Oxa Analogues of Nexturastat A Demonstrate Improved HDAC6 Selectivity and Superior Antileukaemia Activity

Article abstract of DOI:10.1002/cmdc.202001011

The acetylome is important for maintaining the homeostasis of cells. Abnormal changes can result in the pathogenesis of immunological or neurological diseases, and degeneration can promote the manifestation of cancer. In particular, pharmacological interv

Full text of DOI:10.1002/cmdc.202001011

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Oxa Analogues of Nexturastat A Demonstrate Improved
HDAC6 Selectivity and Superior Antileukaemia Activity
Marc Pflieger+,[a] Melf Sönnichsen+,[b] Nadine Horstick-Muche,[a] Jing Yang,[b, c]
Julian Schliehe-Diecks,[b] Andrea Schöler,[d] Arndt Borkhardt,[b] Alexandra Hamacher,[a]
Matthias U. Kassack,[a] Finn K. Hansen,[e] Sanil Bhatia,*[b] and Thomas Kurz*[a]
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The acetylome is important for maintaining the homeostasis of
cells. Abnormal changes can result in the pathogenesis of
immunological or neurological diseases, and degeneration can
promote the manifestation of cancer. In particular, pharmaco-
logical intervention in the acetylome with pan-histone deacety-
lase (HDAC) inhibitors is clinically validated. However, these
drugs exhibit an undesirable risk-benefit profile due to severe
side effects. Selective HDAC inhibitors might promote patient
compliance and represent a valuable opportunity in personal-
ised medicine. Therefore, we envisioned the development of
HDAC6-selective inhibitors. During our lead structure identifica-
tion, we demonstrated that an alkoxyurea-based connecting
unit proves to be beneficial for HDAC6 selectivity and
established the synthesis of alkoxyurea-based hydroxamic acids.
Herein, we report highly potent N-alkoxyurea-based hydroxamic
acids with improved HDAC6 preference compared to nextur-
astat A. We further validated the biological activity of these oxa
analogues of nexturastat A in a broad subset of leukaemia cell
lines and demonstrated their superior anti-proliferative proper-
ties compared to nexturastat A.
Introduction
and class IV (HDAC11). Depending on their cellular localisation,
they influence the condensation state of histones,[2] participate
in the post-translational modifications of cytosolic proteins[3] or
even might act as an epigenetic reader in the case of class IIa
HDACs.[4] Amongst humans, their diverse array of functions
Histone deacetylases (HDACs) are proteases that catalyse the
cleavage of acetylated lysine residues (isopeptide bonds).[1]
Human zinc dependant histone deacetylases (HDACs) are
classified into class I (HDAC1, HDAC2, HDAC3, HDAC8), class IIa
(HDAC4, HDAC5, HDAC7, HDAC9), class IIb (HDAC6, HDAC10)
renders them
a valuable target for the pharmacological
intervention of immunological[5–8] or neurodegenerative
diseases[9,10] and are clinically validated targets for the treatment
of cancer.[11]
[a] Dr. M. Pflieger,+ N. Horstick-Muche, Dr. A. Hamacher,
Prof. Dr. M. U. Kassack, Prof. Dr. T. Kurz
Institut für Pharmazeutische und Medizinische Chemie
Heinrich-Heine-Universität Düsseldorf
Universitätsstr. 1, 40225 Düsseldorf (Germany)
E-mail: thomas.kurz@hhu.de
Whilst CD1 has a high specificity in the substrate recog-
nition of acetylated C-terminal lysine residues, CD2 exhibits a
promiscuity towards a wide range of client proteins.[12,13] In
addition to those domains, HDAC6 displays an inherent zinc-
finger ubiquitin binding domain (ZnFÀ UBP)[13,14] that enables it
to recognise ubiquitylated proteins. The rigidly controlled
localisation of HDAC6 in the cytoplasm is the result of the
interplay between the nuclear export signal (NES), the nuclear
localisation signal (NLS) and the SerÀ Glu-containing tetrapep-
tide (SE14).[15]
HDAC6 catalyses the deacetylation of, for example, α-
tubulin,[16] cortactin[17] and HSP90[18] and is of relevance for the
pathogenesis of cancer[19] as well as in immunological[20] and
neurological diseases.[21–23] Its participation in pathogenesis or
disease progression is tissue dependant and of multifactorial
nature. Despite intensive research, the clinical significance of
HDAC6 selective inhibitors, as single agent, remains
controversial.[24] Increasing evidence suggest, that the anti-
cancer effect of those biologically active compounds is the
result of concentrations at which other HDAC isozymes,
particularly class I HDACs, are also inhibited. Nevertheless, the
pharmacological intervention of cancer by addressing HDAC6,
remains a promising target due to its participation in the
invasiveness of cancer cells and its immunomodulatory proper-
ties.
[b] M. Sönnichsen,+ J. Yang, J. Schliehe-Diecks, Prof. Dr. A. Borkhardt,
Dr. S. Bhatia
Department of Pediatric Oncology, Hematology and Clinical Immunology,
Medical Faculty
Heinrich Heine University Düsseldorf
Universitätsstr. 1, 40225 Düsseldorf (Germany)
E-mail: Sanil.Bhatia@med.uni-duesseldorf.de
[c] J. Yang
Department of Medicine
Yangzhou Polytechnic College
West Wenchang Road 458, Yangzhou, 225009 (P.R. China)
[d] A. Schöler
Institute for Drug Discovery, Medical Faculty
Leipzig University
Brüderstraße 34, 04103 Leipzig (Germany)
[e] Prof. Dr. F. K. Hansen
Pharmaceutical and Cell Biological Chemistry, Pharmaceutical Institute
University of Bonn
An der Immenburg 4, 53121 Bonn (Germany)
[+] These authors contributed equally to this work,
Supporting information for this article is available on the WWW under
https://doi.org/10.1002/cmdc.202001011
© 2021 The Authors. ChemMedChem published by Wiley-VCH GmbH. This is
an open access article under the terms of the Creative Commons Attribution
Non-Commercial NoDerivs License, which permits use and distribution in
any medium, provided the original work is properly cited, the use is non-
commercial and no modifications or adaptations are made.
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Currently approved HDAC inhibitors (HDACi) are vorinostat,
facilitates its selectivity by the exhibition of a branched cap
group.
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belinostat, panobinostat, romidepsin for the treatment of
haematological malignancies and tucidinostat (chidamide) for
the treatment of breast cancer in China. All approved HDACi
exhibit a mutual pharmacophore model: a zinc binding group
(ZBG), a linker, and a cap group.[25–27] Whilst romidepsin[28,29] is
preferential and tucidinostat[30] selective for HDAC class I
isozymes, the remaining are regarded as pan inhibitors as they
do not differentiate between individual isozymes. Since the
elucidation of structural information of HDACs,[31] the design of
selective inhibitors was significantly accelerated. Despite their
high sequence identity inside the catalytic pocket, HDACs
exhibit distinct features that allows a rational design for
isozyme selective inhibitors. Particularly in comparison to
Recently, we demonstrated that the modification of the
functional group that connects the liker and the cap group of
HDACi (connecting unit, CU) can significantly alter the isozyme
profile of unselective HDACi (pan HDACi).[37,38] The reported
alkoxyamide and alkoxyurea derivatives of vorinostat and
panobinostat exhibited a refined isozyme profile that resulted
in a HDAC6 preference.
Here we report a rational derivatisation of Nexturastat A to
improve HDAC6 selectivity (Scheme 1) and compounds with
superior antiproliferative properties.
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HDAC1,[32] HDAC6[31] exhibits a much wider and shallower Results and Discussion
entrance tunnel (Figure 1).
Accumulating structural information of HDACs challenged
the traditional HDACi pharmacophore model (ZBG, linker, cap)
which was insufficient for the design of selective inhibitor and a
revised pharmacophore model was developed. The pharmaco-
phore model for HDAC6 inhibitors comprises of a ZBG, an
aromatic linker and a sterically demanding surface cap group
(S-CAP).[33] Based on these structural features and the revised
pharmacophore model, the selectivity towards HDAC6 can be
governed by a sterically demanding cap group (Figure 2).
Two of the most prominent HDAC6 selective inhibitors are
nexturastat A[35,36] and tubastatin A,[34] of which both exhibit a
sterically demanding cap group. Whilst tubastastin A realises
this steric demand by a bulky cap group, nexturastat A
Nexturastat A is a selective HDAC6 inhibitor with a low-nano-
molar activity and a reported 600-fold selectivity for HDAC6
over HDAC1.[36] Despite its high selectivity reported[36] by
Bergman et al., nexturastat A exhibited only a selectivity index
of 24 in our HDAC enzyme assays (Table 1). The lower
selectivity, compared with the initial selectivity data, is in good
agreement with recently published data by Vergani et al.[39]
To increase the selectivity towards HDAC6, we envisioned
structural modifications of nexturastat A by the introduction of
an alkoxyurea-based connecting unit (CU), whilst maintaining
the potency of nexturastat A.
Molecular docking
The focus of our rational design revolved around structural
modifications of nexturastat A to establish a hydrogen bond
interaction with Ser568 of the HDAC6 L1 loop segment. To
support our initial hypothesis that alkoxyurea derivatives of
nexturastat A could exhibit a hydrogen bond interaction with
HDAC6, we have performed molecular docking studies with
AutoDock 4.2 employing
a previously validated docking
protocol.[38] Compound 4a and nexturastat A were docked in
HDAC6 (PDB ID: 5EDU).[31] The molecular docking results
suggest, that the CU of compound 4a could rotate at the
benzylic position to adapt a conformation to establish a
hydrogen bonding interaction between Ser568 and the oxygen
atom of the alkoxy side chain (Figure 3).
Figure 1. Surface comparison of HDAC1 and HDAC6.[16,33]
Scheme 1. Structural modification of nexturastat A. The pharmacophore
model of HDAC6 inhibitors comprises a zinc binding group (ZBG, red) that
coordinates to the zinc ion in the catalytic centre, a linker that interacts with
the hydrophobic entrance tunnel and a sterically demanding cap group that
is connected to the linker via the connecting group (CU).
Figure 2. Revised pharmacophore model for the design of selective HDAC6
inhibitors.[33–35]
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Table 1. HDAC1-3/6/8 isozyme profiling of compounds 4a and b.
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R’
R’’
IC50 [μM]
HDAC1
SI1/6
37
SI2/6
71
SI3/6
45
SI8/6
232
241
HDAC2
HDAC3
HDAC6
HDAC8
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4a
4b
0.742�0.039
0.299�0.057
1.42�0.082
0.902�0.008
0.020�0.003
4.64�0.84
0.515�0.043
1.14�0.074
0.375�0.084
0.972�0.064
0.014�0.002
0.022�0.002
3.37�0.61
4.46�0.41
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4c
0.715�0.010
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52
44
203
4d
2.89�0.125
3.59�0.410
3.12�0.578
0.341�0.021
0.021�0.001
9.94�1.71
9.91�1.25
8.5
24
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41
9.2
35
29.1
472
nexturastat A
0.504�0.033
0.861�0.008
0.730�0.033
Data are the mean�SD of at least two independent experiments, each carried out in duplicate wells.
Synthesis of branched alkoxyurea based hydroxamic acids
In order to evaluate the influence of the alkoxyurea CU in
respect to HDAC isozyme profile, the direct alkoxyurea deriva-
tive of Nexturastat A was synthesised (4a), based on our
retrosynthetic analysis (Scheme 2).
N-Boc-O-alkylhydroxylamines (1) were synthesised either by
O-alkylation of N-Boc-hydroxylamine with 1-bromopropane (1a)
or by the N-Boc protection of O-benzyl hydroxylamine (1b).
The O-substituted hydroxylamine moiety was introduced to
the benzyl linker by the N-alkylation of 1 with methyl 4-
(bromomethyl)benzoate (Scheme 3). As the purification of this
intermediate was laborious, 2 was accessed after Boc depro-
tection over two steps. Subsequently, the branched alkoxyurea
derivatives 3a, 3b, 3d were synthesised by the conversion of 2
Figure 3. Proposed binding mode of compound 4a (beige) and nexturasta-
t A (cyan) in HDAC6 (PDB ID: 5EDU).
Scheme 2. Retrosynthetic analysis of alkoxyurea based hydroxamic acids.
Scheme 3. Synthesis of branched alkoxyurea-based hydroxamic acids 4. i) 1.10 equiv. methyl 4-(bromomethyl)benzoate 1.20 equiv. NaH; ii) 5.00 equiv.
HCl(dioxane), CH2Cl2. 91–92% (2 steps) iii) 1.00 equiv. R’NCO, 1.00 equiv. DIPEA, CH2Cl2. 73–85%, iv) 1.00 equiv. N,N-dimethyl-p-phenylenediamine, 1.00 equiv. 4-
nitrophenyl chloroformate, 2.00 equiv. DIPEA; v) 30.0 equiv. H2NOH(aq), 10.0 equiv. NaOH, CH2Cl2/MeOH, 34% À 76%.
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with the respective isocyanate. In the case of 3c, N,N-dimethyl-
p-phenylenediamine was coupled with 2a (R’’=Pr) by 4-nitro-
phenyl chloroformate. Finally, the esters were converted into
the corresponding hydroxamic acids 4 by hydroxylaminolysis.
tested leukaemic entities, 4b exhibited antiproliferative activ-
ities in the micromolar range from 1.6 (�0.035) μM for K562 to
3.0 (�0.14) μM for Jurkat.
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The superior activity against the AML cell lines was a
common feature shared by all four derivates except for 4c,
which showed a general weak efficacy against all cell lines
compared to the other tested compounds. The weak antiproli-
ferative properties of 4c were surprising, as it exhibited a similar
HDAC isozyme inhibition profile to the other inhibitors.
HDAC inhibition by branched alkoxyurea based hydroxamic
acids
The branched alkoxyurea based hydroxamic acids 4 were
subjected to HDAC1-3/6/8 isozyme profiling to evaluate their
HDAC6 selectivity and their inhibitory potential. Table 1 depicts
the isozyme profiling of compounds 4. 4a (IC50 =0.020�
0.003 μM), 4b (IC50 =0.014�0.002 μM) and 4c (IC50 =0.022�
0.002 μM) demonstrated similar HDAC6 inhibition potencies to
nexturastat A ((IC50 =0.021�0.001). A benzyl substituent (4d,
IC50 =0.341�0.021 μM) caused a significant loss in HDAC6
inhibition, indicating that aliphatic substituents at R’’ are
beneficial for HDAC6 inhibition.
Similarly to nexturastat A, 4a demonstrated the highest
selectivity (antiproliferative profile) towards myeloid lineage
originated leukaemic cell lines (K562, HL60 and MOLM13),
amongst the tested alkoxyurea based hydroxamic acid deriva-
tives, whereas 4b exhibited pronounced antiproliferative
activity across wide range of tested leukaemia cell lines.
Compound 4 and nexturastat A were evaluated for their in vitro
selectivity towards HDAC isoform (Figure 4B). HL60 (AML) cells
were treated with 4 and nexturastat A in increasing concen-
trations, to compare the dose dependent hyperacetylation
induction of α-tubulin and histone H3. The degree of α-tubulin
hyperacetylation (HDAC6 inhibition marker) upon treatment
with 4a or 4d was in agreement with the HDAC isozyme
profile. Total α-tubulin was not affected by the treatment.
However, nexturastat A and 4c showed slightly higher levels of
H3 acetylation compared to 4a and 4b. The total H3 was not
affected by the treatment. Depetter et al. performed a compre-
hensive analysis of the biochemical and functional impact of
selective HDAC6 inhibitors in a variety of in vitro and in vivo
cancer models.[24] HDAC6 inhibition results in α-tubulin acetyla-
tion but not in the anticipated anti-cancer effects. They have
further demonstrated that selective HDAC6 inhibitor can result
in a reduced cell growth as well as a reduced migratory and
invasive activity at concentration, where other HDAC isozymes
are co-inhibited. Based on these findings, the antiproliferative
effect of selective HDACi is the result of the overall HDAC
isozyme inhibition profile inside a cell.
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In contrast to the reported 600-fold selectivity of nextur-
astat A for HDAC6 over HDAC1, it showed
a moderate
selectivity index of 24 (SI2/6 =41, SI3/6 =35) in our enzyme assay.
The hydroxylamine derivative 4a showed a 1.5-, 1.7-, 1.3-fold
higher SI1/6, SI2/6 and SI3/6 than nexturastat A, respectively. A
higher selectivity towards HDAC6 was anticipated by sterically
demanding substituents such as 3,5-dimethylphenyl (4b) or by
a 4-(N,N-dimethyl amino)-phenyl (4c). However, these substitu-
ent patterns had either no significant impact on the selectivity
(4c) or was even disadvantageous in the case of 4b.
Furthermore, a benzyl substituent at R’’ (4d) resulted in a
significant decreased inhibition of HDAC6 (0.341�0.021 μM)
with a concomitant decrease in selectivity as evidenced by the
comparison with 4b. Compound 4a–4c demonstrated HDAC8
inhibition in the micromolar range (3.37�0.61 μM to 4.64�
0.84 μM) and an approximately twofold lower SI8/6 (SI8/6(4a)=
232, SI8/6(4c)=241, SI8/6(4c)=203) compared to nexturastat A
(SI8/6 =472). 4d, exhibiting a benzyl substituent at R’’, showed
highest HDAC8 inhibitory concentration (IC50 =9.94�1.71 μM)
and the lowest SI8/6 (SI8/6(4d)=29.1), which is mainly the result
of a lower HDAC6 inhibition (IC50 =0.341�0.021 μM).
The obtained data indicate, that 4b exhibits a desirable
isozyme inhibition profile that manifests in antiproliferative
properties and therefore renders it a valuable hit for the
development of active pharmaceutical ingredients for the
treatment of a broad range of haematological malignancies.
Biological evaluation
Conclusion
To analyse the anti-cancer activity of the branched alkoxyurea
based hydroxamic acids 4, the in vitro antiproliferative efficacy
of all four derivatives were tested on a broad range of
leukaemia cell lines. The tested cell lines were HAL01, SUP-B15
(B-cell acute lymphoblastic leukaemia or B-ALL), K562 (chronic
myeloid leukaemia or CML), Jurkat (T-cell acute lymphoblastic
or T-ALL), HL60 and MOLM13 (acute myeloid leukaemia or
AML)). In the performed experiments, nexturastat A was used as
a reference (Figure 4).
Compound 4b showed the highest efficacy with the lowest
IC50 (Figure 4, A) across all tested cell lines. In the AML cell lines
HL60 and MOLM13, 4b demonstrated IC50 values of 0.44 (�
0.024) μM and 0.11 (�0.014) μM, respectively. Against the other
HDAC6 is a major effector in the non-histone mediated
regulation of cellular processes and represents a valuable target
in the pharmacological intervention of immunological as well
as neurological diseases. In addition, HDAC6 selective inhibitors
remain valuable tools to explore the potential participation of
HDAC6 in tumorigenesis. In this study, we developed a
synthetic strategy for the synthesis of alkoxyurea based
hydroxamic acids that exhibited an up to 1.5-fold higher SI1/6
(4a) than the established HDAC6 selective inhibitor nexturasta-
t A, whilst maintaining its potency. Amongst the tested
inhibitors, 4b was identified as the inhibitor with the most
pronounce antiproliferative activity across a selection of AML
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