Synthesis, X-ray crystal structure and anti-tumor activity of calix[n]arene polyhydroxyamine derivatives

Article abstract of DOI:10.1016/j.ejmech.2016.07.016

Calixarene-based compounds are highly effective therapeutic agents against cancer. This study aims to prepare a series of calix [n]arene (n?=?4, 6, 8) polyhydroxyamine derivatives (3a–3m) and to study their potential antitumor activities. The single crystal structure of calixs[4]arene derivative 3a was determined through X-ray diffraction. We assessed the ability of the prepared calix [n]arene polyhydroxyamine derivatives to induce cytotoxicity in six cancer cell lines by performing cancer cell growth inhibition assays. Results demonstrated that compounds 3a–3d achieved IC₅₀values ranging from 1.6?μM to 11.3?μM. Among the different compounds, 3a and 3b exerted the strongest cytotoxic effect in inhibiting the growth of SKOV3 cells. In relation to the underlying mechanisms of cytotoxic effects, cell cycle analysis revealed that the exposure of SKOV3 cells to 3a induced cell cycle arrest in the G0/G1 phase, suggesting a reduction in DNA synthesis. Immunofluorescent staining indicated that the protein expression levels of caspase-3 and p53 in cells significantly increased, whereas that of Bcl-2 was effectively suppressed. Meanwhile, no significant changes in Bax were observed in SKOV3 cells. These results highlight that calixarene 3a can be further studied as a potential anticancer agent.

Full text of DOI:10.1016/j.ejmech.2016.07.016

Accepted Manuscript
Synthesis, X-ray crystal structure and anti-tumor activity of calix[n]arene
polyhydroxyamine derivatives
Lin An, Li-Li Han, You-Guang Zheng, Xian-Na Peng, Yun-Sheng Xue, Xiao-Ke Gu,
Jing Sun, Chao-Guo Yan
PII:
S0223-5234(16)30564-5
10.1016/j.ejmech.2016.07.016
EJMECH 8734
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Revised Date: 8 July 2016
Accepted Date: 9 July 2016
Please cite this article as: L. An, L.-L. Han, Y.-G. Zheng, X.-N. Peng, Y.-S. Xue, X.-K. Gu, J. Sun, C.-
G. Yan, Synthesis, X-ray crystal structure and anti-tumor activity of calix[n]arene polyhydroxyamine
derivatives, European Journal of Medicinal Chemistry (2016), doi: 10.1016/j.ejmech.2016.07.016.
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ACCEPTED MANUSCRIPT

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Synthesis, X-ray crystal structure and anti-tumor activity of
calix[n]arene polyhydroxyamine derivatives
Lin An a,b,c, Li-Li. Han b,c +, You-Guang Zheng b,c, Xian-Na Peng b,c, Yun-Sheng Xue b,c Xiao-Ke
Gu b,c, Jing Suna, Chao-Guo Yana,*
a College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, P. R. China
b College of Pharmacy, Xuzhou Medical University, Xuzhou 221004, P. R. China
c JiangsuKey Laboratory of New Drug Research and Clinical Pharmacy, Xuzhou Medical University, Xuzhou 221004, P. R. China
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a b s t r a c t
Calixarene-based compounds are highly effective therapeutic agents against cancer. This study aims to prepare a series of calix[n]arene (n = 4, 6, 8)
polyhydroxyamine derivatives (3a–3m) and to study their potential antitumor activities. The single crystal structure of calix[4]arene derivative 3a
was determined through X-ray diffraction. We assessed the ability of the prepared calix[n]arene polyhydroxyamine derivatives to induce
cytotoxicity in six cancer cell lines by performing cancer cell growth inhibition assays. Results demonstrated that compounds 3a–3d achieved IC50
values ranging from 1.6 ꢀM to 11.3 ꢀM. Among the different compounds, 3a and 3b exerted the strongest cytotoxic effect in inhibiting the growth
of SKOV3 cells. In relation to the underlying mechanisms of cytotoxic effects, cell cycle analysis revealed that the exposure of SKOV3 cells to 3a
induced cell cycle arrest in the G0/G1 phase, suggesting a reduction in DNA synthesis. Immunofluorescent staining indicated that the protein
expression levels of caspase-3 and p53 in cells significantly increased, whereas that of Bcl-2 was effectively suppressed. Meanwhile, no significant
changes in Bax were observed in SKOV3 cells. These results highlight that calixarene 3a can be further studied as a potential anticancer agent.
Keywords:calix[n]arene, polyhydroxyamine derivative, anti-tumor activity.
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1. Introduction
In the past decades, calix[n]arenes comprising phenol units bridged by methylene linkers have been proven to be an important
family of host molecules in supramolecular chemistry. Moreover, calix[n]arenes could be functionalized at the upper and lower
rims and readily converted into various derivatives such as esters, carboxylates, ethers, and amides [1-3]. In this case,
calix[n]arene and their derivatives exhibit molecular recognition of ions [4, 5], neutral molecules [6], peptides [7], amino acids
[8], proteins [9], and nucleic acids[10], due to their flexible macrocyclic backbones. Recently, their potential applications in
biomedical fields [11-12] as enzymatic inhibitors [13-16], antimicrobials[17-19], and antitumor agents [20-26] have gained
considerable attention. To the best of our knowledge, water-soluble calixarenes modified with anionic sulfonate, phosphate,
carboxylate, or cationic tetra-alkyl ammonium functional groups could solubilize [27, 28]and deliver drugs [29, 30]. Recent
studies have found that p-sulfonato-calix[6]arene and p-sulfonato-calix[8]arene exert antitumor effects on leukemia cell lines[31].
In addition, neutral, hydrophilic substituents has been proposed as an alternative means of rendering calixarenes
water-soluble[32]. Research on neutral water-soluble calixarene-attaching glycosylated or polyhydroxylated groups have been
investigated both in synthesis and biological evaluation[23, 33-37]. From this perspective, searching for new functionalized
calixarenes as antitumor agents and exploring the molecular mechanisms underlying their actions are ongoing challenges. The
present work aimed to report the convenient synthesis of calix[n]arene (n = 4, 6, 8) polyhydroxyamine derivatives 3a–3m and
assess the ability of 3a–3d (Fig.1) to induce cytotoxicity in six cancer cell lines, namely, the human lung cancer cell line A549,
the ovarian cancer cell line SKOV3, the pancreatic cancer cell line SW1990, the cervical cancer cell line Hela, Burkitt’s
lymphoma cell line Raji, and the breast cancer cell line MDA-MB-231. The mechanisms underlying the potential antitumor
properties of these compounds were also elucidated.
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* Corresponding author. College of Chemistry & Chemical Engineering, Yangzhou University, Yangzhou 225002, P. R.
E-mail address: cgyan@yzu.edu.cn (C.-G. Yan).
+L. L. Han contributed equally to this work.
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Figure1. Structural representations of calix[4]arene polyhydroxyamine derivatives 3a–3d
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2. Results and discussion
2.1 Rational anti-tumor agents design based on calixarenes
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Recent years, studies on anti-tumor activity of various functionalized calixarene derivatives have been very modestly
investigated. Dings et al[22, 24-25] reported some calix[4]arene amides were found to be highly effective at inhibiting various
tumor cells. Pelizzaro-Rocha et al [26] revealed that calix[6] arene was potent in reducing Panc-1 cell viability. Thus, the amino
or acylamino groups at the lower rim of calix[n]arene are essential for inhibitory activity. Likewise, alcohols or hydramine are by
far the most used functional groups to improve the physicochemical property of anti-tumor agents. With the goal of developing
novel and effective anti-tumor agents based on calixarenes, we used the tert-butyl calix[4]arene as a model core to design a series
of calixarene polyhydroxyamine derivatives via changing the cavity size, removing the hydrophobic tert-butyl group at the upper
rim and introducing hydrophilic polyhydroxyamine group at the lower rim (Fig.2).
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Figure2. Structural optimization design of calix[n]arene
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2.2 Synthesis of the polyhydroxyamine calix[n]arene derivatives 3a–3m
A convenient procedure for the synthesis of polyhydroxyamine calix[n]arene derivatives 3a–3m is shown in Scheme 1.
Calix[n]arene polyhydroxyamine derivatives can be synthesized through the direct ammonolysis reaction of calixarene esters
with hydroxyamines [2] or through the condensation reaction between calixarene carboxylic acids and hydroxyamines [38].
Considering that ethanolamine and diethanolamine possess less steric hindrance, we selected the first synthetic method. As
shown in Scheme 1, the desired calixarene ester derivatives were prepared by treating p-tert-butylcalix[n]arenes 1a-c (n = 4, 6, 8)
and calix[n]arenes 1d–1f (n = 4, 6, 8) with ethyl chloroacetate in the presence of anhydrous K2CO3 and KI in refluxing acetone.
Ultrasonic irradiation was introduced into this reaction to reduce the reaction time from 3–5 days to approximately 12 h. To our
satisfaction, seven corresponding calix[n]arene ester derivatives (2a–2g) were obtained in 75%–85% yields. Subsequently,
calixarene esters 2a–2g reacted with excess ethanolamine or diethanolamine in a refluxing solvent of 1:1 EtOH/toluene to
produce the desired calixarene polyhydroxyamine derivatives 3a–3m in moderate yields from 25% to 84%. Ethanolamine and
diethanolamine reagents were chosen because they were commercially available, inexpensive, and highly water soluble.
Calix[8]arene polyhydroxyamines 3l–3m were obtained in lower yields (25%, 27%) possibly because of the steric congestion of
eight diethanolamine scaffolds at the lower rim of calix[8]arene. By contrast, calix[4]arene 1,3-disubstituted hydroxyamine
derivatives were successfully prepared in high yields by using calix[4]arene 1,3-diesters as the starting material. The unreacted
hydroxyamines in the reaction were separated from the calixarene polyhydroxyamine derivatives by washing the extracted crude
material with water/chloroform. Further product purification can be achieved by performing column chromatography with silica
as the solid support.

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Scheme1. Synthesis of calix[n]arene polyhydroxyamine derivatives 3a–3m
Reagents and conditions: (i) ClCH2COOC2H5, K2CO3, acetone, ultrasonic irradiation; (ii) NH2CH2CH2OH, ethanol/toluene,reflux; (iii) NH(CH2CH2OH)2,
ethanol/toluene, reflux.
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2.3 X-ray crystal structure of 3a
The single crystal structure of derivative 3a is shown in Fig. 3 and the crystal data and refinements are listed in Table 1.
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Figure 3. Single crystal structure of 3a (hydrogen atoms on the skeletons were omitted for clarity)
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It is clearly seen that calix[4]arene core adopts a cone-conformation. The two N-(2-hydroxyethyl) -acetamide chains exist in
1,3-distal position. In molecules, the two p-t-butylphenol units bearing with acetamide chain stand more perpendicular (dihedral
angle: 82.122(170) ° , 89.500(18) ° ) to the other two p-tert-butylphenol units without substituents (dihedral angle:
40.905(133)°, 41.384(146)°). There are several intramolecular and intermolecular hydrogen bonds formed by the interaction
of the phenolic hydroxy group and N-(2-hydroxyethyl)-acetamide chain.

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Table 1. Crystal data for Compound 3a
Phase
3a
Empirical formula
Formula weight
Temperature
C52H72N2O9
869.12
296(2)
Wavelength/nm
Crystal system
0.71073
Triclinic
space group
P -1
a (Å)
9.937(4)
b (Å)
12.228(4)
c (Å)
22.273(6)
α(°)
87.276(10)
β(°)
82.963(13)
γ(°)
72.794(13)
Volume(Å3)
2565.7(14)
Z
2
F(000)
940
0.36 x 0.34 x 0.28
1.125
Crystal size (mm)
Calculated density (g.cm-3)
Absorption coefficient (mm-1)
θ range (°)
0.076
0.92 to 25.00
Limiting indices
Reflections collected / unique
Completeness to theta
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodness-of-fit on F^2
Final R indices [Iꢂ2sigma(I)]
R indices (all data)
Largest diff. peak and hole(e.Å-3)
-11ꢀꢁhꢀꢁ11, -14ꢀꢁkꢀꢁ14, -26ꢀꢁlꢀꢁ26
29477 / 8702 [R(int.) = 0.1267]
96.3 %
0.979 and 0.973
Full-matrix least-squares on F^2
8702 / 72 / 601
1.038
R1 = 0.1203, wR2 = 0.2627
R1 = 0.2326, wR2 = 0.3168
0.589 and -0.456
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2.4 Compounds 3a–3m reduce the viability of six differenet cancer cells
The efficiencies of the calix[n]arene polyhydroxyamine derivatives 3a–3m as anti-cancer drugs and tools for biological
research were examined. We first evaluated their ability to induce cytotoxicity through preliminary screening in six cell lines,
namely, A549, SKOV3, SW1990, Hela, Raji and MDA-MB-231. Cell viability was assessed 72 h after treatment by conducting
an MTT or SRB assay, and preliminary screening results are shown in Table 2. In general, the different cavities and molecular
footprints of the calixarene scaffold showed different results. Calix[4]arene derivatives 3a–3e, each of which contains four
phenolic units, showed better inhibitory effects on the cell lines than the other derivatives. By contrast, calix[6]arenes and
calix[8]arenes 3f–3m possessing six or eight phenolic units showed poor or even no viability. We assume that large molecular
structures cannot easily permeate the cell membrane. These results indicated that the cavity size of calix[n]arene was a critical
factor for its efficiency against cancer cells. The absence of activity of 3f–3m illustrated that enlarging the cavity of the
calixarenes greatly attenuated the activity. Interestingly, the introduction of a hydrophobic group (e.g., tert-butyl groups) at the
upper rim and a short-chained hydrophilic face at the lower rim of calixarenes improved the activity of these compounds
regardless of calixarene cavity size. For example, 3a and 3c bearing two or four O-acetyl ethanolamine groups at the lower rim
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were effective at inducing cytotoxicity in various cell types; these groups were tested and showed almost 100% single
concentration inhibition. However, 3e showed a low inhibitory effect on the cancer cell types because of the removal of tert-butyl
groups and the introduction of long-chained diethanolamine groups.
Table 2. Single concentration inhibition of calixarenes 3a–3m on cancer cell viability
Single concentration inhibition (%)a
Compound
A549
89
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93
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SKOV3
SW1990
Hela
Raji
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100
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MDA-MB-231
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95
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99
59
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100
99
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95
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3a
3b
3c
3d
3e
3f
86
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100
95
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95
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3g
3h
3i
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0
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3m
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the concentration of the calixarenes 3a–3m was 10 ꢀM
Basing from the above results, we treated with 3a–3d. Dose response curves are shown in Fig. 4 as the percentage of cell
survival relative to that from cultures treated only with the vehicle (100%). Data points represented the means obtained in at least
three independent experiments, each using triplicate cell cultures. Apparent IC50 values (i.e., the concentration required for 50%
cell viability) derived from these curves are given in Table 3.
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Table3. IC50 values (ꢀM) of calixarenes 3a–3d on various cancer cells
IC50 (ꢀΜ)
Cancer cells
3a
4.0
2.8
4.3
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5.0
5.3
3b
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3c
3d
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A549
SKOV3
SW1990
Hela
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3.3
7.1
4.7
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2.3
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--
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8.1
11.2
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Raji
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MDA-MB231
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Figure 4. Cytotoxicity effects of calix[4]arenes 3a–3d. Cell viability of A549, SKOV3, SW1990, Hela, Raji as well as MDA-MB231cells was measured in the
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presence of various concentrations of calixarenes.
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Compounds 3a–3d achieved IC50 values ranging from 1.6 ꢀM to 11.3 ꢀM. Importantly, 3a and 3c exerted great cytotoxic
effects in inhibiting the growth of the six types tested. Compound 3a showed a narrow activity variation from 2.7 ꢀM to 5.3 ꢀM
(the single digit micromolar activity is approximately 4 ꢀM) against all the tested cell types. These results revealed that the
tert-butyl groups introduced by calix[4]arene into the upper rim significantly increased the cytotoxic function of the compounds.
By contrast, 3b was only selectively effective against the SKOV3 and MDA-MB231 cells (IC50 of 2.3 ꢀM against SKOV3 and
1.6 ꢀM against MDA-MB231). Meanwhile, 3d showed a reduced potency over the cytotoxicity profiles of 3a and 3c.
Compounds 3a and 3b were more effective than the other calix[n]arenes in inhibiting the growth of SKOV3. Chemical
antitumor agents can effectively inhibit cell proliferation by inducing cell cycle arrest [25, 26]. To clarify how calix[n]arenes
suppress cell growth, cell cycle progression was analyzed by quantitating DNA content through flow cytometry in SKOV3 cells
[39]. We treated SKOV3 cells with different concentrations (0, 0.3, 1, 3.3, and 10 ꢀM) of calixarene derivatives 3a and 3b after
24 and 48 h, respectively, and measured DNA content through propidium iodide staining (Fig. 5).
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Figure 5. The amount of SKOV3 cells each cell cycle phase (G0/G1 phase, S phase and G2/M) after treatment with specified concentrations of 3a (A) and 3b
(B) for 48 h was determined by propidium iodide staining followed by flow cytometry.
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As shown in Fig. 5A, 64.25% ± 2.26% of cells treated with a low concentration of 3a (3 ꢀM) were arrested in the G0/G1
phase compared with the 49.47% ± 1.91% of the nontreated cells. Accordingly, the percentage of cells in the S phase reduced
from 37.31% ± 3.48% in the nontreated cells to 24.19% ± 2.26% in the cells treated with 3.0 ꢀM 3a. The same profile was
observed in the cells treated with 10 ꢀM 3b (Fig. 5B). Moreover, cell cycle analysis revealed that 3a and 3b induced cell cycle
arrest in the G0/G1 phase. This result was supported by the increased cell count in the G0/G1 phase at 3 and 10 ꢀM for 48 h and
the decreased cell count in the S and G2/M phases.
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2.5 Immunofluorescent staining study of 3a
To further illuminate the molecular of 3a mediated apoptosis in SKOV3 cells, immunofluorescent staining was used to
evaluate apoptotic related proteins Caspase-3, P53, Bcl-2, and Bax protein levels[40, 41]. As shown in Fig.6, after SKOV3 cells
were treated with certain concentration (3ꢀM)of calixarene derivative 3a for 48h, Caspase-3, p53 protein levels in SKOV3 cells
were upregulated significantly, whereas that of Bcl-2 protein was obviously downregulated, meanwhile no significant changes of
Baxprotein was observed in SKOV3 cells, compared to DMSO group. Therefore, these results suggested that 3a might induce
cellular apoptosis through these proteins in SKOV3 cells.

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Fig.6 Effects of apoptotic related proteins expression on the apoptosis of treated and untreated SKOV3 cells. Caspase-3 (A), P53 (C), Bcl-2 (E), Bax (F)
protein antibody and fluorescein-conjugated second antibody were used to visualize corresponding protein, and Hoechst 33342 was used to stain cell nuclei
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(blue).The levels of Caspase-3 (B), P53 (D) are increased in SKOV3 cells, the level of Bcl-2 (E) is decreased with Bax protein no changes.

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3. Conclusion
The results presented in this study showed that calix[4]arene polyhydroxyamine derivatives 3a are highly effective in inducing
cell death in human ovarian carcinoma cells. Our findings provide strong evidence of the large effects of 3a in reducing SKOV3
cell aggressiveness. We also find that 3a could result in apoptotic cell death by up-regulation of Caspase-3, p53 proteins
In SKOV3 cells with keeping Bax no change. Overall, calixarene 3a can be further studied as a potential anticancer agent in the
future.
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4. Experimental section
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4.1. Chemistry synthesis
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p-tert-butylcalix[n]arenes 1a–1c (n = 4, 6, 8) were synthesized according to Gutsche’s method [42]. Calix[n]arenes 1d–1f (n
= 4, 6, 8) were obtained by deakylation of calixarenes 1a–1c with AlCl3 in toluene [43]. calix[n]arenes esters 2a–2g were
prepared by treating 1a–1f with chloroacetate in the presence of K2CO3/KI/acetone under ultrasonic irradiation[44]. Other
reagents and solvents were commercial reagents with analytical grade and utilized without further purification. All compounds
were dissolved in CDCl3, or DMSO-d6 and the 1H and 13C NMR spectra were recorded at 400 MHz on a Bruker AV-400
spectrometer or on a JEOL JNM-ECS400 spectrometer. The chemical shifts were reported as δ values in parts per million (ppm)
and coupling constants (J) were given in hertz (Hz). The peak pattern abbreviations are as follows: s, singlet; brs, broad singlet, d,
doublet; q, quadruplet; dd, doublet of doublet; t, triplet; m, multiplet. IR spectra were obtained on a Nicolet FT-IR 8400
spectrometer (KBr disc). Mass spectra were carried out on (UHR-TOF) maXis 4G mass spectrometers. Purification was
performed by using preparative separations in flash column chromatography. Melting points were determined with capillaries
with an YRT-3 microscope apparatus and were uncorrected. Reactions were monitored by thin layer chromatography (TLC) on
2.5 mm Merck silica gel F254 strips.
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4.1.1 General procedure for the synthesis of 5,11,17,23-tetra-tert -butyl-25,27-bis [N-(2-hydroxyethyl) aminocarbonylmethoxyl]
calix[4]arene-26,28-diol ( 3a)
A mixture of calix[4]arene ester 2a (820mg, 1.0 mmol) and ethanolamine (244mg, 4.0mmol) was dissolved in refluxing
ethanol/toluene (50 mL, v/v = 1:1) for 24h until complete by TLC. The solution was extracted with water/chloroform. The
combined chloroform layers were washed with brine solution, dried over MgSO4 and concentrated invacuo. Then, the oil residue
was dissolved in 95% ethanol and the resulting precipitate was collected by filtration, which was purified by column
chromatography (SiO2, 1:3 petroleum ether/ethyl acetate to give product 3a (637 mg, 75%) as a white solid. Mp =244.6-247.9ꢃ,
1H NMR (CDCl3, 400 MHz) δ: 1.01, 1.29 (2s, 36H), 3.42 (d, J = 13.2 Hz, 4H ), 3.59 (q, J = 4.8 Hz, 4H), 3.81 (t, J = 4.8 Hz, 4H),
4.18 (d, J = 13.2 Hz, 4H), 4.58 (s, 4H), 6.89 (s, 4H), 7.10 (s, 4H), 7.60 (brs, 2H), 9.11 (t, J = 5.2 Hz, 2H); 13C NMR (CDCl3, 100
MHz) δ: 30.9, 31.6, 32.0, 33.9, 34.1, 42.4, 61.7, 74.8, 125.6, 126.2, 127.5, 132.3, 143.3, 148.3, 149.3, 149.4, 169.8; IR (KBr) υ:
3385, 3338, 2961, 2868, 1666, 1558, 1486, 1452, 1363, 872 cm-1; MS m/z: HRMS (ESI) Calcd: for C52H70N2NaO8 ([M+Na]+):
873.5024, found: 873.5066.
25, 27-bis-[N-(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[4]arene-26,28-diol (3b): White powder, purified by column
chromatography (SiO2, 1:3 petroleum ether/ethyl acetate), Mpꢂ250ꢃ, yield: 84 %; 1H NMR (DMSO-d6, 400MHz) δ: 3.43 (q, J
= 4.0 Hz, 4H ), 3.47 (d, J = 8.8 Hz, 4H), 3.56 (brs, 4H), 4.23 (d, J = 8.8 Hz, 4H), 4.53 (s, 4H), 4.72 (brs, 2H), 6.62 (t, J = 5.2 Hz,
2H), 6.81 (t, J = 5.2 Hz, 2H), 7.06 (d, J = 4.8 Hz, 4H), 7.17 (d, J = 4.8Hz, 4H), 8.30 (s, 2H), 8.54 (t, J = 3.6 Hz, 2H); 13C NMR
(DMSO-d6, 100 MHz) δ: 31.0, 41.8, 60.2, 60.3, 61.6, 74.7, 119.8, 119.9, 120.1, 126.1, 127.8, 127.9, 128.1, 128.2, 129.1, 129.2,
129.4, 129.5, 129.6, 129.8, 133.9, 134.0, 134.1, 134.3, 134.4, 152.5, 152.6, 152.7, 152.8, 167.4, 168.4; IR (KBr) υ: 3337, 3318,
2925, 1682, 1667, 1545, 1464, 1439 1343, 1250, 764 cm-1; MS m/z: HRMS (ESI) Calcd: for C36H38N2NaO8 ([M+Na]+):
649.2520, found: 649.2548.
5,11,17,23-tetra-tert-butyl-25,26,27,28-tetra[N-(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[4]arene (3c): White powder,
purified by column chromatography (SiO2, 10:1 ethyl acetate/methanol), Mp =195.8-197.1ꢃ, yield: 67 %; 1H NMR (CDCl3, 400
MHz) δ: 0.83 (s, 18H), 1.33, 1.34 (2s, 18H), 3.34 (q, J = 6.8 Hz, 5H ), 3.48-3.57 (m, 5H), 3.66 (q, J = 4.8 Hz, 2H) 3.72-3.81 (m,
5H), 3.86 (t, J = 4.8 Hz, 2H), 4.27 (q, J = 6.4 Hz, 5H), 4.38 (dd, J = 10.0 Hz, 5H), 4.56 (t, J = 16.4 Hz, 3H), 5.71 ( s, 1H), 6.52 (d,

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J = 2.0 Hz, 2H), 6.58 (d, J = 2.0Hz, 2H), 7.10 (s, 2H), 7.19 (s, 2H), 7.64 (t, J = 5.6 Hz, 2H), 8.39 (t, J = 5.6 Hz, 1H); 13C NMR
(CDCl3, 100 MHz) δ: 30.9, 31.0, 31.2, 31.4, 31.6, 31.7, 33.8, 34.0, 34.3, 42.3, 42.4, 61.5, 75.5, 125.6, 126.6, 129.0, 131.1, 131.3,
135.0, 146.8, 147.6, 149.3, 151.0, 169.0, 171.0; IR (KBr) υ: 3381, 2961, 2868, 1659, 1555, 1481, 1194, 1124, 872 cm-1; MS m/z:
HRMS (ESI) Calcd: for C60H84N4NaO12 ([M+Na]+): 1075.5983, found: 1075.6011.
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5,11,17,23-tetra-tert-butyl-25,27-bis[N,N-di(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[4]arene-26,28-diol (3d): White
powder, purified by column chromatography (SiO2, 1:3 petroleum ether/ethyl acetate), Mpꢂ250ꢃ, yield: 81%; 1H NMR
(DMSO-d6, 400 MHz) δ: 1.05, 1.19 (2s, 36H), 3.50 (q, J = 5.2 Hz, 8H), 3.62 (brs, 8H), 4.36 (d, J = 12.8 Hz, 4H), 4.71 (t, J = 4.8
Hz, 4H), 4.85 (s, 4H), 4.91 (t, J = 4.8 Hz, 4H), 6.92-6.95 (m, 4H), 7.01 (s, 4H), 8.27 (brs, 2H); 13C NMR (DMSO-d6, 100 MHz) δ:
31.3, 31.8, 33.9, 34.2, 59.2, 78.9, 79.2, 79.4, 79.6, 125.5, 125.8, 127.7, 133.2, 169.0; IR (KBr): 3393, 3309, 2961, 2869, 1653,
1636, 1486, 1363, 1193, 971 cm-1; MS m/z: HRMS (ESI) Calcd: for C56H78N2NaO10 ([M+Na]+): 961.5554, found: 961.5598.
25,27-bis[N,N-di(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[4]arene-26,28-diol(3e): White powder, purified by column
chromatography (SiO2, 1:1 petroleum ether/ethyl acetate), Mp=225.7- 227.4 ꢃ, yield: 67 %; 1H NMR (DMSO-d6, 400 MHz) δ:
3.35 (d, J = 8.4 Hz, 4H), 3.44-3.48 (m, 8H ), 3.57-3.59 (m, 8H), 4.40 (d, J = 8.4Hz, 4H), 4.74(t, J=3.6 Hz, 2H), 4.89 (s, 4H), 4.93
(t, J = 3.6 Hz, 2H), 6.54(t, J = 5.2 Hz, 2H), 6.74 (t, J = 5.2 Hz, 2H), 6.98 (d, J = 5.2 Hz, 4H), 7.08 (d, J = 4.8 Hz, 4H), 8.41 (s,
2H); 13C NMR (DMSO-d6, 100 MHz) δ: 31.2, 59.0, 59.1, 128.1, 129.0, 129.2, 134.2, 154.2, 168.9; IR (KBr) υ: 3385, 1653, 1635,
1558, 1466, 1437, 1194, 1050, 755 cm-1; MS m/z: HRMS (ESI) Calcd: for C40H46N2NaO10 ([M+Na]+): 737.3045, found:
737.3087.
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5,11,17,23,29,35-hexa-tert-butyl-37,38,39,40,41,42-hexa[N-(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[6]arene(3f):
White powder, purified by column chromatography (SiO2, 6:1 dichloromethane/methanol), Mpꢂ250ꢃ, yield: 68 %; 1H NMR
(DMSO-d6, 400 MHzꢄδ: 1.00 (s, 54H), 3.16 (brs, 12H), 3.40 (brs, 6H), 4.00 (brs, 24H), 4.72 (t, J = 5.2 Hz, 6H), 6.88 (s, 12H),
7.92 (s, 6H); 13C NMR (DMSO-d6, 100 MHz) δ: 29.9, 31.6, 34.3, 60.3, 72.3, 126.2, 132.8, 146.4, 152.8, 168.5; IR (KBr) υ: 3415,
2961, 2868, 1668, 1653, 1363, 1116, 874 cm-1; MS m/z: HRMS (ESI) Calcd: for C90H126N6NaO18 ([M+Na]+): 1601.9025, found:
1601.9038.
37,38,39,40,41,42-hexa[N-(2-hydroxyethyl)aminocarbonylmethoxyl]calix[6]arene(3g): White powder, purified by column
chromatography (SiO2, 10:1 ethyl acetate/methanol), Mpꢂ250ꢃ, yield: 72 %; 1H NMRꢅDMSO-d6, 400 MHzꢄδ: 3.18 (s, 12H),
3.38 (s, 6H), 4.11 (d, J = 22.8 Hz, 24H), 4.73 (s, 6H), 6.81 (s, 12H), 6.93 (s, 6H), 7.95 (s, 6H); 13C NMR (DMSO-d6, 100 MHz) δ:
29.8, 41.6, 56.5, 60.1, 72.2, 125.2, 129.4, 134.0, 154.7, 168.2; IR (KBr) υ: 3420, 3303, 2960, 1662, 1653, 1558, 1480, 1190,
1041, 974 cm-1; MS m/z: HRMS (ESI) Calcd: for C66H78N6NaO18 ([M+Na]+): 1265.5270, found: 1265.5247.
5,11,17,23,29,35-hexa-tert-butyl-37,38,39,40,41,42-hexa[N,N-di(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[6]arene(3h).
White powder, purified by column chromatography (SiO2, 1:2 dichloromethane/methanol), Mpꢂ250ꢃ, yield: 96.82%; 1H
NMR(DMSO-d6, 400 MHz) δ: 0.94, 1.17 (2s, 54H), 3.30-3.53(m, 42H), 3.92 (d, J = 34.8 Hz, 12H), 4.57-4.87 (m, 16H),
6.74-7.10 (m, 12H); 13C NMR (DMSO-d6, 100 MHz) δ: 30.0, 31.7, 34.2, 34.3, 34.4, 58.4, 59.4, 132.9, 146.0, 169.1; IR (KBr) υ:
3385, 2956, 2867, 1653, 1635, 1482, 1189,1049, 874 cm-1; MS m/z: HRMS (ESI) Calcd: for C78H102N6NaO24 ([M+Na]+):
1529.6843, found: 1529.6818.
37,38,39,40,41,42-hexa[N,N-di(2-hydroxyethyl)aminocarbonylmethoxyl]calix[6]arene (3i): White powder, purified by
column chromatography (SiO2, 1:2 dichloromethane/methanol), Mpꢂ250ꢃ, yield: 58 %; 1H NMRꢅDMSO-d6, 400 MHzꢄδ:
3.36-3.43 (m, 24H), 3.44-3.50 (m, 24H), 4.05 (s, 12H), 4.72-4.76 (m, 12H), 4.94 (t, J = 5.2Hz, 6H), 6.76-6.92 (m, 18H); 13C
NMR (DMSO-d6, 100 MHz) δ: 30.0, 59.1, 59.3, 60.2, 61.0, 71.7, 124.6, 129.2, 134.4, 155.7, 168.6; IR (KBr) υ: 3377, 2955,
1657, 1645, 1481, 1194, 1045, 872 cm-1; MS m/z: HRMS (ESI) Calcd: for C78H102N6Na2O24 ([M+2Na]2+): 1889.0497, found:
945.0259.
5,11,17,23,29,35,41,47-octa-tert-butyl-49,50,51,52,53,54,55,56-octa[N-(2-hydroxyethyl)aminocarbonylmethoxyl]calix[8]
arene(3j). purified by column chromatography (SiO2, 6:1 dichloromethane/methanol), White powder: Mpꢂ250ꢃ, yield: 53 %;
1H NMR (DMSO-d6, 400 MHz) δ: 1.00 (s, 72H), 3.15 (brs, 16H ), 3.38 (s, 16H), 4.00 (brs, 24H), 4.71 (brs, 8H), 6.87 (s, 16H),
7.91 (s, 8H); 13C NMR (DMSO-d6, 100 MHz) δ: 31.5, 34.3, 41.6, 60.1, 72.0, 132.7, 146.3, 152.7, 168.4; IR (KBr) υ: 3413, 2960,

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2865, 1667, 1653, 1558, 1539, 1476, 1363, 1189, 668 cm-1; MS m/z: HRMS (ESI) Calcd: for C120H168N8NaO24 ([M+Na]+):
2128.2069, found: 2128.2132.
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49,50,51,52,53,54,55,56-octa[N-(2-hydroxyethyl)aminocarbonylmethoxyl]calix[8]arene(3k):White powder, purified by
column chromatography (SiO2, 7:1 ethyl acetate/methanol), Mpꢂ250ꢃ, yield: 60%; 1H NMR (DMSO-d6, 400 MHz) δ:
3.20-3.40 (m,32H), 4.00-4.25 (m, 32H), 4.71 (s, 8H), 6.66-6.93 (m, 24H), 7.86-8.39 (m, 8H); 13C NMR (DMSO-d6, 100 MHz) δ:
29.7, 29.9, 30.4, 41.6, 60.1, 60.1, 71.9, 72.2, 72.3, 124.9, 125.1, 127.4, 128.4, 128.8, 129.4, 129.5, 133.2, 134.3, 153.5, 154.4,
154.6, 154.9, 168.2, 168.2, 168.4; IR (KBr) υ: 3409, 2960, 1663, 1637, 1558, 1480, 1362, 1189, 874 cm-1. MS m/z: HRMS (ESI)
Calcd: for C88H104N8Na2O24 ([M+2Na]2+): 1702.6959, found: 851.3514.
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5,11,17,23,29,35,41,47-octa-tert-butyl-49,50,51,52,53,54,55,56-octa[N,N-di(2-hydroxyethyl)aminocarbonylmethoxyl]calix[
8]arene (3l): White powder, purified by column chromatography (SiO2, 1:3 dichloromethane/methanol), Mpꢂ250ꢃ, yield: 25 %;
1H NMR (DMSO-d6, 400 MHz) δ: 1.09, 1.17 (2s, 72H), 3.31-3.54 (m, 40H), 3.92 (d, J =3.9Hz, 16H), 4.79 (d, J =55.6Hz, 16H),
6.82-7.03 (m, 16H); 13C NMR (DMSO-d6, 100 MHz) δ: 30.7, 31.7, 34.2, 58.0 59.6, 60.6, 71.8, 126.2, 133.3, 146.3, 150.9, 153.1,
169.4; IR (KBr) υ: 3411, 2961, 2869, 1648, 1633, 1482, 1363, 1202, 874 cm-1; MS m/z: HRMS (ESI) Calcd: for
C136H200N8Na2O32 ([M+2Na]2+): 2503.4064, found: 1251.7051.
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49,50,51,52,53,54,55,56-octa[N,N-di(2-hydroxyethyl)aminocarbonylmethoxyl]-calix[8]arene(3m).White powder, purified by
column chromatography (SiO2, 1:1 dichloromethane/methanol), Mpꢂ250ꢃ, yield: 37%; 1H NMR (DMSO-d6, 400 MHz) δ:
3.43-3.50 (m, 64H), 3.93-4.03 (m, 16H), 4.75 (brs, 16H), 6.64-7.07 (m, 24H); 13C NMR (DMSO-d6, 100 MHz) δ: 30.2, 57.9, 59.4,
71.7, 73.4, 124.1, 124.7, 129.3, 134.1, 134.4, 134.7, 155.9, 156.4, 169.0, 173.9; IR (KBr) υ: 3331, 2949, 1632, 1362, 1192, 1045,
868 cm-1. MS m/z: HRMS (ESI) Calcd: for C104H136 N8Na2O32 ([M+2Na]2+): 2054.9056, found: 1027.4544.
4.2.Crystallography
The single crystal of 3a was obtained in ethanol and the single crystal structure was determined on Bruker Smart Apex
X-single crystal diffractometer. The data were processed with HKL2000. The structure was solved by direct methods of
SHELX86 and subsequent Fourier-difference synthesis and refined by full-matrix least-squares on F2 with SHELXS-9737[45].
No absorption correction was done. All non-hydrogen atoms were refined with anisotropic displacement parameters.
4.3.Cellular Assay
Cell culture: A549 cells (Human lung cancer cells), SKOV3 cells (Human ovarian cancer), SW1990 cells (Human pancreatic
cancer), Hela cells (Human cervical cancer), Raji cells (Human Burkitt's shower Pakistan tumor), MDA-MB-231 cells (Human
breast cancer) were kindly provided by WeiFang Caleb Pharmaceuticals, Inc. A549 cells were cultured in culture Ham's F12K
medium containing 10% (v/v) fetal bovine serum, 2 mM L-glutamine and 1.5g/L sodium bicarbonate. SKOV3 cells were
cultured on Cell culture flask using 10 % fetal bovine serum, 1.5 mM L-glutamine and 2.2 g/L sodium bicarbonate in McCoy's
5a medium. SW1990 cells and MDA-MB-231 cells were cultured in Leibovitz's L-15 medium with 10% (v/v) fetal bovine serum
and 2 mM L-glutamine. Hela cells were cultured on Cell culture flask using 2 mM L-glutamine adjusted to contain 1.5 g/L
sodium bicarbonate, 4.5 g/L glucose, 10 mM HEPES and 1.0 mM sodium pyruvate in RPMI 1640 medium supplemented with
0.5 mg/ml G418and 10% fetal bovine serum. Raji cells were cultured in RPMI 1640 medium with 1.5 g/L sodium bicarbonate,
2.5 g/L glucose and 0.11 g/L sodium pyruvate, supplemented with 10 % fetal bovine serum.
4.4. Cytotoxicity assay
In this experiment, all the adherent cells used SRB method. All suspension cells used MTT method.
SRB assay: All cells were cultured in culture medium containing 10 % fetal calf serum, and been in the logarithmic growth
phase. All cell types were seeded in 96-well culture plate at a concentration of 1-5×104 cells per well at 37ꢃ in 5% CO2
incubator for 24 h. The cells were then exposed to 10 drug concentrations of calixarene derivatives for 72 h with each
concentration located three wells. Then the cells were fixed with trichloroacetic acid (TCA). After washing, SRB working
solution was added to the cells to clean them. SRB combined with protein was dissolved in Tris base. OD values were measured
for each well with SPECTRA max 190 Cell microplate reader under 565 nm wavelength.
MTT assay: All cells were cultured in culture medium containing 10% fetal calf serum, and been in the logarithmic growth

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phase. All cell types were seeded in 96-well culture platet at a concentration of 1-5 ×104 cells per well at 37ꢃ in 5% CO2
incubator for 24 h. The cells were then exposed to 10 drug concentrations of calixarene derivatives for 72 h with each
concentration located three wells. Then MTT solution was added to each well, the cells were placed in incubator for 4 h. Each
well was removed 100 ul solution, added 100ul of three Joint fluid (HCl + isopropanol + SDS), and placed in the incubator
overnight. Next day, the cells were labeled in SPECTRA max 190Cell microplate reader, and OD values were measured under
550 nm wavelength.
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4.5.Flow cytometry for cell cycle analysis
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fter incubation for 48 h at 37ꢃ in an atmosphere of 5 % CO2, SKOV3 cells were detached by trypsinization, collected,
washed three times with PBS and fixed in ice-cold ethanol for overnight. The cells were washed twice with PBS and resuspended
in working solution (10 ꢀg/mL propidiumiodide (PI), 10 ꢀg /mL RNase) and incubated at 37ꢃ in the dark for 30 min. Cell cycle
analysis was performed by analysis of PI staining using a FACSC alibur flow cytometer.
4.6. Immunofluorescence assay
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SKOV3 cells were grown in 24-well plates, treated with 3a. After 24 h, cells were fixed with 4 % paraformaldehyde for 20
min and perforated with 0.3 % Triton X-100 for 10 min. The cells were blocked with sheep serum followed by incubation with
P53/Bax/Bcl-2/Caspase-3 antibody at 4ꢃovernight. The cells were incubated with the relevant secondary antibody conjugated
with goat antimouse or antirabbit antibody for 1h, and counterstained with Hoechst 33342 for 15 min. Finally, Cells were
mounted on glass slide and images were examined and captured under a fluorescence microscope (Olympus, BX43F).
Acknowledgement
This work was financially supported by the National Natural Science Foundation of China (No. 81202490), Natural Science
Foundation of Jiangsu Province (No. BK20130216), the Innovative Practice Training Program for Students of Jiangsu Higher
Education Institutions (No. KYLX-1335) and the Foundation of Jiangsu Key Laboratory of New Drug Research and Clinical
Pharmacy(No. Z-XY201404). We also thank Dr. Y. J. Song for the guidance of immunofluorescence staining analysis and
Shanghai Huawei Pharmaceutical Com. Ltd. for the assistance of cytotoxicity experiments.
Conflict of Interest
The authors declare no conflict of interest.
Author contributions statement
L. An, L. L. Han and X. N. Peng synthesized all the compounds; J. Sun determined and analyzed the single crystal. Y.S.Xue
optimized the Molecular structure. L. L. Han performed the flow cytometry for cell-cycle analysis and Immunofluorescence
staining. All authors extensively discussed the results; the paper was written by L. An, C. G. Yan, with the help of Y. G. Zheng,
X. K. Gu. All authors reviewed the manuscript.
Additional information
The detailed spectroscopic data including crystallographic data (CIF) of compounds are available. Single crystal data for
compounds 3a has been deposited in the Cambridge Crystallographic Data Center and assigned to the following deposition
number CCDC 1450999.
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Figure1. Structural representations of calix[4]arene polyhydroxyamine derivatives 3a–3d
Figure 2. Structural optimization design of calix[n]arene
Scheme1. Synthesis of calix[n]arene polyhydroxyamine derivatives 3a–3m
Reagents and conditions: (i) ClCH2COOC2H5, K2CO3, acetone, ultrasonic irradiation; (ii) NH2CH2CH2OH, ethanol/toluene,reflux; (iii) NH(CH2CH2OH)2,

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Figure 3. Single crystal structure of 3a (hydrogen atoms on the skeletons were omitted for clarity)
Table 1. Crystal data for Compound 3a
Phase
Empirical formula
Formula weight
Temperature
3a
C52H72N2O9
869.12
296(2)
Wavelength/nm
Crystal system
0.71073
Triclinic
space group
P -1
a (Å)
9.937(4)
b (Å)
12.228(4)
c (Å)
22.273(6)
α(°)
87.276(10)
β(°)
82.963(13)
γ(°)
72.794(13)
Volume(Å3)
2565.7(14)
Z
2
F(000)
940
0.36 x 0.34 x 0.28
1.125
Crystal size (mm)
Calculated density (g.cm-3)
Absorption coefficient (mm-1)
θ range (°)
0.076
0.92 to 25.00
Limiting indices
Reflections collected / unique
Completeness to theta
Max. and min. transmission
Refinement method
Data / restraints / parameters
Goodness-of-fit on F^2
Final R indices [I﹥2sigma(I)]
R indices (all data)
Largest diff. peak and hole(e.Å-3)
-11﹤＝h﹤＝11, -14﹤＝k﹤＝14, -26﹤＝l﹤＝26
29477 / 8702 [R(int.) = 0.1267]
96.3 %
0.979 and 0.973
Full-matrix least-squares on F^2
8702 / 72 / 601
1.038
R1 = 0.1203, wR2 = 0.2627
R1 = 0.2326, wR2 = 0.3168
0.589 and -0.456

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Table 2. Single concentration inhibition of calixarenes 3a–3m on cancer cell viability
Single concentration inhibition (%)a
Compound
A549
89
13
93
16
7
SKOV3
97
SW1990
Hela
100
Raji
100
29
100
77
34
38
18
14
6
MDA-MB-231
95
10
99
59
4
99
57
99
95
22
12
4
3a
3b
3c
3d
3e
3f
86
46
100
95
38
40
6
95
42
34
7
24
4
7
10
3
3g
3h
3i
0
26
26
0
0
0
5
19
9
22
12
27
11
13
8
26
5
23
32
10
18
24
7
3j
2
1
4
3k
3l
0
25
5
11
11
6
25
2
3m
a
the concentration of the calixarenes 3a–3m was 10 ꢀM
Table3. IC50 values (ꢀM) of calixarenes 3a–3d on various cancer cells
IC50 (ꢀΜ)
Cancer cells
3a
4.0
2.8
4.3
2.7
5.0
5.3
3b
--
3c
3d
--
A549
SKOV3
SW1990
Hela
3.7
5.1
3.3
7.1
4.7
3.4
2.3
--
--
11.3
8.1
11.2
5.2
--
Raji
--
MDA-MB231
1.6
“--”means no detected

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A
B
Figure 5. The amount of SKOV3 cells each cell cycle phase (G0/G1 phase, S phase and G2/M) after treatment with specified concentrations of 3a (A) and 3b
(B) for 48 h was determined by propidium iodide staining followed by flow cytometry.

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Figure 4. Cytotoxicity effects of calix[4]arenes 3a–3d. Cell viability of A549, SKOV3, SW1990, Hela, Raji as well as MDA-MB231cells was measured in the
presence of various concentrations of calixarenes.

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E
F
G
Fig.6 Effects of apoptotic related proteins expression on the apoptosis of treated and untreated SKOV3 cells. Caspase-3 (A), P53 (C), Bcl-2 (E), Bax (F)
protein antibody and fluorescein-conjugated second antibody were used to visualize corresponding protein, and Hoechst 33342 was used to stain cell nuclei
(blue).The levels of Caspase-3 (B), P53 (D) are increased in SKOV3 cells, the level of Bcl-2 (E) is decreased with Bax protein no changes
.

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Highlights
Novel calix[n]arene (n = 4, 6, 8) polyhydroxyamine derivatives (3a–3m) were designed
and afforded, including a single crystal of 3a.
Cell viability assay were evaluated in the six cell lines ( A549, SKOV3, SW1990, Hela,
Raji and MDA-MB-231).
Calix[4]arenes 3a–3d indicated good anti-tumor activity with IC50 values ranging from
1.6 μM to 11.3 μM.
Cell cycle analysis and Immunofluorescent staining indicated that 3a can be further
studied as a potential anticancer agent.