Design and Synthesis of Novel, Selective GPR40 AgoPAMs

Article abstract of DOI:10.1021/acsmedchemlett.6b00443

GPR40 is a G-protein-coupled receptor expressed primarily in pancreatic islets and intestinal L-cells that has been a target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion

Full text of DOI:10.1021/acsmedchemlett.6b00443

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Letter
Design and Synthesis of Novel, Selective GPR40 AgoPAMs
Christopher W. Plummer, Matthew J. Clements, Helen Chen, Murali Rajagopalan, Hubert Josien, William
K Hagmann, Michael W Miller, Maria E. Trujillo, Melissa Kirkland, Daniel Kosinski, Joel Mane, Michele
Pachanski, Boonlert Cheewatrakoolpong, Andrew F. Nolting, Robert K. Orr, Melodie Christensen, Louis-
Charles Campeau, Michael J. Wright, Randal Bugianesi, Sarah B. Souza, Xiaoping Zhang, Jerry Di Salvo,
Adam B. Weinglass, Richard Andre Tschirret-Guth, Ravi P Nargund, Andrew D. Howard, and Steven L. Colletti
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.6b00443 • Publication Date (Web): 23 Jan 2017
Downloaded from http://pubs.acs.org on January 24, 2017
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Design and Synthesis of Novel, Selective GPR40 AgoPAMs
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Christopher W. Plummer ,* Matthew J. Clements , Helen Chen , Murali Rajagopalan , Hubert Josien ,
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William K. Hagmann , Michael Miller , Maria E. Trujillo , Melissa Kirkland , Daniel Kosinski , Joel
d
d
d
b
b
Mane , Michele Pachanski , Boonlert Cheewatrakoolpong , Andrew F. Nolting , Robert Orr , Melodie
Christensen , LouisꢀCharles Campeau , Michael J. Wright , Randal Bugianesi , Sarah Souza , Xiaoping
Zhang , Jerry Di Salvo , Adam B. Weinglass , Richard TschirretꢀGuth , Ravi Nargund , Andrew D.
Howard , Steven L. Colletti
b
b
e
e
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e
e
e
c
a
d
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Departments of Discovery Chemistry, Process Chemistry, Drug Metabolism and Pharmacokinetics, In Vivo Pharmaꢀ
e
cology, In Vitro Pharmacology, Merck Research Laboratories, Kenilworth, NJ 07033, USA
KEYWORDS GPR40, FFA1, GPCR, diabetes, insulin secretogogue, agoPAM, chroman
ABSTRACT: GPR40 is a Gꢀproteinꢀcoupled receptor expressed primarily in pancreatic islets and intestinal Lꢀcells that has been a
target of significant recent therapeutic interest for type II diabetes. Activation of GPR40 by partial agonists elicits insulin secretion
only in the presence of elevated blood glucose levels, minimizing the risk of hypoglycemia. GPR40 agoPAMs have shown superiꢀ
or efficacy to partial agonists as assessed in a glucose tolerability test (GTT). Herein, we report the discovery and optimization of a
series of potent, selective GPR40 agoPAMs. Compound 24 demonstrated sustained glucose lowering in a chronic study of Goto
Kakizaki rats, showing no signs of tachyphylaxis for this mechanism.
Type II diabetes is characterized by an inability to maintain
glucose homeostasis due to insulin desensitization and insuffiꢀ
1
cient insulin secretion. GPR40 (FFAR1 or FFA1) is a Gꢀ
proteinꢀcoupled receptor that is primarily expressed in pancreꢀ2
atic beta cells, intestinal enteroendocrine cells, and the brain.
When activated by its endogenous ligands (medium/long chain
fatty acids), GPR40 elicits insulin secretion only in the presꢀ
ence of elevated glucose levels, making it a target of signifiꢀ
cant therapeutic interest due to a potentially reduced risk of
3
hypoglycemia.
Numerous groups have reported clinical candidates as novel
insulin secretagogues via GPR40 agonism as a potential treatꢀ
4ꢀ7
ment for diabetes. To date, all GPR40 modulators that have
been assessed in a clinical setting are partial agonists (Figure
8
ꢀ10
1
). Recently, researchers at Amgen
and BristolꢀMyersꢀ
11
Squibb have reported several GPR40 agonists that operate as
full agonists with positive allosteric modulation (effectively
1
2, 13
agoPAMs or agoꢀallosteric)
on the endogenous ligands
(e.g. DHA). GPR40 agoPAMs exhibit superior levels of recepꢀ
tor activation in vitro (soꢀcalled “full agonists”) and demonꢀ
strate superior in vivo efficacy versus the previously described
partial agonists. In addition to stimulating insulin secretion
via pancreatic action, GPR40 agoPAMs have been reported to
stimulate GLPꢀ1 secretion in the gut, potentially accounting
Figure 1. GPR40 “full agonists” and partial agonists.
1
4
for the observed enhancement in efficacy.
mationally constrained GPR40 agonists with acceptable physiꢀ
Given the promising level of glycemic control from the
agoPAM mode of GPR40 activation, it became our objective
to identify full agoPAM GPR40 modulators with clean offꢀ
target profiles in order to assess the risk of tachyphylaxis (deꢀ
sensitization) in a chronic setting. The medicinal chemistry
strategy deployed in order to achieve this objective was to
improve the offꢀtarget profiles through the design of conforꢀ
cal properties (Figure 2).
Researchers at Amgen have shown that partial agonists and
agoPAMs show differentiated levels of receptor activation in
transiently transfected CHO cells expressing relatively low
10
levels of hGPR40. The primary cellꢀbased assay used for
compound optimization measured
phate(IP1) accumulation in HEK293 cells stably expressing
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human GPR40 (hGPR40/HEK293). This cell line was deꢀ provethe physicochemical profile, we examined smaller, hetꢀ
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signed to clearly differentiate the efficacy of partial agonists
versus AgoPAM’s and assess the impact of plasma proteins on
potency in approaching 100% human serum (HS). Figure 3
shows that while the partial agonist AMG837 delivers a standꢀ
ardized receptor activation of 100%, the full agoPAM
AM1638 demonstrates approximately 400% activation.
Moreover, the potencies of AMG837 and AM1638 are right
shifted in the presence of HS.
eroatomꢀcontaining substituents such as nitrile and and
Table 1. Initial Identification of Chroman agoPAMs
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Figure 2. Medicinal chemistry tactics to identify GPR40 agoꢀ
PAMs with improved ligand efficiency
a
Mean of at least 2 runs.
triflouromethoxy analogs 22 and 23. While these modificaꢀ
tions improved physical properties, both resulted in a loss of
potency as compared to compounds 14 and 15.
Table 2. Exploration of B Ring SAR
Figure 3. AgoPAMs deliver increased % activation in IP1 assay.
We initiated our investigation into the structureꢀactivity reꢀ
lationship (SAR) of agoPAMs by examining the impact of
removing the dimethylcyclopentenyl moiety of AMꢀ1638 (1).
Interestingly, the potency of 7 (Table 1) was retainined, howꢀ
ever the human serumꢀshifted IP1 potency was greatly diminꢀ
ished. Next, in an effort to install conformational constraint,
we cyclized the phenyl propionic acid headpiece onto the benꢀ
zyl ether linker, making a chroman ring system (compounds 8
and 9, which are epimeric at the benzylic chroman position).
This resulted in an improvement in the 100% HS IP1 potency.
Removal of the dimethylcyclopentenyl moiety from this deꢀ
rivative resulted in compounds 10 and 11, which exhibited
improved potency and physical properties (lower molecular
weight and logD) relative to compounds 8 and 9 at the exꢀ
pense of decreased HSꢀshifted IP1 potency. In an effort to
further benchmark the impact of substituents, the 2ꢀflouroꢀ5ꢀ
methoxy phenyl and cyclopropyl moieties were removed, reꢀ
sulting in compounds 12 and 13, respectively. Both modificaꢀ
tions resulted in a significant loss of potency.
We next examined a variety of substituents at the meta posiꢀ
tion of the B ring. Incorporation of the methyl neopentoxy
1
0
fragment known in previous GPR40 agoPAMs led to a
marked improvement in potency and serum shift (Table 2,
cpds 14 and 15, which are diastereomeric at the benzylic
chroman position). Noting the high molecular weight and
HPLC logD of these derivatives, we removed the tꢀbutyl
fragment to give compounds 16 and 17. This resulted in a loss
of potency. Replacing the tꢀbutyl group with a cyclopropyl
group in 18, however, resulted in a log unit improvement in
logD while retaining potency. In an effort to further imꢀ
a
Mean of at least 2 runs.
Returning to the notion of conformational constraint, we
next examined the possibility of restricting the phenyl propiꢀ
onic acid headpiece to its active conformation by installing a
methyl group alpha to the carboxylic acid. We postulated that
vicinal stereocenters at the positions alpha and beta to the carꢀ
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boxylic acid would restrict free rotation, potentially enhancing
potency and selectivity. Incorporation of the Sꢀconfigured
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Scheme 1
methyl substituent (trans relative configuration) resulted in a
remarkable improvement in potency over the desꢀmethyl deꢀ
rivatives 10 and 11. The R diastereomer provided no imꢀ
provement in potency. Finally, in an effort to further improve
the physicochemical profile of our series, we replaced the Bꢀ
ring phenyl with a pyridine ring. This resulted in compounds
2
9 and 30, which were comparable in potency to phenyl derivꢀ
atives 24 and 25, although the impact of serum on potency was
greater.
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Table 3. α-Methyl and B-Ring Pyridyl
Reagents and conditions: (a) TBSCl, imidazole, DMF, 98%; (b)
LDA, MeI, THF; then KOtBu, HCl; (c)TBAF, THF, 70% over 3
steps, 3:1 dr 36/35; (d) paraformaldehyde, MgCl , TEA, MeCN,
2
7
9
4%; (e) NaBH , MeOH, 95%; (f) Pd(PPh ) , K CO , dioxane,
3%; (g) Montmorillonite K10, LiClO , H O, MeNO , 36%; (h)
4 3 4 2 3
4
2
2
o
LiOH, MeOH, H O, THF, 60 C, 96% (i) SFC seperation – ADꢀH
chiralpak, 60% IPA/CO with 0.2% HN(iPr) modifier
2
2
2
established both stereocenters as a single diastereomer in 98%
ee. Ortho iodination with NIS delivered iodophenol 46. Cross
coupling of pꢀbromobenzaldehyde with 2ꢀflouroꢀ4ꢀ
methoxyphenyl boronic acid followed by treatment with viꢀ
nylmagnesium bromide gave allylic alcohol 48. Iodophenol
4
6 and allylic alcohol 48 underwent Pdꢀmediated Heck couꢀ
pling using tꢀbutyl XꢀPhos and N,Nꢀdicyclohexylmethylamine
as base to give ketone 49. Asymmetric reduction of the keꢀ
tone in 49 using the Noyori protocol yielded benzylic alcohol
19
5
0 in 92:8 dr. Ring closure of diol 50 under Mitsunobu condiꢀ
tions afforded chroman 51 in high yield, notably without eroꢀ
sion in dr. Hydrolysis of the ester with aqueous LiOH folꢀ
lowed by SFC purification to remove the minor chroman diaꢀ
stereomer delivered 24 in 36% overall yield from commercialꢀ
ly available 42.
a
Mean of at least 2 runs.
The preparation of biaryl chroman agoPAMs is exemplified
by the synthesis of 24 (Scheme 1). αꢀMethylation of the TBS
Scheme 2
10
ether of 33 resulted in a ~2:1 (R/S) mixture of diastereomers.
Epimerization of this mixture to deliver primarily the (S)ꢀ
configured αꢀMethyl diastereomer was affected via deprotonaꢀ
tion with KOtBu followed by quenching with HCl to deliver a
1
:3 mixture of diasteromers, favoring the desired (S) isomer.
TBS deprotection with TBAF gave a separable mixture of
phenol compounds 35 and 36. The (S)ꢀconfigured methyl
diastereomer 36 was formylated and then reduced to give diol
3
7. Suzuki coupling of pꢀbromostyrene and 2ꢀflouro,4ꢀ
methoxyphenyl boronic acid delivered biaryl styrene 40.
Heating of 37 and 40 under the conditions described by Chiꢀ
15
ba resulted in a hetero Diels Alder cycloaddition reaction to
give chroman compound 41 as a 1:1 mixture of diastereomers
at the newly formed benzylic stereocenter. Hydrolysis with
LiOH followed by SFC separation (ADꢀH chiralpak, 60% IPA
in CO , 0.2% DIPA) gave the final compounds 24 and 25.
2
In order to support acute dose titrations and chronic in vivo
studies, multigram quantities of 24 were needed and an imꢀ
proved synthesis was devised (Scheme 2). Suzuki cross couꢀ
Reagents and conditions: (a) CataxiumꢀPd precat, K PO , MeCN,
3
3
o
8
9%; (b) RuꢀJosiphos, BF HꢀEt O, 500 atm H , 85 , MeOH, 86%,
4 2 2
1
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98% ee; (c) Pd/Pt/C, H gas, MeOH, 104%; (d) NIS, DCM, 91%;
2
pling of vinyl tosylate 43 and 3ꢀbenzyloxyphenyl boronic
acid gave cinnamate 44. Asymmetric hydrogenation of the
(e) Pd(PPh ) , K CO , 1,4ꢀdioxane, 98%; (f) vinylmagnesium
3 4 2 3
1
7, 18
bromide, THF, 67%; (g) tBuXPHOS Pd precat, MeN(Cy) , PhMe,
2
alkene in 44 at 500 psi with 2.5 mol% RuꢀJosiphos
6
8%; (h) RuCl[(R,R)ꢀTsDPEN(mesitylene)], TEA, Formic Acid,
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MeCN, 100%, 92:8 dr; (i) PPh , DIAD, DCM, 83%; (j) LiOH,
THF/MeOH/water, 97% (k) SFC separation ꢀ conditions
4A). To assure that 24 and 29 bind to hGPR40 in a similar
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manner to AM1638, competition binding experiments were
performed in membranes from stably transfected CHOꢀK1
The compounds studied in Table 4 demonstrated acceptable
exposure and halfꢀlife for further profiling in rodent models.
While neopentoxy derivatives 14 and 15 have moderate clearꢀ
ance in rats, cpds 24, 29, and their chroman stereoisomers
showed improved clearance.
3
6, 23
cells. Two different radioligands were used; 1. [ H]L358 , a
thiazolidinedione allosteric partial agonist (see Supplementary
Info for chemical structure), binding in a similar manner to
22
24
3
AMG837 and TAK875 . 2. [ H]25, a study compound utiꢀ
lized following binding studies demonstrating that GPR40
partial allosteric agonists and AgoPAM’s demonstrated identiꢀ
Table 4. Pharmacokinetic Profiles in Wistar Rats
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cal profiles with [ H]AM1638 and [ H]25 (data not shown).
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Like AM1638, 24 and 29 augment [ H]L358, and displace,
H]25 binding (Figure 4B-C). These properties are consistent
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[
Cl
mL/min/kg)
8.2
Vdss
(L/kg)
2.6
T
1/2
%F
Cpd
with an agoPAM binding mode and clearly contrast with those
of AMG837, TAK875, MKꢀ2305 and L358 which augment
(
(h)
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[
H]25 and displace the binding of [ H]L358.
6.6
3.1
3.0
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1.9
1.1
2.9
3.5
2.4
6.2
4.4
Figure 4. (A) IP1 Functional Assay [- or + human serum
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0.41
0.39
0.35
0.56
(HS)] (B) & (C) [ H]L358 and [ H]25 binding assays.
a
Aministered at a dose of 1 mg/kg iv, 2 mg/kg po.
With leaner, more ligand efficient agonists in hand, we beꢀ
gan to assess the selectivity of several analogs. Compounds
14, 15, 24, 25, 29, and 30 were screened against a broad panel
GPCR’s, ion channels, transporters, and enzymes at 10 ꢀM
(Table 5). While compounds with higher logD exhibited
poorer selectivity (# of hits), the conformationallyꢀrestricted
compounds 29 and 30 showed improved selectivity, exhibiting
an excellent correlation between the number of offꢀtarget hits
and logD. We also examined the selectivity of these comꢀ
pounds for GPR40 versus other free fatty acid Gꢀprotein couꢀ
2
0
21
pled receptors ( 41, 43 , and 120 ). None of these comꢀ
pounds displayed discernable agonism of GPR41, GPR43 or
GPR120 up to 3 ꢀM, 10 ꢀM, and 8.3 ꢀM, respectively.
Table 5. Binding Activity Screen Against 40 Receptors
# of hits
Cpd
>50%
inh
>
90% inh
>70% inh
hplc LogD/MW/PSA
3.76 / 546 / 71
3.81 / 546 / 71
2.82 / 460 / 62
2.81 / 460 / 62
2.24 / 461 / 76
2.26 / 461 / 76
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Oral administration of 24 and 29 1h before oral dextrose
challenge in a rodent oGTT (oral glucose tolerability test)
significantly reduced blood glucose levels compared to vehicle
(Figure 5). The efficacy of both agoPAMs examined in this
study was superior to the partial agonist positive control 6
dosed at a maximally efficacious level. 24 was maximally
Detailed results of receptors and %inh are provided in Supportꢀ
ing Information
2
5,26
A more detailed in vitro characterization along the lines of
2
2
Lin et al 2012 reveals that 24 and 29 demonstrate comparaꢀ
ble efficacy to AM1638 in hGPR40/HEK293 cells (Figure
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efficacious at 10 mpk with a total drug plasma level of 3.1
M. 29 was dosed up to 30 mpk, which corresponded to a
drug plasma level of 13.9 ꢀM and resulted in 125% reduction
in glucose AUC.
careful modulation of physicochemical properties. These
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compounds induce GLPꢀ1 secretion through a GPR40ꢀ
mediated mechanism, as demonstrated in a GPR40 KO/WTꢀ
mouse study. In a chronic setting, compound 24 delivered
sustained fasting plasma glucose lowering that was superior to
ꢀ
A)
Blood Glucose Over Time
F
*
Wistar Control
Vehicle
MK-2305 (6) @30 mpk
24 @30 mpk
24 @3 mpk
220
200
*
*
MeO
O
CO
2
H
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F
MeO
Time (days)
O
2
CO H
N
Figure 7. AgoPAM GK rat chronic study with 24
29
the partial agonist control. These preclinical results suggest
GPR40 agoPAMs may have the potential to affect a powerful
new means of enhanced gylcemic control for diabetic patients.
Figure 5. GK Rat oGTT titrations
GPR40 agoPAMs have been previously reported to stimuꢀ
ASSOCIATED CONTENT
1
4
late GLPꢀ1 secretion.
To demonstrate that the enhanced
Supporting Information. Experimental procedures, analytical
data, and receptor screening data. This material is available free
of charge via the Internet at http://pubs.acs.org.
GLPꢀ1 secretion observed with GPR40 agoPAMs was entirely
onꢀtarget, total GLPꢀ1 (ng/mL) was measured in wild type
versus knockout GPR40 mice. Figure 6 shows that no offꢀ
target incretin effects were observed in this study for comꢀ
AUTHOR INFORMATION
Corresponding Author
pounds 24 and 29, while AMꢀ1638 did exhibit offꢀtarget GLPꢀ
27
1
elevation.
*
Email: christopher_plummer@merck.com
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENT
The authors would like to thank Leo Joyce for structural determiꢀ
nation by VCD analysis.
ABBREVIATIONS
GPR40, gꢀproteinꢀcoupled receptor 40; FFAR1, free fatty acid
receptor 1; agoPAM, agoꢀallosteric modulator; DHA, docosahexꢀ
aenoic acid; GLPꢀ1, glucagonꢀlike peptide 1; CHO, chinese hamꢀ
ster ovarian (cell line); HEK293, human embryonic kidney (cell
line); HPLC, high performance liquid chromatography; TBS, tꢀ
butyl dimethylsilyl; TBAF, tetraꢀnꢀbutylammonium flouride;
LDA, lithium diisopropylamide; THF, tetrahydrofuran; SFC, suꢀ
percritical fluid chromatography; NIS, Nꢀiodosuccinimide; DCM,
dichloromethane; DIAD, diisopropylazodicarboxylate; WT/KO,
wildꢀtype/knockout; mpk, milligrams per kilogram; dr, diastereoꢀ
meric ratio; HS, human serum.
Figure 6. GLPꢀ1 secretion for GPR40 KO versus WT mice
In order to assess the duration of effect on fasting plasma
glucose, 24 was dosed at 3 and 30 mg/kg in Goto Kakizaki
rats for 14 days. AgoPAM 24 demonstrated excellent efficacy
with respect to fasting plasma glucose lowering (as compared
to the partial agonist control MKꢀ2305 (6)) with no indication
of tachyphylaxis or desensitization (Figure 7).
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(25) See Supplementary Info for oGTT study protocol
(26) See Supplemmentary Info for potency (nM) and efficacy (%
act) of key GPR40 agonists against the human, mouse, and rat recepꢀ
tors
(27) The authors note that results previously reported by researchꢀ
ers at Amgen (Luo et al, Plos One 2012, e46300) indicate no off tarꢀ
get GLPꢀ1 effects associated with AM1638 in a similar GPR40
WT/KO mouse experiment. We propose that differences in GLPꢀ1
sampling times (30 minutes in the Amgen study and 1 hour for the
study reported herein) is one possible explanation for this discrepanꢀ
cy. Further, it is notable that the GLPꢀ1 secretion induced by
AM1638 is greater than one might predict on the basis of GPR40
target engagement alone compared to other compounds in this class
(based on measured potency and exposure), potentially indicating
additive, nonꢀGPR40ꢀmediated GLPꢀ1 elevation may be operative.
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(
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D.; Conn, P. J. Functional impact of allosteric agonist activity of seꢀ
lective positive allosteric modulators of metabotropic glutamate reꢀ
ceptor subtype 5 in regulating central nervous system function. Mo-
lecular Pharmacology 2012, 81(2), 120ꢀ133.
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