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

Article abstract of DOI:10.1016/j.ejmech.2020.112269

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

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

Journal Pre-proof
Targeting RAS-RAF pathway significantly improves antitumor activity of Rigosertib-
derived platinum(IV) complexes and overcomes cisplatin resistance
Zhikun Liu, Meng Wang, Hengshan Wang, Lei Fang, Shaohua Gou
PII:
S0223-5234(20)30236-1
DOI:
https://doi.org/10.1016/j.ejmech.2020.112269
EJMECH 112269
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 11 December 2019
Revised Date: 22 March 2020
Accepted Date: 22 March 2020
Please cite this article as: Z. Liu, M. Wang, H. Wang, L. Fang, S. Gou, Targeting RAS-RAF pathway
significantly improves antitumor activity of Rigosertib-derived platinum(IV) complexes and overcomes
cisplatin resistance, European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/
j.ejmech.2020.112269.
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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
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
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
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 (IC50 values 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
(
IC50 value 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
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
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
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
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
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
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
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
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
3
1
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
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
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
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
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