Indole-2-carboxamide inhibitors of human liver glycogen phosphorylase [3]

Full text of DOI:10.1021/jm980264k

2934
J . Med. Chem. 1998, 41, 2934-2938
In d ole-2-ca r boxa m id e In h ibitor s of
Hu m a n Liver Glycogen P h osp h or yla se†
Dennis J . Hoover,*,‡ Sheri Lefkowitz-Snow,‡
J ana L. Burgess-Henry,‡,§ William H. Martin,|
Sandra J . Armento,| Ingrid A. Stock,|
F igu r e 1. Selected details of hepatic glycogenolysis and
gluconeogenesis. Abbreviations: HLGPa, human liver glycogen
phosphorylase a (phosphorylated, active form); HLGPb, un-
phosphorylated form; UDP-G, uridine diphosphoglucose; G-1-
P, glucose-1-phosphate; G-6-P, glucose-6-phosphate.
R. Kirk McPherson, Paul E. Genereux,
E. Michael Gibbs, and J udith L. Treadway*,
Departments of Cardiovascular and Metabolic Diseases
Medicinal Chemistry, Exploratory Medicinal Biology, and
Cardiovascular and Metabolic Diseases Biology, Central
Research Division, Pfizer Inc., Groton, Connecticut 06340
glucose-6-phosphate (G-6-P) bind here as inhibitors.21
Synthetic G-6-P analogues22 and a dihydropyridinedi-
carboxylic acid23 also inhibit at the nucleotide activation
site of rabbit muscle glycogen phosphorylase b (RMG-
Pb). Most reported phosphorylase inhibitors10 are
glucose or other sugar-based analogues,24 which have
in numerous instances been shown to bind at the
catalytic site of RMGPb.25-29 Caffeine and other nu-
cleosides inhibit at another allosteric site, termed the
purine inhibitor site.30 No physiologic role or ligand for
the purine inhibitor site is known, although xanthines
may regulate phosphorylase activity here in certain
physiologic states.31,32 Inhibition by caffeine and glucose
is synergistic, because of cooperative ordering of both
binding sites.25 This is an attractive property, for a
therapeutic inhibitor which would lose potency as
glucose levels fall should offer reduced risk of severe
hypoglycemia.
Recently, we reported finding the orally active glyco-
gen phosphorylase inhibitor 1 (Table 1) by high-
throughput screening against recombinant human liver
glycogen phosphorylase a (rHLGPa).18 Other inhibitors
of the human liver isoform33 of glycogen phosphorylase
have not been described. Here, we demonstrate that
compounds in two series derived from 1 (Tables 1 and
2) are also potent inhibitors of rHLGPa and have
cellular and oral activity. Their synthesis and charac-
terization are also described.
The simple, heterocyclic, and asymmetric structure
of 1 was exciting, as was the opportunity to explore the
importance of these features to its high inhibitory
activity. Inhibitor 1 and other (3S,2R)-2-amino-3-hy-
droxy-4-phenylbutyric acid derivatives were prepared
as shown in Scheme 1. Methyl ester 28 was formed on
treatment of cyanohydrin 2734 with anhydrous HCl in
methanol and purified by extraction and recrystalliza-
tion. Carbodiimide-mediated coupling with commer-
cially available indole-2-carboxylic acids gave methyl
ester 10 and the fluoro, bromo, and deschloro analogues
29-31 in high yield. Some of these products contained
up to 10% of the diester resulting from hydroxyl
acylation, and were hydrolyzed with aqueous sodium
hydroxide in methanol, giving acids 11 and 32-34 in
pure form after extraction and trituration. Coupling
with dimethylamine and methylamine hydrochlorides
gave target amides 1 and 5-8 in good yields. The
stereoisomers of 1 (2-4) were also synthesized this way,
from the stereoisomers of methyl ester 28, which were
obtained as their hydrochlorides in quantitative yield
by refluxing the commercial (2S,3R)-, (2R,3R)-, and
Received May 1, 1998
Type 2 diabetes (formerly non-insulin-dependent dia-
betes mellitus) is a severe and prevalent disease.
Diabetics may suffer debilitating eye, kidney, and nerve
damage and are at risk of premature death due to these
and other diabetic complications.1 Tight control of
plasma glucose, achieved by intensive insulin therapy,
reduces the incidence and progression of diabetic com-
plications in type 1 and type 2 diabetics.2,3 Unfortu-
nately, achieving control is often impossible with current
oral hypoglycemic agents. Moreover, their chronic use
often brings, in addition to toleration, fear of dangerous
hypoglycemic episodes. Safer and more effective therapy
is urgently needed.
We targeted liver glycogen phosphorylase (EC 2.4.1.1)
as an approach to controlling elevated glucose levels.
The liver is the predominant source of plasma glucose
in fasting type 2 diabetics.4-7 Figure 1 shows, sche-
matically, selected details of hepatic glucose synthesis,
storage, and release.8 Glycogen phosphorylase9,10 re-
leases glucose-1-phosphate from glycogen. Although
significant evidence exists of an important role for
glycogenolysis in hepatic glucose production,11-14 gly-
cogen phosphorylase is not a widely pursued antidia-
betic target, partly because of conclusions that the high
glucose levels in type 2 diabetics arise independently
from elevated gluconeogenesis.7,15,16 Recent data, how-
ever, showing that much of glucose arising from gluco-
neogenesis has cycled through glycogen,17 exposes a
possibility that inhibition of glycogen phosphorylase
could suppress hepatic glucose arising from both path-
ways.18 Orally active glycogen phosphorylase inhibi-
tors19 would clearly be valuable additions to this re-
search area.
Activity of liver glycogen phosphorylase is regulated
physiologically by hormonal and neuronal signals which
alter phosphorylation at serine 14. Only the phospho-
rylated enzyme has significant activity. The liver
isozyme is also regulated by ligands which bind to a
distant allosteric site, termed the nucleotide activation
site. AMP binds here as an activator,20 while ATP and
† This manuscript is dedicated to the occasion of Professor E. J .
Corey’s 70th birthday.
* To whom correspondence should be addressed.
‡ Department of Cardiovascular and Metabolic Diseases Medicinal
Chemistry.
§ Present address: AXYS Pharmaceuticals, 180 Kimball Way, South
San Francisco, CA 94080.
| Exploratory Medicinal Biology.
Cardiovascular and Metabolic Diseases Biology.
S0022-2623(98)00264-7 CCC: $15.00 © 1998 American Chemical Society
Published on Web 07/30/1998

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 2935
Sch em e 1a
Ta ble 1. Structures and in Vitro Phosphorylase Inhibitory
Activity of 3-[(Indole-2-carbonyl)amino]-2-hydroxy-4-phenyl-
butyric Acid Analogues
rHLGPa
cell
compd
X
R
Y
*
IC50, nMa
IC50, µMb
1
2
3
4
5
6
7
8
9
10
11
12
13
Cl Ph CONMe2
Cl Ph CONMe2
Cl Ph CONMe2
Cl Ph CONMe2
3S,2R 110 ( 12
3R,2S 1800 ( 320
3R,2R 1700 ( 190
3S,2S 8700 ( 1700
3S,2R 170 ( 16
3S,2R 97 ( 11
1.5
F
Ph CONMe2
Br Ph CONMe2
Ph CONMe2
6.2
3.3
>30
>30
a
Key: (a) HCl-MeOH, 60 h, 23 °C, then NaHCO3/H2O, 77%;
(b) 1.0 equiv of 1-(3-(dimethylamino)propyl)-3-ethylcarbodiimide
hydrochloride, 1.5 equiv of 1-hydroxybenzotriazole, dichloromethane,
23 °C, 18 h; (c) 2.0 equiv of NaOH, MeOH, H2O, 25 °C, 2 h; (d), as
for (b), but using also 1.1 equiv of either Me2NH‚HCl or MeNH2‚HCl
and 1.1 equiv of triethylamine in DMF, 18 h, 23 °C.
H
3S,2R 440 ( 48
Cl Ph CONHMe 3S,2R 680 ( 37
Cl Ph CONH2
Cl Ph COOMe
Cl Ph COOH
Cl Ph CH2OH
Cl Cyc CONMe2
3S,2R 1700 ( 140
3S,2R 210 ( 40
3S,2R 330 ( 31
3S,2R 6800 ( 1700
3S,2R 6500 ( 610
13
>30
Sch em e 2a
a
Arithmetic mean ( SEM (n g 3, each in triplicate). b Inhibition
of forskolin-stimulated glycogenolysis in SK-HEP-1 cells (pooled
data from n g 2, each in triplicate). c Cyclohexyl.
Ta ble 2. Structures and in Vitro Phosphorylase Inhibitory
Activity of N-(Indole-2-carbonyl) Phenylalanine Analogues
a
R, Y, and X are defined in Table 2. Key: (a) 1.0-1.1 equiv of
dimethylamine, methylamine, or 4-hydroxy-1-piperidine hydro-
chloride, 1.1 equiv of Et3N, 1.0 equiv of DEC, 1.5 equiv of HOBT,
CH2Cl2, 0-20 °C, 18 h; (b) HCl-dioxane, 23 °C, 1 h; (c) 1.0
X-indole-2-COOH, 1.0 DEC, 1.5 HOBT, 1.0 Et3N, CH2Cl2, 23 °C,
18 h.
rHLGPa cell IC50
,
37 and benzofuran 38 were obtained from (S)-phenyla-
lanine dimethylamide hydrochloride (35a ) and 1-meth-
ylindole-2-carboxylic acid or 5-chlorobenzofuran-2-
carboxylic acid.
compd
X
R
Y
*
IC50, nMa
µMb
1.9
12
5.2
12
1.4
14
15
16
17
18
19
20
21
22
23
24
25
26
Cl
H
H
H
H
H
H
H
H
H
F
CONMe2
CONMe2
CONHMe
CONHMe
CONMe2
CONMe2
CONMe2
CONMe2
CO(1-piperidin-4-ol)
CO(1-piperidin-4-ol)
COOMe
COOH
CH2OH
S
R
S
R
S
S
S
S
S
S
S
S
S
82 ( 10
Cl
Cl
Cl
Br
F
260 ( 33
110 ( 10
220 ( 32
110 ( 8
430 ( 44
400 ( 45
4700 ( 300
260 ( 17
205 ( 16
140 ( 18
2.3
7.8
H
OMe
Cl
Cl
Cl
Cl
Cl
5.1
1.9
>30
H
H
H
1700 ( 490 >30
>10000c
a
Arithmetic mean ( SEM (n g 3, each in triplicate). b Inhibition
of forskolin-stimulated glycogenolysis in SK-HEP-1 cells (pooled
data from n g 2, each in triplicate). c 23% inhibition at 10 µM.
Compounds were assessed for ability to inhibit rHLG-
Pa-catalyzed release of phosphate from glucose-1-
phosphate (reverse direction of the physiological reac-
tion) in the presence of glycogen and a high physiological
concentration of glucose (7.5 mM).18 The (3S,2R) ster-
eochemistry of 1 is optimal as seen in the reduced
inhibitory activity of stereoisomers 2-4 (Table 1). In-
hibitory activity was also sensitive to changes in the
indole 5-substituent. Activity was completely retained
in bromo analogue 6, but diminished 1.5- and 4-fold in
the 5-fluoro and deschloro analogues (5 and 7, respec-
tively). C-Terminally modified analogues showed con-
siderable variation in their inhibitory activity. Monom-
ethylamide 8 and primary amide 9 showed 6- and 15-
fold lower activity than 1. The synthetic intermediates
10 (methyl ester) and 11 (carboxylic acid) were closer
in activity to 1. Diol 12 showed about a 60-fold
reduction of inhibitory activity relative to 1, as did the
cyclohexylmethyl side chain analogue 13.
(2S,3S)-3-amino-2-hydroxy-4-phenylbutyric acids with
chlorotrimethylsilane in methanol.35 Less than 1% of
1 was present in isomers 2-4 by chiral stationary phase
HPLC analysis. Primary amide 9 was obtained by
heating methyl ester 10 with ammonia in methanol.
Diborane reduction of acid 11 gave diol 12. Cyclohexane
13 was prepared by steps b-d of Scheme 1, starting
from (2R,3S)-3-amino-4-cyclohexylmethyl-2-hydroxybu-
tyric acid isopropyl ester.36
Phenylalanine-containing analogues of 1 were also
synthesized (Scheme 2), by stepwise coupling from the
C-terminus using t-Boc-protected phenylalanines. Meth-
yl ester 24 and alcohol 26 were obtained from L-
phenylalanine methyl ester hydrochloride and L-phe-
nylalaninol, respectively. Hydrolysis of 24 with aqueous
lithium hydroxide in THF gave acid 25. N-Methylindole

2936 J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16
Communications to the Editor
F igu r e 3. Time course of glucose lowering following oral
dosing of 1 in diabetic ob/ ob mice. Vertical lines show SEM.
Each point represents 35-66 mice per group. Significance
(***P < 0.001, **P < 0.01) by unpaired t-test relative to vehicle
control groups. Similar results were obtained in an indepen-
dent experiment of the same design.
F igu r e 2. Difference in 14C glycogen content (dpm) of [14C]-
glycogen prelabeled, vehicle-treated human SK-HEP-1 cells
vs those treated with 50 µM forskolin (4 h incubations), at
different concentrations of 1. Mean of 3-5 experiments, each
in triplicate.
Phenylalanine derivatives also have high rHLGPa
inhibitory activity (Table 2). L-Phenylalanine dimethyl-
amide 14 retained the potency of 1, inhibiting rHLGPa
with an IC50 of 82 nM. In contrast to the trend observed
for stereoisomers of 1, D-phenylalanine 15 (260 nM) was
only about 3-fold less active than its enantiomer 14.
Similar moderate dependence of rHLGPa inhibitory
activity on absolute configuration also prevailed be-
tween the enantiomeric monomethylamides 16 and 17.
Potency was retained in methyl ester 24 (140 nM) and
diminished in acid 25 (1700 nM) and in the correspond-
ing alcohol 26 (9300 nM). Changing the indole 5-sub-
stituent in the phenylalanines reduced inhibitory ac-
tivity in the order Cl ≈ Br > F ≈ H > OCH3 (analogues
14, 18-21, respectively). Thus, in the â-amino-R-
hydroxybutyric acid and phenylalanine series, potency
was affected very differently by changes in stereochem-
istry and C-terminal amide functionality, but similarly
by changes to the indole 5-substituent. These results
suggest that compounds in both series inhibit rHLGPa
at the same site, with the 1H-indole-2-carboxamide
moiety making a significant contribution to binding
affinity. Sharply reduced activity of N-methylindole 37
and benzofuran 38 (rHLGPa IC50 > 10 µM) is consistent
with this hypothesis. Structural and kinetic experi-
ments are being performed to understand the inhibition
mechanisms of these compounds.
To evaluate the activity of inhibitors in cells, an assay
was established in a human liver-derived tissue culture
cell line. In the absence of inhibitor, addition of for-
skolin to SK-HEP-1 cells activated glycogen phospho-
rylase (phosphorylation of HLGPb to HLGPa), resulting
in degradation of 14C-labeled glycogen (Figure 2). In-
hibitor 1 blocked the forskolin effect with an IC50 of 1.5
µM (concentration-dependent), a value similar to that
previously obtained in primary cultures of isolated
human hepatocytes (IC50 ) 2.1 µM).18 Likewise, inhibi-
tor 23 showed IC50 values of 1.9 µM (SK-HEP-1, Table
2) and 2.7 µM (human hepatocytes). Other analogues
of 1 in both series are also potent inhibitors of glyco-
genolysis in SK-HEP-1 cells (Tables 1 and 2). These
data indicate that 1 and analogues readily penetrate
Ta ble 3. Hypoglycemic Responses Resulting from Oral Dosing
of Glycogen Phosphorylase Inhibitors in Diabetic ob/ ob Mice
doses
inactive,
mg/kg po
dose
% glucose
lowering at
active dosesb
active,a
mg/kg po
significance
compd
(P value)
1
15, 10
5018
2518
40
32
0.05
0.05
4
5
6
50
10
50
50
25
25
50
50
25
10
5
25
33
0.05
10
16
17
18
19
20
22
23
50
50
49
39
0.01
0.05
50
25
50
25
15
10
40
36
44
45
26
24
0.01
0.05
0.001
0.001
0.01
0.05
24
50
a
Significant (P e 0.05) plasma glucose lowering at 3 h postdose,
relative to vehicle treated controls (n ) 10 per group), in drug
treated ob/ ob mice. Statistical analysis by two-tailed, unpaired
b
Student t-test. Percent decrease in drug-treated plasma glucose
concentration, relative to the vehicle-treated control within the
experiment.
liver cells. Cell potency often correlated with potency
in the purified enzyme assay (e.g., the two pairs of
phenylalanine enantiomers 14-17). Certain potent
inhibitors, however, were less effective inhibitors of
glycogenolysis in cells (cf. 7, 8, 11, 24). These data do
not reveal factors responsible for these differences.
Oral administration of 1 to diabetic (ob/ ob) mice in
the fed state at 50 or 25 mg/kg lowers plasma glucose 3
h later.18 The time course of this effect was determined
using a 50 mg/kg po dose of 1 (Figure 3). Glucose
lowering was statistically significant at 3, 6, and 10 h,
but not at 24 h, with the largest drop occurring at 3 h.
Analogues of 1 were screened for oral activity at this
timepoint (Table 3). The (2S) stereoisomer of 1 (4, not
a potent rHLGPa inhibitor) did not show oral hypogly-
cemic activity at 50 mg/kg, which is consistent with
phosphorylase inhibition being the mechanism of the

Communications to the Editor
J ournal of Medicinal Chemistry, 1998, Vol. 41, No. 16 2937
Su p p or tin g In for m a tion Ava ila ble: Synthetic proce-
dures and characterization data for all compounds, chiral
HPLC method, and biological procedures (28 pages). Ordering
information is given on any current masthead page.
hypoglycemic action of 1 in vivo. Like 1, fluoro analogue
5 also showed oral activity at 25 mg/kg, but not at 10
mg/kg. Bromoindole 6 and the methyl ester 10, both
potent inhibitors in vitro, did not show oral activity at
50 mg/kg. Several phenylalanines also showed oral
activity. Hydroxypiperidines 22 and 23 were active at
25 mg/kg, and unlike 1, 23 produced marked glucose
lowering at 10 mg/kg. Furthermore, under these same
conditions 23 did not alter plasma insulin levels (222
( 26 microunits/mL, vehicle-treated versus 234 ( 32
microunits/mL, 23-treated). Based on this finding,
coupled with our previous observation that 1 did not
affect insulin secretion from isolated rat islets,18 we
conclude that the observed glucose-lowering appears to
be directly due to hepatic glycogenolysis inhibition and
cannot be attributed to a mild insulin secretory activity.
Additional experiments are being performed to better
understand the origin of in vivo potency differences in
these series.
Important questions arise when this approach is
considered for antidiabetic therapy. Two are whether
the inhibitor effects can be confined to the liver and
whether glycogen will accumulate to unacceptably high
levels as in glycogen storage diseases. Experiments to
address these questions have given promising results.
IC50 values for inhibition of the human muscle isoform
of glycogen phosphorylase (rHMGPa)37 were 200 ( 7 nM
(1), 180 ( 22 nM (14), and 430 ( 15 nM (15), which are
2-3-fold higher than for rHLGPa. Additionally, com-
pound 23 is a potent inhibitor of the muscle enzyme (83
( 3 nM), but did not substantially impair glycogen
mobilization in muscle, in situ, under conditions where
liver glycogenolysis should be inhibited.38 The absence
of a muscle effect may be a result of low inhibitor
concentration in muscle. Another question is whether
glucose lowering can be sustained chronically by this
mechanism. To this end, we have observed that 23
produces glucose lowering in diabetic ob/ ob mice after
daily b.i.d. dosing for up to 2 weeks.38
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