Design and synthesis of 2-N-substituted indazolone derivatives as non-carboxylic acid glycogen synthase activators

Article abstract of DOI:10.1016/j.bmcl.2013.03.049

Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen polymer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen levels and decreased muscle glucose uptake is

Full text of DOI:10.1016/j.bmcl.2013.03.049

Bioorganic & Medicinal Chemistry Letters 23 (2013) 2936–2940
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry Letters
journal homepage: www.elsevier.com/locate/bmcl
Design and synthesis of 2-N-substituted indazolone derivatives
as non-carboxylic acid glycogen synthase activators
a
b
a
a
c
Yimin Qian a, , David Bolin , Karin Conde-Knape , Paul Gillespie , Stuart Hayden , Kuo-Sen Huang ,
⇑
Andrée R. Olivier b, Tsutomu Sato a, Qing Xiang c, Weiya Yun a, Xiaolei Zhang c
a Discovery Chemistry, Small Molecule Research, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, United States
b Metabolic and Vascular Diseases, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, United States
c Discovery Technologies, Small Molecule Research, Pharmaceutical Research and Early Drug Development, Hoffmann-La Roche Inc., 340 Kingsland Street, Nutley, NJ 07110, United
States
a r t i c l e i n f o
a b s t r a c t
Article history:
Glycogen synthase (GS) catalyzes the transfer of glucose residues from UDP-glucose to a glycogen poly-
mer chain, a critical step for glucose storage. Patients with type 2 diabetes normally exhibit low glycogen
levels and decreased muscle glucose uptake is the major defect in whole body glucose disposal. There-
fore, activating GS may provide a potential approach for the treatment of type 2 diabetes. In order to iden-
tify non-carboxylic acids GS activators, we designed and synthesized a series of 2-N-alkyl- and 2-N-aryl-
indazolone derivatives and studied their activity in activating human GS.
Received 24 January 2013
Revised 9 March 2013
Accepted 12 March 2013
Available online 22 March 2013
Keywords:
Ó 2013 Elsevier Ltd. All rights reserved.
Glycogen synthase
Enzyme activation
Type 2 diabetes (T2D)
Metabolic stability
Indazolone
N-arylation and cyclization
Glycogen synthase (GS) is one of the key regulatory enzymes in
glycogen synthesis. It catalyzes the addition of glucose from UDP-
glucose to the growing glycogen chain through a 1,4-a linkage and
hyperglycemic–hyperinsulinemic clamp studies with 13C-glucose
revealed a greater than 50% decrease in muscle glycogen synthesis
in NIDDM compared to normal subjects. This decrease paralleled
the decrease in glucose uptake and accounted for essentially the
entire defect in whole-body glucose disposal.6 Using the same
analysis, it was concluded that decreased muscle glycogen synthe-
sis in diabetic subjects was primarily due to impaired glucose
transport.7 On the other hand, in animal models, muscle-specific
disruption of GSK3b (which phosphorylates and inactivates GS)
led to improved glucose tolerance, a twofold increase in glycogen
accumulation, and an increase in GS activation by insulin without
any effect on glucose transport.8 Therefore, it has been suggested
that both glucose transport and GS activity can contribute to the
rate of glycogen accumulation, possibly to different extents that
may vary under different circumstances.2,9
this step is the rate-limiting step for glycogen synthesis under
most circumstances.1,2 GS is regulated covalently through multiple
phosphorylation sites and allosterically activated by glucose-6-
phosphate (G6P).3 Two forms of GS exist in mammals, encoded
by the genes GYS1 and GYS2. The gene products share 69% homol-
ogy, where GYS2 encodes the liver specific form and GYS1 encodes
the form expressed in all other tissues including muscle.2 By con-
verting excess glucose into part of the glycogen polymer chain,
GS plays a key role in glucose storage.
There is genetic and clinical evidence implicating GS in type 2
diabetes (T2D). It has been reported that a polymorphism of the
muscle gene GYS1 is more prevalent in T2D patients than in non-
diabetic controls.4 Both basal and insulin-stimulated GS activity
in muscle cells from diabetic subjects are significantly decreased
relative to cells from non-diabetic subjects.5
In our metabolic disease research, we were interested in identi-
fying small molecule GS activators. High-throughput screening of
our compound library provided a single hit 1 illustrated in Figure 1
The exact molecular mechanism that underlies the decreased
glycogen synthesis in T2D is still under investigation and debate.
On one side, the use of quantitative 13C NMR spectroscopy in
(GYS1 EC50 = 15.1
expansion and lead optimization led to very potent GS activators
as exemplified by 2 (GYS1 EC50 = 0.14 M, 5.4-fold increase at
M, 6.8-fold increase at
M).11–14 However, these carboxylic acids displayed very low
lM, 5.8-fold increase at 75 l
M).10 Further hit
l
75
75
l
l
M) and
3
(GYS1 EC50 = 0.1 l
⇑
Corresponding author. Tel.: +1 973 641 9072; fax: +1 973 235 2448.
E-mail address: yiminqian81@gmail.com (Y. Qian).
exposure in muscle relative to that in liver after oral dosing in
0960-894X/$ - see front matter Ó 2013 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmcl.2013.03.049

Y. Qian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2936–2940
2937
O
O
O
O
O
O
O
O
O
O
O
N
N
N
R
H
N
OH
N
O
F
OH
O
O
F
O
F
F
OH
F
F
6
3
2
1
O
Figure 2. Design of 2-N-substituted indazolones as non-carboxylic acid GS
O
activators.
N
F
O
F
envisioned the synthesis of 6 through an intermolecular N-aryla-
tion reaction between N-Boc-N-alkylhydrazine or N-Boc-N-aryl-
hydrazine 7 and the ester of ortho-bromobenzoic acid 8 followed
by ring closure under acidic conditions as shown in Scheme 2 for
the preparation of 10ah through 10ok. While the intermolecular
N-arylation of N-arylhydrazine with aryl halide has been reported
using palladium acetate and tri-tert-butylphosphonium tetrafluo-
roborate,18 the synthetic method we describe here has not been re-
ported in the literature for the preparation of 2-N-substituted
indazolones. The required N-aryl-N-Boc-hydrazines 7d and 7e
were synthesized according to the literature method through the
N-arylation of N-Boc-hydrazine with an aryl iodide.19 The neces-
sary N-Boc-N-alkylhydrazine was prepared from an alkyl chloride
and hydrazine followed by protection of the secondary amine
rather than the primary amine.20 As described in Scheme 3 for
the preparation of 7c, 7f and 7g, we observed selective N-Boc for-
mation at the alkyl-substituted nitrogen of the hydrazine deriva-
tive 11. The preparation of aryl halides 8h through 8k is also
presented in Scheme 3. For the synthesis of 10mh (Table 2), 2,2-
diethoxyethylhydrazine was used and the aldehyde group in the
indazolone was reduced by sodium borohydride to provide the cor-
responding alcohol 10mh (reaction not shown in the scheme).21
As shown in Scheme 2, this synthetic sequence made it possible
to install different functional groups at the 2-N-position. Selected
reaction yields for step (a) and step (b) in Scheme 2 are listed in Ta-
ble 1. The N-arylation reaction gave decent yields despite the di-
ortho-substitution pattern. The deprotection of the Boc-group and
the subsequent cyclization was completed in one pot with aqueous
hydrochloric acid and THF. This method also made it possible for
one-pot deprotection of other acid-labile protective groups, such
as ester in 9ch and acetonide in 9fh, during the indazolone ring for-
mation step (10lh and 10nh).21 The relatively low yield for 10lh
was due to partial ester hydrolysis under these conditions.
Compounds 10ah through 10ok synthesized according to
Scheme 2 were studied for their activity as activators of human
muscle glycogen synthase (GYS1). The recombinant human muscle
GYS1 expressed and partially purified from sf9 cells was used as
the glycosyl transferase and the assay was carried out by coupling
with pyruvate kinase and lactate dehydrogenase.22 The more de-
tailed enzyme assay procedure is described in the Supplementary
data. The activation potencies are listed in Table 2.
Our earlier work on GS activators was primarily focused on car-
boxylic acids and acid mimetics.11–14,23 Although those compounds
demonstrated good potency in activating muscle glycogen syn-
thase, they have high protein binding and are mainly distributed
in liver when orally dosed in mice. Our rationale to identify non-
carboxylic acid GS activators was based on the assumption that
neutral compounds might have decreased protein binding and im-
proved muscle distribution relative to the liver. We designed the 2-
N-substituted indazolone series to improve the metabolic stability
of 5. As shown in Table 2, all compounds demonstrated GS activa-
O
OH
3
Figure 1. Structures of biphenyl ether derived carboxylic acids as GS activators.
mouse. Therefore, our goal was to identify non-carboxylic acid GS
activators with a potential of improved muscle distribution. In this
Letter, we report the first non-carboxylic acid GS activator from the
2-N-substituted-indazolone chemical class.
Since the major defect of glucose disposal in T2D is due to im-
paired glucose incorporation into glycogen in muscles (as shown
by the clamp study described in the introduction), compounds
with higher muscle exposure could achieve a higher on-target ef-
fect by increasing muscle GS activity. To identify non-carboxylic
acid GS activators to increase potential muscle exposure, we first
investigated derivatives of 3. Esterification of the carboxylic acid
to its corresponding ester abolished GS activation potency. When
the carboxylic acid was reduced to a hydroxyl group, significant
loss of potency was observed (EC50 = 4.1
75 M). Replacing the COOH group in 3 with an ethylenediol also
led to large reduction of potency (EC50 = 10.3 M, 6.6-fold increase
at 75 M). We next decided to replace the right hand phenylcarb-
oxamide fragment with heterocycles. One of the designed hetero-
cycles required an intermediate of ortho-aminobenzoic acid ester.
During the hydrogenation of intermediate 4 (reduction of the nitro
group in 4 to an amine, Scheme 1), benzisoxazolone 5 was isolated
as a side product, probably through partial reduction of the nitro
group to a hydroxylamine followed by cyclization. To our surprise,
this side product displayed good potency in GS activation
lM, 7.3-fold increase at
l
l
l
(EC50 = 1.0 lM, SC200 = 0.41 lM, 4.0-fold increase at 75 lM). How-
ever, when 5 was orally dosed to C57 mice, complete breakdown of
the bicyclic molecule into ortho-aminobenzoic acid was observed,
with the parent compound not detected in the plasma.
To resolve the metabolic stability problem in 5, we explored dif-
ferent bioisosteres to replace the benzisoxazolone. As shown in
Figure 2, a series of indazolones was designed and investigated
as non-carboxylic acid GS activators.
We initially investigated the application of Buchwald–Hartwig
palladium- and copper-catalyzed aromatic amination for the for-
mation of the C–N bond.15,16 Although it has been reported that
copper(I)-catalyzed intramolecular N-arylation of 2-halobenzhyd-
razide provided an efficient synthesis of 1-N-substituted indazol-
ones,17 a general and efficient method is still required for the
synthesis of 2-N-alkyl- and 2-N-aryl indazolones such as 6. We
O
O
H
NO2
F
F
N
H2, Pd/C
O
O
O
O
F
F
F
tion (2.5- to 6.8-fold increase at 75
tested). Results from the dose–response assay indicated a range
of EC50 (0.2–4 M) and SC200 (0.06–2.75 M) value. Compound
lM, the highest concentration
F
4
5
l
l
Scheme 1. Partial reduction of nitro group to form benzisoxazolone.

2938
Y. Qian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2936–2940
(a)
O
O
N
Pd(OAc)2 (5% eq)
F
O
H3CO
R1
F
N
O
O
O
N
P(t-Bu)3BF4 (10% eq)
Br
O
F
O
F
O
N
R1
Cs2CO3 (2 eq), toluene
reflux, 5 hr
R2
R2
9ah-9gk
7a-7g
8h-8k
3N HCl (aq), THF
reflux, 2 hr
R1
(b)
h: R2=F
i: R2=Cl
a: R1=CH3
b: R1=CH2CH2OCH3
c: R1=CH2COOEt
d: R1=3-Pyridyl
j: R2=CH3
F
N
O
k: R2=OCH3
l: R1=CH2COOH
n: R1=(S)-2,3-Dihydroxypropyl
o: R1=(R)-2,3-Dihydroxypropyl
HN
F
O
e: R1=4-F-Phenyl
f: R1=(S)-2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl
g: R1=(R)-2,2-Dimethyl-[1,3]dioxolan-4-ylmethyl
R2
10ah, 10aj, 10ak, 10bh,
10bk, 10dh, 10di, 10ek,
10ej, 10lh, 10lj, 10lk,
10nh, 10ni, 10nj, 10nk,
10oh, 10oi, 10oj, 10ok
Scheme 2. Synthesis of 2-N-substituted indazolones 10ah–10ok through C–N bond formation and subsequent cyclization.
However, when R1 position is substituted with 3-pyridyl or 4-F-
R1
R1
(Boc)2O
MeOH
R1Cl
H2N
N
phenyl group, a decrease in activation potency was observed
(10dh, 10di, 10ek and 10ej). Beside the decrease of activity, these
four compounds also possess undesirable hydrophobicity
(clogP >5.0). The three carboxylic acids resulting from 2-N-substi-
tution (10lh, 10lj and 10lk) revealed potency comparable to the 2-
N-methyl analog, but they displayed much better solubility (data
not shown) due to the ionizable group. To reduce the hydrophobic-
ity of the non-carboxylic acid GS activators, we incorporated polar
groups at the 2-N-position. Substitution with hydroxyethyl at the
H2N NH2
H2N
11
N
H
O
O
7c, 7f and 7g
F
F
F
Br
Br
F
R2
K2CO3
R2
acetone
O
R1 position led to 10mh with
(SC200 = 0.22 M, EC50 = 0.77 M, 5.5-fold increase at the highest
a good activation potency
O
O
Br
O
l
l
OH
8h-8k
concentration). To further decrease hydrophobicity, we investi-
gated 8 diols (10nh through 10nk and 10oh through 10ok). Unlike
the diol derivative from our earlier work, the 8 diols in Table 2 all
displayed good potency with a desirable range of hydrophobicity
(clogP <3.0). It is interesting to note that methoxy substitution at
the R2 position still delivered the best potency (10nk and 10ok).
On the other hand, the absolute stereochemistry of the diol did
not affect activation potency (compare 10nh with 10oh). Results
from Table 2 clearly suggested that it is possible to find potent
non-carboxylic acid GS activators with desirable lipophilicity. Here
we only focused on a limited substitution pattern of the biphenyl
fragment, as the four R2 substitutions (8h–8k) displayed the best
potency/in vivo compound exposure level in the corresponding
carboxylic acid series (chemical class of 2 and 3).11,12
The GS activators from the 2-N-substituted indazolones were
designed to resolve the metabolic instability of the benzisoxazo-
lone. To confirm the stability improvement, two compounds
(10ah and 10mh) were dosed (in aqueous suspension, phosphate
buffer, 0.1 M, pH 7.4, containing 10% PEG 400 by volume) to C57
mice and compound exposure was measured. Unlike 5, which
was completely cleaved after oral dosing, data for these two com-
pounds (Table 3) clearly suggested no stability issue. It is also
interesting to note that the reduction in clogP (clogP of 4.56 for
10ah and 3.25 for 10mh) contributed to compound exposure level
(Cmax of 254 ng/mL and AUC of 746 ng h/mL for 10ah, Cmax of
1350 ng/mL and AUC of 3594 ng h/mL for 10mh), which suggests
the possibility of solubility limited oral exposure. In fact, when
metabolite identification was studied for 10ah, the major metabo-
lite was from the glucuronidation of the NH group on the indazo-
lone ring.
O
Scheme 3. Synthesis of N-alkyl-N-Boc-hydrazines 7 and aryl halides 8.
Table 1
Efficient hydrazine amination and indazolone formation
Hydrazine Aryl
halide
Step (a) product (yield)a Step (b) product (yield)a
7a
7b
7c
7d
7d
7f
8j
9aj (91%)
9bh (87%)
9ch (77%)
9dk (90%)
9di (89%)
9fh (86%)
9gk (92%)
9fi (84%)
10aj (62%)
10bh (70%)
10lh (40%)
10dk (68%)
10di (72%)
10nh (73%)
10ok (71%)
10ni (82%)
8h
8h
8k
8i
8h
8k
8i
7g
7f
a
All yields are based on purified products which were checked for purity by LC/
MS and characterized by 1H NMR.
10ah with 2-N-methyl substitution on the indazolone ring dis-
played EC50 of 1.78
lone was much less active (EC50 >75
table). Therefore, we focused on 2-N-substitution and investigated
different alkyl and aryl groups. When R1 is a simple methyl group,
methoxy substitution at the R2 position afforded the most active
compound (compare 10ak with 10ah and 10aj). Replacing the
methyl at the R1 position with methoxyethyl group did not cause
significant change in activation potency (10bh and 10bk).
l
M, while the 1-N-methyl substituted indazo-
M, data not listed in the
l

Y. Qian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2936–2940
2939
Table 2
Activating human muscle glycogen synthase by 2-N-substituted indazolone derivativesa
F
F
O
HN
O
R2
N
R1
Compounds
R1
R2
SC200
(l
M)
EC50
(lM)
Fold increase @ 75
lM
clogP
10ah
10aj
CH3
CH3
CH3
CH2CH2OCH3
CH2CH2OCH3
3-Pyridyl
3-Pyridyl
4-F-Phenyl
4-F-Phenyl
CH2COOH
CH2COOH
CH2COOH
F
0.49
0.13
0.07
0.52
0.11
2.75
1.14
2.01
1.86
0.69
0.33
0.06
0.22
0.41
0.16
0.11
0.07
0.32
0.25
0.10
0.06
1.78
0.24
0.32
0.79
0.37
4.01
1.86
1.37
1.47
1.20
0.42
0.20
0.77
0.58
0.30
0.34
0.25
0.68
0.26
0.23
0.27
6.8
3.0
5.4
3.0
4.4
5.8
6.1
2.8
2.5
3.6
3.7
3.2
5.5
3.0
3.5
3.3
3.3
3.3
3.6
3.0
3.1
4.56
4.61
3.94
3.97
3.34
5.91
6.23
6.16
6.83
3.14
3.19
2.52
3.25
2.43
2.75
2.48
1.81
2.43
2.75
2.48
1.81
CH3
OCH3
F
OCH3
F
10ak
10bh
10bk
10dh
10di
10ek
10ej
Cl
OCH3
CH3
F
CH3
OCH3
F
F
Cl
CH3
OCH3
F
10lh
10lj
10lk
10mh
10nh
10ni
10nj
10nk
10oh
10oi
10oj
10ok
CH2CH2OH
(S)-CH2CH(OH)CH2OH
(S)-CH2CH(OH)CH2OH
(S)-CH2CH(OH)CH2OH
(S)-CH2CH(OH)CH2OH
(R)-CH2CH(OH)CH2OH
(R)-CH2CH(OH)CH2OH
(R)-CH2CH(OH)CH2OH
(R)-CH2CH(OH)CH2OH
Cl
CH3
OCH3
a
All numbers are the average from the dose–response enzyme activation assay run in triplicate.
Table 3
Oral exposure of 2-N-substituted indazolone in C57 mice
Compounds
clogP
Dose (mg/kg, PO)
Cmax (ng/mL)
AUC (ng h/mL)
AUC /dose
10ah
10mh
4.56
3.25
10
10
254
1350
746
3594
74
350
In summary, we designed a series of 2-N-substituted indazol-
ones as non-carboxylic acid GS activators and identified an
efficient route to synthesize an array of 2-N-alkyl- and 2-N-aryl-
indazolones. These compounds demonstrated potent GS activation
activity as well as drug-like properties (H-bond donors and
acceptors, lipophilicity, and oral exposure). Apart from glucose-
6-phosphate, an endogenous allosteric glycogen synthase
activator, there is no small molecule GS activator described in
the literature except our novel chemical class. Compounds pre-
sented in this report are the first small molecule non-carboxylic
acid GS activators. It still remains to be determined whether
non-carboxylic acids can improve muscle distribution, and
whether activating muscle GS can provide robust efficacy in dia-
betic animal models.
Rondinone for project guidance. We also thank Karen Lackey for
management support of the manuscript preparation.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmcl.2013.
03.049.
References and notes
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Acknowledgments
The authors gratefully acknowledge the following people who
contributed to the GS activator chemistry: Robert Goodnow, Jian-
ping Cai, Agostino Perrotta, Mushtaq Ahmad, Lin Yi, Kshitij Thak-
kar, Bruce Banner, Jianping Lou, Matthew Hamilton and Lee
McDermott. We thank the following people who contributed to
GS activator biological assay: Yingsi Chen, Wen Yang, David Mark,
Mary Lou Gubler, Joseph Sergi, Yonglin Ren, Cheryl Spence and Xi-
lin Liu. We thank Theresa Burchfield for 13C NMR assignment of the
final compounds, Mike Lanyi for IR spectroscopy and optical rota-
tion measurement, Jagdish Racha for metabolite identification, Mei
Liu for in vivo exposure analysis, and Jefferson Tilley and Cristina
8. Patel, S.; Doble, B. W.; MacAulay, K.; Sinclair, E. M.; Drucker, D. J.; Woodgett, J.
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9. Lawrence, J. C., Jr.; Roach, P. J. Diabetes 1997, 46, 541.
10. We used EC50
, SC200 and fold increase at 75 lM (highest compound
concentration assayed) to characterize the activity of our GS activators. The
EC50 is the compound concentration required to reach half of the maximum

2940
Y. Qian et al. / Bioorg. Med. Chem. Lett. 23 (2013) 2936–2940
enzyme activation; the SC200 is the compound concentration required to
21. Bolin, D. R.; Hayden, S.; Qian, Y. U.S. Patent Appl. 0,112,147, 2011.
double enzyme activity.
22. The enzymatic activity of GS was measured in 384-well polystyrene plates (BD
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Goodnow, R.; Gubler, M. L.; Hamilton, M. M.; Hayden, S.; Huang, K. S.; Liu, X.;
Lou, J. P.; McDermott, L.; Olivier, A. R.; Perrotta, A.; Qian, Y.; Rondinone, C. M.;
Rowan, K.; Sergi, J. A.; Tilley, J. W.; Thakkar, K.; Yi, L.; Yun, W.; Xiang, Q, 239th
ACS National Meeting, San Francisco, CA, March 21–25, 2010; MEDI-529.
13. Bolin, D. R. et al. in preparation.
Biosciences) using a coupled enzyme assay in a total volume of 32
An aliquot of activator solution (diluted compounds in 30 mM
glycylglycine, pH 7.3, 40 mM KC1, 20 mM MgC12, 9.2% DMSO, with or
without 20 mM glucose-6-phosphate) was added to an 12 aliquot of
substrate solution containing glycogen (4.32 mg/mL), 2.67 mM UDP-glucose,
21.6 mM phospho(enol)pyruvate and 2.7 mM NADH in 30 mM glycylglycine,
lL per well.
8 lL
l
L
pH 7.3 buffer. The reaction was then started by adding 12
lL of enzyme
14. Uto, Y. Expert Opin. Ther. Pat. 2012, 22, 89.
solution containing glycogen synthase (16.88 g/mL), pyruvate kinase
l
15. Hartwig, J. F.; Kawatsura, M.; Hauck, S. I.; Shaughnessy, K. H.; Alcazar-Roman,
L. M. J. Org. Chem. 1999, 64, 5575.
(0.27 mg/mL), lactate dehydrogenase (0.27 mg/mL) in 50 mM Tris–HCl, pH
8.0, 27 mM DTT and bovine serum albumin (BSA, 0.2 mg/mL), mixed and
incubated at room temperature. The conversion rate of NADH to NAD was
measured every 3 min over a period of 15 min at abs 340 nm on an Envision
reader (Perkin–Elmer). The enzyme activity (with or without compound) was
calculated by the reaction rate per minute.
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