A novel cereblon modulator for targeted protein degradation

Article abstract of DOI:10.1016/j.ejmech.2019.01.023

Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera (PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428, which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is inhibited due to suppressed C-MYC transcription. These results, therefore, firmly suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN modulator that can be used for targeted protein degradation.

Full text of DOI:10.1016/j.ejmech.2019.01.023

Accepted Manuscript
A novel cereblon modulator for targeted protein degradation
Sung Ah Kim, Ara Go, Seung-Hyun Jo, Sun Jun Park, Young Uk Jeon, Ji Eun Kim,
Heung Kyoung Lee, Chi Hoon Park, Chong-Ock Lee, Sung Goo Park, Pilho Kim,
Byoung Chul Park, Sung Yun Cho, Sunhong Kim, Jae Du Ha, Jeong-Hoon Kim, Jong
Yeon Hwang
PII:
S0223-5234(19)30033-9
DOI:
https://doi.org/10.1016/j.ejmech.2019.01.023
EJMECH 11030
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 7 November 2018
Revised Date: 19 December 2018
Accepted Date: 10 January 2019
Please cite this article as: S.A. Kim, A. Go, S.-H. Jo, S.J. Park, Y.U. Jeon, J.E. Kim, H.K. Lee, C.H. Park,
C.-O. Lee, S.G. Park, P. Kim, B.C. Park, S.Y. Cho, S. Kim, J. Du Ha, J.-H. Kim, J.Y. Hwang, A novel
cereblon modulator for targeted protein degradation, European Journal of Medicinal Chemistry (2019),
doi: https://doi.org/10.1016/j.ejmech.2019.01.023.
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ACCEPTED MANUSCRIPT
N
S
H
N
N
O
N
O
NH2
O
O
N
H
O
O
N
O
N
O
NH
O
N
N
N
N
2
H
N
N
Cl
Novel CRBN ligand (TD-106)
-degradation of IKZF1/3
PROTAC (TD-428)
-degradation of BET proteins

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A Novel Cereblon Modulator for Targeted Protein Degradation
Sung Ah Kim1,2,#, Ara Go3,#, Seung-Hyun Jo1,2, Sun Jun Park3, Young Uk Jeon3, Ji
3
Eun Kim3, Heung Kyoung Lee , Chi Hoon Park3, 4, Chong-Ock Lee3, Sung Goo
Park1,2, Pilho Kim3,4, Byoung Chul Park,1,5, Sung Yun Cho3, Sunhong Kim 1,6, Jae Du
Ha*,3, Jeong-Hoon Kim*,1,2, and Jong Yeon Hwang*,3,4
1 Disease Target Structure Research Center, Korea Research Institute of Bioscience
and Biotechnology, Daejeon 34141, Republic of Korea
2
Department of Functional Genomics, University of Science and Technology,
Daejeon 34113, Republic of Korea
3Therapeutics & Biotechnology Division, Korea Research Institute of Chemical
Technology, Daejeon 34114, Republic of Korea
4Department of Medicinal Chemistry and Pharmacology, University of Science and
Technology, Daejeon 34113, Republic of Korea
5Department of Bio-Analytical Science, University of Science and Technology,
Daejeon 34113, Republic of Korea
6
Department of Biomolecular Science, University of Science and Technology,
Daejeon 34113, Republic of Korea
# These authors contributed equally to this study.
*Corresponding author. E‐mail: jdha@krict.re.kr (J.D. Ha), jhoonkim@kribb.re.kr (J.-
H. Kim), and jyhwang@krict.re.kr (J.H. Hwang)
Keywords: CRBN, BET, PROTAC, IMiDs

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Abstract
Immunomodulatory drugs (IMiDs) exert anti-myeloma activity by binding to the
protein cereblon (CRBN) and subsequently degrading IKZF1/3. Recently, their ability
to recruit E3 ubiquitin ligase has been used in the proteolysis targeting chimera
(PROTAC) technology. Herein, we design and synthesize a novel IMiD analog TD-
106 that induces the degradation of IKZF1/3 and inhibits the proliferation of multiple
myeloma cells in vitro as well as in vivo. Moreover, we demonstrate that TD-428,
which comprises TD-106 linked to a BET inhibitor, JQ1 efficiently induce BET protein
degradation in the prostate cancer cell line 22Rv1. Consequently, cell proliferation is
inhibited due to suppressed C-MYC transcription. These results, therefore, firmly
suggest that the newly synthesized IMiD analog, TD-106, is a novel CRBN
modulator that can be used for targeted protein degradation.

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1. Introduction
Immunomodulatory drugs (IMiDs), including thalidomide and its analogues, are a
new class of anticancer agents with the glutarimide group, [1]. Thalidomide was
developed and marketed as a sedative in the 1950s, but, was banned due to serious
teratogenic effects in the early 1960s [2, 3]. Later on, it was demonstrated that
thalidomide possessed anti-angiogenic and anti-inflammatory properties, and was
consequently approved by the FDA for the treatment of multiple myeloma (MM) in
2006 [4, 5]. Although IMiDs were used for MM treatment, the mechanism underlying
their pleotropic effects remained obscure for many years. By using affinity purification
coupled with proteomic analysis, Ito et al., demonstrated that thalidomide specifically
binds to cereblon (CRBN) and DNA damage-binding protein 1 (DDB1). Subsequently,
CRBN, forms a Cullin Ring ligase (CRL) complex together with DDB1, CUL4, and
RBX1 [6, 7]. Once bound to CRBN, IMiD promotes the degradation of IKZF1 and
IKZF3 through the ubiquitination-dependent proteasome pathway [8, 9]. As
thalidomide functioned successfully as an IMiD, next generation IMiDs, such as
lenalidomide, pomalidomide, and CC122, were developed (Figure 1) [10] [1].
Figure 1. Known CRBN modulators (thalidomide, lenalidomide, pomalidomide, and
CC-122).

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The unique ability of IMiDs to bind to CRBN led to the development of the proteolysis
targeting chimeras (PROTACs) technology. PROTAC is a bifunctional molecule with
two ligands–one for the target protein, and the other for E3 ubiquitin ligases that
induce proteasomal degradation of the target protein [11-15]. Several E3 ubiquitin
ligases, including β-TrCP, MDM2, cIAP, VHL, and CRBN have been successfully
applied for the degradation of various targets, such as FKBP12, ERα, AR, BET,
BCR-ABL, BTK, and RIPK2 [16-20]. Of these, BRD4 is the most commonly-targeted
protein in PROTAC technology. BRD4 is a member of the Bromodomain and Extra-
Terminal domain (BET) family, which includes BRD2, BRD3, and BRDT [21-23]. The
BET family recruits transcriptional regulatory complexes to acetylated chromatin, and
control specific gene networks involved in cellular proliferation and cell cycle
progression [24-26]. In particular, deregulation of BET protein activity, such as that of
BRD4, is observed in cancer and inflammatory diseases, thereby making BET
proteins attractive therapeutic targets [27, 28]. Therefore, several reports are
available on the degradation of BET proteins in multiple cancer cells by highly potent
PROTACs with thalidomide analogues or VHL ligands [21, 29-32].
Although PROTAC selectivity is theoretically dependent on the inhibitor warhead, it
can also be influenced by the linker and the recruited E3 ligase [33, 34]. Therefore,
discovery of novel E3 ligase ligands is important and necessary for PROTAC-based
drug discovery. In the present study, we describe the synthesis and biological
evaluation of a novel CRBN modulator (aminobenzotriazino glutarimide) and also
validate its applicability in targeted protein degradation (Figure 2).

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Figure 2. (A) Structure of a novel CRBN modulator (aminobenzotriazino glutarimide).
(B) Application of the CRBN modulator in targeted protein degradation.
2. Results and Discussion
2.1. Chemistry
Scheme 1. Reagents and conditions; (a) 3-aminopiperidine-2,6-dione, EDCI, HOBt,
DIPEA, DMF, rt; (b) NaNO2, AcOH, rt; (c) Fe, AcOH, THF, rt.
Aminobenzotriazino glutarimides (TDs) 4 were synthesized as shown in
Scheme 1. The reagent 2-amino-6-nitrobenzoic acid 1 reacts with 3-aminopiperidine-
2,6-dione in the presence of EDCI, HOBt, and DIPEA at room temperature to give
amide 2. Cyclization of 2 was performed by treatment with NaNO2 in AcOH,
furnishing benzotriazinones 3. The nitro functional group of 3 was reduced by
treatment with iron in the presence of AcOH to give the desired aminobenzotriazino
glutarimides 4.

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Scheme 2. Reagents and conditions; (a) 3-aminopiperidine-2,6-dione, EDCI, HOBt,
DIPEA, DMF, rt; (b) NaNO2, AcOH, rt; (c) DIPEA, DMF, 90 ℃; (d) 4 M HCl in dioxane,
DCM, rt; (e) K2CO3, acetone, reflux; (f) NaN3, DMF, 50 ℃; (g) Pd(OH)2, H2 (g), EtOH,
rt; (h) TFA, DCM, rt; (i) EDCI, HOBt, DIPEA, DMF, rt.
Synthesis of BRD4 degraders is depicted in Scheme 2. For synthesizing a CRBN
ligand, 5-fluorobenzotriazinone 7 was synthesized from 2-amino-6-fluorobenzoic acid

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5 as per the same procedure described above. Nucleophilic substitution of 7 with
different Boc-protected diamines 8 provided compounds 9, which are transformed to
intermediates 10 by TFA treatment. For intermediates 17 with aniline linker, Boc-
aminophenols 11 were alkylated with diiodoalkane 12 to give compound 13.
Substitution reaction of 13 with NaN3 provided azide 14 that was reduced to amine
15. Similarly, condensation of 7 and the subsequent deprotection of Boc group
provided intermediates 17. Finally, reaction of the intermediates 10 and 17 with JQ1-
acid 19 (generated from JQ1 by hydrolysis of tert-butylester) gave the desired BRD4
degraders 20 and 21, respectively, via treatment with EDCI, HOBt, and DIPEA in
DMF.
2.2.
Biology
2.2.1. Identification of a novel CRBN modulator
To determine whether TDs (4a-4d) can induce the degradation of IKZF1/3 like
IMiDs, NCI-H929 cells were treated with increasing amounts of TDs or pomalidomide.
TD-106 (4a) induced the degradation of IKZF1/3 as much as pomalidomide did
(Figure 3A), whereas the other analogs 4b-4d showed no degradation (data not
shown). Binding of target proteins with ligands improve their thermal stability. On this
basis, we determined whether TD-106 directly binds to CRBN. At 53 °C, treatment
with TD-106 and pomalidomide increased thermal stability in a dose dependent
manner whereas treatment with DMSO did not (Figure 3B). Clinically, IMiDs are used
to treat multiple myeloma as it induces cell cytotoxicity in multiple myeloma cell lines
[35]. Therefore, we carried out the cytotoxicity assay with the NCI-H929 myeloma

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cell line to see if TD-106 inhibits proliferation. Treatment of NCI-H929 with TD-106 or
pomalidomide at similar concentrations (CC50 = 0.039 µM and 0.035 µM, respectively,
Figure 3C) inhibited cell proliferation.
Figure 3. TD-106 is a novel CRBN modulator. (A) Degradation of IKZF1/3 by TD-106
in NCI-H929 cells. (B) Cellular thermal shift assay (CETSA) at 53 ℃ in HEK293
expressing CRBN-ePL. (C) Cytotoxicity assay of pomalidomide and TD-106 in NCI-
H929 cells (D) Antitumor activity of TD-106 in TMD-8 xenograft model. Compounds
were administered intraperitoneally (i.p.) to SCID mice at doses of 50 mg/kg q.d. for
14 days. Data are represented as the mean ± standard error (n = 6).

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2.2.2. In vivo evaluation of TD-106 in a xenograft model
To determine the antitumor property of TD-106, an in vivo xenograft study was
performed with TMD8 cell lines, which is an ABC (activated B-cell) subtype of DLBCL
(diffuse large B-cell lymphoma). TMD8 cells were implanted subcutaneously into the
right flanks of female SCID mouse, and drug treatment was initiated after tumor
volumes reached approximately 150 mm3. TD-106 or control (DMSO) was
administered intraperitoneally to tumor-implanted mice in 20% PEG 400 and 3%
Tween 80 in PBS (-) at doses of 50 mg/kg q.d. for the 14-day study duration. The
drug treatment inhibited tumor growth during this duration. However, slight rebound
tumor-growth was observed after discontinuation of the dosage (Figure 3D),
indicating that TD-106 possesses anti-myeloma activity in vivo. No side effects or
changes in body weight were observed during the study (data not shown).
2.2.3. BRD4 PROTACs with TD-106 induce BRD4 degradation and anti-
proliferative effects on 22Rv1 cells
Recently, IMiDs have been used to degrade endogenous target proteins with
PROTACs [36]. To determine if TD-106 could induce targeted protein degradation,
we developed a series of bifunctional compounds containing TD-106 and JQ1, a
BET inhibitor, with various linkers [31, 37]. Based on previous reports [29, 32, 38, 39],
four degraders (20 and 21) were synthesized and, were examined for BRD4
degradation by immunoblotting in 22Rv1, the prostate cancer cell line. A dose
titration experiment in 22Rv1 spanning 24 h showed that compounds 21, bearing a
phenyl ring in the linker, are better at degrading BRD4 than compounds 20, which

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have a linear alkyl linker (Figure 4A). Compounds 21a (TD-188) and 21b (TD-428)
have the same molecular weight, but the linker was attached to different positions of
the phenyl ring. Meta-substituted 21b (DC50 = 0.32 nM) induced a more potent
degradation of BRD4 than did ARV-825 (DC50 = 0.57 nM), a known BET degrader.
However, the degradation activity of para-substituted 21a (DC50 = 3.3 nM) was
weaker than that of ARV-825 (Figure 4B). Indeed, 21b is approximately 50 times
more potent than 21a, indicating that the potency depends not only on the length of
the linker, but also on its attachment location. Next, we examined the effects of the
BRD4 degraders on the cell proliferation in 22Rv1. As with earlier results, cytotoxicity
was highest with 21b (CC50 = 20.1 nM, Figure 4C). Like other BET degraders, 21b
induced the degradation of all the studied BET proteins (Supplementary Figure 1A)
Figure 4. BET PROTACs with TD-106 induce BRD4 degradation (A and B) and

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inhibit proliferation of 22RV1 cells (C).
2.2.4. Mechanistic characterization of BRD4 degradation by TD-428
Next, we investigated the mechanism of BRD4 degradation by TD-428. To
determine if TD-428 mediated BRD4 degradation is dependent on UPS or CRL, the
proteasome inhibitor Bortezomib, or the neddylation inhibitor MLN4924, was added
along with TD-428. This completely inhibited BRD4 degradation, implying that BRD4
degradation by TD-428 is dependent on both UPS and CRL (Figure 5A). When
endogenous CRBN was knocked down by specific siRNA, BRD4 levels remained
unchanged in the presence of TD-428, suggesting that BRD4 degradation by TD-428
requires CRBN (Figure 5B). Moreover, TD-428-mediated BRD4 degradation was
inhibited by addition of excess TD-106 or excess JQ1, indicating that proximity
between BRD4 and CRBN is essential for BRD4 degradation (Figure 5C and 5D). As
reported in other PROTAC compounds, TD-428 also facilitated the physical
interaction between two unrelated proteins, BRD4 and CRBN (Figure 5E and
Supplementary Figure 1B). Furthermore, TD-428 reduced C-MYC levels more
efficiently than JQ1 (Figure 5F).

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Figure 5. Mechanistic characterization of BRD4 degradation by TD-428. (A) UPS
and CRL dependent BRD4 degradation by TD-428. (B) CRBN is required for TD428
mediated BRD4 degradation. (C) TD-428 mediated BRD4 degradation is inhibited by
excessive TD-106 (D) TD-428 mediated BRD4 degradation is inhibited by excessive
JQ1 (E) CRBN interacts with BRD4 in the presence of TD-428 (F) TD-428 reduced
C-MYC levels more efficiently than JQ1
2.2.5. TD-428 is a highly specific BRD4 degrader
Biochemical characterization of TD-428 showed that it has a higher specificity for
BRD4 than does ARV-825. However, the IKZF1/3 degradation efficiency of TD-428 is
approximately 100-fold lower than that of ARV-825 (Figure 6). In addition, TD-428
lacked the hook effect, a typical characteristic of many PROTACs that prevents

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target protein degradation at high doses of the compound [40].
Figure 6. TD-428 is a more specific BRD4 degrader. U266 cells were treated with
increasing concentration of TD188 (up to 10uM), TD428 (up to 10uM), or ARV825
(up to 10uM) for 12h. Cell lysates were prepared and analyzed by immunoblotting for
BRD4, IKZF1, IKZF3 and GAPDH.
3.
Conclusion
Targeted protein degradation by PROTAC is a powerful technology in drug discovery.
Hijacking E3 ubiquitin ligases to target proteins by PROTAC is essential for its
degradation. Although several E3 ubiquitin ligases, including β-TRCP, MDM2, cIAP,
VHL, and CRBN have been successfully used in PROTACs, thalidomide analogs,
CRBN ligands are the most widely used. In the present study, we discovered a novel
CRBN modulator, TD-106. We showed that TD-106 induces the degradation of
IKZF1/3 and inhibits the proliferation of multiple myeloma cells. In addition, we
demonstrated that TD-428, the BRD4 degrader with TD-106, is highly specific for
BET proteins and lacks the hook effect. Collectively, TD-106 as a novel CRBN
modulator can be used for targeted protein degradation.

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4.
Experimental Section
Chemistry
4.1.
All materials were obtained from commercial suppliers and used without further
purification. Solvents in this study were dried using an aluminum oxide column. Thin-
layer chromatography was performed on pre-coated silica gel 60 F254 plates (Merck,
art. 5715). Purification of intermediates was carried out by normal phase column
1
chromatography (MPLC, Silica gel 230-400 mesh). H and 13C NMR spectra were
recorded with Bruker Avance 300 and 500, using CDCl3 or other deuterated solvents
as an internal standard. LC/MS analysis was performed the Agilent Technology 6130
Quadrupole LC/MS with electrospray ionization.The melting point was measured
with Thomas-Hoover melting point apparatus.
4.1.1. Synthesis of 2-amino-N-(2,6-dioxopiperidin-3-yl)-6-nitrobenzamide (2a)
To a solution of 2-amino-6-nitrobenzoic acid (7.0 g, 38 mmol), 3-aminopiperidine-2,6-
dione hydrogen chloride (25.0 g, 152 mmol), EDCl-HCl (8 .0g, 42 mmol), and HOBt
(6.5 g, 42 mmol) in DMF (90 mL) was added DIPEA (21 mL, 121.6 mmol). The
mixture was stirred at room temperature overnight. The reaction mixture was diluted
with water and extracted with ethyl acetate. The organic layer was washed with brine,
dried over Na2SO4, and concentrated. The crude compound 2a was obtained as
1
yellow solid (15 g) and used in the next step without further purification. H NMR
(300 MHz, DMSO-d6) δ 10.99 (s, 1H), 9.02 (d, J = 8.2 Hz, 1H), 7.35–7.25 (m, 1H),
7.25–7.16 (m, 1H), 7.11–6.99 (m, 1H), 6.01 (s, 2H), 4.80–4.66 (m, 1H), 2.92–2.71 (m,
1H), 2.63–2.52 (m, 1H), 2.24–2.05 (m, 1H), 2.04–1.89 (m, 1H). 13C NMR (101 MHz,
DMSO-d6) δ 173.0, 169.8, 151.5, 146.7, 144.0, 137.3, 131.7, 127.0, 111.0, 59.2, 31.1,

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23.0 ;LC/MS (M + H)- (m/z) 292.9 ; LC/MS (M + H)- (m/z) 290.9 ; mp 230 oC
Synthesis of 3-(5-nitro-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)piperidine-2,6-dione (3a)
To a solution of 2a (15 g) in AcOH added was sodium nitrite (2.7 g, 40 mmol) and
stirred at room temperature for 1.5 h. The mixture was diluted with water and the
precipitated white product was collected, washed with water, and dried to afford the
product 3a (7.0 g, 60%, 2 steps overallyield). 1H NMR (300 MHz, DMSO-d6) δ 11.25
(s, 1H), 8.53 (dd, J = 7.8, 1.5 Hz, 1H), 8.44–8.26 (m, 2H), 6.11–5.91 (m, 1H), 3.03–
2.86 (m, 1H), 2.76–2.56 (m, 2H), 2.38–2.22 (m, 1H). 13C NMR (101 MHz, DMSO-d6)
δ 173.6, 173.4, 165.3, 147.6, 147.6, 130.3, 120.9, 116.0, 111.1, 49.9, 31.4,
23.8 ;LC/MS (M + H)- (m/z) 303.9; LC/MS (M + H)- (m/z) 301.9 ; mp 265 oC
4.1.2. Synthesis of 3-(5-amino-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)piperidine-2,6-
dione (4a)
To a suspension of 3a (1.0 g, 3.2 mmol) in a mixture of AcOH (25 mL) and THF (25
mL) was added iron powder (1.0 g). The mixture was stirred at room temperature
overnight. The reaction mixture was filtered and washed with ethyl acetate. The
filtrate was washed with saturated aqueous NaHCO3 and water, dried over MgSO4,
and concentrated under reduced pressure. The residue was purified by column
1
chromatography to afford product 4a (500 mg, 57%) as yellow solid. H NMR (300
MHz, DMSO-d6) δ 11.16 (s, 1H), 7.69 (t, J = 8.1 Hz, 1H), 7.27–7.15 (m, 3H), 7.01 (d,
J = 8.3 Hz, 1H), 5.89–5.79 (m, 1H), 3.05–2.86 (m, 1H), 2.74–2.58 (m, 2H), 2.33–2.18
(m, 1H). 13C NMR (125 MHz, DMSO) δ 172.8, 170.0, 156.8, 150.0, 145.0, 136.2,
116.7, 113.4, 101.6, 58.1 30.8, 22.6. ; LC/MS (M + H)- (m/z) 274.0; LC/MS (M + H)-
(m/z) 272.0 ; mp 245 oC

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4.1.3. Synthesis of 2-amino-N-(2,6-dioxopiperidin-3-yl)-6-fluorobenzamide (6)
To a solution of 2-amino-6-fluoro-benzoic acid (10 g, 64.46 mmol) in DMF (150 mL),
EDCI·HCl (13.6 g, 70.90 mmol ), HOBt (10.85 g , 70.90 mmol), and 3-amino
pyridine-2,6-dione hydrogen chloride (10.85 g , 70.90 mmol ), was added DIPEA
(36.0 mL, 206.2 mmol). The mixture was stirred at room temperature for 9 h and was
quenched with ice cold water. The precipitate was collected and washed with water
and dried to afford 6 (15.6 g, 91%) as a bluish white solid; 1H NMR (300 MHz, DMSO)
δ 10.90 (s, 2H), 8.53 (dd, J = 8.2, 2.6 Hz, 2H), 7.16 – 7.05 (m, 2H), 6.52 (d, J = 8.2
Hz, 2H), 6.38 – 6.24 (m, 2H), 6.00 (s, 4H), 4.84 – 4.65 (m, 2H), 2.88 – 2.70 (m, 2H),
2.62 – 2.54 (m, 1H), 2.22 – 1.83 (m, 4H); LC/MS (M + H)- (m/z) 264.0; LC/MS (M +
H)- (m/z) 274.8 flash point 250 oC.
4.1.4. Synthesis of 3-(5-fluoro-4-oxobenzo[d][1,2,3]triazin-3(4H)-yl)piperidine-2,6-
dione (7)
To a solution of 6 (15 g, 56.6 mmol) in AcOH (400 mL) was added NaNO2 (6.66 g ,
96.1 mmol). The reaction mixture was stirred at room temperature for 2 h. After
completion reaction, the reaction mixture was quenched with water (500 mL). The
precipitant was collected, washed with water, and dried to afford 7 (11.2 g, 42%) as a
white solid. 1H NMR (300 MHz, DMSO-d6) δ 11.21 (s, 1H), 8.24–8.03 (m, 2H), 7.86–
7.72 (m, 1H), 5.97 (dd, J = 12.1, 5.4 Hz, 1H), 2.96 (m, 1H), 2.76–2.58 (m, 2H), 2.35–
2.16 (m, 1H); 13C NMR (125 MHz, DMSO) δ 173.1, 170.1, 160.4, 158.3, 151.8 (d, J =
2.5 Hz), 145.4, 137.6 (d, J = 9.5 Hz), 125.0 (d, J = 4.0 Hz), 120.0 (d, J = 19.7 Hz),
58.0, 31.2, 23.0. flash point 270 oC.
4.1.5. Synthesis
of
tert-butyl
(8-((3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-

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dihydrobenzo[d][1,2,3]triazin-5-yl)amino)octyl)carbamate (9a)
A solution of 7 (232 mg, 0.8 mmol), tert-butyl (8-aminooctyl)carbamate 8a (227 mg,
0.9 mmol), and DIPEA (0.5 mL, 3 mmol) in DMF (10 mL) was reacted at 90 ℃
overnight. The reaction mixture was diluted with water and extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4, and concentrated. The
residue was purified by column chromatography to give the compound 9a (281 mg,
1
61%) as a yellow solid. H NMR (300 MHz, CDCl3) δ 8.35 (s, 1H), 8.24 – 8.11 (m,
1H), 7.70 (t, J = 8.2 Hz, 1H), 7.28 – 7.23 (m, 1H), 6.79 (d, J = 8.4 Hz, 1H), 5.76 –
5.65 (m, 1H), 4.58 (s, 1H), 3.31 – 3.19 (m, 2H), 3.17 – 3.07 (m, 2H), 3.02 – 2.69 (m,
3H), 2.43 – 2.26 (m, 1H), 1.81 – 1.69 (m, 2H), 1.57 – 1.35 (m, 15H); LC/MS (M + H)-
(m/z) 498.9; mp 113–118 oC.
Similar procedure of 9a was performed to give compound 9b (13 mg, 34%) as a
1
yellow solid. H NMR (300 MHz, MeOD) δ 8.53 – 8.41 (m, 1H), 7.77 (t, J = 8.2 Hz,
1H), 7.29 – 7.19 (m, 1H), 6.98 (d, J = 8.5 Hz, 1H), 5.95 – 5.80 (m, 1H), 3.79 (t, J =
5.2 Hz, 2H), 3.74 – 3.61 (m, 4H), 3.61 – 3.51 (m, 2H), 3.51 – 3.39 (m, 2H), 3.28 –
3.18 (m, 2H), 3.02 – 2.76 (m, 3H), 2.41 – 2.30 (m, 1H), 1.41 (s, 9H); LC/MS (M + H)-
(m/z) 498.9; mp 63–72 oC.
4.1.6. Synthesis of 3-(5-((8-aminooctyl)amino)-4-oxobenzo[d][1,2,3]triazin-3(4H)-
yl)piperidine-2,6-dione hydrogen chloride (10a)
To a solution of 9a (281 mg, 0.56 mmol) in DCM (3 mL) was added 4 N HCl in
dioxane (0.1 mL) and stirred at room temperature for 1 h. The reaction mixture was
concentrated under reduced pressure to afford 10a (91%) as yellow solid. The next
step was processed without purification. 1H NMR (300 MHz, MeOD) δ 7.90 – 7.72 (m,

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1H), 7.28 – 7.18 (m, 1H), 7.00 – 6.91 (m, 1H), 5.94 – 5.79 (m, 1H), 3.32 – 3.28 (m,
2H), 3.06 – 2.76 (m, 5H), 2.43 – 2.29 (m, 1H), 1.85 – 1.64 (m, 4H), 1.63 – 1.41 (m,
4H); LC/MS (M + H)+ (m/z) 373.1, (M + H)- (m/z) 371.0; mp 110–125 oC.
Similar procedure of 10a was performed to give compound 10b (90%) as a yellow
solid. 1H NMR (400 MHz, MeOD) δ 7.79 (t, J = 8.2 Hz, 1H), 7.28 – 7.22 (m, 1H), 7.01
(d, J = 8.5 Hz, 1H), 5.91 – 5.81 (m, 1H), 3.81 (t, J = 5.1 Hz, 2H), 3.78 – 3.72 (m, 6H),
3.55 – 3.46 (m, 2H), 3.19 – 3.06 (m, 2H), 3.06 – 2.79 (m, 3H), 2.44 – 2.29 (m, 1H);
LC/MS (M + H)+ (m/z) 405.0, (M + H)- (m/z) 403.0; mp 58–63 oC.
4.1.7. Synthesis
of
tert-butyl
(4-(2-(2-(2-
iodoethoxy)ethoxy)ethoxy)phenyl)carbamate (13a)
A mixture of tert-butyl (4-hydroxyphenyl)carbamate 11 (522 mg, 2.5 mmol), 12
(4.62 g, 13 mmol), and K2CO3 (689 mg, 5.0 mmol) in acetone (15 mL) was refluxed
overnight. The reaction mixture was diluted with water and extracted with EtOAc.
The organic layer was washed with brine, dried over MgSO4, and concentrated. The
residue was purified by column chromatography to give the compound 13a (322 mg,
1
75%) as a pale yellow oil. H NMR (400 MHz, CDCl3) δ 7.32 – 7.21 (m, 4H), 6.90 –
6.81 (m, 4H), 6.42 (s, 2H), 4.14 – 4.07 (m, 4H), 3.88 – 3.83 (m, 4H), 3.79 – 3.67 (m,
12H), 3.26 (t, J = 6.9 Hz, 4H), 1.51 (s, 19H). Similar procedure of 13a was performed
to give compound 13b (13 mg, 34%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ
7.23 – 7.09 (m, 2H), 6.88 – 6.82 (m, 1H), 6.70 – 6.57 (m, 1H), 6.47 (s, 1H), 4.19 –
4.13 (m, 2H), 3.91 – 3.85 (m, 2H), 3.83 – 3.67 (m, 6H), 3.29 (t, J = 6.9 Hz, 2H), 1.54
(s, 9H).
4.1.8. Synthesis
azidoethoxy)ethoxy)ethoxy)phenyl)carbamate (14a)
A solution of 13a (775 mg, 1.7 mmol), NaN3 (335 mg, 5.2 mmol) in DMF (8 mL) was
of
tert-butyl
(4-(2-(2-(2-

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reacted at 50 ℃ overnight. The reaction mixture was concentrated under reduced
pressure to afford 14a. The next step was processed without purification.
4.1.9. Synthesis
of
tert-butyl
(4-(2-(2-(2-
aminoethoxy)ethoxy)ethoxy)phenyl)carbamate (15a)
A solution of crude 14a and Pd(OH)2 /carbon (16 mg) in EtOH (8 mL) was reacted at
room temperature for 4 h under H2 atmosphere. The reaction mixture was filtered
through a pad of Celite and washed with EtOH. The filtrate was concentrated in
1
vacuo to give the compound 15a (424 mg, 73%) as a clear oil. H NMR (300 MHz,
CDCl3) δ 7.28 – 7.22 (m, 2H), 6.90 – 6.83 (m, 2H), 6.41 (s, 1H), 4.19 – 4.04 (m, 2H),
3.93 – 3.81 (m, 2H), 3.76 – 3.70 (m, 2H), 3.69 – 3.64 (m, 2H), 3.54 (t, J = 5.2 Hz, 2H),
2.89 (t, J = 5.2 Hz, 2H), 2.18 (s, 3H), 1.52 (s, 9H); LC/MS (M + H)+ (m/z) 341.1
Similar procedure of 15a was performed to give compound 15b (236 mg, quant.) as
a yellow solid. 1H NMR (300 MHz, CDCl3) δ 7.18 (t, J = 8.1 Hz, 2H), 6.95 – 6.87 (m,
1H), 6.76 (s, 1H), 6.66 – 6.58 (m, 1H), 4.24 – 4.10 (m, 2H), 3.93 – 3.78 (m, 2H), 3.79
– 3.70 (m, 2H), 3.70 – 3.61 (m, 2H), 3.61 – 3.52 (m, 2H), 2.94 – 2.87 (m, 2H), 2.50 (s,
2H), 1.53 (s, 9H); LC/MS (M + H)+ (m/z) 341.1
4.1.10. Synthesis of tert-butyl (4-(2-(2-(2-((3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3]triazin-5-yl)amino)ethoxy)ethoxy)ethoxy)phenyl)carbamate
(16a)
A mixture of 15a (271 mg, 0.8 mmol), 7 (220 mg, 0.8 mmol), DIPEA (0.42 Ml, 2.4
mmol) in DMF (4 mL) was heated at 90 ℃ for 4 h. The reaction mixture was diluted
with water and extracted with EtOAc. The organic layer was washed with brine, dried
over MgSO4, and concentrated. The residue was purified by column chromatography
1
to give the crude compound 16a (115 mg) as a yellow solid. H NMR (300 MHz,

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CDCl3) δ 8.35 (t, J = 5.1 Hz, 1H), 8.25 (s, 1H), 7.67 (t, J = 8.2 Hz, 1H), 7.30 – 7.15
(m, 4H), 6.89 – 6.78 (m, 3H), 6.40 (s, 1H), 5.74 – 5.61 (m, 1H), 4.13 – 4.04 (m, 2H),
3.87 – 3.81 (m, 2H), 3.81 – 3.65 (m, 6H), 3.47 – 3.34 (m, 2H), 3.01 – 2.66 (m, 3H),
2.37 – 2.23 (m, 1H), 1.51 (s, 9H); LC/MS (M + H)+ (m/z) 597.0, (M + H)- (m/z) 594.9;
mp 80–88 oC
Similar procedure of 16a was performed to give compound 16b (259 mg, 62%) as a
yellow solid. 1H NMR (400 MHz, CDCl3) δ 8.42 – 8.34 (m, 1H), 8.31 (s, 1H), 7.69 (t, J
= 8.1 Hz, 1H), 7.29 – 7.25 (m, 1H), 7.18 – 7.12 (m, 1H), 7.10 (s, 1H), 6.88 – 6.81 (m,
2H), 6.68 – 6.56 (m, 2H), 5.77 – 5.65 (m, 1H), 4.20 – 4.09 (m, 2H), 3.94 – 3.85 (m,
2H), 3.83 – 3.66 (m, 6H), 3.50 – 3.35 (m, 2H), 3.01 – 2.70 (m, 4H), 2.38 – 2.29 (m,
1H), 1.53 (s, 9H); LC/MS (M + H)+ (m/z) 597.0, (M + H)- (m/z) 594.9; mp 80–87 oC.
4.1.11. Synthesis of 3-(5-((2-(2-(2-(4-aminophenoxy)ethoxy)ethoxy)ethyl)amino)-4-
oxobenzo[d][1,2,3]triazin-3(4H)-yl)piperidine-2,6-dioneyl)piperidine-2,6-dione (17a)
To a solution of 16a (115 mg, 0.2 mmol) in DCM (5 mL) was added 4 M HCl in
dioxane (2 mL) at room temperature for 1 h. The reaction mixture was concentrated
and dissolved in DCM. The organic layer was neutralized with aqueous NaHCO3,
washed with brine, dried over MgSO4, and concentrated. The residue was purified by
column chromatography to give the compound 17a (28 mg, 29%) as a yellow solid.
1H NMR (300 MHz, CDCl3) δ 9.18 (s, 1H), 8.38 – 8.27 (m, J = 5.0 Hz, 1H), 7.69 –
7.58 (m, J = 8.1 Hz, 1H), 7.26 – 7.13 (m, J = 7.7 Hz, 1H), 6.83 – 6.76 (m, J = 8.5 Hz,
1H), 6.71 (d, J = 8.8 Hz, 2H), 6.59 (d, J = 8.7 Hz, 2H), 5.76 – 5.60 (m, J = 11.5, 5.0
Hz, 1H), 4.11 – 3.94 (m, 2H), 3.87 – 3.77 (m, 2H), 3.77 – 3.65 (m, 6H), 3.56 (s, 2H),
3.47 – 3.34 (m, J = 10.4, 5.1 Hz, 2H), 2.91 – 2.61 (m, 3H), 2.33 – 2.15 (m, J = 11.3,
6.7 Hz, 1H); LC/MS (M + H)+ (m/z) 497.0, (M + H)- (m/z) 494.9; mp 63–74 oC.

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Similar procedure of 17a was performed to give compound 17b (105mg, 49%) as a
yellow solid. 1H NMR (300 MHz, CDCl3) δ 8.52 (s, 1H), 8.37 (s, 1H), 7.68 (t, J = 8.1
Hz, 1H), 7.29 – 7.25 (m, 1H), 7.11 – 7.02 (m, 1H), 7.02 – 6.99 (m, 1H), 6.83 (d, J =
8.5 Hz, 1H), 6.47 – 6.34 (m, 2H), 5.77 – 5.66 (m, 1H), 4.13 – 4.01 (m, 2H), 3.91 –
3.83 (m, 2H), 3.83 – 3.68 (m, 6H), 3.49 – 3.36 (m, 2H), 2.99 – 2.67 (m, 3H), 2.38 –
2.20 (m, 1H); LC/MS (M + H)+ (m/z) 497.0, (M + H)- (m/z) 494.9; mp 52–65 oC.
4.1.12. Synthesis
of
(S)-2-(4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)acetic acid (19)
The JQ1 18 (1 g, 2.2 mmol) was dissolved in 40% TFA/DCM solution (24 mL) and
stirred at room temperature for 5 h. The reaction mixture was concentrated and
dissolved in EtOAc. The organic layer was washed with water and brine, dried over
MgSO4, and concentrated. The residue was purified by column chromatography to
1
give the compound 19 (900 mg, quant.) as a yellow solid. H NMR (300 MHz,
Chloroform-d) δ 7.45 (d, J = 8.6 Hz, 2H), 7.36 (d, J = 8.6 Hz, 2H), 6.36 (s, 2H), 4.63
(t, J = 6.8 Hz, 1H), 3.81 – 3.55 (m, 2H), 2.74 (s, 3H), 2.44 (s, 3H), 1.72 (s, 3H).
LC/MS (M + H)+ (m/z) 400.9, (M + H)- (m/z) 398.9;; mp 90–93 oC
4.1.13. Synthesis
of
2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(8-((3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3]triazin-5-yl)amino)octyl)acetamide (20a, TD-230)
A mixture of 10a (10 mg, 0.03 mmol), 19 (13 mg, 0.03 mmol), EDCI·HCl (6 mg, 0.03
mmol), HOBt (5 mg, 0.03 mmol), DIPEA (0.03 mL, 0.14 mmol) in DMF (1 mL) was
stirred at room temperature for 4 h. The reaction mixture was diluted with water and

ACCEPTED MANUSCRIPT
extracted with EtOAc. The organic layer was washed with brine, dried over MgSO4,
and concentrated. The residue was purified by column chromatography to give the
compound 20a (5 mg, 60%) as a yellow solid. 1H NMR (300 MHz, CDCl3) δ 9.06 (s,
1H), 8.94 (s, 1H), 8.23 – 8.11 (m, 2H), 7.73 – 7.64 (m, 2H), 7.40 (d, J = 8.4 Hz, 4H),
7.36 – 7.28 (m, 5H), 7.23 (d, J = 7.7 Hz, 2H), 6.85 – 6.72 (m, 4H), 5.79 – 5.62 (m,
2H), 4.64 (t, J = 7.0 Hz, 2H), 3.66 – 3.51 (m, 2H), 3.42 – 3.13 (m, 10H), 3.00 – 2.75
(m, 6H), 2.66 (s, 6H), 2.39 (s, 5H), 2.37 – 2.27 (m, 3H), 1.74 – 1.62 (m, 10H), 1.60 –
1.51 (m, 4H), 1.45 – 1.35 (m, 7H); 13C NMR (101 MHz, CDCl3) δ 171.23, 171.17,
170.49, 170.48, 168.28, 163.91, 157.76, 155.67, 149.90, 149.58, 145.70, 145.69,
136.73, 136.64, 136.50, 132.16, 130.89, 130.80, 130.45, 129.84, 128.68, 113.53,
111.60, 102.58, 102.54, 77.40, 77.08, 76.76, 58.28, 58.07, 54.54, 42.93, 42.89,
39.48, 39.42, 39.40, 31.08, 31.04, 29.31, 29.28, 28.44, 28.40, 26.70, 26.66, 26.49,
26.46, 23.13, 14.37, 13.08, 11.81; LC/MS (M + H)+ (m/z) 754.9, (M + H)- (m/z) 752.9;
mp 110–115 oC.
Synthesis
of
2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(2-(2-(2-((3-(2,6-dioxopiperidin-3-yl)-4-
oxo-3,4-dihydrobenzo[d][1,2,3]triazin-5-yl)amino)ethoxy)ethoxy)ethyl)acetamide (20b,
TD-207)
Similar procedure of 20a was performed to give compound 20b (47 mg, 88%) as a
yellow solid. 1H NMR (300 MHz, CDCl3) δ 9.51 (s, 1H), 9.27 (s, 1H), 8.44 – 8.36 (m,
2H), 7.76 – 7.66 (m, 2H), 7.47 – 7.38 (m, 4H), 7.38 – 7.30 (m, 4H), 7.28 – 7.24 (m,
1H), 7.03 – 6.90 (m, 2H), 6.82 (d, J = 8.5 Hz, 2H), 5.72 – 5.52 (m, 2H), 4.67 (q, J =
6.8 Hz, 2H), 3.82 (t, J = 5.0 Hz, 4H), 3.75 – 3.28 (m, 25H), 2.97 – 2.75 (m, 6H), 2.67
13
(d, J = 3.8 Hz, 6H), 2.41 (s, 6H), 2.38 – 2.26 (m, 2H), 1.67 (d, J = 7.5 Hz, 6H); C

ACCEPTED MANUSCRIPT
NMR (101 MHz, CDCl3) δ 171.20, 170.81, 170.71, 168.31, 168.22, 163.99, 163.89,
157.56, 155.61, 149.88, 149.82, 149.43, 149.40, 145.75, 145.71, 136.69, 136.40,
132.23, 130.95, 130.87, 130.73, 130.69, 130.43, 129.90, 129.88, 128.67, 128.65,
113.99, 111.61, 103.10, 77.35, 77.03, 76.72, 70.84, 70.77, 70.35, 70.22, 69.84, 69.79,
69.11, 69.02, 59.12, 58.70, 54.45, 54.41, 42.95, 39.59, 39.19, 39.15, 31.13, 31.10,
22.97, 14.38, 14.35, 13.09, 11.85, 11.83
784.9; mp 115–118 oC
;
LC/MS (M + H)+ (m/z) 786.9, (M + H)- (m/z)
2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-f][1,2,4]triazolo[4,3-
a][1,4]diazepin-6-yl)-N-(3-(2-(2-(2-((3-(2,6-dioxopiperidin-3-yl)-4-oxo-3,4-
dihydrobenzo[d][1,2,3]triazin-5-yl)amino)ethoxy)ethoxy)ethoxy)phenyl)acetamide
(21a, TD-188)
Similar procedure of 20a was performed to give compound 21a (27 mg, 36%) as a
1
yellow solid. H NMR (300 MHz, CDCl3) δ 9.15 (d, J = 11.9 Hz, 1H), 8.97 (s, 0H),
8.87 (s, 1H), 8.46 – 8.34 (m, 1H), 7.67 (t, J = 8.1 Hz, 1H), 7.58 – 7.38 (m, 4H), 7.38 –
7.30 (m, 2H), 7.30 – 7.20 (m, 2H), 6.91 – 6.73 (m, 3H), 5.78 – 5.57 (m, 1H), 4.80 –
4.67 (m, 1H), 4.18 – 4.05 (m, 2H), 3.92 – 3.66 (m, 9H), 3.66 – 3.52 (m, 1H), 3.52 –
3.33 (m, 2H), 2.95 – 2.58 (m, 6H), 2.42 (s, 3H), 2.35 – 2.20 (m, 1H), 1.68 (s, 3H);13C
NMR (101 MHz, CDCl3) δ 171.12, 171.09, 168.50, 168.48, 168.37, 168.33, 157.59,
157.56, 155.46, 155.31, 150.15, 149.50, 145.61, 136.42, 131.73, 131.32, 131.07,
131.05, 130.12, 130.07, 128.76, 121.59, 121.57, 114.95, 114.90, 113.97, 111.71,
102.79, 102.77, 77.37, 77.05, 76.73, 70.87, 70.84, 70.72, 69.77, 69.26, 69.24, 67.83,
67.81, 54.54, 42.94, 39.95, 31.03, 31.00, 23.23, 14.42, 13.14, 11.83; LC/MS (M +
H)+ (m/z) 878.9, (M + H)- (m/z) 876.7; mp 160–165 oC

ACCEPTED MANUSCRIPT
4.1.14. Synthesis
of
2-((S)-4-(4-chlorophenyl)-2,3,9-trimethyl-6H-thieno[3,2-
f][1,2,4]triazolo[4,3-a][1,4]diazepin-6-yl)-N-(4-(2-(2-(2-((3-(2,6-dioxopiperidin-3-yl)-4-
oxo-3,4-dihydrobenzo[d][1,2,3]triazin-5-
yl)amino)ethoxy)ethoxy)ethoxy)phenyl)acetamide (21b, TD-428)
Similar procedure of 20a was performed to give compound 21b (20 mg, 30%) as a
yellow solid. 1H NMR (300 MHz, CDCl3) δ 9.58 – 9.39 (m, 2H), 8.38 – 8.27 (m, 1H),
7.66 – 7.54 (m, 1H), 7.37 (d, J = 8.2 Hz, 2H), 7.29 – 7.24 (m, 2H), 7.21 – 7.15 (m,
1H), 7.15 – 7.01 (m, 2H), 6.75 (d, J = 8.5 Hz, 1H), 6.61 – 6.52 (m, 1H), 5.71 – 5.60
(m, 1H), 4.73 (t, J = 7.0 Hz, 1H), 4.12 – 4.00 (m, 2H), 3.84 – 3.77 (m, 2H), 3.74 –
3.55 (m, 8H), 3.43 – 3.30 (m, 2H), 2.80 – 2.70 (m, 2H), 2.67 (s, 3H), 2.36 (s, 4H),
1.63 (s, 4H), 1.38 – 1.11 (m, 2H); 13C NMR (101 MHz, CDCl3) δ 171.61, 171.56,
169.10, 168.80, 168.67, 164.13, 159.04, 157.52, 155.63, 155.61, 150.11, 150.09,
149.43, 145.54, 139.69, 136.73, 136.45, 136.35, 132.13, 132.12, 131.00, 130.89,
130.43, 130.41, 129.98, 129.39, 128.62, 113.77, 112.35, 111.68, 110.66, 110.61,
106.05, 105.93, 102.71, 77.55, 77.23, 76.92, 70.75, 70.74, 70.68, 70.66, 69.75,
69.17, 67.38, 57.49, 57.39, 54.45, 54.40, 42.84, 40.01, 30.99, 23.19, 23.16, 14.41,
13.09, 11.82; LC/MS (M + H)+ (m/z) 878.9, (M + H)- (m/z) 876.7; mp 109–111 oC
Biology
4.1.15. Antibodies and reagents
Anti-BRD4 antibody (#13440), IKZF1 antibody (#9034) and IKZF3 antibody
(#151035) were obtained from Cell Signaling Technology. Anti-CRBN antibody
(HPA045910) and Anti-Flag M2 Magnetic Bead (M8823) were obtained from Sigma
Aldrich. DBeQ (4417) was obtained from Tocris Bioscience. Anti-HA Magnetic Bead

ACCEPTED MANUSCRIPT
(88837) was obtained from Thermo Scientific. MLN4924 (505477) was obtained from
Calbiochem, Bortezomib (S1013) was obtained from Selleckchem.
4.1.16. Cell culture
HEK293T cell was maintained in Dulbecco’s Modified Eagle’s Medium (DMEM,
Gibco) supplemented with 10% fetal bovine serum (FBS, Gibco), penicillin
100units/ml, streptomycin 100µg/ml and amphotericin B 0.25µg/ml (Gibco) in a
humidified atmosphere of 5% CO2 in air at 37 °C. 22Rv1 cell was maintained in
RPMI-1640 Medium (Gibco) supplemented with 10% fetal bovine serum (FBS,
Gibco), penicillin 100 units/mL, streptomycin 100 µg/mL and amphotericin B 0.25
µg/mL (Gibco) in a humidified atmosphere of 5% CO2 in air at 37 °C. H929 cell was
maintained in RPMI-1640 Medium (Gibco) supplemented with 10% fetal bovine
serum (FBS, Gibco), penicillin 100 units/mL, streptomycin 100 µg/mL, amphotericin
B 0.25 µg/mL (Gibco) and 2-mercaptoethanol 0.05 mM (Sigma) in a humidified
atmosphere of 5% CO2 in air at 37 °C.
4.1.17. Plasmid constructs and transfection
Xpress-CRBN, HA-CRBN and FLAG-BRD4 were cloned using Infusion cloning. Cells
were transfected with various plasmids using X-treamGENE HP DNA Transfection
Reagent (Roche) according to the manufacturer’s instruction.
4.1.18. RNA interference
siRNA against CRBN (51185) and negative control siRNA (SN-1003) were obtained
from Bioneer. For RNA interference, cells were transfected with siRNAs using
Lipofectamine RNAiMAX Transfection Reagent (Invitrogen) according to the
manufacturer’s instruction. After 48h incubation, TD428 was treated for 12h. And
cells were harvested and analyzed.
4.1.19. Immunoprecipitation and immunoblotting

ACCEPTED MANUSCRIPT
Cells were lysed using lysis buffer (50 mM HEPES KOH pH 7.4, 40 mM NaCl, 1 mM
EDTA, 1 mM EGTA, 10 mM sodium pyrophosphate, 10 mM sodium β-
glycerophosphate, 50 mM NaF, 1 mM NaVO4, 1% Triton X-100) containing protease
inhibitor (05056489001, Roche). The lysate was incubated for 10 min on ice and
clarified by centrifuging at 13,000 rpm, 4°C for 10 min. The supernatant was
quantified and incubated with beads at 4°C for 3h. After incubation, beads were
washed twice in lysis buffer and triplets in wash buffer (50 mM HEPES KOH pH 7.4,
500 mM NaCl, 1 mM EDTA, 1 mM EGTA, 10 mM sodium pyrophosphate, 10 mM
sodium β-glycerophosphate, 50 mM NaF, 1 mM NaVO4, 1% Triton X-100, protease
inhibitor). Elution was performed using SDS Sample buffer (40 mM Tris HCl pH 6.8,
2% SDS, 0.05% bromophenol blue, 5% glycerol, 2.8 M mercaptoethanol) and
samples were separated using SDS-PAGE. After transfer to NC membrane,
immunoblotting was performed using the indicated antibodies.
4.1.20. Cellular Thermal Shift Assay (CESTA)
For the cell lysate CETSA experiments, cultured HEK293 cells were harvested and
washed with PBS. The cells were diluted in Assay Medium (DMEM plus 10% FBS) to
1.25*105/mL. 5xDMSO, 5xPomalidomide (up to 10 µM) and 5xTD106 (up to 10 µM)
were split for 10 µL into assay plate. The cell lysates were split for 40 µL into assay
plate with inhibitor. After 1 hour incubation in a humidified atmosphere of 5% CO2 in
air at 37°C, the assay plate were incubate at 53°C for 3 minutes using thermocycler
(ProFlex, ThermoFisher Scientific). Sample in assay plate were transfer to 96-well
white plate by 40 µL. Put the Master mix in each well for 60 µL (10 µL EA Reagent,
10 µL Lysis Buffer and 40 µL Substrate Reagent) and incubate for 1 hour at room
temperature in dark. The 96-well white plate measured luminescence using
luminometer (Victor, PerkinElmer). [44]

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4.1.21. Proliferation Assay
22Rv1 cells (15,000/100 µL) were seeded in 96-well tissue culture white plates
followed by addition of compound at indicated concentrations. After 72 hr, 100 µL per
well of reconstituted CelTiter-Glo reagent (#G7572, Promega) was added and read
on Luminometer (ProFlex, ThermoFisher Scientific). Relative cell growth was
determined by comparing assay readings of treated cells with control DMSO-treated
cells. NCI-H929 cells were seeded in 96-well plates at 30% confluency and exposed
to chemicals the next day. After 72 h, WST-1 reagent was added and absorbance at
450 nm was measured using a Spectramax spectrophotometer (Molecular Devices,
US) according to the manufacturer’s instructions. The IC50s were calculated using
GraphPad Prism version 6 for Windows. The curves were fitted using a nonlinear
regression model with a log (inhibitor) versus response formula.
4.1.22. In vivo xenograft assay
Female SCID (CB-17/IcrCri-scid) mice were obtained from Charles River of Japan.
Animals were maintained under clean room conditions in sterile filter top cages and
housed on high efficiency particulate air-filtered ventilated racks. Animals were
received sterile rodent chow and water ad libitum. All of the procedures were
conducted in accordance with guidelines approved by the Laboratory Animal Care
and Use Committee of Korea Research Institute of Chemical Technology. TMD-8
cells were implanted subcutaneously into the right flank region of each mouse and
allowed to grow to the designated size. Once tumors reached an average volume of
about 150 mm3, mice were dosed via intraperitoneal daily with the indicated doses
of TD-106 for 14 days. Mice were observed daily throughout the treatment period for
signs of morbidity/mortality. Tumors were measured twice weekly using calipers, and
volume was calculated using the formula: length x width2 x 0.5. Body weight was

ACCEPTED MANUSCRIPT
also assessed twice weekly. Statistical significances were evaluated by using Mann-
Whitney U-test (a = 0.01).
Acknowledgements
This work was supported by the grant (CAP-15-11-KRICT) from National Research
Council of Science and Technology, Ministry of Science, ICT, and future planning and
the grant from KRIBB Initiative Program.
Author contributions
AG, SJP, YUK synthesized compounds. SAK, SHJ, JEK, HKL conducted biology
experiments. CHP, COL, SGP, PK, BCP, SYC, SK designed and conducted some
experiments. SAK, JDH, JHK, and JYH wrote the manuscript. JDH, JHK, and JYH
contributed to experimental design and supervision.
Conflict of interest
The authors declare no conflict of interest.
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