Development of a Brigatinib degrader (SIAIS117) as a potential treatment for ALK positive cancer resistance

Article abstract of DOI:10.1016/j.ejmech.2020.112190

EML4-ALK and NPM-ALK fusion proteins possess constitutively activated ALK (anaplastic lymphoma kinase) activity, which in turn leads to the development of non-small cell lung cancer and anaplastic large-cell lymphomas (ALCLs). FDA-approved ALK inhibitor drugs cause significant cancer regression. However, drug resistance eventually occurs and it becomes a big obstacle in clinic. Novel proteolysis targeting chimera (PROTAC) technology platform provides a potential therapeutic strategy for drug resistance. Herein, we designed and synthesized a series of ALK PROTACs based on Brigatinib and VHL-1 conjunction, and screened SIAIS117 as the best degrader which not only blocked the growth of SR and H2228 cancer cell lines, but also degraded ALK protein. In addition, SIAIS117 also showed much better growth inhibition effect than Brigatinib on 293T cell line that exogenously expressed G1202R-resistant ALK proteins. Furthermore, it also degraded G1202R mutant ALK protein in vitro. At last, it has the potentially anti-proliferation ability of small cell lung cancer. Thus, we have successfully generated the degrader SIAIS117 that can potentially overcome resistance in cancer targeted therapy.

Full text of DOI:10.1016/j.ejmech.2020.112190

Journal Pre-proof
Development of a Brigatinib degrader (SIAIS117) as a potential treatment for ALK
positive cancer resistance
Ning Sun, Chaowei Ren, Ying Kong, Hui Zhong, Jinju Chen, Yan Li, Jianshui Zhang,
Yuedong Zhou, Xing Qiu, Haifan Lin, Xiaoling Song, Xiaobao Yang, Biao Jiang
PII:
S0223-5234(20)30157-4
DOI:
https://doi.org/10.1016/j.ejmech.2020.112190
EJMECH 112190
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 17 December 2019
Revised Date: 24 February 2020
Accepted Date: 24 February 2020
Please cite this article as: N. Sun, C. Ren, Y. Kong, H. Zhong, J. Chen, Y. Li, J. Zhang, Y. Zhou, X.
Qiu, H. Lin, X. Song, X. Yang, B. Jiang, Development of a Brigatinib degrader (SIAIS117) as a potential
treatment for ALK positive cancer resistance, European Journal of Medicinal Chemistry (2020), doi:
https://doi.org/10.1016/j.ejmech.2020.112190.
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Development of a Brigatinib Degrader (SIAIS117) as a Potential Treatment for ALK Positive
Cancer Resistance
§
†
§
†
#
§
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∇
Ning Sun , Chaowei Ren , Ying Kong , Hui Zhong , Jinju Chen , Yan Li , Jianshui Zhang ,
#
‡
§£
§
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‡
Yuedong Zhou , Xing Qiu , Haifan Lin *, Xiaoling Song *, Xiaobao Yang * and Biao Jiang *
§
Shanghai Institute for Advanced Immunochemical Studies, ShanghaiTech University, 393 Middle
Huaxia Road, Shanghai 201210, China
‡
CAS Key Laboratory of Synthetic Chemistry of Natural Substances, Shanghai Institute of Organic
Chemistry, Chinese Academy of Sciences, 345 Lingling Road, Shanghai 200032, China
∇Changzhou University, Changzhou, Jiangsu, 213164, China.
£
Yale Stem Cell Center, Yale University, New Haven, CT 06511, USA.
#
Jing Medicine Technology (Shanghai), Ltd., Y building, 230 Haike Road, Shanghai 201210, China
+
These authors contributed equally to this work.
Abstract
EML4-ALK and NPM-ALK fusion proteins possess constitutively activated ALK (anaplastic
lymphoma kinase) activity, which in turn leads to the development of non-small cell lung cancer and
anaplastic large-cell lymphomas (ALCLs). FDA-approved ALK inhibitor drugs cause significant
cancer regression. However, drug resistance eventually occurs and it becomes a big obstacle in
clinic. Novel proteolysis targeting chimera (PROTAC) technology platform provides a potential
therapeutic strategy for drug resistance. Herein, we designed and synthesized a series of ALK
PROTACs based on Brigatinib and VHL-1 conjunction, and screened SIAIS117 as the best degrader
which not only blocked the growth of SR and H2228 cancer cell lines, but also degraded ALK
protein. In addition, SIAIS117 also showed much better growth inhibition effect than Brigatinib on
2
93T cell line that exogenously expressed G1202R-resistant ALK proteins. Furthermore, it also
degraded G1202R mutant ALK protein in vitro. At last, it has the potentially anti-proliferation
ability of small cell lung cancer. Thus, we have successfully generated the degrader SIAIS117 that
can potentially overcome resistance in cancer targeted therapy.
Key words: ALK, Brigatinib, degrader, VHL, PROTAC, NSCLC, ALCL, resistance
1
. Introduction

ALK is a tyrosine kinase that was firstly found in anaplastic large cell lymphoma[1]. It forms
fusion proteins with more than 20 different genes in cancer. ALK fusion proteins can constitutively
activate the ALK signaling pathway, which renders ALK a strong cancer driver gene[2, 3]. In lung
cancer, the EML4-ALK (Echinoderm microtubule-associated protein-like 4) gene fusion is the most
common form of ALK fusion. Cell lines with such mutations, including H2228, are highly
dependent on ALK kinase activity; blocking ALK kinase activity greatly inhibits cancer cell
growth[4-6]. ALK fusions are also present in approximately 60% anaplastic large-cell lymphomas
(ALCLs) patients, and the major form of fusion is NPM-ALK. Different forms of the ALK fusion
have been reported as cancer oncogenes that transform normal cells to cancer cells. The first
generation ALK inhibitor drug Crizotinib was approved for the treatment of ALK-positive NSCLC
patients as a first-in-class drug; however, resistance to Crizotinib occurs after treatment. To
overcome the resistance, the second generation (Ceritinib, Alectinib, and Brigatinib) and third
generation (Lorlatinib) drugs have been approved to treat NSCLC patients carrying an ALK fusion
gene[7-11] (Figure 1). Despite the initial response to the second line use of such inhibitors, the
majority of these patients eventually developed new resistances to these drugs[3, 12-14]. Therefore,
our group construct protein degradation technology platform and devote to discovery of novel small
molecule degraders for overcoming the treatment obstacle.

a
Figure 1 FDA-approved ALK drugs and the co-crystal structure of ALK with Brigatinib
a
The ligand interaction diagram is generated by Schrodinger.
PROTAC is a brand-new technology that has attracted considerable attention for its great
potential as a promising cancer therapeutic strategy[15]. PROTAC molecules are bi-functional small
molecules (Figure 2) that simultaneously bind to a target protein and an E3-ubiquitin ligase to
establish a ternary complex. Because PROTAC molecules trigger ubiquitination and degradation of
the target protein by the proteasome, they can degrade oncoproteins and theoretically can overcome
drug resistance. This technology has been successfully applied to many target proteins, such as AR,
ER, BET, FKBP-12, Bcr-Abl, PARP, BTK, EGFR, FLT-3,[16] TBK1, CDK4/6 and HER-2[17-32]
used as the different E3 ligand including VHL-1, MDM2, CRBN and cIAP ligands (Figure 2).
Figure 2. Reported E3 ligands and sketch of PROTAC
Recently, degraders of ALK also have been developed. Gray group reported an ALK degrader
(A) (Figure 3) by linking ALK inhibitor Ceritinib to the CRBN E3 ligase ligand pomalidomide[24].
Jin and his colleagues also reported a potent Ceritinib-based ALK degrader[4] (B, C) (Figure 3).
Hwang group reported ALK degrader (D) (Figure 3) by using Ceritinib to VHL-based
PROTAC[19]. However, they all used Ceritinib as the ALK-binding moiety. To date,
Brigatinib-based PROTAC targeting ALK has not been reported. There are many advantages of
nd
using Brigatinib over than ALK 2 generation drugs. Brigatinib has more efficacy against most
point mutant ALK than other second generation ALK drugs [33]. Brigatinib also induces
intracranial response in patients, and was approved by FDA for treating patients with metastatic

non-small cell lung cancer[34]. Most importantly, patient received Brigatinib as their treatment
showed higher overall response rate and had longer progression free survival comparing to other
nd
two 2 ALK drugs[35, 36]. Based on these Brigatinib advantages, we selected Brigatinib as the
PROTAC warhead. From co-crystal structure of ALK bound by Brigatinib (PDB ID: 6MX8), the
solvent exposure piperidinyl part of Brigatinib is suitable for PROTAC design[37] (Figure 1,
bottom), so we design a series of Brigatinib-PROTAC based on VHL E3 ligase.
Figure 3. Reported potential ALK degraders
We successfully generated E3 VHL ligand and Brigatinib-based ALK degraders through two
types of linkers. After exploration of the structure-activity relationships, we discovered that
SIAIS117 degrades ALK protein level in a time- and dose-dependent manner. Firstly, this degrader
not only inhibited the growth of ALK fusion positive ALCL cancer cell lines but also the growth of
lung cancer cell line H2228 cells. Secondly, it effectively inhibited the growth of 293T cells that
exogenously express resistant ALK fusion proteins and degraded G1202R mutant ALK protein in a
dose-dependent manner. Thirdly, it inhibited the growth of small cell lung cancer cell lines. In
summary, we successfully developed an ALK degrader and provides a promising tool to overcome
ALK TKI resistance.
2
. Result and discussion
2
.1 Chemical Synthesis. Compounds 1, 2, 3, s-4, s-5, 4-38 were synthesized using the synthetic
routes shown in Schemes 1−3. The preparation of compound 1-3 was started from
-fluoro-2-methoxy-1-nitrobenzene [37]. Nucleophilic aromatic substitution reaction of the amino
group under basic conditions provided intermediate s-1. Next, the NO group on the phenyl ring
4
2
were converted to NH group under reduction reaction conditions, resulting in intermediate s-2,
2
which was subsequently converted to s-3 by microwave promoted Buchwald coupling. Boc

deprotection under acid conditions provided compound 1-3 (Scheme 1). Then, a linker library of
VHL E3 ligase ligand VHL-1 were built (Scheme 2). The amino group of VHL-1 was reacted with
various aliphatic acid to afford the linkers s-4 and s-5 (Scheme 2). Lastly, compound 1-3 condensed
with various linkers to generate the corresponding ALK PROTACs 4-37 (Scheme 3). Furthermore,
the negative control compound 38 (SIAIS117NC) could be synthesized from a similar route
(Scheme 3).
Scheme 1: Syntheses of Brigatinib analogue A、B、C
Scheme 2: Syntheses of VHL ligand based linkers

Scheme 3: Syntheses of ALK PROTACs
2
.2 VHL-based ALK degraders inhibit the growth of ALK-fusion-positive cancer cells
Brigatinib, in vitro, can inhibit the viability of cell lines carrying EML4-ALK fusion protein and
1
7 different ALK mutants conferring crizotinib resistance[37]. It has been approved by FDA for
nd
treating ALK-positive metastasized non-small cell lung cancer. Comparing to other two 2 ALK
drugs, Brigatinib showed better efficacies in improving overall response rate and progression free
survival of crizotinib-refractory NSCLC patients[34-36, 38]. Therefore, we chose Brigatinib as the
ALK binding moiety linking VHL ligands to generate PROTAC molecules.. ALCL SR cell line
carrying NPM-ALK fusion gene is very sensitive to ALK inhibitors in our pre-test and was used in
our screening (Table 1). Indeed, all three forms of Brigatinib can effectively inhibit the growth of
SR cells, but there is no significant difference between Brigatinib analogs and the original drug
(Table 1). In addition, Brigatinib and all three Brigatinib analogs did not degrade ALK proteins
(
Figure S2). These results indicated SR cell line is a good system to test our degraders.
a
compound
structure
SR IC (nM)
50

Brigatinib
2.7±0.4
1
(Brigatinib
1.6±0.6
1.7±0.8
6.8±1.8
A)
2
(Brigatinib
(Brigatinib
B)
3
P
O
O
H
H
N
N
N
C)
N
Cl
N
N
NH
a
Table 1 Chemical Structures of three analogs of Brigatinib and their IC50 in SR cells. IC values were obtained
50
from three independent experiments.
The conjugation of three Brigatinib precursors with an alkyl VHL-1 library can efficiently
generated PROTAC molecules (Figure 4A; see supporting information 1, 2 and 3). The changes of
carbon-chain linkers interfered the growth inhibition effect on SR cell line. With increasing the
length of the linker from C2 to C5, the growth inhibition ability became obviously weaker (as
indicated as increase IC50 value), and then gradually increased from C6 to C8. Finally, Brigatinib B
series showed the best growth inhibition effect with the same length linker compared with Brigatinib
A and C series (Figure 4B). When the alkyl linkers length was increased to C9 to produce
compound 18 (SIAIS117), the inhibitory activity of PROTAC compounds against SR growth was
the strongest (IC50 = 1.7±1.0 nM). However, while using ethylenedioxy (-O-CH2-CH2-O-,
abbreviated as PEG) linkers to generate three series Brigatinib degraders (Figure 4A), those
degraders growth inhibition have sharply reduced in SR cell line (Figure 4C).

Figure 4: Optimization of linker type and length on the antiproliferative activity in SR. (A). Design and synthesis
of Brigatinib A, B and C series degraders. (B). Study on the structure-activity relationship between carbon chain
linker length and SR cell anti-proliferation ability, Cn stands for numbers for CH
2
. (C). Study on the
structure-activity relationship between PEG (-O-CH2-CH2-O-) linkers length and SR cell anti-proliferation ability.
2
.3 ALK degraders effectively reduce ALK protein levels
a
Name
Series
carbon linker length
DC (nM)
50
compound 8
compound 9
compound 10
compound 15
compound 16
compound 17
compound 23
compound 24
compound 25
Brigatinib A
Brigatinib A
Brigatinib A
Brigatinib B
Brigatinib B
Brigatinib B
Brigatinib C
Brigatinib C
Brigatinib C
6
7
8
6
7
8
6
7
8
>50
15
>50
9.1
20.9
7.2
11.4
25.1
7.5

compound 18 (SIAIS117)
Brigatinib B
9
7.0
a
Table 2 Optimization of linker type and length with regard to the degradative activity in SR. DC50 are generated
using gray value quantified by ImageJ.
ALK degraders can degrade ALK proteins at the concentration as low as 7.0 nM, and there was
no obvious correlation between their degradation ability and cell growth inhibition was observed
(
Figure 4 and Table 2).Eventually, addition of PEG series of linker-based VHL compounds to
Brigatinib resulted in loss of the growth inhibition effect on cancer cell lines. This effect did not
change regardless whether A, B or C forms of Brigatinib analogue was used. When examined the
ALK protein level, we found all these compounds cannot degrade ALK proteins. Thus, it indicates
that PEG linkers are not tolerated with ALK fusion protein and VHL ligase.
2
.4 ALK PROTAC degrades ALK proteins through the proteasome-mediated pathway

Figure 5 Mechanism of the ALK degradation induced by compound SIAIS117. (A). Structure of SIAIS117 and
SIAIS117NC (Negative control). (B). The anti-proliferative activity of SIAIS117 and SIAIS117NC in SR. (C).
Time course of SIAIS117 and SIAIS117NC (100 nM) in SR. (D). Concentration course SIAIS117NC in SR for 16
h. (E). SR cell line treated with proteasome inhibitor MG132 (5 µM) or SIAIS117, SIAIS117NC for 16 h. Data are
representative of three independent experiments.
SIAIS117 degrades ALK in a time-dependent manner. To check how fast SIAIS117 will induce
ALK protein degradation, we firstly treated SR cell line with 100 nM of compound SIAIS117, and
then collected protein at indicated time points, and finally checked ALK protein levels (Figure 5C).
After 4 hours treatment of SIAIS117, ALK protein started to be degraded, and 24 hours later, almost
all ALK protein were degraded. To verify degrader works through proteasome-mediated pathway,
we designed and synthesized a negative control compound 38 called as SIAIS117NC, an epimer of
SIAIS117 (Figure 5A), which disturbing the binding at the E3 recruiting region. When the same
amount of SIAIS117NC was used to treat SR cells, we did not detect any ALK protein degradation
even after 24 hours (Figure 5C). We evaluated the degradation effect of Brigatinib in SR cell line,
and found with varied time from 4 h to 24 hours and concentration from 30 nM to 300 uM,
Brigatinib did not induce any reduction of ALK protein (Figure S4). We also found neither of the
brigatinib isoforms degrade ALK proteins (Figure S2). Therefore, considering with a similar cellular
permeability and kinase binding affinity for SIAIS117 and its epimer SIAIS117NC, a better growth
inhibition effect on ALK mutant cancer was resulted from SIAIS117-induced ALK protein
degradation (Fig. 5).
To further examine whether the degradation acted through the proteasome-mediated pathway,
we also used proteasome inhibitors MG132 and MLN4924 to block proteasome function (Figure
5
E). After pretreated cells with MG132 or MLN4924 for 4 hours, compound SIAIS117 or
SIAIS117NC were used to treat SR cell line for 16 hours. Same as above, compound SIAIS117
degraded ALK protein in either case but negative control compound SIAIS117NC could not do so
(
Figure 5D and 5E). The degradation of ALK protein by SIAIS117 was rescued by pretreated cells
with proteasome inhibitor MG132, and ALK protein levels turned back to normal. The same effect
was observed when using MLN4924. This experiment further indicated that ALK
PROTAC-induced ALK protein degradation act through proteasome mediated pathway.
2
.5 ALK degrader retains ALK phosphorylation inhibition capability and inhibits signaling
downstream of ALK.

Figure 6 Compare SIAIS117 with negative control SIAIS117NC on degradative activity in SR. The degradative
activity of SIAIS117 and SIAIS117NC in SR. Data are representative of three independent experiments.
We then studied the effects of SIAIS117 on the phosphorylation of ALK protein and signaling
downstream of ALK. Compound SIAIS117 inhibited all of the phosphorylation of ALK at 10 nM
(
Figure 6). In comparison, it required approximately 50 nM of compound SIAIS117NC to achieve
complete inhibition of the phosphorylation on ALK. We also checked ALK-mediated signaling
after treatment with the two compounds. Compound SIAIS117 showed better inhibition of
pSTAT3 than SIAIS117NC (Figure 6). Furthermore, proteomics data in SR cells (7280 proteins
were identified) showed there was no other Brigatinib target kinase including EGFR mutations,
IGF-1R, FLT3 and ROS1[39] degraded by SIAIS117 besides ALK fusion protein. In addition,
there was also no significant change in the protein level of some kinases like Aurora A that were
reported previously degraded by some ALK PROTAC molecules[24]. Among identified kinases in
our proteomics data, UCK2 and GAK were also downregulated significantly. Conclusively, the
selectivity of SIAIS117 is tending to be good in SR cell line (Figure S5, S6, Table S1).
2
.6 ALK PROTAC compound SIAIS117 degrades ALK proteins and inhibits ALK
phosphorylation in ALK mutant lung cancer cell lines.

Figure 7 Degradative and inhibitory activities of compound SIAIS117 in NSCLC-H2228. (A). The
anti-proliferative activity of Brigatinib, SIAIS117 and SIAIS117NC in H2228. (B). The degradative activity of
Brigatinib and SIAIS117 in H2228. Data are representative of three independent experiments.
NSCLC cell line H2228 carries an EML4-ALK gene fusion and is sensitive to ALK inhibitors.
After treating cells with drug for 72 h, living cells activities were evaluated by using CCK8 reagent
(
Figure 7A). Brigatinib inhibited H2228 cell growth with an IC50 around 58 nM. The designed
degrader SIAIS117 kept the same potency level with Brigatinib. Compound SIAIS117 inhibited cell
growth with an about two folds lower IC value (46 nM) comparing to SIAIS117NC (114 nM).
5
0
We next checked phosphorylation and degradation capability of the ALK protein after treating
H2228 cell line with compound SIAIS117. While Brigatinib itself did not induce ALK protein
degradation, compound SIAIS117 degraded ALK proteins at the concentration starting from 50 nM
(
Figure 7B).
.7 ALK PROTAC compound SIAIS117 inhibited the growth of G1202R-mutant ALK cell
line
2

Figure 8 The ability of compound SIAIS117 in inhibiting the growth of G1202R-mutant ALK cell line and
degrading G1202R-ALK protein. Left: growth inhibition assay; right: western blot. Data are representative of three
independent experiments.
Point mutations of clinically Crizotinib-relevant resistance mutations include G1202R,
G1269A, C1156Y, L1196M etc. G1202R mutation is a highly refractory mutation which is resistant
nd
to Crizotinib and all three 2 generation ALK inhibitors including Ceritinib and Brigatinib[2]. To
further investigate the ability of SIAIS117 to inhibit and degrade clinically relevant mutant
isoforms, we exogenously expressed EML4-ALK with G1202R mutation in 293T cell line (Figure
8
). SIAIS117 showed better growth inhibition than Brigatinib while SIAIS117NC had a similar
growth inhibition compared to Brigatinib in 293T transduced with ALK G1202R. The IC values of
5
0
Brigatinib or SIAIS117NC was approximately three times higher than that of SIAIS117 in this ALK
mutant expressing cell lines (Figure 8). Due to the efficient inhibition effect (IC50 146.4 nM) and
degradation ability of SIAIS117 (DC50 < 200 nM) on ALK drug Crizotinib resistant cell lines,
SIAIS117 provides a potential strategy to overcome G1202R-mediated drug resistance in ALK
targeted therapy.
2
.8 The anti-proliferation research against small cell lung cancer for ALK PROTAC
compound SIAIS117
Transformation to small cell lung cancer is one of the resistant mechanisms in ALK- and EGFR-
targeted therapy [40-43]. Small Cell Lung cancer (SCLC) is the most lethal lung cancer type. It
progresses very fast, with cancer metastasis frequently already occurring before diagnosis. Given
that SIAIS117 can effectively degrade ALK protein, we tested the effect of SIAIS117 in some
SCLC cell lines although their proliferation did not rely on ALK protein expression. We found that
SIAIS117 exerted the cell-killing effect of on small cell lung cancer cell lines (Figure 9). After
treatment with SIAIS117 and Brigatinib, Brigatinib barely inhibited the growth of NCI-H69 cell line
with an IC around 2.6 µM (Figure 9). However, SIAIS117 showed a lot better growth inhibition
5
0

effect on this cell line with an IC50 around 799 nM, which is achievable physiological range for
many FDA approved drugs. To further exclude E3 ligand cell-killing effect, we picked up negative
control E3 linker (s-4h) which had same length with SIAIS117, and found it did not inhibited the
growth of NCI-H69 (IC50 > 40 µM). In another small cell lung cancer cell line NCI-H1688, and
SIAIS117 showed a better anti-proliferation ability (IC = 259 nM) than Brigatinib (IC = 691 nM)
5
0
50
and E3 linker s-4h (IC50 > 45 µM) (Figure 9). Similar rule was also observed for small cell lung
cancer line NCI-H446. The reason why SIAIS117 inhibits growth of small cell lung cancer is under
investigation.
NCI-H69
NCI-H1688
SIAIS117
SIAIS117
1
00
100
Brigatinib
Brigatinib
E3 linker s-4h
E3 linker s-4h
5
0
0
5
0
0
0
1
2
3
4
5
0
1
2
3
4
5
log(10, nM)
log [10, nM]
Figure 9 The ability of compound SIAIS117 to inhibit the growth of small cell lung cancer. Data are
representative from at least three independent experiments. Two different small cell lung cancer cell lines were
tested. Left: NCI-H69; right: NCI-H1688.
2
.9 ALK PROTAC compound SIAIS117 maintained a longer degradative activity after drug
removal
Next, we investigated the long-term degradation activity of SIAIS117. SIAIS117 was removed
from medium after treating SR cells for 24 hours, and ALK protein levels were evaluated at 24h,
8h and 72hs after removal of SIAIS117. Results showed that SIAIS117 caused sustained
degradation of ALK protein in SR cells (Figure 10). This result further illustrates that ALK degrader
4

maintained a longer cellular activity than that of ALK kinase inhibitor after short pulse of drug
treatment. This provides a promising strategy to reduce side effect of Brigatinib drug and improving
patients' quality of life in a more economical way.
Figure 10 Sustained cellular degradative activity induced by SIAIS117 after drug removal in SR cells. Cells was
treated with 100 nM SIAIS117 for 24 hours, following wash-out with drug free medium and then was incubated
with drug-free medium. The ALK protein level was determined by western blot and normalized against β-actin.
3
. Conclusions
We have successfully developed an ALK degrader, SIAIS117 that not only degrades but also
inhibits the activities of oncogenic ALK protein in both non-small cell lung cancer H2228 cell and
ALCL cell line SR cells. In addition, this compound displays a clear advantage in inhibiting the
growth of cell line expressing G1202R-resistant ALK proteins and in degrading G1202R-resistant
proteins. This is the novel reported ALK PROTAC molecule that can inhibit the growth of small
cell lung cancer. Further studies will extend the application of ALK degrader in other ALK-positive
cancer types.
4
. EXPERIMENTAL SECTION
Chemistry. General Experiment and Information. Unless otherwise noted, all purchased
reagents were used as received without further purification. N,N-diisopropylethylamine (DIPEA),
N-methyl-2-pyrrolidinone (NMP), dichloromethane (DCM) and N,N-dimethylformamide (DMF)

were dried by a 4Å MS. Microwave reactions were performed with a CEM Discover single mode
microwave reactor equipped with a 300W source under an atmosphere of dry argon. Flash
1
13
chromatography was carried out on silica gel (200–300 mesh). H NMR and CNMR spectra were
1
recorded on Bruker AVANCE III 500 MHZ (operating at 500 MHz for H NMR, 126 MHz for
1
3
6
1
CNMR), chemical shift were reported in ppm relative to the residual d -DMSO (
δ
2.50 ppm H
1
NMR) or CD OD (
δ
3.31 ppm H NMR). High Resolution Mass spectra were recorded on AB
3
Triple 4600 spectrometer (QTOF) with acetonitrile and water as solvent. Preparative HPLC was
performed on SHIMADZU LC-20AP series with UV detector set to 254 nm. The final compounds
were all purified by C18 reverse phase preparative HPLC column with solvent A (0.5% HCl in
2
H O) and solvent B (MeCN) as eluents. The purity of all the final compounds was confirmed to be
>
95% purity by HPLC (SHIMADZU).
General procedure for synthesis of compounds Brigatinib analogue A、B、C.
Typical
2-((5-chloro-2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)
dimethylphosphine oxide 1 (Brigatinib A). To a solution of 5-fluoro-2-nitroanisole (7 g, 40.9
mmol) in DMF (60 mL) were added K CO (8.4 g, 60.8 mmol) and tert-butyl
procedure
for
the
synthesis
of
(
2
3
piperazine-1-carboxylate (9.1 g, 48.9 mmol) at room temperature under air. After stirring at the same
temperature overnight, then the reaction mixture was quenched with water, extracted with ethyl
2 4
acetate, washed with brine and dried over anhydrous Na SO . The solvent was evaporated under the
reduced pressure and the residue was purified by recrystallization (eluent: petroleum ether / ethyl
acetate = 5:1) to afford tert-butyl 4-(3-methoxy-4-nitrophenyl)piperazine-1-carboxylate s-1a in
1
8
0% yield (11.1 g) as a yellow solid. H NMR (500 MHz, DMSO) δ 7.89 (d, J = 9.3 Hz, 1H), 6.57
(d, J = 9.5 Hz, 1H), 6.52 (s, 1H), 3.90 (s, 3 H), 3.49-3.41 (m, 8H), 1.42 (s, 9H). HRMS (ESI) calcd
+
+
24 3 5
for C16H N O [M+H] , 338.1710; found, 338.1705.
To a stirred solution of s-1a (10 g, 29.6 mmol) in EtOH (90 mL) and H O (30 mL) were added
2
NH
4
Cl (6.3 g, 118.6 mmol) and Fe powder (8.3 g, 148.2 mmol). Then the resulting mixture was
o
refluxed at 80 C for 2 h under an atmosphere of nitrogen. When the reaction was complete, the
crude mixture was cooled down to room temperature, filtered by silica, after solvent evaporation,
extracted with DCM (3 x 50 mL), the combined organic layer was washed with brine (20 mL), then
dried
over
Na SO4
and
concentrated
in
vacuum
to
afford
tert-butyl
2

4
-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate s-2a 7.7 g in 85% yield (11.1 g) as a gray
1
solid. H NMR (500 MHz, CD OD) δ 6.72 (d, J = 8.3 Hz, 1H), 6.63 (d, J = 2.2 Hz, 1H), 6.47 (d, J =
3
7
.0 Hz, 1H), 3.86 (s, 3H), 3.57 (s, 4H), 2.99 (s, 4H), 1.50 (s, 9H). HRMS (ESI) calcd for
+
+
C
16
H
26
N
3
O
3
[M+H] , 308.1969; found, 308.1966.
To a solution of 2,5-dichloro-N-(2-(dimethylphosphoryl)phenyl)pyrimidin-4-amine (2 g, 6.3
mmol) in DMF (30 mL) were added s-2a (2.4 g, 7.8 mmol)，Pd(OAc) (176 mg, 0.78 mmol)，
2
Xantphos (810 mg, 1.4 mmol) and Cs CO (6.4 g, 19.6 mmol) in microwave tube, the solution was
2
3
o
purged and refilled with nitrogen three times. Then the mixture was stirred at 110 C for 2 h in the
microwave. After the starting material was consumed, the reaction mixture was filtered through a
pad of silica gel and most of the solvent of the filtrate was removed under the reduced pressure.
Then the mixture was quenched with water, extracted with ethyl acetate, washed with water and
2 4
brine, dried over anhydrous Na SO . The solvent was evaporated under the reduced pressure and the
residue was purified by reverse phase ISCO (C18) to give the title compound s-3a as a brown solid.
To a solution of above compound s-3a (900 mg) in DCM (6 mL) were added CF COOH (2 mL)
3
under air. The resulting solution was stirred at room temperature for 1 h. Then most of the solvent of
the filtrate was removed under the reduced pressure, and adjusted the pH of the solution to 8-9 with
saturated NaHCO solution, extracted with DCM, washed with brine, dried over anhydrous Na SO .
3
2
4
The solvent was evaporated under the reduced pressure and the residue was purified by reverse
phase ISCO (C18) to afford
2-((5-chloro-2-((2-methoxy-4-(piperazin-1-yl)phenyl)amino)pyrimidin-4-yl)amino)phenyl)
(
dimethylphosphine oxide 1 (Brigatinib A) as a yellow solid (701 mg, 23% yield over two steps).
1
H NMR (500 MHz, CD OD) δ 8.32 (dd, J = 8.2, 4.4 Hz, 1H), 8.04 (s, 1H), 7.70 (d, J = 8.7 Hz, 1H),
3
7
1
=
.61 (dd, J = 14.1, 7.7 Hz, 1H), 7.52 (t, J = 7.9 Hz, 1H), 7.26 (t, J = 7.5, 1H), 6.67 (d, J = 2.5 Hz,
H), 6.45 (dd, J = 8.8, 2.5 Hz, 1H), 3.86 (s, 3H), 3.24 - 3.17 (m, 4H), 3.17 - 3.11 (m, 4H), 1.83 (d, J
+
+
13.5 Hz, 6H). HRMS (ESI) calcd for C23
6 2
H29ClN O P [M+H] , 487.1773; found, 487.1772.
(2-((2-((4-(4-aminopiperidin-1-yl)-2-methoxyphenyl)amino)-5-chloropyrimidin-4-yl)amino)p
henyl)dimethylphosphine oxide (Brigatinib B, 2). (yellow solid, 330 mg, 33% yield over two
1
steps) H NMR (500 MHz, DMSO) δ 8.49 (s, 1H), 8.08 (s, 1H), 8.06 (s, 1H), 7.53 (ddd, J = 14.0,

7
.7, 1.3 Hz, 1H), 7.38 – 7.32 (m, 2H), 7.10 (t, J = 7.1 Hz, 1H), 6.62 (d, J = 2.5 Hz, 1H), 6.46 (dd, J
=
8.7, 2.5 Hz, 1H), 3.75 (s, 3H), 3.65 – 3.61 (m, 2H), 2.78 – 2.67 (m, 3H), 1.82 – 1.79 (m, 2H), 1.78
+
(
s, 3H), 1.75 (s, 3H), 1.42 – 1.34 (m, 2H). HRMS (ESI) calcd for C H ClN O P [M+H] : 501.1913,
2
4
31
6
2
found 501.1905.
(2-((5-chloro-2-((2-methoxy-4-(4-(piperazin-1-yl)piperidin-1-yl)phenyl)amino)pyrimidin-4-y
l)amino)phenyl)dimethylphosphine oxide (Brigatinib C, 3). (yellow solid, 350 mg, 37% yield
1
over two steps). H NMR (500 MHz, CD OD) δ 8.33 (dd, J = 8.2, 4.4 Hz, 1H), 8.03 (s, 1H), 7.69 –
3
7
1
2
.64 (m, 1H), 7.61-7.60 (m, 1H), 7.51 (t, J = 7.9 Hz, 1H), 7.28-7.24 (m, 1H), 6.66 (d, J = 2.4 Hz,
H), 6.45 (dd, J = 8.8, 2.5 Hz, 1H), 3.85 (s, 3H), 3.73 – 3.63 (m, 2H), 3.11 – 3.02 (m, 4H), 2.79 –
.66 (m, 6H), 2.48-2.43 (m, 1H), 1.99 (d, J = 12.5 Hz, 2H), 1.84 (d, J = 13.5 Hz, 6H), 1.72 – 1.63
+
(m, 2H). HRMS (ESI) calcd for C28
H38ClN
7
O
2
P [M+H] : 570.2508, found 570.2506.
General procedure for synthesis of VHL linkers s-4, s-5
To a stirred solution of succinic acid (680 mg, 5.8 mmol) in DMF (10 mL) was added
o
anhydrous DCM (150 mL). Then the mixture was cooled to 0 C, NMM (1.16 g, 11.5 mmol),
VHL-1 (1.0 g, 2.3 mmol), HOAT (63 mg, 0.46 mmol) and EDCI.HCl (530 mg, 2.8 mmol) were
added sequentially. The solution was purged and refilled with nitrogen. The resulting mixture was
stirred at room temperature for 12 h. The reaction mixture was quenched with water (1 mL). After
concentration, the residue was purified reverse phase ISCO (C18) to afford the desired compound
s-4a
(4-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,
3
-dimethyl-1-oxobutan-2-yl)amino)-4-oxobutanoic acid) (s-4a) (0.82 g, 65% yield) as a white
1
solid. H NMR (500 MHz, CDCl ) δ 11.88 (s, 1H), 8.85 (s, 1H), 7.69 (s, 1H), 7.37 – 7.29 (m, 4H),
3
6
1
2
.09 (br, 1H), 4.67 – 4.54 (m, 3H), 4.49 (s, 1H), 4.29 (dd, J = 15.0, 5.0Hz, 1H), 4.05 (d, J = 11.3 Hz,
H), 3.73 – 3.63 (m, 1H), 2.73 – 2.58 (m, 1H), 2.57 – 2.41 (m, 3H), 2.50 (s, 3H), 2.31 – 2.14 (m,
+
+
H), 0.96 (s, 9H). HRMS (ESI) m/z: cal. C H N O S [M+H] , 531.2272; found. 531.2280.
2
6
35
4
6
5-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-y
l)-3,3-dimethyl-1-oxobutan-2-yl)amino)-5-oxopentanoic acid (s-4b). (white solid, 0.85 g, 67%).
1
H NMR (500 MHz, CDCl ) δ 9.08 (s, 1H), 8.10 (s, 1H), 7.38 – 7.29 (m, 4H), 4.72 – 4.64 (m, 3H),
3
4
.52 (s, 1H), 4.25 (dd, J = 15.4, 5.0 Hz, 1H), 4.09 (d, J = 10.5 Hz, 1H), 3.73 (d, J = 10.0 Hz, 1H),
2
.48 (s, 3H), 2.39 – 2.13 (m, 6H), 1.92 – 1.74 (m, 2H), 0.96 (s, 9H). HRMS (ESI) m/z: cal.
+
+
C
27 37 4 6
H N O S [M+H] , 545.2428; found. 545.2436.

6-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-y
1
l)-3,3-dimethyl-1-oxobutan-2-yl)amino)-6-oxohexanoic acid (s-4c). (white solid, 0.79 g, 55%) H
NMR (500 MHz, CDCl ) δ 8.99 (s, 1H), 7.66 (s, 1H), 7.39 – 7.33 (m, 4H), 7.30 (d, J = 7.5 Hz, 1H),
3
7
.14 (br, 1H), 4.67 – 4.61 (m, 3H), 4.52 (s, 1H). 4.28 (dd, J = 15.4, 5.0 Hz, 1H), 4.09 (d, J = 11.4 Hz,
1
H), 3.74 – 3.63 (m, 1H), 2.52 (s, 3H), 2.31 – 2.17 (m, 6H), 1.65 – 1.53 (m, 4H), 0.96 (s, 9H).
+
+
40 4 6
HRMS (ESI) m/z: cal. C28H N O S [M+H] , 559.2585; found. 559.2590.
7-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-y
l)-3,3-dimethyl-1-oxobutan-2-yl)amino)-7-oxoheptanoic acid (s-4d). (white solid, 0.80 g, 57%)
1
H NMR (500 MHz, CDCl
.0 Hz, 1H), 6.50 (br, 1H), 4.79 – 4.46 (m, 3H), 4.55 (s, 1H), 4.28 (dd, J = 15.2, 5.1 Hz, 1H), 4.12 (d,
J = 11.3 Hz, 1H), 3.72 – 3.63 (m, 1H), 2.51 (s, 3H), 2.38 – 2.33 (m, 1H), 2.28 – 2.21 (m, 4H), 2.18 –
.12 (m, 1H), 1.62 – 1.51 (m, 4H), 1.33 – 1.26 (m, 4H), 0.96 (s, 9H). HRMS (ESI) m/z: cal.
3
) δ 8.90 (s, 1H), 7.42 – 7.38 (m, 1H), 7.41 – 7.33 (m, 4H), 7.31 (d, J =
9
2
+
+
C H N O S [M+H] , 573.2741; found. 573.2738.
29
41
4
6
8-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-y
1
l)-3,3-dimethyl-1-oxobutan-2-yl)amino)-8-oxooctanoic acid (s-4e). (white solid, 0.95 g, 68%) H
NMR (500 MHz, CDCl
3
) δ 8.82 (s, 1H), 7.43 (t, J = 6.0 Hz, 1H), 7.36 – 7.34 (m, 4H), 6.98 (d, J =
.5 Hz, 1H), 6.10 (br, 1H), 4.69 – 4.65 (m, 1H), 4.63 – 4.51 (m, 2H), 4.55 – 4.50 (m, 1H), 4.38 –
.27 (m, 1H), 4.11 (d, J = 16.7 Hz, 1H), 3.72 – 3.62 (m, 1H), 2.51 (s, 3H), 2.39 – 2.13 (m, 6H), 1.58
8
4
–
5
+
+
1.54 (m, 4H), 1.33 – 1.21 (m, 4H), 0.95 (s, 9H). HRMS (ESI) m/z: cal. C30
87.2898; found. 587.2907.
43 4 6
H N O S [M+H] ,
9-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-y
1
l)-3,3-dimethyl-1-oxobutan-2-yl)amino)-9-oxononanoic acid (s-4f). (white solid, 0.92 g, 64%). H
NMR (500 MHz, CDCl ) δ 8.82 (s, 1H), 7.35 (s, 4H), 7.00 (d, J = 10.0 Hz, 1H), 5.99 (br, 1H), 4.74
3
–
1
4
4.49 (m, 4H), 4.30 (dd, J = 15.2, 5.1 Hz, 1H), 4.13 (d, J = 11.3 Hz, 1H), 3.67 (dd, J = 11.5, 3.5 Hz,
H), 2.51 (s, 3H), 2.42 – 2.36 (m, 1H), 2.28 (t, J = 7.5 Hz, 2H), 2.24 – 2.12 (m, 3H), 1.67 – 1.48 (m,
+
+
45 4 6
H), 1.35 – 1.22 (m, 6H), 0.95 (s, 9H). HRMS (ESI) m/z: cal. C31H N O S [M+H] , 601.3054;
found. 601.3060.
0-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-
1
yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-10-oxodecanoic acid (s-4g). (white solid, 0.96 g, 66%)
1
H NMR (500 MHz, CDCl ) δ 8.79 (s, 1H), 7.38 (t, J = 6.0 Hz, 1H), 7.35 (s, 4H), 7.01 (d, J = 9.0 Hz,
3
1
H), 5.80 (br, 1H), 4.68 – 4.52 (m, 4H), 4.29 (dd, J = 15.2, 5.0 Hz, 1H), 4.12 (d, J = 11.2 Hz, 1H),

3
1
.72 – 3.62 (m, 1H), 2.51 (s, 3H), 2.41 – 2.33 (m, 1H), 2.32 – 2.23 (m, 2H), 2.23 – 2.11 (m, 3H),
+
.65 – 1.48 (m, 4H), 1.32 – 1.21 (m, 8H), 0.95 (s, 9H). HRMS (ESI) m/z: cal. C H N O S
3
2
47
4
6
+
[
M+H] , 615.3211; found. 615.3215.
11-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-1-
yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-11-oxoundecanoic acid (s-4h). (white solid, 1.0 g, 67%)
1
3
H NMR (500 MHz, CDCl ) δ 8.77 (s, 1H), 7.39 – 7.32 (m, 4H), 7.30 (m, 1H), 7.01 (d, J = 8.8 Hz,
1
3
1
H), 4.69 – 4.59 (m, 3H), 4.53 (s, 1H), 4.29 (dd, J = 15.2, 5.0 Hz, 1H), 4.14 (d, J = 11.3 Hz, 1H),
.68 – 3.64 (m, 1H), 2.51 (s, 3H), 2.44 – 2.40 (m, 1H), 2.29 (t, J = 7.1 Hz, 2H), 2.26 – 2.12 (m, 3H),
+
49 4 6
.68 – 1.48 (m, 4H), 1.30 – 1.20 (m, 10H), 0.95 (s, 9H). HRMS (ESI) m/z: cal. C33H N O S
+
[
M+H] , 629.3367; found. 629.3358.
3-(2-(3-(((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidi
n-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)amino)-3-oxopropoxy)ethoxy)propanoic acid (s-5a).
1
(
white solid, 0.53 g, 44%) H NMR (500 MHz, DMSO) δ 12.17 (s, 1H), 8.99 (s, 1H), 8.57 (t, J = 6.0
Hz, 1H), 7.92 (d, J = 9.3 Hz, 1H), 7.41 (dd, J = 18.5, 8.2 Hz, 4H), 4.55 (d, J = 9.5 Hz, 1H), 4.46 –
4
2
1
6
.40 (m, 2H), 4.36 (s, 1H), 4.23 (dd, J = 15.8, 5.4 Hz, 1H), 3.69 – 3.56 (m, 7H), 3.49 – 3.46 (m, 4H),
.58 – 2.53 (m, 1H), 2.47 – 2.42 (m, 2H), 2.45 (s, 3H), 2.39 – 2.32 (m, 1H), 2.06 – 2.01 (m, 1H),
+
+
.95 – 1.88 (m, 1H), 0.94 (s, 9H). HRMS (ESI) m/z: cal. C H N O S [M +H] , 619.2796; found.
3
0
43
4
8
19.2800.
S)-15-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-ca
(
rbonyl)-16,16-dimethyl-13-oxo-4,7,10-trioxa-14-azaheptadecanoic acid (s-5b). (white solid, 0.63
1
g, 59%) H NMR (500 MHz, DMSO) δ 8.99 (s, 1H), 8.57 (t, J = 6.0 Hz, 1H), 7.92 (d, J = 9.4 Hz,
1
4
–
9
H), 7.41 (dd, J = 18.5, 8.2 Hz, 4H), 4.56 (d, J = 9.4 Hz, 1H), 4.47 – 4.41 (m, 2H), 4.36 (s, 1H),
.23 (dd, J = 15.9, 5.5 Hz, 1H), 3.70 – 3.57 (m, 8H), 3.51 – 3.47 (m, 7H), 2.58 – 2.52 (m, 1H), 2.47
2.42 (m, 2H), 2.45 (s, 3H), 2.39 – 2.32 (m, 1H), 2.08 – 2.00 (m, 1H), 1.94 – 1.88 (m, 1H), 0.94 (s,
+
+
H). HRMS (ESI) m/z: cal. C H N O S [M+H] , 663.3058; found. 663.3067.
3
2
47
4
9
(S)-18-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-ca
rbonyl)-19,19-dimethyl-16-oxo-4,7,10,13-tetraoxa-17-azaicosanoic acid (s-5c). (white solid, 0.53
1
g, 51%) H NMR (500 MHz, DMSO) δ 8.98 (s, 1H), 8.56 (t, J = 6.0 Hz, 1H), 7.91 (d, J = 9.4 Hz,
1
4
2
H), 7.40 (dd, J = 18.8, 8.3 Hz, 4H), 4.55 (d, J = 9.4 Hz, 1H), 4.45 – 4.40 (m, 2H), 4.35 (s, 1H),
.22 (dd, J = 15.8, 5.5 Hz, 1H), 3.69 – 3.54 (m, 10H), 3.48 (d, J = 2.7 Hz, 9H), 2.56 – 2.52 (m, 1H),
.45 – 2.41 (m, 2H), 2.45 (s, 3H), 2.38 – 2.32 (m, 1H), 2.06 – 2.00 (m, 1H), 1.94 – 1.88 (m, 1H),

+
+
0
.93 (s, 9H). HRMS (ESI) m/z: cal. C34
51 4
H N O10S [M+H] , 707.3320; found. 707.3322.
(S)-21-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidine-1-carbon
yl)-22,22-dimethyl-19-oxo-4,7,10,13,16-pentaoxa-20-azatricosanoic acid (s-5d). (white solid,
1
0
.82 g, 85%) H NMR (500 MHz, DMSO) δ 8.98 (s, 1H), 8.56 (d, J = 5.7 Hz, 1H), 7.91 (d, J = 9.3
Hz, 1H), 7.40 (dd, J = 18.6, 7.9 Hz, 4H), 4.55 (d, J = 9.3 Hz, 1H), 4.47 – 4.40 (m, 2H), 4.35 (s, 1H),
4
–
1
.22 (dd, J = 15.7, 5.2 Hz, 1H), 3.68 – 3.56 (m, 11H), 3.51 – 3.49 (s, 9H), 2.56 – 2.53 (m, 1H), 2.45
2.41 (m, 5H), 2.44 (s, 3H), 2.36 (dd, J = 13.4, 7.0 Hz, 1H), 2.08 – 2.00 (m, 1H), 1.94 – 1.86 (m,
+
+
H), 0.93 (s, 9H). HRMS (ESI) m/z: cal. C H N O S [M+H] , 751.3583; found. 751.3579.
3
6
55
4
11
General procedure for synthesis of ALK degraders
In a 25 mL of round-bottom flask, to a stirred solution of ALK inhibitors (0.02 mmol, 1 equiv) in
DMF (2 mL) were added linker s-4 or s-5 (0.02 mmol, 1 equiv), HOAt (0.04 mmol, 2 equiv), EDCI
0.04 mmol, 2 equiv) and NMM (0.2 mmol, 10 equiv) sequentially. Then the resulting mixture was
stirred for 12 h at room temperature. The reaction was quenched with water (1.0 mL), followed by
purification via preparative HPLC [C18 column, eluent (v/v)：MeCN/(H O+0.05%HCl) = 10%
100%] to afford the desired degraders.
2S,4R)-1-((S)-2-(4-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl
amino)-3-methoxyphenyl)piperazin-1-yl)-4-oxobutanamido)-3,3-dimethylbutanoyl)-4-hydrox
(
2
–
(
)
y-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (4). (white solid, 10.1 mg, 37%)
1
H NMR (500 MHz, CD OD) δ 9.98 (s, 1H), 8.22 (s, 1H), 8.18 (s, 1H), 7.80 - 7.45 (m, 8H), 7.39 (s,
3
1
1
1
H), 7.13 (d, J = 8.5 Hz, 1H), 4.66 – 4.35 (m, 5H), 4.05 (s, 4H), 3.96 (s, 3H), 3.89 (d, J = 10.7 Hz,
H), 3.80 (d, J = 8.1 Hz, 1H), 3.70 (s, 2H), 3.61 (s, 2H), 2.91 – 2.63 (m, 4H), 2.61 (s, 3H), 2.23 (s,
+
H), 2.07 (s, 1H), 1.87 (d, J = 13.4 Hz, 6H), 1.05 (s, 9H). HRMS (ESI) calcd for C H ClN O PS
49
61
10
7
+
[
M+H] , 999.3866; found, 999.3865.
2S,4R)-1-((S)-2-(5-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl
amino)-3-methoxyphenyl)piperazin-1-yl)-5-oxopentanamido)-3,3-dimethylbutanoyl)-4-hydro
(
)
xy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (5). (white solid, 9.4 mg, 35%)
1
H NMR (500 MHz, CD OD) δ 10.00 (s, 1H), 8.25 (s, 1H), 8.14 (s, 1H), 7.79 - 7.63 (m, 3H), 7.61 -
3
7
3
2
1
.46 (m, 6H), 7.22 (d, J = 8.6 Hz, 1H), 4.65 - 4.47 (m, 4H), 4.41 (d, J = 15.7 Hz, 1H), 4.07 (s, 4H),
.97 (s, 3H), 3.93 (d, J = 11.1 Hz, 1H), 3.80 (dd, J = 11.0, 3.6 Hz, 1H), 3.74 (s, 2H), 3.66 (s, 2H),
.60 (s, 3H), 2.53 (t, J = 7.4 Hz, 2H), 2.40 (t, J = 7.1 Hz, 2H), 2.28 – 2.20 (m, 1H), 2.11 – 2.02 (m,
H), 2.01 – 1.92 (m, 2H), 1.87 (d, J = 13.6 Hz, 6H), 1.05 (s, 9H). HRMS (ESI) calcd for

+
+
C
50
H
63ClN10
O
7
PS [M+H] , 1013.4023; found, 1013.4022.
(2S,4R)-1-((S)-2-(6-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl
)amino)-3-methoxyphenyl)piperazin-1-yl)-6-oxohexanamido)-3,3-dimethylbutanoyl)-4-hydrox
y-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide. (6). (white solid, 11.4 mg, 42%)
1
H NMR (500 MHz, CD OD) δ 10.02 (d, J = 2.9 Hz, 1H), 8.26 (s, 1H), 8.13 (m 1H), 7.80 – 7.63 (m,
3
3
1
2
2
H), 7.60 – 7.49 (m, 6H), 7.25 (d, J = 8.8 Hz, 1H), 4.64 – 4.46 (m, 4H), 4.40 (dd, J = 15.7, 3.7 Hz,
H), 4.10 (s, 4H), 3.97 (s, 3H), 3.90 (d, J = 11.1 Hz, 1H), 3.80 (dd, J = 11.0, 3.9 Hz, 1H), 3.76 (s,
H), 3.68 (s, 2H), 2.61 (s, 3H), 2.59 – 2.48 (m, 2H), 2.38 – 2.32 (m, 2H), 2.27 - 2.21(m, 1H), 2.10 -
.03 (m, 1H), 1.87 (d, J = 13.6 Hz, 6H), 1.77 – 1.64 (m, 4H), 1.04 (s, 9H). HRMS (ESI) calcd for
+
+
C H ClN O PS [M+H] , 1027.4179; found, 1027.4171.
51
65
10
7
(2S,4R)-1-((S)-2-(7-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl
)amino)-3-methoxyphenyl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylbutanoyl)-4-hydro
xy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (7). (white solid, 12.7 mg,
1
4
7
2
1
–
2
7%) H NMR (500 MHz, CD
3
OD) δ 10.03 (d, J = 1.2 Hz, 1H), 8.26 (s, 1H), 8.13 (s, 1H), 7.78 -
.65 (m, 3H), 7.61 – 7.48 (m, 6H), 7.25 (d, J = 8.6 Hz, 1H), 4.63 (d, J = 7.6 Hz, 1H), 4.59 – 4.53 (m,
H), 4.50 (s, 1H), 4.40 (dd, J = 15.8, 6.4 Hz, 1H), 4.11 (s, 4H), 3.98 (s, 3H), 3.90 (d, J = 11.0 Hz,
H), 3.80 (dd, J = 11.0, 3.7 Hz, 1H), 3.77 - 3.60 (m, 4H), 2.61 (s, 3H), 2.52 (t, J = 7.5 Hz, 2H), 2.36
2.19 (m, 3H), 2.12 - 2.04 (m, 1H), 1.87 (d, J = 13.6 Hz, 6H), 1.72 – 1.57 (m, 4H), 1.47 – 1.29 (m,
+
+
H), 1.03 (s, 9H). HRMS (ESI) calcd for C52
7
H67ClN10O PS [M+H] , 1041.4336; found, 1041.4346.
(2S,4R)-1-((S)-2-(8-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl
)amino)-3-methoxyphenyl)piperazin-1-yl)-8-oxooctanamido)-3,3-dimethylbutanoyl)-4-hydroxy
-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (8). (white solid, 6.8 mg, 25%)
1
H NMR (500 MHz, CD OD) δ 9.99 (d, J = 4.4 Hz, 1H), 8.23 (s, 1H), 8.18 (s, 1H), 7.74 (dd, J =
3
1
1
4
3
3.0, 7.6 Hz, 1H), 7.66 (t, J = 7.5 Hz, 2H), 7.60 – 7.46 (m, 5H), 7.40 (s, 1H), 7.14 (d, J = 8.7 Hz,
H), 4.63 (d, J = 6.1 Hz, 1H), 4.60 -- 4.52 (m, 2H), 4.50 (s, 1H), 4.40 (dd, J = 15.8, 7.7 Hz, 1H),
.05 (s, 4H), 3.96 (s, 3H), 3.91 (d, J = 11.0 Hz, 1H), 3.80 (dd, J = 11.1, 3.7 Hz, 1H), 3.68 (s, 2H),
.62 (s, 2H), 2.61 (s, 3H), 2.51 (t, J = 7.5 Hz, 2H), 2.40 – 2.17 (m, 3H), 2.11 - 2.03 (m, 1H), 1.87 (d,
J = 13.5 Hz, 6H), 1.70 – 1.55 (m, 4H), 1.45 - 1.30 (m, 4H), 1.04 (s, 9H). HRMS (ESI) calcd for
+
+
C
53
H
69ClN10
O
7
PS [M+H] , 1055.4492; found, 1055.4486.
(2S,4R)-1-((S)-2-(9-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl
)amino)-3-methoxyphenyl)piperazin-1-yl)-9-oxononanamido)-3,3-dimethylbutanoyl)-4-hydrox

y-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (9). (white solid, 8.3 mg, 31%)
1
H NMR (500 MHz, CD OD) δ 9.96 (s, 1H), 8.19 (d, J = 14.1 Hz, 2H), 7.73 (dd, J = 14.0, 6.8 Hz,
3
1
4
H), 7.67 – 7.45 (m, 6H), 7.31 (s, 1H), 7.07 (d, J = 8.7 Hz, 1H), 4.64 (s, 1H), 4.60 - 4.52 (m, 2H),
.50 (s, 1H), 4.41 (d, J = 15.8 Hz, 1H), 4.00 (s, 4H), 3.95 (s, 3H), 3.91 (d, J = 11.1 Hz, 1H), 3.80 (dd,
J = 11.0, 3.8 Hz, 1H), 3.63 (s, 2H), 3.57 (s, 2H), 2.60 (s, 3H), 2.50 (t, J = 7.6 Hz, 2H), 2.37 – 2.18
(m, 3H), 2.11 - 2.03 (m, 1H), 1.87 (d, J = 13.6 Hz, 6H), 1.70 – 1.55 (m, 4H), 1.45 - 1.30 (m, 6H),
+
+
1
.02 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] , 1069.4649; found, 1069.4642.
54 71 10 7
(2S,4R)-1-((S)-2-(10-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-
yl)amino)-3-methoxyphenyl)piperazin-1-yl)-10-oxodecanamido)-3,3-dimethylbutanoyl)-4-hydr
oxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (10). (white solid, 10.6 mg,
1
3
1
4
2
1
1
9%) H NMR (500 MHz, CD
3
OD) δ 9.56 (d, J = 4.2 Hz, 1H), 8.35 (s, 1H), 8.08 (s, 1H), 7.70 (s,
H), 7.62 - 7.30 (m, 6H), 6.86 (s, 1H), 6.71 (s, 1H), 4.64 (d, J = 3.5 Hz, 1H), 4.60 - 4.47 (m, 3H),
.39 (dd, J = 15.7, 3.2 Hz, 1H), 4.00 – 3.69 (m, 9H), 3.36 (s, 4H), 2.55 (d, J = 3.8 Hz, 3H), 2.51 –
.40 (m, 2H), 2.38 - 2.18 (m, 3H), 2.12 – 2.02 (m, 1H), 1.89 (dd, J = 13.6, 3.5 Hz, 6H), 1.62 (s, 4H),
+
+
.35 (s, 8H), 1.03 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] , 1083.4805; found,
5
5
73
10
7
083.4802.
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N4-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car
bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)succinamide (11). (white solid, 12.8 mg,
1
6
7
3%) H NMR (500 MHz, CD OD) δ 9.95 (s, 1H), 8.27 (s, 1H), 8.11 (s, 1H), 7.78 – 7.73 (m, 2H),
3
.68 (t, J = 7.5 Hz, 1H), 7.60 – 7.50 (m, 6H), 7.29 (d, J = 8.2 Hz, 1H), 4.62 – 4.47 (m, 4H), 4.41 (d,
J = 15.6 Hz, 1H), 4.19 – 4.11 (m, 1H), 4.00 (s, 3H), 3.91 – 3.89 (m, 1H), 3.81 – 3.78 (m, 5H), 2.67 –
2
.53 (m, 4H), 2.59 (s, 3H), 2.31 – 2.15 (m, 5H), 2.11 – 2.04 (m, 1H), 1.88 (s, 3H), 1.86 (s, 3H), 1.04
+
+
(
7
s, 9H). HRMS (ESI) calcd for C50H63ClN10O PS [M+H] : 1013.4023, found 1013.4017.
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-methox
yphenyl)piperidin-4-yl)-N5-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carba
moyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)glutaramide (12). (white solid, 12.2 mg,
1
5
7
1
9%). H NMR (500 MHz, CD OD) δ 9.96 (s, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 7.78 – 7.73 (m, 2H),
3
.68 (t, J = 7.8 Hz, 1H), 7.61 – 7.56 (m, 3H), 7.55 – 7.49 (m, 3H), 7.30 (d, J = 8.7 Hz, 1H), 4.64 (s,
H), 4.69 – 4.51 (m, 3H), 4.45 – 4.40 (m, 1H), 4.20 – 4.12 (m, 1H), 4.00 (s, 3H), 3.93 (d, J = 11.2
Hz, 1H), 3.86 – 3.73 (m, 5H), 2.60 (s, 3H), 2.38 – 2.17 (m, 9H), 2.11 – 2.05 (m, 1H), 1.96 – 1.91 (m,

+
+
2
1
H), 1.88 (s, 3H), 1.85 (s, 3H), 1.05 (s, 9H). HRMS (ESI) calcd for C51
027.4179, found 1027.4170.
7
H65ClN10O PS [M+H] :
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N6-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car
bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)adipamide
1
(
13). (white solid, 13.1 mg, 63%) H NMR (500 MHz, CD
3
OD) δ 10.01 (s, 1H), 8.28 (s, 1H), 8.11
(s, 1H), 7.78 – 7.74 (m, 2H), 7.69 (t, J = 7.9 Hz, 1H), 7.63 (s, 1H), 7.59 – 7.57 (m, 2H), 7.55 – 7.50
(m, 3H), 7.32 (d, J = 6.9 Hz, 1H), 4.64 (s, 1H), 4.61 – 4.47 (m, 3H), 4.43 – 4.39 (m, 1H), 4.21 – 4.13
(m, 1H), 4.00 (s, 3H), 3.92 (d, J = 11.1 Hz, 1H), 3.85 – 3.71 (m, 5H), 2.61 (s, 3H), 2.36 – 2.17 (m,
9
H), 2.10 – 2.04 (m, 1H), 1.88 (s, 3H), 1.86 (s, 3H), 1.70 – 1.62 (m, 4H), 1.04 (s, 9H). HRMS (ESI)
+
+
7
calcd for C52H67ClN10O PS [M+H] : 1041.4336, found 1041.4339.
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N7-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car
bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)heptanediamide (14). (white solid, 12.3
1
mg, 58%) H NMR (500 MHz, CD OD) δ 9.97 (s, 1H), 8.27 (s, 1H), 8.11 (s, 1H), 7.77 – 7.73 (m,
3
2
H), 7.68 (t, J = 7.8 Hz, 1H), 7.61 – 7.50 (m, 6H), 7.29 (d, J = 8.7 Hz, 1H), 4.64 (s, 1H), 4.61 – 4.48
(m, 3H), 4.44 – 4.38 (m, 1H), 4.18 – 4.12 (m, 1H), 4.00 (s, 3H), 3.91 (d, J = 11.1 Hz, 1H), 3.85 –
3
–
.70 (m, 5H), 2.60 (s, 3H), 2.36 – 2.15 (m, 9H), 2.10 – 2.05 (m, 1H), 1.88 (s, 3H), 1.85 (s, 3H), 1.69
+
7
1.61 (m, 4H), 1.42 – 1.33 (m, 2H), 1.04 (s, 9H). HRMS (ESI) calcd for C53H69ClN10O PS
+
[
M+H] : 1055.4492, found 1055.4491.
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N8-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car
bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)octanediamide
1
(
15). (white solid, 14.8 mg, 69%) H NMR (500 MHz, CD
3
OD) δ 9.91 (s, 1H), 8.27 (s, 1H), 8.11
(s, 1H), 7.77 – 7.73 (m, 2H), 7.69 (t, J = 7.8 Hz, 1H), 7.61 (d, J = 2.1 Hz, 1H), 7.58 – 7.51 (m, 5H),
7
.31 (d, J = 8.7 Hz, 1H), 4.64 (s, 1H), 4.60 – 4.49 (m, 3H), 4.43 – 4.38 (m, 1H), 4.20 – 4.13 (m, 1H),
.00 (s, 3H), 3.92 (d, J = 11.1 Hz, 1H), 3.86 – 3.75 (m, 5H), 2.60 (s, 3H), 2.33 – 2.18 (m, 9H), 2.11
2.05 (m, 1H), 1.88 (s, 3H), 1.85 (s, 3H), 1.68 – 1.59 (m, 4H), 1.41 – 1.34 (m, 4H), 1.04 (s, 9H).
4
–
+
+
7
HRMS (ESI) calcd for C54H71ClN10O PS [M+H] : 1069.4649, found 1069.4661.
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N9-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)car

bamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)nonanediamide (16). (white solid, 12.8
1
mg, 59%) H NMR (500 MHz, CD OD) δ 10.01 (s, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 7.78 – 7.73 (m,
3
2
7
4
–
H), 7.69 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.60 – 7.57 (m, 2H), 7.55 – 7.49 (m, 3H),
.31 (d, J = 8.8 Hz, 1H), 4.64 (s, 1H), 4.60 – 4.49 (m, 3H), 4.44 – 4.38 (m, 1H), 4.21 – 4.13 (m, 1H),
.00 (s, 3H), 3.91 (d, J = 11.1 Hz, 1H), 3.85 – 2.73 (m, 5H), 2.61 (s, 3H), 2.33 – 2.18 (m, 9H), 2.10
2.04 (m, 1H), 1.88 (s, 3H), 1.86 (s, 3H), 1.66 – 1.59 (m, 4H), 1.36 (s, 6H), 1.04 (s, 9H). HRMS
+
+
(
ESI) calcd for C H ClN O PS [M+H] : 1083.4805, found 1083.4797.
55 73 10 7
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N10-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)ca
rbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)decanediamide (17). (white solid, 10.2
1
mg, 46%) H NMR (500 MHz, CD
3
OD) δ 10.02 (s, 1H), 8.28 (s, 1H), 8.11 (s, 1H), 7.79 – 7.73 (m,
2
H), 7.69 (t, J = 7.9 Hz, 1H), 7.63 (d, J = 2.0 Hz, 1H), 7.58 (d, J = 8.3 Hz, 2H), 7.54 – 7.50 (m, 3H),
7
.32 (d, J = 8.6 Hz, 1H), 4.64 (s, 1H), 4.60 – 4.49 (m, 3H), 4.44 – 4.38 (m, 1H), 4.20 – 4.14 (m, 1H),
.00 (s, 3H), 3.91 (d, J = 11.2 Hz, 1H), 3.81 (dd, J = 10.9, 3.9 Hz, 5H), 2.61 (s, 3H), 2.33 – 2.18 (m,
H), 2.10 – 2.05 (m, 1H), 1.88 (s, 3H), 1.86 (s, 3H), 1.67 – 1.57 (m, 4H), 1.35 (s, 9H), 1.04 (s, 9H).
4
9
+
+
7
HRMS (ESI) calcd for C56H75ClN10O PS [M+H] : 1097.4962, found 1097.4953.
N1-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2-yl)amino)-3-meth
oxyphenyl)piperidin-4-yl)-N11-((S)-1-((2S,4R)-4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)ca
rbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobutan-2-yl)undecanediamide (18, SIAIS117).
1
(
white solid, 10.6 mg, 48%) H NMR (500 MHz, CD OD) δ 8.84 (s, 1H), 8.31 (dd, J = 7.5, 3.9 Hz,
3
1
7
1
5
2
H), 8.02 (s, 1H), 7.77 (d, J = 8.6 Hz, 1H), 7.62-7.58 (m, 1H), 7.53 (t, J = 7.8 Hz, 1H), 7.43 (t, J =
.3 Hz, 2H), 7.37 (d, J = 7.9 Hz, 2H), 7.28 (t, J = 7.2 Hz, 1H), 6.84 (s, 1H), 6.64 (s, 1H), 4.64 (s,
H), 4.59 (t, J = 8.3 Hz, 1H), 4.51 (d, J = 15.4 Hz, 2H), 4.34 (d, J = 15.5 Hz, 1H), 3.93 – 3.83 (m,
H), 3.81-3.78 (m, 1H), 3.74 – 3.57 (m, 2H), 3.04-2.85 (m, 2H), 2.44 (s, 3H), 2.28 – 2.18 (m, 4H),
.10 – 2.02 (m, 2H), 1.83 (d, J = 13.5 Hz, 6H), 1.80-1.76 (m, 2H), 1.60 (d, J = 6.4 Hz, 4H), 1.38 (d,
1
3
J = 7.0 Hz, 2H), 1.33-1.28 (m, 10H), 1.03 (s, 9H). C NMR (126 MHz, CD
3
OD) δ 176.24, 176.15,
1
1
4
74.56, 172.16, 159.98, 156.85, 152.34, 142.71, 142.13, 141.92, 141.17, 137.74, 134.32, 132.60,
32.52, 130.44, 129.47, 127.69, 127.11, 114.44, 108.03, 106.63, 71.04, 60.81, 59.04, 57.97, 57.52,
3.55, 38.98, 36.90, 36.54, 30.41, 30.34, 30.24, 30.21, 27.04, 27.00, 18.37, 17.80, 12.95. HRMS
+
(
ESI) calcd for C H ClN O PS [M+H] : 1111.5118, found 1111.5109.
5
7
77
10
7
(2S,4R)-1-((S)-2-(4-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2

-yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-4-oxobutanamido)-3,3-dimethylbu
tanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (19). (white
1
solid, 9.1 mg, 48%) H NMR (500 MHz, CD OD) δ 9.62 (s, 1H), 8.31 (s, 1H), 8.11 (s, 1H), 7.70 (dd,
3
J = 14.0, 9.1 Hz, 1H), 7.60 (s, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.49 (d, J = 8.3 Hz, 2H), 7.43 (t, J = 7.7
Hz, 2H), 6.98 (s, 1H), 6.80 (s, 1H), 4.61 (s, 1H), 4.59-4.54 (m, 2H), 4.50 (s, 1H), 4.40 (d, J = 15.8
Hz, 1H), 4.34 – 4.22 (m, 1H), 3.97 (d, J = 11.9 Hz, 2H), 3.92 – 3.86 (m, 4H), 3.81 (dd, J = 11.0, 3.9
Hz, 1H), 3.67-3.60 (m, 5H), 3.17 (s, 4H), 2.84 – 2.57 (m, 5H), 2.56 (s, 3H), 2.37 (d, J = 10.3 Hz,
2
H), 2.28 – 2.19 (m, 1H), 2.19 – 2.05 (m, 3H), 1.88 (d, J = 13.6 Hz, 6H), 1.03 (s, 9H). HRMS (ESI)
+
calcd for C54
H70ClN11
O
7
PS [M+H] : 1082.4601, found 1082.4593.
(2S,4R)-1-((S)-2-(5-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2
-yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-5-oxopentanamido)-3,3-dimethylb
utanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (20). (white
1
solid, 6.3 mg, 33%) H NMR (500 MHz, CD OD) δ 9.72 (s, 1H), 8.28 (s, 1H), 8.15 (s, 1H), 7.72 (dd,
3
J = 13.9, 7.9 Hz, 1H), 7.67 – 7.60 (m, 1H), 7.59 – 7.35 (m, 6H), 7.14 (s, 1H), 6.91 (s, 1H), 4.69 –
4
.49 (m, 5H), 4.41 (d, J = 15.8 Hz, 1H), 4.31 – 4.16 (m, 1H), 3.95-3.92 (m, 6H), 3.81 (d, J = 7.8 Hz,
1
H), 3.69 (s, 4H), 3.27 – 3.02 (m, 5H), 2.57 (s, 3H), 2.44-2.37 (m, 6H), 2.24-2.08 (m, 4H),
+
1
.93-1.87 (m, 8H), 1.04 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] : 1096.4758,
5
5
72
11
7
found 1096.4748.
(2S,4R)-1-((S)-2-(6-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2
-yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-6-oxohexanamido)-3,3-dimethylbu
tanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (21). (white
1
solid, 8.6 mg, 44%) H NMR (500 MHz, CD OD) δ 9.76 (s, 1H), 8.26 (s, 1H), 8.15 (s, 1H), 7.72 (dd,
3
J = 13.9, 7.8 Hz, 1H), 7.63 (t, J = 8.0 Hz, 1H), 7.59 – 7.43 (m, 6H), 7.17 (s, 1H), 6.95 (s, 1H), 4.64
(s, 1H), 4.61 – 4.49 (m, 3H), 4.41 (d, J = 15.6 Hz, 1H), 4.25 (s, 1H), 3.99 – 3.88 (m, 6H), 3.81 (dd, J
=
10.8, 3.8 Hz, 1H), 3.69 (s, 4H), 3.40-3.31 (m, 3H), 3.17 (s, 3H), 2.58 (s, 3H), 2.50-2.43 (m, 4H),
2
.36-2.22 (m, 5H), 2.13 – 2.03 (m, 1H), 1.88 (d, J = 13.6 Hz, 6H), 1.67 (s, 4H), 1.05 (s, 9H). HRMS
+
(
ESI) calcd for C H ClN O PS [M+H] : 1110.4914, found 1110.4903.
5
6
74
11
7
(2S,4R)-1-((S)-2-(7-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2
-yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-7-oxoheptanamido)-3,3-dimethylb
utanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (22). (white
1
solid, 8.4 mg, 43%) H NMR (500 MHz, CD
3
OD) δ 9.64 (s, 1H), 8.32 (s, 1H), 8.11 (s, 1H), 7.71 (dd,

J = 13.9, 7.7 Hz, 1H), 7.61 (s, 1H), 7.55-7.44 (m, 6H), 7.01 (s, 1H), 6.81 (s, 1H), 4.64 (s, 1H),
.60-4.50 (m, 3H), 4.41 (d, J = 15.7 Hz, 1H), 3.96 (d, J = 10.3 Hz, 2H), 3.92-3.90 (m, 4H), 3.81 (dd,
J = 10.9, 3.9 Hz, 1H), 3.69-3.60 (m, 4H), 3.31-3.17 (m, 7H), 2.56 (s, 3H), 2.47 (t, J = 7.3 Hz, 2H),
4
2
6
.37 (s, 2H), 2.35 – 2.27 (m, 2H), 2.27 – 2.20 (m, 1H), 2.19 – 2.03 (m, 3H), 1.88 (d, J = 13.6 Hz,
H), 1.70 – 1.59 (m, 4H), 1.43-1.37 (m, 2H), 1.03 (s, 9H). HRMS (ESI) calcd for C H ClN O PS
5
7
76
11
7
+
[
M+H] : 1124.5071, found 1124.5070.
2S,4R)-1-((S)-2-(8-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2
yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-8-oxooctanamido)-3,3-dimethylbut
(
-
anoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (23). (white
1
solid, 8.3 mg, 42%) H NMR (500 MHz, CD OD) δ 9.76 (s, 1H), 8.27 (s, 1H), 8.15 (s, 1H), 7.72 (dd,
3
J = 14.0, 7.9 Hz, 1H), 7.63 (s, 1H), 7.59 – 7.39 (m, 6H), 7.19 (s, 1H), 6.94 (s, 1H), 4.64 (s, 1H), 4.62
–
4.46 (m, 3H), 4.41 (d, J = 15.7 Hz, 1H), 4.26 (s, 1H), 4.03 – 3.86 (m, 6H), 3.81 (dd, J = 10.9, 3.8
Hz, 1H), 3.69 (s, 4H), 3.33 (s, 3H), 3.17 (s, 3H), 2.58 (s, 3H), 2.53 – 2.38 (m, 4H), 2.38 – 2.17 (m,
5
4
H), 2.13 – 2.02 (m, 1H), 1.88 (d, J = 13.6 Hz, 6H), 1.64 (d, J = 6.7 Hz, 4H), 1.39 (d, J = 3.5 Hz,
+
H), 1.03 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] : 1138.5227, found 1138.5230.
5
8
78
11
7
(2S,4R)-1-((S)-2-(9-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-2
-yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-9-oxononanamido)-3,3-dimethylbu
tanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (24). (white
1
solid, 8.9 mg, 44%) H NMR (500 MHz, CD
3
OD) δ 9.82 – 9.70 (m, 1H), 8.25 (s, 1H), 8.16 (s, 1H),
7
4
.78 – 7.68 (m, 1H), 7.64 (s, 1H), 7.56-7.47 (m, 6H), 7.23 (s, 1H), 6.98 (s, 1H), 4.64 (s, 1H), 4.61 –
.47 (m, 3H), 4.40 (d, J = 15.7 Hz, 1H), 4.25 (s, 1H), 3.92 (t, J = 8.1 Hz, 6H), 3.81 (dd, J = 11.0, 3.8
Hz, 1H), 3.70 (s, 4H), 3.39-3.32 (m, 3H), 3.30-3.17 (m, 3H), 2.58 (s, 3H), 2.52 – 2.39 (m, 4H), 2.39
–
9
2.17 (m, 5H), 2.13 – 2.03 (m, 1H), 1.88 (d, J = 13.6 Hz, 6H), 1.62 (s, 4H), 1.37 (s, 6H), 1.03 (s,
+
H). HRMS (ESI) calcd for C59
H80ClN11
O
7
PS [M+H] : 1152.5384, found 1152.5388.
(2S,4R)-1-((S)-2-(10-(4-(1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidin-
2
-yl)amino)-3-methoxyphenyl)piperidin-4-yl)piperazin-1-yl)-10-oxodecanamido)-3,3-dimethyl
butanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (25). (white
1
solid, 7.7 mg, 38%) H NMR (500 MHz, CD OD) δ 9.88 – 9.79 (m, 1H), 8.19 (s, 2H), 7.73 (dd, J =
3
1
4
3.9, 7.8 Hz, 1H), 7.65 (s, 1H), 7.57-7.48 (m, 6H), 7.33 (s, 1H), 7.06 (s, 1H), 4.64 (s, 1H), 4.61 –
.46 (m, 3H), 4.44 – 4.37 (m, 1H), 4.27 (s, 1H), 3.95-3.90 (m, 6H), 3.85 – 3.48 (m, 8H), 3.20-3.04
(m, 3H), 2.60 (s, 3H), 2.53 – 2.43 (m, 4H), 2.42 – 2.19 (m, 5H), 2.10-2.05 (m, 1H), 1.88 (d, J = 13.6

Hz, 6H), 1.62 (d, J = 7.0 Hz, 4H), 1.36 (s, 8H), 1.03 (s, 9H). HRMS (ESI) calcd for
+
C H ClN O PS [M+H] : 1166.5540, found 1166.5528.
60
82
11
7
(2S,4R)-1-((S)-2-(3-(2-(3-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)
pyrimidin-2-yl)amino)-3-methoxyphenyl)piperazin-1-yl)-3-oxopropoxy)ethoxy)
propanamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)
1
pyrrolidine-2-carboxamide (26). (white solid, 14.9 mg, 55%) H NMR (500 MHz, CD
3
OD) δ 8.89
(s, 1H), 8.40 (s, 1H), 8.03 (s, 1H), 7.66 (dd, J = 14.0, 7.7 Hz, 1H), 7.55 (s, 1H), 7.46 (d, J = 8.2 Hz,
2
1
3
H), 7.42 - 7.33 (m, 3H), 7.29 (d, J = 7.8 Hz, 1H), 6.72 (s, 1H), 6.58 (d, J = 8.5 Hz, 1H), 4.64 (s,
H), 4.60 – 4.52 (m, 2H), 4.49 (s, 1H), 4.34 (d, J = 15.5 Hz, 1H), 3.88 (d, J = 10.9 Hz, 1H), 3.84 (s,
H), 3.81 – 3.66 (m, 9H), 3.62 – 3.58 (m, 4H), 3.31 (s, 2H), 3.21 (s, 2H), 2.77 – 2.66 (m, 2H), 2.46
(s, 3H), 2.51 – 2.43 (m, 1H), 2.43 – 2.36 (m, 1H), 2.25 - 2.18 (m, 1H), 2.12 - 2.04 (m, 1H), 1.88 (d,
+
+
J = 13.6 Hz, 6H), 1.03 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] , 1087.4390;
5
3
69
10
9
found, 1087.4388.
2S,4R)-1-((S)-2-(tert-butyl)-16-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)py
(
rimidin-2-yl)amino)-3-methoxyphenyl)piperazin-1-yl)-4,16-dioxo-7,10,13-trioxa-3-azahexadec
anoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (27). (white
1
solid, 17.1 mg, 63%) H NMR (500 MHz, CD OD) δ 8.91 (s, 1H), 8.39 (s, 1H), 8.04 (s, 1H), 7.66
3
(dd, J = 14.1, 7.8 Hz, 1H), 7.54 (s, 1H), 7.46 (d, J = 8.2 Hz, 2H), 7.44 – 7.35 (m, 3H), 7.29 (d, J =
8
4
3
.2 Hz, 1H), 6.73 (d, J = 2.1 Hz, 1H), 6.59 (d, J = 8.4 Hz, 1H), 4.64 (s, 1H), 4.59 – 4.51 (m, 2H),
.49 (s, 1H), 4.35 (d, J = 15.5 Hz, 1H), 3.88 (d, J = 11.1 Hz, 1H), 3.85 (s, 3H), 3.82 – 3.73 (m, 7H),
.72 – 3.65 (m, 2H), 3.63 – 3.54 (m, 8H), 3.44 (t, J = 7.0 Hz, 1H), 3.31 (s, 2H),3.24 (s, 2H), 2.71 (t,
J = 6.2 Hz, 2H), 2.60 – 2.50 (m, 1H), 2.47 (s, 3H), 2.49 – 2.40 (m, 1H), 2.25 - 2.18 (m, 1H), 2.11 –
+
2
.05 (m, 1H), 1.88 (d, J = 13.6 Hz, 6H), 1.03 (s, 9H). HRMS (ESI) calcd for C H ClN O PS
55 73 10 10
+
[
M+H] , 1131.4652; found, 1131.4651.
2S,4R)-1-((S)-2-(tert-butyl)-19-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)py
rimidin-2-yl)amino)-3-methoxyphenyl)piperazin-1-yl)-4,19-dioxo-7,10,13,16-tetraoxa-3-azanon
(
adecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(28).
1
(
white solid, 16.7 mg, 62%) H NMR (500 MHz, CD OD) δ 8.89 (s, 1H), 8.39 (s, 1H), 8.04 (s, 1H),
3
7
.66 (dd, J = 13.5, 8.1 Hz, 1H), 7.54 (s, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.42 - 7.35 (m, 3H), 7.31 (d, J
=
8.6 Hz, 1H), 6.73 (s, 1H), 6.58 (d, J = 7.5 Hz, 1H), 4.64 (s, 1H), 4.59 – 4.50 (m, 2H), 4.49 (s, 1H),
4
.35 (d, J = 15.5 Hz, 1H), 3.87 (d, J = 12.2 Hz, 1H), 3.85 (s, 3H), 3.82 – 3.72 (m, 7H), 3.73 - 3.65

(m, 2H), 3.64 – 3.53 (m, 12H), 3.31 (s, 2H),3.24 (s, 2H), 2.72 (t, J = 6.2 Hz, 2H), 2.59 – 2.51 (m,
1
6
H), 2.46 (s, 3H), 2.49 – 2.40 (m, 1H), 2.25 – 2.16 (m, 1H), 2.11 -2.04 (m, 1H), 1.88 (d, J = 13.6 Hz,
+
+
H), 1.03 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] , 1175.4915; found,
57
77
10 11
1
175.4918.
2S,4R)-1-((S)-2-(tert-butyl)-22-(4-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)py
rimidin-2-yl)amino)-3-methoxyphenyl)piperazin-1-yl)-4,22-dioxo-7,10,13,16,19-pentaoxa-3-aza
(
docosanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
(29).
1
(
white solid, 16.5 mg, 61%) H NMR (500 MHz, CD OD) δ 8.90 (s, 1H), 8.39 (s, 1H), 8.03 (s, 1H),
3
7
.67 (dd, J = 14.0, 6.9 Hz, 1H), 7.55 (s, 1H), 7.46 (d, J = 8.1 Hz, 2H), 7.44 – 7.34 (m, 3H), 7.30 (d,
J = 8.5 Hz, 1H), 6.74 (s, 1H), 6.59 (d, J = 8.1 Hz, 1H), 4.64 (s, 1H), 4.59 - 4.51 (m, 2H), 4.49 (s,
1
3
H), 4.35 (d, J = 15.6 Hz, 1H), 3.88 (d, J = 11.1 Hz, 1H), 3.85 (s, 3H), 3.82 - 3.4 (m, 7H), 3.74 –
.67 (m, 2H), 3.65 – 3.54 (m, 16H), 3.31 (s, 2H), 3.25 (s, 2H), 2.72 (t, J = 6.1 Hz, 2H), 2.60 – 2.51
(m, 1H), 2.47 (s, 3H), 2.50 – 2.40 (m, 1H), 2.25 – 2.17 (m, 1H), 2.11 - 2.04 (m, 1H), 1.88 (d, J =
+
+
1
1
3.6 Hz, 6H), 1.03 (s, 9H). HRMS (ESI) calcd for C59
219.5181.
H81ClN10O12PS [M+H] , 1219.5177; found,
(2S,4R)-1-((S)-2-(3-(2-(3-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)pyrimidi
n-2-yl)amino)-3-methoxyphenyl)piperidin-4-yl)amino)-3-oxopropoxy)ethoxy)propanamido)-3,
3
-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
1
(
30). (white solid, 6.1 mg, 28%) H NMR (500 MHz, CD
3
OD) δ 8.93 (s, 1H), 8.30 (dd, J = 7.5, 3.5
Hz, 1H), 8.12 (s, 1H), 7.73 – 7.66 (m, 2H), 7.63 – 7.59 (m, 1H), 7.47 – 7.45 (m, 2H), 7.43 – 7.38 (m,
3
–
H), 7.03 (d, J = 2.0 Hz, 1H), 6.84 (dd, J = 8.8, 2.4 Hz, 1H), 4.66 (s, 1H), 4.60 – 4.54 (m, 1H), 4.51
4.47 (m, 2H), 4.39 – 4.34 (m, 1H), 4.03 – 3.96 (m, 1H), 3.91 – 3.87 (m, 1H), 3.92 (s, 3H), 3.80 (dd,
J = 11.0, 3.8 Hz, 1H), 3.75 – 3.70 (m, 6H), 3.64 – 3.60 (m, 4H), 3.40 – 3.34 (m, 2H), 2.61 – 2.53 (m,
H), 2.49 – 2.44 (m, 3H), 2.47 (s, 3H), 2.25 – 2.20 (m, 1H), 2.16 – 2.05 (m, 3H), 1.88 (s, 3H), 1.85
1
+
+
(
s, 3H), 1.84 – 1.78 (m, 2H), 1.04 (s, 9H). HRMS (ESI) calcd for C H ClN O PS [M+H] :
54 71 10 9
1101.4547, found 1101.4548.
(2S,4R)-1-((S)-2-(tert-butyl)-16-((1-(4-((5-chloro-4-((2-(dimethylphosphoryl)phenyl)amino)p
yrimidin-2-yl)amino)-3-methoxyphenyl)piperidin-4-yl)amino)-4,16-dioxo-7,10,13-trioxa-3-aza
hexadecanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (31).
1
(
white solid, 13.2 mg, 58%) H NMR (500 MHz, CD OD) δ 8.94 (s, 1H), 8.29 (dd, J = 7.5, 3.5 Hz,
3
1
H), 8.12 (s, 1H), 7.73 – 7.67 (m, 2H), 7.62 – 7.58 (m, 1H), 7.47 – 7.44 (m, 2H), 7.43 – 7.37 (m,