A novel series of 2-pyridyl-containing compounds as lysophosphatidic acid receptor antagonists: Development of a nonhydrolyzable LPA3 receptor-selective antagonist

Article abstract of DOI:10.1016/j.bmcl.2004.05.023

A recently reported dual LPA₁/LPA₃ receptor antagonist (1) has been modified so as to modulate the basicity, sterics, and dipole moment of the 2-pyridyl moiety. Additionally, the implications of installing nonhydrolyzable phosphate head group isosteres with regard to antagonist potency and selectivity at LPA receptors is described. This study has resulted in the development of the first nonhydrolyzable and presumably phosphatase-resistant LPA₃-selective antagonist reported to date.

Full text of DOI:10.1016/j.bmcl.2004.05.023

Bioorganic & Medicinal Chemistry Letters 14 (2004) 4069–4074
A novel series of 2-pyridyl-containing compounds as
lysophosphatidic acid receptor antagonists: development
of a nonhydrolyzable LPA3 receptor-selective antagonist
Brian H. Heasley,a,* Renata Jarosz,b Karen M. Carter,a S. Jenny Van,a
Kevin R. Lynchb and Timothy L. Macdonalda
aDepartment of Chemistry, University of Virginia, PO Box 400319, McCormickRoad, Charlottesville, VA 22904, USA
bDepartment of Pharmacology, University of Virginia, PO Box 400319, McCormickRoad, Charlottesville, VA 22904, USA
Received 16 April 2004; revised 11 May 2004; accepted 12 May 2004
Available online 10 June 2004
Abstract—A recently reported dual LPA1/LPA3 receptor antagonist (1) has been modified so as to modulate the basicity, sterics, and
dipole moment of the 2-pyridyl moiety. Additionally, the implications of installing nonhydrolyzable phosphate head group isosteres
with regard to antagonist potency and selectivity at LPA receptors is described. This study has resulted in the development of the
first nonhydrolyzable and presumably phosphatase-resistant LPA3-selective antagonist reported to date.
Ó 2004 Elsevier Ltd. All rights reserved.
Lysophosphatidic acid (LPA 1- or 2-O-acyl-sn-glycero-
3-phosphate) is one of several lipid mediators that
evokes an array of biological responses by binding to
extracellular and intracellular targets.1 Subtype-selec-
tive/tissue-specific LPA receptor blockade has been
proposed to have tremendous therapeutic potential.2
However, the contribution of individual LPA receptors
to various disease states is unknown due, in part, to a
lack of highly potent and selective antagonist ligands.
enzyme assays show that LPPs can also dephosphoryl-
ate dual LPA1/LPA3 receptor antagonist diacyl-glycerol
pyrophosphate (DGPP).6 LPP overexpression can in-
deed functionally antagonize LPA signaling. Manipu-
lation of LPP1levels in fibroblasts suggested that this
enzyme exerts a selective negative effect on LPA1
receptor signaling.7 Additionally, overexpression of
LPP1has been shown to attenuate the mitogenic re-
sponse to LPA.8 Finally, overexpression of each of the
LPPs in HEK293 cells resulted in decreases in acute
responsiveness of MAP kinase activation by LPA.9
Enzymatic hydrolysis of the phosphomonoester polar
head group of phospholipid signaling molecules results
in functional inactivation or conversion to different
mediators. The degradation of LPA may be attributed
to the activity of lipid phosphate phosphatases (LPPs),
also designated type 2 phosphatidic acid phosphatases
(PAP2),3;4 a family of integral membrane glycoproteins
that catalyze the dephosphorylation of a number of
bioactive lipid mediators. LPPs exhibit broad specificity;
Km values for substrates such as LPA, phosphatidic acid,
and N-oleoyl ethanolamine phosphoric acid are in the
upper micromolar range for LPP1, 2, and 3.5 In vitro
The development of metabolically stable LPA receptor
agonists as pharmacological tools has received consid-
erable attention in recent years.8;10 Strategies include
replacement of the bridging oxygen of the phosphate
group with carbon as well as substitution of sulfur for a
bridging or nonbridging oxygen. However, to our
knowledge, no phosphatase-resistant LPA receptor
antagonists exist currently. It is therefore difficult to
employ long-term in vivo models to study the physio-
logical implications of LPA receptor blockade.
We recently reported a high-affinity LPA1/LPA3 recep-
tor antagonist containing a 2-pyridyl moiety (1, Fig.
1).11 We have investigated the importance of the Lewis-
basicity and dipole moment of the N-heterocycle as well
Keywords: Lysophosphatidic acid receptor antagonist.
* Corresponding author. Tel.: +1-434-924-0595; fax: +1-434-982-2302;
e-mail: bhh4x@virginia.edu
0960-894X/$ - see front matter Ó 2004 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2004.05.023

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B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4069–4074
Synthesis of all the compounds listed in Tables 1and 2
O
P
are described in Schemes 1and 2. The synthetic route
outlined in Scheme 1has been described in detail else-
where.11 In brief, a one-pot procedure accomplished
selective acylation of tyrosine methyl ester followed by
O-silylation to provide 4 in acceptable yield. The phenol
6 was efficiently generated in three steps from 4. This
intermediate was O-alkylated by subjecting a series of
benzylic alcohols to standard Mitsunobu conditions.
The benzylic alcohols were either prepared in a straight-
forward manner from commercially available methyl
esters or carboxylic acids (7n–p) or the synthetic meth-
ods have been described elsewhere (7e–f,12 7g–m13). In
most cases, the aryl ether Mitsunobu products were
inseparable from the by-product tri-n-butyl phosphine-
oxide by standard silica gel flash chromatography. This
mixture was therefore subjected to trifluoracetic acid
(TFA) deprotection of the phosphotriester and the pure
HO
HO
O
Ar
X
R1 R2
1: R1 = NHC(O)C17H33; R2 = H; Ar = 2-pyr; X = O
LPA1: Ki = 18 nM; LPA3: IC50= 175 nM
Figure 1. Structure of dual LPA1/LPA3 antagonist (1).
as the steric constraints in this region of the antagonist
binding pocket for the LPA receptors of the endothelial
differentiation gene (Edg) family. Furthermore, we have
elucidated the implications of installing nonhydrolyz-
able phosphate head group mimetics with regard to
antagonist potency and selectivity at LPA receptors.
These studies have resulted in a detailed structure–
activity relationship (SAR) of LPA receptor antagonists
described below.
Table 1. Inhibitory evaluation of aryl ether derivatives 7a–n as compared with lead compound 1
O
H
N
HO
HO
P
*
O
O
RO
Compds
R
S/R
LPA1
LPA3 IC50, nM
IC50, nM
109
Ki
111
R
S
18
175
940
N
1411
604
992
156
N
7a
R
N/D
1688
N
N
7b
7c
S
4500
735
N/D
N/D
6236
2075
R
N
7d
7e
S
>10,000
2840
N/D
N/D
6960
455
N
N
N
R
OMe
OMe
7f
S
>10,000
N/D
1093
OMe
OEt
7g
7h
R
R
6250
143
N/D
26
102
39
N
N

B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4069–4074
4071
Table 1 (continued)
Compds
R
S/R
LPA1
LPA3 IC50, nM
IC50, nM
84
Ki
O
O
O
CF3
7i
R
R
R
19
48
1660
423
N
N
N
7j
141
114
64
35
OMe
7k
Me
Me
OMe
7l
R
S
62
28
39
N
N
Me
OMe
Me
7m
3690
N/D
561
7n
7o
R
R
1430
962
N/D
N/D
533
N
OMe
1156
N
OMe
7p
S
>10,000
N/D
2600
N
Table 2. Inhibitory evaluation of polar head group derivatives 13a–f as compared with lead compound 1
O
H
N
HO
HO
P
R
*
X
O
Ar
O
Compds
X
Ar
R
S/R
LPA1
LPA3,
IC50 nM
IC50, nM
109
Ki, nM
1
–O–
Oleoyl, 17:1b
Oleoyl, 17:1b
Oleoyl, 17:1b
n-C15H31
R
R
S
S
18
175
>10,000
>10,000
1050
N
N
N
N
13a
13b
13c
–CH2–
–CH2–
–CH2–
>10,000
>10,000
>10,000
N/D
N/D
N/D
Me
OMe
Me
13d
–CH2–
Oleoyl, 17:1b
R
>10,000
N/D
150
N
(continued on next page)

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B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4069–4074
Table 2 (continued)
Compds
X
Ar
R
S/R
LPA1
LPA3,
IC50 nM
IC50, nM
>10,000
Ki, nM
Me
OMe
Me
13e
13f
–CH2–
Oleoyl, 17:1b
Oleoyl, 17:1b
S
S
N/D
4000
N
–CH(OH)–a
4230
N/D
232
a To the best of our knowledge, the additional stereocenter (a to P) is racemic. 13f is therefore evaluated as the unseparated mixture of diastereomers.
b 18 carbon chain; cis double bond located between C-9 and C-10 from the carbonyl.
O
O
H
N
NH3Cl
a
MeO
MeO
O
OH
RO
3 (R = H)
b
2
4 (R = TBDMS)
O
H
N
O
O
P
c, d
O
O
5 (R = TBDMS)
6 (R = H)
RO
e
O
P
H
N
HO
HO
O
f, g
O
Ar
O
7a-l
Scheme 1. Synthesis of compounds 7a–l. Reagents and conditions: (a) oleoyl chloride, DIEA, CH2Cl2, 4 h; (b) TBDMSCl, DIEA, DMAP, 2 h;
(c) NaBH4, CaCl2, EtOH–THF (2:1), 18 h, 50% over three steps; (d) di-tert-butyldiisopropyl phosphoramidite, tetrazole, 4 h, then 30% H2O2, 4 h,
83%; (e) TBAFÆ3H2O, THF, 1h, 95%; (f) ROH, PBu 3, DIAD, CH2Cl2, 18 h, 73–88%; (g) TFA–CH2Cl2 (1:2), 1 h, 80–90% after crystallization from
MeOH/Et2O.
O
O
P
O
P
Ph
Ph
Ph
Ph
EtO
EtO
EtO
EtO
N
NH2
N
a
b
BnO
BnO
HO
8
9
10
O
P
O
P
EtO
EtO
RO
RO
OH
O
Ar
c
d, e
HN
HN
O
O
17:1
17:1
12 (R = Et)
13 (R = H)
11
Scheme 2. Synthesis of compounds 13a–f. Reagents and conditions: (a) tetraethyl methylenediphosphonate, n-BuLi, )78 to 0 °C, 3 h, 90%; (b) H2
(100 psi), Pd(OH)2, EtOH, 18 h, 95%; (c) oleoyl chloride, DIEA, CH2Cl2, 4 h, 65%; (d) ROH, PBu3, DIAD, CH2Cl2, 18 h, 73–88%; (e) TMSBr,
CH2Cl2, 4 h, then 95% MeOH, 1h, 80–90% after crystallization from MeOH/Et 2O.

B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4069–4074
4073
phosphoric acids were obtained by recrystallization
from methanol/diethyl ether.
LPA3
4000
3000
2000
1000
0
a-Methylene phosphonates 13a–e were realized from the
N,N-dibenzylamino aldehyde 8, which was easily pre-
pared from tyrosine following the procedure reported by
Reetz et al.14 Next, treatment of tetraethyl meth-
ylenediphosphonate with n-butyllithium at )78 °C gen-
erated the lithiated carbanion, which condensed with 8
to afford the vinylphosphonate 9 in good yield.15 Cata-
lytic dual-hydrogenation/hydrogenolysis using Pearl-
mann’s catalyst provided the amino-phenol 10, which
was converted to the phosphonodiester 12 using the
above-mentioned procedures. Finally, transesterification
of each ester 12 using bromotrimethylsilane (TMSBr)
and subsequent desilylation with aqueous methanol
provided the pure a-methylene phosphonates 13a–e
after crystallization. The detailed synthesis of the dia-
stereomeric a-hydroxy phosphonate 13f will be reported
in a manuscript which is in preparation.
7l + 10uM LPA
1 + 10uM LPA
-10
-9
-8
-7
-6
-5
-4
Log[compd]
Figure 2. Effect of aryl ether analogues on LPA3 receptor GTP[c35S]
binding.11;17
All compounds were characterized by 1H and 13C NMR,
mass spectroscopy and, in most cases, C, H, N analy-
ses.16 A membrane-based GTP[c35S] binding assay was
adapted to assess in vitro activity at the LPA/Edg
receptors.11;17 As in the case of 1, all compounds pre-
sented herein are devoid of any significant activity at the
LPA2 receptor at concentrations below 30 lM. Antag-
onist potency data for 2-pyridyl aryl analogues of 1 (7a–
p) are presented in Table 1in a manner analogous to
previously published LPA receptor antagonist data.11;18
Antagonist potency data for phosphonate derivatives
13a–f are presented in Table 2. a-Methylene phospho-
nate compounds 13a–e do not retain the inhibitory
activity of their phosphate analogues at the LPA1
receptor (Table 2). Hence, by applying this strategy for
selectivity to compound 7l, a truly subtype-selective
LPA3 receptor antagonist was developed (13d). Com-
pound 13d shows improved potency over lead com-
pound 1 at the LPA3 receptor (Fig. 3) and is the first
nonhydrolyzable and presumably phosphatase-resistant
LPA antagonist reported to date. Compound 13d dis-
plays no antagonism at the LPA1 receptor (Fig. 4). Only
13f, an a-hydroxy phosphonate derivative of an early
lead antagonist, retained modest inhibitory activity at
the LPA1 receptor. The divergent properties of phos-
phonic acid antagonist head group analogues (13a–e) at
It is evident that the serendipitously discovered11 2-pyr-
idyl regioisomer in the R configuration exhibits superior
dual antagonism (1 as compared with 7a–d). Addition-
ally, the net dipole moment of the aromatic system may
play a role in LPA receptor antagonist binding, as
compounds 7e, 7g, and 7i exhibit varying inhibitory
potencies at each receptor. Lewis-basicity of the N-het-
erocycle moiety could also potentially account for this
phenomenon. Electron-rich compounds 7g–i, l are more
potent at the LPA3 receptor than the unsubstituted lead
compound 1 (Fig. 2). Indeed, the 4-methoxy-3,5-di-
methyl-pyridine system of 7l has been reported to exhibit
basicity approximately one pKa unit greater than that of
pyridine13;19 (pKa of pyridine in water 5.1720). Of this
series, compound 7g (1.71 pKa units higher than pyri-
dine in acetonitrile21) is particularly potent and LPA3-
selective. Alternatively, for compounds 7j–k, increased
steric hindrance of the 4-alkoxy pyridyl substituent im-
proves selectivity at the LPA1 receptor. n-Propyloxy
derivative 7j displays ꢀ10-fold LPA1 to LPA3 receptor
selectivity. The quinoline series 7n–p exhibits a marked
dual reduction in potency compared to 1 and therefore
may be too sterically demanding for either antagonist
binding pocket. Notably, trifluoroethoxy derivative 7i is
equipotent to 1 at the LPA1 receptor and ꢀthreefold
more potent at LPA3. Indeed, this compound is the most
potent LPA1/LPA3 dual antagonist reported to date.
Thus, we have shown that by altering the sterics and
electronics of the outermost aromatic system, highly
potent and somewhat selective phosphate-bearing
antagonists of LPA receptors may be realized.
LPA3
4000
3000
2000
13d + 10uM LPA
1000
1
+ 10uM LPA
0
-10
-9
-8
-7
-6
-5
-4
Log[compd]
Figure 3. Effect of LPA antagonists on LPA3 receptor GTP[c35S]
binding.11;17 A comparison of a-methylene phosphonate 13d with lead
antagonist 1.

4074
B. H. Heasley et al. / Bioorg. Med. Chem. Lett. 14 (2004) 4069–4074
6. Dillon, D. A.; Chen, X.; Zeimetz, G. M. J. Biol. Chem.
1997, 272, 10361–10366.
LPA1
1000
7. Xu, J.; Love, L. M.; Singh, I.; Zhang, Q.-X.; Dewald, J.;
Wang, D.-A.; Fischer, D. J.; Tigyi, G.; Berthiaume, L. G.;
Waggoner, D. W.; Brindley, D. N. J. Biol. Chem. 2000,
275, 27520–27530.
8. Hooks, S. B.; Santos, W. L.; Im, D.-S.; Heise, C. E.;
Macdonald, T. L.; Lynch, K. R. J. Biol. Chem. 2001, 276,
4611–4621.
9. Alderton, F.; Darroch, P.; Sambi, B.; McKie, A.; Ahmed,
I. S.; Pyne, N.; Pyne, S. J. Biol. Chem. 2001, 276, 13452–
13460.
10. Quian, L.; Xu, Y.; Hasegawa, Y.; Aoki, J.; Mills, G. B.;
Prestwich, G. D. J. Med. Chem. 2003, 46, 5575–5578.
11. Heasley, B. H.; Jarosz, R.; Lynch, K. R.; Macdonald,
T. L. Bioorg. Med. Chem. Lett. 2004, 14, 2735–2740.
12. Haudrechy, A.; Chassaing, C.; Riche, C.; Langlois, Y.
Tetrahedron 2000, 56, 3181–3187.
13. Kuhler, T. C.; Fryklund, J.; Bergman, N.-A.; Weilitz, J.;
Lee, A.; Larsson, H. J. Med. Chem. 1995, 38, 4906–4916.
14. Reetz, M. T.; Drewes, M. W.; Schmitz, A. Angew. Chem.,
Int. Ed. Engl. 1987, 26, 1141–1143.
750
500
250
13d + 5uM LPA
13f + 5uM LPA
1 + 5uM LPA
-10
-9
-8
-7
-6
-5
-4
Log[compd]
15. Blackburn, G. M.; Parratt, M. J. J. Chem. Soc., Chem.
Commun. 1982, 1270–1271.
Figure 4. Effect of LPA antagonists on LPA1 receptor GTP[c35S]
binding.11;17 A comparison of 13d and a-hydroxy phosphonate 13f
with lead antagonist 1.
16. (R)-Phosphoric acid mono-{2-octadec-9-enoylamino-3-[4-
(pyridin-3-ylmethoxy)-phenyl]-propyl} ester (7a): 1H
NMR (300 MHz, CD3OD) d 0.83 (t, 3H, J ¼ 6:9 Hz),
1.14–1.29 (m, 11H), 1.39–1.49 (m, 2H), 1.90–1.99 (m, 4H),
2.07 (t, 2H, J ¼ 7:7 Hz), 2.66 (dd, 1H, J ¼ 8:5, 14.2 Hz),
2.85 (dd, 1H, J ¼ 8:5, 14.2 Hz), 3.82–3.90 (m, 2H), 4.14–
4.23 (m, 1H), 5.23–5.29 (m, 4H), 6.92 (d, 2H, J ¼ 8:5 Hz),
7.16 (d, 2H, J ¼ 8:5 Hz), 7.99 (t, 1H, J ¼ 6:5 Hz), 8.57 (d,
LPA1 may be due to a discrepancy between the second
pKa value of unsubstituted phosphonic acids versus the
relatively more acidic phosphoric acids.22–24 Hence,
the substitution of an electronegative heteroatom onto
the methylene group a to phosphorous (13f) increases
acidity and regains LPA1 antagonism. a-Substituted
phosphonates (Fig. 1, ‘X’ ¼ CH(OH or F)) will therefore
provide a useful platform for the realization of meta-
bolically stable LPA1/LPA3 dual antagonists. Progress
toward this end will be reported in due course.
1H, J ¼ 8:1Hz), 8.74 (d, 1H, J ¼ 5:8 Hz), 8.88 (s, 1H); 13
C
NMR (300 MHz, CD3OD) d 13.51, 21.27, 22.78, 26.05,
27.19, 29.09, 29.25, 29.40, 29.48, 29.66, 29.89, 32.10, 36.00,
36.18, 51.32, 66.15, 67.06, 114.79, 114.96, 127.12, 129.83,
129.91, 130.67, 131.81, 138.61, 141.29, 142.04, 144.28,
157.01, 175.11; MS (ESI) m=z 603.3 [M+Hþ, 100%].
Anal. Calcd for C33H51N2O6P: C, 65.76; H, 8.53; N,
4.65. Found: C, 64.63; H, 8.53; N, 4.58. (S)-Phospho-
ric acid mono-{2-octadec-9-enoylamino-3-[4-(pyridin-3-yl-
methoxy)-phenyl]-propyl} ester (7b): this product had
spectroscopic properties identical to its enantiomer.
17. Im, D. S.; Heise, C. E.; Harding, M. A.; George, S. R.;
O’Dowd, B. F.; Theodorescu, D.; Lynch, K. R. Mol.
Pharm. 2000, 57, 753–759.
18. DNAs encoding LPA1, LPA2, or LPA3 receptors were
transfected into HEK293T cells along with DNAs encod-
ing Gai2, Gb, and Gc2 proteins. After 48 h, microsomes
were prepared and used in a GTPc[35S] binding assay, as
described.14 For determination of IC50 values, a concen-
tration of 1-oleoyl-LPA equal to the EC80 value (LPA1:
1 lM; LPA3: 1 0lM) was collided with antagonists at
concentrations ranging from 0.1to 30,000 nM. Points are
in triplicate and are representative of at least two
experiments.
In summary, a detailed SAR of LPA receptor antago-
nists has been described. A nonhydrolyzable and highly
potent LPA3 receptor-selective antagonist has been
developed. Compound 13d represents a pharmacologic
agent with which to manipulate LPA signaling in a
number of experimental systems so as to define the
LPA3 receptor-specific pathophysiological responses to
LPA stimulation in intact animals.
Acknowledgement
Support by NIH (R01-GM52722).
19. Olbe, L.; Carlsson, E.; Lindberg, P. Nature Rev. Drug
Disc. 2003, 2, 132–139.
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