Synthesis and anticancer properties of phenyl benzoate derivatives possessing a terminal hydroxyl group

Article abstract of DOI:10.1039/c3tb21736a

To assess the cytotoxic effects on A549 human lung cancer cells, we investigated a liquid-crystalline compound possessing a terminal hydroxyl group at concentrations of 0.1-20 μM. The compound, 4-butylphenyl 4-(6-hydroxyhexyloxy)benzoate (2), showed marked cell-growth inhibition at concentrations higher than 5 μM. Cell accumulation in the Sub-G1 phase indicating apoptosis was observed only at the highest concentration. Dynamic light scattering measurements show that the molecules form a spherical nanoparticle with a diameter of 130-170 nm at concentrations of 5-20 μM. We prepared the corresponding dimeric compounds and investigated their anticancer activity. The 1,2-benzene derivative, 1,2-bis[4-(6-hydroxyhexyloxy)benzoyloxy] benzene (4), exhibited cell-growth inhibition without affecting the cell cycle. However, the 1,3-benzene derivative, 1,3-bis[4-(6-hydroxyhexyloxy)benzoyloxy] benzene (5), was found to induce marked cell accumulation in the Sub-G1 phase. Furthermore, we assessed the cytotoxic effects of compounds 2, 4 and 5 on SW480 colon cancer cells and THP1 leukemic cells, as well as on WI-38 normal fibroblast cells. Both compounds 2 and 5 suppressed the growth of the solid cancer cells (A549 and SW480) more strongly compared with that of the hematological cancer cells (THP1). Unexpectedly, they also exhibited a strong cytotoxicity against the normal cells. We discuss the structure-property relationship in the anticancer activity of the mesogenic compounds.

Full text of DOI:10.1039/c3tb21736a

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Materials Chemistry B
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Synthesis and anticancer properties of phenyl
benzoate derivatives possessing a terminal
hydroxyl group
Cite this: J. Mater. Chem. B, 2014, 2,
1335
Yukako Fukushi,a Hironori Yoshino,b Junya Ishikawa,b Masanobu Sagisaka,a
b
a
*
Ikuo Kashiwakura and Atsushi Yoshizawa
To assess the cytotoxic effects on A549 human lung cancer cells, we investigated a liquid-crystalline
compound possessing a terminal hydroxyl group at concentrations of 0.1–20 mM. The compound, 4-
butylphenyl 4-(6-hydroxyhexyloxy)benzoate (2), showed marked cell-growth inhibition at concentrations
higher than 5 mM. Cell accumulation in the Sub-G1 phase indicating apoptosis was observed only at the
highest concentration. Dynamic light scattering measurements show that the molecules form a spherical
nanoparticle with a diameter of 130–170 nm at concentrations of 5–20 mM. We prepared the
corresponding dimeric compounds and investigated their anticancer activity. The 1,2-benzene derivative,
1,2-bis[4-(6-hydroxyhexyloxy)benzoyloxy]benzene (4), exhibited cell-growth inhibition without affecting
the cell cycle. However, the 1,3-benzene derivative, 1,3-bis[4-(6-hydroxyhexyloxy)benzoyloxy]benzene
(5), was found to induce marked cell accumulation in the Sub-G1 phase. Furthermore, we assessed the
cytotoxic effects of compounds 2, 4 and 5 on SW480 colon cancer cells and THP1 leukemic cells, as
well as on WI-38 normal fibroblast cells. Both compounds 2 and 5 suppressed the growth of the solid
cancer cells (A549 and SW480) more strongly compared with that of the hematological cancer cells
(THP1). Unexpectedly, they also exhibited a strong cytotoxicity against the normal cells. We discuss the
structure–property relationship in the anticancer activity of the mesogenic compounds.
Received 6th December 2013
Accepted 19th December 2013
DOI: 10.1039/c3tb21736a
www.rsc.org/MaterialsB
investigated. Many strategies to entrap anticancer agents in
different nanocarriers with a variety of architectures including
polymer–drug conjugates,10 micelles,11 nanogels,12 liposomes,13
Introduction
Numerous studies have been conducted over the past few
decades to develop effective anticancer agents either by
synthesis or from natural products. Most chemotherapeutic
drugs are classiable as alkylating agents, antimetabolites,
anthracyclines, plant alkaloids, topoisomerase inhibitors,
monoclonal antibodies, molecular target drugs, or as other
anticancer agents.1–7 However, the development of anticancer
agents against solid cancer cells such as lung cancer has pre-
sented some difficult problems. To reach cancer cells in optimal
quantity, a therapeutic agent must pass through an imperfect
blood vasculature to the cancer, cross the vessel walls into the
interstitium, and penetrate multiple layers of solid cancer
cells.8,9 Furthermore, most anticancer drugs lack any intrinsic
anticancer selectivity, thereby causing severe side effects
because of the massive destruction of normal tissues. To over-
come these hurdles, many drug carriers designed to deliver
cytostatic agents exclusively at the tumor site have been
dendrimers,14 and nanospheres15,16 have been developed.
Nanocarriers can be taken up by cells via endocytosis. The
suitable size of such a nanocarrier for a tumor targeted by the
enhanced permeability and retention (EPR) effect is known to
be 100–200 nm.17 In addition to these carriers, prodrug systems
were designed.18 The use of non-toxic prodrugs that can be
activated by an enzyme that is naturally overexpressed in the
tumor microenvironment has emerged as a promising strategy
to enhance the selectivity of chemotherapy.
Biological systems have links with liquid crystallinity. Cell
membranes are lamellar bilayer mesophases of phospholipids,
glycolipids and cholesterol. Recently, we reported that some
liquid-crystalline compounds induce cell-growth suppression in
A549 lung cancer cells. Moreover, we described a relationship
between anticancer activity and liquid-crystallinity.19–21 We
surmise that a supramolecular assembly consisting of liquid-
crystalline molecules possessing a biologically active site is
effective not only for cell permeability but also for cytotoxicity
against tumor cells. On the other hand, various polyhydroxy
compounds such as sugar derivatives and avanol derivatives
are known to show anticancer activity.22,23 Conventional cyano-
biphenyl derivatives possessing a primary alcohol showed
aDepartment of Frontier Materials Chemistry, Graduate School of Science and
Technology, Hirosaki University, 3 Bunkyo-cho, Hirosaki, 036-8561, Japan. E-mail:
ayoshiza@cc.hirosaki-u.ac.jp
bDepartment of Radiological Life Sciences, Hirosaki University Graduate School of
Health Sciences, 66-1 Hon-cho, Hirosaki, Japan
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inhibition activity against A549 human lung cancer cells and TMS) dH/ppm: 8.14 (d, 2H, Ar-H, J ¼ 9.2 Hz), 7.22 (d, 2H, Ar-H, J
did not exhibit suppressive effects on WI-38 normal broblast ¼ 8.0 Hz), 7.10 (d, 2H, Ar-H, J ¼ 8.6 Hz), 6.97 (d, 2H, Ar-H, J ¼ 9.2
cells.19 These lead to a design concept that molecular assembly Hz), 4.06 (t, 2H, –OCH2–, J ¼ 6.6 Hz), 3.68 (t, 2H, –CH2–OH, J ¼
of mesogenic compounds possessing a terminal hydroxyl group 6.6 Hz), 2.63 (t, 2H, Ar–CH2–, J ¼ 7.7 Hz), 1.85 (quin, 2H,
can induce aggregation of terminal alcohols exhibiting anti- –OCH2CH2–, J ¼ 7.0 Hz), 1.66–1.45 (m, 8H, aliphatic –CH2–),
cancer activities without severe side effects. Here, we investi- 1.38 (sext, 2H, –CH2CH3, J ¼ 7.5 Hz), 1.28 (br s, 1H, –OH), 0.94 (t,
gated anticancer activities of some phenyl benzoate derivatives 3H, –CH3, J ¼ 7.2 Hz). n/cmꢂ1 (KBr): 3360 (O–H str.), 2933, 2857
possessing a terminal hydroxyl group against A549 human lung (C–H str.), 1725 (C]O str.), 1607, 1514 (C]C str.). Elemental
cancer cells. Furthermore we designed the corresponding analysis. Calculated for C33H40O8: C, 74.56; H, 8.16. Found: C,
dimeric compounds, i.e., 1,2- and 1,3-benzene derivatives with 74.41; H, 8.05%.
different conguration of their terminal hydroxyl groups and
evaluated their anticancer activities.
1,2-Bis[4-(6-hydroxyhexyloxy)benzoyloxy]benzene (4). To a
solution of catechol (110 mg, 1.0 mmol) in dichloromethane
(10 ml), 4-(6-hydroxyhexyloxy)benzoic acid (480 mg, 2.0 mmol),
N,N0-dicyclohexylcarbodiimide (500 mg, 2.4 mmol), and 4-(N,N0-
dimethylamino)pyridine (240 mg, 4.0 mmol) were added. The
resulting solution was stirred at room temperature for 12 h.
Then, the precipitated materials were removed by ltration.
Aer removal of the solvent by evaporation, the residue was
puried by column chromatography using a dichloromethane–
ethyl acetate (1 : 1) mixture as the eluent. Recrystallization from
ethanol gave the desired product. Yield: 290 mg (52%). 1H NMR
(500 MHz, solvent CDCl3, standard TMS) dH/ppm: 8.00 (d, 4H,
Ar-H, J ¼ 9.0 Hz), 7.37–7.31 (m, 4H, Ar-H), 6.83 (d, 4H, Ar-H,
J ¼ 8.9 Hz), 3.98 (t, 4H, –OCH2–, J ¼ 6.5 Hz), 3.66 (t, 4H, –CH2–
OH, J ¼ 6.5 Hz), 1.81 (quin, 4H, –OCH2CH2–, J ¼ 7.0 Hz), 1.61
(quin, 4H, –CH2CH2–OH, J ¼ 7.0 Hz), 1.53–1.41 (m, 8H,
aliphatic –CH2–), 1.37 (br s, 2H, –OH). n/cmꢂ1 (KBr): 3317 (O–H
str.), 2937, 2860 (C–H str.), 1737 (C]O str.), 1605, 1492 (C]C
Experimental
Characterization of materials
The purication of each nal product was conducted using
column chromatography over silica gel (63–210 nm; Kanto
Chemical Co. Inc.) using a dichloromethane–ethyl acetate
mixture as the eluent, followed by recrystallization from
ethanol. The purity was conrmed using elemental analysis (EA
1110; CE Instruments Ltd.). The structures of the nal products
were elucidated using infrared (IR) spectroscopy (Varian 670-IR;
Varian Inc.) and proton nuclear magnetic resonance (1H NMR)
spectroscopy (JNM-ECA 500; JEOL).
Preparation of materials
4-Butylphenyl 4-(6-hydroxyhexyloxy)benzoate (2). Ethyl 4- str.). Elemental analysis. Calculated for C33H40O8: C, 70.19; H,
hydroxybenzoate (3.0 g, 18 mmol) and 6-bromo-1-hexanol were 7.14. Found: C, 70.32; H, 6.80%.
dissolved in cyclohexanone (15 ml). Potassium carbonate (5.0 g,
1,3-Bis[4-(6-hydroxyhexyloxy)benzoyloxy]benzene (5). To a
36 mmol) was then added. The resulting mixture was stirred at solution of resorcinol (110 mg, 1.0 mmol) in dichloromethane
110 ꢀC for 10 h. The resulting mixture was ltered and the (10 ml), 4-(6-hydroxyhexyloxy)benzoic acid (480 mg, 2.0 mmol),
solvent was removed by evaporation under reduced pressure. N,N0-dicyclohexylcarbodiimide (500 mg, 2.4 mmol), and 4-(N,N0-
The product was puried by column chromatography using a dimethylamino)pyridine (240 mg, 4.0 mmol) were added. The
dichloromethane–ethyl acetate (10 : 1) mixture as the eluent. resulting solution was stirred at room temperature for 6 h.
Recrystallization from hexane gave ethyl 4-(6-hydroxyhexyloxy)- Then, the precipitated materials were removed by ltration.
benzoate. Yield: 3.4 g (71%).
Aer removal of the solvent by evaporation, the residue was
Then, ethyl 4-(6-hydroxyhexyloxy)benzoate (530 mg, 2.0 mmol) puried by column chromatography using a dichloromethane–
was added to a solution of KOH (340 mg, 6.0 mmol) in ethanol ethyl acetate (1 : 1) mixture as the eluent. Recrystallization from
(95%, 20 ml). The resulting mixture was stirred under reux for 4 ethanol gave the desired product. Yield: 350 mg (63%). 1H NMR
h. Next, water (40 ml) was added to the mixture. The solution was (500 MHz, solvent CDCl3, standard TMS) dH/ppm: 8.13 (d, 4H,
acidied with HCl (concentrated, 4.0 ml). The aqueous phase was Ar-H, J ¼ 8.7 Hz), 7.45 (t, 1H, Ar-H, J ¼ 8.2 Hz), 7.16–7.13 (m, 3H,
extracted with dichloromethane (5 ꢁ 20 ml). The organic extracts Ar-H), 6.96 (d, 4H, Ar-H, J ¼ 8.8 Hz), 4.05 (t, 4H, –OCH2–, J ¼ 6.5
were combined, dried over Na2SO4, ltered and evaporated. 4-(6- Hz), 3.68 (q, 4H, –CH2–OH, J ¼ 6.2 Hz), 1.85 (quin, 4H,
Hydroxyhexyloxy)benzoic acid was obtained. Yield: 0.34 g (71%).
–OCH2CH2–, J ¼ 7.0 Hz), 1.63 (quin, 4H, – CH2CH2–OH, J ¼ 7.0
4-(6-Hydroxyhexyloxy)benzoic acid (242 mg, 1.0 mmol), N,N0- Hz), 1.55–1.44 (m, 8H, aliphatic –CH2–), 1.26–1.23 (br m, 2H,
dicyclohexylcarbodiimide (500 mg, 2.4 mmol) and 4-butylphe- –OH). n/cmꢂ1 (KBr): 3378 (O–H str.), 2934, 2859 (C–H str.), 1731
nol (154 mg, 1.0 mmol) were added to dichloromethane (12 ml), (C]O str.), 1606, 1512 (C]C str.). Elemental analysis. Calcu-
and then 4-(N,N0-dimethylamino)pyridine (24 mg, 0.2 mmol) lated for C33H40O8: C, 70.19; H, 7.14. Found: C, 70.21; H, 6.87%.
was added. The resulting solution was stirred at room temper-
1,4-Bis-[4-(6-hydroxyhexyloxy)benzoyloxy]benzene (6). To a
ature for 12 h. Then, the precipitated materials were removed by solution of hydroquinone (94 mg, 0.9 mmol) in dichloro-
ltration. Aer removal of the solvent by evaporation, the methane (10 ml), 4-(6-hydroxyhexyloxy)benzoic acid (430 mg,
residue was puried by column chromatography using a 1.8 mmol), N,N0-dicyclohexylcarbodiimide (370 mg, 1.8 mmol),
dichloromethane–ethyl acetate (10 : 1) mixture as the eluent. and 4-(N,N0-dimethylamino)pyridine (22 mg, 0.18 mmol) were
Recrystallization from ethanol gave the desired product. Yield: added. The resulting solution was stirred at room temperature
150 mg (41%). 1H NMR (500 MHz, solvent CDCl3, standard for 10 h. Then, the precipitated materials were removed by
1336 | J. Mater. Chem. B, 2014, 2, 1335–1343
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Table 1 Cell lines, cell type and culture medium
ltration. Aer removal of the solvent by evaporation, the
residue was puried by column chromatography using a
dichloromethane–ethyl acetate (1 : 1) mixture as the eluent.
Recrystallization from an ethanol–hexane (1 : 1) mixture gave
Cell line
Cell type
Culture mediuma
A549
Lung
DMEM
1
the desired product. Yield: 126 mg (25%). H NMR (500 MHz,
SW480
THP 1
WI-38
Colon
Leukemia
Fibroblast
RPMI1640
RPMI1640
MEME
solvent CDCl3, standard TMS) dH/ppm: 8.15 (d, 4H, Ar-H, J ¼ 9.2
Hz), 7.26 (s, 4H, Ar-H), 6.98 (d, 4H, Ar-H, J ¼ 9.1 Hz), 4.06 (t, 4H,
–OCH2–, J ¼ 7.6 Hz), 3.68 (q, 4H, –CH2–OH, J ¼ 6.1 Hz), 1.85
(quin, 4H, –OCH2CH2–, J ¼ 6.9 Hz), 1.63 (quin, 4H, –CH2CH2–
OH, J ¼ 7.0 Hz), 1.54–1.45 (m, 8H, aliphatic –CH2–), 1.24 (t, 2H,
–OH, J ¼ 5.5 Hz). n/cmꢂ1 (KBr): 3402 (O–H str.), 2937, 2857 (C–H
str.), 1730 (C]O str.), 1608, 1511 (C]C str.). Elemental anal-
ysis. Calculated for C33H40O8: C, 70.19; H, 7.14. Found: C, 70.26;
H, 6.85%.
a
DMEM: Dulbecco's MEM (Gibco®, Invitrogen Corp., California, USA),
RPMI: Roswell Park Memorial Institute (Gibco®, Invitrogen Corp.,
California, USA), MEME: Minimum essential medium eagle (Sigma-
Aldrich, Japan).
A549, SW480 and WI-38 cells were refed with fresh medium
supplemented with each compound. Aer culturing (A549: 96 h,
SW480: 96 h, WI-38: 168 h), cells attached to the plates were
released by trypsinization, and cell count was conrmed by the
trypan blue dye exclusion test. However, since some SW480 cells
were oating in solvent (DMSO)-treated control, not only cells
attached to the plates but also oating cells were harvested.
With respect to THP1, each compound was added to the culture
immediately aer seeding the cells. Aer 72 h culturing, THP1
cells were harvested, and cell count was performed. Cell viability
was expressed as a percentage relative to solvent (DMSO)-
treated control incubations.
Liquid-crystalline and physical properties
The initial assignments and corresponding transition tempera-
tures for the nal product were determined by thermal optical
microscopy using a Nikon Optiphoto-pol polarizing microscope
equipped with a Mettler FP82 hot stage and an FP80 contꢂro1l
processor. The heating and cooling rates were 5 ꢀC min
.
Transition temperatures were investigated by differential scan-
ning calorimetry (DSC) using a Seiko Instruments Inc. DSC6200.
The materials were studied at a scanning rate of 5 ꢀC minꢂ1 aer
being encapsulated in aluminum pans. The lyotropic liquid
crystallinity at 37 ꢀC was determined by thermal optical
microscopy using a polarizing microscope (BX-51, Olympus)
equipped with a thermal stage (TS62, INSTEC), and a tempera-
ture controller (STC200, INSTEC). Each compound was dissolved
in DMSO at a concentration of 10 mM. The DMSO solution was
added to a suitable amount of water. Dynamic light scattering
measurements were performed on an Otsuka Electronics FDLS-
3000L equipped with a He–Ne laser beam at a wavelength of
632.8 nm at 37 ꢀC. The scattering angle was xed at 90ꢀ.
Cell cycle analysis
A549 cells (3 ꢁ 103 cells per ml) were placed in a 60 mm dish
(IWAKI, Tokyo, Japan) at 37 ꢀC in a humidied atmosphere with
5% CO2. Aer 24 h incubation, cells were refed with fresh
medium supplemented with each compound. Aer 24 and 48 h
of treatment, cells were washed with phosphate-buffered saline
(PBS). Then, cells attached to the plates were released by trypsi-
nization, and cell count was conrmed by the trypan blue dye
exclusion test. The harvested A549 cells (3 ꢁ 105 cells per ml)
were washed with PBS and xed with 70% ethanol for more than
24 h. The xed cells were washed with PBS and then treated with
RNase (200 mg mlꢂ1) at 37 ꢀC for 30 min. Aer treatment, the cells
were washed with PBS, and stained with 25 mg mlꢂ1 propidium
iodide (Sigma, Tokyo, Japan, PI) for 30 min at room temperature
in the dark. The cell cycle distribution was analyzed using a
Cytomics FC500 (Beckman-Coulter, Fullerton, CA, USA).
Cell line
To evaluate the anticancer activity of some liquid-crystalline
compounds, an A549 human lung cancer cell line, a THP1
human leukemic cancer cell line and an SW480 human colon
cancer cell line were selected. To investigate the toxicity on
normal cells, we used a WI-38 normal broblast cell line. A549,
THP1 and WI-38 cell line was purchased from the RIKEN Bio-
Resource Center (Tsukuba, Japan), and SW480 cell lines were
purchased from the American Type Culture Collection (Rock-
ville, MD, USA). Briey, each cell line was cultured in appro-
priate liquid culture medium including 1% penicillin/
streptomycin and 10% heat-inactivated fetal bovine serum
(Bioserum, UBC, Japan) as shown in Table 1.
Statistical analysis
A test to reject outliers was conducted for results obtained using
the cell proliferation assay. Outliers were dismissed by a 5%
level. Signicant differences between the control and the
experimental groups were determined using Mann–Whitney's
U-test.
Cell growth inhibition assay
Results and discussion
Synthesis and biological activities of phenyl benzoate
Each evaluated compound was dissolved in dimethyl sulfoxide
(Wako, Osaka, Japan, DMSO) at a concentration of 10 mM. Each
cell line (A549: 4 ꢁ 103 cells per ml, THP1: 5 ꢁ 104 cells per ml,
derivatives
SW480: 4 ꢁ 103 cells per ml, WI-38: 4 ꢁ 103 cells per ml) was We prepared phenyl benzoate derivatives 1–3 and evaluated
placed at 1 or 0.5 ml per well in 24-well plates at 37 ꢀC in a their cytotoxicity against A549 human lung cancer cells. Their
humidied atmosphere with 5% CO2. Aer 24 h incubation, molecular structures are shown in Fig. 1. Compounds 1 and 2
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were prepared by a synthetic method as depicted in Scheme 1.
Both compounds were puried by column chromatography and
then by recrystallization from ethanol. Compound 324 was
prepared using a method similar to that described in Scheme 1.
The cell viability of A549 cells was estimated by treatment
with each compound at 10 mM for 96 h. The cell-growth inhi-
bition ratio was 0% for compound 1, 99% for compound 2 and
20% for compound 3, as presented in Fig. 2. Compound 2
showed marked inhibition of the cell proliferation compared to
the other compounds, indicating that both a phenyl benzoate
unit and a terminal hydroxyl group contribute to the cell inhi-
bition. We observed the dose-dependent cytotoxicities of
compound 2 on A549 lung cancer cells. The results are depicted
in Fig. 3. The cell viability decreases with increasing concen-
trations of compound 2. The 50% inhibitory concentration
(IC50) for 96 h was 4.7 mM.
One characteristic of cancer cells is their uncontrolled
growth by mutation or deregulation of cell cycle checkpoints. To
clarify the origin of cell suppression, we investigated the inhi-
bition effects of compound 2 on the cell cycle prole in A549 cell
proliferation. The A549 lung cancer cells were treated with
compound 2 (10 mM and 20 mM). The cell cycle proles were
Fig. 1 Molecular structures of compounds 1–3.
Fig. 2 Effects of compounds 1–3 on human lung carcinoma A549 cell
growth at a concentration of 10 mM for 96 h. *p < 0.05 by Mann–
Whitney's U-test.
Fig. 3 Dose-dependent cytotoxicities of compound 2 on A549 cells
Scheme 1 Synthesis of phenyl benzoate derivatives 1 and 2.
1338 | J. Mater. Chem. B, 2014, 2, 1335–1343
for 96 h.
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analyzed using ow cytometry. Fig. 4(a) shows the cell cycle phase were 33% for 24 h and 61% for 48 h, whereas those for the
distributions for 24 h and those for 48 h including both control were less than 1%. The inhibition ratios of compound 2
adherent cells and oating cells. As a result, compound 2 was were 62% for 24 h and 88% for 48 h. On the other hand, with a
found to induce cell accumulation in the Sub-G1 phase at a concentration of 10 mM, no signicant difference was found in
concentration of 20 mM. The cell accumulations in the Sub-G1 the cell cycle distribution between the control and compound 2.
Fig. 6 (a) Photomicrograph of compound 2 at 100 mM in DMSO–H2O
(1 : 99) solution at 37 ꢀC under crossed polarizers. (b) Dynamic light
scattering profile of the aggregation system of compound 2 at 10 mM in
DMSO–H2O (1 : 999) solution at 37 ꢀC.
Fig. 4 (a) Dose-dependent effects of compound 2 on the cell cycle
distribution in A549 lung cancer cells. (b) Cell cycle distribution in
adherent cells and that in floating cells in the presence of compound 2
at a concentration of 20 mM.
Fig. 5 Effects of compound 2 on cell proliferation of A549 lung cancer
cells, SW480 colon cancer cells, THP1 leukemic cells and WI-38
normal fibroblast cells at 10 mM and 20 mM. Those cells cultured in the
presence of compound 2 were harvested (A549: 96 h, SW480: 96 h,
THP1: 72 h, WI-38: 168 h). Survivor cells were not detected in A549
cells in the presence of compound 2 at 20 mM. They were not detected
in WI-38 cells in the presence of compound 2 at 10 mM and 20 mM. *p <
0.05 by Mann–Whitney's U-test.
Fig. 7 Molecular structures of compounds 4, 5 and 6.
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The inhibition ratios of compound 2 at 10 mM were 32% for 24 h concentration of 10 mM and 20 mM. Compound 2 exhibits a
and 41% for 48 h. The results shown in Fig. 4(a) are the cell cycle marked cell-growth inhibition against SW480 colon cells at a
distributions including both adherent cells and oating cells. In concentration of 10 mM. Although it also shows cytotoxicity
order to clear whether the cell accumulation in the Sub-G1 against THP1 leukemic cancer cells, cell-growth inhibition of
phase is induced in adherent cells or oating cells, effects of the cancer cells is much smaller than that of the solid cancer
compound 2 at a concentration of 20 mM for 24 h on the cell cells (A549 and SW480). These results suggest that compound 2
cycle distribution were evaluated separately in adherent cells tends to suppress the growth of solid cancer cells more strongly
and in oating cells (Fig. 4(b)). With respect to the adherent compared with that of hematological cancer cells. Unexpect-
cells, the cell accumulation increases in the G1 phase compared edly, compound 2 exhibited a strong cytotoxicity against WI-38
with the control, whereas there is no signicant difference in normal broblast cells, indicating that it has severe side effects.
cell accumulation in the Sub-G1 phase between them. With
We evaluated the ability of molecular aggregation for
respect to the oating cells, the cell accumulation in the G1 compound 2. We observed thermotropic phase transition
phase decreases whereas that in the Sub-G1 phase increases behaviour of compound 2 using polarized optical microscopy
markedly. Therefore, compound 2 is thought to induce pro- and DSC. On cooling, compound 2 exhibited an isotropic to
grammed apoptosis following G1 arrest in A549 lung cancer nematic phase transition at 57.1 ꢀC. Its melting point was
cells. On the other hand, the cyanobiphenyl derivatives pos- 64.5 ꢀC. We then investigated the lyotropic liquid-crystallinity of
sessing a terminal alcohol did not induce cell death.19 The compound 2. Fig. 6(a) portrays
phenyl benzoate unit plays an important role in the cytotoxicity compound 2 (100 mM) in DMSO–H2O (1 : 99) solution at 37 C
a
photomicrograph of
ꢀ
of compound 2.
under crossed polarizers. The maltase cross texture suggests
We evaluated the cytotoxicity of compound 2 against the that compound 2 forms a spherical molecular aggregation in
other tumor cells, SW480 colon cancer cells and THP1 leukemic DMSO–H2O mixture solution. The texture was not conrmed by
cells, as well as WI-38 normal broblast cells. Fig. 5 shows POM at a concentration lower than 100 mM because the texture
effects of compound 2 on the growth of those cell lines at a size might be smaller than 1 mm. Therefore, we observed the
formation of particles of compound 2 in DMSO–H2O (1 : 999)
solution using dynamic light scattering. Fig. 6(b) shows the
histogram of compound 2 at 10 mM. The average diameter was
138 nm with a poly-dispersity index (PDI) value of 0.08. The
formation of particles was conrmed at the other concentra-
tions. The diameters were 166 nm at 5 mM with a PDI value of
0.06 and 134 nm at 20 mM with a PDI value of 0.07. However, we
could not estimate the diameter at a concentration of 1 mM due
Table 2 Cell accumulation (%) in the Sub-G1 phase and the inhibition
ratio (%) against control in brackets for compounds 4 and 5 for 96 h
4
5
10 mM
20 mM
10 mM
20 mM
24 h
48 h
0.6 (21)
0.8 (34)
1.5 (41)
0.8 (60)
24.1 (52)
20.7 (81)
46.4 (56)
63.8 (93)
Scheme 2 Synthesis of compound 5.
Fig. 8 Dose-dependent cytotoxicity of compound 4 and that of compound 5 on A549 cells for 96 h.
1340 | J. Mater. Chem. B, 2014, 2, 1335–1343
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Fig. 9 MOPAC models for (a) compound 4 and (b) compound 5.
to a large experimental error. At least we can say that compound 20 mM. Molecular structures are obtained by semi-empirical
2 forms a spherical molecular assembly possessing a suitable calculations using MOPAC-6/PM3, as presented in Fig. 9.
size for EPR effects17 with a concentration range of 5–20 mM.
Compounds 4 and 5 can produce a signicant difference in
The molecular assembly of compound 2 might be taken up by the mechanism of the cytotoxicity against A549 lung cancer
the cells via endocytosis. At present we do not have experi- cells. Compound 4 might interact with the nucleus via the
mental evidence how the molecules penetrate multiple layers of hydroxyl groups whereas compound 5 might interact with it via
the solid cancer cells. We cannot exclude the other both hydroxyl and ester groups. The structure–property rela-
penetration mechanism. There is marked dose-dependent tionship in anticancer activity indicates that compound 2
effects of compound 2 on the cell distribution in A549 lung
cancer cells, indicating that the intracellar interaction between
compound 2 and a nucleus of the tumor depends on the
concentration.
Synthesis and biological activities of compounds possessing
two terminal hydroxyl groups
In order to know how compound 2 interacts with the nucleus,
we designed some dimeric compounds, i.e., 1,2-, 1,3- and 1,4-
benzene derivatives with different congurations of their
terminal hydroxyl groups (Fig. 7). The compounds were
prepared by a synthesis outlined in Scheme 2.
We investigated the anti-proliferative activity of compounds
4 and 5 against A549 lung cancer cells. Unfortunately, 1,4-
derivative 6 could not be tested because of the low solubility in
the culture. Fig. 8 shows the dose-dependent suppressive
effects. The anti-proliferative activity of compound 4 increases
continuously with increasing concentrations, whereas that of
compound 5 shows a discontinuous increase from 5 mM to 10
mM. IC50 values of compounds 4 and 5 for 96 h were 5.8 mM and
8.9 mM, respectively. We investigated the effects of compounds 4
and 5 on the cell cycle prole in A549 cell proliferation. Table 2
presents the cell accumulation in the Sub-G1 phase and the
inhibition ratios for compounds 4 and 5. No signicant differ-
ence in the cell distribution was found between the control and
compound 4. However, marked cell accumulation was observed
in the Sub-G1 phase for compound 5, indicating that compound
5 induced cell death caused by DNA fragmentation. Compound Fig. 10 Effects of compounds 4 and 5 on cell proliferation of A549
lung cancer cells, SW480 colon cancer cells, THP1 leukemic cells and
WI-38 normal fibroblast cells at 10 mM and 20 mM. Those cells cultured
in the presence of each compound were harvested (A549: 96 h,
SW480: 96 h, THP1: 72 h, WI-38: 168 h). Survivor cells were not
4 exhibits cell-growth inhibition without inducing cell death,
which is similar to that observed for compound 2 at a concen-
tration of 10 mM. On the other hand, compound 5 was found to
induce marked cell accumulation in the Sub-G1 phase, which is
detected in A549 and WI-38 cells in the presence of compound 5 at a
similar to that observed for compound 2 at a concentration of concentration of 20 mM. *p < 0.05 by Mann–Whitney's U-test.
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Fig. 11 Schematic illustration of a mechanism for cytotoxic effects of liquid-crystalline compound 2 against A549 lung cancer cells.
interacts with the nucleus via the hydroxyl and ester groups and benzene derivative 5 induced cell death through cell cycle arrest
induces cell death in A549 lung cancer cells at a concentration in the G1 phase similarly to compound 2 at a concentration of
of 20 mM.
20 mM. We propose a mechanism of cytotoxic effects of
We evaluated their cytotoxicities against SW480 colon cancer compound 2 against A549 lung cancer cells as follows: (1) the
cells, THP1 leukemic cells and WI-38 normal broblast cells. liquid-crystalline molecules organize into a spherical particle
Fig. 10 shows the effects of compounds 4 and 5 on cell-growth with a suitable size for EPR effects, (2) the particles can be taken
inhibition of those cells. Compound 4 shows much smaller cell- up by the cells via endocytosis, and (3) the molecules interact
growth inhibition against those cells than compounds 2 and 5. with the nucleus via the hydroxyl and ester groups to induce cell
On the other hand, compound 5 was found to suppress not only death.
the proliferation of SW480 colon cancer cells but also that of WI-
38 normal broblast cells at 10 mM, similarly to compound 2.
Acknowledgements
The structure–property relationship in anticancer activity
suggests the following mechanism for the cytotoxic effects of
This work was supported by a Grant-in-Aid for Scientic
compound 2 against A549 lung cancer cells (Fig. 11): (1) self-
Research (no. 25107702) on the Innovative Areas: “Fusion
assembly of the molecules produces spherical nanoparticles
Materials” (Area no. 22006) from MEXT.
with a suitable size for EPR effects, (2) the nanoparticles might
be taken up by the cells via endocytosis, and (3) the molecules
might interact with the nucleus via the hydroxyl group at a
concentration lower than 10 mM whereas they might interact
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Liquid-crystalline compound 2 possessing a phenyl benzoate
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A549 lung cancer cells with an IC50 value of 4.7 mM. Compound
2 formed a spherical molecular assembly with a suitable
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