Development of Selective, Orally Active GPR4 Antagonists with Modulatory Effects on Nociception, Inflammation, and Angiogenesis

Article abstract of DOI:10.1021/acs.jmedchem.6b01703

A novel, selective, and efficacious GPR4 antagonist 13 was developed starting from lead compound 1a. While compound 1a showed promising efficacy in several disease models, its binding to a H₃ receptor as well as a hERG channel prevented it from further development. Therefore, a new round of optimization addressing the key liabilities was performed and led to discovery of compound 13 with an improved profile. Compound 13 showed significant efficacy in the rat antigen induced arthritis as well as in the hyperalgesia and angiogenesis model at a well-tolerated dose of 30 mg/kg.

Full text of DOI:10.1021/acs.jmedchem.6b01703

Article
pubs.acs.org/jmc
Development of Selective, Orally Active GPR4 Antagonists with
Modulatory Effects on Nociception, Inflammation, and Angiogenesis
Juraj Velcicky,*,† Wolfgang Miltz,† Berndt Oberhauser,† David Orain,† Andrea Vaupel,† Klaus Weigand,†
Janet Dawson King,‡ Amanda Littlewood-Evans,‡ Mark Nash,§ Roland Feifel,∥ and Pius Loetscher‡
‡
§
∥
†Global Discovery Chemistry, Autoimmunity Transplantation Inflammation, Musculoskeletal, Metabolism and Pharmacokinetics,
Novartis Institutes for BioMedical Research, CH-4002 Basel, Switzerland
S
* Supporting Information
ABSTRACT: A novel, selective, and efficacious GPR4 antagonist 13 was developed starting from lead compound 1a. While
compound 1a showed promising efficacy in several disease models, its binding to a H3 receptor as well as a hERG channel
prevented it from further development. Therefore, a new round of optimization addressing the key liabilities was performed and
led to discovery of compound 13 with an improved profile. Compound 13 showed significant efficacy in the rat antigen induced
arthritis as well as in the hyperalgesia and angiogenesis model at a well-tolerated dose of 30 mg/kg.
Another study has shown that GPR4 activation by acidosis
stimulates cyclic adenosine monophosphate/protein kinase A
signaling, leading to the upregulation of the receptor activator
of nuclear factor κ-B ligand (RANKL) cytokine expression in
osteoblasts.7 GPR4, being pro-inflammatory within inflamed
intestinal tissues, has also been demonstrated by studies with
GPR4-deficient mice.8 The intestinal inflammation was reduced
in an acute experimental colitis model. Similarly, the myocardial
infarction-induced injury in mice which is characterized by
decreased tissue pH has been shown to be reduced by blocking
GPR4.9 In addition, GPR4 was shown to be overexpressed in
various types of human cancer.10 The data from the squamous
cell carcinoma of the head and neck indicate that GPR4 induces
angiogenesis via p38-mediated IL6, IL8, and VEGFA secretion
at acidic extracellular pH.11 On the other hand, recent evidence
suggests also a role of GPR4 in the regulation of breathing.12
Genetic deletion of GPR4 disrupted acidosis-dependent
activation of retrotrapezoid nucleus (RTN) neurons, increased
apnea frequency, and blunted ventilatory responses to CO2. To
further explore the biology of GPR4 and to determine the
potential of GPR4 as therapeutic target, we describe here the
development and characterization of small molecules targeting
GPR4.
INTRODUCTION
■
GPR4 is a proton sensing G protein-coupled receptor (GPCR)
belonging to a family of three closely related receptors
including GPR4, OGR1, and TDAG8.1 GPR4 has been
shown to respond to acidic pH by eliciting cAMP via gas
signaling. GPR4 mRNA is expressed in different tissues
including lung, heart, liver, joint, and dorsal root ganglions.
Gene expression profiling studies have shown a strong
correlation between GPR4 mRNA and endothelial marker
genes, suggesting a role for GPR4 in endothelial cell function.2
Although GPR4 is still a relatively poorly characterized
receptor, the proton-sensing properties suggest that GPR4
plays a key role in tissue acidosis.
Acidosis commonly exists in the tissue microenvironment of
various pathophysiological conditions such as inflammation,
tumors, ischemia, metabolic, and respiratory disease.3 For
instance, it is well-known that the acidic extracellular pH at
inflammatory sites affects cellular and humoral immune
functions.4 In fact, it has been reported that activation of
GPR4 by acidic pH augments the overall acidosis response and
particularly stimulates the expression of a wide range of
inflammatory genes like chemokines, cytokines, and adhesion
molecules.5 Furthermore, it has recently been reported that
GPR4 is a novel mediator for endoplasmic reticulum (ER)
stress in response to acidosis in endothelial cells.6 GPR4
activation by acidosis stimulates three arms of the ER stress
pathways (PERK, ATF6, and IRE1) in endothelial cells.
Received: November 21, 2016
© XXXX American Chemical Society
A
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a
Scheme 1. Synthesis of the Key Intermediates 18 and 22
a
Reaction conditions: (a) NaH, THF, 0 °C to rt, 16 h (74%); (b) NaH, NBS, THF, 0 °C to rt, 16 h (37%); (c) Amberlyst 15, PhMe, 80 °C, 16 h
(67%); (d) TsOH (cat.), PhMe, reflux, Dean−Stark, 16 h (74%); (e) phenacyl bromide, CH3CN, reflux, 2 h, then 10% NaOH (aq), reflux, 16 h
(50%); (f) i-Pr2NEt, DCM, 75 °C, 4 h (43%); (g) ethyl propionate, t-pentylOK, 0−25 °C, 20 min (85%); (h) N2H4·H2O, AcOH/EtOH, 140 °C, 15
min, then NaOH, 160 °C, 30 min (76%); (i) acetylacetone, TFA/dioxane (1:1), 100 °C, 16 h (98%).
a
Scheme 2. Synthesis of Compounds with Aliphatic Linkers via Cinnamates 23a,b
a
Reaction conditions: (a) methyl acrylate, cHex2NMe, (t-Bu3P)2Pd (2.5%), dioxane, 130 °C, 5−15 min (58−95%); (b) DIBALH, CH2Cl2, −78 °C,
3 h (25−92%); (c) amine, Zaragoza reagent ((cyanomethyl)trimethylphosphonium iodide), i-Pr2NEt, CH3CH2CN, reflux, 3 h (63−92%); (d) H2,
Pd/C or Pd(OH)2/C, MeOH, rt, 1−2 h (29−48%); (e) HCl in dioxane or Et2O, rt, 0.5−5 h (98−99%); (f) amine or carbonyl, NaBH(OAc)3, i-
Pr2NEt, 23−70 °C, 2−15 h (13−62%); (g) MnO2, CH3CN, rt, 3 h (74%); (h) LiOH, THF/H2O, rt, 60 h (89%); (i) Boc-piperazine, HOBt,
EDC.HCl, Et3N, CH2Cl2, rt, 16 h (73%).
B
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a
Scheme 3. Attachment of Allyl Piperidine/Piperazine Containing Tails to Intermediate 22
a
Reaction conditions: (a) Grignard reagent, Et2O, rt, 1−4 h (30−54%); (b) 22, cHex2NMe, (t-Bu3P)2Pd (2.5%), dioxane, 130−140 °C, 10−15 min
(26−66%); (c) H2, Pd/C, MeOH, rt, 16 h (76%); (d) HCl in dioxane, Et2O or EtOH, rt, 0.5−3 h (94−99%); (e) acetone, NaBH(OAc)3, i-Pr2NEt,
rt, 15 h (66%); (f) NaH, allyl bromide, THF, 0−25 °C, 2.5 h (100%); (g) NaBH4, MeOH, 0 °C, 2 h (42%).
The allyl and propylene linkers were introduced by
attachment of methyl acrylate to intermediates 18 and 22
using Heck reaction (Scheme 2). Reduction of the ester group
in cinnamates 23a,b provided corresponding alcohols 24a,b.
These alcohols were coupled with piperazines under Zaragoza
conditions,19 providing compounds 2, 3, and 8. The latter one
was reached from its Boc-piperazine precursor after Boc
cleavage followed by reductive amination with (S)-2,3-
dihydroxypropanal. Hydrogenation of the allyl double bond
in 3 led to propylene analogue 4. Compound 11 was prepared
via reductive amination between piperazinone and cinnamalde-
hyde obtained by MnO2 oxidation of alcohol 24b. Amide 9 was
reached from the intermediate 23b by ester hydrolysis, coupling
of the obtained acid with N-Boc piperazine, reduction of the
allyl double bond, and acidic cleavage of the Boc protecting
group.
CHEMISTRY
■
All compounds described herein were prepared via key
bromointermediates 18 and 22 (Scheme 1), which provided
the opportunity to readily attach appendages by means of Pd-
catalyzed reactions such as cross-coupling or carbonylation.
The prerequisite bromo substituent was introduced into the
molecules during formation of the corresponding bicyclic
heterocycle. Imidazopyridazine intermediate 18 was prepared
by condensation13 of a known aminopyridazine 1714 with
bromoketone 15 (Scheme 1). The latter intermediate could be
obtained from β-ketoester 1415 by using a method for
regioselective bromination of unsymmetrical ketones16 consist-
ing of three steps: (i) alkylation of 14 by para-bromobenzyl
bromide, (ii) bromination with NBS, and (iii) Amberlyst-
mediated decarboxylation of t-Bu ester. Synthesis of the
pyrazolopyrimidine intermediate 22 was achieved in three
steps (Scheme 1). Acylation17 of nitrile 19 by propionate
provided α-cyanoketone 20. This intermediate underwent a
smooth assembly of the pyrazolopyrimidine ring by two
consecutive cyclizations, the hydrazine mediated formation of
aminopyrazole 21 and its condensation with acetylacetone
leading to compound 22.18
Compounds 6, 7, and 10 were accessed from intermediate 22
via direct attachment of allyl substituted piperidines 25, 29, and
piperazine 27 by means of Heck reaction (Scheme 3). The
Heck product 26 was derived from allylhydroxypiperidine
intermediate 25 and transformed to compound 6 by reduction
of the allyl double bond and removal of Boc protecting group.
C
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a
Scheme 4. Synthesis of the Oxadiazole Linker Containing Compounds 12 and 13
a
Reaction conditions: (a) PdCl2dppf (10%), dppf (1%), CO (1 atm), Et3N, MeOH/DMF, 80 °C, 16 h (52%); (b) N2H4·H2O, MeOH, reflux, 2 h
(87%); (c) CDI, Et3N, THF, rt, 2.5 d (91%); (d) N-Boc-piperazine, BOP, i-Pr2NEt, THF, rt, 2.5 d (75%); (e) HCl/dioxane, rt, 1 h (68−73%); (f)
4-Boc-piperidine carboxylic acid, HOBt, EDC·HCl, Et3N, CH2Cl2, rt, 2.5 d (97%); (g) TsCl, Et3N, DMF/CH2Cl2, rt, 18 h (59%).
Scheme 5. Lead Compound 1a and Initial Derivatization of the Bicyclic Head
The corresponding i-Pr analogue 5 was obtained from
compound 6 by its reaction with acetone under reductive
amination conditions. Reaction of allyl alcohol 29 with
compound 22 under Heck conditions20 led to ketone 30,
which after NaBH4 reduction and Boc deprotection provided
analogue 7. Derivative 10 was built from allylpiperazinone 27 in
a similar manner as described for 6.
Oxadiazoles 12 and 13 were synthesized from a common
intermediate 33 (Scheme 4), obtained by Pd-catalyzed
carbonylation21 of the intermediate 22 followed by reaction
of the formed ester 32 with hydrazine. The obtained hydrazide
33 was cyclized to oxadiazolone 34 using carbonyldiimidazole
(CDI).22 BOP-mediated coupling23 of compound 34 with N-
Boc-piperazine and final removal of the Boc protecting group
delivered piperazine substituted oxadiazole 12. Its piperidine
analogue (13) was built up starting from the hydrazide 33 by its
conversion to bisacylhydrazide 35, followed by ring closure
using tosyl chloride24 and final Boc cleavage.
RESULTS AND DISCUSSION
■
Recently, a series of potent GPR4 antagonists including
compound 1a (Scheme 5) was disclosed by our group.25
After oral dosing, this compound showed encouraging results in
several rodent disease models including antigen induced
arthritis (AIA), angiogenesis, and hyperalgesia. However, 1a
carries safety liabilities such as cross-reactivity with the
histamine H3 receptor.26 H3 receptor acts as a presynaptic
autoreceptor, regulating the synthesis and release of histamine,
but it is also involved in regulation of the release of other
neurotransmitters such as dopamine, noradrenaline, GABA,
acetylcholine, etc. Beside its function in the central nervous
system, it is also widely distributed in various areas of the
peripheral nervous system (e.g., in the gastrointestinal tract, the
airways, and the cardiovascular system). While H3 receptor
antagonists are studied for their role in narcolepsy, cognitive
disorders, obesity, neuropatic pain, and Alzheimer disease,
inhibition of this receptor bears a risk of increased food
D
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Table 1. Exploration of Bicyclic Head in the Lead Structure 1a for Its Impact on Binding to hERG and H3 Receptor
a
b
IC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels. IC50 determined as a mean (n ≥ 2) by a
c
binding assay using radiolabeled 3H-dofetilide binding to HEK293 cell membranes expressing human recombinant HERG K+ channels. Antagonist
IC50 determined as a mean (n ≥ 2) using H3 receptor binding assay with radiolabeled Me-histamine and membranes from CHO-K1 cells expressing
*
human recombinant H3 receptors. Single experiment.
consumption and prolonged wakefulness. In addition to
blocking H3, compound 1a also induced cardiotoxicity (QT
prolongation), which could be rationalized by inhibition of
hERG channel.27 Altogether, compound 1a was prevented from
its further development, and therefore, further optimization
addressing the above-mentioned liabilities was performed.
A similar type of compounds (1b) was recently described by
Fukuda et al.9 While no information on binding of 1b or its
analogues into H3-receptor or hERG channel was provided, the
molecules possess the same type of bicyclic group as 1a,
indicating the importance of this part of the molecule for
binding into GPR4. Therefore, for probing the bicyclic head for
its impact on hERG and H3 inhibition, the 5,6-bicycle was kept
while nitrogen was moved to different positions. This exercise
revealed two derivatives (2 and 3) with slightly improved
potency (Table 1), whereas the binding into H3 and hERG
were influenced only marginally.
Next, the tail bearing the piperazine moiety was explored.
Because of a positive effect on binding to GPR4 and selectivity
against hERG (calculated as a ratio of hERG vs hGPR4
inhibition), the pyrazolopyrimidine (3) was chosen for further
optimization (Table 2). Saturation of the allyl linker (4) was
tolerated by GPR4, but only limited improvement in selectivity
over hERG binding was seen. Out of several possibilities for
disturbing hERG interaction,27 modulation or removal of one
of the basic nitrogens in piperazine ring was probed initially.
While replacement of the internal nitrogen by hydroxyl group
(5) retained GPR4 potency, it showed only a slightly positive
effect on H3- and no effect on hERG binding. However,
removal of the iso-propyl group (6) led to reduced hERG-
binding without any loss in GPR4 potency. Moving the
hydroxyl group into the linker as in compound 7 restored the
hERG binding, thus indicating a beneficial effect of a polar
group close to the amine. The lipophilic nature of substituent at
the terminal piperazine nitrogen (e.g., iso-propyl) appeared to
be essential for binding to H3-receptor. Interestingly, such a
substituent was also preferred by hERG channel, and polar
groups such as dihydroxypropyl (8) in this region were not
tolerated by both off-targets. The effect of polarity on hERG
binding was further explored. Carbonyl next to one of the
nitrogens in the piperazine, aimed to increase the polarity but
also to remove the nitrogen basicity, proved to be favorable
(compounds 9−11), and piperidone analogue 11 was found
with an excellent hERG selectivity of >1000. Because polarity
had such a positive effect on selectivity against hERG, polar
linkers between the phenyl ring and the terminal aliphatic
heterocycle were also investigated. In particular, 1,3,4-
oxadiazole in a combination with piperidine (13) helped to
interrupt its binding into hERG while keeping the potency on
GPR4. Interestingly, the close piperazine analogue 12 was
found to be less tolerated by the GPR4 receptor (Table 2).
Several compounds were also tested for their PK properties
in the rat iv/po cassette (Table 2). The clearance data suggest
that the compounds possessing saturated propylene linker are
metabolically more labile, resulting in lower oral exposure than
the corresponding compounds with the allylic linker (3 vs 4) or
E
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Table 2. Tail Derivatization and Its Influence on Potency, Off-Targets, and PK Properties
a
b
IC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels. IC50 determined as a mean (n ≥ 2) by a
c
binding assay using radiolabeled 3H-dofetilide binding to HEK293 cell membranes expressing human recombinant HERG K+ channels. Antagonist
IC50 determined as a mean (n ≥ 2) using H3 receptor binding assay with radiolabeled Me-histamine and membranes from CHO-K1 cells expressing
d
human recombinant H3 receptors. CL: clearance measured as a mean SD of four animals (female Sprague−Dawley Rat) after iv dosing (1 mg/
F
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Table 2. continued
e
kg) using NMP:PEG200 (30:70) formulation. Exposure (AUC; dn = dose-normalized to 1 mg/kg) measured as a mean of 4 animals (female
*
Sprague−Dawley Rat) after po dosing (3 mg/kg) using CMC:Water:Tween (0.5:99:0.5) formulation. Single experiment.
Table 3. Efficacy of Selected Compounds in the Rat Antigen Induced Arthritis (AIA) and hGPR4 IC50 Measured in the Presence
of Rat Plasma
a
b
c
d
compd hGPR4 [μM] rGPR4 [μM] inhibition in rat AIA [%] hGPR4 with 40% rat plasma [μM] IC50 shift (hGPR4 with 40% plasma IC50/hGPR4)
8
0.021 0.06
0.027 0.05
0.070 0.019
0.86 0.26
0.74 0.20
1.84 0.35
35.9 2.46*
0.8 2.02ns
34.2 6.6*
0.067 0.013
0.89 0.21
3
33
1
11
13
0.067 0.012
a
b
IC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels; IC50 determined as a mean (n ≥ 3) in the
HEK cells expressing rat GPR4 measuring cAMP levels; Determined as % inhibition (compared to effect of dexamethasone, 1 mg/kg qd po) of the
swelling after 30 mg/kg bid po dosing for 7 days to the female Lewis rats (n = 5) after challenge with antigen (methylated albumin bovine), statistics
are One-Way ANOVA followed by Dunnett’s test for multiple comparisons (* p < 0.05, ns p > 0.05); IC50 determined as a mean (n ≥ 3) in the
HELA cells expressing human GPR4 measuring cAMP levels after incubation with 40% rat plasma.
c
d
the oxadiazole 13. While the hydroxy-piperidine derivatives (6
and 7) tend to have further increased clearance, the bishydroxy-
piperazine containing compound 8 displayed good oral
exposure with a low clearance. Within the amide derivatives,
compound 9 with the saturated linker showed again a rather
low oral exposure, whereas the analogue with allyl linker (11)
displayed one of the best exposures after oral dosing observed
for tested GPR4 antagonists.
Selected compounds (8, 11, and 13) were tested in the rat
antigen induced arthritis (AIA) model at 30 mg/kg bid oral
dose (Table 3). Surprisingly, compound 11, the most potent
compound in the rat GPR4 assay with the highest oral exposure
in the rat PK, showed no effect in this model, while compounds
8 and 13 displayed a significant efficacy. To better understand
the discrepancy, we looked at the influence of protein binding
on the potency. To this end, the IC50 data were measured using
40% rat plasma, and indeed, a large shift (factor >30) was
observed for compound 11. Therefore, even the high blood
levels achieved with this compound at 2 h after dosing (23 μM,
Figure 1) were borderline in reaching the required plasma
experiment being above their plasma corrected rat GPR4 IC50
even at 17 h time point, i.e., 9 h after the last dose.
In comparison to 8, compound 13 displayed not only higher
exposures in the rat AIA but also lower plasma protein binding
in rat (95%) and human (93%) (>99% in rat and human for
compound 8) and was therefore selected for further profiling.
With respect to the in vitro safety profile of compound 13, the
internal panel of enzymes, receptors, and ion channels did not
reveal any significant inhibition of the measured off-targets
(>150) with this compound. Compound 13 is also selective
over the related proton sensing receptors OGR1 and TDAG8
(IC50 > 20 μM; determined in HELA cells expressing OGR1 or
TDAG8 measuring cAMP levels). In addition, its low binding
affinity to hERG (19 μM) translated into only a poor effect in
the mechanistic patch clamp assay for hERG K+ channel
interaction (44% inhibition at 60 μM) measured by patch-
clamp electrophysiology using transfected HEK293 cells.
Besides good efficacy of 13 reached in the rat AIA model,
the compound was also tested for its anti angiogenic effect
(Figure 2). Given the expression of GPR4 in endothelial cells
and the pH-induced cAMP formation which can be inhibited by
GPR4 antagonists and on the basis of the data obtained with
WT and KO mice,2 the effect of compound 13 (mouse GPR4
Figure 1. Blood exposure of 8, 11, and 13 measured at 2, 6, and 17 h
on day 7 in the rat AIA (30 mg/kg po bid; mean blood conc SD, n =
5).
Figure 2. Inhibition of angiogenesis by compound 13 in the mouse
chamber implant model. Female FVB mice were implanted
subcutaneously in the flank with a porous chambers containing 2
μg/mL VEGF in 0.8% agar containing 20 U/mL heparin and then
treated with vehicle (no compound) or 30 mg/kg bid po dosing of
compound 13 for 4 days. The vascularized tissue formed around each
implant was removed and weighed. The effect of compound treatment
was expressed as % tissue weight reduction compared to vehicle
treatment. Statistics are One-Way ANOVA followed by Dunnett’s test
for multiple comparisons (* p < 0.05).
corrected rat GPR4 IC50 of ∼24 μM, estimated using rGPR4
and observed shift for hGPR4 with and without addition of rat
plasma in the assay (Table 3). In contrast, the presence of rat
plasma caused no shift for compound 13 and only a limited
shift (factor 3) was seen for compound 8. The calculated
plasma corrected rat GPR4 IC50 were determined to be ∼2.6
μM for 8 and ∼1.8 μM for 13. Thus, the significant inhibition
of the arthritic knee swelling in this model with compounds 8
and 13 can be rationalized by the blood levels reached in this
G
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IC50 of 0.53 0.12 μM was determined using cAMP assay) was
tested in the VEGF-induced implant angiogenesis chamber
model.28 In this model, the addition of VEGF triggers the
formation of a new, well vascularized tissue around the
implanted chamber. Treatment with compound 13 at 30 mg/
kg po bid starting on day 0, the day of the chamber
implantation, showed a statistically significant reduction (46.8
10.6%) of tissue growth by day 4. The blood levels of 13 on
day 4 at 2 and 16 h after compound application in this model
were 9.03 2.87 and 0.09 0.06 μM (Figure 2).
Furthermore, given the expression of GPR4 in dorsal root
ganglion (DRG) tissue and the GPR4 dependence of CGRP4
release from DRG cell cultures,29 the effect of compound 13
was tested for reversing persistent inflammatory pain in the rat
complete Freund’s adjuvant (CFA) model (Figure 3). Intra-
improved safety window to the two main off-targets: H3
receptor and hERG channel. It shows a significant anti-
inflammatory effect in the rat antigen induced arthritis model
after oral administration at 30 mg/kg bid. In addition, at a well
tolerated 30 mg/kg dose, compound 13 also prevents
angiogenesis in the mouse chamber model as well as pain as
demonstrated in the rat complete Freund’s adjuvant model. On
the basis of its good overall profile (potency, selectivity, and
oral pharmacokinetics) as well as efficacy in the arthritis and the
pain model, 13 has been selected for further development as an
anti-inflammatory drug.
EXPERIMENTAL SECTION
■
Chemistry. All reagents and solvents were purchased from
commercial suppliers and used without further purification. All
reactions were performed under inert conditions (argon) unless
1
otherwise stated. H NMR spectra were recorded on a Bruker 400
MHz or a Bruker 600 MHz NMR spectrometer. Chemical shifts are
reported in parts per million (ppm) relative to an internal solvent
reference. Significant peaks are tabulated in the order multiplicity (s,
singlet; d, doublet; t, triplet; q, quartet; quintet; m, multiplet; br,
broad), coupling constants, and number of protons. Final compounds
were purified to ≥95% purity as assessed by analytical liquid
chromatography: Waters UPLC Acquity; column: Acquity HSS T3,
1.8 μm, 2.1 mm × 50 mm, at 60 °C; Eluent A, water +0.05% HCOOH
+ 3.75 mM ammonium acetate; B, ACN + 0.04% HCOOH; gradient,
5−98% B in 9.4 min hold 0.4 min; flow, 1 mL/min.
General Procedures. Heck Coupling. Bromide (1 equiv), alkene (2
equiv), dicyclohexylmethylamine (2 equiv), and Pd(tBu3P)2 (0.025
equiv) in dioxane (0.1 M solution) was stirred under argon at 130 °C
(microwave oven) for 5−10 min. After cooling to rt, the mixture was
concentrated on RV and diluted with ethyl acetate, extracted with satd
NaHCO3 and brine, dried (Na2SO4), and concentrated. The products
were obtained by purification on MPLC.
DIBALH Reduction. A solution of DIBALH in toluene (1 M, 3
equiv) was added under argon at −78 °C to a solution of acrylate (1
equiv) in CH2Cl2 (0.15 M solution). After stirring for 3 h at −78 °C,
the reaction was quenched by addition of water and the solvent was
evaporated. The residue was diluted with ethyl acetate, washed with
water and brine, dried (Na2SO4), and concentrated to provide the
products.
Zaragoza Coupling. Alcohol (1 equiv), amine (1 equiv),
(cyanomethyl)trimethyl phosphonium iodide (Zaragoza reagent)
(2.4 equiv), and i-Pr2NEt (5 equiv) were dissolved in propionitrile
(0.5 M solution), and the mixture was stirred at rt for 16 h. After
dilution with EtOAc, the mixture was washed with brine, dried
(Na2SO4), and concentrated. The crude product was purified by
MPLC to provide the products.
Procedures. (R,E)-3-(4-(3-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)methyl)phenyl)allyl)piperazin-1-yl)propane-1,2-
diol (8). Step 1: According to the general procedure for Zaragoza
coupling, (E)-3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)methyl)phenyl)prop-2-en-1-ol (24b) (6 g, 18.67 mmol) and Boc-
piperazine (3.48 g, 18.67 mmol) after 3 h at 60 °C provided (E)-tert-
butyl 4-(3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
methyl)phenyl)allyl)piperazine-1-carboxylate in 8.4 g (92%) yield as
a yellow oil. MS (ESI): 490.2 [M + H]+. 1H NMR (400 MHz, DMSO-
d6, δ) 7.30 (d, J = 8.1 Hz, 2H), 7.12 (d, J = 8.1 Hz, 2H), 6.74 (s, 1H),
6.45 (d, J = 15.9 Hz, 1H), 6.18 (dt, J = 15.9, 6.6 Hz, 1H), 4.01 (s, 2H),
3.07 (d, J = 6.5 Hz, 2H), 2.58−2.72 (m, 9H), 2.47 (s, 3H), 2.32 (t, J =
4.5 Hz, 4H), 1.38 (s, 9H), 1.12 (t, J = 7.5 Hz, 3H).
̈
Figure 3. Anti-nociceptive effect of compound 13. The naive
withdrawal thresholds of both hind paws were determined in male
Wistar Han rats and then inflammatory mechanical hyperalgesia
induced by intra-plantar injection of 25 μL of Complete Freund’s
Adjuvant (CFA) into one hindpaw with the contralateral paw acting as
the control. After 3 days, compound 13 or diclofenac were
administered po. Paw withdrawal thresholds were re-measured at 1,
3, and 6 h post dosing and the reversal of hyperalgesia calculated using
the formula: Reversal (%) = 100 × (postdose ipsilateral threshold −
̈
predose ipsilateral threshold)/(naive ipsilateral threshold − predose
ipsilateral threshold). Statistics are One-Way ANOVA followed by
Dunnetts test compared to matched vehicle group * p < 0.05, ** p <
0.01, *** p < 0.001
plantar injection of CFA into the hind paw produces an
inflammatory response that excites and sensitizes peripheral
nociceptive neurons, resulting in a long-lasting mechanical
hyperalgesia. Compound 13 induced a dose-related reversal of
established mechanical hyperalgesia induced by CFA. After oral
administration it induced a maximal reversal of 62 8%, with
an ED50 value (50% reversal of predose hyperalgesia) calculated
from data obtained 1 h following administration of 14.7 mg/kg.
The effect of compound 13 was rapid in onset, with maximal
activity achieved within 1 h. At the highest dose tested (30 mg/
kg), significant reversal up to 6 h postdose was measured.
Moreover, its antihyperalgesic activity was comparable to that
of the NSAID diclofenac.
Step 2: (E)-tert-Butyl 4-(3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)methyl)phenyl)allyl)piperazine-1-carboxylate (8.4 g,
17.16 mmol) was dissolved in dioxane (20 mL) and treated with 4 M
HCl in dioxane (8.58 mL, 34.3 mmol). After stirring the mixture for 5
h at rt, the solid was filtered off and washed with ether. After drying
the product, (E)-2-ethyl-5,7-dimethyl-3-(4-(3-(piperazin-1-yl)prop-1-
enyl)benzyl)pyrazolo[1,5-a]pyrimidine was obtained as an yellow solid
CONCLUSIONS
■
In this paper, we have disclosed the discovery of a novel,
selective, and orally bioavailable GPR4 antagonist 13. In
comparison to the lead compound 1a, the new compound
shows a more favorable safety profile with a substantially
H
DOI: 10.1021/acs.jmedchem.6b01703
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
Article
in 6.6 g (99%) yield. MS (ESI): 390.2 [M + H]+. 1H NMR (400 MHz,
DMSO-d6, δ) 12.08 (brs, 1H), 9.82 (brs, 2H), 7.37 (d, J = 8.2 Hz,
2H), 7.22 (d, J = 8.1 Hz, 2H), 6.85 (d, J = 15.9 Hz, 1H), 6.78 (s, 1H),
6.30 (dt, J = 15.8, 7.5 Hz, 1H), 4.07 (s, 2H), 3.94 (d, J = 7.3 Hz, 2H),
3.16−3.61 (m, 8H), 2.68 (q, J = 7.6 Hz, 2H), 2.65 (s, 3H), 2.50 (s,
3H), 1.13 (t, J = 7.6 Hz, 3H).
Step 3: NaBH(OAc)3 (84 mg, 0.40 mmol) was added to a mixture
of (E)-2-ethyl-5,7-dimethyl-3-(4-(3-(piperazin-1-yl)prop-1-enyl)-
benzyl)pyrazolo[1,5-a]pyrimidine (100 mg, 0.26 mmol), (S)-2,3-
dihydroxypropanal (23.1 mg, 0.26 mmol), and ethyldiisopropylamine
(52 μL, 0.30 mmol) in CH2Cl2 (2 mL), and the mixture was stirred at
70 °C for 3 h. After dilution with EtOAc, the mixture was washed with
water and brine, dried (Na2SO4), and concentrated. The crude
product was purified by MPLC (0−100% MeOH in CH2Cl2) to
provide the title product 8 as a white foam in 16 mg (13%) yield.
HRMS (C27H38N5O2) calculated 464.3026 [M + H]+, found 464.3023.
1H NMR (400 MHz, DMSO-d6, δ) 7.37 (d, J = 8.2 Hz, 2 H), 7.21 (d, J
= 8.1 Hz, 2 H), 6.84 (d, J = 15.6 Hz, 1 H), 6.77 (d, J = 0.7 Hz, 1 H),
6.29 (dd, J = 7.2, 15.8 Hz, 1 H), 4.06 (s, 3 H), 3.98−4.04 (m, 4 H),
3.62−3.77 (m, 4 H), 3.46−3.61 (m, 4 H), 3.42 (dd, J = 11.1, 4.7 Hz, 2
H), 3.31 (dd, J = 11.0, 6.2 Hz, 2 H), 3.11 (br s, 1 H), 2.67 (q, J = 7.6
Hz, 2 H), 2.64 (s, 3 H), 2.49 (s, 3 H), 1.12 (t, J = 7.6 Hz, 3 H). 13C
NMR (101 MHz, DMSO-d6, δ) 157.2, 156.8, 146.3, 144.4, 140.5,
134.2, 131.7, 128.1, 126.2, 126.1, 107.5, 104.2, 68.5, 64.9, 61.6, 60.2,
53.5, 52.7, 27.2, 24.2, 20.2, 16.3, 13.3.
(E)-4-(3-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
methyl)phenyl)allyl)piperazin-2-one (11). Step 1: A suspension of
(E)-3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
methyl)phenyl)prop-2-en-1-ol (24b) (3.5 g, 10.9 mmol) and MnO2
(9.47 g, 109 mmol) in acetonitrile (100 mL) was stirred under argon
at rt for 3 h. Then the mixture was filtered through Hyflo and
concentrated to provide (E)-3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)methyl)phenyl)acrylaldehyde as light-yellow crystals
in 2.57 g (74%) yield. MS (ESI): 320 [M + H]+. 1H NMR (400 MHz,
DMSO-d6, δ) 9.65 (d, J = 7.8 Hz, 1H), 7.68 (d, J = 15.9 Hz, 1H), 7.62
(d, J = 8.2 Hz, 2H), 7.29 (d, J = 8.2 Hz, 2H), 6.78 (s, 1H), 6.78 (dd, J
= 15.9, 7.8 Hz, 1H), 4.10 (m, 2H), 2.67 (q, J = 7.5 Hz, 2H), 2.64 (s,
3H), 2.50 (s, 3H), 1.14 (t, J = 7.5 Hz, 3H).
Step 2: NaBH(OAc)3 (313 mg, 1.48 mmol) was added to a mixture
of (E)-3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
methyl)phenyl)acrylaldehyde (236 mg, 0.74 mmol), piperazinone
(81 mg, 0.81 mmol), and acetic acid (51 μL, 0.89 mmol) in DCE (3
mL), and the mixture was stirred at rt for 2 h. After dilution with
CH2Cl2, the mixture was washed with NaHCO3, water, and brine,
dried (Na2SO4), and concentrated. The crude product was then
purified by MPLC (EtOAc/MeOH/NH4OH = 90:9:1 to 80:18:2) to
provide the title product 11 as a white solid in 184 mg (62%) yield.
HRMS (C24H30N5O) calculated 404.2450 [M + H]+, found 404.2447.
MS (ESI): 404.55 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ) 7.70
(br s, 1H), 7.32 (d, J = 8.1 Hz, 2H), 7.13 (d, J = 8.1 Hz, 2H), 6.75 (s,
1H), 6.49 (d, J = 15.9 Hz, 1H), 6.19 (dt, J = 15.9, 6.6 Hz, 1H), 4.01 (s,
2H), 3.08−3.19 (m, 4H), 2.92 (s, 2H), 2.66 (q, J = 7.7 Hz, 2H), 2.63
(s, 3H), 2.56 (t, J = 5.4 Hz, 2H), 2.48 (s, 3H), 1.12 (t, J = 7.6 Hz, 3H).
13C NMR (101 MHz, DMSO-d6, δ) 167.6, 157.2, 156.8, 146.3, 144.4,
H), 3.23 (tt, J = 10.9, 4.0 Hz, 1 H), 2.90−3.05 (m, 2 H), 2.68 (q, J =
7.7 Hz, 2 H), 2.64 (s, 3 H), 2.48 (s, 3 H), 1.99−2.09 (m, 2 H), 1.58−
1.72 (m, 2 H), 1.40 (s, 9 H), 1.12 (t, J = 7.6 Hz, 3 H).
Step 2: tert-Butyl 4-(5-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-a]-
pyrimidin-3-yl)methyl)phenyl)-1,3,4-oxadiazol-2-yl)piperidine-1-car-
boxylate (207 mg, 0.4 mmol) was treated with 4 M HCl in dioxane (4
mL) and stirred at rt for 1 h. The mixture was then evaporated and
triturated with ether and CH2Cl2. The suspension was filtered off,
washed with ether, and dried to give the product 13 as an HCl salt in
144 mg (73%) yield (yellow solid). HRMS (C24H29N6O) calculated
417.2403 [M + H]+, found 417.2398. FTIR: 3417, 2971, 2937, 2804,
2717, 2497, 1881, 1662, 1621, 1588, 1568, 1500, 1452, 1422, 1297,
1
1037, 844. H NMR (400 MHz, DMSO-d6, δ) 9.14 (brs, 1 H), 8.92
(brs, 1 H), 7.87 (d, J = 8.1 Hz, 2 H), 7.40 (d, J = 8.1 Hz, 2 H), 6.79 (s,
1 H), 4.14 (s, 2 H), 3.39−3.46 (m, 1 H), 3.27−3.35 (m, 2 H), 3.00−
3.12 (m, 2 H), 2.68 (q, J = 7.6 Hz, 2 H), 2.64 (s, 3 H), 2.49 (s, 3 H),
2.18−2.28 (m, 2 H), 1.95−2.06 (m, 2 H), 1.12 (t, J = 7.6 Hz, 3 H).
13C NMR (101 MHz, DMSO-d6, δ) 168.9, 163.6, 157.4, 156.9, 146.4,
145.4, 144.6, 128.9, 126.4, 121.2, 107.7, 103.4, 45.1, 33.2, 30.0, 27.5,
24.2, 20.2, 16.3, 13.3.
2-(4-Bromo-benzyl)-3-oxo-pentanenitrile (20). A 1.7 M solution
of potassium i-amylate in toluene (81 mL, 139 mmol) was added
dropwise to a solution of 3-(4-bromo-phenyl)-propionitrile (9.70 g,
46.2 mmol) in THF (200 mL) at rt being followed by addition of ethyl
propionate (21.2 mL, 185 mmol). After stirring for 20 min, the
reaction mixture was quenched by addition of 1N hydrochloric acid
and extracted with ethyl acetate. The organic layer was washed with
water and brine, dried over Na2SO4, and evaporated. The residue was
purified by column chromatography (5−30% EtOAc/n-hexane) to
yield the product 20 as an yellow oil in 10.4 g (85%) yield. MS (ESI):
285.1 [M + NH4]+, 266.0 [M − H]−. 1H NMR (600 MHz, DMSO-d6,
δ) 7.47 (d, J = 8.3 Hz, 2 H), 7.14 (d, J = 8.2 Hz, 2 H), 3.61 (dd, J =
8.2, 5.6 Hz, 1 H), 3.18 (dd, J = 13.9, 5.6 Hz, 1 H), 3.08 (dd, J = 13.9,
8.4 Hz, 1 H), 2.60−2.74 (m, 2 H), 1.09 (t, J = 7.2 Hz, 3 H).
4-(4-Bromo-benzyl)-5-ethyl-2H-pyrazol-3-ylamine (21). A mixture
of 2-(4-bromo-benzyl)-3-oxo-pentanenitrile (20) (10.4 g, 39.1 mmol)
and hydrazine hydrate (1.9 mL, 39.1 mmol) in a 1:1 mixture of
ethanol and acetic acid (35 mL) was heated in a microwave reactor to
140 °C for 15 min. After cooling, the reaction mixture was diluted with
ethyl acetate and washed several times with satd NaHCO3 followed by
brine. The organic layer was dried over Na2SO4 and evaporated to give
a mixture of 21 and its N-acetamide. For conversion of the latter to 21,
the crude product was taken up in 1N sodium hydroxide and heated to
160 °C in a microwave reactor for 30 min. The reaction mixture was
extracted with ethyl acetate, and the organic layer was washed with
brine, dried over Na2SO4, and evaporated to give the product 21 as a
white powder in 8.3 g (76%) yield. The compound was used in the
next step without any further purification. MS (ESI): 280.1 [M + H]+.
1H NMR (400 MHz, DMSO-d6, δ) 7.41 (d, J = 8.3 Hz, 2 H), 7.10 (d, J
= 8.3 Hz, 2 H), 3.55 (s, 3 H), 3.39 (br s, 3 H), 2.33 (q, J = 7.6 Hz, 2
H), 0.98 (t, J = 7.6 Hz, 3 H).
3-(4-Bromo-benzyl)-2-ethyl-5,7-dimethyl-pyrazolo[1,5-a]-
pyrimidine (22). To a solution of 4-(4-bromo-benzyl)-5-ethyl-2H-
pyrazol-3-ylamine (21) (7.9 g, 28.2 mmol) in a 1:1 mixture of dioxane
and trifluoroacetic acid (120 mL) was added acetylacetone (2.9 mL,
28.2 mmol) at rt, and the reaction mixture was stirred at 100 °C for 16
h. The mixture was neutralized with satd NaHCO3, extracted with
ethyl acetate, dried over Na2SO4, and evaporated to dryness to provide
product 22 as an off-white solid in 9.5 g (98%) yield. MS (ESI): 346.0
140.7, 134.1, 132.3, 131.8, 128.1, 127.0, 126.2, 125.2, 107.5, 104.2,
58.9, 56.6, 48.4, 27.2, 24.2, 20.2, 16.3, 13.3.
2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)-
phenyl)-5-(piperidin-4-yl)-1,3,4-oxadiazole (13). Step 1: Et3N (3.93
mL, 28.2 mmol) was added to a mixture of 35 (2.56 g, 4.7 mmol) and
TsCl (1.34 g, 7.1 mmol) in CH2Cl2 (84 mL) and DMF (5 mL). After
stirring at rt for 16 h, more TsCl (445 mg, 2.3 mmol) was added and
the mixture was stirred at rt for additional 2 h. It was then treated with
satd aq NaHCO3 and extracted with CH2Cl2. The organic layers were
washed with water and brine, dried (Na2SO4), and concentrated. The
crude product was purified by MPLC (25−50% EtOAc in cyclo-
hexane) to give tert-butyl 4-(5-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-
a]pyrimidin-3-yl)methyl)phenyl)-1,3,4-oxadiazol-2-yl)piperidine-1-
carboxylate as a white solid in 2.15 g (59%) yield. MS (ESI): 517.3 [M
1
[M]+. H NMR (600 MHz, DMSO-d6, δ) 7.41 (d, J = 8.1 Hz, 1 H),
7.12 (d, J = 8.3 Hz, 1 H), 6.75 (s, 1 H), 3.99 (s, 2 H), 2.65 (q, J = 7.7
Hz, 2 H), 2.61 (s, 3 H), 2.46 (s, 3 H), 1.11 (t, J = 7.6 Hz, 3 H).
(E)-Methyl 3-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-
yl)methyl)phenyl)acrylate (23b). According to the general procedure
for Heck coupling, 3-(4-bromobenzyl)-2-ethyl-5,7-dimethylpyrazolo-
[1,5-a]pyrimidine (22) (3 g, 8.71 mmol), 15 min at 130 °C
(microwave oven), and MPLC (5−50% ethyl acetate in cyclohexane)
provided the product 23b as a white solid in 2.89 g (95%) yield. MS
1
1
+ H]+. H NMR (400 MHz, DMSO-d6, δ) 7.87 (d, J = 8.1 Hz, 2 H),
(ESI): 530.1 [M + H]+. H NMR (400 MHz, CDCl3, δ) 7.64 (d, J =
7.39 (d, J = 8.1 Hz, 2 H), 6.77 (s, 1 H), 4.13 (s, 2 H), 3.85−3.98 (m, 2
16.2 Hz, 1 H), 7.39 (d, J = 8.1 Hz, 2 H), 7.25 (d, J = 8.0 Hz, 2 H), 6.46
I
DOI: 10.1021/acs.jmedchem.6b01703
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Journal of Medicinal Chemistry
Article
(s, 1 H), 6.36 (d, J = 15.9 Hz, 1 H), 4.15 (s, 2 H), 3.78 (s, 3 H), 2.72
(d, J = 7.6 Hz, 2 H), 2.66−2.75 (m, 5 H), 2.54 (s, 3 H), 1.19 (t, J = 7.6
Hz, 3 H).
(HTRF) technology as provided by CisBio Inc. The cells were seeded
in 384-well plates and cultured for 24 h at 37 °C, 5% CO2 before
performing the assay. Medium was removed, and 10 μL of buffer A
(Hepes buffered saline (HBS), 10 mM Hepes, pH 8, 2 mM 3-isobutyl-
1-methylxanthin (IBMX)) was added. For compound testing, buffer A
with 2× concentrated compounds was used. Cells were incubated for
15 min at room temperature. Then 10 μL of buffer B (HBS, 30 mM
Hepes, specific pH) was added to reach the appropriate final pH for
stimulation, and incubation was continued for 15 min at rt. Finally, 10
μL of cAMP-XL 665 and 10 μL of anti-cAMP-cryptate were dispensed
and plates were read on a Pherastar reader after 60 min incubation at
rt. Data were calculated from the 665 nm/620 nm ratio, and % activity
was normalized according to values at minimum and maximum of
GPR4 activation.
hERG Binding Assay. HEK293 cells stably transfected with the
HERG-1 gene (Swiss-Prot: Q12809) were obtained under license
from the Wisconsin Alumni Research Foundation. Membranes
prepared from these cells were used in the binding assay using the
radioligand:[3H]dofetilide with specific activity of 46.5 Ci/mmol. The
assay contains in a 96-well Millipore GF/C filter plate 119 μL of
buffer, 1 μL of compound, 40 μL of 12.5 nM [3H]dofetilide, and 40
μL of crude membrane suspension (15 μg protein). After for 90 min at
room temperature, incubations were terminated by rapid filtration and
three washes with ice-cold buffer. The dried including 40 μL of
scintillant was read in a Wallac MicroBeta Trilux β counter.
Histamine H3 Receptor Binding Assay. Lyophilized PVT PEI-
treated wheat germ agglutinin SPA beads type A (RPNQ0003) were
purchased from GE Healthcare (Buckinghamshire, UK). [3H]-R(−)-
α-Methyl[imidazole-2.5(n)]histamine, an agonist radioligand, was
purchased from GE Healthcare, TRK1017 (specific activity 34 Ci/
mmol, 1mCi/mL). Membrane prepared from CHO-K1 cells stably
expressing the human H3 histamine receptor were purchased from
Euroscreen (ES-392-M). The SPA assay was performed in a final
volume of 50 μL in a 384-well polystyrene plate (10 μL of test
compounds, total binding was determined by adding 10 μL of water,
and nonspecific binding was determined by the addition of 10 μL of
Clobenpropit, 20 μL of 7.5 nM [3H]-R-α-methylhistamine in assay
buffer [50 mM Tris-HCl, 5 mM EDTA, 1 mM EDTA, pH 7.4], 20 μL
of beads, and membranes mixed suspension in assay buffer). The
plates were shaken at room temperature, then allowed to stand for at
least 1 h and then counted using a PerkinElmer TopCount reader.
In Vivo Experiments. All animal studies were performed in
accordance with the animal experimentation guidelines and laws laid
down by the Swiss Federal and Cantonal Authorities. The animal
licenses were granted by the Veterinary Authority of the City of Basel,
Switzerland.
in Vivo Pharmacokinetic Studies in Rats. First, 96−120 h before
administration of the test substance, adult female wild-type Sprague−
Dawley rats (Iffa Credo, France) were anesthetized with isoflurane and
catheters were surgically implanted under aseptic precautions (use of
sterile instruments and surgical material in combination with local
antibiotic prophylaxis) into the femoral artery and vein. Then the
catheters were exteriorized in the neck region, connected to a Harvard
swivel system (Harvard Instruments), and filled with 0.9% saline
containing 100 U·mL−1 heparin. After recovery from anesthesia, the
animals were housed individually in special cages with free access to
food and tap water until and throughout the experiment. Analgesic
treatment with Temgesic (10 μg/kg sc, application volume 1 mL/kg)
was performed the evening following surgery and the next morning.
Compound administration in cassette format was in the morning (6−8
AM). Blood samples were collected at various time points from the
femoral artery catheter into Eppendorf tubes coated with sodium
EDTA. Blood samples were immediately frozen at −20 °C until final
processing (maximum storage was 8 days). Intravenous and oral
dosing was performed in the same animals after a 48 h wash-out
interval between the single dose applications. The test substances were
administered intravenously as a solution in 1-methyl-2-pyrrolidone and
polyethylene glycol 200 (30:70, v/v) at a dose of 1 mg/kg per
compound and orally as a homogeneous aqueous suspension in Tween
(E)-3-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
methyl)phenyl)prop-2-en-1-ol (24b). According to the general
procedure for DIBALH reduction, (E)-methyl 3-(4-((2-ethyl-5,7-
dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)phenyl)acrylate (23b)
(1 g, 2.86 mmol) provided the product 24b as a white solid in 850 mg
(92%) yield. MS (ESI): 322.3 [M + H]+. 1H NMR (400 MHz, CDCl3,
δ)7.25 (d, J = 7.6 Hz, 2 H), 7.18 (d, J = 7.6 Hz, 2 H), 6.55 (d, J = 15.9
Hz, 1 H), 6.45 (s, 1 H), 6.25−6.33 (m, 1 H), 4.28 (d, J = 5.6 Hz, 2 H),
4.12 (s, 2 H), 2.72 (q, J = 7.6 Hz, 2 H), 2.69 (s, 3 H), 2.54 (s, 3 H),
1.19 (t, J = 7.6 Hz, 3 H).
Methyl 4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)-
methyl)benzoate (32). Et3N (167 mL, 1.2 mol) was added to a
solution of 3-(4-bromobenzyl)-2-ethyl-5,7-dimethylpyrazolo[1,5-a]-
pyrimidine (22) (20.65 g, 60 mmol) in dry methanol (300 mL) and
DMF (360 mL), and the mixture was purged with CO. PdCl2dppf
(4.90 g, 6 mmol) and dppf (0.34 g, 0.6 mmol) were added, and the
mixture was stirred under CO atmosphere (balloon) at 80 °C for 16 h.
After cooling to rt, the mixture was filtered through a plug of Hyflo
and washed with methanol, and the filtrate was concentrated on RV.
The residue was treated with satd aq NaHCO3 and extracted with
EtOAc. The organic layers were washed with brine, dried (Na2SO4),
and concentrated on RV. The crude product was purified by MPLC
(25% EtOAc in cyclohexane) to provide the product 32 as a white
1
solid in 10.26 g (52%) yield. MS (ESI): 324.0 [M + H]+. H NMR
(400 MHz, DMSO-d6, δ) 7.84 (d, J = 8.1 Hz, 2 H), 7.31 (d, J = 8.3 Hz,
2 H), 6.77 (s, 1 H), 4.11 (s, 2 H), 3.81 (s, 3 H), 2.65 (q, J = 7.6 Hz, 2
H), 2.63 (s, 3 H), 2.47 (s, 3 H), 1.10 (t, J = 7.6 Hz, 3 H).
4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3-yl)methyl)-
benzohydrazide (33). Hydrazine hydrate (38.5 mL, 840 mmol) was
added to a solution of 32 (7.76 g, 24 mmol) in methanol (78 mL) at
rt, and then the mixture was stirred at reflux for 2 h. The obtained clear
solution was cooled down to rt with stirring, and the formed white
solid was filtered off. The filtrate was then concentrated and treated
with water. The white precipitate was filtered off, washed with water
and Et2O, and finally purified by MPLC (0−50% CH3OH in EtOAc).
The title product 33 was obtained as a white solid in 6.85 g (87%)
yield. MS (ESI): 324.0 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ)
9.64 (brs, 1 H), 7.69 (d, J = 8.1 Hz, 2 H), 7.23 (d, J = 8.1 Hz, 2 H),
6.76 (s, 1 H), 4.43 (brs, 2 H), 4.07 (s, 2 H), 2.68 (q, J = 7.6 Hz, 2 H),
2.63 (s, 3 H), 2.48 (s, 3 H), 1.11 (t, J = 7.6 Hz, 3 H).
tert-Butyl 4-(2-(4-((2-Ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-
3-yl)methyl)benzoyl)hydrazinecarbonyl)piperidine-1-carboxylate
(35). EDC·HCl (345 mg, 1.8 mmol) was added to a mixture of
hydrazide 33 (504 mg, 1.56 mmol), 1-(tert-butoxycarbonyl)piperidine-
4-carboxylic acid (278 mg, 1.2 mmol), HOBt·H2O (195 mg, 1.44
mmol), and Et3N (0.217 mL, 1.56 mmol) in CH2Cl2 (12 mL). After
stirring at rt for 16 h, the mixture was treated with satd aq NaHCO3
and extracted with EtOAc. The organic layers were washed with water
and brine, dried (Na2SO4), and concentrated. The crude product was
purified by flash chromatography (10% MeOH in CH2Cl2) to give the
compound 35 as a white solid in 632 mg (97%) yield. MS (ESI): 535.2
[M + H]+. 1H NMR (400 MHz, DMSO-d6, δ) 10.18 (s, 1 H), 9.83 (s,
1 H), 7.74 (d, J = 8.1 Hz, 2 H), 7.27 (d, J = 8.1 Hz, 2 H), 6.77 (s, 1 H),
4.10 (s, 2 H), 3.89−4.01 (m, 2 H), 2.72−2.92 (m, 2 H), 2.67 (q, J =
7.6 Hz, 2 H), 2.63 (s, 3 H), 2.48 (s, 3 H), 2.43 (tt, J = 11.4, 3.3 Hz, 1
H), 1.64−1.77 (m, 2 H), 1.42−1.52 (m, 2 H), 1.40 (s, 9 H), 1.12 (t, J
= 7.6 Hz, 3 H).
Biology. pH-Dependent cAMP Release Assay (Human GPR4 in
HeLa, Rat, and Mouse GPR4 in HEK Cells). HeLa and HEK cells
stably expressing human, rat, and mouse GPR4, respectively, were
established by transfecting the cells with a construct containing the
respective GPR4 coding sequence. The cells were grown in Dulbecco’s
Modified Eagle Medium (DMEM)/HAM’s tissue culture medium F12
(HAM’s F12) medium supplemented with 10% fetal calf serum (FCS),
100 U/mL penicillin, 100 μg/mL streptomycin, and 400 μg/mL G418
and 10 mM Hepes pH 8.0. pH-induced formation of cAMP was
determined using the homogeneous time-resolved fluorescence
J
DOI: 10.1021/acs.jmedchem.6b01703
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Journal of Medicinal Chemistry
Article
80 and carboxy methyl cellulose sodium 0.5/0.5/99 (w/w) at a dose of
3 mg/kg per compound.
ACKNOWLEDGMENTS
■
We thank Dr. Klaus Seuwen for useful discussions and for
critical reading of the manuscript.
Rat Antigen Induced Arthritis. Female Lewis rats were sensitized
intradermally on the back at two sites to methylated bovine serum
albumin (mBSA) homogenized 1:1 with complete Freund’s adjuvant
on days −21 and −14 (0.1 mL containing 1 mg/mL mBSA). On day
0, the right knee received 50 μL of 10 mg/mL mBSA in 5% glucose
solution (antigen injected knee), while the left knee received 50 μL of
5% glucose solution alone (vehicle injected knee). The diameters of
the left and right knees were then measured using calipers immediately
after the intra-articular injections and again on days 2, 4, and 7.
Treatments were administered daily as follows by oral gavage; vehicle
(saline) at 5 mL/kg, compounds as indicated. Right knee swelling was
calculated as a ratio of left knee swelling, and the R/L knee swelling
ratio plotted against time to give area under the curve (AUC) graphs
for control and treatment groups.
ABBREVIATIONS USED
■
cHex, cyclohexyl; CDI, carbonyldiimidazole; Zaragoza reagent,
(cyanomethyl)trimethylylphosphonium iodide; HOBt, 1-hy-
droxybenzotriazole; EDC.HCl, 1-ethyl-3-(3-(dimethylamino)-
propyl)carbodiimide hydrochloride; BOP, (benzotriazol-1-
yloxy)tris(dimethylamino)phosphonium hexafluorophosphate;
dppf, 1,1′-bis(diphenylphosphino)ferrocene; UPLC, ultraper-
formance liquid chromatography; MPLC, medium pressure
liquid chromatography
Angiogenesis Growth Factor Model. Porous tissue chambers made
of perfluoro-alkoxy-Teflon (Teflon-PFA, 21 mm × 8 mm diameter,
550 μL volume) were filled with 0.8% agar and 20 U/mL heparin
supplemented with or without 2 ug/mL recombinant human
VEGF165. Female FVB mice of 8−10 weeks old were anesthetized
using 3% isoflurane inhalation. For subcutaneous implantation, a small
skin incision was made at the base of the tail to allow the insertion of
an implant trocar. The chamber was implanted under aseptic
conditions through the small incision onto the back of the animal.
The skin incision was closed by wound clips. On the fourth day after
implantation, animals were sacrificed using CO2. Chambers were
excised, and the vascularized fibrous tissue formed around each
implant carefully removed and weighed. Body weight was used to
monitor the general condition of the mice. Compound 13 was dosed
twice a day at 30 mg/kg for 4 days and was started 5 h prior to
implantation of the chamber.
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ASSOCIATED CONTENT
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* Supporting Information
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AUTHOR INFORMATION
■
Corresponding Author
*Phone: +41-61-3243399. E-mail: juraj.velcicky@novartis.com.
ORCID
Juraj Velcicky: 0000-0001-6564-1448
Notes
The authors declare no competing financial interest.
K
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