Pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives as new multi-targeted inhibitors of Aurora A/B and KDR

Article abstract of DOI:10.1016/j.ejmech.2018.09.032

Aurora A, Aurora B and Kinase Insert Domain-containing Receptor (KDR) play essential roles in sustained cancer growth. In the present study, eighteen pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives were designed and synthesized. Most of the prepa

Full text of DOI:10.1016/j.ejmech.2018.09.032

Accepted Manuscript
Pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives as new multi-targeted
inhibitors of Aurora A/B and KDR
Qiumeng Zhang, Qianqian Shen, Lixin Gao, Linjiang Tong, Jia Li, Yi Chen, Wei Lu
PII:
S0223-5234(18)30804-3
10.1016/j.ejmech.2018.09.032
EJMECH 10743
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 22 March 2018
Revised Date: 16 August 2018
Accepted Date: 10 September 2018
Please cite this article as: Q. Zhang, Q. Shen, L. Gao, L. Tong, J. Li, Y. Chen, W. Lu,
Pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives as new multi-targeted inhibitors of Aurora A/B
and KDR, European Journal of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.09.032.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT
Pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives as new
multi-targeted inhibitors of Aurora A/B and KDR
Qiumeng Zhang a, c, Qianqian Shen b, Lixin Gaoc, Linjiang Tong b, Jia Li c,
Yi Chen b, and Wei Lu a,
* *
a Shanghai Engineering Research Center of Molecular Therapeutics and New Drug Development,
School of Chemistry and Molecular Engineering, East China Normal University, 3663 North
Zhongshan Road, Shanghai 200062, P. R. China.
b Division of Anti-Tumor Pharmacology, State Key Laboratory of Drug Research, Shanghai
institute of Materia Medica, Chinese Academy of Sciences, Shanghai 201203, P. R. China
c National Center for Drug Screening, Shanghai Institute of Materia Medica, Chinese Academy of
Sciences, Shanghai 201203, P. R. China
* Corresponding authors: Email: ychen@simm.ac.cn (Y. Chen);
Email: wlu@chem.ecnu.edu.cn (W. Lu)
Abstract:
Aurora A, Aurora B and Kinase Insert Domain-containing Receptor (KDR) play
essential roles in sustained cancer growth. In the present study, eighteen
pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives were designed and
synthesized. Most of the prepared compounds exhibited obviously enzymatic (Aurora
A/B and KDR) activities. Among these analogs, compound 17g displayed significant
Aurora A/B and KDR potencies with IC50 values of 46.2 nM, 37.6 nM and 21.6 nM,
respectively. The results of further biological assays showed that compound 17g
possessed moderate anti-proliferative activities against SNU-5, MKN-45 and
MKN-74 cells lines, induced G2/M cell cycle arrest and apoptosis in MKN-45,
MKN-74, SGC-7901 and SNU-5 cell lines, provided acceptable pharmacokinetic
profiles (F = 63.8%), and inhibited the proliferation of SNU-5 tumors in vivo of mice.
All of the above results suggested that pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine
could be developed as a promising scaffold of multiple Aurora A/B and KDR
inhibitors and 17g was worth of further research as a multi-targeted lead compound.
Keywords: Aurora A; Aurora B; KDR; multi-targeted; anticancer; heterocyclic.
1. Introduction
Human tumor cells have the characteristics of rapid malignant proliferation with
mitotic progression increasing significantly. The Aurora kinases (Aurora A, Aurora B
and Aurora C) belong to a subfamily of serine/threonine kinases that are critical
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regulators of mitosis and cytokinesis [1-3]. Aurora A localizes to centrosomes/spindle
poles and is require for spindle assembly, associates with late S phase and the M
phase [4, 5]. Aurora B localizes to the centromeres in prometaphase, and then
relocates to the spindle center area at anaphase. It is a chromosome passenger protein
required for phosphorylation of histone H3, chromosome segregation and cytokinesis
[5, 6]. Aurora C has been shown localized to spindle poles, its function is appearing to
be restricted mainly to spermatogenesis [5]. The frequent over expression of Aurora A
and B has been observed in a wide range of different human tumors including solid
and hematological malignancies [7-9]. Furthermore, Aurora A has been shown to be
an oncogene in vitro transformation assays [9, 10]. Thereby, targeting Aurora A,
Aurora B or both provide a unique opportunity for anti-cancer therapy. Many Aurora
or pan Aurora inhibitors have been documented for the treatment of cancer [11], such
as Alisertib [12, 13], Tozasertib [14], MK-5108 [15], Barasertib [16], MLN8054[13],
Danusertib [17] and ENMD-2076 [18] (Fig. 1).
Fig. 1. Structures of some Aurora or pan Aurora inhibitors.
Angiogenesis, the growth of new blood vessels, is an essential component in the
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progression and metastasis of tumors [19, 20]. The Vascular Endothelial Growth
Factor Receptor (VEGFR) family, most notably VEGFR2 or Kinase Insert Domain
Receptor (KDR), is one of the most potent angiogenic factors and is closely linked
with growth and metastatic spreading of several types of tumors [21, 22]. A growing
number of studies have demonstrated that inhibition of KDR is an effective therapy
for various indications [23-25]. Several multi-targeted KDR inhibitors have been
approved by the Food and Drug Administration (FDA) for the treatment of cancer,
such as Sunitinib Malate [26], Regorafenib [27], Sorafenib [28], Vandetanib [29],
Cabozantinib [30], Lenvatinib [31], Pazopanib [32] and Axitinib [33] (Fig. 2).
Fig. 2. Structures of some FDA approved multi-targeted KDR inhibitors.
Multi-kinase inhibitors have been exhibited the compelling antitumor effects,
such as Sorafenib [28, 34] and Sunitinib [26, 35]. We aimed to design a series of
derivatives with new scaffold that could impact both proliferation and angiogenesis
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processes by targeting Aurora A, Aurora B, and KDR. Several inhibitors have been
reported for the treatment of cancer by inhibiting Aurora family and KDR. Among
these inhibitors, R1530, a 7,8-disubstituted pyrazolobenzodiazepine, shows potent
inhibitory activities against multi-kinase, especially KDR, Fibroblast Growth Factor
Receptor (FGFR), Platelet-derived Growth Factor Receptor (PDGFR), Fms-Like
Tyrosine Kinase (FLT), Aurora A, and Checkpoint kinase. R1530, also exhibits potent
in vitro and in vivo antiproliferative activities, and high oral bioavailability [36]. The
mitosis/angiogenesis inhibitor R1530 attracts our interesting. Besides, ENMD-2076 is
an Aurora A and FLT3 inhibitor, also displays inhibitory activities against FLT4, KDR,
FGFR1, FGFR2, and PDGFRα [37]. Based on our aim of developing Auroras/KDR
inhibitors with high kinase selectivity,
a
series of derivatives with
pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine scaffold using fragments fused strategy
has been designed (Fig. 3). Herein, we present the design, synthesis and biological
activities of these compounds.
Fig. 3. Design strategy of target compounds.
2. Results and discussion
2.1. Chemistry
Detailed synthetic strategy to derivatives 13a-13k was illustrated in Scheme 1.
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According to published procedures, the pyrazole 3 was prepared from commercially
available 5-methyl-1H-pyrazol-3-amine (1) through acetylation and nitrification [38],
then the pyrazole N-H of compound 3 was protected with PMB by reacting with
o
4-methoxybenzylchloride (PMBCl) in the present of potassium carbonate at 50 C to
1
produce compound 4. The chemical structure of compound 4 was confirmed by H,
13C, HH NOESY NMR techniques. Subsequently, the acetyl protecting of compound
4 was removed by refluxing in 1 M KOH (KOH was dissolved in equal volume
ethanol and water) solution to get the key intermediate 5. On the other hand, the
commercially available pyrimidinone 6 was treated with N-Iodosuccinimide (NIS) in
CHCl3 at 50 o
refluxing to provide
C
for 3 h to afford compound 7. Compound 7 was reacted with POCl3 by
[39]. The newly formed was coped with
8
8
isopropyl-magnesium chloride and then reacted with benzaldehydes 9a-9k to afford
corresponding 10a-10k. Hydroxyl compounds 10a-10k were oxidized by Dess-Martin
Periodinane to give corresponding ketones 11a-11k. Compound 11a-11k reacted
with compound 5 using NaH as base to get pyrazoles 12a-12k. 12a-12k were
dissolved and heated in CF3COOH to remove the protective group of the pyrazole N-H.
Reduction of nitro was through SnCl2•2H2O, which promoted cyclization to afford the
targets 13a-13k (Scheme 1).
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PMB
N
O
O
H
H
N
PMB
N
H2N
N
N
N
N
N
a
N
b
c
d
NH
NH
NH
HN
H2N
O2N
NO2
O
1
2
NO2
3
4
5
H
N
H
S
N
Cl
S
O
I
S
N
O
e
f
N
N
N
I
8
6
7
PMB
N
N
NO2
S
N
Cl
S
N
Cl
O
S
N
NH
N
O
g
i
N
OH
h
8
+
N
O
R
R
R
9a-k
R
11a-k
10a-k
12a-k
H
N
N
NH
j
S
N
9g-13g R=p-Me
9a-13a R=H
9b-13b R=o-Cl
9c-13c R=m-Cl
9d-13d R=p-Cl
9e-13e R=m-F
9f-13f R=o-Me
9h-13h R=o-OMe
9i-13i R=p-OMe
9j-13j R=o-CF3
9k-13k R=p-CF3
N
k
N
R
13a-k
Scheme 1. Synthesis of target compounds 13a-13k. Reagents and conditions: (a) acetyl anhydride,
NaHCO3, H2O, reflux, 16h, 43%; (b) HNO3, H2SO4, 0 oC, 2h, 80%; (c) PMBCl, K2CO3, DMF, 50
oC, 3h, 87%; (d) KOH, ethanol, H2O, 95%; (e) NIS, CHCl3, N2, 50 oC, 3h, 50%; (f) POCl3, reflux,
16h, 98%; (g) isopropyl-magnesium chloride, anhydrous THF, -40 oC~0 oC; (h) Dess-Martin
Periodinane, THF, rt; (i) NaH, anhydrous THF, N2; (j) CF3COOH, reflux, 16h; (k) SnCl2•2H2O,
2-methylfuranidine, reflux.
The general synthetic route used to prepare target compounds 17a-17g was
outlined in the Scheme 2. Compound 12g was oxidized by KMnO4 in HOAc at room
temperature to give the key intermediate 14. Compound 14 was then engaged in a
nucleophilic substitution reaction in the presence of amines 15a-15g (15a acted as
nucleophile and N,N-diisopropylethylamine (DIPEA) acted as base, 15b-15g acted as
nucleophile, base and solvent) to obtain compounds 16a-16g. Deprotection of the
pyrazole N-H and reduction of nitro as the same as the synthesis procedures of
13a-13k to afford the target compounds 17a-17g. The chemical structures of newly
synthesized compounds were confirmed by physicochemical and spectral means.
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Scheme 2. Synthetic approach of target compounds 17a-17g. Reagents and conditions: (a)
KMnO4, H2O, HOAc, 0oC ~ rt, 95%; (b) (i) 15a, DIPEA, THF, 50%; (ii)15b ~ 15g (neat), rt; (c)
CF3COOH, 80 oC, 16h; (d) SnCl2•2H2O, 2-methylfuranidine, reflux.
2.2. Biological evaluation
2.2.1. In vitro enzymatic assay – Aurora A, Aurora B, and KDR
The prepared analogs 13a-13k were assessed for enzymatic activity against
Aurora A, Aurora B, and KDR. Using Staurosporine and Su11248 as the positive
controls, the results of the mean values from three independent determinations,
expressed as IC50 values, were summarized in Table 1.
As presented in Table 1, all of the compounds exhibited great inhibition activity
against Aurora A. Most of the compounds displayed equipotent or more excellent
activity than compound 13a （R = H）, except 13k (R = p-CF3). Among these
derivatives, compound 13g (R = p-CF3) was the most effective Aurora A inhibitor.
Dramatically reduce (6-fold) of Aurora A inhibition activity was observed when 13g
transformed into 13k, indicating that strong electronegative groups were unprofitable.
Compared 13b with 13d and also 13j with 13k, the results showed that the impact of
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ortho electronegative substitution in activity decrease was less than corresponding
para substitution. In addition, most of the compounds exhibited modest Aurora B
activity. It was interesting that, compound 13g provided optimal activity in both
Aurora A and Aurora B.
Table 1. Enzymatic activities of compounds 13a-13k.
Comp.
R
IC50a (nM) ± SD
Aurora B
Aurora A
KDR
13a
H
192.9 ± 25.4
94.0 ± 11.4
101.6 ± 10.3
182.8 ± 26.1
167.0 ± 37.9
75.0 ± 0.8
70.6 ± 17.5
191.0 ± 47.0
93.0 ± 11.8
128.0 ± 22.7
422.9 ± 98.4
3.67 ± 0.61
-
894.0 ± 267.6
278.1 ± 22.5
1003.6±162.6
1620.0 ± 18.3
611.0 ± 62.4
610.5 ± 80.4
99.8 ± 22.4
697.3 ± 158.5
83.7 ± 5.7
13b
o-Cl
m-Cl
p-Cl
m-F
o-Me
p-Me
o-OMe
p-OMe
o-CF3
p-CF3
-
13c
140.9 ± 5.9
317.6 ± 22.8
329.5 ± 95.5
110.1 ± 18.3
156.6 ± 5.7
13d
13e
13f
13g
13h
2320.6 ± 151.8 101.6 ± 7.9
2681.5 ± 109.3 429.8 ± 13.4
13i
13j
307.4 ± 60.2
854.4 ± 22.1
7.80 ± 3.35
-
205.2 ± 29.7
209.3 ± 24.1
-
13k
Staurosporineb
Su11248b
-
4.7±5.9
a Data presented is the mean ± SD value of three independent determinations.
b Used as positive control.
17a-17g were analogs with different R1 substitutions. As shown in Table 2, the
compounds 17b，17c and 17g displayed more excellent activity than compound 13g.
The most potent Aurora A and Aurora B inhibitor was morpholine substituted
compound 17g. Diethylamine substituted compound 17e was the most effective KDR
inhibitor. Because all of the IC50 values of Aurora A, Aurora B, and KDR were below
50 nM，compound 17g was chosen for further evaluation.
Table 2. Enzymatic activities of compounds 17a-17g.
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a Data presented is the mean ± SD value of three independent determinations.
b Used as positive control.
2.2.2. In vitro enzymatic screen assays of 17g.
The inhibitory activities of compound 17g were carried out over a panel of ten
selected kinases. Compound 17g were measured at the concentration of 100 nM and
1000 nM in duplicate. As presented in Table 3, 17g was justified to have a negligible
impact on most of the tested kinases at the concentration of 100 nM. Though the
inhibition rate of RET was 68.7% at the concentration of 1000 nM, the activity of 17g
against RET was weekly than Aurora A, Aurora B and KDR, indicating that
compound 17g had relatively better kinase selectivity.
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Table 3. In vitro enzymatic screen assays of compound 17g.
Inhibition (%)a
Comp.
nM
VEGFR-1
PDGFR-β
RET
EGFR
ErbB2
17g
17g
Su11248b
BIBW2992b
1000
100
28.1
0.0
41.0
13.2
75.7
-
68.7
28.7
76.7
-
37.4
24.5
-
0.0
3.0
-
1000
1000
66.0
c
-
99.7
97.6
ErbB4
c-Src
ABL
EPH-A2
IGF1R
17g
1000
100
17.5
12.0
88.4
-
32.0
0.0
0.0
-
46.3
26.8
1.4
-
17g
0.0
3.9
BIBW2992b
Dasatinibb
AEW541b
1000
1000
1000
-
100.0
-
-
100.0
-
100.0
-
-
-
98.9
a Data presented is the mean value of two independent determinations.
b Used as positive control.
c Not tested.
2.2.3. Cytotoxicity of 17g.
The development of gastric cancer drugs was urgent, we were more interested in
this filed. Base on that, the anti-proliferative activity of compound 17g against human
gastric cancer cells were tested, including SNU-5, MKN-45, MKN-74, SGC-7901 and
BGC-823 cell lines. Using the approved drug Doxorubicin (ADR) as positive controls,
the results of the mean values of experiments from three independent determinations,
expressed as half-maximal inhibitory concentration (IC50) values, were summarized in
Table 4.
As presented in Table 4, compound 17g exhibited moderate anti-proliferative
activities against SNU-5, MKN-45 and MKN-74 cell lines but low activities against
SGC-7901 and BGC-823 cells lines.
Table 4. In vitro anti-proliferative activities of 17g against five cancer cell lines.
Anti-proliferative activities IC50 (µM)a
Comp.
SNU-5
MKN-45
MKN-74
SGC-7901
> 10
BGC-823
> 10
17g
1.27±0.14
1.26 ± 0.56
3.14 ± 0.41
ADRb 0.237±0.022
0.127 ± 0.063 0.204 ± 0.038 0.13 ± 0.039
0.405 ± 0.197
a Data presented is the mean ± SD value of three independent determinations.
b Used as positive control.
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2.2.4. Cell cycle analysis
Inhibition of Aurora A has been documented could induce cell cycle G2/M phase
arrest, while suppression on Aurora kinase B has been proved to cause polyploidy in
cells without cytokinesis [5]. The effects of 17g on cell cycle distribution was then
determined in MKN-74, MKN-45, SGC-7901 and SNU-5 cells (Fig.4). In all the
tested cells, 17g treatment induced an obvious accumulation of cells in G2/M phase
and with >4 N DNA content at 24 h, in a concentration-dependent manner.
Fig. 4. Cell cycle distribution of MKN-74, MKN-45, SGC-7901 and SNU-5 cells after compound
17g treatment for 24 h at different concentration. The 2 N (diploid) and 4 N (tetraploid) DNA
content represented the G1 and G2/M phases of the cell cycle, respectively.
2.2.5. Pharmacokinetic analysis of 17g.
17g showed acceptable PK properties in ICR mouse. The single-dose PK of 17g
was given in Table 5 and Table 6. Following intravenous injection, the volume of
distribution at steady state (Vdss) was generally high (2.01 L/kg), the terminal
elimination half-life (T1/2) was 1.53 h. Following single-dose oral administration, the
terminal elimination half-life (T1/2) was 3.27 h. Besides, 17g was generally well
absorbed (F = 63.8%).
Table 5. Single IV dose PK of 17ga.
Comp. Does
(mg/kg) h﹒ng﹒mL
4724 ± 356
AUC0-tb± SD CL ± SD
MRTIV ± SD
(h)
Vdss ± SD t1/2 ± SD
(mL/kg/h)
(L/kg)
(h)
17g
5
17.3± 1.39 1.93 ± 0.05
2.01 ± 0.17
1.53 ± 0.33
a ICR mouse, male.
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b Data presented is the mean ± SD value of three independent determinations.
Table 6. Single PO dose PK of 17ga.
Comp.
Does
(mg/kg)
25
AUC0-tb± SD Cmax± SD
Tmax± SD
(h)
t1/2 ± SD
(h)
F%
h﹒ng﹒mL
(ng/mL)
17g
15335 ± 69
2783± 881 0.5 ± 0
3.27± 0.66 63.8%
a ICR mouse, male.
b Data presented is the mean ± SD value of three independent determinations.
2.2.6. Antitumor activity in vivo
To further explore the anti-tumor efficacy of 17g in vivo, human gastric cancer
xenograft model SNU-5 was used. Using the approved drug Cabozantinib（XL184）
as positive controls, the results were summarized in Table 7. The p.o. administration
of 17g at dosage of 20 mg/kg per day delayed tumor growth with a T/C value of 61.63%
and no obvious toxicity observed.
Table 7. Anti-tumor activity of 17g in vivo.
TV (mm3,
No.
Weitht (g)
d0 d21
RTV
T/C
mean±SD)
d0 d21
Groups
(mean±SD) (%)
d0 d21
Vehicle
XL184
po 12 12 19.4 19.6 149±52 2144±897 14.40±4.36
30 mg/kg
qd×21
po
po
6
6
6
6
18.5 19.9 157±51
185±65
1.21±0.46** 8.41
20 mg/kg,
qd×21
17g
18.9 18.4 149±47 1426±869 8.88±2.84* 61.63
*p<0.05，**p<0.001
3. Conclusion
In summary, a series of pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine derivatives
were designed and synthesized. Most of the derivatives 13a-13k exhibited obviously
inhibition of Aurora A and KDR below the concentration of 200 nM. The analogs
with different substitutions of R1, 17b, 17c, 17f, and 17g displayed inhibition
activities of Aurora B below 100 nM. Among the derivatives, 17g with significant
Aurora A/B and KDR potencies was chosen for further evaluation. In addition,
compound 17g induced G2/M cell cycle arrest in all tested cell lines as expected.
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Enzymatic assays on ten selected kinases confirmed that compound 17g had relatively
better kinase selectivity. Besides, 17g gave acceptable pharmacokinetic profiles (F =
63.8%). Furthermore, the significant growth inhibition of this compound was found at
dosage of 20 mg/kg. In summary, pyrazolo[4,3-b]pyrimido[4,5-e][1,4]diazepine could
be developed as new scaffold of multiple Auroras and KDR inhibitors and compound
17g was worth of further modification as a new multi-target anticancer potential lead
compound.
4. Experimental
4.1. Chemistry
All reagents and solvents were purchased from the suppliers and purified/dried if
1
13
anhydrous one was necessary. The H NMR spectra and C NMR spectra were
recorded in CDCl3, CH3OD, CD3CN and DMSO-d6 on a Bruker DRX-400 (400 MHz)
using TMS as internal standard. Chemical shifts were reported as δ (ppm) and
spin-spin coupling constants as J (Hz) values. Mass spectra (MS) were recorded on a
Waters SDQ mass spectrometer and high resolution mass spectra (HRMS) were
recorded on a Waters SYNAPT G2 ESI-TOF-MS analyzer. Melting points were taken
on a SGW X-4 melting point apparatus, uncorrected and reported in degrees
Centigrade. Column chromatography was performed with silica gel (200–300 mesh).
The purity of all tested compounds was established by HPLC to be >95.0%. HPLC
analyses were performed on an Agilent Technologies 1200 series. HPLC analysis was
performed on an Agilent Technologies 1200 series using an Agilent Eclipse XDBC18
(250 mm × 4.6 mm) and as a mobile phase gradient from 5% MeCN/H2O (1‰ TFA)
for 1 min, 5% MeCN/H2O (1‰ TFA) to 95% MeCN/H2O (1‰ TFA) for 9 min and 95%
MeCN/H2O (1‰ TFA) for 5 min more, a flow rate of 1.0 mL/min.
4.1.1
．
N-(3-methyl-1H-pyrazol-5-yl)acetamide (2)
5-methyl-1H-pyrazol-3-amine (1) (10g, 103mmol) was dissolved in 100 mL of
distilled water, NaHCO3 (26g, 309 mmol) was slowly added. Acetic anhydride (19.5
mL, 206 mmol) was then added dropwise and the resulting suspension was heated at
reflux for 16 h (monitored by TLC). Then, the mixture was allowed to cool down to
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RT, white crystals precipitated slowly. The precipitate was collected, washed with
water and dried to provide the desired product 2 (6.2g, 44 mmol, 43% yield) without
further purification. Mp: 210~211 °C; 1H NMR (400 MHz, DMSO-d6) δ 11.92 (s, 1H),
10.19 (s, 1H), 6.24 (s, 1H), 2.17 (s, 3H), 1.96 (s, 3H).
4.1.2
．
N-(5-methyl-4-nitro-1H-pyrazol-3-yl)acetamide (3)
Compound 2 (4.5g, 32mmol) was dissolved in 50 mL concentrated H2SO4, and
the solution was cooled to -5 °C. Concentrated HNO3 (2.3 mL, 32mmol) was added
dropwise and the mixture was maintained at -5 °C for 3 h. The reaction mixture was
added to 150 mL ice water slowly, and then white solid appeared with stirring. The
solid washed by water and dried to give pure 3 (4.65 g, 25 mmol, 72% yield). Mp:
1
240~241 °C; H NMR (400 MHz, DMSO-d6) δ 10.24 (s, 1H), 2.45 (s, 3H), 2.13 (s,
3H).
4.1.3
．
N-(1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)acetamide (4)
A solution of compound 3 (4.4 g, 24 mmol) and K2CO3 (6.6 g, 48 mmol) in
DMF (80 ml) under N2 atmosphere was added 4-methoxybenzylchloride (PMBCl, 3.6
mL, 25 mmol) over a time period of 20 minutes under ice bath. The resulting solution
was maintained at 50 °C for 3 h (monitored by TLC). The reaction mixture was then
quenched by the adding 400 ml of ice-water. The resulting solution was extracted
three times with 200 ml of ethyl acetate (EA) and the organic layers combined and
dried over anhydrous sodium sulfate. The residue was purified by eluting through a
silica gel column with a 1:5 EA/PE solvent system to give 4 (6.33 g, 20.8 mmol, 87%
yield), White solid. Mp: 132.2~132.8 °C; 1H NMR (400 MHz, DMSO-d6) δ 10.19 (s,
1H), 7.22 (d, J = 8.2 Hz, 2H), 6.93 (d, J = 8.1 Hz, 2H), 5.28 (s, 2H), 3.74 (s, 3H), 2.60
13
(s, 3H), 2.03 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 168.7, 159.0, 140.5, 140.1,
129.1, 127.34, 125.5, 114.1, 55.1, 52.2, 22.7, 11.2; ESI-MS: m/z 305.1 [M+H]+.
4.1.4. 1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-amine (5)
Compound 4 (6.08 g, 20mmol) was dissolved in 30 mL ethanol, 2M KOH 30 mL
was added subsequently. The resulting solution was heated at reflux for 16 h. Then,
the mixture was allowed to cool down to RT, yellow crystals precipitated slowly. The
precipitate was collected, washed with water and ether, then dried to provide the pure
14

ACCEPTED MANUSCRIPT
5 (4.98 g, 19 mmol, 98% yield). Mp: 155.1~155.5 °C; 1H NMR (400 MHz, CDCl3) δ
7.12 (d, J = 8.5 Hz, 2H), 6.87 (d, J = 8.6 Hz, 2H), 5.16 (s, 2H), 5.04 (s, 2H), 3.79 (s,
3H), 2.58 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 158.8, 151.1, 138.8, 128.9,
127.7, 119.3, 114.0, 55.1, 51.6, 11.6; ESI-MS: m/z 263.11 [M+H]+.
4.1.5. 5-Iodo-4-hydroxy-6-methyl-2-methylthiopyrimidine (7)
A solution of 4-hydroxy-6-methyl-2-methylthiopyrimidine (6) (42.6 g, 300 mmol)
and NIS (74.2g, 330 mmol) in CHCl3 (200 mL) was maintained at 70 °C for 5h under
N2 atmosphere. After cooling, the solvent is removed under reduced pressure. Water
(300 mL) was added to this residue, and the precipitated crystals were collected. The
crystals were washed with water until the color of yellow disappeared, and then dried
with P2O5 in desiccator to afford the desired product 7 (40.3 g, 150 mmol, 50% yield)
1
without further purification, White solid. Mp: 212~213 °C; H NMR (400 MHz,
DMSO-d6) δ 13.13 (s, 1H), 8.30 (s, 1H), 2.46 (s, 3H); ESI-MS: m/z 268.9 [M+H]+.
4.1.6
．
4-chloro-5-iodo-2-(methylthio)pyrimidine (8)
Compound 7 (26.8 g, 100 mmol) was dissolved in POCl3 (100mL, 1.07mol). The
solution was heated to reflux for 8 h (monitored by TLC). The mixture was allowed to
cool down to 40 °C, and then added to 300 mL water slowly. The aqueous layer was
further extracted with DCM (total 1L). The organic layer was washed with saturated
NaHCO3 aqueous solution several times, then with water and brine, dried over
anhydrous sodium sulfate. Filtered and the filtration was concentrated in vacuo and
used in the next step directly (28.1 g, 98mmol, 98% yield), White solid. Mp: >300°C;
1H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 2.55 (s, 3H); ESI-MS: m/z 286.9 [M+H]+.
4.1.7. General procedure of the synthesis of compounds 11a-11k (Procedure 1)
Step 1: To a solution of compound 8 (572mg, 2 mmol) in anhydrous THF (10
mL) at -40 °C under N2 atmosphere was slowly added a solution of 2.0 M
isopropylmagnesium chloride（1 mL, 2 mmol）. After addition was completed, the
resulting mixture was stirred at -40 °C for an additional 30 min. The corresponding
aldehyde (2 mmol) in THF (1 mL) was added dropwise subsequently. The reaction
mixture was stirred for another 2 h at -40 °C and then quenched with saturated
ammonium chloride solution. The mixture was allowed to warm to RT, EA was added,
15

ACCEPTED MANUSCRIPT
and the layers were partitioned and separated. The aqueous layer was extracted with
EA, and the combined organic layers were washed with brine and then dried over
anhydrous sodium sulfate, filtered and concentrated to give corresponding crude
product 10a-10k which was purified by column chromatography furthermore.
Step 2: Corresponding alcohol (1 mmol) was dissolved in anhydrous THF (10
mL), Dess-Martin Periodinane (848 mg, 2 mmol, oxidant) was added to the solution
in portions at 0 °C. After addition was completed, the resulting mixture was allowed
to warm to RT and stirred for 2 h (monitored by TLC). EA (30 mL) and water (30 mL)
were added, and the layers were partitioned and separated. The organic layers were
washed with Na2S2O3 solution, then water and brine, dried over anhydrous sodium
sulfate. Filtered and the filtration was concentrated in vacuum. The residue was
purified through a silica gel column to give corresponding ketone 11a-11k.
4.1.7.1. (4-chloro-2-(methylthio)pyrimidin-5-yl)(phenyl)methanol (10a).
From compound 8 (572mg, 2 mmol) and 9a (212 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10a 288mg. Yield: 54%; Yellow viscous
1
semi-solid; H NMR (400 MHz, CDCl3) δ 8.69 (s, 1H), 7.38 (s, 2H), 7.37 (m, 3H),
13
6.04 (s, 1H), 2.56 (s, 4H，Me + OH); C NMR (100 MHz, CDCl3) δ 172.7, 158.9,
139.1, 134.4, 129.1, 128.2, 70.6, 14.3; ESI-MS: m/z 267.0 [M+H]+.
4.1.7.2. (4-chloro-2-(methylthio)pyrimidin-5-yl)(phenyl)methanone (11a).
From compound 10a (266 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11a 242mg. Yield: 92%;
1
Yellow viscous semi-solid; H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.87 – 7.78
(m, 2H), 7.70 – 7.61 (m, 1H), 7.56 – 7.46 (m, 2H), 2.63 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 191.4, 175.5, 158.1, 157.7, 136.1, 134.3, 129.9, 128.9, 126.9, 14.4; ESI-MS:
m/z 265.0 [M+H]+.
4.1.7.3. (4-chloro-2-(methylthio)pyrimidin-5-yl)(2-chlorophenyl)methanol (10b).
From compound 8 (572mg, 2 mmol) and 9b ( 280 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10b 354 mg. Yield: 59%; Orange-yellow
semi-solid; 1H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 7.46 (d, J = 6.9 Hz, 1H), 7.40
(d, J = 7.0 Hz, 1H), 7.31 (s, 2H), 6.38 (s, 1H), 2.76 (s, 1H), 2.56 (s, 3H); ESI-MS: m/z
16

ACCEPTED MANUSCRIPT
301.0 [M+H]+.
4.1.7.4. (4-chloro-2-(methylthio)pyrimidin-5-yl)(2-chlorophenyl)methanone (11b).
From compound 10b (300 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11b 269mg. Yield: 90%;
1
Yellow viscous semi-solid; H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.49 (d, J =
7.3 Hz, 1H), 7.42 (d, J = 7.1 Hz, 1H), 7.40 – 7.27 (m, 2H), 2.55 (s, 3H); ESI-MS: m/z
299.0 [M+H]+.
4.1.7.5. (4-chloro-2-(methylthio)pyrimidin-5-yl)(3-chlorophenyl)methanol (10c).
From compound 8 (572mg, 2 mmol) and 9c ( 280 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10c 340 mg. Yield: 57%; Orange-yellow
1
semi-solid; H NMR (400 MHz, CDCl3) δ 8.62 (s, 1H), 7.38 (s, 1H), 7.31 – 7.27 (m,
2H), 7.26 – 7.22 (m, 1H), 6.02 (s, 1H), 3.00 (s, 1H), 2.56 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 172.7, 159.0, 156.9, 142.8, 134.8, 130.1, 129.2, 128.6, 126.9, 124.9, 70.4,
14.3; ESI-MS: m/z 301.0 [M+H]+.
4.1.7.6. (4-chloro-2-(methylthio)pyrimidin-5-yl)(3-chlorophenyl)methanone (11c).
From compound 10c (300 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11c 282 mg. Yield: 94%;
Yellow viscous semi-solid; 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.80 (m, 1H),
13
7.69 – 7.64 (m, 1H), 7.64– 7.61(m, 1H), 7.46 (t, J = 7.9 Hz, 1H), 2.64 (s, 3H); C
NMR (100 MHz, CDCl3) δ 190.2, 176.0, 158.1, 157.7, 137.7, 135.4, 134.2, 130.2,
129.6, 128.0, 126.3,14.4; ESI-MS: m/z 299.0 [M+H]+.
4.1.7.7. (4-chloro-2-(methylthio)pyrimidin-5-yl)(4-chlorophenyl)methanol (10d).
From compound 8 (572mg, 2 mmol) and 9d ( 280 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10d 364 mg. Yield: 60%; Orange-yellow
semi-solid; 1H NMR (400 MHz, CDCl3) δ 8.66 (s, 1H), 7.34 (d, J = 2.9 Hz, 4H), 6.03
(d, J = 3.6 Hz, 1H), 2.56 (s, 3H), 2.45 (d, J = 3.7 Hz, 1H); 13C NMR (100 MHz,
CDCl3) δ 174.9, 156.8, 139.2, 134.4, 129.2, 129.1, 128.2, 70.6, 14.3; ESI-MS: m/z
301.0 [M+H]+.
4.1.7.8. (4-chloro-2-(methylthio)pyrimidin-5-yl)(4-chlorophenyl)methanone (11d).
From compound 10d (300 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
17

ACCEPTED MANUSCRIPT
mmol), as that described in Step 2 of Procedure 1, gave pure 11d 270 mg. Yield: 90%;
1
Yellow viscous semi-solid; H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.79 – 7.72
(m, 2H), 7.56 – 7.46 (m, 2H), 2.63 (s, 3H); 13C NMR (100 MHz, CDCl3) δ190.2,
175.8, 158.0, 157.7, 141.0, 134.5, 131.2, 129.3, 126.5, 14.4; ESI-MS: m/z 299.0
[M+H]+.
4.1.7.9. (4-chloro-2-(methylthio)pyrimidin-5-yl)(3-fluorophenyl)methanol (10e).
From compound 8 (572mg, 2 mmol) and 9e (248 mg, 2 mmol), as that described
1
in Step 1 of Procedure 1, gave pure 10e 354 mg. Yield: 62%; Yellow oil; H NMR
(400 MHz, CDCl3) δ 8.61 (s, 1H), 7.33 (m, 1H), 7.12 (m, 2H), 7.01 (m, 1H), 6.04 (s,
1H), 2.55 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 164.2, 161.8, 159.0, 157.0, 143.3 (d,
J = 6.7 Hz), 130.5 (d, J = 8.2 Hz), 129.3, 122.4 (d, J = 3.0 Hz), 115.4 (d, J = 21.1 Hz),
113.8 (d, J = 22.1 Hz ), 70.4, 14.3; ESI-MS: m/z 285.0 [M+H]+.
4.1.7.10. (4-chloro-2-(methylthio)pyrimidin-5-yl)(3-fluorophenyl)methanone (11e).
From compound 10e (285 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11e 261 mg. Yield: 92%;
Yellow oil; 1H NMR (400 MHz, CDCl3) δ 8.51 (s, 1H), 7.53 (m, 2H), 7.51 – 7.45 (m,
13
1H), 7.36 (t, J = 7.7 Hz, 1H), 2.63 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 189.8,
162.9, 161.6, 160.5, 150.8, 148.2, 130.2 (d, J = 7.7 Hz), 125.1, 119.3 (d, J = 22.1 Hz),
115.5 (d, J = 22.2 Hz), 111.8, 13.0; ESI-MS: m/z 283.0 [M+H]+.
4.1.7.11. (4-chloro-2-(methylthio)pyrimidin-5-yl)(o-tolyl)methanol (10f).
From compound 8 (572mg, 2 mmol) and 9f ( 240 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10f 354 mg. Yield: 63%; White solid; Mp:
1
127.1~128.5 °C; H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.33 – 7.28 (m, 1H),
7.26 – 7.22 (m, 2H), 7.22 – 7.18 (m, 1H), 6.21 (s, 1H), 2.57 (s, 3H), 2.36 (s, 1H), 2.34
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 172.3, 159.8, 157.3, 138.2, 135.7, 131.0,
129.0, 128.5, 126.5, 126.4, 68.1, 19.1, 14.3; ESI-MS: m/z 281.0 [M+H]+.
4.1.7.12. (4-chloro-2-(methylthio)pyrimidin-5-yl)(o-tolyl)methanone (11f).
From compound 10f (281 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11f 272 mg. Yield: 97%;
Yellow viscous semi-solid; 1H NMR (400 MHz, CDCl3) δ 8.53 (s, 1H), 7.46 (td, J =
18

ACCEPTED MANUSCRIPT
7.6, 1.1 Hz, 1H), 7.33 (d, J = 7.8 Hz, 2H), 7.24 (d, J = 7.6 Hz, 1H), 2.62 (s, 3H), 2.54
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 194.0, 178.6, 158.5, 139.4, 132.6, 132.1,
130.8, 130.6, 125.9, 124.4, 124.0, 21.0, 14.5; ESI-MS: m/z 279.0 [M+H]+.
4.1.7.13. (4-chloro-2-(methylthio)pyrimidin-5-yl)(p-tolyl)methanol (10g).
From compound 8 (572mg, 2 mmol) and 9g ( 240 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10g 339 mg. Yield: 60%; White solid; Mp:
130.5~132.0 °C; 1H NMR (400 MHz, CDCl3) δ 8.72 (s, 1H), 7.25 (d, J = 7.9 Hz, 2H),
7.17 (d, J = 7.9 Hz, 2H), 6.01 (s, 1H), 2.56 (s, 3H), 2.34 (s, 3H); 13C NMR (100 MHz,
CDCl3) δ 172.2, 159.0, 156.8, 138.5, 137.8, 129.7, 129.6, 126.9, 71.1, 21.2, 14.3;
ESI-MS: m/z 281.0 [M+H]+.
4.1.7.14. (4-chloro-2-(methylthio)pyrimidin-5-yl)(p-tolyl)methanone (11g).
From compound 10g (281 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11g 267 mg. Yield: 96%;
White solid; Mp: 101.1~102.4 °C; 1H NMR (400 MHz, CDCl3) δ 8.49 (s, 1H), 7.71 (d,
13
J = 8.1 Hz, 2H), 7.31 (d, J = 8.0 Hz, 2H), 2.63 (s, 3H), 2.45 (s, 3H); C NMR (100
MHz, CDCl3) δ 190.9, 175.2, 158.0, 157.5, 145.6, 133.6, 130.1, 129.7, 127.1, 21.9,
14.4; ESI-MS: m/z 279.0 [M+H]+.
4.1.7.15. (4-chloro-2-(methylthio)pyrimidin-5-yl)(2-methoxyphenyl)methanol (10h).
From compound 8 (572mg, 2 mmol) and 9h ( 272 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10h 342 mg. Yield: 58%; Yellow viscous
semi-solid; 1H NMR (400 MHz, CDCl3) δ 8.57 (s, 1H), 7.31 (m,1H), 7.20 (d, J = 7.4
Hz, 1H), 6.97 (d, J = 7.4 Hz, 1H), 6.91 (d, J = 8.3 Hz, 1H), 6.21 (s, 1H), 3.87 (s, 1H),
3.85 (s, 3H), 2.57 (s, 3H); 13C NMR (101 MHz, CDCl3) δ 172.0, 159.5, 157.5, 156.7,
129.7, 128.8, 128.3, 127.9, 120.9, 110.8, 67.8, 55.4, 14.3; ESI-MS: m/z 297.0
[M+H]+.
4.1.7.16. (4-chloro-2-(methylthio)pyrimidin-5-yl)(2-methoxyphenyl)methanone (11h).
From compound 10h (297 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11h 275 mg. Yield: 93%;
Yellow viscous semi-solid;1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.73 (d, J = 7.6
Hz, 1H), 7.55 (d, J = 7.7 Hz, 1H), 7.09 (t, J = 7.5 Hz, 1H), 7.06 – 6.97 (m, 1H), 6.94
19

ACCEPTED MANUSCRIPT
(d, J = 8.3 Hz, 1H), 3.69 (s, 3H), 2.63 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ
189.8, 173.6, 158.7, 158.0, 156.5, 135.5, 130.6, 129.0, 125.8, 121.0, 112.6, 56.0, 13.9;
ESI-MS: m/z 295.0 [M+H]+.
4.1.7.17. (4-chloro-2-(methylthio)pyrimidin-5-yl)(4-methoxyphenyl)methanol (10i).
From compound 8 (572mg, 2 mmol) and 9i ( 272 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10i 340 mg. Yield: 57%; White solid; Mp:
135.1~136.9 °C ; 1H NMR (400 MHz, CDCl3) δ 8.73 (s, 1H), 7.28 (d, J = 8.5 Hz, 2H),
6.89 (d, J = 8.3 Hz, 2H), 5.98 (s, 1H), 3.80 (s, 3H), 2.56 (s, 3H), 2.37 (d, J = 2.1 Hz,
13
1H); C NMR (101 MHz, CDCl3) δ 172.2, 159.7, 159.0, 156.7, 132.9, 129.7, 128.3,
114.3, 70.9, 55.3, 14.3; ESI-MS: m/z 297.0 [M+H]+.
4.1.7.18. (4-chloro-2-(methylthio)pyrimidin-5-yl)(4-methoxyphenyl)methanone (11i).
From compound 10i (297 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11i 272 mg. Yield: 92%;
White solid; Mp: 93.2~94.7 °C; 1H NMR (400 MHz, CDCl3) δ 8.48 (s, 1H), 7.80 (d, J
13
= 8.5 Hz, 2H), 6.98 (d, J = 8.5 Hz, 2H), 3.90 (s, 3H), 2.63 (s, 3H); C NMR (101
MHz, CDCl3) δ 189.7, 175.0, 164.7, 158.0, 157.3, 132.5, 129.0, 127.3, 114.2, 55.7,
14.4; ESI-MS: m/z 295.0 [M+H]+.
4.1.7.19. (4-chloro-2-(methylthio)pyrimidin-5-yl)(2-(trifluoromethyl)phenyl)methanol
(10j).
From compound 8 (572mg, 2 mmol) and 9j ( 348 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10j 367 mg. Yield: 55%; White solid;
Mp:126.0~126.9 °C ; 1H NMR (400 MHz, CDCl3) δ 8.50 (s, 1H), 7.74 (d, J = 7.7 Hz,
1H), 7.59 (t, J = 7.6 Hz, 1H), 7.48 (t, J = 8.0 Hz, 2H), 6.44 (s，1H), 2.58 (s, 1H), 2.57
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 172.9, 159.6, 157.0, 138.4, 132.6, 128.8,
128.6, 128.4, 126.6, 126.5, 122.9, 66.9, 14.3; ESI-MS: m/z 335.0 [M+H]+.
4.1.7.20.
(4-chloro-2-(methylthio)pyrimidin-5-yl)(2-(trifluoromethyl)phenyl)methanone (11j).
From compound 10j (335 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11j 310 mg. Yield: 93%;
White solid; Mp: 93.2~94.7 °C ; 1H NMR (400 MHz, CDCl3) δ 8.65 (s, 1H), 7.81 (d,
20

ACCEPTED MANUSCRIPT
J = 6.6 Hz, 1H), 7.70 – 7.57 (m, 2H), 7.44 (d, J = 6.0 Hz, 1H), 2.62 (s, 3H); 13C NMR
(101 MHz, DMSO-d6) δ 191.9, 176.0, 169.2, 160.6, 159.6, 157.9, 139.6, 132.1, 129.9,
127.3, 126.2, 118.1, 13.1; ESI-MS: m/z 333.0 [M+H]+.
4.1.7.21. (4-chloro-2-(methylthio)pyrimidin-5-yl)(4-(trifluoromethyl)phenyl)methanol
(10k).
From compound 8 (572mg, 2 mmol) and 9k ( 348 mg, 2 mmol), as that described
in Step 1 of Procedure 1, gave pure 10k 360 mg. Yield: 54%; White solid; Mp:
152.2~153.7 °C ; 1H NMR (400 MHz, CDCl3) δ 8.63 (s, 1H), 7.64 (d, J = 8.2 Hz, 2H),
7.53 (d, J = 8.2 Hz, 2H), 6.13 (s, 1H), 2.62 (s, 1H), 2.56 (s, 3H); 13C NMR (101 MHz,
CDCl3) δ 173.0, 159.0, 157.0, 144.5, 130.9, 130.5, 129.0, 127.1, 125.9, 70.5, 14.3;
ESI-MS: m/z 335.0 [M+H]+.
4.1.7.22.
(4-chloro-2-(methylthio)pyrimidin-5-yl)(4-(trifluoromethyl)phenyl)methanone (11k).
From compound 10k (335 mg, 1 mmol) and Dess-Martin Periodinane (848mg, 2
mmol), as that described in Step 2 of Procedure 1, gave pure 11k 305 mg. Yield: 92%;
White solid; Mp: 91.0~92.5 °C ; 1H NMR (400 MHz, CDCl3) δ 8.54 (s, 1H), 7.92 (d,
J = 8.1 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H), 2.64 (s, 3H); 13C NMR (100 MHz, CDCl3) δ
190.6, 176.3, 158.1, 158.0, 138.9, 135.8, 135.5, 135.2, 134.9, 130.1, 126.1, 126.0,
124.7, 122.0,14.5; ESI-MS: m/z 333.0 [M+H]+.
4.1.8. General procedure of the synthesis of compounds 13a-k (Procedure 2)
Step 1: Compound 5 (220 mg, 0.45 mmol) was dissolved in anhydrous THF (10
mL), NaH (2.7 mmol) was added to the solution in portions at 0 °C. After addition
was completed, the resulting mixture was allowed to warm to RT and stirred for 1 h.
Corresponding ketone 11a-k (0.5 mmol ) in anhydrous THF (5 mL) was added slowly,
the resulting mixture was stirred at RT for overnight. Water (40 mL) was added, the
aqueous layer was extracted with EA several times. The combined organic layers were
washed with brine solution and then dried over anhydrous sodium sulfate, filtered and
concentrated to give corresponding crude product 12a-k. The residue was purified
through a silica gel column or beating with EA.
21

ACCEPTED MANUSCRIPT
Step 2：Corresponding 12a-k (0.1 mmol) was dissolved in TFA (2 mL) and
toluene (2 mL), the resulting solution was heated to reflux for overnight. The reaction
mixture was monitored by TLC, TFA added until the reaction completely. Then, the
mixture was added to water, NaHCO3 solution was added until pH>8. The aqueous
layer was extracted with EA several times. The combined organic layers were washed
with brine solution and then concentrated to give corresponding crude product, which
used in the next step immediately. The crude product was dissolved in
2-methylfuranidine (5 mL), SnCl2•2H2O (0.3mmol) was added to the solution, and the
resulting mixture was heated to reflux for overnight (monitored by LC-MS). EA (30
mL) and Na2CO3 (30 mL) solution were added, filtered and the filtrate was partitioned
and separated. The aqueous layer was extracted with EA several times. The combined
organic layers were washed with water and brine solution, and then dried over
anhydrous sodium sulfate, filtered and concentrated to give corresponding crude
product 13a-k, which was purified through a silica gel column furthermore.
4.1.8. 1.
(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2-(methylthiopyri
midin-5-yl)(phenyl)methanone (12a).
From compound 5 (220 mg, 0.45 mmol) and 11a (133 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12a 177 mg. Yield: 72%; White solid;
Mp: 189.6~190.2 °C ; 1H NMR (400 MHz, CDCl3) δ 12.19 (s, 1H), 8.57 (s, 1H), 7.75
– 7.66 (m, 2H), 7.60 (t, J = 7.4 Hz, 1H), 7.50 (t, J = 7.5 Hz, 2H), 7.20 (d, J = 8.6 Hz,
2H), 6.89 (d, J = 8.7 Hz, 2H), 5.25 (s, 2H), 3.80 (s, 3H), 2.67 (s, 3H), 2.58 (s, 3H); 13C
NMR (100 MHz, CDCl3) δ 195.8, 177.2, 161.6, 159.8, 157.5, 142.1, 139.6, 137.8,
132.4, 129.4, 128.6, 126.7, 114.5, 109.2, 55.4, 53.8, 14.3, 11.8; ESI-MS: m/z 491.1
[M+H]+.
4.1.8. 2.
3-methyl-8-(methylthio)-5-phenyl-2,10-dihydropyrazolo[4,3-b]pyrimido[4,5-e][1,4]di
azepine (13a).
From compound 12a (49mg, 0.1 mmol) as that described in Step 2 of Procedure
2 gave pure 13a 11 mg. Yield: 34%; Yellow solid; Mp: > 300 °C ; 1H NMR (400 MHz,
22

ACCEPTED MANUSCRIPT
DMSO-d6) δ 11.89 (s, 1H), 9.31 (s, 1H), 7.46 (s, 1H), 7.39 (d, J = 10.0 Hz, 5H), 2.41
13
(s, 3H), 2.07 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 172.2, 164.8, 159.8, 156.9,
143.2, 141.4, 138.8, 128.9, 128.2, 128.1, 118.4, 112.1, 13.3, 8.4; HPLC: room
temperature, tg= 8.50 min, UV254= 96.2%; HRMS (ESI) m/z calcd for C16H14N6S
[M+H]+: 323.1079 ; found: 323.1073.
4.1.8. 3.
(2-chlorophenyl)(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)-amino)-
2-(methylthio)pyrimidin-5-yl)methanone (12b).
From compound 5 (220 mg, 0.45 mmol) and 11b (150 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12b 179 mg. Yield: 68%; White solid;
Mp: 153.5~154.6 °C ; 1H NMR (400 MHz, CDCl3) δ 12.29 (s, 1H), 8.24 (s, 1H), 7.42
(m, 4H), 7.20 (d, J = 8.7 Hz, 2H), 6.89 (d, J = 8.7 Hz, 2H), 5.26 (s, 2H), 3.80 (s, 3H),
2.67 (s, 3H), 2.56 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 194.6, 178.1, 162.4, 159.8,
156.8, 141.9, 139.6, 137.4, 131.5, 131.0, 130.1, 129.0, 128.6, 127.1, 126.6, 123.7,
114.5, 109.2, 55.4, 53.8, 14.4, 11.8; ESI-MS: m/z 525.1 [M+H]+.
4.1.8. 4.
5-(2-chlorophenyl)-3-methyl-8-(methylthio)-2,10-dihydropyrazolo[4,3b]pyrimido[4,5-
e][1,4]diazepine (13b).
From compound 12b (53mg, 0.1 mmol) as that described in Step 2 of Procedure
2 gave pure 13b 13 mg. Yield: 36%; Yellow solid; Mp: 285.4~286.7 °C ; 1H NMR
(400 MHz, DMSO-d6) δ 11.86 (s, 1H), 9.48 (s, 1H), 7.53 – 7.48 (m, 1H), 7.46 – 7.37
13
(m, 3H), 7.02 (s, 1H), 2.38 (s, 3H), 1.98 (s, 3H); C NMR (100 MHz, DMSO-d6) δ
172.7, 162.4, 158.6, 154.2, 142.9, 140.0, 139.7, 130.8, 130.7, 129.9, 129.6, 127.9,
118.0, 112.5, 13.2, 8.3; HPLC: room temperature, tg= 9.21 min, UV290= 98.0%;
HRMS (ESI) m/z calcd for C16H13ClN6S [M+H]+: 357.0689 ; found: 357.0674.
4.1.8.5.
(3-chlorophenyl)(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2
-(methylthio)pyrimidin-5-yl)methanone (12c).
From compound 5 (220 mg, 0.45 mmol) and 11c (150 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12c 181 mg. Yield: 69%; White solid;
23

ACCEPTED MANUSCRIPT
Mp: 221.0~222.5 °C ; 1H NMR (400 MHz, CDCl3) δ 12.16 (s, 1H), 8.54 (s, 1H), 7.69
(s, 1H), 7.56 (t, J = 6.4 Hz, 2H), 7.45 (t, J = 7.8 Hz, 1H), 7.20 (d, J = 8.2 Hz, 2H),
13
6.89 (d, J = 8.2 Hz, 2H), 5.25 (s, 2H), 3.80 (s, 3H), 2.67 (s, 3H), 2.58 (s, 3H); C
NMR (100 MHz, CDCl3) δ 194.3, 177.7, 161.5, 159.7, 157.3, 142.0, 139.6, 139.4,
134.9, 132.3, 129.9, 129.3, 128.6, 127.4, 126.6, 123.5, 114.5, 108.8, 55.4, 53.8, 14.4,
11.8; ESI-MS: m/z 525.2 [M+H]+.
4.1.8.6.
5-(3-chlorophenyl)-3-methyl-8-(methylthio)-2,10-dihydropyrazolo[4,3-b]pyrimido[4,
5-e][1,4]diazepine (13c).
From compound 12c (53mg, 0.1 mmol) as that described in Step 2 of Procedure
2 gave pure 13c 13 mg. Yield: 36%; Yellow solid; Mp: >300 °C ; 1H NMR (400 MHz,
DMSO-d6) δ 11.95 (s, 1H), 9.36 (s, 1H), 7.50 (s, 1H), 7.48 (s, 1H), 7.45 (s, 1H), 7.43
13
– 7.34 (m, 2H), 2.43 (s, 3H), 2.09 (s, 3H); C NMR (100 MHz, DMSO-d6) δ 172.4,
164.7, 159.7, 155.4, 143.5, 143.2, 139.2, 133.0, 130.1, 128.8, 127.9, 126.7, 118.2,
111.9, 13.3, 8.4; HPLC: room temperature, tg= 10.03 min, UV280= 95.0%; HRMS
(ESI) m/z calcd for C16H13ClN6S [M+H]+: 357.0689 ; found: 357.0682.
4.1.8.7.
(4-chlorophenyl)(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2
-(methylthio)pyrimidin-5-yl)methanone (12d).
From compound 5 (220 mg, 0.45 mmol) and 11d (150 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12d 172 mg. Yield: 66%; White solid;
Mp: 220.7~221.3 °C ; 1H NMR (400 MHz, CDCl3) δ 12.13 (s, 1H), 8.53 (s, 1H), 7.65
(d, J = 8.4 Hz, 2H), 7.49 (d, J = 8.4 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 6.89 (d, J = 8.6
13
Hz, 2H), 5.25 (s, 2H), 3.80 (s, 3H), 2.67 (s, 3H), 2.58 (s, 3H); C NMR (100 MHz,
CDCl3) δ 194.4, 177.5, 161.3, 159.8, 157.4, 142.0, 139.6, 138.9, 136.1, 130.8, 128.9,
128.6, 126.6, 123.6, 114.5, 108.9, 55.4, 53.8, 14.4, 11.8; ESI-MS: m/z 525.1 [M+H]+.
4.1.8.8.
5-(4-chlorophenyl)-3-methyl-8-(methylthio)-2,10-dihydropyrazolo[4,3-b]pyrimido[4,5
-e][1,4]diazepine (13d).
From compound 12d (53mg, 0.1 mmol) as that described in Step 2 of Procedure
24

ACCEPTED MANUSCRIPT
2 gave pure 13d 14 mg. Yield: 39%; Yellow solid; Mp: 277.8~279.2 °C ;1H NMR
(400 MHz, DMSO-d6) δ 11.94 (s, 1H), 9.35 (s, 1H), 7.51 (s, 1H), 7.50 – 7.41 (m, 4H),
2.43 (s, 3H), 2.09 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 172.3, 164.9, 159.7,
155.7, 143.2, 140.2, 138.9, 133.5, 130.0, 128.2, 118.3, 111.9, 13.3, 8.4; HPLC: room
temperature, tg= 9.87 min, UV280= 98.1%; HRMS (ESI) m/z calcd for C16H13ClN6S
[M+H]+: 357.0689 ; found: 357.0694.
4.1.8.9.
(3-fluorophenyl)(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2
-(methylthio)pyrimidin-5-yl)methanone (12e).
From compound 5 (220 mg, 0.45 mmol) and 11e (142 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12e 156 mg. Yield: 61%; White solid;
Mp: 162.5~163.7 °C ; 1H NMR (400 MHz, CDCl3) δ 12.13 (s, 1H), 8.55 (s, 1H), 7.49
(d, J = 5.1 Hz, 1H), 7.42 (d, J = 8.8 Hz, 2H), 7.31 (dd, J = 6.7, 2.4 Hz, 1H), 7.19 (d, J
= 8.6 Hz, 2H), 6.89 (d, J = 8.9 Hz, 2H), 5.25 (s, 2H), 3.80 (s, 3H), 2.67 (s, 3H), 2.58
(s, 3H); 13C NMR (100 MHz, CDCl3) δ 194.3, 177.7, 163.8, 161.5, 159.7, 157.3,
150.7, 142.0, 139.6, 130.3, 128.6 (d, J = 11.7 Hz), 126.6, 125.1, 119.4 (d, J = 21.1 Hz),
116.3 (d, J = 22.6 Hz), 114.5, 108.8, 55.4, 53.8, 14.4, 11.8; ESI-MS: m/z 509.1
[M+H]+.
4.1.8.10.
5-(3-fluorophenyl)-3-methyl-8-(methylthio)-2,10-dihydropyrazolo[4,3-b]pyrimido[4,5
-e][1,4]diazepine (13e).
From compound 12e (51mg, 0.1 mmol) as that described in Step 2 of Procedure
1
2 gave pure 13e 12 mg. Yield: 35%; Yellow solid; Mp: 248.5~249.2 °C ; H NMR
(400 MHz, DMSO-d6) δ 11.95 (s, 1H), 9.36 (s, 1H), 7.49 (s, 1H), 7.43 (dd, J = 14.1,
7.6 Hz, 1H), 7.26 (m, 3H), 2.43 (s, 3H), 2.09 (s, 3H); 13C NMR (100 MHz, DMSO-d6)
δ 172.3, 164.8, 163.2, 160.8, 159.7, 155.5, 143.8 (d, J = 7.3 Hz), 139.1, 130.2 (d, J =
8.1 Hz), 124.2, 118.2, 115.8 (d, J = 21.1 Hz), 115.0 (d, J = 21.7 Hz), 111.9, 13.3, 8.4;
HPLC: room temperature, tg= 8.88min, UV280= 97.4%; HRMS (ESI) m/z calcd for
C16H13FN6S [M+H]+: 341.0985, found: 341.0977.
4.1.8.11.
25

ACCEPTED MANUSCRIPT
(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2-(methylthio)pyri
midin-5-yl)(o-tolyl)methanone (12f).
From compound 5 (220 mg, 0.45 mmol) and 11f (140 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12f 151mg. Yield: 66%; White solid; Mp:
1
176.9~178.0 °C ; H NMR (400 MHz, CDCl3) δ 12.36 (s, 1H), 8.29 (s, 1H), 7.42 –
7.37 (m, 1H), 7.29 (dd, J = 8.7, 4.9 Hz, 4H), 7.20 (d, J = 8.6 Hz, 2H), 6.90 (d, J = 8.6
Hz, 2H), 5.26 (s, 2H), 3.81 (s, 3H), 2.68 (s, 3H), 2.55 (s, 3H), 2.33 (s, 3H); 13C NMR
(100 MHz, CD3CN) δ198.2, 162.0, 159.3, 156.8, 141.6, 140.2, 137.8, 135.5, 130.7,
130.0, 128.7, 127.4，127.3, 125.2, 124.5, 113.8, 109.8, 54.7, 52.6, 18.4, 13.5, 10.9;
ESI-MS: m/z 505.1 [M+H]+.
4.1.8.12.
3-methyl-8-(methylthio)-5-(o-tolyl)-2,10-dihydropyrazolo[4,3-b]pyrimido[4,5-e][1,4]
diazepine (13f).
From compound 12f (51mg, 0.1 mmol) as that described in Step 2 of Procedure 2
gave pure 13f 13 mg. Yield: 38%; Yellow solid; Mp: 267.5~268.2 °C ; 1H NMR
(400 MHz, DMSO-d6) δ 11.82 (s, 1H), 9.44 (s, 1H), 7.31 – 7.20 (m, 3H), 7.16 (d, J =
7.1 Hz, 1H), 7.05 (s, 1H), 2.37 (s, 3H), 2.23 (s, 3H), 1.98 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 172.6, 162.7, 159.2, 156.5, 142.8, 140.9, 139.4, 134.9, 130.4,
128.0, 126.0, 118.2, 113.0, 19.0, 13.2, 8.3; HPLC: room temperature, tg= 8.82 min,
UV280= 98.5%; HRMS (ESI) m/z calcd for C17H16N6S [M+H]+: 337.1235, found:
337.1244.
4.1.8.13.
(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2-(methylthio)pyri
midin-5-yl)(p-tolyl)methanone (12g).
From compound 5 (220 mg, 0.45 mmol) and 11g (140 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12f 151mg. Yield: 66%; Yellow solid;
Mp: 206.9~208.5 °C ; 1H NMR (400 MHz, CDCl3) δ 12.16 (s, 1H), 8.58 (s, 1H), 7.61
(d, J = 7.8 Hz, 2H), 7.30 (d, J = 7.8 Hz, 2H), 7.19 (d, J = 8.3 Hz, 2H), 6.89 (d, J = 8.4
Hz, 2H), 5.24 (s, 2H), 3.80 (s, 3H), 2.66 (s, 3H), 2.57 (s, 3H), 2.44 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 195.5, 176.9, 161.4, 159.7, 157.5, 143.3, 142.2, 139.5, 135.1,
26

ACCEPTED MANUSCRIPT
129.7, 129.2, 128.6, 126.7, 114.5, 109.3, 55.4, 53.8, 21.7, 14.3, 11.8; ESI-MS: m/z
505.1 [M+H]+.
4.1.8.14.
3-methyl-8-(methylthio)-5-(p-tolyl)-2,10-dihydropyrazolo[4,3-b]pyrimido[4,5-e][1,4]
diazepine (13g).
From compound 12g (51mg, 0.1 mmol) as that described in Step 2 of Procedure
1
2 gave pure 13g 13 mg. Yield: 38%; Yellow solid; Mp: 245.5~247.2 °C ; H NMR
(400 MHz, DMSO-d6) δ 11.87 (s, 1H), 9.27 (s, 1H), 7.49 (s, 1H), 7.30 (d, J = 7.9 Hz,
2H), 7.16 (d, J = 7.9 Hz, 2H), 2.40 (s, 3H), 2.29 (s, 3H), 2.06 (s, 3H); 13C NMR (100
MHz, DMSO-d6) δ 172.1, 165.1, 159.9, 156.9, 143.2, 138.5, 138.5, 128.7, 128.1,
118.4, 112.1, 20.8, 13.3, 8.4; HPLC: room temperature, tg= 9.64 min, UV254= 98.5%;
HRMS (ESI) m/z calcd for C17H16N6S [M+H]+: 337.1235, found: 337.1241.
4.1.8.15.
(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2-(methylthio)pyri
midin-5-yl)(2-methoxyphenyl)methanone (12h).
From compound 5 (220 mg, 0.45 mmol) and 11h (140 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12h 165 mg. Yield: 70%; Yellow solid;
Mp: 159.6~160.7 °C ; 1H NMR (400 MHz, CDCl3) δ 12.38 (s, 1H), 8.34 (s, 1H), 7.49
(t, J = 7.9 Hz, 1H), 7.39 (d, J = 7.5 Hz, 1H), 7.20 (d, J = 8.4 Hz, 2H), 7.15 – 7.03 (m,
2H), 6.98 (d, J = 8.4 Hz, 1H), 6.90 (d, J = 8.6 Hz, 2H), 5.26 (s, 2H), 3.81 (s, 3H), 3.75
(s, 3H), 2.67 (s, 3H), 2.57 (s, 3H); 13C NMR (100 MHz, CDCl3) δ 195.9, 177.1, 162.7,
159.7, 156.7, 142.2, 139.5, 132.4, 129.6, 128.6, 127.7, 126.7, 121.0, 114.5, 111.2,
110.0, 55.6，55.4, 53.8, 14.4, 11.8; ESI-MS: m/z 521.1 [M+H]+.
4.1.8.16.
5-(2-methoxyphenyl)-3-methyl-8-(methylthio)-2,10-dihydropyrazolo[4,3-b]pyrimido[4
,5-e][1,4]diazepine (13h).
From compound 12h (52mg, 0.1 mmol) as that described in Step 2 of Procedure
1
2 gave pure 13h 11 mg. Yield: 31%; Orange-yellow solid; Mp: 252.1~253.5 °C ; H
NMR (400 MHz, DMSO-d6) δ 11.75 (s, 1H), 9.30 (s, 1H), 7.34 (t, J = 7.6 Hz, 1H),
7.16 (d, J = 7.3 Hz, 1H), 7.09 (s, 1H), 7.05 – 6.94 (m, 2H), 3.70 (s, 3H), 2.34 (s, 3H),
27

ACCEPTED MANUSCRIPT
1.94 (s, 3H); 13C NMR (100 MHz, DMSO-d6) δ 172.2, 162.0, 159.1, 156.1, 154.8,
142.9, 139.5, 130.2, 129.9, 129.7, 120.8, 118.0, 112.8, 111.4, 55.5, 13.2, 8.4; HPLC:
room temperature, tg= 8.51 min, UV280= 98.1%; HRMS (ESI) m/z calcd for
C17H16N6OS [M+H]+ : 353.1185, found: 353.1201.
4.1.8.17.
(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2-(methylthio)pyri
midin-5-yl)(4-methoxyphenyl)methanone (12i).
From compound 5 (220 mg, 0.45 mmol) and 11i (140 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12i 154 mg. Yield: 65%; Yellow solid;
Mp: 248.5~249.2 °C ; 1H NMR (400 MHz, CDCl3) δ 12.09 (s, 1H), 8.58 (s, 1H), 7.73
(d, J = 8.8 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 6.99 (d, J = 8.8 Hz, 2H), 6.89 (d, J = 8.7
Hz, 2H), 5.24 (s, 2H), 3.89 (s, 3H), 3.80 (s, 3H), 2.66 (s, 3H), 2.58 (s, 3H); 13C NMR
(100 MHz, CDCl3) δ 194.2, 176.6, 163.3, 161.0, 159.7, 157.4, 142.2, 139.5, 132.0,
130.3, 128.6, 126.7, 123.4, 114.5, 113.9, 109.4, 55.6, 55.4, 53.7, 14.3, 11.8; ESI-MS:
m/z 521.2 [M+H]+.
4.1.8.18.
5-(4-methoxyphenyl)-3-methyl-8-(methylthio)-2,10-dihydropyrazolo[4,3-b]pyrimido[4
,5-e][1,4]diazepine (13i).
From compound 12i (52mg, 0.1 mmol) as that described in Step 2 of Procedure 2
1
gave pure 13i 12 mg. Yield: 34%; Yellow solid; Mp: 289.1~290.0 °C ; H NMR (400
MHz, DMSO-d6) δ 11.89 (s, 1H), 9.27 (s, 1H), 7.59 (s, 1H), 7.40 (d, J = 8.4 Hz, 2H),
6.94 (d, J = 8.4 Hz, 2H), 3.79 (s, 3H), 2.44 (s, 3H), 2.10 (s, 3H); 13C NMR (100 MHz,
DMSO-d6) δ 172.0, 165.3, 159.9, 156.6, 143.3, 138.1, 133.6, 129.7, 118.4, 113.5,
112.1, 55.2, 13.3, 8.4; HPLC: room temperature, tg= 8.08 min, UV280= 95.7%; HRMS
(ESI) m/z calcd for C17H16N6OS [M+H]+ : 353.1185, found: 353.1197.
4.1.8.19.
(4-((1-(4-methoxybenzyl)-5-methyl-4-nitro-1H-pyrazol-3-yl)amino)-2-(methylthio)pyri
midin-5-yl)(2-(trifluoromethyl)phenyl)methanone (12j).
From compound 5 (220 mg, 0.45 mmol) and 11j (166 mg, 0.5 mmol), as that
described in Step 1 of Procedure 2 gave pure 12j 162 mg. Yield: 58%; White solid;
28