Platinum-Based Modification of Styrylbenzylsulfones as Multifunctional Antitumor Agents: Targeting the RAS/RAF Pathway, Enhancing Antitumor Activity, and Overcoming Multidrug Resistance

Article abstract of DOI:10.1021/acs.jmedchem.9b01223

Inhibiting/disturbing the RAS/RAF pathway may benefit the treatment of cancer and overcome the resistance. Utilizing such a pathway as the target, nine styrylbenzylsulfone derivatives generated from the platinum-based modification of the side chain of Rig

Full text of DOI:10.1021/acs.jmedchem.9b01223

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Article
Platinum-Based Modification of Styrylbenzylsulfones as Multifunctional
Antitumor Agents: Targeting RAS-RAF Pathway, Enhancing
Antitumor Activity and Overcoming Multidrug Resistance
Zhikun Liu, Meng Wang, Heng-Shan Wang, Lei Fang, and Shaohua Gou
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b01223 • Publication Date (Web): 10 Dec 2019
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Platinum-Based Modification of Styrylbenzylsulfones as Multifunctional
Antitumor Agents: Targeting RAS-RAF Pathway, Enhancing Antitumor
Activity and Overcoming Multidrug Resistance
9
Zhikun Liua,1, Meng Wangb,1, Hengshan Wangb, Lei Fanga,*, Shaohua Goua,*
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a
Jiangsu Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University,
Nanjing, 211189, China
b
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
1These authors contributed equally.
* Corresponding authors
E-mail: lei.fang@seu.edu.cn (L. Fang); sgou@seu.edu.cn (S. Gou)
ABSTRACT: Inhibiting/disturbing the RAS-RAF pathway may benefit the treatment of cancer
and overcome the resistance. Utilizing such pathway as the target, nine styrylbenzylsulfone
derivatives generated from the platinum-based modification of the side chain of Rigosertib were
designed. Among them, compound 29 showed the most potent antitumor activity in vitro with IC50
values at nanomolar level against the tested tumor cell lines and 1000-fold higher than cisplatin
against the multidrug resistant cells (A549/CDDP, A549/DOX, and SKOV-3/CDDP cells) while
showed only moderate cytotoxicity against normal cells (HEUVC cells). Compound 29 could
clearly disturb signaling transduction between RAS and CRAF by directly bonding to CRAF and
inhibit CRAF activation. Besides, the enhanced intracellular platinum level made 29 more potent
than cisplatin in DNA damage, ROS accumulation and mitochondrial membrane potential
decrease. Moreover, 29 induced apoptosis by endogenous pathway and efficiently inhibited tumor
growth in A549 xenograft model without side effect.
Key Words: overcoming multidrug resistance, RAS-CRAF interference agent, drug conjugation
INTRODUCTION
The mitogen-activated protein kinase (MAPK) signaling pathway is of great importance to
cellular biological functions, which not only activates proliferative signals but also affects signals
that are responsible for differentiation and apoptosis [1-3]. The signal transmission is achieved by
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the conversation from RAS.GDP (inactive) to RAS.GTP (active) with the help of extracellular
signals and in turn triggering several downstream cascades, including RAF-MEK-ERK pathway [4]
Naturally, the MAPK pathway was frequently dysregulated in many cancers harboring RAS
mutations, resulting in constitutive activation of the pathway, uncontrolled proliferation and
decreased cellular sensitivity to agents that could originally induce apoptosis [5-7]. Therefore, it
will be attractive to inhibit the activation of RAS for the treatment of cancers. However, the lack
of well-defined druggable pockets and cavities on the RAS surface made RAS an ‘undruggable’
target [8-10]. In addition, the RAS genes in cancer cells mutated frequently, which made inhibition
of RAS activation directly more challenging. Thus, alternatively, disturbing the signal
transduction between RAS proteins and downstream effectors could serve as a new approach to
develop novel RAS-driven therapies.
.
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In mammals, RAF family contains three members: ARAF, BRAF and CRAF, binding with
RAS as upstream activator and mediating the MAPK signaling transduction to the MEK as only
downstream effector. Among those three subtypes, CRAF plays an important role as a mediator in
mutant KRAS-driven proliferation and tumor development [11]. On the other hand, CRAF also
[12-14]
affects the anti-tumor activity of BRAF inhibitors and MEK feedback-mediated pathway
.
Therefore, in consideration of inhibiting signaling transduction of RAS, inhibition of CRAF
activation was proved to be an effective method to disturb RAS-driven tumorigenesis and
feedback reactivation [15]. Rigosertib (Figure 1) belongs to the benzylstyryl sulphone compounds
and can inhibit tumor cell proliferation in nanamole ranges, including some resistant cells [16, 17]. In
in vitro study, Rigosertib acted 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 [16]
.
From the structure-activity relationship point of view, the nature, number, and position of
substituents on the two aromatic rings of Rigosertib, as described by Reddy et al. [16], are
conservative and cannot tolerate significant structural modification. Thus, we have paid attention
to the carboxyl group at the end of the side chain. In the past, the therapeutic potential of
metal-based complexes has been widely used and investigated for cancer therapy, because of the
unique characteristics of metals, such as redox activity, lewis acid properties and reactivity toward
the organic substrate [18]. A large number of metal-based complexes have been designed and
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synthesized and among them several complexes were proved to be promising anticancer agents.
For example, Ma et al. found a novel benzofuran-conjugated iridium(III) complex as a STAT3 and
NF-kB dual inhibitor in prostate cancer cells [19]. Leung et al. found an enantiomeric iridium(III)
metal-based compound as an efficient RAS/RAF interaction inhibitor, which could directly
engage both H-Ras and Raf-1-RBD and efficiently disrupt the signaling between
RAF/RAF/MEK/ERK [20]. Zhang et al. also summarized chirality metal-based agents in cancer
research [21]. Therefore, metal-based complexes in cancer therapy has attracted a lot of interest.
Platinum drugs, such as cisplatin (CDDP), carboplatin and oxaliplatin (Figure 1), have been
developed into an important part of clinical chemotherapy. As a kind of cytotoxic agents, platinum
drugs mainly act on DNA and then cause death of tumor cells. This action mechanism is quite
different from that of Rigosertib, suggesting introduction of platinum pharmacophore to
Rigosertib may give additional activity to the parent compound. On the other hand, the platinum
drugs often suffer from the severe toxic side effects and the acquired resistance associated with the
long-term application [22-27]. Given the good performance of RAS inhibitors against the
multiple-resistance tumor cells, it may be anticipated that combination of Rigosertib and platinum
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drugs may overcome the resistance.
O
H2
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H3N
H3N
Cl
H3N
H3N
Pt
Pt
Pt
O
O
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Cl
N
H2
H
ONa
Rigosertib
Cisplatin
Carboplatin
Oxaliplatin
Figure 1. Structures of Rigosertib and current Pt(II) drugs approved for worldwide use.
In this context, we here made structural modifications to Rigosertib by conjugating a
platinum(IV) complex, which can be considered as a stable prodrug of the classic platinum(II)
drug [28, 29], to carboxyl group via different linkers. The resulting conjugates were supposed to be
relative stable under the circulation condition, and the good lipophilicity [30, 31], as compared with
the traditional platinum(II) complex, could be beneficial for the uptake by tumor cells. When
entering the tumor cells, the conjugates could be effectively reduced by the reductive substances
(e.g. glutathione, protein and other biomoleculesn [28-32]) in tumor cells to release Rigosertib and
platinum(II) complex to exert anti-tumor activity via different pathways. Thus, enhancing activity
and the potential to overcome the multidrug resistance can be expected. Our drug design idea is
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outlined in Figure 2.
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O
O
O
O
O
O
O
Ester bond conjugation
Good lipophilicity
S
S
Improved cellular uptake
O
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O
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linker
Excellent cytotoxicity against tumor cells
Overcome multi- drug resistance
Multiple antitumor mechanism
Remarkable antitumor efficacy in vivo
O
N
N
Pt(IV)
9
H
H
ONa
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Rigosertib
Target compounds
Figure 2. The structural modification strategy and the drug design idea of the target compounds.
RESULTS AND DISCUSSION
Chemistry. As shown in Scheme 1 and 2, compound 8 was prepared according to previously
reported procedures [16], which was further esterified and followed by a hydrolysis reaction to give
compounds 13 and 14. Pt(IV) complexes 15, 16 and 17 were obtained by oxidation of the
corresponding Pt(II) complexes following known procedures [32-34]. As for the synthesis of
compound 26 (carboxyl acid form of Rigosertib), compound 8 was firstly treated with ethyl
bromoacetate and then hydrolyzed to offer 26 in high yield. Finally, nine Pt(IV) derivatives were
obtained via combining 15, 16 or 17 with compounds 13, 14 and 26, respectively, in the presence
of O-(benzotriazol-1-yl)-N,N,N',N' -tetramethyluronium tetrafluoroborate (TBTU). All of the
target compounds were characterized with 1H-NMR, 13C-NMR and ESI-MS techniques.
Scheme 1. Preparation of Pt(IV) Complexes 18-23
O
O
O
O
OH
O
Br
S
COOH
+
S
COOH
e
O
O
d
c
a
b
NO2
NO2
NO2
NO2
O
NO2
NO2
O
O
OH
O
2
O
3
O
5
O
4
SM1
1
6
O
O
S
O
O
O
O
O
O
S
S
O
f
g
O
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h
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O
O
O
+
O
O
N
O
O
O2N
O
OH
H2N
n
O
O
8
n
H
O
7
9 n=3; 10 n=4
11 n=3; 12 n=4
O
O
O
S
OH
Pt
OH
Cl
H2
OH
H2
N
O
N
O
O
O
O
O
O
Cl
Cl
H3N
i
Pt
Pt
HO
N
H
Cl
N
n
H3N
O
N
O
H2
H2
Cl
Cl
15
Cl
17
16
13 n=3; 14 n=4
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O
O
S
OH
O
O
O
O
O
O
S
S
j
O
O
O
O
O
O
O
O
O HN
O HN
O HN
O
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H2
N
O
H2
N
O
n
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O
Cl
O
O
n
O
Pt
Cl
H3N
H3N
Cl
Pt
Cl
Cl
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N
Pt
Cl
O
N
O
H2
H2
Cl
18 n=3; 19 n=4
20 n=3; 21 n=4
22 n=3; 23 n=4
Reagents and conditions: a) CH3I, K2CO3, DMF; b) NaBH4, MeOH; c) PBr3, DCM; d) Mercapto acetic acid,
NaOH, MeOH; e) H2O2, AcOH; f) Piperidine, benzoic acid, toluene; g) Fe, NH4Cl, EtOH, H2O; h) Oxalyl chloride,
Et3N, DCM; i) LiOH•H2O, CH3OH, H2O; j) TBTU, Et3N, 15/16/17, DMF.
Scheme 2. Preparation of Pt(IV) Complexes 27-29
O
O
O
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O
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O
O
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O
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a
b
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+
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8
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O
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O
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O
O
O
O
O
O
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c
N
O
H
N
O
H2
N
O
O
N
H
H
Cl
H2
N
O
O
O
O
O
Pt
Cl
H3N
H3N
Cl
Pt
Cl
Cl
N
Pt
H2
O
N
O
Cl
H2
Cl
29
27
28
Reagents and conditions: a) K2CO3, KI, DMF; b) LiOH•H2O, CH3OH, H2O; c) 15/16/17, TBTU, Et3N, DMF.
In Vitro Cytotoxicity Measurement. The cytotoxicity of the synthesized target compounds
was evaluated by MTT methods, using 26 (carboxyl acid form of Rigosertib), cisplatin and
oxaliplatin as references. Different cancer cell lines, including A549 (non-small cell lung cancer
cell line), MGC-803 (gastric cancer cell line), SKOV-3 (ovarian cancer cell line), and NCI-H460
(large cell lung cancer cell line) were used in this study. The results after 72 h drug exposure were
shown in Table 1. All of these compounds tested showed moderate to strong anti-proliferative
activity against the cancer cells. It was found that the cytotoxicity of 19, 21 and 23 containing a
four methylene linker was higher than that of 18, 20 and 22 owning a three methylene linker, but
their IC50 values are only at the level of cisplatin and oxaliplatin. Interestingly, when a glycine
group was employed as the linker, the resulting complexes (i.e. 27, 28, 29) showed much better
activity than the above mentioned compounds. It was observed that all cancer cell lines tested
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were sensitive to complexes 27-29 with the IC50 values comparable to those of compound 26, but
much lower than those of cisplatin and oxaliplatin. Since the target complexes can be regarded as
prodrugs, we assumed that the original styrylbenzylsulfone moiety was mainly responsible for
their antitumor activity. Complexes 27-29 were capable of releasing 26, a known potent antitumor
agent. In contrast, complexes 18-23 were capable of releasing compounds 13 or 14. Our MTT
assay revealed that 13 or 14 showed only moderate cytotoxic activity with IC50 values ranged
from 10.81-17.31 μM against the tested tumor cells (Table 1), much lower than 26 whose IC50
values ranged from 44-83 nM. Thus, it may be understandable why complexes 27-29 showed
much better activity.
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Among all of the target compounds, 29 showed the best activity, with the potency at least
over 1000-fold higher than cisplatin and even 10-fold higher than 26 against A549. In contrast, the
combination administration of 26 with cisplatin (i.e. equal molar 26/cisplatin mixture) didn’t show
significant improvement of the activity relative to the administration of 26 alone. Unexpectedly,
compound 8 showed low cytotoxicity against these cancer cell lines tested, which may be
attributed to the miss of the glycine group as well as its poor solubility in aqueous buffers and
solutions [16]. However, when the amino group was converted to glycine, the resulting compound
26 produced much better cytotoxic activity than compound 8 against all cancer cells used in this
study. The enhanced activity of 26 illustrated that the glycine group was of great importance for
the anti-proliferative activity.
Table 1. IC50 values of the tested compounds toward various cancer cell lines.
aIC50 (μM)
Compd.
A549
MGC-803
>50
SKOV-3
>50
NCI-H460
>50
8
>50
13
14
18
19
20
21
22
23
26
27
10.81 ± 1.24
13.82 ± 1.75
10.09 ± 0.83
6.91 ± 0.63
19.50 ± 1.18
16.75 ± 0.88
20.05 ± 1.74
6.31 ± 0.75
0.083 ± 0.023
0.094 ± 0.040
13.96 ± 1.23
12.58 ± 1.67
5.70 ± 0.19
6.57 ± 0.25
4.03 ± 0.23
4.68 ± 0.34
11.39 ± 0.82
5.83 ± 0.98
0.054 ± 0.090
0.058 ± 0.120
17.31 ± 1.32
14.58 ± 1.43
2.88 ± 0.11
2.81 ± 0.04
6.53 ± 0.32
5.87± 0.25
23.63 ± 1.14
1.37 ± 0.47
0.045 ± 0.190
0.079 ± 0.160
15.14 ± 0.38
14.28 ± 1.12
16.40 ± 1.13
16.33 ± 1.12
23.39 ± 1.78
13.99 ± 0.11
25.55 ± 1.63
4.89 ± 0.37
0.044 ± 0.013
0.053 ± 0.050
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29
0.120 ± 0.130
0.062 ± 0.080
0.014 ± 0.008
0.062 ± 0.049
7.21 ± 0.83
0.087 ± 0.070
0.089 ± 0.200
0.017 ± 0.005
0.029 ± 0.010
9.05 ± 0.55
0.006 ± 0.004
0.076 ± 0.035
8.22 ± 0.91
0.050 ± 0.024
0.050 ± 0.095
5.45 ± 1.26
bCDDP/26
cCDDP
dOxa
11.95 ± 1.43
8.29 ± 0.76
12.06 ± 1.66
16.09 ± 2.44
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aIC50 values were presented as means ± SD of three independent experiments; bCDDP/26, cisplatin/26 mixture (1:1,
n/n); cisplatin; cCDDP, cisplatin; dOxa, oxaliplatin.
In Vitro Antiproliferative Activity of 29 against CDDP/Doxorubicin-Resistant Cancer
Cells and Normal Cells. Development of multidrug resistance to classical chemotherapeutic
agents (e.g. cisplatin, doxorubicin) is the main reason for the fail of most first round chemotherapy.
It was revealed that the overexpression of ATP binding cassette (ABC) transporters is responsible
for the resistance since such transporters could enable the tumor cells to pump out drugs so that
reduce the intracellular drug concentrations. According to the previous report, Rigosertib was
highly active against multidrug resistant tumor cell lines that overexpressing ABC transporters [16]
.
This is probably because the structure of Rigosertib cannot act as the substrate for ABC transport
proteins, thus cannot be moved out the cell by ABC transport. So, it will be interesting to
investigate whether our target compounds which are Rigosertib derivatives could also overcome
the multidrug resistance. Thus, the most active compound 29 was selected to test the cytotoxicity
against CDDP resistant A549 cells (A549/CDDP), doxorubicin resistant A549 cells (A549/DOX)
and CDDP resistant SKOV3 cells (SKOV3/CDDP) as well as human umbilical vein endothelial
cells (HUVEC), using 26 and CDDP as controls, respectively. To verify that those
multidrug-resistant cancer cell lines were successfully constructed, we firstly performed MTT
assays and western blotting assays to identify whether they were drug-resistant or not. As shown
in Figure S1 and Table S1, the IC50 values of A549/CDDP, A549/DOX and SKOV3/CDDP were
significantly increased when compared with those corresponding drug-sensitive A549 and
SKOV-3 cancer cell lines. The resistant factors (RF) of A549/CDDP, A549/DOX and
SKOV3/CDDP improved more than 4, 15 and 4 times than A549 and SKOV-3, respectively.
Besides, western blotting assays also revealed that P-glycoprotein (P-gp) in A549/CDDP,
A549/DOX and SKOV3/CDDP was also improved. Therefore, those drug-resistant cancer cell
lines were successfully constructed. The results of 29 against the drug-resistant cancer cells were
shown in Table 2. After 72 h co-incubation, both 26 and 29 showed high cytotoxicity to the drug
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resistance tumor cells with very low IC50 values from 0.008 to 0.094 μM and the resistant factor
no more than 1.5. In contrast, the activity of CDDP against A549/CDDP cells (IC50 value was
35.77 μM) and SKOV-3/CDDP cells (IC50 value was 28.67 μM) were significantly decreased as
compared to that of non-resistant A549 and SKOV-3 tumor cells (RF values were 4.4 and 5.3,
respectively) (Table 2). These results indicated that the A549/CDDP, A549/DOX and
SKOV3/CDDP cells showed obvious resistance to cisplatin while were sensitive our designed
compound 29, meaning 29 may overcome the multidrug resistance. Furthermore, in order to
compare 29 with the combination administration of 26 with cisplatin, the cytotoxicity of
26/cisplatin mixture (1:1, n/n) was also measured. It was found that its activity was comparable to
that of 26, but lower than 29, especially to the A549 cells. Particularly, the mixture showed
highest toxicity to human normal cells (IC50 0.78 μM against HUVEC cells), suggesting a serious
safety risk. In contrast, compound 29 showed only moderate cytotoxicity to human normal cells
(IC50 8.74 μM against HUVEC cells), at least 100-fold lower than that against tumor cells,
indicating a good safety and a wide therapeutic window for compound 29.
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Table 2. IC50 values in vitro of tested compounds in various CDDP-resistant cancer lines and human normal cells.
aIC50 (μM)
Compounds
A549
26
29
CDDP
8.22 ± 0.91
5.45 ± 1.26
35.77 ± 2.24
28.67 ± 2.24
18.81 ± 0.95
9.03 ± 0.78
4.4
CDDP/26
0.089 ± 0.055
0.051 ± 0.020
0.082 ±0 .035
0.050 ± 0.030
0.076 ± 0 .039
0.78 ± 0.55
0.9
0.083 ± 0.023
0.045 ± 0.009
0.094 ± 0.040
0.041 ± 0.039
0.081 ± 0.020
3.59 ± 0.55
1.1
0.006 ± 0.004
0.050 ± 0.024
0.008 ± 0.011
0.038 ± 0.025
0.009 ± 0.005
8.74 ± 0.44
1.3
SKOV-3
A549/CDDP
SKOV-3/CDDP
A549/DOX
HUVEC
bRF1
cRF2
0.9
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5.3
1.0
dRF3
1.0
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2.3
0.9
b
aIC50, values were presented as means ± SD of three independent experiments; RF1, Resistant Factor = IC50
c
(A549/CDDP)/IC50 (A549); RF2, Resistant Factor = IC50 (SKOV-3/CDDP)/IC50 (SKOV-3); dRF3, Resistant
Factor = IC50 (A549/DOX)/IC50 (A549).
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HPLC Analysis on the Release of Rigosertib Moiety under the Reduction of Ascorbic
Acid. The target compounds, especially compound 29, showed remarkable activity to both normal
and drug-resistant tumor cells. This exciting result might be attributed to that the Pt(IV) part of the
target compound could be reduced to release the corresponding Pt(II) part and the Rigosertib
moiety under reduction, thus a synergistic effect can be achieved. In order to prove this drug
design strategy, HPLC analysis was performed for compound 29 which was co-cultured
with/without ascorbic acid (3.0 equiv.) in a mixture of PBS (pH= 7.4) and acetonitrile (90:10, v/v)
and preserved at 37 °C water bath in dark. Compound 26 and ascorbic acid dissolved in the same
solvent mentioned above were used as controls to match the peaks of compound 29 in the
reduction process. As shown in Figure S2D, compound 29 was stable in 72 h in the PBS (pH=7.4)
solution. In view of the acidic microenvironment of tumor cells, the stability of 29 in the PBS
(pH=5.5) was also investigated. The results in Figure S2E showed that more than 90 % percent of
29 remained stable in the solution within 24 h. However, when mixed with ascorbic acid, two
peaks (i.e. ascorbic acid and compound 26 peaks) appeared as time passed. The peak of compound
29 gradually decreased with an increasing peak at the position of compound 26, indicating that
compound 29 could release compound 26 upon the reduction of ascorbic acid. Unsurprisingly, no
peak of cisplatin was detected because of its weak chromophore under the tested condition. In all,
compound 29 was stable under physiological condition and could be effectively reduced to release
26 within 5 h when exposed to ascorbic acid.
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In order to verify that 29 can release 26 and CDDP in cells and detect the concentration of 26
in cancer cells, HPLC analysis was performed. Briefly, A549 cells was co-incubated with 29 (10
μM, 5 μM, 2 μM) for 24 h, washed with PBS and lysed to obtain cell lysates. Supernatant fraction
gained after centrifugation was recorded and analyzed with RP-HPLC (waters, e2695 system;
2489 UV/Vis detector). The standard curve of 26 was displayed in Figure S3 and Table S2, and
the results of cell lysates were shown in Figure S4 and Table S3. As shown in Figure S4, no peak
of 29 (Rt =6.8 min) but only 26 (Rt =8.1 min) was detected, illustrating that 29 was fully reduced
to 26 in A549 cells in 24 h. The reduction of 29 in A549 cells was further proved with HR-MS in
Figure S4D (26, M+H=452.1359) and Figure S4E (CDDP, M+Na=322.9356). As for the
intracellular concentration of 26, 0.77 μM, 2.88 μM or 5.91 μM of 26 could be detected after
A549 cells co-incubated with 2 μM, 5 μM or 10 μM of 29, respectively. Therefore, we conclude
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that 29 can be fully reduced in A549 cells in 24 h to release 26 and CDDP.
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Cellular Uptake. Compound 29 showed excellent cytotoxicity in the cellular assays, which
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[29-33]
may, according to the former findings, be partly attributed to the improved cellular uptake
.
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Therefore, to testify this hypothesis, we used ICP-MS to measure the intracellular platinum
content. After 12 h co-incubation with 29 (5 µM and 10 µM) or CDDP (5 µM and 10 µM), the
platinum content in A549 cells was determined by ICP-MS after digestion with 65% nitric acid.
As shown in Figure 3, the accumulation of platinum of 29 was evidently enhanced compared with
that of CDDP. Moreover, as the concentration increased, A549 cells internalized more platinum
than those treated with CDDP at equal concentration (Figure 3). This may be associated with the
hydrophobic ligands which increased the lipophilicity of the Pt(IV) complexes and contributed to
the transportation trough cell membrane. The more uptake of 29 also corresponds to the increased
anti-tumor activity in vitro in Table 1.
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Figure 3. Cell uptake of 26 in A549 cells treated with 29 for 12 h at the concentration of 5 µM and 10 µM.
Platinum content was measured by ICP-MS after digestion with 65% HNO3. Data represented the mean ± SD of at
least three different experiments.
Effect of 29 on RAS/RAF Interaction. Originally, Rigosertib was reported to be a non-ATP
competitive Plk1 inhibitor in nanomole concentration [17]. However, subsequent experiments of
Plk1 kinase activity test indicated that Rigosertib, 20 and 26 had little effect on kinase activity
(data was not shown) [17]. Later, Reddy and co-workers reported that Rigosertib acted as RAS
mimetic blocking signals that associated with downstream effector proteins [8]. Therefore, in order
to determine whether our target compounds could also block the RAS/RAF/MEK/ERK signaling
pathway, we conducted a series of western blot to study the effect of 26 and 29 on RAS/RAF
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complex and MAPK signaling cascade. Briefly, A549 cells were co-incubated with 26 (5 μM) and
29 (5 μM) for 24 h, then the coprecipitation obtained using VE-cadherin (containing anti-RAS
antibody) was further purified with protein G agarose and subjected to western blot to determine
the levels of its associated CRAF. For the analysis of effects on the phosphorylation and
expression of MEK and ERK, A549 cells were treated with CDDP (5μM) or different
concentrations of 26 and 29 for 24 h, then the levels of phosphorylation and expression of MEK
and ERK were measured, using GAPDH as loading control. As shown in Figure 4A and 4B, the
levels of CRAF proteins which were associated with RAS proteins were reduced in input group,
whereas little effect on the expression of RAS was observed. This result suggested that 26 and 29
did disturb the interaction between RAS and CRAF, which was consistent with the result reported
previously [8]. Moreover, the levels of RAS/CRAF complexes of cells treated with 26 and 29 in IP
group were significantly decreased when compared to those of control group (Figure 4A and 4B).
Interestingly, the disruption of RAS and CRAF interaction may raise a question whether 29
directly targets either RAS protein or CRAF protein or both. We thereby carried out microscale
thermophoresis (MST) assay and cellular protein thermal shift assay (CETSA) for further studies.
As shown in Figure 4C and 4D, 29 can directly target CRAF protein with high affinity at
nanomolar concentration (affinity constant of Kd value was 149 nM), whereas the Kd value
between 29 and K-RAS, N-RAS or H-RAS cannot be detected, even with the initial concentration
was increased to 125 μM, indicating the poor affinity of 29 to K-RAS, N-RAS or H-RAS (results
are shown in Figure S5C-S5H). The obvious difference of the affinity explained our design
concept that as a derivative of styrylbenzylsulfones compound 29 still possessed the ability to
target CRAF protein and acted as an inhibitor of the protein. Similarly, as the positive control, 26
also displayed high affinity with Kd value of 124 nM (results are shown in Figure S5A and S5B).
Moreover, the results of CETSA assays (Figure 4E-4F) also showed that the level of CRAF
remaining in the soluble fraction quickly decreased when the A549 cell lysates were heated from
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o
47 to 67 C. In contrast, when A549 cell lysates were treated with 26 or 29, the melting
o
temperature of CRAF from 51 to 67 C significantly increased, indicating the tested compounds
can effectively bind and stabilize CRAF. However, as for RAS, the melting temperature of RAS
hardly changed when A549 cell lysates were treated with 26 or 29 as compared with the negative
DMF treatment group (Figure S6A and S6B). Besides, the results of GAPDH in the CETSA assay
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showed that both 26 and 29 had no effect on the thermal stability of GAPDH (Figure S6C and
S6D). From these results, we could conclude that 29 could directly bind to CRAF protein rather
than RAS protein, and disrupt the signaling transferred from RAS to CRAF.
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Figure 4. Compound 29 interfered with the interaction of Ras-Raf and MEK/ERK activation. (A) Cell lysates
collected from A549 cells that were pretreated with 29 (5μM) and 26 (5μM) for 24 h were subjected to
immunoprecipitation with anti-Ras antibodies and determined the associated CRAF with western blot. (B)
Statistics of grey intensity were normalized with input. Data were presented as the mean ± SD of three independent
experiments. (C): MST trace lines of 29 binding to CRAF-RBD protein. (D): MST results of 29 binding to CRAF
protein. Results provided above showed that 29 binds to the CRAF protein (Kd values was 149 nM). (E) Thermal
stability of RAS; (F) Statistics of grey intensity were normalized with grey intensity of 47 oC. Data were presented
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as the mean ± SD of three independent experiments.
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RAF kinases were essential for RAS-RAF-MEK-ERK pathway signaling. The RAF kinases
can form dimers just like other protein kinases, which were RAS-dependent. Under normal
signaling transduction condition, the dimers occurred in plasm and disassociated by the feedback
of activation of ERK [35-37]. Thus we conducted western blotting to detect the activation of BRAF
in A549 cells which were pretreated with CDDP (5 μM), 26 (2μM, 5μM) 29 (2μ, 5μM) for 24 h
by using BRAF antibodies. The results in Figure S7A and S7B showed that 26 and 29 could
inhibit the activation BRAF in a dose-dependent manner while 29 (2 μM) was weaker than 26 (2
μM) in this assay. Thereafter, to detect the effects of 29 on the RAS-mediated signaling,
heterodimerization of endogenous CRAF and BRAF with the stimulation of EGF was investigated
by co-immunoprecipitation assays, using CRAF antibodies to detected the levels of associated
BRAF. The results were displayed in Figure S7C and Figure S7D, from which we could see that
no BRAF protein in IP group was detected when A549 cells were treated with control (DMF
without EGF), though BRAF protein existed in Input group [8]. The formation of CRAF and
BRAF dimers was found upon the stimulation of EGF. However, significant decrease occurred in
their dimers after 24 h co-incubation of A549 cells with 26 (5 μM) or 29 (5 μM), revealing that
both 26 and 29 could also disrupt the interaction of CRAF and BRAF by interrupting the signaling
between RAS and RAF. Not surprisingly, CDDP had no activity in both assays. In all, 29 could
inhabit the activation of BRAF and disrupt the signaling transduction from RAS to CRAF.
As for the classical MAPK signaling cascade, the block signaling between RAS and RAF
usually led to the down-regulation of phosphorylation of MEK and ERK. Indeed, the inhibition of
phosphorylation were observed in our study, as shown in Figure 5A and 5B. As expected,
treatment with 26 or 29 resulted in reduced phospho-MEK and phospho-ERK levels in a
dose-dependent manner (Figure 5A and 5B). However, little effect on phosphorylation of MEK
and ERK was found in CDDP-treated group, which explained indirectly that anti-tumor activity of
CDDP was not involved in this signaling pathway. In summary, these findings supported our
hypothesis that 29, similar to Rigosertib, could disturb the MAPK signaling transduction by
inhibiting the formation of RAS/ RAF complexes.
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Figure 5. (A) Phosphorylation and expression of the indicated proteins determined by western blot in A549 cells
after treatment with CDDP or increasing concentrations of 29 and 26. (B) Signals obtained from western blot and
normalized with GAPDH. Data were presented as the mean ± SD of three independent experiments
A549 Apoptosis Induced by 29. To further investigate whether compound 29 produced
cytotoxicity by inducing apoptosis of tumor cells, cell apoptosis analysis was carried out. A549
cells were firstly treated with compound 29 (5 μM), compound 26 (5 μM) as well as CDDP (5 μM)
which was used as positive control for 24 h. Thereafter, the tumor cells were co-stained with
Annexin-V FITC and PI and the apoptotic cells were analyzed with flow cytometry. The result of
apoptotic value was shown in Figure 6, from which four states of cells were found: necrotic cells
(Q1), late apoptotic(Q2), early apoptotic cells (Q3) and living cells(Q4). A great difference of the
apoptotic value was presented clearly. One hand, compound 29, compound 26 and CDDP could
efficiently induced apoptosis in A549 cells at the concentration of 5 μM and the apoptotic values
(including the early and late apoptosis states) are 40.6 %, 37.2 %, 15.55 %, respectively. On the
other hand, the apoptotic value (40.6%) of compound 29 was more supior to that of compound 26
(37.2 %), and CDDP (15.55%), which revealed that compound 29 could induce more apoptosis of
A549 cells than that of compound 26 and cisplatin at the same concentration. Due to apoptosis
data of A549 cells were obtained after 24 h co-incubation with high concentration of 29 (5 μM),
26 (5 μM) and CDDP (5 μM), we further investigated the apoptosis-inducing ability of 29, 26, and
CDDP with the concentration at the IC50 value level and total 72 h drug exposure. From Table S4,
we could find that 26 and 29 did not cause significant apoptosis in the first 24 h. However, when
A549 were exposed to 29, 26 and CDDP for 72 h much more cells were in apoptotic state even at
the concentration of IC50 values (the results were shown in Figure S8). Altogether, 29 showed a
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similar antitumor behavior by inducing apoptosis of tumor cells as compound 26 and CDDP did,
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but its potency was much higher than the others.
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Figure 6. Annexin V-FITC and PI staining to evaluate apoptosis in A549 cells treated with various compounds.
A549 cells were co-incubated with DMF, cisplatin (5 μM), compound 26 (5 μM) and compound 29 (5 μM),
stained with Annexin V-FITC and PI and analyzed with flow cytometry.
Cell Cycle Arrest Analysis. It was well demonstrated that 29 and cisplatin produced
cytotoxicity by inducing apoptosis. Therefore, to study the effect of the target complexes on tumor
cell cycle, 26 (5 μM) and 29 (5 μM) were incubated with A549 cells overnight using CDDP (5 μM)
and DMF as postive and negative control, respectively. As the results of cell arrest shown in
Figure 7, CDDP mainly arrested A549 cells in S phase when compared to that of control group,
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whose value increaed from 24.25% to 32.47%. However, the cells treated with 26 (5 μM) were
mainly arrested in G2/M phase with the percentage of 68.83%, whereas the values were 12.25%
and 7.72% for control and cisplatin respectively. Intrestingly, when the tumor cells were treated
with the same concentration of compound 29 (5 μM), the percentage of cells arrested in G2/M
phase was larger than that of compound 26 (5 μM) and control. In conclusion, compound 26 and
29 could produce cytotoxicity by introducing cell arrest in G2/M phase.
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Figure 7. Cell cycle arrest was analyzed after 24 h treated with various compounds. A549 cells were treated with
DMF, CDDP (5 μM), 26 (5 μM) and 29 (5 μM), stained with PI and analyzed with flow cytometry.
DNA Damage Induced by 29. It is well known that Pt(IV) complexes exsert anti-tumor
activity mainly denpend on the release of Pt(II) complexes [34-37]. Previously we proved that 29
could release 26 and CDDP under the reductive condition in cells. Therefore, we performed comet
assays and wetern blotting assays to verify whether 29 could also cause DNA damage. As shown
in Figure 8, A549 cells were induced apparent DNA damage after 24 h co-incubation with CDDP
(5 μM), 26 (5 μM) and 29 (5 μM), which was characterized by the tail DNA. Normally, the
number and length of tail DNA were in positive correlation with the digree of DNA damage. As
shown in Figure 8 more and longer tail DNA exsited in A549 cells treated with 29, whereas less
and shorter DNA tails were found in CDDP-treated group and 26-treated group. This indicated
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that 29 induced stronger DNA damage than 26 and CDDP. Usually, the DNA damage induced by
CDDP was along with double-strand break (DSB) [38-40]. Therefore we detected the expression of
H2AX phosphorylation (γH2AX), a hallmark of DNA DSB. As indicated in Figure S9A and S9B,
after 24h co-incubation of A549 cells with 29 (2 μM,5 μM) and CDDP (5 μM) the level of
γH2AX was significantly increased when compared with control group. The 29-treated group
produced higher level of discrete γH2AX in a dose-dependent manner than CDDP-treated group,
which explained that 29 increased the extent of DSB. Subsequently, the increased DSB in A549
cells also promote the expression of p53 protein, which usually occurred in an activation of the
mitotic checkpoint following drug exposure [34a]. In Figure S9A and S9B, higher level of p53 was
induced in A549 cells by 29 (2 μM,5 μM) than CDDP (5 μM) in a dose-dependent manner. This
also incadicated that 29 can not only arrest A549 cells in G2/M phase but induced DNA damage
and finally induced more apoptosis as just shown in Figure 6 and Figure S8.
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Figure 8. After 24 h treatment with vehicle, cisplatin (5 μM), 26 (5 μM) and 29 (5 μM), A549 cells were
transferred onto comet slide and treated according to the method reported previously[38]. Images were analyzed and
recorded with laser confocal microscope. Tail DNA was marked with white arrow.
Detection of Reactive Oxygen Species Active Oxygen. According to previous reports [32-34]
,
metal complex broke the balance of intracellular redox to induce excess ROS accumulation.
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Therefore, in order to study the accumulation of intracellular ROS, A549 cells were incubated
with following compounds respectively ：vehicle (DMF), CDDP (5 μM), 26 (5 μM) and 29 (5
μM). After 24h treatment, the tumor cells were stained with DCFH-DA (Molecular Probe,
Beyotime, Haimen, China) and were observed and recorded with fluorescence microscope. The
results were shown in Figure 9, from which we could see that the treatment with cisplatin
significantly increased the level of intracellular ROS. Similarly, complex 26 and 29 also caused
the increased level of ROS, and the fluorescence intensity of later two was higher than that of
cisplatin, suggesting higher ROS levels caused by 26 and 29. It is widely accepted that high level
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ROS produces high cytotoxicity. Thus, these results were consistent with former findings [34, 41a]
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Generally, mitochondria are responsible for production of ATP and provide the energy using
for cellular biological functions. In mitochondrial electron transport chain, only 1–2% of
[41-43]
molecular oxygen consumed by mammalian cells turned into ROS via electron leakage
.
Therefore, the endogenous ROS was mainly from mitochondria in human and animal cells. Given
the significant accumulation of ROS in A549 cells displayed in Figure 9 after co-incubation with
26, 29 and CDDP, we observed the cellular location of ROS in A549 cells to confirm the
distribution of ROS and to verify the source of ROS. The ROS was traced with DCHF-DA as
green fluorescence (Figure S10A2-D2), while the mitochondria were highlighted by Mito Tracker
RED as red fluorescence (Figure S10A3-D3). In Figure S10A3, without drug treatment, the red
mitochondria were regularly distributed around the cell nucleus, and only a little green
fluorescence was detected. However, much more ROS in A549 cells with green fluorescence was
detected and distributed in the whole cell cytoplasm upon treated with 26, 29 or CDDP with the
same concentration. However, the merging images in Figure S10A4-D4 displayed that the
location of ROS and mitochondria did not overlap well. The Pearson's R value analyzed according
to the 2D intensity histogram by ImageJ lab program was shown in Figure S10A4-1 to D4-1,
from which we could see that ROS was irrelevant to mitochondria. Thus, the results suggested that
the production of ROS may not come from mitochondria itself.
To confirm whether intrinsic luminescence of compounds that would interfere with the
results of the luminescence of ROS tracker, we next tested the fluorescence emission spectra of 26,
29 in various solutions (λex = 315 nm，λex = 494 nm, λex = 535 nm). Not surprisingly, negligible
fluorescence emission spectra were obtained (Figure S11).
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Figure 9. After 24 h treatment with vehicle, cisplatin (5 μM), 26 (5 μM) and 29 (5 μM), A549 cells were incubated
with fluoresce probe DCFH-DA. Images were analyzed and recorded with fluorescence microscope.
The Decreasing of Mitochondrial Membrane Potential Induced by 29. As we have found
that 26 and 29 could induce the accumulation of ROS in cells, it will be interesting to investigate
whether the enhanced formation of ROS in cells could make the decrease of mitochondrial
[41a,
membrane potential (MMP)
44,45], which was the notable hallmark of classical
mitochondrial apoptosis pathway. Therefore, A549 cells were incubated with 26 (5 μM), 29 (5 μM)
or CDDP (5 μM), using equal DMF as negative control. After 24 h treatment, cells were stained
with JC-1 test kit , analysed and recorded with flow cytometry. The result of MMP was shown in
Figure 10, from which we could see that the △Ψm of cells treated with three compounds
mentioned above was decreased relative to control, indicating the damage of the cells which
eventually resulting in apoptosis.
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Figure 10. After 24 h treatment with vehicle, CDDP (5 μM), 26 (5 μM) and 29 (5 μM), A549 cells were stained
with JC-1 probe. Mitochondrial membrane potential (Δψm) were analyzed and recorded with flow cytometry.
Cell Morphology of Apoptosis Induced by 29. In order to investigate cell morphology of
apoptosis, A549 tumor cells were treated with various compounds: 26 (5 μM), 29 (5 μM), CDDP
(5 μM), and vehicle (DMF) for 24 h. Then the cells were co-stained with AO (6 μg/mL) and EB (6
μg/mL) and apoptotic morphology was analyzed with fluorescence microscope. The result was
shown in Figure 11, from which different nuclear morphology could be found. The cells with
different treatment was colored in green or orange, especially for that of cisplatin, 26 and 29
groups, whose brightness was enhanced, indicating the happening of apoptosis induced by these
three compounds. In addition, the tumor cells treated with 26 or 29 showed clear apoptotic state
with decreased cell density, reduced cell volume and concentrated cytoplasm, which was
consistent with that of cells treated with cisplatin at the same concentration. So, compound 29
could induce apoptosis just as 26 and CDDP did.
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Figure 11. Morphological changes of A549 cells induces by 29. After 24 h treatment with DMF, CDDP (5 μM),
26 (5 μM) and 29 (5 μM), A549 cells were stained with AO/EB. Results were anylyzed and recorded with a Nikon
Te2000 deconvolution microscope (magnification 200×).
26 and 29 Regulated Apoptotic Related Proteins. To elucidate the mechanisms involved in
potent apoptotic pathway induced by 26 (2 μM, 5 μM), 29 (2 μM, 5 μM) and CDDP (5 μM)
several apoptotic related proteins, including pro- or/and anti-apoptotic proteins, such as Bax and
Bcl-2 were analyzed with western blot assay. The results were shown in Figure 12A and 12B.
Treatment with 29 (5 μM) for 24 h induced down-regulation of anti-apoptotic protein Bcl-2 and
up-regulation of pro-apoptotic protein Bax in A549 cells, which deeply caused the decrease of
MMP studied above. Then the colapsed MMP actived the release of Cyt c and susequently led to
the activation of downstream caspase cascade. As was shown in Figure 12A and 12B, A549 cells
exposed to 29 showed statistically up-regulated level of cleaved-casepase 9 and cleaved-caspase 3
along with the down-regulated level of pro-caspase 9 and pro-caspase3, which aslo contributed to
the up-regulation of cleaved-parp level. It is noted that this protein-regulating activity of 29 was in
a dose-dependent manner. Not surprisingly, simialr effects were aslo observed when A549 cells
were treated with 26. Besides, as the positive control, CDDP (5 μM) also showed the same
activity to regulate the expression of the tested proteins, however, its potency was not comparable
to that of 26 or 29 at the concentration of 2 μM or 5 μM.
Since Prolyl-isomerase 1 (Pin1), overexpressed in many cancers such as prostate, breast,
and lung cancers, can also regulate the process of apoptosis [20], we detected the expression of Pin
1 in A549 cells after 24 h treatment of 26 (2 μM), 26 (5 μM), 29 (2 μM), 29 (5 μM) and CDDP (5
μM). As shown in Figure S12, the level of Pin 1 changed little when A549 cells were treated with
tested compounds, except for the treatment of higher concentration of 29 which caused moderate
down-regulation of Pin 1. The weak effect of the tested compounds on Pin 1 may be attributed to
the lack of effective binding domain in Pin 1. Thus, the obtained results clearly illustrated that 29
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could effectively bind to CRAF and disrupt the signaling transduction between RAS and CRAF
other than other protein, such as Pin1 -mediated pathway.
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Figure 12. After 24h treatment of A549 cells with 26 (5 μM) and 29 (5 μM), (A) Western blot analysis of Cyt c,
Bcl-2, Bax, Pro-caspase 9, caspase-9, Pro-caspase 3, caspase-3, Total-PARP and cleaved PARP were analyzed,
using β-actin antibody was used as reference control. (B) Expression levels of (A) shown as percentages.
In Vivo Evaluation of 29 on A549 Xenograft Models. On the basis of its superior in vitro
antitumor activities, 29 was selected for an in vivo xenograft mouse study. A549 cells (3×106 in
100 μL) were injected subcutaneously into the flank of the nude mice to establish tumor xenograft
models. Once the tumors reached to 150-250 mm3, those mice were divided randomly into four
different groups (three mice per group) and treated via injecting intravenously following: (1) 29 (6
μmol/kg, on days 0, 2 and 4), Rigosertib (6 μmol/kg, on days 0, 2 and 4), CDDP (6 μmol/kg, on
days 0, 2 and 4) and vehicle (equivalent volume of normal saline injection, on days 0, 2 and 4). As
shown in Figure 13, compound 29 caused statistically significant inhibition of tumor growth with
52.1% (percentage of inhibition ration [IR] value, Table S5) as compared with that of untreated
control. In addition, compound 29 (IR, 52.1%) showed better potential antitumor activity than that
of positive drug CDDP (IR, 36.7%) and Rigosertib (IR, 38.0%) at equal molecular dose. The
curves of Figure 13A and 13B showed the same trendance. It should be noted that though
Rigosertib showed strong antitumor activity at nanomolar concentrations in vitro, it only exhibited
moderate tumor growth inhibition in vivo. This may be due to the low dosage used in the study [16,
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34]. Importantly, no evident side effects such as body weight loss or animal death were observed in
groups treated with compound 29 and Rigosertib, as shown in Figure 13C. On the contrary, the
CDDP-treated group showed serious toxicity as reflected by the big body weight loss, especially
during the days of 6-12 after intravenous injection of CDDP. These results showed that compound
29 was efficacious against A549 tumor xenografts with good safety.
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Figure 13. Antitumor activity of compound 29 on A549 xenograft models. Nude mice implanted with A549 cells
in the flank were dosed via injecting intravenously of 29 (6 μmol/kg), Rigosertib (6 μmol/kg), CDDP (6 μmol/kg),
vehicle (equivalent volume of normal saline injection) on days 0, 2 and 4. The mice were sacrificed and made
mathematical statistics. (A) Tumor pictures obtained after sacrifice of the mice treated with 29, Rigosertib, CDDP
and vehicle. (B) The RTV (relative tumor volume) (± SEM) is graphed with error bars representing the standard
deviation. (c) The average body weights (± SEM) were plotted after sacrifice of the mice treated with 29,
Rigosertib, CDDP and vehicle.
CONCLUSION
Nine styrylbenzylsulfone derivatives generated from the platinum-based modification of the
side chain of Rigosertib were designed, synthesized, and tested for their biological activity. The
results of MTT activity test in vitro showed that all of the synthesized target compounds showed
good anti-proliferative activity against the tested cancer cells; among them compound 29 was
most active with the potency higher than the positive control cisplatin and Rigosertib.
Significantly, compound 29 could also inhibit the multidrug resistant tumor cells at nanomolar
concentration. This unique merit of 29 may be attributed to the enhancement of cellular uptake as
well as its multifunctional antitumor mechanism. As our studies revealed that 29 could effectively
release Rigosertib and the corresponding Pt(II) moiety upon the reducing substances, thus the
active components could exert the antitumor effect via different pathways. Indeed, we have found
that 29 could, on the one hand, disturb signaling transduction between RAS and CRAF via
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directly binding to CRAF protein, and finally induce downregulation of phosphor-MEK and
phosphor-ERK; On the other hand, 29 could also induce apoptosis and clearly arrest the A549
cells at the G2/M phase. Furthermore, the study of apoptotic pathway showed that massive
reactive oxygen species was produced when the cancer cell was treated with 29. All of these
performances of 29 are quite similar to that of platinum-based agents, suggesting that 29
maintained the cisplatin-like activity. Thus, we can summarize that 29 could inhibit the
proliferation of tumor cells via two different pathways: the RAF-MEK-ERK pathway and the
platinum-based cytotoxicity pathway, as outlined in Figure 14. Due to this multifunctional
character, 29 could efficiently inhibit tumor growth in A549 xenograft model, and no obvious side
effects were observed during the test. In all, these excellent performances in the pharmacological
tests both in vitro and in vivo made compound 29 possible for ‘efficient and hypotoxic’
antineoplastic candidate.
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Figure 14. The proposed antitumor action mode of 29.
EXPERIMENTAL SECTION.
(1) Materials and Instruments. All chemical reagents and solvents were purchased
commercially from Aladdin, Energy Chemical or Adamas-beta and used without further
purification. Potassium tetrachloroplatinate(II) was purchased from commercial sources (Lingfeng
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Chemical Ltd.). H and 13C NMR spectra were obtained from with Bruker 300, 400 or 600 MHz
spectrometer in DMSO-d6 or CDCl3 and tetramethylsilane (TMS) was used as reference. Mass
spectra were measured by an Agilent 6224 ESI/TOF MS instrument. All of the compounds
submitted for biological studies were at least 95% purity by RP-HPLC on ODS column (250×4.6
mm, 5 μm) with an eluent of acetonitrile/water (45:55, V/V) (0.1% trifluoroacetic acid in water).
Besides all cells used in this article were obtained from KeyGEN BioTECH Corp. Besides,
Mitochondrion Red (KGMP0071) and ROS tracker were obtained from KeyGEN BioTECH Corp.
Bax, Bcl-2, Cytochrome c, Pro-caspase-3/9, Cleaved-casepase-3/9, Total-PARP and
Cleaved-PARP antibodies were purchased from Imgenex, USA. Antibodies directed against
p-MEK, MEK, p-ERK, ERK, Pin 1 (#3722), p53 were purchased from Cell Signaling Technology.
CRAF antibody was purchased from Affinity Biosciences, whereas RAS antibody, BRAF
antibody (ab124794) was from Abcam. Secondary antibody (1:2000) labeled with horseradish
peroxidase was came from Santa Cruz Biotechnologies
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(2) Synthesis and Characterization of Intermediates.
Compound 1. To a solution of compound SM1 (3.01 g, 18.01 mmol) and anhydrous K2CO3
(6.22 g, 45.03 mmol) in DMF (30 mL) was added CH3I (3.83 g, 27.02 mmol) at room temperature
and stirred overnight at the same temperature. Progress of the reaction was monitored with TLC.
After completion of the reaction, solvent was evaporated under reduced pressure to obtain crude
yellow solid. After that, DCM (300 mL) was added to dissolve the residue and washed with NaOH
solution (1N), sat. NaCl (100 mL）. The organic layer was dried with anhydrous Na2SO4 and
concentrated in vacuum to give pale white solid 3.05 g. Yield: 93.7%. 1H NMR (500 MHz, CDCl3)
δ 9.94 (s, 1H), 8.34 (d, J = 2.0 Hz, 1H), 8.09 (dd, J = 8.7, 2.0 Hz, 1H), 7.25 (d, J = 8.7 Hz, 1H),
4.07 (s, 3H).
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Compound 2. To a solution of compound 1 (1.01 g, 5.58 mmol) in MeOH (20 ml) at 0 C
was added NaBH4 (0.27g, 7.25 mmol) gradually and stirred at the same temperature for 20 min.
Progress of the reaction was monitored with TLC. The reaction was worked up by quenched with
HCl solution (1 N) at 0 oC and removed the solvent to obtain white solid under reduced pressure.
After that, DCM (300 ml) was added to dissolve the residue, washed with sat. NaCl (3×100 mL).
The combined organic layer was dried with anhydrous Na2SO4 and concentrated in vacuum to
give white solid 1.02 g. Yield: 100%. 1H NMR (500 MHz, CDCl3) δ 7.83 (d, J = 2.1 Hz, 1H), 7.54
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(dd, J = 8.6, 2.1 Hz, 1H), 7.07 (d, J = 8.6 Hz, 1H), 4.68 (s, 2H), 3.96 (s, 3H), 2.06 (s, 1H).
Compound 3. To a solution of compound 2 (0.50 g, 2.73 mmol) in DCM (15 mL) at 0 oC was
added PBr3 (1.10 g, 4.10 mmol) gradually and stirred at the same temperature for 2 h. The reaction
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(300 mL) was added to dissolve the residue, washed with sat. NaCl (3×100 mL). The organic
layer was dried with anhydrous Na2SO4 and concentrated in vacuum to give white solid 0.67 g.
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Yield: 99.7%. H NMR (500 MHz, CDCl3) δ 7.88 (d, J = 2.2 Hz, 1H), 7.57 (dd, J = 8.7, 2.2 Hz,
1H), 7.07 (d, J = 8.7 Hz, 1H), 4.46 (s, 2H), 3.97 (s, 3H).
Compound 4. To a solution of NaOH (162.4 mg, 4.06 mmol) in MeOH (15 mL) at 0 oC was
added TGA (224.6 mg, 2.43 mmol) gradually and stirred at the same temperature for 10 min. Then
compound 3 (0.50 g, 2.03 mmol) in 10 MeOH (10 mL) was dropped slowly into the solution. The
reaction was monitored with TLC. After completion of the reaction, the solvent was removed
under reduced pressure to obtain pale yellow solid. After that, water (50 mL) was added to
dissolve the residue and adjusted the PH to 4-5 with HCl (1N) solution. The precipitate was
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filtered to obtain pale yellow solid 469.8 mg. Yield: 90.0%. H NMR (500 MHz, DMSO-d6) δ
12.53 (s, 1H), 7.82 (d, J = 2.1 Hz, 1H), 7.60 (dd, J = 8.6, 2.1 Hz, 1H), 7.32 (d, J = 8.6 Hz, 1H),
3.91 (s, 3H), 3.83 (s, 2H), 3.14 (s, 2H). HR-MS (m/z) (ESI): calcd for C10H11NO5S [M-H]:
256.0358; Found: 256.0359.
Compound 5. To a solution of compound 4 (257.3 mg, 1.94 mmol) in AcOH (15 mL) at
room temperature was added H2O2 (661.0 mg, 19.40 mmol) and stirred at the 50 ℃ for 3h. The
reaction was monitored with TLC. After the completion of the reaction, the solvent was removed
under reduced pressure to attain crude product. The residue was further purified with silica gel
chromatography with the eluent of DCM and MeOH (100:0-30:1) to give pale yellow solid 413.0
mg. Yield: 73.7%. 1H NMR (500 MHz, DMSO) δ 13.48 (s, 1H), 7.92 (d, J = 2.1 Hz, 1H), 7.68 (dd,
J = 8.7, 2.1 Hz, 1H), 7.43 (d, J = 8.7 Hz, 1H), 4.68 (s, 2H), 4.20 (s, 2H), 3.95 (s, 3H). HR-MS (m/z)
(ESI): calcd for C10H11NO5S [M-H]: 288.0250; Found 288.0254.
Compound 7. To a solution of compound 5 (3.0 g, 10.37 mmol) in toluene (30 mL) at 40 ℃
was added piperidine (823.8 μL, 8.30 mmol) and stirred at the same temperature for 10 min. Then
benzoic acid (1.3 g, 10.37 mmol) and compound 6 (2.2 g, 11.42 mmol) were added to the solution
and heated to reflux overnight. Progress of reaction was monitored with TLC. After the
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completion of the reaction, the solution was cooled to room temperature and filter to obtain yellow
crude product. The precipitate was recrystallized with MeOH (20 mL) to get yellow solid 1.5 g.
Yield: 34.1%.1H NMR (300 MHz, DMSO) δ 7.88 (s, 1H), 7.64 (d, J = 8.6 Hz, 1H), 7.51 (d, J =
15.6 Hz, 1H), 7.38 (d, J = 8.6 Hz, 1H), 7.09 (d, J = 15.6 Hz, 1H), 6.29 (s, 2H), 4.54 (s, 2H), 3.93
(s, 3H), 3.85 (s, 9H).
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Compound 8. To a solution of compound 7 (1.80 g, 4.25 mmol) in EtOH (20 mL) was added
H2O (5.0 mL), NH4Cl (136.4 mg, 2.55 mmol), Fe（1.43g，25.50 mmol）and heated to 70 oC for
4 h. Progress of reaction was monitored with TLC. After the completion of the reaction, the
insoluble content was removed by filtration. Solvent of the reaction was finally evaporated under
reduced pressure to give yellow solid 1.5 g. Yield: 88.2%. 1H NMR (300 MHz, CDCl3) δ 7.59 (d,
J = 15.6 Hz, 1H), 7.08 (d, J = 15.6 Hz, 1H), 6.75 (d, J = 8.1 Hz, 1H), 6.64 (s, 1H), 6.50 (d, J = 8.1
Hz, 1H), 6.29 (s, 2H), 4.76 (s, 2H), 4.20 (s, 2H), 3.85 (s, 9H), 3.75 (s, 3H). HR-MS (m/z) (ESI):
calcd for C19H23NO6S [M+H]: 394.1246; Found 394.1230.
Compound 11. (1) To a solution of compound 9 (612.9 mg, 4.21 mmol) in anhydrous DCM
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(10 mL) was added oxalyl chloride (1.8 mL) at 0 C and stirred at room temperature overnight.
After completion of the reaction, the solvent was removed under reduced pressure to obtain
colorless oil. The crude product was used for next step without further treatment. (2) To a solution
of compound 8 (1.50 g, 3.83 mmol) in anhydrous DCM (15 mL) at 0 oC was added the compound
got previously in DCM (5 mL) at the same temperature and stirred at room temperature for 10 min.
After that, Et3N (507.8 mg, 699 μL,) in DCM (5 mL) was added to the solution and stirred for
another 2 h. After completion of the reaction, the residue was dissolved in DCM (300 ml) and
washed with water (100 mL), sat. NaCl (3×100 mL). The organic layer was dried with anhydrous
Na2SO4 and concentrated under reduced pressure. The crude product was further purified with
silica gel chromatography with the eluent of DCM and PE (2:1) to get pale yellow solid (1.62 g).
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Yield: 85.0%. H NMR (300 MHz, DMSO-d6) δ 9.04 (s, 1H), 7.98 (s, 1H), 7.59 (d, J = 15.7 Hz,
1H), 7.11 (d, J = 15.7 Hz, 1H), 7.04 (s, 1H), 7.01 (d, J = 8.4 Hz, 1H), 6.29 (s, 2H), 4.35 (s, 2H),
3.86 (s, 9H), 3.83 (s, 3H), 3.60 (s, 3H), 2.46 – 2.29 (m, 4H), 1.86-1.76 (m, 2H). HR-MS (m/z)
(ESI): calcd for C25H31NO9S [M+H]: 522.1792; Found 522.1721.
Compound 12. Compound 8 (751.0 mg, 1.91 mmol), compound 10 (160.2 mg, 2.10 mmol),
oxalyl chloride (1.0 mL), Et3N (249.5 mg, 344 μL) and DCM 25 mL were used following the
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procedure described for synthesis of compound 11, and attained pale yellow solid (0.85 g). Yield:
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84.9%. H NMR (400 MHz, CDCl3) δ 8.37 (s, 1H), 7.84 (d, J = 15.6 Hz, 1H), 7.72 (s, 1H), 7.15
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2H), 3.88 (s, 3H), 3.84-3.83 (m, 9H), 3.67 (s, 3H), 2.40 - 2.35 (m, 4H), 1.74 - 1.69 (m, 4H).
HR-MS (m/z) (ESI): calcd for C26H33NO9S [M+H]: 536.1954; Found 536.1902.
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Compound 13. To a solution of compound 10 (1.2 g, 2.30 mmol) in EtOH (10 mL) and pure
water (5 mL) was added LiOH•H2O (243.6 mg, 5.75 mmol) at room temperature and stirred at
room temperature overnight. Progress of the reaction was monitored by TLC. After the
completion of reaction, the solvent was removed under reduced pressure to obtain pale yellow
solid. The residue was dissolved with 30 mL pure water and adjusted the PH of the solution to 5
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with HCl solution (1N), then filtered to give pale yellow solid (1.0 g). Yield: 91.0%. H NMR
(500 MHz, DMSO-d6) δ 9.02 (s, 1H), 7.98 (s, 1H), 7.59 (d, J = 15.7 Hz, 1H), 7.10 (d, J = 15.7 Hz,
1H), 7.05 (d, J = 8.2 Hz, 1H), 7.01 (d, J = 8.2 Hz, 1H), 6.29 (s, 2H), 4.34 (s, 2H), 3.85 (s, 9H),
3.82 (s, 3H), 2.43-2.37 (m, 2H), 2.27-2.25 (m, 2H), 1.79-1.76 (m,2H). HR-MS (m/z) (ESI): calcd
for C24H29NO9S [M+H]: 508.1436; Found 508.1460.
Compound 14. Compound 12 (850.0 mg, 1.58 mmol), LiOH•H2O (166.6 mg, 3.97 mmol),
EtOH (10 mL) and pure water (5 mL) were used following the procedure described for synthesis
of compound 11, and got pale yellow solid 746.5 g. Yield: 90.0%. 1H NMR (300 MHz, DMSO-d6)
δ 11.98 (s, 1H), 9.03 (s, 1H), 7.99 (s, 1H), 7.59 (d, J = 15.7 Hz, 1H), 7.11 (d, J = 15.7 Hz, 1H),
7.05 (d, J = 8.5 Hz, 1H), 7.01 (d, J = 8.5 Hz, 1H), 6.30 (s, 2H), 4.34 (s, 2H), 3.86 (s, 9H), 3.83 (s,
3H), 2.43-2.33 (m, 2H), 2.26-2.22 (m, 2H), 1.60-1.49 (m, 4H). HR-MS (m/z) (ESI): calcd for
C25H31NO9S, [M-H]: 520.1647; Found 520.1751.
(3) General Procedures for Preparing Compounds 18-23.
To a solution of compound 13 or 14 (0.33 mmol) in DMF (3 ml) was added compound
TBTU (0.50 mmol) and stirred at room temperature for 5 min. Then, Et3N (68.0 ul, 0.50 mmol)
was added to the solution under the protection of nitrogen and stirred for 5min at the same
temperature. After that, compound 15, 16 or 17(0.36 mmol) was added to the solution under the
protection of nitrogen and stirred for 2 h at 35 ℃. 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 (150 mL). The organic layer was dried with anhydrous Na2SO4 and concentrated
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under reduced pressure to obtain crude product. The crude product was further purified with silica
gel chromatography with the eluent of DCM and MeOH (60:1-20:1) to give target product.
Compound 18. Yield: 36.2%.1H NMR (300 MHz, DMSO-d6) δ 9.58 (s, 1H), 8.94 (s, 1H),
8.13 (s, 1H), 8.02 (s, 1H), 7.78 (s, 1H), 7.59 (d, J = 15.6 Hz, 1H), 7.41 (s, 1H), 7.10 (d, J = 15.6
Hz, 1H), 7.03-6.99 (m, 2H), 6.29 (s, 2H), 4.34 (s, 2H), 3.85 (s, 9H), 3.82 (s, 3H), 2.83-2.71 (m,
2H), 2.44 – 2.39 (m, 2H), 2.32 – 2.28 (m, 2H), 2.21 – 2.16 (m, 1H), 2.08 – 2.04 (m, 1H), 1.81 –
1.76 (m, 2H), 1.56-1.49 (m, 3H), 1.33-1.25 (m, 1H), 1.11-1.03 (m, 2H).13C NMR (75 MHz,
DMSO-d6) δ 183.39, 171.29, 163.99, 160.89 (C×2), 149.89, 133.37, 127.68, 127.50, 124.82,
123.98, 120.88, 111.02, 102.88, 91.40 (C×2), 63.71, 62.91, 60.06, 56.55 (C×2), 56.23, 56.12,
37.12, 35.82, 31.36, 31.30, 24.16 (C×2), 22.28. HR-MS (m/z) (ESI): calcd for C30H42Cl3N3O9PtS,
[M-H]: 920.1480; Found 920.1414.
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25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
58
59
60
Compound 19. Yield: 33.6%.1H NMR (300 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.99 (s, 1H),
8.12 (s, 1H), 8.00 (s, 1H), 7.77 (s, 1H), 7.59 (d, J = 15.5 Hz, 1H), 7.41 (s, 1H), 7.11 (d, J = 15.5
Hz, 1H), 7.02 (s, 2H), 6.30 (s, 2H), 4.34 (s, 2H), 3.85 (s, 9H), 3.83 (s, 3H), 2.39-2.35 (m, 2H),
2.30-2.25 (m, 2H), 2.21-2.02 (m, 3H), 1.55-1.48 (m, 7H), 1.30-1.24 (m, 1H), 1.16 – 0.96 (m, 3H).
13C NMR (75 MHz, DMSO-d6) δ 183.57, 171.94, 164.28, 161.31（C×2), 149.97, 133.34, 127.70,
127.45, 124.91, 124.04, 121.09, 111.17, 103.05, 91.41 (C×2), 63.91, 62.62, 60.35, 56.55 (C×2),
56.23, 56.12, 37.43, 36.18, 31.37, 31.29, 25.53 (C×2), 25.15, 24.00. HR-MS (m/z) (ESI): calcd for
C31H44Cl3N3O9PtS, [M-H]: 934.1282; Found 934.1586.
Compound 20. Yield: 39.0%.1H NMR (300 MHz, DMSO-d6) δ 8.98 (s, 1H), 8.32 (s, 3H),
8.03 (d, J = 28.2 Hz, 1H), 7.59 (d, J = 15.7 Hz, 2H), 7.14 (t, J = 20.0 Hz, 1H), 7.02 (s, 2H), 6.29 (s,
2H), 4.34 (s, 2H), 3.85 (s, 9H), 3.82 (s, 3H), 2.66 - 2.55 (m, 2H), 2.39 - 2.22 (m, 4H), 2.11 -1.92
(m, 2H), 1.801-1.68 (m, 2H), 1.55-1.30 (m, 4H), 1.18-1.04 (m, 2H).13C NMR (75 MHz, DMSO-d6)
δ 180.44, 171.38, 164.11, 163.75, 161.41 (C×2), 160.73, 150.00, 133.38, 127.76, 124.95,
124.00(C×2), 121.07, 111.27, 103.01, 91.40 (C×2), 62.00, 61.78, 60.27, 56.52 (C×2), 56.22, 56.10,
36.40, 35.77, 31.40, 31.04, 23.97 (C×2), 21.95. HR-MS (m/z) (ESI): calcd for C32H42ClN3O13PtS,
[M-H]: 938.1700; Found 938.1862.
Compound 21. Yield: 39.0%.1H NMR (300 MHz, DMSO-d6) δ 9.61 (s, 1H), 8.99 (s, 1H),
8.12 (s, 1H), 8.00 (s, 1H), 7.77 (s, 1H), 7.59 (d, J = 15.5 Hz, 1H), 7.41 (s, 1H), 7.11 (d, J = 15.9
Hz, 1H), 7.02 (s, 2H), 6.30 (s, 2H), 4.34 (s, 2H), 3.85-3.82 (m, 9H), 3.83 (s, 3H), 2.35-2.30 (m,
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