Discovery of Selective Small Molecule Degraders of BRAF-V600E

Article abstract of DOI:10.1021/acs.jmedchem.9b02083

BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations. However, the benefits of BRAF kinase inhibitors are generally short-lived. Small molecule-mediated targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we developed thalidomide-based heterobifunctional compounds that induced selective degradation of BRAF-V600E, but not the wild-type BRAF. Downregulation of BRAF-V600E suppressed the MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the interaction between degraders and cereblon or blocking the UPS significantly impaired the activities of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF mutants and provide a framework to treat BRAF-dependent human cancers.

Full text of DOI:10.1021/acs.jmedchem.9b02083

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Article
Discovery of Selective Small Molecule Degraders of BRAF-V600E
Xiao-Ran Han, Liqun Chen, Yuanqi Wei, Weihua Yu, Yanke Chen,
Chunyan Zhang, Bingyang Jiao, Tingting Shi, Lei Sun, Chao Zhang,
Yang Xu, Matthew R Lee, Ying Luo, Michael B. Plewe, and Jialiang Wang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b02083 • Publication Date (Web): 30 Mar 2020
Downloaded from pubs.acs.org on March 30, 2020
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Discovery of Selective Small Molecule Degraders of BRAF-V600E
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Xiao-Ran Han, Liqun Chen, Yuanqi Wei, Weihua Yu, Yanke Chen, Chunyan Zhang, Bingyang
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Jiao, Tingting Shi, Lei Sun, Chao Zhang, Yang Xu, Matthew R. Lee, Ying Luo, Michael B.
Plewe, * Jialiang Wang †,§,*
†,
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Cullgen Inc., 12671 High Bluff Drive Suite 130, San Diego, California 92130, USA. Cullgen
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Shanghai) Inc., Building 6, 230 ChuanHong Road, Chuansha, Pudong New Area, Shanghai 201202,
People’s Republic of China.
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Abstract
BRAF is among the most frequently mutated oncogenes in human cancers. Multiple small molecule
BRAF kinase inhibitors have been approved for treating melanoma carrying BRAF-V600 mutations.
However, the benefits of BRAF kinase inhibitors are generally short lived. Small molecule-mediated
targeted protein degradation has recently emerged as a novel pharmaceutical strategy to remove
disease proteins through hijacking the cellular ubiquitin proteasome system (UPS). In this study, we
developed thalidomide-based heterobifunctional compounds that induced selective degradation of
BRAF-V600E, but not the wild type BRAF. Downregulation of BRAF-V600E suppressed the
MEK/ERK kinase cascade in melanoma cells and impaired cell growth in culture. Abolishing the
interaction between degraders and cereblon or blocking the UPS significantly impaired the activities
of these degraders, validating a mechanistic role of UPS in mediating targeted degradation of BRAF-
V600E. These findings highlight a new approach to modulate the functions of oncogenic BRAF
mutants and provide a framework to treat BRAF-dependent human cancers.
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Introduction
The rapidly accelerated fibrosarcoma (RAF) family of serine/threonine kinases, including ARAF,
BRAF, and CRAF, transduce signals downstream of RAS to the mitogen-activated protein kinase
(MAPK) cascade. They are the core components of a signal transduction network that supports
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proliferation and survival in mammalian cells. Aberrations of BRAF are widely documented in human
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cancer, accounting for 6-8% of all cases. In particular, almost half of melanoma and virtually all hairy
cell leukemia are driven by BRAF mutations. While many forms of BRAF mutations have been
identified, approximately 90% of these mutations convert the valine residue at codon 600 into a
glutamic acid residue (i.e. V600E), which renders BRAF acting as a constitutively active monomer.3
BRAF-V600E and other oncogenic BRAF mutants result in aberrant activation of the downstream
MEK/ERK kinase cascade and other signaling pathways, thereby promoting oncogenesis.
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To date, three BRAF kinase inhibitors, vemurafenib, dabrafenib, and encorafenib, have been
approved for treating melanoma patients carrying BRAF-V600 mutations. These inhibitors belong to
the second generation of BRAF inhibitors that selectively target BRAF-V600 mutants and block
activity of these proteins in monomeric forms. These BRAF kinase inhibitors have wide therapeutic
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windows because they spare dimeric wild type BRAF in normal tissues. In addition, they are not
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active against other classes of BRAF mutants, such as BRAF fusions and splice variants. Furthermore,
the second-generation BRAF inhibitors are known to enhance formation of BRAF homodimer or
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heterodimers between BRAF and other RAF proteins. This unique mechanism is thought to
compromise clinical efficacy of these drugs, promote acquired resistance, and induce adverse effects,
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particularly induction of secondary skin cancer. Nevertheless, vemurafenib and other approved BRAF
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kinase inhibitors have produced marked response in melanoma patients with BRAF-V600 mutations.10
The clinical benefits of currently approved BRAF kinase inhibitors are greatly restrained by rapid
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emergence of acquired resistance. Combination with MEK inhibitors neutralizes the paradoxical
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activation of wild type BRAF induced by BRAF inhibitors, modestly improves response rate, and
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extends tumor control. However, resistance and tumor recurrence remain inevitable. Approaches
leading to more potent, selective and durable suppression of oncogenic BRAF mutants are expected to
produce better tumor response and provide additional clinical benefits to patients.
Hijacking the ubiquitin proteasome system via heterobifunctional small molecule compounds is an
emerging pharmaceutical strategy aiming to selectively remove disease-causing proteins in affected
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cells. These small molecule degraders are also known as proteolysis-targeting chimeras (PROTACs),
as these compounds comprise two recruiting ligands connected by a linker. One of the ligands binds
to an E3 ubiquitin complex, and the other recruits the protein of interest (POI) to the E3 ligase complex.
Once brought to close proximity of E3 ligases, the POI is polyubiquitinated and subsequently degraded
by the proteasome. Targeted degradation of POIs not only compromises the catalytic activities of these
proteins but also removes their scaffolding and other non-catalytic functions, providing unique
advantages over inhibitors.
A growing list of disease-relevant proteins, mostly implicated in oncology, has been targeted by
PROTAC molecules, such as BRD4, BRD9, ALK, and CDK4/6.13-20 In the current study, we report
two BRAF-V600E degraders generated from the E3 ligase ligand thalidomide and two BRAF kinase
inhibitors, vemurafenib and BI882370.21 These degraders selectively induced downregulation of
BRAF-V600E in melanoma cells at nanomolar concentrations but not wild type BRAF, leading to
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Figure 1. Chemical structures of 1 (vemurafenib), 2 (dabrafenib), 3 (encorafenib), 4 (BI882370) and 5 (Vem-
bisamide-2).
growth arrest and apoptosis in target cells. Degradation of BRAF-V600E was verified to be dependent
on the ubiquitin proteasome system. Thereby we demonstrated a novel therapeutic approach
modulating oncogenic BRAF mutants at protein levels.
Results
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Design and synthesis of BRAF degraders. From multiple reported BRAF kinase inhibitors we
selected vemurafenib and BI882370 as the BRAF binding moiety (Figure 1). Vemurafenib is the first
approved BRAF kinase inhibitor for the treatment of late stage melanoma with threefold selectivity
for BRAF-V600E over wild type BRAF,22 while BI882370 is a more potent pan-RAF kinase
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inhibitor. Co-crystal structures of BRAF in complex with vemurafenib (PDB ID: 3OG7) and with 5
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Vem-bisamide-2) (PDB ID: 5JT2) show that part of the phenyl ring is solvent exposed allowing
attachment of the linker. We connected the linker through an amide bond, with molecular modeling
suggesting that the amide carbonyl group is capable of forming a hydrogen bond with the imidazole
ring of His539 in BRAF-V600E (Supporting Figure S1). In the co-crystal structure of BRAF with
BI882370 (PDB ID: 5CSX), the ethylpiperidinyl group is exposed to solvent and was exploited for
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attachment of the linker. Besides the correct site of attachment, the choice of linker and E3-ligase
ligand is critical for the development of effective PROTACs. In this study, a set of degraders varying
in linkers and E3-ligase ligands was screened against BRAF-V600E (Supporting Figure S2-5), leading
to the identification of two degraders of BRAF-V600E, namely 12 and 23. In 12, vemurafenib is
attached to the E3 ligase ligand thalidomide at the meta-position through a pentanoyl linker. In 23,
using BI882370 as warhead, an 11-atom PEG linker was found to be the better choice (Scheme 1). We
prepared 13 and 24 as the corresponding negative control compounds by N-methylation of the
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thalidomide moiety which disabled binding to cereblon. We developed a convergent synthetic route
for the synthesis of the designed BRAF degraders and their negative control compounds (Scheme 1).
Suzuki coupling of 6 and 7, followed by Boc deprotection afforded the vemurafenib analog 8 as the
BRAF binding warhead. The thalidomide derivatives 11a and 11b were synthesized by nucleophilic
aromatic substitution of the meta-fluoro-substituted thalidomide derivatives 9a and 9b with amino
ester 10, followed by tert-butyl ester deprotection. Amide coupling of 8 with 11a and 11b afforded 12
and its negative control 13 as the desired product. Ullman type C-N coupling of 14 and 15 gave
intermediate 16, which was converted to 17 by iodination. Warhead 19 was made by Suzuki coupling
of 17 with 5-pyrimidinylboronic acid 18, followed by Boc deprotection. The linker attached
pomalidomide analogs 22a and 22b were synthesized following similar procedures as for 11a. Amide
coupling of 22a and 22b with warhead 19 afforded the desired compound 23 and its negative control
24.
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2 and 23 bind BRAF and BRAF-V600E with high affinity. Using the DiscoverX KINOMEscan
platform, the binding affinities of the two degraders to wild type BRAF or BRAF-V600E mutant were
compared with their corresponding warheads. Our results demonstrated that the affinities of 23 were
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Scheme 1. Synthesis of 12 and 23 and their cereblon binding-deficient derivatives.
Reagents and conditions: (a) Pd(dppf)Cl , K PO , dioxane/H O, 80 C, 6 h; (b) HCl/EtOAc, rt, overnight; (c)
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DIEA, NMP, microwave, 85 C, 30 min; (d) TFA, rt, 2 h; (e) EDCI, HOAt, NMM, DMSO, rt, overnight; (f) CuI,
(R,R)-N,N'-dimethyl-1,2-diaminocyclohexane, Cs CO , toluene, 120 C, 48 h; (g) NIS, DMF/THF, rt, 1 h; (h)
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approximately 6-fold lower than those of BI882370 for both BRAF and BRAF-V600E mutant (Figure
). In contrast, the affinities of 12 were 4- to 5-fold higher compared to vemurafenib (Figure 2), most
likely due to the formation of an additional hydrogen bond between the amide carbonyl of 12 and the
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Figure 2. Binding affinities of vemurafenib, BI882370, degraders (12, 23), and negative controls
13, 24) to BRAF or BRAF-V600E. Binding affinities of individual compounds were determined
(
using the KINOMEscan assay (DiscoveryX). The lowest concentration points represent data for
the DMSO samples. Data are shown as mean ± SEM derived from duplicated independent
experiments.
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imidazole ring of His539 in BRAF as predicted by molecular modeling (Supporting Figure S1).
Modifications to the thalidomide moiety by N-methylation (13, 24) did not significantly affect
affinities to either wild type BRAF or BRAF-V600E (Figure 2).
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2 and 23 induce degradation of BRAF-V600E. The ability of 12 and 23 to degrade BRAF-V600E
protein was first examined in A375 cells, a melanoma cell line homogeneously expressing BRAF-
V600E. Cells were incubated with the degraders at various concentrations for 16 hours. For both
degraders, reductions of BRAF-V600E protein levels were detected at 12 nM and intensified at higher
concentrations, with the downstream ERK phosphorylation inhibited accordingly (Figure 3A and 3B).
Moderate hook effects were observed at 1000 nM for both 12 and 23, probably as excessive
compounds undermined formation of the ternary complex of E3-degrader-BRAF-V600E. To
determine the kinetics of BRAF-V600E degradation, we showed that significant depletion was not
detected until 16 hours after degrader treatment (Figure 3C). However, reduction in the
phosphorylation of ERK induced by 23 was detected as early as 2 h after incubation with cells,
suggesting that degradation of BRAF-V600E was substantially delayed after binding of the degraders
and the target protein.
In addition to BRAF-V600E, both vemurafenib and BI882370 are known to bind to the active form of
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wild type BRAF. The KINOMEscan data also suggested that the degraders bound to wild type BRAF
and BRAF-V600E with comparable affinities. Hence, we explored whether these degraders also
degraded wild type BRAF using a lung cancer cell line, A549, in which BRAF is constantly activated
by signals transduced from the oncogenic KRAS mutant. Our results demonstrated that neither 12 nor
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(h)
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Lane
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V600E
p-ERK
Tubulin
Figure 3. 12 and 23 induce degradation of BRAF-V600E. (A, B) A375 cells were treated with
DMSO or serial dilutions of indicated compounds for 16 h. (C) A375 cells were treated with
DMSO, 500 nM BI882370 or 23 for the indicated length of time.
2
3 resulted in significant degradation of wild type BRAF (Figure 4A and 4B), suggesting that binding
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to the targeted protein is insufficient to induce degradation in the case of BRAF. To exclude influences
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from variations in cellular context, we introduced expression of the kinase domain (residues: 448-723)
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Tubulin
B
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BI882370
Cpd.
nM)
Veh.
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(
15 30 62 125 250 500 1000 15 30 62 125 250 500 1000
Lane
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10 11 12 13 14 15
BRAF
Tubulin
A375 (BRAF KD-WT)
A375 (BRAF KD-V600E)
Cpd.
nM)
12
12
Veh.
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FLAG-
BRAF KD
Tubulin
D
A375 (BRAF KD-WT)
A375 (BRAF KD-V600E)
Cpd.
nM)
23
23
Veh.
1
Veh. Veh.
9
Veh.
(
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10 11 12 13 14 15 16 17
FLAG-
BRAF KD
Tubulin
Figure 4. 12 and 23 do not degrade wild type BRAF. (A, B) A549 cells were treated with DMSO
or indicated compounds following a 3-fold serial dilution for 16 h. (C, D) A375 cells stably
expressing the kinase domain of BRAF or BRAF-V600E was treated with DMSO or indicated
compounds for 16 hours following a 3-fold serial dilution. Levels of indicated proteins were
shown using immunoblotting.
of BRAF-V600E or wild type BRAF into A375 cells. In line with the observations with full-length
proteins, 12 and 23 selectively degraded the kinase domain of BRAF-V600E but not the wild type one
(Figure 4C and 4D). These results collectively demonstrate that compounds 12 and 23 are potent and
selective degraders for BRAF-V600E. However, there is a latency of degradation following binding
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of the degraders and the target protein, which appeared to be longer than those for some of the reported
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degraders, such as BRD4 , ALK , CDK4/6 , and BRD7/9 . It is possible that our degraders require
further optimization to facilitate the formation of productive ternary complexes comprising of E3
ligase, the degrader, and BRAF-V600E.
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Degradation of BRAF-V600E is mediated by the ubiquitin-proteasome system. To confirm that
the activities of our degraders were mediated via the cullin family E3 ligases and the UPS system, we
employed multiple approaches to validate the mechanisms of BRAF-V600E targeted degradation. We
generated derivatives of 12 and 23 that were deficient in binding to cereblon (Scheme 1). These
negative control compounds, 13 and 24 respectively, were incapable of inducing degradation of
BRAF-V600E in A375 cells (Figure 5A and 5B). However, their abilities of suppressing the
downstream ERK phosphorylation were impaired but not blocked (Figure 5A and 5B), suggesting that
the impact on downstream signaling for the degraders could result from both kinase inhibitory and
protein degrading activities. We further demonstrated that degradation of BRAF-V600E induced by
12 and 23 could be rescued by pretreatment with proteasome inhibitors MG-132 or bortezomib (Figure
5
C and 5D). Additionally, excessive amount of pomalidomide also counteracted the degrading
activities of 12 and 23, likely through competitive interaction with cereblon (Figure 5C and 5D).
Similarly, pretreatment with MLN4929, an inhibitor of the NEDD8-activating enzyme (NAE) that
suppresses the cullin family E3 ligases, also restored BRAF-V600E levels in the presence of 12 and
23 (Figure 5C and 5D). These results collectively validate a cullin-UPS-mediated mechanism for
targeted degradation of BRAF-V600E.
Degradation of BRAF-V600E impairs melanoma cell growth. We next assessed the cellular
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activities of BRAF-V600E degraders. In line with previous publications, BRAF kinase inhibitors
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vemurafenib and BI882370 reduced viability of A375 cells (Figure 6A and 6B). The IC value of 23
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was approximately 10 times higher than its warhead, BI882370, while the IC of 24, the cereblon
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p-ERK
Tubulin
Cpd.
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BRAF-
V600E
p-ERK
Tubulin
C
12 (500 nM)
23 (500 nM)
D
BRAF-
V600E
BRAF-
V600E
p-ERK
p-ERK
Tubulin
Tubulin
Figure 5. Degradation of BRAF-V600E is mediated by the ubiquitin-proteasome system (UPS).
A, B) A375 cells were treated with serial dilutions of compound 12, 23, or their respective
negative controls 13 or 24 for 16 hours prior to immunoblotting. (C, D) A375 cells were pre-
treated for 1 hour with DMSO, pomalidomide (POM, 10 µM), MG-132 (20 µM), bortezomib
200 nM) or MLN4924 (1 µM), and subsequently incubated with 500 nM 12 or 23 for 6 hours
(
(
prior to immunoblotting.
binding-deficient derivative, was 4 times higher than 23, suggesting that target degradation contributed
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A
B 125
A375
A375
BI882370, IC50=6.3 nM
1
25
Vemurafenib, IC50=116 nM
2
3, IC50=46.5 nM
4, IC50=190 nM
1
2, IC50=500 nM
3, IC50=492 nM
100
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10-10 10-9 10-8 10-7 10-6 10-5
Compounds (M)
1
0-10 10-9 10-8 10-7 10-6 10-5
Compounds (M)
C
D
F
HT-29
HT-29
125
125
100
75
50
25
0
Vemurafenib, IC50=164nM
BI882370, IC50=1.7 nM
23, IC50=51 nM
1
00
12, IC50=124 nM
24, IC50=348 nM
13, IC50=459 nM
7
5
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0
0-10 10-9 10-8 10-7 10-6 10-5
10-10 10-9 10-8 10-7 10-6 10-5
1
Compounds (M)
Compounds (M)
E
100
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*
*
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V
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V
1
2 (nM)
13 (nM) Vemurafenib (nM)
23 (nM)
24 (nM) BI882370 (nM)
Figure 6. Degradation of BRAF-V600E impairs cell viability and colony formation. (A-D)
Compound 12 and 23 reduced viability of A375 and HT-29 cells in a dose-dependent manner. (E,
F) Compounds 12 and 23 inhibited colony formation of A375 cells. Data are shown as mean ±
SD. *: p< 0.05 by Student’s t-test compared with the corresponding control groups.
substantially to the cellular activity of 23 (Figure 6B). In contrast, the IC values of 12 and its non-
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degrading control 13 were similar (Figure 6A), suggesting that target degradation and kinase inhibition
are implicated in the cellular activity of 12 to comparable extents. In the BRAF-V600E-dependent
colon cancer cell line HT-29, these degraders and their negative controls induced similar patterns of
growth inhibition, although the IC value of 12 appeared to be slightly lower in this line (Figure 6C
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and 6D). These compounds as well as their warheads also impaired colony formation by A375 cells in
a fashion resembling the observations in cell viability assays (Figure 6E and 6F). On the contrary, the
degraders and their warheads did not induce significant loss of cell viability at physiologically
concentrations in 3 non-neoplastic human cell lines (Supporting Figure S6). Taken together, our data
suggest that selective degradation of BRAF-V600E by small molecule compounds impairs cell growth
and viability in BRAF-V600E-driven melanoma and colon cancer cells.
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Discussion and Conclusions
BRAF-V600E expression has been reported in a wide variety of human cancers. Mutations of BRAF
2
6
are particularly common in melanoma, colon cancer, and thyroid cancer. Additional aberrations that
activate BRAF, such as alternative splicing and chromosomal translocation, have been increasingly
27
recognized. Multiple approved BRAF-V600E kinase inhibitors provide significant yet short-lived
benefits to melanoma patients. In the current study, we report development of small molecule BRAF
degraders. Degraders generated using VHL-1 as the E3 ligand were modestly active at best. In contrast,
both thalidomide and pomalidomide yielded potent BRAF degraders with either vemurafenib or
BI882370 as the warhead. Linker length had significant yet contrasting impact on the efficacy of
degraders. Shorter linker length was associated with higher potency for degraders with vemurafenib
as the warhead, while BI882370-based degraders required longer linker length in order to induce
significant degradation. The development efforts were culminated in the discovery of two low-
nanomolar degraders, 12 and 23. These compounds selectively induced degradation of BRAF-V600E
but not wild type BRAF, despite binding to both proteins with similar affinities. Mounting evidence
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show that PROTAC compounds differentially degrade closely related target proteins,28-31 suggesting
that forming the ternary complex is necessary but insufficient to induce effective degradation of the
target proteins. It has been well documented that BRAF kinase inhibitors bind to wild type BRAF and
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BRAF-V600E in distinct modes. Hence, our results suggest that 12 and 23 may bind to BRAF-V600E
in a conformation that favors the formation of cooperative ternary complex with the E3 complex,
whereas they may form disruptive ternary complex with wild type BRAF. Following rapid binding to
BRAF-V600E and blockade of the downstream MAPK kinase cascade, there were several hours of
delay before significant degradation of BRAF-V600E could be detected. Therefore, the rate-limiting
step of small molecule-induced proteolysis of BRAF-V600E is likely not the formation of the
degrader-BRAF-V600E binary complex. Additional data, particularly crystal structures, will be
required to fully address how 12 and 23 differentially target wild type BRAF and BRAF-V600E mutant.
In BRAF-V600E-dependent melanoma cells and colon cancer cells, 12 and 23 provoked in vitro
antineoplastic activities comparable to clinical stage BRAF kinase inhibitors, but not in non-neoplastic
human cells. Further studies are required to optimize the physiochemical properties of these novel
degraders for preclinical and clinical development. Their activities should also be characterized in a
wider range of BRAF-V600E-expressing cells. While the kinase activity has been well documented to
be essential for the oncogenic potential of BRAF-V600E, kinase-independent activities of wild type
BRAF and BRAF mutants have been described in various diseases.33-36 Thus, the development of
BRAF-V600E selective degraders provides a platform to systemically investigate the impact of
depleting oncogenic BRAF mutants in human cancers.
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Experimental Section
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General Chemistry Methods. All chemicals and reagents were purchased from commercial suppliers
and used without further purification. LCMS spectra for all compounds were acquired using a
Shimadzu LC-MS 2020 system comprising a pump (LC-20AD) with degasser (DGU-20A3), an
autosampler (SIL-20AHT), a column oven (CTO-20A) (set at 40 C, unless otherwise indicated), a
photo-diode array (PDA) (SPD-M20A) detector, an evaporative light-scattering (ELSD) (Alltech
3
300ELSD) detector. Chromatography was performed on a Shimadzu SunFire C18 (5 m 50 * 4.6mm)
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 2.0 mL/min. Flow from the column was split to a 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 Labsolution data system. Proton Nuclear Magnetic
1
Resonance ( H-NMR) spectra were recorded on a Bruker Avance Ⅲ400 spectrometer. Chemical shifts
are expressed in parts per million (ppm) and reported as 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, m = multiple). Preparative HPLC was performed
on Agilent Prep 1260 series with UV detector set to 254 nm or 220 nm. Samples were injected onto a
Phenomenex Luna 75 x 30 mm, 5 μm, C18 column at room temperature. The flow rate was 40 mL/min.
A linear gradient was used with 10% (or 50%) of MeOH (A) in H O (with 0.1 % TFA) (B) to 100%
2
of MeOH (A). All compounds showed > 95% purity using the LCMS methods described above.
Purities of final compounds were confirmed by UPLC-MS.
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Synthesis of 12, 13, 23, and 24
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N-(3-(5-(4-(Aminomethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-
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difluorophenyl)propane-1-sulfonamide hydrochloride (8). To a solution of N-(3-(5-bromo-1H-
pyrrolo[2,3-b] pyridine-3-carbonyl)-2,4-difluorophenyl) propane-1-sulfonamide (2 g, 4.36 mmol) in
1,4-dixoane (100 mL) were added tert-butyl 4-(4,4,5,5-tetramethyl-1,3,2-dioxaborolan-2-
yl)benzylcarbamate (1.45 g, 4.36 mmol), K PO (2.32 g, 10.91 mmol) in water (30 mL), and [1,1'-
3
4
bis(diphenylphosphino)ferrocene] dichloropalladium(II) (200 mg, 10% wt). After the reaction mixture
was heated to 80 °C for 6 h, it was diluted with EtOAc (50 mL), washed with 5% lithium chloride
aqueous solution (3 x 20 mL), and brine (20 mL). The organic layer was dried over Na SO , filtered,
2
4
and concentrated in vacuo. The resulting residue was purified by silica gel column chromatography
petroleum ether: EtOAc = 1:4) to afford tert-butyl 4-(3-(2,6-difluoro-3-(propylsulfonamido)
benzoyl)-1H-pyrrolo [2,3-b]pyridin-5-yl)benzylcarbamate (1.3 g, yield: 51%) as an off-white solid.
(
+
1
MS (ESI) m/z: 585 [M+H] . HNMR (400 MHz, DMSO-d ) δ 12.98 (s, 1H), 9.76 (s, 1H), 8.70 (d, J =
6
2
.4 Hz, 1H), 8.61 (s, 1H), 8.28 (s, 1H), 7.71 (d, J = 8.0 Hz, 2H), 7.62 – 7.56 (m, 1H), 7.45 (t, J = 6.0
Hz, 1H), 7.39 (d, J = 8.0 Hz, 2H), 7.31 – 7.26 (m, 1H), 4.20 (d, J = 6.0 Hz, 2H), 3.14 – 3.11 (m, 2H),
1
.77 (dd, J = 8.0 Hz, 13.2, 2H), 1.42 (s, 9H), 0.97 (t, J = 7.2 Hz, 3H).
A mixture of tert-butyl 4-(3-(2,6-difluoro-3-(propylsulfonamido)benzoyl)-1H-pyrrolo[2,3-
b]pyridin- 5-yl)benzylcarbamate (1.25 g, 2.14 mmol) and HC1 acid in EtOAc (3 M, 10 mL) was stirred
at room temperature overnight. The reaction mixture was filtered to give the crude product, which was
washed with EtOAc and dried in vacuum to afford the title compound (1.06 g, yield: 95%) as an off-
+
1
white solid. MS (ESI) m/z: 485 [M+H] . HNMR (400 MHz, DMSO-d ) δ 13.10 (s, 1H), 9.80 (s, 1H),
6
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8.74 (d, J = 2.4 Hz, 1H), 8.67 (s, 1H), 8.54 (br, 3H), 8.23 (s, 1H), 7.83 (d, J = 8.4 Hz, 2H), 7.66 (d, J
8.0 Hz, 2H), 7.61 – 7.57 (m, 1H), 7.31 – 7.27 (m, 1H), 4.10 (q, J = 5.6 Hz, 2H), 3.16 – 3.20 (m, 2H),
=
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.78 – 1.72 (m, 2H), 0.97 (t, J = 7.2 Hz, 3H).
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5-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)pentanoic acid (11a).
A
mixture of 2-(2,6-dioxopiperidin-3-yl)-5-fluoroisoindoline-1,3-dione (6.6 g, 24.0 mmol), tert-butyl 5-
aminopentanoate (5.0 g, 26.4 mmol) and DIEA (9.3 g, 72 mmol) in NMP (60 mL) was stirred at 140 °C
in a microwave reactor for 30 min. After cooling down to room temperature, the mixture was poured
into EtOAc (200 mL), washed with water (2 x 200 mL) and brine (200 mL). The organic phase was
dried over anhydrous Na SO , filtered and concentrated. The resulting crude product was purified by
2
4
silica gel chromatography (petroleum ether : EtOAc = 1:1) to give tert-butyl 5-((2-(2,6-dioxopiperidin-
3-yl)-1,3-dioxoisoindolin-5-yl)amino)pentanoate (1.5 g, yield: 13%) as a yellow solid. MS (ESI) m/z:
+
3
74.1 [M+H] . A solution of tert-butyl 5-((2-(2,6-dioxopiperidin-3-yl) -1,3-dioxoisoindolin-5-
yl)amino)pentanoate (1.5 g, 3.5 mmol) in TFA (15 mL) was stirred at room temperature for 1 h. The
reaction mixture was concentrated and the residue was purified by silica gel chromatography (DCM :
MeOH = 20:1) to give the title compound (660 mg, yield: 51%) as a yellow solid. MS (ESI) m/z: 374.1
+
1
[M+H] . H NMR (400 MHz, DMSO-d ) δ 12.03 (br, 1H), 11.05 (s, 1H), 7.55 (d, J = 8.4 Hz, 1H),
6
7
.10 (t, J = 5.2 Hz, 1H), 6.94 (s, 1H), 6.83 (dd, J = 1.6 Hz, 8.4 Hz, 1H), 5.02 (dd, J = 5.6 Hz, 12.8 Hz,
1
H), 3.17 – 3.16 (m, 2H), 2.92 – 2.83 (m, 1H), 2.60 – 2.53 (m, 2H), 2.26 – 2.25 (m, 2H), 2.01 – 1.98
(m, 1H), 1.60 – 1.59 (m, 4H).
-((2-(1-Methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)pentanoic
5
acid
(11b). The title compound (41% yield over 2 steps) was synthesized according to procedures for the
preparation of 11a from 5-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione and tert-
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butyl 5-aminopentanoate followed by tert-butyl ester deprotection. MS (ESI) m/z: 388.5 [M+H]+.
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N-(4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyrrolo[2,3-b]pyridin-5-
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yl)benzyl)-5-((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino)pentanamide (12). A
solution
of
N-(3-(5-(4-(aminomethyl)phenyl)-1H-pyrrolo[2,3-b]pyridine-3-carbonyl)-2,4-
difluorophenyl)propane-1- sulfonamide hydrochloride (10 mg, 0.019 mmol), 5-((2-(2,6-
dioxopiperidin-3-yl)-1,3-dioxoisoindolin- 5-yl)amino)pentanoic acid (8.6 mg, 0.023 mmol), EDCI
(
5.94 mg, 0.031 mmol), HOAt (4.18 mg, 0.031 mmol) and NMM (12.53 mg, 0.12 mmol) in DMSO (3
mL) was stirred at room temperature overnight. The reaction solution was diluted with EtOAc (100
mL), washed with water (100 mL). The aqueous layer was extracted with EtOAc (50 mL). The
combined organic layer was washed with brine twice, dried over Na SO , filtered and concentrated.
2
4
The resulting residue was purified by Prep-TLC (DCM: MeOH = 10:1) to give the title compound (10
1
mg, 63% yield) as a white solid. H NMR (400 MHz, DMSO-d ) δ 8.71 (s, 1H), 8.60 (d, J = 2.0 Hz,
6
1H), 7.98 (s, 1H), 7.63 – 7.71 (m, 3H), 7.51 (d, J = 8.4 Hz, 1H), 7.43 (d, J = 8.4 Hz, 2H), 7.16 (t, J =
8
.4 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 6.81 – 6.84 (m, 1H), 4.98 – 5.03 (m, 1H), 4.44 (s, 2H), 3.23 (t,
J = 7.2 Hz, 2H), 3.11 (t, J = 8.0 Hz, 2H), 2.60 – 2.87 (m, 3H), 2.34 (t, J = 7.2 Hz, 2H), 2.01 – 2.08 (m,
1
3
1
H), 1.74 – 1.90 (m, 4H), 1.66 – 1.73 (m, 2H), 1.03 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSO-d )
6
δ 181.7, 174.4, 173.3, 170.4, 168.3, 167.9, 154.7, 148.5, 143.8, 138.3, 137.9, 137.3, 134.5, 132.3, 129.5,
128.1, 127.1, 124.8, 118.2, 116.6, 116.5, 115.4, 112.0, 111.7, 105.2, 53.8, 48.9, 42.4, 35.2, 35.1, 31.7,
3
0.8, 30.4, 29.4, 29.3, 29.2, 29.1, 28.9, 27.9, 26.7, 25.5, 23.0, 22.4, 16.9, 11.8. HRMS calcd for
+
C H F N O S [M+H ] 840.2622, found 840.2628.
4
2
40
2
7
8
N-(4-(3-(2,6-Difluoro-3-(propylsulfonamido)benzoyl)-1H-pyrrolo[2,3-b]pyridin-5-
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yl)benzyl)-5-((2-(1-methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-
yl)amino)pentanamide (13). The title compound (28% yield) was synthesized according to the
procedures for the preparation of 12 from N-(3-(5-(4-(aminomethyl)phenyl)-1H-pyrrolo[2,3-
b]pyridine-3-carbonyl)-2,4-difluorophenyl)propane-1-sulfonamide hydrochloride and 5-((2-(1-
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methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-5-yl)amino) pentanoic acid. H NMR (400 MHz,
DMSO-d ) δ 8.71 (s, 1H), 8.60 (s, 1H), 7.97 (s, 1H), 7.63 – 7.73 (m, 3H), 7.52 (d, J = 8.4 Hz, 1H),
6
7
.42 (d, J = 8.0 Hz, 2H), 7.16 (t, J = 8.4 Hz, 1H), 6.95 (d, J = 1.6 Hz, 1H), 6.83 (d, J = 9.2 Hz, 1H),
.01 – 5.05 (m, 1H), 4.44 (s, 2H), 3.24 (t, J = 6.8 Hz, 2H), 3.09 – 3.13 (m, 5H), 2.80 – 2.86 (m, 2H),
5
2.59 – 2.68 (m, 1H), 2.34 (t, J = 7.2 Hz, 2H), 2.00 – 2.05 (m, 1H), 1.76 – 1.90 (m, 4H), 1.68 – 1.73
1
3
(
m, 2H), 1.03 (t, J = 7.2 Hz, 3H). C NMR (100 MHz, DMSO-d ) δ 181.7, 174.4, 172.3, 170.1, 168.3,
6
167.9, 154.7, 148.3, 143.6, 138.4, 137.9, 137.3, 134.6, 129.5, 128.2, 128.1, 127.2, 124.8, 118.3, 116.6,
1
16.5, 115.4, 112.0, 111.8, 105.2, 105.1, 53.8, 49.5, 42.4, 35.2, 31.7, 31.1, 30.4, 29.4, 29.3, 29.2, 29.1,
+
2
8.9, 27.9, 26.7, 25.9, 23.0, 22.3, 21.7, 16.9, 13.0, 11.7. HRMS calcd for C H F N O S [M+H ]
4
3
42
2
7
8
854.2778, found 854.2778.
tert-Butyl
4-((1-(2,6-difluoro-3-(propylsulfonamido)phenyl)-1H-pyrrolo[3,2-b]pyridin-5-
yl)(methyl) amino)piperidine-1-carboxylate (16). A mixture of tert-butyl 4-(methyl-(1H-
pyrrolo[3,2-b]pyridin-5-yl) amino)piperidine-1-carboxylate (7.0 g, 21.21 mmol), N-(2,4-difluoro-3-
iodophenyl) propane-1-sulfonamide (11.5 g, 31.82 mmol), cuprous iodide (806 mg, 4.24 mmol), (R,R)-
N,N'-dimethyl-1,2-diaminocyclohexane (1.34 mL, 8.48 mmol), and cesium carbonate (20.8 g, 63.64
mmol) in dry toluene (30 mL) was stirred at 120 °C for 16 hours. After addition of cuprous iodide (806
mg, 4.24 mmol), (R,R)-N,N'-dimethyl-1,2-diaminocyclohexane (1.34 mL, 8.48 mmol), and cesium
carbonate (20.8 g, 63.64 mmol) the reaction mixture was stirred for further 24 h. Upon removal of
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solvent in vacuo, the residue was diluted with DCM and washed with sodium bicarbonate solution.
The organic layer was dried over anhydrous sodium sulfate and filtered. The solvent was removed in
vacuo and the residue was purified by silica gel column chromatography (petroleum ether: EtOAc =
0
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2:1) to afford the title compound (5.5 g, yield: 46%) as a yellow solid. MS (ESI) m/z: 564 [M+H]+.
1
HNMR (400 MHz, CDCl ) δ 7.64 – 7.58 (m, 1H), 7.26 – 7.25 (m, 2H), 7.16 – 7.11 (m, 1H), 6.70 (d,
3
J = 3.2 Hz, 1H), 6.53 (d, J = 9.2 Hz, 1H), 4.82 – 4.74 (m, 1H), 4.35 – 4.17 (m, 2H), 3.14 – 3.10 (m,
2
H), 2.96 – 2.83 (m, 5H), 1.96 – 1.86 (m, 2H), 1.75 – 1.66 (m, 4H), 1.49 (s, 9H), 1.07 (t, J = 7.6 Hz,
3
H).
tert-Butyl
4-((1-(2,6-difluoro-3-(propylsulfonamido)phenyl)-3-iodo-1H-pyrrolo[3,2-
b]pyridin-5-yl) (methyl)amino)piperidine-1-carboxylate (17). To a solution of tert-butyl 4-((1-(2,6-
difluoro-3-(propylsulfonamido)phenyl)-1H-pyrrolo[3,2-b]pyridin-5-yl)(methyl)amino)piperidine-1-
carboxylate (5.5 g, 9.75 mmol) in DMF (50 mL) and THF (1 mL) was added N-iodosuccinimide (2.4
g, 10.73 mmol). After the mixture was stirred at room temperature for 1 h, the reaction was diluted
with DCM (30 mL) and washed with aqueous saturated NaHCO . The organic layer was dried over
3
anhydrous Na SO , filtered and concentrated under reduced pressure. The resulting residue was
2
4
purified by silica gel column chromatography (petroleum ether: EtOAc = 2:1) to afford the title
+
1
compound (5.1 g, yield: 76%) as a yellow solid. MS (ESI) m/z: 690 [M+H] . HNMR (400 MHz,
CDCl ) δ 7.66 – 7.60 (m, 1H), 7.33 (s, 1H), 7.21 (d, J = 9.2 Hz, 1H), 7.14 (t, J = 8.8 Hz, 1H), 6.55 (d,
3
J = 8.8 Hz, 1H), 6.49 (s, 1H), 4.85 – 4.78 (m, 1H), 4.37 – 4.25 (m, 2H), 3.15 – 3.11 (m, 2H), 2.99 –
2
.86 (m, 5H), 1.96 – 1.86 (m, 2H), 1.80 – 1.65 (m, 4H), 1.49 (s, 9H), 1.07 (t, J = 7.6 Hz, 3H).
N-(2,4-Difluoro-3-(5-(methyl(piperidin-4-yl)amino)-3-(pyrimidin-5-yl)-1H-pyrrolo[3,2-
b]pyridin-1-yl)phenyl)propane-1-sulfonamide hydrochloride (19). To a solution of tert-butyl 4-
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((1-(2,6-difluoro-3-(propylsulfonamido)phenyl)-3-iodo-1H-pyrrolo[3,2-b]pyridin-5-
yl)(methyl)amino)piperidine-1-carboxylate (5.7 g, 8.26 mmol) in 1,4-dioxane and water (2:1 mixture,
mL), was added pyrimidin-5-yl-boronic acid (3.07g, 24.78 mmol), [1,1'-
bis(diphenylphosphino)ferrocene]dichloropalladium(II) (604 mg, 0.83 mmol), lithium chloride (1.7 g,
1.3 mmol) and sodium carbonate (2.6 g, 24.78 mmol). After the resulting mixture was stirred at
6
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100 °C for 1 h, it was diluted with DCM and washed with sodium bicarbonate solution. The organic
layer was dried over anhydrous sodium sulfate, filtered and concentrated. The residue was purified by
column chromatography (petroleum ether: EtOAc = 1:9) to afford tert-butyl 4-((1-(2,6-difluoro-3-
(propylsulfonamido)phenyl)-3-(pyrimidin-5-yl)-1H-pyrrolo[3,2-b]pyridin-5-
yl)(methyl)amino)piperidine-1-carboxylate (2.2 g, yield: 41%) as a yellow solid. MS (ESI) m/z: 642
+
1
[M+1] . HNMR (400 MHz, DMSO-d ) δ 9.93 (s, 1H), 9.66 (s, 2H), 9.03 (s, 1H), 8.42 (s, 1H), 7.64 –
6
7
3
.58 (m, 1H), 7.49 – 7.44 (m, 2H), 6.81 (d, J = 9.6 Hz, 1H), 4.59 – 4.53 (m, 1H), 4.16 – 4.13 (m, 2H),
.19 – 3.15 (m, 2H), 2.94 – 2.83 (m, 5H), 1.80 – 1.59 (m, 6H), 1.43 (s, 9H), 0.85 (t, J = 7.2 Hz, 3H).
To a solution of tert-butyl 4-((1-(2,6-difluoro-3-(propylsulfonamido)phenyl)-3-(pyrimidin-5-yl)-
H- pyrrolo[3,2-b]pyridin-5-yl)(methyl)amino)piperidine-1-carboxylate (2.2 g, 3.43 mmol) in
1
DCM/MeOH (5:1, 30 mL) was added HCl (3 M in EtOAc, 20 mL) and the mixture was stirred at room
temperature for 3 h. After evaporation, the residue was diluted with EtOAc. The precipitate was
collected by filtration and dried to give N-(2,4-difluoro-3-(5-(methyl(piperidin-4-yl)amino)-3-
(pyrimidin-5-yl)-1H-pyrrolo[3,2-b]pyridin-1-yl)phenyl) propane-1-sulfonamide hydrochloride (1.9 g,
+
1
yield: 96%) as a yellow solid. MS (ESI) m/z: 542 [M+1] . HNMR (400 MHz, DMSO-d ) δ 10.00 (s,
6
1H), 9.74 (s, 2H), 9.27 – 9.18 (m, 3H), 8.53 (s, 1H), 7.68 – 7.62 (m, 1H), 7.58 (d, J = 9.2 Hz, 1H), 7.51
–
7.46 (m, 1H), 6.94 (d, J = 9.2 Hz, 1H), 4.72 – 4.66 (m, 1H), 3.42 – 3.39 (m, 2H), 3.32 – 3.08 (m,
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Page 29 of 50
Journal of Medicinal Chemistry
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4H), 3.00 (s, 3H), 2.17 – 2.08 (m, 2H), 1.92 – 1.87 (m, 2H), 1.83 – 1.74 (m, 2H), 1.00 (t, J = 7.6 Hz,
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H).
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-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl)amino)ethoxy)ethoxy)
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ethoxy) propanoic acid (22a). The title compound (8% yield over 2 steps) was synthesized by
coupling of 2-(2,6-dioxopiperidin-3-yl)-4-fluoroisoindoline-1,3-dione and tert-butyl 3-(2-(2-(2-
aminoethoxy)ethoxy) ethoxy)propanoate followed by TFA mediated tert-butyl ester deprotection
+
1
according to the procedures for the preparation of compound 11a.MS (ESI) m/z: 478.1 [M+H] . H
NMR (400 MHz, DMSO-d ) δ 7.59 (d, J = 11.2 Hz, 1H), 7.23 (t, J = 6.8 Hz, 1H), 7.04 (d, J = 1.6 Hz,
6
1H), 7.04 (dd, J = 2.4 Hz, 11.2 Hz, 1H), 5.06 (dd, J = 7.2 Hz, 16.8 Hz, 1H), 3.64 – 3.57 (m, 8H), 3.54
–
3.48 (m, 4H), 3.40 – 3.38 (m, 2H), 2.92 – 2.89 (m, 1H), 2.64 – 2.54 (m, 2H), 2.42 – 2.38 (m, 2H),
2.05 – 2.01 (m, 1H).
-(2-(2-(2-((2-(1-Methyl-2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
3
yl)amino)ethoxy)ethoxy)ethoxy)propanoic acid (22b). The title compound (60% yield over 2 steps)
was synthesized by coupling of 4-fluoro-2-(1-methyl-2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione
and tert-butyl 3-(2-(2-(2-aminoethoxy) ethoxy)ethoxy)propanoate followed by tert-butyl ester
deprotection according to the procedures for the preparation of 11a. MS (ESI) m/z: 492.2 [M+H]+.
N-(3-(5-((1-(3-(2-(2-(2-((2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)ethoxy)ethoxy)ethoxy)propanoyl)piperidin-4-yl)(methyl)amino)-3-(pyrimidin-5-yl)-
1H-pyrrolo[3,2-b]pyridin-1-yl)-2,4-difluorophenyl)propane-1-sulfonamide (23). The title
compound (72% yield) was synthesized according to the procedures for the preparation of 12 from N-
(2,4-Difluoro-3-(5-(methyl(piperidin-4-yl)amino)- 3-(pyrimidin-5-yl)-1H-pyrrolo[3,2-b]pyridin-1-
yl)phenyl)propane-1-sulfonamide hydrochloride and 3-(2-(2-(2-((2-(2,6-dioxopiperidin-3-yl)-1,3-
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