Pyrazole Agonist of the Apelin Receptor Improves Symptoms of Metabolic Syndrome in Mice

Article abstract of DOI:10.1021/acs.jmedchem.0c01448

Apelin receptor agonism improves symptoms of metabolic syndrome. However, endogenous apelin peptides have short half-lives, making their utility as potential drugs limited. Previously, we had identified a novel pyrazole-based agonist scaffold. Systematic modification of this scaffold was performed to produce compounds with improved ADME properties. Compound 13 with favorable agonist potency (cAMPi EC50 = 162 nM), human liver microsome stability (T1/2 = 62 min), and pharmacokinetic profile in rodents was identified. The compound was tested in a mouse model of diet-induced obesity (DIO) and metabolic syndrome for efficacy. Treatment with 13 led to significant weight loss, hypophagia, improved glucose utilization, reduced liver steatosis, and improvement of disease-associated biomarkers. In conclusion, a small-molecule agonist of the apelin receptor has been identified that is suitable for in vivo investigation of the apelinergic system in DIO and perhaps other diseases where this receptor has been implicated to play a role.

Full text of DOI:10.1021/acs.jmedchem.0c01448

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Article
Pyrazole Agonist of the Apelin Receptor Improves Symptoms of
Metabolic Syndrome in Mice
Sanju Narayanan, Shaobin Wang, Vineetha Vasukuttan, Ravi Kumar Vyas Devambatla, Donghua Dai,
Chunyang Jin, Rodney Snyder, Lucas Laudermilk, Scott P. Runyon,* and Rangan Maitra*
Cite This: J. Med. Chem. 2021, 64, 3006−3025
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ABSTRACT: Apelin receptor agonism improves symptoms of metabolic syndrome. However, endogenous apelin peptides have
short half-lives, making their utility as potential drugs limited. Previously, we had identified a novel pyrazole-based agonist scaffold.
Systematic modification of this scaffold was performed to produce compounds with improved ADME properties. Compound 13
with favorable agonist potency (cAMPi EC50 = 162 nM), human liver microsome stability (T1/2 = 62 min), and pharmacokinetic
profile in rodents was identified. The compound was tested in a mouse model of diet-induced obesity (DIO) and metabolic
syndrome for efficacy. Treatment with 13 led to significant weight loss, hypophagia, improved glucose utilization, reduced liver
steatosis, and improvement of disease-associated biomarkers. In conclusion, a small-molecule agonist of the apelin receptor has been
identified that is suitable for in vivo investigation of the apelinergic system in DIO and perhaps other diseases where this receptor has
been implicated to play a role.
of the apelin receptor have been under-reported in the
literature.5−11 Furthermore, the ones disclosed have little to
no brain penetration. Both apelin and APJ are expressed in the
central nervous system (CNS) and peripherally.12,13 Strong
expression of APJ is noted in the vasculature within endothelial
cells but expression of APJ mRNA in other types of cells outside
of the vasculature has also been reported.13,14 Within the CNS,
the receptor is expressed in the hypothalamus and other regions
that are associated with hunger, satiety, motivation, and reward
processing.15,16 Therefore, central regulation of this receptor
system could well be important beyond peripheral actions
within the context of metabolic diseases.
INTRODUCTION
■
Metabolic syndrome associated with obesity is a major problem
in the developed world including the United States. Metabolic
syndrome leads to type 2 diabetes, steatohepatitis, and
cardiovascular diseases; these diseases together have a high
mortality rate.1 Therefore, new medications to treat metabolic
syndrome are needed. The apelin receptor has emerged as a
novel target for various important diseases including metabolic
syndrome.2 This class A GPCR is activated by two distinct
classes of peptidesapelin and ELABELA. Activation of the
receptor initiates downstream signaling primarily through the
Gαi-dependent pathway, which inhibits cyclic adenosine
monophosphate (cAMP) production and activates the extrac-
ellular signal-regulated kinase (ERK) signaling cascade.
Termination of receptor signaling is through internalization of
the apelin receptor via β-arrestin 1/2 recruitment.3,4 These
endogenous peptides of the apelin receptor are labile and not
suitable for chronic dosing in patients suffering from metabolic
syndrome and other diseases that require repeated admin-
istration of a therapeutic agent.4 To date, small-molecule ligands
Received: August 20, 2020
Published: March 11, 2021
https://dx.doi.org/10.1021/acs.jmedchem.0c01448
© 2021 American Chemical Society
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Studies on the antiobesity and antidiabetic properties of the
apelinergic system present a very intriguing picture.17,18 Apelin
is an adipokine that is produced and secreted by adipocytes.19
Several reports indicate that circulating apelin levels are higher in
obese and diabetic patients (reviewed in17) but not necessarily
in patients with type 2 diabetes or gestational diabetes. These
data indicate that hyperactivation of the apelinergic system is a
possible consequence of obesity. Apelin is also an insulin
sensitizer. Apelin lowered insulin secretion alone20 or upon
glucose stimulation21 and improved glucose utilization in
skeletal muscle cells of high-fat diet (HFD)-fed mice.22,23
Apelin treatment also decreased body adiposity and serum levels
of insulin and triglycerides in obese HFD-fed mice.24 Apelin
increased the serum adiponectin level and decreased leptin.24
Furthermore, increased muscle fatty acid (FA) oxidation in
response to apelin stimulation has been also described.25 Loss of
function studies support these observations. Apelin knockout
(KO) mice on regular chow exhibit significantly higher insulin
levels and glucose intolerance with increased abdominal fat and
higher bodyweight than littermates (Figure 1). Administration
In agreement with these data, apelin KO mice have vessels that
are more amenable to FA transport.31 Therefore, apelin KO
animals on HFD with abnormal lymphatic and blood vessel
enlargement are obese compared to littermates with normal
apelin expression. Another report has indicated that apelin
transgenic mice are resistant to diet-induced obesity (DIO) by
virtue of increased vascular mass and mitochondrial biogenesis
in skeletal muscles.32
In addition to these data, APJ activation by apelin has many
beneficial effects on the cardiovascular system, which is a major
problem in a large number of patients with metabolic syndrome.
Every APJ KO line described to date suffers from heart failure
due to pressure overload at an advanced age. They also
demonstrate reduced angiogenic potential and develop vascular
abnormalities.29,30,33,34 Furthermore, reports indicate that
apelin promotes recovery from ischemia, which is a hypoxic
and vaso-occlusive disorder.29 In reported clinical studies, acute
Pyr-apelin-13 administration caused peripheral and coronary
vasodilatation and increased cardiac output in human patients
with chronic heart failure and also induced nitric oxide-
dependent arterial vasodilation in healthy human volunteers
without any adverse effects.35−37 Interestingly, there are also
reports suggesting that apelin has a positive ionotropic effect in
the heart.38,39 Taken together, APJ is a novel multimodal target
for metabolic syndrome that is capable of diminishing insulin
resistance, is antihypertensive, and has an inhibitory effect on
DIO.36,37
Recently, we discovered and reported a potent pyrazole
agonist 2 of the apelin receptor (cAMPi EC50 = 238 nM, Figure
2) through focused screening, which was further optimized to 3
(cAMPi EC50 = 97 nM).11 However, compound 3 was not
suitable for in vivo studies since it was rapidly metabolized
(human liver microsome stability (HLM) T1/2 = 6.25 min,
CLint = 199 mL/min/kg), had poor solubility (<1 μM at pH
7.4), and was still too lipophilic. A small-molecule agonist that is
suitable for in vivo investigation in DIO and possibly in animal
models of other diseases modulated by the apelin receptor was
needed.
RESULTS AND DISCUSSION
■
To improve pharmacokinetic (PK) properties of compound 3
(Figure 2) and to identify an in vivo metabolically stable
compound, we started our SAR campaign first by replacing the
lipophilic cyclohexyl moiety of compound 2. Compounds with
substituted alkyl/cycloalkyl amines replacing the cyclohexyl
moiety in 2 were synthesized first (Table 1) to generate
preliminary structure−activity relationships (SARs) and gain an
understanding of receptor tolerances associated with these
modifications and their effects on potency and efficacy. Alkyl
and cycloalkyl amines provide a basic nitrogen that not only
increases polarity but also improves solubility of compounds as
hydrochloride salts. The agonist activity of synthesized
analogues was evaluated using inhibition of forskolin-induced
cAMP accumulation. In addition, recruitment of β-arrestin 2 was
also evaluated as a measure of the desensitization potential of
compounds.
At first, noncyclic diethyl amine-substituted 4 was synthesized
and evaluated to monitor the influence of alkyl amine
substitution on agonist potency (Table 1). The diethyl amine-
substituted analogue 4 (cAMPi EC50 = 1365 nM) exhibited
approximately sixfold reduced potency compared to the
cyclohexyl analogue 2 (cAMPi EC50 = 238 nM). However,
five-membered closed pyrrolidine ring analogue 5 was
Figure 1. Role of an apelinergic system in DIO and metabolic
syndrome. Apelin transgenic (TG) animals are insulin-sensitive and
resistant to HFD-induced obesity compared to apelin knockout
animals. APJ activation by apelin leads to the formation of large
nonleaky vessels that combat hypertension and free FA transport.
Apelin blocks differentiation of preadipocytes to adipocytes and
improves glucose uptake in skeletal muscles. It also increases vascular
mass.
of sucrose to KO animals exacerbates this phenotype, but these
effects can be reversed by infusion of animals with Pyr-apelin-
13.26,27 Additional studies indicate that apelin, through its
interaction with APJ receptor, can inhibit adipogenesis of
preadipocytes and lipolysis in mature adipocytes (Figure 1).28
Interestingly, both apelin and APJ are strongly expressed in
endothelial cells. Endothelial cells play a key role in regulating
FA transport and this process is affected by apelin and APJ.
Activation of APJ by apelin leads to the formation of large,
nonleaky lymphatic and blood vessels29,30 that restrict the
transport of FA and their uptake in adipose tissues (Figure 1).31
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Figure 2. Chemical structure of initial lead 2 identified through focused screen and optimized to lead compound 3.
Table 1. Modification of Cyclohexyl Moiety of 2
*
EC50 values are averages of 2−3 experiments performed in duplicate unless otherwise stated standard error of the mean.
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Table 2. Modification of the Cyclohexyl Moiety of 3
*
EC50 values are averages of 2−3 experiments performed in duplicate unless otherwise stated standard error of the mean, NCnot calculated as
no compound 12 peak detected in wells, NDnot done, HLMhuman liver microsome stability, T1/2half-life, and CLclearance.
equipotent (cAMPi EC50 = 398 nM) compared to 2 (cAMPi
EC50 = 238 nM) indicating a preference for a cyclic ring
structure for enhanced agonist potency. Increasing the hydro-
phobic volume to six carbon piperidine 6, however, reduced the
potency (cAMPi EC50 = 2110 nM) to ∼9-fold. This diminished
potency was then rescued by the introduction of heteroatoms
(O and N-Me) at the para position of the piperidine.
Compounds 7 (cAMPi EC50 = 703 nM) and 8 (cAMPi EC50
= 805 nM) with morpholine and 4-methyl piperazine,
respectively, exhibited ∼threefold improved potency compared
to the piperidine analogue 6 (cAMPi EC50 = 2110 nM).
Similarly, 2,6-dimethyl-substituted piperidine 9 was less potent
(cAMPi EC50 = 2112 nM) indicating reduced receptor tolerance
with compounds having six-membered heterocyclic ring
systems. The cyclohexyl moiety in 2 was also replaced with a
seven-membered bicyclic moiety, 7-azabicyclo[2.2.1]heptane
10, to monitor the effect of steric bulk on agonist activity.
However, the 7-azabicyclo[2.2.1]heptane substitution was not
tolerated and reduced agonist potency (cAMPi EC50 = 7562
nM). Interestingly, the seven-membered azepane ring (11)
exhibited enhanced agonist potency (cAMPi EC50 = 181 nM)
compared to the cyclohexyl-substituted 2 indicating a
preference for seven-membered hydrophobic cycloalkyl ring
systems. Overall, five- or seven-membered cyclic heterocyclic
rings (pyrrolidine and azepane) were well-tolerated and
exhibited comparable or enhanced agonist potency relative to
compound 2. However, six-membered cycloalkyl derivatives (6,
7, 8, and 9) exhibited reduced receptor tolerance and variable
agonist potencies. β-Arrestin recruitment of these compounds
was also investigated as a potential measure of desensitization.40
Additionally, literature evidences also suggest a correlation
between β-arrestin signaling and hypotensive effect in vivo.41,42
Several compounds among 4−11 exhibited weak activity in the
β-arrestin recruitment assay, with some showing no appreciable
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recruitment at 10 μM, suggesting the possibility of low receptor
internalization and desensitization with these analogues. This
could improve the duration of action of these compounds and
might be advantageous in a chronic disease setting requiring
reduced or absent tolerance to an administered agent.
Table 3. Modification of the Carboxylic Acid Moiety of 6
As noted earlier in Figure 2, conversion of cyclohexyl
carboxylic acid 2 (cAMPi EC50 = 238 nM) to cyclobutyl
amide 3 (cAMPi EC50 = 97 nM) enhanced the potency of the
starting carboxylic acid scaffold. To monitor similar effects with
the cycloalkyl amine-substituted cyclobutylamide scaffold,
compounds 12−18 (Table 2) were synthesized. As previously
observed (Figure 2), the five-membered cyclic pyrrolidine
cyclobutylamide 12 (cAMPi EC50 = 167 nM) exhibited twofold
enhanced potency relative to its carboxylic acid precursor 5
(cAMPi EC50 = 398 nM). Furthermore, increasing the
hydrophobic volume to six-membered piperidine 13 (cAMPi
EC50 = 162 nM) resulted in ∼13-fold enhanced potency
compared to its carboxylic acid counterpart 6 (cAMPi EC50
=
2110 nM), indicating enhanced receptor interaction through
cyclobutylamide substitution. However, compound 14 with a
morpholine (cAMPi EC50 = 237 nM) did not show much
improvement in potency relative to the carboxylic acid derivative
7 (cAMPi EC50 = 703 nM). Also, conversion of N-methyl
piperazine carboxylic acid 8 (cAMPi EC50 = 805 nM) to
cyclobutyl amide 15 (cAMPi EC50 = 721 nM) did not enhance
potency suggesting that the presence of an additional H-bond
acceptor did not enhance receptor interaction. Interestingly, 2,6-
dimethyl-substituted piperidine 16 (cAMPi EC50 = 33 nM, ∼64-
fold), 7-azabicyclo[2.2.1]heptane 17 (cAMPi EC50 = 363 nM,
21-fold), and seven-membered azepane cyclobutylamide 18
(cAMPi EC50 = 8 nM, ∼23-fold) substitutions significantly
enhanced potency compared to their carboxylic acid precursors.
β-Arrestin recruitment of these compounds was also inves-
tigated. Compounds with piperidine (13, βarr EC50 = 705 nM),
2,6-dimethyl piperidine (16, βarr EC50 = 459 nM), and azepane
(18, βarr EC50 = 159 nM) also recruited β-arrestin with an EC50
value < 1 μM (Table 2). Since our goal was to develop potent
and metabolically stable compounds suitable for in vivo study,
the stability of select potent compounds 13, 14, 16, and 18 was
evaluated in the HLM stability assay (Table 2). Compound 13
exhibited the most favorable HLM stability (T1/2 = 62 min) and
clearance (CLINT = 20 mL/min/kg) suggesting the importance
of the piperidine moiety for improved metabolic stability.
Therefore, this compound was selected for further evaluation
versus the more potent compounds.
Since compound 13 with a piperidine moiety exhibited the
most favorable HLM stability, we continued with this scaffold
for our next round of optimization to identify compounds with
improved potency and metabolic stability. Diverse polar and
nonpolar substituted amides and nonamide derivatives were
synthesized to monitor the effect in agonist potency and
metabolic stability of these compounds (Table 3). Compound
19 with cyclobutyl ester (Table 3) was synthesized to explore
the importance of the amide nitrogen in 13 for agonist potency.
Substituting NH with O did not reduce the agonist potency
suggesting that the amide nitrogen may not be critical for
preserving the agonist activity. Considering the metabolic
liability of the cyclobutylamide moiety, we synthesized
compounds 20 (3, 3-diflurocyclobutylamide), 21 (N-methyl
cyclobutylamide), 22 (1-methylcyclobutylamide), and 23 (1-
methyloxetanamide) that blocked the possible metabolic sites
with the incorporation of heteroatoms (F and O) and a methyl
group. Compounds 20 (cAMPi EC50 = 9 nM) and 21 (cAMPi
*
EC50 values are averages of 2−3 experiments performed in duplicate
unless otherwise stated standard error of the mean, NDnot done,
HLMhuman liver microsome stability, T1/2half-life; and CL
clearance.
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Table 4. In vitro Screening and ADMET Profiling of 13
calcium
(human) EC50
nM SEM
calcium
(mouse) EC50
nM SEM
a
a
[35S]GTPγS
binding
AT1
MDCK
plasma protein
binding
a
a
EC50 nM SEM (human) Ki
(% Emax nM SEM
hERG
calcium
solubility
A−B
compd.
(% Emax
)
(% Emax
)
)
(10 μM) EC50 (μM) (μM, pH 7.4) ratio (%) (human) (%)b
13
136 14 (91)
22 0.3 (94)
483 44 (116) 90
3
inactive
>10
192
9.6
91
a
b
EC50 and Ki values are averages of 2−3 experiments performed in duplicate unless otherwise stated standard error of the mean. Propanolol was
used as a positive control with ∼61% PPB.
a
Table 5. PK Analyses of 13
species
mouse
route
IP
dose (mg/kg)
15
C
max (ng/mL)
AUCall (h·ng/mL)
CL (mL/min/kg)
T1/2 (h)
plasma
brain
1580
103
2998
1075
83
169
2.7
13.4
a
Cmaxmaximum concentration, T1/2half-life, CLclearance, and AUCarea under the curve.
a
Scheme 1. Synthesis of Compound 13
a
Reagents and conditions: (a) (S)-tert-butyl 3-amino-5-hydroxypentanoate (47, Intermediate 2), HBTU, Et3N, CH3CN, 16 h, 45%; (b) Dess−
Martin periodinane, DCM, 1 h, 98%; (c) piperidine, NaBH(OAc)3, DCE, RT, 16 h, 61%; (d) 4 M HCl in dioxane, DCM, RT, 8 h; (e)
cyclobutylamine, HBTU, CH3CN, RT, 4 h, 87%; (f) 2 M HCl in ether, MeOH, 0 °C to RT, 30 min.
EC50 = 105 nM) exhibited enhanced agonist potency compared
to 13 (cAMPi EC50 = 162 nM), however were surprisingly
metabolized faster (20: HLM T1/2 = 38 min; 21: HLM T1/2 = 37
min) than 13 (HLM T1/2 = 62 min) suggesting emergence of
additional liabilities due to these substitutions.
heterocyclic substitutions (34, 1,2,4-triazole; 35, 1,3,4-oxadia-
zole) were synthesized and evaluated for agonist potency (Table
3). Among these, compound 34 (cAMPi EC50 = 134 nM)
exhibited improved potency, however it was metabolically less
stable (HLM T1/2 = 33 min) than 13 (HLM T1/2 = 62 min). β-
Arrestin recruitment of these compounds was also evaluated.
Compounds with cyclobutyl ester (19, βarr EC50 = 101 nM),
3,3-difluorocyclobutylamide (20, βarr EC50 = 158 nM), N-
methyl, N-cyclobutylamide (21, βarr EC50 = 326 nM), N-
thiazolylamide (29, βarr EC50 = 12 nM), and N-methyl, N-
thiazolylamide (30, βarr EC50 = 14 nM) activated the β-arrestin
pathway with an EC50 value < 0.5 μM (Table 3). Overall, since
compound 13 exhibited favorable agonist potency (cAMPi EC50
= 162 nM) and HLM stability (HLM t1/2 = 62 min, CLINT = 20
mL/min/kg), we proceeded with this compound for broad in
vitro screening and ADMET profiling (Table 4).
Compound 13 exhibited a Ki of 90 nM and high agonist
potency (Ca2+ EC50 = 22 nM) at a mouse APJ receptor (calcium
mobilization CHO-Gαq16 cells stably expressing mouse APJ). In
addition, compound 13 was not active in the hERG assay with
<50% inhibition at 10 μM as determined by displacement of
radiolabeled astemizole using membrane preparation from
PerkinElmer.43 Potential off-target activity of 13 was independ-
Further in our quest to identify more potent and stable
analogues, we synthesized analogues substituting the cyclobutyl
moiety with various alkyl, cycloalkyl, and heterocyclic groups.
Compounds 26 (cAMPi EC50 = 1185 nM) and 25 (cAMPi EC50
= 2411 nM) with 2-methoxyethylamide and N-methyl, 2-
methylethylamide substitutions (Table 3), respectively, did not
enhance potency compared to 13 (cAMPi EC50 = 162 nM).
Similarly, compound 27 with 2-oxa-6-azaspiro[3.3]heptane
amide substitution (cAMPi EC50 = 5892 nM) also exhibited
poor potency compared to 13 (cAMPi EC50 = 162 nM). Diverse
heterocyclic amides (28, 29, 30, 31, 32, and 33) (oxazole,
thiazole, and tetrazole) were also synthesized and evaluated for
agonist potency. Compounds 29 (cAMPi EC50 = 1 nM), 30
(cAMPi EC50 = 5 nM), and 31 (cAMPi EC50 = 93 nM) were
more potent than 13 (cAMPi EC50 = 162 nM), however were
metabolized (29: HLM T1/2 = 27 min; 30: HLM T1/2 = 37 min;
31: HLM T1/2 = 24.2 min) rapidly compared to 13 (HLM T1/2
=
62 min). In addition, analogues lacking the amide with direct
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a
Scheme 2. Synthesis of 1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxylic Acid (Intermediate 1)11
a
Reagents and conditions: (a) Diethyl oxalate, NaOEt, EtOH, reflux, 4 h; 97% (b) cyclopentylhydrazine trifluoroacetate, glacial acetic acid, conc.
HCl, reflux,3 h; 73% (c) LiOH·H2O, MeOH/THF/H2O, RT, 18 h, 98%.
a
Scheme 3. Synthesis of (S)-tert-Butyl 3-Amino-5-hydroxypentanoate (Intermediate 2)46
a
t
Reagents and conditions: (a) Dess−Martin periodinane, DCM, 0 °C to RT, 3 h, 98% (b) (EtO)2POCH2CO2 Bu, BuLi, THF, −78° C to RT, 1 h;
61% (c) (S)-N-benzyl-N-α-methyl-benzylamine, BuLi, THF, −78 °C, 3 h; 60% (d) 10% Pd/C, H2, 50 psi, 20% acetic acid in EtOH, RT, 48 h, 88%.
ently determined by Eurofin PanLabs against several targets
including receptors and ion channels. At 10 μM test
concentration, compound 13 showed minimal cross reactivity
to most receptors tested. Approximately >50% inhibition of
binding was noted at few receptors and ion channels (see the
Supporting Information). Furthermore, IC50 determination at
these receptors revealed weak off-target activity at these
receptors (see the Supporting Information). Plasma protein
binding (human) was also undertaken for compound 13 and was
∼91% bound (Rapid Equilibrium Dialysis method with LC−MS
detection, Thermo Fisher kit).44 This compound demonstrated
limited transport across monolayers of MDCK-mdr1 cells
predicting low but positive brain penetration and was not active
at the AT1 receptor.45 Furthermore, this compound was
negative in Ames assay suggesting low carcinogenic potential
(data not shown).
injection (IP). Samples were collected in multiple time points
(0.5, 1, 2, 4, 8, and 24 h) to obtain plasma and brain maximum
concentrations. Compound 13 demonstrated (Table 5)
reasonable half-life (2.7 h in plasma and 13.4 h in brain),
clearance (∼80 mL/min/kg in plasma), and brain penetration
(>100 ng/mL based on Cmax). Based on cumulative data, a BID
dosing regimen was envisioned with this compound for in vivo
studies in the DIO model of metabolic syndrome.
Chemistry. Compound 13 was synthesized in five steps, as
shown in Scheme 1. Coupling of 1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-1H-pyrazole-3-carboxylic acid 36 (Inter-
mediate 1, Scheme 2) with (S)-tert-butyl 3-amino-5-hydrox-
ypentanoate (Intermediate 2, Scheme 3) using the HBTU
coupling reagent provided the O-tert butyl-protected inter-
mediate 37. Primary alcohol in intermediate 37 was then
subsequently oxidized to aldehyde 38 using the Dess−Martin
periodinane oxidizing reagent. The aldehyde then underwent
reductive amination with piperidine to provide intermediate 39
in good yields. Deprotection of tert-butyl ester in 39 using 4 M
Based on data generated in vitro, 13 was deemed suitable for in
vivo exploration. PK evaluation was performed in mice that were
administered a dose of 15 mg/kg of 13 via intraperitoneal
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a
Scheme 4. Synthesis of Compounds 4−11 and 12−18
a
Reagents and conditions: (a) cycloalkyl amine (R−NH), NaBH(OAc)3, DCE, RT, 16 h; (b) 4 M HCl in dioxane, DCM, RT; (c)
cyclobutylamine, HBTU, CH3CN, RT, 3 h; (d) 2 M HCl in ether, MeOH, 0 °C to rt, 30 min.
a
Scheme 5. Synthesis of 34 and Amide Analogues 20−33
a
Reagents and conditions: (a) ammonium carbonate, Boc2O, pyridine, dioxane, 12 h, 69%; (b) DMF−DMA, 120 °C, 2 h; NH2NH2·H2O,
CH3COOH, 90 °C, 2 h, 57%; (c) R1−NH2, HBTU, MeCN, RT, 4 h.
a
Scheme 6. Synthesis of Analogue 35
a
Reagents and conditions: (a) TFA, DCM, RT, 2 h, crude; (b) MeOH, H2SO4, RT, 15 h, crude; (c) NH2NH2·H2O, EtOH, 80 °C, 3 h, 55%; (d)
CH(OMe)3, PTSA·H2O, 85 °C, 2 h, 59%.
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a
Table 6. Compound 13 Improves Biomarkers Associated with Metabolic Syndrome in DIO Mice
treatment groups
biomarker
ND + vehicle
HFD + vehicle
HFD + comp 13 (5 mg/kg)
HFD + comp 13 (15 mg/kg)
bodyweight (% of baseline)
daily food consumption (g)
fat pad weight (total, g)
fructosamine (μmol)
triglycerides (mg/dL)
cholesterol (mg/dL)
ALT (U/L)
free FAs (mEq/L)
lactate dehydrogenase (u/L)
fasting Glucose
94.0 (1.0)
2.6 (0.2)
98.5 (1.2)
2.5 (0.3)
94.2 (1.3)*
2.5 (0.3)
82.0 (1.8)**
1.8 (0.2)*
0.43 (0.07)*
830.8 (26.4)**
66.5 (3.2)
181.8 (4.3)
16.8 (2.0)
0.80 (0.10)
182.2 (17.4)
235 (7)**
0.87 (0.2)**
1028.4 (46.2)
69.0 (3.4)
138.6 (5.8)**
15.9 (2.2)
0.97 (0.1)
193.3 (12.5)
244 (15)*
1.98 (0.12)
1037.1 (40.8)
74.6 (3.5)
198.2 (5.9)
20.7 (1.2)
0.99 (0.10)
215.1 (24.9)
307 (8)
1.38 (0.38)
904.3 (22.0)**
59.0 (2.6)*
207.2 (4.1)
16.8 (1.2)
0.86 (0.09)
184.4 (18.2)
171 (8)**
a
All data are presented as mean (SEM). Statistical significance determined using t-test versus HFD, * < 0.05, ** < 0.01. Nine to 10 animals per
experimental group.
HCl provided the HCl salt of the carboxylic acid intermediate 6
which was then coupled to cyclobutylamine using the HBTU
coupling reagent to provide the free base of compound 13.
Finally, treatment of 2 M HCl in methanol gave the
hydrochloride salt of 13.
two different doses5 and 15 mg/kg for 28 daysled to
improved metabolic parameters (Table 6) in mice maintained
on a 60% fat-containing diet (HFD) compared to animals
receiving vehicle alone. Animals on 10% fat diet (ND) served as
baseline controls for this experiment. Compared to mice on
HFD alone, several biomarkers associated with metabolic
syndrome showed statistically significant improvement includ-
ing fructosamine levels (the level of fructosamine in the blood is
a reflection of glucose levels over the previous 2−3 weeks similar
to hemoglobin A1c), total fat pad weights, and triglycerides.
Cholesterol, ALT, lactate dehydrogenase (LDH), and free FA
concentrations in plasma trended lower upon treatment as well.
The most pronounced effects were noticed at the higher dose,
including reduced food consumption, suggesting a central role
of APJ in regulation of feeding and associated metabolic
processes. However, certain benefits such as fasting glucose
levels were noted at the lower dose groups in the absence of a
pronounced effect on body weight presumably through the
activation of peripheral APJ receptors in agreement with the past
literature.47,48 The oral GTT data recorded on day 28 indicated
significant blunting of circulating glucose levels in fasted animals
(Figure 3). Degree of liver steatosis was vastly reduced in
animals treated with 13 in a dose-dependent fashion with
minimum fat deposition noted at the high dose group (Figure
3). Taken together, these very promising studies firmly establish
a role of apelin receptor agonists in the treatment of DIO and
metabolic syndrome.
Intermediate 1 (36) was synthesized as shown in Scheme 2.11
2,6-Dimethoxy acetophenone 40 was condensed with diethyl
oxalate to afford the sodium salt of diketone 41 in quantitative
yield. Reaction of 41 with cyclopentylhydrazine trifluoroacetate
in refluxing glacial acetic acid provided 1,5-pyrazoles 42a and
1,3-pyrazoles 42b in a ratio of 4:1. Hydrolysis of ethyl ester 42a
to acid 36 was achieved using lithium hydroxide monohydrate.
Synthesis of intermediate 2 (47) was achieved as described in
Scheme 3 using reported methods.46 3-Benzyloxy-1-propanol
43 was oxidized to 44 using Dess−Martin periodinane, which
then underwent Horner−Wadsworth-Emmons reaction with
tert-butyl diethylphosphonoacetate to provide alkene 45. BuLi-
facilitated addition of (S)-N-benzyl-N-α-methyl-benzylamine to
45 gave intermediate 46 which was then hydrogenated using
10% Pd/C catalyst to provide the intermediate 2 (47).
Target compounds 4−11 and 12−18 were synthesized, as
shown in Scheme 4. Reductive amination of aldehyde 38 with
cycloalkyl amines gave the corresponding tert-butyl ester
intermediates. Deprotection of tert-butyl ester intermediates
with 4 M HCl provided the HCl salt of carboxylic acids 4−11
which were then coupled to cyclobutylamine using the HBTU
coupling reagent to give the free base of compounds 12−18.
Finally, treatment of the free base with 2 M HCl in methanol
gave the hydrochloride salts of 12−18.
CONCLUSIONS
■
Target compound 34 and diverse amide analogues 20−33
were synthesized as described in Scheme 5. Starting compound
6 was converted to the amide 48 using Boc2O and (NH4)2CO3.
Amide 48 was treated with DMF−DMA to obtain the reactive
aldehyde intermediate which was further treated with hydrazine
hydrate to provide the target compound 34. HBTU-facilitated
coupling of 6 with diverse alkyl amines provided the amide
analogues 20−33.
Scheme 6 describes the synthesis of target compound 35.
Starting compound 39 was first converted to the methyl ester 49
which was then treated with hydrazine hydrate to provide the
hydrazide intermediate 50. Hydrazide 50 was then treated with
triethyl orthoformate to provide the target compound 35.
Efficacy Study of 13 in the DIO Mouse Model. The DIO
model of metabolic syndrome is a well-accepted model of this
disorder and has a high degree of correlation to clinical outcome
in humans. Twice daily administration of the compound 13 at
Systematic modification at the amino acid side chain of 2 and 3
and at the carboxylic acid end of 6 led to compound 13 with
favorable agonist potency, HLM stability, and PK profile in
rodents. At first, compounds with substituted alkyl/cycloalkyl
amines replacing the cyclohexyl moiety in 2 were synthesized
and evaluated to gain an understanding of receptor tolerances
and their effects on potency and efficacy. Among these,
compounds with five- (5) or seven-membered (11) heterocyclic
rings (pyrrolidine and azepane) were tolerated well and
exhibited comparable or enhanced agonist potency relative to
compound 2. In contrast, six-membered cyclic piperidine
derivatives (6, 7, 8, and 9) exhibited reduced receptor tolerance
and variable agonist potencies. Several compounds among 4−11
exhibited weak activity in the β-arrestin recruitment assay
suggesting the possibility of low receptor internalization and
desensitization with these analogues. Furthermore, compounds
12−18 with diverse cycloamine substitutions at the amino acid
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compound 13 was deemed suitable for in vivo efficacy study
based on the favorable agonist potency, HLM stability, in vitro
ADMET, and PK properties. Upon evaluation in the mouse
model of DIO, compound 13 exhibited significant weight loss,
improved glucose utilization, and reduced liver steatosis and
associated biomarkers.
In conclusion, we have identified a small-molecule agonist of
the apelin receptor that is suitable for in vivo investigation in
DIO and possibly in animal models of other diseases modulated
by the apelin receptor. Favorable in vivo efficacy demonstrated
by compound 13 in DIO firmly establishes a role of apelin
receptor agonists in the treatment of DIO and metabolic
syndrome.
EXPERIMENTAL SECTION
■
Reagents and starting materials were obtained from commercial
suppliers and were used without purification. Reactions were conducted
under a N2 atmosphere using oven-dried glassware. All solvents and
chemicals used were reagent 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 gold prepacked HP silica columns. Purity and
characterization of compounds were established using a combination of
1
HPLC, TLC, and NMR analytical techniques described below. H
NMR spectra were recorded on a Bruker AVANCE DPX300 (300
MHz) or Variance 200 (200 MHz) spectrometer using CHCl3-d,
MeOH-d4, and 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 constant (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). Analytical HPLC was performed on
a Waters 2695 Separation Module equipped with a Waters 2996
photodiode array detector and a Phenomenex Synergi 4 μm Hydro-RP
80A C18 250 × 4.6 mm column using a flow rate of 1 mL/min starting
with 1 min at 5% solvent B, followed by a 15 min gradient of 5−95%
solvent B, followed by 9 min at 95% solvent B (solvent A, water with
0.1% TFA; solvent B, acetonitrile with 0.1% TFA and 5% water);
absorbance was monitored at 220 and 280 nm. Purity of the target
compounds was determined to be ≥95% by HPLC. Optical rotations
were measured on an Auto Pol IV automatic polarimeter at the sodium
D line. Chemical names were generated using ChemDraw Ultra
(CambridgeSoft, version 10.0).
Figure 3. Treatment with 13 improved glucose tolerance and reduced
hepatic steatosis. (A) Both macro- and microvesicular steatosis of the
liver were reduced in mice receiving 13 as determined using Oil Red O
staining. The representative image is taken from animals receiving 15
mg/kg dose of 13 along with ND and HFD controls receiving vehicle
only. (B) Liver steatosis is decreased by 13 as determined by
quantifying Oil Red O-positive staining in liver samples. Statistical
significance indicated (p < 0.01, ** and p < 0.001, ***) following
ANOVA with post hoc Dunnett test vs HFD group. (C) Oral GTT
testing on day 28 indicated that treatment with 13 reduced fasting
glucose levels and improved glucose utilization in a dose-dependent
manner. Dose effects are significant for both groups (p < 0.01, ** and p
< 0.001, ***) over time compared to HFD as assessed using ANOVA
with a post hoc Dunnett test.
end of 3 were evaluated to examine if these substitutions
enhanced potency. Interestingly, six-membered piperidine 13
(∼13-fold), 2,6-dimethyl-substituted piperidine 16 (∼65-fold),
7-azabicyclo [2.2.1] heptane 17 (21-fold), and seven-membered
azepane cyclobutylamide 18 (∼23-fold) substitutions signifi-
cantly enhanced potency compared to their carboxylic acid
precursors indicating enhanced receptor interaction with the
addition of the cyclobutylamide group. Among these,
compound 13 with a piperidine moiety had the most favorable
HLM stability (T1/2 = 62 min) and clearance (CLINT = 20 mL/
min/kg). Compounds 13, 16, and 18 also recruited β-arrestin
with an EC50 value < 1 μM. To further improve potency and
stability, the cyclobutyl end of 13 was replaced with diverse polar
and nonpolar substituted amides and nonamide moieties. The
compound with thiazolylamide (29) was the most potent
(∼167-fold compared to 13), however it was surprisingly
metabolized faster than 13. Similarly, compounds with 1,1-
difluorocyclobutanamide (20, 18-fold) and N-methylthiazola-
mide (30, 33-fold) were significantly more potent than 13 but
were metabolized more rapidly than 13 suggesting emergence of
additional liabilities due to these substitutions. Ultimately,
Scheme 1: Synthesis of (S)-N-(1-(Cyclobutylamino)-1-oxo-5-
(piperidin-1-yl)pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxy-
phenyl)-1H-pyrazole-3-carboxamide Hydrochloride (13). tert-
Butyl-(S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-
carboxamido)-5-hydroxypentanoate (37). To a solution of (S)-tert-
butyl 3-amino-5-hydroxypentanoate 47 (16 g, 0.084 mol) and 5-(2,6-
dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxylic acid 36
(26.7 g, 0.084 mol) in MeCN (400 mL) were added HBTU (64 g,
0.169 mol) followed by the dropwise addition of triethylamine (35.3
mL, 0.253 mol) at room temperature. After stirring at room
temperature for 16 h, the reaction mixture was quenched using
saturated aqueous sodium bicarbonate (250 mL). The layers were
separated, and the aqueous layer was extracted with EtOAc (3 × 200
mL). The combined organic layers were dried with Na2SO4, filtered,
and concentrated in vacuo to give the crude product. The crude product
was purified by silica gel flash column chromatography (0−100%
EtOAc/hexanes containing 5% triethylamine) to give the purified title
1
compound 37 as light-yellow foam (18.6 g, 45%). H NMR (CDCl3,
300 MHz): δ 1.49 (s, 9H), 1.51−1.75 (m, 4H), 1.84−1.98 (m, 4H),
1.99−2.16 (m, 2H), 2.47−2.59 (m, 1H), 2.62−2.77 (m, 1H), 3.63−
3.71 (m, 2H), 3.73 (s, 3H), 3.75 (s, 3H), 4.23−4.33 (m, 1H), 4.55−
4.67 (m, 1H), 6.63 (dd, J = 8.48, 1.70 Hz, 2H), 6.66−6.72 (m, 1H), 7.38
3015
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(t, J = 8.48 Hz, 1H), 7.68 (d, J = 9.61 Hz, 1H). MS (ESI) m/z: calcd for
C26H37N3O6, 487.27 [M]+; found, 488.7 [M + H]+.
free base of 13 (12.31 g, 87%) as a white solid. 1H NMR (CDCl3, 300
MHz): δ 1.38−1.49 (m, 2H), 1.49−1.72 (m, 10H), 1.78−2.00 (m,
7H), 2.01−2.18 (m, 2H), 2.19−2.33 (m, 2H), 2.34−2.50 (m, 4H),
2.50−2.69 (m, 3H), 3.72 (s, 3H), 3.73 (s, 3H), 4.24 (quin, J = 7.63 Hz,
1H), 4.34−4.49 (m, 2H), 6.63 (d, J = 8.29 Hz, 2H), 6.67 (s, 1H), 7.01
(d, J = 8.67 Hz, 1H), 7.38 (t, J = 8.29 Hz, 1H), 8.09 (d, J = 8.29 Hz, 1H).
MS (ESI) m/z: calcd for C31H45N5O4, 551.35 [M]+; found, 552.8 [M +
H]+.
The free base of (S)-N-(1-(cyclobutylamino)-1-oxo-5-(piperidin-1-
yl)pentan-3-yl)-5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-
3-carboxamide 13 (9.0 g, 16.68 mmol) was mixed with MeOH (100
mL) at 0 °C. To it was added 2 M HCl solution in ether (16.7 mL, 33.37
mmol) dropwise at 0 °C. The reaction mixture was warmed to room
temperature and stirred for 30 min. MeOH was evaporated in vacuo
and dried under high vacuum to remove all the excess HCl. To it was
added ether, triturated, and filtered to obtain a white solid. The solid
was dried under high vacuum for 16 h to give the hydrochloride salt of
13 (10.1 g). 1H NMR (DMSO-d6, 300 MHz): δ 1.46−1.69 (m, 4H),
1.69−1.79 (m, 4H), 1.79−2.05 (m, 9H), 2.07−2.18 (m, 2H), 2.43 (t, J
= 8.85 Hz, 2H), 2.77−2.93 (m, 2H), 2.97−3.12 (m, 2H), 3.34−3.47
(m, 2H), 3.71 (s, 6H), 4.13−4.34 (m, 6H), 6.45 (s, 1H), 6.78 (d, J =
8.29 Hz, 2H), 7.44 (t, J = 8.38 Hz, 1H), 8.04 (d, J = 8.85 Hz, 1H), 8.32
(d, J = 7.54 Hz, 1H), 9.99 (br s, 1H). 13C NMR (75 MHz, DMSO-d6): δ
14.68, 21.35, 22.32, 24.01, 24.05, 28.38, 30.08, 30.19, 32.50, 43.83,
44.26, 51.82, 51.97, 53.54, 55.68, 59.42, 104.20, 106.71, 107.35, 131.46,
136.42, 144.82, 158.18, 158.21, 161.38, 168.83. MS (ESI) m/z: calcd
for free base C31H45N5O4, 551.35 [M]+; found, 552.8 [M + H]+; [α]2D5
−23.56 (c 0.50, MeOH).
Synthesis of Intermediates. Intermediate 1. Synthesis of 1-
Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxylic
Acid. Ethyl 4-(2,6-Dimethoxyphenyl)-2,4-dioxobutanoate (41). To a
solution of sodium ethoxide (21% in EtOH) (5.4 mL, 14.37 mmol) was
added dropwise a mixture of diethyl oxalate (1.85 mL, 13.690 mmol)
and 2,6-dimethoxy acetophenone 40 (2.45 g, 13.690 mmol) in
anhydrous ethanol (15 mL). The resultant mixture was stirred at RT
for 30 min, upon which yellow suspension formed. The reaction
mixture was heated to reflux for 4 h. The reaction mixture was cooled to
room temperature. Ethanol was evaporated in vacuo. The resultant
residue was triturated with diethyl ether (30 mL) and filtered to obtain a
sodium salt of ethyl 4-(2,6-dimethoxyphenyl)-2,4-dioxobutanoate 41
as a yellow solid (4.0 g, 97%). MS (ESI) m/z: calcd for C14H16O6,
280.09 [M]+; found, 279.3 [M − H]−.
tert-Butyl (S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxamido)-5-oxopentanoate (38). tert-Butyl-(S)-
3-(5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxami-
do)-5-hydroxypentanoate 37 (11.19 g, 22.95 mmol) was dissolved in
DCM (saturated with water, 90 mL). Dess−Martin periodinate (20 g,
47.26 mmol) was added, and the reaction mixture was stirred at RT for
1 h, with the addition of 15 mL of DCM (saturated with water) every 15
min. Upon completion, the mixture was diluted with diethyl ether (400
mL). A mixture containing sodium thiosulfate (20 g) dissolved in
saturated sodium bicarbonate (80 mL) and water (20 mL) was added.
This mixture was stirred for 30 min at room temperature. The aqueous
layer was extracted with ether, and the combined organic layers were
washed with ice-cold saturated sodium bicarbonate solution (2 × 150
mL) and brine (150 mL). The mixture was dried (Na2SO4), filtered,
and concentrated to yield 11.0 g (98%) of compound 38 as a tan solid
1
which was used without further purification. H NMR (CDCl3, 300
MHz): δ 1.48 (s, 9H), 1.49−1.62 (m, 2H), 1.83−1.97 (m, 3H), 1.98−
2.14 (m, 3H), 2.70 (d, J = 5.84 Hz, 2H), 2.78−3.01 (m, 2H), 3.73 (s,
6H), 4.26 (quin, J = 7.21 Hz, 1H), 4.82−4.94 (m, 1H), 6.62 (d, J = 8.48
Hz, 2H), 6.66 (s, 1H), 7.37 (t, J = 8.38 Hz, 1H), 7.53 (d, J = 9.23 Hz,
1H), 9.81 (s, 1H). MS (ESI) m/z: calcd for C26H35N3O6, 485.25 [M]+;
found, 486.7 [M + H]+.
tert-Butyl (S)-3-(5-(2,6-Dimethoxyphenyl)-1-cyclopentyl-1H-
pyrazole-3-carboxamido)-5-(piperidin-1-yl)pentanoate (39).
Crude tert-butyl (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxamido)-5-oxopentanoate 38 (11.0 g, 22.61 mmol)
was mixed with DCE (170 mL). To the solution, piperidine (2.8 mL,
28.3 mmol) and NaBH(OAc)3 (9.9 g, 46.57 mmol) were added at RT.
The mixture was stirred at RT for 16 h. The reaction mixture was then
quenched with sat. NaHCO3 and diluted with DCM. The organic layer
was then washed with water and brine, dried with Na2SO4, and
concentrated to give the crude product. The crude residue was purified
by silica gel flash chromatography (0−30% DCM/MeOH/aq. NH4OH
(100:20:2)/DCM) to give the title compound 39 as white foam (7.74 g,
1
61%). H NMR (CDCl3, 300 MHz): δ 1.52−1.64 (m, 5H), 1.46 (s,
9H), 1.51−1.67 (m, 5H), 1.78−1.98 (m, 6H), 1.99−2.14 (m, 2H),
2.35−2.70 (m, 6H), 3.72 (s, 3H), 3.74 (s, 3H), 4.25 (quin, J = 7.63 Hz,
1H), 4.42−4.55 (m, 1H), 6.62 (d, J = 8.48 Hz, 2H), 6.67 (s, 1H), 7.32−
7.40 (m, 1H), 7.69 (d, J = 8.67 Hz, 1H). MS (ESI) m/z: calcd for
C31H46N4O5, 554.35 [M]+; found, 556.1 [M + H]+.
Ethyl 1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-car-
boxylate (42a). Step 1: Preparation of cyclopentylhydrazine
trifluoroacetate: cyclopentanone (2.0 g, 23.77 mmol) and tert-butyl
carbazate (3.1 g, 23.77 mmol) in methanol (20 mL) were stirred at
room temperature for 3 h. The solvent was evaporated, and the
resulting solid was dried in vacuo to give a white solid of tert-butyl 2-
cyclopentylidenehydrazinecarboxylate (4.6 g, 98%). Sodium cyanobor-
ohydride (2.1 g, 34.06 mmol) was added portionwise to a mixture of
tert-butyl 2-cyclopentylidenehydrazinecarboxylate (4.5 g, 22.697
mmol) in 75% of aqueous acetic acid (25 mL) at room temperature.
The resultant solution was stirred for 3 h at room temperature. The
reaction mixture was neutralized with 1 N NaOH and extracted with
CH2Cl2 (3 × 30 mL). The organic layer was washed with saturated
NaHCO3, dried with Na2SO4, filtered, and evaporated to give tert-butyl
2-cyclopentylhydrazinecarboxylate as oil (4.4 g, 97%). 1H NMR
(CDCl3, 300 MHz): δ 1.40−1.55 (m, 4H), 1.46 (s, 9H), 1.63−1.75 (m,
4H), 3.45−3.58 (m, 1H). MS (ESI) m/z: calcd for C10H20N2O2, 200.15
[M]+; found, 223.3 [M + Na]+.
Trifluoroacetic acid (24 mL) was added dropwise to a solution of the
tert-butyl 2-cyclopentylhydrazinecarboxylate (4.4 g, 21.97 mmol) in
CH2Cl2 (24 mL). The reaction mixture was stirred at room
temperature for 2.5 h. The solvent was evaporated to give cyclo-
pentylhydrazine trifluoroacetate as colorless oil in quantitative yield. 1H
NMR (CDCl3, 300 MHz): δ 1.58−1.70 (m, 2H), 1.72−1.92 (m, 4H),
1.95−2.09 (m, 2H), 3.60−3.78 (m, 1H). MS (ESI) m/z: calcd for
C5H12N2, 100.10 [M]+; found, 101.2 [M + H]+.
(S)-N-(1-(Cyclobutylamino)-1-oxo-5-(piperidin-1-yl)pentan-
3-yl)-5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-
carboxamide Hydrochloride (13). tert-Butyl (S)-3-(5-(2,6-dime-
thoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxamido)-5-(piperi-
din-1-yl)pentanoate 39 (2.71 g, 4.88 mmol) was dissolved in anhydrous
DCM (40 mL) and treated with 4 M HCl/dioxane (18.3 mL, 73.2
mmol); this mixture was stirred for 8 h at RT. Upon completion, the
reaction mixture was concentrated and vacuum-dried overnight to
obtain quantitative yield of (S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclo-
pentyl-1H-pyrazole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid
6 as a hydrochloride salt. MS (ESI) m/z: calcd for free base
C27H38N4O5, 498.28 [M]+; found, 499.52 [M + H]+.
To a solution of crude (S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclo-
pentyl-1H-pyrazole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid
hydrochloride 6 (16.6 g, 0.0258 mol) and cyclobutylamine (2.42 mL,
0.0283 mol) in MeCN (200 mL) were added triethylamine (17.95 mL,
0.129 mol) and HBTU (14.6 g, 0.0386 mol). After stirring at room
temperature for 4 h, the reaction mixture was quenched using saturated
aqueous sodium bicarbonate solution (100 mL). The aqueous layer was
separated and extracted with EtOAc (3 × 100 mL). The combined
organic layers were further washed with 50% concentrated NH4OH/
H2O (2 × 50 mL), dried with Na2SO4, filtered, and concentrated in
vacuo to give the crude product. The crude product was purified by
silica gel flash column chromatography (0−30% DCM/MeOH/aq.
NH4OH (100:20:2)/DCM). Purified fractions were combined and
concentrated. The residue was dissolved in DCM (200 mL), washed
with 50% concentrated NH4OH/H2O (50 mL) and brine (50 mL), and
dried with Na2SO4. Removal of the solvent in vacuo afforded the pure
Step 2: Preparation of ethyl 1-cyclopentyl-5-(2,6-dimethoxyphenyl)-
1H-pyrazole-3-carboxylate (42a): Sodium salt of ethyl 4-(2,6-
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dimethoxyphenyl)-2,4-dioxobutanoate (41) (1.2 g, 3.96 mmol) and
cyclopentylhydrazine trifluoroacetate (1.3 g, 5.95 mmol) were mixed
with glacial acetic acid (25 mL) and conc. HCl (0.6 mL). The reaction
mixture was heated to reflux for 3 h. After cooling, the reaction mixture
was poured into water (50 mL). The aqueous layer was extracted with
DCM (3 × 30 mL). The combined DCM layer was washed with
saturated aqueous NaHCO3. The organic layer was then washed with
saturated brine and then dried over Na2SO4, followed by filtration. The
solvent was evaporated in vacuo. The residue was purified by silica gel
flash chromatography (0−40% EtOAc/Hex) to give N1-substituted
title compound 42a (1.0 g, 73%, major product) as a light-yellow solid
and less polar N2-substituted 42b, 2-cyclopentyl-5-(2,6-dimethoxy-
phenyl)-1H-pyrazole-3-carboxylate as colorless oil (50 mg, 5%).
Ethyl 1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-car-
EtOAc in hexanes), and the fractions containing the product were
pooled and evaporated to obtain 152 g (61%) of (E)-tert-butyl 5-
(benzyloxy)pent-2-enoate 45 as a colorless liquid. 1H NMR (DMSO-
d6, 300 MHz): δ 1.45 (s, 9H), 2.40−2.60 (m, 2H), 3.55 (t, J = 6.8 Hz,
2H), 4.50 (s, 2H), 5.80 (s, 1H), 6.80−6.95 (m, 1H), 7.30−7.40 (m,
5H). MS (ESI) m/z: calcd for C16H22O3, 262.16 [M]+; found, 263.1 [M
+ H]+
(S)-tert-Butyl 3-(Benzyl((S)-1-phenylethyl)amino)-5-(benzyloxy)-
pentanoate (46). To a stirred solution of (S)-N-benzyl-N-α-
methybenzylamine (150 g, 0.710 mol) in THF (600 mL) at −78 °C
was added n-BuLi (2.5 M in hexanes) (285 mL, 0.710 mol) using a
dropping funnel. After 30 min, a solution of (E)-tert-butyl 5-
(benzyloxy)pent-2-enoate 45 (152 g, 0.546 mol) in THF (200 mL)
also at −78 °C was transferred via cannula. The resulting solution was
stirred at −78 °C for 3 h, warmed to −10 °C, and quenched with 20%
citric acid solution (900 mL). The organic phase was separated, washed
with brine (500 mL), and dried with Na2SO4, and the solvent was
removed in vacuo to give the crude product. The crude product was
purified by silica gel flash chromatography (0−4% of EtOAc in
hexanes), and the fractions containing the product were pooled and
evaporated to obtain 155 g (60%) of (S)-tert-butyl 3-(benzyl((S)-1-
phenylethyl)amino)-5-(benzyloxy)pentanoate 46 as a pale-yellow
liquid. 1H NMR (DMSO-d6, 300 MHz): δ 1.40−1.80 (m, 12H),
1.60−1.80 (m, 2H), 1.85−1.90 (m, 2H), 3.40−3.80 (m, 6H), 4.52−
4.60 (m, 2H), 7.15−7.40 (m, 15H). MS (ESI) m/z: calcd for
C31H39NO3, 473.29 [M]+; found, 474.3 [M + H]+; [α]2D5 +6.47 (c
0.850, MeOH).
(S)-tert-Butyl 3-Amino-5-hydroxypentanoate (47). (S)-tert-Butyl
3-(benzyl((S)-1-phenylethyl)amino)-5-(benzyloxy)pentanoate 46
(65.4 g, 138 mmol) was dissolved in 20% acetic acid in EtOH (550
mL) and to it was added 10% Pd/C (10.5 g). The mixture was
hydrogenated in a sealed vessel at 50 psi for 48 h. The catalyst was
removed by filtration through celite, and the solvent was evaporated to
obtain the crude product as oil. The crude product was basified at 0 °C
with NH4OH solution (35 mL) to pH 9.0. This crude mixture was
loaded onto a column and purified by silica gel flash chromatography
(0−40% and 40−60% of DCM/MeOH/aq. NH4OH (100:20:2)/
DCM) to give the 23 g (88%) of (S)-tert-butyl 3-amino-5-
hydroxypentanoate 47 as colorless oil. 1H NMR (DMSO-d6, 300
MHz): δ 1.40 (s, 9H), 1.55−1.65 (m, 2H), 2.20−2.40 (m, 2H), 2.50
(br, 2H), 3.25−3.40 (m, 1H), 3.75−3.85 (m, 2H). MS (ESI) m/z:
calcd for C9H19NO3, 189.14 [M]+; found, 190.0 [M + H]+; [α]2D5 −4.21
(c 0.285, MeOH).
(S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(diethylamino)pentanoic Acid Hydrochloride (4).
Crude (S)-tert-butyl (S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclopentyl-
1H-pyrazole-3-carboxamido)-5-oxopentanoate 38 (60 mg, 0.123
mmol) was mixed with DCE (5 mL). To the mixture was added
diethylamine (11 mg, 0.148 mmol) and NaBH(OAc)3 (52 mg, 0.246
mmol) at RT. The reaction mixture was stirred at room temperature for
16 h. The reaction mixture was then quenched with saturated NaHCO3,
diluted with DCM (50 mL), and washed with water followed by brine.
The organic layer was separated, dried with Na2SO4, filtered, and
concentrated to give the crude product. The crude residue was purified
by silica gel flash chromatography (0−30% DCM/MeOH/aq. NH4OH
(100:20:2)/DCM) to give tert-butyl (S)-3-(1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(diethylamino)-
pentanoate as white foam (65 mg, 73%).
1
boxylate (42a). H NMR (CDCl3, 300 MHz): δ 1.38 (t, J = 7.2 Hz,
3H), 1.44−1.57 (m, 2H), 1.84−1.98 (m, 4H), 2.08−2.26 (m, 2H), 3.74
(s, 6H), 4.21−4.31 (m, 1H), 4.39 (q, J = 6.9 Hz, 2H), 6.63 (d, J = 8.3
Hz, 2H), 6.69 (s, 1H), 7.38 (t, J = 8.48 Hz, 1H). MS (ESI) m/z: calcd
for C19H24N2O4, 344.17 [M]+; found, 345.0 [M + H]+.
Ethyl 2-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-car-
1
boxylate (42b). H NMR (CDCl3, 300 MHz): δ 1.36 (t, J = 7.2 Hz,
3H), 1.62−1.74 (m, 2H), 1.85−1.98 (m, 2H), 2.08−2.26 (m, 4H) 3.77
(s, 6H), 4.33 (q, J = 7.16 Hz, 2H), 5.68 (quin, J = 7.49 Hz, 1H), 6.62 (d,
J = 8.48 Hz, 2H), 6.91 (s, 1H), 7.27 (t, J = 8.48 Hz, 1H). MS (ESI) m/z:
calcd for C19H24N2O4, 344.17 [M]+; found, 345.3 [M + H]+.
1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbox-
ylic Acid (36). Lithium hydroxide monohydrate (440 mg, 10.452
mmol) in 2.5 mL of water was added to a solution of ethyl 1-
cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxylate
(42a) (1.2 mg, 3.484 mmol) in MeOH (11 mL) and THF (2.5 mL).
The mixture was stirred at RT for 18 h. The reaction mixture was
concentrated to about half the volume and then extracted with ether (2
× 15 mL). The aqueous layer was acidified with 1 N HCl and extracted
with CH2Cl2 (3 × 25 mL). The combined organic layers were washed
with water and brine and then dried with Na2SO4. The solvent was
evaporated in vacuo to give the title compound as a white solid (1 g,
98% yield). 1H NMR (CDCl3, 300 MHz): δ 1.48−1.64 (m, 2H), 1.84−
2.02 (m, 4H), 2.03−2.20 (m, 2H), 3.75 (s, 6H), 4.30 (quin, J = 7.4 Hz,
1H), 6.64 (d, J = 8.3 Hz, 2H), 6.76 (s, 1H), 7.40 (t, J = 8.3 Hz, 1H). MS
(ESI) m/z: calcd for C17H20N2O4, 316.14 [M]+; found, 317.2 [M +
H]+.
Intermediate 2. Synthesis of (S)-tert-Butyl 3-Amino-5-hydrox-
ypentanoate (47). 3-(Benzyloxy)propanal (44). To a solution of
commercially available 3-benzyloxy-1-propanol 43 (160 g, 0.96 mol) in
CH2Cl2 (2000 mL) at 0 °C was added Dess−Martin periodinane (450
g, 1.06 mol) in 50 g portions, and the reaction mixture was stirred at RT
for 3 h. To the reaction mixture was added solution of 20% Na2S2O3
solution (1 L) and stirred for 15 min. The reaction mixture was poured
into a separating funnel containing Et2O (2 L). The organic phase was
washed with 1:1:1 20% Na2S2O3 solution/sat. NaHCO3/H2O (750
mL) and brine (250 mL). The organic phase was concentrated to give a
crude solid which was filtered over Celite and washed with THF. The
filtrate was concentrated and dried to give 155 g (98%) of 3-
(benzyloxy)propanal 44 as a pale-yellow liquid. 1H NMR (DMSO-d6,
300 MHz): δ 2.70 (t, J = 6.2 Hz, 2H), 3.70 (t, J = 6.2 Hz, 2H), 4.55 (s,
2H), 7.25−7.40 (m, 5H), 9.80 (s, 1H).
(E)-tert-Butyl 5-(Benzyloxy)pent-2-enoate (45). To a stirred
solution of tert-butyl diethylphosphonoacetate (286 g, 1.13 mol) in
THF (1400 mL) at −78 °C was added n-BuLi (2.5 M in hexanes) (453
mL, 1.13 mol) using a dropping funnel over 40 min. After stirring for 30
min at −78 °C, a solution of 3-(benzyloxy)propanal 44 (42.3 g, 0.945
mol) in THF (600 mL) also cooled at −78 °C was transferred via
cannula. The resulting solution was stirred at −78 °C for 40 min before
being allowed to warm at RT and then stirred for 1 h. The solution was
subsequently cooled to −78 °C and quenched with saturated aqueous
NH4Cl (250 mL). The reaction mixture was brought to RT. The layers
were separated, the aqueous layer was extracted in EtOAc (500 mL),
the combined organics were dried over Na2SO4 and filtered, and the
solvent was evaporated in vacuo to give the crude residue. The crude
product was purified by silica gel flash chromatography (0−5% of
4 M HCl in dioxane (0.2 mL) was added to tert-butyl (S)-3-(1-
cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-
(diethylamino)pentanoate (53 mg, 0.097 mmol) in 3 mL of DCM. The
reaction mixture was stirred at RT for 24 h, and the solvent was
evaporated. The solid residue was triturated with ethyl ether and filtered
1
to obtain 45 mg (94%) of the title compound 4 as a white solid. H
NMR (MeOH-d4, 300 MHz): δ 1.35 (t, J = 7.25 Hz, 6H), 1.53−1.69
(m, 2H), 1.83−2.25 (m, 8H), 2.79 (d, J = 6.2 Hz, 2H), 3.12−3.29 (m,
4H), 3.46−3.59 (m, 1H), 3.68 (m, 3H), 3.69−3.65 (m, 1H), 3.72−3.77
(m, 2H), 3.76 (s, 3H), 4.24−4.40 (m, 1H), 4.41−4.58 (m, 1H), 6.56 (s,
1H), 6.77 (d, J = 8.5 Hz, 2H), 7.46 (t, J = 8.4 Hz, 1H). MS (ESI) m/z:
3017
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Article
calcd for free base C26H38N4O5, 486.28 [M]+; found, 487.6 [M + H]+;
[α]2D5 −24.88 (c 0.225, MeOH).
azabicyclo[2.2.1]heptane (15 mg, 0.148 mmol) as described in
procedure 4. 83% yield; white solid; 1H NMR (MeOH-d4, 300
MHz): δ 1.52−1.69 (m, 2H), 1.78−2.00 (m, 7H), 2.00−2.28 (m, 6H),
2.80 (d, J = 6.4 Hz, 2H), 3.14−3.23 (m, 2H), 3.46−3.56 (q, J = 7.0 Hz,
2H), 3.64−3.71 (m, 3H), 3.76 (s, 6H), 4.13−4.19 (br s, 1H), 4.20−
4.26 (m, 1H), 4.28−4.40 (m, 1H), 4.45−4.59 (m, 1H), 6.57 (s, 1H),
6.77 (d, J = 8.5 Hz, 2H), 7.46 (t, J = 8.4 Hz, 1H). MS (ESI) m/z: calcd
for free base C28H38N4O5, 510.28 [M]+; found, 511.5 [M + H]+; [α]2D5
−41.81 (c 0.055, MeOH).
(S)-5-(Azepan-1-yl)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-
1H-pyrazole-3-carboxamido)pentanoic Acid Hydrochloride (11).
Compound 11 (60 mg) was synthesized using tert-butyl (S)-3-(5-(2,6-
dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxamido)-5-oxo-
pentanoate 38 (60 mg, 0.123 mmol) and azepane (11 mg, 0.148 mmol)
as described in procedure 4. 91% yield; white solid; 1H NMR (MeOH-
d4, 300 MHz): δ 1.51−1.68 (m, 2H), 1.69−1.83 (m, 4H), 1.84−2.29
(m, 10H), 2.79 (d, J = 6.40 Hz, 2H), 3.25−3.38 (m, 4H), 3.43−3.58
(m, 3H), 3.63−3.69 (m, 2H), 3.76 (s, 6H), 4.26−4.38 (m, 1H), 4.42−
4.53 (m, 1H), 6.57 (s, 1H), 6.73−6.81 (m, 2H), 7.46 (t, J = 8.3 Hz, 1H).
MS (ESI) m/z: calcd for free base C28H40N4O5, 512.30 [M]+; found,
513.7 [M + H]+; [α]D25 −21.43 (c 0.210, MeOH).
(S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(pyrrolidin-1-yl)pentanoic Acid Hydrochloride (5).
Compound 5 (60 mg) was synthesized using tert-butyl (S)-3-(5-(2,6-
dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxamido)-5-oxo-
pentanoate 38 (60 mg, 0.123 mmol) and pyrrolidine (11 mg, 0.148
mmol) as described in procedure 4. 92% yield; white solid; 1H NMR
(MeOH-d4, 300 MHz): δ 1.51−1.69 (m, 2H), 1.83−1.99 (m, 4H),
1.99−2.26 (m, 7H), 2.78 (d, J = 6.4 Hz, 2H), 3.02−3.18 (m, 1H),
3.21−3.35 (m, 3H), 3.45−3.57 (m, 1H), 3.62−3.71 (m, 4H), 3.76 (s,
6H), 4.26−4.37 (m, 1H), 4.47−4.59 (s, 1H), 6.57 (s, 1H), 6.77 (d, J =
8.5 Hz, 2H), 7.46 (t, J = 8.4 Hz, 1H). MS (ESI) m/z: calcd for free base
C26H36N4O5, 484.27 [M]+; found, 485.1 [M + H]+. [α]2D5 −26.15 (c
0.260, MeOH).
(S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(piperidin-1-yl)pentanoic Acid Hydrochloride (6).
Compound 6 (37 mg) was synthesized using tert-butyl (S)-3-(5-(2,6-
dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxamido)-5-oxo-
pentanoate 38 (42 mg, 0.086 mmol) and piperidine (10 mg, 0.095
mmol) as described in procedure 4. 96% yield; off-white solid; 1H NMR
(CDCl3, 300 MHz): δ 1.37−1.63 (m, 3H), 1.80−1.98 (m, 8H), 1.98−
2.12 (m, 2H), 2.13−2.37 (m, 2H), 2.62−2.78 (m, 2H), 2.82 (d, J = 8.5
Hz, 2H), 3.11−3.28 (m, 2H), 3.54−3.71 (m, 3H), 3.76 (s, 3H), 3.74 (s,
3H), 4.21−4.35 (m, 1H), 4.36−4.55 (m, 1H), 6.64 (d, J = 8.67 Hz,
3H), 7.40 (t, J = 8.5 Hz, 1H), 7.95 (d, J = 9.0 Hz, 1H), 10.01 (br s, 1H).
MS (ESI) m/z: calcd for free base C27H38N4O5, 498.28 [M]+; found,
499.8 [M + H]+; [α]D25 −36.00 (c 0.100, MeOH).
(S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-morpholinopentanoic Acid Hydrochloride (7).
Compound 7 (50 mg) was synthesized using tert-butyl (S)-3-(5-(2,6-
dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxamido)-5-oxo-
pentanoate 38 (60 mg, 0.123 mmol) and morpholine (13 mg, 0.148
mmol) as described in procedure 4. 94% yield; white solid; 1H NMR
(MeOH-d4, 300 MHz): δ 1.53−1.69 (m, 3H), 1.85−2.00 (m, 5H),
2.00−2.14 (m, 2H), 2.16−2.30 (m, 1H), 2.80 (d, J = 6.4 Hz, 2H),
3.11−3.29 (m, 5H), 3.65−3.69 (m, 4H), 3.76 (s, 6H), 3.83−4.08 (m,
3H), 4.24−4.41 (m, 1H), 4.43−4.59 (m, 1H), 6.57 (s, 1H), 6.77 (d, J =
8.5 Hz, 1H), 7.46 (t, J = 8.5 Hz, 1H). MS (ESI) m/z: calcd for free base
C26H36N4O6, 500.26 [M]+; found, 501.9 [M + H]+; [α]2D5 −38.46 (c
0.065, MeOH).
(S)-N-(1-(Cyclobutylamino)-1-oxo-5-(pyrrolidin-1-yl)pentan-3-
yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxa-
mide Hydrochloride (12). To a solution of (S)-3-(1-cyclopentyl-5-
(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(pyrrolidin-1-
yl)pentanoic acid hydrochloride 5 (60 mg, 0.123 mol) and cyclobutyl-
amine (12 mg, 0.161 mmol) in MeCN (5 mL) were added
triethylamine (0.05 mL, 0.558 mmol) and HBTU (70 mg, 0.184
mmol). After stirring at RT for 4 h, the reaction mixture was diluted
with DCM (75 mL) and washed with saturated aqueous sodium
bicarbonate (75 mL). The layers were separated, and the aqueous layer
was extracted with DCM (2 × 50 mL). The combined organic layers
were further washed with brine, dried with Na2SO4, filtered, and
concentrated in vacuo to give the crude product. The crude product was
purified by silica gel flash column chromatography (0−40% DCM/
MeOH/aq. NH4OH (100:20:2)/DCM) to provide (S)-N-(1-(cyclo-
butylamino)-1-oxo-5-(pyrrolidin-1-yl)pentan-3-yl)-1-cyclopentyl-5-
(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamide (42 mg, 64%) as a
white solid. The free base (11 mg, 0.020 mmol) was mixed with MeOH
(2 mL). To it was added 2 M HCl solution in ether (0.05 mL) dropwise
at room temperature and stirred for 45 min. MeOH was evaporated in
vacuo and dried under high vacuum to remove all the HCl. To it was
added ether, followed by trituration and filtration to obtain a white
solid. The solid product was then dried under high vacuum for 16 h to
(S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(4-methylpiperazin-1-yl)pentanoic Acid Dihydro-
chloride (8). Compound 8 (40 mg) was synthesized using tert-butyl
(S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carbox-
amido)-5-oxopentanoate 38 (60 mg, 0.123 mmol) and 1-methylpiper-
azine (15 mg, 0.148 mmol) as described in procedure 4. 95% yield;
1
give the title compound (10 mg, 85%) as a white solid. Free base H
NMR (CDCl3, 300 MHz): δ 1.47−1.72 (m, 3H), 1.73−1.98 (m, 11H),
1.99−2.16 (m, 3H), 2.17−2.38 (m, 3H), 2.44−2.83 (m, 8H), 3.73 (s,
3H), 3.74 (m, 3H), 4.16−4.32 (m, 1H), 4.32−4.50 (m, 2H), 6.63 (d, J
= 8.5 Hz, 2H), 6.66 (s, 1H), 7.01 (d, J = 6.4 Hz, 1H), 7.38 (t, J = 8.4 Hz,
1H), 8.03 (d, J = 7.7 Hz, 1H). MS (ESI) m/z: calcd for free base
C30H43N5O4, 537.33 [M]+; found, 538.5 [M + H]+; [α]2D5 −19.75 (c
0.415, MeOH).
(S)-N-(1-(Cyclobutylamino)-5-morpholino-1-oxopentan-3-yl)-1-
cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamide
Hydrochloride (14). Compound 14 (32 mg) was synthesized using (S)-
3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxami-
do)-5-morpholinopentanoic acid hydrochloride 7 (50 mg, 0.093
mmol) and cyclobutylamine (9 mg, 0.12 mmol) as described in 12.
60% yield; white solid; Free base 1H NMR (CDCl3, 300 MHz): δ 1.46−
1.74 (m, 7H), 1.78−1.99 (m, 7H), 2.01−2.16 (m, 2H), 2.17−3.36 (m,
2H), 2.37−2.71 (m, 8H), 3.70−3.82 (m, 2H), 3.73 (s, 3H), 3.74 (s,
3H), 4.20−4.51 (m, 3H), 6.63 (d, J = 8.5 Hz, 2H), 6.67 (s, 1H), 6.74−
6.83 (m, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.88−7.97 (m, 1H). MS (ESI)
m/z: calcd for free base C30H43N5O5, 553.33 [M]+; found, 555.0 [M +
H]+; [α]2D5 −27.20 (c 0.125, MeOH).
1
white solid; H NMR (MeOH-d4, 300 MHz): δ 1.52−1.68 (m, 2H),
2.10−1.83 (m, 6H), 2.09−2.32 (m, 2H), 2.82 (d, J = 6.4 Hz, 2H), 3.06
(s, 3H), 3.41 (t, J = 7.0 Hz, 2H), 3.48−3.96 (m, 4H), 3.63−3.71 (m,
5H), 3.76 (s, 6H), 4.26−4.37 (m, 1H), 4.47−4.59 (m, 1H), 6.59 (s,
1H), 6.77 (d, J = 8.5 Hz, 2H), 7.46 (t, J = 8.4 Hz, 1H). MS (ESI) m/z:
calcd for free base C27H39N5O5, 513.30 [M]+; found, 514.8 [M + H]+;
[α]2D5 −24.31 (c 0.255, MeOH).
(3S)-3-(1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(2,6-dimethylpiperidin-1-yl)pentanoic Acid Hydro-
chloride (9). Compound 9 (26 mg) was synthesized using tert-butyl
(S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-carbox-
amido)-5-oxopentanoate 38 (60 mg, 0.123 mmol) and 2,6-
dimethylpiperidine (17 mg, 0.148 mmol) as described in procedure
1
4. 90% yield; white solid; H NMR (MeOH-d4, 300 MHz): δ 1.28−
1.50 (m, 6H), 1.51−1.82 (m, 5H), 1.82−2.30 (m, 9H), 2.72 (d, J = 6.4
Hz, 4H), 3.35−3.49 (m, 3H), 3.63−3.71 (m, 2H), 3.76 (s, 6H), 4.26−
4.39 (m, 1H), 4.40−4.54 (m, 1H), 6.46−6.62 (m, 1H), 6.77 (d, J = 8.5
Hz, 2H), 7.46 (t, J = 8.3 Hz, 1H). MS (ESI) m/z: calcd for free base
C29H42N4O5, 526.32 [M]+; found, 527.7 [M + H]+.
(S)-N-(1-(Cyclobutylamino)-5-(4-methylpiperazin-1-yl)-1-oxo-
pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-
3-carboxamide Dihydrochloride (15). Compound 15 (19 mg) was
synthesized using (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxamido)-5-(4-methylpiperazin-1-yl)pentanoic acid di-
(S)-5-(7-Azabicyclo[2.2.1]heptan-7-yl)-3-(1-cyclopentyl-5-(2,6-di-
methoxyphenyl)-1H-pyrazole-3-carboxamido)pentanoic Acid Hy-
drochloride (10). Compound 10 (60 mg) was synthesized using tert-
butyl (S)-3-(5-(2,6-dimethoxyphenyl)-1-cyclopentyl-1H-pyrazole-3-
carboxamido)-5-oxopentanoate 38 (60 mg, 0.123 mmol) and 7-
3018
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hydrochloride 8 (40 mg, 0.068 mmol) and cyclobutylamine (7 mg,
0.088 mmol) as described in 12. 49% yield; white solid; free base H
the aqueous layer was extracted with EtOAc (3 × 50 mL). The
combined organic layers were further washed with brine (50 mL). The
organic layer was separated, dried with Na2SO4, filtered, and
concentrated in vacuo to give the crude product. The crude product
was purified by silica gel flash column chromatography (0−30% DCM/
MeOH/aq. NH4OH (100:20:2)/DCM). Purified fractions were
combined and concentrated to afford pure product 20 as a white
solid (30 mg). 68% yield; white solid; 1H NMR (CDCl3, 300 MHz): δ
1.38−1.50 (m, 2H), 1.51−1.77 (m, 8H), 1.82−1.99 (m, 5H), 2.00−
2.16 (m, 2H), 2.32−2.71 (m, 9H), 2.80−2.99 (m, 3H), 3.73 (s, 2H),
3.74 (s, 3H), 4.16−4.31 (m, 2H), 4.46 (m, 1H), 6.63 (d, J = 8.3 Hz,
2H), 6.67 (s, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.66 (d, J = 6.8 Hz, 1H), 8.20
(d, J = 8.3 Hz, 1H). MS (ESI) m/z: calcd for C31H43F2N5O4, 587.33
[M]+; found, 588.4 [M + H]+; [α]2D5 −23.07 (c 0.065, MeOH).
(S)-N-(1-(Cyclobutyl(methyl)amino)-1-oxo-5-(piperidin-1-yl)-
pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-
3-carboxamide (21). Compound 21 (18 mg) was synthesized from
(S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbox-
amido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35 mg,
0.065 mmol) and N-methylcyclobutanamine (7 mg, 0.084 mmol) using
procedure as described in 20. 49% yield; white solid; 1H NMR (CDCl3,
300 MHz): δ 1.40−1.77 (m, 8H), 1.82−1.98 (m, 6H), 2.00−2.30 (m,
7H), 2.35−2.73 (m, 7H), 2.86−3.05 (m, 2H), 2.91 (s, 3H), 3.72 (s,
6H), 4.18−4.29 (m, 1H), 4.33−4.46 (m, 1H), 4.49−4.66 (m, 1H), 6.62
(d, J = 8.5 Hz, 2H), 6.66 (d, J = 3.0 Hz, 1H), 7.37 (t, J = 8.4 Hz, 1H),
8.05−8.23 (m, 1H). MS (ESI) m/z: calcd for C32H47N5O4, 565.36
[M]+; found, 566.4 [M + H]+; [α]2D5 −5.18 (c 0.135, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-((1-
methylcyclobutyl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1H-
pyrazole-3-carboxamide (22). Compound 22 (30 mg) was synthe-
sized from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochloride
6 (40 mg, 0.074 mmol) and 1-methylcyclobutan-1-amine (8 mg, 0.097
mmol) using procedure as described in 20. 71% yield; off-white solid;
1H NMR (CDCl3, 300 MHz): δ 1.42 (s, 3H), 1.54−1.81 (m, 5H),
1.81−2.13 (m, 12H), 2.17−2.31 (m, 3H), 2.32−2.64 (m, 10H), 3.72
(m, 3H), 3.73 (m, 3H), 4.24 (dt, J = 15.4, 7.7 Hz, 1H), 4.30−4.42 (m,
1H), 6.55 (s, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.67 (s, 1H), 7.37 (t, J = 8.4
Hz, 1H), 8.04 (d, J = 7.9 Hz, 1H). MS (ESI) m/z: calcd for
C32H47N5O4, 565.36 [M]+; found, 566.4 [M + H]+; [α]2D5 −14.00 (c
0.100, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-((3-methyloxe-
tan-3-yl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1H-pyrazole-
3-carboxamide (23). Compound 23 (20 mg) was synthesized from
(S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbox-
amido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (40 mg,
0.075 mmol) and 3-methyloxetan-3-amine (9 mg, 0.097 mmol) using
procedure as described in 20. 48% yield; white solid; 1H NMR (CDCl3,
300 MHz): δ 1.39−1.59 (m, 5H), 1.69 (s, 3H), 1.55−1.75 (m, 5H),
1.80−2.00 (m, 5H), 2.00−2.19 (m, 2H), 2.30−2.52 (m, 5H), 2.52−
2.72 (m, 3H), 3.74 (s, 6H), 4.19−4.30 (m, 1H), 4.37−4.48 (m, 3H),
4.78 (d, J = 6.4 Hz, 2H), 6.63 (d, J = 8.5 Hz, 1H), 6.66 (s, 1H), 7.38 (t, J
= 8.4 Hz, 1H), 7.57−7.70 (m, 1H), 8.23 (d, J = 8.1 Hz, 1H). MS (ESI)
m/z: calcd for C31H45N5O5, 567.34 [M]+; found, 568.7 [M + H]+; [α]2D5
= −18.18 (c 0.110, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-((1-
methylcyclopropyl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-
1H-pyrazole-3-carboxamide (24). Compound 24 (18 mg) was
synthesized from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochlor-
ide 6 (40 mg, 0.075 mmol) and 1-methylcyclopropan-1-amine (11 mg,
0.097 mmol) using procedure as described in 20. 44% yield; white solid;
1H NMR (CDCl3, 300 MHz): δ 0.54−0.62 (m, 2H), 0.66−0.75 (m,
1
NMR (CDCl3, 300 MHz): δ 1.46−1.73 (m, 9H), 1.74−1.99 (m, 6H),
2.00−2.16 (m, 2H), 2.17−2.36 (m, 4H), 2.37−2.67 (m, 9H), 3.73 (s,
3H), 3.74 (s, 3H), 4.18−4.46 (m, 3H), 6.63 (d, J = 8.5 Hz, 2H), 6.67 (s,
1H), 6.71−6.78 (m, 1H), 7.23−7.29 (m, 1H), 7.38 (t, J = 8.4 Hz, 1H),
7.53 (d, J = 8.1 Hz, 1H). MS (ESI) m/z: calcd for free base
C31H46N6O4, 566.36 [M]+; found, 567.5 [M + H]+; [α]2D5 −40.80 (c
0.125, MeOH).
N-((3S)-1-(Cyclobutylamino)-5-(2,6-dimethylpiperidin-1-yl)-1-ox-
opentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carboxamide hydrochloride (16). Compound 16 (15 mg) was
synthesized using (3S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-
1H-pyrazole-3-carboxamido)-5-(2,6-dimethylpiperidin-1-yl)pentanoic
acid hydrochloride 9 (32 mg, 0.057 mmol) and cyclobutylamine (6 mg,
0.074 mmol) as described in 12. 45% yield; white solid. Free base 1H
NMR (CDCl3, 300 MHz): δ 1.15 (m, 6H), 1.22−1.42 (m, 5H), 1.44−
1.75 (m, 8H), 1.75−1.99 (m, 8H), 2.18−2.36 (m, 3H), 2.55 (d, J = 6.6
Hz, 2H), 2.78−3.10 (s, 3H), 3.73 (s, 3H), 3.74 (s, 3H), 4.13−4.30 (s,
2H), 4.32−4.51 (m, 2H), 6.63 (d, J = 8.1 Hz, 2H), 6.66 (s, 1H), 7.38 (t,
J = 8.4 Hz, 1H). MS (ESI) m/z: calcd for C33H49N5O4, 579.38 [M]+;
found, 580.8 [M + H]+; [α]2D5 −21.53 (c 0.065, MeOH).
(S)-N-(5-(7-Azabicyclo[2.2.1]heptan-7-yl)-1-(cyclobutylamino)-1-
oxopentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carboxamide Hydrochloride (17). Compound 17 (13 mg) was
synthesized using (S)-5-(7-azabicyclo[2.2.1]heptan-7-yl)-3-(1-cyclo-
pentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-
pentanoic acid hydrochloride 10 (35 mg, 0.064 mmol) and cyclobutyl-
amine (6 mg, 0.083 mmol) as described in 12. 36% yield; white solid;
free base 1H NMR (CDCl3, 300 MHz): δ 1.18−1.40 (m, 8H), 1.46−
1.72 (m, 7H), 1.72−1.96 (m, 4H), 1.98−2.16 (m, 2H), 2.18−2.37 (m,
2H), 2.45−2.72 (m, 4H), 3.27−3.37 (m, 2H), 3.72 (s, 3H), 3.74 (s,
3H), 4.17−4.30 (m, 2H), 4.31−4.52 (m, 2H), 6.63 (d, J = 8.5 Hz, 2H),
6.67 (s, 1H), 7.04 (d, J = 8.1 Hz, 1H), 7.37 (t, J = 8.2 Hz, 1H), 8.06 (d, J
= 8.3 Hz, 1H). MS (ESI) m/z: calcd for free base C32H45N5O4, 563.35
[M]+; found, 564.5 [M + H]+; [α]2D5 −42.00 (c 0.100, MeOH).
(S)-N-(5-(Azepan-1-yl)-1-(cyclobutylamino)-1-oxopentan-3-yl)-
1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxa-
mide Hydrochloride (18). Compound 18 (30 mg) was synthesized
using (S)-5-(azepan-1-yl)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-
1H-pyrazole-3-carboxamido)pentanoic acid hydrochloride 11 (60 mg,
0.109 mmol) and cyclobutylamine (10 mg, 0.142 mmol) as described in
12. 48% yield; off-white solid; Free base 1H NMR (CDCl3, 300 MHz):
δ 1.44−1.74 (m, 11H), 1.78−1.98 (m, 7H), 1.99−2.18 (m, 3H), 2.18−
2.36 (m, 3H), 2.48−2.78 (m, 8H), 3.73 (s, 6H), 4.18−4.30 (m, 1H),
4.32−4.49 (m, 2H), 6.62 (d, J = 8.5 Hz, 2H), 6.67 (s, 1H), 7.03 (d, J =
8.5 Hz, 1H), 7.38 (t, J = 8.4 Hz, 1H), 8.05 (d, J = 8.3 Hz, 1H). MS (ESI)
m/z: calcd for free base C32H47N5O4, 565.36 [M]+; found, 566.5 [M +
H]+; [α]2D5 −32.00 (c 0.100, MeOH).
(S)-Cyclobutyl 3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyr-
azole-3-carboxamido)-5-(piperidin-1-yl)pentanoate (19). Com-
pound 19 (21 mg) was synthesized from (S)-3-(1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(piperidin-1-yl)-
pentanoic acid hydrochloride 6 (35 mg, 0.065 mmol) and cyclobutanol
(6 mg, 0.084 mmol) using procedure as described in 20. 58% yield;
white solid; 1H NMR (CDCl3, 300 MHz): δ 1.37−1.49 (m, 2H), 1.50−
1.68 (m, 6H), 1.73−1.98 (m, 9H), 2.00−2.18 (m, 4H), 2.27−2.52 (m,
7H), 2.54−2.78 (m, 2H), 3.73 (s, 3H), 3.74 (m, 3H), 4.20−4.30 (m,
1H), 4.45−4.57 (m, 1H), 5.01 (quin, J = 7.6 Hz, 1H), 6.63 (d, J = 8.3
Hz, 2H), 6.67 (s, 1H), 7.37 (t, J = 8.5 Hz, 1H), 7.73 (d, J = 8.9 Hz, 1H).
MS (ESI) m/z: calcd for C31H44N4O5, 552.33 [M]+; found, 553.8 [M +
H]+; [α]2D5 −11.02 (c 0.100, MeOH).
(S)-1-Cyclopentyl-N-(1-((3,3-difluorocyclobutyl)amino)-1-oxo-5-
(piperidin-1-yl)pentan-3-yl)-5-(2,6-dimethoxyphenyl)-1H-pyrazole-
3-carboxamide (20). To a solution of crude (S)-3-(5-(2,6-dimethox-
yphenyl)-1-cyclopentyl-1H-pyrazole-3-carboxamido)-5-(piperidin-1-
yl)pentanoic acid hydrochloride 6 (40 mg, 0.075 mmol) and 3,3-
difluorocyclobutan-1-amine (14 mg, 0.097 mmol) in MeCN (4 mL)
were added triethylamine (0.06 mL, 0.45 mmol) and HBTU (43 mg,
0.112 mmol). After stirring at room temperature for 4 h, the reaction
mixture was diluted with water (50 mL). The layers were separated, and
2H), 0.80−0.98 (m, 1H), 1.34 (s, 3H), 1.40−1.59 (m, 4H), 1.59−1.79
(m, 5H), 1.80−1.99 (m, 5H), 2.00−2.17 (m, 2H), 2.39−2.68 (m, 7H),
3.73 (s, 6H), 4.19−4.40 (m, 2H), 6.63 (d, J = 8.48 Hz, 2H), 6.65 (s,
1H), 6.89 (br s, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.98 (d, J = 7.9 Hz, 1H).
MS (ESI) m/z: calcd for C31H45N5O4, 551.35 [M]+; found, 552.9 [M +
H]+; [α]2D5 = −18.00 (c 0.100, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-((2-
methoxyethyl)(methyl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-
3019
https://dx.doi.org/10.1021/acs.jmedchem.0c01448
J. Med. Chem. 2021, 64, 3006−3025

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
yl)-1H-pyrazole-3-carboxamide (25). Compound 25 (24 mg) was
synthesized from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochlor-
ide 6 (35 mg, 0.065 mmol) and 2-methoxy-N-methylethan-1-amine (7
mg, 0.084 mmol) using procedure as described in 20. 65% yield;
zole-3-carboxamide (30). Compound 30 (7 mg) was synthesized from
(S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbox-
amido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (30 mg,
0.056 mmol) and N-methylthiazol-2-amine (8.3 mg, 0.072 mmol)
1
using procedure as described in 20. 22% yield; white solid; H NMR
1
colorless foam; H NMR (CDCl3, 300 MHz): δ 1.38−1.58 (m, 3H),
(CDCl3, 300 MHz): δ 1.38−1.49 (br s, 2H), 1.49−1.71 (m, 5H), 1.81−
1.98 (m, 5H), 1.98−2.18 (m, 3H), 2.33−2.53 (m, 5H), 2.55−2.68 (m,
1H), 2.77−2.94 (m, 1H), 3.28−3.40 (m, 2H), 3.73 (s, 3H), 3.72 (s,
3H), 3.85 (s, 3H), 4.18−4.32 (m, 1H), 4.50−4.61 (m, 1H), 6.63 (d, J =
8.5 Hz, 2H), 6.68 (s, 1H), 6.99 (d, J = 3.4 Hz, 1H), 7.37 (t, J = 8.4 Hz,
1H), 7.51 (d, J = 3.6 Hz, 1H), 8.31 (d, J = 7.0 Hz, 1H). MS (ESI) m/z:
1.58−1.75 (m, 3H), 1.80−2.21 (m, 10H), 2.35−2.53 (m, 4H), 2.53−
2.69 (m, 2H), 2.88−3.01 (m, 2H), 3.11−3.20 (m, 2H), 3.33 (d, J = 6.0
Hz, 3H), 3.46−3.55 (m, 2H), 3.49 (s, 3H), 3.73 (s, 6H), 4.19−4.29 (m,
1H), 4.37−4.52 (m, 1H), 6.62 (d, J = 8.3 Hz, 2H), 6.66 (s, 1H), 7.37 (t,
J = 8.4 Hz, 1H), 8.03−8.14 (m, 1H). MS (ESI) m/z: calcd for
C31H47N5O5, 569.36 [M]+; found, 570.9 [M + H]+; [α]2D5 = −15.29 (c
0.085, MeOH).
calcd for C31H42N6O4S, 594.30 [M]+; found, 595.6 [M + H]+; [α]2D5
−5.71 (c 0.105, MeOH).
=
(S)-N-(1-((1H-Tetrazol-5-yl)amino)-1-oxo-5-(piperidin-1-yl)-
pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-
3-carboxamide (31). Compound 31 (18 mg) was synthesized from
(S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbox-
amido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35 mg,
0.065 mmol) and 1H-tetrazol-5-amine (7.2 mg, 0.085 mmol) using
procedure as described in 20. 49% yield; white solid; 1H NMR (CDCl3,
300 MHz): δ 1.47−1.61 (m, 3H), 1.73−2.02 (m, 9H), 2.03−2.22 (m,
3H), 2.59−2.91 (m, 4H), 2.99−3.22 (m, 3H), 3.24−3.40 (m, 2H), 3.73
(s, 3H), 3.77 (s, 3H), 4.19−4.31 (m, 2H), 4.64−4.81 (s, 1H), 6.63 (dd,
J = 8.5, 3.20 Hz, 2H), 6.69 (s, 1H), 7.38 (t, J = 8.5 Hz, 1H), 7.61 (s, 1H).
13.63 (br s, 1H). (MS ESI) m/z: calcd for C28H39N9O4, 565.31 [M]+;
found, 566.5 [M + H]+; [α]2D5 = −36.00 (c 0.125, MeOH).
(S)-N-(1-(((1H-Tetrazol-5-yl)methyl)amino)-1-oxo-5-(piperidin-1-
yl)pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carboxamide (32). Compound 32 (32 mg) was synthesized
from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35
mg, 0.065 mmol) and (1H-tetrazol-5-yl)methanamine (8.4 mg, 0.085
mmol) using procedure as described in 20. 84% yield; off-white solid;
1H NMR (CDCl3, 300 MHz): δ 1.47−1.58 (m, 2H), 1.58−1.73 (br s,
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-((2-
methoxyethyl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1H-pyr-
azole-3-carboxamide (26). Compound 26 (19 mg) was synthesized
from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35
mg, 0.065 mmol) and 2-methoxyethan-1-amine (6.4 mg, 0.085 mmol)
using procedure as described in 20. 52% yield; colorless foam; 1H NMR
(CDCl3, 300 MHz): δ 1.39−1.58 (m, 4H), 1.67 (d, J = 4.9 Hz, 5H),
1.81−1.99 (m, 6H), 2.01−2.18 (m, 2H), 2.36−2.75 (m, 7H), 3.29 (m,
3H), 3.38−3.52 (m, 4H), 3.72 (s, 3H), 3.74 (s, 3H), 4.18−4.32 (m,
1H), 4.36−4.49 (m, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.66 (s, 1H), 6.81 (br
s, 1H), 7.37 (t, J = 8.4 Hz, 1H), 8.00 (d, J = 7.9 Hz, 1H). MS (ESI) m/z:
calcd for C30H45N5O5, 555.34 [M]+; found, 557.1 [M + H]+; [α]2D5
−12.29 (c 0.740, MeOH).
=
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-oxo-5-(piperi-
din-1-yl)-1-(2-oxa-6-azaspiro[3.3]heptan-6-yl)pentan-3-yl)-1H-pyr-
azole-3-carboxamide (27). Compound 27 (25 mg) was synthesized
from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35
mg, 0.065 mmol) and 2-oxa-6-azaspiro[3.3]heptane (7.2 mg, 0.085
mmol) using procedure as described in 20. 66% yield; white solid; 1H
NMR (CDCl3, 300 MHz): δ 1.43 (m, 2H), 1.51−1.70 (m, 6H), 1.79−
2.00 (m, 6H), 2.00−2.16 (m, 2H), 2.31−2.45 (m, 6H), 2.48−2.68 (m,
2H), 3.73 (s, 6H), 4.13 (s, 2H), 4.18−4.47 (m, 4H), 4.71 (s, 2H),
4.65−4.77 (m, 2H), 6.67 (s, 1H), 6.63 (d, J = 8.3 Hz, 2H), 7.37 (t, J =
8.4 Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H). MS (ESI) m/z: calcd for
C32H45N5O5, 579.34 [M]+; found, 580.7 [M + H]+; [α]2D5 = −7.87 (c
0.080, MeOH).
2H), 1.81−2.16 (m, 10H), 2.94−3.04 (m, 4H), 3.06−3.34 (m, 4H),
3.49 (s, 2H), 3.74 (m, 6H), 4.17−4.32 (m, 2H), 4.37−4.50 (m, 1H),
4.56−4.70 (m, 2H), 6.64 (d, J = 8.5 Hz, 2H), 6.62 (m, 1H), 7.37 (t, J =
8.4 Hz, 1H), 8.21−8.24 (m, 1H), 8.88−8.98 (m, 1H). MS (ESI) m/z:
calcd for C29H41N9O4, 579.33 [M]+; found, 580.7 [M + H]+; [α]2D5
−21.66 (c 0.060, MeOH).
=
(S)-N-(1-((2-(1H-Tetrazol-5-yl)ethyl)amino)-1-oxo-5-(piperidin-1-
yl)pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyra-
zole-3-carboxamide (33). Compound 33 (25 mg) was synthesized
from (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35
mg, 0.065 mmol) and 2-(1H-tetrazol-5-yl)ethan-1-amine (10 mg, 0.085
mmol) using procedure as described in 20. 64% yield; white solid; 1H
NMR (CDCl3, 300 MHz): δ 1.46−1.58 (m, 2H), 1.60−1.75 (br s, 2H),
1.81−2.16 (m, 8H), 2.37−2.68 (m, 11H), 3.04−3.28 (m, 6H), 3.55−
3.68 (m, 1H), 3.74 (s, 3H), 3.73 (s, 3H), 4.16−4.31 (m, 1H), 4.33−
4.46 (m, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.66 (s, 1H), 7.38 (t, J = 8.4 Hz,
1H), 7.55 (br s, 1H). MS (ESI) m/z: calcd for C30H43N9O4, 593.34
[M]+; found, 594.5 [M + H]+; [α]2D5 = −42.00 (c 0.100, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(4-(piperidin-1-yl)-
1-(4H-1,2,4-triazol-3-yl)butan-2-yl)-1H-pyrazole-3-carboxamide
(34). Step 1: Synthesis of (S)-N-(1-amino-1-oxo-5-(piperidin-1-
yl)pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-
3-carboxamide (48): To a solution of (S)-3-(1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(piperidin-1-yl)-
pentanoic acid hydrochloride 6 (1.0 g, 1.8 mmol) in pyridine (20 mL)
and dioxane (20 mL) were added ammonium carbonate (0.21 g, 2.7
mmol), followed by Boc2O (0.47 g, 2.16 mmol). The progress of the
reaction was monitored by LC−MS. Additional ammonium carbonate
and Boc2O (2 equiv each) were added after 1 h. Stirring was continued
for 12 h, and additional ammonium carbonate (2 equiv) and Boc2O (2
equiv) were added. After further stirring for 1 h, LC−MS indicated the
completion of the reaction. The mixture was concentrated to dryness,
quenched with saturated aqueous NaHCO3 (20 mL), and extracted
with EtOAc (20 mL). The EtOAc layer was separated and dried over
Na2SO4. The solution was filtered, concentrated, and purified using
silica gel flash chromatography using 0−15% MeOH in a DCM (with
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-((oxazol-2-
ylmethyl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1H-pyrazole-
3-carboxamide (28). Compound 28 (27 mg) was synthesized from
(S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carbox-
amido)-5-(piperidin-1-yl)pentanoic acid hydrochloride 6 (35 mg,
0.065 mmol) and oxazol-2-ylmethanamine (8 mg, 0.084 mmol) using
procedure as described in 20. 72% yield; white solid; 1H NMR (CDCl3,
300 MHz): δ 1.37−1.73 (m, 8H), 1.77−1.98 (m, 6H), 2.00−2.18 (m,
2H), 2.33−2.80 (m, 8H), 3.74 (s, 6H), 4.24 (quin, J = 7.6 Hz, 1H),
4.42−4.53 (m, 1H), 4.58 (d, J = 5.7 Hz, 2H), 6.63 (d, J = 8.48 Hz, 2H),
6.65 (s, 1H), 6.99 (s, 1H), 7.38 (t, J = 8.4 Hz, 1H), 7.45−7.54 (m, 1H),
7.71 (t, J = 5.65 Hz, 1H), 8.07−8.21 (m, 1H). MS (ESI) m/z: calcd for
C31H42N6O5, 578.32 [M]+; found, 579.9 [M + H]+; [α]2D5 = −20.95 (c
0.105, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-oxo-5-(piperi-
din-1-yl)-1-(thiazol-2-ylamino)pentan-3-yl)-1H-pyrazole-3-carbox-
amide (29). Compound 29 (19 mg) was synthesized from (S)-3-(1-
cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-
(piperidin-1-yl)pentanoic acid hydrochloride 6 (40 mg, 0.074 mmol)
and thiazol-2-amine (9.7 mg, 0.097 mmol) using procedure as
described in 20. 44% yield; white solid; 1H NMR (CDCl3, 300
MHz): δ 1.40−1.59 (m, 4H), 1.68 (m, 6H), 1.80−1.97 (m, 6H), 2.00−
2.19 (m, 2H), 2.35−2.66 (m, 5H), 2.77 (dd, J = 15.2, 6.1 Hz, 1H), 2.98
(dd, J = 15.3, 5.3 Hz, 1H), 3.73 (s, 6H), 4.19−4.31 (m, 1H), 4.42−4.56
(m, 1H), 6.63 (d, J = 8.5 Hz, 2H), 6.74 (s, 1H), 6.93 (d, J = 3.6 Hz, 1H),
7.38 (t, J = 8.3 Hz, 1H), 7.47 (d, J = 3.6 Hz, 1H), 8.41 (br s, 1H). MS
(ESI) m/z: calcd for C30H40N6O4S, 580.28 [M]+; found, 582.0 [M +
H]+; [α]2D5 = −25.00 (c 0.020, MeOH).
(S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-(methyl-
(thiazol-2-yl)amino)-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1H-pyra-
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1% NH3) solvent system to give the title product 48 (0.64 g, 69% yield)
as a white solid; 1H NMR (CDCl3, 200 MHz): δ 1.50−2.20 (m, 11H),
2.20−2.80 (m, 8H), 3.15−3.50 (m, 2H), 3.72 (s, 6H), 4.15−4.30 (m,
1H), 4.30−4.50 (m, 1H), 5.37 (br s, 1H), 6.62 (d, J = 8.4 Hz, 2H), 6.65
(s, 1H), 7.18 (br s, 1H), 7.20−7.40 (m, 2H), 7.64 (t, J = 7.0 Hz, 1H),
8.13 (d, J = 8.4 Hz, 1H), 8.61 (d, J = 4.0 Hz, 1H). MS (ESI): m/z calcd
for C27H39N5O4, [M]+ 497.30; found, 498.3 [M + H]+.
yl)-1H-pyrazole-3-carboxamide 50 (50 mg, 0.10 mmol) in CH(OMe)3
was added PTSA.H2O (25 mg, 0.13 mmol). The mixture was heated at
85 °C for 2 h, cooled to RT, diluted with EtOAc (10 mL), and washed
with saturated aqueous NaHCO3 (10 mL). The EtOAc solution was
dried over Na2SO4, concentrated, and purified by flash silica gel
chromatography using 0−15% MeOH in DCM (with 1% NH3) to give
the title product 35 (30 mg, 59%) as white foam; 1H NMR (CDCl3, 200
MHz): δ 1.40−2.20 (m, 18H), 2.20−2.80 (m, 4H), 3.15−3.50 (m,
2H), 3.73 (s, 6H), 4.15−4.30 (m, 1H), 4.30−4.50 (m, 1H), 6.62 (d, J =
8.0 Hz, 2H), 6.65 (s, 1H), 7.37 (t, J = 8.4 Hz, 1H), 7.90 (d, J = 8.4 Hz,
1H), 8.36 (s, 1H). MS (ESI): m/z calcd for C28H38N6O4 [M]+, 522.30;
found, 523.2 [M + H]+.
Bioassays. Lance cAMP Accumulation Assay. Stimulation buffer
containing 1× Hank’s Balanced Salt Solution (HBSS), 5 mM HEPES,
0.1% BSA stabilizer, and 0.5 mM final IBMX was prepared and titrated
to pH 7.4 at room temperature. Serial dilutions of the test compounds
and 300 nM forskolin, both prepared at 4× the desired final
concentration in stimulation buffer, were added to a 96-well white 1/
2 area microplate (PerkinElmer). A cAMP standard curve prepared at
4× the desired final concentration in stimulation buffer was added to
the assay plate. The final DMSO concentration in all wells was 1.5%.
Stable hAPJ-CHO cells were lifted with a nonenzymatic solution (Cell-
stripper, Mediatech Inc., Orlando, FL) and spun at 270 × g for 10 min.
The cell pellet was resuspended in stimulation buffer, and 4000 cells
were added to each well except wells containing the cAMP standard
curve. After incubating for 30 min at RT, Eu-cAMP tracer and
uLIGHT-anti-cAMP working solutions were added as per the
manufacturer’s instructions. After incubation at RT for 1 h, the TR-
FRET signal (ex 337 nm, em 620 and 650 nm) was read on a
CLARIOstar multimode plate reader (BMG Biotech, Cary, NC). For
the screen, the same procedure was followed except that instead of
adding serial dilutions of the test compounds, a single concentration (1
μM, prepared at 4× final) of each test compound was added to the assay
plates. Data were analyzed using Prism software (GraphPad, La Jolla,
CA). Nonlinear regression analysis was performed to fit data and obtain
maximum response (Emax), EC50, correlation coefficient (r2), and other
parameters. All experiments were performed in duplicate 2−3 times to
ensure reproducibility, and data are reported as mean s.e.m., unless
noted otherwise.
Step 2: (S)-1-Cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(4-(piperi-
din-1-yl)-1-(4H-1,2,4-triazol-3-yl)butan-2-yl)-1H-pyrazole-3-carboxa-
mide (34): A solution of (S)-N-(1-amino-1-oxo-5-(piperidin-1-yl)-
pentan-3-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamide 48 (40 mg, 0.80 mmol) in DMF−DMA (1 mL) was
heated at 120 °C for 2 h. The solution was concentrated to dryness, and
the residue was dissolved in glacial acetic acid (1.0 mL), followed by the
addition of NH2NH2·H2O (0.10 mL). The resulting solution was
heated at 90 °C for 2 h, cooled to RT, quenched with saturated aqueous
NaHCO3 (20 mL), and extracted with EtOAc (10 mL). The EtOAc
solution was dried with Na2SO4, filtered, and concentrated to give the
crude product. The crude product was then purified by silica gel flash
chromatography using 0−15% MeOH in a DCM (with 1% NH3)
solvent system to give the title product 34 (24 mg, 57% yield) as a white
solid; 1H NMR (CDCl3, 200 MHz): δ 1.40−2.20 (m, 16H), 2.30−2.80
(m, 6H), 3.20−3.40 (m, 2H), 3.72 (s, 6H), 4.15−4.30 (m, 1H), 4.50−
4.70 (m, 1H), 6.62 (d, J = 8.6 Hz, 2H), 6.68 (s, 1H), 7.35 (t, J = 7.5 Hz,
1H), 7.91 (s, 1H), 8.20 (d, J = 8.6 Hz, 1H). MS (ESI): m/z calculated
for C28H39N7O3, [M]+ 521.31; found, 522.3 [M + H]+. [α]2D5 = −15.00
(c 0.060, MeOH).
(S)-N-(1-(1,3,4-Oxadiazol-2-yl)-4-(piperidin-1-yl)butan-2-yl)-1-
cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamide
(35). Step 1: Synthesis of (S)-methyl 3-(1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(piperidin-1-yl)-
pentanoate (49): To a solution of (S)-tert-butyl 3-(1-cyclopentyl-5-
(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(piperidin-1-
yl)pentanoate 39 (1.73 g, 5.5 mmol) in DCM (10 mL) was added TFA
(2 mL, 28 mmol) and stirred at RT for 2 h. The solvent was removed to
provide crude (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyr-
azole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid trifluoroace-
tate as a yellow solid (2.1 g), which was used as is for the next reaction
without further purification; MS (ESI): m/z calcd for C27H38N4O5 [M
+ H+], 498.28; found, 499.5.
Calcium Mobilization Assay. CHO cells stably expressing either the
Gαq16 protein (CHO-RD-HGA16; Molecular Devices) plus the human
apelin receptor (CHO hAPJ), mouse apelin receptor (CHO mAPJ), or
human angiotensin II receptor, type I (CHO hAGTR1; DiscoverX)
were removed from flasks using Cell-stripper and quenched with
DMEM/F12, 10% FBS, and 1× Penicillin/Streptomycin, centrifuged,
and resuspended in the serum-containing media. Cells were counted
with a hemocytometer, and 30,000 cells of CHO hAPJ and 20,000 cells
of CHO hAGTR1 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. The culture media was removed
from the plates, and cells were washed with DPBS and were
subsequently loaded with a fluorescent calcium probe (Calcium 5
dye; Molecular Devices, Sunnyvale, CA) in an HBSS-based buffer
containing 20 mM HEPES, 1% BSA, and 10 μM probenecid (Sigma) in
a total volume of 225 μL with the final DMSO concentration at 1%.
Cells were incubated at 37 °C for 1 h and then stimulated with test
compounds or pyr-apelin-13 (Anaspec, Freemont, CA) at various
concentrations using an FLIPR Tetra plate reader. Agonist-mediated
change in fluorescence (ex 488 nm, em 525 nm) was monitored in each
well at 1 s intervals for 90 s. Data were collected using Softmax version
4.8 (MDS Analytical Technologies) and analyzed using Prism software.
Nonlinear regression analysis was performed to fit data and obtain
maximum response (Emax), EC50, correlation coefficient (r2), and other
parameters. All experiments were performed in duplicate 2−3 times to
ensure reproducibility, and data are reported as mean s.e.m., unless
noted otherwise.
To a solution of (S)-3-(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-
pyrazole-3-carboxamido)-5-(piperidin-1-yl)pentanoic acid trifluoroa-
cetate (120 mg, 0.24 mmol) in MeOH (2 mL) was added sulfuric acid
(con., 0.20 mL) and stirred at RT for 15 h. The solution was quenched
with saturated aqueous NaHCO3 (20 mL), extracted with EtOAc (10
mL), dried over Na2SO4, and concentrated to give crude (S)-methyl 3-
(1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-carboxami-
do)-5-(piperidin-1-yl)pentanoate 49 (0.11 g) as colorless oil, which
was used as is for the next reaction without further purification. MS
(ESI): m/z calcd for C28H40N4O5 [M]+, 512.30; found, 513.1 [M +
H]+.
Step 2: Synthesis of (S)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-N-
(1-hydrazinyl-1-oxo-5-(piperidin-1-yl)pentan-3-yl)-1H-pyrazole-3-car-
boxamide (50): A mixture of (S)-methyl 3-(1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-1H-pyrazole-3-carboxamido)-5-(piperidin-1-yl)-
pentanoate 49 (0.11 g), NH2NH2·H2O (1.0 mL), and EtOH (3.0 mL)
was heated at 80 °C for 3 h. The solution was cooled to RT,
concentrated, and purified by flash silica gel chromatography using 0−
15% MeOH in a DCM (with 1% NH3) solvent system to give (S)-1-
cyclopentyl-5-(2,6-dimethoxyphenyl)-N-(1-hydrazinyl-1-oxo-5-(pi-
peridin-1-yl)pentan-3-yl)-1H-pyrazole-3-carboxamide 50 (60 mg, 55%
yield) as colorless foam; 1H NMR (CDCl3, 200 MHz): δ 1.40−2.20 (m,
18H), 2.20−2.80 (m, 6H), 3.73 (s, 6H), 3.89 (br s, 2H), 4.15−4.30 (m,
1H), 4.30−4.50 (m, 1H), 6.62 (d, J = 8.4 Hz, 2H), 6.66 (s, 1H), 7.37 (t,
J = 8.4 Hz, 1H), 8.06 (d, J = 7.4 Hz, 1H), 8.37 (br s, 1H). MS (ESI): m/
z calcd for C27H40N6O4 [M]+, 512.31; found, 513.1 [M + H]+.
Step 3: Synthesis of (S)-N-(1-(1,3,4-oxadiazol-2-yl)-4-(piperidin-1-
yl)butan-2-yl)-1-cyclopentyl-5-(2,6-dimethoxyphenyl)-1H-pyrazole-3-
carboxamide (35): To a solution of (S)-1-cyclopentyl-5-(2,6-
dimethoxyphenyl)-N-(1-hydrazinyl-1-oxo-5-(piperidin-1-yl)pentan-3-
PathHunter β-Arrestin-2 Recruitment Assay. The stable CHO-K1
human AGTRL1 β-arrestin-2 cell line (DiscoverX) was maintained in
TC-treated flasks with growth media containing DMEM/F12 (Corning
Cellgro, Manassas, VA), 10% FBS, and 1× Penicillin-streptomycin L-
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glutamine (Gibco, Carlsbad, CA). The selection reagents used were
geneticin (800 μg/mL) and hygromycin (300 μg/mL). The cells were
removed from the flasks using a Cell-stripper. Cells were resuspended in
complete media and spun at 300g for 5 min. The supernatant was
discarded, and cells were resuspended in Assay Complete Cell plating 2
Reagent (DiscoverX). Cells were counted using a hemocytometer and
seeded in a white clear bottom TC-treated 96-well plate (DiscoverX) at
10,000 cells per well. The plates were incubated at 37 °C and 5% CO2
for 24 h. Compound dilutions were prepared at 10× final in DPBS (1×)
buffer with 10% DMSO and 1% BSA. Compound dilutions were added
to the 80−90% confluent assay plate, and the plates were incubated at
37 °C and 5% CO2 for 90 min, followed by the addition of PathHunter
Detection Reagent (DiscoverX), prepared as per the manufacturer’s
instructions. The assays were run at 1% DMSO and 0.1% BSA final. The
culture plates were incubated another 60 min at room temperature. The
luminescent signal was detected on an Enspire multimode plate reader
(PerkinElmer CT, USA). Data were collected using EnSpire Software
4.1 and analyzed using GraphPad Prism. Nonlinear regression analysis
was performed to fit data and obtain maximum response (Emax), EC50,
correlation coefficient (r2), and other parameters. All experiments were
performed in duplicate at least two times to ensure reproducibility, and
data are reported as mean s.e.m., unless noted otherwise.
conducted with an Agilent 1100 binary pump with a flow rate of 0.5
mL/min. The mobile phase solvents were 0.1% formic acid in water (A)
and 0.1% formic acid in methanol (B). The solvent conditions were
10% B for 1 min, followed by a gradient to 95% B over 5 min. Data
reported are average values of two to three measurements.
Plasma Protein Binding Assay. Compounds were diluted in plasma
at a concentration of 10 μM. A total of 100 μL of the compound in
plasma was placed into the sample chamber of a Thermo Fisher
Scientific RED Device (Waltham, MA). A total of 350 μL of dialysis
buffer (PBS containing 100 mM sodium phosphate and 150 mM
sodium chloride) was added to the buffer chamber. The plate was
covered with sealing tape and incubated at 37 °C for 4 h at 100 rpm in a
shaking incubator. Fifty microliters of postdialysis samples from the
buffer and the plasma chambers were pipetted into separate microfuge
tubes. Fifty microliters of plasma was added to the buffer sample, and 50
μL of buffer was added to the plasma sample. Three hundred microliters
of ice-cold 90/10 acetonitrile/water with 0.1% formic acid was added to
precipitate protein and release compound. Samples were centrifuged at
13,000g for 10 min and transferred to LC−MS/MS vials for analysis.
Samples were analyzed with an SCIEX 6500 + UHPLC/MS system
(Framingham, MA). Experiments were performed with three
independent samples.
[35S]GPTγS Binding Assay. The [35S]GPTγS assays were conducted
using the methods previously reported.49 Briefly, the binding of the
GTP analogue [35S]GPTγS to the membrane was determined in a
volume of 500 μL. The assay mixture contained 50 mM HEPES (pH
7.4), 100 mM NaCl, 5 mM MgCl2, 1 mM EDTA·2H2O, 1 mM
dithiothreitol, 0.1% BSA, 10 μM GDP, test compounds, and
approximately 132,000 cpm of [35S]-GTPγS (0.1 nM). Human apelin
2 μg (per assay tube) APJ receptor-expressing CHO-K1 cell
membranes were added to each tube. For the apelin receptor assays,
seven-point concentration response curves of the agonist Pyr-aplein-13
(1 μM, 100 nM, 10 nM, 1 nM, 0.1 nM, 0.01 nM, and 0.001 nM) were
prepared, and the dilutions were coincubated with a seven-point
concentration curve of the test compound (31.6 μM, 10 μM, 3.16 μM,
316 nM, 100 nM, 31.6 nM, 3.16 nM, and 0.316 nM final). Following a
60 min shaking incubation at 22 °C, the assay was terminated by
filtration under vacuum on a Brandel (Gaithersburg, MD, USA) 96-well
harvester using presoaked PerkinElmer GF/B glass-fiber filters. The
filters were rinsed three times with 1 mL washes of ice-cold wash buffer
(20 mM HEPES pH 7.4, 100 mM NaCl, and 10 mM MgCl2). Filter
plates were dried for 1 h at 55 °C. Microscint 20 (50 μL) was added to
each well, and filter-bound radioactivity was counted on a Packard Top
Count NXT microplate scintillation and luminescence counter. Total
binding (TB) was determined in the absence of compounds, and
nonspecific binding (NSB) was determined in the presence of 10 μM
final unlabeled GTPγS. Percent specific bound (SB) was calculated
using the equation % SB = (SB/MB) × 100, where maximal binding
(MB) is calculated by subtracting NSB from TB. Percent SB was
plotted against the log of compound concentration. Data were fit to a
three-parameter logistic curve to generate EC50 values (GraphPad
Prism, GraphPad Software, Inc., San Diego, CA). Ke values were
calculated using the equation Ke = [L]/((EC50+/EC50−) − 1) where [L]
is the concentration of the test compound, EC50+ is the EC50 of the
control agonist with the test compound, and EC50− is the EC50 of the
Radioligand Displacement Assay for hERG Interaction.
Competition binding with [3H] astemizole was used to measure
hERG interaction, as has been previously described.43 Preparations of
membranes overexpressing hERG were purchased from PerkinElmer.
The binding assays were performed for 60 min using 4 μg of hERG-
expressing membranes, ∼3 nM [3H]astemizole, and various concen-
trations of the test agent in a pH 7.4 buffer (10 mM HEPES, 130 mM
NaCl, 5 mM KCl, 0.8 mM MgCl2, 1 mM NaEDTA, 10 mM glucose, and
0.1% BSA). Binding was terminated by rapid filtration onto GF/B fiber
filter mats (presoaked in 0.3% polyethyleneimine), followed by rapid
washing (6 × 2 mL) with an ice-cold pH 7.4 buffer (25 mM Tris−HCl,
130 mM NaCl, 5 mM KCl, 0.8 mM MgCl2, 0.05 mM CaCl2, and 0.1%
BSA) using a Brandel harvester. Filters were dried and counted after the
addition of a scintillant. Data were analyzed using nonlinear regression
(GraphPad Prism) and Ki values. All experiments were performed at
least twice in duplicate, and the data reported are the mean values.
Human Microsomal Stability Studies. Human microsomal
stability assays were performed as described previously.50 Briefly, test
compounds were incubated at a 1 μM final concentration with 0.5 mg/
mL pooled human liver microsomes from 200 unidentified donors
(Xenotech, LLC, Lenexa, KS) in a 100 mM phosphate buffer (pH 7.4)
containing 3 mM MgCl2, 1 mM nicotinamide adenine dinucleotide
phosphate (NADPH), 5 mM uridine diphosphate glucuronic acid, and
50 μg/mL alamethicin. Triplicate samples were incubated for up to 120
min. Samples were removed at regular intervals. Reactions were
terminated by the addition of 3 volumes of methanol and processed for
LC−MS by centrifugation. Standard curves were prepared in the blank
matrix for each compound for quantitative assessment. The intrinsic
clearance rate was calculated for each compound using the formula:
Clint (μL/min/mg) = 0.693/(t1/2 × microsomal protein concen-
tration). Data reported are average values from 3 measurements.
Kinetic Solubility Determination. The kinetic solubility of the
test compound was measured in commercial phosphate buffer saline
(PBS, pH 7.4), which consisted of potassium phosphate monobasic 1
mM, sodium phosphate dibasic 3 mM, and sodium chloride 155 mM. A
10 μL of the test compound stock solution (20 mM DMSO) was
combined with 490 μL of PBS buffer to reach a targeted concentration
of 400 μM. The solution was agitated on a VX-2500 multitube vortexer
(VWR) for 2 h at room temperature. Following agitation, the sample
was filtered on a glass-fiber filter (1 μm) and the eluate was diluted 400-
fold with a mixture of acetonitrile/water (1:1). On each experimental
occasion, nicardipine and imipramine were assessed as reference
compounds for low and high solubility, respectively. All samples were
assessed in triplicate and analyzed by LC−MS/MS using electrospray
ionization against standards prepared in the same matrix.
control agonist alone. Ke values were considered valid when the EC50+
/
EC50− ratio was at least 4. All experiments were performed in duplicate
2−3 times to ensure reproducibility, and data are reported as mean
s.e.m., unless noted otherwise.
MDCK-MDR1 Assay. MDCK-mdr1 cells obtained from The
Netherlands Cancer Institute were grown on Transwell type filters
(Corning) for 4 d to confluence in DMEM/F12 media containing 10%
fetal bovine serum and antibiotics. Compounds were added to the
apical side at a concentration of 10 μM in a transport buffer composed
of 1× Hank’s balanced salt solution, 25 mM D-glucose, and buffered
with HEPES to pH 7.4. Samples were incubated for 1 h at 37 °C and
carefully collected from both the apical and basal sides of the filters.
Compounds selected for MDCK-mdr1 cell assays were infused on an
Applied Biosystems API-4000 mass spectrometer to optimize for
analysis using multiple reaction monitoring. The chromatography was
PK Analysis. Male CD-1 mice were ordered from Charles River
Laboratories and allowed to acclimate to the facility for 72 h. Mice were
aged around 10 weeks and were dosed with compound 13 formulated in
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modified citrate buffer (pH: 6) via IP (15 mg/kg). Animals were
sacrificed at multiple time points (0.5, 1, 2, 4, 8, and 24 h), and samples
were removed. PK analyses were performed as described in our
previous publications using Phoenix WinNonlin (Certara).51
Shaobin Wang − Center for Drug Discovery, RTI International,
Durham, North Carolina 27709, United States
Vineetha Vasukuttan − Center for Drug Discovery, RTI
International, Durham, North Carolina 27709, United States
Ravi Kumar Vyas Devambatla − Sterling Pharma Solutions
Limited, Cary, North Carolina 27513, United States
Donghua Dai − Sterling Pharma Solutions Limited, Cary,
North Carolina 27513, United States
Chunyang Jin − Center for Drug Discovery, RTI International,
Durham, North Carolina 27709, United States; orcid.org/
0000-0001-6733-3094
Rodney Snyder − Center for Drug Discovery, RTI International,
Durham, North Carolina 27709, United States
Lucas Laudermilk − Center for Drug Discovery, RTI
International, Durham, North Carolina 27709, United States
DIO Study. All studies were performed in accordance with
guidelines established by the Office of Laboratory Animal Welfare
(OLAW) and protocols were approved by the institutional animal care
and use committee (IACUC). Preconditioned male C57BL6J mice
were purchased from the Jackson Laboratories (Bar Harbor, Maine) at
18 weeks of age. Power analysis was performed to refine study design
and optimize animal usage using SAS software (Cary, NC). We
assumed 9 animals per group. This gave the study >80% statistical
power assuming 40% change effect, 25% deviation, 5% alpha error level
(95% confidence) in a two-tailed design. These animals were
maintained either on ad libitum normal diet with 10% fat (D12450B)
or on 60% fat (D12492) chow from Research Diets (New Brunswick,
NJ). Animals were acclimated to the facility for two weeks and then
randomly assigned to various test groups (9−10 animals per group).
Animals were housed individually in polycarbonate cages. Animals were
dosed with a citrate-buffered vehicle (10 mM citrate buffer with 40 mg/
mL D-mannitol adjusted to pH 3) or 13 dissolved the vehicle BID by
i.p. dosing for 28 consecutive days. Dosing volume was maintained at 5
mL/kg. Body weights were taken at regular intervals and food
consumption tracked. Animals were food-deprived for 12 h prior to
the oral glucose tolerance test (GTT). For GTT, animals were orally
dosed with a 2 g/kg bolus dose of glucose in water. Using a fine scissors,
tails were snipped (<1 mm) once, the same snipped area was used for
subsequent blood collection. A very small sample size of approximately
2−5 μL of blood was used. Measurements were performed using a
glucometer and standard blood glucose measurement strips at 15, 30,
60, 90, 120 min, post challenge. At termination of study, plasma and
vital organs were collected and preserved appropriately. Serum samples
from each animal were analyzed for alanine aminotransferase (ALT),
free FA, and LDH levels by an independent laboratory (MPI Research/
Charles River Laboratories, Mattawan, MI) using good laboratory
practice. Tissue sections (10 μm) from frozen livers were prepared and
analyzed following hematoxylin−eosin staining and Oil Red O staining
using standard procedures. Data were graphed using Prism software
(GraphPad, La Jolla, CA), and statistical analyses were performed using
the same program. Triglyceride levels were measured using a
commercially available colorimetric kit from Cayman Chemicals (Cat
# 10010303, Ann Arbor, MI).
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.0c01448
Funding
This work was supported by NIH grant: 1R01DK103625.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
Authors wish to thank Dr. Elaine Gay, Dr. Ann Decker, Nigel
Edgerton, and Taylor Rosa for help with various assays.
ABBREVIATIONS
■
AT1, angiotensin 1; ALT, alanine aminotransferase; Boc2O, di-
tert-butyl dicarbonate; CHO-K1, chinese hamster ovary cells;
DCE, 1,2-dichloroethane; DCM/CH2Cl2, dichloromethane;
DMF, N,N-dimethylformamide; DMF−DMA, N,N-dimethyl-
formamide dimethyl acetal; DIO, drug-induced obesity; EtOAc,
ethyl acetate; EtOH, ethanol; Et3N, triethylamine; HBTU,
N,N,N′,N′-tetramethyl-O-(1H-benzotriazol-1-yl)uronium hex-
afluorophosphate; HCl, hydrochloric acid; HFD, high-fat diet;
LiOH·H2O, lithium hydroxide monohydrate; MeCN, acetoni-
trile; MeOH, methanol; MDCK-mdr1, Madin−Darby canine
kidney cells transfected with the human MDR1 gene; NaBH-
(OAc)3, sodium triacetoxyborohydride; (NH4)2CO3, ammo-
nium carbonate; Na2S2O3, sodium thiosulfate; NaOH, sodium
hydroxide; NH4OH, ammonium hydroxide; NaHCO3, sodium
bicarbonate; PTSA·H2O, p-toluenesulfonic acid monohydrate;
PPB, plasma protein binding; SAR, structure−activity relation-
ship; TFA, trifluoroacetic acid; THF, tetrahydrofuran; TLC,
thin-layer chromatography
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01448.
■
sı
1H NMR, mass, and HPLC trace of compound 13; safety
screen data of compound 13; PK analysis of compound
13; and HPLC trace of target compounds (PDF)
List of molecular formula strings (CSV)
REFERENCES
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