Synthesis of heterocycle-based analogs of resveratrol and their antitumor and vasorelaxing properties

Article abstract of DOI:10.1016/j.bmc.2010.07.059

New resveratrol (RES) analogs were developed by replacing the aromatic 'core' of our initial naphthalene-based RES analogs with pseudo-heterocyclic (salicylaldoxime) or heterocyclic (benzofuran, quinoline, and benzothiazole) scaffolds. The resulting analo

Full text of DOI:10.1016/j.bmc.2010.07.059

Bioorganic & Medicinal Chemistry 18 (2010) 6715–6724
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of heterocycle-based analogs of resveratrol and their antitumor
and vasorelaxing properties
Simone Bertini a, Vincenzo Calderone b, Isabella Carboni a, Roberta Maffei c, Alma Martelli b,
Adriano Martinelli a, Filippo Minutolo a, Mehdi Rajabi c, Lara Testai b, Tiziano Tuccinardi a,
Riccardo Ghidoni c, Marco Macchia a,
*
a Department of Pharmaceutical Sciences, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
b Department of Psychiatry, Neurobiology, Pharmacology and Biotechnology, University of Pisa, Via Bonanno 6, 56126 Pisa, Italy
c Laboratory of Biochemistry and Molecular Biology, San Paolo Medical School, University of Milan, Via A. Di Rudinì 8, 20142 Milan, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
New resveratrol (RES) analogs were developed by replacing the aromatic ‘core’ of our initial naphthalene-
based RES analogs with pseudo-heterocyclic (salicylaldoxime) or heterocyclic (benzofuran, quinoline, and
benzothiazole) scaffolds. The resulting analogs were tested for their antiproliferative and vasorelaxing
effect, two typical properties shown by RES. Some of the new compounds confirmed strong antiprolifer-
ative activities, comparable to that previously found with the most active naphthalene-based analog. In
particular, 3-(3,5-dihydroxyphenyl)-7-hydroxyquinoline exhibited the most potent antiproliferative
Received 31 March 2010
Revised 23 July 2010
Accepted 24 July 2010
Available online 2 August 2010
Keywords:
Resveratrol
effect (IC50 = 17.4 lM). In vascular assays, the highest levels of potency (pIC50 = 4.92) and efficacy
(Emax = 88.2%) were obtained with 2-(3,5-dihydroxyphenyl)-6-hydroxybenzothiazole. A conformational
analysis of these compounds indicated that the antiproliferative activity on MDA-MB-231 cancer cells
can be correlated to a common sterical profile of the most active compounds and, in particular, to the spa-
tial arrangement of the three phenolic groups. Furthermore, the vasorelaxing properties showed a good
correlation with the electronic properties measured through the electrostatic molecular potential (ESP).
Ó 2010 Elsevier Ltd. All rights reserved.
Antitumor
Vasorelaxing
Potassium channels
Heterocycles
1. Introduction
tive oxygen species (ROS) and to inhibit low density lipoprotein
(LDL) peroxidation leading to atherogenesis.8 More recently, it
Resveratrol (trans-3,5,40-trihydroxystilbene, RES), a dietary pol-
yphenol isolated from edible materials such as red wine, grape
skins, and peanuts, is one of the most widely studied natural stilb-
enoids since it shows remarkable cancer chemopreventive and car-
dioprotective activities.1 We have previously shown that the effect
of RES on cancer cell growth is associated to its ability to interfere
with the sphingolipid pathways. In particular, it was found to in-
duce apoptosis by triggering the de novo biosynthesis of endoge-
nous ceramide, a bioactive sphingolipid.2,3 Some of the biological
effects of RES, such as inhibition of cancer growth, reduction of
cholesterol levels, and prevention of osteoporosis, resemble those
found with many other phytoestrogens and, therefore, have been
was shown that resveratrol can have anti-hypertensive properties
due to both endothelium-independent and endothelium-mediated
vasorelaxing effects. These vasoactive responses are mainly linked
to the activation of large-conductance calcium-activated potas-
sium channels (BK) expressed on vascular smooth muscle and
endothelial cells, respectively.9,10 Furthermore, additional cardio-
protective effects of resveratrol may be related to the NO release
caused by the activation of the BK channels.9 All these beneficial ef-
fects on human health have prompted the use of RES as a therapeu-
tic agent. Unfortunately, RES is characterized by a very low
bioavailability,11 mainly due to extensive phase II metabolism
consisting of in vivo transformation into sulfate and glucuronic
conjugates, mostly on 3-OH.12 Besides, a molecular point of
environmental, chemical, and metabolic instability in RES is consti-
tuted by the stilbene double bond, which may undergo E/Z-isomer-
ization13 or other types of oxidative transformations.14
associated to its ability to bind the estrogen receptors
a
and b.4
Moreover, this molecule proved to efficiently reduce the risk of car-
diovascular diseases.5 This phenomenon, which may be attributed
to the antioxidant activity of resveratrol, has been proven in sev-
eral studies;6,7 it is able to counteract the damages caused by reac-
So far there have been several reports on synthetic analogs of
RES, in an attempt to determine the structural requirements for
resveratrol-like biological activities and, at the same time, to ex-
tend the chemical diversity of this type of polyphenols, in view
of possible improvements of their biopharmacological profiles.
* Corresponding author. Tel./fax: +39 050 2219553.
E-mail address: mmacchia@farm.unipi.it (M. Macchia).
0968-0896/$ - see front matter Ó 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2010.07.059

6716
S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
Antitumor assays with MDA MB 468 cells of some 3-hydroxy-(E)-
stilbenes revealed an apoptosis-inducing activity in the low micro-
molar range.15 Contemporarily, our group developed some
naphthalene-based RES analogs endowed of proapoptotic proper-
ties against MDA-MB-231 breast cancer cells.16 Later, other scien-
tists have extended the series of naphthalene analogs of RES,
displaying strong antitumor effects.17
In an attempt to broaden the chemical diversity of resveratrol
analogs, we now report further transformations of our initial naph-
thalene-based analogs of RES, which leads to the development of
polyphenols containing pseudo-heterocyclic or heterocyclic scaf-
folds. In particular, our design started from our previously found
most active RES analog 1 (Fig. 1),16 whose naphthalene core was re-
placed by the following portions: salicylaldoxime (2), benzofuran
(3), quinoline (4 and 5), and benzothiazole (6–9). It is important
to notice that the tri-hydroxylated pattern of RES was substantially
maintained in all cases. The only compound bearing only two phe-
nol groups was 2, but in this case the presence of the oxime OH
group in the six-membered pseudo-cycle formed by an intramolec-
ular H-bond, can isosterically replace one phenol moiety, as we had
previously verified in the field of ER ligands.18
2. Results and discussion
2.1. Synthetic chemistry
The synthesis of salicylaldoxime 2 (Scheme 1) started from
trans-b-methylstyrene derivative 10,19 which was submitted to a
palladium-catalyzed cross-coupling reaction with 3,5-dim-
ethoxyphenylboronic acid, prepared as previously reported.20 Oxi-
dative cleavage of the double bond of the resulting biphenyl
derivative 11 with sodium periodate in the presence of catalytic
amounts of osmium tetroxide, yielded salicylaldehydes 12. Over-
night treatment of 12 with boron tribromide caused the removal
of the two O-methyl groups and subsequent condensation of the
resulting aldehyde (13) with hydroxylamine hydrochloride pro-
duced final oxime 2, obtained as a single E-diastereoisomeric form;
this is probably due to the fact that only this isomer can form intra-
molecular hydrogen bonds that contribute to the energetic stabil-
ization of the product. This selectivity had already been verified
for other oxime analogs we had developed as estrogen receptor li-
gands.21 Anyway, the E-configuration of 2 was confirmed by the
chemical shift value of the oxime proton, which was found down-
field from the 8 ppm value (d = 8.39 ppm, see Section 4).22
These compounds were submitted to two different biological
evaluations in order to assess both their growth-inhibitory proper-
ties in cancer cells, and their vasorelaxing effects, since these are
some of the most important activities that have been reported to
be associated to RES itself.
The synthesis of benzofuran 3 is shown in Scheme 2 and started
from 2-hydroxy-3-methoxybenzaldehyde 14, which was initially
reduced with sodium borohydride to the corresponding benzyl
alcohol 15. Subsequent reaction with triphenylphosphine
OMe
OH
OH
HO
Br
HO
a
b
OMe
OH
OH
OH
HO
HO
10
11
trans-resveratrol
OH
1
OMe
HO
OHC
HO
c
OMe
OH
OH
O
OHC
O
H
N
OH
OH
12
13
HO
OH
HO
HO
d
2
4
3
2
OH
OH
Scheme 1. Reagents and conditions: (a) 3,5-dimethoxyphenylboronic acid,
Pd(OAc)2, PPh3, aqueous 2 M K3PO4, dioxane, 80 °C, 16 h [77%]; (b) OsO4, NaIO4,
dioxane–H2O, 2 h [68%]; (c) BBr3, CH2Cl2, ꢀ78 °C to rt, overnight [44%]; (d)
NH2OHꢁHCl, MeOH-H2O, rt, 18 h [39%].
OH
N
HO
N
Cl
5
OH
OH
OH
a
b
OH
OH
OH
N
MeO
MeO
CHO
MeO
MeO
N
S
14
15
S
HO
OMe
OMe
OH
OH
OH
PPh3+Br-
O
c
6
7
OH
OH
16
17
S
N
N
d
HO
HO
S
OH
OH
3
8
9
Scheme 2. Reagents and conditions: (a) NaBH4, EtOH, rt, 1 h [85%]; (b) PPh3ꢁHBr
CH3CN, reflux, 1 h [84%]; (c) (1) 3,5-dimethoxybenzoic acid, DCC, DMAP, CH2Cl2,
overnight; (2) Et3N, dioxane, reflux, overnight [47%]; (d) BBr3, CH2Cl2, ꢀ78 °C to rt,
overnight [47%].
Figure 1. Chemical structures of resveratrol analogs containing naphthalene (1),
pseudo-heterocycle (2), or heterocycle-portions (3–9).

S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
6717
hydrobromide formed phosphonium salt 16, which was submitted
OMe
OMe
to an initial condensation with 3,5-dimethoxybenzoic acid, fol-
lowed by a base-promoted thermal cyclization. These sequence
afforded trimethoxy-substituted benzofuran 17, which was then
deprotected with BBr3, leading to final compound 3.
H
N
H
N
a
b
OMe
OMe
21
O
S
Quinoline derivatives 4 and 5 were synthesized as described in
Scheme 3. An initial transformation of (3,5-dimethoxyphenyl)ace-
tic acid 18 into its corresponding acid chloride, followed by its
condensation with m-anisidine in the presence of a polymer-sup-
ported base (PS-DIEA), produced amide 19. Subsequent cyclization
of 19 in the presence of POCl3 formed the quinoline scaffold of
compound 20. Removal of the three O-methyl groups with BBr3
afforded 2-chloquinoline 5. The other quinoline analog 4 was ob-
tained by a catalytic hydrogenation, which caused a proto-dechlo-
rination reaction of 5.
OMe
OMe
34
35
OMe
OMe
OMe
MeO
N
S
N
c
+
S
OMe
OMe
36
37
d
d
The synthesis of benzothiazole 6 (Scheme 4) starts with a con-
densation of 3,5-dimethoxybenzoyl chloride 21 with p-anisidine,
to form amide 22. Treatment of this amide with the Lawesson’s
reagent produced thioamide 23, which was then submitted to an
oxidative cyclization promoted by potassium ferricyanide. The
resulting trimethoxy-substituted benzothiazole 24 was treated
with BBr3 to yield final product 6.
7
decomposition
Scheme 5. Reagents and conditions: (a) m-anisidine, CH2Cl2, DMAP, PS-DIEA, rt,
overnight [31%]; (b) Lawesson’s reagent, 130 °C, 3 h [52%]; (c) (1) K3Fe(CN)6, aq
NaOH 30%, EtOH, 85 °C, 30 min; (2) rt, overnight [70%]; (d) BBr3, CH2Cl2, ꢀ78 °C to
rt, overnight [49%].
Benzothiazole 7 was obtained following the same synthetic se-
quence, as shown in Scheme 5. This time, acid chloride 21 was con-
densed with m-anisidine, thus producing amide 34 and, after
treatment with Lawesson’s reagent, thioamide 35. In this case,
the oxidative cyclization step produced, as predicted, a 1:1 mixture
of two isomeric benzothiazoles 36 and 37. Much to our surprise,
while BBr3-promoted O-demethylation of 36 led to the desired
product 7, the same treatment of regioisomer 37 caused its decom-
position to a complex mixture, so we were not able to obtain the
trihydroxy-substituted regioisomer of 7.
OMe
OMe
a
O
HOOC
OMe
MeO
N
OMe
H
18
19
This reaction sequence was also employed in the synthesis of
benzothiazoles 8 and 9, as shown in Scheme 6. Condensation of
4-methoxybenzoyl chloride 38 with 3,5-dimethoxyaniline afforded
amide 39. Lawesson’s reagent produced the oxygen/sulfur atom
exchange in the amide group and the resulting thioamide 41 was
submitted to ferricyanide-promoted cyclization. Benzothiazole 43
was then deprotected with BBr3, yielding final product 8. The other
benzothiazole analog 9 was obtained following an identical syn-
thetic pathway, with the only modification consisting in the con-
densation of 38 with 2,4-dimethoxyaniline.
OMe
b
c
d
OMe
5
4
MeO
N
Cl
20
Scheme 3. Reagents and conditions: (a) (1) SOCl2, CH2Cl2, 60 °C, 4 h; (2) m-
anisidine, CH2Cl2, DMAP, PS-DIEA, rt, overnight [74%]; (b) POCl3, DMF, 75 °C, 1.5 h
[55%]; (c) BBr3, CH2Cl2, ꢀ78 °C to rt, overnight [84%]; (d) H2 (1 atm), Pd–C, Et3N,
EtOH, 24 h [13%].
R'
OMe
R
O
O
OMe
H
Cl
N
R'
a
b
N
a
b
H
OMe
R
MeO
MeO
Cl
O
OMe
MeO
38
39: R = H; R' = OMe.
40: R = OMe; R' = H.
O
21
22
OMe
R'
R
OMe
OMe
R'
H
R
N
S
N
S
R
c
OMe
S
N
MeO
S
MeO
N
H
R'
c
MeO
R'
MeO
23
24
R
8
9
41: R = H; R' = OMe.
42: R = OMe; R' = H.
43: R = H; R' = OMe.
44: R = OMe; R' = H.
d
d
6
Scheme 6. Reagents and conditions: (a) 3,5-dimethoxyaniline or 2,4-dimethoxy-
aniline, CH2Cl2, DMAP, PS-DIEA, rt, overnight [39: 89%; 40: 74%]; (b) Lawesson’s
reagent, 130 °C, 3 h [41: 70%; 42: 72%]; (c) (1) K3Fe(CN)6, aq NaOH 30%, EtOH, 85 °C,
30 min; (2) rt, overnight [43: 70%; 44: 49%]; (d) BBr3, CH2Cl2, ꢀ78 °C to rt, overnight
[8: 41%; 9: 66%].
Scheme 4. Reagents and conditions: (a) p-anisidine, CH2Cl2, DMAP, PS-DIEA, rt,
overnight [61%]; (b) Lawesson’s reagent, 130 °C, 3 h [67%]; (c) K3Fe(CN)6, aq NaOH
30%, EtOH, 85 °C, 30 min, then rt overnight [74%]; (d) BBr3, CH2Cl2, ꢀ78 °C to rt,
overnight [49%].

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S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
2.2. Antiproliferative properties
of the test compounds evoking a 50% reduction in the contractile
tone induced by 30 mM KCl. The pIC50 values could not be calcu-
lated for compounds with efficacy below 50% at 30 M. Six out of
Resveratrol analogs were compared for their growth-inhibitory
effect on MDA-MB-231 breast cancer cells. Cells were treated with
compounds 1–9 at different concentrations, ranging from 0.25
l
the nine compounds tested exhibited a vasorelaxing effect gener-
ally comparable, albeit slightly less potent, to that of RES, which
in our hands showed a pIC50 of 5.16 and a Emax of 83%. Naptha-
lene analog 116 was tested here for the first time as a vasorelax-
ing agent and it shows a pIC50 of 4.89 with an efficacy of 78.5%.
Among the heterocyclic-based compounds, the highest levels of
potency (pIC50 = 4.92) and efficacy (Emax = 88.2%) were obtained
with benzothiazole 6. Its close analog 7 displayed similar levels
of potency (pIC50 = 4.81) and efficacy (Emax = 76.8%). Dihydroxyl-
ated benzothiazole 8 proved to be a poor vasorelaxing agent in
lM
to 64 M for 6 days. The sulphorhodamine B (SRB) cell proliferation
l
assay was used. The proliferation index at the beginning of the
experiment (time 0) was set at 1. The IC50 for the new resveratrol
analogs 2–9 are reported in Table 1, together with the IC50 of resve-
ratrol (1) and the previously reported naphthalene derivative 1.
Some of the new compounds (2, 4, 6, and 7) confirmed strong anti-
proliferative activities which are comparable to that previously
found with naphthalene-based analog 1. We were pleased to find
that the replacement of the naphthalene scaffold of 1 with a salicyl-
aldoxime-based pseudocycle, as in compound 2, substantially
maintained the IC50 value in the low micromolar range, thus con-
firming in this case the suitability of this isosteric replacement for
phenols.18 Quninoline 4 exhibited the most potent antiproliferative
this assays (Emax <40% at 40 lM), whereas the other dihydroxy-
substituted benzothiazole 9 showed again significant vasorelax-
ing effects (pIC50 = 4.90 and Emax = 80.6%). Oxime 2 displayed a
rather weak activity on the vascular smooth muscle, with an effi-
cacy lower than 50%, thus proving that in this specific pharmaco-
dynamic feature at the vascular level, differently from the case of
the antiproliferative mechanisms, the phenol/salicylaldoxime
effect (IC50 = 17.4 lM) among these new compounds, whereas its 2-
chloro-substituted analog 5 suffered from a nearly threefold reduc-
tion of the activity. Similarly, benzofuran 3 showed a moderate
replacement was not tolerated. Benzofuran
3 showed good
potency, with an IC50 of 46.1
l
M. The two benzothiazoles bearing
vasorelaxing properties, with potency (pIC50 = 4.81) and effi-
ciency (Emax = 77.2%) comparable to those obtained with naph-
thalene derivative 1 and benzothiazole 7. A remarkable efficacy
(Emax = 84.6%) was reached by 2-chloroquinoline 5, which also
displayed a noticeable pIC50 value (4.80), whereas the formal
remotion of the chlorine atom from 5, leading to compound 4,
produced a dramatic reduction of the vasorelaxing effectiveness
(Emax <20%). In order to correlate the vascular effects with the
activation of potassium currents, the synthesised compounds en-
dowed of satisfactory levels of vasorelaxing efficacy (Emax >50%)
were also tested in the presence of tetraethylammonium chloride
(TEA, 10 mM), which acts as a non-selective blocker of BK chan-
nels, as well as other different types of voltage-operated potas-
sium channels.
a single hydroxy group in their bicyclic portion 6 and 7 displayed
noticeable antiproliferative effects, whereas their fellow com-
pound, based on the 4,6-dihydroxybenzothiazole scaffold, was
either less active (8). Finally, 5,7-dihydroxybenzothiazole 9 dis-
played no significant activity in the micromolar range.
2.3. Vasorelaxing properties
The vasorelaxing properties of RES and its analogs 1–9 were
studied on in vitro vascular smooth muscle preparation of iso-
lated rat aortic rings. Data reported in Table 1 concern vasorelax-
ing efficacy of the synthesised compounds and RES, which were
evaluated as the maximal vasorelaxing response at a concentra-
tion of 30
lM, expressed as a percentage of the contractile tone
Benzothiazole derivative 6 showed the highest levels of
induced by 30 mM KCl (Emax, %). Potency is given as the pIC50
calculated as the negative logarithm of the molar concentration
,
vasorelaxing potency and efficacy, nevertheless these effects
were completely unaffected by TEA. This experimental observa-
tion indicates that the activity of 6 on vascular smooth muscle
is not linked to the activation of hyperpolarizing potassium cur-
rents. Hence, an additional experimental protocol was designed
to further support this hypothesis. In particular, it is widely
known that high levels of membrane depolarization due to high
extracellular concentrations of potassium ions can dramatically
and non-specifically reduce the vasorelaxing effects of vasorelax-
ing agents acting through the activation of every type of mem-
brane potassium channels.23 Therefore, the vasorelaxing effect
of 6 was also evaluated in rat aortic rings pre-contracted with
KCl 60 mM and, again, they were completely unaffected by this
experimental condition, confirming that activation of potassium
channels does not play a role in the vascular activity of 6. The
effects of compound 2 were only poorly, and not significantly, af-
fected by TEA, suggesting that the activation of potassium chan-
nels might play only a modest role (if any) in the vascular effects
of this salicylaldoxime derivative.
On the other hand, the vasodilator effects of resveratrol, 1, 3, 5,
7, and 9 were significantly antagonised by TEA, indicating a clear
involvement of potassium channels in their pharmacodynamic
mechanism of action. Compound 5, showing the highest level of
efficacy among these subset of molecules exhibiting TEA-sensitive
effects, was selected for a more accurate mechanistic investigation
and was tested in the presence of iberiotoxin (IbTX, 100 nM), a
highly selective blocker of BK channels. This scorpion toxin signif-
icantly antagonised the effects of 5, thus demonstrating that the
vasodilator effect of this compound is due to the activation of vas-
cular BK potassium channels.
Table 1
Antiproliferative activities and vasorelaxing properties of resveratrol and its analogs
1–9
c
Compound MDA-MB-231
Rat aortic rings Emax at
TEAd
10 mM
a
b
IC50
(
lM)
pIC50
30
lM
Resveratrol
20.5
12.7e
19.2
46.1
17.4
47.4
19.4
18.1
67.6
5.16 0.13
4.89 0.03
n.c.
4.81 0.08
n.c.
4.80 0.02
4.92 0.01
4.81 0.02
n.c.
83.0 5.0
78.5 8.0
47.3 8.6
77.2 19.8
17.2 3.0
84.6 6.6
88.2 2.7
76.8 2.6
39.6 13.7
80.6 5.6
+
+
—
+
n.t.
+
—
+
+
+
1
2
3
4
5
6
7
8
9
g
h
f
>100
4.90 0.04
a
IC50 (lM) was calculated as the mean of the proliferation values obtained at
6 days of treatment with each compound used at different concentrations in three
independent experiments. Standard errors are not shown for the sake of clarity and
were never higher than 15% of the means.
b
Negative logarithm of the molar concentration evoking an half-reduction of the
contractile tone induced by KCl 20 mM (n.c. = not calculable).
c
Maximal vasorelaxing effects induced by the concentration 30 lM, expressed as
a % of the contractile tone induced by KCl 20 mM.
d
The vasorelaxing effects were significantly (+) or were not significantly (ꢀ)
antagonised by TEA 10 mM (n.t. = not tested).
e
See Ref. 16.
f
Additional test: the effects were not affected also by high KCl concentrations
(60 mM).
g
TEA induced modest and not significant influences.
Additional test: IbTX 100 nM antagonised the vasorelaxing effects.
h

S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
6719
2.4. Computational chemistry
The structures of resveratrol and compounds 1–9 were mini-
mized at ‘ab initio’ SCF 6-31G(d) level through GAUSSIAN03 pro-
gram.24 Compounds 3 and 6–9 showed to prefer a completely
planar conformation, while the other derivatives displayed an an-
gle of about 45° between the two planar aromatic systems, with
the exception of compound 5 where this angle was much larger
(70°) due to the presence of the chlorine in alpha position of the
quinoline scaffold. The conformational analysis of compound 2
confirmed the presence of the intramolecular H-bond between
the phenolic OH group and the oxime nitrogen atom, as already
found in estrogen receptor ligands.18,19
With the aim of explaining the activity differences of com-
pounds 1–9 in the two separate pharmacological assays, namely,
the measurements of antiproliferative activity in MDA-MB-231
cancer cells and the vasorelaxing properties in vascular smooth
muscle, their structural superimpositions with resveratrol was ta-
ken into consideration. These superimpositions were obtained
through the ROCS 2.2 software,25 which is a shape-similarity meth-
od based on the Tanimoto-like overlap of volumes and superim-
poses the molecules on this basis. Figure 2 evidences the good
superimpositions found for compounds 1, 2, 4, and 6 with resvera-
trol (Fig. 2A), whereas significant differences are evident for com-
pounds 3, 5, 8, and 9 (Fig. 2B). Therefore, compounds 1, 2, 4, and
6 seem to be able to interact with biological targets in a manner
very similar to resveratrol, since all three phenolic groups are ste-
rically placed in comparable arrangements. These computational
results find a good correlation with the results deriving from the
antiproliferative experiments. In fact, compounds 1, 2, 4, and 6 dis-
play good antiproliferative activities on MDA-MB-231 cells, that
are similar to the activity level associated to resveratrol, whereas
compounds 3, 5, 8, and 9 generally showed lower antiproliferative
activities. In particular, the worst superimposition is observed for
compound 9, which is also the least active derivative. This analysis
does not seem to work for compound 7, which shows a good activ-
ity although the superimposition of one of its three hydroxyls to
the p-OH of resveratrol is rather poor. However, there is still the
possibility that a H-bond acceptor group of an hypothetical biolog-
ical target (still not identified for resveratrol anticancer activity),
indicated as G in Figure 2C, is placed in a position such as it can
interact with both the phenolic group of the resveratrol and the
corresponding OH of the benzothiazole portion of 7.
On the other hand, the results about cardiovascular activity of
resveratrol and compounds 1–9 indicate that they generally pos-
sess similar appreciable activities, with the exception of com-
pounds 2, 4, and 8. Therefore, in this case, the same theoretical
analysis based on structural superimpositions, reported above for
the interpretation of the antiproliferative data, is not able to repre-
sent reliable structure–activity correlations. Hence, we analyzed
electronic properties of resveratrol and compounds 1–9, such as
the electrostatic molecular potential (ESP), which was calculated
through the GAUSSIAN03 program. The ESP was calculated at SCF 6-
31G(d) level for the preferred conformation and it is visualized
through the GAUSSVIEW program on the isosurface corresponding to
a value of the total electron density of 0.0004 atomic unit. All com-
pounds were aligned according to the above mentioned results of
the shape similarity analysis. In Figure 3 the ESP of resveratrol
and compounds 1–9 are reported. Red regions correspond to neg-
ative ESP while blue regions correspond to positive ESP. The results
of this analysis show a common feature present in the three inac-
tive compounds 2, 4, and 8: there is negative ESP in spatially cor-
responding regions, which is otherwise not present in the active
compounds. This negative-potential area is generated in com-
pound 2 by the phenolic oxygen of the salicylaldoxime portion,
Figure 2. (A) Superimposition of resveratrol (gray) with active compounds
1
(green),
2
(light blue),
4
(magenta), and
6
(purple). (B) Superimposition of
resveratrol (gray) with inactive compounds 3 (green), 5 (light blue), 8 (magenta),
and 9 (purple). (C) Superimposition of resveratrol (gray) with compound 7. The
position of an hypothetical H-bond acceptor group G is indicated.
in compound 4 by the nitrogen of the quinoline moiety and in com-
pound 8 by the nitrogen of the benzothiazole system.
As shown by Figure 3, active compounds 1, 3, 5–7, and 9 do not
possess a similar negative ESP in this region. In the case of the ac-
tive chloro-quinoline 5 which is very similar to its inactive non-
chlorinated analog 4, the lack of the negative ESP in the concerned
region of 5 is due to a different conformation that it assumes when
compared to 4, due to the presence of the a-Cl atom in the quino-
line moiety, which forces the dihedral angle between the two pla-
nar aromatic systems to a much larger value (70°), as seen before.
Therefore, these data would suggest that the presence of a negative
ESP in this region could be responsible for the unfavorable vasore-
laxing properties associated to resveratrol analogs 2, 4, and 8.
3. Conclusion
This study was carried out with the aim of finding new deriva-
tives of resveratrol and of its naphthalene-based analog 1, by intro-
ducing heterocyclic systems into their structures, in order to
increase the polarity of these systems which, eventually, may be
useful in the future in improving the poor pharmacokinetic proper-
ties associated to resveratrol itself. The resulting analogs were
tested for two typical properties shown by resveratrol: tumor anti-
proliferative action and vasorelaxing effect. A conformational

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S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
silica gel (60 F254) sheets that were visualized under a UV lamp.
Evaporation was performed in vacuo (rotating evaporator).
4.1.1. 5-(3,5-Dimethoxy)phenyl-2-(prop-1-enyl)phenol (11)
To a solution of 10 (300 mg, 1.41 mmol) in dioxane (7 mL) was
added, under nitrogen flux, 3,5-dimetoxyphenylboronic acid
(615 mg, 3.38 mmol), Pd(PPh3)4 (104 mg, 0.09 mmol) and 3.7 mL
of a 2 M aqueous solution of K3PO4. The resulting mixture was stir-
red at 80 °C overnight in a sealed vial. The crude mixture was then
diluted with water and extracted with ethyl acetate. The organic
phase was dried over sodium sulfate and concentrated under
vacuum. The crude residue was purified by column chromatogra-
phy (n-hexane/ethyl acetate 95:5), affording 11 (295 mg,
1.09 mmol, 77% yield). 1H NMR (CDCl3) d (ppm): 1.94 (dd, 3H,
J = 6.5, 1.6 Hz), 3.84 (s, 6H), 6.25 (m, 1H), 6.45 (t, 1H, J = 2.2 Hz),
6.60 (dd, 1H, J = 14.5, 1.4 Hz), 6.70 (d, 2H, J = 2.2 Hz), 7.01 (d, 1H,
J = 1.5 Hz), 7.11 (dd, 1H, J = 8.1, 1.6 Hz), 7.35 (d, 1H, J = 7.9 Hz).
4.1.2. 4-(3,5-Dimethoxy)phenylsalicylaldehyde (12)
A solution of 11 (120 mg, 0.44 mmol) in dioxane (4.7 mL) was
treated with H2O (2.0 mL), NaIO4 (219 mg, 1.02 mmol) and a solu-
tion of OsO4 in t-BuOH (0.014 mL). The resulting mixture was stir-
red at 50 °C for 2 h, then cooled to rt, diluted with H2O and
extracted with CHCl3. The organic phase was dried over sodium
sulfate and concentrated under vacuum. The crude residue was
purified by column chromatography (n-hexane/ethyl acetate
95:5), affording 12 (77 mg, 0.30 mmol, 68% yield) as a white
powder. 1H NMR (CDCl3) d (ppm): 3.85 (s, 6H), 6.53 (t, 1H,
J = 2.2 Hz), 6.75 (d, 2H, J = 2.2 Hz), 7.20–7.26 (m, 2H), 7.61 (d, 1H,
J = 7.9 Hz), 9.92 (br s, 1H), 11.12 (br s, 1H).
4.1.3. 4-(3,5-Dihydroxy)phenylsalicylaldehyde (13)
A solution of 12 (77 mg, 0.30 mmol) in 5.0 mL of anhydrous
CH2Cl2 was cooled to ꢀ78 °C under nitrogen flux and treated with
a 1 M solution of BBr3 in Et2O (1.8 mL, 1.8 mmol). The mixture was
stirred at rt overnight, then quenched with water and extracted
with ethyl acetate. The organic phase was dried over sodium sul-
fate and concentrated under vacuum. The crude residue was puri-
fied by column chromatography (n-hexane/ethyl acetate 6:4),
affording pure 13 (30 mg, 0.13 mmol, 44% yield). 1H NMR (ace-
tone-d6) d (ppm): 6.45 (t, 1H, J = 2.2 Hz), 6.68 (d, 2H, J = 2.2 Hz),
7.14 (d, 1H, J = 1.6 Hz), 7.29 (dd, 1H, J = 8.1, 1.6 Hz), 7.82 (d, 1H,
J = 8.1 Hz), 10.04 (br s, 1H).
Figure 3. ESP of resveratrol and compounds 1–9. Red regions correspond to
negative ESP, blue region to positive ESP, and green region to neutral ESP. The red
regions of compounds 2, 4, and 8 correlable with a low affinity are evidenced.
analysis of these compounds indicated that the antiproliferative
activity on MDA-MB-231 cancer cells can be correlated to a com-
mon sterical profile of the most active compounds and, in particu-
lar, to the spatial arrangement of the three phenolic groups.
Furthermore, the vasorelaxing properties of these analogs showed
4.1.4. 4-(3,5-Dihydroxy)phenylsalicylaldoxime (2)
a
good correlation with the electronic properties measured
A solution of 13 (25 mg, 0.11 mmol) in methanol (0.2 mL) was
added to a solution of hydroxylamine hydrochloride (15 mg,
0.22 mmol) in water (0.02 mL). The mixture was stirred at rt for
18 h. Partial removal of the solvent, followed by dilution with
water and extraction with AcOEt, afforded a crude product which
was purified by column chromatography (n-hexane/ethyl acetate
1:1), affording pure product 2 (8.9 mg, 0.04 mmol, 39% yield). 1H
NMR (acetone-d6) d (ppm): 6.39 (t, 1H, J = 2.1 Hz), 6.64 (d, 2H,
J = 2.2 Hz), 7.10 (m, 1H), 7.14 (dd, 1H, J = 8.0, 1.7 Hz), 7.41 (d, 1H,
J = 7.9 Hz), 8.39 (s, 2H), 10.12 (br s, 1H), 10.55 (br s, 1H). MS m/z
245 (M+, 100).
through the ESP. These theoretical observations point out that
the structural requirements for mimicking resveratrol anticancer
effects are likely to be different from those needed to obtain a res-
veratrol-like vasorelaxing action, and may indicate the future
directions to be followed in the design of new analogs of this pow-
erful, but still poorly drug-like, natural polyphenol.
4. Experimental section
4.1. Chemistry
NMR spectra were obtained with a Varian Gemini 200 MHz
spectrometer. Chemical shifts (d) are reported in parts per million
downfield from tetramethylsilane and referenced from solvent ref-
erences. Electron impact (EI, 70 eV) mass spectra were obtained on
a ThermoQuest Finnigan GCQ Plus mass spectrometer. Chromato-
graphic separations were performed on silica gel columns by flash
(Kieselgel 40, 0.040–0.06 mm; Merck) or gravity column (Kieselgel
60, 0.063–0.200 mm; Merck) chromatography. Reactions were fol-
lowed by thin-layer chromatography (TLC) on Merck aluminum
4.1.5. 2-Hydroxy-5-methoxybenzyl alcohol (15)
Sodium borohydride (62 mg, 1.65 mmol) was added to a cooled
(ice bath) solution of 2-hydroxy-5-methoxybenzaldehyde 14
(500 mg, 3.29 mmol) in EtOH (5 mL). The reaction mixture was
stirred at rt for 1 h. After the solvent was removed, the residue
was treated with 1 N aqueous solution of HCl and extracted with
Et2O. The organic phase was dried over Na2SO4 and filtered. The fil-
trate was concentrate to give compound 15 (432 mg, 2.80 mmol,

S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
6721
85% yield) as an oil. 1H NMR (CDCl3) d (ppm): 3.72 (s, 3H), 4.72 (s,
2H), 6.59 (s, 1H), 6.73 (m, 2H).
formamide (0.04 mL). After 30 min, amide 19 (100 mg, 0.33 mmol)
was added at the same temperature. The ice bath was then re-
moved and the mixture was heated at 75 °C for 1.5 h. The mixture
was quenched with water and extracted with EtOAc. The organic
phase was dried and concentrated under vacuum. The crude resi-
due was purified by column chromatography (n-hexane/ethyl ace-
tate 9:1), affording pure 20 (198 mg, 0.60 mmol, 55% yield). 1H
NMR (CDCl3) d (ppm): 3.85 (s, 6H), 3.96 (s, 3H), 6.54 (t, 1H,
J = 2.2 Hz), 6.65 (d, 2H, J = 2.2), 7.20 (dd, 1H, J = 9.0, 2.7 Hz), 7.39
(d, 1H, 2.7 Hz), 7.71 (d, 1H, J = 9.0 Hz), 8.03 (s, 1H).
4.1.6. (2-Hydroxy-5-methoxybenzyl)-triphenylphosphonium
bromide (16)
A solution of 15 (351 mg, 2.28 mmol) and triphenylphosphine
hydrobromide (783 mg, 2.28 mmol) in CH3CN (7 mL) was refluxed
for 1 h. The solid was filtered and washed with CH3CN to give 16
(918 mg, 1.92 mmol, 84% yield) that was used for the next step
without further purification. 1H NMR (DMSO-d6) d (ppm): 3.38 (s,
3H), 4.87 (d, 2H, J = 14.7 Hz), 6.33 (s, 1H), 6.65–6.71 (m, 2H),
7.67–7.89 (m, 15H), 9.34 (s, 1H).
4.1.11. 2-Chloro-3-(3,5-dihydroxyphenyl)-7-hydroxyquinoline
(5)
4.1.7. 2-(3,5-Dimethoxy)phenyl-5-methoxybenzofuran (17)
A solution of dicyclohexylcarbodiimide (485 mg, 2.35 mmol) in
dry CH2Cl2 (1 mL) was added to a solution of 16 (343 mg,
1.88 mmol), DMAP (34.4 mg, 0.28 mmol), and 3,5-dimethoxyben-
zoic acid (900 mg, 1.88 mmol) in dry CH2Cl2 (18 mL), under nitro-
gen flux. The mixture was stirred overnight at rt, then concentrated
under vacuum. The residue, dissolved in dry dioxane (9 mL), was
treated with triethylamine (1.5 mL, 10.64 mmol) and the resulting
mixture was refluxed overnight. After cooling to rt, the solution
was filtered and the solvent removed under vacuum. The crude
residue was purified by column chromatography (n-hexane/ethyl
acetate 9:1), affording product 17 (250 mg, 0.88 mmol, 47% yield)
as a solid. 1H NMR (CDCl3) d (ppm): 3.86 (s, 3H), 3.87 (s, 6H),
6.47 (t, 1H, J = 2.3 Hz), 6.89 (dd, 1H, J = 9.0, 2.6 Hz), 6.95 (s, 1H),
7.00 (d, 2H, J = 2.4 Hz), 7.04 (d, 1H, J = 2.6 Hz), 7.41 (d, 1H,
J = 8.8 Hz).
A solution of 20 (80 mg, 0.24 mmol) in 2.6 mL of anhydrous
CH2Cl2 was cooled at ꢀ78 °C, under flux of nitrogen, and treated
with a 1 M solution of BBr3 in Et2O (2.9 mL, 2.90 mmol). The mix-
ture was then stirred at rt overnight, then quenched with water
and extracted with ethyl acetate. The organic phase were dried
and concentrated under vacuum. The crude residue was purified
by column chromatography (n-hexane/ethyl acetate 1:1), affording
5 (60 mg, 0.21 mmol, 84% yield) as a white solid. 1H NMR (acetone-
d6) d (ppm): 6.44 (t, 1H, J = 2.2 Hz), 6.49 (d, 2H, J = 2.2), 7.27 (dd,
1H, J = 7.8 Hz, 2.5 Hz), 7.31 (d, 1H, 2.5 Hz), 7.90 (d, 1H, J = 7.8 Hz),
8.16 (s, 1H), 8.46 (br, 3H). MS m/z 287 (M+, 100).
4.1.12. 3-(3,5-Dihydroxyphenyl)-7-hydroxyquinoline (4)
Compound 5 (50 mg, 0.17 mmol) was dissolved in EtOH (5 mL),
and Et3N (0.07 mL), then Pd/C 10% (22 mg) was added and the mix-
ture was allowed to react under H2 for 24 h. The mixture was fil-
tered on a Celite pad and the solution was evaporated under
vacuum. The crude residue was purified by column chromatogra-
phy (n-hexane/ethyl acetate 9:1), affording product 4 (12 mg,
0.05 mmol, 13% yield) as a white solid. 1H NMR (acetone-d6) d
(ppm): 6.43 (t, 1H, J = 2.2 Hz), 6.75 (d, 2H, J = 2.2 Hz), 7.25 (dd,
1H, J = 8.8, 2.3 Hz), 7.40 (d, 1H, J = 2.2 Hz), 7.89 (d, 1H, J = 8.8 Hz),
8.32 (d, 1H, J = 2.3 Hz), 8.49 (br, 2H), 9.01 (d, 1H, J = 2.3 Hz), 9.10
(br, 1H). MS m/z 253 (M+, 7).
4.1.8. 2-(3,5-Dihydroxy)phenyl-5-hydroxybenzofuran (3)
A solution of 17 (120 mg, 0.43 mmol) in 5 mL of anhydrous
CH2Cl2 was cooled to ꢀ78 °C under nitrogen flux and treated with
a 1 M solution of BBr3 in Et2O (4,5 mL, 4,5 mmol). The mixture was
stirred at rt overnight, then quenched with water and extracted
with ethyl acetate. The organic phase was dried over sodium sul-
fate and concentrated under vacuum. The crude residue was puri-
fied by column chromatography (n-hexane/ethyl acetate 1:1),
affording product 3 (49 mg, 0.20 mmol, 47% yield) as a white solid.
1H NMR (acetone-d6) d (ppm): 6.39 (t, 1H, J = 2.2 Hz), 6.82 (dd, 1H,
J = 8.8, 2.4 Hz), 6.89 (d, 2H, J = 2.2 Hz), 7.01 (d, 1H, J = 2.6 Hz), 7.03
(d, 1H, J = 0.7 Hz), 7.35 (d, 1H, J = 8.8 Hz). MS m/z 242 (M+, 100).
4.1.13. N-(p-Methoxyphenyl)-3,5-dimethoxybenzamide (22)
3,5-Dimethoxybenzoyl chloride 21 (736 mg, 3.67 mmol), PS-
DIEA (1.040 g, 3.67 mmol) and DMAP (catalytic amount) were
added to a solution of p-anisidine (377 mg, 3.06 mmol) in dry
CH2Cl2. The mixture was stirred at rt overnight, then filtered and
evaporated under vacuum. The crude residue was purified by flash
chromatography (n-hexane/ethyl acetate 9:1), affording product
22 (593 mg, 2.06 mmol, 61% yield) as a white solid. 1H NMR
(CDCl3) d (ppm): 3.81 (s, 3H), 3.84 (s, 6H), 6.61 (t, 1H, J = 2.2 Hz),
4.1.9. N-(3-Methoxyphenyl)-3,5-dimethoxyphenylacetamide
(19)
To a solution of 3,5-dimethoxyphenylacetic acid 18 (627 mg,
3.20 mmol) in dry CH2Cl2 (32 mL) was added SOCl2 (0.93 mL,
12.80 mmol). The reaction mixture was stirred at 60 °C for 4 h.
The solution was then concentrated under nitrogen flux and the
residue containing crude acid chloride was treated with a solution
of m-anisidine (328 mg, 2.66 mmol) in a minimum amount of dry
CH2Cl2, PS-DIEA (973 mg, 3.19 mmol) and DMAP (catalytic
amount). The mixture was stirred at room temperature overnight,
then neutralized with aq satd K2CO3 and extracted with CH2Cl2.
The organic phase was dried and evaporated to afford a crude res-
idue that was purified by flash chromatography (n-hexane/ethyl
acetate 8:2), yielding pure 19 (540 mg, 1.79 mmol, 74% yield) as
a white solid. 1H NMR (CDCl3) d (ppm): 3.66 (s, 2H), 3.78 (s, 3H),
3.81 (s, 6H), 6.43 (t, 1H, J = 2.2 Hz), 6.47 (d, 2H, J = 2.2 Hz), 6.64
(ddd, 1H, J = 8.3, 2.4, 1.0 Hz), 6.85 (ddd, 1H, J = 8.3, 2.4, 1.0 Hz),
7.17 (t, 1H, J = 8.24 Hz), 7.23 (m, 1H).
6.91 (AA0XX0, 2H, JAX = 9.0 Hz, JAA /XX = 2.7 Hz), 6.97 (d, 2H,
0
0
J = 2.2 Hz), 7.52 (AA0XX0, 2H, JAX = 9.0 Hz, JAA /XX = 2.7 Hz), 7.70
0
0
(br, 1H).
4.1.14. N-(p-Methoxyphenyl)-3,5-dimethoxythiobenzamide (23)
A solution of 22 (593 mg, 2.06 mmol) and Lawesson’s reagent
(500 mg, 1.24 mmol) in chlorobenzene (3.0 mL) was boiled at
130 °C for 3 h. The mixture was evaporated and the crude residue
was purified by flash chromatography (n-hexane/ethyl acetate
8:2), affording 23 (417 mg, 1.37 mmol, 67% yield) as a solid. 1H
NMR (CDCl3) d (ppm): 3.85 (s, 9H), 6.58 (t, 1H, J = 2.2 Hz), 6.97
(AA0XX0, 2H, JAX = 9.0 Hz, JAA /XX = 2.4 Hz), 6.98 (d, 2H, J = 2.2 Hz),
0
0
7.64 (AA0XX0, 2H, JAX = 9.0 Hz, JAA /XX = 2.4 Hz).
0
0
4.1.15. 2-(3,5-Dimethoxyphenyl)-6-methoxybenzothiazole (24)
Compound 23 (300 mg, 0.99 mmol) was wetted with a little
amount of ethanol, and 30% aqueous NaOH solution (1.3 mL) was
added. The mixture was diluted with water to provide a final
suspension of 10% aqueous NaOH. This mixture was added
4.1.10. 2-Chloro-3-(3,5-dimethoxyphenyl)-7-methoxyquinoline
(20)
Phosphorus oxychloride (0.21 mL, 2.31 mmol) was added,
dropwise, to well stirred and cooled (0 °C, ice bath) N,N-dimethyl-

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S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
portionwise (1 mL, 1 min intervals) to a stirred solution of potas-
sium ferricyanide (1.302 g, 3.96 mmol) in water, at 85 °C. The reac-
tion mixture was heated for a further 30 min and then allowed to
cool to rt and stirred overnight. The mixture was filtered and the
residue dissolved in CHCl3, dried over sodium sulfate and
concentrated under vacuum. The crude residue was purified by
column chromatography (n-hexane/ethyl acetate 9:1), affording
24 (220 mg, 0.73 mmol, 74% yield) as a solid. 1H NMR (CDCl3) d
(ppm): 3.89 (s, 6H), 3.90 (s, 3H), 6.57 (t, 1H, J = 2.3 Hz), 7.09 (dd,
1H, J = 8.8, 2.6 Hz), 7.20 (d, 2H, J = 2.3 Hz), 7.35 (d, 1H, J = 2.6 Hz),
7.95 (d, 1H, J = 8.8 Hz).
4.1.20. 2-(3,5-Dihydroxyphenyl)-7-hydroxybenzothiazole (7)
A solution of the mixture of 37 and 36 (90 mg, 0.30 mmol) in
3.3 mL of anhydrous CH2Cl2 was cooled to ꢀ78 °C under nitrogen
flux and treated with a 1 M solution of BBr3 in Et2O (3.2 mL,
3.56 mmol). The mixture was stirred at rt overnight, then
quenched with water and extracted with ethyl acetate. The organic
phase was dried over sodium sulfate and concentrated under
vacuum. The crude residue was purified by column chromatogra-
phy (n-hexane/ethyl acetate 1:1), affording 7 (19 mg, 0.07 mmol,
49% yield) as a solid, while the trihydroxy-substituted regioisomer
of 7 was not isolated. 1H NMR (CD3COCD3) d (ppm): 6.54 (t, 1H,
J = 2.1 Hz), 6.91 (dd, 1H, J = 7.9, 0.7 Hz), 7.16 (d, 2H, J = 2.1 Hz),
7.35 (t, 1H, J = 7.9 Hz), 7.54 (dd, 1H, J = 7.9, 0.7 Hz), 8.59 (br, 2H),
9.37 (br, 1H). MS m/z 259 (M+, 100).
4.1.16. 2-(3,5-Dihydroxyphenyl)-6-hydroxybenzothiazole (6)
A solution of 24 (210 mg, 0.69 mmol) in 7.6 mL of anhydrous
CH2Cl2 was cooled to ꢀ78 °C under nitrogen flux and treated with
a 1 M solution of BBr3 in Et2O (7.3 mL, 8.33 mmol). The mixture
was stirred at rt overnight, then quenched with water and ex-
tracted with ethyl acetate. The organic phase was dried over so-
dium sulfate and concentrated under vacuum. The crude residue
was purified by column chromatography (n-hexane/ethyl acetate
1:1), affording 6 (89 mg, 0.34 mmol, 49% yield) as a solid. 1H
NMR (CD3COCD3): 6.50 (t, 1H, J = 2.1 Hz), 7.05 (dd, 1H, J = 8.8,
2.3 Hz), 7.08 (d, 2H, J = 2.1 Hz), 7.43 (d, 1H, J = 2.3 Hz), 7.83 (d,
1H, J = 8.8 Hz), 8.56 (br, 2H), 8.75 (br, 1H). MS m/z 259 (M+, 40).
4.1.21. N-(3,5-Dimethoxy)phenyl-4-methoxybenzamide (39)
To a solution of 3,5-dimethoxyaniline (250 mg, 1.63 mmol) in
dry CH2Cl2 (3 mL), p-anisoylchoride 38 (401 mg, 1.35 mmol), PS-
DIEA (425 mg, 1.96 mmol), and DMAP (catalytic amount) were
added. The mixture was stirred at rt overnight, then filtered and
evaporated under vacuum. The crude residue was purified by flash
chromatography (n-hexane/ethyl acetate 9:1), affording 39
(463 mg, 1.61 mmol, 89% yield) as a white solid. 1H NMR (CDCl3)
d (ppm): 3.81 (s, 6H), 3.88 (s, 3H), 6.27 (t, 1H, J = 2.2 Hz), 6.89 (d,
2H, J = 2.2 Hz), 6.98 (AA0XX0, 2H, JAX = 8.8 Hz, JAA /XX = 2.6 Hz),
0
0
7.82 (AA0XX0, 2H, JAX = 8.8 Hz, JAA XX = 2.6 Hz).
0
0
4.1.17. N-(m-Methoxyphenyl)-3,5-dimethoxybenzamide (34)
To a solution of m-anisidine (0.34 mL, 3.06 mmol) in dry dichlo-
romethane, 3,5-dimethoxybenzoyl chloride 21, PS-DIEA (1.04 g,
3.67 mmol) and DMAP (catalytic amount) were added. The mixture
was stirred overnight at rt, then filtered and evaporated under
vacuum. The crude residue was purified by flash chromatography
(n-hexane/ethyl acetate 9:1), affording 34 (300 mg, 1.04 mmol,
31% yield) as a solid. 1H NMR (CDCl3) d (ppm): 3.84 (s, 3H), 3.85
(s, 6H), 6.62 (t, 1H, J = 2.2 Hz), 6.72 (ddd, 1H, J = 8.0, 2.3, 0.8 Hz),
6.97 (d, 2H, J = 2.2 Hz), 7.06 (ddd, 1H, J = 8.0, 2.3, 0.8 Hz), 7.25 (t,
1H, J = 8.0 Hz), 7.44 (t, 1H, J = 2.3 Hz), 7.75 (br, 1H).
4.1.22. N-(3,5-Dimethoxy)phenyl-4-methoxythiobenzamide
(41)
A solution of 39 (200 mg, 0.70 mmol) and Lawesson’s reagent
(170 mg, 0.42 mmol) in chlorobenzene (1.0 mL) was boiled at
130 °C for 3 h. The mixture was evaporated and the crude residue
was purified by flash chromatography (n-hexane/ethyl acetate
8:2), affording 41 (148 mg, 0.49 mmol, 70% yield) as a solid. 1H
NMR (CDCl3) d (ppm): 3.79 (s, 6H), 3.86 (s, 3H), 6.38 (t, 1H,
J = 2.1 Hz), 6.91 (m, 4H), 7.82 (m, 2H).
4.1.18. N-(m-Methoxyphenyl)-3,5-dimethoxythiobenzamide
(35)
4.1.23. 2-(p-Methoxyphenyl)-5,7-dimethoxybenzothiazole (43)
Compound 41 (100 mg, 0.33 mmol) was wetted with a little
amount of ethanol, and 30% aqueous NaOH solution (0.4 mL) was
added. The mixture was diluted with water to provide a final sus-
pension of 10% aqueous NaOH. This mixture was added portion-
wise (1 mL, 1 min intervals) to a stirred solution of potassium
ferricyanide (435 mg, 1.32 mmol) in water at 85 °C. The reaction
mixture was heated for a further 30 min and then allowed to cool
to rt and stirred overnight. The mixture was filtered and the resi-
due dissolved in CHCl3, dried over sodium sulfate and concentrated
under vacuum. The crude residue was purified by column chroma-
tography (n-hexane/ethyl acetate 9:1), affording 43 (60 mg,
0.20 mmol, 70% yield) as a solid. 1H NMR (CDCl3) d (ppm): 3.88
(s, 3H), 3.90 (s, 3H), 3.96 (s, 3H), 6.48 (d, 1H, J = 2.0 Hz), 6.99
A solution of 34 (300 mg, 1.04 mmol) and Lawesson’s reagent
(253 mg, 0.63 mmol) in chlorobenzene (1.5 mL) was boiled at
130 °C for 3 h. The mixture was evaporated and the crude residue
was purified by flash chromatography (n-hexane/ethyl acetate
8:2), affording 35 (163 mg, 0.54 mmol, 52% yield) as a yellow solid.
1H NMR (CDCl3) d (ppm): 3.84 (s, 9H), 6.57 (m, 1H), 6.84 (m, 1H),
6.96 (m, 2H), 7.29 (m, 2H), 7.64 (m, 1H), 8.98 (br, 1H).
4.1.19. 2-(3,5-Dimethoxyphenyl)-5-methoxybenzothiazole (37)
and 2-(3,5-dimethoxyphenyl)-7-methoxybenzothiazole (36)
Compound 35 (163 mg, 0.54 mmol) was wetted with a little
amount of ethanol, and 30% aqueous NaOH solution (0.6 mL) was
added. The mixture was diluted with water to provide a final sus-
pension of 10% aqueous NaOH. This mixture was added portion-
wise (1 mL, 1 min intervals) to a stirred solution of potassium
ferricyanide (709 mg, 2.15 mmol) in water at 85 °C. The reaction
mixture was heated for a further 30 min and then allowed to cool
to rt and stirred overnight. The mixture was filtered and the resi-
due dissolved in CHCl3, dried over sodium sulfate and concentrated
under vacuum. The crude residue was purified by column chroma-
tography (n-hexane/ethyl acetate 9:1), affording a mixture of 37
and 36 (70% yield). 1H NMR (CDCl3) d (ppm), (asterisk denotes
(AA0XX0, 2H, JAX = 9.0 Hz, JAA /XX = 2.6 Hz), 7.16 (d, 1H, J = 2.0 Hz),
0
0
8.01 (AA0XX0, 2H, JAX = 9.0 Hz, JAA /XX = 2.6 Hz).
0
0
4.1.24. 2-(p-Hydroxyphenyl)-5,7-hydroxybenzothiazole (8)
A solution of 43 (60 mg, 0.20 mmol) in 2.2 mL of anhydrous
CH2Cl2 was cooled to ꢀ78 °C under nitrogen flux and treated with
a 1 M solution of BBr3 in Et2O (1.98 mL, 1.98 mmol). The mixture
was stirred at rt overnight, then quenched with water and ex-
tracted with ethyl acetate. The organic phase was dried over so-
dium sulfate and concentrated under vacuum. The crude residue
was purified by column chromatography (n-hexane/ethyl acetate
4:6), affording 8 (21 mg, 0.08 mmol, 41% yield) as a solid. 1H
NMR (acetone-d6) d (ppm): 6.50 (d, 1H, J = 2.1 Hz), 6.98 (d, 1H,
*
*
peaks of compound 36): 3.89 (s, 6H+6H), 3.91 (s, 3H), 4.01
*
(s,3H), 6.59 (t, 1H+1H, J = 2.2 Hz), 6.84 (d, 1H, J = 8.3 Hz), 7.04
*
(dd, 1H, J = 8.3, 2.6 Hz), 7.24 (d, 2H, J = 2.2 Hz), 7.27 (d, 2H,
J = 2.1 Hz), 6.99 (AA0XX0, 2H, JAX = 6.8 Hz, JAA /XX = 2.0 Hz), 7.97
0
0
*
J = 2.2 Hz), 7.44 (t, 1H, J = 8.2 Hz), 7.56 (d, 1H, J = 2.6 Hz), 7.10 (d,
(AA0XX0, 2H, JAX = 6.8 Hz, JAA /XX = 2.0 Hz). MS m/z 259 (M+, 49).
0
0
1H, J = 8.2 Hz), 7.74 (d, 1H, J = 8.2 Hz).

S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
6723
4.1.25. N-(2,4-Dimethoxy)phenyl-4-methoxybenzamide (40)
To a solution of 2,4-dimethoxyaniline (328 mg, 2.66 mmol) in
dry CH2Cl2 (3 mL), p-anisoylchoride 38 (401 mg, 1.35 mmol), PS-
DIEA (510 mg, 2.35 mmol) and DMAP (catalytic amount) were
added. The mixture was stirred at rt overnight, then filtered and
evaporated under vacuum. The crude residue was purified by flash
chromatography (n-hexane/ethyl acetate 9:1), affording 40
(418 mg, 1.45 mmol, 74% yield) as a white solid. 1H NMR (CDCl3)
d (ppm): 3.82 (s, 3H), 3.87 (s, 3H), 3.90 (s, 3H), 6.52 (d, 1H,
J = 2.6 Hz), 6.53 (dd, 1H, J = 9.4, 2.6 Hz), 6.98 (AA0XX0, 2H,
mL each of penicillin and streptomycin (Invitrogen, San Giuliano
Milanese, Italy).
4.2.2. Cell proliferation by sulforhodamine B (SRB) assay
Cells were seeded in 96-wells tissue culture plates at
1.3 ꢂ 103 cells/well and were allowed to adhere for 24 h before
treatment. Cells were grown in the presence of vehicle (EtOH or
DMSO) (five wells) or drugs at a indicated concentrations (five
wells for each treatment). Cellular growth was assessed after
6 days by SRB assay. Briefly, proteins were precipitated with 10%
(final concentration) trichloroacetic acid for 1 h at 4 °C and stained
for 30 min with SRB dye 0.4% w/v in acetic acid 1% v/v. Finally, pre-
cipitated proteins were washed and solubilized in Tris buffer
10 mM. Absorbance (optical density, OD) was measured at
540 nm by using a microplate reader. For each treatment the pro-
liferation index was calculated as OD TC tx/OD TC t0 where ‘OD TC
tx’ is the mean optical density of treated cells at time x and ‘OD TC
t0’ is the value at time zero.
JAX = 8.8 Hz, JAA /XX = 2.4 Hz), 7.85 (AA0XX0, 2H, JAX = 8.8 Hz, JAA /
= 2.6 Hz), 8.26 (br, 1H), 8.38 (d, 1H, J = 9.4 Hz).
0
0
0
XX0
4.1.26. N-(2,4-Dimethoxy)phenyl-4-methoxythiobenzamide
(42)
A solution of 40 (250 mg, 0.87 mmol) and Lawesson’s reagent
(213 mg, 0.52 mmol) in chlorobenzene (1.3 mL), was boiled at
130 °C for 3 h. The mixture was evaporated and the crude residue
was purified by flash chromatography (n-hexane/ethyl acetate
8:2), affording 42 (190 mg, 0.63 mmol, 72% yield) as a solid. 1H
NMR (CDCl3) d (ppm): 3.84 (s, 3H), 3.86 (s, 3H), 3.89 (s, 3H), 6.54
(d, 1H, J = 2.1 Hz), 6.55 (dd, 1H, J = 9.5, 2.1 Hz), 6.93 (AA0XX0, 2H,
JAX = 8.8 Hz), 7.87 (AA0XX0, 2H, JAX = 8.8 Hz), 8.91 (d, 1H,
J = 9.5 Hz), 9.34 (br, 1H).
4.3. Vascular assays
All the experimental procedures were carried out following the
guidelines of the European Community Council Directive 86-609.
To determine a possible vasodilator mechanism of action, the com-
pounds were tested on isolated thoracic aortic rings of male nor-
motensive Wistar rats (250–350 g). The rats were sacrificed by
cervical dislocation under light ether anesthesia and bled. The aor-
tae were immediately excised and freed of extraneous tissues. The
endothelial layer was removed by gently rubbing the intimal sur-
face of the vessels with a hypodermic needle. Five millimeter wide
aortic rings were suspended, under a preload of 2 g, in 20 mL organ
baths, containing Tyrode solution (composition of saline in mM:
NaCl 136.8; KCl 2.95; CaCl2 1.80; MgSO4ꢁ7H2O 1.05; NaH2PO4
0.41; NaHCO3 11.9; Glucose 5.5), thermostated at 37 °C and contin-
uously gassed with a mixture of O2 (95%) and CO2 (5%). Changes in
tension were recorded by means of an isometric transducer (Grass
FTO3), connected with a preamplifier (Buxco Electronics) and with
a software of data acquisition (BIOPAC Systems Inc., MP 100). After
an equilibration period of 60 min, the endothelial removal was
4.1.27. 2-(p-Methoxyphenyl)-4,6-dimethoxybenzothiazole (44)
Compound 42 (190 mg, 0.62 mmol) was wetted with a little
amount of ethanol, and 30% aqueous NaOH solution (0.6 mL) was
added. The mixture was diluted with water to provide a final sus-
pension of 10% aqueous NaOH. This mixture was added portion-
wise (1 mL, 1-min intervals) to a stirred solution of potassium
ferricyanide (822 mg, 2.50 mmol) in water at 85 °C. The reaction
mixture was heated for a further 30 min and then allowed to cool
to rt and stirred overnight. The mixture was then filtered and the
residue dissolved in CHCl3, dried over sodium sulfate and concen-
trated under vacuum. The crude residue was purified by column
chromatography (n-hexane/ethyl acetate 1:1), affording 44
(92 mg, 0.30 mmol, 49% yield) as a solid. 1H NMR (CDCl3) d
(ppm): 3.87 (s, 3H), 3.88 (s, 3H), 4.03 (s, 3H), 6.54 (d, 1H,
J = 2.2 Hz), 6.91 (d, 1H, J = 2.2 Hz), 6.96 (AA0XX0, 2H, JAX = 8.8 Hz, J
confirmed by the administration of acetylcholine (ACh) (10 lM)
= 2.6 Hz), 8.01 (AA0XX0, 2H, JAX = 8.8 Hz, JAA /XX = 2.6 Hz).
to KCl (30 mM)-precontracted vascular rings. A relaxation <10%
of the KCl-induced contraction was considered representative of
an acceptable lack of the endothelial layer, while a relaxation
P70% of the KCl-induced contraction was considered representa-
tive of an acceptable integrity of the endothelium. Forty-five min-
utes after the confirmation of the endothelium removal/integrity,
the aortic preparations were contracted by treatment with a single
concentration of KCl (30 mM) and when the contraction reached a
stable plateau, threefold increasing concentrations of the reference
drug resveratrol were added cumulatively. Preliminary experi-
ments showed that the KCl (30 mM)-induced contractions re-
mained in a stable tonic state for at least 40 min. When required,
in some sets of experiments, the non-selective potassium channel
blocker Tetraethylammonium chloride (TEA 10 mM) or the BK-
selective blocker Iberiotoxin (IbTX, 100 nM) were added to aortae,
after the KCl (30 mM)-induced contraction, followed by the admin-
istration of tested compounds. In another set of experiments, the
behavior of compound 6 in high depolarizing conditions was ob-
served, replacing the standard KCl concentration of 30 mM with
a 60 mM KCl concentration. The reference drug resveratrol (Sigma)
was dissolved (10 mM) in DMSO and further diluted in Tyrode
solution. Acetylcholine chloride (Sigma) was dissolved (100 mM)
in EtOH 95% and further diluted in bidistilled water whereas KCl,
TEA, and IbTX were both dissolved in Tyrode solution. All the solu-
tions were freshly prepared immediately before the pharmacolog-
ical experimental procedures. Previous experiments showed a
0
0
AA0/XX0
4.1.28. 2-(p-Hydroxyphenyl)-4,6-hydroxybenzothiazole (9)
A solution of 44 (92 mg, 0.30 mmol) in 3.3 mL of anhydrous
CH2Cl2 was cooled to ꢀ78 °C under nitrogen flux and treated with
a 1 M solution of BBr3 in Et2O (3.2 mL, 3.20 mmol). The mixture
was stirred at rt overnight, then quenched with water and ex-
tracted with ethyl acetate. The organic phase was dried over so-
dium sulfate and concentrated under vacuum. The crude residue
was purified by column chromatography (n-hexane/ethyl acetate
1:1), affording 9 (52 mg, 0.20 mmol, 66% yield) as a solid. 1H
NMR (acetone-d6) d (ppm): 6.50 (d, 1H, J = 2.2 Hz), 6.88 (d, 1H,
J = 2.2 Hz), 6.96 (AA0XX0, 2H, JAX = 8.8 Hz, JAA /XX = 2.5 Hz), 7.88
0
0
(AA0XX0, 2H, JAX = 8.8 Hz, JAA /XX = 2.5 Hz), 8.76 (br, 3H). MS m/z
0
0
259 (M+, 100).
4.2. Antiproliferative assays
All drugs were dissolved in absolute ethanol (EtOH) before use.
4.2.1. Cell lines
Human breast cancer cell line MDA-MB-231 (American Type
Culture Collection, Rockville, MD, USA), was maintained at 37 °C
in 5% CO2 in Dulbecco’s modified Eagle’s medium (EuroClone Ltd,
Wetherby West Yorkshire, UK), supplemented with 5% fetal bovine
serum (EuroClone Ltd, Wetherby West Yorkshire, UK) and 100 ng/

6724
S. Bertini et al. / Bioorg. Med. Chem. 18 (2010) 6715–6724
9. Calderone, V.; Martelli, A.; Testai, L.; Martinotti, E.; Breschi, M. C. Naunyn
Schmiedebergs Arch. Pharmacol. 2007, 375, 73.
10. Gojkovic-Bukarica, L.; Novakovic, A.; Kanjuh, V.; Bumbasirevic, M.; Lesic, A.;
Heinle, H. A. J. Pharmacol. Sci. 2008, 108, 124.
complete ineffectiveness of the administration of the vehicle. The
vasorelaxing efficacy was evaluated as maximal vasorelaxing re-
sponse, expressed as a percentage (%) of the contractile tone in-
duced by KCl 30 mM (or KCl 60 mM). When the limit
11. Scalbert, A.; Williamson, G. J. Nutr. 2000, 130, 2073S.
12. Yu, C.; Shin, Y. G.; Chow, A.; Li, Y.; Kosmeder, J. W.; Lee, Y. S.; Hirschelman, W.
H.; Pezzuto, J. M.; Mehta, R. G.; Van Breemen, R. B. Pharm. Res. 2002, 19, 1907.
13. Lopez-Hernandez, J.; Paseiro-Losada, P.; Sanches-Silva, A. T.; Lage-Yusty, M. A.
Eur. Food Res. Technol. 2007, 225, 789.
14. (a) Henderson, R. F. Chem. Biol. Interact. 2001, 135–136, 53; (b) Metzler, M.;
Neumann, H. G. Xenobiotica 1977, 7, 117.
15. Lion, C. J.; Matthews, C. S.; Stevens, M. F. G.; Westwell, A. D. J. Med. Chem. 2005,
48, 1292.
16. Minutolo, F.; Sala, G.; Bagnacani, A.; Bertini, S.; Carboni, I.; Placanica, G.; Prota,
G.; Rapposelli, S.; Sacchi, N.; Macchia, M.; Ghidoni, R. J. Med. Chem. 2005, 48,
6783.
concentration 30 lM (the highest concentration, which could be
administered) of resveratrol or test compounds did not reach the
maximal effect, the parameter of efficacy represented the vasore-
laxing response, expressed as a percentage (%) of the contractile
tone induced by KCl 30 mM, evoked by this limit concentration.
The parameter of potency was expressed as pIC50, calculated as
negative logarithm of the molar concentration of the tested com-
pounds, evoking a half reduction of the contractile tone induced
by KCl 30 mM. The pIC50 could not be calculated for those com-
pounds showing an efficacy parameter lower than 50%. The param-
eters of efficacy and potency were expressed as mean standard
error, for 5–10 experiments. Student t test was selected as statisti-
cal analysis, P <0.05 was considered representative of a significant
statistical difference. Experimental data were analyzed by a com-
puter fitting procedure (software: GraphPad Prism 4.0).
17. Jeong, S. H.; Jo, W. S.; Song, S.; Suh, H.; Seol, S.-Y.; Leem, S.-H.; Kwon, T. K.; Yoo,
Y. H. Biochem. Pharmacol. 2009, 77, 1337.
18. Minutolo, F.; Bertini, S.; Papi, C.; Carlson, K. E.; Katzenellenbogen, J. A.;
Macchia, M. J. Med. Chem. 2001, 44, 4288.
19. Minutolo, F.; Bellini, R.; Bertini, S.; Carboni, I.; Lapucci, A.; Pistolesi, L.; Prota, G.;
Rapposelli, S.; Solati, F.; Tuccinardi, T.; Martinelli, A.; Stossi, F.; Carlson, K. E.;
Katzenellenbogen, B. S.; Katzenellenbogen, J. A.; Macchia, M. J. Med. Chem.
2008, 51, 1344.
20. Roberti, M.; Pizzirani, D.; Recanatini, M.; Simoni, D.; Grimaudo, S.; Di Cristina,
A.; Abbadessa, V.; Gebbia, N.; Tolomeo, M. J. Med. Chem. 2006, 49, 3012.
21. Minutolo, F.; Bertini, S.; Granchi, C.; Marchitiello, T.; Prota, G.; Rapposelli, S.;
Tuccinardi, T.; Martinelli, A.; Gunther, J. R.; Carlson, K. E.; Katzenellenbogen, J.
A.; Macchia, M. J. Med. Chem. 2009, 52, 858. and references therein.
22. Karabatsos, G. J.; Hsi, N. Tetrahedron 1967, 23, 1079.
Acknowledgment
Mehdi Rajabi was supported by a Predoctoral fellowships of the
Doctorate School of Molecular Medicine, University of Milan.
23. Magnon, M.; Calderone, V.; Floch, A.; Cavero, I. Naunyn Schmiedebergs Arch.
Pharmacol. 1998, 358, 452.
24. GAUSSIAN 03, Revision A.1, Frisch, M. J.; Trucks, G. W.; Schlegel, H. B.; Scuseria, G.
E.; Robb, M. A.; Cheeseman, J. R.; Montgomery, J. A., Jr., Vreven, T.; Kudin, K. N.;
Burant, J. C.; Millam, J. M.; Iyengar, S. S.; Tomasi, J.; Barone, V.; Mennucci, B.;
Cossi, M.; Scalmani, G.; Rega, N.; Petersson, G. A.; Nakatsuji, H.; Hada, M.;
Ehara, M.; Toyota, K.; Fukuda, R.; Hasegawa, J.; Ishida, M.; Nakajima, T.; Honda,
Y.; Kitao, O.; Nakai, H.; Klene, M.; Li, X.; Knox, J. E.; Hratchian, H. P.; Cross, J. B.;
Adamo, C.; Jaramillo, J.; Gomperts, R.; Stratmann, R. E.; Yazyev, O.; Austin, A. J.;
Cammi, R.; Pomelli, C.; Ochterski, J. W.; Ayala, P. Y.; Morokuma, K.; Voth, G. A.;
Salvador, P.; Dannenberg, J. J.; Zakrzewski, V. G.; Dapprich, S.; Daniels, A. D.;
Strain, M. C.; Farkas, O.; Malick, D. K.; Rabuck, A. D.; Raghavachari, K.;
Foresman, J. B.; Ortiz, J. V.; Cui, Q.; Baboul, A. G.; Clifford, S.; Cioslowski, J.;
Stefanov, B. B.; Liu, G.; Liashenko, A.; Piskorz, P.; Komaromi, I.; Martin, R. L.;
Fox, D. J.; Keith, T.; Al-Laham, M. A.; Peng, C. Y.; Nanayakkara, A.; Challacombe,
M.; Gill, P. M. W.; Johnson, B.; Chen, W.; Wong, M. W.; Gonzalez, C.; Pople, J. A.
GAUSSIAN, Inc, Pittsburgh, PA, 2003.
References and notes
1. Wolter, F. Drugs Future 2002, 27, 949.
2. Scarlatti, F.; Sala, G.; Somenzi, G.; Signorelli, P.; Sacchi, N.; Ghidoni, R. FASEB J.
2003, 17, 2339.
3. Sala, G.; Minutolo, F.; Macchia, M.; Sacchi, N.; Ghidoni, R. Drugs Exp. Clin. Res.
2003, 29, 263.
4. Bowers, J. L.; Tyulmenkov, V. V.; Jernigan, S. C.; Klinge, C. M. Endocrinology
2000, 141, 3657.
5. Renaud, S.; De Lorgeril, M. Lancet 1992, 339, 1523.
6. Stivala, L. A.; Savio, M.; Carafoli, F.; Perucca, P.; Bianchi, L.; Maga, G.; Forti, L.;
Pagnoni, U. M.; Albini, A.; Prosperi, E.; Vannini, V. J. Biol. Chem. 2001, 276,
22586.
7. Belguendouz, L.; Fremont, L.; Linard, A. Biochem. Pharmacol. 1997, 53, 1347.
8. Ou, H.-C.; Chou, F.-P.; Sheen, H.-M.; Lin, T.-M.; Yang, C.-H.; Sheu, W. Clin. Chim.
Acta 2006, 364, 196.
25. ROCS, version 2.2; OpenEye Scientific Software, Inc, Santa Fe, NM, 2006.