Chemically induced degradation of CK2 by proteolysis targeting chimeras based on a ubiquitin–proteasome pathway

Article abstract of DOI:10.1016/j.bioorg.2018.09.005

As a ubiquitous, highly pleiotropic and constitutively active serine/threonine protein kinase, casein kinase 2 (CK2) is closely associated with tumorigenesis by its overexpression in cancer cells. Here we report several proteolysis targeting chimeras (PROTACs) via “click reaction” to connect a CK2 inhibitor (CX-4945) and pomalidomide for degradation of CK2 protein. Among them, compound 2 degraded CK2 in a dose and time-dependent manner, and kept CK2 at a low basal level by recruiting ubiquitin-proteasome system. The degradation of CK2 resulted in the reduced phosphorylation of Akt and the up-regulation of p53. As a CK2 protein degrader, 2 showed the analogous cytotoxicity to CX-4945 but with a quite different mechanism of action from the CK2 inhibitor, hinting that degradation of CK2 proteins by PROTACs is a potential way for cancer treatments.

Full text of DOI:10.1016/j.bioorg.2018.09.005

Accepted Manuscript
Chemically induced degradation of CK2 by proteolysis targeting chimeras based
on a ubiquitin–proteasome pathway
Hong Chen, Feihong Chen, Nannan Liu, Xinyi Wang, Shaohua Gou
PII:
S0045-2068(18)30687-4
DOI:
https://doi.org/10.1016/j.bioorg.2018.09.005
YBIOO 2502
Reference:
Bioorganic Chemistry
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10 July 2018
5 September 2018
6 September 2018
Please cite this article as: 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, Bioorganic Chemistry (2018), doi: https://
doi.org/10.1016/j.bioorg.2018.09.005
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Chemically induced degradation of CK2 by proteolysis targeting
chimeras based on a ubiquitin–proteasome pathway
Hong Chen†, Feihong Chen†, Nannan Liu, Xinyi Wang and 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, (P.R.) China
* Corresponding Author. E-mail: sgou@seu.edu.cn
† These authors contributed equally.
Abstract
As a ubiquitous, highly pleiotropic and constitutively active serine/threonine protein kinase,
casein kinase 2 (CK2) is closely associated with tumorigenesis by its overexpression in cancer
cells. Here we report several proteolysis targeting chimeras (PROTACs) via “click reaction” to
connect a CK2 inhibitor (CX-4945) and pomalidomide for degradation of CK2 protein. Among
them, compound 2 degraded CK2 in a dose and time-dependent manner, and kept CK2 at a low
basal level by recruiting ubiquitin-proteasome system. The degradation of CK2 resulted in the
reduced phosphorylation of Akt and the up-regulation of p53. As a CK2 protein degrader, 2
showed the analogous cytotoxicity to CX-4945 but with a quite different mechanism of action
from the CK2 inhibitor, hinting that degradation of CK2 proteins by PROTACs is a potential way
for cancer treatments.
Keywords: casein kinase 2; protein degradation; proteolysis targeting chimeras; CX-4945,
pomalidomide.
1. Introduction
Casein kinase 2 (CK2), a ubiquitous, highly pleiotropic and constitutively active
serine/threonine protein kinase, is a heterotetramer composed of two catalytic subunits (α

and /or α′) and two regulatory β subunits.[1] It is known that CK2 can phosphorylate more
than 300 substrates, many of which are involved in signal transduction, gene expression
and cell growth.[2,3] Overexpression of CK2 has been documented in a great number of
cancers such as lung cancer, breast carcinoma, prostate and kidney cancers, and elevated
CK2 activity is found to be associated with tumorigenesis.[4,5] Thus, CK2 has been
regarded as a common denominator of diverse cancer cells and can represent a
multi-purpose target for the treatment of different kinds of tumors. So far, there are two
methods to reduce the function of CK2, one is to down-regulate the expression of CK2
protein by gene knockout such as siRNA, the other is to inhibit CK2 activity by small
molecular inhibitors.[6,7] However, the application of gene knockout technology is rather
complicated and limited in clinic, and CK2 inhibitors could only inhibit the activity of the
enzyme without degrading CK2 protein.[8]
Ubiquitin-proteasome system (UPS), responsible for much of the regulated
proteolysis in the cell, mainly contains ubiquitin (Ub), Ub-activating enzyme E1,
Ub-conjugating enzymes E2s (UBCs,) Ub-protein ligands E3s, and 26S proteasome.[9] E3
ligand is known as substrate-specific, which can catalyze transfer of ubiquitin from E2s to
an amide linkage with the substrate or with a polyubiquitin chain already anchored to it,
then the tagged substrate is degraded by the 26S proteasome.[10] Because the UPS
represents a major system controlling many cellular processes, drugs suppressing the UPS
can be efficacious in the treatment of cancers. So far, several small molecules like
bortezomib and carfilzomib have been approved as proteasome inhibitors for clinical
use.[11,12]
Proteolysis targeting chimeras (PROTACs), owning full advantage of the function of protein
degradation by UPS, is a heterobifunctional compound with two recruiting ligands connected via a
linker, in which one ligand is specific to the protein of interest while the other one specifically
recruits E3 ligase.[13-15] Generally, an E3 ligase needs a specific recognition signal to recruit and
ubiquitinate its natural substrate, however, PROTAC acts in a way that can effectively tether the
E3 ligase to the substrate and result in the ubiquitination and subsequent proteasomal degradation
of the target protein (Fig. 1).[16] Typically, a small molecule protein inhibitor is usually selected as

the ligand specific to the protein of interest, and so far thalidomide, lenalidomide, pomalidomide
and their derivatives have been chosen as the recruiters of E3 ligase.[17-20] At present, PROTAC
has been widely applied to induce the degradation of various proteins, such as BRD4, Sirt2, and
BCR-ABL.[21-23]
Fig. 1. Proposed CK2 degradation mechanism for PROTACs.
Based on the above, we hypothesized that a PROTAC strategy would be effective to induce
CK2 protein degradation. Herein reported is an approach for the preparation of novel PROTACs
via “click reaction” for degradation of CK2 protein (Fig. 2). Importantly, “click reaction” is a very
facile, selective and “green” reaction with the aim of binding two molecules together under mild
water-tolerant conditions. CX-4945, a selective CK2 inhibitor with a strong binding force

stemming from hydrophobic interactions of flat tricyclic core with binding pocket residues,
hydrogen bonding of pyridine nitrogen with the NH of Val116 in the hinge region, and an ionic
bridge with Lys68, was first elected as CK2 targeting ligand.[24] And pomalidomide was applied as
a recruiter of E3 ligase to conjugate with CX-4945 via different linkers by click reaction. It was
worth noting that the number of alkyl chains was important for PROTACs, because the length of
the chain has a great influence on the interaction between E3 ligase and CK2 protein.
Fig. 2. Composition of the designed PROTAC.
2. Results and discussion
2.1. Design and synthesis
Compounds 1-4 were designed and synthesized based on the protocol as possible
PROTACs
(
Scheme
1
). CX-4945, prepared according to
a method reported
previously,[24,25] was first esterified with several different alkynols to produce alkyne
derivatives
precursor
6
. Pomalidomide (10) was obtained through reduction of the nitro group of its
9
that was synthesized by the reaction of 4-nitro-2-benzofuran-1,3-dione ( ) with
7
3-aminopiperidine-2,6-dione trifluoroacetate (
refluxing in acetic acid.[26] Then, pomalidomide was used to react with chloroacetyl
chloride and sodium azide subsequently to get compounds 11 and 12 [27]
8) in the presence of sodium acetate under
.
Finally, “click
reaction” was carried out to connect the azide (12) with the resulting alkyne derivatives at

room temperature. All the resulting PROTACs were structurally characterized by
microanalysis, 1H- and 13C-NMR spectroscopy together with ESI-MS spectrometer.
Scheme 1. Synthesis of the PROTACs.
(a) TBTU, TAE, acetone, reflux, yields: 6a: 82.3%, 6b: 91.2%, 6c: 83.7%, 6d: 87.2%; (b)
AcONa, AcOH, reflux, yield: 66.4%; (c) Fe, NH4Cl, EtOH/H2O, rt, yield: 44.4%; (d)
chloroacetyl chloride, THF, reflux, yield: 87.2%; (e) NaN3, acetone, reflux, yield: 75.7%;
(f)
5, CuSO4·5H2O, sodium ascorbate, THF/H2O, rt, yields: 1: 46.5%, 2: 67.2%, 3: 42.3%,
4: 79.7%.

2.2. Interaction between CK2 protein and PROTAC
The interactions between CK2 protein and compounds 1-4 were first detected by CycLex
CK2 Kinase Assay/Inhibitor Screening Kit that was designed to measure the activities of purified
CK2 for the rapid and sensitive evaluation of inhibitors. As shown in Fig. 3, at a concentration of
10 μM, CK2 activity was hardly detected under the action of CX-4945 with an inhibition rate of
98.7%, and all the target compounds showed weaker inhibitory abilities against CK2 than
CX-4945. Compound 2 with an inhibition rate of 88.2% had the best potency of inhibiting CK2
activity among the four PROTACs, and the insertion of an oxygen into the linker in compound 4
diminished its inhibitory ability greatly. The results showed that these compounds still maintained
the binding ability with CK2 protein derived from the component of CX-4945, via hydrophobic
interactions and hydrogen bonding coming from the flat tricyclic core and pyridine nitrogen in
their structures as previously reported.[24,28] It was worth noting that PROTAC is different from the
conventional small molecule based drugs that are typically required to directly bind to a pocket or
active site within the targeting protein.[29] For instance, conjugation of the high-affinity dasatinib
to pomalidomide reduced the binding affinity for BCR-ABL by more than 20-fold, but led to
degradation of BCR-ABL in human leukemic K562 cells at a lower concentration.[23] Despite part
of the CK2 inhibitory ability lost in contrast to CX-4945, the interactions between CK2 protein
and these compounds, especially 2, still maintained, and these PROTACs acting in a catalytic
manner could be more selective for CK2.
Fig. 3. The average inhibition ratio of CK2 protein under the treatment of CX-4945 or compounds
1-4 at a concentration of 10 μM. Results are representative of three independent experiments and
shown as the average values.

2.3. Degradation of CK2
All the targeted compounds were then tested for CK2 degradation in human triple-negative
breast cancer line MDA-MB-231, in which CK2 is over-expressed (Fig. S1). As shown in Fig. 4,
these compounds possessed modest degradation capacity on CK2α at a concentration of 5 μM, and
among them, the effect of compound 2 was a little stronger than others. With the concentration up
to 10 μM, these compounds presented remarkable degradative capacity. Compound 2, with the
best inhibitory ability against CK2 at 10 μM, was also the most effective to degrade CK2 protein
among these compounds, and compound 4 with an oxygen atom in the linker exhibited the
weakest ability to degrade the protein at both concentrations. The results indicated that the
interaction between the PROTAC and CK2 protein was essential to decide the potential of protein
degradation, and the abilities of PROTACs to degrade CK2 were consistent with the CK2 activity
inhibitory abilities. It was noticed that the CK2 inhibitor, CX-4945, was invalid as a protein
degrader even at a high concentration of 10 μM, because CX-4945 is only an ATP site-directed
inhibitor toward CK2 that can only decrease the CK2 activity without the ability of inducing the
down-regulation of the protein as previously reported.[30,31] Consequently, the PROTACs we
designed, especially 2, were effective to degrade CK2 protein in a concentration dependent
manner.
B
D

Fig. 4. The effects of compounds on CK2 protein degradation in MDA-MB-231 cells. The
levels of CK2α after treatment with CX-4945 and compounds 1-4 for 24 h at 5 μM (A, B)
and 10 μM (C, D), respectively.
2.4. Time-dependent research of compound 2
For further study, compound 2 was selected as a representative against related proteins for
time-dependent research (Fig. 5 and Fig. S2). Results showed that compound 2 degraded CK2α
after 12 hours of treatment, and reached a larger extent of degradation before 24 hours. Moreover,
the protein degrader could maintain CK2α at a low level until 36 hour in this study. Conversely,
pomalidomide and CX-4945 could not induce the degradation of CK2 until 24 hour. Since Akt is
one of the most important phosphorylated substrate of CK2, it was used to study with compound 2.
It was found that obvious up-regulation of Akt treated with 2 happened in 24 hour. However, in
contrast, the phosphorylated p-Akt was observed to be reduced time-dependently with a little
p-Akt detected at 24 hour and none detected at 36 hour. While, pomalidomide and CX-4945
hardly made any change of Akt and p-Akt at a concentration of 10 μM at 24 hour. Another
phosphorylated substrate of CK2 is MDM2, and p-MDM2 can accelerate the degradation of p53,
which is a low-expressed tumor suppressor protein or mutant in tumor cells.[32] After treatment
with 2 for 12 hours, a slight time-dependent increase of p53 was also observed, which was
consistent with the degradation of CK2. Pomalidomide and CX-4945 also could slightly increase
the content of p53. Therefore, all the above changes of Akt, p-Akt and p53 can be mainly ascribed
to the CK2 degradation as well as inhibition, because they happened after the change of CK2
treated with compound 2 over 12 hours, and the degradation of CK2 could obviously change the
level of downstream proteins of CK2.

Fig. 5. The levels of CK2α, Akt, p-Akt and p53 after the treatment with pomalidomide (10
μM) and CX-4945 (10 μM) for 24 hours, and compound
2 (10 μM) at different times.
2.5. Stability of compound 2
Based on the fact that compound 2 was the most effective PROTAC, HPLC method was used
to study the stability of compound 2 in PBS with different pH values (7.4 and 5.0) and cell culture
medium (DMEM). As Fig. S3 indicated that compound 2 was stable in PBS with a pH value of
7.4 and DMEM for 24 hours. However, as acidity increased (pH = 5.0), a small amount of
CX-4945 was detected due to the decomposition of compound 2 under acidic condition in PBS.
Owing to the steric hindrance of aromatic unit closed to ester bond in the component of CX-4945,
compound 2 was relatively stable and could effectively induce the degradation of CK2 in
MDA-MB-231 cells during 24 hours. Moreover, as the degradation of CK2 is time dependent,
once the pre-existing repository of cellular CK2 is depleted by compound 2, only a small number
of 2 is needed to degrade the smaller reservoir of de novo re-synthesized CK2.[33]
2.6. CK2 degradation mechanism study
In order to confirm the degradation mechanism of CK2, bortezomib (BTZ, originally
PS-341), the first therapeutic reversible inhibitor of 26S proteasome that degrades
ubiquitinated proteins, was employed for the joint action with 2 [34]
CX-4945 and BTZ could not induce the degradation of CK2, and compound
pre-treatment of BTZ could degrade CK2 protein effectively at 24 h. Whereas,
.
As shown in Fig. 6
,
2
without the

pre-treatment with BTZ could dose-dependently suppress the degradation of CK2α induced
by compound . The results implied that degraded CK2 protein through a ubiquitin and
2
2
proteasome-dependent proteolysis pathway (UPP), which was completely in conformity
with the working principles of PROTAC as described before.
Fig. 6. (A) The levels of CK2α after pre-treatment with BTZ at different concentrations for
1 h and then treated with
2 (10 μM) for 24 h. (B) The level of CK2α after pre-treatment
with BTZ at different concentrations for 1 h.
2.7. In vitro cytotoxicities
In vitro cytotoxicities of compounds 1-4 against MDA-MB-231 cancer cells were
measured by MTT assay together with CX-4945 and pomalidomide as positive drugs
(
Table 1 and Fig. S4). All compounds were cytotoxic to MDA-MB-231 cancer cells in
which CK2 are abnormally overexpressed. Compound as the most effective degrader of
CK2 protein exhibited the most potent anticancer activity that was similar as CX-4945, and
in contrast compound as the weakest CK2 protein degrader displayed the lowest
anticancer activity. The results can be partly attributed to the CK2 degradation ability of
these compounds by a way of PROTAC in addition to inhibition. Although compound
2
4
2
was not highly cytotoxic in comparison with CX-4945, its new action mechanism, namely
obvious influence on the downstream proteins of CK2 and the lasting action time due to
the rebirth of the PROTAC, made it superior to the inhibitor. Interestingly, the cytotoxicity
of compound
A549/DDP, the higher the CK2 level in the cancer cells, the better the anticancer activity
of compound Fig. S5-S7). But compound was less toxic toward normal human
2 was dependent on the CK2 levels of the cancer cells such as A549, and
2
(
2

HUVEC cells, that maybe due to the fact that UPS was more active in cancer cells.[9,12]
Notably, pomalidomide exhibited almost no cytotoxicity toward all the tested cancer cells.
Table 1. Cytotoxicities of compounds 1-4 toward a few cancer cell lines and a normal cell
line for 72 h.
IC50a (μM)
Compound
MDA-MB-231b
A549c
A549/DDPd
HUVECe
22.56 ± 1.89
17.36 ± 1.43
21.32 ± 1.85
31.67 ± 2.97
15.77 ± 1.02
> 200
25.75 ± 0.91
31.19 ± 2.95
29.77 ± 0.84
36.46 ± 0.52
22.24 ± 2.01
> 200
79.51 ± 3.10
57.55 ± 4.20
30.68 ± 2.45
> 200
29.92 ± 1.81
34.39 ± 2.99
33.92 ± 2.84
37.13 ± 2.52
22.31 ± 2.05
> 200
1
2
3
4
CX-4945
Pomalidomide
27.97 ± 2.16
> 200
a IC50 is the drug concentration effective at inhibiting 50% of the cell growth measured by
the MTT assay after 72 h of drug exposure expressed as the mean value ± standard
b
deviation of three independent experiments. MDA-MB-231: human triple-negative breast
c
d
carcinoma cell line. A549: human non-small cell lung cancer cell line. A549/DDP:
e
cisplatin resistant non-small cell lung cancer cell line. HUVEC: human umbilical vein
endothelial cells.
2.8. Apoptosis study
According to the results of cytotoxicity assay, the apoptotic analysis of compound
2
as well as CX-4945 against MDA-MB-231 were carried out by an Annexin V-FITC/PI
assay. The tested compounds were incubated with MDA-MB-231 cells for 24 h at different
concentrations. As showed in Fig. 7, the effect of compound
2 on apoptotic rate (26.2%,
17.3 μM) of MDA-MB-231 cells was higher than that of CX-4945 (16.8%, 15.7 μM) after
24 h incubation at the concentration of IC50 values, respectively. Even at a a higher
concentration of 35 μM, the number of apoptotic cells induced by compound
2 (31.0%)
was also higher than that by CX-4945 (19.3%) for 24 h incubation. Although the in vitro

cytotoxicity of compound
2 for 72 hours was closed to that of CX-4945, its effect on
apoptotic rate for 24 hours was quite higher than that of CX-4945, which may be attributed
to the quick degradation of CK2 and rapid changes of the downstream proteins of CK2.
And the higher apoptosis rate in a shorter time period than that of inhibitor would be
another advantage of the PROTAC over the inhibitor.
Fig. 7. Flow cytometric analysis of the distribution of MDA-MB-231 cells treated with
CX-4945 and compound
2 at the concentration of IC50 values or 35 μM for 24 h.
2.9. Cell cycle analysis
The effect of compound
2 on MDA-MB-231 cell cycle progression was examined by
flow cytometry with propidium iodide (PI) staining, using CX-4945 as a positive control.
As shown in Fig. 8, in the control group, 53.89% cells were arrested at G1 phase, 32.02%
at S phase, and 14.09% at G2/M phase. CX-4945 arrested the cell cycle mainly at G2/M
phase (26.70% at G1 phase, 36.76% at S phase and 36.54% at G2/M phase), and
compound
2 also arrested the cell cycle mainly at G2/M phase, meanwhile, a small amount
of G1 phase cell cycle arrest was observed (60.54% at G1 phase, 13.77% at S phase and
25.69% at G2/M phase). The results could be due to the fact that CX-4945 and compound
2
had the same target of CK2, but worked in different ways.

Fig. 8. Cell cycle distributions upon treatment with CX-4945 and compound 2 at the concentration
of 35 μM for 24 h.
3. Conclusions
We have reported the design and synthesis of four CK2 targeting compounds composed of a
CK2 inhibitor CX-4945 and pomalidomide, which are connected by alkyl linkers via click
reactions. Among them, compound 2 exhibited a strong PROTAC feature. As a CK2 protein
degrader, compound 2 degrades the protein in a dose and time-dependent manner, and can
maintain CK2 at a low basal level. And the degrader degrades the protein through an ubiquitin and
proteasome-dependent proteolysis pathway. The degradation of CK2 by compound 2 resulted in a
series of changes in its downstream proteins which include the reduced phosphorylation of Akt (a
phosphorylated substrate of CK2) and the up-regulation of p53 (a tumor suppressor protein).
Significantly, 2 also possessed similar cytotoxicity to CX-4945 toward CK2 overexpressed cancer
cells like MDA-MB-231, but its mechanism of action is quite different from the inhibitor. Owing
to the overexpression of CK2 in most tumors with highly pleiotropic features, and more obvious
influence on the downstream proteins of CK2 during a shorter time period triggered by PROTAC,

targeting CK2 degradation will have potential applications for cancer treatments.
4. Experimental section
4.1 Reagents and instruments
All regents and solvents were obtained from commercial suppliers, and used as received
unless otherwise stated. CK2 Kinase Assay/Inhibitor Screening Kit was from CycLex Corp. All
antibodies used in this study were purchased from Cell Signaling Technology (CST). CX-4945
1
was synthesized in our lab, purity > 98%. H- and 13C-NMR spectra were recorded on a Bruker
400 MHz or 600 MHz spectrometer, using DMSO-d6 as a solvent. 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 analyzer (Elementary). Bio-Rad’s iMark microplate
absorbance reader was used for MTT and CK2 kinase assays.
4.2 General procedure for preparation
General synthetic procedure of alkyne derivatives 6. A solution of CX-4945 (350.0 mg, 1.0
mmol) and TBTU (385.0 mg, 1.2 mmol) in 20 mL of acetone was stirred at room temperature.
After 15 min, excess TEA was added and the reaction mixture was stirred for another 15 min.
Alkynol 5 (1.2 mmol) was then added and the reaction mixture was kept stirring under reflux for
24 h. The solvent was then removed by evaporation under reduced pressure, and DCM was added.
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 crude
compounds 6a-d as yellow oil. Yields: 6a: 82.3%, 6b: 91.2%, 6c: 83.7%, 6d: 87.2%. The crude
products were used directly for the subsequent reaction without further purification.
Synthesis
of
2-(2,6-dioxopiperidin-3-yl)-4-nitroisoindoline-1,3-dione
(9).
4-Nitro-2-benzofuran -1,3-dione (7) (280.0 g, 1.4 mmol) and AcONa (177.0 mg, 2.1 mmol) were
added to a solution of 3-aminopiperidine-2,6-dione trifluoroacetate (8) (350.0 g, 1.4 mmol) in
acetic acid (30 mL) and the reaction mixture was stirred at refluxing for 6 h. After the mixture was
cooled to room temperature, gray solid precipitated, which was filtered out and washed with water
to give compound 9 (Yield: 290.0 mg, 66.4%). ESI-MS: calcd m/z = 303.05, found [M+H]+ =

304.05, [M+Na]+ = 326.03 and [M+K]+ = 342.00.
Synthesis of pomalidomide (10). 9 (410.0 mg, 1.4 mmol), NH4Cl (360.0 mg, 7.0 mmol) and
iron powders (380.0 mg, 7.0 mmol) were mixed in a EtOH/H2O (v:v = 7:3) solution (40 mL). The
resulting mixture was kept stirring at room temperature overnight. Then saturated NaHCO3
solution was added to the reaction mixture which was then extracted with ethyl acetate. The
organic layer was dried over sodium sulfate, filtered and concentrated under reduced pressure to
give compound 10 as a yellow powder (Yield: 163.0 mg, 44.4%). ESI-MS: calcd m/z = 273.07,
found [M+H]+ = 274.07, [M+Na]+ = 296.05 and [2M+Na]+ = 569.12 . 1H-NMR (ppm, 400 MHz,
DMSO-d6): δ 11.11 (s, 1H), δ 7.47 (q, J = 6.0 Hz, 1H ), δ 7.01 (q, J = 6.0 Hz, 2H ), δ 6.54 (s, 2H ),
δ 5.05 (q, J = 10.0 Hz, 1H ), δ 2.89 (m, 1H ), δ 2.57 (m, 2H ), δ 2.02 (m, 1H ). 13C-NMR (ppm,
100 MHz, DMSO-d6): δ 173.32, 170.63, 169.04, 167.85, 147.19, 135.94, 132.47, 122.18, 111.46,
108.97, 48.95, 31.46, 22.62.
Synthesis of 2-chloro-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide
(11). Chloroacetyl chloride (450 μL) was added to a suspension solution of 10 (500.0 mg, 1.83
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 11 as a white solid (Yield:
558.0 mg, 87.2%). ESI-MS: calcd m/z = 349.05, found [M+H]+ = 350.05 and [M+Na]+ = 372.03.
1H-NMR (ppm, 400 MHz, DMSO-d6): δ 11.19 (s, 1H), δ 10.32 (s, 1H), δ 8.54 (d, J = 4.0 Hz,
1H),δ 7.89 (t, J = 8.0 Hz, 1H ), δ 7.69 (d, J = 4.0 Hz, 1H ), δ 5.18 (q, J = 8.0 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-azido-N-(2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)acetamide (12).
NaN3 (142 mg, 2.2 mmol) was slowly added to a suspension solution of 11 (380.0 mg, 1.1 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 12 as a white solid (Yield:
293.0 mg, 75.7%). ESI-MS: calcd m/z = 356.09, found [M+H]+ = 357.09 and [M+Na]+ = 379.07.
1H-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 ).
General synthetic procedure of compounds 1-4. “Click reaction” was carried out to prepare
compounds 1-4. In brief, equimolar alkyne derivative (5, 6, 7 or 8) and azide (12) were mixed in
THF/H2O (v:v = 1:1). After the mixture was stirred for 5 min, catalytic amount of CuSO4·5H2O
and sodium ascorbate was added under nitrogen atmosphere, then the mixture was kept stirring at
room temperature overnight. Removal of the organic solvent by evaporation under reduced
pressure led to the deposit from the solution. The resulting deposit was filtered out and washed
with water to give crude products. The pure products were obtained by column chromatography
using silica as stationary phase and a solution of 10:1 dichloromethane/methanol as eluent.
2-(1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)-1H-1,2,3-t
riazol-4-yl)ethyl 5-((3-chlorophenyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (1)： Yield:
46.5%. ESI-MS: calcd m/z = 757.18, found [M+Na]+ = 780.16. Anal. Calcd (%) for C38H28ClN9O7:
C 60.20, H 3.72, N 16.63. Found: C 60.01, H 3.81, N 16.46. 1H-NMR (ppm, 600 MHz, DMSO-d6):
δ 11.15 (s, 1H), δ 10.25 (s, 1H), δ 9.97 (s, 1H), δ 9.68 (s, 1H), δ 9.11 (s, 1H), δ 8.81 (d, J = 6.0 Hz,
1H), δ 8.63 (s, 1H), δ 8.35 (d, J = 12.0 Hz, 1H), δ 8.27 (t, J = 6.0 Hz, 1H ), δ 8.23 (d, 1H), δ 8.14
(s, 1H), δ 8.09 (dd, J = 6.0 Hz, 1H), δ 7.95 (dd, J = 6.0 Hz, 1H), δ 7.71 (t, J = 6.0 Hz, 1H ), δ 7.53
(d, J = 6.0 Hz, 1H ), δ 7.45 (t, J = 6.0 Hz, 1H ), δ 7.14 (dd, J = 6.0 Hz, 1H), δ 5.54 (s, 2H), δ 5.12
(q, J = 6.0 Hz, 1H ), δ 4.62 (t, J = 6.0 Hz, 2H ), δ 3.24 (t, J = 6.0 Hz, 2H), δ 2.90 (m, 1H ), δ 2.60
(m, 2H ), δ 2.09 (m, 1H ). 13C-NMR (ppm, 150 MHz, DMSO-d6): δ 173.18, 170.20, 167.67,
166.93, 166.12, 165.92, 150.65, 148.15, 147.92, 147.05, 143.92, 143.74, 142.18, 136.44, 135.83,
133.19, 131.91, 130.88, 130.50, 128.59, 126.88, 125.04, 124.78, 124.71, 124.23, 123.22, 123.09,
122.67, 120.62, 119.62, 119.33, 117.93, 64.48, 52.88, 49.42, 31.61, 25.38, 22.42.
3-(1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)-1H-1,2,3-t
riazol-4-yl)propyl 5-((3-chlorophenyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (2) ：
Yield: 67.2%. ESI-MS: calcd m/z = 771.20, found [M+Na]+ = 794.18. Anal. Calcd (%) for
1
C39H30ClN9O7: C 60.66, H 3.92, N 16.33. Found: C 60.42, H 4.02, N 16.07. H-NMR (ppm, 600
MHz, DMSO-d6): δ 11.17 (s, 1H), δ 10.18 (s, 1H), δ 10.05 (s, 1H), δ 9.66 (s, 1H), δ 9.04 (s, 1H), δ
8.84 (d, J = 12.0 Hz, 1H), δ 8.58 (s, 1H), δ 8.39 (d, J = 6.0 Hz, 1H), δ 8.26 (s, 1H ), δ 8.22 (s, 1H),
δ 8.10 (d, J = 6.0 Hz, 1H), δ 8.03 (s, 1H), δ 7.95 (t, J = 6.0 Hz, 1H), δ 7.81 (t, J = 6.0 Hz, 1H ), δ

7.64 (d, J = 12.0 Hz, 1H ), δ 7.45 (t, J = 6.0 Hz, 1H ), δ 7.14 (t, J = 6.0 Hz, 1H), δ 5.51 (s, 2H), δ
5.14 (q, J = 6.0 Hz, 1H ), δ 4.42 (t, J = 6.0 Hz, 2H ), δ 3.94 (s, 1H), δ 2.89 (t, J = 6.0 Hz, 2H), δ
2.60 (m, 2H ), δ 2.17 (t, J = 6.0 Hz, 2H), δ 2.09 (m, 1H ). 13C-NMR (ppm, 150 MHz, DMSO-d6):
δ 173.20, 170.22, 167.68, 166.98, 166.16, 165.97, 150.54, 148.10, 147.98, 146.65, 143.64, 142.17,
136.57, 135.85, 133.19, 132.02, 130.96, 130.73, 130.48, 128.45, 127.00, 124.29, 124.17, 124.13,
123.06, 122.65, 120.60, 120.54, 119.60, 119.54, 119.47, 118.09, 67.47, 64.87, 52.79, 49.43, 31.41,
25.59, 22.25.
5-(1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)-1H-1,2,3-t
riazol-4-yl)pentyl 5-((3-chlorophenyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate (3)：Yield:
42.3%. ESI-MS: calcd m/z = 799.23, found [M+Na]+ = 822.21. Anal. Calcd (%) for C41H34ClN9O7:
C 61.54, H 4.28, N 15.75. Found: C 61.33, H 4.40, N 15.50. 1H-NMR (ppm, 600 MHz, DMSO-d6):
δ 11.16 (s, 1H), δ 10.01 (s, 1H), δ 9.67 (s, 1H), δ 8.84 (d, J = 6.0 Hz, 1H), δ 8.68 (s, 1H), δ 8.32 (d,
J = 6.0 Hz, 1H), δ 8.28 (s, 1H ), δ 8.24 (s, 1H), δ 8.08 (d, J = 6.0 Hz, 1H), δ 7.96 (d, J = 12.0 Hz,
1H), δ 7.97 (s, 1H ), δ 7.76 (t, J = 6.0 Hz, 1H ), δ 7.61 (d, J = 6.0 Hz, 1H ), δ 7.45 (t, J = 6.0 Hz,
1H), δ 7.14 (d, J = 6.0 Hz, 1H), δ 5.47 (s, 2H), δ 5.14 (q, J = 6.0 Hz, 1H ), δ 4.36 (t, J = 6.0 Hz,
2H ), δ 2.91 (m, 1H), δ 2.72 (t, J = 6.0 Hz, 2H), δ 2.61 (m, 2H ), δ 2.11 (m, 1H), δ 1.83 (m, 2H ), δ
1.73 (m, 2H ), δ 1.52 (m, 2H ). 13C-NMR (ppm, 150 MHz, DMSO-d6): δ 173.20, 170.22, 167.65,
166.96, 166.15, 165.96, 150.71, 143.90, 142.17, 136.52, 135.83, 133.20, 131.97, 131.09, 130.52,
128.48, 126.92, 124.16, 123.13, 122.69, 122.53, 120.64, 120.45, 119.63, 119.47, 119.41, 118.00,
65.34, 52.73, 49.43, 31.42, 29.00, 28.37, 25.50, 25.35, 22.45.
2-((1-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)-2-oxoethyl)-1H-1,2,3
-triazol-4-yl)methoxy)ethyl-5-((3-chlorophenyl)amino)benzo[c][2,6]naphthyridine-8-carboxylate
(4)： Yield: 79.7%. ESI-MS: calcd m/z = 787.19, found [M+Na]+ = 810.17. Anal. Calcd (%) for
1
C39H30ClN9O8: C 59.43, H 3.84, N 15.99. Found: C 59.31, H 3.96, N 15.86. H-NMR (ppm, 600
MHz, DMSO-d6): δ 11.16 (s, 1H), δ 10.14 (s, 1H), δ 10.10 (s, 1H), δ 9.67 (s, 1H), δ 9.00 (d, J =
6.0 Hz, 1H), δ 8.86 (d, J = 12.0 Hz, 1H), δ 8.58 (d, J = 6.0 Hz, 1H), δ 8.31 (d, J = 12.0 Hz, 1H), δ
8.25 (s, 2H ), δ 8.19 (s, 1H), δ 8.11 (d, J = 12.0 Hz, 1H), δ 7.96 (d, J = 12.0 Hz, 1H), δ 7.76 (t, J =
6.0 Hz, 1H ), δ 7.62 (d, J = 6.0 Hz, 1H ), δ 7.46 (t, J = 6.0 Hz, 1H ), δ 7.14 (d, J = 6.0 Hz, 1H), δ
5.76 (s, 2H), δ 5.15 (q, J = 6.0 Hz, 1H ), δ 4.70 (s, 2H ), δ 4.51(t, J = 6.0 Hz, 1H ), δ 3.88 (t, J =

6.0 Hz, 2H), δ 2.91 (m, 1H ), δ 2.61 (m, 2H ), δ 2.09 (m, 1H ). 13C-NMR (ppm, 150 MHz,
DMSO-d6): δ 173.22, 170.24, 167.60, 166.98, 165.99, 165.98, 150.65, 148.21, 148.16, 144.41,
143.68, 142.17, 136.50, 135.77, 133.21, 132.03, 130.88, 130.58, 128.58, 127.20, 127.13, 126.18,
124.49, 124.28, 123.23, 123.16, 122.74, 120.68, 119.70, 119.49, 118.19, 116.80, 67.94, 64.70,
63.88, 52.74, 49.43, 31.41, 22.44.
4.3 CK2 activity assay
The CK2 activity was detected with the CK2 assay kit treated with 10 μM compounds 1-4,
CX-4945, respectively, according to the manufacturer’s instruction. OD values were measured in
each well using a spectrophotometric plate reader at the wavelength of 450 nm. Results are
representative of three independent experiments and shown as the average values.
4.4 Western blotting
After the treatment with the indicated concentration of each sample for some time (12, 24 or
36 h), MDA-MB-231 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 ℃. 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 ℃, followed by overnight incubation at 4 ℃ with primary antibodies diluted in
conjugated secondary antibody diluted 1:1000 in PBST, and the labeled proteins were detected
with an Odyssey Scanning System (Li-COR., Lincoln, Nebraska, USA).
4.5 Stability study by HPLC.

The stability of compound 2 in PBS (pH=5.0 and 7.4) and DMEM (0.05~0.1 mg/mL) were
examined at 25 °C by HPLC. Reversed phase HPLC was implemented on a 250 × 4.5 mm ODS
column and the HPLC profiles were recorded on UV detection at 254 nm. Mobile phase consisted
of methanol/water (70:30, v/v), and flow rate was 1.0 mL/min. The samples were taken for HPLC
analysis after filtration by 0.45 μm filter.
4.6 Cell lines and cell culture.
MDA-MB-231 (human triple-negative breast cancer cells), A549 (human non small cell lung
cancer cells) and A549/DDP (cisplatin-resistant cancer cells) as well as HUVEC (human umbilical
vein endothelial cells) were purchased from Jiangsu KeyGen BioTECH Co. Ltd. MDA-MB-231,
A549 and HUEVC 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.
Cisplatin-resistant A549/DDP cells were cultured and screened in RPMI-1640 supplemented with
10% FBS, streptomycin (100 μg/mL), and ampicillin sodium (100 μg/mL) and 800 ng/mL
cisplatin before use.
4.7 Cytotoxicity measurement.
The in vitro activity of the compounds was determined by the MTT method. 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 and curves of cell viability were drawn by EXCEL.
4.8 Apoptosis analysis.

MDA-MB-231cells with good activity (5 × 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, 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).
4.9 Cell Cycle Measurement.
MDA-MB-231 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, 35 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 1 × 104 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 Jiangsu Province Natural Science Foundation (No. BK20150643) for
financial aids to this work. 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.
Abbreviations
BTZ, bortezomib; CK2, casein kinase 2; DCM, dichloromethane; DMEM, dulbecco's
modified eagle medium; DMSO, dimethyl sulfoxide; FBS, fetal bovine serum; FITC, fluorescein
isothiocyanate; MTT, 3-(4,5-dimethyl-2-thiazolyl)-2,5-diphenyl-2-H-tetrazolium bromide; PBS,
phosphate buffered saline; PROTAC, proteolysis targeting chimera; rt, room temperature; TBTU,
O-(Benzotriazol-1-yl)-N,N,N',N'-tetramethyluronium tetrafluoroborate; TEA, triethylamine; THF,
tetrahydrofuran; UV, ultraviolet and visible spectrum.
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Graphical abstract

Highlights





Chemically induced degradation of CK2 by a proteolysis targeting chimera.
Compound 2 degraded CK2 in a dose and time-dependent manner.
Downstream proteins of CK2 were more susceptible to its degradation.
2 degraded CK2 through the ubiquitin–proteasome pathway.
2 could induce more cell apoptosis during a shorter time period.