Synthesis and biological evaluation of pyrimidine derivatives as novel human Pin1 inhibitors

Article abstract of DOI:10.1016/j.bmc.2018.03.024

Pin1 (Protein interacting with NIMA1) is a cis–trans isomerase and promotes the amide bond rotation of phosphoSer/Thr-Pro motifs in its substrates. Inhibition of Pin1 might be a novel strategy for developing anticancer agents. Herein, a series of pyrimidi

Full text of DOI:10.1016/j.bmc.2018.03.024

Accepted Manuscript
Synthesis and biological evaluation of pyrimidine derivatives as novel human
Pin1 inhibitors
Guonan Cui, Jing Jin, Hualong Chen, Ran Cao, Xiaoguang Chen, Bailing Xu
PII:
S0968-0896(18)30258-X
https://doi.org/10.1016/j.bmc.2018.03.024
BMC 14262
DOI:
Reference:
Bioorganic & Medicinal Chemistry
To appear in:
Received Date:
Revised Date:
Accepted Date:
8 February 2018
12 March 2018
14 March 2018
Please cite this article as: Cui, G., Jin, J., Chen, H., Cao, R., Chen, X., Xu, B., Synthesis and biological evaluation
of pyrimidine derivatives as novel human Pin1 inhibitors, Bioorganic & Medicinal Chemistry (2018), doi: https://
doi.org/10.1016/j.bmc.2018.03.024
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Synthesis and biological evaluation of pyrimidine derivatives as novel
human Pin1 inhibitors
Guonan Cuia,#, Jing Jinb,#, Hualong Chena, Ran Caoa, Xiaoguang Chenb,*, Bailing Xua,*
aBeijing Key Laboratory of Active Substance Discovery and Druggability Evaluation, Institute of
Materia Medica, Chinese Academy of Medical Sciences＆Peking Union Medical College,
Beijing, 100050, China
bState Key Laboratory of Bioactive Substances and Functions of Natural Medicines,
Institute of Materia Medica, Chinese Academy of Medical Sciences＆Peking Union
Medical College, Beijing, 100050, China
Abstract
Pin1 (Protein interacting with NIMA1) is a cis-trans isomerase and promotes the
amide bond rotation of phosphoSer/Thr-Pro motifs in its substrates. Inhibition of Pin1
might be a novel strategy for developing anticancer agents. Herein, a series of
pyrimidine derivatives were synthesized and their Pin1 inhibitory activities were
evaluated. Among them, four compounds (2a, 2f, 2h and 2l) displayed potent
inhibitory activities against Pin1 with IC50 values lower than 3 µM. This series of
pyrimidine-based inhibitors presented time-dependent inhibition against Pin1. The
structure-activity relationships on the 2-, 4- and 5-positions of the pyrimidine ring
were analyzed in details, which would facilitate further exploration of new Pin1
inhibitors.
Keywords: Pyrimidine; Pin1; Pin1 inhibitor; PPIase; Anti-cancer agents
1. Introduction
Pin1 (Peptidyl-prolyl cis-trans isomerase NIMA-interacting 1) is a member of the
Peptidyl-prolyl cis-trans isomerase (PPIase) family1, which is the only known
mammalian isomerase that specifically catalyzes the interconversion of cis-trans
conformations of the amide bond of phosphoSer/Thr-Pro motifs in Pin1 substrates2. It
has been demonstrated that Pin1 can regulate diverse signaling processes in cell by
functioning as a molecular switch and participate in cell cycle progression, apoptosis
and immune responses.3-5 In particular, many research suggested that Pin1 played a
critical role in oncogenesis by upregulation of oncogenes and downregulation of
tumor suppressors.6 Therefore, it was speculated that inhibiting Pin1 might be an
# The first two authors contributed equally.
* Corresponding author. Bailing Xu, Tel: +86 10 63166764; Xiaoguang Chen, Tel: +86 10 63166302
E-mail address: xubl@imm.ac.cn (B. Xu); chxg@imm.ac.cn (X. Chen).

effective way to conquer the aggressive cancers by simultaneously impacting on
multiple oncogenic signaling pathways. Furthermore, Pin1 over-expression was
observed in a variety of human cancer cells, and its over-expression closely correlated
with the occurrence and development of tumors and clinical outcomes.7,8 Noticeably,
cells from Pin1-deficient mice are resistant to the induction of breast cancer by
over-expression of oncogene Ras or ErbB29 and the depletion of Pin1 on various
human cancer cell lines cause mitotic arrest and apoptosis10. Hence, Pin1 is a
promising target for discovering anti-cancer agents with unique mechanism.
A. Juglone
B. IC50 = 0.83 µM
C. IC50 = 3.9 µM
IC50 = 5 µM
GI50 = 13 µM (PC3 cells)
GI50 = 4.7 µM (PC3 cells)
D. Ki = 0.006 µM
E. Ki = 0.89 µM
F. Ki = 0.138 µM
G. Ki = 0.58 µM
H. ATRA IC50 = 0.82 µM
I. IC50 = 2.9 µM
IC50 = 1.9 µM ( HT29 cells)
J. IC50=5.99 µM
K. IC50=26.67 µM
L. IC50=9.05 µM

Figure 1. Chemical structures of several reported Pin1 inhibitors and schematic
pictures of their binding modes
Research efforts for exploring therapeutically useful Pin1 inhibitors have been
made for nearly twenty years since Juglone (A) was identified as an irreversible small
molecule inhibitor of Pin1 in 1998.11 As a result, a number of structurally distinct
Pin1 inhibitors have been disclosed (Fig. 1 A-K).11-24 Juglone (A), discovered by
screening a natural product library, is the first irreversible Pin1 inhibitor, which
inactivates Pin1 enzymatic activity by covalent binding on Cys113 in the active
site.11,12 However, the detailed binding information of Juglone within the catalytic site
has not been resolved. Fragment-based design strategy was exploited by Vernalis and
identified phenyl-imidazole and naphthyl-alanine based inhibitors of Pin1,
exemplified by compounds B and C.13,14 By taking structure-based design approach,
Pfizer achieved several potent Pin1 inhibitors, such as compounds D, E, F and G.15-17
Compound D is the most potent small molecule inhibitor of Pin1 reported so far.
However, due to the poor permeability conferred by the phosphate group, compound
D failed to show cellular effects.15 Although tremendous works have been devoted,
there are no desirable inhibitors with potent activity in cells reported, due to either
poor physicochemical properties or low potency in enzymatic activity. Thus, it is still
an enormous challenge to develop highly potent Pin1 inhibitors with drug-like
properties. Recently, all-trans retinoic acid (ATRA), a therapeutic agent for acute
promyelocytic leukemia, was found to be a potent Pin1 inhibitor, which could inhibit
and degrade active Pin1 in cancer cells, suggesting that it was a viable way for using
Pin1 inhibitors as anti-cancer agents.18
Pin1 poses challenges for drug discovery because of its unique substrate binding
site, consisting of a cluster of basic amino acids such as Lys63, Arg68 and Arg69
residues bound to the negatively-charged phosphate ion and less exploitable
hydrophobic pocket.13-19 Generally, the known Pin1 inhibitors occupied the following
subpockets, including the prolyl pocket formed by His59, His157, Met130 and
Phe134 residues, the slightly shallow hydrophobic shelf lined with His59, Ala118 and
Leu22 residues, and the unique phosphate binding pocket. Interestingly,
phenyl-imidazole-based Pin1 inhibitors such as compound B had a different
orientation in terms of the bulky aromatic group, which contacted a hydrophobic
region lined with the side chain of Arg68.13 It has been suggested that it might be an
alternative strategy to develop desired Pin1 inhibitors by taking advantage of all of the
above mentioned binding regions.
In our previous work, quinazoline-based (I, Fig. 1) and benzophenone-based (J,
Fig. 1) Pin1 inhibitors were discovered by screening our in-house library and further
structure modifications.20,21 Based on the benzophenone-derived human Pin1
inhibitors, a series of novel diarylether derivatives containing an oxalic acid fragment
were synthesized and compound K (Fig. 1) was identified as a hit with an IC50 of
26.67 µM.22 However, to our surprise, it was found by chance that a key intermediate

(compound L, IC50 = 9.05 µM) for synthesis of compound K showed micromolar
potency in the protease-coupled enzyme assay. Due to its low molecular weight and
readily accessible structure, we conducted an extensive investigation on structure
activity relationships (SAR) on compound L. And we found that the pyrimidine
derivatives in this work possessed inhibitory effects on Pin1 with covalent binding
features. Herein, we reported the chemical synthesis of the designed pyrimidine
derivatives and the SAR results of our structure modifications.
2. Chemistry
The synthesis of a variety of pyrimidine derivatives was presented in Schemes
1-5, and the chemical structures of target compounds were presented in Tables 1-3.
The aromatic nucleophilic substitution reactions were performed using
2,4-dichloro-5-nitropyrimidine (1a) and an array of phenols, alcohols or amines as
reactants (Scheme 1). In general, only 4-substituted pyrimidine derivatives were
obtained (yields 30.3%~95.9%), and no corresponding 2-substituted regio-isomers
were isolated. However, when benzyl alcohol or 2-phenylethanol was reacted with
compound 1a, two regio-isomers generated respectively. Interestingly, in contrast to
other reactions, 2-substituted isomers 2m-2 (yield 22.3%) and 2n-2 (yield 35.0%)
were obtained as the major products, and 4-substituted isomers 2m-1 (yield 10.9%)
and 2n-1 (yield 13.0%) were isolated as minor products in these two cases. In order to
confirm the chemical structures of regio-isomers, single crystal X-ray analysis was
conducted on some representative compounds. Among the obtained single-isomers,
the crystal structures of compounds 2a, 2d and 2t were obtained and shown in
Supplementary data (1.1-1.3), which proved to be 4-substituted isomers. As to the
chemical structures of isomers 2m-1 and 2m-2, the structure of compound 2m-1 was
confirmed with X-ray diffraction method, and the crystal structure was presented in
Supplementary data (1.4). Accordingly, by comparing the 1H-NMR data of
compounds 2n-1 and 2n-2 with those of 2m-1 and 2m-2, we confirmed the structures
of these two isomers. The signals of C6-H on pyrimidine ring in 4-substituted isomers
2m-1 and 2n-1 appeared at δ 9.04 and δ 9.02 ppm; while the signals of C6-H in
2-substituted isomers 2m-2 and 2n-2 resonated at δ 9.17 and 9.14 ppm. Compound 2b
was prepared by removing the Boc group of compound 2b’ with TFA. Compound 2b
was further converted into compound 2c by reaction with methylsulfonyl chloride.
The pyridine derivatives 2v-1 and 2w were constructed through nucleophilic aromatic
substitution reaction between compound 1b or 1c and 4-hydroxybenzooxazole25 in
36% and 88% yields, respectively. In fact, 2,6-dichloro-3-nitropyridine 1b was
reacted with 4-hydroxyl benzo[d]oxazole giving two isomers under various conditions.
The chemical structure of the desired compound 2v-1 was confirmed by X-ray
crystallographic analysis (shown in Supplementary data, 1.5). The structure of
compound 2w was confirmed to be 4-substituted isomer according to the literature.26

Scheme 1. Reagents and conditions: (i) K2CO3 or DIEA, acetone/DMF or DCM or
THF, 10.9%~95.9%; (ii) TFA, DCM, 75.2%; (iii) methylsulfonyl chloride, TEA,
DCM, 0 °C, 25.5%.
The synthesis of 2-substituted or 2,4-disubstituted pyrimidines 7a-7e was
outlined in Scheme 2. (Z)-Ethyl 3-(dimethylamino)-2-nitroacrylate 5 was synthesized
by mixing ethyl 2-nitroacetate and 1,1-dimethoxy-N,N-dimethylmethanamine.27
Compound 5 was condensed with amidine reagents 4a or 4b to construct pyrimidine
derivatives 6a and 6b, followed by chlorination with POCl3 affording compounds 7a
and 7b. Compound 7b was converted into 7c by substitution reaction with 4-hydroxy
benzo[d]oxazole (3). Compounds 7d and 7e could be prepared by Suzuki coupling
with compound 2h and phenyl or naphthyl boric acid, although the yields were
somewhat low.

Scheme 2. Reagents and conditions: (i) sodium ethoxide, ethanol, 40 °C,
°
°
48.3%~55.8%; (ii) POCl3, DIEA, 80 C, 14.4%~36.2%; (iii) 3, DIEA, DCM, 0 C,
23.8%; (iv) phenyl or 2-naphthyl boric acid, Pd2(dppf)3, Na2CO3, CsF, toluene, DMF,
16.7%~20.4%.
In order to investigate the effects of substituents on the 5-position of pyrimidine
ring on the inhibitory activity, a number of 5-substituted pyrimidine derivatives were
designed and synthesized, while phenoxyl, benzo[d]oxazole oxyl, phenyl amino
fragments were chosen as R4 group according to obtained SAR results. As shown in
Scheme 3, compounds 9a-9g were readily prepared via nucleophilic aromatic
substitution reaction, and compound 9h was obtained by hydrolysis of compound 9g.
Compounds 9i and 9j were prepared via condensation reaction between aromatic
amine 10 and succinic anhydride or maleic anhydride in 21% and 27% yield,
respectively (Scheme 4). According to the synthetic route shown in Scheme 5, the
oxadiazole or oxazole substituted derivatives (compounds 9k and 9l) were prepared in
3 or 5 steps. The key immediate 13 was synthesized via substitution and hydrolysis
reaction starting from ethyl 2,4-dichloropyrimidine-5-carboxylate 11. Then compound
9k was synthesized via aza-Witting reaction by mixing compound 13 with
(isocyanoimino)triphenylphosphorane 1428 for 24 h. The acid 13 was condensed with
2,2-dimethoxyethanamine 15, followed by cyclization with Eaton’s reagent to give
compound 17. Of note, the chlorine atom on the 2-position was converted into a
hydroxyl group in the presence of Eaton’s reagent29. Finally, compound 17 was
transformed into the desired compound 9l by using POCl3 as a chlorinating reagent.
Scheme 3. Reagents and conditions: (i) K2CO3, acetone, DMF, 87.7-99%; (ii) NaOH,
H2O, THF, 20.9%.

Scheme 4. Reagents and conditions: (i) Fe, NH4Cl, ethyl acetate, EtOH, H2O, 60 °C,
68.9%; (ii) succinic anhydride or maleic anhydride, DMAP, AcOH, 20.6% ~27%.
Scheme 5. Reagents and conditions: (i) DIEA, DCM, 0 °C, 79.6%; (ii) NaOH, THF,
H2O, 96.6%; (iii) DCM, 60.3%; (iv) SO2Cl2, TEA, DCM, r.t., 87.0%; (v) Eaton’s
reagent, argon; (vi) POCl3, DIEA, two-step total yield 12.3%.
3. Biological results and discussion
All the target compounds were screened against Pin1 by a protease-coupled
enzyme assay with Suc-Ala-Glu-Pro-Phe-pNA as the substrate.30,31 The corresponding
results are expressed as IC50 values and presented in Tables 1-3.
Since compound L was identified to be a potent Pin1 inhibitor with IC50 value of
9.05 µM in our group, we immediately set to work on SAR studies around the
pyrimidine scaffold. Initially, the SAR on 4-substituents of pyrimidine ring was
investigated. When phenoxyl or various substituted phenoxyl groups were introduced
at the 4-position, all the resulted compounds (2a-2l) showed inhibitory activities with
IC50 values ranging from 1.68 µM to 34.04 µM. Among them, compounds 2a, 2f, 2h
and 2l displayed more potent activities than other diary ether, they had IC50 values at
low micromolar level (1.68-2.52 µM). It was noticed that compound 2h was more
active than compound 2i and 2j, and compound 2l was more active than compound 2k,
suggesting that heteroaromatic groups were preferred as R4 group. In addition, when
benzyloxy, phenethoxy or phenyl amino moiety was used as R4 group, the obtained

compounds 2m-1, 2n-1 and 2q had inhibitory activities as well. Interestingly,
compound 2r with a phenethylamino group as the 4-substituent showed no inhibition
against Pin1, the corresponding phenethoxy substituted analog (compound 2n-1) had
noticeable activity though. Furthermore, the placement of a cycloalkyloxy or
cycloalkylamino substituent led to loss of inhibition. Taken together, the results
suggested that incorporation of an aromatic group, particularly a heteroaromatic group
on the 4-position of pyrimidine ring was beneficial to the potency, presumably due to
the positive contributions of the aromatic hydrophobic interactions occurred.
With an aim to probe the contributions of the nitrogen atoms on the pyrimidine
ring, two pyridine derivatives (2v-1 and 2w) were prepared. None of them produced
inhibitory effects on Pin1. This result suggested that both nitrogen atoms exerted
strong impacts on the binding affinity. We assumed that the nitrogen atoms probably
formed key hydrogen bonds within the pocket, or the electrophilic properties of the
pyrimidine ring played a role in the binding. Therefore, we retained the pyrimidine as
a scaffold to further vary the substituents on its 2- and 5-positions.
Table 1
The chemical structures and inhibitory activities against hPin1 of 4-substituted
pyrimidine derivatives and substituted pyridine derivatives
2a-2u
R4
2v-1
Compd.
2w
Compd.
L
IC50 (µM)a
9.05±0.06
R4
IC50 (µM)
2.17±0.21
2l
2a
2b
2c
2d
2.24±0.75
33.5±2.81
5.20±0.03
4.03±0.83
2m-1
2n-1
2o
8.33±2.13
4.08±0.09
>100
2p
>100

2e
2f
5.04±0.09
2.52±0.27
3.48±0.50
1.68±0.80
16.55±1.54
2q
2r
2s
2t
6.66±0.01
>100
2g
2h
2ib
>100
>100
2u
>100
2jb
22.6±2.38
2v-1
2w
-
-
>100
>100
2kb
34.04±2.88
a
The measured IC50 for compound A was 5.90 µM; the measured IC50 for compound D was
0.05 µM; the measured IC50 for compound F was 2.96 µM; SD, standard deviation of two
independent assays.
b Compounds 2i, 2j and 2k were obtained in our previous work.22
As shown in Table 2, in comparison with compounds 2m-1 and 2n-1, the
corresponding 2-substituted regio-isomers 2m-2 (IC50 7.28 µM) and 2n-2 (IC50 7.27
µM) had micromolar level inhibitory activity as well. Compound 7a (IC50 13.87 µM)
bearing a phenethyl fragment at 2-position showed somewhat lower activity,
compared with compound 2m-2. However, replacement of the chloro atom in
compound 2h (IC50 1.68 µM), the most potent Pin1 inhibitor of this work, with a
hydrogen atom, benzene ring or naphthyl group (compounds 7c-7e) resulted in
complete loss of activity. These data suggested that the chloro atom on the pyrimidine
core had an important role in the Pin1 inhibitory activity.
Table 2
The chemical structures and inhibitory activities against hPin1 of 2-substituted
pyrimidine and 2,4-disubstituted pyrimidine derivatives
Com
R2
R4
IC50 (µM) a Com
R2
R4
IC50 (µM) a

pd.
pd.
7c
2m-2
Cl
Cl
Cl
7.28±0.42
7.27±1.14
13.87±1.51
H
>100
>100
>100
2n-2
7d
7a
7e
a The measured IC50 for compound A was 5.90 µM; the measured IC50 for compound D was
0.05 µM; the measured IC50 for compound F was 2.96 µM; SD, standard deviation of two
independent assays.
We further explored the SARs on 5-substituents of the 2-chloropyrimidine
scaffold by choosing phenoxyl, benzo-oxazole oxyl or phenyl amino as R4 group. A
number of substituents were tentatively selected as R5 with various electronic
parameters. As shown in Table 3, grafting a CH3 (9a, 9b), CF3 (9c, 9d), Cl (9e) or
COOCH3 (9g) on the 5-position rendered the inhibitory activity totally lost. While a
CN or COOH was presented on the 5-position, the corresponding compounds 9f (IC50
4.36 µM) and 9h (IC50 18.9 µM) produced Pin1 inhibitory activity. In particular,
compound 9f had comparable activity with nitro substituted counterpart 2a. By
examining the Hansch’s π constant, Hammett’s σ constant, and the molar refraction
(MR) parameters of these substituents32, we found out that groups NO2, CN and
COOH had similar properties in terms of hydrophobicity, electronic effects and the
molecular volume. They all had less lipophilicity, strong electron withdrawing ability
in comparison with CH3 and Cl groups. Noticeably, the inhibitory activity enhanced
along with the increase in electron-withdrawing capability of the substituents (IC50
value: 2a < 9f, 2h < 9h). Interestingly, although the CF3 and COOCH3 groups had a
somewhat strong electron-withdrawing ability, the more bulky size of these groups
might cause the loss of activity. These results indicated that a limited space available
around 5-position of the pyrimidine ring.
With regard to compounds 9i-9l bearing a five-membered heterocyclic ring on
5-position, it was delighted that two of them (compounds 9j and 9l) exhibited
inhibitory activity, especially compound 9l with an oxazole moiety on 5-position
produced a comparable potency with compound 2q. The oxazole fragment is a good
substitute for the NO2 group with respect to improving the structural novelty and
drug-like properties of this class of Pin1 inhibitors. Noticeably, compound 9k with an
oxadiazole as R5 group gave no inhibition on Pin1, although the oxadiazole is an
isosteric molecule of oxazole and it has even stronger electron-withdrawing ability.
Together, these results suggested that a strong electron-withdrawing group on
5-position would be beneficial for the potency, and the molecular size and shape of
5-substituents were restricted. The cyano group or oxazole moiety would be the
alternative substitute for NO2 group.

Table 3
The chemical structures and inhibitory activities against hPin1 of compounds 9a-9l
Com
pd.
Com
pd.
R4
R5
IC50 (µM) a
>100
R4
R5
IC50 (µM) a
>100
9a
9b
9c
9d
9e
9f
CH3
9g
9h
9i
COOCH3
CH3
CF3
CF3
Cl
>100
>100
COOH
18.9±0.25
>100
>100
9j
20.2±0.11
>100
>100
9k
9l
CN
4.36±0.97
9.87±0.95
a
The measured IC50 for compound A was 5.90 µM; the measured IC50 for compound D was
0.05 µM; the measured IC50 for compound F was 2.96 µM; SD, standard deviation of two
independent assays.
The electrophilic property of 2- or 4-chloro-pyrimidine was well recognized, and
the obtained SAR results gave us a hint that this series of Pin1 inhibitors possibly
interacted with Pin1 through a covalent binding. Thus, the intrinsic time dependent
nature of a covalent binding was explored tentatively by choosing four representative
inhibitors. As shown in Figure 2, all of the four compounds exhibited time dependent
inhibitory effects at different concentrations. At the same concentration of the
inhibitor, the inhibitory effects became stronger with the increasing incubation time.
The results suggested that these inhibitors might covalent bind with Pin1. The binding
features of pyrimidine derivatives deserved to be further investigated, and that would
be informative for exploring covalent inhibitors of Pin1.

A
2a
B
2 h
120
100
80
60
40
20
0
120
100
80
0
m i n
0 min
15 min
30 min
1 5 m i n
3 0 m i n
60
40
20
0
-7
-6
-5
-4
-3
-8
-7
-6
-5
-4
-3
-20
- 2 0
Concentration [logC(mol/L)]
C o n c e n t r a t i o n [ l o g C ( m o l / L
C 2m-1
D
2m-2
120
120
100
80
60
40
20
0
0 min
0 min
100
80
60
40
20
0
15 min
30 min
15 min
30 min
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-3.5
-6.5
-6.0
-5.5
-5.0
-4.5
-4.0
-3.5
Concentration [logC(mol/L)]
Concentration [logC(mol/L)]
Figure 2. Time-dependent Pin1 PPIase assay of compound 2a (A), 2h (B), 2m-1(C)
and 2m-2 (D) (●) Pin1 protein and inhibitors (varying concentrations in DMSO) were
mixed and tested immediately; (■) Pin1 protein and inhibitors (varying concentrations
in DMSO) were incubated for 15 min; (▲) Pin1 protein and inhibitors (varying
concentrations in DMSO) were incubated for 30 min.
4. Conclusion
In conclusion, a series of pyrimidine derivatives were disclosed as novel Pin1
inhibitors with IC50 values at low micromolar level. The most potent compound 2h
was identified with an IC50 value of 1.68 µM. The SAR results and the
time-dependent inhibition of this series of pyrimidine derivatives indicated that they
possibly inactivate Pin1 by forming a covalent bond within the binding pocket.
Except for Juglone, there are no covalent inhibitors against Pin1 reported, although
many non-covalent inhibitors have presented. The covalent inhibitor of Pin1 deserved
to be explored since it might be an alternative means to address this problematic
target.
5. Experimental
5.1. Chemistry
5.1.1.
General
1H NMR spectra were recorded with a Varian Mercury 300, 400 or 500
spectrometer using tetramethylsilane (TMS) as the internal standard in Acetone-d6,
DMSO-d6 or CDCl3. High resolution mass spectra (HRMS) were recorded on an
Agilent Technologies LC/MSD TOF spectrometer. Melting points were measured on

a Yanaco micro melting point apparatus. All chemicals and solvents used were of
reagent grade without further purification or dried before used. All the reactions were
monitored by thin-layer chromatography (TLC) under a UV lamp at 254 nm. Column
chromatography separations were performed with silica gel (200–300 mesh).
5.1.2.
Chemical synthesis of pyrimidine derivatives
5.1.2.1 2-Chloro-5-nitro-4-phenoxypyrimidine (2a)
To a stirred solution of 2,4-dichloro-5-nitropyrimidine (1a) (970 mg, 5.0 mmol)
°
in DCM (20 mL) at 0 C was added DIEA (0.87 mL, 5.0 mmol) and phenol (470 mg,
°
5.0 mmol) dropwise. After stirring at 0 C for 0.5 h, the reaction mixture was washed
with saturated brine (20 mL), water (20 mL×2), and then dried over anhydrous
magnesium sulfate. The crude product was purified by silica gel column
chromatography (petroleum ether/ethyl acetate = 40:1) to afford the title compound
°
1
2a as a white solid (1.12 g, 88.8%); mp 119-120 C; H NMR (400 MHz, CDCl3) δ
(ppm) 9.16 (s, 1H), 7.48 (t, J = 8.0 Hz, 2H), 7.36 (t, J = 7.6 Hz, 1H), 7.20 (d, J = 8.0
Hz, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm) 162.97, 161.86, 157.21, 150.92,
129.93, 126.99, 121.21; HRMS (ESI): m/z, Calcd. for C10H7O3N3Cl [M+H]+:
252.0171, Found 252.0163.
5.1.2.2
N1-(2-chloro-5-nitropyrimidin-4-yl)benzene-1,2-diamine (2b)
To the stirred solution of compound 2b’ (470 mg, 1.28 mmol) in DCM (20 mL)
was added TFA (1.96 mL, 25.6 mmol) dropwise, the reaction mixture was then
allowed to stir at room temperature overnight. The solvent was washed with
ammonium hydroxide and then evaporated under reduced pressure to afford the title
compound 2b as a yellow solid (257 mg, 75.2%); mp 163-166 °C; 1H NMR (300 MHz,
DMSO-d6) δ (ppm) 10.65 (s, 1H), 10.41 (s, 1H), 9.19 (s, 1H), 8.06 (d, J = 7.8 Hz, 1H),
7.10 (t, J = 7.5 Hz, 1H), 6.98 (d, J = 7.2 Hz, 1H), 6.91 (t, J = 7.8 Hz, 1H); HRMS
(ESI): m/z, Calcd. for C10H8O3N4Cl [M+H]+: 267.0279, Found 267.0274.
5.1.2.3
N-(2-((2-chloro-5-nitropyrimidin-4-yl)oxy)phenyl)methanesulfonamide (2c)
To a stirred solution of compound 2b (100 mg, 0.38mmol) in DCM (10 mL) was
added TEA (0.21 mL, 1.5 mmol) and methanesulfonyl chloride (0.032 mL, 0.40
mmol) dropwise at 0 °C, after 5 min the reaction mixture was quenched with water (2
mL). The organic layer was separated and washed with water (10 mL×2), then dried
over anhydrous magnesium sulfate. The crude product was purified by silica gel
column chromatography (petroleum ether/ethyl acetate = 10:1~5:1) to afford the title
compound 2c as a yellow solid (33 mg, 25.5%); mp 204-208 °C; 1H NMR (400 MHz,
Acetone-d6) δ (ppm) 10.54 (s, 1H), 9.25 (s, 1H), 8.14 (d, J = 8.0 Hz, 1H), 7.57-7.42
(m, 3H), 3.45 (s, 3H); HRMS (ESI): m/z, Calcd. for C11H10O5N4ClS [M+H]+:
345.0055, Found 345.0045.
5.1.2.4 2-chloro-4-(2-methoxyphenoxy)-5-nitropyrimidine (2d)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (217 mg, 1.05 mmol), K2CO3 (262 mg, 1.90

mmol) and 2-methoxyphenol (118 mg, 0.95 mmol) in DMF (1 mL) and acetone (9
°
mL) was stirred at -20 C for 5 h to give the title compound 2d as a white solid (111
°
1
mg, 41.4%); mp 152-153 C; H NMR (400 MHz, Acetone-d6) δ (ppm) 9.32 (s, 1H),
7.35 (t, J = 8.0 Hz, 1H), 7.29 (d, J = 7.6 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 7.06 (t, J=
7.6 Hz, 1H), 3.80 (s, 3H); 13C NMR (100 MHz, Acetone-d6) δ (ppm) 162.72, 162.66,
158.52, 151.59, 140.95, 132.62, 128.69, 122.90, 121.75, 114.00, 56.30; HRMS (ESI):
m/z, Calcd. for C11H9O4N3Cl [M+H]+: 282.0276, Found 282.0266.
5.1.2.5 2-Chloro-4-(3-methoxyphenoxy)-5-nitropyrimidine (2e)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (406 mg, 2.1 mmol), DIEA (0.66 mL, 4.0 mmol)
and 3-methoxyphenol (248 mg, 2.0 mmol) in THF (10 mL) was stirred at -40 °C for 3
°
1
h to give the title compound 2e as a white solid (425 mg, 75.4%); mp 89-92 C; H
NMR (400 MHz, Acetone-d6) δ (ppm) 9.31 (s, 1H), 7.43 (t, J = 7.6 Hz, 1H), 6.96-6.90
(m, 3H), 3.83 (s, 3H); 13C NMR (100 MHz, Acetone-d6) δ (ppm) 162.95, 162.62,
161.81, 158.42, 153.21, 131.11, 114.11, 113.20, 108.23, 55.82; HRMS (ESI): m/z,
Calcd. for C11H9O4N3Cl [M+H]+: 282.0276, Found 282.0270.
5.1.2.6 2-Chloro-4-(2-fluorophenoxy)-5-nitropyrimidine (2f)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (406 mg, 2.1 mmol), DIEA (0.66 mL, 4.0 mmol)
and fluorophenol (224 mg, 2.0 mmol) in THF (10 mL) was stirred at -40 °C for 3 h to
°
1
give the title compound 2f as a white solid (490 mg, 82.6%); mp 103-104 C; H
NMR (400 MHz, Acetone-d6) δ (ppm) 9.39 (s, 1H), 7.49-7.35 (m, 4H); HRMS (ESI):
m/z, Calcd. for C10H6O3N3ClF [M+H]+: 270.0076, Found 270.0073.
5.1.2.7
2-Chloro-5-nitro-4-(pyridin-2-yloxy)-pyrimidine (2g)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (243 mg, 1.25 mmol), K2CO3 (328 mg, 2.38
mmol) and 2-hydroxypyridine (114 mg, 1.19 mmol) in DMF (1 mL) and acetone (9
mL) was stirred at 0 °C for 6 h to give the title compound 2g as a white solid (95 mg,
°
1
31.5%); mp 85-87 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.23 (s, 1H), 8.38-8.37
(m, 1H), 7.92 (t, J = 7.2 Hz, 1H), 7.33 (t, J = 6.4 Hz, 1H), 7.20 (d, J = 8.0 Hz, 1H);
HRMS (ESI): m/z, Calcd. for C9H6O3N4Cl [M+H]+: 253.0123, Found 253.0119.
5.1.2.8
4-(2-Chloro-5-nitro-pyrimidin-4-yloxy)-benzooxazole (2h)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (786 mg, 4.07 mmol), K2CO3 (1.02 g, 7.40 mmol),
4-hydroxyl benzo[d]oxazole (500 mg, 3.70 mmol) in DMF (5 mL) and acetone (45
°
mL) was stirred at -20 C for 1 h to give the title compound 2h as a white solid (886
1
mg, 71%); mp 120-123 °C; H NMR (300 MHz, Acetone-d6) δ (ppm) 9.43 (s, 1H),
8.54 (s, 1H), 7.77 (dd, J1 = 8.1 Hz, J2 = 0.9 Hz, 1H), 7.61 (t, J = 8.1 Hz, 1H), 7.42 (dd,
J1 = 8.1 Hz, J2 = 0.9 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ (ppm) 162.98, 161.52,

157.49, 152.82, 151.65, 141.99, 132.47, 131.26, 126.23, 116.99, 110.13; HRMS (ESI):
m/z, Calcd. for C11H6O4N4Cl [M+H]+: 293.0072, Found 293.0068.
5.1.2.9
2-Chloro-4-((1-methyl-3-phenyl-1H-pyrazol-5-yl)oxy)-5-nitropyrimidine
(2l)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (130 mg, 0.67 mmol), DIEA (0.09 mL, 0.52
mmol) and 1-methyl-3-phenyl-5-hydroxyl-1H-pyrazol33 (90 mg, 0.52 mmol) in THF
°
(5 mL) was stirred at -40 C for 0.5 h to give compound 2l as a yellowish white solid
°
1
(84 mg, 49.1 %); mp 135-137 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.24 (s, 1H),
7.79-7.77 (m, 2H), 7.43-7.39 (m, 2H), 7.33 (tt, J1 = 7.2 Hz, J2 = 1.2 Hz, 1H), 6.69 (s,
1H), 3.89 (s, 3H); 13C NMR (100 MHz, CDCl3) δ (ppm) 163.28, 158.44, 157.76,
149.75, 144.44, 132.93, 128.14, 125.24, 91.73, 35.19; HRMS (ESI): m/z, Calcd. for
C14H11O3N5Cl [M+H]+: 332.0545, Found 332.0537.
5.1.2.10 4-(Benzyloxy)-2-chloro-5-nitropyrimidine (2m-1)
2-(Benzyloxy)-4-chloro-5-nitropyrimidine (2m-2)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (194 mg, 1.0 mmol), DIEA (0.18 mL, 1.0 mmol)
°
and benzyl alcohol (93 mg, 1.0 mmol) in DCM (5 mL) was stirred at 0 C for 5 h to
give compound 2m-1 as a white solid (29 mg, 10.9%); mp 127-129 °C; 1H NMR (400
MHz, CDCl3) δ (ppm) 9.04 (s, 1H), 7.51-7.49 (m, 2H), 7.43-7.35 (m, 3H), 5.65 (s,
2H); 13C NMR (100 MHz, CDCl3) δ (ppm) 162.71, 162.02, 156.64, 133.74, 128.93,
128.75, 128.38, 71.21; HRMS (ESI): m/z, Calcd. for C11H9O3N3Cl [M+H]+: 266.0327,
Found 266.0319; The isomer 2m-2 as a white solid (59 mg, 22.3%); mp 98-99 °C; 1H
NMR (400 MHz, CDCl3) δ (ppm) 9.17 (s, 1H), 7.49-7.47 (m, 2H), 7.42-7.34 (m, 3H),
5.55 (s, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm) 164.48, 158.15, 156.37, 134.34,
128.84, 128.67, 128.54, 71.71; HRMS (ESI): m/z, Calcd. for C11H9O3N3Cl [M+H]+:
266.0327, Found 266.0320.
5.1.2.11 2-Chloro-5-nitro-4-phenethoxypyrimidine (2n-1)
4-Chloro-5-nitro-2-phenethoxypyrimidine (2n-2)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (283 mg, 1.46 mmol), DIEA (0.25 mL, 1.46
mmol) and 2-phenylethanol (178 mg, 1.46 mmol) in DCM (10 mL) was stirred at 0 °C
for 20 h to give compound 2n-1 as a white solid (53 mg, 13%); mp >250 °C; 1H NMR
(400 MHz, CDCl3) δ (ppm) 9.02 (s, 1H), 7.35-7.23 (m, 5H), 4.78 (t, J = 6.8 Hz, 2H),
3.17 (t, J = 6.8 Hz, 2H), 13C NMR (100 MHz, CDCl3) δ (ppm) 162.83, 162.25, 156.54,
136.69, 129.16, 128.64, 126.98, 70.73, 34.84; HRMS (ESI): m/z, Calcd. for
C12H11O3N3Cl [M+H]+: 280.0484, Found 280.0481; The isomer 2n-2 as a white solid
°
1
(144 mg, 35%); mp 215-217 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.14 (s, 1H),
7.34-7.23 (m, 5H), 4.71 (t, J = 7.2 Hz, 2H), 3.15 (t, J = 7.2 Hz, 2H), 13C NMR (100
MHz, CDCl3) δ (ppm) 164.62, 158.09, 156.30, 136.84, 128.99, 128.65, 126.87, 70.75,

34.95; HRMS (ESI): m/z, Calcd. for C12H11O3N3Cl [M+H]+: 280.0484, Found
280.0481.
5.1.2.12 2-Chloro-4-(cyclopentyloxy)-5-nitropyrimidine (2o)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (194 mg, 1.0 mmol), K2CO3 (138 mg, 1.0 mmol),
°
in cyclopentanol (5 mL) was stirred at 40 C for 2 d to give compound 2o as light
1
yellow oil (94 mg, 38.6%); H NMR (400 MHz, CDCl3) δ (ppm) 9.00 (s, 1H),
5.76-5.72 (m, 1H), 2.07-2.00 (m, 2H), 1.97-1.81 (m, 4H), 1.73-1.67 (m, 2H).
5.1.2.13 2-Chloro-4-(cyclohexyloxy)-5-nitropyrimidine (2p)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (194 mg, 1.0 mmol), K2CO3 (138 mg, 1.0 mmol),
°
in cyclohexanol (5 mL) was stirred at 40 C for 23 h to give compound 2p as light
1
yellow oil (78 mg, 30.3%); H NMR (400 MHz, CDCl3) δ (ppm) 9.00 (s, 1H),
5.46-5.40 (m, 1H), 1.98-1.93 (m, 2H), 1.82-1.68 (m, 4H), 1.52-1.35 (m, 4H); HRMS
(ESI): m/z, Calcd. for C10H13O3N3Cl [M+H]+: 258.0640, Found 258.0628.
5.1.2.14 2-Chloro-5-nitro-N-phenylpyrimidin-4-amine (2q)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (250 mg, 1.29 mmol), DIEA (0.22 mL, 1.29
mmol) and aniline (120 mg, 1.29 mmol) in DCM (1 mL) was stirred at 0 °C for 5 min
°
to give the title compound 2q as a yellow solid (277 mg, 85.8%); mp 171-173 C; 1H
NMR (400 MHz, DMSO-d6) δ (ppm) 10.45 (s, 1H), 9.16 (s, 1H), 7.54 (d, J = 8.0 Hz,
2H), 7.45 (t, J = 7.6 Hz, 2H), 7.29 (t, J = 7.2 Hz, 1H); 13C NMR (100 MHz, CDCl3) δ
(ppm) 164.26, 157.60, 153.44, 135.45, 129.30, 126.65, 122.85; HRMS (ESI): m/z,
Calcd. for C10H8O2N4Cl [M+H]+: 251.0330, Found 251.0329.
5.1.2.15 2-Chloro-5-nitro-N-phenethylpyrimidin-4-amine (2r)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (500 mg, 2.59 mmol), DIEA (0.86 mL, 4.92
mmol) and 2-phenylethanamine (299 mg, 2.46 mmol) in THF (15 mL) was stirred at
°
-40 C for 10 min to give the title compound 2r as a light yellow solid (383 mg,
°
1
55.7%); mp 95-96 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.02 (s, 1H), 8.40 (brs,
1H), 7.35 (t, J = 7.6 Hz, 2H), 7.29-7.24 (m, 3H), 3.95-3.90 (m, 2H), 2.99 (t, J = 7.2 Hz,
2H); HRMS (ESI): m/z, Calcd. for C12H12O2N4Cl [M+H]+: 279.0643, Found
279.0639.
5.1.2.16 2-Chloro-N-methyl-5-nitropyrimidin-4-amine (2s)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (388 mg, 2.0 mmol), DIEA (0.4 mL, 2.4 mmol)
and 40% methylamine solution in water (171 mg，2.2 mmol) in DCM (10 mL) was
°
stirred at -60 C for 3 h to give the title compound 2s as a yellow solid (251 mg,
°
1
66.6%); mp 81-83 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.04 (s, 1H), 8.39 (brs,

1H), 3.22 (d, J = 4.8 Hz, 3H);13C NMR (125 MHz, CDCl3) δ (ppm) 164.29, 156.94,
156.13, 126.81, 28.32; HRMS (ESI): m/z, Calcd. for C5H6O2N4Cl [M+H]+: 189.0174,
Found 189.0169.
5.1.2.17 2-Chloro-N-cyclopentyl-5-nitropyrimidin-4-amine (2t)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (500 mg, 2.58 mmol), DIEA (0.51 mL, 3.1 mmol)
°
and cyclopentylamine (171 mg, 2.2 mmol) in DCM (15 mL) was stirred at -40 C for
1 h to give the title compound 2t as a yellowish white solid (444 mg, 71.0%); mp
80-81 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 9.03 (s, 1H), 8.38 (brs, 1H), 4.65-4.57
(m, 1H), 2.21-2.13 (m, 2H), 1.81-1.68 (m, 4H), 1.61-1.53 (m, 2H); 13C NMR (100
MHz, CDCl3) δ (ppm) 164.14, 157.06, 154.95, 126.42, 53.22, 32.98, 23.68; HRMS
(ESI): m/z, Calcd. for C9H12O2N4Cl [M+H]+: 243.0643, Found 243.0638.
5.1.2.18 2-chloro-N-cyclohexyl-5-nitropyrimidin-4-amine (2u)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyrimidine (1a) (540 mg, 2.78 mmol), DIEA (0.51 mL, 3.1 mmol)
and cyclohexanamine (171 mg, 2.2 mmol) in DCM (15 mL) was stirred at -40 °C for 5
min to give the title compound 2u as an off-white solid (685 mg, 95.9%); mp
°
1
120-122 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.03 (s, 1H), 8.34 (brs, 1H),
4.29-4.21 (m, 1H), 2.05-2.02 (m, 2H), 1.81-1.78 (m, 2H), 1.70-1.67 (m, 1H),
1.53-1.25 (m, 5H); 13C NMR (100 MHz, CDCl3) δ (ppm) 164.26, 157.25, 154.63,
126.31, 50.19, 32.37, 25.26, 24.40; HRMS (ESI): m/z, Calcd. for C10H14O2N4Cl
[M+H]+: 257.0800, Found 257.0788.
5.1.2.19 4-(6-Chloro-3-nitro-pyridin-2-yloxy)-benzooxazole (2v-1)
4-(6-Chloro-5-nitropyridin-2-yloxy)-benzooxazole (2v-2)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,6-dichloro-3-nitropyridine (1b) (212 mg, 1.1 mmol), K2CO3 (276 mg, 2.0 mmol)
and 4-hydroxyl benzo[d]oxazole (135 mg, 1.0 mmol) in acetone (3 mL) and DMF (3
mL) was stirred at room temperature for 3 h to give compound 2v-1 as a white solid
°
(104 mg, 35.7%); mp 178-179 C; 1H NMR (300 MHz, Acetone-d6) δ (ppm) 8.67 (d,
J = 8.1 Hz, 1H), 8.47 (s, 1H), 7.70 (dd, J1 = 8.1 Hz, J2 = 0.9 Hz, 1H), 7.57 (t, J = 8.1
Hz, 1H), 7.49 (d, J = 8.4 Hz, 1H), 7.37 (dd, J1 = 8.1 Hz, J2 = 0.9 Hz, 1H); HRMS
(ESI): m/z, Calcd. for C12H7O4N3Cl [M+H]+: 292.0120, Found 292.0118; The isomer
°
1
2v-2 (72 mg, 24.7%) as a light yellow solid; mp 159-162 C; H NMR (300 MHz,
Acetone- d6) δ (ppm) 8.63 (dd, J1 = 8.7 Hz, J2 = 0.9 Hz, 1H), 8.48 (s, 1H), 7.70 (d, J =
8.4 Hz, 1H), 7.57 (t, J = 8.4 Hz, 1H), 7.39-7.34 (m, 2H).
5.1.2.20 4-(2-Chloro-5-nitro-pyridin-4-yloxy)-benzooxazole (2w)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-nitropyridine (1c) (269 mg, 1.39 mmol), K2CO3 (384 mg, 2.78 mmol)
and 4-hydroxyl benzo[d]oxazole (171 mg, 1.27 mmol) in acetone (18 mL) and DMF
(2 mL) was stirred at room temperature for 4 h to give the title compound 2w as a

°
1
white solid (324 mg, 87.8%); mp 195-196 C; H NMR (400 MHz, Acetone-d6) δ
(ppm) 9.07 (s, 1H), 8.54 (s, 1H), 7.77 (d, J = 8.0 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H),
7.43 (d, J = 8.0 Hz, 1H), 7.07 (s, 1H); 13C NMR (100 MHz, Acetone-d6) δ (ppm)
159.52, 156.42, 154.98, 153.03, 148.02, 144.10, 127.68, 117.43, 113.52, 110.71;
HRMS (ESI): m/z, Calcd. for C12H7O4N3Cl [M+H]+: 292.0120, Found 292.0109.
5.1.2.21 4-Chloro-5-nitro-2-phenethylpyrimidine (7a)
To a stirred solution of compound 6a (1.21 g, 4.90 mmol) dissolved in anhydrous
toluene (20 mL) was added phosphorus oxychloride (2.27 g, 14.8 mmol), DIEA (0.85
mL, 4.90 mmol) under argon atmosphere. The reaction was heated to 80°C and stirred
for 2 h. Then the reaction mixture was cooled and quenched with water (10 mL).
Ethyl acetate (40 mL) was added and the organic layer was separated and washed
with water (30 mL×2), dried over anhydrous magnesium sulfate. The crude product
was purified by silica gel column chromatography (petroleum ether/ethyl acetate =
10:1) to afford the title compound 7a as a white solid (470 mg, 36.2%); mp
°
1
102-104 C; H NMR (400 MHz, CDCl3) δ (ppm) 9.16 (s, 1H), 7.29-7.22 (m, 5H),
3.39 (t, J = 8.0 Hz, 2H), 3.19 (t, J = 8.0 Hz, 2H); 13C NMR (100 MHz, CDCl3) δ (ppm)
154.76, 138.85, 138.18, 127.41, 118.25, 118.08, 116.53, 47.90, 42.36; HRMS (ESI):
m/z, Calcd. for C12H11O2N3Cl[M+H]+: 264.0534, Found 264.0526.
5.1.2.22 4-((5-nitropyrimidin-4-yl)oxy)benzo[d]oxazole (7c)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
4-chloro-5-nitropyrimidine (7b) (50 mg, 0.31 mmol), DIEA (0.05 mL, 0.31 mmol)
and 4-hydroxyl benzo[d]oxazole (3) (47 mg, 0.34 mmol) in DCM (10 mL) was stirred
°
at 0 C for 0.5 h to give the title compound 7c as an off-white solid (19 mg, 23.8%);
°
mp 157-159 C;1H NMR (400 MHz, CDCl3) δ (ppm) 9.36 (s, 1H), 8.81 (s, 1H), 8.05
(s, 1H), 7.62 (d, J = 8.4 Hz, 1H), 7.51 (t, J = 8.0 Hz, 1H), 7.29 (d, J = 8.0 Hz, 1H);
HRMS (ESI): m/z, Calcd. for C11H7O4N4 [M+H]+: 259.0462, Found 259.0459.
5.1.2.23 4-(5-Nitro-2-phenyl-pyrimidin-4-yloxy)-benzooxazole (7d)
The reaction mixture of 2h (150 mg, 0.51 mmol), phenylboronic acid (188 mg,
1.54 mmol), sodium carbonate (163 mg, 1.54 mmol), cesium fluoride (77 mg, 0.51
mmol) and Pd(pddf)Cl2 (78 mg, 0.10 mmol) in toluene (9 mL) and DMF (1 mL) was
°
stirred under argon atmosphere at 80 C for 1 h. Then the mixture was diluted with
ethyl acetate (10 mL) and washed with saturated brine (10 mL), water (10 mL×2), and
then dried over anhydrous magnesium sulfate. The crude product was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 20:1) to afford the
°
1
title compound 7d as a white solid (35 mg, 20.4%); mp 164-165 C; H NMR (400
MHz, DMSO-d6) δ (ppm) 9.59 (s, 1H), 8.77 (s, 1H), 7.92 (d, J = 7.6 Hz, 2H), 7.87 (d,
J = 8.4 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.55 (t, J = 7.6 Hz, 1H), 7.48 (d, J = 8.0 Hz,
1H), 7.43 (t, J = 7.6 Hz, 2H);13C NMR (100 MHz, DMSO-d6) δ (ppm) 166.01, 160.84,
157.45, 155.17, 151.49, 142.61, 135.04, 133.33, 132.85, 130.91, 129.48, 129.10,
126.83, 117.91, 110.25; HRMS (ESI): m/z, Calcd. for C17H11O4N4 [M+H]+: 335.0775,
Found 335.0763.

5.1.2.24 4-(2-Naphthalen-2-yl-5-nitro-pyrimidin-4-yloxy)-benzooxazole (7e)
Following the preparation protocol of Section 5.1.2.23, compound 2h (100 mg,
0.34 mmol), 2-naphthaleneboronic acid (177 mg, 1.03 mmol), sodium carbonate (109
mg, 1.03 mmol), cesium fluoride (52 mg, 0.34 mmol) and Pd(pddf)Cl2 (52 mg, 0.07
mmol) in toluene (9 mL) and DMF (1 mL) was stirred under argon atmosphere at
°
80 C for 3.5 h to give the title compound 7e as a light yellow solid (27 mg, 16.7%);
°
1
mp 240-242 C; H NMR (400 MHz, Acetone-d6) δ (ppm) 9.59 (s, 1H), 8.70 (s, 1H),
8.50 (s, 1H), 8.01 (d, J = 8.8 Hz, 1H), 7.93-7.82 (m, 4H), 7.68 (t, J = 8.0 Hz, 1H),
7.63-7.50 (m, 3H);13C NMR (100 MHz, DMSO-d6) δ (ppm) 166.01, 160.86, 157.49,
155.20, 151.55, 142.67, 135.31, 132.92, 132.75, 132.47, 130.83, 130.59, 129.81,
129.16, 129.07, 128.17, 127.53, 126.87, 124.73, 117.97, 110.27; HRMS (ESI): m/z,
Calcd. for C21H13O4N4 [M+H]+: 385.0931, Found 385.0917.
5.1.2.25
2-Chloro-5-methyl-4-phenoxypyrimidine (9a)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-methylpyrimidine (8a) (300 mg, 1.84 mmol), K2CO3 (508 mg, 1.84
mmol) and phenol (173 mg, 1.84mmol) in acetone (20 mL) was stirred overnight
under reflux to give the title compound 9a as a white solid (390 mg, 96.0%); mp
°
59-60 C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.27 (s, 1H), 7.43 (t, J = 7.6 Hz, 2H),
13
7.27 (t, J = 7.6 Hz, 1H), 7.16 (d, J = 7.6 Hz, 2H), 2.30 (s, 3H); C NMR (100 MHz,
CDCl3) δ (ppm) 168.38, 159.24, 157.54, 152.01, 129.61, 125.81, 121.40, 117.26,
12.33; HRMS (ESI): m/z, Calcd. for C11H10ON2Cl [M+H]+: 221.0476, Found
221.0475.
5.1.2.26 4-((2-Chloro-5-methylpyrimidin-4-yl)oxy)benzo[d]oxazole (9b)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-methylpyrimidine (8a) (300 mg, 1.84 mmol), K2CO3 (508 mg, 1.84
mmol) and 4-hydroxyl benzo[d]oxazole (3) (249 mg, 1.84 mmol) in acetone (20 mL)
was stirred overnight under reflux to give compound 9b as a white solid (434 mg,
°
1
90.1%); mp 151-153 C; H NMR (400 MHz, CDCl3) δ (ppm) 8.33 (s, 1H), 8.05 (s,
1H), 7.54 (d, J = 8.4 Hz, 1H), 7.46 (t, J = 8.0 Hz, 1H), 7.22 (d, J = 8.0 Hz, 1H), 2.41 (s,
13
3H); C NMR (100 MHz, CDCl3) δ (ppm) 168.12, 159.62, 157.45, 152.41, 151.62,
143.41, 132.80, 126.00, 117.32, 117.13, 108.96, 12.32; HRMS (ESI): m/z, Calcd. for
C12H9O2N3Cl [M+H]+: 262.0378, Found 262.0376.
5.1.2.27 2-Chloro-4-phenoxy-5-(trifluoromethyl)pyrimidine (9c)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-(trifluoromethyl)pyrimidine (8b) (217 mg, 1.0 mmol), K2CO3 (276 mg,
2.0 mmol) and phenol (94 mg, 1.0 mmol) in acetone (10 mL) was stirred at 0 °C for 1
h to give compound 9c as a white solid (263 mg, 96.0%); mp 87-89 °C; 1H NMR (400
MHz, CDCl3) δ (ppm) 8.70 (s, 1H), 7.46 (t, J = 8.0 Hz, 2H), 7.33 (t, J = 7.6 Hz, 1H),
7.19 (d, J = 8.0 Hz, 2H); HRMS (ESI): m/z, Calcd. for C11H7ON2ClF3 [M+H]+:
275.0194, Found 275.0189.

5.1.2.28 4-((2-Chloro-5-(trifluoromethyl)pyrimidin-4-yl)oxy)benzo[d]oxazole (9d)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloro-5-(trifluoromethyl)pyrimidine (8b) (217 mg, 1.0 mmol), K2CO3 (276 mg,
2.0 mmol) and 4-hydroxyl benzo[d]oxazole (3) (135 mg, 1.0 mmol) in acetone (10
°
mL) was stirred at 0 C for 1 h to give compound 9d as a light pink solid (279 mg,
°
1
88.4%); mp 148-150 C; H NMR (400 MHz, CDCl3) δ (ppm) 8.69 (s, 1H), 8.05 (s,
1H), 7.58 (d, J = 8.4 Hz, 1H), 7.48 (t, J = 8.0 Hz, 1H), 7.25 (d, J = 7.2 Hz, 1H);
HRMS (ESI): m/z, Calcd. for C12H6O2N3ClF3 [M+H]+: 316.0095, Found 316.0092.
5.1.2.29 2,5-Dichloro-4-phenoxypyrimidine (9e)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4,5-trichloropyrimidine (8c) (367 mg, 2.0 mmol), K2CO3 (276 mg, 2.0 mmol) and
phenol (188 mg, 2.0 mmol) in acetone (10 mL) was stirred at room temperature for 6
h to give compound 9e as a white solid (480 mg, 99.0%); mp 82-83 °C; 1H NMR (400
MHz, CDCl3) δ (ppm) 8.47 (s, 1H), 7.46 (t, J = 7.6 Hz, 2H), 7.31 (t, J = 7.6 Hz, 1H),
7.19 (d, J = 8.0 Hz, 2H);13C NMR (125 MHz, CDCl3) δ (ppm) 165.09, 158.15, 157.46,
151.47, 129.74, 126.40, 121.27, 116.97; HRMS (ESI): m/z, Calcd. for C10H7ON2Cl2
[M+H]+: 240.9930, Found 240.9928.
5.1.2.30
2-Chloro-4-phenoxypyrimidine-5-carbonitrile (9f)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
2,4-dichloropyrimidine-5-carbonitrile (8d) (367 mg, 2.0 mmol), K2CO3 (552 mg, 4.0
mmol) and phenol (188 mg, 2.0 mmol) in acetone (10 mL) was stirred at room
temperature for 7 h to give compound 9f as a white solid (408 mg, 88.0%); mp
°
1
94-96 C; H NMR (400 MHz, Acetone-d6) δ (ppm) 9.08 (s, 1H), 7.54 (t, J = 7.2 Hz,
2H), 7.41-7.35 (m, 3H);HRMS (ESI): m/z, Calcd. for C11H7ON3Cl [M+H]+: 232.0272,
Found 232.0270.
5.1.2.31 4-(Benzooxazol-4-yloxy)-2-chloro-pyrimidine-5-carboxylic acid methyl ester
(9g)
Following the preparation protocol of Section 5.1.2.1, the reaction mixture of
methyl 2,4-dichloropyrimidine-5-carboxylate (8e) (228 mg, 1.10 mmol), K2CO3 (276
mg, 2.0 mmol) and 4-hydroxyl benzo[d]oxazole (3) (135 mg, 1.0 mmol) in DMF (1
mL) and acetone (9 mL) was stirred at -40 °C for 6 h to give compound 9g as a white
solid (268 mg, 87.7 %); mp 131-133 °C; 1H NMR (400 MHz, DMSO-d6) δ (ppm) 9.13
(s, 1H), 8.76 (s, 1H), 7.80 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.37 (d, J= 8.0
Hz, 1H), 3.93 (s, 3H); HRMS (ESI): m/z, Calcd. for C13H9O4N3Cl [M+H]+: 306.0276,
Found 306.0273.
5.1.2.32 4-(Benzo[d]oxazol-4-yloxy)-2-chloropyrimidine-5-carboxylic acid (9h)
The reaction mixture of compound 9g (100 mg, 0.36 mmol) and NaOH (72 mg,
1.80 mmol) in water (3 mL) and THF (3 mL) was stirred at room temperature for 3 h.
The organic phase was removed by evaporation and the aqueous phase was cooled in

ice-water bath and acidified to pH 2.0 with 2N aqueous hydrochloric acid. The
resulting precipitate was collected by filtration to give the title compound 9h as a
white solid (20 mg, 20.9 %); mp 118-120oC; 1H NMR (400 MHz, DMSO-d6) δ (ppm)
9.10 (s, 1H), 8.76 (s, 1H), 7.79 (d, J = 8.0 Hz, 1H), 7.56 (t, J = 8.0 Hz, 1H), 7.37 (d, J
= 8.0 Hz, 1H); 13C NMR (100 MHz, DMSO-d6) δ (ppm) 167.77, 164.04, 163.62,
161.37, 155.06 151.50, 142.99, 132.57, 126.85, 117.73, 112.86, 110.05; HRMS (ESI):
m/z, Calcd. for C12H7O4N3Cl [M+H]+: 292.0120, Found 292.0114.
5.1.2.33 1-(2-Chloro-4-(phenylamino)pyrimidin-5-yl)pyrrolidine-2,5-dione (9i)
To a stirred solution of 2-chloro-N4-phenylpyrimidine-4,5-diamine (10) (100 mg,
0.45 mmol) and succinic anhydride (90 mg, 0.9 mmol) dissolved in glacial acetic acid
(5 mL) was added DMAP ( 6 mg, 0.05 mmol) under argon atmosphere. After stirred
°
at room temperature for 1 h, the reaction was heated to 90 C and stirred overnight.
Then the reaction mixture was concentrated and dissolved in ethyl acetate (15 mL),
washed with saturated aqueous sodium bicarbonate (10 mL×3), water (10 mL×2), and
then dried over anhydrous magnesium sulfate. The crude product was purified by
silica gel column chromatography (petroleum ether/ethyl acetate = 3:1) to afford
°
1
compound 9i as a white solid (37 mg, 27%); mp 115-116 C; H NMR (400 MHz,
CDCl3) δ (ppm) 8.04 (s, 1H), 7.37-7.21 (m, 5H), 6.91 (brs, 1H), 2.68 (s, 4H);HRMS
(ESI): m/z, Calcd. for C14H12O2N4Cl [M+H]+: 303.0643, Found 303.0633.
5.1.2.34 1-(2-Chloro-4-(phenylamino)pyrimidin-5-yl)-1H-pyrrole-2,5-dione (9j)
Following the preparation protocol of Section 5.1.2.33, the reaction mixture of
2-chloro-N4-phenylpyrimidine-4,5-diamine (10) (200 mg, 0.91 mmol), maleic
anhydride (178 mg, 1.81 mmol), DMAP (11 mg, 0.09 mmol) in glacial acetic acid (10
°
mL) was stirred at 90 C overnight to give compound 9j as a yellow solid (56 mg,
°
20.6%); mp 104-106 C; 1H NMR (400 MHz, CDCl3) δ (ppm) 8.04 (s, 1H), 7.39-7.32
(m, 4H), 7.18 (t, J = 7.2 Hz, 1H), 6.89 (brs, 1H), 6.85 (s, 2H); HRMS (ESI): m/z,
Calcd. for C14H10O2N4Cl [M+H]+: 301.0487, Found 301.0478.
5.1.2.35 2-Chloro-5-(1,3,4-oxadiazol-2-yl)-N-phenylpyrimidin-4-amine (9k)
To a stirred solution of (isocyanoimino)triphenylphosphorane (14) (302 mg, 1.0
mmol) dissolved in DCM (8 mL) was added the solution of
2-chloro-4-(phenylamino)pyrimidine-5-carboxylic acid (13) (250 mg, 1.0 mmol) in
DCM (7 mL) dropwise. After stirred at room temperature for 24 h, the reaction was
washed with water (10 mL×3), and then dried over anhydrous magnesium sulfate.
The crude product was purified by silica gel column chromatography (petroleum
ether/ethyl acetate = 5:1) to afford compound 9k as a white solid (132 mg, 60.3%);
mp 163-165 °C; 1H NMR (400 MHz, CDCl3) δ (ppm) 10.42 (s, 1H), 8.82 (s, 1H), 8.56
(s, 1H), 7.76 (d, J = 8.0 Hz, 2H), 7.43 (t, J = 8.0 Hz, 2H), 7.23 (t, J = 7.6 Hz, 1H); 13C
NMR (100 MHz, CDCl3) δ (ppm) 162.73, 161.01, 156.83, 156.66, 151.95, 136.99,
129.09, 125.35, 121.86, 100.10; HRMS (ESI): m/z, Calcd. for C12H9ON5Cl [M+H]+:
274.0490, Found 274.0483.

5.1.2.36 2-Chloro-5-(1,3,4-oxadiazol-2-yl)-N-phenylpyrimidin-4-amine (9l)
2-chloro-N-(2,2-dimethoxyethyl)-4-(phenylamino)pyrimidine-5-carboxamide (16)
°
(574 mg, 1.70 mmol) was dissolved in Eaton’s reagent (10 mL) and stirred at 130 C
for 3 h under argon atmosphere. Then the reaction was concentrated and dissolved in
ethyl acetate (20 mL) and methanol (4 mL), washed with water (5 mL). The organic
phase was dried over anhydrous magnesium sulfate. The crude product
2-hydroxyl-5-(oxazol-2-yl)-4-(phenylamino)pyrimidine
(17)
obtained
after
concentration as a white solid which was used in the next step without further
purification.
Following the preparation protocol of Section 5.1.2.21, the reaction mixture of
2-hydroxyl-5-(oxazol-2-yl)-4-(phenylamino)pyrimidine (17), DIEA (0.56 mL, 3.4
°
mmol) in phosphorus oxychloride（5 mL）was stirred at 90 C for 5 h to give
1
compound 9l as a light yellow needle crystal (57 mg, 12.3%); mp 141-142; H NMR
(400 MHz, CDCl3) δ (ppm) 11.10 (s, 1H), 8.84 (s, 1H), 7.79-7.76 (m, 3H), 7.42 (t, J=
8.0 Hz, 2H), 7.35 (s, 1H), 7.19 (t, J = 7.6 Hz, 1H); HRMS (ESI): m/z, Calcd. for
C13H10ON4Cl [M+H]+: 273.0538, Found 273.0530.
5.2. Biological evaluation
5.2.1. Protein expression and purification
The Pet28a-Pin1 plasmid was a gift from Professor Joseph P. Noel (The Salk
Institute for Biological Studies, La Jolla, California). The N-terminally His6-tagged
°
Pin1 was expressed at 22 C in E. coli strain BL21 following induction at an optical
density of 0.6 (600 nm) with 0.5 mM IPTG for 20 h in terrific broth. Cells were
resuspended in 25 mM Tris-Cl, 500 mM NaCl, 10 mM imidazole, 100 g/mL cocktail,
°
0.5 mg/mL lysozyme. Following sonication at 4 C, the soluble supernatant was
loaded onto an Ni-NTA (Qiagen) column and washed with 10 bed volumes of
washing buffer (50 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl). His6-Pin1 was
eluted with 400 mM imidazole, 500 mM NaCl, 20 mM Tris-Cl and condensed by
ultrafiltration (Millipore 5 kDa) with 20 mM Tris-Cl, 100 mM NaCl, 5 mM DTT.
5.2.2. Pin1 PPIase assay and IC50 measurements of Pin1 inhibitors
PPIase activities were measured at 6°C JASCO V-650 spectrophotometer using
protease-coupled
assay
according
to
Wang
et
al.30,31
Suc-Ala-Glu-Pro-Phe-4-nitroanilide in 0.47 M LiCl/trifluoroethanol was used as the
substrate. In brief, the assay buffer (855 μL of 35 mM HEPES at pH 7.8), Pin1 (0.5
μL of 850 μg/mL stock solution), and inhibitors (5 μL of varying concentrations in
DMSO) were pre-equilibrated in the 1.0 mL quartz cuvette in spectrophotometer for
10 min. Then, 100 μL of ice-cooled chymotrypsin (60 mg/mL in 0.001 M HCl) was
added and mixed immediately. Additional 40 μL of substrate (2.5 mM in 0.47 M
LiCl/trifluoroethanol) was added to the cuvette and the reaction was monitored by
absorbance at 390 nm for 90 s. For each compound, three concentrations (100 μM, 10
μM, 1 μM) were chosen, and the assay was performed in duplicate. The data was
analyzed by Graphpad Prism 5.01. The inhibition at each concentration was

calculated according the following equation: Inhibition ratio(%)=[1-(kX-k1)/(kD-k1)]×
100, where kX represents the reaction rate in the presence of tested compound, kD is
the reaction rate of DMSO control without the tested compound, k1 means the reaction
rate of blank control without Pin1 protein, it represents the reaction rate of thermal
isomerization without any catalysis. The IC50 was measured according to the
inhibition ratio at each concentration of tested compound.
5.2.3. Time-dependent Pin1 PPIase assay
Parallel PPIase activities were measured with 96-well UV microplate on the
BECKMAN COULTER-PARADIGM Detection Platform. The substrate
Suc-Ala-Glu-Pro-Phe-4-nitroanilide was dissolved with 0.47 M LiCl/trifluoroethanol.
In brief, the assay buffer (280 μL/well, 35 mM HEPES, 100 mM NaCl at pH 7.8),
Pin1 protein (1.8~2.5 μL/well of 670 μg/mL stock solution), and inhibitors (1.7
μL/well of varying concentrations in DMSO) were added sequentially in the 96-well
UV microplate and incubated for different time (e.g. 0 min, 5 min, 15 min). Then,
ice-cooled chymotrypsin (60 mg/mL in stock buffer with 0.001 M HCl and 0.2 mM
CaCl2) was added in the 96-well UV microplate (33 μL/well). After that, the whole
system was immediately mixed. Finally, the substrate (2.5 mM in 0.47 M
LiCl/trifluoroethanol) was added in the system (14 μL/well) and mixed. During the
whole process any detectable bubbles should be strictly avoided. The reaction was
monitored by detecting the absorbance at 390 nm using kinetic mode (interval 2s,
20~30 cycles) on the BECKMAN COULTER-PARADIGM Detection Platform.
Acknowledgments
This work is supported by National Natural Science Foundation of China (No.
81273380) and “863” Program of China (No. 2012AA020302).
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Graphical Abstract:
Synthesis and biological evaluation of pyrimidine derivatives as novel human
Pin1 inhibitors
Guonan Cui, Jing Jin, Hualong Chen, Ran Cao, Xiaoguang Chen, Bailing Xu
R2 = H, Cl, Ph, naphthyl, PhCH2CH2, PhCH2O,
PhCH2CH2O
R4 = H, Cl, substituted aryloxy or alkyloxy moieties,
substituted arylamino or alkylamino moieties
R5 = small size fragments with various electrostatic
properties
IC50 = 1.68 – 34.0 μM