A Novel Agonist of the Type 1 Lysophosphatidic Acid Receptor (LPA1), UCM-05194, Shows Efficacy in Neuropathic Pain Amelioration

Article abstract of DOI:10.1021/acs.jmedchem.9b01287

Neuropathic pain (NP) is a complex chronic pain state with a prevalence of almost 10% in the general population. Pharmacological options for NP are limited and weakly effective, so there is a need to develop more efficacious NP attenuating drugs. Activation of the type 1 lysophosphatidic acid (LPA₁) receptor is a crucial factor in the initiation of NP. Hence, it is conceivable that a functional antagonism strategy could lead to NP mitigation. Here we describe a new series of LPA₁ agonists among which derivative (S)-17 (UCM-05194) stands out as the most potent and selective LPA₁ receptor agonist described so far (E_max = 118%, EC₅₀ = 0.24 μM, K_D = 19.6 nM; inactive at autotaxin and LPA_2-6 receptors). This compound induces characteristic LPA₁-mediated cellular effects and prompts the internalization of the receptor leading to its functional inactivation in primary sensory neurons and to an efficacious attenuation of the pain perception in an in vivo model of NP.

Full text of DOI:10.1021/acs.jmedchem.9b01287

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Article
A novel agonist of the type 1 lysophosphatidic acid receptor (LPA1),
UCM-05194, shows efficacy in neuropathic pain amelioration
Ines Gonzalez Gil, Debora Zian, Henar Vázquez-Villa, Gloria Hernandez-Torres, R. Fernando
Martinez, Nora Khiar-Fernandez, Richard Rivera, Yasuyuki Kihara, Isabel Devesa, Sakthikumar
Mathivanan, Cristina Rosell del Valle, Emma Zambrana-Infantes, Maria Puigdomenech, Giovanni
Cincilla, Melchor Sanchez-Martinez, Fernando Rodriguez de Fonseca, Antonio Ferrer-Montiel,
Jerold Chun, Ruben Lopez-Vales, María L. López-Rodríguez, and Silvia Ortega-Gutierrez
J. Med. Chem., Just Accepted Manuscript • Publication Date (Web): 02 Dec 2019
Downloaded from pubs.acs.org on December 2, 2019
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A novel agonist of the type 1 lysophosphatidic acid
receptor (LPA ), UCM-05194, shows efficacy in
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neuropathic pain amelioration
1
,‡
1,‡
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Inés González-Gil, Debora Zian, Henar Vázquez-Villa, Gloria Hernández-Torres,
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R. Fernando Martínez, Nora Khiar-Fernández, Richard Rivera, Yasuyuki Kihara,
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Isabel Devesa, Sakthikumar Mathivanan, Cristina Rosell del Valle, Emma Zambrana-
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,
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Infantes, María Puigdomenech, Giovanni Cincilla, Melchor Sanchez-Martinez,
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,5
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Fernando Rodríguez de Fonseca, Antonio V. Ferrer-Montiel, Jerold Chun, Rubén
6
1,*
1,*
López-Vales, María L. López-Rodríguez and Silvia Ortega-Gutiérrez
Dedicated to Professor José M. González on the occasion of his 60th birthday
1Departamento de Química Orgánica I, Facultad de Ciencias Químicas, Universidad
Complutense de Madrid, E-28040 Madrid, Spain
2Sanford Burnham Prebys Medical Discovery Institute, 10901 North Torrey Pines Road,
La Jolla, CA 92037, USA
3IDiBE, Universidad Miguel Hernández de Elche, E-03202 Alicante, Spain
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4Instituto de Investigación Biomédica de Málaga, UGC Salud Mental, Universidad de
Málaga, Hospital Universitario Regional de Málaga, E-29010 Málaga, Spain
5Departamento de Psicobiología, Facultad de Psicología, Universidad Complutense de
Madrid, E-28223 Pozuelo de Alarcón, Madrid, Spain
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6Departament de Biologia Cel·lular, Fisiologia i Immunologia, Institut de Neurociències,
Centro de Investigación Biomédica en Red sobre Enfermedades Neurodegenerativas
(CIBERNED), Universitat Autònoma de Barcelona, E-08193 Bellaterra, Barcelona, Spain
7Molomics, Barcelona Science Park, Baldiri i Reixac 4-8, E-08028 Barcelona, Spain
*Correspondence: mluzlr@ucm.es, siortega@ucm.es
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ABSTRACT
Neuropathic pain (NP) is a complex chronic pain state with a prevalence of almost 10%
in the general population. Pharmacological options for NP are limited and slightly
effective, so there is a need of developing more efficacious NP attenuating drugs.
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Activation of the type 1 lysophosphatidic acid (LPA ) receptor is a crucial factor in the
1
initiation of NP. Hence, it is conceivable that a functional antagonism strategy could
lead to NP mitigation. Here we describe a new series of LPA agonists among which
1
derivative (S)-17 (UCM-05194) stands out as the most potent and selective LPA1
receptor agonist described so far (Emax=118%, EC =0.24 µM, K =19.6 nM; inactive at
50
D
autotaxin and LPA2-6 receptors). This compound induces characteristic LPA -mediated
1
cellular effects and prompts the internalization of the receptor leading to its functional
inactivation in primary sensory neurons and to an efficacious attenuation of the pain
perception in an in vivo model of NP.
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INTRODUCTION
Lysophosphatidic acid (LPA, 1-acyl-sn-glycerol-3-phosphate) is a lipid mediator
required for maintaining the homeostasis of numerous physiological processes but also
participates in the development of various pathological conditions. Although LPA can
denote a variety of lysophospholipids with saturated (16:0, 18:0) and unsaturated (16:1,
18:1, 18:2, 20:4) acyl chains, in the context of LPA as a signaling molecule and thus along
this work, LPA refers to 1-oleoyl-sn-glycerol-3-phosphate. LPA acts through six
different receptors (LPA ) that belong to the G protein-coupled receptor (GPCR)
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superfamily.1, 2 These six receptors can be further divided into two groups, the
endothelial differentiation gene (EDG) family (LPA ) and the phylogenetically distant
1-3
3
, 4
non-EDG family (LPA ).
Genetic deletion studies, over-expression models, and
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–6
other chemical tools have provided insight into some of the many biological effects,
mechanisms and medical application of LPA signaling.5-7 Because LPA signaling has
been implicated in cancer,8, 9 fibrosis,10, 11 and pain,12-14 LPA receptors are regarded as
promising drug targets.15, 16 Among all these effects, LPA has recently attracted
attention because of its emerging role as an important factor in neuropathic pain (NP).13,
1
4, 17, 18
NP is a type of chronic pain caused by a lesion or disease of the somatosensory
system, including peripheral fibers and central neurons. It affects around 10% of the
general population and its incidence is likely to increase. Patients with NP do not
usually respond to standard pain treatments such as acetaminophen, non-steroidal anti-
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inflammatory drugs (NSAIDs) or weak opioids. In general, available pharmacological
treatments for NP are only moderately efficacious with effectiveness in less than 50% of
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patients, and they are also associated with adverse effects. Mechanistically, NP arises
because peripheral sensory and central neurons show a gain of excitation and/or a loss
of inhibition prompting a permanent state of hyperexcitability. In this scenario, the
brain receives abnormal sensory messages that translate into continuous burning pain
and abnormal sensory sensations such as allodynia (pain as a result of non-noxious
stimuli) and hyperalgesia (increased response to a normally painful stimuli). Several
signaling pathways have been described that contribute to NP neuropathy, including
pathways activated through inflammatory molecules, growth factors, and lipid
metabolites. Signaling through these pathways causes changes in the function and
expression of voltage-gated sodium, calcium and potassium channels, and also leads to
abnormal function of second-order nociceptive and inhibitory neurons. As this situation
becomes chronic, it leads to a permanent maladaptive plasticity that none of the
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pharmacological therapies currently available can reverse. NP seriously compromises
the quality of life of affected people, and clearly represents a currently unmet clinical
need. Consequently, there is a pressing demand for the development of efficacious new
drugs which can be used either alone or in combination with existing therapies to
provide a long-term alleviation of chronic pain through novel mechanisms of action.
Among the different factors involved in NP, LPA has been characterized as crucial for
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NP initiation.13, 18 NP is attenuated in Lpar1-deficient mice, indicating that LPA
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signaling through the LPA is responsible for pain transmission. However, direct
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antagonists of LPA such as the compound Ki-16425, have shown efficacy in inhibiting
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NP but only for limited periods of time. This fact suggests that initial LPA signaling
through LPA is not only important for NP initiation, but that LPA signaling may
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exacerbate chronic pain through subsequent signaling mechanisms including: LPA1-
dependent regulation of pain-related molecules, feed forward pain facilitation caused
21
by the LPA -stimulated production of LPA following the initial LPA activation, and
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concomitant LPA mediated activation of the transient receptor potential vanilloid 1
TRPV1) ion channel, which also dramatically contributes to the NP onset.22-24
(
Therefore, we hypothesized that a longer period of LPA desensitization will be a more
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efficacious strategy for ameliorating NP. This strategy has been already successfully
used in the development of fingolimod (FTY720 or Gilenya®), the first oral disease-
modifying drug for treating multiple sclerosis, and a non-selective agonist of
sphingosine 1-phosphate (S1P) that behaves as a functional S1P and S1P antagonist
1
3-5
or receptor modulator, inducing receptor internalization and degradation after binding
and activation.25-27
In this work we describe the development of a potent new LPA selective agonist that
1
behaves as a functional receptor antagonist and shows efficacy in an in vivo mouse
model of NP.
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RESULTS AND DISCUSSION
Very few agonists of the LPA have been reported to date.6, 28, 29 The first LPA-based
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agonist was N-acylethanolamide phosphoric acid (2-[(9Z)-octadec-9-enoylamino]ethyl
dihydrogen phosphate or NAEPA, 1, Figure 1), which was characterized as a dual
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LPA1/2 agonist. Several changes in its structure have led to ligands with improved
activity and, in some cases, such as derivative 2, selectivity for LPA (Figure 1), with
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EC values of 221 nM and 1089 nM at LPA and LPA , respectively. In an attempt to
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identify new structures devoid of the fatty acid moiety, Astellas Pharma reported a
series of indoloquinolizines with agonist activity at the LPA . Among them, compound
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(Figure 1) exhibited the best EC50 value (3.6 µM),32 although selectivity was not
discussed.
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Figure 1. Representative LPA agonists
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Considering this paucity of LPA selective agonists and the lack of an LPA /agonist
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complex crystal structure, we attempted to identify novel LPA agonists using the
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structure of the endogenous ligand as a starting point (Figure 2). Initially, modifications
were made in two parts of the LPA molecule, the acid group and the hydrophobic
moiety, keeping the LPA stereochemistry constant. With respect to the acid group,
several structure-activity relationship studies suggested that changes in the LPA polar
head group would be poorly tolerated.34 In particular, it has been shown that the
presence of free acid groups that retain their negative charge under physiological
conditions is an important requirement for activity. Hence, we decided to replace the
phosphate with carboxylic and boronic acids or with a tetrazole moiety (compounds 4-
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1, Figure 2), all of which have been described as suitable isosteres.35-37 With respect to
the hydrophobic moiety, modifications in the length of the fatty acid chain were studied
(compounds 12-16, Figure 2). In addition, based on our previous results showing that
fatty acid chains can be replaced by phenyl and biphenyl moieties,38, 39 we incorporated
a variety of phenyl and biphenyl-containing subunits (compounds 17-26, Figure 2).
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O
O
P
OH
OH
( )7
O
O
Hydrophobic
subunit
(
)7
Acid group
OH
LPA
(
)
( )
X
n
7
CO2H
O
CO2H
O
O
O
P
n = 3, 5, 6, 8, 9
X
CO2H
Hydrophobic
subunit
X = H, F
( )
7
( )
O
Acid group
O
O
OH
7
OH
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OH
OH
OH
B
H
( )
n
X = H, Ph
n = 6-14
N
OH
N
N
O
N
Figure 2. Design of new LPA agonists 4-26
1
Changes in the polar head of LPA
Compounds 4-11 (Scheme 1) were obtained by coupling of oleoyl chloride with the
corresponding alcohol or glycerol derivatives 27-33 (see Supporting Information for
details) in the presence of 2,4,6-collidine at low temperature. For all the diols employed,
this reaction was completely regioselective, and only the acylation at sn-1 position of the
glycerol moiety was observed. Carboxylic acid derivatives 34-39 were then deprotected
with TFA to yield the final products 4-9. In the case of the malonic acid derivatives 35
and 36, this reaction led to the formation of lactones 5 and 6 as a mixture of
diastereoisomers instead of free dicarboxylic acids. Thus, in order to avoid cyclization,
the hydroxy group was masked with a methyl group (compound 7) or removed
(
derivatives 8 and 9). Boronic acid 10 was obtained by transformation of the pinacolyl
boronate ester 40 into the corresponding trifluoroborate salt, followed by hydrolysis
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with trimethylsilyl chloride to yield the desired boronic acid. Finally, treatment of
nitrile 41 with sodium azide led to tetrazole 11.
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a
Scheme 1. Synthesis of Compounds 4-11
O
(
)7
( )7
O
O
CO R
2
27
OH
t
3
4: R = Bu
: R = H
(b)
4
O
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O
O
( )7
( )7
O
X
CO2H
(b)
O
t
CO Bu
2
O
C17H33
(
a) 28 or 29
5: X = H
6: X = F
(
)7
( )7 Cl
t
O
OH CO Bu
2
35: X = H
O
F
36: X = F
(c)
X = F
CO R
2
(
)7
( )7
O
OMe CO R
2
t
O
37: R = Bu
7: R = H
X
(b)
CO R
30 or 31
2
(
)7
( )7
O
CO R
2
t
38: X = H, R = Bu
t
39: X = F, R = Bu
(b)
8: X = H, R = H
9: X = F, R = H
OR
B
O
OR
32
( )7
( )7
O
O
OH
0: R = C(Me )C(Me )
_
_
4
2
2
O
(d)
10: R = H
(a)
(
)7
( )7 Cl
O
O
(e)
H
N
33
C17H33
O
CN
( )7
( )7
O
N
OH
OH
N
N
41
11
a
Reagents and conditions: (a) Glycerol or alcohol derivatives (27-33), 2,4,6-collidine,
DCM, −78 °C to rt, 24 h, 36-99%; (b) TFA, DCM, rt, 16-18 h, 52−99%; (c)
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
trimethylsilyldiazomethane, HBF , DCM, 0 °C, 90 min, 38%; (d) i) KHF , CH OH:H O,
4
2
3
2
rt, 30 min; 99%, ii) TMSCl, CH CN:H O, rt, 1 h, 60%; (e) NaN , NH Cl, DMF, MW, 160
3
2
3
4
°
C, 45 min, 18%.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
The ability of compounds 4-11 to activate the LPA was assessed by measuring the
1
extent of calcium mobilization in LPA stably transfected RH7777 cells, as the binding of
1
an LPA agonist causes an increase in intracellular calcium levels, which can be
1
quantified using a fluorescence-based assay. Among these compounds, only derivatives
6
and 7 were able to activate the receptor [Emax (6) = 33%; EC (6) = 1.7 µM and Emax (7)
50
=
74%; EC (7) = 6 µM] whereas the rest of the compounds did not induce calcium
5
0
mobilization at the highest concentration of compound tested (10 µM). Although these
results indicate that other polar moieties can mimic the LPA phosphate group, these
derivatives are less potent inducers of calcium mobilization than LPA.
Changes in the hydrophobic moiety of LPA: modification of fatty acid chain length
The hydrophobic moiety plays an important role in LPA receptor activity. To study
the influence of this part of the molecule on receptor activity, comprehensive changes
were made to the overall length of the fatty acid chain (compounds 12-16, Scheme 2)
and aromatic ring were incorporated (compounds 17-26, Scheme 3). Modification of the
fatty acid chain length required the preparation of the non-commercial carboxylic acids
4
2-44 by steroselective cis-hydrogenation of the corresponding alkynyl derivatives (see
1
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Supporting Information for details) and glycerol 45, obtained following a synthetic
40
procedure previously developed in our group. Regioselective esterification between
the corresponding fatty acid and the protected phosphorylated diol 45 yielded
derivatives 46-50 which were deprotected to provide the final compounds 12-16
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(Scheme 2).
a
Scheme 2. Synthesis of Compounds 12-16
O
O
O
P
O
(a)
P
t
+
HO
O
O Bu
( )n
( )7 OH
( )n
( )7
O
O
OR
t
O Bu
OR
OH
OH
n = 3, 5, 6 (42), 8 (43), 9 (44)
45
t
4
6-50: n = 3, 5, 6, 8, 9; R = Bu
(b)
12-16: n = 3, 5, 6, 8, 9; R = H
a
Reagents and conditions: (a) DCC, DMAP, DCM, −20 °C to rt, 16 h, 12−49%; (b) TFA,
DCM, rt, 4−5 h, 90−99%.
The results obtained for LPA agonist activity (Table 1) highlight the tremendous
1
influence exerted by the hydrophobic chain length. In this regard, compounds 12-16
show that variations of just one methylene unit can turn a compound inactive (for
example derivative 15 shows an activity comparable to LPA whereas 16 is inactive) or
increase its activity almost two-fold, as shown by compounds 15 (Emax = 88%; EC = 3.6
50
µM) and 14 (Emax = 202%; EC = 2.1 µM). These data suggest that the optimal chain is
50
that of palmitoleic acid, present in compound 13 (Emax = 205%; EC = 0.45 µM), the most
50
potent derivative within the series and more active than the endogenous ligand LPA
(Emax = 100%; EC = 0.83 µM).
50
1
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1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
Table 1. Agonist activities of compounds 12-16 at LPA receptor
1
O
O
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
P OH
OH
(
)n
( )7
O
O
OH
Cpd n Emax (%)a
EC (µM)
b
50
1
1
1
1
2
3
4
5
3
5
6
8
9
7
127± 1
205 ± 9
202 ± 1
88 ± 2
N.E.c
2.8 ± 0.1
0.45 ± 0.01
2.1 ± 0.3
3.6 ± 0.2
-
16
LPA
100
0.83 ± 0.02
aEmax = maximal efficacy of the drug/maximal efficacy of LPA, expressed as the
b
percentage. For E
> 30%, EC50 values are expressed as mean ± s.e.m, from a
max
c
minimum of two independent experiments, performed in triplicate. N.E., no effect was
observed at the highest concentration of compound tested (10 µM).
Changes in the hydrophobic moiety of LPA: derivatives containing aromatic rings
Previous data from our group showed that unsaturated fatty acid chains can be
replaced by phenyl groups.38, 39 Our results observed with derivatives 12-16 together
4
1
with studies using other lipid-binding GPCRs, show the importance of aliphatic chain
length on activity. To determine if the distance between the aromatic rings and the
phosphorylated glycerol moiety (n) influences activity, compounds 17-26 (Scheme 3)
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were prepared. Their synthesis required the preparation of carboxylic acids 51-56 (see
Supporting Information for details) and the conveniently protected phosphorylated
glycerol. Initially, looking for synthetic simplicity and high yields, the ethyl group was
selected for phosphate protection (intermediate 57). However, using standard
conditions to remove the ethyl groups from intermediate 59 with TMSBr yielded
bromine derivative 17 instead of the expected product. The structure of 17 was
determined by nuclear magnetic resonance (NMR) and high pressure liquid
chromatography coupled to mass spectrometry (HPLC-MS). The benzyl group was
therefore selected as the phosphate protecting group for intermediates 60-68, which,
after hydrogenation, led to the target compounds 18-26 (Scheme 3).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
a
Scheme 3. Synthesis of Compounds 17-26
Br
(b)
O
OH
O
OH
PG = Et
X = H
X
X
( )9
O
P
O
P
O
O
O
P
(a)
17
O
HO
O
OPG
(
)n OH
+
( )
O
O
OPG
OPG
n
OH
OPG
OH
X
X = H; n = 6, 7-9 (51-53), 10, 14
X = Ph; n = 6 (54), 9 (55), 12 (56)
O
O
P
5
7: PG = Et
58: PG = Bn
5
9: X = H; n = 9; PG = Et
(c)
60-65: X = H; n = 6-10, 14; PG = Bn
6-68: X = Ph; n = 6, 9, 12; PG = Bn
( )
PG = Bn
X = H, Ph
O
O
OH
6
n
OH
OH
18-23: X = H; n = 6-10, 14
24-26: X = Ph; n = 6, 9, 12
a
Reagents and conditions: (a) DCC, DMAP, DCM, −20 °C to rt, 16 h, 10−58%; (b) i)
TMSBr, DCM, rt, 4 h; ii) MeOH/H O, rt, 1h, 90%; (c) H , 10% Pd/C, EtOH, rt, 74−99%.
2
2
All the synthesized compounds, including the initially unexpected derivative 17, were
tested for LPA activity. The data obtained (Table 2) confirms that the fatty acid chain
1
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7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
can be replaced by phenyl and biphenyl moieties and that the distance between the
ester group and the aromatic subunit influences agonist activity, as small changes in the
number of methylene units dramatically affects Emax and EC50 values. For example,
compound 19, with n = 7, is inactive, whereas derivative 20, with n = 8, is almost as
active as LPA, or the case of derivative 25, which shows the highest activity when
compared to either compounds 24 or 26. In all the cases the optimal chain resulted to be
the one with n=9. Finally, it is remarkable the excellent activity of compound 17 at the
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
LPA receptor.
1
In summary, from the modifications carried out in the hydrophobic moiety of LPA,
three compounds, derivatives 13, 17, and 21, with Emax values of 205%, 118% and 112%,
respectively, and EC50 values of 0.45, 0.24, and 0.5 µM respectively, have activities
greater than that of the endogenous ligand LPA (Emax = 100%; EC = 0.83 µM) and stand
50
out within the series.
Table 2. Agonist activities of compounds 17-26 at LPA receptor
1
O
O
P
Hydrophobic
Subunit
O
O
OH
OH
OH
Hydrophobic
Subunit
b
Cpd
n
Emax (%)a
EC (µM)
50
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Br
O
OH
O
OH
1
7
118 ± 14
0.24 ± 0.09
( )9
O
P
O
N.E.c
1
1
2
8
9
0
6
-
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
N. E.
-
8
74 ± 4
112 ± 3
135 ± 31
N.E.
2.1 ± 0.3
0.5 ± 0.1
3.2 ± 0.5
-
(
)
n
21
9
2
2
2
2
2
3
4
5
10
14
6
N.E.
-
Ph
9
127 ± 9
37 ± 1
3.3 ± 0.6
19 ± 2
(
)
n
26
12
O
O
P
LPA
( )
( )
O
O
OH
100
0.83 ± 0.02
OH
7
7
OH
aEmax = maximal efficacy of the drug/maximal efficacy of LPA, expressed as the
> 30%, EC50 values are expressed as mean ± s.e.m, from a
minimum of two independent experiments, performed in triplicate. N.E., no effect was
observed at the highest concentration of compound tested (10 µM).
b
percentage. For E
max
c
The possibility of optimizing the structure by combination of the best phosphate free polar
heads identified so far (the fluorine containing moieties of compounds 6 and 7) with the optimal
hydrophobic subunits (the aliphatic chains of palmitoleic acid, in compound 13, and of 10-
phenyldecanoic acid, in derivatives 17 and 21) was assessed with the synthesis of compounds
6
9-72 (Figure 3, see Supporting Information for details). In the case of derivative 71, its
1
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
chromatographic purification allowed the separation of the two diastereoisomers. The absolute
configuration of the chiral carbon bonded to the fluorine atom was determined on the basis of
1
19
NOE experiments and H- and -F-NMR spectra. Compound 72 was obtained as 1:1 mixture of
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
diastereoisomers. Among these derivatives, only compound 69 showed some agonist activity
(
Emax and EC values of 43±6% and 1.4±0.6 µM, respectively), whereas the rest of the
50
compounds were inactive at the highest concentration tested (10 µM).
O
O
F
F
COOH
COOH
(
)5
( )7
O
( )9
O
OMe COOH
OMe COOH
69
70
O
R
O
(
)5
( )7
O
R'
F
(
)9
O
O
COOH
O
O
72
O
(3S, 5S)-71: R = F, R' = COOH
3R, 5S)-71: R = COOH, R' = F
(
Figure 3. Compounds 69-72
From the obtained results, derivative 17, which contains a chiral center, was identified
as the compound with the greatest activity (Emax and EC values of 118% of 0.24±0.09
50
µM, respectively). In order to establish its absolute configuration, the two enantiomers
of 17 were synthesized from the corresponding (S)- or (R)-oxiran-2-ylmethanol
(
glycidol), which was phosphorylated using the conditions previously employed. The
oxirane ring was opened and the resulting intermediates (S)- and (R)-74 coupled with
0-phenyldecanoic acid (53). Final removal of the benzyl groups yielded the target
1
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compounds (S)- and (R)-17 (Scheme 4). Comparison of the specific optical rotation
values revealed that compound 17, obtained in the phosphate deproctection reaction
(Scheme 3) was the S enantiomer. Therefore, after confirming that (S)-17 was indeed the
active enantiomer (Table 3), we carried out some structural exploration around this
compound (Scheme 5). Modifications included changing the length of its methylenic chain
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
(derivatives 76 and 77) and replacing it with palmitoleic and oleic acid chains (compounds 78
and 79). However, none of these compounds had agonist activities that were significantly better
that that obtained with (S)-17, as shown in Table 3.
a
Scheme 4. Synthesis of (S)- and (R)-17
Br
O
P
O
P
O
(a)
(b)
(c)
OH
OH
O
OH
O
OBn
Br
O
OBn
O
( )9
O
P
OBn
OBn
O
OH
O
(
(
S)-75
R)-75
(S)-glycidol
R)-glycidol
(R)-73
(S)-73
(S)-74
(R)-74
(
(d)
(
(
S)-17
R)-17
a
i
Reagents and conditions: (a) i) Pr NP(OBn) , 1H-tetrazole, DCM, rt, 2 h; ii) mCPBA,
2
2
DCM, -30 ºC, 90 min, 86-95%; (b) TBABr, TFA, CHCl , rt, 10 min, 89-99%; (c) 53, DCC,
3
DMAP, DCM, rt, 18 h, 68-73%; (d) H , 10% Pd/C EtOH, rt, 97-99%.
2
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9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
a
Scheme 5. Synthesis of analogues of compound (S)-17
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
Br
O
OR
(
a)
O
OR
(
)n
O
P
PG =Bn
O
O
P
81, 82: n = 8, 10; R = Bn
(c)
Br
O
OPG
76, 77: n = 8, 10; R = H
OPG
OH
(
S)-74: PG = Bn
(a) n = 5
Br
O
t
OR
8
0: PG = Bu
(b) n = 7
O
OR
(
)n
( )7
O
P
t
PG = Bu
O
t
8
3, 84: n = 5, 7; R = Bu
(d)
78, 79: n = 5, 7; R = H
a
Reagents and conditions: (a) 52, 11-phenylundecanoic acid, or palmitoleic acid, DCC,
DMAP, DCM, rt, 5 h, 55-96%; (b) oleoyl chloride, pyridine, DCM, rt, 18 h, 15%; (c) H2,
0% Pd/C, EtOH, rt, 72-80%; (d) TFA, DCM, rt, 5 h, 81-93%.
1
Table 3. Agonist activities of analogs of (S)-17 at LPA receptor
1
Emax (%)a
N.E.c
EC (µM)
b
Cpd
Structure
50
Br
O
OH
7
7
6
7
O
OH
-
(
)
O
P
8
O
Br
O
OH
O
OH
N.E.
-
(
)
O
P
10
O
1
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Br
O
OH
O
O
OH
7
7
8
9
N.E.
-
( )
( )
O
O
P
5
7
O
Br
O
OH
OH
39 ± 3
3.2 ± 0.4
(
)
( )
P
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
7
7
O
Br
O
OH
(
R)-17
S)-17
O
OH
32 ± 3
26 ± 5
(
)
O
P
9
O
Br
O
OH
OH
(
O
118 ± 14
0.24 ± 0.09
( )
O
P
9
O
aEmax = maximal efficacy of the drug/maximal efficacy of LPA, expressed as the
> 30%, EC50 values are expressed as mean ± s.e.m, from a
minimum of two independent experiments, performed in triplicate. N.E., no effect was
observed at the highest concentration of compound tested (10 µM).
b
percentage. For E
max
c
In addition, we analysed the effect of the replacement of the ester for an amide group. Hence,
the synthesis of the corresponding amides (85-87) of three representative LPA agonists
1
identified so far [(S)-17, and phosphate free compounds 7 and 69] was addressed as depicted in
Scheme 6 and their activity at LPA receptor determined (Table 4). However, whereas amides 85
1
and 86 could be readily obtained by coupling of intermediate amine 88 with palmitoleic acid or
oleyl chloride, respectively, access to amide 87 was more difficult than initially expected. In this
case, removal of the Boc group of intermediate 91 followed by condensation of the free amine
with the N-hydroxysuccinimidyl ester of 10-phenyldecanoic acid led to aziridine 92 due to an
intramolecular cyclization instead of formation of the desired amide 87 (Scheme 6). Further
attempts to open the aziridine ring with bromide allowed the isolation of intermediate 93, which
after hydrogenation to remove the remaining benzyl group yielded a complex mixture of
2
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6
unidentified products. This result suggests that target amide 87 has a low intrinsic stability.
Therefore, only compounds 85 and 86 were assessed for LPA activity (Table 4). The obtained
1
results reflect the key influence of the oxygen atom, as the two studied amides displayed a
0
1
2
3
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7
8
9
0
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9
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9
0
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3
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7
8
9
0
dramatic loss of activity both in terms of Emax and EC values when compared with their ester
50
analogues.
a
Scheme 6. Synthesis of amides 85-87
(a)
n = 5
O
O
F
F
F
CO2tBu
t
(c)
CO2 Bu
CO2H
H2N
( )n
( )7
N
( )n
( )7
N
H
H
OMe CO2tBu
t
OMe CO2 Bu
OMe CO2H
(b)
88
n = 7
89, 90: n = 5, 7
85, 86: n = 5, 7
Br
O
OH
O
OH
O
P
X
( )9
N
H
P
(d), (e)
O
Br
O
OBn
87
OBn
NHBoc
Br
Br
O
O
O
OH
(g)
OH
OBn
91
(f)
O
OBn
O
OH
O
OBn
( )9
N
P
( )9
N
H
P
(
)9
N
P
X
H
O
O
O
92
93
87
a
Reagents and conditions: (a) palmitoleic acid, EDC, HOBt, DMF, rt, 18 h, 37%; (b)
oleoyl chloride, Et N, DCM, rt, 18 h, 46%; (c) TFA, DCM, rt, 18 h, 69-99%; (d) TFA,
3
DCM, rt, 16 h, 99%; (e) N-hydroxysuccinimidyl ester of 53, Et N, DCM, rt, 12 h, 48%; (f)
3
MgBr ·OEt , Et O, rt, 4 h, 99%; (g) H , 10% Pd/C, EtOH, rt.
2
2
2
2
Table 4. Agonist activities of amides 85 and 86 at LPA receptor in comparison with
1
their ester analogues
b
Cpd
Structure
X
Emax (%)a
EC (µM)
50
O
85
NH
26 ± 1
5 ± 1
F
COOH
(
)
( )
X
2
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O
43 ± 6
N. E.c
74 ± 14
1.4 ± 0.6
O
NH
-
F
COOH
( )7
( )7
X
7
OMe COOH
O
6 ± 1
1
1
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8
9
0
1
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8
9
0
aEmax = maximal efficacy of the drug/maximal efficacy of LPA, expressed as the
percentage. For E
minimum of two independent experiments, performed in triplicate. N.E., no effect was
observed at the highest concentration of compound tested (10 µM).
b
> 30%, EC50 values are expressed as mean ± s.e.m, from a
max
c
In summary, the results obtained up to this moment identify compound (S)-17, with an
excellent EC value of 0.24 μM, as the most potent within the series, being able to activate
5
0
LPA with a slightly higher intensity (118%) than the endogenous ligand LPA. In addition, we
1
confirmed the binding of (S)-17 to the LPA receptor using back-scattering interferometry
1
(BSI),42-44 a technique which detects changes in refractive index (RI) due to variations in
molecular structure, dipole moment, polarizability, conformation and solvation that occur upon
binding of a ligand to a protein. Thus, BSI can be used to measure binding affinities by detecting
changes in the RI of mixtures of protein and ligand after incubation to reach equilibrium. In this
case, increasing concentrations of (S)-17 were incubated with a constant quantity of LPA1-
expresssing membrane homogenates and the difference between the signals of protein-ligand
complex and free protein solution was plotted versus ligand concentration to obtain a saturation
binding curve from which the dissociation constant (K ) was obtained (Supporting Figure S1),
D
resulting in a K value of 19.9 nM for compound (S)-17 and of 5.6 nM for LPA, used as
D
reference. We also carried out molecular docking studies to propose a plausible binding mode
between the ligand and the receptor. The results showed that (S)-17 can interact with LPA in a
1
3
3, 45
similar manner to LPA and other known LPA agonists.
The phosphate moiety of (S)-17
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establishes three hydrogen bonds with Arg124, Thr113, and Trp121 and the carbonyl moiety of
the ligand forms an additional hydrogen bond with Gln125. Besides, (S)-17 establishes
hydrophobic interactions with Tyr 102, Ile128, Leu132, Tyr202, Trp210, Leu297, and Ala300
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(Figure 4).
Figure 4. Two images (0º-180º views) of the binding mode between (S)-17 and the LPA1
receptor (active conformation) as predicted by docking calculations. Blue lines represent
hydrogen bonds and grey dashed lines hydrophobic interactions. Residues marked in green are
reported in the literature33, 45 to bind to other agonists. The LPA active state model was
1
retrieved from the GPCRdb46, 47 (structure file provided in the Supporting Information).
Due to the high sequence similarity between LPA , a selectivity profile was determined for
1
-3
all compounds with LPA agonist activity (Supporting Table 1). In addition, for compound (S)-
1
1
7, we studied its selectivity against LPA4-6 receptors and also against the enzyme autotaxin
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(
ATX), involved in the biosynthesis of LPA, and its antagonist capacity at the LPA receptor.
1
Remarkably, this compound showed 10-fold selectivity versus LPA receptor and more than 50-
5
fold selectivity versus any of the other analysed targets, making this compound the most potent
1
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and selective LPA agonist described so far. Besides, we assessed the reversibility of the binding
1
and the stability of the compound. To determine whether derivative (S)-17 activates LPA in a
1
reversible manner, we carried out a wash out experiment. The obtained results indicate that, after
incubation and removal of the compound, the receptor recovers its ability for activation, thus
ruling out a covalent binding between LPA and the compound (Supporting Figure S2).
1
Additionally, the compound remains stable (>85%) up to 8 hours at 37 ºC both in PBS and in
culture media (Supporting Figure S3), making (S)-17 an excellent candidate for assessing LPA1-
mediated cellular effects in vitro and for modulation of NP in vivo.
Cellular effects of (S)-17: induction of neurite retraction, cellular migration and
internalization of the LPA receptor
1
We have shown that (S)-17 has the ability to bind to LPA and activate calcium mobilization.
1
Several in vitro assays were performed to determine if (S)-17 has the ability to recapitulate LPA
induce cellular changes. In agreement with its ability to function as a receptor agonist, (S)-17
induced neurite retraction, as indicated by a rounded cellular morphology, in a manner
similar to that of LPA (Figure 5). In addition, (S)-17 induced cell migration of LPA1
overexpressing B103 cells in a transwell chamber assay (Figure 6). Finally, the ability of (S)-17
to induce LPA internalization was assessed in B103 neuroblastoma cells overexpressing
1
an enhanced green fluorescent-LPA fusion receptor [(EGFP)-LPA ]. The results
1
1
obtained clearly show that in the absence of stimulus, the receptor is localized to plasma
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membrane (Figure 7A), whereas in the presence of an agonist (LPA or (S)-17) the
receptor is internalized, as indicated by the presence of the green fluorescence inside the
cell (Figure 7B,C). Consistent with its ability to act as an agonist in vitro, (S)-17 was able to
mimic other LPA mediated cellular effects including, neurite retraction, cellular migration, and
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LPA internalization. Receptor desensitization, through internalization, is of special importance
1
because it is a mechanism of protection against LPA mediated NP that occurs through LPA1
signaling.
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Figure 5. Compound (S)-17 induces neurite retraction. B103 neuroblastoma cells
overexpressing EGFP-LPA receptor were treated with (A, D) vehicle (0.1% fatty acid
1
free bovine serum albumin), (B) LPA, or (C, E) (S)-17. Cells were then fixed and stained
1
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with phalloidin and DAPI, and analysed under fluorescence microscopy for visualization of LPA
(green), cell morphology (red) and nuclei (blue). The number of cells with retracted neurites was
counted and expressed as the percentage of the total number of cells (F). The data shown
correspond to the mean±SEM of the number of cells counted in two independent experiments
and three different slides per experiment. *, p<0.05; **, p<0.01 vs BSA (Student’s t test).
Samples were imaged under the same conditions using a Zeiss fluorescence microscope
(A-C, bars 100 µm) or Zeiss fluorescence confocal microscope (D, E, bars 10 µm).
Figure 6. Compound (S)-17 induces cellular migration. B103 neuroblastoma cells
5
overexpressing LPA receptor were seeded (2.5·10 cells) into the upper chamber of the
1
transwell and vehicle (0.1% fatty acid free bovine serum albumin), LPA 1 µM, or (S)-17
1
µM were added in the lower chamber. After allowing the cells to migrate for 5 h,
migratory cells were stained with 0.1% crystal violet solution and counted. Images
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shown are representative of the migratory cells in the presence of (A) vehicle, (B) LPA 1
µM, or (C) (S)-17 1 µM. (D) Data in the bar graph correspond to the mean±SEM of the
number of cells counted in two independent experiments and three different transwell chambers
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per experiment. *, p<0.05; **, p<0.01 vs BSA (Student’s t test). Samples were imaged under
the same conditions by using a Zeiss microscope (bars 50 µm).
Figure 7. Compound (S)-17 induces internalization of the LPA receptor. B103
1
neuroblastoma cells overexpressing EGFP-LPA were treated with (A) vehicle (0.1%
1
fatty acid free bovine serum albumin), (B) LPA 1 µM, or (C) (S)-17 1 µM. The cells were
then fixed, stained with phalloidin and DAPI, and analysed under fluorescence microscopy for
visualization of LPA (green), cell morphology (red) and nuclei (blue). Green fluorescence in the
1
cytosol in (B) and (C) shows internalization of the LPA , which, in the absence of agonist,
1
appears almost exclusively in the cell membrane. Images shown are representative of two
independent experiments and at least three different fields per experiment. All samples were
imaged under the same conditions using a Zeiss fluorescence confocal microscope (bars
1
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Functional desensitization induced by (S)-17
The observed internalization of the LPA after treatment with the agonist (S)-17
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strongly suggests its associated functional desensitization. We sought to confirm this
aspect in dorsal root ganglia (DRG) primary culture, since sensory neurons express
LPA receptor and thus, represent a more relevant model than cell lines. As expected,
1
Ca2+ microfluorometric measurements show that 10 µM LPA induces Ca2+ influx in
4
8
DRG neurons (Figure 8), as detected by an increase in the Fluo-4 fluorescence. After
the first application of LPA, neurons desensitize and the Ca2+ response is notably
reduced in a second LPA application (Figure 8A). LPA produced intracellular Ca2+
2
+
changes in TRPV1-expressing nociceptors, as capsaicin also triggered a Ca influx in all
LPA responsive neurons, although not all capsaicin responding neurons were activated
by LPA. Importantly, stimulation of LPA/capsaicin responding neurons with 40 mM
2
+
KCl indicate that changes in cytosolic Ca concentrations were recorded in viable,
healthy nociceptors. A similar behaviour was observed for (S)-17, which under the same
experimental paradigm (Figure 8B), activated the LPA receptor TRPV1-sensitive DRG
1
nociceptors. Akin to LPA, repeated instillation of compound (S)-17 induced a reduction
2
+
of the Ca responses, consistent with the desensitization of the LPA receptor (see
1
Supporting Figure S4 for representative calcium microfluorography recordings).
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