Synthesis physicochemical profile and PAMPA study of new NO-donor edaravone co-drugs

Article abstract of DOI:10.1016/j.bmc.2011.11.065

A new class of co-drugs were synthesised by joining antioxidant edaravone with a vasodilating substructure containing NO-donor nitrooxy functions, and characterised for their stability in different media, lipophilicity and permeability profile. The produc

Full text of DOI:10.1016/j.bmc.2011.11.065

Bioorganic & Medicinal Chemistry 20 (2012) 841–850
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis physicochemical profile and PAMPA study of new NO-donor
edaravone co-drugs
Barbara Rolando a, Andrea Filieri a, Konstantin Chegaev a, Loretta Lazzarato a, Marta Giorgis a,
c
c
a
Claudio De Nardi b, Roberta Fruttero a, , Sophie Martel , Pierre-Alain Carrupt , Alberto Gasco
⇑
a Dipartimento di Scienza e Tecnologia del Farmaco, Università degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy
b Nicox Reasearch Institute, Via Ludovico Ariosto 21, 20092 Bresso (Milano), Italy
c School of Pharmaceutical Sciences, University of Geneva, University of Lausanne, 30 Quai Ernest-Ansermet, CH 1211 Geneva 4, Switzerland
a r t i c l e i n f o
a b s t r a c t
Article history:
A new class of co-drugs were synthesised by joining antioxidant edaravone with a vasodilating substruc-
ture containing NO-donor nitrooxy functions, and characterised for their stability in different media, lipo-
philicity and permeability profile. The products display good stability in water/co-solvent at different pH.
Conversely, they are rapidly metabolised into edaravone and NO-donor moieties when incubated in human
serum or rat-liver homogenates. In the latter conditions time dependent production of nitrite/nitrate (NOx)
occurs. The compounds display wide-ranging lipophilicity. PAMPA studies predict good gastrointestinal
absorption for a number of these compounds. The title products are potentially useful for treating ROS-
related conditions accompanied by decreased NO availability.
Received 26 April 2011
Revised 9 November 2011
Accepted 29 November 2011
Available online 8 December 2011
Keywords:
Nitrooxy-acyl derivatives of edaravone
Co-drugs
Lipophilicity profile
In vitro permeability profile
Ó 2011 Elsevier Ltd. All rights reserved.
1. Introduction
Edaravone (MC-186) (1), is an acid pyrazoline drug that can exist
in three tautomeric forms a, b, c (Fig. 1). It was developed in Japan for
treating of patients in the acute stage of cerebral thrombosis or
embolism, and has been reported to be effective also in the acute
stage of stroke. Edaravone displays potent antioxidant properties,
and consequently it might be useful in managing other pathological
processes involving oxidative stress.1 Generally, these processes are
complex diseases which must be treated with a cocktail of drugs. An
alternative could be the use of polyvalent drugs, namely single prod-
ucts active at more than one target, and consequently exercising
more than one action simultaneously.2 The design of a polyvalent
drug is achieved by fusing, or joining through appropriate linkers,
generally two drugs, or crucial parts of them. The linker can be sus-
ceptible to metabolic cleavage (co-drug), or can be a hard linker
(dual drug). Another possibility is to overlap substructures which
are common to the two leads. We recently proposed a new class of
polyvalent drugs: the nitric oxide (NO)-donor antioxidants.3–5 These
compounds are obtained by combining known antioxidants with
moieties able to release NO. All products display both antioxidant
and NO-dependent activities. They are potentially useful in treating
reactive oxygen species (ROS)-related diseases that are accompa-
nied by decreased NO availability. Typical examples of such
Figure 1. Edaravone and its tautomeric forms.
conditions are atherosclerosis and related diseases. Among the hy-
brid products we designed, there is also a series of derivatives ob-
tained by combining edaravone with nitrooxy (ONO2) or furoxan
(1,2,5-oxadiazole 2-oxide) NO-donor moieties through a hard
linker.4
As extension of this work, we describe here a new class of
co-drugs obtained by joining edaravone through a vulnerable car-
bonate (4a–c) or ester (7a–i) linker with alcohols (2a–c) or carbox-
ylic acids (5a–i) containing ONO2 functions (Schemes 1 and 2). In
contrast with the compounds described above, the antioxidant
activity is linked to the edaravone part, recovered after hydrolysis
of the vulnerable linker. This difference may lead to different
in vivo pharmacokinetic and pharmacodynamics profiles. The
synthesis of these products, their stability in different media, their
lipophilicity as well as the prediction of their human gastrointesti-
nal permeation (PAMPA assay) are described. Vasodilator
⇑
Corresponding author.
E-mail address: roberta.fruttero@unito.it (R. Fruttero).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.11.065

842
B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
the unsaturated ester 9 by action of diisoamylborane, prepared in
situ, followed by oxidation with hydrogen peroxide, gave 11. Basic
hydrolysis of this intermediate, followed by the action of sulfoni-
tric mixture in dichloromethane, afforded the corresponding final
nitrooxy substituted acid 5d.
NO-donor acids 5a–i were transformed into the corresponding
acyl chlorides 6a–i by action of thionyl chloride in toluene, in the
presence of a catalytic amount of DMF (Scheme 2). These intermedi-
ates were not purified or characterised, but immediately trans-
formed into the desired products 7a–i by reaction with edaravone,
in the presence of triethylamine. The pyrazole structures assigned
to these final compounds were confirmed by NMR spectroscopy:
13C NMR and 1H NMR spectra of the products show a signal occurring
in the range d 95.6ꢀ95.8, attributable to the 4-C aromatic carbon,
and a signal occurring in the range d 6.06ꢀ6.28 attributable to
4-CH methyne proton. Also for this class of products, additional
evidence of the assigned structures derives from their capacity to
undergo hydrolysis yielding edaravone.
Scheme 1. Carbonates synthesis. Reagents and conditions: (i) (Cl3CO)2CO, Py,
CH2Cl2, 0 °C to rt; (ii) edaravone, Et3N, CH2Cl2, 0 °C to rt.
2.2. Stability and metabolic studies
The stabilities of the co-drugs reported here were assessed by
high-performance liquid chromatography (HPLC), in aqueous med-
ia, in human serum and in rat-liver homogenate.
2.2.1. Stability in aqueous media
The stability of all the compounds was assessed in water/DMSO
5% (v/v) (the experimental conditions of PAMPA permeability stud-
ies), and in acidic medium (0.1 M HCl/MeOH 60/40) to mimic the
gastric environment. In both media, the products underwent
hydrolysis following pseudo-first-order kinetics, affording edarav-
one and the related nitrooxy substituted alcohol and acid moieties.
The observed pseudo-first-order rate constants (kobs) and half-lives
(t1/2, Table 1) were determined by fitting the data with one-phase
exponential decay equation (Graph Pad, Prism software version 5).
Table 1 shows that half-lives are quite different in the two media
used. The highest stabilities are in water/DMSO 5% (v/v). In this
medium t1/2 values of the three carbonates tested lie in the range
25–6 h and rank in the order 4b > 4a > 4c, indicating that the sta-
bility of the products increases on moving from the propyl to the
Scheme 2. Esters synthesis. Reagents and conditions: (i) SOCl2, toluene, DMF, rt;
(ii) edaravone, Et3N, CH2Cl2, 0 °C to rt.
properties of the NO-donor related alcohols and acids used to build
the co-drugs, and of selected co-drugs, are also reported.
2. Results and discussion
2.1. Chemistry
hexyl chain bearing at the
decreases when two of these groups are present on the hexyl chain
at - and -1 positions. The two esters 7a,b are slowly hydrolyzed,
x-position the nitrooxy function, and
x
x
The carbonates 4a–c were obtained by the procedure reported
in Scheme 1. The already-known NO donor alcohols 2a–c (see
Section 4) were transformed into the corresponding chlorofor-
mates 3a–c, by action of triphosgene in dichloromethane, in the
presence of pyridine. Crude compounds thus obtained were imme-
diately treated with edaravone in dichloromethane solution, in the
presence of triethylamine, to give the expected final carbonates as
principal reaction products. The assigned pyrazole structures were
confirmed by NMR spectroscopy: 13C NMR and 1H NMR spectra of
the compounds show a signal occurring at d 95.6–95.8, attributable
to the 4-C aromatic carbon, and a signal occurring at d 6.09 or 6.10
attributable to 4-CH methyne proton. Additional evidence of the
assigned structures derives from the capacity of the products to
undergo hydrolysis, yielding edaravone. Synthesis of the final es-
ters 7a–i required preliminary preparation of the NO-donor acids
5a–i (Scheme 2); a number of these, compounds 5a–c and 5f,g,
are known products (see Section 4), while acids 5d,e were obtained
via the pathway reported in Scheme 3. Methylisobutyrate (8) was
alkylated by action of 4-bromobut-1-ene in the presence of lithium
diisopropylamide, prepared in situ. The resulting methyl 2,2-dim-
ethylhex-5-enoate (9) was treated with AgNO3 and I2 in CH3CN
to give the dinitrooxy substituted ester 10. Finally, acid hydrolysis
of this ester afforded the corresponding acid 5e. Hydroboration of
and again the dinitrooxy compound 7b is less stable than its mono-
substituted analogue 7a. The introduction of two methyl groups at
a-position of the ester function gives rise to products which dis-
play high t1/2 values in the order 7c > 7d > 7e. Their low water sol-
ubility prevented us from studying the aromatic esters 7f–i. In
acidic medium, the compounds are less stable than in water, with
the sole exception of 7d. Also in this medium, dinitrooxy com-
pounds are less stable than their mono-substituted analogues
(compare 4b/4c, 7a/7b, 7d/7e, 7g/7i).
2.2.2. Stability in human serum and in rat-liver homogenate
When incubated in human serum or in rat-liver homogenates, all
products were rapidly hydrolyzed, giving edaravone and the NO-do-
nor moieties. In these conditions, hydrolysis is catalysed by carboxy-
lesterases; these enzymes are ubiquitous and display broad
substrate specificity. Frequently, an ester can be hydrolyzed by more
than one of these enzymes, and as yet a precise classification has not
been proposed.6 Humans express carboxylesterases in several com-
partments, including liver, plasma, small intestine, brain, stomach,
colon macrophages, and monocytes.7 Table 1 shows that carbonates
and unbranched aliphatic esters display t1/2 values <1 min, while
values for branched aliphatic esters and aromatic esters lie in the

B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
843
Scheme 3. Synthesis of NO-donor acids 5d,e. Reagents and conditions: (i) BuLi, DPA, 4-bromobut-1-ene, HMPA, THF dry, ꢁ78 °C; (ii) AgNO3, I2, CH3CN, rt, then 4; (iii) HCl
6 M, dioxane, 4; (iv) NaBH4, BF3ꢃEt2O, 2-methylbut-1-en, THF dry, 0 °C rt; (v) NaOH, H2O2, 40 °C; (vi) NaOH, MeOH, 4; (vii) HCl; (viii) HNO3, H2SO4, CH2Cl2, 0 °C.
Table 1
Stability in aqueous media, in human serum and in rat-liver homogenate
Compound
t1/2
Water (5% DMSO, 0.1 M KCl)a
HCl 0.1 M/MeOH (60/40, v/v)c
Human serumd
Rat-liver homogenated
Edaravone
Stable
13 h
25 h
6 h
31 h
10 h
36 h
22 h
Stable
1.4 h
4.0 h
3.1 h
1.0 h
0.7 h
1.9 h
24 h
10 h
13 h
35 h
8 h
Stable
n.d.
4a
4b
4c
7a
7b
7c
7d
7e
7f
<1 min
<1 min
<1 min
<1 min
<1 min
1.9 min
13.3 min
16.2 min
4.0 min
5.0 min
7.1 min
7.8 min
<1 min
<1 min
<1 min
<1 min
<1 min
<1 min
2 min
16 h
3 min
b
<1 min
<1 min
<1 min
<1 min
b
b
b
7g
7h
7i
25 h
a
b
c
Room temperature; n = 3, SD <1.
Not determined due to low solubility in the experimental conditions.
t = 37 °C; n = 3, SD <0.1.
d
t = 37 °C; n = 3, SD <0.3.
range 1.9–16.2 min in human serum. For all compounds, hydrolysis
is almost immediate in rat-liver homogenate.
tion logkw factors.5 This equation was used for the evaluation of
logP of edaravone and edaravone derivatives from their logkw fac-
tors, measured under the same conditions as the reference
compounds.
In liver homogenate, production of nitrite (NO2ꢁ) and nitrate
(NO3ꢁ), the oxidised metabolites of NO derived from biotransfor-
mation of the organic nitrates, was time-dependent. Figure 2A
and B reports, as an example, the time course of nitrite and nitrate
generated from 7d and 7e over the incubation time. Figure 2C
shows NOx (nitrite + nitrate) produced by all compounds under
study after 5 h incubation. As expected, the highest levels of NOx
were obtained from the dinitrooxy substituted products 4c, 7e,
7h, 7i.
log Poct ¼ 1:098ðꢂ0:022Þ log kw þ 0:335ðꢂ0:047Þ
N = 63, r2 = 0.98, s= 0.15, F = 2531.
ð1Þ
The experimental logPs of edaravone derivatives vary widely
across the series (from 3.35 to 5.14) and are higher than that of
parent compound (edaravone, logPoct = 1.44). Most of them are in
accordance with calculated values (CLOGP, Bio-Loom for Windows
v. 1.5, BioByte Corp., Claremont, CA, USA) within 0.4, while others
only slightly exceed this range.
2.2.3. In vivo study
Release of nitrite was studied preliminarily in vivo for com-
pounds 4a and 7d, following oral administration of 10 mg/kg in
mice (imwitor742/tween80 50/50, vehicle). Production of nitrite
2.4. Permeability studies
The PAMPA assay is a recently-developed procedure for the ra-
pid determination of passive transport permeability, which is
widely accepted in pharmaceutical research circles. In this method,
the donor compartment, containing a buffer solution of the tested
compound, and the acceptor compartment, containing an initial
fresh buffer solution, are separated by a 96-well filter plate coated
with a liquid artificial membrane. To predict gastrointestinal
absorption, phospholipids8 or hexadecane9 have been proposed
as artificial membrane. Although the PAMPA-HDM (hexadecane
membrane) does not contain phospholipids, the formation of a
over 24 h was monitored. Nitrite AUC0–24h was 964
lM min and
1790 M min, respectively. Under the same conditions, AUC0–24h
l
for isosorbide 5-mononitrate (ISMN) was 2352 lM min.
2.3. Lipophilicity studies
The lipophilicity (logPoct) of the new products (Table 2) was
determined by a RP-HPLC method (see Section 4 for details). The
method was calibrated (Eq. 1) by plotting logPoct values of 63 ref-
erence compounds against the corresponding extrapolated reten-

844
B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
50
15
10
5
A
B
40
30
20
10
0
0
0
1
2
3
4
5
0
1
2
3
4
5
time (h)
time (h)
80
C
60
40
20
0
ISDN 4a 4b 4c
7a 7b 7c 7d 7e
7f
7g 7h
7i
Figure 2. The time course of NOx species generated from 7d (A) and 7e (B) over the incubation time in rat-liver homogenate. Data are expressed as %mol NOx/mol compound:
j NOx (nitrite + nitrate); d nitrite; s nitrate. (C) total NOx (nitrite + nitrate) generated after 5 h of incubation of NO-donor edaravone co-drugs and ISDN (isosorbide dintrate,
as reference compound), in rat-liver homogenate; data are expressed as %mol NOx/mol compound. Values are presented as mean SD; number of determinations = 3.
Table 2
(cm/s) can be calculated using the following equation (see Supple-
Calculated and measured logPoct, membrane retention (R) and effective permeability
coefficient (logPe) obtained from PAMPA-HDM experiments
mentary data for details):
ꢀ
ꢁ
2:303Vd
ð1 þ rꢁv 1
Þ CaðtÞ
a
b
Compound
ClogPoct
logPoct
PAMPA-HDM
Pe ¼ ꢁ
log 10 1 ꢁ
ð2Þ
ð1 þ rvÞðt ꢁ sLagÞA
ð1 ꢁ RÞeꢁkt Cdð0Þ
R ( SD)
logPe ( SD)
where rv = Vd/Va, A (cm2) is the filter area, t is the incubation time (s),
sLag is the steady-state time (s), namely the time needed for the
permeant’s concentration gradient to became stabilised, Va and Vd
(cm3) are the volumes in the acceptor and the donor wells, respec-
tively, Ca(t) is the concentration of the compound (mol cmꢁ3) in
the acceptor well at time t, Cd(0) is the concentration of the
compound (mol cmꢁ3) in the donor well at time 0, and k is the
pseudo-first-order rate constant of the hydrolysis. R is the retention
factor defined as the mole fraction of compound that is lost in the
membrane and in the microplates (filters and plate material) and
calculated from Eq. 3.
Edaravone
1.33
2.85
4.15
3.96
3.84
3.65
3.36
4.55
4.36
5.41
5.17
5.37
5.26
1.44
3.35
4.39
4.63
3.79
3.95
3.55
4.30
4.04
4.76
4.74
4.88
5.14
0.12 0.00
0.21 0.03
0.92 0.04
0.50 0.01
0.69 0.08
0.46 0.01
0.60 0.05
0.89 0.02
ꢁ3.52 0.01
ꢁ3.51 0.05
ꢁ4.68 0.24
ꢁ4.40 0.08
ꢁ4.93 0.10
ꢁ3.75 0.08
ꢁ3.56 0.05
ꢁ4.06 0.12
4a
4b
4c
7a
7b
7c
7d
7e
7f
0.88 0.04
ꢁ4.12 0.15
c
c
c
c
c
c
c
c
7g
7h
7i
a
b
c
Bio-Loom for Windows v.1.5, Bio Byte Corp., Claremont, CA, USA.
Determined by RP-HPLC (SE 6 0.05).
Not determined due to low solubility in the experimental conditions.
CdðtÞ
VaCaðtÞ
R ¼ 1 ꢁ
ꢁ
ð3Þ
Cdð0Þeꢁkt VdCdð0Þeꢁkt
where Ca(t)/Cd(0) represents the fraction of compound that reaches
the acceptor compartment after the incubation time t (for Va = Vd).
PAMPA experiments were carried out on edaravone and its new
derivatives, using filters impregnated with hexadecane to evaluate
the gastrointestinal passive permeability of these compounds
(PAMPA-HDM experiments). The logPe and R values calculated
from Eqs. 2 and 3 are in Table 2. The use of a simple solvent,
namely hexadecane, as artificial membrane leads to a more robust
model than using phospholipids. Indeed, for a set of 32 chemically-
diverse drugs, Wohnsland et al. described quite a good sigmoidal
correlation between their logPe value, measured by PAMPA-
HDM, and human absorption.9 The original logPe values of a set
bilayer membrane has never been characterized in the phospho-
lipid-based PAMPA assay. As a consequence, PAMPA-HDM was
used as a simpler and more robust alternative to predict GI
permeation.
Equations to calculate the effective permeability Pe can be de-
duced in several ways, depending on experimental conditions
and on the design of the in vitro assay. When retention must not
be neglected, the no-sink conditions in acceptor wells are fixed,
and compounds degrade following a first-order kinetics in experi-
mental conditions, thus the effective permeability coefficients Pe

B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
845
Figure 3. (A) Correlation between retention (R) and number of CH2 groups. (B) Correlation between permeability coefficient (logPe) from PAMPA experiments and number of
CH2 groups. d compounds with up to 3 free non substituted CH2 groups; N compounds with 4 or more free non substituted CH2 groups; s compounds with substituted CH2
groups. Black symbols correspond to compounds having a unique ONO2 group, and grey symbols to compounds having 2 ONO2 groups.
Figure 4. Correlation between octanol–water partition coefficients and retention (R) from PAMPA experiments (A) and permeability coefficients (logPe) (B). d compounds
with up to 3 free non-substituted CH2 groups; N compounds with 4 or more free non-substituted CH2 groups; s compounds with substituted CH2 groups.
of 9 reference compounds were first compared to logPe values ob-
tained in house. The correlation (data not shown) produced an r2 of
0.83, a Y-intercept of 0.008 and a slope of 1.04. This means that val-
ues of logPe for edaravone derivatives can be compared to those
published in Wohnsland et al.9 The logPe values measured for
these new edaravone NO-donor co-drugs (Table 2) vary across
the series of compounds studied: around ꢁ3.5 for compounds 4a,
7b, and 7c, in the range ꢁ4.06 to ꢁ4.40 for 4c, 7d, and 7e, ꢁ4.68
for 4b, and ꢁ4.93 for 7a.
or less.9 Although membrane retention cannot be determined,
since considerable adsorption onto the microplate probably oc-
curred, and hypothesizing that the loss of compound observed in
the PAMPA experiments is mainly due to this adsorption, edarav-
one derivatives (excepted for compounds 4b, 4c and 7a) might pos-
sess high potential for passive penetration through the intestinal
barrier. Permeability through artificial membrane appears to be
lower for compounds 4b, 7a and, to a lesser extent, for compound
4c. It may therefore be assumed that intestinal absorption is lim-
ited by the increasing number of free CH2 groups (Fig. 3B).
In addition, no clear relationship exists between these
permeation parameters and the logPoct value measured by HPLC
(Fig. 4A and B). In general, permeability through an artificial mem-
brane decreases, and membrane retention increases, with in-
creased lipophilicity. However, a number of compounds deviate
from this tendency, suggesting that permeability may be lowered
by the presence of long linear hydrophobic CH2 chains (compounds
4b, 4c and 7a).
The retention factors R are quite high for most derivatives. The
retention linearly correlates with the number of free CH2 groups
for most compounds, excepted for 7c, 7d and 7e (Fig. 3A). The high-
er retention of most compounds than edaravone may partly be due
to adsorption processes on the plastic material from which the fil-
ter-microplates are made: it is known that molecules bearing
nitrooxy groups can be adsorbed onto plastic materials.10 How-
ever, the variation of retention factors is not correlated with the
number of ONO2 substituents, as shown in Figure 3A, in which
black symbols represent compounds having a single ONO2 group,
and grey symbols to compounds having two ONO2 groups. But
the combination of fairly high lipophilicity (Fig. 4A) and the pres-
ence of ONO2 substituents could explain significant adsorption
onto plastic material.10 However, since Pe is an ‘effective’ perme-
ation value, it does not depend on the loss of compound through
membrane retention or adsorption processes. The logPe values ob-
tained, ranging from ꢁ3.5 to ꢁ4.1 (excepted compounds 4b, 4c and
7a), indicate that these edaravone derivatives can be classified as
‘high permeant’ compounds, compared to reference compounds.
In the reference set, compounds having logPe between ꢁ3.3 and
ꢁ4.1 were those having intestinal absorption above 70%, whereas
compounds with logPe below ꢁ4.8 had 20% intestinal absorption
2.5. Vasodilator studies
The vasodilator activity of the NO-donor nitrooxy substituted
alcohols and acids, which are rapidly generated when edaravone
co-drugs are incubated in human serum, was evaluated on de-
nuded rat aorta strips precontracted with phenylephrine. All com-
pounds relaxed the contracted tissue in a concentration-dependent
manner. Their potencies, expressed as EC50, are in Table 3. The dini-
trooxy substituted compounds 5b, 5h, 5i, 2c were more potent
than their mono-substituted analogues 5a, 5f, 5g and 2b. Alcohols
2b and 2c are better vasodilators than the structurally-related
acids 5a and 5b, which might partly be due to their greater facility

846
B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
Table 3
phy (TLC) on 5 ꢄ 20 cm plates with a layer thickness of 0.20 mm.
Anhydrous magnesium sulphate was used as drying agent for the
organic phases. Organic solvents were removed under vacuum at
30 °C. Preparative HPLC was performed on a LiChrospherÒ C18 col-
Vasodilator activity of nitrooxy-substituted alcohols 2a–c and acids 5a–i generated
from the hydrolysis of edaravone co-drugs by esterases, and of 7d,7e, the most stable
compounds in human serum
Compound
EC50
(
l
M) SEM
EC50 (lM) SEM + 1 lM ODQ
umn (250 ꢄ 25 mm, 10
lm) (Merck Darmstadt, Germany) with a
2a
2b
2c
5a
5b
5c
5d
7d
5e
7e
5f
4.0 0.6
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
>100
Varian ProStar mod-210 with Varian UV detector mod-325. Ele-
mental analyses (C, H, N) of the new compounds dried at 20 °C,
pressure <10 mmHg for 24 h, were performed at the University
of Geneva and the results are within 0.4% of the theoretical val-
ues. Structures 2a, 2b,11 2c,12 5a,13 5b,14 5c,15 5f, 5g,16 5h and
5i17 were synthesized by published methods. Methyl 2,2-dimeth-
ylhex-5-enoate (9) was obtained using a published procedure
starting from methyl isobutyrate.18 Tetrahydrofuran (THF) was dis-
tilled immediately before use from Na and benzophenone under a
positive atmosphere of N2.
1.5 0.4
0.83 0.17
8.3 1.5a
5.8 0.8a
13
8.9 2.2
11
2
5
4.2 1.2
2.5 0.4
0.51 0.08a
0.62 0.07a
0.33 0.06a
0.28 0.04a
5g
5h
5i
66 12
>100
67
6
4.1.1. General procedure for preparation of carbonate
derivatives 4a–c
a
Taken from Ref.12
.
A solution of the appropriate alcohol (2a–c) (2.5 mmol) and
anhydrous pyridine (0.20 mL; 2.5 mmol) in dry CH2Cl2 (10 mL)
was added dropwise to a stirred solution of triphosgene (0.29 g;
1.0 mmol) in dry CH2Cl2 (5 mL) at 0 °C. The reaction mixture was
stirred at 0 °C until reaction completion. Organic phase was
washed with 1 M HCl (10 mL), brine and dried. The CH2Cl2 solution
of the crude chloroformate (3a–c) thus obtained was added drop-
wise to a solution of 1 (0.45 g; 2.6 mmol) and Et3N (0.36 mL;
2.6 mmol) in dry CH2Cl2 (20 mL) at 0 °C. The reaction mixture
was stirred at rt for 1 h, then washed with H2O (20 mL), 0.1 M
HCl (50 mL), brine, dried, and the solvent evaporated. The product
was purified by flash chromatography using the eluents indicated.
Solid substances were further purified by crystallization.
to penetrate into smooth muscle cells, consequent on their higher
lipophilicity. When the experiments were repeated in the presence
of
1
lM
ODQ (1H-[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one),
potencies decreased, in keeping with NO-induced activation of
the sGC being the mechanism underlying the vasodilator effect.
Vasodilator experiments were also carried out on the whole co-
drugs 7d and 7e, the most stable versus serum esterases. As ex-
pected, their vasodilator activity is close to those of the individual
related NO-donor moieties 5d and 5e.
3. Conclusion
4.1.1.1. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 3-(nitrooxy)propyl
In conclusion, we prepared new NO-donor edaravone co-drugs
by joining edaravone, through a vulnerable carbonate or ester lin-
ker, with alcohols or carboxylic acids containing NO-donor nitro-
oxy functions. The resulting products display wide-ranging
lipophilicity values, and for a number of these compounds PAMPA
studies predict good gastrointestinal absorption. They display good
stability in acidic medium; conversely, when incubated in human
serum or in liver homogenate they undergo rapid hydrolysis by
esterases, yielding edaravone and the nitrooxy functions (NO-do-
nor alcohols and acids). In liver homogenate, all NO-donor moieties
deriving from hydrolysis of the co-drugs produce inorganic nitrite
and nitrate in a time-dependant manner. The NO-donor alcohols
and acids relax rat aorta strips precontracted with phenylephrine
in a concentration-dependent manner, through a cGMP dependent
mechanism. The co-drugs described here are a new class of polyva-
lent agents, which combine edaravone-dependent activities with
the NO-dependent activities of organic nitrates. For this reason
they are potentially interesting for the treatment of ROS-related
diseases accompanied by decreased NO availability, such as ath-
erosclerosis and diabetes.
carbonate (4a).
Eluent PE/EtOAc 8/2 v/v; yellow oil which
solidified in the freezer. The solid obtained was crystallized from
i-Pr2O to give the title compound as a white solid, mp = 42–
43 °C; 51% yield. 1H NMR (300 MHz, CDCl3, TMS): d = 2.11 (qi,
2H, CH2), 2.33 (s, 3H, CH3), 4.33 (t, 2H, CH2), 4.51 (t, 2H, CH2),
6.10 (s, 1H, CH), 7.30–7.35 (m, 1H), 7.42–7.47 (m, 2H), 7.54–
7.57 ppm (m, 2H) (C6H5); 13C NMR (75 MHz, CDCl3, TMS):
d = 14.5, 26.2, 65.5, 69.0, 95.4, 122.9, 127.3, 129.2, 137.8, 144.4,
149.0, 150.7 ppm. MS m/z 322 (M+H)+. Anal. Calcd for
C14H15N3O6: C, 52.34; H, 4.70; N, 13.08. Found: C, 52.36; H, 4.73;
N, 13.12.
4.1.1.2. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 6-(nitrooxy)hexyl
carbonate (4b).
Eluent PE/EtOAc 8/2 v/v; yellow oil which
solidified in the freezer. The solid obtained was crystallized from
hexane to give the title compound as a white solid, mp = 39–
40 °C; 60% yield. 1H NMR (300 MHz, CDCl3, TMS): d = 1.36–1.42
(m, 4H, 2CH2), 1.66–1.74 (m, 4H, 2CH2), 2.32 (s, 3H, CH3), 4.22 (t,
2H, CH2), 4.43 (t, 2H, CH2), 6.09 (s, 1H, CH), 7.29–7.34 (m, 1H),
7.41–7.46 (m, 2H), 7.55–7.58 ppm (m, 2H) (C6H5); 13C NMR
(75 MHz, CDCl3, TMS): d = 14.5, 25.1, 25.3, 26.6, 28.2, 69.5, 73.0,
95.4, 122.9, 127.2, 129.1, 137.9, 144.6, 148.9, 151.0 ppm. MS m/z
363 (M+H)+. Anal. Calcd for C17H21N3O6: C, 56.19; H, 5.82; N,
11.56. Found: C, 56.23; H, 5.86; N, 11.58.
4. Experimental section
4.1. Chemistry
1H and 13C NMR spectra were recorded on a Bruker Avance 300;
abbreviations: s, singlet; d, doublet; t, triplet; q, quartet; qi, quin-
tet; m, multiplet; br, broad. Low resolution mass spectra were re-
4.1.1.3. 5,6-Bis(nitrooxy)hexyl 3-methyl-1-phenyl-1H-pyrazol-
5-yl carbonate (4c).
Eluent PE/EtOAc 8/2 v/v; yellow oil; 64%
yield. 1H NMR (300 MHz, CDCl3, TMS): d = 1.43–1.56 (m, 2H, CH2),
1.68–1.79 (m, 4H, 2CH2), 2.32 (s, 3H, CH3), 4.23 (t, 2H, CH2), 4.44
(dd, 1H, CHH), 4.71 (dd, 1H, CHH), 5.23–5.27 (m, 1H, CH), 6.09 (s,
1H, CH), 7.29–7.35 (m, 1H), 7.41–7.47 (m, 2H), 7.54–7.58 ppm
(m, 2H) (C6H5); 13C NMR (75 MHz, CDCl3, TMS): d = 14.5, 21.3,
28.0, 28.9, 68.9, 71.0, 78.7, 95.4, 122.9, 127.2, 129.1, 137.9, 144.5,
corded with
a Finnigan-Mat TSQ-700. Melting points were
determined with a capillary apparatus (Büchi 540). Flash column
chromatography was performed on silica gel (Merck Kieselgel 60,
230-400 mesh ASTM); PE stands for 40–60 petroleum ether. The
progress of the reactions was followed by thin layer chromatogra-

B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
847
148.9, 150.9 ppm. MS m/z 424 (M)+. Anal. Calcd for C17H20N4O9: C,
48.12; H, 4.75; N, 13.20. Found: C, 47.9; H, 4.80; N, 13.09.
The oil obtained was further purified by reverse phase flash chro-
matography (RP-18, eluent MeCN/H2O 6/4 v/v); yellow oil; 45%
yield. 1H NMR (300 MHz, CDCl3, TMS): d = 1.25 (s, 3H, CH3), 1.27
(s, 3H, CH3), 1.45–1.70 (m, 4H, 2CH2), 2.33 (s, 3H, CH3), 4.31 (dd,
1H, CHH), 4.58 (dd, 1H, CHH), 5.06–5.10 (m, 1H, CH), 6.07 (s, 1H,
CH), 7.32–7.51 ppm (m, 5H) (C6H5); 13C NMR (75 MHz, CDCl3,
TMS): d = 14.5, 24.5, 24.9, 25.3, 35.2, 42.4, 70.9, 78.8, 95.6, 124.0,
127.7, 129.0, 137.8, 144.3, 149.1, 172.5 ppm. MS m/z 422 (M)+.
Anal. Calcd for C18H22N4O8: C, 51.18; H, 5.25; N, 13.26. Found: C,
51.01; H, 5.24; N, 13.12.
4.1.2. General procedure for preparation of ester derivatives
7a–i
To a solution/suspension of appropriate acid (5a–i) (3.5 mmol)
in dry toluene (5 mL) anhydrous DMF (two drops) was added, fol-
lowed by SOCl2 (0.3 mL; 4.2 mmol). The reaction mixture was stir-
red at rt until reaction completion (monitored by TLC). Solvent was
evaporated and the acylchloride obtained (6a–i) was dissolved in
dry CH2Cl2 (15 mL). The resulting solution was added dropwise
to a solution of 1 (0.55 g; 3.2 mmol) and Et3N (0.50 mL; 3.2 mmol)
in dry CH2Cl2 (20 mL) at 0 °C. The reaction mixture was stirred at rt
for 1 h, then washed with H2O (20 mL), 0.1 M HCl (50 mL), brine,
dried, and the solvent evaporated. The product was purified by
flash chromatography with the eluent indicated. Solid substances
were further purified by crystallization.
4.1.2.6. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 4-[3-(nitrooxy)pro-
pyl]benzoate (7f).
Eluent PE/EtOAc 8/2 v/v. The oil obtained
was further purified by preparative HPLC (eluent MeCN/H2O 7/3
v/v); colourless oil which solidified in the freezer; 79% yield. The
resulting solid was crystallized from hexane to give the title com-
pound as a white solid, mp = 38.5–39.5 °C. 1H NMR (300 MHz,
CDCl3, TMS): d = 2.06–2.11(m, 2H, CH2), 2.36 (s, 3H, CH3), 2.82
(t, 2H, CH2), 4.45 (t, 2H, CH2), 6.27 (s, 1H, CH), 7.28–7.33 (m, 3H),
7.41–7.46 (m, 2H), 7.58–7.62 (m, 2H), 8.01–8.03 ppm (m, 2H)
(C6H5 + C6H4); 13C NMR (75 MHz, CDCl3, TMS): d = 14.6, 27.9,
31.9, 71.9, 95.8, 123.2, 126.2, 127.2, 128.9, 129.1, 130.8, 138.2,
144.5, 147.3, 149.1, 161.7 ppm. MS m/z 381 (M)+. Anal. Calcd for
4.1.2.1. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 6-(nitrooxy)hexano-
ate (7a).
Eluent PE/EtOAc 9/1 v/v; yellow oil which solidified
in the freezer. The solid obtained was crystallized from hexane to
give the title compound as a white solid, mp = 39–40 °C; 45% yield.
1H NMR (300 MHz, CDCl3, TMS): d = 1.34–1.44 (m, 2H, CH2), 1.64–
1.75 (m, 4H, 2CH2), 2.32 (s, 3H, CH3), 2.52 (t, 2H, CH2), 4.39 (t, 2H,
CH2) 6.08 (s, 1H, CH), 7.29–7.34 (m, 1H), 7.40–7.46 (m, 2H), 7.50–
7.53 ppm (m, 2H) (C6H5); 13C NMR (75 MHz, CDCl3, TMS): d = 14.5,
24.0, 25.0, 26.4, 33.7, 72.8, 95.8, 123.3, 127.2, 129.1, 138.0, 144.3,
149.0, 168.6 ppm. MS m/z 333 (M)+. Anal. Calcd for C16H19N3O5:
C, 57.65; H, 5.74; N, 12.60. Found: C, 57.56; H, 5.66; N, 12.56.
C20H19N3O5: C, 62.98; H, 5.02; N, 11.02. Found: C, 62.99; H, 5.02;
N, 11.04.
4.1.2.7. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 4-[3-(nitrooxy)pro-
poxy]benzoate (7g).
Eluent PE/EtOAc 8/2 v/v. The oil ob-
tained was further purified by preparative HPLC (eluent MeCN/
H2O 6/4 v/v); colourless oil which solidified in the freezer; 79%
yield. The resulting solid was crystallized from hexane to give
the title compound as a white solid, mp = 61.5–62.5 °C. 1H NMR
(300 MHz, CDCl3, TMS): d = 2.25 (qi, 2H, CH2), 2.36 (s, 3H, CH3),
4.14 (t, 2H, CH2), 4.67 (t, 2H, CH2), 6.24 (s, 1H, CH), 6.94 (d, 2H),
7.30–7.32 (m, 1H), 7.40–7.45 (m, 2H), 7.58–7.61 (m, 2H),
8.03 ppm (d, 2H) (C6H5 + C6H4); 13C NMR (75 MHz, CDCl3, TMS):
d = 14.6, 26.9, 63.9, 69.6, 95.8, 114.5, 120.6, 123.2, 127.1, 129.1,
132.6, 138.2, 144.6, 149.1, 161.5, 163.2 ppm. MS m/z 397 (M)+.
Anal. Calcd for C20H19N3O6: C, 60.45; H, 4.82; N, 10.57. Found: C,
60.42; H, 4.84; N, 10.57.
4.1.2.2. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 5,6-bis(nitrooxy)
hexanoate (7b).
Eluent PE/EtOAc 8/2 v/v; yellow oil; 75%
yield. 1H NMR (300 MHz, CDCl3, TMS): d = 1.68–1.82 (m, 4H,
2CH2), 2.32 (s, 3H, CH3), 2.56–2.60 (m, 2H, CH2), 4.40 (dd, 1H,
CHH), 4.67 (dd, 1H, CHH), 5.21–5.22 (m, 1H, CH), 6.08 (s, 1H, CH),
7.35–7.36 (m, 1H), 7.41–7.52 ppm (m, 4H) (C6H5); 13C NMR
(75 MHz, CDCl3, TMS): d = 14.5, 19.9, 28.4, 33.1, 70.9, 78.5, 95.8,
123.3, 127.4, 129.1, 138.0, 144.0, 149.0, 168.1 ppm. MS m/z 394
(M)+. Anal. Calcd for C16H18N4O8: C, 48.73; H, 4.60; N, 14.21.
Found: C, 48.74; H, 4.70; N, 14.16.
4.1.2.3.
(nitrooxy)propanoate (7c).
3-Methyl-1-phenyl-1H-pyrazol-5-yl 2,2-dimethyl-3-
Eluent PE/EtOAc 9/1 v/v; yellow
4.1.2.8.
oxy)propyl]benzoate (7h).
3-Methyl-1-phenyl-1H-pyrazol-5-yl 4-[2,3-bis(nitro-
Eluent PE/EtOAc 8/2 v/v. The oil
oil which solidified on standing. The resulting solid was crystallized
from hexane to give the title compound as a white solid, mp = 35.5–
36.0 °C; 92% yield. 1H NMR (300 MHz, CDCl3, TMS): d = 1.33 (s, 6H,
2CH3), 2.33 (s, 3H, CH3), 4.49 (s, CH2), 6.10 (s, 1H, CH), 7.34–
7.48 ppm (m, 5H) (C6H5); 13C NMR (75 MHz, CDCl3, TMS): d = 14.5,
22.2, 42.5, 76.8, 95.7, 123.7, 127.7, 129.0, 137.7, 143.9, 149.0,
170.0 ppm. MS m/z 319 (M)+. Anal. Calcd for C15H17N3O5: C, 56.42;
H, 5.37; N, 13.16. Found: C, 56.40; H, 5.44; N, 12.99.
obtained was further purified by preparative HPLC (eluent MeCN/
H2O 7/3 v/v); colourless oil; 64% yield. 1H NMR (300 MHz, CDCl3,
TMS): d = 2.37 (s, 3H, CH3), 3.11–3.15 (m, 2H, CH2), 4.45 (dd, 1H,
CHH), 4.73 (dd, 1H, CHH), 5.46–5.49 (m, 1H, CH), 6.28 (s, 1H, CH),
7.37–7.47 (m, 5H), 7.58–7.61 (m, 2H), 8.05–8.07 ppm (m, 2H)
(C6H5 + C6H4); 13C NMR (75 MHz, CDCl3, TMS): d = 14.6, 35.7,
70.0, 78.7, 95.8, 123.3, 127.3, 127.6, 129.1, 129.8, 131.1, 138.1,
140.9, 144.3, 149.1, 161.3 ppm. MS m/z 442 (M)+. Anal. Calcd for
C20H18N4O8: C, 54.30; H, 4.10; N, 12.66. Found: C, 54.32; H, 4.06;
4.1.2.4.
(nitrooxy)hexanoate (7d).
3-Methyl-1-phenyl-1H-pyrazol-5-yl 2,2-dimethyl-6-
Eluent PE/EtOAc 9/1 v/v; yellow
N, 12.58.
oil which solidified on standing. The resulting solid was crystal-
lized from hexane to give the title compound as a white solid,
mp = 51.0–51.5 °C; 90% yield. 1H NMR (300 MHz, CDCl3, TMS):
d = 1.17–1.28 (m, 8H, 2CH3, CH2), 1.54–1.63 (m, 4H, 2CH2), 2.33
(s, 3H, CH3), 4.32 (t, 2H, CH2), 6.06 (s, 1H, CH), 7.30–7.52 ppm (m,
5H) (C6H5); 13C NMR (75 MHz, CDCl3, TMS): d = 14.5, 21.2, 24.9,
27.0, 39.8, 42.7, 72.8, 95.6, 123.8, 127.4, 128.9, 137.9, 144.5,
149.0, 173.0 ppm. MS m/z 361 (M)+. Anal. Calcd for C18H23N3O5:
C, 59.82; H, 6.41; N, 11.63. Found: C, 59.95; H, 6.41; N, 11.65.
4.1.2.9.
oxy)propoxy]benzoate (7i).
3-Methyl-1-phenyl-1H-pyrazol-5-yl 4-[2,3-bis(nitro-
Eluent PE/EtOAc 8/2 v/v. The oil
obtained was further purified by preparative HPLC (eluent MeCN/
H2O 7/3 v/v); colourless oil; 61% yield. 1H NMR (300 MHz, CDCl3,
TMS): d = 2.36 (s, 3H, CH3), 4.31 (d, 2H, CH2), 4.78 (dd, 1H, CHH),
4.92 (dd, 1H, CHH), 5.59–5.65 (m, 1H, CH), 6.25 (s, 1H, CH), 6.97
(d, 2H), 7.26–7.33 (m, H), 7.40–7.45 (m, 2H), 7.57–7.60 (m, 2H)
8.06 ppm (d, 2H) (C6H5 + C6H4); 13C NMR (75 MHz, CDCl3, TMS):
d = 14.6, 64.8, 68.5, 76.3, 95.8, 114.6, 121.7, 123.2, 127.2, 129.1,
132.8, 138.2, 144.5, 149.1, 161.3, 162.0 ppm. MS m/z 458 (M)+.
Anal. Calcd for C20H18N4O9: C, 52.40; H, 3.96; N, 12.22. Found: C,
52.37; H, 4.02; N, 12.09.
4.1.2.5. 3-Methyl-1-phenyl-1H-pyrazol-5-yl 2,2-dimethyl-5,6-
bis(nitrooxy)hexanoate (7e).
Eluent PE/EtOAc 85/15 v/v.

848
B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
4.1.3. Methyl 2,2-dimethyl-5,6-bis(nitrooxy)hexanoate (10)
To a vigorously stirred solution of 9 (1.0 g; 6.4 mmol) and
AgNO3 (3.3 g; 19.0 mmol) in MeCN (20 mL) I2 (1.6 g; 6.4 mmol)
was added in one portion. The reaction mixture was stirred at rt
until all iodine dissolved and then refluxed for 6 h. The precipitate
was filtered and the filtrate poured into H2O (50 mL) and extracted
with EtOAc (100 mL). The organic phase was washed with H2O
(50 mL), brine, dried, and the solvent evaporated. The resulting
oil was purified by flash chromatography (eluent PE/EtOAc 9/1 v/
v) to give the title compound as a colourless liquid; 74% yield. 1H
NMR (300 MHz, CDCl3, TMS): d = 1.21 (m, 6H, 2CH3), 1.61–1.74
(m, 4H, 2CH2), 3.69 (s, 3H, CH3), 4.48 (dd, 1H, CHH), 4.75 (dd, 1H,
CHH), 5.23–5.26 ppm (m, 1H, CH); 13C NMR (75 MHz, CDCl3,
TMS): d = 24.8, 25.2, 25.5, 35.3, 41.9, 52.0, 71.1, 79.3, 177.5 ppm.
MS m/z 281 (M+H)+.
removed and the reaction mixture stirred at rt for 1 h. The organic
phase was decanted, washed with H2O (2 ꢄ 20 mL), 10% urea solu-
tion (15 mL), brine, dried, and the solvent was evaporated. The
resulting oil was purified by flash chromatography (eluent CH2Cl2)
to give the title compound as a colourless oil; 85% yield. 1H NMR
(300 MHz, CDCl3, TMS): d = 1.20 (s, 6H, 2CH3), 1.35–1.46 (m, 2H,
CH2), 1.56–1.61 (m, 2H, CH2), 1.68–1.77 (m, 2H, CH2), 4.46 ppm
(t, 2H, CH2); 13C NMR (75 MHz, CDCl3, TMS): d = 21.3, 24.9, 27.2,
49.8, 42.1, 73.1, 184.7 ppm. MS m/z 206 (M+H)+.
4.2. Stability studies
4.2.1. Stability in water solution
A solution of each compound (10 mM) in DMSO was added to
water (0.1 M KCl) in order to obtain 5% DMSO (v/v) in the mixture.
The suspension was filtered using 4 mm, 0.2 lm nylon filters (Ti-
4.1.4. 2,2-Dimethyl-5,6-bis(nitrooxy)hexanoic acid (5e)
A mixture of 10 (1.3 g; 3.7 mmol) 1,4-dioxane (5 mL), 6 M HCl
(5 mL) and DMSO (1 mL) was heated at reflux for 6 h. The reaction
mixture was then cooled, poured into H2O (20 mL) and extracted
with EtOAc (25 mL). The organic phase was washed with brine,
dried, and the solvent evaporated. The resulting oil was purified
by flash chromatography (eluent CH2Cl2 + 0.1% HCOOH) to give
the title compound as a colourless oil which solidified on standing;
36% yield. The resulting solid was crystallized from hexane/toluene
mixture, mp = 87.5–88.5 °C. 1H NMR (300 MHz, CDCl3, TMS):
d = 1.24–1.25 (m, 6H, 2CH3), 1.65–1.80 (m, 4H, 2CH2), 4.49 (dd,
1H, CHH), 4.75 (dd, 1H, CHH), 5.24–5.26 ppm (m, 1H, CH); 13C
NMR (75 MHz, CDCl3, TMS): d = 24.6, 25.1, 25.3, 35.1, 41.7, 71.0,
79.2, 174.7. MS m/z 267 (M+H)+.
tan), obtaining a solution useful for the experiments. The resulting
solution was maintained at rt and, at appropriate time intervals, a
100 lL aliquot of reaction mixture was analyzed by RP-HPLC. The
reverse-phase HPLC procedure allowed for the separation and
quantitation of compounds and hydrolysis product. HPLC analyses
were performed with a Hitachi Elite LaChrom equipped with a
L-2130 pump, a L-2200 autosampler, a L-2400 UV detector, a L-
7614 degasser and a Gynkotek STH585 oven. Data were analyzed
using the EZChromElite V. 3.1.7. The analytical column was a Zor-
bax Extend C18 (150 ꢄ 4.6 mm, 5
lm particle size). The mobile
phase consisted of methanol/water at 1.0 mL minꢁ1 with gradient
conditions: 70% methanol until 2.5 min, from 70% to 80% methanol
between 2.5 and 5.0 min, 80% methanol for next 3 min and from
80% to 70% methanol between 8 and 12 min. The injection volume
was 100 lL. The column effluent was monitored at 240 nm (for
4.1.5. Methyl 6-hydroxy-2,2-dimethylhexanoate (11)
compounds) referenced against a 600 nm wavelength. Quantita-
tion was done using calibration curves of compounds; the linearity
of the calibration curves was determined in a concentration range
To a vigorously-stirred mixture of NaBH4 (2.9 g; 76 mmol), 2-
methylbut-1-ene (30 mL; 0.28 mol) and dry THF (30 mL) kept un-
der positive N2 pressure, BF3ꢃEt2O (7.1 mL; 56 mmol) was added
dropwise at ꢁ10 °C. The reaction mixture was stirred at rt for
5 h, cooled in an ice-bath, and a solution of 9 (0.84 g; 5.6 mmol)
was added dropwise. The cooling bath was removed and the reac-
tion mixture stirred at rt overnight. The following day, the reaction
was cooled to ꢁ10 °C and, after the sequential addition of H2O
(36 mL), 3 M NaOH (36 mL) and H2O2 30% (54 mL), it was heated
to 40 °C for 2 h. The organic phase was separated and the water
phase extracted with Et2O (2 ꢄ 75 mL). The combined organic ex-
tracts were washed with brine, dried, and the solvent evaporated.
The resulting liquid was purified by flash chromatography (eluent
PE/EtOAc 8/2 v/v) to give the title compound as a colourless liquid;
71% yield. 1H NMR (300 MHz, CDCl3, TMS): d = 1.17 (s, 6H, 2CH3),
1.26–1.34 (m, 2H, CH2), 1.50–1.57 (m, 4H, 2CH2), 1.79 (br s, 1H,
OH), 3.62–3.66 ppm (m, 5H, CH2 + CH3); 13C NMR (75 MHz, CDCl3,
TMS): d = 21.2, 25.2, 33.0, 40.4, 42.3, 51.7, 62.6, 178.6 ppm. MS m/z
175 (M+H)+.
of 1–200 l
M (r2 >0.99).
4.2.2. Stability in acidic medium
A 2 mL aliquot of 0.5 mM solution of each compound in metha-
nol was diluted to 10 mL using a 0.1 M HCl/MeOH 60/40 (v/v) mix-
ture. The resulting solution was maintained at 37 0.5 °C and, at
appropriate time intervals, a 20 lL aliquot of reaction solution
was analysed by RP-HPLC. HPLC analyses were performed with a
HP1100 chromatograph system (Agilent Technologies, Palo Alto,
CA, USA) equipped with a quaternary pump (model G1311A), a
membrane degasser (model G1379A), and a diode-array detector
(DAD) (model G1315B). Data analysis was accomplished using a
HP ChemStation system (Agilent Technologies). The analytical col-
umn was a Zorbax Eclipse C18 (150 ꢄ 4.6 mm, 5
lm particle size).
The mobile phase consisted of acetonitrile/water at 1.2 mL minꢁ1
with gradient conditions: 40% acetonitrile until 2.0 min, from
40% to 70% acetonitrile between 2.0 and 5.0 min, 70% acetonitrile
for next 5 min and from 70% to 40% acetonitrile between 10 and
4.1.6. 2,2-Dimethyl-6-(nitrooxy)hexanoic acid (5d)
12 min. The injection volume was 20 lL (Rheodyne, Cotati, CA).
KOH (0.59 g; 11 mmol) was added to a solution of 11 (0.66 g,
3.8 mmol) in MeOH (15 mL) and H2O (15 mL). The reaction mix-
ture was heated at reflux for 6 h, after which it was cooled to rt,
poured into H2O (30 mL) and extracted with Et2O (2 ꢄ 15 mL).
The water phase was then acidified with 6 M HCl until pH 1 and
extracted again with Et2O (2 ꢄ 25 mL). The organic phases were
washed with brine, dried, and the solvent evaporated. The result-
ing 6-hydroxy-2,2-dimethylhexanoic acid was used further with-
out any purification. To vigorously-stirred fuming HNO3 (2 mL),
cooled in an ice bath, concd H2SO4 (2 mL) was added dropwise at
t <15 °C. To the resulting sulfonitric mixture, CH2Cl2 (10 mL) was
added, followed by the dropwise addition of a solution of the hy-
droxyl derivative in CH2Cl2 (5 mL) at t <10 °C. The ice bath was
The column effluent was monitored at 240 nm referenced against
a 600 nm wavelength. Quantitation was done using calibration
curves of compounds; the linearity of the calibration curves was
determined in a concentration range of 1–200 l
M (r2 >0.99).
4.2.3. Stability in human serum
A solution of each compound (10 mM) in MeOH was added to
human serum (from human male AB plasma, Sigma) preheated
at 37 °C. The final concentration of the compound was 250
The resulting solution was incubated at 37 0.5 °C and, at appro-
priate time intervals, a 300 L aliquot of the reaction mixture
was withdrawn and added to 450 L of acetonitrile containing
0.1% trifluoroacetic acid, in order to deproteinize the serum. The
lM.
l
l

B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
849
sample was sonicated, vortexed, and then centrifuged for 10 min at
2150g. The clear supernatant was filtered by 0.45 m PTFE filters
4.4. Lipophilicity studies
l
(Alltech) and analyzed by RP-HPLC using the HP1100 chromato-
graph system described in the ‘acidic medium stability’
experimental section.
The logPoct of all compounds was determined by a RP-HPLC
method using the HP1100 chromatograph system described in
the ‘acidic medium stability’ experimental section. Retention times
were measured on
a
Discovery RP-amide-C16 column
4.2.4. Stability in rat-liver homogenate
(150 ꢄ 4.6 mm i.d., 5 mm; Supelco, Bellefonte, PA, USA) thermo-
stated at 30 °C. The flow rate was 1.0 mL minꢁ1. The column efflu-
ent was monitored at 226 and 240 nm referenced against a 600 nm
wavelength. Each analysis was performed isocratically using pH
7.0 phosphate buffer and methanol mixtures in proportions vary-
ing from 30 to 70% (v/v), with final ionic strength of 20 mM. Before
use, the mobile phase was filtered under vacuum through a
Liver homogenate from male Wistar rats (200ꢁ250 g) was pre-
pared by centrifuging (8000g, 20 min) a suspension of 20% tissue in
a Tris–HCl/KCl (100 mM/150 mM, pH 7.4) and stored at ꢁ80 °C.
Incubation was at 37 °C in a Tris–HCl/KCl (100 mM/150 mM, pH
7.4) containing a known amount of homogenate (6.4 mg prot/
mL), 2 mM GSH, 1 mM NADPH; a solution of each compound
(10 mM) in acetonitrile was added to incubation mixture pre-
heated at 37 °C, the final concentration of the compound was
0.45
solutions (10 mM) of compounds were prepared in methanol and
diluted (1–0.1 mM) in the mobile phase for injection (20 L). All
lm HA Millipore filter (Millipore, Milford, MA, USA). Stock
200
at fixed times, 700
deproteinized with 700
sonicated, vortexed, and then centrifuged for 10 min at 2150g.
The clear supernatant was filtered through 0.45 m PTFE filters
(Alltech). A 20 L aliquot of filtrate was analyzed by RP-HPLC to
l
M. The resulting solution was incubated at 37 0.5 °C and,
L of incubation mixture were removed and
L of cold acetonitrile; the sample was
l
l
samples were injected at least three times for each mobile phase.
Uracil was used as the non-retained compound. The logarithms
of the capacity factor (logk) for a minimum of four different meth-
anol/buffer ratios were measured for each compound. Logkw,
namely the logarithm of the capacity factor corresponding to 0%
methanol modifier, was obtained by linear extrapolation (r2
>0.99, for all the compounds). LogPoct values of edaravone and
derivatives were determined using the reference line obtained
from the correlation between logPoct and logkw of 63 known struc-
turally-diverse compounds.5
l
l
l
determine the stability of the compound in the homogenate (anal-
yses were performed using the RP-HPLC method described in the
‘acidic medium stability’ experimental section). An 800 lL aliquot
of filtrate was analyzed by UV–vis spectroscopy (UV 2501-PC, Shi-
madzu) to determine nitrate and nitrite production. Nitrite and ni-
trate quantitation was performed by adapting a method described
by Miranda et al.: nitrite and nitrite + nitrate (NOx) concentrations
were determined in parallel.19 Briefly, for nitrite + nitrate (NOx)
4.5. Permeability studies
analysis, a 400
saturated solution (400 mg in 50 mL HCl 1 M), rapidly followed
by the addition of 400 L Griess reagent (200 L NEDD 0.1% w/v
in water + 200 L SULF 2% w/v in 5% HCl v/v); for nitrite analysis,
a 400 L aliquot of filtrate was added to 400 L of HPLC-grade
water and to 400 L of the Griess reagent. The reaction mixtures
l
L aliquot of filtrate was added to 400
l
L of VCl3
Permeation experiments were carried out in 96-well microtiter
polycarbonate filter plates obtained from Millipore AG
(MPC4NTR10, Volketswil, Switzerland). Polycarbonate filter speci-
fications were as follows: 0.45 lm pore size, 10 lm thickness, and
5–20% porosity. An average porosity value of 12.5% was used for
l
l
l
l
l
l
permeability calculations. Each well of the filter plate was impreg-
were incubated at 37 °C for 45 min (NOx detection) and for
10 min (nitrite detection) and in both cases the absorbance at
540 nm was measured. NOx quantitation was done using calibra-
tion curves obtained for nitrite and nitrate standard solutions;
the linearity of the calibration curves was determined in a concen-
nated with 15
for at least 10 min for complete evaporation of the hexane. Subse-
quently, the donor compartments were hydrated with 280 L of
lL of 5% hexadecane dissolved in hexane and shaken
l
solution (i.e., prepared as described in the ‘water solution stability’
experimental section) of test compound in water, containing 5%
DMSO and 100 mM KCl, and placed upon a Teflon acceptor plate
(MSSACCEPTOR, Millipore, Volketswil, Switzerland), which had
been prefilled with water containing 5% DMSO and 100 mM KCl.
The resulting sandwich was incubated at room temperature under
constant light shaking (150 rpm). After appropriate time intervals
(varying between 1 and 6 h), the sandwich was disassembled and
tration range of 1–100 l
M (r2 >0.99).
4.3. In vivo study of nitrite production
Compounds 4a, 7d, and ISMN as reference, were orally admin-
istered to mice (10 mg/kg in imwitor742/tween80 50/50) and ni-
trite blood level over 24 h was monitored. At fixed times blood
was drawn, protein-precipitated using 3 volumes of acetonitrile
and analyzed by adapting a method described by Misko et al.20
a 100 lL aliquot of solutions in the acceptor and donor compart-
ment were analyzed by RP-HPLC. Analyses were performed using
the RP-HPLC method and the Hitachi EliteLaChrom system, de-
scribed in the ‘water solution stability’ experimental section.
Membrane integrity was checked by electrical resistance mea-
surements conducted on the filter plate at the end of the incuba-
tion time, using an electrometer system especially designed for
PAMPA assays (EVOMX and MULTI96, World Precision Instru-
ments, Sarasota FL).
10
200
at room temperature to convert the nitrite to (1H)-naphtotriazole
(NAT); after 20 min the reaction was stopped by adding 10 L of
lL of 1 mM 2,3-diaminonaphtalene (DAN) were added to
l
L of the deproteinized sample and left under gentle shake
l
1 M NaOH. As the conversion of nitrite to NAT in deproteinized
blood is not quantitative, a set of deproteinized blood samples
spiked with Na15NO2 at four different concentrations (0, 0.1, 1.0
and 10
l
M) were added to each batch prior to nitrite conversion.
4.6. Vasodilator studies
Samples and spikes were analyzed by LC–MS/MS instead of the
usual fluorimetric detection against a calibration curve of NAT in
Thoracic aortas were isolated from male Wistar rats weighing
180–200 g. As few animals as possible were used. The purposes
and protocols of these studies were approved by the Ministry for
Health, Rome, Italy. The endothelium was removed, the vessels
were cut helically, and three strips were obtained from each aorta.
The tissues were mounted under 1.0 g tension in organ baths con-
taining 30 mL of Krebs-bicarbonate buffer with the following com-
position (mM): NaCl 111.2, KCl 5.0, CaCl2 2.5, MgSO4 1.2, KH2PO4
the concentration range 0.01–10
lM. Nitrite basal level was ob-
tained from the 0 M spikes while the nitrite-to-naphtotriazole
l
conversion factor was calculated from the ratio between experi-
mental and nominal concentration of NAT in the 0.1, 1.0 and
10 lM spikes. This conversion factor was used to calculate the ori-
ginal nitrite concentration in the samples from the NAT experi-
mental concentration.

850
B. Rolando et al. / Bioorg. Med. Chem. 20 (2012) 841–850
2. Morphy, R.; Rankovic, Z. Curr. Pharm. Des. 2009, 15, 587.
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1.0, NaHCO3 12.0, glucose 11.1, maintained at 37 °C and gassed
with 95% O2–5% CO2 (pH 7.4). The aorta strips were allowed to
equilibrate for 1.5 h and then contracted with 1 lM L-phenyleph-
rine. When the response to the agonist reached a plateau, cumula-
tive concentrations of the vasodilator agent were added. Results
are expressed as EC50 ( SEM)
lM. The effects of 1 lM ODQ on
relaxation were evaluated in a separate series of experiments in
which it was added to the organ bath 5 min before contraction.
In this protocol, the inhibitor is pre-incubated for at least 30 min
before addition of the vasodilator compound. Responses were re-
corded by an isometric transducer connected to the MacLab Sys-
tem PowerLab. Addition of the drug vehicle, DMSO, had no
appreciable effect on contraction. At least five experiments for each
compound were performed.
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Acknowledgements
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This work was supported by a grant from Regione Piemonte
Progetto Ricerca Sanitaria Finalizzata 2009.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at doi:10.1016/j.bmc.2011.11.065.
19. Miranda, K. M.; Espey, M. G.; Wink, D. A. Nitric Oxide 2001, 5(1), 62.
20. Misko, T. P.; Schilling, R. J.; Salvemini, D.; Moore, W. M.; Currie, M. G. Anal.
Biochem. 1993, 214, 11.
References and notes
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