The identification of nonpeptide neurotensin receptor partial agonists from the potent antagonist SR48692 using a calcium mobilization assay

Article abstract of DOI:10.1016/j.bmcl.2009.01.024

In a search for nonpeptide agonists for the neurotensin receptor (NTR1), we replaced the adamantyl amino acid moiety found in the antagonist SR48692 (1a) with leucine and related α-alkylamino acids found in peptide agonists. When tested in a calcium mobil

Full text of DOI:10.1016/j.bmcl.2009.01.024

Bioorganic & Medicinal Chemistry Letters 19 (2009) 1438–1441
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
The identification of nonpeptide neurotensin receptor partial agonists from
the potent antagonist SR48692 using a calcium mobilization assay
*
James B. Thomas , Hernán Navarro, Keith R. Warner, Brian Gilmour
Organic and Medicinal Chemistry, Research Triangle Institute, PO Box 12194, Research Triangle Park, NC 27709-2194, USA
a r t i c l e i n f o
a b s t r a c t
Article history:
In a search for nonpeptide agonists for the neurotensin receptor (NTR1), we replaced the adamantyl
Received 17 November 2008
Revised 8 January 2009
Accepted 12 January 2009
Available online 15 January 2009
amino acid moiety found in the antagonist SR48692 (1a) with leucine and related
found in peptide agonists. When tested in a calcium mobilization assay, we found that both
cine confer partial agonist activity to the pyrazole scaffold with the -enantiomer (3a) providing a signif-
icantly greater response. A brief SAR survey demonstrated that the observed agonist activity was resilient
to changes made to the dimethoxyaryl ring in 3a. The resulting compounds were less potent relative to 3a
but showed greater agonist responses. The partial agonist activity was extinguished when the chloro-
quinoline ring was replaced with naphthalene. Thus, while L-leucine appears to possess a powerful ago-
nist directing affect for the NTR1 receptor, its presence alone in the molecular architecture is not
sufficient to insure agonist behavior.
a
-alkylamino acids
D
- and -leu-
L
L
Keywords:
Neurotensin
Partial agonist
Antagonist
Nonpeptide
Ó 2009 Elsevier Ltd. All rights reserved.
Abuse of methamphetamine represents a major and increasing
threat to public health.1 Yet despite many years of research, no
pharmacotherapies have been identified for psychostimulant
abuse. Neurotensin (NT, pGlu-Leu-Tyr-Glu-Asn-Lys-Pro-Arg-Arg-
Pro-Tyr-Ile-Leu), a tridecapeptide identified over 30 years ago2 is
widely distributed in the central and peripheral nervous system
and functions as both a neurotransmitter and neuromodulator.3–6
It is a key player with regard to dopamine control in an area of
the brain that is central to the mediation of reward behavior. It is
co-localized with mesolimbic dopamine and modulates its trans-
mission,7–9 functionally antagonizing dopamine in the mesolimbic
system while increasing dopaminergic transmission in the nigra-
striatal system.10,11 It modulates both dopaminergic and glutama-
tergic inputs to the nucleus accumbens, a region critical to the
brains response to psychostimulants.12–14 Such an ability to mod-
ulate dopamine has drawn the attention of researchers evaluating
the role that NT plays in a number of maladies including schizo-
phrenia and abuse of psychostimulants.15,16 NT receptor peptide
agonists and nonpeptide antagonists have both received attention
in these efforts. Along similar lines, our interest in identifying
pharmacotherapies for methamphetamine abuse directed us to
the discovery of nonpeptide small-molecule NT receptor agonists,
an area of research that has received little attention over the years.
Neurotensin achieves its effects via three receptor proteins,
NTR1, NTR2, and NTR3.17–22 The first two are seven-transmem-
brane domain G-protein-coupled receptors (GPCRs) while the third
is a single-transmembrane domain sorting protein. Despite the fact
that this receptor system was identified many years ago, very few
nonpeptide ligands have been described for any NT receptor.23–25
For NTR1, the most widely studied small-molecule ligands are
those related to the antagonist SR48692 (1a, Chart 1) that shows
* Corresponding author. Tel.: +1 919 541 6375; fax: +1 919 541 8868.
E-mail address: jbthomas@rti.org (J.B. Thomas).
Chart 1. Standard antagonists for the NTR1 receptor.
0960-894X/$ - see front matter Ó 2009 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2009.01.024

J. B. Thomas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1438–1441
1439
potent antagonist activity at NTR1 and good selectivity versus
NTR2.20,26 Despite the paucity of information regarding nonpeptide
NT receptor compounds, much information supporting their design
was available. Two research groups using point mutation studies
have reported ligand-binding site models for the NTR1 recep-
tor.27–30 The model put forth by Barroso et al. and Labbe-Jullie
et al. was of particular importance for the development of NTR1
nonpeptide small-molecule agonists as they proposed overlapping
binding sites for the peptide agonist neurotensin-(8–13) and the
nonpeptide antagonist 1a. This suggested that it might be possible
to obtain nonpeptide agonists through modification of the nonpep-
tide small-molecule antagonists.27–29
All of the receptor modeling studies to date have demonstrated
that the terminal amino acid in both the agonist and antagonist li-
gands is of primary importance to the ligand/receptor interaction.
Both ligand types were proposed to anchor on three key residues
Arg327 Met,208 and Phe.331 The Arg327 residue was suggested to
bind the Leu13 terminal residue in the peptide agonist and the car-
boxyl group in the nonpeptide antagonist 1a. The side chains of the
amino acids were proposed to interact with Met208 and Phe.331 Ta-
ken together, these findings strongly suggested that replacement
of the amino acids in the small-molecule antagonists (1a and 1b)
with those found in peptide agonists, -leucine and similar -alkyl-
amino acids, was a reasonable strategy for discovery of small-mol-
ecule nonpeptide agonists.
L
L
The synthesis of the target compounds described in this study
was accomplished by coupling pyrazole carboxylic acids 2a–e with
Fmoc-protected amino acids pre-loaded onto Wang resin (Schemes
1–3). The names of the amino acids used to prepare specific target
compounds are listed in Table 1. The pyrazole acids 2a–e were pre-
pared according to the method previously described by Labeeuw
et al.31 In each synthesis, the resin was first Fmoc-deprotected with
piperidine and then coupled to 2a–e using benzotriazole-1-yl-oxy-
tris-(dimethylamino)-phosphonium hexafluorophosphate (BOP)
and triethylamine. Cleavage of the target compounds from the re-
sin was accomplished using 95% trifluoroacetic acid in water to
give the desired products 3a–i (Scheme 1), 4a–c (Scheme 2), and
5 (Scheme 3) in 60–80% yield.32
Instead of using a binding assay to test our compounds 3a–i,
4a–c, and 5, we chose to use a high-throughput functional assay.
To this end, we generated a CHO-K1 cell line stably expressing
NTR1.33 The receptor specificity of agonist activity was determined
Scheme 1. Reagents: (a) 20% piperidine, NMP; (b) BOP, Et3N, NMP, 95:5, TFA:H2O.
Cl
a
Fmoc-Leu-Wang Resin
N
b,c
R1
O
Cl
N
N
H
OH
N
N
R1
R2
4a: R1 = R2 = H
O
N
N
OH
4b: R1 = H; R2 = OCH3
4c: R1 = H; R2 = OCF3
R2
O
2b: R1 = R2 = H
2c: R1 = H; R2 = OCH3
2d: R1 = H; R2 = OCF3
Scheme 2. Reagents: (a) 20% piperdine, NMP; (b) BOP, Et3N, NMP; (c) 95:5, TFA:H2O.

1440
J. B. Thomas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1438–1441
Scheme 3. Reagents: (a) 20% piperdine, NMP; (b) BOP, Et3N, NMP; (c) 95:5, TFA:H2O.
scribed antagonist, compound 1b bearing the amino acid L-cyclo-
Table 1
Fmoc-amino acid Wang resins used in target compound synthesis
hexylglycine, did not stimulate calcium release in our assay.
Interestingly, the amount of activity observed varied with the struc-
ture of the side chain and the isobutyl group appeared to be pre-
ferred. The EC50 data was obtained for compounds 3a and 3b.
Target compound
Fmoc-[amino acid]–Wang
1b
[
[
[
L
-Cyclohexylglycine]
-Leucine]
3a, 4a–c, 5
L
Compound 3a bearing the L-leucine amino acid, was found to be a
3b
3c
3d
3e
3f
3g
3h
3i
D-Leucine]
[Glycine]
partial agonist with an EC50 of 67 nM and an Emax of 51% of NT. Its
enantiomer, 3b, also showed partial agonist activity but was signif-
icantly less potent (EC50 = 986 nM). Both of these are considerably
less potent than NT. Overall, this data set clearly demonstrates that
the amino acid in the test compounds 1a, 1b, and 3a–i has a powerful
directing effect on the compounds intrinsic behavior and can confer
either agonist or antagonist activity depending on its substructure.
The NTR1 specificity of the observed calcium release for 3a and 3b
was demonstrated by blocking with the NTR1 antagonist (1a) as de-
scribed earlier (data not shown).
[L
[L
[L
[L
[L
[L
-a-aminobutyric]
-Norvaline]
-Norleucine]
-Valine]
-Isoleucine]
-Phenylalanine]
by the ability of the NTR1 antagonist 1a to block test compound
NTR1 activation. For these experiments, the cells were pre-incu-
bated with 1 or 10 mM 1a for 15 min at 37 °C prior to the addition
of the test compound at its EC50. The apparent affinity (Ke) of 1a
and b and 5 were determined by measuring the EC50 of neuroten-
sin in their presence and absence.34
The screening data obtained from test compounds 3a–i (Scheme
1) is shown in Table 2. As depicted, many of the compounds demon-
strated agonist behavior while leucine, both L and D isomers, showed
Compounds 4a–c were prepared to determine if the agonist
directing effect of the L-leucine group in 3a would be resilient to
changes made to the substituents in its dimethoxyaryl ring
(Scheme 2). Quéré had previously shown that these groups were
essential to high affinity binding in 1a.35 As depicted in Table 2,
analogs 4a–c retained their partial agonist behavior and, 4b and
4c showed a higher agonist response relative to NT though they
all had lower EC50 values. Compound 4a with no methoxy groups
was significantly less potent than 3a and also less potent than 4b
with a single methoxy group. The lower potency of 4c relative to
4b suggests that the aryl ring prefers a greater degree of electron
density. The NTR1 specificity of the observed calcium release for
4a–c was demonstrated by blocking with the NTR1 antagonist
(1a) as described earlier (data not shown).
the greatest effect on calcium release. In contrast, the previously de-
Table 2
Data obtained for target and standard compounds in an NTR1 agonist-mediated
calcium mobilization assay
Compound Screena
EC50
Emax
Ke
b
Compound 5 was prepared to determine if the agonist direct-
ing effect of the L-leucine group in 3a would be affected with
(10
lM)
(nM SEM)
(%NT SEM)
(nM SEM)
NT
1a
1b
3a
3b
3c
3d
3e
3f
3g
3h
3i
4a
4b
4c
5
0.23 0.04
100
6
changes made to its chloroquinoline ring (Scheme 3). We re-
placed the chloroquinoline group in 3a with a naphthalene ring
system since it is of similar size and had been shown to produce
compounds with high binding affinity.35 Interestingly, this com-
pound (5) showed no agonist activity when screened at 10 mM
but instead was found to be an antagonist with an apparent
affinity (Ke) of 93 nM for the NTR1 receptor (Table 2). This find-
36 10
35
NAc
55 12
57 10
NA
11
18
20
5
NA
NA
62
86
84
NA
9
67 26
986 496
54 18
37
2
1
4
3
1
ing illustrates that the presence of L-leucine in target molecules
derived from 1a is insufficient to guarantee that the resulting li-
gand will display agonist behavior for the NTR1 receptor. When
considered together with the data from 4a–c, the results from 5
suggest that each of the groups pendant to the pyrazole core can
impact ligand activity which in turn suggests that each of these
groups are interacting with the receptor protein, a notion consis-
tent with the receptor modeling studies of Labbe-Jullie et al.29
Taken together, this compound survey demonstrated that chang-
6
7
7
2043 86
220 42
1214 197
56
81
91 16
7
7
93 32
a
Test compounds were screened in triplicate at 10
l
M and data is presented as
percent stimulation relative to NT.
b
Data represent means SE from at least two independent experiments.
Not active.
c
ing the amino acid residue in the antagonist 1a to L-leucine (3a)

J. B. Thomas et al. / Bioorg. Med. Chem. Lett. 19 (2009) 1438–1441
1441
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Figure 1. NTR1 receptor partial agonists disclosed by Wyeth Laboratories.
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was sufficient to convert from antagonist to partial agonist activ-
ity. Beyond this we have shown that this agonist activity was
resilient to some (4a–c) but not all (5) changes to the scaffold.
We have also shown that the presence of L-leucine alone (5) is
insufficient to guarantee partial agonist activity.
During the course of this investigation, Fan and coworkers dis-
closed the structures for two NTR1 partial agonists (6 and 7,
Fig. 1).36 These compounds arose from a campaign to identify NT
small-molecule agonists using virtual screening techniques. The
investigators confirmed the NTR1 receptor partial agonist activity
of 6 and 7 in a calcium release (FLIPR) assay. We found this of inter-
est, and based on our observations, do not believe that it is a coin-
cidence that both of these compounds incorporate the amino acid
leucine. Though there is much more to learn regarding small-mol-
ecule NTR1 compounds, it will be of interest to discover why leu-
cine appears to be a privileged group for promoting agonist
activity in NTR1 ligands.
25. Hong, F.; Zaidi, J.; Cusack, B.; Richelson, E. Bioorg. Med. Chem. 2002, 10, 3849.
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Kitabgi, P.; Labbe-Jullie, C. J. Biol. Chem. 2000, 275, 328.
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Bernassau, J.; Kitabgi, P. J. Biol. Chem. 1998, 273, 16351.
29. Labbe-Jullie, C.; Barroso, S.; Nicolas-Eteve, D.; Reversat, J. L.; Botto, J. M.;
Mazella, J.; Bernassau, J. M.; Kitabgi, P. J. Biol. Chem. 1998, 273, 16351.
30. Pang, Y.; Cusack, B.; Groshan, K.; Richelson, E. J. Biol. Chem. 1996, 271, 15060.
31. Labeeuw, B.; Gully, D.; Jeanjean, F.; Molimard, J.-C.; Boigegrain, R. WO/1996/
032382, 1996.
In conclusion, we have demonstrated that replacement of the
amino acid group in NTR1 antagonists such as 1a or 1b with leu-
cine afforded small-molecule partial agonists (3a and 3b) with
32. All compounds showed satisfactory NMR and combustion data. The NMR
spectrum of 3a was recorded using
a 300 MHz (Bruker AVANCE 300)
spectrometer using tetramethylsilane as the internal standard. 1H NMR
(CDCl3) d 8.8 (d, 1H, J = 4.72 Hz), 8.14 (d, 1H, J = 1.81 Hz), 7.95 (d, 1H, J = 9.1),
7.5 (dd, 1H, J = 1.89 and 9.1 Hz), 7.4 (d, 1H, J = 7.95 Hz), 7.20 (t, 1H, J = 8.42 Hz),
7.10 (s, 1H), 7.08 (d, 1H, J = 4.72 Hz), 6.38 (d, 2H, J = 8.42 Hz), 4.81 (br s, 1H),
3.39 (s, 6H), 1.74 (m, 3H), 0.96 (dd, 6H, J = 5.49 and 10.21 Hz).
the L-leucine enantiomer providing significantly higher potency.
The propensity of this amino acid to confer agonist behavior was
also observed in analogs of 3a (4a–c). In addition to this, we have
also shown that the presence of leucine as part of a molecular
structure (5) is not enough to guarantee an NTR1 partial agonist.
Finally, the results from this study also provide support for the
receptor-binding models that proposed overlapping active sites
for NT agonists and antagonists. Additional studies to identify po-
tent NTR1 small-molecule agonists are currently in progress and
will be reported in due course.
33. Agonist activity was measured in these cells by monitoring increases in
internal calcium concentrations using the calcium-sensitive dye, calcium-4,
and a FlexStation II384 fluorometric imaging plate reader (MDS Analytical
Technologies, Sunnyvale, CA). Cells were plated in 96-well plates at 20,000
cells per well 24 h before assay. Prior to assay, the media was removed and the
cells were washed 1Â with 100
2 mM probenecid) followed by addition of 100
dye was reconstituted in dye loading buffer as per manufacturer’s instructions
and 100 l dye was added to wells. Dye loading was performed for 1 h at 37 °C
l
l dye loading buffer (HBSS plus 20 mM HEPES,
ll dye loading buffer. Calcium-4
l
in 5% CO2. Fluorescent intensities were measured for 17 s (baseline intensity)
before and for 40 s after compound addition. Compound-induced fluorescence
increases were measured as the difference between peak and baseline
Acknowledgments
intensity for each well. Test compounds were initially screened at 10 lM
This research was supported by the National Institute on Drug
Abuse, Grant DA024702-01. We gratefully acknowledge the NIMH
Chemical Synthesis and Drug Supply Program for providing us with
the standard NTR1 antagonist SR48692 (1a).
final concentration and an EC50 was determined for compounds exhibiting at
least 50% neurotensin Emax. EC50 values were determined from 10-point half-
log concentration–response curves.
34. The assays were run as above except that during the last 15 min of the
incubation the cells were pretreated (15 min, in duplicate) with 1a (1 or 4
lM)
or 1b (1 or 10 M) or 5 (1 or 10 M) followed by the addition of one of eight
l
l
References and notes
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dissolved in DMSO (1% final assay concentration). The Ke was determined using
the equation: Ke = [antagonist]/((EC50 Ant/EC50control) À 1). The EC50 values
were determined using Prism (v 5.0, Graph Pad, San Diego, CA).
35. Quéré, L.; Boigegrain, R.; Jeanjean, F.; Gully, D.; Evrard, G.; Durant, F. J. Chem.
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