Discovery of First-In-Class Potent and Selective Tropomyosin Receptor Kinase Degraders

Article abstract of DOI:10.1021/acs.jmedchem.0c01342

We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion protein in KM12 colo

Full text of DOI:10.1021/acs.jmedchem.0c01342

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Article
Discovery of First-In-Class Potent and Selective Tropomyosin
Receptor Kinase Degraders
Liqun Chen, Yanke Chen, Chunyan Zhang, Bingyang Jiao, Sheng Liang, Qiong Tan, Hongyu Chai,
Weihua Yu, Yongzheng Qian, Hui Yang, Wuyi Yao, Jianguo Yu, Ying Luo, Michael Plewe, Jialiang Wang,
Xiao-Ran Han,* and Jing Liu*
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ABSTRACT: We report compounds 5 (CG416) and 6 (CG428) as two first-in-class tropomyosin receptor kinase (TRK) degraders
that target the intracellular kinase domain of TRK. Degraders 5 and 6 reduced levels of the tropomyosin 3 (TPM3)-TRKA fusion
protein in KM12 colorectal carcinoma cells and inhibited downstream PLCγ1 signaling at sub-nanomolar concentrations. Both
degraders also degraded human wild-type TRKA with similar potency. Interestingly, both degraders, especially 6, showed selectivity
for the degradation of endogenous TPM3-TRKA over ectopically expressed ATP/GTP binding protein-like 4 (AGBL4)-TRKB or
ETS variant transcription factor 6 (ETV6)-TRKC fusion proteins in KM12 cells. Global proteomic profiling assays demonstrated
that 5 is highly selective for the intended target. TPM3-TRKA protein degradation induced by 5 and 6 was further confirmed to be
mediated through cereblon and the ubiquitin-proteasome system. Compared with the parental TRK kinase inhibitor, both degraders
exhibited higher potency for inhibiting growth of KM12 cells. Moreover, both 5 and 6 showed good plasma exposure levels in mice.
Therefore, 5 and 6 are valuable chemical tool compounds for investigating the in vivo function of TRK fusion during tumorigenesis.
Our study also paves the way for pharmacological degradation of TRK.
transformation.12−15 Among the different molecular mecha-
INTRODUCTION
■
nisms of TRK activation, NTRK gene chromosomal trans-
The tropomyosin receptor kinase (TRK) receptor family
location has been the most frequently studied.9 The most
comprises three members: TRKA, TRKB, and TRKC that are
common TRK fusions include tropomyosin 3 (TPM3)-TRKA,
encoded by the NTRK1, NTRK2, and NTRK3 genes,
lamin A/C-TRKA, and ETS variant transcription factor 6
respectively.1−5 TRKs are receptor tyrosine kinases primarily
(ETV6)-TRKC.16 Typically, the 3′ regions of the NTRK genes
implicated in the development and function of the neuronal
are joined with the 5′ regions of a partner gene because of
tissues. The main ligands of TRKs are the nerve growth factor
chromosomal rearrangement. The resulting chimeric proteins
(NGF) for TRKA,6,7 brain-derived neurotrophic factor
lead to constitutive activation of the kinase domain and
(BDNF) for TRKB,8 and neurotrophins for TRKC.9,10 The
downstream oncogenic signaling.17 Additionally, expression of
binding of ligands to the extracellular domains of TRKs
the chimeric proteins is driven by the promoters of the fusion
induces dimerization and activation of the intracellular kinase
domains, which provide signaling to downstream pathways,
Received: August 7, 2020
primarily PI3K/AKT, RAF/MEK/ERK, and phospholipase C
gamma (PLCγ).11 Various genetic aberrations of NTRK genes
have been reported, including chromosomal translocation,
amplification, point mutations, and alternative splicing. These
changes lead to constitutive activation of the TRK pathway
that promotes cellular proliferation, survival, and malignant
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A

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Figure 1. Structures of TRK inhibitors. (A) Chemical structures. (B) X-ray cocrystal structure of compound 3 (cyan) in the complex with TRKA
(gray) (PDB: 4YNE). The loop formed by GLY605 and GLY623 residues is highlighted in light blue.
a
Scheme 1. Synthetic Routes for TRK Degraders 5 and 6 and Their Control Analogues 25 and 26
a
Reagents and conditions: (a) KF, DMSO, 100 °C, 12 h; (b) Pd(Ph3P)4, toluene, 110 °C, 12 h; (c) piperazine, KF, DMSO, 130 °C; (d) DIEA,
NMP, microwave, 85 °C, 50 min; (e) formic acid, 30 h; (f) EDCI, HOAt, NMM, DMSO, rt, overnight; and (g) MeI, K2CO3, DMSO, rt, 2 h.
partners, which often result in overexpression.18,19 TRK fusions
have been found across a wide range of human malignancies,
including lung cancer,20,21 colorectal cancer,22 and soft tissue
sarcoma.23 In addition, TRK fusions appear to be the primary
oncological drivers of some rare cancers, such as infantile
fibrosarcoma,24 secretory breast carcinoma,25 and mammary
analogue secretory carcinoma (MASC).26
In the past decade, significant efforts have been devoted
toward the development of TRK inhibitors that can antagonize
TRK’s catalytic function.14,15,27 As a result, two TRK
inhibitors, 1 (larotrectinib) and 2 (entrectinib), have been
approved by FDA to treat certain cancer patients carrying
NTRK fusion genes (Figure 1A).28,29 Although both 1 and 2
showed good safety profiles in the clinic, modest on-target
adverse effects, such as dizziness/ataxia, paresthesia, and
weight gain, have been observed.27 Some of these adverse
effects are believed to result from inhibition of wild-type TRK
signaling in the brain. In addition, acquired drug resistance has
been reported in patients after treatment with these FDA-
approved drugs.14,27 Hence, therapeutic approaches with a new
mechanism of action (MOA) are desired to provide more
durable inhibition of the TRK pathway with fewer adverse
effects.
Molecules capable of chemical downregulation of target
protein levels using proteolysis-targeting chimeras have been
called PROTACs (PROteolysis TArgeting Chimeras), uS-
MITEs (ubiquitin-mediated, small molecule-induced target
elimination), SNIPERs (specific and nongenetic IAP-depend-
ent protein ERasers), DEGRONIMIDs, and other acronyms.
In this paper, we will simply refer to these molecules as
degraders. Numerous degraders have exhibited significant
advantages over enzymatic inhibitors.30−34 For example,
protein degraders not only suppress the enzymatic activity of
the intended targets but also deplete the scaffolding and other
noncatalytic functions of the targeted proteins.32,35,36 De-
graders also have the potential to overcome the acquired
resistance induced by traditional small-molecule drugs.31,37−39
Moreover, because of the catalytic nature of degraders, high
exposure may be avoided to ameliorate potential adverse
effects.40
B
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a
Table 1. SAR of Synthesized Putative TRK Degraders
a
KM12 cells were treated with indicated compounds at 0.1, 1, and 10 nM for 6 h. TPM3-TRKA protein levels were determined by immunoblotting
and normalized against α-tubulin. The experiments were performed in triplicate, and the values are presented as mean SD.
A growing number of protein degraders have been
developed to selectively degrade disease-associated proteins,
of which many are kinases, including RIPK2,41 BCR-ABL,42−44
CDK9,45,46 FLT3,47 BTK,37,47 EGFR,48,49 ALK,50−52 CDK4/
6,53−55 MEK,56,57 and BRAF.58 Recently, a wild-type TRKC
degrader has been reported using a bivalent dipeptidomimetic
which binds wild-type TRKC at the ectodomain.59 Because
TRK activation in human cancers typically includes fusions,
mutations, and deletions at the extracellular domain, TRK
degraders targeting the ectodomain may have limited
application to treat cancers. In this study, we designed,
synthesized, and evaluated a series of TRK degraders derived
from an analogue (3) of a pan TRK inhibitor 4 (GNF-8625)60
and a cereblon (CRBN) E3 ligase ligand, pomalidomide.61
Through a structure−activity relationship (SAR) study, we
identified two potent and selective TRK degraders, 5 (CG416)
and 6 (CG428). Here, we present the discovery and detailed
biological characterization of these two first-in-class TRK
degraders that target the intracellular domain of the kinase.
analogue 3 in complex with the TRKA kinase domain (PDB:
4YNE) (Figure 1B).60 It is worth mentioning that a loop
between GLY605 and GLY623 in the TRKA C-lobe (Figure
1B) together with the N-lobe of the kinase forms a concave
surface at the incoming E3 complex binding site. This surface
shape may be unfavorable for the protein−protein interactions
between TRK and the recruited E3 ligase. However, we
speculated that this loop could be flexible enough to adapt to
another geometry to accommodate the incoming E3 complex.
This hypothesis fueled our curiosity to initiate this TRK
degrader project. Based on the cocrystal structure and the
published cellular activity data obtained from the Ba/F3 assay
(TRKA IC50 = 3 nM for 3 and GNF-8625), we anticipate that
both 3 and GNF-8625 should bind to TRKA.60 Considering
the significant difference in the molecular weights between the
two compounds (MW = 359 for 3 and 536 for GNF-8625), we
decided to choose 3 as the TRK binding moiety for our TRK
degraders. From the X-ray crystal structure, the pyridinyl group
of 3 is at the solvent front and suitable to attach linkers. To
facilitate the synthesis of degraders, we attached a piperazinyl
moiety at the 2-position of the pyridinyl group (compound 7
in Scheme 1). This newly installed piperazinyl group can serve
as a bridge between 3 and the linker region. It is well
documented that the linker region is critical for the
development of degraders.62 Therefore, we designed degraders
bearing a variety of carbon linkers or polyethylene glycol
(PEG) linkers (Scheme 1 and Table 1). In this study, we chose
RESULTS AND DISCUSSION
■
Design and Synthesis of Putative TRK Degraders.
GNF-8625 is a type I pan TRK kinase inhibitor competitively
occupying the ATP binding site.60 Although an X-ray cocrystal
structure of GNF-8625 in complex with TRK does not
currently exist, a cocrystal structure is available for its truncated
C
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Figure 2. Binding affinities of GNF-8625, degraders (5 and 6), and negative controls (25 and 26) to TRKA, TRKB, or TRKC. Binding affinities of
individual compounds were determined using the KINOMEscan assay (DiscoveryX). The lowest concentration points represent data from the
DMSO samples. Data are shown as mean SD derived from duplicated independent experiments.
pomalidomide as the E3 recruiting ligand because of its
relatively low molecular weight (MW = 273) and potentially
favorable PK properties compared with Von Hippel−Lindau
(VHL) recruiting ligands.63−65
We developed a convergent synthetic route to prepare the
designed putative TRK degraders. The synthesis of two key
degraders, 5 and 6, and their corresponding control
compounds, 25 and 26, are outlined in Scheme 1. Briefly, 3-
bromo-6-chloroimidazo[1,2-b]pyridazine 8 reacted with (R)-2-
D
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Figure 3. Compounds 5 and 6 induce degradation of TRKA fusion and wild-type TRKA. (A) KM12 cells harboring TPM3-TRKA fusion were
treated with DMSO or indicated compounds following a threefold serial dilution for 6 h. (B) KM12 cells were treated with 10 nM compound 5 or
6 for indicated time points. (C) HEL cells harboring wild-type TRKA were treated with DMSO or indicated compounds following a threefold serial
dilution for 6 h. Levels of indicated proteins were determined by immunoblotting and normalized against loading controls.
(3-fluorophenyl)pyrrolidine 9 through a nucleophilic aromatic
substitution reaction to yield intermediate 10.60 Palladium-
catalyzed Stille coupling reaction between 10 and 2-fluoro-6-
(tributylstannyl)pyridine 11 provided intermediate 12,60 which
reacted with piperazine through a nucleophilic substitution
reaction to provide the common intermediate 7. The linker-
attached pomalidomide analogues 15a and 15b were prepared
from 2-fluoro-substituted thalidomide 13 through nucleophilic
aromatic substitution reactions with amino esters 14a and 14b,
respectively, followed by acid-mediated tert-butyl ester
deprotection.58 Amide coupling of the common intermediate
7 with 15a and 15b yielded degraders 5 and 6, respectively.
Other putative degraders (16−24) were synthesized similarly
using different linker-attached pomalidomide analogues. It has
been well documented that N-methylation of the glutarimide
moiety could impair the binding of pomalidomide-based
degraders to CRBN, thus diminishing the target protein
degradation capability.50,58 Therefore, the N-methyl gluta-
rimide analogues 25 and 26 were prepared from 5 and 6,
respectively, by N-alkylation reactions with methyl iodide
(Scheme 1).
inhibitor GNF-8625 also decreased TPM3-TRKA protein
levels at 10 nM, albeit to a lesser degree (52 5%
degradation). Compound 17, bearing an ethylene linker,
showed a similar effect as GNF-8625 and reduced protein
levels by 65 5%. The remaining designed compounds appear
to be much more effective than GNF-8625 and 17 at this
concentration with at least 80% degradation at 10 nM. At 1
nM compound concentration, GNF-8625, 17, 20, 21, and 23
did not significantly reduce TPM3-TRKA levels (<30%
degradation). Compounds 18, 19, and 5, bearing propylene,
butylene, and pentylene linkers, respectively, were the most
potent compounds carrying alkyl linkers and led to over 60%
TPM3-TRKA fusion protein downregulation at 1 nM. Among
compounds carrying PEG linkers, 6, bearing a short PEG
linker, showed the best degradation potency with 82
2%
TPM3-TRKA reduction. At 0.1 nM compound concentration,
only compound 6 significantly degraded TPM3-TRKA (46
6%). Compounds 5 and 6 are close analogues with only one
atom difference on the linker moiety. Hence, they are an
excellent pair of degraders for investigating the effects of the
subtle linker chemical structure changes. Therefore, we
selected these two compounds for further biological character-
izations.
Compounds 5 and 6 Bind TRKA, TRKB, and TRKC with
High Affinity. In order to design our degraders, we
significantly modified the chemical structure of the pan TRK
inhibitor, GNF-8625, by replacing the 4-hydroxypiperidinyl-
substituted pyridinyl group with a piperazinyl moiety and
adding the linker and E3 binder moieties. To determine
SAR Study of the Synthesized Putative TRK
Degraders. The TRK degradation capability of the
synthesized compounds was evaluated using an immunoblot-
ting assay in KM12 cells (Table 1, and Figure S3), which are
human colorectal carcinoma cells expressing the TPM3-TRKA
fusion protein.21,66 Our data showed that all of the synthesized
compounds significantly reduced TPM3-TRKA levels at 10
nM after 6 h of treatment. Interestingly, the pan-kinase
E
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Figure 4. Compounds 5 and 6 induce degradation of TRKB and TRKC fusion protein. (A,B) KM12 cells stably expressing AGBL4-TRKB or
ETV6-TRKC fusion proteins were treated with DMSO or indicated compounds following a threefold serial dilution for 6 h. Levels of indicated
proteins were determined by immunoblotting and normalized against loading controls.
whether these structural changes at the solvent exposed area
affected the binding to TRK proteins, we evaluated the binding
affinities of inhibitor GNF-8625, degraders 5 and 6, and the
control compounds 25 and 26 to TRKA, TRKB, and TRKC
using the DiscoverX KINOMEscan platform (Figure 2).
Compared with GNF-8625, the binding affinities of the carbon
linker-bearing degrader 5 were slightly decreased by about
three-, five-, and threefold for TRKA, TRKB, and TRKC,
respectively. The PEG linker-derived degrader 6, however,
showed identical binding affinities to all three TRK family
members. These binding affinity results validated our
hypothesis that modifications of solvent-exposed groups of
GNF-8625 are well tolerated. Importantly, the degrader and
control pairs showed similar binding affinities to all three
TRKs. These data confirmed that N-methylation of the
glutarimide on the pomalidomide moiety does not affect the
binding affinities to TRKs. Considering the close chemical
structural similarities and near-identical target protein binding
affinities to their corresponding degraders, 25 and 26 are
excellent control compounds for 5 and 6, respectively.
GSPT1 protein levels in our experiments and determined that
neither degraders affected GSPT1 levels (Figure 3A).
We further evaluated the kinetics of the induced TPM3-
TRKA degradation process in KM12 cells. As illustrated in
Figure 3B, significant TPM3-TRKA protein degradation and
PLCγ1 phosphorylation inhibition were observed for both 5
and 6 within 0.3 h, and near complete TPM3-TRKA
degradation and PLCγ1 phosphorylation inhibition were
achieved within 4 h post 10 nM compound treatment.
Therefore, both 5 and 6 rapidly induced TPM3-TRKA protein
reduction and inhibited downstream signaling in KM12 cells.
It has been reported that some protein degraders, such as
EGFR degraders48,49 and BRAF degraders,58 have the potential
to selectively downregulate mutated proteins and spare the
wild-type proteins. We evaluated compounds 5 and 6 in HEL
cells expressing endogenous wild-type TRKA (Figure 3C). In
contrast to EGFR degraders and BRAF degraders, our TRK
degraders 5 and 6 did not spare the wild-type TRKA. Instead,
both degraders 5 and 6 degraded wild-type TRKA in HEL cells
with high potency (DC50 values of 1.26 0.25 and 2.23 0.42
nM, respectively). Conversely, the control compounds 25 and
26 did not degrade wild-type TRKA in HEL cells at 30 nM.
To assess the degradation potency of compounds 5 and 6 on
other TRK family members, we ectopically expressed ATP/
GTP binding protein-like 4 (AGBL4)-TRKB or ETV6-TRKC
fusion proteins in KM12 cells. After incubating the cells with
various concentrations of 5 and 6 for 6 h, we assessed the TRK
fusion protein levels (Figure 4). Interestingly, compared with
the TPM3-TRKA degradation results shown in Figure 3A,
compounds 5 and 6 demonstrated much weaker degradation
potency for overexpressed AGBL4-TRKB or ETV6-TRKC in
KM12 cells. This selectivity is more significant for the PEG
linker-derived degrader 6. We believe that such a selectivity
profile within the same TRK family members may be due to
the decreased binding affinities of 5 and 6 for TRKB (Kd = 112
Compounds 5 and 6 are Potent and Selective TRK
Degraders. To quantitatively determine the TRK degradation
capability of compounds 5 and 6, KM12 cells were incubated
with these degraders at various concentrations for 6 h. As
illustrated in Figure 3A, compounds 5 and 6 potently induced
TPM3-TRKA degradation (DC50 = 0.48 0.06 nM for 5 and
0.36
0.06 nM for 6) and inhibited downstream PLCγ1
phosphorylation (IC50 = 0.36 0.06 nM for 5 0.33 0.06 nM
for 6) in a concentration-dependent manner. Similar to GNF-
8625 (Figure S3), the control compounds 25 and 26 also
reduced TPM3-TRKA levels at 30 nM. Although we are still
investigating the degradation mechanisms of GNF-8625 and
the control compounds, we are certain that degraders 5 and 6
are significantly more potent than these compounds for the
degradation of the TPM3-TRKA fusion protein. We further
confirmed that inhibition of PLCγ1 phosphorylation is not due
to the modulation of PLCγ1 protein levels (Figure 3A). It was
recently reported that immunomodulatory imide drug (IMiD)-
derived degraders degrade G1 to S phase transition 1 (GSPT1)
protein, a neo-substrate for CC-885,67 and lead to unexpected
toxicity to certain types of cells.68−70 Therefore, we assessed
21 nM for 5 and 28 5 nM for 6) and TRKC (Kd = 18
nM for 5 and 4.2 1.0 nM for 6) compared with TRKA (Kd =
2.1 0.3 nM for 5 and 1.0 0.2 nM for 6) (Figure 2).
4
However, we do not rule out the possible contributions from
subcellular locations, microenvironments, protein surfaces, and
protein expression levels of these TRK fusion proteins. In
addition, with respect to the degrader-induced AGBL4-TRKB
F
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downregulation experiments, a “hook effect” was observed for
both compounds 5 and 6 at 500 nM compound concen-
tration.48 We also found that the weaker TRKB binder 5
exhibited a superior degradation capability for AGBL4-TRKB
when compared to the stronger TRKB binder 6 (Figure 4A).
This unexpected result highlights the fact that binding affinity
is not the only factor that determines the degradation potency
of degrader molecules. As illustrated in Figure 4B, both 5 and 6
are weak degraders for the overexpressed ETV6-TRKC fusion
proteins.
We further evaluated the selectivity of compound 5 across
the global proteome using tandem mass tag (TMT)-based
quantitative proteomics technology. The samples were
prepared by treating KM12 cells with 100 nM (200-fold of
the DC50 value) of compound 5 or dimethyl sulfoxide
(DMSO) for 6 h. This relatively high compound concentration
was selected for exploring possible off-targets. TPM3-TRKA
downregulation was confirmed by immunoblotting (Figure
S4). Out of the 7903 identified proteins, only 5 proteins,
TRKA, GDF15, ERRFI1, DUSP4, and AREG, were signifi-
cantly downregulated (p-value <0.01, fold change <0.7) and 2
proteins, SOX4 and FZD6, were upregulated (p-value < 0.01,
fold change >1.5), as indicated in the volcano plot (Figure 5,
(UPS), we performed a series of rescue experiments. KM12
cells were pretreated with an excess amount of pomalidomide
(10 μM), MG-132 (20 μM), bortezomib (200 nM), or
MLN4924 (5 μM), prior to the treatment with degraders 5
and 6 for 6 h (Figure 6A,B). Pomalidomide is a CRBN binder
and competes with degraders 5 and 6 at the same binding
site.61 MG-132 and bortezomib are proteasome inhibitors.
MLN4924 is an inhibitor of the NEDD8-activating enzyme
(NAE).71 It inhibits neddylation of the cullin RING family E3
ubiquitin ligases (CRL) and suppresses their activity. As
illustrated in Figure 6A,B, the addition of these inhibitors
significantly impaired TPM3-TRKA degradation induced by
degraders 5 and 6, indicating that targeted degradation of
TPM3-TRKA was mediated by CRBN, proteasome, and CRL.
To further confirm that degrader-induced TPM3-TRKA
reduction is mediated by CRBN, we performed another rescue
assay by knocking down CRBN in KM12 cells (Figure 6C). As
expected, knockdown of CRBN also significantly impaired
degrader 5-induced TPM3-TRKA protein reduction. Based on
these results, we can conclude that 5 and 6 degrade TPM3-
TRKA fusion protein through UPS and CRL4CRBN
.
Compounds 5 and 6 Potently Inhibit Cell Growth. We
also investigated the impact of degraders on the growth of
KM12 cells using GNF-8625 and the N-methyl glutarimide
analogues 25 and 26 as controls (Figure 7). Interestingly,
although compound 5 displayed a threefold weaker binding
affinity to TRKA than GNF-8625, compound 5 (IC50 = 5.4
0.4 nM) showed threefold higher potency to inhibit the growth
of KM12 cells than GNF-8625 (IC50 = 16 1.7 nM) and a
tenfold higher potency than its control compound 25 (IC50
=
52 5.2 nM). Similarly, compound 6 (IC50 = 2.9 0.3 nM),
an equally potent TRKA binder as GNF-8625, was fivefold
more potent at the inhibition of KM12 cell growth than GNF-
8625 and 10-fold more potent than its control compound 26
(IC50 = 28 2.9 nM). These results suggest that event-driven
degraders could exhibit superior cellular activities compared
with the occupancy-driven small-molecule kinase inhibitors,
despite weaker binding affinities.
Compounds 5 and 6 are Bioavailable in Mice. We next
evaluated the pharmacokinetic (PK) properties of compounds
5 and 6 in ICR mice (Figure 8, Table S2A−C). The plasma
concentrations of compounds 5 and 6 were monitored over 8
h after a single intraperitoneal (IP) administration at 10 mg/
kg. Compounds 5 and 6 shared similar PK profiles. Both
compounds reached maximum plasma concentrations (Cmax
=
Figure 5. Compound 5 selectively degrades TPM3-TRKA. The
volcano plot of proteins with significantly differential abundance for
compound 5-treated samples vs DMSO-treated control samples.
KM12 cells were treated with DMSO or compound 5 at 100 nM for 6
h and subsequently analyzed using TMT quantitative proteomic
analyses. The negative log (base 10) of the P-values is plotted on the
y-axis, and the log (base 2) of the fold change is plotted on the x-axis.
The green and red data points indicate proteins with a p-value of
<0.01 and fold changes of <0.7 and >1.5, respectively. Data were
representative of three independent experiments.
2050 120 nM for 5 and 1640 120 nM for 6) at 1 h post
dosing. Although the half-lives of both compounds were not
optimal (T1/2 = 0.9 h), compound plasma concentrations (14.8
3.3 nM for 5 and 12.4 13.6 nM for 6) at the last time
point (8 h) were still well above their DC50 values (DC50
=
0.48 0.06 nM for 5 and 0.36 0.06 nM for 6) in KM12
cells. In addition, no clinical signs were observed during the PK
study, and both compounds were well-tolerated by the treated
mice. These results indicated that both 5 and 6 could be
valuable tool compounds for in vivo study.
Table S1). Although we are still investigating whether these
proteins are directly or indirectly targeted by degrader 5, the
proteomic study results suggest that compound 5 is a highly
selective TRK degrader.
Degrader-Induced TRK Downregulation is Mediated
by the UPS and CRL4CRBN. In an effort to verify that the
observed TPM3-TRKA degradation induced by degraders 5
and 6 is mediated through the ubiquitin-proteasome system
CONCLUSIONS
■
Based on the X-ray cocrystal structure of TRKA in complex
with an analogue of GNF-8625, we have designed and
synthesized a series of CRBN-recruiting putative protein
degrader molecules with different types of linkers. Initially,
we were concerned about the geometry of the loop between
GLY605 and GLY623 residues on TRKA. The data collected
G
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Figure 6. Degradation of TPM3-TRKA fusion is mediated by E3 ligase cereblon and the UPS. (A, B) KM12 cells were pretreated with DMSO,
pomalidomide (POM, 10 μM), MG-132 (20 μM), bortezomib (BTZ, 200 nM), or MLN4924 (5 μM) and subsequently incubated with 10 nM
compounds 5 or 6 for 6 h prior to immunoblotting. (C) KM12 cells were transfected with two distinct siRNAs targeting CRBN gene and then
treated with compound 5 following a threefold serial dilution for 6 h prior to immunoblotting.
Figure 7. Degraders 5 and 6 are more potent than GNF-8625, 25, and 26 to inhibit KM12 cell viability. KM12 cells were seeded in 96-well plates
and treated with test compounds following 11-point serial dilution for 3 days. Cell viability was determined using the CellTiter-Lumi assay. Error
bars represent SD in three independent experiments.
Figure 8. Plasma concentrations of compounds 5 and 6 following a single 10 mg/kg IP injection in male ICR mice. Plasma concentrations reported
at each of the eight time points are the average values from four test animals. Error bars represent + SD.
here reveal that the loop did not undermine interaction with
TRK degraders. Indeed, most of the designed degraders
effectively reduced TPM3-TRKA fusion protein levels at low
nanomolar concentrations in KM12 cells. We selected two
structurally similar degraders 5 and 6 for detailed biological
investigation. Although the overall degradation profiles of these
two degraders were very similar, the weaker TRKB binder
compound 5 turned out to be a better AGBL4-TRKB degrader
than the stronger TRKB binder 6.
The common features of these two key TRK degraders are
summarized as follows: both 5 and 6 exhibited high binding
affinities to TRK family members and degraded TPM3-TRKA
fusion protein levels in KM12 cells in a concentration- and
time-dependent manner with sub-nanomolar DC50 values.
Both 5 and 6 also exhibited single-digit nanomolar potencies
for the degradation of wild-type TRKA in HEL cells.
Interestingly, both degraders, especially 6, showed excellent
selectivity for endogenous TPM3-TRKA over ectopically
H
https://dx.doi.org/10.1021/acs.jmedchem.0c01342
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
over Na2SO4, filtered, and concentrated. The resulting residue was
purified by silica gel column chromatography (50−100% EtOAc in
hexanes) to provide the title compound (2.2 g, yield: 97%) as yellow
oil. MS (ESI) m/z: 378.0 [M + 1]+. 1H NMR (400 MHz, CD3OD): δ
8.09 (s, 1H), 8.01 (br s, 1H), 7.74−7.79 (m, 2H), 7.35−7.41 (m,
1H), 7.15 (d, J = 7.6 Hz, 1H), 7.07 (d, J = 10.0 Hz, 1H), 6.86−6.99
(m, 3H), 5.16 (dd, J = 8.0 Hz, 2.4 Hz, 1H), 3.98−4.03 (m, 1H),
3.71−3.77 (m, 1H), 2.55−2.60 (m, 1H), 2.12−2.17 (m, 2H), 1.99−
2.04 (m, 1H).
expressed AGBL4-TRKB and ETV6-TRKC fusion proteins in
KM12 cells. Global proteomic studies further revealed that 5
was highly selective for TRKA. Next, we confirmed that 5 and
6 induced the degradation of TPM3-TRKA through the UPS
and CRL4CRBN. Moreover, both 5 and 6 showed better
antiproliferation activity than their parental kinase inhibitor
GNF-8625 in KM12 cells, indicating possible advantages of
TRK degraders over TRK kinase inhibitors. In mouse PK
studies, both 5 and 6 showed good plasma exposure levels via
IP injection, suggesting potential use as in vivo tool
compounds. In summary, 5 and 6 are highly potent and
selective TRK degraders. Further optimization is ongoing to
improve their drug properties.
(R)-6-(2-(3-Fluorophenyl)pyrrolidin-1-yl)-3-(6-(piperazin-1-
yl)pyridin-2-yl)imidazo[1,2-b]pyridazine (7). To a solution of
(R)-6-(2-(3-fluorophenyl)pyrrolidin-1-yl)-3-(6-fluoropyridin-2-yl)-
imidazo [1,2-b]pyridazine (1.4 g, 3.7 mmol) 12 in DMSO (40 mL)
was added piperazine (6.4 g, 74 mmol), followed by potassium
fluoride (8.6 g, 148 mmol). The resulting mixture was stirred at 130
°C for 12 h before being poured into water and extracted with EtOAc.
The combined organic layers were washed with brine, dried over
Na2SO4, and concentrated. The resulting residue was purified by silica
gel column chromatography (10−20% MeOH in DCM) to give the
desired product as yellow oil. The oil was dissolved in a HCl solution
in EtOAc (4 M) and stirred for 1 h, at which time the reaction
mixture was concentrated to give the title compound as HCl salt (1.17
EXPERIMENTAL SECTION
■
Chemistry General Procedure. All chemicals and reagents were
purchased from commercial suppliers and used without further
purification. LCMS spectra for all compounds were acquired using a
Waters LC−MS AcQuity H UPLC class system. The Waters LC−MS
AcQuity H UPLC class system comprised a pump (Quaternary
Solvent Manager) with a degasser, an autosampler (FTN), a column
oven (40 °C, unless otherwise indicated), and a photodiode array
PDA detector. Chromatography was performed on an AcQuity UPLC
BEH C18 (1.7 μm, 2.1 × 50 mm) with water containing 0.1% formic
acid as solvent A and acetonitrile containing 0.1% formic acid as
solvent B at a flow rate of 0.6 mL/min. Flow from the column was
split to a mass spectrometry (MS) spectrometer. The MS detector
was configured with an electrospray ionization source. Nitrogen was
used as the nebulizer gas. Data acquisition was performed with a
MassLynx data system. Nuclear magnetic resonance spectra were
recorded on a Bruker Avance III400 spectrometer. Chemical shifts are
expressed in parts per million (ppm) and reported as the δ value
(chemical shift δ). Coupling constants are reported in units of hertz (J
value, Hz; integration and splitting patterns: where s = singlet, d =
double, t = triplet, q = quartet, brs = broad singlet, and m = multiple).
The purification of intermediates or final products was performed on
Agilent Prep 1260 series with a UV detector set to 254 or 220 nm.
Samples were injected onto a Phenomenex Luna C18 column (5 μm,
30 × 75 mm) at room temperature. The flow rate was 40 mL/min. A
linear gradient was used with either 10 or 50% MeOH in H2O
containing 0.1% TFA as solvent A and 100% of MeOH as solvent B.
Alternatively, the products were purified on a CombiFlash NextGen
300 system with a UV detector set to 254, 220, or 280 nm. The flow
rate was 40 mL/min. A linear gradient was used with H2O containing
0.05% TFA as solvent A and 100% of MeOH containing 0.05% TFA
as solvent B. All compounds showed >95% purity using the LCMS
methods described above.
1
g, yield: 66%) as a yellow solid. MS (ESI) m/z: 444.2 [M + 1]+. H
NMR (400 MHz, DMSO-d6): δ 9.62 (s, 2H), 8.63 (s, 1H), 8.21 (s,
1H), 7.62−7.19 (m, 6H), 7.06−7.01 (m, 2H), 5.26−5.25 (m, 1H),
4.07−4.02 (m, 1H), 3.86−3.85 (m, 4H), 3.74−3.72 (m, 1H), 3.16−
3.15 (m, 4H), 2.08−2.07 (m, 2H), 1.92−1.91 (m, 2H).
6-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-
amino)hexanoic Acid (15a). A solution of 2-(2,6-dioxopiperidin-3-
yl)-4-fluoroisoindoline-1,3-dione 13(1.4 g, 5.0 mmol), tert-butyl 6-
aminohexanoate 14a (1.1 g, 5.9 mmol) and N,N-diisopropylethyl-
amine (DIEA, 1.94 g, 15 mmol) in N-methyl-2-pyrrolidone (NMP, 12
mL) was heated at 85 °C in a microwave reactor for 50 min. Four
batches of the reaction mixture were combined and diluted with
EtOAc (200 mL). The mixture was washed with water and brine,
dried over Na2SO4, filtered, and concentrated. The resulting residue
was purified by silica gel chromatography (30% EtOAc in hexanes) to
give tert-butyl 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)hexanoate (1.8 g, yield: 20%) as a yellow solid. A solution of
this intermediate (1.8 g, 4.06 mmol) in formic acid (15 mL) was
stirred at rt for 30 h. The reaction was concentrated and triturated
with DCM. After the resulting mixture was filtered, the solid was
washed with DCM and MTBE and dried to give the title compound
(1.43 g, yield: 91%) as a yellow solid. MS (ESI) m/z: 388.1 [M + 1]+.
1H NMR (400 MHz, DMSO-d6): δ 11.97 (s, 1H), 11.08 (s, 1H), 7.57
(dd, J = 7.2, 8.8 Hz, 1H), 7.08 (d, J = 8.8 Hz, 1H), 7.02 (d, J = 7.2 Hz,
1H), 6.52 (t, J = 6.0 Hz, 1H), 5.05 (dd, J = 5.6, 12.8 Hz, 1H), 3.30 (q,
J = 6.8 Hz, 2H), 2.93−2.83 (m, 1H), 2.61−2.50 (m, 2H), 2.32 (t, J =
7.2 Hz, 2H), 2.07−2.00 (m, 1H), 1.61−1.50 (m, 4H), 1.39−1.33 (m,
2H).
(R)-3-Bromo-6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo-
[1,2-b]pyridazine (10). To a solution of 3-bromo-6-chloroimidazo-
[1,2-b]pyridazine (4.6 g, 20.0 mmol) in DMSO (40 mL) were added
potassium fluoride (20 g, 362 mmol) and (R)-2-(3-fluorophenyl)-
pyrrolidine (3 g, 18.2 mmol). The resulting mixture was stirred at 100
°C for 12 h, before the mixture was diluted with EtOAc, and washed
with brine. The organic layer was dried over Na2SO4, filtered, and
concentrated. The resulting residue was passed through a short silica
gel column (100% EtOAc) to give the title compound (1.8 g, crude
yield: 28%) as a yellow solid, which was used in the next step without
further purification.
(R)-6-(2-(3-Fluorophenyl)pyrrolidin-1-yl)-3-(6-fluoropyridin-
2-yl)imidazo[1,2-b]pyridazine (12). To a solution of (R)-3-
bromo-6-(2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazine
10 (2.17 g, 6.03 mmol) in toluene (50 mL) were added 2-fluoro-6-
(tributylstannyl)pyridine 11 (3.5 g, 9.04 mmol) and tetrakis-
(triphenylphosphine)palladium (566 mg, 0.49 mmol). The resulting
mixture was stirred at 110 °C for 12 h under a nitrogen atmosphere
before being poured into a mixture of EtOAc and aqueous solution of
1 M potassium fluoride. After stirring at rt for 2 h, the mixture was
extracted with EtOAc (3×). The combined organic layers were dried
3-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)-
amino)ethoxy)propanoic Acid (15b). The title compound (18%
yield over two steps) was synthesized according to procedures for the
1
preparation of 15a. MS (ESI) m/z: 390.1 [M + 1]+. H NMR (400
MHz, DMSO-d6): δ 12.18 (s, 1H), 11.08 (s, 1H), 7.58 (dd, J = 7.2
Hz, 8.8 Hz, 1H), 7.13 (d, J = 8.4 Hz, 1H), 7.04 (d, J = 7.2 Hz, 1H),
6.58 (t, J = 5.6 Hz 1H), 5.05 (dd, J = 6.4 Hz, 12.8 Hz, 1H), 3.67−3.58
(m, 4H), 3.47−3.43 (m, 2H), 2.93−2.84 (m, 1H), 2.61−2.45 (m,
4H), 2.07−2.01 (m, 1H).
2-(2,6-Dioxopiperidin-3-yl)-4-((6-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-6-oxohexyl)amino)isoindoline-
1,3-dione (5). A mixture of (R)-6-(2-(3-fluorophenyl)pyrrolidin-1-
yl)-3-(6-(piperazin-1-yl)pyridin-2-yl)imidazo[1,2-b]pyridazine 7
(50.0 mg, 0.113 mmol), 6-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl)amino)hexanoic acid 15a (43.6 mg, 0.113 mmol), 1-
hydroxy-7-aza-benzo-triazole (HOAt, 45.7 mg, 0.339 mmol), and 1-
ethyl-3-(3- dimethylaminopropyl)carbodiimide (EDCI, 65.1 mg,
0.339 mmol) in DMSO (2 mL) was added to N-methylmorpholine
(0.5 mL). After the reaction was stirred at rt for 12 h, it was diluted
I
https://dx.doi.org/10.1021/acs.jmedchem.0c01342
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Journal of Medicinal Chemistry
pubs.acs.org/jmc
Article
with EtOAc (20 mL) and brine (30 mL). The organic layer was
washed with brine, dried over Na2SO4, filtered, and concentrated. The
resulting residue was purified by preparative TLC (10% MeOH in
DCM) to give the title compound (60 mg, 65% yield) as a yellow
solid. MS (ESI) m/z: 813.7 [M + 1]+. 1H NMR (400 MHz, DMSO-
d6): δ 11.03 (s, 1H), 8.40 (s, 1H), 8.06 (d, J = 9.6 Hz, 1H), 7.49 (t, J
= 8.0 Hz, 2H), 7.30−7.36 (m, 1H), 7.10−7.21 (m, 3H), 6.97−7.03
(m, 2H), 6.93 (d, J = 6.8 Hz, 1H), 6.81 (d, J = 8.8 Hz, 1H), 6.47 (br s,
1H), 5.15 (d, J = 6.8 Hz, 1H), 4.95−5.00 (m, 1H), 3.95−3.99 (m,
1H), 3.61−3.68 (m, 1H), 3.50 (br s, 7H), 3.23 (br s, 2H), 2.76−2.82
(m, 1H), 2.40−2.53 (m, 5H), 2.31 (t, J = 7.2 Hz, 2H), 1.92−2.22 (m,
3H), 1.79−1.88 (m, 1H), 1.47−1.57 (m, 4H), 1.28−1.35 (m, 2H).
13C NMR (100 MHz, DMSO-d6): δ 173.3, 171.2, 170.6, 169.4, 164.1,
2.99 (s, 3H), 2.87−2.96 (m, 1H), 2.63−2.74 (m, 3H), 2.45−2.56 (m,
4H), 2.00−2.09 (m, 3H), 1.86−1.96 (m, 1H). 13C NMR (100 MHz,
DMSO-d6): δ 172.2, 170.3, 169.4, 167.7, 164.1, 161.7, 158.4, 153.4,
146.9, 146.8, 146.6, 144.0, 138.6, 136.8, 132.5, 131.0, 130.9, 123.1,
122.2, 117.9, 115.6, 114.2, 114.0, 113.1, 112.9, 111.1, 110.9, 109.7,
107.9, 69.1, 67.2, 62.0, 49.6, 49.1, 45.0, 44.6, 42.2, 41.1, 35.6, 33.3,
31.6, 27.0, 23.0, 21.8.
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-2-oxoethyl)amino)isoindoline-
1,3-dione (16). The title compound (59% yield) was synthesized
according to procedures for the preparation of 5. MS (ESI) m/z:
1
757.6 [M + 1]+. H NMR (400 MHz, DMSO-d6): δ 11.06 (s, 1H),
8.38 (s, 1H), 8.04 (d, J = 8.8 Hz, 1H), 7.45−7.56 (m, 2H), 7.30−7.36
(m, 1H), 6.96−7.11 (m, 6H), 6.84 (d, J = 8.8 Hz, 1H), 5.13 (d, J =
7.2 Hz, 1H), 4.99−5.04 (m, 1H), 4.18 (s, 2H), 3.94−3.97 (m, 1H),
3.46−3.67 (m, 8H), 2.79−2.88 (m, 1H), 2.38−2.56 (m, 5H), 1.93−
2.01 (m, 3H), 1.79−1.85 (m, 1H).
161.7, 158.4, 153.4, 146.9, 146.7, 146.6, 144.0, 138.7, 136.7, 132.7,
131.0, 128.5, 124.8, 123.1, 122.2, 117.6, 115.5, 114.2, 114.0, 113.1,
112.9, 110.8, 109.5, 108.0, 62.0, 49.0, 45.1, 45.0, 44.7, 42.2, 41.1, 35.6,
32.7, 31.5, 29.0, 26.6, 25.0, 23.1, 26.6.
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(3-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethyl)amino)-
isoindoline-1,3-dione (6). The title compound (83% yield) was
synthesized according to procedures for the preparation of 5. MS
(ESI) m/z: 815.8 [M + 1]+. 1H NMR (400 MHz, DMSO-d6): δ 11.04
(s, 1H), 8.38 (s, 1H), 8.06 (d, J = 10.0 Hz, 1H), 7.45−7.49 (m, 2H),
7.31−7.36 (m, 1H), 7.10−7.24 (m, 3H), 6.97−7.05 (m, 2H), 6.91−
6.93 (m, 1H), 6.76 (d, J = 8.0 Hz, 1H), 6.50 (br s, 1H), 5.15 (d, J =
6.8 Hz, 1H), 4.94−4.99 (m, 1H), 3.95−4.02 (m, 1H), 3.65 (t, J = 6.4
Hz, 2H), 3.50−3.56 (m, 10H), 3.39 (br s, 2H), 2.71−2.83 (m, 1H),
2.58 (t, J = 6.8 Hz, 2H), 2.38−2.50 (m, 5H), 1.91−2.02 (m, 3H),
1.79−1.88 (m, 1H). 13C NMR (100 MHz, DMSO-d6): δ 173.2, 170.5,
169.4, 167.7, 164.1, 161.7, 158.4, 153.4, 146.8, 146.7, 146.6, 143.9,
138.6, 136.7, 134.1, 132.5, 131.0, 128.5, 123.0, 122.2, 117.8, 116.8,
115.7, 114.2, 113.1, 112.9, 111.1, 110.9, 109.7, 108.0, 69.2, 67.2, 62.0,
49.0, 45.0, 44.6, 42.2, 41.2, 35.6, 33.3, 31.4, 23.1, 22.6.
4-((6-(4-(6-(6-((R)-2-(3-Fluorophenyl)pyrrolidin-1-yl)-
imidazo[1,2-b]pyridazin-3-yl)pyridin-2-yl)piperazin-1-yl)-6-
oxohexyl)amino)-2-(1-methyl-2,6-dioxopiperidin-3-yl)-
isoindoline-1,3-dione (25). To a solution of 2-(2,6-dioxopiperidin-
3-yl)-4-((6-(4-(6-(6-((R)-2-(3-fluorophenyl)pyrrolidin-1-yl)imidazo-
[1,2-b]pyridazin-3-yl)pyridin-2-yl)piperazin-1-yl)-6-oxohexyl)amino)-
isoindoline-1,3-dione 5 (25 mg, 0.03 mmol) in DMSO were added
K2CO3 (12.24 mg, 0.09 mmol) and MeI (5.07 mg, 0.036 mmol). The
reaction mixture was stirred at rt for 30 min, before it was diluted with
EtOAc (10 mL) and brine (20 mL). The organic layer was washed
with brine, dried over Na2SO4, filtered, and concentrated. The
resulting residue was purified by Prep-TLC (5% MeOH in DCM) to
give the title compound (24 mg, 97% yield) as a yellow solid. MS
(ESI) m/z: 827.8 [M + 1]+. 1H NMR (400 MHz, DMSO-d6): δ 8.02
(s, 1H), 7.84 (d, J = 9.2 Hz, 1H), 7.49 (t, J = 8.0 Hz, 1H), 7.41 (br s,
1H), 7.27−7.33 (m, 1H), 6.92−7.09 (m, 5H), 6.77 (br s, 1H), 6.68
(d, J = 8.4 Hz, 1H), 6.47 (t, J = 6.0 Hz, 1H), 5.02−5.08 (m, 2H),
3.88−3.93 (m, 1H), 3.55−3.61 (m, 1H), 3.50 (br s, 5H), 3.44 (br s,
2H), 3.19−3.24 (m, 2H), 2.94 (s, 3H), 2.82−2.92 (m, 1H), 2.65−
2.70 (m, 1H), 2.35−2.51 (m, 4H), 2.30 (t, J = 11.2 Hz, 2H), 1.91−
2.01 (m, 3H), 1.77−1.81 (m, 1H), 1.46−1.56 (m, 4H), 1.26−1.34
(m, 2H). 13C NMR (100 MHz, DMSO-d6): δ 172.3, 171.2, 170.3,
169.4, 167.8, 164.1, 161.7, 158.4, 152.3, 147.7, 146.9, 145.9, 138.5,
137.8, 136.8, 132.6, 130.9, 127.7, 126.3, 122.1, 117.7, 114.0, 113.8,
113.0, 112.8, 110.9, 109.9, 109.4, 106.5, 61.8, 49.6, 48.9, 45.2, 44.9,
42.2, 41.2, 35.7, 32.7, 31.6, 29.0, 27.1, 26.6, 25.0, 23.2, 21.8.
2-(2,6-Dioxopiperidin-3-yl)-4-((3-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-3-oxopropyl)amino)isoindoline-
1,3-dione (17). The title compound (53% yield) was synthesized
according to procedures for the preparation of 5. MS (ESI) m/z:
1
771.6 [M + 1]+. H NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H),
8.36 (s, 1H), 8.03 (d, J = 9.6 Hz, 1H), 7.47−7.54 (m, 2H), 7.30−7.35
(m, 1H), 7.09−7.15 (m, 4H), 6.95−7.00 (m, 2H), 6.80 (d, J = 8.4 Hz,
1H), 6.72 (s, 1H), 5.14 (d, J = 7.2 Hz, 1H), 4.95−4.99 (m, 1H),
3.94−3.99 (m, 1H), 3.60−3.66 (m, 1H), 3.51 (br s, 9H), 2.75−2.84
(m, 1H), 2.66 (t, J = 6.0 Hz, 2H), 2.37−2.52 (m, 5H), 1.91−2.02 (m,
3H), 1.80−1.85 (m, 1H).
2-(2,6-Dioxopiperidin-3-yl)-4-((4-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-4-oxobutyl)amino)isoindoline-
1,3-dione (18). The title compound (54% yield) was synthesized
according to procedures for the preparation of 5. MS (ESI) m/z:
1
785.6 [M + 1]+. H NMR (400 MHz, DMSO-d6): δ 11.03 (s, 1H),
8.40 (s, 1H), 8.06 (d, J = 10.0 Hz, 1H), 7.42−7.53 (m, 2H), 7.30−
7.36 (m, 1H), 7.10−7.19 (m, 4H), 6.99 (t, J = 8.4 Hz, 1H), 6.94 (d, J
= 6.8 Hz, 1H), 6.82 (d, J = 8.0 Hz, 1H), 6.61 (s, 1H), 5.15 (d, J = 7.6
Hz, 1H), 4.96−5.01 (m, 1H), 3.96−4.01 (m, 1H), 3.61−3.67 (m,
1H), 3.50−3.52 (m, 7H), 3.28 (s, 2H), 2.77−2.86 (m, 1H), 2.38−
2.53 (m, 7H), 1.94−2.02 (m, 3H), 1.73−1.85 (m, 3H).
2-(2,6-Dioxopiperidin-3-yl)-4-((5-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-5-oxopentyl)amino)isoindoline-
1,3-dione (19). The title compound (53% yield) was synthesized
according to procedures for the preparation of 5. MS (ESI) m/z:
1
799.7 [M + 1]+. H NMR (400 MHz, DMSO-d6): δ 11.10 (s, 1H),
8.48 (s, 1H), 8.13 (d, J = 8.8 Hz, 1H), 7.49−7.60 (m, 2H), 7.38−7.43
(m, 1H), 7.17−7.29 (m, 3H), 7.00−7.12 (m, 3H), 6.89 (d, J = 8.4 Hz,
1H), 6.58 (s, 1H), 5.22 (d, J = 7.2 Hz, 1H), 5.03−5.07 (m, 1H),
4.01−4.08 (m, 1H), 3.68−3.75 (m, 1H), 3.58 (s, 7H), 3.33 (s, 2H),
2.83−2.91 (m, 1H), 2.43−2.60 (m, 7H), 2.00−2.10 (m, 3H), 1.88−
1.96 (m, 1H), 1.62 (s, 4H).
2-(2,6-Dioxopiperidin-3-yl)-4-((7-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-7-oxoheptyl)amino)isoindoline-
1,3-dione (20). The title compound (53% yield) was synthesized
according to procedures for the preparation of 5. MS (ESI) m/z:
1
827.7 [M + 1]+. H NMR (400 MHz, DMSO-d6): δ 11.00 (s, 1H),
8.37 (s, 1H), 8.04 (d, J = 9.6 Hz, 1H), 7.48 (d, J = 8.4 Hz, 2H), 7.30−
7.36 (m, 1H), 7.10−7.20 (m, 4H), 6.98 (t, J = 8.0 Hz, 1H), 6.87 (d, J
= 1.2 Hz, 1H), 6.76−6.82 (m, 2H), 5.15 (d, J = 7.2 Hz, 1H), 4.94−
4.98 (m, 1H), 3.95−3.98 (m, 1H), 3.61−3.67 (m, 1H), 3.50 (s, 7H),
3.08 (t, J = 6.8 Hz, 2H), 2.77−2.81 (m, 1H), 2.40−2.52 (m, 5H),
2.30 (t, J = 7.6 Hz, 2H), 1.82−2.22 (m, 4H), 1.44−1.52 (m, 4H),
1.24−1.36 (m, 4H).
2-(2,6-Dioxopiperidin-3-yl)-4-((8-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-8-oxooctyl)amino)isoindoline-1,3-
dione (21). The title compound (54% yield) was synthesized
according to procedures for the preparation of 5. MS (ESI) m/z:
4-((2-(3-(4-(6-(6-((R)-2-(3-Fluorophenyl)pyrrolidin-1-yl)-
imidazo[1,2-b]pyridazin-3-yl)pyridin-2-yl)piperazin-1-yl)-3-
oxopropoxy)ethyl)amino)-2-(1-methyl-2,6-dioxopiperidin-3-
yl)isoindoline-1,3-dione (26). The title compound (74% yield) was
synthesized according to procedures for the preparation of 25. MS
(ESI) m/z: 829.7 [M + 1]+. 1H NMR (400 MHz, DMSO-d6): δ 8.45
(br s, 1H), 8.12 (br s, 1H), 7.54 (t, J = 8.0 Hz, 2H), 7.37−7.43 (m,
1H), 7.17−7.31 (m, 3H), 7.11 (d, J = 8.4 Hz, 1H), 7.06 (t, J = 8.0 Hz,
1H), 6.99 (d, J = 6.8 Hz, 1H), 6.83 (d, J = 8.4 Hz, 1H), 6.57 (br s,
1H), 5.21 (d, J = 6.8 Hz, 1H), 5.07−5.12 (m, 1H), 4.02−4.07 (m,
1H), 3.72 (t, J = 6.4 Hz, 3H), 3.57−3.64 (m, 9H), 3.46 (br s, 2H),
J
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1
841.7 [M + 1]+. H NMR (400 MHz, DMSO-d6): δ 11.10 (s, 1H),
Technology. Antibodies against eRF3/GSPT1 (ab126090) and
mCherry (ab167453) were purchase from Abcam. HRP-conjugated
anti-α-Tubulin (GNI4310-AT) and anti-GAPDH (GNI4310-GH)
antibodies were purchased from GNI. The CellTiter-Lumi assay kit
was purchased from Beyotime Biotechnology, Beijing. The small
interference RNAs (siRNAs) targeting human CRBN were purchased
from GenePharma, Shanghai. The sequences of all siRNAs used in
this study are as follow: siControl, UUCUCCGAACGUGUCAC-
GUTT; siCRBN-1, CCAGCAAGCUAAAGUGCAA; siCRBN-2,
GCGACUUCGCUGUGAAUUA.
Immunoblotting. Cultured cells were washed with cold PBS once
and lysed in cold RIPA buffer supplemented with protease inhibitors
and phosphatase inhibitors (Beyotime Biotechnology). The solutions
were then incubated at 4 °C for 30 min with gentle agitation to fully
lyse cells. Cell lysates were centrifuged at 13,000 rpm for 10 min at 4
°C, and pellets were discarded. Total protein concentrations in the
lysates were determined following BCA assays (Beyotime Biotechnol-
ogy). Cell lysates were mixed with Laemmli loading buffer to 1× and
heated at 99 °C for 5 min. Proteins were resolved on SDS-PAGE and
visualized using Western ECL Substrate kits on a ChemiDoc MP
Imaging system (Bio-Rad). Protein bands were quantitated using
Image Lab software provided by Bio-Rad.
Cell Viability Assay. Cells were seeded at a density of 5000 cells
per well in 96-well assay plates and treated with test compounds
following a 11-point serial dilution for 3 days. Cell viability was
determined using the CellTiter-Lumi assay kit according to the
manufacturer’s instructions.
Statistical Analyses. Data were analyzed using GraphPad Prism
software. Kd (binding affinities), DC50 (concentration that resulted in
50% of protein degradation), and IC50 (concentration that led to 50%
of maximal cell growth inhibition) were calculated using a nonlinear
regression (least squares fit) method. Data presented were mean
standard deviation (SD).
TMT-Based Quantitative Proteomic Analyses. The selectivity
of degraders was determined by MajorBio (Shanghai, China) using
the TMT quantitative proteomic analysis. In brief, total protein was
extracted from treated KM12 cells using a urea lysis buffer (8 M urea
and 1% SDS) supplemented with protease inhibitors. Protein
concentrations were determined following BCA assays. Protein
digestion was performed according to the standard procedure, and
the resulting peptide mixture was labeled using the 16-plex TMT
reagent (Thermo fisher, A44522) according to the manufacturer’s
instructions. Digested peptides were pooled, desalted, and vacuum-
dried. Pooled peptides were fractionated into fractions by ACQUITY
Ultra Performance liquid chromatography (Waters, USA) with an
ACQUITY UPLC BEH C18 column (1.7 μm, 2.1 mm × 150 mm,
Waters, USA) to increase proteomic depth.
The labeled peptide mixture was separated with a nano reverse-
phase C18 column (75 μm × 25 cm, Thermo, USA) connected to
EASY-nLC 1200 (Thermo, USA) and analyzed with a Q Exactive HF-
X Hybrid Quadrupole-Orbitrap mass spectrometer (Thermo, USA).
The RAW data files were analyzed using Proteome Discover software
version 2.4 (Thermo Scientific) against the UniProt human protein
sequence database (UP000005640). The MS/MS search criteria were
as follows: mass tolerance of 20 ppm for MS and 0.02 Da for MS/MS,
trypsin as the enzyme with two missed cleavage allowed, carbamido
methylation of cysteine and the TMT of N- terminus and lysine side
chains of peptides as fixed modification, and methionine oxidation as
dynamic modifications. The false discovery rate (FDR) of peptide
identification was set as FDR ≤0.01. A minimum of one unique
peptide identification was used to support protein identification.
Mouse PK Studies. Standard PK studies were conducted using
male ICR mice. Compounds 5 and 6 were dissolved in 5% NMP, 10%
solutol, 30% PEG 400, and 55% saline as solution formulation. A
single 10 mg/kg IP injection of degraders was evaluated. Plasma
concentrations of degraders reported at each of the eight time points
(5, 15, 30 min, 1, 2, 4, 6, and 8 h post dosing) are the mean values
from four experimental animals. Error bars represent SD.
8.46 (s, 1H), 8.13 (d, J = 10.4 Hz, 1H), 7.48−7.58 (m, 2H), 7.37−
7.43 (m, 1H), 7.17−7.27 (m, 3H), 7.00−7.08 (m, 3H), 6.89 (d, J =
7.6 Hz, 1H), 6.52 (s, 1H), 5.22 (d, J = 7.2 Hz, 1H), 5.03−5.07 (m,
1H), 3.99−4.08 (m, 1H), 3.69−3.75 (m, 1H), 3.57 (s, 7H), 3.29 (s,
2H), 2.84−2.89 (m, 1H), 2.47−2.61 (m, 5H), 2.35 (t, J = 7.2 Hz,
2H), 2.01−2.08 (m, 3H), 1.90−1.92 (m, 1H), 1.52−1.57 (m, 4H),
1.33 (s, 6H).
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(3-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethyl)-
amino)isoindoline-1,3-dione (22). The title compound (72%
yield) was synthesized according to procedures for the preparation of
5. MS (ESI) m/z: 859.9 [M + 1]+. 1H NMR (400 MHz, DMSO-d6):
δ 11.11 (s, 1H), 8.45 (s, 1H), 8.11 (d, J = 9.6 Hz, 1H), 7.50−7.54 (m,
2H), 7.38−7.43 (m, 1H), 7.17−7.27 (m, 3H), 7.04−7.08 (m, 2H),
6.98−7.00 (m, 1H), 6.86 (d, J = 8.8 Hz, 1H), 6.57 (s, 1H), 5.22 (d, J
= 8.0 Hz, 1H), 5.03−5.08 (m, 1H), 4.02−4.06 (m, 1H), 3.64−3.72
(m, 3H), 3.54−3.61 (s, 13H), 3.42 (s, 2H), 2.85−2.89 (m, 1H),
2.46−2.62 (m, 7H), 2.00−2.10 (m, 3H), 1.90−1.93 (m, 1H).
2-(2,6-Dioxopiperidin-3-yl)-4-((2-(2-(2-(3-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
pyridin-2-yl)piperazin-1-yl)-3-oxopropoxy)ethoxy)ethoxy)-
ethyl)amino)isoindoline-1,3-dione (23). The title compound
(49% yield) was synthesized according to procedures for the
preparation of 5. MS (ESI) m/z: 903.7 [M + 1]+. H NMR (400
1
MHz, DMSO-d6): δ 11.11 (s, 1H), 8.46 (s, 1H), 8.12 (d, J = 10.0 Hz,
1H), 7.53 (t, J = 8.0 Hz, 2H), 7.37−7.43 (m, 1H), 7.17−7.27 (m,
3H), 7.00−7.09 (m, 3H), 6.88 (d, J = 7.6 Hz, 1H), 6.56 (s, 1H), 5.21
(d, J = 7.2 Hz, 1H), 5.03−5.08 (m, 1H), 3.99−4.09 (m, 1H), 3.50−
3.72 (m, 19H), 3.42 (s, 2H), 2.84−2.93 (m, 1H), 2.43−2.63 (m, 8H),
2.01−2.09 (m, 3H), 1.89−1.92 (m, 1H).
2-(2,6-Dioxopiperidin-3-yl)-4-((15-(4-(6-(6-((R)-2-(3-
fluorophenyl)pyrrolidin-1-yl)imidazo[1,2-b]pyridazin-3-yl)-
p y r i d i n - 2 - y l ) p i p e r a z i n - 1 - y l ) - 1 5 - o x o - 3 , 6 , 9 , 1 2 -
tetraoxapentadecyl)amino)isoindoline-1,3-dione (24). The
title compound (56% yield) was synthesized according to procedures
for the preparation of 5. MS (ESI) m/z: 947.6 [M + 1]+. H NMR
1
(400 MHz, DMSO-d6): δ 11.16 (s, 1H), 8.58 (s, 1H), 8.21 (d, J = 9.6
Hz, 1H), 7.59 (t, J = 8.0 Hz, 2H), 7.42−7.48 (m, 1H), 7.22−7.35 (m,
3H), 7.05−7.15 (m, 3H), 6.95 (d, J = 8.4 Hz, 1H), 6.62 (s, 1H), 5.27
(d, J = 7.2 Hz, 1H), 5.08−5.13 (m, 1H), 4.07−4.11 (m, 1H), 3.48−
3.79 (m, 26H), 2.89−2.98 (m, 1H), 2.67 (t, J = 6.4 Hz, 2H), 2.50−
2.62 (m, 5H), 2.06−2.15 (m, 3H), 1.95−1.98 (m, 1H).
Binding Affinity Assays. Binding affinities were determined by
DiscoverX (San Diego, USA) using the KINOMEscan platform.
KINOMEscan is based on a competition binding assay that
quantitatively measures the ability of a compound to compete with
an immobilized, active site-directed ligand. The assay is performed by
combining three components: DNA-tagged TRK, an immobilized
ligand, and a test compound. The ability of the test compound to
compete with the immobilized ligand is measured via quantitative
PCR of the DNA tag. Kd values were determined using an 11-point
threefold compound dilution series (the top concentration of 1 μM
for degraders and 0.3 μM for GNF-8625) with three DMSO control
points in duplicate. Some outlier data points were subtracted.
Cell Culture and Transfection. KM12 and HEL cells were
cultured at 37 °C with 5% CO2 in DMEM or RPMI 1640 medium
supplemented with 10% fetal bovine serum (Gibco, Thermo Fisher).
Cells were authenticated using the short tandem repeat (STR) assays
by BioWing Applied Biotechnology, Shanghai. Mycoplasma con-
tamination was excluded following a PCR-based method. Cell
transfection was performed using Lipofectamine 2000 (Invitrogen)
following the manufacturer’s instructions. Stable cell lines were
established by lentivirus transduction, selected, and maintained in a
medium containing 1 μg/mL puromycin (Beyotime Biotechnology).
Plasmids and Reagents. Human AGBL4-TRKB and ETV6-
TRKC fusions were constructed into the pLVX-EF1a-mCherry-C1
lentiviral expression vector for stable transduction. Antibodies against
TRK (pan) (92991S), PLCγ2 (3872S), p-PLCγ2 (Tyr759, 3874S),
and CRBN (71810S) were purchased from Cell Signaling
K
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Article
Notes
ASSOCIATED CONTENT
■
The authors declare the following competing financial
interest(s): All authors are employees of Cullgen. Y. L. is the
president and CEO of Cullgen and GNI Group. GNI group
has a significant financial equity interest in Cullgen.
sı
* Supporting Information
The Supporting Information is available free of charge at
https://pubs.acs.org/doi/10.1021/acs.jmedchem.0c01342.
1H NMR and 13C NMR spectra, and UPLC-MS traces
of compounds 5 and 6; screening of the synthesized
putative TRK degraders; immunoblotting analysis of
samples for proteomic profiling; significantly differential
proteins from proteomic profiling; and raw data of
compounds 5 and 6 (PDF)
ACKNOWLEDGMENTS
■
We thank Seth Goldblum at Cullgen for proof-reading the
manuscript. The design, study conduct, and financial support
for the research were provided by Cullgen.
ABBREVIATIONS
Molecular formula strings and biological data (CSV)
■
TRK, tropomyosin receptor kinase; PROTAC, proteolysis
targeting chimera; uSMITE, ubiquitin-mediated, small mole-
cule-induced target elimination; SNIPER, specific and non-
genetic IAP-dependent protein erasers; DEGRONIMID,
acronym/neologism from C4 Therapeutics used to describe
a protein degrader; TPM3, tropomyosin 3; AGBL4, ATP/GTP
binding protein like 4; ETV6, ETS variant transcription factor
6; NGF, nerve growth factor; BDNF, brain-derived neuro-
trophic factor; PLCγ, phospholipase C gamma; LMNA, lamin
A/C; MOA, mechanism of action; UPS, ubiquitin−protea-
some system; BBB, blood−brain−barrier; CNS, central
nervous system; CRBN, cereblon; MS, mass spectrometry;
VHL, Von Hippel−Lindau; SAR, structure−activity relation-
ship; PEG, polyethylene glycol; GSPT1, G1 to S phase
transition 1; TMT, tandem mass tag; CRL, cullin-RING E3
ubiquitin ligase; PK, pharmacokinetic; IP, intraperitoneal;
DMSO, dimethyl sulfoxide; DIEA, N,N-diisopropylethylamine;
NMP, N-methyl-2-pyrrolidone; EDCI, 1-ethyl-3-(3-
dimethylaminopropyl)carbodiimide; HOAt, 1-hydroxy-7-aza-
benzo-triazole; NMM, N-methylmorpholine
AUTHOR INFORMATION
■
Corresponding Authors
Xiao-Ran Han − Cullgen Inc., San Diego, California 92130,
United States; Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China; Email: xiaoran.han@cullgen.com
Jing Liu − Cullgen Inc., San Diego, California 92130, United
States; orcid.org/0000-0003-4740-243X; Email: jing.liu@
cullgen.com
Authors
Liqun Chen − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Yanke Chen − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Chunyan Zhang − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Bingyang Jiao − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Sheng Liang − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
REFERENCES
■
Qiong Tan − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
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People’s Republic of China
Weihua Yu − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Yongzheng Qian − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Hui Yang − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Wuyi Yao − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Jianguo Yu − Cullgen (Shanghai), Inc., Shanghai 201202,
People’s Republic of China
Ying Luo − Cullgen Inc., San Diego, California 92130, United
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