Structural development of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives as human peroxisome proliferator-activated receptor alpha (PPARα)-selective agonists

Article abstract of DOI:10.1016/j.bmcl.2019.06.062

We previously reported that 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivative 6 is an agonist of human peroxisome proliferator-activated receptor alpha (hPPARα). Here, we prepared a series of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives in order to examine the structure-activity relationships (SAR). SAR studies clearly indicated that the steric bulkiness of the substituent on 1H-pyrazolo-[3,4-b]pyridine ring, the position of the distal hydrophobic tail part, and the distance between the distal hydrophobic tail part and the acidic head part are critical for hPPARα agonistic activity. These SAR results are somewhat different from those reported for fibrate-class hPPARα agonists. A representative compound (10f) was as effective as fenofibrate in reducing the elevated plasma triglyceride levels in a high-fructose-fed rat model.

Full text of DOI:10.1016/j.bmcl.2019.06.062

Accepted Manuscript
Str uctur al development of 1H-pyrazolo-[3,4-b]pyr idine-4-car boxylic acid
derivativesashuman peroxisomeproliferator-activated receptor alpha
(PPAR α)-selective agonists
Hiroyuki Miyachi, Tomohiro Yuzuriha, Ryotaro Tabata, Syohei Fukuda,
Kazuto Nunomura, Bangzhong Lin, Tadayuki Kobayashi, Kenji Ishimoto,
Takefumi Doi, Keisuke Tachibana
PII:
S0960-894X(19)30444-5
https://doi.org/10.1016/j.bmcl.2019.06.062
BMCL 26540
DOI:
Reference:
To appear in:
Bioorganic & Medicinal Chemistry Letters
Received Date:
Revised Date:
Accepted Date:
16 May 2019
19 June 2019
29 June 2019
Please cite this article as: Miyachi, H., Yuzuriha, T., Tabata, R., Fukuda, S., Nunomura, K., Lin, B., Kobayashi, T.,
Ishimoto, K., Doi, T., Tachibana, K., Structural development of 1H-pyr azolo-[3,4-b]pyr idine-4-car boxylic acid
derivatives as human peroxisome proliferator-activated receptor alpha (PPAR α)-selective agonists,
Bioorganic & Medicinal Chemistry Letters (2019), doi: https://doi.org/10.1016/j.bmcl.2019.06.062
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Structural development of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives as human
peroxisome proliferator-activated receptor alpha (PPARα-selective agonists
Hiroyuki Miyachia,1*, Tomohiro Yuzuriha , Ryotaro Tabata , Syohei Fukuda , Kazuto Nunomura ,
b,2
b
b
b
b
b
b
b
b
Bangzhong Lin , Tadayuki Kobayashi , Kenji Ishimoto , Takefumi Doi , Keisuke Tachibana
a
1
b
Graduate School of Medicine, Dentistry and Pharmaceutical Sciences, Okayama University, 1-1-1,
Tsushima-Naka, Kita-ku, Okayama 700-8530, Japan.
(Current address) Lead Exploration Unit, Drug Discovery Initiative, The University of Tokyo, 7-3-1
Hongo, Bunkyo-ku, Tokyo 113-0033, Japan.
Graduate School of Pharmaceutical Sciences, Osaka University, 1-6 Yamadaoka, Suita, Osaka 565-
871, Japan.
0
2
(Current address) Faculty of Pharmaceutical Sciences, Setsunan University, 45-1 Nagaotoge-cho,
Hirakata, Osaka 573-0101, Japan.
Abstract— We previously reported that 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivative 6 is
an agonist of human peroxisome proliferator-activated receptor alpha (hPPAR. Here, we prepared
a series of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid derivatives in order to examine the
structure-activity relationships (SAR). SAR studies clearly indicated that the steric bulkiness of the
substituent on 1H-pyrazolo-[3,4-b]pyridine ring, the position of the distal hydrophobic tail part, and
the distance between the distal hydrophobic tail part and the acidic head part are critical for
hPPAR agonistic activity. These SAR results are somewhat different from those reported for
fibrate-class hPPAR agonists. A representative compound (10f) was as effective as fenofibrate in
reducing the elevated plasma triglyceride levels in a high-fructose-fed rat model.

Peroxisome proliferator-activated receptors (PPARs) are members of the human nuclear
receptor family, functioning as ligand-dependent transcription factors with pleiotropic
biological functions that include regulatory roles in lipid, lipoprotein and glucose homeostasis.
PPARs are activated by various kinds of endogenous saturated and unsaturated fatty acids and
1
their metabolites, as well as by synthetic ligands. Three subtypes have been isolated to date:
PPAR (NR1C1), PPAR (NR1C2) and PPAR (NR1C3). Among these subtypes, hPPAR is
mostly expressed in tissues involved in lipid oxidation, such as liver, kidney, and regulates
genes involved in uptake and oxidation of free fatty acids, triglyceride hydrolysis; it also up-
2
regulates reverse cholesterol transport. Fibrate-class antihyperlipidemic agents, such as
fenofibrate (1) which effectively lower elevated serum triglycerides and moderately increase
high-density lipoproteins (HDL), can bind and activate hPPAR However, their affinity is
3
weak in the high micromolar range), and the subtype selectivity is not high. Recently, a
selective hPPARα modulator (SPPARMα), pemafibrate was launched as a hypolipidemic agent
in Japan.
We have been engaged in structural development studies of ligands for nuclear receptors,
including PPAR subtypes, and have reported some hPPAR-selective agonists, such as
compounds 4 and 5 (APHM-13).4, 5 We also solved the X-ray crystallographic structure of 5
6
complexed with the hPPAR ligand-binding domain (LBD). In addition, we recently discovered
a new structural class of hPPAR agonist (6), whose basic structure, i.e., 1H-pyrazolo[3,4-
b]pyridine-4-carboxylic acid, is different from those of known fibrate-class hPPAR agonists,
using our established screening system of human hepatoblastoma cell lines with luciferase
reporter expression driven by promoters containing peroxisome proliferator-responsive
elements (PPREs) together with tetracycline-regulated expression of full-length hPPAR
(relative hPPAR transactivation activity of 6 to the DMSO control was 150.83± 27.71 at 1 M

)
.7 As an extension of that work, we describe here the preparation of a series of 1H-pyrazolo-
[
3,4-b]pyridine-4-carboxylic acid derivatives in order to examine the structure-activity
relationships (SAR) for hPPAR-agonistic activity
N
O
S
COOH
O
O
O
H
Cl
O
N
O
N
N
O
HN
H
O
O
F
F
3
1
2
F
N
N
O
O
O
F
COOH
COOH
N
H
MeO
N
F
OH
H
N
O
MeO
4
5
6
Figure 1. The chemical structures of hPPAR agonists fenofibrate (1), GW-7647 (3), our reported
compounds 4, APHM-13 (5), and 6, and hPPAR antagonist GW-6471 (2).
The synthetic routes to the present series of 1H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid
derivatives are outlined in Scheme 1. Compounds 10a-10ae were prepared from methyl ester
derivative (7) in 4 steps. Methyl ester derivative (7) was treated with acetonitrile in the
presence of sodium hydride as a base, followed by reaction with aryl hydrazine to afford the
corresponding 1H-pyrazol-5-amine derivative (8). 1H-Pyrazolo-[3,4-b]pyridine ring formation
was performed by the reaction of 8 with ,-diketoester in acetic acid, and subsequent alkaline

hydrolysis afforded the desired 10a-10ae. To confirm the structures of these compounds, we
performed X-ray crystallographic analysis of the precursor ethyl ester derivative of 10f (CCDC
1
900468; an ORTEP drawing is shown in the box in Scheme 1).
1
H-Pyrazolo-[3,4-b]pyridine-5-carboxylic acid derivative 14 was prepared from 11. Michael
addition of 11 with diethyl ethoxymethylene malonate afforded the adduct 12, which was
treated with phosphoryl chloride followed by alkaline hydrolysis to afford 14.
A hybrid-type compound of 4 and 10 was prepared from 10p. The carboxyl group of 10p
was reduced with BH -THF, then oxidized with activated MnO to afford the formyl derivative
3
2
1
5, which was treated with triethyl 2-phosphonobutyrate, followed by hydrogenolysis and
alkaline hydrolysis to afford the desired product 17.
R2
N
R1
COOEt
R2
N
N
R1
COOH
R2
N
R1
N
O
N
a)
b)
c)
R1 OMe
N
N
H2N
R3
R 10a-10ae
3
7
8
9
R1 = Me, i-Pr, s-Bu, t-Bu,
R2 = substituted aryl
R3 = Me, Et, cyclopropyl,
phenyl
cyclopropyl, cyclobutyl,
pentan-3-yl, cyclopentyl
F
F
F
N
N
F
N
N
N
N
N
N
d)
e)
f)
HN
N
Cl
N
Cl
COOH
H2N
EtOOC
COOEt
COOEt
12
1
1
13
14
F
F
N
N
F
F
N
N
N
g)
N
h)
i)
N
N
N
N
COOEt
COOH
N
COOH
N
CHO
10p
15
16
17

Scheme 1. Synthetic routes to the present series of compounds. Reagents and conditions: a) (1)
°
NaH, MeCN, THF reflux, 2 h, (2) arylhydrazines, EtOH, cHCl, 160 C, 50 min (microwave), 35-70% (2
3
°
steps); b) R COCH COCOOEt, AcOH, 170 C, 50 min (microwave), 63-90%; c) 1 mol/L NaOH, EtOH,
2
reflux, overnight, 48-95%; d) diethyl ethoxymethylene malonate, EtOH, reflux, overnight, 80%; e)
POCl , reflux, overnight, 48%; f) 1 mol/L NaOH, EtOH, r.t., overnight, 90-95%; g) (1) BH -THF, THF,
3
3
reflux, 10 h, 85%; (2) MnO CH Cl , r.t., overnight, 90%; h) (1) (EtO) POCH(Et)CO Et, NaH, THF r.t.,
2
,
2
2
2
2
overnight, 76%, (2) H , 10% Pd−C, EtOH, r.t., quant.; i) 1 mol/L NaOH, EtOH, r.t., overnight, 90%.
2
8
Considering the hydrophobic nature of the large binding pocket of the hPPAR LBD, we
prepared 1H-pyrazolo-[3,4-b]pyridine derivatives with various hydrophobic substituents at the
1
2
3
R , R and R positions depicted in Table 1. Broadly speaking, the steric bulkiness of the
1
substituent introduced at the R position, the position of the acidic carboxyl group and the
2
distance between R substituent and the 4-position carboxyl group each greatly affected
1
hPPAR transactivation activity. In the case of a smaller R substituent such as a methyl group
or cyclopropyl group (10a, 10b), the hPPAR transactivation activity was very weak. However,
1
compounds with an isopropyl group at the R position (10c-10r, except 10s) exhibited similar
or greater hPPAR transactivation activity compared with 10 M fenofibrate. Cyclobutyl and
1
cyclopentyl substitution at the R position (10t, 10y-10aa) also resulted in comparable hPPAR
transactivation activity to fenofibrate, while pentan-3-yl substitution (10ab-10ae) resulted in
1
more potent activity. However, compounds with a tert-butyl group at the R position (10u,
1
0v) did not show significant hPPAR transactivation activity.

2
In the case of the R substituent, a phenyl group is preferable, while the pyridine derivative
(10s) did not show hPPAR transactivation activity in our assay system. This result is
2
interesting, but at present we cannot explain it. As for substituted phenyl groups at R , the
steric factor may again be important, because the order of activity for halogen-substituted
phenyl groups was F < Cl > Br = I. The fact that the 3-benzyloxy derivative (10e) was less active
is consistent with this idea.
The optimum location of the acidic head carboxyl group might be the 4-position, as the 5-
carboxyl derivative (14) exhibited poor hPPAR transactivation activity. Surprisingly, 2-
ethylphenyl propanoic acid derivative (17), which is a mimic of compounds 4 and 5, showed
less hPPAR transactivation activity.

R5
N
R1
R4
N
N
R3
R2
1
2
3
4
5
No.
R
R
R
R
R
relative activity
0.00
SD
EC50 (M)
DMSO
fenofibrate (1)
GW-7647 (3)
6
100.00
199.64
43.09
27.71
150.83
1
1
1
1
1
0a
0b
0c
0d
0e
Me
cyclopropyl
i-Pr
phenyl
Me
H
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
COOH
Cl
13.62
24.56
96.72
26.02
47.42
0.56
6.15
35.20
5.18
7.03
N.T.
N.T.
N.T.
N.T.
N.T.
4-Cl-phenyl
3-Cl-phenyl
Me
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
i-Pr
3
4-CF -phenyl
Me
i-Pr
3-BnO-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
3-Cl-phenyl
3-Cl-phenyl
4-Br-phenyl
4-Br-phenyl
4-Br-phenyl
4-I-phenyl
Me
1
0f
i-Pr
Me
187.37
195.94
113.84
192.16
162.93
36.03
44.69
20.14
27.74
28.03
0.66
1.39
1
0g
0h
i-Pr
Et
1
i-Pr
cyclopropyl
>2.74
>2.27
>1.34
1
0i
i-Pr
Et
1
0j
i-Pr
cyclopropyl
10k
i-Pr
Me
85.15
23.32
56.47
>4.01
1
0l
i-Pr
Et
251.16
1.23
1.98
10m
i-Pr
cyclopropyl
198.56
111.03
46.54
3.21
1
0n
0o
0p
0q
i-Pr
Me
2.41
1.76
2.06
N.T.
1.80
N.T.
N.T.
N.T.
N.T.
0.68
0.46
N.T.
N.T.
N.T.
1.45
1.53
>3.57
1.24
N.T.
1
i-Pr
4-I-phenyl
Et
111.04
79.17
50.95
242.71
16.16
88.34
2.26
13.34
10.72
23.97
56.29
8.16
1
1
i-Pr
4-F-phenyl
3,4-diCl-phenyl
4-Cl-phenyl
5-Cl-pyridin-2-yl
4-Cl-phenyl
4-Cl-phenyl
3-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-F-phenyl
4-F-phenyl
4-Cl-phenyl
4-Cl-phenyl
4-F-phenyl
Me
i-Pr
Me
1
0r
0s
0t
i-Pr
phenyl
1
i-Pr
Et
1
cyclobutyl
t-butyl
t-butyl
s-butyl
s-butyl
cyclopentyl
cyclopentyl
cyclopentyl
pentan-3-yl
pentan-3-yl
pentan-3-yl
pentan-3-yl
i-Pr
cyclopropyl
26.09
0.19
1
0u
0v
Me
1
Me
5.75
0.53
10w
Me
175.98
223.73
64.83
78.21
76.78
204.60
250.26
182.88
213.36
6.21
8.01
10x
10y
10z
cyclopropyl
3.17
Me
9.68
Et
19.05
6.14
10aa
10ab
10ac
10ad
10ae
cyclopropyl
Me
Et
6.97
25.79
16.71
19.06
2.65
Me
Et
1
4
H
COOH
CH
COOH
2
CH(Et)
17
i-Pr
4-F-phenyl
Me
H
1.53
0.60
N.T.
Table 1 PPAR transactivation activity of compounds in the present series. HepG2 cells were co-
transfected with 4xUAS-tk-luc and pBIND-hPPAR. Transfected cells were treated with the test
compounds (1 M final concentration each), 10 M fenofibrate, or 0.1% DMSO (Vehicle) for 24 h,
then used for reporter gene assays. Luciferase activities from reporter plasmids were normalized to
Renilla luciferase activity. Values are expressed as fold induction as compared to that obtained with

1
0 M fenofibrate (n = 3).
9
Next, we investigated the PPAR-subtype selectivity of a representative compound, 10f. As
shown in Figure 2 (A)-2 (C), 10f selectively induced hPPAR transactivation in a dose-
dependent manner, and its EC value was 0.66 M, which is lower than that of fenofibrate
5
0
(
EC = 50 M). It slightly enhanced hPPAR transactivation only at the highest concentration
5
0
(30 M), and had no effect on hPPAR (Figure 2 (B), 2(C)). The activity of 10f towards
hPPAR was completely inhibited in the presence of GW6471 (2), a hPPAR-specific
1
0
antagonist, indicating that 10f induced transcriptional activation of hPPAR by binding to the
LBD (Figure 2 (D)).
We then examined the species-selectivity of 10f, because some PPAR agonists show
species-dependent transactivation of PPAR11 The classical PPAR agonist pirinixic acid (WY-
1
4643) is more effective on rodent PPAR rPPAR than on hPPAR. 5,8,11,14-
Eicosatetraynoic acid (ETYA) shows the reverse preference, i.e., it is 10-fold more effective on
hPPAR than rPPAR while fenofibrate does not show apparent species selectivity. We
previously demonstrated the importance of the interaction between the distal hydrophobic
tail part of our phenylpropanoic acid-type hPPAR agonist and amino acid residue 272
(
isoleucine) in the helix-three region of hPPAR LBD for hPPAR-selective activation.12
As can be seen from Figure 2 (A), 2 (E), 2 (F), 10f activated human, mouse and rat PPAR
with EC values of 0.66 M, 11.27 M and 15.93 M, respectively. Thus, 10f showed species
5
0
preference for humans, and its transactivation activity was approximately 20-fold less potent
in mouse and rat.

In order to examine whether the transactivation of hPPAR elicited by 10f was mediated
directly by the binding of 10f to the LBD of hPPAR, we evaluated the binding affinity of 10f to
hPPAR LBD. The results of cell-free time-resolved fluorescence resonance energy transfer
(TR-FRET) hPPAR competitive binding assay demonstrated that the 10f indeed binds to
hPPAR-LBD, and competitively and dose-dependently displaces FluormoneTM pan-PPAR green
as indicated by the reduction in the 518 nm/488 nm ratio; the IC was 4.48 M (Figures 2
5
0
(G)). On the other hand, a transactivation-inactive compound, 10u, did not cause any
decrease in the 518 nm/488 nm ratio (Figures 2 (H)).
In order to confirm the ability of 10f to activate genes having a peroxisome proliferator-
responsive element (PPRE) in the promoter region at the cellular level, we next examined
changes in the expression of a representative hPPAR-mediated gene, the pyruvate
1
3
dehydrogenase kinase 4 (PDK4) gene, in functional liver cell 4 (FLC4) (Figures 2 (I)). Pyruvate
dehydrogenase kinase 4 (PDK4) is a key metabolic enzyme, which regulates cell metabolism by
inhibiting pyruvate dehydrogenase (PDH). When FLC4 cells were treated with 10f and then
analyzed by means of real-time RT-PCR, we found that PDK4 mRNA expression was dose-
dependently up-regulated. In contrast, cyclophilin A, used as an internal control, was
unaffected (data not shown).

(
A)
(B)
(C)
(D)
hPPAR
hPPAR
hPPAR
120
90
60
30
0
0
1
10
GW6471 (µM)
(
E)
(F)
mPPAR
rPPAR
(
G)
(H)
(I)
4.0
3.0
2.0
1.0
0.0
1
0f
10u
DMSO
DMSO
4f
30 M
1 40 f0 M
1
0f

Figure 2. 10f selectively binds to and activates PPAR. Figs 2(A)-2(C). Dose-response relationships
for transcriptional activation of GAL4-hPPAR, GAL4-hPPAR, and GAL4-hPPAR. HepG2 cells were
co-transfected with 4xUAS-tk-luc and pBIND-hPPAR. Transfected cells were treated with 10f for
2
4 h, and then used for reporter gene assays. Luciferase activities from reporter plasmids were
normalized to Renilla luciferase activity. Values are expressed as fold induction compared with the
vehicle, set as 1. Fig 2(D). Dose-dependent repression of the transcriptional activation of 10f by the
PPAR antagonist GW6471 (2). Cells were pretreated with GW6471 (2) for 1 h before addition of
1
0f (10 M final concentration). Percentage inhibition values were calculated based on the activity
in the absence of GW6471 (2) taken as 100%. Figs 2(E), 2(F). Subtype-selective dose-response
relationships for transcriptional activation of GAL4-mPPAR, and GAL4-rPPAR. Figs 2(G), 2(H).
Binding affinity assessment of 10f (active compound) and 10u (inactive compound) to human
PPAR-LBD, evaluated by means of TR-FRET assay. The human PPAR-LBD was incubated with
FluormoneTM pan-PPAR green, LanthaScreenTM terbium-labeled anti-GST antibody, and
compounds, and the TR-FRET emission ratio (518 nm/488 nm) was measured (n = 3). Fig 2(I). 10f
induces the expression of hPPAR target gene. FLC4 cells were treated with various concentrations
of 10f for 48 h. Human PDK4 mRNA level was measured using real-time RT-PCR and normalized to

2-microglobulin mRNA, set as 1 in the vehicle. Values are expressed as the means ± S.D. (error

bars) (n = 3).
Based on synthetic considerations (total yield, chirality, physicochemical properties, cost)
and potency, we selected 10f for further studies. Prior to in vivo experiments, we confirmed
1
4
that both 10f and its sodium salt (10f Na) show acceptable in vitro ADME profiles (Table 2).
Both the molecular form (10f) and the ionic form (10f Na) exhibited high aqueous solubility
and high membrane permeability. They were stable and were recovered almost quantitatively
after incubation with both human and mouse liver microsomes. These compounds also
showed similar levels of protein binding in human and mouse plasma. Thus, they were
considered to be suitable for in vivo pharmacological study.
a)
b)
c)
d)
aqueous solubility
Caco-2 permeability
stability
protein binding
cpd
−
6
(
μM)
(x 10 cm/s)
human liver microsomes (%)
rat liver microsomes (%)
human plasma (%)
rat plasma (%)
99.0
10f
>95
>95
51.8
91
91
87
92
99.7
99.7
1
0f Na
22.5
98.9
Table 2 ADME Profiling of 10f a) Solubility in PBS, pH 7.4. b) A–B permeability at 25 μM 10f. c)
Percent (%) remaining after incubation of 0.5 μM 10f at 37°C for 1h. d) Percent of 10f bound to
protein after incubation of 20 M 10f with human or rat plasma at 37°C for 5h. Final DMSO
concentration was 0.2%.

To elucidate the in vivo effects of 10f on increased serum triglyceride levels, we used a well-
established fructose-fed rat model.15 After fructose feeding (25% fructose in drinking water)
for 3 weeks, serum triglyceride levels of rats fed fructose (25% fructose in drinking water) for 3
weeks were much higher (306 ± 63 mg/dl) than those of rats given normal drinking water (122
±
26 mg/dl) (Figure 3 (A)). The fructose-fed rats were then administered 10f or fenofibrate for
an additional 2 weeks, together with the fructose. As shown in Figure 3 (B), serum triglyceride
levels were lower in rats treated with 10f or fenofibrate under fructose-feeding conditions
than in the vehicle controls after 2 weeks. After the 2-week treatment period, hepatic
expression of PPAR target genes, such as acyl-CoA dehydrogenase medium chain (ACADM),
solute carrier family 25 member 20 (SLC25A20), acyl-CoA Oxidase 1 (ACOX1) and carnitine
palmitoyltransferase 1A (CPT1A), were enhanced in rats administered 10f or fenofibrate
(
(Figures 3 (C)- 3 (F)). These data clearly indicate that the 1H-pyrazolo-
3,4-b]pyridine derivative 10f attenuates hypertriglyceridemia in vivo.
[

(
A)
(B)
5
00
00
00
00
00
0
-2 day
day
13 day
4
3
2
1
00
6
4
3
2
1
00
00
00
0
-
+
Fructose
Vehicle
10f
Fenofibrate
(
C)
(D)
(E)
(F)
3
ACADM
5
SLC25A20
15
ACOX1
6
CPT1A
4
3
2
1
0
12
9
2
1
0
4
2
0
6
3
0
Figure 3. Effect of 10f on in vivo lipid metabolism in fructose-fed rats. Fig 3(A) Fructose feeding
markedly increased serum triglyceride levels, compared with rats consuming normal drinking
water. Male rats received either a 25% fructose challenge in the drinking water or normal drinking

water. Fig 3 (B) Serum triglyceride (TG) levels after 2-week oral treatment with 10f (10 mg/kg/day),
fenofibrate (30 mg/kg/day), or 0.5% methylcellulose (Vehicle). Three weeks later, fructose-fed rats
were orally treated with compound 10f (10 mg/kg/day), fenofibrate (30 mg/kg/day), or 0.5%
methylcellulose (Vehicle) for 2 further weeks. Figs 3(C)-3(F). Hepatic mRNA levels of PPAR target
genes, ACADM (Fig 3(C)), SLC25A20 (Fig 3(D)), ACOX1 (Fig 3(E)) and CPT1A (Fig 3(F)). mRNA levels in
rat liver were measured using real-time RT-PCR and normalized to 2-microglobulin mRNA, set as 1
in the vehicle group. Values are expressed as means ± S.D. (error bars) (n = 3-5).
In conclusion, we have succeeded in creating a new chemotype of hPPAR-selective
activators, as exemplified by 10f. This confirms that the 1H-pyrazolo-[3,4-b]pyridine
framework is an attractive basic structure for hPPAR modulators, which are candidate agents
to control altered lipid homeostasis, such as dyslipidemia. The SAR results in the current series
are somewhat different from those of known fibrate-class hypolipidemic agents. We are
currently conducting an X-ray crystallographic study of 1H-pyrazolo-[3,4-b]pyridine derivatives
complexed with hPPAR LBD to examine the reasons for this difference.
Acknowledgement.
This work was supported in part by a grant for Platform for Drug Discovery, Informatics, and
Structural Life Science from the Ministry of Education, Culture, Sports, Science and
Technology, Japan, the Japan Agency for Medical Research and Development (AMED) under
Grants JP18am0101085, Platform Project for Supporting Drug Discovery and Life Science
Research (Basis for Supporting Innovative Drug Discovery and Life Science Research

(BINDS)) from AMED under Grant Number JP19am0101085, JSPS KAKENHI (grant numbers
1
5H02896, 16K13044 and 18H03190), and Takeda Science Foundation. The authors gratefully
thank Advanced Science Research Center, Okayama University for the single crystal XRD
measurements.
References and Notes
1
2
3
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6
7
. Kuwabara, N.; Oyama, T.; Tomioka, D.; Ohashi, M.; Yanagisawa, J.; Shimizu, T.;
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H. J.Med. Chem. 2012, 55, 893.
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8
. Nolte, R.T.; Wisely, G. B.; Westin, S.; Cobb, J. E.; Lambert, M. H.; Kurokawa, R.;
Rosenfeld, M. G.; Willson, T. M.; Glass, C. K.; Milburn, M. V. Nature. 1998, 395, 137.
1
9
. Physico-chemical properties of compound 10f: H-NMR (300 MHz, DMSO-d ) δ 8.31,(2H,
6
dd, J=6.8, 2.1Hz), 7.56,(1H, s), 7.47,(2H, dd, J=6.9, 2.2Hz), 3.86,(1H, sep, J=6.8Hz ), 2.76,(3H,
1
3
s), 1.42,(6H, d, J=6.7Hz). C-NMR (100 MHz, DMSO-d ) δ 167.27, 159.11, 151.12, 151.04,
6
1
37.95, 135.63, 129.52, 128.99, 121.83, 117.60, 109.29, 27.41, 24.45, 21.96. MS 329
+
(
M+H) . mp. 270.6-271.4 ºC . HPLC purity was estimated to be 99.5% by means of
reversed-phase HPLC, using a Pegasil ODS sp100 column (4.6 mm x 250 mm, Senshu
Chemical, Japan) fitted on a JASCO HPLC system, with CH CN : 0.1% TFA = 2:1 v/v as the
3
eluent and detection at 254 nm.
1
0. Xu, H. E.; Stanley, T. B.; Montana, V. G.; Lambert, M. H.; Shearer, B. G.; Cobb, J. E.;
McKee, D. D.; Galardi, C. M.; Plunket, K. D.; Nolte, R. T.; Parks, D. J.; Moore, J. T.;
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1
1
1
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Gonzalez, F. J.; Sakai, J.; Kodama, T. J. Atheroscler. Thromb. 2015, 22, 754.
1
1
4. Physico-chemical properties of compound 10fNa: H-NMR (300 MHz, CDCl ) δ 8.32,(2H,
3
dd, J=6.9, 2.2Hz), 7.48,(2H, dd, J=7.0, 2.3Hz), 7.08,(1H, s), 3.78,(1H, sep, J=6.9Hz ), 2.65,(3H,
s), 1.40,(6H, d, J=6.9Hz). mp. >300 ºC . HPLC purity was estimated to be 99.8% by means
of reversed-phase HPLC, using a Pegasil ODS sp100 column (4.6 mm x 250 mm, Senshu
Chemical, Japan) fitted on a JASCO HPLC system, with CH CN : 0.1% TFA = 2:1 v/v as the
3
eluent and detection at 254 nm.
1
5. Park, J.; Lemieux, S.; Lewis, G. F.; Kuksis, A.; Steiner, G. J. Lipid Res. 1997, 38, 2529.

Graphical Abstract
Structural development studies of a series of
Leave this area blank for abstract info.
1
H-pyrazolo-[3,4-b]pyridine-4-carboxylic acid
human peroxisome proliferator-activated receptor
(PPAR) -selective agonists
Hiroyuki Miyachi, Tomohiro Yuzuriha, Ryotaro Tabata,
Syohei Fukuda, Kazuto Nunomura, Bangzhong Lin,
Tadayuki Kobayashi, Kenji Ishimoto, Takefumi Doi,
Keisuke Tachibana
COOH
R5
R1
N
R4
N
N
N
N
R3
N
2
R
Cl