Conformational restriction in a series of GPR119 agonists: Differences in pharmacology between mouse and human

Article abstract of DOI:10.1016/j.bmcl.2013.04.006

A series of conformationally restricted GPR119 agonists were prepared based around a 3,8-diazabicyclo[3.2.1]octane scaffold. Examples were found to have markedly different pharmacology in mouse and human despite similar levels of binding to the receptor. This highlights the large effects on GPCR phamacology that can result from small structural changes in the ligand, together with inter-species differences between receptors.

Full text of DOI:10.1016/j.bmcl.2013.04.006

Bioorganic & Medicinal Chemistry Letters 23 (2013) 3175–3179
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Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Conformational restriction in a series of GPR119 agonists:
Differences in pharmacology between mouse and human
⇑
James S. Scott , Katy J. Brocklehurst, Hayley S. Brown, David S. Clarke, Helen Coe, Sam D. Groombridge,
David Laber, Philip A. MacFaul, Darren McKerrecher, Paul Schofield
Cardiovascular & Gastrointestinal Innovative Medicines Unit, AstraZeneca, Mereside, Alderley Park, Macclesfield, Cheshire SK10 4TG, UK
a r t i c l e i n f o
a b s t r a c t
Article history:
A series of conformationally restricted GPR119 agonists were prepared based around a 3,8-diazabicy-
clo[3.2.1]octane scaffold. Examples were found to have markedly different pharmacology in mouse
and human despite similar levels of binding to the receptor. This highlights the large effects on GPCR pha-
macology that can result from small structural changes in the ligand, together with inter-species differ-
ences between receptors.
Received 4 March 2013
Revised 28 March 2013
Accepted 2 April 2013
Available online 10 April 2013
Ó 2013 Elsevier Ltd. All rights reserved.
Keywords:
G-protein coupled receptors
GPR119 agonists
Pharmacology
Partial agonists
G-protein coupled receptor 119 (GPR119) is a class A type
receptor expressed predominantly in pancreatic islets and intesti-
nal enteroendocrine cells.1 Research has demonstrated that agon-
ism of the GPR119 receptor results in secretion of incretins from
the gut (e.g., glucagon-like peptide-1 (GLP-1) and glucose-depen-
dent insulinotropic peptide (GIP) release from L-cells)2 and release
of insulin from b-cells in the pancreas.3 Taken together, these ef-
fects represent a potential mechanism for modulation of glucose
homeostasis and a novel approach to the treatment of type 2
diabetes.4
A number of chemical series displaying GPR119 agonism have
been disclosed from Arena,5 Astellas,6 Boehringer Ingelheim,7
GSK,8 Pfizer,9 Merck,10 and Sanwa Kagaku Kenkyusho.11 Data has
recently been reported from the first example of a GPR119 agonist
to enter trials, APD-597 (JNJ-38431055). Results indicated eleva-
tion of GLP-1, GIP and peptide YY levels although decreases in glu-
cose excursion or insulin secretion were not significant.12
Many of the reported chemical series share a common struc-
tural motif incorporating a carbamate or heterocycle capped piper-
idine linked via a spacing group to an aryl group with a strong
acceptor (pyridyl, sulphone, heterocycle). A pharmacophore model
has been proposed that encapsulates these features.13 Work from
Pfizer,9a,e together with a recent disclosure from researchers from
the College of Pharmacy of Kangwon National University,14 have
highlighted the importance of conformational restriction of the
piperidine portion in controlling human GPR119 pharmacology.
Examples of conformationally restricted piperidines are shown in
Figure 1.
We herein report our own findings in this area around a series
of 3,8-diazabicyclo[3.2.1]octanes and the divergent nature of the
effects on human and mouse pharmacology relative to piperazine
matched pairs. In accordance with previous reports9a we postulate
conformational restriction, leading to an inability of the bridged
compounds to access an ‘agonist conformation’, as a likely source
of these effects in mouse. As part of this work we developed an as-
say that allowed us to assess the strength of the ligand–receptor
binding interaction.16 These results were compared with EC50 val-
ues derived from a functional cAMP assay.17
Initial exploration around the piperazine core 1 had shown that
methyl substitution, piperazine 2, brought increases in both hu-
man and mouse binding potency (Table 1). This was reflected by
an increase in human potency (cAMP EC50) and intrinsic activity
(IA) for this compound although no corresponding improvement
in the mouse. Further exploration around ethylene bridging prox-
imal to each of the nitrogens in the piperazine gave isomeric 3,8-
diazabicyclo[3.2.1]octanes 3 and 4 which showed marked differ-
ences. In particular, compound 4 was around two orders of magni-
tude more potent in both human and mouse binding assays than
the unsubstituted piperazine 1. In the human cAMP assay this
compound had a lower EC50 and a larger intrinsic effect than piper-
azine 1 but in stark contrast, it had no discernible effect in the
mouse cAMP assay and no EC50 could be determined. Similarly
the alternative bridged isomer 3 showed no effect in mouse despite
a full agonist profile in human.
⇑
Corresponding author. Tel.: +44 (0)1625 232567; fax: +44 (0)1625 516667.
E-mail address: jamie.scott@astrazeneca.com (J.S. Scott).
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.04.006

3176
J. S. Scott et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3175–3179
O
O
O
O
O
N
O
N
O
N
NO2
N
F
F
H
N
N
O
O
O
O
N
N
N
N
N
N
S
O O
N
N
N
N
Figure 1. Examples of conformationally restricted piperidine based GPR119 agonists.
Table 1
GPR119 binding and cAMP data for aryl sulphones 1–4
O
N
N
O
S
N
N
O
O
O
Core
Compd Core
Human binding IC50
M)
mouse binding IC50
M)
Human cAMP EC50
M)
Human cAMP IA
(%)
Mouse cAMP EC50
M)
Mouse cAMP IA
(%)
(
l
(
l
(
l
(l
N
N
1
2
0.351
0.017
0.543
0.129
0.065
0.019
83
0.041
0.084
99
N
N
150
101
N
N
3
4
0.092
0.003
0.072
0.005
0.079
0.011
133
166
NA
NA
0
0
N
N
Table 2
GPR119 binding and cAMP data for cyanopyridines 5–16
N
N
N
N
N
N
N
N
N
R
N
N
R
N
R N
N
N
O
O
O
N
N
N
A
B
C
Compd
R
Core Human binding IC50
M)
Mouse binding IC50
M)
Human cAMP EC50
M)
Human cAMP IA Mouse cAMP EC50
Mouse cAMP IA
(%)
(l
(
l
(
l
(%)
(lM)
5
6
7
A
B
C
0.010
0.001
0.001
0.507
0.100
0.009
0.021
0.005
0.004
155
171
250
0.120
0.036
NA
102
97
5
O
O
8
9
10
A
B
C
0.025
0.006
0.009
0.758
0.694
0.065
0.020
0.006
0.007
251
182
183
0.252
0.084
NA
98
54
9
O
O
F
F
F
F
11
12
O
A
B
0.018
0.031
4.985
1.827
0.047
0.020
193
132
0.698
0.246
101
76
13
C
0.008
0.104
0.009
203
0.020
31
O
F3C
14
15
N
A
B
0.012
0.003
2.716
0.568
0.019
0.008
167
269
0.242
0.056
74
92
O
16
N
C
0.004
0.122
0.011
229
0.045
41
Intrigued by the lack of effect in the mouse cAMP assay with the
bridged piperazines, we prepared a series of compounds in a
cyanopyridyl subseries of compounds with a range of capping
groups (5–16) as shown in Table 2.
Compounds 5–7 are matched molecular pairs of 1, 3 and 4 with
the aryl sulphone replaced with a cyanopyridyl group. The same
structure–activity relationship was observed with the bridged
compound 7 having the lowest EC50 value and high intrinsic activ-
ity in the human cAMP assay and minimal effect in the murine
cAMP assay despite being the most potent in the binding assay
in both species.18 Further variation of the carbamate to the tetra-
fluoro cyclobutyl (compounds 8–10) and the trifluoroethyl (com-
pounds 11–13) showed similar trends in both human and mouse.
Compound 13 was notable for the fact that it was the first bridged
compound to show a measurable EC50 in the mouse but with
intrinsic activity that was significantly lower than either the
unsubstituted (11) or methyl (12) piperazine, indicating that this
was only
a partial agonist. Switching to an isopropyl 1,2,

J. S. Scott et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3175–3179
3177
4-oxadiazole (compounds 14–16) maintained the trends previ-
ously observed with compound 16 showing full agonism in human
and only partial agonism in mouse.
For oxadiazole 16 the strategy was modified with the pyridyl
substituted with a bromine rather than a cyano group to avoid
any chemoselctivity issues with the later hydroxylamine addition.
Displacement of the 2-chloro pyrimidine and removal of the Boc
group was carried out as before to give the amine which was trans-
formed into the cyanamide using cyanogen bromide. Addition of
hydroxylamine followed by capping with isobutyric acid and ring
closure gave the oxadiazole. Subsequent conversion of the bromide
to the cyano group was achieved under palladium catalysed condi-
tions with zinc cyanide to afford 16 (Scheme 3).
Compounds 11–13 were profiled further to assess the impact of
the piperazine modifications on the properties of the compounds
(Table 3). The logD7.4 values showed no significant changes for
addition of the methyl group in 12 or the ethylene bridge in 13.
This was reflected in the similar values for free drug levels for
the three compounds measured in both mouse and rat. Notably
the solubility of the methyl compound 12 was higher than either
Synthesis of isomeric bridged compounds in the sulphone series
was achieved by nucleophilic displacement of the 2-chloro substi-
tuent of a pyrimidine.19 In the case of compound 3 this was
achieved directly by displacement of the Boc substituted bridged
piperazine. In the case of compound 4, the regioisomeric acetyl
bridged piperazine was used for the initial displacement. The acet-
yl group was then removed using forcing acidic conditions and the
resultant amine capped with a tert-butyl carbamate (Scheme 1).
In the cyanopyridyl series, a similar strategy was employed
with nucleophilic displacement with the Boc substituted bridged
piperazine affording compound 7 directly. Alternative carbamates
were introduced by removing the Boc group and then reacting
the resultant amines with appropriately substituted phenylcarba-
mates to give 10 and 13 (Scheme 2).
O
N
NH
N
N
N
O
S
Cl
O
R N
O
S
N
O
O
O
N
b
R=Ac
R=H
O
c
N
NH
a
d
O
O
N
O
N
N
O
S
N
O
N
O
S
N
O
O
O
N
O
O
N
3
4
i
Scheme 1. Synthesis of sulphones 3 and 4. Reagents and conditions: (a) K2CO3, nBuCN, 165 °C,
85 °C, 6 h, 70%; (d) (tBuO)2CO, K2CO3, 1,4-dimethylaminopyridine, 20 °C, 4 d, 14%.
l
W, 4 h, 28%; (b) Pr2NEt, K2CO3, nBuCN, 160 °C,
lW, 8 h, 29%; (c) 2 M HCl,
O
N
N
N
a
NH
O
N
N
N
O
N
N
Cl
O
O
O
O
N
N
N
N
7
N
N
b
O
N
F
F
N
N
N
O
O
c
O
N
10
N
N
N
N
N
F
F
d
HN
N
O
N
O
N
N
F3C
13
Scheme 2. Synthesis of cyano pyridines 7, 10 and 13. Reagents and conditions: (a) K2CO3, CH3CN, 150 °C,
C4H3F4OCO2Ph, NEt3, CHCl3, 90 °C, 18 h, 30%; (d) CF3CH2OCO2Ph, NEt3, CHCl3, 100 °C, 36 h, 73%.
lW, 8 h, 81%; (b) HCl, 1,4-dioxane/CH2Cl2, 20 °C, 18 h, 100%; (c)
boc N
NH
N
N
Br
c
N
N
N
N
Br
Br
Br
Cl
O
R N
N
N
N
N
N
O
O
O
N
a
N
N
N
R=Boc
R=H
b
d
N
N
N
N
N
N
N
N
O
O
e
N
N
O
N
N
N
16
Scheme 3. Synthesis of oxadiazole 16. Reagents and conditions: (a) K2CO3, CH3CN, 150 °C,
l
W, 8 h, 50%; (b) HCl, 1,4-dioxane/CH2Cl2, 20 °C, 3 h, 89%; (c) CNBr, NaHCO3,
CH2Cl2/H2O, 0–25 °C, 2 h, 78%; (d) (i) NH2OHÁHCl, Na2CO3, DMF, 80 °C, 1 h; (ii) PrCOOH, Pr2NEt, HOBt, EDAC, DMF, 20 °C, 16 h; (iii) toluene, 120 °C, 1 h, 61%; (e) Zn(CN)2,
Pd(dba)2, Xantphos, DMF, 130 °C, W, 2 h, 46%.
i
i
l

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J. S. Scott et al. / Bioorg. Med. Chem. Lett. 23 (2013) 3175–3179
Table 3
Physical properties for compounds 11–13
a
Compd logD7.4
Mouse PPBb (%
free)
Rat PPBb (%
free)
Solubilityc
M)
MDCK permeabilityd Papp
CYPSe IC50
M)
hERGf IC50
M)
Rat Heps Clint (
lL/min/
(
l
(Â10À6 cm sÀ1
)
(
l
(l
106 ells)
11
12
13
3.0
3.0
3.0
9.4
11
9.6
5.1
4.4
6.5
3.3
23
3.8
32 (A–B); - (B–A)
19 (A–B); 10 (B–A)
25 (A–B); 20 (B–A)
All >30
All >30
All >30
10
12
13
12
27
13
a
b
c
d
e
f
Distribution coefficient between 1-octanol and aqueous phosphate buffer at pH 7.4.
% Free compound measured when dialysed with appropriate plasma proteins.
Solubility of compounds in aqueous phosphate buffer at pH 7.4 after 24 h at 25 °C (
lM).
Compounds were incubated at 10
Inhibition of cytochrome P450 enzymes IC50
Inhibition of hERG channel IC50 M) in a electrophysiology (IonWorks™) assay
lM in cultured MDCK cells. Permeability was measured in both the A–B and B–A direction.
(
lM).
(l
Table 4
Pharmacokinetic parameters for compounds 11–13a
Compd
Clp (mL/min/kg)
Vdss (L/kg)
PO half-life (h)
IV half-life (h)
AUC (
lM h)
Bioavailability (%)
11
12
13
22
14
11
5.3
1.1
1.0
2.1
2.2
2.6
3.9
1.4
1.7
4.0
3.2
2.1
48
27
17
a
Compounds were dosed at 2 mg/kg (IV) and 5 mg/kg (po) in 5% DMSO:95% hydroxylpropyl beta cyclodextrin, and a 0.1% pluronic F127 suspension, respectively at
volumes of 5 mL/kg.
Table 5
Oral glucose tolerance test (OGTT) results for compounds 8 and 10–13a
Compd
Mouse GPR119 EC50
(
lM)
Mouse GPR119 IA (%)
Mouse GPR119 binding (
lM)
% Reduction in OGTT blood glucose AUCa
8
0.252
NA
0.698
0.246
0.020
98
9
101
76
31
0.758
0.065
4.985
1.827
0.104
30
3
21
10
6
10
11
12
13
a
Compounds administered 30 min prior to a glucose load of 2 g/kg and glucose levels monitored out to 90 min.
the piperazine or bridged compound, perhaps reflecting the dis-
ruption to the crystal packing by this axial methyl group observed
in previous work.15a All three compounds were highly permeable
as measured in a Madin-Darby Canine Kidney (MDCK) epithelial
cell line and showed no inhibition against five isoforms of the cyto-
chrome P450 enzymes (CYP1A2, CYP2C9, CYP2C19, CYP2D6 and
CYP3A4). Compounds showed similar activity against the hERG
channel and were moderately stable in rat hepatocytes.
Profiling of the compounds in rat showed similar profiles for all
three matched cores (unsubstituted, methyl and bridged) with low
to moderate clearance and bioavailabilities and a difference in AUC
of less than twofold (Table 4).
development of the binding assays and Rob Garcia and Steve Bloor
are thanked for the generation of in vitro data. Jo Teague and Lar-
aine Gregory are thanked for contributions to in vitro OGTT data
generation.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.04.
006.
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In order to assess the relevance of the mouse cAMP results
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compete for binding sites on the receptor with
a
standard [3H]-labelled
standard GPR119 agonist. The human or mouse receptor was overexpressed
with a Gs protein in a baculovirus system, from which membranes were
prepared. All binding data is the mean of at least two determinations. See
Supplementary data for full details.
17. Full details of the cAMP assay are described in Ref. 15a. The intrinsic activity
was expressed as the percent effect compared to that of the control, 50 lM
oleoylethanolamide, defined as 100%. All human cAMP data is the mean of at
least two determinations. All mouse cAMP data is the mean of at least two
determinations with the exception of compounds 3, 4, 7, 8 and 11 which is
n = 1 data.
A typical standard deviation in logEC50 when a compound is
repeated is 0.20 and 0.27 for the human and mouse assays respectively. This
translates to 95% of EC50 values within 2.5-fold (human) and 3.5-fold (mouse)
of a compound’s ‘true’ EC50
.
18. Some elevation above baseline (<5%) was observed however this was not of
sufficient magnitude to be able to determine an EC50 value. This led us to
conclude that these were not inverse agonists, but we did not confirm their
antagonist status in mouse.
19. The synthesis of compounds 1, 2, 5, 6, 11, 12, 14 and 15 are described in PCT
Int. Appl. (2011), WO 2011030139 A1.