Mitochondria-localizing curcumin-cryptolepine Zn(II) complexes and their antitumor activity

Article abstract of DOI:10.1016/j.bmc.2020.115948

Many metal complexes are potent candidates as mitochondrial-targeting agents. In this study, four novel Zn(II) complexes, [Zn(BPQA)Cl₂] (Zn1), [Zn(BPQA)(Curc)]Cl (Zn2), [Zn(PQA)Cl₂] (Zn3), and [Zn(PQA)(Curc)]Cl (Zn4), containing N,N-bis(pyridin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (BPQA), N-(pyridin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (PQA), and curcumin (H-Curc) were synthesized. An MTT assay showed that Zn1–Zn4 had strong anticancer activities against SK-OV-3/DDP and T-24 tumor cells with IC₅₀ values of 0.03–6.19 μM. Importantly, Zn1 and Zn2 displayed low toxicities against normal HL-7702 cells. Mechanism experiments demonstrated that probe Zn2 showed appreciable fluorescence in the red region of the spectrum, and substantial accumulation of Zn2 occurred in the mitochondria after treatment, indicating increases in Ca²⁺ and reactive oxygen species levels, loss of the mitochondrial membrane potential, and consequent induction of mitochondrial dysfunction at low concentrations. In addition, the probe Zn2 effectively (50.7%) inhibited the growth of T-24 bladder tumor cells in vivo. The probe Zn2 shows potential for use in cancer therapy while retaining the H-Curc as an imaging probe.

Full text of DOI:10.1016/j.bmc.2020.115948

Bioorg. Med. Chem. 30 (2021) 115948
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Mitochondria-localizing curcumin-cryptolepine Zn(II) complexes and their
antitumor activity
,
,
,
,
b
,
,*
Li-Qin Qina 1, Chun-Jie Lianga 1, Zhen Zhoua 1, Qi-Pin Qina *, Zu-Zhuang Weic
,
Ming-Xiong Tana, Hong Liangb
,
*
a Guangxi Key Lab of Agricultural Resources Chemistry and Biotechnology, College of Chemistry and Food Science, Yulin Normal University, 1303 Jiaoyudong Road,
Yulin 537000, PR China
b State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources, School of Chemistry and Pharmacy, Guangxi Normal University, 15 Yucai
Road, Guilin 541004, PR China
c School of Pharmaceutical Sciences, Sun Yat-sen University, Guangzhou 510006, PR China
A R T I C L E I N F O
A B S T R A C T
Keywords:
Many metal complexes are potent candidates as mitochondrial-targeting agents. In this study, four novel Zn(II)
complexes, [Zn(BPQA)Cl2] (Zn1), [Zn(BPQA)(Curc)]Cl (Zn2), [Zn(PQA)Cl2] (Zn3), and [Zn(PQA)(Curc)]Cl
(Zn4), containing N,N-bis(pyridin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (BPQA), N-(pyridin-2-
ylmethyl)benzofuro[3,2-b]quinolin-11-amine (PQA), and curcumin (H-Curc) were synthesized. An MTT assay
showed that Zn1–Zn4 had strong anticancer activities against SK-OV-3/DDP and T-24 tumor cells with IC50
Curcumin-cryptolepine
Zn(II) complexes
Cell apoptosis
Mitochondrial dysfunction
values of 0.03–6.19
μM. Importantly, Zn1 and Zn2 displayed low toxicities against normal HL-7702 cells.
Mechanism experiments demonstrated that probe Zn2 showed appreciable fluorescence in the red region of the
spectrum, and substantial accumulation of Zn2 occurred in the mitochondria after treatment, indicating in-
creases in Ca2+ and reactive oxygen species levels, loss of the mitochondrial membrane potential, and consequent
induction of mitochondrial dysfunction at low concentrations. In addition, the probe Zn2 effectively (50.7%)
inhibited the growth of T-24 bladder tumor cells in vivo. The probe Zn2 shows potential for use in cancer therapy
while retaining the H-Curc as an imaging probe.
1. Introduction
Recent studies suggested that traditional Chinese medicines
composed of metal complexes have selective activity between tumor and
Cis-platinum and its analog are widely used as therapies in a range of
cancers1–6 but exhibit various limitations7–13; thus, compounds based on
other metal ions, such as Zn, Co, Cu, Ru, and Ir14–25 have attracted
attention. Additionally, Zn is considered as an essential element for
numerous cellular processes26–30, and many Zn(II) compounds with
strong anticancer activity have recently been reported31–52 such as
naproxen Zn(II) complexes,27 anthracenyl-linked bis(pyrazolyl)
methane Zn(II) complexes,28 ortho-phenylendiimine Zn(II) and Cu(II)
complexes,31 5,7-dihalo-substituted-8-quinolinoline Zn(II) and copper
normal cells.16,53–61 The use of curcumin (H-Curc) and its complexes in
cancer treatment has shown some success.62 Further, the natural prod-
uct cryptolepine and its Zn complexes exhibited significant effects
against T-24 bladder tumor cells.63–70 However, only two Zn complexes
of cryptolepine have been reported as mitochondrial membrane-
targeting drugs.70 Additionally, a Zn complex containing N,N-bis(pyr-
idin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (BPQA), N-(pyr-
idin-2-ylmethyl)benzofuro[3,2-b]quinolin-11-amine (PQA), and H-Curc
has not been reported. To exploit novel Zn(II) complexes with more
extended planar cryptolepine ligands, two new cryptolepine ligands
(BPQA, PQA) and four novel Zn(II) complexes, [Zn(BPQA)Cl2] (Zn1),
[Zn(BPQA)(Curc)]Cl (Zn2), [Zn(PQA)Cl2] (Zn3), and [Zn(PQA)(Curc)]
Cl (Zn4) were prepared and their biological activities were evaluated.
(II)
complexes,34
zinc-tolfenamato
complexes,36
zinc(II)-
thiosemicarbazone complexes,42 zinc bis(thiosemicarbazone) com-
plexes,43
triapine
(3-aminopyridine-2-carboxaldehyde
thio-
semicarbazone) Zn(II) complexes,28 Zn(II) bis(thiosemicarbazonato)
complexes,48 and zinc(II) phthalocyanine derivatives,49 among others.
* Corresponding authors.
E-mail addresses: qpqin2018@126.com (Q.-P. Qin), weizuzhuang@126.com (Z.-Z. Wei), hliang@mailbox.gxnu.edu.cn (H. Liang).
1
These authors contributed equally to this work.
https://doi.org/10.1016/j.bmc.2020.115948
Received 8 November 2020; Received in revised form 3 December 2020; Accepted 6 December 2020
Available online 19 December 2020
0968-0896/© 2020 Elsevier Ltd. All rights reserved.

L.-Q. Qin et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115948
2. Results and discussion
SK-OV-3/DDP cell lines (Table 1). For example, Zn2 showed low IC50
values (0.03 ± 0.01
μM) in T-24 cells, exhibiting >3.3–1545.0-fold
2.1. Synthesis and stability
higher cytotoxicity than Zn1, Zn3, Zn4, H-Curc, PQA, and BPQA li-
gands, cryptolepine–H-Curc BQ-Zn (5.92 ± 0.35
μ
M),70 and BQCur-Zn
The synthesis of 11-chlorobenzofuro[3,2-b]quinoline (ClQ) has been
reported previously.70 In addition, the new cryptolepine BPQA and PQA
ligands were synthesized via the routes shown in Scheme 1, starting
from o-nitrobenzoic acid. Two mononuclear Zn complexes, [Zn(BPQA)
Cl2] (Zn1) and [Zn(PQA)Cl2] (Zn3), were prepared by reacting ZnCl2
with BPQA in CH3OH (5.0 mL) at 65.0 ◦C for 24 h (yields: 93.6% and
80.2%, respectively). Further, Zn1 and Zn3 were reacted with H-Curc
ligand in the presence of triethylamine (0.1 mL) and methanol (3.0 mL)
at 80.0 ◦C for 24 h to afford [Zn(BPQA)(Curc)]Cl (Zn2) and [Zn(PQA)
(Curc)]Cl (Zn4) in 88.1% and 83.9% yield (Scheme 1); their structures
were determined from extensive spectral data and by elemental analyses
(Figs. S1–S19). The Zn(II) centers in Zn1–Zn4 were coordinated to one
cryptolepine ligand, one H-Curc ligand, or one H2O molecule, creating a
distorted five-coordinate tetragonal-pyramid geometry. Further, the
(1.55 ± 0.04
μ
M),70 and even 610.7-fold higher cytotoxicity than
cisplatin (18.32 ± 1.11 μM) and many other traditional Chinese medi-
cines metal analogues that have been previously evaluated.16,53–61
Complexes Zn1 and Zn2 exhibited obviously higher cytotoxic effects
than previously reported cryptolepine–H-Curc BQ-Zn (5.92 ± 0.35
μ
M)70 and BQCur-Zn (1.55 ± 0.04
μ
M),70 which is correlated with the
key roles of the more extended planar cryptolepine BPQA ligand.
Interestingly, Zn1 ꢀ Zn4 showed low toxicity against normal HL-7702
cells (IC50 > 100 μM).
2.3. Cellular localization behaviors
Fig. 1 shows that the absorption spectra of cryptolepine complex Zn2
(0.03 μM) were ~420.0 and 440.0 nm in Tris-HCl buffer (10 mM, pH
cryptolepine complex Zn2 (0.03 μM) was tested for its stability in Tris-
7.40). In addition, the emission spectra of cryptolepine complex Zn2
(0.03 M) displayed red emission with a maximum wavelength at
approximately 600.0 nm upon excitation at 440 nm. Next, the red probe
HCl buffer (10 mM, pH 7.40) by UV ꢀ vis spectroscopy. As shown in
Fig. 1, the time-dependent (0 and 48 h) UV ꢀ vis spectra of these
μ
compounds indicated that the cryptolepine complex Zn2 (0.03 μM) was
of cryptolepine complex Zn2 (0.03 μM) was used specifically for mito-
stable in Tris-HCl buffer (10 mM, pH 7.40) for 48 h at room temperature.
chondrial membrane imaging (λem = 600 nm) upon excitation at 440 nm
(Fig. 2), with the results indicating that the mitochondrial membrane
potential was destroyed (Fig. 2). In contrast, complex Zn1 (0.10 μM)
2.2. Anticancer activity
showed no obvious effects, which was very similar to the results for
another cryptolepine BQ-Zn complex.70
The cytotoxicity of cryptolepine compounds Zn1 ꢀ Zn4, H-Curc,
ZnCl2, and BPQA was evaluated against the T-24, SK-OV-3/DDP, and
HL-7702 cell lines by 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetra-
zolium bromide (MTT) assay.71–74 Compound Zn2, containing H-Curc
and more extended planar cryptolepine BPQA ligands, showed the
highest cytotoxicity among the tested Zn complexes Zn1, Zn3, Zn4, and
ZnCl2, and also its H-Curc, BPQA and PQA ligands against the T-24 and
2.4. Loss of mitochondrial membrane potential (ΔΨMMP
)
Mitochondria damage, particularly the loss of ΔΨMMP, is a key event
in controlling the intrinsic apoptosis pathway.57,75–79 To further eval-
uate the effects of cryptolepine complexes Zn1 (0.10 μM) and Zn2 (0.03
Scheme 1. Synthetic route of BPQA and PQA ligand and their metal complexes Zn1–Zn4.
2

L.-Q. Qin et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115948
Fig. 1. (A) UV–Vis spectra and (B) emission spectra of cryptolepine complex Zn2 (0.03 μ
M) in Tris-HCl buffer (10 mM, pH 7.40) at 37 ◦C.
(0.03
respectively. Additionally, in the presence of Zn2 (0.03
Curc ligand, a greater increase in ROS levels than those induced by the
μ
M) led to increases in the ROS levels by 31.90% and 40.05%,
Table 1
μ
M), with H-
IC50 (μM) of cryptolepine compounds Zn1–Zn4 towards three human cells.
Compounds
T-24
SK-OV-3/DDP
HL-7702
cryptolepine complex Zn1 (0.10
μM) was observed. Therefore, the
cryptolepine complexes Zn1 and Zn2 induced an increase in intracel-
BPQA
Zn1
20.13 ± 1.09
0.10 ± 0.05
19.22 ± 1.17
0.03 ± 0.01
46.35 ± 0.79
2.06 ± 0.45
0.14 ± 0.08
>150
35.99 ± 1.46
3.13 ± 0.59
32.68 ± 2.35
1.31 ± 0.22
55.03 ± 0.76
6.19 ± 0.52
1.98 ± 0.53
>150
>100
>100
lular ROS levels in T-24 cancer cells.
H-Curc
Zn2
>100
>100
2.6. Determination of intracellular [Ca2+] levels
PQA
>100
Zn3
>100
Zn4
>100
Intracellular [Ca2+] levels have emerged as an important factor in
many cellular processes,76,80,81 and some metallodrugs have been
shown to increase intracellular [Ca2+] levels.76,80–82 To evaluate the
ZnCl2
Cisplatin
>150
12.42 ± 0.45
69.05 ± 1.01
18.32 ± 1.11
effects of the cryptolepine complexes Zn1 (0.10 μM) and Zn2 (0.03 μM)
on intracellular [Ca2+] levels, T-24 cancer cells were exposed to these
complexes for 24 h and then stained with Fluo-3AM. As shown in Fig. 5,
intracellular [Ca2+] levels in T-24 cancer cells increased steadily
μ
M) on ΔΨMMP, the fluorescence probe JC-1 was used. Green fluores-
cence populations of 22.69% and 37.79% were observed in the Zn1 and
Zn2 groups, whereas this value was 6.93% in untreated T-24 cells
(Fig. 3), indicating that cryptolepine complexes Zn1 and Zn2 induced a
decrease in the ΔΨMMP level in the order of Zn2 > Zn1. These results
agree with the cellular localization behaviors determined by confocal
microscopy imaging of T-24 cells.
(~31.52% for Zn1 and 36.48% for Zn2), particularly in Zn2 (0.03 μM)-
treated cells, which agrees with the above results.
2.7. Expression of apoptosis-related proteins
2.5. Analysis of reactive oxygen species (ROS) levels
Mitochondria dysfunction is an important factor in cell death.76,80–84
Upon treatment with cryptolepine complexes Zn1 (0.10 μM) and Zn2
Increases in ROS levels and loss of ΔΨMMP are closely associated with
(0.03
μ
M), the ΔΨMMP was rapidly lost and ROS and [Ca2+] levels were
intrinsic apoptosis in cancer cells.57,75–79 As shown in Fig. 4, treatment
increased (Figs. 2–5), resulting in T-24 cell apoptosis. Tumor-associated
of T-24 cells with the cryptolepine complexes Zn1 (0.10
μ
M) and Zn2
mitochondrial proteins such as bcl-2, caspase-3, apaf-1, caspase-9, and
Fig. 2. Intercellular localization of cryptolepine complex Zn2 (0.03
μ
M) evaluated by confocal microscopy for 24 h (λex: 440 nm; λem: 600 nm).
3

L.-Q. Qin et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115948
Fig. 3. Green fluorescence indicating the ΔΨMMP level was determined by flow cytometry of T-24 cells incubated with cryptolepine complexes Zn1 (0.10
Zn2 (0.03 M) for 24 h.
μM) and
μ
Fig. 4. Intracellular ROS in T-24 cells exposed to cryptolepine complexes Zn1 (0.10 μM) and Zn2 (0.03 μM) for 24 h.
Fig. 5. Effects of cryptolepine complexes Zn1 (0.10
μ
M) and Zn2 (0.03
μ
M) on intracellular [Ca2+] levels in T-24 cells for 24 h.
cytochrome c were inhibited or activated in the mitochondria (Fig. 6),
indicating that the cryptolepine complexes Zn1 and Zn2 induced T-24
cell apoptosis via mitochondria dysfunction.
respiration rate in the treated mice (Tables S1–S3, Fig. 8). In addition,
the cryptolepine complex Zn2 (2.0 mg/kg) significantly reduced the
cancer volume, with growth inhibition by 50.7% (p < 0.01) compared
with 5% dimethyl sulfoxide (DMSO) in the saline group (v/v), which
was greater than previously reported values for cryptolepine–H-Curc
BQ-Zn (43.0%)70 and cisplatin (37.1%).23,70,85–87
2.8. Apoptosis
Fig. 7 shows that when T-24 cells were treated with the cryptolepine
complexes Zn1 (0.10
μM) and Zn2 (0.03
μM), the percentage of
3. Conclusions
apoptotic (UR + LR) cells was increased to 79.71% for Zn1 and 95.36%
for Zn2 compared with the control (9.63%). The ability of Zn1 and Zn2
to induce cell apoptosis was better than that of previously reported
cryptolepine-H-Curc BQ-Zn (~36.23%).70 Clearly, T-24 cell apoptosis
was higher with Zn2, as it bears one deprotonated H-Curc (H-Curc)
ligand as compared with Zn1. Finally, nude mice bearing NCI-H460 cell
xenografts are typically used to evaluate antitumor activity in
vivo.23,70,85–87 Compared to in control mice, there were no effects on the
heart rate, body weight (Tables S1–S3), food consumption, or
In summary, two new cryptolepine BPQA and PQA ligands and their
four cryptolepine-H-Curc Zn(II) complexes Zn1 ꢀ Zn4 were synthesized
and characterized. The MTT assay showed that cryptolepine-H-Curc
Zn1 ꢀ Zn4 have high cytotoxic activity toward T-24 cells, with IC50
values of 0.10 ± 0.05, 0.03 ± 0.01, 2.06 ± 0.45, and 0.14 ± 0.08 μM,
respectively. Interestingly, Zn1 ꢀ Zn4 displayed low toxicity against
normal HL-7702 cells. Additionally, cryptolepine-H-Curc Zn1 and Zn2
entered the mitochondrial membrane and decreased the ΔΨMMP and
4

L.-Q. Qin et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115948
Fig. 6. Expression of five apoptosis-related proteins in T-24 cells treated with cryptolepine complexes Zn1 (0.10 μM) and Zn2 (0.03 μM) for 24 h.
Fig. 7. T-24 cells treated with cryptolepine complexes Zn1 (0.10 μM) and Zn2 (0.03 μM) for 24 h and measured by flow cytometry.
Fig. 8. Tumor volumes and image formation of T-24 xenografts after treatment with the cryptolepine complex Zn2 (2.0 mg/kg) via percutaneous injection every 2
days (q2d). p < 0.01, compound-treated group vs. control.
increased the Ca2+ and ROS levels, further disturbing mitochondrial
function. Further, the probe Zn2 showed appreciable fluorescence in the
red region, indicating effective (50.7%) inhibition of T-24 tumor growth
in vivo. These results can be used to design new probe Zn drugs con-
taining cryptolepine-H-Curc for mitochondrial targeting as anticancer
drugs.
4. Experimental
4.1. Synthesis of ClQ compound
Synthesis and characterization of ClQ were performed as described
in our previous study.70
4.2. Synthesis and characterization of BPQA ligand
ClQ (500 mg, 1.97 mmol, 1.00 eq) was mixed with phenol (5.00 mL)
◦
◦
at 25 C and stirred at 60 C for 30 min. Compound 5 (432 mg, 2.17
5

L.-Q. Qin et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115948
mmol, 1.10 eq) was added dropwise into the mixture and stirred at
120 ◦C for 16 h. Thin-layer chromatography (petroleum ether: ethyl
acetate = 1:1, product Rf = 0.35) indicated that the reactant was
consumed completely and that two new species were formed. Finally,
the mixture was added to ethyl acetate (50.0 mL) and extracted three
times with aqueous NaOH (30.0 mL), followed by drying over anhy-
drous Na2SO4. The residue was purified by column chromatography
(SiO2, petroleum ether: ethyl acetate = from 3:1 to 0:1) and evaporated
to obtain the desired BPQA product (67.3% yield, 99.4% purity). 1H
NMR (400 MHz, CDCl3) δ 8.60 (d, J = 8.2 Hz, 1H), 8.49 (d, J = 4.9 Hz,
2H), 8.36–8.25 (m, 2H), 7.72 (t, J = 7.7 Hz, 1H), 7.60 (br dd, J = 7.5,
12.1 Hz, 2H), 7.56–7.49 (m, 3H), 7.46–7.35 (m, 3H), 7.09 (dd, J = 5.5,
6.8 Hz, 2H), 4.96 (s, 4H). 13C NMR: (101 MHz, CDCl3) δ 158.70, 158.23,
149.40, 147.90, 142.56, 136.60, 136.51, 130.55, 129.75, 127.93,
125.38, 124.63, 124.19, 123.42, 123.10, 122.21 (d, J = 3.7 Hz, 1C),
122.13, 112.12, 59.21. ESI-MS m/z: 417.1 [M + H]+. Elemental analysis
calcd (%) for C27H20N4O: C 77.87, H 4.84, and N 13.45; found: C 77.86,
H 4.86, and N 13.44.
156.88, 149.08, 147.18 (d, J = 15.4 Hz, 1C), 136.76, 134.06 (d, J = 3.7
Hz, 1C), 130.29–129.35 (m, 1C), 127.92, 124.27–123.23 (m, 1C),
123.03, 122.47, 122.01 (d, J = 24.9 Hz, 1C), 120.51, 118.28, 111.88,
49.48. ESI-MS m/z: 326.1 [M + H]+. Elemental analysis calcd (%) for
C21H15N3O: C 77.52, H 4.65, and N 12.91; found: C 77.51, H 4.67, and N
12.90.
4.6. Synthesis of [Zn(PQA)Cl2] (Zn3)
The mononuclear Zn3 complex was prepared by reacting ZnCl2 with
BPQA in CH3OH (5.0 mL) at 65.0 ◦C for 24 h (yield: 80.2%). 1H NMR
(500 MHz, DMSO‑d6) δ 8.58–8.52 (m, 2H), 8.21 (d, J = 7.7 Hz, 1H), 8.02
(d, J = 8.5 Hz, 1H), 7.79 (t, J = 7.8 Hz, 1H), 7.74 (td, J = 7.7, 1.8 Hz,
1H), 7.70–7.65 (m, 1H), 7.65–7.57 (m, 2H), 7.47 (dq, J = 7.7, 4.3 Hz,
2H), 7.26 (dd, J = 7.4, 4.9 Hz, 1H), 5.32 (d, J = 6.3 Hz, 2H). 13C NMR
(126 MHz, DMSO) δ 159.55, 157.61, 149.53, 149.50, 137.49, 137.47,
132.82, 131.54, 130.02, 126.62, 124.57, 124.17, 123.13, 122.76,
122.33, 121.43, 117.79, 112.78, 50.04, 40.47, 40.30, 40.14, 39.97,
39.80, 39.64, 39.47. ESI-MS m/z: 460.1 [Mꢀ H-(H2O)]ꢀ . Elemental
analysis calcd (%) for C21H17Cl2N3OZn: C 52.58, H 3.57, and N 8.76;
found: C 52.56, H 3.60, and N 8.75.
4.3. Synthesis of [Zn(BPQA)Cl2] (Zn1)
The mononuclear Zn complex [Zn(BPQA)Cl2] (Zn1) was prepared by
reacting ZnCl2 with BPQA in CH3OH (5.0 mL) at 65.0 ◦C for 24 h (yield:
93.6%). 1H NMR (500 MHz, DMSO‑d6) δ 8.65 (dd, J = 8.5, 1.4 Hz, 1H),
8.47 (d, J = 4.5 Hz, 2H), 8.22 (dd, J = 24.4, 8.0 Hz, 2H), 7.78 (ddd, J =
8.3, 6.6, 1.4 Hz, 1H), 7.74 (d, J = 8.2 Hz, 1H), 7.72–7.67 (m, 4H), 7.49
(t, J = 7.3 Hz, 1H), 7.45 (d, J = 7.8 Hz, 2H), 7.22 (dd, J = 7.5, 4.8 Hz,
2H), 4.90 (s, 4H). 13C NMR (126 MHz, DMSO) δ 158.49, 158.04, 149.52,
147.76, 147.36, 142.50, 137.37, 136.46, 131.63, 129.87, 128.55,
126.01, 124.76, 124.63, 124.33, 123.04, 122.98, 122.79, 122.23,
112.78, 59.01, 40.49, 40.32, 40.15, 39.99, 39.82, 39.65, 39.48. ESI-MS
m/z: 514.6 [Mꢀ Cl]+. Elemental analysis calcd (%) for C27H20Cl2N4OZn:
C 58.67, H 3.65, and N 10.14; found: C 58.66, H 3.68, and N 10.13.
4.7. Preparation of [Zn(PQA)(Curc)]Cl (Zn4)
In addition, the reactions of Zn3 with H-Curc ligand in the presence
◦
of triethylamine (0.1 mL) and methanol (3.0 mL) at 80.0 C for 24 h
afforded Zn4 in 83.9% yield. 1H NMR (500 MHz, DMSO‑d6) δ 9.44 (s,
2H), 8.52 (s, 1H), 8.49 (d, J = 8.2 Hz, 1H), 8.17 (d, J = 7.5 Hz, 1H), 8.03
(d, J = 8.2 Hz, 1H), 7.98 (s, 1H), 7.69 (t, J = 7.1 Hz, 2H), 7.60 (q, J = 8.0
Hz, 2H), 7.54–7.50 (m, 1H), 7.48–7.38 (m, 5H), 7.27 (s, 2H), 7.23–7.21
(m, 1H), 7.08 (s, 2H), 6.80 (s, 2H), 5.66 (s, 2H), 5.26 (d, J = 6.3 Hz, 2H),
2.61 (s, 6H). 13C NMR (126 MHz, DMSO) δ 160.33, 157.75, 149.47,
149.11, 148.99, 148.37, 147.26, 146.55, 137.30, 135.46, 133.18,
130.66, 129.53, 128.45, 127.48, 127.40, 123.93, 123.73, 123.40,
122.87, 122.72, 122.53, 121.96, 121.13, 118.55, 116.11, 112.52,
111.43, 103.59, 90.00, 56.08, 49.99, 40.49, 40.32, 40.15, 39.99, 39.82,
39.65, 39.49. ESI-MS m/z: 790.20 [M + H]+. Elemental analysis calcd
(%) for C42H34ClN3O7Zn: C 63.57, H 4.32, and N 5.30; found: C 63.55, H
4.34, and N 5.29.
4.4. Preparation of [Zn(BPQA)(Curc)]Cl (Zn2)
The reactions of Zn1 with H-Curc ligand in the presence of trie-
thylamine (0.1 mL) and methanol (3.0 mL) at 80.0 ◦C for 24 h afforded
[Zn(BPQA)(Curc)]Cl (Zn2) in 88.1% yield. 1H NMR (500 MHz,
DMSO‑d6) δ 8.64 (d, J = 8.4 Hz, 2H), 8.42 (d, J = 4.8 Hz, 3H), 8.25 (d, J
= 7.6 Hz, 2H), 8.18 (d, J = 8.4 Hz, 2H), 7.79–7.64 (m, 9H), 7.52–7.39
(m, 6H), 7.28 (s, 1H), 7.20 (dd, J = 7.5, 4.8 Hz, 3H), 7.09 (d, J = 7.4 Hz,
1H), 6.79 (d, J = 8.3 Hz, 2H), 6.67 (d, J = 17.9 Hz, 2H), 4.87 (s, 6H). ESI-
MS m/z: 848.95 [Mꢀ Cl]+. Elemental analysis calcd (%) for
C48H39N4O7Zn: C 67.89, H 4.63, and N 6.60; found: C 67.88, H 4.64, and
N 6.58.
4.8. Other materials and methods
The materials and methods used to prepare cryptolepine complexes
Zn1–Zn4 were similar to those described in our previous work57,70,82. In
addition, the detailed experimental methods for evaluating the in vitro
and in vivo anticancer activities of cryptolepine complexes Zn1–Zn4
were described in the Electronic Supporting Information Materials.
4.5. Synthesis and characterization of PQA ligand
Declaration of Competing Interest
ClQ (2.50 g, 9.85 mmol, 1.00 eq) was added to phenol (20.0 mL) at
◦
◦
25 C. The mixture was stirred at 60 C for 30 min and compound 6
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.
(1.17 g, 10.8 mmol, 1.11 mL, 1.10 eq) was added dropwise into the
◦
mixture. The mixture was stirred at 120 C for 16 h. Thin-layer chro-
matography (petroleum ether: ethyl acetate = 1:1, product Rf = 0.47)
indicated that the reactant was consumed completely, and two new
spots had formed. Finally, the mixture was added to ethyl acetate (50.0
mL) and extracted three times with aqueous NaOH (30.0 mL), pH 13,
and dried over anhydrous Na2SO4. The residue was purified by column
chromatography (SiO2, petroleum ether: ethyl acetate = 3:1 to 1:2) and
evaporated to obtain the desired product. Compound PQA (80.8% yield,
97.8% purity) was obtained as a light-yellow solid and evaluated by
Acknowledgements
This work was supported by the National Natural Science Foundation
of China (21867017), the basic skills improvement project for the young
and middle-aged teachers in Guangxi colleges and universities (No.
2020KY14010) and the Natural Science Foundation of Guangxi
(2018GXNSFBA138021).
1
1
liquid chromatography-mass spectrometry, H NMR and 13C NMR. H
NMR (400 MHz, CDCl3) δ 8.68 (d, J = 4.6 Hz, 1H), 8.36 (d, J = 7.7 Hz,
1H), 8.24–8.06 (m, 2H), 7.76–7.64 (m, 2H), 7.59 (d, J = 3.7 Hz, 2H),
7.50 (dt, J = 1.1, 7.6 Hz, 1H), 7.46–7.39 (m, 2H), 7.26 (s, 1H), 6.79 (br s,
1H), 5.38 (d, J = 4.9 Hz, 2H). 13C NMR: (101 MHz, CDCl3) δ 158.30,
Appendix A. Supplementary material
The supporting information contains the detailed procedures for the
experimental methods and anticancer activities of BPQA, H-Curc, PQA,
6

L.-Q. Qin et al.
Bioorganic & Medicinal Chemistry 30 (2021) 115948
43 Cowley AR, Davis J, Dilworth JR, et al. Chem Commun. 2005:845–847.
ZnCl2, cisplatin, Zn1–Zn4, as well as the ESI-MS, UV-Vis, IR, and NMR
data, in the online version at https://doi.org/10.1016/j.bmc.2020.11
5948.
44 Kowol CR, Trondl R, Arion VB, Jakupec MA, Lichtscheidl I, Keppler BK. Dalton Trans.
2010;39:704–706.
45 Dayal D, Palanimuthu D, Shinde SV, Somasundaram K, Samuelson AG. J Biol Inorg
Chem. 2011;16:621–632.
46 Lim S, Price KA, Chong S-F, et al. J Biol Inorg Chem. 2010;15:225–235.
47 Pascu SI, Waghorn PA, Conry TD, et al. Dalton Trans. 2007:4988–4997.
48 Pascu SI, Waghorn PA, Conry TD, et al. Dalton Trans. 2008:2107–2110.
49 Jia X, Yang F-F, Li J, Liu J-Y, Xue J-P. J Med Chem. 2013;56:5797–5805.
50 Liu J-Y, Jiang X-J, Fong W-P, Ng DKP. Org Biomol Chem. 2008;6:4560–4566.
51 Lau JTF, Lo P-C, Fong W-P, Ng DKP. J Med Chem. 2012;55:5446–5454.
52 Sibata CH, Colussi VC, Oleinick NL, Kinsella TJ. Braz J Med Biol Res. 2000;33:
869–880.
References
1 (a) Wheate NJ, Walker S, Craig GE, Oun R. Dalton Trans. 2010;39:8113–8127;
(b) Theiner S, Kornauth C, Varbanov HP, et al. Metallomics. 2015;7:1256–1264;
(c) Stultz LK, Hunsucker A, Middleton S, et al. Metallomics. 2020;12:876–890;
(d) Bisceglie F, Orsoni N, Pioli M, et al. Metallomics. 2019;11:1729–1742.
2 (a) Qin Q-P, Wang Z-F, Wang S-L, et al. Eur J Med Chem. 2019;170:195–202;
(b) Patra M, Gasser G, Metzler-Nolte N. Dalton Trans. 2012;41:6350–6358;
53 To KKW, Au-Yeung SCF, Ho Y-P. Anticancer Drugs. 2006;17:673–683.
54 Ho Y-P, To KKW, Au-Yeung SCF, Wang X, Lin G, Han X. J Med Chem. 2001;44:
2065–2068.
`
(c) Spano V, Pennati M, Parrino B, et al. J Med Chem. 2016;59:7223–7238;
(d) Lim MH, Song H, Olmon ED, Dervan EE, Barton JK. Inorg Chem. 2009;48:
5392–5397;
55 Zinsmeister HD, Becker H, Eicher T. Angew Chem Int Ed. 1991;30:130–147.
56 Chen Z-F, Liu Y-C, Huang K-B, Liang H. Curr Top Med Chem. 2013;13:2104–2115.
57 (a) Chen Z-F, Qin Q-P, Qin J-L, et al. J Med Chem. 2015;58:2159–2179;
(b) Qin Q-P, Qin J-L, Meng T, et al. Eur J Med Chem. 2016;124:380–392.
58 Chen Z-F, Qin Q-P, Qin J-L, et al. J Med Chem. 2015;58:4771–4789.
59 Qin Q-P, Wang S-L, Tan M-X, et al. Metallomics. 2018;10:1160–1169.
60 Qin Q-P, Zou B-Q, Wang Z-F, et al. Eur J Med Chem. 2019;183, 111727. https://doi.
org/10.1016/j.ejmech.2019.111727.
(e) Nam JS, Kang M-G, Kang J, et al. J Am Chem Soc. 2016;138:10968–10977;
(f) Lim MH, Xu D, Lippard SJ. Nat Chem Biol. 2006;2:375–380.
3 Jakupec MA, Galanski M, Arion VB, Hartinger CG, Keppler BK. Dalton Trans. 2008:
183–194.
4 Zhang H, Gou S, Zhao J, Chen F, Xu G, Liu X. Eur J Med Chem. 2015;96:187–195.
5 Fong TT-H, Lok C-N, Chung CY-S, et al. Angew Chem Int Ed. 1939;55(2016):
11935–11941.
6 Gandioso A, Shaili E, Massaguer A, et al. Chem Commun. 2015;51:9169–9172.
7 Zeng L, Gupta P, Chen Y, et al. Chem Soc Rev. 2017;46:5771–5804.
8 Qin Q-P, Wang Z-F, Huang X-L, Tan M-X, Zou B-Q, Liang H. Eur J Med Chem. 2019;
184, 111751. https://doi.org/10.1016/j.ejmech.2019.111751.
9 Meggers E. Chem Commun. 2009:1001–1010.
61 Qin Q-P, Wang Z-F, Huang X-L, et al. Dalton Trans. 2019;48:15247–15254.
62 (a) Renfrew AK, Bryce NS, Hambley TW. Chem. Sci., 4(2013), pp. 3731-3739 (b)
Pettinari R, Marchetti F, Di Nicola C, et al., Inorg Chem Frontiers, 6(2019), pp. 2448-
2457 (c) Esatbeyoglu T, Huebbe P, Ernst IMA, Chin D, Wagner AE, Rimbach G.
Angew Chem Int Ed, 51(2012), pp. 5308-5332 (d) Salem M, Rohani S, Gillies ER.
RSC Adv, 4(2014), pp. 10815-10829 (e) Prasad S, Tyagi AK, Aggarwal BB, Cancer
Res Treat, 46(2014), pp. 2-18 (f) Banerjee S, Chakravarty AR. Acc Chem Res, 48
(2015), pp. 2075-2083 (g) Wanninger S, Lorenz V, Subhan A, Edelmann FT. Chem.
Soc. Rev., 44(2015), pp. 4986-5002 (h) Caruso F, Rossi M, Benson A, et al., J. Med.
Chem., 55(2012), pp. 1072-1081 (i) Pettinari R, Condello F, Marchetti F, et al., Eur.
J. Inorg. Chem., 2017(2017), pp. 2905-2910 (j) Pettinari R, Petrini A, Marchetti F,
et al., ChemistrySelect, 3(2018), pp. 6696-6700 (k) Pettinari R, Marchetti F, Pettinari
C, et al., Dalton Trans., 44(2015), pp. 20523-20531 (l) Pettinari R, Marchetti F,
Condello F, et al., Organometallics, 33(2014), pp. 3709-3715 [m] Huang S, Li H, Liu
Y, Liang Y, Su W, Xiao Q. Polyhedron, 167(2019), pp. 51-61 (n) Huang S, Xie J, Su
W, Liu Y, Wang X, Hu B, Xiao Q. J. Organomet. Chem., 853(2017), pp. 81-92.
63 Zhou JM, Zhu XF, Lu YJ, et al. Oncogene. 2006;25:503–511.
10 Zheng Y-R, Suntharalingam K, Johnstone TC, Lippard SJ. Chem Sci. 2015;6:
1189–1193.
11 Wong DYQ, Lau JY, Ang WH. Dalton Trans. 2012;41:6104–6111.
12 Noh J, Kwon B, Han E, et al. Nat Commun. 2015;6:6907. https://10.1038/
ncomms7907.
13 Hurley LH. Nat Rev Cancer. 2002;2:188–200.
14 Reed JE, Arnal AA, Neidle S, Vilar R. J Am Chem Soc. 2006;128:5992–5993.
15 Xu L, Chen X, Wu J, Wang J, Ji L, Chao H. Chem Eur J. 2015;21:4008–4020.
16 Qin Q-P, Wang Z-F, Huang X-L, et al. Chem Lett. 2019;10:936–940.
17 Zhong H-J, Lu L, Leung K-H, et al. Chem Sci. 2015;6:5400–5408.
18 Leung C-H, Liu L-J, Lu L, et al. Chem Commun. 2015;51:3973–3976.
19 Ma D-L, Liu L-J, Leung K-H, et al. Angew Chem Int Ed. 2014;53:9178–9182.
20 Gothe Y, Marzo T, Messori L, Metzler-Nolte N. Chem Eur J. 2016;22:12487–12494.
21 Meng T, Qin Q-P, Zou H-H, Wang K, Liang F-P. ACS Med Chem Lett. 2019;10:
1603–1608.
64 Yin R, Zhang M, Hao C, et al. Chem Commun. 2013;49:8516–8518.
65 Ou T-M, Lu Y-J, Zhang C, et al. J Med Chem. 2007;50:1465–1474.
66 Zhou J-L, Lu Y-J, Ou T-M, et al. J Med Chem. 2005;48:7315–7321.
67 Wang X-D, Ou T-M, Lu Y-J, et al. J Med Chem. 2010;53:4390–4398.
68 Ou T-M, Lin J, Lu Y-J, et al. J Med Chem. 2011;54:5671–5679.
69 Bierer DE, Dubenko LG, Zhang P, et al. J Med Chem. 1998;41:2754–2764.
70 Qin Q-P, Wei Z-Z, Wang Z-F, et al. Chem Commun. 2020;56:3999–4002.
71 Pettinari R, Marchetti F, Di Nicola C, et al. Inorg Chem Front. 2019;6:2448–2457.
72 Li M, Du C, Guo N, et al. Eur J Med Chem. 2019;164:640–653.
73 Compain G, Oumata N, Clarhaut J, et al. Eur J Med Chem. 2018;158:1–6.
74 Oumata N, Bettayeb K, Ferandin Y, et al. J Med Chem. 2008;51:5229–5242.
75 Zhao C, Gao W, Chen T. Apoptosis. 2014;19:668–681.
22 Pettinari R, Marchetti F, Condello F, et al. Organometallics. 2014;33:3709–3715.
23 Weiss A, Berndsen RH, Dubois M, et al. Chem Sci. 2014;5:4742–4748.
24 Ott I. Coordin Chem Rev. 2009;253:1670–1681.
´
ˇ´
25 Smolkova R, Zelenak V, Smolko L, et al. Eur J Med Chem. 2018;153:131–139.
¨
´
´
´ˇ
´
´
ˇ
´
´
´
26 Gyoryova, Szunyogova E, Kovarova J, Hudecova D, Mudronova D, Juhaszova E.
J Therm Anal Calorim. 2003;72:587–596.
27 Abu Ali, Fares H, Darawsheh M, Rappocciolo E, Akkawi M, Jaber S. Eur J Med Chem,
89(2015), pp. 67–76.
28 Ruan B-F, Zhu Y-Z, Liu W-D, Song B-A, Tian Y-P. Eur J Med Chem. 2014;72:46–51.
29 Farrell N. Coordin Chem Rev. 2002;232:1–4.
76 (a) Zhang W-Y, Yi Q-Y, Wang Y-J, et al. Eur J Med Chem. 2018;151:568–584;
(b) Tang B, Wan D, Wang Y-J, Yi Q-Y, Guo B-H, Liu Y-J. Eur J Med Chem. 2018;145:
302–314;
30 Walkup GK, Burdette SC, Lippard SJ, Tsien RY. J Am Chem Soc. 2000;122:
5644–5645.
31 Silvestri A, Barone G, Ruisi G, Anselmo D, Riela S, Liveri VT. J Inorg Biochem. 2007;
101:841–848.
(c) Wan D, Tang B, Wang Y-J, et al. Eur J Med Chem. 2017;139:180–190;
(d) Qin L-Q, Zou B-Q, Qin Q-P, et al. New J Chem. 2020;44:7832–7837;
(e) Bhat SS, Revankar VK, Pinjari RV, et al. New J Chem. 2018;42:16846–16854.
77 Chen T, Liu Y, Zheng W-J, Liu J, Wong Y-S. Inorg Chem. 2010;49:6366–6368.
78 Nunnari J, Suomalainen A. Cell. 2012;148:1145–1159.
32 Haddad JJ. Mol Immunol. 2009;47:205–214.
33 Vasto S, Candore G, Listì F, et al. Brain Res Rev. 2008;58:96–105.
34 Liu Y-C, Wei J-H, Chen Z-F, et al. Eur J Med Chem. 2013;69:554–563.
35 Tan, Wang B, Zhu L. Bioorg. Med. Chem., 17(2009), pp. 614–620.
36 Tarushi A, Totta X, Papadopoulos A, et al. Eur J Med Chem. 2014;74:187–198.
37 Finney LA, Halloran TV. Science. 2003;300:931–936.
38 Di Vaira M, Bazzicalupi C, Orioli P, Messori L, Bruni B, Zatta P. Inorg Chem. 2004;43:
3795–3797.
79 Zhou R, Yazdi AS, Menu P, Tschopp J. Nature. 2011;469:221–225.
80 Petersen H, Michalak M, Verkhratsky A. Cell Calcium. 2005;38:161–169.
81 Cheng J-S, Shu S-S, Kuo C-C, et al. Arch Toxicol. 2011;85:1257–1266.
82 (a) Zou H-H, Wang L, Long Z-X, et al. Eur J Med Chem. 2016;108:1–12;
(b) Qin Q-P, Chen Z-F, Shen W-Y, et al. Eur J Med Chem. 2015;89:77–87.
83 Qin Q-P, Wang S-L, Tan M-X, et al. Eur J Med Chem. 2019;161:334–342.
39 Sakurai, Kojima Y, Yoshikawa Y, Kawabe K, Yasui H. Coordin Chem Rev. 2002;226:
187–198.
ˇ
´
ˇ
84 Malugin A, Kopeckova P, Kopecek J. Mol Pharm. 2006;3:351–361.
40 Zhou TW, Hambley BJ, Kennedy PA, et al. Regtop. Inorg Chem. 2000;39:3742–3748.
41 Wen J-H, Li C-Y, Geng Z-R, Ma X-Y, Wang Z-L. Chem Commun. 2011;47:
11330–11332.
85 Wang J-Q, Zhang P-Y, Ji L-N, Chao H. J Inorg Biochem. 2015;146:89–96.
¨
86 Goschl S, Schreiber-Brynzak E, Pichler V, et al. Metallomics. 2017;9:309–322.
87 Gao Y-H, Zhu X-X, Zhu W, et al. Eur J Med Chem. 2020;187, 111959. https://doi.org/
42 Stacy AE, Palanimuthu D, Bernhardt PV, Kalinowski DS, Jansson PJ, Richardson DR.
J Med Chem. 2016;59:4965–4984.
10.1016/j.ejmech.2019.111959.
7