Discovery and investigation of a novel class of thiophene-derived antagonists of the human glucagon receptor

Article abstract of DOI:10.1016/j.bmcl.2005.01.003

A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This SAR e

Full text of DOI:10.1016/j.bmcl.2005.01.003

Bioorganic & Medicinal Chemistry Letters 15 (2005) 1401–1405
Discovery and investigation of a novel class of
thiophene-derived antagonists of the human glucagon receptor
Joseph L. Duffy,a,* Brian A. Kirk,a Zenon Konteatis,a Elizabeth L. Campbell,a Rui Liang,a
Edward J. Brady,b Mari Rios Candelore,c Victor D. H. Ding,c Guoqiang Jiang,c
Frank Liu,c Sajjad A. Qureshi,c Richard Saperstein,b Deborah Szalkowski,c Sharon Tong,a
Lauri M. Tota,c Dan Xie,c Xiaodong Yang,c Peter Zafian,b Song Zheng,a
Kevin T. Chapman,a Bei B. Zhangc and James R. Tataa
aDepartment of Basic Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Pharmacology, Merck Research Laboratories, Rahway, NJ 07065, USA
cDepartment of Metabolic Disorders, Molecular Endocrinology, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 24 November 2004; revised 28 December 2004; accepted 4 January 2005
Available online 23 January 2005
Abstract—A novel class of antagonists of the human glucagon receptor (hGCGR) has been discovered. Systematic modification of
the lead compound identified substituents that were essential for activity and those that were amenable to further optimization. This
SAR exploration resulted in the synthesis of 13, which exhibited good potency as an hGCGR functional antagonist (IC50 = 34 nM)
and moderate bioavailability (36% in mice).
Ó 2005 Elsevier Ltd. All rights reserved.
Diabetes mellitus is a condition characterized by chron-
ically elevated levels of blood glucose. This condition
arises from inappropriate secretion and activity of the
two major hormones that control glucose homeostasis,
insulin, and glucagon. Diabetic insulin resistance and
insulin deficiency impair the bodyÕs ability to utilize glu-
cose from the bloodstream. This condition is commonly
aggravated by chronically elevated levels of the hormone
glucagon, or hyperglucagonemia.1 The peptide glucagon
is normally secreted from pancreatic a cells in response
to depleted plasma glucose levels. This hormone acti-
vates the hepatic glucagon receptor (GCGR) and trig-
gers the synthesis of glucose (gluconeogenesis) and
processing and release of hepatic glycogen stores (glyco-
genolysis) to elevate and restore plasma glucose levels.
In diabetes mellitus the inappropriately high hepatic glu-
cose production from glucagon and the inappropriately
low glucose utilization from insulin both contribute to
chronic hyperglycemia. Many therapeutic approaches
to the treatment of diabetes have focused on the normal-
ization and utilization of plasma insulin.1c,2 An alterna-
tive or additional therapy may be realized by blocking
hepatic glucose production using glucagon antagonists.3
Peptidyl antagonists such as 1 (Table 1) are known,4 and
several small molecule antagonists are under investiga-
tion as well.1a,5
Recently we completed a high throughput screening pro-
gram directed toward discovering compounds that com-
petitively inhibit the binding of 125I-glucagon to the
human glucagon receptor (hGCGR). Among the com-
pounds identified for further investigation were a series
of acylated aminothiophene nitriles exemplified by 2.6
The syntheses of the bicyclic thiophene derivatives 2–9
were accomplished using the Gewald cyclization as illus-
trated in Scheme 1.7 The cyclohexanone derivatives I
were condensed with malononitrile, followed by addi-
tion of elemental sulfur, affording the bicyclic aminothio-
phenes II. Acylation of these intermediates afforded the
desired amides III. The amine-substituted derivatives 5–
9 were prepared from the corresponding dioxolane
derivative IV. Removal of the ketal protecting group
with aqueous acid followed by successive reductive
aminations afforded the final products 5–9.
Keywords: Glucagon; Diabetes; Thiophene; Gewald.
*
Corresponding author. Tel.: +1 732 594 1062; fax: +1 732 594
5350; e-mail: joseph_duffy@merck.com
0960-894X/$ - see front matter Ó 2005 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2005.01.003

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J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1401–1405
Table 1. Binding and functional activity of antagonists of the human glucagon receptor
Compound
Diastereomera
Binding IC50 (nM)b
18
cAMP IC50 (nM)b
18
1
Des-His1[Glu9]glucagon-NH2
2
3
4
181
>20,000
870
129
—
38%c
5
3820
—
6
7
D1
D2
139
118
158
59
8
9
92
63
59
129
10
11
153
41
3210
—
12
368
185
13
89
34
a All compounds containing one or more chiral centers were tested as racemic mixtures.
b Values reported are the mean of a minimum of two experiments with the standard deviation <50% of the mean.
c Percent inhibition of glucagon cAMP accumulation at 10 lM compound concentration.

J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1401–1405
1403
Scheme 1. Reagents and conditions: (a) malononitrile, S8, morpholine, EtOH, 70 °C; (b) 2-ethylbutyryl chloride, DIEA, CH2Cl2; (c) 1.0 N HCl (aq),
THF; (d) R3NH2, NaBH(OAc)3, 1% AcOH in CH2Cl2; (e) R4CHO, NaBH(OAc)3, 1% AcOH in CH2Cl2.
Scheme 2. Reagents and conditions: (a) malononitrile, S8, morpholine, EtOH, 70 °C; (b) 2-ethylbutyryl chloride, DIEA, CH2Cl2 (49%—two steps);
(c) TFA, CH2Cl2 (92%); (d) R1R2NH, EDC, HOBT, DIEA, DMF; (e) EDC, MeOH; (f) NH2NH2, MeOH, 65 °C; (g) R1CO2H, EDC, HOBT, DMF;
(h) SO2Cl, pyridine, Et2O, 0 °C, then 100 °C in toluene; (i) R1C@NHOH(NH2), PyBop (2 equiv), DMF.
These compounds were assayed by measurement of inhi-
bition of binding of 125I-glucagon to the hGCGR ex-
pressed in CHO cell membrane, affording the binding
IC50 values in Table 1. The active compounds were fur-
ther examined for functional antagonist potency, as
measured by the inhibition of glucagon induced cAMP
accumulation in hGCGR transfected CHO cells (cAMP
IC50, Table 1).6
clude empirically that the 3-cyanothiophene amide rep-
resents an optimized pharmacophore.
The cyclohexyl moiety on 2 was more amenable to SAR
optimization. We sought to improve the physical prop-
erties of 2 (logD = 6.6) by incorporating a more hydro-
philic amine substituent. Although the N-methyl
benzylamine 5 was substantially less active in the bind-
ing assay, the activity was restored by constraining the
benzyl substituent with a fused cyclohexyl ring as in
the diastereomers 6 and 7. The diastereomers also exhib-
ited enhanced hydrophilicity (logD = 4.8). Alterna-
tively, gains in the potency of 5 were achieved with the
addition of substituents on the phenyl ring. Extensive
investigation lead to the identification of the 2,4-dichlo-
robenzylamine substituent in 8 and 9, which retained the
antagonist potency of 7 but yielded the gains in hydro-
philicity (8 logD = 6.6).
The thiophene derivative 2 displayed moderate activity
as an antagonist of the human glucagon receptor with
a
binding IC50 of 181 nM and similar potency
(IC50 = 129 nM) in the cAMP accumulation assay. Ali-
phatic amides were tolerated as substituents on the ami-
no moiety, whereas aromatic amides such as 3 were
inactive. The nitrile substituent was also found to be
important for activity, although the related terminal
amide derivative 4 retained measurable potency in the
binding and functional assays. The thiophene heterocy-
cle was found to be crucial for activity, as direct ana-
logues of active thiophene derivatives featuring a
suitably substituted furan, pyrrole, or phenyl ring were
inactive in the binding assay (data not shown). While
we have no information on specific interactions between
these compounds and the glucagon receptor, we con-
The Gewald cyclization is generally applicable to both
cyclic and acyclic ketones, and may employ a variety
of malonyl electrophiles. Thus, using an extension of
this procedure as exemplified in Scheme 2, additional
positions on the aminothiophene ring were indepen-
dently modified to explore the SAR of this class. We

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J. L. Duffy et al. / Bioorg. Med. Chem. Lett. 15 (2005) 1401–1405
Table 2. Pharmacokinetic parameters of glucagon antagonists dosed in mice (n = 3 mice/route of administration)
Compound Species Dose PO/IV (mg/kg) CLp (mL/min/kg) AUCN (PO) (lM h/dose) Vdss (L/kg) t1/2 (h) Cmax (lM) F %
8
Mouse
Mouse
Mouse
2.0/1.0
10.0/5.0
10.0/5.0
50
84
23
0.07
0.21
0.62
3.4
16
8.7
1.9
2.7
5.1
0.08
0.80
0.80
9.2
39
36
12
13
investigated the replacement of the lipophilic cyclohexyl
moiety with alternative functionality that would simi-
larly constrain the conformational freedom of the distal
substituents relative to the thiophene, including the use
of amides and heterocycles as in 10–13 (Table 1). The
syntheses of these derivatives are illustrated in Scheme
2. The Gewald cyclization was performed on b-ketoest-
ers such as VI, followed by acylation of the aminothio-
phene product, to afford VII in moderate yield.
Deprotection of the carboxyl substituent afforded the
carboxy thiophene VIII for derivatization. This interme-
diate was coupled with secondary amines under stan-
dard conditions to afford amides IX. The carboxyl
intermediate VIII was otherwise derivatized to the corre-
sponding oxadiazoles8 X or isoxadiazoles9 XI using pub-
lished methods.
(9.2%), affording rapid clearance (50 mL/min/kg) and a
very low maximal blood concentration of only 80 nM.
While 12 did afford greater overall bioavailability at a
higher dose (39%), the compound was rapidly metabo-
lized in vivo, affording a clearance of 84 mL/min/kg.
The corresponding dichlorobenzyl analogue 13 afforded
substantially lower clearance in vivo (23 mL/min/kg), a
longer half life (5.1 h) and a 3-fold greater oral AUC
than the isobutyl analogue 12.
Finally, the antagonist 13 was tested for the ability to
block glucagon-induced glycogenolysis in vivo using
transgenic mice that exclusively express a functional hu-
man glucagon receptor. The full characterization of
these mice and their response to known antagonists have
been reported previously.11 Oral administration of 13
(100 mpk, n = 9 mice) was followed 45 min later by an
IP injection of glucagon (15 lg/kg). In this experiment
13 did not afford a statistically significant reduction in
glucagon-induced glucose excursion AUC as compared
to administration of glucagon alone. One possible rea-
son for this lack of in vivo efficacy may be the shift to-
ward lower intrinsic potency in the presence of
plasma. Addition of 5% mouse plasma to the functional
assay afforded a 15-fold decrease in the cAMP IC50 of
13.12
The intrinsic potency of the thiophene-derived antago-
nists was maintained with the removal of the cyclohexyl
ring. The N-isopropyl benzyl amide 10 afforded potency
similar to the cyclohexylamine derivative 9, with an
IC50 = 41 nM in the cAMP assay. In order to reduce
the peptidic nature of the compounds, we sought to re-
place the secondary amide with heterocyclic isosteres.
Replacement of the amide substituent with the isobutyl
substituted oxadiazole afforded 11, which lost over an
order of magnitude in potency in the binding assay.
However, the corresponding isoxadiazole 12 was sub-
stantially more potent in the binding assay, and afforded
moderate activity in the functional assay as well, with an
IC50 of 185 nM. Finally, substitution of the isoxadiazole
with the 2,4-dichlorobenzyl substituent, which was
found to be optimal in the amine and amide series, affor-
ded 13 with gains in potency demonstrated in both the
binding assay (IC50 = 89 nM) and the functional cAMP
assay (IC50 = 34 nM).
A novel class of thiophene-derived antagonists of the
hGCGR has been discovered. An investigation of this
class has resulted in the identification of the substituted
isoxadiazole 13 as a lead for further investigation.
Improvements were made in both the intrinsic potency
and in vivo exposure of this class of compounds. The
further optimization of oral exposure and minimization
of plasma interference in activity remain as challenges to
achieving pharmacodynamic efficacy with this class of
antagonists.
The activity of both the initial lead 2 and the more po-
tent derivative 13 toward the related human GIP and
GLP-1 receptors was also examined.10 As these two
receptors represent insulinotropic mechanisms, antago-
nism of these targets should be minimized. The bicyclic
thiophene 2 was indeed inactive against both the hGLP-
1 and hGIP receptors, with a functional IC50 > 20 lM in
the respective cAMP accumulation assays. The more po-
tent isoxadiazole derivative 13 retained over 30-fold
selectivity over the hGLP-1 receptor, with an
IC50 = 1.18 lM in the cAMP assay, and exhibited simi-
larly modest activity (IC50 = 973 nM) against the hGIP
receptor.
Acknowledgements
We are very grateful to Judy Fenyk-Melody, Irene
Capodanno, Xiaolan Shen, John Strauss, and ZuLiang
Yao for in vivo pharmacokinetic studies.
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