Evaluation of the NO-releasing properties of NO-donor linkers

Article abstract of DOI:10.1211/jpp.60.2.0007

This work describes the synthesis of some benzoic (1-4) and alcoholic (5-7) nitrooxy derivatives, which are nitric oxide (NO) donors in themselves, and can also be seen as useful linkers that can be used in multi-target drugs capable of releasing NO. The

Full text of DOI:10.1211/jpp.60.2.0007

JPP 2008, 60: 189–195
© 2008 The Authors
Received January 8, 2007
Accepted October 12, 2007
DOI 10.1211/jpp.60.2.0007
ISSN 0022-3573
Evaluation of the NO-releasing properties of NO-donor
linkers
Vincenzo Calderone, Maria Digiacomo, Alma Martelli, Filippo Minutolo,
Simona Rapposelli, Lara Testai and Aldo Balsamo
Abstract
This work describes the synthesis of some benzoic (1–4) and alcoholic (5–7) nitrooxy derivatives,
which are nitric oxide (NO) donors in themselves, and can also be seen as useful linkers that can be
used in multi-target drugs capable of releasing NO. The NO-mediated vasorelaxing effects of the
compounds were tested on endothelium-denuded isolated rat aortic rings pre-contracted with KCl.
The pharmacological study of these compounds demonstrated that slight structural modification,
such as the insertion of (a) methyl group(s) into the nitrooxymethyl chain or into the aromatic ring,
and a change in the position of this nitrooxymethyl chain, could exert a marked (and potentially
useful) influence on the NO releasing properties.
Introduction
The pharmacological treatment of many pathological states often requires the action of
different and complementary pharmacodynamic mechanisms. In clinical practice, this is
frequently achieved by the administration of ‘cocktails’ of drugs with different mechanisms
of action. However, the benefits of this approach are often compromised by poor compli-
ance, particularly in the treatment of asymptomatic chronic diseases such as hypertension
(Eisen et al 1990).
In the last few years, the ‘one drug–one target’ paradigm has been shelved, and numerous
multi-target drugs have been designed and synthesised. These compounds are designed to
address a particular disease, with the goal of enhancing efficacy and/or improving safety
compared with existing drugs. In this way, a single entity modulates multiple targets
simultaneously by virtue of two (or more) pharmacodynamic properties resulting from the
presence of overlapping or conjugated pharmacophores.
Compared with pharmacological cocktails, the use of a single multi-target drug presents
certain advantages, such as easier prediction of pharmacokinetic/pharmacodynamic rela-
tionships, and improved compliance (Morphy & Rankovic 2006).
The release of nitric oxide (NO) is the mechanism of action underlying the pharma-
cological features and clinical applications of drugs such as the ‘old’ vasodilator nitrites
and nitrates, reflecting the fundamental roles played by this small molecule in the cardio-
vascular system. A large number of important biological activities, principally connected
with homeostasis of the cardiovascular system (Martelli et al 2006), are mediated by the
production of endogenous NO, and could be mimicked by the administration of exoge-
nous NO through NO-releasing drugs. Among these activities, the vasorelaxing effects are
principally due to NO-induced activation of cytosolic guanylate cyclase in the vascular
smooth muscle, with a consequent rise in the intracellular concentration of cGMP, and
direct activation of potassium channels.
Dipartimento di Psichiatria,
Neurobiologia, Farmacologia
e Biotecnologie, Università di
Pisa, Via Bonanno 6, 56126
Pisa, Italy
Vincenzo Calderone, Alma
Martelli, Lara Testai
Dipartimento di Scienze
Farmaceutiche, Università di
Pisa, Via Bonanno 6, 56126
Pisa, Italy
Maria Digiacomo, Filippo
Minutolo, Simona Rapposelli,
Aldo Balsamo
Correspondence: Vincenzo
Calderone, Dipartimento di
Psichiatria, Neurobiologia,
Farmacologia e Biotecnologie,
Università di Pisa, Via Bonanno
6, 56126 Pisa, Italy. E-mail:
calderone@farm.unipi.it
In addition to its vasorelaxing effects, NO controls platelet function (Furlong et al 1987;
Radomski et al 1990). In particular, NO, which is produced by platelets as well as by
endothelial cells, reduces platelet adhesion and aggregation. More recently, a possible role
189

190
Vincenzo Calderone et al
of NO in the process of ischaemic preconditioning has been
debated extensively, and there is clear evidence that the
administration of exogenous NO donors significantly reduces
myocardial damage in ischaemia-injured hearts from different
animal species (Nakano et al 2000; Qin et al 2004).
R2
R3
O2NO
X
R
R1
This type of NO-releasing ability has recently been iden-
tified as an interesting property to be added to molecules
that already possess another fundamental pharmacodynamic
effect. This strategy has led to the development of impor-
tant multi-target drugs (Shaffer et al 1991; Minuz et al 1998;
Villarroya et al 1999; Baraldi et al 2004; Breschi et al 2006) –
interesting pharmacodynamic hybrids that combine a ‘native’
mechanism of action with NO donor activity, aiming to
reduce possible side-effects (e.g. the gastric toxicity of
aspirin) or to improve the therapeutic impact (e.g. to increase
the antiplatelet activity of aspirin, or to increase the antihy-
pertensive activity of the angiotensin receptor antagonists).
In order to obtain a multi-target drug possessing NO donor
properties, it was important to conjugate the NO function to
another drug with an easily cleavable group. The ester-based
linker is a cleavable group which, as a result of the action of
plasma esterases, is capable of releasing two individual drugs
(NO and the native drug), which act independently.
In the last few years, many cleavable conjugates have been
described (e.g. NO–aspirin, NO–sartans) (Shaffer et al 1991;
Minuz et al 1998; Villarroya et al 1999; Baraldi et al 2004;
Breschi et al 2006) containing an NO-releasing functionality
linked via an ester group to a native drug. The chemical
strategies usually employed to add NO donor properties to a
known drug involved conjugation of the drug with different
nitrogen-containing molecular portions. Among them, conju-
gation of a nitrooxy moiety, often via a molecular linker,
probably represents the most convenient approach. In order
to ensure appropriate conjugation between the NO-donor
linker and a native drug, the nitrooxy group was inserted into
different structures possessing carboxylic, phenolic or alco-
holic functions suitable for forming a vulnerable bond such
as the ester one. On this basis, and with the aim of studying
the pharmacologic properties of these NO-donor structures
and evaluating their use for building up new pharmacody-
namic hybrids, the nitrooxymethyl-benzoic acids (1–3) and
the 2-nitrooxymethyl-pyridinecarboxylic acid (4) have been
synthesised; furthermore, as hydroxy-functionalised linkers
the phenol derivative (5) and four nitrooxymethyl-benzylic
alcohols (6a, b and 7a,b) were also synthesised (Figure 1).
Compound
X
C
C
C
N
C
C
C
R
R1
H
H
Me
–
H
R2
H
H
Me
H
H
R3
1a
2a
3
4
5
H
Me
H
H
H
COOH
COOH
COOH
COOH
OH
CH2OH
CH2OH
6a
7a
H
Me
H
H
H
H
R
R2
R3
O2NO
X
R1
Compound
X
R
R1
R2
R3
1b
2b
6b
7b
C
C
C
C
H
Me
H
H
H
H
H
H
H
H
H
COOH
COOH
CH2OH
CH2OH
Me
Figure 1 NO-donor linkers with a carboxy (compounds 1a, b and 4),
phenolic (compound 5) or hydroxymethyl function (compounds 6a, 6b,
7a and 7b).
(Kieselgel 40, 0.040–0.063 mm; Merck (NJ, USA) or gravity
column (Kieselgel 60, 0.063–0.200 mm; Merck) chromatog-
raphy. Reactions were followed by thin-layer chromatography
on Merck aluminum silica gel (60 F254) sheets that were
visualised under a UV lamp. Evaporation was performed in
vacuo (rotating evaporator). Sodium sulfate was used as the
drying agent.
Synthesis
The synthesis of compounds 1–6 has been described previ-
ously (Breschi et al 2004, 2006). Figure 2 shows the general
procedures used to obtain nitrooxy derivatives. Compounds
1, 3, 5 and 6 were obtained by reaction of the corresponding
chloro-derivatives with silver nitrate in acetonitrile (method
A, Figure 2). Compounds 2a, 2b and 4 were obtained by reac-
tion with nitric acid and acetic anhydride at −10ꢀC (method
B, Figure 2). Compounds 7a and 7b were synthesised starting
from the appropriate acetylbenzoic acids (8a and 8b), which
were reduced to the corresponding benzyl alcohols (9a and
9b) with LiAlH4 in THF (Scheme 1). Treatment of a solution
of 9a and 9b in toluene with concentrated HCl afforded the
monochloro derivatives 10a and 10b, which were submitted
to reaction with AgNO3 in CH3CN to yield the corresponding
nitro esters 7a and 7b (Figure 3).
Materials and Methods
Melting points were determined on a Kofler hot-stage appa-
ratus and are uncorrected. NMR spectra were obtained with
a Varian Gemini 200 MHz spectrometer (Palo Alto, CA,
USA). Chemical shifts ꢀꢁꢂ are reported in parts per million
(ppm) downfield from tetramethylsilane and referenced from
solvent references. Mass spectra were obtained on a Hewlett-
Packard 5988 A spectrometer (Palo Alto, CA, USA) using a
direct injection probe and an electron beam energy of 70 eV.
The elemental compositions of the compounds agreed with
3-(2-Hydroxyethyl)benzyl alcohol (9a)
the calculated values to within 0ꢃ4%. Chromatographic A solution of 3-acetylbenzoic acid (8a) (500 mg; 3.05 mmol)
separation was performed on silica gel columns by flash in THF (3 mL) was added to a solution of 1 m LiAlH4 in THF

NO-releasing properties of NO-donor linkers
191
6ꢃ5Hzꢄ CHꢂ; 7.32–7.40 ppm (m, 4H, Ar). Anal. C9H12O (C,
Method A
H). Calcd: C, 71.03; H, 7.95. Found: C, 71.05; H, 7.90.2
R2
R3
R2
R3
Cl
R
O2NO
R
I
3-(2-Chloroethyl)benzyl alcohol (10a)
X
X
To a stirred suspension of compound 9a (783 mg, 5.15 mmol)
in toluene (26 mL), concentrated HCl (2.71 mL) was added
at room temperature. The resulting solution was stirred for
12 h at room temperature. The solution was then washed
with aqueous NaHCO3 and water, the organic phase was
separated and the aqueous layer extracted with CH2Cl . The
organic phase was dried and the solvent was evap2orated
to afford compound 10a (583 mg, 3.42 mmol, 66% yield)
R1
R1
1, 3, 5, 6
I: AgNO3, CH3CN, r.t.
Method B
1
as a colourless oil; H NMR ꢀCDCl3ꢂ ꢁ1ꢃ85 ꢀdꢄ 3Hꢄ J =
R2
R3
R2
R3
HO
O2NO
R
I
6ꢃ9Hzꢄ CH3ꢂꢅ 4ꢃ72 ꢀsꢄ 2Hꢄ CH2OHꢂꢅ 5ꢃ10 ꢀqꢄ 1Hꢄ J =
6ꢃ9Hzꢄ CHꢂ; 7.30–7.43 ppm (m, 4H, Ar). Anal. C9H11OCl
(C, H). Calcd: C, 63.35; H, 6.50. Found: C, 63.50; H, 6.24.
R
X
X
R1
I: HNO3 conc., Ac2O, r.t.
R1
2, 4
4-(2-Chloroethyl)benzyl alcohol (10b)
Compound 10b was synthesised from compound 9b (645 mg,
4.24 mmol) following the same procedure described above for
the preparation of 10a, to give 10b (602 mg, 3.54 mmol, 83%
yield) as a yellow oil; 1H NMR ꢀCDCl3ꢂ ꢁ1ꢃ86 ꢀdꢄ 3Hꢄ J =
6ꢃ7Hzꢄ CH3ꢂꢅ 4ꢃ71 ꢀsꢄ 2Hꢄ CH2OHꢂꢅ 5ꢃ10 ꢀqꢄ 1Hꢄ J =
6ꢃ7Hzꢄ CHꢂ; 7.32–7.45 ppm (m, 4H, Ar). Anal. C9H11OCl
(C, H). Calcd: C, 63.35; H, 6.50. Found: C, 63.50; H, 6.24.
Figure 2 General methods for the synthesis of nitrooxymethyl deriva-
tives starting from halogenated compounds (method A) or hydroxyalkyl
compounds (method B).
(230 mg; 6.09 mmol) cooled to 0ꢀC. The mixture was stirred
at 0ꢀC for 12 h, then water (1.7 mL) and 1 m NaOH (0.4 mL)
were added, and the resulting suspension filtered. The solvent
was evaporated to give 9a as a yellow oil (432 mg, 2.84 mmol,
93% yield); 1H NMR ꢀCDCl3ꢂ ꢁ1ꢃ46 ꢀdꢄ 3Hꢄ J =
6ꢃ5Hzꢄ CH3ꢂꢅ 4ꢃ63 ꢀsꢄ 2Hꢄ CH2OHꢂꢅ 4ꢃ85 ꢀqꢄ 1Hꢄ J =
6ꢃ5Hzꢄ CHꢂ; 7.20–7.35 ppm (m, 4H, Ar). Anal. C9H12O2 (C,
H). Calcd: C, 71.03; H, 7.95. Found: C, 71.34; H, 7.64.
General procedure for preparation of nitro-esters
Method A
A solution of opportune chloride (3.42 mmol) in CH3CN
(9 mL) was added to a stirred solution of AgNO3 (2.33 g,
13.69 mmol) in CH3CN (5 mL). Stirring was continued over
4 h at room temperature in the dark. The precipitate (silver
chloride) was then filtered off and the solvent evaporated.
The crude product was triturated with CHCl3 (20 mL) and
filtered off to remove the unreacted silver nitrate and AgCl.
4-(2-Hydroxyethyl)benzyl alcohol (9b)
Compound 9b was synthesised from 8b (500 mg, 3.05 mmol)
following the same procedure described above for the prepa-
ration of 9a, to give 9b as yellow oil (246 mg, 1.62 mmol,
Method B
53% yield); 1H NMR ꢀCDCl3ꢂ ꢁ1ꢃ49 ꢀdꢄ 3Hꢄ J = A solution of the opportune alcohol (2.0 mmol) in Ac2O
6ꢃ5Hzꢄ CH3ꢂꢅ 4ꢃ68 ꢀsꢄ 2Hꢄ CH2OHꢂꢅ 4ꢃ90 ꢀqꢄ 1Hꢄ J = (2 mL) was added to a solution of HNO3 (0.55 mL) and Ac2O
O
HO
Cl
i
ii
OH
OH
COOH
8a,b
10a,b
9a,b
iii
a: meta
b: para
O2NO
OH
7a,b
i: LiAlH4, THF, r.t.; ii: conc HCl, toluene, r.t., iii: AgNO3, CH3CN, r.t.
Figure 3 Process for the synthesis of compounds 7a, 7b, 8a, 8b, 9a, 9b, 10a and 10b.

192
Vincenzo Calderone et al
(1.15 mL) cooled to −10ꢀC. The mixture was stirred at −10ꢀC 1-[3-(Hydroxymethyl)phenyl]ethyl nitrate (7a)
for 2 h. The solvent was then evaporated to give a crude Compound 7a was synthesised following the general proce-
product (277 mg, 1.4 mmol, yield 69%) which was submitted dure described in method A. The crude product was puri-
to the subsequent reaction without any further purification.
fied by column chromatography using hexane/AcOEt (7:3)
as the eluent to give compound 7a (227 mg, 1.15 mmol, yield
34%) as a yellow oil; 1H NMR ꢀCDCl3ꢂ ꢁ1ꢃ64 ꢀdꢄ 3HꢄJ =
6ꢃ6Hzꢄ CH3ꢂꢅ 4ꢃ73 ꢀsꢄ 2Hꢄ CH2OHꢂꢅ 5ꢃ93 ꢀqꢄ 1Hꢄ J =
6ꢃ6Hzꢄ CHꢂ; 7.31–7.39 ppm (m, 4H, Ar). Anal. C9H11NO
(C, H, N): Calcd: C, 54.82; H, 5.62; N, 7.10. Found: C, 54.684;
H, 5.73; N, 7.07.
3-[(Nitrooxy)methyl]benzoic acid (1a)
Compound 1a was synthesised following the general proce-
dure described in method A (Breschi et al 2004), to
give compound 1a (78% yield) as a white solid; mp
1
129–131ꢀCꢅ H NMR ꢀCDCl3ꢂ ꢁ5ꢃ49 ꢀsꢄ 2Hꢄ CH2ONO2ꢂ;
7.49–7.58 (m, 1H, Ar); 7.67 (d, 1H, J = 7ꢃ8Hzꢄ Ar); 8.14–
8.17 (m, 2H, Ar); 13C NMR ꢀCDCl3ꢂ ꢁ73ꢃ99 ꢀCH2ONO2ꢂ,
{129.34, 130.18, 130.76, 131.29, 133.20, 134.19} (Ar–C),
171.08 (COOH). Anal. C8H7NO5 (C, H, N): Calcd: C, 48.74;
H, 3.58; N, 7.10. Found: C, 48.62; H, 3.73; N, 7.12.
1-[4-(hydroxymethyl)phenyl]ethyl nitrate (7b)
Compound 7b was synthesised and purified as described for
compound 7a, giving compound 7b (269 mg, 1.37 mmol, 39%
yield) as a yellow oil; 1H NMR ꢀCDCl3ꢂ ꢁ1ꢃ62 ꢀdꢄ 3HꢄJ =
6ꢃ7Hzꢄ CH3ꢂꢅ 4ꢃ71 ꢀsꢄ 2Hꢄ CH2OHꢂꢅ 5ꢃ93 ꢀqꢄ 1Hꢄ J =
6ꢃ7Hzꢄ CHꢂ; 7.31–7.42 ppm (m, 4H, Ar). Anal. C9H11NO
(C, H, N): Calcd: C, 54.82; H, 5.62; N, 7.10. Found: C, 54.744;
H, 5.77; N, 7.35.
4-[(Nitrooxy)methyl]benzoic acid (1b)
Compound 1b was synthesised following the general proce-
dure described in method A (Breschi et al 2006), to give
compound 1b (yield 54%) as a white solid; mp 144–146ꢀC;
1H NMR (CDCl3) ꢁ 5.50 (s, 2H, CH42); 7.50 (d, 2H, J = 8.2
Hz, Ar); 8.15 (d, 2H, J = 8.2 Hz, Ar); 13C NMR (CDCl3ꢁ
73.72 (CH2ONO2), 128.65, 130.86, 138.27, 154.56 (Ar – C),
178.00 (COOH). MS (m/z) 197 (M+, 3); 135 (M+−ONO2,
46); Anal. C8H7NO5 (C, H, N): Calcd: C, 48.74; H, 3.58; N,
7.10. Found: C, 48.45; H, 3.34; N, 7.42.
Pharmacological procedures
All the experimental procedures were carried out following
the guidelines of the European Community Council Direc-
tive 86-609. The experimental protocol was approved by the
Animal Care Committee of the University of Pisa.
In-vitro vascular protocols
2,6-Dimethyl-3-[(nitrooxy)methyl]benzoic acid (3)
The compounds were tested on isolated thoracic aortic rings
from male normotensive Wistar rats (250–350 g). After a light
ether anaesthesia, the rats were killed by cervical dislocation
and exsanguination. The aortas were excised immediately
and freed from extraneous tissues, and the endothelial layer
was removed by gently rubbing the intimal surface with a
hypodermic needle. Aortic rings (5 mm wide) were suspended
under a preload of 2 g in 20 mL organ baths containing
Tyrode solution (composition in mm: NaCl 136.8, KCl 2.95,
CaCl2 1ꢃ80ꢄ MgSO4 7H2O 1ꢃ05ꢄ NaH2PO4 0ꢃ41ꢄ NaHCO3
11.9, glucose 5.5), maintained at 37ꢀC and gassed continu-
ously with a mixture of O2 (95%) and CO2 (5%). Changes
in tension were recorded using an isometric transducer
(Grass FTO3; West Warwick, RI, USA), connected to a
preamplifier (Buxco Electronics; Wilmington, NC, USA)
and to data acquisition software (MP 100, BIOPAC Systems
Inc.; Goleta, CA, USA).
After an equilibration period of 60 min, endothelium
removal was confirmed by the administration of acetylcholine
ꢀ10ꢆmꢂ to rings pre-contracted with 30 mm KCl. Relax-
ation that was less than 10% of the KCl-induced contraction
was considered to be indicative of an acceptable lack of the
endothelial layer; rings that showed relaxation of 10% or
more were assumed to have significant amounts of endothe-
lium remaining and were therefore discarded.
Compound 3 was synthesised following the general proce-
dure described in method A (Breschi et al 2006), to
give compound 3 (yield 90%) as a white solid; mp
123–125 Cꢅ H NMR ꢀCDCl3ꢂ ꢁ2ꢃ43 ꢀsꢄ 6Hꢄ CH3ꢂ 5ꢃ47ꢅ
ꢀsꢄ 2Hꢄ CH2ꢂꢅ 7.12 (d, 1H, J = 7ꢃ9Hzꢄ Arꢂꢅ 7.33 (d, 1H,
J = 7ꢃ8Hzꢄ Arꢂꢅ 13C NMR ꢀCDCl3ꢂ ꢁ16ꢃ40 ꢀCH3ꢂ, 20.09
ꢀCH3ꢂꢄ 73ꢃ19 ꢀCH2ONO2ꢂ, 128.23, 128.36, 132.20, 134.48,
134.88, 136.94 (Ar–C), 174.96 (COOH). Anal. C10H11NO5
(C, H, N): Calcd: C, 53.33; H, 4.92; N, 6.22. Found: C, 50.04;
H, 4.67; N, 6.06.
ꢀ
1
3-(Hydroxymethyl)benzyl nitrate (6a)
Compound 6a was synthesised following the general proce-
dure described in method A (Breschi et al 2006), and was
obtained as a yellow oil (yield 91%); 1H NMR (CDCl3)
ꢁ 4.72 (s, 2H, CH2OH); 5.43 (s, 2H, CH2ONO2); 7.32–7.44
(m, 4H, Ar); 13C NMR (CDCl3) ꢁ 52.47 (CH2OH), 73.12
(CH2ONO2), 129.13, 130.16, 130.64, 130.96, 132.82, 133.33
(Ar–C). Anal. C8H9NO4 (C, H, N): Calcd: C, 52.46; H, 4.95;
N, 7.65. Found: C, 52.37; H, 4.82; N, 7.72.
4-(Hydroxymethyl)benzyl nitrate (6b)
Compound 6b was synthesised following the general proce-
dure described in method A (Breschi et al 2006), giving
compound 6b (yield 74%) as a yellow oil; 1H NMR (CDCl3)
ꢁ 4.71 (s, 2H, CH2OH); 5.42 (s, 2H, CH2ONO2); 7.35–7.44
NO-mediated vasorelaxing effect:
(m, 4H, Ar); 13C NMR (CDCl3) ꢁ 64.32 (CH2OH), 73.19 concentration–response curves
(CH2ONO2), 126.79, 128.89, 131.00, 141.82 (Ar–C). Anal. Aortic preparations were contracted with a single applica-
C8H9NO4 (C, H, N): Calcd: C, 52.46; H, 4.95; N, 7.65. Found: tion of 30 mm KCl 30–40 min after confirmation of endothe-
C, 52.52; H, 5.03; N, 7.28.
lium removal. When the contraction reached a stable plateau,

NO-releasing properties of NO-donor linkers
193
the test substance was added, in 3-fold increasing concen- two-way ANOVA. P values below 0.05 were considered to
trations from 1 nm to 100ꢆm. Each aortic ring was used be significant.
for only one concentration–response curve for a given test
compound. Preliminary experiments showed that the KCl-
induced contraction remained in a stable tonic state for at
least 40 min.
Results
The same experiments were carried out in the presence
of the guanylate cyclase inhibitor, 1H-(1,2,4)-oxadiazolo-
(4,3-a)-quinoxalin-1-one (ODQ; 1ꢆm), which was added to
Insertion of the nitrooxymethyl chain in the meta position
(as in compounds 1a, 2a, 6a and 7a) or in the para position
(as in compounds 1b, 2b, 6b and 7b) with respect to the
the aortic preparations after confirmation of endothelium
carboxylic function (in the benzoic series) or to the hydrox-
removal.
ymethyl function (in the benzylic series) was selected in order
to investigate the potential influence played by steric and/or
electronic factors on the NO-releasing properties of these
compounds.
Time course of vasorelaxing effect
Aortic preparations were contracted with a single applica-
tion of 30mm KCl 30–40 min after confirmation of endothe-
lium removal. Once a stable plateau contraction was reached,
a single concentration (1ꢆm, representing the mean of the
pIC50 values (defined below) of all the test substances) of the
compounds was added. The vasorelaxing effect of the added
compounds was monitored for 50 min.
The nitrooxymethyl chain was also inserted into the
aromatic ring (compound 3) or into the carbon atom
(compounds 2a and b and 7a and b) of one (or more) methyl
group(s) in order to determine the potential influence of these
groups on the NO-releasing kinetics of compounds 2, 3 and 7.
All the compounds synthesised were tested as vasodilator
agents on rat aortic rings pre-contracted with a depolar-
ising stimulus (30mm KCl). In this experimental model, all
compounds exhibited full or almost full vasorelaxing activity.
The potency (pIC50) and efficacy of the compounds tested
and of reference drug sodium nitroprusside (SNP) are given
in Table 1. The parameters describing the time-course profile
of the vasorelaxing effect (T25 values), reflecting the NO
release rates, are shown in Table 2.
Data analysis
The vasorelaxing efficacy was expressed as the maximal
vasorelaxing response as a percentage of the contractile tone
induced by 30 mm KCl. The highest concentration of the
test compounds that could be administered ꢀ100ꢆmꢂ did not
necessarily exert the maximal effect, in which case efficacy
was expressed as the vasorelaxing response as a percentage
of the contractile tone induced by 30 mm KCl evoked by this
limit concentration. Potency was expressed as pIC50, calcu-
lated as the negative logarithm of the molar concentration
of the test compound that evoked a 50% reduction of the
contractile tone induced by 30 mm KCl. The pIC50 could
not be calculated for compounds with efficacy below 50%.
The above experimental data were obtained by a computer
fitting procedure from the concentration–response curves
using commercial software GraphPad Prism 4.0 (San Diego,
CA, USA).
In terms of potency, the pIC50 values were always lower
than that of SNP ꢀpIC50 = 8ꢃ73ꢂ, and ranged between 4.83
(compound 7a) and 7.21 (compound 6b). The vasorelaxing
effects of all the test compounds and of SNP were almost
completely abolished by pre-incubation with ODQ (an
Table 1 The nitric oxide (NO)-mediated vasorelaxing efficacy of the
synthesised compounds and sodium nitroprusside (SNP) was evaluated
as the maximal vasorelaxing response, expressed as a percentage of the
contractile tone induced by 30 mm KCl (% EM). Potency is given as
the pIC50, calculated as the negative logarithm of the molar concentra-
tion of the test compounds evoking a 50% reduction in the contractile
In order to describe the time course of the effects, the
effect vs time curves were analysed using commercial soft-
ware (GaphPad Prism 4.0) using the equation: E = ꢀEM × tone induced by 30 mm KCl. The pIC50 could not be calculated for
compounds with efficacy below 50%. Values are given as mean s.e.
for 5–10 experiments
Tꢂ/ꢀKt + Tꢂ, where E is the vasorelaxing effect (expressed
as a percentage of the contractile tone induced by 30 mm
KCl) recorded at each min after administration of the test
Compound
pIC50
% EM
compounds ꢀ1ꢆmꢂ, EM is the maximum vasorelaxing effect
(expressed as a percentage of the contractile tone induced
by 30 mM KCl) induced by the test compounds ꢀ1ꢆmꢂ, T is
SNP
1a
8ꢃ73 0ꢃ04
5ꢃ80 0ꢃ08
6ꢃ47 0ꢃ02
5ꢃ64 0ꢃ04
5ꢃ71 0ꢃ02
5ꢃ30 0ꢃ03
6ꢃ34 0ꢃ02
6ꢃ26 0ꢃ05
7ꢃ03 0ꢃ04
7ꢃ21 0ꢃ03
4ꢃ83 0ꢃ04
5ꢃ98 0ꢃ02
100a
78
9
2
2
the time (in min) correlated to a given E, Kt is the time (in 1b
100a
96
min) required to reach E = 1/ EM. This analysis allowed us
2a
2b
3
4
5
6a
6b
7a
7b
2
100a
98
to calculate the parameter of T25 (time, in min, required to
reach an equi-effective level of vasorelaxing effect = 25%
100a
100a
100a
100a
91
of the contractile tone induced by 30 mm KCl). This T25
parameter could not be calculated for compounds exhibiting
an EM lower than or close to 25%.
The parameters of pIC50 and T25 are given as mean s.e.
for 5–10 experiments, and were analysed statistically using
one-way analysis of variance (ANOVA), followed by the
Bonferroni post-hoc test. The concentration–response curves
and the time–course curves were evaluated statistically using
1
100a
aThese compounds produced 100% vasorelaxation in all the tests.

194
Vincenzo Calderone et al
Table 2 Summary of the time-course profiles of the effects of the
synthesised compounds and sodium nitroprusside. EM is the maximum
vasorelaxing effect (expressed as a percentage of the contractile tone
induced by 30 mm KCl) induced by the test compounds ꢀ1ꢆmꢂ. T25 is
the time (in min) required to reach 25% of the vasorelaxation induced by
30 mM KCl. This parameter could not be calculated for compounds that
had an EM value below 25%. Some compounds (indicated as ineffective)
did not exhibit any significant vasorelaxing effect at 1ꢆm concentration.
Values are expressed as means s.e. for 5–10 experiments
the rate of release of NO from the drug. As expected, SNP
(a well-known rapid NO releasing agent) exhibited a very
low T25 value (0.39 min) and two compounds (6a and 6b)
showed similar T25 values (0.48 min and 0.45 min, respec-
tively). Compound 5 exhibited a slightly higher T25 value
(2.00 min) than that of SNP, but the two values did not differ
significantly. All the other compounds proved to be NO donor
agents possessing a significantly lower rate of NO release,
with T25 values ranging between 3.84 min (compound 1b)
and 8.83 min (compound 2a); Compound 7b gave a modest
vasorelaxing effect ꢀEM = 26%ꢂ, which did not allow calcu-
lation of T25; compounds 3 and 7a did not evoke any signifi-
cant vasorelaxing effect, indicating that these two compounds
were the slowest NO donors of the series.
Compound
% EM
T25
SNP
1a
1b
2a
2b
3
98
39
65 10
42
50
2
8
0ꢃ39 0ꢃ08
8ꢃ25 0ꢃ42
3ꢃ84 0ꢃ48
8ꢃ83 0ꢃ58
6ꢃ25 0ꢃ21
–
2
7
Ineffective
4
5
6a
6b
7a
7b
49
73
90
90
3
1
1
4
4ꢃ68 0ꢃ33
2ꢃ00 0ꢃ27
0ꢃ48 0ꢃ02
0ꢃ45 0ꢃ03
–
Discussion
Although this preliminary study has focused on a limited
number of lead compounds, some aspects of the structure–
activity relationships can be hypothesised in terms of the
influence of slight structural variations on the NO releasing
properties of the nitrooxy group.
Ineffective
26
1
Not calculable
Considering the carboxylic derivatives, a comparison of
compounds 1a vs 1b and 2a vs 2b showed that the para
position ensured more effective NO release. In particular,
compound 1b was more potent than compound 1a; also the
T25 value of 1b was significantly lower than that of 1a,
indicating a more rapid release of NO. Although 2a and 2b
analogues showed almost equal potency and efficacy, release
of NO was more rapid from 2b, indicated by the significantly
lower T25 value of compound 2a. Insertion of a methyl group
into the nitrooxymethyl chain did not seem to have any signif-
icant influence in the meta-substituted analogues 1a and 2a,
and seemed to have a negative impact for the para-substituted
analogues 1b and 2b: compound 2b was less potent than
compound 1b, and, consistent with this, NO release was
slower with this compound. Insertion of two methyl groups
into the aromatic ring (compound 3) led to a decrease in
potency compared with compound 1a, and, consistent with
this, compound 3 ꢀ1ꢆmꢂ did not possess any significant
vasorelaxing effect in the time-course protocol. Replacement
of the benzene ring of compound 1a with a pyridine system
(compound 4), and replacement of its carboxy function with
a hydroxy group (compound 5), caused improvements in
both the vasorelaxing potency and the rate of NO release.
The benzylic derivatives 6a and 6b were the most potent of
the compounds tested, and their NO release rates similar to
that with SNP. In the benzylic series, the para-substituted
compound 7b was a more potent NO donor than the meta-
substituted compound 7a. However, the positive effect due
to this structural feature was not evident for the analogues 6a
and 6b, which had almost equal pIC50 and T25 values. The
negative influence due to the insertion of the methyl group
into the nitrooxymethyl chain observed in the benzoic series
was even more evident in the benzylic compounds (compare
compound 7a vs 6a, and 7b vs 6b). In particular, compound
1a
1a + ODQ 1 µM
6a
6a + ODQ 1 µM
125
100
75
50
25
0
–9
–8
–7
–6
–5
–4
Log concn
Figure 4 Representative examples of the effect of the guanylate cyclase
inhibitor ODQ ꢀ1ꢆmꢂ on the vasorelaxing effects of compounds 1a and
6a. The vasorelaxing efficacy is expressed as the maximal vasorelaxing
response as a percentage of the contractile tone induced by 30 mm KCl.
inhibitor of guanylate cyclase and thus of the NO–cGMP
pathway), indicating a clear involvement of NO release in
their mechanism of action (Figure 4). Although the pIC50
value was clearly influenced by the rate of NO release,
analysis that focused more closely on evaluating the devel-
opment of the vasorelaxing effect over time (through the
time-course protocol and the T25 parameter) seemed to be a
more appropriate approach to describe this aspect. Indeed, the
T25 parameter reflects the time needed to achieve a partic-
ular level of vasorelaxation (25% of the pre-contraction by
KCl) induced by 1ꢆm of the compounds tested. Thus, the 7a was the least potent of all the compounds tested, and
T25 parameter could be viewed as an indirect indicator of did not exhibit any vasorelaxing effect in the time-course

NO-releasing properties of NO-donor linkers
195
protocol, while compound 7b exhibited a poor vasorelaxing Breschi, M. C., Calderone, V., Digiacomo, M., Martelli, A.,
Martinotti, E., Minutolo, F., Rapposelli, S., Balsamo, A. (2004)
NO-sartans: a new class of pharmacodynamic hybrids as cardio-
vascular drugs. J. Med. Chem. 47: 5597–5600
Breschi, M. C., Calderone, V., Digiacomo, M., Macchia, M.,
Martelli, A., Martinotti, E., Minutolo, F., Rapposelli, S.,
Rossello, A., Testai, L., Balsamo, A. (2006) New NO-releasing
pharmacodynamic hybrids of losartan and its active metabolite:
design, synthesis, and biopharmacological properties. J. Med.
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effect which did not allow evaluation of T25.
This study aimed to develop some representative NO-donor
structures that might be useful as linkers to be added to
native drugs, in order to obtain pharmacodynamic hybrids.
The key aim with such hybrids is the presence of the two
mechanisms of action; however, correct balancing of the two
pharmacodynamic properties is another fundamental aspect.
In particular, depending on the characteristics of the native
drug (mechanism of action, posology, therapeutic indication),
the release of NO should be correctly modulated in order
to obtain well-calibrated levels of additive biological effects
of NO itself. The availability of different NO-donor linkers
possessing different NO release rates thus represent a neces-
sary tool for this purpose.
The main findings of this work indicate that the shift of
the nitrooxymethyl chain from the meta to the para posi-
tion, in both benzoic and in benzylic derivatives, signif-
icantly influences the NO donor properties and achieves
more effective release. Furthermore, the insertion of a methyl
group into the nitrooxymethyl chain, as well as the pres-
ence of two methyl groups on the aromatic system, reduced
the rate of NO release. It is reasonable to hypothesise
that in a pharmacodynamic hybrid, the NO releasing rate
of a given NO-donor linker might be influenced by the
presence of other molecular portions (i.e. the native drug).
However, this work indicates that variations in the structure
of the NO-donor linkers described above ensure almost the
same trend of NO-releasing properties as that shown by the
hybrid compounds (Breschi et al 2004, 2006). This hypothesis
seems to be confirmed by previous results concerning NO-
releasing derivatives, in which losartan and its active metabo-
lite EXP3174 were conjugated with some of the linkers
described in this work (Breschi et al 2004, 2006), the NO-
releasing properties of these hybrids were, in some cases,
different from those exhibited by the corresponding NO-
donor linkers alone.
Martelli, A., Rapposelli, S., Calderone, V. (2006) NO-releasing
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