Designing an eEF2K-Targeting PROTAC small molecule that induces apoptosis in MDA-MB-231 cells

Article abstract of DOI:10.1016/j.ejmech.2020.112505

Eukaryotic elongation factor 2 kinase (eEF2K) is a key α-kinase that negatively regulates the extension step of protein synthesis, which consumes most of the energy and amino acids required for protein synthesis. Studies have found that eEF2K protein is related to the breast cancer. However, existing inhibitor effect has not achieved the desired effect in cancer therapy. Proteolysis target chimeric (PROTAC) technology is uses proteasome to degrade target protein to achieve the purpose of inhibiting tumour cell growth. Here, we reported that the use of PROTAC strategy in combining with star eEF2K inhibitor A484954 and its potential derivatives. Consequently, candidate compound 11l was found to degrade eEF2K and induce apoptosis in human breast carcinoma MDA-MB-231 cells. Together, these findings demonstrate that our eEF2K-targeting PROTAC small molecule would be a potential new strategy for future breast cancer therapy.

Full text of DOI:10.1016/j.ejmech.2020.112505

Journal Pre-proof
Designing an eEF2K-Targeting PROTAC Small Molecule That Induces Apoptosis in
MDA-MB-231 Cells
Yao Liu, Yongqi Zhen, Guan Wang, Gaoxia Yang, Leilei Fu, Bo Liu, Liang Ouyang
PII:
S0223-5234(20)30477-3
DOI:
https://doi.org/10.1016/j.ejmech.2020.112505
EJMECH 112505
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 6 February 2020
Revised Date: 7 May 2020
Accepted Date: 21 May 2020
Please cite this article as: Y. Liu, Y. Zhen, G. Wang, G. Yang, L. Fu, B. Liu, L. Ouyang, Designing an
eEF2K-Targeting PROTAC Small Molecule That Induces Apoptosis in MDA-MB-231 Cells, European
Journal of Medicinal Chemistry, https://doi.org/10.1016/j.ejmech.2020.112505.
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Designing an eEF2K-Targeting PROTAC Small Molecule That Induces
Apoptosis in MDA-MB-231 Cells
Yao Liu a, #, Yongqi Zhen b, #, Guan Wang , Gaoxia Yang , Leilei Fu b, *, Bo Liu a, * and Liang Ouyang a, *
a
a
aState Key Laboratory of Biotherapy and Cancer Center, West China Hospital, Sichuan University, and Collaborative
Innovation Center of Biotherapy, Chengdu 610041, P.R.China. E-mail: ouyangliang@scu.edu.cn, liubo2400@163.com
bSchool of Life Science and Engineering, Southwest Jiaotong University, Chengdu 610041, P.R. China, E-mail:
leilei_fu@163.com
#These authors contributed equally to this work.
Abstract
Eukaryotic elongation factor 2 kinase (eEF2K) is a key α-kinase that negatively regulates the
extension step of protein synthesis, which consumes most of the energy and amino acids required for
protein synthesis. Studies have found that eEF2K protein is related to the breast cancer. However,
existing inhibitor effect has not achieved the desired effect in cancer therapy. Proteolysis target chimeric
(PROTAC) technology is uses proteasome to degrade target protein to achieve the purpose of inhibiting
tumor cell growth. Here, we reported that the use of PROTAC strategy in combining with star eEF2K
inhibitor A484954 and its potential derivatives. Consequently, candidate compound 11l was found to
degrade eEF2K and induce apoptosis in human breast carcinoma MDA-MB-231 cells. Together, these

findings demonstrate that our eEF2K-targeting PROTAC small molecule would be a potential new
strategy for future breast cancer therapy.
Key words: Eukaryotic elongation factor 2 kinase; Proteolysis target chimeric; Apoptosis;
MDA-MB-231 Cells, Breast cancer therapy
1
. Introduction
Eukaryotic elongation factor 2 kinase (eEF2K) belonging to the α-kinases family is well-known as a
calcium/calmodulin dependent protein kinase III (CaM kinase III) [1, 2]. eEF2K may catalyse the
phosphorylation of the Thr56 of eEF2 [3], the only known substrate, and make it incapable to bind
ribosomes, which negatively regulates the extension steps of protein synthesis and consumes most of the
energy and amino acids required for protein synthesis [4, 5]. In general, the activation of eEF2K by Ca2+ is
2+
catalyzed by calmodulin (CaM) binding to the N-terminus of four Ca -ions domain, which mechanism
may be related to the removal of an autoinhibitory helical feature from the active site [6, 7]. It is also
regulated by nutrition and energy-related signaling molecules, such as AMPK and mTORC [8-10]. In
poorly-vascularised tumors, eEF2K can save programmed death in the case of energy consumption or
lack of nutrients [11]. Silencing eEF2K can inhibit endoplasmic reticulum stress to promote tumour cell
apoptosis [12]. Studies showed that eEF2K can protect breast cancer cells from nutritional starvation and
energy deficiency [13].
eEF2K is promising potential target at the end of tumour signaling pathway and the discovery of
targeted inhibitors for eEF2K also shows an increasing trend. Rottlerin (1) (Fig. 1A), a known PKCδ

(protein kinase Cδ) inhibitor, has been found to activate calcium and potassium channels and thereby
affect mitochondrial function, and has been used as an inhibitor of eEF2K [14, 15]. The first drug-like
eEF2K inhibitor (2) is an ATP competitive inhibitor, but its inhibition in cells is not ideal [16]. Compound
NH125 (3) has the same phenomenon as 2 [17]. Compound 4 may be covalently bound to the catalytic
domain of cysteine residues to exert kinase inhibition [18]. A high-throughput screening method used by
U.S. scientists and Abbott Laboratories obtained 5 [19] and A484954 (6) [20]. They are all ATP
competitive inhibitors, among which 6, showed better inhibitory activity but can cause cytotoxicity at the
effective concentration [21]. However, the existing inhibitors are difficult to play a better anti-tumour role in
clinical application, which may be related to factors such as no crystal structure of the eEF2K protein to
help scientists find a more proper inhibitor [22]. Based on this situation, there is a demand for a way to
solve the current dilemma of eEF2K inhibitors.
Here, we proposed to use proteolysis target chimeric (PROTAC) to eliminate eEF2K protein
fundamentally rather than inhibit it. The concept of PROTAC was first proposed by Craig M. Crews [23,
24]. The typical PROTAC degrader is composed of a linker and two ligands, one binding to an E3 ligase
degradation complex and the other to the protein of interest (POI) [25, 26]. The POI is recruited to the E3
ligase complex by the PROTAC degradant for ubiquitination and subsequent degradation by the
proteasome [27, 28] (Fig. 1B). The process of protein degradation is similar to catalytic reaction. Drugs
can repeatedly inhibit the target protein and an equimolar amount of the drug is not required. Therefore, it
is possible to obtain highly active drugs and the activity requirements for PROTAC may not be high.

Moreover, PROTACs provide potential for "undruggable", non-enzyme promoted and easily resistant
proteins to develop clinical candidate drugs [29, 30]. Currently, two PROTACs of selective hormone
receptor inhibitors from Arvinas were approved by FDA for Phase I clinical trials [31], and it is foreseeable
that more drugs using PROTAC strategies will enter the clinical trials in the near future.
Fig. 1. (A) Chemical structures of some representative eEF2K inhibitors; (B) Schematic diagram of PROTAC mechanism.
2
. Results
2.1 PROTAC Design and Efficacy
Our goal is to discover eEF2K inhibitors that are used to design as eEF2K degrading agents. Great
efforts have been made to find eEF2K inhibitors, so we focused on the reported eEF2K inhibitors.

Through the analysis and comparison of the existing inhibitors, A484954 successfully attracted our
attention based on a linker connection and enzyme activity. We used Dictyostelium myosin heavy chain
kinase A (MHCK A) as a template for homology modelling to generate the structure of eEF2K. A484954
was combined with the ATP binding pocket of eEF2K and forms hydrogen bond with residues LYS170,
ILE232, GLY234 and TRY236 (Fig. 2). The ethyl group attached to the N atom extended out of the cavity
is very suitable for linker connection [22]. There is also a good enzymatic activity and targeting according
to the previously reported ethyl-substituted cyclopropyl group, whereby a derivative of ethyl-substituted
A484954 is also employed as an inhibitor for targeting eEF2K [22]. We used linkers with different lengths
and types. Meanwhile, thalidomide, a mature E3 ubiquitin ligase ligand [32, 33], was used as a
recruitment element. In this study, the synthesis of compounds 9a-9l is illustrated in Scheme 1., western
blot was used to detect the degradation of eEF2K-PROTACs in MDA-MB-231 cells, and the degradation
rate (Dr) was calculated based on control group and used to characterize the degradation ability of
eEF2K-PROTACs. Compared with the control group and A483954, compounds 9h-9l could degrade
eEF2K to a certain extent (Table 1, Fig. 3A). It means that the small PROTACs designed and synthesized
by us are effective. The results showed that with the increase of linker, the degradation is increasing.
Although the degradation of eEF2K-PROTACs was observed, the value of Dr did not reach an ideal level.
Interestingly, A484954 seems to have an agonistic effect on the eEF2K protein in MDA-MB-231 cells. The
reason may be that the compound mainly works by affecting the phosphorylation of downstream proteins
or feedback regulation which is a compensatory increase in quantity resulting from functional suppression

[20, 22]. The degradation of cyclopropyl substituted eEF2K-PROTACs was not observed, but the
degradation resulting from the increase of chain length may be observed. Based on this result, we used a
3-OH-substituted thalidomide linked to tert-butyl bromoacetate to extend the linker moiety, then
compounds 11a-11l could be synthesized shown in Scheme 2. Unfortunately, the degradation of
eEF2K-PROTACs substituted by cyclopropyl not reach to our expectation (Table 1, Fig. 3B). However,
the degradation of ethyl substituted eEF2K-PROTACs are enhanced and compound 11l has the
r
maximum D value is 56.7%. Because the introduction of carbonyl groups is in the process of increasing
the carbon chain, the carbonyl group may have a positive effect on the enhancement of degradation,
which needs our further study. Next, we tested compound 11l at different concentrations, and compared
them with the corresponding concentration of A484954. Iinhibitions of compound 11l on eEF2K in
different concentration gradients was improved compared with positive-control and the control group (Fig.
3C)
Fig. 2. Design of PROTACs for eEF2K. (A) Compound 6 docked into the ATP-binding pocket of eEF2K.(B) The key four

residues LYS170, ILE232, GLY234 and TRY236 bond with compound 6. (C) Schematic diagram of eEF2K-PROTAC.
Scheme 1. Synthesis of compounds 9a-9l. Regents and conditions: (i) HCl, Potassium cyanate, 0-70 °C; (ii) Ethyl
2 3
cyanoacetate, EtONa, EtOH, reflux; (iii) DMF, COCl , DCM, 0-10 °C; (iv) 2-Cyanoacetamide, Et N, EtOH, r.t.; (v) tert-Butyl
bromoacetate, KOtBu, DMF, r.t.; (vi) THF: DCM= 1:2, r.t.; (vii) NaOAc, AcOH, 90 °C; (viii) NMP, DIPEA, 90 °C; (ix)
THF/DCM= 1:2, r.t.; (x) HATU, DIPEA, DMF, r.t..
3
Scheme 2. Synthesis of compounds 11a-11l. Regents and conditions: (xi) NaOAc, AcOH, 90 °C; (xii) KHCO , KI, DMF,
6
0 °C; (xiii) THF: DCM= 1:2, r.t.; (xiv) HATU, DIPEA, DMF, r.t.; (xv) THF: DCM= 1:2, r.t.; (xvi) HATU, DIPEA, DMF, r.t..

Table 1. Degradation rate of eEF2K-PROTACs candidate compounds 9a-9l.
Compound
R
1
Linker
Dra
N.A.b
N.A.
9a
9b
9c
9d
9e
cyclopropyl
cyclopropyl
cyclopropyl
N.A.
cyclopropyl
N.A.
cyclopropyl
N.A.
9f
cyclopropyl
N.A.
9
g
h
Et
Et
Et
Et
Et
Et
N.A.
9
20.0%
35.0%
24.0%
20.0%
15.0%
9
i
9
j
9
k
9
l
a
r
eEF2K degradation rate (D ) was calculated by quantified western blots after 24 h in MDA-MB-231 cells.
bNot active (degradation rate was negative value).
Table 2. Degradation rate of eEF2K-PROTACs candidate compounds 11a-11l.

Compound
R1
Linker
Dra
1
1a
1b
cyclopropyl
N.A.b
N.A.
1
cyclopropyl
11c
cyclopropyl
N.A.
1
1d
1e
cyclopropyl
N.A.
1
cyclopropyl
N.A.
11f
cyclopropyl
N.A.
1
1g
1h
Et
Et
Et
Et
Et
Et
10.0%
15.4%
26.0%
32.0%
35.3%
56.7%
1
1
1i
1
1j
1
1k
1
1l
a
eEF2K degradation rate (Dr) was calculated by quantified western blots after 24 h in MDA-MB-231 cells.
bNot active (degradation rate was negative value).

Fig. 3. Characterization of eEF2K-PROTACs mediated eEF2K degradation in MDA-MB-231 cells. (A) Western blot of
eEF2K and β-actin following 24 h incubation with DMSO or indicated small molecules 9a-9l at 200 μM. (B) Western blot of
eEF2K and β-actin following 24 h incubation with DMSO or indicated small molecules 11a-11l at 200 μM (C) Western blot
of eEF2K and β-actin following 24 h incubation with DMSO or the indicated concentrations of compound 11l in
MDA-MB-231 cells. *, p<0.05, **, p<0.01, ***, p<0.001, compared with control group.
2.2 PROTACs mechanistic studies and effects on apoptosis in MDA-MB-231 cells
To further explore the degradation mechanism of eEF2K by compound 11l, MDA-MB-231 cells
pretreatment with proteasome inhibitor MG-132 and ubiquitination inhibitors thalidomide to blocked the
degradation of eEF2K. As shown in Figure 4A, the expression levels of eEF2K in co-treated group were
almost no changed, indicating that 11l induced eEF2K degradation is dependent on ubiquitin-proteasome

degradation system. In addition, treatment with thalidomide alone had no effect on the expression of
eEF2K (Fig.4A). At the same time, the inhibition on downstream signal transduction of eEF2K
degradation was also detected. The compound 11l was able to significantly degrade eEF2K and p-eEF2K
in a dose-dependent manner and also had a significant inhibitory effect on phosphorylated eEF2 (Fig.
4B).
Next, to further evaluate the effect of our synthesized PROTACs on cell biological activity, we
performed subsequent activity detection experiments. During the cloning experiment, we found that
treatment of MDA-MB-231 cells with compound 11l at 100 μM could inhibit cell proliferation (Fig. 4C). The
apoptosis alternation was observed by fluorescence microscopy through Hoechst33258 maintaining
method, and obvious apoptosis could be detected (Fig. 4D). Moreover, 6 (A484954) displayed slight
anti-proliferative and apoptosis-inducing effects, but obviously weaker than 11l at the same condition (Fig.
4C, 4D). Therefore, we speculated that compound 11l could induce apoptosis in MDA-MB-231 cells. Next,
the Annexin V/PI co-staining method was used to evaluate the apoptosis rate. Compared with the control,
the apoptosis rate of the MDA-MB-231 cells which was treated with compound 11l increased significantly
with a dose dependent manner (Fig. 4E). Furthermore, in order to detect the toxicity of the compound 11l,
we treated the normal breast cancer cells MCF-10A with the compounds 11l and 6 (A484954), as shown
in Fig. S1，both of the two compounds showed little cytotoxic effects on MCF-10A cells.
Subsequently, we used immunoblotting to detect several key apoptosis-related proteins.
MDA-MB-231 cells were treated with compound 11l with increased concentration for 24 hours. The

results showed that compound 11l could significantly up regulate the expression of Bax and
cleaved-caspase 3 and down regulate the expression of Bcl-2, which indicated that compound 11l played
an anti-tumour role in MDA-MB-231 cells by inducing apoptosis (Fig. 4F).
Fig. 4. Characterization of compound 11l mechanistic studies and effects on apoptosis in MDA-MB-231 cells. (A)
MDA-MB-231 cells were treated with 10 µM of thalidomide only; or pretreated 10 µM of DMSO, thalidomide and MG-132 for
2
h, followed by 8 h treatment with 11l at 100 µM. (B) Immunoblot for eEF2K, p-eEF2K, eEF2 and p-eEF2 after treatment

with DMSO and indicated concentrations of compound 11l. (C) Results of colony-forming assay in MDA-MB-231 cells with
6
(A484954) compound 11l at 100 µM. (D) Fluorescence microscopic appearance of Hoechst 33258 staining nuclei of
MDA-MB-231 cells at 100 µM. Scale bar = 50 µM. (E) MDA-MB-231 cells were treated with DMSO and compound 11l for
2
3
4 h, and apoptosis was assayed by flow cytometry after Annexin V/PI co-staining. (F) Immunoblot for Bcl-2, Bax, caspase
and cleaved-caspase 3 after treatment with DMSO and indicated concentrations of compound 11l, β-actin was measured
as a loading control.
3
. Discussion and Conclusion
In conclusion, eEF2K has the characteristics of high specificity and low side effects and the discovery
of targeted and potent inhibitors for eEF2K has been drawing more attention. To address the current
limitations of eEF2K inhibitors, we developed a protein degrader based on A484954, by using the protein
hydrolysis mechanism of the cell itself to eliminate the target protein in a cell. Consequently, we obtained
PROTAC which could degrade eEF2K and inhibit its autophosphorylation and downstream signalling.
Interestingly, our PROTAC was able to induce MDA-MB-231 cells apoptosis in a dose-dependent manner.
As for the difficulty in α-kinase inhibitor design and discovery[34], there are only six inhibitors that have a
certain inhibitory effect on eEF2K. Among them, A484954 is the most recognized and effective eEF2K
inhibitors (IC50= 0.28 μM) [20], but it also needs a high concentration to exert its inhibitory effect in
MDA-MB-231 cells [22], which may the reason why PROTACs cannot perform degradation at low
concentration solution. The most advantageous aspect of PROTAC is that it can target traditionally
“undruggable” targets, which may account for more than 80% of the human proteome. Moreover, for
many drug-implemented targets, PROTAC is also a strategy to solve the problems of drug resistance and

selectivity. Despite the rapid development of PROTAC technology, there are still problems such as
excessive molecular weight, poor water solubility, asynchronous pharmacokinetics and
pharmacodynamics. However, there are also authoritative articles describing that although the molecular
weight of PROTAC exceeds the 500 Daltons recommended by the “rule of five”, the molecules can still
enter the cell to play their role, possibly because they are recognized by the cell membrane as two small
molecules [27]. This is an attempt to overcome the insufficient inhibitory effect of existing eEF2K inhibitors
by using the PROTAC strategy for the first time, and we will make further optimizations. More importantly,
we will continue to study various types of terminal proteins and develop new potential PROTACs for their
future druggable implications in cancer therapeutics.
4
4
4
. Experimental section
.1. Chemistry
.1.1. Materials and measurements
1H NMR (400 MHz) and 13C NMR (100 MHz) spectra were detected by means of a nuclear magnetic
resonance apparatus (Bruker). All deuterated reagents were produced by Sigma-Aldrich or Cambridge
Isotope Laboratories. The chromatography column was performed using 300-400 mesh silica gel
produced by Qingdao Ocean Chemical Co., Ltd. The chemical shift was recorded in ppm in units of
tetramethylsilane(TMS) as the internal standard. The peak shape is expressed as follows: s, singlet, brs,
broad signal, d, doublet, brd, broad doublet, t, triplet, q, quadruplet, m, multiplet.
4.1.2. Synthesis

4.1.3. General procedure for the preparation of intermediates 7a-7d
The synthesis of 7a. Added the potassium cyanate (120 mmol) to a mixture of alkylamine (40 mmol)
in 1M HCl (40 mL) and stirred at 60 °C for 16 hours. The solution cooled to room temperature. The
resulting precipitate was collected by under reduced pressure and residue washed with 2 L ethyl acetate
2 4
(500 mL for every 10 mmol of amine used), dried over Na SO . The concentrated organic layer to give the
pure urea and was used in next step without further purification (90% yield).
The synthesis of 7b. Dissolved the sodium (2.3 g, 100 mmol) in anhydrous ethanol (100 mL) to
acquire the fresh sodium ethoxide. Added ethyl 2-cyanoacetate (5.7 g, 50 mmol) into the fresh
preparation sodium ethoxide solution to get the white turbid liquid and then the 7a (50 mmol) was added.
Besides the mixture was supposed to stir at reflux for 24 h. The solvent was evaporated under reduced
pressure, and dissolved in a small amount of water. The pH of the solution was adjusted to 7 by using
concentrated HCl. The yellow solid formed and collected by filtration, and washed with water, then dried
under vacuum to afford 7b (92% yield).
The synthesis of 7c. To a solution of dimethylformamide (11 mmol) in dichloromethane (150 mL) was
added oxalyl chloride (11 mmol) dropwise at 0 °C. The reaction mixture was stirred for 30 min at room
temperature. Compound 7b (10 mmol) was added and stirred for another 30 min. The solvent was
removed under reduced pressure and the crude was used in to the next step without further purification.
To the crude in dry ethanol (100 mL) was added cyanoacetamide (10 mmol), stirred at 0 °C, and then
triethylamine (12 mmol) dropwise. The suspension was allowed to room temperature and stirred for 2 h.

Filtered the resulting precipitate and washed with dry ethanol to give the product 7c.
The synthesis of 7d. Added KOtBu (2 mmol) to a solution of compound 7c (1 mmol) in
dimethylformamide (5 mL) at room temperature. Then the tert-Butyl bromoacetate (2 mmol) was added by
dropwise slowly. The mixture was stirred at room temperature for 4 hours, then the solvent was
evaporated under reduced pressure. The mixture was purified by flash column chromatography to get a
light brown solid. A solution of light brown solid (1 mmol) in TFA (trifluoroacetic acid): DCM
(dichloromethane) = 1:2 (6 mL) was stirred at room temperature for 1h. The mixture was evaporated
under reduced pressure to give the product 7d (TFA salt) without further purification.
4.1.4. General procedure for the target compounds 8a-8b
The synthesis of 8a. To a solution of acetic acid was added 3-fluorophthalic anhydride (1 mmol) and
-aminopiperidine-2,6-dione hydrochloride (1 mmol), then put the sodium acetate in the above mixture.
3
The reaction mixture was stirred overnight at 90 °C. The reaction was extracted with dichloromethane
4
(3×50 mL). The combined organic phases were washed with water and dried over MgSO , then
evaporated under reduced pressure to give a dark-brown oil which was purified by flash column
chromatography to yield the product 8a as a yellow solid.
The synthesis of 8b. To a solution of NMP was added compound 8a (1 mmol) and appropriate N-Boc
alkyl amine (1.1 mmol), then DIPEA (2 mmol) was added, the reaction mixture was stirred overnight at
90 °C. The reaction mixture was extracted with dichloromethane and washed with 10% citric acid. The
3
combined organic phases were washed with saturated NaHCO and dried over MgSO4. Then the organic

phases evaporated under reduced pressure to produce a brown oil which was purified by flash column
chromatography to yield a yellow solid. Then a solution of yellow solid (1 mmol) in TFA: DCM= 1:2 (6 mL)
was stirred at room temperature for 1 h. The mixture was evaporated under reduced pressure to give the
product 8b (TFA salt) without further purification.
4.1.5. General procedure for the the target compounds 10a-10c
The synthesis of 10a. To a solution of acetic acid was added 3-hydroxyphthalic anhydride (2 mmol)
and 3-aminopiperidine-2,6-dione hydrochloride (2 mmol), then sodium acetate (2.4 mmol) was added.
The reaction mixture was stirred overnight at 90 °C. The reagent was extracted with dichloromethane
4
(3×50 mL). Washed the combined organic phases were with water and dried over by MgSO , then
evaporated under reduced pressure. The mixture was purified by flash column chromatography to yield
the product 10a as a white solid.
The synthesis of 10b. In a round-bottom flask, compound 10a (2 mmol) was dissolved in DMF (8 mL).
3
KI (0.2 mmol) and KHCO (3 mmol) were added to the stirred solution. Then tert-Butyl bromoacetate (2.4
mmol) was continuously added dropwise. The resulting mixture was stirred at 60 °C for 12h. The reaction
mixture was extracted with dichloromethane (3×50 mL). The combined organic phases were dried over
4
MgSO and evaporated under reduced pressure. The mixture was purified by flash column
chromatography to obtain the product as a white solid. Then a solution of white solid (1 mmol) in TFA:
DCM= 1:2 (6 mL) was stirred at room temperature for 1 h. The mixture was evaporated under reduced
pressure to give the product 10b (TFA salt) without further purification.

The synthesis of 10c. To a solution of 10b (1 eq.) in DMF was added the appropriate acid (1 eq.), and
the solution was stirred at room temperature. Then HATU (1.1 eq.) was added and DIPEA (3 eq.) was
added dropwise. The mixture was stirred at room temperature for 2 h. The solvent was evaporated under
reduced pressure, which was purified by flash column chromatography purification to yield the
intermediates. A solution mixture of the intermediates (1 mmol) in TFA: DCM = 1:2 was stirred at the room
temperature and stirred for 1h. The mixture was evaporated under reduced pressure to obtain the 10c
(TFA salt).
4.1.6. General procedure for the target compounds 9a-9l, 11a-11l
To a solution of amine (1 eq.) in DMF (N, N-dimethylformamide) was added the appropriate acid (1
eq.), and the solution was stirred at room temperature. Then HATU (1.1 eq.) was added and DIPEA (N,
N-diisopropylethylamine) (4 eq.) was added dropwise. The mixture was stirred at room temperature for 2
h. The solvent was evaporated under reduced pressure, which was purified by flash column
chromatography purification to yield the product.
7-amino-1-cyclopropyl-3-(2-((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2
-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9a)
1
Yellow solid; yield: 63.2%; mp: 147.8-149.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.08 (s, 1H), 8.56
(s, 1H), 8.31 (t, J = 5.3 Hz, 1H), 8.24 (brs, 1H), 7.61 (dd, J = 8.6, 7.0 Hz, 1H), 7.36 (brs, 1H), 7.18 (d, J =
8.6 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.77 (t, J = 5.9 Hz, 1H), 5.08-5.03 (m, 1H), 4.43 (s, 2H), 3.38-3.37(m,
2H), 3.27-3.24 (m, 2H), 2.94-2.85 (m, 1H), 2.81-2.77 (m, 1H), 2.62-2.54 (m, 2H), 2.24-2.20 (m, 1H),

2
1
1
.04-2.02 (m, 1H), 1.11-1.08 (m, 2H), 0.79 (brs, 2H); 13C NMR (100 MHz, DMSO-
d ), δ(ppm): 173.28,
6
70.54, 169.27, 169.16, 167.90, 167.78, 161.70, 160.57, 154.19, 151.80, 146.78, 138.90, 136.77, 132.70,
17.60, 111.07, 109.76, 105.99, 99.88, 49.02, 43.64, 41.81, 38.79, 31.46, 26.39, 22.65, 10.06; HRMS
+
+
(
28 9 8
ESI ): calcd for C28H N O [M+H] : 618.2061. Found: 618.2060.
7-amino-1-cyclopropyl-3-(2-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-
2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9b)
1
Yellow solid; yield: 71.0%; mp:159.1-161.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.56
(s, 1H), 8.23 (brs, 1H), 8.11 (brs, 1H), 7.59 (dd, J = 8.5, 7.0 Hz, 1H), 7.35 (brs, 1H), 7.12 (d, J = 8.5 Hz,
1
5
2
1
1
H), 7.01 (d, J = 7.0 Hz, 1H), 6.66 (t, J = 6.0 Hz, 1H), 5.06-5.02 (m, 1H), 4.44 (s, 2H), 3.15-3.14 (brd, J =
.8 Hz, 2H), 2.92-2.83 (m, 1H), 2.79 (brs, 1H), 2.62-2.54 (m, 2H), 2.04-2.01 (m, 1H), 1.68 (t,
J = 6.3 Hz,
6
J d ), δ(ppm): 173.26, 170.55,
= 6.7 Hz, 2H), 0.79 (s, 2H); 13C NMR (100 MHz, DMSO-
H), 1.11 (d,
69.30, 169.27, 167.77, 167.45, 161.69, 160.63, 154.21, 151.87, 146.74, 138.86, 136.73, 132.72, 117.64,
10.87, 109.66, 105.97, 99.95, 49.02, 43.81, 36.43, 31.45, 29.27, 26.38, 22.63, 10.08; HRMS (ESI+):
+
calcd for C29
H
30
N
O
9 8
[M+H] : 632.2217. Found: 632.2214.
7-amino-1-cyclopropyl-3-(2-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-2
-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9c)
1
Yellow solid; yield: 70.6%; mp: 155.5-157.1 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.55
(
s, 1H), 8.23 (brs, 1H), 8.03 (t,
J
= 5.4 Hz, 1H), 7.58 (dd,
J
= 8.6, 6.9 Hz, 1H), 7.35 (brs, 1H), 7.12 (d,
J
J
=
=
8
.7 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.54 (t, J = 5.7Hz, 1H), 5.07-5.02 (m, 1H), 4.41 (s, 2H), 3.10 (q,

6.1 Hz, 2H), 2.93-2.84 (m, 1H), 2.79-2.75 (m, 1H), 2.61-2.53 (m, 2H), 2.04-2.01 (m, 1H), 1.59-1.54 (m,
2H), 1.49-1.46 (m, 2H), 1.26-1.24 (m, 2H), 1.09 (q,
J
= 6.7 Hz, 2H), 0.77 (brs, 2H); 13C NMR (100 MHz,
6
DMSO-d ), δ(ppm): 173.27, 170.56, 169.41, 169.27, 167.77, 167.09, 161.68, 160.60, 154.20, 151.82,
1
2
6
7
2
46.88, 138.86, 136.75, 132.66, 117.72, 110.86, 109.49, 105.93, 99.94, 49.02, 43.72, 42.00, 38.68, 31.46,
+
+
6.95, 26.56, 26.36, 22.63, 10.07; HRMS (ESI ): calcd for C30
H
32
N
O
9 8
[M+H] : 646.2374. Found:
46.2376.
-amino-1-cyclopropyl-3-(2-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)amino)-
-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9d)
1
Yellow solid; yield: 78.2%; mp: 174.7-177.0 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.55
(s,1H), 8.23 (brs, 1H), 8.00 (t, J = 5.4 Hz, 1H), 7.57 (dd, J = 8.6, 7.0 Hz, 1H), 7.36 (brs, 1H), 7.10 (d, J =
8
2
1
0
1
9
.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.53 (t, J = 5.7 Hz, 1H), 5.07-5.02 (m, 1H), 4.41 (s, 2H), 3.29-3.26 (m,
H), 3.06 (q,
J = 6.3 Hz, 2H), 2.93-2.83 (m, 1H), 2.81-2.75 (m, 1H), 2.61-2.53 (m, 2H), 2.04-1.99 (m, 1H),
.62-1.55 (m, 2H), 1.48-1.41 (m, 2H), 1.39-1.34 (m, 2H), 1.26-1.23 (m, 2H), 1.10 (q,
J = 6.9 Hz, 2H),
.80-0.76 (m, 2H); 13C NMR (100 MHz, DMSO-
d ), δ(ppm): 173.27, 170.56, 169.41, 169.27, 167.78,
6
67.02, 161.68, 160.60, 154.19, 151.82, 146.88, 138.86, 136.74, 132.66, 117.65, 110.85, 109.52, 105.93,
+
9.94, 49.02, 43.68, 42.30, 38.91, 31.45, 29.23, 28.80, 26.36, 24.07, 22.63, 10.07; HRMS (ESI ): calcd
+
for C31
H
34
N
O
9 8
[M+H] : 660.2530. Found: 660.2528.
7-amino-1-cyclopropyl-3-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)-
2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9e)

1
6
Yellow solid; yield: 74.1%; mp: 173.1-175.8 °C; H NMR (400MHz, DMSO-d ), δ(ppm): 11.08 (s, 1H), 8.55
(s, 1H), 8.23 (brs, 1H), 7.98 (t, J = 5.5 Hz, 1H), 7.57 (dd, J = 8.7, 7.0 Hz, 1H), 7.35 (brs, 1H), 7.10 (d, J =
8
1
1
0
1
9
.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.53 (t, J = 5.8 Hz, 1H), 5.07-5.02 (m, 1H), 4.41 (s, 2H), 3.29-3.27 (m,
H), 3.04 (q,
J = 6.3 Hz, 2H), 2.93-2,83 (m, 1H), 2.80-2.75 (m, 1H), 2.61-2.53 (m, 2H), 2.05-1,99 (m, 1H),
.59-1.54 (m, 2H), 1.42-1.37 (m, 3H), 1.32 (brs, 3H), 1.27-1.23 (m, 2H), 1.10 (q,
J = 6.9 Hz, 2H),
.79-0.75 (m, 2H); 13C NMR (100 MHz, DMSO-
d ), δ(ppm): 173.27, 170.56, 169.42, 169.27, 167.78,
6
66.98, 161.68, 160.60, 154.19, 151.82, 146.90, 138.85, 136.74, 132.66, 117.64, 110.83, 109.50, 105.93,
+
9.95, 49.02, 43.68, 42.24, 38.97, 31.46, 29.47, 29.10, 26.47, 26.35, 22.63, 10.07; HRMS (ESI ): calcd
+
for C32
H
35
N
9
O
8
Na [M+Na] : 696.2506. Found: 696.2515.
7-amino-1-cyclopropyl-3-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethy
l)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9f)
1
Yellow solid; yield: 65.8%; mp: 167.1-169.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.54
(
s, 1H), 8.22 (brs, 1H), 8.11 (t,
J
= 5.4 Hz, 1H), 7.59 (dd,
J
= 8.6, 7.0 Hz, 1H), 7.36 (brs, 1H), 7.16 (d,
J
J
=
=
8
.6 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.62 (t, J = 5.4 Hz, 1H), 5.08-5.04 (m, 1H), 4.45 (s, 2H), 3.63 (t,
5.2 Hz, 2H), 3.48-3.45 (m, 4H), 3.23 (q,
J = 5.6 Hz, 2H), 2.92-2.83 (m, 1H), 2.80-2.75 (m, 1H), 2.60-2.56
6
m, 2H), 2.04-2.01 (m, 1H), 1.09 (q, J d ),
= 6.8 Hz, 2H), 0.80-0.76 (m, 2H); 13C NMR (100 MHz, DMSO-
(
δ
1
2
(ppm): 173.27, 170.54, 169.44, 169.27, 167.77, 167.40, 161.69, 160.57, 154.17, 151.81, 146.87, 138.86,
36.73, 132.57, 117.91, 111.18, 109.80, 105.96, 99.88, 69.30, 69.11, 49.05, 43.61, 42.11, 39.12, 31.47,
+
+
31 9 9
6.36, 22.60, 10.07; HRMS (ESI ): calcd for C30H N O Na [M+Na] : 684.2142. Found: 684.2141.

7-amino-3-(2-((2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethyl)amino)-2-oxoethyl)-1-e
thyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9g)
1
Yellow solid; yield: 67.3%; mp: 169.2-171.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.08 (s, 1H), 8.59
(
s, 1H,), 8.34 (t,
J
= 5.4 Hz, 1H), 8.24 (brs, 1H), 7.60 (dd,
J
= 8.6, 7.0 Hz, 1H), 7.38 (brs, 1H), 7.18 (d,
J
J
=
=
8
6
1
1
1
.6 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.77 (t, J = 6.1 Hz, 1H), 5.08-5.03 (m, 1H), 4.47 (s, 2H), 4.18 (q,
.8 Hz, 2H), 3.39-3.36 (m, 2H), 3.28-3.25 (m, 2H), 2.94-2.85 (m, 1H), 2.62-2.54 (m, 2H), 2.04-2.01 (m,
6
J d ), δ(ppm): 173.27, 170.54, 169.20, 169.16,
= 6.8 Hz, 3H) 13C NMR (100 MHz, DMSO-
H), 1.20 (t,
67.82, 167.78, 162.08, 160.26, 152.38, 151.03, 146.79, 139.19, 136.75, 132.70, 117.60, 111.06, 109.76,
+
06.07, 99.43, 49.02, 43.60, 41.78, 38.81, 37.31, 31.46, 22.65, 13.50; HRMS (ESI ): calcd for
+
C
27
H
28
N
O
9 8
[M+H] : 606.2061. Found: 606.2054.
7-amino-3-(2-((3-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)propyl)amino)-2-oxoethyl)-1-
ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9h)
1
Yellow solid; yield: 72.7%; mp: 187.5-189.9 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.59
(
s, 1H,), 8.24 (brs, 1H), 8.15 (t,
J
= 5.6 Hz, 1H), 7.59 (dd,
J
= 8.3, 7.3 Hz, 1H), 7.36 (brs, 1H), 7.12 (d,
J
J
=
=
8
.6 Hz, 1H), 7.03 (d,
.8 Hz, 2H), 3.15 (q,
J
J
= 7.0 Hz, 1H), 6.66 (t, J = 6.0 Hz, 1H), 5.07-5.02 (m, 1H), 4.48 (s, 2H), 4.19 (q,
6
= 6.1 Hz, 2H), 2.92-2.83 (m, 1H), 2.61-2.53 (m, 2H), 2.04-2.01 (m, 1H), 1.72-1.65
6
m, 2H), 1.19 (t, J d ), δ(ppm): 173.26, 170.55, 169.30, 169.20,
= 6.8 Hz, 3H); 13C NMR (100 MHz, DMSO-
(
167.77, 167.38, 162.07, 160.32, 152.40, 151.10, 146.74, 139.16, 136.71, 132.73, 117.63, 110.87, 109.66,
+
1
06.05, 99.51, 49.02, 43.75, 37.30, 36.46, 31.45, 29.26, 22.63, 13.51; HRMS (ESI ): calcd for

+
28 29 9 8
C H N O Na [M+Na] : 642.2037. Found: 642.2210.
7-amino-3-(2-((4-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)butyl)amino)-2-oxoethyl)-1-e
thyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9i)
1
Yellow solid; yield: 73.5%; mp:174.3-175.9 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.08 (s, 1H), 8.58
(s, 1H), 8.24 (brs,1H), 8.07 (brs, 1H), 7.57 (dd, J = 8.5, 6.9 Hz, 1H), 7.36 (brs, 1H), 7.12 (d, J = 8.5 Hz, 1H),
7
3
1
1
9
.02 (d, J = 6.9 Hz, 1H), 6.54 (brs, 1H), 5.07-5.03 (m, 1H), 4.46 (s, 2H), 4.17 (brd, J =6.7 Hz, 2H),
.12-3.10 (m, 2H), 2.92-2.85 (m, 1H), 2.62-2.55 (m, 2H), 2.05-2.02 (m, 1H), 1.58 (brs, 2H), 1.49 (brs, 2H),
6
J d ), δ(ppm): 173.27, 170.56, 169.41, 169.20, 167.77,
= 6.7 Hz, 3H); 13C NMR (100 MHz, DMSO-
.18 (t,
67.04, 162.06, 160.29, 152.38, 151.05, 146.88, 139.17, 136.73, 132.66, 117.71, 110.85, 109.49, 106.03,
+
9.50, 49.03, 43.66, 42.01, 38.70, 37.27, 31.46, 26.93, 26.56, 22.63, 13.48; HRMS (ESI ): calcd for
+
C
29
H
32
N
O
9 8
[M+H] : 634.2374. Found: 634.2379.
7-amino-3-(2-((5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)pentyl)amino)-2-oxoethyl)-1-
ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9j)
1
Yellow solid; yield: 72.1%; mp: 194.0-196.2 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.58
(
s, 1H), 8.24 (brs, 1H), 8.04 (t,
J
= 5.3 Hz, 1H), 7.57 (dd,
J
= 8.6, 7.0 Hz, 1H), 7.37 (brs, 1H), 7.10 (d,
J
J
=
=
8
.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.53 (t, J = 5.7 Hz, 1H), 5.07-5.02 (m, 1H), 4.45 (s, 2H), 4.17 (q,
6.8 Hz, 2H), 3.28-3.26 (m, 2H), 3.06 (q,
J = 6.3 Hz, 2H), 2.93-2.83 (m, 1H), 2.61-2.54 (m, 2H), 2.04-2.01
(m, 1H), 1.62-1.55 (m, 2H), 1.47-1.41 (m, 2H), 1.38-1.34 (m, 2H), 1.19 (t, J
= 6.8 Hz, 3H); 13C NMR (100
6
MHz, DMSO-d ), δ(ppm): 173.26, 170.55, 169.41, 169.20, 167.78, 166.95, 162.06, 160.28, 152.38,

1
3
6
7
51.05, 146.89, 139.16, 136.74, 132.67, 117.66, 110.85, 109.53, 106.03, 99.49, 54.07, 49.03, 42.31,
+
34 9 8
H N O
[M+H]+:
8.93, 31.45, 29.21, 28.80, 24.08, 18.56, 17.21, 13.50; HRMS (ESI ): calcd for C30
48.2530. Found: 648.2527.
-amino-3-(2-((6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)hexyl)amino)-2-oxoethyl)-1-e
thyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9k)
1
Yellow solid; yield: 68.3%; mp:194.4-197.0 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.08 (s, 1H), 8.58
(
s, 1H), 8.24 (brs, 1H), 8.02 (t,
J
= 5.5 Hz, 1H), 7.57 (dd,
J
= 8.6, 7.0 Hz, 1H), 7.36 (brs, 1H) 7.10 (d,
J
J
=
=
8
.6 Hz, 1H), 7.02 (d, J = 7.0 Hz, 1H), 6.53 (t, J = 5.9 Hz, 1H), 5.07-5.02 (m, 1H), 4.44 (s, 2H), 4.17 (q,
6.8 Hz, 2H), 3.29-3.27 (m, 1H), 3.05 (q,
J = 6.3 Hz, 2H), 2.92-2.83 (m, 1H), 2.61-2.53 (m, 2H), 2.06-2.01
(m, 1H), 1.59-1.54 (m, 2H), 1.44-1.39 (m, 2H), 1.35-1.30 (m, 3H), 1.27-1.23 (m, 4H), 1.18 (t, J = 6.8 Hz,
3
1
4
6
7
H); 13C NMR (100 MHz, DMSO-
d ), δ(ppm): 173.27, 170.56, 169.42, 169.20, 167.77, 166.91, 162.05,
6
60.28, 152.37, 151.04, 146.90, 139.15, 136.73, 132.67, 117.63, 110.83, 109.50, 106.02, 99.49, 49.02,
+
36 9 8
H N O
[M+H]+:
3.61, 42.24, 38.99, 31.46, 29.46, 29.10, 26.46, 22.63, 13.49; HRMS (ESI ): calcd for C31
62.2687. Found: 662.2683.
-amino-3-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethyl)amino)-2-ox
oethyl)-1-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (9l)
1
Yellow solid; yield: 62.0%; mp: 165.1-167.4 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.07 (s, 1H), 8.57
(
s, 1H), 8.23 (brs, 1H), 8.14 (t,
J
= 5.4 Hz, 1H), 7.59 (dd,
J
= 8.6, 7.0 Hz, 1H), 7.37 (brs, 1H), 7.16 (d,
J
J
=
=
8
.6 Hz, 1H), 7.05 (d, J = 7.0 Hz, 1H), 6.63 (t, J = 5.5 Hz, 1H), 5.08-5.04 (m, 1H), 4.49 (s, 2H), 4.17 (q,

6
1
δ
1
2
7
.9 Hz, 2H), 3.64-3.62 (m, 3H), 3.48-3.45 (m, 4H), 3.26-3.22 (m, 2H), 3.18-3.11 (m, 1H), 2.91-2.82 (m,
6
J d ),
= 6.9 Hz, 3H); 13C NMR (100 MHz, DMSO-
H), 2.60-2.53 (m, 2H), 2.04-1.99 (m, 1H), 1.19 (t,
(ppm): 173.26, 170.54, 169.44, 169.20, 167.77, 167.33, 162.06, 160.26, 152.36, 151.04, 146.88, 139.16,
36.74, 132.57, 117.92, 111.18, 109.80, 106.06, 99.43, 69.30, 69.11, 49.05, 43.55, 42.32, 39.15, 31.47,
+
+
32 9 9
2.60, 13.50, 12.95; HRMS (ESI ): calcd for C29H N O [M+H] : 650.2323. Found: 650.2321.
-amino-1-cyclopropyl-3-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)eth
yl)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11a)
1
Brown solid; yield: 53.7%; mp: 204.2-206.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.09 (s, 1H), 8.54
(s, 1H), 8.22 (brs, 1H), 8.11 (brs, 1H), 8.04 (brs, 1H), 7.80 (dd, J = 8.6, 7.2 Hz, 1H), 7.50 (d, J = 7.2 Hz,
1H), 7.39 (brd, J = 8.5 Hz, 2H), 5.15-5.11 (m, 1H), 4.78 (s, 2H), 4.43 (s, 2H), 3.21-3.15 (m, 4H), 2.94-2,84
(
m, 1H), 2.81-2.75 (m, 1H), 2.62-2.54 (m, 2H), 2.06-2.03 (m, 1H), 1.12-1.08 (m, 2H), 0.78 (brs, 2H); 13C
6
NMR (100 MHz, DMSO-d ), δ(ppm): 173.26, 170.34, 169.25, 167.62, 167.57, 167.21, 165.93, 161.69,
1
4
6
7
60.58, 155.54, 154.17, 151.83, 138.85, 137.41, 135.50, 121.00, 117.36, 116.54, 105.98, 99.87, 68.14,
+
29 9
H N O
10Na [M+Na]+:
9.30, 43.70, 38.65, 38.57 31.44, 26.37, 22.46, 10.07; HRMS (ESI ): calcd for C30
98.1935. Found: 698.1936.
-amino-1-cyclopropyl-3-(2-((3-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)pro
pyl)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11b)
1
Brown solid; yield: 57.9%; mp: 185.6-188.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.10 (s, 1H), 8.55
(s, 1H), 8.23 (brs, 1H), 8.02 (brs, 1H), 7.97 (brs, 1H), 7.80 (dd, J = 8.4, 7.1 Hz, 1H), 7.49 (d, J = 7.1 Hz,

1H), 7.38 (brd, J = 8.4 Hz, 2H), 5.14-5.10 (m, 1H), 4.78 (s, 2H), 4.41 (s, 2H), 3.15 (brd, J = 5.6 Hz, 2H),
3.07 (brd,
J = 5.3 Hz, 2H), 2.93-2.86 (m, 1H), 2.78 (brs, 2H), 2.62-2.58 (m, 1H), 2.06-2.03 (m, 1H), 1.57
6
brs, 2H), 1.10-1.09 (J d ), δ(ppm): 173.25,
= 5.7 Hz, 2H), 0.79-0.75 (m, 2H); 13C NMR (100 MHz, DMSO-
(
1
1
1
7
70.34, 169.26, 167.31, 167.23, 165.92, 161.69, 160.59, 155.55, 154.19, 151.83, 138.85, 137.40, 133.52,
20.89, 117.30, 116.50, 105.96, 99.91, 68.13, 49.29, 43.72, 36.82, 36.71, 31.42, 29.64, 26.37, 22.46,
+
+
35 9
0.08; HRMS (ESI ): calcd for C31H N O10Na [M+Na] : 712.2092. Found: 712.2098.
-amino-1-cyclopropyl-3-(2-((4-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)but
yl)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11c)
1
Brown solid; yield: 69.1%; mp: 187.6-190.3 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.10 (s, 1H), 8.55
(s, 1H), 8.23 (brs, 1H), 8.00 (t, J = 5.5 Hz, 1H), 7.96 (t, J = 5.4 Hz, 1H), 7.82 (dd, J = 8.5, 7.3 Hz, 1H), 7.50
(d,
J = 7.3 Hz, 1H), 7.38 (d, J = 8.5 Hz, 2H), 5.14-5.10 (m, 1H), 4.77 (s, 2H), 4.41 (s, 2H), 3.17-3.13 (m,
3H), 3.07-3.04 (m, 2H), 2.94-2,85 (m, 3H), 2.81-2.75 (m, 1H), 2.61-2.55 (m, 2H), 2.06-2.03 (m, 1H), 1.42
(
brs, 3H), 1.11-1.09 (m, 2H), 0.78 (s, 2H); 13C NMR (100 MHz, DMSO-
d ), δ(ppm): 173.24, 170.34,
6
1
1
1
7
69.27, 167.21, 167.18, 167.04, 165.96, 161.68, 160.59, 155.55, 154.19, 151.83, 138.86, 137.42, 133.51,
20.89, 117.29, 116.52, 105.94, 99.93, 68.13, 49.29, 43.68, 38.73, 38.51, 31.42, 26.89, 26.37, 22.46,
+
+
33 9
0.07; HRMS (ESI ): calcd for C32H N O10Na [M+Na] : 726.2248. Found: 726.2245.
-amino-1-cyclopropyl-3-(2-((5-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)pe
ntyl)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11d)
1
Brown solid; yield: 74.4%; mp: 181.6-183.9 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.09 (s, 1H), 8.55

(s, 1H), 8.23 (brs, 1H), 7.98 (t, J = 5.0 Hz, 1H), 7.93 (t, J = 5.1 Hz, 1H), 7.81 (dd, J = 8.5, 7.2 Hz, 1H), 7.50
(d,
J = 7.2 Hz, 1H), 7.40 (brd, J = 8.5 Hz, 2H), 5.14-5.10 (m, 1H), 4.77 (s, 2H), 4.41 (s, 2H), 3.17-3.14 (m,
3H), 3.05-3.03 (m, 2H), 2.94-2.85 (m, 1H), 2.78 (brs, 1H), 2.62-2.53 (m, 2H), 2.06-2.03 (m, 1H), 1.47-1.38
6
m, 4H), 1.26 (brs, 3H), 1.10 (brd, J d ), δ(ppm):
= 6.6 Hz, 2H), 0.78 (s, 2H); 13C NMR (100 MHz, DMSO-
(
1
1
2
7
7
73.23, 170.34, 169.27, 167.20, 167.13, 167.02, 165.98, 161.67, 160.59, 155.53, 154.18, 151.83, 138.85,
37.39, 133.49, 120.87, 117.29, 116.50, 105.93, 99.94, 68.13, 49.29, 43.68, 38.96, 38.73, 31.41, 29.19,
+
+
9.12, 26.36, 24.05, 22.47, 10.07; HRMS (ESI ): calcd for C33
H
35
N
9
O
10Na [M+Na] : 740.2405. Found:
40.2399.
-amino-1-cyclopropyl-3-(2-((6-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)he
xyl)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11e)
1
Brown solid; yield: 68.4%; mp: 183.8-186.0 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.10 (s, 1H), 8.55
(s, 1H), 8.23 (brs, 1H), 7.97 (t, J = 5.6 Hz, 1H), 7.92 (t, J = 5.7 Hz, 1H), 7.81 (dd, J = 8.5, 7.3 Hz, 1H), 7.50
(d,
J = 7.3 Hz, 1H), 7.40 (brd, J = 8.5 Hz, 2H), 5.14-5.09 (m, 1H), 4.77 (s, 2H), 4.41 (s, 2H), 3.17-3.12 (m,
3
1
δ
1
3
7
H), 3.05-3.01 (m, 2H), 2.94-2.85 (m, 2H), 2.81-2.75 (brs, 1H), 2.62-2.54 (m, 2H), 2.06-2.03 (m, 1H),
.43-1.36 (m, 4H), 1.26-1.25 (m, 4H), 1.11-1.07 (m, 2H), 0.78 (s, 2H); 13C NMR (100 MHz, DMSO-
d ),
6
(ppm): 173.24, 170.33, 169.27, 167.20, 167.11, 166.99, 165.98, 161.68, 160.60, 155.52, 154.19, 151.82,
38.85, 137.39, 133.50, 129.17, 120.85, 117.30, 116.52, 105.93, 99.95, 68.13, 49.30, 43.69, 38.96, 38.76,
+
33 9
H N O
10Na [M+Na]+:
1.42, 29.48, 29.42, 26.43, 26.36, 22.47, 10.07; HRMS (ESI ): calcd for C34
54.2561. Found: 754.2562.

7-amino-1-cyclopropyl-3-(2-((2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)
ethoxy)ethyl)amino)-2-oxoethyl)-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11f)
1
Brown solid; yield: 55.6%; mp: 183.3-185.4 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.09 (s, 1H), 8.54
(s, 1H), 8.22 (brs, 1H), 8.10 (t, J = 5.4 Hz, 1H), 8.01 (t, J = 5.4 Hz, 1H), 7.81 (dd, J = 8.6, 7.2 Hz, 1H), 7.49
(d, J = 7.2 Hz, 1H), 7.40 (brd, J = 8.6 Hz, 2H), 5.14-5.09 (m, 1H), 4.80 (s, 2H), 4.44 (s, 2H), 3.47 (t, J = 5.5
Hz, 2H), 3.42 (t,
J = 5.8 Hz, 2H), 3.23 (q, J = 5.6 Hz, 2H), 2.93-2.84 (m, 1H), 2.81-2.75 (m, 1H), 2.61-2.54
(
m, 2H), 2.05-2.03 (m, 1H), 1.12-1.07 (m, 2H), 0.78 (s, 2H); 13C NMR (100 MHz, DMSO-
d ), δ(ppm):
6
1
1
3
7
73.22, 170.34, 169.26, 167.44, 167.39, 167.20, 165.92, 161.68, 160.57, 155.49, 154.16, 151.82, 138.85,
37.39, 133.51, 120.82, 117.23, 116.49, 105.98, 99.86, 69.24, 69.12, 67.97, 49.29, 43.59, 39.09, 38.82,
+
+
33 9
1.40, 26.36, 22.46, 10.07; HRMS (ESI ): calcd for C32H N O11Na [M+Na] : 742.2197. Found: 742.2191.
-amino-3-(2-((2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)oxy)acetamido)ethyl)amino)-2-ox
oethyl)-1-ethyl-2,4-dioxo-1,2,3,4-tetrahydropyrido[2,3-d]pyrimidine-6-carboxamide (11g)
1
Off-white solid; yield: 71.2%; mp: 175.3-178.0 °C; H NMR (400MHz, DMSO-
d
6
),
δ
(ppm): 11.10 (s, 1H),
8
7
2
1
1
1
.57 (s, 1H), 8.23 (brs, 1H), 8.16 (t, J = 5.0 Hz, 1H), 8.04 (t, J = 5.2 Hz, 1H), 7.81 (dd, J = 8.6, 7.2 Hz, 1H),
.50 (d,
J = 7.2 Hz, 1H), 7.41 (d, J = 8.6 Hz, 2H), 7.38 (brs, 1H), 5.16-5.11 (m, 1H), 4.78 (s, 2H), 4.48 (s,
H), 4.16 (q,
J = 6.9 Hz, 2H), 3.22-3.17 (m, 4H), 2.94-2.85 (m, 1H), 2.63-2.53 (m, 2H), 2.06-2.03 (m, 1H),
6
.19 (J d ), δ(ppm): 173.26, 170.33, 169.19, 167.61, 167.51,
= 6.9 Hz, 3H); 13C NMR (100 MHz, DMSO-
67.22, 165.94, 162.06, 160.27, 155.54, 152.36, 151.05, 139.15, 137.41, 133.50, 121.01, 117.37, 116.54,
+
06.07, 99.42, 68.15, 49.30, 43.63, 38.67, 38.57, 31.43, 22.45, 13.49; HRMS (ESI ): calcd for