Practical access to four stereoisomers of naftidrofuryl and their binding affinity towards 5-hydroxytryptamine 2A receptor

Article abstract of DOI:10.1016/j.bmcl.2012.03.093

Naftidrofuryl oxalate (Praxilene, 1) has been used for the treatment of intermittent claudication for more than 30 years. It selectively blocks vascular and platelet 5-hydroxytryptamine 2 (5-HT₂) receptors. This drug is marketed as a mixture of four stereoisomers, and so far there is no individual biological evaluation on the single isomers. The purpose of this study is to provide an improved method for the preparation of all four stereoisomers of naftidrofuryl, and more importantly, to distinguish them in terms of their binding affinity to 5-hydroxytryptamine 2A (5-HT_2A) receptor. The bioassay results revealed that the C-2S configuration of naftidrofuryl was crucial for the binding affinity with 5-HT_2A receptor, and the C-2′ configuration was less important for binding. In conclusion, our study may pave the way to develop single naftidrofuryl isomers with C-2S configuration as inhibitors of 5-HT_2A receptor that have clinical significance as vasodilators and CNS agents.

Full text of DOI:10.1016/j.bmcl.2012.03.093

Bioorganic & Medicinal Chemistry Letters 22 (2012) 3441–3444
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Practical access to four stereoisomers of naftidrofuryl and their binding
affinity towards 5-hydroxytryptamine 2A receptor
Jia Hao a,b, Bo Chen a, Yiwu Yao a, Murad Hossain c, Takafumi Nagatomo c, Hequan Yao a,
Lingyi Kong a, Hongbin Sun a,
⇑
a State Key Laboratory of Natural Medicines, China Pharmaceutical University, Nanjing 210009, PR China
b Tianjin State Key Lab Modern Chinese Med, Tianjin University of Traditional Chinese Medicine, Tianjin 300193, PR China
c Department of Pharmacology, Faculty of Pharmaceutical Sciences, Niigata University of Pharmacy and Applied Life Sciences, Niigata 956-8603, Japan
a r t i c l e i n f o
a b s t r a c t
Naftidrofuryl oxalate (PraxileneÒ, 1) has been used for the treatment of intermittent claudication for
more than 30 years. It selectively blocks vascular and platelet 5-hydroxytryptamine 2 (5-HT2) receptors.
This drug is marketed as a mixture of four stereoisomers, and so far there is no individual biological eval-
uation on the single isomers. The purpose of this study is to provide an improved method for the prep-
aration of all four stereoisomers of naftidrofuryl, and more importantly, to distinguish them in terms of
their binding affinity to 5-hydroxytryptamine 2A (5-HT2A) receptor. The bioassay results revealed that
the C-2S configuration of naftidrofuryl was crucial for the binding affinity with 5-HT2A receptor, and
the C-20 configuration was less important for binding. In conclusion, our study may pave the way to
develop single naftidrofuryl isomers with C-2S configuration as inhibitors of 5-HT2A receptor that have
clinical significance as vasodilators and CNS agents.
Article history:
Received 27 January 2012
Revised 24 March 2012
Accepted 27 March 2012
Available online 1 April 2012
Keywords:
Naftidrofuryl stereoisomers
5-HT2A receptor
Inhibitors
Resolution
Ó 2012 Elsevier Ltd. All rights reserved.
Configuration
Naftidrofuryl oxalate (PraxileneÒ, 1) is a vasodilator that has
been used for the treatment of intermittent claudication for more
than 30 years in Europe.1 It also can be used for the treatment of
stroke2 and dementia.3 Naftidrofuryl has selective 5-hydroxytryp-
tamine 2 (5-HT2) receptor antagonist activity,4 and this antago-
nism appears to reduce the vasoconstrictive effects of 5-HT
released from endothelia which is damaged by atherosclerosis
and hypoxia. It also inhibits serotonin-induced platelet aggregation
and platelet-induced vasospasm.5 Moreover, naftidrofuryl appears
to increase ATP levels in fibroblast and endothelial cells by improv-
ing the aerobic metabolism in the blood vessel wall.6 This particu-
lar effect towards the aerobic metabolism makes naftidrofuryl a
unique therapeutic choice for the treatment of intermittent claudi-
cation, stroke, and dementia, in comparison with other anti-plate-
let agents. Nevertheless, the action mechanism of naftidrofuryl is
still unclear. Recently, Nagatomo and co-workers7 reported the
assessment of binding affinity of naftidrofuryl to 5-hydroxytrypta-
mine 2A (5-HT2A) receptor in comparison with sarpogrelate, a
well-known selective 5-HT2A receptor antagonist, claiming that
naftidrofuryl showed lower binding affinity than sarpogrelate.
Considering that naftidrofuryl is marketed as a mixture of four
stereoisomers (2–5, Fig. 1), and so far there is no individual biolog-
ical evaluation on the single isomers,8 it is thus desirable to
2
COOH
COOH
3
2'
1
N
O
O
O
1
N
N
O
O
O
O
O
O
3
2R, 2'S
2 2S, 2'S
N
N
O
O
O
O
O
O
4 2R, 2'R
5 2S, 2'R
⇑
Corresponding author. Tel./fax: +86 25 83271198.
E-mail address: hbsun2000@yahoo.com (H. Sun).
Figure 1. Chemical structures of compounds 1–5.
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.093

3442
J. Hao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3441–3444
OH
OTs
CH 2(COOEt) 2
EtONa, 78%
TsCl, Pyr.
92%
O
7
O
6
Cl
COOEt
COOEt
KOH, EtOH
COOEt
COOEt
9
O
DMF, NaH
80%
8
O
Vacuum
130 oC,1h
96% for two steps
COOH
COOH
10
O
O
OH
O
11
1) S-1-Phenylethylamine
2) HCl 35%
O
OH
O
12
1) R-1-Phenylethylamine
2) HCl
23%
O
OH
O
13
Scheme 1. Synthesis of diastereomerically pure carboxylic acids 12 and 13.
Cl
N
HCl
N
O
K2CO3 PTC, 97%
,
O
O
O
OH
O
12
2
COOH
COOH
COOH
COOH
2H2O
Actone
N
O
O
O
14
Cl
N
HCl
K2CO3, PTC, 98%
N
O
O
OH
O
O
O
13
3
COOH
COOH
2H2O
COOH
COOH
Actone
N
O
O
O
15
Scheme 2. Preparation of naftidrofuryl isomers 2, 3, and corresponding oxalates 14, 15.

J. Hao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3441–3444
3443
distinguish these isomers in terms of their in vitro and in vivo
activities. We report herein an improved method for the prepara-
tion of all four stereoisomers of naftidrofuryl (2–5, Fig. 1), and
the biological evaluation of their binding affinity to 5-HT2A
receptor.
CH2(COOEt)2
EtONa, 77%
TsCl, Pyr.
96%
OH
OTs
O
O
17
16
Cl
Descours et al.8 reported the preparation of naftidrofuryl iso-
mers 2–5 via resolution of the corresponding carboxylic acid
resulting from the hydrolysis of naftidrofuryl, or through synthesis
starting with (S)-tetrahydro-2-furanmethanol or (R)-tetrahydro-2-
furanmethanol. However, these procedures required repeated and
tedious recrystallizations, and the yields were very low (10–23%).
In contrast to the previous procedures, we have developed a more
practical access to carboxylic acids 12 and 13 (Scheme 1), which
are the key intermediates of naftidrofuryl isomers 2 and 3, respec-
tively. Therefore, tosylation of S-tetrahydrofurfuryl alcohol (6)9 fol-
lowed by the treatment of diethyl malonate furnished diester 8 in
72% yield. Diester 8 was treated with 1-chloromethyl naphthalene
to yield compound 9 in 80% yield. Hydrolysis of 9 with potassium
hydroxide afforded diacid 10. Under the vacuum decarboxylation
condition, compound 10 was converted in 96% yield to carboxylic
acid 11 as a diastereomeric mixture. After extensive experimenta-
tion on the resolution of 11, we developed a practical access to dia-
stereomerically pure carboxylic acids 12 and 13. Treatment of 11
with S-(ꢀ)-1-phenylethylamine, followed by recrystallization of
the resulting salt and acidification, furnished crude acid 12, which
was further recrystallized to give pure 128 as colorless needle-like
solid in 35% yield. The mother liquor originated from the salt pre-
cipitation by S-(ꢀ)-1-phenylethylamine was recycled, and subse-
quently treated with R-(+)-1-phenylethylamine to afford the
corresponding crude salt. Recrystallization and subsequent
acidification of this salt gave pure 13 as a white solid in 24% yield.
Reaction of 12 and 13 with 2-diethylaminoethylchloride
COOEt
KOH, EtOH
COOEt
COOEt
COOEt
19
O
DMF, NaH
76%
18
O
Vacuum
130 oC, 1h
95% for two steps
COOH
COOH
20
O
OH
O
O
21
1) R-1-Phenylethylamine
2) HCl 30%
O
OH
O
22
1) S-1-Phenylethylamine
2) HCl 24%
O
OH
O
23
Scheme 3. Synthesis of diastereomerically pure carboxylic acids 22 and 23.
Cl
N
HCl
K2CO3, PTC, 97%
N
O
O
O
OH
O
O
22
4
COOH
COOH
2H2O
COOH
COOH
N
Actone
O
O
O
24
Cl
N
HCl
N
K2CO3, PTC, 99%
O
O
O
O
OH
O
5
23
COOH
2H2O
COOH
COOH
COOH
N
Actone
O
O
O
25
Scheme 4. Preparation of naftidrofuryl isomers 4 and 5, and corresponding oxalates 24, 25.

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J. Hao et al. / Bioorg. Med. Chem. Lett. 22 (2012) 3441–3444
the highest binding affinity (pKi = 6.84 0.01), and the binding
affinity of 24 was 14-fold (pKi = 5.64 0.09) lower than 25. The
same trend occured between 14 and 15 (for 14, pKi = 6.55 0.10;
for 15, pKi = 5.77 0.08), indicating that S-configuration at C-2
was crucial for the ligand binding affinity. On the other hand, ste-
reochemical configuration of C-20 at the tetrahydrofuran ring
seemed to be less important for binding affinity (e.g. 14 vs 25;
15 vs 24). This result provides a theoretiacl guidance for the devel-
opment of single naftidrofuryl isomers with C-2S configuration as
inhibitors of 5-HT2A receptor that may have more clinical signifi-
cance than the current mixture of four stereoisomers.
In summary, we have developed a practical access to the four
stereoisomers of naftidrofuryl. All of the naftidrofuryl isomers have
been evaluated as 5-hydroxytryptamine 2A receptor antagonists.
The results reveal that the stereochemical configuration of C-2 is
crucial for the ligand binding affinity, suggesting that naftidrofuryl
isomers 2 and 5 with 2S-configuration may contribute most to the
pharmacological effects of naftidrofuryl. Further pharmacological
and toxicological evaluation of these naftidrofuryl isomers is in
progress in order to find more potent and safer therapeutic agents
against vascular and CNS diseases.
Figure 2. Competition binding studies towards different naftidrofuryl isomers to
wide-type 5-HT2A receptor using [3H] ketanserin.
Table 1
Binding affinities (Ki, pKi) of different naftidrofuryl isomers to wide-type 5-HT2A
receptor
Drugs
Ki (nM)
pKi
1
256.45 10.25
153.00 20.20
972.15 68.85
2073.50 695.50
150.40 6.40
6.48 0.04
6.55 0.10
5.77 0.08
5.64 0.09
6.84 0.01
Acknowledgements
14 (2S, 20S)
15 (2R, 20S)
24 (2R, 20R)
25 (2S, 20R)
This work was supported by the ‘111 Project’ from the Ministry
of Education of China and the State Administration of Foreign Ex-
pert Affairs of China (No. 111-2-07).
Supplementary data
hydrochloride in the presence of potassium carbonate afforded
naftidrofuryl isomers 2 and 3, respectively (Scheme 2). Free bases
2 and 3 were further converted to the corresponding oxalates 14
and 15 for further ligand binding assays.
Supplementary data associated with this article can be found, in
the
online
version,
at
http://dx.doi.org/10.1016/
j.bmcl.2012.03.093.
In the same manner, naftidrofuryl isomers 4 and 5 were
prepared as depicted in Schemes 3 and 4. Therefore, treatment of
R-tetrahydrofurfuryl alcohol 1610 with p-toluenesulfonyl chloride
afford tosylate 17 quantitatively. Alkylation of diethyl malonate
with 17 afforded diester 18 in 77% yield. Alkylation of 18 with
1-chloromethyl naphthalene furnished compound 19 in high yield.
Hydrolysis of 19 with potassium hydroxide, followed by solvent-
free decarboxylation to afford carboxylic acid 21 in 95% yield.
Resolution of 21 with R-(+)-1-phenylethylamine afforded diaste-
reomerically pure carboxylic acid 22.8 Further resolution of the
resulting mother liquor with S-(ꢀ)-1-phenylethylamine provided
pure carboxylic acid 23. Reaction of 22 and 23 with 2-diethylami-
noethylchloride hydrochloride in the presence of potassium car-
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