No
No
88
91
83
78
44
86
87
1
No
28
No
12
1.91
2.41
2.73
3.23
2.41
2.41
2.41
3.23
3.23
3.23
2.08
1.41
1.91
46
91
24
57
No
No
71
100
Nt
60
52.5
No
No
No
55.5
43
15
89
84
5
14
13
7
27
21
2
8
5
2
81
Coumarin
-CH -coumarin
NDGA
Trolox
7
3
No
83
73
63
NDGA, nordihydroguaeretic acid; no, no result under the experimental conditions; each experiment was performed at least in triplicate and the standard deviation of
absorbance was less than 10% of the mean.
calculated values, is not well correlated with the results [36]. Thus,
the nitroblue tetrazolium method [34]. The compounds did not
present scavenging activity at 100 M.
The synthesized derivatives were evaluated for inhibition of
soybean lipoxygenase by the UV-absorbance-based enzyme assay
[34] and the results are presented in Table 1. As far as these deriv-
atives are concerned, their majority did not show significant LO
the presence of a methyl group in 6b compared to 6a (calculated
m
3
log P value 6a ¼ 1.91, 6b ¼ 2.41) does not elevate the activity. The
D
-
pyrroline derivatives 6a, 6b and 11a are more potent than the cor-
responding pyrrolyl derivatives 7a, 7b and 12a. This concept is not
3
followed by
D
-pyrroline derivative 11b (41%), which presents lower
competition than the corresponding pyrrole derivative 12b (88%), as
inhibitory activity at 100 mM. Among them compounds, 11a, 11b and
3
well as
D
-pyrroline derivative 11c (no) compaired to pyrrole
12c exhibit better LO inhibitory activity. Lipophilicity seems to be an
important physicochemical parameter for the increase of LO inhi-
bition of compound 11b (2.41) compared to 6a (1.91). It is interesting
that 7-methylcoumarin inhibits LO, whereas 6a does not. Both
present similar lipophilicity (1.91) regardless the specific moiety of
derivative 12c (91%). Control experiments showed that free
coumarin presents significant scavenging behavior followed by 7-
methylcoumarin. The later with CLOG P value similar to 6a pres-
ents lower activity. Thus, the specific structural characteristics of the
new synthesized derivatives are responsible for the increase of the %
competition with DMSO for hydroxyl radicals.
substitution at position 7. Comparing 11a to 12a and 11b to 12b the
3
biological response of
D
-pyrroline derivatives (11a,b) is better than
The water soluble azo compound AAPH has been extensively
used as a clean and controllable source of alkylperoxyl free radicals.
In our studies AAPH was used as a free radical initiator to follow
oxidative changes of linoleic acid to conjugated diene hydroper-
oxide [31,34]. In our experiments compounds 17,11a, 6a, 6b and 12c
the corresponding of the pyrrole derivatives (12a,b), with the
exception of 12c (55.5%), whereas 11c does not present any result.
Our study indicates that LPO and LO inhibitory activity is not
accompanied by DPPH radical scavenging activity. In our case the
hydroxyl radical scavenging activity is followed by efficient LPO
inhibitory activity.
showed excellent inhibition on lipid peroxidation (LPO) at 100
mM
(Table 1) compared to trolox, used as a standard (73%), whereas
compound 11c and 7b followed with lower inhibition (57% and 46%
respectively) and 11b was found to present much lower activity
4. Conclusion
(
24%). Compounds 7a, 12a, 12b do not seem to influence lipid
In summary, the broad spectrum of the observed antioxidant
activity of the majority of the examined (2,5-dihydro-1H-pyrrol-1-
yl)-2H-chromen-2-ones allows us to propose them as templates in
the design of compounds useful in treating human diseases that
involves reactive oxygen species (ROS). Their synthesis is almost
simple with moderate to high yields. Most of them are potent
hydroxyl radical scavengers and inhibit in vitro lipid peroxidation.
Antioxidant power might be important in the inhibition of lipid
peroxidation. Compounds 11a and 12c presenting higher LO
inhibitory activity among the tested derivatives, as well as
compound 17 were found to present a promising antioxidant and
LO inhibitory profile. Attempts have been made to develop some
preliminary structure activity conclusions based on the compounds
synthesized and tested.
peroxidation under our experimental conditions. No differences
were observed between the 6a and 6b analogues. Thus, the pres-
ence of a methyl group, as well as the increase in lipophilicity (2.41
from 1.91), does not seem to influence the LPO. On the contrary
significant differences are observed among 11a, 11b and 11c.
Although all present similar lipophilicity, the differences in the
substitution’s position led to different responses. Comparing 6a to
1
3
7, it is better the
D
-pyrroline ring to be located at position 4
instead of 7. The small increase in lipophilicity (2.08) enhances the
inhibition. Comparing 11a to 12a as well as 11b to 12b it seems
%
3
that the
activity. Among the 5-, 6- or 7-
substituted is more potent followed by 7-N and 5-N-substituted.
The 6-N (12a) and 7-N (12b) pyrrolyl substituted derivatives did not
show any anti-LPO activity with the exception of 12c (12c > 11c
D -pyrroline derivatives present enhanced anti-LPO
3
D
-pyrroline derivatives the 6-N-
5. Experimental protocol
71% > 57%).
Non-enzymatic superoxide anion radicals were generated. The
superoxide producing system was set up by mixing phenazine
methosulfate (PMS), nicotinamideeadenineedinucleotide NADH
and aireoxygen. The production of superoxide was estimated by
5.1. Chemistry
For the experiments under MW irradiation a Biotage (Initiator
2.0) scientific microwave oven has been used. Melting points were

5898
A. Balabani et al. / European Journal of Medicinal Chemistry 46 (2011) 5894e5901
determined on a Kofler hot-stage apparatus and are uncorrected. IR
spectra were obtained with a PerkineElmer 1310 spectrophotom-
eter as Nujol mulls. NMR spectra were recorded on a Bruker AM 300
5.1.2. Synthesis of 7-(diallylamino)-4-methyl-2H-chromene-2-one
(4b) and 7-(allylamino)-4-methyl-2H-chromene-2-one (3b)
From the reaction of 7-amino-4-methylcoumarin (1b) with allyl
bromide (2) the 7-allylamino-4-methylcoumarin (3b) (41%) and
7-diallylamino-4-methylcoumarin (4b) (56%) were received.
1
13
(
3
300 MHz and 75 MHz for H and C, respectively) using CDCl as
solvent and TMS as an internal standard. J values are reported in Hz.
Mass spectra were determined on a VG-250 spectrometer at 70 eV
under Electron Impact (EI) conditions or on a Shimadzu LCMS-2010
EV system under Electrospray Ionization (ESI) conditions. Micro-
analyses were performed on a PerkineElmer 2400-II Element
analyzer (Table 2). Analyses indicated by the symbols of the
elements or functions were within ꢃ0.4% of the theoretical values.
Silica gel No. 60, Merck A.G. was used for column chromatography.
5
.1.2.1. 7-(Diallylamino)-4-methyl-2H-chromene-2-one (4b). Yellow
ꢂ
crystals, m.p. 50e52 C (DCM-hexane), IR (Nujol): 3080, 1725,
ꢀ1
1
1590 cm ; H-NMR (CDCl
3
)
d
2.34 (s, 3H), 3.98 (d, 4H, J ¼ 4.1 Hz),
.17 (d, 2H, J ¼ 15.3 Hz), 5.21 (d, 2H, J ¼ 8.9 Hz), 5.78e5.91 (m, 2H),
.97 (s, 1H), 6.56 (s, 1H), 6.61 (d, 1H, J ¼ 8.9 Hz), 7.38 (d, 1H,
5
5
13
J ¼ 8.9 Hz); C-NMR (CDCl
26.5, 128.4, 132.4, 138.1, 144.0, 150.3, 161.4; MS (EI): 255 (M ,
00%), 254 (63), 227 (68), 213 (34), 186 (86), 184 (41), 159 (64), 158
47),103 (55); Anal. C16 (C, H, N).
3
) d 18.4, 53.0, 109.1, 109.5, 119.9, 125.5,
þ
ꢁ
1
1
(
5.1.1. General procedure for the allylation of aminocoumarins.
Synthesis of 6-(diallylamino)-2H-chromene-2-one (4a) and 6-
H17NO
2
(
allylamino)-2H-chromene-2-one (3a)
Anhydrous K CO (1.846 g, 13.3 mmol) and allyl bromide (2)
0.6 ml, 0.858 g, 7 mmol) were added to the solution of compound
a [28] (0.536 g, 3.33 mmol) in dry acetone (70 ml) and the mixture
2
3
5
.1.2.2. 7-(Allylamino)-4-methyl-2H-chromene-2-one (3b), m.p.
(
1
ꢂ
ꢂ
107e109 C (DCM/hexane) (lit.[37] m.p. 110 C).
was refluxed for 5 days. The mixture was filtered and the filtrate was
evaporated and separated by column chromatography (silica gel no.
(d) 6-(Diallylamino)-7-methyl-2H-chromene-2-one (10a) (52%) (the
yield is only 25% with equimolar amount of K
2
CO
3
), m.p.
6
0, DCM) to give 4a (0.513 g, 64% yield) and 3a (0.12 g, 18% yield).
ꢂ
ꢀ1
1
23e125 C (DCM-hexane), IR (Nujol): 3045, 1730, 1570 cm
;
1
H-NMR (CDCl
H, J ¼ 9.8 Hz), 5.15 (d, 2H, J ¼ 17.7 Hz), 5.69e5.83 (m, 2H), 6.32
d, 1H, J ¼ 9.8 Hz), 7.02 (s, 1H), 7.15 (s, 1H), 7.60 (d, 1H,
3
)
d
2.40 (s, 3H), 3.57 (d, 4H, J ¼ 5.9 Hz), 5.10 (d,
5
.1.1.1. 6-(Diallylamino)-2H-chromene-2-one (4a). Yellow crystals,
2
(
ꢂ
m.p. 53e55 C (ethyl acetateehexane); IR (Nujol): 3060, 1710,
ꢀ1 1
1
555 cm ; H-NMR (CDCl
J ¼ 11.1 Hz), 5.18 (d, 2H, J ¼ 16.3 Hz), 5.70e5.92 (m, 2H), 6.35 (d, 1H,
J ¼ 9.5 Hz), 6.64 (d, 1H, J ¼ 2.6 Hz), 6.90 (dd, 1H, J ¼2.6 Hz,
3
)
d
3.94 (d, 4H, J ¼ 2.6 Hz), 5.17 (d, 2H,
13
J ¼ 9.8 Hz); C-NMR (CDCl
3
) d 18.7, 55.9, 115.5, 116.7, 117.6,
118.7, 120.3, 134.6, 140.1, 144.3, 146.6, 150.2, 161.2; MS (EI): 255
þ
ꢁ
1
(M , 100%), 254 (65), 228 (39), 214 (49), 199 (35), 187 (76), 158
38),130 (62); Anal. C16 (C, H, N).
13
J
(
1
1
2
¼ 9.4 Hz), 7.17 (d, 1H, J ¼ 9.4 Hz), 7.61 (d, 1H, J ¼ 9.5 Hz); C-NMR
CDCl 53.2, 109.1, 116.4, 116.5, 117.1, 117.8, 119.2, 132.3, 133.2,
42.5, 143.8, 161.4; MS (EI) m/z: 241 (M , 32%), 215 (100), 201 (14),
73 (71), 145 (13), 117 (51), 89 (58); Anal. C15 (C, H, N).
(
H17NO
2
3
) d
þ
ꢁ
(
e) 7-(Diallylamino)-6-methyl-2H-chromene-2-one (10b) (51%), m.p.
H15NO
2
ꢀ1 1
ꢂ
57e60 C (DCM-hexane), IR (Nujol): 3040, 1725, 1600 cm ; H-
NMR (CDCl
3
)
d
2.26 (s, 3H), 3.60 (d, 4H, J ¼ 5.9 Hz), 5.10 (d, 2H,
5
.1.1.2. 6-(Allylamino)-2H-chromene-2-one (3a). Yellow crystals,
J ¼ 9.9 Hz), 5.14(d, 2H, J ¼ 16.7 Hz),5.63e5.77(m, 2H), 6.16(d,1H,
ꢂ
m.p. 88e90 C (DCM-hexane); IR (Nujol): 3335, 3050, 1725,
13
J ¼ 9.8 Hz), 6.81 (s, 1H), 7.15 (s, 1H), 7.51 (d, 1H, J ¼ 9.8 Hz); C-
ꢀ
1 1
1550 cm ; H-NMR (CDCl
3
)
d
3.80 (d, 2H, J ¼ 5.0 Hz), 4.50 (brs, 1H),
.19 (d, 1H, J ¼ 9.5 Hz), 5.30 (d, 1H, J ¼ 17.2 Hz), 5.90e5.91 (m, 1H),
.37 (d, 1H, J ¼ 9.8 Hz), 6.59 (d, 1H, J ¼ 2.8 Hz), 6.83 (dd, 1H,
¼ 2.8 Hz, ¼ 7.3 Hz), 7.16 (d, 1H, J ¼ 7.3 Hz), 7.60 (d, 1H,
NMR (CDCl
3
)
d
18.4, 54.9, 108.7, 113.6, 118.0, 118.2, 129.0, 129.6,
5
6
þ
ꢁ
1
2
C
33.9,143.2,153.1,153.7,161.5; MS (EI): 255 (M ,100%), 254 (65),
28 (39), 214 (49), 199 (35), 187 (76), 158 (38),130 (62); Anal.
(C, H, N).
J
1
J
2
15 2
H15NO
1
3
J ¼ 9.8 Hz); C-NMR (CDCl
19.4, 134.7, 143.4, 144.8, 146.9, 161.3; MS (EI) m/z: 201 (M , 100%),
75 (61), 161 (67), 144 (16), 133 (49), 118 (27), 104 (34), 91 (25);
3
) d 46.8, 108.6, 116.7, 116.8, 117.5, 118.3,
þ
ꢁ
1
1
(
f) 5-(Diallylamino)-6-methyl-2H-chromene-2-one (10c) (51%), m.p.
ꢂ
ꢀ1 1
7
4e76 C (DCM-hexane), IR (Nujol): 3060, 1710, 1578 cm ; H-
2
Anal. C12H11NO (C, H, N).
3
NMR (CDCl ) d
2.35 (s, 3H), 3.72 (d, 4H, J ¼ 5.9 Hz), 5.08 (d, 2H,
J ¼ 9.9 Hz), 5.13 (d, 2H, J ¼ 17.7 Hz), 5.68e5.90 (m, 2H), 6.37 (d,
1
8
H, J ¼ 9.8 Hz), 7.06 (d, 1H, J ¼ 8.9 Hz), 7.29 (d, 1H, J ¼ 8.9 Hz),
Table 2
Elemental analyses.
13
.14 (d, 1H, J ¼ 9.8 Hz); C-NMR (CDCl
3
) d 19.0, 56.5, 113.8,115.3,
117.5, 118.7, 132.9, 134.5, 135.1, 141.7, 146.4, 153.2, 161.0; MS (EI):
Elemental analyses
Calculated
þ
ꢁ
2
55 (M , 11%), 254 (10), 240 (33), 228 (21), 214 (76), 198 (81),
(C, H, N).
Found
186 (85), 170 (95), 158 (69), 130 (100); Anal. C16
H17NO
2
3
4
4
6
6
7
7
1
1
1
1
1
1
1
1
1
1
1
a
a
b
a
b
a
b
(C12
(C15
(C16
(C13
(C14
(C13
(C14
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
11NO
15NO
17NO
11NO
13NO
2
2
2
2
)
)
)
)
)
C% 71.62, H% 5.51, N% 6.96 C% 71.58, H% 5.67, N% 6.59
C% 74.67, H% 6.27, N% 5.81 C% 74.46, H% 6.41, N% 5.65
C% 75.26, H% 6.79, N% 5.49 C% 75.54, H% 6.63, N% 5.32
C% 73.21, H% 5.20, N% 6.57 C% 73.07, H% 5.32, N% 6.50
C% 73.98, H% 5.77, N% 6.17 C% 73.69, H% 5.49, N% 6.36
C% 73.91, H% 4.30, N% 6.63 C% 74.02, H% 4.51, N% 6.49
C% 74.65, H% 4.92, N% 6.22 C% 74.52, H% 4.75, N% 6.38
C% 75.26, H% 6.72, N% 5.49 C% 74.94, H% 6.65, N% 5.48
C% 74.67, H% 6.27, N% 5.81 C% 74.48, H% 6.43, N% 5.62
C% 75.26, H% 6.72, N% 5.49 C% 75.20, H% 6.60, N% 5.29
C% 73.98, H% 5.77, N% 6.17 C% 74.05, H% 5.51, N% 6.06
C% 73.98, H% 5.77, N% 6.17 C% 73.76, H% 5.89, N% 6.32
C% 73.98, H% 5.77, N% 6.17 C% 74.16, H% 5.58, N% 6.02
C% 74.65, H% 4.92, N% 6.22 C% 74.43, H% 4.86, N% 6.13
C% 74.65, H% 4.92, N% 6.22 C% 74.76, H% 4.69, N% 6.45
C% 74.65, H% 4.92, N% 6.22 C% 74.71, H% 4.71, N% 6.29
C% 74.67, H% 6.27, N% 5.81 C% 74.62, H% 6.50, N% 5.89
C% 73.21, H% 5.20, N% 6.57 C% 73.02, H% 5.34, N% 6.34
(
g) 4-(Diallylamino)l-2H-chromene-2-one (16) (from 1.3 equiv. of
ꢂ
K
2
CO
3
, after refluxing for 12 days) (54%), m.p. 66e69 C (DCM-
ꢀ
1
1
2
hexane), IR (Nujol): 3020, 1725, 1583 cm
3
; H-NMR (CDCl )
9
NO
2
)
d
3.97 (d, 4H, J ¼ 4.9 Hz), 5.32e5.37 (m, 4H), 5.69 (s. 1H),
11NO
17NO
15NO
17NO
13NO
13NO
13NO
11NO
11NO
11NO
15NO
11NO
2
2
2
2
2
2
2
2
2
2
2
2
)
)
)
)
)
)
)
)
)
)
)
)
5
7
.83e5.95(m, 2H), 7.23 (d, 1H, J ¼ 7.8 Hz), 7.29e7.54 (m, 2H),
0a (C16
0b (C15
0c (C16
1a (C14
1b (C14
1c (C14
2a (C14
2b (C14
2c (C14
13
.72 (d, 1H, J ¼ 8.6 Hz); C-NMR (CDCl
3
) d 53.9, 104.5, 106.7,
117.1, 118.1, 118.7, 123.2, 132.2, 133.9, 145.6, 150.6, 161.2; MS (EI):
þ
ꢁ
241 (M , 100%), 240 (38), 213 (44), 200 (23), 172 (45), 115 (32),
1 (32); Anal. C15 (C, H, N).
9
H15NO
2
(h) General procedure. RCM reaction 6-(diallylamino)-2H-chromene-
2-one (4a).
6
7
(C15
(C13
(i) Under reflux. The catalyst 5 [38 mg (in seven parts after
9e10 h each), 0.046 mmol, 11 mol %] was added to

A. Balabani et al. / European Journal of Medicinal Chemistry 46 (2011) 5894e5901
5899
ꢀ
a solution of compound 4a (0.104 g, 0.43 mmol) in 30 ml
dry DCM after the removing of the air by a pump and filling
with Argon (in three cycles) and the solution was refluxed
under Argon atmosphere for 3 days. After the evaporation
of the solvent the residue was separated by repeated (two
times) PTLC [silica gel, hexane/ethyl acetate (2:1)] to give
from the faster moving band the unreacted compound 4a
hexane), IR (Nujol): 3060, 1720, 1600 cm 1; 1H-NMR (CDCl
3
)
d
2.50 (s, 3H), 4.36 (s, 4H), 5.93 (s, 2H), 6.09 (d, 1H, J ¼ 9.3 Hz),
13
6.53 (s, 1H), 7.08 (s, 1H), 7.52 (d, 1H, J ¼ 9.3 Hz); C-NMR
3
(CDCl ) d 22.3, 57.4, 101.3, 110.7, 118.0, 121.2, 125.9, 131.1, 143.2,
ꢁ ꢁ
144.8, 153.2, 159.7; MS (ESI): 228 [M þ H], 250 [Mþ þ Na],
þ
þ
ꢁ
266 [M þ K]; Anal. C14
2
H13NO (C, H, N).
(
2
4 mg, 4%) followed by the 6-(1H-pyrrol-1-yl)-2H-chromen-
(m) 5-(2,5-Dihydro-1H-pyrrol-1-yl)-6-methyl–2H-chromen-2-one
(11c) (56% yield or 65% yield under MW irradiation or 46%
yield with the catalyst 13 (12 mol %) under stirring at r.t. for 2
ꢂ
-one (7a) (25 mg, 27% yield), m.p. 128e130 C (DCM-
ꢀ
1
1
hexane), IR (Nujol): 3060, 1720, 1555 cm
;
H-NMR
ꢂ
(
(
CDCl
t, 2H, J ¼ 2.2 Hz), 7.40 (d, 1H, J ¼ 8.8 Hz), 7.46 (d, 1H,
¼ 2.6 Hz, J ¼ 8.8 Hz), 7.72 (d, 1H,
J ¼ 9.6 Hz); C-NMR (CDCl 107.0, 111.2, 118.2, 119.6,
21.7, 124.5, 134.7, 142.7, 150.1, 155.6, 161.1; MS (ESI): 212
3
)
d
6.38 (t, 2H, J ¼ 2.2 Hz), 6.49 (d, 1H, J ¼ 9.6 Hz), 7.06
days), m.p. 105e107 C (DCM-hexane), IR (Nujol): 3050, 1725,
ꢀ
1 1
1580 cm ; H-NMR (CDCl
3
) d 2.26 (s, 3H), 4.12 (s, 4H), 5.97 (s,
J ¼ 2.6 Hz), 7.57 (dd, 1H, J
1
2
2H), 6.35 (d, 1H, J ¼ 9.7 Hz), 7.10 (d, 1H, J ¼ 8.6 Hz), 7.36 (d, 1H,
13
13
3
)
d
J ¼ 8.6 Hz), 7.97 (d, 1H, J ¼ 9.7 Hz); C-NMR (CDCl
3
) d 17.5,
1
60.7, 109.8, 114.3, 115.8, 119.5, 127.4, 134.4, 141.0, 145.6, 153.4,
161.0; MS (ESI): 228 [M þ H], 250 [M þ Na]; Anal.
þ
ꢁ
þ
ꢁ
þ
ꢁ
[M
þ H]; Anal. C13
H
9
NO
2
(C, H, N).
Cl
(
ii) Under MW irradiation. Dry CH
2
2
(2 ml) was placed in
14 2
C H13NO
(C, H, N).
a tube (5 ml) for the Biotage Initiator 2.0, MW oven,
equipped with a Teflon-coated stirring bar; the compound
(n) 4-(2,5-Dihydro-1H-pyrrol-1-yl)-2H-chromen-2-one (17) (75%
ꢂ
yield) (at r.t. for 3 days), m.p. 145e147 C (DCM-hexane), IR
ꢀ
1 1
4
5
a (21 mg, 0.087 mmol) was added followed by the catalyst
3
(Nujol): 3085, 1705, 1580 cm ; H-NMR (CDCl ) d 4.58 (s, 4H),
(1.4 mg, 2 mol %), argon was flushed in the tube and the
5.24 (s, 1H), 6.01 (s, 2H), 7.23 (t, 1H, J ¼ 8.3 Hz), 7.38 (d, 1H,
ꢂ
13
mixture was irradiated at 100 C for 30 min (checked by
J ¼ 8.3 Hz), 7.52 (t, 1H, J ¼ 8.3 Hz), 8.07 (d, 1H, J ¼ 8.3 Hz); C-
TLC). Another portion of 5 (1.1 mg, 1.5 mol %, totally 2.5 mg,
NMR (CDCl ) d 58.4, 87.3, 118.6, 122.9, 125.1, 125.2, 131.3, 137.8,
3
þ
ꢁ
3
.5 mol %) was added and the solution was irradiated for
143.4, 158.1, 160.5; MS (EI): 213 (M ,100%),185 (33),184 (32),170
(20), 118 (39), 90 (29); Anal. C13 (C, H, N).
further 30 min (totally 1 h). After cooling and evaporation
of the solvent the residue was separated by column chro-
matography [silica gel No 60, hexane/EtOAc (3:2)] to give
after the elution of unreacted starting material 4a (1.9 mg,
H11NO
2
(o) 4-Methyl-7-(1H-pyrrol-1-yl)-2H-chromen-2-one
(7b)
(6%
ꢂ
yield) (at r.t. for 5 days), m.p. 169e171 C (DCM-hexane), IR
ꢀ
1 1
(Nujol): 3050, 1705, 1590 cm ; H-NMR (CDCl
3
) d 2.44 (s, 3H),
9
%) compounds 7a (1.1 mg, 6% yield) and 6a (15 mg, 81%
6.25 (s, 1H), 6.40 (t, 2H, J ¼ 2.0 Hz), 7.16 (t, 2H, J ¼ 2.0 Hz),
13
yield).
7.30e7.36 (m, 2H), 7.63 (d, 1H, J ¼ 9.3 Hz); C-NMR (CDCl
3
)
d
18.8, 107.9, 111.9, 114.2, 115.8, 119.1, 126.1, 134.4, 142.3, 151.5,
þ
ꢁ
þ
ꢁ
1
C
55.6, 160.6; MS (ESI): 226 [M þ H], 248 [M þ Na]; Anal.
(
i) 6-(2,5-Dihydro-1H-pyrrol-1-yl)-2H-chromen-2-one
eluted next (61 mg, 66% yield), m.p. 149e151 C (ethyl ace-
(6a)
14 2
H11NO (C, H, N).
ꢂ
(
p) 7-Methyl-6-(1H-pyrrol-1-yl)-2H-chromen-2-one (12a) [7% yield,
ꢀ
1
1
tateehexane), IR (Nujol): 3055, 1725, 1545 cm
CDCl
4.13 (s, 4H), 5.98 (s, 2H), 6.39 (d, 1H, J ¼ 8.9 Hz),
.46 (d, 1H, J ¼ 2.8 Hz), 6.74 (dd, 1H, J ¼2.8 Hz, J ¼ 8.9 Hz),
.23 (d, 1H, J ¼ 8.9 Hz), 7.65 (d, 1H, J ¼ 8.9 Hz); C-NMR
CDCl 54.7, 107.3, 111.1, 115.7, 116.9, 117.5, 126.3, 143.7,
50.9, 153.7, 160.6; MS (EI): 213 (M , 77%), 211 (90), 183
100), 156 (70), 154 (83), 145 (79), 128 (90), 117 (90); Anal.
(C, H, N).
; H-NMR
after refluxing for 2 days with the catalyst 5 (7 mol%) or 9%
yield under MW irradiation], light yellow crystals, m.p.
(
6
7
(
1
(
C
3
) d
1
2
ꢂ
ꢀ1
1
35e137 C (DCM-hexane), IR (Nujol): 3040, 1720, 1595 cm
;
13
1
H-NMR (CDCl
3
)
d
2.27 (s, 3H), 6.34 (t, 2H, J ¼ 1.8 Hz), 6.42 (d,
3
) d
ꢁ
1
7
1
(
H, J ¼ 9.5 Hz), 6.76 (t, 2H, J ¼ 1.8 Hz), 7.26 (s, 1H), 7.37 (s, 1H),
þ
13
.65 (d, 1H, J ¼ 9.5 Hz); C-NMR (CDCl
3
) d 18.2, 106.1, 109.4,
15.1, 116.8, 118.8, 122.2, 125.4, 137.7, 142.5, 155.3, 160.5; MS
13
H
11NO
2
þ
ꢁ
ESI): 226 [M þ H]; Anal. C14
2
H11NO (C, H, N).
The same reaction with the catalyst 5 (6 mol %) at r.t. for 3 days
yielded compounds 6a (12%), 7a (2%) and the unreacted 4a (78%).
(q) 6-Methyl-7-(1H-pyrrol-1-yl)-2H-chromen-2-one (12b) (21% yield)
ꢂ
(at r.t. for 4 days), m.p. 202e204 C (dec.) (DCM-hexane), IR
Nujol): 3020, 1708, 1580 cm ; H-NMR (CDCl ) d 2.30 (s, 3H),
3
ꢀ
1 1
(
(
j) 7-(2,5-Dihydro-1H-pyrrol-1-yl)-4-methyl–2H-chromen-2-one (6b)
6.36 (t, 2H, J ¼ 2.1 Hz), 6.43 (d, 1H, J ¼ 9.3 Hz), 6.83 (t, 2H,
ꢂ
13
(
70% yield) (at r.t. for 5 days), m.p. 144e146 C (DCM-hexane), IR
J ¼ 2.1 Hz), 7.24 (s, 1H), 7.40 (s, 1H), 7.69 (d, 1H, J ¼ 9.3 Hz); C-
ꢀ
1 1
(Nujol): 3050, 1722, 1585 cm ; H-NMR (CDCl
3
)
d
2.35 (s, 3H),
3
NMR (CDCl ) d 18.1, 108.5, 110.2, 114.1, 116.8, 123.0, 124.8, 129.2,
ꢁ ꢁ
137.7,142.5,157.0,161.5; MS (ESI): 226 [M þ H], 248 [Mþ þ Na];
þ
4
.17 (s, 4H), 5.97 (s, 1H), 5.98 (s, 2H), 6.38 (d, 1H, J ¼ 1.7 Hz), 6.47
13
(
(
dd, 1H, J
CDCl
1
¼1.7 Hz, J
2
¼ 8.5 Hz), 7.42 (d, 1H, J ¼ 8.5 Hz); C-NMR
Anal. C H NO (C, H, N).
14
11
2
3
)
d
22.6, 53.8, 98.7, 103.5, 109.2, 113.2, 125.4, 125.9, 146.5,
(r) 6-Methyl-5-(1H-pyrrol-1-yl)-2H-chromen-2-one (12c) (21%
yield or 10% yield under MW irradiation or 20% yield with the
catalyst 13 (12 mol %) under stirring at r.t. for 2 days), light
þ
ꢁ
ꢁ
1
55.1, 157.5,161.3; MS (ESI): 228 [M þ H], 250 [Mþ þ Na]; Anal.
14 2
C H13NO (C, H, N).
ꢂ
(
k) 6-(2,5-Dihydro-1H-pyrrol-1-yl)-7-methyl–2H-chromen-2-one
yellow crystals m.p. 143e145 C (DCM-hexane), IR (Nujol):
ꢀ
1 1
(
(
(
(
11a) [83% yield, after refluxing for 2 days with the catalyst 5
3
3080, 1705, 1580 cm ; H-NMR (CDCl ) d 2.14 (s, 3H), 6.34 (d,
ꢂ
7 mol%) or 75% yield under MW irradiation], m.p. 104e106 C
1H, J ¼ 9.7 Hz), 6.40 (t, 2H, J ¼ 2.1 Hz), 6.69 (t, 2H, J ¼ 2.1 Hz),
ꢀ1
1
DCM-hexane), IR (Nujol): 3020, 1710, 1590 cm
; H-NMR
7.12 (d, 1H, J ¼ 9.7 Hz), 7.30 (d, 1H, J ¼ 8.7 Hz), 7.44 (d, 1H,
1
3
CDCl 2.46 (s, 3H), 4.16 (s, 4H), 5.94 (s, 2H), 6.43 (d, 1H,
3
)
d
J ¼ 8.7 Hz); C-NMR (CDCl )
3
d 16.7, 109.8, 116.7, 117.5, 122.7,
13
J ¼ 8.8 Hz), 6.90 (s, 1H), 7.10 (s, 1H), 7.63 (d, 1H, J ¼ 8.8 Hz); C-
NMR (CDCl 18.2, 53.9, 109.4, 116.9, 117.8, 119.6, 122.2, 125.4,
42.5, 151.3, 157.1, 161.9; MS (EI): 227 (M , 47%), 197 (100), 180
14), 168 (73), 154 (25), 141 (58), 115 (42), 102 (31); Anal.
(C, H, N).
126.6, 127.5, 133.3, 139.3, 150.5, 153.6 160.3; MS (ESI): 226
[M þ H], 248 [Mþ þ Na]; Anal. C H NO (C, H, N).
þ
ꢁ
ꢁ
3
) d
1
4
11
2
þ
ꢁ
1
(
5.2. Determination of lipophilicity as Clog P
14 2
C H13NO
(
l) 7-(2,5-Dihydro-1H-pyrrol-1-yl)-6-methyl–2H-chromen-2-one
Lipophilicity was theoretically calculated as Clog P values in n-
octanol-buffer by CLOG P Programme of Biobyte Corp. [36]
ꢂ
(11b) (64% yield) (at r.t. for 4 days), m.p. 171e173 C (DCM-

5900
A. Balabani et al. / European Journal of Medicinal Chemistry 46 (2011) 5894e5901
5.3. Biological assays
5.3.1.5. Non-enzymatic assay of superoxide radicals e measurement
of superoxide radical scavenging activity. The superoxide producing
system was set up by mixing phenazine methosulfate (PMS), NADH
and aireoxygen [34]. The production of superoxide was estimated
by the nitroblue tetrazolium method. The reaction mixture con-
Materials. All the reagents used were commercially available by
Merck, 1,1-diphenyl-2-picrylhydrazyl (DPPH), nordihydroguairetic
acid (NDGA) were purchased from the Aldrich Chemical Co. Mil-
waukee, WI, USA. Soybean Lipoxygenase, linoleic acid sodium salt,
nicotinamidoeadenineedinucleotide NADH, Nitrotetrazolium Blue
taining 3 mM PMS, 78 mM NADH, and 25 mM NBT in 19 mM phos-
phate buffer pH 7.4 was incubated for 2 min at room temperature
and the absorption measured at 560 nm against a blank containing
PMS. The tested compounds were preincubated for 2 min before
adding NADH.
(
NBT), porcine heme and indomethacin were obtained from Sigma
Chemical, Co. (St. Louis, MO, USA) and carrageenin, type K, was
commercially available. For the in vivo experiments, male and
female Fischer-344 rats (180e240 g) were used. N-methyl-
phenazonium-methyl sulfate and trolox were purchased by Fluka
A.G. For in vitro determination a UVeVis PerkineElmer Lambda
Spectrophotometer was used.
Acknowledgments
The authors gratefully acknowledge Drs. C. Hansch, A. Leo and
Biobyte Corp. 201 West 4th Street, Suite 204, Claremont, CA 91711,
USA for free access to the C-QSAR program.
Part of this research (A. Vronteli) has been co-financed by the
European Union (European Social Fund e ESF) and Greek national
funds through the Operational Program “Education and Lifelong
Learning” of the National Strategic Reference Framework (NSRF) e
Research Funding Program: Heracleitus II. Investing in knowledge
society through the European Social Fund.
5
.3.1. In vitro
In the in vitro assays each experiment was performed at least in
triplicate and the standard deviation of absorbance was less than
0% of the mean.
1
5
1
(
.3.1.1. Determination of the reducing activity of the stable radical 1,
-diphenyl-picrylhydrazyl (DPPH) [34]. To a solution of DPPH
100 mM) in absolute ethanol an equal volume of the compounds
dissolved in ethanol was added. As control solution ethanol was
used. The concentrations of the solutions of the compounds
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