Human glucagon receptor antagonists with thiazole cores. A novel series with superior pharmacokinetic properties

Article abstract of DOI:10.1021/jm8016249

The aim of the work presented here was to design and synthesize potent human glucagon receptor antagonists with improved pharmacokinetic (PK) properties for development of pharmaceuticals for the treatment of type 2 diabetes. We describe the preparation of compounds with cyclic cores (5-aminothiazoles), their binding affinities for the human glucagon and GIP receptors, as well as affinities for rat, mouse, pig, dog, and monkey glucagon receptors. Generally, the compounds had slightly less glucagon receptor affinity compared to compounds of the previous series, but this was compensated for by much improved PK profiles in both rats and dogs with high oral bioavailabilities and sustained high plasma exposures. The compounds generally showed species selectivity for glucagon receptor binding with poor affinities for the rat, mouse, rabbit, and pig receptors. However, dog and monkey glucagon receptor affinities seem to reflect the human situation. One compound of this series, 18, was tested intravenously in an anesthetized glucagon-challenged monkey model of hyperglucagonaemia and hyperglycaemia and was shown dose-dependently to decrease glycaemia. Further, high plasma exposures and a long plasma half-life (5.2 h) were obtained.

Full text of DOI:10.1021/jm8016249

J. Med. Chem. 2009, 52, 2989–3000
2989
Human Glucagon Receptor Antagonists with Thiazole Cores. A Novel Series with Superior
Pharmacokinetic Properties
Peter Madsen,* Ja´nos T. Kodra, Carsten Behrens, Erica Nishimura, Claus B. Jeppesen, Lone Pridal, Birgitte Andersen,
Lotte B. Knudsen, Carmen Valcarce-Aspegren, Mette Guldbrandt, Inge T. Christensen, Anker S. Jørgensen, Lars Ynddal,
Christian L. Brand, Morten Aa. Bagger, and Jesper Lau
NoVo Nordisk A/S, NoVo Nordisk Park, DK-2760 MaaloeV, Denmark
ReceiVed December 22, 2008
The aim of the work presented here was to design and synthesize potent human glucagon receptor antagonists
with improved pharmacokinetic (PK) properties for development of pharmaceuticals for the treatment of
type 2 diabetes. We describe the preparation of compounds with cyclic cores (5-aminothiazoles), their binding
affinities for the human glucagon and GIP receptors, as well as affinities for rat, mouse, pig, dog, and
monkey glucagon receptors. Generally, the compounds had slightly less glucagon receptor affinity compared
to compounds of the previous series, but this was compensated for by much improved PK profiles in both
rats and dogs with high oral bioavailabilities and sustained high plasma exposures. The compounds generally
showed species selectivity for glucagon receptor binding with poor affinities for the rat, mouse, rabbit, and
pig receptors. However, dog and monkey glucagon receptor affinities seem to reflect the human situation.
One compound of this series, 18, was tested intravenously in an anesthetized glucagon-challenged monkey
model of hyperglucagonaemia and hyperglycaemia and was shown dose-dependently to decrease glycaemia.
Further, high plasma exposures and a long plasma half-life (5.2 h) were obtained.
Introduction
(hGluRa) antagonists.27 This class was subsequently developed
into a related isoserine class of hGluR antagonists where
selectivity for the hGluR over the related human glucose-
dependent insulinotropic peptide receptor (hGIPR) was opti-
mized28 (see Figure 1). Here, we present a novel series of
thiazole-based hGluR antagonists with superior pharmacokinetic
properties. These compounds show species selectivities of
glucagon receptor binding, and they only bind to the human,
dog, and monkey receptors. Further, the present compounds also
display selectivity for the hGluR over the hGIPR. A selected
compound was shown to be active in a monkey model of
hyperglucagonemia and hyperglycemia.
Glucagon, a 29-amino acid hormone, secreted from the R-cells
of the pancreas, stimulates hepatic glucose output by increasing
glycogenolysis and gluconeogenesis. A glucagon receptor
antagonist is suggested to decrease the hepatic glucose output
and thus lower hyperglycaemia in type 2 diabetic patients.1-5
Previous studies have shown, in both acute and chronic models,
that in vivo immunoneutralisation of glucagon does lead to
lowered blood glucose levels in normal and STZ-induced
diabetic rats, in normal and alloxan-induced diabetic rabbits,
and in ob/ob mice.6-9 Also, antidiabetic effects have been shown
in db/db mice10,11 and in ob/ob mice11 by reduction of glucagon
receptor expression by antisense oligonucleotides. Thus, the
glucagon receptor is a potential target for a new drug class for
treatment of type 2 diabetes. Additionally, reports of glucagon
receptor gene knockout mice have been published12,13 and these
animals have lower blood glucose levels throughout the day
and improved glucose tolerance compared to control animals.
Moreover, the knockout mice also showed reduced adiposity
and leptin levels but with normal body weight, food intake, and
energy expenditure.13 These data indicate that glucagon action
is involved in regulation of whole body composition and thus
that glucagon antagonism may give additional benefits to type
2 diabetic patients than just blood glucose regulation.
From our previous work27,28 we have a working hypothesis
that the urea linker does not form direct contacts with the
receptor but merely presents the pharmacophoric elements to
the receptor in a well-defined geometric orientation. The initial
idea behind the strategy of changing the scaffold from a linear
one (like ureas and amides) to a cyclic one as in the present
publication was to rigidify the scaffold, hoping that the
conformation(s) thus obtained would be receptor-active. Since
this strategy results in target compounds of quite different
shapes, we adopted (again) the power of numbers in combina-
torial library synthesis in 96- and 384-well formats. The initial
libraries were designed for maximum diversity in order to
increase the chances of finding active compounds. Subsequent
libraries focused more on optimizing the active compounds and
including building blocks that were known from previous series
to be better or optimal for binding because of steric fit and/or
electrostatic properties. The initial libraries were screened at 1
µM, and later targeted libraries were screened at 100 nM.
Several papers but only few classes of non-peptide glucagon
receptor antagonists have been published, primarily by scientists
from Abbott,14 Merck,15-20 Bayer,21,22 Novo Nordisk/Pfizer,23-27
and Novo Nordisk.28 One compound, Bay 27-9955, has been
tested in human phase 1 clinical trials and was shown to blunt
the effect of glucagon on blood glucose in a hyperglucagonaemic
clamp setting.29 Previously we have published the discovery of
a new class of ꢀ-alanine based human glucagon receptor
As affinity for the hGIPR has been demonstrated in the
previous series of ꢀ-alanine urea compounds27 and the selectivity
a Abbreviations: hGluR, human glucagon receptor; GIP, glucose-
dependent insulinotropic peptide; hGIPR, human glucose-dependent insuli-
notropic peptide receptor; GLP-1, glucagon-like peptide-1; hGLP-1R, human
glucagon-like peptide-1 receptor; SPA, scintillation proximity assay.
* To whom correspondence should be addressed. Phone: +4544434894,
+4530754894. Fax: +4544663450. E-mail: pem@novonordisk.com.
10.1021/jm8016249 CCC: $40.75
2009 American Chemical Society
Published on Web 04/22/2009

2990 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Madsen et al.
Figure 1. First examples of ꢀ-alanine and isoserine urea-based benzoylaminopropionic acids as hGluR antagonists.
Scheme 1. Preparation Method Aa
a Reagents: (i) (1) piperidine, DMF, (2) 4-CHO-PhCO2H, HOBt, DIC; (ii) R1-NH2, NaCNBH3; (iii) Fmoc-NCS; (iv) (1) piperidine, (2) R3CHBrCOR2;
(v) TFA.
for glucagon receptor binding subsequently was optimized28 by
scale), and resyntheses of hits were conveniently done by
utilizing the same procedure employing more resin. Com-
mercially available Fmoc-ꢀ-Ala-Wang resin was deprotected
and coupled with 4-formylbenzoic acid using HOBt/DIC
activation. The amine was prepared through reductive amination
of the aldehyde functionality, and the benzylic amine was
subsequently treated with Fmoc-NCS.31,32 Deprotection, reaction
with R-bromoketones,32 and cleavage from the resin furnished
the desired products in both high yields and good purities. By
use of this procedure, several focused libraries comprising more
than 800 analogues were prepared.
Preparation of compounds (including resynthesis of library
hits) was also done using solution phase chemistry as shown
below in Scheme 2, preparation method B. 4-Formylbenzoic
acid (or the corresponding methyl ester) was reductively
aminated, and the benzylic amine was converted to the thiourea.
This conversion was in certain cases problematic, and various
methods were employed depending on the reactivity of the
secondary amine in question (see Experimental Section).
Formation of the aminothiazole was done easily with R-bro-
moketones in acetic acid. The benzoic acids were subsequently
coupled with either ꢀ-alanine or (R)-isoserine methyl esters
followed by alkaline hydrolysis to furnish the desired substituted
ꢀ-alanines or (R)-isoserines. Alternatively, the benzoic acids
were coupled with 5-aminotetrazole to afford the corresponding
tetrazolylbenzamides.
changing the ꢀ-alanine motif to an (R)-isoserine motif, the
affinity for the hGIPR was monitored throughout this work.
Selected compounds were further tested for affinity for the
related human glucagon-like-1 peptide receptor (hGLP-1R).
The primary assay used for screening was the SPA (scintil-
lation proximity assay) hGluR assay, and selected hits and
resynthesised compounds were assayed in SPA assays for hGluR
and hGIPR binding (IC50) in parallel. Selected compounds were
further assayed for hGluR and hGIPR binding (IC50) in the
previously described membrane filter assays26 so that compari-
sons of receptor and species selectivities could be done in similar
assays. Usually, but not always, the binding IC50 values were
better (i.e., lower) in the filter assay than in the SPA assay.
The goal of this work was to find new potent, orally available
hGluR selective antagonists that were efficacious in animal
models of type 2 diabetes and that had both high plasma
exposure and long half-life.
Selected compounds (usually those better than 50 nM for the
hGluR) were tested for rat PK properties. Selected compounds
were assayed also for species selectivities, and it became evident
that these compounds had poor affinities for the rodent glucagon
receptors. This was important when deciding for the animal
model in which to show pharmacodynamic effects.
Chemistry
The aminothiazoles were first prepared on solid phase in a
library format (preparation method A, Scheme 1, on a 50 µmol
Analogues with altered connectivities were prepared as
described in Schemes 3 and 4, on solid support and in solution,

Glucagon Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2991
Scheme 2. Preparation Method Ba
a Reagents and conditions: (i) NaCNBH3, HOAc; (ii) KSCN, HOAc, ∆ or (1) EtO2CNCS, (2) NaOH or (1) AcNCS, (2) NaOH; (iii) R3CHBrCOR2, DMF,
HOAc; (iv) 1 N NaOH, EtOH; (v) (1) EDAC, HOBt, Et3N, (2) H-ꢀ-Ala-OMe·HCl or H-(R)-iso-Ser-OMe·HCl; (vi) 1 N NaOH, EtOH.
Scheme 3. Preparation Method Ca
a Reagents: (i) (1) piperidine, (2) 4-NO2-Ph-COCl, DIPEA; (ii) SnCl2 ·2H2O; (iii) R1-CHO, NaCNBH3; (iv) Fmoc-NCS; (v) (1) piperidine, (2) R3CHBrCOR2;
(vi) TFA.
respectively. The solid supported route (preparation method C)
is very similar to method A; 4-nitrobenzoyl chloride was coupled
to resin-bound ꢀ-alanine, the nitro group serving as a protection
for an amine function, being unmasked with SnCl2 reduction.

2992 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Scheme 4. Preparation Method Da
Madsen et al.
a Reagents: (i) R1-CHO, NaCNBH3; (ii) EtO2C-NCS; (iii) NaOH; (iv) R3CHBrCOR2; (v) (1) EDAC; HOBt, (2) H-ꢀ-Ala-OMe, HCl; (vi) NaOH.
Scheme 5. Preparation Method Ea
a Reagents and conditions: (i) K2CO3, ∆, MeOH; (ii) NaBH4, THF; (iii) (EtO)2P(S)SH; (iv) R3CHBrCOR2; (v) (1) EDAC, HOBt, (2) H-ꢀ-Ala-OMe, HCl
or H-iso-Ser-OMe, HCl; (vi) NaOH.
The amine was reductively alkylated, and the thiourea func-
tionality was introduced with Fmoc isothiocyanate followed by
treatment with piperidine. Cyclization with R-bromoketones
afforded the desired products in high yields and purities.
The solution phase method (preparation method D) is very
similar to method B and is shown in Scheme 4.
Synthesis of analogues wherein the core 5-aminothiazole
functionality has been changed to a 5-alkylthiazole core is shown
in Scheme 5 (preparation method E). Knoevenagel condensa-
tion33 of methyl 4-formylbenzoate with phenylacetonitriles
afforded the corresponding acrylonitriles. Reduction34 of the
double bond (NaBH4/THF) followed by treatment of the
saturated nitrile with dithiophosphoric acid O,O-diethyl ester35
afforded the saturated thioamide. Subsequently, Hantzsch thia-
zole formation36 as before using R-bromoketones followed by
coupling with ꢀ-alanine methyl ester (or (R)-isoserine methyl
ester) and hydrolysis afforded the desired compounds.
Discussion
Early on it became evident that in order to confer hGluR
binding affinity, compounds of the present aminothiazole series
with aliphatic R1 groups were not well tolerated. In Table 1,
statistics for representative aliphatic R1 groups are listed and,
for comparison, two entries with aromatic R1 groups. Numerous

Glucagon Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2993
Table 1. Representative Aliphatic R1 Groups and Their Statistics from
Library Screening
5 (mL/min)/kg versus 13 and 15 (mL/min)/kg, respectively). It
is noteworthy that 10 had an extremely high plasma exposure,
Cmax ) 1180 ng/mL.
We also investigated other aromatic carbon-substituted R1
groups, e.g., 3-methyl-4-isopropylphenyl (12 and 13), 4-tert-
butylphenyl (14), and 1,2,3,4-terahydronaphthalen-1-yl (15), but
they had not increased hGluR affinities.
Aromatic R1 groups with (pseudo)halogen substituents were
similarly investigated, and several promising compounds were
discovered. Of compounds with the R1-group 4-chlorophenyl,
16 and 17 were particularly interesting. They had reasonable
affinities for the hGluR, especially in the membrane filter assay
(both 53 nM) but showed poor bioavailabilities following oral
administration to rats (13% and 16%, respectively).
R1 groups 4-CF3-, 4-OCF3-, and 4-SCF3-phenyl did confer
both good binding affinities (e.g., 18, 19, and 21) and superior
rat PK properties. Especially 18 had an encouraging profile with
high oral bioavailability (58%), low clearance (1 (mL/min)/kg),
long plasma T1/2 (228 min after iv administration), and extremely
high plasma exposure (Cmax ) 2100 ng/mL).
In Figure 2, the rat PK profile of 18 is shown. The
compound may enter enterohepatic circulation, as the po
profile indicates.
10, 16, 17, 18, and 19 were tested for pharmacokinetic
properties in dogs, and all showed encouraging profiles, as can
be seen in Table 5. The high (>100%) bioavailability of 18
indicates that this compound in dogs might enter enterohepatic
circulation. Clearances were generally very low (<5 (mL/min)/
kg), and peroral bioavailabilities were generally very high.
The necessity of the R2 group not being electron rich, as
expected from previous series,27,28 was evident from library
screenings results, i.e., no or few hits were found among those
compounds. This was further substantiated by the low affinity
of 20 (420 nM).
A few compounds were made to investigate the influence of
changing the ꢀ-alanine moiety to an (R)-isoserine moiety, a
modification that in the urea series of glucagon receptor
antagonists proved to enhance selectivity toward GIP receptor
affinity28 while retaining glucagon receptor affinity. This
modification did not give the desired effects, as in the present
series of aminothiazoles glucagon receptor affinity was damaged
substantially by introduction of a (R)-isoserine-moiety (22 and
23), all with affinities >200 nM.
We also investigated the possibility of changing the
connectivities going from a 4-(thiazol-2-ylaminomethyl)ben-
zamide scaffold (as in 1) to a 3- or 4-(thiazol-2-ylamino)ben-
zamide scaffold (as in 24). Screening results from libraries
indicated that meta-substitution on the benzamide was not
tolerated, as no hits were found. We found that compounds
of equal hGluR affinity could be obtained from the series of
4-(thiazol-2-ylamino)benzamides, as 24 had IC50 ) 34 nM
in the hGluR membrane filter assay. The rat PK parameters
of 25 and 26 were, however, not encouraging when compared
to those of 6.
The possibility of changing the exocyclic nitrogen in the
aminothiazoles to a (racemic) sp3-carbon was investigated. This
change results in an altered geometry around the tripod in
question, going from a planar nitrogen atom to a tetrahedral
carbon atom. This change did not result in dramatically different
binding affinities; e.g., compare 30 with 19 (119 versus 92 nM).
A compound of this class, 27, had, however, a very poor PK-
profile with high clearance and low exposure after po dosing,
and consequently this class was abandoned.
R2 and R3 groups were identical or similar in these libraries,
indicating the strong preference for aromatic R1 groups.
Instead, focus was put on compounds with aromatic R1
groups, and in particular compounds with cyclohex(en)ylphenyl
groups were pursued. The SAR (see Table 2) proved to be quite
insensitive regarding R2 and R3, and even the very bulky 5 with
two phenyl substituents on the thiazole ring was among the
compounds with the highest affinities in this class.
Although compounds like 1 and 2 had reasonable affinities
(around 120 nM; see Table 2), they suffered from very poor rat
PK properties with short plasma half-lives (T1/2 of around 30-40
min) high clearances, and poor Fpo values (2-5%) (see Table
4). 3 and 4 with higher binding affinities (IC50 ) 30-50 nM)
were equally poor on the PK parameters. The fluorene analogue
6 also had better hGluR affinity (50 nM), showed improved
T1/2 (148 min), and very low clearance but still associated with
an unacceptable low Fpo (12%).
Compounds with smaller aliphatically substituted phenyl
groups as R1 substituents, such as annelated cyclopentyl- and
cyclohexylphenyl groups proved to be much better. The first
two in this class, 7 and 8, had only moderate hGluR affinities
but had significantly improved rat PK profiles with full oral
bioavailabilities (89% and 87%, respectively) and very high
plasma exposures (Cmax 920 and 704 ng/mL, respectively).
Consequently with these encouraging data, more focus was put
into elaborating this class further. We tried to increase the size
of the annelated ring, from a five-membered to a six-membered,
and affinities were similar; e.g., compare 11 with 7 (77 vs 86
nM) and 10 with 9 (79 vs 99 nM). With respect to rat PK
parameters, 11 and 10 were as good as 7 and 8, although oral
availabilities were lower (27% and 36% versus 89% and 87%,
respectively) and clearances were significantly improved (7 and

2994 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Madsen et al.
Table 2. Structures, Binding Affinities, and Synthetic Methods of Synthesized Compounds

Glucagon Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2995
Table 2. Continued
a Binding affinity of compounds binding to the recombinant human glucagon and GIP receptors on membranes from BHK cells in a scintillation proximity
assay (SPA). Data are expressed as mean IC50. SD was typically within 10-15% of the reported mean, n ) 2-6. b Binding affinity of compounds binding
to the recombinant human glucagon and GIP receptors on membranes from BHK cells in a filtration assay. Data are expressed as mean IC50. SD was
typically within 10-15% of the reported mean, n ) 2-6.
In summary, the SAR regarding hGluR affinities is as follows:
R1 being lipophilic, large, and bulky (like 4-(cyclohex-1-
enyl)phenyls 1-5 or fluorenyl 6) is conferring the highest hGluR
binding affinities. Making R1 smaller (like annelated cyclopen-

2996 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Madsen et al.
Table 3. Binding Affinities (IC50 Values in nM) to Isolated Glucagon Receptors from Various Species and Inhibition of Glucose Production from
Cultures Primary Hepatocytes (IC50 Values in µM)
compd
humana (nM)
rat (nM)
mouse (nM)
rabbit (nM)
pig (nM)
dog (nM)
monkey (nM)
human hepatocytes (µM)
6
7
8
10
11
16
17
18
19
59
64
96
54
48
53
53
93
51
1800
5400
4400
1600
218
830
680
452
62
81
94
55
3.0
51
60
114
72
0.7
<20
8.6
1900
3300
81
4.0
1400
1700
2700
2000
372
418
648
552
943
1200
1600
4000
5700
7000
5300
68
78
155
96
2.7
2.8
6.7
3.6
a The assay for the human receptor is the same as “hGlu binding affinity” from Table 2.
Table 4. Rat pharmacokinetic properties of selected compounds
compd
T1/2, iv (min)
Fpo (%)
Cl ((mL/min)/kg)
Cmax (ng/mL)
Tmax (min)
iv dose (mg/kg)
po dose (mg/kg)
1
2
3
4
6
7
8
10
11
16
17
18
19
25
26
27
37
30
43
5
2
3
35
39
34
44
5
13
15
5
7
5
6
1
94
13
47
90
90
90
30
300
60
240
60
90
90
60
1.9
1.6
1.9
1.9
3.7
3.15
3.7
3.9
2.1
3.34
3.34
3.15
3.25
3.25
3.20
3.04
3.19
3.0
35
3
36
148
131
61
104
74
135
91
228
454
40
12
89
87
36
27
13
16
58
29
13
13
52
471
920
704
1180
615
216
521
2100
638
93
1.1
1.66
1.66
1.6
1.62
1.60
1.59
1.62
1.49
1.66
1.66
1.58
85
>360
2
31
31
28
90
90
30
46
24
82
117
3.0
3.16
tylphenyls 7-9 or 4-(pseudo)halophenyls 16-21) causes a drop
in affinity of about 2-fold. The SAR for R2 (and R3) is of minor
importance as long as the substituents on R2 are lipophilic and
present in the meta or para position of the phenyl ring.
Compounds of the present series were generally quite
selective for the hGluR over the hGIPR by 5- to 10-fold
irrespective of the substitution patterns and even more selective
over the hGLP-1R by >50-fold (data not shown), so receptor
selectivity was not found to be an issue in the present series. It
is not possible to conclude whether or not the isoserine
analogues 22 and 23 are as selective as the isoserines were in
the urea series28 compared to corresponding ꢀ-alanines27 (10-
fold) because of the low hGluR affinity.
The SAR regarding rat PK properties is slightly different
(opposite) from the SAR for hGluR binding. The poorest
properties (very low Fpo and high clearance rates) are obtained
with R1 being lipophilic, large, and bulky (like 4-(cyclohex-1-
enyl)phenyls 1-5 or fluorenyl 6). The highest Fpo (88%) is
obtained with R1 being annelated cyclopentylphenyl (7, 8) but
still associated with relatively high clearance rates (14 (mL/
min)/kg). With R1 being annelated cyclohexylphenyl (10, 11),
clearance rates are improved (6 (mL/kg)/min), but Fpo is lower
(∼30%). With R1 being 4-chlorophenyl (16, 17), clearance rates
are 6 (mL/kg)/min, but Fpo is further lowered (to ∼15%). When
4-chlorophenyl is changed to 4-CF3- or 4-CF3O-phenyl (18, 19),
clearance rates are further lowered (to ∼1 (mL/kg)/min) and
Fpo is increased substantially (58% and 29%, respectively).
Changing the connectivity around the aminothiazole (25, 26
compared with 6) was neither beneficial for binding affinity nor
for PK properties with much higher clearance rates (31 vs 5
(mL/kg)/min).
Figure 2. Rat pharmacokinetic profile of 18. Plasma concentration vs
time plots of 18 after intravenous ([) and peroral (b) dosings to rats
of 1.65 and 3.06 mg/kg, respectively. Selected pharmacokinetic
parameters are given in Table 4.
Table 5. Dog Pharmacokinetic Propertiesa of Selected Compounds
Selected compounds were assayed for binding to isolated
glucagon receptors from various species to select an animal
model to show pharmacodynamic effects. As can be seen from
Table 3, the present compounds bound poorly to most of the
receptors of the species panel. Thus, rodents (mice, rats, and
rabbits) and pigs were not adequate as animal models. On the
contrary, only dog and monkey receptors recognized the tested
compounds with affinities similar to those of the human
T1/2
,
Cl
Cmax
compd iv (min) Fpo (%) ((mL/min)/kg) (ng/mL) Tmax (min)
10
16
17
18
19
124
80
66
104
235
92
16
71
141
55
1.44
4.66
4.64
2.23
0.78
1190
120
554
1253
1000
51
105
90
90
88
a Doses: iv, 0.25 mg/kg; po, 0.5 mg/kg.

Glucagon Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2997
Figure 5. Plasma concentration of 18 vs time plots after intravenous
dosings to monkeys.
Figure 3. Example of inhibition of glucagon-stimulated glucose
production in cultured primary human hepatocytes. The hepatocytes
were cultured as described in the methods section. The hepatocytes
were preincubated with 6 for 10 min, and then the glycogenoly-
sis was induced with 5 nM glucagon for 60 min. Glucose in the medium
was measured (glucagon-induced glycogenolysis (9) and basal glyco-
genolysis (2)). Results are means values ( SEM for four experiments.
The IC50 was calculated to 0.7 µM.
In order to demonstrate acute in vivo efficacy in a nonhuman
primate model, 18 was chosen to be tested in an efficacy model
of glucagon antagonism: intravenous administration in an
anesthetized glucagon-challenged monkey model of hyperglu-
cagonaemia and hyperglycaemia. We would have preferred to
test the compound orally, but because of animal handling issues,
we had to do the dosing to anesthetized animals and, conse-
quently, intravenously. 18 was chosen on the basis of rat PK
data (Table 4) as the best compromise between low clearance,
long T1/2 (iv), high Fpo, and high Cmax. In monkeys 18 was shown
(Figure 4) dose-dependently to decrease glucagon stimulated
glycaemia. At doses of 1 and 3 mg/kg the hyperglycaemic effect
of exogenous administered glucagon was completely abolished.
Further, blood samples were assayed for presence of compound,
and also in the monkey, high plasma exposure was obtained
(Figure 5). Additionally, a long T1/2 was obtained, 314 min.
glucagon receptor, suggesting that dogs or monkeys would be
relevant species in which to show pharmacodynamic effects.
6, 7, 8, 10, 16, 17, 18, and 19 were tested for inhibition of
glucagon stimulated glucose production in human hepatocytes,30
and as can be seen in Figure 3 and Table 3, these compounds
inhibited glucose production in a dose-dependent manner. The
IC50 values were in the range from 0.7 to 10 µM. The same
compounds were also tested in freshly isolated rat hepatocytes,
and none of them showed any activity (data not shown), in
agreement with the rat receptor binding data. It is noted that
the IC50 values in this hepatocyte assay were generally much
higher than in the receptor binding assays. This was at least
partly due to the fact that the compounds bind to albumin that
was present in the hepatocyte assays and not in the binding
assays.
Conclusion
The discovery of and library-assisted optimization of ami-
nothiazole-based glucagon receptor antagonists have been
Figure 4. Plasma glucose concentration vs time plot (upper panel) of intravenous administration of 18 to anesthetized glucagon-challenged monkeys.
The lower panel shows the AUCs (areas under the curves) and statistical p-values.

2998 Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9
Madsen et al.
mmol) was added. The resulting suspension was heated at reflux
temperature for 30 min and subsequently cooled to approximately
35 °C. Acetic acid glacial (50 mL) was added followed by
NaCNBH3 (4.96 g, 80 mmol) in portions. The mixture was stirred
at room temperature for 30 min. The mixture was concentrated in
vacuo to approximately 150 mL by rotary evaporation. Water (150
mL) was added, and the resulting suspension was stirred at room
temperature for 16 h. Filtration, washing with water, and drying
afforded 26.8 g (100%) of 4-[(4-chlorophenylamino)methyl]benzoic
acid as a solid. 1H NMR (DMSO-d6): δ ) 4.31 (2H, d), 6.54 (4H,
“d”), 7.05 (2H, d), 7.45 (2H, d), 7.89 (2H, d), 12.8 (1H, bs).
A mixture of 4-[(4-chlorophenylamino)methyl]benzoic acid (33.3
g, 127 mmol) and KSCN (37 g, 382 mmol) in 1 N HCl (500 mL)
was refluxed for 16 h. The suspension was filtered and washed
with water. The solid was mixed with KSCN (37 g, 382 mmol)
and 1 N HCl (500 mL), and the mixture was again refluxed for
16 h. Filtration, washing with water, and resubmission to the
reaction conditions above afforded 30.2 g (70%) of 4-[1-(4-
described. These compounds are species selective; binding to
human receptors was only paralleled by the dog and monkey
receptors, whereas affinities for the rat, mouse, and rabbit
receptors were low. The compounds displayed selectivity for
the hGluR over both the hGIPR and hGLP-1R. Efficacy of
selected compounds in human hepatocytes was demonstrated.
These compounds also generally showed superior pharmaco-
kinetic properties in both rats and dogs with high plasma
exposures and long plasma half-lives. One compound, 18, was
tested in a glucagon-challenged monkey model of hyperglu-
cagonemia and hyperglycemia and was found to be active. In
addition, this compound was shown also in the monkey to have
high plasma exposure and long plasma half-life.
Experimental Section
General. 1H NMR spectra were recorded in deuterated solvents
at 200, 300, or 400 MHz (DRX 200, DRX 300, and AMX2 400
from Bruker Instruments, respectively). Chemical shifts are reported
as δ values (ppm) relative to internal tetramethylsilane (δ ) 0 ppm).
Elemental analyses were performed by the microanalytical labo-
ratories at Novo Nordisk A/S, Denmark. High resolution mass
spectra (MALDI-TOF) were recorded at the University of Southern
Denmark, Odense. Column chromatography was performed on silica
gel 60 (40-63 µm). Chemicals and solvents used were com-
mercially available and were used without further purification.
Yields refer to pure materials and are not optimized. The preparation
methods are illustrated by single representative experimental
procedures. The purities of the key target compounds (>95%) were
verified by combustion analyses.
1
chlorophenyl)thioureidomethyl]benzoic acid as a solid. H NMR
(DMSO-d6): δ ) 5.44 (2H, s), 7.14 (2H, d), 7.39 (4H, “t”), 7.85
(2H, d), 12.9 (1H, bs).
4-[1-(4-Chlorophenyl)thioureidomethyl]benzoic acid (29 g, 88.8
mmol) was dissolved in DMF (300 mL), and acetic acid (40 mL)
and 2′-bromo-4-trifluoromethoxyacetophenone (25.1 g, 88.8 mmol)
were added. The resulting mixture was stirred at room temperature
for 16 h. EtOAc (500 mL) was added, and the mixture was washed
with water (2 × 500 mL), saturated aqueous NaCl (500 mL), and
saturated aqueous NH4Cl (500 mL). Drying (MgSO4) and concen-
tration in vacuo afforded 48 g (quant) of 4-({(4-chlorophenyl)-[4-
(4-trifluoromethoxyphenyl)thiazol-2-yl]amino}methyl)benzoic acid
1
as an oil. H NMR (DMSO-d6): δ ) 5.35 (2H, s), 7.36 (1H, s),
7.39 (2H, d), 7.47-7.57 (6H, m), 7.90 (2H, d), 7.97 (2H, d), 12.7
(1H, bs).
Preparation Method A. 3-[4-({(4-Chlorophenyl)-[4-(4-trifluoro-
methylphenyl)thiazol-2-yl]amino}methyl)benzoylamino]propio-
nic Acid (16). Fmoc-ꢀ-Ala-Wang resin (0.29 mmol/g, 2 g, 0.58
mmol) was treated with piperidine (20% in NMP, 20 mL) for 40
min, and the resin was drained. This was repeated once. The resin
was washed with NMP (6 × 20 mL), 4-formylbenzoic acid (0.348
g, 2.3 mmol), and HOBt (0.35 g, 2.6 mmol) in NMP (20 mL).
DIC (0.36 mL, 2.3 mmol) in MeCN (5 mL) was added, and the
resulting mixture was shaken for 16 h at room temperature.
The resin was drained and washed with DMF (5 × 20 mL). To the
resulting resin-bound aldehyde was added 4-chloroaniline (0.37 g,
2.9 mmol) dissolved in a mixture of NMP and trimethyl orthofor-
mate (1:1, 20 mL). HOAc (2 mL) was added, and the mixture was
shaken for 4 h at room temperature. NaCNBH3 (0.36 g, 5.8 mmol)
in a mixture of NMP and methanol (1:1, 6 mL) was added, and
the resulting mixture was shaken for 16 h at room temperature,
drained, and washed successively with NMP/MeOH (1:1, 2 × 20
mL), NMP (3 × 20 mL), DCP/DIPEA (7:1, 3 × 20 mL), and DCP
(3 × 20 mL). To the resulting resin-bound amine was added Fmoc
isothiocyanate (1.79 g, 6.38 mmol) in DCP (20 mL), and the mixture
was shaken for 4 h at room temperature and washed with DCM (3
× 20 mL) and NMP (3 × 20 mL). The resin was treated with
piperidine (20% in NMP, 20 mL) for 1 h, and the resin was drained.
The resin was washed with NMP (6 × 20 mL). 2′-Bromo-4-
trifluoromethylacetophenone (1.60 g, 5.8 mmol) in NMP (15 mL)
and acetic acid (4 mL) were added to the resin, and the mixture
was shaken for 16 h at room temperature and washed with NMP
(3 × 20 mL), MeOH (3 × 20 mL), and DCM (10 × 20 mL). The
product was cleaved from the resin by treatment with 50% TFA in
DCM (20 mL) for 30 min. Concentration in vacuo and purification
of the residue by column chromatography afforded 0.32 g (98%)
of 3-[4-({(4-chlorophenyl)-[4-(4-trifluoromethylphenyl)thiazol-2-
yl]amino}methyl)benzoylamino]propionic acid. 1H NMR (DMSO-
d6): δ ) 2.49 (“t”, below DMSO), 3.43 (2H, q), 5.33 (2H, s), 7.43
(2H, d), 7.52 (4H, m), 7.75 (4H, m), 8.07 (2H, d), 8.46 (1H, t),
12.2 (1H, bs). Anal. (C27H21N3ClF3O3S) C, H, N.
4-({(4-Chlorophenyl)-[4-(4-trifluoromethoxyphenyl)thiazol-2-
yl]amino}methyl)benzoic acid (47 g, 93 mmol) was dissolved in
DMF (500 mL), and HOBt (18.9 g, 140 mmol) and EDAC ·HCl
(26.7 g, 140 mmol) were added. The mixture was stirred at room
temperature for 1.5 h. DIPEA (19.1 mL, 111 mmol) and 3-ami-
nopropionic acid methyl ester hydrochloride (15.5 g, 111 mmol)
were added to the mixture, and the resulting mixture was stirred at
room temperature for 16 h. EtOAc (500 mL) was added, and the
mixture was washed with water (500 mL). The aqueous phase was
extracted with EtOAc (500 mL). The combined organic phases were
washed with water (2 × 500 mL) and saturated aqueous NH4Cl (2
× 300 mL), dried (Na2SO4), and concentrated in vacuo to afford
60 g (quant) of crude product. Recrystallization from ethanol
afforded 31.2 g (55%) of pure 3-[4-({(4-chlorophenyl)-[4-(4-
trifluoromethoxyphenyl)thiazol-2-yl]amino}methyl)benzoylamino]-
1
propionic acid methyl ester as a solid. H NMR (DMSO-d6): δ )
2.57 (3H, t), 3.46 (2H, q), 3.59 (3H, s), 5.32 (2H, d), 7.36 (1H, s),
7.41 (4H, m), 7.41 (4H, m), 7.76 (2H, d), 7.97 (2H, d), 8.51 (1H,
t).
3-[4-({(4-Chlorophenyl)-[4-(4-trifluoromethoxyphenyl)thiazol-2-
yl]amino}methyl)benzoylamino]propionic acid methyl ester (18.7
g, 31.7 mmol) was dissolved in ethanol (500 mL), and 1 N sodium
hydroxide (63 mL, 63 mmol) was added. The resulting mixture
was stirred at room temperature for 16 h. The mixture was filtered,
washed with ethanol, and dried in vacuo at 40 °C for 16 h to afford
8.0 g (42%) of 3-[4-({(4-chlorophenyl)-[4-(4-trifluoromethoxyphe-
nyl)thiazol-2-yl]amino}methyl)benzoylamino]propionic acid as the
sodium salt, mp 134-139 °C. 1H NMR (DMSO-d6): δ ) 2.13 (2H,
t), 3.37 (m, below water), 5.31 (2H, s), 7.34-7.39 (5H, m), 7.50
(4H, m), 7.74 (2H, d), 7.97 (2H, d), 8.78 (1H, bs). Anal.
(C27H20N3Cl1F3O4SNa ·2H2O) C, H. N calcd, 6.63%; found, 7.30%.
The above filtrate was added to 1 N HCl (80 mL) and water
(200 mL), and the mixture was extracted with ethyl acetate (300
mL). The organic phase was washed with water (300 mL), dried,
and concentrated in vacuo to afford 6.63 g (36%) of 3-[4-({(4-
chlorophenyl)-[4-(4-trifluoromethoxyphenyl)thiazol-2-yl]amino}me-
Preparation Method B. 3-[4-({(4-Chlorophenyl)-[4-(4-trifluoro-
methoxyphenyl)thiazol-2-yl]amino}methyl)benzoylamino]propio-
nic Acid (17). 4-Formylbenzoic acid (15 g, 100 mmol) was
suspended in methanol (500 mL), and 4-chloroaniline (12.7 g, 100
1
thyl)benzoylamino]propionic acid as a solid. H NMR (DMSO-
d6): δ ) 2.47 (m, below DMSO), 3.43 (2H, q), 5.32 (2H, s), 7.36

Glucagon Receptor Antagonists
Journal of Medicinal Chemistry, 2009, Vol. 52, No. 9 2999
(1H, s), 7.41 (4H, “t”), 7.39 (5H, m), 7.51 (4H, m), 7.76 (2H, d),
7.97 (2H, d), 8.48 (1H, t), 12.2 (1H, bs). Anal. (C27H21N3Cl1F3O4S)
C, H, N.
concentrated in vacuo to afford 14 g (100%) of [(9H-fluoren-2-
yl)-(4-mehoxycarbonylbenzyl)thiocarbamoyl]carbamic acid ethyl
ester. 1H NMR (DMSO-d6): δ ) 10.18 (s, 1H), 7.90-7.80 (m, 4H),
7.56-7.50 (m, 3H), 7.42 (s, 1H), 7.38-7.30 (m, 3H), 7.17 (d, 1H),
5.64 (s, 2H), 3.88-3.80 (m, 5H), 0.92 (t, 3H).
2(R)-Hydroxy-3-[4-({indan-5-yl-[4-(4-trifluoromethoxyphenyl)-
thiazol-2-yl]amino}methyl)benzoylamino]propionic Acid (22). 4-
({Indan-5-yl-[4-(4-trifluoromethoxyphenyl)thiazol-2-yl]amino}me-
thyl)benzoic acid (0.44 g, 0.86 mmol) was dissolved in DMF (10
mL), and HOBt (0.18 g, 1.29 mmol) and EDAC HCl (0.25 g, 1.29
mmol) were added. The resulting mixture was stirred at room
temperature for 1.5 h. 3-Amino-2(R)-hydroxypropionic acid methyl
ester hydrochloride ((R)-isoserine methyl ester hydrochloride),
prepared as previously described28 (0.16 g, 1.03 mmol), and DIPEA
(225 µL, 1.29 mmol) were added, and the resulting mixture was
stirred at room temperature for 16 h. EtOAc (100 mL) was added,
and the mixture was washed with water (2 × 80 mL). The organic
phase was dried (MgSO4) and concentrated in vacuo. The residue
(0.35 g) was purified by column chromatography on silica gel,
eluting with a mixture of EtOAc and heptane (1:1). This afforded
0.10g(19%)of2(R)-hydroxy-3-[4-({indan-5-yl-[4-(4-trifluoromethoxy-
phenyl)thiazol-2-yl]amino}methyl)benzoylamino]propionic acid meth-
[(9H-Fluoren-2-yl)-(4-mehoxycarbonylbenzyl)thiocarba-
moyl]carbamic acid ethyl ester (14 g, 30.36 mmol) was dissolved
in warm EtOH (96%, 160 mL), and 4 N NaOH (76 mL, 304 mmol)
was added to the solution. The resulting mixture was refluxed for
16 h. The mixture was concentrated in vacuo to remove ethanol.
The residue was suspended in water (150 mL), and 4 N HCl (76
mL, 304 mmol) was carefully added. The suspension was stirred
1
for /2 h at 25 °C and filtered, washed with water, and dried in
vacuo at 40 °C for 16 h to afford 11.4 g (100%) of 4-{[1-(9H-
fluoren-2-yl)thioureido]methyl}benzoic acid. 1H NMR (DMSO-d6):
δ ) 12.88 (broad, 1H), 7.88-7.84 (m, 4H), 7.58 (d, 1H), 7.46-7.38
(m, 5H), 7.09 (d, 1H), 5.53 (s, 2H), 3.87 (s, 2H).
3-[4-({(4-Trifluoromethoxyphenyl)-[4-(4-trifluoromethylphe-
nyl)thiazol-2-yl]amino}methyl)benzoylamino]propionic Acid (19).
4-[(4-Trifluoromethoxyphenylamino)methyl]benzoic acid methyl
ester (15 g, 46.11 mmol) was dissolved in DCM (220 mL), and
ethoxycarbonyl isothiocyanate (7.05 mL, 59.95 mmol) was added.
The resulting mixture was stirred at room temperature for 16 h.
The mixture was concentrated in vacuo to afford 21 g (100%) of
[(4-trifluoromethoxyphenyl)-(4-mehoxycarbonylbenzyl)thiocarba-
1
yl ester. H NMR (CDCl3): δ ) 2.10 (2H, p), 2.90 (4H, m), 3.81
(5H, m), 4.39 (1H, bs), 5.26 (2H, s), 6.50 (1H, t), 6.66 (1H, s),
7.04 (1H, dd), 7.14 (1H, s), 7.21 (3H, “d”), 7.46 (2H, d), 7.69 (2H,
d), 7.84 (2H, d).
2(R)-Hydroxy-3-[4-({indan-5-yl-[4-(4-trifluoromethoxyphenyl)-
thiazol-2-yl]amino}methyl)benzoylamino]propionic acid methyl
ester (0.10 g, 0.16 mmol) was dissolved in MeOH (5 mL), and 1
N NaOH (0.16 mL, 0.16 mmol) was added. The resulting mixture
was stirred at room temperature for 16 h. Then 1 N HCl (0.17 mL)
and water (50 mL) were added, and the mixture was extracted with
EtOAc (50 mL). The organic phase was washed with water (50
mL), dried (MgSO4), and concentrated in vacuo. This afforded
0.06 g (61%) of 2(R)-hydroxy-3-[4-({indan-5-yl-[4-(4-trifluo-
romethoxy-phenyl)thiazol-2-yl]amino}methyl)benzoylamino]pro-
1
moyl]carbamic acid ethyl ester. H NMR (DMSO-d6): δ ) 10.44
(s, 1H), 7.89 (dd, 2H), 7.51 (dd, 2H), 7.36-7.28 (m, 4H), 5.61 (s,
2H), 3.88-3.80 (m, 5H), 0.91 (t, 3H).
[(4-Trifluoromethoxyphenyl)(4-mehoxycarbonylbenzyl)thiocar-
bamoyl]carbamic acid ethyl ester (21 g, 46.11 mmol) was dissolved
in warm EtOH (96%, 250 mL), and 4 N NaOH (115 mL, 460
mmol) was added. The resulting mixture was refluxed for 16 h.
After cooling, the mixture was evaporated in vacuo to remove
ethanol, the residue was suspended in water (400 mL), and 4 N
HCl (115 mL, 460 mmol) was carefully added. The water was
decanted from the oil, and the residual oil was dissolved in DCM,
dried (MgSO4), and evaporated. The amorphous residue was partly
dissolved in EtOAc, filtered, and evaporated to afford 17 g (100%)
of 4-[1-(4-trifluoromethoxyphenyl)thioureidomethyl]benzoic acid.
1H NMR (DMSO-d6): δ ) 12.9 (s, 1H), 7.87 (d, 2H), 7.40 (d, 2H),
7.33 (d, 2H), 7.26 (d, 2H), 5.45 (s, 2H).
1
pionic acid. H NMR (CDCl3): δ ) 2.09 (2H, m), 2.88 (4H, m),
3.78 (1H, m), 3.87 (1H, m), 4.37 (1H, t), 5.24 (2H, s, 6.65 (1H, s),
7.00 (1H, t), 7.02 (1H, d), 7.13 (1H, s), 7.19 (3H, “d”), 7.45 (2H,
d), 7.68 (2H, d), 7.82 (2H, d). HR-MS calcd for C30H26F3N3O5S:
598.1618 (M + 1). Found: 598.1647.
In the following, details as to how the thiourea functionality has
been prepared are given.
3-(4-{[[4-(4-Chlorophenyl)thiazol-2-yl]-(4-trifluoromethylphenyl)-
amino]methyl}benzoylamino)propionic Acid (18). 4-[(4-Trifluo-
romethylphenylamino)methyl]benzoic acid methyl ester (1.00 g;
3.23 mmol) was dissolved in DCM (10 mL), and Fmoc isothio-
cyanate (0.91 g; 3.23 mmol) was added. The resulting mixture was
stirred at room temperature for 16 h. The mixture was concentrated
in vacuo, and the residue was purified by flash chromatography
using DCM as eluent. This afforded 1.79 g (94%) of [(4-
trifluoromethylphenyl)-(4-mehoxycarbonylbenzyl)thiocarba-
moyl]carbamic acid 9H-fluoren-9-ylmethyl ester. 1H NMR (CDCl3):
δ ) 3.88 ppm (s, 3H), 4.05 (t, 1H), 4.30 (d, 2H), 5.52 (s, 2H),
7.15-7.30 (m, 5H), 7.30-7.45 (m, 7H), 7.53 (d, 2H), 7.72 (d, 2H),
7.95 (d, 2H).
Acknowledgment. The excellent technical assistance of
Dorthe Egholm Jensen, Annette Nielsen, Paw Bloch, Jette
Bolding Lenstrup, Bent Høgh, Poul Enrico Nielsen, Helle Bach,
Janni Kaja Kastrup Sørensen, Brian S. Hansen, Jeanette Hansen,
Mette Winther, Annette Hansen, Lone Honore´, Pia Justesen,
and Karin Hamborg Albrechtsen is gratefully acknowledged.
The library production/purification team (Dr. Helle Demuth, Dr.
Leif Christensen, Vibeke Rhode, Anne-Mette Jacobsen, and
Lone Riisegaard) was instrumental for the work described here,
and their efforts are highly acknowledged. Dr. Vladim´ıra
Panajotova is acknowledged for supervising the monkey studies.
[(4-Trifluoromethylphenyl)-(4-mehoxycarbonylbenzyl)thiocar-
bamoyl]carbamic acid 9H-fluoren-9-ylmethyl ester (1.70 g, 2.87
mmol) was dissolved in a mixture of piperidine and DCM (1:4, 20
mL). The mixture was stirred at room temperature for 30 min and
then evaporated to dryness in vacuo. The residue was purified by
column chromatography, eluting with dichloromethane to give an
oil, which was subsequently crystallized from ethyl acetate/heptane
to afford 495 mg (46%) of 4-[1-(4-trifluoromethylphenyl)thioure-
idomethyl]benzoic acid methyl ester. 1H NMR (CDCl3): δ ) 3.88
ppm (s, 3H), 5.62 (s, 2H), 5.65 (bs, 2H), 7.20 (d, 2H), 7.40 (d,
2H), 7.65 (d, 2H), 7.96 (d, 2H).
3-[4-({(9H-Fluoren-2-yl)-[4-(4-trifluoromethoxyphenyl)thiazol-
2-yl]amino}methyl)benzoylamino]propionic Acid (6). 4-[(9H-Fluo-
ren-2-ylamino)methyl]benzoic acid methyl ester (10 g, 30.36 mmol)
was dissolved in dichloromethane (160 mL), and ethoxycarbonyl
isothiocyanate (4.65 mL, 39.47 mmol) was added. The resulting
mixture was stirred at room temperature for 16 h. The mixture was
Supporting Information Available: Experimental procedures
for the preparation of compounds according to preparation methods
C, D, and E; purity information for target compounds; in vitro and
in vivo protocols. This material is available free of charge via the
Internet at http://pubs.acs.org.
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