Novel 3,4-Dihydroquinolin-2(1H)-one inhibitors of human glycogen phosphorylase a

Article abstract of DOI:10.1016/j.bmcl.2003.09.022

The preparation of a series of substituted indoles coupled to six- and seven-membered cyclic lactams is described and their role as human glycogen phosphorylase a inhibitors discussed. The SAR of the indole moiety and lactam ring are presented.

Full text of DOI:10.1016/j.bmcl.2003.09.022

Bioorganic & Medicinal Chemistry Letters 13 (2003) 4385–4388
Novel 3,4-Dihydroquinolin-2(1H)-one Inhibitors of Human
Glycogen Phosphorylase a
Keith G. Rosauer,a,* Anthony K. Ogawa,a Chris A. Willoughby,a Kenneth P. Ellsworth,b
Wayne M. Geissler,b Robert W. Myers,b Qiaolin Deng,c Kevin T. Chapman,a
Georgianna Harrisb and David E. Mollerb
aDepartment of Basic Chemistry, Merck Research Laboratories, Rahway, NJ 07065, USA
bDepartment of Metabolic Disorders-Diabetes, Merck Research Laboratories, Rahway, NJ 07065, USA
cDepartment of Molecular Systems, Merck Research Laboratories, Rahway, NJ 07065, USA
Received 9 April 2003; revised 28 August 2003; accepted 13 September 2003
Dedicated to the memory of Chris A. Willoughby
Abstract—The preparation of a series of substituted indoles coupled to six- and seven-membered cyclic lactams is described and
their role as human glycogen phosphorylase a inhibitors discussed. The SAR of the indole moiety and lactam ring are presented.
# 2003 Elsevier Ltd. All rights reserved.
A recent World Health Organization report estimates
that approximately 130 million people worldwide are
afflicted with type 2 diabetes, a number projected to
double over the next two decades.1 Patients suffer from
progressive insulin insensitivity that manifests itself in
both postprandial and fasting hyperglycemia. Current
therapy targets restoration of normal glucose levels with
treatment determined by disease progression. One
investigational approach involves restricting hepatic
glucose output (HGO), a major demonstrated con-
tributor to diabetic hyperglycemia.2 HGO derives from
two sources, gluconeogenesis, and the breakdown of
stored glycogen catalyzed by glycogen phosphorylase a
(GPa). In addition, a portion of the glucose derived
from gluconeogenesis, which is elevated in type 2 dia-
betics relative to normal patients,3 cycles through gly-
cogen via the glycogenolytic pathway.4 Previous reports
indicated GPa inhibition to be efficacious in lowering
blood glucose in diabetic models,5,6 thereby validating it
as an attractive therapeutic target for lowering blood
glucose in the treatment of type 2 diabetes.
potency against both the liver and muscle GPa isoforms
(hLGPa and hMGPa, respectively). The following data
represents our preliminary findings of a novel, bicyclic
lactam scaffold.
Lead structures 1 and 2 resemble an inhibitor class6 that
bind in an allosteric site at the enzyme homodimeric
interface. The reported enzyme-inhibitor co-crystal,
1EXV,8 suggests that both the indole moiety from 1,
and the potential indole replacement 3,4-dichlorophenyl
group from 2 bind in a restricted hydrophobic pocket.
The overall structural homology shared by compound 1
and CP-403700 suggests common enzyme–inhibitor
interactions.
Initial glycogen phosphorylase inhibitor leads 1 and 2
originated from high throughput screening of our com-
pound collection.7 Both inhibitors possessed comparable
*Corresponding author. Tel.: +1-732-594-3236; fax: +1-732-594-
9473; e-mail: keith_rosauer@merck.com
0960-894X/$ - see front matter # 2003 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2003.09.022

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K. G. Rosauer et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4385–4388
Other potentially significant functionality in 1 and 2 are
the carboxamide moiety that links the two ring systems
in each inhibitor and the lactam carbonyl. The corre-
sponding functional groups in 1EXV form a hydrogen
bonding lattice spanning the binding pocket,8a thereby
reinforcing the importance of these hydrogen bonding
groups. In addition, variation of lactam ring size could
alter the lactam phenyl group orientation. Selection
between both lead isosteric scaffolds and subsequent
optimization provided broad SAR for this reported
class of inhibitor.
clear preference for the six-membered lactam rings over
their seven-membered counterparts. The specific stereo-
chemistry preferred by the enzyme was also investi-
gated.9 The data appears to be somewhat ambiguous in
that unalkylated (R)-enantiomer 7 was more active (2-
fold) than the corresponding (S)-enantiomer 8, while
both enantiomers are essentially equally potent upon
lactam alkylation (13 vs 14).
As a result of our findings in Table 1,10 the indole moi-
ety was investigated further. While literature pre-
cedence6,8 establishes the 5-chloroindole moiety as
optimal, we were interested in a more comprehensive
SAR study. The synthesis of inhibitors 18–36 followed
from EDC-mediated coupling of deprotected amino-
lactam with each corresponding indole-2-carboxylic
acid, as outlined in Scheme 1. Although several of the
indole-2-carboxylic acids were commercially available,
the majority were prepared from their substituted ani-
line 16. Directed iodination ortho to the amine group,11
followed by coupling with pyruvic acid and cyclization
at 105 ꢀC afforded the desired indole 17.12 The 5-phe-
nylindole-2-carboxylate used for the synthesis of 27
required Suzuki coupling of phenylboronic acid onto 5-
bromoindole-2-carboxylate prior to amide bond forma-
tion. The related 6-phenyl indole-2-carboxylate leading
to 30 was derived from the reduction of 3-nitrobiphenyl
to afford the aryl amine input relevant to Scheme 2.13
Lastly, the 7-aminoindole-2-carboxylate from 33 was
generated via reduction of the corresponding 7-nitro-
indole-2-carboxylate derivative used for the synthesis of
32.12
The synthesis of the six-membered lactam series was
relatively straightforward, beginning with a Horner–
Emmons condensation of the N-Boc-ethyl-2-(dimethyl-
phosphono)glycinate and 2-nitro-benzaldehyde, followed
by nitro group reduction and subsequent cyclization to
afford Boc-lactam 3. Alkylation, deprotection and cou-
pling to the corresponding aryl carboxylate using stan-
dard methods afforded the substituted products of
general structure, 4a (Scheme 1). The seven-membered
lactam series was synthesized from the commercially
available 3-amino-2,3,4,5-tetrahydro-1-benazepin-2-one
to which was coupled the corresponding aryl carboxylic
acid affording the products of general structure 4b
(Scheme 1). In example 15, the amino-lactam was Boc-
protected, followed by methylation, deprotection, and
coupled to the corresponding carboxylic acid (Scheme 1).
Initial SAR comparison of acyl aromatic groups indi-
cated a strong preference for the indole group (6 and 9)
over the 3,4-dichlorphenyl-substituted compounds (2
and 5). One possible explanation stemmed from the
hydrogen bonding potential of the indole nitrogen,
which contacted a GP backbone carbonyl in the afore-
mentioned X-ray structure.8 The effect of lactam ring
size could be inferred from a comparison of lactams 6
and 12 with 9 and 15, respectively, which indicates a
Analysis of the indole SAR confirmed the limited space
surrounding the heterocyclic scaffold (Table 2).10
Although smaller substituents at C5–C7 were tolerated,
Table 1. In vitro phosphorylase inhibitory activity: six versus seven-
membered lactams10
Compd
n
R1
R2
H
HLGPa
IC50 (nM)
HMGPa
IC50 (nM)
2-rac.
5-rac.
6-rac.
1
2
1
1400
6400
44
2300
21,700
38
H
7-(R)
8-(S)
1
1
2
2
2
1
1
1
2
25
47
105
99
240
25
61
57
14
39
55
83
240
32
58
57
9-rac.
10-(R)
11-(S)
12-rac.
13-(R)
14-(S)
15-rac.
Me
Scheme 1. Reagents and conditions: (i) 2-nitro-benzaldehyde, NaH,
THF, 0 ꢀC to rt, 1 h; (ii) Pd/C, MeOH, H2, 40 psi, 3 h, then EtOAc,
reflux, 24 h; (iii) Boc2O, DCM; (iv) NaOtBu, R2-X, DMF, rt, 1 h;
(v) acetyl chloride, MeOH, 50 ꢀC, 30 min; (vi) R1-CO2H, EDC.
305
105

K. G. Rosauer et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4385–4388
4387
Table 3. In vitro phosphorylase inhibitory activity of N1-sub-
stituted lactams10,14
Scheme 2. Reagents and conditions: (i) Ag2SO4, I2, EtOH, rt, 16 h;
(ii) pyruvic acid, DABCO, Pd(OAc)2, DMF, 105 ꢀC, 4 h.
Table 2. In vitro phosphorylase inhibitory activity of indole analo-
gues10
Compd
R1
HLGPa
IC50 (nM)
Hepatocyte
EC50 (mM)
7-(R)
8-(S)
13-(R)
14-(S)
H
25
47
61
57
1.8
7.8
13.2
8.4
Me
39-rac.
110
20.7
Compd
R
HLGPa
IC50 (nM)
HMGPa
IC50 (nM)
40-(R)
41-(S)
120
140
14.5
11.6
18
19
20
12
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
H
4-Cl
5-Br
5-Cl
5-F
5-Me
5-Et
5-OMe
5-CN
5-CF3
160
1600
30
25
120
56
2100
73
32
89
98
850
1700
110
42-rac.
43-rac.
44-rac.
130
140
160
16.6
1.2
120
2500
1700
180
3.7
1500
>20,000
5300
99
>20,000
88
300
1200
>20,000
>20,000
83
550
5-Ph
>20,000
2700
69
>20,000
88
120
6800
>20,000
>20,000
55
5-CO2Me
6-Cl
6-Ph
of 1EXV suggested that stabilizing van der Waals
interactions in such a polar pocket might enhance
potency. An overlay of compound 12 with CP-403700
(data not shown), reveals the predictably similar orien-
tation of the dihydroquinolone carbonyl and phenyla-
lanine carbonyl, lending support to the idea of the N-
substituents projecting into the water filled cavity of the
homodimer interface.
7-Cl
7-NO2
7-NH2
4-CO2Me, 5-Cl
5-Cl, 6-CO2Me
5-Cl, 7-F
The incorporation of hydrophilic groups may however
alter the physical properties of the inhibitor to positively
impact cell potency. Inhibitors 39–42 were prepared
following the methodology outlined in Scheme 1, while
triazolylmethyl-substituted lactams (38) were generated
from cyclization of hydrazide 37 with various thio-
amides (Scheme 3).
Data analysis indicated no clear SAR for the lactam
N1-substituent in both the enzyme and cell-based assays
(Table 3),14 except for the general trend toward a slight
loss of potency against HGPa observed for the aryl-
methyl substituents. However, the magnitude of the
potency shifts from enzyme to cell-based GP inhibition
tracked with the relative hydrophilicity of the N1-side
chain.15 Specifically, the polar triazoylmethyl lactams
(43–44) possessed 9- and 23-fold shifts in IC50, while the
remaining IC50 values were, at best, 72-fold shifted (7)
for the same comparison. A plausible explanation for
observed effect was improved cell penetration due to
greater amphiphilic character of inhibitors possessing
the more polar lactam sidechains.
Scheme 3. (i) NaOtBu, methyl bromoacetate; (ii) HCl; (iii) 5-chloro-
indole-2-carboxylic acid, EDC; (iv) NH2–NH2, reflux; (v) R-CSNH2,
(4:1) pyr: nBuOH, reflux.
the 5-chloroindole analogue (12) proved to be the most
active.
A number of heterocyclic substituted six-membered
lactam inhibitors were prepared in order to examine the
SAR proximal to the N1-position. Qualitative analysis
In summary, the (R)-six-membered unsubstituted cyclic
lactam coupled to the 5-chloroindole-2-carboxylic acid
(7) was the most potent in vitro GP inhibitor (25 nM).

4388
K. G. Rosauer et al. / Bioorg. Med. Chem. Lett. 13 (2003) 4385–4388
Substitution of the triazolylmethyl group at N1 (43) led
to an improvement in cell-based GP inhibition (1.2 mM)
that revealed an apparent correlation between lactam
substituent polarity and improved cell activity.
McPherson, R. K.; Olson, T. V.; Treadway, J. L.; Hoover,
D. J. Chem. Biol. 2000, 7, 677. (b) Oikonomakos, N. G.;
Skamnaki, V. T.; Tsitsanou, K. E.; Gavalas, N. G.; Johnson,
L. N. Structure 2000, 8, 575.
9. Separation of racemic Boc-lactam 3 by chiral NP-HPLC
[chiral AD column; isocratic (92:8) heptane/iPrOH] afforded
individual enantiomers that were carried on as shown in
Scheme 1. A chiral synthesis of this peptidomimetic will be
reported separately.
Acknowledgements
A special thanks to Anthony Ogawa for his time during
many useful discussions and mentorship.
10. Phosphorolysis of glycogen using recombinant human
liver or muscle enzyme. Glucose-1-phosphate production was
monitored via an enzymatic assay involving phosphogluco-
mutase/glucose-6-phosphate dehydrogenase-mediated NADH
production (ex. 340 nm, em. 465 nm). The standard error for
liver and muscle enzyme activity is 0.17/0.14 (n>8), respec-
tively, based on an internal standard.
Phosphorylase a activity was measured at the sub-saturating
substrate levels in the physiologic glycogenolytic direction by a
modification (manuscript in preparation reference) of the
method in which glucose-1-phosphate production is coupled
to NAD reduction using phosphoglucomutase dehydrogenase.
For further details, see: Maddailh, V. T.; Madsen, N. B. J.
Biol. Chem. 1966, 241, 3873.
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14. Glucagon-stimulated glycogenolysis in primary rat hepa-
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digesting remaining glycogen and quantifying by monitoring
glucose dehydrogenase-mediated NADPH production (ex. 340
nm, em. 465 nm). The standard error for hepatocyte activity is
0.15.
15. Due to incomplete solubility data, global logD calcula-
tions were performed on the compounds in Table 3 that
showed no sensitivity toward lactam N1-side-chain variation,
presumably due to the dominant effect of the 5-chloroindole
carboxamide moiety. Subsequent logD determination for
the truncated N1-substituted-3-acetamido-tetrahydroquinoline
yielded the noted correlative trend.
8. (a) Rath, V. L.; Ammirati, M.; Danley, D. E.; Ekstrom,
J. L.; Gibbs, E. M.; Hynes, T. R.; Mathiowetz, A. M.;