Novel potent HIF-1 inhibitors for the prevention of tumor metastasis: discovery and optimization of 3-aryl-5-indazole-1,2,4-oxadiazole derivatives

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ARTICLE

Novel Potent HIF-1 Inhibitors for the Prevention of Tumor

Metastasis: Discovery and Optimization of 3-Aryl-5-indazole-1,2,4-

oxadiazole Derivatives

Received 00th January 20xx,

Accepted 00th January 20xx

Rong Sheng^†#, Shan Li^†#, Guanyu Lin^‡#, Shihao Shangguan^†, Yongchuan Gu^‖, Ni Qiu^†, Ji Cao^‡*, Qiaojun

He^‡, Bo Yang^‡, Yongzhou Hu^†*

DOI: 10.1039/x0xx00000x

www.rsc.org/

Hypoxia inducible factor-1 (HIF-1) is the key transcription factor of cellular response to hypoxia and plays a critical role in

tumor metastasis. We describe here the discovery and a structure-activity relationship study of a series of 3-aryl-5-

indazole- 1,2,4-oxadiazole derivatives as novel HIF-1 inhibitors. The two most promising compounds 4g and 4h inhibit HIF-

1 transcription with IC₅₀values of 0.62 and 0.55 μM in vitro, respectively, and they exhibit more efficient HIF-1 inhibition in

xenograft tumors than YC-1, a potential anticancer drug targeting HIF-1. In addition, they also remarkably prevent the

hypoxia-driven migration of SKOV3 cells in vitro and tumor metastasis in vivo. Further investigation of the mechanism

revealed that the two inhibitors could decrease HIF-1α and VEGF expression. These results suggest that our newly

synthesized HIF-1 inhibitors 4g and 4h are potential therapeutic agents with which to treat tumor metastasis.

transcription of more than 70 putative target genes, including

glucose transporter 1 (Glut1), carboanhydrase IX (CAIX), vascular

endothelial growth factor (VEGF) and erythropoietin (EPO) via

1 Introduction

Hypoxia is a common characteristic of most solid tumors

resulting from inefficient microvascular systems associated with

rapid tumor growth. Tumor cells within these regions show

resistance to both radiotherapy and chemotherapy, and are

associated with poor prognosis in cancer patients.^{1, 2}The hypoxia

inducible factor-1 (HIF-1) is the key transcription factor activated in

response to intratumoral hypoxia and plays a pivotal role in

adaptation of tumor cells to hypoxia by orchestrating a number of

cellular responses, such as angiogenesis, glycolysis, pH adaptation,

cell proliferation and migration.³

binding to hypoxic response elements or (HRE)-containing promoter

regions. In this way, anaerobic glycolysis, angiogenesis, cell

proliferation and tumor invasion are mediated.^{4, 5}

Increased expression of HIF-1 has been observed in many kinds

of human cancers, including breast, brain, colon, lung and prostate

cancers, and is strongly correlated with aggressive tumor growth,

therapeutic resistance and poor patient prognosis.⁵Consequently,

HIF-1 is regarded as an attractive target for the treatment of tumor

hypoxia. A variety of compounds with different scaffolds have been

identified as HIF-1 inhibitors by HIF-1-targeted cell based screens

and empirical discoveries based on chemical libraries and natural

products,^6-8including the PI3K/mTOR inhibitor wortmannin,⁹the

microtubule inhibitor 2-methoxyestradiol,¹⁰the topoisomerase I

inhibitor topotecan,¹¹the trypanocidal agent acriflavine,¹²and the

arylsulfonamide derivative KCN1^{13, 14}. In particular, the pyrazole-

oxadiazole derivative BAY 87-2243 has been advanced into Phase I

clinical trial as an antitumor agent¹⁴(Figure 1). These HIF-1

inhibitors regulate the HIF-1 signaling pathway through different

molecular mechanisms, including transcriptional regulation, mRNA

translation, nuclear translocation, HIF-1α degradation and HIF-1

dimerization. The majority of HIF-1 inhibitors identified so far are

multi-target compounds and HIF-1 inhibition cannot be easily

separated from other activities. Therefore, design of more specific

HIF-1 targeting agents is of great significance and clinical

importance in the investigation of potential therapeutic agents for

solid tumors.

HIF-1 is a heterodimeric basic helix-loop-helix transcription

factor, with an oxygen sensitive HIF-1α subunit and a constitutively

expressed HIF-1β subunit. Under normoxic conditions, HIF-1α

continuously binds to pVHL, ARD1, SUMO and RACK1 and this leads

to ubiquitination and proteasomal degradation. However, under

hypoxic conditions, HIF-1α is stabilized and rapidly accumulated.

After binding to transcriptional co-activators such as p300/CBP, HIF-

1α is translocated to the nucleus and heterodimerized with HIF-1β

to form an HIF-1 complex. HIF-1 specifically activates the

^†Department of Medicinal Chemistry, College of Pharmaceutical Sciences, Zhejiang

University, Hangzhou 310058, China

^‡Institute of Pharmacology & Toxicology, College of Pharmaceutical Sciences, Zhejiang

University, Hangzhou, 310058, China

^‖Auckland Cancer Society Research Centre, the University of Auckland, Auckland, New

Zealand.

^#These authors contributed equally to this work

* Corresponding authors:

Yongzhou Hu, Phone/Fax: +086-571-8820-8460. E-mail: huyz@zju.edu.cn.

Ji Cao. E-mail: caoji88@163.com

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Figure 1. Structures of selected HIF-1 inhibitors

Figure 2. Diagram for the structural optimization strategy

Tumor metastasis is a complex and multi-step process by

which primary tumor cells invade other tissues or parts of the body,

finally proliferating into macroscopic secondary tumors. It is

estimated that metastasis is responsible for as much as 90% of

2 Results and discussion

2.1 Chemistry

In the compounds 1 and 2a-2l, the 5-(N-benzyl-indazole)-1,2,4-

oxadiazole skeleton was retained and various alkyl and aryl groups

were examined as an R₁moiety to find an optimal substituent.

Condensation of alkyl or aryl nitriles 6a-l with hydroxylamine gave

the intermediates 7a-l.³⁰Esterification of compound 8 with MeOH

and thionyl chloride led to compound 9, benzylation of which with

4-chlorobenzyl chloride gave compound 10. Hydrolysis of 10 gave

11, which was condensed with 7a-l to afford compounds 2a-2l

(Scheme 1).

cancer-associated mortality while only

a few drugs exhibit

metastasis-inhibitory activity in the clinic.^15-17Although the

pathogenesis of cancer metastasis remains unclear, abundant

evidence proves that HIF-1 is the key regulator of tumor

metastasis.^{3, 18, 19}Recent research also has revealed that YC-1 and

HSP90 inhibitor Ganetespib can efficiently inhibit tumor metastasis

by blocking the HIF-1 pathway^{20, 21}and consequently, development

of novel HIF-1 inhibitors will provide specific therapeutic agents

against tumor metastasis.

In the past few years, our group has focused on the

development of small molecules targeting tumor hypoxia and has

identified several hypoxia selective antitumor agents, including the

quinoxaline-1,4-dioxide derivatives Q39 and Q6. In addition to their

excellent hypoxia selective cytotoxic activities against a variety of

tumor cell lines, these two compounds inhibit hypoxia-induced HIF-

1α transcription or protein expression in vitro.^22-25In our search for

novel hypoxia-targeted antitumor agents, we recently designed the

indazole-1,2,4-oxadiazole derivative 1 as a bioisostere analogue of

the indazole-furan derivative YC-1, an attractive lead compound.

This compound has dual reactions with HIF-1, decreasing HIF-1α

protein accumulation and stimulating FIH-dependent p300

dissociation from HIF-1α.^26-28Compound 1 exhibits comparable HIF-

1 inhibitory activity (IC₅₀= 5.72 μM) to that of YC-1 (IC₅₀= 3.97 μM).

Previous SAR study on YC-1 derivatives reveals that the substituted

furan moiety on indazole skeleton as well as its substitution pattern

is crucial for the high HIF-1 inhibition.²⁹To identify more potent HIF-

1 inhibitors for further pharmacological study as well as to verify

the known structure-activity relationships, optimization of

Scheme 1. Synthesis of compounds 2a-2l. Reagents and conditions: (a) NH₂OH-HCl,

Na₂CO₃, EtOH, H₂O, reflux; (b) SOCl₂, CH₃OH, r.t., overnight; (c) p-chlorobenzyl chloride,

NaH, DMF, r.t.; (d) 0.25 mol/L NaOH, THF, r.t., overnight; (e) 7a-l, EDCI, HOBt, DMF, 140

^oC.

While retaining the optimal 4-OCF₃-Ph moiety in the 3-position

of the 1,2,4-oxadiazole, further modifications were made on the

indazole moiety to probe their effect on activity (3a-3c).

Compounds were prepared from the corresponding indole, 1,2,4-

triazole or 2,5-dimethyl pyrrole 3-carboxylic acid ester 12a-12c with

the same synthetic route as was used for compound 2l (Scheme 2).

compound

1 was carried out through four modifications: 1)

modification of the R₁hydroxyethyl moiety of 1,2,4-oxadiazole, 2)

modification or scaffold hopping of the indazole, 3) modification of

R₂or the chlorobenzyl moiety of the indazole and investigation of

the tautomerism possibilities of the indazole (Figure 2).

Scheme 2. Synthesis of compounds 3a-3c. Reagents and conditions: (a) i) p-

Chlorobenzyl chloride, NaH, DMF, r.t.; ii) 0.25 mol/L NaOH, THF, r.t., overnight; (b) 7l,

EDCI, HOBt, DMF, 140 ^oC.

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Variations of the R₂indazole moiety were extensively explored from 1.63 μM to 3.16 μM. Compound 2l was the most active

with substituted aryl groups. First, compounds 4a-4f were prepared compound in this series and the 4-CF₃O moiety was chosen as the

through condensation of compound 14 with substituted benzyl optimal R₁group for the further structural exploration.

chlorides and analogues in the presence of NaH in DMF. Table 1. Structures and biological activities of compounds 2a-2l.

Meanwhile, compounds 5a and 5b were separated as minor

products to probe the effect of tautomerisim on activity (Scheme

3). Benzylation of compound 14 with p- or m-TBDMS-O-benzyl

bromide yielded compounds 15a and 15b, and deprotection with

No.

YC-1

1

R₁

-

HRE IC₅₀(μM) ^a

3.97 ± 0.13

5.72 ± 0.27

> 10

Cell Viability (μM)

TBAF gave 16a and 16b, followed by alkylation with 1,2-

dibromoethane gave compounds 17a and 17b. Subsequently,

amination of 17a and 17b with various amines afforded compounds

4g-4j.

> 10

2a

2b

2c

2d

2e

2f

5.25 ± 0.24

> 10

2.67 ± 0.19

3.16 ± 0.15

5.48 ± 0.31

4.83 ± 0.25

6.80 ± 0.30

5.08 ± 0.23

> 10

2g

2h

2i

2j

2k

2l

8.24 ± 0.52

1.63 ± 0.09

Scheme 3. Synthesis of compounds series 4 and 5. Reagents and conditions: (a) 7l,

EDCI, HOBt, DMF, 140 ^oC; (b) substituted benzyl chloride or analogues, NaH, DMF, r.t.,

major products; (c) 4-fluorobenzyl chloride or 4-chloromethyl pyridine, NaH, DMF, r.t,

minor products were separated; (d) NaH, DMF, r.t. (e) TBAF, THF; (f) K₂CO₃,

BrCH₂CH₂Br, acetone, reflux; (g) secondary amine, K₂CO₃, dry CH₃CN, reflux.

^aIC₅₀values are indicated as the mean ± SD (standard deviation) of three independent

experiments.

Next, compounds 3a-3c were designed to probe the effect of

indazole moiety on activity. Replacement of the indazole with

indole, 1,2,4-triazole or dimethylpyrrole moieties also clearly

attenuated HIF-1 inhibition. In comparison with 2l, compound 3a

(IC₅₀= 5.88 ± 0.41 μM) and 3c (IC₅₀= 6.25 ± 0.38 μM) showed a 3- to

4-fold decrease in activity and compound 3b (IC₅₀> 10.0 μM) was

almost inactive. These results confirmed that the indazole moiety is

essential to HIF-1 inhibitory activity and thus they were retained in

future modifications.

2.2 Biological evaluations

HIF-Luciferase activity in vitro. With a human colon carcinoma

cell line HCT-116 stably expressing a hypoxia-inducible luciferase

reporter gene (HRE element), the synthesized target compounds

were evaluated for their potency in inhibiting HIF-1-mediated

transcription. All of the assays were performed under standard

hypoxic conditions (1% O₂). Cell viability, as measured by

a

sulforhodamine B assay, showed that all compounds effectively

inhibited HIF-1 transcriptionbut had no significant cytotoxicity at

the concentrations at which they were active (cell viability >10 μM).

YC-1 was used as a positive control with IC₅₀value of 3.97 ± 0.13

μM, which was in substantial agreement with the reported value in

reference (IC₅₀= 1.2 μM).³¹

Table 2. Structures and biological activities of compounds 3a-3c.

No.

Structure

HRE IC₅₀(μM) ^a

1.63 ± 0.09

Cell Viability (μM)

> 10

2l

The first series of compounds 2a-2l were designed to assess

the importance of the R₁moiety to HIF-1 inhibition. In general, aryl

substituents are favorable in this position. Except for compounds 2c

and 2j, other aryl substituted compounds demonstrate moderate

HIF-1 inhibitory activities with IC₅₀values ranging from 1.63 to 8.24

μM, while the alkyl substituted compounds 2a showed a dramatic

decrease in activity (Table 1). The results also reveal that the p-

position substitution was more favorable than substitution at the o-

or m-position (2f vs 2j, 2k). Three compounds (2d, 2e, 2l) exhibited

more potent HIF-1 inhibition than YC-1 with IC₅₀values ranging

3a

3b

3c

5.88 ± 0.41

> 10

6.25 ± 0.38

^aIC₅₀values are indicated as the mean ± SD (standard deviation) of three independent

experiments.

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4h

4i

0.55 ± 0.029

1.00 ± 0.074

1.94 ± 0.11

> 10

Compounds 5a and 5b were prepared to investigate the

influence of indazole tautomerism on HIF-1 inhibitory activity.

Compounds substituted at the N-2 position show an approximately

2-fold decrease in activity over those substituted at the N-1 position

(5a vs 4a, 5b vs 4b), indicating the N-1 substitution is more

favorable for HIF-1 inhibition.

4j

^aIC₅₀values are indicated as the mean ± SD (standard deviation) of three independent

experiments.

Compounds 4a-j resulted from modification of the R₂moiety in

2l with various aryl substituents (Table 3). In general, 4a-j all

exhibited good to excellent HIF-1 inhibition, indicating that different

variations of the phenyl group were well tolerated. In particular,

compounds 4g-4j, with IC₅₀values ranging from 0.55 to 1.94 μM in

vitro, were more efficient HIF-1 inhibitors than YC-1. Such

enhancement in HIF-1 inhibitory activity suggests that the

introduction of hydrophilic aminoalkyl ether moiety that aims to

improve physical chemistry property may be beneficial for the cell-

based evaluation and in vivo biological evaluation. The weaker

activity of compound 4j compared to 4h also revealed that

substitution at the m-position is more favorable than at the p-

position. Compounds 4g and 4h were the most potent HIF-1

inhibitors of all the designed compounds with IC₅₀values of 0.62

and 0.55 μM, respectively, and they were selected for further

mechanism studies and in vivo evaluation.

In order to determine the optimal adminstration manner for in

vivo evaluation, the physicochemical properties of compounds 4g

and 4h were calaulated by using ChemBioDraw and ACD/I-Lab

prediction engine (https://ilab.acdlabs.com/iLab2/index.php). The

results in Table 4 revealed that the two compounds possessed

relatively large molecular weight, high LogP and TPSA values, which

were unfavourable for oral admination. Therefore, the intravenous

injection was chosen as the suitable method for them in in-vivo

stdies.

Table 4. Predicted physicochemical properties of compounds 4g and 4h.

Compounds

4g

4h

Molecular Weight

578.6

579.6

Table 3. Structures and biological activities of compounds 4a-4j and 5a, 5b

TPSA : 74.49

CLogP : 5.74

TPSA : 81.68

LogP : 5.56 ± 0.91

TPSA : 91.48

CLogP : 5.62

TPSA : 98.67

LogP : 5.65 ± 0.89

Data predicted by

ChemBioDraw

Data predicted by

ACD/Labs

No.

4a

5a

4b

5b

4c

R₂

HRE IC₅₀(μM)^a

3.84 ± 0.12

7.65 ± 0.55

4.65 ± 0.31

10.3 ± 0.77

1.74 ± 0.11

5.67 ± 0.26

6.01 ± 0.30

5.00 ± 0.19

0.62 ± 0.035

Cell Viability (μM)

> 10

HIF-1 transcriptional activity in vivo. HCT116 colon carcinoma

cells stably transfected with the hypoxia-inducible luciferase

reporter gene (HRE-luciferase) were xenografted in nude mice.

When the tumor volume reached 200 mm³, the mice were divided

into four groups, followed by i.v. administration of vehicle or test

compounds for three consecutive days (50 mg/kg, once daily,

Fig.3A). The luciferase activity was markedly decreased after

treatment with 4g, 4h or YC-1 for 3 days compared to that of

vehicle control group (Fig. 3B). These data demonstrate that 4g, 4h

and YC-1 also inhibit the HIF-1 activity in tumor xenografts. More

importantly, consistent with in vitro results, the inhibitory activities

of 4g and 4h on HIF-1 transcriptional activity in vivo are much better

than the positive control YC-1 (Fig. 3C).

4d

4e

4f

4g

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Figure 3. Compounds 4g and 4h inhibit HIF-1 transcriptional activity in vivo. (A) The scheme for evaluation of the inhibitory activity of the compound on the HIF-1 transcriptional

activity in vivo. (B-C) Mice bearing HCT-116 xenografts expressing HRE-luciferase were treated with vehicle or compounds (50mg/kg) once daily for three days. (B) The HRE-driven

luciferase activity was determined by Xenogen imaging after three days’ treatment. The representative images in different groups are shown. (C) The intensity of luminescence

was calculated in software, and the data are shown as the ratio vs. control group and expressed as the mean ± SD. *, vs. control (n≧3, *, p<0.05, ***, p<0.001)

Figure 4. The effect of 4g and 4h on HIF-1 accumulation and hypoxia-driven cell migration. (A) The accumulation of HIF-1α, HIF-1β and VEGF was examined in HCT116 cells treated

with 4g, 4h and YC-1 (5 μM) by Western blots analysis. β-actin was used as a loading control. The density of immunoreactive bands was calculated by Quantity One software, and

the data are shown as the ratio versus β-Actin. (B) HCT116 cells were exposed to 4g, 4h and YC-1 (5 μM). Then, the total RNA was extracted and analyzed for HIF-1α mRNA

expression by RT-PCR, using GAPDH as a control gene. (C-D) the effect of compounds on hypoxia-driven cell migration basing on transwell model was conducted. (C) The

representative images in different groups are shown. (D) The number of cells was calculated, and the data are expressed as the mean ± SD. (n=3, *, p<0.05, ***, p<0.001)

Western blotting and RT-PCR analysis. For further mechanistic transcriptional level, RT-PCR analysis was used to study the effect of

studies, the structural based effects of compounds 4g and 4h were compounds on the accumulation of HIF-1α mRNA. As shown in Fig.

evaluated using biochemical techniques with YC-1 as positive 4B, the HIF-1α mRNA levels are not significantly changed after 4g

control. Since most of the HIF-1 regulation usually occurs at the and 4h treatment in HCT116 cells. Of note, the inhibitory activity on

protein level, we investigated the effect of compounds on HIF-1α, HIF-1α downstream VEGF caused by 4g and 4h is better than that

HIF-1β and its downstream target protein VEGFR using Western resulting from YC-1 (Fig. 4A), and this is consistent with the fact that

blotting under normoxia or hypoxic conditions in HCT116 cells. As 4g and 4h show more efficient inhibitory activities on HIF-1

expected, the protein levels of HIF-1α are markedly increased under transcription than does YC-1 both in vitro and in vivo. Therefore,

hypoxia compared with those under normoxia, while the protein these data further suggest that the decrease of HIF-1α protein

levels of HIF-1β remained unchanged (Fig. 4A). Meanwhile, maybe not the only reason for their strong activity. Additional work

compounds 4g and 4h exhibit remarkable inhibitory activity on HIF- in this area is in progress and will be reported in due course.

1α protein accumulation, similar to the effect of YC-1. To ascertain

Transwell assay. To confirm the activities of compounds 4g

whether the reduction of HIF-1α by these compounds occurs at the and 4h against tumor cell invasion and tumor metastasis, a cell

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migration assay based on transwell was carried out using human the following two weeks (Fig. 5A). After two weeks’ treatment, a

ovarian cancer cells SKOV3. As shown in Fig. 4C, compared to the transient decrease of the body weight of mice is only observed in

control group, 4g, 4h and YC-1 significantly reduce the number of the YC-1 treatment group (Fig. 5B). The mice were sacrificed on day

cells passing through the polycarbonate filter. In addition, 4g and 4h 30 and the lungs were removed and fixed in paraformaldehyde. For

are more efficient than YC-1 in preventing cell migration with weak analysis of pulmonary metastasis, lungs were cut into 5 μm sections

cytotoxicity (Fig. 4D and data not shown).

and stained for HE. As shown in Fig. 5C and 5D, the numbers of

pulmonary nodules in lungs are significantly decreased in treatment

Pulmonary metastasis model of SKOV3 in nude mice. SKOV3 groups 4g and 4h, but not in the YC-1 group. These in vivo results

cells were i.v. injected into nude mice, and the mice started to are further confirmation that the newly synthesized HIF-1 inhibitors

receive i.v. injections of the compounds with dose of 50 mg/kg on 4g and 4h are potential therapeutic agents for tumor metastasis.

Figure 5. The effect of 4g and 4h on the development of pulmonary metastasis in vivo. (A) The scheme for evaluating the effect of 4g and 4h on the development of pulmonary

metastasis in vivo. (B) The changes of body weight in the pulmonary metastasis model of SKOV3 in nude mice. (C) The representative images of lung in different groups are shown.

(D) The numbers of pulmonary nodules in lung were calculated, and the data are expressed as the mean ± SD. (n=5)

4.1 General Methods for Chemistry.

3 Conclusion

All reagents and solvents used were reagent grade and

In summary,

a series of 3-aryl-5-indazole-1,2,4-oxadiazole

purchased from commercial resources. Flash chromatography was

performed using silica gel (200-300 mesh). All reactions were

monitored by TLC, using silica gel plates with fluorescence F254 and

UV light visualization. Melting points were determined with a B-540

Buchi melting-point apparatus. ¹H NMR was recorded on a Brüker

Advance DMX 500 MHz spectrometer. Coupling constants (J) are

expressed in hertz (Hz) and chemical shifts (δ) of NMR are reported

in parts per million (ppm) units relative to an internal control (TMS).

Low resolution mass spectra (ESI-MS) were gathered on a Finnigan

LCQ DecaXP ion trap mass spectrometer and HRMS spectra on an

Agilent 6224 TOF LC/MS. The purity of compounds was determined

by Agilent 1260 HPLC system and analysis to be over 95%. (Column:

Eclipse XDB-C18, 5.0 μm, 4.6 × 250mm (Agilent); Flow rate: 1.0

mL/min; Mobile phase: A: MeOH, B: H₂O).

derivatives has been designed, synthesized and identified as specific

HIF-1 inhibitors. All the synthesized compounds show weak

cytotoxic activities (IC₅₀> 10 μM) and a majority of them exhibit

moderate to potent HIF-1 transcriptional inhibition. The two most

potent HIF-1 inhibitors were 4g and 4h with IC₅₀values of 0.62 and

0.55 μM respectively, which is 6- to 7-fold more effective than YC-1.

Consistent with the in vitro results, the two compounds 4g and 4h

demonstrate much better HIF-1 inhibitory activities in xenografts

tumor than YC-1. A cell migration assay based on transwell

conducted in hypoxic conditions disclosed that compounds 4g and

4h can significantly reduce the number of SKOV3 cells passing

through the polycarbonate filter, and are more efficient than YC-1.

Furthermore, the in vivo cell metastasis experiment reveals that

they can decrease the numbers of pulmonary nodules in lungs

remarkably compared to vehicle control and YC-1. The mechanistic

studies indicate that these two inhibitors influence the protein

expression levels of HIF-1α and its downstream target gene VEGF.

Our results indicate the newly synthesized HIF-1 inhibitors 4g and

4h are potential therapeutic agents against tumor metastasis.

General Procedure for the Synthesis of Compounds 7a-7l. The

substituted cyanide (6a-6l, 4.0 mmol) was added to a solution of

hydroxylamine hydrochloride (0.69 g, 10 mmol), Na₂CO₃(0.64 g, 6.0

mmol) in 8 mL H₂O and 4 mL EtOH. The mixture was refluxed for 6 h

and extracted with EtOAc (3×20 mL). The combined organic layer

was washed with saturated saline, dried over anhydrous Na₂SO₄

and concentrated in vacuo to give 7a-7l

.

4 Experimental Section

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N

-hydroxy-2-phenylacetimidamide (7a). White solid, 70% yield. Mp temperature overnight and concentrated. The residue was

65-66 ^oC. (lit.³²65-67 ^oC).

partitioned with EtOAc (30 mL) and H₂O (30 mL) and the organic

N

-hydroxybenzimidamide (7b). White solid, 73% yield. Mp 73-75 layer was discarded. The aqueous layer was acidified with 2.0 mol/L

^oC. (lit.³⁰76-78 ^oC).

HCl until the pH was adjusted to 2. The precipitate formed was

N

-hydroxy-4-(trifluoromethyl)benzimidamide (7c)

.

White solid, collected by suction filtration, washed with H₂O and dried in vacuo

o

1

92% yield. Mp 113-114 C. H NMR (500 MHz, DMSO-d₆): δ 9.92 (s, to afford 11 as a white solid with 69% yield. Mp 196-197 ^oC (lit.³⁹

1H, OH), 7.90 (d, J = 8.5 Hz, 2H, ArH), 7.75 (d, J = 8.0 Hz, 2H, ArH), 196 ^oC).

5.98 (s, 2H, NH); MS (ESI), m/z: 205 [M+H]⁺.

General Procedure for the Synthesis of Compounds 2a-2l.

N

-hydroxy-4-tert-butylbenzimidamide (7d). White solid, 73% yield. Compound 7a-7l (0.32 mmol) was added to a solution of 11 (72 mg

Mp 147-149 ^oC. ¹H NMR (500 MHz, CDCl₃)：δ 7.57 (d, J = 8.5 Hz, 2H, 0.25 mmol), EDCI (62 mg, 0.32 mmol) and HOBt (44 mg, 0.32 mmol)

ArH), 7.43 (d, J = 8.5 Hz, 2H, ArH), 4.90 (brs, 2H, NH), 1.33 (s, 9H, in 8 mL dry DMF. The mixture was stirred at room temperature for

3×CH₃); MS (ESI), m/z: 193 [M+H]⁺.

1h and then heated to 140 ^oC for 1 h. The completion of reaction

N

-hydroxy-4-methoxybenzimidamide (7e). White solid, 65% yield. was confirmed by TLC and the DMF was removed in vacumm. The

Mp 119-120 ^oC. (lit.³³116-117 ^oC).

residue was extracted with EtOAc (15 mL×3) and the combined

4-Chloro-N-hydroxybenzimidamide (7f). White solid, 81% yield. Mp organic layer was washed with H₂O and saturated saline, and dried

133-134 ^oC. (lit.³³133-134 ^oC).

over anhydrous Na₂SO₄. The solvent was removed under vacuum

-hydroxyisonicotinimidamide (7g). White solid, 90% yield. Mp and the residue was purified by silica gel chromatography to get 2a-

197-198 ^oC. (lit.³⁴197-199 ^oC).

2l

N

.

N

-hydroxythiophene-2-carboximidamide (7h). White solid, 73% 3-Benzyl-5-(1-(4-chlorobenzyl)-1

H

-indazol-3-yl)-1,2,4-oxadiazole

yield. Mp 86-88 ^oC. (lit.³⁵90 ^oC).

(2a). White solid, 23% yield. Mp 120-121 ^oC. ¹H NMR (500 MHz,

N

-hydroxyfuran-2-carboximidamide (7i). White solid, 44% yield. CDCl₃): δ 8.17 (d, J = 8.5 Hz, 1H, ArH), 7.95 (d, J = 8.5 Hz, 1H, ArH),

Mp 52-53 ^oC. (lit.³⁶57 ^oC).

7.58 (t, J = 7.5 Hz, 1H, ArH), 7.44 (d, J = 7.5 Hz, 1H, ArH), 7.37-7.41

3-Chloro-N-hydroxybenzimidamide (7j). White solid, 96% yield. Mp (m, 6H, ArH), 7.32 (d, J = 8.5 Hz, 2H, ArH), 7.28 (t, J = 7.5 Hz, 1H,

111-112 ^oC. (lit.³⁰116-117 ^oC). ArH), 5.86 (s, 2H, benzylic-CH₂), 4.23 (s, 2H, benzylic-CH₂); ¹³C NMR

2-chloro-N-hydroxybenzimidamide (7k). White solid, 84% yield. Mp (100 MHz, CDCl₃): δ 170.92, 169.94, 140.40, 135.59, 134.20, 133.97,

116-118 ^oC. (lit.³⁷119-121 ^oC).

130.47, 129.12, 128.70, 127.73, 127.11, 123.66, 123.09, 121.86,

N

-hydroxy-4-(trifluoromethoxy)benzimidamide (7l). White solid, 109.96, 53.42, 32.45; MS (ESI), m/z: 401 [M+H]⁺; HRMS (TOF) m/z

o

1

89% yield. Mp 107-109 C. H NMR (500 MHz, DMSO-d₆): δ 9.76 (s, calcd for C₂₃H₁₇ClN₄O [M+H]⁺: 401.1164, found 401.1166. HPLC

1H, OH), 7.79 (d, J = 8.5 Hz, 2H, ArH), 7.38 (d, J = 8.5 Hz, 2H, ArH), purity = 98.72%, Rt 8.57 min.

5.90 (s, 2H, NH); MS (ESI), m/z: 221 [M+H]⁺.

5-(1-(4-Chlorobenzyl)-1

-indazole-3-carboxylate (9). To a stirred solution of 1H- (2b). White solid, 77% yield. Mp 144-145 ^oC. ¹H NMR (500 MHz,

indazole-3-carboxylic acid ( , 1.5 g, 9.3 mmol) in 33 mL MeOH was CDCl₃): δ 8.37 (d, J = 8.0 Hz, 1H, ArH), 8.19 (dd, J₁= 8.0 Hz, J₂= 2.0

H-indazol-3-yl)-3-phenyl-1,2,4-oxadiazole

Methyl 1

H

8

added SOCl₂(2.2 g, 18.6 mmol) at 0 ^oC, the mixture was stirred Hz, 2H, ArH), 7.99 (d, J = 8.5 Hz, 1H, ArH), 7.59-7.65 (m, 4H, ArH),

overnight at room temperature. The solvent was removed in vacuo 7.50 (t, J = 7.5 Hz, 1H, ArH), 7.42 (d, J = 9.0 Hz, 2H, ArH), 7.37 (d, J =

and the residue was partitioned between 20 mL EtOAc and 15 mL 9.0 Hz, 2H, ArH), 5.91 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz,

H₂O. The organic layer was separated, washed with saturated CDCl₃): δ 170.96, 168.91, 140.57, 134.32, 134.09, 131.39, 130.64,

saline, dried over anhydrous Na₂SO_4.The solvent was removed 129.24, 128.94, 128.82, 127.89, 127.86, 126.90, 123.83, 123.33,

under vacuum to give

^oC. (lit.³⁸162-163 ^oC).

9

as a white solid in 93% yield. Mp 163-165 122.07, 110.13, 53.56; MS (ESI), m/z: 387 [M+H]⁺; HRMS (TOF) m/z

calcd for C₂₂H₁₅ClN₄O [M+H]⁺: 387.1007, found 387.1005. HPLC

Methyl 1-(4-chlorobenzyl)-1

(0.08 g, 1.8 mmol) was added to a solution of 1H-indazole-3- 5-(1-(4-Chlorobenzyl)-1

carboxylate (

, 0.30 g, 1.7 mmol) in dry DMF (5 mL) at 0 ^oC. The l)-1,2,4-oxadiazole (2c). White solid, 62% yield. Mp 185-186 ^oC. ¹H

H

-indazole-3-carboxylate (10)

.

NaH purity = 100.00%, Rt 10.96 min.

H

-indazol-3-yl)-3-(4-(trifluoromethyl)pheny

9

mixture was stirred at room temperature for 30 min and 4- NMR (500 MHz, CDCl₃): δ 8.40 (d, J = 8.0 Hz, 2H, ArH), 8.37 (d, J =

chlorobenzyl chloride (0.30 g, 1.8 mmol) was added. After stirring 8.5 Hz, 1H, ArH), 7.98-8.01 (m, 3H, ArH), 7.63 (t, J = 7.5 Hz, 1H, ArH),

for 4 h at room temperature, the reaction was quenched with H₂O 7.51 (t, J = 7.5 Hz, 1H, ArH), 7.42 (d, J = 8.5 Hz, 2H, ArH), 7.38 (d, J =

(20 mL) and the mixture was extracted with EtOAc (3×20 mL). The 8.5 Hz, 2H, ArH), 5.91 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz,

combined organic layer was washed with saturated saline, dried CDCl₃): δ 171.33, 167.81, 140.50, 134.30, 133.88, 130.25, 130.20,

over anhydrous Na₂SO₄. The solvent was removed under vacuum 129.17, 128.72, 128.09, 127.89, 125.86 (q, J₁= 7.2 Hz, J₂= 3.4 Hz),

and the residue was purified by silica gel chromatography to give 10 125.18, 123.89, 123.23, 122.47, 121.83, 110.11, 53.53; MS (ESI),

as white solid with 57% yield. Mp 156-157 ^oC. ¹H NMR (500 MHz, m/z: 455 [M+H]⁺; HRMS (TOF) m/z calcd for C₂₃H₁₄ClF₃N₄O [M+H]⁺:

CDCl₃): δ 8.28 (d, J = 8.0 Hz, 1H, ArH), 7.43 (t, J = 7.5 Hz, 1H, ArH), 455.0881, found 455.0871. HPLC purity = 99.03%, Rt 12.78 min.

7.36 (m, 2H, ArH), 7.30 (d, J = 8.0 Hz, 2H, ArH), 7.18 (d, J = 8.0 Hz, 3-(4-(Tert-butyl)phenyl)-5-(1-(4-chlorobenzyl)-1H-indazol-3-yl)-

2H, ArH), 5.69 (s, 2H, benzylic-CH₂), 4.07 (s, 3H, OCH₃); MS (ESI), 1,2,4-oxadiazole (2d). White solid, 61% yield. Mp 162-163 ^oC. ¹H

m/z: 301 [M+H]⁺.

NMR (500 MHz, CDCl₃): δ 8.35 (d, J = 8.0 Hz, 1H, ArH), 8.10 (d, J =

1-(4-Chlorobenzyl)-1

H

-indazole-3-carboxylic acid (11)

.

Aqueous 8.5 Hz, 2H, ArH), 7.98 (d, J = 8.5 Hz, 1H, ArH), 7.65 (d, J = 8.5 Hz, 2H,

NaOH (0.25 mol/L, 25.0 mL) was added to a solution of compound ArH), 7.62 (t, J = 7.5 Hz, 1H, ArH), 7.50 (t, J = 7.5 Hz, 1H, ArH), 7.42

10 (0.85 g, 2.9 mmol) in 25 mL THF. The mixture was stirred at room (d, J = 8.5 Hz, 2H, ArH), 7.36 (d, J = 8.5 Hz, 2H, ArH), 5.91 (s, 2H,

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benzylic-CH₂), 1.34 (s, 9H, 3×CH₃); ¹³C NMR (100 MHz, CDCl₃): δ ¹³C NMR (100 MHz, CDCl₃): δ 170.86, 161.71, 145.29, 142.42,

170.72, 168.75, 154.72, 140.47, 134.20, 134.02, 130.64, 129.14, 140.46, 134.26, 133.89, 130.16, 129.16, 128.73, 127.84, 123.84,

128.70, 127.76, 127.54, 125.80, 123.94, 123.68, 123.25, 122.01, 123.17, 121.88, 114.35, 111.89, 110.06, 53.52; MS (ESI), m/z: 377

110.00, 53.45, 35.02, 31.23; MS (ESI), m/z: 443 [M+H]⁺; HRMS (TOF) [M+H]⁺; HRMS (TOF) m/z calcd for C₂₀H₁₃ClN₄O₂[M+H]⁺: 377.0800,

m/z calcd for C₂₆H₂₃ClN₄O [M+H]⁺: 443.1633, found 443.1639. HPLC found 377.0802. HPLC purity = 99.01%, Rt 6.88 min.

purity = 96.78%, Rt 12.78 min.

5-(1-(4-Chlorobenzyl)-1 -indazol-3-yl)-3-(4-methoxyphenyl)-1,2,4-

5-(1-(4-Chlorobenzyl)-1

H-indazol-3-yl)-3-(3-chlorophenyl)-1,2,4-

H

oxadiazole (2j). White solid, 67% yield. Mp 136-137 ^oC. ¹H NMR

oxadiazole (2e). White solid, 57% yield. Mp 157-159 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.44 (d, J = 8.0 Hz, 1H, ArH), 8.30 (t, J = 2.0 Hz,

(500 MHz, CDCl₃): δ 8.35 (d, J = 8.5 Hz, 1H, ArH), 8.12 (d, J = 8.5 Hz, 1H, ArH), 8.16-8.18 (m, 1H, ArH), 7.50-7.53 (m, 1H, ArH), 7.46-7.49

2H, ArH), 7.98 (d, J = 8.5 Hz, 1H, ArH), 7.62 (t, J = 7.5 Hz, 1H, ArH), (m, 2H, ArH), 7.41-7.45 (m, 2H, ArH), 7.31 (d, J = 8.5 Hz, 2H, ArH),

7.49 (t, J = 7.5 Hz, 1H, ArH), 7.42 (d, J = 8.5 Hz, 2H, ArH), 7.36 (d, J = 7.23 (d, J = 8.5 Hz, 2H, ArH), 5.76 (s, 2H, benzylic-CH₂); ¹³C NMR (100

8.5 Hz, 2H, ArH), 7.17 (d, J = 9.0 Hz, 2H, ArH), 5.90 (s, 2H, benzylic- MHz, CDCl₃): δ 171.22, 167.92, 140.59, 135.07, 134.37, 134.03,

CH₂), 3.86 (s, 3H, OCH₃); ¹³C NMR (100 MHz, CDCl₃): δ 170.61, 131.43, 130.42, 130.28, 129.27, 128.83, 128.63, 127.95, 125.87,

168.50, 162.04, 140.47, 134.20, 134.02, 130.64, 129.37, 129.13, 123.94, 123.31, 122.00, 110.17, 53.61; MS (ESI), m/z: 421 [M+H]⁺;

128.70, 127.75, 123.66, 123.22, 122.00, 119.26, 114.24, 109.99, HRMS (TOF) m/z calcd for C₂₂H₁₄Cl₂N₄O [M+H]⁺: 421.0617, found

55.40, 53.44; MS (ESI), m/z: 417 [M+H]⁺; HRMS (TOF) m/z calcd for 421.0620. HPLC purity = 100.00%, Rt 13.95 min.

C₂₃H₁₇ClN₄O₂[M+H]⁺: 417.1113, found 417.1107. HPLC purity = 5-(1-(4-Chlorobenzyl)-1

H-indazol-3-yl)-3-(2-chlorophenyl)-1,2,4-

97.72%, Rt 10.59 min.

oxadiazole (2k). White solid, 67% yield. Mp 132-133 ^oC. ¹H NMR

(500 MHz, CDCl₃): δ 8.44 (d, J = 8.0 Hz, 1H, ArH), 8.17 (dd, J₁= 8.0

5-(1-(4-Chlorobenzyl)-1H-indazol-3-yl)-3-(4-chlorophenyl)-1,2,4-

oxadiazole (2f). White solid, 71% yield. Mp 175-176 ^oC. ¹H NMR Hz, J₂= 2.0 Hz, 1H, ArH), 7.61 (dd, J₁= 8.0 Hz, J₂= 1.5 Hz, 1H, ArH),

(500 MHz, CDCl₃): δ 8.34 (d, J = 8.0 Hz, 1H, ArH), 8.19 (d, J = 8.5 Hz, 7.41-7.51 (m, 5H, ArH), 7.33 (d, J = 8.5 Hz, 2H, ArH), 7.25 (d, J = 9.0

2H, ArH), 7.99 (d, J = 8.5 Hz, 1H, ArH), 7.70 (d, J = 8.5 Hz, 2H, ArH), Hz, 2H, ArH), 5.77 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz, CDCl₃):

7.62 (t, J = 7.5 Hz, 1H, ArH), 7.50 (t, J = 7.5 Hz, 1H, ArH), 7.42 (d, J = δ 170.41, 167.60, 140.47, 134.24, 133.97, 133.65, 132.04, 131.78,

8.5 Hz, 2H, ArH), 7.37 (d, J = 8.5 Hz, 2H, ArH), 5.91 (s, 2H, benzylic- 130.96, 130.33, 129.15, 128.75, 127.81, 126.88, 126.11, 123.80,

CH₂); ¹³C NMR (100 MHz, CDCl₃): δ 171.05, 168.01, 140.48, 137.44, 123.26, 121.91, 110.03, 53.48; MS (ESI), m/z: 421 [M+H]⁺; HRMS

134.26, 133.92, 130.36, 129.17, 129.05, 128.72, 127.85, 125.28, (TOF) m/z calcd for C₂₂H₁₄Cl₂N₄O [M+H]⁺: 421.0617, found

123.81, 123.20, 121.89, 110.08, 53.50; MS (ESI), m/z: 421 [M+H]⁺; 421.0619. HPLC purity = 99.14%, Rt 10.08 min.

HRMS (TOF) m/z calcd for C₂₂H₁₄Cl₂N₄O [M+H]⁺: 421.0617, found 5-(1-(4-Chlorobenzyl)-1

H-indazol-3-yl)-3-(4-(trifluoromethoxy)

421.0619. HPLC purity = 100.00%, Rt 13.85 min.

5-(1-(4-Chlorobenzyl)-1 -indazol-3-yl)-3-(pyridin-4-yl)-1,2,4-oxadi

phenyl)-1,2,4-oxadiazole (2l). White solid, 74% yield. Mp 133-135

^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.37 (d, J = 8.0 Hz, 1H, ArH), 8.32 (d,

H

azole (2g). White solid, 66% yield. Mp 182-184 ^oC. ¹H NMR (500 J = 7.5 Hz, 2H, ArH), 8.00 (d, J = 8.5 Hz, 1H, ArH), 7.60-7.64 (m, 3H,

MHz, CDCl₃): δ 8.86 (d, J = 6.0 Hz, 2H, ArH), 8.37 (d, J = 8.5 Hz, 2H, ArH), 7.51 (t, J = 7.5 Hz, 1H, ArH), 7.43 (d, J = 8.5 Hz, 2H, ArH), 7.38

ArH), 8.11 (d, J = 6.0 Hz, 2H, ArH), 8.00 (d, J = 8.5 Hz, 1H, ArH), 7.63 (d, J = 8.5 Hz, 2H, ArH), 5.92 (s, 2H, benzylic-CH₂); ¹³C NMR (100

(t, J = 7.5 Hz, 1H, ArH), 7.51 (t, J = 7.5 Hz, 1H, ArH), 7.42 (d, J = 8.5 MHz, CDCl₃): δ 171.26, 167.90, 151.51, 140.61, 134.40, 134.03,

Hz, 2H, ArH), 7.37 (d, J = 9.0 Hz, 2H, ArH), 5.92 (s, 2H, benzylic-CH₂); 130.47, 129.61, 129.29, 128.84, 127.98, 125.53, 123.95, 123.35,

¹³C NMR (100 MHz, CDCl₃): δ 171.61, 167.31, 150.70, 140.50, 121.99, 121.82 (q, J = 256.6 Hz), 121.19, 110.21, 53.63; MS (ESI),

134.32, 134.23, 133.82, 130.08, 129.19, 128.73, 127.94, 123.97, m/z: 471 [M+H]⁺; HRMS (TOF) m/z calcd for C₂₃H₁₄ClF₃N₄O₂[M+H]⁺:

123.21, 121.77, 121.51, 110.15, 53.56; MS (ESI), m/z: 388 [M+H]⁺; 471.0830, found 471.0826. HPLC purity = 98.63%, Rt 12.98 min.

HRMS (TOF) m/z calcd for C₂₁H₁₄ClN₅O [M+H]⁺: 388.0960, found 2-(5-(1-(4-Chlorobenzyl)-1

388.0958. HPLC purity = 96.11%, Rt 7.59 min. nol (1) Ethyl 2-(5-(1-(4-chlorobenzyl)-1H-indazol-3-yl)-1,2,4-

5-(1-(4-Chlorobenzyl)-1 -indazol-3-yl)-3-(thiophen-2-yl)-1,2,4-oxa oxadiazol-3-yl)acetate (120 mg, 3.0 mmol) was added to a solution

H-indazol-3-yl)-1,2,4-oxadiazol-3-yl)etha

.

H

diazole (2h). White solid, 72% yield. Mp 173-174 ^oC. ¹H NMR (500 of LiAlH₄(152 mg, 3.27 mmol) in 10 mL of dry Et₂O at 0 ^oC. After

MHz, CDCl₃): δ 8.43 (d, J = 8.5 Hz, 1H, ArH), 8.01 (dd, J₁= 4.0 Hz, J₂= stirring for 1 h, H₂O (2 mL) and saturated aqueous NH₄Cl (4 mL)

1.5 Hz, 1H, ArH), 7.58 (dd, J₁= 5.0 Hz, J₂= 1.0 Hz, 1H, ArH), 7.44- were added to quench the reaction. The mixture was extracted with

7.51 (m, 1H, ArH), 7.42-7.45 (m, 2H, ArH), 7.32 (d, J = 8.5 Hz, 2H, EtOAc (2×15 mL) and the organic layer was separated, washed with

ArH), 7.21-7.24 (m, 3H, ArH), 5.77 (s, 2H, benzylic-CH₂); ¹³C NMR saturated saline, dried over anhydrous Na₂SO₄. The solvent was

(100 MHz, CDCl₃): δ 170.78, 164.92, 140.46, 134.24, 133.94, 130.27, removed under vacuum and the residue was purified by silica gel

130.05, 129.41, 129.16, 128.71, 128.33, 127.94, 127.83, 123.81, chromatography to give

1 as pale yellow solid with 60% yield. Mp

123.22, 121.93, 110.05, 53.50; MS (ESI), m/z: 393 [M+H]⁺; HRMS 118-119 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.33 (d, J = 8.0 Hz, 1H,

(TOF) m/z calcd for C₂₀H₁₃ClN₄OS [M+H]⁺: 393.0571, found ArH), 7.47-7.50 (m, 1H, ArH), 7.39-7.44 (m, 2H, ArH), 7.32 (d, J = 8.5

393.0573. HPLC purity = 100.00%, Rt 9.44 min.

5-(1-(4-Chlorobenzyl)-1 -indazol-3-yl)-3-(furan-2-yl)-1,2,4-oxadiaz

ole (2i). White solid, 67% yield. Mp 191-192 C. ¹H NMR (500 MHz, m/z: 355 [M+H]⁺; HRMS (TOF) m/z calcd for C₁₈H₁₅ClN₄O₂[M+H]⁺:

CDCl₃): δ 8.41 (d, J = 9.0 Hz, 1H, ArH), 7.67 (d, J = 1.0 Hz, 1H, ArH), 355.0956, found 355.0955. HPLC purity = 100.00%, Rt 3.96 min.

7.46-7.50 (m, 1H, ArH), 7.40-7.43 (m, 2H, ArH), 7.34 (d, J = 3.0 Hz, 1-(4-chlorobenzyl)-1H-indole-3-carboxylic acid (13a). With the

Hz, 2H, ArH), 7.23 (d, J = 8.5 Hz, 2H, ArH), 5.73 (s, 2H, benzylic-CH₂),

4.17 (t, J = 5.5 Hz, 2H, CH₂), 3.19 (t, J = 6.0 Hz, 2H, CH₂); MS (ESI),

H

o

2H, ArH), 7.31 (d, J = 8.5 Hz, 2H, ArH), 7.23 (d, J = 8.5 Hz, 2H, ArH), same synthetic route to compound 11

, compound 13a was

6.62 (dd, J₁= 3.5 Hz, J₂= 2.0 Hz, 1H, ArH), 5.74 (s, 2H, benzylic-CH₂); prepared as white solid. Mp 204-206 ^oC. (lit.⁴⁰205-207 ^oC).

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1-(4-chlorobenzyl)-1H-1,2,4-triazole-3-carboxylic acid (13b). White was removed under vacuum and the residue was purified by silica

1

solid, Mp 213-216 ^oC. H NMR (500 MHz, DMSO-d₆)：13.30 (s, 1H, gel chromatography to get 14 as white solid with 52% yield. Mp

COOH), 8.79 (s, 1H, ArH), 7.45 (d, J = 8.5 Hz, 2H, ArH), 7.35 (d, J = 8.5 209-210 ^oC. ¹H NMR (500 MHz, DMSO-d₆): δ 14.31 (s, 1H, NH), 8.36-

Hz, 2H, ArH), 5.48 (s, 2H, CH₂). MS (ESI), m/z: 238 [M+H]⁺.

8.32 (m, 2H, ArH), 7.79 (d, J = 8.5 Hz, 2H, ArH), 7.65 (d, J = 8.5 Hz,

1-(4-chlorobenzyl)-2,5-dimethyl-1H-pyrrole-3-carboxylic acid (13c). 2H, ArH), 7.59 (t, J = 8.0 Hz, 1H, ArH), 7.47 (t, J = 8.0 Hz, 1H, ArH).

White solid, Mp 176-179 ^oC. ¹H NMR (500 MHz, DMSO): δ 11.55 ( s, MS (ESI), m/z: 347 [M+H]⁺.

1H, COOH), 7.41 (d, J = 8.5 Hz, 2H, ArH), 6.92 (d, J = 8.5 Hz, 2H, ArH), General Procedure for the Synthesis of Compounds 4a-4f NaH

6.16 (d, J = 1.0 Hz, 1H, pyrrole-H), 5.11 (s, 2H, CH₂), 2.34 (s, 3H, CH₃), (0.60 mmol) was added to a mixture of intermediate 14 (0.50

2.05 (s, 3H, CH₃). MS (ESI), m/z: 264 [M+H]⁺.

mmol) in dry DMF (5 mL) at 0 ^oC and the mixture was stirred at

General Procedure for the Synthesis of Compounds 3a-3c. room temperature for 30 min. Substituted benzyl chloride (0.60

Compound 7l (0.50 mmol) was added to a solution of 13a 13b or mmol) was added to the above solution and stirred for 4-6 h at

,

13c (0.60 mmol), EDCI (0.65mmol), HOBt (0.65 mmol) in dry DMF (5 room temperature. After quenching with 10 mL H₂O, the mixture

mL). The mixture was stirred at room temperature for 1 h and then was extracted with EtOAc (2×15 mL) and the organic layer was

heated to 140 ^oC for 1 h. The mixture was partitioned between 25 separated, washed with saturated saline, dried over anhydrous

mL EtOAc and 15 mL H₂O. The organic layer was separated, washed Na₂SO₄. The solvent was removed under vacuum and the residue

with H₂O and saturated saline, dried over anhydrous Na₂SO₄. The was purified by silica gel chromatography.

solvent was removed under vacuum and the residue was purified 5-(1-(4-Fluorobenzyl)-1H-indazol-3-yl)-3-(4-(trifluoromethoxy)

by silica gel chromatography.

5-(1-(4-Chlorobenzyl)-1 -indol-3-yl)-3-(4-(trifluoromethoxy)pheny

phenyl)-1,2,4-oxadiazole (4a). White solid, 47% yield. Mp 128-130

^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.36 (d, J = 8.5 Hz, 1H, ArH), 8.32 (d,

H

l)-1,2,4-oxadiazole (3a). White solid, 44% yield. Mp 121-124 ^oC. ¹H J = 9.0 Hz, 2H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.60-7.63 (m, 3H,

NMR (500 MHz, CDCl₃): δ 8.74 (s, 1H, ArH), 8.23-8.27 (m, 3H, ArH), ArH), 7.50 (t, J = 7.5 Hz, 1H, ArH), 7.40-7.43 (m, 2H, ArH), 7.20 (t, J =

7.67 (d, J = 8.5 Hz, 1H, ArH), 7.62 (d, J = 8.5 Hz, 2H, ArH), 7.42 (d, J = 9.0 Hz, 2H, ArH), 5.90 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz,

9.0 Hz, 2H, ArH), 7.38 (d, J = 8.5 Hz, 2H, ArH), 7.35 (t, J = 6.0 Hz, 2H, CDCl₃): δ 171.30, 167.90, 163.96 (d, J = 245.7 Hz), 151.50, 140.60,

ArH), 5.61(s, 2H, benzylic-CH₂); ¹³C NMR (125 MHz, CDCl₃): δ 173.38, 131.35 (d, J = 3.0 Hz), 130.38, 129.61, 129.36 (d, J = 8.1 Hz), 127.92,

167.49, 151.25, 136.84, 134.44, 134.26, 131.98, 129.46, 129.39, 125.56, 123.91, 123.37, 121.98, 121.18, 116.17 (d, J = 22.0 Hz),

128.55, 126.20, 125.82, 124.00, 122.78, 121.80, 121.59 (q, J = 256.8 110.25, 53.64; MS (ESI), m/z: 455 [M+H]⁺; HRMS (TOF) m/z calcd for

Hz), 121.17, 110.61, 101.86, 50.43; MS (ESI), m/z: 469 [M+H]⁺; C₂₃H₁₄F₄N₄O₂[M+H]⁺: 455.1126, found 455.1128. HPLC purity =

HRMS (TOF) m/z calcd for C₂₄H₁₅ClF₃N₃O₂[M+H]⁺: 470.0878, found 99.42%, Rt 11.23 min.

470.0879. HPLC purity = 99.40%, Rt 13.31 min.

5-(1-(Pyridin-4-ylmethyl)-1H-indazol-3-yl)-3-(4-(trifluoromethoxy)

5-(1-(4-chlorobenzyl)-1H-1,2,4-triazol-3-yl)-3-(4-(trifluoromethoxy) phenyl)-1,2,4-oxadi-azole (4b). White solid, 50% yield. Mp 162-164

phenyl)-1,2,4-oxadi-azole (3b). White solid, 65% yield. Mp 132-135 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.55 (d, J = 6.0 Hz, 2H, ArH), 8.40 (d,

℃. ¹H NMR (500 MHz, DMSO-d₆)：8.43 (s, 1H, ArH), 8.26 (d, J = 9.0 J = 8.5 Hz, 1H, ArH), 8.33 (d, J = 8.5 Hz, 2H, ArH), 7.97 (d, J = 8.5 Hz,

Hz, 2H, ArH), 7.64 (d, J = 8.5 Hz, 2H, ArH), 7.43-7.47 (m, 4H, ArH), 1H, ArH), 7.62-7.65 (m, 3H, ArH), 7.54 (t, J = 7.5 Hz, 1H, ArH), 7.23

6.00 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz, CDCl₃): δ 167.94, (d, J = 5.5 Hz, 2H, ArH), 6.01 (s, 2H, benzylic-CH₂); ¹³C NMR (100

165.76, 151.81, 145.00, 140.26, 134.82, 133.06, 129.56, 129.39, MHz, CDCl₃): δ 170.97, 167.81, 151.39, 150.46, 144.31, 140.73,

129.19, 124.28, 121.64 (q, J = 257.4 Hz), 121.05, 53.65; MS (ESI), 130.84, 129.48, 128.17, 125.32, 124.03, 123.12, 122.01, 121.74,

m/z: 422 [M+H]⁺; HRMS (TOF) m/z calcd for C₁₈H₁₁ClF₃N₅O₂[M+H]⁺: 121.07, 109.77, 52.74; MS (ESI), m/z: 438 [M+H]⁺; HRMS (TOF) m/z

422.0626, found 422.0625. HPLC purity = 97.80%, Rt 9.18 min.

calcd for C₂₂H₁₄F₃N₅O₂[M+H]⁺: 438.1172, found 438.1175. HPLC

purity = 100.00%, Rt 7.63 min.

5-(1-(4-chlorobenzyl)-2,5-dimethyl-1H-pyrrol-3-yl)-3-(4-(trifluoro

methoxy)phenyl)-1,2,4-oxadiazole (3c). White solid, 45% yield. Mp 5-(1-(4-Nitrobenzyl)-1H-indazol-3-yl)-3-(4-(trifluoromethoxy)pheny

127-129℃.¹H NMR (500 MHz, CDCl₃)：8.20 (d, J = 9.0 Hz, 2H, ArH), l)-1,2,4-oxadiazole (4c). Yellowish solid, 46% yield. Mp 173-176 ^oC.

7.29-7.33 (m, 4H, ArH), 6.87 (d, J = 8.5 Hz, 2H, ArH), 6.56 (s, 1H, ¹H NMR (500 MHz, CDCl₃): δ 8.40 (d, J = 8.0 Hz, 1H, ArH), 8.33 (d, J =

ArH), 5.76 (s, 2H, benzylic-CH₂), 2.60 (s, 3H, CH₃), 2.18 (s, 3H, CH₃)； 9.0 Hz, 2H, ArH), 8.24 (d, J = 8.5 Hz, 2H, ArH), 8.02 (d, J = 8.5 Hz, 1H,

¹³C NMR (100 MHz, CDCl₃): δ 174.33, 167.09, 150.97, 135.23, ArH), 7.63-7.65 (m, 3H, ArH), 7.57 (d, J = 8.5 Hz, 2H, ArH), 7.54 (t, J =

133.50, 133.47, 129.63, 129.20, 129.15, 126.92, 126.35, 121.70 (q, J 7.5 Hz, 1H, ArH), 6.11 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz,

= 256.2 Hz), 120.96, 106.79, 105.41, 46.54, 12.20, 11.65; MS (ESI), CDCl₃): δ 170.93, 167.85, 151.44, 147.92, 142.50, 140.65, 130.98,

m/z: 448 [M+H]⁺; HRMS (TOF) m/z calcd for C₂₂H₁₇ClF₃N₃O₂[M+H]⁺: 129.49, 128.26, 128.02, 125.30, 124.24, 124.09, 123.20, 122.10,

448.1034, found 448.1035. HPLC purity = 98.03%, Rt 10.52 min.

5-(1 -indazol-3-yl)-3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazo

121.69 (q, J = 256.6 Hz), 121.08, 109.67, 53.15; MS (ESI), m/z: 482

[M+H]⁺; HRMS (TOF) m/z calcd for C₂₃H₁₄F₃N₅O₄[M+H]⁺: 482.1071,

H

le (14). N-hydroxy-4- (trifluoromethoxy)-benzimidamide (7l, 0.87 g, found 482.1072. HPLC purity = 97.11%, Rt 10.11min.

3.9 mmol) was added to a solution of 1H-indazole-3-carboxylic acid 5-(1-(4-Methoxybenzyl)-1 -indazol-3-yl)-3-(4-(trifluoromethoxy)p

, 0.7 g, 4.3 mmol), EDCI (0.90 g, 4.7 mmol) and HOBt (0.64g, 4.7 henyl)-1,2,4-oxadi- azole (4d). White solid, 48% yield. Mp 143-145

H

(8

mmol) in dry DMF (10 mL). The mixture was stirred at room ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.34 (d, J = 8.0 Hz, 1H, ArH), 8.31 (d,

temperature for 1h and then heated to 140 ^oC for 1h. After removal J = 9.0 Hz, 2H, ArH), 8.00 (d, J = 8.5 Hz, 1H, ArH), 7.63 (d, J = 8.5 Hz,

of DMF under vacumm, the residue was partitioned with EtOAc (35 2H, ArH), 7.59 (d, J = 8.0 Hz, 1H, ArH), 7.48 (t, J = 7.5 Hz, 1H, ArH),

mL) and H₂O (25 mL). The organic layer was separated, washed with 7.34 (d, J = 8.5 Hz, 2H, ArH), 6.91 (d, J = 9.0 Hz, 2H, ArH), 5.81 (s, 2H,

H₂O and saturated saline, dried over anhydrous Na₂SO₄. The solvent benzylic-CH₂), 3.70 (s, 3H, OCH₃); ¹³C NMR (100 MHz, CDCl₃): δ

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171.30, 167.74, 159.98, 151.33, 140.43, 129.91, 129.48, 128.88, General Procedure for the Synthesis of Compounds 15a, 15b. To a

127.56, 127.48, 125.49, 123.63, 123.28, 121.72, 121.04, 119.13, mixture of 14 (4.6 mmol) in dry DMF (15 mL) was added NaH (5.0

114.30, 110.40, 55.27, 53.92; MS (ESI), m/z: 467 [M+H]⁺; HRMS mmol) at 0 ^oC, and the mixture was stirred at room temperature for

(TOF) m/z calcd for C₂₄H₁₇F₃N₄O₃[M+H]⁺: 467.1326, found 30 min. (3 or 4-(bromomethyl)phenoxy)(tert-butyl)dimethylsilane⁴⁵

467.1324. HPLC purity = 97.21%, Rt 11.59 min.

5-(1-(3-Fluorobenzyl)-1 -indazol-3-yl)-3-(4-(trifluoromethoxy)ph

(5.0 mmol) was added to the above solution and stirred for 4-6 h at

room temperature. After quenching with H₂O (5 mL), the mixture

H

o

enyl)-1,2,4-oxadiazole (4e). White solid, 46% yield. Mp 126-129 C. was extracted with EtOAc (2×15 mL) and the organic layer was

¹H NMR (500 MHz, CDCl₃): δ 8.39 (d, J = 8.0 Hz, 1H, ArH), 8.34 (d, J = separated, washed with saturated saline, dried over anhydrous

8.5 Hz, 2H, ArH), 8.02 (d, J = 8.5 Hz, 1H, ArH), 7.62-7.65 (m, 3H, Na₂SO₄. The solvent was removed under vacuum and the residue

ArH), 7.53 (t, J = 7.5 Hz, 1H, ArH), 7.39-7.43 (m, 1H, ArH), 7.22 (d, J = was purified by silica gel chromatography.

9.5 Hz, 1H, ArH), 7.14-7.18 (m, 2H, ArH), 5.96 (s, 2H, benzylic-CH₂); 5-(1-(3-((Tert-butyldimethylsilyl)oxy)benzyl)-1H-indazol-3-yl)-3-(4-

¹³C NMR (100 MHz, CDCl₃): δ 171.18, 167.79, 164.27 (d, J = 246.0 (trifluoromethoxy)- phenyl)-1,2,4-oxadiazole (15a). White solid,

o

Hz), 151.37, 140.59, 137.93 (d, J = 7.0 Hz), 130.63 (d, J = 8.2 Hz), 31% yield. Mp 137-140 C. ¹H NMR (500 MHz, CDCl₃): δ 8.42 (d, J =

130.42, 129.48, 127.89, 125.42, 123.84, 123.20, 122.89 (d, J = 3.0 8.0 Hz, 1H, ArH), 8.35 (d, J = 9.0 Hz, 2H, ArH), 7.46-7.41 (m, 2H,

Hz), 121.88, 121.70 (q, J = 256.8 Hz), 121.05, 115.42 (d, J = 20.8 Hz), ArH), 7.39 (d, J = 8.5 Hz, 2H, ArH), 7.19 (t, J = 8.0 Hz, 1H, ArH), 6.89

114.45 (d, J = 22.0 Hz), 110.06, 53.57; MS (ESI), m/z: 455 [M+H]⁺; (d, J = 8.0 Hz, 1H, ArH), 6.76 (d, J = 8.0 Hz, 1H, ArH), 6.72 (s, 1H,

HRMS (TOF) m/z calcd for C₂₃H₁₄F₄N₄O₂[M+H]⁺: 455.1126, found ArH), 5.74 (s, 2H, benzylic-CH₂), 0.91 (s, 9H, 3×CH₃), 0.09 (s, 6H,

455.1123. HPLC purity = 97.98%, Rt 11.48 min.

5-(1-(Pyridin-2-ylmethyl)-1 -indazol-3-yl)-3-(4-(trifluoromethoxy)

2×CH₃). MS (ESI), m/z: 567 [M+H]⁺.

H

5-(1-(4-((Tert-butyldimethylsilyl)oxy)benzyl)-1H-indazol-3-yl)-3-(4-

phenyl)-1,2,4-oxadi- azole (4f). White solid, 52% yield. Mp 167-169 (trifluoromethoxy) phenyl)-1,2,4-oxadiazole (15b). White solid,

o

^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.51 (d, J = 4.0 Hz, 1H, ArH), 8.38 (d, 38% yield. Mp 141-143 C. ¹H NMR (500 MHz, CDCl₃): δ 8.41 (d, J =

J = 8.0 Hz, 1H, ArH), 8.33 (d, J = 8.5 Hz, 2H, ArH), 7.95 (d, J = 8.5 Hz, 8.0 Hz, 1H, ArH), 8.34 (d, J = 9.0 Hz, 2H, ArH), 7.46 (d, J = 8.0 Hz, 2H,

1H, ArH), 7.82 (t, J = 7.5 Hz, 1H, ArH), 7.60-7.64 (m, 3H, ArH), 7.52 (t, ArH), 7.42-7.39 (m, 1H, ArH), 7.38 (d, J = 9.0 Hz, 2H, ArH), 7.19 (d, J

J = 7.5 Hz, 1H, ArH), 7.30-7.34 (m, 2H , ArH), 6.02 (s, 2H, benzylic- = 8.5 Hz, 2H, ArH), 6.79 (d, J = 8.5 Hz, 2H, ArH), 5.72 (s, 2H, benzylic-

CH₂); ¹³C NMR (100 MHz, CDCl₃): δ 171.20, 167.77, 155.41, 151.37, CH₂), 0.95 (s, 9H, 3×CH₃), 0.16 (s, 6H, 2×CH₃). MS (ESI), m/z: 567

149.58, 140.96, 137.18, 130.47, 129.48, 127.86, 125.42, 123.85, [M+H]⁺.

123.11, 123.08, 121.76, 121.69 (q, J = 256.5 Hz), 121.65, 121.05, General Procedure for the Synthesis of Compounds 16a

110.58, 55.94; MS (ESI), m/z: 438 [M+H]⁺; HRMS (TOF) m/z calcd for solution of tetrabutyl ammonium fluoride (1.2 mmol) in THF (5 mL)

C₂₂H₁₄F₃N₅O₂[M+H]⁺: 438.1172, found 438.1174. HPLC purity = was added to a solution of intermediate 15a

15b (1.0 mmol) in THF

96.76%, Rt 8.58 min.

(10 mL) at 0 ^oC and the mixture was stirred at room temperature for

, 16b. A

,

General Procedure for the Synthesis of Compounds 5a, 5b. When 30 min. The completion of reaction was confirmed by TLC. The

4-fluorobenzyl chloride and 4-chloromethyl pyridine were used as mixture was partitioned between 35 mL EtOAc and 15 mL H₂O. The

starting materials, the minor products were collected through silica organic layer was separated, washed with H₂O and saturated saline,

gel chromatography.

dried over anhydrous Na₂SO₄. The solvent was removed under

vacuum and the residue was purified by silica gel chromatography.

5-(2-(4-Fluorobenzyl)-2H-indazol-3-yl)-3-(4-(trifluoromethoxy)

phenyl)-1,2,4-oxadiazole (5a). White solid, 27% yield. Mp 150-152 3-((3-(3-(4-(Trifluoromethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-1H-

^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.55 (d, J = 8.5 Hz, 1H, ArH), 8.46 (d, indazol-1-yl)methyl) phenol (16a). White solid, 95% yield. Mp 171-

J = 9.0 Hz, 2H, ArH), 8.00-7.95 (m, 3H, ArH), 7.79 (d, J = 8.5 Hz, 1H, 174 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.39 (d, J = 8.0 Hz, 1H, ArH),

ArH), 7.58 (t, J = 7.5 Hz, 1H, ArH), 7.42-7.46 (m, 2H, ArH), 7.13 (t, J = 8.28 (d, J = 9.0 Hz, 2H, ArH), 7.43 (d, J = 8.5 Hz, 2H, ArH), 7.40-7.37

9.0 Hz, 2H, ArH), 6.03 (s, 2H, benzylic-CH₂); ¹³C NMR (100 MHz, (m, 1H, ArH), 7.36 (d, J = 8.5 Hz, 2H, ArH), 7.23 (t, J = 7.5 Hz, 1H,

CDCl₃): δ 168.31, 167.31, 163.86 (d, J = 246.0 Hz), 151.55, 148.35, ArH), 6.89 (d, J = 8.0 Hz, 1H, ArH), 6.78-6.76 (m, 1H, ArH), 6.69 (s,

131.60 (d, J = 2.9 Hz), 129.87 (d, J = 8.2 Hz), 129.33, 127.27, 126.00, 1H, ArH), 5.73 (s, 2H, benzylic-CH₂). MS (ESI), m/z: 453 [M+H]⁺.

125.07, 123.66, 121.68 (q, J = 257.2 Hz), 121.25, 120.64, 118.99, 4-((3-(3-(4-(trifluoromethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-1H-

118.63, 115.82 (d, J = 20.9 Hz), 55.98; MS (ESI), m/z: 455 [M+H]⁺; indazol-1-yl)methyl) phenol (16b). White solid, 92% yield. Mp 220-

HRMS (TOF) m/z calcd for C₂₃H₁₄F₄N₄O₂[M+H]⁺: 455.1126, found 221 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.41 (d, J = 8.5 Hz, 1H, ArH),

455.1125. HPLC purity = 99.06%, Rt 14.10 min.

5-(2-(Pyridin-4-ylmethyl)-2 -indazol-3-yl)-3-(4-(trifluoromethoxy)

8.34 (d, J = 9.0 Hz, 2H, ArH), 7.47 (d, J = 2.5 Hz, 2H, ArH), 7.43-7.41

(m, 1H, ArH), 7.39 (d, J = 8.5 Hz, 2H, ArH), 7.28 (d, J = 8.5 Hz, 2H,

H

phenyl)-1,2,4-oxadi- azole (5b). White solid, 25% yield. Mp 185-188 ArH), 6.88 (d, J = 9.0 Hz, 2H, ArH), 5.73 (s, 2H, benzylic-CH₂). MS

^oC. ¹H NMR (500 MHz, CDCl₃)：δ 8.63 (d, J = 6.0 Hz, 2H, ArH), 8.55 (ESI), m/z: 453 [M+H]⁺.

(d, J = 8.5 Hz, 1H, ArH), 8.12 (d, J = 8.5 Hz, 2H, ArH), 7.96-8.00 (m, General Procedure for the Synthesis of Compounds 17a and 17b.

3H, ArH), 7.71 (d, J = 8.5 Hz, 1H, ArH), 7.59 (t, J = 8.0 Hz, 1H, ArH), anhydrous K₂CO₃(3.0 mmol) was added to

a mixture of

7.21 (d, J = 5.5 Hz, 2H, ArH), 6.13 (s, 2H, benzylic-CH₂); ¹³C NMR (100 intermediate 16a

,

16b (0.5 mmol), 1,2-dibromoethane (5.0 mmol)

MHz, CDCl₃): δ 168.04, 167.33, 151.60, 150.31, 148.53, 144.63, in dry acetone (20 mL) , and the mixture was refluxed for 24 h. The

129.30, 127.60, 126.31, 124.89, 123.58, 122.08, 121.27 (q, J = 216.5 completion of the reaction was confirmed by TLC. The solvent was

Hz), 121.22, 120.63, 119.52, 118.67, 55.71; MS (ESI), m/z: 438 removed under vacuum and the residue was dispartitioned

[M+H]⁺; HRMS (TOF) m/z calcd for C₂₂H₁₄F₃N₅O₂[M+H]⁺: 438.1172, between 35 mL EtOAc and 15 mL H₂O. The organic layer was

found 438.1173. HPLC purity = 100.00%, Rt 9.17 min.

separated, washed with H₂O and saturated saline, dried over

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anhydrous Na₂SO₄. The solvent was removed under vacuum and m/z: 580 [M+H]⁺; HRMS (TOF) m/z calcd for C₃₀H₂₈F₃N₅O₄[M+H]⁺:

the residue was purified by silica gel chromatography.

5-(1-(3-(2-bromoethoxy)benzyl)-1 -indazol-3-yl)-3-(4-(trifluorome

580.2166, found 580.2164. HPLC purity = 98.84%, Rt 9.64 min.

H

4-(2-(3-((3-(3-(4-(Trifluoromethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-

thoxy)phenyl)-1,2,4-oxadiazole (17a). White solid, 52% yield. Mp 1H-indazol-1-yl)methyl)phenoxy)ethyl)morpholine (4i)

.

White

162-165 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.43 (d, J = 8.5 Hz, 1H, solid, 61% yield. Mp 61-63 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.41 (d, J

ArH), 8.34 (d, J = 9.0 Hz, 2H, ArH), 7.48-7.45 (m, 2H, ArH), 7.43-7.40 = 8.0 Hz, 1H, ArH), 8.33 (d, J = 9.0 Hz, 2H, ArH), 7.49-7.41 (m, 2H,

(m, 1H, ArH), 7.39 (d, J = 8.0 Hz, 2H, ArH), 7.26-7.24 (m, 1H, ArH), ArH), 7.42-7.39 (m, 1H, ArH), 7.38 (d, J = 8.0 Hz, 2H, ArH), 6.89 (d, J

6.92 (d, J = 7.5 Hz, 1H, ArH), 6.85-6.83 (m, 2H, ArH), 5.76 (s, 2H, = 7.5 Hz, 1H, ArH), 6.84-6.82 (m, 2H, ArH), 5.74 (s, 2H, benzylic-CH₂),

benzylic-CH₂), 4.24 (t, J = 7.5 Hz, 2H, CH₂), 3.60 (t, J = 7.5 Hz, 2H, 4.05 (t, J = 5.5 Hz, 2H, CH₂), 3.71 (t, J = 4.5 Hz, 4H, morpholine), 2.75

CH₂). MS (ESI), m/z: 559 [M+H]⁺.

(t, J = 5.5 Hz, 2H, CH₂), 2.54 (t, J = 4.5 Hz, 4H, morpholine); ¹³C NMR

5-(1-(4-(3-Bromoethoxy)benzyl)-1H-indazol-3-yl)-3-(4-(trifluorome

(125 MHz, CDCl₃): δ 171.24, 167.76, 159.14, 151.33, 140.56, 136.95,

thoxy)phenyl)-1,2,4-oxadiazole (17b). White solid, 46% yield. Mp 130.06, 130.02, 129.48, 127.71, 125.40, 123.75, 123.20, 122.45 (q, J

132-134 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.42 (d, J = 8.0 Hz, 1H, = 261.2Hz), 121.74, 121.07, 119.84, 114.20, 113.76, 110.37, 66.83,

ArH), 8.34 (d, J = 8.5 Hz, 2H, ArH), 7.49-7.45 (m, 2H, ArH), 7.43-7.41 65.68, 57.50, 54.23, 54.03；MS (ESI), m/z: 566 [M+H]⁺; HRMS (TOF)

(m, 1H, ArH), 7.39 (d, J = 8.5 Hz, 2H, ArH), 7.28 (d, J = 8.5 Hz, 2H, m/z calcd for C₂₉H₂₆F₃N₅O₄[M+H]⁺: 566.2010, found 566.2014. HPLC

ArH), 6.88 (d, J = 9.0 Hz, 2H, ArH), 5.73 (s, 2H, benzylic-CH₂), 4.27 (t, purity = 96.35%, Rt 10.74 min.

J = 6.0 Hz, 2H, CH₂), 3.62 (t, J = 7.5 Hz, 2H, CH₂). MS (ESI), m/z: 559 1-(2-(4-((3-(3-(4-(Trifluoromethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-

[M+H]⁺.

1H-indazol-1-yl)methyl) phenoxy)ethyl)piperidin-4-ol (4j). White

General Procedure for the Synthesis of Compounds 4g-4j. solid, 62% yield. Mp 141-144 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.41

Anhydrous K₂CO₃(0.42 mmol) and secondary amine (0.42 mmol) (d, J = 8.0 Hz, 1H, ArH), 8.34 (d, J = 9.0 Hz, 2H, ArH), 7.47 (d, J = 8.5

was added to a solution of 17a or 17b (0.072 mmol) in dry CH₃CN (4 Hz, 2H, ArH), 7.42-7.39 (m, 1H, ArH), 7.39 (d, J = 8.5 Hz, 2H, ArH),

mL), then the mixture was refluxed for 24 h. The completion of 7.26 (d, J = 8.5 Hz, 2H, ArH), 6.86 (d, J = 9.0 Hz, 2H, ArH), 5.72 (s, 2H,

reaction was confirmed by TLC. Then the mixture was partitioned benzylic-CH₂), 4.07 (s, 2H, CH₂), 2.87 (s, 2H, CH₂), 2.80 (s, 2H,

between 15 mL EtOAc and 5 mL H₂O. The organic layer was piperidine), 2.30 (s, 2H, piperidine), 1.62 (s, 4H, piperidine), 1.39 (s,

separated, washed with H₂O and saturated saline, and dried over 1H, piperidine); ¹³C NMR (100 MHz, CDCl₃): δ 171.30, 167,75,

anhydrous Na₂SO₄. The solvent was removed under vacuum and 158.76, 151.36, 140.44, 129.92, 129.48, 128.86, 127.62, 127.56,

the residue was purified by silica gel chromatography.

125.49, 123.64, 123.28, 121.72, 121.04, 119.13, 114.97, 110.40,

5-(1-(3-(2-(4-Methylpiperazin-1-yl)ethoxy)benzyl)-1H-indazol-3-yl)- 67.66, 66.16, 56.96, 53.90, 51.49, 34.40; MS (ESI), m/z: 580 [M+H]⁺;

3-(4-(trifluoro-methoxy)phenyl)-1,2,4-oxadiazole (4g)

solid, 52% yield. Mp 103-105 ^oC. ¹H NMR (500 MHz, CDCl₃): δ 8.41 580.2169. HPLC purity = 98.70%, Rt 9.50 min.

.

Yellowish HRMS (TOF) m/z calcd for C₃₀H₂₈F₃N₅O₄[M+H]⁺: 580.2166, found

(d, J = 8.0 Hz, 1H, ArH), 8.33 (d, J = 8.5 Hz, 2H, ArH), 7.47-7.43 (m,

4.2 Biological Materias and Methods

2H, ArH), 7.42-7.40 (m, 1H, ArH), 7.38 (d, J = 8.5 Hz, 2H, ArH), 7.24

(t, J = 7.5 Hz, 1H, ArH), 6.91-6.84 (m, 2H, ArH), 6.83 (d, J = 8.0 Hz,

HIF-Luciferase Assay. Human colon carcinoma cell line HCT-116

1H, ArH), 5.74 (s, 2H, benzylic-CH₂), 4.03 (t, J = 6.0 Hz, 2H, CH₂), 2.76

(t, J = 6.0 Hz, 2H, CH₂), 2.57-2.46 (brd, 8H, piperazine), 2.70 (s, 3H,

CH₃); ¹³C NMR (125 MHz, CDCl₃): δ 171.25, 167.75, 159.21, 151.33,

140.56, 136.91, 130.04, 130.00, 129.49, 127.70, 125.43, 123.74,

123.20, 122.79 (q, J = 258.2Hz), 121.73, 121.06, 119.76, 114.20,

113.78, 110.39, 65.89, 57.03, 55.01, 54.97, 54.25, 53.53, 45.99；MS

(ESI), m/z: 579 [M+H]⁺; HRMS (TOF) m/z calcd for C₃₀H₂₉F₃N₆O₃

[M+H]⁺: 579.2326, found 579.2328. HPLC purity = 97.90%, Rt 11.08

min.

provided by Zhejiang University was revived in DMEM containing

10% heat-inactivated fetal bovine serum, Penicillin (100 IU/mL) and

Streptomycin (100 μg/mL). HCT-116 cells transfected with

a

luciferase report gene under the control of HIF-responsive element

and a renilla luciferase (internal control for transfection efficiency)

were seeded in 96-well plates with 6000 cells per well and

incubated overnight under normoxic conditions (20% O₂). After 24

hours, test compounds with different concentrations were added

into wells, and the cells were incubated under normoxic condition

(20% O₂) for 1 hour followed by 24 hours in the hypoxic condition

(1% O₂). Finally, the medium was aspirated, cells were lysed and

reporter activities were measured using the Dual-Luciferase

reporter assay system (Promega, Madison, Wis., USA). IC₅₀was

calculated with Bliss method.

1-(2-(3-((3-(3-(4-(Trifluoromethoxy)phenyl)-1,2,4-oxadiazol-5-yl)-

1H-indazol-1-yl)methyl) phenoxy)ethyl)piperidin-4-ol (4h). White

solid, 86% yield. Mp 107-110 ^oC. ¹H NMR (500 MHz, DMSO-d₆): δ

8.37 (d, J = 8.5 Hz, 1H, ArH), 8.33 (d, J = 8.5 Hz, 2H, ArH), 8.00 (d, J =

8.5 Hz, 1H, ArH), 7.65-7.60 (m, 3H, ArH), 7.50 (t, J = 7.5 Hz, 1H, ArH),

7.26 (t, J = 8.0 Hz, 1H, ArH), 6.94 (t, J = 1.5 Hz, 1H, ArH), 6.88-6.86

(m, 2H, ArH), 5.87 (s, 2H, benzylic-CH₂), 4.53 (d, J = 4.0 Hz, 1H, OH),

4.02 (t, J = 5.5 Hz, 2H, CH₂), 3.44-3.39 (m, 1H, piperidine), 2.74-2.72

(m, 2H, CH2), 2.61-2.59 (m, 2H, piperidine), 2.09-2.05 (m, 2H,

piperidine), 1.68-1.65 (m, 2H, piperidine), 1.37-1.32 (m, 2H,

piperidine); ¹³C NMR (125 MHz, CDCl₃): δ 171.23, 167.75, 158.93,

151.33, 140.56, 136.98, 130.06, 129.48, 127.74, 125.38, 123.76,

123.18, 122.12 (q, J = 256.4Hz), 121.73, 121.07, 119.92, 114.13,

113.81, 110.38, 65.39, 56.58, 54.19, 50.94, 50.91, 33.50；MS (ESI),

Western Blotting Assay. Human colon cancer cell line HCT116 was

cultured under normoxia (20% O₂) or hypoxia (1% O₂) in the

absence or presence of test compounds for 24 hours. Proteins were

then extracted with lysis buffer and the lysates were centrifuged to

get equivalent amounts of proteins which were analyzed by SDS-

polyacrylamide gel electrophoresis. Proteins were visualized using

enhanced chemiluminescence detection reagents (GE Healthcare,

Buckinghamshire, UK).

RT-PCR. Total RNA from HCT116 cells was isolated using the Trizol

reagent (Sangon Biotech Co., Ltd), and cDNA was synthesized using

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2 µg of total RNA with random hexamer primers and the Moloney Statistical analysis. For all parameters measured, the values for all

murine leukemia virus reverse transcriptase (M-MuLV RT) samples in the different experiment conditions were averaged and

(Fermentas International Inc., Burlington, Ontario, Canada). The the SD of the mean was calculated. ANOVA followed by Student’s t

conditions used for reverse transcription-PCR were as follows: 10 test was used for the statistical analysis.

min at 25 °C, 60 min at 42°C and 15 min at 72 °C. The cDNA was

Acknowledgments

subjected to PCR amplification using the following forward and

This study was financially supported by the National Natural Science

Foundation of China (21072171, 81072575) and the Science and

Technology Department of Zhejiang Province (2014C33181). We

thank Mrs Jianyang Pan (Pharmaceutical Informatics Institute,

reverse

primer

sets:

HIF-1α,

forward

primer:

5′-

TCACCACAGGACAGTACAGGATGC-3′ and reverse primer: 5′-

CCAGCAAAGTTAAAGCATCAGGTTCC-3′; GAPDH, forward primer: 5′-

GTCATCCATGACAACTTTGG-3′

and

reverse

primer:

5′-

Zhejiang University) for performing the NMR and MS spectrometry

.

GAGCTTGACAAAGTGGTCGT-3′. The housekeeping gene GAPDH was

used as the internal standard. Quantitative real-time RT-PCR

analysis was performed by TAKARA SYBR Premix EXTaqTM. The

reaction mixtures containing SYBR Green were composed following

the manufacturer's protocol. The cycling program was 95 °C for 30

s, 58 °C or 70 °C (GADD153) for 20 s, and 72 °C for 30 s followed by

40 cycles using an Eppendorf epGradient Mastercycler (Eppendorf,

Hamburg, Germany).

Notes

The authors declare no competing financial interest.

Abbreviations

HIF-1, hypoxia inducible factor-1; pVHL, Von Hippel–Lindau

tumor suppressor; ARD1, arrest defective 1; SUMO, small ubiquitin-

related modifier; RACK1, receptor for activated C kinase 1; PI3K,

phosphatidylinositol-4,5-bisphosphate 3-kinase; mTOR, mammalian

target of rapamycin; HDAC, histone deacetylase; HRE, hypoxic

response element; HSP90, heat shock protein 90; VEGF, vascular

endothelial growth factor; VEGFR, vascular endothelial growth

factor receptor; CAIX, carboanhydrase IX; EPO, erythropoietin; SD,

standard deviation; SDS, Sodium Dodecyl Sulfonate; RT-PCR,

reverse transcription-polymerase chain reaction; PBS, phosphate

Cell Migration Assay. A human ovarian cancer cell SKOV3 migration

assay was performed in a Transwell Boyden chamber containing a

polycarbonate filter with a pore size of 8 μm. SKOV3 cells (10000

cells/ml) were maintained in the upper chamber with serum-free

medium before treated with test compounds. The lower chamber

contained medium plus 10% FBS as chemoattractant. The chamber

was incubated under normoxia (20% O₂) for

1 hour before

transferring to hypoxia (1% O₂), or continuously incubated under

normoxia for another 24 hours. All nonmigrant cells were then

removed by Phosphate Buffered Saline (PBS) washing. The migrated

cells were fixed with 90% EtOH and stained with 0.1% crystal violet

in 2% MeOH. The stained cells were subsequently photographed

and the number of cells passed through the polycarbonate filter

was calculated in five randomly selected, nonoverlapping fields

under a light microscope.

buffered

saline;

GAPDH,

glyceraldehyde-3-phosphate

dehydrogenase ; DMEM, dulbecco's modified eagle medium; IC₅₀

,

the half maximal (50%) inhibitory concentration of a substance;

THF, tetrahydrofuran; DMSO, dimethyl sulfoxide; rt, room

temperature;

DCM,

dichloromethane;

DMF,

N,N-

dimethylformamide;

EDCI,

1-ethyl-3-(3-dimethylaminopropyl)

carbodiimide hydrochloride; HOBT, hydroxybenzotriazole; TBDMS,

tert-butyldimethylsilane; TBAF, tetrabutyl ammonium fluoride;

DIPEA, N,N-Diisopropylethylamine.

HIF-1 transcriptional activity assay in vivo. HCT116 colon

carcinoma cells stably transfected with the hypoxia-inducible

luciferase reporter gene (HRE-luciferase) were implanted in twenty

nude mice. When the tumor volume reached 200 mm³, the mice

were divided into five groups followed by i.v. administration of

vehicle or test compounds for three consecutive days (50 mg/kg,

once daily). HRE-driven FLuc activity was determined by Xenogen

imaging and the intensity of luminescence was calculated by

software. Animal study was approved by the Animal Research

Committee at Zhejiang University (log number Zju2013101032), and

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Novel potent HIF-1 inhibitors for the prevention of tumor metastasis: discovery and optimization of 3-aryl-5-indazole-1,2,4-oxadiazole derivatives

DOI: 10.1039/c5ra15191k

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