Synthesis, regioselectivity, and DFT analysis of new antioxidant pyrazolo[4,3-c]quinoline-3,4-diones

Article abstract of DOI:10.1007/s00706-016-1660-7

The condensation of hydrazine, N-methylhydrazine, and N-phenylhydrazine with ethyl 4-chloro-2-oxo- 1,2-dihydroquinoline-3-carboxylate derivatives has been investigated. As a result, 12 new antioxidant pyrazolo[4,3- c]quinolin-3,4-diones were obtained with good to high yields. When two cross-products could be possible, only one isomer bearing the methyl or the phenyl group at the N1 position is isolated and unequivocally characterized using 1D and 2D NMR techniques, FT-IR, and combustion analyses. DFT analysis of the reaction mechanism was carried out in the Pearson’s hard soft acid base framework, confirming the assigned structure to the observed pyrazolo[4,3-c]quinolin-3,4-diones. These calculations indicate a favored kinetic control for the synthesized pyrazolo[4,3- c]quinolin-3,4-diones compared to its possible regioisomer.

Full text of DOI:10.1007/s00706-016-1660-7

Monatsh Chem
DOI 10.1007/s00706-016-1660-7
ORIGINAL PAPER
Synthesis, regioselectivity, and DFT analysis of new antioxidant
pyrazolo[4,3-c]quinoline-3,4-diones
1
,4
2
2
1,5
•
Isabelle Tomassoli
Oscar M. Bautista-Aguilera
•
Guillaume Herlem
1
•
Fabien Picaud
1
•
Mohamed Benchekroun
3
•
Vincent Luzet
1
•
Mar ´ı a-Luisa Jimeno
•
2
1
Tijani Gharbi
•
Bernard Refouvelet
•
Lhassane Ismaili
Received: 14 November 2015 / Accepted: 7 January 2016
Ó Springer-Verlag Wien 2016
Abstract The condensation of hydrazine, N-methylhy-
drazine, and N-phenylhydrazine with ethyl 4-chloro-2-oxo-
Graphical abstract
1
,2-dihydroquinoline-3-carboxylate derivatives has been
investigated. As a result, 12 new antioxidant pyrazolo[4,3-
c]quinolin-3,4-diones were obtained with good to high
yields. When two cross-products could be possible, only
one isomer bearing the methyl or the phenyl group at the
N1 position is isolated and unequivocally characterized
using 1D and 2D NMR techniques, FT-IR, and combustion
analyses. DFT analysis of the reaction mechanism was
carried out in the Pearson’s hard soft acid base framework,
confirming the assigned structure to the observed pyra-
zolo[4,3-c]quinolin-3,4-diones. These calculations indicate
a favored kinetic control for the synthesized pyrazolo[4,3-
c]quinolin-3,4-diones compared to its possible regioisomer.
Keywords Isomers Á Kinetic control Á
Pyrazoloquinolines Á Density functional theory Á
Antioxidants
Introduction
&
Lhassane Ismaili
lhassane.ismaili@univ-fcomte.fr
Over the past two decades, functionalized pyrazoloquinoli-
nes have attracted much attention due to their considerable
biological and pharmacological activities [1, 2]. Particularly,
the pyrazolo[4,3-c]quinoline heterocyclic ring system is a
very attractive scaffold in medicinal chemistry which has
been incorporated in high-affinity benzodiazepine receptor
ligands [3, 4], interleukin inhibitors [5], selective
cyclooxygenase-2 (COX-2) inhibitors [6], phosphodi-
esterase 4 (PDE4) inhibitors [7], as well as anticancer [8] and
anti-inflammatory agents [9]. Accordingly, several synthetic
approaches have been reported, based on the annulation of
the pyrazole ring onto a quinoline motif [10–12] or the
quinoline ring onto a pyrazole scaffold [13–19].
1
Neurosciences Int e´ gratives et Cliniques EA 481, Laboratoire
de Chimie Organique et Th e´ rapeutique, UFR SMP,
Universit e´ de Franche-Comt e´ , Universit e´ Bourgogne
Franche-Comt e´ , 19 Rue Ambroise Par e´ , 25000 Besan c¸ on,
France
2
3
NanoMedicine, Imagery and Therapeutics Lab EA 4662,
UFR Sciences and Techniques, Universit e´ de Franche-
Comt e´ , Universit e´ Bourgogne Franche-Comt e´ , 16, Route de
Gray, 25030 Besan c¸ on, France
Laboratorio de RMN, Centro de Qu ´ı mica Org a´ nica «Lora-
Tamayo» , CSIC, C/Juan de la Cierva, 3, 28006 Madrid,
Spain
4
5
Department of Radiology, University of Pittsburgh,
Pittsburgh PA 15213-2582, USA
Recently, Moyano et al. reported a new synthetic
approach toward 2-aryl-2H-pyrazolo[4,3-c]quinolin-3-
ones. They found that the reaction of arylhydrazines with
Centre de recherche de Gif-sur-Yvette, Institut de Chimie des
Substances Naturelles, UPR 2301, CNRS, Avenue de la
Terrasse, 91198 Gif-sur-Yvette, France
123

I. Tomassoli et al.
ethyl 4-chloroquinolin-3-carboxylates in DMF at
30–140 °C, furnished only the N2 isomer of the corre-
sponding pyrazolo[4,3-c]quinolin-3-one adduct, but when
the condensation was carried out with benzylhydrazine, in
the presence of sodium methoxide, a mixture of the N1 and
N2 regioisomers was detected and conveniently charac-
terized (Scheme 1; Eq. (1)) [20].
was the preparation of the two isatoic anhydrides 3 bearing
a hydrogen atom or a chlorine atom at C5. Accordingly,
while isatoic anhydride (3a) is commercially available,
compound 3b was obtained from the corresponding
2-amino-6-chlorobenzoic acid (1) after reaction with
triphosgene in dioxane at 0 °C [25].
Next, compound 3a was submitted to N-alkylation with
benzyl bromide in the presence of di-iso-propylethylamine
(DIPEA) as a base, in dimethylacetamide (DMA) as a
solvent, to provide the benzylated isatoic anhydride 4a
(Scheme 2) [26]. N-Methylisatoic anhydride (4b,
Scheme 2) is commercially available, and used as received.
In the next steps, these two intermediates were submitted to
the same synthetic sequence starting the reaction with
diethyl malonate in the presence of NaH in DMA, a process
that afforded the expected quinolones 5a, 5b [21–24, 27]
1
Based on this report and the recent synthetic develop-
ments using the related, diversely substituted ethyl 4-chloro-
2
-oxo-1,2-dihydroquinoline-3-carboxylate derivatives I [21–
4], we describe in this paper the results we have obtained in
2
the condensation of compounds of type I with hydrazine, N-
methylhydrazine, and N-phenylhydrazine (Scheme 1;
Eq. (2)). As a result, we have synthesized and characterized
1
2 pyrazolo[4,3-c]quinolin-3,4-diones 7a–7l, whose antiox-
idant properties are reported herein. In addition, in-depth
NMR studies and mechanistic DFT-analysis have been
carried out in order to shed light into the mechanism of this
reaction, to justify the regioselective formation of the N1-
substituted pyrazolo[4,3-c]quinolin-3,4-diones (isomer II-
N1) versus the N2-substituted pyrazolo[4,3-c]quinolin-3,4-
diones (isomer II-N2) (Scheme 1; Eq. (2)) in the reaction of
whose reaction with POCl provided the required chloride
3
derivatives 6a, 6b (Scheme 2). Similarly, the reaction with
diethyl malonate followed by treatment with POCl applied
3
to precursor 3a, provided the required chloride 6c via
compound 5c (Scheme 2).
Afterward, the isatoic anhydride 3b was submitted to the
same synthetic procedures as shown for compound 3a,
which afforded the expected key intermediates 6d–6f via
anhydrides 4c, 4d and hydroxy-derivatives 5d–5f
ethyl
4-chloro-2-oxo-1,2-dihydroquinoline-3-carboxylate
derivatives with N-methylhydrazine and N-phenylhydrazine.
(Scheme 3) [21–24, 28]. All new compounds gave ana-
Results and discussion
Chemistry
lytical and spectroscopic data in full agreement with their
structures (see ‘‘Experimental’’ section).
Finally, the tricyclic pyrazolo[3,4-c]quinolin-3,4-
diones 7a–7l were obtained by treatment of chlorides 6a–
6f with hydrazine, N-methylhydrazine, and N-phenylhy-
drazine (Scheme 4), in yields ranging between 44 and
83 % (Table 1). As shown, only the N1-substituted (Me
The key 4-chloro-2-oxo-1,2-dihydroquinoline-3-carboxy-
late intermediates 6 have been prepared as shown in
Schemes 2 and 3. The first step in the synthetic sequence
Scheme 1
1
23

Synthesis, regioselectivity, and DFT analysis of new antioxidant pyrazolo[4,3-c]quinoline-3,…
Scheme 2
Scheme 3
hydrogen of the N-methyl group of the quinoline ring
Scheme 4
(3.57 ppm), the carbonylic carbon signal (157.22 ppm) at
the position 4, carbon C-5a (139.18 ppm), and carbon C-6
(115.86 ppm). In the ROESY experiment, two significant
signals corresponding to interactions through space
between protons of the methyl group of the hydrazide
(4.13 ppm) and proton H-9 (8.17 ppm) were observed: the
second interaction shows correlation between hydrogen of
the N-methyl group (3.57 ppm) of the quinoline ring and
proton H-6 (7.55 ppm). These findings allowed us to con-
firm the structure of compound 7e and the conclusion was
also extended to the other derivatives 7.
or Ph) pyrazolo[3,4-c]quinolin-3,4-dione regioisomers
were isolated.
A complete elucidation of the structures was achieved
by HMBC and ROESY 2D NMR experiments (Fig. 1).
HMBC analysis of compound 7e showed correlations
between hydrogen of methyl group of hydrazide and the
C-9b at 138.74 ppm and three correlations between
Computational studies and reaction mechanism
Since the total electronic energy of isomers II-N1 is less
negative than the other possible regioisomer II-N2, the
123

I. Tomassoli et al.
Table 1 Synthesis of compounds 7a–7l
the four centers, there are two possible pathways D and
I
1
2
3
D :
II
Compound
R
R
R
Yield/
%
DI ¼ ðsþ
À
2
Þ þ ðsþ
À s
ðunsubstituted NÞ
ðC esterÞ
ðsubstitutedNÞ
À
2
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
H
H
H
79
46
72
66
77
78
83
44
52
71
61
51
À s
Þ
ðC of CÀClÞ
H
H
Me
Ph
H
DII ¼ ðsþ
À s
À
Þ þ ðsþ
2
H
H
ðsubstituted NÞ
ðC esterÞ
ðunsubstitutedNÞ
À
2
H
Me
Me
Me
Bn
Bn
Bn
H
À s
Þ
ðC of CÀClÞ
H
Me
Ph
H
These calculations indicate a preferable mechanism DI
D \ D ) leading to the unique product 7b and further
H
(
I
II
g
h
i
H
confirmed experimentally by HMBC and ROESY 2D NMR
experiments. According to the DFT-based descriptors, the
bond formation between the atoms concerns the ester carbon
H
Me
Ph
Me
Me
Me
H
j
9-Cl
9-Cl
9-Cl
(
(
from quinolinone) with the non-substituted nitrogen atom
from methylhydrazine) having antibonding molecular
k
l
Me
Bn
orbitals (MOs) and the carbon atom bonded to the
chlorine atom (from quinolinone) with the substituted
nitrogen atom (from methylhydrazine) having bonding
MOs [32]. Moreover, the charges carried by these atoms
show that the two nitrogen centers act as nucleophilic site
and on the contrary, the two carbon atoms (from
quinolinone) involved in the reaction are electrophilic
sites. Therefore, a larger orbital overlap occurs explaining
a single major product as confirmed experimentally.
The reaction path calculations allowed us to verify the
connection between the reactant and the product via the
transition structures. The resulting potential energy profile
of the reaction is illustrated by Fig. 2. The energy profile
for the synthesized pyrazolo[4,3-c]quinolin-3,4-diones 7a–
Fig. 1 HMBC and ROESY (500 MHz, 298 K, DMSO-d
tions for compound 7e
6
) correla-
7l compared to its possible regioisomer underlines a kinetic
control of the reaction between selected quinolinone and
hydrazine (and derivatives) since the more energetically
stable isomer II-N2 is not obtained.
elucidation of their regioselectivity cannot be studied on
the basis of this global descriptor. In the HSAB framework,
local descriptors such as Fukui functions f(r) and local
chemical softness s(r) and electrophilicity (x) are more
appropriate and considered as the reactivity index. In this
context, successful prediction criteria have been proposed
on rigorous theoretical basis [29–31]. When applied to our
molecules, all these elements provide a better understand-
ing for the formation of a single product between the
quinolinones 6 and hydrazine or derivatives.
The preferable mechanism of the reaction between quino-
lone and hydrazine derivatives (Scheme 5) consists in the
approach of hydrazine towards 6b near the ester function; this
constitutes a transition state leading to an energy barrier. Then,
a rearrangement occurs with the departure of ethanol (step 2)
and finally a nucleophilic attack of the substituted nitrogen
atom on the halogenated carbon atom (step 3). This step
corresponds to a second energy transition state in gas phase
which is found to disappear in 2-ethoxyethanol explaining
why the solvent can affect the kinetic or thermodynamic
control of the reaction. The departure of hydrochloric acid
causes the reaction to evolve towards the final product (step 4).
The regioselectivity and reactivity of compounds 6 with
hydrazine and its derivatives have been studied by calculating
the local Fukui functions and then the corresponding softnesses
and electrophilicity indexes of electrophilic and nucleophilic
?
-
?
-
?
-
centers (f and f , then s and s respectively and x and x ).
In addition to these chemical indicators, the effect of solvent
Antioxidant activity study
(i.e. ethoxyethanol) as a polarized continuum was taken into
consideration since error can rise from reactivity difference of
molecules in gas and liquid phases (Table 2).
Free radical scavenging is considered to be one of the
major mechanisms by which antioxidants halt lipid per-
oxidation, thus mitigating any damages caused to the cell
membrane lipids and mitochondrial machinery. In our
By applying the site selectivity criteria to the reaction
between compound 6b and methylhydrazine and between
1
23

Synthesis, regioselectivity, and DFT analysis of new antioxidant pyrazolo[4,3-c]quinoline-3,…
Table 2 Condensed Fukui functions, local softnesses for C and N centers of molecules solvated in 2-ethoxyethanol and electrophilicity indexes
?
-
?
s
-
?
-
f
f
s
x
x
6
b
Carbon atom of C–Cl
Carbon atom of ester
0.11374
0.00961
0.03329
0.01825
0.00154
0.00534
0.222
0.019
0.065
-0.00589
-0.00094
-0.011
Methyl hydrazine
Non-substituted nitrogen atom
Substituted nitrogen atom
-0.10075
-0.07652
0.24100
0.24967
-0.02532
-0.01923
0.06057
0.06274
-0.030
-0.023
0.072
0.075
The global softnesses calculated from the neutral molecules are S(quinolinone 6b) = 0.16041 (a.m.u.) and S(methylhydrazine) = 0.25131
a.m.u.). x(6b) = 1.952 eV and x(methylhydrazine) = 0.300 eV
(
Fig. 2 Energy profile DE (in
kJ/mol) of the reaction between
compound 6b and
methylhydrazine versus the
reaction coordinates. Dash line
molecules in vacuum; solid line
molecules solvated in
2
-ethoxyethanol
picryl-hydrazyl (DPPH) and hydroxyl radicals (standard
assays) as probes to determine the radical scavenging
activity (RSA) of the aforementioned compounds.
Scheme 5
DPPH radical scavenging
In a methanolic solution, the DPPH radical displays a deep
purple color with a maximum absorption at 517 nm. This
color vanishes gradually in the presence of various concen-
tration of antioxidant in the medium (see Scheme 6). Thus,
the DPPH radical can be quenched by the capture of a labile
hydrogen atom or by electron donation from the antioxidant,
probably via a free-radical attack on the DPPH molecule. This
process provides then a blanched solution [33, 34]. RSA
DPPH values of compound 7a–7l (Table 3) correspond to a
percentage of the ratio of the decrease in optical density at
517 nm over the absorbance of a DPPH control solution
which does not contain the compounds 7a–7l.
quest to find potent antioxidants, we were then interested in
assessing the antioxidant power of pyrazolo[4,3-c]quinolin-
3
,4-diones 7a–7l using the well-known 2,2-diphenyl-1-
123

I. Tomassoli et al.
Scheme 6
4-hydroxybenzoic acid in the presence of the hydroxyl
radical generated during the Fenton reaction. The fluores-
cence emission, measured at 407 nm with excitation
wavelength set at 305 nm, decreases if there is any
antioxidant in the medium. Potent antioxidant product
impedes the hydroxylation of benzoic acid by providing
hydrogen atom. The hydroxyl RSAs of the adducts 7a–7l
are summarized in Table 3. We can conclude that all the
3
products presented at 50 lmol/dm good hydroxyl radical
scavenging activities ranging between 53.3 and 73.9 %
3
compared to quercetol at 50 lmol/dm (88.9 %), used here
as reference compound.
Table 3 Decreasing DPPH absorbance and decreasing fluorescence
3
at 50 lmol/dm of compounds 7a–7l
Compound
Decreasing
absorbance DPPH/%
Decreasing
fluorescence/%
Conclusions
1
13
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
15.2
13.8
10.8
6.9
66.7
65.8
53.3
68.2
69.2
67.8
64.3
71.6
73.9
72.7
72.4
73.1
–
The spectroscopic (1D NMR H/ C and 2D NMR HMBC
and ROESY) and the computational analyses (DFT) con-
firms that the reaction between ethyl 4-chloro-2-oxo-1,2-
dihydroquinoline-3-carboxylate derivatives and the selec-
ted hydrazine derivatives yields a unique product,
kinetically favored but disadvantaged in comparison to
other possible regioisomers. Compared to conventional
frontier molecular orbital description, any of the DFT-
based descriptors predicted the correct regioselectivity in
favor of the N1-II isomer. Our results showed also the
important role played by 2-ethoxyethanol during this type
of condensation. All new compounds gave interesting
hydroxyl radical scavenging activities and slight capacity
to inhibit DPPH radical. Based on these outcomes, com-
pound 7a bearing three NH moieties was found to be the
most balanced antioxidant. Further synthetic studies are
underway to enhance the antioxidant potency of the pyra-
zolo[4,3-c]quinolin-3,4-dione backbone.
5.7
7.3
g
h
i
6.4
6.9
7.6
j
10.1
5.0
k
l
5.9
Ascorbic acid
Quercetol
75.9
–
88.9
From the results gathered in Table 3, we can say that the
pyrazolo[4,3-c]quinolin-3,4-diones 7a–7l scavenging
effect on DPPH radicals increase was suitable for com-
3
pounds 7a–7c (10.8–15.2 % at 50 lmol/dm ), but lower
than RSA DPPH value found for ascorbic acid (37.9 % at
Experimental
3
5
0 lmol/dm ) used as a reference. N-Substituted deriva-
All reactions were controlled by TLC using precoated sil-
ica gel aluminum plates (Macherey–Nagel) and revealed
by UV light at 254 nm. Melting points were determined on
a K o¨ fler bench. Infrared spectra were recorded on a Shi-
tives 7d–7g, 7i, and 7k have shown moderate activity while
derivatives 7h and 7l presented the poorest activity com-
pared to the standard one. Thus, in order to display a good
antioxidant activity, the pyrazolo[4,3-c]quinolin-3,4-dione
scaffold may embed the N–H moiety in the quinolone or
pyrazole ring. Accordingly, compound 7a behaves as a
better antioxidant with three N–H moieties in its structure.
-
1
madzu FTIR-8201 PC spectrometer in KBr (m in cm ).
NMR spectra were recorded on Bruker AC 300, AC 250
spectrometers or on a Varian System-500 spectrometer.
CDCl and DMSO-d were used as deuterated solvents.
3 6
Chemical shifts are reported in parts per millions relative to
tetramethylsilane (TMS) signal and coupling constants
Hydroxyl radical scavenging
(
J) are given in Hertz (Hz). The following abbreviations
The capacity of our compounds to scavenge the hydroxyl
radical was evaluated using the benzoic acid method [35].
In brief, the benzoic acid is converted to salicylic acid or
are employed: s, singlet; br s, broad singlet; d, doublet;
dd, doublet of doublets; t, triplet; q, quadruplet; m, multi-
plet. Microanalyses were performed at Service Central
1
23

Synthesis, regioselectivity, and DFT analysis of new antioxidant pyrazolo[4,3-c]quinoline-3,…
d’Analyses (CNRS, Vernaison, France). All reagents are of
analytical grade pure and used without further purification.
Isatoic anhydride (3a) and N-methylisatoic anhydride (4b)
are commercially available. The compounds 5-chlor-
oisatoic anhydride (3b) [25], N-benzylisatoic anhydride
(29CH, benzyl), 124.33 (C7), 123.63 (C4-a), 123.27 (C6),
112.46 (C8), 102.33 (C3), 61.13 (CH , ester), 46.28 (CH
benzyl), 15.63 (CH ) ppm.
,
2
2
3
General procedure for preparation of 6d and 6e
(
4a) [26], 5-chloro-N-methylisatoic anhydride (4d) [21],
ethyl 1-benzyl-4-hydroxy-2-oxo-1,2-dihydroquinoline-3-
The corresponding quinolines 5d, 5e suspended in POCl
3
3
carboxylate (5a) [27], ethyl 4-hydroxy-1-methyl-2-oxo-
(2–6 cm ) were heated at 100 °C for 1 h. Then, the mixture
was cooled to 0 °C and neutralized with water and NaOH
(10 N). The resulting solid was filtered and dried.
1
,2-dihydroquinoline-3-carboxylate (5b) [21], ethyl 4-hy-
droxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (5c) [21],
ethyl 5-chloro-4-hydroxy-1-methyl-2-oxo-1,2-dihydro-
quinoline-3-carboxylate (5e) [22], ethyl 5-chloro-4-
hydroxy-2-oxo-1,2-dihydroquinoline-3-carboxylate (5f)
21], ethyl 1-benzyl-4-chloro-2-oxo-1,2-dihydroquinoline-
Ethyl 1-benzyl-4,5-dichloro-2-oxo-1,2-dihydroquinoline-3-
carboxylate (6d, C H Cl NO )
3
1
9
15
2
Following the general procedure, 0.50 g compound 5d
(
[
3
1.40 mmol) and 2 cm POCl gave white solid 6d (0.31 g,
3
3
1
4
-carboxylate (6a) [23], ethyl 4-chloro-1-methyl-2-oxo-
,2-dihydroquinoline-3-carboxylate (6b) [21], and ethyl
5
8 %). M.p.: 115 °C; IR (KBr): m = 1715, 1656,
-1 1
1
550 cm ; H NMR (CDCl , 500 MHz): d = 7.40–7.24
3
-chloro-2-oxo-1,2-dihydroquinoline-3-carboxylate
(6c)
(
m, 6H), 7.23 (d, J = 7.5 Hz, 2H), 5.57 (s, 2H), 4.53 (q,
[
28] had been previously described.
1
J = 7.1 Hz, 2H), 1.46 (t, J = 7.1 Hz, 3H) ppm; C NMR
3
N-Benzyl-5-chloroisatoic anhydride (4c, C H ClNO )
3
(CDCl , 125 MHz): d = 164.25 (CO), 158.10 (CO),
3
1
5
10
5
-Chloroisatoic anhydride (3b, 1 g, 5.06 mmol) was sus-
141.52 (C8-a), 140.24 (C, benzyl), 135.51 (C5), 134.18
(C4), 132.04 (CH, benzyl), 130.13 (C4-a), 129.47 (29CH,
benzyl), 128.16 (C7), 128.02 (C6), 126.94 (29CH, benzyl),
3
pended in 10 cm DMA with 0.654 g DIPEA (5.06 mmol).
The solution was stirred 10 min and then, 1.09 g benzyl
bromide (6.07 mmol) was added and the mixture was
116.23 (C3), 115.44 (C8), 62.92 (CH
benzyl), 14.54 (CH ) ppm.
, ester), 47.82 (CH ,
2
2
3
heated for 3 h. After cooling to room temperature, 10 cm
3
water was added and the solution was vigorously stirred for
Ethyl 4,5-dichloro-1-methyl-2-oxo-1,2-dihydroquinoline-3-
3
0 min; the precipitate was filtered, washed with water and
carboxylate (6e, C H Cl NO )
3
1
3
11
2
diethyl ether to give white product 4c (0.70 g, 48 %). M.p.:
Following the general procedure, 1 g compound 5e
(
-
1 1
1
15 °C; IR (KBr): m = 1768, 1700, 1590 cm ; H NMR
CDCl3, 300 MHz): d = 7.87 (m, 2H), 7.44 (d,
J = 8.2 Hz, 1H), 7.25–7.35 (m, 5H), 4.80 (s, 2H) ppm;
3
3.74 mmol) was suspended in 3 cm POCl3 and gave
(
white product 6e (0.43 g, 40 %). M.p.: 106 °C; IR (KBr):
-
1
1
m = 1710, 1660, 1573 cm
;
H NMR (DMSO-d6,
1
3
C NMR (DMSO-d , 75 MHz): d = 156.15 (CO), 151.20
6
5
00 MHz): d = 7.70 (m, 2H), 7.51 (dd, J = 6.6, 2.1 Hz,
H), 4.36 (q, J = 7.0 Hz, 2H), 1.31 (t, J = 7.0 Hz, 3H)
(
CO), 139.42 (C8-a), 130.88 (C, benzyl), 129.11 (C5),
1
1
1
28.14 (C7), 126.13 (2CH, benzyl), 124.13 (2CH, benzyl),
23.77 (CH, benzyl), 123.28 (C6), 121.21 (C4-a), 112.44
1
3
ppm; C NMR (DMSO-d , 125 MHz): d = 163.18 (CO),
6
156.34 (CO), 141.51 (C8-a), 137.14 (C5), 132.50 (C7),
(
C8), 43.52 (CH , benzyl) ppm.
2
131.41 (C4), 129.13 (C4-a), 127.17 (C6), 115.71 (C8),
Ethyl 1-benzyl-5-chloro-4-hydroxy-2-oxo-1,2-
113.90 (C3), 61.81 (CH
ester) ppm.
), 30.74 (N-CH ) 13.80 (CH
2 3 3
dihydroquinoline-3-carboxylate (5d, C H ClNO )
4
1
9
16
To a solution of 1 g 5-chloro-N-benzylisatoic anhydride
3
Ethyl 4,5-dichloro-2-oxo-1,2-dihydroquinoline-3-carboxy-
(
4c, 3.47 mmol) in 10 cm DMF at 0 °C were added
late (6f, C H Cl NO )
1
2
9
2
3
slowly 0.17 g sodium hydride (7.0 mmol) and 2.78 g
diethyl malonate (17.6 mmol). The reaction was heated at
3
Compound 5f (0.8 g, 3 mmol) and 2 cm POCl3 were
stirred at 110 °C for 6 h. After cooling, the solvent was
concentrated. The residue was dissolved in a small amount
of AcOEt and the mixture was poured into ice water
followed by extraction with AcOEt. The extract was
washed with 1 M NaOH, water, and brine, dried over
8
5 °C for 5 h. After cooling, water was added and the
mixture obtained was acidified with conc. HCl. The
resultant solid was filtered, washed with water, and dried
to afford a yellow product 5d (0.78 g, 63 %). M.p.:
-
1 1
1
24 °C; IR (KBr): m = 1672, 1630, 1570 cm ; H MMR
MgSO , and concentrated in vacuum. This mixture was
4
(
300 MHz, DMSO-d ): d = 12.9 (s, 1H, OH), 7.53 (d,
6
3
then stirred with 0.27 g AcONa (3.3 mmol) in 2 cm
J = 7.3 Hz, 1H), 7.15–7.28 (m, 7H), 5.02 (s, 2 H), 4.16 (q,
AcOH at 120 °C for 20 h. The reaction mixture was then
added to water, the precipitated solid was collected and
washed with water to give the product 6f (0.52 g, 60 %) as
a white solid. M.p.: 102–104 °C; IR (KBr): m = 2995,
1
3
J = 7.0 Hz, 2H), 1.30 (t, J = 7.0 Hz, 3 H) ppm; C NMR
(
DMSO-d , 75 MHz): d = 162.45 (CO), 161.22 (CO),
6
1
39.45 (C8-a), 138.67 (C4), 135.67 (C, benzyl), 130.32
(
C5), 127.14 (CH, benzyl), 126.68 (29CH, benzyl), 126.32
123

I. Tomassoli et al.
-
1 1
1
735, 1615 cm ; H NMR (CDCl , 300 MHz): d = 12.74
300 MHz): d = 10.61 (br s, 1H, NH), 7.95–7.97 (m, 3H),
7.47 (m, 2H), 7.39 (m, 1H), 7.23 (m, 2H), 7.12 (m, 1H)
3
(
s, 1H), 7.59 (t, J = 8.1 Hz, 1H), 7.46 (dd, J = 8.3,
.8 Hz, 1H), 7.59 (dd, J = 7.8, 1.0 Hz, 1H), 4.34 (q,
1
J = 7.1 Hz, 2H), 1.30 (t, J = 7.1 Hz, 3H) ppm; C NMR
1
3
0
ppm; C NMR (DMSO-d , 75 MHz): d = 161.44 (CO),
6
3
160.18 (CO), 147.32 (C, C5-a), 139.32 (C9-b), 135.12 (C,
phenyl), 129.80 (C7), 129.22 (29CH, phenyl), 122.21
(C9), 121.76 (C8), 121.47 (CH, phenyl), 121.39 (29CH,
phenyl), 116.21 (C6), 116.18 (C9-a), 107.09 (C3-a) ppm.
(
DMSO-d , 125 MHz): d = 163.28 (CO), 156.69 (CO),
6
1
1
6
40.73 (C8-a), 138.42 (C5), 130.73 (C4), 132.45 (C7),
24.26 (C4-a), 126.27 (C6), 112.95 (C3), 116.27 (C8),
1.83 (CH ), 13.89 (CH ) ppm.
2
3
5
3
-Methyl-1,2-dihydro-3H-pyrazolo[4,3-c]quinoline-
,4(5H)-dione (7d, C H N O )
General procedure for preparation of compounds
a–7l
11 9 3 2
Following the general procedure, the reaction of 0.3 g
3
compound 6b (1.13 mmol) and 0.3 cm hydrazine
7
3
9.45 mmol) in 25 cm ethoxyethanol gave yellow com-
(
The 4-choroquinolines were added to a solution of the
3
corresponding hydrazine (5–7 equiv) in 5–25 cm ethox-
pound 7d (0.16 g, 66 %). M.p.: [260 °C; IR (KBr):
m = 3328, 1651, 1585, 1554 cm ; H NMR (DMSO-d ,
-
1 1
yethanol. The solution was heated for 2–6 h and allowed
cool to rt. The precipitate obtained was filtered, washed
with diethyl ether, and dried to yield the desired compound.
6
3
00 MHz): d = 7.95 (dd, J = 1.5 Hz, 7.7 Hz, 1H), 7.45 (t,
J = 7.0 Hz, 1H), 7.36 (d, J = 8.1 Hz, 1H), 7.18 (t,
1
3
J = 7.2 Hz, 1H), 3.50 (s, 3H) ppm; C NMR (DMSO-
d6, 75 MHz): d = 161.18 (CO), 159.35 (CO), 143.57 (C,
C5-a), 139.49 (C, C9-b), 129.49 (CH, C7), 122.16 (CH,
C9), 121.81 (CH, C8), 115.79 (C, C6), 114.89 (CH, C9-a),
1
,2-Dihydro-3H-pyrazolo[4,3-c]quinoline-3,4(5H)-dione
(
7a, C H N O )
0 7 3 2
1
Following the general procedure, the reaction of 0.3 g
3
compound 6c (1.19 mmol) and 0.6 cm hydrazine mono-
3
hydrate (8.33 mmol) in 10 cm ethoxyethanol gave off-
94.76 (C, C3-a), 28.48 (CH ) ppm.
3
white compound 7a (0.19 g, 79 %). M.p.: [260 °C; IR
1
3
,5-Dimethyl-1,2-dihydro-3H-pyrazolo[4,3-c]quinoline-
-
1
1
(
(
(
(
KBr): m = 3320, 2990, 1651, 1585 cm
;
H NMR
,4(5H)-dione (7e, C H N O )
1
2 11 3 2
DMSO-d , 300 MHz): d = 10.52 (br s, 1H, NH), 7.87
6
Following the general procedure, the reaction of 0.5 g
3
compound 6b (1.88 mmol) and 0.5 cm methylhydrazine
3
(9.55 mmol) in 20 cm ethoxyethanol gave yellow com-
1
d, J = 6.2 Hz, 1H), 7.09–7.32 (m, 3H) ppm; C NMR
3
DMSO-d , 62.5 MHz): d = 161.53 (CO), 160.36 (CO),
6
1
1
9
44.72 (C, C5-a), 138.49 (C, C9-b), 128.84 (CH, C7),
21.67 (CH, C9), 121.44 (CH, C8), 115.96 (2C, C6, C9-a),
5.22 (C, C3-a) ppm.
pound 7e (0.33 g, 77 %). M.p.: [260 °C; IR (KBr):
-
1 1
m = 3256, 1647, 1596, 1542 cm ; H NMR (DMSO-d ,
6
500 MHz): d = 8.17 (d, J = 8.0 Hz, 1H), 7.62 (t,
J = 7.9 Hz, 1H) 7.55 (d, J = 8.5 Hz, 1H), 7.33 (t,
1
1
3
-Methyl-1,2-dihydro-3H-pyrazolo[4,3-c]quinoline-
3
J = 7.6 Hz, 1H), 4.13 (s, 3H), 3.57 (s, 3H) ppm;
C
,4(5H)-dione (7b, C H N O )
1
1 9 3 2
NMR (DMSO-d , 125 MHz): d = 157.31 (CO), 157.22
6
Following the general procedure, the reaction of 0.3 g
3
compound 6c (1.19 mmol) and 0.44 cm methylhydrazine
(CO), 139.18 (C5-a), 138.74 (C9-b), 129.77 (C7), 123.30
(C9), 121.71 (C8), 115.86 (C6), 111.94 (C9-a), 97.90 (C3-
a), 39.5 (CH , overlapped with DMSO), 28.39 (CH ) ppm.
3
9.45 mmol) in 10 cm ethoxyethanol gave off-white
(
3
3
compound 7b (0.12 g, 46 %). M.p.: [260 °C; IR (KBr):
m = 3330, 2995, 1675, 1590 cm ; H NMR (DMSO-d ,
-
1 1
6
5-Methyl-1-phenyl-1,2-dihydro-3H-pyrazolo[4,3-c]quino-
3
00 MHz): d = 11.11 (br s, 1H, NH), 10.58 (br s, 1H, NH),
.10 (d, J = 7.8 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.37
line-3,4(5H)-dione (7f, C H N O )
1
7 13 3 2
8
Following the general procedure, the reaction of 0.5 g
3
compound 6b (1.88 mmol) and 1 cm phenylhydrazine
1
3
(
m, 1H), 7.22 (d, J = 7.3 Hz, 1H), 4.11 (s, 3H) ppm;
C
NMR (DMSO-d , 75 MHz): d = 158.41 (CO), 157.69
6
3
10.1 mmol) in 20 cm ethoxyethanol gave light brown
(
(
(
CO), 140.41 (C5-a), 139.02 (C9-b), 129.94 (C7), 123.44
C9), 122.07 (C8), 116.63 (C6), 111.40 (C9-a), 98.89 (C3-
compound 7f (0.094 g, 78 %). M.p.: 189 °C; IR (KBr):
-
1 1
m = 1627, 1593, 1569, 1542 cm ; H NMR (DMSO-d ,
6
a), 39.5 (CH , overlapped with DMSO) ppm.
3
300 MHz): d = 8.10 (d, J = 7.5 Hz, 1H), 7.83 (d,
1
3
-Phenyl-1,2-dihydro-3H-pyrazolo[4,3-c]quinoline-
J = 7.8 Hz, 2H), 7.62–7.48 (m, 4H), 7.31 (m, 1H), 7.29
1
3
,4(5H)-dione (7c, C H N O )
1
(t, J = 7.5 Hz, 1H), 3.57 (s, 3H) ppm; C NMR (DMSO-
d6, 75 MHz): d = 158.19 (2xCO), 146.53 (C, C5-a),
140.13 (C9-b), 138.10 (C, phenyl), 130.97 (C7), 129.53
(29CH, phenyl), 127.29 (CH, phenyl), 122.91 (29CH,
phenyl), 122.74 (C9), 122.48 (C8), 116.23 (C6, C9-a),
6 11 3 2
Following the general procedure, the reaction of 0.3 g
3
compound 6c (1.19 mmol) and 0.82 cm phenylhydrazine
3
8.33 mmol) in 10 cm ethoxyethanol gave light brown
(
compound 7c (0.24 g, 72 %). M.p.: [260 °C; IR (KBr):
m = 3327, 3015, 1666, 1588 cm ; H NMR (DMSO-d ,
-
1 1
6
96.30 (C3-a), 28.11 (CH ) ppm.
3
1
23

Synthesis, regioselectivity, and DFT analysis of new antioxidant pyrazolo[4,3-c]quinoline-3,…
3
(0.87 mmol) in 5 cm ethoxyethanol gave yellow com-
5
3
-Benzyl-1,2-dihydro-3H-pyrazolo[4,3-c]quinoline-
,4(5H)-dione (7g, C H N O )
1
pound 7j (0.031 g, 71 %). M.p.: [260 °C; IR (KBr):
7 13 3 2
-
1
1
Following the general procedure, the reaction of 0.05 g
3
compound 6a (0.15 mmol) and 25 mm hydrazine
m = 3002, 1680, 1590 cm
;
H NMR (DMSO-d6,
250 MHz): d = 11.29 (br s, 1H), 7.46 (m, 1H),
7.33–7.26 (m, 2H ? 1NH), 3.96 (s, 3H) ppm; C NMR
3
0.78 mmol) in 5 cm ethoxyethanol gave yellow com-
13
(
pound 7g (0.038 g, 83 %). M.p.: [260 °C; IR (KBr):
(DMSO-d , 62.5 MHz): d = 158.94 (CO), 158.00 (CO),
141.71 (C5-a), 141.29 (C9-b), 130.82 (C9), 127.99 (C7),
6
-
1
1
m = 1643, 1554 cm ; H NMR (DMSO-d , 250 MHz):
6
d = 13.00 (br s, 1H, NH), 8.06 (d, J = 7.0 Hz, 1H), 7.43
124.24 (C9-a), 115.58 (C8), 110.42 (C6), 100.84 (C3-a),
(
t, J = 7.1 Hz, 1H), 7.10–7.33 (m, 7H), 5.49 (s, 2H) ppm;
3
44.07 (CH ) ppm.
3
1
C NMR (DMSO-d , 62.5 MHz): d = 159.53 (CO),
6
9-Chloro-1,5-dimethyl-1,2-dihydro-3H-pyrazolo[4,3-c]-
1
58.43 (CO), 141.97 (C5-a), 138.47 (C9-b), 137.95 (C,
quinoline-3,4(5H)-dione (7k, C H ClN O )
1
2
10
3 2
benzyl), 130.32 (CH, benzyl), 128.99 (29CH, benzyl),
27.28 (C7), 126.79 (29CH, benzyl), 122.80 (C9), 122.34
Following the general procedure, the reaction of 0.5 g
3
compound 6e (0.17 mmol) and 45 mm methylhydrazine
1
(
(
C8), 116.73 (C6), 112.31 (C9-a), 96.73 (C3-a), 44.14
3
0.87 mmol) in 5 cm ethoxyethanol gave yellow com-
(
CH , benzyl) ppm.
2
pound 7k (0.027 g, 61 %). M.p.: [260 °C; IR (KBr):
m = 3290, 1667, 1610 cm
-
1
1
5
-Benzyl-1-methyl-1,2-dihydro-3H-pyrazolo[4,3-c]-
;
H NMR (DMSO-d6,
quinoline-3,4(5H)-dione (7h, C H N O )
1
300 MHz): d = 10.80 (br s, 1H), 7.63–7.44 (m, 2H),
7.42 (d, J = 7.5 Hz, 1H), 3.93 (s, 3H), 3.55 (s, 3H) ppm;
13
8 15 3 2
Following the general procedure, the reaction of 0.05 g
3
compound 6a (0.15 mmol) and 39 mm methylhydrazine
C NMR (DMSO-d , 75 MHz): d = 159.21 (CO), 157.52
6
3
0.75 mmol) in 5 cm ethoxyethanol gave yellow com-
(
(CO), 141.86 (C5-a), 140.84 (C9-b), 131.09 (C9), 128.55
(C7), 124.49 (C9-a), 115.27 (C8), 111.39 (C6), 100.41 (C3-
a), 43.78 (CH ), 29.68 (CH ) ppm.
pound 7h (0.021 g, 44 %). M.p.: [260 °C; IR (KBr):
-
1
1
m = 3355, 1651, 1573 cm
;
H NMR (DMSO-d6,
3
3
3
1
7
00 MHz): d = 12.55 (s, 1H, NH), 8.18 (d, J = 8.1 Hz,
H), 7.47 (t, J = 7.5 Hz, 1H), 7.36 (d, J = 8.4 Hz, 1H),
.26–7.21 (m, 3H), 7.21–7.21 (m, 3H), 5.51 (s, 2H), 4.16
5
-Benzyl-9-chloro-1-methyl-1,2-dihydro-3H-pyrazolo[4,3-
c]quinoline-3,4(5H)-dione (7l, C H ClN O )
14
1
8
3 2
Following the general procedure, the reaction of 0.05 g
3
compound 6d (0.14 mmol) and 36 mm methylhydrazine
1
3
(
(
s, 3H) ppm; C NMR (DMSO-d , 75 MHz): d = 158.04
2xCO), 139.42 (C5-a), 138.75 (C9-b), 137.77 (C, benzyl),
6
3
0.70 mmol) in 5 cm ethoxyethanol gave yellow com-
(
1
30.21 (CH, benzyl), 129.06 (29CH, benzyl), 127.35 (C7),
26.78 (29CH, benzyl), 124.12 (C9), 122.41 (C8), 116.95
pound 7l (0.023 g, 51 %). M.p.: [260 °C; IR (KBr):
mn = 3300, 1675, 1580 cm
1
-
1
;
1
H NMR (DMSO-d6,
(
C6), 112.83 (C9-a), 98.16 (C3-a), 44.26 (CH , benzyl),
2
3
00 MHz): d = 7.31–7.24 (m, 8H), 5.40 (s, 2H), 3.53 (s,
3
9.5 (CH , overlapped with DMSO) ppm.
3
13
3
H) ppm; C NMR (DMSO-d , 75 MHz): d = 161.27
6
5
-Benzyl-1-phenyl-1,2-dihydro-3H-pyrazolo[4,3-c]quino-
(2xCO), 142.31 (C5-a), 140.36 (C9-b), 137.90 (C, benzyl),
129.90 (C9), 128.92 (CH, benzyl), 127.29 (29CH, benzyl),
126.88 (29CH, benzyl), 124.67 (C7), 123.96 (C9-a),
115.50 (C8), 114.88 (C6), 104.01 (C3-a), 44.32 (CH2,
line-3,4(5H)-dione (7i, C H N O )
2
3 17 3 2
Following the general procedure, the reaction of 0.05 g
3
compound 6a (0.15 mmol) and 74 mm phenylhydrazine
3
0.75 mmol) in 5 cm ethoxyethanol gave light brown com-
(
benzyl), 32.57 (CH ) ppm.
3
pound 7i (0.030 g, 52 %). M.p.: [260 °C; IR (KBr):
-
1
1
m = 3200, 1667, 1580 cm ;
00 MHz): d = 8.10 (d, J = 7.4 Hz, 1H), 7.96 (d,
J = 7.8 Hz, 2H), 7.61–7.40 (m, 3H), 7.40–7.15 (m, 8H),
H
NMR (DMSO-d6,
Computational studies
3
DFT calculations at the B3LYP level of theory using
6-311G** basis set were performed with the Gaussian09
package. Full geometry optimizations of reactants and
products (in gas and liquid phases) were done at 298 K, for
ground state as well as for transition state searches
including vibrational analysis to locate stationary point and
zero point energy (zpe). The effect of solvation in
2-ethoxyethanol as included in the code was carried out
using PCM method [36, 37]. Transition state (TS) struc-
tures were determined by searching the synchronous
transit-guided quasi-Newton (STQN) method (QST2 key-
word) for first-order saddle points on the potential energy
13
7
7
1
.18–7.20 (m, 5H), 5.48 (s, 2H) ppm; C NMR (DMSO-d6,
5 MHz): d = 160.09 (CO), 159.97 (CO), 146.22 (C5-a),
39.07 (C9-b), 138.07 (C, benzyl), 130.28 (C, phenyl), 129.43
(
29CH, benzyl), 129.33 (C7), 129.02 (29CH, phenyl), 127.31
CH, benzyl), 126.89 (29CH, benzyl), 122.72 (CH, phenyl),
22.53 (29CH, phenyl), 121.90 (C9), 121.85 (C8), 121.79
(
1
(C6), 116.73 (C9-a), 95.20 (C3-a), 43.95 (CH , benzyl) ppm.
2
9
-Chloro-1-methyl-1,2-dihydro-3H-pyrazolo[4,3-c]quino-
line-3,4(5H)-dione (7j, C H ClN O )
3 2
1
1
8
Following the general procedure, the reaction of 0.05 g
3
compound 6f (0.17 mmol) and 45 mm methylhydrazine
123

I. Tomassoli et al.
surface (PES) via frequency calculations. The Fukui
function and local softness were computed from anionic
and cationic forms of reactants with the same geometry as
the neutral one using gross electronic population [38].
3. Savini L, Chiasserini L, Pellerano C, Biggio G, Maciocco E,
Serra M, Cinone N, Carrieri A, Altomare C, Carotti A (2001)
Bioorg Med Chem 9:431
4
. Carotti A, Altomare C, Savini L, Chiasserini L, Pellerano C,
Mascia MP, Maciocco E, Busonero F, Mameli M, Biggio G,
Sanna E (2003) Bioorg Med Chem 11:5259
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. Skotnicki JS, Gilman SC, Steinbaugh BA, Musser JH (1988)
Pyrazolo[4,3-c]quinolines as anti inflammatories. US Patent
Hydroxyl radical scavenging activity assay
4
,748,246, May 31, 1988; Chem Abstr 109:110425
3
In a screw-capped test tube, 0.2 cm sodium benzoate
6. Baruah B, Dasu K, Vaitilingam B, Vanguri A, Rao Casturi S, Rao
Yeleswarapu K (2004) Bioorg Med Chem Lett 14:445
. Crespo MI, Gr a` cia J, Puig C, Vega A, Bou J, Beleta J, Dom e´ nech
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3
(
10 mmol), 0.2 cm FeSO Á7H O (10 mmol), and EDTA
4
2
7
(
10 mmol) were added. Then a phosphate buffer (pH 7.4,
3
.1 mol) was added to give a total volume of 1.8 cm .
0
3
A H O solution (0.2 cm , 10 mmol) was added, and the
2
2
8. Wentland MP (1994) Preparation of 5-cyclopropyl-8-fluoro-7-(4-
pyridyl)-3H-pyrazolo[4,3-c]quinolin-3-one topoisomerase-in-
mixture was then incubated at 37 °C for 2 h. The fluores-
cence was measured at 407 nm emission and excitation
hibiting anticancer agents. US Patent 5,334,595, Aug 02, 1994;
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. Suzuki F, Nakasato Y, Ohmori K, Tamura T, Hosoe H, Kubo K,
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1. Ghotekar BK, Ghagare MG, Toche RB, Jachak MN (2010)
Monatsh Chem 141:169
3
05 nm. RSA OH°% = Absorbance in the presence of
sample divided by the absorbance in the absence of sample
100. Mean values from three independent samples were
9
9
calculated and showed for each compound a standard
deviations less than 5 %. Quercetol was used as a positive
control.
1
1
1
2. Isma ¨ı li L, Refouvelet B, Robert JF (1999) J Heterocycl Chem
6:719
3
DPPH radical scavenging activity assay
1
3. Truong AP, Aubele DL, Probst GD, Neitzel ML, Semko CM,
Bowers S, Dressen D, Hom RK, Konradi AW, Sham HL,
Garofalo AW, Keim PS, Wu J, Dappen MS, Wong K, Goldbach
E, Quinn KP, Sauer J-M, Brigham EF, Wallace W, Nguyen L,
Hemphill SS, Bova MP, Bard F, Yednock TA, Basi G (2009)
Bioorg Med Chem Lett 19:4920
For this assay the method of Hatano et al. [39] was used to
evaluate the capacity of compounds to scavenge the ‘‘stable’’
free radical DPPH. Different concentrations of methanolic
3
solutions (0.3 cm ) of the compounds were mixed with a
1
4. Beshore DC, DiPardo RM, Kuduk SD (2010) Tetrahedron Lett
51:970
-4
3
3
DPPH methanolic solution (1.5 9 10 mol/dm , 2.7 cm ).
The solution was mixed vigorously and kept for 2 h in the
dark, until stable absorption values were obtained. The
quenching of the DPPH radical was determined by measuring
the absorption at 517 nm. The RSA was calculated as a per-
centage of disappearance of DPPH purple color using the
equation % RSA ¼ ½ðADPPH À ASÞ=ADPPHŠ  100; where
15. Kalita PK, Baruah B, Bhuyan PJ (2006) Tetrahedron Lett
7:7779
4
6. Shawali AS (1993) Chem Rev 93:2731
7. Daou B, Soufiaoui M (1989) Tetrahedron 45:3351
1
1
18. G a´ l M, Feh e´ r O¨ , Tihanyi E, Horv a´ th G, Jerkovich G, Argay G,
Ka
´
lm a´ n A (1980) Tetrahedron Lett 21:1567
¨
1
9. G a´ l M, Feh e´ r O, Tihanyi E, Horv a´ th G, Jerkovich G (1982)
Tetrahedron 38:2933
A
DPPH
is the absorbance of the DPPH solution and A is the
S
2
0. L o´ pez Rivilli MJ, Moyano EL, Yranzo GI (2010) Tetrahedron
Lett 51:478
absorbance of the solution when the compound has been
added at a given concentration. Mean values from three
independent samples were calculated and showed for each
compound a standard deviations less than 5 %. Ascorbic acid
was used as a positive control.
´
21. Tomassoli I, Ismaili L, Pudlo M, de Los Rıos C, Soriano E,
Colmena I, Gand ´ı a L, Rivas L, Samadi A, Marco-Contelles J,
Refouvelet B (2011) Eur J Med Chem 46:1
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