Design, Synthesis, and Biological Evaluation of Lysine Demethylase 5 C Degraders

Article abstract of DOI:10.1002/cmdc.202000933

Lysine demethylase 5 C (KDM5C) controls epigenetic gene expression and is attracting great interest in the field of chemical epigenetics. KDM5C has emerged as a therapeutic target for anti-prostate cancer agents, and recently we identified triazole 1 as an inhibitor of KDM5C. Compound 1 exhibited highly potent KDM5C-inhibitory activity in in vitro enzyme assays, but did not show strong anticancer effects. Therefore, a different approach is needed for the development of anticancer agents targeting KDM5C. Here, we attempted to identify KDM5C degraders by focusing on a protein-knockdown strategy. Compound 3 b, which was designed based on compound 1, degraded KDM5C and inhibited the growth of prostate cancer PC-3 cells more strongly than compound 1. These findings suggest that KDM5C degraders are more effective as anticancer agents than compounds that only inhibit the catalytic activity of KDM5C.

Full text of DOI:10.1002/cmdc.202000933

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Very Important Paper
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Design, Synthesis, and Biological Evaluation of Lysine
Demethylase 5 C Degraders
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Tetsuya Iida,[a, b] Yukihiro Itoh,*[a, b] Yukari Takahashi,[b] Yasunobu Yamashita,[a]
Takashi Kurohara,[a] Yuka Miyake,[a] Makoto Oba,[b] and Takayoshi Suzuki*[a, b]
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Lysine demethylase 5 C (KDM5C) controls epigenetic gene
expression and is attracting great interest in the field of
chemical epigenetics. KDM5C has emerged as a therapeutic
target for anti-prostate cancer agents, and recently we identi-
fied triazole 1 as an inhibitor of KDM5C. Compound 1 exhibited
highly potent KDM5C-inhibitory activity in in vitro enzyme
assays, but did not show strong anticancer effects. Therefore, a
different approach is needed for the development of anticancer
agents targeting KDM5C. Here, we attempted to identify
KDM5C degraders by focusing on a protein-knockdown strat-
egy. Compound 3b, which was designed based on compound
1, degraded KDM5C and inhibited the growth of prostate
cancer PC-3 cells more strongly than compound 1. These
findings suggest that KDM5C degraders are more effective as
anticancer agents than compounds that only inhibit the
catalytic activity of KDM5C.
Introduction
number of KDM inhibitors have been reported to date.[3,5]
Several KDM1 inhibitors have already been clinically tested and
are expected to be available for use in drug markets in the near
future.[6] However, although many biological studies using
genetic deletion techniques, such as RNA interference (RNAi),
have suggested that KDM2-7 enzymes are promising therapeu-
tic targets[3,7] and several KDM2-7 inhibitors have exhibited
highly potent inhibitory activity in in vitro enzyme assays, these
inhibitors are currently lagging in drug development. This may
be because these conventional enzyme inhibitors only inacti-
vate the “catalytic function” of KDMs but do not disturb their
“scaffolding function” (Figure 1B). In contrast, RNAi decreases
protein expression levels and inhibits both these functions of
the target KDMs and can completely block the epigenetic
mechanism related to the targets (Figure 1C). In this context,
we decided to use a protein degradation strategy, which is a
method to decrease the levels of the target protein, to inhibit
both “catalytic function” and “scaffolding function” of the target
enzyme (Figure 1D).
In the protein knockdown strategy, chimeric compounds,
which are often called proteolysis-targeting chimeras (PROTACs)
or specific and nongenetic inhibitors of apoptosis protein-
dependent protein erasers (SNIPERs), have been used.[8] In
general, these are designed by linking a target protein ligand to
a ubiquitin ligase (E3) ligand and can thereby hijack the
ubiquitin-proteasome system, which is a key protein degrada-
tion process in cells. This induces the ternary complex and
ubiquitylation at lysine residues of the target protein by the
recruitment of ubiquitin molecules from ubiquitin conjugate
enzymes (E2). Target proteins conjugated to a polyubiquitin
chain are then degraded by proteasomes (Figure 1D). This
strategy should be applicable for targeting epigenetic proteins
such as KDMs, which have both “catalytic function” and
“scaffolding function,” because KDM degraders are expected to
inhibit both these functions by degradation of the KDMs. In this
study, we focused on KDM5C and attempted to identify KDM5C
degraders.
Epigenetic mechanisms are gene expression processes that
occur independent of changes in the primary DNA sequence
and play important roles in various biological functions.[1] In
epigenetic mechanisms, histone and DNA modifications have
been well studied and their alterations are associated with
several diseases, including cancer. Therefore, controlling epige-
netic changes is a reasonable strategy for treating these
diseases.[2]
Histone demethylases (KDMs) regulate epigenetic gene
expression by their “catalytic function,” where methyl groups
are oxidatively removed from histone lysine residues.[3] In
addition, KDMs control epigenetic gene expression by a
“scaffolding function” where they interact with transcription
factors to form protein complexes (Figure 1A).[4] The KDM family
proteins are divided into two classes according to a difference
in demethylation mechanisms: one class consists of FAD-
dependent enzymes (KDM1 A and KDM1B), which are also
called lysine-specific demethylases (LSDs), whereas the other
class consists of Fe(II)/α-ketoglutarate-dependent enzymes
(KDM2-7), which contain a jumonji C domain.[3] Both KDM
classes have been viewed as therapeutic targets for diseases
such as cancers and neurodegenerative disorders, and a
[a] T. Iida, Dr. Y. Itoh, Dr. Y. Yamashita, Dr. T. Kurohara, Dr. Y. Miyake,
Prof. T. Suzuki
The Institute of Scientific and Industrial Research
Osaka University
8-1 Mihogaoka, Ibaraki, Osaka 567-0047 (Japan)
E-mail: itohy@sanken.osaka-u.ac.jp
tkyssuzuki@sanken.osaka-u.ac.jp
[b] T. Iida, Dr. Y. Itoh, Y. Takahashi, Prof. M. Oba, Prof. T. Suzuki
Graduate School of Medical Science
Kyoto Prefectural University of Medicine
1-5 Shimogamohangi-cho, Sakyo-ku, Kyoto 606-0823 (Japan)
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202000933
This article belongs to the joint Special Collection with ChemBioChem,
“Chemical Epigenetics”.
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Figure 1. A) Catalytic function and scaffolding function of KDMs. B) KDM inhibition by conventional inhibitors. C) Genetic KDM deletion by RNAi. D) KDM
degradation by protein knockdown strategy.
KDM5 family members (KDM5A–D) demethylate tri- or di-
methylated lysine 4 at histone H3 (H3K4me3/2)[9] and interact
with chromatin remodeling NuRD complex or histone deacety-
lase (HDAC) complexes.[10] KDM5 enzymes are believed to be
associated with cancer cell growth, drug resistance of cancer
cells, and neurodegenerative disorders via epigenetic
mechanisms.[11] To date, several KDM5 inhibitors have been
reported. The representative inhibitors are CPI-455[11a] and
indicated that defects in KDM5 had anticancer effects.[11b,14]
Accordingly, we hypothesized that KDM5 degraders that inhibit
both “catalytic function” and “scaffolding function” of KDM5s
are more effective on cancer cells than conventional KDM5
inhibitors. In this paper, we report KDM5C degraders designed
based on a KDM5C inhibitor 1.
KDM5-C49.[12] We have also recently identified several KDM5 Results and Discussion
inhibitors, including compound 1 (Figure 2).[13] These com-
pounds exhibited strong inhibitory activity against KDM5 in
in vitro enzyme assays. However, they did not show potent
anticancer effects, even though experiments using RNAi
Design and synthesis of KDM5C degraders
We recently reported compound 1 as a KDM5C inhibitor
(Figure 2).[13b] This inhibitor is assumed to coordinate with the
catalytic Fe2+ ion of KDM5C at the 2-(1,2,3-triazol-4-yl) pyridine
moiety and most strongly inhibits KDM5C among the KDM5
family proteins. In this study, we designed KDM5C degraders
based on compound 1. To design the linker structures of
KDM5C degraders, we initially performed a docking study on
compound 1a using Moregro virtual docker 6.0 software. We
docked compound 1a into the catalytic pocket of KDM5C, as it
is thought to coordinate with the catalytic Fe2+ ion (Figure 3A).
The docking pose obtained as the lowest energy conformer
suggested that the terminal carbon of the hexyl chain in
compound 1a is located near the entrance of the catalytic site
(Figure 3B). In addition, at least eight lysine residues were
positioned at the side of the entrance of the catalytic site
(Figure 3C). Therefore, we assumed that the terminal carbon is
an appropriate position for linker introduction. Considering the
access to the solvent surface, the linker would need at least two
or three more carbon lengths for the E3 ligase approach
(Figure 3B). Taken together, we planned to synthesize com-
Figure 2. Structure of representative KDM5 inhibitors. Compound 1b is a
prodrug form of compound 1a.
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Figure 3. Docking pose of compound 1a (green, ball-and-stick model) in KDM5C and subsequent design of KDM5C degraders based on the docking result.
The X-ray structure of KDM5C (PDB ID: 5FWJ) was used for the docking study. A) Overview of compound 1a docked into KDM5C. B) The surface of the
catalytic site. C) View of the side at the entrance of catalytic site. The stick models indicate lysine residues. D) Design of KDM5C degraders.
pounds by inserting a 2–6 carbon linker at the terminal carbon
of the hexyl chain. We used a thalidomide structure as an E3
ligase recruiter, which is frequently used for protein
knockdown.[15] Thus, we designed compounds 2–4 (Figure 3D);
compounds 2a–4a were tested in in vitro assays and their
methyl ester prodrugs were assessed 2b–4b in cellular
assays.[13b,c,16]
The routes used for the synthesis of compounds 2–4 are
shown in Schemes 1 and 2. Scheme 1 presents the preparation
of intermediates 19–21. Bromide 5 was converted to amine 8
with a three-step sequence: SN2 reaction with methylamine, Boc
protection for purification, and removal of the Boc group under
Tosylation of compounds 12–14 gave compounds 15–17. The
coupling reaction of compounds 15–17 with compound 18
afforded intermediates 19–21.
Scheme 2 shows the synthesis of compounds 2–4. Sonoga-
shira coupling between compound 22 and (trimethylsilyl)
acetylene yielded compound 23. Removal of the trimethyl silyl
group of compound 23 provided alkyne 25. Cycloaddition
between alkynes 24[13b]/25 and intermediates 19–21 furnished
compounds 2b, 2c, 3b, 3c, 4b, and 4c. Finally, compounds
2a–4a were synthesized by deprotection of the t-butyl ester of
compounds 2c–4c.
acidic conditions. Amines
9 and 10 were derived from
compounds 6 and 7 by treatment with Boc2O, followed by
reduction in the presence of LiAlH4. Amines 8–10 were allowed
to react with mesylate 11[13b] to form compounds 12–14.
Scheme 1. Compounds 19–21. a) MeNH2, MeOH, RT, overnight; b) Boc2O, NaHCO3, H2O, THF, RT, overnight; c) trifluoroacetic acid, CH2Cl2, RT, 3 h; 36% for 8 (3
°
steps); d) LiAlH4, THF, reflux temperature, 3 h, 46% for 9 (2 steps) or 45% for 10 (2 steps); e) Et3N, THF, 80 C, overnight, 53% for 13 or 47% for 14; f) p-
°
toluenesulfonyl chloride, Et3N, CH2Cl2, RT, overnight, 5.4% for 15 (2 steps), 71% for 16, or 90% for 17; g) KHCO3, KI, DMF, 80 C, 4 h, 32% for 19, 73% for 20, or
14% for 21.
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Scheme 2. Compounds 2–4. a) (Trimethylsilyl)acetylene, Pd(PPh3)2Cl2, CuI, Et3N, MeCN, RT, overnight, 53%; b) KF, MeOH, RT, 3 h, 96%; c) CuSO4·5H2O, sodium
ascorbate, tBuOH, THF, H2O, RT, overnight, 67% for 2b, 57% for 2c, 19% for 3b, 28% for 3c, 64% for 4b or 59% for 4c; d) trifluoroacetic acid, CH2Cl2, RT, 4 h,
84% for 2a, 98% for 3a or 47% for 4a.
In vitro biological evaluation
inhibited KDM5C with IC50 values of 0.089–0.255 μM, which are
superior to CPI-455 (IC50 =3.01 μM),[13a] although their activities
were weaker than their parent compound 1a (IC50 =
0.0088 μM).[13b] Next, we tested compound 20, which is an
intermediate for the synthesis of compound 3a. The purpose of
this test was to examine whether the E3 ligase-recruiting moiety
influences KDM5C enzymatic activity. As a result, we found that
compound 20 did not show KDM5C-inhibitory activity (Table 1).
This suggests that KDM5C-inhibition by compounds 2a–4a is
dependent on the 2-(1,2,3-triazol-4-yl) pyridine moiety for Fe2+
chelation, but not the E3 ligase-recruiting moiety. These results
suggested the possibility of compounds 2–4 as KDM5C
degraders.
Subsequently, we evaluated the KDM5C-inhibitory activity of
compounds 2a–4a using the AlphaLISA screen assay system to
confirm whether they can bind to KDM5C.[13b] As shown in
Table 1, compounds 2a–4a almost completely inhibited
KDM5C at 10 μM and exhibited 70%–86% inhibition even at
1 μM. To determine their IC50 values, we tested compounds 2a–
4a at several concentrations (Figure 4). Compounds 2a–4a
Table 1. KDM5C-inhibitory activity of compound 2a–4a, 3b and 20.
Compound
% Inhibition a
at 10 μM
at 1 μM
2a
3a
4a
20
3b
95�1.4%
95�1.7%
99�1.3%
no inhibition
14�4.2%
70�1.4%
Cellular KDM5C degradation assay
78�2.8%
86�2.6%
no inhibition
no inhibition
We then assessed the cellular KDM5C degradation activity of
compounds 2b–4b, which are prodrugs of compounds 2a–
4a.[13b,c,16] We used prostate cancer cell line PC-3, where KDM5C
is expressed.[14a] PC-3 cells were treated with the test com-
pounds, and we detected KDM5C levels in cells after 24 h of
incubation via western blot analysis. Treatment with compound
3b decreased the levels of KDM5C in cells; this suggested that
it enabled KDM5C degradation in cells (Figure 5A). However,
compounds 2b and 4b did not induce substantial degradation
of KDM5C compared with that induced by compound 3b.
Based on previous reports that the linker structure is crucial for
protein degraders,[17] these results indicate that the linker length
was too short for compound 2b and too long for compound
4b to degrade KDM5C. Because compound 3b was the most
active of the three compounds, we selected compound 3 for
further experiments. Compound 3a, a carboxylate form, did not
decrease the levels of KDM5C (Figure 5B). Combined with the
fact that compound 3b did not show potent KDM5C-inhibitory
activity (Table 1), it is suggested that compound 3a is an active
form generated by the hydrolysis of compound 3b after being
[a] Values are the mean�SD of at least three experiments.
Figure 4. IC50 curves for the inhibition of KDM5C by compounds 2a, 3a, and
4a. Values are the mean � S.D. of at least three experiments.
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Figure 5. Western blot-detection of KDM5C levels in PC-3 cells after 24 h of
treatment with test compounds. A) 2b–4b. B) 3a. C) 3b, 1b, and 20. D) Co-
treatment with 3b and proteasome inhibitor MG-132 (10 μM).
Figure 6. Western blot-detection of A) H3K4me3 and B) H3K27Ac levels in
PC-3 cells after 24 h of treatment with compounds 3b, 1b, and 20. C)
Antiproliferative activity of 3b, 1b, and 20 in PC-3 cells after 72 h of
treatment. Values are the mean � SD of at least three experiments.
taken into the cells. Next, we confirmed whether compound 3b
induced a decrease in the levels of KDM5C through the protein
degradation mechanism. KDM5C levels did not decrease
following treatment with parent compounds 1a and 20, which
lack the E3 ligase-recruiting moiety and the KDM5C-inhibiting
moiety, respectively, compared with those following treatment
with compound 3b (Figure 5C). In addition, treatment of the
cells with proteasome inhibitor MG132 disturbed the decrease
in KDM5C levels by compound 3b (Figure 5D). These results
indicate that compound 3b reduces KDM5C levels by the
formation of a ternary complex of KDM5C/E3/3a and proteaso-
mal degradation.
would increase if compound 3b degrades KDM5C and disturbs
the interaction of KDM5C with HDAC1/2. Analysis of H3K27Ac
levels via western blotting showed that 20 μM of compound 3b
increased the amount of H3K27Ac, whereas compounds 1b and
20 did not (Figure 6B). We also confirmed that compound 3b
did not inhibit HDAC1/2 enzymatic activity at 100 μM (data not
shown). These results indicate that KDM5C degradation controls
H3K27Ac by inhibiting complex formation between KDM5C and
HDAC1/2. In other words, the induction of H3K27Ac suggests
that compound 3b inhibits the function of NuRD complex by
the degradation of KDM5C, although further detailed analyses
are needed.
Finally, the anticancer effects of compound 3b were tested.
Inhibition of cell growth was evaluated after treatment of PC-3
cells with compound 3b that suppressed the growth of PC-3
cells in a dose-dependent manner (Figure 6C). However, treat-
ment with compounds 1b and 20 did not significantly affect
cell growth at <150 μM compared with that following treat-
ment with compound 3b. These results suggest that KDM5C
degraders are more useful for prostate cancer treatment than
conventional enzyme inhibitors.
Effects of a KDM5C degrader on histone modifications and
cancer cell growth
Next, we examined the effects of compound 3b on histone
modifications and cancer cell growth. KDM5C catalyzes the
demethylation of H3K4me3/2, and KDM5C inhibitors have been
reported to accumulate H3K4me3 in cells.[13b] Therefore, we
expected that KDM5C degrader 3b would increase H3K4me3
levels, similar to KDM5C inhibitors, and we investigated
H3K4me3 accumulation in PC-3 cells treated with compound
3b. As shown in Figure 6A, compound 3b induced the Conclusion
accumulation of H3K4me3 more potently than its parent
compound 1b. In addition, compound 20 did not change the
amount of H3K4me3. These results are consistent with the
KDM5C degradation activities shown in Figure 5C. We also
investigated the effects of compound 3b on acetylated Lys27 at
histone H3 (H3K27Ac) because KDM5C is suggested to form a
complex with HDAC1/2 and cooperatively regulate deacetyla-
tion of H3K27Ac.[10,18] We expected that the amount of H3K27Ac
KDM5C modulates epigenetic mechanisms via the “catalytic
function”, which demethylates H3K4me3/2. In addition, KDM5C
can also regulate epigenetic gene expression depending on the
“scaffolding function” to form epigenetic protein complexes.
Therefore, conventional KDM5C inhibitors may be insufficient to
disrupt gene expression mediated by KDM5C. However, the
protein knockdown strategy induces inhibition of both the
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and dried in vacuum to produce tert-butyl (10-hydroxydecyl)(meth-
yl)carbamate (1.50 g, 95%) as a white solid; 1H NMR ([D6]DMSO,
400 MHz, δ; ppm) 6.73 (br s, 1H), 4.29 (t, J=5.2 Hz, 1H), 3.37 (q, J=
6.5 Hz, 2H), 2.88 (q, J=6.5 Hz, 2H), 1.43–1.17 (m, 16H), 1.37 (s, 9H).
catalytic activity and complex formation with other proteins
because this decreases the amount of the target protein in cells.
Therefore, this protein knockdown methodology should be
useful in inhibiting the functions of KDM5C in cells.
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Step 2: 10-(methylamino)decan-1-ol (9). A solution of tert-butyl (10-
hydroxydecyl)(methyl)carbamate (1.50 g, 5.49 mmol) in dry THF
(15 mL) was added dropwise to a suspension of LiAlH4 (1.04 g,
27.4 mmol) in dry THF (15 mL) at room temperature. After the
exothermic bubbling ceased, the mixture was heated at reflux for
In this study, we attempted to identify KDM5C degraders.
We designed and synthesized three chimeric compounds by
conjugating our reported KDM5C inhibitor with thalidomide as
an E3 recruiter. Compound 3b showed KDM5C-inhibitory
activity and induced KDM5C degradation in cells. Further
cellular studies showed that compound 3b increased levels of
both H3K4me3 and H3K27Ac, which are thought to be induced
by the direct inhibition of the “catalytic function” of KDM5C and
by indirect HDAC inhibition through the suppression of the
“scaffolding function” of KDM5C, respectively. Furthermore,
compound 3b inhibited the growth of PC-3 cells more strongly
than that of the conventional KDM5C inhibitor 1b. To the best
of our knowledge, compound 3b is the first KDM degrader, and
our data provide new insights into drug discovery studies on
KDM inhibitors as KDM family proteins generally have both
catalytic and scaffolding functions.
°
3 h under N2. After the reaction mixture had cooled to 0 C, water
(1.1 mL), followed by 4 N aqueous NaOH solution (1.1 mL), and
water (3.1 mL) were added. The resulting suspension was stirred for
30 min at room temperature. Subsequently, MgSO4 was added and
stirred for another 30 min. The mixture was filtered through a celite
pad, washed with THF, and concentrated to give 9 (500 mg, 49%)
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1
as a white solid; H NMR ([D6]DMSO, 400 MHz, δ; ppm) 4.31 (br s,
1H), 3.37 (t, J=6.5 Hz, 2H), 2.41 (t, J=6.9 Hz, 2H), 2.24 (s, 3H), 1.44–
1.18 (m, 16H); HRMS (ESI) calcd. for C11H26NO+: 188.2014, found:
188.2006.
Step 3: 10-[(2-Azidoethyl)(methyl)amino]decan-1-ol (13). Et3N
(0.740 mL, 5.34 mmol) was added to a solution of 9 (500 mg,
2.67 mmol) and 2-azidoethyl methanesulfonate (11)[13b] (440 mg,
°
2.67 mmol) in dry THF (5.0 mL). The mixture was stirred at 85 C
overnight under N2. The reaction was quenched with water, and
the reaction mixture was extracted with EtOAc. The organic layer
was washed with brine and dried over anhydrous MgSO4. After
filtration and concentration under reduced pressure, the residue
was purified by flash chromatography (n-hexane/EtOAc 1:1 to
Experimental Section
General information
1
EtOAc only) to produce 13 (361 mg, 53%) as an oil; H NMR ([D6]
Proton nuclear magnetic resonance spectra (1H NMR: 300 or
400 MHz) and carbon nuclear magnetic resonance spectra (13C
NMR: 175 MHz) were recorded on AVANCE300 AV (Bruker), ECS-400
(JEOL), and Avance III 700 (Bruker) spectrometers in the indicated
solvents, respectively. Chemical shifts (δ) are reported in parts per
million (ppm) relative to the internal standard tetramethylsilane.
Electrospray ionization (ESI) mass spectrometry measurements were
performed on an LTQ Orbitrap XL (THERMO) and AccuTOF-DART
(JEOL) mass spectrometers. Reagents and solvents were purchased
from Aldrich, Tokyo Kasei Kogyo, Kishida Kagaku, Kanto Kagaku,
Wako Pure Chemical Industries, or Nacalai Tesque, and used
without purification. Flash column chromatography was performed
using silica gel (particle size 200–440 mesh) supplied by Toyotakako
Silica Gel (#AP300D). HPLC was performed with a Shimadzu
instrument equipped with a COSMOSIL packed column (5 C18-AR-II,
4.6 ID ×150 mm, Nacalai) for analysis, and the eluents were water
containing 0.1% trifluoroacetic acid (TFA) (A) and MeCN containing
0.1% TFA (B). The conditions for analytical HPLC were as follows:
flow rate 1.0 mL/min, detection wavelength 254 nm, gradient A/B 0
to 20 min (90:10 to 10:90), 20 to 30 min (10:90), and 30 to 40 min
(10:90 to 90:10). The NMR spectra (1H and 13C) and HPLC charts of
test compounds are presented in Supporting Information.
DMSO, 400 MHz, δ; ppm) 4.29 (t, J=5.2 Hz, 1H), 3.36 (dd, J=11.8,
6.5 Hz, 2H), 3.32–3.28 (m, 2H), 2.53–2.49 (m, 2H), 2.32 (t, J=5.2 Hz,
2H), 2.17 (s, 3H), 1.44–1.35 (m, 4H), 1.30–1.22 (m, 12H).
Step
4:
10-[(2-Azidoethyl)(methyl)amino]decyl
4-meth-
ylbenzenesulfonate (16). p-Toluenesulfonyl chloride (322 mg,
1.69 mmol) was added at room temperature to a solution of 13
(361mg, 1.41mmol) in dry CH2Cl2 (3.6mL) and Et3N (0.240mL,
1.69mmol). The mixture was stirred at room temperature overnight
under N2. The reaction was quenched with water, and the reaction
mixture was extracted with EtOAc. The organic layer was washed
with brine and dried over anhydrous MgSO4. After filtration and
concentration under reduced pressure, the residue was purified by
flash chromatography (n-hexane/EtOAc 1:1 to EtOAc only) to
1
produce 16 (410 mg. 71%) as an oil; H NMR ([D6]DMSO, 300 MHz,
δ; ppm) 7.78 (d, J=8.3 Hz, 2H), 7.48 (d, J=8.1 Hz, 2H), 3.99 (t, J=
6.3 Hz, 2H), 3.31–3.28 (m, 2H), 2.51–2.48 (m, 2H), 2.42 (s, 3H), 2.31 (t,
J=7.1 Hz, 2H), 2.16 (s, 3H), 1.59–1.48 (m, 2H), 1.48–1.31 (m, 2H),
1.30–1.10 (m, 12H).
Step 5: 4-({10-[(2-Azidoethyl)(methyl)amino]decyl}oxy)-2-(2,6-dioxopi-
peridin-3-yl)isoindoline-1,3-dione (20). Potassium iodide (17.0 mg,
0.100mmol) followed by KHCO3 (150 mg, 1.50 mmol) was added to
a solution of 16 (410mg, 1.00mmol) and 2-(2,6-dioxopiperidin-3-yl)-
4-hydroxyisoindoline-1,3-dione (18) (274mg, 1.00mmol) in DMF
Preparation of 4-({10-[(2-azidoethyl)(methyl)amino]decyl}
oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (20)
°
(4.1mL). The mixture was stirred at 80 C for 4 h under N2. After
cooling to room temperature, the reaction was quenched with
water, and the reaction mixture was extracted with EtOAc. The
organic layer was washed with brine and dried over anhydrous
MgSO4. After filtration and concentration under reduced pressure,
the residue was purified by flash chromatography (CHCl3/EtOAc 1:1
to CHCl3/EtOAc/MeOH 9:1:0.1) to produce 20 (373 mg, 73%) as a
Step 1: tert-Butyl (10-hydroxydecyl)(methyl)carbamate. Boc2O
(2.52 g, 11.5 mmol) was added at room temperature to a solution
of 10-aminodecan-1-ol (6) (1.00 g, 5.77 mmol) in dry tetrahydrofur-
an (THF)/saturated aqueous NaHCO3 solution (5.0 mL/5.0 mL). The
mixture was stirred at room temperature overnight. The reaction
was quenched with water, and the reaction mixture was extracted
with EtOAc. The organic layer was washed with brine and dried
over anhydrous MgSO4. The residue obtained via filtration and
concentration under reduced pressure was washed with n-hexane/
EtOAc (20:1), and the insoluble material was collected via filtration
1
white solid; H NMR ([D6]DMSO, 400 MHz, δ; ppm) 11.09 (br s, 1H),
7.80 (dd, J=8.4, 7.3 Hz, 1H), 7.51 (d, J=8.3 Hz, 1H), 7.44 (d, J=
7.2 Hz, 1H), 5.07 (dd, J=12.7, 5.3 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H),
3.35–3.27 (m, 4H), 2.93–2.83 (m, 1H), 2.62–2.53 (m, 2H), 2.31 (t, J=
6.4 Hz, 2H), 2.16 (s, 3H), 2.05–1.99 (m, 1H), 1.78–1.72 (m, 2H), 1.48–
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ChemMedChem
1.23 (m, 14H); 13C NMR ([D6]DMSO, 175 MHz, δ; ppm) 172.72,
169.88, 166.76, 165.21, 155.89, 136.93, 133.12, 119.62, 116.05,
115.00, 68.63, 56.97, 56.21, 48.59, 47.65, 41.46, 30.84, 28.91, 28.85,
28.56, 28.30, 26.60, 26.55, 25.17, 21.88; HRMS (ESI) calcd. for
C26H37N6O5+: 513.2825, found: 513.2816; HPLC tR =13.94 min, purity
97.8%.
Step 2: tert-Butyl 2-ethynylisonicotinate (25). Potassium fluoride
(186 mg, 3.20 mmol) was added at room temperature to a solution
of 23 (689 mg, 2.50 mmol) in MeOH (6.8 mL). The mixture was
stirred at room temperature for 3 h under N2. The reaction was
quenched with water, and the reaction mixture was extracted with
EtOAc. The organic layer was washed with brine and dried over
anhydrous MgSO4. After filtration and concentration under reduced
pressure, the residue was purified by flash chromatography (n-
hexane/EtOAc 9:1) to afford 25 (488 mg, 96%) as a pale yellow
1
2
3
4
5
6
7
8
Preparation of 4-({8-[(2-azidoethyl)(methyl)amino]octyl}
oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (19)
1
solid; H NMR ([D6]DMSO, 400 MHz, δ; ppm) 8.76 (d, J=5.1 Hz, 1H),
7.85 (s, 1H), 7.79 (dd, J=5.1, 1.6 Hz, 1H), 4.49 (s, 1H), 1.56 (s, 9H);
9
Step 1: 8-(Methylamino)octan-1-ol (8). 2 M MeNH2 in MeOH solution
(50 mL, 100 mmol) was added to 8-bromooctan-1-ol 5 (5.00 g,
22.8 mmol) at room temperature. The mixture was stirred at room
temperature overnight under N2. The solvent was removed under
reduced pressure. To the resulting residue, 2 N aqueous NaOH
solution and EtOAc were added. The organic layer was washed with
brine and dried over anhydrous MgSO4. Filtration and concentration
under reduced pressure produced 8 as a crude product. This crude
product was directly used in the next reaction without further
purification.
HRMS (ESI) calcd. for C12H13NO2Na+: 226.0844, found: 226.0833.
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
Step 3: tert-Butyl 2-(1-{2-[(10-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoi-
soindolin-4-yl]oxy}decyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinate (3c). Sodium ascorbate (20.8 mg, 0.105 mmol) and
CuSO4 ·5H2O (26.2 mg, 0.105 mmol) was added at room temper-
ature to a solution of 25 (21.3 mg, 0.105 mmol) and 20 (54.8 mg,
0.105 mmol) in tert-BuOH/H2O/THF (1.0 mL/1.0 mL/0.10 mL). The
mixture was stirred at room temperature overnight under N2. The
reaction was quenched with water, and the reaction mixture was
extracted with EtOAc. The organic layer was washed with brine and
dried over anhydrous MgSO4. After filtration and concentration
under reduced pressure, the residue was purified by flash
chromatography (CHCl3/EtOAc 4:1 to CHCl3/EtOAc/MeOH 9:1:0.1)
4-({8-[(2-Azidoethyl)(methyl)amino]octyl}oxy)-2-(2,6-dioxopiperidin-3-
yl)isoindoline-1,3-dione (19): Compound 19 was synthesized from
crude compound 8 using a similar procedure to that described for
the preparation of compound 20 (steps 3–5); a white solid was
produced (yield, 59 mg, 0.62% from compound 5); 1H NMR ([D6]
DMSO, 400 MHz, δ; ppm) 11.11 (br s, 1H), 7.81 (t, J=7.8 Hz, 1H),
7.51 (d, J=8.7 Hz, 1H), 7.44 (d, J=7.3 Hz, 1H), 5.08 (dd, J=12.8,
5.5 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 3.43–3.28 (m, 4H), 2.93–2.84 (m,
1H), 2.62–2.47 (m, 2H), 2.32 (t, J=7.1 Hz, 2H), 2.16 (s, 3H), 2.04–1.99
(m, 1H), 1.79–1.72 (m, 2H), 1.50–1.24 (m, 10H).
1
to afford 3c (20.8 mg, 28%) as an oil; H NMR ([D6]DMSO, 400 MHz,
δ; ppm) 11.11 (br s, 1H), 8.77 (d, J=5.0 Hz, 1H), 8.64 (s, 1H), 8.37 (s,
1H), 7.80 (dd, J=8.2, 7.3 Hz, 1H), 7.71 (dd, J=5.0, 1.8 Hz, 1H), 7.50
(d, J=8.7 Hz, 1H), 7.44 (d, J=7.3 Hz, 1H), 5.08 (dd, J=12.8, 5.5 Hz,
1H), 4.52 (t, J=6.0 Hz, 2H), 4.17 (t, J=6.0 Hz, 2H), 2.93–2.84 (m, 1H),
2.77 (t, J=6.0 Hz, 2H), 2.62–2.53 (m, 2H), 2.28 (t, J=6.0 Hz, 2H), 2.21
(s, 3H), 2.06–2.00 (m, 1H), 1.73–1.66 (m, 2H), 1.57 (s, 9H), 1.39–1.30
(m, 2H), 1.29–0.99 (m, 12H); MS (ESI) m/z 716 (MH+).
Preparation of 4-({12-[(2-azidoethyl)(methyl)amino]dodecyl}
oxy)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione (21)
Step 4: 2-(1-{2-[(10-{[2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl]oxy}decyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)isonicotinic acid
dihydrochloride (3a·2HCl). Trifluoroacetic acid (22 μL, 0.290 mmol)
4-({12-[(2-Azidoethyl)(methyl)amino]dodecyl}oxy)-2-(2,6-dioxopiperi-
din-3-yl)isoindoline-1,3-dione (21): Compound 21 was synthesized
from compound 7 by using a similar procedure to that described
for the preparation of compound 20 (Steps 1–5); a white solid was
produced (yield, 522 mg, 2.7% from compound 7); 1H NMR ([D6]
DMSO, 400 MHz, δ; ppm) 11.09 (br s, 1H), 7.80 (dd, J=8.4, 7.3 Hz,
1H), 7.51 (d, J=8.6 Hz, 1H), 7.44 (d, J=7.2 Hz, 1H), 5.07 (dd, J=12.8,
5.4 Hz, 1H), 4.20 (t, J=6.4 Hz, 2H), 3.37–3.28 (m, 4H), 2.88 (ddd, J=
18.2, 12.9, 4.3 Hz, 1H), 2.67–2.54 (m, 2H), 2.31 (t, J=7.2 Hz, 2H), 2.16
(s, 3H), 2.06–1.98 (m, 1H), 1.78–1.71 (m, 2H), 1.49–1.21 (m, 18H).
°
was added at 0 C to a solution of 3c (20.8 mg, 0.0290 mmol) in dry
CH2Cl2 (22 μL). The reaction mixture was stirred at room temper-
ature for 4 h. The solvent was removed under reduced pressure,
and 4 N HCl in EtOAc was added to the residue. Thereafter, the
solvent was removed under reduced pressure, and azeotroped with
toluene several times to afford 3a·2HCl (20.8 mg, 98%) as a brown
solid; 1H NMR ([D6]DMSO, 400 MHz, δ; ppm) 11.09 (br s, 1H), 9.75 (br
s, 1H), 8.85 (s, 1H), 8.81 (t, J=2.4 Hz, 1H), 8.45 (s, 1H), 7.83–7.78 (m,
2H), 7.51 (d, J=8.3 Hz, 1H), 7.44 (d, J=7.2 Hz, 1H), 5.07 (dd, J=12.7,
5.3 Hz, 1H), 4.93 (t, J=6.2 Hz, 2H), 4.19 (t, J=6.5 Hz, 2H), 3.73–3.65
(m, 2H), 3.17–3.06 (m, 2H), 2.94–2.84 (m, 1H), 2.83 (d, J=4.6 Hz, 3H),
2.63–2.54 (m, 2H), 2.07–1.99 (m, 1H), 1.78–1.70 (m, 2H), 1.66–1.54
(m, 2H), 1.50–1.37 (m, 2H), 1.36–1.15 (m, 10H); 13C NMR ([D6]DMSO,
175 MHz, δ; ppm) 172.74, 169.90, 166.75, 165.82, 165.22, 155.87,
150.75, 150.58, 146.72, 139.17, 136.95, 133.11, 124.42, 121.87,
119.63, 118.14, 116.03, 115.03, 68.61, 55.05, 53.01, 48.57, 44.28,
30.83, 28.74, 28.63, 28.53, 28.36, 28.28, 25.80, 25.16, 22.82, 21.87;
HRMS (ESI) calcd. for C34H42N7O7+: 660.3146, found: 660.3137; HPLC
tR =13.11 min, purity 95.5%.
Preparation of
2-(1-{2-[(10-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoli-
n-4-yl]oxy}decyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinic acid dihydrochloride (3a·2HCl)
Step 1: tert-Butyl 2-[(trimethylsilyl)ethynyl]isonicotinate (23). CuI
(90.0 mg, 0.400 mmol) in Et3N (0.95 mL) and MeCN (9.50 mL) was
added (trimethylsilyl)acetylene (0.800 mL, 5.00 mmol) to a mixture
of tert-Butyl 2-bromoisonicotinate (22) (1.21 g, 4.69 mmol), bis
(triphenylphosphine)palladium(II) dichloride (165 mg, 0.200 mmol).
The resulting mixture was stirred at room temperature overnight
under N2. The reaction was quenched with water, and the reaction
mixture was extracted with EtOAc. The organic layer was washed
with brine and dried over anhydrous MgSO4. After filtration and
concentration under reduced pressure, the residue was purified by
flash chromatography (n-hexane/EtOAc 9:1) to generate 23
Preparation of
2-(1-{2-[(8-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4--
yl]oxy}octyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinic acid dihydrochloride·2HCl (2a·2HCl)
2-(1-{2-[(8-{[2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}
octyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)isonicotinic acid dihy-
drochloride (2a·2HCl): Compound 2a·2HCl was synthesized from
compounds 19 and 25 using a similar procedure to that described
for the preparation of compound 3a·2HCl (Steps 3–4); an
1
(689 mg, 53%) as a colorless oil; H NMR ([D6]DMSO, 400 MHz, δ;
ppm) 8.75 (dd, J=5.0, 0.8 Hz, 1H), 7.80 (t, J=1.3 Hz, 1H), 7.77 (dd,
J=4.9, 1.6 Hz, 1H), 1.56 (s, 9H), 0.27 (s, 9H).
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ChemMedChem
amorphous solid (yield, 15.0 mg, 48% from compound 25); 1H NMR
([D6]DMSO, 400 MHz, δ; ppm) 11.10 (br s, 1H), 10.03 (br s, 1H), 8.85
(s, 1H), 8.81 (dd, J=5.0, 0.8 Hz, 1H), 8.45 (s, 1H), 7.83–7.77 (m, 2H),
7.50 (d, J=8.6 Hz, 1H), 7.44 (d, J=7.2 Hz, 1H), 5.07 (dd, J=12.6,
5.4 Hz, 1H), 4.94 (t, J=6.4 Hz, 2H), 4.19 (t, J=6.4 Hz, 2H), 3.71–3.64
(m, 2H), 3.19–3.01 (m, 2H), 2.93–2.82 (m, 1H), 2.82 (d, J=4.4 Hz, 3H),
2.64–2.53 (m, 2H), 2.06–1.99 (m, 1H), 1.77–1.71 (m, 2H), 1.67–1.59
(m, 2H), 1.48–1.39 (m, 2H), 1.37–1.22 (m, 6H); 13C NMR ([D6]DMSO,
175 MHz, δ; ppm) 172.75, 169.91, 166.74, 165.83, 165.23, 155.86,
150.77, 150.59, 146.73, 139.17, 136,96, 133.11, 124.43, 121.87,
119.61, 118.13, 116.03, 115.04, 68.59, 55.11, 53.05, 48.58, 44.28,
Preparation of methyl
1
2
3
4
5
6
7
8
9
2-(1-{2-[(8-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4--
yl]oxy}octyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinate (2b)
Methyl 2-(1-{2-[(12-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl]oxy}dodecyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)isonicotinate
(2b): Compound 2b was synthesized from compounds 19 and
24[13b] using
a similar procedure to that described for the
preparation of compound 3a·2HCl (Step 3); an amorphous solid
1
(yield, 17.0 mg, 67%); H NMR (DMSO d6, 400 MHz, δ; ppm) 11.09
(br s, 1H), 8.80 (d, J=5.1 Hz, 1H), 8.66 (s, 1H), 8.43 (s, 1H), 7.80 (t, J=
7.9 Hz, 1H), 7.74 (d, J=4.2 Hz, 1H), 7.47 (d, J=8.8 Hz, 1H), 7.44 (d,
J=7.4 Hz, 1H), 5.07 (dd, J=12.7, 5.3 Hz, 1H), 4.55–4.50 (m, 2H), 4.12
(t, J=6.5 Hz, 2H), 3.91 (s, 3H), 2.94–2.83 (m, 1H), 2.81–2.76 (m, 2H),
2.68–2.52 (m, 2H), 2.34–2.26 (m, 2H), 2.21 (s, 3H), 2.06–1.99 (m, 1H),
1.67–1.59 (m, 2H), 1.36–1.06 (m, 10H); 13C NMR ([D6]DMSO, 175 MHz,
δ; ppm) 172.73, 169.89, 166.75, 165.19, 164.86, 155.84, 151.21,
150.81, 145.99, 137.66, 136.92, 133.11, 124.06, 121.23, 119.56,
117.69, 116.02, 114.99, 68.57, 56.69, 56.03, 52.77, 48.58, 47.50, 41.47,
30.83, 28.32, 28.29, 28.22, 25.73, 25.07, 22.81, 21.87; HRMS (ESI)
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39
40
41
42
43
44
45
46
47
48
49
50
51
52
53
54
55
56
57
+
calcd. for C32H38N7O7
: 632.2833, found: 632.2824; HPLC tR =
11.83 min, purity 96.5%.
Preparation of
2-(1-{2-[(12-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoli-
n-4-yl]oxy}dodecyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinic acid dihydrochloride·2HCl (4a·2HCl)
30.83, 30.60, 28.76, 28.60, 28.24, 26.43, 25.06, 21.87; HRMS (ESI)
+
2-(1-{2-[(12-{[2-(2,6-Dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-yl]oxy}
decanoyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)isonicotinic acid
dihydrochloride (4a·2HCl): Compound 4a·2HCl was synthesized
from compounds 21 and 25 using a similar procedure to that
described for the preparation of compound 3a·2HCl (Steps 3–4); as
a white solid (yield, 36.0 mg, 28% from compound 25); 1H NMR
([D6]DMSO, 400 MHz, δ; ppm) 11.09 (br s, 1H), 10.40 (br, s, 1H), 8.86
(s, 1H), 8.81 (d, J=4.6 Hz, 1H), 8.45 (s, 1H), 7.85–7.76 (m, 2H), 7.51
(d, J=8.6 Hz, 1H), 7.44 (d, J=7.2 Hz, 1H), 5.08 (dd, J=12.6, 5.2 Hz,
1H), 5.00–4.94 (m, 2H), 4.25–4.16 (m 2H), 3.87–3.74 (m, 2H), 3.12–
3.00 (m, 2H), 2.93–2.84 (m, 1H), 2.80 (d, J=4.2 Hz, 3H), 2.64–2.54 (m,
2H), 2.07–1.98 (m, 1H), 1.79–1.70 (m, 2H), 1.66–1.56 (m, 2H), 1.49–
1.38 (m, 2H), 1.36–1.14 (m, 14H); 13C NMR ([D6]DMSO, 175 MHz, δ;
ppm) 172.26, 169.40, 166.26, 165.34, 164.73, 155.39, 150.30, 150.12,
146.28, 138.64, 136.48, 132.60, 123.91, 121.37, 119.14, 117.63,
115.52, 114.54, 68.13, 54.57, 52.54, 48.08, 43.80, 30.34, 28.37, 28.33,
28.30, 28.20, 28.06, 27.89, 27.79, 25.30, 24.67, 22.32, 21.38; HRMS
(ESI) calcd. for C36H46N7O7+: 688.3459, found: 688.3450; HPLC tR =
14.35 min, purity 98.5%.
calcd. for C33H40N7O7
: 646.2989, found: 646.3302; HPLC tR =
13.09 min, purity 98.1%.
Preparation of methyl
2-(1-{2-[(12-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoli-
n-4-yl]oxy}dodecyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinate (4b)
Methyl 2-(1-{2-[(12-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl]oxy}dodecyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)isonicotinate
(4b): Compound 4b was synthesized from compounds 21 and
24[13b] using
a similar procedure to that described for the
preparation of compound 3a·2HCl (Step 3); a white amorphous
solid (yield, 16.7 mg, 64%); H NMR (DMSO d6, 400 MHz, δ; ppm)
1
11.09 (br s, 1H), 8.80 (d, J=5.1 Hz, 1H), 8.65 (s, 1H), 8.44 (d, J=
0.7 Hz, 1H), 7.80 (dd, J=8.4, 7.3 Hz, 1H), 7.76 (dd, J=5.1, 1.6 Hz, 1H),
7.51 (d, J=8.6 Hz, 1H), 7.44 (d, J=7.2 Hz, 1H), 5.07 (dd, J=12.8,
5.4 Hz, 1H), 4.52 (t, J=5.8 Hz, 2H), 4.19 (t, J=6.4 Hz, 2H), 3.93 (s,
3H), 2.93–2.83 (m, 1H), 2.78 (t, J=5.5 Hz, 2H), 2.62–2.52 (m, 2H),
2.28 (t, J=6.8 Hz, 2H), 2.21 (s, 3H), 2.04–1.99 (m, 1H), 1.77–1.70 (m,
2H), 1.45–1.37 (m, 2H), 1.30–0.99 (m, 16H); 13C NMR; [D6]DMSO,
175 MHz, δ; ppm) 172.73, 169.89, 166.76, 165.21, 164.89, 155.89,
151.26, 150.78, 145.98, 137.64, 136.94, 133.11, 124.08, 121.21,
119.61, 117.69, 116.03, 115.01, 68.61, 56.70, 56.01, 52.78, 48.58,
47.52, 41.49, 30.84, 30.61, 29.06, 28.86, 28.56, 28.29, 26.55, 26.49,
25.14, 21.88; HRMS (ESI) calcd. for C37H48N7O7+: 702.3615, found:
702.3602; HPLC tR =15.53 min, purity 96.5%.
Preparation of methyl
2-(1-{2-[(10-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindoli-
n-4-yl]oxy}decyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)
isonicotinate (3b)
Methyl 2-(1-{2-[(10-{[2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl]oxy}decyl)(methyl)amino]ethyl}-1H-1,2,3-triazol-4-yl)isonicotinate
(3b): Compound 3b was synthesized from compounds 20 and
24[13b] using
a similar procedure to that described for the
preparation of compound 3a·2HCl (Step 3); ; an oil (yield, 13.6 mg,
Molecular docking
1
19%); H NMR ([D6]DMSO, 400 MHz, δ; ppm) 11.09 (br s, 1H), 8.80
The docking study was performed using Molegro Virtual Docker 6.0
software. The structure of compound 1a bound to KDM5C (PDB
code: 5FWJ) was constructed using MolDock, which uses a heuristic
search algorithm that combines differential evolution with a cavity
prediction algorithm. The docking parameters were as follows: grid
resolution, 0.30; maximum iterations:, 1500; population size, 50;
energy threshold, 100.00; simplex evolution, 300 (max steps) and
1.00 (neighbor distance factor); search space, (X, Y, Z)=(23.88,
62.85, 3.61) with radius 15; and distance constraints, 2-(1,2,3-triazol-
4-yl) pyridine moiety of 1a as an Fe2+ chelator constraint center (X,
Y, Z)=(20.49, 63.73, À 3.43) for the N atom of the pyridine ring and
(X, Y, Z)=(21.68, 64.81, À 5.59) for the N3 atom of the triazole ring
with a hard constraint between minimum 0.0 and maximum 0.5.
(d, J=4.9 Hz, 1H), 8.65 (s, 1H), 8.44 (s, 1H), 7.80 (t, J=8.4 Hz, 1H),
7.76 (dd, J=5.0, 1.7 Hz, 1H), 7.50 (d, J=8.6 Hz, 1H), 7.44 (d, J=
7.4 Hz, 1H), 5.07 (dd, J=12.6, 5.4 Hz, 1H), 4.52 (t, J=6.0 Hz, 2H), 4.17
(t, J=6.5 Hz, 2H), 3.93 (s, 3H), 2.94–2.82 (m, 1H), 2.78 (t, J=5.8 Hz,
2H), 2.67–2.54 (m, 2H), 2.28 (t, J=6.9 Hz, 2H), 2.21 (s, 3H), 2.07–2.00
(m, 1H), 1.73–1.66 (m, 2H), 1.40–1.30 (m, 2H), 1.30–0.97 (m, 12H); 13
C
NMR ([D6]DMSO, 175 MHz, δ; ppm) 172.73, 169.89, 166.75, 165.20,
164.89, 155.87, 151.25, 150.79, 145.97, 137.65, 136.93, 133.11,
124.07, 121.21, 119.61, 117.69, 116.03, 115.00, 68.60, 56.70, 56.03,
52.78, 48.58, 47.52, 41.49, 30.83, 30.60, 28.96, 28.85, 28.57, 28.27,
+
26.54, 26.50, 25.12, 21.87; HRMS (ESI) calcd. for C35H44N7O7
:
674.3302, found: 674.3302; HPLC tR =14.30 min, purity 97.4%.
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cells were exposed to the test compounds by adding solutions (50
μL per well) of the compounds at various concentrations in a
Enzyme assay
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Activity assays of KDM5C were performed using the Alpha screen
assay system. Inhibitor solution (2.5 μL; 3.0% DMSO in assay buffer)
or assay buffer (50 mM HEPES pH 7.5, 0.1% w/v bovine serum
albumin, 0.01% v/v Tween-20 containing 3% DMSO as control or
blank) were placed in wells of a white opaque OptiPlateTM-384.
Thereafter, 5.0 μL of 1×10À 5 g/mL KDM5C solution (BPS Bioscience
Inc., #50112) was added to each well (for the blank, 5.0 μL of assay
buffer was added instead), and 2.5 μL of substrate peptide (1 μM)/
2-OG (50 μM)/FeII (5 μM)/Asc (100 μM) mixture in buffer was added
to each well. The plate was incubated for 2 h at room temperature
with gentle shaking (250 rpm). Next, 5.0 μL of 100 μg/mL acceptor
beads (PerkinElmer, #AL116 C) in epigenetic buffer (PerkinElmer,
#AL008) was added to each well and the plate was incubated for
1 h at room temperature with gentle shaking (250 rpm). Thereafter,
10 μL of 100 μg/mL donor beads in epigenetic buffer (PerkinElmer,
#676002) was added to each well, and incubation was continued
for 30 min at room temperature under shielding from light. The
alpha signal of the wells was measured with Ensight® readers
(PerkinElmer) with excitation at 615 nm and emission at 655 nm.
The % inhibition was calculated from the alpha signal readings of
inhibited wells relative to those of control wells. The concentration
°
medium at 37 C under 5% CO2 in air for 72 h. The mixtures were
treated with 10 μL of AlamarBlue® (AbD Serotec, #BUF012 A), and
°
incubation was continued at 37 C for 3 h. The fluorescence in each
well was measured with an ARVO™ X3 microplate reader (λex =
540 nm, λem =590 nm). Percentage cell growth was calculated from
fluorescence readings.
Acknowledgements
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The authors would like to thank the members of the Comprehen-
sive Analysis Center, ISIR, Osaka University, for HRMS analysis. This
work was supported by Scientific Research on Innovative Areas
“Ubiquitin new frontier driven by Chemo-technologies”
(19H05295) and for Scientific Research (C) (17K08367; Y.I.),
“Dynamic Alliance for Open Innovation Bridging Human, Environ-
ment and Materials” in “Network Joint Research Center for
Materials and Devices” (Y.I.), Public Promoting Association Asano
Foundation for Studies on Medicine (Y.I.), the JST CREST program
(JPMJCR14L2; T.S.), “Dynamic Alliance for Open Innovation
Bridging Human, Environment and Materials” from the Ministry of
Education, Culture, Sports, Science and Technology of Japan
(MEXT; T.S.), the Project for Cancer Research and Therapeutic
Evolution (T.S.) and the Naito Foundation (T.S.).
of
a compound that resulted in 50% inhibition (IC50) was
determined by plotting log[Inh] versus the logit function of %
inhibition. IC50 values were determined via regression analysis of
the concentration/inhibition data.
Western blotting
Human prostate cancer PC-3 cells (provided by the Japanese
Collection of Research Bioresources; #JCRB9110) were cultured in
Ham’s F-12 K (Kaighn’s) medium (Gibco, #21127022) containing
10% heat-inactivated fetal bovine serum (FBS; Sigma, #173012),
Conflict of Interest
°
The authors declare no conflict of interests.
penicillin and streptomycin mixture (Nacalai, #02892-54) at 37 C in
a humidified atmosphere of 5% CO2 in air. PC-3 cells (5×105 cells/
2 mL/dish) were treated for 24 h with test compounds in the
presence or absence of 10 μM of MG-132 (Calbiochem, #474790) at
the indicated concentrations in 10% FBS-supplemented cell culture
medium as indicated, and cells were collected and extracted with
SDS sampling buffer. Protein concentrations of the lysates were
determined using a BCA protein assay. Equivalent amounts of
protein from each lysate were resolved in 5%–20% SDS-polyacryla-
mide gels and bands were transferred onto PVDF membranes
(Millipore, #IPVH00010 for KDM5C and β-actin detection,
#ISEQ10100 for H3, H3K4me3, and H3K27Ac detection). After
blocking with TBSÀ T containing 5% skimmed milk, the transblotted
membranes were probed with primary antibodies: rabbit mono-
clonal KDM5C antibody (CST #5361, 1:1000 dilution); mouse
monoclonal β-actin antibody (Santa Cruz, #sc-47778, 1:2000
dilution); rabbit polyclonal H3K4me3 antibody (Abcam, #ab8580,
1:5000 dilution); rabbit polyclonal H3K27Ac antibody (Abcam,
#ab4729, 1:1000 dilution); or rabbit polyclonal histone H3 antibody
(Abcam, #ab1791, 1:200000 dilution). The probed membranes were
washed three times with TBSÀ T, incubated with HRP-linked
secondary antibody (ECL rabbit IgG, HRP-linked whole antibody (GE
Healthcare Life Science, #NA934, 1:2500 dilution) or ECL mouse IgG
HRP-linked whole antibody (GE Healthcare Life Science, #NA931,
1:2500 dilution) and again washed three times with TBSÀ T. The
immunoblots were visualized using enhanced chemiluminescence
with chemiluminescent HRP substrate (Millipore, #P90718).
Keywords: drug discovery · epigenetics · lysine methylation ·
medicinal chemistry · protein knockdown
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Manuscript received: November 30, 2020
Accepted manuscript online: January 20, 2021
Version of record online: ■■■, ■■■■
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T. Iida, Dr. Y. Itoh*, Y. Takahashi,
Dr. Y. Yamashita, Dr. T. Kurohara,
Dr. Y. Miyake, Prof. M. Oba, Prof. T.
Suzuki*
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Chimeric compound: Compound 3 is
a KDM5C degrader based on a KDM5C
inhibitor and a ubiquitin E3 ligase
recruiter. The prodrug form induces
proteasomal degradation of KDM5C in
prostate cancer PC-3 cells and inhibits
cell growth. KDM5C degraders are
expected to be small molecules that
modulate the epigenetic mechanism
via KDM5C and may be therapeutic
candidates for prostate cancer.
Design, Synthesis, and Biological
Evaluation of Lysine Demethylase
5 C Degraders
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