Development of a Potent and Selective G2A (GPR132) Agonist

Abstract

G protein-coupled receptor G2A was postulated to be a promising target for the development of new therapeutics in neuropathic pain, acute myeloid leukemia, and inflammation. However, there is still a lack of potent, selective, and drug-like G2A agonists to be used as a chemical tool or as the starting matter for the development of drugs. In this work, we present the discovery and structure–activity relationship elucidation of a new potent and selective G2A agonist scaffold. Systematic optimization resulted in (3-(pyridin-3-ylmethoxy)benzoyl)-d-phenylalanine (T-10418) exhibiting higher potency than the reference and natural ligand 9-HODE and high selectivity among G protein-coupled receptors. With its favorable activity, a clean selectivity profile, excellent solubility, and high metabolic stability, T-10418 qualifies as a pharmacological tool to investigate the effects of G2A activation.

Introduction

The G protein-coupled receptor GPR132, also termed G2A (G2 accumulation protein), was first described by Witte et al. in 1998.¹ In 2003, Ludwig et al. introduced the concept of proton-sensing GPCRs and originally assigned the four orphan receptors of the class A GPCRs: GPR4, TDAG8 (GPR65), OGR1 (GPR68), and G2A into the family.²⁻⁵ The physiological and pathophysiological role of proton-sensing GPCRs was initially speculated to be sensing tissue injury, inflammation, or tumor growth where local pH decreases.^6,7 However, proton sensitivity of G2A is under controversial discussion in the literature.⁸⁻¹⁰ Under physiological conditions, GPR4, OGR1, and G2A are expressed ubiquitously, with G2A showing the strongest expression in leukocytes, neutrophils, and macrophages.^8,11,12 G2A is expressed in peripheral sensory neurons that coexpress the transient receptor potential vanilloid 1 channel (TRPV1).¹³ In this environment, G2A is essentially connected with development and persistence of oxaliplatin-induced neuropathic pain (OINP) through protein kinase C (PKC) activation of and subsequent sensitization of TRPV1. Therefore, inhibition of G2A was proposed as a promising novel approach to attenuate OINP.¹⁴ G2A has also been identified as a factor promoting breast cancer metastasis. Since G2A is transcriptionally repressed upon activation of peroxisome proliferator-activated receptor γ, its repression potentially contributes to the antitumor effects of rosiglitazone and suggests inhibition of G2A as a new strategy for treatment of breast cancer.^15,16

G2A has also been considered to be a receptor for lipids, and lysophosphatidylcholine (LPC) was described as the first activator.¹² However, LPC as a G2A agonist has not gained widespread acceptance. One study claiming G2A activation by LPC was retracted,¹⁷ several other studies confirmed lack of G2A agonism of LPC,¹⁸ and one study even concluded that LPC is actually a weak antagonist of G2A (IC₅₀ > 10 μM).⁸ Oxidized lipids derived from linoleic acid (hydroxyoctadecadienoic acids, HODEs) or from arachidonic acid (hydroxyeicosatrienoic acids, HETEs) have also been described as G2A ligands exhibiting weak to moderate G2A agonism among which the linoleic acid metabolite 9-HODE has been reported as the most potent endogenous G2A agonist (Figure 1A).¹⁹ Notably, the structure of G2A in complex with its endogenous agonist 9-HODE has been recently resolved by means of cryo-electron microscopy.²⁰ Also the natural bacterial product commendamide²¹ as well as N-palmitoylglycine and N-linoleoylglycine have been discovered as G2A-activators strengthening the concept of G2A being a receptor for signaling lipids rather than a proton sensor. Compounds SB-583831 (Figure 1B) and SKF-95667 (Figure 1C) were identified as potent synthetic agonists, with the EC₅₀ value of SB-583831 being in the nanomolar range. On the other hand, compound SB-583355 (Figure 1D) and the telmisartan analogue GSK1820795A (Figure 1E) were reported as potent G2A antagonists.¹⁸

Figure 1.

Structures of 9(S)-HODE (A), SB-583831 (B), SKF-95667 (C), SB-583355 (D), GSK1820795A (E), ONC201 (F), and ONC212 (G).

G2A activation has been suggested to offer potential in hematopoiesis,²² sepsis,²³ and acute myeloid leukemia (AML).²⁴ The latter indication is supported by the observation that the anticancer agents ONC201 and ONC212 (Figure 1F,G) activate G2A in a β-arrestin recruitment assay. ONC212 also exerted prominent proapoptotic effects in AML but not in healthy bone marrow cells and reduced AML growth in vivo, suggesting that G2A activation may be a promising approach for the treatment of AML.

Taken together, current evidence points to the potential of both G2A activation and inhibition for the development of new therapeutics. In this work, we present the discovery and SAR elucidation of a novel potent and selective G2A agonist scaffold. Optimized analogues exceeded the reference agonist ±9-HODE in G2A activation efficacy and have excellent solubility as well as metabolic stability, thus emerging as a pharmacological tool for studying G2A.

Results and Discussion

High-Throughput Screening for Identification of G2A Agonists

In order to identify novel G2A agonists, approx. 25,000 compounds from the Enamine library were screened in an unbiased high-throughput-screening (HTS) setup. Compounds were evenly sourced from all subsets of the library, and library plates with a higher number of compounds containing carboxylic acids were prioritized, resulting in approximately 3000 carboxylic acids in the screened set. The compounds were tested at 50 μM in a functional cell-based assay with detection of accumulation of inositol monophosphate (IP-1) as a measure for activation of human G2A. Efficacy was compared to the reference agonist 9-HODE (EC₅₀ = 7.5 μM). The most promising hit (1, Figure 2) in terms of potency (EC₅₀ = 1.74 μM) and synthetic accessibility was selected for further optimization.

Figure 2.

Compound 1, the most promising candidate from the HTS campaign.

Hit Validation in Mouse Primary Sensory Neurons

To validate the hit, compound 1 was applied to a calcium-imaging-based TRPV1 sensitization assay. As described previously, activation of G2A in sensory neurons causes enhanced activity of TRPV1 in a G_q-PKC-dependent manner;¹⁴ we therefore stimulated mouse primary sensory neurons twice with two identical capsaicin stimuli (100 nM, 30 s) and preincubated compound 1 prior to the second capsaicin stimulus. Analysis of the amplitude of calcium responses [Δ_ratio(F340/F380)] revealed that compound 1 could potently sensitize for capsaicin-induced TRPV1 responses and was comparable to the reference G2A agonist 9-HODE (Figure 3A,B).

Figure 3.

Compound 1 potently sensitized capsaicin-induced TRPV1 responses in mouse primary sensory neurons. (A) Representative traces of capsaicin-induced calcium responses in mouse sensory neurons. Neurons were incubated with vehicle [DMSO, 0.013% (v/v) black], (±)9-HODE (200 nM, 2 min, gray), or compound 1 (1 μM, 2 min, light gray) 2 min prior to the second capsaicin stimulus. Stimulations with capsaicin (100 nM) took place for 30 s as indicated. (B) Statistical analysis of the amplitudes of the second capsaicin responses with the respective conditions. Data are shown as mean ± SEM from n = 44 to 81 neurons, **p < 0.01, ***p < 0.001, one-way ANOVA with Tukeýs post hoc test.

Chemistry

To explore the SAR of the selected scaffold, the synthetic efforts were divided in two parts. A set of 32 compounds was prepared to study the role of the phenylalanine motif (compounds 2–33). This set of compounds was prepared in a rapid amide coupling and ester hydrolysis procedure in one pot as depicted in Scheme 1. Final structures and activity results are presented in Table 1.

Scheme 1. Synthetic Procedure Used for the Synthesis of Compounds 2–33.

Conditions. (a) HOBt, TEA, DMF. (b) KOH/H₂O, DMSO.

Table 1. First Set of Compounds (1–33) with EC₅₀ for Human G2A Activation as Determined in the IP-One Assay and Efficacy at 20 μM of Compound Relative to Maximal Activation Observed with ±9-HODE (for Compounds with Observable Activity and for 9(S)-, 9(R)-, and Racemic 9-HODE the Dose–Response Curves Are Shown in Supporting Information Figure S1).

Further synthetic efforts were carried out to investigate the activity of both enantiomers of the most promising compound with aliphatic substitution on the amino acid residue (41, 42) and to explore more substituents in para position of the benzylic ring (43, 44). Additionally, variations on the left side of the scaffold were studied by exchanging the position of the nitrogen in the pyridine ring (49, 50) and by replacing the pyridine ring by benzene (51). The synthesis of these sets of final compounds could be achieved by two different synthetic routes (Scheme 2). For the optimization study of the amino acid moiety, synthetic route A was employed, whereas for optimization of the left side of the molecule, route B was preferred. Route A consisted of 4 synthetic steps. First, 3-(bromomethyl)pyridine was coupled with methyl 3-hydroxybenzoate in the presence of cesium carbonate in dimethylformamide (DMF) to provide compound 34. Then, the ester was hydrolyzed under basic conditions and the resulting carboxylic acid 35 reacted in the next step with the corresponding protected amino acid to give intermediates 36–39. Finally, the ester moiety was hydrolyzed again to provide new final products 40–43. Route B consisted of 3 steps starting with amide coupling between 3-hydroxybenzoic acid and phenylalanine methyl ester. The resulting intermediate 44 was reacted with the corresponding aryl bromide providing 45–47, before ester hydrolysis afforded the final products 48–50. Finally, compound 54 with a para configuration of the substituents in the central aromatic ring was prepared in a similar way to route A (Scheme 3).

Scheme 2. Conditions: (a) Cs₂CO₃, DMF, 85% for 34, 56–70% for 45–47. (b) LiOH/H₂O, THF, MeOH, 71% for 35, 30–63% for, 40–43 and 48–50. (c) HBTU, EDCl, 4-Methylmorpholine, 52%-Quant.

For 36–39, 79% for 44.

Scheme 3. Conditions: (a) Cs₂CO₃, DMF, 81%. (b) LiOH/H₂O, THF, MeOH, 70% for 51, 61% for 54. (c) HBTU, EDCl, 4-Methylmorpholine, 62%.

For EC₅₀ determination, the compounds were tested in a functional cell-based assay with detection of accumulation of IP-1 as a measure for activation of G2A. For concentrations of 9-HODE exceeding 100 μM, we did regularly observe a substantial decrease from the maximally reached IP-1 concentration. Also, for several compounds with EC₅₀ values exceeding 10 μM, the upper plateau could not be determined with optimal precision. Henceforth, as a statement of efficacy, it was more meaningful to determine the relative activation of G2A observed with 20 μM of compound vs the maximal activation by 9-HODE. For the first set of compounds, the EC₅₀ and efficacy values are presented in Table 1. All compounds were in addition tested in the antagonist mode vs 12.5 μM 9-HODE (ca. EC₈₀). Compounds lacking agonistic activity did also not show signs of antagonism or neutral modulation (data not shown).

First, the two enantiomers of the original racemic hit (1) from the HTS campaign were synthesized, leading to the identification of the (R) eutomer (3 henceforth named T-10418, EC₅₀ = 0.82 μM), which is more than 30 times more active than the (S) distomer (2, EC₅₀ = 31.1 μM). Chromatograms from chiral high-performance liquid chromatography (HPLC) analysis for all three compounds are shown in Supporting Information Figure S7. Next, compounds with homobenzyl and phenyl residues were studied (4–7), which were significantly less active than the original compound with benzylic residue but showed the same tendency in terms of stereochemistry. A small set of aromatic substituents were tested in compounds 8–11. The results showed that para substitution (8, EC₅₀ = 1.96; 9, EC₅₀ = 3.34 μM) seems to be better tolerated than meta (10, inactive) or ortho substitution (11, EC₅₀ = 13.5 μM). Also compounds bearing a pyridine ring (13 and 14) showed only weak potency. Methyl substituents in the benzylic position or in α-position of the carboxyl group were tolerated (12 and 15) but did not improve the activity. Next, a series of aliphatic substituents with or without heteroatoms (16–32) was analyzed. In general, the compounds from this series were weakly active (EC₅₀ > 10 μM) or inactive with the exception of compounds 19 (EC₅₀ = 7.33 μM) and 22 (EC₅₀ = 6.87 μM). Interestingly, in case of compound 19 that has (S)-configuration, the corresponding enantiomer (R) was less active in terms of potency (20, EC₅₀ = 64.8 μM). However, compound 19 showed only very limited efficacy at >20 μM, and in this regard was clearly inferior to the enantiomer (R) (Supporting Information Figure S1D), which again was in line with the observation of the R-configuration being preferred. Finally, compound 33 with an indane substituent was only mildly active.

Taking into consideration the results obtained with the first set of compounds (2–33), a second set of compounds was designed and synthesized. The G2A activity results for the second set of compounds are presented in Table 2.

Table 2. Final Product Structures and EC₅₀ Values for Activation of Human G2A in IP-One Assay^a.

^a(Dose–response curves are shown in Supporting Information Figure S2).

From the previous series, the racemic compound 22 had been the most promising derivative among those with an aliphatic substituent replacing the phenyl ring. Henceforth, its enantiomers were synthesized and tested as well. In this case, as in the pair of compounds 19 and 20, the (S) enantiomer (41) with an EC₅₀ = 2.92 μM showed a higher potency than its (R) counterpart (40, EC₅₀ = 40.6 μM). Compounds 42 and 43 are both in R-configuration and possess para substituents in the phenyl ring on the right side of the molecule. These para substituents were tolerated just as it had been shown in the previous set of compounds, but the substituents did not bring any improvement regarding EC₅₀ although 43 showed the highest maximum activation. Next, racemic compounds with a different position of the nitrogen on the pyridine ring were studied. 48 with the nitrogen in para was potent showing an EC₅₀ = 2.87 μM while compounds 49 with the nitrogen in ortho and 50 with a phenyl ring were only weakly active. Finally compound 54 with a para disposition of the substituents in the central ring led to loss of potency. The SAR results are summarized in Figure 4.

Figure 4.

SAR summary.

In order to use it as a reference tool compound, the described antagonist SB-583355 (56) was also prepared in two steps (Scheme 4). First, a Suzuki reaction between 4-methoxyphenyl boronic acid and 3-bromo-4-hydroxybenzoic acid following the conditions described by Carter et al.²⁵ took place in good yield. Next, an amide coupling reaction between 55 and the commercially available N-cyclohexyl-l-phenylalaninamide produced the known inhibitor 56.

Scheme 4. Conditions: (a) Pd(OAc)₂, Cs₂CO₃, DMF, H₂O, 45 °C 83%. (b) HBTU, EDCl, 4-Methylmorpholine, DMF 20%.

In the IP-One assay, it was then investigated whether the described G2A antagonist 56 (SB-583355) could counteract activation of G2A by compound T-10418; Figure 5A. 56 did indeed completely suppress activation by T-10418 in the same dose–response dependent manner as observed versus reference agonist 9-HODE.

Figure 5.

Target engagement in human G2A expressing CHO-K1 cells by second messenger and β-arrestin recruitment. (A) Antagonist 56 (SB-583355) blocked activation of G2A mediated either by 9-HODE or T-10418. Activity was determined in a cell-based functional assay with detection of accumulation of IP-1 (IP-One assay). CHO-K1 cells stably expressing human G2A in combination with human GNA 11 were stimulated with the indicated concentrations of compounds. Data represent mean ± SD (3 technical replicates, N = 3). Even when T-10418 and (±)9-HODE were applied at circa EC_80., which corresponds to 3 and 12.5 μM, respectively, 56 completely suppressed activation of G2A. (B) NanoLuc βarr2 recruitment assay. CHO-K1 expressing human G2A-SmBit and LgBit-βarr2 were treated with T-10418 (red), which resulted in a dose-dependent complementation of NanoLuc only in cells expressing G2A-SmBit; EC₅₀ = 7.7 μM. Treatment with 9(S)-HODE resulted in a comparably strong response. Racemic 9-HODE was unable to stimulate the same degree of β-arrestin recruitment, which can be explained by the observation that 9(R)-HODE did not result in any observable NanoLuc complementation. As an additional control, cells expressing LgBit-βarr2 alone did not show any response when stimulated with T-10418 (gray; no G2A). Presented data were recorded 15 min after addition of compound. Data are fold RLU (relative light units) as mean ± SD; N = 3.

Direct Binding to G2A and β-Arrestin-2 Recruitment

Direct binding of T-10418 to G2A was analyzed by means of LC–MS in membrane preparations obtained from CHO-K1 cells stably expressing human G2A. Membrane preparations from wild-type CHO-K1 served as the control. Albeit the affinity of T-10418 likely is in the mid to upper nM range, which makes such experiments challenging, a small but statistically significant difference between membrane preparations from transfected vs untransfected cells at higher concentrations of T-10418 (600 nM shown in Supporting Information Figure S3) indicated direct interaction with G2A. To further characterize T-10418 as a G2A ligand, we utilized a split-luciferase complementation assay reporting on β-arrestin-2 [βarr2; uniprot entry P32121; also known as (nonvisual) arrestin-3] recruitment to G2A upon activation of the receptor, which supported direct target engagement in cells (Figure 5B).

Target Engagement in Mouse Sensory Neurons

Next, T-10418 was tested in the TRPV1 sensitization assay on mouse sensory neurons; Figure 6. 100 nM T-10418 on average resulted in 244% TRPV1 sensitization, on par or exceeding the sensitization observed after treatment with 400 nM 9-HODE. T-10418 was thus more active than the racemic compound 1, 1 μM of which resulted in TRPV1 sensitization comparable to that observed with 200 nM (±)9-HODE (Figure 3). Furthermore, the TRPV1 sensitizing effect of both agonists, T-10418 as well as 9-HODE, was completely abolished following cotreatment with G2A antagonist 56, which provided additional proof for target engagement of T-10418.

Figure 6.

Compound T-10418 potently sensitized capsaicin-induced TRPV1 responses in primary mouse sensory neurons. (A) Representative traces of capsaicin-induced calcium responses in mouse sensory neurons for T-10418. Neurons were incubated for 4 min with vehicle (DMSO, 0.01% (v/v) black), T-10418 (100 nM, gray), or a combination of 100 nM T-10418 and 10 μM 56 (light gray) 4 min prior to the second capsaicin stimulus. Stimulations with capsaicin (50 nM) took place for 20 s as indicated. (B) Statistical analysis of the amplitudes of the second capsaicin responses with the respective conditions; (±)9-HODE (400 nM), T-10418 (100 nM), 56 (10 μM), a combination, or DMSO (0.01%) alone as vehicle control. Data are shown as mean ± SEM from n = 32 to 72 neurons. Stimulation with agonists vs vehicle: ##p < 0.01, ####p < 0.0001; reduction observed with antagonist: ****p < 0.0001, Kruskal–Wallis with Dunn’s post hoc test.

GPCR Selectivity

To evaluate the potential of xx as chemical tool, T-10418 was tested for GPCR selectivity in the PRESTO-Tango assay that measures association of a receptor with β-arrestin. The assay panel is covering more than 300 nonolfactory GPCRs. Apart from the expected G2A activation, T-10418 showed activity only on one other receptor, GPR1 (human protein sequence), which suggests excellent GPCR selectivity (Figure 7). However, it should be noted that such a GPCR screen has its limitations. The more than 300 GPCRs in the PRESTO-Tango panel include a large number of orphan receptors, and for some of the receptors that have already been characterized, activation by reference agonists has not yet been demonstrated in this particular assay. In addition, antagonist activity will not be observed because β-arrestin recruitment is generally only expected if the compound tested acts as agonist for the receptor. Henceforth, compound T-10418 was also profiled in the SafetyScreen44 Panel from Eurofins showing inhibition of specific binding by <25% across the entire set (Supporting Information, Figure S4).

Figure 7.

PRESTO-Tango GPCR selectivity screen conducted with 10 μM compound T-10418.

The GPCR 1 (GPR1; Chemerin-like receptor 2, uniprot P46091) is activated by chemerin, a 136 amino acid protein also named chemerin-S157, together with two other GPCRs from the chemerin receptor family: chemokine-like receptor 1 (CMKLR1) and CC-motif chemokine receptor-like 2 (CCRL2). The C-terminal nonapeptide of chemerin-S157, named chemerin-9 (C9) was identified to be able to activate CMKLR1²⁶ as well as GPR1.²⁷ For GPR1, only arrestin recruitment is detectable, whereas for CMKLR1, also G protein signaling is measurable after ligand stimulation. Thus, the influence on internalization and βarr2 recruitment at GPR1 and CMKLR1 were determined for T-10418.

Conjugation of C9 to the fluorescent dye TAMRA (tetramethylrhodamine) allows visualization of its localization and cellular uptake. Cells expressing either CMKLR1-eYFP or GPR1-eYFP were treated with a dilution series of TAMRA-(EG)₄-C9, which resulted in an increase in TAMRA specific red fluorescence due to binding and cellular uptake of the ligand. This was then challenged by cotreatment with 30 μM T-10418. Whereas the chemerin receptor CMKLR1 was not affected by T-10418 (Figure 8A), HEK293 cells stably transfected with GPR1-eYFP displayed a 2-fold shift of the EC₅₀ compared to the control without compound, demonstrating a minor effect of T-10418 on GPR1 internalization (Figure 8B,C). Figure 8D visualizes the GPR1 internalization stimulated with TAMRA-(EG)₄-C9 in comparison to TAMRA-(EG)₄-scr2C9 (scrambled C9 sequence) which was used as a negative control peptide.²⁷

Figure 8.

Influence of T-10418 on receptor internalization at CMKLR1 and GPR1. HEK293 cells stably express CMKLR1-eYFP (A) or GPR1-eYFP (B). They were treated with 30 μM of T-10418 (green) or 0.3% DMSO (black, control) and a serial dilution of TAMRA-labeled chemerin-9 [TAMRA-(EG)₄-C9]. The uptake of TAMRA-(EG)₄-C9 was detected as an increase in FI. Presence of 30 μM T-10418 resulted in a shift in TAMRA-(EG)₄-C9 concentration dependent uptake (B) whereas no change was observed at CMKLR1 (A) compared to control. The calculated values of this experiment are summarized in table (C). All data points represent mean ± SEM from at least three independent experiments performed in duplicate. The results were analyzed using GraphPad prism and nonlinear fit of log(agonist) vs response (three parameter). Internalization of GPR1-eYFP (green fluorescence) stimulated by 1 μM of TAMRA-(EG)₄-C9 (red fluorescence) is illustrated in (D). TAMRA-(EG)₄-C9 colocalized with GPR1-eYFP. An experiment involving a negative control peptide named TAMRA-(EG)₄-scr2C9 (red fluorescence; scrambled C9 sequence) showed that the control peptide was removed in the wash step and that it did not stimulate internalization of GPR1 (D, upper panel). Data about the TAMRA-(EG)4-scr2C9 have been published previously.²⁷ Hoechst33324 was used to stain the nuclei. Scale bar = 50 μm.

Modulation by T-10418 is stronger in the βarr2 recruitment bioluminescence resonance energy transfer (BRET) assay, which measures association with βarr2 as a readout for ligand-dependent activation of the receptor (Figure 9). T-10418 shows only partial agonism on GPR1 (Figure 9E), and this limited stimulation of βarr2 recruitment led us to test its effect again indirectly. T-10418 caused a significant shift of the C9-induced activation of GPR1 in contrast to CMKLR1 (Figure 9A,B). T-10418 at a 60 μM concentration attenuated C9 stimulated βarr2 recruitment with a 232-fold shift in EC₅₀ of C9 compared to the control without compound. When no endogenous ligand was applied to GPR1, partial agonism of 60 μM T-10418 is reaching approximately 50% of maximal βarr2 recruitment observed upon full activation with C9 (Figure 9B, w/o). In controls, 4 nM C9 was required for half-maximal arrestin-3 recruitment.

Figure 9.

Influence of T-10418 on βarr2 recruitment BRET at CMKLR1 and GPR1. HEK293 cells expressing either GPR1-eYFP or CMKLR1-eYFP and βarr2-Nluc. Stimulation was performed with 60 μM of T-10418 (green) or 0.6% DMSO (black, control) and a serial dilution of chemerin-9 (C9). No influence of T-10418 at CMKLR1 (A) was observed but for GPR1, a lag in full βarr2 recruitment was determined (B). With a submaximal concentration of C9 (EC₈₀), a reduction of βarr2 recruitment to only 50% was observed after adding the highest concentrations of T-10418, and consequently on C9-EC₅₀ stimulated cells, βarr2 recruitment was not affected by T-10418. Remarkably, T-10418 showed partial agonism when GPR1-containing cells are costimulated with EC₂₀ of C9 (C). Table (D) summarizes the calculated values of the βarr2 recruitment experiments shown in (A,B). All data points represent mean ± SEM from at least two independent experiments performed in triplicate. The results were analyzed by using GraphPad prism for nonlinear fit of log(agonist) vs response (three parameter). (E) demonstrates the partial agonism of T-10418 saturated at high concentrations compared to much lower concentration (1 μM) for C9 stimulation of GPR1 in the βarr2 recruitment BRET. As a control, only buffer and DMSO were added instead of peptide C9 or compound T-10418. ANOVA with the posthoc Dunnett’s multiple comparison test was used for significance evaluation to define the variance compared to control. *p < 0.05, ***p < 0.001.

Finally, a displacement binding assay was performed as developed recently²⁸ (Figure 10). Here, we demonstrate that T-10418 was not able to displace bound TAMRA-labeled C9 at the Nluc-GPR1 construct, whereas C9 showed full displacement with a K_i of 35.4 nM. Thus, either T-10418 did not interfere at all with the binding of C9 to GPR1 or this compound is a low affinity binder (agonist) at the orthosteric C9 site with an affinity too low to show an effect in this experimental setup and concentration range. T-10418-mediated inhibition of the C9-induced internalization of GPR1 (Figure 8B) was limited although substantial stimulation of β-arr2 recruitment was observed in the βarr2 NanoBRET assay (Figure 9B,C), which is in agreement with the identification of GPR1 in the PRESTO-Tango selectivity screen (Figure 7). However, we also demonstrate that in the NanoBRET β-arr2 recruitment assay, T-10418 is counteracting the effect of C9 [at > EC₅₀(C9); Figure 9C], and hence, T-10418 vs C9 has a moderating effect on β-arr2 recruitment. When using T-10418 as a pharmacological tool, a notable modulation of GPR1 would only be expected at ≥10 μM. Further investigations of T-10418 and related compounds at GPR1 will allow for further characterization of its mode of action.

Figure 10.

Displacement of bound TAMRA-labeled C9 in orthosteric binding pocket at Nluc-GPR1. HEK293 cells containing Nluc-GPR1-eYFP were stimulated with a constant concentration of 10 nM TAMRA-labeled chemerin-9 (TAMRA-C9). Different concentrations of T-10418 (green) or chemerin-9 (C9, black, control) were used to displace TAMRA-C9. Data represent mean ± SEM from three independent experiments performed in triplicate. The results were analyzed by using GraphPad prism and nonlinear fit of log(inhibitor) vs response (three parameter).

Effects of in Leukemic Cell Lines

Two studies reported on the proapoptotic effects of imipridones ONC201 and ONC212 on AML cells^24,29 presumably through the induction of G2A.²⁴ Encouraged by these observations, we tested the effects of T-10418 on AML cells alongside the prototypical imipridone ONC201. We incubated the AML cell lines Molm-13 and ML-2 with ONC201 or T-10418 alone or in combination with the Bcl2-Inhibitor ABT-199 (known as Venetoclax). The rationale behind the latter was a report on a synergistic effect between ONC201 and ABT-199.²⁹ After 72 h, we observed/detected a strong reduction in viable Molm-13 and ML-2 cells in the presence of ONC201, while T-10418 did not influence cell expansion nor viability; Figure 11. Furthermore, we confirmed an even stronger effect on cell viability in the combination treatment of ONC201 and ABT-199. The induction of apoptosis could be confirmed by AnnexinV/7-AAD measurements using flow cytometry. The discrepancy on the sensitivity of AML cells toward ONC201 and T-10418 and our findings on the selectivity of T-10418 for G2A suggest that these cell lines may not respond to G2A activation and that ONC201 induced the strong antileukemic effect independent of G2A.

Figure 11.

No antileukemic effect induced by T-10418. (A,B) Cell numbers of the AML cell lines Molm-13 (A) and ML2 (B) after 72 h of treatment in vitro. Single compounds or combinations were used as indicated with the following concentrations: ONC201, 30 μM; T-10418, 30 μM; ABT199, 200 nM. (C,D) AnnexinV/7-AAD staining for early (AnnexinV+/7-AAD-) and late apoptotic cells (AnnexinV+/7-AAD+) in Molm-13 (C) and ML2 (D) after 24 h of treatment in vitro. Single compounds or combinations were used as indicated at following concentrations: ONC201, 30 μM; T-10418, 30 μM; ABT199, 100 nM, one-way ANOVA with Tukey multiple comparisons test, ns, not significant; **P < 0.01; ***P < 0.001; ****P < 0.0001.

We therefore questioned whether the compounds activated the G_q pathway in AML cell lines and tested this in the IP-One assay. In order to follow up on the previous experiments, we first tested the imipridones ONC201 and ONC212 on G2A + GNA11 expressing CHO-K1 cells. In agreement with the report by Nii et al.,²⁴ ONC201 (EC₅₀ = 38.6 μM) showed limited activity and was inferior to ONC212 (EC₅₀ = 3.92 μM). In order to test for any response that is not mediated by the receptor, control experiments were conducted on CHO-K1 cells overexpressing only GNA11 but not G2A. At ≥ 30 μM ONC201 and ONC212 both stimulated a substantial increase in IP-1. Such had previously not been observed with 9-HODE, nor with T-10418 or with any of the derivatives listed in Tables 1 and 2. It indicates that ONC201 and ONC212 address some other target in CHO-K1 cells that can activate the G_q pathway. We then went on and tested the response of Molm-13 and ML-2 cells in the IP-One assay. We could not detect any activation of the G_q pathway after treatment with 9-HODE, T-10418 or the imipridone ONC212 (more active on G2A) (Supporting Information Figure S5). The strong effect of ONC201 in AML cells is, hence, most likely independent of G2A, which may explain the inability of T-10418 to inhibit AML cell survival. There is a clear additive effect of ONC201 and ABT199, as reported by Nii et al.²⁴ However, we detected no additional inhibitory effect when combining ABT-199 and T-10418.

Cell Toxicity, Aqueous Solubility, Metabolic Stability, and In Vivo Pharmacokinetic Studies

The toxicological profiles of compounds 1 (racemic) and T-10418 (enantiomer R) were tested in hepatocellular carcinoma cells (HepG2; DSMZ #ACC 180) using CellTiter-Glo as a global measure for metabolic activity and cell survival, which were both unaffected (threshold ≥90%) after 72 h of treatment with either compound at up to 100 μM (Supporting Information Figure S6).

To evaluate whether T-10418 also qualified as a chemical tool for in vivo studies, we first studied its aqueous solubility and metabolic stability as key factors for good absorption and low clearance of the drug. Solubility was determined in phosphate-buffered saline (PBS) buffer and in vitro metabolic half-life was studied using rat liver microsomes (Table 3). T-10418 showed very high metabolic stability and excellent aqueous solubility greater than 3 mM.

Table 3. Aqueous Solubility and Metabolic Stability of Compound T-10418.

cmpd	aq. solubilitya, (μM)	rat liver microsomesb (remaining after 60 min)	CLint (μL/min mg)
T-10418	≥3000	98%c	1.68d

^aSolubility of compound in PBS buffer, pH 7.4, containing 1% DMSO.

^bMicrosome mix from the liver of Sprague–Dawley rats.

^cPercentage of remaining compound after 60 min.

^dIntrinsic clearance calculated according to formula 1, see Experimental Section.

For its attractive characteristics with high in vitro potency and excellent physicochemical properties, T-10418 was selected for a PK study in male C57Bl/6N mice. The compound was applied intravenously (iv) at 1 mg/kg and subcutaneously (sc) at 10 mg/kg in PBS. Plasma concentrations were measured at 15 min, 30 min, 1 h, 2 h, 4 h, and 8 h for the iv study and at 30 min, 1 h, 2 h, 4 h, 8 h, and 12 h for the sc study. The resulting plasma concentration profiles obtained as well as the calculated PK parameters are shown in Table 4. The iv route resulted in a maximum plasma concentration after 15 min before the concentration dropped exponentially until the last measurement at 8 h. On the other hand, when T-10418 was applied sc, the peak plasma concentration was achieved at 30 min, hence, again the earliest time point recorded. The AUC upon sc application was close to 10-fold compared to the iv application, which perfectly fits to the ratio between the sc and iv dose. Overall, the compound presented high plasma concentrations and a promising PK profile, which makes T-10418 a suitable candidate for in vivo studies on therapeutic potential of G2A agonism.

Table 4. PK Parameters of Compound T-10418.

cmpd	Cmax (ng/mL)	Tmax (h)	Cfinal (ng/mL)	CL/F [mL/(h·kg)]	CL [mL/(h·kg)]	AUC (ng·h/mL)
T-10418 (1 mg/kg iv)	45.3	15 min	2.4	16451		47.3a
T-10418 (10 mg/kg sc)	755	30 min	2.9		8451	591b

^aAUC_0–8 h.

^bAUC_0–12 h.

Conclusions

The orphan GPCR G2A is emerging as an attractive target in various potential therapeutic applications, but experimental validation of therapeutic effects by pharmacological G2A activation is pending. In this study, we have discovered and optimized a new G2A agonist scaffold. The most active descendant T-10418 activates G2A with strong efficacy and is characterized by high selectivity within the GPCR family. T-10418 failed to induce apoptosis in leukemic cell lines, suggesting that G2A agonism does not contribute to the antileukemic effects of imipridones. Moreover, favorable physicochemical properties and PK profile of T-10418 suggest suitability for in vivo application. T-10418 therefore presents as very valuable tool to study the biology and therapeutic potential of G2A.

Experimental Section

General

Chemicals for the synthesis of 2–56 were purchased from Acros Organics (Geel, Belgium), Alfa Aesar GmbH & Co KG (Karlsruhe, Germany), BLD PharmaTtech GmbH (Kaiserslautern, Germany), Enamine Ltd. (Kyiv, Ukraine), Sigma-Aldrich Chemie GmbH (Steinheim, Germany), and TCI Deutschland (Eschborn, Germany).

Reactions were monitored via thin-layer chromatography using ALUGRAM from Merck (Darmstadt, Germany). To record NMR-spectra, compounds were dissolved in DMSO-d₆ or CDCl₃ and measured on DPX250, Avance 300, and Avance 400 from Bruker Corporation (Massachusetts, USA) using tetramethylsilane as an internal standard. All chemical shift values are reported in ppm, the multiplicity of the signals was assigned as follows: s (singlet), d (duplet), t (triplet), and m (multiplet). Mass spectrometry analysis was performed in positive ion mode by electrospray-ionization (ESI) on a LCMS-2020 single quadrupole MS from Shimadzu (Duisburg, Deutschland). Precision mass was measured using MALDI Orbitrap XL from Life Technologies GmbH (Darmstadt, Germany). For purity estimation of the synthesized compounds, a reverse phase HPLC (RP-HPLC) was performed using the Luna 10 μm C18(2) 100 Å, LC Column 250 × 4.6 mm from Phenomenex LTD (Aschaffenburg, Germany), and the analysis was conducted using the Shimadzu prominence module from Shimadzu. Acetonitrile and aqueous formic acid 0.1% were used as eluents. The established method for purity determination was initiated with 90% water (0.1% formic acid), then a linear gradient from 90 to 5% water (0.1% formic acid) for 13 min was chosen, finally additional 7 min 5% water (0.1% formic acid). The flow rate was adjusted to 1.0 mL/min and the UV–vis detection occurred at 254 and 280 nm, respectively. Purity of all tested compounds was determined higher than 95%.

Synthetic Methods

Altogether, 41 new final products were synthesized, analyzed, and tested. Pure compounds (≥95% purity) were obtained after purification by RP-HPLC (see Experimental Section). The structures of all final products were confirmed by ¹H, ¹³C NMR spectroscopy and HPLC analysis coupled to electrospray ionization mass spectrometry (HPLC/ESI–MS), which was also used to determine the purity. Additionally, high-resolution mass spectrometry (HRMS) was also performed.

General Procedures

GP1. One-Pot Amide Coupling with Ester Hydrolysis

An ester of amino acid (100 mg), 1% hydroxybenzotriazole (HOBt) in DMF, a carboxylic acid (1 mole equiv to the ester), and triethylamine (TEA, 1 or 2 mole equiv to the amino acid in case the latter is used as a salt) were place in a vial with a screw cap. The vial was left shaking for 24 h at room temperature (RT), and chloroform (3 mL) and water (1 mL) were added. After 15 min, the organic phase was separated and dried over Na₂SO₄. Then, DMSO (0.5 mL) was added and the solution was left shaking overnight at RT followed by the addition of 4 M KOH (2 mole equiv to the ester). The solution was left shaking for 12 h at RT and further neutralized with formic acid. The solvent and volatile components were evaporated under reduced pressure to give the crude product. The product was further purified by HPLC.

GP2. Nucleophilic Substitution

The corresponding phenol (1.0 equiv) and cesium carbonate (2.3 equiv) were suspended in DMF and warmed at 60 °C for 15 min. After cooling down at rt, the corresponding benzyl bromide (1.3 equiv) was added dissolved in DMF and the resulting reaction mixture was stirred overnight at rt. DMF was evaporated under reduced pressure and the crude was dissolved in water and ethyl acetate. The aqueous phase was extracted with ethyl acetate (3×) and the organic layer was evaporated. Flash chromatography was performed to purify the crude product.

GP3. Amide Coupling

The corresponding benzoic acid (1.0 equiv) and HBTU (1.3 equiv) were dissolved in DMF under argon and stirred at rt for 10 min. Then, the corresponding aminoester hydrochloride (1.2 equiv) and 4-methylmorpholine (1.3 equiv) were added at 0 °C, and the reaction mixture was stirred at this temperature for 30 min. EDCCl (1.3 equiv) was then added at 0 °C, and the reaction mixture was allowed to stir at rt overnight.

Solvents were evaporated, and the residue was dissolved in water and ethyl acetate. The aqueous phase was extracted with ethyl acetate (3×) and the combined organic layers were dried over MgSO₄, filtered, and evaporated under reduced pressure. The crude product was purified by flash chromatography.

GP4. Ester Hydrolysis

GP3. The corresponding methyl ester was dissolved in THF and 10 equiv of LiOH (dissolved in a few uL of water) was added. Methanol was added until a monophasic solution was obtained and the reaction was heated at 60 °C overnight. The solvents were evaporated under reduced pressure and crude product was purified by preparative HPLC.

(S)-3-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (2)

The product was synthesized according to GP1. Yield: 44%. ¹H NMR (300 MHz, DMSO-d₆): δ 12.75 (bs, 1H), 8.71–8.67 (m, 2H), 8.57 (d, J = 4.8 Hz, 1H), 7.83 (dt, J = 7.9, 1.9 Hz, 1H), 7.48–7.15 (m, 10H), 5.20 (s, 2H), 4.66–4.59 (m, 1H), 3.20 (dd, J = 14.1, 4.6 Hz, 1H), 3.07 (dd, J = 13.8, 10.6 Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆): δ 173.6, 166.3, 158.4, 156.9, 149.4, 149.2, 138.6, 136.5, 135.8, 133.0, 129.9, 129.5, 128.6, 126.8, 124.2, 120.5, 118.3, 114.0, 67.5, 54.6, 36.7; R_f HPLC: 7.8 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 100.0% purity; HRMS (MALDI) m/z; found, 377.1497: [M + H]⁺ (calcd C₂₂H₂₁N₂O₄⁺, 377.1496).

(R)-3-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (3, T-10418)

The product was synthesized according to GP1. Yield: 35%. ¹H NMR (300 MHz, DMSO-d₆): δ 12.70 (bs, 1H), 8.70–8.67 (m, 2H), 8.56 (dd, J = 4.8, 1.6 Hz, 1H), 7.88 (dt, J = 7.8, 1.8 Hz, 1H), 7.46–7.14 (m, 10H), 5.19 (s, 2H), 4.66–4.58 (m, 1H), 3.20 (dd, J = 14.1, 4.6 Hz, 1H), 3.07 (dd, J = 13.8, 10.6 Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆): δ 173.6, 166.3, 158.4, 156.9, 149.4, 149.2, 138.6, 136.5, 135.8, 133.0, 129.9, 129.5, 128.6, 126.8, 124.2, 120.5, 118.3, 114.0, 67.5, 54.6, 36.7; R_f HPLC: 7.6 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 100.0% purity; HRMS (MALDI) m/z; found, 377.1490: [M + H]⁺ (calcd C₂₂H₂₁N₂O₄⁺, 377.1496).

(R)-4-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)butanoic Acid (4)

The product was synthesized according to GP1. Yield: 50%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.61 (s, 1H), 8.71–8.63 (m, 2H), 8.54 (d, J = 4.8 Hz, 1H), 7.89 (dt, J = 7.9, 2.0 Hz, 1H), 7.58–7.48 (m, 2H), 7.46–7.37 (m, 2H), 7.27 (t, J = 7.5 Hz, 2H), 7.25–7.13 (m, 4H), 5.21 (s, 2H), 4.31 (q, J = 7.6 Hz, 1H), 2.72 (dt, J = 14.3, 7.2 Hz, 1H), 2.68–2.57 (m, 1H), 2.07 (q, J = 7.4, 7.0 Hz, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₃H₂₁N₂O₄, 389.2; found, 389.2.

(S)-4-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)butanoic Acid (5)

The product was synthesized according to GP1. Yield: 54%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.60 (s, 1H), 8.71–8.63 (m, 2H), 8.54 (d, J = 4.7 Hz, 1H), 7.89 (dt, J = 7.9, 1.9 Hz, 1H), 7.58–7.48 (m, 2H), 7.46–7.37 (m, 2H), 7.27 (t, J = 7.5 Hz, 2H), 7.19 (q, J = 8.7, 8.0 Hz, 4H), 5.21 (s, 2H), 4.31 (q, J = 7.5 Hz, 1H), 2.72 (dt, J = 14.4, 7.3 Hz, 1H), 2.68–2.57 (m, 1H), 2.07 (q, J = 7.8 Hz, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₃H₂₁N₂O₄, 389.2; found, 389.0.

(S)-2-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)acetic Acid (6)

The product was synthesized according to GP1. Yield: 41%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.91 (s, 1H), 9.03 (d, J = 7.5 Hz, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.54 (dd, J = 4.8, 1.6 Hz, 1H), 7.87 (dt, J = 7.9, 2.0 Hz, 1H), 7.58 (t, J = 2.0 Hz, 1H), 7.50 (dd, J = 22.0, 7.4 Hz, 3H), 7.45–7.28 (m, 5H), 7.19 (dd, J = 8.1, 2.6 Hz, 1H), 5.58 (d, J = 7.4 Hz, 1H), 5.19 (s, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₁H₁₇N₂O₄, 361.1; found, 361.2.

(R)-2-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)acetic Acid (7)

The product was synthesized according to GP1. Yield: 57%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.90 (s, 1H), 9.02 (d, J = 7.5 Hz, 1H), 8.67 (s, 1H), 8.54 (d, J = 4.9 Hz, 1H), 7.87 (dd, J = 7.8, 2.0 Hz, 1H), 7.58 (t, J = 2.0 Hz, 1H), 7.50 (dd, J = 21.8, 7.6 Hz, 3H), 7.38 (tq, J = 18.2, 7.2, 6.0 Hz, 5H), 7.19 (dd, J = 8.2, 2.4 Hz, 1H), 5.58 (d, J = 7.5 Hz, 1H), 5.19 (s, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₁H₁₇N₂O₄, 361.1; found, 361.0.

3-(4-Fluorophenyl)-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (8)

The product was synthesized according to GP1. Yield: 48%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 8.67 (d, J = 2.2 Hz, 1H), 8.57–8.51 (m, 1H), 8.32 (s, 1H), 7.86 (dt, J = 7.8, 2.0 Hz, 1H), 7.45–7.28 (m, 4H), 7.24 (dd, J = 8.4, 5.7 Hz, 2H), 7.15 (dt, J = 7.7, 1.8 Hz, 1H), 7.01 (t, J = 8.9 Hz, 2H), 5.18 (s, 2H), 4.38 (d, J = 12.3 Hz, 1H), 3.16 (dd, J = 13.7, 4.6 Hz, 1H), 3.01 (dd, J = 13.6, 8.9 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₂₂H₁₈FN₂O₄, 393.1; found, 393.1.

2-(3-(Pyridin-3-ylmethoxy)benzamido)-3-(4-(trifluoromethoxy)phenyl)propanoic Acid (9)

The product was synthesized according to GP1. Yield: 49%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.81 (s, 1H), 8.73–8.62 (m, 2H), 8.54 (d, J = 4.9 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.45–7.32 (m, 6H), 7.24 (d, J = 8.1 Hz, 2H), 7.18 (dt, J = 5.9, 2.8 Hz, 1H), 5.18 (s, 2H), 4.60 (ddd, J = 12.5, 8.5, 4.5 Hz, 1H), 3.20 (dd, J = 13.9, 4.6 Hz, 1H), 3.07 (dd, J = 13.9, 10.6 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₂₃H₁₈F₃N₂O₅, 459.1; found, 459.0.

3-(3-Chlorophenyl)-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (10)

The product was synthesized according to GP1. Yield: 33%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.81 (s, 1H), 8.72–8.64 (m, 2H), 8.54 (d, J = 4.7 Hz, 1H), 7.86 (d, J = 7.9 Hz, 1H), 7.46–7.33 (m, 5H), 7.31–7.19 (m, 3H), 7.18 (dt, J = 5.7, 3.2 Hz, 1H), 5.18 (s, 2H), 4.65–4.56 (m, 1H), 3.19 (dd, J = 13.8, 4.5 Hz, 1H), 3.04 (dd, J = 13.7, 10.7 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₂₂H₁₈ClN₂O₄, 409.1; found, 409.2.

3-(2-Chlorophenyl)-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (11)

The product was synthesized according to GP1. Yield: 44%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.82 (s, 1H), 8.73 (d, J = 8.5 Hz, 1H), 8.67 (s, 1H), 8.54 (d, J = 4.7 Hz, 1H), 7.87 (dd, J = 7.9, 2.0 Hz, 1H), 7.46–7.32 (m, 6H), 7.19 (ddd, J = 15.2, 7.0, 3.3 Hz, 3H), 5.18 (s, 2H), 4.72 (ddd, J = 10.9, 8.4, 4.3 Hz, 1H), 3.38 (dd, J = 13.9, 4.4 Hz, 1H), 3.11 (dd, J = 13.9, 10.9 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₂₂H₁₈ClN₂O₄, 409.1; found, 409.0.

3-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)butanoic Acid (12)

The product was synthesized according to GP1. Mixture of isomers. Yield: 47%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.78 (s, 1H), 12.48 (s, 1H), 8.54 (s, 1H), 8.43 (d, J = 8.5 Hz, 1H), 7.86 (ddt, J = 19.3, 8.0, 2.0 Hz, 1H), 7.52–7.38 (m, 2H), 7.42–7.30 (m, 1H), 7.34–7.18 (m, 6H), 7.22–7.13 (m, 1H), 7.16–7.09 (m, 1H), 5.21 (s, 1H), 5.14 (s, 1H), 4.57 (dd, J = 10.1, 8.5 Hz, 1H), 3.29 (s, 1H), 1.28 (t, J = 7.6 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₂₃H₂₁N₂O₄, 389.2; found, 389.0.

2-(3-(Pyridin-3-ylmethoxy)benzamido)-3-(pyridin-4-yl)propanoic Acid (13)

The product was synthesized according to GP1. Yield: 68%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 13.00 (s, 1H), 8.81 (d, J = 8.4 Hz, 1H), 8.78–8.67 (m, 3H), 8.61 (d, J = 4.9 Hz, 1H), 7.99 (d, J = 7.9 Hz, 1H), 7.79 (d, J = 5.7 Hz, 2H), 7.54 (dd, J = 7.8, 4.9 Hz, 1H), 7.40 (dd, J = 17.1, 3.6 Hz, 3H), 7.23–7.16 (m, 1H), 5.20 (s, 2H), 4.82 (ddd, J = 12.6, 8.4, 4.4 Hz, 1H), 3.43 (dd, J = 14.0, 4.5 Hz, 1H), 3.25 (dd, J = 14.0, 10.9 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₂₁H₁₈N₃O₄, 376.1; found, 376.0.

2-(Pyridin-3-yl)-2-(3-(pyridin-3-ylmethoxy)benzamido)acetic Acid (14)

The product was synthesized according to GP1. Yield: 42%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 13.12 (s, 1H), 9.15 (d, J = 7.5 Hz, 1H), 8.67 (d, J = 2.5 Hz, 2H), 8.53 (dd, J = 10.6, 4.8 Hz, 2H), 7.88 (ddd, J = 6.6, 4.4, 2.2 Hz, 2H), 7.56 (d, J = 2.5 Hz, 1H), 7.51 (d, J = 7.7 Hz, 1H), 7.45–7.36 (m, 3H), 7.21 (dd, J = 8.2, 2.5 Hz, 1H), 5.66 (d, J = 7.4 Hz, 1H), 5.19 (s, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₀H₁₆N₃O₄, 362.1; found, 362.2.

2-Phenyl-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (15)

The product was synthesized according to GP1. Yield: 37%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.61 (s, 1H), 8.68 (d, J = 2.5 Hz, 2H), 8.54 (dd, J = 4.9, 1.6 Hz, 1H), 7.88 (dt, J = 7.8, 2.0 Hz, 1H), 7.56–7.46 (m, 4H), 7.45–7.32 (m, 4H), 7.28 (t, J = 7.3 Hz, 1H), 7.20 (dd, J = 8.2, 2.6 Hz, 1H), 5.20 (s, 2H), 1.86 (s, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₂₂H₁₉N₂O₄, 375.1; found, 375.1.

(S)-3-Cyclohexyl-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (16)

The product was synthesized according to GP1. Yield: 36%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 8.68 (d, J = 2.1 Hz, 1H), 8.57–8.47 (m, 2H), 7.88 (dd, J = 7.8, 1.9 Hz, 1H), 7.55–7.45 (m, 2H), 7.46–7.35 (m, 2H), 7.19 (dd, J = 8.2, 2.5 Hz, 1H), 5.20 (s, 2H), 4.43 (ddd, J = 11.2, 8.0, 4.5 Hz, 1H), 2.38 (s, 1H), 1.72 (dt, J = 10.2, 5.1 Hz, 2H), 1.70–1.54 (m, 6H), 1.35 (s, 1H), 1.11 (qt, J = 19.8, 8.2 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₂₂H₂₅N₂O₄, 381.2; found, 381.2.

2-(3-(Pyridin-3-ylmethoxy)benzamido)propanoic Acid (17)

The product was synthesized according to GP1. Yield: 38%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.52 (s, 1H), 8.70–8.61 (m, 2H), 8.54 (dd, J = 4.8, 1.7 Hz, 1H), 7.88 (dt, J = 8.0, 2.0 Hz, 1H), 7.54 (t, J = 2.0 Hz, 1H), 7.48 (d, J = 7.7 Hz, 1H), 7.46–7.35 (m, 2H), 7.19 (dd, J = 8.2, 2.5 Hz, 1H), 5.19 (s, 2H), 4.39 (p, J = 7.2 Hz, 1H), 1.37 (d, J = 7.3 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₁₆H₁₅N₂O₄, 299.1; found, 299.2.

(R)-2-(3-(Pyridin-3-ylmethoxy)benzamido)propanoic Acid (18)

The product was synthesized according to GP1. Yield: 35%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.51 (s, 1H), 8.70–8.61 (m, 2H), 8.54 (dd, J = 4.8, 1.7 Hz, 1H), 7.88 (dd, J = 7.8, 1.9 Hz, 1H), 7.54 (d, J = 2.3 Hz, 1H), 7.48 (d, J = 7.8 Hz, 1H), 7.46–7.35 (m, 2H), 7.19 (dd, J = 8.2, 2.4 Hz, 1H), 5.19 (s, 2H), 4.45–4.35 (m, 1H), 1.37 (dd, J = 7.2, 1.4 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₁₆H₁₅N₂O₄, 299.1; found, 299.1.

(S)-3-Methyl-2-(3-(pyridin-3-ylmethoxy)benzamido)butanoic Acid (19)

The product was synthesized according to GP1. Yield: 37%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.60 (s, 1H), 8.68 (d, J = 2.1 Hz, 1H), 8.54 (dd, J = 4.7, 1.6 Hz, 1H), 8.41 (d, J = 8.2 Hz, 1H), 7.88 (dt, J = 7.8, 2.0 Hz, 1H), 7.55–7.46 (m, 2H), 7.45–7.35 (m, 2H), 7.19 (dd, J = 8.2, 2.5 Hz, 1H), 5.20 (s, 2H), 4.26 (t, J = 7.6 Hz, 1H), 2.16 (h, J = 6.9 Hz, 1H), 0.94 (dd, J = 9.3, 6.8 Hz, 6H). LC/MS (APSI) m/z: [M – H] calcd for C₁₈H₁₉N₂O₄, 327.1; found, 327.0.

(R)-3-Methyl-2-(3-(pyridin-3-ylmethoxy)benzamido)butanoic Acid (Z1455329281, 20)

The product was synthesized according to GP1. Yield: 40%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.61 (s, 1H), 8.68 (d, J = 2.2 Hz, 1H), 8.54 (dd, J = 4.9, 1.8 Hz, 1H), 8.40 (d, J = 8.3 Hz, 1H), 7.88 (dt, J = 7.8, 2.1 Hz, 1H), 7.55–7.46 (m, 2H), 7.45–7.35 (m, 2H), 7.19 (dd, J = 8.1, 2.6 Hz, 1H), 5.20 (s, 2H), 4.26 (t, J = 7.5 Hz, 1H), 2.16 (h, J = 6.9 Hz, 1H), 0.94 (td, J = 7.9, 6.7, 2.3 Hz, 6H). LC/MS (APSI) m/z: [M – H] calcd for C₁₈H₁₉N₂O₄, 327.1; found, 327.1.

2-(3-(Pyridin-3-ylmethoxy)benzamido)butanoic Acid (21)

The product was synthesized according to GP1. Yield: 45%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.56 (s, 1H), 8.68 (d, J = 2.3 Hz, 1H), 8.54 (t, J = 5.4 Hz, 2H), 7.88 (dt, J = 7.8, 2.0 Hz, 1H), 7.56–7.46 (m, 2H), 7.46–7.35 (m, 2H), 7.19 (dd, J = 8.1, 2.6 Hz, 1H), 5.20 (s, 2H), 4.27 (ddd, J = 9.3, 7.7, 5.0 Hz, 1H), 1.91–1.68 (m, 2H), 0.93 (t, J = 7.4 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₁₇H₁₇N₂O₄, 313.1; found, 313.2.

2-(3-(Pyridin-3-ylmethoxy)benzamido)pentanoic Acid (22)

The product was synthesized according to GP1. Yield: 32%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.56–12.51 (m, 1H), 8.68 (d, J = 2.1 Hz, 1H), 8.57–8.51 (m, 2H), 7.88 (d, J = 7.8 Hz, 1H), 7.56–7.46 (m, 2H), 7.46–7.35 (m, 2H), 7.19 (dd, J = 8.2, 2.5 Hz, 1H), 5.20 (s, 2H), 4.35 (q, J = 7.5 Hz, 1H), 1.75 (q, J = 7.6 Hz, 2H), 1.37 (ddp, J = 28.7, 14.3, 7.4 Hz, 2H), 0.88 (t, J = 7.3 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₁₈H₁₉N₂O₄, 327.1; found, 327.1.

3-Methyl-2-(3-(pyridin-3-ylmethoxy)benzamido)pentanoic Acid (23)

The product was synthesized according to GP1. Yield: 47%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.59 (s, 1H), 8.68 (s, 1H), 8.54 (d, J = 4.7 Hz, 1H), 8.40 (d, J = 8.1 Hz, 1H), 7.88 (dt, J = 7.9, 2.0 Hz, 1H), 7.54–7.45 (m, 2H), 7.45–7.34 (m, 2H), 7.19 (dd, J = 8.3, 2.6 Hz, 1H), 5.20 (s, 2H), 4.30 (t, J = 7.6 Hz, 1H), 1.93 (dtd, J = 11.1, 7.1, 3.8 Hz, 1H), 1.48 (dtd, J = 14.8, 7.5, 4.1 Hz, 1H), 1.31–1.18 (m, 1H), 0.91 (d, J = 6.8 Hz, 3H), 0.85 (t, J = 7.4 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₁₉H₂₁N₂O₄, 341.2; found, 341.1.

3-(Piperidin-1-yl)-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (24)

The product was synthesized according to GP1. Yield: 43%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 8.68 (s, 1H), 8.57–8.51 (m, 1H), 8.41 (d, J = 7.4 Hz, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.50 (t, J = 1.9 Hz, 1H), 7.42 (dq, J = 12.0, 7.7 Hz, 3H), 7.20 (dd, J = 8.1, 2.5 Hz, 1H), 5.20 (s, 2H), 4.53 (q, J = 7.7 Hz, 1H), 2.98 (dd, J = 12.5, 6.9 Hz, 1H), 2.86 (dd, J = 12.6, 8.4 Hz, 3H), 2.74 (dt, J = 11.3, 5.1 Hz, 2H), 2.45 (s, 2H), 1.57 (q, J = 6.0, 5.6 Hz, 4H), 1.44 (p, J = 5.8 Hz, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₁H₂₄N₃O₄, 382.2; found, 382.0.

2-(3-(Pyridin-3-ylmethoxy)benzamido)-2-(tetrahydro-2H-pyran-4-yl)acetic Acid (25)

The product was synthesized according to GP1. Yield: 34%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 8.68 (s, 1H), 8.54 (d, J = 4.8 Hz, 1H), 8.45 (d, J = 8.0 Hz, 1H), 7.88 (dt, J = 7.9, 2.0 Hz, 1H), 7.55–7.45 (m, 2H), 7.45–7.34 (m, 2H), 7.19 (dd, J = 8.2, 2.6 Hz, 1H), 5.20 (s, 2H), 4.26 (t, J = 7.7 Hz, 1H), 3.84 (dt, J = 10.6, 4.5 Hz, 2H), 3.25 (dt, J = 11.8, 9.3 Hz, 2H), 2.47 (s, 1H), 1.61–1.51 (m, 2H), 1.36 (dqd, J = 29.4, 12.3, 4.4 Hz, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₀H₂₁N₂O₅, 369.2; found, 369.0.

(S)-3-Methoxy-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (26)

The product was synthesized according to GP1. Yield: 49%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 8.67 (d, J = 2.2 Hz, 1H), 8.53 (dd, J = 4.7, 1.7 Hz, 1H), 8.09 (d, J = 6.9 Hz, 1H), 7.88 (dd, J = 7.8, 2.0 Hz, 1H), 7.57–7.40 (m, 2H), 7.43–7.34 (m, 3H), 7.17 (dt, J = 7.5, 2.1 Hz, 1H), 5.20 (s, 2H), 4.21 (d, J = 7.9 Hz, 1H), 3.66 (qd, J = 9.8, 4.8 Hz, 2H). LC/MS (APSI) m/z: [M + H] calcd for C₁₇H₁₉N₂O₅, 331.1; found, 331.2.

2-Cyclopropyl-2-(3-(pyridin-3-ylmethoxy)benzamido)acetic Acid (27)

The product was synthesized according to GP1. Yield: 51%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.50 (s, 1H), 8.77 (d, J = 7.2 Hz, 1H), 8.68 (s, 1H), 8.54 (d, J = 4.8 Hz, 1H), 7.88 (d, J = 7.9 Hz, 1H), 7.56 (t, J = 1.9 Hz, 1H), 7.50 (d, J = 7.7 Hz, 1H), 7.46–7.31 (m, 2H), 7.19 (dd, J = 8.2, 2.5 Hz, 1H), 5.20 (s, 2H), 3.69 (dd, J = 9.2, 7.1 Hz, 1H), 2.45 (s, 1H), 1.22 (tq, J = 8.6, 4.9, 4.3 Hz, 1H), 0.58 (tt, J = 8.5, 4.3 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₁₈H₁₇N₂O₄, 325.1; found, 325.2.

2-Methyl-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (28)

The product was synthesized according to GP1. Yield: 34%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.15 (s, 1H), 8.67 (d, J = 2.2 Hz, 1H), 8.54 (dd, J = 4.8, 1.6 Hz, 1H), 8.41 (s, 1H), 7.87 (dt, J = 7.9, 2.0 Hz, 1H), 7.50 (t, J = 2.0 Hz, 1H), 7.48–7.39 (m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.18 (dd, J = 8.1, 2.6 Hz, 1H), 5.19 (s, 2H), 1.43 (s, 6H). LC/MS (APSI) m/z: [M – H] calcd for C₁₇H₁₇N₂O₄, 313.1; found, 313.0.

(1R,4R)-4-Methyl-1-(3-(pyridin-3-ylmethoxy)benzamido)cyclohexanecarboxylic Acid (29)

The product was synthesized according to GP1. Yield: 36%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.07 (s, 1H), 8.67 (s, 1H), 8.54 (d, J = 4.7 Hz, 1H), 8.24 (s, 1H), 7.87 (d, J = 7.9 Hz, 1H), 7.46 (s, 1H), 7.43 (d, J = 7.5 Hz, 2H), 7.36 (t, J = 7.9 Hz, 1H), 7.17 (dd, J = 8.2, 2.5 Hz, 1H), 5.19 (s, 2H), 2.25 (d, J = 12.9 Hz, 2H), 1.65–1.54 (m, 4H), 1.46 (s, 1H), 1.26 (q, J = 12.8, 11.3 Hz, 2H), 0.87 (d, J = 6.5 Hz, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₂₁H₂₃N₂O₄, 367.2; found, 367.2.

1-(3-(Pyridin-3-ylmethoxy)benzamido)cyclohexanecarboxylic Acid (30)

The product was synthesized according to GP1. Yield: 39%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.11 (s, 1H), 8.67 (d, J = 2.2 Hz, 1H), 8.54 (dd, J = 4.9, 1.7 Hz, 1H), 8.16 (s, 1H), 7.87 (dt, J = 8.0, 2.0 Hz, 1H), 7.48–7.39 (m, 3H), 7.37 (t, J = 7.9 Hz, 1H), 7.19 (dd, J = 8.0, 2.5 Hz, 1H), 5.20 (s, 2H), 2.10 (d, J = 13.6 Hz, 2H), 1.72 (dt, J = 14.0, 7.0 Hz, 2H), 1.51 (h, J = 7.1, 5.5 Hz, 5H), 1.26 (d, J = 10.8 Hz, 1H). LC/MS (APSI) m/z: [M – H] calcd for C₂₀H₂₁N₂O₄, 353.2; found, 353.1.

1-(3-(Pyridin-3-ylmethoxy)benzamido)cyclopropanecarboxylic Acid (31)

The product was synthesized according to GP1. Yield: 57%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.38 (s, 1H), 8.93 (s, 1H), 8.67 (s, 1H), 8.54 (d, J = 4.8 Hz, 1H), 7.87 (dt, J = 7.8, 2.0 Hz, 1H), 7.50 (t, J = 2.0 Hz, 1H), 7.48–7.32 (m, 3H), 7.18 (dd, J = 8.2, 2.5 Hz, 1H), 5.19 (s, 2H), 1.38 (q, J = 4.5 Hz, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₁₇H₁₅N₂O₄, 311.1; found, 311.1.

1-(3-(Pyridin-3-ylmethoxy)benzamido)cyclopentanecarboxylic Acid (32)

The product was synthesized according to GP1. Yield: 42%; purity, >95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.12 (s, 1H), 8.67 (d, J = 2.1 Hz, 1H), 8.54 (dd, J = 4.7, 1.7 Hz, 1H), 8.47 (s, 1H), 7.87 (dt, J = 7.9, 1.9 Hz, 1H), 7.49 (t, J = 2.0 Hz, 1H), 7.48–7.39 (m, 2H), 7.37 (t, J = 7.9 Hz, 1H), 7.18 (dd, J = 8.1, 2.5 Hz, 1H), 5.19 (s, 2H), 2.16–2.06 (m, 2H), 2.02 (dt, J = 14.3, 5.8 Hz, 2H), 1.72–1.61 (m, 2H), 1.66 (s, 3H). LC/MS (APSI) m/z: [M – H] calcd for C₁₉H₁₉N₂O₄, 339.1; found, 339.0.

2-(3-(Pyridin-3-ylmethoxy)benzamido)-2,3-dihydro-1H-indene-2-carboxylic Acid (33)

The product was synthesized according to GP1. Yield: 29%; purity, 95% (assessed by LC/MS). ¹H NMR (500 MHz, DMSO-d₆): δ 12.42 (s, 1H), 8.79 (s, 1H), 8.66 (s, 1H), 8.53 (d, J = 4.6 Hz, 1H), 7.86 (dd, J = 7.8, 1.9 Hz, 1H), 7.49 (t, J = 1.9 Hz, 1H), 7.48–7.32 (m, 3H), 7.24–7.10 (m, 5H), 5.17 (s, 2H), 3.56 (d, J = 16.7 Hz, 2H), 3.38 (d, J = 16.8 Hz, 2H). LC/MS (APSI) m/z: [M – H] calcd for C₂₃H₂₀N₂O₄, 387.1; found, 387.0.

Methyl 3-(Pyridin-3-ylmethoxy)benzoate (34)

The product was synthesized according to GP2 from 3-hydroxybenzoic acid methyl ester (2.30 mmol, 350 mg), 3-(bromomethyl)pyridine hydrobromide (2.53 mmol, 640 mg), and cesium carbonate (5.29 mmol, 1.73 g) in DMF (20.0 mL). Purification by flash chromatography (hexane/EtOAc 7:3) yielded 476 mg (85%) of 34. ¹H NMR (250 MHz, CDCl₃): δ 8.63 (d, J = 2.0 Hz, 1H), 8.55 (dd, J = 4.8, 1.4 Hz, 1H), 7.45 (dt, J = 7.8, 1.8 Hz, 1H), 7.63–7.57 (m, 2H), 7.33–7.26 (m, 2H), 7.12–7.07 (m, 1H), 5.06 (s, 2H), 3.85 (s, 3H).

3-(Pyridin-3-ylmethoxy)benzoic Acid (35)

The product was synthesized according to GP4 from methyl 3-(pyridin-3-ylmethoxy)benzoate (5.82 mmol, 1.42 g) and lithium hydroxide (58.2 mmol, 1.42 g) in THF (10.0 mL) to obtain 940 mg (71%) of 35. ¹H NMR (250 MHz, CDCl₃): δ 13.03 (brs, 1H), 8.68 (d, J = 2.0 Hz, 1H), 8.55 (dd, J = 4.8, 1.6 Hz, 1H), 7.90 (dt, J = 7.8, 1.8 Hz, 1H), 7.58–7.54 (m, 2H), 7.47–7.40 (m, 2H), 7.31–7.27 (m, 1H), 5.22 (s, 2H).

Methyl (R)-2-(3-(Pyridin-3-ylmethoxy)benzamido)pentanoate (36)

The product was synthesized according to GP3 from 3-(pyridin-3-ylmethoxy)benzoic acid (0.872 mmol, 200 mg), HBTU (1.13 mmol, 434 mg), methyl (R)-2-aminopentanoate (1.05 mmol, 176 mg), 4-methylmorpholine (1.13 mmol, 125 μL), and EDCl (1.08 mmol, 207 mg) in DMF (6.0 mL). Purification by flash chromatography (hexane/EtOAc 1:1) yielded 263 mg (88%) of 36. ¹H NMR (250 MHz, DMSO-d₆): δ 8.70–8.67 (m, 2H), 8.56–8.54 (m, 1H), 7.89 (dq, J = 7.8, 1.7 Hz, 1H), 7.54–7.38 (m, 4H), 7.23–7.20 (m, 1H), 5.21 (s, 2H), 4.42 (q, J = 7.2 Hz, 1H), 3.64 (s, 3H), 3.19–3.06 (m, 2H), 1.77 (q, J = 7.2 Hz, 2H), 1.44–1.32 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H).

Methyl (S)-2-(3-(Pyridin-3-ylmethoxy)benzamido)pentanoate (37)

The product was synthesized according to GP3 from 3-(pyridin-3-ylmethoxy)benzoic acid (0.872 mmol, 200 mg), HBTU (1.13 mmol, 434 mg), methyl (S)-2-aminopentanoate (1.05 mmol, 176 mg), 4-methylmorpholine (1.13 mmol, 125 μL), and EDCl (1.08 mmol, 207 mg) in DMF (6.0 mL). Purification by flash chromatography (hexane/EtOAc 1:1) yielded 229 mg (77%) of 37. ¹H NMR (250 MHz, DMSO-d₆): δ 8.70–8.67 (m, 2H), 8.56–8.54 (m, 1H), 7.89 (dq, J = 7.8, 1.7 Hz, 1H), 7.54–7.38 (m, 4H), 7.23–7.20 (m, 1H), 5.21 (s, 2H), 4.42 (q, J = 7.3 Hz, 1H), 3.64 (s, 3H), 3.19–3.06 (m, 2H), 1.77 (q, J = 7.2 Hz, 2H), 1.44–1.32 (m, 2H), 0.89 (t, J = 7.2 Hz, 3H).

Methyl (R)-3-(4-Chlorophenyl)-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoate (38)

The product was synthesized according to GP3 from 3-(pyridin-3-ylmethoxy)benzoic acid (0.872 mmol, 200 mg), HBTU (1.31 mmol, 501 mg), 4-chloro-d-phenylalanine methyl ester hydrochloride (0.959 mmol, 240 mg), 4-methylmorpholine (5.23 mmol, 575 μL), and EDCl (1.31 mmol, 251 mg) in DMF (8.0 mL Purification by flash chromatography (hexane/EtOAc, from 6:4 to 3:7) and yielded 370 mg (quantitative) of 38. ¹H NMR (250 MHz, DMSO-d₆): δ 8.84 (d, J = 8.0 Hz, 1H), 8.60–8.57 (m, 1H), 7.84 (td, J = 7.7, 1.8 Hz, 1H), 7.52 (d, J = 7.8 Hz, 1H), 7.45–7.43 (m, 1H), 7.40–7.37 (m, 2H), 7.36–7.29 (m, 4H), 7.22–7.17 (m, 1H), 5.23 (s, 2H), 4.71–4.62 (m, 1H), 4.09 (br s, 1H), 3.64 (s, 3H), 3.21–3.02 (m, 2H).

Methyl (R)-2-(3-(Pyridin-3-ylmethoxy)benzamido)-3-(4-(trifluoromethyl)phenyl)propanoate (39)

The product was synthesized according to GP3 from 3-(pyridin-3-ylmethoxy)benzoic acid (0.833 mmol, 191 mg), HBTU (1.25 mmol, 479 mg), methyl (R)-2-amino-3-(4-(trifluoromethyl)phenyl)propanoate (0.833 mmol, 206 mg), 4-methylmorpholine (5.00 mmol, 550 μL), and EDCl (1.25 mmol, 240 mg) in DMF (8.0 mL). Purification by flash chromatography (hexane/EtOAc, from 7:3 to 3:7) yielded 382 mg (quantitative) of 39. ¹H NMR (250 MHz, DMSO-d₆): δ 8.89 (d, J = 8.0 Hz, 1H), 8.60–8.57 (m, 1H), 7.84 (td, J = 7.7, 1.8 Hz, 1H), 7.63 (d, J = 8.3 Hz, 2H), 7.51 (d, J = 8.3 Hz, 2H), 7.44–7.32 (m, 4H), 7.22–7.18 (m, 1H), 5.22 (s, 2H), 4.78–4.68 (m, 1H), 3.65 (s, 3H), 3.30–3.13 (m, 2H).

(R)-2-(3-(Pyridin-3-ylmethoxy)benzamido)pentanoic Acid (40)

The product was synthesized according to GP4 from 36 (0.768 mmol, 263 mg), LiOH (7.68 mmol, 188 mg) in THF (6.0 mL). Purification by preparative HPLC yielded 75.0 mg (30%) of 40. ¹H NMR (300 MHz, DMSO-d₆): δ 12.54 (bs, 1H), 8.70 (s, 1H), 8.55 (d, J = 7.8, 2H), 7.90 (d, J = 7.9 Hz, 1H), 7.56–7.37 (m, 4H), 7.20 (ddd, J = 8.1, 2.5, 0.9 Hz, 1H), 5.21 (s, 2H), 4.37 (q, J = 7.4 Hz, 1H), 1.77 (q, J = 7.2 Hz, 2H), 1.44–1.33 (m, 2H), 0.89 (t, J = 7.3 Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆): δ 174.3, 166.5, 158.4, 149.6, 149.5, 136.2, 135.9, 129.9, 124.1, 120.6, 118.3, 114.1, 67.6, 55.3, 52.7, 33.0, 19.5, 13.9; R_f HPLC: 7.2 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 97.1% purity; HRMS (MALDI) m/z; found, 329.1496: [M + H]⁺ (calcd C₁₈H₂₁N₂O₄⁺, 329.1502).

(S)-2-(3-(Pyridin-3-ylmethoxy)benzamido)pentanoic Acid (41)

The product was synthesized according to GP4 from 37 (0.669 mmol, 229 mg), LiOH (6.69 mmol, 163 mg) in THF (6.0 mL). Purification by preparative HPLC yielded 113 mg (51%) of 41. ¹H NMR (300 MHz, DMSO-d₆): δ 12.56 (bs, 1H), 8.72 (s, 1H), 8.58 (d, J = 7.8, 2H), 7.90 (dt, J = 7.8, 1.9 Hz, 1H), 7.58–7.40 (m, 4H), 7.23 (ddd, J = 8.1, 2.5, 0.9 Hz, 1H), 5.24 (s, 2H), 4.37 (q, J = 7.4 Hz, 1H), 1.79 (q, J = 7.2 Hz, 2H), 1.51–1.31 (m, 2H), 0.91 (t, J = 7.3 Hz, 3H); ¹³C NMR (75 MHz, DMSO-d₆): δ 174.3, 166.5, 158.4, 149.4, 149.3, 136.4, 135.9, 129.9, 124.2, 120.7, 118.3, 114.2, 67.5, 55.3, 52.7, 33.0, 19.5, 13.9; R_f HPLC: 7.3 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 98.1% purity; HRMS (MALDI) m/z; found, 329.1502: [M + H]⁺ (calcd C₁₈H₂₁N₂O₄⁺, 329.1502).

(R)-3-(4-Chlorophenyl)-2-(3-(pyridin-3-ylmethoxy)benzamido)propanoic Acid (42)

The product was synthesized according to GP4 from 38 (0.291 mmol, 123 mg), LiOH (2.91 mmol, 71.0 mg) in THF (4.0 mL). Purification by preparative HPLC yielded 75.5 mg (63%) of 42. ¹H NMR (300 MHz, DMSO-d₆): δ 12.81 (bs, 1H), 8.71 (d, J = 8.3 Hz, 1H), 8.60 (d, J = 4.8, 1H), 7.86 (dt, J = 7.8, 1.9 Hz, 1H), 7.54 (d, J = 7.8 Hz, 1H), 7.46–7.28 (m, 8H), 7.21–7.18 (m, 1H), 5.24 (s, 2H), 4.66–4.58 (m, 1H), 3.19 (dd, J = 13.7, 4.6 Hz, 1H), 3.05 (dd, J = 13.7, 10.7, Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆): δ 172.9, 165.9, 158.0, 156.3, 148.9, 137.3, 137.2, 135.3, 131.0, 130.9, 129.5, 128.1, 123.1, 121.7, 120.0, 117.9, 113.5, 70.2, 53.9, 35.6; R_f HPLC: 10.9 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). > 99.9% purity; HRMS (MALDI) m/z; found. 411.1104: [M + H]⁺ (calcd C₂₂H₂₀ClN₂O₄⁺, 411.1106).

(R)-2-(3-(Pyridin-3-ylmethoxy)benzamido)-3-(4-(trifluoromethyl)phenyl)propanoic Acid (43)

The product was synthesized according to GP4 from 39 (0.208 mmol, 95.5 mg), LiOH (2.08 mmol, 51.0 mg) in THF (4.0 mL). Purification by preparative HPLC yielded 53.0 mg (57%) of 43. ¹H NMR (300 MHz, DMSO-d₆): δ 12.83 (bs, 1H), 8.76 (d, J = 8.0 Hz, 1H), 8.61 (d, J = 4.9 Hz, 1H), 7.90 (t, J = 7.7 Hz, 1H), 7.62 (d, J = 8.2 Hz, 2H), 7.58–7.51 (m, 3H), 7.46–7.35 (m, 4H), 7.21–7.18 (m, 1H), 5.25 (s, 2H), 4.72–4.64 (m, 1H), 3.30 (dd, J = 13.9, 4.7 Hz, 1H), 3.16 (dd, J = 13.9, 10.4 Hz, 1H); ¹³C NMR (75 MHz, DMSO-d₆): δ 172.8, 165.9, 158.0, 156.0, 143.1, 137.7, 135.3, 129.9, 129.6, 127.1 (q, J = 31.5 Hz), 125.0 (q, J = 3.7 Hz), 124.4 (q, J = 270.3 Hz), 123.3, 121.9, 120.1, 117.9, 113.5, 70.0, 53.7, 36.0; R_f HPLC: 11.4 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). > 99.9% purity; HRMS (MALDI) m/z; found. 445.1367: [M + H]⁺ (calcd C₂₃H₂₀F₃N₂O₄⁺, 445.1370).

Methyl (3-Hydroxybenzoyl)phenylalaninate (44)

The product was synthesized according to GP3 from 3-hydroxybenzoic acid (14.5 mmol, 2.0 g), HBTU (18.8 mmol, 7.21 g), methyl 2-amino-3-phenylpropanoate hydrochloride (17.4 mmol, 3.75 g), 4-methylmorpholine (18.8 mmol, 2.07 mL), and EDCl (18.8 mmol, 3.61 g) in DMF (20.0 mL). Purification by flash chromatography (hexane/EtOAc 1:2) yielded 3.45 g (79%) of 44. ¹H NMR (250 MHz, CDCl₃): δ 7.45 (t, J = 2.0 Hz, 1H), 7.28–7.21 (m, 4H), 7.14–7.10 (m, 3H), 7.00 (ddd, J = 1.0, 2.5, 8.0 Hz, 1H), 6.70 (d, J = 7.5 Hz, 1H), 5.02–4.94 (m, 1H), 3.76 (s, 3H), 3.24 (t, 2H).

Methyl (3-(Pyridin-4-ylmethoxy)benzoyl)phenylalaninate (45)

The product was synthesized according to GP2 from 44 (0.668 mmol, 200 mg), 4-(bromomethyl)pyridine hydrobromide (0.735 mmol, 192 mg), and cesium carbonate (1.54 mmol, 501 mg) in DMF (6.0 mL). Purification by flash chromatography (hexane/EtOAc 1:3) yielded 183 mg (70%) of 45. ¹H NMR (250 MHz, CDCl₃): δ 8.64 (d, J = 5.3 Hz, 2H), 7.44–7.23 (m, 7H), 7.14–7.08 (m, 3H), 6.56 (d, J = 7.6 Hz, 1H), 5.15 (s, 2H), 5.11–5.03 (m, 1H), 3.77 (s, 3H), 3.33–3.17 (m, 2H).

Methyl (3-(Pyridin-2-ylmethoxy)benzoyl)phenylalaninatee (46)

The product was synthesized according to GP2 from 44 (0.668 mmol, 200 mg), 2-(bromomethyl)pyridine hydrobromide (0.735 mmol, 192 mg), and cesium carbonate (1.54 mmol, 501 mg) in DMF (6.0 mL). Purification by flash chromatography (hexane/EtOAc 1:3) yielded 147 mg (56%) of 46. ¹H NMR (250 MHz, CDCl₃): δ 8.61 (d, J = 5.2 Hz, 1H), 7.74 (td, J = 7.6 Hz, 1H), 7.54–7.24 (m, 7H), 7.14–7.11 (m, 3H), 6.56 (s, 1H), 5.25 (s, 2H), 5.11–5.03 (m, 1H), 3.77 (s, 3H), 3.27–3.22 (m, 2H).

Methyl (3-(Benzyloxy)benzoyl)phenylalaninate (47)

The product was synthesized according to GP2 from 44 (0.668 mmol, 200 mg), benzyl bromide (0.720 mmol, 87.8 μL), and cesium carbonate (1.33 mmol, 435 mg) in DMF (6.0 mL). Purification by flash chromatography (hexane/EtOAc 7:3) yielded 141 mg (54%) of 47. ¹H NMR (250 MHz, DMSO-d₆): δ 8.82 (d, J = 7.9 Hz, 1H), 7.49–7.32 (m, 8H), 7.31–7.23 (m, 4H), 7.22–7.14 (m, 2H), 5.14 (s, 2H), 4.71–4.61 (m, 1H), 3.64 (s, 3H), 3.21–3.03 (m, 2H).

(3-(Pyridin-4-ylmethoxy)benzoyl)phenylalanine (48)

The product was synthesized according to GP4 from 45 (0.469 mmol, 183 mg), LiOH (4.69 mmol, 115 mg) in THF (4.0 mL). Purification by preparative HPLC yielded 90.0 mg (52%) of 48. ¹H NMR (300 MHz, DMSO-d₆): δ 12.79 (bs, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.58 (d, J = 5.6 Hz, 2H), 7.45–7.14 (m, 11H), 5.23 (s, 2H), 4.66–4.58 (m, 1H), 3.64 (s, 3H), 3.22–3.02 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ 173.6, 166.4, 158.3, 150.2, 146.5, 138.6, 135.9, 130.0, 129.5, 128.7, 126.8, 122.3, 120.6, 118.4, 114.1, 68.1, 54.7, 36.8; R_f HPLC: 7.2 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). > 99.9% purity; HRMS (MALDI) m/z; found. 377.1496: [M + H]⁺ (calcd C₂₂H₂₁N₂O₄⁺, 377.1496).

(3-(Pyridin-2-ylmethoxy)benzoyl)phenylalanine (49)

The product was synthesized according to GP4 from 46 (0.376 mmol, 147 mg), LiOH (3.76 mmol, 91.9 mg) in THF (4.0 mL). Purification by preparative HPLC yielded 47.0 mg (34%) of 49. ¹H NMR (300 MHz, DMSO-d₆): δ 12.76 (bs, 1H), 8.69 (d, J = 8.0 Hz, 1H), 8.58 (dq, J = 4.8, 0.8 Hz, 1H), 7.83 (td, J = 7.7, 1.8 Hz, 1H), 7.53–7.14 (m, 11H), 5.22 (s, 2H), 4.66–4.58 (m, 1H), 3.22–3.02 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ 173.6, 166.4, 158.4, 156.9, 149.6, 138.6, 137.4, 135.8, 129.9, 129.5, 128.6, 126.8, 123.4, 122.0, 120.5, 118.3, 114.0, 70.8, 54.6, 36.7; R_f HPLC: 9.5 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). > 99.9% purity; HRMS (MALDI) m/z; found. 377.1496: [M + H]⁺ (calcd C₂₂H₂₁N₂O₄⁺, 377.1496).

2-(3-(Benzyloxy)benzamido)-3-phenylpropanoic Acid (50)

The product was synthesized according to GP4 from 47 (0.362 mmol, 141 mg), LiOH (3.62 mmol, 88.5 mg) in THF (4.0 mL). Purification by preparative HPLC yielded 80.0 mg (59%) of 50. ¹H NMR (500 MHz, DMSO-d₆): δ 12.76 (bs, 1H), 8.70 (d, J = 8.2 Hz, 1H), 7.47–7.44 (m, 3H), 7.41–7.33 (m, 5H), 7.31 (d, J = 7.4 Hz, 2H), 7.28–7.14 (m, 2H), 7.19–7.15 (m, 2H), 5.15 (s, 2H), 4.65–4.60 (m, 1H), 3.19 (dd, J = 13.6, 4.4 Hz, 1H), 3.07 (dd, J = 13.7, 10.8 Hz, 1H); ¹³C NMR (126 MHz, DMSO-d₆): δ 173.2, 166.0, 158.2, 138.2, 136.9, 135.3, 129.5, 129.1, 128.5, 128.2, 127.9, 127.7, 126.4, 119.9, 117.9, 113.6, 69.4, 54.3, 36.3; R_f HPLC: 13.0 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 97.4% purity; HRMS (MALDI) m/z; found. 376.1546: [M + H]⁺ (calcd C₂₃H₂₂NO₄⁺, 376.1543).

Methyl 4-(Pyridin-3-ylmethoxy)benzoate (51)

The product was synthesized according to GP2 from 4-hydroxybenzoic acid methyl ester (4.23 mmol, 650 mg), 3-(bromomethyl)pyridine hydrobromide (4.65 mmol, 1.18 g), and cesium carbonate (9.73 mmol, 3.19 g) in DMF (20.0 mL). Purification by flash chromatography (hexane/EtOAc 1:1) yielded 836 mg (81%) of 51. ¹H NMR (250 MHz, CDCl₃): δ 8.69 (s, 1H), 8.60 (d, J = 5.0 Hz, 1H), 8.00 (dt, J = 9.0, 2.1 Hz, 2H), 7.79 (dt, J = 7.8, 1.9 Hz, 1H), 7.34 (dd, J = 7.8, 5.0 Hz, 1H), 6.99 (dt, J = 8.9, 2.1 Hz, 2H), 5.13 (s, 2H), 3.88 (s, 3H).

4-(Pyridin-3-ylmethoxy)benzoic Acid (52)

The product was synthesized according to GP4 from methyl 4-(pyridin-3-ylmethoxy)benzoate (3.44 mmol, 836 mg) and lithium hydroxide (34.4 mmol, 840 mg) in THF (10.0 mL) to obtain 552 mg (70%) of 52. ¹H NMR (250 MHz, CDCl₃): δ 12.64 (bs, 1H), 8.69 (s, 1H), 8.59 (d, J = 4.2 Hz, 1H), 7.92–7.88 (m, 3H), 7.43 (dd, J = 4.9, 2.9 Hz, 1H), 7.12 (d, J = 8.7 Hz, 2H), 5.23 (s, 2H).

Methyl (4-(Pyridin-3-ylmethoxy)benzoyl)phenylalaninate (53)

The product was synthesized according to GP3 from 4-(pyridin-3-ylmethoxy)benzoic acid (0.872 mmol, 200 mg), HBTU (1.13 mmol, 434 mg), phenylalanine methyl ester hydrochloride (1.05 mmol, 226 mg), 4-methylmorpholine (1.13 mmol, 125 μL), and EDCl (1.13 mmol, 217 mg) in DMF (6.0 mL). Purification by flash chromatography (hexane/EtOAc 1:1) yielded 210 mg (62%) of 53. ¹H NMR (250 MHz, DMSO-d₆): δ 8.69 (d, 2H), 8.56 (d, J = 5.0 Hz, 1H), 7.88 (dt, J = 7.8, 1.7 Hz, 2H), 7.79 (d, J = 8.7 Hz, 2H), 7.43 (dd, J = 7.7, 4.7 Hz, 1H), 7.31–7.14 (m, 5H), 7.09 (d, J = 8.7 Hz, 2H), 5.21 (s, 2H), 4.67–4.58 (m, 1H), 3.62 (s, 3H), 3.19–3.06 (m, 2H).

(4-(Pyridin-2-ylmethoxy)benzoyl)phenylalanine (54)

The product was synthesized according to GP4 from methyl (4-(pyridin-4-ylmethoxy)benzoyl)phenylalaninate (0.538 mmol, 210 mg), LiOH (5.38 mmol, 131 mg) in THF (4.0 mL). Purification by preparative HPLC yielded 122 mg (61%) of 54. ¹H NMR (300 MHz, DMSO-d₆): δ 12.72 (bs, 1H), 8.68 (s, 1H), 8.55 (d, J = 7.7 Hz, 2H), 7.87 (td, J = 8.0, 1.9 Hz, 1H), 7.82–7.77 (m, 2H), 7.42 (dd, J = 7.8, 4.8 Hz, 1H), 7.32–7.23 (m, 4H), 7.19–7.14 (m, 1H), 7.11–7.06 (m, 2H), 5.21 (s, 2H), 4.63–4.56 (m, 1H), 3.21–3.02 (m, 2H); ¹³C NMR (75 MHz, DMSO-d₆): δ 173.6, 166.3, 158.4, 156.9, 149.4, 149.2, 138.6, 136.5, 135.8, 133.0, 129.9, 129.5, 128.6, 126.8, 124.2, 120.5, 118.3, 114.0, 67.5, 54.6, 36.7; R_f HPLC: 6.5 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 99.7% purity; HRMS (MALDI) m/z; found. 377.1499: [M + H]⁺ (calcd C₂₂H₂₁N₂O₄⁺, 377.1496).

6-Hydroxy-4′-methoxy-[1,1′-biphenyl]-3-carboxylic Acid (55)

3-Bromo-4-hydroxybenzoic acid (1.0 mmol, 217 mg), 4-methoxyphenylboronic acid (1.0 mmol, 157 mg, cesium carbonate (3.0 mmol, 997 mg), and palladium acetate (0.03 mmol, 6.74 mg) were dissolved in DMF (5.0 mL) and water (2.0 mL) under Ar. The resulting solution was heated at 45 °C overnight. After this time, the reaction mixture was diluted with water and the pH adjusted to 3 with HCl 1M. The aqueous phase was then extracted with ethyl acetate (3×). The combined organic layers were dried over magnesium sulfate, filtered, and evaporated. Purification by flash chromatography (hexane/EtOAc 1:1) yielded 202 mg (83%) of 55. ¹H NMR (400 MHz, DMSO-d₆): δ 12.49 (br s, 1H), 10.34 (br s, 1H), 7.80 (d, J = 2.2 Hz, 1H), 7.74 (dd, J = 8.4, 2.2 Hz, 1H), 7.48 (d, J = 8.8 Hz, 2H), 7.00–6.96 (m, 3H), 3.79 (s, 3H).

(S)-N-(1-(Cyclohexylamino)-1-oxo-3-phenylpropan-2-yl)-6-hydroxy-4′-methoxy-[1,1′-biphenyl]-3-carboxamide (56, SB-583355)

The product was synthesized according to GP3 from 55 (0.409 mmol, 100 mg), HBTU (0.614 mmol, 235 mg), N-cyclohexyl-l-phenylalaninamide (0.45 mmol, 111 mg), 4-methylmorpholine (2.45 mmol, 269 μL), and EDCl (0.614 mmol, 118 mg) in DMF (5.0 mL). Purification by preparative HPLC yielded 38.9 mg (20%) of 56. ¹H NMR (500 MHz, CDCl₃): δ 7.79 (br s, 1H), 7.66 (s, 1H), 7.49 (d, J = 8.0 Hz, 1H), 7.39 (d, J = 8.6 Hz, 1H), 7.25–7.18 (m, 5H), 7.09 (d, J = 4.9 Hz, 1H), 6.94–6.91 (m, 3H), 6.17 (s, 1H), 4.84 (q, J = 6.1 Hz, 1H), 3.81 (s, 3H), 3.65–3.55 (m, 1H), 3.20 (dd, J = 13.3, 5.6 Hz, 1H), 3.07 (dd, J = 12.9, 8.4 Hz, 1H), 1.71 (d, J = 10.2 Hz, 1H), 1.61 (d, J = 11.5 Hz, 1H), 1.56–1.48 (m, 3H), 1.26–1.18 (m, 2H), 1.03–0.90 (m, 2H), 0.83 (q, J = 11.2 Hz, 1H); ¹³C NMR (126 MHz, CDCl₃): δ 170.3, 166.9, 159.2, 156.6, 136.7, 130.3, 129.9, 129.4, 129.0, 128.6, 128.2, 127.5, 126.9, 125.5, 115.9, 114.2, 55.3, 48.4, 39.1, 32.6, 32.4, 25.2, 24.6; R_f HPLC: 12.8 min (13 min from 10 to 95% MeCN in water (0.1% formic acid), then 7 min 95% MeCN). 99.7% purity; HRMS (ESI) m/z; found. 473.2456: [M + H]⁺ (calcd C₂₉H₃₃N₂O₄⁺, 473.2435).

Cell Culture

HepG2 cells were maintained at 5% CO₂ and 37 °C in Dulbecco’s modified Eagle’s medium (DMEM) high-glucose with phenol red (Gibco #41965) supplemented with 20% fetal bovine serum (FBS), 1 mM sodium pyruvate (Gibco #11360), penicillin (100 units/ml), and streptomycin (100 μg/mL) (Gibco #15140). For the last passage before the cells were used in the CellTiter-Glo assay, the tissue culture (TC) flask was coated with collagen G. Therefore, 10 mL of PBS supplemented with 0.01 mg/mL collagen G (Merck, #L7213) was incubated for 30 min at 37 °C in the new 175 cm² TC flask and then immediately replaced with new growth medium.

CHO-K1 cells (DSMZ, ACC 110) were maintained in Ham’s F-12 medium (ThermoFisher) supplemented with 10% FBS, 100 units/ml Penicillin and 100 μg/mL Streptomycin (Gibco), and harvested using Trypsin/EDTA.

Stable CHO-K1 Cell Lines for IP-One and LC–MS Binding Experiments

A CHO-K1-derived stable cell line that constitutively overexpresses human G2A (uniprot Q9UNW8-1) and human G alpha subunit 11 (GNA11; uniprot Q9NPC1-1) was generated using the sleeping beauty method.³⁰ In brief, the native coding DNA sequence (CDS) for Homo sapiens (h.s.) G2A was cloned into the plasmid vector pSBbi-Bla (addgene #60526), which procures resistance to Blasticidin, and the h.s. cDNA for GNA11 was cloned into pSBbi-GP (addgene #60511), which procures resistance to Puromycin. Stable insertion of the expression cassettes into the host cell genome was facilitated by the transposase expressed from the cotransfected plasmid pCMV(CAT)T7-SB100 (addgene #34879). Expression is governed by the constitutively active EF1a promoter. Double-positive cells were selected for 10 days with 10 μg/mL Blasticidin S HCl and 50 μg/mL Puromycin dihydrochlorid.

Accumulation of inositol monophosphate in response to activation of the G_q pathway can be enhanced by overexpression of a GNA to which the respective GPCR efficiently couples. We have successfully used this strategy before in recent studies directed at another fatty acid receptor, namely, the GPCR BLT2 (leukotriene B4 receptor type 2), and its relevant off-targets BLT1 and AT1 (type-1 angiotensin II receptor).³¹⁻³³ For G2A, we had tested the combination with GNAQ, GNA11, GNA14, GNA15/16 or none, and identified the combination with GNA11 as the one enabling the best assay window. Several single cell clones with G2A + GNA11 were isolated and their response to 9-HODE evaluated in comparison to the pool of G2A + GNA11 expressing cells. A single cell clone with representative response to 9-HODE was selected and used throughout the HTS campaign as well as for all experiments presented in this study. A cell line stably transfected with only GNA11 served as control for any target independent cellular response. This was not observed for any of the investigated compounds (data not shown).

Stable CHO-K1 Cell Lines for the NanoBit βarr2 Recruitment Assay

Using pBiT2.1-C [TK/SmBiT] (Promega #N197) as the template, the section from two base pairs (bp) upstream of the XhoI site at the beginning of the Glycine–Serine linker until the end of the SmBit open reading frame (ORF) was amplified by PCR. Thereby, the forward primer was designed in such a way that it attached 27 bp homologous to the 3′ end of the human G2A CDS. The CDS for G2A and [Gly–Ser linker SmBit] were fused in another PCR and then subsequently cloned into the plasmid vector pSBbi-Bla (addgene #60526). The expressed fusion protein is [human G2A full length]-GS(SGGGG)₂SSGVTGYRLFEEIL.

The CDS for βarr2 (uniprot entry P32121, also known as (nonvisual) arrestin-3) with N-terminal fusion to split NanoLuc large bit (LgBit) was amplified by PCR from LgBit-ARRB2 fusion vector (Promega #CS1603B122), and the stop codon was changed to TGA. The ORF for LgBit-βarr2 was then subsequently cloned into pSBtet-Neo (addgene #60509).

CHO-K1 were cotransfected with G2A-SmBit in pSBbi-Bla and LgBit-βarr2 in pSBtet-Neo. Insertion into the genome was facilitated as described above, and cells were selected for 10 days with 10 μg/mL Blasticidin S HCl and 250 μg/mL Geneticin G418. A control cell line without the G2A expression cassette was generated in parallel (only Geneticin resistance). Expression of LgBit-βarr2 is controlled by the tet-On system and induced by addition of Doxycycline.

IP-One Assay for G2A

Activation of human G2A was probed in a functional cell-based assay in which accumulation of IP-1 (inositol monophosphate) as a downstream effect of G2A signaling was detected in a displacement assay based on homogeneous time-resolved FRET (HTRF) between the FRET acceptor-coupled IP-1 and Terbium cryptate-coupled anti-IP-1 antibody (IP-One assay kit, cisbio, part of PerkinElmer).

CHO-K1 cells stably overexpressing human G2A in combination with human GNA11, or as control, cells overexpressing only GNA11 were seeded in full growth medium into white 384-well TC plates (Greiner bio-One) at 12,500 cells/well, respectively. After overnight incubation at 37 °C and 5% CO₂, the medium was removed using a Tecan HydroSpeed plate washer, and cells were washed four times with stimulation buffer [146 mM NaCl, 4.2 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 50 mM LiCl₂, 5.5 mM d-glucose, 0.1%_(w/v) fatty acid free bovine serum albumin fraction V (Carl Roth, Karlsruhe, Germany) buffered with 10 mM HEPES at pH 7.4 (NaOH)]. Thereafter, the synthesized end compounds and a total of 0.5% DMSO were added to the cells, and the plate was sealed and incubated at 37 °C for 90 min. After this stimulation period, the cells were lysed by addition of the detection agents prepared in lysis buffer according to the manufacturer’s instructions. Plates were stored at RT overnight, and afterward HTRF was recorded on a Tecan Spark equipped with enhanced fluorescence module. In a filter-based method, fluorescence intensity (FI) at 665 nm (d2, FRET acceptor) and at 620 nm (Terbium, FRET donor) was recorded following excitation at 340 nm. According to the convention FI₆₆₅ was multiplied by 10,000 and divided by FI₆₂₀ to give the dimensionless HTRF value. The concentration of IP-1 produced by the cells was calculated from a standard curve using dilutions of unlabeled IP-1 in buffer without cells. Experiments were always conducted with three technical replicates (N = 3). Mean IP-1 and standard error values were calculated in Excel and exported to GraphPrism 8.3.1 for determination of dose–response using the four-parameter curve fitting protocol (variable hill slope). Experiments with activation of G2A by reference agonist ±9-HODE (cayman chemicals, # 38400) were always conducted in parallel as a control for assay performance and as a reference for calculation of relative efficacy of 20 μM of compound vs maximal activation observed with ±9-HODE. Dose–response curves are shown in Supporting Information Figure S1.

NanoBit βarr2 Recruitment Assay

Recruitment of βarr2 in response to activation of human G2A was probed in a split-luciferase complementation assay. The receptor is expressed as a fusion protein with the NanoLuc small bit (SmBit) at its C-terminus, while the split NanoLuc large bit (LgBiT) is fused to the N-terminus of βarr2. Expression of G2A-SmBit is governed by the constitutively active EF1a promoter, while the expression of LgBit-βarr2 is induced by addition of doxycycline. One day ahead of the experiment, cells were harvested and transferred into Opti-MEM (Gibco; #51985) supplemented with 100 units/ml Penicillin and 100 μg/mL Streptomycin (Gibco). Cells were seeded in 90 μL at 10,000 cells/well into 96-well half area white PS (polystyrol) flat bottom TC plates (Greiner bio-One, #675083), and expression of LgBit-βarr2 was induced by addition of 10 μL doxycycline in Opti-MEM for a final concentration of 50 ng/mL doxycycline. The plates were covered with Aera-Seal foil (Merck #Z721573) for overnight incubation at 37 °C and 5% CO₂. The medium was removed using a Tecan HydroSpeed plate washer, and cells were washed four times with βarr stimulation buffer [146 mM NaCl, 4.2 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 5.5 mM d-glucose, 0.1% (w/v) fatty acid free bovine serum albumin fraction V (Carl Roth, Karlsruhe, Germany) buffered with 10 mM HEPES at pH 7.4 (NaOH)], leaving 20 μL/well.

Thereafter, the Nano-Glo live cell assay system (Promega N2012) was used according to the manufacturer’s instructions with an addition of 5 μL of premixed (1–20) substrate in Nano-Glo LCS dilution buffer. Afterward the baseline luminescence was measured on a Tecan Spark (450 ms integration time; for 5 measurements with an offset of 100 s). Subsequently, the compound at the indicated concentration and a total of 0.5% DMSO were added to the cells (5 μL/well). In order to prevent dilution of the substrate through the addition of the compound, the compound dilution was also supplemented with 20%(v/v) premixed substrate. Luminescence was further recorded every 100 s.

Aqueous Solubility

Aqueous solubility was evaluated as previously described.²⁴ Final concentrations of 42, 56, 75, 100, 134, 178, 237, 316, 422, 563, 750, 1000, 1688, 2250, and 3000 μM of T-10418 were prepared in PBS pH 7.4 solution containing 1% DMSO, in a 96-well transparent flat bottom microtiter plate. Precipitation of the compound was measured at 600 and 800 nm after 1 and 24 h at RT using a microplate reader (Infinite M200, Tecan Group Ltd., Crailsheim, Germany), and solution clarity was confirmed by eye.

Metabolic Stability

Compounds were tested according to the following method: a solution of compound (final concentration 1 mM) was prepared in 100% DMSO. 432 μL of phosphate buffer (0.1 M, pH 7.4) together with 50 μL of NADPH-regenerating system (30 mM glucose-6-phosphate, 4 U/mL glucose-6-phosphate dehydrogenase, 10 mM NADP, 30 mM MgCl₂) and 5 μL of the corresponding test compound were preincubated at 37 °C. After 5 min, the reaction was started by the addition of 13 μL of the microsome mix from the liver of Sprague–Dawley rats (Thermo Fisher Scientific, Darmstadt, Germany; 20 mg protein/ml in 0.1 M phosphate buffer). The incubation was performed in a shaking water bath at 37 °C. The reaction was stopped by the addition of 500 μL of ice-cold methanol at 0, 15, 30, and 60 min. The samples were centrifuged at 5000g for 5 min at 4 °C. The supernatants were analyzed and quantified by HPLC. Control samples were always studied to check the stability of the compounds in the reaction mixture. The first control was without NADPH, which is needed for the enzymatic activity of the microsomes. The second control was with inactivated (microsomes which had been incubated for 20 min at 90 °C). The third control was without test compounds (to determine the baseline). The amounts of the test compounds were quantified by an external calibration curve. The in vitro intrinsic clearance was calculated by eq 1, wherein k represents the -gradient of the ln peak area ratio plotted against time.

1

Animal Experiments

All animal experiments were approved by the local Ethics Committees for AnimalResearch (Darmstadt, Germany) under the approval code FK/1113, approved on 29 March 2019. In addition, all animal experiments were performed according to the recommendations of the Working Group PPRECISE (Preclinical Pain Research Consortium for Investigating Safety and Efficacy) (https://pubmed.ncbi.nlm.nih.gov/26683237) and the recommendation of the Guide of the Care and Use of Laboratory Animals of the National Institutes of Health and the ARRIVE guidelines 2.0 (https://pubmed.ncbi.nlm.nih.gov/32663219/).

All experimental C57BL/6NRj animals were purchased from the commercial breeding company Janvier (Le Genest-Saint-Isle, France). They were housed in a day/night cycle of a 12 h rhythm and food and water were available ad libitum. In addition, all tissues were isolated from 8- to 12-week-old C57BL/6NRj male mice.

Isolation of Sensory Neurons

For tissue isolation, mice were euthanized by isoflurane anesthesia (2–2.5% isoflurane in carbogen), cardio puncture, and cervical dislocation. The lumbar (L1–L6) dorsal root ganglia were dissected and cultivated overnight for calcium-imaging experiments.

Calcium Imaging

For calcium imaging, cultured mouse sensory neurons were stained with Fura-2-AM (Thermo Fisher) for 60 min at 37 °C and washed afterward twice with fresh Ringeŕs solution consisting of: 145 mM NaCl, 1.25 mM CaCl₂ × 2H₂O, 1 mM MgCl₂ × 6H₂O, 5 mM KCl, 10 mM d-glucose and 10 mM HEPES adjusted to a pH of 7.3. During the experiments, Ringer’s solution was also used for baseline measurements and for washing out agonists and compounds between stimulations. For investigating the effect of compounds in comparison to 9-HODE-mediated TRPV1-sensitization, the sensory neurons were stimulated twice with capsaicin (100 nM) for 30 s and preincubated with 9-HODE or compounds at the indicated concentrations for 2 min prior to the second capsaicin stimulus. Finally, KCl (50 mM, 1 min) was used as a positive control to depolarize neurons and to activate all voltage-gated calcium channels at the end of each experiment. All stimulating compounds were dissolved in Ringer’s solution to their final concentration. The measurements were performed using a DMI4000 B Microscope, a compact light source CTR550 HS (Leica), and a ValveBank II perfusion system (AutoMate Scientific). Fluorescence was evoked by 340 and 380 nm excitation wavelengths, collected at 510 nm, and reported as change in the ratio of F₃₄₀/F₃₈₀. Image acquisition occurred every 2 s in both wavelengths. Changes in intracellular calcium concentrations were determined as Δ_ratio of the fluorescence intensities F₃₄₀/F₃₈₀.

Cell Toxicity

Cell viability was probed using CellTiter-Glo (Promega) according to the manufacturer’s instructions with minor modifications. Hep-G2 cells were harvested from Collagen-G coated TC flasks using Trypsin and recovered in white DMEM high-glucose medium (Gibco #31053) supplemented with 10% FBS, 100 units/mL Penicillin, and 100 μg/mL Streptomycin as well as 2 mM l-Glutamin (Gibco). The cell suspension was then passed through a 40 μm cell strainer (pluriselect #43-50040) in order to remove cell clots, and cell density was adjusted to 100,000 cells/mL. Then, 30 μL/well, equivalent to 3000 cells/well, were seeded into 96-well half area white PS (polystyrol) flat bottom TC plates (Greiner bio-One, #675083). Compounds were added in 10 μL of medium with 2% DMSO to give the indicated compound concentrations and 0.5% DMSO during treatment at 37 °C and 5% CO₂. For detection, CellTier-Glo reagent mix was applied at 20 μL/well and protected from light evolved for 30 min at RT before luminescence was recorded using the standard attenuation protocol on a Tecan SPARK. Wells with medium alone and wells with cells treated with only DMSO were used as 0 and 100% cell viability control, respectively. Treatment with 50 μM Paclitaxel was conducted in parallel as an assay performance control.

Direct Binding

CHO-K1 cells expressing human G2A were cultured as described above to a confluence of 80–90%. After trypsinization and washing in PBS, cells were homogenized in G2A buffer (10 mM HEPES, pH 7.4 (NaOH), 146 mM NaCl, 4.2 mM KCl, 1 mM CaCl₂, 0.5 mM MgCl₂, 5.5 mM d-Glucose) with a Polytron. Aliquots of 1 mL (resulting from about 1 × 10⁷ cells) were frozen and stored at −80 °C. In parallel, membrane material from nontransfected CHO-K1 cells at the same density was generated accordingly. The same amounts of G2A and CHO-K1 membrane preparations (from at least 2 × 10⁵ cells) were incubated with varying concentrations of T-10418 in G2A buffer at 25 °C for 1 h. Subsequently, the binding samples were subjected to vacuum filtration over 96-well filter plates (AcroPrep Advance, glass fiber, 1.0 μm, 350 μL; Pall Corporation, Port Washington, New York, US, pretreated with 0.5% polyethylenimine) for separation of nonbound ligand and rapidly washed with 154 mM ice-cold ammonium acetate solution. After drying at 50 °C, the filter plates were eluted three times with 100 μL of acetonitrile per well. Aliquots of the eluates were spiked with 50 nM Oxazepam (as internal standard), supplemented with 0.1% formic acid (A) and acetonitrile (B, resulting in A/B, 65/35, v/v), and subjected to LC–MS/MS quantification. For this purpose, a QTRAP5500 triple quadrupole mass spectrometer with a TurboV ESI-source coupled to an Agilent 1260 HPLC system and a SIL-20A/HT autosampler controlled by the Analyst software v. 1.6.3 was used. An Agilent Zorbax C8 (3.5 μm, 50 mm × 2.1 mm, with a C8 precolumn (4 mm × 2, Phenomenex, Aschaffenburg, Germany) and two in-line filters (0.5 and 0.2 μm, Idex, Oak Harbor, Washington, US) in combination with A/B (65/35, v/v) as mobile phase at a flow rate of 500 μL/min were employed for LC. MS detection was performed under ESI-pos conditions in the MRM mode detecting T-10418 at m/z 377/212 and Oxazepam at m/z 287/241. Quantification of T-10418 was achieved for injected samples of 5 μL based on calibration curves using Oxazepam as internal standard (LLOQ: 10 pM).

β-arrestin Assay Screening of T-10418

5000 HTLA cells were seeded in a white, transparent, and poly-l-Lysin coated 384-well plates from PerkinElmer (cat-no: 6007480). The cells were transfected after 6 h with a plasmid from the PRESTO-Tango kit (Addgene, cat-no: Kit #1000000068). We used a mixture of 10 ng of plasmid and 0.04 μL of Lipofectamine 2000 per well and we used a transfection plan as described by Kroeze in et al.³⁴ As a transfection control, we used GFP and as an assay control we used 100 μM carbachol at the muscarinic M5 receptor. After 24 h, the medium was aspirated and replaced by 45 μL of serum-free medium. The ligand (5 μL) was then added at a final concentration of 10 μM for approximately 24 h. Thereafter, the medium was aspirated and the cells were lysed using 50 μL of a mixture of bright-Glo reagent (Promega cat-no: E2610) diluted 10× with PBS. After 15 min of incubation with the lysis buffer the luminescence (end point, 1500 ms integration time) was measured using a Flexstation 3 (Molecular Devices).

CMKLR1 and GPR1 Experiments

Cell Lines

HEK293 cells were cultivated in DMEM/Ham’s F12 supplemented with 15% FBS. HEK293-hCMKLR1b_eYFP + Gα_Δ6qi4myr cells and HEK293-hGPR1_eYFP cells (h = human protein sequence) were cultivated in DMEM/Ham’s F12 supplemented with 15% FBS and 100 μg/μL Hygromycin. The stable HEK293-hCMKLR1b_eYFP + Gα_Δ6qi4myr cell line was used for previous Ca²⁺ experiments but the chimeric G protein is not needed for βarr2 recruitment NanoLuc-BRET experiments described in this paper. The G protein was kindly provided by Evi Kostenis.³⁵ All cells were maintained in T75 cell culture flasks at 37 °C, 95% humidity, and 5% CO₂ (standard conditions).

Peptide Synthesis

The peptide chemerin-9 was synthesized by using an orthogonal 9-fluorenylmethoxycarbonyl/tert-butyl (Fmoc/tBu) solid phase peptide synthesis strategy (SPPS). The synthesis was performed on a Syro II peptide synthesizer (MultiSynTech, Bochum, Germany) on a scale of 15 μmol per resin. A Wang-resin, which was preloaded with serine for chemerin-9 (C9), was used for automated SPPS. Coupling was performed twice with 8 equiv of the respective Fmoc-protected amino acid activated in situ with equimolar amounts of oxyma and N,N′-diisopropylcarbodiimide in DMF for 30 min. By incubation with 40% piperidine in DMF (v/v) for at least 3 min and 20% piperidine in DMF (v/v) for 10 min, Fmoc was deprotected. Both peptides were cleaved from the resin by incubation with 90% trifluoroacetic acid and the scavenger thiocresol/thioanisol (1:1) for 3 h at RT and precipitated from ice-cold diethyl ether at −20 °C for at least 2 h and washed with diethyl ether. The peptide was purified by using preparative RP-HPLC. Peptide identity was confirmed by MALDI–MS and purity was confirmed to be >95% by RP-HPLC on two different systems Phenomenex Jupiter Peptide Proteo C-12, 250 × 4.6 mm, 90 Å, 4 μm; 2 Phenomenex Aeris Peptide XB-C18, 250 × 4.6 mm, 100 Å, 3.6 μm.

Quantification of Peptide Uptake Using a High-Content Imaging System at CMKLR1 and GPR1

The influence of different compound concentrations on the intracellular accumulation of TAMRA-labeled chemerin-9 (C9) peptide after receptor internalization was observed. HEK293 cells stably transfected with hCMKLR1-eYFP + Gα_Δ6qi4myr or hGPR1_eYFP were seeded into 96 black well plates (100,000 cells/well) coated with poly d-lysine and incubated at 37 °C, 95% humidity, and 5% CO₂ (standard conditions) overnight. Prior to the experiment, cells were incubated with Hoechst 33342 in OptiMEM for 30 min at standard conditions. The solution was replaced by 0.3% DMSO or 30 μM compound in OptiMEM. Additionally, cells were stimulated with different concentrations of TAMRA-labeled peptide, followed by washing with acidic wash (50 mM glycine, 100 mM NaCl, pH 3.0) in HBSS. ImageXpress Micro Confocal High-Content Imaging System (Molecular Devices, San José, CA, USA) was applied using the respective filters for the nuclei (excitation 352 nm, emission 455 nm), TAMRA (excitation 549 nm, emission 577 nm) and eYFP (excitation 514 nm, emission 526 nm). The FI per cell was automatically analyzed for each well by a module detecting the nuclei (5–30 μm in diameter and 20 gray levels above background) and the granules by TAMRA-peptide fluorescence (2–6 μm in diameter, 100 gray levels above background). The assay was performed at least three times in duplicate and then analyzed by using GraphPad prism version 5.03 and nonlinear fit of log(agonist) vs response (three parameter).

βarr2-Recruitment BRET

HEK293 cells were grown in a 25 cm² cultivation flask at 37 °C, 95% humidity, and 5% CO₂ (standard conditions) overnight to 70% confluence and were transiently cotransfected using Metafectene Pro transfection reagent (Biontex Laboratories GmbH) according to manufacturer’s protocol, whereby 11700 ng of CMKLR1-eYFP-pVitro2 or GPR1-eYFP-pVitro2 plasmid and 300 ng of βarr2-Nluc plasmid were used. On the next day, white clear-bottom 96-well plates (Greiner Bio-one) were coated with poly-d-lysine and transfected cells were reseeded with phenolred-free HEK293 medium (DMEM/Ham’s F12 supplemented with 15% FBS). 2 days after transfection, the cell medium was replaced with 0.6% DMSO or 60 μM compound solution diluted in BRET buffer (25 mM HEPES in HBSS, pH 7.3). Afterward, a serial dilution of chemerin-9 (C9) was added as well as the luciferase substrate coelenterazine h (Nanolights; final concentration 8.4 μM). For submaximal arr3-recruitment BRET, cells were incubated with different compound concentrations and an EC₈₀ value of C9. For both set-ups, measurements were performed after 15 min incubation at 37 °C with the plate reader Tecan Spark (filter: luminescence 400–440 nm, fluorescence 505–590 nm). The BRET ratio was calculated as fluorescence divided by bioluminescence values. By subtracting BRET signals of unstimulated cells, the netBRET signal was determined. At least two independent experiments were performed in triplicate and analyzed by using GraphPad prism and nonlinear fit of log(agonist) vs response (three parameters).

Displacement of BRET at Nluc-GPR1

A displacement assay was performed to determine the influence of the compound on the orthosteric binding of C9 at GPR1. Therefore, HEK293 cells were transfected with Nluc-GPR1-eYFP and incubated overnight at standard conditions. One day prior to the assay, cells were seeded into poly-d-lysine coated, black, solid 96-well plates. For the assay, cells were stimulated with a constant concentration of 10 nM TAMRA-labeled chemerin-9 (TAMRA-C9) and different concentrations of compound or chemerin-9 (C9) as positive control. The cells were incubated for 1 h on a tumbler. Afterward, coelenterazine h was added (final concentration 8.4 μM) and measured directly with a plate reader (Tecan Spark, luminescence 430–470 nm, fluorescence 550–700 nm). BRET and netBRET ratios were calculated in the same way as described in βarr2-recruitment of BRET. The experiments were done in triplicate and at least three times. The results were analyzed by using GraphPad prism version 5.03 and nonlinear fit of log(inhibitor) vs response (three parameter).

Leukemia Cell Experiments

Cell Lines

The AML cell lines Molm-13 and ML-2 were acquired from DSMZ and cultured in RPMI supplemented with 10% fetal calf serum, 1% Glutamine, 1% Pen/Strep (Life Technologies, Carlsbad, USA) in a 5% CO2 37 °C incubator.

Cell Viability

Cells were cultured in a 96-well culture plate and treated with different compounds for 72 h. Cell counts were determined using trypan blue exclusion and a Neubauer counting chamber.

Apoptosis Assay

To determine induction of apoptosis in cells after 24 h of incubation with the compounds, an AnnexinV/7-AAD staining (Thermo Fisher Scientific, Waltham, USA) was performed and measured by flow cytometry. Viable cells were both Annexin-V and 7-AAD negative. AnnexinV+/7-AAD- cells reflected early apoptosis. Late-apoptotic cells were positive for AnnexinV and 7-AAD. Cells were measured on an BD FACSFortessa (BD Biosciences, San Diego, USA).

Pharmacokinetics

All experimental procedures were approved by and conducted in accordance with the regulations of the local Animal Welfare authorities (Landesamt für Gesundheit and Verbraucherschutz, Abteilung Lebensmittel- and Veterinärwesen, Saarbrücken). The pharmacokinetics studies were performed by CRO Pharmacelsus GmbH (Saarbrücken Germany). The studies were performed in adult male C57Bl/6N mice (weight range 24–27 g). T-10418 was dissolved in PBS and applied either iv or sc. All animals showed a normal behavior and there were no clinical signs observed after dosing. The plasma concentration was determined via LC–MS.

Glossary

Abbreviations Used

βarr2

β-arrestin-2 also known as (nonvisual) arrestin-3 (arr-3)

CDS

coding DNA sequence

GPR132

G protein-coupled receptor 132

G2A

G2 accumulation protein

GPR4

G protein-coupled receptor 4

HETEs

hydroxyeicosatrienoic acids

HODEs

hydroxyoctadecadienoic acids

IP-1

inositol monophosphate

LPC

lysophosphatidylcholine

OGR1

ovarian cancer G protein-coupled receptor 1

OINP

oxaliplatin-induced neuropathic pain

ORF

open reading frame

PKC

protein kinase C

TDAG8

T-cell death-associated gene 8

TRPV1

transient receptor potential cation channel subfamily V member 1

Supporting Information Available

The Supporting Information is available free of charge at https://pubs.acs.org/doi/10.1021/acs.jmedchem.3c02164.

• IP-One assay representative curves; direct binding of compound 3 to G2A measured by LC-ESI-MS/MS; IP-One assay in leukemia cell lines MOLM13 and ML-2; CellTiter-Glo Tox assay for compounds 1 and 3; Eurofins SafetyPanel 44 results; chiral HPLC chromatograms and HPLC traces of key compounds; And synthesis methods and characterization of intermediate compounds (PDF)

• Molecular formula strings (CSV)

Author Contributions

VHO and JH contributed equally to the manuscript. The manuscript was written through contributions of all authors. All authors have given approval to the final version of the manuscript.

The authors declare the following competing financial interest(s): Y.S.M. is a CEO at Chemspace LLC and a scientific advisor at Enamine Ltd. V.V.I. is a Chief Sales Officer at Enamine Ltd.