Synthesis and Activity Evaluation of Nasopharyngeal Carcinoma Inhibitors Based on 6-(Pyrimidin-4-yl)-1H-indazole

Article abstract of DOI:10.1002/cbdv.201800598

Human nasopharyngeal carcinoma is a common head and neck malignancy with high incidence in Southeast Asia and Southern China. It is necessary to develop safe, effective and inexpensive anticancer agents to improve the therapeutics of patients with nasopharyngeal carcinoma. A series of small molecular compounds based on 6-(pyrimidin-4-yl)-1H-indazole were synthesized and evaluated for antiproliferative activities against human nasopharyngeal carcinoma cell lines SUNE1. Compounds 6b, 6c, 6e and 6l showed potent antiproliferative activities similar to positive control drug cisplatin in vitro with lower nephrotoxicity than it. N-[4-(1H-Indazol-6-yl)pyrimidin-2-yl]benzene-1,3-diamine (6l) was selected for further study. It was found that 6l induced mitochondria-mediated apoptosis and G₂/M phase arrest in SUNE1 cells. Furthermore, compound 6l at 10 mg/kg can suppress the growth of an implanted SUNE1 xenograft with a TGI% (tumor growth inhibition) value of 50 % and did not cause serious side effects in BALB/c nude mice. This study suggests that 6-(pyrimidin-4-yl)-1H-indazole derivatives are a series of small molecule compounds with anti-nasopharyngeal carcinoma activities.

Full text of DOI:10.1002/cbdv.201800598

Title: Synthesis and Activity Evaluation of Nasopharyngeal Carcinoma
Inhibitors Based on 6-(pyrimidin-4-yl)-1H-indazole
Authors: Bohong Liao, Jin Zhou, Zike Yang, Xiaowen Guo, Peiquan
Zhang, Xin Zhang, zhibo zhu, Lingrong Peng, Huiting Mo, and
Jialan Zhao
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content of this Accepted Article.
To be cited as: Chem. Biodiversity 10.1002/cbdv.201800598
Link to VoR: http://dx.doi.org/10.1002/cbdv.201800598

Chemistry & Biodiversity
10.1002/cbdv.201800598
Synthesis and Activity Evaluation of Nasopharyngeal Carcinoma
Inhibitors Based on 6-(pyrimidin-4-yl)-1H-indazole
a#
b#
a
a
a
a
Bohong Liao , Lingrong Peng , Jin Zhou , Huiting Mo , Jialan Zhao , Zike Yang ,
a
c
a
a*
Xiaowen Guo , Peiquan Zhang , Xin Zhang , Zhibo Zhu
aIntegrated Hospital of Traditional Chinese Medicine, Southern Medical University,
Guangzhou, Guangdong 510315, P. R. China
bDepartment of Radiology, The Third Affiliated Hospital of Sun Yat-Sen University,
Guangzhou, Guangdong 510630, P.R. China
cGuangdong Provincial Key laboratory of New drug screening, School of
Pharmaceutical Sciences, Southern Medical University, Guangzhou, Guangdong
5
10515, P.R. China
*Corresponding to: Z. Zhu, Integrated Hospital of Traditional Chinese Medicine,
#
Southern Medical University, 13 Shiliugang Road, Haizhu district, Guangzhou
5
10315, China. Tel: 86 (20) 61650510; Fax: 86 (20) 61650039. E-mail:
zhuzb676@smu.edu.cn
#
These authors contributed equally
Abstract: Human nasopharyngeal carcinoma is a common head and neck malignancy
with high incidence in Southeast Asia and Southern China. It is necessary to develop
safe, effective and inexpensive anti-cancer agents to improve the therapeutics of
patients with nasopharyngeal carcinoma. A series of small molecular compounds
based on 6-(pyrimidin-4-yl)-1H-indazole were synthesized and evaluated for
antiproliferative activities against human nasopharyngeal carcinoma cell line SUNE1.
Compound 6b, 6c, 6e and 6l showed potent antiproliferative activities similar to
positive control drug cisplatin in vitro with lower nephrotoxicity than it. 6l was
selected for further study. It was found that 6l induced mitochondria-mediated
2
apoptosis and G /M phase arrest in SUNE1 cells. Furthermore, 6l at 10 mg/kg can
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Chemistry & Biodiversity
10.1002/cbdv.201800598
suppress the growth of an implanted SUNE1 xenograft with a TGI% (tumor growth
inhibition) value of 50% and did not cause serious side effects in BALB/c nude mice.
This study suggests that 6-(pyrimidin-4-yl)-1H-indazole derivatives are a series of
small molecule compounds with anti-nasopharyngeal carcinoma activities.
Keywords: nasopharyngeal carcinoma, indazole, antiproliferative, apoptosis, phase
arrest
Introduction
Human nasopharyngeal carcinoma (NPC) is a common head and neck
malignancy prominently in Southeast Asia and Southern China. Though trends in
declining incidence and mortality of NPC were reported, age-standardized rates in
incidence remained 20/100,000 in southern China in 2012.[1] Due to its high radio
sensitivity, the standard treatment for NPC is radiotherapy, in fact local radiation and
surgery may provide good control of NPC. However, the prognosis of patients with
NPC still stays poor due to the advanced stage at the time of diagnosis, regional
[
2]
relapse, and distant metastasis. Conventionally, chemotherapy is given concurrently
with radiotherapy for treating locally advanced disease or distant metastasis. The main
chemotherapeutic agents of nasopharyngeal carcinoma are cisplatin, fluorouracil,
paclitaxel, docetaxel and so on. The curative effects of chemotherapy for advanced
NPC patients remain unsatisfactory and the overall survival of recurrent or advanced
NPC patients is usually poor, with reported median survival range from 7.2 to 22
[
3]
months. Moreover, adverse effects, including upper gastrointestinal impairment and
bone marrow suppression, depressed the toleration and limited the clinical use of
chemotherapeutic agents.[4] These urge us to explore new strategies based on
molecular mechanisms and the disease characteristics to improve the therapeutics of
patients with NPC.
Indazole is an important intermediate, derivatives of which have shown a wide
spectrum of biological activities such as anti-bacterial, anti-schizophrenia, cytotoxic
effect against cancer cell lines and anti-tumor.[5-9] Recently, cytotoxic effect against
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Chemistry & Biodiversity
10.1002/cbdv.201800598
cancer cell lines and anti-tumor activity of a variety of indazole derivatives were
reported worldwide (Fig. 1). A indazole-based covalent inhibitor 1 which targeted
drug-resistant EGFR exhibited potent inhibition to non-small cell lung cancers cells
H1975 (the EGFR-L858R/T790M drug-resistant cell line) and HCC827 (the
EGFR-delE746_A750 activating mutation cell line) with IC50 values of 694 nM and
4
59 nM, respectively.[10] Its suppression on phosphorylation of EGFR and
downstream cascade was demonstrated by Western blot analysis. Fibroblast growth
factor receptor inhibitor 2 based on 1H-indazol-3-amine showed wonderful
antiproliferative activity to KG1 cell lines and SNU16 cell lines, IC50 of which were
2
5.3 ± 4.6 nM and 77.4 ± 6.2 nM respectively.[11] Multi-target RTKs inhibitor 3
bearing 1H-indazole-3-amine displayed antiproliferative activity to a broad spectrum
of cancer cells with IC50 values ranging from 1.80 μM to 13.26 μM, such as HepG2,
SMMC-7721 and MIAPaCa-2.[9] In addition, some skeletons based on indazole or
containing indazole with anti-tumor activities have been reported.[12-16] These studies
show that indazole derivatives have promising potential for applications in
pharmaceutical industry.
Aminoindazole compound 4 identified by researchers at GlaxoSmithKline was
reported as a potent leadlike PDK1 inhibitor.[17] Compound 5 (GSK2334470) with
higher potency and selectivity against PDK1 was developed after further optimization
and its cytotoxic effect against cancer cell lines was demonstrated (IC50 against
MM.1R = 4.89 µM).[18-21] Based on its antiproliferative activities and our effort to
develop safe and effective nasopharyngeal carcinoma inhibitors, the antiproliferative
activity to human nasopharyngeal carcinoma cell line SUNE1 of GSK2334470 was
measured. It only displayed moderate antiproliferative activities to SUNE1 cell line
(IC50 = 24.52 ± 2.73 µM). Making it as a lead compound, we attempted to improve its
potency against SUNE1 cell line. It was found that introduction of substituent on
nitrogen site at position 2 of pyrimidine ring slightly improve cytotoxic effect (The
IC50 of 6d is 20.28 ± 1.12 µM). Therefore we introduced a variety of substituents on
this position to improve potency of lead compound, and synthesized a series of
derivatives 6a-m and 7a. It was demonstrated that indazole derivatives we
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Chemistry & Biodiversity
10.1002/cbdv.201800598
synthesized can suppress the growth of SUNE1 cell line in vitro with lower
nephrotoxicity than cisplatin. Preliminary research indicates that these compounds
inhibited the growth of SUNE1 cells by mitochondria-mediated apoptosis and G /M
2
phase arrest. And 6l can suppress the growth of an implanted SUNE1 xenograft
without serious side effects in BALB/c nude mice. Herein, we will report the
synthesis and activity evaluation of these compounds.
Fig. 1. Chemical structures of indazole derivatives 1-5 with cytotoxic effect and new
compounds 6a-m and 7a.
Results and discussion
Chemistry
The general synthesis for compound 6a-m is shown in Scheme 1. For protecting
the 1-amino group, 6-bromoindazole (8) was heated to reflux with dihydropyran (9),
pyridinium 4-toluenesulfonateto and p-toluenesulfonic acid monohydrate in
dichloromethane to obtain compound 10.[22] Compound 12 was generated from 10 and
bis(pinacolato)diboron (11) with potassium acetate as a base via classic
palladium-catalyzed Miyaura borylation.[23,24] The Suzuki reaction was performed on
compound 12 and 2,4-dichloropyrimidine (13) using Pd(PPh
3
4
) as the catalyst to
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Chemistry & Biodiversity
10.1002/cbdv.201800598
generate the intermediate 14.[25] To produced compounds 16a-m, coupling of the
intermediate 14 with various amines (15a-m) was performed under one of the typical
Buchwald-Hartwig amination reaction conditions with
tris(dibenzylideneacetone)dipalladium(0) (Pd (dba) ) and RuPhos as a ligand in the
2
3
presence of caesium carbonate in refluxing 1,4-dioxane.[23,26] The THP group was
removed from 16a-m with HCl in ethanol to give the target compound 6a-m.[27]
Scheme 2 shows the synthesis of compound 7a. Nucleophilic substitution of
3
,5-dibromopyrazin-2-ol (17) with aniline (18) produced compound 19.[23,26] Finally,
compound 7a was prepared from 19 and 12 via Suzuki reaction.[25]
Reagents and conditions: (a) PPTS, p-toluenesulfonic acid monohydrate, dichloromethane, 60
ºC reflux; (b) 1,1'-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride dichloromethane
complex, KOAc, 1,4-dioxane, 110 ºC reflux; (c) Pd(PPh
3
)
4
, saturated sodium bicarbonate
solution, 1,4-dioxane, 95 ºC; (d) Pd
ethanol; 70 ºC.
2
(dba)
3
, RuPhos, Cs
2
CO , 1,4-dioxane, 110 ºC; (e) HCl,
3
Scheme 1. Synthesis of 6a-m
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Chemistry & Biodiversity
10.1002/cbdv.201800598
3 4
Reagents and conditions: (f) camphorsulfonic acid, isopropanol, 82 ºC; (g) i) Pd(PPh ) ,
saturated sodium bicarbonate solution, 1,4-dioxane, 110 ºC reflux; ii) HCl, ethanol; 70 ºC.
Scheme 2. Synthesis of 7a
Antiproliferative activity and nephrotoxicity
The synthesized derivatives 16b-d, 6a-m and 7a were evaluated for
antiproliferative activities against SUNE1 cells using CCK8 assay. Cisplatin was
included as the positive control drug. Cytotoxicity of these compounds against
MDCK cells were also determined to evaluate nephrotoxicity. The results were listed
in Table 1. The most active compounds were 6b, 6c, 6e and 6l. Similar to cisplatin,
they displayed antiproliferative potency in the low micromolar range (IC50
=
4
.34-9.27 μM). Compounds 16b, 16c, 16d, 6d, 6f, 6i and 6m possessed modest
activity with IC50 range 20.28-34.98 μM. The rest of the compounds showed no
activities on SUNE1 (IC50 > 100 μM). CC50 Values in Table 1 showed that most of the
compounds demonstrated lower cytotoxicity than cisplatin (CC50 = 8.45 ± 0.68 μM)
on MDCK cell line, which indicated that they possess low nephrotoxicity.
Table 1. Antiproliferative activity and nephrotoxicity of cisplatin, 16b-d, 6a-m and 7a in vitro
compounds
cisplatin
R
1
R
2
IC50 (μM)a
CC50 (μM)b
6.16 ± 0.73c
8.45 ± 0.68
1
6b
m-tolyl
Tetrahydro-2H-pyran-2-yl
20.91 ± 0.85
NDd
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1
6c
1-methyl-1H-pyrazol-4-yl
2-fluorophenyl
p-tolyl
Tetrahydro-2H-pyran-2-yl
24.78 ± 1.53
34.98 ± 2.76
> 100
ND
ND
1
6d
Tetrahydro-2H-pyran-2-yl
6
a
H
H
H
H
H
H
H
H
H
H
H
H
H
> 200
6
b
m-tolyl
4.34 ± 0.26
8.28 ± 0.99
20.28 ± 1.12
9.27 ± 0.54
33.72 ± 2.85
> 100
44.63 ± 5.57
33.19 ± 3.37
> 100
6
c
pyridin-3-yl
6
d
2-fluorophenyl
6
e
43.70 ± 3.68
> 200
6
f
4-methylthiazol-2-yl
3-fluorophenyl
3-chlorophenyl
phenyl
6
6
g
> 200
h
> 100
> 200
6
i
26.08 ± 0.90
> 100
58.32 ± 4.84
> 200
6
j
pyrimidin-2-yl
5-methylpyridin-2-yl
3-aminophenyl
thiazol-2-yl
6
k
> 100
> 200
6
l
6.37 ± 0.93
26.23 ± 1.63
24.55 ± 3.02
19.38 ± 2.68
> 200
6
m
7
a
68.80 ± 2.05
a
b
c
Antiproliferative activities against SUNE1 cells. Cytotoxicity against MDCK cells. Values
d
shown are the mean ± SD (n =3). ND is not determined.
The structure-activity relationship
The structure-activity relationship of these compounds can be analyzed by IC50
values and chemical structure (Table 1). As for R
1
substituent, phenyl (6b),
pyridin-3-yl (6c), isoindolin-2-yl (6e) and 3-aminophenyl (6l) contributed most to
antiproliferative activities. Group thiazol-2-yl (6m) contribute to activities. But
pyrimidin-2-yl (6j) made compound lose its activities. Compounds with a substitution
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Chemistry & Biodiversity
10.1002/cbdv.201800598
at the 3-position of the benzene ring (6b, 6l) were superior to those with substitutions
at the 2- (6d) and 4-positions (6a, 6k) on the phenyl group. Compounds containing
electron-donating group (6b, 6l) indicated more potent activity than compounds
containing electron-withdrawing group (6g, 6h). As for R
tetrahydropyran ring with hydrogen enhances the activity (6b > 16b, 6c > 16c, 6d >
6d).
2
substituent, substitution of
1
Dynamic changes of cell morphology after compound 6l administration
l was selected for further study. In order to pinpoint the onset time and process
6
of compound 6l, we observed the morphological changes of SUNE1 cells dynamically
with YeeSpec Automatic microscopic imaging system. Cell death began to occur at 17
h after compound 6l administration compared with before treatment. More than half
cells died by 24 h and all cells died by 48 h. A large number of cell debris can be
found in the cell culture medium (Fig. 2). These results suggested that the duration of
compound 6l induced SUNE1 cell death was 17-48 h after compound 6l
administration.
Fig. 2. Dynamic changes of cell morphology were observed by YeeSpec Automatic microscopic
imaging system at the time point of 17 h, 24 h and 48 h after 6l administration.
Dynamic monitoring antiproliferative activity of compound 6l via xCELLigence
system
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To further characterize compound 6l, SUNE1 cells exposed to selected
compounds were dynamically monitored for over 70 h using real-time electrical
impedance as a measure of viable cell number. After compound 6l administration, the
CI (cell index) value of SUNE1 cells slightly increase within 24 h, slowly decreased
over time and dropped down to baseline by 82 h (Fig. 3). The antiproliferative activity
of compound 6l was strengthened with the increase of concentration. These data
suggested that compound 6l kill tumor cells in a time-and dose-dependent manner.
Fig. 3. SUNE1 cells exposed to compound 6l were dynamically monitored over 70 h using
real-time electrical impedance as a measure of viable cell number.
Compound 6l induced apoptosis of nasopharyngeal carcinoma cell
To further evaluate the cytotoxicity of compound 6l, we tested whether 6l
induces the cell death via apoptosis. SUNE1 cells were treated with compound 6l at
different concentrations of 2 μM, 4 μM, 6 μM, and 8 μM (Fig. 4). The apoptotic cells
were measured by double staining of Annexin-V and propidium iodide (PI) at
different time of 12 h, 24 h, 36 h, and 48 h. No obvious change of the percentage of
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Chemistry & Biodiversity
10.1002/cbdv.201800598
early apoptotic cells were observed at different time point, but the percentage of late
apoptotic cells gradually increased over time, with measured values 11.8%, 16.7%,
8.5% and 23.5% at the time point of 12 h, 24 h, 36 h, and 48 h, respectively.
1
Moreover, the percentage of apoptotic cells rose gradually with the increase of 6l
concentration. 15.6%, 14.5%, 25.4% and 43.5% early apoptotic cell were induced at
the different dose of 2 μM, 4 μM, 6 μM, and 8 μM, respectively. Therefore, the
antiproliferative effect of compound 6l is caused by inducing cell apoptosis.
Fig. 4. SUNE1 cells were treated with compound 6l at the different concentrations of 2 μM, 4 μM,
6
μM, and 8 μM. The apoptotic cells were measured by double staining of Annexin-V and PI at
different time points 12 h, 24 h, 36 h, and 48 h.
Compound 6l induced mitochondria-mediated apoptosis in SUNE1 cells
To determine whether 6l treatment could induce apoptosis through the
mitochondrial pathway, normally growing SUNE1 cells were treated with different
concentrations of 6l. After 48 h, the cells were stained with Rhodamine 123. The
mitochondrial membrane potential decreased significantly with the increase of 6l
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Chemistry & Biodiversity
10.1002/cbdv.201800598
concentration compared with the untreated group (Fig. 5). These results suggested
that 6l may induce apoptosis through the mitochondrial pathway.
Fig. 5. The mitochondrial membrane potential (MMP) was analyzed using the fluorescent dye,
Rhodamine 123. SUNE1 cells were seeded on glass coverslips and treated with different
concentrations of 6l. Twenty-four hours after incubation, the cells were fixed with rhodamine 123
for 30 min, the mean fluorescence intensities of the granulosa cells were determined using Flow
Cytometer.
Compound 6l induce G /M phase arrest in SUNE1 cells dose-dependently
2
Cell cycle arrest is an important sign for the inhibition of proliferation and the
series of events take place in cells, leading to cell division and replication. Cells were
washed with PBS, fixed with ice-cold 70% ethanol overnight, stained with PI, and
analyzed for cell cycle distribution by flow cytometry. As shown in Fig. 6, the percent
of G /M-phase cells rose gradually with the increase of concentration. The results
2
indicate that compound 6l can induce G /M phase arrest in SUNE1 cells
2
dose-dependently.
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Chemistry & Biodiversity
10.1002/cbdv.201800598
Fig. 6. SUNE1 cells treated with compound 6l at the different concentrations of 2 μM, 4 μM, 6
μM, and 8 μM were analyzed for cell cycle distribution by flow cytometry.
In vivo anti-tumor activity of 6l
To evaluate the in vivo anti-tumor activity of compound 6l, a human
nasopharyngeal carcinoma xenograft model established by inoculating
subcutaneously BALB/c nude mice with SUNE1 cells was used. Mice in treatment
group were intraperitoneally treated with 6l at a dose of 10 mg/kg everyday for 14
consecutive days (Fig. 7). As shown in Fig. 7 a) and b), 6l at 10 mg/kg could
significantly suppress the growth of tumor in vivo (p = 0.003 < 0.05), and the TGI%
(tumor growth inhibition) value was 50% at day 14. In addition, weight change and
other serious side effects were not observed during 6l administration (Fig. 7 c)).
Therefore, these results demonstrate the safety and efficacy of 6l in vivo.
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Chemistry & Biodiversity
10.1002/cbdv.201800598
Fig. 7. In vivo anti-tumor activity of 6l. a) The average tumor volume of treatment group and
vehicle group mice during 6l administration. b) The average tumor weight of treatment group and
vehicle group mice at day 14. c) The average body weight of treatment group and vehicle group
mice during 6l administration. All the values were shown as mean ± SD (n = 7 per group), * P <
0
.05.
Conclusion
In summary, we synthesized a series of indazole derivatives. In this research, we
not only evaluated the inhibition of these compounds against nasopharyngeal
carcinoma in vitro and vivo, but also preliminarily investigated the action mechanism.
Some compounds (6b, 6c, 6e, 6l) showed potent inhibition against SUNE1 cells in
vitro .with lower nephrotoxicity than cisplatin. Compound 6l was found to induce
SUNE1 cell death at the time point of 17 h, 24 h and 48 h after administration and
exert antiproliferative activity in a time- and dose-dependent manner. Further study
demonstrated that 6l induced mitochondria-mediated apoptosis and G /M phase arrest
2
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Chemistry & Biodiversity
10.1002/cbdv.201800598
in SUNE1 cells. To our delight, 6l at 10 mg/kg showed moderate anti-tumor activity
without serious side effects in vivo. Therefore, 6-(pyrimidin-4-yl)-1H-indazole
derivatives are a series of small molecule compounds with potent anti-nasopharyngeal
carcinoma activities.
Experimental Section
Chemical material and equipment
Reagents for synthesis were obtained commercially, unless otherwise noted. Pyran
was dried by distillation from sodium. Flash chromatography was performed on silica
1
gel 60 (230-240 mesh, 60 Å) with the indicated eluents. H NMR was recorded at 400
1
3
or 500 MHz and C NMR at 100 or 125 MHz on a Bruker DPX-400 or DPX-500
instrument. Mass spectra were recorded with Bruker MicroTof. The purity of all
compounds was determined by a Shimadzu LC-20AT HPLC system with detection at
2
54 and 365 nm and C18-bonded silica column (InertSustain, 4.6 × 150 mm). The
column was perfused at a flow rate of 1 mL/min with methanol, and a linear gradient
from 10 to 90% of water over 120 min was adopted for elution.
Chemistry
Synthesis of 6-bromo-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (10): To a solution
of 6-bromoindazole (10 mmol, 1970 mg), PPTS (1.5 mmol, 380 mg), and
p-toluenesulfonic acid monohydrate (2.5 mmol, 430 mg) in dichloromethane (30 mL)
was added dihydropyran (6 mL) and the mixture was refluxed under argon at 60 ºC
overnight. After cooling, the mixture was extracted with dichloromethane, washed
with saline. The organic layer was collected and the solvent was removed under
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Chemistry & Biodiversity
10.1002/cbdv.201800598
vacuum. The crude product was purified by silica gel column chromatography
hexane / ethyl acetate 5 : 1) to give yield white solid as product (1886 mg, yield
7%).
Synthesis of 1-(tetrahydro-2H-pyran-2-yl)-6-(4,4,5,5-tetramethyl-1,3,2-
(
6
dioxaborolan-2-yl)-1H-indazole (12): To a solution of 6-bromo-1-(tetrahydro-2H-
pyran-2-yl)-1H-indazole (10.62 mmol, 2987 mg), bis(pinacolato)diboron (12.75
mmol,
3238
mg),
KOAc
(49.83
mmol,
4900
mg)
and
1
,1'-Bis(diphenylphosphino)ferrocene-palladium(II)dichloride
dichloromethane
complexsaline (1.00 mmol, 817 mg) in 1,4-dioxane (30 mL) was refluxed at 110 ºC
for 3 h under argon atmosphere. Then, the reaction mixture was extracted with ethyl
acetate, washed with saline. The organic phase was collected and the solvent was
removed under vacuum. The crude product was purified by silica gel chromatography
(hexane / ethyl acetate 5 : 1) to give yield white solid as product (2728 mg, yield
7
8.34%).
Synthesis of 6-(2-chloropyrimidin-4-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-
indazole (14): To a solution of 1-(tetrahydro-2H-pyran-2-yl)-6-(4,4,5,5-tetramethyl-
1
,3,2-dioxaborolan-2-yl)-1H-indazole (12.18 mmol, 4 g), 2,4-dichloropyrimidine
(
18.32 mmol, 2710 mg) and Pd(PPh (600 mg) in 1,4-dioxane (30 mL) was added
3 4
)
saturated sodium bicarbonate solution (18 mL) and the reaction mixture protecting
with argon was stirred at 95 ºC overnight. After cooling, the mixture was extracted
with ethyl acetate, washed with saline. The organic phase was collected and the
solvent was removed under vacuum. The crude product was purified by silica gel
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Chemistry & Biodiversity
10.1002/cbdv.201800598
chromatography (hexane / ethyl acetate 3 : 1) to give yield white solid as product
2.913 g, yield 76.17%).
General procedure
-(2-chloropyrimidin-4-yl)-1-(tetrahydro-2H-pyran-2-yl)-1H-indazole (0.15 mmol, 48
mg), corresponding compound 15a-m (0.30 mmol), Pd (dba) (0.025 mmol, 23 mg),
RuPhos (0.028 mmol, 13 mg) and Cs CO (0.5 mmol, 163 mg) in 1,4-dioxane (5 mL)
(
for
synthesis
of
16a-m:
A
mixture
of
6
2
3
2
3
protecting with argon was heated at 110 ºC overnight. After cooled to room
temperature, the reaction mixture was extracted with ethyl acetate, washed with saline.
The organic phase was collected and the solvent was removed under vacuum. All the
crude product except 16l was purified by silica gel chromatography (hexane / ethyl
acetate 3 : 1). 16l was purified by silica gel chromatography (hexane / ethyl acetate 1 :
1
4
3
).
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-p-tolylpyrimidin-2-amine (16a).
1
2 mg of 16a as yellow solid, yield 55%. Purity 98.80%. Mp 218-219 ºC. H NMR
(
500 MHz, DMSO) δ 9.59 (s, 1H), 8.56 (d, J = 5.2 Hz, 1H), 8.52 (s, 1H), 8.19 (s, 1H),
.97 (d, J = 8.5 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.72 (d, J = 8.4 Hz, 2H), 7.51 (d, J
5.2 Hz, 1H), 7.13 (d, J = 8.3 Hz, 2H), 5.97 (dd, J = 9.6, 2.1 Hz, 1H), 3.93 (d, J =
1.3 Hz, 1H), 3.78 (td, J = 11.4, 4.5 Hz, 1H), 2.47 – 2.41 (m, 1H), 2.27 (s, 3H), 2.10 –
7
=
1
1
1
3
.99 (m, 2H), 1.78 (dd, J = 24.3, 12.3 Hz, 1H), 1.64 – 1.58 (m, 2H), C NMR (151
MHz, DMSO) δ:164.13, 160.71, 159.56, 140.00, 138.49, 135.49, 134.03, 130.75,
1
2
29.36, 126.02, 121.77, 120.42, 119.71, 109.54, 108.65, 84.69, 67.18, 29.45, 25.28,
+
2.79, 20.89; MS (ESI-TOF), for C23
H N
23 5
O [M+H] calculated 386.20, found
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Chemistry & Biodiversity
10.1002/cbdv.201800598
3
4
4
86.17.
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-m-tolylpyrimidin-2-amine (16b).
1
0 mg of 16b as yellow solid, yield 69%. Purity 99.13%. Mp 73-74 ºC. H NMR (500
MHz, DMSO) δ 9.63 (s, 1H), 8.58 (d, J = 5.2 Hz, 1H), 8.52 (s, 1H), 8.19 (s, 1H), 7.99
d, J = 8.5 Hz, 1H), 7.92 (d, J = 8.5 Hz, 1H), 7.69 (d, J = 7.2 Hz, 2H), 7.53 (d, J = 5.2
Hz, 1H), 7.20 (t, J = 8.1 Hz, 1H), 6.80 (d, J = 7.4 Hz, 1H), 5.98 (dd, J = 9.5, 2.3 Hz,
(
1
2
H), 3.91 (d, J = 11.3 Hz, 1H), 3.83 – 3.73 (m, 1H), 2.48 – 2.41 (m, 1H), 2.32 (s, 3H),
1
3
.10 – 1.98 (m, 2H), 1.82 – 1.72 (m, 1H), 1.64 – 1.55 (m, 2H), C NMR (151 MHz,
DMSO) δ:164.23, 160.68, 159.54, 140.99, 140.07, 138.02, 135.51, 134.08, 128.79,
1
2
3
4
26.00, 122.67, 121.79, 120.46, 120.00, 116.67, 109.47, 108.92, 84.39, 67.06, 29.43,
+
5.28, 22.72, 21.91; MS (ESI-TOF), for C23
H
23
N
5
O [M+H] calculated 386.20, found
86.15.
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-(pyridin-3-yl)pyrimidin-2-amine
1
(
16c). 34 mg of 16c as yellow solid, yield 61%. Purity 98.02%. Mp 177-178 ºC. H
NMR (500 MHz, DMSO) δ 9.92 (s, 1H), 9.00 (d, J = 2.4 Hz, 1H), 8.63 (d, J = 5.2 Hz,
H), 8.53 (s, 1H), 8.31 (d, J = 8.4 Hz, 1H), 8.19 (s, 2H), 8.02 – 7.96 (m, 1H), 7.92 (d,
J = 8.4 Hz, 1H), 7.61 (d, J = 5.2 Hz, 1H), 7.36 (dd, J = 8.3, 4.6 Hz, 1H), 5.98 (dd, J =
1
9
2
.6, 2.2 Hz, 1H), 3.93 (d, J = 11.4 Hz, 1H), 3.85 – 3.75 (m, 1H), 2.48 – 2.41 (m, 1H),
1
3
.10 – 1.98 (m, 2H), 1.85 – 1.70 (m, 1H), 1.65 – 1.56 (m, 2H), C NMR (151 MHz,
DMSO) δ:164.44, 160.49, 159.71, 142.81, 141.44, 140.01, 137.75, 135.23, 134.05,
1
26.09, 125.92, 123.78, 121.86, 120.42, 109.65, 84.61, 67.13, 29.44, 25.28, 22.73;
+
MS (ESI-TOF), for C21
H
20
N
6
O [M+H] calculated 373.18, found 373.13.
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Chemistry & Biodiversity
10.1002/cbdv.201800598
N-(2-fluorophenyl)-4-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)pyrimidin-2-ami
1
ne (16d). 7 mg of 16d as yellow solid, yield 12%. Purity 96.93%. Mp 92-93 ºC. H
NMR (500 MHz, DMSO) δ 9.18 (s, 1H), 8.54 (d, J = 5.2 Hz, 1H), 8.47 (s, 1H), 8.17
(s, 1H), 7.93 (dd, J = 11.9, 4.5 Hz, 1H), 7.89 – 7.84 (m, 2H), 7.54 (d, J = 5.2 Hz, 1H),
7
1
1
1
1
.30 – 7.24 (m, 1H), 7.23 – 7.13 (m, 2H), 5.92 (dd, J = 9.6, 2.0 Hz, 1H), 3.89 (t, J =
1.0 Hz, 1H), 3.75 (td, J = 11.2, 3.7 Hz, 1H), 2.47 – 2.39 (m, 1H), 2.06 – 1.96 (m, 2H),
1
3
.81 – 1.71 (m, 1H), 1.63 – 1.55 (m, 2H), C NMR (151 MHz, DMSO) δ:164.20,
60.91, 159.65, 156.41, 154.78, 139.98, 135.27, 134.02, 126.04, 125.64, 125.14 ,
24.56, 121.72, 120.38, 116.00 , 109.51, 109.15, 84.64, 67.18, 29.43, 25.28, 22.77;
+
MS (ESI-TOF), for C22H20FN
5
O [M+H] calculated 390.17, found 390.00.
1
-(tetrahydro-2H-pyran-2-yl)-6-(2-(isoindolin-2-yl)pyrimidin-4-yl)-1H-indazole (16e).
1
4
0 mg of 16e as yellow solid, yield 67%. Purity 97.15%. Mp 119-121 ºC. H NMR
(500 MHz, DMSO) δ 8.68 (d, J = 5.2 Hz, 1H), 8.58 (s, 1H), 8.47 (d, J = 7.9 Hz, 1H),
8
1
.20 (s, 1H), 8.03 (d, J = 8.5 Hz, 1H), 7.94 (d, J = 8.5 Hz, 1H), 7.59 (d, J = 5.2 Hz,
H), 7.29 – 7.20 (m, 2H), 6.94 (t, J = 7.4 Hz, 1H), 6.04 – 5.98 (m, 1H), 4.30 (t, J = 8.6
Hz, 2H), 3.94 (d, J = 11.3 Hz, 1H), 3.85 – 3.76 (m, 1H), 3.20 (t, J = 8.6 Hz, 2H), 2.44
(dd, J = 12.8, 3.5 Hz, 1H), 2.05 (dt, J = 16.5, 4.1 Hz, 2H), 1.79 (dt, J = 16.3, 11.9 Hz,
1
3
1
1
1
H), 1.60 (dd, J = 17.5, 4.7 Hz, 2H), C NMR (126 MHz, DMSO) δ:164.08, 159.36,
43.79, 140.02, 135.50, 134.03, 132.90, 127.37, 126.11, 125.30, 121.88, 120.46,
15.25, 109.71, 108.50, 84.71, 67.13, 49.09, 29.43, 27.11, 25.32, 22.71; MS
+
(
ESI-TOF), for C24
H
23
N
5
O [M+H] calculated 398.20, found 398.16.
4
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-(4-methylthiazol-2-yl)pyrimidin-
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Chemistry & Biodiversity
10.1002/cbdv.201800598
2
-amine (16f). 44 mg of 16f as yellow solid, yield 75%. Purity 98.94%. Mp 212-213
1
ºC. H NMR (600 MHz, DMSO) δ 11.59 (s, 1H), 8.81 – 8.64 (m, 2H), 8.21 (s, 1H),
8
1
2
.06 (d, J = 8.4 Hz, 1H), 7.95 (d, J = 8.4 Hz, 1H), 7.70 (d, J = 5.2 Hz, 1H), 7.13 (s,
H), 6.04 – 5.93 (m, 1H), 3.92 (t, J = 15.5 Hz, 1H), 3.78 (dd, J = 14.7, 10.1 Hz, 1H),
.35 (d, J = 16.2 Hz, 1H), 2.09 – 2.02 (m, 2H), 1.81 – 1.71 (m, 1H), 1.62 (d, J = 3.4
1
3
Hz, 2H), C NMR (151 MHz, DMSO) δ:157.78, 140.03, 134.79, 134.10, 126.22,
1
21.95, 120.63, 110.15, 84.81, 67.23, 29.36, 25.29, 22.76, 11.74; MS (ESI-TOF), for
+
C
20
H
20
N
6
OS [M+H] calculated 393.15, found 393.09.
N-(3-fluorophenyl)-4-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)pyrimidin-2-
amine (16g). 32 mg of 16g as yellow solid, yield 55%. Purity 98.43%. Mp 146-147 ºC.
1H NMR (500 MHz, DMSO) δ 9.97 (s, 1H), 8.63 (d, J = 5.2 Hz, 1H), 8.53 (s, 1H),
8
1
1
1
1
1
.19 (s, 1H), 8.01 – 7.90 (m, 3H), 7.59 (t, J = 6.9 Hz, 2H), 7.33 (dd, J = 15.4, 8.0 Hz,
H), 6.78 (td, J = 8.4, 2.4 Hz, 1H), 5.97 (dd, J = 9.6, 2.2 Hz, 1H), 3.92 (d, J = 11.3 Hz,
H), 3.82 – 3.73 (m, 1H), 2.48 – 2.41 (m, 1H), 2.11 – 1.96 (m, 2H), 1.83 – 1.70 (m,
1
3
H), 1.63 – 1.54 (m, 2H), C NMR (151 MHz, DMSO) δ:164.38, 163.67, 162.08,
60.36, 159.63, 142.99, 140.03, 135.31, 134.08, 130.45, 126.07, 121.88, 120.44,
15.07, 109.59, 108.01, 105.84, 105.66, 84.53, 67.11, 29.46, 25.28, 22.76; MS
+
(
ESI-TOF), for C22
H
20FN
5
O [M+H] calculated 390.17, found 390.69.
N-(3-chlorophenyl)-4-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)pyrimidin-2-am
ine (16h). 27 mg of 16h as yellow solid, yield 44%. Purity 97.58%. Mp 147-148 ºC.
1H NMR (500 MHz, DMSO) δ 9.96 (s, 1H), 8.62 (d, J = 5.2 Hz, 1H), 8.50 (s, 1H),
8
.19 (s, 1H), 8.15 (t, J = 1.8 Hz, 1H), 8.00 – 7.94 (m, 1H), 7.91 (d, J = 8.5 Hz, 1H),
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Chemistry & Biodiversity
10.1002/cbdv.201800598
7
.75 (dd, J = 8.2, 1.3 Hz, 1H), 7.57 (d, J = 5.2 Hz, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.01
(dd, J = 7.8, 1.6 Hz, 1H), 5.97 (dd, J = 9.5, 2.1 Hz, 1H), 3.90 (d, J = 11.3 Hz, 1H),
3
.77 (td, J = 11.6, 6.9 Hz, 1H), 2.48 – 2.41 (m, 1H), 2.08 – 1.95 (m, 2H), 1.80 – 1.69
1
3
(
m, 1H), 1.58 (t, J = 6.3 Hz, 2H), C NMR (151 MHz, DMSO) δ:164.38, 160.32,
1
1
59.59, 142.68, 140.05, 135.30, 134.09, 133.48, 130.54, 126.06, 121.83, 121.30,
20.45, 118.53, 117.66, 109.66, 109.45, 84.34, 67.06, 29.44, 25.28, 22.72; MS
+
(
ESI-TOF), for C22
H
20ClN
5
O [M+H] calculated 406.14, found 406.14.
4
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-methyl-N-phenylpyrimidin-2-
amine (16i). 21 mg of 16i as yellow solid, yield 35%. Purity 96.67%. Mp 57-58 ºC.
1H NMR (500 MHz, DMSO) δ 8.48 (d, J = 5.2 Hz, 1H), 8.40 (s, 1H), 8.15 (s, 1H),
7
.89 (dd, J = 8.5, 1.0 Hz, 1H), 7.83 (d, J = 8.5 Hz, 1H), 7.47 – 7.41 (m, 5H), 7.24
(ddd, J = 10.3, 5.3, 3.3 Hz, 1H), 5.86 (dd, J = 9.8, 2.3 Hz, 1H), 3.89 (d, J = 11.4 Hz,
1
–
H), 3.73 (td, J = 11.0, 3.3 Hz, 1H), 3.58 (s, 3H), 2.40 (qd, J = 13.1, 4.0 Hz, 1H), 2.08
1
3
1.93 (m, 2H), 1.81 – 1.69 (m, 1H), 1.65 – 1.50 (m, 2H), C NMR (151 MHz,
DMSO) δ:163.50, 161.86, 159.28, 145.83, 139.97, 135.43, 134.01, 129.12, 126.81,
1
2
4
25.99, 125.56, 121.62, 120.28, 109.39, 107.61, 84.63, 67.23, 38.52, 29.43, 25.27,
+
2.82; MS (ESI-TOF), for C23
H
23
N
5
O [M+H] calculated 386.20, found 386.04.
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-(pyrimidin-2-yl)pyrimidin-2-
amine (16j). 40 mg of 16j as yellow solid, yield 71%. Purity 96.49%. Mp 121-123 ºC.
1H NMR (500 MHz, DMSO) δ 10.36 (s, 1H), 8.77 – 8.55 (m, 3H), 8.19 (s, 1H), 8.04
(d, J = 8.3 Hz, 1H), 7.91 (d, J = 8.5 Hz, 1H), 7.24 (dd, J = 14.6, 7.3 Hz, 1H), 7.19 –
7
.08 (m, 1H), 6.73 (d, J = 5.4 Hz, 1H), 5.96 (d, J = 8.5 Hz, 1H), 3.90 (t, J = 15.3 Hz,
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Chemistry & Biodiversity
10.1002/cbdv.201800598
1
1
1
1
H), 3.77 (t, J = 12.5 Hz, 1H), 2.44 (dd, J = 18.0, 9.1 Hz, 1H), 2.06 – 1.96 (m, 2H),
1
3
.83 – 1.73 (m, 1H), 1.64 – 1.56 (m, 2H), C NMR (151 MHz, DMSO) δ:160.33 –
60.06, 159.17, 157.12, 140.04, 134.09, 129.37, 128.67, 126.14, 125.78, 121.76,
20.55, 115.73, 110.61, 109.24 – 108.53, 84.56, 67.18, 29.43, 25.28, 22.77; MS
+
(
ESI-TOF), for C20
H
19
N
7
O [M+H] calculated 374.17, found 374.15.
4
2
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-(5-methylpyridin-2-yl)pyrimidin-
-amine (16k). 30 mg of 16k as white solid, yield 52%. Purity 99.24%. Mp 165-166
1
ºC. H NMR (500 MHz, DMSO) δ 9.78 (s, 1H), 8.64 (d, J = 5.2 Hz, 1H), 8.56 (s, 1H),
8
8
1
.27 (d, J = 8.5 Hz, 1H), 8.21 – 8.15 (m, 2H), 8.00 (d, J = 8.5 Hz, 1H), 7.92 (d, J =
.5 Hz, 1H), 7.66 – 7.60 (m, 2H), 5.98 (dd, J = 9.7, 2.1 Hz, 1H), 3.93 (d, J = 11.3 Hz,
H), 3.84 – 3.74 (m, 1H), 2.48 – 2.41 (m, 1H), 2.26 (s, 3H), 2.09 – 1.99 (m, 2H), 1.79
1
3
(
ddd, J = 18.2, 14.2, 6.6 Hz, 1H), 1.65 – 1.56 (m, 2H), C NMR (151 MHz, DMSO)
δ:164.23, 159.88, 159.63, 151.41, 148.12, 140.01, 138.58, 135.16, 134.06, 126.77,
1
26.11, 121.84, 120.43, 113.06, 109.72, 84.67, 67.18, 29.45, 25.29, 22.78, 17.72; MS
+
(
ESI-TOF), for C22
H
22
N
6
O [M+H] calculated 387.19, found 387.17.
2
N -(4-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)pyrimidin-2-yl)pyridine-2,4-
1
diamine (16l). 40 mg of 16l as brown liquid, yield 68%. Purity 98.96%. H NMR (500
MHz, DMSO) δ 9.39 (s, 1H), 8.58 – 8.50 (m, 2H), 8.19 (s, 1H), 7.98 (dd, J = 8.5, 0.9
Hz,1H), 7.90 (d, J = 8.5 Hz, 1H), 7.48 (d, J = 5.2 Hz, 1H), 7.15 (d, J = 1.8 Hz, 1H),
7
7
.00 (d, J = 8.2 Hz, 1H),6.95 (t, J = 7.9 Hz, 1H), 6.64 (t, J = 7.8 Hz, 2H), 6.23 (d, J =
.8 Hz, 1H), 6.00 (dd, J = 9.5, 2.3 Hz, 1H),4.97 (d, J = 32.6 Hz, 2H), 3.89 (d, J = 11.5
Hz, 1H), 3.84 – 3.76 (m, 1H), 2.44 (dd, J = 12.6, 3.6 Hz, 1H),2.07 – 1.96 (m, 2H),
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Chemistry & Biodiversity
10.1002/cbdv.201800598
1
3
1
.78 (td, J = 7.8, 3.1 Hz, 1H), 1.60 (dd, J = 10.0, 6.4 Hz, 2H), C NMR (151 MHz,
DMSO) δ 164.01, 160.79, 159.47, 149.35, 141.55, 140.09, 135.47, 134.05, 129.17,
1
2
3
4
25.96, 121.74, 120.49, 109.48, 108.50 (d, J = 11.8 Hz), 108.02, 105.50, 84.33, 67.03,
+
9.43, 25.31, 22.72; MS (ESI-TOF), for C22
H
22
N
6
O [M+H] calculated 387.19, found
87.19.
-(1-(tetrahydro-2H-pyran-2-yl)-1H-indazol-6-yl)-N-(thiazol-2-yl)pyrimidin-2-amine
1
(
16m). 37 mg of 16m as yellow solid, yield 65%. Purity 96.74%. Mp 206-208 ºC. H
NMR (600 MHz, DMSO) δ 11.83 (s, 1H), 8.76 (d, J = 16.8 Hz, 1H), 8.22 (s, 1H),
.11 (d, J = 7.0 Hz, 1H), 7.97 (d, J = 7.4 Hz, 1H), 7.75 (d, J = 17.5 Hz, 1H), 7.48 (d, J
18.5 Hz, 1H), 7.21 (s, 1H), 6.65 (d, J = 7.1 Hz, 1H), 5.99 (d, J = 8.5 Hz, 1H), 3.99
s, 1H), 3.82 (s, 1H), 2.12 (t, J = 33.2 Hz, 3H), 1.78 (d, J = 33.5 Hz, 1H), 1.54 (d, J =
8
=
(
1
3
5
1
2
3
3.1 Hz, 2H), C NMR (151 MHz, DMSO) δ:160.04, 157.79, 156.62, 139.92, 138.60,
34.68, 133.99, 126.31, 121.94, 120.62, 112.61, 110.35, 107.59, 85.19, 67.39, 29.47,
+
5.33, 22.21; MS (ESI-TOF), for C19
H
18
N
6
OS [M+H] calculated 379.13, found
79.07.
General procedure for synthesis of 6a-m: 16a-m (0.45 mmol) was dissolved in
ethanol (5 mL) in a 25 mL round-bottom flask. Then, 4 mol/L HCl in 1,4-dioxane (0.3
mL) was added to the mixture and the mixture was heated at 70 ºC for 2 h. After
cooled to room temperature, NaHCO solution was added to the reaction mixture until
3
pH = 8. The mixture was extracted with ethyl acetate, washed with saline. The organic
phase was collected and the solvent was removed under vacuum. The crude product
was purified by silica gel chromatography.
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Chemistry & Biodiversity
10.1002/cbdv.201800598
4
-(1H-indazol-6-yl)-N-p-tolylpyrimidin-2-amine (6a).
The crude product was purified by silica gel chromatography (cyclohexane / ethyl
acetate 2 : 1).
1
8
8 mg of 6a as yellow solid, yield 65%. Purity 96.63%. Mp 222-224 ºC. H NMR
(400 MHz, DMSO) δ:13.33 (s, 1H), 8.53 (d, J = 5.2 Hz, 1H), 8.35 (s, 1H), 8.14 (s,
1
7
H), 7.89 (s, 2H), 7.72 (d, J = 8.4 Hz, 2H), 7.65-7.58 (m, 2H), 7.57-7.51 (m, 1H),
13
.74 (d, J = 5.2 Hz,1H), 7.13 (d, J = 8.3 Hz, 2H), 2.27 (s,3H), C-NMR (101 MHz,
DMSO) δ 109.89, 110.66, 120.94, 122.72, 126.10, 130.52, 130.64, 130.75, 132.01,
33.27, 133.37, 133.84, 135.43, 136.46, 139.92, 141.98, 160.81, 162.09, 165.77; MS
ESI-TOF), for C18 [M] calculated 301.13, found 301.25.
-(1H-indazol-6-yl)-N-m-tolylpyrimidin-2-amine (6b).
1
(
H
15
N
5
4
The crude product was purified by silica gel chromatography (cyclohexane / ethyl
acetate 2 : 1).
1
1
02 mg of 6b as yellow solid, yield 75%. Purity 98.64%. Mp 191-192 ºC. H NMR
(500 MHz, DMSO) δ:13.38 (s, 1H), 9.61 (s, 1H), 8.55 (d, J = 5.2 Hz, 1H), 8.36 (s,
1
H), 8.15 (s, 1H), 7.90 (s, 2H), 7.70 (d, J = 8.1 Hz, 1H), 7.47 (d, J = 5.2 Hz, 1H), 7.19
1
3
(
t, J = 7.8 Hz, 1H), 6.79 (d, J = 7.4 Hz, 1H), 2.32 (s, 3H), 1.38 (s, 2H); C NMR (126
MHz, DMSO) δ 164.38, 160.68, 159.49, 141.04, 140.59, 138.05, 135.06, 134.09,
28.82, 124.77, 122.61, 121.39, 119.95, 119.60, 116.63, 109.29, 108.74, 21.92; MS
ESI-TOF), for C18 [M] calculated 301.13, found 301.74.
-(1H-indazol-6-yl)-N-(pyridin-3-yl)pyrimidin-2-amine (6c).
1
(
H
15
N
5
4
The crude product was purified by silica gel chromatography (hexane / ethyl acetate
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Chemistry & Biodiversity
10.1002/cbdv.201800598
1
1
: 1).
1
09 mg of 6c as white solid, yield 52.43%. Purity 97.38%. Mp 239-240 ºC. H NMR
(
500 MHz, DMSO) δ:13.39 (s, 1H), 9.90 (s, 1H), 9.02 (d, J = 2.5 Hz, 1H), 8.59 (d, J =
.2 Hz, 1H), 8.36 (s, 1H), 8.27 (ddd, J = 8.4, 2.5, 1.5 Hz, 1H), 8.19 (dd, J = 4.6, 1.3
Hz, 1H), 8.16 (s, 1H), 7.92–7.87 (m, 2H), 7.55 (d,J = 5.2 Hz, 1H), 7.35 (dd, J = 8.3,
5
4
1
1
2
.6 Hz, 1H), 13C-NMR (DMSO, 125.67 MHz): 109.40, 109.53, 119.61, 121.48,
23.83, 124.82, 125.86, 134.10, 134.32, 137.30, 140.57, 141.29, 142.73, 159.64,
+
60.49, 164.67; MS (ESI-TOF), for C16
H N
12 6
[M+H] calculated 289.12, found
89.12.
N-(2-fluorophenyl)-4-(1H-indazol-6-yl)pyrimidin-2-amine (6d).
The crude product was purified by silica gel chromatography (cyclohexane / ethyl
acetate 3 : 1).
1
9
6 mg of 6d as yellow solid, yield 70%. Purity 96.28%. Mp 154-155 ºC. H NMR
(500 MHz, DMSO) δ:13.34 (s, 1H), 9.16 (s, 1H), 8.50 (d, J = 5.2 Hz, 1H), 8.30 (s,
1
1
1
1
H), 8.13 (s, 1H), 7.89-7.80 (m, 3H), 7.48 (d, J = 5.2 Hz, 1H), 7.26 (ddd, J = 11.0, 8.0,
1
3
.5 Hz, 1H), 7.20 (td, J = 7.6, 1.5 Hz, 1H), 7.18-7.13 (m, 1H), C-NMR (DMSO,
25.67 MHz): 109.05, 109.31, 115.95, 116.11, 121.33, 124.60, 124.62, 124.75, 125.17,
25.66, 134.06, 134.88, 140.56, 154.63, 156.58, 159.57, 160.95, 164.47; MS
(
ESI-TOF), for C17
H
12FN
5
[M] calculated 305.11, found 305.87.
6
-(2-(isoindolin-2-yl)pyrimidin-4-yl)-1H-indazole (6e).
The crude product was purified by silica gel chromatography (hexane / ethyl acetate
: 1).
3
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Chemistry & Biodiversity
10.1002/cbdv.201800598
1
9
2 mg of 6e as yellow solid, yield 65%. Purity 95.72%. Mp 96-98 ºC. H NMR (500
MHz, DMSO) δ:13.34 (s, 1H), 8.65 (d, J = 5.2 Hz, 1H), 8.44 (d, J = 8.0 Hz, 1H),
8
2
.39(s, 1H), 8.16(s, 1H), 7.93(p, J=8.5Hz, 2H), 7.53(d, J=5.2Hz, 1H), 7.27-7.20(m,
H), 6.93(t, J = 7.3 Hz, 1H), 4.30 (t, J = 8.7 Hz, 2H), 3.20 (t, J = 8.6 Hz, 2H), 1.38
(
s, 1H), 13C-NMR (DMSO, 125.67 MHz): 27.09, 49.12, 108.43, 109.43, 115.23,
1
1
3
4
19.67, 121.50, 121.82, 124.81, 125.30, 127.46, 132.85, 134.10, 135.13, 140.64,
+
43.82, 159.31, 159.38, 164.32; MS (ESI-TOF), for C19
H N
15 5
[M+H] calculated
14.14, found 314.83.
-(1H-indazol-6-yl)-N-(4-methylthiazol-2-yl)pyrimidin-2-amine (6f).
The crude product was purified by silica gel chromatography (dichloromethane /
methanol 10 : 1).
1
1
28 mg of 6f as yellow solid, yield 92%. Purity 97.68%. Mp > 300 ºC. H NMR (500
MHz, DMSO) δ:13.52 (s, 1H), 11.54 (s, 1H), 10.34 (s, 2H), 8.66 (d, J = 5.2 Hz, 1H),
8
7
.53 (s, 1H), 8.16 (s, 1H), 7.98 (dd, J = 8.5, 1.2 Hz, 1H), 7.93 (d, J = 8.5 Hz, 1H),
1
3
.64 (d, J = 5.2 Hz, 1H), 7.12(d, J = 1.2 Hz, 1H), 2.40 (d, J = 0.9 Hz, 3H), C-NMR
(DMSO, 125.67 MHz): 11.69, 45.73, 110.07, 119.76, 121.49, 124.86, 125.40, 134.00,
1
34.34, 135.45, 157.76, 158.29, 159.71, 164.66; MS (ESI-TOF), for C15
H
12
N S
6
+
[
M+H] calculated 309.09, found 308.93.
N-(3-fluorophenyl)-4-(1H-indazol-6-yl)pyrimidin-2-amine (6g).
The crude product was purified by silica gel chromatography (cyclohexane / ethyl
acetate 2 : 1).
1
9
9 mg of 6g as white solid, yield 72%. Purity 98.41%. Mp 244-245 ºC. H NMR (400
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Chemistry & Biodiversity
10.1002/cbdv.201800598
MHz, DMSO) δ:13.38 (s, 1H), 9.93 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.35 (s, 1H),
8
7
1
1
1
.16 (s, 1H), 7.95–7.85 (m, 3H), 7.60 (d, J = 8.2 Hz, 1H), 7.53 (d, J = 5.2 Hz, 1H),
1
3
.33 (dd, J = 15.4, 8.0 Hz, 1H), 6.77 (td, J = 8.4, 2.4 Hz, 1H), C-NMR (DMSO,
00.61 MHz): 106.94, 107.20, 119.21, 110.78, 110.89, 116.38, 120.98, 122.82, 126.18,
31.87, 135.46, 136.27, 141.95, 144.35, 144.47, 160.84, 161.75, 163.03, 165.42,
+
66.07; MS (ESI-TOF), for C17
H
12FN
5
[M+H] calculated 306.12, found 306.83.
N-(3-chlorophenyl)-4-(1H-indazol-6-yl)pyrimidin-2-amine (6h).
The crude product was purified by silica gel chromatography (cyclohexane / ethyl
acetate 3 : 1).
1
1
10 mg of 6h as white solid, yield 76%. Purity 97.34%. Mp 240-241 ºC. H NMR
(400 MHz, DMSO) δ:13.38 (s, 1H), 9.92 (s, 1H), 8.60 (d, J = 5.2 Hz, 1H), 8.35 (s,
1
H), 8.13 (d, J = 25.2 Hz, 2H), 7.90 (s, 2H), 7.77 (d, J = 8.1 Hz, 1H), 7.54 (d, J = 5.2
1
3
Hz, 1H), 7.33 (t, J = 8.1 Hz, 1H), 7.00 (d, J = 7.9 Hz, 1H), C-NMR (DMSO, 100.61
MHz): 110.81, 110.92, 118.95, 119.86, 120.98, 122.62, 122.81, 126.19, 131.94,
1
34.86, 135.46, 136.23, 141.95, 144.09, 160.85, 161.71, 166.06; MS (ESI-TOF), for
+
C
17
H
12ClN
5
[M+H] calculated 322.79, found 322.05.
4
-(1H-indazol-6-yl)-N-methyl-N-phenylpyrimidin-2-amine (6i).
The crude product was purified by silica gel chromatography (hexane / ethyl acetate
3
8
: 1).
4 mg of 6i as white solid, yield 62%. Purity 96.73%. Mp 168169 ºC. H NMR (500
1
MHz, DMSO) δ:13.29 (s, 1H), 8.44 (d, J = 5.2 Hz, 1H), 8.25 (s, 1H), 8.12 (s, 1H),
7
.86–7.79 (m, 2H), 7.44–7.39 (m, 5H), 7.25–7.20 (m, 1H), 3.58 (s, 3H), 1.38 (s, 1H),
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Chemistry & Biodiversity
10.1002/cbdv.201800598
13C-NMR (DMSO, 125.67 MHz): 26.81, 38.65, 107.60, 109.17, 119.53, 121.26,
1
1
4
24.71, 125.57, 126.77, 129.16, 134.03, 135.11, 140.56, 145.87, 159.20, 161.88,
+
63.88; MS (ESI-TOF), for C18
H
15
N [M+H] calculated 302.14, found 302.13.
5
-(1H-indazol-6-yl)-N-(pyrimidin-2-yl)pyrimidin-2-amine (6j).
The crude product was purified by silica gel chromatography (dichloromethane /
methanol 10 : 1).
1
8
3 mg of 6j as white solid, yield 64%. Purity 97.13%. Mp 273-274 ºC. H NMR (400
MHz, DMSO) δ:13.34 (s, 1H), 10.29 (s, 1H), 8.65 (d, J = 5.2 Hz, 1H), 8.62 (d, J = 4.8
Hz, 2H), 8.42 (s, 1H), 8.14 (s, 1H), 7.91 (dt, J = 20.9, 4.7 Hz, 2H), 7.68 (d, J = 5.3 Hz,
1
3
1
1
1
2
4
H), 7.07 (t, J = 4.8 Hz, 1H), C-NMR (DMSO, 100.61 MHz): 111.01, 112.62,
16.94, 121.05, 122.69, 126.18, 135.44, 136.04, 141.99, 142.20, 142.49, 159.93,
+
60.88, 160.95, 161.01, 165.82; MS (ESI-TOF), for C15
H N
11 7
[M+H] calculated
90.12, found 290.11.
-(1H-indazol-6-yl)-N-(5-methylpyridin-2-yl)pyrimidin-2-amine (6k).
The crude product was purified by silica gel chromatography (cyclohexane / ethyl
acetate 3 : 1).
1
8
8 mg of 6k as white solid, yield 65%. Purity 96.48%. Mp > 300 ºC. H NMR (500
MHz, DMSO) δ:13.52 (s, 1H), 9.85 (s, 1H), 8.61 (d, J = 5.2 Hz, 1H), 8.39 (s, 1H),
8
8
.25 (d, J = 8.5 Hz, 1H), 8.17–8.09 (m, 2H), 7.90 (d, J = 0.8 Hz, 2H), 7.66 (dd, J =
.5, 2.1 Hz, 1H), 7.57 (d, J = 5.2 Hz, 1H), 3.15 (s, 2H), 2.25 (s, 3H), 1.20 (s, 1H),
13C-NMR (DMSO, 125.67 MHz): 17.71, 109.59, 109.73, 113.07, 119.59, 121.43,
1
24.80, 126.71, 134.00, 134.67, 138.94, 140.60, 147.74, 151.33, 159.50, 159.82,
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Chemistry & Biodiversity
10.1002/cbdv.201800598
+
1
64.59; MS (ESI-TOF), for C17
H
14
N [M+H] calculated 303.14, found 303.10.
6
2
N -(4-(1H-indazol-6-yl)pyrimidin-2-yl)pyridine-2,4-diamine (6l).
The crude product was purified by silica gel chromatography (hexane / ethyl acetate
1
8
: 1).
5 mg of 6l as white solid, yield 62%. Purity 98.15%. Mp > 300 ºC. H NMR (500
1
MHz, DMSO) δ:13.35 (d, J = 10.3 Hz, 1H), 9.38 (s, 1H), 8.51 (d, J = 5.2 Hz, 1H),
8
1
2
2
1
.36 (s, 1H), 8.15 (s, 1H), 7.89 (s, 2H), 7.42 (d, J = 5.2Hz, 1H), 7.12 (d,J = 1.7 Hz,
H), 7.00 (d,J = 8.2 Hz, 1H), 6.94 (t, J = 7.9 Hz, 1H), 6.24–6.20 (m, 1H), 4.97 (s, 2H),
1
3
.09 (d, J = 16.3 Hz, 4H), 1.79 (s, 1H), 1.38 (s, 3H), C-NMR (DMSO, 100.61 MHz):
8.18, 32.51, 106.68, 109.36, 109.74, 109.84, 110.68, 121.03, 122.70, 126.09, 126.44,
30.57, 135.42, 136.49, 141.99, 143.01, 150.64, 160.69, 162.19, 165.72, 165.91; MS
(ESI-TOF), for C17H N
14 6
[M] calculated 302.12, found 302.97.
4
-(1H-indazol-6-yl)-N-(thiazol-2-yl)pyrimidin-2-amine (6m).
The crude product was purified by silica gel chromatography (dichloromethane /
methanol 10 : 1).
1
1
12 mg of 6m as white solid, yield 85%. Purity 95.42%. Mp > 300 ºC. H NMR (500
MHz, DMSO) δ:8.71 (d, J = 5.4 Hz, 1H), 8.54 (s, 1H), 8.18 (s, 1H), 8.03 (d, J = 8.5
Hz, 1H), 7.95 (d, J = 8.5 Hz, 1H), 7.73 (d, J = 5.4 Hz, 1H), 7.51 (d, J = 3.7 Hz, 1H),
1
3
7
1
.22 (d, J = 3.7 Hz, 1H), C-NMR (DMSO, 125.67 MHz): 110.22, 110.42, 112.83,
19.950, 121.62, 125.04, 134.04, 134.09, 140.65, 157.33; MS (ESI-TOF), for
+
C
14
H
10
N
6
S [M+H] calculated 295.08, found 295.03.
Synthesis of 5-bromo-3-(phenylamino)pyrazin-2-ol (19):
A
mixture of
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Chemistry & Biodiversity
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3
,5-dibromo-2-hydroxypyrazine (0.2 mmol, 50 mg), aniline (0.2 mmol, 20 mL), and
camphorsulfonic acid (25 mg) in isopropanol (50 mL) was heated at 82 ºC for 48 h.
After cooled to room temperature, the solvent was removed under vacuum. The crude
product was purified by silica gel chromatography (hexane / ethyl acetate 1 : 1) to
give 5-bromo-3-(phenylamino)pyrazin-2-ol (19). 34 mg of 19 as yellow solid, yield
1
6
3%. Purity 96.25%. Mp > 300 ºC. H NMR (500 MHz, DMSO) δ 12.15 (s, 1H), 9.34
(s, 1H), 7.89 (d, J = 7.9 Hz, 2H), 7.31 (t, J = 7.9 Hz, 2H), 7.03 (t, J = 7.4 Hz, 1H), 6.98
1
3
(
s, 1H). C NMR (126 MHz, DMSO) δ 151.05, 148.19, 139.45, 129.04, 123.42,
20.03, 115.59, 112.15.
Synthesis of 7a: To a solution of 1-(tetrahydro-2H-pyran-2-yl)-6-(4,4,5,5-tetramethyl-
1
1
5
1
,3,2-dioxaborolan-2-yl)-1H-indazole
(0.3
mmol,
98
mg),
-bromo-3-(phenylamino)pyrazin-2-ol (0.3 mmol, 83 mg) and Pd(PPh
)
3 4
(0.01 mmol,
2 mg) in 1,4-dioxane (30 mL) was added saturated sodium bicarbonate solution (18
mL) and the reaction mixture was refluxed at 110 ºC for 16 h under argon atmosphere.
After cooling, the mixture was extracted with ethyl acetate,washed with saline. The
organic phase was collected and the solvent was removed under vacuum. Ethanol (5
mL) and 4 mol/L HCl in 1,4-dioxane (0.3 mL) were added to the mixture and the
mixture was heated at 70 ºC for 2 h. After cooled to room temperature, NaHCO
3
solution was added to the reation mixture until pH = 8. The mixture was extracted
with ethyl acetate, washed with saline. The organic phase was collected and the
solvent was removed under vacuum. The crude product was purified by silica gel
chromatography
(hexane
/
ethyl
acetate
1
:
1)
to
afford
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