Synthesis and anticancer activity evaluation of novel azacalix[2]arene[2]pyrimidines

Article abstract of DOI:10.1016/j.ejmech.2018.02.079

A series of novel azacalix[2]arene[2]pyrimidines were synthesized, and evaluated for their antiproliferative activities against A549, MCF7, SH-SY5Y and CNE human cancer cell lines in vitro by using the CCK-8 assay. A number of compounds showed low micromolar antiproliferative activities against MCF7 cell line. Compound 4j, containing a pyrrolidine moiety, exhibited the strongest inhibitory activity with an IC₅₀ value of 0.58 μM. Furthermore, breast cancer cells were used to explore the inhibition mechanism of these azacalix[2]arene[2]pyrimidines. The results suggested these compounds were involved in inducing cell apoptosis via up-regulation of caspase-3 and caspase-9 protein expression, and the cell cycle was arrested at the S phase. Our reports here represent the first studies on the biological activities of azacalix[2]arene[2]pyrimidines.

Full text of DOI:10.1016/j.ejmech.2018.02.079

Accepted Manuscript
Synthesis and anticancer activity evaluation of novel azacalix[2]arene[2]pyrimidines
Yesu Addepalli, Xiaohong Yang, Minghui Zhou, D. Prabhakar Reddy, Shao-Lin
Zhang, Zhen Wang, Yun He
PII:
S0223-5234(18)30216-2
10.1016/j.ejmech.2018.02.079
EJMECH 10256
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 19 September 2017
Revised Date: 23 February 2018
Accepted Date: 25 February 2018
Please cite this article as: Y. Addepalli, X. Yang, M. Zhou, D.P. Reddy, S.-L. Zhang, Z. Wang, Y. He,
Synthesis and anticancer activity evaluation of novel azacalix[2]arene[2]pyrimidines, European Journal
of Medicinal Chemistry (2018), doi: 10.1016/j.ejmech.2018.02.079.
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Graphical abstract
Novel azacalix[2]arene[2]pyrimidines were designed and synthesized. Compound 4j exhibited
strong inhibitory activity against MCF7 cells, and its mechanisms were involved in inducing
apoptosis via up-regulation of caspase-3 and caspase-9 cell and cycle arrest at the S phase.

ACCEPTED MANUSCRIPT
Synthesis and anticancer activity evaluation of novel
azacalix[2]arene[2]pyrimidines
Yesu Addepalli,+ Xiaohong Yang,+ Minghui Zhou, D. Prabhakar Reddy
, Shao-Lin Zhang, Zhen
Wang,* and Yun He*
+School of Pharmaceutical Sciences and Chongqing Key Laboratory of Natural Drug Research, Chongqing
University, 55 Daxuecheng South Road, Shapingba, Chongqing 401331, P. R. China
E-mail: wangz1114@cqu.edu.cn and yun.he@cqu.edu.cn.
ABSTRACT:
A series of novel azacalix[2]arene[2]pyrimidines were synthesized, and evaluated for their
antiproliferative activities against A549, MCF7, SH-SY5Y and CNE human cancer cell lines in
vitro by using the CCK-8 assay. A number of compounds showed low micromolar
antiproliferative activities against MCF7 cell line. Compound 4j, containing a pyrrolidine
moiety, exhibited the strongest inhibitory activity with an IC50 value of 0.58 µM. Furthermore,
breast cancer cells were used to explore the inhibition mechanism of these
azacalix[2]arene[2]pyrimidines. The results suggested these compounds were involved in
inducing cell apoptosis via up-regulation of caspase-3 and caspase-9 protein expression, and the
cell cycle was arrested at the S phase. Our reports here represent the first studies on the
biological activities of azacalix[2]arene[2]pyrimidines.
Keywords: 2,4-diaminopyrimidine, calixarene, azacalix[2]arene[2]pyrimidines, anticancer
activity, antiproliferation.
1. Introduction
Cancer is a huge group of diseases characterized by uncontrolled growth and spread of the
abnormal cells, and the second leading disease after cardiovascular [1]. The World Health
Organization (WHO) has estimated 15 million deaths due to cancer worldwide by 2030. Over
two hundred different types of cancers affecting major organs such as lung, brine, breast, colon,
kidney and stomach have been identified thus far and more are emerging. Although steady
progresses have been made in reducing mortality for cancer patients, in particular with
immunotherapies in the recent years, cancer still accounts for nearly one in every four deaths in
the world [2-4]. The search for novel, effective and safe chemotherapeutic agents for cancer
treatment remains a challenge in pharmaceutical research.

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Calixarene is a class of macrocyclic molecules with unique three-dimensional architectures,
and one of the best known host molecules along with cyclodextrins, cucurbiturils, cryptands, and
crown ethers [5]. Due to their ready availability and convenient functionalization at either the
upper and/or lower rim of the molecular skeleton, calixarenes have become important synthetic
receptors and supramolecular platforms for molecular recognition, sensing and self-assembly,
catalysis, nanotechnology, and drug discovery [6-13]. In particular, calixarenes exhibit various
biological activities, serving as potential anti-tuberculosis agents [14], antiviral agents [15], anti-
thrombotic agents [16], and anticancer agents [17]. Pyrimidine is an important class of
heterocyclic compounds with wide applications in pharmaceutical research due to their broad
range of biological effects such as anticancer [18-24], antimicrobial, [25] anti-inflammatory,
analgesic and anthelmintic activities [26-29]. Among the diverse pyrimidine derivatives, 2,4-
diaminopyrimidines have attracted significant attention due to their highly effective therapeutic
activities against cancers and favorable pharmaceutical properties [30-41]. Several drugs
containing 2,4-diaminepyrimidine nucleus such as ceritinib (Zykadia®) and brigstinib (Alunbrig®)
have been approved by the U.S Food and Drug administration (FDA) as anticancer agents (Fig.
1a). Recently, Breslin et. al. designed a macrocycle to rigidly lock an energy-minimized
bioactive conformation of the diaminopyrimidine (DAP) scaffold. These macrocyclic 2,4-
diaminepyrimidine derivative showed promising inhibitory activities against ALK enzyme (IC50
= 0.5 nM) and cells (IC50 = 10 nM) [42, 43] (Fig. 1a, ALK inhibitor).
As part of our efforts in searching for novel anticancer agents as well as special interest in
macrocyclic compounds, [44-46] we designed and synthesized a series of novel
azacalix[2]arene[2]pyrimidines, and evaluated their potential as anticancer agents (Fig. 1b). Our
efforts represent the first studies on the biological properties of azacalix[2]arene[2]pyrimidines.

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a) Selected anticancer 2,4-diaminopyrimidines
O
Cl
N
Cl
N
N
NH2
HN
N
NH
O
P
HN
N
NH
O
HN
N
NH
O
O
S
O
N
N
N
Ceritinib
(LDK-378)
Brigatinib
(AP-26113)
Cerdulatinib
(PRT-062070)
N
H
N
N
O
S
Novartis, launched
Ariad, launched
Portola, Phase II
O
CF3
NH
Cl
N
N
CH3
N
HN
N
HN
N
NH
O
HN
N
NH
O
N
SO2Me
NH
O
S
NH
O
N
N
N
N
O
O
N
Fedratinib
(SAR-302503)
Rociletinib
(CO-1686)
ALK inhibitor
O
Sanofi, Phase lll
Celgene, NDA Filing
b) This work
R1
N
R1
N
HN
N
NH
Aza macrocyclization
Dimerization
HN
N
NH
R2
R2
HN
R1
N
NH
R'
R'
N
Designed azacalix[2]arene[2]pyrimidines
Fig. 1. Reported anticancer 2,4-diaminopyrimidines and our design of novel
azacalix[2]arene[2]pyrimidines.
2. Results and discussion

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2.1. Chemistry
The synthesis of azacalix[2]arene[2]pyrimidines with various substituents is shown in
Schemes 1-3. As illustrated in Scheme 1, substituted 2,4-dichloropyrimidines (1) reacted with
various benzene-1,3-diamines (2) in the presence of DIPEA in n-butanol at rt-120 °C to furnish
3-((2-chloropyrimidin-4-yl)amino)anilines 3a-j. For derivatives 3k-r bearing methyl or methoxy
o
substituents, microwave at 120 C were applied (See the experimental section for details).
Compounds 3a-r were then heated in 2-butanol at 120 oC for 12-18 hours in acetic acid to afford
calixarenes 4a-r in 18-54% yields via dimerization. Interestingly, the mixed
azacalix[2]arene[2]pyrimidines 5a and 5b could be prepared by reacting compounds 3a and 3d
with compounds 3b and 3e respectively. The cross-coupling products were found as major
products, although the yields were low due to dimerization by itself (Scheme 2). The 4-sulfoxide
substituted azacalix[2]arene[2]pyrimidine derivative 10 was synthesized as shown in Scheme 3.
Palladium acetate catalyzed coupling reaction between 2,5-dichloropyrimidin-4-amine 6 and 2-
bromo-1-(isopropylsulfonyl)-4-nitrobenzene 7 gave intermediate 8. Intermediate 8 could be
reduced smoothly with tin chloride to furnish 9, followed by the pTSA catalyzed dimerization
reaction in 2-butanol at 120 °C to produce the target calixarene 10 (Scheme 3).

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Scheme 1. Synthesis of azacalix[2]arene[2]pyrimidines (4a-r). Reagents and conditions, X-ray
crystal structure of 4a: (i) DIPEA, n-butanol, r.t, 2-6 h (R1 = Cl, F, H), (42-65%) or DIPEA, n-
butanol, 120 °C, microwave, 45 min., R1 = Me, OMe, 36-45 %. (ii) AcOH, 2-butanol, 120 °C,
12-18 h (18-54%).
Scheme 2. Synthesis of mixed azacalix[2]arene[2]pyrimidinedes 5a and 5b: (i) AcOH, 2-
butanol, 120 °C, 18 h, 5a (18%), 5b (22%).
Scheme 3. Synthesis of azacalix[2]arene[2]pyrimidine (10). Reagents and conditions: (i)
Xantphos, Pd(OAc)2, t-BuOK, dioxane, reflux, 4h, 36%. (ii) SnCl2·2H2O, EtOH, reflux, 3 h,
78%. (iii) pTSA, 2-butanol, 120 °C, 18 h, 48%.

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2.2. 1H NMR spectra
Table 1 Selected 1H NMR data (δ / ppm) of compounds 4a-r and 10
1H NMR ( δ / ppm)
Ha
Hb
Hc
Compounds
8.07
8.18
8.13
8.05
8.16
6.18
6.29
6.24
6.15
6.14
7.84
7.93
7.79
7.76
7.80
7.89
7.78
7.75
8.33
7.49
7.81
7.74
7.66
7.97
7.95
8.25
8.00
7.53
7.38
7.57
7.89
7.70
7.01
7.83
8.04
7.47
7.30
7.92
6.75
7.11
7.35
6.72
7.07
6.72
7.10
6.95
6.37
6.07
6.72
7.02
6.30
5.97
6.71
7.04
6.35
6.01
-
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
10
1
The H NMR spectra of symmetrical NH bridged azacalix[2]arene[2]pyrimidines 4a-r and
10 show the presence of two N-H signals in the range of 9.76 - 8.44 ppm. As shown in Table 1,
the azacalix[2]arene[2]pyrimidine derivatives with methyl (4k-n), methoxy (4o-r) and halogen
(4a-c) substituent (R1) on the pyrimidine displayed downfield shifts for protons Ha with a singlet
peak at 8.05 - 7.75 ppm, but hydrogen substituent (R1 = H) on the pyrimidine ring exhibited
upfield shifts at the region of 6.18 - 6.14 ppm. These facts manifested that methyl, methoxy and
halogen groups caused shielding effect on Ha in these compounds. In addition, protons Hb and Hc

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in the diaminophenyl ring with electron-withdrawing groups (R2) (4b, 4c) displayed downfield
shifts in comparison with those with electron-donating substituents (4i, 4j) which exhibited
upfield shifts. It was presumably due to the electron-withdrawing character of CF3 and CO2Et
substituted phenyl moieties.
2.3 In vitro anticancer evaluations
Azacalix[2]arene[2]pyrimidines 4a-r, 5a-b and 10 were evaluated for their anticancer
activities against human lung adenocarcinoma cell (A549), human breast cancer cell (MCF-7),
human neuroblastoma cell (SH-SY5Y), and human nasopharyngeal carcinoma cell (CNE) using
the CCK-8 assay. The IC50 for each compound with respect to the four human cancer cell lines
and one human normal liver cell line L-02 were calculated and the results are summarized in
Table 2. These values represent the concentration at which 50% decrease in cell growth is
observed after 72 h incubation in presence of the drug and compared with control cells treated
with DMSO and positive control Doxorubicin under similar conditions.
The in vitro anticancer activities of these azacalix[2]arene[2]pyrimidines are shown in
Table 2. Compounds 4g, 4h, 4j, 4k, 4n and 4r showed good activities against all four human
cancer cell lines with IC50 values ranging from 0.58 µM to 46.49 µM. Among these compounds,
compound 4j showed the best anticancer activity against MCF-7 cell with an IC50 value of 0.58
µM (Table 2, entry 11). Compound 4g showed very good activity against A549 and SH-SY5Y
cell lines while compound 4h showed moderate activities. Compound 4r showed good activity
against A549, MCF7 and SH-SY5Y cells with IC50 values of 1.60 µM, 1.45 µM and 2.05 µM,
respectively. However, most other azacalix[2]arene[2]pyrimidines sharing very similar structures
did not show any significant anticancer activity even at 50 µM concentration, suggesting their
anticancer activities are very sensitive to the structural perturbations. All
azacalix[2]arene[2]pyrimidines were screened for their toxicity against normal human liver cell
line L-02. Compounds 4g, 4h, 4j, 4k, 4n and 4r displayed relative poor inhibitory activities
against L-02 cells as compared with the cancer cells, indicating the compounds have good
therapeutic indexes (Table 2, entries 8, 9, 11, 12, 15, 19).
The structure activity relationship (SAR) studies revealed that a number of
azacalix[2]arene[2]pyrimidines derivatives (4a-r) exhibited good anticancer activities.
Compound 4j bears a pyrrolidine substituent and showed promising antiproliferative activities

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against A549, MCF7 and SHSY5Y cell lines, but did not show any effect against non-cancerous
L02 cell (IC50 > 25.6 µM). Compounds 4n and 4r containing a methyl and methoxy substituent
on the pyrimidine skeleton respectively showed reduced activities compared to 4j, which has no
substituent on the pyrimidine ring. The analogue with a CF3 substituent on the phenyl ring (4g)
also showed good activity against all four human cancer cell lines. Compound 4h with a bromine
substituent on the phenyl ring showed moderate activity. Among all the tested compounds, those
bearing a pyrrolidine group on the phenyl ring (4j, 4n, 4r) exhibited promising cytotoxicity
against all four tested cancer cell lines. It’s probable that these pyrrolidine-containing analogues
possess the right conformations and sizes to fit the presumed target’s three-dimensional binding
pocket. The mixed azacalix[2]arene[2]pyrimidines 5a and 5b, and sulfoxide derivative 10 did not
show obvious activity up to 50 µM concentration, which are consistent with the test results for
their corresponding homodimers (4a, 4d, 4b and 4e). The target identification efforts are
ongoing, which could eventually help further understanding these SAR results.
Then, MCF7 cell line was selected for further biological studies as compound 4j showed the
best anti-proliferative effect on MCF7 cell. As shown in Fig. 2, compound 4j inhibited MCF7
cell proliferation in concentration-dependent and time-dependent manners.
Table 2. Inhibitory activities of 4a-r & 5a-b and 10 against the cancer cell lines.
IC50 (µM)a
Entry Comp
-ound
R1
R2
A549
MCF7
0.48 ± 0.10 0.89 ± 0.06 0.22 ± 0.04
>50 >50 >50
SH-SY5Y
CNE
L02
0.88 ± 0.05
>50
-
-
1
2
DOX
4a
0.63 ±
0.05
Cl
H
>50

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3
4
5
6
7
8
Cl
Cl
F
CF3
CO2Et
H
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
4b
4c
4d
4e
4f
F
CF3
H
H
H
CF3
2.05 ±
0.10
4g
5.38 ± 0.07 1.43 ± 0.22 15.61 ± 0.14 20.43 ± 0.03
9
H
H
Br
6.55 ±
0.04
47.92 ±
4h
4i
9.24 ± 0.04 2.96 ± 0.02
>50
>50
0.03
>50
10
>50
>50
>50
11
H
4j
0.94 ±
0.16
0.58 ± 0.21 1.82 ± 0.09 46.49 ± 0.03 25.57 ± 0.08
12
13
14
Me
Me
Me
H
7.42±0.03 18.75±0.09 17.48±0.02 23.67±0.02
9.89±0.02
>50
4k
4l
CF3
>50
>50
>50
>50
>50
>50
>50
>50
4m
>50
15
Me
4.25 ±
0.08
4n
4.90 ± 0.09 7.21 ± 0.03
>50
30.84 ± 0.04
16
17
18
OMe
OMe
OMe
H
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
4o
4p
4q
CF3
>50
>50
>50
>50
>50
19
OMe
1.60 ±
0.19
4r
1.45 ± 0.20 2.05 ± 0.07 35.71 ± 0.04 15.81 ± 0.09
20
21
>50
>50
>50
>50
>50
>50
>50
>50
>50
>50
5a
5b
-
-
-
-

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22
>50
>50
>50
>50
>50
10
-
-
a
Values are average of three determinations and deviation from the average is less than 5% of
the average value.
Fig. 1. Antiproliferative activities of Doxorubicin, 4h, 4j, 4n and 4r against A549, MCF7, and
SH-SY5Y cell lines
.
Fig. 2. Cytotoxic effects of compound 4j against MCF7 cells. (A) MCF7 cells were treated with
compound 4j at concentrations ranging from 0 to 10 µM for 72 h; cell viabilities were
determined by using the CCK-8 assay. (B) MCF7 cells were treated with compound 4j at

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concentration of 0.58 µM for 0, 12, 24, 36, 48, and 72 h, respectively, and then the cell viability
(% control) was determined by using the CCK-8 assay. Data were expressed as mean ± SD (n =
3).
2.4 Cell cycle arrest
Then MCF7 cells were treated with increased concentrations of 4j (0-1.2 µM) or control
diluents for 24 h, and cell cycle distributions were determined by staining with propidium iodide
(PI). Compound 4j lead to significant accumulation of cells at the S phase from 6.75% to 11.54%
(0.3 µM), 17.49% (0.6 µM) and 38.08% (1.2 µM); and reduced cells at the G2 phase from
35.08% to 34.8% (0.3 µM), 25.13% (0.6 µM) and 7.88% (1.2 µM) (Fig. 3). These data suggest
that compound 4j induced MCF7 cell cycle arrest at the S phase in a concentration-dependent
manner, compared to untreated cells.
Fig. 3 Compound 4j induced cell cycle arrest in MCF7 cells. Cells were treated with 0.3, 0.6,
and 1.2 µM of compound 4j for 24ꢀh. The cells were harvested, washed with PBS, resuspended
in PBS, and then fixed in ice cold 70% ethanol at 4ꢀ°C. After overnight incubation, the cell

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pellets were collected by centrifugation, resuspended in 50ꢀµg/mL of RNase A in PBS, and
incubated at 37ꢀ°C for 1 h. Next, propidium iodide dye (50ꢀµg/mL) was added, and the mixture
was incubated at 37ꢀ°C for 15ꢀmin. Cell cycle analysis was evaluated via PI fluorescence and
flow cytometry.
2.5 Cell apoptosis
To explore whether the antitumor activity of compound 4j was associated with apoptosis,
MCF7 cells were stained with Hoechst 33258 to investigate the nuclear morphological changes.
As can be seen from the Fig. 4A, MCF7 cells exhibited obvious apoptosis features like improved
brightness, nuclear condensation and nuclear fragmentation, after treatment with compound 4j
(0.3, 0.6, 1.2 µM). This pro-apoptotic effect of 4j on MCF7 cells was further confirmed by
staining with Annexin V-FITC/PI followed by flow cytometry analysis. As shown in Fig. 4B,
after treatment with various concentrations of compound 4j (0, 0.3, 0.6, 1.2 µM) apoptotic cells
were significantly increased with apoptotic rate of 1.1%, 7.1%, 26.1% and 30.7%, respectively.
These results indicated that the anti-proliferative effect of compound 4j might be involved in the
induction of apoptosis.
Fig. 4 Pro-apoptotic effect of compound 4j on MCF7 cells. (A) Apoptotic assay by Hoechst
33342. MCF7 cells were treated with compound 4j at 0.3, 0.6, and 1.2 µM for 24 h, and then
cells were stained with Hoechst 33342 and visualized under a fluorescent microscope (×100). (B)

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Apoptotic assay by flow cytometry. MCF7 cells were treated with compound 4j at 0.3, 0.6, and
1.2 µM for 24 h. Then cells were stained with Annexin V-FITC/PI and were detected by flow
cytometry analysis.
2.6 Protein expressions of Caspase-3 and Caspase-9
To further explore the mechanism by which compound 4j induces apoptosis in MCF7 cells,
we determined the protein expression of C-caspase-3 and C-caspase-9. As can be seen from the
Fig. 5, MCF7 cells exhibited comparable degrees of caspase-3 and caspase-9 cleavage/activation
in response to 4j treatment. Apoptosis is a regulated cell death process which requires the
cascaded activation and execution of a series of regulatory molecules and caspases [47, 48], a
family of cysteine proteases, are the central regulators of apoptosis. Initiator caspases like
caspase-9 are closely coupled to pro-apoptotic signals. Once activated, caspase-9 cleave and
activate downstream effector caspases like caspase-3, which in turn execute apoptosis by
cleaving cellular proteins following specific Asp residues. Caspase-3 is a crucial activated death
protease and caspase-3 activation is recognized as a bio-marker for cells undergoing apoptosis
[49, 50]. Therefore, it is possible that the pro-apoptotic effect of compound 4j on MCF7 cells
was mediated by activating caspase-3 - caspase-9 signalling pathway
Fig. 5. Effects of compound 4j on expressions of caspase family proteins. Cells were treated with
compound 4j for 72 h; then, the total proteins were extracted and subjected to western blot
analysis using antibodies against c-caspase-3 and c-caspase-9, and β-actin was used an internal
control. Data were expressed as mean ± SD (n= 3). **p < 0.01, compared with the control
group.
3. Conclusion

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In summary, a series of novel azacalix[2]arene[2]pyrimidines were designed and synthesized by
azamacrocyclization of 5-substituted benzene pyrimidine of monomer derivatives under acidic
condition. All azacalix[2]arene[2]pyrimidines (4a-r, 5a-b and 10) were screened for their
antiproliferative activities against a panel of four different human cancer cell lines (A549,
MCF7, SH-SY5Y and CNE). A number of these compounds possessed moderate to potent
antiproliferative activities. Among these, the derivatives bearing a pyrrolidine group showed
excellent activity, and 4j was the more potent. Further study suggested that the anti-proliferative
activity could be related to apoptosis via up-regulation of C-caspase-3 and C-caspase-9
expression, and cell cycle arrest at the S phase. These results indicated that this novel class of
azacalix[2]arene[2]pyrimidines may find potential medicinal applications with further structural
modulations and biological studies.
4. Experimental section
4.1 Material and methods
All chemicals were purchased from commercial source and used without further purification
unless otherwise stated. The reactions were monitored by TLC using Merck Kieselgel 60 F254
plates and visualized under UV light at 254 nm. Column chromatography was generally
1
performed on silica gel (60-120 mesh size). H NMR spectra were obtained on an Agilent
400MR or 600MR DD2 spectrometer at ambient temperature. Data were reported as follows:
chemical shift on the δ scale using residual proton solvent as internal standard [δ 0.00 (TMS)],
multiplicity (br = broad, s = singlet, d = doublet, t = triplet, q = quartet, m = multiplet),
integration, and coupling constant(s) in hertz. 13C NMR spectra were obtained with proton
decoupling on an Agilent 400MR DD2 (100 MHz) spectrometer and were reported in ppm with
residual solvent for internal standard [δ 77.16 (CDCl3)]. The NMR data was processed by
software Mest Re-Nova (Ver. 9.0.0.12821, mestrelab research S.L.). High resolution mass
spectra were obtained on a Bruker SolariX 7.0T spectrometer. Melting point was determined by
WRS-2A Digital Melting Point Apparatus. IR spectra were recorded on a Bruker 100 FT-IR
spectrometer and are reported in terms of frequency of absorption (cm–1).
4.2. Chemical synthesis
General procedure for the preparation of compounds 3a-j:
To a mixture of 5-substituted-2,4-dichloropyrimidines 1a-j (1.00 g, 5.52 mmol) and
benzene-1, 3-diamines 2a-j (0.59 g, 5.52 mmol) was dissolved in n-butanol (10 mL) and DIPEA

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(1.42 g, 11.04 mmol) was added and the reaction mixture was stirred at room temperature. The
reaction was continued until complete consumption of starting material, monitored by TLC, for
2-8 h. The solvent was removed under reduced pressure and the residue was dissolved in EtOAc.
Organic layer was washed with water, dried over Na2SO4 and evaporated to dryness. The residue
was purified by flash column chromatography (SiO2, Hexanes/EtOAc) to afford the compounds
3a-j.
4.2.1. N1-(2,5-Dichloropyrimidin-4-yl) benzene-1,3-diamine (3a)
1
A white solid; 45 % yield; m.p.: 132–134 °C; H NMR (400 MHz, DMSO-d6) δ: 9.26 (s, 1H),
8.32 (s, 1H), 7.02 (t,
J = 8.0 Hz, 1H, 6.73 (s, 1H), 6.70 (d, J = 8.0 Hz, 1H), 6.43 (d, J = 8.0 Hz,
1H), 5.31 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 157.3, 157.0, 155.1, 148.7, 137.8, 128.8,
113.4, 111.7, 111.4, 109.5. LC-MS (ESI, 70ev): tR = 4.84 min, m/z 255.0[M+H] +.
4.2.2. N1-(2,5-Dichloropyrimidin-4-yl)-5-(trifluoromethyl) benzene-1,3-diamine (3b)
A white solid; 56% yield; m.p.: 155–158 °C; 1H NMR (400 MHz, CDCl3) δ: 8.23 (s, 1H), 7.42 (s,
1H), 7.22 (s, 1H), 7.06 (s, 1H), 6.71 (s, 1H), 4.00 (s, 2H). 13C NMR (100 MHz, CDCl3) δ: 158.2,
156.3, 155.0, 147.9, 138.5, 132.3 (q,
J = 32.4 Hz), 123.8 (q, J = 272.5), 114.0, 109.9, 108.0,
107.6, (q,
J
= 3.7); LC-MS (ESI, 70ev): tR = 4.50 min, m/z 323.0 [M+H] +.
4.2.3. Ethyl 3-amino-5-((2,5-dichloropyrimidin-4-yl) amino) benzoate (3c)
1
A white solid; 65% yield; m.p.: 145–146 °C; H NMR (400 MHz, CDCl3) δ: 8.21 (s, 1H), 7.53
(s, 1H), 7.42 (s, 1H), 7.25 (s, 1H), 7.16 (s, 1H), 4.37 (q, J = 7.11 Hz, 2H), 3.94 (s, 2H), 1.40 (t, J
= 7.16 Hz, 3H).13C NMR (100 MHz, CDCl3) δ: 166.4, 158.2, 156.4, 154.8, 147.6, 138.1, 132.3,
113.1, 112.5, 112.0, 111.3, 61.3, 14.4, LC-MS (ESI, 70ev): tR = 4.01 min, m/z 327.0 [M+H] +.
4.2.4. N1-(2-chloro-5-fluoropyrimidin-4-yl) benzene-1, 3-diamine (3d)
1
Light yellow solid; 52% yield; m.p.: > 300 °C; H NMR (400 MHz, DMSO-d6) δ: 9.67 (s, 1H),
8.22 (d, J = 3.5 Hz, 1H), 6.97 (t, J = 7.9 Hz, 1H), 6.84 – 6.75 (m, 2H), 6.34 (d, J = 8.0, Hz, 1H),
5.12 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 153.0, 151.3, 151.2, 149.1, 143.9, 141.3, 141.1,
138.1, 128.9, 110.6, 109.8, 107.5. LC-MS (ESI, 70ev): tR = 2.40 min, m/z 239.1[M+H] +.
4.2.5. N1-(2-Chloro-5-fluoropyrimidin-4-yl)-5-(trifluoromethyl) benzene-1, 3-diamine (3e)

ACCEPTED MANUSCRIPT
A off white solid; 61% yield; m.p. 164–166 °C; 1H NMR (400 MHz, CDCl3) δ: 8.10 (d,
J = 2.6
Hz, 1H), 7.45 (d,
(100 MHz, CDCl3) δ: 154.07 (d,
141.10 (d, = 20.7 Hz), 138.9, 131.9 (q,
J
= 2.0 Hz, 1H), 7.05 (s, 1H), 6.95 (s, 1H), 6.70 (s, 1H), 4.01 (s, 2H). 13C NMR
= 3.8 Hz), 151.02 (d, = 10.5 Hz), 147.9, 146.5, 143.9,
= 32.1 Hz), 123.93 (q, = 272.4 Hz), 109.73, 107.3,
J
J
J
J
J
LC-MS (ESI, 70ev): tR = 4.29 min, m/z 307.0 [M+H] +.
4.2.6. N1-(2-Chloropyrimidin-4-yl) benzene-1, 3-diamine (3f)
A off white solid; 48% yield; m.p.: 181–182 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.72 (s, 1H),
8.07 (d, = 5.9 Hz, 1H), 6.96 (t, = 7.9 Hz, 1H), 6.68 (d, = 6.0 Hz, 3H), 6.33 (d, = 8.02 Hz,
J
J
J
J
1H), 5.19 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 161.7, 159.5, 157.1, 149.3, 139.0, 129.3,
110.1, 108.7, 106.3, 105.2. LC-MS (ESI, 70ev): tR = 2.231 min, m/z 221.1 [M+H] +.
4.2.7. N1-(2-Chloropyrimidin-4-yl)-5-(trifluoromethyl) benzene-1, 3-diamine (3g)
A off white solid; 52% yield; m.p.: > 300 °C; 1H NMR (400 MHz, CDCl3) δ: 8.18 (d,
1H), 6.94 (s, 1H), 6.88 (d, = 12.8 Hz, 2H), 6.74 (s, 1H), 6.62 (d, = 5.9 Hz, 1H), 4.00 (s, 2H).
13C NMR (100 MHz, DMSO-d6) δ: 161.4, 159.3, 157.3, 149.9, 140.1, 130.17 (q,
= 30.9 Hz),
125.70 (q, = 270.0Hz), 108.1, 106.1, 105.2, 103.9. LC-MS (ESI, 70ev): tR = 4.22 min, m/z
J = 5.9 Hz,
J
J
J
J
289.0 [M+H] +.
4.2.8. 5-Bromo-N1-(2-chloropyrimidin-4-yl) benzene-1, 3-diamine (3h)
A off white solid; 56% yield; m.p.: > 300 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.89 (s, 1H),
8.15 (d, = 5.9 Hz, 1H), 7.04 (s, 1H), 6.74 (d, = 5.9 Hz, 1H), 6.70 (s, 1H), 6.50 (s, 1H), 5.63 (s,
J
J
2H). 13C NMR (100 MHz, DMSO-d6) δ: 161.4, 159.3, 157.3, 150.5, 140.6, 122.0, 111.8, 110.4,
105.9, 104.3. LC-MS (ESI, 70ev): tR = 4.18 min, m/z 298.0 [M+H] +.
4.2.9. N1-(2-Chloropyrimidin-4-yl)-5-morpholinobenzene-1, 3-diamine (3i)
A brown solid; 38% yield; m.p.: 144–146 °C; 1H NMR (400 MHz, CDCl3) δ: 8.09 (d,
J = 5.6 Hz
1H), 6.83 (s, 1H), 6.53 (d, = 6.0 Hz, 1H), 6.21 (s, 1H), 6.13 (s, 1H), 6.09 (s, 1H), 3.87 – 3.80
J
(m, 4H), 3.74 (s, 2H), 3.18 – 3.09 (m, 4H). 13C NMR (100 MHz, CDCl3) δ: 162.9, 160.9, 158.1,
153.6, 148.6, 138.8, 102.8, 101.8, 101.3, 99.8, 66.9, 49.1. LC-MS (ESI, 70ev): tR = 2.28 min, m/z
306.1 [M+H] +.

ACCEPTED MANUSCRIPT
4.2.10. N1-(2-Chloropyrimidin-4-yl)-5-(pyrrolidin-1-yl) benzene-1, 3-diamine (3j)
A brown solid; 36 % yield; m.p.: 152–153 °C; 1H NMR (400 MHz, CDCl3) δ: 8.07 (d,
J
= 5.9 Hz,
1H), 6.83 (s, 1H), 6.72 (d,
J = 6.0 Hz, 1H), 5.91 (s, 1H), 5.86 (s, 1H), 5.77 (s, 1H), 3.67 (s, 2H),
3.29 – 3.12 (m, 4H), 2.09 – 1.85 (m, 4H). 13C NMR (100 MHz, CDCl3) δ: 163.1, 160.7, 157.8,
149.9, 148.5, 138.6, 102.8, 98.1, 97.9, 96.2, 47.7, 25.5. LC-MS (ESI, 70ev): tR = 2.92 min, m/z
290.1 [M+H] +.
General procedure for the preparation of compounds (3k-r):
To a solution of 5-substituted-2,4-dichloropyrimidines 1k-r (1.00 g, 6.21 mmol) and benzene-1,
3-diamines 2k-r (0.67 g, 6.21 mmol) and DIPEA (1.61 g, 12.42 mmol) was dissolved in n-
butanol was subjected to microwave irradiation at 120 °C for 30 mins. The solvent was removed
under reduced pressure and the residue was dissolved in EtOAc. Organic layer was washed with
water, dried over Na2SO4 and evaporated to dryness. The residue was purified by flash column
chromatography (SiO2, Hexanes/EtOAc) to afford the compounds 3k-r.
4.2.11. N1-(2-Chloro-5-methylpyrimidin-4-yl) benzene-1, 3-diamine (3k)
1
A brown solid, 45% yield; m.p.: 158–160 °C; H NMR (400 MHz, DMSO-d6) δ: 8.58 (s, 1H),
7.98 (s, 1H), 6.98 (t,
J = 7.9 Hz, 1H), 6.75 (d, J = 8.1 Hz, 2H), 6.35 (d, J = 6.8 Hz, 1H), 5.10 (s,
2H), 2.13 (s, 3H). LC-MS (ESI, 70 ev): tR = 4.18 min, m/z 235.0 [M+H] +.
4.2.12. N1-(2-Chloro-5-methylpyrimidin-4-yl)-5-(trifluoromethyl) benzene-1, 3-diamine (3l)
A off white solid; 48% yield; m.p.: 161–163 °C; 1H NMR (400 MHz, DMSO-d6) δ: 8.79 (s, 1H),
8.06 (s, 1H), 7.15 (d,
MHz, DMSO-d6) δ: 160.2, 156.7, 156.5, 149.7, 140.0, 129.71 (q,
J
= 10.9 Hz, 2H), 6.62 (s, 1H), 5.66 (s, 2H), 2.16 (s, 3H). 13C NMR (100
J
= 31.1 Hz), 124.45 (q, J =
272.2 Hz), 114.7, 110.4, 105.9, 105.4, 13.5. LC-MS (ESI, 70ev): tR = 4.18 min, m/z 303.1[M+H]
+
.
4.2.13. N1-(2-Chloro-5-methylpyrimidin-4-yl)-5-morpholinobenzene-1, 3-diamine (3m)
A brown solid; 38% yield; m.p.: 186–187 °C; 1H NMR (400 MHz, DMSO-d6) δ: 8.47 (s, 1H),
7.98 (s, 1H), 6.42 (d, J = 11.1 Hz, 2H), 5.99 (s, 1H), 4.98 (s, 2H), 3.73 (t, J = 4.8 Hz, 4H), 3.02 (t,

ACCEPTED MANUSCRIPT
J
= 4.8 Hz, 4H). 2.13 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 160.5, 156.9, 155.8, 151.9,
149.1, 139.5, 114.1, 100.8, 99.1, 97.6, 66.1, 48.8, 13.5. LC-MS (ESI, 70ev): tR = 2.32 min, m/z
320.1 [M+H] +.
4.2.14. N1-(2-Chloro-5-methylpyrimidin-4-yl)-5-(pyrrolidin-1-yl) benzene-1, 3-diamine (3n)
1
A brown solid; 36% yield; m.p.: 142–145 °C; H NMR (400 MHz, CDCl3) δ: 7.96 (s, 1H), 6.38
(s, 1H), 6.34 (s, 1H), 6.32 (s, 1H), 5.71 (s, 1H), 3.65 (s, 2H), 3.32 – 3.19 (m, 4H), 2.14 (s, 3H),
2.03 – 1.92 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ: 160.6, 156.9, 155.7, 148.8, 148.6, 139.4,
114.1, 97.8, 96.3, 94.5, 47.3, 24.9, 13.5. LC-MS (ESI, 70ev): tR = 2.88 min, m/z 304.1 [M+H] +.
4.2.15. N1-(2-Chloro-5-methoxypyrimidin-4-yl) benzene-1, 3-diamine (3o)
A brown solid; 42% yield; m.p. 126–128 °C; 1H NMR (400 MHz, DMSO-d6) δ: 8.92 (s, 1H),
7.88 (s, 1H), 6.97 (t, J = 8.0 Hz, 1H), 6.90 (s, 1H), 6.84 (d, J = 8.2 Hz, 1H), 6.33 (d, J = 7.4 Hz,
1H), 5.08 (s, 2H), 3.92 (s, 3H). 13C NMR (100 MHz, DMSO-d6) δ: 152.7, 149.6, 148.8, 139.7,
138.8, 135.1, 128.8, 110.1, 109.8, 107.4, 56.5. LC-MS (ESI, 70ev): tR = 2.34 min, m/z 251.0
[M+H] +.
4.2.16. N1-(2-Chloro-5-methoxypyrimidin-4-yl)-5-(trifluoromethyl) benzene-1, 3-diamine
(3p)
A off white solid; 46% yield; m.p.: 157–158 °C; 1H NMR (400 MHz, DMSO-d6) δ: 9.22 (s, 1H),
7.94 (s, 1H), 7.24 (d,
J
= 21.4 Hz, 2H), 6.58 (s, 1H), 5.60 (s, 2H), 3.92 (s, 3H). 13C NMR (101
MHz, DMSO-d6) δ: 152.4, 149.7, 149.3, 139.9, 139.8, 129.7 (q,
J
= 30.8 Hz), 128.5, 123.1 (q,
J
= 270.0 Hz), 109.5, 105.2, 105.2, 56.6. LC-MS (ESI, 70ev): tR=4.18 min, m/z 319.1[M+H] +.
4.2.17. N1-(2-Chloro-5-methoxypyrimidin-4-yl)-5-morpholinobenzene-1, 3-diamine (3q)
A brown solid; 38% yield; m.p.: 196–198 °C; 1H NMR (400 MHz, DMSO-d6) δ: 8.75 (s, 1H),
7.87 (s, 1H), 6.54 (d, = 8.9 Hz, 2H), 5.96 (s, 1H), 4.96 (s, 2H), 3.92 (s, 3H), 3.72 (t, = 4.7 Hz,
4H), 3.01 (t,
= 4.8 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ: 152.7, 152.0, 149.6, 149.3,
J
J
J
139.7, 139.3, 135.0, 99.7, 98.1, 97.0, 66.2, 56.5, 48.7. LC-MS (ESI, 70ev): tR = 2.38 min, m/z
336.1 [M+H] +.

ACCEPTED MANUSCRIPT
4.2.18. N1-(2-Chloro-5-methoxypyrimidin-4-yl)-5-(pyrrolidin-1-yl) benzene-1, 3-diamine
(3r)
1
A brown solid; 35% yield; m.p.: 181–184°C; H NMR (400 MHz, DMSO-d6) δ: 8.63 (s, 1H),
7.83 (s, 1H), 6.29 (s, 1H), 6.22 (s, 1H), 5.59 (s, 1H), 4.80 (s, 2H), 3.17 – 3.05 (m, 4H), 1.97 –
1.85 (m, 4H). 13C NMR (100 MHz, DMSO-d6) δ: 152.7, 149.6, 149.1, 148.6, 139.6, 139.2, 134.8
96.5, 95.1, 94.1, 56.4, 47.3, 24.9. .LC-MS (ESI, 70ev): tR = 2.90 min, m/z 320.1 [M+H] +.
4.2.19. 2, 5-Dichloro-N-(2-(isopropylsulfonyl)-5-nitrophenyl) pyrimidin-4-amine (8)
To a solution of 2, 5-dichloropyrimidin-4-amine 6 (500 mg, 3.08 mmol) in 1, 4-dioxane (40 mL)
was added 2-bromo-1-(isopropylsulfonyl)-4-nitrobenzene 7 (1.04 g, 3.39 mmol), potassium tert
-
butoxide (692 mg, 6.1 mmol), Pd(OAc)2 (69 mg, 0.30 mmol) and xanthphos (357 mg, 0.61
mmol). The reaction mixture was heated under reflux for 4 h under N2 atmosphere. The mixture
was filtered and filtrate washed with brine, dried over Na2SO4, concentrated and purified by
column chromatography to give title compound 8 (577 mg, 48%) as pale yellow solid; m.p.:
181–184 °C; 1H NMR (400 MHz, CDCl3) δ: 10.22 (s, 1H), 9.65 (d,
8.10 (s, 1H), 3.32 – 3.22 (m, 1H), 1.35 (d,
= 6.9 Hz, 6H). 13C NMR (100 MHz, CDCl3) δ:
J = 1.8 Hz, 1H), 8.39 (s, 1H),
J
158.0, 156.5, 156.0, 151.8, 139.0, 132.9, 129.1, 118.1, 117.6, 115.6, 56.5, 15.4. LC-MS
(ESI70ev): tR = 4.75 min, m/z 391.0 [M+H] +.
4.2.20. N1-(2, 5-Dichloropyrimidin-4-yl)-6-(isopropylsulfonyl) benzene-1, 3-diamine (9)
.
A mixture of compound 8 (400 mg, 1.02 mmol) and SnCl2 2H2O (690 mg, 3.06 mmol) in EtOH
(10 mL) was heated to reflux for 4 h. The solvent was removed under reduced pressure. The
residue was dissolved in EtOAc, washed with saturated aq. NaHCO3. The crude product was
purified by column chromatography to give title compound 9 (287 mg, 78%); a white solid; m.p.:
174–176 °C; 1H NMR (400 MHz, CDCl3) δ: 10.14 (s, 1H), 8.26 (s,1H), 7.95 (s, 1H), 7.64 (d,
8.7 Hz, 1H), 6.47 (d, = 8.8 Hz, 1H), 4.38 (s, 2H), 3.18 – 3.10 (m, 1H), 1.28 (dd, = 1.77, 6.83
J =
J
J
Hz, 6H).13C NMR (100 MHz, CDCl3) δ: 157.7, 156.5, 155.5, 152.7, 139.2, 133.6, 115.5, 112.3,
109.6, 106.8, 56.7, 15.7.LC-MS (ESI, 70ev): tR = 4.15 min, m/z 361.0 [M+H]+.
General procedure for the synthesis of azacalix[2]arene[2]pyrimidines: 4a-r, 5a, 5b and 10

ACCEPTED MANUSCRIPT
To a solution of compounds 3a-r (1.0 mmol) in 2-butanol (25 mL) was added acetic acid (1.0
mL) (for compound 10 with 1.5 equivalent pTSA) and heated in seal tube at 120°C for 12-18 h.
The reaction mixture was cooled to ambient temperature, the solvent was evaporated and the
crude product was purified by column chromatography on silica gel (1-6 % MeOH in DCM with
0.01% TEA) to give compounds 4a-r, 5a, 5b and 10.
4.2.21. 15,55-Dichloro-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-dibenzena Cyclo
Octaphane (4a)
1
A white solid; 44% yield; m.p.: > 300 °C; IR (KBr cm-1): 3416, 1628, 1588, 1562, 1476; H
NMR (400 MHz, DMSO-d6) δ 8.94 (s, 2H), 8.44 (s, 2H), 8.07 (s, 2H), 7.49 (s, 2H), 7.17 (t,
J =
7.94 Hz, 2H), 6.75 (d,
J
= 7.91 Hz, 4H),13C NMR (100 MHz, DMSO-d6) δ: 158.4, 156.7, 154.6,
140.4, 138.5, 128.5, 119.3, 118.9, 118.5, 103.7. LC-MS (ESI, 70ev): tR = 2.54 min, m/z 437.0
+
[M+H] +. HRMS (m/z): [M+H] + calcd for C20H15Cl 2N8 437.07926, found 437.07912.
4.2.22. 15,55-Dichloro-35,75-bis(trifluoromethyl)-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-
3,7(1,3)-dibenzenacyclooctaphane (4b)
1
A white solid; 52% yield; m.p.: > 300 °C; IR (KBr cm-1): 3407, 1593, 1516, 1475, 1402; H
NMR (400 MHz, DMSO-d6) δ: 9.31 (s, 2H), 8.81 (s, 2H), 8.18 (s, 2H), 7.81 (s, 2H), 7.11 (s, 2H),
7.05 (s, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 157.8, 156.8, 154.9, 141.5, 139.6, 129.58 (q,
J =
32.3 Hz), 123.4 (q,
J = 270.0 Hz), 121.5, 115.3, 113.9, 104.9. LC-MS (ESI, 70ev): tR = 5.23 min,
m/z 573.0 [M+H] +.
4.2.23. Diethyl15,55dichloro2,4,6,8tetraaza1(2,4),5(4,2)dipyrimidina3,7(1,3)dibenzenacyclo
octaphane-35,75-dicarboxylate (4c)
1
A off white solid; 18% yield; m.p.: > 300 °C; IR (KBr cm-1): 3413, 1721, 1588, 1446, 1401; H
NMR (400 MHz, DMSO-d6) δ: 9.17 (s, 2H), 8.73 (s, 2H), 8.13 (s, 2H), 7.77 (s, 2H), 7.35 (s, 4H),
13
4.28 (q,
J
= 7.17 Hz, 4H), 1.28 (t,
J
= 7.16 Hz, 6H). C NMR (100 MHz, DMSO-d6) δ: 165.1,
158.2, 156.9, 154.9, 141.0, 139.1, 130.5, 123.4, 119.9, 118.8, 104.4, 60.8, 14.2. LC-MS (ESI,
70ev): tR = 4.26 min, m/z 583.1 [M+H] +.
4.2.24. 15,55-Difluoro-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-dibenzenacyclo-
octaphane (4d)

ACCEPTED MANUSCRIPT
1
A white solid; 35% yield; m.p.: > 300 °C; IR (KBr cm-1): 3386, 1627, 1593, 1547, 1443; H
NMR (400 MHz, DMSO-d6) δ: 8.90 (s, 2H), 8.82 (s, 2H), 8.05 (d, = 3.3 Hz, 2H), 7.66 (s, 2H),
7.14 (t, = 7.9 Hz, 2H), 6.72 (d,
= 7.9 Hz, 2H). 13C NMR (100 MHz, DMSO-d6) δ: 156.1,
150.7 (d, = 9.7 Hz), 142.0, 141.3, 140.7 (d, = 19.3 Hz), 139.6, 138.5, 128.6, 117.1 (d,
44.3 Hz), 115.6. LC-MS (ESI, 70ev): tR = 2.03 min, m/z 405.1 [M+H] +.
J
J
J
J
J
J =
4.2.25. 15,55-Difluoro-35,75-bis(trifluoromethyl)-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-
3,7(1,3)-dibenzenacyclooctaphane (4e)
A white solid; 55% yield; m.p.: > 300 °C; IR (KBr cm-1): 3429, 1602, 1575, 1482, 1365, 1365;
1H NMR (400 MHz, DMSO-d6) δ: 9.23 (s, 4H), 8.16 (d,
= 1.88 Hz, 4H),13C NMR (100 MHz, DMSO-d6) δ: 155.5, 150.6 (d,
(d, = 19.3 Hz), 140.0, 139.7, 129.6 (d, = 31.7), 123.9 (q, = 272.6), 117.6, 113.1, 112.3. LC-
MS (ESI, 70 ev): tR = 4.49 min, m/z 541.1 [M+H] +.
J
= 3.15 Hz, 2H), 7.97 (s, 2H), 7.07 (d,
= 10.9 Hz), 142.5, 141.3
J
J
J
J
J
4.2.26. 2, 4,6,8-Tetraaza-1(2,4), 5(4,2)-dipyrimidina-3,7(1,3)-dibenzenacyclooctaphane (4f)
1
A white solid; 36% yield; m.p.: > 300 °C; IR (KBr cm-1): 3452, 1590, 1537, 1483, 1439; H
NMR (400 MHz, DMSO-d6) δ 8.94 (s, 2H), 8.77 (s, 2H), 7.95 (d,
J
= 5.18 Hz, 4H), 7.12 (t,
J =
7.92 Hz, 2H), 6.72 (d, = 7.98 Hz, 2H), 6.59 (d, = 7.98 Hz, 2H), 6.18 (d, J
J
J
= 5.63 Hz, 2H);13C
NMR (100 MHz, DMSO-d6) δ: 161.5, 160.3, 155.9, 141.2, 140.0, 128.6, 115.7, 115.0, 113.5,
99.4. LC-MS (ESI, 70ev): tR = 1.88 min, m/z 369.1 [M+H] +.
4.2.27. 35,75-Bis(trifluoromethyl)-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-diben-
zenacyclooctaphane (4g)
1
A off white solid; 54% yield; m.p.: > 300 °C; IR (KBr cm-1): 3441, 1591, 1558, 1478, 1446; H
NMR (400 MHz, DMSO-d6) δ: 9.28 (s, 2H), 9.21 (s, 2H), 8.25 (s, 2H), 8.06 (d,
7.10 (s, 2H), 6.91 (s, 2H), 6.29 (d,
= 5.6 Hz, 2H) ;13C NMR (100 MHz, DMSO-d6) δ:161.4,
159.9, 156.4, 142.4, 141.1, 129.55 (q, = 31.8, 30.9 Hz), 124.71(q, = 271.0 Hz), 115.1, 111.0,
J = 5.6 Hz, 2H),
J
J
J
110.5, 100.4. LC-MS (ESI, 70ev): tR = 2.78 min, m/z 505.1 [M+H] +; HRMS (m/z): [M+H] +
+
calcd for C22H15F6N8 505.13189, found 505.13184.
4.2.28. 35,75-Dibromo-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-dibenzenacyclo
Octaphane (4h)

ACCEPTED MANUSCRIPT
1
A white solid; 38% yield; m.p.: > 300 °C; IR (KBr cm-1): 3409, 1584, 1488, 1419, 1342; H
NMR (400 MHz, DMSO-d6) δ: 9.10 (s, 2H), 9.04 (s, 2H), 8.00 (d, = 5.6 Hz, 2H), 7.98(s, 2H),
6.95 (s, 2H), 6.79 (s, 2H), 6.24 (d,
= 5.2Hz, 2H); 13C NMR (100 MHz, DMSO-d6) δ: 161.3,
J
J
159.9, 156.3, 142.8, 141.6, 120.7, 117.1, 116.7, 110.9, 100.2 ; LC-MS (ESI,70ev):tR = 2.55 min,
+
m/z 525.0 [M+H]+; HRMS (m/z): [M+H] + calcd for C20H15Br2N8 524.97819, found 524.97809.
4.2.29
zena cyclooctaphane (4i)
A brown solid; 44% yield; m.p.: > 300 °C; IR (KBr cm-1): 3454, 1586, 1489, 1450, 1415; H
NMR (400 MHz, DMSO-d6) δ: 8.83 (s, 2H), 8.55 (s, 2H), 7.91 (d, = 5.67 Hz, 2H), 7.54 (s, 2H),
6.37 (s, 2H), 6.19 (s, 2H), 6.15 (d, = 5.66 Hz, 2H), 3.70 (t, = 4.8 Hz, 8H), 3.00 (t, = 4.78 Hz,
.
35,75-Dimorpholino-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-diben-
1
J
J
J
J
8H);13C NMR (101 MHz, DMSO-d6) δ: 161.5, 160.3, 155.7, 151.2, 141.7, 140.6, 105.1, 102.6,
101.9, 99.2, 66.0, 48.4. LC-MS (ESI, 70ev): tR = 2.00 min, m/z 539.2 [M+H] +.
4.2.30
zenacyclooctaphane (4j)
A brown solid; 41% yield; m.p.: > 300°C; IR(KBr cm-1): 3451, 1582, 1496, 1417, 1348; H
NMR (400 MHz, DMSO-d6) δ: 8.75 (s, 2H), 8.44 (s, 2H), 7.88 (d, = 5.6 Hz, 2H), 7.37 (s, 2H),
6.12 (d, = 5.67 Hz, 2H), 6.01 (s, 2H), 5.83 (s, 2H), 3.19 – 3.07 (m, 8H), 1.96 – 1.84 (m,
.
35,75-Di(pyrrolidin-1-yl)-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-diben
1
J
J
8H);13C NMR (101 MHz, DMSO-d6) δ 161.5, 160.5, 155.4, 147.8, 141.7, 140.5, 101.7, 99.5,
99.1, 98.8, 47.3, 24.9. LC-MS (ESI, 70ev): tR = 2.93 min, m/z 507.2 [M+H] +, HRMS (m/z): [M+
H] + calcd for C28H31N10+ 507.2727, found 507.2727.
4.2.31.
15,55-Dimethyl-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-dibenzena
Cyclooctaphane (4k)
A off white solid; 46% yield; m.p.: > 300 °C; IR (KBr cm-1): 3434, 1585, 1537, 1429, 1231;1H
NMR (400 MHz, DMSO-d6) δ: 8.84 (s, 2H), 8.19 (s, 2H), 7.84(s, 2H), 7.57 (s, 2H), 7.15 (t,
J =
7.94 Hz, 2H), 6.72 (m, 4H), 2.07 (s, 6H). 13C NMR (100 MHz, DMSO-d6) δ: 160.5, 157.5, 140.8,
139.3, 128.5, 118.3, 117.5, 117.2, 109.6, 105.9, 13.7. LC-MS (ESI, 70ev): tR = 1.86 min, m/z
397.2 [M+H] +; HRMS (m/z): [M+H] + calcd for C22H21N8 397.18786, found 397.18837.
+

ACCEPTED MANUSCRIPT
4.2.32. 15,55-Dimethyl-35,75-bis(trifluoromethyl)-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-
3,7(1,3)-dibenzenacyclooctaphane (4l)
1
A white solid; 51% yield; m.p.: > 300 °C; IR (KBr cm-1): 3449, 1598, 1563, 1471, 1362; H
NMR (400 MHz, DMSO-d6) δ: 8.97 (s, 2H), 8.18 (s, 2H), 7.93 (s, 2H), 7.89 (s, 2H), 7.02 (s, 4H),
2.11(s, 6H); 13C NMR (100 MHz, DMSO-d6) δ: 160.1, 158.1, 156.1, 142.7, 140.8, 129.4 (q,
31.6 Hz), 124.01 (q, = 272.3 Hz), 117.9, 112.9, 111.6, 107.0, 13.6. LC-MS (ESI, 70ev): tR =
2.70 min, m/z 533.1 [M+H] +.
J =
J
4.2.33. 4,4'-(15,55-Dimethyl-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-dibenzena-
cyclooctaphane-35, 75-diyl)dimorpholine (4m)
1
A brown solid, 36% yield; m.p.: > 300°C ; IR(KBr cm-1): 3446, 1601, 1427, 1268, 1210; H
NMR (400 MHz, DMSO-d6) δ: 8.34 (s, 2H), 7.79 (s, 2H), 7.70 (s, 2H), 7.22 (s, 2H), 6.31 (s, 4H),
3.77 – 3.60 (m, 8H), 3.13 – 2.87 (m, 8H), 2.05 (s, 6H) ; 13C NMR (100 MHz, DMSO-d6) δ:
160.1, 158.9, 155.5, 151.2, 142.0, 140.2, 107.5, 105.4, 104.5, 103.1, 66.0, 48.6, 13.7. LC-MS
(ESI, 70ev): tR = 2.08 min, m/z 567.3 [M+H] +.
4.2.34. 15,55-Dimethyl-35,75-di(pyrrolidin-1-yl)-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-
3,7(1,3)-dibenzenacyclooctaphane (4n)
1
A brown solid, 42% yield; m.p
.
: > 300°C ; IR ( KBr cm-1 ): 3451, 1600, 1455, 1425, 1352; H
NMR (400 MHz, DMSO-d6) δ: 8.22 (s, 2H), 7.76 (s, 2H), 7.63 (s, 2H), 7.01 (s, 2H), 5.97 (s, 4H),
3.13-3.26 (m, 8H), 1.94 – 1.86 (m, 8H); 13C NMR (100 MHz, DMSO-d6) δ: 160.2, 159.11, 155.3,
148.00, 141.9, 140.1, 105.1, 104.6, 101.4, 100.2, 47.4, 24.9, 13.7. LC-MS (ESI, 70ev) m/z 535.3
[M+H] +; HRMS (m/z): [M+H] + calcd for C30H35N10+ 535.30413, found 535.30407.
4.2.35. 15, 55-dimethoxy-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-diben zena
cyclooctaphane (4o)
A white solid; 46% yield; m.p.: > 300 °C; IR (KBr cm-1): 3383, 1587, 1520, 1455, 1420; 1H
NMR (400 MHz, DMSO-d6) δ: 8.51 (s, 2H), 8.22 (s, 2H), 7.83 (s, 2H), 7.80 (s, 2H), 7.08 (t,
J =
7.9 Hz, 2H), 6.71 (d, = 8.1 Hz, 2H), 6.64 (d,
J
J
= 8.1 Hz, 2H), 3.85 (s, 6H).; 13C NMR (100
MHz, DMSO-d6) δ: 154.1, 152.8, 142.5, 139.2, 136.5, 134.9, 128.4, 115.9, 115.0, 112.7, 56.8.
LC-MS (ESI, 70ev): tR = 2.82min, m/z 429.2 [M+H] +.

ACCEPTED MANUSCRIPT
4.2.36. 15,55-dimethoxy-35,75-bis(trifluoromethyl)-2,4,6,8-tetraaza-1(2,4),5(4,2)- dipyrim-
Idina-3, 7(1, 3)-dibenzenacyclooctaphane (4p)
1
A white solid; 48% yield; m.p.: > 300 °C; IR (KBr cm-1): 3419, 1597, 1561, 1480, 1441; H
NMR (400 MHz, DMSO-d6) δ: 8.93 (s, 2H), 8.66 (s, 2H), 8.04 (s, 2H), 7.89 (s, 2H), 7.04 (d,
14.70 Hz, 4H), 3.87 (s, 6H), 13C NMR (100 MHz, DMSO-d6) δ: 153.2, 152.67, 143.5, 140.3,
136.8, 135.6, 129.4 (q, = 31.5 Hz), 124.04 (q, = 272.3 Hz), 115.0, 111.9, 110.5, 56.8. LC-MS
(ESI, 70ev): tR = 2.97 min, m/z 565.1 [M+H] +.
J
=
J
J
4.2.37. 4,4'-(15,55-dimethoxy-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-dibenzena-
cyclooctaphane-35, 75-diyl) dimorpholine (4q)
1
A brown solid, 38% yield; m.p.: 278-280 °C; IR (KBr cm-1): 3347, 1585, 1548, 1423, 1337; H
NMR (400 MHz, DMSO-d6) δ: 8.30 (s, 2H), 8.03 (s, 2H), 7.78 (s, 2H), 7.47 (s, 2H), 6.35 (d,
J =
7.20 Hz, 4H), 3.74 (s, 6H), 3.72 (t, = 4.74 Hz, 8H), 3.03 (t,
J
J
= 4.80 Hz, 8H); 13C NMR (100
MHz, DMSO-d6) δ: 154.2, 152.6, 151.1, 142.9, 139.7, 136.4, 134.7, 104.0, 103.0, 101.7, 66.0,
56.8, 48.5. LC-MS (ESI, 70ev): tR = 2.23 min, m/z 599.3 [M+H] +.
4.2.38. 15,55-dimethoxy-35,75-di(pyrrolidin-1-yl)-2,4,6,8-tetraaza-1-(2,4),5(4,2)- dipyrim-
idina-3, 7(1, 3)-dibenzenacyclooctaphane(4r)
1
A brown solid; 45% yield; m.p.: > 300°C ; IR (KBr cm-1): 3391, 1610, 1577, 1448, 1355; H
NMR (400 MHz, DMSO-d6) δ: 8.20 (s, 2H), 7.94 (s, 2H),7.75(s, 2H), 7.30 (s, 2H), 6.01 (s, 2H),
5.96 (d, 2H), 3.83 (s, 6H), 3.20 – 3.09 (m, 8H), 1.96 -189 (m, 8H); 13C NMR (100 MHz, DMSO-
d6)δ: 154.4, 152.7, 147.9, 142.9, 139.6, 136.1, 134.6, 100.9, 100.0, 98.7, 56.8, 47.3, 24.9. LC-MS
+
+
(ESI, 70ev): tR = 3.26 min, m/z 567.3[M+H] +. HRMS (m/z): [M+H] calcd for C30H35N10O2
567.29320, found 567.29390.
4.2.39. 15-chloro-55-fluoro-2,4,6,8-tetraaza-1(2,4),5(4,2)-dipyrimidina-3,7(1,3)-diben-
zena Cyclooctaphane (5a)
1
A white solid; 18% yield; m.p.: > 300 °C; IR (KBr cm-1): 3444, 1632, 1595, 1563, 1473; H
NMR (400 MHz, DMSO-d6) δ: 8.96 (s, 1H), 8.91 (s, 1H), 8.81 (s, 1H), 8.44 (s, 1H), 8.09 (s, 1H),
8.03 (d,
J = 3.41 Hz, 1H), 7.59 (d, J = 2.56 Hz, 1H), 7.53 (s, 1H), 7.15 (t, J = 7.91 Hz, 2H), 6.73
(d,
J
=8.00, Hz, 4H); 13C NMR (100 MHz, DMSO-d6) δ: 158.3, 156.8, 156.2, 154.6, 150.6, 141.1,

ACCEPTED MANUSCRIPT
140.8, 140.6, 139.5, 138.5 (d,
J = 4.7 Hz), 128.6 (d, J = 8.1 Hz), 118.7, 118.1, 118.0, 117.5,
116.6, 103.9. LC-MS (ESI, 70ev): tR = 2.23 min, m/z 421.1 [M+H] +.
4.2.40. 15-Chloro-55-fluoro-35,75-bis(trifluoromethyl)-2,4,6,8-tetraaza-1(2,4),-5(4,2)-dipyrim
Idina-3,7(1,3)-dibenzenacyclooctaphane (5b)
1
A white solid; 22% yield; m.p.: > 300 °C; IR (KBr cm-1): 3436, 1598, 1572, 1478, 1369; H
NMR (400 MHz, DMSO-d6) δ: 9.31 (s, 1H), 9.20 (d,
1H), 8.14 (d,
= 3.15 Hz, 1H), 7.91 (s, 1H), 7.85 (s, 1H), 7.14 – 7.00 (m, 4H); 13C NMR (100
MHz, DMSO-d6) δ: 157.8, 156.8, 155.5, 155.0, 150.6 (d, = 11.1 Hz), 142.3, 142.1, 141.8,
141.4, 141.2, 139.9, 139.6, 139.6, 129.8, 129.7, 129.4, 125.2, 122.5(d, = 15.2Hz), 114.6, 113.7,
J = 13.27 Hz, 2H), 8.78 (s, 1H), 8.19 (s,
J
J
J
113.3, 112.8, 105.2. LC-MS (ESI, 70ev): tR = 4.834 min, m/z 557.1[M+H] +.
4.2.41. 15, 55-Dichloro-34,74-bis(isopropylsulfonyl)-2,4,6,8-tetraaza-1(2,4), -3,7(1,3)-
dibenzenacyclooctaphane (10)
A light brown solid; 48% yield; m.p.: >300°C; IR (KBrcm-1): 3447, 1631, 1432, 1361, 1126; 1H
NMR (400 MHz, DMSO-d6) δ: 9.77 (s, 2H), 8.90 (s, 2H), 8.34 (s, 2H), 7.92 (s, 2H), 7.70 (d,
J =
8.71 Hz, 2H), 6.98 (d, = 8.0 Hz, 2H), 3.18 (dt, = 13.4, 6.7 Hz, 2H), 1.08 (d, = 6.76 Hz,
J
J
J
12H); 13C NMR (100 MHz, DMSO-d6) δ: 157.0, 156.4, 155.2, 146.8, 138.3, 131.8, 119.6, 115.7,
115.1, 106.9, 54.7, 14.9. LC-MS (ESI, 70ev): tR = 4.91 min, m/z 649.1 [M+H] +. HRMS (m/z):
+
[M + H] + calcd for C26 H27Cl2N8 O4 S2 649.0974, found 649.0968.
4.3 Cell culture
All of the five cell lines were purchased from Shanghai cell bank of Chinese Academy of
Sciences (Shanghai, China). A549, MCF7 and SH-SY5Y were cultured in DMEM medium
supplemented with 10% FBS, 1% penicillin and streptomycin. CNE and L-02 cells were
maintained in RPMI-1640 medium supplemented with 10% FBS, 1% penicillin and
streptomycin. The cells were cultured at 37 °C in a 5% CO2 humidified atmosphere.
4.4 Cell viability assay
Cell viability was assessed by Cell Counting Kit - 8 (Beyotime, Jiangsu, China) following
the manufacturer’s protocol. Compounds were dissolved in DMSO and diluted with culture
medium. Cells at density of 5 × 103 per well were seeded in 96-well plates and treated with either

ACCEPTED MANUSCRIPT
vehicle (DMSO) or desired concentrations of compounds for 72 h at 37°C in a 5% CO2
incubator. After that, cell viability was measured by CCK-8 and the absorbance was measured
using a 96-well plate reader (Bio-Rad Laboratories, Shanghai, USA) at 450 nm. Cell viability
was calculated as the ratio of the absorbance of the treated cells tothe absorbance of the control
groups. IC50 values were calculated by GraphPad Prism 6. All experiments were performed in
triplicate in three independent.
4.5 Cell cycle assay
MCF7 cells and compound 4j were chosen for cell cycle analysis. Cells were seeded in 6-
well plates at density of 1 × 105 cells/well and treated with different concentrations of compound
4j (0.3, 0.6 and 1.2 µM) for 24 h at 37°C in a 5% CO2 incubator. Then, cells were harvested and
fixed in 70 % precooled ethanol. Finally, cells were stained by PI / RNase A and measured by
flow cytometry (Accuri C6, BD Biosciences).
4.6. Cell apoptosis assay
4.6.1 Hoechst 333258 staining
MCF7 cells were treated with 4j at concentrations of 0.3, 0.6 and 1.2 µM. After 24h
incubation, cells were incubated with 5µg/mL Hoechst 33342 for 20 min at 37°C under 5% CO2.
The morphological changes of apoptotic cell in nuclei and nuclear chromatin were carried
outusinga fluorescence microscope (Olympus, IX51, Tokyo, Japan).
4.6.2 Annexin V/PI detection
Annexin V - FITC Apoptosis Detection Kit (Beyotime, Jiangsu, China) was used to
determine the effect of 4j on MCF7 cells apoptosis. 1 × 105 cells/well were seeded in 6-well
plates. After treatment with or without desired concentrations of 4j ( 0.3, 0.6 and 1.2 µM) for 24
h, cells were collected and then were stained byannexin-V-fluorescein isothiocyanate (FITC) in
binding buffer for 15 min at room temperature protected from light. Subsequently, cells were
labled by PI and the apoptotic cells were measured by Flow Cytometer (Accuri C6, BD
Biosciences). Cells undergoing early and late apoptosis were labeled by annexin-V and both
annexin-V and PI, respectively.

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4.7. Western blotting
After treatment with increased concentrations of 4j (0.3, 0.6 and 1.2 µM) for 72 h, total cellular
protein was extracted by western blot & IP cell lysis buffer kit. The protein concentration was
quantified using the BCA Protein Assay Reagent (Beyotime, Jiangsu, China). Total protein (30
µg) was separated by sodium dodecyl sulfate-polyacrylamide electrophoresis gels (SDS-PAGE)
and transferred to polyvinylidene fluoride (PVDF) membrane. Membranes were blocked with 5%
fat-free dry milk in 1
Õ Tris-buffered saline (TBS) containing 0.05% Tween 20 for 2 h at room
temperature. After that, membranes were incubated with anti-cleaved-Caspase-3, Caspase-9 and
β-actin antibodies at 4 °C overnight. The horseradish-peroxidase-conjugated anti-rabbitIgG were
used as secondary antibodies and the immune reactive band was visualized with ECL-detecting
reagents.
4.8. Statistical analysis
All results were expressed as mean ± standard deviation (SD) of three independent
experiments performed in triplicates. Statistical analyses were carried out using SPSS 19.0
software package and the one way analysis of variance (ANOVA) with Dunnett’s test was used
to compare the means between two groups with a significance level of P < 0.05.
AUTHOR INFORMATION
Corresponding Author
* E-mail: wangz1114@cqu.edu.cn.
* E-mail: yun.he@cqu.edu.cn.
Notes
+ These authors contributed equally.
Acknowledgement
We are grateful for financial support from the National Natural Science Foundation of China
(Nos. 21572027, 21372267 and 21602023) and Chongqing Research and Frontier Technology
(cstc2016jcyjA0403).
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