Development of Multifunctional Histone Deacetylase 6 Degraders with Potent Antimyeloma Activity

Article abstract of DOI:10.1021/acs.jmedchem.9b00516

Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from the side chain of acetylated lysine residues in cytoplasmic proteins such as α-tubulin and HSP90. HDAC6 is involved in multiple disease-relevant pathways. Based on the proteolysis targeting chimera strategy, we previously developed the first HDAC6 degrader by tethering a pan-HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new generation of multifunctional HDAC6 degraders by tethering selective HDAC6 inhibitor Nexturastat A with CRBN ligand that can synergize with HDAC6 degradation for the antiproliferation of multiple myeloma (MM). This new class of degraders exhibited improved potency and selectivity for the degradation of HDAC6. After the optimization of the linker length and linking positions, we discovered potent HDAC6 degraders with nanomolar DC₅₀ and promising antiproliferation activity in multiple myeloma (MM) cells.

Full text of DOI:10.1021/acs.jmedchem.9b00516

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Article
Development of Multi-Functional Histone Deacetylase
Degraders with Potent Anti-Myeloma Activity
6
Hao Wu, Ka Yang, Zhongrui Zhang, Eric Leisten, ZIYUAN LI, Haibo Xie,
Jin Liu, Kerry A. Smith, Zora Novakova, Cyril Barinka, and Weiping Tang
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.9b00516 • Publication Date (Web): 04 Jul 2019
Downloaded from http://pubs.acs.org on July 5, 2019
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Development of Multi-Functional Histone
Deacetylase 6 Degraders with Potent Anti-Myeloma
Activity
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‡
‡
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,
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Hao Wu , Ka Yang , Zhongrui Zhang , Eric D. Leisten , Ziyuan Li , Haibo Xie , Jin Liu ,
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1,2*
Kerry A. Smith, Zora Novakova, Cyril Barinka, and Weiping Tang
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School of Pharmacy, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
Department of Chemistry, University of Wisconsin-Madison, Madison, Wisconsin 53705, USA
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Institute of Biotechnology of the Czech Academy of Sciences, BIOCEV, Prumyslova 595, 252
0 Vestec, Czech Republic
5
Keywords: Histone Deacetylase, HDAC6, Targeted Protein Degradation, PROTAC, Multiple
Myeloma
Abstract: Histone deacetylase 6 (HDAC6) primarily catalyzes the removal of acetyl group from
the side chain of acetylated lysine residues in cytoplasmic proteins such as α-Tubulin and HSP90.
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HDAC6 is involved in multiple disease-relevant pathways. Based on the proteolysis targeting
chimera (PROTAC) strategy, we previously developed the first HDAC6 degrader by tethering a
pan HDAC inhibitor with cereblon (CRBN) E3 ubiquitin ligase ligand. We herein report our new
generation of multi-functional HDAC6 degraders by tethering selective HDAC6 inhibitor Next-A
with CRBN ligand that can synergize with HDAC6 degradation for the anti-proliferation of
multiple myeloma. This new class of degraders exhibited improved potency and selectivity for
the degradation of HDAC6. After the optimization of the linker length and linking positions, we
discovered potent HDAC6 degraders with nanomolar DC50 and promising anti-proliferation
activity in multiple myeloma (MM) cells.
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Introduction
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Post translational modification of proteins is essential in all cellular pathways . Among them,
lysine acetylation is regulated by histone acetyltransferases (HATs) and histone deacetylases
(HDACs). Most members of HDACs serve as epigenetic “erasers” to remove the acetyl groups of
lysine residues of histone tails and promote chromatin condensation and gene suppression within
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,3
the nucleus . HDAC inhibitors can reverse this modulation by inhibiting HDAC activity and re-
2
activate the transcription of important genes including tumor suppressors . There are 11 zinc-
+
dependent HDACs and 7 nicotinamide adenine dinucleotide (NAD ) dependent deacetylase
sirtuins (SIRTs) in the HDAC superfamily. Among them, HDAC6 is a unique member of class IIb
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,5
HDACs. The cellular location of HDAC6 is in the cytoplasm rather than nuclei. HDAC6 also
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has a number of non-histone substrates such as α-Tubulin , cortactin and HSP90 . HDAC6 is
responsible for regulating diverse cellular functions such as cell motility , immunoregulation9,10,
7,8
and aggresome formation11,12. Abnormal expression of HDAC6 has been observed in cancers such
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as oral squamous cell carcinoma, acute myeloid leukemia, ovarian cancer, and hepatocellular
carcinomas13–16. Selective inhibitors of HDAC6 have been developed and emerged as promising
therapeutic agent for cancer treatment13,17
.
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A
B
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Figure 1. PROTAC strategy and HDAC6 degraders. (A) Illustration of general PROTAC
induced protein ubiquitination and degradation. (B) Our previously reported HDAC6 degrader
1. (C) “Warhead” HDAC inhibitors 2 for our previous degraders and 3 for the new degraders.
Cellular knockdown and genetic silencing of certain functional proteins are important methods
in basic biological research and drug discovery. CRISPR and siRNA knockdown proteins by
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changing the content of DNA or degrading mRNA. Recently, proteolysis targeting chimeras
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(
PROTACs) emerged as a technique to induce efficient degradation of targeted protein (Figure
1A). PROTACs are rationally designed bi-functional small molecules composed of an E3 ubiquitin
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ligase ligand, a ligand for the protein of interest (POI), and a linker connecting them. The chemical
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chimera binds to either POI or E3 ligase to form binary complex first . A subsequent ternary
complex formation recruits E3 ligase to be adjacent to the POI and promotes its degradation
through the ubiquitination-proteasome system (UPS)18,20,21. To date, many disease-associated
proteins have been degraded by PROTACs using three E3 ligases including cereblon (CRBN),
Von Hippel-Lindau (VHL), and inhibitor of apoptosis proteins (IAPs)22–29. By targeting the
degradation of MDM230 or BET25,31,32, PROTACs exhibited excellent antitumor activity in
leukemia models. These pre-clinical data underlined the significance of PROTACs in drug
26
discovery. Furthermore, PROTACs are also useful chemical probes for target validation and
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modulation of cellular functions .
The degradation of Sirt2 by PROTACs was the first published example of degraders serving as
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epigenetic “erasers” . We previously reported degrader 1 as the first PROTAC for zinc-dependent
HDACs by conjugating HDAC inhibitors with pomalidomide-linked aldehydes34,35 (Figure 1B).
Since there was no report on the degradation of any of these Zn-dependent HDACs prior to our
studies, we used pan-HDAC inhibitor 236,37 (Figure 1C) as the PROTAC “warhead” to examine
which one of these HDACs could be degraded by PROTACs. Surprisingly, we found that
compound 1 selectively degraded HDAC6 among the HDACs we examined. However, there are
several limitations for these HDAC6 degraders. For example, the hydrazone linker is not
hydrolytically stable and therefore not ideal for further studies. Although compound 1 selectively
degrades HDAC6, other members of HDACs are still inhibited by the pan-inhibitor “warhead” as
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demonstrated by the elevated level of acetylated histones. Clearly, the selectivity and potency of
HDAC6 degraders need to be improved by using a completely different scaffold for further
biological and pharmacological studies. A number of selective HDAC6 inhibitors have been
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developed in the past decade38–44. Among them, potent inhibitor Nexturastat A45 (Next-A, 3,
Figure 1C) showed great selectivity for HDAC6 over all other HDACs. We therefore designed a
new generation of selective HDAC6 degraders by attaching E3 ligase ligand
pomalidomide18,23,30,31,34 to the solvent-exposing benzene ring of HDAC6 selective inhibitor Next-
A. During our investigation of the new generation HDAC6 selective degraders, Rao’s group
reported a class of PROTACs in 2019 by attaching a E3 ligase ligand to the alkyl chain of Next-
4
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A. However, their PROTACs did not show any improved anti-proliferation activity over the
parent HDAC6 inhibitor Next-A.
Despite the revolution in myeloma therapy in the last two decades, many patients are resistant
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to currently approved agents . HDAC6 selective inhibitors have been used in combination with
proteasome inhibitors48,49, immunomodulatory drugs (IMiDs, e.g. pomalidomide and its related
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analogues) and anti-PD-L1 antibody in anti-myeloma therapeutic treatment. HDAC6 selective
inhibitors showed synergy with IMiDs for the treatment of multiple myeloma in animal models
and human clinical trials, though the mechanism is still not clear. Upon binding to CRBN,
pomalidomide analogues are known to activate CRBN’s E3 ligase activity towards ikaros family
of zinc fingers (IKZFs) and promote their ubiquitination and subsequent degradation52–54. IKZFs
become the neo-substrates of ligand-bound CRBN. The induced degradation of IKZFs by
pomalidomide and its analogues are believed to be responsible for their significant anti-
proliferation effect in multiple myeloma. Interestingly, PROTACs with IKZF degradation activity
have also been reported in a number of cases when pomalidomide was employed as the ligand for
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CRBN E3 ligase26,55–57. The degradation of IKZFs is often considered as undesired during the
development of PROTACs. We hypothesize that multifunctional HDAC6 degraders that retain
the degradation activity of IKZFs would have enhanced anti-myeloma activity. We herein report
that our new generation of HDAC6 selective degraders have distinct advantages in degradation
efficiency and selectivity over our previous compounds. Our new HDAC6 degraders also have
significantly more potent anti-proliferation effects than the HDAC6 inhibitor Next-A in multiple
myeloma cancer cell lines.
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Results and Discussion
We synthesized 18 different degraders by tethering HDAC6-selective inhibitor Next-A (3) with
CRBN E3 ligand Pomalidomide. The general synthetic route is illustrated in Scheme 1.
Pomalidomide analogues 4a-e containing alkyne group were synthesized by a S
N
Ar reaction
between racemic fluoro-thalidomide (3a and 3b) and aminoalkynes according to literature
2
5
procedures . Alkylation of N-Boc protected 4-aminophenol (5) with different α,ώ-
dibromoalkanes provided intermediates 6a-e. Subsequent nucleophilic substitution reaction with
sodium azide generated compounds 7a-e which were deprotected under boiling water or acidic
5
8
conditions to give the amines 8a-e . These amines reacted with secondary amine 9 in the presence
4
5
of carbonyldiimidazole (CDI) to generate urea esters 10a-e . The hydroxamic acid group was
introduced to 10a-e by utilizing hydroxylamine under basic conditions to afford hydroxamic acids
1
1a-e. Finally, PROTACs 12a-r with varying linker lengths were obtained by click reactions
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between azides 11a-e and alkynes 4a-e under typical conditions .
a
Scheme 1. Synthesis of Compounds 12a-r
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a
Reaction conditions: (a) DIPEA, DMF, 90 °C, 17-62%; (b) K
2
CO , MeCN, reflux, 45-70%;
3
o
o
(
c) NaN
3
o
, DMF, 50 C, 66-90%; (d) H
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O, Ar reflux or TFA, DCM, 0 C-rt, 94-97%; (e) CDI,
o
THF, 0 C-rt, 13-88%; (f) NaOH, NH
2
OH/H
2
O, THF, MeOH, 0 C-rt, 87-92%; (g) CuSO
4
,
sodium ascorbate, TBTA, H O/t-BuOH (1:1.5), rt, 14-80%. For compounds 3a-b and 4a-e, C4
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means 4-substitution and C5 means 5-substitution.
These degraders are divided into to two series based on their linking position of the amino group
on the phthalimide ring of Pomalidomide: C4- or C5-linked series (Table 1). Within each series,
the degraders are different from each other by the numbers of carbon atoms between Next-A and
the triazole ring (n) or between Pomalidomide and the triazole ring (m). It is known that both C-4
and C-5 positions of Pomalidomide are exposed to solvent and can be the position to place the
linker for PROTACs 53,60. The para position of the aniline in Next-A was chosen to place the linker
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because it is well known that the aromatic ring “cap” of HDAC inhibitors is exposed to the
solvent61,62
.
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ns
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C o m p o u n d s
Figure 2. Screening of compounds for HDAC6 degradation activity by in-cell ELISA. MM1S
cells were treated with compounds at 100 nM or 10 nM for 6 h. Data was normalized to vehicle
(DMSO) treated group and bar graph represented as mean of relative HDAC6 protein level (n =
3
) with ± SD as error bar. Statistical significance was analyzed by one-way ANOVA. Not
significant (ns) P > 0.05, **P ≤ 0.01, ****P ≤ 0.0001.
Table 1. Screening of compounds for HDAC6 degradation activity
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Degradation (%)a
Degradation (%)a
Cpd
n
m
Cpd
n
m
1
00 nM
10 nM
100 nM
10 nM
Next-A
Poma
1
N/A
21.2 ± 3.5 -0.4 ± 3.9
22.1 ± 11.2 -2.0 ± 4.8
73.5 ± 0.3 47.9 ± 0.9
81.3 ± 2.3 62.1 ± 1.0
84.1 ± 1.0 67.3 ± 0.8
85.0 ± 1.2 64.3 ± 3.2
82.7 ± 1.7 74.9 ± 1.2
77.8 ± 1.7 66.1 ± 1.1
81.8 ± 0.9 49.3 ± 0.6
84.2 ± 0.4 64.8 ± 1.0
79.2 ± 0.9 71.7 ± 0.5
83.1 ± 1.0 70.6 ± 1.1
77.3 ± 1.5 45.6 ± 1.6
75.7 ± 0.8 50.9 ± 2.2
-
-
N/A
N/A
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12l
12m
12n
12o
12p
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81.4 ± 0.6 57.1 ± 1.4
81.2 ± 0.6 60.3 ± 1.3
80.9 ± 0.4 63.3 ± 0.8
84.7 ± 1.0 69.9 ± 1.3
83.5 ± 0.5 70.8 ± 0.4
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80.4 ± 0.3 47.2 ± 4.8
81.9 ± 0.4 60.9 ± 0.4
1
2j
1
2k
a
Degradation percentage calculated from Figure 2. Degradation percentage represents [100%
mean (± SD) of HDAC6 relative expression of biological replicates (n = 3) ].
-
To screen the new series of degraders, we first developed and validated a high throughput in-
cell ELISA assay (Supplementary Figure S1). We treated the compounds at 100 nM and 10 nM
in MM1S cells for 6 h (Figure 2A). Resulting cells were then fixed and analyzed by in-cell ELISA.
Comparing with vehicle, Next-A and Pomalidomide didn’t affect the expression of HDAC6 at 10
nM and had very minimal effects at 100 nM. At both concentrations, all degraders decreased
significant amount of HDAC6 protein. The degradation level was calculated and listed in Table
1
. Degraders including 1 at 100 nM degraded 73.5% to 85.0% of HDAC6 with minor difference.
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At 10 nM concentration of compounds, obvious structure-activity-relationship (SAR) was
observed. While only 47.9% of HDAC6 expression was suppressed by our previously reported
degrader 1, several new degraders are much more potent. For example, 12d, 12h, 12i, 12o and 12p
all achieved about 70% degradation. Among the C4-linked series, 12d with medium length linker
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(
5 + 1) achieved most degradation in sub-series 12a-e (m = 1). For sub-series 12f-h (m = 2), the
degradation potency increased when linkers were elongated and 12h (4 + 2) was the best. Within
sub-series 12i-k (m = 3), 12i with the shortest linker (2 + 3) turned out to be the most potent
degrader. Among C5-linked series, the potency of sub-series 12l-p (m = 1) increased with the
linker length. Compounds 12o (5 + 1) and 12p (6 + 1) have the similar potency for the degradation
of HDAC6. Sub-series 12q-r (m = 3) showed relative low effects for the degradation of HDAC6.
The above results suggest the optimal total number of methylene units in the linker is about 6 and
the C4-linked series are slightly more potent than the C5-series, which might relate to the
accessibility of the degrader-recruited E3 ligase to the available ubiquitination site(s) of HDAC6.
It appeared that both the distance and linking positions contributed to the degradation efficiency.
We prepared and tested the C4-linked series of compounds first. After learning the optimal length
of the linker, only selected C5-linked series of compounds were prepared and tested for SAR
studies.
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Figure 3. Western blot analysis of MM1S cells treated with selected candidates from C4-lined
series (A) and C5-lined series (B) for 6 h.
After the identification of the most potent candidates from each sub-series, we incubated selected
compounds at 100 nM, 10 nM and 1 nM for western blot analysis (Figure 3) to confirm the ELISA
results. Class II HDAC4 was selected for comparison. For selected compounds from C4- or C5-
linked series, all of them presented maximal effects at 100 nM and degraded significant amount of
HDAC6 at 10 nM. Clearly, our previous HDAC6 degrader 1 degraded much less HDAC6 at 10
nM concentration. The results from Western blot are consistent with the screening results by
ELISA.
As discussed before, pomalidomide and its analogues are known to activate CRBN’s E3 ligase
activity for the degradation of IKZFs. Interestingly, among the candidates we examined, only 12d
retained the induced degradation of IKZF1/3 by pomalidomide moiety at 100 nM. IKZFs regulate
the expression of interferon regulatory factor 4 (IRF4) and c-Myc to affect the proliferation of
5
2
multiple myeloma (MM) . The downregulation of IKZF 1 and 3 by pomalidomide and its
analogues are believed to be responsible for their significant anti-proliferation effect in multiple
myeloma. PROTACs with IKZF degradation activity have been reported in a number of cases26,55–
5
7
and the IKZFs are often considered as “off-targets” during the development of these PROTACs.
Our results indicate that the IKZF degradation activity can be impacted by the linker position and
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the functional group next to the phthalimide ring. In our case, the triazole ring linked to C4 position
likely contributed to the induced interaction between CRBN and IKZFs.
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1000 10000
C o m p o u n d C o n c e n tra tio n (n M )
Figure 4. Dose response of selected degrader candidates. MM1S cells treated in 6 h and
analyzed by in-cell ELISA. Data was normalized to vehicle (DMSO) treated group and dot plot
represented as mean relative expression (n = 3) with ± SD as error bar. Nonlinear fitting of
2
[
Iinhibitor] vs. response (three parameters) was generated by GraphPad Prism with R from 0.97
to 0.99.
a
b
Table 2. DC50 and Dmax of Selected Degraders
Cpd
DC50 (nM)
9.12 ± 1.64
3.41 ± 0.52
1.64 ± 0.24
2.54 ± 0.32
D
max (Vehicle%)
84.07 ± 2.41
88.01 ± 2.23
86.26 ± 1.70
86.30 ± 1.67
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2d
1
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a
The concentration at which half-maximal
b
degradation was achieved. The maximum
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percentage of degradation. a,bValues with ± SD
obtained from nonlinear fitted data in Figure 4.
To examine the potency of the selected candidates, we used in-cell ELISA to analyze the
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HDAC6 content in MM1S cells treated with 12a, 12d and 12i and compared them with degrader
1
. We measured the amount of cellular HDAC6 in response to concentrations of compounds from
0
.1 nM to 1 µM (Figure 4). DC50 and Dmax were calculated and listed in Table 2. Targeted protein
degradation is often attenuated at higher PROTAC concentrations, due to competitive formation
of POI-PROTAC or E3 ligase-PROTAC binary complexes instead of the desired POI-PROTAC-
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E3 ligase ternary complex, which is termed as “hook effect”. All degraders achieved 84-88%
maximal degradation and no “hook effect” was observed at concentrations ≤ 1 µM. Our new
degraders had single-digit nanomolar DC50, which was about 3 to 5 fold improvement from DC50
of 9.1 nM for degrader 1. Among these degraders, 12d showed excellent potency with a DC50 at
around 1.6 nM. As discussed above, most PROTACs were designed to avoid degrading other
proteins except the targeted POIs. However, in our study, we hypothesized that HDAC6 degraders
with IKZF degradation will have enhanced anti-proliferation effect in MM1S cells. Since
compound 12d showed promising IKZF degradation activity (Figure 3A), we further
characterized its activity and mechanism of action.
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Figure 5. Degrader 12d selectively promote HDAC6 degradation in MM1S cells. (A) Dose
response of 12d at concentration from 0.3 nM to 10 µM. (B) Time-course change of proteins
expression under treatment of 12d at 100 nM. See Supplementary Figure S2A and S2B for full
blot of (A) and (B) with other loading controls. (C) Comparison of HDAC6 degraders and
HDAC inhibitors.
To evaluate the activity and selectivity of 12d, we first performed a full dose response
experiment with compound concentrations ranging from 0.3 nM to 10 µM in MM1S cells (Figure
5
A) for several representative HDACs, IKZFs, and Ac-Tubulin. The result indicated that only the
expression of HDAC6 was affected among the HDACs we examined. Degrader 12d reduced
HDAC6 protein content at as low as 3 nM and achieved maximal effects around 30 nM. The “Hook
63
effect” was observed at 3 µM or above due to formation of binary complexes . The degradation
of IKZFs started from 30 nM and was dose-dependent. It is interesting to see the more efficient
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degradation of HDAC6, which requires the binding of both ligands to their protein targets, than
that of IKZFs, which only requires the binding of Pomalidomide motif to CRBN. In addition to
the distinct mechanism, the different turnover rates of HDAC6 and IKZFs may also contribute to
the observed results. The acetylated Tubulin level was also dose-dependent. At higher
concentrations (3 µM and 10 µM), Tubulin acetylation was not decreased in response to recovered
HDAC6 expression by “hook effect”. The elevated Tubulin acetylation at higher concentrations
of 12d was likely due to more significant HDAC6 inhibition because the degrader contained a
potent HDAC6 inhibitor motif.
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To probe the efficiency of 12d, we treated MM1S with 100 nM 12d and monitored the change
of HDAC6 protein level by time (Figure 5B). The HDAC6 degradation started around 1 h and
reached maximal degradation effect at 4 h. The degradation of IKZFs was only observed after 4 h.
Meanwhile, HDAC1 was not affected and the acetylation of Tubulin was accumulated by time. To
study the re-synthesis rate of HDAC6 after degradation, we performed “wash-out” experiment
(
Supplementary Figure S2D). Cells were treated with 100 nM 12d for 6 h and then washed with
PBS to remove remaining degraders. HDAC6 expression was monitored for 48 h. HDAC6 was
not fully recovered within 48 h, suggesting the slow turnover rate of HDAC6. Interestingly, IKZF3
was quickly recovered after 12 h, indicating the fast re-synthesis rate of IKZFs, which might be
one of the reasons for the requirement of higher concentrations of compounds for the degradation
of IKZF. We also tested selected degraders in other cell lines (Supplementary Figure S3) and
they are effective among all tested cancer cell lines.
To further examine the selectivity of our new generation of HDAC6 degraders, we compared
compound 12d with SAHA, Next-A, degrader 1 and pan-inhibitor 2 for the change of acetylated
α-Tubulin and acetylated histone H3 (Figure 5C). Compound 12d increased the acetylated
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Tubulin at 100 nM while SAHA and Next-A didn’t, indicating that the elevated acetylated Tubulin
was primarily due to HDAC6 degradation rather than the inhibition by “warhead” Next-A. We
didn’t observe any increase of acetylated histone H3 by the treatment 12d, which is in sharp
contrast to the strong acetylated histone H3 signal induced by SAHA, suggesting high selectivity
of the degrader. Comparing to our previously developed degrader 1, compound 12d also showed
significantly improved selectivity for increasing the level of acetylated Tubulin over acetylated
histone H3, indicating the advantage of replacing a pan-HDAC inhibitor by HDAC6 selective
inhibitor as the ligand of HDAC6 for PROTACs. We also compared the activity of 12d and Next-
A in biochemical assays (Supplementary Table S3). Indeed, 12d has an IC50 of 8.7±1.9 against
HDAC6, which is similar to Next-A (IC50 = 3.8±2.1). Their IC50s against other HDACs are in the
range of 300 to >30,000 nM.
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Figure 6. Mechanistic studies of compound 12d. Western blot analysis of MM1S cells with pre-
treatment of E3 ligase ligand Pomalidomide, HDAC6 inhibitor Next-A or NAE inhibitor MLN
4924 (A) or pre-treatment of proteasome inhibitor MG132 or Bortezomib (B) or vehicle
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(
DMSO) for 1 h and it is followed by the treatment of 100 nM 2d or vehicle (DMSO) for 6 h.
See Supplementary Figure S2C for full blot of (B) with IKZFs and Tubulin.
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To support the involvement of ubiquitination-proteasome system for the decrease of HDAC6
protein level, we co-treated the degrader with binding competitors or pathway blockers (Figure
6
). The co-treatment of degrader with Pomalidomide or Next-A recovered HDAC6 level while the
degradation of IKZFs was preserved (Figure 6A), indicating that the binding of the degrader to
both HDAC6 and CRBN E3 ligase are required for induced protein degradation. Moreover, it also
confirmed the role of Pomalidomide moiety in modulating IKZFs. Neddylation of cullin RING
ligase (CRL) by NEDD8-Activating Enzyme (NAE) regulated CRL’s activity as E3 ligase32,64
.
Inhibiting neddylation by NAE inhibitor MLN4924 resulted in abolishing the degradation of both
HDAC6 and IKZFs (Figure 6A). We also used proteasome inhibitor MG132 and Bortezomib to
block the down-stream proteasome degradation (Figure 6B). Under the co-treatment of both
inhibitors, no degradation of HDAC6 or of other HDACs was observed. Acetylated Tubulin level
was suppressed as well. This confirms that HDAC6 degradation is responsible for the increased
acetylation of its substrates rather than inhibition by the “warhead” moiety. To exclude the possible
transcriptional impact by degraders, we used qRT-PCR to examine the mRNA level of HDAC6
and related genes (Supplementary S4). We observed little effects on HDACs and IKZFs after 6
h treatment of degrader 12d. It is consistent with the hypothesis that the cellular knockdown of
HDAC6 is due to direct protein degradation, not by transcriptional downregulation.
a
Scheme 2. Synthetic Route to Deactivated Degraders 13 and 15
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aReaction conditions: (a) CuSO
Cs CO , CH I, DMF, 44%; (c) CuSO
, sodium ascorbate, TBTA, H
O/t-BuOH (1:1.5), rt, 93%; (b)
O/t-BuOH (1:1.5), rt, 70%.
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, sodium ascorbate, TBTA, H
2
Our HDAC6 degrader 12d thus has three functions: inhibition of HDAC6 by the Next-A motif,
degradation of IKZFs by the pomalidomide moiety, and the degradation of HDAC6 through the
formation of the ternary complex. The former two functions can be achieved by the combination
of HDAC6 selective inhibitor and pomalidomide. The third function is unique for HDAC6
degraders. Since HDAC6 selective inhibitors showed synergy with pomalidomide and its related
analogues for the treatment of multiple myeloma in animal models and human clinical trials50,65,66
,
we envision that HDAC6 degrader 12d would have enhanced anti-myeloma activity over the
combination of HDAC6 selective inhibitor and pomalidomide.
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To rule out the potential cell-permeability issue, we synthesized compounds 13 and 15, which
have similar molecular weight and size comparing to 12d, for better comparison. We blocked the
binding of 13 to HDAC6 by replacing the hydroxamic acid with a methyl ester. We also blocked
the binding of 15 to CRBN by attaching a methyl group to the key imide NH in 15. Compounds
1
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1
3 and 15 were synthesized according to the routes shown in Scheme 2. Compound 13 was
synthesized using the procedure described for the synthesis of compounds 12a-r with ester 10d
and alkyne 4a as the starting materials. Replacing the hydroxamic acid group on Next-A by a
methyl ester group was expected to prevent the binding of the resulting product to zinc co-factor
of HDAC6. N-methylation of the glutarimide ring of the pomalidomide moiety in 4a yielded
compound 14, which was converted to product 15 using click reaction. The N-methylated
Pomalidomide moiety has been frequently used as the negative control for Pomalidomide-based
2
3
PROTACs . Western blot analysis proved that both compounds 13 and 15 were inactive in MM1S
(Supplementary Figure S5A). Compound 13 induced limited HDAC6 degradation which might
due to hydrolysis of carboxylic ester to carboxylic acid, which is a very weak ligand for zinc.
We performed single treatment or co-treatment as combined therapy (1 µM) for 72 hours in MM1S
cells (Figure 7A). Next-A had minor effects on cell growth while 15 was totally inactive.
Pomalidomide, 13 and degrader 1 shared similar antiproliferation effects at this concentration. We
didn’t observe statistically significant synergy of dual treatment with 13 + 15 (1:1) or dual
treatment with Pomalidomide + Next-A (1:1) comparing with single treatment of 13 or
Pomalidomide (P > 0.05). The dual treatment of Pomalidomide + Next-A (1:1) is slightly more
potent than that of 13 + 15 (1:1). However, the single treatment of 12d improved about 19% (P =
0
.0017) and 11% (P = 0.0213) growth inhibition comparing with the combination sets of 13 + 15
(1:1) and Pomalidomide + Next-A (1:1), respectively. To further confirm the observed
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enhancement, we studied the anti-proliferation in response to 12d from 0.3 nM to 3 µM (Figure
7
B). We compared this single treatment with dual treatment of 13 and 15 to rule out the potential
complication derived from cell permeability issue. The resulting EC50 and maximal inhibition was
0
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listed in Table 3. Although the EC50 of 12d (74.9 ± 11.3) appeared higher than the combination of
1
3+15 (23.5 ± 6.3) or 13 (17.7 ± 2.8) alone, it is clear that the relatively higher EC50 for 12d is due
to its lower bottom or the higher maximal growth inhibition. The three curves almost overlap at
concentrations lower than 100 nM. At concentrations higher than 100 nM, degrader 12d starts to
inhibit the growth of the cell much more significantly than 13 alone or the combination of 13 and
1
5. The maximal inhibition of 12d (63.1 ± 1.8) is much higher than that of the combination of 13
and 15 (42.6 ± 1.9) or 13 alone (44.0 ± 1.2).
The combination of 13 and 15 failed to work synergistically, indicating that the HDAC6
degradation, other than inhibition, was crucial to the enhanced antiproliferation by degrader 12d
in MM1S cells. Hence, we concluded that the HDAC6 degradation and IKZF degradation had
synergistic effects at higher concentration of the degrader. For 48 h treatment of 12d in MM1S,
we observed the cleavages of Caspase-3 and PARP in dose dependent manner (Supplementary
Figure S5B), suggesting that the synergy was derived from degrader-promoted cell apoptosis.
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A
B
A n tip ro life ra tio n a t 1  M (7 2 h o u rs )
1
50
A n tip ro life ra tio n (7 2 h o u rs )
125
100
75
ns
12d
100
****
*
*
1
1
3
3
+ 1 5 (1 :1 )
**
*
5
0
0
15
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0
5
2
0
5
0
*
*
*
***
****
)
)
le
d
t
:1
:1
1
a
3
A
5
ic
2
1
(1
m
1
t
x
e
1
1
(
o
h
e
A
5
P
V
1
N
0.1
1
10
100
1000
10000
x
+
e
N
3
1
C o m p o u n d C o n c e n tra tio n (n M )
+
a
m
o
P
Figure 7. Antiproliferation of 12d in multiple myeloma. (A) Cell viability of MM1S cells treated
with compounds at 1 µM for 72 h. (B) Cell viability of MM1S cells treated with compounds at
concentrations from 0.3 nM to 3 µM for 72 h. All data was normalized to vehicle (DMSO)
treated group and dot plot represented as mean of relative viability (n = 3) with ± SD as error
bar. For curve in (B), nonlinear fitting of [Inhibitor] vs. response (three parameters) was
2
generated by GraphPad Prism with R from 0.92 to 0.97. Statistical significance was analyzed
by one-way ANOVA for (A) and two-way ANOVA for (B). See Supplementary Table S2 for
details of (B). Not significant (ns) P > 0.05, P ≤ 0.05, P ≤ 0.01, ***P ≤ 0.001, ****P ≤
*
**
0
.0001.
Table 3. EC50 of Antiproliferation in MM1S
EC50 (nM) a Maximal Inhibition (%) b
12d
74.9 ± 11.3
23.5 ± 6.3
17.7 ± 2.8
63.1 ± 1.8
42.6 ± 1.9
44.0 ± 1.2
1
3 + 15 (1:1)
1
3
a
The concentration at which half-maximal growth
b
inhibition was achieved. The maximum percentage of
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growth inhibition. a,bValues with ± SD obtained from
nonlinear fitted data in Figure 7B.
CONCLUSION
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In this study, we reported the development of a new generation of HDAC6 degraders by
tethering Pomalidomide and HDAC6 selective inhibitor Next-A. By varying the linker length and
linking positon, we discovered potent and selective HDAC6 degrader 12d that retains the
degradation activity of IKZFs. Further investigation confirmed its mechanism of action. The
antiproliferation study demonstrated the advantage of our HDAC6 degraders over HDAC6
inhibitor alone, IMiD alone, or its combination, presumably because of the multi-functions of the
degrader. Our results highlighted the power and utility of PROTACs as a novel strategy for the
development of therapeutics against multiple myeloma.
Experimental Section
General Information in Synthetic Chemistry. All reactions were conducted under a positive
pressure of dry argon in glassware that had been oven dried prior to use. Anhydrous solutions of
reaction mixtures were transferred via an oven dried syringe or cannula. All solvents were dried
prior to use unless noted otherwise. Thin layer chromatography (TLC) was performed using
1
precoated silica gel plates. Flash column chromatography was performed with silica gel. H and
13C nuclear magnetic resonance spectra (NMR) were recorded on Bruker 400 MHz and 500 MHz
1
spectrometers. H NMR spectra were reported in parts per million (ppm) referenced to 7.26 ppm
of CDCl
patterns were described as singlet (s), doublet (d), triplet (t), quartet (q), quintet (quint), or multiplet
m), with coupling constants (J) in hertz. High resolution mass spectra (HRMS) were performed
3
or referenced to the center line of a septet at 2.50 ppm of DMSO-d . Signal splitting
6
(
on an Electron Spray Injection (ESI) TOF mass spectrometer. The liquid chromatography mass
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spectrometry LC-MS analysis of final products was processed on Agilent 1290 Infinity II LC
system using Poroshell 120 EC-C18 column (5 cm × 2.1 mm, 1.9 μm) for chromatographic
separation. Agilent 6120 Quadrupole LC/MS with multimode Electrospray Ionization plus
atmospheric pressure chemical ionization (MM-ES+APCI) was used for detection. The mobile
phases were 5.0% methanol and 0.1% formic acid in purified water (A) and 0.1% formic acid in
methanol (B). The gradient was held at 5% (0-0.2 min), increased to 100% at 2.5 min, then held
at isocratic 100% B for 0.4 min and then immediately stepped back down to 5% for 0.1 min re-
equilibration. The flow rate was set at 0.8 mL/min. Column temperature was set at 40 °C. The
purities of all the final compounds were determined to be over 95% by LC-MS. See Supporting
1
1
1
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0
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1
13
Information for H and C NMR spectrums and LC-MS purity analysis of all compounds.
Pomalidomide analogues. Alkyne materials 4a-e were made according to literature
procedures25,34
.
2-(2,6-Dioxopiperidin-3-yl)-4-(prop-2-yn-1-ylamino)isoindoline-1,3-dione 4a (30% yield):
1H NMR (500 MHz, CDCl
3
): δ 2.12-2.16 (m, 1H), 2.27 (t, J = 2.3 Hz, 1H), 2.70-2.93 (m, 3H),
4
.10 (dd, J = 6.1, 2.4 Hz, 2H), 4.92 (dd, J= 12.3, 5.4 Hz, 1H), 6.45 (t, J = 5.8 Hz, 1H), 7.03 (d, J
1
3
=
8.5 Hz, 1H), 7.20 (d, J = 7.2 Hz, 1H), 7.56-7.59 (m, 1H), 7.97 (s, 1H). C NMR (126 MHz,
DMSO): δ 22.7, 31.6, 32.3, 49.0, 72.5, 79.7, 111.3, 112.2, 117.4, 132.5, 136.0, 145.5, 167.6, 169.2,
+
+
1
69.4, 172.3. LC−MS(ESI) m/z (M + H) : 312.1; calcd for C16
-(But-3-yn-1-ylamino)-2-(2,6-dioxopiperidin-3-yl)isoindoline-1,3-dione 4b (59% yield): H
NMR (400 MHz, DMSO): δ 1.98-2.07 (m, 1H), 2.43-2.49 (m, 2H), 2.53-2.65 (m, 2H), 2.82-2.95
H
13
N
3
O
4
(M + H) : 312.1.
1
4
(m, 2H), 3.43-3.53 (m, 2H), 4.96-5.17 (m, 1H), 6.65-6.75 (m, 1H), 6.98-7.10 (m, 1H), 7.16 (d, J =
1
3
8
.6 Hz, 1H), 7.53-7.65 (m, 1H), 11.09 (s, 1H). C NMR (100 MHz, DMSO): δ 172.8, 170.1,
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1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
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5
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1
68.8, 167.3, 146.0, 136.3, 132.2, 117.3, 110.8, 109.4, 81.9, 72.9, 48.6, 40.7, 31.0, 22.1, 18.6.
+
+
LC−MS(ESI) m/z (M + H) : 326.0; calcd for C17
H
15
N
3
O (M + H) : 326.1.
4
2-(2,6-Dioxopiperidin-3-yl)-4-(pent-4-yn-1-ylamino)isoindoline-1,3-dione 4c (62% yield):
1H NMR (400 MHz, CDCl
): δ 1.84-1.92 (m, 2H), 2.09-2.16 (m, 1H), 2.30-2.38 (m, 2H), 2.68-
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
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8
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0
3
2
7
1
.99 (m, 4H), 3.40-3.49 (m, 2H), 4.96-4.83 (m, 1H) 6.23-6.35 (m, 1H), 6.98-6.88 (m, 1H) 7.20-
1
3
.04 (m, 1H), 7.47-7.55 (m, 1H), 8.12 (brs, 1H). C NMR (100 MHz, DMSO): δ 172.8, 170.1,
67.3, 162.3, 146.3, 136.3, 132.3, 117.3, 110.5, 109.3, 83.8, 71.7, 48.5, 43.0, 41.0, 35.8, 27.5, 15.3.
+
+
LC−MS(ESI) m/z (M + H) : 340.1; calcd for C18
H
17
N
3
O (M + H) : 340.1.
4
2
-(2,6-Dioxopiperidin-3-yl)-5-(pent-4-yn-1-ylamino)isoindoline-1,3-dione 4d (17% yield):
1H NMR (400 MHz, DMSO): δ 1.99-2.02 (m, 1H), 2.53-2.60 (m, 2H), 2.83-2.93 (m, 1H), 3.19 (s,
1
7
H), 4.07 (br, 2H), 5.03-5.06 (m, 1H), 6.94 (d, J = 8.4 Hz, 1H), 7.04 (br, 1H), 7.41-7.44 (m, 1H),
1
3
.62 (d, J = 8.4 Hz, 1H), 11.07 (br, 1H). C NMR (100 MHz, DMSO): δ 173.3, 170.6, 168.1,
167.6, 153.9, 134.4, 125.4, 117.9, 117.1, 106.4, 81.2, 74.3, 49.2, 32.2, 31.5, 22.7. LC−MS(ESI)
+
+
m/z (M + H) : 312.0; calcd for C16
H
13
N
3
O (M + H) : 312.1.
4
2
-(2,6-Dioxopiperidin-3-yl)-5-(prop-2-yn-1-ylamino)isoindoline-1,3-dione 4e (20% yield):
1H NMR (400 MHz, DMSO): δ 1.67-1.74 (m, 2H), 1.94-1.98 (m, 1H), 2.22-2.26 (m, 2H), 2.46
(br, 2H), 2.78-2.88 (m, 2H), 3.18-3.21 (m, 2H), 4.99 (dd, J = 5.6, 12.8 Hz, 1H), 6.81 (d, J = 8.4
13
Hz, 1H), 6.92 (s, 1H), 7.06-7.16 (m, 1H), 7.53 (d, J = 8.4 Hz, 1H), 11.02 (br, 1H). C NMR (100
MHz, DMSO): δ 172.8, 170.2, 167.7, 167.1, 154.3, 134.2, 125.1, 116.1, 83.9, 71.6, 48.6, 41.3,
+
+
31.0, 27.2, 22.2, 15.4. LC−MS(ESI) m/z (M + H) : 340.0; calcd for C18
H
17
N
3
O (M + H) : 340.1.
4
General Procedure for Preparing PROTACs 12a-r
A mixture of compound 5 (500 mg, 2.4 mmol), 1,2-dibromoethane (0.82 mL, 9.6 mmol) and
potassium carbonate (1.3g, 9.6 mmol) in acetonitrile (5 mL) was stirred at 90℃ overnight. The
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mixture was cooled to room temperature and poured into water (10 mL), then extracted with ethyl
acetate (10 mL x 3). The combined organic phases were washed with brine (20 mL), dried over
anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified by
silica gel flash column chromatography (eluted with 5% ethyl acetate in hexane) to afford
compound 6a (482 mg, 64%).
1
1
1
1
1
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1
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A suspension of compound 6a (300 mg, 0.95 mmol) and sodium azide (308 mg, 4.7 mmol) in
DMF (5 mL) was stirred at 50 ℃for 4h. Then EtOAc and water were added. The organic layer
was separated and washed once with water. The resulting aqueous layer was extracted once with
EtOAc. The combined organic layer was dried over with Na
2
SO and concentrated in vacuo. The
4
residue was purified by silica gel chromatograph to give the product 7a (224 mg, 85%).
For the syntheses of 8a-c (n=2-4): In a 250 mL round bottle flask filled with 100 mL of water,
7a-c was added. Then the flask topped with a condenser was dipped in a 110 ℃ oil bath. TLC was
used to monitor the progress of the reaction. The reaction mixture was cooled down after 12h and
was extracted with ethyl acetate. The extract was washed with brine, dried over with anhydrous
Na
2
SO , and then concentrated in vacuo. The residue was purified by column chromatography on
4
silica gel with ethyl acetate/hexane (1:1, v/v) to afford the free amine 8a-c (95-97%).
For the syntheses of 8d-e (n=5, 6): To a solution of azide (7d-e) in DCM (45 ml) was added
TFA (30 eq) dropwise at 0℃. The resulting mixture was stirred at room temperature for 4h. Upon
completion as indicated by TLC, the reaction was quenched by aqueous NaHCO , then extracted
3
with ethyl acetate (10 mL x 3). The combined organic layers were washed with brine (20 mL),
dried over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was
purified by silica gel flash column chromatography (eluted with 20% ethyl acetate in hexane) to
afford compounds 8d-e (94-96%).
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6
A solution of 8a (140 mg, 0.78 mmol) in THF (2 mL) was added carbonyldiimidazole (CDI:
1
53 mg, 0.94 mmol) at room temperature under an atmosphere of Ar, and the resulting solution
was stirred for 2h. Then 9 (173 mg, 0.78 mmol) in THF (2 ml) was added dropwise to the reaction
mixture and the resulting mixture was stirred overnight. The reaction was quenched with saturated
aqueous sodium bicarbonate (10 mL) and extracted with DCM (3 × 10 mL). The combined
organics were washed with brine (15 mL), dried over sodium sulfate, concentrated in vacuo, and
purified via silica gel chromatography, affording the urea ester 10a as a brown oil (253 mg, 76%).
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
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9
0
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9
0
NaOH (188 mg, 4.7 mmol) was dissolved in an aqueous solution of NH
2
OH (50 wt %, 1 mL) at
0
℃. Then a solution of 10a (250 mg, 0.59 mmol) in THF/MeOH (1:1, 3 mL total) was added
dropwise where the biphasic solution became homogeneous upon compete addition. The resulting
solution was stirred 1h at room temperature. The reaction was quenched with AcOH (0.35 mL,
5.64 mmol). Water (10 ml) was added and the aqueous layer was extracted three times with DCM
(
10 mL × 3). The combined organic layers were washed with brine (15 ml), dried over sodium
sulfate, concentrated in vacuo, and purified via silica gel chromatograph, affording 11a (231 mg,
9
2%).
-((3-(4-(2-Azidoethoxy)phenyl)-1-butylureido)methyl)-N-hydroxybenzamide 11a: 1H
4
NMR (400 MHz, CDCl
3
): δ 0.85 (t, J = 7.2 Hz, 3H), 1.16-1.24 (m, 1H), 1.48 (br, 2H), 1.95 (brs,
1
H), 3.16 (br, 2H), 3.50 (br, 2H), 4.02 (br, 2H), 4.42 (br, 2H), 6.63-6.64 (m, 3H), 6.95-7.23 (m,
13
4H), 7.55 (br, 2H), 8.02 (br, 2H). C NMR (100 MHz, CDCl
3
): δ166.1, 156.3, 154.6, 142.2, 132.7,
1
30.2, 127.6, 127.1, 122.9, 114.9, 67.4, 50.3, 50.1, 47.4, 30.4, 20.2, 14.0. LC−MS(ESI) m/z (M +
+
+
H) : 427.1; calcd for C21
H
26
N
6
O (M + H) : 427.2.
4
Following the procedures, 11b-e with different linker lengths were obtained.
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-((3-(4-(3-Azidopropoxy)phenyl)-1-butylureido)methyl)-N-hydroxybenzamide 11b (87%
1
yield): H NMR (400 MHz, CDCl
3
): δ 0.86 (t, J = 7.2 Hz, 3H), 1.16-1.24 (m, 1H), 1.43-1.56 (m,
2H), 1.90-2.04 (m, 3H), 3.17 (br, 2H), 3.45 (t, J = 7.2 Hz, 2H), 3.87-4.01 (m, 2H), 4.44 (br, 2H),
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
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4
5
6
7
8
9
0
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3
4
5
6
7
8
9
0
13
6
.61-6.82 (m, 3H), 6.79-7.22 (m, 4H), 7.56 (br, 2H), 8.48 (br, 2H). C NMR (100 MHz, CDCl
3
):
δ 166.0, 156.2, 154.5, 142.1, 132.6, 130.1, 127.5, 127.0, 122.8, 114.8, 67.3, 50.2, 50.0, 47.3, 30.3,
+
+
2
0.1, 13.9. LC−MS(ESI) m/z (M + H) : 441.1; calcd for C22
H
28
N
6
O
4
(M + H) : 441.2.
4
-((3-(4-(4-Azidobutoxy)phenyl)-1-butylureido)methyl)-N-hydroxybenzamide 11c (92%
1
yield): H NMR (400 MHz, CDCl
3
): δ 0.86 (t, J = 7.3 Hz, 3H), 1.16-1.33 (m, 2H), 1.42-1.57 (m,
2
H), 1.68-1.86 (m, 4H), 1.95 (s, 1H), 3.16 (s, 2H), 3.31 (t, J = 6.5Hz, 2H), 3.81-3.97 (m, 2H), 4.43
1
3
(s, 2H), 6.60-6.85 (m, 3H), 6.92-7.23 (m, 4H), 7.75 (br, 1H), 8.51 (br, 2H). C NMR (100 MHz,
CDCl
3
): δ 165.9, 156.3, 155.2, 142.1, 131.9, 130.1, 127.5, 127.0, 122.9, 114.6, 67.5, 51.2, 50.0,
+
4
7.3, 30.3, 26.5, 25.7, 20.1, 13.9. LC−MS(ESI) m/z (M + H) : 455.1; calcd for C23
H
30
N
6
O
4
(M +
+
H) : 455.2.
4
-((3-(4-((5-Azidopentyl)oxy)phenyl)-1-butylureido)methyl)-N-hydroxybenzamide
11d
1
(
(
(
79% yield): H NMR (400 MHz, CDCl
3
): δ 0.87 (t, J = 7.2 Hz, 3H), 1.13-1.40 (m, 2H), 1.43-1.56
m, 4H), 1.58-1,69 (m, 2H), 1.70-1.81 (m, 2H), 3.19 (s, 2H), 3.27 (t, J = 6.8 Hz, 2H), 3.79-3.95
1
3
m, 2H), 4.47 (s, 2H), 6.51-6.86 (m, 3H), 6.90-7.23 (m, 4H), 7.59 (br, 2H). C NMR (100 MHz,
CDCl
3
): δ 165.9, 156.3, 155.4, 142.0, 131.7, 130.0, 127.5, 127.0, 122.9, 114.7, 67.9, 51.3, 50.1,
+
4
7.5, 30.3, 28.8, 28.6, 23.4, 20.1, 13.9. LC−MS(ESI) m/z (M + H) : 469.1; calcd for C24
H
32
N
6
O
4
+
(M + H) : 469.3.
4
-((3-(4-((6-Azidohexyl)oxy)phenyl)-1-butylureido)methyl)-N-hydroxybenzamide
11e
1
(
90% yield): H NMR (400 MHz, CDCl
3
): δ 0.75-0.97 (m, 3H), 1.11-1.27 (m, 2H), 1.33-1.52 (m,
6H), 1.55-1.66 (m, 2H), 1.69-1.82 (m, 2H), 2.97-3.40 (m, 4H), 3.86 (s, 2H), 4.45 (s, 2H), 6.44-
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Journal of Medicinal Chemistry
Page 28 of 57
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5
5
5
5
5
5
5
6
13
6
1
.80 (m, 3H), 6.89-7.22 (m, 4H), 7.61 (br, 2H). C NMR (100 MHz, CDCl
3
): δ 165.9, 156.3,
55.4, 142.0, 131.8, 130.1, 127.5, 127.0, 122.9, 114.7, 68.0, 51.4, 50.0, 47.4, 30.3, 29.2, 28.8, 26.5,
25.7, 20.1, 13.9.
0
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2
3
4
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6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
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9
0
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8
9
0
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2
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6
7
8
9
0
A mixture of 11a (20 mg, 0.05 mmol) and compound 4a (15 mg, 0.05 mmol), tris[(1-benzyl-
1
H-1,2,3-triazol-4-yl)methyl]amine (5 mg, 0.001 mmol), CuSO
ascorbate (12 mg, 0.06 mmol) in t-BuOH:H O (1.5:1) (2 mL) was stirred at room temperature for
6 h. The reaction mixture was then quenched with water (5 mL) and extracted with
4
(1.5 mg, 0.01 mmol), sodium
2
1
dichloromethane (10 mL x 3). The combined organic phases were washed brine (50 mL), dried
over anhydrous sodium sulfate, and concentrated under reduced pressure. The residue was purified
by silica gel flash column chromatography (eluted with 30% methanol in dichloromethane) to
afford the 12a (14 mg, 40%).
4-((1-Butyl-3-(4-(2-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)methyl)-1H-1,2,3-triazol-1-yl)ethoxy)phenyl)ureido)methyl)-N-
1
hydroxybenzamide 12a (m=1, n=2, C4 position): H NMR (400 MHz, DMSO): δ 0.70-0.90 (m,
3
1
2
1
H), 1.22-1.32 (m, 2H), 1.33-1.56 (m, 2H), 1.91-2.14 (m, 1H), 2.52-2.70 (m, 2H), 2.79-2.95 (m,
H), 3.36-3.58 (m, 3H), 4.12-4.86 (m, 7H), 4.96-5.17 (m, 1H), 6.51-7.43 (m, 8H), 7.47-7.79 (m,
13
H), 7.87-8.64 (m, 2H), 11.10 (brs, 1H). C NMR (100 MHz, DMSO): δ 173.3, 170.5, 169.2,
67.7, 164.4, 155.8, 153.5, 146.3, 145.0, 142.9, 136.6, 134.4, 132.6, 127.4, 123.9, 122.3, 118.1,
114.8, 111.4, 110.2, 66.9, 55.4, 49.5, 49.0, 46.5, 38.1, 31.5, 30.4, 29.5, 22.6, 19.9, 14.2. HRMS
+
(EI) calcd. for C37
H
39
N O
9 8
(M + H) 738.3000, found 738.2974. Purity: >98% (LCMS).
Following the procedure for 12a, compounds 12b-r were prepared.
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Journal of Medicinal Chemistry
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4
-((1-Butyl-3-(4-(3-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
yl)amino)methyl)-1H-1,2,3-triazol-1-yl)propoxy)phenyl)ureido)methyl)-N-
1
hydroxybenzamide 12b (m=1, n=3, C4 position, 40% yield): H NMR (400 MHz, DMSO): δ
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
0
2
2
7
1
1
.85 (t, J = 7.3 Hz, 3H), 1.24-1.31 (m, 2H), 1.41-1.53 (m, 2H), 1.97-2.07 (m, 1H), 2.16-2.28 (m,
H), 2.51-2.64 (m, 2H), 2.81-2.96 (m, 1H), 3.21-3.27 (m, 1H), 3.83-3.91 (m, 2H), 4.44-4.53 (m,
H), 4.49 (t, J = 7.0 Hz, 1H), 4.57-4.62 (m, 3H), 4.98-5.13 (m, 1H), 6.78 (d, J = 9.0 Hz, 1H), 7.00-
.11 (m, 2H), 7.16 (d, J = 8.6 Hz, 1H), 7.27-7.42 (m, 4H), 7.51-7.60 (m, 1H), 7.72 (d, J = 8.0 Hz,
13
H), 8.06 (s, 1H), 8.22 (s, 1H), 10.97-11.39 (m, 2H). C NMR (100 MHz, DMSO): δ 172.8, 170.1,
68.8, 167.3, 164.1, 155.5, 153.6, 145.8, 144.5, 142.5, 136.2, 133.7, 132.1, 131.4, 127.0, 123.0,
122.0, 117.6, 114.2, 110.9, 109.7, 105.4, 64.6, 49.0, 48.6, 46.6, 46.1, 37.7, 31.0, 30.0, 29.6, 22.2,
+
1
9.5, 13.8.HRMS (EI) calcd. for C38
LCMS).
4-((1-Butyl-3-(4-(4-(4-(((2-(2,6-dioxopiperidin-3-yl)-1,3-dioxoisoindolin-4-
H
41
N
9
O
8
(M + H) 752.3156, found 752.3160. Purity: 98%
(
yl)amino)methyl)-1H-1,2,3-triazol-1-yl)butoxy)phenyl)ureido)methyl)-N-
1
hydroxybenzamide 12c (m=1, n=4, C4 position, 45% yield): H NMR (400 MHz, DMSO): δ 0.85
(
(
(
(
br, 3H), 1.14-1.22 (m, 1H), 1.34-1.72 (m, 4H), 1.78-2.04 (m, 3H), 2.52-2.66 (m, 2H), 2.81-2.96
m, 1H), 3.39-3.47 (m, 1H), 3.83-3.94 (m, 2H), 4.13-4.28 (m, 2H), 4.32-4.44 (m, 2H), 4.53-4.67
m, 3H), 5.01-5.15 (m, 1H), 6.69-6.88 (m, 2H), 7.00-7.21 (m, 3H), 7.24-7.44 (m, 4H), 7.52-7.64
1
3
m, 1H), 7.66-7.77 (m, 1H), 8.05 (br, 1H), 8.21 (br, 1H), 10.98-11.32 (m, 2H). C NMR (100
MHz, DMSO): δ 172.8, 170.1, 168.8, 167.3, 155.5, 153.8, 145.8, 136.1, 133.4, 132.1, 128.8, 127.0,
1
2
22.9, 122.0, 117.6, 114.1, 110.9, 109.7, 66.9, 49.0, 46.1, 37.7, 31.0, 30.7, 30.0, 26.6, 25.8, 22.2,
+
2.2, 19.5, 13.8. HRMS (EI) calcd. for C39
H
43
N O
9 8
(M + H) 766.3313, found 766.3309. Purity:
96% (LCMS).
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