Identification of a novel conformationally constrained glucagon receptor antagonist

Article abstract of DOI:10.1016/j.bmcl.2013.12.090

Identification of orally active, small molecule antagonists of the glucagon receptor represents a novel treatment paradigm for the management of type 2 diabetes mellitus. The present work discloses novel glucagon receptor antagonists, identified via confo

Full text of DOI:10.1016/j.bmcl.2013.12.090

Accepted Manuscript
Identification of a Novel Conformationally Constrained Glucagon Receptor
Antagonist
Esther C.Y. Lee, Meihua Tu, Benjamin D. Stevens, Jianwei Bian, Gary Aspnes,
Christian Perreault, Matthew F. Sammons, Stephen W. Wright, John Litchfield,
Amit S. Kalgutkar, Raman Sharma, Mary T. Didiuk, David C. Ebner, Kevin J.
Filipski, Janice Brown, Karen Atkinson, Jeffrey A. Pfefferkorn, Angel Guzman-
Perez
PII:
S0960-894X(13)01474-1
DOI:
http://dx.doi.org/10.1016/j.bmcl.2013.12.090
BMCL 21198
Reference:
Bioorganic & Medicinal Chemistry Letters
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 November 2013
18 December 2013
19 December 2013
Please cite this article as: Lee, E.C.Y., Tu, M., Stevens, B.D., Bian, J., Aspnes, G., Perreault, C., Sammons, M.F.,
Wright, S.W., Litchfield, J., Kalgutkar, A.S., Sharma, R., Didiuk, M.T., Ebner, D.C., Filipski, K.J., Brown, J.,
Atkinson, K., Pfefferkorn, J.A., Guzman-Perez, A., Identification of a Novel Conformationally Constrained
Glucagon Receptor Antagonist, Bioorganic & Medicinal Chemistry Letters (2013), doi: http://dx.doi.org/10.1016/
j.bmcl.2013.12.090
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Identification of a Novel Conformationally Constrained Glucagon
Receptor Antagonist
Leave this area blank for abstract info.
Esther C. Y. Lee,* Meihua Tu, Benjamin D. Stevens, Jianwei Bian, Gary Aspnes, Christian Perreault, Matthew F.
Sammons, Stephen W. Wright, John Litchfield, Amit S. Kalgutkar, Raman Sharma, Mary T. Didiuk, David C. Ebner,
Kevin J. Filipski, Janice Brown, Karen Atkinson, Jeffrey A. Pfefferkorn, and Angel Guzman-Perez.

Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com
Identification of a Novel Conformationally Constrained Glucagon Receptor
Antagonist
Esther C. Y. Lee,1,§ Meihua Tu,§ Benjamin D. Stevens,§ Jianwei Bian,¥ Gary Aspnes,¥ Christian Perreault,¥
Matthew F. Sammons,§ Stephen W. Wright, ¥ John Litchfield,§ Amit S. Kalgutkar,§ Raman Sharma,¥ Mary
T. Didiuk,¥ David C. Ebner,§ Kevin J. Filipski,§ Janice Brown,¥ Karen Atkinson,¥ Jeffrey A. Pfefferkorn,§
and Angel Guzman-Perez¥
§Pfizer Worldwide Research & Development, 620 Memorial Drive, Cambridge, Massachusetts 02139, United States
¥ Pfizer Worldwide Research & Development, Eastern Point Rd., Groton, Connecticut 06340, United States
ARTICLE INFO
ABSTRACT
Article history:
Received
Revised
Identification of orally active, small molecule antagonists of the glucagon
receptor represents a novel treatment paradigm for the management of type 2
diabetes mellitus.
The present work discloses novel glucagon receptor
Accepted
antagonists, identified via conformational constraint of current existing literature
antagonists. Optimization of lipophilic ligand efficiency (LLE or LipE)
culminated in enantiomers (+)-trans-26 and (-)-trans-27 which exhibit good
physicochemical and in vitro drug metabolism profiles. In vivo, significant
pharmacokinetic differences were noted with the two enantiomers, which were
primarily driven through differences in clearance rates. Enantioselective oxidation
by cytochrome P450 was ruled out as a causative factor for pharmacokinetic
differences.
Available online
Keywords:
Glucagon receptor antagonist
Diabetes mellitus
Physicochemical properties
Metabolite
2013 Elsevier Ltd. All rights reserved.
———
1 Corresponding author. Tel: +1 (617) 551-3508 (E. C. Y. L.)
Email address: esther.lee2@pfizer.com (E. C. Y. Lee)

Type 2 Diabetes Mellitus (T2DM), a chronic disease
prevalent worldwide, is characterized by impaired insulin
secretion and insulin resistance in muscle and liver, resulting
in higher blood glucose levels. As insulin resistance and the
resulting hyperinsulinemia worsen over time, patients
eventually experience -cell failure. Despite the existence of
several therapies, there remains a need to identify novel
mechanisms of restoring glucose homeostasis.1 Glucagon is a
29-amino acid peptide secreted in the -cells of the pancreas
that binds to the glucagon receptor in the liver to modulate
hepatic glycogenolysis and gluconeogenesis. Blockade of the
glucagon receptor has been proposed as an avenue to reduce
hepatic glucose output, thus enabling improved glycemic
control.2
the lipophilic ligand efficiency, (LLE or LipE, as it will be
presented from here on).11
In an effort to identify the conformational preference
of glucagon receptor antagonists,
a
computational
conformational search of literature glucagon antagonists 2,5
3,6 and 58b was conducted in MacroModel (Version 9.0.
Schrodinger Inc., New York). To simplify the analysis, the
-alanine amide (Figure 1: highlighted in red) was kept
constrained during the calculation.
The lowest energy
conformer of compound 3 was used as the preferred binding
conformation, because of its relative rigidity. The measured
low energy conformers (within 3 Kcal/mol of relative
conformational energy) of compounds 2 and 5 were then
manually overlaid. The second lowest energy conformer of
compound 2 and the lowest energy conformer of compound 5
provided the best overlay with the template (Figure 2), and
were selected as the possible binding conformations for
In the last two decades, various small molecule
glucagon receptor antagonists have been identified.3 Most of
these candidates contain an acidic moiety with the exception
of Bay 27-9955 (1)4 (Figure 1), which demonstrated a clinical
proof of mechanism via blockade of glucose excursion
following
a
glucagon challenge in healthy subjects.
optimal glucagon antagonism.
The proposed binding
Throughout several lead optimization programs, the -alanine
side-chain has emerged as an important motif for interaction
with the glucagon receptor (see Figure 1, highlighted in red).
Novo-Nordisk was the first to report this motif5a and
subsequent optimizations culminated in NNC 25-0926 (2),
which showed reduction in hepatic glucose production during
a glucagon challenge in dogs.5c Merck and Lilly have also
disclosed clinical candidates, MK-0893 (3)6a and LY2409021
(structure undisclosed),6b respectively, which demonstrated
robust clinical HbA1c reductions in diabetic patients.
conformation of compound 5 was later proved to be identical
to the X-ray structure of the small molecule alone.8b
Another common theme from these glucagon
antagonists are high molecular weight and high lipophilicity,
which is similar to the endogenous ligands for class B
GPCRs.7 In continuation of our previous work to identify
novel glucagon receptor antagonists (Figure 1, compounds 48a
and 58b) that possess drug-like attributes (MW <500, logD
<3), we pursued the incorporation of a cyclic core in our
previously reported series8b as a tactic to further increase
antagonist activity against the glucagon receptor.
Figure 2. Overlay of low energy conformers of compound 2
(purple), 3 (blue) and 5 (orange).
The proposed binding conformation led to the
hypothesis that the conformation of compound 5 might be
reinforced by the introduction of appropriate cyclic
constraints, for example, the pyrrolidine structure (6) in
Figure 3, or other 5-member rings which will be discussed.
Given synthetic considerations, we decided to initially test the
hypothesis using the des-methyl pyrrolidine analogue ((+/-)-
11) (Scheme 1).
Constrain
6
5
Figure 3. Introducing a cyclic constraint in compound 5 to provide
compound 6.
Figure 1. Literature glucagon receptor antagonists and their
molecular weight and calculated logD (clogD).
The pyrrolidine analogue ((+/-)-11) was synthesized
according to the route shown in Scheme 1. Pyrrolidine
intermediate (+/-)-9 was prepared via a Suzuki reaction
between pyrrole boronic acid 7 and 4-bromobenzoate 8
followed by hydrogenation of the pyrrole and removal of the
Conformational restraint is an established strategy
for improving potency and selectivity against pharmacologic
targets,9 including the glucagon receptor.10 In the present
case, the rationale for introducing a cyclic core was to
improve potency without increasing logD, thereby increasing

Boc group. N-arylation of pyrrolidine (+/-)-9 with 4-bromo-
4'-trifluoromethylbiphenyl provided the bis-substituted
The synthesis of cyclopentanes (+)-trans-26 and (-)-
trans-27 are illustrated in Scheme 3. Iodopyrimidine 17 was
cross-coupled with 4-trifluoromethylphenyl boronic acid (18)
to yield bromide 19, which was subsequently converted to
intermediate boronate 20 via a palladium-mediated coupling
pyrroldine 10 as
a
racemic mixture.
Base-mediated
hydrolysis of racemic ester 10 afforded the corresponding
carboxylic acid which was then subjected to a standard amide
coupling with -alanine methyl ester to provide the
homologated ester, which was hydrolyzed to (+/-)-11.
with bis(pinacolato)diboron.
A
Suzuki reaction with
commercially available 2-iodocyclopent-2-enone afforded -
aryl cyclopentenone 21, which represents the first appendage
onto the cyclopentane core. A conjugate addition was
performed using a cuprate generated from the Knochel-type
Grignard reagent prepared from ethyl-4-iodobenzoate and
copper(I) iodide under Lewis acid conditions to provide the
bis-substituted cyclopentanone (+/-)-2213 in the trans
configuration as shown. In order to reduce the ketone, (+/-)-
22 was protected with ethane-1,2-dithiol and the dithiolane
was subjected to hydrogenolysis with Raney nickel to provide
the cyclopentane core (+/-)-24 in 72% yield. Saponification
generated the carboxylic acid that subsequently was treated
with -alanine ethyl ester to secure the fully elaborated side-
chain (+/-)-25. Ethyl ester (+/-)-25 was purified by chiral
supercritical fluid chromatography to deliver the separated
enantiomers. Each enantiomer was hydrolyzed separately to
provide the final compounds (+)-trans-26 and (-)-trans-27,
each at 99% enantiomeric excess.
Scheme 1. Synthetic method for the synthesis of pyrrolidine
analogue (a) Pd(PPh3)4, Na2CO3, DME/water, 3 h, reflux, 88%; (b)
30 psi H2, 5% Pt/C, AcOH, 25 °C, 24 h, 84%; (c) 4 M HCl,
dioxane/CH2Cl2,
25
°C,
2.5
h;
(d)
4-bromo-4'-
trifluoromethylbiphenyl, Pd2(dba)3, DavePhos, NaHMDS, THF, 65
°C, 20 h, 21%; (e) LiOH, THF/H2O/MeOH, 60 °C, 20 h; (f) -
alanine methyl ester hydrochloride, EDC, Et3N, HOAt, CH2Cl2, 25
°C, 17 h, 80%, 2 steps; (f) LiOH, THF/H2O, 25 °C, 3 h, 62%.
The pyrrolidinone analogue ((+/-)-16) was prepared
as shown in Scheme 2.
Condensation between 4’-
(trifluoromethyl)biphenyl-4-amine (12) and methyl 4-
formylbenzoate (13) provided imine 14. A nickel-catalyzed
regioselective reductive coupling between imine 14 and
methyl acrylate afforded an intermediate that cyclized to the
bis-substituted pyrrolidinone (+/-)-15 upon heating with p-
TsOH.12 Standard side chain elaborations as displayed in
Scheme 1 were performed to produce analogue (+/-)-16.
Scheme 3. Synthetic method for the synthesis of cyclopentyl
analogs: (a) Pd(dppf)Cl2, Na2CO3 (2 M), CH3CN, 0 °C, 16 h, 90%;
(b) bis(pinacolato)diboron, PdCl2(dppf), KOAc, DMF/H2O, 75 °C, 3
h, 84%; (c) 2-iodocyclopent-2-enone, Pd(dppf)Cl2, K3PO4,
DME/H2O, 80 °C,
1
h, 70%; (d) ethyl-4-iodobenzoate,
iPrMgCl•LiCl, -45 °C, 1 h, then CuI, -20 °C , 30 min., then
BF3•OEt2, THF, -40 to -20 °C, 1 h, 44%; (e) ethane-1,2-dithiol, p-
TsOH, PhMe, 100 °C, 7 h, 69%; (f) Raney Ni, EtOH, 100 °C, 3 h,
72%; (g) 1 N NaOH, THF/MeOH, 25 °C, 18 h; (h) ethyl-3-
aminopropanonate, EDC, HOAt, Et3N, CH2Cl2, 25 °C, 75% over 2
steps; (i) 1 N NaOH, THF/MeOH, 25 °C, 18 h, (+)-trans-26, 91%,
99% ee and (-)-trans-27, 93%, 99% ee, respectively.
Scheme 2. Synthetic method for the synthesis of pyrrolidinone
analogue (a) MeOH, reflux, 1 h, 62%; (b) methyl acrylate, Zn,
NiBr2(phen), CH3CN/H2O, 80 °C, 18 h, then p-TsOH, n-BuOH, 120
°C, 18 h, 21%; (c) NaOH, THF/H2O, 25 °C, 18 h, 97%; (d) -alanine
methyl ester hydrochloride, EDC, Et3N, HOAt, CH2Cl2, 25 °C, 18 h;
(e) LiOH, THF/H2O, 25 °C, 30 min., 34% yield over 2 steps.

Binding and functional assays were utilized to assess
in vitro pharmacology of the glucagon receptor antagonists
prepared in these studies. The binding affinity of test
compounds for the human glucagon receptor was assessed by
their ability to displace [125I] Glucagon-Cex from membranes
containing the human glucagon receptor.14 The functional
activity was determined by the ability to inhibit glucagon-
induced cAMP production in a cell line expressing the human
glucagon receptor. Binding and functional assays were
performed in the presence of 0.2% and 4% bovine serum
albumin (BSA), respectively, to understand the impact of
protein binding on compound potency.
As seen in Table 1, (+/-)-11, a close-in compound to
the modeled analogue (6), displays moderate binding and
functional activity for the human glucagon receptor compared
to compound 5, which provided satisfactory proof of the
conformational restraint concept. However, apart from the
modest potency, (+/-)-11 also possesses unfavorable
physicochemical properties. (+/-)-11 is lipophilic (logD =
3.92) leading to a low LipE (1.1) and demonstrates poor
passive absorptive permeability (0.48 x 10-6 cm/sec) in the
Madin-Darby canine kidney-low efflux (MDCKII-LE)
assay.15 Furthermore, (+/-)-11 displays oxidative metabolic
turnover as judged from its intrinsic clearance (Clint) in human
liver microsomes (HLM). The metabolic clearance is a
possible manifestation of its high logD. Encouraged by the
activity of compound (+/-)-11, we decided to try to increase
LipE by adding polarity on the core ring. A change of
constraint from pyrrolidine to pyrrolidin-2-one (+/-)-16
retained binding affinity but not functional activity. The LipE
gain (1.1 to 3.2) was solely from reducing logD and not by
increasing potency. Compound (+/-)-16 was hypothesized to
be resistant to metabolic turnover in HLM due to low
lipophilicity (logD < 3). From these two examples, the
nitrogen-containing ring motif would have a low probability
of delivery of a potent, high LipE compound.
The distal para-trifluoromethylbiphenyl and the
central phenyl substituents in compound (+/-)-28 served as
starting points for incorporation of polarity. Replacement of
the distal aryl ring with a trifluoromethylpyrazole group ((+/-
)-trans-31) and placement of a nitrogen in the central phenyl
ring of (+/-)-trans-31 to yield compound (+/-)-trans-32
decreased both lipophilicity and antagonist potency (Table 2).
Since altering the distal ring seemed to have a smaller chance
of success, we aimed for manipulation of the central ring.
Previously, we have shown beneficial effects via addition of
dimethyls ortho to the pyrazole. The dimethyls increase
Further modeling effort showed that the trans
substitution of a cyclopentane core would provide a similar
desired overlap to the pyrrolidine (structure overlay provided
in the supporting material). Indeed, a change of the constraint
to all-carbon resulted in improved binding and functional
affinities as shown in racemic cyclopentyl variant (+/-)-28 (Ki
= 0.022 M, Kb = 0.495 M). Although, the logD is high
(3.68), which may explain the higher CLint in HLM (25.5
mL/min/kg), the LipE is higher than (+/-)-11 which has
comparable lipophilicity and clearance. Therefore, from a
lipophilic efficiency standpoint, the cyclopentane core of
enantiomers 29 and 30 held higher promise as a lead in terms
of its likelihood to retain potency when changes were made to
reduce logD.
potency and have
a minimal effect on lipophilicity
presumably due to a change in the conformation of the
terminal pyrazole.8b The logD of (+/-)-trans-33 is similar to
(+/-)-trans-31 and the potency improved 3-fold leading to a
LipE gain of 0.6. Unfortunately, the HLM turnover increased
significantly to 132 mL/min/kg. Not surprisingly, the cis
analogue, (+/-)-cis-34, was devoid of functional activity, as it
did not overlay well with the binding conformation (structure
overlay provided in the supporting material). In contrast,
replacement of the central phenyl ring in 29 and 30 with a
pyrimidine led to enantiomers (+)-trans-26 and (-)-trans-27
with a reduced logD value. Gratifyingly, they also retained
the glucagon receptor antagonist potency observed with 29
and 30 with good LipE (3.5-3.6).

of (+)-trans-26 and (-)-trans-27. Interestingly, examination of
the dog pharmacokinetics of (+)-trans-26 and (-)-trans-27
revealed significant differences in clearance for the individual
enantiomers; compound (+)-trans-26 showed higher clearance
in dogs compared to (-)-trans-27 in two experiments. Both
enantiomers demonstrate little to no metabolic turnover in
DLM and dog hepatocytes suggesting a lack of chiral bias in
metabolic elimination. Likewise, metabolic profiling revealed
only trace amounts of cyclopentyl ring oxidation (possibly
mediated by cytochrome P450 enzymes) as a metabolic fate
for both compounds.
No metabolites derived from
glucuronidation were observed in these studies. Considering
that biliary excretion appears to be a major pathway for the
clearance of (+)-trans-26 and (-)-trans-27 in rats, we
speculate that the clearance differences in the dog possibly
arise through an enantiospecific interaction of (+)-trans-26
(relative to (-)-trans-27) with a transporter(s) responsible for
biliary efflux in the dog.
Both enantiomers (+)-trans-26 and (-)-trans-27 were
resistant to oxidative metabolism in HLM, rat liver
microsomes (RLM), and dog liver microsomes (DLM) (Table
3). Likewise, little to no metabolic turnover was observed
upon incubation of (+)-trans-26 and (-)-trans-27 in
cryopreserved hepatocytes from human, rat and dog.
In conclusion, a pharmacophore model of potent
glucagon antagonists guided the development of a new
structural series with good physicochemical and
pharmacokinetic properties. Through ring constraint, we
hypothesized that we could lock the desired side chains to
provide optimal interactions with the receptor. As a result, we
found that both good properties and potency can be obtained
in the cyclopentane core motif. These changes point to the
possibility of further improving both potency and
physicochemical properties for the glucagon receptor
antagonist, yielding higher LipE and potentially better PK
profiles in vivo. Finally, we have also identified an interesting
disconnect between the in vivo PK of two enantiomers in dog
that is worth further investigation.
In vivo pharmacokinetics of (+)-trans-26 and (-)-trans-27
were also assessed in Wistar-Han rats following intravenous
administration at 1 mg/kg (Table 4). Unfortunately, a
disconnect was noted between the low predicted clearance
from RLM and rat hepatocytes and the observed high plasma
clearance for these compounds (in particular, compound (+)-
trans-26). Upon reexamination of the pharmacokinetics in
bile-duct cannulated rats, ~ 40-45% of unchanged dosed
parent compounds were measured in rat bile, which implies
that biliary excretion is a major contributor to the elimination
Supporting information
See attached files.

Tata, J. R.; Parmee, E. R. Bioorg. Med. Chem. Lett. 2007,
17, 587.
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