Synthesis and evaluation of multi-functional NO-donor/insulin-secretagogue derivatives for the treatment of type II diabetes and its cardiovascular complications

Article abstract of DOI:10.1016/j.bmc.2014.12.043

Although there is a significant effort in the discovery of effective therapies to contrast both the pathological endocrine and metabolic aspects of diabetes and the endothelial dysfunction associated with this disease, no hypoglycemic drug has been proven

Full text of DOI:10.1016/j.bmc.2014.12.043

Bioorganic & Medicinal Chemistry xxx (2015) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and evaluation of multi-functional NO-donor/
insulin-secretagogue derivatives for the treatment of type II
diabetes and its cardiovascular complications
Maria Digiacomo , Alma Martelli , Lara Testai , Annalina Lapucci, Maria C. Breschi,
⇑
⇑
Vincenzo Calderone , Simona Rapposelli
Dipartimento di Farmacia, Università di Pisa, Via Bonanno, 6, 56126 Pisa, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Although there is a significant effort in the discovery of effective therapies to contrast both the patholog-
ical endocrine and metabolic aspects of diabetes and the endothelial dysfunction associated with this
disease, no hypoglycemic drug has been proven to defeat the cardiovascular complications associated
with type II diabetes. The aim of this research was to design new compounds exhibiting a double profile
of hypoglycemic agents/NO-donors. The synthesis of molecules obtained by the conjunction of NO-donor
moieties with two oral insulin-secretagogue drugs (repaglinide and nateglinide) was reported.
NO-mediated vasorelaxing effects of the synthesized compounds were evaluated by functional tests on
isolated endothelium-denuded rat aortic rings. The most potent molecule (4) was tested to evaluate
the hypoglycemic and the anti-ischemic cardioprotective activities. This study indicates that 4 should
represent a new insulin-secretagogue/NO-donor prodrug with an enhanced cardiovascular activity,
which may contrast the pathological aspects of diabetes and endowed of cardioprotective activity.
Ó 2014 Published by Elsevier Ltd.
Received 4 September 2014
Revised 12 December 2014
Accepted 17 December 2014
Available online xxxx
Keywords:
Hybrid drugs
NO-releasing drugs
NO-donor
Insulin-secretagogue drug
Hypoglycemic
Cardioprotection
1. Introduction
particular, it is well known that diabetes is associated with a signif-
icant reduction in the biosynthesis and release of endothelial nitric
Type II diabetes mellitus is a multifactorial disease character-
ized by a combination of insulin resistance and reduced insulin
secretion. The impact of this disease on social health is closely
linked to the co-existence of both metabolic and cardiovascular
disorders.1 It is well-known that the endothelial dysfunction is
responsible for important macro-vascular problems such as
myocardial ischemia, hypertension and peripheral vasculopathy.
Lately, heart diseases and stroke are the main causes of death
and disability among people with type 2 diabetes.2
Structural and functional alterations of vascular structures, such
as the glycation of wall components of blood vessels, are strongly
involved in the pathogenesis of cardiovascular complications asso-
ciated with diabetes. One of the major vascular alterations is the
endothelial dysfunction, resulting in a relevant imbalance in the
production of endothelium-derived endogenous factors pivotally
involved in the regulation of the cardiovascular function. In
oxide (NO).3
NO is an important endothelium-derived mediator endowed of
vasodilator, anti-platelet, anti-proliferative, permeability-decreas-
ing and anti-inflammatory properties.4 An impairment of endothe-
lium-dependent vasorelaxation caused by a reduced NO activity,
worsens the diabetic metabolic alterations (dyslipidaemia, glyca-
tion end-products, oxidative stress), thus resulting in a dramati-
cally prevalence of atherosclerosis, thrombosis, vascular
inflammation and remodeling, hypertension, coronaropathy and
stroke.5,6
Therefore, in order to reduce the cardiovascular risk, diabetic
patients usually follow additional pharmacological treatments tar-
geting hypertension, platelet aggregation, and dyslipidaemia.6,7
In the last years, the development of new ‘chimeras’, with the
double pharmacodynamic profile of hypoglycemic agents and, at
the same time, of slow NO donors, has been first reported by us
and then widely investigated.8–10 NO-sulfonylureas and NO-meg-
litinides2,8,11 are interesting examples of drugs able to contrast
both the endocrine and the cardiovascular complications of diabe-
tes mellitus. These ‘chimeric drugs’ conserved the antidiabetic
properties, due to their insulin-secretagogue activity; moreover,
they were endowed of additional NO-releasing property. Such an
⇑
Corresponding authors. Tel.: +39 050 2219589; fax: +39 050 2219577
(V. Calderone); tel.: +39 050 2219582; fax: +39 050 2219577 (S. Rapposelli).
E-mail addresses: calderone@farm.unipi.it (V. Calderone), simona.rapposelli@
farm.unipi.it (S. Rapposelli).
These authors contributed equally.
http://dx.doi.org/10.1016/j.bmc.2014.12.043
0968-0896/Ó 2014 Published by Elsevier Ltd.
Please cite this article in press as: Digiacomo, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.043

2
M. Digiacomo et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
Figure 1. General structures of multifunctional NO-donor/insulin secretagogue derivatives NO-Rep and NO-Nat.
ability to release exogenous NO was considered as an intriguing
pharmacological approach, aimed at counterbalancing the reduced
availability of endogenous NO in the diabetic patients, and thus at
attenuating the diabetes-associated cardiovascular complications.
Indeed, the meglitinides repaglinide (REP) and nateglinide (NAT)
are non-sulfonylurea insulin secretagogue drugs, effective in treat-
ing type 2 diabetes.12 Like sulfonylureas, these drugs stimulate
insulin secretion by blocking the ATP-sensitive potassium channels
(KATP) in pancreatic b cells, thus improving overall glycemic con-
trol. REP and NAT inhibit also KATP channels of cardiomyocytes,
and vascular smooth muscle cells,13,14 and this may contribute to
the onset of cardiovascular complications.15
As a further development of our work on the design of new NO-
donor antidiabetics,8 we describe a new class of NO-donor hybrids
obtained by coupling REP and NAT with appropriate NO-releasing
moieties,16 aiming at improving the pharmacological profile
(Fig. 1). In particular, this paper describes the synthesis of new
multifunctional insulin-secretagogue/NO-donor derivatives and
the evaluation of both the NO-mediated vasorelaxing effects on
isolated rat aortic rings and the in vivo hypoglycemic properties.
Moreover, the cardioprotective and hypoglycemic activities of the
most active compound 4 (namely, NO-NAT) were evaluated.
an in vivo hydrolysable ester bond. The NO-releasing groups
involved in this study have already been used for the synthesis
of other class of NO-donor drugs.16 In particular 3-nitrooxymeth-
ylbenzyl alcohol-(8a), 4-nitrooxymethylbenzyl alcohol-(8b),
3-[1-(nitrooxy)ethyl]benzyl alcohol, and 4-[1-(nitrooxy)ethyl]benzyl
alcohol were chosen as linker groups because of their different
NO-releasing rate.16 The NO donor-meglitinide derivatives
NO-REP (1,2) and NO-NAT (3,4), were synthesized by condensation
of REP or NAT, respectively, with the appropriate nitrooxymethylb-
enzyl alcohol (8a,b)17 in the presence of DCC and a catalytic
amount of DMAP in DCM (Scheme 1). The meta- and para-[(1-
nitrooxy)ethyl]benzyloxy derivatives 5 and 6 were prepared, as
shown in Scheme 2. The 3-(1-hydroxyethyl)benzyl alcohol (9a)
or 4-(1-hydroxyethyl)benzyl alcohol (9b), obtained by reduction
of the appropriate acetyl benzoic acid, was condensed with REP
in the presence of DCC and a catalytic amount of DMAP affording
the product (10,11). The subsequent reaction of the alcohol 10,11
with HCl in toluene gave the corresponding chloride and the nitra-
tion with AgNO3 afforded to the products 5 and 6. Compound 7
was obtained starting from NAT and following the same synthetic
procedure described above for compounds 5 and 6.
2.2. Pharmacology
2. Results and discussion
2.1. Chemistry
2.2.1. Evaluation of NO-releasing properties
The prodrugs 2 and 4 induced almost full vasorelaxing effects
(Emax = 94 4 and 93 1, for 2 and 4, respectively; Table 1) which
were strongly inhibited by 1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-
The new compounds were prepared by the coupling of the car-
boxylic function of REP and NAT with a NO-donor moiety through
1-one (ODQ) 1 lM, an inhibitor of guanylate cyclase, as expected in
Scheme 1. Synthetic route for the preparation of the NO-releasing/insulin secretagogues hybrids 1–4. Reagents and conditions: (a) DCC, DMAP, DCM, 3 h, rt.
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M. Digiacomo et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
3
N
N
N
OH
b
c
H
N
H
N
H
N
HO
OH
Cl
+
O
OH
O
O
O
O
O
O
O
O
O
O
9a,b
10,11
12,13
a
d
O
N
HO
H
N
ONO2
Me
O
O
O
O
O
5,6
Cl
OH
O
O
OH
O
OH
O
b
c
O
N
N
H
N
H
H
O
HO
+
O
O
14
15
9a
d
ONO2
O
O
N
H
O
7
Scheme 2. Synthetic route for the preparation of the NO-releasing/meglitinides hybrids 5–7. Reagents and conditions: (a) LiAlH4 1 M, THF, 0 °C (yield >90%); (b) DCC, DMAP,
DCM, 3 h, rt (yield >90%); (c) HClconcd, 20 h, rt (yield: 60–80%); (d) AgNO3, MeCN, 4 h, rt (yield 16–25%).
Table 1
levels, following the administration of glucose. The glycemic pat-
tern shown by diabetic animals submitted to acute pre-treatment
The NO-mediated vasorelaxing efficacy (Emax%) and potency values (pIC50) of 1–7
Compd
pIC50
Emax
with NAT or with an equivalent dose of 4 was almost superimpos-
able to that exhibited by normoglycemic animals, indicating
that NAT and the hybrid compound 4 are endowed with almost
equivalent insulin-secretagogue activity (Fig. 2).
1
2
3
4
5
6
7
4.88 0.03
5.09 0.03
n.c.a
79
94
45
93
26
84
58
4
1
2
4
6
3
6
5.30 0.04
n.c.a
2.2.3. Cardio-protective anti-ischemic effects
5.30 0.04
4.63 0.03
In order to evaluate if the new multifunctional insulin-
secretagogue/NO-donor derivative 4 was provided of additional
beneficial cardiovascular activity, the effect induced by 4 on
Langendorff-perfused diabetic rat hearts subjected to an ische-
mia-reperfusion cycle was evaluated. As indicated in Figure 3,
the ischemia/reperfusion injury in the hearts from diabetic rats
(decrease of RPP and presence of ischemic areas) was almost
equivalent to that observed in hearts from normoglycemic animals.
Pre-treatment with nateglinide determined a slight and not signif-
The parameters of efficacy and potency are expressed as mean SEM.
The pIC50 value was not calculable (n.c.) because of the low vasorelaxing effi-
cacy (lower than 50%).
a
the case of an NO-mediated effect. The shifting of the nitrooxym-
ethyl-group from the para (2,4) to the meta position (1,3) in both
NAT and REP derivatives reduced the vasorelaxing efficacy; such
a reduction is more marked in the NAT-derivatives with respect
to the REP-derivatives (Table 1). The same effect has been also
observed for 5–7 compounds in which a methyl group has been
added at the alpha-position of nitrooxy-group.
icant improvement of the parameters. Pre-treatment with
4
promoted a cardio-protective effect, determining an evident (albeit
not statistically significant) reduction of the ischemic areas and a
marked and a statistically significant (P <0.01) improvement of
the post-ischemic recovery of functional parameter (RPP).
2.2.2. Evaluation of hypoglycemic properties
Since the essential issue for an insulin-secretagogue/NO-donor
hybrid is the retention of its hypoglycemic activity, we selected
the NO-NAT derivative 4, which showed the best vasorelaxing pro-
file, to further investigate its activity on the glucose metabolism. As
shown in Figure 2, when compared to the normoglycemic animals,
the diabetic rats showed a significantly higher increase of glycemic
3. Conclusion
In the last decade, the development of ‘chimeric drugs’ has been
one of the most exciting fields of investigation. The addition of
NO-releasing properties has been a widely used strategy in the
design of such ‘chimeras’, in order to improve the overall
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4
M. Digiacomo et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
NO-releasing properties to antidiabetic agents seems to be a win-
ning strategy for projecting ‘chimeric’ drugs able to target both
the metabolic dysfunction and, at the same time, the vascular com-
plications.2,8 Indeed, this work reported effective NO-antidiabetic
molecules, combining the hypoglycemic activity of meglitinides
and NO-mediated cardiovascular properties, due to the conjuga-
tion of the insulin secretagogue drugs NAT and REP with oppor-
tunely spaced nitrooxy function, which is one of the most
versatile and reliable NO-releasing moiety. In particular, the
NO-NAT derivative 4, selected as the lead compound in this exper-
imental work, was endowed of satisfactory hypoglycemic effects,
comparable to those exhibited by the ‘parent drug’ NAT. Moreover,
the presence of the NO-releasing feature conferred on this dual
drug vasorelaxing and cardioprotective effects, which seem to be
an extremely useful improvement of the pharmacological profile.
Besides the improved cardiovascular profile, it is noteworthy that
the risk of excessive hypoglycaemia (i.e., the typical side-effect of
insulin-secretagogues) is likely to be lower in meglitinides than
in sulfonylureas.18 However, no specific test has been carried out
in this study. The evaluation of the possible toxicity of the new
NO-meglitinides reported in this study, as well as the investigation
of further NO-mediated useful effects, such as the antiplatelet and
the antihypertensive ones, will be the object of future experimen-
tal work.
4. Experimental section
4.1. Chemistry
1H NMR spectra of all compounds were obtained with a Gemini
200 spectrometer operating at 200 MHz (for compounds 6, 9–15)
or with a Bruker TopSpin 3.2 400 MHz spectrometer (for com-
pounds 1–5, 7). 13C NMR spectra were fully decoupled. The follow-
ing abbreviations are used: singlet (s), doublet (d), triplet (t),
multiplet (m) and broad signal (br s). Chemical shifts (d) are
reported in parts per million downfield from tetramethylsilane
and referenced from solvent references. Analytical TLCs were car-
ried out on 0.25 mm layer silica gel plates containing a fluorescent
indicator; spots were detected under UV light (254 nm). Column
Figure 2. (a) Vasorelaxing effect (%) of 4 alone and in the presence of ODQ. (b)
Hypoglycemic effects of 4. Non-fasting glycemic levels recorded in normoglycemic
animals and in streptozotocin-induced diabetic rats after intraperitoneal adminis-
tration of Nateglinide (50 mg/kg, ip), or an equimolar dose of 4 (75.8 mg/kg ip) or
vehicle.
pharmacotherapeutic impact of a given drug and/or to reduce the
adverse effects.9,10 Since the reduced biosynthesis of endogenous
NO is a typical feature in diabetes (leading to the well-known dia-
betes-associated cardiovascular complications),1 the addition of
Figure 3. (a) Functional recovery (RPP %) and (b) morphological evidence of myocardial injury (Ai/Avs %) recorded in Langendorff-perfused hearts exposed to ischemia-
reperfusion. The hearts were isolated from normoglycemic and diabetic rats, submitted to different pharmacological treatments. The data are expressed as mean SEM.
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M. Digiacomo et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
5
chromatography was performed using 70–230 mesh silica gel.
Infrared (IR) spectra were obtained using a Agilent 660 with ATR
Pike spectrophotometer. Data are presented as frequency of
absorption (cmꢀ1). Mass spectra were obtained on a Hewlett-
Packard 5988 A spectrometer using a direct injection probe and
an electron beam energy of 70 eV. Evaporation was performed in
vacuo (rotating evaporator); sodium sulfate was always used as
drying agent. Elemental analyses were performed in our analytical
laboratory and agreed with the theoretical values to within 0.4%.
organic layer evaporated to give a crude residue which was puri-
fied by column chromatography eluting with hexane/AcOEt (7:3).
The product was then crystallized from AcOEt/hexane obtained
compound 3 (98 mg, 0.20 mmol, 32%). Mp: 93–95 °C, 1H NMR
(CDCl3):
d 0.85 (d, 6H, J = 4.0 Hz, CH3), 0.94–1.04 (m, 3H,
cyclohexyl), 1.25–1.42 (m, 3H, cyclohexyl), 1.75–2.03 (m, 5H,
cyclohexyl), 3.07–3.17 (m, 2H, CH2Ph), 4.90–4.96 (m, 1H, CHNH),
5.11–5.18 (m, 2H, CH2O), 5.42 (s, 2H, CH2ONO2), 5.87 (d, 1H,
J = 8.0 Hz, NH), 6.95–7.05 (m, 2H, Ar), 7.21–7.39 (m, 7H, Ar). 13C
NMR (CDCl3):
d 19.85 (CH3), {29.02, 29.10, 29.63, 29.88}
4.1.1. 3-Nitrooxymethyl-benzoate of repaglinide 1
(cyclohexyl), 32.89 (CH), 38.03 (CH2Ph), 43.34 (CH), 45.60 (CHCO),
52.89 (CHCOO), 66.74 (CH2O), 74.48 (CH2ONO2), {127.23, 128.66,
129.14, 129.24, 129.36, 129.45, 129.67, 132.87, 135.87, 136.09}
A solution of repaglinide (300 mg, 0.66 mmol), 3-(nitrooxym-
ethyl)-benzylalcohol 8a (121 mg, 0.66 mmol), DCC (164 mg,
0.79 mmol) and DMAP (7 mg) in CH2Cl2 (13 mL) was stirred at
room temperature for 3 h. The suspension was then filtered off
and the solvent evaporated to give a crude residue which was puri-
fied by column chromatography eluting with hexane/AcOEt (7:3).
The solid obtained was then mashed with hexane provided com-
pound 1 (162 mg, 0.26 mmol, 39%). 1H NMR (CDCl3): d 0.91 (d,
6H, J = 4.0 Hz, CH3), 1.37 (t, 3H, J = 7.0 Hz, OCH2CH3), 1.49–1.70
(m, 9H, CH2, CH), 2.54–2.65 (m, 2H, CH2N), 2.86–2.97 (m, 2H,
CH2N), 3.53 (s, 2H, CH2CONH), 3.94–4.07 (m, 2H, OCH2CH3),
5.30–5.38 (m, 1H, CHNH), 5.34 (s, 2H, CH2O), 5.43 (s, 2H,
CH2ONO2), 6.69 (d, 1H, J = 8.6 Hz, Ar), 6.78–6.86 (m, 2H, Ar),
7.05–7.07 (m, 2H, Ar), 7.16–7.19 (m, 2H, Ar), 7.37–7.48 (m, 4H,
Ar), 7.78 (d, 1H, J = 7.7 Hz, NH). 13C NMR (CDCl3): d 14.73 (CH3),
22.62 (CH3), 22.86 (CH), 24.22 (CH2), 25.43 (CH2, pip), 26.84
(CH2, pip), 44.32 (CH2CO), 46.77 (CH2O), 49.90 (CHNH), 64.60
(CH2N, pip), 66.02 (OCH2), 74.64 (CHONO2), {113.91, 118.70,
120.92, 122.90, 125.17, 127.48, 128.03, 128.65, 128.78, 129.15,
129.23, 132.44, 132.61, 137.25, 138.75, 141.70, 152.61, 159.12}
(Ar), 166.03 (COO), 168.81 (CONH) ppm. Anal. (C35H43N3O7) C, H,
N Calcd: 68.07 (C); 6.97 (H); 6.81 (N). Found: 68.28 (C); 7.23 (H);
6.77 (N).
(Ar), 171.76 (CONH), 175.78 (COO) ppm.
[a] (in MeOH
1.0%) = ꢀ1.7. Anal. (C27H34N2O6) C, H, N. Calcd: 67.20 (C); 7.10
(H); 5.80 (N). Found: 67.08 (C); 6.87 (H); 5.55 (N).
4.1.4. 4-(Nitroxymethyl)benzoate of nateglinide 4
The nateglinide 4-(nitrooxymethyl)benzoate was synthesized
from 4-(nitrooxymethyl)benzylalcohol 8b (231 mg, 1.26 mmol)
following the same procedure described above for the preparation
of the nateglinide 3-(nitrooxymethyl)benzoate (3). This compound
was purified by column chromatography eluting with AcOEt/hex-
ane (8:2) and then by precipitation with n-hexane from AcOEt to
give compound 4 (140 mg, 0.29 mmol, 23%). Mp: 115–117 °C, IR
(neat):
m
3299 (NH), 2928 (Ar), 1737 (CO), 1640 (NO2), 1277
;
(NO2) cmꢀ1
1H NMR (CDCl3): d 0.85 (d, 6H, J = 4.0 Hz, CH3),
0.90–1.06 (m, 3H, cyclohexyl), 1.26–1.42 (m, 3H, cyclohexyl),
1.75–1.78 (m, 2H, cyclohexyl), 1.83–1.89 (m, 2H, cyclohexyl),
1.96–2.04 (m, 1H, CH), 3.06–3.17 (m, 2H, CH2Ph), 4.90–4.95 (m,
1H, CHNH), 5.12 (d, 1H, J = 14.0 Hz, CH2O), 5.17 (d, 1H,
J = 14.0 Hz, CH2O), 5.43 (s, 2H, CH2ONO2), 5.89 (d, 1H, J = 8.0 Hz,
NHCH), 6.99–7.01 (m, 2H, Ar), 7.21–7.23 (m, 3H, Ar), 7.32 (d, 2H,
J = 8.0 Hz, AA0XX0), 7.39 (d, 2H, J = 8.0 Hz, AA0XX0) ppm. 13C NMR
(CDCl3): d 19.89 (CH3), {29.15, 29.22, 29.75, 30.00} (cyclohexyl),
33.01 (CH), 38.11 (CH2Ph), 43.46 (CH), 45.72 (CHCO), 53.00
(CHCOO), 66.79 (CH2O), 74.51 (CH2ONO2), {127.36, 128.78,
129.13, 129.51, 129.57, 132.81, 135.98, 136.77} (Ar), 171.86
(CONH), 175.90 (COO) ppm. MS (m/z): 482 (M+, 70%), 439
(M+ꢀ(CH3)2CH, 100%). Anal. (C27H34N2O6) C, H, N. Calcd: 67.20
(C); 7.10 (H); 5.80 (N). Found: 67.14 (C); 6.99 (H); 5.67 (N).
4.1.2. 4-Nitrooxymethyl-benzoate of repaglinide 2
The repaglinide 4-(nitrooxymethyl)benzoate was synthesized
from 4-(nitrooxymethyl)benzylalcohol 8b (121 mg, 0.66 mmol)
following the same procedure described above for the preparation
of the repaglinide 3-(nitrooxymethyl)benzoate (1). This compound
was purified by column chromatography eluting with hexane/
AcOEt (7:3) and then by precipitation with n-hexane from AcOEt
to give compound 2 (154 mg, 0.25 mmol, 38%). FTIR (neat)
3273 (NH), 2931 (Ar), 1699 (CO), 1640 (NO2), 1277 (NO2), 1252
(OEt) cmꢀ1 1H NMR (CDCl3): d 0.91 (d, 6H, J = 4.0 Hz, CH3), 1.37
m
4.1.5. 3-[1-(Nitrooxy)ethyl]benzoate of repaglinide 5
AgNO3 (201 mg, 1.19 mmol) was added to a stirred solution of
of 3-[1-(chloroethyl]benzoate of repaglinide (12) (184 mg,
0.30 mmol) in CH3CN (1.1 mL). Stirring was continued over 4 h at
rt in the dark, and then the precipitate (silver chloride) was filtered
off and the solvent was evaporated. The crude product was purified
by column chromatography eluting with hexane/AcOEt (7:3) to
give 5 (30 mg, 0.05 mmol, 16% yield) as a white solid: 1H NMR
(CDCl3): d 0.91 (d, 6H, J = 4.0 Hz, CH3), 1.22–1.26 (m, 2H, CH2),
1.37 (t, 3H, J = 7.0 Hz, OCH2CH3), 1.50–1.70 (m, 10H, CH2, CH,
CH3), 2.55–2.65 (m, 2H, CH2N), 2.86–2.97 (m, 2H, CH2N), 3.53 (s,
2H, CH2CONH), 3.97–4.06 (m, 2H, OCH2CH3), 5.30–5.42 (m, 1H,
CHNH), 5.34 (s, 2H, CH2O), 5.94 (q, 1H, J = 4.0 Hz, CHCH3), 6.71
(d, 1H, J = 8.0 Hz, Ar), 6.81–6.86 (m, 2H, Ar), 7.05–7.07 (m, 2H,
Ar), 7.16–7.22 (m, 2H, Ar), 7.33–7.46 (m, 4H, Ar), 7.79 (d, 1H,
J = 8.0 Hz, NH) ppm. 13C NMR (CDCl3): d 14.79 (CH3), 20.52 (CH3),
22.68 (CH3), 22.91 (CH2), 24.26 (CH), 25.47 (CH2, pip), 26.90
(CH2, pip), 29.85 (CH), 44.43 (CH2CO), 46.80 (CH2O), 64.66 (CH2N,
pip), 66.19 (OCH2), 81.91(CHONO2), {113.95, 120.98, 122.82,
125.26, 125.98, 126.03, 127.75, 127.77, 128.09, 128.82, 129.21,
132.50, 137.20, 138.77, 139.17, 141.67, 152.66, 159.16} (Ar),
165.96 (COO), 168.70 (CONH). Anal. (C31H36N2O7) C, H, N. Calcd:
68.44 (C); 7.18 (H); 6.65 (N). Found: 68.57 (C); 7.46 (H); 6.98 (N).
;
(t, 3H, J = 8.0 Hz, OCH2CH3), 1.49–1.59 (m, 9H, CH2, CH),
2.57–2.65 (m, 2H, CH2N), 2.86–2.98 (m, 2H, CH2N), 3.53 (s, 2H, CH2-
CONH), 3.95–4.10 (m, 2H, OCH2CH3), 5.30–5.39 (m, 1H, CHNH),
5.34 (s, 2H, CH2O), 5.43 (s, 2H, CH2ONO2), 6.73–6.74 (m, 1H, Ar),
6.82–6.86 (m, 2H, Ar), 7.05–7.08 (m, 2H, Ar), 7.18–7.20 (m, 2H,
Ar), 7.40 (d, 2H, J = 8.0 Hz, AA0XX0), 7.49 (d, 2H, J = 8.0 Hz, AA0XX0),
7.78 (d, 1H, J = 8.0 Hz, NH). 13C NMR (CDCl3): d 14.79 (CH3), 22.66
(CH3), 22.88 (CH), 24.25 (CH2), 25.46 (CH2, pip), 26.87 (CH2, pip),
44.38 (CH2CO), 46.79 (CH2O), 49.96 (CHNH), 64.63 (CH2N, pip),
66.98 (OCH2), 74.55 (CHONO2), {113.90, 118.74, 120.94, 122.95,
125.20, 127.82, 128.05, 128.51, 129.32, 132.11, 132.48, 137.90,
138.78, 141.70, 152.64, 159.15} (Ar), 166.04 (COO), 168.78 (CONH)
ppm. Anal. (C35H43N3O7) C, H, N. Calcd: 68.07 (C); 6.97 (H); 6.81
(N). Found: 67.82 (C); 6.68 (H); 6.45 (N).
4.1.3. 3-Nitrooxymethylbenzoate of nateglinide 3
A
solution of nateglinide (200 mg, 0.63 mmol) in CH2Cl2
(12 mL), 3-(nitroxymethyl)benzylalcohol 8a (115 mg, 0.63 mmol),
DCC (156 mg, 0.75 mmol) and DMAP (6 mg) was stirred at room
temperature for 3 h. The mixture was then filtered off and the
Please cite this article in press as: Digiacomo, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.043

6
M. Digiacomo et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
4.1.6. 4-[1-(Nitrooxy)ethyl]benzoate of repaglinide 6
4.1.10. 3-[1-(Hydroxyethyl]benzoate of repaglinide 10
AgNO3 (827 mg, 4.87 mmol) was added to a stirred solution of
of 4-[1-(chloroethyl]benzoate of repaglinide (11) (752 mg,
1.24 mmol) in CH3CN (10 mL). Stirring was continued over 4 h at
rt in the dark, and then the precipitate (silver chloride) was filtered
off and the solvent was evaporated. The crude product was purified
by column chromatography eluting with hexane/AcOEt (7:3) to
give 6 (156 mg, 0.25 mmol, 20% yield): 1H NMR (CDCl3): d 0.91
(d, 6H, J = 6.7 Hz, CH3); 1.25–1.64 (m, 12H, CH2, CH, CH3); 1.37 (t,
3H, J = 7.0 Hz, OCH2CH3); 2.55–2.65 (m, 2H, CH2N); 2.86–2.97 (m,
2H, CH2N); 3.53 (s, 2H, CH2CONH); 3.94–4.06 (m, 2H, OCH2CH3);
5.30–5.42 (m, 1H, CHNH); 5.33 (s, 2H, CH2O); 5.93 (q, 1H,
J = 6.7 Hz, CHCH3); 6.70 (d, 1H, J = 8.1 Hz, Ar); 6.81–6.85 (m, 2H,
Ar); 7.02–7.10 (m, 2H, Ar); 7.16–7.50 (m, 6H, Ar); 7.79 (d, 1H,
J = 7.7 Hz, NH). 13C NMR (CDCl3): d 14.87 (CH3), 22.75 (CH3),
22.97 (CH3), 24.39 (CH2), 25.63 (CH2, pip), 26.99 (CH2, pip), 34.15
(CH), 44.94 (CH2CO), 46.93 (CH2O), 49.97 (CHNH), 55.26 (CH2N,
pip), 58.57 (OCH2), 64.87 (CHONO2), {113.54, 114.23, 120.99,
122.84, 125.19, 125.61, 126.41, 126.48 127.27, 127.78, 128.03,
128.42, 132.35, 138.90} (Ar), 159.50 (COO) 169.00 (CONH). Anal.
(C31H36N2O7) C, H, N. Calcd: 68.44 (C); 7.18 (H); 6.65 (N). Found:
68.72 (C); 7.39 (H); 6.74 (N).
To a solution of 3-(1-hydroxyethyl)benzyl alcohol (9a) (168 mg,
1.11 mmol) in CH2Cl2 (22 mL) was added repaglinide (500 mg,
1.11 mmol), DCC (273 mg, 1.32 mmol) and DMAP (11 mg). The
resulting suspension was stirred at rt for about 4 h, then the pre-
cipitate was removed by filtration and the filtrate was concen-
trated, to give 10 (583 mg, 0.99 mmol, 90% yield) as a colorless
oil: 1H NMR (CDCl3): d 0.90 (d, 6H, J = 6.2 Hz, CH3); 1.37 (t, 3H,
J = 7.0 Hz, OCH2CH3); 1.48–1.89 (m, 12H, CH2, CH, CH3);
2.54–2.65 (m, 2H, CH2N); 2.86–2.97 (m, 2H, CH2N); 3.52 (s, 2H,
CH2CONH); 3.94–4.10 (m, 2H, OCH2CH3); 4.91 (q, 1H, J = 6.2 Hz,
CHCH3); 5.28–5.38 (m, 1H, CHNH); 5.33 (s, 2H, CH2O); 6.68 (d,
1H, J = 8.2 Hz, Ar); 6.80–6.84 (m, 2H, Ar); 7.05–7.44 (m, 8H, Ar);
7.70–7.80 (m, 1H, NH). Anal. (C36H46N2O5) C, H, N. Calcd: 73.69
(C); 7.90 (H); 4.77 (N). Found: 73.58 (C); 7.95 (H); 5.05 (N).
4.1.11. 4-[1-(Hydroxyethyl]benzoate of repaglinide 11
To a solution of 4-(1-hydroxyethyl)benzyl alcohol (235 mg,
1.55 mmol) in CH2Cl2 (5 mL) was added repaglinide (700 mg,
1.55 mmol), DCC (383 mg, 1.86 mmol) and DMAP (15 mg). The
resulting suspension was stirred at rt for about 4 h, then the pre-
cipitate was removed by filtration and the filtrate was concen-
trated, to give 11 (817 mg, 1.39 mmol, 90% yield) as a colorless
oil: 1H NMR (CDCl3): d 0.90 (d, 6H, J = 6.3 Hz, CH3); 1.37 (t, 3H,
J = 6.6 Hz, OCH2CH3); 1.16–1.80 (m, 12H, CH2, CH, CH3);
2.54–2.65 (m, 2H, CH2N); 2.86–2.97 (m, 2H, CH2N); 3.52 (s, 2H,
CH2CONH); 3.93–4.05 (m, 2H, OCH2CH3); 4.91 (q, 1H, J = 6.3 Hz,
CHCH3); 5.29–5.42 (m, 1H, CHNH); 5.31 (s, 2H, CH2O); 6.71 (d,
1H, J = 8.4 Hz, Ar); 6.79–6.83 (m, 2H, Ar); 7.01–7.10 (m, 2H, Ar);
7.15–7.23 (m, 2H, Ar); 7.35–7.44 (m, 4H, Ar); 7.77 (m,1H, NH).
Anal. (C36H46N2O5) C, H, N. Calcd: 73.69 (C); 7.90 (H); 4.77 (N).
Found: 73.47 (C); 8.02 (H); 4.89 (N).
4.1.7. 4-[1-(Nitrooxy)ethyl]benzoate of nateglinide 7
AgNO3 (991 mg, 5.84 mmol) was added to a stirred solution of
of 4-[1-(chloroethyl]benzoate of nateglinide (15) (700 mg,
1.49 mmol) in CH3CN (8 mL). Stirring was continued over 4 h rt
in the dark, and then the precipitate (silver chloride) was filtered
off and the solvent was evaporated. The crude product was purified
by column chromatography eluting with hexane/AcOEt (7:3) to
give 7 (185 mg, 0.37 mmol, 25% yield) as a white solid: mp: 80–
82 °C, 1H NMR (CDCl3): d 0.85 (d, 6H, J = 4.0 Hz, 2CH3), 0.91–1.06
(m, 3H, cyclohexyl), 1.33–1.42 (m, 3H, cyclohexyl), 1.63 (d, 3H,
J = 4.0 Hz, CH3), 1.75–1.78 (m, 2H, cyclohexyl), 1.83–1.88 (m, 2H,
cyclohexyl), 1.96–2.03 (m, 1H, CHCH3), 3.07–3.17 (m, 2H, CH2Ph),
4.90–4.96 (m, 1H, CHNH), 5.12 (d, 1H, J = 14.0 Hz, CH2O), 5.17 (d,
1H, J = 14.0 Hz, CH2O), 5.89 (d, 1H, J = 8.0 Hz, NHCH), 5.94 (q, 1H,
J = 4.0 Hz, CHONO2), 6.96–7.01 (m, 2H, Ar), 7.21–7.22 (m, 3H, Ar);
7.31 (d, 2H, J = 8.2 Hz, AA0XX0); 7.38 (d, 2H, J = 8.2 Hz, AA0XX0)
ppm. 13C NMR (CDCl3): d 19.91 (CH3), 20.54 (CH3), {29.08, 29.16,
29.68, 29.94} (cicloesil), 32.95 (CH), 38.04 (CH2Ph), 43.40 (CH),
45.66 (CH-CONH), 52.91 (CHCOO), 66.79 (CH2O), 81.71 (CHONO2),
{126.67, 127.28, 128.69, 129.17, 129.52, 135.92, 136.11, 139.29}
(Ar), 171.80 (CONH), 175.80 (COO) ppm. Anal. (C28H36N2O6) C, H,
N. Calcd: 67.34 (C); 6.91 (H); 5.82 (N). Found: 67.58 (C); 6.95
(H); 6.05 (N).
4.1.12. 3-[1-(Chloroethyl]benzoate of repaglinide 12
HClconcd (0.32 mL) was added at room temperature to a stirred
suspension of 3-[1-(hydroxyethyl]benzoate of repaglinide (10)
(356 mg, 0.61 mmol) in toluene (3 mL). The resulting solution
was stirred for 20 h at room temperature. Then, the solvent was
evaporated to afford 12 as a colorless oil (295 mg, 0.48 mmol,
80% yield): 1H NMR (CDCl3): d 0.87–0.99 (m, 6H, CH3); 1.20–2.11
(m, 15H, CH2, CH, CH3); 2.62–2.70 (m, 2H, CH2N); 3.07–3.43 (m,
2H, CH2N); 3.67–3.82 (m, 2H, CH2CONH); 4.00–4.17 (m, 2H, OCH2-
CH3); 4.60–4.71 (m, 1H, CHNH); (q, 1H, J = 7.0 Hz, CHCH3); 5.30
(s, 2H, CH2O); 6.70–6.73 (m, 1H, Ar); 6.90–7.68 (m, 10H, Ar);
7.89–8.00 (m,1H, NH). Anal. (C36H45Cl N2O4) C, H, N. Calcd: 71.44
(C); 7.49 (H); 4.63 (N). Found: 71.56 (C); 7.71 (H); 4.80 (N).
4.1.8. 3-(1-Hydroxyethyl)benzyl alcohol 9a
4.1.13. 4-[1-(Chloroethyl]benzoate of repaglinide 13
A solution of 3-acetylbenzoic acid (500 mg; 3.05 mmol) in THF
(3 mL) was added to a solution of LiAlH4 1 M in THF (230 mg;
6.09 mmol) cooled at 0 °C. The mixture was stirred at 0 °C for
12 h, then water (1.7 mL) and NaOH 1 M (0.4 mL) was added, and
the resulting suspension filtrated. The solvent was evaporated to
give 9a (432 mg, 2.84 mmol, 93% yield) as a yellow oil: 1H NMR
(CDCl3): d 1.46 (d, 3H, J = 6.5 Hz, CH3); 4.63 (s, 2H, CH2OH); 4.85
(q, 1H, J = 6.5 Hz, CH); 7.20–7.35 ppm (m, 4H, Ar).
HClconcd (1 mL) was added at room temperature to a stirred sus-
pension of 4-[1-(hydroxyethyl]benzoate of repaglinide (11) (1.12 g,
1.91 mmol) in toluene (7 mL). The resulting solution was stirred for
20 h at room temperature. Then, the solvent was evaporated to
afford 13 as a colorless oil (752 mg, 1.24 mmol, 65% yield): 1H
NMR (CDCl3): d 0.85–0.99 (m, 6H, CH3); 1.30–2.08 (m, 15H,
CH2, CH, CH3); 2.58–2.68 (m, 2H, CH2N); 3.00–3.08 (m, 2H,
CH2N); 3.75 (s, 2H, CH2CONH); 3.94–4.08 (m, 2H, OCH2CH3);
4.60–4.71 (m, 1H, CHNH); 5.08 (q, 1H, J = 6.8 Hz, CHCH3); 5.28 (s,
2H, CH2O); 6.71–6.74 (d, 1H, J = 6.2 Hz, Ar); 6.89 (d, 1H,
J = 7.7 Hz, Ar); 7.08 (s, 1H, Ar); 7.17–7.66 (m, 8H, Ar); 7.83–7.86
(m, 1H, NH). Anal. (C36H45Cl N2O4) C, H, N. Calcd: 71.44 (C); 7.49
(H); 4.63 (N). Found: 71.22 (C); 7.19 (H); 4.53 (N).
4.1.9. 4-(1-Hydroxyethyl)benzyl alcohol 9b
A solution of 4-acetylbenzoic acid (1 g; 6.10 mmol) in THF
(6 mL) was added to a solution of LiAlH4 1 M in THF (691 mg;
18.29 mmol) cooled at 0 °C. The mixture was stirred at 0 °C for
12 h, then water (5 mL) and NaOH 1 M (1.3 mL) was added, and
the resulting suspension filtrated. The solvent was evaporated to
give 9b (899 mg, 5.92 mmol, 97% yield) as a yellow oil: 1H NMR
(CDCl3): d 1.48 (d, 3H, J = 6.4 Hz, CH3); 4.66 (s, 2H, CH2OH); 4.88
(q, 1H, J = 6.4 Hz, CH); 7.29–7.38 ppm (m, 4H, Ar).
4.1.14. 4-[1-(Hydroxyethyl]benzoate of nateglinide 14
To a solution of 4-(1-hydroxyethyl)benzyl alcohol (335 mg,
2.21 mmol) in CH2Cl2 (5 mL) was added nateglinide (700 mg,
2.21 mmol), DCC (547 mg, 2.65 mmol) and DMAP (22 mg). The
Please cite this article in press as: Digiacomo, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.043

M. Digiacomo et al. / Bioorg. Med. Chem. xxx (2015) xxx–xxx
7
resulting suspension was stirred at rt for about 4 h, then the
precipitate was removed by filtration and the filtrate was concen-
trated, to give 14 (966 mg, 2.14 mmol, 97% yield) as a colorless oil:
1H NMR (CDCl3): d 0.84 (d, 6H, J = 6.8 Hz, 2CH3), 0.96–1.01 (m, 2H,
CH2), 1.29–1.58 (m, 4H, CH2, 2CH), 1.50 (d, 3H, J = 6.4 Hz, CH3);
1.65–2.00 (m, 5H, 2CH2, CH), 3.04–3.21 (m, 2H, CH2Ph), 4.86–
4.94 (m, 1H, CHNH), 5.06–5.19 (m, 3H, CH2O, CHOH), 5.89 (d, 1H,
J = 7.9 Hz, NH), 6.95–7.02 (m, 2H, Ar), 7.07–7.41 (m, 7H, Ar). Anal.
(C28H37NO4) C, H, N. Calcd: 74.47 (C); 8.26 (H); 3.10 (N). Found:
74.36 (C); 8.11 (H); 2.98 (N).
reported as increase from the basal one. The corresponding areas
under curve (AUC) were also calculated.
4.2.3. Evaluation of cardio-protective anti-ischemic effects
Male Wistar rats (250–300 g) were treated with streptozotocin
(50 mg/kg ip). After 4 days, animals showing a fasting glycemic
level of 100–150 mg/dl (representative of mild type 2 diabetes)
were used for the study. 11 days after the administration of strep-
tozotocin, diabetic animals were treated with Nateglinide (12 mg/
kg/day, ip), or an equimolar dose of 4 (18.2 mg/kg/day ip) or vehi-
cle, for 7 days. Vehicle-treated normoglycemic male Wistar rats
(showing fasting glycemia between 30 and 40 mg/dl) were used
as reference animals. On the day of the experiment, the animals
were heparinised and anaesthetised with sodium pentobarbital.
The hearts were rapidly removed and perfused in a Langendorff
apparatus. After 30 min of equilibration time, the hearts were sub-
mitted to a ischemia/reperfusion cycle (30 min and 120 min,
respectively). The inotropic and chronotropic parameters were
monitored with a latex balloon inserted in the left ventricle
through the mitral valve and expressed as rate pressure product
(RPP = left ventricle developed pressure x heart rate). The RPP
value recorded at the end of reperfusion (120th min) is reported
as a % of the pre-ischemic one. After 120 min of reperfusion,
2 mm-wide slices of left ventricles were treated with triphenyltet-
razolium chloride, in order to allow a planimetric evaluation of
damaged areas, expressed as ischemic area % of the whole area
(Ai/Atot).
4.1.15. 4-[1-(Chloroethyl]benzoate of nateglinide 15
HClconcd (1.2 mL) was added at room temperature to a stirred
suspension of 4-[1-(hydroxyethyl]benzoate of nateglinide (14)
(1.04 g, 2.31 mmol) in toluene (10 mL). The resulting solution
was stirred for 20 h at room temperature. Then, the solvent was
evaporated to afford 15 as a colorless oil (1.02 mg, 2.17 mmol,
94% yield): 1H NMR (CDCl3): d 0.83 (d, 6H, J = 6.6 Hz, 2CH3),
0.97–1.01 (m, 2H, CH2), 1.25–1.42 (m, 4H, CH2, 2CH), 1.58–2.00
(m, 8H, 2CH2, CH, CH3), 3.10–3.24 (m, 2H, CH2Ph), 4.81–4.96 (m,
1H, CHNH), 5.04–5.20 (m, 3H, CH2O, CHCl), 5.94 (d, 1H, J = 7.7 Hz,
NH), 6.94–7.00 (m, 2H, Ar), 7.10–7.44 (m, 7H, Ar). Anal.
(C28H36ClNO3) C, H, N. Calcd: 71.55 (C); 7.72 (H); 2.98 (N). Found:
71.68 (C); 7.84 (H); 3.09 (N).
4.2. Pharmacology
4.2.1. Evaluation of NO-releasing properties
As
a
preliminary pharmacological investigation, the
Acknowledgment
NO-mediated vasorelaxing effects of the synthesized compounds
were evaluated by functional tests on isolated endothelium-
denuded rat aortic rings.
This study has been partially supported by NICOX SpA.
As concerns the vasorelaxing responses, increasing concentra-
tions (10 nM–30 lM) of the tested compounds were added cumu-
Supplementary data
Supplementary data (1H NMR and 13C NMR spectra) of final
compounds) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmc.2014.12.043.
latively to aortic rings pre-contracted by KCl 30 mM. The index of
potency was expressed as pIC50, representing the negative Loga-
rithm of the concentration of the tested compound evoking an
half-reduction of the contractile tone induced by KCl.
The index of efficacy (Emax) indicated the maximal vasorelaxing
response induced by the highest concentration (30 lM) of the
References and notes
1. Schalkwijk, C. G.; Stehouwer, C. D. Clin. Sci. 2005, 109, 143.
2. Kaur, J.; Bhardwaj, A.; Huang, Z.; Narang, D.; Chen, T. Y.; Plane, F.; Knaus, E. E. J.
Med. Chem. 2012, 55, 7883.
3. Drexler, H.; Hornig, B. J. Mol. Cell. Cardiol. 1999, 31, 51.
4. Kawashima, S. Endothelium 2004, 11, 99.
tested compounds and expressed as a % of the contractile tone
induced by KCl. The pIC50 value could not be calculated for those
compounds, exhibiting Emax < 50%. The inhibition of guanylate
cyclase by ODQ 1 lM was used as a tool to correlate the vasorelax-
ing activity with the release of NO.
5. Torlone, E.; Rambotti, A. M.; Perriello, G.; Botta, G.; Santeusanio, F.; Brunetti, P.;
Bolli, G. B. Diabetologia 1991, 34, 119.
6. Storey, A. M.; Perry, C. J.; Petrie, J. R. Br. J. Diabetes Vasc. Dis. 2001, 1, 22.
7. Scheen, A. J. Acta Clin. Belg. 2003, 58, 3184.
4.2.2. Evaluation of hypoglycemic properties
8. 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.
9. Martelli, A.; Rapposelli, S.; Calderone, V. Curr. Med. Chem. 2006, 13, 609.
10. Martelli, A.; Breschi, M. C.; Calderone, V. Curr. Pharm. Des. 2009, 15, 614.
11. Tamboli, Y.; Lazzarato, L.; Marini, E.; Guglielmo, S.; Novelli, M.; Beffy, P.;
Masiello, P.; Fruttero, R.; Gasco, A. Bioorg. Med. Chem. Lett. 2012, 22, 3810.
12. DeFronzo, R. A. Ann. Intern. Med. 1999, 131, 281.
13. Dabrowski, M.; Wahl, P.; Holmes, W. E.; Ashcroft, F. M. Diabetologia 2001, 44,
747.
14. Stephan, D.; Winkler, M.; Kuhner, P.; Russ, U.; Quast, U. Diabetologia 2006, 49,
2039.
15. Quast, U.; Stephan, D.; Bieger, S.; Russ, U. Diabetes 2004, 53, S156.
16. Calderone, V.; Digiacomo, M.; Martelli, A.; Minutolo, F.; Rapposelli, S.; Testai,
L.; Balsamo, A. J. Pharm. Pharmacol. 2008, 60, 189.
17. Breschi, M. C.; Calderone, V.; Digiacomo, M.; Macchia, M.; Martelli, A.;
Martinotti, E.; Minutolo, F.; Rapposelli, S.; Rossello, A.; Testai, L.; Balsamo, A.
J. Med. Chem. 2006, 49, 2628.
Male Wistar rats (250–300 g) were treated with streptozotocin
(50 mg/kg ip). After 4 days, animals showing a fasting glycemic
level of 100–150 mg/dl (representative of mild type 2 diabetes)
were used for the study. Normoglycemic male Wistar rats (show-
ing fasting glycemia between 30 and 40 mg/dl) were used as refer-
ence animals. Diabetic animals were deprived of food for 24 h, and
then they received Nateglinide (50 mg/kg, ip), or an equimolar
dose of 4 (75.8 mg/kg ip) or vehicle. 10 min after drug administra-
tion, the animals received glucose (1 g/kg ip). While, normoglyce-
mic animals were deprived of food for 24 h, and then they
received only vehicle. 10 min after the drug or vehicle administra-
tion, all the animals received glucose (1 g/kg ip). The glycemic lev-
els were recorded for two h (at intervals of 0, 15, 30, 45, 60, 90 and
120 min following the glucose administration) by tail puncture
and the use of commonly used sensors (New Glucocard G sensor
and Glucocard Gmeter, Arkray, A. Menarini diagnostics), and
18. Guardado-Mendoza, R.; Prioletta, A.; Jiménez-Ceja, L. M.; Sosale, A.; Folli, F.
Arch. Med. Sci. 2013, 9, 936.
Please cite this article in press as: Digiacomo, M.; et al. Bioorg. Med. Chem. (2015), http://dx.doi.org/10.1016/j.bmc.2014.12.043