Synthesis and biological evaluation of phosphonate derivatives as autotaxin (ATX) inhibitors

Article abstract of DOI:10.1016/j.bmcl.2006.12.114

Autotaxin (ATX) is an autocrine motility factor that promotes cancer cell invasion, cell migration, and angiogenesis. ATX, originally discovered as a nucleotide phosphodiesterase, is known now to be responsible for the lysophospholipid-preferring phosphol

Full text of DOI:10.1016/j.bmcl.2006.12.114

Bioorganic & Medicinal Chemistry Letters 17 (2007) 1634–1640
Synthesis and biological evaluation of phosphonate derivatives
as autotaxin (ATX) inhibitors
Peng Cui,a,* Jose L. Tomsig,b William F. McCalmont,a Sangderk Lee,b
Christopher J. Becker,a Kevin R. Lynchb and Timothy L. Macdonalda
aDepartment of Chemistry, University of Virginia, McCormick Road, PO Box 400319, Charlottesville, VA 22904, USA
bDepartment of Pharmacology, University of Virginia, 1300 Jefferson Park Avenue, PO Box 800735, Charlottesville, VA 22908, USA
Received 6 December 2006; accepted 22 December 2006
Available online 13 January 2007
Abstract—Autotaxin (ATX) is an autocrine motility factor that promotes cancer cell invasion, cell migration, and angiogenesis.
ATX, originally discovered as a nucleotide phosphodiesterase, is known now to be responsible for the lysophospholipid-preferring
phospholipase D activity in plasma. As such, it catalyzes the production of lysophosphatidic acid (LPA) from lysophophatidylch-
oline (LPC). ATX is thus an attractive drug target; small molecular inhibitors might be efficacious in slowing the spread of cancers.
With this study we have generated a series of b-keto and b-hydroxy phosphonate derivatives of LPA, some of which are potent ATX
inhibitors.
Ó 2007 Elsevier Ltd. All rights reserved.
The autocrine motility factor autotaxin (ATX) was
originally isolated from melanoma cell supernatants as
a 125-kDa glycoprotein that stimulated tumor cell
motility.1 In vivo experiments documented that forced
expression of ATX augments tumor cell invasion and
metastasis.2 Further, ATX promotes angiogenesis and
may act in concert with other angiogenic factors to
facilitate new blood vessel formation.3 These biological
properties require enzymatic activity.
in ascitic fluid characteristic of ovarian cancer patients.
Elevated levels of LPA are present both at early and late
stages in ovarian cancer and may play a role in tumor
cell proliferation and invasion.12,13 LPA mediates its ef-
fects through the activation of G protein-coupled recep-
tors (GPCR).14 Thus, great efforts have been made on
the study of LPA receptor antagonists and agonists
due to their therapeutic potential.15–21 In aggregate,
these data suggest that ATX is an attractive pharmaco-
logical target; blockage of LPA production via ATX
inhibition by small molecules could be a useful antican-
cer chemotherapy.22,23
ATX belongs to the nucleotide pyrophosphatase and
phosphodiesterase (NPP) family of enzymes, which
hydrolyze phosphodiester and diphosphate bonds, typi-
cally found in ATP and ADP.4 Interest in ATX was
stimulated by the identification of this enzyme as the
long elusive plasma lysophospholipase D activity, which
is responsible for the cleavage of choline group of lys-
ophophatidylcholine (LPC) to form lysophosphatidic
acid (LPA) (Fig. 1).5,6 This is a major pathway of bio-
synthesis of LPA in plasma.7,8 LPA is an intercellular
lipid mediator that influences many biochemical pro-
cesses including cell proliferation, smooth muscle con-
traction, platelet aggregation, and apoptosis.9–11 For
example, LPA is the ‘‘ovarian cancer activating factor’’
O
OH
O
P
O
O
C17H33
+
N
OH
O
LPC
lysoPLD/ATX
O
OH
HO
P
O
O
C17H33
OH
+
N
+
OH
O
Keywords: Autotaxin; ATX; Phosphonate; Choline; LPA.
*
LPA
Corresponding author. Tel.: +1 434 924 0595; fax: +1 434 982
2302; e-mail: pc3n@virginia.edu
Figure 1. Hydrolysis of LPC by lysoPLD/ATX.
0960-894X/$ - see front matter Ó 2007 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2006.12.114

P. Cui et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1634–1640
1635
A lead towards developing ATX inhibitors was provided
by the discovery that this enzyme undergoes end prod-
uct inhibition by, for example, LPA.24 Indeed a limited
number of ATX inhibitors that are LPA analogs have
been reported to date. Recently, a series of fatty alcohol
phosphate analogs were identified as LPA receptor
ligands.20 Some of the analogs showed ATX inhibition
activity. A series of phosphatidic acid derivatives were
investigated and only two acyl thiophosphates showed
autotaxin inhibition.21 A group of Darmstoff analogs
were reported as weak ATX inhibitors recently.25 Most
recently 3-carba analogs of cyclic phosphatidic acid were
reported.26 Although lacking significant activity at LPA
receptors, they were potent inhibitors of ATX activity.
In this report, we developed a series of b-hydroxy and
b-keto phosphonate derivatives of LPA as ATX
inhibitors.
b-keto phosphonate g.27 Sodium borohydride reduction
of d proceeded to give two possible diastereometic
b-hydroxy phosphonate dimethyl esters which were sep-
arated by column chromatography. The stereochemistry
determination is ongoing. The b-hydroxy phosphonate f
was obtained by using the same deprotection method
(for compounds f41 and f42, pyridine was used in the
deprotection).
The phosphonate derivatives were tested in choline
detection assay for ATX inhibition.28 The ATX activity
was measured in the presence of the compounds under
different concentrations (100, 10, and 1 lM). The ATX
activity without compounds was used as the standard
(100% activity). Most b-hydroxy phosphonate deriva-
tives inhibited ATX activity at only the highest concen-
tration tested. However, f17 and f18 exhibited
significant inhibition at 1lM (Table 1). These two com-
pounds were synthesized from protected L-tyrosine and
they are diastereomers in terms of the b-hydroxy groups.
The less polar isomer, f17, (also known as VPC8a202)
was able to inhibit 73% of ATX activity at 1 lM. Com-
pounds f15 and f16, which were synthesized from D-ty-
rosine, did not potently inhibit ATX although they
contained the same 4-methoxy-3,5-dimethyl-pyridyl
structure moiety. Other groups including some alkyl
chains and aromatic rings were also investigated. How-
ever, these compounds were not potent ATX inhibitors.
Synthesis of the phosphonate derivatives is described in
Scheme 1. It began with the acylation of the ammonium
hydrochloride salt of tyrosine O-methyl ester a with
appropriate acyl chlorides followed by etherification of
the free phenol with appropriate mesylates to afford
the fully protected tyrosine c. Next, was the base medi-
ated addition onto the methyl ester with the lithium
anion of dimethyl methylphosphonate to achieve b-keto
phosphonate dimethyl ester d. A bromotrimethylsaline
mediated deprotection of the ester ensued to afford the
O
O
O
NH Cl
3
NH
R
1
NH
R
1
MeO
MeO
MeO
ii
i
iii
O
O
HO
R O
2
HO
c
b
a
O
P
O
O
P
OH
O
OH
MeO
MeO
NH
R
MeO
MeO
HO
HO
1
NH
R
1
P
NH
R
1
O
O
v
O
iv
R O
2
R O
2
R O
2
d
e
f
v
O
O
HO
HO
P
NH
R
1
O
R O
2
g
Scheme 1. Synthesis of compounds f and g. Reagents and conditions: (i) appropriate acyl chloride, Et3N, CH2Cl2, 0 °C, 3 h, 70–80%; (ii) appropriate
mesylate, K2CO3, 18-crown-6, acetone, reflux overnight, 90–95%; (iii) n-BuLi, dimethyl methylphosphonate, then add in ester c, À78 °C, 3 h,
50–60%; (iv) NaBH4, THF, EtOH, 0 °C, 2 h, 70–80%; (v) bromotrimethylsaline, w/wo pyridine, CH2Cl2, rt, 4 h, then H2O and MeOH, overnight,
90–95%.

1636
P. Cui et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1634–1640
Table 1. ATX inhibitory evaluation of compound f1–f34
O
P
OH
H
N
HO
HO
*
O
R
O
Compound
R
*S/R
ATX activity (%)
1 lM
10 lM
100 lM
R (a1)
92
84
55
O
CF3
f1
N
O
O
CF3
CF3
f2
f3
R (b1)
S (a)
N/D
92
84
80
64
32
N
N
O
CF3
f4
f5
f6
S (b)
R (a)
R (b)
103
83
83
82
81
56
83
83
N
80
f7
S (a)
97
77
68
f8
f9
S (b)
R (a)
76
83
76
78
60
N
N
N
N
103
f10
f11
f12
f13
f14
f15
R (b)
S (a)
S (b)
R (b)
S (b)
R (a)
92
104
N/D
84
78
81
74
80
80
95
69
52
65
67
80
63
80
N
108
O
N
f16
f17
f18
R (b)
S (a)
S (b)
69
27
63
64
13
21
47
6
O
N
O
N
8
O

P. Cui et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1634–1640
1637
Table 1 (continued)
Compound
R
*S/R
ATX activity (%)
1 lM
10 lM
100 lM
f19
f20
f21
f22
f23
f24
f25
f26
f27
f28
f29
f30
f31
f32
f33
f34
CH3
R (a)
R (b)
S (a)
S (b)
R (a)
R (b)
S (a)
S (b)
R (a)
R (b)
S (a)
S (b)
R (a)
R (b)
S (a)
S (b)
100
78
57
82
36
69
72
90
77
83
80
70
67
70
72
94
82
91
CH3
95
84
56
CH3
77
CH3
84
80
n-C3H7
n-C3H7
n-C3H7
n-C3H7
n-C5H11
n-C5H11
n-C5H11
n-C5H11
80
79
108
110
100
81
108
97
100
85
86
82
98
83
94
90
103
N/D
104
106
104
104
104
101
1 a, refers to the diastereomer that elutes first; b, refers to the diastereomer that elutes second.
The corresponding b-keto phosphonate derivatives were
also tested (Table 2). At the concentration of 100 lM,
some compounds inhibited 50–70% of ATX activity.
Further structure optimization was attempted based
on the two lead compounds f17 and f18. We kept the
4-methoxy-3,5-dimethyl-pyridyl moiety and investigated
a series of b-hydroxy phosphonate derivatives with a
variety of lipophilic tails. These data are presented in
Table 3. None of these compounds were as potent as
f17 or f18. All of the compounds were tested as potential
antagonists at the recombinant LPA1, LPA2, and LPA3
receptors, but none showed significant blockade at con-
centrations up to 10 lM (data not shown).
A potential complication of our studies is the reported
inhibition of ATX by its product, LPA,24 which is gen-
erated in our assay using LPC as a substrate.28 There,
we measured the inhibition of ATX using the artificial

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P. Cui et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1634–1640
Table 2. ATX inhibitory evaluation of compound g1–g15
O
P
O
H
N
HO
HO
*
O
R
O
Compound
R
*S/R
ATX activity (%)
1 lM
10 lM
100 lM
O
O
CF3
CF3
g1
R
85
70
57
N
N
g2
S
R
S
R
S
S
R
S
R
S
R
S
R
S
79
N/D
N/D
78
68
82
44
g3
76
g4
71
61
N
N
g5
68
52
g6
102
71
72
50
N
g7
74
26
O
g8
CH3
CH3
84
71
32
g9
96
68
45
g10
g11
g12
g13
g14
g15
n-C3H7
n-C3H7
n-C5H11
n-C5H11
104
N/D
93
97
75
97
72
84
N/D
N/D
76
95
83
97
101
104
104
85
substrate, para-nitrophenol-thymidylic acid.5,6 Using this
substrate, we found the same rank order potency of our
most potent inhibitors, including f17 and f18 (data not
shown).
activity was determined for these compounds. Two
b-hydroxy phosphonates, which originated from pro-
tected L-tyrosine, were identified as the lead com-
pounds. The stereochemistry of original tyrosines and
4-methoxy-3,5-dimethyl-pyridyl moiety proved to be
important to the activity. Further SAR studies are
ongoing.
In summary, we developed a series of b-hydroxy
and b-keto phosphonate derivatives. ATX inhibitory

P. Cui et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1634–1640
Table 3. ATX inhibitory evaluation of compound f35–f44
1639
O
P
OH
H
N
HO
HO
R
*
O
N
O
O
Compound
R
*S/R
ATX activity (%)
1 lM
10 lM
100 lM
f35
f36
f37
f38
f39
f40
f41
f42
f43
f44
n-C9H19
n-C9H19
n-C13H27
n-C13H27
n-C17H35
n-C17H35
n-C17H33
n-C17H33
n-C19H39
n-C19H39
R (a1)
R (b1)
R (a)
R (b)
S (a)
S (b)
S (a)
S (b)
S (a)
S (b)
N/D
N/D
N/D
N/D
101
86
30
N/D
99
N/D
67
N/D
91
35
81
N/D
N/D
77
90
45
62
15
2
2
68
45
58
10
64
N/D
N/D
N/D
1 a, refers to the diastereomer that elutes first, b refers to the diastereomer that elutes second.
2 cis double bond located between C9 and C10 from the carbonyl.
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Acknowledgment
This work was supported by a Grant from the NIH
(R01 GM052722).
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P. Cui et al. / Bioorg. Med. Chem. Lett. 17 (2007) 1634–1640
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containing 2.7 mM TOOS (N-ethyl-N-2-hydroxy-3-sulfo-
propyl)-m-toluidine,2.7 mM 4-AAP (4-aminoantipyrine),
47.7 U/ml horseradish peroxidase, 18 U/ml choline oxi-
dase, and 5 mM MgCl2. After 30 min incubation at 37 °C,
light absorbance (550 nm) was determined, and the
amount of choline release was calculated from a standard
curve.
28. ATX activity assay: in 0.1 ml, buffer: (in mM: Tris–HCl
100, pH9, NaCl 150, MgCl2 5, CoSO4 0.03, 0.05%
Triton X-100), 1 mM 18:1 LPC, recombinant human
ATX with or without inhibitors; incubation was for 16 h
at 37 °C. Released choline was detected as follows:
samples were mixed with 0.15 ml of 50 mM Tris–HCl