Discovery of 2-pyridylpyrimidines as the first orally bioavailable GPR39 agonists

Article abstract of DOI:10.1021/ml500240d

The identification of highly potent and orally bioavailable GPR39 agonists is reported. Compound 1, found in a phenotypic screening campaign, was transformed into compound 2 with good activity on both the rat and human GPR39 receptor. This compound was fu

Full text of DOI:10.1021/ml500240d

Letter
pubs.acs.org/acsmedchemlett
Discovery of 2‑Pyridylpyrimidines as the First Orally Bioavailable
GPR39 Agonists
Stefan Peukert,* Richard Hughes, Jill Nunez, Guo He, Zhao Yan, Rishi Jain, Luis Llamas,
Sarah Luchansky, Adam Carlson, Guiqing Liang, Vidya Kunjathoor, Mike Pietropaolo, Jeffrey Shapiro,
Anja Castellana, Xiaoping Wu, and Avirup Bose
Novartis Institutes for BioMedical Research, 250 Massachusetts Avenue, Cambridge, Massachusetts 02139, United States
S
* Supporting Information
ABSTRACT: The identification of highly potent and orally
bioavailable GPR39 agonists is reported. Compound 1, found
in a phenotypic screening campaign, was transformed into
compound 2 with good activity on both the rat and human
GPR39 receptor. This compound was further optimized to
improve ligand efficiency and pharmacokinetic properties to
yield GPR39 agonists for the potential oral treatment of type 2
diabetes. Thus, compound 3 is the first potent GPR39 agonist
(EC50s ≤ 1 nM for human and rat receptor) that is orally bioavailable in mice and robustly induced acute GLP-1 levels.
KEYWORDS: GPR39 agonists, GPR39 receptor, Zn2+-sensing receptor, antidiabetic treatment, β-cell protection, GLP-1 secretion,
insulin secretion
PR39 is a putative zinc-sensing G protein-coupled
receptor (GPCR) related to the ghrelin/neurotensin
screening of GPCR-focused compound libraries.13 However,
the reported chemical probes are moderately potent, and their
properties are not suitable for in vivo studies.
G
peptide receptor subfamily.1,2 The functional full-length
receptor is widely expressed in peripheral, endocrine, and
metabolic organs such as the stomach, gastrointestinal (GI)
tract, pancreas, liver, kidney and the white adipose tissue.3 Zn2+
is the only known stimulator of GPR39 activity4,5 via the Gαq,
Gα12/13, and Gαs pathways with an EC50 value in the
micromolar range. GPR39 signaling is implicated in cellular
processes such as insulin secretion, protection from cell death,6
gastric emptying,7 and epithelial repair.8 Initial reports about
obestatin as the natural ligand for this receptor have not been
verified.4 Information relating to the pancreatic function of
GPR39 has come from multiple mouse knockout studies.9−11
Deletion of the GPR39 gene is associated with impaired insulin
secretion and glucose homeostasis. These knockout studies
suggested that GPR39 may play a role in the development of
type 2 diabetes, and therefore, activating the receptor could
exert a β-cell protective effect. This hypothesis was tested with a
transgenic mouse strain with inducible overexpression of
human GPR39 selectively in pancreatic β-cells.12 Because of
the high degree of constitutive activity, increased expression
leads to increased receptor signaling mimicking the action of an
endogenous agonistic ligand. This study by Egerod et al.
showed that GPR39 overexpression in the β-cells by itself
impaired glucose tolerance slightly and decreased β-cell mass,
but it completely protected against gradual hyperglycemia in
streptozotocin-treated animals. This suggests that GPR39
functions in a β-cell protective manner and could be a target
for antidiabetic drug treatment with long-term benefits. GPR39
agonists would help to test this hypothesis pharmacologically.
The first GPR39 agonists have been recently identified by
Herein, we report the first highly potent and orally
bioavailable GPR39 agonists. Compound 1 was discovered
through a reporter gene assay aimed at identifying inhibitors of
Hedgehog signaling. The compound was employed in a
chemogenomics approach to identify GPR39 as its molecular
target.14 Compound 1 had good activity via the Gαq pathway
in an assay that measured inositol-1-phosphate (IP1) in
HEK293 cells overexpressing rat or mouse GPR39; however,
it showed moderate cross-reactivity with the human receptor.
Whereas the sequence identity between rat and mouse receptor
is very high (93.6%), the overall homology for human GPR39
with mouse (80.5%) and rat (79.6%) is lower.15 Additionally, 1
suffered from high metabolic clearance (Cl(h) = 51.2 mL min−1
kg−1) in rat liver microsomes and low aqueous solubility (<5
μM at pH 6.8). Our objective was to optimize the potency of
these compounds against human and rodent receptors and
improve their pharmacokinetic properties in order to validate
the target in vivo and ultimately to utilize them as therapeutic
agents.
Compound 2, a pyrimidine with a regioisomeric presentation
of the central pyrimidine ring compared to 1, resulted in
improved activity on all three receptors and a much smaller
shift in potency between the human and rodent receptors. This
finding prompted us to investigate more thoroughly the role of
Received: June 8, 2014
Accepted: August 4, 2014
© XXXX American Chemical Society
A
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ACS Medicinal Chemistry Letters
Letter
the biaryl moiety (boxed area of compound 2, Figure 1) for
potency on human and rodent receptors. None of the
1 and 2, the rapid clearance of compound 15 in rat liver
microsomes (RLM) did not predict sufficient exposure upon
oral dosing in rats. Prediction of metabolic sites for compound
15 indicated the methylamino substituent and the benzylic
position as preferred sites for oxidative metabolism.17 None of
the attempts to block these metabolic weak spots by replacing
the methylamino group with a trifluoroethylamino group
(compound 16), methyl group (compound 17), or blocking
the benzylic position with a cyclobutyl group (compound 18),
provided the desired outcome as these compounds continued
to have high in vitro clearance in RLMs. Comparison of various
molecular descriptors such as clogP, clogD, and polar surface
area (PSA) against in vitro clearance revealed an interesting
correlation useful for further optimization of metabolic stability:
Compounds 15 to 20 with lower polar surface area (PSA ≤ 72
Å2) had high clearance, but compound 20, with a PSA of 96.9
Å2, showed a reduced clearance in RLM (Cl(h) = 34.1 mL
min−1 kg−1). A pharmacokinetic study in Sprague−Dawley rats
confirmed the improved clearance in vivo (18 mL min−1 kg−1
after a single iv dose of 1 mg/kg, F = 45% after a 3 mg/kg oral
dose) and supporting further use of rat liver microsome stability
for optimization. We utilized this hypothesis to synthesize a
number of compounds (21 to 23) with PSAs of ≥91.8 Å2.
Gratifyingly, we observed improved in vitro clearance in all
three compounds without compromising their ability to cross
cell membranes. For example, compound 21 demonstrated
high permeability in Caco-2 cells (Papp A−B = 6.9 × 10−6 cm
s−1) and the parallel artificial membrane permeability assay
(calculated fraction absorbed = 89%). To further improve
potency, we made use of an earlier observation that substituents
in 2-position on the phenyl ring in R1 increased potency as seen
by the comparison between compounds 12 and 15.
Compounds 24 and 3 show increased potency while retaining
their in vitro metabolic stability.
Figure 1. Discovery of 2-pyridylpyrimidines as GPR39 agonists.
investigated biaryl compounds including regioisomeric pyr-
idylpyrimidines matched the potency of compound 2 and
further work focused on the 4-(pyridin-2-yl)pyrimidines
described below.
Although compound 2 had very good potency, the
pharmacokinetic properties (clearance of 42 mL min−1 kg−1
in Sprague−Dawley rats after a single iv dose of 2 mg/kg in a
formulation of 10% NMP, 30% PEG200, and 5% Solutol in
water and a bioavailability of 25% after a oral dose of 5 mg/kg
of the above formulation) were not favorable for in vivo studies.
As this molecule has a molecular weight of 497 and a clogP of
5.6 we focused on “deconstructing” this lead with the goal of
identifying the minimal pharmacophore and increasing the
apparent ligand efficiency (LE).16 In a second step we planned
to build upon this and further optimize relevant drug
properties. Compound 4 with a low molecular weight of 255
was found to be the most ligand efficient molecule with a LE =
0.49 for the human GPR39 receptor compared to compound 2
with a LE = 0.3. “Reconstruction” with the goal to maintain the
high apparent ligand efficiency involved two small compound
libraries, one with modifications to the cyclopropylmethylami-
no substituent R1 and one with variations of the methylamino
substituent R2 (Table 1). As demonstrated by representative
compounds 5−9, changes to the methylamino group (R2)
resulted in partial or significant loss of activity. By contrast,
modifications to substituent R1 leaving the methylamino
substituent R2 unchanged resulted mostly in improvements in
EC50 values: Whereas a neopentyl group (compound 10)
increased potency slightly (EC50 = 73 nM), this improvement
was more distinct for the compounds containing the cyclo-
hexylmethyl (11) and para-chlorobenzyl (12) substituent at
this position (EC50s = 11 and 15 nM, respectively). The 4-
pyridyl group in compound 13, instead of a phenyl moiety,
resulted in reduced activity (EC50 = 87 nM). Direct attachment
of an aryl substituent, exemplified by compound 14, led to a
significant drop in activity into the micromolar range.
Exploration of the regioisomeric positions on the phenyl ring
of R1 resulted in a >10-fold increase in potency for compound
15, which exhibits an additional 2-chloro substituent in
comparison to compound 12. Remarkably, compound 15
combines the high LE of our starting compound 4 (LE = 0.49
and 0.50 for compounds 4 and 15, respectively) with
subnanomolar potency. However, as observed for compounds
Some of the most potent compounds, with Zn2+ for
comparison, were evaluated in additional cell assays to further
assess their potency. GPR39 is known to signal through Gαs in
addition to Gαq.4 Thus, we tested compounds in a HEK293
cell line overexpressing the human GPR39 receptor and used
forskolin as a reference compound to measure the maximal
cAMP release. All compounds were active in Gαs signaling,
with compound 3 as the most active both in terms of EC50
value and maximal efficacy. Both the IP1 assay and the cAMP
assay relied on recombinant cell lines. In order to more
accurately assess the functional activity we employed a mouse
GLP-1 secreting enteroendocrine cell line (STC-1) that
expresses endogenous GPR39.18 Additionally, we used a rat
insulinoma cell line (INS-1E)19 that has a beta cell phenotype
and tested for protection against cytokine-induced cell death.
All four compounds showed activity in both assays with a rank-
order as seen in the cAMP assay (Table 2). Compound 22 had
an unexpectedly high EC50 value in the STC-1 assay, yet
achieved the highest maximal GLP-1 secretion of all four
compounds, suggesting that it might be working through an
additional target on these cells. Compound 3 was the most
potent compound across the assays. Rat and mouse plasma
protein binding for 3 was measured as 99.3% and 99.1%,
respectively. Considering off-targets, 2,4-diamino-6-(pyridin-2-
yl) pyrimidines have been described as kinase inhibitors,20 but
3 and other compounds described in this letter did not show
inhibitory effects (IC50s > 10 μM) on a panel of kinases nor did
they exhibit relevant binding affinity for the related ghrelin and
neurotensin-1 receptors (IC50s > 30 μM) and other enzymes,
B
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Table 1. SAR of 2-Pyridylpyrimidine Analogues
a
Values derived from a 11-point dose−response curve performed in triplicate. The number of replicates range from 1 to 4 as indicated, with standard
b
c
deviation reported in parentheses where available. HEK293 cells expressing human or mouse GPR39. Predicted hepatic clearance based on
intrinsic clearance in rat liver microsomes. Scaling factors for Cl(int) → Cl(h): 45 mg of microsomal protein/g of liver; 40 g of liver/kg of body
weight; and 55 mL of portal blood flow/min/kg. N.D. = not determined.
C
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yl)pyrimidine23 yielded compound 28 as the major re-
gioisomer, which after displacement of the second chlorine
with methylamine furnished compound 3 in 61% yield for the
two displacement reactions.
Since compound 3 exhibited promising in vitro properties,
the pharmacokinetic properties were evaluated in male C57BL/
6 mice. Upon single oral doses of 10, 30, and 100 mg/kg of
aqueous suspensions in 0.5% methylcellulose/0.1% Tween 80,
compound 3 achieved, between 1 and 1.5 h, maximal exposures
of 1.4, 6.1, and 25.3 μM, respectively, indicating slightly
overproportional exposures. A half-life of 7 h for compound 3
in mice was calculated from a separate study with 10 mg/kg.
The potency of compound 3 and the less active analogue 21
were assessed in mice by measuring a pharmacodynamic
marker, GLP-1 levels (Figure 2). Consistent with the EC50s
Table 2. In Vitro Potencies of Selected Compounds in
Various Cell Assays
a
EC50, μM
c
EC50, μM
EC50, μM
d
EC50, nM
b
hcAMP
GLP1/STC
protection
e
compd rGPR39
[% efficacy]
[fold max]
/INS-1E
ZnCl2
4038
3.6 [48%]
N.D.
N.D.
21
3 (1.5)
n = 3
0.18 (0.05)
n = 3 [55%]
1.3 (0.37)
n = 16 [3×]
0.4 (0.13)
n = 3
22
24
3
2 (0.5)
0.55 (0.08)
4.4 (0.32)
1.2
n = 2
n = 3 [48%]
n = 2 [5.5×]
n = 1
0.6
n = 1
0.35 (0.07)
n = 2 [55%]
0.2
0.04
n = 1
n = 1 [3.5×]
0.4
n = 1
0.06 (0.03)
n = 3 [64%]
0.06 (0.03)
n = 2 [3.5×]
0.02
n = 1
a
Values derive from a dose−response curve performed in triplicate.
The number of replicates range from 1 to 16 as indicated, with
standard deviation reported in parentheses where available. HEK293
cells expressing rat GPR39. HEK293 cells expressing human GPR39.
b
c
The percent efficacy is reported relative to the cAMP response with 25
d
μM forskolin. Mouse enteroendocrine STC-1 cells secreting GLP-1.
The maximal activity of the test sample was expressed as fold increase
e
of GLP-1 compared with the DMSO control. Caspase 3/7 activity
was measured in cytokine-treated rat insulinoma INS-1E cells. N.D. =
not determined.
transporters, and GPCRs (data given in Supporting Informa-
tion). On the basis of mutagenesis studies, Zn2+ has been
proposed to act as a GPR39 agonist by binding to His-17 and
His-19 located in the extracellular domain.21 Mutagenesis
studies and pharmacological characterization of the mutants
with the 2-pyridylpyrimidines described herein are not yet
available. However, studies with sub-EC50 concentrations of
Zn2+ in the presence of a potent 2-pyridylpyrimidine GPR39
agonist resulted in generation of IP1 greater than the amount
produced by either substance alone (data given in Supporting
Information). This suggests that our compounds and Zn2+ do
not compete for binding to the receptor, which is most likely
due to binding to separate sites resulting in a receptor response.
More detailed data to this point will be reported in due course.
Compound 3 was prepared as described in Scheme 1.
Mesylation of 4-amino-2-chlorobenzonitrile (25) in pyridine
provided 26, which was reduced to the benzylamine 2722 by
reaction with lithium aluminum hydride in 54% yield over the 2
steps. Reaction of amine 27 with 2,4-dichloro-6-(pyridin-2-
Figure 2. Induction of a PD Marker, active GLP-1, for compounds 21
and 3.
observed in vitro, compound 3 induced very high levels of
active GLP-1 (6-fold), whereas the less potent compound 21
modestly induced (<2-fold) active GLP-1 levels at comparable
plasma exposure for both compounds (1.6 and 2.0 μM,
respectively).
Male C57BL/6 mice were concurrently dosed with DPP-IV
inhibitor (S)-1-(2-(adamantan-2-ylamino)acetyl)pyrrolidine-2-
carbonitrile24 and the indicated compound. Animals were
challenged after 1 h with a glucose bolus (3 g/kg, po), and
active GLP-1 levels were measured 30 min later. Data were
statistically evaluated with one-way ANOVA followed by
Dunnett’s post-test. P < 0.001 versus the vehicle group.
The current study demonstrates that a low molecular weight
agonist with drug-like properties can be found for the GPR39
receptor. We identified the first GPR39 agonists with high
potency for the human and rodent receptor and excellent
functional activity in physiologically relevant rodent cells and in
vivo. An acute study in normal mice with orally administrated
GPR39 agonists confirmed in vitro findings by demonstrating
an increase of the relevant pharmacodynamic marker GLP-1.
a
Scheme 1. Synthesis of 3
ASSOCIATED CONTENT
■
S
* Supporting Information
a
Experimental procedures and characterization data for com-
pounds 1−24, additional biological data, PK and PD study
information, and experimental details for the in vitro assays.
This material is available free of charge via the Internet at
http://pubs.acs.org.
Reagents and conditions: (a) MsCl, pyridine, 0 °C, 80% yield; (b)
LiAlH4, THF, 0 °C, 68% yield; (c) 2,4-dichloro-6-(pyridin-2-
yl)pyrimidine, MeCN, triethylamine 60 °C, chromatographic separa-
tion on silica gel, 70% yield; (d) 15% MeNH2 in EtOH, 130 °C,
microwave heating, 87% yield.
D
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ACS Medicinal Chemistry Letters
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Schwartz, T. W. G protein-coupled receptor 39 deficiency is associated
with pancreatic islet dysfunction. Endocrinology 2009, 150, 2577−2585.
(10) Tremblay, F.; Richard, A. M.; Will, S.; Syed, J.; Stedman, N.;
Perreault, M.; Gimeno, R. E. Disruption of G protein-coupled receptor
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2595.
(11) Verhulst, P. J.; Lintermans, A.; Janssen, S.; Loeckx, D.;
Himmelreich, U.; Buyse, J.; Tack, J.; Depoortere, I. GPR39, a receptor
of the ghrelin receptor family, plays a role in the regulation of glucose
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Plonsky, S.; Lee, L.; Ruffner, H.; Grandi, P.; Schirle, M.; Jenkins, J.;
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AUTHOR INFORMATION
Corresponding Author
*(S.P.) Tel: 617 871 3644. Fax: 617 871 4081. E-mail: stefan.
peukert@novartis.com.
■
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank the whole GPR39 team for stimulating scientific
discussions and in particular Heather Burks, Robert Damon,
MooJe Sung, and Scott Tria for valuable contributions to the
medicinal chemistry strategy as well as Lawrence Hamann for
the strong support of the program. We thank Fred Bassilana for
providing HEK293 cells expressing human GPR39 and Martin
Marro for discussions in regards to in vitro assays.
ABBREVIATIONS
■
HEK, human embryonic kidney; EC50, half maximal effective
concentration; IP1, inositol-1-phosphate; NMP, N-methyl-2-
pyrrolidone; PEG, polyethylene glycol; DMSO, dimethyl
sulfoxide; LE, ligand efficiency, calculated from −RT ln-
(EC50)/number of heavy atoms in the molecule; RLM, rat
liver microsome; Cl(h), hepatic clearance; F, bioavailability;
PSA, polar surface area; cAMP, cyclic adenosine mono-
phosphate; GLP-1, glucagon-like peptide-1; PD, pharmacody-
namic; DPP-IV, dipeptidyl peptidase IV
(15) Sequence homology was calculated in UniProt (www.uniprot.
org) using the Clustal W algorithm and entries O43194, Q5U431, and
E9PTT1 for human, mouse, and rat GPR39 protein, respectively.
(16) Hopkins, A. L.; Groom, C. R.; Alex, A. Ligand-efficiency: a
useful metric for lead selection. Drug Discovery Today 2004, 9, 430−
431.
(17) Cytochrom P450 mediated metabolism has been predicted
using Metasite from Molecular Discovery. See also Cruciani, G.;
Carosati, E.; De Boeck, B.; Ethirajulu, K.; Mackie, C.; Howe, T.;
Vianello, R. MetaSite: Understanding metabolism in human
cytochromes from the perspective of the chemist. J. Med. Chem.
2005, 48, 6970−6979.
(18) Synthesis and secretion of intestinal proglucagon-derived
peptides by the STC-1 enteroendocrine cell line. Brubaker, P. L.;
Izzo, A.; Rocca, A. S. Can. J. Diabetes 2003, 27, 141−148.
(19) Merglen, A.; Theander, S.; Rubi, B.; Chaffard, G.; Wollheim, C.
B.; Maechler, P. Glucose sensitivity and metabolism-secretion coupling
studied during two-year continuous culture in INS-1E insulinoma cells.
Endocrinology 2004, 145, 667−678.
(20) Nagarathnam, D.; Dumas, J.; Hatoum-Mokdad, H.; Boyer, S.;
Pluempe, H. Preparation of pyrimidine derivatives for use in
pharmaceutical compositions as Rho-kinase inhibitors. PCT patent
application WO2003062227, 2003.
(21) Storjohann, L.; Holst, B.; Schwartz, T. W. Molecular mechanism
of Zn2+ agonism in the extracellular domain of GPR39. FEBS Lett.
2008, 582, 2583−2588.
(22) Ding, Y.; Longdregan, A. T.; Marino, J. P., Jr. Preparation of 2-
amino-1,3,5-triazine derivatives as soluble epoxide hydrolase (sEH)
inhibitors and their use. PCT patent application WO2009049154,
2009.
(23) Harden, D. B.; Mokrosz, M. J.; Strekowski, L. Addition and
substitution reactions of chloropyrimidines with lithium reagents. J.
Org. Chem. 1988, 53, 4137−4140.
(24) Akarte, A. S.; Srinivasan, B. P.; Gandhi, S. A novel long acting
DPP-IV inhibitor PKF-275-055 stimulates β-cell proliferation resulting
in improved glucose homeostasis in diabetic rats. Biochem. Pharmacol.
2012, 83, 241−252.
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