Identification of 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3- yl]carbonyl}amino)cyclohexane carboxylic acid as a selective nonpeptide neurotensin receptor type 2 compound

Article abstract of DOI:10.1021/jm5003843

Compounds active at neurotensin receptors (NTS1 and NTS2) exert analgesic effects on different types of nociceptive modalities, including thermal, mechanical, and chemical stimuli. The NTS2 preferring peptide JMV-431 (2) and the NTS2 selective nonpeptide compound levocabastine (6) have been shown to be effective in relieving the pain associated with peripheral neuropathies. With the aim of identifying novel nonpeptide compounds selective for NTS2, we examined analogues of SR48692 (5a) using a FLIPR calcium assay in CHO cells stably expressing rat NTS2. This led to the discovery of the NTS2 selective nonpeptide compound 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl] carbonyl}amino)cyclohexane carboxylic acid (NTRC-739, 7b) starting from the nonselective compound 5a.

Full text of DOI:10.1021/jm5003843

Article
pubs.acs.org/jmc
Identification of 1‑({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)‑
1H‑pyrazol-3-yl]carbonyl}amino)cyclohexane Carboxylic Acid as a
Selective Nonpeptide Neurotensin Receptor Type 2 Compound
James B. Thomas,*,† Angela M. Giddings,† Robert W. Wiethe,† Srinivas Olepu,† Keith R. Warner,†
Philippe Sarret,‡ Louis Gendron,‡ Jean-Michel Longpre,‡ Yanan Zhang,† Scott P. Runyon,†
and Brian P. Gilmour†
†Center for Organic and Medicinal Chemistry, Research Triangle Institute, P.O. Box 12194, Research Triangle Park,
North Carolina 27709, United States
‡Department of Physiology and Biophysics, Faculty of Medicine and Health Sciences, Universite
12th Avenue North, Sherbrooke, Quebec J1H 5N4, Canada
́
de Sherbrooke, 3001,
S
* Supporting Information
ABSTRACT: Compounds active at neurotensin receptors (NTS1 and NTS2) exert anal-
gesic effects on different types of nociceptive modalities, including thermal, mechanical, and
chemical stimuli. The NTS2 preferring peptide JMV-431 (2) and the NTS2 selective non-
peptide compound levocabastine (6) have been shown to be effective in relieving the pain
associated with peripheral neuropathies. With the aim of identifying novel nonpeptide com-
pounds selective for NTS2, we examined analogues of SR48692 (5a) using a FLIPR calcium
assay in CHO cells stably expressing rat NTS2. This led to the discovery of the NTS2 selective
nonpeptide compound 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]-
carbonyl}amino)cyclohexane carboxylic acid (NTRC-739, 7b) starting from the nonselective
compound 5a.
respiratory depression. NT-mediated analgesia has been
demonstrated with compounds selective for both the NTS1
and NTS2 receptors as well as nonselective compounds.23−28
Beyond this, there is now substantial evidence that both NTS1
and NTS2 can mediate relief from chronic or neuropathic
pain.29 This type of pain is difficult to treat with current drugs
and does not always respond well to opioid therapy.30 Taken
together with their neuroleptic activity, it is easy to understand
why the development of compounds acting via the NT network
has engendered so much interest.
The NTS1 receptor has received the most attention since the
discovery of the NT system as it binds NT with high affinity,
hence the name NTRH (NT receptor high affinity), and is well
behaved in heterologous expression systems. The NTS2 re-
ceptor, on the other hand, has received less attention by com-
parison, and as a consequence fewer compounds have thus
far been identified that interact with this receptor. As is true for
NTS1, the most potent and selective NTS2 compounds
discovered to date have been obtained via modification of the
NT(8−13) (1) fragment.31 Discovery of potent and selective
nonpeptide compounds however has proven elusive and
remains as a significant challenge.
INTRODUCTION
■
Neurotensin (NT) is a tridecapeptide (Glu-Leu-Tyr-Glu-Asn-
Lys-Pro-Arg-Arg-Pro-Tyr-Ile-Leu) that was identified 40 years
ago from bovine hypothalamus.1 NT functions as a neurotrans-
mitter and neuromodulator demonstrating a range of biological
actions.2 In the CNS, NT is colocalized with and regulates
the action of mesolimbic and nigrastriatal dopamine3−7 as well
as mediating nonopioid analgesia and hypothermia.8,9 It is
believed that NT operates primarily through interaction with
two G-protein coupled receptors NTS1 and NTS2 (also
referred to as NTR1, NTR2, NTRH, NTRL) to regulate these
activities. A third receptor, NTS3, also known as sortilin, is a
type I receptor bearing a single transmembrane domain.10−14
The ability of the NT receptor system to regulate CNS
dopamine led researchers to postulate that NT might be an
endogenous neuroleptic15 and that drugs acting via the NT
system might therefore be useful as antipsychotic agents.16,17
Methamphetamine also produces profound disruption of
dopamine flow in the mesolimbic and nigrastriatal dopamine
networks with chronic methamphetamine exposure, eliciting
behaviors resembling schizophrenia.18,19 It is thus no surprise
that compounds acting via the NT network are now being inves-
tigated as potential treatments for methamphetamine abuse.20,21
The NT system also plays an important role in the pain
processing network.8,22 The nonopioid analgesia mediated by
the NT system has also generated much interest over the years
as a potential means of circumventing the side effects produced
by opioid analgesics including addiction, constipation, and
Several important milestone compounds from NT receptor
research are depicted in Chart 1. The 8−13 fragment of NT,
compound 1, is as potent as the full length peptide, and several
Received: March 11, 2014
© XXXX American Chemical Society
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Chart 1
hexapeptide variants of 1 have been reported over the years that
either favor NTS2 over NTS1 or are selective for NTS2 versus
NTS1. The first of these is JMV-431 (2) that was produced
via reduction of the tyrosine 11 amide bond and shows a clear
preference for NTS2.32,33 While it is not bioavailable, it is active
in several models of chronic pain when delivered intrathecally.28,29
The peptide NT79 (3), reported more recently,34 is selective
for NTS2 (>100-fold) and has provided a wealth of information
regarding the role of NTS2 in animal models of pain, anti-
psychotic activity, thermoregulation, and regulation of blood
pressure.34 Like peptide 2, this compound attained NTS2
selectivity via modification of the tyrosine 11 residue. The most
recent additions to the rolls of NTS2-selective compounds are
the potent peptide−peptoid hybrids 4a and 4b.35,36 These com-
pounds are ultraselective for NTS2, with selectivity ratios re-
aching 12000- and 22000-fold, respectively, with 4b also de-
monstrating excellent plasma stability.
AlThough few in number, there are three prominent non-
peptide compounds that interact with NTS2 that have been
used extensively in the characterization of the NT receptors.
These include the two pyrazole-based compounds 5a
(SR48692) and SR142948a (5b) and the histamine blocker
levocabastine (6).37−39 Pyrazole compound 5a prefers NTS1 to
NTS2 while 5b is nonselective, but they both antagonize the
activity of NT at NTS1. Compound 6 is selective for NTS2
versus NTS1, but it is also a potent antagonist at histamine
receptor 1 (H1). These compounds (5a,b and 6) highlight the
fact that while NTS2-selective peptides exist, selective non-
peptide compounds are rare.
discovering novel NTS2 selective ligands, we undertook this
endeavor by adapting a previously described calcium mobiliza-
tion assay to our high throughput FLIPR Tetra system and
confirmed the functional results of active compounds using a
radioligand binding assay.40 This effort led to the identification
of a nonpeptide compound that is selective for NTS2 versus
NTS1, 1-({[1-(4-fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-
3-yl]carbonyl}amino)cyclohexane carboxylic acid (7b), starting
from the nonselective compound 5a. The newly discovered
compound (7b) displays neither calcium nor binding activity at
NTS1, but is a potent partial agonist at NTS2 (EC50 of 12
6 nM and an Emax of 7% relative to 5b) that gave a Ki of 153
10 nM in competition with [125I]NT. The details of this effort
are presented herein.
CHEMISTRY
■
The target compounds, depicted in Tables 2 and 3, were syn-
thesized as described in Schemes 1−2. Commercially available
materials were used when available. Those not commercially
available were prepared according to literature precedent.
Scheme 1 illustrates the synthesis of the key intermediate
pyrazole carboxylic acids (11a−j). These were prepared as
described by Labeeuw,41 although improved methods were
recently described by Jiang et al.42 and also Baxendale et al.43
This employed a Knorr [3 + 2]-cyclization reaction between
4-aryl-2,4-diketoesters (8a−f) and arylhydrazines 9a−c in acetic
acid at reflux. The resulting esters 10a−j were hydrolyzed using
LiOH and dioxane to give 11a−j. The 4-aryl-2,4-diketoesters
were commercially available with the exception of 8b. The 2,6-
dimethoxy-butyrophenone used to synthesize compound 8b
was prepared exactly as described by Lindh.44
The SAR of pyrazole compounds 5a and 5b has been
described at NTS1 but, to our knowledge, no systematic survey
of SAR has ever been described at NTS2. With the goal of
B
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a
Scheme 1. Synthesis of Key Pyrazole Intermediates 11a−j
a
Reagents and conditions: (i) HOAc, HCl, and 9a (7-chloroquinolin-4-yl)hydrazine·HCl)) or 9b (1-naphthylhydrazine·HCl) or 9c (4-fluorophenylhydrazine·
HCl), reflux 4 h; (ii) LiOH 3 equiv, dioxane, RT 16 h.
a
Scheme 2. Synthesis of Target Compounds 7b, 13, 14b−29b, and 30
a
Reagents and conditions: (i) HBTU, Et3N, CH2Cl2, 2-aminoadamantane·HCl, RT, 16 h (12e); (ii) SOCl2, toluene, reflux, 3 h; (iii) NaOH, THF,
12f, RT, 16 h; (iv) HBTU, Et3N, CH2Cl2, amino acid ester 12d, RT, 16 h; (v) TFA, CH2Cl2, RT, 16 h; (vi) HBTU, Et3N, CH2Cl2, amino acid ester
12a−c, RT, 16 h; (vii) LiOH, dioxane, RT, 16 h.
In Scheme 2, the synthetic methods used to produce target
compounds 7b, 13, 14b−29b, and 30 are described. Thus,
a given pyrazole carboxylic acid (11a−j) and amino acid
ester (12a−d) or amine (12e) from Chart 2 were coupled
using O-benzotriazol-1-yl-N,N,N′,N′-tetramethyluronium
hexafluorophosphate (HBTU) and triethylamine to give ester
intermediates 7a, and 14a−29a and the descarboxy target
compound 13. The ester intermediates 7a, and 14a−29a were
hydrolyzed to give final products using either basic (LiOH and
dioxane) or acidic (trifluoroacetic acid (TFA) in CH2Cl2) con-
ditions as shown. We used the tert-butyl rather than the methyl
esters of L-cyclohexylglycine (12d) to avoid racemization of the
chiral amino acid. We used the basic conditions for hydrolysis
of achiral amino acid esters. The synthesis of target compound
Chart 2. Amines, Amino Acids, and Amino Acid Esters Used
to Prepare Target Compounds 7b, 13, and 14b−29b
30 was accomplished according to the method of Quer
́
e,45
́
wherein pyrazole acid 11i was converted to its acid chloride
using SOCl2 in toluene, followed by coupling of the acid
chloride intermediate with the adamantyl amino acid 12f under
Schotten−Bauman conditions. The adamantyl amino acid 12f
was prepared as described by Nagasawa.46
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Table 1. Functional and Radioligand Binding Data Obtained for NT, 1, 5a, 5b, and 6 at the NTS1 and NTS2 Receptors
a
FLIPR assay
binding
NTS2
Ki
NTS1
NTS2
b
c
d
compd
EC50
Emax
Ke
EC50
Emax
IC50
e
NT
1
0.04 0.012
0.01 0.002
100
114
3
7
NA
18.5 1.2
5.4 0.6
18.9
3
NA
33 11
62 35
5a
5b
6
4.7 0.8
1.5 0.6
120 20
100
3
5
20
5
100
16
6
2
5
NA
NA
28
4
3
33
a
b
c
d
e
[
125I]NT, EC50, Ke, IC50, and Ki values are nM SEM. Emax value is % NT. Emax value is % 5b. Not active.
NTS2, we believed that each system would produce internally
consistent (reproducible) data. We moved forward with the
working hypothesis that the calcium mobilization described by
these various laboratories was indeed NTS2-mediated and
chose one system to study in greater detail. As we already had
the CHO-k1-rNTS2 cell line available from previous work, this
became the obvious choice.40 We studied the calcium release
produced by analogues of the nonpeptide compound 5a by
measuring their ability to either stimulate release of calcium or
to block the calcium release stimulated by 5b and found that it
responded to changes in structure (SAR), Table 2. This
information was compared with that obtained for (NT and 1)
as well as levocabastine, which revealed that this assay was
capable of identifying a range of activities from potent agonist
(5a and 5b) to partial agonist (6) to antagonist (NT and 1),
Table 1 below, which implied that the structure of the
compound made a direct impact on the resulting mobilization
of calcium. This indicated that our hypothesis was correct and
that we could identify NTS2 agonists directly and NTS2
antagonists indirectly against the agonist activity of 5b.
BIOLOGICAL RESULTS
■
The binding affinities of the test compounds for the rNTS1
and rNTS2 receptors listed in Tables 1 through 3 were deter-
mined using previously reported competitive binding assays.40
The NTS1 and 2 receptors were labeled using [125I]NT. The
cells used in the binding assay were CHO-k1 cells (American
Type Culture Collection) engineered to overexpress either
the rNTS1 or rNTS2 receptor. Measures of functional agonist
and antagonist activity were obtained by measuring changes in
intensity of a calcium-sensitive fluorescent dye as an indirect
measure of changes in internal calcium concentrations. These
measurements were performed using a FLIPR Tetra (NTS2) or
a FlexStation II plate reader for NTS1 (Molecular Devices) and
were analyzed using GraphPad Prism software. Antagonist activity
was measured as inhibition of NT-induced calcium release
(NTS1) or inhibition of 5b-induced calcium release (NTS2).
RESULTS AND DISCUSSION
■
In this article, we provide the details of our effort to identify
nonpeptide compounds that are selective for NTS2 starting
from the nonselective compound 5a. This included developing
an assay capable of detecting activity at the NTS2 receptor
based upon literature reports that compound 5a is an agonist at
NTS2 (induced calcium mobilization at the NTS2 receptor). A
summary of the key SAR elements of 5a discovered using the
NTS2 calcium mobilization assay that led to the identification
of the potent partial agonist 7b (NTRC-739) are provided in
Table 2 and includes the NTS2 EC50 as well as the NTS1 Ke
and the NTS2 binding affinity (Ki). Each of the appended
molecular regions of 5a is discussed herein and includes:
the dimethoxyphenyl ring, the amino acid side chain, the
7-chloroquinoline ring, and the 4-position of the pyrazole ring.
The data obtained from the testing of NT reference com-
pounds in this assay, including NT, 1, 5a,b and levocabastine
(6), are provided in Table 1.
In attempting to develop a method of screening for NTS2
activity, we observed that the literature reports dealing with
functional assays of NTS2 receptors yielded contradictory data,
exhibiting cell-type expression- and species-dependent pharma-
cological properties with opposing patterns. Indeed, NT has
been reported to be an agonist, an inverse agonist, and a neutral
antagonist depending upon the cell expression system.13,47−49
Similar findings were reported for compounds 5a, 5b, and 6 as
well when tested alongside NT in these systems. Specifically,
the potent NTS1 antagonists 5a and 5b were found to be
agonists at NTS2 as they mobilized calcium release while
levocabastine (6) was found to behave either as an agonist,
antagonist, or even as a partial inverse agonist.47−49
Further support for our hypothesis was obtained by
collecting the binding affinity data in competition with
[
125I]NT for our study set of analogues of 5a,Table 2. These
data revealed that compounds that modulated calcium release
also competed with NT for binding at NTS2. Involvement of
NTS2 was implicated further by the observation that
compounds that mobilized calcium release in the CHO-k1-
rNTS2 cell line did not produce calcium mobilization in the
parent CHO line. Taken together, these data indicated that this
assay method could be used to steer SAR studies in search of
novel NTS2 selective compounds.
As described above, the baseline for our study was
established using the compounds common to NT receptor
research. In Table 1, we have summarized the calcium release
and binding data obtained for NT, 1, 5a, and 5b, and the
NTS2-selective compound levocabastine (6) in both our
rNTS1 and rNTS2 cell lines. In our CHO-k1-NTS2 cell line,
neither NT nor 1 showed any calcium release in our FLIPR
assay as previously reported.49 Furthermore, we did not observe
constitutive activity in this expression system as has been
reported by other investigators using alternate expression
systems.35,50 On the other hand, both 5a and 5b stimulated
calcium release with EC50 values of 120 and 20 nM, respec-
tively. Compound 5b was more potent than compound 5a in
the calcium mobilization assay but equally efficacious, and was
designated as our NTS2 full agonist standard (the use of the
term agonist here refers to a compound that stimulates calcium
release in this assay). Levocabastine (6), a compound used for
years to distinguish NTS1 from NTS2, was also tested and
found to be a potent partial agonist with an EC50 of 28 nM and
While the information above illustrated that there was much
inconsistency between cell lines and expression systems for
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Table 2. Antagonism of NT Induced Calcium Release at NTS1 Compared to Target Compound Induced Calcium Mobilization
and Binding Affinity at NTS2 for 7-Chloroquinolyl (A), Naphthyl (B), and 4-F-Phenyl (C) Substituted Pyrazole Carboxamides
at the NTS1 and NTS2 Receptors
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Table 2. continued
a
b
c
d
[
125I]NT. EC50, Ke, IC50, and Ki values are all nM SEM. Emax value is % 5b. Not active.
an Emax of 16% relative to 5b. The activity of 5b and 6 are
illustrated in Figure 1.
Neurotensin and 1 did not stimulate calcium release, but
they both blocked the calcium release stimulated by compound
5b in an insurmountable manner as they showed a rightward
shift in the dose−response curve with an accompanying dose-
dependent drop in the Emax value of 5b.51,52 This is illustrated
for NT in Figure 2. We thus determined and report here the
IC50 for both NT and 1 in competition with 5b and found these
to be 18.9 and 5.4 nM, respectively. The IC50 curve generated
for NT against 5b is depicted in Figure 3.
Corroboration of the functional data was obtained in the
binding assay. We found that compound 5a gave a Ki of 62 nM,
while 5b showed a 10-fold higher affinity with a Ki of 6 nM. As
in the FLIPR assay, compound 5b has a higher affinity than 5a.
NT gave a Ki of 18.5 nM. Compound 1 showed similar affinity
to NT, while levocabastine (6) gave a Ki of 33 nM. The binding
affinities found for NT, 1, and levocabastine are slightly higher
than those found in other laboratories, while those for 5a and b
are similar. We attribute this to our use of an in-plate whole
cell binding method40 as opposed to the use of membrane
preparations.
Figure 1. Dose−response curves for 5b and 6 in CHO-k1-rNTS2
cells.
antipsychotic activity and mediates nonopioid analgesia,
while both NT and levocabastine (6) are active in models of
persistent pain.29,34 The pyrazole compounds 5a and 5b, on
the other hand, are not analgesics in vivo but instead block
the analgesic activity of NT in animal models of pain.28,53,54
This is also observed in animal models based on runaway
mesolimbic dopamine where the neuroleptic action of NT is
Relating the functional behaviors observed in vitro for these
reference compounds to their demonstrated in vivo behaviors is
instructive from a drug discovery perspective. NT possesses
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We chose to use compound 14b as a surrogate for com-
pound 5a because it was easier to produce compound libraries
using the L-cyclohexyl glycine amino acid compared with the
adamantyl amino acid. Compound 14b, like 5a, was previously
́ ́
reported to be a potent NTS1 antagonist by Quer and thus
e60
was expected to be an agonist in the NTS2. The L-isomer was
chosen as it is known to be more active than the R isomer.61
The data from 14b indicated that it would be a suitable stand in
for 5a as it provided comparable agonist potency and efficacy
for NTS2 (EC50 of 217 nM and Emax 86% of 5b) and NTS1
antagonist activity (Ke of 23 nM). The 10-fold difference in its
relative binding affinity at NTS2 illustrated to us that the
structure of the amino acid side chain significantly impacts
binding affinity.
A comparison of the data obtained for compounds 14b−18b
(Table 2) illustrates the SAR realized from changing the
position of the 6-methoxy group (14b, 15b, and 16b), elimina-
tion of the 6-methoxy group (17b), and replacing the 2,6-
methoxy groups with fluorine atoms (18b). We found that the
NTS1 antagonist activity decreased across the series of posi-
tional isomers 14b−16b with Ke values of 23 nM, 1275 nM,
and >10 μM, respectively. This trend continued for 17b and
the difluoro derivative (18b), with Ke values of 1682 nM and
>10 μM. Their NTS2 efficacy (Emax) also fell across this series
of compounds while the functional potency (EC50) was little
varied. The binding affinities showed surprisingly little variation
compared with the change seen going between 5a and 14b and
reinforced the notion that the structure of the amino acid side
chain has a strong impact on binding affinity and appeared to
set the range of observed activity within the study group.
Taken together, we found that the potency of antagonist
activity for 14b at the NTS1 receptor relied heavily upon the
2,6-dimethoxyphenyl ring for its activity. At NTS2, however, it
was the efficacy of calcium release that was most significantly
affected by alteration of the 2,6-dimethoxyphenyl ring. The
binding affinity data and NTS2 potency were much less affected
by changes to this molecular region. This in turn suggested
that an NTS2 potent partial agonist would not possess the 2,6-
dimethoxyphenyl ring.
The data from compound 19b illustrated the effect on in
vitro activity produced by alkylation of the 4-position of the
pyrazole ring. The changes seen here resembled the previous
set of compounds as 19b showed lower NTS1 antagonist re-
lative to 14b and decreased efficacy at NTS2 with less effect on
NTS2 potency and binding affinity. Overall, this example
illustrated that alkylation of this position provided compounds
that favored NTS2 activity over NTS1 but with only modest
levels of affinity at NTS2. Collectively, the data suggested that
this was not a substitution that would lead in the right direction.
The amino acid side chain is known to have a strong
influence on ligand behavior, presumably due to its proximity to
the carboxyl group, the primary anchoring point of the ligand to
the receptor. The data obtained for compounds 5a, 14b, and
20b−23b in Table 2 illustrate the SAR of the large amino acid
side chains. This group of compounds demonstrated that large
cyclic (cycloheptyl, 20b), large bicyclic (adamantyl, 5a), and
large pendant alicyclic (14b) structures are potent NTS1
antagonists with Ke values of 42, 4.7, and 23 nM, respectively.
Compounds with smaller cyclic rings were somewhat less
potent at NTS1, Ke = 222 nM for the five-membered cyclic ring
(22b) and 157 nM for the six-membered cyclic ring (21b).
According to these data then, the compounds with smaller
cyclic rings (21b, 22b) trended toward NTS2 selectivity.
Figure 2. NT is an insurmountable antagonist of 5b mediated calcium
mobilization.
Figure 3. IC50 curve for NT versus 5b.
blocked by compounds 5a and 5b.21,38,55,56 According to the
functional data in Table 1, the proven analgesic and
antipsychotic compounds appeared as either antagonists of
calcium mobilization (NT and peptide 1) or potent partial
agonists of calcium mobilization (levocabastine, 6), while the
compounds that possess neither analgesic nor antipsychotic
activity in vivo, compounds 5a and 5b, appeared as potent
agonists. This point of reference provided a means of parsing in
vitro active compounds into two categories, those that might
possess desirable behaviors in animal models and those that
might not. As this data set is small, we set out to buttress these
relationships using our functional assay to identify additional
examples of novel NTS2-selective antagonists and potent
partial agonists. This article focuses attention on the latter.
The results obtained in our SAR study to find a potent partial
agonist based on compound 5a were centered on the three
pyrazole scaffolds A−C, Table 2. We began by examining the
effect of deleting the carboxylic acid group, compound 13,
as the carboxyl group is known to be a primary attachment
point for ligands of the NT receptors.57−59 We found that the
descarboxy derivative 13 was inactive in both NTS1 and NTS2
FLIPR assays and gave a Ki > 11 μM in the radioligand binding
assay. This behavior stands in stark contrast to the activity
found for 5a and provided additional evidence that the calcium
release observed for 5a (and 5b) results from these ligands
interacting with NTS2.
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Compounds 20b−22b also showed increased NTS2 agonist
potency (lower EC50), but this was accompanied by full efficacy
(high Emax relative to 5b) at NTS2, exactly opposite of that
found in the potent partial agonist levocabastine (6).
with that seen for the similarly substituted 7-chloroquinolyl-
substituted compounds, with the 2-methoxy derivatives
showing lower affinity (higher Ki values) than the 2,6-
dimethoxy counterparts. As before though, the compounds
bearing the L-cyclohexyl glycine side chain (24b,25b) were less
potent than those with the 1-aminocyclohexancarboxylic side
chain (26b,27b).
Toward obtaining an NTS2 selective potent partial agonist,
the calcium data profile for compound 26b looked promising as
it moved closer to that of levocabastine (6). Compound 26b
showed 2-fold greater NTS2 potency and comparable efficacy.
The binding data was also similar, but in this case, compound 6
was more potent, showing a Ki 3.5-fold greater than that for
26b. The similarity between these two compounds ended,
however, when comparing receptor selectivity, as compound
26b showed substantial NTS1 antagonist activity while
levocabastine (6) displayed no activity at NTS1. The 2-
methoxy naphthyl-substituted analogue 27b was not able to
compensate for this deficit, for while its NTS1 antagonist
activity was lowered by 20-fold, the NTS2 potency and binding
affinity also suffered substantial losses.
Fortunately, the pyrazole scaffold (C) bearing the 4-
fluorophenyl-substituent seen in compounds 30, 28b, 29b,
and 7b overcame the deficiencies found in scaffold B to provide
selective potent partial agonists. Comparison of the adamantyl-
substituted compounds 5a and 30 highlights the contribution
of the 4-fluorophenyl ring. These data show that the NTS1
antagonist activity was diminished by 40-fold for 30 versus 5a,
with Ke values of 191 and 4.7 nM, respectively. The NTS2
potency, on the other hand, was doubled (EC50 values of
120 versus 68 nM) while the efficacy was less than half relative
to 5a (Emax values of 100 versus 34% of 5b). This phenomenon
was also observed in the data for compounds 28b, 29b, and 7b.
These compounds are all far less active at NTS1 compared with
their corresponding 7-chloroquinolyl (14b, 21b, and 23b) or
naphthyl (24b, 26b, and 27b) analogues and thereby attain
enhanced NTS2 selectivity with respect to calcium release. The
4-fluorophenyl-substituted compounds (29b and 7b), bearing
the 1-aminocyclohexancarboxylic acid substituent, showed the
most levocabastine-like profiles whether they had a 2,6-
dimethoxyphenyl ring (29b) or a 2-methoxyphenyl ring (7b).
In keeping with previous observations, the transition to the
2-methoxyphenyl ring (7b) from the 2,6-dimethoxyphenyl ring
(29b) led to a nearly 50% reduction in NTS2 efficacy, with
NTS2 Emax values of 15 and 8% of 5b, respectively.
The NTS2 binding affinity observed for 29b and 7b was
consistent with similarly substituted 7-chloroquinolyl (21b,
23b) or naphthyl-substituted (26b, 27b) analogues bearing the
1-aminocyclohexanecarboxylic acid, with one important ex-
ception; for 7b, the binding affinity did not decrease in going
from the 2,6-dimethoxyphenyl (29b) to the 2-methoxyphenyl
ring substitution (7b) as seen in the Ki values of 140 and 153
nM, respectively. We imagine that this change in SAR for 29b
and 7b could result from a change in binding mode at NTS2
that is permitted for 4-fluorophenyl but not 7-chloroquinolyl or
naphthyl-substituted analogues. In the end, the 4-fluorophenyl-
substituted pyrazole scaffold C was able to overcome the
deficits found in scaffolds A and B to provide pyrazole-based
compounds (29b and 7b) with enhanced NTS2 potency and
binding affinity with significantly lower efficacy compared to 5a
at the NTS2 receptor.
In their favor, however, these same compounds (20b−22b)
showed improved binding affinity at NTS2 versus 14b (Ki =
170, 151, and 102 nM versus 644 nM for 14b), more in line
with 5a (Ki = 62 nM). For these compounds, the binding
affinity and NTS2 potency improvement worked in concert
with the lower NTS1 activity to provide enhanced NTS2
affinity and potency while maintaining low efficacy and lower
NTS1 activity. This comparison of NTS2 agonist potency to
the NTS1 antagonist potency for compounds 21b and 23b was
an important clue to achieving selectivity in this series of
compounds as this data reinforced the data from the previous
series, which showed that the NTS1 receptor relied much more
heavily upon the 2,6-dimethoxyphenyl ring for its activity
compared with NTS2 and that the amino acid side chain could
work in concert with the methoxyphenyl ring to retain these
desired properties. This trend was further advanced in
compounds that did not possess the chloroquinoline group.
Two key substitutions for the 7-chloroquinoline group (A)
were the naphthyl (B) and 4-fluorophenyl (C) groups, Table 2.
These substitutions were included in our compound libraries
because the naphthyl compounds were known to yield NTS1
antagonists45 and the 4-fluorophenyl group was found in
levocabastine (6). Like their 7-chloroquinolyl counterparts, the
naphthyl-substituted 2,6-dimethoxyphenyl derivatives 24b and
26b possess potent NTS1 antagonist activity and also NTS2
agonist activity; however, distinct differences were observed
that led us in the right direction.
Most apparent was that these compounds were inherently
less potent at NTS1 and less efficacious at NTS2. The Ke
values for NTS1 antagonist activity and the NTS2 Emax
values were roughly half of that found for similarly substituted
chloroquinoline-based compounds. This was observed for both
the L-cyclohexyl glycine (24b) and the 1-aminocyclohexancar-
boxylic acid side chains (26b). Comparing the naphthyl (24b)
and the 7-chloroquinolyl (14b) compounds, we found Ke
values of 58 versus 23 nM and NTS2 Emax values of 45 and
86% of 5b, respectively. Comparing compounds 26b and 21b,
we found Ke values of 230 versus 157 nM and NTS2 Emax
values of 35 and 78%, respectively.
As seen in the 7-chloroquinolyl series (A), the naphthyl-
substituted 2-methoxyphenyl derivatives 25b and 27b were less
potent NTS1 antagonists than their 2,6-dimethoxyphenyl
substituted counterparts. More surprising was the observation
that the NTS2 efficacies for these compounds were nearly half
again as low as that found in similarly substituted 7-
chloroquinolyl compounds 17b and 23b. This is readily
revealed in a comparison of the NTS2 efficacy data obtained
for compound 21b, 23b, and the 2-methoxy naphthyl
substituted compound 27b, with NTS2 Emax values of 78, 35,
and 18%, respectively.
The naphthyl-substituted compounds with the 2,6-dimeth-
oxyphenyl ring were more potent at NTS2 than their
chloroquinoline-based counterparts (14b, 17b, 21b, and
23b), but naphthyl compounds bearing the 2-methoxyphenyl
ring were equally potent. This was observed for com-
pounds with the L-cyclohexyl glycine as well as those with
1-aminocyclohexancarboxylic acid as in 26b and 27b.
The NTS2 binding data observed for the naphthyl-
substituted compounds 24b−27b was found to be in line
From the standpoint of calcium mobilization at NTS1 and
NTS2, compounds (29b and 7b) appear to be selective for the
H
dx.doi.org/10.1021/jm5003843 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Most other reagents were obtained from either Aldrich Chemical
Corp. or Fischer Scientific and used without further purification. Flash
column chromatography was carried out using a Teledyne ISCO
Combiflash Rf system and Redisep Rf gold prepacked HP silica
columns. Evaporation of solvents was accomplished using rotary
evaporators. The purity of target compounds was determined to be
≥95% by combustion analysis. Characterization of compounds was
accomplished by a combination of TLC, combustion analysis, mass
NTS2 receptor. However, this was not a fair comparison, as one
assay measures calcium mobilization (NTS2) while the other
blockade of calcium mobilization (NTS1). We therefore
acquired the radioligand binding data for both 29b and 7b at
NTS1 in order to compare like measurements. As seen in
Table 3, comparison of the relative binding affinities at NTS1
1
spectrometry (MS) as well as H and 13C NMR. NMR spectra were
Table 3. Selectivity Ratios for 29b and 7b at the NTS1 and
NTS2 Receptors Determined Using [125I]NT Radioligand
Binding
recorded on a Bruker Avance DPX-300 (300 MHz). Chemical shifts
are reported in ppm relative to the tetramethylsilane, and coupling
constant (J) values are reported in hertz (Hz). Low-resolution mass
spectra were obtained using a Waters Alliance HT/Micromass ZQ
system (ESI) in both positive and negative mode. Optical rotations
were measured on an Auto Pol III polarimeter at the sodium D line.
Thin layer chromatography (TLC) was performed on EMD precoated
silica gel 60 F254 plates, and spots were visualized with UV light and I2
or phosphomolybdic acid stain. CHO-k1 cells were from the American
Type Culture Collection, 125I−neurotensin was from PerkinElmer,
Calcium 5 dye was from Molecular Devices, and cell culture reagents
were from Life Technologies. The graphs provided in Figures 1−3
were obtained using Graphpad Prism Software.
compd
NTS1 Ki nM SEM
NTS2 Ki nM SEM
NTS2/NTS1
29b
7b
3210 879
140 29
153 10
23
>25 μM
161
and NTS2 showed that compound 7b is 161-fold selective for
NTS2 versus NTS1 while the dimethoxy analogue 29b shows
only a 23-fold preference for NTS2 over NTS1. These data
point out that while the calcium assay described herein is useful
for identifying compounds that are active at NTS2, conclusions
about selectivity require the use of the binding assay.
General Methods. General Method for Preparation of Pyrazole
Carboxylic Acid Esters (10a−j). A magnetically stirred solution of a
4-aryl-2,4-diketoester sodium salt (8a−f, 5 mmol) and arylhydrazine
hydrochloride (9a−c, 5 mmol) in glacial acetic acid (35 mL) and
concd HCl (1.5 mL) was heated under reflux for 5 h and then cooled
to rt. This was then poured into 300 mL of water and extracted five
times with CH2Cl2. The extracts were washed carefully with satd
NaHCO3 and then water and brine then dried over Na2SO4 and
concentrated to give a crude material. This material was purified using
flash chromatograph with a gradient of 0−100% EtOAc in hexanes
over 30 min collecting peaks only. Flow rates are automatically set based
on column size chosen for separation. Combination of desired fractions
and evaporation provided methyl esters 10a−j as foamy solids.
General Method for Preparation of Pyrazole Carboxylic Acids
(11a−j). Pyrazole carboxylic acids 11a−j were prepared from the esters
(10a−j) using the methyl ester hydrolysis method described below.
General Amide Coupling. To a magnetically stirred suspension
of the appropriate pyrazole carboxylic acid (11a−j, 0.122 mmol) in
anhydrous CH2Cl2 (20 mL) were added successively triethylamine
(0.366 mmol), HBTU (0.146 mmol), and the appropriate amino acid
ester hydrochloride salt (12a−e, 0.122 mmol). After stirring for 16 h,
the resulting mixture was concentrated and the residue purified by
flash chromatography using a gradient of 0−100% EtOAc in hexanes
over 30 min collecting peaks only. Flow rates are automatically set
based on column size chosen for separation. This gave an intermediate
ester that was taken directly to the hydrolysis step.
The identification of 7b as an NTS2 selective compound
demonstrated that this calcium assay was useful for driving SAR
studies in the pyrazole carboxamide series of compounds.
However, the low efficacy of 7b, less than half of that found for
levocabastine, prompted us to revisit mobilization of calcium in
the parent CHO cell line as a negative control. We thus carried
out a final study and examined all of the compounds reported
herein that showed NTS2 Emax values less than 15% of 5b. This
was done collectively by comparing all of the low efficacy com-
pounds together in the same assay, under identical conditions,
in our CHO-k1-NTS2 cells and simultaneously in the parent
CHO line. The results of these experiments confirmed that the
calcium mobilization observed was only found in the CHO cells
stably expressing the NTS2 receptor.
CONCLUSIONS
■
In summary, we tested the compounds common to neurotensin
research and have identified their associated calcium mobi-
lization patterns at NTS1 and 2 using a FLIPR tetra using
NT as our agonist standard for NTS1 and 5b as our agonist
standard for NTS2. We found that compounds known to
possess analgesic and antipsychotic activity in vivo appear as
antagonists (NT and 1)) or potent partial agonists (levocabas-
tine (6)) in this NTS2 in vitro assay. Using the assays described
above in combination with binding data, we identified structural
changes to compound 5a that led to the discovery of the NTS2
selective compound 7b, a pyrazole-based compound with
in vitro properties similar to levocabastine in this assay, a com-
pound known to be active against chronic pain. We are now
working to identify the relevance of the functional data
produced using this method by investigating compound 7b in
recognized models of neuropathic pain in an attempt to
establish an in vitro/in vivo correlation.
General Methyl Ester Hydrolysis. To a magnetically stirred solution
of methyl ester (7a,20−23a, 26a, 27a, and 29a) from the coupling
reaction (0.1 mmol) in 1,4-dioxane (5 mL) was added 1N LiOH
(2 mL), followed by stirring at room temperature overnight. The
mixture was then concentrated, and the residue was taken up in water
(2 mL) and extracted with ethyl acetate (15 mL). After this, 2 N HCl
was added to the aqueous layer to precipitate the carboxylic acid final
product. This was extracted twice with CH2Cl2, and the combined
extracts were dried over Na2SO4 and concentrated to give solid final
products.
General tert-Butyl Ester Hydrolysis. To a magnetically stirred
solution of the tert-butyl ester intermediate (14−19a,24a,25a, and
28a) obtained from the coupling reaction (0.08 mmol) in CH2Cl2
(10 mL) was added excess TFA (10 mL) at room temp. After stirring
for 16 h, the mixture was concentrated and then triturated with ethyl
ether to give a solid product that was isolated by vacuum filtration,
washed with ether, and then dried under high vacuum overnight.
Methyl 1-(1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazole-
3-carboxamido)cyclohexanecarboxylate (7a). Following the general
amide coupling procedure, 7a was prepared from 11j and 12b as an
EXPERIMENTAL SECTION
■
Reactions were conducted under a nitrogen atmosphere using oven-
dried glassware as required. All solvents and chemicals used were
reagent grade. Anhydrous tetrahydrofuran (THF), dichloromethane
(DCM), and N,N-dimethylformamide (DMF) were purchased from
VWR and used without further purification. (7-Chloroquinolin-4-yl)-
hydrazine·HCl (9a) was purchased from Aldrich Chemical Corp.
1-Naphthylhydrazine·HCl (9b) was purchased from TCI America.
4-Fluorophenylhydrazine·HCl (9c) was purchased from Alfa Aesar.
1
amorphous off-white solid (55% yield). H NMR (CDCl3) δ 7.36 (t,
J = 7.8 Hz, 1H) 7.23−7.31 (m, 3H), 7.18 (s, 1H), 6.94−7.04 (m, 3H),
I
dx.doi.org/10.1021/jm5003843 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
1
6.80 (d, J = 8.5 Hz, 1H), 3.75 (s, 3H), 3.48 (s, 3H), 2.15−2.25 (m,
2H), 1.88−2.02 (m, 2H), 1.46−1.56 (m, 6H).
to the general method (off-white solid, 40%). H NMR (CDCl3) δ
7.78−7.88 (m, 2H), 7.62−7.71 (m, 1H), 7.44−7.53 (m, 2H), 7.29−7.36
(m, 1H), 7.24−7.29 (m, 1H), 7.10−7.24 (m, 3H), 6.81 (dt, J = 0.8, 7.5
Hz, 1H), 6.64 (d, J = 8.2 Hz, 1H), 3.97 (s, 3H), 3.13 (s, 3H).
Methyl 5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyra-
zole-3-carboxylate (10i). Ester 10i was prepared from 8a and 4-
fluorophenyl hydrazine hydrochloride (9c) according to the general
method (white amorphous powder, 55%). 1H NMR (CDCl3) δ 7.22−
7.33 (m, 3H), 6.89−7.00 (m, 3H), 6.51 (d, J = 8.3 Hz, 2H), 3.96 (s,
3H), 3.59 (s, 6H).
1-({[1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazol-3-yl]-
carbonyl}amino)cyclohexanecarboxylic Acid (7b). Following the
general methyl ester hydrolysis procedure, 7b was obtained from 7a as
1
a white solid (85%). H NMR (300 MHz, CDCl3) δ 7.33−7.42 (m,
1H), 7.18−7.31 (m, 4H), 6.94−7.05 (m, 4H), 6.81 (d, J = 8.2 Hz,
1H), 3.44 (s, 3H), 2.28 (d, J = 13.9 Hz, 2H), 1.94−2.08 (m, 2H),
1.33−1.83 (m, 6H). 19F NMR (282 MHz, CDCl3) δ −113.42. 13C
NMR (CDCl3) δ 175.00, 163.76, 163.45, 160.16, 156.36, 145.66,
142.48, 136.54, 136.50, 131.24, 131.16, 125.81, 125.70, 120.87, 118.66,
115.71, 115.41, 111.24, 109.45, 60.25, 54.94, 32.20, 25.13, 21.30. MS
(ESI) m/z: 436.7 (M − H)−. Anal. (C24H24FN3O4) C, H, N.
Methyl 1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazole-3-
carboxylate (10j). Ester 10j was prepared from 8f and 9c according
to the general method (off-white powder, 45%). H NMR (CDCl3) δ
7.32−7.43 (m, 1H), 7.23−7.32 (m, 3H), 6.92−7.04 (m, 4H), 6.81 (d,
1
Methyl 1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxylate (10a).45 Ester 10a was prepared via the
general procedure starting from methyl 4-(2,6-dimethoxyphenyl)-2,4-
dioxobutanoate sodium salt (8a) and 7-chloro-4-hydrazinylquinoline
J = 8.5 Hz, 1H), 3.97 (s, 3H), 3.44 (s, 3H).
1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-
3-carboxylic Acid (11a).45 Pyrazole acid 11a was prepared from ester
10a according to the general methyl ester hydrolysis method (pale-
1
hydrochloride (9a) to give 10a as a yellow foam (68%). H NMR
1
yellow powder, 78%). H NMR (300 MHz, DMSO-d6) δ 8.90 (d, J =
(CDCl3) δ 8.98 (d, J = 5.2 Hz, 1H), 8.88 (d, J = 1.8 Hz, 1H), 8.37 (d,
J = 9.2 Hz, 1H), 7.82 (dd, J = 1.8, 9.2 Hz, 1H), 7.45 (d, J = 5.2 Hz,
1H), 7.34 (t, J = 8.4 Hz, 1H), 7.15−7.24 (m, 1H), 6.50 (d, J = 8.4 Hz,
2H), 4.01 (s, 3H), 3.48 (s, 6H).
4.7 Hz, 1H), 8.17 (s, 1H), 7.73 (s, 2H), 7.26 (t, J = 8.4 Hz, 1H),
7.20 (d, J = 4.5 Hz, 1H), 6.99 (s, 1H), 6.54 (d, J = 8.5 Hz, 2H), 3.39
(s, 6H).
1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-4-ethyl-1H-
pyrazole-3-carboxylic Acid (11b). Pyrazole acid 11b was prepared
from ester 10b according to the general methyl ester hydrolysis
method (pale-yellow solid, 80%). H NMR (CDCl3) δ 10.36−11.51
Methyl 1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-4-
ethyl-1H-pyrazole-3-carboxylate (10b). Ester 10b was prepared
from methyl 3-[(2,6-dimethoxyphenyl)carbonyl]-2-oxopentanoate,
sodium salt (8b), and 9a according to the general method (pale-
yellow solid, 45%). 1H NMR (CDCl3) δ 8.87 (d, J = 4.8 Hz, 1H), 8.18
(d, J = 1.9 Hz, 1H), 7.95 (d, J = 9.1 Hz, 1H), 7.50 (d, J = 2.0 Hz, 1H),
7.12−7.27 (m, 2H), 6.42 (d, J = 8.5 Hz, 2H), 3.76 (s, 3H), 3.50 (s,
6H), 2.69 (q, J = 7.4 Hz, 2H), 1.17 (t, J = 7.4 Hz, 3H).
1
(m, 2H), 8.87 (d, J = 4.8 Hz, 1H), 8.19 (d, J = 2.0 Hz, 1H), 7.94 (d,
J = 9.0 Hz, 1H), 7.49 (dd, J = 2.0, 9.0 Hz, 1H), 7.19−7.32 (m, 1H),
7.17 (d, J = 4.7 Hz, 1H), 6.42 (d, J = 8.4 Hz, 2H), 3.50 (s, 6H), 2.68
(q, J = 7.4 Hz, 2H), 1.16 (t, J = 7.4 Hz, 3H). MS (ESI) m/z: 436.5
(M − H)−.
Methyl 1-(7-Chloroquinolin-4-yl)-5-(2,5-dimethoxyphenyl)-1H-
pyrazole-3-carboxylate (10c). Ester 10c was prepared from methyl
4-(2,5-dimethoxyphenyl)-2,4-dioxobutanoate sodium salt (8c) and 9a
1-(7-Chloroquinolin-4-yl)-5-(2,5-dimethoxyphenyl)-1H-pyrazole-
3-carboxylic Acid (11c). Pyrazole acid 11c was prepared from ester
10c according to the general methyl ester hydrolysis method (amorphous,
1
according to the general method (pale-yellow solid, 57%). H NMR
1
pale-yellow solid, 94%). H NMR (CDCl3) δ 8.80 (d, J = 4.7 Hz, 1H),
(CDCl3) δ 8.77 (d, J = 4.5 Hz, 1H), 8.14 (d, J = 2.1 Hz, 1H), 7.91 (d,
J = 9.0 Hz, 1H), 7.54 (dd, J = 2.1, 8.95 Hz, 1H), 7.14 (s, 1H), 7.01 (d,
J = 4.5 Hz, 1H), 6.78−6.91 (m, 2H), 6.57 (d, J = 8.9 Hz, 1H), 3.99 (s,
3H), 3.73 (s, 3H), 2.91 (s, 3H).
8.18 (d, J = 1.9 Hz, 1H), 7.91 (d, J = 9.2 Hz, 1H), 7.56 (dd, J = 1.9, 9.0
Hz, 1H), 7.19 (s, 1H), 7.02 (d, J = 4.7 Hz, 1H), 6.91−6.82 (m, 2H), 6.57
(d, J = 9.0 Hz, 1H), 3.75 (s, 3H), 2.92 (m, 3H).
1-(7-Chloroquinolin-4-yl)-5-(2,4-dimethoxyphenyl)-1H-pyrazole-
3-carboxylic Acid (11d). Pyrazole acid 11d was prepared from ester
10d according to the general methyl ester hydrolysis method (pale-
Methyl 1-(7-Chloroquinolin-4-yl)-5-(2,4-dimethoxyphenyl)-1H-
pyrazole-3-carboxylate (10d). Ester 10d was prepared from methyl
4-(2,4-dimethoxyphenyl)-2,4-dioxobutanoate sodium salt (8d) and
7-chloro-4-hydrazinylquinoline hydrochloride (9a) according to the
1
yellow solid, 78%). H NMR (DMSO-d6) δ 8.91 (d, J = 4.7 Hz, 1H),
1
8.22 (d, J = 1.7 Hz, 1H), 7.78 (s, 1H), 7.76 (d, J = 2.0 Hz, 1H), 7.32
(d, J = 8.4 Hz, 1H), 7.24 (d, J = 4.7 Hz, 1H), 7.04 (s, 1H), 6.56 (dd,
J = 2.2, 8.4 Hz, 1H), 6.34 (d, J = 2.2 Hz, 1H), 3.72 (s, 3H), 2.92 (s, 3H).
1-(7-Chloroquinolin-4-yl)-5-(2,6-difluorophenyl)-1H-pyrazole-3-
carboxylic Acid (11e). Pyrazole acid 11e was prepared from ester 10e
according to the general methyl ester hydrolysis method (pale-yellow
general method (yellow amorphous solid, 45%). H NMR (CDCl3) δ
8.78 (d, J = 4.6 Hz, 1H), 8.14 (d, J = 1.8 Hz, 1H), 7.87 (d, J = 9.0 Hz,
1H), 7.52 (dd, J = 2.0, 9.0 Hz, 1H), 7.20 (d, J = 8.4 Hz, 1H), 7.09 (s,
1H), 7.02 (d, J = 4.6 Hz, 1H), 6.47 (dd, J = 2.2, 8.43 Hz, 1H), 6.19 (d,
J = 2.1 Hz, 1H), 3.98 (s, 3H), 3.77 (s, 3H), 2.99 (s, 3H).
Methyl 1-(7-Chloroquinolin-4-yl)-5-(2,6-difluorophenyl)-1H-pyra-
zole-3-carboxylate (10e). Ester 10e was prepared from methyl 4-(2,6-
difluorophenyl)-2,4-dioxobutanoate sodium salt (8e) and 9a according
to the general method (yellow amorphous solid, 52%). 1H NMR
(CDCl3) δ 8.87 (d, J = 4.5 Hz, 1H), 8.14 (d, J = 1.9 Hz, 1H), 7.61−
7.75 (m, 1H), 7.51 (dd, J = 1.9, 9.0 Hz, 1H), 7.41 (dd, J = 0.9, 8.7 Hz,
1H), 7.25−7.35 (m, 1H), 7.22 (d, J = 4.5 Hz, 1H), 6.77−6.89 (m,
2H), 4.01 (s, 3H).
Methyl 1-(7-Chloroquinolin-4-yl)-5-(2-methoxyphenyl)-1H-pyra-
zole-3-carboxylate (10f). Ester 10f was prepared from methyl 4-(2-
methoxyphenyl)-2,4-dioxo-butanoate sodium salt (8f) and 9a
according to the general method (yellow solid, 43%). 1H NMR
(CDCl3) δ 8.78 (d, J = 4.7 Hz, 1H), 8.15 (s, 1H), 7.91 (d, J = 9.0 Hz,
1H), 7.50 (td, J = 1.0, 9.0 Hz, 1H), 7.27−7.32 (m, 2H), 6.96−7.01 (m,
2H), 6.65 (d, J = 8.5 Hz, 2H), 4.0 (s, 3H), 4.0 (s, 3H).
1
solid, 94%). H NMR (CD3OD) δ 8.90 (d, J = 4.7 Hz, 1H), 8.10 (s,
1H), 7.80 (d, J = 9.0 Hz, 1H), 7.62 (d, J = 9.0 Hz, 1H), 7.23−7.45 (m,
2H), 7.04 (s, 1H), 6.54 (d, J = 8.5 Hz, 2H).
1-(7-Chloroquinolin-4-yl)-5-(2-methoxyphenyl)-1H-pyrazole-3-
carboxylic Acid (11f).63 Pyrazole acid 11f was prepared from ester 10f
according to the general methyl ester hydrolysis method (pale-yellow
solid, 92%). 1H NMR (DMSO-d6) δ 8.80 (d, J = 4.7 Hz, 1H), 8.15 (d,
J = 2.1 Hz, 1H), 7.98 (d, J = 9.0 Hz, 1H), 7.69 (dd, J = 2.1, 9.0 Hz,
1H), 7.25−7.39 (m, 2H), 7.04 (d, J = 4.7 Hz, 1H), 6.97 (t, J = 7.4 Hz,
1H), 6.79 (d, J = 8.1 Hz, 1H), 6.70 (s, 1H), 2.91 (s, 3H).
5-(2,6-Dimethoxyphenyl)-1-naphthalen-1-yl-1H-pyrazole-3-car-
boxylic Acid (11g).62 Pyrazole acid 11g was prepared from ester 10g
according to the general methyl ester hydrolysis method (off-white
1
solid, 70%). H NMR (CDCl3) δ 7.78−7.86 (m, 2H), 7.72 (dd, J =
3.5, 6.3 Hz, 1H), 7.44−7.52 (m, 2H), 7.22−7.35 (m, 2H), 7.10−7.19
(m, 2H), 6.35 (d, J = 8.3 Hz, 2H), 3.41 (s, 6H).
Methyl 5-(2,6-Dimethoxyphenyl)-1-naphthalen-1-yl-1H-pyra-
zole-3-carboxylate (10g).62 Ester 10g was prepared from 8a and
1-naphthylhydrazine hydrochloride (9b) according to the general
5-(2-Methoxyphenyl)-1-naphthalen-1-yl-1H-pyrazole-3-carbox-
ylic Acid (11h).62 Pyrazole acid 11h was prepared from ester 11h
according to the general methyl ester hydrolysis method (off-white
powder, 92%). 1H NMR (CDCl3) δ 7.81−7.91 (m, 2H), 7.68 (dd, J =
3.4, 6.2 Hz, 1H), 7.46−7.56 (m, 2H), 7.28−7.37 (m, 1H), 7.14−7.24
(m, 3H), 6.83 (t, J = 7.4 Hz, 1H), 6.65 (d, J = 8.3 Hz, 1H), 3.12 (s,
3H). MS (ESI) m/z: 343.2 (M − H)−.
1
method (colorless foam, 55%). H NMR (CDCl3) δ 7.67−7.83 (m,
3H), 7.38−7.49 (m, 2H), 7.22−7.33 (m, 2H), 7.05−7.17 (m, 2H),
6.34 (d, J = 8.3 Hz, 2H), 3.96 (s, 3H), 3.41 (br s, 6H).
Methyl 5-(2-Methoxyphenyl)-1-naphthalen-1-yl-1H-pyrazole-3-
carboxylate (10h).62 Ester 10h was prepared from methyl 4-(2-
methoxyphenyl)-2,4-dioxobutanoate sodium salt (8f) and 9b according
J
dx.doi.org/10.1021/jm5003843 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-car-
boxylic Acid (11i). Pyrazole acid 11i was prepared from ester 10i
according to the general methyl ester hydrolysis method (off-white
powder, 93%). H NMR (CDCl3) δ 7.23−7.36 (m, 3H), 6.91−7.03
(m, 3H), 6.52 (d, J = 8.5 Hz, 2H), 3.60 (s, 6H).
(2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,4-dimethoxyphenyl)-1H-
pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoic Acid (16b). Fol-
lowing the general tert-butyl ester hydrolysis procedure, 16b was
obtained as a yellow solid in 86% yield from 16a. 1H NMR (CDCl3) δ
9.11 (br s, 1 H), 8.48 (s, 1 H), 8.27 (d, J = 9.1 Hz, 1 H), 7.80 (d, J =
8.9 Hz, 1 H), 7.46 (d, J = 8.4 Hz, 1 H), 7.42−7.35 (m, 1 H), 7.31 (d,
J = 8.4 Hz, 1 H), 7.12 (s, 1 H), 6.59 (d, J = 8.4 Hz, 1 H), 6.23 (d, J =
1.7 Hz, 1 H), 4.73 (dd, J = 5.4, 8.4 Hz, 1 H), 3.81 (s, 3 H), 3.02 (s, 3
H), 1.87−1.61 (m, 5 H), 1.37−1.04 (m, 6 H). MS (ESI) m/z: 548.5
1
1-(4-Fluorophenyl)-5-(2-methoxyphenyl)-1H-pyrazole-3-carbox-
ylic Acid (11j). Pyrazole acid 11j was prepared from ester 10j
according to the general methyl ester hydrolysis method (off-white
1
powder, 91%). H NMR (CDCl3) δ 7.34−7.44 (m, 1H), 7.22−7.33
(M − H)−. [α]25 +3.1° (c 0.45, MeOH). Anal. (C29H29ClN4O5)
(m, 3H), 6.95−7.07 (m, 4H), 6.82 (d, J = 8.3 Hz, 1H), 3.44 (s, 3H).
1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-N-tricyclo-
[3.3.1.13,7]dec-2-yl-1H-pyrazole-3-carboxamide (13). Compound
13 was prepared from 11a and 2-aminoadamantane hydrochloride
(12e) following the general amide coupling method (off-white
D
C, H, N.
tert-Butyl (2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2-methoxyphenyl)-
1H-pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoate (17a). Fol-
lowing the general amide coupling procedure, 17a was obtained from
1
1
powder, 65%). H NMR δ (CDCl3) 8.77 (d, J = 4.7 Hz, 1H), 8.16
11f and 12d in 94% yield as a pale-yellow solid. H NMR (CDCl3) δ
(d, J = 1.9 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 7.55 (dd, J = 1.9, 9.0 Hz,
1H), 7.24−7.37 (m, 2H), 7.13 (s, 1H), 6.92−7.02 (m, 2H), 6.64 (d,
J = 8.3 Hz, 1H), 4.28 (d, J = 8.1 Hz, 1H), 2.98 (s, 6H), 2.02−2.11 (m,
2H), 1.80−1.95 (m, 9H), 1.70−1.79 (m, 2H), 1.59−1.70 (m, 2H). MS
(ESI) m/z: 544.4 (M+H)+. Anal. (C31H31ClN4O3) C, H, N.
8.77 (d, J = 4.7 Hz, 1H), 8.16 (d, J = 1.9 Hz, 1H), 7.98 (d, J = 9.0 Hz,
1H), 7.55 (dd, J = 2.0, 9.1 Hz, 1H), 7.41 (d, J = 9.1 Hz, 1H), 7.23−
7.35 (m, 2H), 7.09−7.16 (m, 1H), 6.89−7.03 (m, 2H), 6.64 (d, J = 8.1
Hz, 1H), 4.66 (dd, J = 5.0, 9.1 Hz, 1H), 2.98 (s, 3H), 1.53−1.99 (m,
7H), 1.49 (s, 9H), 1.01−1.35 (m, 6H).
tert-Butyl (2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxy-
phenyl)-1H-pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoate
(14a). Following the general amide coupling procedures, 14a was
obtained from 11a and tert-butyl (2S)-amino(cyclohexyl)ethanoate
hydrochloride (12d). The material was purified by flash chromatog-
raphy (0−100% EtOAc/hexanes) to afford 14a (84%) as a pale-yellow
film. 1H NMR (CDCl3) δ 8.78 (d, J = 4.7 Hz, 1H), 8.12 (d, J = 2.0 Hz,
1H), 7.97 (d, J = 9.0 Hz, 1H), 7.50 (dd, J = 2.1, 9.0 Hz, 1H), 7.42 (d,
J = 9.0 Hz, 1H), 7.21 (t, J = 8.4 Hz, 1H), 7.01−7.12 (m, 2H), 6.39 (d,
J = 8.2 Hz, 2H), 4.66 (dd, J = 5.1, 9.1 Hz, 1H), 3.40 (br s, 6H), 1.54−
1.99 (m, 6H), 1.44−1.53 (m, 9H), 1.01−1.32 (m, 5H).
(2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2-methoxyphenyl)-1H-pyra-
zol-3-yl]carbonyl}amino)(cyclohexyl)ethanoic Acid (17b). Following
the general tert-butyl ester hydrolysis procedure, 17b was obtained as a
pale-yellow film in 74% yield from 17a. 1H NMR (CDCl3) δ 9.09 (br
s, 1 H), 8.76 (br s, 1 H), 8.33 (d, J = 8.9 Hz, 1 H), 7.82 (d, J = 8.9 Hz,
1 H), 7.57−7.32 (m, 4 H), 7.22 (s, 1 H), 7.10 (t, J = 6.9 Hz, 1 H), 6.70
(d, J = 7.7 Hz, 1 H), 4.78 (dd, J = 4.8, 8.2 Hz, 1 H), 3.04 (br s, 3 H),
2.10−1.90 (m, 1 H), 1.89−1.60 (m, 5 H), 1.25−1.07 (m, 5 H). MS
(ESI) m/z: 518.0 (M − H)−. [α]25 −24.3° (c 2.04, MeOH). Anal.
D
(C28H27ClN4O4) C, H, N.
tert-Butyl (2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-difluorophenyl)-
1H-pyrazol-3-yl]carbonyl}-amino)(cyclohexyl)ethanoate (18a). Fol-
lowing the general amide coupling procedure, 18a was obtained from
11e and 12d as a pale-yellow foam in 53% yield. 1H NMR (CDCl3) δ
8.89 (d, J = 4.7 Hz, 1H), 8.15 (d, J = 4.7 Hz, 1H), 7.75 (d, J = 9.0 Hz,
1H), 7.75 (d, J = 9.0 Hz, 1H), 7.52 (dd, J = 1.9, 9.0 Hz, 1H), 7.4 (d,
J = 9.0 Hz, 1H), 7.32−7.25 (m, 3H), 7.2 (d, J = 9.0 Hz, 1H), 6.80 (t,
J = 7.6 Hz, 1H), 4.69−4.63 (m, 1H), 1.90−1.85 (m, 1H), 1.84−1.60
(m, 5H), 1.50 (s, 9H), 1.32−1.06 (m, 5H).
(2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-
pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoic Acid (14b).58
Following the general tert-butyl ester hydrolysis procedure, 14b was
obtained as a pale-yellow solid in 91% yield from 14a. 1H NMR
(CDCl3) δ 9.10 (br s, 1 H), 8.74 (br s, 1 H), 8.31 (d, J = 8.9 Hz, 1 H),
7.78 (d, J = 8.9 Hz, 1 H), 7.60−7.41 (m, 2 H), 7.39−7.25 (m, 1 H),
6.48 (d, J = 7.5 Hz, 2 H), 4.80 (dd, J = 4.7, 7.9 Hz, 1 H), 3.47 (br s, 6
H), 2.10−1.92 (m, 1 H), 1.90−1.60 (m, 5 H), 1.32−1.09 (m, 5 H).
MS (ESI) m/z: 548.4 (M − H)−. [α]25 −18.1° (c 1.94, MeOH).
D
(2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-difluorophenyl)-1H-pyra-
zol-3-yl]carbonyl}amino)(cyclohexyl)ethanoic Acid (18b). Following
the general tert-butyl ester hydrolysis procedure, 18b was obtained as a
white foam (90%) from 18a. 1H NMR (CDCl3) δ 8.91 (d, J = 4.7 Hz,
1H), 8.18 (s, 1H), 8.17 (d, J = 9.2 Hz, 1H), 7.74 (d, J = 9.0 Hz, 1H),
7.53 (dd, J = 1.9, J = 9.0 Hz, 1H), 7.40 (d, J = 9.0 Hz, 1H), 7.20−7.05
(m, 4H), 6.80 (t, J = 7.6 Hz, 1H), 4.82−4.74 (m, 1H), 2.08−1.94 (m,
1H), 1.89−1.60 (m, 5H), 1.37−1.05 (m, 5H). MS (ESI) m/z: 523.6
Anal. (C29H29ClN4O5) C, H, N.
tert-Butyl (2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,5-dimethoxy-
phenyl)-1H-pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoate
(15a). Following the general amide coupling procedure, 15a was
1
obtained from 11c and 12d as a pale-yellow film (70%). H NMR
(CDCl3) δ 8.78 (d, J = 4.7 Hz, 1H), 8.16 (d, J = 1.9 Hz, 1H), 8.01 (d,
J = 9.2 Hz, 1H), 7.55 (dd, J = 2.3, 9.0 Hz, 1H), 7.40 (d, J = 9.0 Hz,
1H), 7.14−7.11 (m, 1H), 7.00 (d, J = 4.7 Hz, 1H), 6.90 (d, J = 3.0 Hz,
1H), 6.83 (dd, J = 3.2, 9.0 Hz, 1H), 6.55 (d, J = 9.0 Hz, 1H), 4.69−
4.62 (m, 1H), 3.72 (s, 3H), 2.89 (m, 3H), 2.05−1.60 (m, 6H), 1.50 (s,
9H), 1.39−1.11 (m, 5H).
(M − H)−. [α]25 −13.3° (c 0.44, MeOH). Anal. (C27H23ClF2N4O3)
D
C, H, N.
tert-Butyl (2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxy-
phenyl)-4-ethyl-1H-pyrazol-3-yl]carbonyl}amino)(cyclohexyl)-
ethanoate (19a). Following the general amide coupling procedures,
(2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,5-dimethoxyphenyl)-1H-
pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoic Acid (15b). Fol-
lowing the general tert-butyl ester hydrolysis procedure, 15b was
1
19a was obtained as a pale-yellow film (86%) 11b and 12d. H NMR
1
(CDCl3) δ 8.76 (d, J = 4.6 Hz, 1H), 8.09 (d, J = 2.1 Hz, 1H), 7.98 (d,
J = 9.1 Hz, 1H), 7.48 (td, J = 2.2, 9.1 Hz, 2H), 7.21 (t, J = 8.4 Hz, 1H),
7.09 (d, J = 4.6 Hz, 1H), 6.40 (dd, J = 8.4, 15.6 Hz, 2H), 4.64 (dd, J =
4.9, 9.0 Hz, 1H), 3.53 (s, 3H), 3.42 (s, 3H), 2.54−2.82 (m, 2H), 1.61−
1.95 (m, 6H), 1.49 (s, 9H), 1.17−1.36 (m, 5H), 1.06−1.15 (m, 3H).
MS (ESI) m/z: 633.7 (M + H)+.
obtained (92%) as a white foam from 15a. H NMR (CDCl3) δ 9.00
(d, J = 5.3 Hz, 1H), 8.39 (d, J = 1.8 Hz, 1H), 8.17 (d, J = 9.2 Hz, 1H),
7.7 (dd, J = 1.9, 9.0 Hz, 1H), 7.30 (d, J = 9.0 Hz, 1H), 7.27−7.16 (m,
2H), 6.90 (d, J = 3.0 Hz, 1H), 6.83 (dd, J = 3.2, 9.0 Hz, 1H), 6.58 (d,
J = 9.0 Hz, 1H), 4.81−4.72 (m, 1H), 3.78 (s, 3H), 2.90 (m, 3H),
2.05−1.60 (m, 6H), 1.37−1.03 (m, 5H). MS (ESI) m/z: 547.8 (M −
H)−. [α]25 −2.6° (c 0.45, MeOH). Anal. (C29H29ClN4O5) C, H, N.
(2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-4-
ethyl-1H-pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoic Acid
(19b). Following the general tert-butyl ester hydrolysis procedure,
D
tert-Butyl (2S)-({[1-(7-Chloroquinolin-4-yl)-5-(2,4-dimethoxy-
phenyl)-1H-pyrazol-3-yl]carbonyl}amino)(cyclohexyl)ethanoate
(16a). Following the general amide coupling procedure, 16a was
1
19b was obtained as a pale-yellow solid in 96% yield from 19a. H
1
NMR (CDCl3) δ 9.12 (d, J = 5.7 Hz, 1 H), 8.43 (s, 1 H), 8.38 (d, J =
9.2 Hz, 1 H), 7.77 (dd, J = 1.3, 9.3 Hz, 1 H), 7.56 (d, J = 8.7 Hz, 1 H),
7.48 (d, J = 5.7 Hz, 1 H), 7.34 (t, J = 8.4 Hz, 1 H), 6.51 (dd, J = 8.5,
15.5 Hz, 2 H), 4.77 (dd, J = 5.2, 8.7 Hz, 1 H), 3.67−3.45 (m, 6 H),
2.66 (dd, J = 3.2, 7.3 Hz, 2 H), 2.12−1.91 (m, 1 H), 1.90−1.60 (m, 6
H), 1.38−1.16 (m, 4 H), 1.15−1.01 (m, 3 H). MS (ESI) m/z: 575.8
(M − H)−. [α]25D +2.3° (c 0.98, MeOH). Anal. (C31H33ClN4O5) C, H, N.
obtained as a pale-yellow film (86%) from 11d and 12d. H NMR
(CDCl3) δ 8.79 (d, J = 4.7 Hz, 1H), 8.16 (d, J = 2.0 Hz, 1H), 7.96 (d,
J = 9.0 Hz, 1H), 7.54 (dd, J = 2.1, 9.0 Hz, 1H), 7.40 (d, J = 9.0 Hz,
1H), 7.21 (d, J = 8.4 Hz, 1H), 7.07 (s, 1H), 7.01 (d, J = 4.6 Hz, 1H),
6.47 (dd, J = 2.3, 8.4 Hz, 1H), 6.18 (d, J = 2.3 Hz, 1H), 4.66 (dd, J =
5.1, 9.0 Hz, 1H), 3.77 (s, 3H), 2.96 (s, 3H), 1.56−1.81 (m, 6H), 1.48
(d, J = 4.5 Hz, 9H), 1.00−1.34 (m, 5H).
K
dx.doi.org/10.1021/jm5003843 | J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
Methyl 1-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-
1H-pyrazol-3-yl]carbonyl}amino)cycloheptanecarboxylate (20a).
Following the general amide coupling procedure, 20a was obtained
from 11a and methyl 1-aminocycloheptanecarboxylate hydrochloride
(d, J = 8.7 Hz, 1H), 2.98 (s, 3H), 2.30−2.16 (m, 2H), 2.10−1.96 (m,
2H), 1.76−1.53 (m, 6H). MS (ESI) m/z: 503.6 (M − H)−. Anal.
(C28H27ClN4O4) C, H, N.
tert-Butyl (2S)-Cyclohexyl({[5-(2,6-dimethoxyphenyl)-1-naphtha-
len-1-yl-1H-pyrazol-3-yl]carbonyl}amino)ethanoate (24a). Follow-
ing the general amide coupling procedures, 24a was obtained from 11g
and 12d as a white solid in 53% yield. 1H NMR (CDCl3) δ 7.88−7.72
(m, 3H), 7.51−7.38 (m, 3H), 7.36−7.21 (m, 2H), 7.11 (t, J = 8.4 Hz,
1H), 7.07−7.03 (m, 1H), 6.33 (d, J = 5.9 Hz, 2H), 4.66 (dd, J = 5.4,
9.1 Hz, 1H), 3.40 (br s, 6H), 1.96−1.52 (m, 8H), 1.47 (s, 9H), 1.33−
1.02 (m, 6H).
(2S)-Cyclohexyl({[5-(2,6-dimethoxyphenyl)-1-naphthalen-1-yl-
1H-pyrazol-3-yl]carbonyl}amino)ethanoic Acid (24b).45 Following
the general tert-butyl ester hydrolysis procedure 24b was obtained as a
slightly yellow solid in 53% yield from 24a. 1H NMR (CDCl3) δ 7.82
(d, J = 6.8 Hz, 2 H), 7.73 (d, J = 6.4 Hz, 2 H), 7.48 (dd, J = 3.2, 6.2
Hz, 2 H), 7.38−7.21 (m, 2 H), 7.19−7.01 (m, 2 H), 6.33 (d, J = 8.1
Hz, 2 H), 4.73 (dd, J = 5.8, 8.3 Hz, 1 H), 3.40 (br s, 6 H), 1.93 (br s, 1
H), 1.83−1.55 (m, 5 H), 1.23−0.99 (m, 5 H). MS (ESI) m/z:
512.4 (M − H)−. [α]25D −26.7° (c 0.24, MeOH). Anal. (C30H31N3O5)
C, H, N.
tert-Butyl (2S)-Cyclohexyl({[5-(2-methoxyphenyl)-1-naphthalen-
1-yl-1H-pyrazol-3-yl]carbonyl}amino)ethanoate (25a). Following
the general amide coupling procedure, 25a was obtained from 11h
and 12d in 81% yield as a colorless film. 1H NMR (CDCl3) δ 7.84 (s,
1H), 7.81 (s, 1H), 7.75 (d, J = 3.5 Hz, 1H), 7.55−7.46 (m, 2H), 7.43
(d, J = 9.1 Hz, 1H), 7.36−7.28 (m, 1H), 7.23−7.14 (m, 3H), 7.12 (s,
1H), 6.85−6.75 (m, 1H), 6.61 (d, J = 8.6 Hz, 1H), 4.66 (dd, J = 5.4,
9.1 Hz, 1H), 3.07 (s, 3H), 1.96−1.57 (m, 7H), 1.47 (s, 9H), 1.32−1.05
(m, 6H).
(2S)-Cyclohexyl({[5-(2-methoxyphenyl)-1-naphthalen-1-yl-1H-
pyrazol-3-yl]carbonyl}amino)ethanoic Acid (25b). Following the
general tert-butyl ester hydrolysis procedure, 25b was obtained from
25a as a white foam (43%). 1H NMR (CDCl3) δ 7.91−7.81 (m, 2 H),
7.73−7.61 (m, 2 H), 7.56−7.47 (m, 2 H), 7.39−7.29 (m, 1 H), 7.25−
7.18 (m, 2 H), 7.15 (s, 2 H), 6.80 (t, J = 7.4 Hz, 1 H), 6.63 (d, J = 8.3
Hz, 1 H), 4.74 (dd, J = 5.7, 8.8 Hz, 1 H), 3.12 (s, 3 H), 1.93 (br s, 1
H), 1.83−1.53 (m, 5 H), 1.24−1.02 (m, 5 H). MS (ESI) m/z:
482.3 (M − H)−. [α]25D −32.7° (c 0.56, MeOH). Anal. (C29H29N3O4)
C, H, N.
1
(12c) as an off-white powder in 65% yield. H NMR (CDCl3) δ 8.77
(d, J = 4.7 Hz, 1H), 8.16 (d, J = 1.9 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H),
7.55 (dd, J = 1.9, 9.0 Hz, 1H), 7.24−7.37 (m, 2H), 7.13 (s, 1H), 6.92−
7.02 (m, 2H), 6.64 (d, J = 8.3 Hz, 1H), 4.28 (d, J = 8.1 Hz, 1H), 2.98
(s, 6H), 2.02−2.11 (m, 2H), 1.80−1.95 (m, 9H), 1.70−1.79 (m, 2H),
1.59−1.70 (m, 2H).
1-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zol-3-yl]carbonyl}amino)cycloheptanecarboxylic Acid (20b). Fol-
lowing the general methyl ester hydrolysis procedure, 20b was
obtained from 20a as a white powder in 86% yield. 1H NMR (CDCl3)
δ 8.80 (d, J = 4.7 Hz, 1H), 8.14 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 9.0
Hz, 1H), 7.50 (dd, J = 2.1, 9.0 Hz, 1H), 7.24−7.17 (m, 1H), 7.11 (s,
1H), 7.07 (d, J = 4.7 Hz, 1H) 6.40 (d, J = 8.7 Hz, 2H), 3.42 (s, 6H),
2.42−2.28 (m, 2H), 2.24−2.13 (m, 2H), 1.74−1.53 (m, 10H). MS
(ESI) m/z: 519.5 (M − H)−. Anal. (C29H29ClN4O5) C, H, N.
Methyl 1-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-
1H-pyrazol-3-yl]carbonyl}amino)cyclohexanecarboxylate (21a).
Following the general amide coupling procedures, 21a was obtained
from 11a and ethyl 1-aminocyclohexanecarboxylate hydrochloride
1
(12b) as a white solid in 87% yield. H NMR (CDCl3) δ 8.74−8.84
(m, 1H), 8.14 (d, J = 2.1 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 7.52 (dd,
J = 2.1, 9.0 Hz, 1H), 7.15−7.25 (m, 1H), 7.04−7.15 (m, 2H), 6.39 (d,
J = 8.5 Hz, 2H), 4.25 (q, J = 7.2 Hz, 2H), 3.41 (s, 6H), 2.18 (d,
J = 13.6 Hz, 2H), 1.86−2.02 (m, 2H), 1.43−1.74 (m, 6H), 1.23−1.34
(m, 3H).
1-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zol-3-yl]carbonyl}amino)cyclohexanecarboxylic Acid (21b). Follow-
ing the general methyl ester hydrolysis procedure, 21b was obtained
1
from 21a in 87% yield as a white solid. H NMR (DMSO-d6) δ 8.92
(d, J = 4.7 Hz, 1H), 8.15 (d, J = 2.1 Hz, 1H), 7.86−7.75 (m, 2H), 7.69
(dd, J = 2.1, 9.0 Hz, 1H), 7.30−7.21 (m, 2H), 6.95 (s, 1H), 6.53 (d,
J = 8.7 Hz, 1H), 3.43 (s, 6H), 2.17−2.06 (m, 2H), 1.85−1.72 (m, 2H),
1.62−1.25 (m, 6H). MS (ESI) m/z: 533.8 (M − H)−. Anal.
(C28H27ClN4O5) C, H, N.
Methyl 1-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-
1H-pyrazol-3-yl]carbonyl}amino)cyclopentanecarboxylate (22a).
Following the general amide coupling procedures, 22a was obtained
from 11a and methyl 1-aminocyclopentanecarboxylate hydrochloride
Methyl 1-({[5-(2,6-Dimethoxyphenyl)-1-naphthalen-1-yl-1H-pyr-
azol-3-yl]carbonyl}amino)cyclohexanecarboxylate (26a). Following
the general amide coupling procedure, 26a was obtained from 11g and
12b as a white solid in 98% yield. 1H NMR (CDCl3) δ 7.73−7.89 (m,
3H), 7.45−7.52 (m, 2H), 7.27−7.37 (m, 2H), 7.06−7.16 (m, 2H),
7.02 (s, 1H), 6.32 (d, J = 8.3 Hz, 2H), 3.78 (s, 3H), 3.23−3.63 (m,
6H), 2.08−2.32 (m, 4H), 1.56 (br s, 8H).
1
(12a) as a white powder in 85% yield. H NMR (CDCl3) δ 8.78 (d,
J = 4.7 Hz, 1H), 8.12 (d, J = 2.1 Hz, 1H), 7.94 (d, J = 9.0 Hz, 1H),
7.52 (dd, J = 2.1, 9.0 Hz, 1H), 7.29−7.22 (m, 1H), 7.21−7.16 (m,
1H), 7.11−7.05 (m, 1H), 6.39 (d, J = 8.5 Hz, 2H), 3.78 (s, 3H), 3.41
(s, 6H), 2.42−2.28 (m, 2H), 2.17−2.05 (m, 2H), 1.90−1.80 (m, 4H).
1-({[1-(7-Chloroquinolin-4-yl)-5-(2,6-dimethoxyphenyl)-1H-pyra-
zol-3-yl]carbonyl}amino)cyclopentanecarboxylic Acid (22b). Fol-
lowing the general methyl ester hydrolysis procedure, 22b was
obtained from 22a as a white powder in 72% yield. 1H NMR (CDCl3)
δ 8.80 (d, J = 4.7 Hz, 1H), 8.14 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 9.0
Hz, 1H), 7.53 (dd, J = 2.1, J = 9.0 Hz, 1H), 7.30−7.18 (m, 1H), 7.11
(s, 1H), 7.07 (d, J = 4.7 Hz, 1H) 6.40 (d, J = 8.7 Hz, 2H), 3.42 (s,
6H), 2.54−2.40 (m, 2H), 2.20−2.07 (m, 2H), 1.90−1.77 (m, 4H). MS
(ESI) m/z: 547.8 (M − H)−. Anal. (C28H27ClN4O5) C, H, N.
Methyl 1-({[1-(7-Chloroquinolin-4-yl)-5-(2-methoxyphenyl)-1H-
pyrazol-3-yl]carbonyl}amino)cyclohexanecarboxylate (23a). Fol-
lowing the general amide coupling procedure, 23a was obtained
from 11f and ethyl 1-aminocyclohexanecarboxylate hydrochloride
1-({[5-(2,6-Dimethoxyphenyl)-1-naphthalen-1-yl-1H-pyrazol-3-
yl]carbonyl}amino)cyclohexanecarboxylic Acid (26b).45 Following
the general ethyl ester hydrolysis procedure, 26b was obtained from
1
26a as an off-white powder in 35% yield. H NMR (CDCl3) δ 7.88−
7.78 (m, 2 H), 7.77−7.67 (m, 1 H), 7.54−7.45 (m, 2 H), 7.37−7.29
(m, 1 H), 7.28−7.22 (m, 4 H), 7.15 (t, J = 8.4 Hz, 1 H), 7.08 (s, 1 H),
6.34 (d, J = 8.5 Hz, 2 H), 3.41 (br s, 6 H), 2.24 (d, J = 14.1 Hz,
2 H), 2.10−1.94 (m, 2 H), 1.79−1.48 (m, 6 H). MS (ESI) m/z: 498.3
(M − H)−. Anal. (C30H31N3O5) C, H, N.
Methyl 1-({[5-(2-Methoxyphenyl)-1-naphthalen-1-yl-1H-pyrazol-
3-yl]carbonyl}amino)cyclohexanecarboxylate (27a). Following the
general amide coupling procedures, 27a was obtained from 11h and
1
12b as a colorless film in 73% yield. H NMR (CDCl3) δ 7.93−7.80
1
(12b) as a white solid in 63% yield. H NMR (CDCl3) δ 8.78 (d, J =
(m, 2H), 7.80−7.71 (m, 1H), 7.59−7.46 (m, 2H), 7.40−7.29 (m, 1H),
7.25−7.12 (m, 4H), 7.10 (s, 1H), 6.87−6.74 (m, 1H), 6.62 (d, J = 8.2
Hz, 1H), 3.77 (s, 3H), 3.09 (s, 3H), 2.13 (br s, 2H), 1.96 (br s, 3H),
1.71−1.30 (m, 10H).
4.7 Hz, 1H), 8.17 (d, J = 1.9 Hz, 1H), 7.96 (d, J = 9.0 Hz, 1H), 7.56
(dd, J = 2.2, 9.1 Hz, 1H), 7.24−7.34 (m, 2H), 7.10 (s, 1H), 6.90−7.01
(m, 1H), 6.64 (d, J = 8.7 Hz, 2H), 3.78 (s, 3H), 2.99 (s, 3H), 2.18 (d,
J = 13.8 Hz, 2H), 1.88−2.01 (m, 2H), 1.63−1.74 (m, 6H).
1-({[1-(7-Chloroquinolin-4-yl)-5-(2-methoxyphenyl)-1H-pyrazol-
3-yl]carbonyl}amino)cyclohexanecarboxylic Acid (23b). Following
the general methyl ester hydrolysis procedure, 23b was obtained from
23a as a white solid in 77% yield. 1H NMR (CDCl3) δ 8.80 (d, J = 4.7
Hz, 1H), 8.17 (d, J = 2.1 Hz, 1H), 7.90 (d, J = 9.0 Hz, 1H), 7.5 (dd,
J = 2.1, J = 9.0 Hz, 1H), 7.35−7.21 (m, 1H), 7.20−7.11 (m, 2H), 6.63
1-({[5-(2-Methoxyphenyl)-1-naphthalen-1-yl-1H-pyrazol-3-yl]-
carbonyl}amino)cyclohexanecarboxylic Acid (27b). Following the
general ethyl ester hydrolysis procedure, 27b was obtained from 27a as
1
a white foam (66%). H NMR (CDCl3) δ 7.92−7.82 (m, 2 H), 7.68
(d, J = 3.4 Hz, 1 H), 7.53 (dd, J = 3.3, 6.3 Hz, 2 H), 7.38−7.30 (m,
1 H), 7.25−7.09 (m, 5 H), 6.82 (t, J = 7.4 Hz, 1 H), 6.63 (d, J = 8.2
Hz, 1 H), 3.10 (s, 3 H), 2.22 (d, J = 13.9 Hz, 2 H), 2.01 (t, J = 11.2 Hz,
L
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2 H), 1.77−1.19 (m, 8 H), 0.97 (br s, 1 H). MS (ESI) m/z: 503.6
(M − H)−. Anal. (C28H27N3O4) C, H, N.
intensity was measured on a FlexStation II fluorometric imaging plate
reader (Molecular Devices). Relative fluorescence units (RFU) were
measured before (20 readings) and after (40 readings) the agonist
compound addition for a total 60 s read time (excitation = 485 nm,
emission = 525 nm, cutoff = 515 nm). For antagonist assays, cells
were treated with Calcium 5 dye for 45 min at 37 °C, 5% CO2 as
with the agonist assays. Then 1/10th volume of 10× final concen-
tration of the test antagonist compounds were added in 1% DMSO
in assay buffer, and the plate was incubated for 15 min at 37 °C,
5% CO2. For Ke assays, the cells were pretreated with a single
concentration of test compound and the FlexStation II added serial
dilutions of the control agonist NT. For IC50 assays, cells were
pretreated with serial dilutions of test compound and the FlexStation
II added 1 nM NT.
Calcium Mobilization Assay for NTS2 Receptor. CHO-k1-rNTS2
cells were maintained in DMEM/F12 supplemented with 10% FBS,
pen/strep, 100 μg/mL normocin, and 400 μg/mL geneticin. For
calcium mobilization assays, cells were plated at 25000 cells/well in
black, clear-bottom 96-well plates the day before the assay and
incubated at 37 °C, 5% CO2. Then 100 μL of reconstituted Calcium 5
dye (Molecular Devices, diluted 1:20 in assay buffer (1× HBSS,
20 mM HEPES, 2.5 mM Probenicid, pH 7.4)) was added per well and
plates were incubated for 45 min at 37 °C, 5% CO2. For agonist assays,
cells were pretreated with 1:10 addition of 10% DMSO and the plates
were returned to 37 °C, 5% CO2. Full-log serial dilutions of the test
compounds were made at 10× the desired final concentration in 1%
DMSO assay buffer and warmed to 37 °C. After the pretreatment
incubation, fluorescence intensity was measured on a FLIPR Tetra
fluorometric imaging plate reader (Molecular Devices). Relative
fluorescence units (RFUs) were measured every second for 100 s
(14 readings before compound addition to establish baseline
fluorescence, 85 after), exposure time 0.53 s, gain = 2000, excitation
intensity = 30%, gate open = 10%. For antagonist assays, 1/10 volume
of 10× concentration of the test compound dilutions in 10% DMSO in
assay buffer was added to cells in lieu of the 10% DMSO only in assay
buffer for the pretreatment. Ke assays were run against dose−response
curves of the control agonist 5b, and IC50 assays were run against the
EC80 of 5b (73 nM final concentration).
Competitive Binding Assays. Relative binding affinity was
evaluated using 125I labeled neurotensin and CHO-k1 cell lines
overexpressing either the rNTS1 or rNTS2 receptor essentially as
described by Gendron.40 Briefly, cells were plated at 100000 cells/well
in 24-well plates in complete DMEM/F12 medium supplemented with
10% fetal bovine serum, 100 units of penicillin/mL, 100 mg of
streptomycin/mL, 0.1 mg/mL of normocin, and 250 mg/mL
geneticin. Cells were incubated at 37 °C with 5% CO2 and 95%
humidity for 48 h. Cells were then equilibrated for 10 min in Earle’s
buffer (130 mM NaCl, 5 mM KCl, 1.8 mM CaCl2, 0.8 mM MgCl2, and
20 mM HEPES, pH 7.4) supplemented with 0.1% BSA and 0.1%
glucose and then incubated with or without 10 nM test compound in
0.1 nM [125I]NT at 37 °C for 30 min. Cells were washed twice in
Earle’s buffer, extracted in 1 mL of 0.1N NaOH and counted in a
Packard Cobra II gamma counter for 1 min. Total binding was
determined in the absence of test compound, and nonspecific binding
was determined in the presence of 1.0 μM nonradiolabeled
neurotensin.
tert-Butyl (2S)-Cyclohexyl({[5-(2,6-dimethoxyphenyl)-1-(4-fluoro-
phenyl)-1H-pyrazol-3-yl]carbonyl}amino)ethanoate (28a). Follow-
ing the general amide coupling procedure, 28a was obtained from 11i
1
and 12d as an off-white powder in 94% yield. H NMR (CDCl3) δ
7.42−7.54 (m, 1H), 7.21−7.35 (m, 3H), 6.90−7.03 (m, 3H), 6.50 (dd,
J = 8.5, 13.9 Hz, 2H), 4.67 (dd, J = 5.09, 9.14 Hz, 1H), 3.62 (s, 3H),
3.52 (s, 3H), 1.60−1.92 (m, 7H), 1.48−1.52 (m, 9H), 1.16−1.33 (m,
5H). MS (ESI) m/z: 536.5 (M − H)−.
(2S)-Cyclohexyl({[5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-
1H-pyrazol-3-yl]carbonyl}amino)ethanoic Acid (28b). Following the
general tert-butyl ester hydrolysis procedure, 28b was obtained as a
1
white foam (83%) from 28a. H NMR (CD3OD) δ 7.23−7.42 (m,
3H), 6.99−7.15 (m, 2H), 6.76−6.86 (m, 1H), 6.62 (d, J = 8.29 Hz,
2H), 4.57 (d, J = 6.03 Hz, 1H), 3.60 (s, 6H), 1.74−2.10 (m, 7H),
1.21−1.44 (m, 4H). MS (ESI) m/z: 480.6 (M − H)−. [α]25 −19.7°
D
(c 0.33, MeOH). Anal. (C26H26FN3O5) C, H, N.
Methyl 1-({[5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyr-
azol-3-yl]carbonyl}amino)cyclohexanecarboxylate (29a). Following
the general amide coupling procedure, 29a was prepared from 11i and
1
12b as a colorless solid in 50% yield. H NMR (CDCl3) δ 7.33−7.25
(m, 4H), 7.00−6.90 (m, 2H), 6.50 (d, J = 8.5 Hz, 2H), 3.75 (s, 3H),
3.58 (s, 6H), 2.25−2.13 (m, 2H), 2.01−1.89 (m, 2H), 1.74−1.52
(m, 6H).
1-({[5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazol-3-
yl]carbonyl}amino)cyclohexanecarboxylic Acid (29b). Following the
general methyl ester hydrolysis procedure, 29b was obtained from 29a
1
as a white solid (89%). H NMR (CDCl3) δ 7.36−7.21 (m, 4H),
7.03−6.95 (m, 2H), 6.50 (d, J = 8.5 Hz, 2H), 3.60 (s, 6H), 2.34−2.20
(m, 2H), 2.10−1.96 (m, 2H), 1.79−1.47 (m, 6H). MS (ESI) m/z:
466.4 (M − H)−. Anal. (C25H26FN3O5) C, H, N.
2-({[5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazol-3-
yl]carbonyl}amino)tricycle-[3.3.1.13,7]decane-2-carboxylic Acid
́ ́
(30). Following the method of Quer 30 was obtained from 11i
e,45
and 9-aminobicyclo[3.3.1]nonane-9-carboxylic acid (12f). Briefly, 11i
was heated under reflux in SOCl2 for 2 h, followed by cooling and
concentration on a rotavap. The residue was then dissolved in toluene
and evaporated 3× to remove HCl. The residue was then taken
up in dry THF and added to a well agitated mixture of 5:1 THF:water
(15 mL/g of acid chloride) containing 1.1 equiv of 2-amino-
adamantane-carboxylic acid and 2.2 equiv of NaOH at 5 °C. Following
the addition, the stirring was continued for 18 h at ambient temp. After
this time, the mixture is made acidic with 1 N HCl and extracted
3× with CH2Cl2, dried over sodium sulfate, and evaporated. The
residue was chromatographed using a 0−15% CH2Cl2:MeOH
gradient. Evaporation provided the desired compound as an off-
1
white solid (71%). H NMR (CDCl3) δ 7.20−7.36 (m, 4H), 6.90−
7.04 (m, 3H), 6.51 (d, J = 8.5 Hz, 2H), 3.54−3.63 (m, 6H), 2.71−2.81
(m, 2H), 2.23 (d, J = 12.8 Hz, 2H), 2.07 (d, J = 12.2 Hz, 2H), 1.68−
1.95 (m, 8H). MS (ESI) m/z: 518.9 (M − H)−. Anal. (C29H30FN3O5)
C, H, N.
Pharmacological Methods. Calcium Mobilization Assay for
NTS1 Receptor. CHO-k1-rNTS1 cells were maintained in DMEM/
F12 medium supplemented with 10% fetal bovine serum (Gibco),
100 U/mL penicillin/100 μg/mL streptomycin, 100 μg/mL normocin
(Invivogen), and 250 μg/mL geneticin. For calcium mobilization
assays, cells were plated at 30000 cells/well in black, clear-bottom
96-well plates the day of the assay and incubated at 37 °C, 5% CO2.
Prior to the assay, Calcium 5 dye (Molecular Devices) was
reconstituted according to manufacturer instructions. The reconsti-
tuted dye was diluted 1:40 in assay buffer (1× HBSS, 20 mM HEPES,
and 2.5 mM Probenicid (Sigma), pH 7.4). Growth media was
removed, and 200 μL of this diluted dye was added to each well. Plates
were incubated for 45 min at 37 °C, 5% CO2 after dye addition. For
agonist assays, cells were pretreated with 1:10 addition (20 μL) of 10%
DMSO in assay buffer, and the plates were returned to 37 °C, 5% CO2
for 15 min. Full-log serial dilutions of the test compounds were made
at 10× the desired final concentration in 1% DMSO assay buffer and
warmed to 37 °C. After the pretreatment incubation, fluorescence
Data Analysis. To determine EC50, IC50, and Ke values, data were
fit to a three-parameter logistic equation using GraphPad Prism
software. Ki values for radioligand binding assays were determined
from IC50 values using the equation of Cheng and Prusoff. All data
are from at least three independent experiments run in duplicate
wells.
ASSOCIATED CONTENT
■
S
* Supporting Information
Elemental analysis results for test compounds. This material is
available free of charge via the Internet at http://pubs.acs.org.
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AUTHOR INFORMATION
Corresponding Author
*Phone: (919)-541-6375. Fax: (919) 541-6499. E-mail:
jbthomas@rti.org.
■
Author Contributions
The manuscript was written through contributions of all
authors. All authors have given approval to the final version of
the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
This research was supported by the National Institute on Drug
Abuse, grant DA29961. We gratefully acknowledge the NIMH
Chemical Synthesis and Drug Supply Program for providing us
with the samples of 5b and 5a.
ABBREVIATIONS USED
■
NT, neurotensin; NTS1, neurotensin receptor 1; NTS2,
neurotensin receptor 2; CNS, central nervous system 5; H1,
histamine receptor 1; FLIPR, fluorometric imaging plate reader;
CHO, Chinese hamster ovary
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