Discovery of a novel small molecule agonist scaffold for the APJ receptor

Article abstract of DOI:10.1016/j.bmc.2016.06.018

The apelinergic system includes a series of endogenous peptides apelin, ELABELA/TODDLER and their 7-transmembrane G-protein coupled apelin receptor (APJ, AGTRL-1, APLNR). The APJ receptor is an attractive therapeutic target because of its involvement in cardiovascular diseases and potentially other disorders including liver fibrosis, obesity, diabetes, and neuroprotection. To date, pharmacological characterization of the APJ receptor has been limited due to the lack of small molecule functional agonists or antagonists. Through focused screening we identified a drug-like small molecule agonist hit 1 with a functional EC₅₀value of 21.5?±?5?μM and binding affinity (K_i) of 5.2?±?0.5?μM. Initial structure–activity studies afforded compound 22 having a 27-fold enhancement in potency and the first sub-micromolar full agonist with an EC₅₀value of 800?±?0.1?nM and K_iof 1.3?±?0.3?μM. Preliminary SAR, synthetic methodology, and in vitro pharmacological characterization indicate this scaffold will serve as a favorable starting point for further refinement of APJ potency and selectivity. [Figure presented]

Full text of DOI:10.1016/j.bmc.2016.06.018

Accepted Manuscript
Discovery of a Novel Small Molecule Agonist Scaffold for the APJ Receptor
Sanju Narayanan, Rangan Maitra, Jeffery R. Deschamps, Katherine Bortoff,
James B. Thomas, Yanyan Zhang, Keith Warner, Vineetha Vasukuttan, Ann
Decker, Scott P. Runyon
PII:
S0968-0896(16)30441-2
DOI:
http://dx.doi.org/10.1016/j.bmc.2016.06.018
BMC 13074
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
13 April 2016
7 June 2016
9 June 2016
Please cite this article as: Narayanan, S., Maitra, R., Deschamps, J.R., Bortoff, K., Thomas, J.B., Zhang, Y., Warner,
K., Vasukuttan, V., Decker, A., Runyon, S.P., Discovery of a Novel Small Molecule Agonist Scaffold for the APJ
Receptor, Bioorganic & Medicinal Chemistry (2016), doi: http://dx.doi.org/10.1016/j.bmc.2016.06.018
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Discovery of a Novel Small Molecule Agonist Scaffold
for the APJ Receptor
†
†
†
†
‡
Sanju Narayanan , Rangan Maitra , Jeffery R. Deschamps , Katherine Bortoff , James B. Thomas ,
Yanyan Zhang†, Keith Warner†,Vineetha Vasukuttan†, Ann Decker†, and Scott P. Runyon*,†
†Center for Drug Discovery, RTI International, Research Triangle Park, NC 27709, United States
‡Center for Bio/Molecular Science and Engineering, Naval Research Laboratory, Code 6930, 4555 Overlook Avenue SW, Washington,
DC 20375, United States
KEYWORDS: APJ small molecule agonist, pyrazole, apelin, AGTRL1, APLNR
ABSTRACT: The apelinergic system includes a series of endogenous peptides apelin, ELABELA/TODDLER and their 7-
transmembrane G-protein coupled apelin receptor (APJ, AGTRL-1, APLNR). The APJ receptor is an attrac-
tive therapeutic target because of its involvement in cardiovascular diseases and potentially other disorders
O
including liver fibrosis, obesity, diabetes, and neuroprotection. To date, pharmacological characterization of
O
N
H
the APJ receptor has been limited due to the lack of small molecule functional agonists or antagonists.
N
N
O
OH
Through focused screening we identified a drug-like small molecule agonist hit 1 with a functional EC50
value of 21.5 5 µM and binding affinity (Ki) of 5.2 0.5 µM. Initial structure-activity studies afforded
compound 22 having a 27-fold enhancement in potency and the first sub-micromolar full agonist with an
EC50 value of 800 0.1 nM and Ki of 1.3 0.3 µM. Preliminary SAR, synthetic methodology, and in vitro
pharmacological characterization indicate this scaffold will serve as a favorable starting point for further
refinement of APJ potency and selectivity.
O
F
22, EC50 = 800 ± 0.1 nM,
Ki = 1.3 ± 0.3 M,
1. Introduction
mouse model wherein nuclear lacZ was knocked into the
endogenous APLNR locus indicated that lacZ expression
was largely restricted to cardiomyocytes and small capillaries
of most tissues.14 Thus, highly vascularized tissues and myo-
cardium in rodents are expected to express APJ in agreement
with past observations.15,16
The APJ (APLNR gene product) receptor is a 380 ami-
no acid class A G protein-coupled receptor (GPCR).1 The
amino acid sequence of the APJ receptor is highly conserved
across a number of species including human, bovine, rat and
mouse suggesting a critical role for this receptor in physiolo-
gy.2 The APJ receptor shares moderate sequence homology
with the angiotensin 2 receptor 1 (AT1). However, none of
the known AT1 ligands or angiotensin II bind APJ.1
However, studies in humans with apelin peptides have
produced some interesting and contradictory data. Japp et al.
conducted studies in healthy human volunteers to investigate
direct vascular effects of apelin-36 and pyr-apelin-13 infu-
sion into forearm vessels.17 Both apelin isoforms caused re-
producible vasodilation in arterial vessels but no such effect
was noted in dorsal hand veins. The vasodilatory effect of
pyr-apelin-13 infusion within the arteries was attributable to
nitric oxide production. The lack of effect within the venous
bed was in sharp contrast to what could be predicted based
upon rodent studies discussed above and possibly points to
species differences and/or disease related activation of the
apelinergic system and underscores the need to test more
stable and drug-like pharmacological agents. Follow-up
studies by the same group in heart disease patients indicated
that acute apelin administration caused peripheral and coro-
nary vasodilation and increased cardiac output.18 Additional
studies in humans indicated that APJ agonism produced
sustained cardiovascular effects that were preserved in the
presence of renin-angiotensin system activation or heart fail-
In 1998, the APJ receptor was de-orphanized when its
first endogenous ligand, apelin, was identified by Tatemoto
and coworkers in bovine stomach extracts.3 Several forms of
apelin peptides are obtained by cleavage of pre-pro-apelin, a
77 amino acid precursor peptide.4-7 These peptides include
apelin-36, apelin-17, apelin-13, apelin-12 and pyr-apelin-13.
Pyr-apelin-13 is the most abundant form in plasma and binds
with high affinity to APJ and functions as a full agonist.4
Recently, a second endogenous peptidergic agonist of APJ
named TODDLER/ELABELA was identified.8,9
In CNS, APLNR mRNA is expressed in neurons of the
paraventricular
and
supraoptic
nucleus
of
the
hypothalamus.10-12 Messenger RNA (mRNA) encoding both
APLNR and apelin are highly expressed peripherally in tis-
sues including gastrointestinal tract, adipose, kidney, liver,
lung and heart.2,5,13 However, questions remain related to
APJ expression levels in various cell types within these or-
gans. Interestingly, in recent studies using a transgenic

ure.19 Taken together, APJ has emerged as an intriguing tar-
get for cardiovascular diseases.
throughput calcium mobilization assay.45 Approximately
100 compounds from our focused library were screened and
four hits belonging to one structural family were identified.
Compound 1 was identified as a functional APJ agonist
with EC50 value of 21.5 + 5 µM and Ki of 5.2 + 0.5 µM us-
ing 125I radiolabeled pyr-apelin-13 (pyr-apelin-13) (Figure
1).40,41 Radioligand displacement studies indicated that 1 was
a competitive orthosteric ligand of APJ that produced 100%
displacement of 125I radiolabeled pyr-apelin-13 (pyr-apelin-
13) as indicated in Figure 1A. However, compound 1 also
had off-target NTR2 receptor activity (EC50 = 271 nM, Ki =
622 nM).47 After confirmation of 1 as a valid hit we began
scaffold modifications at sites A-C in order to determine
receptor tolerances at each site and enhance selectivity for
APJ versus NTR2.
APJ has also emerged as an attractive target because of
its possible involvement in fluid homeostasis,20,21 liver fibro-
sis,22 obesity,23-28 diabetes29-31, neuroprotection,32-34 immune
response,35,36 nociception37 and others. Pathophysiological
roles played by APJ are currently being elucidated using
various apelin peptides.21,29,38,39 However, in vivo pharmaco-
logical studies are difficult due to the short half-life of apelin-
13 and limited availability of drug-like small molecule
probes.
A non-apelin based APJ agonist E339-3D6 has been re-
cently described. The molecular weight of this compound is
>1000 kDa and contains peptidergic fragments. E339-3D6
also contains a fluorescent probe that has been shown to be
critical for potency at the receptor.40 Further structure activity
relationships on E339-3D6 afforded more potent agonists (Ki
= 0.089 µM) biased toward cAMP production that retain the
critical fluorophore.41 In addition to E339-3D6, a low molec-
ular weight small molecule APJ full agonist ML233 (EC50 =
3.7 µM) was reported to be selective over AT1.42 Current
attempts to improve agonist potency have proven challenging
despite two rounds of optimization.42 Recent patent disclo-
sures from Sanofi Aventis and Sanford-Burnham Medical
Research Institute reported small molecule agonists based on
the substituted benzimidazole and triazole core scaffold re-
spectively.43,44 These are the only reports of drug like small
molecule agonists to date. Therefore, novel small molecule
agonists of APJ with improved pharmacological properties
are needed.
A.
B.
Considering the limitations of available tool compounds
to study the APJ receptor, our laboratory initiated a program
to discover novel, drug-like, small molecule agonists using a
primary calcium mobilization assay45 and a secondary
radioligand binding assay.40,41 Based on protein sequence, the
AT1 receptor shares the highest overall homology with the
APJ receptor.1 Using this information, we hypothesized that
some of the structural features common to AT1 receptor lig-
ands might be a good starting point for identification of APJ
small molecules. Further refinement of our compound selec-
tion was based on an evaluation of the putative binding site
homology among related GPCR’s.
Chemogenomic analysis of 30 critical GPCR residues
within the transmembrane cavity places APJ in the
chemoattractant receptor cluster.46 Using binding site cavity
alignment Surgand and colleagues depict a very hydrophobic
APJ core with conserved aromatic residues on helix 6 (F6.44
,
W6.48, and Y6.51) in addition to a positively charged K6.55 that
resides toward the extracellular face of helix 6. Other resi-
dues on helix 3 that surround the binding cavity include a
series of hydrophobic and lipophilic residues (F3.33, M3.36, and
V3.40). Coupling this data with common structural features of
AT1 ligands suggested a more focused screen of ligands
having a lipophilic core with anionic features would be ap-
propriate.
Our initial evaluation focused on AT1 antagonists in-
cluding losartan and valsartan. Since none of these analogs
possessed agonist activity we proceeded to an in house li-
brary of targeted peptidergic GPCR compounds possessing
lipophilic cores with a carboxylic acid moiety using a high-
Figure 1. A. Chemical structure of initial hit 1, identified
through HTS screen and sites of chemical modification ex-
plored that resulted in new lead 22; B. Radioligand binding
of compound 1 using using 125I radiolabeled pyr-apelin-13
2. Chemistry
A general scheme for the synthesis of analog 22 is
shown in Scheme 1. 2,6-Dimethoxy acetophenone 32 was
condensed with diethyl oxalate to afford the sodium salt of
diketone 33 in quantitative yield. Reaction of 33 with 4-
fluorophenyl hydrazine hydrochloride and concentrated hy-
drochloric acid in refluxing acetic acid provided 1, 5-
pyrazoles 34a and 1,3-pyrazoles 34b in a ratio of 4:1. Trans-
2

formation of 34a to acid 2 was achieved by alkaline hydroly-
sis using lithium hydroxide monohydrate. Carboxamides
were obtained through parallel solution-phase synthesis by
coupling acids with selected amines in presence of HBTU as
coupling reagent. Intermediate 35 was obtained by coupling
tert-butyl (S)-3-amino-5-cyclohexylpentanoate with 2. Tert-
butyl ester in 35 was simply deprotected by trifluoroacetic
acid to provide 22.
Figure 2. Crystal structure of compound 2
Evaluation of compound 1a and 3 for calcium mobiliza-
tion demonstrated movement of the 4-Fluoro-Phenyl group to
the N2 position and isomerization of the R-cyclohexyl gly-
cine significantly reduce activity. Methyl ester 4 and com-
pound 5 lacking a carboxylic acid group were also evaluated
and did not possess activity confirming the significance of
carboxylic acid group for APJ potency.
Scheme 1: Synthesis of compound 22
1a, EC50 > 30 µM
3, EC50 > 30 µM
Scheme 1. Reagents and conditions: a) Diethyl oxalate,
MeONa, MeOH, reflux, 6 h, 90%; b) 4-Fluorophenyl hydra-
zine hydrochloride, glacial acetic acid, conc. HCl, reflux, 3.5
h, 61%; c) LiOH, MeOH/THF/H2O, rt, 18 h, 87%; d) Tert-
butyl (S)-3-amino-5-cyclohexylpentanoate, HBTU, Et3N,
CH3CN, rt, 3 h, 79%; e) TFA, DCM, rt, 4 h, 80%
3. Results and Discussion
4, EC50 > 30 µM
5, EC50 > 30 µM
Modification of Site A: As shown in Scheme 1 the synthe-
sis of 2 provides a mixture of isomers at N1 (34a) and N2
(34b) in a ratio of 4:1 that are readily separable using normal
phase chromatography. Confirmation that 2 bears the 4-
Fluoro-Phenyl ring substituted on the N1 nitrogen was ob-
tained through X-Ray crystallography (Figure 2).
Figure 3. Structures of compound 1a, 3, 4 and 5
Many potential therapeutic indications have been postulated
for apelin agonists. Of those, neuroprotection32-34 is one that
may require therapeutic exposure to the central nervous sys-
tem (CNS). In general, carboxylic acids do not penetrate the
CNS well. Therefore, a small set of amides were prepared to
determine if replacement of the terminal carboxylic acid
would be detrimental to APJ potency. Primary amide 6 was
devoid of activity as was dimethyl amide 7, and N-benzyl
amide 8. The addition of more acidic protons from
hydroxamic acid 9 was also not tolerated. Additional analogs
having glycine methyl ester substitution 10 and glycine acid
11 were also inactive. These data demonstrated that the car-
boxyl OH group is an important mediator of receptor activa-
tion in this series.
Table 1. Structural modification at site A
3

O
OH
N
H
N
O
R/Ar
N
F
EC50 (µM)
Compd.
R
>30
>30
6
7
EC50(µM
Compd.
Ar/R
)
8
9
>30
>30
>30
>30
>30
12
10
13
11
>30
14
15
Ph
>30
>30
Modification of Site B: Based on the results from our initial
screen of structurally related scaffolds, the 2,6-
dimethoxyphenyl substituent appeared critical. Confirmation
of this facet was obtained through the synthesis of analogs
12-15 (Table 2). Removal of both methyl groups from the
dimethoxy group (12) caused a complete loss of agonist po-
tency. Similarly, removal of just one methyl groups (13) also
resulted in a loss of potency. In order to evaluate if the in-
crease in polarity from the phenolic groups was responsible
for the loss of activity, unsubstituted phenyl (14) was evalu-
ated. This suggested that the 2,6-dimethoxy phenyl group
was critical for activity. Moreover, replacement of the aryl
group with methyl 15 was inactive. Overall, this suggests the
2, 6-dimethoxy phenyl moiety is an important contributor of
agonist activity at the APJ receptor.
CH3
Modification of Site C: A chemically diverse set of amino
acid scaffolds were substituted for cyclohexyl glycine (Table
3) to explore the effects of side chain hydrophobic and polar
requirements for receptor activity. These results indicated
that polar side-chains were not tolerated (16, 17, 18) and
hydrophobic substituents such as isobutyl 19 did not retain
APJ potency. We then substituted β-Leu (20) for Leu (19) to
determine if elongation of the carboxylic acid group by one
methylene would enhance binding and agonist potency. This
modification resulted in a two-fold enhancement in potency
(20, EC50 = 9.4 µM) with a roughly 9-fold increase in affinity
(20, Ki = 0.60 µM) compared to 1. Similarly, substitution of
β-cyclohexyl glycine 21 for cyclohexyl glycine (1) resulted in
a similar potency enhancement (EC50 = 9.9 µM) and an im-
proved Ki of 1.4 µM. Interestingly, extending the hydropho-
bic cyclohexyl ring by two methylene units improved the
potency by 12 fold (22, EC50 = 0.80 µM) compared to 21.
Since the original hit came from an NTR2 library, we exam-
Table 2. Structural modification at Site B
4

ined the activity of 21 and 22 at both NTR1 and NTR2. Nei-
ther compound had any significant NTR1 activity either as
an agonist or antagonist. These compounds behaved as par-
tial agonists at NTR2 with 21 having an EC50 value of ~345
nM with 57% Emax and 22 having an EC50 value of ~3406
nM with Emax of ~23%. Therefore, these compounds demon-
strated significantly reduced activity at NTR2 compared with
1 and these data indicate that selectivity over NTR1 and 2
can be further increased with additional structural modifica-
tions. An additional series of analogs that altered placement
of the carboxyl and saturation of the hydrophobic group pos-
sessed limited activity at the APJ receptor (23-31). Overall
the SAR modifications on the core scaffold indicate that 22
can serve as a new lead for further refinement as a selective
APJ agonist.
9.4 +
3.4
O
0.6 +
0.10
20
21
OH
9.9 +
2.6
1.4 +
0.2
O
OH
0.8 +
0.1
1.3 +
0.3
Table 3. Structural modification at Site C
22
O
O
R
N
H
N
N
F
O
>30
>30
23
24
*
*
EC50
(µM)
Ki
(µM) +
Compd.
R
+SEM
SEM
O
NH2
OH
16
>30
>30
O
25
>30
>30
>30
>30
17
26
27
28
NH2
OH
18
19
>30
>30
O
O
OH
5

grade. Anhydrous tetrahydrofuran (THF), dichloromethane
(DCM), and N, N-dimethylformamide (DMF) were
purchased from Fisher Scientific and used as such. Flash
column chromatography was carried out using a Teledyne
ISCO Combiflash Rf system and Redisep Rf pre-packed HP
silica columns. Purity and characterization of compounds were
established by combination of HPLC, TLC, LC-MS, and
>30
>30
29
30
1
NMR analytical technique described below. H NMR spectra
were recorded on a Bruker Avance DPX300 (300 MHz)
spectrometer using CHCl3-d, MeOH-d4, DMSO-d6 with
tetramethylsilane (TMS) (0.00 ppm) as the internal reference.
Chemical shifts are reported in ppm relative to the solvent
signal, and coupling constants (J) values are reported in Hertz
(Hz). Thin-layer chromatography (TLC) was performed on
precoated silica gel GF Uniplates from Analtech and spots
were visualized with UV light or I2 detection or
phosphomolybdic acid stain. Low-resolution mass spectra
were obtained using a Waters Alliance HT/Micromass ZQ
system (ESI). Optical rotations were measured on an Auto Pol
III polarimeter at the sodium D line. Analytical and
preparative HPLC was performed on an automated Varian
ProStar HPLC system equipped with a Xterra® C18 RP18 (4.6
× 100 mm i. d., 3.5 µm) column, with a flow rate of 1
mL/min.; λmax = 254 nm; mobile phase A: H2O containing
0.1% TFA and mobile phase B: CH3CN containing 0.1%
TFA. Purity of the target compounds was determined to be ≥
95% by HPLC. Chemical names were generated using
ChemDraw Ultra (CambridgeSoft, version 10.0).
3 + 0.2
31
3 + 0.6
* EC50 and Ki values are averages of multiple experiments
(where n = 3, unless otherwise stated) standard error of the
mean.
4. Conclusion
In summary, compound 1 was found through focused
screening to be a specific APJ receptor ligand that served as
novel scaffold to explore the importance of various structural
features (sites A-C). Conversion of the carboxylic acid in 1 to
a variety of amides, (6-11) resulted in analogs with no activity
at the APJ receptor. This suggests the carboxylic acid plays an
important role in receptor interactions and potentially activa-
tion. Modification of site B (12-15) demonstrated the signifi-
cance of a 2,6-dimethoxyphenyl substitution on APJ agonist
potency. Modification of site C demonstrated that polar side-
chains were not tolerated (16, 17, 18) and hydrophobic sub-
stituents such as isobutyl (19) did not retain APJ potency.
Elongation of the carboxylic acid by one methylene retained
agonist potency for compounds 20 (EC50 = 9.4 µM) and 21
(EC50 = 9.9 µM). Interestingly, extending the hydrophobic
cyclohexyl ring by two methylene (22) improved the potency
by 12 fold (EC50 = 0.80 µM). More importantly, radioligand
binding studies on compounds 1 (Ki = 5.2 µM), 21 (Ki = 1.4
µM), 22 (Ki = 1.3 µM), and 20 (Ki = 0.60 µM) indicate these
ligands specifically act at the APJ receptor and the affinity can
be enhanced through structural modification. In conclusion,
we have identified and characterized one of the first drug-like
small molecule (mw <500) APJ agonists (1, 20, 22) that
serves as an ideal starting scaffold for the development of
more potent and selective APJ agonists having improved
drug-like properties.
S)-2-Cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)acetic acid (1)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (400 mg, 0.168 mmol) was dissolved in
THF (15 mL). To the solution was added HATU (444 mg,
0.168 mmol) and triethylamine (0.80 mL, 5.482 mmol). The
resulting mixture was stirred at room temperature for 15
minutes. L-Cyclohexylglycine-tert-butyl ester hydrochloride
(320 mg, 1.285 mmol) was added and stirred at room temper-
ature for 2 h. THF was evaporated in vacuo, water was added
to the residue and the aqueous layer was extracted with
CH2Cl2 (3 x 20 mL). The combined organic layers were
washed with water, brine and then dried with Na2SO4. The
solvent was evaporated in vacuo to give the crude residue. The
residue was purified by silica gel flash chromatography
(EtOAc:Hex) to give (S)-tert-butyl 2-cyclohexyl-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetate as white solid (75 mg, 79 %). Following
the tert-butyl ester deprotection procedure 22, the title com-
pound 1 was obtained from (S)-tert-butyl 2-cyclohexyl-2-(5-
(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
25
carboxamido)acetate. 94% yield; white solid; [α]D = + 9.03
1
(c 0.15, MeOH); H NMR (MeOH-d4 , 300 MHz) δ 1.11 -
5. Experimental Procedures
5.1 Chemistry
1.41 (m, 5 H), 1.60-1.86 (m, 5 H), 1.89-2.04 (m, 1 H), 3.60
(br. s., 6 H), 4.57 (d, J=6.03 Hz, 1 H), 6.62 (d, J=8.29 Hz, 2
H), 6.80 (s, 1 H), 7.02-7.10 (m, 2 H), 7.25 - 7.38 (m, 4 H).
MS (ESI) m/z: Calcd. for C26H28FN3O5 481.20 [M]+, found
482.5 [M+H]+.
5.1.1 General
Reagents and starting materials were obtained from
commercial suppliers and were used without purification.
Reactions were conducted under N2 atmosphere using oven-
dried glassware. All solvents and chemicals used were reagent
(S)-2-Cyclohexyl-2-(3-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-5-carboxamido)acetic acid (1a)
6

Using the procedure described in 1, the title compound 1a
was obtained from 3-(2,6-dimethoxyphenyl)-1-(4-
carboxylic acid (2) (700 mg, 2.045 mmol) was dissolved in
THF (17 mL). To the solution was added HATU (778 mg,
2.045 mmol) and triethylamine (0.850 mL, 6.13 mmol). The
resulting mixture was stirred at room temperature for 15
minutes. L-Cyclohexylglycine methyl ester hydrochloride (425
mg, 2.045 mmol) was added and stirred at room temperature
for 16 h. THF was evaporated in vacuo, water was added to
the residue and the aqueous layer was extracted with CH2Cl2
(3 x 30 mL). The combined organic layers were washed with
water, brine and then dried with Na2SO4. The solvent was
evaporated in vacuo to give the crude residue. The residue was
purified by silica gel flash chromatography (EtOAc:Hex) to
give (S)-methyl 2-cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carboxamido)acetate (4) as
white solid. 94% yield; 1H NMR (CDCl3 , 300 MHz) δ 1.06 -
1.37 (m, 5 H), 1.61 - 1.82 (m, 5 H), 1.84 - 1.99 (m, 1 H), 3.53
(s, 3 H), 3.62 (s, 3 H), 3.76 (s, 3 H), 4.76 (dd, J=9.04, 6.03
Hz, 1 H), 6.47 - 6.54 (m, 2 H), 6.94 (s, 1 H), 6.97 – 7.01 (m, 2
H), 7.25-7.33 (m, 3 H), 7.43 (d, J=9.04 Hz, 1 H). MS (ESI)
m/z: Calcd. for C27H30FN3O5 495.22 [M]+, found 496.5
[M+H]+.
fluorophenyl)-1H-pyrazole-5-carboxylic acid (34b). white
1
solid. HNMR (CDCl3 , 300 MHz) δ 0.78 - 1.20 (m, 5 H),
1.53 - 1.79 (m, 5 H), 1.81 - 1.94 (m, 1 H), 3.79 (s, 6 H), 4.61
(dd, J=8.29, 4.90 Hz, 1 H), 6.40 (d, J=8.67 Hz, 1 H), 6.64 (d,
J=8.29 Hz, 2 H), 6.88 (s, 1 H), 7.11 (t, J=8.48 Hz, 2 H), 7.26 -
7.34 (m, 1 H), 7.54 (dd, J=8.85, 4.71 Hz, 2 H). MS (ESI) m/z:
Calcd. for C26H28FN3O5 481.20 [M]+, found 482.5 [M+H]+.
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-
3-carboxylic acid (2) Lithium hydroxide monohydrate (324
mg, 7.717 mmol) in 2 mL of water was added to a solution of
methyl
5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylate (34a) (1.1 g, 3.086 mmol) in MeOH
(11 mL) and THF (3 mL). The mixture was stirred at room
temperature for 3 h. The reaction mixture was then concentrat-
ed to about half the volume and then extracted with ether (2 x
25 mL). The aqueous layer was acidified with 1 N HCl and
extracted with CH2Cl2 (3 x 25 mL). The combined organic
layers were washed with water, brine and then dried with
Na2SO4. The solvent was evaporated in vacuo to give the title
compound 2 as white solid (872 mg, 87 %). 1HNMR (CDCl3 ,
300 MHz) δ 3.60 (s, 6 H), 6.51 (d, J=9.0 Hz, 2 H), 7.01 (s,
1H). 7.00-6.97 (m, 2 H), 7.27-7.31 (m, 3 H). MS (ESI) m/z:
Calcd. for C18H15FN2O4 342.10 [M]+, found 343.3 [M+H]+,
341.6 [M-H]-.
.
N-(Cyclohexylmethyl)-5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamide (5) Following
the procedure described in 4, compound 5 was obtained using
5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) and cyclohexyl methylamine. 82% yield;
1
white solid; H NMR (CDCl3 , 300 MHz) δ 0.93 - 1.07 (m, 2
(R)-2-Cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)acetic acid (3)
Following the procedure described in 1, the title compound 3
H), 1.14 - 1.32 (m, 3 H), 1.52 - 1.87 (m, 6 H), 3.30 (t, J=6.40
Hz, 2 H), 3.58 (s, 6 H), 6.50 (d, J=8.67 Hz, 2 H), 6.92 - 7.00
(m, 3 H), 7.05 (t, J=6.03 Hz, 1 H), 7.22 - 7.33 (m, 3 H). MS
(ESI) m/z: Calcd. for C25H28FN3O3 437.21 [M]+, found 438.5
[M+H]+.
was
obtained
using
5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxylic acid (2) and D-
cyclohexylglycine methyl ester hydrochloride .
For hydrolysis of methyl ester, (R)-methyl 2-cyclohexyl-2-(5-
(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetate (50 mg, 0.100 mmol) was dissolved in
1,2-dichloroethane (2 mL) and after addition of trimethyltin
hydroxide (55 mg, 0.300 mmol), the mixture was heated at
80 ˚C until TLC analysis indicated a complete reaction. After
completion of the reaction, the mixture was concentrated in
vacuo, and the residue was taken up in ethyl acetate (15 mL).
The organic layer was washed with HCl (5%) (3 x 5–15
mL). The organic layer was then washed with brine (5–15
mL) and dried over sodium sulfate. Removal of the solvent in
vacuo afforded the crude residue. The residue was purified by
silica gel flash chromatography (MeOH:DCM) to give (R)-2-
Cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-
1H-pyrazole-3-carboxamido)acetic acid (3) as white solid.
37% yield; [α]D25 = -9.52 (c 0.10, MeOH); 1H NMR (CDCl3
, 300 MHz) δ 1.06 - 1.37 (m, 5 H), 1.58 - 1.90 (m, 5 H), 1.92
- 2.06 (m, 1 H), 3.54 (s, 3 H), 3.61 (s, 3 H), 4.73 (dd, J=8.67,
5.65 Hz, 1 H), 6.50 (t, J=8.67 Hz, 2 H), 6.93 - 7.04 (m, 3 H),
7.22 - 7.36 (m, 3 H), 7.48 (d, J=8.67 Hz, 1 H). MS (ESI)
m/z: Calcd. for C26H28FN3O5 481.20 [M]+, found 482.4
[M+H]+.
(S)-N-(2-Amino-1-cyclohexyl-2-oxoethyl)-5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamide (6) Following the procedure described in 4,
compound 6 was obtained using (S)-2-cyclohexyl-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetic acid (1) and 2 M ammonia in methanol.
50 % yield; white solid; 1H NMR (CDCl3 , 300 MHz) δ 1.03 -
1.40 (m, 5 H), 1.63 - 1.92 (m, 5 H), 1.93-2.09 (m, 1 H), 3.54
(s, 3 H), 3.62 (s, 3 H), 4.48 (dd, J=8.67, 7.16 Hz, 1 H), 5.44
(br. s., 1 H), 6.16 (br. s., 1 H), 6.51 (t, J=9.23 Hz, 2 H), 6.90 -
7.03 (m, 2 H), 7.24 - 7.34 (m, 4 H), 7.42 (d, J=9.04 Hz, 1 H).
MS (ESI) m/z: Calcd. for C26H29FN4O4 480.22 [M]+, found
481.4 [M+H]+.
(S)-N-(1-Cyclohexyl-2-(dimethylamino)-2-oxoethyl)-5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamide (7) Following the procedure described in 4,
compound 7 was obtained from (S)-2-cyclohexyl-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetic acid (1) and N, N’-dimethyl hydrochlo-
1
ride. 69% yield; white solid; H NMR (CDCl3 , 300 MHz) δ
1.02 - 1.35 (m, 5 H), 1.61 - 1.82 (m, 5 H), 1.83-1.94 (m, 1 H),
2.99 (s, 3 H), 3.20 (s, 3 H), 3.50 (s, 3 H), 3.64 (s, 3 H), 4.98 -
5.07 (m, 1 H), 6.47 (d, J=8.29 Hz, 1 H), 6.53 (d, J=8.29 Hz, 1
H), 6.89 – 6.99 (m, 2 H), 7.22 - 7.34 (m, 4 H), 7.59 (d, J=9.42
(S)-Methyl 2-cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)acetate (4) 5-
(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
7

(12) Following the procedure described in 1, the title com-
pound 12 was obtained from 5-(2,6-dihydroxyphenyl)-1-(4-
Hz, 1 H). MS (ESI) m/z: Calcd. for C28H33FN4O4 508.25
[M]+, found 509.5 [M+H]+.
fluorophenyl)-1H-pyrazole-3-carboxylic
acid
and
L-
(S)-N-(2-(Benzylamino)-1-cyclohexyl-2-oxoethyl)-5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamide (8) Following the procedure described in 4,
compound 8 was obtained from (S)-2-cyclohexyl-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
Cyclohexylglycine-tert-butyl ester hydrochloride. 1H NMR
(MeOH-d4 , 300 MHz) δ 1.08 - 1.41 (m, 5 H), 1.61-1.86 (m,
5 H), 1.89-2.05 (m, 1 H), 4.48-4.64 (m, 1 H), 6.24 - 6.32 (m,
2 H), 6.85 (s, 1 H), 6.96 - 7.09 (m, 3 H), 7.37-7.45 (m, 2 H).
MS (ESI) m/z: Calcd. for C24H24FN3O5 453.17 [M]+, found
454.4 [M+H]+.
carboxamido)acetic acid (1a) and benzylamine. 79 % yield;
1
white solid; H NMR (CDCl3 , 300 MHz) δ 1.00 - 1.40 (m, 5
H), 1.62 – 1.91 (m, 5 H), 1.93 - 2.16 (m, 1 H), 3.53 (s, 3 H),
3.62 (s, 3 H), 4.39 - 4.56 (m, 3 H), 6.43 - 6.58 (m, 3 H), 6.92
- 7.02 (m, 3 H), 6.90 (s, 1 H), 7.20 - 7.37 (m, 7 H), 7.45 (d,
J=8.67 Hz, 1 H). MS (ESI) m/z: Calcd. for C33H35FN4O4
570.26 [M]+, found 571.9 [M+H]+.
2-Cyclohexyl-2-(1-(4-fluorophenyl)-5-(2-hydroxy-6-
methoxyphenyl)-1H-pyrazole-3-carboxamido)acetic acid
(13)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylic acid (2) (150 mg, 0438 mmol) was
dissolved in DCM (6 mL). To the solution was added 1M
BBr3 solution in DCM dropwise at 0 ̊C. Reaction mixture
was then stirred at room temperature for 18 hrs. Water was
added to the reaction mixture and the aqueous layer was ex-
tracted with EtOAc (3 x 20 mL). The combined organic
layers were washed with water, brine and then dried with
Na2SO4 .The solvent was evaporated in vacuo to give the
crude residue. Crude residue had a mixture of 1-(4-
fluorophenyl)-5-(2-hydroxy-6-methoxyphenyl)-1H-pyrazole-
3-carboxylic acid and 5-(2,6-dihydroxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxylic acid. Following the
procedure described in 1, the title compound 13 was obtained
after purification with preparative TLC (Ether/Hexane (8:2).
1H NMR (MeOH-d4 , 300 MHz) δ 1.10 - 1.43 (m, 5 H), 1.63
- 1.88 (m, 5 H), 1.90-2.06 (m, 1 H), 3.53 (s, 3 H), 4.53 - 4.60
(m, 1 H), 6.44 (t, J=8.67 Hz, 2 H), 6.83 (s, 1 H), 7.01 - 7.10
(m, 2 H), 7.18 (t, J=8.29 Hz, 1 H), 7.30 - 7.41 (m, 2 H), 7.94
(d, J=9.0 Hz, 1 H). MS (ESI) m/z: Calcd. for C25H26FN3O5
467.19 [M]+, found 468.5 [M+H]+.
(S)-N-(1-Cyclohexyl-2-(hydroxyamino)-2-oxoethyl)-5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamide (9) Following the procedure described in 4
compound 9 was obtained from (S)-2-cyclohexyl-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetic acid (1a) and hydroxylamine hydrochlo-
1
ride. 27 % yield; white solid; HNMR (MeOH-d4 , 300 MHz)
δ 1.02 - 1.38 (m, 5 H), 1.62-1.72 (m, 1 H), 1.73 - 1.95 (m, 5
H), 3.61 (s, 6 H), 5.45 - 5.54 (m, 1 H), 6.62 (d, J=8.29 Hz, 2
H), 6.78 (s, 1 H), 7.00 - 7.09 (m, 2 H), 7.23 - 7.39 (m, 4 H).
MS (ESI) m/z: Calcd. for C26H29FN4O5 496.21 [M]+, found
495.4 [M-H]-.
(S)-Methyl 2-(2-cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetamido)acetate (10) Following the proce-
dure described in 4, compound 10 was obtained using (S)-2-
cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-
1H-pyrazole-3-carboxamido)acetic acid (1) and glycine me-
(S)-2-Cyclohexyl-2-(1-(4-fluorophenyl)-5-phenyl-1H-
pyrazole-3-carboxamido)acetic acid (14) Following the
procedure described in 1, compound 14 was obtained using
1-(4-fluorophenyl)-5-phenyl-1H-pyrazole-3-carboxylic acid.
85% yield; white solid; 1H NMR (CDCl3 , 300 MHz) δ 1.06 -
1.40 (m, 5 H), 1.59-1.90 (m, 5 H), 1.93-2.07 (m, 1H), 4.73
(dd, J=8.67, 5.65 Hz, 1 H), 7.04 (s, 1 H), 7.06 - 7.12 (m,
2H), 7.16 - 7.23 (m, 2 H) 7.28 - 7.37 (m, 5 H), 7.42 (d,
J=8.67 Hz, 1 H). MS (ESI) m/z: Calcd. for C24H24FN3O3
421.18 [M]+, found 422.5 [M+H]+.
1
thyl ester hydrochloride. 85% yield; white solid; H NMR
(CDCl3 , 300 MHz) 1.04 - 1.36 (m, 5 H), 1.62 - 1.92 (m, 5
H), 1.94 - 2.08 (m, 1 H), 3.54 (s, 3 H), 3.62 (s, 3 H), 3.75 (s, 3
H), 4.07 (t, J=5.09 Hz, 2 H), 4.49 (dd, J=8.85, 7.35 Hz, 1 H),
6.51 (t, J=9.80 Hz, 2 H), 6.58 (t, J=5.09 Hz, 1 H), 6.92 - 6.95
(m, 1 H), 6.97 (d, J=8.67 Hz, 1 H), 7.21 - 7.33 (m, 4 H), 7.43
(d, J=8.67 Hz, 1 H). MS (ESI) m/z: Calcd. for C29H33 FN4O6
552.24 [M]+, found 553.4 [M+H]+.
(S)-2-(2-Cyclohexyl-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-
carboxamido)acetamido)acetic acid (11) Following the
lithium hydroxide hydrolysis procedure 2, compound 11 was
(S)-2-Cyclohexyl-2-(1-(4-fluorophenyl)-5-methyl-1H-
pyrazole-3-carboxamido)acetic acid (15) Following the
procedure described in 1, compound 15 was obtained using
1-(4-fluorophenyl)-5-methyl-1H-pyrazole-3-carboxylic acid.
74% yield; white solid; 1H NMR (CDCl3 , 300 MHz) δ 1.05
- 1.35 (m, 5 H), 1.59 - 1.87 (m, 5 H), 1.90–2.05 (m, 1 H),
2.31 (s, 3 H), 4.66 (dd, J=8.67, 5.65 Hz, 1 H), 6.73 (s, 1 H),
7.16 - 7.25 (m, 2 H), 7.35 (d, J=8.67 Hz, 1 H), 7.40 - 7.48
(m, 2 H). MS (ESI) m/z: Calcd. for C19H22FN3O3 359.16
[M]+, found 360.2 [M+H]+.
obtained
from
(S)-methyl
2-(2-cyclohexyl-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)acetamido)acetate (10). 95% yield; white solid;
1H NMR (CDCl3 , 300 MHz) δ 1.07-1.40 (m, 5 H), 1.62 -
1.72(m, 1 H), 1.73 - 1.97 (m, 5 H), 3.59 (s, 3 H), 3.62 (s, 3H),
3.86-4.04 (s, 2H), 4.49-4.58 (m, 1 H), 6.62 (d, J=9.0 Hz, 2 H),
6.80 (s, 1H), 7.00-7.10 (m, 2H), 7.25-7.39 (m, 3H), 7.97 (d,
J=9.0 Hz, 1 H ), 8.55 (br. s., 1 H). MS (ESI) m/z: Calcd. for
C28H31FN4O6 538.22 [M]+, found 537.5 [M-H]-.
(S)-5-Amino-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)-5-
(S)-2-Cyclohexyl-2-(5-(2,6-dihydroxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)acetic
oxopentanoic acid (16) 5-(2,6-Dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxylic acid (2) (100 mg,
acid
8

0.292 mmol) was dissolved in THF (7 mL). To the solution
was added HATU (111 mg, 0.292 mmol) and triethylamine
(0.20 mL, 1.461 mmol). The resulting mixture was stirred at
(S)-6-((tert-butoxycarbonyl)amino)-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
1
carboxamido)hexanoic acid (17). 78% yield; white solid; H
NMR (MeOH-d4 , 300 MHz) δ 1.49-1.62 (m, 2 H), 1.65-1.79
(m, 2 H), 1.85-1.97 (m, 1 H), 2.01-2.13 (m, 1 H), 2.81 (bs,
2H), 2.93 (t, J=9.0 Hz, 3 H), 3.62 (s, 6 H), 4.64-4.71 (m, 1
H), 6.63 (d, J=9.0 Hz, 2 H), 6.81 (s, 1 H), 7.06 (t, J=9.0 Hz,
2 H), 7.27-7.32 (m, 2 H), 7.36 (t, J=9.0 Hz, 1H). MS (ESI)
m/z: Calcd. for C24H27FN4O5 470.20 [M]+, found 471.5
[M+H]+.
room temperature for 15 minutes.
L-glutamine α-4-
nitrobenzyl ester hydrobromide (116 mg, 0.32 mmol) was
added and stirred at room temperature for 2 h. THF was
evaporated in vacuo, water was added to the residue and the
aqueous layer was extracted with CH2Cl2 (3 x 20 mL). The
combined organic layers were washed with water, brine and
then dried with Na2SO4 .The solvent was evaporated in vacuo
to give the crude residue. The residue was purified by silica
gel flash chromatography (EtOAc:Hex) to give (S)-4-
(S)-2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-4-methylpentanoic acid (19) 5-
(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (167 mg,
0.438 mmol) and triethylamine (0.18 mL, 1.314 mmol). The
resulting mixture was stirred at room temperature for 20
minutes. L-leucine methyl ester hydrochloride (80 mg, 0.438
mmol) was added and stirred at room temperature for 4 h.
THF was evaporated in vacuo, water was added to the resi-
due and the aqueous layer was extracted with CH2Cl2 (3 x 20
mL). The combined organic layers were washed with water,
brine and then dried with Na2SO4 .The solvent was evapo-
rated in vacuo to give the crude residue. The residue was
purified by silica gel flash chromatography (EtOAc:Hex) to
nitrobenzyl
5-amino-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)-5-oxopentanoate
as white solid (113 mg, 65 %). Following the trimethyltin
hydroxide hydrolysis procedure 3, compound 16 was ob-
tained
from
(S)-4-nitrobenzyl
5-amino-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
1
carboxamido)-5-oxopentanoate. 75 % yield; white solid. H
NMR (CDCl3 , 300 MHz) δ 2.18 - 2.46 (m, 2 H), 2.47 - 2.65
(m, 2 H), 3.55 (s, 3 H), 3.60 (s, 3 H), 4.73 - 4.87 (m, 1 H),
6.42-6.61 (m, 3 H), 6.90 - 7.03 (m, 2 H), 6.93 (s, 1 H), 7.20 -
7.37 (m, 4 H), 7.88 (d, J=7.16 Hz, 1 H). MS (ESI) m/z:
Calcd. for C23H23FN4O6 470.16 [M]+, found 471.4 [M+H]+.
(S)-6-((Tert-butoxycarbonyl)amino)-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
give
(S)-methyl
2-(5-(2,6-dimethoxyphenyl)-1-(4-
carboxamido)hexanoic
acid
(17)
5-(2,6-
fluorophenyl)-1H-pyrazole-3-carboxamido)-4-
Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
methylpentanoate as white solid (135 mg, 66 %). Using the
trimethyltin hydroxide hydrolysis procedure 3, compound 19
was obtained from (S)-methyl 2-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carboxamido)-4-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (167 mg,
0.438 mmol) and triethylamine (0.30 mL, 2.19 mmol). The
resulting mixture was stirred at room temperature for 20
minutes. H-Lys(Boc)-OMe hydrochloride (143 mg, 0.482
mmol) was added and stirred at room temperature for 3 h.
THF was evaporated in vacuo, water was added to the resi-
due and the aqueous layer was extracted with CH2Cl2 (3 x 20
mL). The combined organic layers were washed with water,
brine and then dried with Na2SO4 .The solvent was evapo-
rated in vacuo to give the crude residue. The residue was
purified by silica gel flash chromatography (EtOAc:Hex) to
give (S)-methyl 6-((tert-butoxycarbonyl)amino)-2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)hexanoate as white solid (145 mg, 57%).
Using the trimethyltin hydroxide hydrolysis procedure 3,
compound 17 was obtained from (S)-methyl 6-((tert-
butoxycarbonyl)amino)-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)hexanoate. 93 %
yield; white solid; 1H NMR (CDCl3 , 300 MHz) δ 1.12 - 1.39
(m, 2 H), 1.42 (s, 9 H), 1.45-1.62 (m., 2 H), 1.74-195 (m, 1
H), 1.96-2.16 (m, 1 H), 3.00-3.21 (m, 2 H), 3.60 (s, 3 H),
3.56 (s, 3 H), 4.56-4.84 (m, 1 H), 6.50 (d, J=5.27 Hz, 2 H),
6.91 - 7.00 (m, 3 H), 7.20 - 7.33 (m, 4 H), 7.49 (br. s., 1 H).
MS (ESI) m/z: Calcd. for C29H35FN4O7 570.25 [M]+, found
571.8 [M+H]+.
methylpentanoate. 82 % yield; white solid. 1HNMR (CDCl3 ,
300 MHz) δ 0.99 (dd, J=6.03, 2.64 Hz, 6 H), 1.69 - 1.92 (m,
3 H), 3.61 (s, 3 H), 3.55 (s, 3 H), 4.74 – 4.86 (m, 1 H), 6.50
(t, J=6.78 Hz, 2 H), 6.93 - 7.01 (m, 3 H), 7.23 - 7.36 (m, 4
H). MS (ESI) m/z: Calcd. for C24H26FN3O5 455.19 [M]+,
found 456.4 [M+H]+.
(S)-2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-4-methylpentanoic acid (20) 5-
(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (6 mL). To the solution was added benzotriazol-1-yl-
oxy-tris(dimethylamino)phosphonium hexafluorophosphate
(BOP) (213 mg, 0.482 mmol) and triethylamine (0.18 mL,
1.314 mmol). The resulting mixture was stirred at room tem-
perature for 15 minutes. (S)-Tert-butyl 3-amino-5-
methylhexanoate (97 mg, 0.482 mmol) in 0.5 mL of THF
was added dropwise, and stirred at room temperature for 2 h.
THF was evaporated in vacuo, water was added to the resi-
due and the aqueous layer was extracted with CH2Cl2 (3 x 15
mL). The combined organic layers were washed with water,
brine and then dried with Na2SO4, followed by filtration. The
solvent was evaporated in vacuo to give the crude residue.
The residue was purified by silica gel flash chromatography
(EtOAc:Hex) to give the title compound as white solid (145
mg, 63 %). Using the tert-butyl ester deprotection procedure
22, the title compound 20 was obtained from (S)-tert-butyl 3-
(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
(S)-6-Amino-2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)hexanoic acid
trifluoroacetate (18)
Following the tert-butyl ester
deprotection procedure 22, compound 18 was obtained from
9

carboxamido)-5-methylhexanoate. 81% yield; white solid; 1H
NMR (CDCl3 , 300 MHz) δ 0.96 (t, J=6.78 Hz, 6 H), 1.40 -
1.51 (m, 1 H), 1.62 - 1.82 (m, 2 H), 2.70 (d, J=5.27 Hz, 2
H), 3.52 (s, 3 H), 3.59 (m, 3 H), 4.47-4.60 (m, 1 H), 6.50 (d,
J=8.67 Hz, 2 H), 6.93 - 7.02 (m, 2 H), 7.22 - 7.35 (m, 5 H).
MS m/z: Calcd. for C25H28FN3O5 469.20 [M]+, found 470.6
[M+H]+.
1-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)cyclohexanecarboxylic acid (23)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (167 mg,
0.438 mmol) and triethylamine (0.30 mL, 2.19 mmol). The
resulting mixture was stirred at room temperature for 20
minutes. Ethyl 1-aminocyclohexane-1-carboxylate hydro-
chloride (100 mg, 0.482 mmol) was added and stirred at
room temperature for 2 h. THF was evaporated in vacuo,
water was added to the residue and the aqueous layer was
extracted with CH2Cl2 (3 x 20 mL). The combined organic
layers were washed with water, brine and then dried with
Na2SO4 .The solvent was evaporated in vacuo to give the
crude residue. The residue was purified by silica gel flash
chromatography (EtOAc:Hex) to give (S)-methyl 6-((tert-
butoxycarbonyl)amino)-2-(5-(2,6-dimethoxyphenyl)-1-(4-
(S)-3-Cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)propanoic
acid (21) 5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylic acid (2) (123 mg, 0.359 mmol) was
dissolved in THF (6 mL). To the solution was added BOP
(146 mg, 0.329 mmol) and triethylamine (0.150 mL, 1.077
mmol). The resulting mixture was stirred at room tempera-
ture for 15 minutes. (S)-Tert-butyl 3-amino-3-
cyclohexylpropanoate (75 mg, 0.329 mmol) was added and
stirred at room temperature for 2 h. THF was evaporated in
vacuo, water was added to the residue and the aqueous layer
was extracted with CH2Cl2 (3 x 20 mL). The combined or-
ganic layers were washed with water, brine and then dried
with Na2SO4 .The solvent was evaporated in vacuo to give
the crude residue. The residue was purified by silica gel flash
chromatography (EtOAc:Hex) to give (S)-tert-butyl 3-
cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-
1H-pyrazole-3-carboxamido)propanoate as colorless foam
(115 mg, 58 %). Using the tert-butyl ester deprotection pro-
cedure 22, the title compound 21 was obtained from (S)-tert-
fluorophenyl)-1H-pyrazole-3-carboxamido)hexanoate
white solid (145 mg, 67%). Using the lithium hydroxide
hydrolysis procedure 2, compound 23 was obtained from
as
ethyl
1-(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)cyclohexanecarboxylate. 83% yield;
white solid; 1H NMR (CDCl3 , 300 MHz) δ 1.29 - 1.46 (m, 2
H), 1.47 - 1.81 (m, 4 H), 1.95 - 2.13 (m, 2 H), 2.21-2.34 (m,
2 H), 3.59 (s, 6 H), 6.52 (d, J=8.29 Hz, 2 H), 6.95 - 7.02 (m,
2 H), 7.17 - 7.36 (m, 5 H). MS (ESI) m/z: Calcd. for
C25H26FN3O5 467.19 [M]+, found 468.5 [M+H]+.
Trans-4-(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)cyclohexanecarboxylic acid (24)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (7 mL). To the solution was added BOP (193 mg, 0.438
mmol) and triethylamine (0.182 mL, 1.314 mmol). The re-
sulting mixture was stirred at room temperature for 20
butyl
3-cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)propanoate. 31%
yield; white solid; 1H NMR (CDCl3, 300 MHz) δ 1.03 - 1.29
(m, 5 H), 1.60-1.94 (m, 6 H), 2.62-2.79 (m, 2 H), 3.56 (s, 3
H), 3.59 (s, 3 H), 4.22-4.31 (m, 1 H), 6.50 (d, J=9.0 Hz, 2
H), 6.93-6.99 (m, 2 H), 6.95 (s, 1 H), 7.23-7.41 (m, 4 H).
MS (ESI) m/z: Calcd. for C27H30FN3O5 495.22 [M]+, found
494.8 [M-H]-.
minutes.
Trans-methyl-4-aminocyclohexanecarboxylate
hydrochloride (93 mg, 0.482 mmol) was added and stirred at
room temperature for 2 h. THF was evaporated in vacuo,
water was added to the residue and the aqueous layer was
extracted with CH2Cl2 (3 x 20 mL). The combined organic
layers were washed with water, brine and then dried with
Na2SO4 .The solvent was evaporated in vacuo to give the
crude residue. The residue was purified by silica gel flash
chromatography (EtOAc:Hex) to give trans-methyl-4-(5-
(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)cyclohexane-1-carboxylate. Following the lithi-
um hydroxide hydrolysis procedure 2, compound 24 was
obtained from trans-methyl-2-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carboxamido)benzoate. 80%
yield; white solid; 1H NMR (CDCl3 , 300 MHz) δ 1.21 - 1.42
(m, 2 H), 1.52 - 1.75 (m, 2 H), 2.01 - 2.24 (m, 4 H), 2.26 -
2.39 (m, 1 H), 3.58 (s, 6 H), 3.92 - 4.08 (m, 1 H), 6.50 (d,
J=8.67 Hz, 2 H), 6.87 (d, J=8.67 Hz, 1 H), 6.93 - 7.01 (m, 3
H), 7.22 - 7.33 (m, 3 H). MS m/z: MS (ESI) m/z: Calcd. for
C25H26FN3O5 467.19 [M]+, found 468.5 [M+H]+.
(S)-5-Cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)pentanoic
acid (22) Following the procedure described in 17, (S)-Tert-
butyl
fluorophenyl)-1H-pyrazole-3-carboxamido)pentanoate (35)
was obtained using 5-(2,6-dimethoxyphenyl)-1-(4-
5-cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxylic acid (2) and tert-
butyl (S)-3-amino-5-cyclohexylpentanoate. Trifluoroacetic
acid (0.7 mL) was added dropwise to a solution of (S)-tert-
butyl5-cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)pentanoate (35)
(70 mg, 0.120 mmol) in CH2Cl2 (1.5 mL). The reaction mix-
ture was stirred at room temperature for 1.5 h and the solvent
was evaporated in vacuo. The residue was purified by silica
gel flash chromatography (EtOAc:Hex) to give the title com-
1
pound 22 as white solid. 80% yield; H NMR (CDCl3, 300
MHz) δ 0.77–0.98 (m, 2 H), 1.08-1.40 (m, 6 H), 1.57-1.78
(m, 7 H), 2.70 (d, J=5.46 Hz, 2 H), 3.57 (s, 3 H), 3.58 (s, 3
H), 4.32-4.44 (m, 1 H), 6.50 (d, J=8.10 Hz, 2 H), 6.90 - 7.05
(m, 3 H), 7.24 (d, J=5.09 Hz, 2 H), 7.27 - 7.36 (m, 2 H). MS
(ESI) m/z: Calcd. for C29H34FN3O5 523.60 [M]+, found
522.7 [M-H]-.
(S)-1-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carbonyl)pyrrolidine-3-carboxylic acid (25)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.305 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (174 mg,
10

0.305 mmol) and triethylamine (0.32 mL, 1.525 mmol). The
resulting mixture was stirred at room temperature for 15
minutes. L-Proline methyl ester hydrochloride (83 mg, 0.335
mmol) was added and stirred at room temperature for 2 h.
THF was evaporated in vacuo, water was added to the resi-
due and the aqueous layer was extracted with CH2Cl2 (3 x 20
mL). The combined organic layers were washed with water,
brine and then dried with Na2SO4 .The solvent was evapo-
rated in vacuo to give the crude residue. The residue was
purified by silica gel flash chromatography (EtOAc:Hex) to
purified by silica gel flash chromatography (EtOAc:Hex) to
give (S)-methyl 2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)-3-
methylbutanoate as white solid (135 mg, 68 %). Following
the trimethyltin hydroxide hydrolysis procedure 3, compound
27
was
obtained
from
(S)-methyl
2-(5-(2,6-
dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)-3-methylbutanoate. 62 % yield; white solid.
1H NMR (MeOH-d4 , 300 MHz) δ 1.04 (t, J=6.59 Hz, 6 H),
2.24 - 2.38 (m, 1 H), 3.61 (s, 6 H), 4.55 (d, J=5.27 Hz, 1 H),
6.62 (d, J=8.67 Hz, 2 H), 6.80 (s, 1 H), 6.99 - 7.10 (m, 2 H),
7.25 - 7.38 (m, 3 H). MS (ESI) m/z: Calcd. for C23H24FN3O5
441.17 [M]+, found 442.3 [M+H]+.
give
(S)-methyl
1-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carbonyl)pyrrolidine-3-
carboxylate as white solid (115 mg, 58 %). Using the
trimethyltin hydroxide hydrolysis procedure 3, compound 25
was obtained from (S)-methyl 1-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carbonyl)pyrrolidine-3-
2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)benzoic acid (28)
1
carboxylate. 45 % yield; white solid. H NMR (MeOH-d4 ,
5-(2,6-
300 MHz) δ 1.86 - 2.14 (m, 1 H), 2.16 - 2.48 (m, 1 H), 3.54
(s, 3 H), 3.60 (s, 3 H), 3.69 - 3.90 (m, 2 H), 4.15 - 4.24 (m, 1
H), 4.56-4.67 (m, 1 H), 5.35 (dd, J=8.67, 2.64 Hz, 1 H), 6.54
- 6.68 (m, 2 H), 6.81 (s, 1 H), 6.94 - 7.10 (m, 2 H), 7.21 -
7.40 (m, 3 H). MS (ESI) m/z: Calcd. for C23H22FN3O5
439.15 [M]+, found 440.4 [M+H]+.
Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (100 mg, 0.292 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (111 mg,
0.292 mmol) and triethylamine (0.122 mL, 0.876 mmol).
The resulting mixture was stirred at room temperature for 20
minutes. Methyl 2-aminobenzoate trifluoroacetate (85 mg,
0.321 mmol) was added and stirred at room temperature for
18 h. THF was evaporated in vacuo, water was added to the
residue and the aqueous layer was extracted with CH2Cl2 (3
x 20 mL). The combined organic layers were washed with
water, brine and then dried with Na2SO4.The solvent was
evaporated in vacuo to give the crude residue. The residue
was purified by silica gel flash chromatography (EtOAc:Hex)
2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)adamantane-2-carboxylic acid
(26)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylic acid (2) was heated under reflux in
SOCl2 for 2 hr followed by cooling and concentration in vac-
uo. 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 eq 2-
aminoadamantane-carboxylic acid and 2.2 eq of NaOH at 5
°C. Following the addition, the stirring was continued for 18
hr at ambient temp. After this time, the mixture is made acid-
ic with 1 N HCl and extracted (3×) with CH2Cl2, dried over
sodium sulfate and evaporated. The residue was chromato-
graphed using a 0-15% CH2Cl2:MeOH gradient. Evaporation
provided the title compound 26. 71% yield; off-white solid;
1H NMR (CDCl3) δ 7.20-7.36 (m, 4 H), 6.90-7.04 (m, 3 H),
6.51 (d, J = 8.5 Hz, 2 H), 3.54-3.63 (m, 6 H), 2.71-2.81 (m,
2 H), 2.23 (d, J = 12.8 Hz, 2 H), 2.07 (d, J = 12.2 Hz, 2 H),
1.68-1.95 (m, 8 H). MS (ESI) m/z: Calcd. for C29H30FN3O5
519.22 [M]+, found 518.9 (M-H)-.
to
give
methyl
2-(5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxamido)benzoate as white
solid (18 mg, 13%). Using the lithium hydroxide hydrolysis
procedure 2, compound 28 was obtained from methyl 2-(5-
(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxamido)benzoate. 70% yield; white solid; 1H NMR
(CDCl3 , 300 MHz) δ 3.59 (s, 6 H), 6.54 (d, J=6.10, 2 H),
7.00 (t, J=6.10, 2 H), 7.05 (s, 1 H), 7.14 - 7.21 (m, 1 H),
7.31 - 7.41 (m, 3 H), 7.62 - 7.71 (m, 1 H), 8.10 (dd, J=8.10,
1.70 Hz, 1 H), 9.00 (dd, J=8.67, 0.75 Hz, 1 H), 12.38 (s, 1
H). MS (ESI) m/z: Calcd. for C25H20FN3O5 461.14 [M]+,
found 462.6 [M+H]+.
(S)-2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-2-phenylacetic acid (29) 5-(2,6-
Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (167 mg,
0.438 mmol) and triethylamine (0.30 mL, 2.191 mmol). The
resulting mixture was stirred at room temperature for 20
minutes. (S)-(+)-2-phenylglycine methyl ester hydrochloride
(90 mg, 0.482 mmol) was added and stirred at room temper-
ature for 2 h. THF was evaporated in vacuo, water was added
to the residue and the aqueous layer was extracted with
CH2Cl2 (3 x 20 mL). The combined organic layers were
washed with water, brine and then dried with Na2SO4 .The
solvent was evaporated in vacuo to give the crude residue.
The residue was purified by silica gel flash chromatography
(EtOAc:Hex) to give (S)-methyl 2-(5-(2,6-dimethoxyphenyl)-
1-(4-fluorophenyl)-1H-pyrazole-3-carboxamido)-2-
(S)-2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-3-methylbutanoic acid (27) 5-
(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (150 mg, 0.438 mmol) was dissolved in
THF (7 mL). To the solution was added HATU (167 mg,
0.438 mmol) and triethylamine (0.30 mL, 0.2.19 mmol). The
resulting mixture was stirred at room temperature for 20
minutes. L-Valine methyl ester hydrochloride (80 mg, 0.482
mmol) was added and stirred at room temperature for 3 h.
THF was evaporated in vacuo, water was added to the resi-
due and the aqueous layer was extracted with CH2Cl2 (3 x 20
mL). The combined organic layers were washed with water,
brine and then dried with Na2SO4 .The solvent was evapo-
rated in vacuo to give the crude residue. The residue was
11

phenylacetate as white solid (145 mg, 68 %). Using the
trimethyltin hydroxide hydrolysis procedure 3, compound 29
was obtained from (S)-methyl 2-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carboxamido)-2-
Sodium salt of methyl 4-(2,6-dimethoxyphenyl)-2,4-
dioxobutanoate (33) Following the procedure as reported
in the thesis titled – Quéré, L.
Etude de l`interaction ligand-récepteur neurotensinergique.
University of Namur. 1995
phenylacetate. 60 % yield; white solid. 1H NMR (MeOH-d4 ,
300 MHz) δ 3.60 (s, 6 H), 5.51-5.59 (m, 1 H), 6.61 (d,
J=8.67 Hz, 2 H), 6.78 (s, 1 H), 7.00 - 7.09 (m, 2 H), 7.22 -
7.40 (m, 6 H), 7.51 (d, J=7.54 Hz, 2 H). MS (ESI) m/z:
Calcd. for C26H22FN3O5 475.15 [M]+, found 476.5 [M+H]+.
Methyl 5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxylate (34a) Sodium salt of methyl 4-
(2,6-dimethoxyphenyl)-2,4-dioxobutanoate (33) (1.5 g, 5.204
mmol) and commercially available 4-fluorophenylhydrazine
hydrochloride (0.85 g, 5.204 mmol) was mixed with glacial
acetic acid (25 mL) and conc. HCl (0.6 mL). The reaction
mixture was heated to reflux for 3.5 h. After cooling, reaction
mixture was poured into water (25 mL). The aqueous layer
was extracted with CH2Cl2 (3 x 30 mL) and the combined
CH2Cl2 layer was washed with saturated aqueous NaHCO3.
The organic layer was then washed with saturated brine,
dried over Na2SO4, followed by filteration. The solvent was
evaporated in vacuo to give crude residue. The residue was
purified by silica gel flash chromatography (EtOAc:Hex) to
give desired isomer methyl 5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxylate (34a) as light yel-
low solid (1.1 g, 61 %). Methyl 3-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-5-carboxylate (34b) was ob-
(S)-2-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-3-phenylpropanoic acid (30)
The title compound 30 was synthesized on a wang resin by
standard protocol. Wang resin (100 mg, 1.11 mmol/ g, 0.11
mmol) was swollen in CH2Cl2/DMF (8:2) for 30 minutes,
and the amino acid was coupled to the resin by use of Fmoc-
L-Phe (5.0 equiv), HBTU (2.0 equiv), and HOBt (2.0 equiv)
in DMF with a reaction time of 1 h. The Fmoc group was
cleaved with piperidine in DMF (20 %). After deprotection
of the Fmoc group, 5-(2,6-dimethoxyphenyl)-1-(4-
fluorophenyl)-1H-pyrazole-3-carboxylic acid (2) was coupled
using HBTU (2.0 equiv), and HOBt (2.0 equiv) in DMF
with a reaction time of 1 h. Cleavage of the product from the
resin
was
achieved
by
treatment
with
TFA/H2O/triisopropylsilane (95:2.5:2.5) for 1 h and precipi-
tation by adding cold ether to the solution. The solid title
compound was filtered and washed with cold ether and dried
(21 mg). white solid. 1H NMR (MeOH-d4 , 300 MHz) δ 3.13
- 3.20 (m, 1 H), 3.59 (s, 6 H), 4.76 - 4.92 (m, 2 H), 6.61 (d,
J=8.29 Hz, 2 H), 6.75 (s, 1 H), 6.95 - 7.09 (m, 2 H), 7.16 -
7.29 (m, 7 H), 7.35 (t, J=8.48 Hz, 2 H). MS (ESI) m/z:
Calcd. for C27H24FN3O5 489.17 [M]+, found 490.6 [M+H]+.
1
tained as as light yellow foam (120 mg). H NMR (CDCl3 ,
300 MHz) δ 3.59 (s, 6 H), 3.96 (s, 3 H), 6.50 (d, J=9.0 Hz, 2
H), 6.90-6.97 (m, 2 H), 6.96 (s, 1H), 7.34-7.24 (m, 3 H).
MS (ESI) m/z: Calcd. for C19H17FN2O4 356.12 [M]+, found
357.4 [M+H]+.
(S)-Tert-butyl 5-cyclohexyl-3-(5-(2,6-dimethoxyphenyl)-1-
(4-fluorophenyl)-1H-pyrazole-3-carboxamido)pentanoate
(35)
5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
(S)-3-(5-(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-
pyrazole-3-carboxamido)-5-phenylpentanoic acid (31) 5-
(2,6-Dimethoxyphenyl)-1-(4-fluorophenyl)-1H-pyrazole-3-
carboxylic acid (2) (45 mg, 0.131 mmol) was dissolved in
THF (6 mL). To the solution was added BOP (69 mg, 0.157
mmol) and triethylamine (0.05 mL, 0.327 mmol). The result-
ing mixture was stirred at room temperature for 15 minutes.
(S)-Tert-butyl 3-amino-5-phenylpentanoate (34 mg, 0.144
mmol) was added and stirred at room temperature for 3 h.
THF was evaporated in vacuo, water was added to the resi-
due and the aqueous layer was extracted with CH2Cl2 (3 x 20
mL). The combined organic layers were washed with water,
brine and then dried with Na2SO4 .The solvent was evapo-
rated in vacuo to give the crude residue. The residue was
purified by silica gel flash chromatography (EtOAc:Hex) to
pyrazole-3-carboxylic acid (2) (1g, 3.286 mmol) was dis-
solved in acetonitrile (30 mL). To the solution was added
HBTU (1.75 g, 4.63 mmol) and tert-butyl (S)-3-amino-5-
cyclohexylpentanoate (0.88 g, 3.450 mmol) followed by
dropwise addition of triethylamine (0.911 mL, 6.572 mmol).
The resulting mixture was stirred at room temperature for 3
h. Reaction mixture was quenched with saturated NaHCO3
solution (10 mL) and acetonitrile was removed in vacuo.
Ethyl acetate was added to the residue and washed with satu-
rated NaHCO3 solution. The aqueous layer was re-extracted
with EtOAc (2 x 30 mL). The combined organic layers were
washed with brine and then dried with Na2SO4, followed by
filtration. The solvent was evaporated in vacuo to give the
crude residue. The residue was purified by silica gel flash
chromatography (EtOAc:Hex) to give the title compound as
give
(S)-tert-butyl
3-(5-(2,6-dimethoxyphenyl)-1-(4-
1
white solid (1.5 g, 79 %). H NMR (CDCl3 , 300 MHz) δ
fluorophenyl)-1H-pyrazole-3-carboxamido)-5-
0.80-0.96 (m, 2 H), 1.11-1.88 (m, 6 H), 1.46 (s, 9 H), 1.58 -
1.76 (m, 7 H), 2.54 (dd, J=5.46, 1.41 Hz, 2 H), 3.56 (s, 3 H),
3.59 (s, 3 H), 4.35-4.47 (m, 1 H), 6.50 (d, J=7.72 Hz, 2 H),
6.89 - 7.03 (m, 3 H), 7.22 -7.26 (m, 2 H), 7.27-7.37 (m, 2
H). MS (ESI) m/z: Calcd. for C33H42FN3O5 579.70 [M]+,
found 580.7 [M+H]+.
phenylpentanoate. Using the tert-butyl ester deprotection
procedure 22, the title compound 31 was obtained from (S)-
tert-butyl
3-(5-(2,6-dimethoxyphenyl)-1-(4-fluorophenyl)-
77%
1H-pyrazole-3-carboxamido)-5-phenylpentanoate.
1
yield; white solid; H NMR (CDCl3, 300 MHz) δ 1.92–2.16
(m, 2 H), 2.67-2.84 (m, 2 H), 2.74 (d, J=5.46 Hz, 2 H), 3.57
(s, 3 H), 3.59 (s, 3 H), 4.46-4.56 (m, 1 H), 6.51 (d, J=8.10
Hz, 2 H), 6.86 - 7.05 (m, 3 H), 7.13 - 7.25 (m, 5 H), 7.27 -
7.35 (m, 3 H), 7.44 (d, J=8.67 Hz, 1 H). MS (ESI) m/z:
Calcd. for C29H28FN3O5 517.55 [M]+, found 516.8 [M-H]-.
5.2 X-ray Crystallographic Analysis
X-ray Crystallographic Analysis. Single-crystal x-ray diffrac-
tion data on compound (2) was collected using MoKα radia-
12

tion and a Bruker APEX 2 CCD area detector. The crystal
was prepared for data collection by coating with high vis-
cosity microscope oil. The oil coated crystal was mounted on
a micromesh mount (Mitergen, Inc.) and transferred to the
diffractometer, and a data set was collected at 150 K. The
0.529 × 0.422 × 0.368 mm3 crystal was triclinic in space
group P-1, with unit cell dimensions a = 8.550(5), b =
12.619(7), c = 16.694(10) Å, a = 91.803(9)°, β = 95.702(8)°,
and g = 109.750(8)°. Data was 96.3% complete to 25.53°θ
(∼0.82 Å) with an average redundancy of 2.12. The data
crystal was twinned with three components in a 14.6:3.9:0.9
ratio (based on mean I/s), and only non-overlapped reflec-
tions from the major twin component were used in structure
solution and refinement. The structure was solved by direct
methods and refined by full-matrix least-squares on F2 val-
ues using the programs found in the SHELXTL suite
(Bruker, SHELXTL v6.10, 2000, Bruker AXS Inc., Madison,
WI). Corrections were applied for Lorentz, polarization, and
absorption effects. Parameters refined included atomic coor-
dinates and anisotropic thermal parameters for all non-
hydrogen atoms. Hydrogen atoms on carbons were included
using a riding model [coordinate shifts of C applied to H
atoms] with C−H distance set at 0.96 Å. The final aniso-
tropic full matrix least-squares refinement on F2 with 463
variables and two restraints converged at R1 = 6.81% for the
observed data and wR2 = 19.00% for all data. Atomic coor-
dinates for (2) have been deposited with the Cambridge
Crystallographic Data Centre (deposition number 1048741).
Copies of the data can be obtained, free of charge, on appli-
cation to CCDC, 12 Union Road, Cambridge, CB2 1EZ, UK
[fax:+44(0)-1223-336033 or e-mail: depos-
EC50, correlation coefficient (r2), and other parameters. All
experiments were performed in duplicate at least three times
to ensure reproducibility and data reported as mean
standard error, unless noted otherwise.
Assays for rat neurotensin receptors 1 and 2 (NTR1 and
NTR2) followed a very similar format and have been de-
scribed.47,48
5.4 Radioligand displacement
Competitive inhibition binding was performed using
[
125I]-apelin-13 (Perkin Elmer, Akron, OH). Eleven concen-
trations (ranging from 10-11 M to 3.16X10-5 M) of compound
were incubated in assay buffer (0.025 M HEPES, (pH=7.4),
0.01 M MgCl2, 0.001 M CaCl2, 0.5% fatty acid-free BSA)
with 7.5X10-11 M [125I]-apelin-13 (Perkin-Elmer) in the pres-
ence of 0.125 µg of CHO-K1 cell membrane homogenates
expressing the human apelin receptor (Perkin Elmer). Non-
specific binding (NSB) was determined in the presence of 10-
6
M unlabeled apelin-13 (Anaspec). The assays were run in
1.4 mL polypropylene tubes (Thermo Scientific, Waltham,
MA) in a 96-well format. All assays were incubated at room
temperature on a shaking platform for two hours. Incubation
was terminated and bound radioligand was separated from
free radioligand by rapid vacuum filtration over 0.5% PEI
treated GF/C filter plates using ice-cold 50 mM Tris-HCl
(pH=7.4) in a Brandel Scientific (Gaithersburg, MD) 96-well
harvester. Filter plates were allowed to dry and Microscint
20 scintillation cocktail (Perkin-Elmer) was added to each
well. Bound radioactivity was determined with a TopCount
12-detector instrument (Packard Instruments) using standard
scintillation counting techniques. The binding data from each
assay plate were normalized to NSB and total counts per
minute (CPM) bound values prior to analysis. The Ki values
were calculated from a three-parameter logistic curve fit to
the data with Prism (version 6.0, GraphPad Software, Inc.,
San Diego, CA) using 2.1X10-10 M as the Kd for [125I]-
apelin-13.
it@ccdc.cam.ac.uk
5.3 Calcium mobilization assay for APJ, NTR1 and
NTR2
Cells stably expressing human APJ-Gαq16 were removed
from the flasks using a non-enzymatic solution (Cell-
stripper, Mediatech Inc., Orlando, FL) and quenched with
DMEM/F12, 10 % FBS, centrifuged, and re-suspended in
the serum-containing media. Cells were counted with a
hemocytometer and 30,000 cells were transferred to each
well of a black Costar 96-well optical bottom plate (Corning
Corporation, Corning, NY). Each plate was incubated at 37
C for 24 h to confluence. The culture media was removed
from the plates and cells were subsequently loaded with a
ASSOCIATED CONTENT
AUTHOR INFORMATION
Corresponding Author
̊
* E-mail: srunyon@rti.org. Tel: 919-316-3842. Fax: 919 541
8868.
fluorescent calcium probe (Calcium 5 dye; Molecular Devic-
es, Sunnyvale, CA) in an HBSS-based buffer containing 20
mM HEPES, 1% BSA and 10 µM probenecid (Sigma) in a
Funding Sources
total volume of 225 µL. Cells were incubated at 37 ̊ C for 1 h
This work was supported NICHD grant 1R01HD079547-
01A1 to Rangan Maitra and Scott Runyon.
and then stimulated with various compounds or pyr-apelin-
13 (Anaspec, Freemont, CA) at various concentrations using
a FLIPR Tetra plate-reader, which automatically added the
agonist at 10x concentration to each well after reading
baseline values for ~17 sec. Agonist-mediated change in
fluorescence (488 nm excitation, 525 nm emission) was
monitored in each well at 1 sec intervals for 90 sec. Data
were collected using Softmax version 4.8 (MDS Analytical
Technologies) and analyzed using Prism software
(GraphPad, La Jolla, CA). Nonlinear regression analysis was
performed to fit data and obtain maximum response (Emax),
ACKNOWLEDGMENT
The authors thank Dr. Angela Giddings, for help with vari-
ous assays and to Dr. Danni Harris for detailed discussion of
data. The X-ray crystallographic work was supported by
NIDA through Interagency Agreement #Y1-DA1101 with
the Naval Research Laboratory (NRL).
13

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