Synthesis and preliminary biological profile of new NO-donor tolbutamide analogues

Article abstract of DOI:10.1016/j.bmcl.2012.03.103

We describe a new class of NO-donor hypoglycemic products obtained by joining tolbutamide, a typical hypoglycemic sulfonylurea, with a NO-donor moiety through a hard link. As NO-donors we chose either furoxan (1,2,5-oxadiazole 2-oxide) derivatives or the classical nitrooxy function. A preliminary biological characterization of these compounds, including stimulation of insulin release from cultured rat pancreatic β-cells and in vitro vasodilator and anti-aggregatory activities, is reported.

Full text of DOI:10.1016/j.bmcl.2012.03.103

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3810–3815
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Synthesis and preliminary biological profile of new NO-donor
tolbutamide analogues
Yasinalli Tamboli a, Loretta Lazzarato a, Elisabetta Marini a, Stefano Guglielmo a, Michela Novelli b,
a
Pascale Beffy c, Pellegrino Masiello b, Roberta Fruttero a, , Alberto Gasco
⇑
a Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, via Pietro Giuria 9, 10125 Torino, Italy
b Dipartimento di Patologia Sperimentale, Biotecnologie Mediche, Infettivologia ed Epidemiologia, Scuola Medica, Università degli Studi di Pisa, via Roma 55, 56126 Pisa, Italy
c Istituto di Fisiologia Clinica, Centro Nazionale delle Ricerche, Area della Ricerca, via Moruzzi 1, 56127 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
We describe a new class of NO-donor hypoglycemic products obtained by joining tolbutamide, a typical
hypoglycemic sulfonylurea, with a NO-donor moiety through a hard link. As NO-donors we chose either
furoxan (1,2,5-oxadiazole 2-oxide) derivatives or the classical nitrooxy function. A preliminary biological
characterization of these compounds, including stimulation of insulin release from cultured rat
pancreatic b-cells and in vitro vasodilator and anti-aggregatory activities, is reported.
Ó 2012 Elsevier Ltd. All rights reserved.
Received 8 February 2012
Revised 25 March 2012
Accepted 28 March 2012
Available online 11 April 2012
Keywords:
Diabetes mellitus
NO-donor
Multitarget drugs
Anti-aggregatory activity
Insulin release
Diabetes mellitus is a metabolic disorder characterized by
chronic hyperglycemia, with disorders of the carbohydrate, fat,
and protein metabolism. It causes long-term complications in target
organs, including retina, kidney, peripheral nerves, and the cardio-
vascular system. From the etiological standpoint, diabetes mellitus
is caused by defects in the secretion and/or action of insulin. Two
main types of diabetes are recognized: type 1 (T1DM), which is pri-
marily due to pancreatic islet beta-cell autoimmune destruction,
and typically causes ketoacidosis, and type 2 (T2DM), the commoner
form of diabetes, which results from defects of insulin secretion and
is almost always associated with the body cells’ inability to ade-
quately respond to insulin (insulin resistance).1 Diabetes is now
one of the most significant public health problems, due to a world-
wide increase in this disease.2 This increase foreshadows a rapid
increase in chronic diabetic complications, including blindness, re-
nal failure, peripheral neuropathy, and multiple athero-thrombotic
lesions, involving coronary, cerebral, and peripheral arteries. There
is general agreement that arterial damage is the most important
factor involved in the high incidence of morbidity and reduced
life-expectancy in diabetic patients. T2DM, in particular, is charac-
terized by a complex profile of cardiovascular risk, due to the pres-
ence of hyperglycemia, dyslipidemia, arterial hypertension,
endothelial dysfunction, and multiple hemostatic alterations.3–5 It
is recognized that impaired formation and/or availability of endo-
thelium-derived nitric oxide (NO, EDRF) may be a crucial factor in
the development of cardiovascular events in diabetes mellitus.6
NO is an endogenous messenger, believed to play a significant role
in the maintenance of micro- and macro-vascular homeostasis. It
inhibits platelet adherence and aggregation, decreases leukocyte
chemotaxis, promotes endothelial regeneration and angiogenesis,
reduces vascular smooth-muscle cell (VSMC) constriction, migra-
tion and proliferation, and enhances VSMC apoptosis.7 NO triggers
a complex cascade of events in target cells, by activating soluble
guanylate cyclase (sGC), which induces synthesis of 3,5-cyclic gua-
nosine monophosphate (cGMP) leading to activation of the cGMP-
dependent protein kinase PKG (NO/cGMP/PKG pathway).8
In T2DM, therapeutic guidelines recommend an aggressive mul-
tiple intervention policy, including hypoglycemic, hypotensive and
anti-platelet aggregation drugs, in the setting of cardiovascular pre-
vention. Thus also in diabetes, as for other complex diseases requir-
ing the administration of multiple drugs, there is today great
interest in the availability of hybrid drugs, known also as polyvalent
or multifunctional drugs, which simultaneously modulate more
than one target.9,10 In particular, compounds deriving from the
hybridization of a hypoglycemic drug with NO-donor moieties
would appear to be of great interest for the treatment of T2DM.
Compounds of this type have recently been characterized, namely
NO-donor pro-drugs of the hydroxylated active metabolite of
glibenclamide, a well-known hypoglycemic agent.11 As a further
⇑
Corresponding author.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.103

Y. Tamboli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3810–3815
3811
development of our work on NO-donor hybrid compounds, we here
describe a new class of NO-donor hypoglycemic products, which
are not co-drugs, obtained by joining tolbutamide 1 (Chart 1), a typ-
ical hypoglycemic sulfonylurea, with a number of NO-donor moie-
ties (NO-donor tolbutamides), through a hard link. As NO-donors,
we chose either furoxan (1,2,5-oxadiazole 2-oxide) derivatives, in
view of their remarkable ability to induce anti-aggregatory effects12
and vasodilation without inducing tolerance,13 or the classical
nitrooxy function (ONO2). It is generally accepted that furoxans
release NO in the presence of thiol co-factors,14 while nitrooxy
derivatives require enzymatic bioactivation to do so.15 In order to
modulate the amount of exogenous NO supplied by the constructs,
we used either differently-substituted furoxan derivatives, or moi-
eties containing one or two nitrate groups. The synthesis of these
products is reported and discussed, together with a preliminary
biological characterization, including the stimulation of insulin
release from cultured rat pancreatic b-cells (INS-1E line) and
in vitro vasodilator and anti-aggregatory activities. Since both
inhibitory and stimulatory effects, on glucose-induced insulin
secretion by INS-1E cells, have been ascribed to NO, the simple
NO-donor substructures (see Schemes 4 and 5) present in these
products were also tested, as controls.16
The final furoxan 11 was obtained starting from 10, following the
procedure used to prepare the nitrates 5 and 8. The 4-bromo-
methyl-3-phenylfuroxan 13 was obtained by action of SOBr2 on
the corresponding hydroxyderivative 12. The 4-bromomethylfu-
roxans, appropriately substituted at the 3-position, were subjected
to reaction with 2 in the presence of NaOH or Na2CO3, to give the
intermediate solfonamides 16–18. These products were trans-
formed into the corresponding target compounds (19, 20, 21) via
the usual reaction with CuCl and butyl isocyanate. The general
procedure to prepare the target compounds 5, 8, 11, 19, 20, 21
and their spectral characterization are reported in reference.17
The simple substructures containing NO-donor nitrooxy func-
tions 24, 26, used for control purposes, were obtained via the path-
ways outlined in Scheme 4. The mononitrate 24 was prepared
under Mitsunobu conditions,18 namely by treating the adduct of
Ph3P and diisopropylazodicarboxylate (DIAD) in THF solution with
phenol 22, followed by addition of the alcohol 23. The dinitrate 26
was synthesized by treating a mixture of silver nitrate and (allyl-
oxy)benzene (25) with iodine in acetonitrile at room temperature,
followed by reflux. The simple NO-donor furoxans 27–29 were pre-
pared by procedures reported in Scheme 5. Furoxans 27, 28 were
obtained from the related (bromomethyl)furoxans 13, 14 through
nucleophilic displacement of bromine by sodium salt of phenol. Fi-
nally, the cyano substituted furoxan 29 was prepared by trifluoro-
acetic anhydride dehydration of the parent amide 28 in THF
solution, in the presence of pyridine. The 3-phenylsulfonyl substi-
tuted furoxan 30 (Chart 1) was synthesized as reported in the
literature.19
Synthesis of the target products and of the intermediates used
for their preparation is summarized in Schemes 1–3. Briefly, the
intermediate mononitrate
4 was obtained by action of the
4-hydroxybenzenesulfonamide (2) on the previously-described
tosylate of 3-hydroxypropyl nitrate 3, in the presence of NaOH
(Scheme 1). The final mononitrate 5 was prepared by treating
4 with CuCl and butyl isocyanate in DMF. The dinitrate target com-
pound 8 was synthesized in a similar manner, starting from dini-
trate 7, in turn obtained by action of iodine and AgNO3 on 6. The
sulfonamide 6 is the product of the nucleophilic attack of the so-
dium salt of 2 on allyl bromide, in ethanol.
The ability of the NO-donor hybrids to release NO was assessed
by detecting nitrite, the principal final product of NO oxidative
metabolism, using the Griess reaction. After incubation in rat liver
homogenate, nitrates 5, 8 generated nitrite (8 > 5). The furoxan-
tolbutamides (11, 20, 21) produce nitrite when incubated with
Nucleophilic attack of 2 on 3,4-bisphenylsulfonylfuroxan (9), in
the presence of Na2CO3 in DMF, afforded the 3-phenylsulfonyl
substituted furoxan 10 in a regioselective manner (Scheme 2).
an excess of L-cysteine in buffer solution (pH = 7.4). Under these
conditions, the NO release from compound 19 was undetectable.
Parallel experiments, on INS-1E cells exposed to the NO-donor hy-
brids, showed a significant production of nitrite for compounds 8,
11, 19, and 21 (see Supplementary data).
The ability of the synthesized products to supply exogenous NO
to blood vessels was evaluated by assessing their capacity to in-
duce relaxation of rat aorta strips pre-contracted with phenyleph-
rine, following a procedure described elsewhere.20 Both nitrate and
furoxan derivatives of tolbutamide were found to determine relax-
ation of the contracted strips in a concentration dependent
Chart 1. Structure of Tolbutamide (1) and of compound 30.
Scheme 1. (a) Tosylpropylnitrate (3), NaOH, EtOH; (b) CuCl, butylisocyanate, DMF; (c) allylbromide, NaOH, EtOH; (d) I2, AgNO3, CH3CN, rt then reflux.

3812
Y. Tamboli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3810–3815
Scheme 2. (a) Na2CO3, DMF; (b) CuCl, butylisocyanate, DMF.
Scheme 3. (a) SOBr2, DMF; (b) NaOH, EtOH for R = Ph, CONH2; Na2CO3, DMF for R = CN; (c) CuCl, butylisocyanate, DMF.
Scheme 4. (a) Ph3P, DIAD, THF; (b) I2, AgNO3, CH3CN, rt then reflux.
Figure 1. Concentration response curves for vasodilator activity of compound 21 in
the absence and in the presence of ODQ.
manner, as reported for the example in Figure 1. The vasodilator
potencies of the products, expressed as EC50, are given in Table 1.
When the experiments were repeated in the presence of 1H-
[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one (ODQ),
a well-known
inhibitor of sGC, a significant reduction in the vasodilator effect
of the products occurred (Table 1). This strongly supports the
Scheme 5. (a) Na, THF, 0 °C to rt; (b) (CF3CO)2O, Py, THF, 0 °C to rt.

Y. Tamboli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3810–3815
3813
Table 1
Anti-aggregatory and vasodilator activity of compounds 1, 5, 8, 11, 19, 20, 21
Compound
Anti-aggregatory activity
M ODQ]
Vasodilator activity
EC50 M) SEM [+1 lM ODQ]
IC50
(l
M)(CL 95%) [+50
l
% inhibition SEM at 300
6.4 4.3
l
Ma
(l
1
5
Inactive
1.6 0.2
1.1 1.1
5.6 4.5
b
0.90 0.10
8
[61 16]
10 (8.9–12)
[34 (32–37)]
0.0024 0.0001
11
19
20
21
[0.22 0.04]
2.4 0.2
13
4
b
0.74 0.09
5.2 1.0
[57 1]
0.010 0.001
26 (22–30)
[209 (188–232)]
[0.90 0.06]
a
Due to the low activity of the compound, IC50 could not be calculated. In this case the percent of inhibition is reported at 300 lM.
In the presence of 1 lM ODQ, EC50 values were >100 lM.
b
Figure 2. Insulin release from INS-1E cell line exposed to 1, 5, 8, 11, 19, 20, and 21
at 200 M concentration in the presence of 2 mM glucose (control). Data are mean
Figure 3. Insulin release from INS-1E cell line exposed to 1, 24, 26, 27, 28, 29, and
30 at 200 M concentration in the presence of 2 mM glucose (control). Data are
l
l
values SEM of 8–12 determinations for tolbutamide derivatives and 20–24
determinations for control and 1. ⁄p <0.05 at least versus control; § p<0.05 at least
versus 1.
mean values SEM of 8–12 determinations for NO-donor derivatives and 20–24
determinations for (control) and 1. ⁄p <0.05 at least versus (control).
concentration tested (300 lM), are in Table 1. Analysis of the data
shows that the potent vasodilator hybrid furoxans 11 and 21 are also
potent anti-aggregatory agents. This finding is in agreement with
the hypothesis that platelets possess the appropriate machinery
for inducing NO release from furoxans.12 The involvement of NO in
the anti-aggregatory effect is demonstrated by the substantial loss
of this property when the experiments are carried out in the pres-
ence of ODQ. The low activity of the remaining two hybrid furoxans,
19 and 20, is presumably due to their lower capacity to release NO,
and in the case of 20 also to a hydrophilic–lipophilic balance that
is not suitable to penetrate the platelets. The lack of antiaggregatory
action displayed by hybrid nitric acid esters 5 and 8 is not surprising,
in view of the established poor capacity of platelets to produce NO
from nitrates.23
involvement of NO in the vasodilator action. Analysis of the data
shows that the most potent vasodilator is the 3-phenylsulfonylf-
uroxan derivative 11, which is active in the nM range, followed
in order by the 3-CN substituted compound 21 and the 3-carbam-
oyl analogue 20. The 3-phenylfuroxan derivative is the least potent
vasodilator of the furoxan series, having an EC50 (2.4) in the low
lM range. The potency ranking parallels the electron-withdrawing
properties of substituents at the 3-position of the ring. This is in
keeping with the hypothesis that NO release by the furoxan system
is triggered by nucleophilic attack of a thiol cofactor at the 3-posi-
tion of the ring.14,21 The nitrates also display potencies in the low
lM range, the di-ester 8 being somewhat more potent than the
mono-ester 5.
Tolbutamide is a well-known hypoglycemic drug belonging to
the class of sulfonylureas, which act by stimulating insulin release
from pancreatic b cells, through a specific interaction with the
SUR-1 subunit of the ATP-dependent K+ channel, leading to the
channel’s closure, beta-cell depolarization, opening of voltage-
dependent Ca2+ channels, increased Ca2+ fluxes into beta cells,
The anti-aggregatory effects of the compounds were evaluated
by measuring collagen-induced platelet aggregation of human
platelet-rich plasma (PRP), following
a procedure reported
elsewhere.22 The results, expressed as either IC50 or, when IC50 could
not be calculated, as the percentage of inhibition at the maximal

3814
Y. Tamboli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3810–3815
in terms of insulin secretion, although there may be some change
with respect to tolbutamide.
One problem with hybrid drugs, in which the stoichiometry be-
tween the two pharmacophores present is necessarily 1:1, is that
these pharmacophores must display their biological activities in
the same concentration range. This is a difficult goal to achieve,
and success can only be evaluated through in vivo experiments.
The results of this in vitro study indicate that the most interesting
products for in vivo testing are compounds 5, 19, 20 in which the
tolbutamide pharmacophore is combined with substructures en-
dowed with low/moderate NO dependent activity. Compounds
11 and 21 are too potent NO-donors to give rise to a balanced
hybrid drug in vivo; however, a dedicated study will be necessary
to confirm this, in view of the probable different pharmacokinetic
properties of the products.
In conclusion, we successfully obtained new hybrid compounds,
which are not co-drugs, that combine the insulin secretagogue
properties of tolbutamide with the ability to restore endothelial
function, due to their ability to activate the NO/cGMP/PKG path-
way. The products were obtained by combining tolbutamide with
NO-donor nitrooxy and furoxan moieties endowed with different
NO-releasing capabilities. Compounds 5, 19, and 20, in which the
tolbutamide pharmacophore is combined with substructures that
display low or moderate NO-dependent vasodilator activity,
emerged as models for which additional in vivo studies would be
worthwhile, as they are potentially useful as drugs to treat
diabetes.
Figure 4. Insulin release from INS-1E cell line exposed to 1, 8, 11, 19, 21 at 200 lM
concentration in the presence of different glucose concentrations (G1, G2, G4). Data
are mean values SEM of at least 5 determinations for tolbutamide derivatives and
15–20 determinations for glucose alone and for 1. ⁄p <0.05 at least versus
corresponding glucose concentrations.
and ultimately insulin secretion.24 In order to verify whether the
NO-donor tolbutamide analogues described here retain the insulin
secretagogue properties of the native drug, we studied all the prod-
ucts, as well as the corresponding simple NO-donor substructures
(i.e., the two nitric esters 24, 26 and the four furoxans 27–30), for
their ability to stimulate insulin secretion from the well-differenti-
ated rat pancreatic b cell line INS-1E, which releases insulin in a glu-
cose-regulated manner.25,26 INS-1E cells were incubated in KRB/
Hepes buffer in the presence of various glucose concentrations
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.
103.
and 200 lM of product; the results are in Figures 2, 3 and 4. Tolbu-
tamide, which was tested as a reference compound, enhanced
insulin release by approximately 2.5 times versus control cells
(Fig. 2). Under the same conditions, all synthesized hybrid products
likewise significantly increased insulin release versus 2 mM glu-
cose, although to different extents. Some products increased insulin
secretion as much as tolbutamide (namely compounds 8, 19, 21) or
to a greater extent (compound 11), whereas others (compounds 5
and 20), were less effective than tolbutamide. In parallel experi-
ments we also tested some compounds at a concentration of
References and notes
1. Textbook of Diabetes; Holt, R. I. G., Cockram, C. S., Flyvbjerg, A., Goldstein, B. J.,
Eds., 4th ed.; Blackwell Publishing Ltd, 2010.
2. Boyle, J. P.; Thompson, T. J.; Gregg, E. W.; Barker, L. E.; Williamson, D. F. Popul.
Health Metr. 2010, 8, 29.
3. Haffner, S. M.; Letho, S.; Ronnemaa, T.; Pyorala, K.; Laakso, M. N. Engl. J. Med.
1998, 339, 229.
4. Dominguez, H.; Flyvbjerg, A. Horm. Metab. Res. 2008, 40, 583.
5. Dandona, P.; Aljada, A.; Chaudhuri, A.; Mohanty, P.; Garg, R. Circulation 2005,
111, 1448.
6. Toda, N.; Imamura, T.; Okamura, T. Pharmacol. Ther. 2010, 127, 189.
7. Nitric Oxide and the Cardiovascular System; Loscalzo, J., Vita, J. A., Eds.; Humana
Press: Totowa, 2000.
8. Jin, R. C.; Loscalzo, J. J. Blood Med. 2010, 1, 147.
9. Morphy, R.; Kay, C.; Rankovic, Z. DDT 2004, 9, 641.
10. Morphy, R.; Rankovic, Z. J. Med. Chem. 2005, 48, 6523.
11. Calderone, V.; Rapposelli, S.; Martelli, A.; Digiacomo, M.; Testai, L.; Torri, S.;
Marchetti, P.; Breschi, M. C.; Balsamo, A. Bioorg. Med. Chem. 2009, 17, 5426.
12. Turnbull, C. M.; Cena, C.; Fruttero, R.; Gasco, A.; Rossi, A. G.; Megson, I. L. Br. J.
Pharmacol. 2006, 148, 517.
13. Bohn, H.; Brendel, J.; Martorana, P. A.; Schonafinger, K. Br. J. Pharmacol. 1995,
114, 1605.
14. Gasco, A.; Schoenafinger, K. The NO-releasing heterocycles. In Nitric Oxide
Donors; Wang, P. G., Cai, T. B., Taniguchi, N., Eds.; WILEY-VCH Verlag GmbH &
Co KGaA: Weinheim, 2005.
100 lM which, in most cases significantly increased insulin secre-
tion with respect to the control, but to a lesser extent than the high-
er concentration (see Supplementary data).
As the simple NO-donor moieties (24, 26–30), tested at 200 lM
under the same experimental conditions, did not significantly in-
crease insulin release (Fig. 3), it is reasonable to assume that the
role of NO, if any, on glucose-induced insulin secretion from INS-
1E cells by the NO-tolbutamides, must be negligible.
The hybrid NO-donor compounds showing insulin-secretory ef-
fects similar to or higher than that of tolbutamide, at 2 mM glu-
cose, were also challenged at various glucose concentrations. As
shown in Figure 4, the results substantially confirm that all NO-
releasing compounds (i.e., 8, 11, 19, 21) exhibit patterns of insulin
secretion similar to that of tolbutamide, characterized by an
increasing effect depending on glucose concentrations up to
4 mM. It should be pointed out that when INS-1E cells are exposed
to glucose concentrations higher than 4 mM (e.g., 5.6 or 11 mM),
tolbutamide as well as its NO-donor derivatives are unable to fur-
ther increase glucose-stimulated insulin secretion (see Supple-
mentary data).
15. Daiber, A.; Wenzel, P.; Oelze, M.; Munzel, T. Clin. Res. Cardiol. 2008, 97, 12.
16. Ding, Y.; Rana, R. S. Biochem. Bioph. Res. Co. 1998, 251, 699. and references
therein.
17. To a solution of the appropriate benzensulfonamide derivative (1.06 mmol) in
DMF (10 mL), stirred under inert atmosphere, butylisocyanate (0.24 mL,
2.17 mmol) and CuCl (0.01 mmol) were added. After 24 h the reaction
mixture was poured into ice water (50 mL) under vigorous stirring and 2N
HCl was added until the precipitation of a white solid that was filtered.
Compound 5: yield: 91%. M.p. = 101 °C (from EtOH/H2O 1/1 v/v). 1H-NMR
(DMSO-d6) d 0.86 (3H, t, -CH2CH2CH2CH3), 1.10–1.34 (4H, m, -CH2CH2CH2CH3),
2.17 (2H, t, -CH2CH2ONO2), 2.93 (2H, q, -CH2CH2CH2CH3), 4.16 (2H, t, -OCH2-),
4.69 (2H, t, -CH2ONO2), 6.39–6.43 (1H, m,-NHCH2-), 7.11 (2H, d, Arom), 7.82
(2H, d, Arom), 10.39 (1H, s, -SO2NH-); 13C NMR (DMSO-d6) d 13.4, 19.2, 26.0,
31.2, 38.7, 64.5, 70.7, 114.4, 129.4, 131.9, 151.2, 161.6; MS (CI) m/z 376 (M+1)+.
Compound 8: yield: 89%. M.p. = 135 °C (from EtOH). 1H NMR (DMSO-d6) d 0.81
Thus, in most cases, the structural modifications induced, with
the goal of obtaining tolbutamide analogues endowed with NO-
releasing properties, do not hamper the compounds’ effectiveness

Y. Tamboli et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3810–3815
3815
(3H, t, -CH2CH2CH2CH3), 1.13–1.32 (4H, m, -CH2CH2CH2CH3), 2.93 (2H, q, -
CH2CH2CH2CH3), 4.41–4.56 (2H, m, -OCH2-), 4.91–5.10 (2H, m, -CH2ONO2),
5.81–5.85 (1H, m, -CHONO2), 6.42 (1H, t, -NHCH2-), 7.15 (2H, d, Arom), 7.86
(2H, d, Arom), 10.42 (1H, s, -SO2NH-); 13C NMR (DMSO-d6) d 14.0, 19.8, 31.7,
39.1, 65.9, 70.3, 78.2, 115.1, 130.0, 133.2, 151.8, 166.7; MS (CI) m/z 437 (M+1)+.
Compound 11: yield: 15%. M.p. = 160 °C (from EtOH). 1H NMR (DMSO-d6) d
0.80 (3H, t, -CH2CH2CH2CH3), 1.10–1.33 (4H, m, -CH2CH2CH2CH3), 2.95 (2H, q, -
CH2CH2CH2CH3), 6.52–6.56 (1H, m, -NHCH2-), 7.67 (2H, d, Arom), 7.76 (2H, t,
Arom), 7.89–8.02 (5H, m, Arom), 10.67 (1H, s, -SO2NH-); 13C-NMR (DMSO-d6) d
13.8, 19.6, 31.5, 39.1, 111.7, 119.9, 128.9, 130.10, 130.2, 136.6, 137.0, 138.2,
151.5, 156.2, 157.6; MS (CI) m/z 497 (M+1)+. Compound 19: yield: 89%.
M.p. = 156.5 °C (from EtOH/H2O 1/1 v/v). 1H NMR (DMSO-d6) d 0.81 (3H, t, -
J.; Simeonov, A.; Maloney, D. J.; Williams, D. L.; Thomas, C. J. J. Med. Chem. 2009,
52, 6474.
22. Venous blood samples were obtained from healthy volunteers who had not
taken any drugs for at least 2 weeks. Volunteers, who were treated according to
the Helsinki protocol for biomedical experimentation, gave their informed
consent to the use of blood samples for research purposes. PRP was prepared
by centrifugation of citrated blood at 210g for 20 min. Aliquots (500 lL) of PRP
were added into aggregometer (Chrono-log 4902D) cuvettes, and aggregation
was recorded as increased light transmission under continuous stirring
(1000 rpm) at 37 °C for 10 min after the addition of the stimulus. Collagen at
submaximal concentrations (0.8–1.5 lg/mL) was used as the platelet activator
in PRP. Compounds under study were preincubated with PRP 10 min before the
addition of the stimulus (collagen). Vehicle alone (0.5% DMSO) added to PRP
did not affect platelet function in control samples. The role of NO and sGC in
CH2CH2CH2CH3), 1.10–1.34 (4H, m, -CH2CH2CH2CH3), 2.93 (2H, q,
-
CH2CH2CH2CH3), 5.57 (2H, s, -OCH2-), 6.44 (1H, t, -NHCH2-), 7.23 (2H, d,
Arom), 7.57–7.62 (3H, m, Arom), 7.83–7.86 (4H, m, Arom), 10.47 (1H, s, -
SO2NH-); 13C NMR (DMSO-d6) d 13.4, 19.2, 31.2, 38.7, 61.3, 114.3, 115.0, 121.9,
127.6, 129.0, 129.4, 130.7, 133.2, 151.0, 153.7, 160.4; MS (CI) m/z 447 (M+1)+.
Compound 20: yield: 27%. M.p. = 182 °C (from EtOH). 1H NMR (DMSO-d6) d
0.90 (3H, t, -CH2CH2CH2CH3), 1.11–1.35 (4H, m, -CH2CH2CH2CH3), 2.94 (2H, q, -
CH2CH2CH2CH3), 5.53 (2H, s, -OCH2-), 6.43 (1H, t, -NHCH2-), 7.24 (2H, d, Arom),
7.78 (1H, s, -SO2NH-), 7.80 (2H, d, Arom), 8.50 (1H, s br, -CONH2), 10.44 (1H, s
br, -CONH2); 13C NMR (DMSO-d6) d 13.6, 19.3, 31.3, 38.7, 61.5, 110.5, 114.9,
129.5, 133.0, 151.4, 154.9, 155.7, 160.9; MS (CI) m/z 414 (M+1)+. Compund 21:
yield: 86%. M.p. = 160 °C (from EtOH/H2O 1/1 v/v). 1H NMR (DMSO-d6) d 0.92
(3H, t, -CH2CH2CH2CH3), 1.11–1.34 (4H, m, -CH2CH2CH2CH3), 2.94 (2H, q, -
CH2CH2CH2CH3), 5.62 (2H, s, -OCH2-), 6.44 (1H, t, -NHCH2-), 7.32 (2H, d, Arom),
7.92 (2H, d, Arom), 10.47 (1H, s, -SO2NH-); 13C NMR (DMSO-d6) d 13.4, 19.2,
31.2, 38.7, 60.8, 98.2, 106.2, 114. 9, 129.5, 133.4, 151.2, 154.4, 160.2; MS (CI) m/
z 396 (M+1)+.
the inhibitory effect was investigated using ODQ (50 lM). At least four
experiments for each compound were performed. The anti-aggregatory activity
of tested compounds is evaluated as percent inhibition of platelet aggregation
compared to control samples. For most active compounds, IC50 values could be
calculated by nonlinear regression analysis; otherwise, percent inhibition at
the maximal concentration tested (300 lM) is reported.
23. Weber, A. A.; Neuhaus, T.; Seul, C.; Dusing, R.; Schror, K.; Sachinidis, A.; Vetter,
H. Eur. J. Pharmacol. 1996, 309, 209.
24. Proks, P.; Reimann, F.; Gribble, F.; Ashcroft, F. Diabetes 2002, 51(Suppl 3), S368.
25. Merglen, A.; Theander, S.; Rubi, B.; Chaffard, G.; Wollheim, C. B.; Maechler, P.
Endocrinology 2004, 145, 667.
26. Cell culture: INS-1E cells were cultured in a humidified atmosphere containing
5% CO2 in complete medium composed of RPMI 1640 supplemented with 10%
heat-inactivated fetal calf serum, 1 mM sodium pyruvate, 50
mercaptoethanol, 2 mM glutamine, 10 mM HEPES, 100 U/ml penicillin and
100 g/ml streptomycin. The maintenance culture was passaged once a week
lM 2
18. Mitsunobu, O. Synthesis 1981, 1, 1.
l
19. Fruttero, R.; Crosetti, M.; Chegaev, K.; Guglielmo, S.; Gasco, A.; Berardi, F.; Niso,
M.; Perrone, R.; Panaro, M. A.; Colabufo, N. A. J. Med. Chem. 2010, 53, 5467.
20. Thoracic aortas were isolated from male Wistar rats weighing 180–200 g
(Harlan Italy Laboratories, S. Pietro al Natisone, Italy). As few animals as
possible were used. The purposes and the protocols of our studies have been
approved by Ministero della Salute, Rome, Italy. The endothelium was
removed; the vessels were helically cut and four to six strips were obtained
from each aorta. The tissues were mounted under 1.0 g of tension in organ
baths containing 30 mL of Krebs-bicarbonate buffer with the following
composition: 111.2 mM NaCl, 5.0 mM KCl, 2.5 mM CaCl2, 1.2 mM MgSO4,
1.0 mM KH2PO4, 12.0 mM NaHCO3, and 11.1 mM glucose, maintained at 37 °C
and gassed with 95:5% O2/CO2 (pH 7.4). The aortic strips were allowed to
by gentle trypsinisation.
Insulin secretion: The secretory response to glucose and other secretagogues
was tested in INS-1E cells between passages 54 and 65. Cells were seeded in
24-well plates at a density of 4 Â 104 cells/cm2 in RPMI medium. After 48 h,
medium was removed and cells were pre-incubated for 1 h at 37 °C in Krebs-
Ringer bicarbonate buffer with HEPES (KRBH) containing 0.5% bovine serum
albumin (BSA, fraction V, Sigma) and basal glucose concentrations (1 or 2 mM,
according to the protocol). At the end of the pre-incubation time, cells were
washed and then incubated for 1 h at 37 °C in 1 mL fresh KRBH buffer
containing 0.5% BSA and suitable glucose concentrations, in the presence or
absence of tolbutamide and its derivatives, used at concentrations of 100 or
200 lM. At the end of the incubation, the buffer was collected for insulin
equilibrate for 120 min and then contracted with 1
the response to the agonist reached a plateau, cumulative concentrations of the
vasodilating agent were added. Results are expressed as EC50 SEM ( M). The
effects of 1 M ODQ upon relaxation were evaluated in a separate series of
lM
L-phenylephrine. When
determination. Finally, 1 mL of cold acidified ethanol (150:47:3, v/v, absolute
ethanol/H2O/concentrated HCl) was added to the cells and left for 24 h in order
to extract their insulin content.
Insulin was measured in the buffer and in the acid-ethanol extract by
radioimmunoassay according to Herbert et al. (Herbert V.; Lau K. S.; Gottlied
C.W.; Bleicher S. J. Coated charcoal immunoassay of insulin. J. Clin. Endocrinol.
1965, 25, 1375), using rat insulin as a standard. The sensitivity and the
coefficient of variation of the radioimmunoassay were as follows: detection
limit 0.13 ng/mL, intra-assay variation 3.3%, inter-assay variation 10.5%.
l
l
experiments, in which ODQ was added to the organ bath 5 min before the
contraction. Responses were recorded by an isometric transducer connected to
the MacLab System PowerLab. The addition of the drug vehicle (DMSO) had no
appreciable effect on the contraction level. At least five experiments for each
compound were perfomed.
21. Rai, G.; Sayed, A. A.; Lea, W. A.; Luecke, H. F.; Chakrapani, H.; Prast-Nielsen, S.;
Jadhav, A.; Leister, W.; Shen, M.; Inglese, J.; Austin, C. P.; Keefer, L.; Arner, E. S.