Development of selective mono or dual PROTAC degrader probe of CDK isoforms

Article abstract of DOI:10.1016/j.ejmech.2019.111952

Cyclin-dependent kinase (CDK) family members are promising molecular targets in discovering potent inhibitors in disease settings, they function differentially. CDK2, CDK4 and CDK6, directly regulate the cell cycle, while CDK9 primarily modulates the transcription regulation. In discovering inhibitors of these CDKs, toxicity associated with off-target effect on other CDK homologs often posts as a clinical issue and hinders their further therapeutic development. To improve efficacy and reduce toxicity, here, using the Proteolysis Targeted Chimeras (PROTACs) approach, we design and further optimize small molecule degraders targeting multiple CDKs. We showed that heterobifunctional compound A9 selectively degraded CDK2. We also identified a dual-degrader, compound F3, which potently induced degradation of both CDK2 (DC₅₀: 62 nM) and CDK9 (DC₅₀: 33 nM). In human prostate cancer PC-3 cells, compound F3 potently inhibits cell proliferation by effectively blocking the cell cycle in S and G2/M phases. Our preliminary data suggests that PROTAC-oriented CDK2/9 degradation is potentially an effective therapeutic approach.

Full text of DOI:10.1016/j.ejmech.2019.111952

Journal Pre-proof
Development of selective mono or dual PROTAC degrader probe of CDK isoforms
Fei Zhou, Luyu Chen, Chaoguo Cao, Jiang Yu, Xiaojiao Luo, Peiting Zhou, Lifeng
Zhao, Wu Du, Jijun Cheng, Yongmei Xie, Yuanwei Chen
PII:
S0223-5234(19)31104-3
DOI:
https://doi.org/10.1016/j.ejmech.2019.111952
EJMECH 111952
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 9 October 2019
Revised Date: 27 November 2019
Accepted Date: 3 December 2019
Please cite this article as: F. Zhou, L. Chen, C. Cao, J. Yu, X. Luo, P. Zhou, L. Zhao, W. Du, J. Cheng,
Y. Xie, Y. Chen, Development of selective mono or dual PROTAC degrader probe of CDK isoforms,
European Journal of Medicinal Chemistry (2020), doi: https://doi.org/10.1016/j.ejmech.2019.111952.
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2019 Published by Elsevier Masson SAS.

Development of selective mono or dual PROTAC degrader probe of CDK
isoforms
a
a
a
a
a
a
Fei Zhou , Luyu Chen , Chaoguo Cao , Jiang Yu , Xiaojiao Luo , Peiting Zhou ,
b
c
c
a
a, c, *
Lifeng Zhao , Wu Du , Jijun Cheng , Yongmei Xie , Yuanwei Chen
State Key Laboratory of Biotherapy and Cancer Center, West China Hospital,
Sichuan University and Collaborative Innovation Center of Biotherapy, Chengdu
a
6
10041, China
b
Sichuan Industrial Institute of Antibiotics, Chengdu University, Chengdu 610052,
China
c
Hinova Pharmaceuticals Inc, 4th Floor, Building RongYao A, No. 5, Keyuan South
Road, Chengdu, 610041, China
*
Corresponding author.
E-mail address: ywchen@scu.edu.cn
Highlights
1
2
3
4
. We designed and synthesized two series PROTAC compounds by tethering CDK
inhibitors with CRBN ligands.
. We identified compounds differentially induced dual CDK2/9 degradation, or
selective to CDK2 or to CDK9.
. Compound F3 is a potent dual degrader for CDK2 (DC : 62 nM) and CDK9
5
0
(
DC : 33 nM).
50
. Compound F3 suppresses prostate cancer PC-3 cell proliferation by interfering
with cell cycle progression.
Abstract
Cyclin-dependent kinase (CDK) family members are promising molecular
targets in discovering potent inhibitors in disease settings, they function differentially.
CDK2, CDK4 and CDK6, directly regulate the cell cycle, while CDK9 primarily
modulates the transcription regulation. In discovering inhibitors of these CDKs,
toxicity associated with off-target effect on other CDK homologs often posts as a
clinical issue and hinders their further therapeutic development. To improve efficacy
and reduce toxicity, here, using the Proteolysis Targeted Chimeras (PROTACs)
approach, we design and further optimize small molecule degraders targeting multiple
CDKs. We showed that heterobifunctional compound A9 selectively degraded CDK2.
We also identified a dual-degrader, compound F3, which potently induced
degradation of both CDK2 (DC : 62 nM) and CDK9 (DC : 33 nM). In human
5
0
50
prostate cancer PC-3 cells, compound F3 potently inhibits cell proliferation by
effectively blocking the cell cycle in S and G2/M phases. Our preliminary data
suggests that PROTAC-oriented CDK2/9 degradation is potentially an effective
therapeutic approach.
Key words: PROTAC, Cell cycle, CDK2, CDK9, Prostate cancer.
1
. Introduction
Cyclin dependent kinases (CDKs) are serine/threonine protein kinases encoded
by 21 genes in human [1, 2]. One class of CDKs, CDKs 1, 2, 4 and 6, play extensive
roles in eukaryotic cell cycle checkpoint regulation. A second class of the sub-branch
of the family, CDKs 7, 8, 9, 12 and 13, were identified as transcriptional regulators [3].

Of particular interest is CDK4/6, which, in the cell cycle forms an active complex
with cyclin D that which phosphorylates the retinoblastoma protein (pRb) to reduce
the inhibition of transcription factor activity of the E2F family. Later during late G1
phase, CDK2 takes turn, upon binding to cyclin E, to further phosphorylate the Rb
protein, potently relieving the E2F suppression to allow cell cycle entry into S phase.
During the entire S phase, CDK2 in complex with cyclin A controls the progression of
DNA synthesis [4, 5]. To further understand the activation mechanism of CDKs, the
crystal structure of CDK2 was first identified within the CDK family [6,7], followed
by structure resolution of the cyclinA-CDK2 complex, providing a computational
model to understand the mechanism of CDKs activation [8]. In cancers where cell
proliferation is deregulated, overexpression of CDKs is often the driver of cancer
pathogenesis, and thus, targeting CDKs has become of much interest to combat cancer.
[
9].
Small molecule CDK inhibitors have been used in clinical studies to treat various
cancers, including, but not limited to, acute myeloid leukemia (AML), breast cancer
BC), non-small cell lung cancer (NSCLC), and prostate cancer (PC) [10-13].
(
However, poor therapeutic efficacy and serious toxicity response have hindered their
clinical development [14]. Emerging Proteolysis Targeted Chimeras (PROTACs)
technique, which tethers a small molecule to a ligand for the E3 ubiquitin ligase,
converts target inhibitors into advantageous target degraders [15, 16]. Determination
of crystal structure of the DDB1–CRBN bound to the drug thalidomide, and studies of
phthalimide binding to the CRL4CRBN E3 complex for ubiquitination and
subsequent proteasome-mediated degradation, have recently helped to extend the
capacities of the PROTAC technique [17-20]. The PROTAC technique has been
shown to selectively degrade specific homologous proteins, including members of the
bromodomain-containing proteins, as well as CDKs [21-23]. Out of the few reported
CDK9 degraders, THAL-SNS-032 stand out as a highly efficient degrader for CDK9
[
23], and showed differential pharmacological effects between inhibitors and
degraders. Recently, degraders designed based on three FDA approved CDK4/6
inhibitors palbociclib, ribociclib, and abemaciclib, also achieved selective degradation
of CDK4 and CDK6 or dual CDK4/6 [24] (Figure 1).
Figure 1. Representative CDK PROTACs
To date, PROTAC technique has been applied to a number of kinase targets
[
25-27]. CDK2 has not been identified as a degradable target. The ubiquitination of
CDK2 could suppress tumor cell growth effectively and safely [28], and the
degradation of CDK2 could eliminate the block of tumor cell differentiation [29].
Development of degrader probe targeting CDK2 may help to understand the CDK2
function as well as to explore potential therapeutic intervention to cancer. In this study,

for the first time we report the design and synthesis of novel CDK small molecule
PROTAC degraders which degrade CDK2 solely or CDK2/9 dually. Some of them
strongly inhibit proliferation of prostate cancer PC-3 cells through mechanism of
down-regulating the CDK2/9 signaling pathways including proto-oncogene products,
suggesting potential therapeutic usage.
2
2
. Results and discussion
.1. Binding-mode analysis
Two pan CDK inhibitors which designed based on CDK2 crystal structure are
proposed to conjugate to cereblon (CRBN) ligands [30, 31]. To identify suitable
attachment points, we modeled the structure of CDK2 complexed with AT-7519 and
FN-1501 respectively. The best poses were visualized with DS3.5 (Figure 2A). As
expected, hydrophobic end of the inhibitor should be attached with linkers. Linker
length and the attachment points of CRBN ligands are uncertain factors [32]. Thus,
we designed series compounds and predicted that the chemical structures would not
affect the ability of the degraders to bind the target proteins (Figure 2B).
Figure 2. Cocrystal structures of CDK2 complexed with AT-7519 and FN-1501 (PDB code:
2
VTH). (A) The best pose of AT-7519 (top panel) and FN-1501 (bottom panel) in CDK2 crystal
structure. (B) Chemical structures of designing PROTAC molecules based on AT-7519 and
FN-1501.
2
.2. Chemistry
AT-7519-based (A1-A10) and FN-1501-based (F1-F10) compound series share
the same flexible chains connecting CDK inhibitors with pomalidomide or 3-hydroxy
thalidomide. In addition to alkyl linkers with different lengths, we employed two
hydrophilic alkyl ether chains (Figure 3). CDKs ligands 1 and 2 and CRBN ligands 9
and 16 were prepared as previously described [24, 30, 31]. Intermediates 3 and 4 were
prepared from 1 and 2 with succinic anhydride. Compounds 5-8 were prepared from
compounds 1 and 2 by acylation with various linkers. Compounds 12 and 14 were
obtained from 9 through three steps synthesis. A mixture of compound 16 and
Mono-Boc protected alkyl diamines in 1-methyl-2-pyrrolidinone (NMP) and DIPEA
was stirred at 90 °C for 12 hours to give compound 17. Finally, target compounds
were prepared by connecting the intermediates after Boc-deprotection (Scheme 1).

Scheme 1. Reagents and conditions: (a) Succinic anhydride, dichloromethane (DCM), Et N, rt, 24
3
h; (b) N-Boc-γ-aminobutyric acid or N-Boc-aminohexanoic acid, HATU, DIPEA, DMF, rt, 12 h;
(c) 8-tert-butyloxycarbonylamino-3,6-dioxaoctanoic acid, HATU, DIPEA, DMF, rt, 12 h; (d)

Tert-butyl bromoacetate, KI, KHCO , DMF, 60 °C, 12 h; (e) CF COOH, DCM, rt, 2 h; (f) HATU,
3
3
DIPEA, DMF, rt, 12 h; (g) NMP, DIPEA, 90 °C, 12 h.
Figure 3. List of linkers used in designed degraders.
2
.3. Western blotting and cellular activity analysis
We first evaluated the ability of 20 target compounds to induce degradation of
the primary CDK targets of AT-7519 in AR-negative human prostate cancer PC-3 cells
by Western blotting. Cells were treated with A1-A10 and F1-F10 respectively for 12
hours. Western blotting showed that compounds A2 and A9 induced selective CDK2
degradation at 1 µM effectively while sparing CDK5 and CDK9 (Figure 4A).
Compound A2 showed degradation activity against both CDK2 and CDK9 at
concentration of 5 µM. Compound A9 induced a modest reduction of CDK9 similar
to inhibition with AT-7519 treatment at concentration of 5 µM (Figure 4B).
Compounds F1-F3 achieved dual CDK2/9 degradation at 1 µM; compounds F5, F6,
and F9 have longer linker connecting the same CDK and CRBN ligands as F1-F3,
achieving selective CDK9 degradation (Figure 4C). This selectivity of CDK is due to
the differential distance between different CDK subunits and CRBN and the
redundant chains affect ternary complex formation. Interestingly, different from low
concentration, F4 could not induce CDK2 degradation at 1 µM and 30µM,
demonstrating a ‘hook effect’. On the other hand, we did not find any degraders that
lost the ability to degrade CDK9 at 30 µM (Figure 4C and Figure 4E). Thus, it is
feasible to degrade CDK9 by F series PROTAC molecules with linkers containing
8
-12 atoms. The chain length of 10 atoms is the maximum linker length for achieving
degradation of CDK2 (Figure 4C and Figure 4D). Compound F6 has a linker length
between those of compounds F4 and F5, but its degradation ability is weaker than
both of them, indicating that pomalidomide recruits E3 ubiquitin ligase better than
4
-hydroxy thalidomide (Figure 4D). To find the most effective degrader, compounds
F2, F3, F4, F5 and F9 were evaluated at 50 nM concentration (Figure 4F), and we
found that F3 is the best whereas all other compounds have little degradation. To
further investigate the correlation between CDKs degradation and anti-proliferative,
all of target compounds were evaluated against PC-3 cells using the cck-8 assay
(
Table 1). The CDK2 degrader A9 has an IC value of 0.84 µM, showing comparable
50
potency to that of AT-7519. The most potent CDK2/9 degrader compound F3 achieves

an IC of 0.12 µM and is 4-20 times more potent than other CDK2/9 degraders F1,
5
0
F2 and F4. Compounds F5，F6 and F9 with selective CDK9 degradation activity
showed weaker cell activity than CDK2/9 degraders F1-F4 generally. In contrast,
compounds F7, F8, and F10 failed to decrease the level of CDK2/9 and thus
displayed decreased inhibitory activities, with IC values ranging from 7.30 to 18.34
5
0
µM. In general, the cell activity result correlated well with CDKs degradation activity.
The data obtained are summarized in Table 1.
Figure 4. Degradation of CDK2, CDK5 and CDK9 by degraders. PC-3 cells were treated with (A)
Compounds A1-A10 at 1 µM, (B) Compounds A1-A10 at 5 µM, (C) Compounds F1-F10 at 1 µM,
(D) Compounds F1-F10 at 500 nM, (E) Compounds AT-7519, FN-1501, A2, A9, F1-F4, F5, F6
and F9 at 30 µM, (F) Compounds F2-F5 and F9 at 50 nM, for 12 hours, followed by Western
blotting analysis of CDK2, CDK5, CDK9, and loading control β-tubulin.
Table 1 Activity of compounds in vitro
a
Compd
PC-3 IC ±SD (µM)
Degradation
CDK9
5
0
CDK2
AT-7519
A1
0.71±0.13
NT
–
–
+
–
–
+
A2
NT
A3
5.95±1.16
23.32±3.17
14.56±1.63
NT
–
-
-
A4
-
A5
-
-
A6
-
-
A7
NT
-
-
A8
1.31±0.02
-
-

A9
A10
FN-1501
F1
0.84±0.13
2.02±0.55
0.69±0.02
2.21±0.36
0.54±0.02
0.12±0.02
1.38±0.15
3.60±0.78
8.03±1.95
13.39±1.56
7.30±1.14
3.54±0.71
18.34±0.69
+
-
-
+
+
+
+
-
-
-
-
-
-
-
-
-
+
+
+
+
+
+
-
-
+
-
F2
F3
F4
F5
F6
F7
F8
F9
F10
a
IC50 values were the average of three separate determinations (Mean ± SD). NT, indicates not
tested. (-) indicates not degradation detected, (+) indicates degradation detected
2
.4. PROTACs Mechanistic studies
To further assess the mechanism of action of these PROTACs beyond CDK
degradation, we first performed dose-response and time-course studies with
compound F3. As shown in Figure 5A, compound F3 showed clearly
concentration-dependent CDK2 and CDK9 degradation activity, achieving DC50
values as 62 nM and 33 nM, respectively. To determine whether compound F3
degrades CDK2 and CDK9 synchronously, we evaluated their degradation kinetics in
PC-3 cells by treatment with 500 nM compound F3 in a time course. As shown in
Figure 5B, degradation ratio of CDK9 increased dramatically by >80% in only 4
hours while CDK2 degradation is minimal. This suggests F3 preferentially degrade
CDK9 over CDK2. Meanwhile, we did not find significant CDK4/6 downregulation,
which indicates selective CDK2/9 isoforms degradation. Transcription factor c-Myc
stimulates the cell cycle progression and the cellular proliferation. Uncontrolled
expression of c-Myc confers cells immortalization. Given c-Myc is historically
undruggable, depletion of CDKs may actually be an effective strategy for
down-regulating its expression [33]. While studying the kinetics of CDK2/9
degradation, we investigated changes in the protein level of c-Myc (Figure 5B). Our
data suggests that our PROTAC caused a significant down-regulation of c-Myc in
response to CDK2/9 downregulation. Similarly, compound F3 also down-regulated
the Mcl-1 protein level in PC-3 cells (Figure 5B). Next, we further explored the
mechanism of CDK2/9 degradation induced by compound F3. CDK2/9 degradation
was inhibited by pretreatment with Pomalidomide and FN-1501, indicating that
degradation required engagement of both CRBN and CDK2/9. Furthermore, the
degradation was blocked by pretreatment with proteasome inhibitor MG-132, which
is consistent with that PROTAC-induced protein degradation is proteasome dependent
(
Figure 5C). Compound A9 was investigated under similar conditions. We found that
it’s degradation of CDK2 is also CDK2, CRBN, and proteasome dependent. (Figure
D).
5

Figure 5. Kinetics and mechanisms of CDK2/9 degradation by compound F3. (A) CDK2 and
CDK9 degradation dose-response for compound F3. PC-3 Cells were treated with compound F3
at different doses as indicated for 24 hours, followed by Western botting of CDK2, CDK9 and
loading control β-tubulin; DC data represent the mean of 2 determinations. (B) Time-dependent
5
0
degradation of CDK2, CDK4, CDK6, CDK9, c-Myc and Mcl-1. PC-3 cells were treated with 500
nM of compound F3 for times as indicated, followed by Western botting analysis. (C) CDK2/9,
CRBN, and proteasome-dependent CDK2/9 degradation by compound F3. PC-3 cells were
pretreated 2 h with 5 µM DMSO, FN-1501, Pomalidomide and MG-132, respectively, followed by
8
h treatment with compound F3 at 250 nM, followed by Western botting of CDK2, CDK9 and
loading control β-tubulin. (D) CDK2, CRBN, and proteasome-dependent CDK2 degradation by
compound A9. PC-3 cells were pretreated 2 h with 10 µM DMSO, AT-7519, Pomalidomide and
MG-132, respectively, followed by 8 h treatment with compound A9 at 1 µM, followed by
Western botting of CDK2 and loading control β-tubulin.
2
.5. Development of analogues of compound F3
To further study the structure–degradation activity relationship, we synthesized
additional analogs compounds F11 and F12 by replacing pomalidomide of compound
F3 with lenalidomide and 4-amino thalidomide, and prepared compound F13 as a

control analog which lack of ability to bind CRBN (Figure 6A) (Scheme S1).
Compounds F11 and F12 significantly reduced CDK2/9 levels, similar to that of
compound F3, whereas CDK levels were unaffected in the presence of compound
F13 (Figure 6B, Figure 6C and Figure 6D). Compared to FN-1501 and its analogs,
compound F3 showed the best anti-cancer activities against PC-3 cells with IC50
values of 0.12 µΜ, and weak activity against non-tumor LO2 cells (IC : 7.99 µM),
5
0
indicating good selectivity (Figure 6E). Interestingly, the anti-proliferation activity of
F13 decreased strikingly, indicating that compound F3 inhibits cell growth by acting
as a CDK degrader but not as a CDK inhibitor (Figure 6E). These data suggests that
CDK2/9 elimination was critical to inhibit cancer cell proliferation. The cell-free
kinase assay confirmed that F3 inhibits both CDK2 and CDK9, with IC as 7.42 nM
5
0
and 14.50 nM, respectively (Figure 6F). Compound F3 was also able to effectively
degrade CDK2/9 in cell lines MCF-7, HCT-116 and 22Rv1, which all have high
CDK2/9 expression. (Figure 6G).

Figure 6. Further characterization of compound F3. (A) Chemical structures of compounds
F11-F13. (B) Dose-dependent CDK2/5/9 degradation studies by compound F13, (C) compound
F11 and (D) compound F12; PC-3 cells were treated with compounds F11, F12and F13 at
indicated concentrations for 12 hours, followed by Western blotting analysis of CDK2, CDK5,
CDK9 and β-tubulin. Compound F13 and FN-1501 at 1 µM used as a control. (E) Growth
inhibitory curves of indicated compounds in PC-3 cells and LO2 cells. IC50 values were the
average of three separate determinations. (F) Inhibitory activities of compound F3 toward CDK2
and CDK9 kinase in vitro. (G) Degradation of CDK2 and CDK9 by F3 in different cell lines as
indicated. The cells were treated with compound F3 at indicated concentrations for 12 hours,
followed by Western botting of CDK2, CDK9 and loading control β-tubulin.
2
.6. F3 induced cell cycle arrest
To investigate mechanisms of F3 anti-proliferation activity, we utilized the
CFSE-labeling. After PC-3 cells were treated with 2 µM compound F3 for 24 hours,
the fluorescence signal significantly different from that of the DMSO treated control
group, demonstrating that compound F3 severely affect the proliferation of PC-3 cells
(
Figure 7A). To further investigate whether the anti-proliferation activities were due
to cell cycle arrest, we performed cell cycle arrest assay by flow cytometry (Figure
B). Compound F3 obviously prolonged the S phase by 9.8% at 250 nM
7
concentration, and, when the concentration increases, induced cell cycle blockage at
the G2/M phase.

Figure 7. Compound F3 inhibits PC-3 cell proliferation and arrest cell cycle in G2/M phases. (A)
Flow cytometry analysis of CFSE-labeled PC-3 cells; PC-3 cells were treated with compound F3
at 2 µM for 24 hours. CFSE was added to the single cell suspension to achieve a final
concentration of 5 µM, and then incubated for 15 min at room temperature in the dark, followed
by flow cytometry analysis (B-C) Effects of F3 on PC-3 cell cycle progress. PC-3 cells were
incubated with compound F3 or FN-1501 at the indicated concentrations for 48 hours, and further
incubated with 50 mg/mL propidium iodide for 20 minutes in the dark, followed by flow
cytometry analysis.
3
. Conclusion
In this study, we designed and synthesized two series of compounds, and
evaluated their degradation activity against CDKs by Western blotting. In one series,
we identified a CDK2 selective degrader A9, which are valuable as starting
prototypes in designing more potent CDK2 degraders. Most compounds in
FN-1501-based series achieved either dual CDK2/9 degradation or selective CDK9
degradation. Structure–degradation relationship studies led to compound F3, which
showed potent and rapid degradation of CDK2 and CDK9. Cell cycle analysis
revealed that compound F3 suppressed PC-3 proliferation by delaying/arresting the
cell cycle in S/G2/M phases. Degradation of CDK2/9 activities of compound F3 in
three different cancer cell lines suggests that it has potential to treat multiple cancer
types. In summary, targeted degradation of CDK2/9 in cancers is a promising
therapeutic method, and compound F3 and its analogs worth further investigations.
4
. Experimental section
4
.1. Chemistry
All commercially obtained reagents and solvents were used as received without
further purification. Flash chromatography was performed using Biotage Isolera One
apparatus with Agela normal-phase silica cartridges. Nuclear Magnetic Resonance
(NMR) spectra were recorded on a Bruker AMX 400 spectrometer. The spectra were
referenced against the deuterated solvents with tetramethylsilane (TMS). In the
spectral data reported, the format (δ) chemical shift (multiplicity, J values in Hz,

integration) was used with the following abbreviations: s = singlet, d = doublet, t =
triplet, q = quartet, m = multiplet. HRMS spectra were measured on Q-Tofmicro
Premier mass spectrometer (Micromass, Manchester, UK) with an electrospray
ionization (ESI) source.
4
4
.1.1. 4-(4-(4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamido)piperidin-1-yl)-
-oxobutanoic acid (3)
Et N (150 µL, 1.1mmol,
-(2,6-dichlorobenzamido)-N-(piperidin-4-yl)-1H-pyrazole-3-carboxamide(1)
3
eq) was added to
a
solution of
(180
3
4
mg, 0.37 mmol, 1.0 eq) and succinic anhydride (44 mg, 0.44 mmol, 1.2 eq) in DCM.
The solution was stirred at room temperature for 24 h. After concentration, the residue
was purified by flash column chromatography to afford the title compound (3) as a
1
white solid (140 mg, 80% yield). H NMR (400 MHz, DMSO-d ) δ 10.17 (s, 1H),
6
8
3
2
.38 (d, J = 8.0 Hz, 1H), 8.34 (s, 1H), 7.61 – 7.52 (m, 3H), 4.34 (d, J = 12.8 Hz, 1H),
.97 – 3.89 (m, 2H), 3.04 (t, J = 12.4 Hz, 1H), 2.63 – 2.50 (m, 3H), 2.42 – 2.93 (m,
+
H), 1.80 – 1.71 (m, 2H), 1.58 – 1.39 (m, 2H). HRMS (ESI ): calcd for
+
C H Cl N O Na [M + Na] 504.0817, found 504.0812.
20
21
2
5
5
4
.1.2. 4-(4-(4-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole-3-carboxam
ido)benzyl)piperazin-1-yl)-4-oxobutanoic acid (4)
Compound 4 (927 mg, 90% yield) was obtained according to similar synthetic
1
procedures for 3. H NMR (400 MHz, DMSO-d ) δ 13.42 (s, 1H), 11.88 (s, 1H),
6
1
7
2
5
4
0.25 (s, 1H), 9.53 (s, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.34 –
.26 (m, 3H), 6.50 (dd, J = 3.2, 2.0 Hz, 1H), 3.47 – 3.45 (m, 6H), 3.18 (s, 2H), 2.46 –
+
+
.27 (m, 6H). HRMS (ESI ): calcd for C H N O [M + H] 518.2264, found
2
5
28
9
4
18.2263.
.1.3. tert-butyl(4-(4-(4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamido)piperi
din-1-yl)-4-oxobutyl)carbamate(5a)
DIPEA (46 mg, 0.36 mmol, 2.0 eq) and HATU (102 mg, 0.27 mmol, 1.5 eq)
were added to a solution of 1 (70 mg, 0.18 mmol, 1.0 eq) and N-Boc-γ-aminobutyric
acid (47 mg, 0.23 mmol, 1.3 eq) in DMF (5 mL). The reaction mixture was quenched
with H O and extracted with EtOAc. The organic layer was separated, washed with
2
brine, dried, and evaporated. The residue was purified by flash column
1
chromatography to afford 5a (60 mg, 58% yield). H NMR (400 MHz, CDCl ) δ 9.90
3
(s, 1H), 8.48 (d, J = 2.8 Hz, 1H), 7.35 – 7.27 (m, 3H), 4.91 – 4.80 (m, 1H), 4.63 –
4
2
.53 (m, 1H), 4.25 – 4.07 (m, 1H), 3.94 – 3.81 (m, 1H), 3.23 – 3.08 (m, 2H), 2.80 –
.66 (m, 1H), 2.39 (t, J = 7.2 Hz, 2H), 2.11 – 1.78 (m, 4H), 1.65 – 1.48 (m, 2H), 1.43
+
+
(s, 9H). HRMS (ESI ): calcd for C H Cl N O Na [M + Na] 589.1709, found
25 32 2 6 5
5
4
89.1703.
.1.4. tert-butyl(6-(4-(4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamido)piperi
din-1-yl)-6-oxohexyl)carbamate(5b)
Compound 5b (70 mg, 38% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, DMSO-d ) δ 10.18 (s, 1H), 8.40 (d, J = 8.4
6
Hz, 1H), 8.35 (s, 1H), 7.60 – 7.50 (m, 3H), 6.75 (t, J = 5.2 Hz, 1H), 4.38 (d, J =
1
2
2.8Hz, 1H), 4.01 – 3.92 (m, 1H), 3.87 (d, J = 13.2 Hz, 1H), 3.10 – 2.97 (m, 1H),
.90 (q, J = 6.8 Hz, 2H), 2.65 – 2.53 (m, 1H), 2.28 (t, J = 7.2 Hz, 2H), 1.85 – 1.68 (m,

+
2
H), 1.60 – 1.41 (m, 4H), 1.41 – 1.34 (m, 11H), 1.28 – 1.21(m, 2H). HRMS (ESI ):
+
calcd for C H Cl N O Na [M + Na] 617.2022, found 617.2026.
2
7
36
2
6
5
4
.1.5. tert-butyl(4-(4-(4-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole-3
-carboxamido)benzyl)piperazin-1-yl)-4-oxobutyl)carbamate(6a)
Compound 6a (58 mg, 38% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, DMSO-d ) δ 13.46 (s, 1H), 11.90 (s, 1H),
6
1
7
2
0.27 (s, 1H), 9.54 (s, 1H), 8.59 (s, 1H), 8.40 (s, 1H), 7.81 (d, J = 8.4 Hz, 2H), 7.35 –
.25 (m, 3H), 6.79 (t, J = 5.2 Hz, 1H), 6.50 (d, J = 3.2 Hz, 1H), 3.51 – 3.39 (m, 6H),
.94 (q, J = 6.8 Hz, 2H), 2.40 – 2.24 (m, 6H), 1.60 (p, J = 6.8 Hz, 2H), 1.38 (s, 9H).
+
+
HRMS (ESI ): calcd for C H N O Na [M + Na] 625.2975, found 625.2971.
30
38 10
4
4
.1.6. tert-butyl(6-(4-(4-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-1H-pyrazole-3
-carboxamido)benzyl)piperazin-1-yl)-6-oxohexyl)carbamate(6b)
Compound 6b (100 mg, 52% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, DMSO-d ) δ 13.42 (s, 1H), 11.87 (s, 1H),
6
1
7
6
1
+
0.25 (s, 1H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.35 –
.24 (m, 3H), 6.74 (t, J = 5.6 Hz, 1H), 6.48 (dd, J = 3.2, 1.6 Hz, 1H), 3.51 – 3.40 (m,
H), 2.88 (q, J = 6.8 Hz, 2H), 2.40 – 2.22 (m, 6H), 1.50 – 1.42 (p, J = 6.8 Hz, 2H),
+
.39 – 1.32 (m, 11H), 1.28 – 1.22 (m, 2H). HRMS (ESI ): calcd for C H N O [M
3
2
43 10
4
+
H] 631.3469, found 631.3463.
.1.7.tert-butyl(2-(2-(2-(4-(4-(2,6-dichlorobenzamido)-1H-pyrazole-3-carboxamido)p
4
iperidin-1-yl)-2-oxoethoxy)ethoxy)ethyl)carbamate(7)
Compound 7 (80 mg, 37% yield) was obtained according to the synthetic
1
procedure for 5a. H NMR (400 MHz, CDCl ) δ 9.87 (s, 1H), 8.46 (s, 1H), 7.35 – 7.28
3
(m, 3H), 7.20 – 7.12 (m, 1H), 5.17 (s, 1H), 4.52 (d, J = 12.8 Hz, 1H), 4.33 – 4.08 (m,
2
3
1
6
4
H), 3.87 (d, J = 12.0 Hz, 1H), 3.78 – 3.64 (m, 4H), 3.54 (t, J = 5.2 Hz, 2H), 3.36 –
.25 (m, 2H), 3.16 – 3.10 (m, 1H), 2.83 – 2.77 (m, 1H), 2.11 – 1.95 (m, 2H), 1.63 –
+
+
.47 (m, 2H), 1.42 (s, 9H). HRMS (ESI ): calcd for C H Cl N O Na [M + Na]
27
36
2
6
7
49.1920, found 649.1919.
.1.8. tert-butyl(2-(2-(2-(4-(4-(4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-1H-pyraz
ole-3-carboxamido)benzyl)piperazin-1-yl)-2-oxoethoxy)ethoxy)ethyl)carbamate(8)
Compound 8 (66 mg, 50% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, MeOD-d ) δ 8.62 (s, 1H), 8.38 (s, 1H), 7.71 (d,
4
J = 8.4 Hz, 2H), 7.32 (d, J = 8.4 Hz, 2H), 7.22 (d, J = 3.6 Hz, 1H), 6.61 (d, J = 3.6 Hz,
1
2
6
4
H), 4.22 (s, 2H), 3.67 – 3.56 (m, 6H), 3.54 – 3.45 (m, 6H), 3.22 (t, J = 5.6 Hz, 2H),
+
+
.47 – 2.41 (m, 4H), 1.43 (s, 9H). HRMS (ESI ): calcd for C H N O Na [M + Na]
3
2
42 10
6
85.3186, found 685.3167.
.1.9. tert-butyl 2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetate
(10)
2
-(2,6-dioxopiperidin-3-yl)-4-hydroxyisoindoline-1,3-dione (9) (274 mg, 1 mmol,
.0 eq) was dissolved in DMF (10 mL) at room temperature. Potassium bicarbonate
150 mg, 1.5 mmol, 1.5 eq) and tert-butyl bromoacetate (175 µL, 1.2 mmol, 1.2 eq)
1
(
were then added. The resulting mixture was stirred at 60 °C for 12 h. The reaction
mixture was quenched with H O and extracted with EtOAc. The organic layer was
2
separated, washed with brine, dried, and evaporated. The residue was purified by flash

1
column chromatography to afford 10 (260 mg, 67% yield). H NMR (400 MHz,
CDCl ) δ 8.00 (s, 1H), 7.67 (t, J = 8.4 Hz, 1H), 7.52 (d, J = 7.2 Hz, 1H), 7.11 (d, J =
3
8
1
4
4
.4 Hz, 1H), 5.02 – 4.92 (m, 1H), 4.79 (s, 2H), 2.94 – 2.68 (m, 3H), 2.18 – 2.08 (m,
+
+
H), 1.48 (s, 9H). HRMS (ESI ): calcd for C H N O Na [M + Na] 411.1168, found
1
9
20
2
7
11.1170.
.1.10. tert-butyl(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acet
amido)ethyl)carbamate(12a)
Compound 12a (93 mg, 72% yield) was given according to the synthetic
1
procedures for 5a. H NMR (400 MHz, CDCl ) δ 8.96 (s, 1H), 7.80 – 7.66 (m, 2H),
3
7
=
.54 (d, J = 7.2 Hz, 1H), 7.22 (d, J = 8.4 Hz, 1H), 5.16 (t, J = 5.6 Hz, 1H), 5.00 (dd, J
12.8, 5.6 Hz, 1H), 4.67 (s, 2H), 3.56 – 3.40 (m, 2H), 3.38 – 3.23 (m, 2H), 2.92 –
+
2
.74 (m, 3H), 2.22 – 2.10 (m, 1H), 1.40 (s, 9H). HRMS (ESI ): calcd for
+
C H N O Na [M + Na] 497.1648, found 497.1644.
22
26
4
8
4
.1.11. tert-butyl(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acet
amido)butyl)carbamate(12b)
Compound 12b (130 mg, 94% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, CDCl ) δ 9.22 (s, 1H), 7.74 (t, J = 7.6 Hz,
3
1
4
4
5
4
H), 7.63 (s, 1H), 7.55 (d, J = 7.2 Hz, 1H), 7.23 (d, J = 8.4 Hz, 1H), 5.10 – 4.59 (m,
H), 3.65 – 3.07 (m, 4H), 2.94 – 2.69 (m, 3H), 2.23 – 2.07 (m, 1H), 1.66 – 1.50 (m,
+
+
H), 1.44 (s, 9H). HRMS (ESI ): calcd for C H N O Na [M + Na] 525.1961, found
2
4
30
4
8
25.1962.
.1.12. tert-butyl(6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acet
amido)hexyl)carbamate(12c)
Compound 12c (113 mg, 80% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, CDCl ) δ 9.13 (s, 1H), 7.73 (t, J = 8.0 Hz,
3
1
(
2
H), 7.54 (d, J = 7.2 Hz, 1H), 7.45 (t, J = 5.6 Hz, 1H), 7.21 (d, J = 8.4 Hz, 1H), 5.00
m, 1H), 4.72 (s, 1H), 4.64 (s, 2H), 3.46 – 3.27 (m, 2H), 3.20 – 3.00 (m, 2H), 2.95 –
.74 (m, 3H), 2.25 – 2.10 (m, 1H), 1.60 (p, J = 6.8 Hz, 2H), 1.54 – 1.32 (m, 15H).
+
+
HRMS (ESI ): calcd for C H N O Na [M + Na] 553.2274, found 553.2279.
26
34
4
8
4
.1.13. tert-butyl(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2-oxo
-7,10,13-trioxa-3-azahexadecan-16-yl)carbamate(14)
Compound 14 (146 mg, 77% yield) was obtained according to the synthetic
1
procedures for 5a. H NMR (400 MHz, CDCl ) δ 9.16 (s, 1H), 7.74 (t, J = 8.0 Hz,
3
1
4
3
2
H), 7.60 – 7.51 (m, 2H), 7.22 (d, J = 8.4 Hz, 1H), 5.05 (t, J = 5.6 Hz, 1H), 5.02 –
.95 (m, 1H), 4.65 (s, 2H), 3.72 – 3.43 (m, 14H), 3.29 – 3.13 (m, 2H), 2.93 – 2.74 (m,
H), 2.28 (s, 1H), 2.21 – 2.11 (m, 1H), 1.88 (p, J = 6.4 Hz, 2H), 1.74 (p, J = 6.4 Hz,
+
+
H), 1.43 (s, 9H). HRMS (ESI ): calcd for C H N O Na [M + Na] 657.2748,
30 42 4 11
found 657.2748.
4
.1.14. tert-butyl(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethy
l)carbamate(17a)
DIPEA (199 µL, 1.2 mmol, 3 eq) was added to a solution of
-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione 16 (110 mg, 0.4 mmol, 1 eq)
2
and N-Boc-Ethylenediamine (70 mg, 0.44 mmol, 1.1 eq) in NMP (5 mL). The mixture
was heated to 90 °C for 12 h. Then, the mixture was diluted with EtOAc and washed

with 10% citric acid (aq), saturated sodium bicarbonate, water and three times with
brine. The residue was purified by flash column chromatography to afford 17a (137
1
mg, 82% yield). H NMR (400 MHz, DMSO-d ) δ 11.08 (s, 1H), 7.58 (t, J = 7.6 Hz,
6
1
(
6
H), 7.14 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz, 1H), 6.99 (t, J = 5.6 Hz, 1H), 6.70
t, J = 6.4 Hz, 1H), 5.05 (dd, J = 12.8, 5.6 Hz, 1H), 3.43 – 3.37 (m, 2H), 3.13 (q, J =
.0 Hz, 2H), 2.90 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.68 – 2.52 (m, 2H), 2.08 – 1.98
+
+
(m, 1H), 1.37 (s, 9H). HRMS (ESI ): calcd for C H N O Na [M + Na] 439.1594,
20 24 4 6
found 439.1592.
4
.1.15. tert-butyl(4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)buty
l)carbamate (17b)
Compound 17b (120 mg, 81% yield) was obtained according to the synthetic
1
procedures for 17a. H NMR (400 MHz, DMSO-d ) δ 11.08 (s, 1H), 7.57 (t, J = 7.6
6
Hz, 1H), 7.10 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.82 (t, J = 5.6 Hz, 1H),
6
2
.54 (t, J = 6.4 Hz, 1H), 5.05 (dd, J = 12.8, 5.6 Hz, 1H), 3.32 – 3.25 (q, J = 6.8 Hz,
H), 2.95 (q, J = 6.0 Hz, 2H), 2.92 – 2.83 (m, 1H), 2.66 – 2.51 (m, 2H), 2.11 – 1.97
(
m, 1H), 1.55 (p, J = 7.2 Hz, 2H), 1.46 (p, J = 7.2 Hz, 2H), 1.37 (s, 9H). HRMS
+
+
(ESI ): calcd for C H N O Na [M + Na] 467.1907, found 467.1912.
22 28 4 6
4
.1.16. tert-butyl(6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hex
yl)carbamate (17c)
Compound 17c (110 mg, 68% yield) was obtained according to the synthetic
1
procedures for 17a. H NMR (400 MHz, CDCl ) δ 8.61 (s, 1H), 7.48 (t, J = 7.6 Hz,
3
1
(
2
H), 7.08 (d, J = 7.2 Hz, 1H), 6.87 (d, J = 8.4 Hz, 1H), 6.24 (t, J = 5.6 Hz, 1H), 4.92
dd, J = 12.0, 5.6 Hz,, 1H), 3.25 (q, J = 6.8 Hz, 2H), 3.11 (q, J = 6.8 Hz, 2H), 2.93 –
.68 (m, 3H), 2.18 – 2.06 (m, 1H), 1.65 (p, J = 7.2 Hz, 2H), 1.53 – 1.33 (m, 15H).
+
+
HRMS (ESI ): calcd for C H N O Na [M + Na] 495.2220, found 495.2216.
24
32
4
6
General procedure for synthesis of compounds A1-A10 and F1-F13. A solution
of compound 5a (119 mg, 0.21 mmol, 1.0 eq) in 1:1 TFA/DCM was stirred at rt for 2
h. The solvents were evaporated under reduced pressure to give the corresponding
de-protected intermediate, which was added to a solution of DIPEA (74 µL, 0.42
mmol, 2.0 eq), HATU (120 mg, 0.31 mmol, 1.5 eq), and compound 11 (70 mg, 0.21
mmol, 1.0 eq) in DMF (5 mL). The resulting mixture was stirred at rt for 12 h. The
reaction mixture was quenched with H O and extracted with EtOAc. The organic
2
layer was separated, washed with brine, dried, and evaporated. The residue was
purified by flash column chromatography to afford A1 (127 mg, 77% yield) as white
solid. Compounds A1-A10 and F1-F13 were obtained using similar procedures for
A1.
4
.1.17. 4-(2,6-dichlorobenzamido)-N-(1-(4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo
isoindolin-4-yl)oxy)acetamido)butanoyl)piperidin-4-yl)-1H-pyrazole-3-carboxamide
(
A1)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.10 (s, 1H), 10.16 (s, 1H), 8.38 (s,
6
1
4
H), 8.35 (s, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.81 (t, J = 7.6 Hz, 1H), 7.62 – 7.47 (m,
H), 7.40 (d, J = 8.4 Hz, 1H), 5.12 (dd, J = 12.8, 5.6 Hz, 1H), 4.78 (s, 2H), 4.36 (d, J
=
12.8 Hz, 1H), 4.01 – 3.89 (m, 1H), 3.81 (d, J = 13.2 Hz, 1H), 3.17 (q, J = 6.4 Hz,
2
H), 3.00 (t, J = 12.4 Hz, 1H), 2.90 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.63 – 2.53 (m,

3
2
1
H), 2.31 (t, J = 7.0 Hz, 2H), 2.08 – 1.97 (m, 1H), 1.81 –1.62 (m, 4H), 1.59 – 1.37 (m,
1
3
H). C NMR (101 MHz, DMSO-d ) δ 173.19 , 170.39 , 170.31 , 167.20 , 167.19 ,
6
65.94 , 163.09 , 160.78 , 155.54 , 137.37 , 135.82 , 133.51 , 133.33 , 132.33 , 131.73
(
4
2C) , 128.88 (2C) , 121.97 , 121.19 , 120.82 , 117.30 , 116.49 , 68.14 , 49.28 (2C) ,
+
6.46 , 44.41 , 40.73 , 38.61 , 32.15 , 31.41 , 30.08 , 25.25 , 22.48 . HRMS (ESI ):
+
calcd for C H Cl N O Na [M + Na] 803.1723, found 803.1724.
3
5
34
2
8
9
4
.1.18. 4-(2,6-dichlorobenzamido)-N-(1-(6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxo
isoindolin-4-yl)oxy)acetamido)hexanoyl)piperidin-4-yl)-1H-pyrazole-3-carboxamide
(
A2)
1
H NMR (400 MHz, CDCl ) δ 11.99 (s, 1H), 10.46 (s, 1H), 9.90 (s, 1H), 8.46 (s, 1H),
3
7
3
4
2
1
1
1
1
3
.72 (t, J = 8.0 Hz, 1H), 7.62 – 7.47 (m, 2H), 7.34 – 7.24 (m, 4H), 7.19 (dd, J = 8.4,
.2 Hz, 1H), 5.00 (p, J = 5.6 Hz, 1H), 4.67 (s , 2H), 4.52 (d, J = 13.2 Hz, 1H), 4.20 –
.03 (m, 1H), 3.83 (d, J = 12.0 Hz, 1H), 3.43 –3.30 (m, 2H), 3.10 (t, J = 12.4 Hz, 1H),
.90 – 2.68 (m, 5H), 2.39 – 2.26 (m, 2H), 2.15 (m, 1H), 2.06 – 1.96 (m, 1H), 1.89 (m,
1
3
H), 1.68 – 1.44 (m, 6H), 1.42 – 1.33 (m, 2H). C NMR (101 MHz, CDCl ) δ
3
72.27 , 171.81 , 169.20 , 167.14 , 166.67 , 166.15 , 163.02 , 161.55 , 154.66 , 137.12 ,
35.37 , 133.45 , 132.75 , 132.47 (2C) , 131.00 , 128.13 (2C) , 122.67 , 121.60 ,
20.08 , 118.12 , 117.44 , 68.46 , 49.20 , 46.34 , 44.73 , 40.87 , 39.09 , 32.92 , 32.51 ,
+
1.32 , 29.68 , 28.88 , 26.61 , 25.13 , 22.76 . HRMS (ESI ): calcd for
+
C H Cl N O Na [M + Na] 831.2036, found 831.2039.
37
38
2
8
9
4
.1.19. 4-(2,6-dichlorobenzamido)-N-(1-(4-((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-diox
oisoindolin-4-yl)amino)ethyl)amino)-4-oxobutanoyl)piperidin-4-yl)-1H-pyrazole-3-ca
rboxamide (A3)
1
H NMR (400 MHz, DMSO-d ) δ 13.45 (s, 1H), 11.09 (s, 1H), 10.17 (s, 1H), 8.39(d,
6
J = 8.0 Hz, 1H), 8.35 (s, 1H), 8.08 (t, J = 5.6 Hz, 1H), 7.63 – 7.52 (m, 4H), 7.18 (d, J
=
8.4 Hz, 1H), 7.03 (d, J = 6.8 Hz, 1H), 6.73 (t, J = 6.2 Hz, 1H), 5.06 (dd, J = 12.8,
5
3
1
1
1
1
.6 Hz, 1H), 4.35 (d, J = 12.8 Hz, 1H),4.03 – 3.92 (m, 1H), 3.88 (d, J = 13.2 Hz, 1H),
.42 – 3.37 (m, 2H), 3.25 (q, J = 6.0 Hz, 2H), 3.03 (t, J = 12.8 Hz, 1H), 2.90 (ddd, J =
7.6, 14.0, 5.2 Hz, 1H), 2.66 –2.53 (m, 5H), 2.31 (t, J = 7.2 Hz, 2H), 2.09 – 1.08 (m,
1
3
H), 1.83 –1.68 (m, 2H), 1.64 – 1.37 (m, 2H). C NMR (101 MHz, DMSO-d ) δ
6
73.25 , 172.54 , 170.54 , 170.00 , 169.18 , 167.76 , 163.10 , 160.81 , 146.85 , 136.64 ,
35.83 , 132.67 (2C) , 132.33 , 131.72 (2C) , 128.88 (2C) , 121.99 (2C) , 117.66 ,
1
3
11.00 , 109.71, 49.00 (2C) , 46.48 , 44.28 , 41.87 , 40.80 , 38.62 , 32.09 , 31.45 ,
0.99 , 28.33 , 22.65 . HRMS (ESI ): calcd for C H Cl N O [M + H] 780.2064,
35 36 2 9 8
+
+
found 780.2067.
4
.1.20. 4-(2,6-dichlorobenzamido)-N-(1-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-diox
oisoindolin-4-yl)amino)butyl)amino)-4-oxobutanoyl)piperidin-4-yl)-1H-pyrazole-3-ca
rboxamide (A4)
1
H NMR (400 MHz, DMSO-d ) δ 13.43 (s, 1H), 11.08 (s, 1H), 10.17 (s, 1H), 8.39(d,
6
J = 8.4 Hz, 1H), 8.35 (s, 1H) , 7.82 (t, J = 5.6 Hz, 1H), 7.61 – 7.52 (m, 4H), 7.10 (d, J
=
8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.54 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 12.8,
5.6 Hz, 1H), 4.34 (d, J = 13.2 Hz, 1H), 4.01 – 3.92 (m, 1H), 3.88 (d, J = 13.6 Hz, 1H),
3.31 – 3.26 (m, 2H), 3.07 – 3.01 (m, 3H), 2.90 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.61
– 2.52 (m, 5H), 2.30 (t, J = 7.2 Hz, 2H), 2.02 – 1.98 (m, 1H), 1.83 – 1.68 (m, 2H),

1
3
1
1
1
.60 – 1.39 (m, 6H). C NMR (101 MHz, DMSO-d ) δ 173.25 , 171.73 , 170.54 ,
6
70.08 , 169.40 , 167.76 , 163.13 , 160.78 , 146.87 , 136.72 , 135.83, 132.66 (2C) ,
32.34 , 131.72 (2C) , 128.89 (2C) , 121.97 (2C) , 117.70 , 110.85 , 109.54, 49.01
(
2C) , 46.49 , 44.29 , 42.03 , 40.78 , 38.59 , 32.10 , 31.44 , 30.98 , 28.35 , 27.01 ,
6.65 , 22.63 . HRMS (ESI ): calcd for C H Cl N O Na [M + Na] 830.2196, found
37 39 2 9 8
30.2192.
.1.21. 4-(2,6-dichlorobenzamido)-N-(1-(4-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-diox
+
+
2
8
4
oisoindolin-4-yl)amino)hexyl)amino)-4-oxobutanoyl)piperidin-4-yl)-1H-pyrazole-3-ca
rboxamide(A5)
1
H NMR (400 MHz, DMSO-d ) δ 10.17 (s, 1H), 8.41 (d, J = 8.0 Hz, 1H), 8.35 (s, 1H),
6
7
.77 (t, J = 5.6 Hz, 1H), 7.62 – 7.52 (m, 4H), 7.09 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2
Hz, 1H), 6.53 (t, J = 6.0 Hz, 1H), 5.06 (dd, J = 12.8, 5.6 Hz, 1H), 4.34 (d, J = 12.8 Hz,
1
2
7
H), 4.00 – 3.93 (m, 1H), 3.88 (d, J = 13.6 Hz, 1H), 3.29 (q, J = 6.4 Hz, 2H), 3.09 –
.95 (m, 3H), 2.89 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.52 (m, 5H), 2.30 (t, J =
1
3
.2 Hz, 2H), 2.11 – 1.97 (m, 1H), 1.85 – 1.66 (m, 2H), 1.63 – 1.26 (m, 10H).
C
NMR (101 MHz, DMSO-d ) δ 173.25 , 171.63 , 170.54 , 170.10, 169.41 , 167.76 ,
6
1
1
4
2
8
4
62.93 , 160.81 , 146.89 , 136.72 , 135.85 , 132.65 (2C) , 132.31 , 131.72 (2C) ,
28.87 (2C) , 122.02 , 117.62 (2C) , 110.83 , 109.49 , 49.02 (2C) , 46.48 , 44.28 ,
2.26 , 40.78 , 38.92 , 32.10 , 31.45 , 30.98 , 29.56 , 29.11 , 28.37 , 26.59 , 26.52 ,
+
+
2.63 . HRMS (ESI ): calcd for C H Cl N O Na [M + Na] 858.2509, found
3
9
43
2
9
8
58.2515.
.1.22. 4-(2,6-dichlorobenzamido)-N-(1-(4-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-di
oxoisoindolin-4-yl)oxy)acetamido)ethyl)amino)-4-oxobutanoyl)piperidin-4-yl)-1H-pyr
azole-3-carboxamide (A6)
1
H NMR (400 MHz, DMSO-d ) δ 13.44 (s, 1H), 11.11 (s, 1H), 10.17 (s, 1H), 8.41 (d,
6
J = 8.4 Hz, 1H), 8.35 (s, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.89 (t, J = 5.6 Hz, 1H), 7.81 (t,
J = 7.6 Hz, 1H), 7.61 – 7.47 (m, 4H), 7.41 (d, J = 8.4 Hz, 1H), 5.13 (dd, J = 12.8, 5.6
Hz, 1H), 4.78 (s, 2H), 4.33 (d, J = 12.8Hz, 1H), 4.04 –3.90 (m, 1H), 3.87 (d, J = 13.6
Hz, 1H), 3.22 (q, J = 5.6 Hz, 2H), 3.14 (q, J = 5.6 Hz, 2H), 3.02 (t, J = 12.8 Hz, 1H),
2
2
.90 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.53 (m, 5H), 2.30 (t, J = 7.2 Hz, 2H),
.10 – 1.99 (m, 1H), 1.82 – 1.68 (m, 2H), 1.61 – 1.38 (m, 2H). C NMR (101 MHz,
13
DMSO-d ) δ 173.22 , 172.25 , 170.32 , 170.11 , 167.51 , 167.20 , 165.91 , 163.07 ,
6
1
1
3
60.81 , 155.55 , 137.40 , 135.83 , 133.49 (2C) , 132.33 , 131.73 (2C) , 128.88 (2C) ,
21.98 (2C) , 120.93 , 117.27 , 116.50 , 68.07 , 49.28 (2C) , 46.47 , 44.27 , 40.79 ,
+
8.73 , 38.66 , 32.08 , 31.42 , 31.00 , 28.29 , 22.48 . HRMS (ESI ): calcd for
+
C H Cl N O Na [M + Na] 860.1938, found 860.1937.
37
37
2
9
10
4
.1.23. 4-(2,6-dichlorobenzamido)-N-(1-(4-((4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-di
oxoisoindolin-4-yl)oxy)acetamido)butyl)amino)-4-oxobutanoyl)piperidin-4-yl)-1H-pyr
azole-3-carboxamide (A7)
1
H NMR (400 MHz, DMSO-d ) δ 10.17 (s, 1H), 8.40 (d, J = 8.0 Hz, 1H), 8.34 (s, 1H),
6
7.97 (t, J = 5.6 Hz, 1H), 7.85 – 7.77 (m, 2H), 7.61 – 7.47 (m, 4H), 7.40 (d, J = 8.4 Hz,
1H), 5.13 (dd, J = 12.8, 5.6 Hz, 1H), 4.78 (s, 2H), 4.34 (d, J = 12.4 Hz, 1H), 4.02 –
3.92(m, 1H), 3.89 (d, J = 13.2 Hz, 1H), 3.15 (q, J = 6.4 Hz, 2H), 3.07 – 2.98 (m, 3H),
2.90 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.51 (m, 5H), 2.30 (t, J = 7.2 Hz, 2H),

13
2
.10 – 2.00 (m, 1H), 1.83 – 1.70 (m, 2H), 1.57 – 1.37 (m, 6H). C NMR (101 MHz,
DMSO-d ) δ 173.22 , 171.70 , 170.33 , 170.10 , 167.19 , 167.14 , 165.97 , 162.93 ,
6
1
1
3
60.81 , 155.53 , 137.40 , 135.83 , 133.50 (2C) , 132.32 , 131.73 (2C) , 128.87 (2C) ,
22.00 , 120.88 (2C) , 117.29 , 116.51 , 68.14 , 49.29 (2C) , 46.48 , 44.29 , 40.79 ,
+
8.63 , 38.58 , 32.10 , 31.42 , 30.98 , 28.35 , 26.99 , 26.97 , 22.47 . HRMS (ESI ):
+
calcd for C H Cl N O Na [M + Na] 888.2251, found 888.2248.
3
9
41
2
9
10
4
.1.24. 4-(2,6-dichlorobenzamido)-N-(1-(4-((6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-di
oxoisoindolin-4-yl)oxy)acetamido)hexyl)amino)-4-oxobutanoyl)piperidin-4-yl)-1H-py
razole-3-carboxamide(A8)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.12 (s, 1H), 10.17 (s, 1H), 8.39 (d,
6
J = 8.4 Hz, 1H), 8.36 (s, 1H), 7.93 (t, J = 5.6 Hz, 1H), 7.81 (t, J = 8.0 Hz, 1H), 7.76 (t,
J = 5.6 Hz, 1H), 7.62 – 7.47 (m, 4H), 7.40 (d, J = 8.4 Hz, 1H), 5.13 (dd, J = 12.8, 5.6
Hz, 1H), 4.78 (s, 2H), 4.36 (d, J = 12.8 Hz, 1H), 4.04 – 3.94 (m, 1H), 3.89 (d, J = 13.2
Hz, 1H), 3.16 (q, J = 6.4 Hz, 2H), 3.05 – 2.98 (m, 3H), 2.91 (ddd, J = 17.6, 14.0, 5.2
Hz, 1H), 2.64 – 2.54 (m, 5H), 2.30 (t, J = 7.2 Hz, 2H), 2.09 – 2.01 (m, 1H), 1.84 –
1
3
1
.68 (m, 2H), 1.59 – 1.33 (m, 6H), 1.33 – 1.25 (m, 4H). C NMR (101 MHz,
DMSO-d ) δ 173.22 , 171.65 , 170.32 , 170.10 , 167.18 , 167.08 , 165.98 , 163.12 ,
6
1
60.75 , 155.48 , 137.37 , 135.82 , 133.49 , 133.36 , 132.31 , 131.73 (2C) , 128.87
(2C) , 121.98 , 121.17 , 120.84 , 117.31 , 116.51 , 68.14 , 49.29 (2C) , 46.49 , 44.31 ,
4
2
9
4
0.81 , 38.89 , 38.74 , 32.10 , 31.42 , 30.98 , 29.57 , 29.42 , 28.37 , 26.53 , 26.46 ,
+
+
2.48 . HRMS (ESI ): calcd for C H Cl N O Na [M + Na] 916.2564, found
4
1
45
2
9
10
16.2536.
.1.25. 4-(2,6-dichlorobenzamido)-N-(1-(2-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3
-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)ethoxy)acetyl)piperidin-4-yl)-1H-pyrazol
e-3-carboxamide (A9)
1
H NMR (400 MHz, DMSO-d ) δ 13.39 (s, 1H), 11.10 (s, 1H), 10.15 (s, 1H), 8.38 (d,
6
J = 8.0 Hz, 1H), 8.35 (s, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.80 (t, J = 8.0 Hz, 1H), 7.60 –
7
4
.46 (m, 4H), 7.39 (d, J = 8.4 Hz, 1H), 5.11 (dd, J = 12.8, 5.6 Hz, 1H), 4.79 (s, 2H),
.29 (d, J = 12.8 Hz, 1H), 4.22 – 4.05 (m, 2H), 4.03 – 3.92 (m, 1H), 3.79 (d, J = 13.2
Hz, 1H), 3.61 – 3.52 (m, 4H), 3.48 (t, J = 5.6 Hz, 2H), 3.38 – 3.33 (m, 2H), 3.05 –
2
1
1
1
.83 (m, 2H), 2.64 – 2.53 (m, 3H), 2.08 – 1.99 (m, 1H), 1.78 – 1.68 (m, 2H), 1.60 –
1
3
.40 (m, 2H). C NMR (101 MHz, DMSO-d ) δ 173.21 , 170.31 , 167.52 , 167.36 ,
6
67.18 , 165.90 , 163.09 , 160.75 , 155.44 , 137.38 , 135.82 , 133.50 , 133.35 , 132.34 ,
31.72 (2C) , 128.89 (2C) , 121.97 , 121.17 , 120.82 , 117.24 , 116.49 , 70.21 , 69.91
(
2C) , 69.28 , 68.01 , 49.29 (2C) , 46.33 , 43.84 , 40.85, 38.92 , 32.08 , 31.42 , 22.48 .
+
+
HRMS (ESI ): calcd for C H Cl N O Na [M + Na] 863.1935, found 863.1934.
37
38
2
8
11
4
.1.26. 4-(2,6-dichlorobenzamido)-N-(1-(1-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoiso
indolin-4-yl)oxy)-2,18-dioxo-7,10,13-trioxa-3,17-diazahenicosan-21-oyl)piperidin-4-y
l)-1H-pyrazole-3-carboxamide(A10)
1
H NMR (400 MHz, CDCl ) δ 12.16 (s, 1H), 10.15 (s, 1H), 9.90 (s, 1H), 8.45 (s, 1H),
3
7.71 (t, J = 8.0 Hz, 1H), 7.67 – 7.60 (m, 1H), 7.52 (d, J = 7.2 , 1H), 7.35 –7.27 (m,
4H), 7.21 (d, J = 8.4 Hz, 1H), 6.86 – 6.78 (m, 1H), 5.01 (dd, J = 11.6 Hz, 6.0 Hz 1H),
4.67 (s, 2H), 4.49 (d, J = 12.8 Hz, 1H), 4.16 – 4.03 (m, 1H), 3.87 (d, J = 13.6 Hz, 1H),
3.66 – 3.54 (m, 10H), 3.51 (t, J = 6.0 Hz, 2H), 3.46 (q, J = 6.4 Hz, 2H), 3.31 (q, J =

6
.4 Hz, 2H), 3.10 (t, J = 12.4 Hz, 1H), 2.85 – 2.49 (m, 8H), 2.18 – 2.10 (m, 1H), 2.06
1.89 (m, 2H), 1.85 (p, J = 6.4 Hz, 2H), 1.74 (p, J = 6.4 Hz, 2H), 1.58 – 1.44 (m, 2H).
–
1
3
C NMR (101 MHz, CDCl ) δ 172.60 , 172.02 , 170.46 , 168.99 , 167.09 , 166.69 ,
3
1
1
7
3
66.07 , 163.10 , 161.49 , 154.62 , 137.05 , 135.41 , 133.49 , 132.70 , 132.47 (2C) ,
30.96 , 128.12 (2C) , 122.56 , 121.62 , 119.93 , 118.01 , 117.35 , 70.38 , 70.35 ,
0.10 , 69.99 , 69.46 , 68.75 , 68.28 , 49.28 (2C) , 46.32 , 44.34 , 41.00 , 37.54 , 36.63 ,
+
2.20 , 31.38 , 29.67 , 29.24 , 28.92 , 28.56 , 22.70 . HRMS (ESI ): calcd for
+
C H Cl N O Na [M + Na] 1020.3038, found 1020.3038.
45
53
2
9
13
4
.1.27. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-(2-((2-(2,6-dioxopip
eridin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butanoyl)piperazin-1-yl)methyl)p
henyl)-1H-pyrazole-3-carboxamide (F1)
1
H NMR (400 MHz, DMSO-d ) δ 13.52 (s, 1H), 11.89 (s, 1H), 11.12 (s, 1H), 10.29 (s,
6
1
3
H), 9.52 (s, 1H), 8.56 (s, 1H), 8.38 (s, 1H), 8.01 (t, J = 5.6 Hz, 1H), 7.84 – 7.76 (m,
H), 7.50 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.33 – 7.24 (m, 3H), 6.48 (d, J
=
3.2 Hz, 1H), 5.12 (dd, J = 12.8, 5.6 Hz, 1H), 4.78 (s, 2H), 3.50 – 3.38 (m, 6H), 3.17
(
2
q, J = 6.8 Hz, 2H), 2.90 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.52 (m, 2H), 2.39 –
.19 (m, 6H), 2.10 – 1.99 (m, 1H), 1.67 (p, J = 7.2 Hz, 2H). C NMR (101 MHz,
1
3
DMSO-d ) δ 173.21 , 170.51 , 170.31 , 167.21 , 167.19 , 165.94 , 163.28 , 155.54 ,
6
1
1
6
2
4
52.16 , 151.65 , 151.03 , 137.60 , 137.37 , 133.63 , 133.50 , 132.76 , 129.60 (2C) ,
25.14 , 123.50 , 121.09 (2C) , 120.81 , 120.57 , 117.29 , 116.48 , 103.56 , 97.19 ,
8.13 , 61.93 , 53.22 , 52.70 , 49.29 , 45.29 , 41.51 , 38.57 , 31.42 , 29.98 , 25.13 ,
+
+
2.48 . HRMS (ESI ): calcd for C H N O [M + H] 817.3170, found 817.3165.
4
0
41 12
8
.1.28. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(6-(2-((2-(2,6-dioxopip
eridin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexanoyl)piperazin-1-yl)methyl)
phenyl)-1H-pyrazole-3-carboxamide (F2)
1
H NMR (400 MHz, DMSO-d ) δ 13.42 (s, 1H), 11.87 (s, 1H), 11.11 (s, 1H), 10.24 (s,
6
1
3
H), 9.52 (s, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 7.93 (t, J = 5.6 Hz, 1H), 7.86 – 7.78 (m,
H), 7.50 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.34 – 7.25 (m, 3H), 6.49 (dd,
J = 3.2, 1.6 Hz, 1H), 5.13 (dd, J = 12.8, 5.6 Hz, 1H), 4.77 (s, 2H), 3.50 – 3.37 (m, 6H),
3
2
.15 (q, J = 6.4 Hz, 2H), 2.91 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.54 (m, 2H),
.38 – 2.23 (m, 6H), 2.10 – 2.01 (m, 1H), 1.55 – 1.39 (m, 4H), 1.34 – 1.25 (m, 2H).
1
3
C NMR (101 MHz, DMSO-d ) δ 173.20 , 170.90 , 170.31 , 167.18 , 167.08 ,
6
1
1
1
3
65.98 , 163.25 , 155.52 , 152.12 , 151.65 , 151.04 , 137.60 , 137.39 , 133.66 , 133.49 ,
32.76 , 129.58 (2C) , 125.08 , 123.49 , 121.12 (2C) , 120.91 , 120.57 , 117.32 ,
16.52 , 103.56 , 97.17 , 68.19 , 61.94 , 53.32 , 52.80 , 49.30 , 45.41 , 41.46 , 38.70 ,
+
2.66 , 31.43 , 29.32 , 26.52 , 24.95 , 22.48 . HRMS (ESI ): calcd for C H N O Na
4
2
44 12
8
+
[
M + Na] 867.3303, found 867.3307.
4
.1.29. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((2-((2-(2,6-dioxopi
peridin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-4-oxobutanoyl)piperazin-1
-
yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (F3)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.08 (s, 1H), 10.24 (s,
6
1
H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 8.05 (t, J = 5.6 Hz, 1H), 7.80 (d, J = 8.4
Hz, 2H), 7.58 (t, J = 7.6 Hz, 1H), 7.34 – 7.25 (m, 3H), 7.18 (d, J = 8.8 Hz, 1H), 7.03
d, J = 7.2 Hz, 1H), 6.73 (t, J = 6.0 Hz, 1H), 6.48 (dd, J = 3.2, 1.6 Hz, 1H), 5.05 (dd, J
(

=
12.8, 5.6 Hz, 1H), 3.48 – 3.39 (m, 6H), 3.36 (q, J = 6.0 Hz, 2H), 3.31 (s, 2H), 3.23
(
q, J = 6.0 Hz, 2H), 2.89 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.64 – 2.53 (m, 2H), 2.40 –
1
3
2
1
1
.25 (m, 6H), 2.07 – 1.97 (m, 1H). C NMR (101 MHz, DMSO-d ) δ 173.24 ,
6
72.50 , 170.53 , 170.14 , 169.17 , 167.76 , 163.25 , 152.12 , 151.66 , 151.04 , 146.85 ,
37.60 , 136.65 , 133.65 , 132.77 , 132.68 , 129.59 (2C) , 125.08 , 123.50 , 121.14
(
4
2C) , 120.58 , 117.65 , 110.99 , 109.73 , 103.56 , 97.18 , 61.95 , 53.17 , 52.73 , 49.01 ,
+
5.20 , 41.88 , 41.61 , 38.61 , 31.46 , 30.93 , 28.31 , 22.65 . HRMS (ESI ): calcd for
+
C H N O [M + H] 816.3330, found 816.3326.
40
42 13
7
4
.1.30. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((4-((2-(2,6-dioxopi
peridin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-4-oxobutanoyl)piperazin-1
-
yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (F4)
1
H NMR (400 MHz, DMSO-d ) δ 13.42 (s, 1H), 11.87 (s, 1H), 11.09 (s, 1H), 10.24 (s,
6
1
1
6
4
2
2
1
1
1
3
+
H), 9.52 (s, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 7.85 – 7.75 (m, 3H), 7.57 (t, J = 7.6 Hz,
H), 7.35 – 7.24 (m, 3H), 7.10 (d, J = 8.4 Hz, 1H), 7.02 (d, J = 7.2 Hz, 1H), 6.57 –
.46 (m, 2H), 5.05 (dd, J = 12.8, 5.6 Hz, 1H), 3.52 – 3.40 (m, 6H), 3.33 – 3.23 (m,
H), 3.07 (q, J = 6.4 Hz, 2H), 2.89 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.53 (m,
H), 2.40 – 2.25 (m, 6H), 2.08 – 1.98 (m, 1H), 1.64 – 1.53 (m, 2H), 1.52 – 1.41 (m,
1
3
H). C NMR (101 MHz, DMSO-d ) δ 173.25 , 171.71 , 170.54 , 170.23 , 169.40 ,
6
67.76 , 163.24 , 152.13 , 151.65 , 151.04 , 146.87 , 137.59 , 136.71 , 133.65 , 132.74 ,
32.65 , 129.58 (2C) , 125.09 , 123.50 , 121.12 (2C) , 120.52 , 117.69 , 110.85 ,
09.48 , 103.56 , 97.18 , 61.95 , 53.16 , 52.74 , 49.02 , 45.21 , 42.04 , 41.60 , 38.58 ,
+
1.46 , 30.94 , 28.33 , 27.01 , 26.65 , 22.64 . HRMS (ESI ): calcd for C H N O [M
4
2
46 13
7
+
H] 844.3643, found 844.3639.
.1.31. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((6-((2-(2,6-dioxopi
peridin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)-4-oxobutanoyl)piperazin-
-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (F5)
4
1
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.07 (s, 1H), 10.24 (s,
6
1
H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 7.80 (d, J = 8.4 Hz, 2H), 7.75 (t, J = 5.6
Hz, 1H), 7.57 (t, J = 7.6 Hz, 1H), 7.34 – 7.24 (m, 3H), 7.08 (d, J = 8.8 Hz, 1H), 7.02
(
=
2
2
d, J = 6.8 Hz, 1H), 6.52 (t, J = 6.0 Hz, 1H), 6.48 (dd, J = 3.2, 1.6 Hz, 1H), 5.05 (dd, J
12.8, 5.6 Hz, 1H), 3.51 – 3.39 (m, 6H), 3.33 – 3.25 (m, 4H), 3.01 (q, J = 6.4 Hz,
H), 2.88 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.65 – 2.52 (m, 2H), 2.41 – 2.25 (m, 6H),
13
.07 – 1.97 (m, 1H), 1.62 – 1.50 (m, 2H), 1.44 – 1.29 (m, 6H). C NMR (101 MHz,
DMSO-d ) δ 173.26 , 171.65 , 170.54 , 170.27 , 169.41 , 167.77 , 163.25 , 152.13 ,
6
1
1
6
2
8
4
51.65 , 151.02 , 146.89 , 137.57 , 136.74 , 133.64 , 132.78, 132.64 , 129.60 (2C) ,
25.07 , 123.50 , 121.12 (2C) , 120.60 , 117.63 , 110.84 , 109.48 , 103.56 , 97.20 ,
1.94 , 53.15 , 52.73 , 49.02 , 45.21 , 42.24 , 41.59 , 38.90 , 31.44 , 30.94 , 29.54 ,
+
+
9.10 , 28.33 , 26.55 , 26.50 , 22.63 ..HRMS (ESI ): calcd for C H N O [M + H]
4
4
50 14
7
72.3956, found 872.3953.
.1.32. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((2-(2-((2-(2,6-dioxo
piperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)amino)-4-oxobutanoyl)p
iperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (F6)
1
H NMR (400 MHz, DMSO-d ) δ 13.43 (s, 1H), 11.88 (s, 1H), 11.11 (s, 1H), 10.25 (s,
6
1
H), 9.52 (s, 1H), 8.58 (s, 1H), 8.39 (s, 1H), 7.99 (t, J = 5.6 Hz, 1H), 7.88 (t, J = 5.6

Hz, 1H), 7.85 – 7.76 (m, 3H), 7.50 (d, J = 7.2 Hz, 1H), 7.41 (d, J = 8.8 Hz, 1H), 7.34
–
2
2
2
1
1
1
4
7.24 (m, 3H), 6.49 (dd, J = 3.2, 1.6 Hz, 1H), 5.13 (dd, J = 12.8, 5.6 Hz, 1H), 4.77 (s,
H), 3.59 – 3.40 (m, 6H), 3.31 (s, 2H), 3.22 (q, J = 6.0 Hz, 2H), 3.15 (q, J = 6.0 Hz,
H), 2.91 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.67 – 2.53 (m, 2H), 2.40 – 2.24 (m, 6H),
1
3
.10 – 2.00 (m, 1H). C NMR (101 MHz, DMSO-d ) δ 173.22 , 172.25 , 170.31 ,
6
70.26 , 167.51 , 167.20 , 165.91 , 163.25 , 155.55 , 152.14 , 151.65 , 151.03 , 137.60 ,
37.40 , 133.63 , 133.48 , 132.76 , 129.60 (2C) , 125.09 , 123.50 , 121.13 (2C) ,
20.96 , 120.57 , 117.28 , 116.51 , 103.56 , 97.20 , 68.09 , 61.93 , 53.13 , 52.71 ,
+
9.29 , 45.18 , 41.59 , 38.74 , 38.64 , 31.43 , 30.94 , 28.28 , 22.49 . HRMS (ESI ):
+
calcd for C H N O Na [M + Na] 896.3204, found 896.3204.
4
2
43 13
9
4
.1.33. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((4-(2-((2-(2,6-dioxo
piperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)butyl)amino)-4-oxobutanoyl)p
iperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide(F7)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.10 (s, 1H), 10.24 (s,
6
1
4
H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 7.94 (t, J = 5.6 Hz, 1H), 7.87 – 7.73 (m,
H), 7.49 (d, J = 7.2 Hz, 1H), 7.39 (d, J = 8.4 Hz, 1H), 7.33 – 7.24 (m, 3H), 6.48 (dd,
J = 3.2, 1.6 Hz, 1H), 5.12 (dd, J = 12.8, 5.6 Hz, 1H), 4.77 (s, 2H), 3.50 – 3.37 (m, 6H),
3
1
–
1
1
1
4
.31 (s, 2H), 3.15 (q, J = 6.0 Hz, 2H), 3.02 (q, J = 6.0 Hz, 2H), 2.90 (ddd, J = 17.6,
4.0, 5.2 Hz, 1H), 2.69 – 2.55 (m, 2H), 2.43 – 2.26 (m, 6H), 2.09 – 1.98 (m, 1H), 1.48
13
1.35(m, 4H). C NMR (101 MHz, DMSO-d ) δ 173.22 , 171.69 , 170.32 , 170.26 ,
6
67.19 , 167.14 , 165.96 , 163.24 , 155.53 , 152.14 , 151.65 , 151.03 , 137.59 , 137.41 ,
33.63 , 133.49 , 132.76 , 129.61 (2C) , 125.08 , 123.50 , 121.13 (2C) , 120.89 ,
20.63 , 117.29 , 116.52 , 103.56 , 97.19 , 68.15 , 61.94 , 53.17 , 52.73 , 49.29 , 45.21 ,
+
1.60 , 38.62 , 38.57 , 31.42 , 30.93 , 28.32 , 26.98 , 26.95 , 22.47 . HRMS (ESI ):
+
calcd for C H N O Na [M + Na] 924.3517, found 924.3518.
4
4
47 13
9
4
.1.34. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((6-(2-((2-(2,6-dioxo
piperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)hexyl)amino)-4-oxobutanoyl)
piperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (F8)
1
H NMR (400 MHz, DMSO-d ) δ 13.42 (s, 1H), 11.88 (s, 1H), 11.12 (s, 1H), 10.25 (s,
6
1
3
7
2
H), 9.53 (s, 1H), 8.59 (s, 1H), 8.39 (s, 1H), 7.92 (t, J = 5.6 Hz, 1H), 7.84 – 7.78 (m,
H), 7.75 (t, J = 5.6 Hz, 1H), 7.50 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.34 –
.25 (m, 3H), 6.49 (dd, J = 3.2, 1.6 Hz, 1H), 5.13 (dd, J = 12.8, 5.6 Hz, 1H), 4.77 (s,
H), 3.52 – 3.40 (m, 6H), 3.31 (s, 2H), 3.15 (q, J = 6.0 Hz, 2H), 3.01 (q, J = 6.0 Hz,
H), 2.91 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.66 – 2.53 (m, 2H), 2.39 – 2.25 (m, 6H),
2
1
3
2
.09 – 2.00 (m, 1H), 1.48 – 1.24 (m, 8H). C NMR (101 MHz, DMSO-d ) δ 173.22 ,
6
1
1
1
5
71.63 , 170.32 , 170.25 , 167.19 , 167.07 , 165.97 , 163.25 , 155.48 , 152.12 , 151.65 ,
51.03 , 137.59 , 137.37 , 133.65 , 133.48 , 132.76 , 129.58 (2C) , 125.08 , 123.49 ,
21.13 (2C) , 120.84 , 120.57 , 117.30 , 116.51 , 103.56 , 97.19 , 68.14 , 61.95 , 53.18 ,
2.75 , 49.29 , 45.21 , 41.60 , 38.88 , 38.74 , 31.42 , 30.93 , 29.57 , 29.42 , 28.34 ,
+
+
2
6.52 , 26.46 , 22.48 . HRMS (ESI ): calcd for C H N O [M + H] 930.4011,
46 52 13 9
found 930.4005.
4
.1.35. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(2-(2-(2-(2-((2-(2,6-dio
xopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethoxy)ethoxy)acetyl)piper
azin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide(F9)

1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.11 (s, 1H), 10.24 (s,
6
1
3
H), 9.51 (s, 1H), 8.58 (s, 1H), 8.38 (s, 1H), 8.00 (t, J = 5.6 Hz, 1H), 7.84 – 7.76 (m,
H), 7.49 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 8.4 Hz, 1H), 7.32 – 7.27 (m, 3H), 6.48 (dd,
J = 3.2, 1.6 Hz, 1H), 5.12 (dd, J = 12.8, 5.6 Hz, 1H), 4.79 (s, 2H), 4.13 (s, 2H), 3.55
br, 4H), 3.48 (t, J = 5.6 Hz, 2H), 3.46 – 3.35 (m, 8H), 2.90 (ddd, J = 17.6, 14.0, 5.2
(
1
3
Hz, 1H), 2.66 – 2.52 (m, 2H), 2.39 – 2.27 (m, 4H), 2.10 – 2.01 (m, 1H). C NMR
101 MHz, DMSO-d ) δ 173.21 , 170.32 , 167.63 , 167.34 , 167.18 , 165.90 , 163.24 ,
(
6
1
55.42 , 152.12 , 151.65 , 151.04 , 137.60 , 137.39 , 133.58 , 133.50 , 132.74 , 129.59
(2C) , 125.08 , 123.50 , 121.12 (2C) , 120.83 , 120.57 , 117.24 , 116.51 , 103.55 ,
9
4
8
4
7.19 , 70.23 , 69.93 , 69.81 , 69.26 , 68.01 , 61.92 , 53.14 , 52.63 , 49.29 , 44.74 ,
+
+
1.58 , 38.93 , 31.42 , 22.48 . HRMS (ESI ): calcd for C H N O Na [M + Na]
4
2
44 12 10
99.3201, found 899.3209.
.1.36. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(1-((2-(2,6-dioxopiperi
din-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)-2,18-dioxo-7,10,13-trioxa-3,17-diazahenicosa
n-21-oyl)piperazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide (F10)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.10 (s, 1H), 10.24 (s,
6
1H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 7.94 (t, J = 5.6 Hz, 1H), 7.83 – 7.77(m,
3H), 7.75 (t, J = 5.6 Hz, 1H), 7.49 (d, J = 7.2 Hz, 1H), 7.40 (d, J = 8.8 Hz, 1H), 7.34 –
7.25 (m, 3H), 6.48 (d, J = 3.6 Hz, 1H), 5.12 (dd, J = 12.8, 5.6 Hz, 1H), 4.77 (s, 2H),
3.53 – 3.36 (m, 18H), 3.30 (s, 2H), 3.25 – 3.17 (m, 2H), 3.11 – 3.02 (m, 2H), 2.90
(
2
ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.66 – 2.53 (m, 2H), 2.43 – 2.25 (m, 6H), 2.10 –
.00 (m, 1H), 1.67 (p, J = 6.4 Hz, 2H), 1.59 (q, J = 6.4 Hz, 2H). C NMR (101 MHz,
1
3
DMSO-d ) δ 173.22 , 171.75 , 170.31 , 170.26 , 167.19 (2C) , 165.95 , 163.243 ,
6
1
1
7
4
55.49 , 152.12 , 151.65 , 151.02 , 137.40 (2C) , 133.49 (2C) , 132.69 , 129.64 (2C) ,
25.06 , 123.50 , 121.13 (2C) , 120.87 , 120.58 , 117.29 , 116.54 , 103.55 , 97.20 ,
0.21 (2C) , 70.03 , 69.96 , 68.53 , 68.40 , 68.14 , 61.87 , 53.13 , 52.70 , 49.29 , 45.18 ,
+
1.53 , 39.66 , 36.26 , 31.41 , 30.90 , 29.82 , 29.68 , 28.30 , 22.47 . HRMS (ESI ):
+
calcd for C H N O Na [M + Na] 1056.4304, found 1056.4299.
5
0
59 13 12
4
.1.37. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((2-((2-(2,6-dioxopi
peridin-3-yl)-1-oxoisoindolin-4-yl)amino)ethyl)amino)-4-oxobutanoyl)piperazin-1-yl)
methyl)phenyl)-1H-pyrazole-3-carboxamide(F11)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.00 (s, 1H), 10.24 (s,
6
1
H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 7.97 (d, J = 5.2 Hz, 1H), 7.80 (d, J = 8.4
Hz, 2H), 7.35 – 7.25 (m, 4H), 6.95 (d, J = 7.2 Hz, 1H), 6.83 (d, J = 8.0 Hz, 1H), 6.48
dd, J = 3.2, 1.6 Hz, 1H), 5.64 (t, J = 4.8 Hz, 1H), 5.11 (dd, J = 12.8, 5.6 Hz, 1H),
.28 – 4.10 (m, 2H), 3.49 – 3.41 (m, 6H), 3.32 (s, 2H), 3.27 – 3.19 (m, 4H), 2.93 (ddd,
J = 17.6, 14.0, 5.2 Hz, 1H), 2.69 – 2.53 (m, 2H), 2.39 – 2.27 (m, 6H), 2.14 – 1.94 (m,
(
4
1
3
1
1
1
1
3
8
H). C NMR (101 MHz, DMSO-d ) δ 173.34 , 172.41 , 171.66 , 170.24 , 169.32 ,
6
63.25 , 152.13 , 151.66 , 151.03 , 143.92 , 137.59 , 133.63 , 132.77 , 132.57 , 129.72 ,
29.61 (2C) , 127.04 , 125.37, 123.51 , 121.14 (2C) , 120.60 , 112.21 , 110.69 ,
03.56 , 97.19 , 61.94 , 53.15 , 52.73 , 52.01 , 46.11 , 45.21 , 42.75 , 41.61 , 38.37 ,
+
+
1.71 , 30.97 , 28.30 , 23.29 . HRMS (ESI ): calcd for C H N O [M + H]
4
0
44 13
6
02.3532, found 802.3539.

4
.1.38. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((3-((2-(2,6-dioxopi
peridin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)propyl)amino)-4-oxobutanoyl)piperazin
-
1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide(F12)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 11.05 (s, 1H), 10.25 (s,
6
1
H), 9.51 (s, 1H), 8.57 (s, 1H), 8.38 (s, 1H), 7.98 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 8.0
Hz, 2H), 7.57 (d, J = 8.4 Hz, 1H), 7.35 – 7.25 (m, 3H), 7.08 (t, J = 5.2 Hz, 1H), 6.99
(
(
d, J = 2.0 Hz, 1H), 6.88 (dd, J = 8.4, 2.0 Hz, 1H), 6.48 (dd, J = 3.2, 1.6 Hz, 1H), 5.03
dd, J = 12.8, 5.2 Hz, 1H), 3.55 – 3.39 (br, 6H), 3.29 (s, 2H), 3.27 – 3.20 (br, 4H),
2
–
1
1
1
3
8
4
6
.88 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.64 – 2.54 (m, 2H), 2.43 – 2.23 (m, 6H), 2.09
1
3
1.89 (m, 1H). C NMR (101 MHz, DMSO-d ) δ 173.27 , 172.43 , 170.60 , 170.25 ,
6
68.11 , 167.59 , 163.25 , 154.82 , 152.14 , 151.66 , 151.03 , 137.59 , 134.66 , 133.64 ,
32.73 , 129.62 (2C) , 125.57 , 125.09 , 123.51 , 121.13 (2C) , 120.60 , 116.73 ,
16.05 , 105.95 , 103.56 , 97.21 , 61.93 , 53.15 , 52.73 , 49.12 , 45.20 , 42.41 , 41.63 ,
+
+
8.17 , 31.46 , 30.97 , 28.31 , 22.71 . HRMS (ESI ): calcd for C H N O [M + H]
4
0
42 13
7
16.3330, found 816.3325.
.1.39. 4-((7H-pyrrolo[2,3-d]pyrimidin-4-yl)amino)-N-(4-((4-(4-((2-((2-(1-methyl-2,
-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-4-oxobutanoyl)pi
perazin-1-yl)methyl)phenyl)-1H-pyrazole-3-carboxamide(F13)
1
H NMR (400 MHz, DMSO-d ) δ 13.41 (s, 1H), 11.87 (s, 1H), 10.24 (s, 1H), 9.52 (s,
6
1
H), 8.58 (s, 1H), 8.39 (s, 1H), 8.06 (t, J = 5.6 Hz, 1H), 7.81 (d, J = 8.0 Hz, 2H), 7.59
(t, J = 7.2 Hz, 1H), 7.33 – 7.26 (m, 3H), 7.18 (d, J = 8.4 Hz, 1H), 7.03 (d, J = 7.2 Hz,
1
1
2
–
1
1
H), 6.73 (t, J = 6.4 Hz, 1H), 6.49 (dd, J = 3.2, 1.6 Hz, 1H), 5.12 (dd, J = 12.8, 5.2 Hz,
H), 3.52 – 3.41 (m, 6H), 3.41 – 3.34 (m, 2H), 3.24 (d, J = 6.4 Hz, 2H), 3.02 (s, 3H),
.95 (ddd, J = 17.6, 14.0, 5.2 Hz, 1H), 2.81 – 2.71 (m, 1H), 2.60 – 2.51 (m, 2H), 2.42
1
3
2.25 (m, 6H), 2.10 – 1.99 (m, 1H). C NMR (101 MHz, DMSO-d ) δ 172.50 ,
6
72.25 , 170.27 , 170.15 , 169.15 , 167.73 , 163.25 , 152.12 , 151.65 , 151.03 , 146.89 ,
37.62 , 136.68 , 133.59 , 132.76 , 132.66 , 129.61 (2C) , 125.08 , 123.50 , 121.13
(
4
2C) , 120.56 , 117.69 , 111.02 , 109.69 , 104.49 , 97.17 , 61.93 , 53.15 , 52.73 , 49.56 ,
+
5.19 , 41.89 , 41.60 , 38.60 , 31.59 , 30.95 , 28.32 , 27.07 , 21.87 . HRMS (ESI ):
+
calcd for C H N O [M + H] 816.3325, found 830.3486.
4
1
43 13
7
4
.2. Molecular modeling
Molecular docking studies were performed using DS 3.5 (Discovery studio 3.5). The
crystal structure of CDK2 complex (PDB code: 2VTH) was obtained from the RCSB
Protein Data Bank. With the water deleted, ligand removed and force field applied,
the receptor was used for the subsequent docking studies. Ligand structure was
protonated and conformation searched with DS 3.5. The other parameters were as
default. The best output poses of the ligands generated according to CDOCKER
ENERGY.
4
.3. Cell culture
PC-3(SCSP-532) and MCF-7(SCSP-531) cells were purchased from the Cell
Bank of Chinese Academy of Sciences (Shanghai, China). Cells were cultured in a
7 °C incubator with 5% CO . PC-3 were maintained in DMEM and F12 (1:1)
3
2
nutrient medium supplemented with 10% (v/v) fetal bovine serum (Gibco) and 1%
v/v) penicillin/streptomycin (HyClone, SV30010). LO2, MCF-7, HCT-116 and
(

2
2Rv1 cells were cultured in Roswell Park Memorial Institute (RPMI) 1640 medium
supplemented with 10% (v/v) fetal bovine serum and 1% (v/v)
penicillin/streptomycin.
4
.4. Western Blot Analysis.
5
Cells (5 × 10 cells/well) were plated in 6-well plates and treated with
compounds under the indicated conditions. After 24 hours, the cells were washed with
ice-cold PBS, lysed and collected with RIPA buffer and protease inhibitors. The lysate
was centrifuged at 12000 rpm for 15 min at 4 °C, the concentration of the
supernatants was determined using the BCA Protein Assay Kit (beyotime#p0012s).
Total proteins (50 µg) were subjected to 10% SDS−PAGE and were then transferred
onto PVDF membranes (Millpore). Membranes were blocked for 30 min, then
incubated with primary antibodies overnight at 4 °C, then washed with 1 × TBST 3
times, then incubated with secondary HRP antibody for 2 h at room temperature, then
washed with 1 × TBST 3 times, followed by washing and detection with
chemiluminescence system (Clinx Science Instruments, Chemiscope 5300). The band
intensities were quantified using GelQuant.NET software. Skimmed (5%) milk was
dissolved in 0.1% TBS-T (10 mM Tris-HCl and Tween 20) and was then used for
blocking membranes and diluting antibodies. Primary antibodies used were
anti-CDK2 (1/1000 dilution, CST#78B2), anti-CDK5 (1/1000 dilution, CST#D1F7M),
anti-CDK9 (1/1000 dilution, CST#C12F7), anti-CDK4 (1/1000 dilution,
CST#D9G3E), anti-CDK6 (1/1000 dilution, CST#D4S8S), anti-βTubulin (1/1000
dilution, CST#2148), anti-c-Myc (1/1000 dilution, CST#D84C12), anti-Mcl-1
(1/1000 dilution, D2W9E).
4
.5. CCK-8 assay
3
Cells (80 µL) were seeded in 96-well plates at a density of 3 × 10 per well for
2
4 h. Compound was tested in 10-dose IC mode with a 3-fold serial dilution starting
5
0
at 10 µM. After 5 days, 10 µL CCK-8 reagents (SAB, CP002) was added to each well
and the plates were incubated at 37 °C for another 4 h. The experiment was repeated
at least twice independently with duplicate measurements. The luminescence was
measured at 450 nm via spectrophotometry (Thermo Multiskan MK3). Data was
analyzed using GraphPad Prism 5 software.
4
.6. Kinase IC tests.
50
The in vitro kinase enzymatic inhibition assays were carried out by Reaction
Biology Corporation (USA). HEK293 cells were seeded into the wells of 384-well
TM
plates. The cells were pretreated with the NanoBRET Tracer K-10 and then treated
with compound for 1 hour. Reactions were carried out at 10 µM ATP. The BRET
signal was measured on an Envision 2104 Multilabel Reader. IC50 value was
calculated and IC curve was plotted using the GraphPad Prism 4 program based on a
5
0
sigmoidal dose response equation. Compound was tested in 10-dose IC mode with a
5
0
3
-fold serial dilution starting at 1 µM. Control compound (staurosporine) was tested
in 10-dose IC50 mode with 4-fold serial dilution starting at 20 µM.
http://www.reactionbiology.com)
.7. Flow cytometry assay
(
4

CFSE assay was performed by BD Horizon™ CFSE (565082). Cell cycle
distribution was determined by Cell Cycle Kit (KeyGEN#KGA512). The
experimental conditions are described in the notes and the experimental methods
follow the protocol of the instruction. Data analysis was performed using FlowJo and
Modfit software.
Conflicts of interest
The authors declare no conflicts of interest.
Acknowledgments
This work was supported by the National Natural Science Foundation of China
(
No. 81672951).
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[

Highlights
1
2
3
4
. We designed and synthesized two series PROTAC compounds by tethering CDK
inhibitors with CRBN ligands.
. We identified compounds differentially induced dual CDK2/9 degradation, or
selective to CDK2 or to CDK9.
. Compound F3 is a potent dual degrader for CDK2 (DC : 62 nM) and CDK9
5
0
(
DC : 33 nM).
50
. Compound F3 suppresses prostate cancer PC-3 cell proliferation by interfering
with cell cycle progression.