Synthesis and Biological Investigation of Oxazole Hydroxamates as Highly Selective Histone Deacetylase 6 (HDAC6) Inhibitors

Article abstract of DOI:10.1021/acs.jmedchem.5b01493

Histone deacetylase 6 (HDAC6) catalyzes the removal of an acetyl group from lysine residues of several non-histone proteins. Here we report the preparation of thiazole-, oxazole-, and oxadiazole-containing biarylhydroxamic acids by a short synthetic proce

Full text of DOI:10.1021/acs.jmedchem.5b01493

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Article
Synthesis and biological investigation of oxazole hydroxamates
as highly selective histone deacetylase 6 (HDAC6) inhibitors
Johanna Senger, Jelena Melesina, Martin Marek, Christophe
Romier, Ina Oehme, Olaf Witt, Wolfgang Sippl, and Manfred Jung
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01493 • Publication Date (Web): 10 Dec 2015
Downloaded from http://pubs.acs.org on December 12, 2015
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Synthesis and biological investigation of oxazole
hydroxamates as highly selective histone deacetylase
6 (HDAC6) inhibitors
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Johanna Senger†, Jelena Melesina‡, Martin Marek§, Christophe Romier§, Ina Oehme┴, Olaf
Witt┴, Wolfgang Sippl‡ and Manfred Jung*,†
†Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Albertstraße 25,
79104 Freiburg, Germany
‡Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Wolfgang-Langenbeck-
Straße 4, 06120 Halle (Saale), Germany
§Département de Biologie Structurale Intégrative, Institut de Génétique et Biologie Moléculaire
et Cellulaire (IGBMC), Université de Strasbourg (UDS), CNRS, INSERM, 1 rue Laurent Fries,
67404 Illkirch Cedex, France
┴Clinical Cooperation Unit Pediatric Oncology, German Cancer Research Centre (DKFZ), Im
Neuenheimer Feld 280, 69120 Heidelberg, Germany
*Corresponding author: Jung, M. (manfred.jung@pharmazie.uni-freiburg.de)
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ABSTRACT. Histone deacetylase 6 (HDAC6) catalyzes the removal of an acetyl group from
lysine residues of several non-histone proteins. Here we report the preparation of thiazole-,
oxazole- and oxadiazole-containing biaryl hydroxamic acids by a short synthetic procedure. We
identified them as selective HDAC6 inhibitors by investigating the inhibition of recombinant
HDAC enzymes and the protein acetylation in cells by western blotting (tubulin vs. histone
acetylation). The most active compounds exhibited nanomolar potency and high selectivity for
HDAC6. For example an oxazole hydroxamate inhibits HDAC6 with an IC50 of 59 nM and has a
selectivity index of >200 against HDAC1 and HDAC8. This is the first report showing that the
nature of a heterocycle directly connected to a zinc binding group (ZBG) can be used to
modulate subtype selectivity and potency for HDAC6 inhibitors to such an extent. We rationalize
the high potency and selectivity of the oxazoles by molecular modelling and docking.
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INTRODUCTION
Protein lysine acetylation is a reversible posttranslational modification which is balanced by
histone deacetylases (HDACs) and histone acetyltransferases (HATs). Histone deacetylase 6
(HDAC6) catalyzes the cleavage of the acetyl group of ε-amino groups of lysine residues in
proteins.1 The substrates of HDAC6 are mainly non-histone substrates as tubulin2, cortactin3,
HSP904 and other proteins.5-6 HDAC6 can shuttle between cytoplasm and nucleus but is mainly
localized in cytoplasm where it is associated with microtubules.6 In nucleus it was found in
protein complexes with HDAC11.7 It consists of 1216 aa and is the only HDAC which has two
functional catalytic domains8. Additionally there is a nuclear export signal (NES) and a serine-
glutamic acid-containing tetradecapeptide (SE14) which implicate the cytoplasmic arrest and a
C-terminal zinc-finger domain which binds ubiquitinated proteins.1 With the help of this domain
it is involved in the process of misfolded protein degradation and represents a promising target
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for the cotreatment with proteasome inhibitors to avoid the appearance of drug resistance.9
Additionally it is involved in many other cellular processes as cell motility, cell adhesion, cell
migration, growth of cells, immune synapse formation and stress granule formation.10 HDAC6
was found to be highly expressed in testis, as well as in liver, kidney and brain.11 Selective
inhibition of HDAC6 has arisen as a promising target for a wide range of diseases. It was shown
to have influences on several kinds of neurological diseases as Alzheimer’s disease12,
Huntington’s disease13-14 and Parkinson’s disease15. Furthermore overexpression of HDAC6 is
associated with the appearance of several types of cancer, solid cancers as well as hematological
malignancies,16 polycystic kidney disease17 and other diseases.5 As pan HDAC inhibitors often
cause severe side effects, the development of selective HDAC6 inhibitors is of great interest
because they are less toxic against normal cells and therefore are thought to have an improved
safety profile.11
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Several selective HDAC6 inhibitors have been reported (Chart 1).5 Tubacin was discovered in
the scope of a chemical genetics screen and was shown to inhibit cell motility.18 Because of its
high lipophilicity, it was not applicable as a drug.5 Tubastatin A is a carbazole hydroxamic acid
with an IC50 of 15 nM for HDAC6. It was shown to exhibit anti-hepatitis C virus activity19 and to
have neuroprotective effects without displaying the neuronal toxicity of unselective HDAC
inhibitors.18, 20 In addition it was demonstrated to have anti-inflammatory and anti-rheumatic
effects.21 Ricolinostat is a very potent but only moderately selective HDAC6 inhibitor with 10-
12fold selectivity towards class I HDAC enzymes. It is part of phase I/II clinical trials in
combination with dexamethasone and either bortezomib, lenalidomide or pomalidomide for the
treatment of multiple myeloma.22 A series of biaryl-23 and pyridylalanine24 containing
hydroxamic acids was reported to inhibit HDAC6 selectively. Additionally the pyridyl
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compounds showed synergistic antiproliferative effects in combination with bortezomib in breast
cancer cells. Nexturastat A, a hydroxamic acid containing inhibitor with a substituted urea linker
was demonstrated to exhibit low nanomolar potency on HDAC6 and very high selectivity
(>600fold) towards HDAC1. It inhibited the growth of B16 melanoma cells and was shown to be
the first HDAC6 inhibitor that possessed antiproliferative effects against melanoma cells.25
ACY775 has a similar potency and selectivity profile as Tubastatin A but was shown to have a
better brain bioavailability. It showed antidepressant effects which were linked to HDAC6
inhibition.26 A common theme among many selective HDAC6 inhibitors is that they achieve
their subtype selectivity by a large ring or a branched respectively Y-shaped structure and the
point of branching is further away from the zinc binding hydroxamate, usually at the rim of the
catalytic tunnel25. However, the fragment-like cyclopentenyl hydroxamate27 also showed very
good HDAC6 selectivity.
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Based on initial data28 we wanted to get insights into the structure-activity relationships (SAR)
of HDAC6 inhibitors with a biaryl hydroxamate structure that do not have such a branching.
Here we report that changes of the heterocycle attached to the hydroxamate have a huge impact
on HDAC6 potency and selectivity and report oxazoles as such potent and selective inhibitors in-
vitro and in cell culture.
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Chart 1. Structures of selective HDAC6 inhibitors
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RESULTS AND DISCUSSION
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2-(Pyridin-3-yl)-1,3-thiazole-4-carbohydroxamic acid (1) was identified by virtual screening as
a selective HDAC6 inhibitor.28 Based on this structure, we set out for structure-activity
relationships to improve selectivity and inhibitory potency.
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Chart 2. Lead structure (1)28
Chemistry. The first modification was the introduction of different aryl groups at the thiazole
of the lead structure (Scheme 1) to get an overview on their impact on HDAC6 inhibition. The
synthesis consisted of three steps: the formation of the thiazole, the cleavage of the ester and the
conversion into the hydroxamic acid. In order to prepare the thiazole heterocycle, ethyl
bromopyruvate and an amide were refluxed in ethanol for about 4 h. The thiazole ethylester was
hydrolyzed with 1 M LiOH in THF and in the last step the carboxylic acid was activated with
ethyl chloroformate and N-methylmorpholine to the mixed anhydride, which was converted in
situ to the hydroxamic acid with hydroxylamine base. The latter was freshly prepared from
hydroxylamine hydrochloride with KOH in methanol.29
Scheme 1. Synthesis of thiazole derivativesa,28
aReagents and conditions: a) EtOH, 70 °C b) 1 M LiOH, THF/H2O, RT c) C3H6ClO2, N-MM,
dry THF, 0 °C d) NH2OH, MeOH, RT29.
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In a second series we then tended to the modification of the heterocycle. The synthesis of
oxazoles was performed in two different ways and is described in Scheme 2. Due to the less
nucleophilic oxygen the reaction had to be run under microwave irradiation and adding silver
hexafluoroantimonate as a catalyst.30 Because of the limitation to electron withdrawing amides,
another synthesis strategy was performed additionally, the Suzuki coupling. The starting material
consisted of a 2-bromo-oxazole and a boronic acid with different substituents. With this method
it was possible to introduce a variety of different aryl groups.
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Scheme 2. Synthesis of oxazole derivativesa
aReagents and conditions: a1) µw, AgSbF6, dry DCM, 90 °C,30 1 h, a2) Pd(PPh3)4, Na2CO3,
toluene, EtOH, 120 °C, b) 1 M LiOH, THF/H2O, RT, c) C3H6ClO2, N-MM, dry THF, 0 °C, d)
NH2OH, MeOH, RT29.
The third heterocycle that was introduced was an oxadiazole (Scheme 3). A carboxylic acid
was activated to the acylchloride followed by the condensation with ethyl
aminohydroxyiminoacetate31 (5) and the formation of the hydroxamic acid as described above.
Scheme 3. Synthesis of oxadiazole derivativesa
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OH
N
O
O
5
6
NH2
5
O
H
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O
O
O
a)
b)
5
R
N
O
R
OH
R
O
Cl
O
N
OH
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O
c)
d)
N
N
OH
R
R
N
O
N
O
3s-u
2s-u
O
e)
f)
OH
N
N
H
R
N
O
4s-u
aReagents and conditions: a) SOCl2, 90 °C b) Et3N, DCM c) 140 °C, DMF, 4 h31 d) 1 M LiOH,
THF/H2O, RT e) C3H6ClO2, N-MM, dry THF, 0 °C f) NH2OH, MeOH, RT29.
Inhibition of hHDAC1, hHDAC6 and hHDAC8. All the synthesized compounds were
evaluated for their inhibitory activity against human HDAC6, HDAC1 and HDAC8 in a
biochemical In-vitro-assay.32 The data is summarized in Table 1. Vorinostat (SAHA)33 was
tested as a pan-HDAC reference inhibitor and cyclopentenyl hydroxamate was tested to compare
the published values27 with the ones obtained in our assay. Results are always depending on the
assay and only comparable within one test system. It turned out to be 3-5 times less potent and
also less selective for HDAC6 over HDAC1 when tested in our system. These outcomes indicate
that our compounds are at least in our system more selective HDAC6 inhibitors in comparison to
cyclopentenyl hydroxamate.
Regarding the thiazole-containing compounds 4a-e they all showed an inhibitory effect on
HDAC6 in the low micromolar region. Compared to the potency on HDAC1 and HDAC8 they
were slightly more active on HDAC6. Compared to SAHA they were a bit more selective against
HDAC1 but equally as or less selective against HDAC8.
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In contrast, introduction of the oxazole heterocycle 4f-r led to a huge increase in potency and
selectivity. Exceptions to that trend are 4i with a diphenylmethyl-group, 4k with an ortho-
substituted phenyl-residue and 4r with a meta-substituted phenyl-residue that diverge from the
more linear structure of the more potent analogues. They were in the inhibitory range of the
thiazole compounds 4a-e and showed approximately the same selectivity profile. All the other
oxazole compounds exhibited a HDAC6 inhibition in the lower nanomolar range and showed a
huge increase in selectivity towards HDAC1 and HDAC8, all of them more than 100fold.
Regarding the substitution pattern of these potent and selective compounds the phenyl residue
was substituted in para-position which seemed to be an important factor to gain selectivity. The
results of compounds 4g with a para-bromo-phenyl-residue and 4l, 4m with a para-halogen-
phenyl-residue and 4h with a 4-biphenyl-residue are prominent examples for this. They all
exhibited an IC50 on HDAC6 in the low nanomolar range (≤100 nM) and showed a very high
selectivity towards HDAC1 and HDAC8 (from 200-400fold). There was a distinct difference
between the varying heterocycles in the molecule. The thiazoles 4a-e were much less potent and
selective than the oxazole containing compounds. For the oxadiazoles 4s-u the results were
somewhat less consistent. The oxadiazole with a phenyl-residue 4s was very potent and even
more selective than the comparable oxazole compound 4f. But in case of the oxadiazole, the
increase of the aryl residue with a substitution in para-position did not lead to an increase but
rather to loss in potency. Thus, the different factors that govern potency and selectivity don´t add
in a strict additive manner.
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Table 1. In vitro inhibition of HDAC1, HDAC6 and HDAC8
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General structure:
hHDAC1a hHDAC6a hHDAC8a SF
SF
compound
R=
X
Y
IC50 [µM] IC50 [µM] IC50 [µM] 6/1b 6/8c
36% @
0.30 28
n.t.
>50
-.
1
10 µM28
0.119
0.053
0.147
1.89
n.t.
2
36
-
SAHA
1.90
13
21
Cyclopentenyl
hydroxamate27
0.63827
82.25
0.03027
8.85
1.0927
61.42
363
S
C
9
7
4a
S
S
C
C
17.46
83.58
1.06
2.54
36.36
35.00
16
33
34
14
4b
4c
S
C
47.66
3.69
12.86
13
3
4d
S
O
O
C
C
C
10.27
29.87
14.47
1.91
0.270
0.059
12.12
41.90
14.37
5
6
4e
4f
111
245
155
244
4g
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O
O
C
C
29.48
93.89
0.089
9.37
27.33
2.70
331
10
307
0.3
4h
4i
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O
O
C
C
36.51
22.63
0.311
2.18
8.13
117
10
26
9
4j
20.24
4k
O
O
O
C
C
C
19.56
23.75
32.89
0.081
0.110
0.195
30.76
43.35
50.94
241
216
128
380
394
108
4l
4m
4n
F
O
O
C
C
12.21
22.47
0.089
0.182
26.16
29.08
137
123
294
160
4o
4p
O
C
40.07
0.313
33.89
169
261
4q
O
O
C
N
19.08
25.00
0.500
0.112
10.08
38.50
38
20
4r
4s
223
344
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O
O
N
N
33.45
14.87
2.77
1.31
20.04
12
11
7
4t
62% @
100 µM
>60
4u
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aIC50 values are the means of at least two experiments. Standard deviation is <10% of the IC50.
bSF6/1: selectivity factor for HDAC6 over HDAC1 (SF6/1 = IC50 HDAC1/IC50 HDAC6),
cSF6/8: selectivity factor for HDAC6 over HDAC8 (SF6/8 = IC50 HDAC8/IC50 HDAC6)
Ligand efficiency (LE) was determined of all the compounds for HDAC6 to evaluate the
possibility for future improvement. LE is defined as the relative free binding energy in kcal/mol
per number of heavy atoms (HA). It is a useful tool to predict whether the potency of a
compound derives from optimal fit with the target protein or just by making many contacts.34
Table 2. IC50 values [µM] and ligand efficiencies (LE) [kcal/mol/non-H-atom] on HDAC6
Tubastatin
A
Compd
HA
4a
4b
4c
4d
4e
4f
4g
4h
4i
4j
25
15
16
16
22
15
15
16
21
22
14
IC50
HDAC6
LE
0.01520
0.44
8.85 1.06 2.54 3.69 1.91 0.270 0.059 0.089 9.37 0.311
0.48 0.53 0.50 0.35 0.54 0.62 0.64 0.48 0.32 0.66
HDAC6
Compd
HA
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
4u
16
16
16
17
19
21
16
21
16
17
21
IC50
2.18 0.081 0.110 0.195 0.089 0.182 0.313 0.500 0.112 2.77 1.31
HDAC6
LE
HDAC6
0.50 0.63 0.62 0.56 0.53
0.46 0.58
0.43 0.62 0.46 0.40
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Regarding the compounds with the highest ligand efficiency value, six compounds had a LE of
>0.6. 4g, 4l and 4m were the halogen-substituted compounds that are also very potent. Two
compounds (4f and 4j) are not among the most potent ones but still have high LE values because
of their low molecular weight. Tubastatin A, a known selective HDAC6-inhibitor shows a LE-
value of 0.44. Compared to this, several compounds had a higher LE and could therefore be
further enlarged to improve their potency.
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Docking study. To rationalize the inhibitory activities for HDAC1, 6 and 8 a docking study
was performed. In case of HDAC1 and HDAC8 available X-ray structures were used, whereas in
case of HDAC6 a homology model was generated as described in the methods part. All
compounds show similar docking poses in the three target proteins: the hydroxamic acid group is
coordinating the zinc ion and hydrogen-bonded to a conserved tyrosine residue (Y303 in
HDAC1, Y782 in HDAC6 and Y306 in HDAC8). The thiazole, oxazole and oxadiazole linker is
located in the acetyl lysine tunnel interacting with two conserved aromatic residues observed in
all three HDACs (F150 and F205 in HDAC1, F620 and F680 in HDAC6, F152 and F208 in
HDAC8). The nitrogen atom of the heteroaromatic ring is located nearby the conserved tyrosine
of the catalytic site. The aromatic capping group is placed between a conserved aspartate and a
hydrophobic residue that is different in the three isoforms (Leu271 in HDAC1, Leu749 in
HDAC6, and Met272 in HDAC8). The suggested binding mode of 4b and 4g is shown
exemplarily in Figure 1.
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a)
b)
c)
Figure 1. Docking poses of HDAC inhibitors 4b (orange) and 4g (cyan) in HDAC6 (a), HDAC1
(b) and HDAC8 (c). The zinc ion is shown as cyan ball, Interactions between the hydroxamic
acid and the zinc ion are shown as green dashed lines. Distances are given in Å.
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Due to the more narrow binding tunnel observed in HDAC1 the hydroxamic acid is not able to
make a perfect coordination with the zinc ion which might explain the low inhibitory activity of
thiazoles, oxazoles as well as oxadiazoles (Figure 1). It has been already suggested by Butler et
al. that HDAC1 has a more narrow binding tunnel than HDAC6.20 In case of HDAC6 and
HDAC8 the aromatic hydroxamic acid group is able to coordinate perfectly with the zinc ion.
However, in case of HDAC8 the substituted phenyl ring is sticking out of the pocket whereas in
HDAC6 the phenyl ring is interacting with residues from the loop Asp499-Pro501 that is not
observed in the HDAC8 structure. In addition the shape of the HDAC6 pocket is different
compared to HDAC8 mainly due to the substitution of Leu789 by Met272 in HDAC8.
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To understand the selectivity of the oxazoles compared to thiazole derivatives for HDAC6 we
compared the docking results of corresponding inhibitors pairs (e.g. 4b and 4g). Due to the more
bulky sulphur in the thiazole ring and the different ring geometry the orientation of the
bromophenyl ring is different for both inhibitors. In case of the oxazole the bromo (4g) or phenyl
(4h) group is interacting with Phe566 and Phe520 (Figure 1a and 3) whereas in case of the
thiazole (4b) the bromophenyl ring is not in close proximity to both residues. This might explain
the high HDAC6 selectivity of para-substituted phenyl-oxazoles. For the oxadiazole based
inhibitors similar docking poses were obtained as for the oxazoles. Here, a structural explanation
for the decreased selectivity is missing. However, it should be taken into account that an
interplay between two catalytic pockets of HDAC6 contributes to the final binding affinity of
ligands, which makes it difficult to model these effects. Furthermore, absence of an X-ray
structure of HDAC6 introduces additional uncertainty to this problem.
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Figure 2. Comparison of HDAC6 and HDAC8 with docked inhibitor 4g (cyan). In case of
HDAC6 (brown ribbon) the bromophenyl ring of the inhibitor is stabilized by residues from an
additional loop (D497-P501, shown in orange) that is not observed in HDAC8 (green ribbon).
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Figure 3. Molecular surface of the HDAC6 binding pocket. The orientation of two docked
inhibitors, thiazole 4b (orange) and oxazole 4g (cyan) is shown. Only for the oxazole the phenyl
ring is close to Phe520 and the bromo group in close proximity to Phe566. The surface is colored
according to the lipophilicty (magenta = polar, green = hydrophobic).
Cellular data. To confirm the results obtained in the In-vitro-assay, western blotting
experiments were performed with two of the best compounds (4g and 4h) in two different cell
lines. The pan-HDAC inhibitor SAHA was used as a reference compound. HeLa and HL60 cells
were incubated for 4 hours with SAHA, 4g or 4h in three different concentrations (10 µM, 1 µM
and 0.1 µM). Cell lysates were blotted against Ac-α-tubulin, Ac-histone H3 and GAPDH. As
seen clearly in Figures 4 and 5 the pan-HDAC inhibitor SAHA induced both increase of tubulin
acetylation and of histone acetylation in HeLa cells as well as in HL60 cells compared to DMSO
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control. 4g and 4h in contrast only induced the acetylation of tubulin but not of histone H3
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indicating that the selectivity observed in vitro is also retained in the cellular setting.
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Figure 4. Western blots of acetyl-α-tubulin, acetyl-H3 and GAPDH after treatment of HeLa cells
with SAHA, 4g and 4h. GAPDH was the loading control, DMSO the negative control.
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Figure 5. Western blots of acetyl-α-tubulin, acetyl-H3 and GAPDH after treatment of HL60
cells with SAHA, 4g and 4h. GAPDH was the loading control, DMSO the negative control.
Because of varying GAPDH levels the western blots were quantified simply by determining the
ratio of acetylated tubulin versus GAPDH and of acetylated histone 3 versus GAPDH (table 3).
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Table 3. Quantification of western blots in HeLa and HL60 cells
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HeLa cells
(Figure 4, WB 1)
HL60 cells
(Figure 5, WB 1)
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Ac
Ac
Com-
pound
Ac H3 /
tubulin /
GAPDH
GAPDH
Selectivity
Indexa
Ac H3 /
tubulin /
GAPDH
GAPDH
Selectivity
Indexa
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DMSO
1.00b
1.00b
-
1.00b
1.00b
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SAHA
10 µM
8.99
9.34
5.86
3.45
0.77
1.76
4.34
0.56
1.53
18.36
3.50
5.25
SAHA
1 µM
2.71
4.69
9.11
3.72
1.71
14.23
2.59
2.94
1.77
1.32
1.14
1.11
4.84
1.46
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1.33
0.76
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3.61
4g
10 µM
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16.13
0.96
12.39
1.52
4g
1 µM
4g
0.1 µM
0.84
HeLa cells
(Figure 4, WB 2)
HL60 cells
(Figure 5, WB 2)
DMSO
1.00 a
1.00 a
-
1.00 a
1.00 a
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SAHA
10 µM
6.89
3.79
1.82
97.38
4.83
20.16
SAHA
1 µM
5.10
1.89
7.03
8.99
1.56
2.01
0.59
0.83
1.61
0.51
2.54
3.20
8.47
5.58
3.06
51.53
5.49
5.39
1.94
2.10
1.46
1.11
9.56
2.83
29.05
9.29
3.18
SAHA
0.1 µM
4h
10 µM
61.01
13.56
3.53
4h
1 µM
4h
0.1 µM
aSelectivity index defined as quotient of Ac Tub/GAPDH divided by Ac H3/GAPDH.
bAll values have been normalized to DMSO.
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It can be seen that 4g and 4h induced the acetylation of tubulin at concentrations of 10 µM and
1 µM to a greater extent as compared to the acetylation of histone H3. In comparison to SAHA
the accumulation of acetylated tubulin versus acetylated histone H3 is more pronounced,
especially at the higher concentrations. This indicated and confirmed the HDAC6 selectivity of
these compounds also on a cellular level.
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Similar results were obtained with western blot experiments in the neuroblastoma cell line
BE(2)-C. Cell lysates of 4b and 4g were blotted against acetyl-tubulin, acetyl-histone H4 and
actin as a loading control (see Figure S1). It could be clearly seen that the acetylation of tubulin
with 10 µM of 4g was induced stronger than the acetylation with 10 µM of 4b.
This confirmed the results obtained in the in-vitro testing where activity and selectivity of the
oxazole 4g was clearly superior to the thiazole 4b. To analyze the antiproliferative effects of
these compounds the CellTiter 96 AQueous Non-Radioactive Cell Proliferation Assay (Promega)
was performed on HL60 cells with compounds SAHA, 4b, 4f, 4g, 4h, 4m, 4n and 4o (Table 4).
It was shown before that HDAC6 plays a role as a target for antileukemic drugs in acute myeloid
leukemia.35 SAHA as a pan HDAC inhibitor is very effective with a GI50 of 80 nM. All the other
compounds showed an antiproliferative effect on HL60 cells in the micromolar range. The four
most effective compounds were 4h with a GI50 of 5.60 µM, 4o with a GI50 of 6.30 µM, 4b with a
GI50 of 11.06 µM and 4g with 10.22 µM.
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Table 4. Antiproliferative effects on HL60 cells determined with the CellTiter 96 AQueous Non-
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Radioactive Cell Proliferation Assay (Promega)
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compound GI50 [µM]a
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SAHA
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10.22
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4g
4h
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6.30
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4n
4o
aGI50 values are the means of at least two experiments. Standard deviation is <10% of the GI50.
Experiments were performed in triplicates.
Additionally, the cell proliferation assay was performed on HeLa cells with compound 4g. The
GI50 value of 4g was determined with a value of 23.69 µM. Compared to the reference substance
SAHA with a value of 1.68 µM, the selective HDAC6 inhibitor seemed to be less toxic in HeLa
cells.
It is difficult to dissect whether antiproliferative potency is caused by selective HDAC6
inhibition or residual class I HDAC inhibition. Several compounds showed an in-vitro efficacy
on HDAC6 similar to SAHA but the antiproliferative effects on HL60 cells were less
pronounced with selective HDAC6 compounds compared to pan-HDAC inhibitors as SAHA.
This may additionally be confounded by differences in cellular uptake or stability. The in-vitro
selectivity was additionally confirmed in two different cell lines with western blot experiments
with clearly visible effects at concentrations of 10 µM and 1 µM.
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CONCLUSION
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Based on 1, found by virtual screening, we explored a new biaryl HDAC6 inhibitor scaffold
with three different heterocycles attached to the hydroxamate. We demonstrated that oxazole-
containing inhibitors showed the best activity and selectivity for HDAC6 compared to thiazole-
and oxadiazole-containing ones. The variation of the aryl-substituent attached to the heterocycle
indicated that in order to gain the best selectivity and potency results, the phenyl-group should be
substituted at 4-position with a halogen- or another phenyl-ring. Substitution in ortho- or meta-
position had a diminishing effect on potency and selectivity on HDAC6. All the synthesized
molecules showed HDAC6 inhibition and selectivity in vitro. The most potent and selective
compounds were 4g, 4h and 4o. We also confirmed the HDAC6 selectivity of 4g and 4h in cell
culture with western blotting of tubulin versus histone acetylation. Several compounds were
tested for their antiproliferative activity on HL60 cells and showed effects in the low micromolar
range. Thus, this is the first example that just the exchange of the heteroatom from sulfur to
oxygen in the aryl ring adjacent to the zinc binding modules is sufficient to modulate the activity
towards very high HDAC6 potency and selectivity. The good ligand efficiency shows that these
compounds can be optimized further, probably by attaching the common branched substitution
patterns further outwards from the zinc ion.
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EXPERIMENTAL SECTION
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Chemistry. All reagents were obtained from commercial sources and used without further
purification. 1H-NMR and 13C-NMR spectra were recorded on Bruker Avance DRX
spectrometer at 400 MHz and 100 MHz. Chemical shifts were reported in parts per million (ppm,
δ) with TMS as internal standard. Compounds were dissolved in DMSO-d6 or in D3COD.
Standard abbreviations indicating multiplicity were used as follows: s = singlet, bs = broad
singlet, d = doublet, t = triplet, q = quartet and m = multiplet. Aromatic 1H-NMR proton signals
were generally described as multiplets. Thin-layer chromatography (TLC) was performed using
Merck 60 F254 silica gel plates. All yields were not optimized. Flash chromatography was
performed using Isolera One (Biotage) with a linear solvent gradient.
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A purity of >95% for all the final compounds was determined with HPLC (Agilent 1260
Infinity, Agilent) and UV detection at 210 nm. Elution was performed under gradient conditions:
Eluent A: H2O containing 0.05% TFA, Eluent B: acetonitrile (MeCN) containing 0.05% TFA.
Linear gradient conditions were as follows: 0−4 min, A = 90%, B = 10%; 4−29 min, linear
increase to 100% of B; 29−31 min, B = 100%; 31−40 min, A = 10%, B = 90%. Method 1 (M1),
analytical column, Phenomenex Synergi 4 µm MAX-RP 80 Å, 150 x 4.6 mm, flow rate 1
mL/min. Method 2 (M2), analytical column, Phenomenex Synergi 4 µm POLAR-RP 80 Å, 150 x
4.6 mm, flow rate 1 mL/min. Method 3 (M3), analytical column, Phenomenex Synergi 4 µm
HYDRO-RP 80 Å, 250 x 4.6 mm, flow rate 1 mL/min.
Mass spectra were recorded with a TSQ700 from Thermo Fisher using APCI (NH3) or ESI.
Melting ranges were determined in glass capillary tubes with the Stuart Melting Point Apparatus
SMP2.
Infrared spectra were recorded with a Spectrum TwoTM FT-IR Spectrometer with a diamond
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universal ATR sampling accessory from PerkinElmer and analysed with the Spectrum 10TM
software.
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Method A. General synthesis of ethyl aryl-1,3-thiazole-4-carboxylates (2a-e). Thioamide
(1 equiv) and ethyl bromopyruvate (1.2 equiv) were dissolved in ethanol and heated to 70 °C.
The reaction was monitored with TLC until completion. The solvent was evaporated and the
ethylester (2a-e) was purified by flash column chromatography (ethyl acetate : cyclohexane 3:7
(V/V)).
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Ethyl 2-(pyrazin-2-yl)-1,3-thiazole-4-carboxylate (2a). 557 mg (4 mmol) pyrazine-2-
thiocarboxamide and 602 µL (4.8 mmol) ethyl bromopyruvate were used according to method A.
1H-NMR (DMSO-d6, δ [ppm]): 9.33-9.32 (m, 1H, 3’-H), 8.80-8.79 (m, 1H, 5’-H), 8.75-8.74 (m,
3
1H, 6’-H), 8.71 (s, 1H, 5-H), 4.38-4.33 (q, 2H, O-CH2-CH3, J = 7.1 Hz), 1.36-1.32 (t, 3H, O-
3
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CH2-CH3, J = 7.1 Hz). C-NMR: (δ [ppm]): 166.9 (2-C), 160.9 (COOEt), 148.1 (4-C), 146.7
(5’-C), 145.6 (2’-C), 145.0 (6’-C), 141.0 (3’-C), 132.4 (5-C), 61.5 (O-CH2-CH3), 14.6 (O-CH2-
CH3)
Method B. General synthesis of aryl-1,3-thiazole-4-carboxylic acids (3a-e). The ethylester
(2a-e) was hydrolysed by dissolving it in THF/H2O, adding 1 M LiOH (4 equiv) and stirring at
RT for 0.5-4 hours controlled by TLC. Upon completion of the reaction the solvent was
evaporated in vacuum and the reaction mixture was extracted with ethyl acetate. The aqueous
layer was acidified with 2 M HCl to pH 4 until the product precipitated. The suspension was
extracted with ethyl acetate three more times. The combined organic layers were dried over
Na2SO4 and evaporated in vacuum obtaining the carboxylic acids (3a-e).
2-(pyrazin-2-yl)-1,3-thiazole-4-carboxylic acid (3a). 371 mg (1.6 mmol) 2a and 6.3 mL (6.3
mmol) LiOH (1 M) were used according to method B. 1H-NMR (DMSO-d6, δ [ppm]): 13.30 (bs,
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1H, COOH), 9.33 (m, 1H, 3’-H), 8.81-8.80 (m, 1H, 5’-H), 8.76-8.75 (m, 1H, 6’-H), 8.67 (s, 1H,
5-H). 13C-NMR: (δ [ppm]): 166.6 (2-C), 162.3 (COOH), 149.3 (4-C), 146.8 (5’-C), 145.7 (2’-C),
145.0 (6’-C), 141.1 (3’-C), 132.0 (5-C).
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Method C. General synthesis of aryl-1,3-thiazole-4-carbohydroxamic acids (4a-e). The
carboxylic acid (3a-e) was dissolved in THF under nitrogen atmosphere and activated by adding
ethyl chloroformate (1.2-1.5 equiv) and N-methylmorpholine (1.3-1.5 equiv) at 0 °C. After
stirring the reaction for 10 min, the precipitated salts were filtered off. Hydroxylamine was
freshly prepared by stirring hydroxylamine hydrochloride (1.5-20 equiv) and potassium
hydroxide (1.5-20 equiv) in methanol at 0 °C for 15 minutes and filtering off the resulting
potassium chloride. The two filtrates were combined and stirred for 30 minutes at room
temperature. Afterwards the solvent was evaporated and the reaction mixture was purified by
flash column chromatography (dichloromethane : methanol = 95:5 (V/V)) and crystallisation or
by preparative reversed phase chromatography (acetonitrile : water = 45:55 (V/V))
2-(pyrazin-2-yl)-1,3-thiazole-4-carbohydroxamic acid (4a). 91 mg (0.4 mmol) 3a, 51 µL
(0.53 mmol) ethyl chloroformate, 63 µL, (0.6 mmol) N-methylmorpholine, 46 mg (0.7 mmol)
hydroxylamine hydrochloride and 37 mg (0.7 mmol) potassium hydroxide were used according
1
to method C. Overall yield : 85 mg (10%). Mp: 179-181 °C. H-NMR (DMSO-d6, δ [ppm]):
11.42 (bs, 1H, NH-OH), 9.49 (m, 1H, 3’-H), 9.30 (bs, 1H, NH-OH), 8.79-8.78 (m, 1H, 5’-H),
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8.74-8.73 (m, 1H, 6’-H), 8.45 (s, 1H, 5-H). C-NMR: (δ [ppm]): 166.2 (2-C), 158.4 (CONH),
150.2 (4-C), 146.6 (5’-C), 145.9 (2’-C), 144.5 (6’-C), 141.7 (3’-C), 126.8 (5-C). MS (APCI,
m/z): 223.0283 [M + H]+. Purity >99% (10.98 min, M3).
Method D and E: General synthesis of Ethyl aryl-1,3-oxazole-4-carboxylates.
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Method D (2f-n): Amide (1 equiv), ethyl bromopyruvate (1.2-1.5 equiv) and silver
hexafluoroantimonate (1 equiv) were mixed in dry methylene chloride in a 15 mL microwave
tube. The tube was sealed and put in the microwave for one hour at 30 watt, 5 bar and 90 °C.
Afterwards, saturated NaHCO3 solution was added and the reaction mixture was extracted with
ethyl acetate. The combined organic layers were washed with brine, dried over Na2SO4 and the
solvent was evaporated off in vacuum. The product was purified by flash column
chromatography (ethyl acetate : cyclohexane 3:7 (V/V)). Hydrolysis of the ester (3f-n) and
formation of the hydroxamic acid (4f-n) was performed as described in methods B and C.
Ethyl 2-phenyl-1,3-oxazole-4-carboxylate (2f). 827 g (6.8 mmol) benzamide, 1.0 mL (8.2
mmol) ethyl bromopyruvate and 2.3 g (6.8 mmol) silver hexafluoroantimonate were used
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according to method D. H-NMR (DMSO-d6, δ [ppm]): 8.95 (s, 1H, 5-H), 8.04-8.01 (m, 2H, 2’-
,6’-H), 7.59-7.57 (m, 3H, 3’-,4’-,5’-H), 4.35-4.30 (q, 2H, O-CH2-CH3, 3J = 7.1 Hz), 1.33-1.30 (t,
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3H, O-CH2-CH3, J = 7.1 Hz). C-NMR (δ [ppm]): 161.7 (2-C), 161.1 (COOEt), 146.1 (5-C),
134.1 (4-C), 131.8 (4’-C), 129.7 (3’-,5’-C), 126.7 (2’-,6’-C), 126.4 (1’-C), 61.1 (O-CH2-CH3),
14.6 (O-CH2-CH3).
Method E (2o-r): Suzuki-coupling. Pd(PPh3)4 (0.1 equiv) was dissolved in 5 mL/mmol
toluene and stirred together with ethyl 2-bromo-1,3-oxazole-4-carboxylate (1 equiv) in a round
bottom flask. The aryl-boronic acid (1.5 equiv) was dissolved in 5 mL/mmol EtOH and added to
the mixture. Finally, aqueous Na2CO3 solution 2 M (10 equiv) was added. After heating the
mixture to 120 °C for 2-5 hours 50 mL methylene chloride were added and the organic layer was
separated, dried over Na2SO4 and evaporated. The product was purified by flash column
chromatography (ethyl acetate : cyclohexane 3:7 (V/V)). Hydrolysis of the ester (3o-r) and
formation of the hydroxamic acid (4o-r) was performed as described in method B and C.
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Ethyl 2-(naphthalene-2-yl)-1,3-oxazole-4-carboxylate (2o). 225 mg (1.0 mmol) ethyl 2-
bromo-1,3-oxazole-4-carboxylate, 264 mg (1.5 mmol) 2-naphthyl-boronic acid, 118 mg (0.1
mmol) Pd(P(Ph)3)4 and 5.1 mL (10.0 mmol) Na2CO3 (2 M) were used according to method E.
1H-NMR (DMSO-d6, δ [ppm]): 8.95 (s, 1H, 5-H), 8.63 (m, 1H, 1’-H), 8.12-8.11 (m, 1H, Ar-H),
8.09-8.08 (m, 2H, Ar-H), 8.01-7.98 (m, 1H, Ar-H), 7.64-7.61 (m, 2H, Ar-H), 4.36-4.30 (q, 2H,
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O-CH2-CH3, J = 7.1 Hz), 1.34-1.30 (t, 3H, O-CH2-CH3, J = 7.1 Hz). 13C-NMR (δ [ppm]):
161.9 (COOEt), 161.3 (2-C), 146.2 (5-C), 134.3 (4-C), 134.3 (Ar-C), 132.9 (Ar-C), 129.5 (Ar-
C), 129.2 (C-Ar-H), 128.4 (Ar-C), 128.2 (Ar-C), 127.6 (Ar-C), 127.0 (1‘-C), 123.7 (2‘-C), 123.3
(3‘-C), 61.2 (O-CH2-CH3), 14.6 (O-CH2-CH3).
Method F: General synthesis of ethyl aryl-1,2,4-oxadiazole-3-carboxylates (2s-u). Thionyl
chloride (3 mmol) was added dropwise to the carboxylic acid (1 equiv) in dry methylene
chloride. After completion of the addition the mixture was heated to 90 °C for 2-3 hours.
Remaining thionyl chloride and solvent were removed with a stream of nitrogen. The acyl
chloride was directly processed further by adding ethyl 2-oximinooxamate (1 equiv) and
dissolving it in methylene chloride (5 mL) at 0 °C. Triethylamine (1.5 equiv) was added
dropwise. The reaction mixture was stirred for 1 to 3 hours. After completion (TLC-control) the
solvent was evaporated and dimethylformamide (2-3 mL) was added to the reaction. The mixture
was heated to 150 °C until completion (TLC-control) of the formation of the oxadiazole. After
completion the mixture was extracted with ethyl acetate and washed with water. The product was
purified by flash column chromatography (ethyl acetate : cyclohexane 3:7 (V/V)). Hydrolysis of
the ester (3s-u) and formation of the hydroxamic acid (4s-u) was performed as described in
method B and C.
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Ethyl 5-phenyl-1,2,4-oxadiazole-3-carboxylate (2s). 485 mg (4.0 mmol) benzoic acid, 864
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µL (12.0 mmol) thionyl chloride, 525 mg (4.0 mmol) ethyl 2-oximinooxamate and 825 µL (6.0
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mmol) triethylamine were used according to method F. H-NMR (DMSO-d6, δ [ppm]): 8.18-
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8.16 (m, 2H, 2’-,6’-H), 7.78-7.74 (m, 1H, 4’-H), 7.69-7.64 (m, 2H, 3’-,5’-H), 4.48-4.42 (q, 2H,
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O-CH2-CH3, J = 7.1 Hz), 1.38-1.34 (t, 3H, O-CH2-CH3, J = 7.1 Hz). 13C-NMR (δ [ppm]):
176.9 (5-C), 162.5 (COOEt), 157.6 (3-C), 134.3 (4’-C), 130.1 (3’-,5’-C), 128.5 (2’-,6’-C), 123.2
(1’-C), 63.0 (O-CH2-CH3), 14.3 (O-CH2-CH3).
In-vitro-testing32. OptiPlate-96 black microplates (Perkin Elmer) were used. Assay volume
was 60 µL. 52 µL of human recombinant HDAC1 (BPS Bioscience, Catalog #: 50051) or human
recombinant HDAC6 (BPS Bioscience, Catalog #: 50006) in incubation buffer (50 mM Tris–
HCl, pH 8.0, 137 mM NaCl, 2.7 mM KCl, 1 mM MgCl2 and 1 mg/mL BSA) were incubated
with 3 µL of different concentrations of inhibitors in DMSO and 5 µL of the fluorogenic
substrate ZMAL (Z-(Ac)Lys-AMC)36 (126 µM) for 90 min at 37 °C. After incubation time 60
µL of the stop solution, comprising 33 µM Trichostatin A (TSA) and 6 mg/mL trypsin in trypsin
buffer (Tris–HCl 50 mM, pH 8.0, NaCl 100 mM), were added. The plate was incubated again at
37 °C for 30 min and fluorescence was measured on a BMG LABTECH POLARstar OPTIMA
plate reader (BMG Labtechnologies, Germany) with an excitation wavelength of 390 nm and an
emission wavelength of 460 nm.
Inhibition of human HDAC8 was measured in ½ AREAPLATE-96 F microplates (Perkin
Elmer) with an assay volume of 30 µL. HDAC8 enzyme was obtained as described before.37
22.5 µL of enzyme in incubation buffer (50 mM KH2PO4, 15 mM Tris, pH 7.5, 3 mM MgSO4*7
H2O, 10 mM MgSO4) were mixed with 2.5 µL of inhibitor in DMSO and 5 µL of Z-L-Lys(ε-
trifluoroacetyl)-AMC (150 µM). The plate was incubated at 37 °C for 90 min. 30 µL of the stop
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