Targeting RAS-RAF pathway significantly improves antitumor activity of Rigosertib-derived platinum(IV) complexes and overcomes cisplatin resistance

Graphical abstract

Targeting RAS-RAF pathway significantly improves antitumor

activity of Rigosertib-derived platinum(IV) complexes and

overcomes cisplatin resistance

a,b,1

c,1

c

a,b

Zhikun Liu , Meng Wang , Hengshan Wang , Lei Fang *, Shaohua Gou

*

Total 11 Pt(IV) compounds by introducing ON013100 onto hydroxyl ligand of Pt(IV)

complexes derived from CDDP and oxaliplatin through ester bond were designed and

synthesized. The conjugate 26 could effectively release Pt(II) complexes and RAS

analogues to exert anti-tumor activity in novel pathway. The following biological

studies revealed that 26 could efficiently disrupt the interaction between RAS and

CRAF by specifically interacting with CRAF. Furthermore, 26 could apparently

induce DNA damage and initiate endogenous caspase apoptosis pathway. Moreover,

2

6 also showed excellent anti-tumor effect in vivo. All the findings suggested that 26

may be considered as a novel antitumor candidate for further exploration.

Targeting RAS-RAF pathway significantly improves antitumor

activity of Rigosertib-derived platinum(IV) complexes and

overcomes cisplatin resistance

a,b,1

c,1

c

a,b

Zhikun Liu , Meng Wang , Hengshan Wang , Lei Fang *, Shaohua Gou

*

a

Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,

Nanjing, 211189, China

b

Pharmaceutical Research Center, School of Chemistry and Chemical Engineering, Southeast

University, Nanjing 211189, China

c

State Key Laboratory for the Chemistry and Molecular Engineering of Medicinal Resources,

School of Chemistry and Pharmaceutical Sciences of Guangxi Normal University, Guilin, 541004,

China

Abstract

RAS-RAF pathway presents a valuable target for the cancer treatment due to its

important roles in the regulation of tumor proliferation, apoptosis and the obtained

resistance. To explore such target a RAS/CRAF interference agent, was therefore

conjugated with Pt(IV) prodrugs via ester bond, resulting in total eleven

multifunctional Pt(IV) complexes. The complexes could target genomic DNA and

disrupt the signaling transduction from RAS protein to CRAF so that block the

mitogen-activated protein kinase (MAPK) signaling pathway. Experiments in vitro

indicated that all of the Pt(IV) complexes showed potent anti-tumor activity with IC₅₀

values ranged from 8 nM to 22.55 μM, which were significantly improved as

compared with cisplatin (CDDP) whose IC values ranged from 5.45 μM to 9.05 μM.

5

0

Among them, 26 exerted the best anti-tumor activity in vitro, which not only

exhibited excellent cytotoxicity against normal tumor cells, but also against

CDDP-resistance cell lines (e.g. A549/CDDP and SKOV-3/CDDP). Importantly, 26

1

These authors contributed equally.

*

Corresponding authors

1

E-mail: lei.fang@seu.edu.cn (L. Fang); sgou@seu.edu.cn (S. Gou)

only showed little effect on normal cell lines such as HUEVC and LO2. Besides, the

following biological mechanisms studies demonstrated that 26 could efficiently enter

A549 cells, significantly arrest cell cycle at G2/M phase, disrupt the signaling

pathway and trigger endogenous caspase apoptosis pathway. Furthermore, results of a

xenograft subcutaneous model of A549 tumor showed that 26 could effectively

decrease tumor growth rates without causing loss of bodyweight.

Keywords: Pt(IV) complexes, RAS-RAF interaction disruptors, RAS-signaling and

mitochondrial apoptosis pathways, Overcoming CDDP resistance.

1

. Introduction

RAS proteins are members of membrane-bound guanine nucleotide-binding

proteins which have a crucial role in regulation of transducing extracellular signals

into diverse physiological activity, including cell proliferation, differentiation and

survival [1-4]. RAS functions as molecular switches in regulation of GDP/GTP

transformation with the help of extracellular signals and subsequently results in

activation of several downstream cascades, including RAF-MEK-ERK pathway [5-8].

Upon extracellular stimulus (e.g. GTPase-activating proteins, GAPs), RAS switches

from active GTP-bound signaling state to inactive GDP-bound signaling state with the

function of its intrinsic GTPase [9,10]. However, mutations frequently occurred in

HRAS, NRAS or KRAS (three isoforms of the RAS family) – mutating about 20% -

3

0% of all human tumors [11,12]. The mutation of RAS results in the attenuation

GTPase activity or insensitivity of GAPs, which in turn constitutively activates RAS

proteins, therefore, leading to cell growth out of control and induction of

carcinogenesis [13,14]. These facts indicate that RAS has played important roles in

occurrence and development of human cancer, thus should be a crucial target for the

cancer treatment. In the past decades, the development of RAS inhibitors proved to be

challenging due to the lack of druggable pockets [1,15-17]. Moreover, many tumors

frequently develop sharing the same signaling pathway, although lack RAS mutations

2

[

18]. Therefore, to develop a novel approach to inhibit mutant RAS-mediated cancer

is in great need.

Due to the enormous obstacles in seeking RAS inhibitors, exploiting therapeutic

agents which can disrupt RAS signaling pathways transduction provides a novel

therapy to treat mutant RAS-driven cancers [16,19]. As downstream effectors of RAS

proteins, proteins serine/threonine kinase RAF are activated by GTP-bound RAS and

subsequently activate mitogen-activated protein kinase kinases 1 and 2 (MEK1 and

MEK2) to regulate cell proliferation and apoptosis [18, 5-8]. Therefore, it should be

valuable to disrupt the signaling transduction between RAS proteins and RAF

proteins to achieve the same efficiency of RAS inhibitors in treatment of cancers.

Previously, Rigosertib (Figure 1) which possessed specific styrylbenzylsulfone

structures acted as RAS-mimetic to block signaling transduction to RAS downstream

effectors [15, 20, 21]. Interestingly, ON 013100 (Figure 1) also contained the same

specific structure, which suggested that it may probably possess the same effect on

disrupting the signaling transduction between RAS and CRAF.

Cisplatin, carboplatin and oxaliplatin (Figure 1) were FDA-approved Pt(II)

agents which were widely used in clinical chemotherapy [22-26]. Despite the

excellent anti-tumor activity in vitro and in vivo, toxicity, acquired or inherent

drug-resistance occurred frequently, limiting their chemotherapeutic efficiency greatly

[

27-30]. Pt(IV) complexes, possessing insert physicochemical properties compared to

Pt(II) complexes, were considered as promising candidates for overcoming the

drawbacks of classical platinum(II) drugs [31-34]. The desired biological properties,

such as lipophilicity, tumor-targeting property and improved cellular uptake, could be

obtained from the structural modification of the two extra ligands [35-39]. Besides,

the insert Pt(IV) complex could release Pt(II) with the help of reduction agents of

intracellular reducing substance (e.g. ascorbic acid and GSH, etc.). Thus, the Pt(IV)

complex could be considered as the prodrugs of the classic Pt(II) complexes. In this

context, it will be interesting to introduce the RAS/CRAF interference agent such as

ON 013100 to the two extra ligands of Pt(IV) complex. We and others have employed

3

one strategy by construct insert Pt(IV) prodrugs which could release Pt(II) with the

help of reduction agents [28, 33-35]. In our assumption, the resulting Pt(IV)

complexes may act as interference agents to disturb the signaling transduction

between RAS protein and RAF protein, and the block of the RAS-RAF pathway may

be beneficial for antitumor activity of Pt-based drugs since such complex could

simultaneously act on two different targets. More importantly, the block of the

RAS-RAF pathway could help to overcome the resistance to platinum-based drugs as

reported from former studies.

Based on the information aforementioned, we here constructed total 11 Pt(IV)

compounds by introducing ON013100 into hydroxyl ligand of Pt(IV) complexes

derived from CDDP and oxaliplatin through ester bond. We expected that the

conjugates could be transported into tumor cells, and then be reduced in cells to

release their corresponding Pt(II) complexes and RAS analogues to exert anti-tumor

activity in novel pathway.

2

. Results and discussion

2

.1 Chemistry

The synthetic routs for preparation of target Pt(IV) compounds were shown

Scheme 1 and 2. Compound 9 was obtained following previously reported procedures

40,41]. In detail, compound 9 was reacted with ethyl bromoacetate in the presence of

anhydrous K CO and catalytic amount of KI, to get compound 10. Complete

[

2

3

hydrolysis of 10 in a solution of MeOH/H O with LiOH H O as base gave compound

•

2

1

1. Compounds 19 and 20 were obtained by acylation of 9 with succinic anhydride or

glutaric anhydride in the presence of anhydrous K CO . Finally, condensation

2

3

reaction was carried out between 11/19/20 and 15/16/17/18 in DMF using TBTU as

condensation agent and Et N as organic base to give the final target Pt(IV)

3

compounds 12-14 and 21-28, respectively.

2

.2. In vitro anti-tumor activity assay

The anti-proliferative effects of the target Pt(IV) compounds on tumour cells

including A549 (human non-small cell lung cancer) cells, OVCaR-3 (human ovarian

4

cancer) cells, SGC-7901 (human gastric cancer) cells and HCT-116 (human colon

cancer) cells were firstly measured in vitro. The 50% growth inhibitory concentration

(

IC ) values were calculated and summarized in Table 1 after 72 h co-incubation

50

with these compounds. All the compounds showed apparent cytotoxic activity toward

the selected human tumour cells, with IC values ranging from 8 nM to 22.55 μM.

5

0

However, the compounds using glycolic acid as the linker (i.e. compounds 12, 13, 14)

showed relative low cytotoxicity (IC₅₀values ranged from 3.82 to 22.55 μM) as

compared with the positive control 9 (IC values ranged from 0.026 to 0.031 μM)

5

0

and other target compounds. The low cytotoxicity may be attributed to stability of

ether bond which could hardly be hydrolysed to release ON013100. In contrast,

compounds 19-28, which used cleavable phenolic ester bond to connect Pt(IV)

complexes and ON013100, obtained excellent anti-tumor activity (IC values ranged

5

0

from 8 to 39 nM) against all cancer cells, over 100-fold higher than that of CDDP and

oxaliplatin. In particular, 26 was most effective in inhibiting A549 cells proliferation

with IC value of 8 nM, which was even over 3-fold lower than that of ON 013100

5

0

(

IC was 29 nM).

50

2

.3. In vitro cytotoxicity assays against cisplatin-resistant cells and normal cells

The acquired drug-resistance occurred in therapy severely hindered CDDP to

fight cancer, therefore easily caused the treatment failure. It is believed that the

overexpressing P-glycoprotein is responsible for the drug-resistance since it could

pump out the therapeutic drug out of the cells and finally reduce the intracellular drug

concentrations [42-44]. Previously, Reddy and co-workers reported that ON013100

was active against a wide variety of human tumor cell lines, including

multidrug-resistance tumor cell lines [40,41]. So it may be possible that our target

compounds may also overcome the multidrug resistance. In consideration of excellent

anti-tumor activity of 26, we further determined the activity of 26 against

CDDP-resistance non-small lung cancer (A549/CDDP) cells and CDDP-resistance

ovarian cancer (SKOV-3/CDDP) cells, selecting CDDP and 20 as the controls.

Besides, to determine the cytotoxicity of 26 to human normal cell lines, human

5

umbilical vein endothelial cells (HUVEC) and normal liver (LO2) cells were also

used for the study. The IC values were calculated and summarized in Table 2. Cell

50

viability measurements showed that CDDP showed moderate cytotoxicity to A549

cells and SKOV3 cells (IC values were 8.22 μM and 7.45 μM, respectively), but

5

0

only weak activity to A549/CDDP (IC value was 34.55 μM) and SKOV-3/CDDP

5

0

(

IC₅₀value was 40.55 μM), with resistance factor as 4.2 and 5.4, respectively. In

contrast, both normal and CDDP-resistant tumor cells were almost equally sensitive to

2

0 and 26 when those tumor cells were co-incubated with 20 or 26, with RF values of

1

.3, 1.2, 1.1 and 1.6, respectively. The results strongly support that our target

compounds could overcome the CDDP-resistance. Interestingly, the RF values raised

slightly when CDDP-resistance cells were treated with combination administration of

CDDP and 20, indicating that our target compounds had better performance than the

combination administration of the active components. More importantly, the

combination administration caused obviously toxicity to normal cell lines (IC values

5

0

of 0.45 µM and 1.45 µM for HUVEC and LO2, respectively), while 20 and 26

showed only a moderate cytotoxicity to the normal cells with IC values ranging

5

0

from 7.86 to 182.80 µM, more than 100-fold higher than the IC values for tumor

5

0

cells. Obviously, our target compounds had much wider therapeutic window than the

combination administration. Therefore, the potent activity of 26 to both normal and

resistant tumor cells as well as the excellent safety profile made 26 valuable for the

following bioactivity studies.

2

.4. HPLC analysis of activation of 26 by reduction

Based on the concept of prodrug strategy, compound 26 could be reduced by

intracellular reducing substance to release their biological compound 20 and the

corresponding Pt(II) complex [42-44]. Therefore, we used HPLC chromatography to

investigate the stability of compound 26 in physiological condition (PBS, pH = 7.4)

and reductive (i.e. ascorbic acid) condition. Compound 20 or ascorbic acid was used

as reference to match the peaks appeared in the chromatography of 26 after the

treatment. The results were shown in Figure S1 (see supporting information). It was

6

found that 26 was stable within 36 h in PBS (pH =7.4). However, when treated with

ascorbic acid (3 eqiv.) compound 26 gradually decomposed as reflected by the

decreasing peak of compound 26 and the increasing peak which appeared just right at

the position of compound 20, indicating that compound 26 was successfully reduced

to its corresponding product compound 20. Surprisingly, no peak of cisplatin was

detected, perhaps for its weak chromophore under this test condition [37-39]. In all,

compound 26 was stable under physiological condition and could be effectively

reduced to compound 20 within 7 h when exposed to ascorbic acid. Moreover,

HR-MS assays were performed to investigate the reduction character of the Pt(IV)

complex. After 24 h co-incubation with 26, A549 cells were lysed and the lysates

were filtered with millipore filtration (0.22 μm). As shown in Figure S1F,

‘[CDDP+NH4] +=317.9834’ could be detected clearly in A549 cells lysates, which

revealed the existence of CDDP in A549 cells and validated the reduction of 26. This

result, together with the finding of the HPLC analysis, gave solid evidences of the

reduction of the Pt(IV) complex. Besides, we also tested the stability of 26 in

DMSO-d solution in every 24 h for one week. The results in Figure S1G showed

6

that 26 was stable in DMSO-d solution at least in 7 days and the characteristic peaks

6

1

(

6.30-6.12, 6H) of cisplatin (oxidation state) in H-NMR spectra showed no

significant change. Besides, the NMR spectrum in DMSO-d of 26 showed no

6

difference with that of DMF-d (Figure S1G).

7

2

.5. Cellular uptake, DNA platination and gel electrophoretic assays

According to previous reports [38, 39], increased cellular uptake plays important

roles in the improved activity of the Pt(IV) complexes. Therefore, the most active

compound 26 was selected to investigate whether the intracellular Pt content was

increased. A549 cells were treated and co-incubated with 26 (5 and 10 µM) for 12

hours, digested with 65% HNO and measured the Pt content with inductively

3

coupled plasma mass spectrometry (ICP-MS) [37-39]. Similarly, CDDP with the same

concentrations were carried out as positive control groups. As shown in Figure 2,

6

total 56 ng/10 cells Pt were found when the cells were treated with 5 µM CDDP, and

7

6

the Pt content was increased to 114 ng/10 cells when the concentration of CDDP was

enhanced to 10 µM. When compound 26 was employed in the test, much higher Pt

accumulation was observed. At equal dosage, the intracellular Pt content of 26 group

was at least 2-fold higher than that of CDDP group. The enhanced cellular uptake

could be attributed to the hydrophobic ligands of the Pt(IV) complex which increased

the lipophilicity of the platinum complex, therefore, contributing to the transportation

trough cell membrane and making 26 exerted better cytotoxicity than CDDP.

The enhanced cellular uptake of 26 motivated us to investigate whether the

intracellular 26 shared the similarly DNA-binding properties like cisplatin. Therefore,

we firstly performed ICP-MS analysis to determine the platinum contents in DNA

extracted from A549 cells after 12 h co-incubation with CDDP (2.5, 5 and 10 µM)

and 26 (2.5, 5 and 10 µM). As revealed in Figure S2B, the Pt content in DNA

accordingly increased as the dosage of CDDP and 26 increased from 2.5 to 10.0 µM.

The results clearly indicated that DNA in A549 cells was platinated after 12 h

co-incubation with 26. It was also found that the contents of platinum in A549 cells

were in a dose-dependent manner (Figure S2B). Interestingly, the extent of DNA

platination in 26-treated A549 cells was lower than the CDDP-treated group at the

same concentration. This may be attributed to the fact that the Pt(IV) prodrug need to

be reduced to the Pt(II) form to interact with DNA and this process was revealed

previously in Figure S1E.

Moreover, to further investigate the effect of 26 on biological function of DNA,

gel electrophoretic assays of 26 interacting with pBR322 plasmid DNA under the

reduction of ascorbic acid were also carried out. Briefly, pBR322 plasmid DNA was

co-incubated for 3 h at 37 °C with increasing concentration of CDDP (1.25, 2.5, 5, 10,

2

0 and 40 µM) or 26 (1.25, 2.5, 5, 10, 20 and 40 µM) which was pre-treated with

ascorbic acid for 7 h at 37 °C and analysed with electrophoresis in agarose gel.

Similarly, 20 and 26 without the pre-treatment with ascorbic acid at various

concentrations were used as control groups. From the electrophoretograms shown in

Figure S2C–S2F, we found that the untreated pBR322 plasmid DNA in each group

8

mainly presented in two states, form I bands (closed circular) and form II bands (open

circular) [45]. However, the mobility of form I bands of pBR322 plasmid DNA

decreased gradually when co-incubated with CDDP and 26 (pre-treated with ascorbic

acid), especially at the concentrations of 20 or 40 µM. On the contrary, the results in

Figure S2C and S2D indicated that the mobility of form II bands of pBR322 plasmid

DNA was accelerated. Naturally, the bands of coalescence in electrophoretograms

were interpreted as a strong unwinding of the supercoiled DNA [46]. Not surprisingly,

both 20 or 26 without the pre-treatment with ascorbic acid did not interacted with

DNA (Figure S2E and S2F). Therefore, these findings proved that 26 could bind to

DNA by inducing DNA platination, change the conformations of plasmid DNA and

consequently affect the biological function of DNA, whereas 20 did not show such

activity. Therefore, those results mentioned above proved that 26 could induce DNA

damage and disrupt DNA biological function in A549 cells.

2

.6. Apoptosis test

The strong antiproliferative activity of compounds 19-26 raised a question

whether the inhibition of growth of tumour cells was promoted by apoptosis.

Therefore, we chose 26 to asses apoptosis of A549 cells by annexin V−propidium

iodide assays. In early apoptosis, phosphatidylserine (PS) jumped into outer leaflet of

cell membrance and could be identified and marked with annexin V−FITC, which

was shown in Q3 of cytometry assay. In contrast, necrotic cells or late apoptotic were

both stained with PI, which was showed in Q1 and Q2. Similialy, normal cells were

shown in Q4. As shown in Figure 3, for the control group few cells were in apoptotic

state (4.57%). In contrast, the apoptosis population was significantly inreased to

4

5.4% after treatment with 26 (2 μM) for 24 h and further increased to 52.5% when

the concentration of 26 was increased to 5 μM. As far as CDDP (5 μM) was

concerned, it only induced apoptosis with a population of 15.43 %, clearly lower than

that of 26. Similarly, 20 also could induce apoptosis in a dose-dependent manner but

was more moderate than 26. This finding is similar to their performance in the

cytotoxicity assay in Table 1. Thus, a conclusion can be drawn that 26 could clearly

9

induce cell apoptosis and this ability closely correlates to its anti-tumor activity.

Therefore, in order to obtain better interpretation of design concepts and quickly

reveal the related biological mechanism, we chose the 20 (2 μM or 5 μM) and 26 (2

μM or 5 μM) of 24 h treatment for further studies.

2

.7. Effect of 26 on RAS/RAF interaction and MEK/ERK activation

Previosly, Reddy and co-worhers reported that Rigosertib could act as RAS

analogues to bind to RAS effectors, resulting in their inability to bind to RAS,

disrupting RAF activation, and inhibiting activation of the RAS-RAF-MEK pathway

[

15]. Becaeuse of the similarity in structure between 20 and Rigosertib, we

hypothesized that 20 and its relative Pt(IV) prodrug 26 could show the same

biological activity. Therefore, to determine whether 26 disturbed the interaction of

RAS and RAF, cell lysates of A549 cells that were pre-incubated with 20 (5 μM) or

2

6 (5 μM) for 24h were incubated with VE-cadherin (anti-RAS). The resulting

complexes were precipitated with protein G agarose and subjected to western blotting

to determine the levels of its assosiated p-CRAF. As shown in Figure 4A and 4B,

treatment of A549 cells with 20 and 26 induced significant decrease of the ratio of

p-CRAF between input groups and IP groups in comparison with untreated groups,

which elucidated that both 20 and 26 could inhibit the formation of p-CRAF. Besides,

treatment of cell lysates with protein G agarose that contained anti-RAS antibody

resulted in disability of CRAF protein to bind to RAS protein, which was observed

apparently in input group in Figure 4A and Figure 4B. These results supported our

hypothesis that 26 did inhibit the formation of RAS/CRAF complexes.

In order to further investigate wheteher 26 dirrectly targetted RAS protein or

CRAF protein or both, we further carried out microscale thermophoretic (MST)

assays and cellular thermal shift assays (CETSA). As illustrated in Figure S3E and

S3F, 26 could bind to CRAF RBD protein with high affinity constant (Kd value was

3

.93 μM) while 26 could not bind to KRAS (Figure S3C and Figure S3D) even when

the initial concentration of 26 was increased to 62.5 μM, indicating that 26 disrupted

the signaling transduction between RAS and CRAF by binding to CRAF rather than

1

0

RAS or both [15-17]. As an analogue of Regosertib, 20 could also bind to CRAF

RBD protein with Kd value of 721.3 nM (Figure S3A and S3B). Moreover, results of

CETSA shown in Figure S4A and S4B revealed that the melting temperature of

CRAF RBD protein from 47 to 67 °C significant increased, especially for CRAF

RBD protein at the temperature from 47 to 55 °C. It was found that more than 90% of

protein remained with the rising of temperature from 47 to 55 °C. As for the negative

group, the level of CRAF remaining in the soluble fraction quickly decreased when

o

the A549 cell lysates were heated from 47 to 67 C. This phenomenon suggested that

both 20 and 26 could bind and stabilize CRAF protein. Thus, both MST and CETSA

assays showed that 26 was able to engage CRAF protein rather than RAS protein.

With these results obtained, we further studied the effect of 26 on the MAPK

signals by measuring phosphorylation of MEK (p-MEK) and ERK (p-ERK). Briefly,

cell lysates collected from A549 cells that were co-incubated with 20 (2 μM and 5

μM ) and 26 (2 μM and 5 μM ) for 24 h were subjected to western blot to evaluated

the levels of p-MEK and p-ERK. CDDP (5 μM) and DMF were used as controls,

respectively. As expected, the results in Figure 4C and 4D showed that treatment with

both 20 and 26 led to reduced p-MEK and p-ERK levels with dose-dependent manner.

On the other hand, when A549 cells were treated with CDDP (5 μM), little effect on

p-MEK or p-ERK were observed. In sum, these results supported that 26 could inhibit

MAPK signals pathway by inhibiting phosphorylation of MEK/ERK.

2

.8. Effect on cell cycle arrest

As compound 26 was found to be able to induce cell apoptosis, it is nessasary to

investigate its effect on tumor cell cycle. Therefore, 26 and its corresponding ligand

0 were chosen to carry out floresence activited cell sorting (FACS) analysis with

2

A549 cells, using DMF and CDDP as negative and positive control respectively. The

results were shown in Figure 5. After 24 h co-incubation with the tested compound,

we found that 26 did affect the progression of cell cycycle. The results in Figure 5

showed that the positive drug CDDP caused a moderate arrest in S phase as compared

with cells without any treatment. However, cells cycle block inducced by 26 or 20

11

were completely different from that of CDDP. A549 cells treated with 20 or 26 were

gradually accumulated in G2/M phase of the cell cycle (cell population of G2/M

phase increased to 39.04%, 67.36% for 2 μM and 5 μM of 26; 35.60%, 60.32% for 20

when compared with 6.03% of negative control) and appeared to be unable to exit

from this phase, resulting in the activation of apoptotic pathways, as reflected with the

increased p53 and cleaved PARP proteins (see western blotting experiments) which

was a marker of caspase activation. In conclusion, compound 26 could induce A549

cells apoptosis by blocking cell cycle at G2/M phase just like 20 did.

2

.9. DNA damage induced by 26

To investigate whether 26 could induce DNA damage, comet assays and relative

western blotting assays were performed. Briefly, A549 cells were co-incubated with

0 (5 µM), 26 (5 µM) or CDDP (5 µM) for 24 h and used for further biological

2

studies. As revealed in Figure 6, DNA damage occurred clearly in both CDDP-treated

and 26-treated groups, which was characterized by the tail DNA. Usually, tail DNA

was in positive correlation with the degree of DNA damage. In our test 26 induced

longer and more tail DNA than both CDDP-treated and 20-treated groups, which

indicated that 26 induced much stronger DNA damage after 24 h co-incubation with

A549 cells. The finding was further confirmed by the fact that 26 induced high

generation of phosphorylation H2AX (γH2AX) in A549 cells, which was a biomarker

of double-strand breaks (DSBs) [47-49] (Figure S5). Moreover, the expression of

γH2AX induced by 26 was in a dose-dependent manner as compared with control

group. Naturally, the DNA damage could induce the up-regulation expression of p53

protein [38, 50, 51]. Results in Figure S5 illustrated that 20 (5 µM), 26 (5 µM) or

CDDP (5 µM) enhanced the level of p53 in A549 cells after 24 h co-incubation. In

sum, 26 could significantly induce DNA damage with the characteristics of platinum

drugs by leading to endogenous DSBs.

2

.10. Reactive oxygen species (ROS) assays

After knowing that 26 showed the most potential anti-tumor activity, we carried

out studies to investigate the intracellular induction mechanism of apoptosis. Previous

reports have proved that metal complexes exerted anti-tumor efficiency through

1

2

increasing generation of ROS [52, 53]. Therefore, we investigated whether treatment

with 26 led to significant production of ROS in A549 cells. To evaluate the level of

ROS accumulation, A549 cells were incubated with 20 (5 µM), 26 (5 µM) or CDDP

(5 µM) for 24 h, using dichloro-dihydro-fluorescein diacetate (DCFH-DA) as the

ROS probe and observed with fluorescence microscopy. Results in Figure 7 showed

that 26 (5µM) treated A549 cells produced a remarkable increase in generation of

ROS (65.56% population of whole A549 cells), which led to irreversible cell death. In

contrast, no evident green fluorescence of A549 cells (5.24%) was found in negative

control group. When the cells were treated with CDDP (5 µM), the green fluorescence

were ascendant (32.70 %), lower than that of the result of 26 (58.10 %). This

phenomenon illustrated that 26 might on one hand induce tumor apoptosis by

increasing production of intracellular ROS just like CDDP did. Moreover,

overproduction of ROS often dissipated the mitochondrial membrane potential and

led to mitochondrial dysfunction, which finally resulted in tumor death.

It was reported that DCFH-DA is not a reliable indicator for ROS measurement

as many other cellular pathways can oxidize DCF-DA [54-56]. Besides, mitochondria

are mainly responsible for intracellular ROS [57, 58]. Therefore, in order to give more

reliable information about the ROS production in treated cells, Mito-sox was further

used as probe to detect superoxide radicals in mitochondrial compartment [59]. The

results in Figure S6A1-S6C4 showed that 20, 26 and CDDP all induced oxidative

stress and 26 was the most active one in increasing the levels of ROS in mitochondria

of A549 cells. In contrast, 20 could only moderately enhance the level of superoxide

radicals in mitochondrial in comparison to CDDP-treated and 26-treated groups.

Besides, the irregular arrangement of mitochondria in cytoplasm induced by 26 also

suggested the depolarization of mitochondrial membrane potential, which was further

confirmed by the following biological studies. These findings are consistent with the

results from the DCFH-DA fluorescent probe. Therefore, we can clearly confirm that

both 20 and 26 could increase the level of ROS in tumor cells.

2

.11. Mitochondrial Membrane Potential (Δψm) depolarization induced by 26

1

3

The generation and accumulation of ROS was closely related to the

mitochondrial membrane potential (Δψm), and the depolarization of mitochondrial

membrane potential (MMP) was considered to be a common feature of cell death.

Besides, many reports proved that Pt(IV) complexes disrupted Δψm. In normal cells,

mitochondria exhibit a high Δψm while in apoptotic cells Δψm dissipated [53].

Therefore, we further investigated whether Δψm was involved in the cells treated with

our target compounds, using JC-1 fluorescent probe. A549 cells were co-incubated

with 20 (5 μM), 26 (5 μM) or CDDP (5 μM) for 24 h, stained with lipophilic cation

JC-1 and analyzed with confocal laser scanning microscope evaluate Δψm. As shown

in Figure 8, after 24 h treatment with 26, the population of cells with red fluorescence

largely decreased as compared with untreated cells (Figure 8D1-8D4). Similarly, the

Δψm of CDDP- and compound 20-treated cells also decreased respectively (Figure

8

B1-8C4), as reflected by decreased red fluorescence of JC-1 aggregate and the

increased green fluorescence from the JC-1 monomer. The evident collapsed red

fluorescence intensity indicated that Δψm has decreased sharply, suggesting that

mitochondrial function changes caused by 26 were closely associated with

cytotoxicity.

2

.12. Cell morphology study

To explore the changes of cell morphology induced by the target compounds,

A549 cells were treated with 26 (5 μM) or 20 (5 μM), using CDDP (5 μM) and

solvent vehicle as positive and negative control, respectively. After 24 h incubation

with the tested compounds, the cells were stained with hoechst 33258 and the results

were recorded with fluorescence microscope. As shown in Figure 9A1-9D1, A549

cells treated with 5 μM of 26 or 5 μM of 20 isolated from cells around, which were

significantly different from that cells treated with CDDP or without treatment. Besides,

there were larger population of cells treated with 26 with apoptotic nuclei containing

condensed chromatin of cells whereas the cells in control culture showed regular and

round outline of nuclei. To further investigate cells morphology changes, we stained

A549 cells with calcein-AM/PI double staining solution after 24 h incubation of cells

1

4

with these compounds mentioned above. In Figure 9A2-9D3, A549 cells without

drug treatment looked in spindle outline and were stained in green fluorescence, while

cells became oval and irregular nucleus was observed after the treatment with CDDP,

2

0 and 26, respectively. The difference of the cell morphology indicated the difference

of the apoptotic inducing ability of the compounds. As was known, calcein-AM

entered cells in green fluorescence, while PI could only enter apoptotic or necrotic

cells and interacted with DNA in red fluorescence. Moreover, the proportion of dead

cells in 20- and 26-treated groups (Figure 9C3 and 9D3) increased significantly as

compared with CDDP-treated group (Figure 9B3), as reflected with enhanced red

fluorescence from PI and the decrease of green fluorescence from calcein-AM, which

indicated that both compounds were more potent in inducing apoptosis than CDDP.

Therefore, characteristic color reaction also revealed that 26-treated cells were in

apoptotic status, which showed red fluorescence and differed from green fluorescence

in living cells without any treatment. In conclusion, 26 exhibited better

antiproliferative activity than CDDP at the same concentration after 24 h

co-incubation with A549 cells.

2

.13. Wound-healing assay

Metastatic cancers have several important characteristics including migration and

invasion of malignant cells in the development of cancer. Therefore, we conducted a

wound healing assay to detect the ability of the complex to inhibit the cell migration

and invasion. A549 cells were scratched with a pipette tip and co-incubated with

control (DMF), CDDP (5 μM), 20 (1 μM) and 26 (1 μM) for 24 h. In order to avoid

the influence of cell proliferation on the test result, the experiments were carried out

under serum-starved conditions. Figure 10 displayed the wound gaps at the time of

wound generation, wound after 24 h drug-treatment, respectively. As illustrated,

compound 20 and 26 significantly inhibit the wound healing at the tested

concentration as compared with the negetive control, whereas the CDDP-treated

group obtained narrower gaps. These results indicated that both 20 and 26 colud

efficiently surpress A549 cells migration and further metastasis, while CDDP did not

1

5

obviously show the activity.

2

.14. Western blotting study

Previous studies demonstrated that Pt(IV) complexes induced apoptosis of tumor

cells by regulation of pro-/anti-apoptotic protein in a mitochondria-mediated

apoptosis pathway [38], such as Bcl family proteins. Therefore, we investigated the

effects of compound 26 on the expression of apoptosis-related proteins of A549 cell,

uing CDDP as positive control. As shown in Figure 11A, we could see that treatment

with 26 decreased the levels of antiapoptotic protein Bcl-2 but led to the up-regulation

of the proapoptotic protein Bax, indicating that 26 induced apotosis by altering the

ratio of Bcl-2 to Bax and resulting in the collapse of mitochondrial membrane

potential which was previouly discussed. Moreover, the evelented ratio of

proapoptotic and antiapoptotic proteins promoted the release Cyt c to cytosol, finally

activated the caspase cascade and induced tumor cells apoptosis. As shown in Figure

11B, when compared to control cells, caspase 9/3 were significantly activated and

overexpressed in A549 cells treated with 20 or 26, which was a critical event in the

initiation and execution of apoptosis in cells. Meanwhile, the western blot data

revealed that 26 also enhanced the expression of cleaved-PARP (89 KD) in

comparison with untreated control. In combination with the decrease of mitochondrial

membrane potential induced by 26, we could conclude that 26 promoted apoptosis in

mitochondria- mediated pathway and caspase cascade.

2

.15. Antitumor Activity in Vivo

Finally, we investigated the therapeutic potential of compound 26 in A549

6

xenograft models to determine its antitumor efficacy in vivo. A549 cells (3×10 ) in

1

00 μL of sterile PBS were injected subcutaneously in the flank of six-week-old

female BALB/c athymic nude mice to established A549 xenograft models. Thereafter,

the mice were divided into four different groups (n=3) after the tumor volume reached

3

to 150-250 mm . Compounds of 20, 26, CDDP and saline were administrated

intravenously every other day for a total of 3 injections at a dose of 6 μmol/kg. The

tumor volume and body weight were measured and recorded 2-3 times a week. As

1

6

shown in Figure 12A and 12B, mice in control group showed a rapid increase in the

3

average RTV (relative tumor volume) (7.1 mm , Table S1). On the contrary, 20, 26

and CDDP were able to suppress the growth of tumor to some extent, with IR

(inhibition ratio) of 41.9%, 59.2% and 36.7%, respectively. (see Figure 12A and 12B,

Table S1). Consistent with the results of in vitro test, compound 26 showed the most

potential antitumor activity in A549 xenograft mice, which may be attributed to high

intracellular accumulation of 26 (Figure 2) and the release of CDDP (Figure S1).

Among the tested substances, CDDP exhibited the lowest antitumor activity with RTV

3

of 4.5 mm . In addition, CDDP showed obvious side effect as reflected by the

decreasing body weight, especially during days 6-12 after the first administration

(Figure 12C). Notably, the results in Figure 12C strongly demonstrated that 20, 26

did not show obvious side effects as the body weight kept increasing during the

treatment. In sum, 26 did not only show significant anti-proliferative effect in vitro,

but showed excellent antitumor activity in vivo, which qualified it as a ‘high

efficiency and low toxic’ antitumor candidate.

3

. Conclusion.

In conclusion, we here presented eleven novel Pt(IV) compounds conjugating

with RAS/MAPK signaling disrupting agents through ester bond. Our cellular data

provided evidence that most of the target Pt(IV) compounds showed higher

cytotoxicity against all tested cancer lines than CDDP. Among those 26 exerted the

best anti-tumor activity against A549 cells but showed only moderate cytotoxicity to

normal LO2 and HUEVC cells. Moreover, 26 could overcome the multi-drug

resistance and exert potent cytotoxicity to A549/CDDP and SKOV-3/CDDP cells.

This unique property may be attributed to its ability to release Pt (II) complex as well

as ON013100. Our stability study showed that 26 was stable in physiological

environment (pH = 7.4) but was rapidly reduced and released its corresponding 20

and Pt(II) complex within 7 h under the reduction of ascorbic acid, indicating that 26

acted as a multi-functional Pt(IV) prodrug. Further biological mechanism studies

indicated that 26 could efficiently disturb the signaling transduction between RAS and

1

7

MAPK pathway, by directly binding to CRAF protein. Besides, the superior cellular

uptake made 26 dramatically arrest A549 cells at G2/M phase and heavily elevate

apoptosis level, whereas CDDP arrested A549 cells at S phase and induced much less

apoptosis. Moreover, the following mechanism studies revealed that 26 induced

massive ROS, induced DNA damage, induced DNA platination and conformational

changes, triggered mitochondrial membrane depolarization by inhibiting

anti-apoptotic Bcl-2 and activating caspase cascade, indicating that 26 evoked

apoptosis through mitochondrial apoptotic pathway. Finally, in vivo anti-tumor

activities indicated that 26 efficiently inhibited tumor growth in A549 xenograft

mouse model without inducing severe side effects, whereas the equimolar CDDP only

gave a moderate inhibition of the tumor and caused obvious loss of body weight. The

proposed antitumor action mode of 26 is outlined in Figure 13. Overall, we have

obtained a multi-functional Pt(IV) which could inhibit the proliferation of tumor cells

via two different pathways: the RAF-MEK-ERK pathway and the platinum-based

cytotoxicity pathway. This unique character may qualify it as a ‘high efficiency and

low toxic’ antitumor candidate.

4

. Experimental section

4

.1. Materials and measurements

All analytical or chemical pure reagents were obtained from commercial source

unless otherwise noted. Solvents were dried over standard agents and freshly distilled

1

13

prior to use. Deionized water was used in this article. The H and C NMR spectra

were recorded on a Bruker Avance NMR spectrometer (300, 400 or 500 MHz for

1

13

195

H ,100 or 125 MHz for C,129 MHz for Pt spectra) and chemical shifts reported

(

in ppm) relative to the indicated solvents. Deuterated component of CHCl or

3

DMSO-d was used as NMR solvent. Mass spectra was obtained on an Agilent Dual

6

ESI Q TOF 6520, in positive-ion or negative-ion model, with methanol as solvent.

The thermogravimetric analysis was perform on thermogravimetry (TG 209 F3)

(heating rate, 20 °C/min; starting temperature, 30 °C; finishing temperature, 800 °C;

nitrogen environment). Thin-layer chromatography (TLC) was carried on

glass-backed silica gel GF plates (obtained from Qingdao Marine Chemical Co., Ltd.)

1

8

and visualized by short wavelength UV light. Target compounds and intermediates

were purified with silica gel (200-300 mesh) column chromatography or

recrystallization. Purity of prepared compounds were determined by HPLC-UV

analysis (waters, e2695 system; 2489 UV/Vis detector), using acetonitrile/water

(45:55, V/V) (0.1% trifluoroacetic acid in water) as eluent except for 14 and 22 using

acetonitrile/water (50:50, V/V) (0.1% trifluoroacetic acid in water) as eluent. All

compounds used in biological assays were above 95%. Bax, Bcl-2, cytochrome c,

cleaved-caspase-9, -3, cleaved-PARP antibodies were purchased from Imgenex, USA.

Antibodies directed against p-MEK, MEK, p-ERK and ERK were purchased from

Cell Signaling Technology. RAF antibody was purchased from Affinity Biosciences,

whereas RAS, γh2AX and p53 antibodies was from Abcam. Secondary antibody

(1:2000) labeled with horseradish peroxidase was came from Santa Cruz

Biotechnologies. TIANamp Genomic DNA Kit was purchased from TIANGEN.

Mito-sox fluorescence probe was from KeyGEN BioTECH. The standard solution of

platinum was from Perkin Elmer.

4

.2. General methods for preparation of compounds

.2.1. 5-formyl-2-methoxyphenyl-4-methylbenzenesulfonate (2)

To a solution of compound 1 (3.0 g, 19.8 mmol) in DMF (15 mL), DCM (30 mL)

and Et N (4.0 g, 39.6 mmol), was added TsCl (4.5g, 23.8 mmol) at 0 °C and stirred at

3

room temperature for 2 h. Progress of the reaction was monitored the reaction with

TLC. After the completion of the reaction, DCM (350 mL) was added to the solution

and washed with sat. NaHCO (150 mL) and sat. NaCl (150 mL). The organic layer

3

was dried with anhydrous Na SO and concentrated under reduced pressure to get

2

4

pale white solid. Purified with silica gel chromatography with the eluent of PE and

EA (100:0 - 5:1, V/V) and dried at 45 °C to obtain white solid 5.8 g. Yield 95.0%; mp

1

43-145 °C; H NMR (300 MHz, CDCl ) δ 9.82 (s, 1H), 7.77-7.74 (m, 3H), 7.62 (d, J

3

=

1.9 Hz, 1H), 7.32 (d, J = 8.1 Hz, 2H), 6.97 (d, J = 8.5 Hz, 1H), 3.69 (s, 3H), 2.45 (s,

3

H).

4

.2.2. 5-(hydroxymethyl)-2-methoxyphenyl-4-methylbenzenesulfonate (3)

1

9

To a solution of compound 2 (5.8 g, 18.9 mmol) in EtOH (30 mL) and DCM

(

15.0 mL) was added NaBH (715.0 mg, 18.9 mmol) at 0 °C and stirred at room

4

temperature for 20 min. Progress of the reaction was monitored with TLC. The

reaction was worked up followed by quenching with 1 M HCl solution at 0 °C. The

solvent was removed under reduced pressure and obtained crude white solid. Then

DCM (300 mL) was added to dissolve the residue, washed with sat. NaCl (150 mL×3).

The combined organic layer was dried with anhydrous Na SO and concentrated in

2

4

1

vacuum to give white solid 5.6 g. Yield 96.6%; mp 144-145 °C; H NMR (300 MHz,

1

DMSO-d ) H NMR (300 MHz, CDCl ) δ 7.75 (d, J = 8.1 Hz, 2H), 7.31 (d, J = 8.1

6

3

Hz, 2H), 7.24-7.20 (m, 1H), 7.14 (d, J = 1.9 Hz, 1H), 6.80 (d, J = 8.4 Hz, 1H), 4.40 (s,

2

H), 3.59 (s, 3H), 2.45 (s, 3H).

4

.2.3. 5-(bromomethyl)-2-methoxyphenyl-4-methylbenzenesulfonate (4)

To a solution of compound 3 (5.7 g, 18.5 mmol) in DCM (30 mL) at 0 °C was

added slowly PBr (6.0 g, 22.2 mmol) in DCM (10 mL) and stirred at the same

3

temperature for 2 h. Progress of the reaction was monitored with TLC. After

completion, the reaction was quenched with NaHCO (1 M) solution at 0 °C. After

3

that, DCM (300 mL) was added to dissolve the residue, washed with sat. NaCl (150

mL×3). The organic layer was dried with anhydrous Na SO and concentrated in

2

4

1

vacuum to give white solid 6.7 g. Yield 98.0%; mp 112-114 °C; H NMR (300 MHz,

CDCl ) δ 7.75 (d, J = 8.1 Hz, 2H), 7.30 (d, J = 8.1 Hz, 2H), 7.23-7.20 (m, 1H), 7.14

3

(d, J = 2.0 Hz, 1H), 6.79 (d, J = 8.4 Hz, 1H), 4.40 (s, 2H), 3.59 (s, 3H), 2.44 (s, 3H).

4

.2.4. 2-((4-methoxy-3-(tosyloxy)benzyl)thio)-acetic acid (5)

To a solution of compound TGA (2.2 g, 23.3 mmol) in MeOH (25 mL) at 0 °C,

was added NaOH (12.0 g, 49.6 mmol) gradually and stirred for 10 min at the same

temperature. Then compound 4 (5.7 g, 15.5 mmol) in 10 MeOH (25 mL) and DCM

(30 mL) was dropped into the solution slowly at 0 °C. Progress of the reaction was

monitored with TLC. After completion of reaction, the solvent was removed under

reduced pressure to obtain pale white solid. After that, the residue was dissolved in

water (70 mL), adjusted the pH to 4-5 with HCl (1 M), extracted with DCM (150

2

0

mL×3) and dried with anhydrous Na SO . The solvent was removed and concentrated

2

4

1

under reduced pressure to obtain white solid 2.5 g. Yield 60.0%; mp 120-122 °C; H

NMR (400 MHz, DMSO) δ 12.64 (s, 1H), 7.68 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2

Hz, 2H), 7.18 (dd, J = 8.5, 2.1 Hz, 1H), 7.03 (d, J = 2.1 Hz, 1H), 6.99 (d, J = 8.5 Hz,

1

H), 3.72 (s, 2H), 3.49 (s, 3H), 3.05 (s, 2H), 2.41 (s, 3H). HR-MS (m/z) (ESI) called

for [C H O S -H] = 381.0545, found, 381.0561.

17

18

6 2

4

.2.5. 2-((4-methoxy-3-(tosyloxy)benzyl)sulfonyl)-acetic acid (6)

To a solution of compound 5 (2.5 g, 6.5 mmol) in acetic acid (20 mL) at room

temperature was added H O (3.7 g, 32.7 mmol) partly and stirred at the 50 °C for 24

2

h. Process of the reaction was monitored with TLC. Solvent of the reaction was

removed under reduced pressure to obtain pale yellow crude solid. The solid was

filtered and purified with silica gel chromatography with the eluent of DCM and

MeOH (100:0-50:1, V/V) to get pale white solid 1.8 g. Yield 66.7%; mp 138-140 °C;

1

H NMR (300 MHz, DMSO-d ) δ 7.70 (d, J = 8.2 Hz, 2H), 7.43 (d, J = 8.2 Hz, 2H),

6

7

4

=

.29 (dd, J = 8.5 Hz, 1.7 Hz, 1H), 7.19 (s, 1H), 7.09 (d, J = 8.5 Hz, 1H), 4.55 (s, 2H),

.08 (s, 2H), 3.53 (s, 3H), 2.41 (s, 3H). HR-MS (m/z) (ESI) called for [2C H O S -H]

1

7

18

8 2

827.0982, found, 827.0984.

.2.6. (E)-2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenyl-4-

methylbenzenesulfonate (8)

4

To a solution of compound 6 (2.0 g, 4.8 mmol) in toluene (35 mL) at 40 °C was

added piperidine (621.7 uL, 6.3 mmol) and stirred at the same temperature for 10 min.

Then benzoic acid (876.4 mg, 7.2 mmol) and 7 (1.0 g, 5.3 mmol) were added to the

solution slowly and heated to reflux for overnight. The reaction was monitored with

TLC. After the completion of the reaction, the solution was cooled to room

temperature and removed the solvent to obtain pale yellow crude product.

Subsequently, the crude product was purified with silica gel chromatography with the

eluent of DCM and PE (2:1, V/V) and recrystallized with DCM and PE (50 mL, 3:1,

1

V/V) to get white solid 1.0 g. Yield 38.5%; mp 168-169 °C; H NMR (300 MHz,

DMSO-d ) δ 7.62 (d, J = 8.2 Hz, 2H), 7.55 (d, J = 15.6 Hz, 1H), 7.35 (d, J = 8.2 Hz,

6

2

1

2

H), 7.24 (d, J = 8.4 Hz, 1H), 7.20 (d, J = 1.6 Hz, 1H), 7.07 (d, J = 15.6 Hz, 1H), 7.02

(d, J = 8.4 Hz, 1H), 6.30 (s, 2H), 4.42 (s, 2H), 3.84 (s, 9H), 3.48 (s, 3H), 2.39 (s, 3H).

4

.2.7. (E)-2,4,6-Trimethoxystyryl-3-hydroxy-4-methoxybenzylsulfone (9)

To a solution of compound 8 (1.0 g, 1.8 mmol) in MeOH (20 mL) and H O (4

2

mL) was added NaOH (364.6 mg, 9.1 mmol) at room temperature and stirred at 90 °C

for overnight. The reaction was monitored with TLC. After the completion of the

reaction, the solution was cooled to room temperature and removed the solvent to

obtain pale yellow crude product. The residue was further purified with silica gel

chromatography with the eluent of DCM and PE (2:1, V/V) to get pale white solid

1

6

1

6

3

4

01.0 mg. Yield 84.5%; M. p.= 128-130 °C; H NMR (300 MHz, DMSO-d ) δ 8.99 (s,

6

H), 7.57 (d, J = 15.6 Hz, 1H), 7.08 (d, J = 15.7 Hz, 1H), 6.88 (d, J = 8.2 Hz, 1H),

.80 (s, 1H), 6.73 (d, J = 8.1 Hz, 1H), 6.30 (s, 2H), 4.28 (s, 2H), 3.85 (s, 9H), 3.76 (s,

H).

.2.8. Methyl-(E)-2-(2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)

acetate (10).

To a solution of compound 9 (800.0 mg, 2.03 mmol) in DMF (15 mL) at room

temperature was added slowly compound K CO (846.9 mg, 5.07 mmol) and ethyl

2

3

bromide (846.9 mg, 5.07 mmol) and stirred at 80 °C for overnight. The reaction was

monitored with TLC. After completion of the reaction, the solvent was removed under

reduced pressure to obtain pale yellow crude solid. The residue was dissolve in DCM

(

50 mL), washed with sat. NaCl (100 mL) and dried with anhydrous Na SO . The

2 4

crud product was purified with silica gel chromatography with the eluent of EA/PE

(5:1, V/V) and DCM/MeOH (30:1, V/V) to get pale white solid 778.07mg. Yield

1

8

7

6

2

4

0.0%; mp 104-106 °C; H NMR (400 MHz, DMSO-d ) δ 7.53 (d, J = 15.7 Hz, 1H),

6

.08 (d, J = 15.7 Hz, 1H), 6.98 (d, J = 8.3 Hz, 1H), 6.93 (dd, J = 8.3, 1.7 Hz, 1H),

.87 (d, J = 1.7 Hz, 1H), 6.30 (s, 2H), 4.61 (s, 2H), 4.35 (s, 2H), 4.13 (q, J = 7.1 Hz,

H), 3.85 (d, J = 2.2 Hz, 9H), 3.77 (s, 3H), 1.20 (t, J = 7.1 Hz, 3H).

.2.9. (E)-2-(2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-acetic

acid (11).

2

To a solution of compound 10 (485.0 mg, 1.0 mmol) in EtOH (10 mL) and pure

water (5 mL) at room temperature was added LiOH·H O (106.0 mg, 2.5 mmol) and

2

stirred at room temperature for overnight. After the completion of the reaction, the

solvent was removed under reduced pressure to obtain pale yellow solid. Added 30

mL pure water to dissolve the residue and adjusted the pH of the solution to 5 with

HCl (1M). The residue was filtered and dried at 45 °C to get pale yellow solid (407.2

1

g, yield 90.0%); mp 112-114 °C; H NMR (300 MHz, DMSO-d ) δ 12.88 (s, 1H),

6

7

.54 (d, J = 15.7 Hz, 1H), 7.09 (d, J = 15.7 Hz, 1H), 6.97 (d, J = 8.1 Hz, 1H), 6.91 (d,

J = 8.1 Hz, 1H), 6.89 (s, 1H), 6.29 (s, 2H), 4.51 (s, 2H), 4.34 (s, 2H), 3.85 (s, 9H),

3

4

.77 (s, 3H). HR-MS (m/z) (ESI) called for [C H O S-H] = 451.1163, found,

21 24 9

51.1170.

.2.10. General procedure for preparing compounds 12-14

To a solution of compound 11 (148 mg, 0.33 mmol) in DMF (3 mL) was added

compound TBTU (158.9 mg, 0.50 mmol) and stirred at room temperature for 5 min.

Then, Et N (68.0 uL, 0.50 mmol) was added to the solution under the protection of

3

nitrogen and stirred for 5min at the same temperature. After that, compound 15, 16, 17

(

0.36 mmol) was added to the solution under the protection of nitrogen and stirred for

h at 35 °C. Progress of the reaction was monitored with TLC. After the completion

of the reaction, DCM (250 mL) was added to the solution and washed with sat. NaCl

100 mL). The organic layer was dried with anhydrous Na SO and concentrated

2

(

2

4

under reduced pressure to obtain crude product. DCM (50 mL) was added to dissolve

the residue and purified with silica gel chromatography with the eluent of DCM and

MeOH (60:1-20:1, V/V) to get target compounds.

4

.2.11. (OC-6-33)-diamminetrichlorido((E)-2-(2-methoxy-5-(((2,4,6-trimethoxystyryl)

sulfonyl)methyl)phenoxy)acetate)-platinum(IV) (12). Pale yellow solid. Yield: 30.1%;

1

mp (decomp.) 169-172 °C; H NMR (300 MHz, DMSO-d ) δ 7.54 (d, J = 15.7 Hz,

6

1

H), 7.09 (d, J = 15.7 Hz, 1H), 6.94 (d, J = 8.2 Hz ,1H), 6.93 (s, 1H), 6.88 (d, J = 8.2

13

Hz, 1H), 6.25 (m, 8H), 4.50 (s, 2H), 4.33 (s, 2H), 3.86 (s 9H), 3.76 (s, 3H); C NMR

(

75 MHz, DMSO-d ) δ 175.74, 164.40,161.72 (2×Ar-OCH , overlapped signals),

6

3

2

3

1

49.34, 147.35, 133.81, 124.31, 123.62, 121.56, 116.94, 112.24, 102.89, 91.40

(

2×Ar-H, overlapped signals), 65.98, 60.37, 60.29(2×CH O-, overlapped signals),

3

1

95

5

6.64, 56.00. Pt NMR (129 MHz, DMSO-d ): δ/ppm 551.96; HR-MS (m/z) (ESI):

6

called for [C H Cl N O PtS-H] = 785.0214, found, 785.0334.

2

1

29

3

2

9

4

.2.12. (OC-6-34)-chlorido(dicarboxylate)(cyclohexane-1R,2R-diamine)((E)-2-(2-met

hoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)acetate)-platinum(IV) (13).

1

Pale yellow solid. Yield: 60.2%; mp (decomp.) 178-180 °C; H NMR (500 MHz,

DMSO-d ) δ 8.35 (s, 1H), 8.26 (s, 1H), 8.03 (s, 1H), 7.64 (s, 1H), 7.55 (d, J = 15.7 Hz,

6

1

H), 7.10 (d, J = 15.7 Hz, 1H), 6.96 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.82

(s, 1H), 6.30 (s, 2H), 4.55 (s, 2H), 4.34 (s, 2H), 3.85 (s, 9H), 3.76 (s, 3H), 2.56 (s, 1H),

2

0

.49 – 2.43 (m, 1H), 2.02 (m, 2H), 1.52 (m, 1H), 1.45 (m, 2H), 1.31 (m, 1H), 1.18 –

13

.95 (m, 2H); C NMR (125 MHz, DMSO-d6) δ 175.52, 164.15, 163.71(2×-COO-,

overlapped signals), 161.41(2×Ar-OCH , overlapped signals), 149.38, 147.25, 133.55,

3

1

6

3

24.65, 123.74, 121.55, 116.31, 112.45, 103.09, 91.43 (2×Ar-H, overlapped signals),

6.59, 62.01, 61.59, 60.02, 56.54 (2×CH O-, overlapped signals), 56.10, 56.06, 31.38,

3

195

0.94, 23.99, 23.88. Pt NMR (129 MHz, DMSO-d ): δ/ppm 995.55; HR-MS (m/z)

6

(

ESI): [C H ClN O PtS-H] = 883.1377, found 883.1385.

29 37 2 13

4

.2.13. (OC-6-33)-trichlorido(cyclohexane-1R,2R-diamine)((E)-2-(2-methoxy-5-(((2,4

,

6-trimethoxystyryl)sulfonyl)methyl)phenoxy)acetate)-platinum(IV) (14). Pale yellow

1

solid. Yield: 30.0%; mp (decomp.) 176-178 °C; H NMR (500 MHz, DMSO-d ) δ

6

9

.08 (s, 1H), 8.16 (s, 1H), 7.88 (s, 1H), 7.53 (d, J = 15.7 Hz, 2H), 7.09 (d, J = 15.6 Hz,

H), 6.95 (d, J = 8.3 Hz, 1H), 6.90 (d, J = 8.3 Hz, 1H), 6.86 (s, 1H), 6.29 (s, 2H), 4.54

1

(s, 2H), 4.32 (q, J = 13.6 Hz, 2H), 3.86 (s, 9H), 3.76 (s, 3H), 2.75 (s, 1H), 2.15 (m,

13

1

H), 2.06 (m, 1H), 1.54 (m, 1H), 1.46 (m, 2H), 1.24 (m, 2H), 1.03 (m, 2H); C NMR

(

75 MHz, DMSO) δ 178.01, 164.15, 161.40 (2×Ar-OCH , overlapped signals), 149.32,

3

1

47.16, 133.64, 124.48, 123.69, 121.58, 116.59, 112.40, 102.99, 91.45 (2×Ar-H,

overlapped signals), 66.82, 63.89, 62.90, 60.24, 56.62 (2×CH O-, overlapped signals),

3

1

95

5

6.10, 56.05, 31.41, 31.29, 23.93 (2×-CH CH -, overlapped signals). Pt NMR (129

2 2

2

4

MHz, DMSO -d ): δ/ppm 404.15; HR-MS (m/z) (ESI) called for [C H Cl N O PtS

6

27 37

3

2

9

-H] = 865.0943, found 865.0967.

4

.1.14. General procedure for preparing compounds 19,20

To a solution of compound 9 (500.0 mg, 1.27 mmol) in DMF (10 mL) was added

K CO (692.0 mg, 5.07 mmol) and stirred at room temperature for 5 min. Then,

2

3

succinic anhydride (5.07 mmol) or slutaric anhydride (5.07 mmol) was added to the

solution under the protection of nitrogen and stirred for 30 min at 50 °C. Progress of

the reaction was monitored with TLC. After the completion of the reaction, the

solvent of the reaction was removed under reduced pressure. After that, water (100

mL) was added to dissolve the residue, adjusted the pH to 4-5 and extracted with

DCM (50 mL×3). The combined organic layer was washed with sat. NaCl (100 mL),

dried with anhydrous Na SO , concentrated under reduced pressure to obtain crude

2

4

product. Finally, the crud product was further purified by silica gel chromatography

with the eluent of DCM and MeOH (100:1-60:1) to get white solid.

4

.2.15. (E)-4-(2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-4-oxo

1

butanoic acid (19). White solid. Yield: 31.0%; mp 155-157 °C; H NMR (600 MHz,

DMSO) δ 12.31 (s, 1H), 7.58 (d, J = 15.6 Hz, 1H), 7.21 (dd, J = 8.5 Hz, 2.1 Hz, 1H),

7

2

4

.11 (d, J = 8.5 Hz, 1H), 7.08 (d, J = 15.6 Hz, 1H), 7.06 (d, J = 2.1 Hz, 1H), 6.29 (s,

H), 4.41 (s, 2H), 3.85-3.75 (m, 9H), 3.75 (s, 3H), 2.79-2.76 (m, 2H), 2.57 – 2.55 (m,

H). HR-MS (m/z) (ESI): [C H O S-C H O ] =393.1008, found 393.1189.

2

3

26 10

4

5

3

.2.16. (E)-5-(2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-5-oxo

1

pentanoic acid (20). White solid. Yield 75.2%; mp 152-154 °C; H NMR (400 MHz,

DMSO-d ) δ 12.18 (s, 1H), 7.58 (d, J = 15.6 Hz, 1H), 7.22 (dd, J = 8.5, 2.0 Hz, 1H),

6

7

2

7

=

.12 (d, J = 8.5 Hz, 1H), 7.09 (d, J = 15.6 Hz, 1H), 7.08 (d, J = 2.0 Hz, 1H), 6.30 (s,

H), 4.42 (s, 2H), 3.85-3.86 (m, 9H), 3.77 (s, 3H), 2.60 (t, J = 7.4 Hz, 2H), 2.35 (t, J =

.4 Hz, 2H), 1.87-1.80 (m, 2H). HR-MS (m/z) (ESI) called for [2C H O S-H]

2

4

28 10

1015.2650, found 1015.2913.

4

.2.17. (OC-6-33)-trichlorido(cyclohexane-1R,2R-diamine)((E)-4-(2-methoxy-5-(((2,

,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-4-oxobutanoicate)-platinum(IV) (21).

2

5

1

Yield: 49.0%. Pale yellow solid; mp (decomp.) 180-184 °C; H NMR (400 MHz,

DMSO) δ 9.43 (s, 1H), 8.06 (s, 1H), 7.86 (s, 1H), 7.58 (d, J = 15.6 Hz, 1H), 7.42 (s,

1

9

2

H), 7.21 (d, J = 7.4 Hz, 1H), 7.13-7.07 (m, 3H), 6.30 (s, 2H), 4.39 (s, 2H), 3.86 (s,

H), 3.76 (s, 3H), 2.73 (s, 1H), 2.69 (s, 2H), 2.63 (s, 2H), 2.16-2.14 (m, 1H),

1

3

.01-1.98 (m, 1H), 1.54 – 1.34 (m, 3H), 1.26-1.01 (m, 3H), 0.91-0.82 (m, 1H); C

NMR (100 MHz, DMSO-d ) δ 181.54, 170.96, 164.19, 161.44 (2×Ar-OCH ,

6

3

overlapped signals), 151.39, 139.34, 133.76, 130.12, 125.74, 123.68, 121.83, 113.01,

1

03.02, 91.44 (2×Ar-H, overlapped signals), 63.51, 62.62, 59.61, 56.57 (2×CH O-,

3

195

overlapped signals), 56.39, 56.13, 32.38, 32.34, 31.31, 29.89, 24.05, 23.75.

Pt

NMR (129 MHz, DMSO-d ): δ/ppm 418.66; HR-MS (m/z) (ESI) called for

6

[

C H Cl N O PtS +H] =909.2290, found 909.2282.

29 39 3 2 10

4

.2.18. (OC-6-33)-trichlorido(cyclohexane-1R,2R-diamine)((E)-5-(2-methoxy-5-(((2,

,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-5-oxopentanoicate)-platinum(IV) (22).

1

Yield: 51.0%. Pale yellow solid; mp (decomp.) 153 -155 °C; H NMR (400 MHz,

DMSO-d ) δ 9.58 (s, 1H), 8.18 (s, 1H), 7.84 (s, 1H), 7.57 (d, J = 15.6 Hz, 1H), 7.53 –

6

7

.42 (m, 1H), 7.22 (d, J = 8.1 Hz, 1H), 7.14 – 7.05 (m, 3H), 6.30 (s, 2H), 4.42 (s, 2H),

.86 (s, 9H), 3.77 (s, 3H), 2.76-2.72 (m, 1H), 2.62 (t, J = 6.6 Hz, 2H), 2.39 (d, J = 2.2

3

Hz, 2H), 2.21 – 2.15 (m, 1H), 2.08-2.05 (m, 1H), 1.88 – 1.80 (m, 2H), 1.56 – 1.45 (m,

1

3

1

H), 1.33 – 1.25 (m, 1H), 1.11-1.02 (m, , 3H); C NMR (100 MHz, DMSO-d ) δ

6

82.92, 171.24, 164.19, 161.44 (2×Ar-OCH , overlapped signals), 151.32, 139.35,

3

33.77, 130.13, 125.67, 123.66, 121.95, 113.01, 103.02, 91.44 (2×Ar-H, overlapped

signals), 63.90, 62.72, 59.60, 56.58 (2×CH O-, overlapped signals), 56.39, 56.14,

3

1

95

3

8.72, 36.69, 32.87, 31.40, 31.33, 24.04, 21.46. Pt NMR (129 MHz, DMSO-d₆):

δ/ppm 417.02; HR-MS (m/z) (ESI) called for [C H Cl N O PtS-H] =921.1106,

30

41

3

2

10

found 921.1192.

.2.19. (OC-6-34)-chlorido(dicarboxylate)(cyclohexane-1R,2R-diamine)((E)-4-(2-met

4

hoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-4-oxobutanoicate)-platinu

1

m(IV) (23). Yield: 61.0%. Pale yellow solid; mp (decomp.) 176 -178 °C; H NMR

(

400 MHz, DMSO-d ) δ 8.39-8.37 (m, 1H), 8.29 – 8.24 (m, 2H), 7.66-7.64 (m, 1H),

6

2

6

7

.57 (d, J = 15.6 Hz, 1H), 7.21 (d, J = 8.5 Hz, 1H), 7.12 – 7.10 (m, 2H), 7.08 – 7.07

m, 1H), 6.30 (s, 2H), 4.40 (s, 2H), 3.85 (s, 9H), 3.75 (s, 3H), 2.77-2.72 (m, 2H),

.67-2.66 (m, 2H), 2.07 (d, J = 11.3 Hz, 1H), 1.96 (d, J = 10.7 Hz, 1H), 1.56 – 1.50

(

2

1

3

(

m, 1H), 1.43-1.28 (m, 3H), 1.33-1.30 (m, 2H), 1.00-0.86 (m, 2H); C NMR (100

MHz, DMSO-d ) δ 179.37, 170.98, 164.19, 163.74, 163.69, 161.44 (2×Ar-OCH ,

6

3

overlapped signals), 151.35, 139.31, 133.72, 130.12, 125.76, 123.71, 121.89, 113.01,

1

03.03, 91.45 (2×Ar-H, overlapped signals), 61.90, 61.62, 59.58, 56.55 (2×CH O-,

3

195

overlapped signals), 56.38, 56.11, 31.61, 31.32, 31.03, 29.74, 23.99, 23.81.

Pt

NMR (129 MHz, DMSO-d ): δ/ppm 1001.05; HR-MS (m/z) (ESI) called for

6

[

C H ClN O PtS+H] =927.2550, found 927.2759.

31 39 2 14

4

.2.20. (OC-6-34)-chlorido(dicarboxylate)(cyclohexane-1R,2R-diamine)((E)-5-(2-me

thoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenoxy)-5-oxopentanoicate)-platin

1

um(IV) (24). Yield: 50.9%. Pale yellow solid; mp (decomp.) 173 -175 °C; H NMR

(

400 MHz, DMSO-d ) δ 8.40-8.35 (m, 3H), 7.68-7.66 (m, 1H), 7.58 (d, J = 15.6 Hz,

6

1

3

2

H), 7.21 (dd, J = 8.5, 1.8 Hz, 1H), 7.13 – 7.06 (m, 3H), 6.30 (s, 2H), 4.41 (s, 2H),

.85 (s, 9H), 3.75 (s, 3H), 2.59-2.56 (m, 4H), 2.42 (t, J = 7.4 Hz, 2H), 2.11-2.01 (m,

1

3

H), 1.83-1.80 (m, 2H), 1.56-1.46 (m, 4H), 1.18-1.06 (m, 2H); C NMR (100 MHz,

DMSO-d ) δ 180.43, 171.17, 164.19, 163.72 (2×-COO-, overlapped signals), 161.44

6

(

2×Ar-OCH , overlapped signals), 151.31, 139.32, 133.77, 130.15, 125.64, 123.66,

3

1

5

21.94, 112.99, 103.01, 91.44 (2×Ar-H, overlapped signals), 61.99, 61.85, 59.58,

6.55 (2×CH O-, overlapped signals), 56.33, 56.12, 35.84, 32.90, 31.37, 31.05, 24.00

3

1

95

(

2×-CH CH -, overlapped signals), 21.27. Pt NMR (129 MHz, DMSO-d ): δ/ppm

2

6

1

9

4

001.30; HR-MS (m/z) (ESI) called for [C H ClN O PtS-H] = 939.1615, found

32 41 2 14

39.1629.

.2.21. (OC-6-33)-diamminetrichlorido((E)-4-(2-methoxy-5-(((2,4,6-trimethoxystyryl)

sulfonyl)methyl)phenoxy)-4-oxobutanoicate))-platinum(IV) (25). Yield: 45.1%, Pale

1

yellow solid; mp (decomp.) 170 -172 °C; H NMR (400 MHz, DMSO-d ) δ 7.57 (d, J

6

=

15.6 Hz, 1H), 7.22-7.20 (m, 1H),7.12- 7.07 (m, 3H), 6.40 – 6.05 (m, 8H), 4.41 (s,

2

H), 3.86-3.85 (m, 9H), 3.76 (s, 3H), 2.74 (t, J = 7.2 Hz, 2H), 2.62 (t, J = 7.2 Hz,

2

7

1

3

2

H); C NMR (100 MHz, DMSO-d ) δ 179.10, 170.92, 164.19, 161.44 (2×Ar-OCH ,

6 3

overlapped signals), 151.34, 139.36, 133.76, 130.10, 125.78, 123.68, 121.86, 113.02,

03.01, 91.44 (2×Ar-H, overlapped signals), 59.61, 56.58 (2×CH O-, overlapped

1

3

1

95

signals), 56.40, 56.15, 31.44, 30.10. Pt NMR (129 MHz, DMSO-d ): δ/ppm 531.92;

6

HR-MS (m/z) (ESI) called for [C H Cl N O PtS+H] = 829.0466, found 829.1240.

23

31

3

2

10

4

.2.22. (OC-6-33)-diamminetrichlorido((E)-5-(2-methoxy-5-(((2,4,6-trimethoxystyryl)

sulfonyl)methyl)phenoxy)-5-oxopentanoicate)-platinum(IV) (26). Yield: 50.7%. Pale

1

yellow solid; mp (decomp.) 169 -171 °C; H NMR (400 MHz, DMSO-d6) δ 7.58 (d, J

=

15.3 Hz, 1H), 7.21 (s, 1H), 7.10-1.07 (m, 3H), 6.30-6.20 (m, 8H), 4.41 (s, 2H),

1

3

.86-3.77 (m, 12H), 2.63 (br. s, 2H), 2.37 (br. s, 2H), 1.82 (br. s, 2H); C NMR (100

MHz, DMSO-d ) δ 180.17, 171.42, 164.18, 161.44 (2×Ar-OCH , overlapped signals),

6

3

1

51.34, 139.35, 133.77, 130.12, 125.68, 123.66, 121.93, 113.00, 103.02, 91.45

(

2×Ar-H, overlapped signals), 59.58, 56.59 (2×CH O-, overlapped signals), 56.39,

3

1

95

5

6.16, 35.49, 32.99, 21.42. Pt NMR (129 MHz, DMSO-d ): δ/ppm 529.87; HR-MS

6

(

m/z) (ESI) called for [C H Cl N O PtS-H] = 841.0578, found 841.0570.

24 33 3 2 10

4

.2.23. (OC-6-33)-dichlorido(hydroxyl)((E)-4-(2-methoxy-5-(((2,4,6-trimethoxystyryl)

sulfonyl)methyl)phenoxy)-4-oxobutanoicate))-platinum(IV) (27). Yield: 35.1%. Pale

1

yellow solid; mp (decomp.) 168 -170 °C; H NMR (400 MHz, DMSO-d ) δ 7.58 (d, J

6

=

15.6 Hz, 1H), 7.21 (dd, J = 8.5, 2.0 Hz, 1H), 7.14 – 7.06 (m, 3H), 6.30 (s, 2H),

6

2

1

5

.09-5.83 (m, 6H), 4.41 (s, 2H), 3.86=3.85 (m, 9H), 3.77 (s, 3H), 2.72 (t, J = 7.2 Hz,

13

H), 2.55 (d, J = 7.2 Hz, 2H); C NMR (100 MHz, DMSO-d ) δ 179.53, 171.11,

6

64.18, 161.43 (2×Ar-OCH , overlapped signals), 151.35, 139.39, 133.74, 130.06,

3

25.80, 123.67, 121.84, 113.00, 103.00, 91.44 (2×Ar-H, overlapped signals), 59.60,

195

6.58 (2×CH O-, overlapped signals), 56.39, 56.14, 31.59, 30.27. Pt NMR (129

3

MHz, DMSO-d ): δ/ppm 1221.32; HR-MS (m/z) (ESI) called for [C H Cl N O PtS

6

23 32

2

11

+H] = 811.0811, found 811.0964.

4

.2.24. (OC-6-33)-dichlorido(hydroxyl)((E)-4-(2-methoxy-5-(((2,4,6-trimethoxystyryl)

sulfonyl)methyl)phenoxy)-5-oxopentanoicate))-platinum(IV) (28). Yield: 38.1%. Pale

1

yellow solid; mp (decomp.) 175 -177 °C; H NMR (400 MHz, DMSO-d ) δ 7.57 (d, J

6

2

8

Article Doi

Targeting RAS-RAF pathway significantly improves antitumor activity of Rigosertib-derived platinum(IV) complexes and overcomes cisplatin resistance

DOI: 10.1016/j.ejmech.2020.112269

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Full text of DOI:10.1016/j.ejmech.2020.112269

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