Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as potential Aurora A inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.11.006

The Aurora kinases are a family of serine/threonine kinases that interact with components of the mitotic apparatus and serve as potential therapeutic targets in oncology. Herein, we reported a series of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl with selective inhibition of Aurora A in either enzymatic assays or cellular phenotypic assays, and displaying more potent anti-proliferation compared with that of VX-680. The most potent compound 10a forms better interaction with Aurora A than Aurora B in molecular docking. Mechanistic studies revealed that 10a disrupt the spindle formation, block the cell cycle progression in the G2/M phase and induce apoptosis in HeLa cell. These results suggested that the produced series of compounds are potential anticancer agents for further development as selective Aurora A inhibitors.

Full text of DOI:10.1016/j.bmc.2018.11.006

Accepted Manuscript
Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetra-
methylpiperidine-N-oxyl as potential Aurora A inhibitors
Chun-Yan Sang, Wen-Wen Qin, Xiu-Juan Zhang, Yu Xu, You-Zhen Ma, Xing-
Rong Wang, Ling Hui, Shi-Wu Chen
PII:
S0968-0896(18)31634-1
https://doi.org/10.1016/j.bmc.2018.11.006
BMC 14610
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
19 September 2018
16 October 2018
5 November 2018
Please cite this article as: Sang, C-Y., Qin, W-W., Zhang, X-J., Xu, Y., Ma, Y-Z., Wang, X-R., Hui, L., Chen, S-
W., Synthesis and identification of 2,4-bisanilinopyrimidines bearing 2,2,6,6-tetramethylpiperidine-N-oxyl as
potential Aurora A inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://doi.org/10.1016/j.bmc.
2018.11.006
This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers
we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and
review of the resulting proof before it is published in its final form. Please note that during the production process
errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.

Synthesis and identification of 2,4-bisanilinopyrimidines bearing
2,2,6,6-tetramethylpiperidine-N-oxyl as potential Aurora A
inhibitors
a
a
a
Chun-Yan Sang , Wen-Wen Qin , Xiu-Juan Zhang a, Yu Xu a, You-Zhen Ma ,
Xing-Rong Wang a, Ling Hui b, Shi-Wu Chen a, *
a School of Pharmacy, Lanzhou University, Lanzhou 730000, China
b Experimental Center of Medicine, General Hospital of Lanzhou Military Command, Lanzhou 730050,
China
__________________
* Corresponding author.
E-mail addresses: chenshw@lzu.edu.cn (S.-W. Chen)
1

Abstract: The Aurora kinases are a family of serine/threonine kinases that interact with
components of the mitotic apparatus and serve as potential therapeutic targets in
oncology. Herein, we reported a series of 2,4-bisanilinopyrimidines bearing
2,2,6,6-tetramethylpiperidine-N-oxyl with selective inhibition of Aurora A in either
enzymatic assays or cellular phenotypic assays, and displaying more potent
anti-proliferation compared with that of VX-680. The most potent compound 10a forms
better interaction with Aurora A than Aurora B in molecular docking. Mechanistic
studies revealed that 10a disrupt the spindle formation, block the cell cycle progression in
the G2/M phase and induce apoptosis in HeLa cell. These results suggested that the
produced series of compounds are potential anticancer agents for further development as
selective Aurora A inhibitors.
Keywords: Aurora kinases; Stable nitroxides; Pyrimidine; Anticancer agents
2

1. Introduction
The Aurora kinase family is a subfamily of serine/threonine kinases that is essential
for the regulation of centrosome maturation, mitotic spindle formation, chromosome
segregation and cytokinesis during mitosis.1,2 The family includes three kinases
designated as Aurora A, B and C, which have sequences that are about 70% identity in
the kinase domain. However, these kinases have quite different and nonoverlapping
functions during mitosis.3 Aurora A regulates the cell cycle and is associated with G2
phase and entry into the M phase, and associates with the spindle poles and is involved in
both centrosomal and acentrosomal spindle assembly.4,5 Aurora B has functions
associates with histone phosphorylation and chromatin condensation in prophase,
chromosome alignment and segregation, regulation of a mitotic checkpoint at metaphase
and a role in cytokinesis.6 Aurora C has similar functions as Aurora B, and plays
important roles in regulating mitotic chromosome alignment, segregation and possibly
cytokinesis. 7
The role of Aurora kinase A and B in mitosis, coupled with evidence that
amplification or over-expression of these kinases and other functions such as stabilization
of N-myc by Aurora A drives tumorigenesis, has led to the development of numerous
inhibitors that display selectivity to either Aurora A or Aurora B, or that are dual
inhibitors.8−10 Some of Aurora kinases inhibitors have been or are currently in clinical
development, such as VX-680,11,12 MLN8054,13 ENMD-2076,14 AMG 900,15 CYC11616
and PF-0381473517,18 (Fig. 1).
3

Fig. 1. Some representative Aurora kinases inhibitors.
Nitroxides are stable free radical species that possess a disubstituted nitrogen atom
linked to a univalent oxygen atom and have a wide range of activities in biology.19,20
Recently, with the development of cancer detection techniques, electron paramagnetic
resonance (EPR) spin labeling using paramagnetic molecules, such as nitroxide free
radicals, have gained considerable attention.21 Previous studies have shown that the
introduction of the nitroxyl moiety lead to a series of positive effects, such as higher
alkylating, lower carbamoylating activity, better antimelanomic activity and lower
general toxicity.22 Recently, there are a number of studies that have successfully
connected nitroxides and active molecules by appropriate linkers to find new antitumor
agents.23−25 We are also interested in nitroxide compounds, which have been found to be
an available antitumor agents and their application has progressed significantly in the
treatment of tumors.26,27
The bisanilinopyrimidine scaffold (such as compound 1) has been reported
4

previously as a scaffold to develop inhibitors of Aurora kinases28,29 and other kinases,
such as JNK30 , FAK31, EphB432,33 , ALK34,35, VEGFR36 , CDK2 and CDK437. However,
the generally lower potency of these compounds in cellular assays compared with that
observed in enzymatic assays might reflect problems with the physical properties. We
hypothesized that stable nitroxides labeled 2,4-bisanilinopyrimidines may be favorable
Aurora kinase inhibitors with improved anticancer potency. Thus, we designed a series of
2,4-bisanilinopyrimidines bearing
a
nitroxyl group (Fig. 2), in which
(4-NH2-TMPO) substituted the
4-amino-2,2,6,6-tetramethylpiperidine-N-oxyl
4-acetylpiperazine at the pyrimidine 2-position of activity compound 1 in the literature.29
Herein, we reported the synthesis of these compounds and identification of their
antitumor effects as Aurora A kinase inhibitors.
Fig. 2. Design of target compounds.
2. Results and discussion
2.1. Chemistry
Intermediates p-aminobenzamides 3a−e were prepared by the protocols as illustrated
in Scheme 1. Specifically, reaction of 4-((tert-butoxycarbonyl)amino)-benzoic acid with
the appropriate amines (such as 4-NH2-TMPO, o-, m-, p-chloride aniline, or
N-methyl-4-amino-piperidine) in the presence of 1-(3-dimethylaminopropyl)-3-ethyl
carbodiimide hydrochloride (EDCI), 1-hydroxybenzyltriazole hydrate (HOBt) in dry
5

dichloromethane (DCM) yielded the series of amides 2a‒e. Deprotection of 2a‒e with a
solution of trifluoroacetic acid in dichloromethane generated the primary amines 3a‒e.38
Scheme 1. Reagents and conditions: (i) EDCI, HOBt, DCM; (ii) 30% TFA in DCM.
Various
libraries
of
2,4-bisanilinopyrimidines
bearing
2,2,6,6-tetramethylpiperidine-N-oxy were prepared in a straightforward way from
corresponding 2,4-dichloropyrimidine (4a−d) as outlined in Scheme 2.39,40 Briefly, the
chlorine atoms at C4 and C2 of 4a−d were sequentially displaced with the appropriate
nucleophiles under different conditions to yield the desired target compounds 8‒10. For
example, the intermediates 5a‒c, 6a‒c and 7a were prepared by reaction of
corresponding 4a‒c and appropriate amine (3a, or 2-chlorophenylamine, or 3b) in the
presence of N,N-diisopropylethylamine (DIPEA) in refluxing methanol with moderate
yields. In contrast, the substitution of 2,4-dichloro-5-nitropyrimidine (4d) with
appropriate amine (3a‒d, or chlorophenylamine, or aminophenol) were easily completed
in DCM at room temperature to provide intermediates 5d, 6d‒i and 7b‒d in high yield.
Subsequently, the chlorine atom at the 2-position of intermediates 5‒7 was displaced with
the appropriate nucleophiles (p-aminobenzoic acid for 8a‒d, 3a for 9a‒i and 10a‒d) in
refluxing 1,4-dioxoane under diluted HCl catalysis conditions to yield the corresponding
target compounds 8‒10. In addition, reference compound 11, which did not contain stable
nitroxyl radicals, was also synthesized by treatment of 7a with 3e as synthetic procedure
6

for 10a, as outlined in Scheme 3.
Scheme 2. Reagents and conditions: (i) 3a (or 2-chlorophenylamine, or 3b), DIPEA,
MeOH, 70℃; (ii) 3a‒d, (or chlorophenylamine, or aminophenol), DCM, rt; (iii)
p-aminobenzoic acid (or 3a), refluxing 1,4-dioxoane, diluted HCl (4N HCl for 8a−c,
9a−c and 10a; 1N HCl for 8d, 9d−i and 10b−d).
7

Scheme 3. Reagents and conditions: (i) 3e, refluxing 1,4-dioxoane, 4N HCl.
The target synthetic compounds 8−11 were characterized by NMR, HRMS, ESR
and IR based on the HPLC purity >95%. In NMR of free-radical labeled compounds
8−10, some signals appear broadened and other signals are missing.25
2.2. Biological profiling
2.2.1. Anti-proliferative activities of compounds 8−11
All the target compounds 8−11 were tested for anti-proliferative activities against
four human tumor cell lines (cervical carcinoma HeLa, lung carcinoma A-549, hepatic
carcinoma HepG2 and human colorectal adenocarcinoma LoVo cells) by MTT assays,
with VX-680 as the reference compound.38 The GI50 values of these compounds, which
are the concentrations corresponding to 50% cells growth inhibition, are summarized in
Tables 1.
Table 1 Anti-proliferative activities of target compounds 8−11 in human cancer cell lines
8

Anti-proliferation (GI50, μM)a
HeLa A549 HepG2
21.32.3 18.52.1 26.83.5 65.35.0
Compounds
X
R
LoVo
F
--
8a
Cl
--
>100
>100
8b
56.53.5
48.24.5 56.64.0
>100
35.82.5
Br
--
>100
8c
NO2
F
--
27.31.5 29.62.5 11.82.6 27.42.5
8d
2-Cl
2-Cl
2-Cl
2-Cl
3-Cl
4-Cl
2-OH
3-OH
4-OH
2-Cl
2-Cl
3-Cl
4-Cl
--
8.41.2 2.30.5
10.21.1 3.60.6
9.62.1 3.80.8
53.13.6 2.81.0
1.20.5
1.60.4
2.10.8
2.72.0
3.21.5
3.21.5
9a
Cl
9b
Br
9c
NO2
NO2
NO2
NO2
NO2
NO2
F
3.71.0 35.22.5
9d
43.22.5 31.33.5 42.12.5 67.85.5
33.32.5 5.60.5 13.01.2 27.84.5
9e
9f
9g
13.91.5 11.20.8
3.71.3 28.33.5
36.52.2 13.21.0 21.72.1 39.62.0
34.31.8 14.31.5 21.53.5 43.83.0
9h
9i
0.40.1
1.10.6
1.20.9
7.31.5
10a
10b
10c
10d
11
NO2
NO2
NO2
--
23.22.2 2.50.8
3.41.2 28.52.0
49.53.6 14.22.3 12.52.5 39.62.0
53.04.5 17.23.5 24.12.5 46.33.5
11.31.0 8.92.8 13.42.3 12.40.5
46.25.5 35.83.4 53.35.5 45.34.5
VX680
--
--
a Data are the mean of three independent experiments.
b drug exposure for 48 h.
Previous SAR studies have indicated that polar substituent at the para position of
the benzene-ring at the pyrimidine 2-position is critical for potent anti-proliferative
activity.29 However, compounds 8a‒d, in which p-aminobenzamides of 4-NH2-TMPO at
the pyrimidine 4-position and polar substituent p-aminobenzoic acid at the pyrimidine
9

2-position, showed moderate even to weak anti-proliferation against these four cell lines.
We speculated that the strong polarity of carboxyl group in these compounds resulted in
their poor penetration and show weak anti-proliferative activity. So we adjusted the
position of free radicals from 4-position to 2-position of pyrimidine as well as introduced
the chlorophenylamine or aminophenol (which were the favorable groups to hold the
anti-tumor activities of 2,4-bisanilinepymidines29) at the 4-position of pyrimidine to
provide target compounds 9a‒i.
Inspiring, compounds 9a−d showed stronger anti-proliferative activities against the
four human tumor cell lines compared to VX-680. In particular, the GI50 values of 9a for
HeLa, A549, HepG2 and LoVo were 8.41.2, 2.30.5, 1.20.5 and 2.72.0 μM,
respectively. The group (F, Cl, Br and NO2) at the pyrimidine 5-position in these
compounds (9a−d) gave no significant change to their proliferative inhibition. In the
meantime, comparison of the GI50 values of compounds 9d−i showed that hydroxy and
chlorine in ortho position of the benzene ring gave more potent anti-proliferations than
those of substitutions at the meta or para positions at the pyrimidine 4-position (9d vs 9e
and 9f, 9g vs 9h and 9i).
Then we extended the substitutes at 4-position of pyrimidines with the
4-amino-N-(chlorophenyl) benzamides (3b‒c) to yield target compounds 10a‒d. We
found that introduction of 4-amino-N-(chlorophenyl) benzamide at 4-position of
pyrimidine slightly improved their anti-proliferation than those of chlorophenylamine
(10a vs 6a, 10b vs 9d, 10c vs 9e, 10d vs 9f). In addition, the ortho-chlorobenzene at
4-position of pyrimidine is critical for their ant-proliferation was obtained (10b vs 10c
and 10d). Especially, compound 10a showed the most potent anti-proliferative activity
10

in the series compounds against HeLa, A549, HepG2 and LoVo cells with GI50 values of
0.40.1, 1.10.6, 1.20.9 and 7.31.5 μM, respectively. Meanwhile, compound 10a
possesses more potent anti-proliferation than that of compound 11, in which the
4-NH2-TEMPO in 10a was substituted with N-methyl-4-amino-piperidine. These results
indicated that introduction of stable nitroxyl radical at the pyrimidine 2-position is
favorable for generation of compounds that are retard cancer cell growth.
2.2.2. Aurora kinases inhibition of compounds 9‒11 in vitro
Many pyrimidine compounds, such as VX-680, ENMD-2076, CYC-116 and
PF-03814735, have entered clinical trials as Aurora kinases inhibitors.10 Compounds
9‒11, which showed stronger anti-proliferative activities against the tested four human
cancer cell lines, were tested for their Aurora kinases inhibitory activity using Kinase-Glo
Plus luminescence kinase assay kit that measures ADP formation by Aurora kinases
phosphorylation of the synthetic peptide LRRASLG, as described in the methods section,
with VX-680 as the reference compound.41
As shown in Table 2, all the stable nitroxides labeled compounds 9a‒i and 10a‒d
showed more excellent or comparable Aurora kinases inhibition in comparison to
VX-680 and non-nitroxides labeled compound 11. Moreover, most of this series
compounds (except 9f, 10b and 10d) exhibited selective inhibition of Aurora A over
Aurora B. Especially, the most potent compound 10a, with the IC50 values for Aurora A
and Aurora B were 0.06 and 2.55 nM, respectively, showed 42-fold selectivity for Aurora
A over Aurora B.
Aurora kinases inhibitory activities of compounds 9‒10 showed various consistent
with their anti-proliferation in different cell lines. For example, the Aurora A inhibitory
11

activities of compounds 9a-c and 10a-d were consistent with their anti-proliferation
against HeLa and LoVo cells. In contrast, the Aurora B inhibitory activity of compounds
9d-f was more consistent with anti-proliferative activity against A549 and HepG2 cells,
which is in correlation with previous publication42. Considered compound 10a showed
the most potent inhibitory activities in either cell lines or Aurora kinases, following
compound 10a was selected to further study its action mechanism in HeLa cells.
Table 2. The Aurora kinases inhibitory activities of compounds 9‒11 in vitro.
(IC50, nM)a
(IC50, nM)a
Compounds
Compounds
Aurora A
Aurora B
Aurora A
Aurora B
0.18
0.19
0.46
1.86
0.62
1.61
8.50
0.14
1.11
0.29
2.67
3.29
19.5
0.34
11.3
3.50
0.59
0.06
5.48
0.11
20.0
2.28
0.95
3.50
2.55
3.07
27.0
0.67
49.1
14.7
9a
9b
9c
9d
9e
9f
9i
10a
10b
10c
10d
11
VX680
9g
9h
a The IC50 values are the means of two experiments.
2.2.3. Compound 10a selectively inhibits Aurora A over Aurora B in HeLa cells
Aurora A kinase activity depends on autophosphorylation of Aurora A on Thr288
(p-Thr288) in the activation loop,43 and Aurora B kinase activity bases on
phosphorylation of Aurora B on Thr232 (p-Thr232).44 Thus, evaluating the levels of
p-Thr288 and p-Thr232 reflect the inhibitory activities of 10a on the kinases in HeLa
cells.
12

Firstly, HeLa cells were treated with various concentrations of compound 10a (0,
1.25, 2.5, and 5.0 μM) for 12 h, and detected the expression of p-Thr288 and p-Thr232 in
the cells by western blotting. As shown in Fig. 3A and Fig. 3B, compound 10a induced
the gradual decline of p-Thr288 and p-Thr232 HeLa cells in a dose dependent manner,
and the degree of reduction of p-Thr288 was more than that of p-Thr232. These results
revealed that 10a possessed the selectivity for Aurora A over Aurora B. Furthermore, the
inhibition effects of 10a on either Aurora A or Aurora B were obviously higher than
those of VX-680, which were consistent with their inhibition of Aurora kinases in vitro.
The selectivity of 10a for Aurora A over Aurora B in HeLa cells were also
confirmed by ELISA.38 When HeLa cells were treated with less than 10 nM of compound
10a for 12 h, the phosphorylation levels of Aurora A and B showed no significant
changes. However, the concentration of 10a was increased to more than 60 nM, the
phosphorylation levels of Aurora A and B protein rapidly decreased, and the reduction of
Aurora A was more than that of Aurora B in HeLa cell lysates. From Fig. 3C, we easily
obtained the IC50 value of 10a for Aurora A and B was 0.008 μM and 0.538 μM,
respectively. However, the IC50 value of VX-680 for Aurora A and B was 0.013 μM and
0.148 μM, respectively (Fig. 3D). The data from the Aurora A and Aurora B cell-based
assays suggest that 10a has a 67-fold greater potency against Aurora A compared with
Aurora B, and the selectivity of VX-680 for Aurora A over Aurora B was less than
12-fold.
13

Fig. 3. Compound 10a selectively inhibits Aurora A over Aurora B in HeLa cells. (A)
Western blots of HeLa cells treated with various concentrations of 10a probed for
p-Thr288 and p-Thr232. (B) The results of western blots are expressed as the mean ± SD;
*P <0.05, **P<0.001 versus control. (C‒D) ELISA analyzes of 10a and VX-680 inhibits
phosphorylation of Aurora A and Aurora B in HeLa cells.
2.2.4. Compound 10a disrupts spindle formation in HeLa cells
Aurora A plays a pivotal role in centrosome maturation and spindle formation
during mitosis, and over-expression of Aurora A provokes abnormalities in spindle
formation, and the failure to complete spindle-microtubule attachment.4,5 Thus, the
morphology of mitotic spindles was examined in HeLa cells treated with 10a at various
concentrations and VX-680 for 24 h. As shown in Fig. 4, the DMSO treated control cells
14

displayed normal bipolar mitotic spindles. However, Compound 10a at 0.1 μM or 1 μM
induced the formation of abnormal mitotic spindles. Especially, when the concentration
of compound 10a was increased to 1 μM, the microtubule spindles wrapped around the
cell nucleus have significantly shrunk and disorder formations. In contrast, VX680
treated cells still retained a spindle shaped microtubule network. These show that
formation of abnormal mitotic spindles is consistent with a known Aurora A inhibition
phenotype.45
Fig. 4. Representative immunofluorescent images of HeLa cells treated with DMSO, 10a
(0.1 μM, or 1 μM), and VX680 (1 μM) for 24 h. Overlapped images were obtained from
cells stained with anti-α-tubulin mouse antibody (tubulin; green) and DAPI (DNA; blue)
15

(Scale bars: 20 μm).
2.2.5. Compound 10a blocks the cell cycle in the G2/M phase in HeLa cells
The Aurora inhibitors including VX-680 often induce common phenotypic effects of
cancer cells such as cell cycle arrest at the G2/M phase. Thus, we also tested the effects
of 10a on cell cycle progression using fluorescence-activated cell sorting analysis of
propidium iodide-stained in HeLa cells.46 As shown in Fig. 5, treatment of HeLa cells
with 10a resulted in a dose-dependent accumulation of cells at the G2/M phase with a
concomitant decrease in the population of G1 phase cells. Exposure to compound 10a at
1 μM and 5 μM for 12 h, the percentages of cells at the G2/M phase arrest were 40.67%
(Fig. 5B) and 60.33% (Fig. 5C), respectively, compared with 23.57% (Fig. 5A) in
untreated cultures and 38.96% (Fig. 5D) in VX-680 treated cultures. Furthermore,
cyclinB1 and cdc2 are two key regulators in the cell cycle progression,47 to confirm the
effects of 10a in cell cycle arrest, we also measured their expression levels in HeLa cells
after treatment with 10a and VX-680. As shown in Fig. 5E and 5F, the levels of cyclinB1
and cdc2 protein marked decreased in HeLa cells with increasing treatment concentration
of 10a and VX-680 compared with that of control group. These results demonstrated that
10a blocks the cell cycle in G2/M phase and displayed phenotypic characteristics similar
to that of VX-680 in HeLa cells.
16

Fig. 5. Effects of 10a on cell cycle progression. (A‒D) HeLa cells were treated with
DMSO, 10a (1 μM, 5 μM), and VX680 (5 μM) for 12 h. Fixed and propidium iodide
17

(PI)-stained cells were analyzed by flow cytometry. (E) Western blot analysis of
cyclinB1 and cdc2 in HeLa cells treated with 10a or VX-680 for 12 h. (F) The results are
expressed as the mean ± SD; *P <0.05, **P <0.001 versus control.
2.2.6. Compound 10a induced apoptosis in HeLa Cell
Previous publication48 has revealed that inhibition of Aurora kinases not only inhibit
proliferation but also induce apoptosis. To further certify whether 10a induce apoptosis in
HeLa cells, an annexin V-FITC/PI binding assay was performed. Specifically, HeLa cells
were treated with 10a at indicated concentrations, following the percentages of apoptotic
cells were measured by flow cytometry. As shown in Fig. 6A‒D, treatment of 10a for 12
h gave substantial rise to the apoptotic ratios from 3.07% of vehicle control to 4.06% (0.1
μM), 11.34% (1 μM) and 28.9% (5 μM). These results demonstrated that 10a exerted its
anti-proliferative effects possibly by inducing apoptosis in HeLa cells.
Mitochondria represent the central checkpoint in propagating apoptotic signaling
pathways, upon exposure to apoptotic stimuli, it can activate apoptosis-related proteins
such as Bax, Bcl-2, and BAD to enter mitochondria, which induce the mitochondria to
release cytochrome c and, in turn, activate the caspase-3 and -9, finally triggering the
execution of apoptosis.48 To explore the apoptotic mechanism of 10a in HeLa cells,
correspondent western blot analysis of apoptosis-related proteins were performed, and
results were summarized in Fig. 6E‒F. Treatment of 10a for 12 h significantly
up-regulated the expression of BAD, Bax, caspase-3, -9, together with down-regulation
of Bcl-2. Thus, it was illuminated that 10a induced cell apoptosis via the
mitochondria-dependent pathway.
18

Fig. 6. Effects of 10a induce apoptosis in the HeLa cells. (A‒D) HeLa cells were treated
with DMSO, 10a (0.1 μM, 1 μM), and 10a (5 μM ) for 12 h, an annexin V-FITC/PI
binding assay were performed by Annexin V-FITC apoptosis detection kit. (E) Western
blot analysis of apoptosis-related proteins in HeLa cells treated with 10a at 0.1 μM, 1 μM,
19

and 5 μM for 12 h. (F) The significantly expression change of the apoptotic proteins was
analysed by immunoblotting (n=3; *P<0.05; #P<0.01).
2.2.7. Binding model of compound 10a with Aurora A
To gain insight into the interaction of compound 10a with Aurora A and Aurora B,
docking simulation was performed using Schrödinger software (Maestro, version 10.1,
Schrödinger, LLC, New York, NY, 2015).49 All the figures displaying the docking results
were obtained using the scientific software Pymol.50 Grid was used to produce grids
based on the position of the ligand in the proteins (PDB code 3E5A for Aurora A and
4AF3 for Aurora B). In the docking process, the protein was considered to be rigid, while
the ligand was considered flexible. The ligand 10a was docked into the appropriate
binding pocket of Aurora A and Aurora B using the dock module and the calculated
binding energy was –44.40 kcal/mol for Aurora A and –31.01 kcal/mol for Aurora B,
respectively. The resulting docking poses are shown in Figure 7. According to the
binding energy, 10a is more sensitive to Aurora A than Aurora B.
Overall, the binding pockets of Aurora A and Aurora B are highly hydrophobic.
Thus, hydrophobic interactions are the main driving force for the binding of 10a to
Aurora A and Aurora B. In Aurora A, 10a can form several H-bonds, such as the nitrogen
atom of N-1 in pyrimidine with hinge region Ala213, the oxygen atom of carbonyl group
in C-2 of pyrimidine with hinge region Try212, and the oxygen atom of nitroxide with
Lys162. Simultaneously the hydrophilic region is very well occupied by nitroxide in 10a
(Fig. 7A). However, in Aurora B, 10a only can form one H-bonds of the oxygen atom of
carbonyl group connected to p-chloride aniline with Pro158 (Fig. 7C). Additionally, from
the shape of the binding pocket, 10a can fit much better with the binding pocket of
20

Aurora A than Aurora B (Fig. 7B and 7D). These differences may explain why
compound 10a binds better with Aurora A compared with Aurora B.
Fig. 7. Binding modes of 10a with Aurora A and Aurora B. The dashed lines indicate
hydrogen bonding interactions between the ligand and the protein. (A) The interaction
between 10a and Aurora A (PDB ID: 3E5A). (B) The binding pocket of Aurora A was
shown in surface. (C) The interaction between 10a and Aurora B (PDB ID: 4AF3). (D)
The binding pocket of Aurora B was shown in surface.
3. Conclusion
In summary, Aurora kinases have been of interest as potential therapeutic targets in
oncology. In this work,
a
series of stable nitroxyl radicals labeled
2,4-bisanilinopyrimidines as small molecule Aurora kinases inhibitors exert potent
21

anticancer activities. We specifically demonstrated that compound 10a was selective
inhibition of Aurora A over Aurora B either in vitro or in HeLa cells, arrested cell cycle in
the G2/M phase, and induced cell apopotosis. The present investigation indicates that
compound 10a possesses potent antitumor activity as an Aurora A inhibitor for further
development.
4. Experimental protocols
4.1. Chemistry
General. Melting points were determined with a XT4-100 apparatus and are
1
uncorrected. H NMR and 13C NMR spectra were recorded using a Agilent NMR
inova600 spectrometer. EPR spectra were recorded with a Bruker A300 X-band EPR
spectrometer. Mass spectra were recorded on a Bruker Dalton APEXII 49e, micrOTOF
and Esquire6000 spectrometer with ESI source as ionization, respectively. IR spectra
were obtained with a Nicolet iS5-IR spectrometer on neat samples placed between KBr
plates. The purities of all of the biologically tested compounds were estimated by HPLC,
and in each case, the major peak accounted for ≥95% of the combined total peak area
when monitored by a UV detector. HPLC analysis using a UltiMate300 DAD HPLC
system equipped with a PU-2089 Plus quaternary gradient pump and a UV-2075 Plus
UV−vis detector, using an Alltech Kromasil C18 column with dimensions of 250
mm×4.6 mm and 5 μm particle size. Analytical thin-layer chromatography was conducted
on silica gel GF254 plates (0.25 mm thick), and compounds were visualized with UV light
at 254 nm. Silica gel flash chromatography was performed using Silica Gel 60 (200–300
mesh, Qingdao Ocean Chemical Ltd., China).
22

4.1.1. General procedures for preparation of 4-Aminobenzenamides 3a‒e
To a solution of 4-((tert-butoxycarbonyl)amino)-benzoic acid (1.8 g, 7.2 mmol) in
dry CH2Cl2 (20 mL) were added EDCI (1.8 g, 9 mmol), HOBt (1.22 g, 9 mmol). After
stirring at room temperature for 1 h, 4-NH2-TEMPO (or 2-, 3-, 4-chloroaniline, or
N-methyl-4-amino-piperidine) (9 mmol) was added and the reaction continued for
another 24 h at room temperature under argon. Water (200 mL) was then added and the
mixture stirred for 5 min. The product was then extracted with ethyl acetate (3×30 mL).
The combined organic extracts were washed with brine (30 mL), dried over sodium
sulfate, filtered, and the solvent removed. Purification was achieved by flash
chromatography (dichloromethane/acetone 20:1 by volume) to yield pure 2a−e.
4.1.1.1. tert-Butyl (4-((2,2,6,6-tetramethyl-1-oxyl)carbamoyl)phenyl)carbamate (2a)
1
Yield: 90%; Pink solid; H NMR (600 MHz, DMSO-d6) (*note compound is a
free-radical, some signals appear broadened and other signals are missing) δ 9.63 (s, 1 H),
7.78 (s, 2 H), 7.53 (s, 2 H), 1.47 (s, 9 H).
4.1.1.2. tert-Butyl (4-((2-chlorophenyl)carbamoyl)phenyl)carbamate (2b) Yield: 62%;
White solid; 1H NMR (600 MHz, CDCl3) δ 8.20 (d, J = 8.4 Hz, 2 H), 8.10 (d, J = 8.4 Hz,
1 H), 7.61 (d, J = 8.4 Hz, 2 H), 7.55 (t, J = 8.4 Hz, 1 H), 7.48 (d, J = 7.8 Hz, 1 H), 7.45 (t,
J = 7.8 Hz, 1 H), 6.97 (s, 1 H), 6.94 (s, 1 H), 1.55 (s, 9 H).
4.1.1.3. tert-butyl (4-((3-chlorophenyl)carbamoyl)phenyl)carbamate (2c) Yield: 64%;
White solid; 1H NMR (600 MHz, DMSO-d6) δ 10.25 (s, 1 H), 9.75 (s, 1 H), 7.97 (s, 1 H),
7.90 (d, J = 9.0 Hz, 2 H), 7.70 (d, J = 7.8 Hz, 1 H), 7.60 (d, J = 8.4 Hz, 2 H), 7.37 (t, J =
8.4 Hz, 1 H), 7.14 (d, J = 7.8 Hz, 1 H), 1.49 (s, 9 H).
23

4.1.1.4. tert-butyl (4-((4-chlorophenyl)carbamoyl)phenyl)carbamate (2d) Yield: 59%;
White solid; 1H NMR (600 MHz, DMSO-d6) δ 10.02 (brs, 1 H), 9.32 (s, 1 H), 7.88 (d, J =
8.4 Hz, 2 H), 7.80 (d, J = 9.0 Hz, 2 H), 7.60 (d, J = 8.4 Hz, 2 H), 7.27 (d, J = 8.4 Hz, 1 H),
1.49 (s, 9 H).
4.1.1.5. tert-Butyl (4-((1-methylpiperidin-4-yl)carbamoyl)phenyl)carbamate (2e) Yield:
53%; White solid; 1H NMR (600 MHz, DMSO-d6) δ 9.58 (s, 1 H), 8.16 (d, J = 7.8 Hz, 1
H), 7.77 (d, J = 8.4 Hz, 2 H), 7.50 (d, J = 8.4 Hz, 2 H), 3.84‒3.80 (m, 1 H), 2.99‒2.96 (m,
2 H), 2.45‒2.34 (m, 5 H), 1.83‒1.79 (m, 2 H), 1,70‒1.67 (m, 2 H), 1.47 (s, 9 H).
A solution of 2a−e (5 mmol) in 30% dichloromethane solution of trifluoroacetic
acid (20 mL) was stirred at room temperature for 1 h. The solvent was evaporated in
vacuo, and diethyl ether (15 mL) was added. The precipitate was collected, washed with
ether and dried to provide white solid 3a−e and be used in the next reaction without
further purification.
4.1.2. General procedure for the synthesis of intermediates 5a−d, 6a−i, 7a−d
Method 1: To a solution of 2,4-dichloro-5-fluoropyrimidine (4a, 0.23 g, 1.4 mmol)
in MeOH (10 mL) at room temperature was added 3a (0.35 g, 1.2 mmol) and DIPEA (0.2
mL, 1.2 mmol). The resulting mixture was refluxed for another 12 h (monitored by TLC).
After cooling to room temperature, the precipitate 5a was filtrated and washed with
MeOH. Similar synthetic procedures were operated to give pure 5b−c, 6a−c and 7d.
Method 2: To a solution of 2,4-dichloro-5-nitropyrimidine (4d, 0.27 g, 1.4 mmol) in
DCM (20 mL) at rt was added 3a (0.35 g, 1.2 mmol), and stirred for another 12 h
(monitored by TLC). The precipitate 5d was filtrated and washed with DCM. Similar
procedures were operated to provide pure 6d−i and 7a−c.
24

4.1.2.1.
4-((2-Chloro-5-fluoropyrimidin-4-yl)amino)-N-(2,2,6,6-tetramethyl-1-oxyl)
1
benzamide (5a). Yield: 67%; White solid; H NMR (600 MHz, DMSO-d6) (*note
compound is a free-radical, some signals appear broadened and other signals are missing)
δ 9.63 (s, 1 H), 7.78 (s, 2 H), 7.53 (s, 2 H); MS (ESI) m/z: 422.4 ([M+2]+, 100%).
4.1.2.2. 4-((2,5-Dichloropyrimidin-4-yl)amino)-N-(2,2,6,6-tetramethyl-1-oxyl) benzamide
1
(5b). Yield: 55%; White solid; H NMR (600 MHz, DMSO-d6) (*note compound is a
free-radical, some signals appear broadened and other signals are missing) δ 9.63 (s, 1 H),
7.78 (s, 2 H), 7.53 (s, 2 H); MS (ESI) m/z: 440.1 ([M+4]+, 100%); 438.1 ([M+2]+, 85%).
4.1.2.3.
4-((5-Bromo-2-chloropyrimidin-4-yl)amino)-N-(2,2,6,6-tetramethyl-1-oxyl)
1
benzamide (5c). Yield: 58%; White solid; H NMR (600 MHz, DMSO-d6) (*note
compound is a free-radical, some signals appear broadened and other signals are missing)
δ 9.63 (s, 1 H), 7.78 (s, 2 H), 7.53 (s, 2 H); MS (ESI) m/z: 484.2 ([M+4]+, 100%), 482.2
([M+2]+, 84%).
4.1.2.4.
4-((2-Chloro-5-nitropyrimidin-4-yl)amino)-N-(2,2,6,6-tetramethyl-1-oxyl)
1
benzamide (5d). Yield: 63%; Yellow solid; H NMR (600 MHz, DMSO-d6) (*note
compound is a free-radical, some signals appear broadened and other signals are missing)
δ 9.63 (s, 1H), 7.78 (s, 2H), 7.53 (s, 2H); MS (ESI) m/z: 449.5 ([M+2]+, 100%).
4.1.2.5. 2-Chloro-N-(2-chlorophenyl)-5-fluoropyrimidin-4-amine (6a). Yield: 76%; White
solid; 1H NMR (600 MHz, CDCl3) δ 8.51 (d, J = 8.4 Hz, 1 H), 8.13 (d, J = 2.4 Hz, 1 H),
7.56 (s, 1 H), 7.44 (d, J = 8.4 Hz, 1 H), 7.37 (t, J = 7.2 Hz, 1 H), 7.11 (t, J = 7.2 Hz, 1 H).
4.1.2.6. 2,5-Dichloro-N-(2-chlorophenyl)pyrimidin-4-amine (6b). Yield: 69%; White
solid;1H NMR (600 MHz, CDCl3) δ 8.52 (d, J = 8.4 Hz, 1 H), 8.26 (s, 1 H), 7.97 (brs, 1
H), 7.44 (d, J = 7.8 Hz, 1 H), 7.37 (t, J = 7.2 Hz, 1 H), 7.11 (t, J = 7.2 Hz, 1 H).
25

4.1.2.7. 5-Bromo-2-chloro-N-(2-chlorophenyl)pyrimidin-4-amine (6c) Yield: 75%; White
solid;1H NMR (600 MHz, CDCl3) δ 8.51 (d, J = 7.8 Hz, 1 H), 8.36 (s, 1 H), 8.01 (brs, 1
H), 7.44 (d, J = 8.4 Hz, 1 H), 7.37 (t, J = 7.8 Hz, 1 H), 7.11 (t, J = 7.8 Hz, 1 H).
4.1.2.8. 2-Chloro-N-(2-chlorophenyl)-5-nitropyrimidin-4-amine (6d). Yield: 89%;
Yellow solid;1H NMR (600 MHz, CDCl3) δ 10.68 (brs, 1 H), 9.22 (s, 1 H), 8.33 (d, J =
7.8 Hz, 1 H), 7.50 (d, J = 7.8 Hz, 1 H), 7.39 (t, J = 7.8 Hz, 1 H), 7.23 (t, J = 7.8 Hz, 1 H).
4.1.2.9. 2-Chloro-N-(3-chlorophenyl)-5-nitropyrimidin-4-amine (6e). Yield: 68%;
Yellow solid;1H NMR (600 MHz, DMSO-d6) δ 10.52 (s, 1 H), 9.19 (s, 1 H), 7.68 (s, 1 H),
7.53 (d, J = 8.4 Hz, 1 H), 7.47 (t, J = 8.4 Hz, 1 H), 7.36 (d, J = 8.4 Hz, 1 H).
4.1.2.10. 2-Chloro-N-(4-chlorophenyl)-5-nitropyrimidin-4-amine (6f). Yield: 82%;
Yellow solid;1H NMR (600 MHz, DMSO-d6) δ 10.51 (s, 1 H), 9.17 (s, 1 H), 7.56 (d, J =
8.4 Hz, 2 H), 7.51 (d, J = 9.0 Hz, 2 H).
4.1.2.11. 2-((2-Chloro-5-nitropyrimidin-4-yl)amino)phenol (6g). Yield: 76%; Red solid;
1H NMR (600 MHz, DMSO-d6) δ 9.43 (brs, 1 H), 9.05 (s, 1 H), 8.83 (s, 1 H), 8.59 (d, J =
8.4 Hz, 1 H), 7.09 (t, J = 7.8 Hz, 1 H), 6.94 (d, J = 8.4 Hz, 1 H), 6.86 (t, J = 7.8 Hz, 1 H);
MS (ESI) 265.3 ([M‒1]‒).
4.1.2.12. 3-((2-Chloro-5-nitropyrimidin-4-yl)amino)phenol (6h). Yield: 86%; Yellow
solid;1H NMR (600 MHz, DMSO-d6) δ 10.60 (s, 1 H), 8.97 (s, 1 H), 8.47 (d, J = 7.8 Hz,
1 H), 6.98 (s, 2 H), 6.83 (t, J = 8.4 Hz, 1 H); MS (ESI) 265.5 ([M‒1]‒).
4.1.2.13. 4-((2-Chloro-5-nitropyrimidin-4-yl)amino)phenol (6i). Yield: 74%; Red solid;
1H NMR (600 MHz, DMSO-d6) δ 10.29 (s, 1 H), 9.58 (brs, 1 H), 9.10 (s, 1 H), 7.28 (d, J
= 9.0 Hz, 2 H), 6.81 (d, J = 9.0 Hz, 2 H); MS (ESI) 265.6 ([M‒1]‒).
4.1.2.14. 4-((2-Chloro-5-fluoropyrimidin-4-yl)amino)-N-(2-chlorophenyl)benzamide (7a).
26

Yield: 54%; White solid;1H NMR (600 MHz, DMSO-d6) δ 10.26 (s, 1 H), 9.95 (s, 1 H),
8.41 (d, J = 3.6 Hz, 1 H), 8.02 (d, J = 7.2 Hz, 2 H), 7.88 (d, J = 7.2 Hz, 2 H), 7.63 (d, J =
7.8 Hz, 1 H), 7.56 (d, J = 8.4 Hz, 1 H), 7.39 (t, J = 7.8 Hz, 1 H), 7.29 (t, J = 7.8 Hz, 1 H).
4.1.2.15. 4-((2-Chloro-5-nitropyrimidin-4-yl)amino)-N-(2-chlorophenyl)benzamide (7b).
Yield: 49%; Yellow solid;1H NMR (600 MHz, DMSO-d6) δ 10.59 (s, 1 H), 10.10 (s, 1
H), 9.19 (s, 1 H), 8.04 (d, J = 8.4 Hz, 2 H), 7.72 (d, J = 9.0 Hz, 2 H), 7.59 (d, J = 8.4 Hz,
1 H), 7.56 (d, J = 8.4 Hz, 1 H), 7.39 (t, J = 7.8 Hz, 1 H), 7.30 (t, J = 7.8 Hz, 1 H).
4.1.2.16. 4-((2-Chloro-5-nitropyrimidin-4-yl)amino)-N-(3-chlorophenyl)benzamide (7c).
Yield: 79%; Yellow solid;1H NMR (600 MHz, DMSO-d6) δ 10.61 (s, 1 H), 10.47 (s, 1
H), 9.21 (s, 1 H), 8.03‒7.99 (m, 3 H), 7.75‒7.71 (m, 3 H), 7.39 (t, J = 7.8 Hz, 1 H), 7.30
(d, J = 7.8 Hz, 1 H).
4.1.2.17. 4-((2-Chloro-5-nitropyrimidin-4-yl)amino)-N-(4-chlorophenyl)benzamide (7d).
Yield: 86%; Yellow solid;1H NMR (600 MHz, DMSO-d6) δ 10.58 (s, 1 H), 10.41 (s, 1
H), 9.19 (s, 1 H), 8.00 (d, J = 8.4 Hz, 2 H), 7.82 (d, J = 9.0 Hz, 2 H), 7.72 (d, J = 8.4 Hz,
2 H), 7.41 (d, J = 9.0 Hz, 2 H).
4.1.3. General synthetic procedures for compounds 8-11
Intermediate 5a (210 mg, 0.5 mmol) and p-aminobenzoic acid (137 mg, 1 mmol)
were dissolved in 25 mL 1,4-dioxoane, following 4N HCl (0.4 mL) was added, and the
mixture
was
refluxed
for
24
h.
After
cooling
to
room
temperature, the precipitate was filtrated and washed with DCM. The precipitate was
further purified by column chromatography on silica gel gave desired product 8a. Similar
procedures were operated to provide pure 8b‒d, 9a‒i, 10a‒d and 11.
4.1.3.1.
4-((5-Fluoro-4-((4-((2,2,6,6-tetramethyl-1-oxyl)carbamoyl)phenyl)amino)
27

pyrimidin-2-yl)amino)benzoic acid (8a). Yield: 39%; White solid; m.p.: 226‒228℃; IR
(KBr, cm-1) 3334, 3194, 2965, 1721, 1614, 1526, 1424, 1387, 1327, 1202, 1172, 843, 763;
1H NMR (600 MHz, DMSO-d6) (*note compound is a free-radical, some signals appear
broadened and other signals are missing) δ 9.65 (s, 1H), 9.60 (s, 1H), 8.22 (s, 1H), 7.96 (s,
13
2H), 7.79 (s, 2H); C NMR (150 MHz, DMSO-d6) δ 164.7, 154.4, 148.7, 142.9, 141.0,
140.7, 139.5, 130.7, 128.6, 127.3, 119.5, 116.8. ESR (DMSO): g = 2.006, An (G) = 15.94,
△H (G) = 2.64. LC-MS (ESI) m/z (rel intensity) 523.2 ([M+2H]+, 100); HRMS (ESI)
523.2457 for [M+2H]+ (calcd 523.2464 for C27H32N6O4F). HPLC purity 97.1% (MeOH:
H2O (0.1%TFA) = 60:40, 0.8 mL/min, tR = 10.03 min).
4.1.3.2.
4-((5-Chloro-4-((4-((2,2,6,6-tetramethyl-1-oxyl)carbamoyl)phenyl)amino)
pyrimidin-2-yl)amino)benzoic acid (8b) Yield: 57%; White solid; m.p.: 245‒247℃; IR
(KBr, cm-1) 3334, 3189, 2945, 1718, 1659, 1616, 1579, 1531, 1501, 1447, 1424, 1329,
1
1200, 1170, 1085, 843, 766; H NMR (600 MHz, DMSO-d6) (*note compound is a
free-radical, some signals appear broadened and other signals are missing) δ 9.68 (s, 1H),
13
9.09 (s, 1H), 8.24 (s, 1H), 7.86 (s, 5H), 7.75 (s, 3H); C NMR (150 MHz, DMSO-d6) δ
164.3, 156.1, 154.5, 141.8, 140.2, 126.8, 126.7, 121.0, 116.8, 104.0; ESR (DMSO): g =
2.006, An (G) = 15.96, △H (G) = 3.15. LC-MS (ESI) m/z (rel intensity) 539.2 ([M+2H]+,
100); HRMS (ESI) 539.2162 for [M+2H]+ (calcd 539.2168 for C27H32N6O4Cl). HPLC
purity 97.6% (MeOH: H2O (0.1%TFA) = 50:50, 0.8 mL/min, tR = 12.44 min).
4.1.3.3.
4-((5-Bromo-4-((4-((2,2,6,6-tetramethyl-1-oxyl)carbamoyl)phenyl)amino)
pyrimidin-2-yl)amino)benzoic acid (8c) Yield: 50%; White solid; m.p.: 267‒269℃; IR
(KBr, cm-1) 3336, 3189, 2990, 1718, 1656, 1616, 1569, 1526, 1422, 1327, 1207, 1172,
1
843, 763; H NMR (600 MHz, DMSO-d6) (*note compound is a free-radical, some
28

signals appear broadened and other signals are missing) δ 9.67 (s, 1H), 8.84 (s, 1H), 8.32
(s, 1H), 7.92 (s, 1H), 7.80 (s, 2H), 7.71 (s, 5H); 13C NMR (150MHz, DMSO-d6) δ 164.3,
157.5, 156.7, 155.5, 141.9, 140.4, 131.2, 128.7, 126.9, 126.8, 121.5, 116.9; ESR (DMSO):
g = 2.006, An (G) = 15.94, △H (G) = 3.05. LC-MS (ESI) m/z (rel intensity) 585.1
([M(Br81)+2H]+, 100), 583.1 ([M(Br79)+2H]+, 88); HRMS (ESI) 583.1656 for [M+2H]+
(calcd 583.1663 for C27H32N6O4Br). HPLC purity 96.7% (MeOH: H2O (0.1%TFA) =
56:44, 0.8 mL/min, tR = 16.72 min).
4.1.3.4.
4-((4-((4-((2,2,6,6-Tetramethyl-1-oxyl)carbamoyl)phenyl)amino)-
5-nitropyrimidin-2-yl)amino)benzoic acid (8d). Yield: 53%; Yellow solid; m.p.:
196‒198℃; IR (KBr, cm-1) 3234, 2970, 2616, 1723, 1656, 1604, 1531, 1440, 1329, 1175,
1
851, 771; H NMR (600 MHz, DMSO-d6) (*note compound is a free-radical, some
signals appear broadened and other signals are missing) δ 9.42 (s, 1H), 8.11 (s, 2H), 8.04
(s, 2H), 7.83 (s, 2H), 7.63 (s, 2H); 13C NMR (150 MHz, DMSO-d6) δ 157.8, 153.8, 139.1,
132.4, 132.1, 131.9, 127.8, 126.4, 125.8, 124.2, 116.2, 115.1; ESR (DMSO): g = 2.006,
An (G) = 15.98, △H (G) = 2.79. LC-MS (ESI) m/z (rel intensity) 550.4 ([M+2H]+, 100);
HRMS (ESI) 550.2407 for [M+2H]+ (calcd 550.2409 for C27H32N7O6). HPLC purity
98.4% (MeOH: H2O (0.1% TFA) = 68:32, 0.8 mL/min, tR = 28.56 min).
4.1.3.5.
4-((4-((2-Chlorophenyl)amino)-5-fluoropyrimidin-2-yl)amino)-
N-(2,2,6,6-tetramethyl-1-oxyl)benzamide (9a) Yield: 57%; White solid; m.p.: 137‒139℃;
IR (KBr, cm-1) 3384, 3277, 2980, 2942, 2628, 2496, 1678, 1606, 1574, 1511, 1494, 1444,
1399,1327, 1190, 1038, 850, 776; 1H NMR (600 MHz, DMSO-d6) (*note compound is a
free-radical, some signals appear broadened and other signals are missing) δ 9.46 (s, 1H),
9.33 (s, 1H), 8.18 (s, 1H), 7.64 (s, 3H), 7.60 (s, 3H), 7.48 (s, 1H), 7.41 (s, 1H); 13C NMR
29