Synthesis of some novel organic nitrates and comparative in vitro study of their vasodilator profile

Article abstract of DOI:10.1021/jm9002236

Synthesis and structural characterization of the 4-phenylbutane-1,2-diyl dinitrate and of the erythro and threo diastereoisomers of 4-phenylbutane-1,2,3- triyl trinitrate as well as the HPLC chiral separation of the corresponding racemic mixtures are repo

Full text of DOI:10.1021/jm9002236

4020
J. Med. Chem. 2009, 52, 4020–4025
Synthesis of Some Novel Organic Nitrates and Comparative in Vitro Study of Their
Vasodilator Profile
Konstantin Chegaev,† Loretta Lazzarato,† Paolo Marcarino,† Antonella Di Stilo,† Roberta Fruttero,† Nicolas Vanthuyne,‡
Christian Roussel,‡ and Alberto Gasco*,†
Dipartimento di Scienza e Tecnologia del Farmaco, UniVersita` degli Studi di Torino, Via Pietro Giuria 9, 10125 Torino, Italy, UMR ISM2,
Chirosciences UniVersite´ Paul Ce´zanne Aix-Marseille III, Case A62, 13397 Marseille Cedex 20, France
ReceiVed February 20, 2009
Synthesis and structural characterization of the 4-phenylbutane-1,2-diyl dinitrate and of the erythro and
threo diastereoisomers of 4-phenylbutane-1,2,3-triyl trinitrate as well as the HPLC chiral separation of the
corresponding racemic mixtures are reported. Vasodilator activity of the single enantiomers of these products,
of 4-phenylbutyl nitrate, and of the previously described phenylpropyl analogues were assessed on rat aorta
strips precontracted with phenylephrine. The compounds were able to relax the contracted tissue in a
concentration dependent manner. In the couples of antipodes, a complete lack of enantioselectivity was
observed as far as the vasodilator potency is concerned. The concentration response curves of the products,
with the exception of those of all the trinitrooxy substituted models, were rightward shifted in the presence
of ALDH-2 inhibitors. Mono and dinitrates, but not trinitrates, displayed in vitro cross-tolerance with GTN.
This new series of nitric acid esters is an interesting tool that can help to shed light on the unresolved
puzzle of nitrate pharmacology. Selected members are worthy of additional study as potential drugs.
Chart 1. Novel Organic Nitrates
A variety of organic nitrates are commonly used in medicine
because of their ability to display relaxing effects on vasculature
smooth muscle.1,2 The generally accepted mechanism of this
action involves their conversion in the smooth muscle cell into
nitric oxide (NOa), which is able to activate soluble guanylate
cyclase (sGC). As a consequence, an increase in intracellular
cGMP concentration occurs, triggering activation of a protein
kinase (cGK-1) that phosphorylates Ca2+ transporters, causing
Ca2+ sequestration and, consequently, vasodilation. The exact
mechanism whereby NO is generated from organic nitrates is
still a topic of dispute. It has been suggested that both
nonenzymatic pathways, involving endogenous thiol groups or
hemoglobin, and several enzymes play roles in the generation
of NO.3 Frequently, repeated or continuous exposure to high
doses of nitrates leads to a marked attenuation of many of their
effects. This phenomenon, known as tolerance, sets a limit to
the practical application of these drugs.4 The prototype of
organic nitrate is glyceryl trinitrate (GTN). It was introduced
in therapy for the relief of angina pectoris pain in 1876 and it
is also the basis of Nobel’s potent detonator.5 In light of the
importance of nitrates in medicine, it is of undoubted interest
to make new products available both as potential drugs which
may increase the effectiveness of those employed in therapy
and for use as probes to examine further the mode of action of
drugs of this class. In a previous work,6 we described synthesis
and configuration assignment of 1-phenylpropane-1,2,3-triyl
trinitrate stereomers (+)1a, (-)1a, (+)1b, (-)1b, and of
3-phenylpropane-1,2-diyl dinitrate stereomers (+)2, (-)2 (Chart
1). In this paper, we report synthesis and chiral HPLC resolution
of the related butyl derivatives (+)5a, (-)5a, (+)5b, (-)5b,
(+)7, and (-)7 (Chart 1). The in vitro vasodilating activity of
all these products, together with the vasodilator profile of the
simple 3-phenylpropyl nitrate (8) and 4-phenylbutyl nitrate (9)
(Chart 1), was evaluated on rat thoracic aorta strips precontracted
with phenylephrine and compared with GTN. Finally, the results
of a preliminary in vitro study of cross-tolerance with GTN are
reported.
* To whom correspondence should be addressed. Phone: +39-011-670-
7670. Fax: +39-011-670-7286. E-mail: alberto.gasco@unito.it.
Results and Discussion
†
Dipartimento di Scienza e Tecnologia del Farmaco, Universita` degli
Chemistry. The new target compounds 5, 7 were synthesized
according to the pathway reported in Scheme 1. The racemic
mixture 5a was prepared starting from the Z-stereomer of
4-phenylbut-2-en-1-ol (3a) containing a small amount of the
E-stereomer 3b (less than 2%, HPLC detection). Following an
old procedure to prepare nitrates,7 the product was treated with
Studi di Torino.
‡
UMR ISM2, Chirosciences Universite´ Paul Ce´zanne Aix-Marseille III.
a Abbreviations: ALDH-2, aldehyde dehydrogenase 2; GTN, glyceryl
trinitrate; NO, nitric oxide; sGC, soluble guanylate cyclase; cGMP, cyclic
guanosine monophosphate; cGK-1, cGMP-dependent protein kinase type
I; ERP, energy release potential; ODQ, 1H-[1,2,4]oxadiazolo[4,3-a]qui-
noxalin-1-one.
10.1021/jm9002236 CCC: $40.75  2009 American Chemical Society
Published on Web 05/13/2009

Organic Nitrates and Their Vasodilator Profile
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4021
Scheme 1. Preparation of Organic Nitrates 5 and 7a
Figure 1. Concentration-response curves of GTN in the absence (b)
and in the presence of inhibitors (1 µM benomyl (O), 1 mM chloral
hydrate ([), or 1 µM ODQ (2)).
(ERP), a parameter that evaluates the explosive hazard of a
compound, calculated with the ASTM Program CHEAT AH
8.0, was found to be HIGH for all these products. Consequently,
we used only small quantities of these substances and even then
treated them with great caution. We never observed violent
decomposition induced by thermal or mechanical shock.
Vasodilator Activity. The vasodilator activities of all ni-
trooxyderivatives, focus of this work, as well as of GTN, taken
as a reference, were assessed on endothelium denuded rat aorta
strips precontracted with 1 µM phenylephrine. All the products
were able to dilate the strips in a concentration-dependent
manner. Their potencies as vasodilators, expressed as EC50, are
collected in Table 1. In our hands, the concentration-response
curve for the GTN relaxant effects was biphasic with a distinct
region of reduced responsiveness at 1 µM (Figure 1), as already
observed by other authors.9-11 The EC50 value was found to
be 0.029 ( 0.004 µM. As previously discussed, it is commonly
accepted that GTN induces vasorelaxation by generating NO
in vascular smooth muscle cells, which in turn activates the
target enzyme soluble guanylate cyclase (sGC) following
interaction with its heme site. Indeed, the vasorelaxing response
of GTN is nearly abolished by the presence of 1 µM ODQ (1H-
[1,2,4]oxadiazolo[4,3-a]quinoxalin-1-one), a well-known heme
site inhibitor of sGC.12 Both enzymatic and nonenzymatic
mechanisms have been proposed for NO production from
GTN.3,4b,c,13 Recently, it was shown that the mitochondrial
isoform of aldehyde dehydrogenase (ALDH-2) catalyzes the
transformation of GTN into 1,2-dinitrate and nitrite, which in
turn is converted to NO through pathways not fully understood.
a Reagent and conditions: (a) AgNO3, I2, CH3CN, rt, then reflux; (b)
AgNO3, PPh3, NBS, CH3CN, -15 °C to rt.
AgNO3 and I2 in CH3CN at room temperature, followed by
reflux, to give the intermediate racemic mixture of the dinitrooxy
substituted alcohol 4a. This product was only partly purified
by column chromatography and then immediately treated with
AgNO3 and triphenylphosphine (PPh3) in acetonitrile solution,
in the presence of N-bromosuccinimide (NBS), to afford the
expected racemic mixture 5a. The racemic mixture 5b was
synthesized in a similar manner starting from the E-stereomer
3b through the intermediate formation of 4b. The erythro and
threo configurations were assigned to 4a and 4b, and conse-
quently to 5a and 5b, respectively, on the basis of the knowledge
that the reaction in a suitable solvent of AgNO3 and iodine with
olefins affords cis-dinitrates.8 The racemic mixture of the
dinitrooxy substituted compound 7 was prepared from olefin 6
following the same procedure used to prepare 4 from 3.
1
Elemental analyses and H- and 13C NMR spectra of these
products are in keeping with the proposed structures. The
racemic mixtures of 5a, 5b, and 7 were resolved into the
corresponding enantiomers by chiral chromatography using a
Chiralcel OD column to a high degree of optical purity (see
Experimental Section). The overall energy release potential
Table 1. Vasodilating Activity of Compounds 1, 2, 5, 7, 8, 9, and GTN
EC50 [µM]a
compd
+benomylb
+chloral hydratec
+ODQd
in tolerant tissuee
EC50beno/EC50
EC50CH/EC50
EC50tol/EC50
(+)1a
(-)1a
(+)1b
(-)1b
(+)5a
(-)5a
(+)5b
(-)5b
(+)2
(-)2
(+)7
(-)7
8
0.29 ( 0.09
0.21 ( 0.06
0.28 ( 0.06
0.22 ( 0.05
0.18 ( 0.02
0.25 ( 0.05
0.11 ( 0.02f
0.18 ( 0.02
0.061 ( 0.010
0.063 ( 0.010
0.049 ( 0.003
0.049 ( 0.006
0.16 ( 0.03
0.15 ( 0.02
0.029 ( 0.004
0.36 ( 0.07
0.32 ( 0.08
0.32 ( 0.06
0.35 ( 0.08
0.23 ( 0.06
0.33 ( 0.07
0.14 ( 0.04
0.23 ( 0.05
0.60 ( 0.13
0.63 ( 0.13
0.16 ( 0.04
0.12 ( 0.02
1.5 ( 0.5
21 ( 2
18 ( 2
30 ( 1
22 ( 1
21 ( 2
18 ( 2
10 ( 1
12 ( 1
>100
0.78 ( 0.06
0.61 ( 0.10
1.0 ( 0.2
1.2
1.5
1.1
1.6
1.3
1.3
1.3
1.3
9.8
10
3.3
2.4
9.4
3.4
14
2.7
2.9
3.6
3.0
2.7
2.7
2.0
2.7
100
102
49
0.21 ( 0.11
0.27 ( 0.02
0.75
1.2
0.65 ( 0.05
0.48 ( 0.09
0.68 ( 0.07
0.22 ( 0.02
0.49 ( 0.10
6.1 ( 1.0
1.2 ( 0.2
1.2 ( 0.3
0.37 ( 0.09
0.27 ( 0.08
3.0 ( 0.6
1.1 ( 0.2
0.94 ( 0.30
20
19
>100
6.4 ( 1.1
30 ( 5
29 ( 3
>100
66 ( 4
>100
2.4 ( 0.7
7.6
5.5
19
7.3
32
1.6 ( 0.3
33
125
30
20 ( 4
9
0.51 ( 0.11
0.42 ( 0.07
4.5 ( 0.8
GTN
4.4 ( 1.3
152
a Data expressed as means ( SE, n ) 3-15. b Experiments performed in the presence of 1 µM benomyl. c Experiments performed in the presence of 1
mM chloral hydrate (CH). d Experiments performed in the presence of 1 µM ODQ. e Vessels were preincubated with 0.55 mM GTN for 1 h followed by
washing. f P < 0.05 when compared to EC50 of (-)5b; Student’s t test for unpaired values.

4022 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
ChegaeV et al.
Figure 2. Concentration-response curves of (-)1b in the absence
(b) and in the presence of inhibitors (1 µM benomyl (O), 1 mM chloral
hydrate ([), or 1 µM ODQ (2)).
Figure 3. Concentration-response curves of (+)2 in the absence (b)
and in the presence of inhibitors (1 µM benomyl (O), 1 mM chloral
hydrate ([), or 1 µM ODQ (2)).
It seems that NO can be produced directly by ALDH-2 also.4c
Inhibitors of ALDH-2, such as benomyl (methyl 1-(butylcar-
bamoyl)-2-benzimidazole carbamate) and chloral hydrate, shift
the concentration-response curve of GTN to higher concentra-
tion.4b,c,13 Because in ALDH-2 deficient animals or in the
presence of ALDH-2 inhibitors the GTN maximal response is
only shifted but not suppressed,11 two independent pathways
of GTN bioactivation have been postulated: the high affinity
pathway dependent on ALDH-2 activity and the low affinity
one dependent on P450 enzyme(s).4b,11
shift about doubled. The related butyl derivatives (+)7 and (-)7
show a vasodilator profile near that of propyl analogues. They
are slightly more potent, but the principal difference is a less
pronounced shift of the concentration-response curves under
the action of benomyl, chloral hydrate, and ODQ. In addition,
in the presence of this latter inhibitor, the products are able to
trigger almost entirely the maximal relaxation. Finally 3-phe-
nylpropyl and 4-phenylbutyl nitrates (8) and (9), respectively,
are about three times less potent than the related dinitrooxy
analogues but they show a similar vasodilator profile. Recently
it was suggested, on the basis of a study of a limited number of
nitrates, that three or more nitrooxy groups must be present in
the vasodilator in order for it to be bioactivated by ALDH-2
because organic nitrates with less than three nitrate groups
should not be sufficiently reactive to undergo nucleophilic attack
by thiol groups present in the active cleft of enzyme.11 All the
data here reported clearly shows that the presence of three
nitrooxy groups is a condition neither necessary nor sufficient
for the bioactivation of the nitrovasodilator by ALDH-2.
Therefore, other molecular descriptors, in addition to the
electrophilicity of the -ONO2 group, must play important roles
in making bioactivation by this enzyme possible.
Cross-Tolerance. In vitro cross tolerance with GTN of all
the nitrates described in the present work was evaluated by
studying their vasodilator activity after incubation of the aortic
strips with a high concentration (0.55 mM) of GTN for 1 h
followed by washout. The results expressed as EC50tol are
reported in Table 1. The two stereomers (+)2, (-)2 and the
mononitrooxy substituted compound 8, whose vasodilator
potencies are reduced by about 10-fold and 20-fold in the
presence of benomyl and chloral hydrate, respectively, display
The introduction of a phenyl group into the C-1 of GTN
modifies the in vitro vasodilator profile of the drug. In fact,
both the erythro enantiomers (+)1a, (-)1a and threo enanti-
omers (+)1b, (-)1b show a monophasic concentration-response
curve. An example of this behavior is reported in Figure 2. In
the two pairs of antipodes, a complete lack of enantioselectivity
is observed as far as the vasodilating potency is concerned, and
the erythro enantiomers are as potent as the threo ones (Table
1). EC50 values of the products are about 10-fold higher than
the GTN value. No shift or a nonsignificant shift of the
concentration-response curves occurred when the experiments
were repeated in the presence of 1 µM benomyl and for (+)1b
and (-)1b also in the presence of 1 mM chloral hydrate (see
Figure 2 for an example). This suggests that the GTN high
affinity pathway (ALDH-2) is not responsible for the bioacti-
vation of these products. A strong rightward shift of the
concentration-response curves was observed when the experi-
ments were repeated in the presence of 1 µM ODQ. Further-
more, the maximal response was maintained and no further
changes occurred, even raising ODQ concentration to 100 µM.
This finding suggests that these nitrates, unlike GTN, can elicit
vasodilation at high concentration by a pathway independent
of the activation of the heme site of sGC. The trinitrooxy
substituted stereomers of the butyl series (+)5a, (-)5a, (+)5b,
and (-)5b behave very similarly to those of the propyl series
(see Table 1). The principal difference is only a slightly higher
potency of the threo stereomer (+)5b compared with all the
other trinitrooxy substituted products. 3-Phenylpropane-1,2-diyl
dinitrate stereomers (+)2, (-)2 behave in a different manner.
They show the same biphasic concentration-response curves
as GTN (Figure 3). The two enantiomers display the same
potency, and they are about 2-fold less potent than GTN and
4-fold more potent than the phenyl trinitrooxy stereomers (Table
1). Also, in the presence of the inhibitors used in our experi-
ments, they display behavior reminiscent of GTN. Indeed, in
tissues preincubated with ODQ, the vasodilator response is
largely suppressed while in the presence of benomyl the
concentration-response curve is shifted rightwards about 10-
fold. When chloral hydrate was used to inhibit ALDH-2, this
similar behavior to that of GTN. Indeed, they show high EC50tol
/
EC50 values, which range from 100 to 125 (see Figure 4 for an
example). The related butyl derivatives (+)7, (-)7, and 9, which
were less sensitive to benomyl and chloral hydrate inhibition,
show lower EC50tol/EC50 values that range from 30 to 49. Finally,
all trinitrooxy substituted products (+)1a, (-)1a, (+)1b, (-)1b,
(+)5a, (-)5a, (+)5b, and (-)5b, whose potency is not
significantly influenced by the presence of benomyl, show a
very modest GTN cross-tolerance (see Figure 5 for an example).
To conclude, in this study, only the products that exhibit high
potencies as vasodilators and whose activity is decreased in the
presence of ALDH-2 inhibitors display in vitro cross-tolerance
with GTN. These results support the hypothesis that ALDH-2
plays a central role in the development of in vitro tolerance.14
Conclusion
This work describes the vasodilator profiles of new esters of
nitric acid, obtained by introducing at C-1 of GTN a benzyl or

Organic Nitrates and Their Vasodilator Profile
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13 4023
were synthesized according to published methods. Compound
3b was obtained by the same procedure used to obtain 3a starting
from trans-but-2-en-1,4-diol,18 and its spectral characteristics
correspond to those published.19
Chiral Separation. Analytical chiral HPLC experiments on
Chiralcel OD-H column (250 mm × 4.6 mm, 5 µm) (Daicel Co.,
Tokyo) were performed with LaChrom (Merck) screening unit
equipped with an L-7100 pump, an L-7200 autosampler, an L-7360
oven that accommodates 12 columns alimented by a Valco valve,
an L-7400 UV detector, and a Jasco OR-1590 polarimeter detector.
Analyses were performed at 1 mL/min, at a controlled temperature
(25 °C) with UV (254 nm) and polarimetric detection, eluting with
hexane/iPrOH 7/3 v/v. Retention times Rt in minutes, retention
factor ki ) (Rti - Rt0)/Rt0 and enantioselectivity factor R ) k2/k1
are given. Semipreparative separations were performed with a
Merck-Hitachi LiChrograph model L-6000 HPLC pump and a
Merck-Hitachi LiChrograph L-4000 UV detector (254 nm). For
semipreparative separations, a Chiralcel OD column (250 mm ×
10 mm, 10 µm) was used, eluting with hexane/iPrOH 7/3 v/v at a
flow rate of 4.5 mL/min. The solvents were HPLC grade from SDS
(Peypin, France) and were filtered on a Millipore membrane of 0.45
µm and degassed before use.
Figure 4. Concentration-response curves of (+)2 (circles) and GTN
(triangles) in vessels with or without treatment with 0.55 mM GTN
for 1 h followed by washout (open or solid symbols, respectively).
rac-erythro-4-Phenylbutane-1,2,3-triyl Trinitrate (5a). To the
solution of 3a (1.10 g, 7.6 mmol) and AgNO3 (3.90 g, 23.0 mmol)
in CH3CN (50 mL), I2 (1.90 g, 7.6 mmol) was added in one portion
at rt. The reaction mixture was vigorously stirred until all iodine
dissolved, and then it was heated at reflux for 12 h. The obtained
mixture was filtered, the precipitate was washed with EtOAc, and
the filtrate was diluted with EtOAc (100 mL). The organic phase
was washed with H2O (2 × 50 mL) and brine, dried and evaporated.
The obtained dark-brown oil was partly purified by flash chroma-
tography (eluent PE/EtOAc 9/1 v/v) to give crude 4a (0.56 g) as a
yellow oil. The obtained oil was dissolved in CH3CN (15 mL),
then PPh3 (0.81 g, 3.1 mmol) and AgNO3 (0.70 g, 4.1 mmol) were
added. The resulting solution was cooled in an ice-salt bath, and
NBS (0.55 g, 3.1 mmol) was added in small portions. The cooling
bath was removed, and the reaction was vigorously stirred at rt for
2 h. The precipitate was filtered and washed with EtOAc. The filtrate
was evaporated and purified by flash chromatography (eluent PE/
CH2Cl2 8/2 v/v) to give the title compound as a colorless oil, which
solidifies at -24 °C and melts at rt. Yield 15%; mp < rt. IR (film)
3034, 2919, 1645, 1497, 1456, 1431, 1358, 1275, 1009, 899, 839,
Figure 5. Concentration-response curves of (-)1b (squares) and GTN
(triangles) in vessels with or without treatment with 0.55 mM GTN
for 1 h followed by washout (open or solid symbols, respectively).
a phenyl group. Further compounds, formally derived from these
leads by elimination of one or two nitrooxy functions, were
studied as well. All trinitrooxy substituted compounds are about
4-10 fold less potent than GTN as vasodilators when tested
on rat aorta strips precontracted with phenylephrine and their
vasodilator profile in the presence of ODQ, benomyl and chloral
hydrate is quite different from that of GTN. By contrast, all the
related dinitrooxy and mononitrooxy substituted products display
a vasodilator profile similar to that of GTN, but their potencies
are 2-fold (dinitrooxy series) and 5-fold (mononitrooxy series)
lower than that of GTN. Unlike the trinitrooxy analogues, they
display evidence of in vitro cross-tolerance with GTN. These
new series of nitrates are interesting tools for shedding new
light on the unresolved puzzle of nitrate pharmacology. Selected
members of these compounds are worthy of additional study
as potential drugs.
1
750, 702 cm-1. H NMR (CDCl3) δ: 3.02-3.18 (2H, m, PhCH2),
2
3
4.55 (1H, dd, JHH ) 13.3 Hz, JHH ) 7.4 Hz, CHHONO2), 4.77
2
3
(1H, dd, JHH ) 13.3 Hz, JHH ) 3.1 Hz, CHHONO2), 5.36-5.40
(1H, m, CHONO2), 5.52-5.58 (1H, m, CHONO2), 7.23-7.39 (5H,
m, C6H5). 13C NMR (CDCl3) δ: 35.6, 67.7, 77.3, 80.3, 128.2, 129.2,
129.4, 133.6. MS (EI): 317 (M)+. Anal. (C10H11N3O9) C, H, N.
The enantiomers (-)5a and (+)5a were separated by semi-
preparative HPLC ((+)5a, first eluted); 350 mg of racemic mixture
were separated. The reported signs are the signs given by the Jasco
OR-1590 polarimeter in the mobile phase used.20 Optical purities
((+)5a, 100%; (-)5a, 99.99%) were assessed on analytical HPLC;
Rt(+) ) 10.91, Rt(-) ) 17.55, k(+) ) 2.54, k(-) ) 4.70, R )
1.85, Rs ) 6.34.
Experimental Section
Chemistry. 1H and 13C NMR spectra were recorded on a
Bruker Avance 300 instrument, with Si(CH3)4 as an internal
standard. The following abbreviations were used to indicate the
peak multiplicity: s ) singlet; d ) doublet; t ) triplet; q )
quartet; m ) multiplet. Low resolution mass spectra were
recorded with a Finnigan-Mat TSQ-700 instrument. IR spectra
were recorded on a Shimadzu FT-IR 8101 M. Flash column
chromatography was performed on silica gel (Merck Kieselgel
60, 230-400 mesh ASTM) using the indicated eluents. Petro-
leum ether 40-70 °C (PE) was used as coeluent. The progress
of the reactions was followed by thin layer chromatography
(TLC) on 5 cm × 20 cm plates with a layer thickness of 0.2
mm. Anhydrous magnesium sulfate was used as a drying agent
for the organic phases. Organic solvents were removed under
vacuum at 35-40 °C. Analysis (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 values. Structures 1,6 2,6 3a,15 8,16 and 917
rac-threo-4-Phenylbutane-1,2,3-triyl Trinitrate (5b). The prod-
uct was synthesized by the same procedure used to obtain 5a,
starting from 3b. Colorless oil. Yield 13%. IR (film) 3034, 2921,
1651, 1645, 1497, 1456, 1485, 1358, 1269, 1030, 1001, 839,
750, 702 cm-1. 1H NMR (CDCl3) δ: 3.08-3.12 (2H, m, PhCH2),
4.58 (1H, dd, 2JHH ) 12.9 Hz, 3JHH ) 6.9 Hz, CHHONO2), 4.77
(1H, dd, 2JHH ) 12.9 Hz, 3JHH ) 3.9 Hz, CHHONO2), 5.37-5.39
(1H, m, CHONO2), 5.49-5.51 (1H, m, CHONO2), 7.20-7.36
(5H, m, C6H5). 13C NMR (CDCl3) δ: 35.6, 68.6, 75.9, 79.9,
128.0, 129.0, 129.3, 133.6. MS (EI): 317 (M)+. Anal.
(C10H11N3O9) C, H, N.
The enantiomers (-)5b and (+)5b were separated by semi-
preparative HPLC ((-)5b, first eluted); 300 mg of racemic mixture
were separated. The reported signs are the signs given by the Jasco
OR-1590 polarimeter in the mobile phase used. Optical purities
((-)5b, 99.7%; (+)5b, 99.8%) were assessed on analytical HPLC;

4024 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 13
ChegaeV et al.
Rt(-) ) 9.96, Rt(+) ) 11.92, k(-) ) 2.23, k(+) ) 2.87, R )
1.28, Rs ) 2.38.
Supporting Information Available: Elemental analyses. IR
spectra of 5a, 5b, and 7. Concentration-response curves of all the
products. This material is available free of charge via the Internet
at http://pubs.acs.org.
rac-4-Phenylbutane-1,2-diyl Dinitrate (7). To the solution of
6 (0.40 mL, 2.7 mmol) and AgNO3 (1.25 g, 7.3 mmol) in CH3CN
(15 mL), I2 (0.68 g, 2.7 mmol) was added in one portion. The
reaction mixture was vigorously stirred at rt until all iodine dissolved
and then it was heated at reflux for 12 h. The obtained mixture
was filtered, and the precipitate was washed with EtOAc. The filtrate
was diluted with EtOAc (30 mL), and the organic phase was washed
with H2O (2 × 25 mL) and brine, dried and evaporated. The
obtained oil was purified by flash chromatography (eluent PE/
CH2Cl2 8/2) to give the title compound as a pale-yellow liquid.
Yield 57%. IR (film) 3088, 3065, 3030, 2930, 1954, 1659, 1651,
1645, 1634, 1497, 1456, 1435, 1383, 1271, 1180, 1157, 1113, 1045,
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1
1001, 855, 752, 700 cm-1. H NMR (CDCl3) δ: 1.97-2.18 (2H,
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)
3
2
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3
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Acknowledgment. This work was supported by a MIUR
grant (PRIN 2005). We are indebted to Dr. Hansjorg Eder,
Universite´ de Gene`ve Section de Pharmacie Service de Mi-
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Organic Nitrates and Their Vasodilator Profile
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