Design, synthesis, and biological evaluation of 1,2,4-oxadiazole-containing pyrazolo[3,4-b]pyridinones as a new series of AMPKɑ1β1γ1 activators

Received: 8 December 2020

Revised: 5 February 2021

Accepted: 12 February 2021

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DOI: 10.1002/ardp.202000458

F U L L P A P E R

Design, synthesis, and biological evaluation of

1,2,4‐oxadiazole‐containing pyrazolo[3,4‐b]pyridinones

as a new series of AMPKɑ1β1γ1 activators

Zhihong Xiao | Yajun Peng | Bifeng Zheng

| Qi Chang | Yating Guo |

Zhuo Chen | Qianbin Li

| Gaoyun Hu

Department of Medicinal Chemistry, Xiangya

School of Pharmaceutical Sciences, Central

South University, Changsha, China

Abstract

Adenosine monophosphate‐activated protein kinase (AMPK) plays a key role in

maintaining whole‐body homeostasis and has been regarded as a therapeutic target

for the treatment of diabetic nephropathy (DN). Herein, a series of 1,2,4‐oxadiazole‐

containing pyrazolo[3,4‐b]pyridinone derivatives is reported as AMPKɑ1β1γ1

activators. The in vitro biological assay demonstrated that compounds 12k

(EC₅₀[AMPKα1γ1β1] = 180 nM) and 13q (EC₅₀[AMPKα1γ1β1] = 2 nM) displayed

significant enzyme activation. Mechanism studies indicated that both compounds

reduced the levels of reactive oxygen species in a rat kidney fibroblast cell line

(NRK‐49F) stimulated by transforming growth factor‐β and induced early apoptosis

of NRK‐49F cells at 10 μM. Molecular docking studies suggested that 13q exhibited

critical hydrogen‐bond interactions with the critical amino acid residues Lys29,

Lys31, Asn111, and Asp88 at the binding site of the AMPK protein. These results

enrich the structure pool of AMPK activators and provide novel lead compounds for

the subsequent development of compounds with a promising therapeutic potential

against DN.

Correspondence

Qianbin Li and Gaoyun Hu, Department of

Medicinal Chemistry, Xiangya School of

Pharmaceutical Sciences, Central South

University, 410013 Changsha, China.

Email: qbli@csu.edu.cn and

hugaoyun@csu.edu.cn

Funding information

National Natural Science Foundation of China,

Grant/Award Numbers: 81573287,

81773640; Natural Science Foundation of

Hunan Province, Grant/Award Number:

2016JJ2162

K E Y W O R D S

activator, AMPK, apoptosis, pyrazolo[3,4‐b]pyridinones, reactive oxygen species (ROS)

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1

INTRODUCTION

leads to DNA damage in mitochondria, which, in turn, causes

mitochondrial dysfunction and produces more ROS. Increases in

Diabetic nephropathy (DN) is one of the major complications of

diabetes, with about 40% incidence in diabetic patients.^[1]As the

early symptoms of DN are often ignored, when the disease pro-

gresses to end‐stage renal disease, it will greatly increase the

burden on the patient's life. The pathogenesis of DN includes

metabolic disorder (chronic hyperglycemia and dyslipidemia),

oxidative stress, inflammation, and fibrosis.^[2–6]A growing body

of evidence demonstrates that oxidative stress plays a major role

in the pathogenesis of DN.^[7–9]Glucose metabolism disorder in

renal cells will increase the production of free radicals such as

reactive oxygen species (ROS). Oxidative stress induced by ROS

oxidative stress can stimulate the production of inflammatory

cytokines.^[10]For example, ROS can activate tumor necrosis

factor‐α (TNF‐α), which further activates nuclear factor‐κB

(NF‐κB), leading to the aggravation of inflammation in the dia-

betic kidney. ROS also activates transforming growth factor‐β

(TGF‐β), which stimulates the deposition of extracellular matrix

(ECM) and increases renal fibrosis. The current clinical treatment

strategy for DN includes hypoglycemic agents that control the

blood glucose^[11]and angiotensin‐converting enzyme inhibitors

and angiotensin receptor blockers that lower the blood

pressure.^[12]However, these strategies are still not efficient for

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preventing disease progression to end‐stage renal disease. The

novel drugs for DN in research are sodium/glucose co‐

transporter 2 (SGLT2) inhibitor that controls blood glucose,

endothelin‐1 (ET‐1) receptor antagonist that controls blood

of AMPK α‐Thr172 phosphorylation induced by drugs that increase

the AMP/ADP ratio in cells, resulting in the phosphorylation of

Ser108 in the ADaM site, which, in turn, leads to AMPK allosteric

activation.^[45]Therefore, the ADaM site is an attractive site for drug

design.

pressure,

drugs.^[13–15]These drugs can alleviate the pathological state of

DN to certain extent. However, most of them have not

anti‐inflammatory

drugs,

and

anti‐fibrotic

A great number of compounds have been reported to indirectly

activate AMPK by increasing the ratio of AMP/ATP, such as the an-

tidiabetic drug metformin (1) (Figure 2),^[46]and natural products such

as resveratrol (2).^[26]AICAR (3) was converted to 5‐aminoimidazole‐4‐

carboxamide‐1‐β‐^D‐ribofuranosyl 5'‐monophosphate (ZMP) (4) in cells,

which acts as AMP mimics.^[47]ZMP can bind to AMPK γ‐subunits to

allosterically activate AMPK and hinder the dephosphorylation of

AMPK, which is independent of the change of the AMP/ATP

ratio.^[48]2‐{2‐[(4‐(Trifluoromethyl)phenyl)amino]thiazol‐4‐yl} acetic

a

been used in clinical practice due to some potential side

effects.^[16,17]Therefore, it is a viable strategy to find novel

therapeutic targets and develop drugs for the treatment of DN.

Adenosine 5'‐monophosphate‐activated protein kinase (AMPK) is a

heterotrimeric serine/threonine‐protein kinase that plays a vital role in

maintaining whole‐body energy metabolic processes.^[18]In response to

the increased ratio of AMP/ATP (adenosine triphosphate) in cells, acti-

vated AMPK stimulates ATP‐generating cellular processes by promoting

glucose uptake and fatty acid oxidation while inhibiting ATP‐consuming

cellular processes by suppressing protein and fatty acid synth-

esis.^[19,20]AMPK is strongly expressed in kidney, and AMPKɑ1β1γ1 is the

predominant isoform.^[21–24]However, low AMPK had been reported in

diabetic kidneys, where it is involved in diverse physiological and pa-

thologic processes in DN, including ECM deposition in the kidney par-

enchyma, podocyte autophagy, diabetic renal hypertrophy, and

glomerulosclerosis.^[25–28]As a regulator of cellular energy, AMPK can

relieve inflammation and oxidative stress and promote apoptosis of

fibroblasts in DN. AMPK activates tuberous sclerosis complex protein‐2

(TSC‐2) involved in cell growth and autophagy, and peroxisome

proliferator‐activated receptor‐γ coactivator 1α (PGC‐1α) that sup-

presses protein and cholesterol synthesis and stimulates mitochondrial

biogenesis, followed by a reduction in the expression of downstream

NF‐κB signaling and inhibition of the inflammatory response

(Figure 1).^[21]It has been recognized that ROS plays a critical role in renal

fibrosis.^[7–9]Enhanced ROS production leads to renal fibrosis by trig-

gering multiple signaling pathways, including the ones involved in me-

sangial cell hypertrophy, renal tubular epithelial cell migration and

transformation, and fibroblast proliferation/activation.^[29–34]AMPK also

reduces the overexpression of ROS by inhibiting nicotinamide adenine

dinucleotide phosphate oxidase^[35]or increases the expression of su-

peroxide dismutase through the AMPK–FoxO pathway to improve the

cellular redox state and reduce the effects of ROS on cells.^[7,9]In addition,

AMPK can induce apoptosis of fibroblasts by activating apoptosis signal‐

regulating kinase/c‐Jun N‐terminal kinase pathways, thereby improving

the glomerular fibrosis state, which can be a new strategy for the

management of DN.^[36]Together, it is reasonable to see AMPK as a

potential therapeutic target for DN.

acid (activator‐3) (5) was reported to be

a potent pan‐AMPK

activator, which AMP likely share a common activation mode for

AMPK activation.^[49]C2 (6) was found to be a potent allosteric acti-

vator of α1 complexes and protected against Thr172 depho-

sphorylation like AMP, and its cell‐permeable prodrug C13 (7) was

also

a

selective activator of α1 complexes in mammalian

cells.^[40,50,51]Abbott Laboratories proposed the first small‐molecule

direct AMPK activator A‐769662 (EC₅₀[AMPKα1γ1β1] = 700 nM) (8),

which is now known to bind to the ADaM site.^[52]Pfizer disclosed an

indole acid‐based AMPK activator PF‐06409577 (EC₅₀[AMP-

Kα1γ1β1] = 7.0 nM) (9), which showed beneficial effects in improving

the renal function of the DN mouse model.^[53]It further reported

PF‐06685249 (EC₅₀[AMPKα1γ1β1] = 12.0 nM) (10), which ex-

hibited robust activation of AMPK in rat kidneys as well as de-

sirable oral absorption.^[54]We previously reported a structure‐

based virtual screening based on our “in‐house” compounds li-

brary that led to the discovery of compound 17f (11), which po-

tently activated AMPKα1γ1β1 (EC₅₀= 420 nM).^[55]However,

compound 17f has insufficient activation capacity as compared

As a conserved heterotrimeric protein kinase, AMPK is highly

expressed in eukaryocytes, comprised of a catalytic α‐subunit in

complex with regulatory β‐ and γ‐subunits.^[37–41]Structural biology

studies have revealed an allosteric binding site between the α‐ and

β‐subunits, which is termed an allosteric drug and metabolite (ADaM)

site and presents a novel binding pocket.^[42,43]It has been reported

that long‐chain fatty acid (LCFA)‐CoA metabolites act as direct en-

dogenous AMPK β1‐selective activators and promote LCFA oxida-

tion.^[44]Exogenous molecules that bind to this site increase the level

FIGURE 1 A schematic representation of the pathological roles

of AMPK in DN. AMPK, adenosine monophosphate‐activated protein

kinase. DN, diabetic nephropathy; JNK, c‐Jun N‐terminal kinase;

NF‐κB, nuclear factor‐κB; ROS, reactive oxygen species

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FIGURE 2 Chemical structures of reported adenosine monophosphate‐activated protein kinase activators

with PF‐06409577, which may be related to their different binding

modes with AMPK. As shown in Figure 3, the phenyl‐containing

side‐chain of PF‐06409577 (Figure 3a) can better stretch in the

hydrophobic pocket of AMPK than that of compound 17f

(Figure 3c). The carboxyl group of PF‐06409577 and PF‐

06685249 (Figure 3b) is exposed to the hydrophilic region, which

acts as a hydrogen‐bond acceptor to form hydrogen‐bond inter-

action with the critical amino acid residues Lys29. The cyano

group at the C‐3 position of compound 17f produces electrostatic

interaction with Lys29 (−8.8 kcal/mol), which is weaker than the

hydrogen‐bond interaction between the carboxyl group of PF‐

06409577 and Lys29 (−17.1 kcal/mol).

hydrophilic region or trifluoromethyl to explore its influence on

binding with critical amino acids. (iv) R₃and R₄were designed to

explore the influence of the substitution of hydroxyl group at the

C‐6 position and the N–H at N‐1 position on the activity (PMB

refers to p‐methoxybenzyl).

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2

RESULTS AND DISCUSSION

Chemistry

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2.1

The target compounds were synthesized following the synthetic

routes shown in Scheme 1. The synthesis of compounds occurred

mainly through the coupling of Part 1 and Part 2. Part 1 is obtained

According to the docking results of the compounds above, we

designed and synthesized a series of novel AMPK activators by mod-

ifying PF‐06409577 (Figure 4). On the basis of retaining the scaffold

pyrazole[3,4‐b]pyridone of compound 17f, (i) a smaller five‐membered

ring (1,2,4‐oxadiazole) was used to replace the benzene ring at the C‐3

position of compound 17f as the linking group, which may allow it to

better penetrate into the narrow binding cavity of AMPK. (ii) The

introduction of the R₁substituents on the side‐chain is used to

explore the effect of different substituents on the potency. (iii)

Given that R₂substituents are carboxylic acid, this moiety might

enhance the interaction with the key amino acids in the

from

a substituted benzonitrile. Ethyl trifluoroacetate (3) and

4‐methoxybenzylhydrazine hydrochloride (4) were used as starting

materials to yield PMB‐protected 3‐(trifluoromethyl)‐1H‐pyrazol‐5‐

ol (5). The Vilsmeier–Haack reaction of 5 yielded aldehyde derivative

6, which was substituted by NaN₃and then reduced to amine group

(7) with Fe/NH₄Cl. The pyrazolo[3,4‐b]pyridine scaffold was con-

structed from 7 and diethyl malonate, followed by alkylation with

PMB (9) and then hydrolysis of ethyl ester to afford 10 in the form of

3‐carboxylic acid.

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FIGURE 3 Predicted binding mode of (a)

PF‐06409577 (red), (b) PF‐06685249

(orange), (c) compound 17f (yellow) with

adenosine monophosphate‐activated protein

kinase (AMPK), (d) overlay of PF‐06409577,

PF‐06685249, and compound 17f in the

AMPK active pocket (PDB ID: 5KQ5)

FIGURE 4 Structural modification on the pyrazolo[3,4‐b]pyridine scaffold

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Compounds 11a–s were synthesized with the cyclization of

N‐hydroxyl‐amidine in Part 1 and carboxyl group in Part 2. Target

compounds 12a–s and 13a–r were obtained with the deprotection of

the PMB group and hydrolysis of the trifluoromethyl group into the

carboxyl group in 1 N HCl.

2.2

Structure–activity relationship (SAR) study

All synthesized compounds were evaluated by using the human AMP-

Kα1β1γ1 isoform that is predominantly expressed in kidney^[21–24]with

PF‐06409577 as a positive control. The potency of test compounds in

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SCHEME 1 Synthesis of compounds 11a–s, 12a–s, and 13a–r. Reagents and conditions: (a) NH₂OH·HCl, DIEA, EtOH, reflux, 5 h; (b) EtOH,

reflux, 8 h; (c) POCl₃, DMF, 100°C, 4 h; (d) NaN₃, TBAI, DMSO, rt, 18 h; (e) Fe, NH₄Cl, H₂O, EtOAc, rt, 24 h; (f) diethyl malonate, pyridine, EtOH,

reflux, 10 h; (g) 4‐methoxybenzyl chloride, Cs₂CO₃, rt, 20 h; (h) (1) 1 mol/l NaOH, H₂O/EtOH, reflux, 5 h, (2) 1 mol/l HCl; (i) HOBt, EDCI, DMSO,

rt, 6 days; (j) (NH₄)₂Ce(NO₃)₆, CH₃CN/H₂O = 5:1, reflux, 15 h; (k) i. 1 mol/l NaOH, H₂O/EtOH, reflux, 18 h, ii. 1 mol/l HCl

different concentrations was compared with the baseline activity of the

enzyme to calculate the activation efficiency. As shown in Table 1, when

position 3 of the pyrazole ring was substituted with carboxylic acid, the

benzene ring that was substituted by chlorine (13i–k) or trifluoromethyl

(13q and 13r) showed better activation, among which meta‐chlorine (13j,

EC₅₀= 20 nM) and trifluoromethyl (13q, EC₅₀=2 nM)‐substituted

compounds had a significantly improved potency and ortho‐ and para‐

substituted compounds had a reduced activity. An increase in the num-

ber of chlorine atoms substituted on the benzene ring (13l and 13m) led

to a significant decrease in activation potency. Trifluoromethoxy (13n–q)

and fluorine (13f–h)‐substituted compounds also resulted in a decreased

potency. Electron‐donating groups such as methyl (13c–e) or methoxy

(13a,b)‐substituted compounds showed no potency.

The compounds whose N–H at N‐1 position and hydroxyl group at

C‐6 position were substituted with para‐methoxybenzyl (PMB) showed

a significant decrease in activation potency. Compared with the double

PMB‐substituted compound 11c, the PMB‐monosubstituted compound

11c‐2, and the non‐PMB‐substituted compound 12c, the unalkylated O

at the C‐6 position of the pyrazole[3,4‐b]pyridone had less effect on the

enzyme activity and the unsubstituted N–H at N‐1 position was critical

to the enzyme activity.

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2.3

Predicted absorption, distribution,

metabolism, excretion, and toxicity (ADMET) and

physicochemical properties of compounds 12c–e, 12j,

12k, 12m–r, 13j, 13k, and 13m–r

A partial loss of activity was observed when position C‐3 was oc-

cupied by trifluoromethyl as compared with a carboxylic acid. In addi-

tion, the potency profiles of different groups on the benzene ring in

trifluoromethyl derivatives are similar to that of carboxylic derivatives.

ADMET and physicochemical properties of compounds 12c–e,

12j, 12k, 12m–r, 13j, 13k, and 13m–r, which showed good

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TABLE 1 In vitro AMPK activation activity of compounds 11a–s, 12a–s, and 13a–r

Efficacy (%)^a

EC⁵⁰(nM)/AMPKα1β1γ1

Compounds

R

1

R

2

R3

R4

11a

11b

11c

11c‐2

11d

11d‐2

11e

11f

m‐OCH₃

p‐OCH₃

o‐CH₃

m‐CH₃

p‐CH₃

o‐F

CF₃

PMB^a

PMB

H

PMB

H

16

7

ND

1470

2770

700

ND

460

180

11

29

24

26

4

PMB

H

PMB

H

7

11g

11h

11i

m‐F

20

2

p‐F

o‐Cl

8

11j

m‐Cl

4

11k

11l

p‐Cl

11

64

30

3

2,4‐Dichloro

3,5‐Dichloro

o‐OCF₃

m‐OCF₃

p‐OCF₃

o‐CF₃

m‐CF₃

p‐CF₃

m‐OCH₃

p‐OCH₃

o‐CH₃

m‐CH₃

p‐CH₃

o‐F

11m

11n

11o

11p

11q

11r

11s

12a

12b

12c

12d

12e

12f

24

29

32

12

64

17

2

H

108

70

93

52

43

12

40

128

120

H

12g

12h

12i

m‐F

H

p‐F

H

o‐Cl

H

12j

m‐Cl

H

12k

p‐Cl

H

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Efficacy (%)^a

EC⁵⁰(nM)/AMPKα1β1γ1

Compounds

R

1

R

2

R3

R4

12l

2,4‐Dichloro

3,5‐Dichloro

o‐OCF₃

m‐OCF₃

p‐OCF₃

o‐CF₃

CF₃

H

–

H

–

56

ND

12m

12n

12o

12p

12q

12r

CF₃

72

4000

330

16010

270

1380

650

ND

CF₃

77

CF₃

81

CF₃

102

131

117

46

CF₃

m‐CF₃

p‐CF₃

CF₃

12s

CF₃

13a

13b

13c

m‐OCH₃

p‐OCH₃

o‐CH₃

COOH

–

10

ND

12

ND

25

ND

13e

13f

p‐CH₃

10

ND

o‐F

7

ND

13g

13h

13i

m‐F

32

ND

p‐F

45

ND

o‐Cl

60

ND

13j

m‐Cl

132

118

37

20^b

13k

13l

p‐Cl

3240

ND

2,4‐Dichloro

3,5‐Dichloro

o‐OCF₃

m‐OCF₃

p‐OCF₃

m‐CF₃

p‐CF₃

13m

13n

13o

13p

13q

13r

88

2280

370

16820

26430

2

68

122

126

84

61

4740

7

PF‐06409577

–

110.2

Abbreviations: AMPK, adenosine monophosphate‐activated protein kinase ND, not determined; PMB, p‐methoxybenzyl.

^aPercent maximum activation referred to enzyme activity level treated with different compounds at 50 μM.

^bAll experiments were performed in duplicate.

potency for AMPK, were further predicted, and the results are

presented in Table 2. All the compounds obeyed Lipinski's rule of

five, which indicates that these compounds have moderate lipo-

philicity and low molecular weight. Furthermore, the values of

blood–brain barrier (BBB+) and % human intestinal absorption

(HIA+) are in the acceptable range, which demonstrates that they

could be absorbed by human intestine. Last but not least, they

exhibited negative toxicity and negative carcinogenicity test.

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FIGURE 5 Putative binding mode of compounds (a) PF‐06409577, (b) 13q, and (c) 12k bound to AMPKα1β1γ1 (PDB ID: 5KQ5). The blue

line represents the hydrogen‐bond interaction and the purple line represents the H–π interaction. AMPK, adenosine monophosphate‐activated

protein kinase

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2.4

Predicted binding mode of compounds 11q,

be the reason for its reduced activity. The oxadiazole ring of compound

11q (Figure 5d) formed hydrogen‐bond interaction with Lys29 and tri-

fluoromethyl of pyrazole[3,4‐b]pyridone formed H–π interaction with

Asn111. However, it lacked interaction with the key amino acids Lys31

and Asp88, which may be the reason for its loss of activity. In general,

Lys31, Lys29, Asn111, and Asp88 are the key amino acids that bind to

the compound and the increased interaction between the compound and

the pocket leads to a significant improvement in activation potency.

13q, and 12k with AMPK

To figure out the interaction between compounds and AMPKα1β1γ1,

compounds 11q, 13q, and 12r were synthesized and docked into the

active pocket of AMPKα1β1γ1 protein (PDB ID: 5KQ5), respectively

(Figure 5). Compound 13q (Figure 5b) showed the best potency, whose

interaction pattern with the active site of AMPK is similar to that of the

positive control PF‐06409577 (Figure 5a). The carboxyl group of

compound 13q displayed hydrogen‐bond interactions with Lys29 and

Asn111, and the hydroxyl group of pyrazole[3,4‐b]pyridone acted as a

hydrogen‐bond donor forming a hydrogen bond with Asp88. In addi-

tion, the trifluoromethylphenyl group on the side‐chain formed H–π

interaction with Lys31, and the oxadiazole ring formed H–π interaction

with Val113, which may be the reason why compound 13q exhibited

enhanced potency as compared with the positive control. A 2018 study

identified Asn111 as a key binding residue and showed it to re-

duce EC₅₀by threefold,^[56]supporting the modeling. A research in 2013

also identified Lys29, Lys31, and Asp88 as critical drug interaction

residues,^[40]once again supporting the modeling in this study. Compound

12k (Figure 5c) retained the hydrogen‐bond interactions of the carboxyl

group with Lys29 and N–H of pyrazole with Asp88 as well as the H–π

interaction of side‐chain benzene ring and oxadiazole ring with Lys31.

However, it lacked hydrogen‐bond interaction with Asn111, which may

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2.5

Compounds 12k and 13q reduce ROS levels

in NRK‐49F cells

The measurement of ROS level is a method that plays an im-

portant role in revealing the correlation between the occurrence

and development of diabetic nephropathy and oxidative

stress.^[57]On the basis of the results of the AMPK enzyme ex-

periment, compounds 12k and 13q were used for the detection of

ROS levels in rat renal fibroblast cell line (NRK‐49F) under high‐

glucose conditions. As shown in Figure 6, under the stimulation

of TGF‐β, the intensity of green fluorescence emitted by

dichloro‐fluorescein diacetate (DCFH‐DA) fluorescent probe in-

creased, which indicated the increase of the intracellular ROS

level. Both compounds can reduce the production of ROS in a

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FIGURE 6 Compounds 12k and 13q reduce ROS levels induced by TGF‐β in NRK‐49F cells. (a) Representative photomicrographs of

fluorescence in NRK‐49F cells treated with 12k or 13q. NRK‐49F cells were treated with 3 or 10 μM 12k/13q under TGF‐β induction in the presence

or absence of 0.1 μM compound C, respectively. The ROS level was measured by DCFH‐DA fluorescence. Dimethyl sulfoxide was used as a control.

(b) A bar graph of ROS level assay for 12k. (c) A bar graph of ROS level assay for 13q. Values shown are means SD. DCFH‐DA, dichloro‐fluorescein

diacetate; DMSO, dimethyl sulfoxide; ROS, reactive oxygen species; TGF‐β, transforming growth factor‐β. *p < .05 and **p < .01

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Compounds 12k and 13q induce cell

apoptosis in the NRK‐49F cell line

dose‐dependent manner; the potency of 12k is slightly weaker

than that of 13q, and the potency of 12k at 3 μM is equivalent to

that of 13q at 10 μM. When compound C (an AMPK inhibitor) was

added, the ability of 12k and 13q to reduce ROS levels was lost,

suggesting that 12k and 13q may reduce ROS levels induced by

TGF‐β in NRK‐49F cells through activating AMPK.

2.6

Phosphatidylserine (PS) on the cell membrane of NRK‐49F evagi-

nated and combined with annexin V to be stained green after

treatment with compounds 12k (Figure 7b) and 13q (Figure 7c) at

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cells and induced early apoptosis of NRK‐49F cells. In summary, our

study developed a novel series of AMPKα1β1γ1 direct activators, pro-

viding novel lead compounds for the subsequent development of

compounds with a promising therapeutic potential against DN.

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4

EXPERIMENTAL

Chemistry

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4.1

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4.1.1

General

All chemicals, reagents, and solvents were purchased from commercial

sources and used without further purification. All reactions except

those in aqueous media were carried out with the use of standard

techniques for the exclusion of moisture. A NOVA‐2S upper microwave

reactor (PreeKem; Shanghai Yiyao Technology Co., Ltd.) was used for

microwave reaction. Flash chromatography was performed by using

silica gel (200–300 mesh and 80–100 mesh). All reactions were mon-

itored by thin‐layer chromatography with 0.25 mm precoated silica gel

plates (60GF‐254) and visualized with ultraviolet light at 254 nm or

365 nm. Melting points (MP) were determined with a WRS‐2 Shen

Guang melting‐point apparatus. ¹H NMR (nuclear magnetic re-

sonance) and ¹³C NMR spectra (see the Supporting Information) were

recorded on a Bruker Advance III 500 MHz spectrometer or a Bruker

Advance III 400 MHz spectrometer. Coupling constants (J) were ex-

pressed in hertz (Hz) and chemical shifts (δ) of NMR were reported in

parts permillion (ppm) units, relative to internal control (tetra-

methylsilane). The purity of compounds was determined by Shimadzu

SPD‐20A LC solution and analyzed to be over 95% (column: ZORBAX

Extend‐C18, 5.0 μm, 4.6 × 250 mm (Agilent); flow rate: 1.0 ml/min;

mobile phase: A: MeOH, B: H₂O).

FIGURE 7 Compounds 12k and 13q induce cell apoptosis in

NRK‐49F cells. (a) Representative photomicrographs of fluorescence

in NRK‐49F cells treated with DMSO, which was used as a control.

(b) Representative photomicrographs of fluorescence in NRK‐49F

cells treated with 13q. NRK‐49F cells were treated with 10 μM 13q

under TGF‐β induction in the presence or absence of 0.1 μM

compound C, respectively. The result of apoptosis was measured by

annexin V‐ FITC/PI fluorescence. (c) Representative

photomicrographs of fluorescence in NRK‐49F cells treated with

12k. NRK‐49F cells were treated with 10 μM 12k under TGF‐β

induction in the presence or absence of 0.1 μM compound C,

respectively. The result of apoptosis was measured by annexin

V‐FITC/PI fluorescence. DMSO, dimethyl sulfoxide; FITC, fluorescein

isothiocyanate; PI, propidium iodide; TGF‐β, transforming growth

factor‐β

The InChI codes of the investigated compounds, together with

some biological activity data, are provided as Supporting Information.

10 μM, suggesting that compounds 12k and 13q could promote the

early apoptosis of fibroblasts at 10 μM. Also, no cell nucleus staining

to red was detected, indicating that the cells did not undergo ad-

vanced apoptosis. When compound C was added, the green signal

decreased, indicating that the compound's proapoptotic effect was

reduced. This result revealed that 12k and 13q may induce cell

apoptosis by TGF‐β in NRK‐49F cells through AMPK activation.

|

4.1.2

General procedure for the synthesis of

compounds 2a–s

A solution of 1 (1.33 g, 0.01 mol), hydroxylamine hydrochloride

(2.08 g, 0.03 mol), N,N‐diisopropylethylamine (3.88 g, 0.03 mol), and

ethanol (25 ml) was heated to reflux for 4 h and then cooled to

ambient temperature. The mixture was concentrated under vacuum,

followed by addition of water (20 ml), and it was then filtered to give

a crude residue, which was subjected to chromatography on a col-

umn of SiO₂eluted with petroleum ether/EtOAc 5:1 to afford 2a–s.

|

3

CONCLUSIONS

In this study, a series of 1,2,4‐oxadiazole‐containing pyrazolo[3,4‐b]

pyridinone derivatives was designed, synthesized, and evaluated as

AMPKα1β1γ1 activators. Enzymatic assay demonstrated that

compound 12k in which R₂was substituted by trifluoromethyl and

compound 13q in which R₂was substituted by carboxyl group showed

potent enzyme activation. Further mechanistic studies indicated that

they both reduced ROS levels stimulated by TGF‐β in NRK‐49F

1‐(4‐Methoxybenzyl)‐3‐(trifluoromethyl)‐1H‐pyrazol‐5‐ol (5)

A

mixture of ethyl trifluoroacetoacetate (39.0 g, 0.21 mol), (4‐

methoxyphenyl)hydrazine hydrochloride (40.00 g, 0.21 mol), and ethanol

(50 ml) was heated to reflux for 8 h. The reaction mixture was cooled to

room temperature, concentrated under vacuum, and poured into a mixed

solution of petroleum ether and ethyl acetate (100 ml, petroleum ether/

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XIAO ET AL.

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EtOAc 4:1). It was then filtered and concentrated to give a crude residue,

which was recrystallized with ethyl acetate and subjected to chromato-

graphy on a column of SiO₂eluted with petroleum ether/EtOAc 8:1 to

afford the product (white solid, yield: 72.5%). MP: 188.3–190.6°C. ¹H

NMR (500 MHz, DMSO‐d₆) δ 11.83 (s, 1H), 7.17 (d, J = 8.8, 2.5 Hz, 2H),

6.91 (d, 2H), 5.76 (s, 1H), 5.08 (s, 2H), and 3.73 (s, 3H). ¹³C NMR

(125 MHz, DMSO‐d₆) δ 158.74, 152.85, 138.56, 128.92, 128.69, 122.55,

120.42, 113.96, 84.50, 55.07, and 49.49. High‐resolution mass spectro-

metry (HRMS) (electrospray ionization [ESI]) m/z calcd for

[C₁₂H₁₁F₃N₂O₂–H]^–271.0700; found: 271.0745.

114.50, 102.40, 55.53, and 49.92. HRMS (ESI) m/z calcd for

[C₁₃H₁₂F₃N₃O₂+H]⁺300.0954; found: 300.0955.

Ethyl 1‐(4‐methoxybenzyl)‐6‐oxo‐3‐(trifluoromethyl)‐6,7‐dihydro‐

1H‐pyrazolo[3,4‐b]pyridine‐5‐carboxylate (8)

A mixture of 7 (6.00 g, 0.020 mol), diethyl malonate (9.60 g, 0.060 mol),

piperidine (3.90 g, 0.060 mol), and absolute ethanol (24 ml) was heated

to reflux for 10 h. The reaction solution was concentrated under va-

cuum to give an oily liquid. Water was added to the oily liquid to

remove piperidine. The mixture was extracted with ethyl acetate

(3 × 100 ml). The combined organic layers were dried over sodium

sulfate, filtered, and concentrated. The resulting solution was purified

by column chromatography (petroleum ether/EtOAc 5:1, then by

CH₂Cl₂/CH₃OH 50:1) to afford 8 (white solid, yield: 73.3%), MP:

124.2–125.9°C. ¹H NMR (500 MHz, chloroform‐d) δ 12.06 (s, 1H), 8.60

(s, 1H), 7.29 (d, 2H), 6.73 (d, 2H), 5.46 (s, 2H), 4.41 (q, 2H), 3.66 (s, 3H),

and 1.37 (t, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 169.14, 164.79,

159.42, 150.82, 135.30, 134.83, 129.85, 127.49, 119.77, 113.95,

107.30, 106.07, 62.73, 61.53, 55.10, 50.71, and 14.02. HRMS (ESI) m/z

calcd for [C₁₈H₁₆F₃N₃O₄+H]⁺396.1166; found: 396.1201.

5‐Chloro‐1‐(4‐methoxybenzyl)‐3‐(trifluoromethyl)‐1H‐pyrazole‐4‐

carbaldehyde (6)

POCl₃(45.00 g, 0.29 mol) was cautiously added to dimethylforma-

mide (DMF) (17.36 g, 0.23 mol) under an ice bath and stirred for

10 min. The resulting mixture was heated to reflux for 4 h after 5

(20.00 g, 74 mmol) was added. The mixture was cooled to room

temperature, poured into 400 ml of ice water, and extracted with

ethyl acetate (3 × 100 ml). The combined organic layers were alka-

lized to pH 8 by addition of saturated NaHCO₃aqueous solution

(50 ml) and separated. The aqueous layer was further extracted with

ethyl acetate (50 ml). The combined organic layers were dried over

sodium sulfate, filtered, and concentrated to produce oil. The oil was

passed through a pad of silica gel eluted with petroleum ether/EtOAc

10:1. Product fractions were concentrated to afford 6 (yellow oil,

yield: 40%). ¹H NMR (500 MHz, DMSO‐d₆) δ 9.82 (s, 1H), 7.26

(d, J = 8.0 Hz, 2H), 6.94 (d, J = 7.8 Hz, 2H), 5.47 (s, 2H), and 3.74 (s, 3H).

¹³C NMR (125 MHz, DMSO‐d₆) δ 181.94, 159.78, 140.00, 134.86,

129.91, 126.63, 120.54, 114.74, 55.60, and 53.19. HRMS (ESI) m/z calcd

for [C₁₃H₁₀ClF₃N₂O₂+H]⁺319.0456; found: 319.0467.

Ethyl 1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridine‐5‐carboxylate (9)

A mixture of 8 (6.00 g, 0.015 mol), p‐methoxybenzyl chloride (3.00 g,

0.019 mol), cesium carbonate (6.4 g, 0.020 mol), and DMF solution

(30 ml) was stirred at room temperature for 3 days. The reaction

solution was poured into water (90 ml), stirred for 30 min, and fil-

tered to give yellow solid. The yellow solid was recrystallized with

ethanol to afford 9 (white solid, yield: 75%). MP: 154.6–156.7°C. ¹H

NMR (500 MHz, DMSO‐d₆) δ 8.53 (s, 1H), 7.46 (d, J = 8.5 Hz, 2H),

7.28 (d, J = 8.5 Hz, 2H), 6.92 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H),

5.61 (s, 2H), 5.56 (s, 2H), 4.29 (q, J = 7.1 Hz, 2H), 3.75 (s, 3H), 3.71 (s,

3H), and 1.28 (t, J = 7.1 Hz, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ

164.39, 161.65, 159.48, 149.16, 134.56, 133.29, 130.09, 129.95,

128.68, 128.34, 122.60, 114.52, 114.22, 114.18, 113.98, 106.41,

68.66, 61.68, 55.52, 50.95, and 14.46. HRMS (ESI) m/z calcd for

[C₂₆H₂₄F₃N₃O₅+H]⁺516.1741; found: 516.1738.

5‐Amino‐1‐(4‐methoxybenzyl)‐3‐(trifluoromethyl)‐1H‐pyrazole‐4‐

carbaldehyde (7)

A mixture of 6 (10.00 g, 0.031 mol), NaN₃(2.5 g, 0.038 mol), TBAI

(2.37 g, 0.0064 mol), and DMSO solution (25 ml) was stirred at room

temperature for 15 min and poured into ice water (200 ml). The re-

sulting mixture was extracted with ethyl acetate (2 × 100 ml). The

combined organic layers were dried over sodium sulfate, filtered, and

concentrated to produce oil. The oil was purified by column chro-

matography (petroleum ether/EtOAc 15:1) to afford a yellow oil

(yield: 85%). The product was unstable and directly used for the next

1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐(trifluoromethyl)‐

1H‐pyrazolo[3,4‐b]pyridine‐5‐carboxylic acid (10)

A solution of 9 (8.00 g, 20 mmol), NaOH (0.40 g, 10 mmol), ethanol

(2.5 ml), and water (7.5 ml) was heated to reflux for 4 h. The resulting

mixture was cooled to room temperature and was acidified to pH ~2

by addition of hydrochloric acid solution (80 ml, 1 mol/l). The mixture

was filtered to afford 10 (white solid, yield: 90.5%). MP:

168.4–170.1°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 13.22 (s, 1H), 8.52

(s, 1H), 7.45 (d, J = 8.6 Hz, 2H), 7.27 (d, J = 8.6 Hz, 2H), 6.92

(d, J = 8.6 Hz, 2H), 6.87 (d, J = 8.7 Hz, 2H), 5.61 (s, 2H), 5.57 (s, 2H),

3.75 (s, 3H), and 3.71 (s, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ

165.89, 161.80, 159.45, 149.09, 133.34, 132.88, 130.06, 129.94,

129.80, 128.85, 128.39, 121.58, 115.18, 114.52, 114.25, 106.45,

68.53, 55.52, and 50.95. HRMS (ESI) m/z calcd for

[C₂₄H₂₀F₃N₃O₅+Na]⁺510.1247; found: 510.1252.

step. To

a solution of the above‐mentioned product (6.00 g,

0.018 mol), ethyl acetate (70 ml), and saturated NH₄Cl aqueous

(4.94 g, 0.092 mol), iron powder (3.1 g, 0.055 mol) was added. The

resulting solution was stirred at room temperature for 3 days and

filtered through celite. The filtrate was extracted with ethyl acetate

(3 × 100 ml). The combined organic layers were neutralized with sa-

turated aqueous NaHCO₃and separated. The organic layers were

dried over sodium sulfate, filtered, and concentrated to afford 7

(white solid, yield: 87.1%). MP: 137.4–139.3°C. ¹H NMR (400 MHz,

DMSO‐d₆) δ 9.64 (s, 1H), 7.35 (s, 1H), 7.19 (d, J = 8.6 Hz, 1H), 6.92

(d, J = 8.6 Hz, 1H), 5.18 (s, 1H), and 3.72 (s, 2H). ¹³C NMR (100 MHz,

DMSO‐d₆) δ 182.01, 159.34, 151.28, 139.50, 129.47, 127.96, 121.26,

XIAO ET AL.

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5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

69.08, 55.27, 51.37, and 22.13. HRMS (ESI) m/z calcd for

[C₃₂H₂₆F₃N₅O₄+H]⁺602.2010; found: 602.1985.

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐(3‐

methoxyphenyl)‐1,2,4‐oxadiazole (11a)

A mixture of 10 (1.00 g, 2.1 mmol), EDCI (0.43 g, 2.3 mmol), HOBt

(0.38 g, 2.8 mmol), and DMF solution (20 ml) was stirred at room tem-

perature for 30 min to give a white precipitate. To the mixture, 2a

(0.34 g, 2.1 mmol) was added, which was then stirred at room tem-

perature for 5 days. The resulting solution was poured into water

(50 ml) and extracted with ethyl acetate (3 × 50 ml). The combined or-

ganic layers were dried over sodium sulfate, filtered, and concentrated.

The solution was recrystallized with methanol to give a crude residue.

The crude residue was purified by column chromatography (petroleum

ether/EtOAc 3:1) to afford 11a (white solid, yield: 41.0%). MP:

186.7–188.5°C. ¹H NMR (500 MHz, chloroform‐d) δ 8.95 (s, 1H), 8.03

(s, 1H), 8.01 (d, J = 7.7 Hz, 1H), 7.57 (d, J = 7.8 Hz, 2H), 7.43 (t, J = 7.5 Hz,

1H), 7.37 (d, J = 7.5 Hz, 3H), 6.95 (d, J = 7.8 Hz, 2H), 6.88 (d, J = 7.8 Hz,

2H), 5.68 (s, 2H), 5.63 (s, 2H), 3.84 (s, 3H), 3.81 (s, 3H), and 2.48 (s, 3H).

¹³C NMR (125 MHz, chloroform‐d) δ 173.54, 168.68, 161.04, 159.63,

149.61, 138.70, 134.89, 134.74, 132.05, 129.76, 129.33, 128.80,

128.11, 127.60, 126.67, 124.67, 122.09, 114.18, 114.00, 107.43,

106.99, 69.09, 55.27, 51.38, and 21.38. HRMS (ESI) m/z calcd for

[C₃₂H₂₆F₃N₅O₅+H]⁺618.1959; found 618.1934.

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐(m‐tolyl)‐1,2,4‐

oxadiazole (11d)

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2d as the starting mate-

rial (white solid, yield: 62.1%). MP: 154.5–157.0°C. ¹H NMR

(500 MHz, chloroform‐d) δ 8.94 (s, 1H), 7.80 (d, J = 7.6 Hz, 1H),

7.73 (s, 1H), 7.57 (d, J = 8.4 Hz, 2H), 7.44 (t, J = 8.0 Hz, 1H), 7.37

(d, J = 8.4 Hz, 2H), 7.10 (dd, J = 8.2, 2.2 Hz, 2H), 6.95 (d, J = 8.4 Hz,

2H), 6.88 (d, J = 8.4 Hz, 2H), 5.68 (s, 2H), 5.62 (s, 2H), 3.92 (s, 3H),

3.84 (s, 3H), and 3.81 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ

173.60, 168.52, 161.04, 159.97, 159.65, 159.54, 149.62, 134.89,

133.34, 129.99, 129.76, 129.35, 128.11, 128.01, 127.60, 121.58,

119.96, 117.70, 113.97, 112.23, 107.43, 106.92, 69.10, 55.48,

55.26, 51.38, and 29.70. HRMS (ESI) m/z calcd for

[C₃₂H₂₆F₃N₅O₄+H]⁺602.2010; found: 602.1985.

5‐(1‐{4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐(p‐tolyl)‐1,2,4‐

oxadiazole (11e)

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2e as the starting mate-

rial (white solid, yield: 53.5%). MP: 178.6–180.9°C. ¹H NMR

(500 MHz, chloroform‐d) δ 8.92 (s, 1H), 8.07 (d, J = 7.6 Hz, 2H),

7.54 (d, J = 8.0 Hz, 2H), 7.33 (dd, J = 12.9, 8.1 Hz, 4H), 6.92

(d, J = 8.0 Hz, 2H), 6.85 (d, J = 8.0 Hz, 2H), 3.82 (s, 3H), 3.78 (s, 3H),

and 2.44 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.46,

168.57, 161.04, 159.63, 159.51, 149.60, 141.60, 135.04, 134.86,

129.76, 129.59, 129.32, 128.16, 127.62, 127.48, 124.00, 119.96,

114.00, 107.42, 107.04, 69.06, 55.27, 51.37, and 21.59. HRMS

(ESI) m/z calcd for [C₃₂H₂₆F₃N₅O₄+H]⁺602.2010; found:

602.1981.

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐(4‐

methoxyphenyl)‐1,2,4‐oxadiazole (11b)

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2b as the starting material (white

solid, yield: 62.1%). MP: 142.6–144.3°C. ¹H NMR (500 MHz,

chloroform‐d)

δ 8.93 (s, 1H), 8.15 (d, J = 7.9 Hz, 2H), 7.56

(d, J = 7.8 Hz, 2H), 7.37 (d, J = 7.8 Hz, 2H), 7.05 (d, J = 7.9 Hz, 2H), 6.94

(d, J = 7.7 Hz, 2H), 6.87 (d, J = 7.7 Hz, 2H), 5.68 (s, 2H), 5.62 (s, 2H),

3.92 (s, 3H), 3.84 (s, 3H), and 3.81 (s, 3H). ¹³C NMR (125 MHz,

chloroform‐d) δ 172.54, 167.00, 162.85, 161.08, 159.59, 159.47,

158.18, 149.59, 134.94, 134.67, 132.44, 131.50, 129.77, 129.47,

128.15, 127.63, 122.35, 120.73, 115.75, 114.15, 113.97, 111.68,

107.40, 107.06, 69.05, 56.05, 55.28, and 51.35. HRMS (ESI) m/z calcd

for [C₃₂H₂₆F₃N₅O₅+H]⁺618.1959; found: 618.1963.

3‐(2‐Fluorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)‐

oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐1,2,4‐

oxadiazole (11f)

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐(o‐tolyl)‐1,2,4‐

oxadiazole (11c)

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2f as the starting ma-

terial (white solid, yield: 60.3%). MP: 141.8–143.5°C. ¹H NMR

(400 MHz, chloroform‐d) δ 8.92 (s, 1H), 8.19 (td, J = 7.6, 1.8 Hz,

1H), 7.57–7.50 (m, 3H), 7.35 (d, J = 8.5 Hz, 2H), 7.33–7.28 (m, 1H),

7.30–7.23 (m, 1H), 5.66 (s, 2H), 5.60 (s, 2H), 3.82 (s, 3H), and 3.78

(s, 3H). ¹³C NMR (100 MHz, chloroform‐d) δ 173.31, 165.55,

165.42, 162.24, 162.19, 161.05, 159.61, 159.52, 149.72, 135.04,

134.69, 132.89, 132.81, 130.89, 129.78, 129.45, 128.04, 127.57,

124.51, 124.49, 122.31, 119.69, 116.90, 116.59, 115.19, 114.98,

114.17, 114.00, 107.34, 106.73, 69.14, 55.29, and 51.39. HRMS

(ESI) m/z calcd for [C₃₁H₂₃F₄N₅O₄+H]⁺606.1759; found:

606.1685.

The title compound was prepared in a manner similar to the proce-

dures described in 12a by utilizing 2c as the starting material (white

solid, yield: 62.1%). MP: 137.6–139.8°C. ¹H NMR (500 MHz,

chloroform‐d)

δ 8.91 (s, 1H), 8.11 (d, J = 7.8 Hz, 1H), 7.54

(d, J = 8.1 Hz, 2H), 7.44–7.38 (m, 2H), 7.34 (d, J = 7.7 Hz, 5H), 6.92

(d, J = 8.1 Hz, 2H), 6.86 (d, J = 8.1 Hz, 2H), 5.67 (s, 2H), 5.60 (s, 2H),

3.82 (s, 3H), 3.79 (s, 3H), and 2.69 (s, 3H). ¹³C NMR (125 MHz,

chloroform‐d) δ 172.59, 169.20, 161.05, 159.64, 159.55, 149.60,

138.32, 135.04, 133.34, 131.41, 130.64, 130.20, 129.74, 129.45,

128.19, 127.62, 125.99, 121.58, 114.19, 114.00, 107.44, 107.00,

14 of 22

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3‐(3‐Fluorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)‐

oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐1,2,4‐

oxadiazole (11g)

54.6%). MP: 142.7–144.5°C. ¹H NMR (500 MHz, chloroform‐d) δ 8.90

(s, 1H), 8.18 (s, 1H), 8.06 (d, J = 7.6 Hz, 1H), 7.54 (d, J = 7.9 Hz, 2H), 7.49

(d, J = 7.9 Hz, 1H), 7.44 (t, J = 7.8 Hz, 1H), 7.35 (d, J = 7.9 Hz, 2H), 6.93

(d, J = 7.7 Hz, 2H), 6.85 (d, J = 7.8 Hz, 2H), 5.65 (s, 2H), 5.60 (s, 2H), 3.82

(s, 3H), and 3.78 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.83,

167.61, 160.99, 159.64, 159.55, 149.65, 134.99, 134.92, 134.78, 131.30,

130.22, 129.77, 129.28, 128.56, 128.02, 127.67, 127.54, 125.57, 122.06,

119.92, 114.18, 114.02, 107.43, 106.65, 69.16, 55.27, and 51.39. HRMS

(ESI) m/z calcd for [C₃₁H₂₃ClF₃N₅O₄+H]⁺622.1463; found: 622.1423.

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2g as the starting material (white

solid, yield: 53.5%). MP: 151.3–154.1°C. ¹H NMR (500 MHz,

chloroform‐d)

δ 8.94 (s, 1H), 8.00 (d, J = 7.7 Hz, 1H), 7.92

(d, J = 9.3 Hz, 1H), 7.57 (d, J = 7.7 Hz, 2H), 7.52 (q, J = 7.1 Hz, 1H), 7.37

(d, J = 7.7 Hz, 2H), 7.26 (t, J = 8.3 Hz, 1H), 6.96 (d, J = 7.5 Hz, 2H), 6.88

(d, J = 7.6 Hz, 2H), 5.68 (s, 2H), 5.63 (s, 2H), 3.85 (s, 3H), and 3.81

(s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.83, 167.77, 163.93,

161.97, 161.01, 159.64, 149.65, 135.11, 134.94, 130.64, 130.58,

129.77, 129.35, 128.92, 128.85, 128.05, 127.56, 123.23, 119.92,

118.16, 114.76, 114.57, 114.18, 114.01, 107.44, 106.71, 69.15, 55.28,

and 51.40. HRMS (ESI) m/z calcd for [C₃₁H₂₃F₄N₅O₄+Na]⁺628.1578;

found: 628.1560.

3‐(4‐Chlorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)‐

oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐1,2,4‐

oxadiazole (11k)

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2k as the starting ma-

terial (white solid, yield: 57.2%). MP: 157.9–158.6°C. ¹H NMR

(500 MHz, chloroform‐d) δ 8.91 (s, 1H), 8.12 (d, J = 7.9 Hz, 2H),

7.53 (d, J = 7.9 Hz, 2H), 7.48 (d, J = 7.9 Hz, 2H), 7.35 (d, J = 7.9 Hz,

2H), 6.92 (d, J = 7.9 Hz, 2H), 6.86 (d, J = 7.9 Hz, 2H), 5.66 (s, 2H),

5.60 (s, 2H), 3.82 (s, 3H), and 3.78 (s, 3H). ¹³C NMR (125 MHz,

chloroform‐d) δ 173.83, 167.76, 161.01, 159.65, 159.56, 149.64,

137.43, 134.94, 134.77, 129.77, 129.36, 129.21, 128.85, 128.07,

127.56, 125.33, 119.94, 114.19, 114.00, 107.44, 106.75,

69.12, 55.27, and 51.39. HRMS (ESI) m/z calcd for

[C₃₁H₂₃ClF₃N₅O₄+H]⁺622.1463; found: 622.1432.

3‐(3‐Fluorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)‐

oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐1,2,4‐

oxadiazole (11h)

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2h as the starting material (white

solid, yield: 49.7%). MP: 140.1–141.1°C. ¹H NMR (500 MHz,

chloroform‐d) δ 8.91 (s, 1H), 8.19 (dd, J = 8.8, 5.4 Hz, 2H), 7.53

(d, J = 8.6 Hz, 2H), 7.35 (d, J = 8.6 Hz, 2H), 7.21 (t, J = 8.6 Hz, 2H), 6.92

(d, J = 8.6 Hz, 2H), 6.85 (d, J = 8.7 Hz, 2H), 5.66 (s, 2H), 5.60 (s, 2H), 3.82

(s, 3H), and 3.79 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.75,

167.75, 165.66, 163.66, 161.02, 159.65, 159.56, 149.63, 135.08,

134.94, 129.76, 129.68, 129.37, 128.10, 127.57, 123.06, 119.53,

116.17, 115.99, 114.18, 114.00, 107.43, 106.83, 69.11, 55.27, and

51.39. HRMS (ESI) m/z calcd for [C₃₁H₂₃F₄N₅O₄+H]⁺606.1759;

found: 606.1730.

3‐(2,4‐Dichlorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐

methoxybenzyl)oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐

5‐yl}‐1,2,4‐oxadiazole (11l)

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2l as the starting material (white

solid, yield: 47.4%). MP: 164.5–165.7°C. ¹H NMR (500 MHz,

chloroform‐d) δ 8.88 (s, 1H), 8.00 (d, J = 8.4 Hz, 1H), 7.58 (s, 1H), 7.50

(d, J = 8.1 Hz, 3H), 7.40 (d, J = 8.5 Hz, 2H), 7.33 (d, J = 8.2 Hz, 3H), 6.89

(d, J = 8.1 Hz, 3H), 6.84 (d, J = 8.2 Hz, 3H), 5.64 (s, 2H), 5.59 (s, 2H),

3.81 (s, 3H), and 3.77 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ

173.32, 166.66, 161.02, 159.57, 149.67, 137.37, 135.68, 135.06,

134.37, 132.63, 129.74, 129.53, 128.01, 127.53, 127.38, 124.65,

121.08, 114.18, 114.00, 107.43, 106.55, 69.17, 55.27, and 51.39.

HRMS (ESI) m/z calcd for [C₃₁H₂₂Cl₂F₃N₅O₄+H]⁺656.1074; found:

656.1064.

3‐(2‐Chlorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)‐

oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐1,2,4‐

oxadiazole (11i)

The title compound was prepared in a manner similar to the procedures

described in 11a by utilizing 2i as the starting material (white solid,

yield: 46.3%). MP: 146.4–148.1°C. ¹H NMR (500 MHz, chloroform‐d) δ

8.91 (s, 1H), 8.03 (d, J = 7.5 Hz, 1H), 7.57 (d, J = 7.8 Hz, 1H), 7.53

(d, J = 7.8 Hz, 2H), 7.46 (t, J = 7.6 Hz, 1H), 7.42 (t, J = 7.5 Hz, 1H), 7.34

(d, J = 7.9 Hz, 2H), 6.91 (d, J = 7.7 Hz, 2H), 5.66 (s, 2H), and 5.60 (s, 2H).

¹³C NMR (125 MHz, chloroform‐d) δ 173.23, 167.38, 161.05, 159.64,

159.55, 149.66, 135.06, 133.58, 131.84, 131.76, 130.99, 129.74,

129.50, 128.09, 127.57, 126.91, 126.12, 119.78, 114.18, 114.01,

107.43, 106.76, 69.13, 55.27, and 51.39. HRMS (ESI) m/z calcd for

[C₃₁H₂₃ClF₃N₅O₄+Na]⁺644.1283; found: 644.1255.

3‐(3,5‐Dichlorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐

methoxybenzyl)oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐

5‐yl}‐1,2,4‐oxadiazole (11m)

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2m as the starting material (white

solid, yield: 38.5%). MP: 168.4–170.8°C. ¹H NMR (500 MHz,

chloroform‐d) δ 8.91 (s, 1H), 8.07 (s, 2H), 7.55 (d, J = 8.0 Hz, 2H), 7.50

(s, 1H), 7.36 (d, J = 8.0 Hz, 2H), 6.95 (d, J = 8.0 Hz, 2H), 6.86

(d, J = 8.0 Hz, 2H), 5.65 (s, 2H), 5.61 (s, 2H), 3.83 (s, 3H), and 3.78

(s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 174.04, 166.76, 160.97,

159.67, 159.60, 149.69, 135.74, 134.95, 134.85, 131.13, 129.79,

3‐(3‐Chlorophenyl)‐5‐{1‐(4‐methoxybenzyl)‐6‐[(4‐methoxybenzyl)‐

oxy]‐3‐(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐1,2,4‐

oxadiazole (11j)

The title compound was prepared in a manner similar to the procedures

described in 11a by utilizing 2j as the starting material (white solid, yield:

XIAO ET AL.

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129.67, 129.23, 127.93, 127.51, 125.88, 119.91, 114.20, 114.05,

107.45, 106.39, 69.24, 55.29, and 51.42. HRMS (ESI) m/z calcd for

[C₃₁H₂₂Cl₂F₃N₅O₄+H]⁺656.1074; found: 656.1119.

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐[2‐

(trifluoromethyl)phenyl]‐1,2,4‐oxadiazole (11q)

The title compound was prepared in a manner similar to the procedures

described in 11a by utilizing 2q as the starting material (white solid,

yield: 23.5%). MP: 197.1–199.0°C. ¹H NMR (500 MHz, chloroform‐d) δ

8.91 (s, 1H), 7.88 (d, J = 7.1 Hz, 2H), 7.69 (p, J = 7.5 Hz, 2H), 7.52

(d, J = 7.9 Hz, 2H), 7.33 (d, J = 7.9 Hz, 2H), 5.67 (s, 2H), and 5.60 (s, 2H).

¹³C NMR (125 MHz, chloroform‐d) δ 173.65, 167.72, 161.04, 159.63,

159.54, 149.68, 135.14, 134.82, 131.92, 131.87, 130.78, 129.71,

129.51, 129.16, 128.10, 127.58, 126.98, 126.94, 125.69, 124.54,

122.03, 114.18, 113.94, 107.46, 106.64, 69.11, 55.26, and 51.38. HRMS

(ESI) m/z calcd for [C₃₂H₂₃F₆N₅O₄+Na]⁺678.1546; found: 678.1523.

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐[2‐

(trifluoromethoxy)phenyl]‐1,2,4‐oxadiazole (11n)

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2n as the starting ma-

terial (white solid, yield: 42.7%). MP: 129.1–132.2°C. ¹H NMR

(500 MHz, chloroform‐d) δ 8.92 (s, 1H), 8.22 (d, J = 7.7 Hz, 1H),

7.58 (t, J = 7.8 Hz, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.48 (t, J = 8.1 Hz,

2H), 7.33 (d, J = 7.7 Hz, 2H), 6.91 (d, J = 7.7 Hz, 2H), 6.85

(d, J = 7.7 Hz, 2H), 5.67 (s, 2H), 5.60 (s, 2H), 3.82 (s, 3H), and 3.79 (s,

3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.28, 165.82, 161.02,

159.64, 159.54, 149.65, 147.18, 135.05, 134.80, 132.27, 131.45,

129.73, 129.45, 128.16, 127.58, 127.23, 123.61, 122.15, 121.04,

119.92, 114.18, 113.99, 107.47, 106.73, 77.27, 69.07, 55.26, and

51.37. HRMS (ESI) m/z calcd for [C₃₂H₂₃F₆N₅O₅+H]⁺672.1676; found:

672.1684.

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐[3‐

(trifluoromethyl)phenyl]‐1,2,4‐oxadiazole (11r)

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2r as the starting

material (white solid, yield: 42.7%). MP: 128.9–130.2°C. ¹H NMR

(500 MHz, chloroform‐d)

δ 8.93 (s, 1H), 8.47 (s, 1H), 8.38

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(d, J = 7.7 Hz, 1H), 7.80 (d, J = 7.7 Hz, 1H), 7.66 (t, J = 7.8 Hz, 1H),

7.54 (d, J = 7.9 Hz, 2H), 7.36 (d, J = 7.9 Hz, 2H), 6.93 (d, J = 7.8 Hz,

2H), 6.86 (d, J = 7.8 Hz, 2H), 5.67 (s, 2H), 5.61 (s, 2H), 3.83 (s, 3H),

and 3.79 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 174.07,

167.60, 161.02, 159.66, 159.60, 149.68, 135.03, 134.83, 131.71,

131.41, 130.66, 129.78, 129.49, 129.34, 127.97, 127.85, 127.78,

127.54, 124.53, 122.70, 122.06, 114.03, 107.46, 106.60, 69.21,

55.27, and 51.42. HRMS (ESI) m/z calcd for [C₃₂H₂₃F₆N₅O₄+-

Na]⁺678.1546; found: 678.1536.

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐[3‐

(trifluoromethoxy)phenyl]‐1,2,4‐oxadiazole (11o)

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2o as the starting material (white

solid, yield: 46.3%). MP: 146.0–148.6°C. ¹H NMR (500 MHz,

chloroform‐d) δ 8.92 (s, 1H), 8.13 (d, J = 7.8 Hz, 1H), 8.06 (s, 1H), 7.55

(dt, J = 7.7, 3.7 Hz, 3H), 7.38 (dd, J = 12.1, 8.2 Hz, 3H), 6.93

(d, J = 8.1 Hz, 2H), 6.87 (d, J = 8.2 Hz, 2H), 5.66 (s, 2H), 5.61 (s, 2H), 3.82

(s, 4H), and 3.79 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.98,

167.52, 161.00, 159.60, 149.66, 134.95, 134.79, 130.45, 129.78,

129.33, 128.87, 128.01, 127.56, 125.83, 123.58, 121.53, 120.20,

119.95, 119.48, 114.19, 114.01, 107.44, 106.63, 69.17, 55.24, and

51.39. HRMS (ESI) m/z calcd for [C₃₂H₂₃F₆N₅O₅+H]⁺672.1676; found:

672.1623.

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐[4‐

(trifluoromethyl)phenyl]‐1,2,4‐oxadiazole (11s)

The title compound was prepared in a manner similar to the proce-

dures described in 11a by utilizing 2s as the starting material (white

solid, yield: 52.0%). MP: 160.4–163.1°C. ¹H NMR (400 MHz,

5‐{1‐(4‐Methoxybenzyl)‐6‐[(4‐methoxybenzyl)oxy]‐3‐

chloroform‐d) δ 8.93 (s, 1H), 8.31 (d, J = 8.1 Hz, 3H), 7.78

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐5‐yl}‐3‐[4‐

(d, J = 8.1 Hz, 3H), 7.53 (d, J = 8.5 Hz, 2H), 7.35 (d, J = 8.5 Hz, 2H), 6.92

(d, J = 8.5 Hz, 2H), 6.86 (d, J = 8.5 Hz, 3H), 5.67 (s, 2H), 5.61 (s, 2H),

3.83 (s, 3H), and 3.79 (s, 3H). ¹³C NMR (100 MHz, chloroform‐d) δ

172.54, 167.00, 162.85, 161.08, 159.59, 159.47, 158.18, 149.59,

134.94, 134.67, 132.44, 131.50, 129.77, 129.47, 128.15, 127.63,

122.35, 120.73, 115.75, 114.15, 113.97, 111.68, 107.40, 107.06,

69.05, 56.05, 55.28, and 51.35. HRMS (ESI) m/z calcd for

[C₃₂H₂₃F₆N₅O₄+Na]⁺678.1546; found: 678.1489.

(trifluoromethoxy)phenyl]‐1,2,4‐oxadiazole (11p)

The title compound was prepared in a manner similar to the

procedures described in 11a by utilizing 2p as the starting ma-

terial (white solid, yield: 46.3%). MP: 158.6–160.9°C. ¹H NMR

(500 MHz, chloroform‐d) δ 8.94 (s, 1H), 8.25 (d, J = 7.8 Hz, 2H),

7.56 (d, J = 8.1 Hz, 2H), 7.42–7.33 (m, 6H), 6.94 (d, J = 7.8 Hz, 2H),

6.88 (d, J = 8.0 Hz, 2H), 5.68 (s, 2H), 5.63 (s, 2H), 3.85 (s, 3H), and

3.81 (s, 3H). ¹³C NMR (125 MHz, chloroform‐d) δ 173.93, 167.53,

161.01, 159.65, 159.56, 151.35, 149.64, 135.40, 135.09, 134.98,

134.78, 134.46, 129.39, 129.29 (q), 125.44, 121.11, 120.93 (q),

114.18, 113.99, 107.44, 106.72, 69.13, 55.27, and 51.39. HRMS

(ESI) m/z calcd for [C₃₂H₂₃F₆N₅O₅+H]⁺672.1676; found:

672.1652.

5‐[3‐(3‐Methoxyphenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐

1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12a)

A mixture of 11a (0.45 g, 0.46 mmol), cerium ammonium nitrate

(3.20 g, 5.83 mmol), 15 ml of acetonitrile, and 3 ml of water was he-

ated to reflux for 20 h. The reactive solution was cooled to room

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temperature. The resulting precipitate was collected by filtration,

washed with 3 ml of solution (acetonitrile/water 5:1), and dried un-

5‐[3‐(2‐Fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo[3,4‐b]pyridin‐6‐ol (12f)

der vacuum to afford 12a (white solid, yield: 40.1%). MP: >300°C. ¹

H

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11f as the starting material (white solid,

yield: 37.3%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 14.35

(s, 1H), 13.45 (s, 1H), 8.73 (s, 1H), 8.13 (td, J = 7.6, 1.6 Hz, 2H), 7.80–7.59

(m, 2H), 7.50–7.45 (m, 2H), and 7.44–7.40 (m, 1H). ¹³C NMR (100 MHz,

DMSO‐d₆) δ 174.21, 164.91, 161.52, 159.04, 136.10, 135.79, 134.06,

131.24, 125.60, 117.37, 117.17, 114.87, 114.71, and 40.37. HRMS (ESI)

m/z calcd for [C₁₅H₇F₄N₅O₂+H]⁺366.0609; found: 366.0611.

NMR (400 MHz, DMSO‐d₆) δ 8.69 (s, 1H), 8.00 (d, J = 8.6 Hz, 2H),

7.09 (d, J = 8.6 Hz, 2H), and 3.83 (s, 3H). ¹³C NMR (100 MHz, DMSO‐

d₆) δ 174.30, 167.62, 162.13, 135.45, 129.23, 118.87, 115.01, and

55.82. HRMS (ESI) m/z calcd for [C₁₆H₁₀F₃N₅O₃+H]⁺378.0809;

found: 378.0811.

5‐[3‐(4‐Methoxyphenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐

1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12b)

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11b as the starting material (white solid,

yield: 43.2%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 14.38

(s, 1H), 13.31 (s, 1H), 8.71 (s, 1H), 8.24–7.71 (m, 4H), 7.57–7.33 (m, 6H),

and 2.41 (s, 3H). ¹³C NMR (100 MHz, DMSO‐d₆) δ 174.49, 167.89,

138.98, 135.60, 132.51, 129.43, 127.83, 126.50, 124.63, 122.77, 119.42,

and 21.28. HRMS (ESI) m/z calcd for [C₁₆H₁₀F₃N₅O₃+H]⁺378.0809;

found: 378.0811.

5‐[3‐(2‐Fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo[3,4‐b]pyridin‐6‐ol (12g)

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11g as the starting material (white solid,

yield: 40.6%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 14.33

(s, 1H), 13.34 (s, 1H), 8.71 (s, 1H), 7.91 (d, J = 7.7 Hz, 1H), 7.80

(d, J = 9.3 Hz, 1H), 7.68–7.55 (m, 1H), and 7.45 (td, J = 8.5, 2.4 Hz, 2H). ¹³

C

NMR (100 MHz, DMSO‐d₆) δ 174.92, 167.15, 163.95, 161.52, 135.69,

132.04, 128.79, 123.75, 119.05, 118.85, 114.33, and 114.10. HRMS (ESI)

m/z calcd for [C₁₅H₇F₄N₅O₂+H]⁺366.0609; found: 366.0611.

5‐[3‐(o‐Tolyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐pyrazolo‐

[3,4‐b]pyridin‐6‐ol (12c)

The title compound was prepared in a manner similar to the

procedures described in 12a by utilizing 11c as the starting ma-

terial (white solid, yield: 45.7%). MP: >300°C. ¹H NMR (500 MHz,

DMSO‐d₆) δ 14.37 (s, 1H), 13.26 (s, 1H), 8.72 (s, 1H), 8.01

(d, J = 7.6 Hz, 1H), 7.48 (t, J = 7.4 Hz, 1H), 7.40 (dd, J = 16.3, 7.8 Hz,

2H), and 2.60 (s, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 173.60,

170.36, 168.56, 160.36, 137.95, 135.38, 131.81, 131.25, 130.27,

126.63, 126.00, 122.65, 120.53, 114.82, and 22.01. HRMS (ESI)

m/z calcd for [C₁₆H₁₀F₃N₅O₂+H]⁺362.0859; found: 362.0873.

5‐[3‐(4‐Fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo[3,4‐b]pyridin‐6‐ol (12h)

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11h as the starting material (white solid,

yield: 41.5%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 14.33

(s, 1H), 13.37 (s, 1H), 8.72 (s, 1H), 8.13 (td, 2H), and 7.41 (t, J = 8.9 Hz,

2H). ¹³C NMR (100 MHz, DMSO‐d₆) δ 174.74, 167.15, 165.63, 163.18,

130.18, 130.09, 123.20, 123.17, 116.95, 116.73, and 40.58. HRMS (ESI)

m/z calcd for [C₁₅H₇F₄N₅O₂+H]⁺366.0609; found: 366.0610.

5‐[3‐(m‐Tolyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo‐[3,4‐b]pyridin‐6‐ol (12d)

5‐[3‐(2‐Chlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo[3,4‐b]pyridin‐6‐ol (12i)

The title compound was prepared in a manner similar to the procedures

described in compound 12a by utilizing 11d as the starting material

(white solid, yield: 37.7%). Mp: >300°C. ¹H NMR (500 MHz, DMSO‐d₆)

δ 13.92 (s, 2H), 8.74 (s, 1H), 7.69 (d, J = 7.5 Hz, 1H), 7.59 (s, 1H), 7.52

(t, J = 7.9 Hz, 1H), 7.19 (d, J = 8.3 Hz, 1H), and 3.87 (s, 4H). ¹³C NMR

(125 MHz, DMSO‐d₆) δ 174.74, 167.93, 160.15, 135.68, 131.00, 127.93,

122.69, 120.54, 120.00, 117.96, 112.46, and 55.86. HRMS (ESI) m/z

calcd for [C₁₆H₁₀F₃N₅O₂+H]⁺362.0859; found: 362.1003.

The title compound was prepared in a manner similar to the proce-

dures described in 12a by utilizing 11i as the starting material (white

solid, yield: 53.2%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

14.23 (s, 1H), 13.42 (s, 1H), 8.73 (s, 1H), 8.01 (d, J = 7.6 Hz, 1H), 7.71

(d, J = 8.0 Hz, 1H), 7.64 (t, J = 7.6 Hz, 2H), and 7.57 (t, J = 7.5 Hz, 1H).

¹³C NMR (125 MHz, DMSO‐d₆) δ 174.22, 166.87, 133.05, 132.63,

132.36, 131.25, 128.15, 126.03, and 122.68. HRMS (ESI) m/z calcd

for [C₁₅H₇ClF₃N₅O₂+H]⁺382.0313; found: 382.0314.

5‐[3‐(p‐Tolyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐pyrazolo‐

[3,4‐b]pyridin‐6‐ol (12e)

5‐[3‐(3‐Chlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo[3,4‐b]pyridin‐6‐ol (12j)

The title compound was prepared in a manner similar to the

procedures described in 12a by utilizing 11e as the starting ma-

terial (white solid, yield: 35.6%). MP: >300°C. ¹H NMR (500 MHz,

DMSO‐d₆) δ 8.67 (s, 1H), 7.99 (d, J = 7.7 Hz, 2H), 7.39 (d, J = 7.6 Hz,

2H), and 2.40 (s, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 167.77,

141.85, 135.39, 131.11, 130.23, 127.54, 124.05, 121.08, and

21.57. HRMS (ESI) m/z calcd for [C₁₆H₁₀F₃N₅O₂+Na]⁺384.0679;

found: 384.0660.

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11j as the starting material (white solid,

yield: 47.3%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 14.33

(s, 1H), 13.31 (s, 1H), 8.71 (s, 1H), 8.03 (d, J = 6.4 Hz, 3H), 7.66

(d, J = 6.7 Hz, 2H), and 7.63–7.55 (m, 1H). ¹³C NMR (125 MHz, DMSO‐d₆)

δ 174.94, 166.90, 160.49, 131.87, 131.72, 128.62, 127.07, 126.21, and

120.48. HRMS (ESI) m/z calcd for [C₁₅H₇ClF₃N₅O₂+H]⁺382.0313; found:

382.0350.

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5‐[3‐(4‐Chlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐1H‐

pyrazolo[3,4‐b]pyridin‐6‐ol (12k)

5‐{3‐[4‐(Trifluoromethoxy)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12p)

The title compound was prepared in a manner similar to the proce-

dures described in 12a by utilizing 11k as the starting material (white

solid, yield: 43.9%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

14.25 (s, 1H), 13.41 (s, 1H), 8.69 (s, 1H), 8.06 (d, J = 7.9 Hz, 3H), and

7.61 (d, J = 7.8 Hz, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.80,

167.12, 160.05, 136.71, 135.47, 129.77, 129.31, 125.47, 122.63, and

120.49. HRMS (ESI) m/z calcd for [C₁₅H₇ClF₃N₅O₂+H]⁺382.0313;

found: 382.0314.

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11p as the starting material (white solid,

yield: 45.6%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 8.71 (s,

1H), 8.20 (d, J = 8.8 Hz, 2H), and 7.55 (d, J = 8.1 Hz, 3H). ¹³C NMR

(125 MHz, DMSO‐d₆) δ 174.93, 166.95, 150.86, 135.70, 129.84, 125.77,

122.63, 122.01, 121.45, 120.49, 119.41, and 117.36. HRMS (ESI) m/z

calcd for [C₁₆H₇F₆N₅O₃+H]⁺432.0526; found: 432.0540.

3‐(Trifluoromethyl)‐5‐{3‐[2‐(trifluoromethyl)phenyl]‐1,2,4‐oxadiazol‐

5‐yl}‐1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12q)

5‐[3‐(2,4‐Dichlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐

1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12l)

The title compound was prepared in a manner similar to the proce-

dures described in 12a by utilizing 11p as the starting material (white

solid, yield: 32.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

14.31 (s, 1H), 13.45 (s, 1H), 8.71 (s, 1H), 8.00 (d, J = 7.5 Hz, 1H), 7.96

(d, J = 7.3 Hz, 1H), 7.90 (t, J = 7.0 Hz, 1H), and 7.87 (t, J = 7.1 Hz, 1H).

¹³C NMR (125 MHz, DMSO‐d₆) δ 174.51, 167.24, 135.73, 133.40,

132.53, 132.06, 128.34, 128.09, 127.47, 127.43, 127.39, 127.35,

125.43, 125.04, 122.87, and 120.23. HRMS (ESI) m/z calcd for

[C₁₆H₇F₆N₅O₂+H]⁺416.0577; found: 416.0588.

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11l as the starting material (white solid,

yield: 53.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 14.36

(s, 0H), 13.39 (s, 0H), 8.70 (s, 1H), 8.04 (d, J = 8.2 Hz, 1H), 7.86 (s, 1H), and

7.64 (d, J = 7.9 Hz, 1H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.26, 166.10,

160.32, 136.95, 135.45, 133.71, 133.55, 130.86, 128.42, 124.94, and

122.61. HRMS (ESI) m/z calcd for [C₁₆H₇F₆N₅O₃+H]⁺415.9923; found:

415.9933.

5‐[3‐(3,5‐Dichlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐3‐(trifluoromethyl)‐

1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12m)

3‐(Trifluoromethyl)‐5‐{3‐[3‐(trifluoromethyl)phenyl]‐1,2,4‐oxadiazol‐

5‐yl}‐1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12r)

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11m as the starting material (white solid,

yield: 32.5%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d6) δ 8.72 (s,

1H), 8.00 (s, 2H), and 7.87 (s, 1H). ¹³C NMR (100 MHz, DMSO‐d₆) δ

175.18, 166.38, 135.55, 131.47, 130.82, 129.85, 125.96, 122.68, and

120.16. HRMS (ESI) m/z calcd for [C₁₆H₇F₆N₅O₃+H]⁺415.9923; found:

415.9929.

The title compound was prepared in a manner similar to the proce-

dures described in 12a by utilizing 11r as the starting material (white

solid, yield: 41.2%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

13.96 (s, 2H), 8.66 (s, 1H), 8.04 (d, 2H), 7.89 (s, 1H), 7.77–7.64 (m,

1H), and 7.56 (d, 1H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 173.57,

165.39, 148.99, 147.74, 134.15, 130.60, 128.28, 127.32, 125.17,

123.00, 120.06, 118.28, and 118.01. HRMS (ESI) m/z calcd for

[C₁₆H₇F₆N₅O₂+H]⁺416.0577; found: 416.0643.

5‐{3‐[2‐(Trifluoromethoxy)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12n)

3‐(Trifluoromethyl)‐5‐{3‐[4‐(trifluoromethyl)phenyl]‐1,2,4‐oxadiazol‐

5‐yl}‐1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12s)

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11n as the starting material (white solid,

yield: 37.4%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 14.37 (s,

1H), 13.35 (s, 1H), 8.72 (s, 1H), 8.20 (d, J = 8.7 Hz, 1H), 7.77–7.71 (m,

1H), and 7.66–7.57 (m, 2H). ¹³C NMR (100 MHz, DMSO‐d₆) δ 174.70,

165.08, 159.73, 146.41, 138.75, 133.63, 131.84, 128.85, 123.62,

123.03, 121.57, 120.92, 119.52, 117.48, and 105.84. HRMS (ESI) m/z

calcd for [C₁₆H₇F₆N₅O₃+H]⁺432.0526; found: 432.0537.

The title compound was prepared in a manner similar to the procedures

described in 12a by utilizing 11s as the starting material (white solid,

yield: 33.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 8.68 (s,

1H), 8.24 (d, J = 7.9 Hz, 3H), and 7.89 (d, J = 8.2 Hz, 3H). ¹³C NMR

(125 MHz, DMSO‐d₆) δ 175.05, 166.91, 143.77, 135.57, 131.90, 131.65,

130.41, 128.35, 126.57, 126.54, 125.35, 123.19, 122.58, and 120.45.

HRMS (ESI) m/z calcd for [C₁₆H₇F₆N₅O₂+H]⁺416.0577; found:

415.9933.

5‐{3‐[3‐(Trifluoromethoxy)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐3‐

(trifluoromethyl)‐1H‐pyrazolo[3,4‐b]pyridin‐6‐ol (12o)

6‐Hydroxy‐5‐[3‐(3‐methoxyphenyl)‐1,2,4‐oxadiazol‐5‐yl]‐1H‐

pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13a)

The title compound was prepared in a manner similar to the proce-

dures described in 12a by utilizing 11n as the starting material (white

solid, yield: 45.6%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

14.03 (s, 2H), 8.66 (s, 1H), 8.20–8.00 (m, 2H), 7.89 (s, 1H), 7.80–7.64

(m, 1H), and 7.65–7.40 (m, 1H). ¹³C NMR (125 MHz, DMSO‐d₆) δ

174.98, 166.79, 160.56, 149.19, 135.68, 131.95, 128.76, 126.53,

124.34, 122.59, 121.51, 120.45, 119.66, 119.46, and 117.42. HRMS

(ESI) m/z calcd for [C₁₆H₇F₆N₅O₃+H]⁺432.0526; found: 432.0534.

A mixture of 12a (50 mg, 0.14 mmol), NaOH (0.40 g, 0.10 mol), 15 ml

of acetonitrile, and 3 ml of water was heated to 70°C for 18 h. The

reactive solution was cooled to room temperature. The resulting

mixture was cooled to room temperature and was acidified by the

addition of hydrochloric acid solution (8 ml, 1 mol/l). The mixture was

filtered to afford 13a (white solid, yield: 87.6%). MP: >300°C. ¹H

NMR (500 MHz, DMSO‐d₆) δ 8.88 (s, 1H), 8.00 (d, J = 7.3 Hz, 3H), and

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7.09 (d, J = 7.4 Hz, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.66,

167.58, 162.12, 159.70, 138.11, 129.24, 119.02, 115.05, and 55.84.

HRMS (ESI) m/z calcd for [C₁₆H₁₁N₅O₅+H]⁺354.0833; found:

354.0827.

(s, 1H), 7.93 (d, J = 7.9 Hz, 1H), 7.81 (d, J = 9.5 Hz, 1H), 7.64 (q, J = 7.4 Hz,

1H), and 7.51–7.39 (m, 1H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 175.38,

167.03, 167.01, 163.74, 161.79, 159.69, 138.92, 132.08, 132.02,

128.88, 123.83, 118.98, 118.82, 114.28, and 114.09. HRMS (ESI) m/z

calcd for [C₁₅H₈FN₅O₄+H]⁺342.0633; found: 342.0633.

6‐Hydroxy‐5‐[3‐(4‐methoxyphenyl)‐1,2,4‐oxadiazol‐5‐yl]‐1H‐

pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13b)

5‐[3‐(4‐Fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐pyrazolo‐

[3,4‐b]pyridine‐3‐carboxylic acid (13h)

The title compound was prepared in a manner similar to the procedures

described in 13a by utilizing 12b as the starting material (white solid,

yield: 86.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 8.94 (s,

1H), 7.88 (d, 2H), 7.44 (d, 2H), and 2.40 (s, 3H). ¹³C NMR (125 MHz,

DMSO‐d₆) δ 175.03, 167.92, 161.44, 159.71, 149.79, 139.09, 138.67,

132.58, 129.58, 127.92, 126.71, 124.73, and 21.35. HRMS (ESI) m/z

calcd for [C₁₆H₁₁N₅O₅+H]⁺354.0833; found: 354.0781.

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12h as the starting material (white

solid, yield: 87.5%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.91 (s, 1H), 8.12 (t, 2H), and 7.40 (t, J = 8.2 Hz, 2H). ¹³C NMR

(125 MHz, DMSO‐d₆) δ 175.09, 167.07, 165.38, 163.40, 159.46,

138.43, 132.77, 130.16, 130.08, 123.32, 116.90, and 116.73. HRMS

(ESI) m/z calcd for [C₁₅H₈FN₅O₄+H]⁺342.0633; found: 342.0626.

6‐Hydroxy‐5‐[3‐(o‐tolyl)‐1,2,4‐oxadiazol‐5‐yl]‐1H‐pyrazolo[3,4‐b]‐

pyridine‐3‐carboxylic acid (13c)

5‐[3‐(2‐Chlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐pyrazolo‐

[3,4‐b]pyridine‐3‐carboxylic acid (13i)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12c as the starting material (white

solid, yield: 80.8%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.92 (s, 1H), 7.99 (d, J = 7.5 Hz, 1H), 7.46 (t, J = 7.2 Hz, 2H), 7.39 (dt,

J = 11.1, 5.3 Hz, 4H), and 2.59 (s, 3H). ¹³C NMR (125 MHz, DMSO‐d₆)

δ 174.01, 168.44, 159.76, 149.98, 138.50, 137.93, 131.84, 131.24,

130.26, 126.71, 126.11, 105.53, and 22.07. HRMS (ESI) m/z calcd for

[C₁₆H₁₁N₅O₄+H]⁺338.0884; found: 338.0909.

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12i as the starting material (white

solid, yield: 83.7%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.90 (s, 1H), 7.98 (dd, J = 7.6, 1.4 Hz, 2H), 7.68 (d, J = 7.4 Hz, 1H), 7.61

(td, J = 7.8, 1.6 Hz, 2H), and 7.56 (td, 2H). ¹³C NMR (125 MHz,

DMSO‐d₆) δ 174.53, 166.72, 159.64, 138.55, 132.99, 132.59, 132.31,

131.21, 128.15, 126.06, and 105.64. HRMS (ESI) m/z calcd for

[C₁₅H₈ClN₅O₄+Na]⁺380.0157; found: 380.0094.

6‐Hydroxy‐5‐[3‐(p‐tolyl)‐1,2,4‐oxadiazol‐5‐yl]‐1H‐pyrazolo[3,4‐b]‐

pyridine‐3‐carboxylic acid (13e)

5‐[3‐(3‐Chlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐pyrazolo‐

[3,4‐b]pyridine‐3‐carboxylic acid (13j)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12e as the starting material (white

solid, yield: 83.3%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.93 (s, 1H), 7.97 (d, J = 7.6 Hz, 3H), 7.37 (d, J = 7.5 Hz, 3H), and 2.38

(s, 4H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.93, 174.35, 167.79,

159.78, 141.85, 138.62, 130.24, 129.82, 127.50, 123.95, 114.59, and

21.56. HRMS (ESI) m/z calcd for [C₁₆H₁₁N₅O₄+H]⁺338.0884; found:

338.0902

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12j as the starting material (white

solid, yield: 89.8%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.95 (s, 1H), 8.29–7.82 (m, 3H), and 7.82–7.26 (m, 3H). ¹³C NMR

(125 MHz, DMSO‐d₆) δ 175.39, 166.81, 159.68, 139.05, 134.37,

131.84, 131.80, 128.73, 127.03, 126.27, and 105.58. HRMS (ESI) m/z

calcd for [C₁₅H₈ClN₅O₄+Na]⁺380.0157; found: 380.0106.

5‐[3‐(2‐Fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐pyrazolo‐

[3,4‐b]pyridine‐3‐carboxylic acid (13f)

5‐[3‐(4‐Chlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐pyrazolo‐

[3,4‐b]pyridine‐3‐carboxylic acid (13k)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12f as the starting material (white

solid, yield: 86.1%). MP: >300°C. 1H NMR (500 MHz, DMSO‐d₆) δ

8.92 (s, 1H), 8.11 (t, J = 7.3 Hz, 1H), 7.66 (q, J = 6.9 Hz, 2H), and

7.52–7.34 (m, 3H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.59, 164.74,

164.70, 161.33, 159.75, 159.30, 138.58, 138.53, 133.97, 133.90,

131.19, 125.61, 117.34, 117.18, 114.92, and 114.83. HRMS (ESI) m/z

calcd for [C₁₅H₈FN₅O₄+Na]⁺364.0453; found: 364.0449.

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12k as the starting material (white

solid, yield: 82.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

14.47 (s, 1H), 12.82 (s, 1H), 8.93 (s, 1H), 8.09 (d, J = 7.7 Hz, 2H), and

7.64 (d, J = 7.4 Hz, 2H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 175.26,

167.07, 161.28, 159.70, 149.82, 138.65, 136.67, 129.87, 129.40,

125.61, and 105.65. HRMS (ESI) m/z calcd for [C₁₅H₈ClN₅O₄+-

Na]⁺380.0157; found: 380.0390.

5‐[3‐(3‐Fluorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐pyrazolo‐

[3,4‐b]pyridine‐3‐carboxylic acid (13g)

5‐[3‐(2,4‐Dichlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐

pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13l)

The title compound was prepared in a manner similar to the procedures

described in 13a by utilizing 12g as the starting material (white solid,

yield: 83.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 8.95

The title compound was prepared in a manner similar to the procedures

described in 13a by utilizing 12l as the starting material (white solid,

yield: 85.1%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ 14.45

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(s, 1H), 12.81 (s, 1H), 9.02–8.87 (m, 2H), 8.01 (s, 2H), and 7.88 (s, 1H).

¹³C NMR (125 MHz, DMSO‐d₆) δ 175.60, 165.96, 161.12, 159.62,

149.80, 139.01, 135.57, 131.42, 130.03, 126.00, and 105.68. HRMS

(ESI) m/z calcd for [C₁₅H₆C_l2F₃N₅O₂+H]⁺391.9948; found: 391.9949.

solid, yield: 79.8%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.91 (s, 1H), 8.35 (d, J = 7.8 Hz, 1H), 8.26 (s, 1H), 7.95 (d, J = 7.9 Hz,

1H), and 7.82 (t, J = 7.8 Hz, 1H). ¹³C NMR (125 MHz, DMSO‐d₆) δ

175.41, 166.78, 159.58, 138.62, 131.51, 131.14, 130.50, 130.24,

128.52, 127.77, 127.46, 125.30, 123.68, 123.65, 123.13, and 120.97.

HRMS (ESI) m/z calcd for [C₁₆H₈F₃N₅O₄+Na]⁺413.9767; found:

414.0445.

5‐[3‐(3,5‐Dichlorophenyl)‐1,2,4‐oxadiazol‐5‐yl]‐6‐hydroxy‐1H‐

pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13m)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12m as the starting material

(white solid, yield: 81.7%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐

d₆) δ 8.95 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), and

7.68–7.59 (m, 2H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.98, 166.79,

160.56, 149.19, 135.68, 131.95, 128.76, 126.53, 124.34, 122.59,

121.51, 120.45, 119.66, 119.46, and 117.42. HRMS (ESI) m/z calcd

for [C₁₆H₈F₃N₅O₅+H]⁺408.0550; found: 408.0550.

6‐Hydroxy‐5‐[3‐[4‐(trifluoromethyl)phenyl]‐1,2,4‐oxadiazol‐5‐yl]‐

1H‐pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13r)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12r as the starting material (white

solid, yield: 81.4%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ

8.97 (s, 1H), 8.30 (d, 2H), and 8.12–7.52 (m, 2H). ¹³C NMR (100 MHz,

DMSO‐d₆) δ 176.36, 166.70, 159.84, 138.35, 131.85, 131.70, 130.18,

128.46, 126.66, 125.54, and 105.55. HRMS (ESI) m/z calcd for

[C₁₆H₈F₃N₅O₄+H]⁺392.0601; found: 392.0547.

6‐Hydroxy‐5‐{3‐[2‐(trifluoromethoxy)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐

1H‐pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13n)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12n as the starting material (white

solid, yield: 81.7%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

8.95 (s, 1H), 8.19 (d, J = 7.7 Hz, 1H), 7.76 (t, J = 7.9 Hz, 1H), and

7.68–7.59 (m, 2H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 174.98, 166.79,

160.56, 149.19, 135.68, 131.95, 128.76, 126.53, 124.34, 122.59,

121.51, 120.45, 119.66, 119.46, and 117.42. HRMS (ESI) m/z calcd

for [C₁₆H₈F₃N₅O₅+H]⁺408.0550; found: 408.0550.

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4.2

Biological activity

|

4.2.1

Cell culture

The NRK‐49F cell line was obtained from American Type Culture

Collection. The plasticware was purchased from Corning Inc. ATP,

MgCl₂, and dithiothreitol (DTT) were obtained from Promega.

Phosphorylated human recombinant AMPK (α1β1γ1) was pur-

chased from Carna Biosciences Inc. HTRF® KinEASE™‐STK1 Kit

was obtained from Cisbio. High‐glucose DMEM was purchased

from Hyclone. Fetal bovine serum was purchased from Germini.

Annexin V‐FITC/PI Apoptosis Detection Kit was purchased from

Solarbio. Active Oxygen Detection Kit was obtained from

Beyotime.

6‐Hydroxy‐5‐{3‐[3‐(trifluoromethoxy)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐

1H‐pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13o)

The title compound was prepared in a manner similar to the procedures

described in 13a by utilizing 12o as the starting material (white solid,

yield: 84.6%). MP: >300°C. ¹H NMR (400 MHz, DMSO‐d₆) δ 14.48

(s, 1H), 12.82 (s, 1H), 8.92 (s, 1H), 8.11 (d, J = 7.6 Hz, 1H), 7.94 (s, 1H),

7.73 (t, J = 7.4 Hz, 1H), and 7.61 (d, J = 8.1 Hz, 2H). ¹³C NMR (100 MHz,

DMSO‐d₆) δ 175.40, 166.76, 159.70, 149.58, 138.94, 132.11, 128.87,

126.70, 124.47, 121.65, 119.68, 118.59, and 105.47. HRMS (ESI) m/z

calcd for [C₁₆H₈F₃N₅O₅+H]⁺408.0550; found: 408.0569.

|

4.2.2

Activation of human recombinant AMPK

α1β1γ1 protein assay

6‐Hydroxy‐5‐{3‐[4‐(trifluoromethoxy)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐

1H‐pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13p)

A previously described HTRF activation assay was used to assess

AMPK activation.^[58]Human recombinant AMPK (α1β1γ1) protein

was prephosphorylated by CaMKKβ. The enzyme reaction was per-

formed in 384‐well plates, which contained 0.16 μM STK substrate 1‐

biotin, 4 mmol MgCl₂, 0.8 mmol DTT, 4 μM ATP, and corresponding

compound. The reaction was initiated by adding 1 ng/μl of pAMPK

(α1β1γ1) protein into the well. After incubation at 37°C for 1 h, the

reaction was terminated by an additional detection reagent con-

taining 57.5 nmol/l XL‐665 and STK‐antibody labeled with Eu3+

Cryptate, followed by incubation at room temperature for another

1 h. The fluorescence was measured at 665 nm (XL665) and 620 nm

(Eu3+ Cryptate). A ratio was calculated ([665/620] × 10,000) for

each well, which represents the activity of AMPK. Compounds' EC₅₀

values were evaluated by GraphPad Prism 6.0 software as long as

their efficacy exceeded 50% at 60 μM.

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12p as the starting material (white

solid, yield: 78.4%). MP: >300°C. ¹H NMR (500 MHz, DMSO‐d₆) δ

14.42 (s, 1H), 12.78 (s, 0H), 9.01 (s, 1H), 8.23 (d, J = 7.9 Hz, 2H), and

7.57 (d, J = 8.2 Hz, 2H). ¹³C NMR (125 MHz, DMSO‐d₆) δ 175.55,

166.86, 161.60, 159.71, 150.84, 149.99, 139.54, 136.08, 129.88,

125.99, 123.53, 121.48, 120.95 (q), and 105.32. HRMS (ESI) m/z

calcd for [C₁₆H₈F₃N₅O₅+H]⁺408.0550; found: 408.0522.

6‐Hydroxy‐5‐{3‐[3‐(trifluoromethyl)phenyl]‐1,2,4‐oxadiazol‐5‐yl}‐

1H‐pyrazolo[3,4‐b]pyridine‐3‐carboxylic acid (13q)

The title compound was prepared in a manner similar to the proce-

dures described in 13a by utilizing 12q as the starting material (white

20 of 22

XIAO ET AL.

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4.2.3

Measurement of intracellular ROS level

were predicted using admetSAR and Molinspiration web‐based tools

to check whether they possess druglike properties or not.^[60,61]

DCFH‐DA was used to detect the generation of ROS. NRK‐49F cells in

logarithmic growth phase were counted after digestion and plated into

a 24‐well plate at a density of 20,000 cells/well. After 24 h, the cells

were adhered and then incubated with a fresh medium containing

5 ng/ml of TGF‐β at 37°C in a 5% CO₂atmosphere. After 1 h, 0.1 μM

compound C was added. After incubation for 1 h, cells were pretreated

with synthesized compounds (0, 1, 3, and 10 μM) and incubated for

another 24 h. The culture medium was removed and washed three

times with a serum‐free medium. The cells were loaded with 200 μl of

DCFH‐DA (10 μmol/L) dissolved in serum‐free medium at 37°C for

20 min and washed three times with serum‐free medium. DCF fluor-

escence was measured at 488 nm excitation and 520 nm emissions

using a microplate reader.

ACKNOWLEDGMENTS

The authors would also like to thank the Nuclear Magnetic Re-

sonance Laboratory of Advanced Research Center in Central South

University for the technological assistance in chemical character-

ization. This study was supported by the National Natural Science

Foundation of China (Nos. 81573287, 81773640) and the Natural

Science Foundation of Hunan Province (No. 2016JJ2162).

CONFLICTS OF INTEREST

The authors declare that there are no conflicts of interest.

ORCID

Bifeng Zheng

Qianbin Li

https://orcid.org/0000-0002-4652-3195

https://orcid.org/0000-0003-1244-7013

|

4.2.4

Cell apoptosis assay

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Design, synthesis, and biological evaluation of 1,2,4-oxadiazole-containing pyrazolo[3,4-b]pyridinones as a new series of AMPKɑ1β1γ1 activators

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