Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological activity toward autotaxin

Article abstract of DOI:10.1016/j.bmcl.2011.05.083

The efficient synthesis of 3-O-thia-cPAs (4a-d), sulfur analogues of cyclic phosphatidic acid (cPA), has been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic thiophosphate moiety. The present synthetic route enables the synthesis of 4a-d in only four steps from the commercially available glycidol. Preliminary biological experiments showed that 4a-d exhibited a similar inhibitory effect on autotaxin (ATX) as original cPA.

Full text of DOI:10.1016/j.bmcl.2011.05.083

Bioorganic & Medicinal Chemistry Letters 21 (2011) 4180–4182
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Efficient synthesis of 3-O-thia-cPA and preliminary analysis of its biological
activity toward autotaxin
Ryo Tanaka a, Masaru Kato a, Takahiro Suzuki a, Atsuo Nakazaki a, Emi Nozaki b, Mari Gotoh b,
a,
Kimiko Murakami-Murofushi b, , Susumu Kobayashi
⇑
⇑
a Faculty of Pharmaceutical Sciences, Tokyo University of Science (RIKADAI), 2641 Yamazaki, Noda-shi, Chiba 278-8510, Japan
b Department of Biology, Faculty of Science, Ochanomizu University, 2-1-1 Ohtsuka, Bunkyo-ku 112-8610, Tokyo, Japan
a r t i c l e i n f o
a b s t r a c t
Article history:
The efficient synthesis of 3-O-thia-cPAs (4a–d), sulfur analogues of cyclic phosphatidic acid (cPA), has
been achieved. The key step of the synthesis is an intramolecular Arbuzov reaction to construct the cyclic
thiophosphate moiety. The present synthetic route enables the synthesis of 4a–d in only four steps from
the commercially available glycidol. Preliminary biological experiments showed that 4a–d exhibited a
similar inhibitory effect on autotaxin (ATX) as original cPA.
Received 11 April 2011
Revised 16 May 2011
Accepted 23 May 2011
Available online 27 May 2011
Ó 2011 Elsevier Ltd. All rights reserved.
Keywords:
Cyclic phosphatidic acid
3-O-thia-cPA
Autotaxin
Intramolecular Arbuzov reaction
PHYLPA 1, which was discovered in 1992 by Murakami-Mur-
ofushi et al. from myxoamoebae of a true slime mold, Physarum
polycephalum, is the first example of a naturally occurring cyclic
phosphatidic acid (cPA) (Fig. 1).1 Later, cPAs were found in mam-
malian organs such as human sera and porcine brain.2a,b cPA is re-
garded as a cyclic derivative of lysophosphatidic acid (LPA), which
elicits a number of physiological effects such as cell proliferation,
invasion and metastasis of cancer cells. Previously, we demon-
strated that cPAs 2a–d induce biological effects that are contrary
to those elicited by LPA. These observations suggest that cell prolif-
eration, invasion and metastasis of cancer cells are controlled by
LPA and cPA. We focused on the synthesis of enzymatically and
chemically stable derivatives of cPA-oriented molecules in order
to develop novel agents of medicinal importance. The concentra-
tion of LPA is controlled through product inhibition of autotaxin
(ATX, nucleotide pyrophosphate/phosphodiesterase-2) by LPA.3
Thus, it seemed likely that cPA could interact with ATX resulting
in the inhibition of cancer cell invasion and metastasis. In this
context, we previously prepared carba analogues of cPA, 2-O-ccPA
3a–d and 3-O-ccPA 30a–d. These carba analogues were found to be
potent inhibitors of ATX activity and to exhibit more potent activ-
ities than cPAs 2a–d in the suppression of cancer cell invasion and
metastasis.4a,b We were also interested in the preparation and
biological activity of the sulfur analogue of cPA, 3-O-thia-cPA 4 (
Fig. 1). In the field of nucleic acid, it is well known that the replace-
ment of a phosphate oxygen atom with a sulfur atom results in an
increase in stability to nuclease-mediated and base-catalyzed
hydrolysis.5 We report here the preparation of 4 and its prelimin-
ary biological activity toward ATX.
In order to synthesize several analogues of 3-O-thia-cPAs, a con-
cise approach toward the target molecules was required. For this
purpose, 3-O-thia-cyclicphosphate III was selected as a common
intermediate, and we planned to construct the cyclic thiophos-
phate moiety via the intramolecular Arbuzov reaction of the phos-
phate II, which could readily be prepared from hydroxythiol I
(Scheme 1).
We first investigated a synthetic route in a racemic form. Thus,
the synthesis of 3-O-thia-cPAs commenced with glycidyl ether 5,
which is derived from ( )-glycidol (Scheme 2). The epoxide 5
was converted to the disulfide 8, the precursor of the intramolecu-
lar Arbuzov reaction, in three steps: (1) epoxide opening with
thioacetic acid, (2) methanolysis, and (3) sulfenylation with 2,4-
dinitrobenzenesulfenyl chloride. When disulfide 8 was treated
with PCl(OMe)2 the initially formed phosphite 9 was found to un-
dergo a simultaneous intramolecular Arbuzov reaction to give the
desired cyclic thiophosphate 10.6 The chemical structure of 10 was
confirmed by means of NMR (1H NMR, 31P NMR and HH-COSY) and
mass spectrometry.
⇑
Corresponding authors. Tel./fax: +81 3 5978 5362 (K.M.-M.); tel./fax: +81 4
7121 3671 (S.K.).
We next attempted a deprotection of the PMB group of 10. We
investigated a range of conditions, such as oxidative cleavage using
DDQ or CAN (Table 1, entries 1–3), Lewis acid-mediated
E-mail addresses: murofushi.kimiko@ocha.ac.jp (K. Murakami-Murofushi), ko-
bayash@rs.noda.tus.ac.jp (S. Kobayashi).
0960-894X/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2011.05.083

R. Tanaka et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4180–4182
4181
Figure 1. Structure of PHYLPA 1 and its analogues 2–4.
Table 1
Deprotection of PMB group
Entry
Reagent
Solvent
Temp.
Result
Scheme 1. Construction of cyclic thiophosphate moiety.
1
2
3
4
5
6
7
DDQ
DDQ
CAN
BBr3
BCl3
MgBr2, Me2S
TFA, anisole
CH2Cl2/H2O
CH2Cl2/buffer
MeCN/H2O
CH2Cl2
CH2Cl2
CH2Cl2
rt
rt
rt
Decomposition
Decomposition
Decomposition
Decomposition
Decomposition
Decomposition
Decomposition
ꢀ78 °C
ꢀ78 °C
ꢀ78 °C
rt
CH2Cl2
MeO
MeO
O
OPMB
P
8
H2, Pd/C
MeOH
rt
O
HS
Scheme 2. Synthesis of cyclic thiophosphate 10.
deprotection (entries 4–7) and hydrogenolysis (entry 9), but were
unable to obtain the desired compound 11. In each case, the cyclic
thiophosphate moiety decomposed resulting in the formation of a
complex mixture of products.
As an alternative approach, we examined the construction of
the cyclic thiophosphate with as a 1-O-acyl derivative (Scheme
3). At first, racemic glycidol 12 was esterified with oleic acid using
WSC and DMAP. Epoxide 13a was cleaved with sodium thiolace-
tate, and the thiol ester was reductively cleaved with Et3SiH and
Pd/C to give thiol 15.7 Thiol 15 was sulfenylated, and the resulting
disulfide 16 was treated with PCl(OMe)2 in a similar manner to the
case of PMB ether 8. Although the desired cyclic thiophosphate 17
was detected in the crude mixture, we were unable to isolate 17 in
a pure form due to the presence of several polar by-products that
proved to be inseparable. We reasoned that the relatively high
nucleophilicity of the thiolate anion might cause undesirable side
reactions. Therefore, we investigated using an alternative leaving
group.
Scheme 3. Synthesis of cyclic thiophosphate 17.
thiocyanate. The cyanide group was then anticipated to act as a
leaving group in the Arbuzov reaction.8 We intended to employ
the trimethylsilyl phosphite 20 as an Arbuzov reaction precursor
in order to obtain the free 3-O-thia-cPA directly. Preparation of
the Arbuzov reaction using the thiocyanate derivative is summa-
rized in Scheme 4.
Next, we examined the thiocyanide group. Thiocyanide could be
obtained from the epoxide 13a in one step by the reaction with
Treatment of the epoxide 13a with HSCN in acetic acid afforded
the thiocyanate 18a in 89% yield. Epoxide opening was found to

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R. Tanaka et al. / Bioorg. Med. Chem. Lett. 21 (2011) 4180–4182
Figure 2. Dose–response relationship of ATX inhibition by 3-O-thia-cPAs 4a–d, 2-
O-ccPA 3c and cPA 2a.
In summary, we have established an efficient route to synthe-
size the sulfur-analogues of cPA, 3-O-thia-cPAs 4a–d, from glyc-
idol. The key feature of the present synthesis is an intramolecular
Arbuzov reaction to construct the cyclic thiophosphate moiety.
Preliminary biological experiments showed that 3-O-thia-cPAs ex-
hibit a similar inhibitory effect on ATX as original cPA. The synthe-
sis of enantiopure 3-O-thia-cPA and detailed evaluation of its
biological activities is now underway.
Acknowledgments
This work was supported in part by the Princess Takamatsu
Cancer Research Fund to K.M.-M. and a Grant-in-Aid for Scientific
Research (KAKENHI, No. 22790203) from the Ministry for
Education, Culture, Sports, Science and Technology of Japan to M.G.
Scheme 4. Synthesis of 3-O-thia-cPA 4a.
proceed smoothly under acidic conditions. Then, the resulting 18a
was reacted with salicylchlorophosphite, followed by hydrolysis
with TEAB buffer to give monoalkylphosphite 19a.9 H-Phosphonate
moiety of 19a was confirmed by the characteristic large P–H cou-
pling constant (631 Hz) in 1H NMR spectroscopy. We were de-
lighted to obtain the desired cyclic thiophosphate 4a in 80% yield
as a triethylammonium salt by the treatment of 19a with trimeth-
ylsilyl chloride and triethylamine in pyridine, followed by hydroly-
sis in acetonitrile. The reaction proceeded with the initial
formation of bistrimethylsilyl phosphite 20a, and the latter, in
turn, underwent a simultaneous intramolecular Arbuzov reaction.
The cyanide group was found to act as an excellent leaving group
in the present Arbuzov reaction. The chemical structure of oleoyl
ester 4a was confirmed by means of NMR (1H NMR, 31P NMR and
HH-COSY) and mass spectrometry.
We were thus able to establish an efficient route to 3-O-thia-
cPA starting from commercially available glycidol.10 In a similar
way, stearoyl derivative 4b, palmitoleoyl derivative 4c and palmi-
toyl derivative 4d were synthesized from corresponding glycidyl
esters 13b–d in four steps with an overall yield of 32%, 40% and
26%, respectively.
These 3-O-thia-cPAs 4a–d were subjected to an ATX inhibition
assay as previously described.11 The catalytic activity of ATX in
conditioned medium (CM) from MDA-MB-231 cells was signifi-
cantly inhibited by 3-O-thia-cPAs in a dose-dependent manner.
Moreover, variation in the acyl chain of these derivatives did not
significantly affect the degree of ATX inhibition (Fig. 2). 2-O-ccPA
3c was reported to be the most potent ATX inhibitor among natu-
rally occurring cPA and chemically synthesized carba-analogues of
cPA.4a,12 Oleoyl derivatives of cPA 2a, 3-O-thia-cPAs 4a–d and
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data; 1H and 13C NMR spectra for new compounds) associated
with this article can be found, in the online version, at doi:10.1016/
j.bmcl.2011.05.083.
References and notes
1. Murakami-Murofushi, K.; Shioda, M.; Kaji, K.; Yoshida, S.; Murofushi, H. J. Biol.
Chem. 1992, 267, 21512.
2. (a) Mukai, M.; Imamura, F.; Ayaki, M.; Shinkai, K.; Iwasaki, T.; Murakami-
Murofushi, K.; Murofushi, H.; Kobayashi, S.; Yamamoto, T.; Nakamura, H.;
Akedo, H. Int. J. Cancer 1999, 81, 918; (b) Murakami-Murofushi, K.; Mukai, M.;
Kobayashi, S.; Kobayashi, T.; Tigyi, G.; Murofushi, H. Ann. N. Y. Acad. Sci. 2000,
905, 319.
3. van Meeteren, L. A.; Ruurs, P.; Christodoulou, E.; Goding, J. W.; Takakusa, H.;
Kikuchi, K.; Perrakis, A.; Nagano, T.; Moolenaar, W. H. J. Biol. Chem. 2005, 280,
21155.
4. (a) Baker, D. L.; Fujiwara, Y.; Pigg, K. R.; Tsukahara, R.; Kobayashi, S.; Murofushi,
H.; Uchiyama, A.; Murakami-Murofushi, K.; Koh, E.; Bandle, R. W.; Byun, H.-S.;
Bittman, R.; Fan, D.; Murph, M.; Mills, G. B.; Tigyi, G. J. Biol. Chem. 2006, 281,
22786; (b) Gupte, R.; Siddam, A.; Lu, Y.; Li, W.; Fujiwara, Y.; Panupinthu, N.;
Pham, T.-C.; Baker, D. L.; Parrill, A. L.; Gotoh, M.; Murakami-Murofushi, K.;
Kobayashi, S.; Mills, G. B.; Tigyi, G.; Miler, D. D. Bioorg. Med. Chem. Lett. 2010, 20,
7525.
5. Inoue, N.; Minakawa, N.; Matsuda, A. Nucleic Acids Res. 2006, 34, 3476.
6. Li, X.; Cosstick, R. J. Chem. Soc., Perkin Trans. 1 1993, 1091.
7. Fukuyama, T.; Lin, S.-C.; Li, L. J. Am. Chem. Soc. 1990, 112, 7050.
8. Erian, A. W.; Sherif, S. M. Tetrahedron 1999, 55, 7957.
9. Marugg, J. E.; Tromp, M.; Kuyl-Yeheskiely, E.; van der Marel, G. A.; van Boom, J.
H. Tetrahedron Lett. 1986, 27, 2661.
10. Since both enantiomer of glycidol is commercially available, the present
methodology would provide 3-O-thia-cPA in an enantioselective manner.
11. Nozaki, E.; Gotoh, M.; Hotta, H.; Hanazawa, S.; Kobayashi, S.; Murakami-
Murofushi, K. Biochim. Biophys. Acta 2011, 1811, 271.
palmitoleoyl derivative of 2-O-ccPA 3c at 10 lM were found to give
25%, 55% and 66% inhibition, respectively. So the order of ATX
inhibitory potency by cPA analogues was as follows: 2-O-ccPA
3c > 3-O-thia-cPAs 4a–d > cPA 2a.
12. Nozaki, E.; Gotoh, M.; Hanazawa, S.; Mori, H.; Kobayashi, S.; Murakami-
Murofushi, K. Cytologia 2011, 76, 73.