Discovery of Highly Potent and Selective IRAK1 Degraders to Probe Scaffolding Functions of IRAK1 in ABC DLBCL

Article abstract of DOI:10.1021/acs.jmedchem.1c00103

MyD88 gene mutation has been identified as one of the most prevalent driver mutations in the activated B-cell-like diffuse large B-cell lymphoma (ABC DLBCL). The published literature suggests that interleukin-1 receptor-associated kinase 1 (IRAK1) is an essential gene for ABC DLBCL harboring MyD88 mutation. Importantly, the scaffolding function of IRAK1, rather than its kinase activity, is required for tumor cell survival. Herein, we present our design, synthesis, and biological evaluation of a novel series of potent and selective IRAK1 degraders. One of the most potent compounds, Degrader-3 (JNJ-1013), effectively degraded cellular IRAK1 protein with a DC50 of 3 nM in HBL-1 cells. Furthermore, JNJ-1013 potently inhibited IRAK1 downstream signaling pathways and demonstrated strong anti-proliferative effects in ABC DLBCL cells with MyD88 mutation. This work suggests that IRAK1 degraders have the potential for treating cancers that are dependent on the IRAK1 scaffolding function.

Full text of DOI:10.1021/acs.jmedchem.1c00103

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Article
Discovery of Highly Potent and Selective IRAK1 Degraders to Probe
Scaffolding Functions of IRAK1 in ABC DLBCL
Liqiang Fu,△ Jing Zhang,△ Bin Shen, Linglong Kong, Yingtao Liu, Wangyang Tu, Wenqian Wang,
Xin Cai, Xiaotao Wang, Na Cheng, Mingxuan Xia, Tianyuan Zhou, Qian Liu, Yanping Xu, Jennifer Yang,
Paul Gavine, Ulrike Philippar, Ricardo Attar, James P. Edwards, Jennifer D. Venable,* and Xuedong Dai*
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ABSTRACT: MyD88 gene mutation has been identified as one of the most prevalent driver mutations in the activated B-cell-like
diffuse large B-cell lymphoma (ABC DLBCL). The published literature suggests that interleukin-1 receptor-associated kinase 1
(IRAK1) is an essential gene for ABC DLBCL harboring MyD88 mutation. Importantly, the scaffolding function of IRAK1, rather
than its kinase activity, is required for tumor cell survival. Herein, we present our design, synthesis, and biological evaluation of a
novel series of potent and selective IRAK1 degraders. One of the most potent compounds, Degrader-3 (JNJ-1013), effectively
degraded cellular IRAK1 protein with a DC50 of 3 nM in HBL-1 cells. Furthermore, JNJ-1013 potently inhibited IRAK1
downstream signaling pathways and demonstrated strong anti-proliferative effects in ABC DLBCL cells with MyD88 mutation. This
work suggests that IRAK1 degraders have the potential for treating cancers that are dependent on the IRAK1 scaffolding function.
Diffuse large B-cell lymphoma (DLBCL) is the most
common aggressive non-Hodgkin lymphoma (NHL), account-
ing for about 30−35% of NHL cases around the world.
DLBCL can be divided into germinal center B-cell-like (GCB)
and activated B-cell-like (ABC) subtypes.3 ABC DLBCL is
associated with poor prognosis compared to the GCB subtype.
MyD88 gain-of-function mutation frequently appears in ABC
DLBCL, resulting in constitutive activation of the IRAK4- and
IRAK1-mediated signaling.4 However, in our previous efforts
to inhibit the MyD88 pathway, neither a selective IRAK4
kinase inhibitor nor a new series of potent and selective IRAK4
degraders that dismantle the IRAK4 scaffolding function
demonstrated noticeable anti-proliferation effects. These
INTRODUCTION
■
Interleukin-1 receptor-activated kinases (IRAK1, IRAK2,
IRAK3, and IRAK4) are serine/threonine kinases that mediate
innate immune and inflammatory responses. IRAKs play
important roles in transducing inflammatory signals down-
stream of the toll-like receptors (TLRs) and interleukin-1
receptors (IL-1Rs).1 When activated, TLRs or IL-1Rs dimerize
and recruit the myeloid differentiation primary response 88
(MyD88) adaptor protein through their intracellular domains.
MyD88 further recruits IRAK4 and IRA1/2 to form a
multiprotein complex, namely, the Myddosome. IRAK4
phosphorylates IRAK1 to first prime its activity, leading to
further activation, hyperphosphorylation, and dissociation from
the Myddosome and subsequent interaction with the E3
ubiquitin ligase, TNF receptor-associated factor 6 (TRAF6).
The activated TRAF6 complex in turn drives downstream
events, including the IκB kinase (IKK)-nuclear factor-κB (NF-
κB) and mitogen-activated protein kinase (MAPK) signaling
pathways that ultimately result in the upregulation of
proinflammatory cytokines.2
Received: January 20, 2021
https://doi.org/10.1021/acs.jmedchem.1c00103
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A

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Figure 1. Chemical structures of the IRAK1 kinase inhibitors and the co-crystal structure of JH-I-25 (green) with IRAK1 kinase (PDB: 6BFN).
The linker attachment point is highlighted by the white arrow.
Figure 2. Design of IRAK1-targeting PROTACs and the chemical structure of Degrader-1.
results suggest that IRAK4 is dispensable for ABC DLBCL cell
survival.5 On the other hand, genome-wide CRISPR screens
pointed to IRAK1 as an essential gene for the survival of ABC
DLBCL cells harboring MyD88 hotspot mutation.6,7 In a
recent publication, Gray’s group demonstrated the anti-
proliferative activity of an IRAK1 covalent inhibitor in ABC
DLBCL cell lines.8 Interestingly, this IRAK1 kinase inhibitor
JH-X-119-01 (Figure 1), which is very potent and selective in
enzymatic assays (IRAK1 IC50 of 8 nM), only modestly
inhibited the proliferation of ABC DLBCL cell lines with IC50
values in the micromolar range.8 These results suggested that
kinase inhibition alone was not sufficient to fully disrupt
myddosome signaling and effectively suppress lymphoma cell
growth. We therefore hypothesize that the kinase-independent
activities of IRAK1 are engaged and that the removal of the
protein scaffold functions may be required to achieve maximal
antagonism of the signaling pathway in MyD88 L265P mutant
DLBCL cells.
Here, we sought to eliminate the IRAK1 scaffolding function
using bivalent chemical degraders. Degraders of this type, often
represented as proteolysis targeting chimeras (PROTACs), are
heterobifunctional small molecules that can induce degradation
of a target protein by bringing it into proximity with an E3
ligase. When a ternary complex is formed, the E3 ligase
ubiquitinates the target protein, leading to its proteasomal
degradation.9−12 In this study, we report the design, synthesis,
and biological evaluation of a series of IRAK1-targeting
degraders. Several of these heterobifunctional molecules
could efficiently and selectively degrade IRAK1 protein at
low concentrations, providing new pharmacological tools to
probe for the IRAK1 scaffolding function. It was demonstrated
that IRAK1 PROTACs effectively inhibited IRAK1 down-
stream signaling pathways and led to strong growth inhibition
of MyD88-dependent ABC DLBCL cells.
RESULTS AND DISCUSSION
■
A set of published IRAK1 inhibitors were chosen as a starting
point to develop IRAK1 PROTACs. Compound JH-I-25
(Figure 1) was identified as a potent IRAK1/4 dual inhibitor
with the first IRAK1 co-crystal structure (PDB: 6BFN)
reported recently.13,14 This compound was also investigated
to understand and verify its IRAK1 kinase inhibition activity.
The crystal structure shows that the morpholine ring is in a
solvent-exposed region, indicating a suitable site for linker
attachment. Therefore, morpholine was replaced with piper-
azine to facilitate linker conjugation via amine alkylation
(Figure 1).
For E3 ligase ligand selection, cereblon (CRBN) ligand
thalidomide derivatives and the von Hippel−Lindau (VHL)
ligand VH032 are the most frequently used ligands in
PROTAC design.10 Previous studies indicated that thalido-
mide derivatives could potentially induce degradation of
multiple neo-substrates, such as Ikaros (IKZF1), Aiolos
(IKZF3), GSPT1, SALL4, and p63.15−18 Thalidomide
derivatives that induce degradation of Ikaros and Aiolos
could potentially lead to strong proliferation inhibition and
apoptosis in ABC DLBCL cell lines, confounding the
B
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a
Scheme 1. Synthesis of Degrader-1
a
Reagents and conditions: (a) 1-(tetrahydro-2H-pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyrazole, Pd(dppf)Cl2, Cs2CO3,
dioxane/H2O. (b) (i) NaOH, MeOH/H2O. (ii) HCl, dioxane. (c) tert-Butyl piperazine-1-carboxylate, K2CO3, DMF. (d) H2, Pd/C, MeOH. (e)
HATU, TEA, DMF. (f) HCl, EtOAc. (g) Methyl 3-bromopropanoate, TEA, KI, NMP, microwave. (h) NaOH, THF/H2O. (i) EDCI, HOBt,
DIPEA, DMF.
Table 1. Biochemical and Protein Degradation Data of Degrader-1 and Warhead Compound JH-I-25
a
a
b
c
compounds
IRAK1 IC50 (nM)
IRAK4 IC50 (nM)
IRAK1 Dmax(% of control)
IRAK1 DC50 (nM)
Degrader-1
JH-I-25
80
23
368
24
88
NA
134
NA
a
b
IRAK1 kinase inhibition potency (IC50) determined by a Caliper biochemical assay. Maximal IRAK1 and IRAK4 protein degradation (Dmax
)
c
determined by immunoblotting 24 h after treatment. IRAK1 degradation was calculated from quantified Western blots. Degradation potency
(DC50) of IRAK1 PROTACs determined by immunoblotting 24 h after treatment with degraders in the 293T cell line.
Figure 3. IRAK1 protein degradation profile in HEK293T cells for Degrader-1, Degrader-2, and Degrader-3. (A) Degradation effects of
Degrader-1 on IRAK1 proteins in HEK293T cells by Western blotting after 24 h treatment by Degrader-1 at illustrated concentrations. (B)
Characterization of Degrader-1 for its concentration- and time-dependent IRAK1 protein degradation by Western blot analysis. (C) Degrader-2
and Degrader-3 dose-dependently degrades IRAK1 protein after 24 h incubation. (D) Quantitation of DC50 values of Degrader-1, Degrader-2,
and Degrader-3 after 24 h incubation.
interpretation of IRAK1-dependent effects.19 To avoid
potential off-target degradation from the CRBN E3 ligase
and enable conclusive data for IRAK1 degradation validation,
the VHL E3 ligase ligand VH032 was selected for IRAK1
PROTAC design and connected to the anchor point of the
piperazine ring moiety from the JH-1-25 IRAK1 inhibitor via
C
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a
Scheme 2. Synthesis of Degrader-2, Degrader-3, and Degrader-4
a
Reagents and conditions: (a) tert-butyl 2-bromoacetate, NaH, THF; (b) Pd/C, H2 (30 psi), MeOH; (c) HATU, DIPEA, DMF; (d) HCl (4 M in
dioxane); and (e) EDCI, HOBt, DIPEA, DMF.
Table 2. Profiles of IRAK1 PROTACs with Different Linker Lengths and Compositions
a
a
b
c
d
compound
Degrader-1
Degrader-2
Degrader-3 (JNJ-1013)
Degrader-4
JH-I-25
IRAK1 IC50 (nM)
IRAK4 IC50 (nM)
VHL FP IC50 (nM)
IRAK1 Dmax (% of control)
IRAK1 DC50 (nM)
80
60
72
79
23
368
260
443
320
24
1646
1400
1071
>10,000
NA
88
95
96
18
NA
134
16
3.3
>10,000
NA
a
b
IRAK1 kinase inhibition potency (IC50) determined by a Caliper biochemistry assay. Binding affinity of degraders to VHL protein (IC50)
c
determined by an FP assay. Maximal IRAK1 and IRAK4 protein degradation (Dmax) determined by immunoblotting 24 h after treatment with
degraders. Degradation potency (DC50) of IRAK1 degraders determined by immunoblotting 24 h after treatment.
d
different lengths and composition linkers (e.g., alkyl linkers and
polyethylene glycol linkers, Figure 2).20,21
for IRAK1 was achieved through heterobifunctional degraders
over functional inhibition selectivity by JH-I-25 and its
degrader analogues. This suggests that the basis of degradation
selectivity may reside in protein−protein interactions between
the VHL E3 ubiquitin ligase and the targeted protein, that is,
the ternary complex.24 In other words, the engaged proteins
may not lead to a productive ternary complex in the case of
IRAK4. Independent experiments are in progress to confirm
this hypothesis.
To evaluate the kinetics of IRAK1 degradation, we
performed time course experiments in HEK293T cells by
Western blot analysis using different concentrations of
Degrader-1. Degradation occurred rapidly, with over 50%
IRAK1 degradation achieved in 2 h across all ≥ 1 μM doses,
and over 90% of protein degradation was achieved after 8 h
(Figure 3B). At 10 μM, reduced degradation of the IRAK1
protein was observed, which is consistent with the “hook
effect” commonly observed at high concentrations of
PROTACs where individual binary complexes become
saturated and compete for the formation of the E3 ligase−
substrate−ligand ternary complex, causing the loss of
degradation efficiency.25
To generate the degrader compounds, a convergent
synthesis method (Scheme 1) was developed to connect the
piperazine group of the IRAK1 warhead with different linkers
and a VHL amine intermediate (Figure 2).22,23 After library
synthesis, the degradation potency (Dmax and DC50) of these
PROTAC degraders was assessed by immunoblotting assays in
HEK293T cells after 24 h of treatment. Interestingly,
Degrader-1 with the shortest linker (two methylene units)
showed the most promising profile, demonstrating a DC50 of
134 nM and a Dmax of 88% at 10 μM (Table 1, Figures 2 and
3A). We also profiled the in vitro biochemical properties of
both Degrader-1 and JH-I-25. Table 1 summarizes the
biochemical activity, determined by a Caliper kinase activity
assay, of the designed PROTACs and the parent ligand to
IRAK1 and IRAK4. The degrader molecule maintained a high-
double-digit nM IRAK1 biochemical potency that was
comparable to that of its parent compound JH-I-25,
confirming the success of the rationally designed attachment
vector. In an IRAK4 biochemical assay, however, the potency
of compound Degrader-1 dropped 15-fold, presumably
because the front pocket of IRAK4 is largely occluded, while
the front pocket of IRAK1 is open and accessible for binding.14
Interestingly, no IRAK4 protein degradation was observed with
Degrader-1 (Figure S4) even though it exhibited reasonable
IRAK4 biochemical potency with an IC50 of 368 nM. Despite
the high sequence similarity (>90%) of IRAK1 and IRAK4 in
their ATP binding pockets, significant degradation selectivity
To our delight, through the replacement of the N-linked
piperazine ring in the linker region of Degrader-1 with an O-
linked piperidine version (Scheme 2), the DC50 of Degrader-2
was improved to a low-double-digit nM range with a Dmax of
95% (Table 2). Meanwhile, the addition of a methyl group in
the S-configuration in the VHL binder moiety of Degrader-2
yielded a 5-fold more potent compound, Degrader-3 (JNJ-
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Figure 4. Mechanistic study of Degrader-3 in HEK293T cells by Western blotting analysis. (A) Degrader-3-mediated IRAK1 degradation is
abrogated by pretreatment with the proteasome inhibitor MG-132. (B) Degrader-3-mediated IRAK1 degradation is abrogated by pretreatment
with the VHL ligand VH298.
Figure 5. TREEspot interaction map of kinases for JH-I-25, Degrader-3, and Degrader-4 from the KinomeScan competition binding assay profiled
at 1 μM concentration.
1013, Table 2). Both PROTACs effectively degraded IRAK1
protein in a dose-dependent manner (Figure 3C,D).
Interestingly, in a direct comparison, Degrader-1, Degrader-
2, and Degrader-3 did not exhibit distinct potency differences
in IRAK1 biochemical activity or in the VHL fluorescence
polarization (FP) binding assay (Table 2). This again indicates
that there is no significant correlation between the binary
binding potency and degradation potency. In addition,
Degrader-4, the diastereoisomer of Degrader-3 with an
inverted hydroxyl stereocenter, was designed as a reference.
through the proteasome mediated by the VHL E3 ubiquitin
ligase.
To compare biochemical selectivity profiles, we submitted
JH-I-25, Degrader-3, and Degrader-4 for the KinomeScan
competition binding assay panel (DiscoverX), which comprises
469 kinases. JH-I-25 is a relatively selective IRAK1 inhibitor as
a limited number of off-targets were inhibited at 1 μM, as
shown in Figure 5. Both Degrader-3 and Degrader-4
degraders demonstrated higher selectivity toward IRAK1
compared to JH-I-25 as fewer off-targets were inhibited in
the KinomeScan assay. Conversion of JH-I-25 into a
PROTAC retained the IRAK1 binding but significantly
reduced the binding affinity for the other members of the
kinase superfamily that had been inhibited by JH-I-25.
Compared to the 17 off-targets with more than 90% inhibition
at 1 μM from JH-I-25, only 3 off-targets were identified for
Degrader-3 (BIKE, DRAK2, and YSK4). It is worth
mentioning that the VHL epimer control compound
Degrader-4 showed an almost identical kinome profile to
that of Degrader-3.
As expected, Degrader-4 lost the VHL binding affinity (IC50
>
10 μM in a VHL FP assay) and did not show significant
IRAK1 degradation activity (DC50 > 10 μM).
Next, Degrader-3 was selected as a representative IRAK1
PROTAC for further evaluation. To verify that the degradation
of IRAK1 protein was mediated through the ubiquitination and
proteasome system, we utilized a proteasome inhibitor, MG-
132, to disrupt proteasome function and a VHL ligand,
VH298, to compete for VHL binding.21 As shown in Figure
4A,B, IRAK1 degradation was completely prevented by either
the proteasome inhibitor MG-132 or competitive VHL ligand
VH298, confirming that the degradation of IRAK1 protein is
To determine the global proteome-wide degradation profiles
of the IRAK1 degraders, an unbiased quantitative mass
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spectrometry-based approach was employed. A GCB DLBCL
cell line (Toledo) was selected due to sufficient expression of
endogenous IRAK1 within this line, allowing simultaneous
detection of IRAK1 loss with other key components of the NF-
κB machinery. In the experiment, Toledo cells were treated
with either DMSO (control) or Degrader-3 at 1 μM for 6 h
before the cell lysates were collected (Figure S1). Proteome-
wide quantitative mass spectrometric analysis was then
performed as described in the Supporting Information.
Significance cutoff was set at a 1.5-fold change with a
minimum of two unique peptide measurements and p-values
of less than 0.05. In total, 7550 proteins were successfully
quantified in this experiment, among which IRAK1, cyclin G-
associated kinase (GAK), and NmrA-like family domain-
containing protein 1 (NMRAL1) were the only three proteins
which exhibited a significant decrease in abundance after
Degrader-3 treatment (Figure 6). The proteome analysis
that Degrader-3 acts as a potent and selective IRAK1
degrader.
Next, utilizing the highly selective IRAK1 PROTAC
Degrader-3 and its epimer control Degrader-4, we aimed to
investigate the biological consequence of IRAK1 depletion in
ABC DLBCL cell lines harboring MyD88 L265P mutation and
dependent on the MyD88 signaling pathway for survival, such
as HBL-1 and OCI-LY10. We initially evaluated the IRAK1
degradation effect by immunoblotting analysis after 24 h
treatment with degrader compounds in both ABC DLBCL cell
lines. Although Degrader-3 demonstrated comparable IRAK1
degradation potency in HBL-1 cell lines compared to
HEK293T cells with DC50 values of 3.0 and 3.3 nM,
respectively, a decreased degradation potency was observed
in the OCI-LY10 cell line with a DC50 of 20 nM (Figure 7A).
To evaluate the effects of IRAK1 depletion on the IRAK1
downstream signaling pathway in ABC DLBCL cell lines,
HBL-1 cells were treated with Degrader-3 and its epimer
Degrader-4 for 24 h before lysis and immunoblotting to detect
phosphorylation events downstream of IRAK1 signaling. As
shown in Figure 7B, Degrader-3 resulted in a significant
decrease in p-IκBα and p-STAT3 and minimal changes in total
STAT3 levels, consistent with the notion that oncogenic
MyD88/IRAK signaling converges on NF-κB activation and
STAT3 phosphorylation and contributes to the survival of
DLBCL cells.26,27 Not surprisingly, although the epimer
control compound Degrader-4 binds to the IRAK1 kinase
domain with the same affinity (Table 2) and thus may act as an
IRAK1 kinase inhibitor, it was unable to degrade IRAK1 at all
doses tested. Minimal changes in cell signaling were observed
by Degrader-4, supporting the hypothesis that the IRAK1
protein scaffolding function, as opposed to its kinase activity, is
important in maintaining constitutive NF-κB activation in ABC
DLBCL cells.4,6
We then evaluated the effects of targeted IRAK1 degradation
on cellular apoptosis. Accordingly, a 24 h treatment of
Degrader-3 in HBL-1 cell lines increased apoptosis, as
monitored by PARP cleavage in a Western blot assay. As
expected, treatment with equivalent doses of Degrader-4 did
not induce IRAK1 protein degradation and produced no
change in PARP cleavage (Figure 7C). These data suggest that
the IRAK1 protein scaffolding function plays an important role
in maintaining constitutive NF-κB and STAT3 activation in
ABC DLBCL cells.
Next, we sought to determine the effect of IRAK1 depletion
on ABC DLBCL cell proliferation. HBL-1 and OCI-LY10 cells
with MyD88 L265P mutation were treated with Degrader-3
for 12 days, and cell viability was determined by CTG assays.
In line with the requirement for VHL engagement, we also
profiled the control epimer Degrader-4. In the HBL-1 cell line,
Degrader-3 exhibited significantly enhanced cytotoxicity as
compared with Degrader-4 (60 nM vs > 1.5 μM); similar
trends were observed in OCI-LY10 cell lines (170 nM vs > 3
μM) (Figure 7D). In contrast, JH-I-25, a dual kinase inhibitor
of IRAK1 and IRAK4, showed marginal cell growth inhibition
in HBL1 with an IC50 of 2.8 μM (Figure S3). The results
demonstrate the distinct advantage of IRAK1 degradation over
kinase inhibition as the degrader showed significantly
improved anti-proliferative effects (over 40-fold) compared
to the kinase inhibitor in HBL-1. Taken together, these data
suggest that the IRAK1 scaffolding function, rather than its
kinase activity, is required for ABC DLBCL cell survival and
proliferation.
Figure 6. Volcano plots of abundance proteomics data of Degrader-3
by proteomics analysis. Log fold change in abundance of protein with
two unique peptide measurement using quantitative mass spectrom-
etry after 6 h treatment of Toledo cells with 1 μM Degrader-3.
showed that the degradation profile was far more selective than
the kinase binding profile, where only the kinases IRAK1 and
GAK were decreased, while the changes for other kinase off-
targets were negligible. It is noteworthy to point out that
similar proteomics assays were carried out in the ABC-DLBCL
cell line HBL1, where low endogenous levels of IRAK1 were
detectable only by Western blotting but were beyond the
sensitivity of mass spectrometry. GAK and NMRAL1 proteins
were also the only two off-targets which were observed after
treatment of Degrader-3 at 1 μM for 6 h (Figure S2).
Surprisingly, though Degrader-3 did not exhibit a strong GAK
binding affinity in the KinomeScan competition binding assay
(63% inhibition at 1 μM), it degraded GAK to 50% at 1 μM
after 6 h treatment in the quantitative mass spectroscopy
experiment. Our efforts to confirm the finding is limited by the
availability of a specific antibody. On the other hand,
degradation of NMRAL1 by Degrader-3 was reproduced in
both HEK293T and HBL-1 cells by Western blotting (data not
shown). The degradation mechanism for NMRAL1 is
inconclusive since the protein is not a kinase, and degradation
may be mediated through other mechanisms which are under
further investigation. Consistent with the findings from the
proteomics study, Degrader-3 degraded only IRAK1, not
IRAK4, as shown by Western blotting analysis (Figure S4).
Collectively, the KinomeScan and proteomics data support
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Figure 7. Characterization of Degrader-3 and Degrader-4 in ABC DLBCL cell lines. (A) DLBCL cell lines were treated with Degrader-3 at the
indicated concentrations for 24 h. IRAK1 protein levels were determined by Western blotting. (B) HBL-1 cells were treated with Degrader-3 or its
epimer Degrader-4 at the indicated doses for 24 h. Phosphorylated IkBα and STAT3 protein levels were determined by Western blotting. (C)
Cleaved PARP was determined by Western blotting. (D) Cell proliferative assay in HBL-1 and OCI-LY10 cells with MyD88 mutation.
specifically indicated, chromatography refers to flash chromatography
on a silica gel column.
6-(1H-Pyrazol-5-yl)picolinic Acid Hydrochloride (2). Step 1:
Ethyl 6-bromopicolinate (1) (15.0 g, 65.2 mmol), 1-(tetrahydro-2H-
CONCLUSIONS
■
We have discovered a novel series of VHL E3 ligase-based
IRAK1 PROTAC degraders. One of the most potent
compounds, Degrader-3 (JNJ-1013), effectively degrades
pyran-2-yl)-5-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-yl)-1H-pyra-
IRAK1 with a DC50 of 3 nM in HBL-1 cells. In ABC
DLBCL cell lines with MyD88 L265P mutation, JNJ-1013
potently inhibits IRAK1 downstream signaling pathways,
induces apoptosis, and demonstrates strong anti-proliferative
effects, suggesting a critical role for the IRAK1 scaffolding
function in ABC DLBCL cell survival. This class of highly
selective IRAK1 degraders offers a pharmacological approach
to evaluate the biological roles of IRAK1 beyond its enzymatic
activity in various disease indications.
zole (19.9 g, 71.5 mmol), and cesium carbonate (63.7 g, 196 mmol)
in 1,4-dioxane (200 mL) and H2O (50 mL) were added to a 500 mL
round-bottomed flask. The mixture was sparged with nitrogen three
times and then treated with Pd(dppf)Cl2 (3.62 g, 6.53 mmol). Then,
the mixture was sparged with nitrogen three times and heated to 90
°C for 16 h. The reaction mixture was filtered and diluted with ethyl
acetate (100 mL). The aqueous solution was acidified to pH = 5−6 by
citric acid, extracted with dichloromethane (100 mL × 5), washed
with brine, and dried over Na2SO4. The organic layer was filtered and
concentrated under reduced pressure to give the title compound as a
gray solid (15.0 g, 76%). LCMS (ESI+): calcd for C16H19N3O3 =
301.1, found; [M + H]+ = 302.2, tR = 1.10 min.
EXPERIMENTAL SECTION
■
Chemistry. All commercially obtained solvents and reagents were
used as received. All solvents used for chemical reactions were of
anhydrous grade, unless specifically indicated. Structures of the target
compounds in this work were assigned by the use of NMR
spectroscopy and mass spectroscopy (MS). The purities of all non-
salt compounds were >95%, as determined on an Agilent 1200 HPLC
system, an XTerra 3.5 μm 4.6 × 150 mm MS C18 column, using 0.04
(v/v) TFA in water and 0.02 (v/v) TFA in acetonitrile as the mobile
Step 2: Ethyl 6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-5-yl)-
picolinate (15.0 g, 49.8 mmol) and NaOH (2.39 g, 59.8 mmol) in
MeOH (45 mL) and H2O (45 mL) were added to a 250 mL round-
bottomed flask. The mixture was stirred at 20 °C for 2 h. The reaction
mixture was concentrated, and the residue was diluted with H2O (50
mL). The mixture was acidified to pH = 5−6 by citric acid, extracted
with dichloromethane (100 mL × 5), washed with brine, and dried
over Na2SO4. The organic layer was filtered and concentrated under
reduced pressure to give 6-(1-(tetrahydro-2H-pyran-2-yl)-1H-pyrazol-
5-yl)picolinic acid (12.0 g, 75.0%) as a gray solid. The crude product
was then dissolved in dichloromethane (20 mL) and HCl/dioxane
(50 mL, 4 M) and added to a 250 mL round-bottomed flask. The
reaction mixture was stirred at 25 °C for 1 h. Then, the mixture was
filtered, and the filter cake was dried to give the title product as a
1
phase. H NMR spectra were recorded on a Bruker ADVANCE III
(400 MHz) or a Varian 400MR (400 MHz) NMR spectrometer.
Chemical shifts are reported in parts per million (ppm, δ) using the
residual solvent line as an internal reference. Splitting patterns are
reported as s (singlet), d (doublet), t (triplet), q (quartet), m
(multiplet), br m (broad multiplet), or br s (broad singlet). Coupling
constants (J) are reported in Hertz (Hz). Mass spectrometric analyses
for nucleosides were performed on an Agilent 1200 HPLC system
with an Agilent 6110/6140/1956C MSD mass spectrometer using
ESI as ionization, a Phenomenex Luna C18 5 μm 5.0 × 20 mm
column, mobile phase: 0.04%(v/v) TFA in water and 0.02%(v/v)
TFA in acetonitrile, 40 °C, and a flow rate of 0.4 mL/min. Unless
1
white solid (9.8 g, 98.3%). H NMR (400 MHz, DMSO-d6): δ 10.67
(br s, 2H), 8.17 (dd, J = 0.8, 7.6 Hz, 1H), 8.05 (t, J = 7.6 Hz, 1H),
7.99−7.94 (m, 1H), 7.81 (d, J = 2.0 Hz, 1H), 7.01 (d, J = 2.0 Hz,1H).
LCMS (ESI+) calcd for C9H7N3O2 = 189.0, found; [M + H]+ = 189.9,
tR = 0.84 min.
G
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1
tert-Butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-car-
boxylate (4). Step 1: 4-Fluoro-2-methoxy-1-nitrobenzene (3) (10.0
g, 58.4 mmol), tert-butyl piperazine-1-carboxylate (11.4 g, 61.2
mmol), potassium carbonate (20.2 g, 87.7 mmol), and DMF (80 mL)
were added to a 250 mL round-bottomed flask. The reaction mixture
was stirred at 80 °C for 16 h. The reaction mixture was poured into
150 mL water and stirred at 25 °C for 0.5 h. The mixture was filtered,
the cake was washed with petroleum ether (60 mL × 2), and the
resultant mixture was concentrated to dryness under reduced pressure
H]+ = 465.2, tR = 0.67 min. H NMR (400 MHz, DMSO-d6): δ =
13.88−13.23 (br s, 1H), 10.58−10.40 (br s, 1H), 8.30−7.45 (m, 5H),
7.06−6.91 (m, 1H), 6.78−6.63 (m, 1H), 6.54 (d, J = 8.8 Hz, 1H),
4.02−3.82 (m, 3H), 3.61 (s, 3H), 3.18−3.12 (m, 4H), 2.66−2.59 (m,
2H), 2.54 (d, J = 6.1 Hz, 4H), 2.20−2.16 (m, 2H).
3 - ( 4 - ( 4 - ( 6 - ( 1 H - P y r a z o l - 5 - y l ) p i c o l i n a m i d o ) - 3 -
methoxyphenyl)piperazin-1-yl)propanoic Acid (7). Methyl 3-
(4-(4-(6-(1H-pyrazol-5-yl)picolinamido)-3-methoxyphenyl)-
piperidin-1-yl)propanoate (6) (150 mg, 0.323 mmol), aqueous
NaOH (6 mL, 1 M), and THF (3 mL) were added to a 50 mL
round-bottomed flask. The reaction mixture was stirred at 20 °C for 1
h. The reaction mixture was concentrated with pH adjusted to 6−7
with aqueous HCl (1 M), and a pale-yellow precipitate was formed.
The precipitate was filtrated and dried to give the title product as a
yellow solid (120 mg, 82.5%). LCMS (ESI+): calcd for C23H26N6O4 =
450.2, found; [M + H]+ = 451.0, tR = 0.77 min. 1H NMR (400 MHz,
DMSO-d6): δ = 13.83−12.25 (m, 1H), 10.70−10.42 (m, 1H), 8.23−
7.98 (m, 4H), 7.92−7.79 (m, 1H), 7.02−6.96 (m, 1H), 6.84−6.77
(m, 1H), 6.65−6.57 (m, 1H), 4.06−3.92 (m, 3H), 3.35−3.03 (m,
11H), 2.93−2.78 (m, 2H).
(2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-
(4-(4-methylthiazol-5-yl)benzyl)pyrrolidine-2-carboxamide
Hydrochloride (8). The free amine compound was prepared
according to the procedure published.28 tert-Butyl ((S)-1-((2S,4R)-
4-hydroxy-2-((4-(4-methylthiazol-5-yl)benzyl)carbamoyl)pyrrolidin-
1-yl)-3,3-dimethyl-1-oxobutan-2-yl)carbamate (1.00 g, 1.88 mmol)
and 4 M HCl in dioxane (8 mL) were added into a 50 mL round-
bottomed flask. The mixture was stirred at 25 °C for 30 min. The
reaction mixture was concentrated to dryness under reduced pressure
to afford the title product as a pale white solid (880 mg, crude). Data
match the previous reports.
N-(4-(4-(3-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-
5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobu-
tan-2-yl)amino)-3-oxopropyl)piperazin-1-yl)-2-methoxyphen-
yl)-6-(1H-pyrazol-3-yl)picolinamide (Degrader-1). 3-(4-(4-(6-
(1H-Pyrazol-3-yl)picolinamido)-3-methoxyphenyl)piperazin-1-yl)-
propanoic acid (7) (100 mg, 0.212 mmol), 1-hydroxybenzotriazole
(43.0 mg, 0.318 mmol), N-ethyl-N-isopropylpropan-2-amine (138
mg, 1.07 mmol), 1-(3-dimethylaminopropyl)-3-ethylcarbodiimide
hydrochloride (61.0 mg, 0.318 mmol), and DMF (3 mL) were
added to a 50 mL round-bottomed flask. (2S,4R)-1-((S)-2-Amino-
3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methylthiazol-5-yl)benzyl)-
pyrrolidine-2-carboxamide·HCl (8) (99.0 mg, 0.212 mmol) was
added to the mixture. The reaction mixture was stirred at room
temperature for 8 h. The mixture was then poured into water (8 mL)
and extracted with ethyl acetate (15 mL × 2). The organic extracts
were washed with brine (8 mL), dried over anhydrous Na2SO4,
filtered, and concentrated under reduced pressure to afford the crude
product, which was purified by prep-TLC (dichloromethane:metha-
nol = 10:1, Rf = 0.4) to afford the title product as a yellow powder
(50.2 mg, 27.1% yield). 1H NMR (400 MHz, DMSO-d6): δ 13.90 (br
s, 0.3H), 13.24 (br s, 0.7H), 10.66−10.21 (m, 1H), 9.02−8.87 (m,
1H), 8.73−8.43 (m, 2H), 8.32−7.51 (m, 5H), 7.48−7.27 (m, 4H),
7.07−6.90 (m, 1H), 6.81−6.62 (m, 1H), 6.58−6.46 (m, 1H), 5.17 (d,
J = 3.4 Hz, 1H), 4.58 (d, J = 9.5 Hz, 1H), 4.50−4.32 (m, 3H), 4.28−
4.16 (m, 1H), 4.03−3.77 (m, 3H), 3.72−3.59 (m, 2H), 3.31−3.14
(m, 4H), 2.71−2.53 (m, 6H), 2.49−2.44 (m, 1H), 2.40 (s, 3H),
2.37−2.29 (m, 1H), 2.07−2.00 (m, 1H), 1.96−1.87 (m, 1H), 0.96 (s,
9H). 13C NMR (100 MHz, DMSO-d6): δ 172.5, 171.4, 170.1, 160.9,
151.7, 151.6, 150.4, 149.9, 149.5, 148.7, 148.1, 139.9, 139.5, 131.6,
130.9, 130.1, 129.1, 128.5, 127.8, 122.4, 120.5, 120.0, 119.6, 107.2,
103.5, 100.1, 69.4, 59.2, 56.9, 56.7, 56.6, 54.5, 52.8, 48.9, 42.1, 40.6,
38.4, 35.9, 32.8, 26.9, 16.4. LCMS (ESI+): calcd for C45H54N10O6S =
862.4, found, [M + H]+ = 863.4, tR = 0.4 min. HRMS (ESI): m/z [M
+ H]+ Calcd for C45H54N10O6S, 863.4027; found, 863.4036.
1
to afford the desired product as a yellow solid (19.0 g, 91.2%). H
NMR (400 MHz, DMSO-d6): δ 7.89−7.82 (m, 1H), 6.58−6.51 (m,
1H), 6.50−6.45 (m, 1H), 3.87 (s, 3H), 3.45−3.40 (m, 8H), 1.38 (s,
9H). LCMS (ESI+): calcd for C16H23N3O5 = 337.2, found; [M + H]+
= 338.1, tR = 0.88 min.
Step 2: tert-Butyl 4-(3-methoxy-4-nitrophenyl)piperazine-1-carbox-
ylate (56.0 g, 166 mmol), wet Pd/C (5.00 g, 10% on activated
carbon), and MeOH (1 L) were added to a 2 L reaction flask. The
suspension was degassed under vacuum and purged with H2 three
times. The mixture was stirred under H2 (30 psi) at 25 °C for 12 h.
The mixture was filtered through a pad of celite, and the filter cake
was washed with MeOH (50 mL). The filtrate was evaporated to
dryness to give the title compound as a purple solid (55.0 g, 97.0%).
1H NMR (400 MHz, DMSO-d6): δ 6.57−6.47 (m, 2H), 6.35−6.26
(m, 1H), 4.51−4.10 (m, 2H), 3.74 (s, 3H), 3.50−3.39 (m, 4H),
2.93−2.81 (m, 4H), 1.41 (s, 9H). LCMS (ESI+): calcd for
C16H25N3O3 = 307.2, found; [M + H]+ = 308.3, tR = 0.74 min.
tert-Butyl 4-(4-(6-(1H-pyrazol-5-yl)picolinamido)-3-
methoxyphenyl)piperazine-1-carboxylate (5). A solution of
tert-butyl 4-(4-amino-3-methoxyphenyl)piperazine-1-carboxylate (4)
(10.0 g, 32.5 mmol), 6-(1H-pyrazol-5-yl)picolinic acid hydrochloride
(2) (6.15 g, 32.5 mmol), HATU (14.8 g, 38.9 mmol), and TEA (13.6
mL, 97.6 mmol) in DMF (100 mL) was stirred at 25 °C for 8 h. The
reaction mixture was diluted with water (200 mL), extracted with
ethyl acetate (300 mL × 2), washed with brine (200 mL × 3), and
dried over Na2SO4. The organic layer was filtered and concentrated
under reduced pressure to give a crude product, which was purified by
chromatography on silica gel (eluentpetroleum ether:ethyl acetate
= 1:0 to 0:1) to give the title compound as a gray solid (11.0 g,
1
70.7%). H NMR (400 MHz, DMSO-d6): δ 13.94−13.17 (m, 1H),
10.65−10.36 (m, 1H), 8.29−8.18 (m, 1H), 8.14−8.00 (m, 2H), 7.94
(s, 1H), 7.67−7.57 (m, 1H), 7.04−6.96 (m, 1H), 6.79−6.71 (m, 1H),
6.61−6.55 (m, 1H), 3.99 (s, 2H), 3.86 (s, 1H), 3.48 (br s, 4H), 3.13
(d, J = 4.5 Hz, 4H), 1.43 (s, 9H). LCMS (ESI+): calcd for
C25H30N6O4 = 478.2, found; [M + H]+ = 479.1, tR = 0.925 min.
Methyl 3-(4-(4-(6-(1H-Pyrazol-5-yl)picolinamido)-3-
methoxyphenyl)piperazin-1-yl)propanoate (6). Step 1: tert-
Butyl 4-(4-(6-(1H-pyrazol-5-yl)picolinamido)-3-methoxyphenyl)-
piperazine-1-carboxylate (8.50 g, 17.8 mmol) and HCl in EtOAc
(80 mL, 4 M) were added to a 250 mL round-bottomed flask. The
reaction mixture was stirred at room temperature for 2 h. The mixture
was concentrated under reduced pressure to give the title compound
1
as a white solid (7.00 g, 93.9%). H NMR (400 MHz, DMSO-d6): δ
10.58 (s, 1H), 9.68−9.31 (m, 2H), 8.20−8.00 (m, 4H), 7.91−7.85
(m, 1H), 7.00 (s, 1H), 6.87−6.80 (m, 1H), 6.69−6.63 (m, 1H), 3.96
(s, 3H), 3.53−3.39 (m, 4H), 3.32−3.12 (m, 4H). LCMS (ESI+):
calcd for C20H22N6O2 = 378.2, found; [M + H]+ = 379.2, tR = 0.69
min.
Step 2: Methyl 3-bromopropanoate (100 mg, 0.599 mmol), N-(2-
methoxy-4-(piperidin-4-yl)phenyl)-6-(1H-pyrazol-5-yl)picolinamide
hydrochloride (249 mg, 0.600 mmol), triethylamine (0.8 mL, 5.74
mmol), potassium iodide (99.0 mg, 0.596 mmol), and 1-methyl-2-
pyrrolidinone (2 mL) were added to a 10 mL sealed tube. The
mixture was heated and stirred at 100 °C under a microwave initiator
for 2 h. The mixture was then poured into water (10 mL) and
extracted with ethyl acetate (20 mL × 2). The organic extracts were
dried over anhydrous Na2SO4, filtered, and concentrated under
reduced pressure to afford the crude product, which was purified by
chromatography on silica gel (eluentdichloromethane:methanol =
1:0 to 10:1) to afford the title product as a brown oil (150 mg,
48.5%). LCMS (ESI+): calcd for C24H28N6O4 = 464.2, found; [M +
tert-Butyl 2-((1-(3-methoxy-4-nitrophenyl)piperidin-4-yl)-
oxy)acetate (10). 1-(3-Methoxy-4-nitrophenyl)piperidin-4-ol (500
mg, 1.98 mmol) (9) and THF (20 mL) were added to a 50 mL
round-bottomed flask at 0 °C before being treated with sodium
hydride (119 mg, 2.97 mmol), and the mixture was stirred at 0 °C for
H
https://doi.org/10.1021/acs.jmedchem.1c00103
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Article
5 min. The reaction mixture was treated with tert-butyl 2-
bromoacetate (770 mg, 3.96 mmol) at 0 °C, warmed, and stirred at
25 °C for 16 h. Brine (10 mL) was added to the mixture, and the
mixture was concentrated under reduced pressure to remove THF
before being diluted with H2O (10 mL); then, the mixture was
extracted with EtOAc (30 mL × 3). The combined organic extracts
were dried over anhydrous Na2SO4, filtered, and concentrated to
dryness under reduced pressure to give the crude product, which was
purified by chromatography on silica gel (eluentpetroleum
ether:ethyl acetate = 100:0 to 1:1) to afford the desired compound
was prepared according to the procedure of J. Med. Chem. 2019, 62,
1420−1442. Data matched previous reports.
N-(4-(4-(2-(((S)-1-((2S,4R)-4-Hydroxy-2-((4-(4-methylthiazol-
5-yl)benzyl)carbamoyl)pyrrolidin-1-yl)-3,3-dimethyl-1-oxobu-
tan-2-yl)amino)-2-oxoethoxy)piperidin-1-yl)-2-methoxyphen-
yl)-6-(1H-pyrazol-5-yl)picolinamide (Degrader-2). 2-((1-(4-(6-
(1H-pyrazol-5-yl)picolinamido)-3-methoxyphenyl)piperidin-4-yl)-
oxy)acetic acid hydrochloride (13) (100 mg, 0.205 mmol), (2S,4R)-
1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide hydrochloride (8)
(95.7 mg, 0.205 mmol), 1-hydroxybenzotriazole (41.5 mg, 0.307
mmol), DIPEA (79.5 mg, 0.615 mmol), and DMF (2 mL) were
added to a 50 mL round-bottomed flask. Then, 1-(3-dimethylamino-
propyl)-3-ethylcarbodiimide hydrochloride (58.9 mg, 0.307 mmol)
was added to the mixture. The reaction mixture was stirred at 40 °C
for 2 h. The mixture was purified by prep-HPLC (Column: Agela
Durashell C18 150*25 5 μm, mobile phase A: water (10 mM
NH4HCO3)−ACN, mobile phase B: acetonitrile, flow rate: 25 mL/
min, gradient condition from 30% B to 60%). The pure fractions were
collected, and the solvent was evaporated under vacuum to give a
residue, which was lyophilized to give the title product as a yellow
1
as a brown oil (650 mg, 76%). H NMR (400 MHz, DMSO-d6): δ
7.87 (d, J = 9.6 Hz, 1H), 6.59 (dd, J = 2.4, 9.2 Hz, 1H), 6.52−6.49
(m, 1H), 4.05 (s, 2H), 3.90 (s, 3H), 3.81−3.73 (m, 2H), 3.66−3.59
(m, 1H), 3.28−3.19 (m, 2H), 1.95−1.86 (m, 2H), 1.56−1.46 (m,
2H), 1.43 (s, 9H).
tert-Butyl 2-((1-(4-Amino-3-methoxyphenyl)piperidin-4-yl)-
oxy)acetate (11). tert-Butyl 2-((1-(3-methoxy-4-nitrophenyl)-
piperidin-4-yl)oxy)acetate (10) (650 mg, 1.77 mmol), wet Pd/C
(200 mg), and MeOH (20 mL) were added to a hydrogenation bottle
under a N2 atmosphere. The suspension was degassed under vacuum,
purged with N2 three times, and then purged with hydrogen another
three times. The resulting mixture was stirred under hydrogen (30
psi) at 25 °C for 16 h. The mixture was filtered through a pad of
celite, and the filter cake was washed with methanol (30 mL × 3).
The combined filtrates were concentrated under reduced pressure to
give the product as a brown oil (450 mg, 64%). 1H NMR (400 MHz,
DMSO-d6): δ 6.52−6.47 (m, 2H), 6.31−6.26 (m, 1H), 4.26−4.13
(m, 2H), 4.01 (s, 2H), 3.73 (s, 3H), 3.45−3.40 (m, 1H), 3.27−3.21
(m, 2H), 2.68−2.61 (m, 2H), 1.92 (m, 2H), 1.60−1.51 (m, 2H), 1.43
(s, 9H). LCMS (ESI+): calcd for C18H28N2O4 = 336.2, found; [M +
H]+ = 337.2, tR = 0.70 min.
tert-Butyl 2-((1-(4-(6-(1H-pyrazol-5-yl)picolinamido)-3-
methoxyphenyl)piperidin-4-yl)oxy)acetate (12). tert-Butyl 2-
((1-(4-amino-3-methoxyphenyl)piperidin-4-yl)oxy)acetate (11) (250
mg, 0.743 mmol), 6-(1H-pyrazol-5-yl)picolinic acid hydrochloride
(2) (140 mg, 0.740 mmol), HATU (171 mg, 0.892 mmol), DIPEA
(301 mg, 2.23 mmol), and DMF (3 mL) were added to a 10 mL
round-bottomed flask. The reaction mixture was stirred at 25 °C for 2
h. The reaction mixture was concentrated under reduced pressure to
obtain a residue, which was diluted with water (30 mL) and extracted
with ethyl acetate (50 mL × 3). The combined organic layer was
dried over anhydrous Na2SO4, filtered, and concentrated to dryness
under reduced pressure to afford the crude product, which was
purified by chromatography on silica gel (eluentpetroleum
ether:ethyl acetate = 1:4 to 1:2) to give the desired product as a
brown−green oil (216 mg, 57%). 1H NMR (400 MHz, DMSO-d6): δ
13.12 (s, 1H), 10.44 (s, 1H), 8.09−7.71 (m, 5H), 6.89 (d, J = 2.0 Hz,
1H), 6.62 (d, J = 2.0 Hz, 1H), 6.46 (dd, J = 2.4, 8.8 Hz, 1H), 4.02−
3.90 (m, 2H), 3.85 (s, 3H), 3.43 (td, J = 4.2, 8.3 Hz, 2H), 2.87−2.73
(m, 3H), 1.93−1.80 (m, 2H), 1.53−1.42 (m, 2H), 1.34 (s, 9H).
LCMS (ESI+): calcd for C27H33N5O5 = 507.2, found, [M + H]+ =
508.2, tR = 0.85 min.
1
powder (20.7 mg, 11.7%). H NMR (400 MHz, DMSO-d6): δ 13.87
(br s, 0.3H), 13.22 (br s, 0.8H), 10.66−10.30 (m, 1H), 9.03−8.92 (m,
1H), 8.77−8.55 (m, 1H), 8.23−7.92 (m, 4H), 7.68−7.17 (m, 6H),
7.04−6.93 (m, 1H), 6.78−6.65 (m, 1H), 6.60−6.49 (m, 1H), 5.17 (d,
J = 3.2 Hz, 1H), 4.56 (d, J = 9.5 Hz, 1H), 4.49−4.21 (m, 4H), 4.09−
3.91 (m, 4H), 3.84 (br s, 1H), 3.73−3.46 (m, 5H), 3.04−2.85 (m,
2H), 2.47−2.40 (m, 3H), 2.13−1.83 (m, 4H), 1.73−1.54 (m, 2H),
0.99−0.91 (m, 9H). 13C NMR (100 MHz, DMSO-d6): δ 172.2, 171.8,
169.6, 169.2, 161.3, 152.0, 151.9, 149.5, 148.2, 139.9, 139.6, 131.6,
130.2, 129.4, 129.2, 128.6, 128.0, 122.6, 120.7, 108.4, 108.2, 108.2,
103.8, 101.0, 75.3, 75.3, 75.2, 69.4, 67.5, 67.3, 59.2, 57.1, 56.5, 56.2,
47.5, 47.4, 42.2, 38.4, 36.7, 36.4, 30.9, 30.8, 26.8, 26.7,16.4, 16.4.
LCMS (ESI+): calcd for C45H53N9O7S = 863.4, found, [M + H]+ =
864.4, tR = 2.09 min. HRMS (ESI): m/z [M + H]+ Calcd for
C45H53N19O7S, 864.3859; found, 864.3855.
N-(4-(4-(2-(((S)-1-((2S,4R)-4-Hydroxy-2-(((S)-1-(4-(4-methyl-
thiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-di-
methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)piperidin-1-yl)-
2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (De-
grader-3). 2-((1-(4-(6-(1H-Pyrazol-5-yl)picolinamido)-3-
methoxyphenyl)piperidin-4-yl)oxy)acetic acid hydrochloride (13)
(650 mg, 1.33 mmol), (2S,4R)-1-((S)-2-amino-3,3-dimethylbutano-
yl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)-
pyrrolidine-2-carboxamide hydrochloride (14) (641 mg, 1.33 mmol),
HOBt (269 mg, 1.99 mmol), DIPEA (516 mg, 3.99 mmol), and DMF
(5 mL) were added to a 50 mL round-bottomed flask. Then, EDCI.
HCl (381 mg, 1.99 mmol) was added to the mixture. The reaction
mixture was stirred at 40 °C for 2 h. The reaction mixture was purified
by prep-HPLC (column: Phenomenex Synergi Max-RP 250*50
mm*10 μm, mobile phase A: water (0.225%FA), mobile phase B:
acetonitrile, flow rate: 60 mL/min, and gradient condition: from 35%
B to 65% B). The pure fractions were collected, and the solvent was
evaporated under vacuum to give a residue, which was lyophilized to
give the product as a yellow powder (218.5 mg, 18.7%).1H NMR
(400 MHz, DMSO-d6): δ 13.97−13.03 (m, 1H), 10.58 (br s, 1H),
8.98 (s, 1H), 8.48 (d, J = 7.6 Hz, 1H), 8.27−7.87 (m, 5H), 7.47−7.33
(m, 5H), 6.99 (br s, 1H), 6.74 (br s, 1H), 6.61−6.54 (m, 1H), 5.21−
5.00 (m, 1H), 4.94−4.84 (m, 1H), 4.59−4.41 (m, 2H), 4.28 (br s,
1H), 4.10−3.90 (m, 5H), 3.63−3.49 (m, 5H), 3.00−2.88 (m, 2H),
2.45 (s, 3H), 2.11−1.95 (m, 3H), 1.80−1.57 (m, 3H), 1.50−1.34 (m,
3H), 0.94 (s, 9H). 13C NMR (100 MHz, DMSO-d6): δ 170.9, 170.0,
169.5, 169.2, 168.5, 163.5, 161.2, 161.2, 151.9, 149.6, 148.2, 145.3,
145.0, 139.6, 131.6, 130.2, 130.1, 129.4,129.3, 126.8, 122.5, 120.7,
107.8, 103.8, 103.8, 100.7, 75.5, 69.3, 67.4, 67.3, 59.2, 59.0, 57.1, 56.5,
56.1, 48.3, 47.0, 38.2, 36.7, 36.4, 31.0, 30.9, 26.8, 26.7,23.0, 22.6, 16.5.
LCMS (ESI+): calcd for C46H55N9O7S = 877.4, found, [M + H]+ =
878.4, tR = 4.5 min. HRMS (ESI): m/z [M + H]+ Calcd for
C46H55N9O7S, 878.4023; found, 878.4036.
2 - ( (1 - ( 4 - ( 6 - ( 1 H - P y r a z o l - 5 - y l ) p i c o l i n a m i d o ) - 3 -
methoxyphenyl)piperidin-4-yl)oxy)acetic Acid Hydrochloride
(13). tert-Butyl 2-((1-(4-(6-(1H-pyrazol-5-yl)picolinamido)-3-
methoxyphenyl)piperidin-4-yl)oxy)acetate (12) (210 mg, 0.414
mmol) and HCl (4 mL, 4 M in dioxane) were added to a 50 mL
round-bottomed flask. The resultant mixture was stirred at 25 °C for 2
h and then concentrated to dryness under reduced pressure to afford
1
the title product as a white solid. (250 mg, crude). H NMR (400
MHz, DMSO-d6): δ 13.19−12.79 (br m, 2H), 10.53 (s, 1H), 8.18−
8.12 (m, 1H), 8.12−7.98 (m, 3H), 7.90−7.81 (m, 1H), 6.99 (d, J =
2.0 Hz, 1H), 6.72 (d, J = 2.0 Hz, 1H), 6.55 (dd, J = 2.0, 8.8 Hz, 1H),
4.08 (s, 2H), 3.94 (s, 3H), 3.60−3.48 (m, 3H), 2.94−2.82 (m, 2H),
2.02−1.91 (m, 2H), 1.63−1.52 (m, 2H). LCMS (ESI+): calcd for
C23H25N5O5 = 451.2, found, [M + H]+ = 452.2, tR = 2.11 min.
(2S,4R)-1-((S)-2-Amino-3,3-dimethylbutanoyl)-4-hydroxy-N-
((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-car-
boxamide (14) and Epimer (2S,4S)-1-((S)-2-Amino-3,3-dime-
thylbutanoyl)-4-hydroxy-N-((S)-1-(4-(4-methylthiazol-5-yl)-
phenyl)ethyl)pyrrolidine-2-carboxamide (15). The compound
N-(4-(4-(2-(((S)-1-((2S,4S)-4-Hydroxy-2-(((S)-1-(4-(4-methyl-
thiazol-5-yl)phenyl)ethyl)carbamoyl)pyrrolidin-1-yl)-3,3-di-
I
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Article
methyl-1-oxobutan-2-yl)amino)-2-oxoethoxy)piperidin-1-yl)-
2-methoxyphenyl)-6-(1H-pyrazol-5-yl)picolinamide (De-
grader-4). 2-((1-(4-(6-(1H-Pyrazol-5-yl)picolinamido)-3-
methoxyphenyl)piperidin-4-yl)oxy)acetic acid (12) (46 mg, 0.102
mmol), (2S,4S)-1-((S)-2-amino-3,3-dimethylbutanoyl)-4-hydroxy-N-
((S)-1-(4-(4-methylthiazol-5-yl)phenyl)ethyl)pyrrolidine-2-carboxa-
mide (15) (49.5 mg, 0.103 mmol), HOBt (20.7 mg, 0.153 mmol),
DIPEA (39.6 mg, 0.306 mmol), and DMF (1 mL) were added to a 50
mL round-bottomed flask. Then, EDCI.HCl (29.4 mg, 0.153 mmol)
was added to the mixture. The reaction mixture was stirred at 40 °C
for 12 h. The mixture was poured into water (10 mL) and extracted
by ethyl acetate (30 mL × 3). The organic layer was collected and
dried over Na2SO4, filtered, and concentrated to give the residue,
which was purified by prep-HPLC (column: DuraShell 150*25 mm*5
μm, mobile phase A: water (0.2% FA), mobile phase B: acetonitrile,
flow rate: 25 mL/min, and gradient condition: from 28% B to 58%).
The pure fractions were collected, and the solvent was evaporated
under vacuum to give a residue, which was lyophilized to give the
Jing Zhang − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Bin Shen − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Linglong Kong − Discovery Chemistry, Janssen Research &
Development, Shanghai 201210, China
Yingtao Liu − Discovery Chemistry, Janssen Research &
Development, Shanghai 201210, China
Wangyang Tu − Discovery Chemistry, Janssen Research &
Development, Shanghai 201210, China
Wenqian Wang − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Xin Cai − Department of Biomarker, Janssen Research &
Development, Shanghai 201210, China
Xiaotao Wang − Department of Biomarker, Janssen Research
& Development, Shanghai 201210, China
Na Cheng − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Mingxuan Xia − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Tianyuan Zhou − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Qian Liu − Discovery Chemistry, Janssen Research &
Development, Shanghai 201210, China
Yanping Xu − Discovery Chemistry, Janssen Research &
Development, Shanghai 201210, China
Jennifer Yang − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
Paul Gavine − Department of Biology, Janssen Research &
Development, Shanghai 201210, China
1
product as a yellow powder (20.9 mg, 23.4%). H NMR (400 MHz,
DMSO-d6): δ 13.88 (br s, 0.2H), 13.22 (br s, 0.6H), 10.63−10.35 (m,
1H), 8.97 (s, 1H), 8.47−7.50 (m, 6H), 7.47−7.33 (m, 5H), 6.98 (br
s, 1H), 6.73 (br s, 1H), 6.61−6.54 (m, 1H), 5.38−5.31 (m, 1H),
4.96−4.87 (m, 1H), 4.52 (d, J = 9.0 Hz, 1H), 4.44−4.31 (m, 1H),
4.26−4.17 (m, 1H), 4.09−3.74 (m, 6H), 3.60−3.43 (m, 4H), 3.00−
2.87 (m, 2H), 2.46−2.43 (m, 3H), 2.39−2.31 (m, 1H), 2.05−1.91
(m, 2H), 1.72−1.54 (m, 3H), 1.38 (m, 3H), 1.03−0.88 (m, 9H). 13C
NMR (100 MHz, DMSO-d6): δ 171.3, 170.1, 169.7, 169.4, 161.2,
151.9, 149.6, 148.2, 144.8, 139.6, 131.6, 130.3, 129.3, 126.8, 122.5,
120.7, 107.8, 100.7,75.5, 69.4, 67.8, 67.2, 58.9, 56.5, 56.2, 56.0, 48.3,
47.0, 37.3, 36.6, 35.9, 31.0, 30.9, 26.9, 26.7, 22.8, 22.4, 16.4. LCMS
(ESI+): calcd for C46H55N9O7S = 877.4, found, [M + H]+ = 878.4, tR
= 4.22 min. HRMS (ESI): m/z [M + H]+calcd for C46H55N9O7S,
878.4023; found, 878.4009.
Ulrike Philippar − Janssen Pharmaceutical Research &
ASSOCIATED CONTENT
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.1c00103.
■
Development, Beerse 2340, Belgium
sı
Ricardo Attar − Janssen Pharmaceutical Research &
Development, Spring House, Pennsylvania 19477, United
States
Western blot validation of proteomics studies of
Degrader-3; global proteomic data of Degrader-3 in
HBL-1 cells; anti-proliferation potency of JH-I-25 in
HBL-1 cells; Western blot of IRAK1 and IRAK4 protein
degradation in HEK293T cells; kinase activity assay;
VHL FP assay; immunoblotting protocol; cell culture
and cell viability assay; global proteomics assay protocol;
1H NMR and 13C NMR spectra for conjugates
Degrader-1, Degrader-2, Degrader-3, and Degrader-
4; and KinomeScan raw data of JH-I-25, Degrader-3,
and Degrader-4 (PDF)
James P. Edwards − Discovery Sciences, Janssen Research &
Development, San Diego, California 92121, United States
Complete contact information is available at:
https://pubs.acs.org/10.1021/acs.jmedchem.1c00103
Author Contributions
△L.F. and J.Z. contributed equally. L.F wrote the original
draft, J.Z., L.K., X.W., T.Z., P.G., J.P.E., J.D.V, and X.D revised
the draft, and all authors reviewed and edited the manuscript.
All authors have given approval to the final version of the
manuscript.
Proteomics raw data of Degrader-3 in Toledo (CSV)
Proteomics raw data of Degrader-3 in HBL-1 (CSV)
Notes
The authors declare no competing financial interest.
AUTHOR INFORMATION
Corresponding Authors
ACKNOWLEDGMENTS
■
■
The authors would like to thank the members of the Discovery
Science and Oncology Translational Research team at Janssen
including Charu Chaudhry and Haopeng Rui for insightful
discussion and Yan Wang for help with graphical abstract
design.
Jennifer D. Venable − Discovery Sciences, Janssen Research &
Development, San Diego, California 92121, United States;
Email: jvenable@its.jnj.com
Xuedong Dai − Discovery Sciences, Janssen (China) Research
& Development, Shanghai 201210, P.R. China; Discovery
Chemistry, Janssen Research & Development, Shanghai
201210, China; orcid.org/0000-0003-4358-0319;
Email: xdai18@its.jnj.com
ABBREVIATIONS
■
ABC, activated B-cell-like; CRBN, cereblon; DIPEA, N, N-
diisopropylethylamine; DLBCL, diffuse large B-cell lymphoma;
DMF, dimethylformamide; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; EtOAc, ethyl acetate;
GAK, cyclin G-associated kinase; GCB, center B-cell-like;
Authors
Liqiang Fu − Discovery Chemistry, Janssen Research &
Development, Shanghai 201210, China
J
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Article
(10) Churcher, I. Protac-induced protein degradation in drug
discovery: breaking the rules or just making new ones? J. Med. Chem.
2018, 61, 444−452.
HATU, 1-[bis(dimethylamino)methylene]-1H-1,2,3- triazolo-
[4,5-b]pyridinium 3-oxide hexafluorophosphate; NHL, non-
Hodgkin lymphoma; IL-1R, interleukin-1 receptor; IRAK1,
interleukin-1 receptor-associated kinase 1; MAPK, mitogen-
activated protein kinase; MyD88, myeloid differentiation
primary response 88; NF-κB, IκB kinase (IKK)-nuclear
factor-κB; NMRAL1, NmrA-like family domain-containing
protein 1; PROTACs, proteolysis-targeting chimeras; TEA,
triethanolamine; TLRs, toll-like receptors; TRAF6, TNF
receptor-associated factor 6; VHL, von Hippel−Lindau.
(11) Pettersson, M.; Crews, C. M. PROteolysis TArgeting Chimeras
(PROTACs)past, present and future. Drug Discov. Today Technol.
2019, 31, 15−27.
(12) Verma, R.; Mohl, D.; Deshaies, R. J. Harnessing the power of
proteolysis for targeted protein inactivation. Mol. Cell 2020, 77, 446−
460.
(13) Buckley, G. M.; Gowers, L.; Higueruelo, A. P.; Jenkins, K.;
Mack, S. R.; Morgan, T.; Parry, D. M.; Pitt, W. R.; Rausch, O.;
Richard, M. D.; Sabin, V.; Fraser, J. L. IRAK-4 inhibitors. Part 1: a
series of amides. Bioorg. Med. Chem. Lett. 2008, 18, 3211−3214.
(14) Wang, L.; Qiao, Q.; Ferrao, R.; Shen, C.; Hatcher, J. M.;
Buhrlage, S. J.; Gray, N. S.; Wu, H. Crystal structure of human
IRAK1. Proc. Natl. Acad. Sci. U. S. A. 2017, 114, 13507−13512.
(15) Hansen, J. D.; Correa, M.; Nagy, M. A.; Alexander, M.;
Plantevin, V.; Grant, V.; Whitefield, B.; Huang, D.; Kercher, T.;
Harris, R.; Narla, R. K.; Leisten, J.; Tang, Y.; Moghaddam, M.;
Ebinger, K.; Piccotti, J.; Havens, C. G.; Cathers, B.; Carmichael, J.;
Daniel, T.; Vessey, R.; Hamann, L. G.; Leftheris, K.; Mendy, D.;
Baculi, F.; LeBrun, L. A.; Khambatta, G.; Lopez-Girona, A. Discovery
of CRBN E3 ligase modulator CC-92480 for the treatment of relapsed
and refractory multiple myeloma. J. Med. Chem. 2020, 63, 6648−
6676.
(16) Matyskiela, M. E.; Lu, G.; Ito, T.; Pagarigan, B.; Lu, C.-C.;
Miller, K.; Fang, W.; Wang, N.-Y.; Nguyen, D.; Houston, J.; Carmel,
G.; Tran, T.; Riley, M.; Nosaka, L. A.; Lander, G. C.; Gaidarova, S.;
Xu, S.; Ruchelman, A. L.; Handa, H.; Carmichael, J.; Daniel, T. O.;
Cathers, B. E.; Lopez-Girona, A.; Chamberlain, P. P. A novel cereblon
modulator recruits GSPT1 to the CRL4(CRBN) ubiquitin ligase.
Nature 2016, 535, 252−257.
REFERENCES
■
(1) De Nardo, D.; Balka, K. R.; Cardona Gloria, Y.; Rao, V. R.; Latz,
E.; Masters, S. L. Interleukin-1 receptor-associated kinase 4 (IRAK4)
plays a dual role in myddosome formation and Toll-like receptor
signaling. J. Biol. Chem. 2018, 293, 15195−15207.
(2) Lin, S.-C.; Lo, Y.-C.; Wu, H. Helical assembly in the MyD88-
IRAK4-IRAK2 complex in TLR/IL-1R signalling. Nature 2010, 465,
885−890.
(3) Roschewski, M.; Staudt, L. M.; Wilson, W. H. Diffuse large B-cell
lymphoma-treatment approaches in the molecular era. Nat. Rev. Clin.
Oncol. 2014, 11, 12−23.
(4) Ngo, V. N.; Young, R. M.; Schmitz, R.; Jhavar, S.; Xiao, W.; Lim,
K.-H.; Kohlhammer, H.; Xu, W.; Yang, Y.; Zhao, H.; Shaffer, A. L.;
Romesser, P.; Wright, G.; Powell, J.; Rosenwald, A.; Muller-
Hermelink, H. K.; Ott, G.; Gascoyne, R. D.; Connors, J. M.;
Rimsza, L. M.; Campo, E.; Jaffe, E. S.; Delabie, J.; Smeland, E. B.;
Fisher, R. I.; Braziel, R. M.; Tubbs, R. R.; Cook, J. R.; Weisenburger,
D. D.; Chan, W. C.; Staudt, L. M. Oncogenically active MYD88
mutations in human lymphoma. Nature 2011, 470, 115−119.
(5) Zhang, J.; Fu, L.; Shen, B.; Liu, Y.; Wang, W.; Cai, X.; Kong, L.;
Yan, Y.; Meng, R.; Zhang, Z.; Chen, Y.-N. P.; Liu, Q.; Wan, Z.-K.;
Zhou, T.; Wang, X.; Gavine, P.; Del Rosario, A.; Ahn, K.; Philippar,
U.; Attar, R.; Yang, J.; Xu, Y.; Edwards, J. P.; Dai, X. Assessing IRAK4
functions in ABC DLBCL by IRAK4 kinase inhibition and protein
degradation. Cell Chem. Biol. 2020, 27, 1500−1509.
(17) Matyskiela, M. E.; Couto, S.; Zheng, X.; Lu, G.; Hui, J.; Stamp,
K.; Drew, C.; Ren, Y.; Wang, M.; Carpenter, A.; Lee, C.-W.; Clayton,
T.; Fang, W.; Lu, C.-C.; Riley, M.; Abdubek, P.; Blease, K.; Hartke, J.;
Kumar, G.; Vessey, R.; Rolfe, M.; Hamann, L. G.; Chamberlain, P. P.
SALL4 mediates teratogenicity as a thalidomide-dependent cereblon
substrate. Nat. Chem. Biol. 2018, 14, 981−987.
(6) Phelan, J. D.; Young, R. M.; Webster, D. E.; Roulland, S.; Wright,
G. W.; Kasbekar, M.; Shaffer, A. L., 3rd; Ceribelli, M.; Wang, J. Q.;
Schmitz, R.; Nakagawa, M.; Bachy, E.; Huang, D. W.; Ji, Y.; Chen, L.;
Yang, Y.; Zhao, H.; Yu, X.; Xu, W.; Palisoc, M. M.; Valadez, R. R.;
Davies-Hill, T.; Wilson, W. H.; Chan, W. C.; Jaffe, E. S.; Gascoyne, R.
D.; Campo, E.; Rosenwald, A.; Ott, G.; Delabie, J.; Rimsza, L. M.;
Rodriguez, F. J.; Estephan, F.; Holdhoff, M.; Kruhlak, M. J.; Hewitt, S.
M.; Thomas, C. J.; Pittaluga, S.; Oellerich, T.; Staudt, L. M. A
multiprotein supercomplex controlling oncogenic signalling in
lymphoma. Nature 2018, 560, 387−391.
(7) Reddy, A.; Zhang, J.; Davis, N. S.; Moffitt, A. B.; Love, C. L.;
Waldrop, A.; Leppa, S.; Pasanen, A.; Meriranta, L.; Karjalainen-
Lindsberg, M.-L.; Nørgaard, P.; Pedersen, M.; Gang, A. O.; Høgdall,
E.; Heavican, T. B.; Lone, W.; Iqbal, J.; Qin, Q.; Li, G.; Kim, S. Y.;
Healy, J.; Richards, K. L.; Fedoriw, Y.; Bernal-Mizrachi, L.; Koff, J. L.;
Staton, A. D.; Flowers, C. R.; Paltiel, O.; Goldschmidt, N.; Calaminici,
M.; Clear, A.; Gribben, J.; Nguyen, E.; Czader, M. B.; Ondrejka, S. L.;
Collie, A.; Hsi, E. D.; Tse, E.; Au-Yeung, R. K. H.; Kwong, Y.-L.;
Srivastava, G.; Choi, W. W. L.; Evens, A. M.; Pilichowska, M.; Sengar,
M.; Reddy, N.; Li, S.; Chadburn, A.; Gordon, L. I.; Jaffe, E. S.; Levy,
S.; Rempel, R.; Tzeng, T.; Happ, L. E.; Dave, T.; Rajagopalan, D.;
Datta, J.; Dunson, D. B.; Dave, S. S. Genetic and functional drivers of
diffuse large B cell lymphoma. Cell 2017, 171, 481−494.
(18) Asatsuma-Okumura, T.; Ando, H.; De Simone, M.; Yamamoto,
J.; Sato, T.; Shimizu, N.; Asakawa, K.; Yamaguchi, Y.; Ito, T.;
Guerrini, L.; Handa, H. p63 is a cereblon substrate involved in
thalidomide teratogenicity. Nat. Chem. Biol. 2019, 15, 1077−1084.
(19) Hagner, P. R.; Man, H.-W.; Fontanillo, C.; Wang, M.; Couto,
S.; Breider, M.; Bjorklund, C.; Havens, C. G.; Lu, G.; Rychak, E.;
Raymon, H.; Narla, R. K.; Barnes, L.; Khambatta, G.; Chiu, H.; Kosek,
J.; Kang, J.; Amantangelo, M. D.; Waldman, M.; Lopez-Girona, A.;
Cai, T.; Pourdehnad, M.; Trotter, M.; Daniel, T. O.; Schafer, P. H.;
Klippel, A.; Thakurta, A.; Chopra, R.; Gandhi, A. K. CC-122, a
pleiotropic pathway modifier, mimics an interferon response and has
antitumor activity in DLBCL. Blood 2015, 126, 779−789.
(20) Galdeano, C.; Gadd, M. S.; Soares, P.; Scaffidi, S.; Van Molle,
I.; Birced, I.; Hewitt, S.; Dias, D. M.; Ciulli, A. Structure-guided
design and optimization of small molecules targeting the protein-
protein interaction between the von Hippel-Lindau (VHL) E3
ubiquitin ligase and the hypoxia inducible factor (HIF) alpha subunit
with in vitro nanomolar affinities. J. Med. Chem. 2014, 57, 8657−
8663.
(21) Soares, P.; Gadd, M. S.; Frost, J.; Galdeano, C.; Ellis, L.;
Epemolu, O.; Rocha, S.; Read, K. D.; Ciulli, A. Group-based
optimization of potent and cell-active inhibitors of the von Hippel-
Lindau (VHL) E3 ubiquitin ligase: structure-activity relationships
leading to the chemical probe (2S,4R)-1-((S)-2-(1-cyanocyclopropa-
necarboxamido)-3,3-dimethylbutanoyl)-4-hydroxy-N-(4-(4-methyl-
thiazol-5-yl)benzyl)pyrrolidine-2-carboxamide (VH298). J. Med.
Chem. 2018, 61, 599−618.
(8) Hatcher, J. M.; Yang, G.; Wang, L.; Ficarro, S. B.; Buhrlage, S.;
Wu, H.; Marto, J. A.; Treon, S. P.; Gray, N. S. Discovery of a selective,
covalent IRAK1 inhibitor with antiproliferative activity in MYD88
mutated B-cell lymphoma. ACS Med. Chem. Lett. 2020, 11, 2238−
2243.
(9) Burslem, G. M.; Crews, C. M. Small-molecule modulation of
protein homeostasis. Chem. Rev. 2017, 117, 11269−11301.
(22) Hu, J.; Hu, B.; Wang, M.; Xu, F.; Miao, B.; Yang, C.-Y.; Wang,
M.; Liu, Z.; Hayes, D. F.; Chinnaswamy, K.; Delproposto, J.; Stuckey,
J.; Wang, S. Discovery of ERD-308 as a highly potent proteolysis
K
https://doi.org/10.1021/acs.jmedchem.1c00103
J. Med. Chem. XXXX, XXX, XXX−XXX

Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
targeting chimera (PROTAC) degrader of estrogen receptor (ER). J.
Med. Chem. 2019, 62, 1420−1442.
(23) Gutschow, M.; Steinebach, C.; Voell, S. A.; Vu, L. P.; Bricelj,
̈
̌
A.; Sosic, I.; Schnakenburg, G. A facile synthesis of ligands for the von
Hippel−Lindau E3 ligase. Synthesis 2020, 52, 2521−2527.
(24) Bondeson, D. P.; Smith, B. E.; Burslem, G. M.; Buhimschi, A.
D.; Hines, J.; Jaime-Figueroa, S.; Wang, J.; Hamman, B. D.;
Ishchenko, A.; Crews, C. M. Lessons in PROTAC design from
selective degradation with a promiscuous warhead. Cell Chem. Biol.
2018, 25, 78−87.
(25) Kostic, M.; Jones, L. H. Critical assessment of targeted protein
degradation as a research tool and pharmacological modality. Trends
Pharmacol. Sci. 2020, 41, 305−317.
(26) Lenz, G. Insights into the molecular pathogenesis of activated
B-cell-like diffuse large B-cell lymphoma and its therapeutic
implications. Cancers (Basel) 2015, 7, 811−822.
(27) Ferreiro, D. U.; Komives, E. A. Molecular mechanisms of
system control of NF-kappaB signaling by IkappaBalpha. Biochemistry
2010, 49, 1560−1567.
(28) Buckley, D. L.; Raina, K.; Darricarrere, N.; Hines, J.; Gustafson,
J. L.; Smith, I. E.; Miah, A. H.; Harling, J. D.; Crews, C. M.
HaloPROTACS: use of small molecule PROTACs to induce
degradation of HaloTag fusion proteins. ACS Chem. Biol. 2015, 10,
1831−1837.
L
https://doi.org/10.1021/acs.jmedchem.1c00103
J. Med. Chem. XXXX, XXX, XXX−XXX