The chemical synthesis of metabolically stabilized 2-OMe-LPA analogues and preliminary studies of their inhibitory activity toward autotaxin

Article abstract of DOI:10.1016/j.bmcl.2012.03.008

The chemical synthesis of five new metabolically stabilized 2-OMe-LPA analogues (1a-e) possessing different fatty acid residues has been performed by phosphorylation of corresponding 1-O-acyl-2-OMe-glycerols which were prepared by multistep process from racemic glycidol. The now analogues were subjected to biological characterization as autotaxin inhibitors using the FRET-based, synthetic ATX substrate FS-3. Among tested compounds 1-O-oleoyl-2-OMe-LPA (1e) appeared to be the most potent, showing ATX inhibitory activity similar to that of unmodified 1-O-oleoyl-LPA. Parallel testing showed, that similar trend was also observed for corresponding 1-O-acyl-2-OMe-phosphorothioates (2a-e, synthesized as described by us previously). 1-O-oleoyl-2-OMe-LPA (1e) was found to be resistant toward alkaline phosphatase as opposed to unmodified 1-O-oleoyl-LPA.

Full text of DOI:10.1016/j.bmcl.2012.03.008

Bioorganic & Medicinal Chemistry Letters 22 (2012) 2698–2700
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The chemical synthesis of metabolically stabilized 2-OMe-LPA analogues
and preliminary studies of their inhibitory activity toward autotaxin
⇑
Edyta Gendaszewska-Darmach , Edyta Laska, Przemysław Rytczak, Andrzej Okruszek
Institute of Technical Biochemistry, Faculty of Biotechnology and Food Sciences, Technical University of Lodz, Stefanowskiego 4/10, 90-924 Lodz, Poland
a r t i c l e i n f o
a b s t r a c t
Article history:
The chemical synthesis of five new metabolically stabilized 2-OMe-LPA analogues (1a–e) possessing dif-
ferent fatty acid residues has been performed by phosphorylation of corresponding 1-O-acyl-2-OMe-gly-
cerols which were prepared by multistep process from racemic glycidol. The now analogues were
subjected to biological characterization as autotaxin inhibitors using the FRET-based, synthetic ATX sub-
strate FS-3. Among tested compounds 1-O-oleoyl-2-OMe-LPA (1e) appeared to be the most potent, show-
ing ATX inhibitory activity similar to that of unmodified 1-O-oleoyl-LPA. Parallel testing showed, that
similar trend was also observed for corresponding 1-O-acyl-2-OMe-phosphorothioates (2a–e, synthe-
sized as described by us previously). 1-O-oleoyl-2-OMe-LPA (1e) was found to be resistant toward alka-
line phosphatase as opposed to unmodified 1-O-oleoyl-LPA.
Received 11 January 2012
Revised 1 March 2012
Accepted 2 March 2012
Available online 10 March 2012
Keywords:
Autotaxin
Lysophospholipase D
Lysophosphatidic acids
Phosphatases
Ó 2012 Elsevier Ltd. All rights reserved.
Numerous studies conducted over last decades by several re-
search groups have shown that lysophospholipids are not only
structural components of cellular membranes but they also show
important biological activity, influencing a broad variety of meta-
bolic processes. Their prominent representatives, lysophosphatidic
acids (LPA) are potent modulators of many hallmarks of cancer
including cell proliferation, survival, migration, invasion, and neo-
vascularization. A major step towards understanding of the biolog-
ical activities of LPA was made by the unexpected discovery that
the lysophospholipase D, the enzyme responsible for biosynthesis
of LPA in serum was identical with autotaxin (ATX, also known
as NPP2), a widely expressed nucleotide pyrophosphatase/phos-
phodiesterase (NPP).1,2 There are several lines of evidence that
ATX is capable of producing not only LPA, but also cyclic phospha-
tidic acids (cPA) containing a dioxaphospholane ring spanning the
sn-2 and sn-3 positions of glycerol.3
Considering the fact that autotaxin is coded by one of the 40
most upregulated genes in invasive cancers, ATX became an attrac-
tive pharmacological target.4 Following the discovery of ATX’ feed-
back inhibition by LPA, several groups have reported the synthesis
of metabolically stabilized analogues of LPA and cPA that could
lead to novel anticancer chemotherapies.5 However, a limited
number of ATX inhibitors with LPA structure have been reported
to date.6–9 To address the need of longer biological half-lives, the
phosphate group was replaced with charged or neutral phosphor-
omimetics including methylene phosphonates, -X methylene
a
phosphonates (where X equals F, Br or OH), phosphorothioates or
phosphonothioates.10 These modifications make such analogues
resistant against lysophospholipid phosphatases (LPP). Recently,
we have also described the chemical synthesis of new sulfur ana-
logues of lysophospholipids, including phosphorothioate/phospho-
rodithioate derivatives of 2-OMe-LPA and phosphorothioate/
phosphorodithioate derivatives of cPA.11 The most potent lipid-
based inhibitor S32826 was described in 2008.12 Its polar head
featured
-substituted methylene phosphonate analogues of LPA. Subse-
quently, the -fluoromethylene, -bromomethylene, and hydroxy-
a metabolically-stable phosphonate, similar to the
a
a
a
methylene phosphonate analogues of S32826 were described to
increase low solubility of the parent molecule.13 However, numer-
ous non-lipid inhibitors have been also reported and very recently
they have been amply reviewed by Albers and Ovaa.14 Such small
molecules targeting ATX activity include boronic acid-based deriv-
atives,15,16 and an N,N-disubstituted piperidine.17 The latter inhib-
itors have a long linear and flexible structure, which is also
reflected in the structure of LPA and LPC.
In this report, we describe the chemical synthesis of a series of
2-OMe-LPA analogues 1a–e (without sulfur), unable to undergo
1?2 acyl migration by anchoring the acyl chain in the 1 position
of glycerol. We have subjected them to biological characterization
as autotaxin inhibitors using the FRET-based, synthetic ATX sub-
strate FS-3. Among tested compounds 1-O-oleoyl-2-OMe-LPA ap-
peared to be the most potent. Since one major catabolic fate of
LPA is the hydrolysis of its phosphate head group by ubiquitous
Abbreviations: ATX, autotaxin; cPA, cyclic phosphatidic acid; LPA, lysophospha-
tidic acid; NMR, nuclear magnetic resonance; MS, mass spectrometry.
⇑
Corresponding author.
E-mail address: edarmach@wp.pl (E. Gendaszewska-Darmach).
0960-894X/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2012.03.008

E. Gendaszewska-Darmach et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2698–2700
2699
O
C
The first steps of the synthesis were realized as described in our
previous report starting from racemic glycidol (Scheme 1).11 Thus,
glycidol was incubated for 5 h at ca. 85 °C with equimolar amounts
of corresponding fatty acids in the presence of catalytic quantity of
tri-n-butylamine.20 Crude 1-O-acylglycerols 3a–e were crystallized
from ether to give chromatographically pure products in 42–68%
isolated yield. The functionalization of 1-O-acylglycerols was
achieved by selective protection of primary hydroxyl function in
the presence of a secondary one with tert-butyldimethylsilyl group
(TBDMS) in 42–78% yield.21 The resulting 3-O-silyl ethers were fur-
ther methylated at 2-hydroxyl group with trimethylsilyldiazome-
thane (TMSCHN2) in the presence of 40% aqueous fluoroboric
acid (HBF4).22 The TBDMS group was removed by standard method
employing tetra(n-butyl)ammonium fluoride (TBAF) in THF solu-
tion21 to give 1-O-acyl-2-OMe-glycerols 4a–e in 64–92% yield.11
Compounds 4a–e were phosphitylated with N,N-diisopropyl-
amino-O,O-(bis)-2-cyanoethylphosphoramidite23 in the presence
of S-ethylthiotetrazole, to give, after standard water-iodine oxida-
tion, 1-O-acyl-2-OMe-3-O,O-(bis)-2-cyanoethylphosphorylglyce-
rols 5a–e in 65–73% yield. The O-2-cyanoethyl groups were
removed by the action of DBU on 5a–e, after its silylation with
N,O-bis(trimethylsilyl)acetamide (BSA).24 The final 1-O-acyl-2-
OMe-glycerol-3-O-phosphates (1a–e) were isolated as ammonium
-
O
O
X
OMe
R
O
P
-
O
2-OMe-LPA analogues 1a-e (X=O)
2-OMe-phosphorothioate-LPA analogues 2a-e (X=S)
R-C(O)- : a-lauroyl, b-myristoyl, c-palmitoyl,
d-stearoyl, e-oleoyl
Figure 1. Structure of 2-OMe analogues of LPA (1a–e) and their phosphorothioate
congeners (2a–e).
O
C
O
C
O
R
O
R
1. iPr2N-P(OCH2CH2CN)2
S-Ethylthiotetrazole
OMe
OH
2. I2/H2O/Py/THF
OH
OH
3a-e
4a-e
O
C
O
O
R
salts in 18-23% yield, after ion-exchange on AmberlystÒ-NH4 re-
+
O
C R
1. BSA
2. DBU
sin. The compounds 1a–e were isolated after lyophilization in a
form of white solids, and were stable when stored at -20 °C. They
were found to be soluble in alcohols (methanol, ethanol) and in
water. The ammonium phosphates 1a–e and all intermediate com-
pounds were isolated by silica gel flash chromatography (FC). Their
purity and identity was confirmed by TLC, NMR (1H and 31P) and
mass spectrometry (MALDI TOF and HR ESI). The details are given
as Supplementary data. The series of 1-O-acyl-2-OMe-glycerol-3-
O-phosphorothioates (2a–e) was synthesized from 4a–e by oxa-
thiaphospholane approach as described in detail in our previous
paper.11
OMe
OMe
-
O
CH CH CN
O
O
O
2
2
O
P
P
-
O
CH CH CN
2
2
O
O
5a-e
1a-e
Scheme 1. Synthesis of 2-OMe analogues of LPA (1a–e).
phosphatases, we have additionally shown resistance of our ana-
logues to the action of alkaline phosphatase.18 In addition, the
autotaxin inhibitory properties of 1a–e were compared with those
of previously synthesized11 phosphorothioate derivatives of 2-
OMe-LPA (2a–e).
For the studies of inhibition of autotaxin, five new 2-OMe-LPA
analogues (1a–e) were synthesized, possessing in sn-1 position
the residues of lauric, myristic, palmitic, stearic and oleic acid,
respectively (Fig. 1). Thus, a series of compounds was synthesized,
bearing five different fatty acid residues both saturated (12:0, 14:0,
16:0, 18:0) and unsaturated (18:1). Similarly, an analogous series
of phosphorothioate derivatives of 2-OMe-LPA (2a–e) was synthe-
sized as described earlier.11 The concept of ‘metabolic stabilization’
by introduction of 2-OMe group was invented by the research team
that synthesized 1-O-oleoyl-2-OMe-sn-glycerophosphorothioate
(OMPT) and studied its interactions with various LPA receptors.19
Compounds 1a–e and 2a–e were examined for their ability to
block ATX-mediated hydrolysis of lysophosphatidylcholine
analogue FS-3 as the substrate, using an assay based on fluores-
cence resonance energy transfer (FRET). The ATX activity was mea-
sured in the presence of the lysophospholipid analogues added
under different concentrations and was compared with the ATX
inhibitory effects of unmodified LPA (18:1) and
a-bromomethyl-
ene phosphonate LPA (BrP-LPA). The ATX activity measured with-
out LPA analogues was used as the standard (100% activity).
Time courses of FS-3 hydrolysis were linear over the time scale
examined (60 min). The results showed that all of the 2-OMe-LPA
analogues (1a–e) inhibited ATX activity to some extent (Table 1).
Comparing ATX preferences for hydrophobic tail length (12:0,
14:0, 16:0, 18:0) and degree of unsaturation (18:0 vs 18:1) the
least potent 2-OMe-LPA analogues appeared to be those containing
lauroyl and stearoyl acyl chains, 1a and 1d (IC50 = 1200.6 nM and
Table 1
Inhibition of autotaxin (ATX) activity by LPA analogues
Compound
Chemical structure
Inhibition (%) at 1
lM conc. SD
IC50 SD (nM)
1a
1b
1c
1d
1e
2a
2b
2c
1-O-lauroyl-2-OMe-LPA
1-O-mirystoyl-2-OMe-LPA
1-O-palmitoyl-2-OMe-LPA
1-O-stearoyl-2-OMe-LPA
1-O-oleoyl-2-OMe-LPA
1-O-lauroyl-2-OMe-thioLPA
1-O-mirystoyl-2-OMe-thioLPA
1-O-palmitoyl-2-OMe-thioLPA
1-O-stearoyl-2-OMe-thioLPA
1-O-oleoyl-2-OMe-thioLPA
BrP-LPA
45.5 1.5
57.7 1.7
74.5 7.1
49.7 0.6
81.3 6.7
36.7 1.8
62.3 1.8
64.7 0.4
35.5 1.3
77.8 11.5
86.1 1.6
65.6 1.7
1200.6 72.7
704.5 79.5
361.1 41.8
1012.8 25.4
220.2 36.0
2410.1 452.2
595.8 49.9
512.0 66.6
2418.0 70.2
355.6 79.5
170.3 10.4
499.8 68.2
2d
2e
BrP-LPA
1-O-Oleoyl-LPA
1-O-Oleoyl-LPA

2700
E. Gendaszewska-Darmach et al. / Bioorg. Med. Chem. Lett. 22 (2012) 2698–2700
1012.8 nM, respectively). The same preference was demonstrated
in the case of corresponding sulfur analogues (2a and 2d), however
their inhibitory potency was even lower (IC50 = 2410.1 nM and
2418.0 nM, respectively). The most significant ATX inhibition
(IC50 = 220.2 nM) was observed for 1e (1-O-oleoyl-2-OMe-LPA). It
Acknowledgement
This work was supported by a Grant (PBZ-MNiSW-07/I/2007)
from the Polish Ministry of Science and Higher Education and a
Grant (2011/01/B/ST5/06383) from the National Science Centre.
was comparable with that of known ATX inhibitors, a-bromometh-
ylene phosphonate LPA (BrP-LPA) and unmethylated 1-O-oleoyl-
LPA.6 Replacement of one phosphate oxygen atom with sulfur
(2e) resulted in a slightly higher IC50 value (355.6 nM).
Supplementary data
Supplementary data (experimental procedures, characteriza-
tion data) associated with this article can be found, in the online
version, at http://dx.doi.org/10.1016/j.bmcl.2012.03.008.
Recent studies have demonstrated that several ectoenzymes lo-
cated on the extracellular side of the cell plasma membrane cata-
lyze hydrolysis of LPA thus limiting its potential usefulness as
drugs. The degradation of extracellular LPA can be mainly attrib-
uted to the ectophosphatase activity of plasma membrane lipid
phosphate phosphatases (LPPs). The second LPA conversion path-
way involves the action of lysophosphatidic acid acyltransferases
(LPAAT) catalyzing the transfer of an acyl group from acyl-CoA to
LPA to form phosphatidic acid (PA).10 It was hypothesized that
thiophosphate substitution may confer resistance to LPP-mediated
degradation in the case of the ester-linked thiophosphate deriva-
tive (1-oleoyl-2-O-methyl-rac-glycerophosphothionate, OMPT).19a
It is known that the rate of hydrolysis of a phosphate ester by alka-
line phosphatase is independent of ester group and for example,
nucleoside 50-phosphorothioates are degraded 2000 times slower
than the corresponding phosphates.25 It was also proved that an-
other phosphorothioate synthetic analogue of LPA, octadecenyl
thiophosphate (OTP) was resistant to LPP1 phosphatase activity
derived from mouse embryonic fibroblasts.26 Recently, we have
also described the chemical synthesis of new sulfur analogues of
lysophospholipids, including phosphorothioate/phosphorodithio-
ate derivatives of 2-OMe-LPA.11 Therefore, we have examined
References and notes
1. Umezu-Goto, M.; Kishi, Y.; Taira, A.; Hama, K.; Dohmae, N.; Takio, K.; Yamori,
T.; Mills, G. B.; Inoue, K.; Aoki, J.; Arai, H. J. Cell. Biol. 2002, 158, 227.
2. Tokumura, A.; Majima, E.; Kariya, Y.; Tominaga, K.; Kogure, K.; Yasuda, K.;
Fukuzawa, K. J. Biol. Chem. 2002, 277, 39436.
3. Tsuda, S.; Okudaira, S.; Moriya-Ito, K.; Shimamoto, C.; Tanaka, M.; Aoki, J.; Arai,
H.; Murakami-Murofushi, K.; Kobayashi, T. J. Biol. Chem. 2006, 281, 26081.
4. Euer, N.; Schwirzke, M.; Evtimova, V.; Burtscher, H.; Jarsch, M.; Tarin, D.;
Weidle, U. H. Anticancer Res. 2002, 22, 733.
5. van Meeteren, L. A.; Ruurs, P.; Christodoulou, E.; Goding, J. W.; Takakusa, H.;
Kikauchi, K.; Perrakis, A.; Nagano, T.; Moolnaar, W. H. J. Biol. Chem. 2005, 280,
21155.
6. Jiang, G.; Xu, Y.; Fujiwara, Y.; Tsukahara, R.; Tsukahara, T.; Gajewiak, J.; Tigyi,
G.; Prestwich, G. D. Chem. Med. Chem. 2007, 2, 679.
7. Cui, P.; Tomsig, J. L.; McCalmont, W. F.; Lee, S.; Becker, C. J.; Lynch, K. R.;
Macdonald, T. L. Bioorg. Med. Chem. Lett. 2007, 17, 1634.
8. Cui, P.; McCalmont, W. F.; Tomsig, J. L.; Lynch, K. R.; Macdonald, T. L. Bioorg.
Med. Chem. 2008, 16, 2212.
9. Zhang, H.; Xu, X.; Gajewiak, J.; Tsukahara, R.; Fujiwara, Y.; Liu, J.; Fells, J. I.;
Perygin, D.; Parrill, A. L.; Tigyi, G.; Prestwich, G. D. Cancer Res. 2009, 69, 5441.
10. Gendaszewska-Darmach, E. Acta Biochim. Pol. 2008, 55, 227.
11. Rytczak, P.; Koziołkiewicz, M.; Okruszek, A. New J. Chem. 2010, 34, 1008.
12. Ferry, G.; Moulharat, N.; Pradere, J. P.; Desos, P.; Try, A.; Genton, A.; Giganti, A.;
Beucher-Gaudin, M.; Lonchampt, M.; Bertrand, M.; Saulnier-Blache, J. S.;
Tucker, G. C.; Cordi, A.; Boutin, J. A. J. Pharmacol. Exp. Ther. 2008, 327, 809.
13. Jiang, G.; Madan, D.; Prestwich, G. D. Bioorg. Med. Chem. Lett. 2011, 21, 5098.
14. Albers, H. M.; Ovaa, H. Chem. Rev. 2012, [Epub ahead of print].
15. Albers, H. M.; Dong, A.; van Meeteren, L. A.; Egan, D. A.; Sunkara, M.; van
Tilburg, E. W.; Schuurman, K.; van Tellingen, O.; Morris, A. J.; Smyth, S. S.;
Moolenaar, W. H.; Ovaa, H. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 7257.
16. Albers, H. M.; Hendrickx, L. J.; van Tol, R. J.; Hausmann, J.; Perrakis, A.; Ovaa, H.
J. Med. Chem. 2011, 54, 4619.
17. Gierse, J.; Thorarensen, A.; Beltey, K.; Bradshaw-Pierce, E.; Cortes-Burgos, L.;
Hall, T.; Johnston, A.; Murphy, M.; Nemirovskiy, O.; Ogawa, S.; Pegg, L.; Pelc,
M.; Prinsen, M.; Schnute, M.; Wendling, J.; Wene, S.; Weinberg, R.; Wittwer, A.;
Zweifel, B.; Masferrer, J. J. Pharmacol. Exp. Ther. 2010, 334, 310.
18. Mills, G. B.; Moolenaar, W. H. Nat. Rev. Cancer 2003, 3, 582.
19. (a) Hasegawa, Y.; Erickson, J. R.; Goddard, G. J.; Yu, S.; Liu, S.; Cheng, K. W.;
Aoki, J.; Jarosz, R.; Schrier, A. D.; Lynch, K. R.; Mills, G. B.; Fang, X. J. Biol. Chem.
2003, 278, 11962; (b) Qian, L.; Xu, Y.; Hasegawa, Y.; Aoki, J.; Mills, G. B.;
Prestwich, G. D. J. Med. Chem. 2003, 46, 5575.
20. Lok, C. M.; Mank, A. P. J.; Ward, J. P. Chem. Phys. Lipids 1985, 36, 329.
21. Xu, Y.; Qian, L.; Prestwich, G. D. Org. Lett. 2003, 5, 2267.
22. Aoyama, T.; Shiori, T. Tetrahedron Lett. 1990, 38, 5507.
23. Uhlman, E.; Engels, J. Tetrahedron Lett. 1986, 27, 1023.
24. Sekine, M.; Tsuruoka, H.; Iimura, S.; Wada, T. Nat. Prod. Lett. 1994, 5, 41.
25. Eckstein, F. Angew. Chem. Int. Ed. Engl. 1975, 14, 160.
26. Deng, W.; Shuyu, E.; Tsukahara, R.; Valentine, W. J.; Durgam, G.; Gududuru, V.;
Balazs, L.; Manickam, V.; Arsura, M.; VanMiddlesworth, L.; Johnson, L. R.;
Parrill, A. L.; Miller, D. D.; Tigyi, G. Gastroenterology 2007, 132, 1834.
27. Murphy, J.; Riley, J. P. Analytica Chim. Acta 1962, 27, 31.
unmodified 1-O-oleoyl-LPA,
a-bromomethylene phosphonate LPA
and the most active 1-O-acyl-2-OMe-derivative of LPA (1e) for their
resistance toward degradation by alkaline phosphatase isolated
from bovine intestinal mucosa. We have measured the amount of
inorganic phosphate liberated from the substrate according to
modified phosphomolybdate blue assay.27 Unfortunately, we
couldn’t apply this method to 1-O-oleoyl-2-OMe-phosphorothioate
(2e) since inorganic phosphorothioate does not form a blue ammo-
nium molybdate complex.25 Unmethylated 1-O-oleoyl-LPA was
completely dephosphorylated after 24 h of incubation at 37 °C
with the enzyme. However, 1-O-oleoyl-2-OMe-LPA (1e) was found
to be completely resistant toward alkaline phosphatase under
identical conditions, similarly to BrP-LPA. These observations sug-
gest that introduction of methyl group into sn-2 position of LPA
may also protect LPA analogues against the action of lysophospho-
lipid phosphatases in vivo.
In summary, our studies demonstrate that the most significant
ATX inhibition was observed for 1-O-oleoyl-2-OMe-LPA (1e), and
this compound was also resistant to the action of alkaline phospha-
tase what makes it the best candidate as autotaxin inhibitor among
2-OMe-LPA analogues studied.