Structure–Activity Relationship of Propargylamine-Based HDAC Inhibitors

Article abstract of DOI:10.1002/cmdc.201700550

As histone deacetylases (HDACs) play an important role in the treatment of cancer, their selective inhibition has been the subject of various studies. These continuous investigations have given rise to a large collection of pan- and selective HDAC inhibitors, containing diverse US Food and Drug Administration (FDA)-approved representatives. In previous studies, a class of alkyne-based HDAC inhibitors was presented. We modified this scaffold in two previously neglected regions and compared their cytotoxicity and affinity toward HDAC1, HDAC6, and HDAC8. We were able to show that R-configured propargylamines contribute to increased selectivity for HDAC6. Docking studies on available HDAC crystal structures were carried out to rationalize the observed selectivity of the compounds. Substitution of the aromatic portion by a thiophene derivative results in high affinity and low cytotoxicity, indicating an improved drug tolerance.

Full text of DOI:10.1002/cmdc.201700550

Accepted Article
Title: Structure-Activity Relationship of Propargylamine-Based HDAC
Inhibitors
Authors: Matthias Wünsch, Johanna Senger, Philipp Schultheisz,
Sabrina Schwarzbich, Karin Schmidtkunz, Carmela Michalek,
Michaela Klaß, Stefanie Goskowitz, Philipp Borchert, Lucas
Praetorius, Wolfgang Sippl, Manfred Jung, and Norbert
Sewald
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the VoR from the journal website shown below when it is published
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To be cited as: ChemMedChem 10.1002/cmdc.201700550
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10.1002/cmdc.201700550
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Structure-Activity Relationship of Propargylamine-Based HDAC Inhibitors
Matthias Wünsch[a], Johanna Senger[b], Philipp Schultheisz[a], Sabrina Schwarzbich[a], Karin
Schmidtkunz[b], Carmela Michalek[a], Michaela Klaß[a], Stefanie Goskowitz[a], Philipp Borchert[a], Lucas
Praetorius[c], Wolfgang Sippl[c], Manfred Jung[b], Norbert Sewald[a],*
.
[a]
M. Wünsch (ORCID 0000-0002-3244-8374), P. Schultheisz, S. Schwarzbich, C. Michalek, M. Klaß, S. Goskowitz, P. Borchert, Prof. Dr. N. Sewald (ORCID
0000-0002-0309-2655)
Organic and Bioorganic Chemistry
Bielefeld University
Universitätsstrasse 25, 33615 Bielefeld, Germany
Norbert.Sewald@uni-bielefeld.de
[b]
Dr. J. Senger, K. Schmidtkunz, Prof. Dr. M. Jung (ORCID 0000-0002-6361-7716)
Institute of Pharmaceutical Sciences
Albert-Ludwigs-Universität Freiburg
Albertstrasse 25, 79104 Freiburg im Breisgau, Germany
L. Praetorius, Prof. Dr. W. Sippl (ORCID 0000-0002-5985-9261)
Institute of Pharmacy
[c]
Martin-Luther-Universität Halle-Wittenberg
Wolfgang-Langenbeck-Str. 4, 06120 Halle/Saale, Germany
Supporting information for this article is given via a link at the end of the document.
Abstract: As histone deacetylases (HDACs) play an important role
in cancer treatment, their selective inhibition has been subject of
various studies. The continuous investigations have spawned a large
collection of pan- and selective HDAC inhibitors, containing diverse
FDA approved representatives. In former studies, a class of alkyne
based inhibitors of HDACs was presented. We modified this scaffold
in two previously neglected regions and compared cytotoxicity and
affinity towards HDAC1, HDAC6 and HDAC8. We could show that
(R)-configured propargylamines contribute increased selectivity on
HDAC6, while the size of the substituents decreases their affinity.
Docking studies on available HDAC crystal structures were carried
out to rationalize the observed selectivity of the compounds. Substi-
tution of the aromatic part by a thiophene derivative results in high
affinity and low cytotoxicity, indicating an improved drug tolerance.
do KO mice show abnormal development or problems with
organ functions.[20] In addition to the modulation of transcription,
HDAC6 uniquely deacetylates α-tubulin,[21] decreasing the speed
of vesicle transport, what makes selective inhibitors of HDAC6 a
promising drug candidates against neurodegenerative diseases
like for example Huntington’s disease.[22–24]
N
O
H
H
N
N
N
OH
OH
H
O
O
O
Vorinostat
Trichostatin A
HN
O
O
S
N
H
H
N
H
N
H
N
OH
OH
Panobinostat
Oxamflatin
O
O
N
H
H
N
N
N
H
N
S
OH
OH
O
O
O
Introduction
O
Belinostat
Tubastatin A
Controlling diverse cellular functions by deactivating transcrip-
tion makes histone deacetylases important effector molecules
for epigenetic regulation and also a therapeutic target. They are
divided into four classes according to their function, localization
and sequence homology.[1] The inhibition of certain HDACs is an
approach for versatile pharmaceutical applications like the regu-
Figure 1. Chemical structures of a selection of important HDAC inhibitors.[7,14]
[3]
lation of autoimmunity,[2] influence on neurological processes
or suppression of tumor growth.[4–6] The development of non-
selective HDACis (Figure 1) including Vorinostat (SAHA),[7]
Trichostatin A,[8] Belinostat,[9] Panobinostat,[10] Oxamflatin,[11]
[12]
[13]
Tubastatin A
and Romidepsin (depsipeptide FK228)
has
already proceeded very far and some have even been clinically
established (Vorinostat, Belinostat, Panobinostat, Romidepsin
and Tucidinostat).[7,14] The benzamide Tucidinostat has achieved
particular relevance in clinical studies, as it is selective on
HDAC 1, 2, 3 and 10.[15]
Figure 2. X-ray structure analysis of the active site of HDAC6 occupied by
However, the inhibition of class I and class IIA HDACs may lead
to serious side effects that have not been reported for class IIB,
Trichostatin A (TSA).[25]
class III, and IV HDACs. For example HDAC1 knockout leads to
[16]
embryonic lethality
and HDAC2, -5, and -9 knockouts pro-
The active sites of HDAC1, 6, and 8 consist of a hydrophobic
channel that is terminated by a bound Zn2+ ion, which catalyzes
deacetylation of lysine residues.[26–28] The active site structure of
voke cardiac defects.[17,18] HDAC6 selective inhibitors do neither
show a change in gene expression in microarray analysis,[19] nor
1
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HDAC6 has been recently elucidated (Figure 2),[25,29] allowing
accurate design of inhibitor topology.
imines direct the alkyne addition from the re-face, while R-
configured N-sulfinylimines induce si-face additions.[35] Therefore,
the N-sulfinyl propargylamines are either R,R or S,S-con-
figured.[34] A diastereomeric excess of more than 99% is ob-
tained, no other diastereomer was detected by 1H NMR spec-
troscopy.
After cleavage of the sulfinamide, the terminal propargylamine is
linked to a benzofuranoyl cap group according to Sendzik et
al.[32] The aromatic methyl ester is finally converted to the zinc
complexing hydroxamic acid by reaction of the methyl ester with
hydroxylamine.
For the Sonogashira cross-coupling 1.6 equivalents of the aro-
matic halide were applied in order to achieve full conversion of
the precious enantiomerically pure propargylamines. The prod-
ucts were formed in a mixture of THF/piperidine (3:1) in the
presence of CuI (2%) and Cl2Pd(PPh3)2 (1%) as catalyst. The
application of pyridine, DIPEA, or 2,4,6-collidine or the applica-
tion of Pd(Ph3)4 lead to a reduced yield. The progress of the
reaction can be monitored by the precipitation of piperidinium
iodide, which forms as a byproduct. Aqueous workup and purifi-
cation by column chromatography provides the linker scaffolds
5a-h in good yields (Table 1). Due to the strict exclusion of oxy-
gen and the excess of aromatic halide, the commonly reported
Glaser homocoupling under Sonogashira conditions can be
excluded, as proven by mass spectrometry.
Potent inhibitors consist of a Zn2+ binding group like a ketone,
benzamide, carboxylic acid, or hydroxamic acid at one end that
is terminated by an inflexible, rod-like linker with a cap group at
the other end.[30,31] Sendzik et al. proposed an achiral propargyl-
amine scaffold for a new generation of HDAC inhibitors and
modified the cap group (Figure 3), which is the most promising
part to induce selectivity.[32]
residues
groups:
Acyl
cap
of
O
cap
R
linker Zinc binding
group
group
N
=
R
S
N
H
Cl
O
O
H
N
OH
N
H
O
X
=
X
NH
O, S,
Figure 3. Canonical structures of potent HDAC inhibitors based on an achiral
propargylamine with different cap groups at the N-terminus.[32]
We became interested in the influence of chirality in the propar-
gylic position and introduced different linker regions with respect
to the aromatic moiety, the configuration and size of the substit-
uents.
substituent of
propargylamine
a)
b)
cap
R'
group
O
R
O
Zinc
binding
group
aromatic
moiety
N
H
R'
N
*
H
ar
H
H
N
N
Results and Discussion
OH
OH
=
or
*
R
S
( )
(
)
linker
rigid
O
O
ar = aromatics
=
R
R'
substituents
aliphatic
We synthesized an array of potential HDAC inhibitors by com-
bining different propargylamines linking the benzofuran cap
group with aromatic hydroxamic acids.
=
O
The cap group of HDAC inhibitors is considered as the most
important moiety to enhance selectivity and activity and has,
therefore, been varied extensively.[32] Compounds 6a-g vary with
respect of configuration and size of the linker region and 6i
slightly deviates from linearity by incorporation of a five-
membered heteroaromatic compound (Figure 4).
Figure 4. Design of inhibitors. a) Patent-registered inhibitor scaffold, used as
model compound with extensively varied cap groups.[32] b) Variations of the
model compound investigated in this work.
The distance between the hydroxamic acid and the propargylic
position of our scaffold is almost equal to the length of the regu-
lar substrate of HDACs, the lysine side chain. That makes chiral
analogues of other propargylamines with hydrophobic substitu-
ents promising alternatives. Combining three separately variable
building blocks by click-reactions (as defined by Sharpless),[33] a
large diversity of inhibitors can easily be achieved.
The building blocks of the linker regions are obtained by a con-
vergent synthesis, linking an enantiopure propargylamine to a
halogenated benzoic acid derivative in a Sonogashira cross-
coupling (Table 1). Variations of the configuration and size of the
substituent in propargylic position can be introduced by the
application of substituted propargylamines.[34] The angle of the
rigid linker can be modified by the use of heteroaromatic moie-
ties (ar) like thiophene derivatives to 156°.
The Sonogashira cross-coupling of the heteroaromatic building
blocks proceeds much more slowly and in lower yields, because
halides 2b and 2c were only available as bromides instead of
iodides. Furthermore, heteroaromatics like thiophene or pyridine
are suspected to coordinate and poison the catalyst. In an effort
to shorten the synthesis, methyl bromothiophene carboxylate 2c
was converted with aqueous hydroxylamine into hydroxamic
acid derivative 3. Dilution of the reaction mixture with cold Et2O
leads to precipitation of hydroxamic acid 3 in pure form. Howev-
er, 5-bromothiophene-2-hydroxamate 3 did not react at all under
Sonogashira cross-coupling conditions.
The corresponding amines are readily formed by acidic meth-
anolysis of the sulfinamides 5a-i. Water has to be excluded to
avoid formation of tert-butylsulfinic acid. The methylsulfinate can
be co-evaporated with DCM to quantitatively yield the amine
hydrochlorides. The free amines can be converted without fur-
ther purification with 2-benzofuranoyl chloride in dry DCM under
basic conditions.
The propargylamines 4 are easily prepared by diastereoselec-
tive nucleophilic addition of trimethylsilylethynyl lithium to N-
sulfinylimines. The N-sulfinyl group serves as the chiral auxiliary
in the propargylamine preparation, as S-configured N-sulfinyl-
2
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The hydroxamic acids 6 are obtained by reaction of the ester
with an excess of aqueous hydroxylamine (50%). The reaction
was monitored by mass spectrometry, which also showed for-
mation of the acid as by-product. After aqueous workup and
precipitation, the crude product was purified by preparative RP-
HPLC. The low solubility in water, acetonitrile or any solvent
(except for DMSO) leads to significant losses during purification.
The inhibitory activity of the synthesized compounds was deter-
mined (Table 1) in an assay developed by Schwienhorst et
al.,[36,37] using a fluorescent substrate, invented by Jung et al.,[38]
which is only cleaved in the enzymatic detection step when
deacetylated. The HDAC activity depending on inhibitor concen-
tration can be quickly determined by quantification of the fluo-
rescence, caused by the released aminomethylcoumarin.[36,37]
Table 1. Synthesis of the inhibitor scaffolds upon reaction of enantiopure propargylamines with aromatic halides under Sonogashira cross-coupling conditions,
acylation with the cap group and transformation of the methyl ester into a hydroxamic acid. Reaction conditions (a): PdCl2(PPh3)2 (1%), CuI (2%), piperidine/THF
(1:3), rt, 2-12 h. (b): 1) HCl (4 M in dioxane, 4 eq). 2) 2-benzofuranoyl chloride (1.5 eq), NEt3 (6 eq), DCM, 0 °C, 6-14 h. 3) H2NOH/H2O/MeOH/THF (1:1:4:4),
NaOH (pH = 10-11), rt, 16 h.
R1 R2
O
R1 R2
R1 R2
O
N
pg
X
N
H
pg
ar
H
H
N
ar
N
(b)
Me
CO2
H
ar
(a)
OH
5a-i
4a-g
Me
CO2
1,
6a-i
2a-c
O
Propargyl-
amine
entry
pg
R1
R2
Ar-X
5, yield
6, yield
O
a)
b)
4a
4b
4c
1
H
Cyclohexyl
5a, 66%
5b, 94%
5c, 83%
5d, 58%
5e, 64%
5f, 95%
5g, 74%
6a, 24%
6b, 43%
6c, 16%
6d, 3%
6e, 37%
6f, 9%
S
H
H
CH2CHMe2
I
c)
CHMe2
(S)-Bus
d) a
e)
Boc
H
H
H
H
H
R
CO2
4e
4f
CHMe2
2a R = Me, Et
f)
CH2CHMe2
Cyclohexyl
g)
4g
6g, 6%
O
S
Br
h)
4g
Cyclohexyl
H
5h, 41%
6h, 25%
6i, 16%
N
Me
CO2
(R)-Bus
2b
Br
S
i)
4g
Cyclohexyl
H
5i, 23%
Me
CO2
2c
a The synthesis of 6d, entry d) had already been established by Sendzik et al.[32] and serves in this work as reference compound.
Table 2. Cytotoxicity and affinity of compounds 6a-i to HDAC1, HDAC6, and
HDAC8.
HDAC1 a
HDAC6 a Selectivity HDAC8 a Selectivity
Cytotoxicity
IC50 ± SEM IC50 ± SEM HDAC1/ IC50 ± SEM HDAC8/
EC50 [µM] b
[nM]
[nM]
HDAC6
1.35
[nM]
HDAC6
18.4
6a 161.5±30.6 119.4 ± 30.4
6b 80.2±10.2 64.4 ± 6.7
2200 ± 300
29±1.3
13±0.2
8.2±0.4
1.08±0.3
4±0.7
7.4±1.5
29
1.25
1.51
0.52
8.20
7.40
8.76
16.00
12.30
803 ± 111
916 ± 93
12.5
15.5
6.1
6c 89.3±14.6 59.2 ± 9.2
6d
19.6±1.4 37.9 ± 4.1
232 ± 22
c
6e 127.9±19.3 15.6 ± 0.9
6f 190.9±30.5 21.8 ± 1.6
6g 268.5±95.3 25.3 ± 5.1
6h 1150±150 72.1 ± 15.2
6i 137.8±26.2 11.2 ± 1.1
417 ± 53
26.7
39.5
34.6
45.8
142.9
862 ± 107
876 ± 125
3300 ±400
1600 ± 200
Figure 5. Inhibition (IC50 ± SEM) of HDAC1 and HDAC6 by compounds 6a-i.
100
44
In comparison to HDAC6, the inhibition of HDAC8 by the hy-
droxamic acids 6a-i is generally weaker with IC50 values in the
range of 0.4-3.3 µM (6-142 times higher than for HDAC6).
The S-configured compounds 6a-c are generally not very active
against the HDAC tested, while potency towards HDAC6 de-
creases with increasing the size of the substituent in propargylic
position. The R-configured compounds 6e-i are potent inhibitors
a The affinity values were determined in an assay developed by Schwienhorst
et al.[36,37] using a fluorescent substrate, invented by Jung et al.,[37] which is
only cleaved enzymatically when deacetylated. The values of each inhibitor
concentration were determined as triplicates for HDAC1 and HDAC6 and as
technical and biological duplicates for HDAC8.
b Cytotoxicity values refer to cell line KB-3-1[40] and were determined as sixfold
replicates for 6a-f (± SEM) and as triplicates for 6g-h.
c Compound 6d published by Sendzik et al.[32] is used as reference compound.
3
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of HDAC6 without significant influence of the substituent size R1.
However, selectivity towards HDAC6 is largely enhanced for
R-configured compounds 6e-i in comparison to the achiral par-
ent compound 6d, that even has a higher affinity to HDAC1
(Figure 5, Table 2).
activities. As observed for the HDAC6 inhibition values, a signifi-
cant correlation with the calculated GlideSP score was obtained
(r2 = 0.71, RMSE = 0.24, q2LOO = 0.46, Figure S4 and Table S1,
Supporting Information).
As shown by X-ray structure analysis of the complex of HDAC6
and its inhibitor TSA (Figure 2), the hydroxamate occupies the
binding site of the acetylated lysine side chain, the native sub-
strates of the enzyme. Considering the CIP priority of the sub-
stituents, R-configured inhibitors with the described scaffold 6
mimic S-configured lysine derivatives.
In order to modify the linker, the aromatic moiety was changed
to introduce another complexation site in form of a pyridine (6h)
or a thiophene unit (6i) able to introduce a slightly bent structure.
Apparently, introduction of an additional hydrogen bond acceptor
or complexation site by replacing a phenyl by a pyridine ring
leads to a reduced activity of the inhibitors. In conclusion, non-
polar linker units are important for optimal interaction with the
hydrophobic channel of histone deacetylases. As the inhibition
of HDAC1 by compound 6h was reduced manifold compared to
HDAC6, this inhibitor is more selective for HCAC6. Interestingly,
a slight modification of the angle of the rigid linker from 180° to
156° by replacing the phenyl ring by a thiophene ring (6i) en-
hances the inhibition significantly with good selectivity.
Docking studies of all compounds to available crystal structures
of human HDAC1, 6 and 8 were performed to understand the
observed in vitro inhibition data (see Experimental Section for
details). As expected, the aromatic hydroxamate group of all
inhibitors is coordinating the catalytic zinc ion and is hydrogen
bonded to the conserved tyrosine and histidine residues as
observed for the co-crystallized hydroxamates (e.g. Trichostatin
A in HDAC6). The aromatic ring (phenyl, pyridine, or thiophene)
is interacting with two conserved phenylalanines in the HDAC
pocket (F150 and F205 in HDAC1, F620 and F680 in HDAC6,
F152 and F208 in HDAC8).[40] The hydrophobic substituents in
the propargylic position, which confer HDAC isoform selectivity,
interact with residues located at the rim of the acetyl-lysine
channel. The docking results for the most potent HDAC6 inhibi-
tor 6i showed that the benzofuran ring is interacting with the
aromatic sidechains of W496 and H500 in the HDAC6 binding
pocket whereas the cyclohexyl ring is located nearby the hydro-
phobic residues P501 and L749 (Figure 6). An additional hydro-
gen bond is observed between the amide of the capping group
and S568. Due to the different angle of the rigid linker of com-
pounds 6a-6h, the hydrophobic capping group adopts a slightly
different orientation whereas the benzofuran ring is interacting
as observed for 6i. In the case of the S-configured capping
groups, the benzofuran ring is shifted away from W496 (Figure
S1 Supporting Information). A significant correlation between the
calculated docking scores (Glide SP) and the HDAC6 inhibitory
Figure 6. Binding mode of the most potent HDAC6 inhibitors 6i (colored
green) and 6e (colored cyan) derived by the docking study. Hydrogen bonds
between inhibitor and HDAC6 are shown as orange dashed lines. Water
molecules are shown as red spheres.
Figure 7. Binding mode of the most potent HDAC1 inhibitor 6d (colored cyan)
derived by the docking study. Hydrogen bonds between inhibitor and HDAC1
are shown as orange dashed lines.
activities was observed (r2 = 0.82, RMSE = 0.13, q2
Figure S2 and Table S1, Supporting Information).
= 0.72,
LOO
[41]
Docking into the HDAC1 crystal structure (PDB ID 4BKX)
gave the best docking score for the unsubstituted derivative 6d
(Figure 7 and Table S1, Supporting Information). The benzofu-
ran ring of the capping group is interacting with the aromatic side
chains of Y204, F205 as well as with the backbone of P206. The
hydrophobic substitutions of the capping group (6a-6c and 6e-i)
showed a similar orientation in the HDAC1 binding pocket but
with less favourable docking scores (Figure S3 and S4, Support-
ing Information) which might explain their decreased inhibitory
The docking of the inhibitors into the HDAC8 binding pocket
showed that the capping group is pointing out of the more open
HDAC8 pocket resulting in fewer contacts between the capping
group and the pocket residues (Figure S5, Supporting Infor-
mation). The rigid propargyl linker does not allow interactions
with the side pocket of HDAC8 that have been shown to be
important for highly potent “linkerless” HDAC8 inhibitors.[42]
4
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Ethyl 4-((S)-3-(((S)-tert-Butylsulfinyl)amino)-5-methylhex-1-yn-1-
Cytotoxicity tests were performed with the cervix carcinoma cell
line KB-3-1. The cytotoxicity of all investigated compounds cor-
relates qualitatively with the affinity to HDAC1, but not at all with
the affinity to HDAC6 which is in accordance to previous publica-
tions.[19,20] (Table 2). This observation confirms the previous
study of Lagger et al. who elucidated the effect of HDAC1 inhibi-
tion by microarray analysis and knockout mice.[16] Interestingly,
the thiophene based compound 6i is much less cytotoxic, con-
sidering its activity to inhibit HDAC1. Decreased general cytotox-
icity of HDAC6 selective compounds like 6h or 6i makes them
potentially interesting for non-cancer indications, like neuropro-
tection.[43]
yl)benzoate (5b). Instead of the methyl ester 2a, ethyl 4-iodobenzoate
was applied. Yield: 23.0 mg, 63.3 mmol, 94%. 1H NMR (500 MHz, CDCl3)
δ = 7.94 (d, 3J = 8.4 Hz, 2H, ar-2-H, ar-6-H), 7.47 (d, 3J = 8.4 Hz, 2H, ar-
3-H, ar-5-H), 4.35 (q, 3J = 7.1 Hz, 2H, CO2-CH2), 4.27 (q, 3J = 7.52 Hz,
1H, CαH), 3.36 (d, 3J = 7.3 Hz, 1H, NH), 1.91 (m, 1H, Me2CH), 1.67 (t, 3J
= 7.4 Hz, 2H, HNCαH-CH2), 1.37 (t, 3J = 7.13 Hz, 3H, CO2CH2CH3), 1.22
(s, 9H, SC(CH3)3), 0.95 (d, 3J = 6.7 Hz, 3H, MeCHCH3), 0.95 (d, 3J =
6.7 Hz, 3H, MeCHCH3). 13C NMR (126 MHz, CDCl3) δ = 166.2 (CO2Et),
131.7 (ar-C-3, ar-C-5), 130.0 (ar-C-1), 129.4 (ar-C-2, ar-C-6), 127.4 (ar-
C-4), 92.4 (Cα-C≡C-ar), 84.2 (Cα-C≡C-ar), 61.2 (CO2CH2), 56.4 (C(CH3)3),
47.1 (NHCα), 46.2 (CαCH2), 25.1 ((CH3)2CH), 22.6 (SC(CH3)3), 22.3
(CH(CH3)2), 14.4 (CO2CH2CH3). C20H29NO3S (363.52 g mol-1), MS (ESI):
m/z = 386.17499 (calcd. 386.17604 [M+Na]+). TLC: Rf (EtOAc/PE, 1:1) =
0.26.
Conclusions
Methyl 4-((S)-3-(((S)-tert-Butylsulfinyl)amino)-4-methylpent-1-yn-1-
yl)benzoate (5c). Yield: 69.0 mg, 0.206 mmol, 83%. 1H NMR (500 MHz,
CDCl3) δ = 7.93 (d, 3J = 8.3 Hz, 2H, ar-2-H, ar-6-H), 7.46 (d, 3J = 8.3 Hz,
2H, ar-3-H, ar-5-H), 4.09 (dd, 3J = 7.0 Hz, 3J = 5.2 Hz, 1H, HNCαH), 3.87
(s, 3H, CO2CH3), 3.41 (d, 3J = 7.0 Hz, 1H, NH), 2.00 (m, 1H, (CH3)2CH),
By coupling enantiomerically pure propargylamines to aromatic
hydroxamate derivatives a versatile type of compounds has
been obtained and used as a linker scaffold for potent HDAC
inhibitors. R-configured propargylamines that mimic S-con-
figured lysine based substrates display higher activity and selec-
tivity towards HDAC6. In particular, thiophene derivative 6i
emerged as a selective low nM HDAC6 inhibitor with good
HDAC selectivity while retaining low cytotoxicity. The observed
structure-activity relationship data, in particular with respect to
the influence of chirality, are clearly supported by molecular
docking studies.
3
1.22 (s, 9H, C(CH3)3), 1.04 (d, 3J = 6.8 Hz, 3H, MeCHCH3), 1.04 (d, J =
6.8 Hz, 3H, MeCHCH3). 13C NMR (126 MHz, CDCl3)
δ = 166.6
(CO2(CH3)), 131.8 (ar-C-3), 129.6 (ar-C-1), 129.4 (ar-C-2), 127.5 (ar-C-4),
90.9 (Cα-C≡C-ar), 85.0 (Cα-C≡C-ar), 56.4 (C(CH3)3), 54.4 (HNCα), 52.3
(CO2CH3), 33.9 (CH(CH3)2), 22.7 (C(CH3)3), 19.1 (MeCHCH3), 17.7
(MeCHCH3). C18H25NO3S (335.46), MS (ESI): m/z = 358.1452 (calcd.
358.1447 [M+Na]+), [α]D20 = 13.7 (c = 0.325, CHCl3). TLC: Rf (EtOAc/PE,
1:1) = 0.28. IR (ATR): ν̃
[cm-1] = 2958 (CH3/CH3), 2927-2873 (CH3/CH2),
1720 (CO2Me), 1603 (N-H), 1280 (S=O), 1442/1306/1173 (ar, C=C), 770
(S-C).
Methyl 4-((R)-3-(((R)-tert-Butylsulfinyl)amino)-4-methylpent-1-yn-1-
yl)benzoate (5e). Yield: 105.7 mg, 0.315 mmol, 64%. 1H NMR (600 MHz,
CDCl3) δ = 7.96 (d, 3J = 8.3 Hz, 2H, ar-2-H, ar-6-H), 7.50 (d, 3J = 8.3 Hz,
2H, ar-3-H, ar-5-H), 4.13 (t, 3J = 5.7 Hz, 1H, NH-CαH), 3.91 (s, 3H,
CO2CH3), 3.38 (d, 3J = 6.9 Hz, 1H, NH), 2.04 (dqq, 3J = 6.6 Hz, 3J =
6.7 Hz, 3J = 6.7 Hz, 1H, (CH3)2CH), 1.26 (s, 9H, C(CH3)3), 1.07 (d, 3J =
6.8 Hz, 3H, CHCH3), 1.07 (d, 3J = 6.7 Hz, 3H, CHCH3). 13C NMR
(126 MHz, CDCl3) δ = 166.6 (CO2Me), 131.8 (ar-2-C, ar-6-C), 129.6 (ar-
4-C), 129.4 (ar-3-C, ar-5-C), 127.5 (ar-1-C), 90.9 (Cα-C≡C-ar), 85.0 (Cα-
C≡C-ar), 56.4 (SC(CH3)3), 54.4 (HNCαH), 52.3 (CO2CH3), 33.9
((CH3)2CH), 22.7 (C(CH3)3), 19.1 ((CH3)CH-CH3), 17.7 ((CH3)CH-CH3).
MS (ESI): m/z = 358.1452 (calcd. 358.1447 [M+Na]+), TLC: Rf (EtOAc/PE,
Experimental Section
A detailed description of the techniques, experiments, characterizations,
spectra and chromatograms is given in the Supporting Information.
Compounds 1-4 are described in the Supporting Information.
General procedure for the synthesis of propargylic aromatics 5. The
aromatic halide 2 or 3 (1.6 eq) was added to a solution of propargylamine
4a-g (1 eq) in a mixture of dry THF and piperidine (3:1, 6 eq piperidine)
and the solution was thoroughly degassed by freeze-pump-thaw cycles
(3 x 10-2 mbar). Afterwards the catalysts PdCl2(PPh3)2 (1 mol%) and CuI
(2 mol%) were added and the solution warmed to rt. While the slightly
yellow solution was stirred for 0.5 to 14 h at ambient temperature, a
colourless precipitate formed. The suspension was diluted with saturated
NH4Cl solution (ca. 10 mL) and neutralized with aqueous HCl (2 M). After
separation of the phases, the aqueous layer was extracted with Et2O (3 x
20 mL) and the combined organic phases dried over Na2SO4. The crude
product was purified by column chromatography (EtOAc/PE, 1:1) and the
title compound isolated in form of a faint green oil.
1:1) = 0.28. IR (ATR): ν̃
[cm-1] = 2958 (CH3/CH3), 2927-2873 (CH3/CH2),
1720 (CO2Me), 1603 (N-H), 1280 (S=O), 1442/1306/1173 (ar, C=C), 770
(S-C).
Ethyl 4-((R)-3-(((R)-tert-Butylsulfinyl)amino)-5-methylhex-1-yn-1-yl)ben-
zoate (5f). Instead of the methyl ester 2a, ethyl 4-iodobenzoate was
applied. Yield: 671.3 mg, 1.847 mmol, 95%. 1H NMR (500 MHz, CDCl3) δ
= 7.97 (d, 3J = 8.3 Hz, 2H, ar-2-H, ar-6-H), 7.49 (d, 3J = 8.4 Hz, 2H, ar-3-
H, ar-5-H), 4.37 (q, 3J = 7.1 Hz, 2H, CO2CH2), 4.29 (q, 3J = 7.5 Hz, 1H,
HNCαH), 3.39 (d, 3J = 7.3 Hz, 1H, NH), 1.92 (m, 1H, (CH3)2CH), 1.70 (t,
3J = 7.4 Hz, 2H, HNCH-CH2), 1.39 (t, 3J = 7.1 Hz, 3H, CO2CH2CH3), 1.25
(s, 9H, C(CH3)3), 0.98 (d, 3J = 6.7 Hz, 3H, (CH3)CHCH3), 0.98 (d, 3J =
6.7 Hz, 3H, (CH3)CHCH3). 13C NMR (126 MHz, CDCl3) δ = 166.2 (CO2Et),
131.7 (ar-C-2, ar-C-6), 130.0 (ar-C-1), 129.4 (ar-C-3, ar-C-5), 127.4 (ar-
C-4), 92.4 (Cα-C≡C-ar), 84.2 (Cα-C≡C-ar), 61.2 (CO2CH2CH3), 56.4
(C(CH3)3), 47.1 (CαNH), 46.2 (CαCH2), 25.1 ((CH3)2CH), 22.6 (C(CH3)3),
22.3 (CH(CH3)2), 14.4 (CO2CH2CH3). C20H29NO3S (363.52 g mol-1), MS
(ESI): m/z = 386.17645 (calcd. 386.17604 [M+Na]+). TLC: Rf (EtOAc/PE,
1:1) = 0.26.
Methyl 4-((S)-3-(((S)-tert-Butylsulfinyl)amino)-3-cyclohexylprop-1-yn-1-
yl)benzoate (5a). Yield: 102.2 mg, 0.27 mmol, 66%. 1H NMR (500 MHz,
CDCl3) δ = 7.93 (d, 3J = 8.2 Hz, 2H, ar-2-H, ar-6-H), 7.47 (d, 3J = 8.1 Hz,
2H, ar-3-H, ar-5-H), 4.06 (dd, 3J = 7.5 Hz, 3J = 5.8 Hz, 1H, HNCαH), 3.88
(s, 3H, CO2CH3), 3.36 (d, 3J = 7.4 Hz, 1H, NH), 1.93-1.82 (m, 2H, cy-H),
1.83-1.72 (m, 2H, cy-H), 1.70-1.61 (m, 2H, cy-H), 1.22 (s, 9H, C(CH3)3),
1.19-1.06 (m, 5H, cy-H). 13C NMR (126 MHz, CDCl3) δ = 166.6 (CO2),
131.8 (ar-C-2, ar-C-6), 129.6 (ar-C-3, ar-C-5), 129.4 (ar-C-4), 127.6 (ar-
C-1), 91.4 (Cα-C≡C-ar), 85.0 (Cα-C≡C-ar), 56.5 (SC(CH3)3), 53.8
(CO2CH3), 52.3 (HNCα), 43.5 (cy-C-1), 29.7 (cy-C-2), 28.6 (cy-C-6), 26.4
(cy-C-4), 26.0 (cy-C-5), 25.9 (cy-C-3), 22.7 (C(CH3)3). C21H29NO3S
(375.53 g mol-1)
TLC: Rf (EtOAc/PE, 1:1) = 0.25.
.
MS (ESI): m/z = 398.1804 (calcd. 398.1760 [M+Na]+).
Methyl 4-((R)-3-(((R)-tert-Butylsulfinyl)amino)-3-cyclohexylprop-1-yn-1-
yl)benzoate (5g). Yield: 114.8 mg, 0.306 mmol, 74%. 1H NMR (500 MHz,
CDCl3) δ = 7.93 (d, 3J = 8.2 Hz, 2H, ar-2-H, ar-6-H), 7.47 (d, 3J = 8.1 Hz,
2H, ar-3-H, ar-5-H), 4.06 (dd, 3J = 7.5 Hz, 3J = 5.8 Hz, 1H, HNCαH), 3.88
5
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700550
ChemMedChem
FULL PAPER
(s, 3H, CO2CH3), 3.36 (d, 3J = 7.4 Hz, 1H, NH), 1.93-1.82 (m, 2H, cy-H),
1.83-1.72 (m, 2H, cy-H), 1.70-1.61 (m, 2H, cy-H), 1.22 (s, 9H, C(CH3)3),
1.19-1.06 (m, 5H, cy-H). 13C NMR (126 MHz, CDCl3) δ = 166.6 (CO2),
131.8 (ar-C-2, ar-C-6), 129.6 (ar-C-4), 129.4 (ar-C-3, ar-C-5), 127.6 (ar-
C-1), 91.4 (Cα-C≡C-ar), 85.0 (Cα-C≡C-ar), 56.5 ((CH3)3C), 53.8 (CO2CH3),
52.3 (HNCαH), 43.5 (HNCHCH2), 29.7 (cy-C-2), 28.6 (cy-C-6), 26.4 (cy-
C-4), 26.0 (cy-C-5), 25.9 (cy-C-3), 22.7 (C(CH3)3). C21H29NO3S
(375.53 g mol-1), MS (ESI): m/z = 398.1804 (calcd. 398.1760 [M+Na]+).
TLC: Rf (EtOAc/PE, 1:1) = 0.25.
of the solution with aqueous HCl (1 M, 6-8 drops) and addition of Et2O
(ca. 10 mL), the hydroxamic acids precipitated in form of a colourless
solid. The solvent was evaporated under reduced pressure and the
remainder was dissolved in acetonitrile (1 mL) and the inhibitors 6 were
purified by preparative RP-HPLC in form of a colourless solid.
(S)-N-(1-Cyclohexyl-3-(4-(hydroxaminocarbonyl)phenyl)prop-2-yn-1-
yl)benzofuran-2-carboxamide (6a). Yield: 25.9 mg, 62 µmol, 24%.
1H NMR (600 MHz, CD3OD) δ = 11.29 (s, 1H, CONHOH), 9.18 (d, 3J =
8.5 Hz, 1H, benzofuran-CONHOH), 7.77 (d, 3J = 8.4 Hz, 1H, benzofuran-
8-H), 7.73 (d, 3J = 8.4 Hz, 2H, ar-2-H, ar-6-H), 7.67 (t, 3J = 8.4 Hz, 1H,
benzofuran-5-H), 7.54 (d, 4J = 0.7 Hz, 1H, benzofuran-3-H), 7.52 (d, 3J =
8.3 Hz, 2H, ar-3-H, ar-5-H), 7.47 (dd, 3J = 6.8 Hz, 3J = 7.2 Hz, 1H, benzo-
furan-7-H), 7.34 (t, 3J = 7.5 Hz, 1H, benzofuran-6-H), 4.86 (d, 3J = 7.9 Hz,
1H, HNCαH), 2.04 (m, 1H, cy-H), 1.86-1.67 (m, 3H, cy-H), 1.61 (m, 1H,
cy-H), 1.29-1.11 (m, 5H, cy-H), 1.05 (m, 1H, cy-H). 13C NMR (151 MHz,
DMSO) δ = 163.5 (CONHOH), 157.7 (CONHCα), 154.4 (benzofuran -C-
9), 148.6 (benzofuran-C-2), 132.5 (ar-C-4), 131.5 (ar-C-1), 127.3 (ar-C-3,
ar-C-5), 127.1 (ar-C-2, ar-C-6), 127.1 (ar-C-4), 125.1 (benzofuran-C-7),
123.8 (benzofuran-C-6), 122.9 (benzofuran-C-5), 112.0 (benzofuran-C-8),
110.2 (benzofuran-C-3), 90.7 (Cα-C≡C-ar), 82.4 (Cα-C≡C-ar), 46.3 (Cα),
41.7 (cy-C-1), 29.3 (cy-C-2), 29.2 (cy-C-6), 25.9 (cy-C-4), 25.4 (cy-C-5),
Methyl 5-((R)-3-(((R)-tert-Butylsulfinyl)amino)-3-cyclohexylprop-1-yn-1-
yl)picolinate (5h). Picolinate 2b was used as aromatic halide. This reac-
tion was performed at 60 °C for 16 h. Yield: 64.6 mg, 0.172 mmol, 41%.
1H NMR (300 MHz, CDCl3) δ = 8.71 (dd, 4J = 2.1 Hz, 5J = 0.9 Hz, 1H, py-
6-H), 8.04 (dd, 3J = 8.1 Hz, 5J = 0.9 Hz, 1H, py-3-H), 7.86 (dd, 3J = 8.1 Hz,
4J = 2.1 Hz, 1H, py-4-H), 4.07 (dd, 3J = 7.9 Hz, 3J = 5.9 Hz, 1H, HNCαH),
3.98 (s, 3H, CO2CH3), 3.37 (d, 3J = 7.9 Hz, 1H, NH), 1.94-1.83 (m, 3H,
cy-H), 1.81-1.72 (m, 3H, cy-H), 1.71-1.62 (m, 2H, cy-H), 1.22 (s, 9H,
C(CH3)3), 1.18-1.07 (m, 3H, cy-H). 13C NMR (151 MHz, CDCl3) δ = 165.3
(CO2(CH3)), 152.1 (py-C-6), 146.3 (py-C-2), 139.9 (py-C-4), 124.5 (py-C-
3), 123.5 (py-C-5), 95.1 (Cα-C≡C-ar), 82.0 (Cα-C≡C-ar), 56.7 (C(CH3)3),
54.0 (CO2CH3), 53.1 (NHCα), 43.5 (cy-C-1), 29.6 (cy-C-2), 28.8 (cy-C-6),
26.3 (cy-C-4), 26.0 (cy-C-5), 25.9 (cy-C-3), 22.7 (C(CH3)3). C20H28N2O3S
(376.51 g mol-1), MS (ESI): m/z = 399.1625 (calcd. 399.1713 [M+Na]+),
25.4 (cy-C-3).
439.16283 (calc. 439.1638 [M+Na]+). [α]D20 = 58.3 (c = 0.105, MeOH). IR
(ATR): ν
[cm-1] = 3224 (CONHO-H), 2920 (N-HCO), 2851 (CON-HOH),
C
25H24N2O4 (417.47 gmo-1). MS (nanoESI): m/z =
IR (ATR): ν
̃
[cm-1] = 2927 (CH2), 2854 (CH2, NH), 1724 (CO2Me), 1448
̃
(ar, C=C), 1302 (ar, C=C). TLC: Rf (EtOAc/PE, 1:2) = 0.05.
1730 (HO-NH-C=O), 1632 (NH-C=O), 1591 (Car=Car), 1581 (Car=Car),
1502 (Car=Car), 1445 (Car=Car).
Methyl 5-((R)-3-(((R)-tert-Butylsulfinyl)amino)-3-cyclohexylprop-1-yn-1-
yl)thiophene-2-carboxylate (5i). Thiophene derivative 2c was applied as
aromatic halide. Yield: 36.6 mg, 95.9 nmol, 23%. 1H NMR (500 MHz,
CDCl3) δ = 7.62 (d, 3J = 3.9 Hz, 1H, thiophene-2-H), 7.12 (d, 3J = 3.9 Hz,
1H, thiophene-3-H), 4.06 (d, 3J = 6.2 Hz, 1H, NHCαH), 3.87 (s, 3H,
CO2CH3), 1.91-1.84 (m, 2H, cy-H), 1.83-1.74 (m, 2H, cy-H), 1.73-1.62 (m,
2H, cy-H), 1.29 (s, 9H, C(CH3)3), 1.26-1.02 (m, 5H, cy-H). 13C NMR
(126 MHz, CDCl3) δ = 162.1 (CO2(CH3)), 133.9 (thiophene-C-5), 133.3
(thiophene-C-3), 132.7 (thiophene-C-4), 129.1 (thiophene-C-1), 94.1
(CαHC≡C-ar), 78.8 (CαHC≡C-ar), 57.6 (C(CH3)3), 55.1 (CO2CH3), 52.5
(HNCαH), 43.6 (cy-C-1), 29.6 (cy-C-2), 28.9 (cy-C-6), 26.3 (cy-C-4), 25.9
(cy-C-5), 25.8 (cy-C-3), 22.8 (C(CH3)3). C19H27NO3S2 (381.55 g mol-1).
MS (ESI): m/z = 404.1298 (calcd. 404.1325 [M+Na]+), [α]D22 = -11.3 (c =
(S)-N-(1-(4-(Hydroxaminocarbonyl)phenyl)-5-methylhex-1-yn-3-yl)benzo-
furan-2-carboxamide (6b). Yield: 10.88 mg, 27.87 μmol, 43%. 1H NMR
(600 MHz, CD3OD) δ = 7.74 (t, 3J = 5.2 Hz, 1H, benzofuran-8-H), 7.72 (d,
3J = 8.4 Hz, 2H, ar-6-H, ar-2-H), 7.62 (d, 3J = 8.4 Hz, 1H, benzofuran-5-
H), 7.55 (s, 1H, benzofuran-3-H), 7.52 (d, 3J = 8.3 Hz, 2H, ar-5-H, ar-3-
H), 7.47 (t, 3J = 7.8 Hz, 1H, benzofuran-6-H), 7.33 (t, 3J = 7.5 Hz, 1H,
benzofuran-7-H), 5.23 (t, 3J = 7.7 Hz, 1H, HNCαH), 1.88 (m, 1H,
(H3C)2CH), 1.84 (t, 3J = 6.9 Hz, 2H, CH2), 1.04 (d, 3J = 6.5 Hz, 3H, CH3),
1.04 (d, 3J = 6.5 Hz, 3H, CH3). 13C NMR (151 MHz, CD3OD) δ = 167.3
(ar-CONHOH), 160.3 (benzofuran-CONH), 156.5 (benzofuran-C-9),
149.6 (benzofuran-C-2), 133.2 (ar-C-1), 132.8 (ar-C-3, ar-C-5), 128.7
(benzofuran-C-6), 128.3 (ar-C-4), 128.2 (ar-C-2, ar-C-6), 127.6 (benzofu-
ran-C-4), 124.9 (benzofuran-C-7), 123.8 (benzofuran-C-8), 112.9 (benzo-
furan-C-5), 111.8 (benzofuran-C-3), 91.9 (Cα-C≡C-ar), 82.8 (Cα-C≡C-ar),
45.4 (HNCαH), 41.2 (CH2), 26.4 ((CH3)2CH), 22.8 (CH3), 22.5 (CH3).
0.40, MeOH). TLC: Rf (EtOAc/PE, 1:1) = 0.16. IR (ATR): ν̃
[cm-1] = 2927
(cy, CH2), 2844 (CH3), 1711 (CO2CH3), 1448 (HNS=O), 1258 (ar, C=C),
1173 (ar, C=C), 1097 (ar, C=C).
C
23H22N2O4 (390.44 g mol-1), MS (nanoESI): m/z = 391.1623 (calc.
391.1652 [M+H]+). [α]D22 = 26.3 (c = 0.065, MeOH). IR (ATR): ν [cm-1] =
̃
General procedure for the preparation of hydroxamic acids 6 from 5:
Acidic methanolysis of the Bus or Boc group: HCl (4 M in dioxane,
4 eq) was slowly added under vigorous stirring to a solution of 5 (1 eq) in
methanol (20 mL). After 2-4 h, the solvent of the slightly yellow reaction
mixture was evaporated to yield a yellow solid. This solid was treated
with DCM and the solvent coevaporated (3 x). The ammonium chloride
salt of the desired amine was isolated quantitatively in form of a colour-
less, odourless solid.
Acylation with benzofuran-2-carbonyl chloride: The propargylammo-
nium chloride salt (65.0 mg, 0.24 mmol, 1.0 eq) and benzofuran-2-
carbonyl chloride (1.1 eq) were dissolved in THF (1.5 mL) at -30 °C
under inert gas atmosphere. Triethylamine (2.5 eq) was added and the
mixture was slowly warmed up to room temperature. Afterwards the
mixture was diluted with ethyl acetate (10 mL) and washed with hydro-
chloric acid (0.5 M, 10 mL), sodium bicarbonate solution (10 mL) and
brine (10 mL). The organic layer was dried over sodium sulfate, the
solvent evaporated and the crude product was purified by column chro-
matography (EtOAc/Petrolether, 1:1) to yield carboxamide as a colorless,
crystalline solid (20-45%).
3237 (CONHOH), 2955 (N-HCO), 2924 (CH3), 2867 (CON-HOH), 1644
(C=ONHOH), 1597 (NHC=O), 1521-1505 (Car=Car).
(S)-N-(1-(4-(Hydroxaminocarbonyl)phenyl)-4-methylpent-1-yn-3-yl)ben-
zofuran-2-carboxamide (6c). Yield: 19.1 mg, 50.7 μmol, 16%. 1H NMR
(600 MHz, CD3OD): δ = 7.73 (d, 3J = 8.4 Hz, 1H, benzofuran-8-H), 7.72
(d, 3J = 8.4 Hz, 2H, ar-2-H, ar-6-H), 7.62 (d, 3J = 8.4 Hz, 1H, benzofuran-
5-H), 7.56 (s, 1H, benzofuran-3-H), 7.54 (d, 3J = 8.3 Hz, 2H, ar-3-H, ar-5-
H), 7.47 (t, 3J = 8.2 Hz, 1H, benzofuran-6-H), 7.33 (t, 3J = 7.5 Hz, 1H,
benzofuran-7-H), 4.97 (d, 3J = 7.3 Hz, 1H, CαH), 2.17 (dqq, 3J = 6.3 Hz,
3J = 6.7 Hz, 3J = 6.7 Hz, 1H, (CH3)2CH), 1.19 (d, 3J = 6.7 Hz, 3H, CH3),
1.11 (d, 3J = 6.7 Hz, 3H, CH3). 13C NMR (151 MHz, CD3OD): δ = 165.9
(CONHOH), 159.1 (benzofuran-CONH), 155.1 (benzofuran-C-9), 148.2
(benzofuran-C-2), 131.8 (ar-C-1), 131.4 (ar-C-3, ar-C-5), 127.3 (benzofu-
ran-C-4), 126.9 (ar-C-4), 126.8 (ar-C-2, ar-C-6), 126.2 (benzofuran-C-6),
123.5 (benzofuran-C-7), 122.4 (benzofuran-C-8), 111.5 (benzofuran-C-5),
110.4 (benzofuran-C-3), 89.2 (Cα-C≡C-ar), 82.6 (Cα-C≡C-ar), 47.8 (Cα),
33.2 ((CH3)2HC), 18.2 (CH3), 17.8 (CH3). C22H20N2O4 (376.41 g mol-1. MS
(nanoESI): m/z = 377.1516 (calcd. 377.1496 [M+H]+). [α]D22 = 58.9 (c =
Hydroxamate formation: Hydroxylamine (0.5 mL, 50% in H2O) was
added dropwise to a vigorously stirred solution of ester (1 eq) in a mix-
ture of THF and MeOH (1:1, 4 mL). Aqueous NaOH (1 M, ca. 4 drops)
was added until a pH value of 10-11 was reached and the reaction mix-
ture was stirred further 14 h at ambient temperature. After neutralization
0.425, MeOH), IR (ATR): ν̃
[cm-1] = 3231 (CONHO-H), 2962 (N-HCO),
2930 (CH3), 2870 (CON-HOH), 1641 (C=ONHOH), 1597 (NHC=O), 1423
(Car=Car).
6
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700550
ChemMedChem
FULL PAPER
(R)-N-(1-(4-(Hydroxaminocarbonyl)phenyl)-4-methylpent-1-yn-3-yl)ben-
zofuran-2-carboxamide (6e). Yield: 15.2 mg, 40.4 μmol, 37%. 1H NMR
(600 MHz, CD3OD): δ = 7.71 (d, 3J = 8.4 Hz, 1H, benzofuran-8-H), 7.71
(d, 3J = 8.4 Hz, 2H, ar-2-H, ar-6-H), 7.60 (d, 3J = 8.4 Hz, 1H, benzofuran-
5-H), 7.53 (s, 1H, benzofuran-3-H), 7.52 (d, 3J = 8.3 Hz, 2H, ar-3-H, ar-5-
H), 7.45 (td, 3J = 7.9 Hz, 4J = 1.3 Hz, 1H, benzofuran-6-H), 7.31 (t, 3J =
3H, cy-H), 1.70 (d, 2J = 11.9 Hz, 1H, cy-4-H), 1.40-1.10 (m, 5H, cy-H).
13C NMR (126 MHz, CD3OD) δ = 163.5 (CONHOH), 160.5 (CONH),
156.5 (benzofuran-C-9), 152.2 (py-C-2), 149.5 (py-C-6), 149.4 (benzofu-
ran-C-2), 141.1 (py-C-3), 128.7 (benzofuran-C-4), 128.4 (benzofuran-C-
6), 125.0 (py-C-5), 124.1 (benzofuran-C-7), 123.8 (benzofuran-C-8),
122.6 (py-C-4), 112.9 (benzofuran-C-5), 111.9 (benzofuran-C-3), 94.3
(Cα-C≡C-ar), 81.1 (Cα-C≡C-ar), 48.2 (NHCα), 43.7 (cy-C-1), 30.7 (cy-C-2),
30.7 (cy-C-6), 27.3 (cy-C-4), 26.9 (cy-C-5), 26.9 (cy-C-3). C24H23N3O4
3
7.5 Hz, 1H, benzofuran-7-H), 4.95 (d, J = 7.3 Hz, 1H, NHCαH), 2.16 (m,
1H, (CH3)2CH), 1.17 (d, 3J = 6.7 Hz, 3H, CH3), 1.09 (d, 3J = 6.7 Hz, 3H,
CH3). 13C NMR (151 MHz, CD3OD): δ = 165.8 (CONHOH), 159.1 (benzo-
furan-CONH), 155.1 (benzofuran-C-9), 148.2 (benzofuran-C-2), 131.8
(ar-C-1), 131.4 (ar-C-3), 127.3 (benzofuran-C-4), 126.9 (ar-C-4), 126.8
(ar-C-2, ar-C-6), 126.2 (benzofuran-C-6), 123.5 (benzofuran-C-7), 122.4
(benzofuran-C-8), 111.5 (benzofuran-C-5), 110.4 (benzofuran-C-3), 89.2
(Cα-C≡C-ar), 82.6 (Cα-C≡C-ar), 47.8 (NHCα), 33.2 ((CH3)2HC), 18.2
(CH3), 17.8 (CH3). C22H20N2O4 (376.41 mol-1). MS (nanoESI): m/z =
399.1347 (calcd. 399.1315 [M+Na]+). [α]2D2 = -61.4 (c = 0.355, MeOH). IR
(417.47 g mol-1). MS (nanoESI): m/z
[M+Na]+). [α]2D2 = -56.7 (c = 0.07, MeOH), IR (ATR): ν
=
440.1574 (calcd. 440.1581
[cm-1] = 3262
̃
(CONHO-H), 2926 (N-HCO), 2850 (CON-HOH), 2359 (C≡C), 2341 (C≡C),
1648 (NH-C=O), 1594 (Car=Car), 1553 (Car=Car), 1505 (Car=Car), 1448
(Car=Car).
(R)-N-(1-Cyclohexyl-3-(5-(hydroxaminocarbonyl)thiophen-2-yl)prop-2-yn-
1-yl)benzofuran-2-carboxamide (6i). Yield: 5.92 mg, 14 μmol, 16%.
1H NMR (300 MHz, DMSO-d6) δ = 11.34 (s, 1H, CONHOH), 9.23 (d, 3J =
8.6 Hz, 1H, CONH), 7.78 (dd, 3J = 7.8 Hz, 4J = 1.3 Hz, 1H, benzofuran-8-
H), 7.69 (d, 3J = 7.9 Hz, 1H, benzofuran-5-H), 7.66 (s, 1H, benzofuran-3-
H), 7.52 (d, 1H, thiophen-3-H), 7.48 (ddd, 3J = 8.5 Hz, 3J = 7.4 Hz, 4J =
1.4 Hz, 1H, benzofuran-6-H), 7.35 (td, 3J = 7.7 Hz, 4J = 0.7 Hz, 1H,
benzofuran-7-H), 7.31 (d, 1H, 3J = 4.0 Hz, thiophen-4-H), 4.88 (t, 3J =
8.4 Hz, 1H, NHCαH), 3.47 (s, 1H, CONHOH), 1.99 (m, 1H, cy-1-H), 1.89-
1.68 (m, 4H, cy-H), 1.62 (d, 2J = 9.0 Hz, 1H, cy-4-H), 1.30-1.10 (m, 4H,
cy-H), 1.02 (m, 1H, cy-H). 13C NMR (126 MHz, DMSO-d6) δ = 158.4
(CONHOH), 157.6 (CONH), 154.3 (benzofuran-C-9), 148.4 (benzofuran-
C-2), 138.3 (thiophene-C-3), 133.1 (thiophene-C-4), 127.5 (benzofuran-
C-6), 127.1 (thiophene-C-2), 127.0 (benzofuran-C-4), 125.3 (benzofuran-
C-6), 123.8 (benzofuran-C-7), 122.9 (benzofuran-C-8), 111.9 (benzofu-
ran-C-5), 110.1 (benzofuran-C-3), 99.5 (thiophene-C-5), 94.7 (Cα-C≡C-
ar), 75.7 (Cα-C≡C-ar), 46.3 (NHCα), 41.5 (cy-C-1), 29.2 (cy-C-2), 29.1
(cy-C-6), 25.8 (cy-C-4), 25.3 (cy-C-5), 25.3 (cy-C-3). C23H22N2O4S
(422.50). MS (nanoESI): m/z = 445.1190 (calcd. 445.1193 [M+Na]+). [α]D22
(ATR): ν̃
[cm-1] = 3231 (CONHO-H), 2962 (N-HCO), 2930 (CH3), 2870
(CON-HOH), 1641 (C=ONHOH), 1597 (NHC=O), 1423 (Car=Car).
(R)-N-(1-(4-(Hydroxaminocarbonyl)phenyl)-5-methylhex-1-yn-3-yl)benzo-
furan-2-carboxamide (6f). Yield: 5.48 mg, 14.0 μmol, 9%. 1H NMR
(600 MHz, CD3OD): δ = 7.71 (d, 3J = 7.2 Hz, 1H, benzofuran-8-H), 7.68
(d, 3J = 8.3 Hz, 2H, ar-6-H, ar-2-H), 7.58 (d, 3J = 8.2 Hz, 1H, benzofuran-
5-H), 7.51 (s, 1H, benzofuran-3-H), 7.49 (d, 3J = 7.9 Hz, 2H, ar-5-H, ar-3-
H), 7.44 (t, 3J = 7.6 Hz, 1H, benzofuran-6-H), 7.29 (t, 3J = 7.3 Hz, 1H,
benzofuran-7-H), 5.19 (t, 3J = 7.6 Hz, 1H, NHCαH), 1.89 (m, 1H,
(CH3)2CH), 1.79 (t, 3J = 7.2 Hz, 2H, CαCH2), 1.00 (d, 3J = 6.3 Hz, 6H,
C(CH3)2). 13C NMR (151 MHz, CD3OD): δ = 165.9 (CONHOH), 158.9
(benzofuran-CONH), 155.1 (benzofuran-C-9), 148.2 (benzofuran-C-2),
131.8 (ar-C-1), 131.4 (ar-C-3, ar-C-5), 127.3 (benzofuran-C-6), 126.9 (ar-
C-4), 126.8 (ar-C-2, ar-C-6), 126.2 (benzofuran-C-4), 123.5 (benzofuran-
C-7), 122.4 (benzofuran-C-8), 111.5 (benzofuran-C-5), 110.4 (benzofu-
ran-C-3), 90.5 (CαHC≡C-ar), 81.4 (CαHC≡C-ar), 44.0 (NHCαH), 39.8
(CαCH2), 25.0 ((CH3)2CH), 21.4 (CH3), 21.2 (CH3). C23H22N2O4
= -25.7 (c = 0.07, MeOH). IR (ATR): ν̃
[cm-1] = 3227 (CONHO-H), 2924
(N-HCO), 2851 (CON-HOH), 2357 (C≡C), 2341 (C≡C), 1730 (OHNH-
(390.44 g mol-1). MS (nanoESI): m/z
[M+H]+). [α]2D2 = -34.2 (c = 0.1, MeOH). IR (ATR): ν
=
391.1623 (calcd. 391.1652
[cm-1] = 3237 (CON-
̃
C=O), 1648 (NH-C=O), 1594 (Car=Car), 1515 (Car=Car), 1448 (Car=Car).
HO-H), 2955 (N-HCO), 2924 (CH3), 2867 (CON-HOH), 1644
(C=ONHOH), 1597 (NHC=O), 1521, 1505 (Car=Car).
In vitro testing [36–38]
(R)-N-(1-Cyclohexyl-3-(4-(hydroxaminocarbonyl)phenyl)prop-2-yn-1-yl)-
benzofuran-2-carboxamide (6g). Yield: 4.8 mg, 12 μmol, 6%. 1H NMR
(600 MHz, CD3OD) δ = 7.74 (d, 3J = 8.1 Hz, 1H, benzofuran-8-H), 7.72 (d,
3J = 8.5 Hz, 2H, ar-C-2, ar-C-6), 7.62 (d, 3J = 8.4 Hz, 1H, benzofuran-5-
H), 7.54 (s, 1H, benzofuran-3-H), 7.53 (d, 3J = 8.4 Hz, 2H, ar-C-3, ar-C-5),
7.46 (ddd, 3J = 8.4 Hz, 3J = 7.1 Hz, 4J = 1.3 Hz, 1H, benzofuran-7-H),
7.33 (t, 3J = 7.5 Hz, 1H, benzofuran-6-H), 4.96 (d, 3J = 7.9 Hz, 1H,
NHCαH), 2.12 (m, 1H, cy-H), 1.92 (m, 1H, cy-H), 1.89-1.77 (m, 3H, cy-H),
1.71 (m, 1H, cy-H), 1.41-1.13 (m, 5H, cy-H). 13C NMR (151 MHz,
CD3OD) δ = 163.5 (CONHOH), 157.7 (CONHC), 154.4 (benzofuran-C-9),
148.6 (benzofuran-C-2), 132.5 (ar-C-1), 131.5 (ar-C-2, ar-C-6), 127.3 (ar-
C-3, ar-C-5), 127.1 (benzofuran-C-4), 127.1 (ar-C-4), 125.1 (benzofuran-
C-7), 123.8 (benzofuran-C-6), 122.9 (benzofuran-C-5), 112.0 (benzofu-
ran-C-8), 110.2 (benzofuran-C-3), 90.7 (Cα-C≡C-ar), 82.4 (Cα-C≡C-ar),
46.3 (Cα), 41.7 (cy-C-1), 29.3 (cy-C-2), 29.2 (cy-C-6), 25.9 (cy-C-4), 25.4
(cy-C-5), 25.4 (cy-C-3). C25H24N2O4 (416.48 g mol-1). MS (nanoESI): m/z
= 439.1639 (calcd. 439.1628 [M+Na]+). [α]D22 = -55.8 (c = 0.069, MeOH).
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)
[44]
(126 µM) for 90 min at
37 °C. After the 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 Labtechnolo-
gies, Germany) with an excitation wavelength of 390 nm and an emission
wavelength of 460 nm.
IR (ATR): ν̃
[cm-1] = 3224 (CONHO-H), 2920 (N-HCO), 2851 (CON-HOH),
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.[45] 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 solution (see
HDAC1 and HDAC6) were added and the plate was incubated again at
37 °C for 30 min. Measurement was performed as described for
HDAC1/6.
1730 (HO-NH-C=O), 1632 (NH-C=O), 1591 (Car=Car), 1581 (Car=Car),
1502 (Car=Car), 1445 (Car=Car).
(R)-N-(1-Cyclohexyl-3-(2-(hydroxaminocarbonyl)pyridin-5-yl)prop-2-yn-1-
yl)benzofuran-2-carboxamide (6h). Yield: 9.99 mg, 23.9 μmol, 25%.
1H NMR (500 MHz, CD3OD) δ = 8.63 (s, 1H, py-6-H), 8.01 (m, 1H, py-4-
H), 7.97 (m, 1H, py-3-H), 7.73 (d, 3J = 7.7 Hz, 1H, benzofuran-8-H), 7.61
(d, 3J = 8.4 Hz, 1H, benzofuran-5-H), 7.55 (s, 1H, benzofuran-3-H), 7.46
(t, 3J = 7.7 Hz, 1H, benzofuran-6-H), 7.32 (t, 3J = 7.5 Hz, 1H, benzofuran-
7-H), 4.99 (d, 3J = 8.0 Hz, 1H, CαH), 3.35 (s, 1H, CONHOH), 2.12 (d, 2J =
12.3 Hz, 1H, cy-1-H), 1.92 (d, 2J = 12.5 Hz, 1H, cy-4-H), 1.89-1.77 (m,
7
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700550
ChemMedChem
FULL PAPER
Cytotoxicity assays
References
The cytotoxicity of the inhibitors was tested as previously described.[39]
The KB-3-1 cells were cultivated as a monolayer in DMEM (Dulbecco’s
modified Eagle medium) with glucose (4.5 g L-1), L-glutamine, sodium
pyruvate and phenol red, supplemented with 10% fetal bovine serum
(FBS). The cells were maintained at 37 °C and 5.3% CO2-humidified air.
On the day before the test, the cells (70% confluence) were detached
with trypsin-ethylenediamine tetraacetic acid (EDTA) solution (0.05%;
0.02% in DPBS) and placed in sterile 96-well plates in a density of 10
000 cells in 100 μL medium per well. The dilution series of the com-
pounds were prepared from stock solutions in DMSO of concentrations of
1 mM or 10 mM. The stock solutions were diluted with culture medium at
least 50 times. Some culture medium was added to the wells to adjust
the volume of the wells to the wanted dilution factor. The dilution pre-
pared from stock solution was added to the wells. Each concentration
was tested in six replicates. Dilution series were prepared by pipetting
liquid from well to well. The control contained the same concentration of
DMSO as the first dilution. After incubation for 72 h at 37 °C and 5.3%
CO2-humidified air, 30 μL of an aqueous resazurin solution (175 μM) was
added to each well. The cells were incubated at the same conditions for
6 h. Subsequently, the fluorescence was measured. The excitation was
effected at a wavelength of 530 nm, whereas the emission was recorded
at a wavelength of 588 nm. The EC50 values as the drug concentrations
resulting in 50% cell viability were calculated as a sigmoidal dose re-
sponse curve using GRAPHPAD PRISM 4.03.
[1]
M. Dokmanovic, C. Clarke, P. A. Marks, Mol. Cancer Res. MCR 2007,
5, 981-989.
[2]
[3]
J. M. Greer, P. A. McCombe, Biol. Targets Ther. 2012, 6, 307-327.
C. Cook, T. F. Gendron, K. Scheffel, Y. Carlomagno, J. Dunmore, M.
DeTure, L. Petrucelli, Hum. Mol. Genet. 2012, 21, 2936-2945.
G. I. Aldana-Masangkay, K. M. Sakamoto, J. Biomed. Biotechnol.
2011, 2011, 875824.
Y.-S. Lee, K.-H. Lim, X. Guo, Y. Kawaguchi, Y. Gao, T. Barrientos, P.
Ordentlich, X.-F. Wang, C. M. Counter, T.-P. Yao, Cancer Res. 2008,
68, 7561-7569.
A. A. Lane, B. A. Chabner, J. Clin. Oncol. Off. J. Am. Soc. Clin. Oncol.
2009, 27, 5459-5468.
B. S. Mann, J. R. Johnson, M. H. Cohen, R. Justice, R. Pazdur, The
Oncologist 2007, 12, 1247-1252.
T. Vanhaecke, P. Papeleu, G. Elaut, V. Rogiers, Curr. Med. Chem.
2004, 11, 1629-1643.
J. A. Plumb, P. W. Finn, R. J. Williams, M. J. Bandara, M. R. Romero,
C. J. Watkins, N. B. La Thangue, R. Brown, Mol. Cancer Ther. 2003, 2,
721-728.
J. Arts, P. Angibaud, W. Floren, B. Janssen, P. King, J. van Dun, L.
Janssen, T. Geerts, R. W. Tuman, D. L. Johnson, L. Andries, M. Jung,
M. Janicot, K. van Emelen, Br. J. Cancer 2007, 97, 1344-1353.
Y. B. Kim, K. H. Lee, K. Sugita, M. Yoshida, S. Horinouchi, Oncogene
1999, 18, 2461-2470.
K. V. Butler, J. Kalin, C. Brochier, G. Vistoli, B. Langley, A. P. Ko-
zikowski, J. Am. Chem. Soc. 2010, 132, 10842-10846.
H. Ueda, H. Nakajima, Y. Hori, T. Fujita, M. Nishimura, T. Goto, M.
Okuhara, J. Antibiot. (Tokyo) 1994, 47, 301-310.
W. K. Kelly, O. A. O’Connor, L. M. Krug, J. H. Chiao, M. Heaney, T.
Curley, B. MacGregore-Cortelli, W. Tong, J. P. Secrist, L. Schwartz, S.
Richardson, E. Chu, S. Olgac, P. A. Marks, H. Scher, V. Richon, J.
Clin. Oncol. Off. J. Am. Soc. Clin. Oncol. 2005, 23, 3923-3931.
Z.-Q. Ning, Z.-B. Li, M. J. Newman, S. Shan, X.-H. Wang, D.-S. Pan, J.
Zhang, M. Dong, X. Du, X.-P. Lu, Cancer Chemother. Pharmacol.
2012, 69, 901-909.
G. Lagger, D. O’Carroll, M. Rembold, H. Khier, J. Tischler, G. Weitzer,
B. Schuettengruber, C. Hauser, R. Brunmeir, T. Jenuwein, C. Seiser,
EMBO J. 2002, 21, 2672-2681.
C. M. Trivedi, Y. Luo, Z. Yin, M. Zhang, W. Zhu, T. Wang, T. Floss, M.
Goettlicher, P. R. Noppinger, W. Wurst, V. Ferrari, C. Abrams, P. J.
Gruber, J. Epstein, Nat. Med. 2007, 13, 324-331.
C. L. Zhang, T. A. McKinsey, S. Chang, C. L. Antos, J. A. Hill, E. N.
Olson, Cell 2002, 110, 479-488.
S. J. Haggarty, K. M. Koeller, J. C. Wong, C. M. Grozinger, S. L.
Schreiber, Proc. Natl. Acad. Sci. U. S. A. 2003, 100, 4389-4394.
Y. Zhang, S. Kwon, T. Yamaguchi, F. Cubizolles, S. Rousseaux, M.
Kneissel, C. Cao, N. Li, H.-L. Cheng, K. Chua, D. Lombard, A. Mi-
zeracki, G. Matthias, F. Alt, S. Kochbin, P. Matthias, Mol. Cell. Biol.
2008, 28, 1688-1701.
Y. Zhang, N. Li, C. Caron, G. Matthias, D. Hess, S. Khochbin, P.
Matthias, EMBO J. 2003, 22, 1168-1179.
S. Dallavalle, C. Pisano, F. Zunino, Biochem. Pharmacol. 2012, 84,
756-765.
[4]
[5]
[6]
[7]
[8]
[9]
[10]
[11]
[12]
[13]
[14]
Docking studies
[15]
[16]
[17]
The resolved crystal structure of human HDACs complexed with inhibi-
[46]
tors were taken from the Protein Data Bank (PDB¸ www.rcsb.org)
HDAC6 complexed with TSA (PDB ID 5EDU, resolution 2.79 Å)
HDAC8 complexed with Trapoxin A (PDB ID 5VI6, resolution 1.24 Å)
:
,
[25]
[40]
and HDAC1 complexed with acetate (PDB ID 4BKX, resolution 3.0 Å). [41]
All protein structures were prepared using Schrödinger's Protein Prepara-
[18]
[19]
[20]
[47]
tion Wizard
by adding hydrogen atoms, assigning protonation states
and minimizing the protein. All inhibitors were prepared for docking using
[48]
the LigPrep tool
as implemented in Schrödinger’s software, where all
possible tautomeric forms as well as stereoisomers were generated and
energy minimized using the OPLS force field. Conformers of the pre-
pared inhibitor structures were calculated with ConfGen using the default
[21]
[22]
[23]
settings and allowing minimization of the output conformations. Inhibitor
[49]
docking was performed using the program Glide
in the Standard
Precision mode. In HDAC6 and HDAC8, the conserved water molecule
bound to His180 (HDAC8, His651 in HDAC6) was considered during the
docking procedure. The chosen docking protocol could perfectly repro-
duce the co-crystallized inhibitors in HDAC1, HDAC6 and HDAC8 as
demonstrated in former studies.[50–52] Top ranked HDAC-inhibitor com-
plexes were visually analyzed in MOE2012.10.[53]
J. P. Dompierre, J. D. Godin, B. C. Charrin, F. P. Cordelières, S. J.
King, S. Humbert, F. Saudou, J. Neurosci. Off. J. Soc. Neurosci. 2007,
27, 3571-3583.
E. Hahnen, J. Hauke, C. Tränkle, I. Y. Eyüpoglu, B. Wirth, I. Blümcke,
Expert Opin. Investig. Drugs 2008, 17, 169-184.
Y. Hai, D. W. Christianson, Nat. Chem. Biol. 2016, 12, 741-747.
M. S. Finnin, J. R. Donigian, A. Cohen, V. M. Richon, R. A. Rifkind, P.
A. Marks, R. Breslow, N. P. Pavletich, Nature 1999, 401, 188-193.
J. R. Somoza, R. J. Skene, B. A. Katz, C. Mol, J. D. Ho, A. J. Jennings,
C. Luong, A. Arvai, J. J. Buggy, E. Chi, J. Tang, B.-C. Sang, E. Verner,
R. Wynands, E. M. Leahy, D. Dougan, G. Snell, M. Navre, M. W.
Knuth, R. Swans, D. McRee, L. W. Tari, Structure 2004, 12, 1325-
1334.
A. Schuetz, J. Min, A. Allali-Hassani, M. Schapira, M. Shuen, P.
Loppnau, R. Mazitschek, N. P. Kwiatkowski, T. A. Lewis, R. L. Magla-
thin, T. McLean, A. Bochkarev, A. Plotnikov, M. Vedadi, C. Arrowsmith,
J. Biol. Chem. 2008, 283, 11355-11363.
[24]
[25]
[26]
[27]
[28]
Acknowledgements
Financial support by Deutsche Forschungsgemeinschaft
(SE609/10-1, JU295/13-1, and SI846/13-1) is gratefully
acknowledged.
[29]
[30]
[31]
[32]
Y. Liu, L. Li, J. Min, Nat. Chem. Biol. 2016, 12, 660-661.
K. V. Butler, A. P. Kozikowski, Curr. Pharm. Des. 2008, 14, 505-528.
T. Suzuki, Chem. Pharm. Bull. (Tokyo) 2009, 57, 897-906.
M. Sendzik, Acetylene Derivatives as Inhibitors of Histone Deacety-
lase, 2006, EP1656348 A1.
Conflict of interest
[33]
[34]
H. C. Kolb, M. G. Finn, K. B. Sharpless, Angew. Chem. Int. Ed Engl.
2001, 40, 2004-2021.
M. Wünsch, D. Schröder, T. Fröhr, L. Teichmann, S. Hedwig, N.
Janson, C. Belu, J. Simon, S. Heidemeyer, P. Holtkamp, J. Rudlof, L.
Klemme, A. Hinzmann, B. Neumann, H.-G. Stammler, N. Sewald,
Beilstein J. Org. Chem. 2017, in press.
The authors declare no conflict of interest.
Keywords: cancer | heterocycle | inhibitor | propargylamine |
histone deacetylase
[35]
D. A. Cogan, G. Liu, J. Ellman, Tetrahedron 1999, 55, 8883-8904.
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201700550
ChemMedChem
FULL PAPER
[36]
[37]
[38]
[39]
[40]
[41]
D. Wegener, F. Wirsching, D. Riester, A. Schwienhorst, Chem. Biol.
2003, 10, 61-68.
D. Wegener, C. Hildmann, D. Riester, A. Schwienhorst, Anal. Biochem.
2003, 321, 202-208.
B. Heltweg, J. Trapp, M. Jung, Methods San Diego Calif 2005, 36,
332-337.
B. Sammet, T. Bogner, M. Nahrwold, C. Weiss, N. Sewald, J. Org.
Chem. 2010, 75, 6953-6960.
N. J. Porter, D. W. Christianson, ACS Chem. Biol. 2017, 12, 2281-
2286.
P. J. Watson, C. J. Millard, A. M. Riley, N. S. Robertson, L. C. Wright,
H. Y. Godage, S. M. Cowley, A. G. Jamieson, B. V. L. Potter, J. W. R.
Schwabe, Nat. Commun. 2016, 7, 1-13.
[42]
[43]
[44]
[45]
A. A. Tabackman, R. Frankson, E. S. Marsan, K. Perry, K. E. Cole, J.
Struct. Biol. 2016, 195, 373-378.
X. Xu, A. P. Kozikowski, L. Pozzo-Miller, Front. Cell. Neurosci. 2014, 8,
68.
B. Heltweg, F. Dequiedt, E. Verdin, M. Jung, Anal. Biochem. 2003,
319, 42-48.
M. Marek, S. Kannan, A.-T. Hauser, M. M. Mourão, S. Caby, V. Cura,
D. A. Stolfa, K. Schmidtkunz, J. Lancelot, L. Andrade, J. P. Renaud, G.
Oliveira, W. Sippl, M. Jung, J. Cavarelli, R. Pierce, C. Romier, PLOS
Pathog. 2013, 9, 1-15.
[46]
[47]
H. M. Berman, J. Westbrook, Z. Feng, G. Gilliland, T. N. Bhat, H.
Weissig, I. N. Shindyalov, P. E. Bourne, Nucleic Acids Res. 2000, 28,
235-242.
Schrödinger Release 2014-2: Schrödinger Suite 2014-2 Protein
Preparation Wizard; Epik version 2.8, Schrödinger, LLC, New York,
NY, 2014; Impact version 6.3, Schrödinger, LLC, New York, NY, 2014;
Prime version 3.6, Schrödinger, LLC, New York, NY, 2014.
Schrödinger Release 2014-2: LigPrep, Schrödinger, LLC, New York,
NY, 2014.
[48]
[49]
[50]
[51]
Small-Molecule Drug Discovery Suite 2014-2: Glide, version 9.8,
Schrödinger, LLC, New York, NY, 2014.
S. Vojacek, K. Beese, Z. Alhalabi, S. Swyter, A. Bodtke, C. Schulzke,
M. Jung, W. Sippl, A. Link, Arch. Pharm. (Weinheim) 2017, 350, 1-19.
T. Heimburg, A. Chakrabarti, J. Lancelot, M. Marek, J. Melesina, A.-T.
Hauser, T. B. Shaik, S. Duclaud, D. Robaa, F. Erdmann, M. Schmidt,
C. Romier, R. Pierce, M. Jung, W. Sippl, J. Med. Chem. 2016, 59,
2423-2435.
[52]
[53]
J. Senger, J. Melesina, M. Marek, C. Romier, I. Oehme, O. Witt, W.
Sippl, M. Jung, J. Med. Chem. 2016, 59, 1545-1555.
Molecular Operating Environment (MOE), 2014.09; Chemical Compu-
ting Group Inc., 1010 Sherbooke St. West, Suite #910, Montreal, QC,
Canada, H3A 2R7, 2014.
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Entry for the Table of Contents
O
R
R'
N
H
X
O
H
N
OH
R'
O
R
=
X
C=N
C=C
S
HDAC6
HDAC1
affinity
Chirality and angle matter: R-configured propargyl amides and a thiophene linker inducing a slight kink in the linker moiety provide
inhibitors of HDAC6 with significantly increased affinity, selectivity and reduced cytotoxicity.
10
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