Development of Selective, Orally Active GPR4 ... - ACS Publications

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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.

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INTRODUCTION

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.

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. © XXXX American Chemical Society

Received: November 21, 2016

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DOI: 10.1021/acs.jmedchem.6b01703 J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry

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Scheme 1. Synthesis of the Key Intermediates 18 and 22a

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,ba

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, iPr2NEt, 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%). a

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Scheme 3. Attachment of Allyl Piperidine/Piperazine Containing Tails to Intermediate 22a

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%).

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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 Pdcatalyzed 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 consisting of three steps: (i) alkylation of 14 by para-bromobenzyl bromide, (ii) bromination with NBS, and (iii) Amberlystmediated 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

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)-2,3dihydroxypropanal. Hydrogenation of the allyl double bond in 3 led to propylene analogue 4. Compound 11 was prepared via reductive amination between piperazinone and cinnamaldehyde 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. 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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Scheme 4. Synthesis of the Oxadiazole Linker Containing Compounds 12 and 13a

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

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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 NBoc-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

IC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels. bIC50 determined as a mean (n ≥ 2) by a binding assay using radiolabeled 3H-dofetilide binding to HEK293 cell membranes expressing human recombinant HERG K+ channels. cAntagonist 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. a

effect on H3- and no effect on hERG binding. However, removal of the iso-propyl group (6) led to reduced hERGbinding 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, 1,3,4oxadiazole 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

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 E



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Table 2. Tail Derivatization and Its Influence on Potency, Off-Targets, and PK Properties

IC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels. bIC50 determined as a mean (n ≥ 2) by a binding assay using radiolabeled 3H-dofetilide binding to HEK293 cell membranes expressing human recombinant HERG K+ channels. cAntagonist 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. dCL: clearance measured as a mean ± SD of four animals (female Sprague−Dawley Rat) after iv dosing (1 mg/ a

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Table 2. continued kg) using NMP:PEG200 (30:70) formulation. eExposure (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 compd

hGPR4a [μM]

rGPR4b [μM]

inhibition in rat AIAc [%]

hGPR4 with 40% rat plasmad [μM]

IC50 shift (hGPR4 with 40% plasma IC50/hGPR4)

8 11 13

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 0.067 ± 0.012

3 33 1

IC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels; bIC50 determined as a mean (n ≥ 3) in the HEK cells expressing rat GPR4 measuring cAMP levels; cDetermined 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); dIC50 determined as a mean (n ≥ 3) in the HELA cells expressing human GPR4 measuring cAMP levels after incubation with 40% rat plasma. a

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 patchclamp 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

the oxadiazole 13. While the hydroxy-piperidine derivatives (6 and 7) tend to have further increased clearance, the bishydroxypiperazine 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

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).

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

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). 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 antiinflammatory 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.

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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 otherwise stated. 1H 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[1,5a]pyrimidin-3-yl)methyl)phenyl)allyl)piperazin-1-yl)propane-1,2diol (8). Step 1: According to the general procedure for Zaragoza coupling, (E)-3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5-a]pyrimidin-3yl)methyl)phenyl)prop-2-en-1-ol (24b) (6 g, 18.67 mmol) and Bocpiperazine (3.48 g, 18.67 mmol) after 3 h at 60 °C provided (E)-tertbutyl 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, DMSOd6, 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). Step 2: (E)-tert-Butyl 4-(3-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5a]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-1enyl)benzyl)pyrazolo[1,5-a]pyrimidine was obtained as an yellow solid

Figure 3. Anti-nociceptive effect of compound 13. The naiv̈ e 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 − ̈ ipsilateral threshold − predose predose ipsilateral threshold)/(naive 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.

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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



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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)-2,3dihydroxypropanal (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. 1 H 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[1,5a]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). 13 C NMR (101 MHz, DMSO-d6, δ) 167.6, 157.2, 156.8, 146.3, 144.4, 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 cyclohexane) to give tert-butyl 4-(5-(4-((2-ethyl-5,7-dimethylpyrazolo[1,5a]pyrimidin-3-yl)methyl)phenyl)-1,3,4-oxadiazol-2-yl)piperidine-1carboxylate as a white solid in 2.15 g (59%) yield. MS (ESI): 517.3 [M + H]+. 1H NMR (400 MHz, DMSO-d6, δ) 7.87 (d, J = 8.1 Hz, 2 H), 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

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-carboxylate (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, 1037, 844. 1H 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). 13 C 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]+. 1 H 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-2Hpyrazol-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 [M]+. 1H 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-3yl)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 (ESI): 530.1 [M + H]+. 1H NMR (400 MHz, CDCl3, δ) 7.64 (d, J = 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



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(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). (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-5,7dimethylpyrazolo[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 solid in 10.26 g (52%) yield. MS (ESI): 324.0 [M + H]+. 1H 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]pyrimidin3-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)piperidine4-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

(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-isobutyl1-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 PEItreated 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 J



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80 and carboxy methyl cellulose sodium 0.5/0.5/99 (w/w) at a dose of 3 mg/kg per compound. 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. 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. ̈ withdrawal thresholds of both Mechanical Hyperalgesia. Naive hind paws were determined in male Wistar Han rats by using an increasing pressure stimulus placed onto the dorsal surface of each paw using an analgesimeter. Delayed inflammatory pain was then induced by intraplantar injection of 25 μL of Complete Freund’s Adjuvant (CFA) into one hindpaw with the contralateral paw acting as the control. After 3 days, 13 (3, 10, and 30 mg/kg) or diclofenac (30 mg/ kg) were administered by gavage as suspension in methylcellulose 5%. One hour later, paw withdrawal thresholds were remeasured on both the ipsilateral (CFA-injected) and contralateral (uninjected) paw; measurements were repeated at 3 and 6 h post dosing. The reversal of hyperalgesia was calculated using the following formula: Reversal (%) = 100 × (postdose ipsilateral threshold − predose ipsilateral ̈ ipsilateral threshold − predose ipsilateral threshthreshold)/(naive old).

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ACKNOWLEDGMENTS We thank Dr. Klaus Seuwen for useful discussions and for critical reading of the manuscript. ABBREVIATIONS USED cHex, cyclohexyl; CDI, carbonyldiimidazole; Zaragoza reagent, (cyanomethyl)trimethylylphosphonium iodide; HOBt, 1-hydroxybenzotriazole; EDC.HCl, 1-ethyl-3-(3-(dimethylamino)propyl)carbodiimide hydrochloride; BOP, (benzotriazol-1yloxy)tris(dimethylamino)phosphonium hexafluorophosphate; dppf, 1,1′-bis(diphenylphosphino)ferrocene; UPLC, ultraperformance liquid chromatography; MPLC, medium pressure liquid chromatography

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The Supporting Information is available free of charge on the ACS Publications website at DOI: 10.1021/acs.jmedchem.6b01703. Synthesis procedures and characterization data and HPLC and NMR charts for compounds 8, 11, and 13 (PDF) Molecular formula strings (CSV)

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