First orally bioavailable prodrug of proteolysis targeting chimera (PROTAC) degrades cyclin-dependent kinases 2/4/6 in vivo

Article abstract of DOI:10.1016/j.ejmech.2020.112903

A growing number of reports suggested that the inhibitor targeting cyclin-dependent kinases (CDK) 2/4/6 can act as a more feasible chemotherapy strategy. In the present paper, a novel PROTAC molecule was developed based on the structure of Ribociclib's derivative. In malignant melanoma cells, the degrader can not only degrade CDK 2/4/6 simultaneously and effectively, but also remarkably induce cell cycle arrest and apoptosis of melanoma cells. Moreover, PROTAC molecules with CRBN ligands always have poor oral bioavailability. We developed the orally bioavailable prodrug for the first time. It would provide general solution for oral administration of the PROTAC molecules, derived from CRBN ligands, for animal test conveniently.

Full text of DOI:10.1016/j.ejmech.2020.112903

Journal Pre-proof
First Orally Bioavailable Prodrug of Proteolysis Targeting Chimera (PROTAC)
Degrades Cyclin-Dependent Kinases 2/4/6 in vivo
Mingming Wei, Rui Zhao, Yuting Cao, Yujiao Wei, Ming Li, Zhiqiang Dong, Yulin Liu,
Hao Ruan, Ying Li, Sheng Cao, Zhiwen Tang, Yuanyuan Zhou, Wei Song, Yubo
Wang, Jiefu Wang, Guang Yang, Cheng Yang
PII:
S0223-5234(20)30875-8
DOI:
https://doi.org/10.1016/j.ejmech.2020.112903
EJMECH 112903
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 28 June 2020
Revised Date: 17 September 2020
Accepted Date: 1 October 2020
Please cite this article as: M. Wei, R. Zhao, Y. Cao, Y. Wei, M. Li, Z. Dong, Y. Liu, H. Ruan, Y. Li,
S. Cao, Z. Tang, Y. Zhou, W. Song, Y. Wang, J. Wang, G. Yang, C. Yang, First Orally Bioavailable
Prodrug of Proteolysis Targeting Chimera (PROTAC) Degrades Cyclin-Dependent Kinases 2/4/6 in vivo,
European Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112903.
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© 2020 Published by Elsevier Masson SAS.

First Orally Bioavailable Prodrug of Proteolysis Targeting Chimera
(PROTAC) Degrades Cyclin-Dependent Kinases 2/4/6 in vivo
‡
‡
‡
‡
Mingming Wei, ‡ Rui Zhao, Yuting Cao, Yujiao Wei, Ming Li,
Zhiqiang Dong, Ying Li, Zhiwen Tang, Yuanyuan Zhou, Wei Song, Yubo
Wang, Jiefu Wang, * Guang Yang, * Cheng Yang *

First Orally Bioavailable Prodrug of Proteolysis Targeting Chimera (PROTAC) Degrades
Cyclin-Dependent Kinases 2/4/6 in vivo
‡, a
‡, a
‡, b
‡, a
Mingming Wei, ‡, a Rui Zhao,
Yuting Cao,
a
Yujiao Wei, ‡, a Ming Li,
Zhiqiang Dong,
Yulin Liu, ‡, a Hao Ruan, Ying Li, Sheng Cao, Zhiwen Tang, Yuanyuan Zhou, Wei Song, a
Yubo Wang, a Jiefu Wang, *, c Guang Yang, *, a Cheng Yang *, a
a
a
a
a
a
The State Key Laboratory of Medicinal Chemical Biology, College of Pharmacy, Nankai
University, Tianjin, 300071, P.R. China.
b
c
Cangzhou Institutes for Food and Drug Control, Cangzhou, 061000, P.R. China.
Tianjin Medical University Cancer Institute and Hospital, National Clinical Research Center
for Cancer, Tianjin's Clinical Research Center for Cancer, Key Laboratory of Cancer Prevention
and Therapy, Tianjin, 300060, P.R. China.
ABSTRACT
A growing number of reports suggested that the inhibitor targeting cyclin-dependent kinases
(CDK) 2/4/6 can act as a more feasible chemotherapy strategy. In the present paper, a novel
PROTAC molecule was developed based on the structure of Ribociclib’s derivative. In malignant
melanoma cells, the degrader can not only degrade CDK 2/4/6 simultaneously and effectively, but
also remarkably induce cell cycle reset and apoptosis of melanoma cells. Moreover, PROTAC
molecules with CRBN ligands always have poor oral bioavailability. We developed the orally
bioavailable prodrug for the first time. It would provide general solution for oral administration of
the PROTAC molecules, derived from CRBN ligands, for animal test conveniently.
Cyclin-dependent kinases (CDKs) are critical cellular enzymes in regulating eukaryotic cell
[1]
division and cell proliferation. CDKs catalytic units are activated by regulatory subunits, i.e.,
cyclins. At least 16 mammalian cyclins have been identified, among which Cyclin B/CDK1,
cyclin A/CDK2, cyclin E/CDK2, cyclin D/CDK4 and cyclin D/CDK6 act as vital regulators of cell
[2]
cycle progression.
Additional functions of cyclin/CDKs (e.g., transcription regulation, DNA
repair, differentiation and apoptosis) have been reported as well. [3-5]
Enhanced activity or temporally abnormal activation of CDKs leads to the development of
many types of tumors. Accordingly, cyclin-dependent kinases inhibitors have been suggested to be
[6]
powerful in the treatment of cancer.
To be specific, CDK4/6 inhibitors have exhibited
noticeably high clinical efficacy in breast and other cancers as single agents or combined with
other therapeutics. [7] For instance, Palbociclib and Ribociclib have been approved by FDA for the
treatment of HR-positive and HER2-negative advanced or metastatic breast cancer in combination
with aromatase inhibitors. Nevertheless, with the development of primary or acquired drug
resistance, CDK4/6 inhibitors are also limited over time.
Recent results revealed that over-expression of CDK2 and its complex with cyclins is highly
[8]
correlated with abnormal regulation of cell-cycle.
The cyclin E/CDK2 complex is critical to
regulate the G1/S transition, histone biosynthesis and centrosomes duplication. Through the
progressive phosphorylation of Rb by cyclin D/CDK4/6 and cyclin E/CDK2, the G1 transcription
[9]
factor, E2F, is released, which can significantly promote S-phase entry.
Activation of cyclin
A/CDK2 during early S-phase also facilitates DNA replication and inactivation of E2F to

complete S-phase. [9] Cyclin E refers to the regulatory cyclin with CDK2. Recent studies have also
reported that cyclin E/CDK2 inhibition restore sensitivity to tamoxifen or CDK4/6 inhibitors in
[10, 11]
breast cancer cells.
Amplification or over-expression of cyclin E/CDK2 has long been
related to poor outcomes in breast cancer [12-14] and other types of cancers. [15-18]
[19]
To the best of our knowledge, there has been no approved drug targeting CDK2 thus far.
However, two small molecules, Dinaciclib (MK-7965) and Seliciclib (CYC202), inhibiting
CDK1/2/5/9 and CDK2/7/9, respectively, are being clinically developed for the treatment of
[19]
advanced tumors as single reagents and in conjunction with chemotherapies.
Very recently, a
novel molecule labeled as PF069736, a CDK2/4/6 inhibitor, has been recruited for phase I study
now. In brief, it has been indicated that combination therapies targeting CDK2 are arousing huge
attention by both medicinal and pharmaceutical areas. At this stage, a novel chemotherapeutical
strategy was proposed by chemical knock-down of CDK2/4/6 simultaneously for tumor therapies.
The technique of Proteolysis-targeting chimeras (PROTACs) was first reported by Crews. [20]
In practice, PROTAC molecules are hetero-bifunctional small molecules conjugated by a proper
chemical linker. They can drag the target proteins close to an E3 ligase, thus causing consequent
degradation of the target proteins by proteasome. Thus far, PROTAC molecules have become a
valuable tool for the chemical knockdown of specific proteins for the treatment of tumor.
[21]
[22]
[23]
[24]
[25]
Numerous inhibitors of target proteins (e.g., FLT3,
AR,
MDM2,
CDK6,
CDK9,
[26]
[27]
[28]
BRDs,
molecules.
BET,
ALK,
as well as PARP-1, [29]) have been developed into PROTAC
Nevertheless, most of the previously reported PROTAC molecules only degrades single one
target. There have been few publications on a PROTAC molecule, which can mitigate proteasomal
down-regulation of multiple proteins simultaneously. In this paper, a novel compound was
synthesized and screened by linking a derivative of Ribociclib and Pomalidomide. This PROTAC
molecule can degrade CDK 2/4/6 in various cancer cells. It also down-regulated the
phosphorylation of Rb, exhibiting potent anti-proliferation of malignant melanomas both in vitro
and in vivo.
The structure of Ribociclib 1 was taken as the CDKs binding moiety for its high binding
potency for CDK 4/6. [30] However, Ribociclib has no effect on inhibiting CDK2. Great efforts
have been made to synthesize and screen appropriate analogue (data are not shown in this paper).
Fortunately, core structure 2, delivered from the straightforward structural modification of
Ribociclib, exhibited enhanced potency for CDK2. The results of kinase activity assays suggested
that compound 2, provided significant improvement its binding capability to CDK 2, though its
affinity to CDK 4/6 slightly decreased. We therefore hypothesized that an extended linker attached
as amides will exert minimal effects on binding to CDK 2/4/6.
Scheme 1. Structure of Ribociclib 1 and its derivative 2
Table 1. Kinase activity of Ribociclib 1 and analogue 2

IC50 value (nM)
Cyclin
Cyclin
E/CDK2
>20000
390
Cyclin
D/CDK6
73
Compounds
D/CDK4
29
1
2
112
101
In accordance with the principle of designing PROTAC molecules, 23 compounds were
synthesized (for details, please refer to the ESI files). After carefully screened, compound 3 was
proven as the most efficient one to degradation of CDK 2/4/6 in various cancer cells, exhibiting
remarkable inhibiting effect of tumor growth. Control assays clearly demonstrated that Ribociclib,
thalidomide or their combination at equimolar could not induce the cleavage of CDK 2/4/6 in
cancer cells or induce the apoptosis of cancer cells.
The forwards synthesis of compound 3 was straightforward and efficient. One hand, the main
core 2 was obtained from commercially available materials 4 and 5 under Buchwald coupling
conditions. Hydrolysis of the methyl ester delivered carboxylic acid 6 as coupling precursor. On
the other hand, 2-fluoro-thalidomide 9, prepared from the commercially available starting
materials 7 and 8, reacted with N-Boc-hexane-1,6-diamine under basic conditions to delivery
amide 10. Removal Boc protecting group by TFA, and subsequential amide coupling with
intermediate 6 furnished the compound 3 required in moderate yield in gram-scale.
Reaction reagents and conditions: a Pd(OAc)2, BINAP, Cs2CO3, dioxane, reflux, 79%; b
LiOH, THF/H2O, rt, 98%; c AcONa, HOAc, reflux, 80%; d N-Boc-hexane-1,6-diamine, DIPEA,
DMF, 80 oC, 55%; e TFA, CH2Cl2, 0 oC to rt; f compound 6, EDCI, HOBt, DIPEA, MeCN, rt, 43%
over 2 steps.
Scheme 2. Design of PROTAC molecule and Synthesis of Compound 3
For all PROTACs, the effect on the cell viability in a panel of cancer cell lines was examined

by standard MTT assay. Ribociclib (RIB), Pomalidomide (POM) and their mixture acted as
reference control. Compound 3 exhibited prominent anti-proliferative activity against a broad
spectrum of human cancer cell lines (Table 1, ESI), in particular for Melanomas (Table 2). IC50 of
B16F10 and A375 was ascertained as 0.09761±0.021 and 0.1659±0.150, respectively.
Table 2. Cytotoxic Activity of Compound 3 Against Melanoma Cells
IC50 value (μM)
Compounds
B16F10
0.09761±0.021
>10
A375
3
0.1659±0.150
>10
RIB
POM
>10
>10
RIB+POM (1:1 ratio)
>10
>10
IC50 > 10 μmol/L indicated no significant inhibition; IC50 was determined from three
independent experiments. MTT assay was employed to ascertain the cytotoxic effect of
compound 3 after 72 h incubation.
Subsequently, compound 3 was tested for CDK2/4/6 degradation in melanoma cells (Figure
1). It was reported that the compound 3 induced significant intracellular cleavage of all CDK 2,
CDK 4 and CDK 6 proteins in both concentration-dependent and time-dependent manners (Figure
1A and 1B). It also effectively mitigated downstream p-Rb signaling pathway activation of
Melanoma Cells (Figure 1A and 1B). To verify whether the cleavage effect of CDK2/4/6 by the
treatment of compound 3 was PROTAC-mediated degradation, the B16F10 cells were first
pre-treated by Pomalidomide, MG132 as well as compound 2 (Figure 1C). Degrading effect of
CDK2, 4 and 6 was totally impeded. Note that pre-treatment with Ribociclib, CDK4/6 degradation
was dramatically inhibited, while CDK2 was down-regulated as normal. These results were
consistent with our original finding and design.
Figure 1. Compounds 3 Induced CDK2/4/6 Protein Degradation and Inhibited the Downstream

Rb Signaling Was Mitigated by Hijacking the E3 Ubiquitin Ligase CRBN. (A) Compounds 3
significantly down-regulated CDK2/4/6 protein levels and inhibited the Rb downstream signaling
in a concentration-dependent manner. B16F10 and A375 cells were treated with DMSO or serial
dilutions of compounds 3 for 12 h. (B) Compounds 3 down-regulated CDK2/4/6 proteins and
inhibited the p-Rb downstream signaling in a time-dependent manner. B16F10 andA375 cells
were treated with DMSO or compound 3 for the indicated length of time. (C) Compounds 3
induced CDK2/4/6 protein degradation was proteasomal degradation. Before being treated with
the 100 nM compounds 3 for 6 h, cells were pre-treated with DMSO, Pomalidomide (10 μM),
MG132 (20 μM) for 2 h. The CDK2/4/6 protein were determined by western blot.
To biologically assess the anti-proliferative activities of compound 3 against two melanoma
cancer cells (A375 and B16F10), colony formation assay was performed (Figure 2). Compound 3
exerted a remarkable inhibiting effect on colony formation in A375 and B16F10, two malignant
melanoma cancer cells, in low nano molar concentration.
Figure 2. Inhibiting Effects of Compound 3 on Colony Formation of Malignant Melanoma Cancer
Cells. Colony formation assay was performed to ascertain the proliferation capacity of A375 and
B16F10 cellstreated with compound 3. (A) Representative image of long-term growth assay of
A375 and B16F10 cells following continuous compound 3 for 7 days. (B) Quantification of
clonogenic growth illustrated in A. Student's T-test; mean ± SD, n = 3. **p<0.01;*** p<0.001.
Since cell apoptosis was distinctly observed under a microscope when cancer cells were
treated with compound 3, both A375 and B16F10 cells were treated with vehicle alone as control
and with 3 at various concentrations for 48 h; then, they were stained with FITC-Annexin V and
propidium iodide (PI). The percentages of apoptotic A375 and B16F10 cells were measured by
flow cytometry. Figure 3A and 3B suggest that compound 3 significantly induced apoptosis of
A375 and B16F10 cells in a dose-dependent manner.

Figure 3. Compound 3 Induced of apoptosis in malignant melanoma cancer cells. (A) Flow
cytometry analysis of apoptotic A375 and B16F10 cells induced by compound 3 at various
concentrations. (B) Quantitative analysis of apoptosis illustrated in A. Student's T-test; mean ± SD,
n= 3. * P < 0.05; **P < 0.01; ***P < 0.001.
To identify the potential mechanism of apoptosis induced by compound 3, several cell
death-related proteins (e.g., PARP, Caspase-3, Bax, Bcl-2 and P53) were ascertained by western
blot in A375 and B16F10 cells. Figure 4 suggests that cleaved Casepase-3 and cleaved PARP, two
critical hallmarks of apoptosis, were noticeably up-regulated by the treatment with compound 3 in
a dose-dependent manner. In the meantime, compound 3 also significantly induced up-regulation
of the pro-protein level of P-53 and Bax. In contrast, the anti-apoptotic protein level of Bcl-2 was
down-regulated following the treatment with compound 3. On the whole, compound 3 induced
Caspase-dependent apoptosis via the common P53/Bcl-2/Bax apoptotic pathway in melanoma
cells.
Figure 4. Apoptosis Induced by Treatment of Compound 3 through Caspase-dependent Manner.
A375 and B16F10 cells were incubated with compound 3 for 48 h, cleaved-Caspase-3,
cleaved-PARP, total expression of P53 and Bcl-2 members (Bcl-2 and Bax) were assessed by
Western blot. β-Actin was used as an internal control.
To verify whether cytotoxic activity of compound 3 results from cell cycle arrest which

induced via degradation of CDK2/4/6, the cell cycle kinetics in human cancer cell lines, including
A375 and B16F10 cells, were then investigated by treating those cancer cell lines with compound
3 (Figure 5). Tumor cells were incubated with complete medium supplemented by compound 3
and DMSO at various concentrations for 48 h; then they underwent flow cytometry to ascertain
the proportion of cells in some phase of cell cycle. As shown in Fig.5, tumor cells were treated
with DMSO normally distributed in each phase of cell cycle. In contrast, treatment of compound 3
increased accumulation of A375 cell population in G0/G1 pahse, but G2/M phase for B16F10 cell.
These results indicated that compound 3 could induce cell cycle arrest via the degradation of cell
cycle-related proteins CDK2/4/6 in tumor cells.
Figure 5. Effect of Compound 3 on Cell Cycle Progression of Tumor Cells. (A) Flow cytometry
of the DNA content of A375 and B16F10 cells following staining with propidium iodide. (B) The
bar graph illustrates the proportions of tumor cells in G0/G1, S and G2/M phase, respectively.
Student's T-test; mean ± SD, n= 3. * P < 0.05; **P < 0.01; ***P < 0.001; ****P < 0.0001.
However, similar as most reported PROTAC molecules with CRBN ligands, the degrader 3
had alomst no oral bioavailblity (<1%) proparbly due to its amphotericity. Therefore, our
hypothesized solution was to prepare a prodrug by adding a lipophilic group at active site of
CRBN ligands (Scheme 3). It might provide convenient oral administration of the PROTAC
molecule in animal test. Chemically, modification of the prodrug was very straightforward. As
shown in scheme 3, a one-step reaction with chloromethyl pivalate under mild conditions
converted degrader 3 into prodrug 11 in gram-scale. Based on the results of preliminary PK study
(For more details, please see ESI), the orally bioavaiblility of compound 11 is up to 68%.
Compound 3 was exposed to plasm of rats with a useful concentration within 4-8 hours. To our
best of konwledge, this is the first time to report the successful development of orally bioavailble
prodrug of PROTAC molecules based on the CRBN ligands.

Reaction reagents and conditions: a Chloromethyl pivalate, Cs2CO3, DMF, TBAI, rt, 65%.
Scheme 3. Synthesis of Prodrug 11
Plasma concentration−time curves for compound 11 after a single dose in rats were shown in
Figure 6. Based on the results, after PO administration (PO, Figure 6B) of the compound 11,
which could steadily convert into active ingredient 3 in rat plasma, its blood concentration rapidly
reached peak, and then remarkably declined in 5 hours. The pharmacokinetic of compound 11 was
also investigated by tail intravenous injection (IV, Figure 6A). The pharmacokinetic parameters
determined with a non-compartmental model were shown in table 2.
Figure 6. PK study of compound 11 in rats. (A) Plasma concentration−time curve of Compounds
11 and 3 in SD rats after a single IV administration of drug (10 mg/Kg, n = 3); (B) Plasma
concentration−time curve of Compound 11 and 3 in SD rats after a single PO administration of
drug (200 mg/Kg, n = 3);
Table 2. Preliminary Pharmacokinetic (PK) Parameters of Compound 11 and 3
PO (200 mg/Kg)
Cmax/ng.mL-1
692268
Tmax/h
0.25
AUC0-12/ng.mL-1
F/%
NA
Compound
11
3
822803.9667
2261897
0.25
2810197.617
NA
IV (10 mg/Kg)
Cmax/ng.mL-1
Tmax/h
AUC0-12/ng.mL-1
F/%
68%
NA
Compound
11
3
59851
0.75
NA
160704.5
95665.81
110809
With the encouraging PK parameters of prodrug 11 in hand, its anticancer activity in vivo was
assessed for its suppression of B16F10 tumor growth. As shown in Figure 7, tumor sizes were
significantly suppressed in mice treated with compound 11. There was no noticeable difference in
body weight between the two groups. These results revealed that oral administration of compound
11 can distinctly inhibit B16F10 tumor growth and mediate degradation of CDK2/4/6 in vivo.

Figure 7. Effect of Compound 11 on B16F10 Tumor Growth Delay and Regression in vivo
through oral administration. C57BL/6J mice intravenously injected with B16F10 cells. (A)
Variations in the tumor volume of B16F10 xenografts. (B) Tumors were weighted on the15th day.
(C) Body weights of animals. (D) The representative images of tumors dissected from vehicle-and
compound 3 and 11-treated mice are presented. Student's T-test;; mean ± SD, n = 6, **P < 0.001.
(E) Tumor tissue lesions (n = 5) from B16F10 xenograft model were stained with monoclonal
antibody against CDK2/4/6 and then photographed (100X).
In conclusion, a novel PROTAC degrader, compound 3, based on a Ribociclib derivative and
CRBN ligand has been developed. The small molecule degraded CDK 2/4/6 as well as their
complex in malignant melanomas simultaneously and effectively. This compound could also
rapidly reset the cell cycles and induce cell apoptosis in various cancer cells, in particular for
melanomas. The mechanism should be interpreted that deficiency CDK 2/4/6 might lead to
synthetic lethal effects to malignant melanomas in the presence of compound 3. These results
suggested that the combination of CDK 2/4/6 would become promising kinase targets for
treatment of solid tumors. Furthermore, an orally bioavailable prodrug 11 was developed for oral
drug delivery in animal test with high bioavailblity, for the first time. It might also provide a
general solution for oral administration of the PROTAC molecules, which were derived from
CRBN ligands.
ASSOCIATED CONTENT
The Supporting Information is available free of charge on the website.
The synthetic procedure and copies of 1H/13C NMR Spectrum of all compounds and intermediates.
Biological evaluation of all selected compounds (PDF)
AUTHOR INFORMATION
Corresponding Author
* jwang05@tmu.edu.cn
* Guang.yang@nankai.edu.cn
* Cheng.yang@nankai.edu.cn
Author Contributions
§ These authors contributed equally to this work.

Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
This work was supported by the National Natural Science Foundation of China (NSFC, No.
81703343 and 81503019), the Fundamental Research Funds for the Central Universities, Nankai
University (NO. 735-63201235); National Science & Technology Major Project “Key New Drug
Creation and Manufacturing Program”, China (No. 2019ZX09201001), Key Research and
Development Program of Tianjin City (No. 19YFZCSY00160).
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Highlights
Compound 3 is an efficient PROTAC molecule which can degrade CDK 2/4/6
simultaneously and effectively.
Compound 3 Remarkably inhibit the growth of melanoma cells
.
The first orally bioavailable prodrug was developed for the PROTAC molecules with
CRBN ligands.
The oral bioavailability of compound 11 is up to 68%.

Declaration of interests
☒ The authors declare that they have no known competing financial interests or personal relationships
that could have appeared to influence the work reported in this paper.
☐The authors declare the following financial interests/personal relationships which may be considered
as potential competing interests: