Nitric oxide donor β2-agonists: Furoxan derivatives containing the fenoterol moiety and related furazans

Article abstract of DOI:10.1021/jm0704595

The structure of fenoterol, a β₂-adrenoceptor agonist used in therapy, has been joined with furoxan NO-donor moieties to give new NO-donor β₂-agonists. The furazan analogues, devoid of the property to release NO, were also synthesized for comparison. All the compounds retained β₂-agonistic activity at micromolar or submicromolar concentration when tested on guinea pig tracheal rings precontracted with carbachol. Among the furoxan derivatives, the NO contribution to trachea relaxation was evident with product 15b at micromolar concentrations. All the new NO-donor hybrids were able to dilate rat aortic strips precontracted with phenylephrine. Both furoxan and furazan derivatives displayed antioxidant activity greater than that of fenoterol.

Full text of DOI:10.1021/jm0704595

J. Med. Chem. 2007, 50, 5003-5011
5003
Nitric Oxide Donor â2-Agonists: Furoxan Derivatives Containing the Fenoterol Moiety and
Related Furazans
M. Federica Buonsanti, Massimo Bertinaria, Antonella Di Stilo, Clara Cena, Roberta Fruttero, and Alberto Gasco*
Dipartimento di Scienza e Tecnologia del Farmaco, UniVersita` degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy
ReceiVed April 18, 2007
The structure of fenoterol, a â2-adrenoceptor agonist used in therapy, has been joined with furoxan NO-
donor moieties to give new NO-donor â2-agonists. The furazan analogues, devoid of the property to release
NO, were also synthesized for comparison. All the compounds retained â2-agonistic activity at micromolar
or submicromolar concentration when tested on guinea pig tracheal rings precontracted with carbachol.
Among the furoxan derivatives, the NO contribution to trachea relaxation was evident with product 15b at
micromolar concentrations. All the new NO-donor hybrids were able to dilate rat aortic strips precontracted
with phenylephrine. Both furoxan and furazan derivatives displayed antioxidant activity greater than that of
fenoterol.
Chart 1. Structures of (RR,SS)-Fenoterol (1a) and
(RS,SR)-Fenoterol (1b)
Introduction
Bronchial constriction is characteristic of asthmatic airway
obstruction and of chronic obstructive pulmonary diseases
(COPD). Selective â2-adrenoceptor agonists are certainly among
the most effective and safest drugs to treat these pathologies.
By activating the â2-receptors positioned on the smooth muscle
bronchial cells, they induce an increase in cAMP with conse-
quent muscle relaxation. A wide number of these drugs,
characterized by having different pharmacokinetic properties,
have been developed in the past.1 These products also display
in vitro antioxidant properties with respect to reactive oxygen
species-mediated cytotoxicity.2 Nitric oxide (NO) is a physi-
ological messenger that triggers a variety of actions in several
systems;3 they include relaxation of vascular smooth muscle,
inhibitory effects on smooth muscle proliferation, inhibition of
platelets aggregation and adhesion, and bronchodilation. NO
triggers all these actions through a mechanism which involves
the activation of the soluble guanylate cyclase (sGC). NO plays
relevant roles in the respiratory system, and its absence or
dysfunction is implicated in a number of airway diseases.4,5 The
therapeutic potential of NO donors in the treatment of respiratory
diseases has recently been discussed.5-8 During the past few
years, a link between â-adrenergic receptors and NO generation
in the cardiovascular system has been evidenced.9 In particular,
the role of â2-adrenoceptors under this aspect has been
discussed.10 There is evidence that â2-mediated vasodilation is
partly or completely NO-mediated in resistance vasculature in
vivo as well as the effects of â2-agonists on arterial pulse wave
reflection. Fenoterol is a short-acting â2-selective agonist
characterized by a prompt onset action, currently used in
therapy.11 It has two chiral centers and exists in two diastere-
oisomers, 1a and 1b, each of them is a 50/50 mixture of two
enantiomers (Chart 1). The RR,SS diastereoisomer 1a is the
marketed product. As development of our research on multi-
functional drugs containing NO-donor moieties, we designed
the new NO-donor â2-agonists 15a, 34a, 36a, 15b, 34b, and
36b, deriving from the conjugation of diastereoisomers 1a and
1b, respectively, to NO-donor furoxan substructures, endowed
with different capabilities of producing NO. This paper describes
the synthesis, structural characterization, dilator, and antioxidant
properties of these products. The furazan analogues 16a, 35a,
37a, 16b, 35b, and 37b, devoid of the property to release NO,
are also considered for comparison.
Results and Discussion
Chemistry. The synthesis of the final drugs required pre-
liminary preparation of the oxirane derivative 6 and of the amino
derivative 10 (Scheme 1). The reaction of 3,5-diacetoxyac-
etophenone 2 with bromine in CHCl3 solution, followed by
heating with HBr in ethanol solution, gave the crude bromo
derivative 3. The protection of the hydroxyl groups of 3 was
accomplished with tert-butyldimethylchlorosilane (TBDMSCl)
in DMF in the presence of diisopropylethylamine (DIPEA).
Chloride 4 was the principal product of this reaction, not the
expected bromide, because a halogen exchange reaction occurs
with the excess of TBDMSCl used. Chloride 4 was treated with
NaBH4 to give alcohol 5, which was transformed into 6 by
action of NaH in THF. Amine 10 was obtained by starting with
p-methoxybenzyl methyl ketone 7. This ketone was heated in
ethanol with NH2OH to obtain the corresponding oxime 8 that,
in turn, was reduced to amine 9 in NH3-saturated methanol
solution, using H2 in the presence of Ni W-612 as the catalyst.
The hydrochloride of amine 10 was obtained by modified
literature method,13 hydrolyzing the methoxy group of 9 with
37% HCl at 140 °C in a closed vessel.
Oxirane derivative 6 was treated with the amine hydrochloride
10 in methanol solution in the presence of triethylamine to give
the 1:1 mixture of the two diastereoisomers 11 (Scheme 2).
Reaction of this mixture with tert-butyldicarbonate ((Boc)2O)
* To whom correspondence should be addressed. Phone: +39 011
6707670. Fax: +39 011 6707286. E-mail: alberto.gasco@unito.it.
10.1021/jm0704595 CCC: $37.00 © 2007 American Chemical Society
Published on Web 09/11/2007

5004 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Scheme 1. Synthesis of Intermediates 6 and 10a
Buonsanti et al.
Scheme 4. Synthesis of Intermediates 22-25a
a Reagents and conditions: (a) Et3N, (Boc)2O, H2O, CH3CN; (b) PPh3,
DIAD, compound 18-21, dry THF, 0 °C to rt; (c) TFA, CH2Cl2, 10% aq
Na2CO3.
Scheme 5. Synthesis of Final Compounds 34a-37a and
34b-37ba
a Reagents and conditions: (a) Br2, CHCl3; (b) 5 N HBr, EtOH, reflux;
(c) TBDMSCl, DIPEA, DMF; (d) NaBH4, MeOH; (e) NaH, dry THF, N2;
(f) NH2OH‚HCl, 10% aq NaOH, EtOH, reflux; (g) H2, 10% Ni W-6,
MeOH(NH ), 30 bar, 68 °C; (h) Et2O(HCl); (i) 37% aq HCl, 140 °C, closed
3
vessel.
Scheme 2. Synthesis of Fenoterol Diastereoisomers 1a and 1ba
a Reagents and conditions: (a) Et3N, MeOH, reflux; (b) (Boc)2O, CH2Cl2,
rt; (c) HPLC separation; (d) 1 N HBr, MeOH.
Scheme 3. Synthesis of Final Compounds 15a, 15b, 16a, and
16ba
a Reagents and conditions: (a) MeOH, reflux; (b) (Boc)2O, CH2Cl2, rt;
(c) HPLC separation; (d) 1 N HCl; (e) 10% aq NaHCO3, H2C2O4/EtOAc.
16b, which were isolated as oxalates. The protection of the
amino group of 10 was accomplished with (Boc)2O to give 17
(Scheme 4). Finally, the appropriately substituted furoxan and
furazan derivatives 18-21 were coupled to the protected amine
17 in THF solution in the presence of PPh3 and diisopropyl
azodicarboxylate (DIAD; Mitsunobu reaction), affording 22-
25. The reaction of these latter amines with 6 in refluxing
methanol yielded the intermediate mixtures of diastereoisomers
26a-29a + 26b-29b, which were treated with (Boc)2O in CH2-
Cl2 to give the corresponding mixtures 30a-33a + 30b-33b
(Scheme 5). Each mixture was resolved into the two corre-
sponding diastereoisomers by direct phase HPLC method (see
Experimental Section), giving 30a-33a and 30b-33b. The
single diastereoisomers were deprotected in acidic conditions,
affording the final pure diastereoisomers 34a-37a and 34b-
37b.
a Reagents and conditions: (a) 50% aq NaOH, dry THF, N2; (b)
MeOH(HCl); (c) 10% aq NaHCO3, H2C2O4/EtOAc.
gave mixture 12, which was subsequently separated by HPLC
to give 12a (first eluted) and 12b. Treatment with 1 N HBr
gave rise from 12a to the fenoterol diastereoisomer 1a, identical
to the RR,SS marketed drug (mixed mp, HPLC, 13C NMR
spectra) and from 12b to the diastereoisomer 1b to which the
RS,SR configuration was consequently assigned. The protected
fenoterol diastereoisomers 12a and 12b were reacted with either
bis(phenylsulfonyl)furoxan 13 or the analogue furazan 14 in
THF solution in the presence of 50% NaOH (Scheme 3).
Treatment with HCl-saturated methanol and then with 10%
NaHCO3 gave rise to the final target compounds 15a, 16a, 15b,
The assignment of the configurations to the couples of
diasteroisomers 15a, 15b and 16a, 16b follows immediately

Nitric Oxide Donor â-Agonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 5005
Table 1. Pharmacological Profile and Nitrite Formation of Fenoterol
Derivatives and References 1a, 1b, 18, 20, and CHF 2363
from the synthetic pathway used to prepare these products from
RR,SS 12a and RS,SR 12b. The configurations of the other
diastereoisomer fenoterol derivative pairs were assigned on the
basis of a comparison of their 13C NMR spectra with those of
1a and 1b. On this subject, the signals belonging to CHOH,
CHCH3, and CH3 groups, spatially close to the chiral centers,
are of particular utility. In the case of 1a, 1b, and in the related
stereoisomers 15a, 16a, 15b, and 16b, the carbon chemical shifts
of all these groups are always upfield in the RR,SS forms with
respect to RS,SR forms (see Experimental Section). Conse-
quently, the configurations of all other diastereoisomers were
assigned on the basis of this statement.
NO Release. The ability of the furoxan diastereoisomers 15a,
15b, 34a, 34b, 36a, and 36b to release NO was indirectly
evaluated through their capacity to produce nitrite in 37 °C water
solution, at physiological pH (7.4), in the presence of a strong
excess of cysteine. Nitrite is the most important product of the
oxidation of NO in aerobic aqueous solution. It was detected
by Griess reaction, according to the procedure we previously
used.14 The results, expressed as NO2-% (mol/mol) are de-
scribed in Table 1. The ability of the products to generate nitrite
generates the rank order 15a, 15b > 34a, 34b > 36a, 36b.
NO release was also evaluated on 15a, 34a, and 36a under
conditions more similar to physiological ones, using a Clark-
type electrode. The experiments were carried out in phosphate
buffer (pH 7.4) in the presence of ascorbate (1 mM) and
glutathione (3 mM), following the release over 10 min. The
release profile for 15a is reported in Figure 1. The product when
tested at 1 µM concentration showed a peak concentration of
0.31 ( 0.03 µM after about half a minute, and after about 2
min, the release was complete. The NO release from 34a was
evaluated under the same conditions. When 34a was tested at
1, 10, and 100 µM concentrations, the NO released was not
detectable, however, when the product was tested at 500 µM
concentration a signal corresponding to the NO release was
detected (Figure 1). The peak concentration was 0.64 ( 0.04
µM after about 10 min, and the release, after this time, was not
complete. NO release from 36a (500 µM) was undetectable
when the product was tested under the aforementioned condi-
tions. In conclusion, 15a appears to be a more potent and faster
NO donor with respect to 34a.
Biological Results. Myorelaxing Activity. The myorelaxing
effects of the two diastereoisomers of fenoterol 1a and 1b, the
target furoxan products 15a, 34a, 36a, 15b, 34b, and 36b, and
related furazans 16a, 35a, 37a, 16b, 35b, and 37b were assessed
on guinea pig tracheal rings precontracted with 1 µM carbachol.
All the products were able to relax the contracted tissue in a
concentration-dependent manner and produced the same maxi-
mum response as (-)-isoprenaline. The potencies of the products
expressed as EC50 value are described in Table 1. The RR,SS
diastereoisomer 1a, the marketed form of fenoterol, displays a
potent relaxant effect, about 450 times higher than that triggered
by the RS,SR diastereoisomer 1b. As expected, the higher
activity of the RR,SS diasteroisomer with respect to the RS,SR
diastereoisomer occurs in all of the other diastereoisomer couples
studied, but in a less-pronounced manner. Both in the furoxan
and in the furazan series, the potencies of the RR,SS diastere-
oisomers are lower than the potency of 1a, with the only
exception of phenylsulfonylfuroxan derivative 15a, which is
equipotent. By contrast, the potencies of the RS,SR diastereoi-
somers are higher than the potency of 1b. The ratio between
these figures (EC50b/EC50a) ranges from about 6 to 46. The
different binding modes of the two diastereoisomers should be
responsible for this behavior.
a Determined on guinea pig tracheal rings precontracted with 1 µM
carbachol. b Determined on rat thoracic aorta precontracted with 1 µM
phenylephrine. c Measured as the ability of the compounds 15a, 34a, 35a,
36a, and 37a tested as the hydrochlorides to inhibit lipid peroxidation in
rat hepatic microsomial membranes. d A solution of the appropriate
compound (100 µM) in 50 mM phosphate buffer (pH 7.4) and 1% DMSO
containing 5 mM L-cys was incubated at 37 °C for 1 h; 1 mL of the reaction
mixture was treated with the Griess reagent (250 µL).14 No production of
nitrite was observed in the absence of l-cys. e Calculated with Bio-Loom
for Windows v. 1.5, BioByte Corp. f P < 0.01 when compared to EC50
value in the absence of ODQ, Student’s t-test for unpaired values.
g Myorelaxation did not reach 50% at maximal concn tested (100 µM).
The furoxan derivatives 15a, 15b, 34a, 34b, 36a, and 36b
and fenoterol diastereoisomer 1a (as the reference) were then
tested for their myorelaxing activity in the presence of 10 µM
1H-[1,2,4]oxadiazolo-[4,3-a]quinoxalin-1-one (ODQ), a well-
known inhibitor of soluble guanylate cyclase (sGC), to evidence
the possible contribution to the relaxation exerted by the released
NO.15 No variation in the concentration-response curves was
observed when products 15a, 34a, 34b, 36a, and 36b and the
reference 1a were employed; this excludes any valuable
contribution to the relaxation by NO. The only exception is the

5006 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Buonsanti et al.
sidered a “well-balanced” hybrid in vitro in a limited concentra-
tion range.
Antioxidant Properties. As outlined above, â2-agonists have
in vitro antioxidant functions. These are strictly correlated to
the presence of phenol groups within their structures. Phenols
are easily oxidized; this capacity is higher for the products
containing more than an aromatic hydroxyl group. The anti-
oxidant function of â2-agonists may be of interest for their
application in the treatment of a number of airways diseases.17
As far as fenoterol is concerned, its ability to inactivate
superoxide anion, hydrogen peroxide, hydroxyl radical, and
hypochlorous acid has been established.2 Consequently, we
evaluated the antioxidant properties of fenoterol diastereoisomers
1a, 1b, and of all the fenoterol derivatives of series a, possessing
the same configuration of marketed fenoterol, through their
ability to inhibit the ferrous salt/ascorbate-induced peroxidation
of lipids present in microsomial membranes of rat hepatocytes.
The progress of this reaction was followed using visible
spectroscopy to detect the thiobarbituric reactive species
(TBARS) produced in the oxidation. This is a very common
technique used in this kind of work.18 The antioxidant potencies
of the two diastereoisomers of fenoterol and of the RR,SS
diastereoisomer of each furazan and furoxan derivative of
fenoterol, expressed as IC50, namely, the concentration able to
inhibit 50% of the TBARS formation, are collected in Table 1
together with the calculated log P. As expected, the antioxidant
potencies of 1a and 1b are the same. All the other products
display antioxidant activities in a range roughly near to that of
fenoterol, with the exception of 15a and 16a. It is likely that
the higher antioxidant potencies of these two products are
principally connected with their definitively higher lipophilicity.
Direct measurements of log P, as well as evaluation of other
molecular descriptors that could influence the antioxidant
behavior of a phenol derivative carried out on a more extended
series of products, are needed to confirm this hypothesis.
Figure 1. Release of NO from 15a (1 µM) and 34a (500 µM). The
NO electrode was inserted into a 10 mL (final volume) aliquot of
phosphate buffer, pH 7.4. Arrows indicate the time points of consecutive
additions of tested compound (a), sodium ascorbate (1 mM; b) and
glutathione (3 mM; c).
Figure 2. Concentration-response curves for myorelaxing activity of
1a (diamond), 15b (circle), 15b in the presence of 10 µM ODQ (square),
and 15b in the presence of 100 µM propranolol (triangle) on
precontracted guinea pig trachea.
Vasodilator Activity. The vasodilator effects of target NO
donor furoxan derivatives were assessed on denuded rat aorta
strips precontracted with phenylephrine. The vasodilator poten-
cies of the products are described in Table 1. Within a fixed
couple of diastereoisomers, the vasodilator potencies are the
same, according to the presence of the same NO donor moiety.
The most potent vasodilators were the couple of diastereoiso-
mers containing the phenylsulfonylfuroxan substructure 15a and
15b, followed by the furoxancarboxamide diasteroisomers 34a
and 34b, and then by the methylfuroxan ones 36a and 36b,
keeping with the vasodilator potency of the simple related
furoxans CHF 2363, 18, and 20. This sequence parallels their
capacity to produce nitrite in 37 °C aqueous solution, at
physiological pH (7.4), in the presence of a relevant excess of
cysteine. When the vasodilation experiments were repeated in
the presence of ODQ, a strong decrease in the potencies
occurred. This is in keeping with the NO-mediated activation
of the sGC.
RS,SR diastereoisomer 15b, for which a decrease in the relaxing
potency was observed. To determine whether there was any NO-
dependent myorelaxant activity, the NO-donor hybrids were then
studied in the presence of 100 µM propranolol, a well-known
potent â-antagonist.16 In our experimental conditions, this high
concentration of propranolol proved to be able to almost
completely abolish the â2-mediated relaxant activity on guinea
pig trachea of fenoterol 1a and of the furazan derivatives 16a,
16b, 35a, 35b, 37a, and 37b, which are structurally related to
furoxan analogues but devoid of the ability to release NO.
Under these experimental conditions, the activity of furoxan
hybrids was not completely abolished, and EC50 values in the
µM range were obtained. This residual activity is likely to be
NO-dependent because similar myorelaxing potency (EC50s) was
obtained for the simple NO donor furoxan models 18 and CHF
2363. To further confirm this hypothesis, we repeated the
experiments in the presence of both 100 µM propranolol and
10 µM ODQ. Under these conditions, EC50 values were not
calculable for all the compounds because myorelaxation did not
reach 50% at maximal concentration tested (100 µM).
Analysis of the concentration-response curves obtained under
different conditions used showed that there is no overlapping
between the concentration range in which the two relaxations,
â2-receptor-dependent and NO-dependent, respectively, occur.
The only exception is product 15b in which partial overlapping
occurs (Figure 2). Consequently, this compound can be con-
Conclusion
We have synthesized a series of diasteroisomer couples of
fenoterol analogues containing NO donor furoxan moieties and
a series of the related furazans. The configuration of the single
diastereoisomers was assigned by chemical or spectroscopic
correlations with the two diastereoisomers of fenoterol. Almost
all the products were able to relax precontracted guinea pig
tracheal rings through â2-mediated receptor mechanism at
submicromolar concentrations. They were full â2-agonists in
this preparation possessing the same efficacy as (-)-isoprena-

Nitric Oxide Donor â-Agonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 5007
t, Ph-4-H, J ) 2.2 Hz), 4.65 (2H, s, CH2Cl), 1.00 (18H, s, C(CH3)3),
0.24 (12H, s, CH3Si). 13C NMR (CDCl3) δ 190.4, 160.4, 136.0,
117.7, 113.4, 46.1, 25.6, 18.2, -4.4. MS (CI) m/z 413-415 (M +
H)+. Anal. (C20H35ClO3Si2‚1/3H2O, 421.06) C, H.
line. The RR,SS series were more potent than the RS,SR ones.
In the case of the NO donor furoxan derivatives, NO-dependent
relaxation also occurs at higher concentrations and in equipotent
manner in the two diastereoisomers of each couple. The only
exception is the furoxan derivative 15b, which triggers relaxation
simultaneously through both of the mechanisms in a limited
concentration range. All the analogues of fenoterol 1a, the
marketed drug, display antioxidant activity particularly marked
in the quite lipophilic phenylsulfonyl-substituted models. The
products containing NO donor furoxan moieties were able to
dilate precontracted rat aorta strips in keeping with a NO-
mediated activation of the sGC. The NO-donor analogues of
fenoterol described in the present paper represent a new
interesting class of polyvalent drugs potentially useful in a
number of airways and cardiovascular disorders.
tert-Butyl-(3-{[tert-butyl(dimethyl)silyl]oxy}-5-oxiran-2-ylphe-
noxy)dimethylsilane (6). To a solution of 4 (4.42 g; 10.7 mmol)
in MeOH (50 mL), NaBH4 (0.1 g; 2.7 mmol) was added at 0 °C,
and the reaction was stirred at room temperature for 30 min. The
solvent was evaporated in vacuo, and the residue was taken up
with brine (100 mL) and extracted with EtOAc (3 × 20 mL). The
organic layer was dried and concentrated in vacuo to leave an oil
that was dissolved in dry THF (100 mL), treated with 60% NaH in
mineral oil (0.35 g; 8.8 mmol), and stirred under nitrogen for 24 h.
The reaction mixture was cooled to 0 °C and diluted with water
(20 mL), and the THF was evaporated in vacuo. The residual
product was taken up with brine (50 mL), extracted with Et2O (3
× 10 mL), dried, and concentrated in vacuo to afford crude 6 as a
cream-colored oil. The product was purified by FC on silica gel
(eluent, petroleum ether/EtOAc ) 9.5/0.5) to yield pure 6 as a
colorless oil (1.64 g; 60%): 1H NMR (CDCl3) δ 6.39 (2H, d, Ph-
2,6-H, J ) 2.2 Hz), 6.26 (1H, t, Ph-4-H, J ) 2.2 Hz), 3.73 (1H, q,
PhCH, J ) 4.1, 2.5 Hz), 3.08 (1H, dd, oxirane-cis, Jgem) 5.6, Jcis
) 4.1 Hz), 2.71 (1H, dd, oxirane-trans, Jgem ) 5.6, Jtrans ) 2.5
Hz), 0.97 (18H, s, C(CH3)3), 0.18 (12H, s, CH3Si); 13C NMR
(CDCl3) δ 156.8, 139.8, 111.9, 110.4, 52.2, 51.1, 25.7, 18.2, -4.2;
MS (CI) m/z 381 (M + H)+. Anal. (C20H36O3Si2, 380.67) C, H.
1-(4-Hydroxyphenyl)propan-2-amonium Chloride (10). To a
stirred solution of 7 (4.68 mL; 30.5 mmol) in EtOH (40 mL),
hydroxylamine hydrochloride (4.92 g; 71.3 mmol) in water (25 mL)
was added, and the reaction pH was adjusted to 8 by the addition
of 10% NaOH. The mixture was refluxed for 15 min, cooled, and
treated with water (100 mL) to obtain a white solid, which was
collected. After drying, the oxime 8 was dissolved in NH3-saturated
methanol solution (50 mL) and transferred in the Parr hydrogenator.
Ni catalyst12 (10%) was added, and the mixture was hydrogenated
for 24 h at 30 bar, keeping the temperature at 68 °C. The mixture
was filtered to remove the catalyst, and the solvent was evaporated
in vacuo. The residual semisolid material was taken up with HCl-
saturated MeOH and precipitated by treatment with Et2O to obtain
the hydrochloride salt (9). This salt was dissolved in concd HCl
(50 mL) and heated at 140 °C in a closed vessel for 24 h. The dark
solution was evaporated in vacuo, the residual solid was dissolved
in abs EtOH (100 mL), treated with decolorizing charcoal, and
refluxed for an additional 24 h. After filtering through celite, the
solvent was evaporated in vacuo to leave a cream-colored solid,
which was recrystallized from abs EtOH/Et2O to give 10 (4.25 g;
75%) as a white solid, mp 172.9-173.8 °C (lit.13 171-172 °C);
1H NMR (CD3OD) δ 7.08 (2H, d, Ph-2,6-H, J ) 8.4 Hz), 6.78
(2H, d, Ph-3,5-H, J ) 8.4 Hz), 3.45 (1H, m, CH), 2.93 (1H, dd,
(CH)H, J ) 13.6, 8.1 Hz), 2.71 (1H, dd, (CH)H, J ) 13.6, 8.3
Hz), 1.25 (3H, d, CH3, J ) 6.6 Hz). 13C NMR (CD3OD) δ 158.3,
132.0, 128.5, 117.2, 51.1, 41.5, 18.8.
Experimental Section
Chemistry. Melting points were determined with a capillary
apparatus (Bu¨chi B-540). 1H and 13C NMR spectra were obtained
on a Bruker Avance 300, at 300 and 75 MHz, respectively; δ in
ppm relative to SiMe4 as the internal standard; coupling constants
J in Hz. 13C NMR spectra were fully decoupled. The following
abbreviations are used: s, singlet; d, doublet; t, triplet; q, quartet;
dd, double doublet; m, multiplet. Mass spectra were recorded on a
Finnigan-Mat TSQ-700.
HPLC measurements were carried out on a Varian Pro-Star 210
chromatograph equipped with a variable wavelength detector (Pro-
Star 325). The chromatography was performed using a 7 µm particle
size Hibar prepacked column (250 mm × 10 mm), with a flow
rate of 20 mL/min.; the detection was performed at 280 nm. Samples
of the mixtures were dissolved (ca 0.5%) in mobile phase and used
in all chromatographic measurements.
Silica gel 60 F254 precoated plates on glass from Merck was
used for thin-layer chromatography. Flash chromatography (FC)
was performed on BDH silica gel (particle size 40-63 µm). When
not otherwise specified, anhydrous magnesium sulfate (MgSO4) was
used as the drying agent of organic phases. Analysis (C, H, N) of
the new compounds was performed by REDOX (Monza), the
analytical results obtained were within (0.4% of the theoretical
value. Compounds 13,19 14,20 19,21 20,22 21,23 and CHF 236319 were
synthesized according to literature. Product 18 (CAS 1609) was a
kind gift from Sanofi-Aventis Deutschland GmbH. rac-Fenoterol
hydrobromide was purchased from Sigma Aldrich. The NO released
was measured by means of an ISO-NO meter equipped with a 2
mm diameter shielded microsensor ISO-NOP and a ISO-NO Mark
II data recording system from World Precision Instrument (Sarasota,
FL).
1-(3,5-Bis{[tert-butyl(dimethyl)silyl]oxy}phenyl)-2-chloroet-
hanone (4). To a stirred solution of 2 (5 g; 21.1 mmol) in CHCl3
(500 mL), bromine (1.29 mL; 25.3 mmol) was added, after 30 min,
the solution was washed with 10% NaHCO3 (5 × 30 mL) and then
with brine (2 × 30 mL), and the organic layer was dried and
concentrated in vacuo to leave an oily residue, which was taken
up with EtOH (50 mL) and treated with 5 N HBr (20 mL). The
mixture was refluxed for 30 min, cooled, poured into water (500
mL), and then extracted with Et2O (5 × 20 mL). The organic phase
was worked up as described above to afford a pale yellow oil, which
was readily dissolved in DMF (5 mL). The obtained solution was
added dropwise into a solution of TBDMSCl (6.87 g; 45.6 mmol)
and DIPEA (9.9 mL; 57 mmol) in DMF (20 mL) kept at 0 °C.
When the addition was over, the reaction mixture was stirred at
room temperature for 1 h, diluted with water (200 mL), and
extracted with Et2O (5 × 20 mL). The ethereal layer was washed
with 10% NaOH (3 × 30 mL), 10% aqueous citric acid (3 × 30
mL), and brine (2 × 30 mL) and the organic phase was dried and
evaporated to obtain the crude product as a yellow oil. The product
was purified by FC on silica gel (eluent, petroleum ether/CH2Cl2
) 8/2 then 7/3) to give pure 4 as a pale yellow oil (7.00 g; 78%):
1H NMR (CDCl3) δ 7.04 (2H, d, Ph-2,6-H, J ) 2.2 Hz), 6.59 (1H,
General Procedure for the Synthesis of Fenoterol Diastere-
oisomers (1a,b). To a solution of 10 (1.19 g; 6.4 mmol) and Et3N
(0.88 mL; 6.4 mmol) in MeOH (15 mL), oxirane 6 (0.8 g; 2.1
mmol) was added and the mixture was refluxed for 24 h. The
solvent was removed in vacuo to leave a yellowish oil (11), and
the product, without further purification, was reacted with (Boc)2O
in CH2Cl2 at room temperature to obtain 12a + 12b as a
diastereoisomeric mixture (RR,SS; RS,SR). The separation of mixture
into both racemic pairs (RR,SS)a and (RS,SR)b was carried out with
a direct phase HPLC method, eluting with CH2Cl2/MeOH 99:1%
v/v. The first eluted compound 12a (tR: 7.1) was then hydrolyzed
in MeOH/1 N HBr to give 1a (RR,SS); with the same acidic
treatment, the second eluted compound 12b (tR: 8.3) was hydro-
lyzed to give 1b (RS,SR). The obtained compounds were freeze-
dried.
rac-(2R)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
(4-hydroxyphenyl)propan-2-amonium Bromide (1a). White solid
(1.02 g, 46%); mp 223.5-225 °C (lit.24 223.8-224.5 °C); 1H NMR
(CD3OD) δ 7.08 (2H, d, H-2,6-PhOH, J ) 8.4 Hz), 6.76 (2H, d,

5008 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Buonsanti et al.
H-3,5-PhOH, J ) 8.4 Hz), 6.40 (2H, d, Ph-2,6-H, J ) 2.1 Hz),
6.23 (1H, m, Ph-4-H), 4.90 (1H, d, CHOH, J ) 3.3 Hz), 3.52-
3.48 (1H, m, CHCH3), 3.24-3.08 (3H, m, CH2NH, H(CH)PhOH),
PhSO2, J ) 7.6 Hz), 7.34 (2H, d, 2,6-H aromatics phenylpropy-
laminic substructure, J ) 8.5 Hz), 7.24 (2H, d, 3,5-H aromatics
phenylpropylaminic substructure, J ) 8.5 Hz), 6.40 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.20 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 4.86 (1H, m, CHOH
overlapping with HOD), 3.55 (1H, m, CHCH3), 3.34-3.00 (3H,
m, CH2NH, H(CH)PhO-), 2.79 (1H, m, H(CH)PhO-), 1.21 (3H, d,
CH3, J ) 6.4 Hz). 13C NMR (CD3OD) δ 169.1, 162.2, 159.9, 154.5,
151.3, 144.9, 139.2, 137.1, 136.4, 132.2, 131.1, 130.3, 120.6, 105.3,
103.2, 70.3, 56.7, 52.7, 39.6, 15.5. Anal. (C25H25N3O7S‚H2C2O4‚
2/3H2O, 613.6) C, H, N.
rac-(2S)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
(4-[(3-phenylsulfonylfurazan-4-yl)]oxy}phenyl)propan-2-amo-
nium Oxalate (16b). White solid (0.66 g; 46%), mp 76.1-
76.6 °C; 1H NMR (CD3OD) δ 8.17 (2H, d, H-2,6-PhSO2, J ) 7.6
Hz), 7.88 (1H, t, H-4-PhSO2, J ) 7.6 Hz), 7.74 (2H, t, H-3,5-
PhSO2, J ) 7.6 Hz), 7.38 (2H, d, 2,6-H aromatics phenylpropy-
laminic substructure, J ) 8.0 Hz), 7.29 (2H, d, 3,5-H aromatics
phenylpropylaminic substructure, J ) 8.0 Hz), 6.41 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.24 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 4.86 (1H, m, CHOH
overlapping with HOD), 3.58 (1H, m, CHCH3), 3.31-3.15 (3H,
m, CH2NH, H(CH)PhO-), 2.80 (1H, m, H(CH)PhO-), 1.26 (3H,
m, CH3). 13C NMR (CD3OD) δ 163.5, 162.2, 160, 154.6, 151.3,
144.7, 139.2, 137.1, 136.3, 132.2, 131.1, 130.1, 120.7, 105.3, 103.3,
70.5, 57.0, 52.6, 39, 16. Anal. (C25H25N3O7S‚H2C2O4‚H2O, 619.6)
C, H, N.
General Procedure for the Synthesis of Amines 22-25. To a
stirred solution of 10 (5.34 mmol) in CH3CN/water (2/1) (15 mL)
at 0 °C, Et3N (13.4 mmol), and (Boc)2O (5.34 mmol) were added.
After 1 h at room temperature, the solvent was removed in vacuo,
and the residual oil was taken up with water (20 mL) and extracted
with EtOAc (3 × 20 mL). The organic phase was dried and the
solvent was removed in vacuo to leave a white solid. The solid
was dissolved in dry THF (50 mL), and the appropriate alcohol
18-21 (8 mmol) and PPh3 (10.7 mmol) were added. The mixture
was cooled to 0 °C, and a solution of DIAD (10.7 mmol) in dry
THF (2 mL) was added dropwise. The mixture was stirred at room
temperature for 24 h, and the solvent was removed in vacuo to
leave the product as a yellow oil, which was dissolved in CH2Cl2
(6 mL) and TFA (2 mL) and stirred for an additional 1 h. The
solvent was removed in vacuo, and the residue was taken up with
water (30 mL) and extracted with EtOAc (2 × 20 mL). The aqueous
layer was treated with 10% Na2CO3 (20 mL) and extracted with
EtOAc (5 × 20 mL), and the organic phase was dried over
anhydrous K2CO3 and concentrated in vacuo to afford a white solid.
The solid was purified by FC on silica gel (eluent, CH2Cl2/MeOH
2.71-2.63 (1H, m, H(CH)PhOH), 1.22 (3H, d, CH3, J ) 6.6). 13
C
NMR (CD3OD) δ 159.9, 157.7, 144.7, 131.4, 128.2, 116.6, 105.3,
103.3, 70.1, 57.2, 52.5, 39.6, 15.5 (chemical shifts, which are
italicized, were used for assigning the diastereoisomers).
rac-(2S)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
(4-hydroxyphenyl)propan-2-amonium Bromide (1b). White solid
(1.02 g, 46%), mp 187-188.4 °C (lit.24 187.4-188.2 °C); 1H NMR
(CD3OD) δ 7.07 (2H, d, H-2,6-PhOH, J ) 8.4 Hz), 6.76 (2H, d,
H-3,5-PhOH, J ) 8.1 Hz), 6.38 (2H,m, Ph-2,6-H), 6.22 (1H, m,
Ph-4-H), 4.83-4.79 (1H, m, CHOH), 3.45-3.34 (1H, m, CHCH3),
3.23-3.18 (1H, m, H(CH)PhOH), 3.09-2.99 (2H, m, CH2NH),
2.69-2.61 (1H, m, H(CH)PhOH), 1.20 (3H, d, CH3, J ) 5.7). 13
C
NMR (CD3OD) δ 160.2, 157.8, 145.3, 131.8, 128.6, 116.9, 105.7,
103.6, 71.1, 57.5, 53.2, 39.9, 16.3. Anal. (C17H21NO4‚HBr‚1/3H2O,
390.9) C, H, N. (chemical shifts, which are italicized, were used
for assigning the diastereoisomers).
General Procedure for the Synthesis of Derivatives 15a, 15b,
16a, and 16b. To a solution of 12a or 12b (0.94 mmol) and furoxan
13 or furazan 14 (1.13 mmol) in distilled THF (30 mL), stirred
under nitrogen, 50% NaOH (0.22 g; 2.8 mmol) was added dropwise.
After 1 h at room temperature, the solvent was evaporated in vacuo,
and the residue was taken up with water (50 mL) and extracted
with EtOAc (4 × 10 mL). The organic phase was dried (K2CO3),
and the solvent was concentrated in vacuo to give a pale yellow
oil. This oil was readily dissolved in HCl-saturated MeOH (30 mL),
stirred for 2 h, and concentrated in vacuo to leave a yellow foam.
The product was taken up with 10% NaHCO3 and extracted with
EtOAc (5 × 10 mL). The organic layer was dried, the solvent was
evaporated in vacuo, and the obtained residue was purified by FC
on silica gel (eluent, CH2Cl2/MeOH(NH ) ) 9.5/0.5) to give the
3
expected product as the free base. The product was converted into
the corresponding oxalate, recrystallized from MeOH/Et2O, and
freeze-dried.
rac-(2R)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
(4-[(3-phenylsulfonylfuroxan-4-yl)]oxy]phenyl)propan-2-amo-
nium Oxalate (15a). White solid (0.61 g; 42%), mp 128.3-
129.6 °C; 1H NMR (CD3OD) δ 8.03 (2H, d, H-2,6-PhSO2, J ) 7.5
Hz), 7.81 (1H, t, H-4-PhSO2, J ) 7.5 Hz), 7.66 (2H, t, H-3,5-
PhSO2, J ) 7.5 Hz), 7.29 (4H, m, H aromatics phenylpropylaminic
substructure, J ) 8.7 Hz), 6.39 (2H, m, 2,6-H aromatics phenyle-
thanolaminic substructure), 6.20 (1H, m, 4-H aromatic phenyle-
thanolaminic substructure), 4.79 (1H, m, CHOH overlapping with
HOD), 3.54 (1H, m, CHCH3), 3.32-3.09 (3H, m, CH2NH, H(CH)-
PhO-), 2.81-2.73 (1H, m, H(CH)PhO-), 1.20 (3H, d, CH3, J )
6.3 Hz). 13C NMR (CD3OD) δ 168.7, 159.9, 159.8, 153.3, 144.8,
139.2, 137.1, 136.4, 132.1, 131, 129.7, 121.2, 112.2, 105.3, 103.3,
70.3, 56.7, 52.7, 39.6, 15.4. Anal. (C25H25N3O8S‚H2C2O4‚2/3H2O,
629.48) C, H, N.
) 9.5/0.5 then CH2Cl2/MeOH(NH ) ) 9.5/0.5) to give the expected
3
product.
4-[4-(2-Aminopropyl)phenoxymethyl]furoxan-3-carboxam-
1
ide (22). Colorless oil (66%); H NMR (CD3OD) δ 7.13 (2H, d,
Ph-2,6-H, J ) 8.5 Hz), 6.97 (2H, d, Ph-3,5-H, J ) 8.5 Hz), 5.37
(2H, s, CH2-furoxan), 3.09-3.00 (1H, m, CHCH3), 2.64-2.51 (2H,
m, CH2Ph), 1.06 (3H, d, CH3, J ) 6.3 Hz). 13C NMR (CD3OD) δ
158.1 (two overlapping peaks), 156.6, 134.0, 131.4, 116.1, 111.8,
62.6, 49.6, 45.8, 22.5. MS (CI) m/z 293 (M + H)+. Anal.
(C13H16N4O4‚0.3H2O, 297.69) C, H, N.
rac-(2S)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
(4-[(3-phenylsulfonylfuroxan-4-yl)]oxy]phenyl)propan-2-amo-
nium Oxalate (15b). White solid (0.61 g; 42%), mp 94.4-
95.6 °C; 1H NMR (CD3OD) δ 8.06 (2H, d, H-2,6-PhSO2, J ) 7.6
Hz), 7.84 (1H, t, H-4-PhSO2, J ) 7.6 Hz), 7.69 (2H, t, H-3,5-
PhSO2, J ) 7.6 Hz), 7.33 (4H, m, H aromatics phenylpropylaminic
substructure, J ) 8.5 Hz), 6.40 (2H, m, 2,6-H aromatics phenyle-
thanolaminic substructure), 6.22 (1H, m, 4-H aromatic phenyle-
thanolaminic substructure), 4.79 (1H, m, CHOH overlapping with
HOD), 3.57 (1H, m, CHCH3), 3.27-3.22 (2H, m, CH2NH), 3.16-
3.08 (1H, m, H(CH)PhO-), 2.78 (1H, m, H(CH)PhO-), 1.24 (3H,
d, CH3, J ) 6.2 Hz). 13C NMR (CD3OD) δ 168.5, 159.9, 159.8,
153.4, 144.8, 139.3, 137.1, 136.3, 132.1, 131, 129.7, 121.2, 112.2,
105.3, 103.3, 70.5, 56.9, 52.7, 39.1, 16.1. Anal. (C25H25N3O8S‚
H2C2O4‚1.5H2O, 644.59) C, H, N.
3-[4-(2-Aminopropyl)phenoxymethyl]furazan-4-carboxam-
1
ide (23). Colorless oil (60%); H NMR (CD3OD) δ 7.13 (2H, d,
Ph-2,6-H, J ) 8.5 Hz), 6.96 (2H, d, Ph-3,5-H, J ) 8.5 Hz), 5.46
(2H, s, CH2-furazan), 3.09-3.03 (1H, m, CHCH3), 2.59-2.54 (2H,
m, CH2Ph), 1.06 (3H, d, CH3, J ) 6.3 Hz). 13C NMR (CD3OD) δ
160.9, 158.1, 153.5, 149.8, 133.8, 131.4, 116.1, 60.8, 49.6, 45.6,
22.4. MS (CI) m/z 277 (M + H)+. Anal. (C13H16N4O3‚1/2 H2O,
285.3) C, H, N.
1-Methyl-2-[4-(3-methylfuroxan-4-ylmethoxy)phenyl]ethy-
lamine (24). Colorless oil (40%); 1H NMR (CD3OD) δ 7.19 (2H,
d, Ph-2,6-H, J ) 8.5 Hz), 7.02 (2H, d, Ph-3,5-H, J ) 8.5 Hz), 5.22
(2H, s, CH2-furoxan), 3.15-3.09 (1H, m, CHCH3), 2.64-2.62 (2H,
m, CH2Ph), 2.22 (3H, s, CH3-furoxan) 1.11 (3H, d, CH3, J ) 6.4
Hz). 13C NMR (CD3OD) δ 156.7, 155.6, 133.1, 130.5, 115.0, 113.2,
rac-(2R)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
(4-[(3-phenylsulfonylfurazan-4-yl)]oxy}phenyl)propan-2-amo-
nium Oxalate (16a). White solid (0.66 g; 46%), mp 100.4-
101.0 °C; 1H NMR (CD3OD) δ 8.14 (2H, d, H-2,6-PhSO2, J ) 7.6
Hz), 7.85 (1H, t, H-4-PhSO2, J ) 7.6 Hz), 7.72 (2H, t, H-3,5-

Nitric Oxide Donor â-Agonists
Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20 5009
61.3, 48.6, 44.5, 21.3, 6.8. MS (CI) m/z 264 (M + H)+. Analytical
sample was obtained as the oxalate (C13H17N3O3‚H2C2O4, 353.33)
C, H, N.
1-Methyl-2-[4-(4-methylfurazan-3-ylmethoxy)phenyl]ethy-
lamine (25). Colorless oil (35%); 1H NMR (CD3OD) δ 7.14 (2H,
d, Ph-2,6-H, J ) 8.7 Hz), 6.97 (2H, d, Ph-3,5-H, J ) 8.7 Hz), 5.28
(2H, s, CH2-furazan), 3.10-3.03 (1H, m, CHCH3), 2.60-2.57 (2H,
m, CH2Ph), 2.42 (3H, s, CH3-furazan) 1.06 (3H, d, CH3, J ) 6.4
Hz). 13C NMR (CD3OD) δ 158.1, 153.2, 153.0, 134.3, 131.9, 116.3,
60.5, 50.0, 45.9, 22.7, 8.6. MS (CI) m/z 248 (M + H)+. Anal.
(C13H17N3O2‚1/3H2O, 253.24) C, H, N.
phenylethanolaminic substructure), 5.48 (2H, s, CH2-furazan), 4.70
(1H, m, CHOH overlapping with HOD), 3.51 (1H, m, CHCH3),
3.30-3.15 (3H, m, CH2NH, H(CH)PhO-), 2.72 (1H, m, H(CH)-
PhO-), 1.29-1.22 (3H, m, CH3). 13C NMR (CD3OD) δ 166.2,
160.9, 160.1, 158.8, 153.5, 149.8, 144.8, 131.6, 130.5, 116.5, 105.3,
103.4, 70.3, 60.8, 57.1, 52.5, 39.6, 15.6. Anal. (C21H24N4O6‚H2C2O4‚
0.2H2O, 522.08) C, H, N.
rac-4-(4-{(2R)-[2-(3, 5-Dihydroxyphenyl)-(2S)-
hydroxyethylamino]propyl}phenoxymethyl)furazan-3-carboxa-
mide Oxalate (35b). White solid (60%), mp 111.3-112.1 °C; 1H
NMR (CD3OD) δ 7.18 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.5 Hz), 6.98 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.5 Hz), 6.38 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.21 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.46 (2H, s, CH2-
furazan), 4.84 (1H, m, CHOH), 3.48 (1H, m, CHCH3), 3.25-3.04
(3H, m, CH2NH, H(CH)PhO-), 2.72-2.65 (1H, m, H(CH)PhO-),
1.23 (3H, d, CH3, J ) 6.3 Hz). 13C NMR (CD3OD) δ 169.6, 160.9,
160.0, 158.6, 153.4, 149.8, 144.8, 131.6, 130.8, 116.4, 105.3, 103.3,
70.5, 60.8, 57.2, 52.7, 39.1, 16.2. Anal. (C21H24N4O6‚H2C2O4,
518.48) C, H, N.
General Procedure for the Synthesis of Derivatives 34a-37a
and 34b-37b. To a stirred solution of the appropriate amine 22-
25 (4.10 mmol) in MeOH (15 mL), oxirane 6 (1.64 mmol) was
added, and the mixture was refluxed for 24 h. The solvent was
removed in vacuo to leave a yellowish oil; the product without
further purification was reacted with (Boc)2O (2.46 mmol) in CH2-
Cl2 at room temperature to obtain 30a + 30b, 31a + 31b, 32a +
32b, and 33a + 33b as a diastereoisomeric mixtures (RR,SS +
RS,SR). The separation of mixtures into both racemic pairs (RR,SS)-
a and (RS,SR)b was carried out with a direct phase HPLC method.
The mobile phase used for the mixture 30a + 30b and 31a + 31b
was CH2Cl2/MeOH 99.5:0.5% v/v, while hex/EtOAc 85:15% v/v
was used for derivatives 32a + 32b, 33a + 33b. The first eluted
enantiomeric pair 30a (tR: 18), 31a (tR: 20), 32a (tR: 12), and
33a (tR: 11) was then hydrolyzed with 1 N HCl (10 mL) and stirred
at room temperature for 1 h. With the same acidic treatment, the
second eluted compounds 30b (tR: 25), 31b (tR: 26), 32b (tR: 14),
and 33b (tR: 13) were then hydrolyzed. The aqueous solutions were
basified with 10% NaHCO3 and extracted with EtOAc (4 × 15
mL), and the organic layers were dried and concentrated in vacuo
to leave colorless oils. The crude products were purified by FC on
silica gel (eluent, CH2Cl2/MeOH(NH ) ) 9.5/0.5) to give the desired
products as the free base. The pro3ducts were converted into the
corresponding oxalates, recrystallized from MeOH/Et2O, and freeze-
dried to afford the desired product 34a-37a and 34b-37b.
rac-4-(4-{(2R)-[(2-(3,5-Dihydroxyphenyl)-(2R)-
hydroxyethylamino]propyl}phenoxymethyl)furoxan-3-carboxa-
rac-(2R)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
{4-[(3-methylfuroxan-4-yl)methoxy]phenyl}propan-2-amoni-
1
um Oxalate (36a). White solid (40%), mp 125.5-126.9 °C; H
NMR (CD3OD) δ 7.21 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.7 Hz), 6.99 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.7 Hz), 6.38 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.20 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.18 (2H, s, CH2-
furoxan), 4.88-4.85 (1H, m, CHOH), 3.55-3.48 (1H, m, CHCH3),
3.23-3.07 (3H, m, CH2NH, H(CH)PhO-), 2.75-2.67 (1H, m,
H(CH)PhO-), 2.18 (3H, s, CH3-furoxan), 1.20 (3H, d, CH3, J )
6.3 Hz). 13C NMR (CD3OD) δ 168.0, 160.0, 158.3, 156.5, 144.9,
131.8, 131.1, 116.3, 114.2, 105.3, 103.2, 70.3, 62.2, 57.0, 52.7,
39.5, 15.5, 7.8. Anal. (C21H25N3O6‚H2C2O4‚1/2H2O, 514.48) C, H,
N.
rac-(2S)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
{4-[(3-methylfuroxan-4-yl)methoxy]phenyl}propan-2-amoni-
um Oxalate (36b). White solid (40%), mp 93.5-94.1 °C; 1H NMR
(CD3OD) δ 7.21 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.7 Hz), 6.99 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.7 Hz), 6.37 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.20 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.18 (2H, s, CH2-
furoxan), 4.85-4.81 (1H, m, CHOH), 3.53-3.46 (1H, m, CHCH3),
3.27-3.02 (3H, m, CH2NH, H(CH)PhO-), 2.74-2.67 (1H, m,
H(CH)PhO-), 2.18 (3H, s, CH3-furoxan), 1.23 (3H, d, CH3, J )
6.6 Hz). 13C NMR (CD3OD) δ 168.4, 160.0, 158.3, 156.5, 144.8,
131.8, 131.2, 116.4, 114.2, 105.3, 103.3, 70.4, 62.3, 57.2, 52.7,
39.0, 16.3, 7.8. Anal. (C21H25N3O6‚H2C2O4‚1/2H2O, 514.48) C, H,
N.
1
mide Oxalate (34a). White solid (46%), mp 131.1-132 °C; H
NMR (CD3OD) δ 7.20 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.1 Hz), 7.02 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.1 Hz), 6.37 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.22 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.40 (2H, s, CH2-
furoxan), 4.80 (1H, m, CHOH), 3.51 (1H, m, CHCH3), 3.16-3.12
(3H, m, CH2NH, H(CH)PhO-), 2.72-2.64 (1H, m, H(CH)PhO-),
1.22 (3H, d, CH3, J ) 6.3 Hz). 13C NMR (CD3OD) δ 166.2, 160,
158.8, 158.1, 156.5, 144.7, 131.6, 130.6, 116.5, 111.7, 105.3, 103.3,
70.3, 62.6, 57.1, 52.5, 39.5, 15.6. Anal. (C21H24N4O7‚H2C2O4‚1/
2H2O, 543.48) C, H, N.
rac-4-(4-{(2R)-[2-(3, 5-Dihydroxyphenyl)-(2S)-
hydroxyethylamino]propyl}phenoxymethyl)furoxan-3-carboxa-
mide Oxalate (34b). White solid (46%), mp 116.5-117.5 °C; 1H
NMR (CD3OD) δ 7.19 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.5 Hz), 7.00 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.5 Hz), 6.37 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.21 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.39 (2H, s, CH2-
furoxan), 4.81 (1H, m, CHOH), 3.49 (1H, m, CHCH3), 3.28-3.05
(3H, m, CH2NH, H(CH)PhO-), 2.73-2.66 (1H, m, H(CH)PhO-),
1.23 (3H, d, CH3, J ) 6.4 Hz). 13C NMR (CD3OD) δ 167.4, 160,
158.7, 158.1, 156.5, 144.7, 131.6, 130.7, 116.5, 111.7, 105.3, 103.3,
70.5, 62.6, 57.3, 52.6, 39.1, 16.2. Anal. (C21H24N4O7‚H2C2O4‚1/
2H2O, 543.48) C, H, N.
rac-4-(4-{(2R)-[2-(3,5-Dihydroxyphenyl)-(2R)-
hydroxyethylamino]propyl}phenoxymethyl)furazan-3-carboxa-
mide Oxalate (35a). White solid (60%), mp 230 °C dec; 1H NMR
(CD3OD) δ 7.19 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.5 Hz), 7.02 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.5 Hz), 6.37 (2H, t, 2,6-H aromatics
phenylethanolaminic substructure), 6.22 (1H, t, 4-H aromatic
rac-(2R)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
{4-[(3-methylfurazan-4-yl)methoxy]phenyl}propan-2-amoni-
1
um Oxalate (37a). White solid (42%), mp 108.2-109.2 °C; H
NMR (CD3OD) δ 7.20 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.6 Hz), 6.99 (2H, d, 3,5-H aromatics phenyl-
propylaminic substructure, J ) 8.6 Hz), 6.38 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.20 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.29 (2H, s, CH2-
furazan), 4.87-4.83 (1H, m, CHOH), 3.54-3.47 (1H, m, CHCH3),
3.22-3.07 (3H, m, CH2NH, H(CH)PhO-), 2.74-2.67 (1H, m,
H(CH)PhO-), 2.42 (3H, s, CH3-furazan), 1.20 (3H, CH3, d, J )
6.3 Hz). 13C NMR (CD3OD) δ 168.1, 160.0, 158.4, 152.8, 152.7,
144.9, 131.7, 130.9, 116.3, 105.3, 103.3, 70.3, 60.3, 57.0, 52.6,
39.5, 15.5, 8.3. Anal. (C21H25N3O5‚H2C2O4‚0.7H2O, 502.09) C, H,
N.
rac-(2S)-N-[(2R)-2-(3,5-Dihydroxyphenyl)-2-hydroxyethyl]-1-
{4-[(3-methylfurazan-4-yl)methoxy]phenyl}propan-2-amoni-
1
um Oxalate (37b). White solid (42%), mp 103.6-104.3 °C; H
NMR (CD3OD) δ 7.20 (2H, d, 2,6-H aromatics phenylpropylaminic
substructure, J ) 8.6 Hz), 7.00 (2H, d, 3,5-H aromatics phenyl-

5010 Journal of Medicinal Chemistry, 2007, Vol. 50, No. 20
Buonsanti et al.
propylaminic substructure, J ) 8.6 Hz), 6.37 (2H, m, 2,6-H
aromatics phenylethanolaminic substructure), 6.20 (1H, m, 4-H
aromatic phenylethanolaminic substructure), 5.29 (2H, s, CH2-
furazan), 4.85-4.81 (1H, m, CHOH), 3.53-3.46 (1H, m, CHCH3),
3.27-3.03 (3H, m, CH2NH, H(CH)PhO-), 2.74-2.63 (1H, m,
H(CH)PhO-), 2.41 (3H, s, CH3-furazan), 1.23 (3H, d, CH3, J )
6.6 Hz). 13C NMR (CD3OD) δ 168.6, 160.2, 158.7, 153.1, 152.9,
145.1, 132.0, 131.2, 116.6, 105.6, 103.5, 70.7, 60.5, 57.5, 53.0,
39.3, 16.5, 8.5. Anal. (C21H25N3O5‚H2C2O4‚1/2H2O, 498.48) C, H,
N.
Amperometric Detection of NO Release from Derivatives 15a,
34a, and 36a. The membrane-covered tip of the measuring electrode
was inserted into a solution containing phosphate buffer (50 mM/
pH 7.4). The solution was constantly mixed by a magnetic stirrer
and kept at 37 ( 0.5 °C in a closed glass vial. The current was
recorded for 15 min to allow for baseline stabilization, and then a
solution of the appropriate compound 15a (1 µM), 34a (500 µM),
or 36a (500 µM) in 100 µL of DMSO (1% v/v in the final solution)
was added via a gastight syringe. Consecutive additions of sodium
ascorbate (1 mM) in HPLC-grade water (50 µL) and glutathione
(3 mM) in phosphate buffer (100 µL) were performed via a gastight
syringe. The final volume of the tested mixture was 10 mL. Change
in the current was recorded as a function of time, and data were
elaborated with a MacLab System PowerLab. Experiments were
run at least in triplicate after appropriate calibration of the electrode
with NaNO2.25
Functional Studies. Tracheas were isolated from male guinea
pigs weighing 200-250 g, while aortae were isolated from male
Wistar rats weighing 200-250 g. The animals, treated humanely
in accordance with recognized guidelines on experimentation, were
anaesthetized with CO2 and killed by decapitation. As few animals
as possible were used. The purposes and the protocols of our studies
have been approved by the Ministero della Salute, Rome, Italy.
The tissues were mounted in organ baths containing 30 mL of
Krebs-bicarbonate buffer of the following composition (mM):
NaCl, 111.2; KCl, 5.0; CaCl2, 2.5; MgSO4, 1.2; KH2PO4, 1.0;
NaHCO3, 12; glucose, 11.1. The solution was maintained at 37 °C
and continuously gassed with 95% O2-5% CO2 (pH 7.4).
Trachea Preparation. The tracheal tube was quickly excised,
cleaned free of excess tissues, and then was cut into single rings.
Four to five rings were joined together by thread to form a chain
and mounted in organ baths. After 2 h equilibration period, 1 µM
carbachol was added to the organ bath to induce a spasm of the
trachea rings, and when a constant level was reached, cumulative
concentration-response curves to compounds 15a, 15b, 16a, 16b,
34a-37a, and 34b-37b and the references were determined.
Effects of 10 µM ODQ16 and 100 µM propranolol were evaluated
in separate series of experiments in which they were added 40 min
before the start of the curve. EC50 values are the mean of 4-8
determinations. Responses were recorded by an isometric transducer
connected to the MacLab System PowerLab.
Aorta Preparation. The aorta endothelium was removed and
the vessels were helically cut: three strips were obtained from each
aorta. The tissues were mounted under 1.0 g of tension in organ
baths. The aortic strips were allowed to equilibrate for 2 h and
then contracted with 1 µM l-phenylephrine. When the response to
the agonist reached a plateau, cumulative concentrations of the
vasodilating agent were added. Effects of 10 µM ODQ on relaxation
were evaluated in a separate series of experiments in which it was
added 5 min before the contraction. EC50 values are the mean of
4-8 experiments. Responses were recorded by an isometric
transducer connected to the MacLab System PowerLab.
experiments. Lipid peroxidation was initiated by adding 2.5 µM
FeSO4. Aliquots were taken from the incubation mixture at 5, 15,
and 30 min and treated with 10% w/v trichloroacetic acid (TCA).
Lipid peroxidation was assessed by spectrophotometric (543 nm)
determination of the TBARS, consisting mainly of malondialdehyde
(MDA), and TBARS concentrations (expressed in nmol/mg protein)
were obtained by interpolation with a MDA standard curve. The
antioxidant activity of compounds, tested as hydrochloride salts,
was evaluated as the percentage of inhibition of TBARS production
with respect to control samples, using the plateau values obtained
after 30 min of incubation. IC50 values were calculated by nonlinear
regression analysis.
Acknowledgment. Financial support from Italian MIUR
(COFIN 2004) is gratefully acknowledged.
Supporting Information Available: Tables of elemental
analyses of all new compounds. This material is available free of
charge via the Internet at http://pubs.acs.org.
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