Discovery of LY3104607: A Potent and Selective G Protein-Coupled Receptor 40 (GPR40) Agonist with Optimized Pharmacokinetic Properties to Support Once Daily Oral Treatment in Patients with Type 2 Diabetes Mellitus

Article abstract of DOI:10.1021/acs.jmedchem.7b01411

As a part of our program to identify potent GPR40 agonists capable of being dosed orally once daily in humans, we incorporated fused heterocycles into our recently disclosed spiropiperidine and tetrahydroquinoline acid derivatives 1, 2, and 3 with the intention of lowering clearance and improving the maximum absorbable dose (Dabs). Hypothesis-driven structural modifications focused on moving away from the zwitterion-like structure. and mitigating the N-dealkylation and O-dealkylation issues led to triazolopyridine acid derivatives with unique pharmacology and superior pharmacokinetic properties. Compound 4 (LY3104607) demonstrated functional potency and glucose-dependent insulin secretion (GDIS) in primary islets from rats. Potent, efficacious, and durable dose-dependent reductions in glucose levels were seen during glucose tolerance test (GTT) studies. Low clearance, volume of distribution, and high oral bioavailability were observed in all species. The combination of enhanced pharmacology and pharmacokinetic properties supported further development of this compound as a potential glucose-lowering drug candidate.

Full text of DOI:10.1021/acs.jmedchem.7b01411

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Article
Discovery of LY3104607: A Potent and Selective G Protein-Coupled Receptor
40 (GPR40) Agonist with Optimized Pharmacokinetic Properties to Support
Once Daily Oral Treatment in Patients with Type 2 Diabetes Mellitus
Chafiq Hamdouchi, Pranab Maiti, Alan M. Warshawsky, Amy C. DeBaillie, Keith A. Otto, Kelly
L. Wilbur, Steven D. Kahl, Anjana Patel Lewis, Guemalli R. Cardona, Richard W. Zink, Keyue
Chen, Siddaramaiah CR, Jayana P. Lineswala, Grace L. Neathery, Cecilia Bouaichi, Benjamin
A. Diseroad,, Alison N Campbell, Stephanie A. Sweetana, Lisa A Adams, Over Cabrera, Xiaosu
Ma, Nathan P. Yumibe, Chahrzad Montrose-Rafizadeh, Yanyun Chen, and Anne Reifel Miller
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.7b01411 • Publication Date (Web): 13 Dec 2017
Downloaded from http://pubs.acs.org on December 13, 2017
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Discovery of LY3104607: A Potent and Selective G Protein-Coupled Receptor 40
(GPR40) Agonist with Optimized Pharmacokinetic Properties to Support Once Daily
Oral Treatment in Patients with Type 2 Diabetes Mellitus
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Chafiq Hamdouchi,*,a Pranab Maiti,b Alan M. Warshawsky,a Amy C. DeBaillie,a Keith A. Otto,a Kelly L.
16 Wilbur,a Steven D. Kahl,a Anjana Patel Lewis,a Guemalli R. Cardona,a Richard W. Zink,a Keyue Chen,a
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Siddaramaiah CR,b Jayana P. Lineswala,a Grace L. Neathery,a Cecilia Bouaichi,a Benjamin A. Diseroad,a
Alison N. Campbell,a Stephanie A. Sweetana,a Lisa A. Adams,a Over Cabrera,a Xiaosu Ma,a Nathan P.
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Yumibe,a Chahrzad MontroseꢀRafizadeh,a Yanyun Chen,a Anne Reifel Millera
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aLilly Research Laboratories, A division of Eli Lilly and Company, Lilly Corporate Center, DC: 0540,
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Indianapolis, IN 46285
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bJubilant Biosys Research Center, Bangalore, India
ABSTRACT:
Insert Abstract Figure here
As part of our program to identify potent GPR40 agonists capable of being dosed orally once daily in
humans, we incorporated fused heterocycles into our recently disclosed spiropiperidine and
tetrahydroquinoline acid derivatives 1, 2 and 3 with the intention of lowering clearance and improving the
maximum absorbable dose (Dabs). Hypothesisꢀdriven structural modifications focused on moving away from
the zwitterionꢀlike structure and mitigating the Nꢀdealkylation and Oꢀdealkylation issues led to
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triazolopyridine acid derivatives with unique pharmacology and superior pharmacokinetic properties.
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Compound 4 (LY3104607) demonstrated functional potency and glucose dependent insulin secretion (GDIS)
in primary islets from rats. Potent, efficacious, and durable dose dependent reductions in glucose levels were
seen during glucose tolerance test (GTT) studies. Low clearance and volume of distribution and high oral
bioavailability were observed in all species. The combination of enhanced pharmacology and
pharmacokinetic properties supported further development of this compound as a potential glucoseꢀlowering
drug candidate.
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INTRODUCTION
Type 2 diabetes mellitus (T2DM) has become a serious global health care problem with an estimated
422 million adults living with diabetes worldwide in 2014, compared to 108 million in 19801. While several
oral treatments for T2DM are available, some are limited by undesired side effects such as hypoglycemia,
liver damage, gastrointestinal symptoms, and weight gain. Therefore, alternative therapeutics with novel
mechanisms are desired. The G proteinꢀcoupled receptor GPR40 is highly expressed in pancreatic βꢀcells.
Upon binding endogenous medium and long chain unsaturated fatty acids, GPR40 promotes insulin secretion
only when glucose levels are elevated2ꢀ6. By this glucose dependent insulin secretion (GDIS) mechanism,
GPR40 offers an attractive target toward efficacious and safe T2DM therapies. Although the GPR40
mechanism of action is not completely understood, GPR40 has been reported to interact predominantly with
the G protein αꢀsubunit of the Gq family (Gαq).7ꢀ9 In this signaling cascade, activation of Gαq proteinꢀ
coupled receptors triggers an increase in phospholipase C (PLC) activity. Subsequent inositol 1,4,5ꢀ
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linked to increased insulin secretion from pancreatic βꢀcells10ꢀ13. Thus, small molecule GPR40 agonists have
been targeted to treat T2DM.7ꢀ8 Synthetic GPR40 agonists have been reported to stimulate insulin secretion
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in a glucoseꢀdependent manner and to correct impaired glucose tolerance in rodents.14ꢀ25 Recently, we
reported the discovery and development of 1 (LY2881835), 2 (LY2922083) and 3 (LY2922470) as potent
and selective GPR40 agonists. Compound 3 was taken into a 28ꢀday multiple ascending dose (MAD) study
in patients with T2DM. This clinical study provided proof of concept for 3 as a potential glucoseꢀlowering
therapy.26 However, the duration of the pharmacodynamic (PD) effect of 3 did not allow for QD dosing.
Toward this goal, subsequent discovery efforts focused on identifying compounds with lower predicted
human clearance and higher fraction of dose absorbed. Metabolic pathways in hepatocytes were identified
during our previous efforts; namely, oxidation at multiple sites, dealkylation (N and O), and conjugation
(glucuronide and taurine). Therefore, hepatocyte clearance measurements were important to the design and
prioritization of new compounds. Herein, we describe the identification of triazolopyridine acid derivatives
as selective GPR40 agonists with unique pharmacology and superior pharmacokinetic (PK) properties
relative to our previously developed compounds. Through optimization of key variables that influence free
drug concentration after oral administration, such as fraction of dose absorbed and intrinsic clearance,
compound 4 was identified as a suitable candidate for clinical study.
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CHEMISTRY
The syntheses of [1,2,4]triazolo[1,5–a]pyridine acid derivatives are detailed in Schemes 1 and 2.
Condensation of 6ꢀaminopyridineꢀ3ꢀcarboxylate 5 with ethyl Nꢀ(thioxomethylene)cabamate followed by
cyclization in the presence of hydroxylamine hydrochloride yielded the 2ꢀaminoꢀ[1,2,4]triazolo[1,5ꢀ
a]pyridine 7 (Scheme 1). The amino group was converted into the bromide 8 using Sandmeyer reaction
conditions. Reduction of the ester using excess DIBALꢀH followed by treatment with thionyl chloride
afforded chloride 9. Reaction of [1,2,4]triazolo[1,5–a]pyridine 9 with (S)ꢀethyl 3ꢀ(4ꢀhydroxyphenyl)hexꢀ4ꢀ
ynoate 10 in the presence of cesium carbonate afforded 11. This bromide was coupled with boronic acids
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under SuzukiꢀMiyaura conditions to give the corresponding esters 12a-c. Hydrolysis of the esters 12a and
12b in the presence of potassium trimethylsilanoate or 5N NaOH afforded the final [1,2,4]triazolo[1,5–
a]pyridine acid derivatives 13a and 13b. Alternatively, alkylation of the phenol 12c under basic conditions
using cesium carbonate followed by ester hydrolysis of the ester yielded carboxylic acids 15a-c.
Insert Scheme 1 here
Compounds 4 and 27b (Scheme 2) were synthesized by a slightly different route. Amino 2,4,6ꢀ
trimethylbenzenesulfonate 19 was prepared by reaction of the oxime 16 with the arylsulfonyl chloride 17 and
subsequent hydrolysis of oxime sulfonate 18 in the presence HClO4. Compound 19 was reacted with 6ꢀ
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20. Treatment with aldehydes 21a and 21b effected triazolo[1,5–a]pyridine ring formation yielding 22a and
22b, respectively. The phenol group of 22b was alkylated under basic conditions to give methanesulfonylꢀ
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propoxy triazolopyridine 23b. Reduction of esters 22a and 23b with excess DIBALꢀH followed by reaction
with PBr3 yielded bromides 25a and 25b. Reaction of these bromides with (S)ꢀethyl 3ꢀ(4ꢀ
hydroxyphenyl)hexꢀ4ꢀynoate 10 in the presence of cesium carbonate afforded the corresponding coupling
products 26a and 26b. Finally, ester hydrolysis in the presence of 5N NaOH afforded the desired
[1,2,4]triazolo[1,5–a]pyridine acids 4 and 27b.
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Insert Scheme 2 here
This discovery chemistry route was acceptable to deliver gram quantities of 4. Due to the safety
concerns associated with compound 1927 an alternative approach was desired to provide kilogram quantities
to support toxicological and clinical studies (Scheme 3). The process chemistry synthesis of 4 began with
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compound 8, which was prepared as described in Scheme 1 with optimization of reagent stoichiometry,
solvent volumes, operating temperatures and reaction times. The bromide was coupled with (2,6ꢀ
dimethylphenyl)magnesium bromide (28) under Negishi conditions to give ester 29, which was reduced with
DIBALꢀH to yield alcohol 30. Treatment of alcohol 30 with thionyl chloride provided chloride 31. Reaction
of 31 with (S)ꢀethylꢀ3ꢀ(4ꢀhydroxyphenyl)hexꢀ4ꢀynoate 10 in the presence of potassium carbonate afforded
ethyl ester 26a. Ester hydrolysis of 26a in the presence of NaOH afforded 4, which was recrystallized from
ethanol and nꢀheptane to provide the desired polymorph of compound 4 in a 56% overall yield from
compound 8 with 99.8% purity and 99.5% chiral purity. Xꢀray crystallography data confirmed the isomer
was of the R configuration.
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Insert Scheme 3 here
RESULTS AND DISCUSSION
Since GPR40 is a Gαqꢀcoupled receptor, in vitro functional activity of our compounds was initially
determined with a calcium flux primary assay using Fluorescence Imaging Plate Reader (FLIPR) technology.
The calcium flux measurements were useful to characterize these new compounds as partial agonists with
efficacies comparable to LY compounds 1ꢀ3. However, these data did not correlate with subsequent in vivo
activities.26 Therefore, we examined the connection between in vivo results and data from a number of in
vitro assays. The best in vitro to in vivo correlation was noted between βꢀarrestin signaling and glucose
lowering, most likely driven by receptor binding kinetics and the equilibrium properties of the β ꢀarrestin
assay.26 With a longer compound incubation time, this nonꢀGꢀprotein mediated signaling assay was used to
drive structure activity relationship (SAR) efforts. Additionally, competitive radioligand binding assays
confirmed that these compounds bind specifically to the allosteric 1 site like compounds 1ꢀ3. Affinities (Ki)
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of compounds in Tables 1were determined by measuring competitive inhibition of the tritiated Fasiglifam
([3H]ꢀTAK875) binding to membranes prepared from cells overexpressing recombinant human GPR40.26
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In the current effort, we sought to improve the overall PK profiles of our potent GPR40 agonists
primarily by minimizing the risk of O and Nꢀdealkylation and by improving solubility. Having established
the Sꢀβꢀ1ꢀpropynyl benzenepropanoic acid as an optimal headpiece in previous efforts by us26 by us and
others,20, 23 we turned our attention toward variations to the central linker and the hydrophobic tail. A number
of heterocycles were evaluated at the central linker region. Fused heterocycles such as 2ꢀphenylꢀ
[l,2,4]triazolo[1,5ꢀa]pyridines proved to be the most interesting (Table 1). In the tail region, phenyls
substituted with mediumꢀsized lipophilic groups led to compounds with potent binding and functional
activity, such as 4 with a 2,6ꢀdimethyl phenyl group. The overall physicochemical properties of these ligands
could be modulated effectively through modification of this tail group. Efforts to increase the polar surface
area (PSA) to improve metabolism and solubility parameters resulted in 13a, 15a, 15b and 7b (tPSA = 101,
95, 106, and 129, respectively) with only slightly decreased βꢀarrestin activity.
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Triazolopyridines with various substitutions at the 2 position showed good in vitro activity across
species using human and rat GPR40 assays (Table 1). All GPR40 compounds tested in an equilibrium
dialysis assay were highly protein bound in plasma (> 99%). However, no significant shift in potency was
noted in a modified Ca2+ mobilization assay with 0.1% of serum albumin added. Since plasma protein
binding (PPB) shifts in these in vitro assays did not predict the in vivo outcomes, SAR efforts did not try to
increase free drug fraction (fu). Instead, structural modifications focused on optimizing the key variables that
influence free drug concentration after oral administration, namely fraction of dose absorbed and intrinsic
clearance.28 Finally, the incorporation of the triazolopyridine moiety into these compounds minimized the
risk of PPAR crossꢀreactivity. Compounds in Table 1 demonstrated a lack of significant PPAR binding or
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functional activity when tested at concentrations up to 30 ꢀM. These compounds were also inactive in an
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adipogenesis assay in 3T3ꢀL1 cells.
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From this set of triazolopyridineꢀbased GPR40 ligands, 4 emerged as a candidate for further
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characterization.
Insert Table 1 here
As shown in Table 3, 4 demonstrated significant improvement in physicochemical and
biopharmaceutical properties relative to our previous compounds for development (1ꢀ3). Compound 4
showed good solubility in a DMSO/aqueous precipitation assay (last soluble concentration >100 ꢀM, pH =
7.4) and the most stable developable form of the crystalline free acid anhydrate exhibited very good water
solubility (0.173 mg/mL, pH = 8.1). Preclinical absorption modeling of rat and dog PK data supported a high
effective permeability in humans (calculated human passive permeability, cPeff = 5.20 x 10ꢀ4 cm/sec). The
maximum absorbable dose (Dabs, MiMBa modeling) for 4 of 593 mg in the fasted state and 919 mg in the
fed state was calculated using measured solubility and calculated human passive permeability; these values
were comparable to those for 3 and much higher than for 1 and 2. Under controlled particle size, clinical
absorption was predicted to be high (> 80% Fa) for doses up to ~350 mg.
From in vitro ADME studies, 4 showed minimal risk for potential drugꢀdrug interactions mediated by
CYP enzymes (IC50 values > 60 ꢀM against human CYP isoforms 3A4, 2D6, 2C9). The in vitro metabolism
of 4 was determined in rat, dog, and human cryopreserved hepatocytes. Plasma, urine, and bile samples from
rats and plasma and urine samples from dogs were used to characterize the in vivo metabolism of 4. Overall,
the in vitro data predicted the major metabolic pathways observed in vivo. Compound 4 was metabolized by
both CYP and nonꢀCYP metabolic processes in all species. In rat, oxidation to metabolite A was the
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prominent metabolic pathway, while in dog, direct glucuronide conjugation to form metabolite B
predominated (Figure 1). From human recombinant CYP phenotyping studies, the predominant metabolizing
CYP isoform was found to be CYP3A4 with minor involvement of CYP2J2. These data demonstrated
significant optimization in the metabolism of 4 compared to 1ꢀ3, which suffered from extensive O/Nꢀ
dealkylation and from oxidation at multiple sites. The [1,2,4]triazolo[1,5–a]pyridine linker in 4 lacks the
aromaticity believed to be responsible for benzylic oxidation and subsequent Oꢀdealkylation. As shown in
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Table 3, the human microsomal metabolic stability of 4 improved dramatically from our previous compounds
(5% metabolized for compound 4 vs 38, 44, and 24% for 1ꢀ3). Human microsomal and hepatocyte intrinsic
clearances were also dramatically lower for compound 4 (<0.0009 and 0.099 mL/min/kg, respectively).
These in vitro ADME data translated into a significant improvement in total clearances in preclinical animal
models and consequently into a much lower predicted human clearance (vida infra).
Insert Figure 1 here
Pharmacokinetic studies with 4 in Beagle dogs and Sprague Dawley rats are summarized in Table 2.
Compound 4 displayed low total clearances and low volumes of distribution in both species. Following oral
administration to fasted rats and dogs at doses of 1 and 3 mg/kg, respectively, 4 was rapidly absorbed (Tmax
< 1 hr) and showed high oral bioavailabilities, 93% (rats) and 83% (dogs). At higher oral doses, absorption
appeared to be delayed and less than proportional increases in exposure were observed from 10 to 500 mg/kg
in rats and from 30 to 300 mg/kg in dogs.
Insert Table 2 here
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Compound 4 had remarkably lower clearances (3 to17ꢀfold) in preclinical species compared to our
previous clinical candidates 1, 2, and 3 (Table 3). These significantly lower clearances were anticipated from
the higher metabolic stability of 4 across species and its improved physical properties. Introduction of
triazolopyridine linker fixed the O and Nꢀdealkylation, reduced the zwitterionic character, and increased the
polar surface area of our previous spiropiperidineꢀbased candidates 1-3.
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The PK data predicted 4 to have more sustained pharmacodynamic effect than 3. Human clearance
(CL) and volume of distribution (Vd) parameters were projected using the allometric scaling method. The
projected human clearance of 1 L/hr for 4 was substantially lower than the observed human clearance of 11ꢀ
14 L/hr for 3. The human PK profiles of 4 and 3 were simulated using projected PK parameters for 4 and
measured PK parameters from the SAD study for 3. Through this simulation, 4 is expected to show a smaller
peakꢀtoꢀtrough ratio than 3 (Figure 2 and Table 3). While no significant compound accumulation was
observed in the clinical study with 3, approximately 50% accumulation is predicted for 4 to provide better
coverage during later part of the day with QD dosing.
Insert Figure 2 here
As part of our optimization and selection process for the identification of preferred (potential) clinical
candidates, enhanced pharmacology and prolonged durability were key criteria. We examined the ability of 4
to induce insulin secretion in rat islets and provide durable glucose control during a chronic study in a rat
disease model. The target selectivity of 4 was assessed against over 100 different GPCRs, kinases, enzymes
and nuclear receptors and showed minimal activity (<50%) at 10 ꢀM concentration; no activity was noted
against the hERG channel at 100 ꢀM.
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Because activation of GPR40 is known to result in glucose dependent insulin secretion, primary islet
assays were developed to characterize compounds that increase intracellular calcium in the GPR40 primary
assays. While 4 did not enhance insulin secretion from primary rodent islets incubated in 2.8 mM glucose
(data not shown), significant increases in insulin secretion were produced by primary rat islets using static
culture conditions in 11.2 mM glucose (Figure 3).
Insert Figure 3 here
Oral glucose tolerance tests (OGTTs) in a rodent model of insulin resistance were used to assess the
pharmacodynamics effects (potency, efficacy, and durability) of our GPR40 agonists in male Zucker fa/fa
27 rats (10 weeks of age), glucose tolerances following an oral glucose challenge were determined 1 day and 21
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days after daily oral administration of 4 or positive control compound 1 (Figure 4). On days 1 and 21, the
area under the curves (AUCs) for glucose lowering were statistically significant for all doses of 4 tested (1, 3,
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and 10 mg/kg) and for the positive control. Weight and food consumption were not altered by any treatment
during the study (data not shown). The similar glucose lowering ED90 values for 4 on day 1 (4.1 mg/kg) and
on day 21 (5.0 mg/kg) suggest that GPR40 is not desensitized by 4 after 21 days of oral administration.
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Insert Figure 4 here
In these preclinical pharmacology studies, 4 demonstrated the robust potency, efficacy, durability and
selectivity to justify clinical testing. GDIS was shown in primary rat islets confirming the GPR40 agonist
mechanism of 4. Potent glucose lowering plus the lack of hypoglycemia during glucose tolerance tests in
rodents after chronic dosing reflect the efficacy, durability, and safety of this compound. Compound 4
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demonstrated an in vivo pharmacology similar to compound 3 (glucose lowering in Zucker fa/fa rats on day
21: ED90 = 5.0 mg/kg for 4 and 3.3 mg/kg for 3). The human efficacious dose of 88 mg [90% PI, 39ꢀ138]
once daily for 4 was projected busing Zucker fa/fa rats efficacious exposure (EAUC90), allometricallyꢀ
projected human clearance, and the projected bioavailability.
Insert Table 3 here
CONCLUSION
Through the GDIS mechanism GPR40 activation provides an excellent approach for developing
effective and safe therapies for the treatment of T2DM. Herein we have described our efforts culminating in
27 the identification of a potent GPR40 agonist suitable for once daily oral treatment in humans. Our
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hypothesisꢀdriven structural modifications focused on mitigating Nꢀdealkylation and Oꢀdelakylation issues
and on moving away from zwitterionꢀlike structures. We identified triazolopyridineꢀcarboxylic acid
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derivatives as GPR40 agonists with unique pharmacology, selectivity, and superior PK properties.
Compound 4 emerged as the leading molecule through optimization of key variables that influence free drug
concentration after oral administration; namely, fraction of dose absorbed and intrinsic clearance. Compound
4 demonstrated acceptable potency and efficacy in G proteinꢀ and βꢀarrestinꢀmediated signaling assays and
GDIS in primary islets from rats. Potent, efficacious, and durable doseꢀdependent reductions in glucose
levels were seen during glucose tolerance test (GTT) studies in insulinꢀresistant rats. In addition, high oral
bioavailability along with low clearance and volume of distribution were observed in all species. Compound
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once daily oral treatment in humans based on preclinical data. Therefore, on the basis of its excellent in vitro,
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ex vivo, and in vivo pharmacology and pharmacokinetic profile, 4 was advanced as a clinical candidate for
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the treatment of T2DM.
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EXPERIMENTAL SECTION
Chemistry. Unless otherwise noted, all materials were obtained from commercial suppliers and used as
15 obtained. Organic solvents were also purchased from commercial sources on scale, without additional
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purification. Reactions were monitored using Agilent 1100 or 1200 Series LCMS with ultraviolet light (UV)
detection at 215 and 254 nm and a low resolution electrospray mode (ESI). HRMS data were recorded on an
Agilent LCꢀMS TOF (timeꢀofꢀflight), Model G1969A instrument. Purity was measured using Agilent 1100
or 1200 Series high performance liquid chromatography (HPLC) with UV detection at 215, 254, and 280 nm
(15 min; 1.5 mL/min flow rate), eluting with a binary solvent system A and B using a gradient elution (A,
water with 0.1% TFA; B, MeCN with 0.1% TFA). Unless otherwise noted, the purity of all compounds was
≥98%. Enantiomeric excess for compounds bearing a stereogenic center were determined using analytical
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HPLC. HNMR spectra were recorded on a Bruker AVꢀ400 (400 MHz) spectrometer at ambient
temperature. Chemical shifts are reported in parts per million (δ) and are calibrated using residual signal
38 from undeuterated solvent as an internal reference. Data for 1H NMR spectra are reported as follows:
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chemical shift (δ ppm) (multiplicity, coupling constant (Hz), integration). Multiplicities are reported as
follows: s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet, br = broad, or combinations thereof.
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Differential scanning calorimetry (DSC) was performed on a TA Instruments Q100 calorimeter in an
aluminum Tzero pan under dry N2 flowing at 50 mL/min.
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We previously reported a patent application that described the discovery route to the synthesis and
characterization of all the compounds that we report in this manuscript (4, 13a, 13b, 15a, 15b, 15c and
27b).29 Herein we describe our process and development route to prepare the clinical candidate 4 in
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kilogram quantities. The process for the preparation of 4 required modifications of the discovery synthesis to
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make the route scalable.
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Process synthesis of [1,2,4]triazolo[1,5–a]pyridine acid 4
Methyl 6-(ethoxycarbonylcarbamothioylamino)pyridine-3-carboxylate (6). To a solution of methyl 6ꢀ
aminopyridineꢀ3ꢀcarboxylate (24.3kg, 160.1 mol) in THF (242 L) was added ethyl Nꢀ
(thioxomethylene)carbamate (25.6 kg, 195.2 mol, 1.2 equiv). The reaction was heated to 45 to 50 °C and
stirred for 15 hr. The reaction was cooled to 20 to 30 °C, concentrated, and solvent exchanged into MTBE.
The resulting solids were filtered, rinsed with MTBE (39 L), and dried at 40 to 50 °C to provide the title
compound as a yellow solid (41.4 kg, 91%). MS (ES+) [M + H]+: calcd, 283.1; found, 283.2. 1H NMR (400
MHz, DMSO, δ): 12.4 (bs, 1H), 12.0(bs, 1H), 8.9(d, J= 2.4 Hz, 1H), 8.75 (b, 1H), 8.38 (d, J= 2.4, 8.8 Hz,
1H), 4.24 (q, J= 7.1 Hz, 2H), 3.88 (s, 3H), 1.28 (t, J= 7.1 Hz, 3H).
Methyl 2-amino-[l,2,4]triazolo[l,5-a]pyridine-6-carboxylate (7). To a 40 to 45 °C solution of
Hydroxylamine hydrochloride (16.6 kg , 238.9 mol, 1.6 equiv ) and methyl 2ꢀaminoꢀ[1,2,4]triazolo[1,5ꢀ
a]pyridineꢀ6ꢀcarboxylate (41.4 kg, 146.5 mol) in ethanol (827 L) was added diisopropylethylamine (38.8 kg,
300 mol, 2.05 equiv ). The reaction was warmed to 55 to 60 °C and stirred for 8 hr. The reaction was cooled
to 30 to 40 °C and concentrated. The resulting solid was filtered and slurried in water (216 L) at 20 to 30 °C
for 3 hr. The solids were filtered and dried at 45 to 55 °C to provide the title compound as a white solid (26.8
kg, 96%). MS (ES+) [M + H]+: calcd, 192.1; found, 192.2.1H NMR (400 MHz, DMSO, δ): 9.02 (d, J = 1.0
Hz, 1H), 7.85 (dd, J = 1.7, 9.2 Hz, 1H), 7.41 (d, J = 9.3 Hz, 1H), 6.38 (s, 2H), 3.87 (s, 3H).
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Methyl 2-bromo-[ 1,2,4]triazolo [ 1,5 -a]pyridine-6-carboxylate (8). To a stirred solution of cupric bromide
(60.0 kg, 280 mol, 1.9 equiv) in acetonitrile (267 L) was added /tertꢀbutyl nitrite (27.1 kg, 280 mol, 1.9
equiv). The reaction mixture was heated to 45 to 55 °C. Methyl 2ꢀaminoꢀ[l,2,4]triazolo[l,5ꢀa]pyridineꢀ6ꢀ
carboxylate (26.8 kg, 140 mol) was added portion wise and the reaction mixture was heated at 45 to 55 °C
for 2 hr. The reaction mixture was cooled to 20 to 30 °C and quenched with water (605 L). The resulting
suspension was filtered through celite (5kg) and the layers were separated. The organic layer was washed
with water (214 L), concentrated and solvent exchanged into ethyl acetate. nꢀHeptane (302 L) was added
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dropwise and the resulting solids were filtered, washed with nꢀHeptane (48 L) and slurried with celite (4 kg)
and DCM (537 L) at 20 to 30 °C for 30 min. The suspension was filtered over silica gel (28 kg) and the
filtrate was concentrated and then diluted with EtOAc (296 L). The EtOAc solution was concentrated and nꢀ
heptane (315 L) was added dropwise. The resulting solids were filtered, washed with nꢀheptane (37 L) and
dried at 50 to 55 °C to give the title compound as an off white solid (22.6 kg, 63%). LCMS m/z [M + H]+:
calcd, 256.0; found, 256.0. 1H NMR (400 MHz, DMSO, δ): 9.49 (dd, J = 0.9, 1.6 Hz, 1H), 8.13 (dd, J = 1.7,
9.4 Hz, 1H), 7.92 (dd, J = 0.8, 9.4 Hz, 1H), 3.93 (s, 3H).
Methyl 2-(2,6-dimethylphenyl)-[1,2,4]triazolo[1,5-a]pyridine-6-carboxylate (29). To an argon inerted
mixture of methyl 2ꢀbromoꢀ[ 1,2,4]triazolo [ 1,5 ꢀa]pyridineꢀ6ꢀcarboxylate (39.0 kg, 152.3 mol) and zinc
chloride (25.1 kg, 1.2 equiv) in THF (24 L) was added Pd (Ph3)4 (9.4 kg, 0.05 equiv). The mixture was
heated to 50 to 55 °C. To the mixture was added a THF solution of (2,6ꢀdimethylphenyl)magnesium bromide
(28) (251 kg with 20.2% assay, 228.99 mol, 1.5 equiv). The mixture was stirred at 50 – 55 °C for 2 to 3 hr
and then cooled to 20 to 30 °C. The reaction mixture was quenched with an aqueous 10% NH4Cl solution
(207 L). The mixture was solvent exchanged into MTBE and then filtered through celite (10 kg). The layers
were separated, and the organic layer was washed with 10% NH4Cl solution (234 L), concentrated and
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solvent exchanged into DCM to provide the title compound as a solution in DCM that was used directly in
the next step (513 kg, 7.9% assay). LCMS m/z [M + H]+: calcd, 281.3; found, 281.0. 1H NMR (400.13 MHz,
DMSO, δ): 9.54 (s, 1H), 8.10 (dd, J = 1.7, 9.3 Hz, 1H), 7.98 (d, J = 9.4 Hz, 1H), 7.31 (t, J = 7.6 Hz, 1H), 7.19
(d, J = 7.6 Hz, 2H), 3.94 (s, 3H), 2.12 (s, 6H).
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[2-(2,6-Dimethylphenyl)-[1,2,4]triazolo[1,5-a]pyridin-6-yl]methanol (30). To a ꢀ15 to ꢀ5 °C solution of
methyl 2ꢀ(2,6ꢀdimethylphenyl)ꢀ[1,2,4]triazolo[1,5ꢀa]pyridineꢀ6ꢀcarboxylate (513 kg as a 7.9% assay) in
DCM was added DIBALꢀH (1.5 M solution toluene, 194 kg, 366.68 mol, 2.4 equiv). The reaction mixture
was warmed to ꢀ5 to 0 °C and stirred for 2 hr. To the reaction mixture was charged DCM (340 L) and water
(13.9 L). The mixture was stirred for 15 – 30 min and then a 15% aqueous solution of NaOH (16 L) was
added followed by additional water (33 L). The reaction mixture was warmed to 25 to 30 °C and was stirred
for 2ꢀ3 hr. The mixture was filtered through celite (10 kg) and the cake was washed with DCM (302 L). The
filtrate was concentrated and a 10% potassium sodium tartrate solution (420 L) was added. The layers were
separated and the organic layer was washed 2x with 2N HCl (410 L and 200 L, respectively). The combined
aqueous layers were treated with 20% aqueous sodium carbonate solution (448 L). The resulting solid was
filtered, washed with water (40 L), and dried under vacuum at 45 to 50 °C to provide the title compound as a
yellow solid (30.8 kg, 80% over two steps). LCMS m/z [M + H]+: calcd, 253.3; found, 253.0. 1H NMR
(400.13 MHz, DMSO, δ): 8.85 (s, 1H), 7.85 (d, J = 9.2 Hz, 1H), 7.67 (dd, J = 1.4, 9.1 Hz, 1H), 7.29 (t, J =
7.6 Hz, 1H), 7.17 (d, J = 7.7 Hz, 2H), 5.57ꢀ5.48 (m, 1H), 4.62 (s, 2H), 2.10 (s, 6H).
6-(Chloromethyl)-2-(2,6-dimethylphenyl)-[1,2,4]triazolo[1,5-a]pyridine (31). To a ꢀ5 to 0 °C solution of
thionyl chloride (33.0kg, 277.4 mol, 2.3 equiv) in THF (167 L) was added [2-(2,6-dimethylphenyl)-
[1,2,4]triazolo[1,5-a]pyridin-6-yl]methanol (30.6 kg, 121.0 mol) in THF (326 L). The solution was warmed
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to 20 to 30 °C and stirred for 2 hr. The reaction mixture was concentrated and solvent exchanged into nꢀ
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Heptane. The solid was filtered, washed with nꢀheptane (47 L) and dried at 35 °C to yield the title compound.
The title compound was slurried in water (297 L) at 20 ꢀ30 °C, filtered, washed with water (31 L), and dried
at 35 to 45 °C to give the title compound as an offꢀwhite solid (29.1 kg, 88%). LCMS m/z [M + H]+: calcd,
271.7; found, 271.1. 1H NMR (400.13 MHz, DMSO, δ): 9.19 (s, 1H), 7.92 (d, J = 9.2 Hz, 1H), 7.78 (dd, J =
1.6, 9.2 Hz, 1H), 7.30 (t, J = 7.6 Hz, 1H), 7.17 (d, J = 7.6 Hz, 2H), 4.95 (s, 2H), 2.10 (s, 6H).
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Ethyl (3 S)-3 -[4- [ [2-(2,6-dimethylpheny 1)- [ 1,2,4]triazolo[ 1,5-a]pyridin-6-
yl]methoxy]phenyl]hex-4-ynoate (26a). To a solution of (S)ꢀethyl 3ꢀ(4ꢀhydroxyphenyl) hexꢀ4ꢀynoate (29.0
kg, 106.78 mol) and 6ꢀ(Bromomethy1)ꢀ2ꢀ(2,6ꢀdimethylphenyl)ꢀ [l,2,4]triazolo[l,5ꢀa]pyridine (25.8 kg,
111.18 mol, 1.04 equiv ) in DMF (167 L) was added potassium carbonate (29.8 kg, 215.94 mol, 2.02 equiv).
The mixture was heated to 35 °C ꢀ45 °C and was stirred for 20 hr. The reaction mixture was cooled to 20 °C
ꢀ25 °C and EtOAc (336 L) and water (502 L) was added. The layers were separated, and the organic layer
was washed with water (170 L). The organic layer was concentrated and the solvent exchanged into ethanol
to give the title compound as a solution in ethanol that was used directly in the next step. LCMS m/z [M +
H]+: calcd, 467.5; found, 467.2. 1H NMR (399.45 MHz, CDCl3, δ): 8.73 (d, J = 0.6 Hz, 1H), 7.89 (d, J = 9.1
Hz, 1H), 7.64 (dd, J = 1.7, 9.1 Hz, 1H), 7.34ꢀ7.31 (m, 2H), 7.27ꢀ7.23 (m, 1H), 7.11 (d, J = 7.5 Hz, 2H), 6.96ꢀ
6.94 (m, 2H), 5.14 (s, 2H), 4.15ꢀ4.08 (m, 3H), 2.77ꢀ2.61 (m, 2H), 2.17 (s, 6H), 1.81 (d, J = 2.4 Hz, 3H), 1.20
(t, J = 7.1 Hz, 3H).
(3S)-3-[4-[[2-(2,6-Dimethylphenyl)-[l,2,4]triazolo[l,5-a]pyridin-6-yl]methoxy]phenyl]hex-4-ynoic acid (4).
To a solution of ethyl (3S)ꢀ3ꢀ[4ꢀ[[2ꢀ(2,6ꢀdimethylphenyl)ꢀ[l,2,4]triazolo[l,5ꢀa]pyridinꢀ6ꢀ
yl]methoxy]phenyl]hexꢀ4ꢀynoate (106.8 mol) in ethanol was added aqueous 10% NaOH (78 L, 195 mol,
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1.83 equiv) drop wise at a rate to maintain the reaction temperature below 30 °C. The reaction mixture was
stirred for 4 hr and then concentrated. Water (333 L) was added, and the reaction mixture was concentrated.
MTBE (170 L) was charged, and the layers were separated. EtOAc (352 L) was charged to the aqueous layer
and the pH was adjusted to 4ꢀ5 with 1M HCl (234 L) at 15 – 20 °C. The layers were separated and the
combined organic layers were washed with water (164 L), filtered through a CUNO filter, and concentrated.
The reaction mixture was heated to 70ꢀ80 °C until a clear solution was obtained and then cooled to 65ꢀ75 °C.
nꢀHeptane (409 L) was charged dropwise to maintain an internal temperature of 65ꢀ75 °C, and then the
solution was slowly cooled to 5 °C. The resulting solids were filtered and washed with nꢀHeptane (91 L) and
dried under vacuum at 40ꢀ45 °C to yield the title compound (42.15 kg, 88% over two steps). To the title
compound (40.0 kg, 89.46 mol) was added ethanol (247 L), and the slurry was heated to 60ꢀ65 °C until a
clear solution was obtained. The resulting solution was cooled to 45ꢀ55 °C and compound 4 (0.217 kg) was
added. The solution was slowly cooled to 20ꢀ25 C, and then nꢀHeptane (724 L) was added dropwise. The
slurry was cooled to 5ꢀ10 °C and allowed to stir for 2ꢀ3 hr. The solids were filtered, washed with nꢀHeptane
(118 L), and dried under vacuum at 45ꢀ50 °C to yield the title compound as white solid (35.76 kg, 91%).
LCMS m/z [M + H]+: calcd, 439.5; found, 439.2. 1H NMR (399.80 MHz, DMSO, δ): 12.22 (s, 1H), 9.13 (dd,
J = 0.8, 1.5 Hz, 1H), 7.88 (dd, J = 0.8, 9.2 Hz, 1H), 7.75 (dd, J = 1.7, 9.2 Hz, 1H), 7.29ꢀ7.24 (m, 3H), 7.14ꢀ
7.12 (m, 2H), 7.01ꢀ6.99 (m, 2H), 5.18 (s, 2H), 3.96ꢀ3.91 (m, 1H), 2.58 (d, J = 7.7 Hz, 2H), 2.06 (s, 6H), 1.75
(d, J = 2.4 Hz, 3H).
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Crystalline form of compound 4 and X-ray structure characterization. (3S)ꢀ3ꢀ[4ꢀ[[2ꢀ(2,6ꢀDimethylphenyl)ꢀ
[l,2,4]triazolo[l,5ꢀa]pyridinꢀ6ꢀ yl]methoxy]phenyl]hexꢀ4ꢀynoic acid 4 was be prepared as a crystalline
anhydrous form by dissolving (3S)ꢀ3ꢀ[4ꢀ[[2ꢀ(2,6ꢀDimethylphenyl)ꢀ[l,2,4] triazolo[l,5ꢀa]pyridinꢀ6ꢀ
yl]methoxy]phenyl]hexꢀ4ꢀynoic acid (580 mg, 132 mmol) in EtOH (1.2 mL) with stirring at 80 °C for 10
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min. The solution was filtered and cooled to 70 °C, and treated with seeds. The mixture was cooled slowly
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to ambient temperature while stirring overnight. The resulting solid was triturated with heptane and the solids
are recovered by vacuum filtration and dried under vacuum at 60 °C to give the crystalline title product (438
mg, 75.5%). The XRD patterns of crystalline solids were obtained on a Bruker D4 Endeavor Xray powder
diffractometer, equipped with a CuKa source (λ = 1.54060 A) and a Vantec detector, operating at 35 kV and
50 mA. The sample was scanned between 4 and 40° with a step size of 0.0087° and a scan rate of 0.5
seconds/step, and with 0.6 mm divergence, 5.28mm fixed antiꢀscatter, and 9.5 mm detector slits. The dry
powder was packed on a quartz sample holder and a smooth surface was obtained using a glass slide.
Confirmation of a crystal form may be made based on any unique combination of distinguishing peaks (in
units of ° 20), typically the more prominent peaks. The crystal form diffraction patterns collected at ambient
temperature and relative humidity were adjusted based on NIST 675 standard peaks at 8.85 and 26.77
degrees 2ꢀtheta. A prepared sample of (3S)ꢀ3ꢀ[4ꢀ[[2ꢀ(2,6ꢀDimethylphenyl)ꢀ[l,2,4]triazolo[l,5ꢀa]pyridinꢀ6ꢀ
yl]methoxy]phenyl]hexꢀ4ꢀynoic acid 4 was characterized by an XRD pattern using CuKa radiation as having
diffraction peaks (2ꢀtheta values). The pattern contains a peak at 17.55 in combination with one or more of
the peaks selected from the group consisting of 5.82, 10.78, 19.65, 21.31, and 24.33 with a tolerance for the
diffraction angles of 0.2 degrees.
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Biological Methods. All in vitro assays including, binding, calcium flux, βꢀarrestin agonist and peroxisome
proliferatorꢀactivated receptor (PPAR) α, δ, γ assays were described previously in detail26. The use of
animals was in accordance with international guidelines (NIH 85ꢀ23) and was approved by the local animal
ethics committee at Lilly Research Laboratories.
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Glucose Dependent Insulin Secretion (GDIS) in Rat Islet: GDIS assays were performed in primary
islets. Pancreatic islets of Langerhans were isolated from male SD (Sprague Dawley) rats by collagenase
digestion and Histopaque density gradient separation. The islets were cultured overnight in RPMIꢀ1640
medium with GlutaMAXn to facilitate recovery from the isolation process. Insulin secretion was determined
using 90 min incubation in EBSS (Earle’s Balances Salt Solution) buffer in a 48ꢀwell plate. Islets were first
preincubated in EBSS with 2.8 mM glucose for 60 min then transferred to a 48ꢀwell plate (four islets/well)
containing 150 ꢀL 2.8 mM glucose, and incubated with 150 ꢀL of EBSS with 2.8 or 11.2 mM glucose in the
presence or absence of test compounds for 90 min. The buffer was removed from the wells at the end of the
incubation period and assayed for insulin levels using a rat insulin ELISA kit (Mercodia).
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Oral Glucose Tolerance Test (OGTT) in Zucker fa/fa Rats: OGTTs were performed in Male Zucker
fa/fa rats (10 weeks of age), a rodent model of insulin resistance, after 1 and 21 days of oral administered.
Compounds were administered orally at various doses to provide a dose response efficacy curve. Compound
1 served as the positive control and reference standard for the study. OGTTs were performed one hour after
36 compound administration with blood samples taken for determination of glucose and insulin levels at 0, 10,
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20, 40, and 60 minutes post glucose administration (2g/kg).
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ASSOCIATED CONTENT
Supporting information
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Molecular formula strings and the associated biochemical and biological data as a CSV file. This material is
available free of charge via the Internet at http://pubs.acs.org.
AUTHOR INFORMAITON
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Corresponding Author
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*Phone: 317ꢀ797ꢀ4751. Eꢀmail: hamdouchi_chafiq@lilly.com or chafiq.hamdouchi@gmail.com
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Author Contributions
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The manuscript was written through contribution of all authors. All authors have given approval to the final
version of the manuscript.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGEMENT
We thank the GPR40 team at both Lilly and Jubilant for their dedicated efforts. We also thank Dr. Ruth
Gimeno, Dr. Guoxin Zhu, Dr. Thomas Hardy and Dr, Timothy Grese for helpful discussions.
ABBREVIATIONS USED
GPR40, G proteinꢀcoupled receptor 40; FFAR1, Free Fatty Acid Receptor 1; GDIS, glucose dependent
insulin secretion; PK, pharmacokinetics; AUC, area under curve; CL, plasma clearance; T2DM, type 2
diabetes mellitus; SAR, structure activity relationship; PPAR, peroxisome proliferator activator receptor;
FLIPR, fluorescence imaging plate reader.
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(15) Burant, C. F.; Viswanathan, P.; Marcinak, J.; Cao, C.; Vakilynejad, M.; Xie, B.; Leifke, E. TAKꢀ875
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(26) Hamdouchi, C.; Kahl, S. D.; Patel Lewis, A.; Cardona, G. R.; Zink, R. W.; Chen, K.; Eessalu, T. E.;
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Scheme 1. Synthesis of [1,2,4]triazolo[1,5–a]pyridine acid derivatives (route A)a
5
6
7
8
9
O
O
O
N
N
O
N
7
O
S
a
O
b
O
H2
N
O
N
H
N
N
H
H2
N
N
10
11
12
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60
5
6
O
O
O
N
N
Cl
10
N
HO
N
N
c
O
d, e
N
Br
Br
f
9
8
O
O
OH
O
O
N
N
h
O
N
g
N
N
O
O
N
R
R
N
N
O
N
Br
11
N
R =
N
R =
12a
12b
13b
13a
HO
12c
O
O
O H
O
N
O
N
N
N
O
N
k
i
O
O
N
12c
A
A
O
O
F
O H
14a
O
A =
OH
F
O
O
A =
O
15c
15a
15b
14b
F
j
A =
F
14c
aReagents and conditions: (a) Ethyl Nꢀ(thiomethylene)carbamate, methyl 6 aminopyridineꢀ3ꢀcarboxyalte, 1:4 dioxane, rt, 7 h, 88%;
(b) Hydroxylamine hydrochloride, DIPEA, methanol, rt, 40 hr, 76%; (c) CuBr2, tꢀBuCN, 60 oC, 1h, 48%; (d) DCM, DIBALꢀH, ꢀ78
oC, 1h, 58%; (e) SOCl2, 0 oC to rt,1h, 100% (used without purification for the next step); (f) CsCO3, rt, 14h, 80%; (g) 4ꢀsubstituted
phenylboronic acids, K2CO3ꢀ2M, Pd(PPh3)2Cl2, microwave 100 oC, 4 to 12 h, 57ꢀ74%; (h) KOSiMe3, THF, 2h, rt, or NaOHꢀ5N,
EtOH, 80 oC, 30min; (i) Cs2CO3, ACN, rt, 2 to 12 h, 64 to 100%; (j) sodium chlorodifluroacetate, DMF, Cs2CO3, 80 oC, 4h, 56%;
(k) 5 N NaOH, ethanol, rt to 80 oC, 30 min to 16 h, 60ꢀ90%.
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Scheme 2. Synthesis of [1,2,4]triazolo[1,5–a]pyridine acid derivatives (route B)a
4
5
6
7
8
9
17
5
10
11
12
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c
a
b
16
18
19
21a,b
e
d
20
A = H, 22a
A = OH, 22b
Y= H
22a
23b
10
h
f, g
Y= H
25a
Y= H Y=
26a 26b
Y=
25b
l
Y= H
4
Y=
27b
aReagents and conditions: (a) DMF, TEA, rt, 16 h, 64%; (b) HClO4, 0 oC to rt, 1 h, 100% (used without purification for the next
step); (c) DCM, rt, 16 h, 41%; (d) TEA, methanol , rt 18ꢀ48 h, 18ꢀ40%; (e) K2CO3, ACN, 100 oC, 16 h, 70%; (f) DIBALꢀH DCM,
0 oC to rt, 2 h, 57%; (g) PBr3, DCM, rt, 2 h, 76%; (h) CsCO3, DMF, rt, 16 h, 57%; (i) NaOH, EtOH/H2O, rt, 16 h, 93 and 55%.
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Scheme 3. Process chemistry synthesis of [1,2,4]triazolo[1,5–a]pyridine acid 4 on 36 kg scalea
5
6
7
8
9
c
b
a
10
11
12
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60
7
5
6
28
d
f
e
8
29
30
h
10
g
26a
31
i
4
4
aReagents and conditions: (a) Ethyl Nꢀ(thiomethylene)carbamate, methyl 6 aminopyridineꢀ3ꢀcarboxyalte, THF, 45 to 50 oC, 15 h,
91%; (b) Hydroxylamine hydrochloride, DIPEA, ethanol, 55 to 60 oC, 8 hr, 96%; (c) CuBr2, ACN/tꢀBuCN, 45 to 50 oC, 1h, 63%;
(d) THF, ZnCl2, Pd(Ph3)4, 50ꢀ55 oC; (e) DCM, toluene, DIBALꢀH, ꢀ10 oC to ꢀ15 oC, 80% over two steps; (f) SOCl2, THF, 20 to
30oC, 88%; (g) DMF, K2CO3, 35ꢀ45 oC; (h) NaOH, EtOH, 25 oC; 88% over two steps; (i) EtOH, seed with compound 4; nꢀ
heptane; 91%.
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Table 1. In vitro activities of triazolopyridine phenyl propanoic acids
6
7
8
9
Human
GPR40
Rat
GPR40
β-arrestin 1% FBSc
Human
GPR40
Human
GPR40
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
Calculated
propertiese
β-arrestin 1% FBSb
Calciumd
Bindinga
Ki, nM
(SEM, n)
EC50, nM
(SEM, n)
% Stim Max
(SEM, n)
EC50, nM
(SEM, n)
% Stim Max
EC50, nM
(SEM, n)
% Stim Max
(SEM, n)
LSN
Cmpd
PSA
77
R
(SEM, n)
15
(4, n=4)
108
124
14
(5, n=9)
130
(6, n=9)
11
54
3104607
4
(34, n=23)
(6, n=23)
(7, n=3)
(10, n=3)
102
(12, n=3)
681
121
562
(273, n=5)
133
(14, n=5)
16
74
3199272
3199271
3199270
15a
13b
15b
95
77
(132, n=8)
(6, n=8)
(13, n=3)
(5, n=3)
32
67
154
30
(8, n=5)
123
(6, n=5)
2
65
(11.5, n=3)
(15, n=8)
(8, n=8)
(1, n=3)
(6, n=3)
37
(8, n=3)
301
132
68
(33, n=5)
121
(13, n=5)
72
89
106
(116, n=8)
(14, n=8)
(3, n=3)
(2, n=3)
63
(5, n=3)
1490
114
95
(31, n=5)
140
(5, n=5)
117
71
3156275
27b
129
(809, n=6)
(10, n=6)
(38, n=3)
(1, n=3)
35
(6, n=3)
443
124
216
(62, n=5)
125
(7, n=5)
9
65
3203801
3199273
13a
15c
101
86
(86, n=8)
(6, n=8)
(4, n=4)
(8, n=4)
60
(17, n=4)
166
127
98
(54, n=5)
142
(12, n=5)
1.0
64
(47, n=8)
(8, n=8)
(0.3, n=3)
(1, n=3)
aCompetitive inhibition with human GPR40 membranes.26 b,cβꢀarrestin recruitment with human and rat
GPR40 in the presence of 1% fetal bovine serum (FBS). dCalcium flux with human GPR40; the percent of
46 stimulation is relative to the 100 uM of the natural ligand, linoleic acid, response. ePSA values were
47
48
computed using Chemaxon software.
49
50
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