The discovery of N-((2 H-tetrazol-5-yl)methyl)-4-((R)-1-((5 r,8 R)-8-(tert-butyl)-3-(3,5-dichlorophenyl)-2-oxo-1,4-diazaspiro[4.5]dec-3-en-1-yl) -4,4-dimethylpentyl)benzamide (SCH 900822): A potent and selective glucagon receptor antagonist

Article abstract of DOI:10.1021/jm401858f

A novel series of spiroimidazolone-based antagonists of the human glucagon receptor (hGCGR) has been developed. Our efforts have led to compound 1, N-((2H-tetrazol-5-yl)methyl)-4-((R)-1-((5r,8R)-8-(tert-butyl)-3-(3, 5-dichlorophenyl)-2-oxo-1,4-diazaspiro[4.5]dec-3-en-1-yl)-4,4-dimethylpentyl) benzamide (SCH 900822), a potent hGCGR antagonist with exceptional selectivity over the human glucagon-like peptide-1 receptor. Oral administration of 1 lowered 24 h nonfasting glucose levels in imprinting control region mice on a high fat diet with diet-induced obesity following single oral doses of 3 and 10 mg/kg. Furthermore, compound 1, when dosed orally, was found to decrease fasting blood glucose at 30 mg/kg in a streptozotocin-treated, diet-induced obesity mouse pharmacodynamic assay and blunt exogenous glucagon-stimulated glucose excursion in prediabetic mice.

Full text of DOI:10.1021/jm401858f

Article
pubs.acs.org/jmc
The Discovery of N‑((2H‑Tetrazol-5-yl)methyl)-4-((R)‑1-((5r,8R)‑8‑(tert-
butyl)-3-(3,5-dichlorophenyl)-2-oxo-1,4-diazaspiro[4.5]dec-3-en-1-
yl)-4,4-dimethylpentyl)benzamide (SCH 900822): A Potent and
Selective Glucagon Receptor Antagonist
Duane DeMong,*,†,§ Xing Dai,*,†,§ Joyce Hwa,† Michael Miller,† Sue-Ing Lin,† Ling Kang,†
Andrew Stamford,† William Greenlee,† Wensheng Yu,† Michael Wong,† Brian Lavey,† Joseph Kozlowski,†
Guowei Zhou,† De-Yi Yang,† Bhuneshwari Patel,† Aileen Soriano,† Ying Zhai,† Christopher Sondey,†
Hongtao Zhang,† Jean Lachowicz,† Diane Grotz,† Kathleen Cox,† Richard Morrison,† Teresa Andreani,†
Yang Cao,† Mark Liang,† Tao Meng,† Paul McNamara,† Jesse Wong,† Prudence Bradley,† Kung-I Feng,†
Jitendra Belani,‡ Ping Chen,‡ Peng Dai,‡ Jolicia Gauuan,‡ Peishan Lin,‡ and He Zhao‡
†Discovery and Preclinical Sciences, Merck Research Laboratories, 2015 Galloping Hill Road, Kenilworth, New Jersey 07033, United
States
‡Medicinal Chemistry Department, AMRI, 26 Corporate Circle, P.O. Box 15098, Albany, New York 12212-5098, United States
S
* Supporting Information
ABSTRACT: A novel series of spiroimidazolone-based
antagonists of the human glucagon receptor (hGCGR) has
been developed. Our efforts have led to compound 1, N-((2H-
tetrazol-5-yl)methyl)-4-((R)-1-((5r,8R)-8-(tert-butyl)-3-(3,5-
dichlorophenyl)-2-oxo-1,4-diazaspiro[4.5]dec-3-en-1-yl)-4,4-
dimethylpentyl)benzamide (SCH 900822), a potent hGCGR
antagonist with exceptional selectivity over the human
glucagon-like peptide-1 receptor. Oral administration of 1
lowered 24 h nonfasting glucose levels in imprinting control
region mice on a high fat diet with diet-induced obesity
following single oral doses of 3 and 10 mg/kg. Furthermore,
compound 1, when dosed orally, was found to decrease fasting blood glucose at 30 mg/kg in a streptozotocin-treated, diet-
induced obesity mouse pharmacodynamic assay and blunt exogenous glucagon-stimulated glucose excursion in prediabetic mice.
To date, three separate small-molecule glucagon receptor
antagonists have been reported to show efficacy in human
subjects. The first human validation of glucagon receptor
blockade as a viable method of decreasing glucose output was
accomplished with (+)-BAY 27-9955 (Figure 1).15 This acute
study in healthy human subjects demonstrated that blockade of
INTRODUCTION
■
Diabetes is a chronic disease that afflicted an estimated 382
million people in the year 2013, according to the International
Diabetes Federation.1 The number of people suffering from
diabetes is expected to rise to 592 million in less than 25 years.
While multiple drugs exist for the treatment of type 2 diabetes
mellitus (T2DM), there is still a need for additional therapeutic
agents to combat the disease.
Glucagon is a 29-amino acid peptide hormone that was
discovered by Kimball and Murlin in 1923.2,3 This hormone,
which is counterregulatory to insulin, is generated in the α-cells
of the islets of Langerhans present in the pancreas by selective
cleavage of the pro-glucagon polypeptide.4 Binding of glucagon
to the glucagon receptor (a class B GPCR present primarily in
the liver) stimulates hepatic glucose production via glyco-
genolysis and gluconeogenesis.5−9 Given its critical role in the
regulation of plasma glucose levels, antagonism of the glucagon
receptor has been an area of interest for the potential treatment
of T2DM.10−14
Figure 1. (+)-BAY 27-9955 and MK-0893.
Received: December 7, 2013
Published: February 14, 2014
© 2014 American Chemical Society
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a
the glucagon receptor led to a decrease in glucose output upon
administration of exogenous glucagon. More recently, the
preclinical development and human clinical trial results for MK-
0893 were disclosed.16,17 As in the case of (+)-BAY 27-9955,
MK-0893 was shown to block exogenous glucagon-stimulated
glucose excursion in humans [200 mg q.d. (po), 59% blockade;
1000 mg q.d. (po), 100% blockade].18 In a study of patients
with T2DM for 4 years or more, MK-0893 at 60 and 80 mg q.d.
(po) demonstrated superior efficacy in both fasting plasma
glucose levels and HbA1c reduction when compared with a
standard metformin dose of 1000 mg b.i.d.19 Analogous to
(+)-BAY 27-9955 and MK-0893, LY2409021 (structure not
disclosed) blocked exogenous glucagon-stimulated glucose
excursion in humans.20 In human subjects with T2DM, after
a 28 day dosing period, statistically significant reductions in
HbA1c were observed at 60 and 90 mg doses of LY2409021.21
These independent clinical results validate antagonists of the
glucagon receptor as a therapeutic approach to treat T2DM.
Previously published reports described the preparation of
acyclic (2) and spirocyclic (3a,b) urea-based antagonists of the
human glucagon receptor (Figure 2).22−24 It was our desire to
Scheme 1. Phenylglycine-Based Synthetic Approach
a
Conditions: (a) methyl 4-(aminomethyl)benzoate hydrochloride,
PyBOP, i-Pr2NEt, CH2Cl2 (70%); (b) TFA, CH2Cl2 (99%); (c) 4-tert-
butylcyclohexanone (1 equiv), Et3N, 4 Å molecular sieves, MeOH,
microwave, 130 °C, 2 h (66%); (d) NBS (2 equiv), CH2Cl2, rt (80%)
or t-BuOCl, CH2Cl2, rt, 1.5 h then Et3N, 1.5 h; (e) 1 M NaOH,
MeOH, 1,4-dioxane, 65 °C (53%); (f) tert-butyl 3-aminopropanoate
hydrochloride, PyBOP, i-Pr2NEt, MeCN (71%); (g) TFA, CH2Cl2
(87%).
(Scheme 2). Methyl 4-(aminomethyl)benzoate hydrochloride
7 was coupled with N-Boc-glycine, and the resulting material
a
Scheme 2. Bromoimidazolone-Based Core Synthesis
Figure 2. Genesis of spiroimidazolone-based glucagon receptor
antagonists.
prepare human glucagon receptor (hGCGR) antagonists that
possessed a novel core structure. We chose to evaluate a
cyclohexanone-based spiroimidazolone such as (11h) as an
isostere for the spirocyclic urea 3a.
a
Conditions: (a) N-Boc-glycine, PyBOP, i-Pr2NEt, MeCN; (b) TFA,
CH2Cl2, (c) 4-tert-butylcyclohexanone, Et3N, 4 Å molecular sieves,
MeOH, microwave, 130 °C, 15% (three steps); (d) m-CPBA, CH2Cl2,
rt, 87%; (e) Br2, PPh3, Et3N, CH2Cl2, 70%; (f) 4-fluorophenylboronic
acid, Pd(dppf)Cl2·CH2Cl2, 2 M Na2CO3, DME, microwave, 100 °C, 5
min, 76%.
RESULTS
■
The preparation of the spiroimidazolone core was initiated by
coupling of the Boc-protected phenylglycine 4 with methyl 4-
(aminomethyl)benzoate hydrochloride (Scheme 1). Removal
of the Boc group afforded the amino amide 5, which was then
subjected to microwave-assisted cyclization with 4-tert-
butylcyclohexanone to afford the dihydrospiroimidazolone. It
is interesting to note that the cyclization afforded exclusively
the trans-isomer. Treatment of the cyclization product with N-
bromosuccinimide afforded compound 6. Hydrolysis of the
methyl ester was followed by coupling with tert-butyl 3-
aminopropanoate hydrochloride. Removal of the tert-butyl ester
with TFA afforded the desired compound 11a.
was treated with TFA to afford the desired amino amide.
Formation of the dihydrospiroimidazolone was accomplished
by condensing the amino amide with 4-tert-butylcyclohexanone.
Treatment of the cyclized product with m-CPBA in dichloro-
methane afforded the spiroimidazolone N-oxide 8 in 87%
yield.25 We hypothesized that compound 7 would undergo
conversion to the 4-bromoimidazolone 9 if properly activated
in the presence of a bromide source. This was realized by
treatment of 7 with triphenylphosphine and bromine to afford
9 in 70% yield.26 The resulting bromoimidazolone was then
converted to 10 via Suzuki coupling with 4-fluorophenylbor-
onic acid. The conversion of 8 to 10 via the 4-
Since it was desired, for the purpose of SAR exploration, to
possess a means to rapidly access compounds with a variety of
aryl groups, a second synthetic approach was developed
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bromoimidazolone represents a novel method for the
preparation of 4-arylimidazolones.
for 12a. The enantiomer 12b was determined to be at least an
order of magnitude less potent than 11a and 12a in the
hGCGR functional assay. The phenethyl-linked compound 12c
possessed a similar potency profile to 12a at the hGCGR and
improved selectivity over the hGLP-1R. Addition of a methyl
group to either methylene of 12c (compounds 12d and 12e,
both racemic) did not improve potency at the hGCGR and
decreased selectivity over the hGLP-1R.
The results observed for compounds 12a and 12b prompted
us to further probe the SAR around this new core structure
(Table 3). Overall the introduction of the (R)-methyl
substituent led to modest hGCGR functional potency improve-
ments (e.g., 12a vs 11a, 13b vs 11b, 13e vs 11i). A significant
improvement in both hGCGR and mouse liver membrane
binding potency was also observed. Select compounds in this
series afforded hGLP-1R functional antagonism in the single
digit micromolar range. While the most potent compounds
maintained from 20- to >60-fold functional selectivity between
hGCGR antagonism and hGLP-1R antagonism, further
selectivity improvements were sought.
As our initial in vitro potency screen we used a human
glucagon receptor (hGCGR) functional assay which measured
inhibition of cAMP production. Select compounds were then
tested in both human and mouse glucagon receptor binding
assays. The mouse liver membrane binding assay was used as a
means to assess compound potency at the murine glucagon
receptor. A functional hGLP-1 receptor assay was used as a
counterscreen, as we wanted to avoid antagonism of this
receptor.
Utilizing one of the methods outlined in Schemes 1 and 2, a
number of β-alanine benzamide-containing analogs were
prepared (Table 1). Compound 11h (structurally related to 1
Table 1. β-Alanine Benzamide SAR
It was at this point that it was decided to investigate
bioisosteres of the β-alanine benzamide (Table 4). In doing so,
some interesting and unexpected results arose. Regarding
potency at the hGCGR, the aminomethyltetrazole benzamide-
cotaining compound 14a was shown to be equipotent to 12a.
Furthermore, 14a did not significantly inhibit the hGLP-1R
(9% at 10 μM), providing us with the functional selectivity we
desired. In addition, 14a possessed an improved mouse liver
membrane binding potency, which we anticipated would enable
in vivo evaluation of antidiabetic effects in mice. Also
interesting was the fact that the minor change resulting in the
aminotetrazole amide 14b afforded an equipotent antagonist of
the hGCGR and hGLP-1R. Replacement of the β-alanine
residue of 12a with isoserine afforded derivatives 14c and 14d,
which possessed favorable hGCGR membrane binding potency
and selectivity over hGLP-1R. However, no hGCGR functional
potency was observed with these analogues.
To further probe the aminomethyltetrazole benzamide SAR,
it was decided to replace the chiral methyl group with a variety
of alkyl groups (Table 5). A wide variety of alkyl groups were
tolerated, with several possessing potency and selectivity similar
to that of compound 14a (e.g., 15c, 15d, and 15h). As we
began to add branching to the longer alkyl chains, we observed
a further increase in potency, with no loss in selectivity over the
hGLP-1R. This was exemplified most noticeably by compound
1.
With the tert-butylethyl group in compound 1 having
resulted in a significant improvement in functional potency
relative to 14a, it was decided to expand the aryl SAR in this
series. To enable this effort, we developed a scalable synthesis
of the requisite benzylamine (Scheme 3). Our approach
centered around a diastereoselective Grignard addition to a
chiral sulfinimine.27 Formation of the isopropyl ester of 4-
formylbenzoic acid was achieved in 79% yield. The aldehyde
was then converted to the (S)-tert-butyl sulfinimine 17 via the
method outlined by Nakata et al.28 The resulting sulfinimine
was treated with (3,3-dimethylbutyl)magnesium bromide to
afford the desired product as a mixture of diastereomers. The
isopropyl ester was chosen to allow for selective addition to the
sulfinimine over the ester. Silica gel chromatography, followed
by recrystallization in i-PrOH/heptane, afforded the diaster-
eomerically pure 18. The relative and absolute stereochemistry
of 18 was confirmed by X-ray crystallographic analysis (see
a
IC50
hGCGR
cAMP
hGCGR
binding
mouse liver membrane
binding
compd
R
11a
11b
11c
11d
11e
11f
3,5-diCl
3-CF3
2-F
290
520
>10000
10000
>10000
>10000
9600
>10000
−
−
−
−
−
510
−
730
>10000
3-Cl
770
770
4-CF3
4-Cl
800
1400
1600
1900
1900
3200
6800
940
−
−
−
−
11g
11h
11i
3,4-diCl
4-OCF3
4-F
11j
H
11k
2-Cl
−
a
IC50’s are reported in nM to two significnat figures. Results are
reported from single determinations.
and 2a) did not possess robust functional potency at the human
glucagon receptor. Generally, substitution at the 2- and 4-
positions was not well tolerated. The mouse liver membrane
binding potency was shown to be significantly diminished
relative to the hGCGR binding potency. One positive note
from this series of compounds was that at 10 μM no significant
inhibition (<50%) of human glucagon-like peptide-1 receptor
(hGLP-1R) was observed. Considering the profile of
compound 11a, these initial investigations provided us with a
promising series from which to build on.
We next decided to look at the effect of varying the linker
between the β-alanine benzamide and the imidazolone core
(Table 2). Generation of a chiral center via installation of a
methyl group onto the benzylic methylene present in
compound 11a afforded enantiomers 12a and 12b, which
demonstrated significantly different profiles from one another.
While the functional potency of 12a at the hGCGR was not
significantly different from that of 11a, both the hGCGR
binding and mouse liver membrane binding were improved. A
detectable amount of inhibition of hGLP-1R was also observed
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a
Table 2. β-Alanine Benzamide Linker SAR
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
Table 3. (R)-Methylbenzyl Core SAR
a
IC50
compd
R
hGCGR cAMP
hGCGR binding
mouse liver membrane binding
hGLP-1R cAMP
13a
12a
13b
13c
13d
13e
13f
3-Cl, 5-F
3,5-diCl
3-CF3
3-c-Pr
2-F
110 16 (2)
6
810
5600 960 (2)
6400 5200 (2)
6300
140
96
1200
280 170 (3)
720 610 (4)
2200 920 (2)
1700
340
56
>10000
350
1100
85 30 (2)
44
>10000
2500 1200 (2)
2300
>10000
4-F
430 170 (5)
7800 860 (2)
4300
3-Cl, 4-F
3-Cl
440
13g
13h
13i
480
130
150
95
3300
3900
3-Me
490
1700
>10000
3,4-diCl
3-F
550
3200
4400
13j
560
490
360
620
340
330
170
67
4400
>10000
13k
13l
H
630 190 (2)
630
5300
>10000
3-Cl, 2-F
3-Cl, 6-F
3-OCF3
4-CF3
4-OCF3
2-Cl
4000
>10000
13m
13n
13o
13p
13q
750
4600
>10000
840 100 (2)
910 350 (2)
1100
1500 1100 (3)
2800
2200
>10000
8000
>10000
2900
520
5400
>10000
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
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a
Table 4. β-Alanine Benzamide Bioisosteres
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
a
Table 5. Chiral Alkyl Benzamide Linkers
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
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a
hydrolysis of the isopropyl ester and subsequent peptide
coupling with aminomethyltetrazole hydrobromide afforded
22k.
Scheme 3. Chiral Benzylamine Synthesis
With a robust method in place for late-stage aryl
incorporation, the aryl SAR around compound 1 was probed
(Table 6). Functional potency was maintained or slightly
decreased when the chlorines present in 1 were systematically
replaced with fluorines (22a and 22b). Removal of one of the
halogens present in 22a (Compounds 22d and 22e) resulted in
a 3−4-fold decrease in functional potency. The unsubstitued
phenyl (22g) was approximately 4-fold less potent in the
functional assay. Incorporation of a nitrogen atom into the aryl
ring (e.g., 22l vs 22d) also afforded a decrease in functional
potency.
Having settled on the 3,5-dichlorophenyl group (compound
1) as the preferred aryl group, we then probed the
spirocyclohexane SAR (Table 7). Replacement of the tert-
butyl group on the cyclohexane ring with either isopropyl (23a)
or ethyl (23b) afforded compounds with in vitro profiles similar
to that of 1. Decreasing in size from ethyl to methyl (23c)
results in a 5-fold drop in function potency compared to that of
1, while the unsubstituted cyclohexane (23d) demonstrated a
10-fold decrease compared to 1. Conversion of the
spirocyclohexane moiety to a gem-dimethylimidazolone 23e
resulted in a near 100-fold drop in functional potency relative
to that of 1.
As a final SAR iteration, we chose to, once again, survey
bioisosteres for the aminomethyltetrazole present in compound
1 (Table 8). Extension of the tetrazole group present in 1 by
one methylene (compound 25j) unit resulted in a significant
decrease in functional potency. As was seen in the chiral methyl
series, use of the β-alanine group (25a) resulted in functional
potency similar to that of 1, but a significant degradation in
selectivity over the hGLP-1R. Interestingly, incorporation of the
aminotetrazole group (25b) resulted in a compound with
greater functional potency against the hGLP-1R than against
the hGCGR. The taurine-derived benzamide 25i was a poor
functional antagonist of the hGCGR, and attempts to
incorporate substituted β-alanines or aminomethyltetrazoles
resulted in loss in functional potency.
a
Conditions: (a) i-PrI, K2CO3, THF, DMF, 70 °C, 64 h (79%); (b)
(S)-2-methylpropane-2-sulfinamide, Cs2CO3, CH2Cl2, 42 °C, 30 h
(99%); (c) (3,3-dimethylbutyl)magnesium bromide (0.5 M in Et2O),
CH2Cl2, −78 °C to rt (32%); (d) 4 N HCl in dioxane, MeOH, rt
(84%).
Supporting Information). Deprotection of 18 with HCl in
dioxane and MeOH afforded the enantiopure 19 in 84% yield.
Attempts to use a method similar to that outlined in Scheme
2 to enable late-stage aryl introduction were unsuccessful due to
rapid hydrolysis of the bromoimidazolone to the imidazolidine-
4,5-dione. These results led us to use an imidazolidine-4,5-
dione as a precursor to a triflylimidazolone, which would serve
as an analogous coupling partner to the bromoimidazolone
(Scheme 4). The N-Boc-glycinamide of 19 was prepared in
a
Scheme 4. Synthesis of 22k
DISCUSSION AND CONCLUSIONS
■
On the basis of their in vitro potency and selectivity profiles,
compounds 1, 22d, 22e, and 23a were selected for
pharmacokinetic (PK) profiling in rat, dog, and monkey.
Compound 1 showed low plasma clearance, acceptable half-life,
and moderate oral bioavailability across all species with the
exception of rat (Table 9; PK data for compounds 22d, 22e,
and 23a are shown in the Supporting Information). These
results led us to select compound 1 for further profiling.
Compound 1 was subjected to a 55-member CEREP
counterscreen panel. At a concentration of 10 μM, compound
1 inhibited antagonist binding to AT1 at 93% and inhibited
agonist binding to A3 at 96% (Ki = 2429 nM), κ-opioid
receptor at 75%, and VPAC (VIP1) at 75%. All other hits were
below 75% inhibition of the control specific binding.
Compound 1 was also ∼100-fold less potent in a human
glucose dependent insulinotropic peptide receptor (hGIPR)
functional assay (IC50 = 2100 nM), which is a class B GPCR
that is closely related to the hGCGR.
a
Conditions: (a) N-Boc-glycine, EDCI, HOBt, i-Pr2NEt, MeCN, rt
(quant); (b) TFA, CH2Cl2, rt (97%); (c) 4-tert-butylcyclohexanone,
Et3N, MeOH, reflux, (48%); (d) t-BuOCl, CH2Cl2, 0 °C, then Et3N
(73%); (e) m-CPBA, CH2Cl2, rt, 18 h (74%); (f) Tf2O, Et3N, CH2Cl2,
−10 °C (67%); (g) 4-methoxyphenylboronic acid, Pd(PPh3)2Cl2, 2 M
Na2CO3, DME, microwave, 100 °C, 45 min (30%); (h) 1 M NaOH
(aq), THF, MeOH, rt, 16 h (52%); (i) (2H-tetrazol-5-yl)methanamine
hydrobromide, PyBOP, i-Pr2NEt, DMF (65%).
quantitative yield. Removal of the Boc group was followed by
cyclization with 4-tert-butylcyclohexanone. Treatment of the
imidazolidinone with tert-butylhypochlorite followed by elim-
ination of the resulting N-chlorinated compound with triethyl-
amine afforded compound 20. Oxidation of 20 with m-CPBA
afforded the imidazolidinedione, which was converted to the
triflate 21 with triflic anhydride and triethylamine. Suzuki
coupling with 4-methoxyphenyl boronic acid followed by
The acceptable oral exposure in mouse led us to probe the in
vivo effects of 1 via oral administration (Figure 3). Imprinting
control region mice on a high fat diet with diet-induced obesity
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Table 6. Aryl Substituent SAR
a
IC50
compd
R
hGCGR cAMP
hGCGR binding
mouse liver membrane binding
hGLP-1R cAMP
1
3,5-di-Cl-phenyl
3-Cl,5-F-phenyl
3,5-di-F-phenyl
3-Cl-phenyl
46 12 (9)
42
24 15 (15)
130 106 (10)
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
>10000
22a
22b
22d
22e
22f
22g
22h
22i
22j
22k
22l
22m
22n
10
160
110 28 (5)
130 55 (5)
140 38 (3)
160
23 18 (9)
140 100 (10)
14 6 (5)
130 57 (4)
3-F-phenyl
21 19 (7)
100 51 (4)
4-CF3-phenyl
4-F-phenyl
7
190
240 57 (3)
180
13 4.4 (3)
90 90 (3)
3,4-di-F-phenyl
3-CF3-phenyl
3-OMe-phenyl
4-OMe-phenyl
5-Cl-pyridin-3-yl
3-CF3-pyridin-2-yl
4-CF3-pyridin-3-yl
13 2.1 (2)
170 0 (2)
180
13
15
26
38
−
140
−
−
230
360
480
84
1600
−
96
1200
22
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
a
Table 7. Spirocyclohexane SAR
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
(ICR-DIO mice) were administered a single dose of compound
1 at doses of 3 and 10 mg/kg po. At 4 h fasting blood glucose
and at 24 h nonfasting blood glucose levels were measured via
glucometer. Compound 1, at both dose levels caused a
statistically significant (P < 0.01) decrease in blood glucose
concentrations at both the 4 and 24 h time points.
Given its ability to decrease blood glucose levels in the ICR-
DIO mouse model, compound 1 was further evaluated in ICR-
DIO mice with streptozotocin-induced type 2 diabetes (STZ-
DIO mice). During this pharmacodynamic assay, fasting blood
glucose was measured via tail bleeds at 8 h after oral dosing of
vehicle, 3, 10, or 30 mg/kg of compound 1 3 h after onset of
light cycle (Figure 4). When compared to vehicle controls, 1
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a
Table 8. Aminomethyltetrazole Isosteres
a
IC50’s are reported in nM to two significnat figures as the mean SD with the number of assay runs in parentheses. In the cases where no SD is
reported, the number of assay runs is one.
a
Table 9. Pharmacokinetic Profile of 1
iv
po
species
dose
ClP
Vdss
T1/2
dose
dnAUC
%F
mouse
rat
3
3
1
3
1.4
1.3
11
3
3
5
3
6
31
1.8 0.48
0.3 0.1
0.6 0.1
1.3 0.21
0.1 0.05
0.4 0.07
8.3 0.97
6.1 3.3
7.9 0.20
2.2 0.38
30 4.0
22 7.0
15 2.7
32 4.2
49 15
dog
monkey
a
Units: dose, mg kg−1; Clp, mL min−1 kg−1; Vdss, L kg−1; T1/2, h; and dnAUC (dose-normalized AUC), μM h mg−1 kg−1. Results reported as mean
SD.
caused a statistically significant decrease in 8 h fasting glucose
levels following the 30 mg/kg oral dose. Plasma concentrations
(1 M) of compound 1 for each dose at 8 h post-treatment were
as follows: 3 mg/kg, 0.43 0.20; 10 mg/kg, 0.81 0.18; 30
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selectivity over the related receptors hGLP-1R and hGIPR.
Moreover, 1 demonstrated a significant glucose-lowering effect
in an STZ-DIO mouse model of type II diabetes at 30 mg/kg
and blunted exogenous glucagon-induced hyperglycemia in
prediabetic mice.
ASSOCIATED CONTENT
■
S
* Supporting Information
Chemical and biological experimental procedures and com-
pound characterization data. This material is available free of
charge via the Internet at http://pubs.acs.org.
Figure 3. Acute in vivo efficacy of 1 in ICR-DIO mice. Fasting blood
glucose was determined at 4 h post-administration of 1 and nonfasting
blood glucose was determined at 24 h post-administration of 1. Plasma
levels of 1 were determined at 24 h post-administration of drug.
Plasma levels (μM) for each dose: 3 mg/kg, 0.43 0.20; 10 mg/kg,
0.81 0.18.
AUTHOR INFORMATION
■
Corresponding Authors
*E-mail: duane.demong@merck.com. Phone: 1-908-740-4410.
*E-mail: xing.dai@merck.com. Phone: 1-908-740-2607.
Author Contributions
§These authors contributed equally.
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
The authors would like to thank Mary Senior, Alexei Buevich,
Tze-Ming Chan, and Andy Evans for their NMR spectroscopy
support and Matthew Lamm for his X-ray crystallography
support.
ABBREVIATIONS USED
■
hGCGR,human glucagon receptor; hGLP-1R,human glucagon-
like peptide 1 receptor; hGIPR,human gastrointestinal
polypeptide receptor; ICR mice,imprinting control region
mice; PK,pharmacokinetics; AUC,area under the curve; STZ-
DIO,streptozotocin-treated, diet-induced obesity
Figure 4. Acute effect of 1 on fasting blood glucose in STZ-DIO mice.
Normalized change in blood glucose and plasma levels of 1 were
determined at 8 h post-administration of 1. Plasma levels of 1 (μM)
for each dose: 3 mg/kg, 1.1 0.38; 10 mg/kg, 4.5 1.4; 30 mg/kg,
8.5 7.3.
mg/kg, 8.5 7.3. Compound 1 had no effect on body weight
24 h after oral administration.
Figure 5 displays the reduction, by compound 1, of glucagon-
induced glucose excursion in ICR-DIO mice. At both the 10
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