Development of small-molecule BRD4 degraders based on pyrrolopyridone derivative

Article abstract of DOI:10.1016/j.bioorg.2020.103817

Bromodomain-containing protein 4 (BRD4) plays a crucial role in the epigenetic regulation of gene transcription and some BRD4 inhibitors have been advanced to clinical trials. Nevertheless, the clinical application of BRD4 inhibitors could be limited by drug resistance. As an alternative strategy, the emerging Proteolysis Targeting Chimeras (PROTACs) technology has the potential to overcome the drug resistance of traditional small-molecule drugs. Based on PROTACs approaches, several BRD4 degraders were developed and have been proved to degrade BRD4 protein and inhibit tumor growth. Herein, we present the design, synthesis, and biological evaluation of pyrrolopyridone derivative-based BRD4 degraders. Four synthesized compounds displayed comparative potence against BRD4 BD1 with IC₅₀ at low nanomolar concentrations. Anti-proliferative activity of 32a against BxPC3 cell line (IC₅₀ = 0.165 μM) was improved by about 7-fold as compared to the BRD4 inhibitor ABBV-075. Furthermore, degrader 32a potently induced the degradation of BRD4 and inhibited the expression of c-Myc in BxPC3 cell line in a time-dependent manner. The exploration of intracellular antitumor mechanism showed 32a induced cell cycle arrest and apoptosis effectively. All the results demonstrated that compound 32a could be considered as a potential BRD4 degrader for further investigation.

Full text of DOI:10.1016/j.bioorg.2020.103817

Journal Pre-proofs
Development of small-molecule BRD4 degraders based on pyrrolopyridone
derivative
Jian Zhang, Pan Chen, Peiyu Zhu, Peiyuan Zheng, Tao Wang, Lixun Wang,
Changliang Xu
PII:
S0045-2068(19)32264-3
DOI:
https://doi.org/10.1016/j.bioorg.2020.103817
YBIOO 103817
Reference:
To appear in:
Bioorganic Chemistry
Received Date:
Revised Date:
Accepted Date:
31 December 2019
31 March 2020
2 April 2020
Please cite this article as: J. Zhang, P. Chen, P. Zhu, P. Zheng, T. Wang, L. Wang, C. Xu, Development of small-
molecule BRD4 degraders based on pyrrolopyridone derivative, Bioorganic Chemistry (2020), doi: https://
doi.org/10.1016/j.bioorg.2020.103817
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Development of small-molecule BRD4 degraders based on pyrrolopyridone
derivative
Jian Zhanga, Pan Chenb, Peiyu Zhua, Peiyuan Zhenga, Tao Wanga, Lixun Wanga, Changliang Xuc,,
orda Center of Drug Discovery, Jiangsu Key Laboratory of Drug Discovery for Metabolic Disease, China
Pharmaceutical University, Nanjing 210009, PR China
b Department of Medicinal Chemistry, China Pharmaceutical University, Nanjing 210009, PR China
c Jiangsu Collaborative Innovation Center of Traditional Chinese Medicine Prevention and Treatment of Tumor,
Nanjing University of Chinese Medicine, Nanjing 210023, PR China
Abstract
Bromodomain-containing protein 4 (BRD4) plays a crucial role in the epigenetic regulation of
gene transcription and some BRD4 inhibitors have been advanced to clinical trials. Nevertheless,
the clinical application of BRD4 inhibitors could be limited by drug resistance. As an alternative
strategy, the emerging Proteolysis Targeting Chimeras (PROTACs) technology has the potential to
overcome the drug resistance of traditional small-molecule drugs. Based on PROTACs approaches,
several BRD4 degraders were developed and have been proved to degrade BRD4 protein and inhibit
tumor growth. Herein, we present the design, synthesis, and biological evaluation of
pyrrolopyridone derivative-based BRD4 degraders. Four synthesized compounds displayed
comparative potence against BRD4 BD1 with IC50 at low nanomolar concentrations. Anti-
proliferative activity of 32a against BxPC3 cell line (IC50 = 0.165 μM) was improved by about 7-
fold as compared to the BRD4 inhibitor ABBV-075. Furthermore, degrader 32a potently induced
the degradation of BRD4 and inhibited the expression of c-Myc in BxPC3 cell line in a time-
dependent manner. The exploration of intracellular antitumor mechanism showed 32a induced cell
cycle arrest and apoptosis effectively. All the results demonstrated that compound 32a could be
considered as a potential BRD4 degrader for further investigation.
1. Introduction
Lysine acetylation of histones is a momentous post-translational modification of gene
transcription [1,2]. By recognizing the acetylated lysine (KAc) residues on histone tails,
bromodomain-containing proteins (BCPs) participate in the epigenetic regulation of the gene
expression as epigenetic “readers” and they are related to various diseases such as cancer,
neurological disorders, inflammation and metabolic disorders [2-4]. Among these BCPs, the
bromodomain and extra-terminal domain (BET) family proteins (BRD2, BRD3, BRD4, and BRDT)
have gained much importance in recent years for their essential physiological functions [5-8].
Bromodomain-containing protein 4 (BRD4) is the most extensively studied protein of BET family
and plays an essential role in different signaling pathways. BRD4 promotes transcriptional
elongation by recruiting of the positive transcription elongation factor b (P-TEFb) for stimulating
phosphorylation of RNA polymerase II [5,9,10]. The c-Myc protein is abnormally expressed in most
malignant tumors as a pivotal factor in regulating cell proliferation, and blocking the binding of
BRD4 and acetyl histone can significantly down-regulate the transcription of c-Myc gene [11-13].
Prior research generally confirms that BRD4 is an ideal target for a great many of cancers such as
leukemia, lymphoma, nuclear protein in testis (NUT) midline carcinoma, prostate cancer, and
pancreatic cancer [12-16].
With the identification of first potent BRD4 inhibitor 1 ((+)-JQ-1) (Fig. 1) [14], a great number

of researches have been devoted to the development of BRD4 inhibitors and numerous candidates
with different scaffolds including 2 (I-BET762), 3 (OTX-015), 4 (CPI-0610), and 5 (ABBV-075),
have been put into clinical research for the treatment of human cancers (Fig. 1) [17-20]. In addition
to single administration, combination strategy of BRD4 inhibitors with other antineoplastic drugs
such as histone deacetylase inhibitor or BCL2 inhibitor brings significant improvement to the
efficacy of chemotherapy [16,21]. Single or combined administration of BRD4 inhibitors has
significant effects in the treatment of many kinds of tumors, and BRD4 has become a promising
target for the development of cancer therapeutic drugs. While preclinical and clinical studies have
demonstrated the enormous therapeutic potential of BRD4 inhibitors, the tumour resistance to
BRD4 inhibitor was found in certain contexts, for example, BRD4 promotes transcription and cell
proliferation in BET-resistant TNBC cells in a bromodomain-independent mode [22,23].
Furthermore, BRD4 inhibitors lead to significant BRD4 protein accumulation and inefficient c-Myc
suppression [24]. Thus, alternative therapeutic modalities are needed which can address these
problems.
Fig. 1. Chemical structures of representative BRD4 inhibitors (1-5 ) and BRD4 degraders (6-8)
A novel method to induce the degradation of pathogenesis-related proteins using the
Proteolysis Targeting Chimeras (PROTACs) has attracted broad attention as a promising strategy
in drug discovery [24-28]. PROTACs (also known as degraders) are reasonably designed
bifunctional molecules that consist of a ligand of the protein of interest (POI), a ligand capable of
recruiting E3 ubiquitin ligase, and an applicable linker that connects two ligands. By simultaneously
binding of POI and E3 ubiquitin ligase, the heterobifunctional molecule induces the formation of a
ternary complex, followed by ubiquitination and proteasome-dependent degradation of the target
protein [24,29]. Owing to the irreversible degradation instead of inhibition, PROTACs have
exhibited comparative advantages over small-molecule inhibitors including targeting the
undruggable proteins, overcoming drug resistance, increasing in vitro/in vivo potency, and
improving pharmacodynamic effect in vivo [30,31]. The rapid development of targeted protein
degradation benefit from the identification of several small-molecule ligands of E3 ubiquitin ligase
especially cereblon (CRBN)，a component of the E3 ubiquitin ligase complex CRL4CRBN, was

identified as the target protein of thalidomide and its analogues (9, pomalidomide and 10,
lenalidomide) (Fig. 2) which have been approved by the FDA as immunomodulatory drug (IMiDs)
[32,33]. Recently, a number of BRD4 degraders were proved to be powerful in the degradation of
BRD4 protein and degradation-based antitumor activity including 6 (ARV-771) [34], 7 (dBET1)
[25], and 8 (BETd-260) (Fig. 1) [35]. ARV-771, a BRD4 degrader derived from (+)-JQ-1, showed
significant anti-proliferation and pro-apoptosis effects on castration-resistant prostate cancer (CRPC)
cells, resulted in rapid BRD4 protein degradation with DC50 values < 1 nM, and had an excellent
potency on the treatment of CRPC [34]. BETd-246 and its analogue BETd-260 potently degraded
BRD4 protein and showed strong antitumor effects in vivo against triple-negative breast cancers,
which are lack of recognized therapeutic targets [36].
Fig. 2. Chemical structures of pomalidomide and lenalidomide
PROTAC BRD4 degraders exhibited increased anti-tumor activities in vitro/in vivo and
favorable pharmacokinetic properties. New BRD4 degraders with different scaffolds are worthy to
explore their therapeutic potential in different cancers. Herein, we would like to describe our work
on the design, synthesis and biological evaluation of BRD4 degraders based on pyrrolopyridone
derivative. All the synthesized compounds were evaluated for their binding affinities against BRD4
BD1 and anti-proliferative effects on several tumor cell lines. Then the degradation of BRD4 and
influence on the expression of c-Myc that induced by degraders 32a and 32b was performed on
BxPC3 cell line using Western blot analysis. Moreover, analyses of cell cycle progression and
apoptosis also were carried out to elucidate the intracellular mechanism of cell viability caused by
the depletion of BRD4. The preferred degrader 32a with excellent BRD4 inhibition activity and
anti-proliferative activity was found to be the most effective and worthy of further investigation.
2. Results and discussion
2.1 Design strategy
The clinical candidate 5 (ABBV-075), which have been effectively applied to the therapy of
various cancers (e.g. castration-resistant prostate cancer, triple negative breast cancer, small-cell
lung cancer, and acute myeloid leukemia) [21,37-39], was selected as the BRD4 binding moiety to
construct our desired BRD4 degraders. To understand the binding mode between BRD4 with
ABBV-075, we performed docking studies with Glide docking. The X-ray cocrystal structure of
pyrrolopyridone derivative A-1359643 in complex with BRD4-BD2 (PDB ID: 5UVX) revealed the
important interactions of the pyrrolopyridone core with the conserved Asn433. The pyridone
carbonyl formed a hydrogen bond to the NH2 of Asn433, and the pyrrole NH accepted a hydrogen
bond from the Asn433 carbonyl (Fig. S1A) [38]. A-1359643 was docked into BRD4-BD1 crystal
complex (PDB ID: 5EI4) and the valuable interactions of the pyrrolopyridone core with the
conserved Asn140 were maintained (Fig. S1C). Consistent with the above results, docking of
ABBV-075 to BRD4-BD2 (PDB ID: 5UVX) or BRD4-BD1 (PDB ID: 5EI4) indicated a similar
binding mode for the interaction of the pyrrolopyridone core with the conserved Asn residue (Fig.
S1B and S1D). We assumed that the pyrrole ring of ABBV-075 was a suitable modification site.

Furthermore, the previous studies on the derivation of the pyrrole ring have demonstrated that the
side chain substitution of pyrrole ring is tolerable to maintain the affinity against BRD4 [40,41]. We
hypothesized it is also acceptable that the linkers connected with the E3 ligase ligand were
introduced in the pyrrole ring. Thus, the pyrrolopyridone derivative 25 was obtained by amide
condensation of intermediate 24 with the propargylamine (Scheme 1), which was used to yield the
structure of triazole. A PROTAC-mediated ternary complex depends on the minimum linker length
so that the proteins can bind together without incurring steric conflicts, and different composition
and length of each linker determine the strength of PROTAC to bind to and stabilize the ternary
complex, thus affecting ubiquitination and proteasome-dependent degradation of the target protein
[42]. We explored different PEG lengths considering the importance of the linker length to exert
degradation activity of degraders. Since immunomodulatory drugs were utilized to effectively
generate PROTACs, lenalidomide (10) was employed as a recruiting moiety for E3 ligase CRBN.
2.2. Chemistry
As shown in Scheme 1, intermediate 25 was synthesized according to the references [40,41].
Nucleophilic substitution reaction of commercially available 11 with 2,4-difluorophenol afforded
compound 12. The intermediate 14 was obtained by reduction of 12 with iron powder, followed by
sulfonylation of amine 13. The intermediate 17 was prepared by reduction of compound 16
employing iron powder, which was derived from condensation of 5-bromo-2-methoxy-4-methyl-3-
nitropyridine (15) with diethyl oxalate. N-benzyl protection of 17 provided intermediate 18. The
compound 20 was synthesized by demethylation of 18 in the presence of 4 N HCl, followed by N-
methylation with dimethyl sulfate. The pinacol arylboronate (21) was prepared by coupling reaction
of 20 with bis- (pinacolato) diboron. Suzuki coupling reaction of 21 with intermediate 14 gave
compound 22. The ester 23 was yielded by N-deprotection of 22. Hydrolysis of 23 delivered
compound 22, which afforded intermediate 25 by the amide condensation of compound 24 with
propargylamine.

Scheme 1. The synthesis of intermediate 25. Reagents and conditions: (a) 2,4-difluorophenol, K2CO3, DMF, 85°C;
(b) Fe, NH4Cl, EtOH/THF, 100°C; (c) (i) ethanesulfonyl chloride, triethylamine, DCM, 0°C to rt; (ii) 10% NaOH,
1,4-dioxane, 100°C; (d) diethyl oxalate, C2H5OK, C2H5OH/ether, 40°C; (e) Fe, C2H5OH/CH3COOH, 80°C; (f)
benzyl bromide, K2CO3, DMF, 80°C; (g) 4 N HCl, 1,4-dioxane, 80°C; (h) Me2SO4, K2CO3, acetonitrile, 70°C; (i)
bis(pinacolato)diboron, tris(dibenzylideneacetone)dipalladium, X-PHOS, KOAc, 1,4-dioxane, 80°C; (j) 14,
Pd(dppf)Cl2·CH2Cl2, K2CO3, toluene/MeOH/DMF, 80°C; (k) anisole, conc. H2SO4, CF3COOH, 60°C; (l)
LiOH·H2O, MeOH/H2O, 60°C; (m) propargylamine, EDCI, HOBT, TEA, DMF, rt.
The synthetic procedures of target compounds 32a-32d are outlined in Scheme 2. Treatment
of commercially available 26a-26d with 4-tosyl chloride delivered compounds 27a-27d, which
afforded intermediates 28a-28d by nucleophilic substitution with sodium azide. Analogues 30a-30d
were prepared by deprotection of compounds 29a-29d with trifluoroacetic acid, which was derived
from nucleophilic substitution of tert-butyl bromoacetate with 28a-28d. The intermediates 30a-30d
were reacted with thionyl chloride to provide chloride intermediates, which were converted to
derivatives 31a-31d by amide condensation with lenalidomide. Finally, target compounds 32a-32d
were synthesized by “Click Reaction” of 31a-31d with intermediate 25 in the presence of copper
sulfate pentahydrate and sodium ascorbate.

Scheme 2. The synthesis of target compounds 32a-32d. Reagents and conditions: (a) 4-tosyl chloride, pyridine, rt;
(b) sodium azide, propanone/H2O, 60°C; (c) tert-butyl bromoacetate, sodium hydride, THF, 0°C to rt; (d) CF3COOH,
DCM, rt; (e) (i) thionyl chloride, DMF, DCM; (ii) lenalidomide, 1-methyl-2-pyrrolidinone, rt; (f) 25, CuSO45H2O,
sodium ascorbate, MeOH/DCM/H2O, rt.
2.3 In vitro BRD4 BD1 inhibitory activities
The binding affinities against BRD4 BD1 of the synthesized target compounds were evaluated
by an AlphaScreen assay, and ABBV-075 was selected as the reference compound. The inhibitory
activities are summarized in Table 1. All the degraders exhibited robust potency against BRD4 BD1
with the values of IC50 at low nanomole levels, which showed comparative efficiency with the
positive control (ABBV-075: IC50 = 2.5 nM). Degrader 32a, with the shortest PEG linker, showed
the greatest efficacy for the inhibition of BRD4 BD1 (32a: IC50 = 2.7 nM). Compounds 32b and
32c were obtained by increasing the length of the PEG linker, and their binding affinities were also
maintained (32b: IC50 = 3.4 nM; 32c: IC50 = 3.7 nM). The IC50 of compound 32d was decreased by
more than four-fold when 4-PEG was introduced into the linker (32d: IC50 = 12.0 nM). In general,
the screening of protein–ligand binding affinities demonstrated our hypothesis that the introduction
of side chains to the pyrrole ring is feasible to maintain the BRD4 BD1 inhibitory activity of the
synthesized degraders.
Table 1
The IC50 values of BRD4 BD1 for target compounds 32a-32d and their anti-proliferation activities against BxPC3
cell line
Compound
BRD4 BD1 Alpha Screen IC50 (nM)a
IC50 (BxPC3, μM)a
32a
32b
2.7
3.4
0.165
0.245
4.138
> 10
32c
3.7
32d
12.0
2.5
ABBV-075
1.216
a The date presented are the mean values of at least three independent determinations.

2.4 Anti-proliferative effects against cancer cell lines in vitro
To determine the effect of degraders on the anti-proliferation activity of cancer cells, we
performed MTT assay of synthesized compounds on several different solid tumor cell lines
including DLD-1, HCT-116, BxPC3, MDA-MB-231, A549 and PC-9. Among them, human
pancreatic cancer cell line (BxPC3) showed the best sensitivity to our PROTACs. The semi-
inhibitory concentration (IC50) values of degraders 32a-32d on BxPC3 were outlined in Table 1.
Compared to the BET inhibitor ABBV-075 (IC50 = 1.216 μM), the degraders 32a and 32b exhibited
stronger anti-proliferative potency (32a: IC50 = 0.165 μM; 32b: IC50 = 0.245 μM) against BxPC3
cell line. As illustrated in Fig. S2, the degraders 32a and 32b showed potent anti-proliferation
activity in a concentration-dependent manner, and those compounds were subsequently selected to
investigate the ability to induce BRD4 degradation. While compounds 32c had moderately reduced
inhibitory activity against BxPC3 cells, the anti-proliferative effect of 32d dropped significantly,
which underlined that the length of linker is crucial for the physiological activity of PROTACs. As
showed in Table S1, in some tumor cell lines, the anti-proliferation effect of 32c was even better
than that of 32a and 32b. We speculated that the inability of 32c to efficiently poly-ubiquitinate
BRD4 protein in BXPC3 cells might underlie the reduced activity.
2.5 Degradation of BRD4 and influence on expression of c-Myc
To elucidate the degradation potency of synthesized compounds, the degradation of BRD4
induced by degraders 32a and 32b was identified on BxPC3 cell line using Western blot analysis.
10 (lenalidomide), 5 (ABBV-075), and their combination were used as the reference groups. Cells
were treated with compounds as indicated at the concentration of 1 μM for 48 h. Compared to the
control groups, 32a and 32b almost completely degraded BRD4 protein as shown in Fig. 3A and
3B. The reference groups had no significant influence on the degradation of BRD4 except ABBV-
075, which indicated a slight decrease in the level of BRD4. These results confirmed that the
degradation of the target protein was induced by the designed degraders. As our compounds were
proved to be efficacious in binding to BRD4, we also performed an evaluation on the expression of
c-Myc, a downstream protein of BRD4-mediated signaling pathway. The expression level of c-Myc
was reduced to varying degrees in the presence of PROTACs or 5. Especially, degraders 32a and
32b showed superior regulatory effect on the downregulation of c-Myc. Interestingly, the expression
of c-Myc was increased by more than two-fold in the group treated with 10 (lenalidomide), which
could be induced by negative feedback. Because of the inhibitory effect of ABBV-075 on the
expression of c-Myc, the high expression of cMyc for lenalidomide only was reduced by the
combination of lenalidomide and ABBV-075.
(A)
(B)

Fig. 3. (A) Western blot analysis of BRD4 and c-Myc in BxPC3 cells treated with indicated compounds at the
concentration of 1 μM for 48 h; (B) Grayscale analysis of protein levels of BRD4 and c-Myc measured by Image J.
In order to gain insights into the degradation of BRD4, we characterized the degradation
efficacy of the preferred compound 32a at different indicated time points. BxPC3 cells were treated
with degrader 32a at the concentration of 1 μM for 48 h. As illustrated in Fig. 4A and 4B, the
significant degradation of BRD4 was detected as early as 1 h after the treatment of 32a, followed
by a maximum degradation of BRD4 while the cells were treated for 8 h. Degrader 32a gradually
inhibited the expression of c-Myc from 2 h after the administration, and had the strongest repressive
effect after 48-hour treatment. The degradation of BRD4 by PROTACs sustained during the
administration as well as the inhibition of c-Myc expression. Cells were washed with PBS and the
new drug-free medium was added after treatment of 8 h. Interestingly, the level of BRD4 and c-
Myc both had a moderate recovery, and failed to restore the initial level. The decrease of c-Myc
expression at 48 h post washout might be related to the stationary phase of cell proliferation in the
culture medium. In brief, the degrader 32a time-dependently degraded BRD4 protein and inhibited
the expression of c-Myc.
(A)
(B)
Fig. 4. (A) Western blot analysis of BRD4 and c-Myc in BxPC3 cells treated with compound 32a (1 μM) at different

indicated time points; (B) Grayscale analysis calculated by Image J for protein levels of BRD4 and c-Myc at different
indicated time points.
2.6 Analysis of cell cycle progression and apoptosis
To explore the intracellular mechanism of anti-proliferation activity generated by our designed
degraders, we performed cell cycle progression and apoptosis analysis in BxPC3 cell line through
flow cytometry. As mentioned above, BxPC3 cells were treated with 32a, 32b and other reference
groups for 48 h. Except the lenalidomide-treated group, treatment with compounds resulted in cell
cycle change, including an increase in G0/G1 and G2/M phase cells, and a decrease of S phase cells
as shown in Fig. 5A. Obviously, the degrader 32a exhibited a superior potency on G0/G1 phase cell
cycle arrest. Subsequently, cell apoptosis in diverse degrees were observed in the presence of
degrader and 5 (ABBV-075) as outlined in Fig. 5B. The PROTACs 32a and 32b were more potent
than BRD4 inhibitor in inducing apoptosis on BxPC3 cells.
(A)
(B)
Fig. 5. Analysis of cell cycle progression (A) and apoptosis (B) in BxPC3 cells treated with all indicated compounds
at the concentration of 1 μM for 48 h. The figures are representative of three separate experiments.

To further verify the effect of compounds on cell apoptosis, the live-dead staining of BxPC3
cells was carried out. As illustrated in Fig. 6A and S3, the degrader 32a showed the optimal potency
in inducing apoptosis, followed by a moderate apoptosis induced by 32b and 5 (ABBV-075). We
also explored the expression of apoptosis-related proteins as shown in Fig. 6B and S4. Degrader
32a and 32b significantly downregulated the expression of anti-apoptotic protein Bcl-xl and
promoted the apoptosis of BxPC3 cells. Meanwhile, early apoptotic events were already apparent
as the activation of key apoptosis protease (caspase 3 and cleaved-caspase 3) was induced by 32a
and 32b. These results demonstrated that the synthesized degraders potently induced cell apoptosis,
which might contribute to the robust anti-proliferative effects against BxPC3 cell line.
(A)
(B)
Fig. 6. (A) Live-dead cell staining of the BxPC3 cells treated with indicated compounds at the concentration of 1
μM for 48 h; (B) Western blot analysis of apoptosis-related proteins in BxPC3 cells treated with indicated
compounds at the concentration of 1 μM for 48 h. The figures are representative of three separate experiments.
3. Conclusion
In summary, we designed and synthesized a series of novel small-molecule BRD4 degraders
based on pyrrolopyridone derivative employing PROTACs technique, and their biological effects
were evaluated. All degraders maintained potent binding affinities against BRD4 BD1. The
compounds 32a and 32b exhibited better anti-proliferative activities on BxPC3 cell line compared
to BRD4 inhibitor, and almost completely degraded BRD4 protein. The degradation of BRD4 was
significantly induced by the preferred compound 32a in a time-dependent manner, which resulted
in the continuous decrease of c-Myc expression. Further mechanism study indicated that degrader
32a could effectively induce the cell cycle arrest and apoptosis. These results highlighted the
therapeutic potential of PROTACs on human pancreatic cancer. The optimization of BRD4
degraders as well as the other physiological effects will be administered in the future investigation.
4. Experimental section

4.1 Chemistry
All commercial solvents or reagents were analytically or chemically pure products and were
directly used without further treatment unless otherwise specified. All reactions were monitored by
thin layer chromatography (TLC) under 254 nm and 365 nm. TLC and column chromatography
were performed on HG/T2354-92 GF254 thin layer chromatography silica gel plate and silica gel
(200-300 mesh) respectively, both of which are produced by Qingdao Ocean Chemical Co., Ltd..
Melting points of the synthesized compounds were measured by RY-1 melting point meter of
Tianjin Analytical instrument Factory. Mass spectrometry was recorded by Shimadzu LCMS-2020
mass spectrometer. 1H NMR and 13C NMR spectra were determined by Bruker AV-300 or Bruker
Avance NEO 400 NMR spectrometer with TMS (tetramethylsilane) as the internal standard.
4.1.1 2-bromo-1-(2,4-difluorophenoxy)-4-nitrobenzene (12)
A solution of compound 11 (0.3 g, 1.36 mmol), 2,4-difluorophenol (0.266 g, 2.05 mmol) and
potassium carbonate(0.376 g, 2.73 mmol) in DMF (3 mL) was stirred at 85°C for 3 h. After cooling
to the room temperature, the reaction mixture was diluted with water and the precipitate was filtered.
The filter cake was washed with water and dried to afford 12 (0.38 g, 84%) as a light yellow solid
1
without further purification. H NMR (300 MHz, CDCl3) δ (ppm): 8.55 (d, J = 2.7 Hz, 1H), 8.10
(dd, J = 9.1, 2.7 Hz, 1H), 7.21 (td, J = 8.9, 5.5 Hz, 1H), 7.07-6.94 (m, 2H), 6.73 (d, J = 9.1 Hz, 1H).
4.1.2 3-bromo-4-(2,4-difluorophenoxy)aniline (13)
Iron powder (0.193 g, 3.45 mmol) was added in portions to a stirred solution of 12 (0.38 g,
1.15 mmol) in ethanol/THF/saturated ammonium chloride solution (15 mL, 1/1/1) at room
temperature. The mixture was heated at 100°C for 2 h. After cooling to the room temperature, the
reaction mixture was filtered through a celite pad and the filtrate was concentrated in vacuo. The
residue was diluted with water and extracted with EtOAc. The combined organic layers were
washed with brine, dried over Na2SO4, and concentrated to give compound 13 (0.312 g, 90%) as a
1
brown solid without further purification. H NMR (300 MHz, DMSO-d6) δ (ppm): 7.40 (ddd, J =
11.7, 7.3, 3.0 Hz, 1H), 7.03-6.94 (m, 1H), 6.92-6.83 (m, 2H), 6.76 (td, J = 9.4, 5.5 Hz, 1H), 6.58
(dd, J = 8.7, 2.6 Hz, 1H), 5.33 (s, 2H).
4.1.3 N-(3-bromo-4-(2,4-difluorophenoxy)phenyl)ethanesulfonamide (14)
To a mixture of compound 13 (1.30 g, 4.33 mmol) and triethylamine (2.41 mL, 17.33 mmol)
in DCM (5 mL) cooled to 0°C was added ethanesulfonyl chloride (1.23 mL, 13.00 mmol) in DCM
dropwise and then stirred at room temperature for 3 h. The reaction mixture was evaporated in vacuo,
diluted with water, and extracted with EtOAc. The combined organic layers were washed with brine,
dried over Na2SO4, and concentrated in vacuo. The resulting intermediate was used without further
purification and suspended in 1,4-dioxane/10% NaOH (10 mL, 1/1), then the mixture was heated at
100°C for 2 h. The reaction mixture was evaporated in vacuo, diluted with water, and extracted with
EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated.
The residue was purified by column chromatography on silica gel (EtOAc/petroleum ether, 1/10) to
obtain the title compound 14 (1.45 g, 84%) as a light yellow solid. 1H NMR (300 MHz, CDCl3) δ
(ppm): 7.52 (d, J = 2.6 Hz, 1H), 7.13 (dd, J = 8.8, 2.7 Hz, 1H), 7.06-6.93 (m, 2H), 6.91-6.82 (m,
1H), 6.75 (d, J = 8.8 Hz, 1H), 6.65 (s, 1H), 3.14 (q, J = 7.4 Hz, 2H), 1.40 (t, J = 7.4 Hz, 3H).
4.1.4 ethyl (Z)-3-(5-bromo-2-methoxy-3-nitropyridin-4-yl)-2-hydroxyacrylate (16)
To a solution of compound 15 (3.90 g, 15.79 mmol) and diethyl oxalate (3.22 mL, 23.68 mmol)
in ethanol/ether (32 mL, 1/1) was added potassium ethoxide (1.59g, 18.94mmol) and then refluxed
for 30 min. The reaction mixture was concentrated in vacuo, diluted with water, and extracted with

EtOAc. The combined organic layers were washed with brine, dried over Na2SO4, and concentrated
in vacuo. The residue was purified by column chromatography on silica gel (EtOAc/petroleum ether,
1
1/10) to give 16 (1.72 g, 31%) as a light yellow solid. H NMR (300 MHz, DMSO-d6) δ (ppm):
10.56 (s, 1H), 8.65 (d, J = 13.3 Hz, 1H), 6.22 (s, 1H), 4.48 (s, 1H), 4.34-4.22 (m, 2H), 3.98 (d, J =
7.1 Hz, 3H), 1.35-1.24 (m, 3H).
4.1.5 ethyl 4-bromo-7-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (17)
In a solution of compound 16 (1.72 g, 4.96 mmol) in ethanol/acetic acid (30 mL, 1/1/) was
added iron powder (0.83 g, 14.87 mmol) in portions at room temperature. The mixture was heated
at 100°C for 2 h. After cooling to the room temperature, the reaction mixture was diluted with
EtOAc and filtered through a celite pad. The filtrate was evaporated in vacuo, diluted with water,
and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by column chromatography on silica gel
1
(EtOAc/petroleum ether, 1/8) to obtain 17 (1.38 g, 93%) as a white solid. H NMR (300 MHz,
DMSO-d6) δ (ppm): 13.06 (s, 1H), 7.84 (d, J = 1.4 Hz, 1H), 7.01 (s, 1H), 4.34 (q, J = 7.2 Hz, 2H),
4.02 (d, J = 1.2 Hz, 3H), 1.34 (dd, J = 7.8, 6.4 Hz, 3H).
4.1.6 ethyl 1-benzyl-4-bromo-7-methoxy-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (18)
Benzyl bromide (0.822 mL, 6.92 mmol) was added to a stirred solution of compound 17 (1.38
g, 4.61 mmol) and potassium carbonate (1.28 g, 9.23 mmol) in DMF (10 mL) at room temperature.
The solution was then heated to 80°C for 5 h. After cooling to the room temperature, the reaction
solution was poured into ice water. The precipitated product was filtered, washed with water, and
dried to afford the title compound 18 (1.75 g, 97%) as a light yellow solid without further
purification. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 7.90 (s, 1H), 7.31-7.18 (m, 4H), 6.95 (d, J =
6.9 Hz, 2H), 6.06 (s, 2H), 4.30 (q, J = 7.0 Hz, 2H), 3.94 (s, 3H), 1.28 (t, J = 7.1 Hz, 3H).
4.1.7 ethyl 1-benzyl-4-bromo-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (19)
A solution of compound 18 (1.75 g, 4.50 mmol) in 1,4-dioxane/4 N HCl (20 mL, 1/1) was
stirred at 80°C for 2h. After cooling to the room temperature, the mixture was filtered. The filter
cake was washed with water and dried to give the crude compound 19 (1.65 g, 98%) as a light
yellow solid without further purification. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 11.57 (s, 1H),
7.31-7.17 (m, 4H), 6.98 (s, 3H), 6.24 (s, 2H), 4.24 (q, J = 6.7 Hz, 2H), 1.24 (t, J = 6.5 Hz, 3H).
4.1.8
ethyl
1-benzyl-4-bromo-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-
carboxylate (20)
In a solution of compound 19 (1.65 g, 4.40 mmol) and potassium carbonate (1.22 g, 8.79 mmol)
in acetonitrile (30 mL) was added dimethyl sulfate (0.625 mL, 6.60 mmol) and then stirred at 70°C
for 4 h. After cooling to the room temperature, the reaction mixture was filtered through a celite pad
and washed with DCM. The filtrate was concentrated in vacuo and purified by column
chromatography on silica gel (EtOAc/petroleum ether, 1/8) to afford compound 20 (1.47 g, 86%)
as a white solid. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 7.71 (s, 1H), 7.23 (dt, J = 13.4, 6.9 Hz,
3H), 6.96 (d, J = 6.5 Hz, 3H), 6.26 (s, 2H), 4.24 (q, J = 7.1 Hz, 2H), 3.47 (s, 3H), 1.24 (t, J = 7.1
Hz, 3H).
4.1.9 ethyl 1-benzyl-6-methyl-7-oxo-4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-6,7-dihydro-
1H-pyrrolo[2,3-c]pyridine-2-carboxylate (21)
A mixture of compound 20 (1.47 g, 3.76 mmol), bis(pinacolato)diboron (1.91 g, 7.53 mmol),
tris(dibenzylideneacetone)dipalladium (0.172 g, 0.188 mmol), X-PHOS (0.179 g, 0.376 mmol) and
potassium acetate (0.812 g, 0.828 mmol) in 1,4-dioxane (30 mL) was refluxed under nitrogen

atmosphere for 12 h. After cooling to the room temperature, the reaction mixture was diluted with
EtOAc and water, and filtered through a celite. The aqueous layer was extracted with EtOAc. The
combined organic layer of the filtrate was washed with water and brine, dried over Na2SO4, and
concentrated in vacuo. The resultant was purified by column chromatography on silica gel
1
(EtOAc/petroleum ether, 1/6) to obtain 21 (0.696 g, 42%) as a white solid. H NMR (300 MHz,
DMSO-d6) δ (ppm): 7.65 (s, 1H), 7.29-7.14 (m, 4H), 6.92 (d, J = 7.0 Hz, 2H), 6.26 (s, 2H), 4.25 (q,
J = 7.0 Hz, 2H), 3.52 (s, 3H), 1.31 (s, 12H), 1.23 (t, J = 7.1 Hz, 3H).
4.1.10 ethyl 1-benzyl-4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-6-methyl-7-oxo-
6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (22)
A solution of 21 (0.695 g, 1.59 mmol), 14 (0.521g，1.33mmol) , Pd(dppf)Cl2·CH2Cl2 (0.054 g,
0.066 mmol) and potassium carbonate (0.367 g, 2.65 mmol) in toluene/MeOH/DMF (14 mL, 9/3/2)
was heated to 80°C and stirred under nitrogen atmosphere for 12 h. After cooling to the room
temperature, the reaction mixture was diluted with EtOAc and water, and filtered through a celite
pad. The aqueous layer was extracted with EtOAc. The combined organic layers were washed with
brine, dried over Na2SO4, and evaporated in vacuo. The residue was purified by column
chromatography on silica gel (EtOAc/petroleum ether, 1/3) to give the title compound 22 (0.537 g,
65%) as a white solid. 1H NMR (300 MHz, DMSO-d6) δ (ppm): 9.83 (s, 1H), 7.44 (s, 1H), 7.35 (d,
J = 2.6 Hz, 2H), 7.30-7.19 (m, 4H), 7.08-6.94 (m, 6H), 6.28 (s, 2H), 4.20 (q, J = 7.1 Hz, 2H), 3.52
(s, 3H), 3.13 (q, J = 7.4 Hz, 2H), 1.23 (q, J = 7.2 Hz, 6H).
4.1.11
ethyl
4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-6-methyl-7-oxo-6,7-
dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxylate (23)
Compound 22 (0.537 g, 0.863 mmol) was dissolved in trifluoroacetic acid (3mL). Anisole
(0.188 mL, 1.73 mmol) and concentrated sulfuric acid (0.23 mL, 4.32 mmol) were then added. The
solution was stirred at 60°C for 6 h. After cooling to the room temperature, the reaction mixture was
poured into ice water, quenched with saturated sodium bicarbonate solution and extracted with
EtOAc. The combined organic layers were washed with brine, dried over Na2SO4 and concentrated
in vacuo. The resultant was purified by column chromatography on silica gel (MeOH/DCM, 1/120)
1
to give compound 23 (0.225 g, 49%) as a white solid. H NMR (300 MHz, DMSO-d6) δ (ppm):
12.90 (s, 1H), 9.82 (s, 1H), 7.36 (dd, J = 8.4, 4.5 Hz, 3H), 7.23 (dd, J = 8.8, 2.7 Hz, 1H), 7.08-6.93
(m, 3H), 6.84 (s, 1H), 4.26 (q, J = 7.1 Hz, 2H), 3.54 (s, 3H), 3.12 (q, J = 7.3 Hz, 2H), 1.26 (dt, J =
17.7, 7.2 Hz, 6H).
4.1.12 4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-6-methyl-7-oxo-6,7-dihydro-1H-
pyrrolo[2,3-c] pyridine-2-carboxylic acid (24)
A solution of compound 23 (0.128 g, 0.241 mmol)) and lithium hydroxide (0.030 g, 0.722
mmol) in MeOH/H2O (3 mL, 2/1) was heated at 60°C for 2 h. After cooling to the room temperature,
the reaction mixture was concentrated in vacuo, diluted with water and acidified with 1N HCl. The
precipitated product was filtered, washed with water, and dried to give the crude compound 24
(0.115 g, 95%) as a white solid without further purification. 1H NMR (400 MHz, DMSO-d6) δ (ppm):
13.01 (s, 1H), 12.67 (s, 1H), 9.84 (s, 1H), 7.37 (dd, J = 14.4, 4.1 Hz, 3H), 7.23 (dd, J = 8.6, 2.7 Hz,
1H), 7.09-6.91 (m, 3H), 6.88-6.77 (m, 1H), 3.54 (s, 3H), 3.19-3.06 (m, 2H), 1.23 (t, J = 6.5 Hz, 3H).
4.1.13 4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-6-methyl-7-oxo-N-(prop-2-yn-1-
yl)-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (25)
A mixture of compound 24 (1.60 g, 3.18 mmol), EDCI (0.731 g, 3.81 mmol), HOBT (0.515 g,
3.81 mmol) and triethylamine (0.883 mL, 6.6 mmol) in DMF was stirred at room temperature for

30 min and then added propargylamine (0.262 mL, 3.81 mmol). The reaction mixture was stirred at
room temperature for 2h and then diluted with saturated ammonium chloride solution. The
precipitated product was filtered, washed with water, and dried to give the crude compound 25 (1.47
g, 86%) as a off-white solid without further purification. 1H NMR (300 MHz, DMSO-d6) δ (ppm):
12.38 (s, 1H), 9.83 (s, 1H), 8.80 (s, 1H), 7.34 (d, J = 7.0 Hz, 3H), 7.24 (d, J = 8.6 Hz, 1H), 6.98 (dd,
J = 18.4, 9.3 Hz, 3H), 6.89 (s, 1H), 4.05 (s, 2H), 3.53 (s, 3H), 3.19 (s, 1H), 3.11 (d, J = 7.0 Hz, 2H),
1.23 (s, 3H).
4.1.14 Synthetic procedures of the target compound 32a
The target compound 32a was synthesized from 26a according to the following procedures,
which can also be applied to the synthesis of the target compounds 32b-32d.
4.1.14.1 2-hydroxyethyl 4-methylbenzenesulfonate (27a)
To a solution of compound 26a (3.91 g, 62.95 mmol) in pyridine (5mL) was added
paratoluensulfonyl chloride (6 g, 31.47 mmol) in portions and then stirred at room temperature for
4h. The reaction mixture was acidified with 6N HCl and extracted with EtOAc. The combined
organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo. The residue
was purified by column chromatography on silica gel (EtOAc/petroleum ether, 1/10) to afford 27a
(2 g, 29%) as a colorless liquid. 1H NMR (300 MHz, CDCl3) δ (ppm): 7.81 (d, J = 8.3 Hz, 2H), 7.36
(d, J = 8.0 Hz, 2H), 4.16-4.12 (m, 2H), 3.85 –3.78 (m, 2H), 2.45 (s, 3H), 2.01 (d, J = 19.0 Hz, 1H).
4.1.14.2 2-azidoethan-1-ol (28a)
A solution of compound 27a (1.2 g, 5.55 mmol) and sodium azide (0.72 g, 11.1 mmol) of
propanone/H2O (10 mL, 1/1) was heated to reflux for 16h. The reaction mixture was diluted with
water and extracted with EtOAc. The combined organic layers were washed with saturated sodium
carbonate solution and brine, dried over Na2SO4, and evaporated in vacuo. The residue was purified
by column chromatography on silica gel (EtOAc/petroleum ether, 1/30) to afford 28a (0.41 g, 85%)
as a colorless liquid. 1H NMR (300 MHz, CDCl3) δ (ppm): 3.82-3.74 (m, 2H), 3.49-3.39 (m, 2H),
2.01 (s, 1H).
4.1.14.3 tert-butyl 2-(2-azidoethoxy)acetate (29a)
To a solution of compound 28a (0.41 g, 4.71 mmol) and tert-butyl bromoacetate (1.1 g, 5.65
mmol) in THF (15 mL) cooled to 0°C was added sodium hydride (0.38 g, 9.42 mmol) in small
portions and kept at 0°C for 30 min. The slurry was then stirred at room temperature for 3h. The
reaction mixture was quenched with methanol, diluted with water and extracted with EtOAc. The
combined organic layers were washed with brine, dried over Na2SO4, and concentrated in vacuo.
The residue was purified by column chromatography on silica gel (EtOAc/petroleum ether, 1/20) to
obtain 29a (0.41 g, 43%) as a colorless liquid. 1H NMR (300 MHz, CDCl3) δ (ppm): 4.06-4.00 (m,
2H), 3.76-3.70 (m, 2H), 3.48-3.41 (m, 2H), 1.49 (d, J = 5.8 Hz, 9H).
4.1.14.4 2-(2-azidoethoxy)acetic acid (30a)
In a solution of compound 29a (0.41 g，2.04 mmol) in DCM (4 mL) was added trifluoroacetic
acid (1 mL) and then stirred at room temperature for 3h. The reaction mixture was concentrated in
vacuo to give 30a (0.29 g, 98%) as a brown liquid without further purification.
4.1.14.5 2-(2-azidoethoxy)-N-(2-(2,6-dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)acetamide (31a)
To a solution of 30a (0.29 g, 2.03 mmol) in DCM were added thionyl chloride (1 mL) and two
drops of DMF at room temperature. The mixture was then refluxed for 4h. The reaction mixture
was evaporated in vacuo and the resulting acyl chloride was used without further purification. To a
solution of lenalidomide (0.3 g, 1.16 mmol) in 1-methyl-2-pyrrolidinone (3 ml) was added the acyl

chloride and then stirred at room temperature for 4 h. The reaction mixture was diluted with water
and extracted with EtOAc. The combined organic layers were washed with brine, dried over Na2SO4,
and concentrated in vacuo. The residue was purified by column chromatography on silica gel
(MeOH/DCM, 1/100) to afford 31a (0.26 g, 58%) as a yellow solid. 1H NMR (400 MHz, DMSO-
d6) δ (ppm): 11.02 (s, 1H), 9.74 (s, 1H), 7.77 (d, J = 7.4 Hz, 1H), 7.54 (dt, J = 15.3, 7.5 Hz, 2H),
5.23-5.10 (m, 1H), 4.37 (q, J = 17.3 Hz, 2H), 4.18 (s, 2H), 3.75 (d, J = 4.4 Hz, 2H), 3.52 (d, J = 4.3
Hz, 2H), 3.02-2.85 (m, 1H), 2.60 (d, J = 17.9 Hz, 1H), 2.35 (d, J = 12.8 Hz, 1H), 2.03 (s, 1H).
4.1.14.6
4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-N-((1-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2-oxoethoxy)ethyl)-1H-1,2,3-triazol-4-
yl)methyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (32a)
A solution of compound 25 (0.06 g, 0.111 mmol), 31a (0.06 g, 0.111 mmol) and copper sulfate
pentahydrate (0.009 g, 0.044 mmol) in DCM/MeOH/H2O was stirred at room temperature for 10
min, and sodium ascorbate (0.006 g, 0.022 mmol) was added. Then the mixture was stirred at room
temperature for 4h. The reaction mixture was diluted with water and extracted with DCM. The
combined organic layers were washed with brine, dried over Na2SO4, and evaporated in vacuo. The
residue was purified by column chromatography on silica gel (MeOH/DCM, 1/30) to give the target
1
compound 32a (0.069 g, 59%) as a light yellow solid. m.p.: 186-190 °C; H NMR (400 MHz,
DMSO-d6) δ (ppm) : 12.34 (s, 1H), 11.01 (s, 1H), 9.76 (d, J = 51.5 Hz, 2H), 8.88 (s, 1H), 8.07 (s,
1H), 7.74 (s, 1H), 7.51 (d, J = 20.4 Hz, 2H), 7.28 (d, J = 48.0 Hz, 4H), 6.97 (dd, J = 40.7, 23.7 Hz,
4H), 5.14 (s, 1H), 4.61 (s, 2H), 4.50 (s, 2H), 4.38 (d, J = 12.0 Hz, 2H), 4.14 (s, 2H), 3.96 (s, 2H),
3.53 (s, 3H), 3.10 (s, 2H), 2.91 (s, 1H), 2.58 (s, 1H), 2.42-2.32 (m, 1H), 2.02 (s, 1H), 1.22 (s, 3H);
13C NMR (101 MHz, DMSO-d6) δ (ppm): 173.38, 171.49, 168.54, 168.27, 159.96, 154.63, 150.76,
144.86, 140.23, 135.15, 134.63, 133.63, 133.21, 130.52, 129.59, 129.26, 127.93, 126.68, 124.83,
124.10, 123.65, 122.15, 121.35, 120.21, 118.69, 112.27, 110.54, 106.05, 105.71, 69.91, 69.61, 52.02,
49.76, 46.91, 45.58, 36.29, 34.89, 31.65, 23.01, 8.49; MS (ESI) m/z: calculated for C43H40F2N10O10S
[M-H]-: 925.26, found: 925.00.
4.1.15
4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-N-((1-(2-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2-oxoethoxy)ethoxy)ethyl)-1H-1,2,3-triazol-4-
yl)methyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (32b)
1
Compound 32b (0.138g, 66%) as a light yellow solid. m.p.: 176-180 °C; H NMR (400 MHz,
DMSO-d6) δ (ppm) : 12.35 (s, 1H), 11.01 (s, 1H), 9.83 (s, 1H), 9.68 (s, 1H), 8.87 (s, 1H), 7.99 (s,
1H), 7.72 (d, J = 7.9 Hz, 1H), 7.54 (d, J = 7.0 Hz, 1H), 7.52-7.45 (m, 1H), 7.34 (d, J = 6.6 Hz, 3H),
7.22 (d, J = 8.7 Hz, 1H), 7.06 (d, J = 6.0 Hz, 1H), 7.00 (d, J = 7.8 Hz, 1H), 6.95-6.81 (m, 2H), 5.13
(d, J = 8.5 Hz, 1H), 4.50 (d, J = 12.7 Hz, 4H), 4.43-4.30 (m, 2H), 4.09 (s, 2H), 3.84 (s, 2H), 3.62
(d, J = 4.5 Hz, 4H), 3.52 (s, 3H), 3.10 (d, J = 7.1 Hz, 2H), 2.89 (d, J = 12.6 Hz, 1H), 2.60 (s, 1H),
2.35 (d, J = 13.8 Hz, 1H), 2.00 (d, J = 10.0 Hz, 1H), 1.24-1.20 (t, 3H); 13C NMR (101 MHz, DMSO-
d6) δ (ppm): 173.34, 171.51, 168.83, 168.25, 159.93, 154.62, 150.75, 144.77, 140.30, 135.25, 134.65,
133.64, 133.27, 130.53, 129.36, 129.09, 127.96, 126.77, 124.86, 123.93, 123.62, 122.30, 121.28,
120.16, 118.70, 112.26, 110.48, 106.32, 105.72, 70.66, 70.39, 69.84, 69.23, 52.01, 49.75, 46.90,
45.56, 36.27, 34.88, 31.66, 23.03, 8.51; MS (ESI) m/z: calculated for C45H44F2N10O11S [M-H]-:
969.29, found: 969.06.
4.1.15
4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-N-((1-(2-(2-(2-(2-((2-(2,6-
dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-2-oxoethoxy)ethoxy)ethoxy)ethyl)-1H-1,2,3-

triazol-4-yl)methyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (32c)
Compound 32c (0.085 g, 70%) as a yellow solid. m.p.: 144-148 °C; 1H NMR (300 MHz, DMSO-
d6) δ (ppm) : 12.33 (s, 1H), 11.00 (s, 1H), 9.82 (s, 1H), 9.67 (s, 1H), 8.86 (s, 1H), 7.96 (s, 1H), 7.73
(d, J = 7.5 Hz, 1H), 7.52 (dt, J = 15.0, 7.5 Hz, 2H), 7.40-7.30 (m, 3H), 7.23 (dd, J = 8.8, 2.6 Hz,
1H), 7.13-6.97 (m, 2H), 6.94 (d, J = 8.7 Hz, 1H), 6.89 (d, J = 2.2 Hz, 1H), 5.14 (dd, J = 13.3, 5.0
Hz, 1H), 4.48 (dd, J = 9.3, 4.9 Hz, 4H), 4.37 (d, J = 6.7 Hz, 2H), 4.11 (s, 2H), 3.77 (t, J = 5.0 Hz,
2H), 3.64-3.61 (m, 2H), 3.54 (d, J = 4.7 Hz, 2H), 3.53 (s, 3H), 3.50 (s, 4H), 3.11 (d, J = 7.4 Hz, 2H),
2.96-2.86 (m, 1H), 2.59 (d, J = 17.7 Hz, 1H), 2.36 (d, J = 9.1 Hz, 1H), 2.02 (s, 1H), 1.22 (t, J = 5.6
Hz, 3H); 13C NMR (126 MHz, DMSO-d6) δ (ppm): 172.86, 170.98, 168.47, 167.83, 159.50, 154.20,
150.33, 144.30, 139.90, 134.84, 134.19, 133.18, 132.88, 132.72, 130.05, 128.97, 128.68, 127.51,
126.36, 124.37, 123.39, 123.24, 121.66, 120.94, 119.79, 118.28, 111.79, 110.12, 105.65, 70.38,
69.96, 69.62, 69.54, 68.72, 51.63, 49.32, 46.50, 45.23, 35.80, 34.42, 31.19, 22.56, 8.01; MS (ESI)
m/z: calculated for C47H48F2N10O12S [M-H]-: 1013.31, found: 1013.05.
4.1.15
4-(2-(2,4-difluorophenoxy)-5-(ethylsulfonamido)phenyl)-N-((1-(14-((2-(2,6-
dioxopiperidin-3-yl)-1-oxoisoindolin-4-yl)amino)-14-oxo-3,6,9,12-tetraoxatetradecyl)-1H-1,2,3-
triazol-4-yl)methyl)-6-methyl-7-oxo-6,7-dihydro-1H-pyrrolo[2,3-c]pyridine-2-carboxamide (32d)
Compound 32d (0.091 g, 84%) as a light yellow solid. m.p.: 130-134 °C; 1H NMR (300 MHz,
DMSO-d6) δ (ppm) : 12.34 (s, 1H), 11.00 (s, 1H), 9.82 (s, 1H), 9.67 (s, 1H), 8.86 (s, 1H), 7.95 (s,
1H), 7.72 (d, J = 7.2 Hz, 1H), 7.52 (dt, J = 15.1, 7.4 Hz, 2H), 7.34 (s, 3H), 7.23 (d, J = 8.9 Hz, 1H),
7.03 (dd, J = 17.9, 8.3 Hz, 2H), 6.96-6.87 (m, 2H), 5.14 (d, J = 8.6 Hz, 1H), 4.48 (s, 4H), 4.37 (d, J
= 6.4 Hz, 2H), 4.12 (s, 2H), 3.77 (s, 2H), 3.65 (s, 2H), 3.54 (s, 2H), 3.53 (s, 3H), 3.43 (d, J = 11.6
Hz, 8H), 3.10 (d, J = 7.4 Hz, 2H), 2.91 (t, J = 12.9 Hz, 1H), 2.59 (d, J = 17.7 Hz, 1H), 2.34 (t, J =
13.6 Hz, 1H), 1.99 (d, J = 4.4 Hz, 1H), 1.23-1.21 (t, 3H); 13C NMR (101 MHz, DMSO-d6) δ (ppm):
173.35, 171.50, 168.93, 168.27, 167.40, 159.93, 154.65, 150.76, 144.75, 140.33 (d, J = 11.7 Hz),
135.35, 134.66, 133.66, 133.28, 131.95, 130.52, 129.39, 129.12, 127.96, 126.84, 124.86, 123.86,
123.64, 122.13, 121.31, 120.24, 118.68, 112.38, 112.15, 110.53, 106.11, 70.84, 70.59, 69.18, 61.80,
52.03, 49.79, 46.93, 45.59, 36.28, 34.89, 31.67, 23.04, 8.50; MS (ESI) m/z: calculated for
C49H52F2N10O13S [M+Na]+: 1081.34, found: 1081.20.
4.2. Biological evaluation
4.2.1 BRD4 BD1 binding affinity assay
The binding affinity assay was administered by Shanghai ChemPartner Co. Firstly 1× assay
buffer was prepared using modified HEPES Buffer. Compounds were transferred to assay plate by
Echo and DMSO’s final concentration is 0.1%. The preparation of protein solution and substrate
solution was performed in 1x assay buffer. After that, 5 μL of protein solution or 5 μL of 1x assay
buffer was transferred to assay plate with an incubation at room temperature for 15 min. Then 5 μL
of substrate solution was added to each well to start reaction, followed by an incubation at room
temperature for 60 min. Next, 15 μL acceptor and donor solution were added and incubated at room
temperature for 60 min under subdued light. Finally, the IC50 values were calculated from datas
readed by EnSpire with Alpha mode.
4.2.2 Cell culture
BxPC3 cell line from Key laboratory of SATCM for Empirical Formulae Evaluation and
Achievements Transformation was cultured in the Dulbecco’s Modified Eagle Medium (DMEM)
medium (YUANPEI, CHINA) supplemented with 10% fetal bovine serum (FBS) (BI, USA) and
routinely maintained at 37℃，5% CO2 incubator.

4.2.3 Cell proliferation assay
The cell proliferation assay was evaluated by MTT assay. BxPC3 cells (4x103 cells) were
seeded in 96-well plates with 100 μL of culture medium and incubated at 37℃ and an atmosphere
of 5% CO2 for 24 h. The cells were then treated with medium containing various concentrations of
compounds and 1‰ DMSO, with every concentration was repeated in four wells. 1‰ DMSO
solution was set to the control group. After that, cells were further incubated for 72 h. Then each
well of the cells was added 20 μL of MTT reagent and incubated for 3 h at 37℃ in standard culture
conditions. The absorbance was measured in a microplate reader at 490 nm. The data were
normalized to the DMSO control. The IC50 was calculated by GraphPad Prism 5 statistical software.
4.2.4 Western blotting analysis
BxPC3 cells were lysed with RIPA lysis buffer containing 1% PMSF and protease and
phosphatase inhibitors. The protein concentration was determined with a BCA assay kit. Equal
amounts of protein for each sample were separated using SDS-PAGE gels and transferred onto
PVDF membranes. The membranes were subsequently blocked in 5% BSA for 1h at room
temperature, followed by incubation with primary antibody overnight at 4℃. The membranes were
then washed with 1X PBST and incubated with a secondary antibody for 1h. Finally, the membranes
were treated with ECL detection reagents and exposed in the chemiluminescent imaging system.
4.2.5 Cell cycle progression analysis
BxPC3 cells were incubated in the absence or presence of compounds for 48h. Then cells were
collected and fixed by 70% alcohol for 8 h at -20℃. The percentage of cells cycles was analyzed
by staining with PI for 20 min at room temperature, followed by flow cytometry.
4.2.6 Cell apoptosis analysis
For the cell apoptosis assay, BxPC3 cells were incubated in the absence or presence of
compounds for 48h. The percentage of apoptotic cells was analyzed by staining with fluorescein
isothiocyanate-conjugated Annexin V and propidium iodide for 20 min at room temperature,
followed by flow cytometry.
4.2.7 Live-dead staining
BxPC3 cells were seeded at 4x104 cells/well in 12-well plates. After 48 h of compounds
treatment, each well of the cells was stained with Live-Dead dye and incubated for 30 min at 37℃
in standard culture conditions. The fluorescence was photographed by an Inverted fluorescence
Microscope.
Acknowledgments
This study was supported by the Natural Science Foundation of Jiangsu Province (No. BK
20141349) and the China National Key HiTech Innovation Project for the R&D of Novel Drugs
(No.2013ZX09301303-002).
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Authors have no conflict of interest to declare.
Several pyrrolopyrridone derivative-based BRD4 degraders were synthesized and evaluated in
AlphaScreen assay and cell-based assays.
Degraders 32a and 32b effectively inhibited the proliferation of pancreatic cancer cell line BxPC3.
Degrader 32a potently degraded BRD4 protein and inhibited the expression of c-Myc in a time-
dependent manner.
Degrader 32a induced the cell cycle arrest and apoptosis of BxPC3 cells.