New polyazamacrocyclic 3-hydroxy-4-pyridinone based ligands for iron depletion antitumor activity

Article abstract of DOI:10.1016/j.bioorg.2020.103574

Iron depletion is an efficient strategy for the development of anticancer agents. In an effort to develop efficient chelators, two new 3-hydroxy-4-pyridinone based polyazamacrocycles 1e and 2e were designed and synthesized. A preliminary study of the liga

Full text of DOI:10.1016/j.bioorg.2020.103574

Journal Pre-proofs
New polyazamacrocyclic 3-hydroxy-4-pyridinone based ligands for iron de-
pletion antitumor activity
Xiaoguang Liu, Xiuxiu Dong, Chuanchuan He, Xiaojuan Zhang, Guangya
Xiang, Xiang Ma
PII:
S0045-2068(19)31550-0
DOI:
https://doi.org/10.1016/j.bioorg.2020.103574
YBIOO 103574
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
11 October 2019
6 January 2020
7 January 2020
Please cite this article as: X. Liu, X. Dong, C. He, X. Zhang, G. Xiang, X. Ma, New polyazamacrocyclic 3-
hydroxy-4-pyridinone based ligands for iron depletion antitumor activity, Bioorganic Chemistry (2020), doi:
https://doi.org/10.1016/j.bioorg.2020.103574
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New polyazamacrocyclic 3-hydroxy-4-pyridinone based ligands for iron depletion
antitumor activity
Xiaoguang Liu a, Xiuxiu Dong b, Chuanchuan He a, Xiaojuan Zhang a, Guangya Xiang
a,* and Xiang Ma a,*
a
School of Pharmacy, Tongji Medical College, Huazhong University of Science and
Technology, 13 Hangkong Road, Wuhan 430030, P. R. China
b I Ward of Urology, Tongji Hospital, Tongji Medical College, Huazhong University
of Science and Technology, 1095 Jiefang Avenue, Wuhan 430030, P. R. China
* Corresponding authors.
Email addresses: gyxiang1968@hotmail.com (G. X.) and xiangma@hust.edu.cn (X.
M.)
1

Abstract
Iron depletion is an efficient strategy for the development of anticancer agents. In an
effort to develop efficient chelators, two new 3-hydroxy-4-pyridinone based
polyazamacrocycles 1e and 2e were designed and synthesized. Herein, a preliminary
study of the two ligands was carried out to investigate their iron chelating capability
and anti-tumor activity. Chelating kinetics revealed that the ligands exhibited excellent
iron depletion capacity in neutral and acidic NH4OAc buffer solution. Moreover, MTT
assay showed that the new ligands displayed potent inhibitory activity in the
proliferation of HepG2 cells. In particular, the increment of hydroxypyridione units on
the polyazamacrocycle promote iron chelating capability and improved the anti-tumor
ability by offering additional chelating sites and lipophilicity. These results indicate that
two novel compounds may possess the therapeutic potential in the treatment of cancer
through scavenging cellular iron.
Keywords: iron depletion; hydroxypyridinones; chelating; antiproliferative activity
2

1. Introduction
Iron is an essential metal for most live organisms as it plays an important role in
many cellular processes [1]. For instance, appropriate iron promotes energy generation
and oxygen transport in living cells [2,3]. However, overloaded iron can also damage
tissues via the Haber-Weiss reaction that result in produce deleterious reactive oxygen
species (ROS) when present in excess [4]. Previous studies have shown that iron-
overloading caused diseases including β-thalassemia Friedreich’s Ataxia and cancer [5].
In addition, excess iron may facilitate the rate of DNA synthesis in neoplastic cells due
to they demand more iron than normal cells [6].
Iron is insoluble in the natural environment, and some natural iron chelators,
known as siderophores, have been used to fight against overloaded iron in organisms.
Desferrioxamine (DFO, Fig. 1), a bacterial siderophore separated from Streptomyces
pilosus, is one of the most effective iron clinical application chelators on treating iron-
overload diseases [7]. Moreover, DFO has been found to possess antiproliferative
activity in previous literatures [8]. Nevertheless, the efficiency of DFO was limited by
the high hydrophilicity and short plasma half-life that lead to low membrane
permeability [9]. Desferrithiocin (DFT, Fig. 1), a tridentate siderophore isolated from
Streptomyces antibioticus, shows excellent activity on the treatment of iron-overload
diseases [10]. Unlike DFO, DFT is a tridentate chelator that binds Fe (III) with the
atomic donors through its phenolic oxygen, carboxylate oxygen, and thiazole nitrogen.
Despite much promise of DFT as a medicine to treat iron-overloaded disease, a previous
study has reported that it presented severe nephrotoxicity [11]. The limitations led to
3

search for new iron chelators as potential anti-cancer agents.
N
O
O
OH
HN
OH
NH
O
HO
N
O
N
N
N
O
S
COOH
OH
NH2
DFO
DFT
COOH
O
OH
N
HOOC
N
N
COOH
N
HOOC
COOH
DTPA
DFP
OH
N
O
O
O
OH
N
N
HO
N
N
N
N
N
N
N
HO
N
N
O
N
HO
OH
O
N
OH
O
O
HP-TACD
HP-TACN
Fig.1 Chemical structures of iron chelators, including DFO, DFT, DTPA, Deferiprone, HP-TACD
and HP-TACN
Different from inartificial siderophores, a vast number of artificial iron chelators
has been synthesized, and they also exhibited remarkable iron clearing activity and
excellent antiproliferative activity. Such as Diethylenetriamine pentaacetic acid (DTPA,
4

Fig. 1), a polyaminocarboxylate chelator which has been demonstrated to possess
anticancer properties against neuroblastoma and ovarian carcinoma cell lines [12]. In
contrast to the membrane permeable iron chelator DFO, diethylenetriamine pentaacetic
acid (DTPA) shows high membrane impermeability [12]. Deferiprone (DFP) (Fig. 1),
a bidentate HOPO iron depletion agent, is the only clinically available peroral agent for
the treatment of β-thalassemia in Europe. However, there exists an inevitable
controversy between preclinical and clinical on the safety and efficiency of this agent
[13].
HOPOs and their derivatives are ideal chelator which shows high affinity for iron
[14]. With the current interest in HOPO-based iron chelator for the treatment of cancer,
and in attempt to promote their chelating properties with iron, we designed and
synthesized two new 3-hydroxy-4-pyridinone based polyazamacrocycles ligands.
HOPOs are a class of benzene-counterpart with hexahydroxy N-containing heterocyclic,
which plays an important role in the development of coordination chemistry, especial
their utilization in the field of therapeutics [15]. There are three prototype HOPOs (Fig.
2) that have long been studied over the past decade: 3-hydroxy-2-pyridinone (3,2-
HOPO), 1-hydroxy-2-pyridinone (1,2-HOPO) and 3-hydroxy-4-pyridinone (3,4-
HOPO). Due to the virtue of their two anionic oxygen donors and their ability to form
dπ-pπ bonds, HOPO ligands show the superb ability to bind with ferric, zirconium,
gallium as well as actinide ions [15].
Among the three types of hydroxypyridinones isomers, 3,4-HOPO exhibits
relatively higher pKa and shows the strongest chelating capability with metal iron at
5

physiological pH (Fig. 2) [16,17]. Besides, 3,4-HOPO possesses a higher affinity for
hard acid metal ions than bio-related divalent soft acid metal ions [18,19]. Meanwhile,
3,4-HOPO stays fairly steady under physiological conditions, and it can be easily
derived because of the Nitrogen atom in the ring. Compared with DOTA, DTPA like
carboxylic-acid based ligands, HOPOs should be more likely to have good oral activity,
because of the higher lipophilicity. Lipophilicity could alter the cell permeability,
biodistribution, and metabolism, and more lipophilic ligands are more likely to act in
intracellular metal ion pools in the cancer cells.
OH
OH
OH
O
O
O
O
O
pKa2 = -0.8
pKa1 = 5.8
N
N
N
N
1,2-HOPO
OH
OH
OH
O
O
O
O
O
pKa2 = 0.1
pKa1 = 8.7
N
H
N
N
N
H
H
H
3,2-HOPO
OH
OH
O
O
O
O
O
pKa2 = 3.4
HN
pKa1 = 9.0
N
HN
N
H
OH
H
3,4-HOPO
Fig. 2 Chemical structures of HOPOS and their pKa values [16,17]
Extending the focus to the functionalization of 3,4-HOPO, the preferred properties
of the chelators which contain three or four HOPO molecular scaffolds give us
considerable interest in synthesis of nitrogen-heterocyclic hydroxypyridinone to
investigate their potential in the treatment of cancer through depleting cellular iron.
Herein, for the first time, we report an efficient and simplified way to the synthesis of
6

TACD
tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(ethane-2,1-diyl))tetrakis(3-hydroxy-2-
methylpyridin-4(1H)-one) (HP-TACD) and 1,1',1''-((1,4,7-triazonane-1,4,7-
triyl)tris(ethane-2,1-diyl))tris(3-hydroxy-2-methylpyridin-4(1H)-one) (HP-TACN)
and
TACN
based
hydroxypyridinones
1,1',1'',1'''-((1,4,7,10-
(Fig. 1) and their metal chelating kinetics and antiproliferative activity for the potential
uses in tumor therapy and the other biomedical applications.
2. Experimental
2.1. Chemistry
Unless otherwise stated, all reagents were purchased from Bide Pharmatech Ltd.
(Bide, Shanghai, China) and used without further purification. Column chromatography
was performed with silica gel (200-300 mesh) purchased from Qingdao Haiyang
Chemical Co. Ltd. Thin-layer chromatography was carried out with Merck silica gel
GF254 plates and visualized by exposure to UV light (254 nm). All the products are
1
characterized by H NMR, 13C NMR and ESI-MS, which were compared with the
1
previously reported data. H NMR and 13C NMR spectra were recorded at room
temperature on a Bruker III HD 400 instrument at 400 MHz. Mass spectra were
recorded on Advion Expression L TLC-MS (Bohui China).
4.1.1 General procedure for the synthesis of 1c-2e
Block 1a (3-(benzyloxy)-2-methyl-4H-pyran-4-one) and 1b (3-(benzyloxy)-1-(2-
hydroxyethyl)-2-methylpyridin-4(1H)-one) were prepared according to a published
literature [20].
7

3-(benzyloxy)-1-(2-bromoethyl)-2-methylpyridin-4(1H)-one (1c)
NBS was added to a solution of 1b (3g, 1.0 equiv) and PPh3 (3.7g, 1.1 equiv) in
50 ml MeCN at 0 oC, in portion, over 1h. The mixture was warmed to r.t. and stirred
for 3h. The reaction mixture was concentrated in vacuo and purified by flash column
chromatograph on silica gel (dichloromethane /methanol as the eluent) to provide 1c
brown oil (3.16 g, 85% yield). 1H NMR (400 MHz, DMSO-d6): δ 7.64 (d, J = 7.6 Hz,
1H), 7.39 (m, 5H), 6.17 (d, J = 7.6 Hz, 1H), 5.01 (s, 2H), 4.29 (t, J = 6.5 Hz, 2H), 3.74
(t, J = 6.5 Hz, 2H), 2.20 (s, 3H). 13C NMR (101 MHz, DMSO-d6): δ 171.9 (s), 140.8
(s), 134.0 (s), 137.7 (s), 128.3, 127.8 (s), 115.7, 71.9, 53.0, 40.15, 39.9, 39.7, 39.5, 39.3,
+
39.1, 38.9, 31.6, 12.0. ESI-MS: calculated for C15H16NO2Br, [M+H] m/z = 322.0,
found 322.0.
1,1',1'',1'''-((1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(ethane-
2,1-diyl))tetrakis(3-(benzyloxy)-2-methylpyridin-4(1H)-one) (1d)
1,4,7,10-tetraazacyclododecane (445 mg, 1.0 equiv) and K2CO3 (3.6 g, 10.0equiv)
was dissolved in 100 ml MeCN, a solution of 1c (4.0 g, 4.8 equiv) in 30 ml MeCN was
o
slowly added at 0 C, then the mixture was heated under gentle reflux for 48h. The
mixture was filtered and the filtrate concentrated in vacuo and purified by flash column
chromatograph on silica gel (dichloromethane /methanol as the eluent) to provide 1d
light yellow solid (2.01 g, 68% yield). 1H NMR (400 MHz, MeOD): δ 7.53 (d, J = 7.4
Hz, 4H), 7.38 (m, 20H), 6.43 (d, J = 7.4 Hz, 4H), 5.06 (s, 8H), 4.14 (t, J = 6.4 Hz, 8H),
3.75 (t, J = 6.4 Hz, 8H), 2.68 (d, J = 6.0 Hz, 16H), 2.25 (s, 12H). 13C NMR (101 MHz,
MeOD): δ 179.2, 175.2, 145.1, 141.4, 138.6, 130.1, 129.6, 117.39, 74.6, 52.1, 49.6,
8

49.4, 49.2, 49.0, 48.8, 48.6, 48.4, 38.7, 28.9, 12.8. ESI-MS: calculated for C68H80N8O8,
[M+H] + m/z = 1137.6, found 1137.6.
1,1',1'',1'''-((1,4,7,10-tetraazacyclododecane-1,4,7,10-tetrayl)tetrakis(ethane-
2,1-diyl))tetrakis(3-hydroxy-2-methylpyridin-4(1H)-one) (1e)
To a solution of 1d in 2 ml MeOH (1.137 g, 1.0 equiv) was added 10% Pd/C (430
mg, 40% equiv). The reaction vessel was bubbled with H2 and stirred vigorously at
room temperature for 5h. The reaction mixture was filtered and the filtrate was
1
concentrated in vacuo to provide 1e a white solid (699.24 mg, 90% yield). H NMR
(400 MHz, MeOD): δ 7.45 (d, J = 7.2 Hz, 4H), 6.35 (d, J = 7.2 Hz, 4H), 4.21 (t, J = 6.5
13
Hz, 8H), 3.82 (t, J = 6.5 Hz, 8H), 2.68 (s, 16H), 2.49 (s, 12H). C NMR (101 MHz,
MeOD): δ 179.2, 139.2, 112.7, 52.0, 49.6, 49.4, 49.2, 49.0, 48.8, 48.6, 48.4, 38.9, 28.99,
11.9. ESI-MS: calculated for C40H56N8O8, (M-H) - m/z = 775.4, found 775.4.
1,1',1''-((1,4,7-triazonane-1,4,7-triyl)tris(ethane-2,1-diyl))tris(3-(benzyloxy)-
2-methylpyridin-4(1H)-one) (2d)
1,4,7-triazonane (129 mg, 1.0 equiv) and K2CO3 (3.6 g, 10.0equiv) was dissolved
in 100 ml MeCN, a solution of 1c (1.16 g, 3.6 equiv) in 15 ml MeCN was slowly added
o
at 0 C, then the mixture was heated under gentle reflux for 48h. The mixture was
filtered and the filtrate concentrated in vacuo and purified by flash column
chromatograph on silica gel (dichloromethane /methanol as the eluent) to provide 2d
light yellow solid (545.9 mg, 64% yield). 1H NMR (400 MHz, MeOD): δ 7.54 (d, J =
7.4 Hz, 3H), 7.43 (dd, J = 7.8, 1.6 Hz, 6H), 7.34 (dd, J = 7.9, 1.8 Hz, 9H), 6.43 (d, J =
7.4 Hz, 3H), 5.06 (s, 6H), 4.14 (t, J = 6.4 Hz, 6H), 3.75 (t, J = 6.4 Hz, 6H), 2.67 (s,
9

12H), 2.25 (s, 9H). 13C NMR (101 MHz, MeOD): δ 179.2, 175.2, 147.4, 145.1, 141.4,
138.5, 130.2, 129.4, 117.4, 74.6, 52.2, 38.7, 29.0, 12.8. ESI-MS: calculated for
C51H60N6O6, [M+H] + m/z = 853.5, found 853.5.
1,1',1''-((1,4,7-triazonane-1,4,7-triyl)tris(ethane-2,1-diyl))tris(3-hydroxy-2-
methylpyridin-4(1H)-one) (2e)
To a solution of 2d in 2 ml MeOH (426.5 mg, 1.0 equiv) was added 10% Pd/C
(159.6 mg, 30% equiv). The reaction vessel was bubbled with H2 and stirred vigorously
at room temperature for 5h. The reaction mixture was filtered and the filtrate was
concentrated in vacuo to provide 2e a white solid (279.7 mg, 96% yield). 1H NMR (400
MHz, MeOD): δ 7.41 (d, J = 7.2 Hz, 4H), 6.31 (d, J = 7.2 Hz, 4H), 4.17 (t, J = 6.5 Hz,
6H), 3.78 (t, J = 6.5 Hz, 6H), 2.64 (s, 12H), 2.45 (s, 9H). 13C NMR (101 MHz, MeOD):
δ 179.2, 139.2, 112.73 (s), 52.0, 38.9, 20.0, 11.9. ESI-MS: calculated for C30H42N6O6,
[M-H]- m/z = 581.3, found 581.3.
2.2. General Procedure for Complexation Kinetics Using AAIII
All reagents were purchased and used as metals basis grade. NH4OAc buffer was
prepared using ultrapure water (18 MΩ cm). Metal-free stock solutions of all NH4OAc
buffers (0.15 M, pH 4, 6 and 7) were prepared using Chelex-100 resin. Chelex resin (5
g) was added into the buffer solution (500 mL), and the mixture was shaken for 1 h in
a shaker (Eberbach), stored in the refrigerator overnight, and filtered through
microfiltration membrane (0.22 µM). Fifteen micromolar Arsenazo III (AAIII)
solutions in 0.15 M NH4OAc (pH 4, 6 and 7) were prepared by adding AAIII (5.82 mg)
to the NH4OAc buffer (500 mL).
10

To a solution of AAIII (15 µM, 50 mL) in NH4OAc buffers (0.15 M, pH 4, 6 and
7) was added freshly prepared ferric chloride solution (60.8 µL, 1 mg/mL) and then
shaken for 2 h in a shaker, then obtain the Fe (III)-AAIII solution with a concentration
of 7.5 µM. 2 mL of AAIII solution in a sample cell served as blank control group which
was zeroed in the UV-visible spectrometer. 2 mL of Fe (III)-AAIII solution was then
added to the other sample cells, and the absorbance of the solution at 625 nm was
measured for 0.5 h. After this step completed, 10 µL of Fe (III)-AAIII solution from
each sample cell was removed, and then 10 µL of prepared solution of the ligands DFO,
DFP, HP-TACD and HP-TACN was added into the sample cells, respectively, and
slightly shake. All kinetics spectra were collected every 30 s at 625 nm for 0.5h by
PerkinElmer UV/VIS spectrophotometer Lambda 365(PerkinElmer, Waltham, MA,
USA). All experiment was performed at least in triplicate.
2.3. Solution Stability
All reagents were purchased and used as metals basis grade. Fe(III)-complexes
were prepared in NH4OAc buffer (0.1M pH∼4) at molar ratios of Fe(III):chelator=1:1
at 60 μM concentrations, after incubated at room temperature for 30 min, the complexes
was blended with a same volume of PBS solution, the final concentration of the
complexes under study was 30 μM. The absorption spectra of theses complexes in PBS
buffer were then monitored at 0 h, 24 h, 48 h and 72 h by PerkinElmer UV/VIS
spectrophotometer Lambda 365(PerkinElmer, Waltham, MA, USA). All experiment
was performed at least in triplicate.
2.4. Antiproliferative Assay
11

2.4.1. Cell culture
Human hepatoma cell line HepG2 and hepatocyte cell line LO2 were purchased
from the China Center for Type Culture Collection at Wuhan Unicersity (Wuhan,
China). The cells were cultured in dulbecco's modified eagle medium (DMEM)
supplemented with 10% fetal bovine serum (FBS) and 1% antibiotic/antimycotic
solution in a humidified atmosphere with 5% CO2 at 37°C. Culture medium was
changed every 3-4 days. The cells were sub-cultured when 80% confluent.
2.4.2. Antiproliferative activity
The cells were seeded in 96-well plates at a density of 10000 cells/well in 0.1 ml
complete medium and left to attach 24 h. Varying concentrations of the test compounds
in the final volume of 0.1 mL complete medium were then added in at least six series
dilutions and incubated for 72 h. In addition, control samples contained complete
medium only without test compounds. To measure cell proliferation, the MTT (3-(4,5-
dimethylthiazol-2-yl)-2,5 diphenyl tetrazolium) reduction assay was used according to
the published procedures [21]. Briefly, at the end of incubation period, an aliquot (20
µL) of the MTT (5 mg/mL) was added into all the wells and the plate was incubated for
additional 4 h at 37°C. After the incubation, the generation of formazan crystals in wells
were dissolved with 0.15 mL of dimethylsulfoxide, and optical absorbance at 490 nm
was then recorded with an enzyme-linked immunosorbent assay (ELISA) microtiter
plate reader (Biotek). Each experiment was performed at least in triplicate.
Antiproliferative activity of the test compounds was expressed as the fraction of optical
densities of treated cells relative to the untreated solvent controls.
12

2.4.3. Metal ion supplementation studies
HepG2 cells were seeded in 96-well plates at a density of 10000 cells/well in 0.1
ml complete medium and incubated for 24 h. The cells were treated with 50 µM of test
compounds in the presence or absence of 50 µM of KCl, NaCl, MgCl2 and FeCl3 in the
final volume of 0.1 mL complete medium and incubated for 72 h. All experiment was
performed at least in triplicate. At the end of the incubation, cell proliferation rates were
determined by MTT assay.
3. Results and discussion
3.1. Synthesis
Initially, the functionalized 3,4-HOPO was synthesized via a direct aminolysis of
3-hydroxy-2-methyl-4-pyrone (maltol) [20]. However, the yield was quite low even
after a prolonged reaction time of 96h, which could be due to the large hydrocarbon
chains substituted on the amine and the difficulties in the following purification [22].
In order to increase the yield, a protection method was applied using benzyl group to
protect the amines with large hydrocarbon chains. As depicted in Scheme.1, the
synthesis of 1a was carried out via a Williamson ether synthetic method [23]. The
hydroxyl group of maltol was functionalised by introducing benzyl to afford 3-
(benzyloxy)-2-methyl-4H-pyran-4-one (1a) as a yellow oil in 87% yield. After
converting the benzyl protected pyrone to pyridone with 2-aminoethanol, an expected
pyridine product 1b was obtained in a yield of 30%. The low yield might be due to the
presence of methyl functional group and the increased steric hindrance which affects
13

the ring-opening process. The following bromination reaction was carried out at room
temperature and the reaction was monitored by TLC and completed within 3 h to give
1c in 85% yield.
With the fragment 1c in hand, the next nucleophilic substitution reaction was
carried out based on the previous literature report [24]. However, the conversion ratio
of this reaction was rather low. Thus, we screened the impact factors of bases,
temperature, reaction time and solvent on the nucleophilic substitution reaction, as
shown in Table 1. And the optimal reaction condition was found to be using K2CO3 as
the base at 60 oC in MeCN for 48h to obtain 1d in the yield up to 68%. The following
reduction reaction occurs in one step with Pd/C under a hydrogen atmosphere to afford
1e in an excellent yield without purification [20].
O
O
O
O
O
O
O
O
c
a
b
Br
OH
O
HO
+
+
NH2
N
N
OH
Br
1a
1b
1c
O
O
O
O
O
N
N
O
N
OH
N
N
HO
N
N
N
N
N
N
N
N
d
e
N
NH HN
NH HN
N
O
O
O
N
O
N
HO
OH
O
O
O
O
1d
1e
HN
Br
NH
OH
N
O
O
N
H
e
N
O
d
O
N
N
N
O
N
HO
N
N
N
N
O
N
O
N
OH
O
O
2d
2e
Scheme 1 Synthesis of HP-TACD and HP-TACN. (a) NaOH, MeOH/H2O, reflux 24h; (b)
EtOH/H2O, 2-aminoethanol, reflux 48h; (c) MeCN, PPh3, NBS, 0 oC-r.t.; (d) MeCN, K2CO3, 60 oC,
14

48h; (e) MeOH, Pd/C, H2, 5h.
The synthesis of 2e (Scheme.1) were similar to that of 1e. 2d was obtained in the
o
optimal conditions consist of K2CO3, 1c and TACN at 60 C in MeCN for 48h. The
following reduction occurs in one step with Pd/C under a hydrogen atmosphere to
afford 2e in 96% yield without purification.
Table 1 Optimization of reaction timea
Entry
Base
Solvent
MeCN
MeCN
MeCN
MeCN
MeCN
MeOH
MeOH
MeOH
MeOH
MeOH
MeOH
T/oC
r.t.
40
60
80
60
60
60
60
60
60
60
t/h
12
12
12
12
12
12
12
12
24
48
72
Yield/%a
trace
trace
36
1
DIEA
DIEA
DIEA
DIEA
Et3N
2
3
4
28
5
23
6
Et3N
40
7
Na2CO3
K2CO3
K2CO3
K2CO3
K2CO3
43
8
47
9
55
10
11
68
67
a Reaction conditions: 1f (0.1 mmol, 1.0 equiv), 1c (0.48 mmol, 4.8 equiv), base (1mmol, 10
equiv), solvent (2 mL)
3.2. Kinetic and stability studies
3.2.1. AAIII complexation kinetics study
According to the procedure reported previously [25], the kinetics of
complexation between metal cation with new ligands could be measured by a
spectroscopy. As known, 2,7-bis(2-Arsonophenylazo)-1,8-dihydroxynaphthalene-3,6-
disulfonic acid (Arsenazo III, AAIII), a diazo dye, was known to chelate with different
metals to form weak complexes, which have UV–vis absorbances maximum between
15

620-670 nm (Fig. 3A). However, the uncoordinated dye AAIII itself showed little
absorbance at this wavelength range. Upon added to an AAIII-metal complex-
contained solution, the ligand could have a competing reaction with AAIII for the metal.
Once the ligand has a stronger capacity of metal binding than AAIII, the metal
dissociates from the AAIII complex and form a complex with the added ligand,
resulting in the decrease of the absorbance at ∼625 nm. The visible absorption
spectrum for the Fe (III) - AAIII solution was carried out in the absence and in the
presence of HP-TACD and HP-TACN by time scan measurements on a
spectrophotometer at ∼625 nm in NH4OAc buffers solution respectively. For
comparison, the chelation kinetics for DFO and Deferiprone with Fe (III) were also
measured, which are known as widely used iron-chelating agents in the clinic
application.
Acidic microenvironment is one of the main characteristics of solid tumors [26],
thus it is important to find out the complexation reaction kinetics of our newly
synthesized ligand in both tumor microenvironment comparable acidic condition at pH
6 and the extremely acidic condition at pH 4, as well as in neutral pH 7. The kinetic
measurement was observed through a direct reaction of HP-TACD, HP-TACN, DFP
and DFO with Fe (III) - AAIII solution. Since the number of donor atoms differ from
chelator to chelator, the reaction rate present relatively large differences. As shown in
Fig. 3B, as a preeminent iron chelator, DFO displayed pertinent chelating properties
with Fe (III) at pH ∼7 in NH4OAc buffer solution but reached the reaction equilibration
in 10mins. DFP was revealed to exhibit slight enhanced complexation with Fe (III) and
16

the equilibration time of the reaction was up to 17mins. However, HP-TACD and HP-
TACN react about 2∼3 times faster than DFO and 5∼7 times faster than Deferiprone
under the same condition, the absorbance measurement for HP-TACD and HP-TACN
with Fe (III) - AAIII were 0.004 and 0.02 respectively, the fast kinetics measurement
of the new ligands might be due to the rigid structure of the macrocyclic that make a
more stable Fe-chelate. At lower pH 6 (Fig. 3C), little changes on equilibrium time and
absorbance measurement were observed when compared with the kinetics at pH 7. Both
HP-TACD and HP-TACN exhibited consistently better complexation kinetics than
DFO and DFP. The advantages of the newly synthesized ligands were slightly enhanced
when the kinetics was carried out at pH 4 (0.15 M, NH4OAc, pH 4, Fig. 3D). While all
the chelators showed increased complexation kinetics with lower equilibrium
absorbance and faster complexation reaction, the new ligands exhibited faster
equilibrium time at 2.5 min and 5.5 min for HP-TACD and HP-TACN, producing
saturation curve at A625 0.003 and 0.014 respectively. Under the same condition, DFO
and DFP reached the reaction equilibrium at 8 min and 13 min with higher absorbances
(A625 0.04 and 0.07 for DFO and DFP respectively). On the basis of these findings
indicates that the ring size and the coordination number control the complexation rate.
For the new ligands, complexation kinetic rate increases with an increase in ring size.
Our data indicate the new ligands displayed rapid binding to Fe (III).
A
B
17

C
D
Fig. 3 A UV–vis absorption spectra of AAIII, Fe (III)-AAIII and Fe (III) (50 μM) in NH4OAc buffer
(0.15 M) at pH 7; Plot of absorbance at 625 nm versus time of Fe(III)-AAIII( ◇ ), Fe(III)-
Deferiprone (◆), Fe(III)- DFO (□), Fe(III)- HP-TACN (▼) and Fe(III)- HP-TACD (▲): B 0.15M
NH4OAc, r.t., pH 7; C 0.15M NH4OAc, r.t., pH 6; D 0.15M NH4OAc, r.t., pH 4
3.2.2. Solution Stability
Due to the fact that hydrolysis reaction is prone to occur in metal complexes, a
UV-vis spectroscopy method was developed to evaluate the stability of the Fe (III)-
complexes in phosphate buffer saline (PBS) [27]. The Fe (III)-complex samples were
prepared in 0.1 M NH4OAc (5mL, pH 4) at molar ratios of 1:1 with concentrations of
60 μM. After the incubation at room temperature for 30 min, the solution was mixed
with PBS solution. The absorption spectra of these complexes in PBS buffer were
monitored at 0 h, 24 h, 48 h and 72 h. As seen in Fig. 4, wavelength-absorption curves
18

of Fe (III)-HP-TACD and Fe (III)-HP-TACN samples did not show any significant
changes even after 72h, revealing that the new Fe (III)-complexes have quite good
stabilities in PBS buffer. By contrast, significant dissociations were observed for Fe
(III)-DFP and Fe (III)-DFO complexes in FBS after 48 h and 24 h incubation,
respectively. Significant absorbance drop was observed at 72 h for Fe (III)-DFP. And
Fe (III)-DFO seemed to be even less stable than Fe (III)-DFP, which could maintain its
structure for only 24 h. Hence, our data indicate that Fe (III)-HP-TACD and Fe (III)-
HP-TACN were stable to maintain their structure for 72 h in PBS solution. However,
the reference Fe (III)-DFP only showed high stability up to 48h and the Fe (III)-DFO
were the least stable complex among them.
Fig. 4 UV-vis spectra of Fe(III)-complexes (0.03 mM) in presence of PBS (0.01M).
19

3.3. Antiproliferative study
Based on the complexation kinetics and stability studies above, the HOPO-based
macrocyclic ligands HP-TACD and HP-TACN display strong capability on iron
chelation and high stability in PBS solution, which makes a hypothesis that the ligands
may have the potential capacity for the treatment of cancer through depleting cellular
iron. According to the previous research [21], the macrocyclic chelator was found
conductive to the treatment of cancer, which were demonstrated by using MTT assay
in cancer and normal cell lines. Therefore, cell cytotoxicity of the two chelators was
determined by MTT assay in vitro. Under the same condition, DFO and DFP were set
as control group. HepG2 cells were used as the cell lines for the evaluation. The
impacts of different concentrations (0-100 μM) of DFO, DFP, HP-TACD and HP-
TACN on the proliferation inhibition of HepG2 cancer cells were assessed by MTT
assay (Fig. 5A). The IC50 of DFO and DFP was estimated to be >100 and 33 μM, while
the IC50S for HP-TACD and HP-TACN were observed at 18 and 27 μM, respectively.
To investigate the iron saturation effect on cancer cell growth, the influences of Fe
(III) concentration on anti-proliferative activity of chelators were investigated by
carrying out iron supplementation studies [12,28]. HepG2 cells were studied with 50
μM of DFO, DFP, HP-TACD, and HP-TACN in the presence of different
concentrations of Fe (III) (0-100 μM). As shown in Fig. 5B, addition of Fe (III) to
HepG2 cells decreased the anti-proliferative effect of each chelator. The effect was
iron concentration dependent, and the cell viability increased to about 95% when
chelators were saturated with equal molar amount of Fe (III) at 50 μM or higher. In
20

addition, with various concentrations of the chelators, the influence of Fe (III) with
different concentration of chelators (0-100 μM) on anti-proliferative activity of the
chelators was also investigated. As seen in Fig. 5C, the inhibitory effects of the
chelators were suppressed at the concentration range between 0-50 μM with iron (50
μM) incubation. However, inhibitory effects of the chelators were revived along with
higher concentrations of chelator in the presence of Fe (III) (50 μM).
A
B
C
D
Fig.5 Effects of (A) chelators DFO(■), DFP(●), HP-TACD(▼) and HP-TACN(▲) , (B)different
concentrations of iron in presence of 50 μM of each chelator, (C) different concentrations of
chelator in presence of 50 μM of Fe (III) and (D) addition of 50 μM K+, Na+, Mg2+ or Fe3+ in
presence of 50 μM of each chelator on viability of HepG2 cancer cells.
The specificity for the high Fe (III) affinity of the new ligands was evaluated by
addition of different metal ions (K+, Na+, Mg2+, and Fe3+, 50 μM) to HepG2 cultures.
As seen in Fig. 5D, addition of excess exogenous K+, Na+, Mg2+ did not affect anti-
21

proliferative of the chelator (50 μM) versus the blank control with addition of K+, Na+,
Mg2+ alone, while cell proliferation for HP-TACD or HP-TACN was restored in the
presence of Fe3+. The data, which was in accordance with the iron supplementation
studies, indicated that the new chelators showed higher affinity for Fe (III), and the
antiproliferative activity of the new ligands was mainly due to chelation of cellular Fe
(III). The cytotoxicity assay above are consistent with the observation from Fe (III)-
AAIII assay, which support our hypothesis that the rigid structure of
polyazamacrocycles is benefit to iron complexation, and the different antiproliferative
activity of the new ligands on HepG2 cells may be due to the fundamental differences
between the size of the ring.
On the basis of the research conclusions above, the ligands shows promising
antiproliferative activity on HepG2 cells, a further study was carried out to investigate
the influence of the chelator on normal cell growth. Therefore, normal LO2 cells were
used as the cell lines for the evaluation, while HepG2 cells were set as the control. As
shown in Fig. 6, when treated the cells with DFO, DFP, HP-TACD and FP-TACN at
the concentration of 50 μM, both HP-TACD and HP-TACN exhibited significant
antiproliferative activity and reduce HepG2 cell viability to about 90% and 85%,
respectively. DFO and DFP produced about 40% and 55% inhibitory effect respectively
in the same condition. In normal hepatocytes cell lines (LO2), the new synthesized
ligands were not significantly cytotoxic at the concentration of 50 μM, which are
comparable to those of DFO and Deferiprone (50 μM). The result indicates that the
ligands have certain degree of selectivity on cancer cells over normal cells. Besides, the
22

new chelators exhibit no or slight toxic effect on normal cells. In addition, our data also
confirm the previous study that the iron-depletion therapy is an efficient way to inhibit
the proliferation of cancer cells. The result indicates that the ligands have certain degree
of selectivity on cancer cells over normal cells. Besides, the new chelators exhibit no
or slight toxic effect on normal cells.
Fig. 6 Cytotoxicity of iron chelators against HepG2 and LO2 cell lines
4. Conclusion
In this study, we have established for the synthesis a class of macrocyclic ligands
multiple substituted with 3,4-HOPO and investigated the kinetics with Fe (III) by AAIII
assay. These ligands perform outstanding complexation with Fe (III). The Fe (III)-
complexes were show high stability against PBS at least up to 72h. Also, their
cytotoxicity using HepG2 and LO2 cell lines and specificity towards iron have been
evaluated. The MTT results show that HP-TACD and HP-TACN exhibited notably
23

antiproliferative activity than DFO and Deferiprone in HepG2 cells, and the
antiproliferative activity decline significantly after chelating with Fe (III), which is also
a convincing proof of the AAIII assay. To normal cells, the new chelator showed no or
slight cytotoxicity. Additional, both HP-TACD and HP-TACN were more affinity for
Fe(III) in the presence of different metal ions. Along with the simple protocol of
synthesis and decent metal complexation, we expect that the two novel compounds can
be potentially applied in the field of cancer therapy. In vivo antitumor efficacy studies
of these chelators with their mechanisms of action are underway.
Acknowledgements
We gratefully thank the Natural Science Foundation of China grants (No.81603046,
No.81673368, No. 81703446 and No. 81973257), Independent Innovation Foundation
of Higher Education of China (2016JCTD109, 2016YXMS140 and 2016YXMS147),
National Postdoctoral Program for Innovative Talents (BX201600057), China
Postdoctoral Science Foundation Funded Project (2017M612474), the Fundamental
Research Funds for the Central Universities, HUST: 2018JYCXJJ008 for their financial
support. The authors thank Analytical & Testing Center of Huazhong University of
Science & Technology.
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Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal
relationships that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be
considered as potential competing interests:
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