Human glucagon receptor antagonists based on alkylidene hydrazides.

Article abstract of DOI:10.1016/S0960-894X(01)00819-8

A series of alkylidene hydrazide derivatives containing an alkoxyaryl moiety was optimized. The resulting hydrazide-ethers were competitive antagonists at the human glucagon receptor. Pharmacokinetic experiments showed fast clearance of most of the compou

Full text of DOI:10.1016/S0960-894X(01)00819-8

Bioorganic & Medicinal Chemistry Letters 12 (2002) 663–666
Human Glucagon Receptor Antagonists Based
on Alkylidene Hydrazides
Anthony Ling,a,* Michael Plewe,a Javier Gonzalez,a Peter Madsen,b Christian K. Sams,b
Jesper Lau,b Vlad Gregor,c Doug Murphy,a Kimberly Teston,a Atsuo Kuki,a
Shenghua Shi,a Larry Truesdale,a Dan Kiel,a John May,a James Lakis,a Kenna Anderes,a
Eugenia Iatsimirskaia,a Ulla G. Sidelmann,b Lotte B. Knudsen,b Christian L. Brandb and
Alex Polinskya
aPfizer Global Research and Development, 10770 Science Center Dr., San Diego, CA 92121, USA
bNovo Nordisk, Novo Nordisk Park, 2760 Maaloev, Denmark
cAnadys Pharmaceuticals Inc., 9050 Camino Santa Fe, San Diego, CA 92121, USA
Received 4 October 2001; accepted 5 December 2001
Abstract—A series of alkylidene hydrazide derivatives containing an alkoxyaryl moiety was optimized. The resulting hydrazide-
ethers were competitive antagonists at the human glucagon receptor. Pharmacokinetic experiments showed fast clearance of most
of the compounds tested. A representative compound [4-hydroxy-3-cyanobenzoic acid (4-isopropylbenzyloxy-3,5-dimethoxy-
methylene)hydrazide] with an IC50 value of 20 nM was shown to reduce blood glucose levels in fasted rats. # 2002 Elsevier Science
Ltd. All rights reserved.
Glucagon stimulates glycogenolysis and gluconeogenesis
resulting in increased levels of glucose in the blood.1,2 In
diabetes, the bihormonal hypothesis implicates not only
the lack of an insulin effect but also a paradoxically
elevated relative level of circulating glucagon. It has
been demonstrated that immunoneutralizing both exo-
genously administered as well as endogenous glucagon
in various animal species effectively alleviates glucagon-
stimulated hyperglycemia.3À5 Thus, small molecule glu-
cagon receptor antagonists6À9 may be useful agents for
the treatment of diabetes.
through alkylation of the naphthylhydroxy group. In
this Letter we report further optimization of a series of
alkylidene hydrazides having high affinity for the
human glucagon receptor, representative in vitro meta-
bolism, in vivo pharmacokinetic and pharmacodynamic
results.
Previously we reported the discovery of a class of
human glucagon receptor antagonists exemplified by
3-chloro-4-hydroxybenzoic acid (4-hydroxy-1-naphthyl-
methylene)hydrazide (1) prepared from the condensation
of 3-chloro-4-hydroxybenzoylhydrazide and 4-hydro-
xynaphthaldehyde.10 Loss of hydrogen-bond donation
capability of 1 (IC50=200 nM) by methylation of the
naphthylhydroxy group to compound 2 (IC50=870 nM)
did not result in substantial loss of affinity. This obser-
vation prompted us to further elaborate structure 1
The alkylidene hydrazides were prepared either indivi-
dually or in parallel as illustrated by the example in
Scheme 1. Alkoxyarylaldehydes or ketones, obtained by
alkylation of hydroxyarylaldehydes or ketones in the
presence of potassium carbonate in acetonitrile, were
condensed with hydrazides in DMSO. The starting
hydrazides were prepared either by hydrazinolysis of the
corresponding esters, or by amide-bond formation with
t-butyl carbazate followed by deprotection. Indoles 18
and 19 were prepared by alkylation of formylindoles
under sodium hydride conditions followed by hyd-
razone formation. Hydrazide 20 was obtained by
reduction of hydrazone 9 with NaBH4/cat. TFA.
*Corresponding author. Tel.: +1-858-526-4660; fax: +1-858-678-
8156; e-mail: anthony.ling@agouron.com
0960-894X/02/$ - see front matter # 2002 Elsevier Science Ltd. All rights reserved.
PII: S0960-894X(01)00819-8

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A. Ling et al. / Bioorg. Med. Chem. Lett. 12 (2002) 663–666
Coupling of alkoxydimethoxyphenethylamine with
hydroxychlorobenzoic acid or the corresponding Pinner
imidate11 gave 21 or 22, respectively. Compound 23 was
prepared from the condensation of hydroxychloroani-
line with the corresponding alkoxyphenylacrylic acid via
HBTU activation. The sulfonylhydrazone 24 was
obtained from the condensation of the corresponding
sulfonylhydrazide12,13 with the alkoxybenzaldehyde.
groups were preferred by the receptor. Structure–activity
relationships of representative resynthesized pure com-
pounds are summarized in Table 1. Compounds alkyl-
ated with para-substituted benzyl groups were among
the most potent (3–4 and 9–11), whereas compounds
alkylated with hydrophilic groups such as 5 and 12 were
less potent. Addition of two ethyl groups to the primary
amide 5 resulted in compound 6, displaying a 10-fold
increase in binding affinity, indicating that lipophilic
groups were preferred by the receptor. Incorporation of
groups containing heteroatoms with hydrogen bond
acceptor properties into this region of the molecules
also resulted in compounds with modest affinity. For
example, the pyranyl 7 and 1,2,4-oxadiazolyl 8 had IC50
values of 140 and 160 nM, respectively. The combina-
tion of 4-isopropylbenzyl with 3,4-dimethoxy-4-
hydroxybenzaldehyde as in compound 9 (IC50=23 nM)
led to a 10-fold improvement in binding affinity com-
pared to 1 (IC50=200 nM).
A targeted library consisting of hydrazide-ethers prepared
according to Scheme 1 was produced. Screening of the
library at 1 mM using 127I-labeled glucagon resulted in
identification of compounds with low nanomolar affi-
nity for the human glucagon receptor with preference
for the 4-hydroxynaphthyl and the 3,5-dimethoxy-4-
hydroxybenzyl groups as the core moiety of the mole-
cules. Among the alkylating agents used for O-alkylation
of the core aryl moieties, hydrophobic aromatic
Modifications of the naphthalene core moiety in 3 or
the 3,5-dimethoxyphenylene moiety in 9 are shown in
Table 2. Replacement of both methoxy groups in 9
(IC50=23 nM) with methyl groups in 13 (IC50=330
nM) resulted in a 15-fold decrease in affinity. Removal
of one or both methoxy groups from 9 resulted in 14
and 15, respectively, exhibiting 50-fold loss of affinity.
Replacement of one methoxy for a carboxy group (16)
resulted in further loss of affinity. Truncation of the
naphthalene in 3 (IC50=53 nM) for a dimethylphenyl-
ene core 17 (IC50=740 nM) likewise exhibited a loss of
affinity. On the other hand, compounds containing a
1,3- or 1,4-disubstituted indole bicyclic system (18 or 19)
were well tolerated by the receptor, the 1,4-substituted
Scheme 1. Reagents and conditions: (a) K2CO3, CH3CN, 4-iso-
propylbenzyl chloride, reflux 18 h (87%); (b) ICl, AcOH (30%); (c)
CuCN, DMF, reflux 2 h (37%); (d) KOH, H2O/THF, then HCl
(87%); (e) t-BuOCONHNH2, DIEA, PYBOP, then TFA (56%); (f)
DMSO (82%).
Table 1. In vitro activities of glucagon antagonists containing a
naphthalene or dimethoxyphenylene central moiety
Table 2. In vitro activities of glucagon antagonists with modified
naphthalene or dimethoxyphenylene moieties
No.
IC50 (mM) No.
IC50 (mM)
No.
W
IC50 (mM)
No.
W
IC50 (mM)
3
4
5
0.053
0.058
1.2
9
10
11
0.023
0.18
0.15
9
3
0.023
0.053
13
14
15
16
0.33
1.3
17
18
19
0.74
0.39
0.084
6
0.13
12
0.69
7
8
0.14
0.16
1.5
30

A. Ling et al. / Bioorg. Med. Chem. Lett. 12 (2002) 663–666
Table 3. Explorations of alkylidene hydrazide isosteres
665
No.
W
IC50 (mM)
Figure 1. Sites of metabolism for 28.
0.88
20
21
22
23
24
25
1.90
5.20
6.40
78.30
>100
Figure 2. Compound 28 significantly reduced blood glucose at 10 and
30 min post injection. At 60 min post injection blood glucose was not
different from control. *p<0.05 versus vehicle.
methylation of the hydrazone carbon to 25 led to
compounds with decreased activities. It appeared
that the position of the carbonyl and the nitrogen
functionalities, and overall geometry of this region, were
crucial to the overall recognition of the molecule by the
receptor.
Table 4. Effect of R1 and R2 substituents in the phenol ring
In vitro metabolism studies in rat liver microsomes
indicated that the hydroxyl groupin the benzoyl ring in
9 underwent rapid glucuronidation. To better under-
stand the effect of this site on receptor affinity and the
metabolic stability of this class of compounds, analo-
gues containing various R1 and R2 substituents in the
phenolic ring were prepared (Table 4). Previously we
suggested10 that the presence of an electron-with-
drawing substituent lowered the pKa of the hydroxy
functionality, thus enhancing its ability to hydrogen-
bond with the receptor. As expected, electron-with-
drawing substituents such as chloro 9, fluoro 26, nitro
27, and cyano 28 at the R1 position resulted in com-
pounds with much improved affinity compared to the
unsubstituted phenol 29. The chloro 9 and the cyano 28
were equipotent. Compound 28 displayed a mean IC50
value of 20 nM in a competition-binding assay using
127I-radiolabeled glucagon and membranes from cells
transfected with the human glucagon receptor. Com-
pound 28 had a mean IC50 value of 1.0 nM for the rat
receptor. Addition of a second halogen atom at R2 (30
and 31) did not significantly affect the potency of the
compounds. Electron-donating substituents at R1 such
as the amino groupin 32 or the hydroxy groupin
33 resulted in compounds with significantly less affinity
for the receptor. In vitro metabolism experiments
indicated a significantly diminished glucuronidation
rate for 28 (27 pmol/min/mg protein) compared to 9
No.
R1
R2
IC50 (mM)
9
Cl
F
NO2
CN
H
Cl
F
NH2
OH
H
H
H
H
H
Cl
F
0.023
0.11
0.050
0.020
0.69
0.031
0.048
0.94
26
27
28
29
30
31
32
33
H
H
2.7
indole 19 being more potent (84 nM). These results
indicated that the arylene core moiety needs to be
hydrophobic and electron-rich in order to give com-
pounds with high affinity towards the human glucagon
receptor.
The alkylidene hydrazide region proved to be sensitive
to modification. Several alkylidene hydrazide equiva-
lents were explored (Table 3). The isosteres consisted of
a four atom system possessing the ability to exist in an
iminol-like tautomeric form and containing at least one
sp2 carbon center. The isosteres investigated were at
least one order of magnitude less active than the hydra-
zone 9. Reduction of the sp2 hydrazone carbon in 9 to
the sp3 hydrazide 20, replacement of the planar amide
moiety with the tetrahedral sulfonamide 24, and

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A. Ling et al. / Bioorg. Med. Chem. Lett. 12 (2002) 663–666
(169 pmol/min/mg protein). In vivo pharmacokinetic
experiments indicated that 28 had a significantly longer
half-life than 9 (t1=2=60 min vs 18 min).
Acknowledgements
We thank Dr. Marvin Brown, Dr. Behrend Lundt and
Dr. Niels Fiil for the many instrumental discussions and
for weaving this collaboration together.
Compound 28 was investigated further with respect to
the major metabolites formed in vitro and it was found
that the compound was metabolized in both human and
rat liver microsomes via both phase I (oxidation) and
phase II (glucuronidation) pathways (Fig. 1). The major
metabolic pathways in rat liver microsomes were iden-
tified to be hydroxylation, further oxidation to a car-
bonyl, or elimination of the alcohol to an alkene in the
isopropyl group. Glucuronidation of the phenol moiety
was found to be minor. Replacement of the chlorine
atom by a cyano grouptherefore resulted in a metabolic
swap from phase II to phase I metabolism within this
series of compounds.
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The nonpeptide glucagon antagonist 28 significantly
lowered blood glucose in fasted rats (Fig. 2). Under the
experimental conditions, maintenance of blood glucose
levels in fasted rats is largely dependent on glucagon.
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rats (data not shown).
In summary, a series of alkylidene hydrazides was
optimized to low nanomolar affinities at the human
glucagon receptor. Compound 28 had an IC50 value of
20 nM and significantly lowered blood glucose in fasted
rats. Replacement of the chlorine substituent with a
cyano groupin the C3 position of the benzoyl ring led
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Further in vitro metabolism and PK optimization of
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