High in vitro and in vivo antitumor activities of luminecent platinum(II) complexes with jatrorrhizine derivatives

Journal Pre-proof High in vitro and in vivo antitumor activities of luminecent platinum(II) complexes with jatrorrhizine derivatives Qi-Pin Qin, Bi-Qun Zou, Zhen-Feng Wang, Xiao-Ling Huang, Ye Zhang, Ming-Xiong Tan, Shu-Long Wang, Hong Liang PII:

S0223-5234(19)30879-7

DOI:

https://doi.org/10.1016/j.ejmech.2019.111727

Reference:

EJMECH 111727

To appear in:

European Journal of Medicinal Chemistry

Received Date: 4 August 2019 Revised Date:

19 September 2019

Accepted Date: 20 September 2019

Please cite this article as: Q.-P. Qin, B.-Q. Zou, Z.-F. Wang, X.-L. Huang, Y. Zhang, M.-X. Tan, S.-L. Wang, H. Liang, High in vitro and in vivo antitumor activities of luminecent platinum(II) complexes with jatrorrhizine derivatives, European Journal of Medicinal Chemistry (2019), doi: https://doi.org/10.1016/ j.ejmech.2019.111727. This is a PDF file of an article that has undergone enhancements after acceptance, such as the addition of a cover page and metadata, and formatting for readability, but it is not yet the definitive version of record. This version will undergo additional copyediting, typesetting and review before it is published in its final form, but we are providing this version to give early visibility of the article. Please note that, during the production process, errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain. © 2019 Published by Elsevier Masson SAS.

Graphical abstract

High in vitro and in vivo antitumor activities of luminecent platinum(II) complexes with Jatrorrhizine derivatives Qi-Pin Qin*,a,b,1, Bi-Qun Zoub,c,1, Zhen-Feng Wanga, Xiao-Ling Huanga, Ye Zhangc,d, Ming-Xiong Tana,b, Shu-Long Wanga,b, Hong Liang*,b

The jatrorrhizine luminecent platinum(II) complexes are designed and synthesized to target tumor-specific p53 and telomerase, and acted as potential anti-tumor agents.

High in vitro and in vivo antitumor activities of luminecent platinum(II) complexes with Jatrorrhizine derivatives Qi-Pin Qin*,a,b,1, Bi-Qun Zoub,c,1, Zhen-Feng Wanga, Xiao-Ling Huanga, Ye Zhangc,d, Ming-Xiong Tana,b, Shu-Long Wanga,b, Hong Liang*,b

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.

[email protected]

(Q.-P.

Qin).

Tel./Fax.:

+86-775-2623650. 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. E-mail: [email protected] (H. Liang). c

Department of Chemistry, Guilin Normal College, 9 Feihu Road, Guilin 541001,

China. d

School of Pharmacy, Guilin Medical University, Guilin 541004, China.

1

These authors contributed equally to this work.

1

ABSTRACT Two highly active anticancer Pt(II) complexes, [Pt(Jat1)Cl]Cl (Pt1) and [Pt(Jat2)Cl]Cl (Pt2), containing jatrorrhizine derivative ligands (Jat1 and Jat2) are described. Cell intake study showed high accumulation in cell nuclear fraction. Pt1 and Pt2 exhibited high selectivity for HeLa cancer cells (IC50=15.01±1.05 nM and 1.00±0.17 nM) comparing with HL-7702 normal cells (IC50>150 µM), by targeting p53 and telomerase. Pt2 containing Jat2 ligand was more potent and showed high selectivity for telomerase. It also caused mitochondria and DNA damage, sub-G1 phase arrest, and a high rate of cell apoptosis at the low dose of 1.00 nM. The HeLa tumor inhibition rate (TIR) of Pt2 was 48.8%, which was even higher than cisplatin (35.2%). In addition, Pt2 displayed green luminescent property and potent telomerase inhibition. Our findings demonstrated the promising development of platinum(II) complexes containing jatrorrhizine derivatives as novel Pt-based anti-cancer agents.

KEYWORDS: platinum(II) complex, Jatrorrhizine derivatives, p53, telomerase, antitcancer activity, apoptosis

2

1. Introduction Cisplatin and its derivatives such as oxaliplatin and carboplatin have been successfully used to treat different tumors in the past decades [1−7]. However, these chemotherapeutic agents have high toxicity and side effects [8−15]. To overcome these side effects, different platinum complexes have been made, including cationic Pt(II) complexes [3,17−20], iminophosphorane organometallic Pt(II) complexes [3], 8-aminoquinoline triamineligated

monofunctional Pt(II)

Pt(II)

complexes

[17],

cis-[Pt(NH3)2(phenanthridine)Cl]NO3

complexes

[16],

monofunctional

(phenanthriplatin) [18], monofunctional pyriplatin platinum agent [19], and N-alkyl imidazole cationic Pt(II) complex [20]. In addition, a large number of metal complexes with natural products, such as liriodenine, oxoglaucine, plumbagin, matrine, oxoisoaporphine and oxoaporphine have been prepared as anti-cancer compounds [21−27], which showed selective activity against tumor cells. (Ox)oisoaporphine and 3-(2′-benzimidazolyl)-7methoxycoumarin Pt(II)/Ru(II) complexes have been reported as telomerase inhibitors targeting telomeric G-quadruplex DNA (G4 DNA), and exhibited higher effective inhibitory

effects

[24−27].

Recent

studies

indicated

that

p53-dependent

down-regulation of hTERT and telomerase levels induced apoptosis in cancer cells as a result of drug treatments [28−44]. Telomerase was over-expressed in 85−90% of cancer cells but has undetectable activity in the normal cell line [45−52], thus its specificity has prompted the design of next-generation p53- and telomerase-targeting Pt(II) anti-cancer complexes. More recently, Jatrorrhizine and its derivatives are isolated from Tinospora capillipes Gagnep as the major bioactive components [53−59]. They are important compounds due to their wide range of antifungal, anti-tumor, antibacterial, and

3

parasite-fighting activities [53−59]. However, no metal complexes with jatrorrhizine derivatives have been reported in the literature, and the detailed in vitro and in vivo anti-cancer mechanisms of p53- and telomerase-targeting jatrorrhizine Pt(II) complexes remain to be explored. In this report, we explored the selective and effective activities of Pt(II) complexes containing jatrorrhizine derivatives. We showed the high in vitro and in vivo anticancer activities of Pt(II) complexes, [Pt(Jat1)Cl]Cl (Pt1) and [Pt(Jat2)Cl]Cl (Pt2), with jatrorrhizine derivatives. The two novel jatrorrhizine derivatives Pt(II) complexes Pt1 and Pt2 exhibited desirable green luminescent property for cellular applications, and showed potent p53 and telomerase inhibiting activities. Furthermore, the in vitro and in vivo mechanism of actions of the novel jatrorrhizine Pt(II) anti-cancer agents were explored.

2. Results and Discussion 2.1 Synthesis and Characterization Jatrorrhizine (1, Figure 1) was an alkaloid isolated from Tinospora capillipes Gagnep [53−59]. The trifluoro-acetate3-[5-(bis-pyridin-2-ylmethyl-amino)-pentyloxy]-2,9,10trimethoxy-5,6-dihydro-isoquino[3,2-a]isoquinolinylium

(Jat1)

and

trifluoro-

acetate3-[7-(bis-pyridin-2-ylmethyl-amino)-heptyloxy]-2,9,10-trimethoxy-5,6-dihydro -isoquino[3,2-a]isoquinolinylium (Jat2) were synthesized (Figure 1, Scheme S1 and S2), starting from jatrorrhizine. Next, cis-Pt(DMSO)2Cl2 (1.0 mmol) was mixed with 1.0 mmol Jat1 and Jat2 in CH3CN (5.0 mL) at 60 °C for 4.0 h to yield yellow product of [Pt(Jat1)Cl]Cl (Pt1) and [Pt(Jat2)Cl]Cl (Pt2), respectively (Figure 1), which were isolated and characterized. In addition, the stabilities of jatrorrhizine platinum(II) complexes Pt1 and Pt2 in Tris-HCl solution (pH= 7.35, 10 mM) are presented in

4

Figures S1−S22 (Electro Supporting Information Materials, ESI†). In the jatrorrhizine platinum(II) complexes Pt1 and Pt2, the Pt(II) center was four coordinated with the Jat1 or Jat2 ligand (Jat1-N∧N∧N or Jat2-N∧N∧N) via three N atoms and one Cl atom, to form a square-planar geometry (Figure 1).

Figure 1. Synthetic routes for jatrorrhizine derivative ligands (Jat1 and Jat2) and their Pt(II) complexes Pt1 and Pt2. 2.2 Crystal Structures of Pt3 The synthesis involved reacting cis-Pt(DMSO)2Cl2 (1.0 mmol) with 1.0 mol equiv of 2,2′-dipicolylamine (DPA) in p-xylene (0.1 mL) and CH3CN (2.5 mL) at 60 °C for 4.0 h to produce [Pt(DPA)Cl]⋅(p-xylene) (Pt3). Thus, Pt3 was orthorhombic, as well as its analogues jatrorrhizine platinum(II) complexes Pt1 and Pt2, with each molecule unit containing one 2,2′-dipicolylamine (N1∧N2H∧N3-DPA) ligand, one p-xylene molecule and one Cl ion (Figure 2, Tables S1−S3). In addition, all the bond lengths (Table S2) and bite angles (Table S3) were within the normal range.

5

Figure 2. X-ray crystal structure of [Pt(DPA)Cl]⋅(p-xylene) (Pt3). 2.3 In Vitro Cytotoxicity The cytotoxic of jatrorrhizine platinum(II) complexes Pt1, Pt2, Jat1 and Jat2 in comparison to TFA, cis-Pt(DMSO)2Cl2, Pt3, DPA, cisplatin and p-xylene were tested with human cervical (HeLa), cisplatin-resistant SK-OV-3 (SK-OV-3/DDP), bladder (T-24), lung (A549) and normal liver (HL-7702) cell lines, by MTT assay[60−65] after a 6.0 h treatment (Table 1). In general, the jatrorrhizine platinum(II) complexes Pt1 and Pt2, especially Pt2 complex showed higher anti-cancer activity in comparison

with

the

2,2′-dipicolylamine

(DPA)

Pt3

complex,

TFA,

cis-Pt(DMSO)2Cl2, DPA, cisplatin and p-xylene (Table 1). More significantly, the cervical (HeLa) cells were sensitive to the jatrorrhizine platinum(II) complexes Pt1 and Pt2, and the jatrorrhizine Pt(II) complexes showed satisfactory cytotoxicity with nanomolar IC50 values of 15.01±1.07nM and 1.00±0.17nM, respectively, which represented approximately 896−20180-fold increases in comparison with Jat1, 6

cisplatin and Jat2. However, the 2,2′-dipicolylamine (DPA) Pt3 complex showed cytotoxic effect in four cancer cell lines in concentrations (IC50 values) of up to 30.45±0.19 µM. HeLa cells appeared to be more sensitive (18.04±1.17 µM) in comparison to bladder (T-24), cisplatin-resistant SK-OV-3 (SK-OV-3/DDP), and lung (A549) cell lines, indicating the specificity for HeLa cells. Remarkably, the jatrorrhizine

platinum(II)

complexes

Pt1

and

Pt2

displayed

remarkable

anti-proliferation activity against HeLa cells, which was much higher than that of previously studied compounds[16−27]. Indeed, the least cytotoxic compounds were the jatrorrhizine platinum(II) complexes Pt1 and Pt2 with IC50 values of >150 µM, 9.1-fold higher against normal liver (HL-7702) cells in comparison with cisplatin. In addition, the LogP of each compound is assessed by using the "calculate molecular properties" tool of the Discovery Studio Client 4.0. [66] As shown in the Figure S23, the observed lower antitumor activity of Pt3 in cellular assays is likely due to reduced hydrophobicity [66], as indicated by lower calculated partition coefficient (LogP= -0.568) values compared to Pt2 (LogP= 7.667). Table 1. In vitro cytotoxicity of jatrorrhizine platinum(II) complexes Pt1 and Pt2 ( IC50±SD in µM (or nM)) , comparing with Pt3 and cisplatin for 6.0 h. Compounds

HeLa

SK-OV-3/DDP

T-24

A549

HL-7702

Jat1

20.18±1.05

41.52±0.54

25.06±0.71

25.16±0.58

>150

Pt1

15.01±1.07nM

10.57±0.19

0.19±0.11

2.88±1.74

>150

Jat2

15.06±1.35

30.11±1.05

19.41±1.07

15.02±1.01

>150

Pt2

1.00±0.17nM

1.25±0.34

0.05±0.02

0.12±0.06

>150

TFA

>100

>100

>100

>100

>100

DPA

>100

>100

>100

>100

>100

p-xylene

>100

>100

>100

>100

>100

Pt3

18.04±1.17

30.45±0.19

20.14±1.09

19.41±1.02

75.21±1.35

7

cis-Pt(DMSO)2Cl2

>150

>150

>150

>150

>100

cisplatin

13.50±1.18

15.01±0.39

13.14±1.55

15.09±1.55

16.41±0.99

2.4 Cellular Uptake and Telomeres Damage Table 2 shows that the cellular platinum(II) amount for Pt2 was (35.16±0.10 ng of Pt)/106 cells, 8.7- and 5.2-times higher than that of Pt3 ((4.05±0.02 ng of Pt)/106 cells) and cisplatin ((6.70±0.05 ng of Pt)/106 cells), after treatment for 6 h. As expected, the distribution of Pt2 (1.0 nM) in nuclear or mitochondria fraction (Table 2) was higher than those of Pt3 (18.0 µM) and cisplatin (13.5 µM). Furthermore, confocal microscopy analysis indicated that Pt2 (1.0 nM) exhibited 525−530 nm emission in HeLa cells under ambient conditions upon excitation at 490−495 nm (Figure 3), which acted as a green luminescent agent for cellular applications. These images also suggested that Pt2 (1.0 nM) could be effectively taken up by HeLa cells and was mainly retained within the nuclear fraction and targeted 53BP1, TRF1 and TRF2 after 6.0 h of incubation (Figure 3 and Table 2). Our results also indicated that the levels of TRF1, 53BP1 and TRF2 expression were more remarkably increased after treated with

Pt2

(1.0

nM),

which

also

induced

TRF1-

and

TRF2-telomeres

damage[24,25,45−52]. However, Pt3 (18.0 µM) did not display such obvious effects on these cancer cells, which is related to the key role of jatrorrhizine derivatives Jat2 ligand in Pt2 complex. Table 2. Cellular distribution (ICP-MS test) of Pt2 (1.0 nM), cisplatin (13.5 µM) and Pt3 (18.0 µM) in HeLa cells after 6.0 h of incubation.

Pt2

total

nuclear fraction

mitochondrial fraction

(35.16±0.10 ng of

(8.17±0.05 ng of

(7.01±0.11 ng of

Pt)/106 cells

Pt)/106 cells

Pt)/106 cells

8

Pt3

cisplatin

(4.05±0.02 ng of

(1.02±0.10 ng of

(2.24±0.03 ng of

Pt)/106 cells

Pt)/106 cells

Pt)/106 cells

(6.70±0.05 ng of

(0.86±0.02 ng of

(3.05±0.08 ng of

Pt)/106 cells

Pt)/106 cells

Pt)/106 cells

9

Figure 3. Pt2 (1.0 nM) induced telomere dysfunction in HeLa cells. The HeLa cells were incubated with Pt2 (1.0 nM) at 37 oC for 6.0 h, and then processed for 53BP1 (red), TRF1 (red) or TRF2 (red) and the nuclei were stained with DAPI (blue). Excitation wavelength (λex) of Pt2: 490−495 nm; Emission filters (λem): 525−530 nm. 10

The excitation wavelength (λex) and emission filters (λem) of Pt2 were determined with Zeiss LSM710 microscope and UV-vis spectroscopy. 2.5 p53 contributes to Pt2-mediated telomerase inhibition and apoptotic induction After transfection with siRNA-p53-270, siRNA-p53-1043, negative control and siRNA-p53-1157 plasmid vector (Table S4), it was found that siRNA-p53-1157 plasmid vector was most effective in inhibiting p53 expression in HeLa cells for 24 h (Figures 4 and S24). Next, we thus checked whether the p53 status in HeLa cells can affect apoptotic induction by Pt2 (1.0 nM) in comparison with Pt3 (18.0 µM). After treated with Pt2 (1.0 nM) for 6.0 h, the expression of p53 was significantly increased in HeLa cells (Figure 5a). In contrast, Pt3 at 18.0 µM significantly decreased the accumulation of p53 in HeLa cells (Figure 5), which was different from Pt2 (1.0 nM). As expected, Pt2 (1.0 nM) and Pt3 (18.0 µM) caused more apoptotic cells (populations) in HeLa (+siRNA-p53-1157) cells than in normal HeLa cells, as determined by Annexin-V APC (Figure 6) and 7AAD staining. Double-staining suggested that Pt2 (1.0 nM) and Pt3 (18.0 µM) induced 29.35% and 25.52% apoptotic cells at 6.0 h, respectively, in HeLa (+siRNA-p53-1157) cells, whereas 20.75% and 15.04% apoptotic cells were observed in HeLa cells (control group) (Figure 6). The results indicated that the p53-mediated pathway in HeLa cells enhanced apoptosis in the following order: Pt2 (1.0 nM) > Pt3 (18.0 µM). The different biological activities of Pt2 (1.0 nM) and Pt3 (18.0 µM) are likely due to the presence of jatrorrhizine derivative Jat2 ligand in Pt2. Telomerase inhibition can lead to cell cycle arrest at sub-G1 phase, and thereby causing DNA damage in tumour cells [45−52,66−72]. To this end, TRAP-silver staining, Western blot, transfection, cell cycle assay and immunofluorescence (DNA 11

damage) assay were carried out [24,25,45−52,67−73]. As shown in Figures 7 and 8, following Pt2 (1.0 nM) treatment for 6.0 h, both telomerase inhibition (60.42%) and cell cycle arrest at sub-G1 phase (31.48%) in HeLa (+siRNA-p53-1157) cells were more significantly decreased and increased in comparison with the HeLa cells (control group). Such observations on telomerase related-proteins (c-myc, hTERT) levels and DNA damage in Pt2 (1.0 nM)- and Pt3 (18.0 µM)- treated cells were further confirmed by the immunofluorescence (Figures 3 and 9) and Western blot (Figure 5) analysis. The results supported the requirement of p53 (siRNA-p53-1157) for telomerase inhibition, c-myc/hTERT inhibition and DNA damage (activation of 53BP1, H2A.X, TRF1, and TRF2), and may explain the mechanism underlying Pt2 (1.0 nM)-induced apoptosis in the following order: Pt2 (1.0 nM) > Pt3 (18.0 µM), which is related to the key role of jatrorrhizine derivative Jat2 ligand in Pt2 complex.

Figure 4. Western blot images (A) and analysis (B) of p53 level in HeLa for 24 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA negative control plasmid vector; 3: HeLa cells + siRNA-p53-270; 4: HeLa cells + siRNA-p53-1043; 5: HeLa cells + siRNA-p53-1157.

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Figure 5. Western blot images (A) and analysis (B) of c-myc, p53 and hTERT levels in HeLa incubated with Pt2 (1.0 nM) and Pt3 (18.0 µM) at 37 oC for 6.0 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA-p53-1157; 3: HeLa cells + siRNA-p53-1157 + Pt3 (18.0 µM); 4: HeLa cells + siRNA-p53-1157 + Pt2 (1.0 nM); 5: HeLa cells + Pt2 (1.0 nM); 6: HeLa cells + Pt3 (18.0 µM); 7: HeLa cells + siRNA negative control plasmid vector.

13

Figure 6. Pt2 (1.0 nM)-induced apoptotic in HeLa cells mediated by p53 at 37 oC for 14

6.0 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA-p53-1157; 3: HeLa cells + siRNA-p53-1157 + Pt3 (18.0 µM); 4: HeLa cells + siRNA-p53-1157 + Pt2 (1.0 nM); 5: HeLa cells + Pt2 (1.0 nM); 6: HeLa cells + Pt3 (18.0 µM); 7: HeLa cells + siRNA negative control plasmid vector. Theses apoptotic cells were determined by FCM (flow cytometry) with Annexin-V APC (red, λex= 633 nm; λem= 660 nm) and 7AAD (red, λex= 546 nm; λem= 647 nm) staining.

Figure 7. Pt2 (1.0 nM)-inhibited telomerase in HeLa cells mediated by p53 at 37 oC for 6.0 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA-p53-1157; 3: HeLa cells + siRNA-p53-1157 + Pt3 (18.0 µM); 4: HeLa cells + siRNA-p53-1157 + Pt2 15

(1.0 nM); 5: HeLa cells + Pt2 (1.0 nM); 6: HeLa cells + Pt3 (18.0 µM); 7: HeLa cells + siRNA negative control plasmid vector.

16

Figure 8. Cell cycle after Pt2 (1.0 nM) treatment in HeLa cells mediated by p53 at 37 o

C for 6.0 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA-p53-1157; 3: HeLa

cells + siRNA-p53-1157 + Pt3 (18.0 µM); 4: HeLa cells + siRNA-p53-1157 + Pt2 (1.0 nM); 5: HeLa cells + Pt2 (1.0 nM); 6: HeLa cells + Pt3 (18.0 µM); 7: HeLa cells + siRNA negative control plasmid vector.

17

18

Figure 9. Pt2 (1.0 nM) induced DNA damage in HeLa cells mediated by p53 at 37 oC for 6.0 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA-p53-1157; 3: HeLa cells + siRNA-p53-1157 + Pt3 (18.0 µM); 4: HeLa cells + siRNA-p53-1157 + Pt2 (1.0 nM); 5: HeLa cells + Pt2 (1.0 nM); 6: HeLa cells + Pt3 (18.0 µM); 7: HeLa cells + siRNA negative control plasmid vector.

2.6 p53 contributes to Pt2-mediated mitochondria dysfunction Hwang, et al. showed that wild-type p53 dysfunction is strongly associated with mitochondria dysfunction, cell aging, apoptotic and affected metabolism.[73,74,77,78] Thus, to gain insight into whether p53 inhibition contributes to Pt2 (1.0 nM) induced mitochondria dysfunction, we established a p53 down-regulated HeLa cells (+siRNA-p53-1157), as shown in Figures 4 and S22. We found that the induction of mitochondrial membrane potential (∆Ψm, MMP) was more obviously decreased in HeLa (+siRNA-p53-1157) cells after Pt2 (1.0 nM)- and Pt3 (18.0 µM)-treatment in comparison with the normal HeLa cells (Figure 10), indicating that p53 contributed to Pt2 (1.0 nM)- and Pt3 (18.0 µM)- caused decrease of ∆Ψm level. The apoptosis-inducing ability was in the following order: Pt2 (1.0 nM) > Pt3 (18.0 µM), indicating the key role of jatrorrhizine derivative Jat2 ligand in Pt2 complex.

19

20

Figure 10. Pt2 (1.0 nM)-decreased ∆Ψm level in HeLa cells mediated by p53 at 37 o

C for 6.0 h. 1: HeLa cells (control group); 2: HeLa cells + siRNA-p53-1157; 3: HeLa

cells + siRNA-p53-1157 + Pt3 (18.0 µM); 4: HeLa cells + siRNA-p53-1157 + Pt2 (1.0 nM); 5: HeLa cells + Pt2 (1.0 nM); 6: HeLa cells + Pt3 (18.0 µM); 7: HeLa cells + siRNA negative control plasmid vector. The HeLa cells were fixed, stained with a JC-1 for 1, 2, 3, 6, 7 or Mito-Red (red, Ex/Em=579nm/600nm; working concentration was 500nM) for 4, 5, respectively, and subsequently detected by flow cytometry.

2.7 In Vivo Antitumor Activity To investigate the in vivo tumor inhibition efficacy of Pt2 (2.0 mg/kg), the HeLa tumour-xenograft mouse model was constructed. Strong inhibition (IR= 48.8%, p<0.05, p vs control group) on HeLa tumor growth was observed after treatment with Pt2 (2.0 mg/kg per 2 days) for 21 days (Figure 11, Table S5–S7), and the IR value was increased by ca. 1.4-fold as compared with that of cisplatin (35.2%) at the dose of 2.0 mg/kg per 2 days [65,79–83]. Most strikingly, after 21 days of treatment with Pt2 (2.0 mg/kg per 2 days), the body weight of the treated group was hardly affected (mstart= 18.6±0.5g, mend= 20.1±0.5g) in comparison with the control group (mstart= 18.7±1.2g, mend= 20.7±1.4g), suggesting the low systemic toxicity of Pt2 (2.0 mg/kg per 2 days).

Figure 11. HeLa tumor progression (B) and changes of tumor volume (A) after 21 21

days of treatment with the jatrorrhizine platinum(II) complex Pt2 (2.0 mg/kg) in comparison with the control group (5.0% v/v DMSO/saline vehicle). 2.7 Structure-Activity Relationships (SARs) Based on our results, certain SARs among the jatrorrhizine platinum(II) complexes Pt1 and Pt2, Jat1, Jat2, TFA, cis-Pt(DMSO)2Cl2, DPA, 2,2′-dipicolylamine (DPA) Pt3 complex, cisplatin, and p-xylene can be established (Figure 12). i) The proliferation inhibitory activity follows the order of Pt2 > Pt1 > cisplatin > Pt3 > Jat2 > Jat1 > DPA～TFA > cis-Pt(DMSO)2Cl2. ii) The in vitro anti-tumor ability follows the order of Pt2 > Pt3. iii) The inhibition on HeLa tumor growth in vivo in the order of Pt2 > cisplatin was observed.

Figure 12. Proposed antitumor mechanisms for jatrorrhizine platinum(II) complex Pt2.

3 Conclusion Two novel jatrorrhizine and Pt(II) complexes Pt1 and Pt2, especially Pt2, showed

22

remarkable anti-tumor activity and lower toxicity in vitro and in vivo in comparison with cisplatin and 2,2′-dipicolylamine (DPA) Pt3 complex. The different biological activities of Pt2 and Pt3 are likely due to the jatrorrhizine derivative Jat2 ligand. The mechanisms of the jatrorrhizine platinum(II) complex Pt2-induced apoptosis included telomerase inhibition, mitochondrial dysfunction, DNA damage and sub-G1 phase arrest in HeLa cells. Importantly, Pt2 showed green luminescent property and potent telomerase inhibition. Taken together, platinum(II) complexes with the jatrorrhizine derivatives are promising candidates for the development new Pt-based anti-tumor drugs.

4 Experimental methods 4.1 Synthesis of the jatrorrhizine derivatives Jat1 and Jat2 4.1.1 General procedure for preparation of compound 3a 2,9,10-Trimethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-3-ol (0.500 g, 1.33 mmol, 1.00 eq, HCl), 1,5-dibromopentane (644 mg, 2.80 mmol, 379 uL, 2.10 eq) and 2a (0.500 g, 1.33 mmol, 1.00 eq) were dissolved in MeCN (12.0 mL), and K2CO3 (553 mg, 4.00 mmol, 3.00 eq) was added to the mixture at 25°C, then warmed at 60°C for 16 hrs. TLC (dichloromethane : methanol = 5 : 1, Rf = 0.56) indicated the starting material was consumed completely and new spots were formed. The reaction was used directly in the next step without purification. ESI-MS m/z: 486.1 [M-Cl]+ (Tris-HCl buffer solution containing 5% DMSO as solvent). Elemental analysis calcd (%) for C25H29BrClNO4: C 57.43, H 5.59, and N 2.68; found: C 57.42, H 5.61, and N 2.67. 4.1.2 General procedure for preparation of compound 3b 2,9,10-Trimethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinolin-3-ol (0.500 g, 1.33 mmol, 1.00 eq, HCl), 1,7-dibromoheptane (757 mg, 2.93 mmol, 2.20 eq) and 2b (0.500 g, 1.33 mmol, 1.00 eq) were dissolved in MeCN (15.0 mL), and K2CO3 (553 mg, 4.00 mmol, 3.00 eq) was added to the mixture at 25°C, then warmed to 60°C for 16 hrs. TLC (dichloromethane : methanol = 5:1, Rf = 0.67) indicated the starting material was consumed completely and new spots were formed. The reaction was 23

used directly in the next step without purification. ESI-MS m/z: 516.1 [M-Cl]+ (Tris-HCl buffer solution containing 5% DMSO as solvent). Elemental analysis calcd (%) for C27H33BrClNO4: C 58.86, H 6.04, and N 2.54; found: C 58.84, H 6.06, and N 2.53. 4.1.3 General procedure for preparation of Jat1 A mixture of 1-(2-pyridyl)-N-(2-pyridylmethyi)methanamine (495 mg, 2.04 mol, 2.00 eq),

KI

(165

mg,

993

umol,

0.800

eq)

and

3-(5-bromopentoxy)-2,9,10-trimethoxy-5,6-dihydroisoquinolino[2,1-b]isoquinline (0.650 g, 1.24 mmol, 1.00 eq, HCl) were dissolved in MeCN (6.00 mL) at 25°C and warmed at 90°C for 4.0 hrs. The reaction was filtered to obtain the filtrate, which was evaporated to obtain the crude product. The crude product was purified by reversed-phase HPLC (column: Phenomenex Luna C18, 250×50mm×10 um; mobile phase: [water (0.1%TFA)-acetonitrile (ACN)]; B%: 5%-35%, 20min). Compound N,N-bis(2-pyridylmethyl)-5-[(2,9,10-trimethoxy-5,6-dihydroisoquinolino[3,2-a]isoqui nolin-7-ium-3-yl) oxy]pentan-1-amine (1.20 g, 1.43 mmol, 19.3% yield) was obtained as a yellow oil. 1H NMR (400 MHz, METHANOL-d4) δ 9.78 (s, 1H), 8.82 (s, 1H), 8.69 (d, J = 4.8 Hz, 2H), 8.18 - 8.12 (m, 1H), 8.03 (d, J = 9.1 Hz, 1H), 7.93 (dt, J = 1.6, 7.7 Hz, 2H), 7.69 (s, 1H), 7.53 (d, J = 7.8 Hz, 2H), 7.48 (dd, J = 5.2, 7.3 Hz, 2H), 7.05 (s, 1H), 4.95 (br t, J = 6.3 Hz, 2H), 4.63 (s, 4H), 4.23 (s, 3H), 4.15 - 4.09 (m, 5H), 4.02 (s, 3H), 3.33 (br s, 2H), 3.29 (s, 2H), 1.92 - 1.81 (m, 4H), 1.59 - 1.49 (m, 2H), 1.38 (br s, 8H). ESI-MS m/z: 605.3 [M-(TFA-H)]+ (Tris-HCl buffer solution containing 5% DMSO as solvent). Elemental analysis calcd (%) for C39H41F3N4O6: C 65.17, H 5.75, and N 7.79; found: C 65.16, H 5.77, and N 7.78. 19F NMR (471 MHz, CHCl3) δ -77.316. 4.1.4 General procedure for preparation of Jat2 A mixture of 3-(7-bromoheptoxy)-2,9,10-trimethoxy-5,6-dihydroisoquinolino[2,1-b]

24

isoquinoline (688 mg, 1.25 mmol, 1.00 eq, HCl), 1-(2-pyridyl)-N-(2-pyridylmethyl) methanamine (546 mg, 2.74 mmol, 2.20 eq) and KI (165 mg, 997 umol, 0.80 eq) were dissolved in MeCN (6.00 mL) at 25°C and warmed at 90°C for 4.0 hrs. The mixture was filtered to obtain the filtrate, which was evaporated to obtain the crude product. The crude product was purified by reversed-phase HPLC (column: Phenomenex Luna C18,

250×50mm×10

um;

mobile

phase:

[water(0.1%TFA)-ACN];

B%:

8%-38%,20min). Compound N,N-bis(2-pyridylmethyl)-7-[(2,9,10-trimethoxy-5,6dihydroisoquinolino[3,2-a]isoqui-nolin-7-ium-3-yl)oxy]heptan-1-amine (1.20 g, 1.30 mmol, 17.4% yield) was obtained as a yellow oil.

1

H NMR (400 MHz,

METHANOL-d4) δ 9.78 (s, 1H), 8.82 (s, 1H), 8.69 (br s, 2H), 8.14 (d, J = 9.1 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.93 (t, J = 1.6 Hz, 2H), 7.69 (s, 1H), 7.54 (s, 2H), 7.48 (br t, J = 2.9 Hz, 2H), 7.05 (s, 1H), 4.95 (br t, J = 6.3 Hz, 2H), 4.64 (s, 4H), 4.23 (s, 3H), 4.15 - 4.10 (m, 5H), 4.01 (s, 3H), 3.34 (br s, 2H), 3.29 - 3.27 (m, 2H), 1.87 (br d, J = 6.5 Hz, 2H), 1.79 - 1.72 (m, 2H), 1.58 - 1.52 (m, 2H), 1.44 (br s, 1H), 1.39 - 1.37 (m, 2H). ESI-MS m/z: 633.4 [M-(TFA-H)]+ (Tris-HCl buffer solution containing 5% DMSO as solvent). Elemental analysis calcd (%) for C41H45F3N4O6: C 65.94, H 6.07, and N 7.50; found: C 65.93, H 6.09, and N 7.49.

19

F NMR (471 MHz, CHCl3) δ

-77.316. 4.2 Synthesis of Pt1–Pt3. Synthesis of Pt1 and Pt2. The cis-Pt(DMSO)2Cl2 (1.0 mmol) was mixed with 1.0 mmol Jat1 and Jat2 in CH3CN (5.0 mL) at 60 °C for 4.0 h to yield yellow products of [Pt(Jat1)Cl]Cl (Pt1) and [Pt(Jat2)Cl]Cl (Pt2), respectively, which were isolated and characterized. Data for Pt1. Yield: 85.71%. ESI-MS: m/z = 947.7 for [M-Cl]+ (Tris-HCl buffer solution containing 5% DMSO as solvent), m/z = 874.4 for [M-(TFA-H)]+, TFA= trifluoroacetic acid. Elemental analysis: calcd (%) for C39H41Cl2F3N4O6Pt: C 47.57, H 25

4.20, N 5.69; found: C 47.54, H 4.22, N 5.66. 1H NMR (500 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.04 (s, 1H), 8.83 – 8.73 (m, 2H), 8.28 (td, J = 7.8, 1.6 Hz, 2H), 8.21 (d, J = 9.2 Hz, 1H), 8.04 (d, J = 9.1 Hz, 1H), 7.84 – 7.77 (m, 2H), 7.69 (s, 1H), 7.66 (tt, J = 5.6, 1.0 Hz, 2H), 7.00 (s, 1H), 5.34 (d, J = 15.9 Hz, 2H), 4.94 (t, J = 6.3 Hz, 2H), 4.85 (d, J = 15.9 Hz, 2H), 4.10 (s, 3H), 4.08 (s, 3H), 3.93 (t, J = 6.3 Hz, 2H), 3.90 (s, 3H), 3.20 (t, J = 6.4 Hz, 2H), 3.13 – 3.01 (m, 2H), 1.66 – 1.53 (m, 4H), 1.36 (p, J = 7.3, 6.8 Hz, 2H). 13C NMR (126 MHz, DMSO-d6) δ 166.29, 161.49, 158.82, 158.55, 158.29, 158.02, 151.18, 150.71, 149.51, 149.23, 145.93, 144.10, 141.79, 138.17, 133.57, 129.03, 127.27, 125.83, 123.92, 121.82, 120.62, 120.34, 119.36, 118.26, 115.90, 113.53, 112.54, 109.33, 68.59, 68.34, 64.50, 62.38, 57.52, 56.60, 55.85, 40.50, 40.34, 40.17, 40.00, 39.84, 39.67, 39.50, 28.64, 27.14, 26.40, 23.01.

19

F NMR (471 MHz,

DMSO-d6) δ -74.03. Data for Pt2. Yield: 90.85%. ESI-MS: m/z = 975.9 for [M-(TFA-H)]+ (Tris-HCl buffer solution containing 5% DMSO as solvent). Elemental analysis: calcd (%) for C41H45Cl2F3N4O6Pt: C 48.62, H 4.48, N 5.53; found: C 48.60, H 4.51, N 5.51. 1H NMR (500 MHz, DMSO-d6) δ 9.89 (s, 1H), 9.06 (s, 1H), 8.81 – 8.78 (m, 2H), 8.31 – 8.28 (m, 2H), 8.20 (d, J = 9.3 Hz, 1H), 8.05 (d, J = 9.1 Hz, 1H), 7.83 – 7.80 (m, 2H), 7.71 (s, 1H), 7.66 (td, J = 5.4, 4.6, 2.4 Hz, 2H), 7.04 (s, 1H), 5.34 (t, J = 7.9 Hz, 2H), 4.95 (t, J = 6.4 Hz, 2H), 4.87 – 4.82 (m, 2H), 4.11 (s, 3H), 4.08 (s, 3H), 3.98 (t, J = 6.5 Hz, 2H), 3.93 (s, 3H), 3.21 (t, J = 6.4 Hz, 2H), 3.05 – 3.00 (m, 2H), 1.66 (p, J = 6.6 Hz, 2H), 1.54 – 1.48 (m, 2H), 1.26 – 1.15 (m, 6H). 13C NMR (126 MHz, DMSO-d6) δ 166.34, 161.56, 158.82, 158.56, 158.30, 158.04, 151.33, 150.69, 149.49, 149.23, 145.90, 144.08, 141.80, 138.18, 133.59, 129.05, 127.23, 125.83, 123.90, 121.82, 120.78, 120.34, 119.27, 118.42, 116.05, 113.68, 112.52, 109.36, 68.79, 68.35, 64.67, 62.37, 57.51, 56.66, 55.85, 40.51, 40.34, 40.17, 40.01, 39.84, 39.67, 39.51, 32.70, 32.14, 28.90, 28.09, 26.08, 25.72. 19F NMR (471 MHz, DMSO-d6) δ -73.93. Synthesis of [Pt(DPA)Cl] ⋅(p-xylene) (Pt3). This synthesis involved reacting cis-Pt(DMSO)2Cl2 (1.0 mmol) with 1.0 mol equiv of 2,2′-dipicolylamine (DPA) in p-xylene (0.1 mL) and CH3CN (2.5 mL) at 60 °C for 4.0 h to produce [Pt(DPA)Cl]⋅(p-xylene) (Pt3). Yield: 80.17%. Elemental analysis: calcd (%) for C20H23Cl2N3Pt: C 42.04, H 4.06, N 7.35; found: C 42.02, H 4.10, N 7.33. 4.3 In Vivo HeLa-Xenograft Model Assay 26

The healthy male nude mice (pathogen-free BALB/C, 6 weeks, Beijing HFK Bioscience Co., Ltd, Beijing, China, Approval No. SCXK 2014-004) were used to establish the human cervical (HeLa)-xenograft model. The animal procedures were performed at the Institute of Radiation Medicine Chinese Academy of Medical Sciences (Tian Jin, China, approval No. SYXK 2014-0002). In addition, the healthy male nude mice were raised under 60−85% humidity at 24 °C (dark and light cycle at 12 h). HeLa-xenograft model was constructed by subcutaneous/skin injection of HeLa cancer cells (5 × 106 cells) into the male nude mice’ flank region (n = 6). The HeLa tumor-bearing mice (vehicle and drug treated-groups) were treated ip with control/vehicle group (v/v, 5.0% DMSO in saline) or with 2.0 mg/kg of the jatrorrhizine anticancer Pt(II) complexes Pt2 per 2 days. The body weight (g) and tumor volume/size (mm3) of the HeLa-xenograft model mice were accurately measured three times per seven days. The tumor size was defined as measuring the width (w) and length (l) and, thereby calculating the volume (V = l×w2/2)[24,25,32]. 4.4 The Other Experimental Methods The in vitro and in vivo anti-tumor activities of Jatrorrhizine derivatives platinum(II) complexes

were

assessed

using

similar

methods

as

in

previous

works

[24,25,32,60–84]. 4.5 Statistical Analysis All the data were used as mean ±SD (standard deviation), which were determined by two-tailed Student t tests, and p <0.05 was considered statistically significant. 4.6 Abbreviations used MTT, 3-[4, 5-dimethylthiazol-2-yl]-2, 5 diphenyl tetrazolium bromide; SD, standard deviation; IR, tumor growth inhibition rate.

27

Acknowledgments The authors were grateful for the National Natural Science Foundation of China (Nos. 21867017 and 21761033), the Natural Science Foundation of Guangxi (No. 2018GXNSFBA138021,

2018GXNSFBA281188

and

2016GXNSFAA380300),

Guangxi New Century Ten, Hundred and Thousand Talents Project ([2017]42) as well as the Innovative Team & Outstanding Talent Program of Colleges and Universities in Guangxi (2014-49 and 2017-38).

Supporting Information. The characterization and HPLC data of Jatrorrhizine derivatives and their Pt(II) complexes, and growth inhibition of HeLa xenograft in vivo.

This

material

is

available

free

of

charge

via

the

Internet

at

https://doi.org/xxx.xxxx. The CCDC numbers for 2,2′-dipicolylamine Pt3 complex was 1935611.

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Highlights: • Two highly active anticancer Pt(II) complexes, Pt1 and Pt2, with jatrorrhizine derivatives were synthesized. • Pt2 exhibited highly selective and strong cytotoxicity towards HeLa cells (1.0 nM) comparing with normal HL-7702 cells, by targeting p53 and telomerase. • Pt2 displayed green luminescent property. • Pt2 exhibited higher in vivo anticancer activity than cisplatin.