Novel Quinoline-based Ir(III) Complexes Exhibit High Antitumor Activity in Vitro and in Vivo

Article abstract of DOI:10.1021/acsmedchemlett.9b00337

Eight novel Ir(III) complexes listed as [Ir(H-P)₂(P)]PF₆ (PyP-Ir), [Ir(H-P)₂(dMP)]PF₆ (PydMP-Ir), [Ir(H-P)₂(MP)]PF₆ (PyMP-Ir), [Ir(H-P)₂(tMP)]PF₆ (PytMP-Ir), [Ir(MPy)₂(P)]PF₆ (MPyP-Ir), [Ir(MPy)₂(dMP)]PF₆ (MPydMP-Ir), [Ir(MPy)₂(MP)]PF₆ (MPyMP-Ir), [Ir(MPy)₂((tMP)]PF₆ (MPytMP-Ir) with 2-phenylpyri-dine (H-P) and 3-methyl-2-phenylpyridine (MPy) as ancillary ligands and pyrido-[3,2-a]-pyrido[1′,2′:1,2]imidazo[4,5-c]phenazine (P), 12,13-dimethyl pyrido-[3,2-a]-pyrido[1′,2′:1,2]-imidazo-[4,5-c]-phenazine (dMP), 2-methylpyrido [3,2-a]-pyrido-[1′,2′:1,2]-imidazo-[4,5-c]-phenazine (MP), and 2,12,13-trimethylpyrido-[3,2-a]-pyrido-[1′,2′:1,2]-imidazo-[4,5-c]-phenazine (tMP) as main ligands, respectively, were designed and synthesized to fully characterize and explore the effect of their toxicity on cancer cells. Cytotoxic mechanism studies demonstrated that the eight Ir(III) complexes exhibited highly potent antitumor activity selectively against cancer cell lines NCI-H460, T-24, and HeLa, and no activity against HL-7702, a noncancerous cell line. Among the eight Ir(III) complexes, MPytMP-Ir exhibited the highest cytotoxicity with an IC₅₀ = 5.05 ± 0.22 nM against NCI-H460 cells. The antitumor activity of MPytMP-Ir in vitro could be contributed to the steric or electronic effect of the methyl groups, which induced telomerase inhibition and damaged mitochondria in NCI-H460 cells. More importantly, MPytMP-Ir displayed a superior inhibitory effect on NCI-H460 xenograft in vivo than cisplatin. Our work demonstrates that MPytMP-Ir could potentially be developed as a novel potent Ir-based antitumor drug.

Full text of DOI:10.1021/acsmedchemlett.9b00337

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Letter
Novel quinoline-based Ir(III) complexes exhibit
high antitumor activity in vitro and in vivo
Yan Yang, Yi-Dong Bin, Qi-Pin Qin, Xu-Jian Luo, Bi-Qun Zou, and Hua-Xin Zhang
ACS Med. Chem. Lett., Just Accepted Manuscript • DOI: 10.1021/acsmedchemlett.9b00337 • Publication Date (Web): 06 Nov 2019
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Novel quinoline-based Ir(III) complexes exhibit high antitumor
activity in vitro and in vivo
Yan Yang,a,b,1 Yi-Dong Bin,a,b,1 Qi-Pin Qin,a,d* Xu-Jian Luo,a Bi-Qun Zou,c,d* and Hua-Xin Zhangb*
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a Guangxi Key Lab of Agricultural Resources Chemistry and Biotechnology, College of Chemistry and Food Science, Yulin
Normal University, 1303 Jiaoyudong Road, Yulin 537000, P. R. China.
b School of Chemistry and Chemical Engineering, Guangxi University, 100 Daxuedong Road, Nanning 530004, P. R. China.
c Department of Chemistry, Guilin Normal College, 9 Feihu Road, Gulin 541001, China.
d 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, P. R. China.
KEYWORDS: Ir(III) complexes, telomerase inhibition, antitumor activity, damaging mitochondria
ABSTRACT: Eight novel Ir(III) complexes listed as [Ir(H-P)2(P)]PF6 (PyP-Ir), [Ir(H-P)2(dMP)]PF6 (PydMP-Ir), [Ir(H-
P)2(MP)]PF6 (PyMP-Ir), [Ir(H-P)2(tMP)]PF6 (PytMP-Ir), [Ir(MPy)2(P)]PF6 (MPyP-Ir), [Ir(MPy)2(dMP)]PF6 (MPydMP-Ir),
[Ir(MPy)2(MP)]PF6 (MPyMP-Ir), [Ir(MPy)2((tMP)]PF6 (MPytMP-Ir) with 2-phenylpyri-dine (H-P) and 3-methyl-2-
phenylpyridine (MPy) as ancillary ligands and pyrido [3,2-a] pyrido[1',2':1,2]imidazo[4,5-c]phenazine (P), 12,13-dimethyl
pyrido[3,2-a]pyrido [1',2':1,2] imidazo[4,5-c] phenazine(dMP), 2-methylpyrido [3,2-a] pyrido [1',2':1,2] imidazo [4,5-c] phenazine
(MP), and 2,12,13-trimethylpyrido [3,2-a] pyrido [1',2':1,2 ] imidazo [4,5-c] phenazine (tMP) as main ligands, respectively, were
designed and synthesized to fully characterize and explore the effect of their toxicity on cancer cells. Cytotoxic mechanism studies
demonstrated that the eight Ir(III) complexes exhibited highly potent antitumor activity selectively against cancer cell lines NCI-
H460, T-24, and HeLa, and no activity against HL-7702, a non-cancerous cell line. Among the eight Ir(III) complexes, MPytMP-
Ir exhibited the highest cytotoxicity with an IC50 = 5.05 ± 0.22 nM against NCI-H460 cells. The antitumor activity of MPytMP-Ir
in vitro could be contributed to the steric or electronic effect of the methyl groups, which induced telomerase inhibition and
damaged mitochondria in NCI-H460 cells. More importantly, MPytMP-Ir displayed a superior inhibitory effect on NCI-H460
xenograft in vivo than cisplatin. Our work demonstrates that MPytMP-Ir could potentially be developed as a novel potent Ir-based
anti-tumor drug.
The capacity of tumor cells to grow, invade, and metastasize
to other organs are the driving forces in cancer development.1-3
Identifying and developing new anticancer drugs and
exploring their putative antitumor mechanisms is an urgent
need. Since the discovery of cisplatin, platinum compounds
were the most successful representative drugs in clinical
treatment.4 However, due to their undesired side effects,
considerable efforts have been made to develop non-platinum
compounds.5,6 Recently, RuII/III, IrIII, RhIII, PdII, and AuIII, and
other complexes have received significant attention because of
their various cellular targets and mechanisms of action.7-13 The
octahedral polypyridyl Ir(III) cyclometalated complexes could
induce apoptosis via regulating protein kinases interactions,14-
La(III)35 complexes have shown activity against senile
dementia, intestinal amebiasis, and cancer.29-36
Inspired by the previous research and the high anticancer
activity of quinoline derivatives, here we used easily acquired
8-hydroxylquinoline
derivative
6,7-
dichloro-5,8-
quinolinequinone with an AI50 = 1.3 ± 0.9 µM and MIC = 6.3-
12.5 lg/mL37 to modify and synthesize four quinoline
derivatives
as
main
ligands:
pyrido
[3,2-a]
pyrido[1',2':1,2]imidazo[4,5-c]phenazine (P), 12,13-dimethyl
pyrido[3,2-a]pyrido [1',2':1,2] imidazo[4,5-c] phenazine
(dMP), 2-methylpyrido [3,2-a] pyrido [1',2':1,2] imidazo [4,5-
c] phenazine (MP), and 2,12,13-trimethylpyrido [3,2-a] pyrido
[1',2':1,2] imidazo [4,5-c] phenazine (tMP) (Chart S1). We
studied and characterized their antineoplastic activity and
explored the underlying mechanisms. Based on previously
reported efficacy of [Ir(C^N)2(N^N)]+,38-41 we synthesized
eight novel quinoline based organoiridium(III) complexes and
fully characterized them (Figures S1-S48, structures shown in
Chart 1). Furthermore, we also noted through HPLC
experiments, that MPydMP-Ir and MPytMP-Ir were stable
for 48 h in Tris-HCl buffer (Figure S47 and S48).
17
mitochondrial membrane disruption,18,19 or DNA binding.20-
22
Heterocyclic compounds have been widely studied and
developed as potent biological compounds.23 The 5,8-
quinolinediones derived from 8-hydroxyquinoline have
demonstrable anticancer activity,23 and are used as anticancer,
antifungal, antimalarial agents.24-28 Additionally, several 7-
aminoquinoline quinones, Streptonigrin, and clioquinol
derivatives29-31, and their Ni(II)32, Yb(III)33, Sm(III)34, and
Chart 1. Chemical Structures of the eight IrIII complexes.
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We studied the cytotoxic effects of the eight novel Ir(III)
complexes and the free ancillary ligands against NCI-H460
(non-small lung), T-24 (bladder), HeLa (cervical), and HL-
7702 (hepatocyte) using the MTT assay.42-44 The results are
shown in Table S1. We found that the IC50 values of the free
ancillary ligands against cancer cells were >50 µM except in
HeLa cells. In contrast, the novel Ir(III) complexes exhibited
much superior cytotoxic activities against all the tested human
cancer cell lines except HL-7702 (non-cancerous cells)
compared to the free ancillary ligands and cisplatin (positive
control). Significantly, their cytotoxic activity decreased in the
following order: MPytMP-Ir, MPydMP-Ir, PytMP-Ir,
PydMP-Ir, MPyMP-Ir, PyMP-Ir, MPyP-Ir, PyP-Ir against
all tested cancer cells except the non-cancerous HL-7702.
Furthermore, among the eight novel Ir(III) complexes,
MPytMP-Ir exhibited significantly superior and sensitive
cytotoxic activity with a much lower IC50 (5.05 ± 0.22 nM)
against NCI-H460 cells. Of course, the methyl electron-
donating group does enhance the cytotoxic activity via a
combination of steric and electronic effects, such as
polypyridyl, pyrazine, and pyrazole ring.4,16,40,45,46 Owing to its
highly efficient and selective cytotoxic activity against the
NCI-H460 cancer cells compared to cisplatin, we performed
further in-depth studies using MPytMP-Ir.16,40 We also
compared its effectiveness to another synthesized compound
MPydMP-Ir and used the sensitive NCI-H460 cell line to
further study the potential interactive mechanisms of its
biological cytotoxic influences.40-51
Figure 1. MPydMP-Ir (125 nM) and MPytMP-Ir (5 nM)
induced NCI-H460 cell apoptosis after 24 h treatment.
Following the treatment of NCI-H460 cells with MPydMP-
Ir and MPytMP-Ir at the corresponding IC50 concentration
for 24 h (Figure 1), apoptosis induction was seen in 38.7% of
the MPydMP-Ir treated cells, while 92.6% cells were
apoptotic following treatment with MPytMP-Ir. These results
underscore the greatly superior apoptosis induction capacity of
MPytMP-Ir.
Figure 2. Telomerase inhibition in NCI-H460 cells treated
with MPydMP-Ir (125 nM) and MPytMP-Ir (5 nM).
Previous studies have shown that telomerase inhibition was
related to cell cycle arrest in cancer cells.43,59-66 We observed
that 68.70 % of the untreated NCI-H460 tumor cells were in
the G1 phase, while this increased to 84.09% after treatment
with MPytMP-Ir (Figure 3). However, MPydMP-Ir
treatment resulted in a 1.38% decrease in cells at the G1 phase.
We could conclude that the antineoplastic activity of
MPytMP-Ir was at least partly due to decreased proliferation
as the cells were arrested in G1 phase. 43,59-66 The expression
level of CDK2, a key player in cell cycle progression,
decreased slightly after treatment with MPydMP-Ir, while a
slight increase in Cyclin D1 was observed due to G1 arrest
(Figure S2). Of note, treatment with MPytMP-Ir resulted in a
A 24 h treatment of NCI-H460 cells with MPydMP-Ir and
MPytMP-Ir resulted in 19.75% and 49.40% inhibition of
telomerase activity, respectively. This clearly demonstrates
that MPytMP-Ir could induce much stronger telomerase
inhibition than MPydMP-Ir. This provides more evidence
that increasing the methyl electron-donating groups could
enhance the telomerase inhibition via a combination of steric
and electronic effects.45,52-58 As expected, c-myc and/or hTERT
proteins were effectively downregulated in MPydMP-Ir and
MPytMP-Ir treated cells (Figure S1) and led to telomerase
inhibition. 52-58
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significant decrease in cyclin D1-CDK2 complex.66 These
results are consistent with our previous expectations and
investigation.
displayed effective inhibition of tumor growth in NCI-H460
models.
In conclusion, we first synthesized eight novel quinoline-
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8
based Ir(III) complexes and demonstrated their high cytotoxic
activity. Based on the first set of cytotoxicity experiments, we
chose two representative complexes to further explore the
effect of their toxicity in selected cancer cell lines. MPytMP-
Ir exhibited the most potent in vitro cytotoxicity against
human cancer cells. MPytMP-Ir also showed the highest
selective cytotoxic against NCI-H460 cancer cells and was
most potent in inhibiting telomerase activity. MPytMP-Ir-
induced cytotoxicity in the NCI-H460 tumor cells was exerted
via G1 cell cycle arrest, leading to inhibition of cell
proliferation. Additionally, MPytMP-Ir could induce cellular
apoptosis by activating the mitochondrial dysfunction pathway
in the NCI-H460 tumor cells. Last but not least, MPytMP-Ir
displayed effective inhibition of tumor growth in NCI-H460
xenograft models and exhibited less toxicity and even better
safety profile than cisplatin. Through this study, we have
identified a novel potent antitumor drug, MPytMP-Ir, and
have demonstrated its superior efficacy compared to cisplatin.
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Figure 3. NCI-H460 cell cycle analysis following MPydMP-
Ir (125 nM) and MPytMP-Ir (5 nM) treatment.
Figure 4. The degradation of MMP in MPydMP-Ir (125
nM)- and MPytMP-Ir (5 nM) treated cells.
We next assessed the effect of MPytMP-Ir treatment on
mitochondria using the JC-1 fluorescent mitochondrial probe.
We saw that the green JC-1 fluorescence (damaged
mitochondria, decrease in mitochondrial membrane potential
[MMP]) was observed in 13.3% of NCI-H460 cells treated
with MPydMP-Ir. Strikingly, 92.2% of the cells treated with
MPytMP-Ir showed green fluorescence, i.e., damaged
mitochondria (Figure 4). Thus, the high methyl electron-
donating levels in MPytMP-Ir could dramatically increase the
effectiveness of this compound on MMP and, in turn, on
apoptosis induction.67-70
Figure 5. The tumor volumes (A, mm3±SD), tumor weights
(B, g±SD), body weights (C, g±SD), and photograph (D) of
NCI-H460 xenograft following MPytMP-Ir treatment (n = 6).
**p<0.05 relative to control.
ASSOCIATED CONTENT
Supporting Information
Experimental procedures for the quinoline-based Ir(III)
complexes are available free of charge on the ACS Publications
website at DOI: xxxxxx.
In addition, we observed that MPytMP-Ir resulted in a
much more significant (Figure S2) accumulation of apaf-1 and
cytochrome than MPydMP-Ir. Taken together, these results
demonstrate that MPytMP-Ir could cause apoptosis in NCI-
H460 cells by inducing mitochondrial dysfunction.64-78
AUTHOR INFORMATION
Corresponding Author
*Q.-P.
Qin:
qpqin2018@126.com;
B.-Q.
Zou:
Treatment with MPytMP-Ir (10.0 mg/kg per 2 days) led to
a 47.1% tumor growth inhibition on day 12.0, significantly
higher than that reported for cisplatin (25.5%) (Figure 5 and
Tables S2-S4).71-77 Moreover, MPytMP-Ir treatment did not
adversely affect body weight (average body weight pre- and
post-treatment, control group: 18.47 ± 1.08 g and 20.37 ± 0.52
g, treated group: 18.03 ± 1.28 and 20.28 ± 0.47g). Thus,
MPytMP-Ir exhibited less toxicity and better safety profile
than that reported for cisplatin.64,71-77 In summary, MPytMP-Ir
zoubiqun@163.com; H.-X. Zhang: 1271050198@qq.com.
Author Contributions
1These authors made an equal contribution to this work.
Funding Sources
We thank the National Natural Science Foundation of China (Nos.
51463023, 51962035, 21867017 and 21461028), Guangxi Natural
Science Foundation (No. 2018GXNSFBA138021), Yulin Normal
University for High-level Talents (No. G2019ZK04), and the
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and Universities in Guangxi (2014-49 and 2017-38) for funding
this work.
Notes
The author claims no competing economic interests.
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