Multicomponent Synthesis and Binding Mode of Imidazo[1,2- a]pyridine-Capped Selective HDAC6 Inhibitors

Article abstract of DOI:10.1021/acs.orglett.8b01118

The multicomponent synthesis of a mini-library of histone deacetylase inhibitors with imidazo[1,2-a]pyridine-based cap groups is presented. The biological evaluation led to the discovery of the hit compound MAIP-032 as a selective HDAC6 inhibitor with promising anticancer activity. The X-ray structure of catalytic domain 2 from Danio rerio HDAC6 complexed with MAIP-032 revealed a monodentate zinc-binding mode.

Full text of DOI:10.1021/acs.orglett.8b01118

Letter
pubs.acs.org/OrgLett
Cite This: Org. Lett. XXXX, XXX, XXX−XXX
Multicomponent Synthesis and Binding Mode of
Imidazo[1,2‑a]pyridine-Capped Selective HDAC6 Inhibitors
Marcel K. W. Mackwitz,† Alexandra Hamacher,‡ Jeremy D. Osko,§ Jana Held,∥ Andrea Scholer,†
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David W. Christianson,§ Matthias U. Kassack,‡ and Finn K. Hansen*,†
†Institut fur Pharmazie, Universitat Leipzig, Bruderstraße 34, 04103 Leipzig, Germany
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‡Institut fur Pharmazeutische und Medizinische Chemie, Heinrich-Heine-Universitat Dusseldorf, Universitatsstr. 1, 40225 Dusseldorf,
Germany
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§Roy and Diana Vagelos Laboratories, Department of Chemistry, University of Pennsylvania, Philadelphia, Pennsylvania 19104-6323,
United States
∥Institut fur Tropenmedizin, Eberhard Karls Universitat Tubingen, Wilhelmstr. 27, 72074 Tubingen, Germany
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* Supporting Information
ABSTRACT: The multicomponent synthesis of a mini-library
of histone deacetylase inhibitors with imidazo[1,2-a]pyridine-
based cap groups is presented. The biological evaluation led to
the discovery of the hit compound MAIP-032 as a selective
HDAC6 inhibitor with promising anticancer activity. The X-ray
structure of catalytic domain 2 from Danio rerio HDAC6
complexed with MAIP-032 revealed a monodentate zinc-
binding mode.
he acetylation/deacetylation interplay is a key modification
multiple HDAC isoforms, leading to alteration of the acetylation
Tin human cells to control and regulate many important status of many different substrates, which can result in serious
unwanted side effects.3a,b,e Therefore, it has been hypothesized
biological processes, such as gene transcription and protein
functions.1 Histone deacetylases (HDACs) play an important
that isoform-selective HDACi may enable a more precise
regulation and, thus, offer decreased side effects.3a,e HDAC6
role in these posttranslational modifications by cleaving acetyl
modulates the function of many nonhistone proteins, including
α-tubulin, Hsp90, cortactin, and many more, thus participating in
numerous diseases.4 For instance, HDAC6 is highly expressed in
several cancer types such as oral squamous cell cancer, advanced
stage breast cancer, and acute myeloid leukemia, ensuring
migration of cancer cells and angiogenesis.3a,4 The knock out of
HDAC6 in mice produces a viable phenotype with no significant
defects, which may indicate an improved safety profile for
HDAC6 selective inhibitors.3a Furthermore, it has recently been
reported that HDAC6 differs distinctly in its structural features
compared to other isoforms, giving space for bulky rigid cap
groups.5 In addition, the structural analysis suggests that HDAC6
uses HDAC6-specific loops for the recognition of HDAC6-
selective substrates and inhibitors.5 Consequently, designing
HDAC6i seems to be more approachable compared to other
isoforms and several HDAC6 selective inhibitors have been
developed (Figure 1).4,5 In this context, we report here on the
rational design and synthesis of a series of preferential HDAC6
inhibitors containing imidazo[1,2-a]pyridine-based cap groups.
Selective HDAC6 inhibitors typically possess a rigid (hetero)-
cyclic or branched cap group combined with a short benzyl or 4-
groups from histone and nonhistone proteins possessing N-
acetyl-lysine residues.2 HDACs are also involved in many
diseases such as cancer, neurodegenerative diseases, and
inflammation, making these enzymes promising targets for
therapeutic approaches.3 Classical HDAC inhibitors (HDACi)
can be described by a widely accepted cap-linker-chelator
pharmacophore model (Figure 1).3b To date, there are four
FDA-approved anticancer HDACi (vorinostat, belinostat,
romidepsin, and panobinostat).3b These inhibitors target
Figure 1. Selected HDAC6 preferential inhibitors and the imidazo[1,2-
a]pyridine-based target compounds.
Received: April 9, 2018
© XXXX American Chemical Society
A
DOI: 10.1021/acs.orglett.8b01118
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Letter
aminophenyl linker (Figure 1).4,5 In this project, we rationally
designed bicyclic imidazo[1,2-a]pyridine-based hydroxamic
acids (Figure 1) and first investigated the potential of these
new compounds to inhibit HDAC6 by molecular docking
utilizing AutoDock 4.2.6 Docking studies were performed in the
active site of the catalytic domain 2 of human HDAC6 (PDB ID:
5EDU) using an aryl-substituted (4a), alkyl-substituted (4l), and
unsubstituted (4i) ligand as representative examples (see Figure
S1, Supporting Information (SI)). All three ligands were found to
fit smoothly in the active site, and we thus decided to synthesize a
mini-library of imidazo[1,2-a]pyridine-capped HDACi. The
target compounds 4a−m are accessible by the Groebke−
Table 1. Activities of 4a−m, Vorinostat, HPOB, and
Tubastatin A against HDAC1 and HDAC6
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Blackburn−Bienayme three-component reaction as the key
step, followed by hydroxylaminolysis (Scheme 1).7 In addition to
Scheme 1. Synthesis of Imidazo[1,2-a]pyridine-Based HDACi
4a−m
the widely commercially available aldehydes and 2-amino-
pyridines, the isocyanide 2 is required for the synthesis of our
target compounds. Thus, we first synthesized isocyanide 2 in a
two-step synthesis, starting with the formamidation of methyl 4-
aminobenzoate, followed by dehydration (2, SI). Subsequently,
we optimized the preparative conditions for the Groebke−
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Blackburn−Bienayme three-component reaction using inter-
mediate 3a (R1 = 4-Me2N-Ph; R2 = H) as model compound. We
investigated different catalysts, solvents, temperatures, and
reaction times that are described in the literature to identify an
efficient procedure for the synthesis of our target compounds.7
The best result was achieved by microwave irradiation (150 W)
at 85 °C for 3 h with acetic acid as the catalyst and methanol as
the solvent with 52% yield for the model compound 3a (see SI).
Furthermore, we were able to synthesize the intermediate 3a
using an isocyanide-less protocol (see SI, 39% yield) adapting the
a
Selectivity factor (SF = IC50 (HDAC1)/IC50 (HDAC6)).
recently published method by Domling and co-workers.8 Due to
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the slightly higher yield and the efficient access to 2, we decided
to use the classical isocyanide-based protocol for the synthesis of
esters 3a−m (Scheme 1). For the synthesis of the 2-
unsubstituted esters 3i−k, glyoxylic acid was used as a
formaldehyde source.7c Pure compounds were obtained either
by simple filtration of the crude reaction mixture, if crystallization
of the products occurred, or by flash column chromatography in
33−73% yields. Although the possible formation of regioisomers
the nature of substituent R1 seems to be important for the
selectivity profile. Aryl-substituted compounds revealed either
moderate (4b−c; selectivity factor (SF) ≥ 10) or no noteworthy
(4a,e−h; SF < 10) preference over HDAC1, depending on
further substitutions on the phenyl residue. In particular,
compounds with a 4-dimethylamino-substituted aryl ring tend
to be nonselective inhibitors (see compounds 4a and 4e).
Compounds 4i−k with no R1 substitution displayed moderate
preference with selectivity factors of 11−15. Interestingly,
compounds 4l and 4m, which bear an alkyl group as the R1
substituent, showed the highest preference over HDAC1.
Compound 4l exhibited the best selectivity factor (SF = 38),
comparable to other well-known preferential HDAC6 inhibitors
such as HPOB (SF = 25). In order to evaluate its selectivity
profile further, 4l was selected for a screening against the
remaining class I isoforms HDAC2, HDAC3, and HDAC8.
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during the Groebke−Blackburn−Bienayme three-component
reaction has been reported, the desired regioselective formation
of imidazo[1,2-a]pyridines 3a−m was confirmed for selected
compounds through 2D-NOESY-NMR (Figure S2, SI).7b The
final step of the synthesis included the hydroxylaminolysis of the
esters, which was accomplished in 43−90% yield (4a−m) using
an excess of aqueous hydroxylamine solution in the presence of
sodium hydroxide in methanol/dichloromethane (3:1).
All synthesized hydroxamic acids were evaluated in regards to
their inhibitory activity in a biochemical assay against HDAC1
and HDAC6 using ZMAL (Z-Lys(Ac)-AMC)9 as a substrate, as
previously published (Table 1).10 First, the HDAC isoform
profiling revealed that the majority of compounds showed potent
double-digit nanomolar activity against HDAC6. In particular,
Strikingly, 4l was inactive against HDAC2 and HDAC3 (IC50 >
10 μM) and displayed only moderate activity against HDAC8
(IC50: 1.58 0.21 μM; SFHDAC8/HDAC6: 27).
In addition, the preferential inhibition of HDAC6 was studied
by Western blotting experiments using the human tongue
squamous cell carcinoma cell line Cal27 (Figure S3, SI).
B
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Compounds 4a and 4l were selected as representative
compounds for aryl and alkyl (R1) substituted derivatives,
whereas the pan-inhibitor vorinostat, the HDAC6 selective
HDACi nexturastat, and tubastatin A were used as reference
compounds. As expected, vorinostat induced an increase in
acetylation of both α-tubulin and histone H3 compared to the
control, indicating the inhibition of HDAC6 and class I HDACs.
In contrast, compounds 4a, 4l, nexturastat, and tubastatin A
showed preferential hyperacetylation of α-tubulin. Notably,
compound 4l only induced the acetylation of α-tubulin but not of
histone H3, demonstrating that its selective HDAC6 inhibition is
retained in a cellular environment.
substrate acetyl-L-lysine.5a Since S531 is unique to HDAC6, a
hydrogen bond with this residue will contribute to HDAC6
inhibitor selectivity. The pendant propyl substituent on the
capping group of 4l is oriented toward P464, and the bicyclic
aromatic ring of the capping group is accordingly oriented in the
opposite direction toward F643 (Figure 2). Interestingly,
aromatic capping groups of many hydroxamate inhibitors are
observed to bind adjacent to P464.5a,c However, the region
occupied by the bicyclic aromatic ring of 4l can be exploited
further in the design of HDAC6-selective inhibitors, due to the
region being occupied by HDAC6 bound substrates. We note
that the imidazo nitrogen atom in the heteroaromatic ring of the
capping group is presumably protonated, since it is within
hydrogen bonding distance to the C-terminal carboxylate of
R798 from another HDAC6 molecule in the crystal lattice
(Figure 2). Although this interaction is poorly oriented for a
hydrogen bond, it could still comprise an electrostatic
interaction. Since this interaction could conceivably occur
regardless of the orientation of the capping group of 4l, this
interaction is unlikely to significantly influence the binding
conformation of 4l.
To investigate the binding mode of 4l, cocrystallization
experiments were performed with the CD2 domain of Danio rerio
HDAC6 (henceforth, simply “HDAC6”). The 2.50 Å-resolution
crystal structure of the HDAC6−4l complex (Figure 2) reveals
To explore the biological activity on a cellular level, all
compounds were tested for antiplasmodial as well as anticancer
activity (Table S1, SI and Table 2). Besides their promising
potency, antiplasmodial HDACi are often disadvantageous as a
result of their toxicity against human cells. Therefore, we, and
others, hypothesized that selective human HDAC6 inhibitors
might be a better starting point for the development of parasite-
selective antiplasmodial HDACi due to a usually lower
toxicity.10,11 Compared to the reference HDACi vorinostat
(Pf 3D7 IC50: 0.209 μM; Pf Dd2 IC50: 0.297 μM), the majority of
compounds revealed only moderate activity against the drug
sensitive 3D7 (Pf 3D7 IC50: 0.9−1.9 μM) and the multidrug
resistant Dd2 (Pf Dd2 IC50: 1.4−6.8 μM) strain. Nonetheless,
compounds 4e (0.524 μM) and 4m (0.517 μM) showed
promising submicromolar antiplasmodial activity against the
3D7 strain.
Next, all synthesized HDACi 4a−m were assessed in a whole
cell HDAC assay using the cell line Cal27 and the class I/IIb
selective substrate Boc-Lys(Ac)-AMC (Table 2). All com-
pounds, except 4g and 4h, exhibited HDAC inhibition in the
whole cell assay, thus confirming the inhibition of cellular histone
deacetylase activity. In agreement with the cellular HDAC data,
all compounds, except 4g and 4h, showed encouraging
cytotoxicity in a MTT cytotoxicity assay against Cal27 cells
with IC50 values ranging from 3.22 to 11.9 μM (Table 2). The
highest anticancer activity was observed for compounds 4a, 4e,
4j, 4l, and 4m (IC50: 3−4 μM). Flow cytometric analysis using
propidium iodide (PI) staining showed that the cytotoxic effect
was mediated by induction of apoptosis (Figure S5, SI).
Compounds 4a and 4l were chosen as representative HDACi.
Both compounds increased the amount of apoptotic nuclei in a
concentration-dependent manner after 48 h of incubation.
Notably, the apoptotic effect was more pronounced for the alkyl
substituted HDACi 4l, even at 1 μM, which is approximately 4-
fold below IC50 of the MTT assay.
Figure 2. Polder omit maps (stereoview, contoured at 3.0 σ each) for 4l
(green) and the Zn2+-bound water molecule (magenta) in the active site
of HDAC6. Atom color codes are as follows: C = orange (4l) or light
blue (protein), N = blue, O = red; Zn2+ appears as a gray sphere, and the
Zn2+-bound water molecule is shown as a small red sphere. Metal
coordination and hydrogen bond interactions are indicated by solid and
dashed black lines, respectively.
no major conformational changes between the inhibitor-bound
and unliganded states of the enzyme, and the root-mean square
deviation is 0.20 Å for 311 Cα atoms between the two structures
(unliganded HDAC6, PDB accession code 5EEM) (Figure S4,
SI). The catalytic Zn2+ ion exhibits square pyramidal
coordination geometry, with D612, D705, H2O, and the ionized
hydroxamate N−O− group serving as equatorial ligands and
H614 serving as an apical ligand (coordination distance range
2.0−2.4 Å). Several hydrogen bond interactions stabilize the
bound inhibitor: the Zn2+-bound hydroxamate N−O− group
accepts a hydrogen bond from Y745, the Zn2+-bound water
molecule forms hydrogen bonds with H573 and H574, and the
hydroxamate carbonyl group accepts a hydrogen bond from the
Zn2+-bound water molecule. Interestingly, the monodentate
hydroxamate-Zn2+ coordination mode observed for 4l is also
observed for other sterically bulky inhibitors such as HPOB,
HPB, and ACY-1083.5a,c The monodentate hydroxamate-Zn2+
coordination mode is only 0.5 kcal/mol less stable than the
canonical bidentate hydroxamate-Zn2+ coordination mode
observed for inhibitors with less steric bulk adjacent to the
hydroxamate moiety, such as ricolinostat.5c The aromatic ring of
the phenylhydroxamate is nestled in an aromatic crevice formed
by F583 and F643. The para-substituted secondary amino group
of 4l forms a hydrogen bond with S531 on the L2 loop (N−O
separation = 3.1 Å).
In conclusion, we have developed a multicomponent approach
for the synthesis of a mini-library of novel imidazo[1,2-
a]pyridine-based HDAC6i. Most notably, we show that 4l,
hereafter named MAIP-032, is a selective HDAC6 inhibitor with
promising anticancer activity. In addition, the crystal structure of
MAIP-032 bound to the second catalytic domain of zebrafish
HDAC6 demonstrates a monodentate binding mode. Taken
S531 plays an important role in substrate binding to HDAC6
by accepting a hydrogen bond from the backbone NH group of
C
DOI: 10.1021/acs.orglett.8b01118
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Organic Letters
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ORCID
Table 2. Whole-Cell HDAC Inhibition (HDAC) and
Cytotoxic Activity (MTT) against the Human Tongue
Squamous Cell Carcinoma Cell Line Cal27 of 4a−m,
Vorinostat, Nexturastat A, and Tubastatin A
David W. Christianson: 0000-0002-0194-5212
Finn K. Hansen: 0000-0001-9765-5975
Notes
The authors declare no competing financial interest.
ACKNOWLEDGMENTS
■
We thank Nicholas Porter of the University of Pennsylvania for
helpful scientific discussions. Additionally, we thank Tzanko
Doukov, Ana Gonzalez, and the beamline 12-2 staff at the
Stanford Synchrotron Radiation Lightsource (SSRL), SLAC
National Accelerator Laboratory, for assistance with data
collection. SLAC is supported by the U.S. Department of Energy
(DOE), Office of Science, Office of Basic Energy Sciences under
Contract No. DE-AC02-76SF00515. The SSRL Structural
Molecular Biology Program is supported by the DOE Office of
Biological and Environmental Research, and by the National
Institutes of Health, National Institute of General Medical
Sciences (Grant P41GM103393). This research was supported
by NIH Grant GM49758 to D.W.C. and by the Deutsche
Forschungsgemeinschaft (DFG) (HA 7783/1-1 to F.K.H. and
HE 7607/1-1 to J.H.).
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ASSOCIATED CONTENT
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AUTHOR INFORMATION
Corresponding Author
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*E-mail: finn.hansen@uni-leipzig.de.
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DOI: 10.1021/acs.orglett.8b01118
Org. Lett. XXXX, XXX, XXX−XXX