Synthesis and collateral dilator activity of nitroxyalkylamides having direct or latent sulfhydryl moieties

Article abstract of DOI:10.1016/S0960-894X(03)00199-9

To develop an orally active, long-acting nitrate that does not induce tolerance, nitroxyalkyl compounds were prepared and their activities evaluated by the use of carotid collaterals in anesthetized dogs. A compound having a favorable pharmacological profile, that is, long-lasting collateral vasodilatation and little hypotension, and lack of nitrate tolerance, was chosen for further evaluation.

Full text of DOI:10.1016/S0960-894X(03)00199-9

Bioorganic & Medicinal Chemistry Letters 13 (2003) 1527–1530
Synthesis and Collateral Dilator Activity of Nitroxyalkylamides
Having Direct or Latent Sulfhydryl Moieties
a,
b
a
a
a
Sadao Ishihara, * Fujio Saito, Yasuo Ohhata, Marie Kanai, Hiroshi Mizuno,
a
a
a
Michio Fujisawa, Ryosuke Yorikane and Hiroyuki Koike
aResearch Laboratories, Sankyo Co., Ltd., Hiromachi, Shinagawa-ku Tokyo 140, Japan
bChemtech Labo., Inc., Hiromachi, Shinagawa-ku Tokyo 140, Japan
Received 7 October 2002; accepted 22 February 2003
Abstract—To develop an orally active, long-acting nitrate that does not induce tolerance, nitroxyalkyl compounds were prepared
and their activities evaluated by the use of carotid collaterals in anesthetized dogs. A compound having a favorable pharmaco-
logical profile, that is, long-lasting collateral vasodilatation and little hypotension, and lack of nitrate tolerance, was chosen for
further evaluation.
#
2003 Elsevier Science Ltd. All rights reserved.
4
Introduction
(mtALDH) has been identified by Stamlar et al. as an
enzyme involved in the bio-activation of nitroglycerin.
The activity of this enzyme has been shown to be
dependent on the presence of sulfhydryl as a reductant.
They have also demonstrated that desensitization of
mtALDH due to a continuous exposure to nitroglycerin
might account for tolerance development.
1
Organic nitrates have been widely used as antianginal
2
drugs. Since nitroglycerin (1) and isosorbide dinitrate
undergo a hepatic first-pass effect and have a short
duration of action, they are administered by the sub-
lingual route for the treatment of angina pectoris or by
the transdermal route for prophylaxis. An orally active
sustained-release preparation of isosorbide 5-mono-
nitrate is also widely used. Nitrates have the dis-
advantage of causing adverse reactions such as
dizziness, headache or tachycardia, which are induced
by their potent hypotensive actions. Another dis-
advantage of these drugs is the development of nitrate
tolerance following their continuous use, which is
regarded to be the most serious clinical limitation
shared by all of the nitrates under clinical use. We,
therefore, set a goal of developing a new nitrate com-
pound having the following pharmacological properties:
We designed and synthesized nitrate compounds that
simultaneously release NO and thiols in the cell. In the
present study, focusing on the cysteinyl group of gluta-
thione, cysteine derivatives (5) and 2-oxothiazolidine
5
derivatives (7) were synthesized. 2-Oxothiazolidine-4-
carboxylic acid is a latent cysteine since it is converted in
vivo into cysteine by the action of an enzyme, 5-oxoproli-
6
nase. The cysteine derivatives and 2 -oxothiazolidine
derivatives are expected to prevent the development of
tolerance by generating sulfhydryl group in vivo.
(
1) a long-lasting collateral vasodilator effect, (2) minimal
In the present study, the pharmacological activity of
cysteine derivatives and 2-oxothiazolidine derivatives
was evaluated for their vasorelaxant effect on carotid
collateral vessels in anesthetized dogs.
hypotensive effect and tachycardia, (3) no hepatic first-
pass effect and (4) lack of tolerance development. Nitrate
drugs are mostly prodrugs which release nitric oxide
(NO), the active intermediate, in vascular smooth muscle
cells. Recently, mitochondrial aldehyde dehydrogenase
3
Synthesis
The synthetic method of compounds 5a–j, 7a–t having a
nitroxyalkylamide group is shown in Scheme 1.
*Corresponding author. Tel.:+81-3-3492-3131; fax:+81-3-5436-8570;
e-mail: isiwar@shina.sankyo.co.jp
0
960-894X/03/$ - see front matter # 2003 Elsevier Science Ltd. All rights reserved.
doi:10.1016/S0960-894X(03)00199-9

1528
S. Ishihara et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1527–1530
we have developed a new method to evaluate the vaso-
dilator effect of a test compound on collateral arteries.
The details of the method will soon be published else-
where, but here is a brief description.
A polyethylene catheter (2F) was inserted into one of
the branches of the left thyrocervical artery for recording
the left carotid arterial pressure (CAP). The common left
carotid artery was occluded for 1 min, proximal to the
insertion site of the polyethylene catheter, and CAP was
measured immediately before and after occlusion. The
decrease in CAP (ꢀP) was measured during occlusion.
Prior to the administration of a test substance, the car-
otid occlusion was repeated every 15 min until the same
response was obtained at least for three successive mea-
surements. The test substance was intravenously admi-
nistered at a dose of 0.1 or 0.3 mg/kg, and the left
carotid artery was occluded for 1 min at 5, 15, 30, 45
and 60 min after the administration. At each time point,
Scheme 1.
0
Compounds 5a–j and 7a–t were synthesized from the
corresponding cysteine derivatives (4) and 2-oxothiazo-
lidine-4-carboxylic acid derivatives (6) with nitroxyalkyl-
amine derivatives (3) by a condensation reaction with
CAP immediately before occlusion (P ) and the decrease
0
in CAP after occlusion (DP ) were measured. The vaso-
dilator activity of the compound in the collateral vessels
(CI=collateral index) was calculated at each measure-
ment time point according to the following equation.
DEPC or DPPA in the presence of triethylamine (Et N).
3
0
0
CI=100ꢀ(ꢀP /P )ꢁ100/(ꢀP/P).
Nitroxyalkylamine derivatives (3) were prepared by add-
ing aminoalcohol derivatives (2) to fuming nitric acid.
Thereafter, the AUC (CIAUC) over a period of 60 min
was also calculated. As a reference substance, nitro-
glycerin was used. It was administered intravenously at
a dose of 0.03 mg/kg, and the CIAUC was calculated.
From the values obtained above, the ratio of the CIAUC
value of each test substance to that of nitroglycerin
The synthesized compounds are shown in Tables 1 and 2.
Screening Procedure
(NTG ratio) was calculated.
Male adult beagle dogs were used in this experiment.
Animals were anesthetized with sodium pentobarbital
Table 2. The synthesized compounds (7a–t)
(
40 mg/kg iv) and artificially ventilated. The femoral
arterial blood pressure and heart rate were monitored
throughout the experiment.
The characteristics of collateral vessels in the carotid vas-
cular bed were investigated in detail in the anesthetized
dogs by Iriuchijima and Koike. Based on their results,
7
,8
Compd
R1
R2*1
*2
A
mp
7
7
7
7
7
7
7
7
7
7
7
7
7m
7n
7o
7p
7q
7r
a
b
c
d
e
f
g
h
i
H
H
H
H
H
H
H
H
H
H
H
H
Me
Me
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
H
H
H
H
H
H
—
—
—
—
—
—
—
—
—
—
—
—
R
R
S
(CH
(CH
(CH
2
2
2
2
3
3
3
3
)
)
)
)
2
2
3
4
133–134
129–130
83–85
68–70
125
102–103
102–103
119
Table 1. The synthesized compounds (5a–j)
R
R
R
R
S
(CH
CH(CH
CH(CH
CH(CH
CH(CH
2
CH CH(CH
CH(Et)CH
CH(Pr)CH
)CH
)CH
)CH
)CH
2
2
2
2
S
R
R
R
R
R
R
3
)
oil
j
k
l
2
106–107
99–100
110–112
oil
98–100
138–140
123
117–118
120
151
Compd
R1
R2A
mp
2
CH(Bu)CH
2
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
H
H
Ac
iByr
Piv
Boz
Nic
Etc
jBuc
Boc
Ac
Nic
Ac
iByr
Ac
Ac
Ac
Ac
Ac
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
2
2
2
2
2
2
2
2
2
2
)
)
)
)
)
)
)
)
)
)
2
2
2
2
2
2
2
2
2
2
103–104
99–100
95–97
(CH
(CH
(CH
(CH
(CH
(CH
(CH
(CH
2
2
2
2
2
2
2
2
)
)
)
)
)
)
)
)
2
2
2
2
2
2
2
2
—
RS RS
RS RS
RS RS
RS RS
RS RS
RS RS
64–66
Bz
101–103
137–138
110–112
119–120
92–93
2-Furyl
2-Thienyl
1-Naphtyl
3-Pyridyl
7s
7t
189
j
Ac
96–97
*1 and *2indicate the stereochemistry of asymmetric carbon atoms.
The asymmetric center of the A moieties of 7e, 7g and 7j–l are S, that
of 7f and 7h are R, and that of 7i are R, S. Me: methyl, Ph: phenyl,
Bz: benzyl.
Ac: acetyl; iByr: isobutyryl, Piv: pivaloyl, Boz: benzoyl, Nic: nicotinoyl,
Etc: ethoxycarbonyl, iBuc: isobutyloxy-carbonyl, Boc: t-buoxycarbonyl.

S. Ishihara et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1527–1530
1529
The vasodilator activity of each test substance in the
collateral vessels was evaluated based on (1) the max-
imum dilatation effect (max CI), (2) CI value at 60 min
after administration, (3) ratio of AUC of each test sub-
stance to that of nitroglycerin and (4) the maximum
decrease in femoral arterial blood pressure (maximum
hypotension, as shown in Tables 3 and 4).
Among 10 cysteine derivatives examined, 8 showed col-
lateral dilator action that was as potent as NTG, but the
duration of the action was relatively short. In addition,
these cysteine derivatives induced marked hypotension.
Thus, the pharmacological profile of each of the
cysteine derivatives was very similar to that of NTG and
the collateral dilator action of the cysteine derivatives
seemed to be inseparable from their hypotensive action.
The effects of 2-oxothiazolidine derivatives (7a–t) on
collateral vessels and systemic blood pressure are shown
in Table 4, when the derivative compounds were given
at doses of 0.1 or 0.3 mg/kg, iv The collateral dilator
activity of most 2-oxothiazolidine derivatives was less
than that of NTG or cysteine derivatives at the peak
response. However, their action was much longer than
cysteine derivatives, such as compounds 7a, 7e, 7f, 7k
and 7l. The hypotensive action of these 5 compounds
was negligible.
Dilatation Activity on Carotid Collaterals
NTG (1) was administered intravenously at a dose of
0
.03 mg/kg. After 5 min, the dilatation of collateral
vessel reached a maximum (CImax=33) and then,
diminished rapidly (CI =4 in Table 3, and Fig. 1).
6
0
The femoral arterial blood pressure fell markedly
immediately after administration of NTG. This was also
accompanied by marked tachycardia.
The collateral dilator and hypotensive actions of
cysteine derivatives (5a–j) are summarized in Table 3.
Of compounds 7a–t, compounds in which the R and R
1
2
were hydrogen atoms exhibited the highest activity and
introduction of alkyl, phenyl or heterocyclic groups at
R1 diminished the activity. Regarding the alkylene
group shown as moiety A, compound 7a having a
straight chain alkylene group showed the highest activ-
ity, and the activity of compounds 7c–d with a longer
straight chain was low, compared with that of 7a.
Introduction of branched alkyl groups gave rise to an
asymmetric center and decreased the activity, as seen
with 7e–j. However, a much more bulky alkyl group
seemed to increase the activity.
Table 3. Collateral Index of nitroxyalkyl compounds (5a–j) in anes-
thetized dog
Compd
Dose
mg/kg, iv)
Max
CI
CI
(at 60 min) ratio hypotension (%)
NG
Max
(
1
0.03
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
0.1
33
39
20
526.0
25
45
49
20
4
9
0.0
2
9
8
5
9
16
6
11
1.0
1.9
ꢀ55
ꢀ52
ꢀ3
5
5
5
5
5
5
5
5
5
5
a
b
c
d
e
f
g
h
i
ꢀ44
ꢀ14
ꢀ23
ꢀ28
ꢀ24
ꢀ18
ꢀ12
ꢀ8
1.5
2.5
2.1
1.1
2.4
1.3
0.8
39
25
21
j
Table 4. Collateral Index of nitroxyalkyl compounds (7a–t) in anes-
thetized dog
Compd
Dose
mg/kg, iv)
Max
Cl
Cl
(at 60 min) ratio hypotension (%)
NG
Max
(
1
0.03
0.1
0.3
0.3
0.1
0.3
0.3
0.3
0.3
0.1
0.3
0.3
0.3
0.1
0.3
0.3
0.1
0.3
0.1
0.1
0.1
33
23
14
4
20
16
12120.9
5
6
0
4
16
11
6
0.0
15
1.0
1.9
0.8
0.1
ꢀ55
ꢀ8
ꢀ6
0
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
7
a
b
c
d
e
f
g
h
i
j
k
l
m
n
o
p
q
r
0
1.1
ꢀ5
ꢀ4
ꢀ1
ꢀ3
0
ꢀ1
ꢀ7
ꢀ4
ꢀ2
ꢀ2
ꢀ6
ꢀ3
ꢀ3
0
3
5
0
0.3
0.4
0.0
0.5
1.3
1.6
0.5
0.6
1.0
7
7
18
21
9
17
17
4
8
9
1.0
3
3
9
0.1
9
18
127
6
8
9
20
0.4
0.5
0.5
Figure 1. Effects of nitroglycerin and compound 7a on hemodynamics
and carotid collateral vessels in anesthetized dogs. HR and MBP are
expressed as absolute changes from baseline values. All data are
meansꢂstandard errors of 7 experiments. Abbreviations, HR: heart
rate, MBP: mean blood pressure, CI: collateral index.
s
t
ꢀ2
ꢀ4

1530
S. Ishihara et al. / Bioorg. Med. Chem. Lett. 13 (2003) 1527–1530
(
CI =16). In contrast to NTG, compound 7a pro-
60
duced little hypotension and reflex tachycardia. Thus,
compound 7a had a potent and long-lasting dilator
activity of collateral vessels and a favorable hemo-
dynamic effect.
Furthermore, we examined whether 2-oxothiazolidine
derivatives developed nitrate tolerance in isolated rat
aorta or not. Tolerance was induced by pre-incubation
ꢀ
4
for 60 min with a high concentration of NTG (10 M)
ꢀ
3
or compound 7a (10 M), followed by a washout per-
iod of 60 min. Pre-incubation with each drug did not
change the degree of contraction induced by phenyl-
ephrine (data not shown). As shown in Figure 2A, the
vasorelaxant effects of NTG in aortic strips pretreated
with 10 M NTG were markedly attenuated as com-
pared with those pretreated with the vehicle, suggesting
the development of tolerance. On the contrary, the
vasorelaxant activity of compound 7a was not affected
by pre-incubation, not even at the highest concentration
ꢀ
4
ꢀ
3
of 10 M, as shown in Figure 2B.
In summary, we synthesized organic nitrates with direct
(cysteine derivatives) and latent (2-oxothiazolidine deri-
vatives) sulfhydryl moieties. Some nitrates with latent
sulfhydryls seemed to have favorable pharmacological
properties as antianginal drug: (1) a long-lasting col-
lateral vasodilator effect, and (2) minimal hypotensive
effect and tachycardia. In particular, among these deri-
vatives, compound 7a induced no nitrate tolerance.
Figure 2. Vasorelaxant effects of nitroglycerin (NTG: A) or com-
pound 7a (7a: B) in isolated rat aortic ring segments pretreated with
the vehicle (control) and nitroglycerin or compound 7a, respectively.
The aortic ring segments were pre-incubated for 60 min with the
ꢀ4
vehicle and NTG or compound 7a at a concentration of 10 M or
ꢀ3
1
6
3
0
M, respectively, washed repeatedly with drug-free fresh buffer for
0 min, and pre-contracted with phenylephrine at a concentration of
References and Notes
:
:7
ꢁ10 M, followed by cumulative administration of NTG or com-
1
2
3
4
. Bertolini, G. Curr. Pharm. Des. 1996, 2, 463.
. Abrams, J. Arch. Intern. Med. 1995, 155, 357.
. Moncada, S. Nature 1987, 327, 524.
pound 7a, respectively. Data are meansꢂSE of 3–5 experiments.
. Chen, Z.; Zhang, J.; Stamler, J. S. Proc. Natl. Acad. Sci.
The stereoconfiguration at 4-position of the thiazolidine
ring of compounds 7a–t affected the activity; R stereo-
isomers derived from natural amino acids generally
showed more potent activity than S isomers.
U.S.A. 2002, 99, 8306.
. Ishihara, S.; Saito, F.; Yoshioka, T.; Koike, H.; Miyake, S.;
Mizuno, H.(sankyo): Eur. Pat. EP506, 434; Chem. Abstr. 1993
5
1
6
18, 59703q.
. Meister, A.; Williamson, J. M. Proc. Natl. Acad. Sci.
The vasodilator effects of compound 7a on collateral
vessels developed slowly and reached a maximum
U.S.A. 1981, 78, 936.
. Iriuchijima, J.; Koike, H. Am. J. Pysiol. 1970, 211, 876.
7
(CImax=23) at 15 min after the administration, but the
dilator action lasted until the end of the experiment
8. Iriuchijima, J.; Koike, H.; Kurihara, M. Phlu¨gers Arch.
1971, 322, 304.