Synthesis and evaluation of the antitumor activity of Calix[4]arene L-proline derivatives

Article abstract of DOI:10.1016/j.bioorg.2019.103207

The unique conformational properties, functionality, low toxicity, and low cost make calixarene-based compounds a valuable candidate against cancer. The aim of the present study is the synthesis of the upper rim and lower rim-functionalized L-proline-based calix[4]arene derivatives and evaluation of their cytotoxic potential for human cancerous cells as well as to determine the death mechanism. Synthesized calix[4]arene (3, 8a, 8b 13a, and 13b) derivatives were characterized by different spectroscopic techniques such as ¹HNMR, ¹³CNMR, and FTIR. In vitro effects of compounds 3, 8a, 8b, 13a and 13b were tested on human cancerous cells (HEPG2, PC-3, A-549, and DLD-1) as well as human healthy epithelium cell (PNT1A). Results show that compounds 3, 8a, 8b and 13b have cytotoxic potential on human colorectal carcinoma cells (DLD-1) with IC₅₀ values of 43 μM, 45.2 μM, 64.57 μM, and 29.35 μM respectively. Apoptosis ratios of cell death were investigated with flow cytometer using 7-AAD and Annexin-V as markers. Cytotoxic potential of 8a was found to be higher due to increased apoptosis, when compared with healthy cells the apoptotic cell death was significantly (p < 0.0001) increased up to 1.7-fold and 2.4-fold in DLD-1 and A549 cells, respectively. In conclusion, these L-proline derived calix[4]arenes with their selective cytotoxic potential on human cancerous cells may be a potential candidate for the treatment of human CRC and lung cancer.

Full text of DOI:10.1016/j.bioorg.2019.103207

Bioorganic Chemistry xxx (xxxx) xxxx
Contents lists available at ScienceDirect
Bioorganic Chemistry
journal homepage: www.elsevier.com/locate/bioorg
Synthesis and evaluation of the antitumor activity of Calix[4]arene L-proline
derivatives
a,b
a
c,⁎
a,⁎
Mehmet Oguz , Alev Gul , Serdar Karakurt , Mustafa Yilmaz
b
a
Selcuk University, Department of Chemistry, 42075 Konya, Turkey
Department of Advanced Material and Nanotechnology, Selcuk University, 42075 Konya, Turkey
c
Selcuk University, Department of Biochemistry, Konya 42075, Turkey
A R T I C L E I N F O
A B S T R A C T
Keywords:
The unique conformational properties, functionality, low toxicity, and low cost make calixarene-based com-
pounds a valuable candidate against cancer. The aim of the present study is the synthesis of the upper rim and
lower rim-functionalized L-proline-based calix[4]arene derivatives and evaluation of their cytotoxic potential for
human cancerous cells as well as to determine the death mechanism. Synthesized calix[4]arene (3, 8a, 8b 13a,
Calixarene
L-Proline
Cytotoxicity
Apoptosis
1
13
and 13b) derivatives were characterized by different spectroscopic techniques such as HNMR, CNMR, and
FTIR. In vitro effects of compounds 3, 8a, 8b, 13a and 13b were tested on human cancerous cells (HEPG2, PC-3,
A-549, and DLD-1) as well as human healthy epithelium cell (PNT1A). Results show that compounds 3, 8a, 8b
and 13b have cytotoxic potential on human colorectal carcinoma cells (DLD-1) with IC50 values of 43 µM,
Anticancer agent
4
5.2 µM, 64.57 µM, and 29.35 µM respectively. Apoptosis ratios of cell death were investigated with flow cyt-
ometer using 7-AAD and Annexin-V as markers. Cytotoxic potential of 8a was found to be higher due to in-
creased apoptosis, when compared with healthy cells the apoptotic cell death was significantly (p < 0.0001)
increased up to 1.7-fold and 2.4-fold in DLD-1 and A549 cells, respectively. In conclusion, these L-proline derived
calix[4]arenes with their selective cytotoxic potential on human cancerous cells may be a potential candidate for
the treatment of human CRC and lung cancer.
1
. Introduction
Cancer is the major cause of death in the world, and especially in
causes the formation of a hydrophobic gap capable of complexing with
various guest molecules [17–22]. Beside these gaps, the molecules
carried by the lower and upper rim of calixarene may also interact with
the proteins and nucleic acids, which modulates the activity of many
enzymes, the proliferation of cancerous cells and metabolic pathways
[23–26]. The exceptional structural properties make calixarene a va-
luable candidate against cancer [27–29]. Recent studies proved that
calixarenes are capable of use in pharmaceutical applications as an anti-
cancer drug, the drug carrier, antiviral and antithrombotic activities
[30–33]. Some calixarene derivatives have been reported for its effec-
tive activity against bacteria, fungi, antitumor, antimicrobials, can-
cerous cells, and enveloped viruses, but also against thrombosis or fi-
brosis diseases [34–36]. Ding et al., prepared calix[4]arene derivatives
functionalized 2-dimethylaminoethyl groups on its hydrophilic face vis-
à-vis the larger and somewhat more polar N-(2-dimethylamino)ethyl
acetamide groups and tested their anti-tumor activities. They observed
effectiveness of inhibiting the growth of several human cancer cell
lines, as well as drug-resistant cancer cells [37]. Alkyl acid and alkyl
ester groups from lower rim position containing calix[4]arenes were
developing countries, the mortality rate has been increased sig-
nificantly [1–3]. According to the world health organization, about 9.6
million new cases have been reported in 2018 and it is expected to rise
by about 70% over the next two decades [4]. Increased morbidity and
mortality rates of cancer lead investigators to find new and active
strategies against cancer. Although there are many synthetic drugs used
for the treatment of the several types of cancers, the severe side effects
and low cytotoxicity makes them ineffective for the cure [5–8]. Indeed,
one of the biggest challenges for pharmaceutical industries is to explore
new anticancer agents that are safe and effective [9–12].
One of the important third generation supramolecular compounds is
calixarene possesses ring and flexible conformation, which make them
suitable for adding functional groups including amino acids, amide,
amine, alcohol, ester, aldehyde and alkyl derivatives [13–16]. Phenolic
rings of calixarenes interact with each other by methylene bridges from
the o-position of the phenolic structures to the hydroxyl groups, which
⁎
Corresponding authors.
E-mail addresses: kserdar1@yahoo.com (S. Karakurt), myilmaz42@yahoo.com (M. Yilmaz).
https://doi.org/10.1016/j.bioorg.2019.103207
Received 19 February 2019; Received in revised form 10 July 2019; Accepted 15 August 2019
0045-2068/ © 2019 Elsevier Inc. All rights reserved.
Please cite this article as: Mehmet Oguz, et al., Bioorganic Chemistry, https://doi.org/10.1016/j.bioorg.2019.103207

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
synthesized as new potential iron chelators, and have been found to be
more potent inhibitor then commercially available compound; ICL670
on the viability of HepaRG cells [38,39]. Another study calix[n]arene
polyhydroxyamine derivatives were synthesized and examined their
cytotoxicity in six cancer cell lines, The results presented in this study
showed that calix[4]arene polyhydroxyamine derivative is highly ef-
fective in inducing cell death in human ovarian carcinoma cells [40].
Proline, the sole proteinogenic secondary amino acid, possess a
crucial role in special biological functions including human physiologic
and pathologic situations [41,42]. Increased level of proline postponed
cell aging by activating redox cycle and maintenance of pyridine nu-
cleotides [43]. One of the main steps for cancer progression and inva-
sion is upregulation of matrix metalloproteinases (MMPs) that catalysis
the degradation of ECM and releasing of proline into the matrix. As a
result of proline metabolism (P5C cycle), glutamate and ornithine are
produced, which further converted to α-ketoglutarate (α-KG) entering
the tricarboxylic acid (TCA) cycle and arginine entering the urea cycle,
respectively [44,45]. Fu et al. synthesized calix[4]arene modified with
L- or D-proline and used them against human cervical cancer, the second
cause of death in the female in the world. They observed that the chiral
proline modified calix[4]arene prevented L1 pentamer formation of
HPV [46].
were synthesized according to the literature [47–50]. Compound 3 was
also synthesized following the reported method with little modification
as depicted below [51]. Lower rim L-proline functionalized calixarene
(7b, 8a, 8b, 12a, 12b, 13a, 13b) as illustrated in Scheme 1–3 were
synthesized according to the methods given below.
2.3. Synthesis of compound 3
Calix[4]arene (1a) (4.0 g, 9.6 mmol) was dissolved in tetra-
hydrofuran (60 mL), glacial acetic acid (11.2 mL) and formaldehyde
(
4.0 mL, 37% w/v, 54 mmol) were added into this solution and left
stirring for 72 h at room temperature. The reaction was completed
when water-insoluble material was not detected. The liquid was re-
moved and drained. The remaining solid was washed with acetone and
the solution was filtered. The precipitate was washed with acetone
several time and then recrystallized from ethanol/water (1:1) to yield a
−1
− 1
white solid (7.4 g; yield, 82%). IR: 1612 cm COO . HNMR (D
2
O): δ,
1
2
3
3
.40–1.82 (broad m, 12H CHCH
.75–2.98 (broad m, 4H, CH N), 3.18–3.35 (broad m, 4H, CH
.54–3.67 (broad t, 4H, NCH), 3.67–3.82 (broads, 8H, ArCH
.83–3.88 (broads, 8H, ArCH Ar), 7.03 (s, 8H, ArH). 13C NMR (D
), 28.7 (ArCH Ar), 52.4, 55.9 (2 × CH
CHN), 121.4, 127.8, 129.5, 149.4 [3], 171.8 (C]O).
2
), 1.82–2.20 (broad m, 4H, CHCH
2
),
2
2
N),
2
N),
2
O):
2
δ, 20.9, 29.9 (2 × CH
2
2
2
N), 66.1
In regards of these studies, we designed and synthesized upper and
lower rim-functionalized L-proline-based calix[4]arene derivatives, and
investigated their cytotoxic potential for various human cancerous cells
and determined the mechanism of cell death.
(
2.4. General procedure for the synthesis of compounds 7a and 7b
Compound 6a and 6b (0.95 g, 1 mmol) dissolved in dry THF
2
. Materials and methods
(
100 mL) in two separate flaks and the solution of L-proline methyl ester
hydrochloride (0.99 g 6 mmol) added dropwise containing pyridine
2.1. Instruments/materials
(
1 mL) and stirred for 48 h at room temperature. The reaction was
monitored by TLC. When the reaction finished excess of the solvent was
removed off in vacuo. The residue was extracted with chloroform/water
All materials and reagents used in the experiments were supplied
from various commercial sources and used without further purification.
several times. Organic layer was separated and dried with MgSO . After
4
NMR measurements obtained from Varian (400 MHz),
D O and
2
filtration of magnesium sulfate, the solvent was evaporated. 7a: Yield
DMSO‑d
6
for calix[4]arene derivatives were used as deuterated sol-
1
7
4%; H NMR (400 MHz, DMSO): δ (ppm) 1.07 (s, 36H, But), 1.83–1.97
vents. Infrared spectra were measured using a Bruker spectrometer
transform-infrared (FT-IR) spectrometer. HR-MS spectra were per-
formed by Synapt G1 High Definition Mass Spectrometer. Elisa plate
reader (Thermo multiscan-90), Flow cytometer (BD FACS Aria III).
(
m, 8H, CH
2
(proline)), 2.13–2.22 (m, 8H, CH
2
(proline)) 3.24–3.35 (m,
and 4H, ArCH Ar)),
), 5.08 (s, 4H, OCH ),
8
4
5
H, CH
2
(proline)), 3.69–3.78 (m, 16H, (12H, OCH
3
2
.48–4.62 (m, 4H, ArCH
2
Ar), 4.93 (s, 4H, OCH
2
2
.20–5.29 (m, 4H, CH(proline)), 6.75 (s, 8H, ArH).
1
7
b: Yield 68%; H NMR (400 MHz, DMSO) δ (ppm): 1.73–1.89 (m,
2.2. Synthesis
8H, CH
CH (proline)), 3.59 (s, 12H, OCH
(m, 8H, OCH ), 4.61 (d, 4H, J = 15.4 Hz, ArCH
(proline)), 6.44–6.58 (m, 4H, ArH), 6.71–6.83 (m, 8H, ArH).
2
(proline)), 2.03–2.19 (m, 8H, CH
), 3.68 (s 4H, ArCH
Ar), 4.93 (d, 4H, CH
2
(proline)) 3.23–3.38 (m, 8H,
2
3
2
Ar), 4.23–4.31
The different derivatives of calixarenes presented in Scheme 1–3
2
2
(
1a, 1b, 4a, 4b, 5a, 5b, 6a and 6b, 7a, 9a, 9b, 10a, 10b, 11a, 11b)
Scheme 1. The schematic route for the synthesis of water-soluble L-proline based calix[4]arene derivative. Reagents and conditions: (i) AlCl3, Toluene; (ii) L-proline,
tetrahydrofuran, glacial acetic acid, formaldehyde.
2

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Scheme 2. The schematic route for the synthesis of L-proline based calix[4]arene derivatives. Reaction conditions; (i) BrCH2COOCH3, K2CO3, Acetone; (ii) KOH,
ethanol/water; (iii) SOCl2, THF; (iv) pyridine; L-Proline methyl ester hydrochloride, THF; v) KOH, ethanol/water.
Scheme 3. The schematic route for the synthesis of L-proline based calix[4]arene derivatives. Reaction conditions; (i) BrCH2COOCH3, K2CO3, Acetone; (ii) KOH,
ethanol/water; (iii) SOCl2, THF; (iv) pyridine; L-Proline methyl ester hydrochloride, THF; (v) KOH, ethanol/water.
3

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
1
3
2.5. Synthesis of compounds 8a and 8b
(proline)), 6.53–7.22 (m, 12H, ArH). C NMR (100 MHz, DMSO): δ
(
ppm) 173.6, 168.2, 155.8, 153.24, 134.9, 134.1, 129.3, 127.9, 124.38,
Compounds 7a and 7b (0.95 g) were taken in two separate flasks
123.4, 73.3, 59.1, 45.9, 31.5, 31.2, 29.0, 25.0. The HR-MS data of 13a
and 13b (959.5515 and 733.2795, respectively) supported a molecular
and dissolved in ethanol (50 mL) containing aqueous KOH solution. The
reaction mixture was then stirred and heated at reflux for 8 h. After the
Solvent was removed under reduced pressure, the residue was pre-
cipitated with 0.1 M HCl. The solid material was then filtered and
washed till neutralization with water several time. The target com-
pounds 8a and 8b were obtained as white solid. 8a: Yield 85%. IR:
formula 13a: C58
H
74
N
2
O
10 and 13b: C42
H
42
2
N O10 (Figs. S15 and S19).
2.8. Cell culture
Human cancerous cell lines; A-549 (Lung cancer), PC-3 (Prostate
Cancer), DLD-1 (Colon Cancer), HePG2 (Liver Cancer) cell lines were
supplied from ATCC (American Type Culture Collection) and healthy
epithelium cell line, PNT1A was obtained from Sigma Aldrich
(Missouri, ABD). The cells were incubated with DMEM, Ham’s F-12,
RPMI 1640 and EMEM mediums, respectively and supplemented with
10% FBS (fetal bovine serum), 1% L-glutamine and 1% penicillin/
−
1
−1
1
1
732 cm
C]O, 1650 cm
NeC]O. H NMR (400 MHz, DMSO): δ
(
(
ppm) 1.16 (s, 36H, But), 1.96–2.16 (m, 8H, CH (proline), 2.18–2.29
2
m, 8H, CH
2
(proline)) 3.27–3.39 (m, 8H, CH
2
(proline)), 3.51–3.63 (m,
4
H, ArCH
2
Ar)), 3.96 (d, 4H, J = 14.6 Hz, ArCH
2
Ar), 4.38 (s, 4H,
OCH
2
), 4.82 (s, 4H, OCH ), 4.94–5.10 (m, 4H, CH(proline)), 7.17 (s,
2
1
3
8
H, ArH). C NMR (100 MHz, DMSO): δ (ppm) 172.7, 168.1, 152.2,
1
47.5, 134.7, 125.3, 4.6, 59.0, 45.6, 34.54, 31.4, 30.8, 29.0, 24.8. 8b:
streptomycin at 37 °C in a 5% CO atmosphere and 95% humidity.
2
−
1
−1
1
Yield 80%. IR: 1729 cm
C]O, 1658 cm
NeC]O. H NMR
Following the confluence, the cells were transferred to 96-well plates
and 6-well plates for cytotoxicity studies and apoptosis/comet assay
studies, respectively.
(
400 MHz, DMSO) δ (ppm): 1.86–2.49 (m, 16H, CH
2
(proline)),
2
.91–3.73 (m, 8H, CH
J = 14.1 Hz, ArCH Ar), 4.55–4.73 (m, 8H, OCH
CH(proline)), 6.86–7.14 (m, 4H, ArH), 7.28 (m, 8H, ArH). C NMR
100 MHz, DMSO): δ (ppm) 174.0, 168.1, 156.5, 135.18, 128.9, 122.4,
2.2, 59.1, 45.6, 31.6, 29.0, 25.0. The HR-MS data of 8a and 8b
1267.6733 and 1043.4077, respectively) supported a molecular for-
mula 8a: C72 16 and 8b: C56 16 (Figs. S7 and S11).
2
(proline) ve 4H ArCH
2
Ar), 3.97 (d, 4H,
2
2
), 4.93–5.31 (m, 4H,
1
3
2.9. Determination of IC50 values for chemical treatment
(
7
The cells were counted with the BioRad TC20 automatic cell counter
and death/live ratio was calculated by using 0.04% “Trypan Blue”.
(
4
H
92
N
4
O
H
60
N
4
O
1 × 10 of the cells were incubated for 24 hr. then treated with various
concentration of L-proline-based calix[4]arenes (3, 8a, 8b.13a, and
13b) ranging between 0 and 200 µM. The viability of the cells and
cytotoxicity of the compounds were determined spectrophotometrically
by using Alamar Blue reagent as an indicator (Invitrogen, Thermo
Fischer Scientific, Waltham, MA, USA) [52]. The IC50 value was cal-
culated from the sigmoidal plot of cell inhibition and statistical analyses
were done by GraphPad Prism 5.0.
2.6. Synthesis of compounds 12a and 12b
Compound 11a and 11b (0.95 g, 1 mmol) were dissolved in dry
THF (50 mL) in two separate flasks and the solution of L-proline methyl
ester hydrochloride (0.66 g 4 mmol) was added dropwise in the pre-
sence of pyridine (0.5 mL). The mixture was allowed to stir for 30 h at
room temperature. The reaction was monitored by TLC. After formation
of products, excess of the solvent was removed off at low pressure. The
residue was extracted with chloroform/water several times. Organic
2.10. Apoptosis assays
5
layer was separated and dried with MgSO
4
. After filtration the solvent
2.5 × 10 of the cells from PNT1A, DLD-1, and A549 cell lines were
was evaporated, and residue was dried at room temperature, 12a: Yield
transferred into 6-well plates and incubated at 37 °C, 5% CO for 24 hr.
2
1
7
1
5%. H NMR (400 MHz, DMSO): δ (ppm) 1.05 (s, 18H, But), 1.16 (s,
Then, PNT1A, DLD-1, and A549 cells were treated with equivalent
concentration as IC50 values of compound 3, 8a, 8b.13a and 13b.
Following 48 hr incubation, the cells were incubated with Annexin V
and 7-AAD and analyzed by flow cytometer (BD FACS Aria III, software:
FACS Diva software).
8H, But), 1.85–2.11 (m, 8H, CH
(proline)), 3.65 (s, 6H, OCH
Ar), 4.49–4.63 (m, 4H, OCH
2
CH (proline)), 3.38 (m, 4H,
2
CH
2
3
), 3.68 (bs, 4H, ArCH Ar), 4.38–4.46
2
(
(
m, 4H, ArCH
2
2
), 4.76–4.83 (m, 2H, CH
1
proline)), 7.05 (s, 4H, ArH), 7.10 (s, 4H, ArH). 12b: Yield 65%.
H
NMR (400 MHz, DMSO): δ (ppm) 1.71–1.96 (m, 8H, CH
2
CH
ve ArCH
), 5.06–5.17 (m,
2
(proline)),
3
4
2
.13 (b, 4H, CH
2
(proline)), 3.41–3.67 (m, 10H, (OCH
3
2
Ar)),
3. Results and discussion
.19–4.31 (m, 4H, ArCH
2
Ar), 4.76–4.82 (m, 4H, OCH
2
H, CH(proline)), 6.41–7.08 (m, 12H, ArH).
The target of this work is to prepare an effective antitumor agent
using calix[4]arene by using L-proline amino acid (Fig. 1). In this re-
gard, compound 3 was designed and readily prepared using simple
reaction conditions (Scheme 1). First of all, p-tert-butylcalix[4]arene (1)
2.7. Synthesis of compound 13a and 13b
Compound 12a and 12b (0.95 g) were dissolved in ethanol (50 mL)
as the precursor was treated with Lewis acid (AlCl ) in the presence of
3
in two separate flasks containing aqueous KOH solution. The reaction
mixture was then stirred and heated at reflux for 8 h. Solvent was re-
moved under reduced pressure, the residue was precipitated with 0.1 M
HCl. The solid material was then filtered and washed till neutralization
with water several time. The target compounds 13a, b was obtained as
phenol to remove upper rim tert-butyl groups to obtain calix[4]arene
(2). Then, the calix[4]arene (2) was reacted with L-proline (Mannich
reaction) in the presence of glacial acetic acid and formaldehyde to
synthesize compound 3 as L-proline functionalized calix[4]arene (3)
reported in published work [46,51].
1
white solid. 13a: Yield 85%. H NMR (400 MHz, DMSO): δ (ppm) 1.07
Compound 8a, 8b, 13a, and 13b were synthesized using an acid
chloride method. The L-proline-substituted calix[4]arene derivatives
8(a,b) and 13(a,b) were obtained in five steps (Scheme 2 and 3). The
parent compounds 1, 4, 5 and 6 were synthesized according to litera-
ture [47–49]. Calix[4]arene tetraester derivative (4) was synthesized in
one step from p-tert-butylcalix[4]arene (1) with a yield of 79% by re-
(
s, 18H, But), 1.13 (s, 18H, But), 1.80–2.27 (m, 8H, CH
2
CH (proline)),
2
3
.33 (m, 4H, CH
2
(proline)), 3.62–3.87 (m, 4H, ArCH Ar), 4.36 (d, 4H,
2
J = 12.05 Hz, ArCH
2
Ar), 4.53–4.72 (m, 4H, OCH ), 4.86–4.91 (m, 2H,
2
13
CH(proline)), 7.12 (s, 4H, ArH), 7.18 (s, 4H, ArH). C NMR (100 MHz,
CDCl3): δ (ppm) 173.6, 167.1, 152.0, 150.6, 147.0, 141.1, 133.5,
1
3
28.8, 127.4, 125.6, 74.19, 59.25, 45.93, 34.35, 33.99, 31.86, 31.36,
acting with methyl bromoacetate and K
2
CO in acetone under reflux
3
1
0.9, 29.1, 24.9, 22.3. 13b: Yield 80. H NMR (400 MHz, DMSO): δ
conditions. Base hydrolysis of tetraester derivative (4) in ethanol pro-
duced acid derivative (5) in 85% yield. In the next step, carboxylic acid
derivative (5) was converted into acid chloride (6) via thionyl chloride
in THF at inert conditions. Compound 7 (a.b) was synthesized using an
(
ppm) 1.71–1.93 (m, 8H, CH
2
CH (proline)), 3.17–3.24 (m, 4H,
2
CH
2
(proline)), 3.48–3.52 (m, 4H, ArCH
2
Ar), 3.96–4.11 (m, 4H,
ArCH
2
Ar), 4.25–4.43 (m, 4H, OCH ), 4.78–4.91 (m, 4H, 2H, CH
2
4

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 1. The structure of L-proline based calix[4]arene derivatives (3, 8a, 8b, 13a, and 13b).
acid chloride derived from calixarene (6) and L-proline methyl ester
HEPG2, A549 and PNT1A cells, the cells were treated with various
concentration of the compounds. Different functional group on calix[4]
arene behave differently on the viability of the cells. They have selec-
tively and dose-dependently inhibited proliferation and viability of the
cancerous cells as well as healthy epithelium cell (Table 1).
hydrochloride in the presence of pyridine in THF. Finally, compound
7
8
(a.b) was hydrolyzed with KOH in ethanol to furnish final product
(a,b). The chemical structures of all derivatives were completely
1
13
characterized by H, C NMR, and IR spectroscopic techniques.
−
In functional group analysis of 3, presence of C]O (COO
)
As shown in Table 1, when the compounds were compared, com-
pound 13b was found to be the highest cytotoxic potential on human
CRC cell (DLD-1) and human hepatocarcinoma cell (HEPG2) with IC50
values as 29.35 µM and 64.45 µM, respectively (Fig. S23). On the other
hand, when the healthy epithelium cell (PNT1A) was considered, al-
though 13b has lower IC50 value on DLD-1 than PNT1A, the best choice
seems compound 3 which has IC50 value as 43 µM for DLD-1, but no
cytotoxic potential on human epithelial cells (> 200 µM) (Fig. 2). The
molecular mechanism of this effect lies in the genetic difference be-
tween two cells. The BRAF, KRAS, p53, and SMAD genes that act as key
enzymes in the proliferation of DLD-1 cells are mutated and their ex-
pressions are much lower than in healthy PNT1A cells. Therefore, it is
not a good candidate for the treatment of other cancerous cells. The pH
of cancerous cells is around 6.0–6.5 and at that pH the L-proline groups
(pI = 6.3) in the upper rim of the compound 3 form zwitterion at this
pH, and makes it soluble in water. Besides, compound 13a was also
found a potent inhibitor for human lung carcinoma cell (A549) and
prostate cells (PC-3) with the IC50 values as 15.7 µM and 23.38 µM,
respectively (Fig. S22). When compared with other compounds, com-
pound 13a seems the perfect candidate against the proliferation of
A549 since it has the lowest ratio between A549 vs PNT1A (Healthy
cell) as 0.39. In addition, when the compounds 8a and 8b were com-
pared to healthy epithelium cells, the best toxicity has been seen in the
DLD-1 cells with 45.2 µM and 64.5 µM IC50 values, respectively (Figs.
S20 and S21).
−
1
stretching band at 1612 cm
confirmed the derivatization of calix-
1
arene with L-proline (Fig. S1). In H NMR spectrum (Fig. S2), the ap-
pearance of different peaks of methyl protons of L-proline from 1.63 to
3
.38 ppm further confirmed the purity of the synthesized compound. In
addition protons of ArCH
2
Ar at 3.86 and 3.98 ArCH Ar, the presence of
2
singlet aromatic protons at 7.07 (s, 8H, ArH) were confirmed the
structure of compound 3 (Fig. S2).
1
13
HNMR, CNMR, and FTIR spectroscopy are used to elucidate the
structure of compound 8 (a,b) and 13 (a, b). In FTIR spectrum of
compounds 8 (a, b), presence of stretching bands at 1732 and
−
1
−1
1
729 cm
for C]O, 1650 and 1658 cm
for NeC]O defines the
presence of L-proline functionality on calix[4]arene (Figs. S4 and S8).
Presence of the signal at 1.16 ppm for singlet t-butyl group and the
peaks from 1.96 to 3.27 ppm for L-proline methylene, at 4.38 ppm show
the presence of OCH protons of ester linkage and finally singlet aro-
2
matic signal (8Hs) at 7.17 ppm for aromatic protons explain the com-
plete structure of 8a (Fig. S5). The synthesis of 8b was confirmed by the
appearance of proline protons at from 1.86 to 3.73 ppm and the pre-
sence of aromatic signals at 7.81 ppm and at 7.28 ppm for aromatic
1
3
rings (Fig. S9). The C NMR spectra of 8a and 8b showed the ap-
pearance of the carbon signals at 173.7 and at 174.0 ppm belong to C]
O groups. In addition, carbon signals at 168.1 confirmed the presence of
NeC]O groups (Figs. S6 and S10).
The formation of 13a and 13b was confirmed by the appearance of
−1
−1
the characteristic carbonyl bands at about 1732 cm
and 1738 cm
,
To clarify the molecular mechanism of cell death, the cells were
subject to flow cytometric analysis with 7-AAD and Annexin-V dyes.
When compared the compound 8a, 8b, 13a, and 13b, it was shown
that compound 3 does not progress the cells through the apoptosis
(p > 0.05). On the other hand, Compound 8a significantly inhibited
the viability of DLD-1 and A549 cells via apoptosis. Apoptotic cell death
was increased as 1.93-fold and 2.02-fold in DLD-1 and A549 cells, re-
spectively (Fig. S20). Furthermore, compound 8b and 13a were also
increased the apoptotic cell death of DLD-1 cells as 4.3-fold and 15.3-
fold, respectively (Figs. S21 and S22).
−1
−1
amide bands at 1650 cm and 1654 cm in the FTIR spectra (Figs. S12
and S16). The structure of 13a was also confirmed by the appearance of
1
proline methyl protons from 1.80 to 3.33 ppm in the H NMR spectra. In
addition, the appearance of p-tert butyl protons at 1.07 (18H) and 1.13
(
18H), aromatic protons at 7.12 (4H) and 7.18 (4H) confirmed the
13
structure of 13a (Fig. S13). The C NMR spectra of 13a showed the
appearance of the carbon signal at 173.6 ppm belong to C]O group and
signal at δ167.1 belong to NeC]O (Fig. S14). The appearance of proline
1
methyl protons at from 1.71 to 3.24 in the H NMR spectrum, the pre-
sence of carbon signals at 173.6 ppm and 168.2 ppm (C]O and NeC]
Activation of death receptors signaling and Bcl-2 (pro-apoptotic
13
O) in C NMR spectrum confirmed the structure of compound 13b (Figs.
S17 and S18). The spectral data were in agreement with the desired
structures. Besides, the HR-MS spectra results also supported the struc-
tures of the synthesized compounds (Figs. S7, S11, S15 and S19).
Table 1
Calculated IC50 values (µM) for L-proline-based calix[4]arene derivatives.
PNT1A
A549
PC-3
DLD-1
HEPG2
3
.1. Effects of L-proline-based Calix[4]arene derivatives on viability and
3
8
8
1
1
> 200
66.10
105.6
40.06
65.91
> 200
49.50
80.95
15.70
108.7
94.50
55.90
91.61
23.38
92.69
43.00
45.20
64.57
59.55
29.35
147.0
105.5
107.7
73.94
64.45
a
proliferation of human cancerous and healthy cells
b
3a
3b
In order to determine the cytotoxic effects of compound 3, 8a, 8b,
1
3a, and 13b on proliferation and viability of human DLD-1, PC-3,
5

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
Fig. 2. Effects of compound 3 on viability, proliferation, and apoptosis of the A549, DLD-1, and PNT1A cells. The cells were treated with compounds (0–200 µM) and
incubated 48 h at 37 °C, 5% CO . (a) Alamar Blue assay was used to investigate viability and cytotoxicity. Viability of PNT1A and DLD-1 cells after treatment with
2
different concentrations (0–200 µM) of compound 3 for 48 h. (b) IC50 value of compound 3 on A549 and DLD-1 cells; (c) and (e) A549 and DLD-1 cells treated with
compound 3; (d) and (f) comparison of histogram plots of compound 3 treated cells with non-treated cells of A549 and DLD-1, respectively. Overlapping plots of
apoptotic cells of NT (Non-treated cells) and Compound 3 (43 µM) treated A549 and DLD-1 cells. The results represent mean ± SD of three independent experiments
(
n = 4).
Fig. 3. Effect of compound 3, 8a and 13a on A549 and DLD-1 cell abpoptosis. Apoptosis was examined by flow cytometry using Annexin V and 7-AAD staining. The
scatterplots illustrate the effects of Compound 3, 8a (S16), 8b (S17), and 13a (S18). The gate was obtained from annexin-V negative and 7-AAD negative cells. The
+
−
+
+
lower left quadrant represents the early apoptosis (annexin V , 7-AAD ), the upper left quadrant represents the late apoptosis (annexin V , 7-AAD ), the lower
right quadrant represents the alive cells (annexin V , 7-AAD ), and the upper right quadrant represents the necrosis (annexin V , 7-AAD+). The bars represent the
−
−
−
percentage of cells in early apoptotic, late apoptotic and necrotic stages.
gene) pathways may increase the early apoptosis which clarifies the
extrinsic or intrinsic pathways of apoptosis. On the other hand, the
intrinsic and extrinsic pathways were merged in the late phase of
apoptosis that is characterized by DNA fragmentation. In both stages of
apoptosis, morphological and phenotypical changes occur [53].
When the cells were treated with 8a, 8b and 3, 1.9%, 4.3% and
18.3%, 9.3%, and 5.3%, and increased late apoptosis as 12.9%, 9.5%
and 7.8%, respectively (Fig. 3).
4
. Conclusion
In conclusion, calix[4]arene molecule was successfully functiona-
0
8
.9% of the DLD-1 cells were death due to early apoptosis and 7.9%,
.8% and 4.8% of the cells were death due to late apoptosis, respec-
lized with L-proline at upper and lower rims. The lower rim L-proline
derivative (8a) has been shown the highest toxicity against human CRC
tively. Besides, 8a, 13a and 3 increased early apoptosis of A549 cells as
(
DLD-1) and lung carcinoma (A549) cells. Compound 8a leads
6

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
apoptotic cell death especially the early stage of apoptosis. Besides this,
other compounds also showed proliferation of various human can-
cerous cells including, liver, prostate, lung and colorectal carcinomas in
a dose-dependent manner. The present study opens a way to develop
selective and cost-effective inhibitors as new antitumor agents.
racemic naproxen methyl ester by lipase encapsulated within iron oxide nano-
particles coated with calix [8] arene valeric acid complexes, Org. Biomol. Chem. 12
(
34) (2014) 6634–6642.
[
23] E. Ozyilmaz, M. Bayrakci, M. Yilmaz, Improvement of catalytic activity of Candida
rugosa lipase in the presence of calix [4] arene bearing iminodicarboxylic/phos-
phonic acid complexes modified iron oxide nanoparticles, Bioorg. Chem. 65
(
2016) 1–8.
[
[
24] A. Yousaf, S.A. Hamid, N.M. Bunnori, A. Ishola, Applications of calixarenes in
cancer chemotherapy: facts and perspectives, Drug Des., Develop. Therapy 9 (2015)
Declaration of Competing Interest
The authors declare no conflict of interest.
Acknowledgements
2
831.
25] P.L. Padnya, E.A. Andreyko, O.A. Mostovaya, I.K. Rizvanov, I.I. Stoikov, The
synthesis of new amphiphilic p-tert-butylthiacalix [4] arenes containing peptide
fragments and their interaction with DNA, Org. Biomol. Chem. 13 (21) (2015)
5894–5904.
[
26] M. Durmaz, M. Yilmaz, A. Sirit, Synthesis of chiral calix [4] arenes bearing ami-
nonaphthol moieties and their use in the enantiomeric recognition of carboxylic
acids, Org. Biomol. Chem. 9 (2) (2011) 571–580.
We would like to thank the Scientific and Technological Research
Council of Turkey, Turkey (TUBITAK-Grant Number 116Z173) and the
Research Foundation of Selcuk University (SUBAP-Grant Number:
[27] T. Läppchen, R.P. Dings, R. Rossin, J.F. Simon, T.J. Visser, M. Bakker, P. Walhe,
T. Van Mourik, K. Donato, J.R. Van Beijnum, Novel analogs of antitumor agent
calixarene 0118: synthesis, cytotoxicity, click labeling with 2-[18 F] fluor-
oethylazide, and in vivo evaluation, Eur. J. Med. Chem. 89 (2015) 279–295.
[28] V. Bagnacani, F. Sansone, G. Donofrio, L. Baldini, A. Casnati, R. Ungaro,
Macrocyclic nonviral vectors: high cell transfection efficiency and low toxicity in a
lower rim guanidinium calix [4] arene, Org. Lett. 10 (18) (2008) 3953–3956.
1
7201066 and 17201064) for financial support of this work and is a
part of Mehmet Oguz Ph.D. thesis and Alev Gul master thesis.
Appendix A. Supplementary material
[
29] D. Santos, J. Medeiros-Silva, S. Cegonho, E. Alves, F. Ramilo-Gomes, A.O. Santos,
S. Silvestre, C. Cruz, Cell proliferation effects of calix [4] arene derivatives,
Tetrahedron 71 (40) (2015) 7593–7599.
Supplementary data to this article can be found online at https://
doi.org/10.1016/j.bioorg.2019.103207.
[
[
30] Â. de Fátima, S.A. Fernandes, A.A. Sabino, Calixarenes as new platforms for drug
design, Curr. Drug Discov. Technol. 6 (2) (2009) 151–170.
31] V. Kuete, S.B. Tankeo, M.E. Saeed, B. Wiench, P. Tane, T. Efferth, Cytotoxicity and
modes of action of five Cameroonian medicinal plants against multi-factorial drug
resistance of tumor cells, J. Ethnopharmacol. 153 (1) (2014) 207–219.
32] V. Saluja, B.S. Sekhon, Calixarenes and cucurbiturils: pharmaceutial and biomedical
applications, J. Pharm. Educat. Res. 4 (1) (2013) 16.
References
[
[
[
[
[
1] E. Salvati, F. Stellacci, S. Krol, Nanosensors for early cancer detection and for
therapeutic drug monitoring, Nanomedicine 10 (23) (2015) 3495–3512.
[33] L. Barbera, G. Gattuso, F.H. Kohnke, A. Notti, S. Pappalardo, M.F. Parisi, I. Pisagatti,
S. Patanè, N. Micali, V. Villari, Self-assembly of amphiphilic anionic calix [4] arenes
and encapsulation of poorly soluble naproxen and flurbiprofen, Org. Biomol. Chem.
13 (23) (2015) 6468–6473.
2] R.L. Siegel, K.D. Miller, A. Jemal, Cancer statistics, 2015, CA: Cancer J. Clin. 65 (1)
(
2015) 5–29.
3] Y.-E. Choi, J.-W. Kwak, J.W. Park, Nanotechnology for early cancer detection,
Sensors 10 (1) (2010) 428–455.
[
34] H. Zhou, D.-A. Wang, L. Baldini, E. Ennis, R. Jain, A. Carie, S.M. Sebti,
A.D. Hamilton, Structure–activity studies on a library of potent calix [4] arene-
based PDGF antagonists that inhibit PDGF-stimulated PDGFR tyrosine phosphor-
ylation, Org. Biomol. Chem. 4 (12) (2006) 2376–2386.
4] F. Bray, J. Ferlay, I. Soerjomataram, R.L. Siegel, L.A. Torre, A. Jemal, Global cancer
statistics 2018: GLOBOCAN estimates of incidence and mortality worldwide for 36
cancers in 185 countries, CA: Cancer J. Clin. 68 (6) (2018) 394–424.
5] K. Kalogerakos, C. Sofoudis, N. Baltayiannis, Early breast cancer: a review, Cancer
Therapy 6 (2) (2008).
[
[
[
[35] P. Sreedevi, J.B. Nair, P. Preethalayam, B.S. Jeeja, C.H. Suresh, K.K. Maiti,
R.L. Varma, Calix [4] arene based redox sensitive molecular probe for SERS guided
recognition of labile iron pool in tumor cells, Anal. Chem. (2018).
6] M. Perfezou, A. Turner, A. Merkoçi, Cancer detection using nanoparticle-based
sensors, Chem. Soc. Rev. 41 (7) (2012) 2606–2622.
[
36] X.M. Chen, Y. Chen, Q. Yu, B.H. Gu, Y. Liu, Supramolecular assemblies with near-
infrared emission mediated in two stages by Cucurbituril and amphiphilic calix-
arene for lysosome-targeted cell imaging, Angew. Chem. Int. Ed. 57 (38) (2018)
7] F.R. Hirsch, W.A. Franklin, A.F. Gazdar, P.A. Bunn, Early detection of lung cancer:
clinical perspectives of recent advances in biology and radiology, Clin. Cancer Res.
7
(1) (2001) 5–22.
1
2519–12523.
[
[
8] M.M. Naseer, M. Ahmed, S. Hameed, Functionalized calix[4]arenes as potential
therapeutic agents, Chem. Biol. Drug Des. 89 (2) (2017) 243–256.
9] S.B. Nimse, T. Kim, Biological applications of functionalized calixarenes, Chem. Soc.
Rev. 42 (1) (2013) 366–386.
[37] R.P. Dings, J.I. Levine, S.G. Brown, L. Astorgues-Xerri, J.R. MacDonald, T.R. Hoye,
E. Raymond, K.H. Mayo, Polycationic calixarene PTX013, a potent cytotoxic agent
against tumors and drug resistant cancer, Invest. New Drugs 31 (5) (2013)
1142–1150.
[
[
[
10] F.N. Pur, Calixdrugs: calixarene-based clusters of established therapeutic drug
agents, Mol. Diversit. 20 (3) (2016) 781–787.
[
38] P. Rouge, V.S. Pires, F. Gaboriau, A. Dassonville-Klimpt, J. Guillon,
S.D. Nascimento, J.-M. Leger, G. Lescoat, P. Sonnet, Antiproliferative effect on
HepaRG cell cultures of new calix [4] arenes, J. Enzyme Inhib. Med. Chem. 25 (2)
(2010) 216–227.
11] F. Perret, A.W. Coleman, Biochemistry of anionic calix[n]arenes, Chem. Commun.
4
7 (26) (2011) 7303–7319.
12] G.M.L. Consoli, G. Granata, G. Fragassi, M. Grossi, M. Sallese, C. Geraci, Design and
synthesis of a multivalent fluorescent folate–calix [4] arene conjugate: cancer cell
penetration and intracellular localization, Org. Biomol. Chem. 13 (11) (2015)
[
39] L. Latxague, F. Gaboriau, O. Chassande, J.-M. Leger, V. Pires, P. Rouge,
A. Dassonville-Klimpt, S. Fardeau, C. Jarry, G. Lescoat, Antiproliferative effect on
HepaRG cell cultures of new calix [4] arenes Part II, J. Enzyme Inhibition Med.
Chem. 26 (2) (2011) 204–215.
3
298–3307.
[
[
13] S. Karakurt, T.F. Kellici, T. Mavromoustakos, A.G. Tzakos, M. Yilmaz, Calixarenes in
lipase biocatalysis and cancer therapy, Curr. Org. Chem. 20 (10) (2016) 1043–1057.
14] E. Akceylan, A. Uyanik, S. Eymur, O. Sahin, M. Yilmaz, Calixarene-proline func-
tionalized iron oxide magnetite nanoparticles (Calix-Pro-MN): an efficient recycl-
able organocatalyst for asymmetric aldol reaction in water, Appl. Catal. A: Gen. 499
[40] 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 poly-
hydroxyamine derivatives, Eur. J. Med. Chem. 123 (2016) 21–30.
[41] E. Adams, Metabolism of proline and of hydroxyproline, International Review of
Connective Tissue Research, Elsevier, 1970, pp. 1–91.
(
2015) 205–212.
[42] J.M. Phang, The regulatory functions of proline and pyrroline-5-carboxylic acid,
Current Topics in Cellular Regulation, Elsevier, 1985, pp. 91–132.
[43] J.M. Phang, Proline metabolism in cell regulation and cancer biology: recent ad-
vances and hypotheses, Antioxidants Redox Signal. (2017).
[
[
[
[
15] A. Uyanik, M. Bayrakci, S. Eymur, M. Yilmaz, Upper rim-functionalized calix [4]
arene-based l-proline as organocatalyst for direct asymmetric aldol reactions in
water and organic media, Tetrahedron 70 (49) (2014) 9307–9313.
16] S. Eymur, E. Akceylan, O. Sahin, A. Uyanik, M. Yilmaz, Direct enantioselective aldol
reactions catalyzed by calix [4] arene-based L-proline derivatives in the water,
Tetrahedron 70 (30) (2014) 4471–4477.
[44] J.M. Phang, W. Liu, O. Zabirnyk, Proline metabolism and microenvironmental
stress, Annu. Rev. Nutr. 30 (2010) 441–463.
[
45] S.N. Dixit, J.M. Seyer, A.H. Kang, Covalent structure of collagen: amino-acid se-
quence of chymotryptic peptides from the carboxyl-terminal region of α2-CB3 of
chick-skin collagen, Eur. J. Biochem. 81 (3) (1977) 599–607.
17] S.D. Brown, J.A. Plumb, B.F. Johnston, N.J. Wheate, Folding of dinuclear platinum
anticancer complexes within the cavity of para-sulphonatocalix [4] arene, Inorg.
Chim. Acta 393 (2012) 182–186.
[46] D.Y. Fu, T. Lu, Y.X. Liu, F. Li, M.I. Ogden, Y. Wang, Y. Wu, M. Mocerino,
Enantioselective Inhibition of human papillomavirus L1 pentamer formation by
chiral-proline modified Calix [4] arenes: targeting the protein interface,
ChemistrySelect 1 (19) (2016) 6243–6249.
18] M. Oguz, A.A. Bhatti, S. Karakurt, M. Aktas, M. Yilmaz, New water soluble Hg2+
selective fluorescent calix [4] arenes: synthesis and application in living cells
imaging, Spectrochim. Acta Part A: Mol. Biomol. Spectrosc. 171 (2017) 340–345.
19] P. Neri, J.L. Sessler, M.-X. Wang, Calixarenes and Beyond, Springer, 2016.
20] R.V. Rodik, V.I. Boyko, V.I. Kalchenko, Calixarenes in bio-medical researches, Curr.
Med. Chem. 16 (13) (2009) 1630–1655.
[
[
[
47] E.M. Collins, M.A. McKervey, E. Madigan, M.B. Moran, M. Owens, G. Ferguson,
S.J. Harris, Chemically modified calix [4] arenes. Regioselective synthesis of 1, 3-
(
distal) derivatives and related compounds. X-Ray crystal structure of a diphenol-
[
21] M. Shinde, R. Khurana, N. Barooah, A. Bhasikuttan, J. Mohanty, Metal ion-induced
supramolecular p K a tuning and fluorescence regeneration of ap-sulfonatocalix-
arene encapsulated neutral red dye, Org. Biomol. Chem. 15 (18) (2017) 3975–3984.
22] S. Sayin, E. Akoz, M. Yilmaz, Enhanced catalysis and enantioselective resolution of
dinitrile, J. Chem. Soc., Perkin Trans. 1 (12) (1991) 3137–3142.
[48] F. Arnaud-Neu, E.M. Collins, M. Deasy, G. Ferguson, S.J. Harris, B. Kaitner,
A.J. Lough, M.A. McKervey, E. Marques, Synthesis, X-ray crystal structures, and
cation-binding properties of alkyl calixaryl esters and ketones, a new family of
[
7

M. Oguz, et al.
Bioorganic Chemistry xxx (xxxx) xxxx
macrocyclic molecular receptors, J. Am. Chem. Soc. 111 (23) (1989) 8681–8691.
functionalised calix [4] arene: an anion-triggered hydrogelator, Chem. Commun. 33
[
[
49] A. Yilmaz, B. Tabakci, E. Akceylan, M. Yilmaz, Synthesis and dichromate anion
(2008) 3900–3902.
extraction ability of p-tert-butylcalix [4] arene diamide derivatives with different
binding sites, Tetrahedron 63 (23) (2007) 5000–5005.
[52] S. Karakurt, Modulatory effects of rutin on the expression of cytochrome P450s and
antioxidant enzymes in human hepatoma cells, Acta Pharmaceut. 66 (4) (2016)
491–502.
50] K.J. Smith, J.D. Wilcox, G.E. Mirick, L.S. Wacker, N.S. Ryan, D.A. Vensel,
R. Readling, H.L. Domush, E.P. Amonoo, S.S. Shariff, Calix [4] arene, calix [4]
resorcarene, and cyclodextrin derivatives and their lanthanide complexes as chiral
NMR shift reagents, Chirality: Pharmacol. Biol., Chem. Consequences Mol.
Asymmet. 15 (S1) (2003) S150–S158.
[53] I. Vermes, C. Haanen, H. Steffens-Nakken, C. Reutellingsperger, A novel assay for
apoptosis flow cytometric detection of phosphatidylserine expression on early
apoptotic cells using fluorescein labelled annexin V, J. Immunol. Methods 184 (1)
(1995) 39–51.
[
51] T. Becker, C.Y. Goh, F. Jones, M.J. McIldowie, M. Mocerino, M.I. Ogden, Proline-
8