Azo-protac: Novel light-controlled small-molecule tool for protein knockdown

Article abstract of DOI:10.1021/acs.jmedchem.9b02058

Reversibly altering endogenous protein levels are persistent issues. Herein, we designed photoswitchable azobenzene-proteolysis targeting chimeras (Azo-PROTACs) by including azobenzene moieties between ligands for the E3 ligase and the protein of interest

Full text of DOI:10.1021/acs.jmedchem.9b02058

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Article
Azo-PROTAC: novel light-controlled small-
molecule tool for protein knockdown
Yuhui Jin, Mengchen Lu, Yan Wang, Wenxin Shan, Xuanyu Wang, Qi-Dong You, and Zhengyu Jiang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02058 • Publication Date (Web): 10 Mar 2020
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Azo-PROTAC: novel light-controlled small-
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Yu-Hui Jin,a,b,‡ Meng-Chen Lu, a,b,‡ Yan Wang, a,b Wen-Xin Shan, a,b Xuan-Yu Wang,b Qi-Dong
You, a,b,* and Zheng-Yu Jiang, a,b,*
a
State Key Laboratory of Natural Medicines and Jiang Su Key Laboratory of Drug Design and
Optimization, China Pharmaceutical University, Nanjing 210009, China.
b
Department of Medicinal Chemistry, School of Pharmacy, China Pharmaceutical University,
Nanjing 210009, China.
KEYWORDS: protein knockdown; photoswitchable PROTAC; protein modifications; ubiquitin-
proteasome system.
ABSTRACT: Reversibly altering endogenous protein levels are persistent issues. Herein, we
designed photoswitchable Azo-PROTACs by including azobenzene moieties between ligands for
the E3 ligase and the protein of interest. Azo-PROTACs are light-controlled small-molecule tools
for protein knockdown in cells. The light-induced configuration change can switch the active state
to induce protein degradation activity, which can be reversely controlled by light exposure in intact
cells. We compared the protein degradation abilities of Azo-PROTACs with different
configurations and linker lengths. Using the stable form with the best degradation ability against
the BCR-ABL fusion and ABL proteins in myelogenous leukemia K562 cells, we showed that
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Azo-PROTAC combines the potent protein knockdown and facile cell uptake properties of the
small-molecule PROTAC with a reversible photo-switch ability, offering a promising chemical
knockdown strategy based on the light-induced reversible on/off property.
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Introduction
Conditional knockdown tools are fundamental to reveal specific protein functions in complex
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biological systems. Two main approaches are currently used to disrupt protein function : DNA-
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modifying methods can knockout proteins at the genome level and RNA interference can be used
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to knockdown protein expression by disrupting the mRNA level . Recently, several protein-
targeting technologies have been applied for inducing protein degradation. For example,
Proteolysis Targeting Chimeras (PROTACs) can recruit the E3 ligase to a protein of interest,
leading to its degradation via the ubiquitin-proteasome system.5-8 Low-molecular-weight
hydrophobic tags (HyTs) can promote labeled protein degradation since exposed hydrophobic
regions are a hallmark of unfolded proteins, signaling their elimination by the ubiquitin-
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proteasome system or autophagy. TRIM-Away is another protein-targeted knockdown strategy
that can harness the cellular protein degradation machinery to remove unmodified native proteins
within only minutes. However, these approaches cannot achieve the simultaneous potent
downregulation activity, facile cell uptake, and reversible control for protein knockdown that are
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required in research applications. Thus, how to find a reversible and controllable small-molecule
protein depletion method that acts exclusively at the protein level currently needs to uncover. Such
a method would not only allow for the depletion of proteins but also allow artificial termination of
the degradation process if necessary.
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Cellular activities are mediated by multiple biochemical pathways involving a network of
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biomolecules that communicate in a temporally and spatially well-defined fashion. Conventional
approaches to knocking out a specific biomolecule in cells and traditional inhibitor-based
therapeutics may have undesired effects because systemic application can affect untargeted
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tissue. Introducing a switchable element within molecular biology tool can allow reversible
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degradation in a spatiotemporal manner. A typical design strategy has emerged based on the
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fusion of a sensory domain with an effector domain to create a chimera such as photo-caging and
photosensitive degrons13-16. Here, we sought to develop an adjustable protein depletion method
based on controllable small-molecular devices. Diverse triggers17-21 or switch modules22-25 have
been introduced in various biofunctional molecules to regulate different targets. Among these
modules, using a photoswitch has the advantages of reversibility, speed, and facile modulation of
the energies involved.26 Over the past decade, various photoswitches with excellent
pharmacodynamic and pharmacokinetic properties, such as azobenzene (Azo), have been applied
to a wide range of biological targets. These studies include transmembrane proteins (G protein-
coupled receptors27-30, ion channels31-34, transporters35, 36, and receptor-linked enzymes ), soluble
proteins (kinases38-40, proteases41-43, and factors involved in epigenetic regulation44-46), lipid
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membranes , and nucleic acids48-50. Therefore, we designed photoswitchable Azo-PROTACs by
including azobenzene moieties between ligands for the E3 ligase and the protein of interest for
targeted and reversible protein degradation.
47
The ABL and BCR-ABL proteins are expressed in many cases of chronic myelogenous leukemia
(
CML)51, 52 and have thus emerged as pioneering targets for PROTAC-based research. In 2015,
Crews group first designed DAS-CRBN and BOS-CRBN PROTACs to knockdown the BCR-ABL
fusion protein and found that these PROTACs could effectively degrade BCR-ABL and ABL
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proteins even at extremely low levels. In 2016, Naito group developed PROTACs by conjugating
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Imatinib and Dasatinib to IAP ligands, which induced the reduction of BCR-ABL protein in
K562 cells. In 2019, Jiang group designed PROTACs targeting BCR-ABL which connected
Dasatinib and ligand of E3 ligase Von Hippel-Lindau (VHL).55 Meanwhile, Crews group
developed PROTACs that allosterically target BCR-ABL1 protein and recruit the VHL E3
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ubiquitin ligase, resulting in degradation of the fusion protein. ABL has also been implicated in
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the response to growth factors , cytokines , cell adhesion , DNA damage , oxidative stress ,
and other physiologically important signals. Therefore, excessive knockdown of ABL may also
affect the normal physiological function of the body. Accordingly, the aim of the present study
was to develop a controllable PROTAC targeting BCR-ABL as a conditional and reversible small-
molecule protein knockdown tool, which can further be used to explore the therapeutic potential
of BCR-ABL degraders.
RESULTS AND DISCUSSION
Design of Azo-PROTACs
Lenalidomide, a derivative of pomalidomide, is frequently used in PROTACs to interact with
the ubiquitin E3 ligase cereblon.62-64 Analysis of the X-ray crystal structure of cereblon in complex
with Lenalidomide (Figure 1A, PDB ID: 4TZ4) revealed that the Lenalidomide-binding pocket
was extremely small, and showed strong steric hindrance at the solvent boundary. Therefore, we
attached the Azo unit at the 3-position of the phenyl in Lenalidomide. This strategy assumed that
the configuration changes of the Azo unit might cause a great difference in degradation activity
after conjugating protein-targeting ligands at the other side of the linker.
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Dasatinib, targeting to ABL protein (Figure 1B), was the second-generation tyrosine kinase
inhibitor developed to treat CML patients with acquired resistance to imatinib, and it has ever been
used in DAS-CRBN PROTAC degrading BCR-ABL fusion protein. The docking results showed
that after conjugating dasatinib at the other side of the linker, only the trans-configuration was
combinative due to steric hindrance (Figure 1C). This implied that the trans and cis configurations
of Lenalidomide-Azo might show great differences in degradation activity after conjugating
dasatinib at the other side of the linker.
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Figure 1．A) Crystal structure of Human Cereblon in complex with DDB1 and Lenalidomide
(PDB ID: 4TZ4); B) Cocrystal structure of Dasatinib bound ABL protein (PDB ID:2GQG); C)
Docking simulation of Azo-PROTAC-4C-trans and Cereblon.
Screening for a linker of suitable length via immunoblotting
To find the suitable length of the linker, we first synthesized Azo-PROTAC-2C (Figure 2A),
the shortest of all dasatinib-Azo-lenalidomide trifunctional molecules, and tested its ability for
ABL and BCR-ABL degradation in cell culture via immunoblotting. For this purpose, we chose
the myelogenous leukemia cell line K562, which has been proven to express ABL and BCR-ABL
together with the E3 ligase CRBN.52, 54, 65 Indeed, Azo-PROTAC-2C induced a dramatic decrease
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in the levels of both ABL protein and the BCR-ABL fusion protein at 100 nM after 36-hours
treatment (Figure SI 2A). We then extended the linker between dasatinib and the Azo unit
(Scheme 1), and found that Azo-PROTAC-4C exhibited the best activity in degrading the BCR-
ABL fusion protein among the various PROTACs tested (Figure 2B).
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Scheme 1. Synthetic Route of Azo-PROTAC
O
O
H2N
Cl
OH
O
Br
O
a, b, c
, d
n
+
O
N
N
N
N
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Cl
HO
O
4a-e
n
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O
O
NH
OH
O
e
f, g
N
N
NH
N
N
N
O
Cl
Cl
O
O
n
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n
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O
O
O
O
NH
Cl
HN
O
Cl
HN
h
N
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NH
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S
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S
+
N
O
N
Cl
NH2
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H
N
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H
O
NH
n
O
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i, j
O
Cl
O
HN
S
N
N
NH
O
4a, 5a, 6a, 9a: n=1
4b, 5b, 6b, 9b: n=2
4c, 5c, 6c, 9c: n=3
d, 5d, 6d, 9d: n=4
e, 5e, 6e, 9e: n=5
N
NH
N
N
N
N
H
N
O
N
4
4
O
n
9a-e
O
Reagents and conditions: a) HCl, NaNO /H O, 1h; b) Cs CO , H O, rt, 3h; c) AcOH, H O, pH=4;
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d) K CO , DMF; e) LiOH, THF : H O = 1 : 1, r.t., overnight; f) (COCl) , DMF(cat), DCM; g)
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2
Lenalidomide, DIEA, THF, rt., 8h; h) Piperazine, DIEA, Dioxane, reflux, 24h; i) NaI, Acetone,
reflux, 24h; j) DIEA, DMF, 16h.
Photoisomerization kinetics
To explore whether Azo-PROTAC-4C can switch the configurations in a stable and controllable
manner, we investigated its photoisomerization kinetics characteristic through analysis of UV-
visible absorption spectra. The trans isomer of Azo-PROTAC-4C exhibited maximal absorption
(λmax) of the Azo unit at 361 nm. When exposed to UV-C light, the peak at 361 nm decreased by
varying degrees over time, indicating formation of the cis isomer (Figure 2E). The plot of
absorbance at 361 nm versus time indicated that 1-hour exposure was sufficient to convert 4C-
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trans into 4C-cis (Figure 2F). We next exposed 4C to white light, which transformed into the cis-
configuration within 4 hours (Figure 2G). In addition, as 4C is a T-type photo-switch, we further
investigated its spontaneous thermal relaxation (from cis to trans) in dark conditions. The
measured half-life of spontaneous thermal relaxation was approximately 620 min at 25°C, which
was sufficiently long to conduct the subsequent experiments. (Figure SI 1A). Moreover, 4C was
stable across five switching cycles (Figure SI 1C). We also tested the stability of 4C to reduction
by glutathione (GSH). 4C was incubated in 20 mM of GSH reduced in a dimethyl sulfoxide
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(DMSO)/phosphate-buffered saline (PBS) buffer (1:1) solution, and no obvious difference was
observed in 48 hours (Figure SI 1B), demonstrating its good stability.
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Figure 2. A) Structure of photoswitchable PROTACs; B) Western blot analysis of BCR-ABL and
ABL after treating different compounds at 100 nM for 24 hours; C) Cell proliferation assay of
Azo-PROTAC-4C-trans for K562 cell line; D) Cell viability assay of Azo-PROTAC-4C-trans for
A549, HCT116, MCF-7, HEK293T and K562 cell lines; E) UV−visible absorption spectroscopy
of 4C-trans and 4C-cis; F) UV−visible absorption spectroscopy of Azo-PROTAC-4C-trans
exposed to UV-C light at different time; G) UV−visible absorption spectroscopy of Azo-
PROTAC-4C-cis exposed to white light at different time points.
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Azo-PROTAC-4C acts selectively on a BCR-ABL-driven K562 cell line
We next evaluated the cellular effects of the PROTAC. Azo-PROTAC-4C showed great potency
against BCR-ABL-driven K562 cells with a half-maximal inhibitory concentration (IC ) of 68
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nM in a cell proliferation assay (Figure 2C) and a half-maximal response concentration (EC ) of
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8 nM in a cell viability assay (Figure 2D). Furthermore, 4C did not affect any of the non-BCR-
ABL-driven cell lines tested, such as A549 pulmonary carcinoma cells, HCT116 colorectal
carcinoma cells, and MCF-7 breast carcinoma cells (Figure 2D). Thus, this PROTAC retains
selective activity against the BCR-ABL-driven cell line K562.
Azo-PROTAC-4C can induce CRBN ubiquitination and proteasome-mediated
degradation of BCR-ABL and ABL
The mechanism of action of BCR-ABL protein degradation by Azo-PROTAC-4C was further
investigated. Addition of the ABL inhibitor Dasatinib effectively blocked the degradation of BCR-
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ABL and ABL proteins induced by Azo-PROTAC-4C (Figure 3C), confirming that the
degradation of ABL proteins by Azo-PROTAC-4C requires its binding to ABL proteins. To
establish the specificity of Azo-PROTAC-4C, we synthesized Azo-PROTAC-4C (-) (Scheme 2),
with N-methylated modification of the glutarimide, as a negative control in this experiment.
Compared to Azo-PROTAC-4C (+), no degradation of BCR-ABL and ABL proteins were
observed at different concentrations of Azo-ROTAC-4C (Figure 3A). Similarly, addition of
Lenalidomide also effectively hindered the degradation induced by Azo-PROTAC-4C for all
proteins (Figure 3D), indicating that degradation of BCR-ABL proteins by Azo-PROTAC-4C is
cereblon-dependent. The NEDD8-activating enzyme (NAE) inhibitor MLN4924 also completely
blocked the degradation of BCR-ABL proteins by Azo-PROTAC-4C (Figure 3B), indicating that
BCR-ABL and ABL protein degradation by Azo-PROTAC-4C depends upon NAE. These
mechanistic data constitute clear evidence that Azo-PROTAC-4C is a bona fide CRBN-dependent
ABL degrader.
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Scheme 2. Synthetic Route of Azo-PROTAC-4C（-）
O
O
O
O
OH
a
N
O
b
N
O
+
c, d, e
N
O
N
NH
N
NH
N
O
O
O
NH
NH
Cl
NH2
Boc
Boc
O
10
11
12
3
5c
O
O
O
Cl
NH
HN
O
N
N
f, g
S
N
N
NH
N
O
N
O
N
N
O
Cl
N
N
H
N
N
O
N
O
3
O
N
O
3
14
15
Reagents and conditions: a) (Boc) O, DMF, 6h; b) Cs CO , CH I, DMF, overnight; c) HCl/EA,
2
2
3
3
DCM, 4h; d) (COCl) , DMF(cat), DCM; e) DIEA, THF, r.t. , 8h; f) NaI, Acetone, reflux, 24h; g)
2
DIEA, DMF, 16h.
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Trans- and cis-configurations of Azo-PROTAC-4C show great difference in degrading
targeting proteins
Next, we tested the difference of ABL and BCR-ABL degradation activity between the 4C-cis
and 4C-trans configurations. For 4C-trans, slight degradation of the BCR-ABL fusion protein was
observed at a concentration of 25 nM, and remarkable reductions of BCR-ABL and ABL were
observed at a 4C concentration of 100 nM in a dose-effect evaluation (Figure 3G). However,
under the same condition, no notable degradation of BCR-ABL was observed with 4C-cis, even at
the highest concentration of 500 nM (Figure 3H). Time-course analysis to assess the temporal
degradation of ABL and BCR-ABL proteins showed that with 4C-trans, the ABL protein clearly
decreased after 4-h treatment, both BCR-ABL and ABL proteins were significantly degraded after
1
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0-h treatment, and more than 90% degradation of BCR-ABL was observed after 32-h treatment
(Figure 3E). By contrast, no noticeable reduction of BCR-ABL was observed until 32 h of
treatment with the 4C-cis group (Figure 3F). In addition, the results of RT-qPCR revealed that
Azo-PROTAC-4C did not affect the expression of ABL gene (Figure S4A & S4B in the SI).
Collectively, these results proved that the trans and cis configurations of 4C show substantially
different degradation activities and that only the trans-configuration was effective.
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Figure 3. A) Western blotting analysis of BCR-ABL and ABL protein after treating Azo-
PROTAC-4C (+)/(-); Western blotting analysis of BCR-ABL and ABL proteins after a 2-hour
pretreatment with a NEDD8-activating enzyme (NAE) inhibitor MLN4924MLN-4924 (B),
Dasatinib (C) or Lenalidomide (D), followed by a 24-hour treatment with Azo-PROTAC-4C at
2
50 nM in K562 cells; Time-course Western blot of Azo-PROTAC-4C-trans (E) or Azo-
PROTAC-4C-cis (F) at 250 nM PROTAC concentration; Western blot of Azo-PROTAC-4C-trans
G) and Azo-PROTAC-4C-trans (H) for K562 cell line after 24h-treatment in concentration
gradient.
(
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Active state of Azo-PROTAC switched by UV irradiation in live cells
Finally, to evaluate the reversible character of the photoswitchable Azo-PROTAC-4C, we
simulated the light control process. After treating K562 cells with 4C-trans for 24 hours, the cells
were transferred to fresh medium and divided into two groups: one group that was harvested and
exposed to UV-C light every 4 hours and the other group that was harvested for 0, 4, 8, 12, 16, and
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4 hours as a control. In the UV-irradiated group, the levels of ABL and BCR-ABL increased over
time (Figure 4B UV-Group), whereas in the white light control group, the BCR-ABL fusion
protein and ABL protein were maintained at low levels (Figure 4B VIS-Group). Similarly, we
tried to trigger Azo-PROTAC-4C-cis in K562 cells. After preincubating 4C-cis shielded from light，
the cells were transferred to fresh medium and exposed to visible light. Whereafter, BCR-ABL
was degraded with time (Figure 4A). These results strongly supported that the UV light-induced
configuration change of 4C could control its degradation activity, which can make the PROTAC-
induced protein knockdown a reversible process.
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Figure 4. A) Western blotting alaysis of light-triggerring process in K562 cells; B) Western
blotting alaysis of Azo-PROTAC-4C in white light (VIS-Group) and exposed to UV every 4 hours
(UV-Group) for K562 cells after 24h-treatment.
Conclusion
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We developed a novel small-molecule tool, Azo-PROTAC, to adjust the protein degradation
process simply using UV light. Utilizing the lenalidomide-Azo-dasatinib trifunctional system, we
demonstrated that the trans and cis isomers of Azo-PROTAC have marked differences in protein
degradation activity, and we could control the degradation of ABL and BCR-ABL proteins by
changing the configuration of Azo-PROTAC with UV-C light. On this basis, we further confirmed
that the active state of Azo-PROTAC can be switched by UV irradiation in live cells.While in the
process of preparing this manuscript, we became aware that Grews’ s Group had reported bistable
PhotoPROTACs.11, 66 Besides, many scientific studies undertaken similar investigations and have
achieved great processes.67-69 As is shown in these studies, the combination of PROTACs and
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photopharmacology has led us to develop the concept of photoswitchable, stable degraders with
potentially far reaching implications for manifold applications.11
EXPERIMENTAL SECTION
Chemistry. General Methods. All reactions were carried out under an atmosphere of dry
nitrogen. Glassware was oven-dried prior to use. Unless otherwise indicated, common reagents or
materials were obtained from commercial source and used without further purification. N, N-
Diisopropylethylamine (DIPEA) was obtained anhydrous by distillation over potassium hydroxide.
Tetrahydrofuran (THF), Dichloromethane (CH Cl ), acetonitrile (CH CN) and dimethylforamide
2
2
3
(DMF) was 99.5%, Extra Dry, with molecular sieves, Water≤50 ppm (by K.F.) obtained from
Energy Chemical. Flash column chromatography was performed using silica gel 60 (100-200
mesh). Analytical thin layer chromatography (TLC) was carried out on Huanghai Chromatography
silica gel plate HSGF254 indicator and visualized by UV. The purity (≥95%) of the compounds
was verified by the HPLC study performed on Agilent C18 (4.6 mm × 150 mm, 3.5 μm) column
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2
2
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4
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4
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5
5
5
5
5
5
5
5
5
5
6
1
using a mixture of solvent methanol/water at a flow rate of 1.0 mL/min. The H NMR spectra were
measured on a Bruker AV-300 instrument using deuterated solvents with tetramethylsilane (TMS)
as internal standard. ESI-mass and high-resolution mass spectra (HRMS) were recorded on a
Water Q-Tof micro mass spectrometer.
0
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methyl (E)-4-((4-hydroxyphenyl)diazenyl)benzoate (3). The suspension of compound 2
(11.54 g, 76.1 mmol) in water (135 ml) was cooled to 5℃ and HCl (15.8 ml, 190 mmol) was added
to the mixture. To the mixture was added the cooled (5℃) solution of sodium nitrate (5.03 g, 79.8
mmol) in water (35 ml) and stirred at 5℃. After 1h, the solution of phenol (1) (7.52 g, 79.9 mmol),
K CO (15.0 g, 108.5 mmol) in water (120 ml) dropwised to the reaction mixture for 10 min and
2
3
stirred at room temperature for 3h. After the reaction was completed, dilute acetic acid was added
to the mixture and adjusted to pH = 4.0. The product was filtered, and washed with water, methanol
1
to give compound 3 (15.65 g, 80.3%) in a brown solid. H NMR (300 MHz, DMSO-d ) δ 10.57 (s,
6
1
H), 8.18 – 8.09 (m, 2H), 7.96 – 7.80 (m, 4H), 6.97 (d, J = 8.4 Hz, 2H), 3.89 (s, 3H).
General Procedures for the Synthesis of Intermediates 4a-4e. Stir a solution of methyl (E)-
-((4-hydroxyphenyl)diazenyl)benzoate (3) (2.56 g, 10 mmol), 1-bromo-n-chlorohydrocarbon (12
4
mmol, 1.2 eq) and potassium carbonate (4.14 g, 30 mmol), in N,N-dimethylforamide (20 mL)
under room temperature for 20 hours. 200 mL water was added to the mixture with stirring. The
product was filtered, then washed with water and dry in 60℃ to give compound 4 in a yellow
solid.
1
methyl (E)-4-((4-(2-chloroethoxy)phenyl)diazenyl)benzoate (4a). H NMR (300 MHz,
DMSO-d ) δ 8.22 – 8.06 (m, 2H), 7.98 – 7.81 (m, 4H), 7.29 – 7.09 (m, 2H), 4.38 (t, J = 11.2 Hz,
6
2
H), 4.08 – 3.94 (m, 2H), 3.90 (s, 3H).
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methyl (E)-4-((4-(3-chloropropoxy)phenyl)diazenyl)benzoate (4b). H NMR (300 MHz,
Chloroform-d) δ 8.24 – 8.12 (m, 2H), 8.01 – 7.86 (m, 4H), 7.09 – 6.97 (m, 2H), 4.21 (t, J = 5.8
Hz, 2H), 3.95 (s, 3H), 3.77 (t, J = 6.2 Hz, 2H), 2.28 (p, J = 6.0 Hz, 2H).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
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3
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3
4
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5
5
5
5
5
6
0
1
2
3
4
5
6
7
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9
0
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2
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0
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2
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4
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1
methyl (E)-4-((4-(4-chlorobutoxy)phenyl)diazenyl)benzoate(4c). H NMR (300 MHz,
Chloroform-d) δ 8.17 (d, J = 8.3 Hz, 2H), 7.92 (dd, J = 11.4, 8.5 Hz, 4H), 7.01 (d, J = 8.5 Hz, 2H),
4
.09 (d, J = 5.2 Hz, 2H), 3.95 (s, 3H), 3.63 (d, J = 5.9 Hz, 2H), 2.01 (p, J = 2.9 Hz, 4H).
1
methyl (E)-4-((4-((5-chloropentyl)oxy)phenyl)diazenyl)benzoate(4d). H NMR (300 MHz,
Chloroform-d) δ 8.17 (d, J = 8.4 Hz, 2H), 7.99 – 7.85 (m, 4H), 7.06 – 6.96 (m, 2H), 4.07 (t, J =
.3 Hz, 2H), 3.95 (s, 3H), 3.59 (t, J = 6.6 Hz, 2H), 1.95 – 1.80 (m, 4H), 1.66 (ddt, J = 14.5, 9.7,
5.7 Hz, 2H).
6
1
methyl (E)-4-((4-((6-chlorohexyl)oxy)phenyl)diazenyl)benzoate (4e). H NMR (300 MHz,
DMSO-d ) δ 8.15 (d, J = 8.0 Hz, 2H), 7.93 (d, J = 8.2 Hz, 4H), 7.15 (d, J = 8.4 Hz, 2H), 4.17 –
6
4.05 (m, 2H), 3.90 (s, 3H), 3.65 (t, J = 5.8 Hz, 2H), 1.84 – 1.66 (m, 4H), 1.56 – 1.38 (m, 4H).
General Procedures for the Synthesis of Intermediates 5a-5e. Stir a solution of methyl
compound 4 (5 mmol), lithium hydroxide (600 mg, 25 mmol) in water and Tetrahydrofuran (1:1,
2
0 mL) under room temperature overnight. 200 mL water was added to the mixture with stirring,
and then dilute hydrochloric acid was added and adjusted to pH = 2. The product was filtered, then
washed with water and dry in 60℃ to give compound 5 in orange solid.
1
(E)-4-((4-(2-chloroethoxy)phenyl)diazenyl)benzoic acid (5a). H NMR (300 MHz, DMSO-d )
6
δ 13.40 – 12.80 (s, 1H), 8.32 – 8.10 (m, 2H), 8.06 – 7.81 (m, 4H), 7.29 – 7.09 (m, 2H), 4.38 (t, J
=
10.8 Hz, 2H), 4.12 – 3.92 (m, 2H).
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(
E)-4-((4-(3-chloropropoxy)phenyl)diazenyl)benzoic acid (5b). H NMR (300 MHz, DMSO-
d6) δ 8.06 (d, J = 8.1 Hz, 2H), 7.90 (d, J = 8.5 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H), 7.16 (d, J = 8.5
Hz, 2H), 5.76 (s, 2H), 4.21 (t, J = 6.1 Hz, 2H), 3.82 (t, J = 6.5 Hz, 2H), 2.22 (p, J = 6.4 Hz, 2H).
0
1
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4
5
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9
0
1
2
3
4
5
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8
9
0
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6
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0
1
(
E)-4-((4-(4-chlorobutoxy)phenyl)diazenyl)benzoic acid (5c). H NMR (300 MHz, DMSO-d )
6
δ 8.04 (d, J = 8.0 Hz, 2H), 7.90 (d, J = 8.6 Hz, 2H), 7.78 (d, J = 8.1 Hz, 2H), 7.14 (d, J = 8.5 Hz,
H), 4.13 (d, J = 5.2 Hz, 2H), 1.97 – 1.77 (m, 4H).
2
1
(
E)-4-((4-((5-chloropentyl)oxy)phenyl)diazenyl)benzoic acid (5d). H NMR (300 MHz,
DMSO-d ) δ 8.09 (d, J = 8.0 Hz, 2H), 7.86 (dd, J = 25.2, 8.2 Hz, 4H), 7.13 (d, J = 8.5 Hz, 2H),
6
4
.10 (t, J = 6.4 Hz, 2H), 3.68 (d, J = 13.2 Hz, 2H), 1.79 (dq, J = 12.5, 5.6 Hz, 4H), 1.57 (q, J = 7.9
Hz, 2H).
1
(
E)-4-((4-((6-chlorohexyl)oxy)phenyl)diazenyl)benzoic acid (5e). H NMR (300 MHz,
DMSO-d ) δ 8.04 (d, J = 7.9 Hz, 2H), 7.88 (d, J = 8.4 Hz, 2H), 7.77 (d, J = 7.9 Hz, 2H), 7.12 (d,
6
J = 8.5 Hz, 2H), 4.07 (d, J = 6.6 Hz, 2H), 3.64 (t, J = 6.7 Hz, 2H), 1.88 – 1.62 (m, 4H), 1.57 – 1.31
(
m, 4H).
General Procedures for the Synthesis of Intermediates 6a-6e. Stir a solution of compound 5
2.0 mmol), oxalyl chloride (338 μL, 4.0 mmol) in 10mL extra dry THF under room temperature,
(
then add one drop of N,N-Dimethylformamide as catalyst and stir for 30 min at room temperature.
The solvent was evaporated to dry, then the mixture was re-dissolved with 5mL extra dry THF and
added to the solution of lenalidomide and DIEA in extra dry THF dropwise with stirring. After the
reaction completely, the solvent was evaporated to dry, and the mixture was recrystallized with
methanol to get compound 6 in orange solid.
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(
E)-4-((4-(2-chloroethoxy)phenyl)diazenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
1
oxoisoindolin-4-yl)benzamide (6a). H NMR (300 MHz, DMSO-d ) δ 10.96 (s, 1H), 10.48 (s,
6
1H), 8.18 (d, J = 8.2 Hz, 2H), 7.96 (dt, J = 8.9, 4.2 Hz, 4H), 7.76 (d, J = 7.5 Hz, 1H), 7.65 – 7.55
1
1
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(
m, 2H), 7.26 – 7.14 (m, 2H), 5.15 (dd, J = 13.2, 5.2 Hz, 1H), 4.47 (d, J = 2.6 Hz, 2H), 4.40 (t, J
=
5.1 Hz, 2H), 4.00 (t, J = 5.1 Hz, 2H), 2.99 – 2.81 (m, 1H), 2.67 – 2.54 (m, 1H), 2.45 – 2.27 (m,
+
1
H), 2.07 – 1.93 (m, 1H). ESI-HRMS: calcd for C H ClN O , 545.1466; m/z: [M] = 546.15387.
28 24
5
5
(E)-4-((4-(3-chloropropoxy)phenyl)diazenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
1
oxoisoindolin-4-yl)benzamide (6b). H NMR (300 MHz, DMSO-d ) δ 10.99 (s, 1H), 10.51 (s,
6
1
H), 8.19 (d, J = 8.2 Hz, 2H), 7.97 (dd, J = 8.4, 5.8 Hz, 4H), 7.77 (d, J = 7.5 Hz, 1H), 7.60 (dt, J
= 15.2, 7.5 Hz, 2H), 7.19 (d, J = 8.6 Hz, 2H), 5.17 (dd, J = 13.2, 5.0 Hz, 1H), 4.54 – 4.38 (m, 2H),
4
2
.23 (t, J = 6.0 Hz, 2H), 3.83 (t, J = 6.5 Hz, 2H), 2.99 – 2.82 (m, 1H), 2.64 – 2.54 (m, 1H), 2.45 –
.34 (m, 1H), 2.23 (p, J = 6.5 Hz, 2H), 2.04 – 1.97 (m, 1H). ESI-HRMS: calcd for C H ClN O ,
2
9
26
5
5
+
559.1622; m/z: [M] = 560.16952.
(E)-4-((4-(4-chlorobutoxy)phenyl)diazenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
1
oxoisoindolin-4-yl)benzamide (6c). H NMR (300 MHz, DMSO- d ) δ 11.01 (s, 1H), 10.53 (s,
6
1
H), 8.15 (dd, J = 18.7, 8.2 Hz, 2H), 7.94 (dd, J = 11.3, 8.3 Hz, 4H), 7.77 (d, J = 7.4 Hz, 1H), 7.60
dt, J = 14.9, 7.4 Hz, 2H), 7.16 (dd, J = 9.2, 2.8 Hz, 2H), 5.17 (dd, J = 13.5, 4.9 Hz, 1H), 4.47 (s,
H), 4.15 (s, 2H), 3.73 (d, J = 6.5 Hz, 2H), 2.99 – 2.85 (m, 1H), 2.66 – 2.55 (m, 1H), 2.48 – 2.33
m, 1H), 2.07 – 1.98 (m, 1H), 1.96 – 1.79 (m, 4H). MS(ESI): calcd for C H ClN O , 573.1779;
(
2
(
30 28
5
5
+
m/z: [M] = 574.18517.
(E)-4-((4-((5-chloropentyl)oxy)phenyl)diazenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
1
oxoisoindolin-4-yl)benzamide (6d). H NMR (300 MHz, DMSO- d ) δ 10.90 (s, 1H), 10.42 (s,
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H), 8.09 (d, J = 8.2 Hz, 2H), 7.87 (t, J = 7.9 Hz, 4H), 7.67 (d, J = 7.5 Hz, 1H), 7.51 (dt, J = 15.1,
.5 Hz, 2H), 7.07 (d, J = 8.5 Hz, 2H), 5.07 (dd, J = 13.1, 5.2 Hz, 1H), 4.43 – 4.33 (m, 2H), 4.03 (t,
J = 6.3 Hz, 2H), 3.59 (t, J = 6.5 Hz, 2H), 2.90 – 2.80 (m, 1H), 2.62 – 2.48 (m, 1H), 2.46 – 2.32 (m,
0
1
2
3
4
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7
8
9
0
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H), 1.99 – 1.87 (m, 1H), 1.70 (d, J = 9.7 Hz, 4H), 1.47 (t, J = 7.8 Hz, 2H). ESI-HRMS: calcd for
+
C H ClN O , 587.1935; m/z: [M] = 588.20082.
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30
5
5
(E)-4-((4-((6-chlorohexyl)oxy)phenyl)diazenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
1
oxoisoindolin-4-yl)benzamide (6e). H NMR (300 MHz, DMSO-d ) δ 11.00 (s, 1H), 10.52 (s,
6
1
7
H), 8.18 (d, J = 8.3 Hz, 2H), 7.95 (t, J = 8.2 Hz, 4H), 7.76 (d, J = 7.4 Hz, 1H), 7.60 (dt, J = 15.0,
.4 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 5.16 (dd, J = 13.1, 5.1 Hz, 1H), 4.54 – 4.40 (m, 2H), 4.10 (t,
J = 6.5 Hz, 2H), 3.65 (t, J = 6.6 Hz, 2H), 2.96 – 2.84 (m, 1H), 2.68 – 2.52 (m, 1H), 2.46 – 2.33 (m,
1
H), 2.04 – 1.98 (m, 1H), 1.88 – 1.62 (m, 4H), 1.47 (d, J = 7.1 Hz, 4H). ESI-HRMS: calcd for
+
C H ClN O , 601.2092; m/z: [M] = 602.21647.
3
2
32
5
5
N-(2-Chloro-6-methylphenyl)-2-(2-methyl-6-(piperazin-1-yl)pyrimidin-4-
ylamino)thiazole-5-carboxamide (8). 2-(6-Chloro-2-methylpyrimidin-4-ylamino)-N-(2-chloro-
6
-methylphenyl)thiazole-5-carboxamide, 7 (1.00 g, 2.54 mmol), piperazine (2.19 g, 25.4 mmol),
and N,N-diisopropylethylamine (0.84 mL, 5.07 mmol) were dissolved in 30 mL of extra dry 1,4-
dioxane and refluxed overnight. The solvent was stripped and the residue was triturated several
times with water/MeOH, MeOH/ether, and then ether. The white solid was dried under high
1
vacuum to give precursor 4 (0.82 g, 73%). H NMR (300 MHz, DMSO-d6) δ 9.88 (s, 1H), 8.21
(s, 1H), 7.40 (d, J = 7.3 Hz, 1H), 7.26 (d, J = 8.2 Hz, 2H), 6.02 (s, 1H), 3.48 – 3.37 (m, 4H), 2.78
–
2.70 (m, 4H), 2.40 (s, 3H), 2.23 (s, 3H).
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General Procedures for the Synthesis of Intermediates 9a-9e. To a solution of 6 (0.11 mmol)
in Acetone (10 ml) was added NaI (83mg, 0.55 mmol). The reaction mixture was stirred at reflux
temperature for 24 h, then remove Acetone by vacuum. Add 8 (43 mg, 0.10 mmol), DIEA (96 μL,
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.57 mmol) in DMF (1 ml) and the resulting solution stirred for 16 h at 80 °C. Then the mixture
was cooled down to rt. The solvent was evaporated, and the residue subjected to Prep TLC
purification (MeOH/DCM from1/50 to 1/25) to give the desired product as orange solid.
(E)-N-(2-chloro-6-methylphenyl)-2-((6-(4-(2-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)carbamoyl)phenyl)diazenyl)phenoxy)ethyl)piperazin-1-yl)-2-
1
methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (9a). H NMR (300 MHz, DMSO-d ) δ
6
11.47 (s, 1H), 10.99 (s, 1H), 10.51 (s, 1H), 9.87 (s, 1H), 8.26 – 8.13 (m, 3H), 8.06 – 7.85 (m, 4H),
7
.75 (d, J = 7.4 Hz, 1H), 7.68 – 7.46 (m, 2H), 7.38 (d, J = 7.4 Hz, 1H), 7.32 – 7.15 (m, 4H), 6.05
s, 1H), 5.15 (dd, J = 13.2, 5.2 Hz, 1H), 4.43 (d, J = 16.4 Hz, 2H), 4.25 (d, J = 5.1 Hz, 2H), 3.59 –
3.46 (m, 4H), 2.99 – 2.88 (m, 1H), 2.77 – 2.67 (m, 1H), 2.63 – 2.53 (m, 4H), 2.40 (s, 3H), 2.36 –
.30 (m, 1H), 2.22 (s, 3H), 2.06 – 1.91 (m, 1H). ESI-HRMS: calcd for C H ClN O S, 952.2994;
(
2
4
8
45
12
6
+
m/z: [M] = 953.3036. Purity: 96.77% by HPLC (MeCN/H O =70:30).
2
(E)-N-(2-chloro-6-methylphenyl)-2-((6-(4-(3-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)carbamoyl)phenyl)diazenyl)phenoxy)propyl)piperazin-1-yl)-2-
1
methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (9b). H NMR (300 MHz, DMSO-d ) δ
6
1
1.47 (s, 1H), 10.99 (s, 1H), 10.51 (s, 1H), 9.87 (s, 1H), 8.25 – 8.11 (m, 3H), 7.95 (dd, J = 8.5, 5.9
Hz, 4H), 7.75 (d, J = 7.4 Hz, 1H), 7.60 (dd, J = 13.7, 7.4 Hz, 2H), 7.38 (dd, J = 7.3, 2.1 Hz, 1H),
7
.31 – 7.12 (m, 4H), 6.04 (s, 1H), 5.15 (dd, J = 13.2, 5.2 Hz, 1H), 4.46 (s, 2H), 4.23 – 4.12 (m,
2
H), 3.59 – 3.46 (m, 4H), 2.96 – 2.85 (m, 1H), 2.63 – 2.60 (m, 1H), 2.57 – 2.49 (m, 4H), 2.39 (s,
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H), 2.32 – 2.25 (m, 1H), 2.22 (s, 3H), 2.07 – 1.88 (m, 3H). ESI-HRMS: calcd for C H ClN O S,
4
9
47
12
6
+
66.3251; m/z [M] = 967.3325. Purity: 98.85% by HPLC (MeCN/H O =70:30).
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(E)-N-(2-chloro-6-methylphenyl)-2-((6-(4-(4-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1-
0
1
2
3
4
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oxoisoindolin-4-yl)carbamoyl)phenyl)diazenyl)phenoxy)butyl)piperazin-1-yl)-2-
1
methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (9c). H NMR (300 MHz, DMSO-d ) δ
6
1
1.41 (s, 1H), 10.96 (s, 1H), 10.44 (d, J = 15.3 Hz, 1H), 9.83 (s, 1H), 8.30 – 8.09 (m, 3H), 7.95 (t,
J = 7.4 Hz, 4H), 7.80 (dd, J = 18.2, 7.5 Hz, 1H), 7.58 (h, J = 8.5, 7.9 Hz, 3H), 7.38 (d, J = 7.4 Hz,
1
H), 7.25 (d, J = 8.2 Hz, 2H), 7.16 (d, J = 8.8 Hz, 2H), 6.05 (d, J = 6.7 Hz, 1H), 5.15 (dd, J = 13.6,
.2 Hz, 1H), 4.50 (d, J = 17.5 Hz, 2H), 4.13 (d, J = 6.6 Hz, 2H), 3.55 – 3.49 (m, 4H), 3.05 – 2.79
5
(m, 1H), 2.66 – 2.54 (m, 1H), 2.44 (s, 3H), 2.43 – 2.33 (m, 4H), 2.32 – 2.26 (m, 1H), 2.25 – 2.19
(s, 3H), 2.10 – 1.95 (m, 1H), 1.89 – 1.71 (m, 2H), 1.63 (t, J = 7.9 Hz, 2H). MS(ESI): calcd for
+
C H ClN O S, 980.3307; m/z: [M] = 981.33728. Purity: 96.20% by HPLC (MeCN/H O
5
0
49
12
6
2
=70:30).
(E)-N-(2-chloro-6-methylphenyl)-2-((6-(4-(5-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)carbamoyl)phenyl)diazenyl)phenoxy)pentyl)piperazin-1-yl)-2-
1
methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (9d). H NMR (300 MHz, DMSO-d ) δ
6
1
4
1.46 (s, 1H), 10.99 (s, 1H), 10.51 (s, 1H), 9.86 (s, 1H), 8.25 – 8.12 (m, 3H), 7.95 (t, J = 7.7 Hz,
H), 7.75 (d, J = 7.4 Hz, 1H), 7.60 (dd, J = 13.6, 7.2 Hz, 2H), 7.38 (d, J = 7.4 Hz, 1H), 7.25 (d, J
=
7.9 Hz, 2H), 7.15 (d, J = 8.6 Hz, 2H), 6.03 (s, 1H), 5.15 (dd, J = 12.2, 5.2 Hz, 1H), 4.50 – 4.37
(m, 2H), 4.09 (d, J = 6.7 Hz, 2H), 3.60 – 3.41 (m, 4H), 2.98 – 2.82 (m, 1H), 2.64 – 2.53 (m, 1H),
2
1
.42 (s, 3H), 2.41 – 2.34(m, 4H), 2.33 – 2.27 (m, 1H), 2.22 (s, 3H), 2.01 – 1.94 (m, 1H), 1.85 –
.70 (m, 2H), 1.57 – 1.43 (s, 4H). MS(ESI): calcd for C H ClN O S 994.3464; m/z:
5
1
51
12
6
+
[M] =995.3535. Purity: 99.13% by HPLC (MeCN/H O =70:30).
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(
E)-N-(2-chloro-6-methylphenyl)-2-((6-(4-(6-(4-((4-((2-(2,6-dioxopiperidin-3-yl)-1-
oxoisoindolin-4-yl)carbamoyl)phenyl)diazenyl)phenoxy)hexyl)piperazin-1-yl)-2-
1
methylpyrimidin-4-yl)amino)thiazole-5-carboxamide (9e). H NMR (300 MHz, DMSO-d ) δ
6
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
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3
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5
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5
5
5
6
0
1
2
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4
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9
0
1
2
3
4
5
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8
9
0
1
2
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8
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0
1
2
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4
5
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0
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8
9
0
1
4
1.39 (s, 1H), 10.92 (s, 1H), 10.41 (s, 1H), 9.80 (s, 1H), 8.25 – 8.02 (m, 3H), 7.90 (t, J = 8.1 Hz,
H), 7.73 (p, J = 10.2, 8.7 Hz, 1H), 7.54 (dt, J = 15.6, 7.8 Hz, 2H), 7.34 (d, J = 7.3 Hz, 1H), 7.20
(
d, J = 8.3 Hz, 2H), 7.09 (d, J = 8.6 Hz, 2H), 6.00 (s, 1H), 5.11 (dd, J = 13.1, 5.2 Hz, 1H), 4.50 –
.34 (m, 2H), 4.04 (t, J = 6.4 Hz, 2H), 3.46 (d, J = 6.7 Hz, 4H), 2.96 – 2.78 (m, 1H), 2.61 – 2.53
m, 1H), 2.41 – 2.31 (m, 4H), 2.26 (s, 3H), 2.21 – 2.11 (m, 4H), 2.01 – 1.87 (m, 1H), 1.79 – 1.64
m, 2H), 1.42 (q, J = 7.1 Hz, 4H), 1.34 (d, J = 9.6 Hz, 2H). MS (ESI):calcd for C H ClN O S
4
(
(
52 53
12
6
+
1008.3620; m/z: [M] = 1009.36956. Purity: 95.51% by HPLC (MeCN/H O =70:30).
2
1
Tert-butyl (2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate (11). H NMR (300
MHz, DMSO-d ) δ 11.04 (s, 1H), 9.26 (s, 1H), 7.80 (dd, J = 6.3, 2.4 Hz, 1H), 7.51 – 7.41 (m, 2H),
6
5.16 (dd, J = 13.2, 5.0 Hz, 1H), 4.51 – 4.32 (m, 2H), 2.94 (d, J = 7.5 Hz, 1H), 2.68 (s, 1H), 2.38
(
tt, J = 13.2, 6.7 Hz, 1H), 2.08 (s, 1H), 1.52 (s, 9H).
1
Tert-butyl (2-(1-methyl-2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)carbamate (12). H
NMR (300 MHz, DMSO-d ) δ 9.24 (s, 1H), 7.78 (s, 1H), 7.48 (s, 2H), 5.21 (dd, J = 12.9, 4.3 Hz,
6
1
1
H), 4.56 – 4.25 (m, 2H), 3.04 (s, 3H), 2.98 (s, 1H), 2.79 (d, J = 18.5 Hz, 1H), 2.48 – 2.29 (m,
H), 2.05 (d, J = 19.3 Hz, 1H), 1.51 (s, 9H).
(E)-4-((4-(4-chlorobutoxy)phenyl)diazenyl)-N-(2-(2,6-dioxopiperidin-3-yl)-1-
1
oxoisoindolin-4-yl)benzamide (13). H NMR (300 MHz, DMSO-d ) δ 10.59 (s, 1H), 8.21 (d, J =
6
8
.2 Hz, 2H), 7.99 (t, J = 8.1 Hz, 4H), 7.79 (d, J = 7.5 Hz, 1H), 7.71 – 7.60 (m, 2H), 7.20 (d, J =
.6 Hz, 2H), 5.28 (dd, J = 13.4, 5.1 Hz, 1H), 4.60 – 4.38 (m, 2H), 4.19 (d, J = 5.6 Hz, 2H), 3.76
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(
q, J = 10.5, 8.0 Hz, 2H), 2.97 (d, J = 5.0 Hz, 1H), 2.77 (d, J = 16.8 Hz, 1H), 2.43 (q, J = 12.2, 8.3
Hz, 1H), 2.10 – 2.00 (m, 1H), 1.97 – 1.85 (m, 4H). ESI-HRMS: calcd for C H ClN O , 587.1935;
3
1
30
5
5
+
m/z: [M] = 588.20082.
0
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(E)-N-(2-chloro-6-methylphenyl)-2-((2-methyl-6-(4-(4-(4-((4-((2-(1-methyl-2,6-
dioxopiperidin-3-yl)-1-oxoisoindolin-4-
yl)carbamoyl)phenyl)diazenyl)phenoxy)butyl)piperazin-1-yl)pyrimidin-4-
yl)amino)thiazole-5-carboxamide (14). 1H NMR (300 MHz, DMSO-d ) δ 11.53 (s, 1H), 10.58
6
(
s, 1H), 9.92 (s, 1H), 8.29 – 8.17 (m, 3H), 8.00 (dd, J = 8.6, 6.5 Hz, 5H), 7.79 (d, J = 7.5 Hz, 1H),
.71 – 7.60 (m, 2H), 7.43 (dd, J = 7.3, 2.3 Hz, 1H), 7.30 (t, J = 4.8 Hz, 2H), 7.20 (d, J = 8.7 Hz,
2H), 6.09 (s, 1H), 5.28 (dd, J = 13.5, 5.1 Hz, 1H), 4.61 – 4.39 (m, 2H), 4.18 (t, J = 6.4 Hz, 2H),
7
3
–
.56 (s, 4H), 3.04 (s, 4H), 2.77 (d, J = 16.2 Hz, 1H), 2.47 (s, 3H), 2.44 (s, 4H), 2.27 (s, 3H), 2.10
2.01 (m, 1H), 1.83 (d, J = 7.6 Hz, 2H), 1.69 (s, 2H). ESI-HRMS: calcd for C H ClN O S,
5
1
51
12
6
+
994.3464; m/z: [M] = 995.35365. Purity: 96.28 % by HPLC (MeCN/H O =80:20).
2
ASSOCIATED CONTENT
Supporting Information.
SI-Experimental section, NMR spectra and HPLC traces (PDF)
SI-Comma delimited.csv (CSV)
AUTHOR INFORMATION
Corresponding Author
*
youqd@163.com
* jiangzhengyucpu@163.com
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Author Contributions
‡Yu-Hui Jin and Meng-Chen Lu contributed equally.
Notes
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The authors declare no competing financial interests.
Funding Sources
This study was supported by projects, [81773581], [81773639], [81803363] and [81930100] of
the National Natural Science Foundation of China; [BK20160746] of the Natural Science
Foundation of Jiangsu Province of China; [CPU2018GY02] of Double First Class Innovation
Team of China Pharmaceutical University; National Science & Technology Major Project ’Key
New Drug Creation and Manufacturing Program’, China(No: 2018ZX09711002); China
Postdoctoral Science Foundation-funded Project (1600010006 and 2018T110576);The Program
for Outstanding Scientific and Technological Innovation Team of Jiangsu Higher Education; the
“
Qing Lan” Project of Jiangsu Province and the Young Elite Scientists Sponsorship Program by
CAST.
ABBREVIATIONS
PROTACs, Proteolysis-Targeting Chimeras; CRISPR, Clustered regularly interspaced short
palindromic repeats; HyTs, Hydrophobic Tags.
REFERENCES
1
.
Daniel, K.; Icha, J.; Horenburg, C.; Muller, D.; Norden, C.; Mansfeld, J. Conditional
Control of Fluorescent Protein Degradation by an Auxin-Dependent Nanobody. Nat. Commun.
018, 9, 3297.
2
ACS Paragon Plus Environment
2
5

Journal of Medicinal Chemistry
Page 26 of 36
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
2
.
Clift, D.; McEwan, W. A.; Labzin, L. I.; Konieczny, V.; Mogessie, B.; James, L. C.; Schuh,
M. A Method for the Acute and Rapid Degradation of Endogenous Proteins. Cell 2017, 171, 1692-
1706.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
3. Fan, X.; Jin, W. Y.; Lu, J.; Wang, J.; Wang, Y. T. Rapid and Reversible Knockdown of
Endogenous Proteins by Peptide-Directed Lysosomal Degradation. Nat. Neurosci. 2014, 17, 471-
4
80.
4
.
Elbashir, S. M.; Harborth, J.; Lendeckel, W.; Yalcin, A.; Weber, K.; Tuschl, T. Duplexes
of 21-Nucleotide RNAs Mediate RNA Interference in Cultured Mammalian Cells. Nature 2001,
11, 494-498.
4
5
.
Lai, A. C.; Crews, C. M. Induced Protein Degradation: An Emerging Drug Discovery
Paradigm. Nat. Rev. Drug. Discov. 2017, 16, 101-114.
6. Sun, Y.; Zhao, X.; Ding, N.; Gao, H.; Wu, Y.; Yang, Y.; Zhao, M.; Hwang, J.; Song, Y.;
Liu, W.; Rao, Y. PROTAC-Induced BTK Degradation as a Novel Therapy for Mutated BTK
C481s Induced Ibrutinib-Resistant B-Cell Malignancies. Cell. Res. 2018, 28, 779-781.
7
.
Gadd, M. S.; Testa, A.; Lucas, X.; Chan, K. H.; Chen, W.; Lamont, D. J.; Zengerle, M.;
Ciulli, A. Structural Basis of PROTAC Cooperative Recognition for Selective Protein
Degradation. Nat. Chem. Biol. 2017, 13, 514-521.
8
.
Powell, C. E.; Gao, Y.; Tan, L.; Donovan, K. A.; Nowak, R. P.; Loehr, A.; Bahcall, M.;
Fischer, E. S.; Janne, P. A.; George, R. E.; Gray, N. S. Chemically Induced Degradation of
Anaplastic Lymphoma Kinase (ALK). J. Med. Chem. 2018, 61, 4249-4255.
ACS Paragon Plus Environment
2
6

Page 27 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
9
.
Neklesa, T. K.; Tae, H. S.; Schneekloth, A. R.; Stulberg, M. J.; Corson, T. W.; Sundberg,
T. B.; Raina, K.; Holley, S. A.; Crews, C. M. Small-Molecule Hydrophobic Tagging-Induced
Degradation of Halotag Fusion Proteins. Nat. Chem. Biol. 2011, 7, 538-543.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
10. Beharry, A. A.; Woolley, G. A. Azobenzene Photoswitches for Biomolecules. Chem. Soc.
Rev. 2011, 40, 4422-4437.
1
1. Pfaff, P.; Samarasinghe, K. T. G.; Crews, C. M.; Carreira, E. M. Reversible Spatiotemporal
Control of Induced Protein Degradation by Bistable Photoprotacs. ACS. Cent. Sci. 2019, 5, 1682-
690.
1
1
2. Delacour, Q.; Li, C.; Plamont, M. A.; Billon-Denis, E.; Aujard, I.; Le Saux, T.; Jullien, L.;
Gautier, A. Light-Activated Proteolysis for the Spatiotemporal Control of Proteins. ACS. Chem.
Biol. 2015, 10, 1643-1647.
13. Mills, E.; Truong, K. Photoswitchable Protein Degradation: A Generalizable Control
Module for Cellular Function? Chem. Biol. 2013, 20, 458-460.
1
4. Renicke, C.; Schuster, D.; Usherenko, S.; Essen, L. O.; Taxis, C. A LOV2 Domain-Based
Optogenetic Tool to Control Protein Degradation and Cellular Function. Chem. Biol. 2013, 20,
619-626.
1
5. Scheffer, J.; Hasenjager, S.; Taxis, C. Degradation of Integral Membrane Proteins
Modified with the Photosensitive Degron Module Requires the Cytosolic Endoplasmic Reticulum-
Associated Degradation Pathway. Mol. Biol. Cell. 2019, 30, 2558-2570.
16. Taxis, C. Development of a Synthetic Switch to Control Protein Stability in Eukaryotic
Cells with Light. Methods. Mol. Biol. 2017, 1596, 241-255.
ACS Paragon Plus Environment
2
7

Journal of Medicinal Chemistry
Page 28 of 36
1
2
3
4
5
6
7
8
9
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
1
7. Akinboye, E. S.; Rosen, M. D.; Denmeade, S. R.; Kwabi-Addo, B.; Bakare, O. Design,
Synthesis, and Evaluation of Ph-Dependent Hydrolyzable Emetine Analogues as Treatment for
Prostate Cancer. J. Med. Chem. 2012, 55, 7450-7459.
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
18. Hansen, M. J.; Feringa, F. M.; Kobauri, P.; Szymanski, W.; Medema, R. H.; Feringa, B. L.
Photoactivation of MDM2 Inhibitors: Controlling Protein-Protein Interaction with Light. J. Am.
Chem. Soc. 2018, 140, 13136-13141.
1
9. Legigan, T.; Clarhaut, J.; Renoux, B.; Tranoy-Opalinski, I.; Monvoisin, A.; Berjeaud, J.
M.; Guilhot, F.; Papot, S. Synthesis and Antitumor Efficacy of a Beta-Glucuronidase-Responsive
Albumin-Binding Prodrug of Doxorubicin. J. Med. Chem. 2012, 55, 4516-4520.
2
0. Sakabe, M.; Asanuma, D.; Kamiya, M.; Iwatate, R. J.; Hanaoka, K.; Terai, T.; Nagano, T.;
Urano, Y. Rational Design of Highly Sensitive Fluorescence Probes for Protease and Glycosidase
Based on Precisely Controlled Spirocyclization. J. Am. Chem. Soc. 2013, 135, 409-414.
2
1. Wu, X.; Sun, X.; Guo, Z.; Tang, J.; Shen, Y.; James, T. D.; Tian, H.; Zhu, W. In Vivo and
in Situ Tracking Cancer Chemotherapy by Highly Photostable Nir Fluorescent Theranostic
Prodrug. J. Am. Chem. Soc. 2014, 136, 3579-3588.
22. Jia, C.; Migliore, A.; Xin, N.; Huang, S.; Wang, J.; Yang, Q.; Wang, S.; Chen, H.; Wang,
D.; Feng, B.; Liu, Z.; Zhang, G.; Qu, D. H.; Tian, H.; Ratner, M. A.; Xu, H. Q.; Nitzan, A.; Guo,
X. Covalently Bonded Single-Molecule Junctions with Stable and Reversible Photoswitched
Conductivity. Science 2016, 352, 1443-1445.
ACS Paragon Plus Environment
2
8

Page 29 of 36
Journal of Medicinal Chemistry
1
2
3
4
5
6
7
8
9
2
3. Mogaki, R.; Okuro, K.; Aida, T. Adhesive Photoswitch: Selective Photochemical
Modulation of Enzymes under Physiological Conditions. J. Am. Chem. Soc. 2017, 139, 10072-
10078.
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
24. Mourot, A.; Fehrentz, T.; Le Feuvre, Y.; Smith, C. M.; Herold, C.; Dalkara, D.; Nagy, F.;
Trauner, D.; Kramer, R. H. Rapid Optical Control of Nociception with an Ion-Channel
Photoswitch. Nat. Methods. 2012, 9, 396-402.
2
5. Velema, W. A.; Szymanski, W.; Feringa, B. L. Photopharmacology: Beyond Proof of
Principle. J. Am. Chem. Soc. 2014, 136, 2178-2191.
2
6. Hull, K.; Morstein, J.; Trauner, D. In Vivo Photopharmacology. Chem. Rev. 2018, 118,
1
0710-10747.
2
7. Agnetta, L.; Kauk, M.; Canizal, M. C. A.; Messerer, R.; Holzgrabe, U.; Hoffmann, C.;
Decker, M. A Photoswitchable Dualsteric Ligand Controlling Receptor Efficacy. Angew. Chem.
Int. Ed. Engl. 2017, 56, 7282-7287.
2
8. Broichhagen, J.; Podewin, T.; Meyer-Berg, H.; von Ohlen, Y.; Johnston, N. R.; Jones, B.
J.; Bloom, S. R.; Rutter, G. A.; Hoffmann-Roder, A.; Hodson, D. J.; Trauner, D. Optical Control
of Insulin Secretion Using an Incretin Switch. Angew. Chem. Int. Ed. Engl. 2015, 54, 15565-15569.
2
9. Donthamsetti, P. C.; Winter, N.; Schonberger, M.; Levitz, J.; Stanley, C.; Javitch, J. A.;
Isacoff, E. Y.; Trauner, D. Optical Control of Dopamine Receptors Using a Photoswitchable
Tethered Inverse Agonist. J. Am. Chem. Soc. 2017, 139, 18522-18535.
30. Westphal, M. V.; Schafroth, M. A.; Sarott, R. C.; Imhof, M. A.; Bold, C. P.; Leippe, P.;
Dhopeshwarkar, A.; Grandner, J. M.; Katritch, V.; Mackie, K.; Trauner, D.; Carreira, E. M.; Frank,
ACS Paragon Plus Environment
2
9