Pomalidomide hybrids act as proteolysis targeting chimeras: Synthesis, anticancer activity and B-Raf degradation

Article abstract of DOI:10.1016/j.bioorg.2019.03.035

As the first intracellular signaling molecule and the most frequently mutated oncogene, B-Raf represents an important target in cancer therapy. Here we report several pomalidomide hybrids acting as proteolysis targeting chimeras (PROTACs) for the degradat

Full text of DOI:10.1016/j.bioorg.2019.03.035

Accepted Manuscript
Pomalidomide hybrids act as proteolysis targeting chimeras: Synthesis, anti-
cancer activity and B-Raf degradation
Hong Chen, Feihong Chen, Sinan Pei, Shaohua Gou
PII:
S0045-2068(19)30093-8
DOI:
https://doi.org/10.1016/j.bioorg.2019.03.035
YBIOO 2861
Reference:
Bioorganic Chemistry
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3 March 2019
14 March 2019
Please cite this article as: H. Chen, F. Chen, S. Pei, S. Gou, Pomalidomide hybrids act as proteolysis targeting
chimeras: Synthesis, anticancer activity and B-Raf degradation, Bioorganic Chemistry (2019), doi: https://doi.org/
10.1016/j.bioorg.2019.03.035
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Pomalidomide hybrids act as proteolysis targeting chimeras:
Synthesis, anticancer activity and B-Raf degradation
Hong Chen1, Feihong Chen1, Sinan Pei, Shaohua Gou*
Pharmaceutical Research Center and School of Chemistry and Chemical Engineering, Jiangsu
Province Hi-Tech Key Laboratory for Biomedical Research, Southeast University, Nanjing
211189, PR China
* Corresponding Author. E-mail address: sgou@seu.edu.cn (S. Gou).
1 These authors contributed equally.
Abstract
As the first intracellular signaling molecule and the most frequently mutated oncogene,
B-Raf represents an important target in cancer therapy. Here we report several pomalidomide
hybrids acting as proteolysis targeting chimeras (PROTACs) for the degradation of B-Raf. Due to
its high expression of B-Raf, MCF-7 cells are sensitive to these compounds. Among them,
compound 2 can effectively kill cancer cells via inducing cells apoptosis. As a B-Raf degrader,
compound 2 can accelerate the degradation of B-Raf by recruiting ubiquitin-proteasome system,
and further affects the expression of Mcl-1, a downstream protein of B-Raf. The anticancer
mechanism of compound 2 is quite different from its mother compound and cancer cells seem to
be more sensitive to the degrader, hinting that degradation of B-Raf by PROTAC is a potential
way for cancer treatment.
Keywords: Pomalidomide, Proteolysis targeting chimeras, B-Raf, Anticancer activity.
1. Introduction
Immunomodulatory drugs (IMiDs, Fig.1) are a class of orally available anticancer agents
derived from thalidomide, which eventually turned out to be responsible for infants with
phocomelia when used as a sedative for pregnant women[1,2]. Despite the serious defect,

thalidomide was serendipitously found to be potent in the treatment of multiple myeloma (MM)
due to its immune-modulation, anti-angiogenic and anti-inflammatory effects[3,4]. Until 2010, Ito
and co-workers successfully identified cereblon (CRBN) as the thalidomide-binding protein[5].
CRBN is a subunit of the cullin-4-containing E3 ubiquitin ligase complex CRL4, which can
promote the neighboring protein to degrade via ubiquitination[6].
Fig. 1. Three immunomodulatory drugs: thalidomide, lenalidomide and pomalidomide.
As the third generation IMiDs, pomalidomide approved by US FDA in 2013 for patients with
MM who have received at least two prior therapies was more effective and less toxic than
thalidomide and lenalidomide[7,8]. Like thalidomide, pomalidomide has the comparable affinity
for CRBN[9]. However, the difference is that, the presence of the substituent at benzene provides
the ability to mediate the interaction of CRBN with neo-substrates that are not typically targeted
by CRL4[6,10,11]. These findings opened a new field of selective ubiquitination and degradation
of specific targets by regulating the interaction of CRBN-based E3 ubiquitin ligase with target
protein.
Proteolysis targeting chimera (PROTAC) is a heterobifunctional molecule consisting of an E3
ligase-binding group and another ligand binging to the protein of interest (POI), which are
conjugated via a linker[12,13]. A number of small molecular-based PROTACs using thalidomide
derivative as the recruiter of E3 ubiquitin ligase had been developed[14-16]. As shown in Fig.2, a
conjugate (DAS-6-2-2-6-CRBN) of pomalidomide with dasatinib, a chemotherapy drug used to
treat chronic myelogenous leukemia (CML) and acute lymphoblastic leukemia (ALL) via
targeting oncogenic BCR-ABL, was designed by Crews and co-workers, the compound could
effectively induce the degradation of BCR-ABL despite some loss in affinity for the target[17].
Another representative PROTAC, named as dBET1, synthesized by Bradner and co-workers could

selectively mediate the degradation of BET protein through a CRBN-dependent manner[18]. Our
group also developed a CK2 targeting PROTAC that could degrade CK2 protein in a dose and
time dependent manner, and kept CK2 at a low basal level by recruiting ubiquitin-proteasome
system[19]. As an emerging and promising approach for drug discovery, PROTAC strategy has
been accelerated greatly in recent years, and it is worthy of exploring novel PROTACs that target
different protein for degradation.
Fig. 2. Several reported PROTACs.
As the most frequently mutated oncogene in Raf kinase superfamily, B-Raf represents an
important target in cancer, in part because of its overexpression in most of tumor cells, and also
for it is the first intracellular signaling molecule that is responsible for cancer cell proliferation.
[20-22]. Rigosertib (RGS), a small molecule anticancer candidate, was firstly developed as a
non-ATP-competitive inhibitor of PLK1[23-25]. Until recent years, it was finally identified as a
Ras-mimetic and the direct target of RGS was the Ras-binding domains (RBDs) of Raf
protein[26,27]. More importantly, the most common B-Raf mutations, such as the V600E
mutation, located in the kinase domain (KD) rather than RBDs. This means that, under most of
conditions, the interaction of RGS with B-Raf was not influenced by the mutations. In contrast,
most of the kinase inhibitors of B-Raf were susceptible to the mutations resulting in drug
resistance[28]. In this study, RGS was selected as the ligand that bound to the POI and several
PROTACs targeting B-Raf kinase were synthesized and studied on their anticancer activity and

the ability of inducing protein degradation.
2. Results and discussion
2.1. Design and synthesis
It has been reported that RGS (10) binds B-Raf RBD in two different orientations, but
it occupies the same interface. The intermolecular interaction between RGS and B-Raf is
primarily hydrophobic in nature, and the contacts are primarily from the trimethoxy styryl
portion and the methoxy benzyl sulfone portion of RGS (Fig.3). Although the glycine
portion of RGS can potentially make a salt link with the guanidino group of R188 amino
acid, the portion is exposed in solvent, and the ionic reaction is just observed in one of the
binding orientations. Moreover, conjugating of RGS with biotin via the carboxyl in the
glycine portion did not influence the interaction between RGS and B-Raf protein, and the
biotin-RGS conjugate also could effectively bind to B-Raf [26]. Based on the above
findings, the trimethoxy styryl portion and the methoxy benzyl sulfone portion of RGS
were strictly retained in the design of the target compounds, and the selection of the linker
between RGS moiety and pomalidomide was variable. Due to the protocol that ligands that
bind the target anywhere on its surface can be used to make PROTACs that degrade it, this
design strategy would be feasible[16,17,19].
Fig. 3. Structural basis for the design of target compounds.
Compounds 1-4 were synthesized based on the protocol as possible PROTACs
(
Scheme 1). Pomalidomide (
5
), RGS (10) and its intermediate (
9) were prepared according
to a method reported previously[19,23].
5
was firstly used to react with bromoacetyl

chloride and sodium azide subsequently to get
with Pd/C catalyst was carried out to produce
esterification with corresponding acyl chloride followed by hydrolysis. Finally, compound
was synthesized through the reaction of with in methanol solution under refluxing
condition in the presence of sodium acetate, and compounds and were obtained
6
and
7. Then, catalytic hydrogenation of 7
8
. Besides, 11 and 12 were obtained via
1
6
9
2
,
3
4
through the esterification of
8
with 10
,
11 or 12, respectively. All the target compounds 1-4
1
were structurally characterized by elemental analysis, H-NMR and 13C-NMR together
with ESI-MS spectrometer.
Scheme 1. Synthesis of the target compounds 1-4. (a) bromoacetyl chloride, acetone,
reflux for 24 h; (b) NaN3, acetone, reflux for 24 h; (c) H2, Pd/C, rt for 6 h; (d)
acetate, methanol, reflux; (e) , TBTU, TAE, DMF, 50 C for 24 h; (f) ethyl
4-chloro-4-oxobutanoate or ethyl 5-chloro-5-oxopentanoate, DCM, reflux for 24 h; (g) i:
6, sodium
8


LiOHH2O, ethanol/H2O, rt, overnight; ii: 1 M HCl; (h) 8, TBTU, TEA, DMF, 50 C for
24 h.
2.2. The level of B-Raf in different cell lines
As B-Raf, the primary target of RGS moiety, was closely related to the tumorigenesis,
its expression level in several cell lines was firstly analyzed before the study of in vitro
anticancer activity. As shown in Fig.4, human MCF-7 breast cancer cell line had the
highest level of B-Raf in the tested five cell lines, while the expression level of B-Raf in
another human breast cancer cell line MDA-MB-231 was apparently lower than that in
MCF-7 cells. In human HepG2 hepatoma cells, the expression level of B-Raf was surpassed
only by MCF-7 cells. And human normal LO2 liver cells also expressed high level of B-Raf
which was in third place in the tested cell lines, and this maybe owe to the rapid
regeneration capacity and higher proliferation rate of hepatocytes[29], as well as the
irreplaceable role of B-Raf in cell proliferation[30]. It was noteworthy that murine B16
melanoma cells expressed a small amount of B-Raf although its mutations were more common in
certain clinical and pathologic subsets of melanoma[31].
Fig. 4. (A) Western blot and (B) statistic analysis of the expression level of B-Raf in different cell
lines based on three independent experiments: human breast cancer cell lines (MCF-7 and
MDA-MB-231), human hepatoma cell line (HepG2), murine melanoma cell line (B16) and human
normal liver cell line (LO2).
2.3. In vitro anticancer activity

In vitro cytotoxicities of compounds 1-4 against the above five cell lines were
measured by MTT assay together with pomalidomide ( ), RGS (10) and its derivative (
5
9)
as positive references. As shown in Table 1, MCF-7 cancer cells with the highest level of
B-Raf were the most sensitive to all the tested compounds targeting B-Raf except for
compound 4. Besides, the cytotoxicities of all tested compounds targeting B-Raf toward
human MCF-7 and MDA-MB-231 breast cancer cells were dependent on the level of B-Raf,
and the higher the B-Raf level in the cancer cells, the better the anticancer activities of the
compounds. On the other hand, we could find that all tested compounds targeting B-Raf
possessed the selectivity between cancer cells and normal cells when compared their
cytotoxicities toward human HepG2 hepatoma cells with that toward human normal LO2 liver
cells. The selectivity may be due to the fact that ubiquitin-proteasome system (UPS), as the action
foundation of PROTAC, was more active in cancer cells[19]. It was worth noting that, although
the level of B-Raf in B16 cells was obviously lower than that in MDA-MB-231 cells, there
was no significant difference in the cytotoxicities of tested compounds. And this in part
because B-Raf mutations were closely related to the occurrence and development of
melanoma. All above results implied that both the level of B-Raf and the cell types have great
impacts on the in vitro anticancer activity of our designed PROTACs. Notably, compound
with an IC50 value of 2.70 μM toward MCF-7 cells could more effectively kill cancer cells
than its mother compounds and 10, and this presented the potential of compound for
) exhibited almost no
2
9
2
the treatment of breast cancer. In addition, pomalidomide (
cytotoxicity toward all the tested cell lines.
5
Table 1. Cytotoxicities of tested compounds toward different cell lines.
IC50 (μM)
Compound
MCF-7
MDA-MB-231
23.12 ± 2.04
21.21 ± 1.95
36.96 ± 3.22
46.23 ± 4.37
21.33 ± 1.96
HepG2
LO2
B16
12.80 ± 0.98
2.70 ± 0.19
6.14 ± 0.50
38.09 ± 3.45
4.17 ± 0.31
19.25 ± 1.73
18.70 ± 1.65
11.91 ± 1.02
14.58 ± 1.31
10.59 ± 0.94
> 200
23.67 ± 2.09
22.68 ± 2.03
28.53 ± 2.65
29.29 ± 2.74
14.49 ± 1.28
1
2
3
4
9
41.11 ± 3.70
41.43 ± 3.72
> 200
35.57 ± 2.81

4.46 ± 0.34
> 200
25.26 ± 2.28
> 200
17.72 ± 1.58
> 200
40.27 ± 3.65
> 200
12.75 ± 1.01
> 200
10
5
IC50 is the drug concentration effective in inhibiting 50% of the cell growth measured by the MTT assay
after 72 h of drug exposure expressed as the mean value ± standard deviation of three independent
experiments.
2.4. Degradation of B-Raf
Degradation of B-Raf induced by compound 2 was investigated in MCF-7 cells using
pomalidomide (5) and compound 9 as references. As shown in Fig. 5, pomalidomide had no
influence on the level of B-Raf even at a high concentration of 10 μM, because there was no
interaction between pomalidomide and B-Raf. Although compound 9 could directly target B-Raf,
it only could disrupt the Ras-Raf interaction and also had little effect on the level of B-Raf.
Obviously, the level of B-Raf was dramatically decreased under the treatment of compound 2,
only 28.3% was left at a concentration of 5 μM and 19.0% at 10 μM. The results implied that
compound 2 could effectively induce the degradation of B-Raf. It was well known that oncogenic
B-Raf can promote the transcriptional upregulation of Mcl-1[32], as the downstream protein of
B-Raf, its level was also detected. Results showed that pomalidomide also could not obviously
influence the level of Mcl-1, in contrast, compounds 2 and 9 had great influences on the
expression of Mcl-1. Because of the disruption of Ras/Raf pathway, modest downregulation of
Mcl-1 was observed under the treatment of compound 9 at a concentration of 5 μM, when its
concentration increased to 10 μM, the level of Mcl-1 was remarkably decreased. Whereas,
compound 2 could dramatically inhibit the expression of Mcl-1 even at the low concentration of 5
μM. Consequently, compound 2 as an effective PROTAC could induce the degradation of B-Raf
and impact the expression of Mcl-1.

(B)
Fig. 5. (A) Western blot image of B-Raf and Mcl-1 in MCF-7 cells after treatment with
pomalidomide (
5 μM or 10 μM. (B) Statistic analysis of B-Raf or Mcl-1 level based on three independent
experiments
5), compounds 2 or its mother compound 9 for 24 h at the concentration of
.
2.5. Mechanism of B-Raf degradation
Ubiquitin-proteasome system (UPS) was the action foundation of protein degradation
induced by PROTAC, and proteasome as a multicatalytic enzyme could mediate many
cellular regulatory signals by degrading ubiquitinated proteins. To confirm the mechanism
of the degradation of B-Raf, the first proteasome inhibitor bortezomib (BTZ, originally
PS-341) was used for the joint research with compound
2[33]. As shown in Fig. 6,
compound without the pre-treatment of BTZ could degrade B-Raf effectively. After the
2
pre-treatment with BTZ, the degradation of B-Raf was dose-dependently suppressed.
Whereas, BTZ had little influence on the level of B-Raf. All above results implied that

compound
2 degraded B-Raf through the ubiquitin-proteasome pathway (UPP), and that
was completely in conformity with the working principles of PROTAC as expected.
Fig. 6. (A) The levels of B-Raf after pre-treatment with BTZ at different concentrations for
1 h and then treated with compound
2 (5 μM) for 24 h. (B) Statistic analysis of B-Raf level
based on three independent experiments.
2.6. Apoptosis study
According to the results of cytotoxicity assay, apoptotic analysis of compound
against MCF-7 cells were carried out by an Annexin V-FITC/PI assay using its mother
compound as reference. As showed in Fig. 7, compounds and owned similar capacity
of inducing MCF-7 cells apoptosis at their IC50 value respectively for 72 h. When the
concentration increased to 20 μM, compound achieved an apoptosis rate of 76.70%
(64.00% early apoptosis, 12.70% late apoptosis) after 24 h incubation. While compound
only attained an apoptosis rate of 25.80% (25.50% early apoptosis, 0.30% late apoptosis)
after 24 h incubation. Apparently, compound was much more effective in promoting
2
9
2
9
2
9
2

apoptosis of MCF-7 cells and the cancer cells seemed to be more sensitive to the PROTAC
that worked as a protein degrader.
Fig. 7. Flow cytometric analysis of the distribution of MCF-7 cells treated with compounds
2
and 9 at the concentration of IC50 values for 72 h or 20 μM for 24 h.
2.7. Cell cycle analysis
The influence of compound
2
on MCF-7 cell cycle progression was studied by flow
as a positive control. As
shown in Fig. 8, in the control group, 58.27% cells were arrested at G1 phase, 22.31% at S
phase, and 19.42% at G2/M phase. Obviously, compounds and arrested cell cycle at the
cytometry with propidium iodide (PI) staining using compound
9
2
9
same G2/M phase, and that would attribute to the fact that they had the same target of
B-Raf.

Fig. 8. Cell cycle distributions upon treatment with compounds 2 and 9 at the concentration of 20
μM for 24 h.
3. Conclusions
We have reported the design and synthesis of four pomalidomide hybrids acting as
proteolysis targeting chimeras (PROTACs) for the degradation of B-Raf. Human MCF-7 breast
cancer cell line was the most sensitive to most of the tested compounds because it had the highest
level of B-Raf in the tested cell lines. Among the four PROTACs, compound 2 with an IC50 value
of 2.70 μM toward MCF-7 cells could more effectively kill cancer cells than its mother
compounds. Moreover, it could effectively induce the degradation of B-Raf via the
ubiquitin-proteasome pathway, and obviously downregulated the level of Mcl-1, a downstream
protein of B-Raf. As a B-Raf degrader, compound 2 was much more potent to promote cancer cell
apoptosis and the cancer cells seemed to be more sensitive to the PROTAC. Owing to the facts
that B-Raf represents an important target in cancer and the anticancer mechanism of PROTAC
was quite different from traditional small molecules, compound 2 possessed the potential for the
treatment of breast cancer.
4. Experimental section
4.1 Reagents and instruments
All regents and solvents were obtained from commercial suppliers, and used as received
unless otherwise stated. All antibodies used in this study were purchased from Cell Signaling
Technology (CST). Mass spectra were measured on an Agilent 6224 TOF LC/MS instrument.
Elemental analyses of C, H, and N were carried out using a Vario MICRO CHNOS elemental

1
analyzer (Elementary). H-NMR and 13C-NMR spectra were recorded on a Bruker 400 MHz or
600 MHz spectrometer, using DMSO-d6 as a solvent. Bio-Rad’s iMark microplate absorbance
reader was used for MTT assay. Cell cycle and apoptosis experiments were measured by flow
cytometry (FAC Scan, Becton Dickenson) and analyzed by Cell Quest software.
4.2 Synthesis and characterization
Synthesis of 2-bromo-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (6).
Bromoacetyl chloride (250 μL) was added to a suspension solution of 5 (273.5 mg, 1.0 mmol) in
THF, then the mixture was kept stirring under reflux overnight. The mixture was concentrated
under reduced pressure, and solids began to deposit after adding diethyl ether. The solid was
filtered out and washed with diethyl ether to give compound 6 as a white solid (Yield: 383.0 mg,
1
97.2%). ESI-MS: [M + Na]+ = 417.98, [2M + Na]+ = 810.98. H-NMR (ppm, 600 MHz,
DMSO-d6): δ 11.11 (s, 1H), δ 10.27 (s, 1H), δ 8.47 (d, J = 6.0 Hz, 1H), δ 7.88 (t, J = 12.0 Hz, 1H ),
δ 7.69 (d, J = 6.0 Hz, 1H ), δ 5.18 (q, J = 12.0 Hz, 1H ), δ 4.34 (s, 2H ), δ 2.90 (m, 1H ), δ 2.55 (m,
2H ), δ 2.08 (m, 1H ).
Synthesis of 2-azido-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (7).
NaN3 (99.0 mg, 1.52 mmol) was slowly added to a suspension solution of 6 (300.0 mg, 0.76 mmol)
in acetone, then the mixture was kept stirring under reflux overnight. The mixture was
concentrated under reduced pressure, and solids began to deposit after adding diethyl ether. The
solid was filtered out and washed with diethyl ether to give compound 7 as a white solid (Yield:
1
234.2 mg, 86.5%). ESI-MS: [M+H]+ = 357.09, [M+Na]+ = 379.07. H-NMR (ppm, 400 MHz,
DMSO-d6): δ 11.19 (s, 1H), δ 10.17 (s, 1H), δ 8.51 (d, J = 6.0 Hz, 1H), δ 7.88 (t, J = 8.0 Hz, 1H ),
δ 7.68 (d, J = 8.0 Hz, 1H ), δ 5.17 (q, J = 10.0 Hz, 1H ), δ 4.55 (s, 2H ), δ 2.89 (m, 1H ), δ 2.57 (m,
2H ), δ 2.02 (m, 1H ).
Synthesis of 2-amino-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (8).
Pd/C (36.0 mg) was added to a suspension solution of 7 (356.3 mg, 1.0 mmol) in methanol, then
the mixture was kept stirring for 6 h under pressurized hydrogen atmosphere at room temperature.
Removal of the reaction solvent by evaporation under reduced pressure led to the deposit from the
solution. The resulting deposit was dissolved in DMF, then Pd/C was removed by filtration. Next,

DMF solution was concentrated under reduced pressure and solids began to deposit after adding
diethyl ether. The solid was filtered out and washed with diethyl ether to give compound 8 as an
off-white powder (Yield: 210.0 mg, 63.6%). ESI-MS: [M + H]+ = 331.10，[M + Na]+ = 353.08,
[2M + H]+ = 661.19，[2M + Na]+ = 683.17. 1H-NMR (ppm, 600 MHz, DMSO-d6): δ 11.16 (s, 1H),
δ 8.82 (d, J = 6.0 Hz, 1H), δ 7.86 (m, 1H ), δ 7.60 (d, J = 6.0 Hz, 1H ), δ 5.16 (q, J = 6.0 Hz, 1H ),
δ 3.34 (s, 2H ), δ 2.90 (m, 1H ), δ 2.53 (m, 2H ), δ 2.07 (m, 1H ).
Synthesis
of
(E)-4-((2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenyl)amino)-4-oxobutanoic
acid (11). Ethyl 4-chloro-4-oxobutanoate (1.52 mmol) was added to a suspension solution of 9
(300.0 mg, 0.76 mmol) in DCM, then the mixture was kept stirring under reflux overnight. The
organic layer was washed with saturated NaHCO3 solution twice and water once, and dried over
sodium sulfate. The filtrate was concentrated under reduced pressure to give the oily intermediate
product. Then, the oily product was dissolved in ethanol / H2O (2 : 1, v : v) solution and
LiOHH2O was added. The mixture was kept stirring at room temperature overnight. After
removal of ethanol, solids began to deposit after adding 0.1 M HCl solution. The solid was filtered
out and washed with water to give compound 11 as a white powder (Yield: 256.2 mg, 68.3%).
1
ESI-MS: [M + Na]+ = 516.13, [2M + Na]+ = 1009.27. H-NMR (ppm, 600 MHz, DMSO-d6): δ
12.11 (s, 1H), δ 9.14 (d, J = 18.0 Hz, 1H), δ 8.02 (d, J = 24.0 Hz, 1H), δ 7.57 (t, J = 18.0 Hz, 1H ),
δ 7.10 (m, 3H ), δ 6.27 (d, J = 12.0 Hz, 2H), δ 4.33 (d, J = 12.0 Hz, 2H ), δ 3.85 (m, 12H), δ 3.02
(d, J = 18.0 Hz, 2H), δ 2.61 (d, J = 18.0 Hz, 2H ).
Synthesis
of
(E)-5-((2-methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenyl)amino)-5-oxopentanoic
acid (12). Same synthetic method of 11 and ethyl 5-chloro-5-oxopentanoate was used to get
compound 12 as a white powder (Yield: 241.0 mg, 62.5%). ESI-MS: [M + Na]+ = 530.14, [2M +
Na]+ = 1037.29. 1H-NMR (ppm, 600 MHz, DMSO-d6): δ 12.08 (s, 1H), δ 9.08 (s, 1H), δ 7.98 (s,
1H), δ 7.59 (d, J = 12.0 Hz, 1H ), δ 7.12 (d, J = 18.0 Hz, 1H ), δ 7.04 (m, 2H ), δ 6.29 (s, 2H), δ
4.35 (s, 2H ), δ 3.85 (m, 12H), δ 2.40 (t, J = 6.0 Hz, 2H), δ 2.26 (t, J = 6.0 Hz, 2H), δ 1.77 (m,
2H ).
Synthesis
of

(E)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-((2-methoxy-5-(((2,4,6-trimethoxy
styryl)sulfonyl)methyl)phenyl)amino)acetamide (1). 9 (196.7 mg, 0.5 mmol) was added to a
solution of 6 (300.0 mg, 0.76 mmol) and sodium acetate (62.5 mg, 0.76 mmol) in methanol and
the reaction mixture was kept stirring under reflux. After the reaction was complete, column
chromatography (DCM : methanol = 1 : 1, v : v) gave compound 1 as a white solid (Yield: 162.0
mg, 45.9%). ESI-MS: [M + Na]+ = 729.18，[2M + Na]+ = 1435.37. Anal. Calcd (%) for
1
C34H34N4O11S: C 57.78, H 4.85, N 7.93. Found: C 57.91, H 4.76, N 7.88. H-NMR (ppm, 600
MHz, DMSO-d6): δ 11.12 (s, 1H), δ 10.45 (s, 1H), δ 8.73 (d, J = 12.0 Hz, 1H), δ 7.84 (t, J = 6.0
Hz, 1H ), δ 7.59 (d, J = 6.0 Hz, 1H ), δ 7.51 (d, J = 18.0 Hz, 1H), δ 7.07 (d, J = 12.0 Hz, 1H), δ
6.88 (d, J = 12.0 Hz, 1H), δ 6.67 (d, J = 6.0 Hz, 1H), δ 6.51 (s, 1H), δ 6.25 (s, 2H), δ 5.89 (s, 1H),
δ 5.08 (q, J = 6.0 Hz, 1H ), δ 4.26 (s, 2H ), δ 3.83 (t, J = 6.0 Hz, 14H), δ 2.83 (m, 1H ), δ 2.55 (m,
2H ), δ 2.00 (m, 1H ). 13C-NMR (ppm, 150 MHz, DMSO-d6): δ 173.18, 171.42, 170.23, 168.35,
167.14, 163.98, 161.32, 147.59, 137.63, 136.80, 136.70, 133.20, 131.74, 125.09, 124.03, 121.96,
121.13, 118.61, 116.41, 113.52, 110.63, 102.97, 91.34, 60.61, 56.48, 56.21, 56.03, 49.33, 49.19,
31.34, 22.39.
General synthetic procedure of compounds 2-4. To a solution of 10, 11 or 12 (0.33 mmol) in
DMF (7.0 mL), TBTU (106.0 mg, 0.33 mmol) and excess TEA were added. The resulting solution
was stirred at room temperature for 15 min, then 8 (100.0 mg, 0.30 mmol) was added. After the
above mixture was kept under stirring at 50 C for 24 h, column chromatography (DCM :
methanol = 1 : 1, v : v) gave compounds 2-4 as white solids.
(E)-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-2-(2-((2-methoxy-5-(((2,4,6-tri
methoxystyryl)sulfonyl)methyl)phenyl)amino)acetamido)acetamide (2). Yield: 106.0 mg, 46.5%.
ESI-MS: [M + Na]+ = 786.19. Anal. Calcd (%) for C36H37N5O12S: C 56.61, H 4.88, N 9.17. Found:
1
C 56.48, H 4.81, N 9.23. H-NMR (ppm, 600 MHz, DMSO-d6): δ 11.15 (s, 1H), δ 9.94 (s, 1H), δ
8.69 (t, J = 6.0 Hz, 1H), δ 8.57 (d, J = 6.0 Hz, 1H), δ 7.84 (t, J = 6.0 Hz, 1H ), δ 7.62 (d, J = 6.0
Hz, 1H ), δ 7.56 (d, J = 12.0 Hz, 1H), δ 7.08 (d, J = 18.0 Hz, 1H), δ 6.82 (d, J = 12.0 Hz, 1H), δ
6.61 (d, J = 6.0 Hz, 1H), δ 6.48 (s, 1H), δ 6.27 (s, 2H), δ 5.35 (s, 1H), δ 5.12 (q, J = 6.0 Hz, 1H ),
δ 4.26 (s, 2H ), δ 3.98 (d, J = 6.0 Hz, 2H), δ 3.85 (m, 14H), δ 2.88 (m, 1H ), δ 2.53 (m, 2H ), δ
2.07 (m, 1H ). 13C-NMR (ppm, 150 MHz, DMSO-d6): δ 173.19, 171.61, 170.23, 169.26, 168.55,

167.13, 164.04, 161.35, 147.06, 137.64, 136.68, 136.54, 133.10, 131.79, 125.56, 124.03, 121.79,
120.04, 118.88, 117.60, 117.00, 112.76, 109.91, 103.00, 91.38, 60.83, 56.53, 56.50, 56.07, 55.90,
49.44, 46.96, 44.00, 31.41, 22.33.
(E)-N1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)-N4-(2-
methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenyl)succinamide (3). Yield: 101.0 mg,
42.0%. ESI-MS: [M + Na]+ = 828.21. Anal. Calcd (%) for C38H39N5O13S: C 56.64, H 4.88, N 8.69.
Found: C 56.51, H 4.93, N 8.74. 1H-NMR (ppm, 600 MHz, DMSO-d6): δ 11.16 (s, 1H), δ 10.00 (s,
1H), δ 9.15 (s, 1H), δ 8.77 (s, 1H), δ 8.62 (d, J = 6.0 Hz, 1H), δ 8.03 (s, 1H), δ 7.85 (t, J = 6.0 Hz,
1H ), δ 7.62 (t, J = 6.0 Hz, 1H ), δ 7.58 (s, 1H), δ 7.13 (d, J = 18.0 Hz, 1H), δ 7.00 (m, 2H), δ 6.29
(s, 2H), δ 5.16 (q, J = 6.0 Hz, 1H ), δ 4.33 (s, 2H ), δ 3.85 (m, 14H), δ 2.88 (m, 1H ), δ 2.57 (m,
6H ), δ 2.05 (m, 1H ). 13C-NMR (ppm, 150 MHz, DMSO-d6): δ 173.22, 173.20, 170.84, 170.24,
169.59, 168.65, 167.12, 164.09, 161.41, 149.65, 136.95, 136.61, 133.37, 131.77, 127.74, 127.32,
125.22, 124.47, 123.99, 121.06, 118.83, 116.77, 111.18, 103.03, 91.39, 60.34, 56.52, 56.20, 56.08,
49.43, 44.03, 31.74, 31.72, 30.68, 22.37.
(E)-N1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)-N5-(2-
methoxy-5-(((2,4,6-trimethoxystyryl)sulfonyl)methyl)phenyl)glutaramide (4). Yield: 145.0 mg,
59.0%. ESI-MS: [M + Na]+ = 842.23. Anal. Calcd (%) for C39H41N5O13S: C 57.14, H 5.04, N 8.54.
Found: C 57.28, H 4.97, N 8.63. 1H-NMR (ppm, 600 MHz, DMSO-d6): δ 11.16 (s, 1H), δ 10.01 (s,
1H), δ 9.08 (s, 1H), δ 8.70 (t, J = 6.0 Hz, 1H), δ 8.23 (d, J = 12.0 Hz, 1H), δ 8.02 (s, 1H), δ 7.86 (t,
J = 6.0 Hz, 1H ), δ 7.62 (t, J = 6.0 Hz, 1H ), δ 7.59 (s, 1H), δ 7.13 (d, J = 12.0 Hz, 1H), δ 7.04 (m,
2H), δ 6.29 (s, 2H), δ 5.15 (q, J = 6.0 Hz, 1H ), δ 4.36 (s, 2H ), δ 3.85 (m, 14H), δ 2.86 (m, 1H ), δ
2.59 (m, 2H ), δ 2.42 (t, J = 6.0 Hz, 2H ), δ 2.34 (t, J = 6.0 Hz, 2H ), δ 2.05 (m, 1H ), δ 1.86 (t, J =
6.0 Hz, 2H ). 13C-NMR (ppm, 150 MHz, DMSO-d6): δ 173.62, 173.25, 172.62, 171.40, 170.26,
169.66, 168.70, 167.13, 164.09, 161.46, 161.40, 155.05, 149.93, 136.98, 136.64, 133.36, 131.78,
127.66, 127.50, 125.17, 124.00, 121.05, 118.82, 116.66, 111.23, 103.00, 91.37, 60.27, 59.63,
56.52, 56.20, 56.07, 49.44, 44.04, 35.92, 34.89, 32.75, 31.37, 22.36.
4.3 Cell lines and cell culture.
Human breast cancer cell lines (MCF-7 and MDA-MB-231), human hepatoma cell line

(HepG2), murine melanoma cell line (B16) and human normal liver cell line (LO2) were
purchased from Jiangsu KeyGen BioTECH Co. Ltd. Cells were incubated carefully in RPMI-1640
medium or Dulbecco’s Modified Eagle Medium (DMEM) supplemented with 10% fetal bovine
serum (FBS), streptomycin (100 μg/mL), and ampicillin sodium (100 μg/mL) in an atmosphere of
5% CO2 and 95% air at 37 C.
4.4 Western blotting
After the treatment with the indicated concentration of each sample for 24 h, cells were
collected and lysed in lysis buffer (100 mM Tris-Cl, pH 6.8, 4% (m/v) sodium dodecylsulfonate,
20% (v/v) glycerol, 200 mM β-mercaptoethanol, 1 mM phenylmethylsulfonyl fluoride, and 1
g/mL aprotinin). Lysates were centrifuged at 12,000 g for 0.5 h at 4 C. The concentrations of total
proteins were measured using the BCA assay method with Varioskan spectrofluorometer and
spectrophotometer (Thermo, Waltham, MA) at 562 nm. Protein (20-100 μg) prepared from the
indicated cells was loaded per lane and electrophoresed in 8% or 10% sodium dodecyl sulfate
polyacrylamide gel electrophoresis (SDS-PAGE), and then transferred onto polyvinylidene
difluoride (PVDF) Immobilon-P membrane (Bio-Rad, USA) using a transblot apparatus (Bio-Rad,
USA). The membranes were blocked with 5% (w/v) non-fat milk at 0.5 h at 37 C, followed by
overnight incubation at 4 C with primary antibodies diluted in PBST. After washing with PBST,
1:1000 in PBST, and the labeled proteins were detected with an Odyssey Scanning System
(Li-COR., Lincoln, Nebraska, USA).
4.5 Cytotoxicity measurement.
The cultured cells with better vitality were transferred to a 96-well plate so that the density of
the cells was 5000 per well, and they were incubated overnight. Then, the compounds were
dissolved in DMSO and diluted with medium to various concentrations (the final concentration of
DMSO was less than 0.4%). After being incubated at 37 °C for 72 h, cells were stained with
3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2Htetrazolium bromide (MTT, 5 mg/mL) for another 4
h, and then dissolved with 150 μL of DMSO. The UV absorption intensity was detected with an

ELISA reader at 490 nm. The IC50 values were calculated by SPSS software after three parallel
experiments.
4.6 Apoptosis analysis.
MCF-7 cells with good activity (1 × 105 cells per plate) were transferred to six-well plates
and cultured overnight in 5% CO2 at 37 °C. The tested compounds were diluted to the specified
concentration. After 24 h or 72 h, the cells were digested with trypsin and washed with cold PBS,
and collected by centrifugation (2000 rpm, 5 min). After that, cells were resuspended in binding
buffer (10 mM HEPES, 140 mM NaCl, 2.5 mM CaCl2, pH 7.4) and incubated with annexin
V-FITC (100 ng/mL) and then with propidium iodide (2 μg/mL) for 15 min in the dark at room
temperature. At last, the fluorescence was measured using a flow cytometer (FACScan, Becton
Dickson, USA).
4.7 Cell Cycle Measurement.
MCF-7 cells with good vitality were transferred into six-well plates, with a density of 10000
cells per well, and cultured overnight at 37 °C. Then, 20 μM of the tested compounds were
incubated with cells for 24 h. All adherent and floating cells were collected and washed twice with
PBS. Then, the cells were fixed with 70% EtOH at 4 °C for 24 h. After that, fixed cells were
washed with PBS. After being centrifuged, cells were stained with 50 μg/mL propidium iodide
solution containing 100 μg/mL RNase at 37 °C for 0.5 h. The samples (at least 10000 cells) were
measured by flow cytometry (FAC Scan, Becton Dickenson) using Cell Quest software and
recording propidium iodide (PI) in the FL2 channel.
Acknowledgments
We are grateful to the National Natural Science Foundation of China (Nos. 21571033
and 81503099) and Southeast University “Perfect Young Scholars” Support Program.
Fundamental Research Funds for the Central Universities (Projects 2242016K30020 and
2242017K41024) and Priority Academic Program Development of Jiangsu Higher
Education Institutions for the construction of fundamental facilities are also appreciated.

Notes
The authors declare no competing financial interest.
Appendix A. Supplementary material
Supplementary data to this article can be found online at
References
[1] C. Galustian, M. C. Labarthe, J. B. Bartlett, A. G. Dalgleish, Thalidomide-derived
immunomodulatory drugs as therapeutic agents, Expert Opin. Boil. Ther. 4 (2004)
1963-1970.
[2] C. Galustian, B. Meyer, M. C. Labarthe, K. Dredge, D. Klaschka, J. Henry, S. Todryk, R.
Chen, G. Muller, D. Stirling, P. Schafer, J. B. Bartlett, A. G. Dalgleish, The anti-cancer agents
lenalidomide and pomalidomide inhibit the proliferation and function of T regulatory cells,
Cancer Immunol. Immun. 58 (2009) 1033-1045.
[3] H. Quach, D. Ritchie, A. K. Stewart, P. Neeson, S. Harrison, M. J. Smyth, H. M Prince,
Mechanism of action of immunomodulatory drugs (IMiDS) in multiple myeloma, Leukemia
24 (2010) 22-32.
[4] R. J. D'Amato, M. S. Loughnan, E. Flynn, J. Folkman, Thalidomide is an inhibitor of
angiogenesis, P. Natl. Acad. Sci. USA 91 (1994) 4082-4085.
[5] T. Ito, H. Ando, T. Suzuki, T. Ogura, K. Hotta, Y. Imamura, Y. Yamaguchi, H. Handa,
Identification of a primary target of thalidomide teratogenicity, Science 327 (2010)
1345-1350.
[6] E. S. Fischer, K. Böhm, J. R. Lydeard, H. Yang, M. B. Stadler, S. Cavadini, J. Nagel, F.
Serluca, V. Acker, G. M. Lingaraju, R. B. Tichkule, M. Schebesta, W. C. Forrester, M. Schirle,

U. Hassiepen, J. Ottl, M. Hild, R. E. J. Beckwith, J. Wade Harper, J. L. Jenkins, N. H. Thomä,
Structure of the DDB1–CRBN E3 ubiquitin ligase in complex with thalidomide. Nature, 512
(2014) 49-53.
[7] M. Q. Lacy, A. R.McCurdy, Pomalidomide, Blood 122 (2013) 2305-2309.
[8] A. A. Chanan-Khan, A Swaika, A. Paulus, S. K. Kumar, J. R. Mikhael, S. V. Rajkumar, A.
Dispenzieri, M. Q. Lacy, Pomalidomide: the new immunomodulatory agent for the treatment
of multiple myeloma, Blood Cancer J. 3 (2013) e143.
[9] A. Lopez-Girona, D. Mendy, T. Ito, K. Miller, A. K. Gandhi, J. Kang, S. Karasawa, G.
Carmel, P. Jackson, M. Abbasian, A. Mahmoudi, B. Cathers, E. Rychak, S. Gaidarova, R.
Chen, P. H. Schafer, H. Handa, T. O. Daniel, J. F. Evans, R. Chopra, Cereblon is a direct
protein target for immunomodulatory and antiproliferative activities of lenalidomide and
pomalidomide, Leukemia 26 (2012) 2445-2445.
[10] G. Petzold, E. S. Fischer, N. H. Thomä. Structural basis of lenalidomide-induced CK1α
degradation by the CRL4CRBN ubiquitin ligase, Nature 532 (2016) 127-130.
[11] J. Krönke, N. D. Udeshi, A. Narla, P. Grauman, S. N. Hurst, M. McConkey, T. Svinkina, D.
Heckl, E. Comer, X. Li, C. Ciarlo, E. Hartman, N. Munshi, M. Schenone, S. L. Schreiber, S.
A. Carr, B. L. Ebert, Lenalidomide causes selective degradation of IKZF1 and IKZF3 in
multiple myeloma cells, Science 343 (2014) 301-305.
[12] K. M. Sakamoto, K. B. Kim, A. Kumagai, F. Mercurio, C. M. Crews, R. J. Deshaies, Protacs:
Chimeric molecules that target proteins to the Skp1-Cullin-F box complex for ubiquitination
and degradation, P. Natl. Acad. Sci. USA 98 (2001) 8554-8559.
[13] I. Churcher, Protac-induced protein degradation in drug discovery: breaking the rules or just

making new ones? J. of Med. Chem. 61 (2018) 444-452.
[14] M. Toure, C. M. Crews, Small-molecule PROTACS: new approaches to protein degradation,
Angew. Chem. Int. Edit. 55 (2016) 1966-1973.
[15] P. M. Cromm, C. M. Crews, Targeted protein degradation: from chemical biology to drug
discovery, Cell Chem. Biol. 24 (2017) 1181-1190.
[16] K. Raina, C. M. Crews, Targeted protein knockdown using small molecule degraders, Curr.
Opin. Chem. Biol. 39 (2017) 46-53.
[17] A.C. Lai, M. Toure, D. Hellerschmied, J. Salami, S. Jaime-Figueroa, E. Ko, J. Hines, C. M.
Crews, Modular PROTAC design for the degradation of oncogenic BCR-ABL, Angew. Chem.
Int. Edit. 55 (2016) 807-810.
[18] G.E. Winter, D.L. Buckley, J. Paulk, J.M. Roberts, A. Souza, S. Dhe-Paganon, J. E. Bradner,
Phthalimide conjugation as a strategy for in vivo target protein degradation, Science 348
(2015) 1376-1381.
[19] H. Chen, F. Chen, N. Liu, X. Wang, S. Gou, Chemically induced degradation of CK2 by
proteolysis targeting chimeras based on a ubiquitin-proteasome pathway, Bioorg. Chem. 81
(2018) 536-544.
[20] H. Davies, G. R. Bignell, C. Cox, P. Stephens, S. Edkins, S. Clegg, J. Teague, H. Woffendin,
M. J. Garnett, W. Bottomley, N. Davis, E. Dicks, R. Ewing, Y. Floyd, K. Gray, S. Hall, R.
Hawes, J. Hughes, V. Kosmidou, A. Menzies, C. Mould, A. Parker, C. Stevens, S. Watt, S.
Hooper, R. Wilson, H. Jayatilake, B. A. Gusterson, C. Cooper, J. Shipley, D. Hargrave, K.
Pritchard-Jones, N. Maitland, G. Chenevix-Trench, G. J. Riggins, D. D. Bigner, G. Palmieri, A.
Cossu, A. Flanagan, A. Nicholson, J. W. C. Ho, S. Y. Leung, S. T. Yuen, B. L. Weber, H. F.

Seigler, T. L. Darrow, H. Paterson, R. Marais, C. J. Marshall, R. Wooster, M. R. Stratton, P.
Andrew Futreal, Mutations of the BRAF gene in human cancer, Nature 417 (2002) 949-954.
[21] K. T. Flaherty, G. Mcarthur, BRAF, a target in melanoma: Implications for solid tumor drug
development, Cancer 116 (2010) 4902-4913.
[22] T. Rajakulendran, M. Sahmi, M. Lefrançois, F. Sicheri, M. Therrien,
A
dimerization-dependent mechanism drives RAF catalytic activation, Nature 461 (2009)
542-545.
[23] M. V. R. Reddy, P. Venkatapuram, M. R. Mallireddigari, V. R. Pallela, S. C. Cosenza, K. A.
Robell, B. Akula, B. S. Hoffman, E. Premkumar Reddy, Discovery of a clinical stage
multi-kinase inhibitor sodium (E)-2-{2-Methoxy-5-[(2’,4’,6’-trimethoxystyrylsulfonyl)
methyl] phenylamino}acetate (ON 01910.Na): synthesis, structure-activity relationship, and
biological activity, J. Med. Chem. 54 (2011) 6254-6276.
[24] K. Gumireddy, M.V. Ramana Reddy, S. C. Cosenza, R. Boomi Nathan, S. J. Baker, N.
Papathi, J. Jiang, J. Holland, E. Premkumar Reddy, ON01910, a non-ATP-competitive small
molecule inhibitor of Plk1, is a potent anticancer agent, Cancer Cell 7 (2005) 275-286.
[25] D. W. Bowles, J. R. Diamond, E. T. Lam, C. D. Weekes, D. P. Astling, R. T. Anderson, S.
Leong, L. Gore, M. Varella-Garcia, B. W. Vogler, S. B. Keysar, E. Freas, D. L. Aisner, C. Ren,
A. C. Tan, F. Wilhelm, M. Maniar, S. Gail Eckhardt, W. A. Messersmith, A. Jimeno, Phase I
study of oral rigosertib (ON 01910.Na), a dual inhibitor of the PI3K and Plk1 pathways, in
adult patients with advanced solid malignancies, Clin. Cancer Res. 20 (2014) 1656-1665.
[26] S. K. Athuluri-Divakar, R.Vasquez-Del Carpio, K. Dutta, S. J. Baker, S. C. Cosenza, I. Basu,
Y. K. Gupta, M. V. Ramana Reddy, L. Ueno, J. R. Hart, P. K. Vogt, D. Mulholland, C. Guha,

A. K. Aggarwal,E. Premkumar Reddy, A small molecule RAS-mimetic disrupts RAS
association with effector proteins to block signaling, Cell 165 (2016) 643-655.
[27] E. P. Reddy, S. K. Divakar, R. Vasquez-Del Carpio, K. Dutta, S. J. Baker, R. Reddy, A. K.
Aggarwal, Rigosertib blocks RAS signaling by acting as a small molecule RAS mimetic that
binds to the RAS-Binding domains of RAS effector proteins, Blood 124 (2014) 5616-5616.
[28] J. Villanueva, A. Vultur, J. T. Lee, R. Somasundaram, M. Fukunaga-Kalabis, A. K. Cipolla, B.
Wubbenhorst, X. Xu, P. A. Gimotty, D. Kee, A. E. Santiago-Walker, R. Letrero, K. D'Andrea,
A. Pushparajan, J. E. Hayden, K. D. Brown, S. Laquerre, G. A. McArthur, J. A. Sosman, K. L.
Nathanson, M. Herlyn, Acquired resistance to BRAF inhibitors mediated by a RAF kinase
switch in melanoma can be overcome by cotargeting MEK and IGF-1R/PI3K, Cancer cell 18
(2010) 683-695.
[29] S. Lee, RAF-targeted therapy for hepatocellular carcinoma in the regenerating liver, J. Surg.
Oncol. 107 (2013) 393-401.
[30] D. N. Chadee, J. M. Kyriakis, A novel role for mixed lineage kinase 3 (MLK3) in B-Raf
activation and cell proliferation, Cell Cycle 3 (2004) 1227-1229.
[31] K. T. Flaherty, M. B. Grant McArthur, BRAF, a target in melanoma, Cancer 116 (2010)
4902-4913.
[32] T. M. Becker, S. C. Boyd, B. Mijatov, K. Gowrishankar, S. Snoyman, G. M. Pupo, R. A.
Scolyer, G. J .Mann, R. F. Kefford, X. D. Zhang, H. Rizos, Mutant B-RAF-Mcl-1 survival
signaling depends on the STAT3 transcription factor, Oncogene 33 (2014) 1158-1166.
[33] J. Adams, M. Kauffman, Development of the proteasome inhibitor Velcade™(Bortezomib),
Cancer Invest. 22 (2004) 304-311.

Graphical abstract

Highlights
1. Several pomalidomide hybrids were synthesized to act as PROTACs.
2. Compound 2 can effectively kill cancer cells via inducing cells apoptosis.
3. Compound 2 can accelerate the degradation of B-Raf.
4. Compound 2 degraded B-Raf by recruiting ubiquitin-proteasome system.