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Anticancer Platinum(IV) Prodrugs Containing Monoaminophosphonate Ester as a Targeting Group Inhibit Matrix Metalloproteinases and Reverse Multidrug Resistance Xiaochao Huang, Rizhen Huang, Shaohua Gou, Zhimei Wang, and Heng-Shan Wang Bioconjugate Chem., Just Accepted Manuscript • DOI: 10.1021/acs.bioconjchem.7b00117 • Publication Date (Web): 09 Mar 2017 Downloaded from http://pubs.acs.org on March 11, 2017

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Bioconjugate Chemistry

Anticancer

Platinum(IV)

Prodrugs

Containing

Mono-aminophosphonate Ester as a Targeting Group Inhibit Matrix Metalloproteinases and Reverse Multidrug Resistance Xiaochao Huang, †, ‡ Rizhen Huang, †, ‡ Shaohua Gou,†, ‡* Zhimei Wang,†, ‡ and Hengshan Wang§, * †

Pharmaceutical Research Center and School of Chemistry and Chemical Engineering

and ‡Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing 211189, China §

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal

Resources (Ministry of Education of China), School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin 541004, China Supporting Information

ACS Paragon Plus Environment


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Table of Contents Graphic TOC


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Abstract A novel class of platinum(IV) complexes comprising a mono-aminophosphonate ester moiety, which can not only act as a bone-targeting group but also inhibit matrix metalloproteinases (MMPs), were designed and synthesized. Biological assay of these compounds showed that they had potent antitumor activities against the tested cancer cell lines compared with cisplatin and oxaliplatin and indicated low cytotoxicity to human normal liver cells. Particularly, the platinum(IV) complexes were much sensitive to cisplatin resistant cancer cell lines. The corresponding structure-activity relationships were studied and discussed. Related mechanism study revealed that the typical complex 11 caused cell cycle arrest at S phase and induced apoptosis in Bel-7404 cells via a mitochondrial-dependent apoptosis pathway. Moreover, complex 11 had potent ability to inhibit the tumor growth in the NCI-H460 xenograft model comparable to cisplatin.



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INTRODUCTION Natural aminophosphonate, found in a number of organisms as components of proteins, polysaccharides and lipids, has attracted increasing interests in an opportunity to understand the biosynthesis with the unique formation mechanism of C-P bonds.1,2 It was known that most of natural and synthetic aminophosphonate compounds displayed a broad spectrum of biological properties and applications in the pharmacological and agrochemical fields.3-7 As the structural analogue of amino acids, their biological activity is mainly presented through metabolic regulation, restraint of metalloproteinases, and inhibition of enzymes related to tumor genesis and invasions.8-12 Since alkaline phosphatase is overexpressed in the extracellular space of specific cancer cells like hepatic carcinoma and ovarian tumor cells13,14, introduction of a phosphate ester group to anticancer drugs for the targeted delivery seems to be a reasonable strategy to improve the solubility of drugs and increase transport through cellular membrane.15,16 In facts, some phosphate esters have been applied to obtain targeted drugs for bone cancer due to their high affinity to calcium ions.15,16 It has been reported that phosphonate esters can be hydrolyzed under physiological conditions, thus, application of aminophosphonate esters is a good choice in designing targeted anticancer drug.17 Platinum agents such as cisplatin (CDDP), carboplatin and oxaliplatin (OXP) are first-line antitumor drugs for chemotherapy. Among them, cisplatin is one of the most widely used chemotherapeutic agent in clinical treatment of numerous types of cancer.18-25 However, its clinical drawbacks including serious toxicity, non-specificity and


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the development of tumor resistance to chemotherapy have confined the application of cisplatin.26-29 Recently, platinum(II) complexes conjugated with aminobisphosphonate and diethyl[(methylsulfinyl)methyl]phosphonate (SMP) ligands have also been reported to achieve bone tissue specificity.30-32 In addition, some researches indicated that platinum(II) complexes containing SMP and diethyl(aminomethyl)phosphonate (AMP) as carrier groups can significantly inhibit the activity of matrix metalloproteinases in vitro (Figure 1).32,33 Moreover, several research groups disclosed that platinum(II) complexes containing phosphonate or bisphosphonate as non-leaving groups have excellent solubility in both organic and aqueous solutions and could potentially provide anticancer agents with bone-targeting ability (Figure 2).34-37 However, unfortunately, these platinum(II) complexes exhibited lower antitumor activity than cisplatin, partly because the phosphate ligand was not easy to depart from the metal atom. In recent years, platinum(IV) complexes have been found to show great promise with kinetic inertness to act as prodrugs, since they can be effectively reduced to active platinum(II) species by intracellular reducing molecules (ascorbic acid or glutathione).38-42 So far, a few of platinum(IV) complexes containing active pharmacophores have been reported as prodrugs to improve the antitumor activity and overcome the side effect of platinum(II) anticancer agents (Figure 3).38-42 In this paper, a series of platinum(IV) prodrugs containing aminophosphonate as an axial ligand in the octahedrally geometric platinum(IV) atom were deliberately designed and obtained. The introduction of mono-aminophosphonate ester species to the platinum(IV) complexes is expected to



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target tumor tissue and inhibit matrix metalloproteinases in addition to presenting dual anticancer activities. Their in vitro cytotoxicity against a number of tumor cell lines and a human normal liver cell line were evaluated. Moreover, the mechanism of apoptotic pathway induced apoptosis in Bel-7404 cells by the representative target complex was also investigated.

Figure 1. Platinum(II) complexes containing phosphonate as a inhibitor of matrix metalloproteinase.


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Figure 2. Platinum(II) complexes bearing bone-targeting groups.

Figure 3. Chemical structures of some platinum(IV) prodrugs. RESULTS AND DISCUSSION



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Synthesis and Characterization. The intermediate of platinum(IV) complexes 1-3 were synthesized according to the reported procedures.43 Compounds 4 and 5 were prepared by the treatment of 2-(4-aminophenyl)acetic acid or 4-(4-aminophenyl)butyric acid with 3,4,5-trimethoxybenzaldehyde in methanol. Compounds 6 and 7 were then obtained in good yields by addition reaction of compounds 4 and 5 treated with diethyl phosphate, respectively. Complexes 8-13 were prepared by the treatment of compounds 6 and 7 with TBTU, Et3N, and complexes 1-3, respectively, in DMF at room temperature. All platinum(IV) complexes were characterized by 1H,

13

C,

31

P and

195

Pt NMR spectra

together with high resolution mass spectroscopy (HR-MS). The synthetic route of the target platinum(IV) complexes is shown in Scheme 1. HPLC Analyses on the Stability of Complex 11 and its Released Ability. The stability of complex 11 was examined by HPLC technique at room temperature in solution of acetonitrile/water (v/v = 30% : 70% at different times (at 0, 3, 6, 12 and 24 h), respectively. As illustrated in Figure S1 (See Supporting Information), complex 11 was stable under the same condition, which was observed in HPLC chromatograms at different time. To further confirm whether the activity of platinum(IV) complex 11 was reduced to its platinum(II) equivalent in the presence of ascorbic acid, complex 11 in a solvent of acetonitrile/water (30% : 70%) to release compound 7 and cisplatin under reduction with ascorbic acid (5 mmol/L) was investigated. As shown in Figure S2 (See Supporting Information), complex 11 was gradually reduced as the time passed, accompanied by the falling down peak of complex 11 and the rising peak of compound 7, respectively. It was noted that cisplatin was not observed in the HPLC chromatogram due to its weak chromophore in the ultraviolet detecting condition. Taken together, these data


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demonstrated that the platinum(IV) complex 11 was stable in solution and easily reduced to generate the corresponding platinum(II) equivalent and compound 7 under the ascorbic acid at the room temperature, displaying its potential biological activity. Scheme 1.

Synthetic route of the platinum(IV) complexes. Reagents and conditions:

(a) CH3OH, reflux; (b) diethyl phosphite, 50 oC; (c) TBTU, Et3N, DMF, room temperature. O

O

CHO H2N

COOH n

O

N

a

n=1, 3

O

COOH n

O O

n=1, 3

4 (n=1) 5 (n=3)

O HN

COOH n

O

b

O

O

P O O

1-3

n=1, 3

platinum(IV) complexes 8-13

c

6 (n=1) 7 (n=3)

H3N

OH

H2 N

Cl

Cl

Pt H3 N

H2 N

OH

Cl

Cl

N H2

N H2

Cl

1

O O

Cl Cl

Cl

Cl

H3N

O

O O

Cl

Cl Cl Cl

Cl 11

O

O

O

H2 N

Cl

H2 N

N H2 10 O

O P

O O

N H

O O O

Pt

O Cl

O Pt

N H2

O

O

N H

Pt H3N

O O

O O P O

O P O

O O

9

O

O P O NH

O

O

H2 N

8

O

O

Pt

Cl

O

O

NH O

Cl

Pt H3N

O

O

O

H3N

O P O

O

NH

O

O 3

O

O P O

O

Cl

2

O

O Pt

Pt

Cl

OH

N H2 12

O

O

H2 N

O Cl

O O O

Pt O

O

N H

N H2 13

In Vitro Cytotoxicity Assay. The cytotoxic potency of platinum(IV) complexes 8−13



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were evaluated by MTT assay against HepG-2 (hepatoma), BEL-7404 (hepatoma), NCI-H460 (lung), MCF-7 (breast) and MG-63 (osteosarcoma) human cancer cell lines as well as three human normal cells HL-7702 (human normal liver cell line), BEAS-2B (human normal lung cell line), NCM460 (human normal colon mucosal epithelial cell line), with CDDP, OXP and DACHPt (dichloro(1R,2R-diaminocyclohexane)platinum(II)) as positive controls. The corresponding IC50 values, obtained after 72 h exposure, are given in Table 1, Table S1 and S2. It is noted that complexes 8-13, the corresponding platinum(IV)

derivatives

of

CDDP,

OXP,

and

DACHPt

with

one

mono-aminophosphonate ester in the axial position, exhibited significant anticancer activity against all tested cell lines. Among them, complexes 8 and 11 were significantly more potent than cisplatin with IC50 values in the range of 2.66–4.65 and 0.62–2.67 µM, and had low cytotoxicity against the human normal HL-7702, BEAS-2B, and NCM460 cells with IC50 values of 46.32±2.35, 39.17±3.51, 44.75±3.24 and 44.36±2.09, 37.29±4.15, 40.63±3.21 µM compared with that of cisplatin (7.16±0.35, 25.26±3.14, 19.31±2.51 µM). In addition, we have further evaluated the ligand 6/CDDP mixture and the ligand7/CDDP mixture against the test human cancer cell lines by MTT assay. As shown in Table S1, two mixtures showed weak cytotoxicity against the test cancer cells (except MCF-7 cells) compared with complexes 8 and 11, while displayed potent cytotoxicity toward three human normal cells compared with complexes 8 and 11. The similar behaviors were also observed in complexes 9, 10, 12 and 13. Despite compounds 6 and 7 showed low cytotoxicity against all tested cell lines with IC50 values in the range


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of 59.43–135.21 and 35.56–62.53 µM, introduction of the mono-aminophosphonate ester in the axial position of the resulting platinum(IV) complexes can effectively enhance the antitumor activity in contrast to their platinum(II) counterparts. In addition, it is easy to make reliable conclusion about the relation between the structures and the biological activity. Complexes (e.g., 8–10 and 11–13), different in the carbon chain length, displayed different antitumor activities that are enhanced with an increase of the carbon chain length. The resulting platinum(IV) complexes showed high antitumor potency against a representative panel of human tumor cell lines, which can be attributed to the “synergistic effect” of both the platinum moiety and the aminophosphonate ester, respectively. Interestingly, all platinum(IV) complexes exhibited lower cytotoxicity than their corresponding platinum(II) positive controls (cisplatin, oxaliplatin and DACHPt) against three human normal cell lines, suggesting that these platinum(IV) complexes have a selective toxicity for the cancer cells. Table 1. Biological Activity of Compounds 6–13 Against Different Cancer Cell Lines. IC50 (µM) d

Compd. HepG-2

Bel-7404

NCI-H460

MCF-7

MG-63

HL-7702

6

75.25±4.11

59.43±5.62

130.55±4.65

135.21±5.41

61.41±3.32

165.24±6.20

7

55.56±2.12

35.56±3.16

40.88±2.35

62.53±3.32

36.56±2.19

112.56±5.02

8

2.66±0.54

4.65±1.14

3.34±0.47

4.36±1.03

2.74±0.45

46.32±2.35

9

1.64±0.58

6.78±0.66

1.49±1.06

3.95±0.72

3.09±1.02

48.63±3.31

10

4.21±0.63

8.88±0.94

5.15±1.05

9.23±1.21

4.55±0.78

50.25±4.01

11

1.54±0.15

0.66±0.21

0.72±0.24

2.67±0.36

1.89±0.67

44.36±2.09

12

1.63±0.22

1.23±0.13

0.89±0.33

3.21±0.24

2.95±0.44

46.36±4.04



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13

2.57±0.55

5.34±0.32

3.25±0.45

7.54±0.81

4.23±0.55

50.75±3.39

CDDP a

4.76±0.41

6.38±0.52

5.29±0.62

5.90±0.35

7.80±0.21

7.16±0.35

OXP b

14.28±1.11

17.90±2.01

14.36±1.25

15.37±2.17

6.89±0.56

9.24±0.77

DACHPt c

11.47±2.53

10.75±1.75

9.51±1.81

8.46±1.95

7.47±0.27

4.47±0.53

a

Cisplatin,

b

Oxaliplatin,

c

Dichloro(1R,2R-diaminocyclohexane)platinum(II).

d

IC50 is

the drug concentration effective in introducing 50% of cell death measured by MTT assay after 72 h drug exposure and mean values ± standard deviation depend on three independent experiments. Effects of Complexes 6-13 on Cisplatin Resistant Cancer Cell Lines. Drug resistance has largely limited the efficacy of cisplatin in clinical applications for treatment of solid tumors. According to the above biological assay, we further investigated cytotoxicity of the platinum(IV) complexes toward two pairs of cisplatin sensitive and resistant cancer cells including SK-OV-3 (human ovarian cancer cells) and A549 (human lung epithelial cells). As shown in Table 2, the IC50 values of cisplatin against SK-OV-3 and SK-OV-3/CDDP cancer cell lines were 3.50 and 35.05 µM, respectively, and those against A549 and A549/CDDP cancer cell lines were 8.25 and 46.45 µM, respectively. It was of much significance to observe that these platinum(IV) complexes presented almost the same antitumor activities as cisplatin against the cisplatin sensitive cancer cell lines (0.89-3.57 µM for SK-OV-3, 3.95-5.93 µM for A549), while they showed much more potent cytotoxicity than cisplatin against the cisplatin resistant cancer cell lines (1.16-3.09 µM for SK-OV-3/CDDP, 4.10-6.73 µM for A549/CDDP). Interestingly, the


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antitumor activity of all the platinum(IV) complexes against cisplatin resistant cancer cell lines did not change to much compared with them against the non-resistant cancer cell lines. It was of much significance to observe that these platinum(IV) complexes had smaller resistance factors (1.18-1.44 for SK-OV-3/CDDP and 1.09-1.25 for A549/CDDP) than cisplatin (10.01 for SGC-7901/CDDP and 5.63 for A549/CDDP). The results indicated that these platinum(IV) complexes might be useful in the treatment of drug refractory tumors resistant to platinum(II) anticancer agents. Table 2. Biological Activity of Compounds 6–13 Against Cisplatin Sensitive and Resistant Cancer Cell Lines SKOV-3 and A549. Compd.

IC50 (µM)

resistant

IC50 (µM)

resistant

SK-OV-3

SKOV-3/CDDP

factor

A549

A549/CDDP

factor

6

53.42±4.42

56.13±5.02

1.05

89.05±4.71

95.92±5.06

1.08

7

33.74±2.55

36.24±3.61

1.07

75.35±3.54

80.95±4.75

1.07

8

2.01±1.13

2.91±1.04

1.44

5.42±1.03

6.17±1.19

1.14

9

2.29±0.42

3.09±0.72

1.35

5.93±1.40

6.73±1.40

1.13

10

3.57±1.03

5.07±1.21

1.42

5.52±0.93

6.92±1.30

1.25

11

0.89±0.27

1.16±0.30

1.30

3.95±0.42

4.10±0.32

1.04

12

1.12±0.19

1.53±0.12

1.37

5.25±0.37

5.75±0.68

1.09

13

2.57±0.35

3.02±0.34

1.18

5.20±0.29

6.18±0.26

1.19

CDDP a

3.50±0.22

35.05±3.35

10.01

8.25±0.59

46.45±0.75

5.63

OXP b

5.43±0.57

21.45±2.26

3.95

10.41±0.82

31.06±0.59

2.98

DACHPt c

3.21±0.15

23.72±3.11

7.39

9.35±0.38

34.21±0.36

3.67

a

Cisplatin,

b

Oxaliplatin,

c

Dichloro(1R,2R-diaminocyclohexane)platinum(II).

d

IC50 is

the drug concentration effective in introducing 50% of cell death measured by MTT assay



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after 72 h drug exposure and mean values ± standard deviation depend on three independent experiments. Cellular Uptake. For comparison, complexes 8 and 11 were typically selected to carry out the cellular uptake test in Bel-7404 cells by using the inductively coupled plasma mass spectrometry (ICP-MS). As shown in Figure 4 and Table 3, treating Bel-7404 cells with the platinum(IV) complexes (10.0 and 20.0 µM) for 12 h resulted in a considerably increase in the content of cellular platinum in a concentration dependent manner, suggesting facile internalization of the complexes within 12 h. Especially, the uptakes of complexes 8 and 11 were markedly higher than those of cisplatin. After exposure to 20.0 µM of complexes 8 and 11 for 12 h, the concentration of cellular platinum rose to 626 and 780 ng/106 cells, respectively, which is more than two times as much as that of cisplatin. Upon the results from the cytotoxicity assay and cellular uptake tests, it seems that the enhanced cellular uptake can result in the increase of the cytotoxicity. Table 3. Cellular Uptake of 8 and 11 in Bel-7404 Cells after 12 h of Incubation Complexes

Pt content (ng/106 cells) Bel-7404

8 (10 µM)

422±45

8 (20 µM)

626±63

11 (10 µM)

572±56

11 (20 µM)

780±75

CDDP (10 µM)

200±21

CDDP (20 µM)

290±30


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Figure 4. (A) Intracellular accumulation of cisplatin, 8 and 11 (10, 20 µM) in Bel-7404 cells after 12 h. Each value is in nanograms of platinum per 106 cells. (B) In vitro cytotoxicity of 8, 11 and CDDP toward Bel-7404 cancer cells at the same concentration for 72 h. Results are expressed as the mean ± SD for three independent experiments. P < 0.05. Cell viability. The effect of complexes 8 and 11 on cell proliferation after 72 h treatment was checked in Bel-7404 cancer cells by MTT assay. Our results indicated that the complexes 8 and 11 can significantly inhibit cell proliferation in Bel-7404 cancer cells compared with cisplatin on the same conditions (10 and 20 µM). (Figure 4 B). Inhibition of Matrix Metalloproteinases (MMPs) Activity. Since MMPs play an important role in stages of initiation, angiogenesis, primary tumor growth, and metastasis.44-46 In the past few years, there have been some reports about the inhibition of cell migration and invasion by MMP inhibitors that have been recognized as promising target tumors agents. 47-49 In previous work, it has been found that aminophosphonates and its derivatives have shown potent inhibition activities towards MMPs.50 Therefore,



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the synthesized platinum(IV) complexes with one mono-aminophosphonate ester in the axial position, were further investigated in vitro against the activated MMP-3, MMP-9 and MMP-11 using synthetic fluorogenic substrates according to a previously reported procedure.51 The IC50 values obtained in the performed in vitro inhibition assays of the target compounds are summarized in Table 4 and Figure.5. As shown in Table 4, the newly synthesized platinum(IV) complexes are potent MMPs inhibitors, with IC50 values mostly in the micromole range. From these data, the test compounds were found to preferentially inhibited MMP-11 than MMP-3 and MMP-9, respectively. In the MMPs studies, both compounds 6 and 7 are capable to inhibit MMP-11 with calculated IC50 values of 11.97 and 8.14 µM, respectively. Notably, it seems that complex 11, bearing a longer carbon chain length, effectively inhibited MMP-11 than complex 8, with IC50 values of 9.45 and 12.36 µM, respectively. Interestingly, cisplatin did not inhibit the activity of the selected MMPs even up to 100 µM. It was established that the length of the carbon chain significantly affected MMPs inhibitory activity and selectivity. According to the result of MMPs assay, the cisplatin did not inhibit MMPs, while the resulting Pt(IV) compounds 8 and 11 were found to effectively inhibit MMPs with IC50 values close to compounds 6 and 7. So we postulate that such inhibitory effects on MMP protein of compounds 8-11 arose from the conjugating aminophosphonate moiety in the Pt(IV) complexes.


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Table 4. Inhibitory Activities against MMP-3, -9 and -11 of Target Compounds. IC50 (µM) a

Compounds

a

MMP-3

MMP-9

MMP-11

6

35.97±3.17

27.63±3.74

11.97±2.25

7

23.41±2.83

19.84±2.02

8.14±2.42

8

36.13±2.43

28.36±2.84

12.36±2.26

11

24.56±2.15

20.96±1.55

9.45±2.31

CDDP

NI b

NI b

NI b

IC50 against MMP-3, MMP-9 and MMP-11. b NI: no inhibition. Each value is the mean

± SD from triplicate assay in a single experiment.

Figure 5. Inhibition activities of complexes toward MMPs. Molecular docking. To gain better understanding on the potency of the synthesized aminophosphonate derivatives (6 and 7), we conducted computational docking of compounds 6 and 7 into the active sites of MMP-3, -9, and -11 to gain insight into the differences in activities against these MMPs using SYBYL-X 2.0 software. The results obtained from compounds 6 and 7 were ranging from 8.27-8.47, 9.34-10.28, 10.56-11.88



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for MMP-3, -9, -11, respectively, as summarized in Figure 6 with histogram. The binding mode of compound 7, with the best docking score and interaction in the binding site of the MMPs, is described in the present work, as illustrated in Figure 7. Some key residues as well as hydrogen bonds between the selected compound and the residues were also labeled. The S1’ cavities of these MMPs vary by the non-conserved amino acid residues lining up the pocket, which in turn alter the conformational state of the S1’ loop. Additionally, the amino acids that reside deep in the pocket have been shown to undergo significant conformational adjustment upon inhibitor binding.52 The carboxyl group extends into the pocket, interacting differently with the loop residues of MMP-11, MMP-3, and MMP-9. In MMP-11 binding mode, as depicted in Figure 7 A, the carboxyl group of compound 7 was nicely bound to the S1’ loop of ATP binding site including three hydrogen bonds with the backbone NH of Thr242, Arg244 and CO of Ala235. Besides, the polar hydrogen of NH formed a hydrogen bond with the backbone carbonyl of Glu220 and the oxygen atom of trimethoxybenzene formed a hydrogen bond with the active site residues (Ala184), which increased the binding affinity dramatically in theory, as displayed in Figure 7 A. There was the binding mode of compound 7 interacting with MMP-3 in Figure 7 B, in which two amino acids, Ala165 and Thr223, were of significance in the binding of the ligand with enzyme of MMP-3. Moreover, the Thr223 formed two hydrogen bonds with 7. In comparison, compound 7 was bonded with the bulkier loop residue Arg424 in MMP-9 by a hydrogen bond (Figure 7 C). In general, the weakest inhibition was observed for MMP-3 compared with MMP-11 and MMP-9 due to


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the carboxyl group unfavorable with the S1’ loop forming part of the S1’ deep cavity. Specifically, the crucial electrostatic interactions between the PO of compound 7 and the zinc ion of MMPs protein were observed in the binding pocket. In short, the molecular docking results along with the MMPs assay data suggested that the synthesized aminophosphonate derivatives (6 and 7) were evidently inhibitors of MMPs.

Figure 6. The histogram about docking score of compounds 6 and 7 for MMPs.



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Figure 7. Details of the crystallographic structures of compound 7 with the MMP-11 (A), MMP-3 (B), MMP-9 (C) biting site. Inhibitors and MMPs residues are shown as stick models, ionic bonds to the zinc atom (gray) and intermolecular hydrogen bonds are shown as broken lines. Migration Assay. Metastasis and invasion play an important role in later period of cancer progression. Migration is a key step during metastasis. Hence, in this study, wound healing assay was designed to evaluate the inhibition effect of complex 11 on cell migration in vitro using human umbilical vein endothelial (HUVEC) cells. As shown in


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Figure. 8, twenty-four hours after wounding, untreated HUVEC cells could be seen to have migrated into the denuded area. As expected, complex 11 and CDDP were able to inhibit the basal migration of HUVEC cells at the indicated concentrations after 24 h of treatment. Significantly, complex 11 strongly suppressed the migration of HUVEC cells compared with CDDP after 24 h at the same concentration.

Figure 8. Complex 11 inhibited the migration of HUVEC cells in vitro. At the same concentration of complex 11 (10 µM) and CDDP (10 µM) suppressed HUVEC cells migration. Values expressed were means ± SD of three independent tests. Effect on Cell Cycle Arrest. It is well-known that the cell cycle is divided into G1, G2, S and M stages. In order to confirm that the inhibition of cancer cell growth by the platinum(IV) complex was caused by cell cycle arrest, untreated cells were used as a negative control and the Bel-7404 cells treated with complex 11 (5 and 10 µM) and cisplatin (10 µM) as positive control for 48 h, respectively, and the cell cycle distribution was investigated by flow cytometry analysis after staining of DNA with propidium iodide



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(PI). As shown in Figure 9, G1 period cells gradually decreased while S phase cells gradually increased compared with the control group, S population of 27.84% was observed in cisplatin group, while 32.07% (5 µM) and 39.54% (10 µM) in complex 11 group compared with the control. On the basis of these results, cell cycle regulatory protein expression, including those of p53, p21, cyclin A and CDK2, was further examined to illuminate the molecular mechanism underlying the S phase arrest induced by complex 11 in Bel-7404 cells. In Figure 10, the expression of CDK2, cyclin A were significantly down-regulated, while p21 and p53 protein levels were markedly enhanced in Bel-7404 cells after treatment with complex 11. These results displayed that the cells are effectively arrested at S phase of the cell cycle.

Figure 9. Investigation of cell cycle distribution by flow cytometry analysis. Untreated Bel-7404 cells as a control. Bel-7404 cells were treated with complex 11 (5 and 10 µM) and CDDP (10 µM) as positive control for 48 h, respectively. Data are expressed as the mean ± SEM of three independent experiments. P < 0.05.


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Figure 10. Western blot analysis of cyclin A, CDK2, p21 and p53 after treatment of Bel-7404 cells with 11 at the indicated concentrations and for the indicated times. β-actin antibody was used as reference control. Data are expressed as the mean ± SEM of three independent experiments. P < 0.05. Complex 11 Induced Apoptotic Cell Death. Because complex 11 was found to exhibit broad spectrum antitumor activity against all tested human cancer cell lines and the best activity against Bel-7404 cell line in vitro, it was chosen to be further investigated on the mechanism of action. Bel-7404 cells were co-stained with PI and Annexin-V FITC, and the number of apoptotic cells was estimated by flow cytometry. Four quadrant images were observed by flow cytometry analysis: quadrants of Q1, Q2, Q3, and Q4 represented necrosis, late apoptosis, early apoptosis and viable cells, respectively. Apoptosis ratios (including the early and late apoptosis ratios) were presented after treatment with complex 11 and cisplatin for 12 h at the concentrations of 5 and 10 µM, respectively. As shown in Figure 11, a small number of apoptotic cells (3.59%) were present in the control, in contrast, the apoptosis ratio rose to 13.26% and 23.74% at different



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concentrations (5 and 10 µM) after treatment with cisplatin for 12 h as positive control. However, after the Bel-7404 cells were treated with complex 11 under the same condition, the apoptosis ratio was conspicuously increased from 19.71% to 36.87%. The results clearly exhibited that complex 11 effectively induced apoptosis in Bel-7404 cells in comparison with control and displayed better antitumor activity than cisplatin in a concentration-dependent manner.

Figure 11. Apoptosis ratio detection of complex 11 by Annexin V/PI assay. Bel-7404 cells were used as control. Bel-7404 cells were treated with complex 11 at 5 and 10 µM and treated with cisplatin at 5 and 10 µM as positive controls for 12 h, respectively. Data are expressed as the mean ± SEM of three independent experiments.


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Morphological Examination. In order to further validate the morphological changes of cell death, complex 11 and cisplatin treated with Bel-7404 cells were stained with Hoechst 33258. As illustrated in Figure 12, Bel-7404 cells with smaller nuclei and condensed chromatin were rarely observed in the control group. After the treatment with 5 µM of complex 11 and 10 µM of cisplatin (as positive control) for 12 h, nuclei morphology of the cells slightly changed, most of Bel-7404 cells emitted brilliant blue fluorescence and revealed typical apoptotic morphology (in the web version), respectively. Our experimental observation revealed that complex 11 could induce apoptosis toward Bel-7404 cell lines. To further confirm the Bel-7404 cells apoptosis induced by treatment with complex 11, the dual staining with Acridine Orange/Ethidium Bromide was used to evaluate the nuclear morphology of apoptotic cells. Bel-7404 cells were stained with AO/EB after treatment with 5 µM of complex 11 and 10 µM of cisplatin for 12 h, and visualized immediately under a fluorescence microscope. The live cells will be evenly stained as green and early apoptotic cells will be thickly stained as green yellow or show green yellow fragments (in the web version). The Bel-7404 cells showed a large number of apoptotic cells with nuclei stained green yellow or bright orange compared with the control cells (green) after treatment with complex 11 and cisplatin for 12 h, respectively, as shown in Figure 13.



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Control

CDDP

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Complex 11

Figure 12. Hoechst 33258 staining of complex 11 in Bel-7404 cells. Bel-7404 cells were treated with 5 µM complex 11 and 10 µM cisplatin (as positive control) for 12 h, respectively. Data are expressed as the mean ± SEM of three independent experiments.

Control

CDDP

Complex 11

Figure 13. AO/EB staining of complex 11 in Bel-7404 cells. Bel-7404 cells were treated with 5 µM complex 11 and 10 µM cisplatin (as positive control) for 12 h, respectively. Data are expressed as the mean ± SEM of three independent experiments. Complex 11 Treatment Induces Intracellular ROS Generation in Bel-7404 Cells. Many studies have revealed that ROS play an important role in the regulation of cell apoptosis.53,54 Thus, complex 11 was tested to explore whether it triggers ROS generation in Bel-7404 cells to induce apoptosis or not. The ROS level was measured with and without (control) treatment of complex 11 (5 and 10 µM) for 12 h, respectively, using the fluorescent probe DCF-DA by flow cytometry with cisplatin (5 and 10 µM) as a positive control. As shown in Figure 14, the cells treated with different concentrations of complex


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11 displayed ROS generation increased in a dose-dependent manner compared with control, separately. Taken together, this data suggested that complex 11 causes oxidative imbalance in Bel-7404 cells.

Figure 14. Assessment of the ROS production in Bel-7404 cells. After 12 h incubation with 11 and CDDP, cells were stained with DCF-DA and analyzed by flow cytometry. Data are expressed as the mean ± SEM of three independent experiments. P < 0.05. Effect of Complex 11 on Mitochondrial Depolarization. The loss of mitochondrial membrane potential is regarded as a characteristic of apoptosis. In order to further confirm the apoptosis-inducing effect of complex 11, mitochondrial membrane potential were detected, using the fluorescent probe JC-1. In non-apoptotic cells, JC-1 existed in the cytosol as aggregates in the mitochondria (orange-red), while in the apoptotic cells, JC-1 existed in the cytosol as a monomer in the mitochondria (orange-green). Bel-7404



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cells treated with complex 11 (5, 10 µM) and cisplatin (5, 10 µM) as positive control for 12 h were stained with JC-1, and not treated with complex 11 was used as control. In Figure 15 A, most of Bel-7404 cells showed green fluorescence, indicating that they were apoptotic cells with 5.0 µM complex 11 and 10 µM CDDP treatment, respectively. In addition, flow cytometry was used to quantitatively analyze changes in red/green fluorescence intensity. As shown in Figure 15 B, the green fluorescence in complex 11-treated cells significantly increased, indicating the loss of mitochondrial membrane potential in Bel-7404 cells.


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Figure 15. JC-1 mitochondrial membrane potential staining of complex 11 in Bel-7404 cells. Blank cells were used as control, cells were treated with complex 11 (5, 10 µM) and cisplatin (5, 10 µM) as positive control for 12 h, respectively. The lost of mitochondrial membrane potential was detected using inverted fluorescence microscope (A) or flow cytometry (B). Data are expressed as the mean ± SEM of three independent experiments. Complex 11 Induced Apoptosis via the Regulation of Apoptosis-Related Protein Expression. It is well-known that antitumor agents can promote apoptotic signaling through two major pathways, one is extrinsic (death receptor) and the other is intrinsic (mitochondrial) pathway.55,56 To further investigate whether complex 11 induced Bel-7404

cells

apoptosis

via

a

mitochondrial

signaling

pathway,

six

mitochondrial-mediated apoptotic proteins of Bax, Bcl-2, Cyt c, Apaf-1, caspase-9 and



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caspase-3 were tested in Bel-7404 cells and examined by Western blot analysis. The cells were treated with 5 µM of complex 11 and 10 µM of cisplatin (as positive control) for 48 h, respectively. Compared with the control group, the level of Cyt c in cytosol was increased after treatment with complex 11 and cisplatin (Figure 16). Modern studies have revealed that the released cytochrome c in cytosol interacted with apoptotic protease factor 1 (Apaf-1), which in turn proteolytically activated downstream caspase-9 and -3.57,58 Western blot analysis showed that expressions of Apaf-1, caspase-9 and caspase-3 were significantly enhanced after treatment of Bel-7404 cells with complex 11 compared with the control group (Figure 16). In addition, the induction of apoptosis by complex 11 was also observed to be associated with changes in the expression of Bax/Bcl-2 proteins ratio of the Bcl-2 family, the results exhibited that the anti-apoptotic protein Bcl-2 was significantly down-regulated, while the pro-apoptotic protein Bax was significantly up-regulated after 48 h of treatment, respectively.

Figure 16. Western blot analysis effect of complex 11 on Cyt c release in cytosol and levels of Bax, Bcl-2, Apaf-1, caspase-9 and caspase-3 expression in Bel-7404 cells.


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Bel-7404 cells were treated with at 5 µM of complex 11 and 10 µM of cisplatin for 48 h, respectively. Equal amount of protein was loaded on SDS-PAGE gel for western blot analysis as described in experimental section. β-actin was used as an internal control. Data are expressed as the mean ± SEM of three independent experiments. P < 0.05. Anti-tumor Effect of Complex 11 in Vivo. To further evaluate the in vivo antitumor activity of complex 11, the NCI-H460 tumor xenograft nude mice were used, in which two doses were adopted for complex 11 once per week. As shown in Figure 17, complex 11 significantly suppressed tumor growth at a dose-dependent manner, and the tumor inhibitory rates on day 28 after treatment reached 46.55% and 61.07%, comparable to that of cisplatin, after iv administration of the complex at 5 mg/kg ( equal mass dose to cisplatin) and 14 mg/kg ( equal mole dose to cisplatin), respectively. Complex 11 exhibited a little lower toxicity than cisplatin as time passed by, as the weight of the mice after iv administration at 5mg/kg or 14 mg/kg of complex 11 reflected in the fourth week. Taken together, complex 11 displayed potent anticancer activity both in vitro and in vivo, indicating that it might be used as a drug candidate for further research on the therapy of lung carcinoma.



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Figure 17. In vivo antitumor activity of complex 11 in mice bearing NCI-H460 xenograft. (A-B) After administered with complex 11 at doses of 5 mg/kg (equal mass dose to cisplatin) and 14 mg/kg (equal mole dose to cisplatin), cisplatin at a dose of 5 mg/kg for 28 days, the mice were sacrificed and weighed the tumors. (C) The tumor volume of the mice in each group during the observation period. (D) The body weight of the mice from each group at the end of the observation period. (E) The weight of the excised tumors of each group. The data were presented as the mean ± SEM. *P < 0.05. CONCLUSION We have designed and synthesized a series of novel platinum(IV) complexes containing a mono-aminophosphonate ester, which were confirmed by HR-MS, 1H NMR,


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13

C NMR and

31

P NMR. Cytotoxicity assay of platinum(IV) complexes 8-13 against

HepG-2, BEL-7404, NCI-H460, MCF-7, MG-63, SK-OV-3, A549, SKOV-3/CDDP, A549/CDDP, HL-7702, BEAS-2B and NCM460 cells showed that these platinum(IV) complexes displayed considerable antitumor activities and low cytotoxicity compared with positive drugs in vitro. Structure-activity relationships were thoroughly studied by changing in carbon chain length of the ligand. It was found that the resulting complexes could not only potentially provide anticancer agents with bone-targeting ability but also inhibit matrix metalloproteinases. Among these compounds, complex 11 exhibited promising in vitro antitumor activities against the tested cancer cell lines including cisplatin resistant cell lines and indicated low cytotoxicity to three human normal cells compared with cisplatin, respectively. Moreover, complex 11 remarkably exhibited inhibition of cell migration against HUVEC cells in vitro compared with cisplatin. Mechanism study revealed that complex 11 increased the S cell population and induced apoptosis in cells via a mitochondrial-dependent apoptosis pathway. In addition, complex 11 exhibited significant antitumor efficacy in the NCI-H460 xenograft mouse model in vivo. Consequently, platinum(IV) complexes containing a mono-aminophosphonate ester moiety were able to effectively enhance the anticancer activity and overcome the side effect of platinum(II) anticancer agents. This rational design offers a significant potential for the discovery of a new class of platinum(IV) anticancer agents. Materials and Instruments. All chemicals and solvents were of analytical reagent grade



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and used without further purification, unless noted specifically. The purity of all target compounds were used in the biophysical and biological studies was ≥95%. The Acitn, Bax, Bcl-2, cytochrome c, caspase-9, caspase-3, Apaf-1, cyclin A, CDK2, p21 and p53 antibodies were purchased from Imgenex, USA. All tumor cell lines were obtained from the Shanghai Institute for Biological Science (China). NMR spectra were recorded on a BRUKER AV-400 (400 MHz for 1H NMR, 100 MHz for 13C NMR and 160 MHz for 31P NMR) and BRUKER AV-600 (600 MHz for 195Pt NMR), using CDCl3 or DMSO-d6 as a solvent. Mass spectra were measured on an Agilent 6224 TOF LC/MS instrument. Elemental analyses of C, H, and N was carried out using a Vario MICRO CHNOS elemental analyzer (Elementary). General Syntheses of Compounds 6-7. A solution of 3,4,5-trimethoxybenzaldehyde (1.10 g, 5.58 mmol) and 4-(4-aminophenyl)acetic acid (0.84 g, 5.58 mmol) or 4-(4-aminophenyl)butyric acid (1.00 g, 5.58 mmol) in dry methanol (30 mL) was stirred and refluxed for overnight. After completion of reaction, the solvent was removed under reduced pressure to give the desired compound 4 or 5, which was added in diethyl phosphite (10 mL) and stirred at 50 °C for 2 h. After completed, the whole mixture was added diethyl ether (100 mL), the solvent was extracted three times with water (100 mL). The organic phase was dried over anhydrous NaSO4 and concentrated under vacuum. The residue was purified on silica gel column eluted with a mixing solvent (petroleum ether: ethyl acetate =1:1) to give compound 6 (1.7 g, yield 65.3%) or 7 (2.1g, yield 77.8%) as a yellow solid. The resulting compounds were characterized by 1H-NMR,

13

C NMR,

31

P

NMR and HR-MS techniques. Compound 6: 1H NMR (400 MHz, DMSO-d6) δ 12.11 (s,


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1H), 6.92 – 6.88 (m, 4H), 6.76 (d, J = 8.4 Hz, 2H), 6.29 – 6.25 (m, 1H), 4.94 – 4.89 (m, 1H), 3.92 – 3.88 (m, 4H, 2×-CH2CH3), 3.74 (s, 6H, 2×-OCH3), 3.61 (s, 3H, -OCH3), 3.33 (s, 2H), 1.20 (t, J = 7.0 Hz, 3H, 2×-CH2CH3), 1.07 (t, J = 7.0 Hz, 3H, 2×-CH2CH3). 13C NMR (100 MHz, DMSO-d6) δ 173.71, 152.98, 152.96, 146.60, 146.45, 137.17, 133.11, 130.07, 123.63, 113.91, 106.37, 106.31, 62.95, 62.6, 60.40, 56.38, 55.39, 53.87, 16.85, 16.61. 31P NMR (160 MHz, CDCl3) δ 22.86. HR-MS (m/z) (ESI): calcd for C20H30NO8P [M-H+]: 466.16308; found: 466.16482. Elem Anal. Calcd (%) for C, 56.53; H, 6.47; N, 3.00; found: C, 56.31; H, 6.51; N, 2.72. Compound 7: 1H NMR (400 MHz, CDCl3) δ 6.95 (d, J = 8.2 Hz, 2H), 6.70 (d, J = 2.1 Hz, 2H), 6.60 (d, J = 8.3 Hz, 2H), 4.67 (d, J = 24.2 Hz, 1H), 4.20 – 3.91 (m, 3H), 3.82 (d, J = 5.0 Hz, 9H, 3×-OCH3), 3.75 – 3.67 (m, 1H), 2.53 (t, J = 7.5 Hz, 2H), 2.33 (t, J = 7.2 Hz, 2H), 1.99 – 1.79 (m, 2H), 1.28 (t, J = 7.1 Hz, 3H, -CH2CH3), 1.14 (t, J = 7.0 Hz, 3H, -CH2CH3).

13

C NMR (100 MHz, CDCl3) δ 177.80, 153.31, 153.29, 144.83,

144.67, 137.58, 131.55, 131.28, 129.17, 114.01, 104.95, 104.89, 63.64, 63.58, 60.84, 57.11, 56.11, 55.60, 34.17, 33.24, 26.68, 16.46, 16.27.

31

P NMR (160 MHz, CDCl3) δ

22.79. HR-MS (m/z) (ESI): calcd for C24H33NO8P [M-H+]: 494.19438; found:494.18534. Elem Anal. Calcd (%) for C, 58.18; H, 6.92; N, 2.83; found: C, 58.03; H, 6.95; N, 2.55. General Synthesis of Compounds 8-13. To a solution of 6 or 7 (1.0 equiv), TBTU (1.5 equiv) and Et3N (1.5 equiv) in dry DMF (4 mL), compound 1, 2 or 3 (1.0 equiv) was added in portions and the mixture was stirred at room temperature for overnight. After completion of reaction, the whole mixture was added CH2Cl2 (120 mL), the solvent was extracted two times with water (100 mL). The organic phase was dried over anhydrous Na2SO4 and concentrated under reduced pressure. The residue was purified on silica gel



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column eluted with a mixing solvent (CH2Cl2:MeOH = 50:1) to give the desired complexes 8-13 as a yellow solid. The resulting compounds were confirmed by HR-MS, 1

H-NMR, 13C-NMR and 31P-NMR spectra. Compound 8. Yield: 45.2%. 1H NMR (400 MHz, DMSO-d6) δ 6.94 (d, J = 8.3 Hz,

2H), 6.88 (s, 2H), 6.73 (d, J = 8.4 Hz, 2H), 6.26 – 6.06 (m, 7H), 4.98 – 4.89 (m, 1H), 4.10 – 4.88 (m, 4H, 2×-CH2CH3), 3.74 (s, 6H, 2×-OCH3), 3.61 (s, 3H, -OCH3), 3.38 (s, 2H), 1.20 (t, J = 7.0 Hz, 3H, -CH2CH3), 1.07 (t, J = 7.0 Hz, 3H, -CH2CH3).

13

C NMR (101

MHz, DMSO-d6) δ 179.75, 152.96, 152.94, 146.26, 146.11, 137.13, 133.21, 130.24, 125.08, 113.76, 106.39, 106.33, 62.96, 62.67, 60.42, 56.40, 55.45, 53.92, 16.88, 16.62. 31

P NMR (160 MHz, DMSO-d6) δ 22.94.

195

Pt NMR (129 MHz, DMSO-d6) δ 552.01.

HR-MS (m/z) (ESI): calcd for C22H35Cl3N3O8PPt [M-H+]:799.07968; found:800.00789. Elem Anal. Calcd (%) for C, 32.99; H, 4.28; N, 5.25; found: C, 32.75; H, 4.35; N, 5.09. Compound 9. Yield: 75.0%.1H NMR (400 MHz, DMSO-d6) δ 9.42 (d, J = 9.4 Hz, 1H), 8.08 (d, J = 3.9 Hz, 1H), 7.77 (s, 1H), 7.52 – 7.32 (m, 1H), 6.96 (d, J = 8.2 Hz, 2H), 6.88 (s, 2H), 6.74 (d, J = 8.2 Hz, 2H), 6.26 – 6.22 (m, 1H), 4.98 – 4.89 (m, 1H), 4.13 – 3.78 (m, 4H, 2×-CH2CH3), 3.74 (s, 6H, 2×-OCH3), 3.61 (s, 3H, -OCH3), 3.38 (s, 2H), 2.68 (d, J = 9.2 Hz, 1H), 2.36 (d, J = 10.5 Hz, 1H), 2.13 – 1.97 (m, 2H), 1.54 – 1.36 (m, 3H), 1.20 (t, J = 7.0 Hz, 3H, -CH2CH3), 1.12 – 0.79 (m, 6H).

13

C NMR (100 MHz,

DMSO-d6) δ 182.07, 152.97, 152.94, 146.45, 146.31, 137.16, 133.15, 129.98, 124.63, 113.82, 106.38, 106.32, 63.71, 62.95, 62.67, 62.54, 62.50, 60.40, 56.39, 55.39, 53.86, 31.39, 31.19, 24.00, 23.97, 16.87, 16.61. 31P NMR (160 MHz, DMSO-d6) δ 22.91.

195

Pt

NMR (129 MHz, DMSO-d6) δ 412.90. HR-MS (m/z) (ESI): calcd for C28H42Cl3N3O8PPt


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[M-H+]:879.41228; found: 880.14076. Elem Anal. Calcd (%) for C, 38.17; H, 4.81; N, 4.77; found: C, 37.90; H, 4.93; N, 4.61. Compound 10. Yield: 63.5%. 1H NMR (400 MHz, DMSO-d6) δ 8.49 – 8.14 (m, 3H), 7.63 – 7.52 (m, 1H), 6.95 – 6.85 (m, 4H), 6.73 (d, J = 8.5 Hz, 2H), 6.26 – 6.23 (m, 1H), 4.96 – 4.87 (m, 1H), 4.11 – 3.77 (m, 4H, 2×-CH2CH3), 3.74 (s, 6H, 2×-OCH3), 3.61 (s, 3H, -OCH3), 3.39 (s, 2H), 2.33 (d, J = 10.6 Hz, 1H), 2.06 – 1.90 (m, 2H), 1.59 – 1.16 (m, 8H), 1.11 – 0.91 (m, 5H).

13

C NMR (100 MHz, DMSO-d6) δ 179.94, 163.64, 152.96,

152.94, 146.52, 146.37, 137.14, 133.14, 130.04, 124.34, 113.85, 106.36, 106.31, 62.96, 62.68, 61.78, 60.40, 60.39, 56.38, 55.43, 53.91, 31.40 31

P NMR (160 MHz, DMSO-d6) δ 22.87.

195

30.92, 23.94, 23.89, 16.86, 16.61.

Pt NMR (129 MHz, DMSO-d6) δ 1004.05.

HR-MS (m/z) (ESI): calcd for C30H42ClN3O12PPt [M-H+]:897.18124; found: 898.18252. Elem Anal. Calcd (%) for C, 40.12; H, 4.71; N, 4.68; found: C, 39.88; H, 4.85; N, 4.50. Compound 11. Yield: 43.5%. 1H NMR (400 MHz, DMSO-d6) δ 6.88 (s, 2H), 6.86 (s, 2H), 6.73 (d, J = 7.8 Hz, 2H), 6.38 – 5.92 (m, 7H), 4.96 – 4.85 (m, 1H), 4.20 – 3.83 (m, 4H, 2×-CH2CH3), 3.74 (s, 6H, 2×-OCH3), 3.61 (s, 3H, -OCH3), 2.40 (t, J = 6.9 Hz, 2H), 2.17 (t, J = 7.0 Hz, 2H), 1.71 – 1.58 (m, 2H), 1.19 (t, J = 7.0 Hz, 3H, -CH2CH3), 1.07 (t, J = 6.9 Hz, 3H, -CH2CH3).

13

C NMR (100 MHz, DMSO-d6) δ 180.98, 152.97, 152.95,

144.82, 144.67, 137.14, 133.25, 130.84, 129.22, 114.08, 106.43, 106.37, 62.94, 62.66, 60.41, 56.42, 55.56, 54.03, 36.18, 34.15, 28.29, 16.85, 16.60. DMSO-d6) δ 22.93. DMSO-d6)

δ

31

P NMR (160 MHz, CDCl3) δ 22.79.

551.79.

HR-MS

(m/z)

(ESI):

calcd


31

195

P NMR (160 MHz,

Pt NMR (129 MHz,

for

C24H39Cl3N3O8PPt


1 2 3 4 5 6 7 8 9 10 11 12 13 14 15 16 17 18 19 20 21 22 23 24 25 26 27 28 29 30 31 32 33 34 35 36 37 38 39 40 41 42 43 44 45 46 47 48 49 50 51 52 53 54 55 56 57 58 59 60

[M+H+]:829.12663; found:830.11908. Elem Anal. Calcd (%) for C, 34.73; H, 4.74; N, 5.06; found: C, 34.40; H, 4.99; N, 4.45. Compound 12. Yield: 63.5%. 1H NMR (400 MHz, DMSO-d6) δ 9.79 – 9.42 (m, 1H), 8.27 – 7.93 (m, 1H), 7.85 – 7.60 (m, 1H), 7.55 – 7.24 (m, 1H), 6.88 (s, 2H), 6.82 (s, 2H), 6.73 (d, J = 8.0 Hz, 2H), 6.15 – 6.06 (m, 1H), 4.91 (dd, J = 24.2, 10.5 Hz, 1H), 4.15 – 3.77 (m, 4H), 3.75 (s, 6H, 2×-OCH3), 3.62 (s, 3H, -OCH3), 2.41 (t, J = 7.1 Hz, 2H), 2.25 – 1.98 (m, 4H), 1.66 (t, J = 10.1 Hz, 2H), 1.58 – 1.38 (m, 3H), 1.30 – 1.17 (m, J = 19.8, 6.5 Hz, 5H), 1.11 – 1.10 (m, 4H).

13

C NMR (100 MHz, DMSO-d6) δ 183.56, 152.97,

152.95, 145.87, 145.72, 137.18, 133.24, 130.64, 129.25, 114.07, 106.43, 106.38, 63.87, 62.93, 62.66, 60.42, 56.41, 55.54, 54.02, 37.24, 33.98, 31.39, 31.23, 28.43, 24.10, 23.95, 16.85, 16.60. 31P NMR (160 MHz, DMSO-d6) δ 22.93. 195Pt NMR (129 MHz, DMSO-d6) δ 412.47. HR-MS (m/z) (ESI): calcd for C30H47Cl3N3O8PPt [M+H+]:909.18923; found:910.18617. Elem Anal. Calcd (%) for C, 39.59; H, 5.21; N, 4.62; found: C, 39.35; H, 5.36; N, 4.45. Compound 13. Yield: 63.5%.1H NMR (400 MHz, DMSO-d6) δ 8.68 – 8.09 (m, 3H), 7.81 – 7.51 (m, 1H), 6.87 (s, 2H), 6.82 (d, J = 8.2 Hz, 2H), 6.72 (d, J = 8.2 Hz, 2H), 6.15 – 6.10 (m, 1H), 4.96 – 4.84 (m, 1H), 4.15 – 3.76 (m, 4H), 3.74 (s, 6H, 2×-OCH3), 3.62 (s, 3H, -OCH3), 2.33 (t, J = 7.0 Hz, 2H), 2.21 (t, J = 6.9 Hz, 2H), 2.11 – 1.98 (m, 2H), 1.68 – 1.57 (m, 2H), 1.54 – 1.33 (m, 4H), 1.19 (t, J = 7.0 Hz, 4H), 1.07 (t, J = 7.0 Hz, 4H). 13C NMR (100 MHz, DMSO-d6) δ 181.51 , 163.58, 152.97, 152.95, 145.90, 145.75, 137.18, 133.23, 130.33, 129.12, 114.07, 106.43, 106.38, 62.93, 62.66, 62.03, 61.89, 60.42, 56.41,


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55.52, 54.00, 36.44, 33.96, 31.37, 31.05, 27.98, 23.97, 23.95, 16.84, 16.60. (160 MHz, DMSO-d6) δ 22.91.

31

P NMR

195

Pt NMR (129 MHz, DMSO-d6) δ 1003.17. HR-MS

(m/z) (ESI): calcd for C32H47ClN3O12PPt [M+H+]:927.23119; found: 928.23241. Elem Anal. Calcd (%) for C, 40.07; H, 4.82; N, 4.67; found: C, 39.84; H, 4.96; N, 4.48. HPLC Analyses on the Stability of Platinum(IV) Complex 11 and Its Released Ability. The released ability of platinum(IV) complexes in a solution of acetonitrile/water (30% : 70%, v:v) was examined by HPLC chromatograms. The test compounds were made by adding ascorbic acid (5 mmol/L), compound 7 (1 mmol/L) and platinum(IV) complex 11 (1 mmol/L), respectively, to a solvent containing 30.0% acetonitrile and 70.0% water. Reversed-phase HPLC was carried out on a 250×4.5 mm ODS column. HPLC profiles were recorded on UV detection at 210 nm. Mobile phase consisted of acetonitrile /Water (30% : 70%, v/v), and flow rate was 1.0 mL/min. The samples were taken for HPLC analysis after filtered by 0.45 µm filter. Cell Culture. The HepG-2 (human hepatoma cancer cell), Bel-7404 (human hepatoma cancer cell), NCI-H460 (human lung cancer cell), MCF-7 (human breast cancer cell), MG-63 (human osteosarcoma cancer cell), SK-OV-3 (human ovarian cancer cell), SK-OV-3/CDDP, A549 (human lung cancer cell line) and A549/CDDP cancer cell lines and HL-7702 humor normal liver cell line used in this study were all obtained from the Institute of Biochemistry and Cell Biology, China Academy of Sciences. They were maintained in DMEM medium; all were supplemented with 10% heat-inactivated fetal bovine serum in a humidified atmosphere of 5% CO2 at 37 oC.



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Cytotoxicity Analysis. All tested compounds (except for CDDP and OXP dissolved in water) were dissolved in DMF (N,N-dimethylformamide) to a final concentration of 2 mmol/L and subsequently diluted in culture medium at final concentration of 2.5, 5, 10, 20, 40 µmol/L, respectively. HepG-2, Bel-7404, NCI-H460, MCF-7, MG-63, SK-OV-3, SK-OV-3/CDDP, A549, A549/CDDP, HL-7702, BEAS-2B and NCM460 cell lines were grown on 96-well plates at a density of 5 × 103 cells per well with 180 µL of the proper culture medium and treated with the compounds at 20 µL for 72 h. In parallel, the cells treated with 0.1% DMF served as negative control and those treated with CDDP and OXP as positive controls. The cells were stained with 10 µL of MTT at incubator for about 4 h. After removal of the supernatant, DMSO (100 mL) was added to dissolve the formazan crystals. The O. D. value was read by enzyme labeling instrument with 570/630 nm double wavelength measurement. Cellular Uptake Test. Bel-7404 cells were grown on 6-well plates. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. After the cells reached about 80% confluence, the test compounds were added at the same concentrations (10, 20 µM). After completion of 12 h incubation, cells were collected and washed three times with ice-cold PBS, then centrifuged at 1000×g for 10 min and resuspended in 1 mL PBS. A volume of 100 µL was taken out to determine the cell density. The remaining cells were spun down and digested at 65 oC in 200 µL 65% HNO3 for 12 h. The Pt level in cells were measured by ICP-MS.


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Inhibition of Matrix Metalloproteinase Activity assay. MMPs inhibition assays were carried out in 50 mM Tris/HCl buffer, pH 6.8, 10 mM CaCl2 at 25 °C as described previously.59 The compounds 8-11 were dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. Assays were performed with a fluorogenic substrate McaPro-Leu-Gly-Leu-Dpa-Ala-Arg-NH2 (13 µM) and human MMPs (nanomolar range concentration) from R&D Systems, except for human MMP-3, MMP-9, and MMP-11 described above. Substrate and enzyme concentrations were kept well below 10% substrate utilization to ensure evaluation of initial rates. Briefly, MMP-3, -9, or -11 and test complexes at varying concentrations (2.5, 5, 10, 20, 40, 80 µM) were pre-incubated at 37 °C for 30 min. Continuous assays were performed by recording the increase in fluorescence induced by the cleavage of fluorogenic substrates. Black, flat-bottomed, 96-well nonbinding surface plates (Corning-Costar, Schiphol-RijK, Netherlands) were used for this test. Fluorescence changes were monitored using a Fluoroskan Ascent microplate reader (Infinite M1000 Pro, Tecan US, Morrisville, NC) equipped with excitation and emission wavelengths of 325 and 393 nm, respectively. IC50 values of inhibitors were obtained with iterative fitting package (GraphPad Prism software). Molecular docking. All the docking studies were carried out using Sybyl-X 2.0 on a windows workstation. The crystal structure of the MMPs proteins were retrieved from the RCSB Protein Data Bank (MMP-11:1HV5, MMP-3: 1SLN and MMP-9: 1GKC).32,33,60 The synthetic analogues, including compounds 6 and 7, were selected for the docking



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studies. The 3D structures of these selected compounds were first built using Sybyl-X 2.0 sketch followed by energy minimization using the MMFF94 force field and Gasteiger-Marsili charges. We employed Powell’s method for optimizing the geometry with a distance dependent dielectric constant and a termination energy gradient of 0.005 kcal/mol. All the selected compounds were automatically docked into the binding pocket of MMPs by an empirical scoring function and a patented search engine in the Surflex docking program. Before the docking process, the natural ligand was extracted; the water molecules were removed from the crystal structure. Subsequently, the protein was prepared by using the Biopolymer module implemented in Sybyl. The polar hydrogen atoms were added. The automated docking manner was applied in the present work. Other parameters were established by default to estimate the binding affinity characterized by the Surflex-Dock scores in the software. Surflex-Dock total scores, which were expressed in -log10 (Kd) units to represent binding affinities, were applied to estimate the ligand-receptor interactions of newly designed molecules. A higher score represents stronger binding affinity. The optimal binding pose of the docked compounds was selected based on the Surflex scores and visual inspection of the docked complexes. Migration assay. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. The migration effects of complex 11 on HUVEC cells were detected by wound-heal assay. HUVEC cells were grown into six-well plates and treated with the same concentration (10 µM) of the test complex 11 and CDDP for 24 h, respectively. The extent of wound heal was observed after 24 h by


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imaging with fluorescence microscope. Cell Cycle Analysis. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. In cell cycle analysis, the Bel-7404 cells were treated with complex 11 (5 and 10 µM) and cisplatin (10 µM) as positive control, and maintained with the proper culture medium in 5% CO2 at 37 °C. After 24 h of incubation, cells were washed twice with ice-cold PBS, fixed and permeabilized with ice-cold 70% ethanol at -20 °C for overnight. The cells were treated with 100 µg /mL RNase A at 37 °C for 20 minutes after washed with ice-cold PBS, and finally stained with 1 mg/ml propidium iodide (PI) in the dark at 4 °C for 30 minutes. Analysis was performed with the system software (Cell Quest; BD Biosciences). Apoptosis Analysis. Apoptosis was detected by flow cytometry analysis of annexin V staining. Bel-7404 cells were grown on 6-well plates at the density of 2×106 cells/mL of the DMEM medium with 10% fetal bovine serum to the final volume of 2 mL. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. The plates were incubated for overnight and then treated with different concentrations of compound 11 (5, 10 µM) and cispaltin (5, 10 µM) for 12 h. Briefly, cells were harvested and washed with PBS twice, and then suspended cells in the annexin-binding buffer at a concentration of 1× 106 cells /ml. Then the cells were incubated with 5 µL of annexin V-FITC and 5 µL of PI for 30 minutes at room temperature in the dark. The apoptosis ratio was quantified by system software (Cell Quest; BD Biosciences).



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Hoechst 333258 Staining. The Hoechst 33258 molecular probes were also used to detect apoptotic cells. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. Bel-7404 cells (1×106 cells) were seeded in six-well tissue culture plates and treated with 5 µM of complex 11 and 10 µM of cisplatin for 12 h. Then the cells Bel-7404 were fixed in 4% paraformaldehyde for 15 minutes followed by the medium was discarded. Cells were then washed twice with ice-cold PBS and incubated with 0.5 mL of Hoechst 33258 at dark for 20 minutes. After for 20 minutes incubation, cells were washed three with ice-cold PBS and the results were analysis by a Nikon ECLIPSETE2000-S fluorescence microscope using 350 nm excitation and 460 nm emissions. AO/EB Staining. The AO/EB molecular probes were also used to detect apoptotic cells. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. Bel-7404 cells (1×106 cells) were seeded in six-well tissue culture plates. Following incubation, the medium was removed and replaced with fresh medium plus 10% fetal bovine serum and treated with 5 µM of complex 11 and 10 µM of cisplatin for 12 h. After the treatment period, briefly, the cells were harvested, suspended in PBS, stained with with 20 µL of AO/EB stain (100 mg/mL) at room temperature for 20 minutes. Fluorescence was read on an Nikon ECLIPSETE2000-S fluorescence microscope (OLYMPUS Co., Japan). Determination of Mitochondrial Membrane Potential. The loss of mitochondrial membrane potential (∆ψ) was evaluated using a JC-1 fluorescent probe. Bel-7404 cells


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were seeded at the density of 1×106 cells/mL of the DMEM medium with 10% fetal bovine serum on 6-well plates to the final volume of 2 mL and incubated for overnight. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. Then the Bel-7404 cells were treated with complex 11 (5 and 10 µM) and cisplatin (5 and 10 µM). After 12 h, the JC-1 fluorescent probe was added 20 minutes after replacing with fresh medium. Cells were harvested at 2000 rpm, rinsed twice with ice-cold PBS and mitochondrial membrane potential were analyzed in FL-1channel by flow cytometer and examined by fluorescence microscopy. The emission fluorescence for JC-1 was monitored at 530 and 590 nm, under the excitation wavelength at 488 nm, respectively. ROS Assay. Reactive oxygen species production of complex 11 treated and untreated Bel-7404 cells were detected using the fluorescent probe DCFH-DA (Beyotime, Haimen, China). The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. Bel-7404 cells were seeded at the density of 1×106 cells/mL of the DMEM medium with 10% fetal bovine serum on 6-well plates to the final volume of 2 mL and incubated for overnight. After cultivation overnight, cells were stimulated with complex 11 (5 and 10 µM) and cisplatin (5 and 10 µM) for 24 h. Cells (about 1×106 /well) were incubated with 10 mM DCFH-DA in darkness at 37 for 30 °C minutes. After being washed with PBS for three times and cellular fluorescence was analyzed immediately by flow cytometry at an excitation of 485 nm and an emission of 538 nm.



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Western Blot Analysis. The compound 11 was dissolved in DMF and CDDP was dissolved in water to a final concentration of 2 mmol/L. Bel-7404 cells were incubated in the presence of complex 11 and, after different times, were collected, centrifuged, and washed three times with ice-cold PBS. The pellet was then resuspended in lysis buffer. After the cells were lysed on ice for 20 min, lysates were centrifuged at 20000 g at 4 °C for 5 min and stored at -20 °C for future use. The protein concentrations were detected with Bradford method (BIO-RAD) using Multimode varioscan instrument (Thermo Fischer Scientifics). Equal amounts of protein per lane was separated on 12% SDS polyacrylamide gel for electrophoresis and transferred to polyvinylidine difluoride (PVDF) membrane (Amersham Biosciences). The membranes were incubated with 5% skim milk in Tris-buffered saline with Tween 20 (TBST) buffer for 1 h, and various primary antibody was added and incubated at 4 °C for overnight. After three TBST washes, the membrane was incubated with appropriate secondary antibody (1:2000) (Santa Cruz) with horse radish peroxidase

at room temperature for 2 h. Then the

membranes were washed with TBST three times for 15 min and the protein blots were detected with chemiluminescence reagent (Thermo Fischer Scientifics Ltd.). The X-ray films were developed with developer and fixed with fixer solution. Antitumor Activity in Vivo. The in vivo antitumor activity of compound 11 was evaluated using a human lung carcinoma cell line NCI-H460 in BALB/c nude mice. Five week-old male BALB/c nude mice were housed purchased from Shanghai Ling Chang biotechnology company (China), tumors were induced by a subcutaneous injection in


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their dorsal region of 1 × 107 cells in 100 µL of sterile PBS. Animals were casually divided into four groups, and starting on the second day. When the tumors reached a volume of 100−150 mm3 in all mice on day 15, the first group was injected with an equivalent volume of 5% dextrose injection via a tail vein injection as the vehicle control mice. The second group was treated with cisplatin at the doses of 5 mg/kg body weight once a week for four weeks, respectively. No.3 and No.4 groups were treated with complex 11 at the doses of 5 mg/kg (equal mass dose to cisplatin) or 14 mg/kg (equal mole dose to cisplatin) body weight once a week for four weeks, respectively. All compounds were dissolved in vehicle. Tumor volume and body weights were recorded every other day after drug treatment. All mice were sacrificed after four weeks of treatment and the tumor volumes were measured with electronic digital calipers and examined by measuring length (A) and width (B) to calculate volume (V = AB2/2). Supporting Information. Cytotoxicity of the positive drugs mixture with ligands against human cancer cell lines were evaluated by MTT assay, and the IC50 values were listed in Table S1. Cytotoxicity of the target platinum(IV) complexes 8-13 toward other two normal BEAS-2B and NCM460 cells were examined by MTT assay and the corresponding IC50 values were listed in Table S2. HPLC analyses on the stability of complex 11 (Figure S1) and the released ability of complex 11 under reduction with ascorbic acid (Figure S2). The binding modes of 6 in complex with MMP-11, MMP-3 and MMP-9 were carried out using Sybyl-X 2.0 on a windows workstation (Figure S3) and 1H NMR,

13

C,

195

Pt NMR and HR-MS of the target compounds. This material is



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available free of charge via the Internet at http://pubs.acs.org. AUTHOR INFORMATION Corresponding Author *E-mail: [email protected]. *E-mail: [email protected] Author Contributions ∥Xiaochao Huang and Rizhen Huang contributed equally to this work. Notes The authors declare no competing financial interest.

ACKNOWLEDGEMENTS We are grateful to the National Natural Science Foundation of China (Grant No. 21571033) and the New Drug Creation Project of the National Science and Technology Major Foundation of China (Grant No. 2015ZX09101032) for financial aids to this work. The authors would also like to thank the Fundamental Research Funds for the Central Universities (Project 2242013K30011) for supplying basic facilities to our key laboratory. We also want to express our gratitude to the Priority Academic Program Development of Jiangsu Higher Education Institutions for the construction of fundamental facilities (Project 1107047002). The research was also supported by the Scientific Research Foundation of Graduated School of Southeast University (YBJJ1677). KeyGen Biotech Company (China) was appreciated for completing the in vivo tests.


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