Ultrasound promoted synthesis of 2-imidazolines in water: A greener approach toward monoamine oxidase inhibitors

Article abstract of DOI:10.1016/j.bmcl.2008.03.001

A series of sixteen 2-substituted-2-imidazolines (where the substituent R = Ph, Me-4-Ph; MeO-4-Ph; (MeO)₂-3,4-Ph; (MeO)₃-3,4,5-Ph; Ph-4-O-C(O)-Ph; Cl-4-Ph; Cl-2-Ph; Cl₂-2,4-Ph; NO₂-4-Ph; NO₂-3-Ph; Naphth-2-yl; Fur-2-yl; Benzofur-2-yl; Pyridin-2-yl; Quinolin-2-yl) has been synthesized from the reaction of the substituted-aldehydes and ethylenediamine by ultrasound irradiation with NBS in an aqueous medium in high yields (80-99%). The 2-imidazoline ability to inhibit the activity of the A and B isoforms of monoamine oxidase (MAO) was investigated and some of them showed potent and selective MAO inhibitory activity especially for the MAO-B isoform and could become promising candidates for future development.

Full text of DOI:10.1016/j.bmcl.2008.03.001

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 19 (2009) 546–549
Ultrasound promoted synthesis of 2-imidazolines in water:
A greener approach toward monoamine oxidase inhibitors
Gabriela da S. Sant’ Anna,b Pablo Machado,a Patricia D. Sauzem,b Fernanda A. Rosa,a
Maribel A. Rubin,b Juliano Ferreira,b Helio G. Bonacorso,a
Nilo Zanattaa and Marcos A. P. Martinsa,*
a
ˆ
Nu´cleo de Quı´mica de Heterociclos (NUQUIMHE), Departamento de Quı´mica, Centro de Ciencias Naturais e Exatas,
Universidade Federal de Santa Maria, 97.105-900, Santa Maria, RS, Brazil
b
ˆ
Laborato´rio de Neurotoxicidade e Psicofarmacologia, Departamento de Quı´mica, Centro de Ciencias Naturais e Exatas,
Universidade Federal de Santa Maria, 97.105-900, Santa Maria, RS, Brazil
Received 27 December 2007; revised 29 February 2008; accepted 3 March 2008
Available online 6 March 2008
Abstract—A series of sixteen 2-substituted-2-imidazolines (where the substituent R = Ph, Me-4-Ph; MeO-4-Ph; (MeO)2-3,4-Ph;
(MeO)3-3,4,5-Ph; Ph-4-O-C(O)-Ph; Cl-4-Ph; Cl-2-Ph; Cl2-2,4-Ph; NO2-4-Ph; NO2-3-Ph; Naphth-2-yl; Fur-2-yl; Benzofur-2-yl; Pyri-
din-2-yl; Quinolin-2-yl) has been synthesized from the reaction of the substituted-aldehydes and ethylenediamine by ultrasound irra-
diation with NBS in an aqueous medium in high yields (80–99%). The 2-imidazoline ability to inhibit the activity of the A and B
isoforms of monoamine oxidase (MAO) was investigated and some of them showed potent and selective MAO inhibitory activity
especially for the MAO-B isoform and could become promising candidates for future development.
ꢀ 2008 Elsevier Ltd. All rights reserved.
Monoamine oxidase (MAO), localized in the outer
mitochondrial membrane, is a flavin adenine dinucleo-
tide (FAD)-containing enzyme responsible for the oxi-
dative deamination of amines in the brain and the
peripheral tissues, regulating their level.1 This reaction
produces the corresponding aldehyde and free amine,
with the generation of hydrogen peroxide.1 MAO exists
in two isoforms, namely, MAO-A and MAO-B, which
differ according to their substrate specificity and their
selectivity to the inhibitor.2 MAO-A preferentially
metabolizes serotonin and noradrenaline and is inhib-
ited by low concentrations of clorgyline.3 MAO-B acts
preferentially on 2-phenylethylamine and benzylamine
and is inhibited by selegiline (L-deprenyl).4 Dopamine,
tyramine, and tryptamine are the substrates for both iso-
forms of MAO.4
an important target for treating psychiatric and neuro-
degenerative disorders. Currently, compounds able to
inhibit these enzymes have been used in the therapy of
Parkinson’s and Alzheimer’s disease, depression syn-
drome and panic disorders.5 In fact, Parkinson’s and
Alzheimer’s disease have been associated with oxidative
stress and increasing MAO-B activity in the CNS.6
Over the last 15 years, since the demonstration that
I2-imidazoline sites are associated with the mitochon-
drial fraction of membranes,7 several studies have pro-
vided evidence that these sites represent regions on
MAOs.8 In fact, I2-binding sites have been identified
on both MAO-A and MAO-B isoforms as regulatory
sites are able to modulate MAO activity through a not
yet fully understood inhibitory mechanism.8 Moreover,
because of the different tissue sensitivities to amiloride,
Due to their role in neurotransmitter metabolism, the
regulation of MAO-A and MAO-B activity has been
a guanidine able to discriminate between I2A- and I2B-
subtypes, it has been proposed that I2A-sites are located
on MAO-A and I2B-sites on MAO-B.9 These I2-binding
sites do not appear to be located within the enzyme cat-
alytic site, or the prosthetic group, or the binding do-
main of classical MAO inhibitors.8a,10 However, most
recent reports have demonstrated that the majority of
amino acid residues identified as I2-sites on MAO-B
Keywords: Imidazolines; Ultrasound; MAO inhibitory activity; Ethy-
lenediamine; Greener reaction conditions.
*
Corresponding author. Tel./fax: +55 55 3220 8756; e-mail:
mmartins@base.ufsm.br
0960-894X/$ - see front matter ꢀ 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.001

G. S. Sant’ Anna et al. / Bioorg. Med. Chem. Lett. 19 (2009) 546–549
547
(149–222) are obtained within the active site or entrance
cavity of the enzyme according to the crystallographic
studies.11 Recently, it was also demonstrated that imi-
dazoline compounds are able to bind to the MAO-A ac-
tive site.12
opportunities for clean processing and pollution preven-
tion. In addition, because organic reactions often dis-
play unique reactivity and selectivity, performing them
in an aqueous medium at or slightly above room tem-
perature has become of great interest in order to exploit,
for example, their so-called hydrophobic effects.20a
Therefore, some reports from the literature have demon-
strated that imidazoline and guanidinium derivative
compounds are able to inhibit MAO activities.10a,13 This
effect has been attributed to a high affinity I2 binding site
on MAO-B (I2B) and a similar lower affinity site on
MAO-A (I2A).14 In this context, the limited accessibility
of the I2 imidazoline binding domain on MAO-B in var-
ious tissues indicates the existence of a distinct subpop-
ulation of the enzyme.15 Thus, the use of imidazoline
derivates such as MAO inhibitors may allow for the
development of new therapeutic agents that target the
enzyme in a cell-type selective manner.
The synthesis of 2-imidazolines (3a–p) was carried out
by the previously reported method of condensation
involving aldehydes (1a–p) and ethylenediamine (2) in
the presence of N-bromosuccinimide.16 The reaction
was performed in water as solvent under ultrasonic irra-
diation furnishing the products (3a–p) in high yields and
significantly shorter times (Scheme 1).
The method for forming 2-imidazolines under ultra-
sonic irradiation offers several advantages including
faster reaction rates, higher purity, and higher yields.
In comparison with conventional methods, the main
goal of ultrasound application was the significant de-
crease of reaction time. While conventional methods
require agitation overnight,16 under ultrasonic irradia-
tion the products were obtained in 12–18 min.21,22
Moreover, this approach does not require the use of
any halogenated solvent or an additional method for
product purification. In contrast to other methods,23
the synthesis of 2-imidazolines did not require the
use of any catalyst.
We are particularly interested in the therapeutic role of
MAO-B inhibitors in Parkinson’s and Alzheimer’s dis-
ease. In this paper, in an attempt to identify novel imi-
dazoline derivatives endowed with MAO inhibitory
activity and selectivity we have synthesized a series of
2-imidazolines combining a greener synthetic approach
with assessment of their in vitro activities.
The energy provided by ultrasound has been utilized re-
cently to accelerate a number of synthetically useful
reactions.17 Its effect observed on organic reactions is
due to cavitation, a physical process that creates, en-
larges, and implodes gaseous and vaporous cavities in
an irradiated liquid, thus enhancing the mass transfer.17a
Compared with traditional methods, this technique is
more appropriate in the consideration of green chemis-
try concepts. Ultrasound application is considered a
processing aid (or auxiliary) in terms of energy conser-
vation and waste minimization.18 Recently, we have
demonstrated the use of this form of energy in heterocy-
clic preparations providing a remarkable rate of
enhancement and a dramatic reduction in reaction
time.19
All compounds 3 showed physical and spectrometric
properties corresponding to the proposed structures
and in accordance with the literature.16,25
The in vitro inhibitory activity of compounds 3a-p
against MAOs was determined as previously de-
scribed24 and the results are shown in Table 1. The
compounds obtained were considered potent when Ki
was less than 10 lM and were particularly selective
when their selectivity index (Ki MAO-A/Ki MAO-B)
was greater than 10. Among the synthesized com-
pounds that inhibited preferentially MAO-A (entries
3c–e, 3j) only compound 3d was found to be selective,
presenting a Ki for MAO-A of approximately 73-fold
lower than its Ki for MAO-B. Imidazoline 3p also
showed a potent inhibitory effect (Ki 4.86 lM) for this
MAO isoform, however, this compound was not selec-
tive (0.91).
In recent decades, the use of water as solvent in organic
reactions has been reinvestigated.20 Water is a nontoxic
solvent and readily available at low cost. It is also non-
flammable and environmentally benign, providing
R
Time (min) Yield (%)
R
Time (min) Yield (%)
Entry
Entry
Ph
Me-4-Ph
12
18
15
15
15
15
18
15
99
95
92
90
86
84
98
92
Cl2-2,4-Ph
NO2-4-Ph
NO2-3-Ph
Naphth-2-yl
Fur-2-yl
Benzofur-2-yl
Pyridin-2-yl
Quinolin-2-yl
12
18
15
15
18
15
15
18
93
82
96
91
80
87
95
97
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
MeO-4-Ph
(MeO)2-3,4-Ph
(MeO)3-3,4,5-Ph
Ph-4-O-C(O)-Ph
Cl-4-Ph
Cl-2-Ph
Scheme 1. Reagents and conditions: (i) H2O, NBS, , 65–70 ꢁC, 12–18 min.

548
G. S. Sant’ Anna et al. / Bioorg. Med. Chem. Lett. 19 (2009) 546–549
Table 1. Monoamine oxidase inhibitory activity of 2-imidazolines
3a–p
MAO with significant selectivity. Reports from the liter-
ature have affirmed that there are no significant correla-
tions between the potencies of imidazoline derivatives at
I2-sites and their values for inhibition of MAO-A or
MAO-B activities. In general, these compounds show
two to three times less activity for MAO inhibition than
their reported values of I2-site affinity.13 Thus, it has
been suggested that I2-sites are not directly related to
the site of action of these drugs against MAO activity.
Entry
Ki MAO-Aa (lM)
Ki MAO-Ba (lM) SIb
3a
3b
28.75 (21.0–39.4)
102.10 (64.4–162)
15.75 (10.2–24.2)
13.57 (9.1–20.2)
114.40 (75.0–174.4)
12.84 (8.6–19.2)
21.27 (14.7–30.7)
13.09 (8.1–21.0)
32.67 (20.9–51.1)
ꢀ1000
1.35
7.79
3c
3d
0.48
0.013
0.114
9.27
3e
ꢀ1000
3f
3g
1.38 (0.97–1.97)
166.30 (102.3–270.5) 5.35 (3.4–8.3)
31.05
>40.70
10.52
0.181
>51.89
9.32
3h
3i
>1000
24.57 (15.2–39.8)
3.98 (2.9–5.3)
>1000
The nature of the interaction of imidazolines with MAO
has been described as either noncompetitive8b and mix-
ed,10a or competitive for MAO-A inhibition and mixed
for MAO-B inhibition.13 Our preliminary kinetics stud-
ies showed that compound 3g, obtained as a lead-like
compound, inhibited MAO-B in a competitive manner.
However, additional studies are necessary to clarify this
point.
28.75 (20.9–39.4)
181.40 (115–286.2)
>1000
3j
3k
19.27 (11.1–33.5)
1.49 (0.4–5.1)
>1000
3l
3m
13.89 (6.02–32.04)
>1000
—
6.86
3n (2-BFI) 24.94 (14.3-43.6)
3o >1000
3p (BU224) 4.86 (3.6–6.6)
3.63 (0.3–4.0)
25.28 (11.8–54.0)
5.32 (3.8–7.4)
>39.55
0.91
a Each value represents the mean (confidance interval) of three or four
independent experiments.
In summary, we have reported the preparation of 2-imi-
dazolines using a highly efficient and environmentally
benign synthetic protocol to obtain novel compounds
endowed with MAO inhibitory activity. The simplicity,
the use of water instead of organic solvents as reaction
medium, the high yields (80–99%), and the short
reaction times (12–18 min) make this reaction highly
attractive. In addition, the existence of a distinct sub-
population of MAO-B with a different accessibility to
the I2-site may allow access to inhibitors of this isoform
in a tissue-specific manner, avoiding numerous side ef-
fects attributed to these classes of compounds. Our con-
tribution toward this understanding was to obtain novel
2-imidazoline derivatives as active MAO inhibitors in
the lM range with good selectivity. Studies on the use
of 2-imidazoline compounds in models of Parkinson’s
and Alzheimer’s disease are in progress and these data
will be communicated hereafter.
b In vitro selectivity index = (Ki MAO-A/Ki MAO-B).
Among the compounds obtained that selectively inhib-
ited MAO-B (entries 3g–i, 3k, 3o), only imidazolines
3g and 3i were shown to be potent with Ki values of
5.35 lM and 3.98 lM, respectively. One can conclude
that the replacement of chlorine at the ortho position
in the 2-imidazoline 3h with the para position on com-
pound 2g seemed to decrease selectively for MAO-B
and increase the inhibitory potency against this isoform.
The dichloro-substituted compound 3i lost selectivity
with a slightly increasing inhibitory activity against
MAO-B. An interesting finding was the inversion of
selectivity of 2-imidazoline 3k from the replacement of
an electron-withdrawing group (NO2) at the meta posi-
tion of benzene with an electron-donating group
(OMe) in compound 3d. While the imidazoline 3k was
able to selectively inhibit MAO-B, compound 3d
selectively inhibited MAO-A. This suggests that elec-
tronic effects can govern the recognition process for
2-imidazolines on both MAOs.
Acknowledgments
The authors thank the Conselho Nacional de Desen-
´
volvimento Cientıfico e Tecnologico (CNPq/PRONEX)
and the Fundac¸a˜o de Amparo a Pesquisa do Estado do
Rio Grande do Sul (FAPERGS) for financial support.
The fellowships from CNPq, CAPES and FAPERGS
are also acknowledged.
´
`
Some of the synthesized compounds were shown to
exert potent and selective binding at I2-sites, such as 3l
(benazoline), 3n (2-BFI), and 3p (BU224).25 In fact,
these imidazolines showed good inhibitory activity espe-
cially against MAO-B. The 2-BFI and BU224 IC50 val-
ues reported in the literature range from 11 to 16.5 and
4.8 to 10.7 lM for MAO-A and from 23 to 27.9 and 44.8
to 51.4 lM for MAO-B, respectively.13,29,30 Our ob-
tained 2-BFI and BU224 IC50 values were 65 and
12.7 lM for MAO-A and 9.1 and 13.3 lM, respectively.
Thus, in our conditions 2-BFI and BU224 seems to be
less potent to inhibit MAO-A and more potent to inhibit
MAO-B when compared with the literature values.
However, when Ki values were compared, the 2-BFI po-
tency on MAO-A obtained in our study (29.94 lM) are
similar with the literature data (26 lM).12 As Ki values
are more appropriated for potency comparison among
different studies, our results are consistent with the liter-
ature data. However, none of the compounds endowed
with high affinity for the I2-site were able to inhibit
References and notes
1. Youdim, B. H.; Bakhle, Y. S. Br. J. Pharmacol. 2006, 147,
287.
2. Shih, J. C.; Chen, K.; Ridd, M. J. Annu. Rev. Neurosci.
1999, 22, 197.
3. Weyler, W.; Hsu, Y. P.; Breakefield, X. O. Pharmacol.
Ther. 1990, 47, 391.
4. Kalgutkar, A. S.; Dalvie, D. K.; Castagnoli, N., Jr.;
Taylor, T. J. Chem. Res. Toxicol. 2001, 14, 1139.
5. Youdim, B. H.; Edmondson, D.; Tipton, K. F. Nat. Rev.
Neurosci. 2006, 7, 295.
6. (a) Volz, H. P.; Gleiter, C. H. Drugs Aging 1998, 13, 341;
(b) Good, P. F.; Werner, P.; Hsu, A.; Olanow, C. W.; Perl,

G. S. Sant’ Anna et al. / Bioorg. Med. Chem. Lett. 19 (2009) 546–549
549
D. P. Am. J. Pathol. 1996, 149, 21; (c) Drukarch, B.;
Muiswinkel, F. L. Biochem. Pharmacol. 2000, 59, 1023.
7. Tesson, F.; Parini, A. Eur. J. Pharmacol. 1991, 208, 81.
8. (a) Raddatz, R.; Parini, A.; Lanier, S. J. Biol. Chem. 1995,
270, 27961; (b) Tesson, F.; Limon-Boulez, I.; Urban, P.;
Puype, M.; Vardekerckhove, J.; Coupry, I.; Pompon, D.;
Parini, A. J. Biol. Chem. 1995, 270, 9856; (c) Raddatz, R.;
Parini, A.; Lanier, S. M. Mol. Pharmacol. 1997, 52, 549;
(d) Raddatz, R.; Savic, S. L.; Bakthavachalam, V.;
Lesnick, J.; Jasper, J. R.; Mcgrath, C. R.; Parini, A.;
Lanier, S. J. Pharmacol. Exp. Ther. 2000, 292, 1135.
9. Parini, A.; Moudanos, C. G.; Pizzinat, N.; Lanier, S. M.
Trends Pharmacol. Sci. 1996, 17, 13.
was removed under reduced pressure to afford the 2-
imidazolines 3 with excellent purity.
22. 1H and 13C NMR spectra were acquired on a Bruker DPX
200 or Bruker DPX 400 spectrometer (1H at 200.13 MHz
or 400.13 MHz and 13C at 50.32 MHz or 100.63 MHz,
respectively) at 300 K, in 5 mm sample tubes, and with a
digital resolution of 0.01 ppm. CDCl3 was used as
solvents containing TMS as internal standard. Mass
spectra were registered in a HP 5973 MSD connected to
a HP 6890 GC and interfaced by a Pentium PC. The GC
was equipped with a split-splitless injector, autosampler
cross-linked HP-5 capillary column (30 m, 0.32 mm of
internal diameter), and helium was used as the carrier gas.
1
´ ´
10. (a) Carpene, C.; Collon, P.; Remaury, A.; Cordi, A.;
Select data for compound 3f: mp 116–118 ꢁC; H NMR
Hudson, A.; Nutt, D.; Lafontan, M. J. Pharmacol. Exp.
Ther. 1995, 272, 681; (b) Limon-Boulez, I.; Tesson, F.;
Gargalidis-Moudanos, C.; Parini, A. J. Pharmacol. Exp.
Ther. 1996, 276, 359.
(CDCl3, 200 MHz) d 3.80 (s, 4H, 2CH2), 3.98 (br, 1H,
NH), 7.25–7.69 (m, 5H, Ph), 7.84–8.22 (m, 4H, Ph); 13C
NMR (CDCl3, 100 MHz) d 49.8 (2CH2), 121.7, 127.5,
128.5, 128.6, 129.0, 130.0, 133.7, 152.8 (Ph), 164.1 (C2),
164.7 (C@O); MS EI (70 ev) m/z (%): 266.1 (M+, 48), 237.1
(4), 132.1 (4), 105.1 (100), 77.1 (62), 51.0 (11); Anal. calcd
for C16H14N2O2: C 72.16%, H 5.30%, N 10.52%; found: C
72.03%, H 5.17%, N 10.47%.
11. (a) Binda, C.; Newton-Vinson, P.; Hubalek, F.; Edmondo-
son, D. E.; Mattevi, A. Nat. Struct. Biol. 2002, 9, 22; (b)
Ma, J.; Yoshimura, M.; Yamashita, E.; Nakagawa, A.;
Ito, A.; Tsukihara, T. J. Mol. Biol. 2004, 338, 103.
12. Jones, T. Z. E.; Giurato, L.; Guccione, S.; Ramsay, R. R.
FEBS J. 2007, 274, 1567.
23. Mirkhani, V.; Moghadam, M.; Tangestaninejad, S.; Kar-
gar, H. Tetrahedron Lett. 2006, 47, 2129.
13. Ozaita, A.; Olmos, G.; Boronat, M. A.; Lizcano, J. M.;
24. MAO inhibitions assay. The synthesized compounds were
tested for their inhibitory activity on MAO-A and -B in rat
brain mitochondrial homogenates.26 The protein concen-
tration was determined according to Bradford.27 The
mitochondrial fractions were preincubated at 37 ꢁC for
5 min with the irreversible and selective inhibitor selegiline
(250 nM) or clorgyline (250 nM) to assay MAO-A or
MAO-B activity, respectively. The imidazoline com-
pounds were dissolved in dimethyl sulfoxide (DMSO)
and added to the reaction mixture from 0 to 103 lM. After
5 min, kynuramine was added as a nonselective substrate
at concentrations equal to the corresponding KM value
(90 lM for MAO-A and 60 lM for MAO-B). The
solutions were incubated at 37 ꢁC for 30 min. Addition
of three chloroacetic acids ended the reaction and the
samples were centrifuged at 16,000g for 5 min. The
supernatant fractions were removed and the concentra-
tions of the MAO generated product, 4-hydroxyquinoline
(4-OHQ), which was measured spectrophotometrically at
314 nm. When there was interference of the compounds
tested on the spectrophotometric methods, the fluorescent
product of the reaction was measured by fluorimetric
method, using 315 nm (excitation) and 380 nm (emission).
Apparent Ki values were calculated using the equation for
competitive inhibitors.28
´
Unzeta, M.; Garcıa-Sevilla, J. A. Br. J. Pharmacol. 1997,
121, 901.
14. Paterson, L. M.; Tyacke, R. J.; Robinson, E. S. J.; Nutt,
D. J.; Hudson, A. L. Neuropharmacology 2007, 52, 395
(and references therein).
15. Raddatz, R.; Parini, A.; Lanier, S. M. J. Biol. Chem. 1995,
270, 27961.
16. (a) Fujioka, H.; Murai, K.; Ohba, Y.; Hiramatsu, A.;
Kita, Y. Tetrahedron Lett. 2005, 46, 2197; (b) Fujioka, H.;
Murai, K.; Kubo, O.; Ohba, Y.; Kita, Y. Tetrahedron
2007, 63, 638.
17. (a) Manson, T. J. Chem. Soc. Rev. 1997, 26, 443; (b) Cella,
R.; Stefani, H. A. Tetrahedron 2006, 62, 5656; (c) Guzen,
´
K. P.; Guarezemini, A. S.; Orfa˜o, A. T. G.; Cella, R.;
Pereira, C. M. P.; Stefani, H. A. Tetrahedron Lett. 2007,
48, 1845.
18. Cintas, P.; Luche, J.-L. Green Chem. 1999, 1, 115.
19. Martins, M. A. P.; Pereira, C. M. P.; Cunico, W.;
Moura, S.; Rosa, F. A.; Peres, R. L.; Machado, P.;
Zanatta, N.; Bonacorso, H. G. Ultrason. Sonochem.
2006, 13, 364.
20. (a) Dallinger, D.; Kappe, C. O. Chem. Rev. 2007, 107,
2563; (b) Lindstro¨m, U. M. Chem. Rev. 2002, 102, 2751.
21. Synthesis of 2-imidazolines 3a–p (General procedure): a
mixture of aldehyde 1 (1 mmol) and ethylenediamine 2
(0.072 g, 1.2 mmol) in water (12 mL) was added to a glass
tube of 15 mL. With aid of adapter, a titanium microtip
(Ø 6 mm) was attached to the reaction glass container and
the mixture was sonicated by 2 min (Vibra Cell VC50,
Sonics & Material Inc.). After this time, NBS (0.213 g,
1.2 mmol) was added to the mixture and the resulting
solution was sonicated for the appropriate time (Scheme
1). The reaction temperature was raised to 65–70 ꢁC after
sonication for 8–10 min. After cooling to room tempera-
ture NaOH (20%) aq was added to the reaction mixture.
Finally, the solution was extracted with ethyl acetate (3·
15 mL); after combined, the organics layers were washed
with water (2· 10 mL) and dried with Na2SO4. The solvent
25. Anastassiadou, M.; Danoun, S.; Crane, L.; Baziard-
Mouysset, G.; Payard, M.; Caignard, D.-H.; Ret-
tori, M.-C.; Renard, P. Bioorg. Med. Chem. 2001,
9, 585.
´
26. Soto-Otero, R.; Mendez-Alvarez, E.; Hermida-Ameijeiras,
A.; Sanchez-Sellero, I.; Cruz-Landeira, A.; Lamas, M. L.
´
Life Sci. 2001, 69, 879.
27. Bradford, M. M. Anal. Biochem. 1976, 72, 248.
28. Cheng, W. C.; Prusoff, W. H. Biochem. Pharmacol. 1973,
22, 3099.
29. Tesson, F.; Prippbuus, C.; Lemoine, A.; Pegorier, J. P.;
Parini, A. J. Biol. Chem. 1991, 266, 155.
30. Lalies, M. D.; Hibell, A.; Hudson, A. L.; Nutt, D. J. Ann.
NY Acad. Sci. 1999, 881, 114.