Discovery of a potent, highly selective, and orally efficacious small-molecule activator of the insulin receptor

Article abstract of DOI:10.1021/jm000285q

A series of 3,6-diaryl-2,5-dihydroxybenzoquinones were synthesized and evaluated for their abilities to selectively activate human insulin receptor tyrosine kinase (IRTK). 2,5-Dihydroxy-6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h) was identified

Full text of DOI:10.1021/jm000285q

J . Med. Chem. 2000, 43, 3487-3494
3487
Expedited Articles
Discover y of a P oten t, High ly Selective, a n d Or a lly Effica ciou s Sm a ll-Molecu le
Activa tor of th e In su lin Recep tor
Kun Liu,* Libo Xu, Deborah Szalkowski, Zhihua Li, Victor Ding, Gloria Kwei, Su Huskey, David E. Moller,
J ames V. Heck, Bei B. Zhang, and A. Brian J ones
Merck Research Laboratories, P.O. Box 2000, Rahway, New J ersey 07065
Received J une 29, 2000
A series of 3,6-diaryl-2,5-dihydroxybenzoquinones were synthesized and evaluated for their
abilities to selectively activate human insulin receptor tyrosine kinase (IRTK). 2,5-Dihydroxy-
6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h ) was identified as a potent, highly
selective, and orally active small-molecule insulin receptor activator. It activated IRTK with
an EC50 of 300 nM and did not induce the activation of closely related receptors (IGFIR, EGFR,
and PDGFR) at concentrations up to 30 000 nM. Oral administration of the compound to
hyperglycemic db/db mice (0.1-10 mg/kg/day) elicited substantial to nearly complete correction
of hyperglycemia in a dose-dependent manner. In ob/ob mice, the compound (10 mg/kg) caused
significant reduction in hyperinsulinemia. A structurally related compound 2c, inactive in IRTK
assay, failed to affect blood glucose level in db/db mice at equivalent exposure levels. Results
from additional studies with compound 2h , aimed at evaluating classical quinone-related
phenomena, provided sufficient grounds for optimism to allow more extensive toxicologic
evaluation.
In tr od u ction
lin. However, dangerously low levels of plasma glucose
can result from this treatment.11 Thiazolidinediones
(glitazones) have been recently described as a class of
compounds that ameliorates many symptoms of
NIDDM.12-16 These agents substantially increase insu-
lin sensitivity in muscle, liver, and adipose tissue,
resulting in the correction of elevated plasma level of
glucose without the occurrence of hypoglycemia. How-
ever, undesirable effects associated with glitazones have
occurred in animal and human studies, including car-
diac hypertrophy, hemodilution, and liver toxicity.17,18
Accordingly, there exists a continuing demand for novel
antidiabetic agents.
Insulin is a hormone that is necessary for normal
carbohydrate, protein, and fat metabolism in mam-
mals.1,2 All known actions of insulin are initiated by its
binding to the extracellular domain of its specific
receptor.3,4 Following insulin binding, conformational
changes in the insulin receptor lead to autophosphor-
ylation of the intracellular â-subunits and stimulation
of the receptor’s intrinsic tyrosine kinase activity.5,6 The
activated insulin receptor tyrosine kinase (IRTK) phos-
phorylates several intermediate substrates, which leads
to the activation of downstream signaling molecules.7
Considerable evidence suggests that IRTK activity is
essential for many, if not all, of the biological effects of
insulin.1,8 However, the precise biochemical mechanism
linking receptor kinase-mediated tyrosine phosphory-
lation to the regulation of cellular metabolic pathways
is not completely defined.
Insulin resistance is a characteristic feature of non-
insulin-dependent diabetes mellitus (NIDDM) and is
also a contributing factor in atherosclerosis, hyperten-
sion, lipid disorders, and polycystic ovarian syndrome.9,10
NIDDM accounts for more than 90% of diabetes in the
United States. This metabolic disorder afflicts an esti-
mated 6% of the adult U.S. population. Conventional
treatments for NIDDM have significant limitations.
While physical exercise and caloric restriction could
improve the diabetic condition, compliance with this
treatment is generally poor. Sulfonylureas have been
widely used to increase the plasma level of insulin by
stimulating the pancreatic â-cells to secrete more insu-
We recently reported the discovery of an antidiabetic
fungal metabolite, 1 (Figure 1).19 It was isolated from a
culture broth of Pseudomassaria following an extensive
screening effort for small molecules that activate human
IRTK. This compound acted as an insulin mimetic in
several biochemical and cellular assays. Oral adminis-
tration of 1 to two mouse models of diabetes resulted
in a significant decrease in blood glucose levels. The
preliminary studies on 1 demonstrated the feasibility
of discovering novel insulin receptor activators that may
lead to new therapy for diabetes. However, to support
realistic drug development, improvements in potency
and selectivity were clearly necessary. It should be noted
at the outset that safe, modern quinone-containing
drugs do exist. Most notable of these is the antiparasitic
Mepron (atovaquone) which has been used in children20
and even infants21 and has recently received additional
indication for malaria prophylaxis.22 We herein describe
the evaluation of a core set of substituted 2,5-dihydroxy-
quinones based on the lead compound 1. 2,5-Dihydroxy-
* To whom correspondence and reprint request should be addressed.
Tel: (732) 594 7445. Fax: (732) 594 9556. E-mail: kun_liu@merck.com.
10.1021/jm000285q CCC: $19.00 © 2000 American Chemical Society
Published on Web 08/25/2000

3488 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Liu et al.
homology in the tyrosine kinase domain.4 Thus, to
determine the specificity of test compounds for insulin
receptor relative to other selected homologous receptors,
similar cell-based assays were established and used to
counterscreen insulin receptor activators against insulin-
like growth factor receptor (IGFIR), epidermal growth
factor receptor (EGFR), and platelet-derived growth
factor receptor (PDGFR). Maximizing selectivity over
such homologous growth factor receptors would clearly
be critically important.
Primary in vitro data for a core set of compounds are
presented in Table 1. These sketch the deconstructive
path taken to define the minimal structural require-
ment for IRTK activation and some key elements
driving selectivity. Data for the previously reported
natural product lead 1 is included for comparison. The
lead compound 1 activated IRTK with an EC50 of 5 µM
and weakly activated IGFIR and EGFR at concentra-
tions greater than 30 µM. It was quickly discovered that
the prenyl and the reverse prenyl substitutions on the
indolyl rings of 1 are superfluous and can be removed
without sacrificing in vitro activity on insulin receptor
activation. The simplest nonsymmetrical bis-indole di-
hydroxyquinone 2a induced 50% of the maximal effect
of insulin (100 nM) on IRTK activity at a concentration
of 7 µM. Encouraged by this result, 2a was further
simplified by replacing one of the indole moiety with a
phenyl ring to give 2b. Compound 2b showed a 20-fold
increase in IRTK activity with an EC50 of 0.3 µM, but
it still activated IGFIR and EGFR at higher concentra-
tions. Further simplification to 3,6-bis-phenyl-2,5-dihy-
droxyquinones resulted in dramatic decrease in potency.
Indeed 2c was inactive in IR activation assay even at a
concentration of 100 µM. This demonstrates that the
presence of a hydroxyquinone alone is clearly not
sufficient for whole-cell insulin receptor activation.
Replacement of indole moiety with other classical iso-
steres such as benzofuran and benzothiophene retained
the potency. Compounds 2d ,e had EC50s of 1 and 1.5
µM, respectively. In addition, they showed improved
selectivity against IGFIR and EGFR. Both compounds
weakly activated IGFIR and EGFR at a concentration
of 100 µM, but not at 30 µM. 1-Naphthyl analogue 2f
also retains much of the ability to activate IRTK with
an EC50 of 6 µM, indicating that the heteroatom is not
essential for potency. In contrast, the 2-naphthyl ana-
logue 2g was some 5 times less potent than 2f, with an
F igu r e 1. Structures of 1, 2h , and atovaquone.
6-(1-methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h )
was identified as a potent, highly selective, and orally
active antihyperglycemic agent (Figure 1).
Resu lts a n d Discu ssion
Ch em istr y. A series of 3,6-diaryl-2,5-dihydroxyben-
zoquinones were prepared using the general route
previously described for the synthesis of our natural
product lead 1 (Scheme 1).23 The individual compounds
2a -h described herein are recorded in Table 1.
In Vitr o In su lin Recep tor Activa tion a n d Selec-
tivity. Insulin binds to two asymmetric sites on the R
subunits of the insulin receptor and causes conforma-
tional changes of the receptor which lead to autophos-
phorylation of the â subunit and activation of the
receptor’s intrinsic tyrosine kinase activity.1-6 A cell-
based functional assay using stably transfected Chinese
hamster ovary cells expressing human insulin receptors
was established and employed as the primary assay for
small-molecule insulin receptor activators.19 In this
assay, the ability of test compounds to independently
stimulate activation of IRTK was measured. The activi-
ties of test compounds were expressed as percentage of
control (percent maximal activity achieved with 100 nM
insulin). Insulin receptor belongs to a superfamily of
receptor tyrosine kinases with high degree of sequence
Sch em e 1. General Synthesis of 3,6-Diaryl-2,5-dihydroxybenzoquinones

Small-Molecule Activator of Insulin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3489
Ta ble 1. IRTK Assay and Counterscreen Results of 3,6-Diaryl-2,5-dihydroxyquinones
a
b
50% of maximal activity achieved with 100 nM insulin. Percentage of control (100 nM IGFI). c Percentage of control (100 nM EGF).
d
Percentage of control (100 nM PDGF). NA, not active up to 100 µM; NT, not tested; NS, not significant (less than 5% activation).
EC50 of 30 µM. This is of note from the point of view of
establishing the existence of a precisely defined SAR.
The N-methyl analogue 2h was among the most potent
insulin receptor activators in this series. It activated the
human IRTK with an EC50 of 300 nM. More impor-
tantly, it did not activate IGFIR, EGFR, and PDGFR
at concentrations up to 30 µM.
Some of these data are graphically represented in
Figure 2. Two key aspects deserve mention. First, the
existence of a spectrum of in vitro IRTK activities among
structurally similar hydroxyquinones (Figure 2A) ar-
gues against a generic redox activation and provides a
critical dyad of compounds (2h ,c) for validation of the
in vivo relevance of the in vitro observation. Second, the
dramatic separation of activities at homologous recep-
tors is noteworthy (Figure 2B). For example, IR and
IGFIR are structurally related and highly homologous
at the amino acid level,3,4,24 and clear discrimination of
these receptors might, a priori, have been envisaged to
be a rather difficult task.
P h a r m a cok in etics (P K). Pharmacokinetics of com-
pound 2h in preclinical species are presented in Table
2. In the rat, plasma clearance, volume of distribution
at steady state, and terminal half-life (t1/2) were 0.04
mL/min/kg, 0.1 L/kg, and 32 h, respectively. After oral
dosing at 0.5 mpk, the peak plasma concentration was
∼4880 ng/mL, reached 10 h postdose, and oral bioavail-
ability was estimated to be 79%. In dogs and monkeys,
the profiles were similar with high Cmax, high AUC’s,
slow clearance, and long t1/2 values (Table 2). Oral
bioavailability remained high in these species at 59%
and 67%, respectively.

3490 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Liu et al.
F igu r e 3. Comparison of glucose-lowering effect of 2h ,
insulin, and glyburide in db/db mice or normal lean mice. Food
was withheld during the experiment. Blood glucose levels were
measured at 1 and 3 h postdosing. *p < 0.05, comparing
treatment groups with the vehicle control group at the same
time point using Student’s t-test.
animal model of NIDDM and is characterized by severe
insulin resistance and marked hyperglycemia. Under
an acute protocol, a single oral dose of compound was
administered and blood glucose levels monitored over
24 h. Food was removed during the study to avoid the
complicating effect of food intake on blood glucose level.
Under these conditions 2h significantly reduced blood
glucose in a dose-dependent manner when monitered
at 1-3 h postdosing (35% correction at 0.2 mpk and 50%
at 1 mpk) (Figure 3). In a head-to-head experiment, the
acute effect of 2h on hyperglycemia in db/db mice was
compared to that of insulin and glyburide (a sulfonyl-
urea insulin secretagogue). The correction of hypergly-
cemia produced by 2h is comparable to ip injection of
insulin at 2 U/kg. Oral dosing with glyburide at 10 mpk
exhibited only a slight glucose-lowering effect.29 When
the same experimental protocol was applied to normal
lean, euglycemic mice, 2h was without effect on glucose
levels at 1 and 10 mpk (Figure 3). The lower dose of
insulin (0.5 U/kg) produced significant glucose lowering,
while severe hypoglycemia was noted at 1 h postinjec-
tion of 2 U/kg insulin followed by an expected rebound
at 3 h. Oral dosing of glyburide also resulted in a
significant glucose-lowering effect.
F igu r e 2. Stimulation of tyrosine kinase activities in CHO
cells: (A) dose-response curves for compounds 1 and 2h ,c on
IRTK activity, (B) selectivity of 2h , (C) selectivity of 1.
Ta ble 2. Pharmacokinetic Parameters of 2h in Preclinical
Species
Intravenous Dose
dose
AUC
Clp
Vdss
t1/2
species
(mg/kg)
(ng‚h/mL) (mL/min/kg) (L/kg) (h)
rat
dog
0.05 (n ) 2)
0.1 (n ) 2)
21770
89810
8374
0.038
0.019
0.02
0.1
0.56
0.31
32
93.5
66.0
monkey 0.01 (n ) 2)
Oral Dose
dose
AUC0-∞
Cmax
Tmax
F
species
(mg/kg)
(ng‚h/mL)
(ng/mL)
(h)
(%)
rat
dog
monkey
0.5 (n ) 2)
0.5 (n ) 2)
0.5 (n ) 2)
17600
265840
278421
4879
5270
7588
10
2.5
8
79.3
59.2
66.5
Of importance is the fact that dihydroxyquinone 2c,
which was inactive in the in vitro IRTK assay, failed to
affect hyperglycemia in db/db mice at 30 mpk under the
above-mentioned acute protocol.30 It has been estab-
lished that compounds 2h ,c have very similar pharma-
cokinetic profiles in rats; therefore, they should have
comparable in vivo exposure levels at comparable dos-
ages in the db/db study. These results demonstrated a
definitive correlation between in vitro IRTK activity and
in vivo glucose-lowering efficacy in animal models.
Thus, high and sustained levels of compound are
observed, a profile that proved uniform among com-
pounds of this class. It is interesting to note that
preclinical and human PK data for other hydroxyquino-
nes are analogous.25-28
In Vivo P h a r m a cology: A. Com p a r ison w ith
In su lin a n d Glybu r id e for Acu te Effica cy in d b/
d b a n d Lea n Mice. The db/db mouse is an obese
B. Ch r on ic Effica cy in d b/d b Mice. Under a
chronic dosing protocol, compounds were administered

Small-Molecule Activator of Insulin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3491
F igu r e 4. (A) Chronic effect of oral dosing of 2h on hyperglycemia in db/db mice and lean control mice (n ) 9/group). Lean
control mice and db/db mice were dosed once a day by oral gavage with vehicle or the indicated doses of 2h . Blood glucose levels
were measured 24 h after dosing on the preceding day. p values (one-way ANOVA) for 2h vs vehicle were: 0.034 (0.1 mpk),
<0.001 (1 and 10 mpk). (B) Comparison of chronic effect of 2h ,c. *p < 0.01, comparing treatment groups with vehicle control
groups (n ) 7-8/group).
once daily for 8-14 days by gavage to ad libitum fed
animals and blood glucose was monitored immediately
prior to the next compound dose at days 0, 3, 7, and 11
and at the termination of the study. Such administra-
tion of 2h resulted in significant reduction of blood
glucose level in a dose-dependent manner: 29% at 0.1
mpk, 47% at 1 mpk, and 84% at 10 mpk (Figure 4A).
No significant effect on blood glucose levels was ob-
served in normal lean mice treated with 2h at 1 or 10
mpk, and no significant effect on food intake or body
weight gain in db/db or lean mice was observed.
To further demonstrate the relevance of in vitro and
F igu r e 5. Effect of 2h on hyperinsulinemia in ob/ob mice (n
in vivo observations, the chronic effect of 2h on hyper-
) 10/group). Ob/ob mice were dosed once a day by oral gavage
glycemia in db/db mice was compared to that of 2c. The
with vehicle or the indicated doses of 2h for 2 days. The mice
results are displayed in Figure 4B. In this experiment,
were fed ad libitum. Plasma samples were obtained 24 h after
db/db mice were treated with a daily dosage of 2h ,c for
8 days. At day 8, treatment with 2h resulted in
correction of hyperglycemia of 35% at 1 mpk dosage and
76% at 10 mpk dosage. Once again, compound 2c, which
was inactive in the in vitro IRTK assay and had no acute
glucose-lowering efficacy in the db/db mice, failed to
affect hyperglycemia in db/db mice at 10 mpk in this
8-day chronic study.
Effect on Eleva ted In su lin Level in ob/ob Mice.
The ob/ob mouse is an alternative obese rodent model
of insulin resistance that is characterized by extreme
hyperinsulinemia. Following single oral dosing of 2h at
10 mpk, significant decreases in elevated plasma insulin
levels were observed 6 h postdosing (food withheld).
Further decreases in plasma insulin level were observed
6 h postdosing on day 2 (reaching a net 65% reduction,
data not shown). The insulin-lowering effect was sus-
tained at 24 h post-second-dosing (Figure 5). The ob/ob
mice were only modestly hyperglycemic at the age tested
(12 weeks). Treatment with 2h (10 mpk) also resulted
in a decrease in mean blood glucose concentrations from
233 ( 15 mg/dL (predosing) to 150 ( 6 mg/dL at 24 h
dosing on the preceding day and insulin concentrations were
measured. *p < 0.00001, compared to the vehicle group.
postdosing (n ) 10/group). The blood glucose level value
for the age- and sex-matched ob/+ lean mice was 143 (
4 mg/dL (n ) 10).
Ad d ition a l Stu d ies. That these compounds are
quinones raises inevitable concerns regarding their
potential for non-mechanism-based toxicity, though as
bis-aryl bis-hydroxy tetrasubstituted quinones, the chemi-
cal properties of this particular series would be expected
to be rather different from those of unsubstituted
ones.31-37 Quinones are widely distributed in nature and
have been isolated from plants, bacteria, and fungi,
including many of the foods we eat.38 Compounds
containing the quinone core have long been used in folk
medicine39 and, more recently, as anticancer agents40
and parasiticides.41
The capacity of quinones to redox cycling is perhaps
the most obvious pharmaceutical concern.31,32 Com-
pounds 1 and 2h did not sustain redox cycling in
aqueous DMF as assessed by cyclic voltammetry. How-

3492 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Liu et al.
Laboratories (Madison, NJ ). All air-sensitive reactions were
run under an nitrogen atmosphere.
ever we had established early in the SAR that the
quinone may be reduced by hydrogen sulfide and back-
oxidized in air. Among the most obvious targets of
aberrant redox activity are interference with the res-
piratory chain through uncoupling of mitochondrial
oxidative phosphorylation,42 catalysis of the formation
of reactive oxygen species,43 and hemolysis through
heme oxidation.44 In this regard, compound 2h (10 µM)
did not uncouple mitochondrial respiration in rat liver
mitochondria (data not shown; m-chlorocarbonyl cya-
nide phenylhydrazone (CCCP) as positive control), was
negative in both a non-GLP (good laboratory practice)
microbial mutagenesis assay (3-10 000 µg/plate, data
not shown) and an in vitro alkaline elution (100-350
µM), and caused no detectable hemolytic anemia in
rodents.
Many quinones are also capable of reacting with
nucleophiles, in particular thiols.32 This is clearly a
lesser concern with electron-rich, tetrasubstituted quino-
nes.33 Indeed compound 2h failed to generate detectable
protein modification when incubated with GST-IRTK
protein (a glutathione S-transferase fusion protein
containing the intracellular domain of the insulin recep-
tor) at 5-fold molar excess (data not shown; iodoacetate
as positive control).45
3,4-De h yd r o-3-h yd r oxy-5-oxo-4-p h e n yl-δ-va le r ola c-
ton e (6a ). To a mixture of phenylacetyl chloride (3.1 g, 20
mmol) and tris(trimethylsilyloxy)ethylene (13.5 g, 44 mmol)
at room temperature was added 3 drops of neat TiCl4 via
syringe. An exothermic reaction occurred, and the color of the
reaction mixture turned to reddish. The reaction mixture was
stirred for 3 h before it was poured into a mixture of dioxane
(25 mL) and 0.6 N HCl aqueous solution (10 mL). The mixture
was stirred at 90 °C for 10 min, cooled to room temperature,
and extracted twice with Et2O. The combined organic layers
were washed with saturated solution of NaHCO3 and brine,
dried over anhydrous MgSO4, filtered, and concentrated in
vacuo. The residue was crystallized from hexanes to give 2.47
g (82%) of 1-hydroxy-3-phenylpropan-2-one (4a ) as a white
solid: 1H NMR (CDCl3, 400 MHz) δ 7.35-7.15 (m, 5H, C6H5),
4.26 (d, J ) 7.0 Hz, 2H, CH2O), 3.70 (s, 2H, CH2CO), 3.00 (t,
J ) 7.0 Hz, 1H, OH); MS (CI) m/e 151 (M + H).
To a solution of 4a (2.47 g, 16.4 mmol) in THF (120 mL) at
0 °C was added Et3N (2.7 mL, 19 mmol), followed by ethyl
oxalyl chloride (1.9 mL, 17 mmol). The mixture was stirred at
0 °C for 3 h, poured into EtOAc (200 mL), washed with water
and brine, dried over anhydrous MgSO4, filtered, and concen-
trated in vacuo to give 3.9 g of ethyl 2,5-dioxo-3-oxy-6-
phenylhexanoate (5a ) as a slightly yellow oil, which was used
in the next step without further purification: 1H NMR (CDCl3,
400 MHz) δ 7.35-7.15 (m, 5H, C6H5), 4.85 (s, 2H, CH2O), 4.37
(q, J ) 7.0 Hz, 2H, COOCH2), 3.77 (s, 2H, PhCH2CO), 1.38 (t,
J ) 7.0 Hz, 3H, CH3); MS (CI) m/e 251 (M + H).
Con clu sion
To a solution of DBU (4.9 mL, 32.8 mL) in DMF (16 mL) at
-20 °C was added dropwise a solution of the crude intermedi-
ate 5a from the previous step (3.9 g, 16 mmol) in DMF (16
mL). The reaction mixture was stirred at -15 °C for 2.5 h
before it was poured slowly into an ice-cold 1.0 N HCl solution
(100 mL). The crystalline product 6a (1.85 g) was collected by
filtration, washed thoroughly with water, and dried under high
vacuum. The mother liquid was extracted with EtOAc. The
extract was washed with water and brine, dried over MgSO4,
filtered and concentrated. The residue was purified by recrys-
tallization from CH2Cl2/hexanes to give another 0.57 g of 6a
as slightly yellow solid. The total yield was 2.42 g (two steps,
72%): mp 174-176 °C; 1H NMR (acetone-d6, 400 MHz) δ 7.50-
7.30 (m, 5H, C6H5), 5.11 (s, 2H, OCH2); 13C NMR (acetone-d6,
400 MHz) δ 189.30, 160.98, 150.96, 130.58, 129.47, 128.53,
127.90, 121.98, 73.39; MS (CI) m/e 205 (M + H). Anal. Calcd
for C11H8O4: C, 64.71; H, 3.95. Found: C, 64.84; H, 3.86.
SAR studies around 1, an antidiabetic fungal metabo-
lite, resulted in the discovery of 2,5-dihydroxy-6-(1-
methylindol-3-yl)-3-phenyl-1,4-benzoquinone (2h ). This
compound is a potent and specific activator of human
IRTK in intact mammalian cells. Oral administration
of 2h to diabetic db/db mice elicited substantial correc-
tion of elevated levels of blood glucose in a dose-
dependent manner. In ob/ob mice, 2h caused significant
decrease in plasma insulin concentration and correction
of mild hyperglycemia. Acute or chronic administration
of 2h , up to 10 mpk, did not provoke hypoglycemia in
normal mice, which is a very desirable property. A
structurally related compound 2c was inactive in the
IRTK assay and also failed to affect blood glucose level
in db/db mice at analogous exposure levels. This obser-
vation provided a critical validation of the in vivo
relevance of the in vitro activity. Despite the concern
that the potential value of 2h may be compromised by
features of its chemical structure and its apparently long
serum half-life, this compound is chemically well-
behaved and demonstrated no overt toxicity at phar-
macologic doses in our animal models. It did not poison
mitochondrial respiration and did not modify proteins
covalently. The discovery of compounds with improved
IR activating potency and selectivity as described in this
report suggests that future efforts in this field may
ultimately lead to a potential new therapy for NIDDM
with a mechanism distinct from all other agents cur-
rently known.
3,4-Deh yd r o-3-h yd r oxy-4-(3-in d olyl)-5-oxo-δ-va ler ola c-
ton e (6b). The title compound was prepared following the
general procedure described above for the preparation of 6a :
1
mp 174-176 °C; H NMR (acetone-d6, 400 MHz) δ 7.74 (d, J
) 2.0 Hz, 1H), 7.58 (d, J ) 8.4 Hz, 1H), 7.44 (d, J ) 8.4 Hz,
1H), 7.13 (t, J ) 8.5 Hz, 1H), 7.03 (t, J ) 8.5 Hz, 1H), 5.14 (s,
2H); MS (CI) m/e 244 (M + H). Anal. Calcd for C13H9NO4: C,
64.20; H, 3.73; N, 5.76. Found: C, 64.54; H, 3.96; N, 5.31.
2,5-Dih yd r oxy-3-(1-m eth ylin d ol-3-yl)-6-p h en yl-1,4-ben -
zoqu in on e (2h ). A mixture of pyrandione 6a (10.03 g, 49.1
mmol) and 1-methylindole-3-carboxaldehyde (8.0 g, 50.3 mmol)
in acetic acid (120 mL) was heated at 60 °C until a clear
solution was formed. To this solution was added 2.5 mL of
concentrated HCl. The resultant reddish solution was heated
at 90 °C for 3 h, during which time red crystalline solid
precipitated out. After cooling to room temperature, the
reaction was diluted with a 1:1 mixture of ether/hexanes (200
mL), then stirred at 0 °C for 10 min. The precipitate was
collected by filtration, washed thoroughly with cold ether/
hexanes (1:1) to afford 16.6 g (98%) of 3-hydroxy-6-(1-meth-
ylindol-3-ylmethylene)-4-phenyl-2H-pyran-2,5(6H)-dione (7h )
as red crystals: 1H NMR (acetone-d6, 400 MHz) δ 8.16 (s, 1H),
7.95 (d, J ) 7.5 Hz, 1H), 7.55 (m, 3H), 7.48 (s, 1H), 7.44 (m,
2H), 7.38 (m, 1H), 7.32 (t, J ) 7.2 Hz, 1H), 7.27 (t, J ) 7.2 Hz,
1H), 4.03 (s, 3H); MS (CI) m/e 346 (M + H).
Exp er im en ta l Section
All reagents were obtained from commercial suppliers and
are used without further purification. Melting points were
determined on a Thomas-Hoover capillary apparatus and are
uncorrected. NMR data were recorded on a Varian XL-400 or
Oxford 500 instrument. Chemical shifts are reported in ppm
(δ) downfield relative to external TMS (0 ppm) as standard.
IR spectra were taken on a Perkin-Elmer 1600 FT-IR spec-
trometer. Elemental analyses were obtained from Robertson
To a suspension of the intermediate obtained above (16.4
g, 47.5 mmol) in methanol (200 mL) at room temperature was

Small-Molecule Activator of Insulin Receptor
J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19 3493
added a solution of NaOMe in MeOH (25 wt %, 60 mL). The
mixture was stirred at room temperature for 3 h before it was
cooled to 0 °C. 1.5 N HCl aqueous solution (350 mL) was added
slowly under stirring. After the addition of HCl was completed,
the suspention was stirred at 0 °C for another 15 min. The
precipitate was collected by filtration, washed thoroughly with
water and hexanes, then dried under high vacuum to give 16.0
g of the crude 2h as greenish powder. The product was further
purified by recrystallization from THF/hexanes to give 15.7 g
(96%) of 2h : mp 267-269 °C; IR (neat) 3303.3, 1631.3, 1333.5,
d6, 400 MHz) δ 7.95 (d, J ) 8.5 z, 2H), 7.85 (d, J ) 8.2 Hz,
1H), 7.60-7.55 (m, overlapping signals, 3H), 7.54-7.42 (m,
overlapping signals, 5H), 7.36 (m, 1H); 13C NMR (acetone-d6,
400 MHz) δ 134.00, 132.20, 130.84, 130.75, 128.87, 128.73,
128.48, 128.00, 127.87, 126.19, 126.15, 125.97, 125.49; MS (CI)
m/e 343 (M + H). Anal. Calcd for C22H14O4: C, 77.18; H, 4.12.
Found: C, 77.06; H, 4.42.
2,5-Dih ydr oxy-6-ph en yl-3-(2-n aph th yl)-1,4-ben zoqu in o-
n e (2g). The title compound was prepared in 90% yield from
6a and 2-naphthaldehyde: mp 287-289 °C; IR (neat) 3318.3,
1234.9 cm-1 1H NMR (acetone-d6, 400 MHz) δ 7.6-7.5 (m,
;
1611.6, 1320.1 cm-1 1H NMR (acetone-d6, 400 MHz) δ 8.08
;
4H), 7.43 (m, 3H), 7.35 (m, 1H), 7.19 (t, J ) 7.2 Hz, 1H), 7.05
(t, J ) 7.2 Hz, 1H), 3.91 (s, 3H); 13C NMR (DMSO-d6, 400 MHz)
δ 136.89, 132.04, 131.57, 131.20, 128.25, 128.08, 127.46,
122.38, 121.76, 119.52, 115.99, 111.88, 110.36, 104.07, 33.33;
MS (CI) m/e 346 (M + H). Anal. Calcd for C21H15NO4: C, 73.03;
H, 4.38; N, 4.06. Found: C, 73.11; H, 4.36; N, 4.13.
(s, 1H), 8.00-7.90 (m, overlapping signals, 3H), 7.66 (d, J )
8.5 z, 1H), 7.59-7.52 (m, overlapping signals, 4H), 7.44 (t, J
) 8.0 Hz, 2H), 7.36 (m, 1H); 13C NMR (DMSO-d6, 400 MHz) δ
133.19, 132.86, 131.50, 131.16, 130.26, 129.16, 129.09, 128.64,
128.29, 128.15, 127.41, 126.97, 126.75, 116.38, 116.23; MS (CI)
m/e 343 (M + H). Anal. Calcd for C22H14O4: C, 77.18; H, 4.12.
Found: C, 77.06; H, 4.42.
In su lin Recep tor (IR) Activa tion Assa y. CHO.IR cells
were cultured in 96-well plates at a density of 150 000 cell/
well for 24 h and then serum-starved for 2 h prior to treatment
with test compounds or insulin for 20 min at 37 °C. The cells
were then lysed and cell lysates were added to 96-well poly-
(vinyl chloride) plates previously coated with a monoclonal
antibody against IR. After incubation for 16 h at 4 °C, the wells
were washed three times with TBST. The kinase reaction
mixtures were added to each well and the incubation was
continued at 25 °C for 40 min. After the addition of phosphoric
acid, the reaction mixtures were transferred to multiscreen
phosphocellulose plates. The plates were washed with ice-cold
phosphoric acid and the radioactivity associated with the filters
was determined. In this assay, the EC50 for insulin is ∼1 nM.
The activities of test compounds were expressed as percent
(%) of control (maximal activity achieved with 100 nM insulin).
The following compounds were prepared following the
general procedure described above for the preparation of 2h .
2,5-Dih yd r oxy-6-(3-in d olyl)-3-(7-m eth ylin d ol-3-yl)-1,4-
ben zoqu in on e (2a ). The title compound was prepared in 70%
yield from 6b and 7-methylindole-3-carboxaldehyde: mp 172-
174 °C; IR (neat) 3328.9, 1629.7, 1341.0 cm-1 1H NMR
;
(acetone-d6, 400 MHz) δ 9.40 (s, 1H), 7.69 (d, J ) 3.0 Hz, 1H),
7.64 (m, 3H), 7.48 (d, J ) 8.0 Hz), 7.13 (t, J ) 7.2 Hz, 1H),
7.05 (t, J ) 7.2 Hz, 1H), 6.95 (m, 2H), 2.52 (s, 3H); HRMS
(ESI) m/z calcd for C23H16N2O4 (M + H) 385.1183, found
385.1193.
2,5-Dih yd r oxy-3-(3-in d olyl)-6-p h en yl-1,4-ben zoqu in o-
n e (2b). The title compound was prepared in 85% yield from
6a and indole-3-carboxaldehyde: mp 237-239 °C; IR (neat)
3312.0, 1630.6, 1340.9, 1232.5 cm-1; 1H NMR (acetone-d6, 400
MHz) δ 7.66 (d, J ) 2.5 z, 1H), 7.59 (d, J ) 8.0 Hz, 1H), 7.56
(d, J ) 8.0 Hz, 2H), 7.48-7.41 (m, overlapping signals, 3H),
7.36 (m, 1H), 7.14 (t, J ) 7.0 Hz, 1H), 7.05 (t, J ) 7.0 Hz,
1H);13C NMR (acetone-d6, 400 MHz) δ 136.40, 131.60, 131.21,
128.25, 128.07, 127.08, 122.16, 121.65, 119.35, 115.94, 112.22,
112.12, 104.92; MS (CI) m/e 332 (M + H). Anal. Calcd for
The protocols for IGTIR, EGFR, and PDGFR counterscreens
were similar to the one described above for IR.19
Deter m in a tion of An tih yp er glycem ic Activity of Test
Com p ou n d in d b/d b Mice. Male db/db (7-8 weeks of age)
and nondiabetic db/+ (lean) mice from J ackson Laboratories
were housed 7-9 mice/cage and provided ad libitum access to
milled rodent chow (Purina #5008) and water. Food consump-
tion and body weight were monitored during the study. Blood
glucose levels were measured using a Lifescan One Touch basic
glucometer with small blood samples obtained from the tail.
For the acute dosing protocol, 8-week-old mice (n ) 8/group)
were dosed via oral gavage with vehicle (0.5% methylcellulose),
compound, glyburide, or via ip injection with vehicle (saline)
or insulin. Food was withheld immediately following dosing
and blood glucose concentrations were monitored during a 3-h
period. For the chronic dosing protocol, mice (n ) 9/group)
received once-a-day oral dosing by gavage with vehicle (0.5%
methylcellulose) or compound. The mice were given free access
to food and water throughout the study. Blood glucose was
monitored immediately prior to the next dose at days 0, 3, 7,
11, and 15. At the termination of the study, blood samples were
collected for measurement of hematocrit and serum chemistry
determinations.
Deter m in a tion of Effect on Eleva ted In su lin Level in
ob/ob Mice. Male ob/ob mice (12 weeks of age) and control
ob/+ (lean) mice from J ackson Laboratories were housed 7-9
mice/cage and provided ad libitum access to rodent chow
(Purina #5008) and water. Food consumption and body weight
were monitored during the study. The mice were given daily
gavage of vehicle (0.5% methylcellulose) or the indicated doses
of compound. Food was removed 0-6 h postdosing and
reintroduced thereafter on days 1 and 2. Free access to water
was allowed throughout the study. Plasma samples were
collected at 6 h postdosing on days 1 and 2 (food withheld) or
24 h postdosing on day 3 (fed ad libitum). The plasma sample
were frozen and analysis of insulin was performed using a
commercial RIA kit.
C
20H13NO4: C, 72.50; H, 3.95; N, 4.23. Found: C, 72.26; H,
4.04; N, 4.21.
2,5-Dih yd r oxy-3-(4-m eth oxyp h en yl)-6-p h en yl-1,4-ben -
zoqu in on e (2c). The title compound was prepared in 92%
yield from 6a and p-anisaldehyde: mp 269-271 °C; 1H NMR
(acetone-d6, 400 MHz) δ 7.40-7.28 (m, overlapping signals,
7H), 7.96 (d, J ) 8.5 z, 2H), 3.77 (s, 3H); 13C NMR (acetone-
d6, 400 MHz) δ 159.22, 132.38, 131.46, 131.12, 128.25, 128.09,
123.37, 116.13, 116.00, 113.78, 55.79; MS (CI) m/e 323 (M +
H). Anal. Calcd for C19H14O5: C, 70.80; H, 4.38. Found: C,
70.84; H, 4.37.
2,5-Dih yd r oxy-3-(3-ben zofu r a n yl)-6-p h en yl-1,4-ben zo-
qu in on e (2d ). The title compound was prepared in 90% yield
from 6a and benzofuran-3-carboxaldehyde: mp 202-204 °C;
IR (neat) 3148.7, 1797.0, 1632.5, 1346.6 cm-1 1H NMR
;
(DMSO-d6, 400 MHz) δ 8.09 (s, 1H), 7.60 (d, J ) 8.4 Hz, 1H),
7.53 (d, J ) 8.0 Hz, 1H), 7.42-7.36 (m, overlapping signals,
4H), 7.30 (m, 2H), 7.22 (t, J ) 8.0 Hz, 1H); 13C NMR (DMSO-
d6, 400 MHz) δ 154.82, 146.53, 131.48, 131.16, 128.28, 128.15,
127.35, 124.93, 123.28, 123.15, 116.44, 111.90, 111.33, 108.03;
MS (CI) m/e 333 (M + H); HRMS (ESI) m/z calcd for C20H13O5
(M + H) 333.0758, found 333.0762.
2,5-Dih yd r oxy-3-(3-b en zot h iop h en eyl)-6-p h en yl-1,4-
ben zoqu in on e (2e). The title compound was prepared in 88%
yield from 6a and benzothiophene-3-carboxaldehyde: mp 251-
253 °C; IR (neat) 3285.2, 1630.0, 1343.4 cm-1 1H NMR
;
(DMSO-d6, 400 MHz) δ 7.99 (d, J ) 7.6 Hz, 1H), 7.65 (s, 1H),
7.56 (d, J ) 6.4 Hz, 1H), 7.42-7.38 (m, overlapping signals,
7H); 13C NMR (DMSO-d6, 400 MHz) δ 169.00 (broad), 139.41,
138.92, 131.71, 131.15, 128.36, 128.25, 128.03, 126.90, 124.79,
124.48, 124.44, 123.28, 116.39, 111.11; MS (CI) m/e 349 (M +
H). Anal. Calcd for C20H12O4S: C, 68.95; H, 3.47; S, 4.20.
Found: C, 68.69; H, 3.39; S, 9.07.
2,5-Dih ydr oxy-6-ph en yl-3-(1-n aph th yl)-1,4-ben zoqu in o-
n e (2f). The title compound was prepared in 91% yield from
6a and 1-naphthaldehyde: mp 149-151 °C; 1H NMR (acetone-
P h a r m a cok in etic Stu d ies. Male rats, beagle dogs and
monkeys (rhesus macaque) were dosed iv or orally with
compound. The iv doses were prepared in ethanol/PEG400/

3494 J ournal of Medicinal Chemistry, 2000, Vol. 43, No. 19
Liu et al.
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in ethanol/PEG400/water (1:3:6, v/v/v). Plasma samples were
extracted and the concentrations of 2h in plasma were
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