Structure-Based Design, Synthesis, and Biological Evaluation of Triazole-Based smHDAC8 Inhibitors

Article abstract of DOI:10.1002/cmdc.201900583

Schistosomiasis is a neglected tropical disease caused by parasitic flatworms of the genus Schistosoma, which affects over 200 million people worldwide and leads to at least 300,000 deaths every year. In this study, initial screening revealed the triazole

Full text of DOI:10.1002/cmdc.201900583

Accepted Article
Title: Structure-Based Design, Synthesis, and Biological Evaluation of
Triazole-Based smHDAC8 Inhibitors
Authors: Ralph Holl, Dmitrii V. Kalinin, Sunit Kumar Jana, Maxim
Pfafenrot, Alokta Chakrabarti, Jelena Melesina, Tajith Baba
Shaik, Julien Lancelot, Raymond J. Pierce, Wolfgang Sippl,
Christophe Romier, and Manfred Jung
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Structure-Based Design, Synthesis, and Biological Evaluation of
Triazole-Based smHDAC8 Inhibitors
Dmitrii V. Kalinin [a][b][c][d] Sunit K. Jana,[c][e] Maxim Pfafenrot,[c] Alokta Chakrabarti,[f] Jelena
,
Melesina,[g] Tajith B. Shaik,[h] Julien Lancelot,[i] Raymond J. Pierce,[i] Wolfgang Sippl,[g]
[a][b]
Christophe Romier,[h] Manfred Jung,[f] and Ralph Holl
*
[a]
Dr. D. V. Kalinin, Prof. Dr. R. Holl*
Department of Chemistry
Institute of Organic Chemistry
University of Hamburg
Martin-Luther-King Platz 6, 20146 Hamburg (Germany)
E-mail: ralph.holl@chemie.uni-hamburg.de
Dr. D. V. Kalinin, Prof. Dr. R. Holl*
German Center for Infection Research (DZIF)
partner site Hamburg-Lübeck-Borstel-Riems
Dr. D. V. Kalinin, Dr. S. K. Jana, M. Pfafenrot
Institute of Pharmaceutical and Medicinal Chemistry
University of Münster
[b]
[c]
Corrensstr. 48, 48149 Münster (Germany)
Dr. D. V. Kalinin
Cells-in-Motion Cluster of Excellence (EXC 1003 - CiM)
University of Münster (Germany)
Dr. S. K. Jana
NRW Graduate School of Chemistry
University of Münster (Germany)
Dr. A. Chakrabarti, Prof. Dr. M. Jung
Institute of Pharmaceutical Sciences
Albert-Ludwigs-Universität Freiburg
[d]
[e]
[f]
Albertstr. 25, 79104 Freiburg (Germany)
Dr. J. Melesina, Prof. Dr. W. Sippl
Institute of Pharmacy
Martin Luther University of Halle-Wittenberg
Wolfgang-Langenbeck Str. 4, 06120 Halle/Saale (Germany)
Dr. T. B. Shaik, Dr. C. Romier
[g]
[h]
Département de Biologie Structurale Intégrative
Institut de Génétique et Biologie Moléculaire et Cellulaire (IGBMC)
Université de Strasbourg, CNRS, INSERM
1 rue Laurent Fries, 67404 Illkirch Cedex (France)
Dr. J. Lancelot, Dr. R. J. Pierce
[i]
Université de Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille
U1019 - UMR 8204 - CIIL - Centre d’Infection et d’Immunité de Lille
59000 Lille (France)
Supporting information for this article is given via a link at the end of the document.
Abstract: Schistosomiasis is a neglected tropical disease caused by
parasitic flatworms of the genus Schistosoma, which affects over 200
million people worldwide and leads to at least 300,000 deaths every
year. In this study, initial screening revealed the triazole-based
Collectively, this study reveals specific interactions between
smHDAC8 and the synthesized triazole-based inhibitors and
demonstrates that these small molecules represent promising lead
structures, which could be further developed in the search for novel
drugs for the treatment of schistosomiasis.
hydroxamate
2b
(N-hydroxy-1-phenyl-1H-1,2,3-triazole-4-
carboxamide) exhibiting potent inhibitory activity toward the novel
antiparasitic target Schistosoma mansoni histone deacetylase 8
(smHDAC8) and promising selectivity over the major human HDACs.
Subsequent crystallographic studies of the 2b/smHDAC8 complex
revealed key interactions between the inhibitor and the enzyme’s
active site, thus explaining the unique selectivity profile of the inhibitor.
Further chemical modifications of 2b led to the discovery of 4-
fluorophenoxy derivative 21 (1-[5-chloro-2-(4-fluorophenoxy)phenyl]-
Introduction
Schistosomiasis, or bilharzia, is a neglected tropical disease,
which is caused by the trematode Schistosoma mansoni and
other platyhelminth parasites of the same genus.[1-3] The disease
is prevalent in Africa, the Middle East, South America, and Asia,
affecting over 200 million people worldwide and causing at least
300,000 deaths every year.[4-6] Currently, praziquantel is the only
drug available for treatment and control of schistosomiasis.[7] The
N-hydroxy-1H-1,2,3-triazole-4-carboxamide),
a
nanomolar
smHDAC8 inhibitor (IC50 = 0.5 µM), exceeding the smHDAC8
inhibitory activity of 2b and SAHA (vorinostat), while exhibiting an
improved selectivity profile over the investigated human HDACs.
1
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intensive use of this drug increases the probability of the
emergence of praziquantel resistant parasite strains and
worrisome data on reduced efficacy of the drug have already been
reported, thus rendering the search for potential drug targets as
well as novel drugs a strategic priority.[5, 8-10]
these hydroxamic acids contain a polar triazole ring, which could
possibly interact with H292 of smHDAC8, these compounds,
along with other triazole derivatives, exhibiting further variations
of the substituent in position 1 of the heterocycle, were
synthesized, assayed for their inhibitory activity toward smHDAC8,
and tested for their selectivity toward hsHDAC1 and hsHDAC8 as
well as the class IIb histone deacetylase hsHDAC6.
The treatment of S. mansoni with small-molecule histone
deacetylase (HDAC) inhibitors was shown to cause dose-
dependent mortality of schistosomula as well as adult worms,
making HDACs potential targets for the treatment of
schistosomiasis.[11-13] In eukaryotes, HDACs, which belong to the
epigenetic machinery of the cells, catalyze the deacetylation of ε-
amino groups of lysine residues in histone tails, leading in
consequence to a more compact chromatin structure, which
usually results in an inhibition of transcription.[14-17] Being drug
targets in cancer therapy, human histone deacetylases
(hsHDACs) were intensively studied and various HDAC inhibitors,
like e.g. SAHA (1, Figure 1), were described.[18-21] The 18 human
HDACs, which have been discovered so far, are grouped into 4
classes.[18, 22] Whereas classes I, II, and IV comprise the Zn2+-
dependent HDACs, the class III enzymes require NAD+ for
catalysis. In S. mansoni, three class I HDACs are known,
representing orthologues of the human class I enzymes HDAC1,
HDAC3, and HDAC8.[12] These three S. mansoni class I HDACs
are expressed in the parasite at all stages of its life-cycle.[11] In
contrast to hsHDAC8, showing in humans the lowest level of
expression of the class I enzymes, in S. mansoni smHDAC8 is
the most abundantly expressed class I HDAC at all life-cycle
stages and was validated as drug target for schistosome-specific
inhibitors. Down-regulation of smHDAC8 expression in
schistosomula caused a decrease in their capacity to survive and
mature in infected mice. In addition, the tissue egg burden was
reduced by 45%.[5, 12, 23] Like its human orthologue, smHDAC8
folds into a single α/β domain being composed of a central parallel
β-sheet, which is sandwiched between several α-helices.[5-6, 24]
The active sites of the enzymes consist of a long narrow tunnel,
accommodating the incoming acetylated lysine side chain of the
substrate, which leads to a cavity containing the catalytic Zn2+-ion.
The active site residues of the two enzymes are highly conserved,
with only M274 in hsHDAC8, being substituted by H292 in
smHDAC8.[6] The replacement of this hydrophobic residue by a
polar one modifies the physicochemical properties of the active
site, which could be exploited for the development of smHDAC8-
specific inhibitors.[5-6] Additionally, at the entrance region of the
binding tunnel, F151 of smHDAC8 (corresponding to F152 in
hsHDAC8) can adopt both a flipped-in and a flipped-out
conformation, whereas in hsHDACs due to steric constriction,
only the flipped-in conformation of this highly conserved residue
has been observed so far. The flipped-out conformation of F151
leads to a wider catalytic pocket in smHDAC8, which hence is
able to accommodate bulkier inhibitors.[5-6] These differences
should allow the development of inhibitors that are selective for
the schistosome enzyme, thereby minimizing off-target effects
Figure 1. Chemical structures of pan-HDAC inhibitor SAHA (vorinostat, 1),
smHDAC8 inhibitors J1038 and TH65, and triazole derivatives 2c, 2f and 2g.
Results and Discussion
Synthesis of triazole derivatives 2b-j
The reported triazole derivatives 2c, 2f, and 2g comprise a phenyl
ring linked to the triazole core via a flexible alkyl chain (from one
to three methylene groups, respectively). To vary length and
flexibility of the side chain, besides phenyl derivative 2b lacking a
methylene linker, diphenylacetylene and diphenylbutadiyne
derivatives 2h-j exhibiting a rigidified side chain should be
synthesized and tested for their inhibitory profile. Furthermore, to
study the effect of an additional substituent in the para-position of
the terminal phenyl ring, triazole derivatives 2d, 2e, and 2i should
be accessed.
The synthesis of triazole derivatives 2b, 2c, 2f, and 2g had been
reported in the literature before.[31-33] However, the envisaged
triazole derivatives were synthesized via an alternative route,
requiring fewer steps compared to the described procedures. In
the first step of the synthetic route, the triazole ring was
established
by
performing
Cu(I)-catalyzed
[3+2]-
cycloadditions.[34] The reaction of methyl propiolate (3) with
various azides (4a-g)[31,
was carried out at ambient
35-39]
temperature in a 1:1 mixture of water and tert-butanol in the
presence of copper(II) sulfate and sodium ascorbate giving
access to triazole derivatives 5a-g (Scheme 1).
caused by interactions with the human (host) orthologues.[25-26]
A
Scheme 1. Reagents and conditions: (a) sodium ascorbate, CuSO4·5 H2O,
tBuOH : H2O = 1 : 1, RT, 16 h, 5a 98 %, 5b 85 %, 5c 96 %, 5d 95 %, 5e 93 %,
5f 85 %, 5g 78 %.
few smHDAC8 inhibitors have been described in the literature so
far, such as J1038 and TH65 (Figure 1).[5, 27-30] These inhibitors
are often aromatic hydroxamic acids and many exploit a hydrogen
bond to the aforementioned histidine in the active site, whereas
the methionine, which the human orthologue has in the same
place, cannot be addressed in a similar fashion.
In order to further vary the substituent in position 1 of the triazole
ring by establishing a linear and rigid side chain, C-C coupling
reactions with 4-iodophenyl derivative 5a were performed
(Scheme 2). Sonogashira couplings with phenylacetylene (6h)
Several triazole derivatives like 2c, 2f, and 2g (Figure 1) have
been reported to weakly inhibit hsHDAC1 and hsHDAC8.[31] As
2
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and its morpholin-4-ylmethyl-substituted derivative 6i[40] were
conducted in a mixture of triethylamine and DMF at 70 °C in the
presence of Pd(PPh3)4 and CuI to obtain diphenylacetylene
derivatives 5h and 5i, respectively.[41]
Scheme 3. Reagents and conditions: (a) NH2OH·HCl, NaOMe, MeOH, rt, 20 h,
2b 66 %, 2c 72 %, 2d 55 %, 2e 48 %, 2f 67 %, 2g 61 %, 2h 91 %, 2i 76 %, 2j
68 %.
HDAC inhibitory activity of triazole derivatives 2b-j
All of the synthesized triazole derivatives were pretested for
inhibitory activity against smHDAC8. The most active compounds
(≥50 % inhibition at 10 µM) were further analyzed and their IC50
values against smHDAC8 and hsHDAC8 were determined in a
homogenous fluorescence assay using a commercially available
oligopeptide (Fluor de Lys) with an acetyl residue, which had also
been employed for pretesting. Additionally, the inhibitory activity
of the selected compounds was tested against hsHDAC1 and
hsHDAC6, using ZMAL with an acetylated lysine as substrate.[43]
Pretesting of triazole derivatives 2b-j at a concentration of 10 µM
revealed phenyl-substituted triazole 2b, trifluoromethylbenzyl
derivative 2e, phenylethyl- and phenylpropyl-substituted
compounds 2f and 2g to be the most potent smHDAC8 inhibitors
of this series of hydroxamic acids (Table 1). Whereas benzyl
derivatives 2c and 2d as well as the morpholinomethyl substituted
compound 2i showed only slightly weaker inhibition of smHDAC8,
compounds 2h and 2j, possessing an unpolar, linear, and rigid
side chain, were found to exhibit nearly no inhibitory activity
against the enzyme.
The IC50 values of the most potent compounds against smHDAC8
were in the low micromolar range, with phenyl derivative 2b being
the most potent inhibitor, possessing an IC50 value of 4.44 µM.
When being tested for hsHDAC8 inhibition, phenylalkyl
derivatives 2e, 2f and 2g showed slightly increased inhibitory
activity against the human orthologue. In contrast, phenyl
derivative 2b was found to inhibit hsHDAC8 with a higher IC50
value, exhibiting a threefold selectivity for smHDAC8. Against
hsHDAC1 and hsHDAC6 all compounds exhibited considerably
lower inhibitory activities.
Scheme 2. Reagents and conditions: (a) Pd(PPh3)4, CuI, Et3N, DMF, 70 °C, 16
h, 5h 96 %, 5i 98 %; (b) trimethylsilylacetylene, Pd(PPh3)4, CuI, Et3N, CH3CN,
70 °C, 3 h, 97 %; (c) i) TBAF, CH2Cl2, rt, 30 min, ii) phenylacetylene, Cu(OAc)2,
pyridine, MeOH, rt, 16 h, 62 %.
Additionally, the butadiyne derivative 5j was synthesized
employing the Eglinton reaction.[42] At first, an acetylene moiety
was introduced to the phenyl ring of triazole derivative 5a.
Performing a Sonogashira coupling with trimethylsilylacetylene
resulted in acetylene derivative 7. In the next step, the
trimethylsilyl protecting group of
7 was cleaved off with
tetrabutylammonium fluoride (TBAF). As TLC indicated a clean
conversion of trimethylsilyl protected compound 7 into the
corresponding terminal alkyne, after aqueous work up, the crude
product of the reaction was directly subjected to a subsequent
Eglinton reaction. This coupling reaction was performed with
phenylacetylene in a mixture of methanol and pyridine in the
presence of copper(II) acetate giving access to diacetylene
derivative 5j.
In the final step, the ester moiety of triazole derivatives 5b-j was
transformed into a Zn2+-chelating hydroxamate moiety. Therefore,
the esters were reacted with hydroxylamine hydrochloride and
sodium methanolate in dry methanol to obtain hydroxamic acids
2b-j (Scheme 3).
Table 1. In vitro inhibition of smHDAC8, hsHDAC8, hsHDAC6 and hsHDAC1. The employed substrate is given in brackets. nd: not determined. Number of replicates:
all pretests n = 1; all IC50 n = 2.
smHDAC8
(% inhibition,
Fluor de Lys)
smHDAC8
IC50 [µM]
(Fluor de Lys)
hsHDAC8
IC50 [µM]
(Fluor de Lys)
hsHDAC6
IC50 [µM]
(ZMAL)
hsHDAC1
(% inhibition,
ZMAL)
compound
SAHA (1)
72.0 @ 10 µM
57.9 @ 10 µM
39.6 @ 10 µM
36.1 @ 10 µM
52.5 @ 10 µM
49.5 @ 10 µM
50.5 @ 10 µM
-9.2 @ 10 µM
48.0 @ 10 µM
6.8 @ 10 µM
1.17 ± 0.33
0.64 ± 0.21
0.14 ± 0.12
97.3 ± 1.3 @ 10 µM
2b
2c
2d
2e
2f
4.44 ± 1.52
12.43 ± 1.08
42.50 ± 3.64
8.1 ± 1.0 @ 10 µM
nd
nd
nd
nd
nd
nd
5.12 ± 0.64
10.67 ± 1.88
8.81 ± 1.89
nd
nd
nd
3.47 ± 0.53
5.54 ± 0.64
5.37 ± 0.68
nd
2.2 ± 0.5 % @ 10 µM
3.3 ± 2.5 @ 10 µM
12.6 ± 9.6 % @ 10 µM
1.7 ± 3.4 @ 10 µM
2g
2h
2i
31.79 ± 6.18
5.4 ± 7.0 @ 10 µM
nd
nd
nd
nd
nd
nd
nd
nd
2j
nd
nd
3
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smHDAC8/2b X-ray crystal structure
different HDAC isoforms helped to rationalize the observed
selectivity. The crystal structure of 2b with smHDAC8 showed that
the inhibitor exhibits a classical binding mode characteristic to
many other hydroxamic acid-based HDAC inhibitors.[44] Namely,
its zinc-binding group chelates the catalytic zinc ion in bidentate
fashion and interacts with conserved residues H141, H142, and
Y341. The triazole linker is placed in the lysine-binding channel
while the phenyl ring is pointed to the entrance of the pocket.
Interestingly molecular docking studies suggested that a similar
binding mode of this ligand could be possible also in human
HDAC isoforms HDAC8, HDAC1, and HDAC6 (Figure 3).
Figure 2. Crystal structure of the smHDAC8/2b complex (PDB ID 6TLD). (A)
Overall view of the complex. The protein is shown as ribbons whereas the
catalytic zinc (orange) and inhibitor 2b (carbon, magenta; nitrogen, blue; oxygen,
red) atoms are shown as spheres. 2b adopts a canonical pan-HDAC binding
mode with a straight conformation, its capping group pointing toward the solvent.
(B) Close-up view of 2b binding into the active site of smHDAC8. 2b (magenta
carbon atoms) and specific side chains of smHDAC8 (grey carbon atoms)
interacting with the inhibitor are shown as sticks (nitrogen, blue; oxygen, red).
Interactions are shown with broken lines. Residues are labelled.
Due to the promising biological activity of the 1-phenyl substituted
triazole derivative 2b, the compound was crystallized in complex
with smHDAC8 (Figure 2, Table S1, Supporting Information). The
structure reveals that compound 2b binds in the smHDAC8 active
site in a straight conformation, its capping group pointing toward
the solvent, whereas its hydroxamate moiety forms a bidentate
interaction with the catalytic zinc ion as well as hydrogen bonds
with the side chains of histidines H141 and H142 and the hydroxyl
group of catalytic tyrosine Y341. This binding mode is reminiscent
of those of many pan-HDAC inhibitors binding to HDACs.
Interestingly, the triazole ring of 2b is observed almost stacked
between the side chains of phenylalanines F151 and F216 that
form the active site tunnel normally accommodating the aliphatic
part of the incoming acetylated lysine side chain. Yet, the F151
side chain is observed in two conformations, one turned toward
the active site, participating to the tunnel mentioned previously,
and one turned away from the active site. This suggests that the
stacking of the triazole ring between the two phenylalanines
observed in the structure is not absolutely essential for the
interaction. In addition, the nitrogen atoms of the triazole ring are
not facing the smHDAC8-specific H292 side chain but turned
toward the Cα carbon atom of glycine G150. Finally, the capping
group of 2b does not appear to make extensive interactions with
smHDAC8. Yet, the rather small size of the inhibitor allows it to fit
nicely into the smHDAC8 active site pocket. Replacement of
smHDAC8 H292 by the somewhat bulkier human methionine
M274 could potentially explain the increased IC50 for 2b measured
with the human enzyme.
Figure 3. Binding modes of compound 2b (predicted – magenta carbon atoms,
experimental – cyan carbon atoms) in: (A) smHDAC8 (pale pink carbons), (B)
hsHDAC8 (pale blue carbons), (C) hsHDAC6 (pale yellow carbons), and (D)
hsHDAC1 (pale green carbons). Catalytic zinc ion is shown as orange sphere.
Nitrogens are colored dark blue, oxygens – red, sulphur – dark yellow. Metal
interactions and hydrogen bonds are shown as dashed yellow lines with
distances in Å.
Despite similar binding modes, slight differences of the binding
pockets could explain the selectivity of 2b. Analysis of the binding
mode of 2b in smHDAC8 suggested a number of possible
interactions. As seen in Figure 3 A, the triazole ring of the
compound makes a weak hydrogen bond with the smHDAC8
unique residue H292 and it is able to form an aromatic interaction
with its imidazole ring. Similar interactions have been also
reported in the literature.[45-46] Furthermore, the triazole ring of 2b
forms a π-cation interaction with K20 of smHDAC8 whereas in the
case of the human enzyme the corresponding K33 is flipped out
of the binding pocket (Figure 3 B). Furthermore, in hsHDAC8 an
H-bond interaction of the triazole ring of 2b with M274 was not
observed, probably due to non-optimal orientation of this residue
and weaker hydrogen bond accepting properties of M274. It was
also observed that the favorable orientation of the triazole ring in
smHDAC8 allows π-stacking interaction between the phenyl ring
of 2b and F216, whereas in hsHDAC8 this interaction with
corresponding F208 is not detected. The lack of interactions
Docking of the synthesized triazole derivatives and design of
novel inhibitors
Although being
a slightly less potent smHDAC8 inhibitor
compared to pan-HDAC inhibitor SAHA (1), the phenyl-
substituted triazole derivative 2b was considered as a promising
lead structure due to its selectivity toward the platyhelminth
enzyme. Analysis of possible binding modes of this compound in
4
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between the triazole and surrounding amino acid residues and the
absence of π-stacking interaction between the phenyl ring and
Phe208 in hsHDAC8 explains why the compound is slightly
selective toward smHDAC8. In hsHDAC6 and hsHDAC1 (Figure
3 C-D) the bulkier L749/L271 residues are responsible for a more
closed side pocket and, in addition, no H-bond interactions can
ether syntheses giving access to phenyl alkyl ethers 14 and 15.
The obtained ethers were finally reacted with hydroxylamine to
yield hydroxamic acids 16 and 17. Additionally, Chan-Lam
couplings of phenol 11 with 4-tolylboronic acid and 4-
fluorophenylboronic acid were performed, leading to diphenyl
ethers 18 and 19,[47] which were finally transformed into
be formed between the triazole and the leucine residue. Therefore, hydroxamic acids 20 and 21. The synthesis of the latter
the inhibitory activity of 2b for hsHDAC1/6 is reduced.
compound has been briefly reported by us in a previous
publication.[28]
To understand the contribution of the cap group to the activity of
the compounds, docking poses of 2c-2j were analyzed (Figure S1,
Supporting Information). It was observed that the benzyl, 4-
fluorobenzyl, and 4-(trifluoromethyl)benzyl caps of compounds
2c-2e do not show any direct interactions with surrounding
residues. The phenylethyl and phenylpropyl groups of 2f and 2g
are able to form aromatic interactions with F216 or Y341. Similar
binding modes are observed for these compounds in hsHDAC8.
In the series of compounds with a larger and rigid scaffold (2h-2j),
all three ligands fitted into the smHDAC8 binding pocket, but a
significant part of the compound was sticking out into the solvent.
The more active compound 2i formed favorable cation-π
interactions between its positively charged nitrogen of the
morpholine scaffold and the aromatic ring of the Y99 amino acid
residue.
In order to improve the affinity toward smHDAC8 and to further
enhance the selectivity of the triazole derivatives, molecular
docking studies of various suggested derivatives of 2b were
performed utilizing the obtained crystal structure of smHDAC8
with this compound. Based on these studies, further 1-phenyl
substituted triazole derivatives were envisaged possessing a
phenyl ether or a benzophenone moiety. The substitutions were
introduced to the ortho-position of the phenyl ring to address the
unique side pocket of HDAC8 with L-shaped inhibitors. As shown
by us previously, this should further increase selectivity against
HDAC6 and HDAC1.[28] Also since the side pockets of smHDAC8
and hsHDAC8 are different, we hoped to further increase
selectivity against hsHDAC8. Thus, on the one hand, these
substituents should lead to better interactions with smHDAC8. On
the other hand, they should render the relatively polar triazole
derivatives more lipophilic, as it is established, that a logP above
2.5 is beneficial for antiparasitic activity.[27]
Scheme 4. Reagents and conditions: (a) methyl propiolate, sodium ascorbate,
CuSO4·5H2O, tBuOH : H2O = 1 : 1, rt, 10 92%, 11 89%; (b) NH2OH·HCl, NaOMe,
MeOH, rt, 12 45%, 13 43%; (c) Cs2CO3, DMF, 90 °C, 14 56%, 15 59%; (d)
NH2OH·HCl, NaOMe, MeOH, rt, 16 83%, 17 52%; (e) Cu(OAc)2, CH2Cl2, Et3N,
rt, 18 11%, 19 13%; (f) NH2OH·HCl, NaOMe, MeOH, rt, 20 99%, 21 95%.
In contrast, diphenyl ethers 28 and 29 were synthesized from
commercially available 2-phenoxyanilines 22 and 23, respectively
(Scheme 5). From these primary aromatic amines, via a
diazotisation and the subsequent reaction of the resulting
diazonium salts with azide ions, azides 24 and 25 were
obtained.[48] Subsequently, these azides were subjected to a
copper(I)-catalyzed [3+2]-cycloaddition[49] with methyl propiolate
and a final aminolysis with hydroxylamine yielding diphenyl ethers
28 and 29. In principally the same way, benzophenone derivative
32 was obtained from azide 30 (Scheme 6).
Molecular docking studies also showed that an additional small
substituent (e.g. chlorine) at position 5 of the aromatic ring fits to
the pocket and stabilizes the rotation of the ring required for the
expected orientation of the ortho-substituent (pushes the ligand
into the side pocket). Introduction of a small substituent to the
meta-position without ortho-substitution or with
a
small
substituent at ortho-position does not induce the rotation of the
ring. Therefore, it was suggested to simultaneously introduce
chlorine to position 5 of the phenyl ring in addition to a large
aromatic substituent in position 2.
Synthesis of ortho-substituted 1-phenyl-1H-1,2,3-triazole
derivatives
The envisaged ether derivatives were synthesized from azides 8
and 9 (Scheme 4). Copper(I)-catalyzed [3+2]-cycloadditions of
azides 8 and 9 with methyl propiolate led to triazole derivatives 10
and 11. Phenols 10 and 11 were subsequently subjected to an
aminolysis with hydroxylamine, which gave hydroxamic acids 12
and 13, bearing a polar substituent in position 2 of the phenyl ring.
Alternatively, phenol derivative 11 was subjected to Williamson
Scheme 5. Reagents and conditions: (a) i) NaNO2, HCl, H2O, 0 °C, ii) NaN3,
NaOAc, 24 83%, 25 63%; (b) methyl propiolate, sodium ascorbate,
CuSO4·5H2O, tBuOH : H2O = 1 : 1, rt, 26 72%, 27 69%; (c) NH2OH·HCl, NaOMe,
MeOH, rt, 28 52%, 29 70%.
5
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Also the synthesized series of ortho-substituted 1-phenyl-1H-
1,2,3-triazole derivatives was pretested for inhibitory activity
against smHDAC8 at concentrations of 50 µM and 5 µM (Table
2). Among the tested triazole derivatives, hydroxamic acids 16
and 17, bearing an arylmethoxy substituent in position 2 of their
phenyl ring, were the least active compounds. The polar phenols
12 and 13 showed moderate activity and were almost as active
as benzophenone 32. The highest inhibitory activity was found for
diphenyl ethers 20, 21, 28, and 29. Among these compounds, the
4-fluorophenoxy derivative 21 was shown to be the most potent
smHDAC8 inhibitor. With an IC50 value of 0.50 µM, its inhibitory
activity against smHDAC8 exceeds those of 2b and SAHA (1),
whereas the compound exhibits only low activity against
hsHDAC1 and hsHDAC6 (Table 3). Although its inhibitory activity
against hsHDAC8 is increased with respect to 2b, diphenyl ether
21 shows an even greater selectivity for smHDAC8 than the
unsubstituted phenyl derivative 2b. Its selectivity over all tested
human HDACs makes triazole derivative 21 a promising starting
point for the development of smHDAC8-specific inhibitors.
Scheme 6. Reagents and conditions: (a) methyl propiolate, sodium ascorbate,
CuSO4·5H2O, tBuOH : H2O = 1 : 1, rt, 42%; (b) NH2OH·HCl, NaOMe, MeOH, rt,
29%.
HDAC inhibitory activity of the ortho-substituted 1-phenyl-
1H-1,2,3-triazole derivatives
Table 2. In vitro inhibition of smHDAC8 (Fluor de Lys) by the ortho-substituted
1-phenyl-1H-1,2,3-triazole derivatives.
% inhibition @ 5 µM
compound
% inhibition @ 50 µM
63.5
67.3
43.5
21.0
91.1
93.4
87.9
91.5
75.4
19.5
14.5
-1.5
12
13
16
17
20
21
28
29
32
Comparison of the obtained crystal structures
-9.3
Comparison of the smHDAC8/2b and smHDAC8/21 structures
shows that both inhibitors bind in a similar way into the smHDAC8
active site (Figure 4). However, 21 is slightly tilted within the
smHDAC8 active site pocket compared to 2b, its triazole ring
coming closer to histidine H292. We have previously shown that
compound 21, like many HDAC8-selective inhibitors, was able to
bind to a HDAC8-selective pocket formed by HDAC8 loops L1 and
L6 and the side chain of the catalytic tyrosine (smHDAC8
Y341/human HDAC8 Y306).[28] Specifically, the fluoro-phenyl
capping group of 21 is stacked onto the Y341 side chain,
49.8
66.2
49.1
51.4
23.4
Table 3. In vitro inhibition of smHDAC8, hsHDAC8, hsHDAC6 and hsHDAC1. The employed substrate is given in brackets.
smHDAC8
IC50 [µM]
(Fluor de Lys)
hsHDAC8
IC50 [µM]
(Fluor de Lys)
hsHDAC6
IC50 [µM] / % inhibition
(ZMAL)
hsHDAC1
IC50 [µM] / % inhibition
(ZMAL)
compound
0.117 ± 0.0056
97.3 ± 1.3 % @ 10 µM
SAHA (1)[5, 50]
1.17 ± 0.33
4.44 ± 1.52
0.64 ± 0.21
12.43 ± 1.08
2.2 ± 0.68
0.14 ± 0.12
2b
21
42.50 ± 3.64
8.1 ± 1.0 % @ 10 µM
34.5 % @ 50 µM
-1.5 % @ 5 µM
38.2 % @ 50 µM
11 % @ 5 µM
0.504 ± 0.046
Figure 4. Comparison of inhibitors 2b (magenta carbon atoms) and 21 (cyan carbon atoms) binding to smHDAC8. Close-up views of 2b (A) and 21 (C) binding to
smHDAC8 active site and their superposition (B). Representation and coloring is as in Figure 1 B. Only inhibitor 21 is observed binding to the HDAC8-selectivity
pocket. This specific binding mode could be responsible for the movement observed for the triazole ring of 21 compared to 2b.
6
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10.1002/cmdc.201900583
ChemMedChem
FULL PAPER
an interaction that appears essential for the binding of many
HDAC8-selective inhibitors to HDAC8. We postulate that the
tilting observed for 21 compared to 2b is caused by the
maximization of the stacking interaction between H292 and the
21 capping group. This would also be in agreement with the lower
IC50 for 21 compared to 2b measured for smHDAC8. Once again,
the bulkier human HDAC8 M274 in replacement of smHDAC8
H292, notably considering the rapprochement of the triazole ring
toward this residue, could explain the higher selectivity of 21 for
smHDAC8. Interestingly, the lower IC50 for 21 compared to 2b for
human HDAC8 could be explained by the stacking interaction
between the catalytic tyrosine and 21 capping group,
demonstrating once again how this latter interaction is essential
for the selective inhibition of HDAC8 enzymes.
are not observed. The central aromatic chlorophenyl ring is
rotated 90 degrees in comparison to phenyl ring of 2b and makes
an edge-to-face aromatic interaction with F216 and the
corresponding F208 in smHDAC8 and hsHDAC8 respectively.
The oxygen atom of the linker is able to make a hydrogen bond
with K20 in smHDAC8, but not in hsHDAC8, which might
contribute to the selectivity of the compound. The distal aromatic
fluorophenyl ring is placed comfortably in the side pocket of
parasitic and human HDAC8 isoform above the conserved Y341
and Y303 respectively, which probably contributes the most to the
increased activity of 21 in comparison to 2b. The binding modes
of 21 in hsHDAC6 and hsHDAC1 are completely different (Figure
5 C-D). The L-shaped form of the inhibitor prevents it from fitting
into the binding pocket of these isoforms due to the absence of
the side pocket and it can only reach the zinc ion with one oxygen
atom of the hydroxamic acid losing a number of H-bond
interactions.
Finally, the predicted binding modes of other 2b derivatives were
analyzed to understand why some compounds were more active
than the others. Compounds 20, 21, 28 and 29 having an aryloxy
moiety showed the highest activity and their binding modes were
similar to 21. In contrast, the least active compounds 16 and 17
with an arylmethoxy scaffold showed shifted positions of the distal
and central aromatic rings due to a longer linker between them
and, as a consequence, lost the hydrogen bond with K20 and did
not form an aromatic interaction with Y341. The benzophenone
derivative 32 interacted with K20 via its carbonyl group, but
showed lower activity than diphenyl ethers probably due to
penalty from the distortion of the conjugated planar system
required for this interaction. The phenols 12 and 13 showed
binding modes similar to 2b with an additional interaction between
the phenol group and D100. However, they were less active than
diphenyl ethers due to the absence of a large aryl group
embedded into the side pocket.
Docking of the novel 1-phenyl substituted triazole
derivatives
Schistosome viability testing
Selected compounds (Table 4) were tested for their effects on the
viability of S. mansoni larvae (schistosomula) using a resazurin-
based fluorescence assay that measures mitochondrial activity.
Assays were carried out in triplicate on two separate batches of
schistosomula and the results are expressed as the percentage
of the value obtained with the solvent (DMSO). Initial testing was
done at 10 µM as part of a high-throughput screening procedure
and compounds provoking at least a 25% reduction in viability
were tested at 20 µM to determine a dose-response. Of the
triazole-based smHDAC8 inhibitors tested, only compound 29
provoked a significant reduction in viability at 10 and 20 µM (Table
4). However, the level of reduction obtained was mediocre and
this compound was not considered for further testing. We have
previously shown[27] that some potent inhibitors of smHDAC8
were inactive or had feeble effects on the parasite in our viability
assay. We consider that this is most likely due to a poor level of
transport across the complex barrier represented by the parasite
tegument for the inactive compounds, since some hydroxamate-
based inhibitors have extremely potent effects on the parasite in
the same assay.[27] This despite an apparently favorable
lipophilicity index. A variety of schistosome viability assays are
currently used in the field [e.g. Panic et al.[51], Singh et al.[52]] that
do not always concur. However, since we have compared
compounds that a priori have the same biological target, it is
Figure 5. Binding modes of compound 21 (predicted – dark magenta carbon
atoms, experimental – dark cyan carbon atoms) in: (A) smHDAC8 (pale pink
carbons), (B) hsHDAC8 (pale blue carbons), (C) hsHDAC6 (pale yellow
carbons) and (D) hsHDAC1 (pale green carbons). Catalytic zinc ion is shown
as orange sphere. Nitrogens are colored dark blue, oxygens – red, sulphur –
dark yellow, chlorine – green, fluorine – pale cyan. Metal interactions and
hydrogen bonds are shown as dashed yellow lines.
Biological testing of the synthesized 2b derivatives 12, 13, 16, 17,
20, 21, 28, 29, and 32 showed that indeed all compounds are
active on smHDAC8 (Schemes 4-6, Tables 2 and 3). The most
active compound 21 not only showed increased activity, but also
an improved selectivity profile, which could be explained by
analysis of its binding modes (Figure 5). In smHDAC8 and
hsHDAC8 (Figure 5 A-B) the inhibitor chelates the zinc ion similar
to 2b (Figure 5). The triazole ring is also placed in a similar way
and it interacts with unique H292 in smHDAC8. However, as
discussed previously for 2b, in the case of hsHDAC8 the
hydrogen bond with M274 and the π-cation interaction with K20
7
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10.1002/cmdc.201900583
ChemMedChem
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unlikely that they will have different effects on the parasite and
that different viability assay, for example based on
morphological characteristics,[52] would give different results.
Nevertheless, in view of the potency of the triazole-based
smHDAC8 inhibitors on the enzyme, their further development,
aimed at improving their bioavailability, is justified.
improved selectivity profile over the investigated human HDACs.
Comparative analysis of the crystal structures of smHDAC8/2b
and smHDAC8/21 revealed that the triazole ring of 21 resides
even closer to the smHDAC8 specific residue H292 and the
fluoro-phenyl capping group of 21 acquires an additional
interaction with the catalytic Y341, collectively resulting in a more
potent smHDAC8 inhibitory activity of 21 compared to 2b.
Although the effects of the compounds on the viability of S.
mansoni larvae was mediocre to low, our findings give an insight
into the specific interactions between smHDAC8 and triazole-
based inhibitors and reveal these small molecules as promising
lead structures for the development of potent and selective
smHDAC8 inhibitors as so far only limited data is available on
inhibitors that are more potent on smHDAC8 over the human
orthologue.
a
Table 4. Effects on the viability of S. mansoni larvae (schistosomula)
determined in a resazurin-based fluorescence assay. nd: not determined.
10 µM
% viability
20 µM
% viability
compound
± SEM
± SEM
92.54
81.95
64.21
5.70
nd
nd
nd
2b
21
29
4.54
1.28
nd
49.88
3.18
Experimental Section
Chemistry, general
Summary and Conclusion
Unless otherwise mentioned, THF was dried with sodium/benzophenone
and was freshly distilled before use. Thin layer chromatography (tlc): Silica
gel 60 F254 plates (Merck). Flash chromatography (fc): Silica gel 60, 40 –
64 µm (Macherey-Nagel); parentheses include: diameter of the column,
fraction size, eluent, Rf value. Melting point (m.p.): Melting point apparatus
SMP 3 (Stuart Scientific), uncorrected. 1H NMR (400 MHz), 13C NMR (100
MHz): Mercury plus 400 spectrometer (Varian); δ in ppm related to
tetramethylsilane; coupling constants are given with 0.5 Hz resolution. IR:
IR Prestige-21(Shimadzu). HRMS: MicrOTOF-QII (Bruker). HPLC
methods for the determination of product purity: Method 1: Merck Hitachi
Equipment; UV detector: L-7400; autosampler: L-7200; pump: L-7100;
The present study experimentally demonstrates the potential of
triazole-based small-molecules as potent and selective S.
mansoni HDAC8 inhibitors, which could find an application in the
treatment of schistosomiasis after improving the bioavailability.
As we reported previously, HDAC8 active site residues and loops
demonstrate high flexibility, thereby making the development of
HDAC8 selective inhibitors a complex task.[28] In this study,
however, structural modifications of weak inhibitors of hsHDAC8
allowed us to identify the lead structure 2b, exhibiting promising
selectivity for the parasitic smHDAC8 over the human enzymes
hsHDAC8, hsHDAC1, and hsHDAC6. Crystallographic studies of
the smHDAC8/2b complex as well as docking studies revealed
key interactions between the triazole ring of 2b and the enzyme’s
active site, being crucial for the selectivity profile of 2b. The
triazole moiety of 2b was found to form a weak hydrogen bond
and an aromatic interaction with the smHDAC8 specific residue
H292 as well as a π-cation interaction with K20 of the enzyme.
These two interactions are not observed between 2b and the
respective amino acids of the human orthologue hsHDAC8, due
to the replacement of H292 by M274 and the flipped out
conformation of K33. Furthermore, the favorable orientation of the
triazole ring in smHDAC8 allows a π-stacking interaction between
the phenyl ring of 2b and F216, whereas for hsHDAC8 this
interaction with the corresponding F208 is not detected. As
evidenced by additionally performed docking studies, in
hsHDAC6 and hsHDAC1 the bulkier L749/L271 residues are
responsible for a more closed side pocket and, in addition, no H-
bond interactions can be formed between the triazole and the
leucine residue. Therefore, the inhibitory activity of 2b toward
hsHDAC1/6 is reduced.
degasser: L-7614; column: LiChrospher® 60 RP-select
B (5 µm);
LiChroCART® 250-4 mm cartridge; flow rate: 1.00 mL/min; injection
volume: 5.0 µL; detection at λ = 210 nm for 30 min; solvents: A: water with
0.05 % (V/V) trifluoroacetic acid; B: acetonitrile with 0.05 % (V/V)
trifluoroacetic acid: gradient elution: (A %): 0 – 4 min: 90 % , 4 – 29 min:
gradient from 90 % to 0 %, 29 – 31 min: 0 %, 31 – 31.5 min: gradient from
0 % to 90 %, 31.5 – 40 min: 90 %. Method 2: Merck Hitachi Equipment;
UV detector: L-7400; pump: L-6200A; column: phenomenex Gemini® 5 µm
C6-Phenyl 110 Å; LC Column 250 × 4.6 mm; flow rate: 1.00 mL/min;
injection volume: 5.0 µL; detection at λ = 254 nm for 20 min; solvents: A:
acetonitrile : 10 mM ammonium formate = 10 : 90 with 0.1 % formic acid;
B: acetonitrile : 10 mM ammonium formate = 90 : 10 with 0.1 % formic acid;
gradient elution: (A %): 0 – 5 min: 100 % , 5 – 15 min: gradient from 100 %
to 0 %, 15 – 20 min: 0 %, 20 – 22 min: gradient from 0 % to 100 %, 22 –
30 min: 100 %.
Synthetic procedures and analytical data of compounds 5c-g,i, 2c-g,i, 10,
12, 14, 16, 18, 20, 24, 26, 28 are given in the Supporting Information.
Methyl 1-(4-iodophenyl)-1H-1,2,3-triazole-4-carboxylate (5a): Methyl
propiolate (0.36 mL, 4.0 mmol) and 1-azido-4-iodobenzene (1.47 g, 6.0
mmol) were dissolved in a 1:1 mixture of tBuOH and H2O (10 mL). Sodium
ascorbate (79 mg, 0.4 mmol) and copper(II) sulfate pentahydrate (20 mg,
0.08 mmol) were added and the mixture was stirred overnight. Then water
was added and the mixture was extracted ethyl acetate (3×). The
combined organic phases were dried (Na2SO4), filtered and evaporated.
The residue was purified by flash column chromatography (Ø = 2 cm, h =
15 cm, V = 10 mL, cyclohexane/ethyl acetate = 2:1, Rf = 0.31) to give 5a
as colorless solid (1.29 g, 3.9 mmol, 98% yield). m.p. = 189 °C; 1H NMR
In an attempt to further improve the affinity of 2b toward
smHDAC8 and to further enhance selectivity, a series of ortho-
substituted 1-phenyl-1H-1,2,3-triazole-based hydroxamates was
designed and synthetically accessed via Cu(I)-catalyzed [3+2]-
cycloaddition reactions, Chan-Evans-Lam couplings, and
aminolyses with hydroxylamine. Among the synthesized
compounds, 4-fluorophenoxy derivative 21 was found to be the
most potent smHDAC8 inhibitor showing IC50 value of 0.50 µM,
thereby exceeding the smHDAC8 inhibitory activity of the initial
lead compound 2b as well as of SAHA (1), while exhibiting an
(CDCl3): δ [ppm] = 4.00 (s, 3H, CO2CH3), 7.51 – 7.54 (m, 2H, 2'-H4-iodophenyl
6'-H4-iodophenyl), 7.88 – 7.91 (m, 2H, 3'-H4-iodophenyl, 5'-H4-iodophenyl), 8.51 (s, 5-
triazole); 13C NMR (CDCl3): δ [ppm] = 52.6 (1C, CO2CH3), 94.8 (1C, C-4'4-
iodophenyl), 122.4 (2C, C-2'4-iodophenyl, C-6'4-iodophenyl), 125.5 (1C, C-5triazole),
136.0 (1C, C-1'4-iodophenyl), 138.3 (1C, C-4triazole), 139.2 (2C, C-3'4-iodophenyl
,
H
,
8
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10.1002/cmdc.201900583
ChemMedChem
FULL PAPER
C-5'4-iodophenyl), 161.0 (1C, CO2CH3); IR (neat): ꢀꢁ [cm-1] = 1728, 1551, 1493,
observed; IR (neat): ꢀꢁ [cm-1] = 3140, 2959, 1717, 1555, 1516, 1265, 1254,
1157, 1042, 841, 760, 679; HRMS (m/z): [M+H]+ calcd for C15H18N3O2Si,
300.1163; found, 300.1173; HPLC (method 1): tR = 21.3 min, purity 98.7%.
1435, 1400, 1342, 1250, 1196, 1150, 1038, 984, 768; HRMS (m/z): [M+H]+
calcd for C10H9IN3O2, 329.9734; found, 329.9704; HPLC (method 1): tR
18.3 min, purity 95.8%.
=
Methyl
1-[4-(4-phenylbuta-1,3-diynyl)phenyl]-1H-1,2,3-triazole-4-
Methyl 1-phenyl-1H-1,2,3-triazole-4-carboxylate (5b): Methyl propiolate
(0.18 mL, 2.0 mmol) and azidobenzene (357 mg, 3.0 mmol) were dissolved
in a 1:1 mixture of tBuOH and H2O (10 mL). Sodium ascorbate (40 mg, 0.2
mmol) and copper(II) sulfate pentahydrate (10 mg, 0.04 mmol) were added
and the mixture was stirred overnight. Then water was added and the
mixture was extracted ethyl acetate (3×). The combined organic phases
were dried (Na2SO4), filtered and evaporated. The residue was purified by
flash column chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL,
cyclohexane/ethyl acetate = 2:1, Rf = 0.30) to give 5b as colorless solid
(345 mg, 1.7 mmol, 85% yield). m.p. = 123 °C; 1H NMR (CDCl3): δ [ppm]
= 3.99 (s, 3H, CO2CH3), 7.47 – 7.52 (m, 1H, 4'-Hphenyl) 7.53 – 7.58 (m, 2H,
3'-Hphenyl, 5'-Hphenyl), 7.73 – 7.77 (m, 2H, 2'-Hphenyl, 6'-Hphenyl), 8.52 (s, 1H,
5-Htriazole); 13C NMR (CDCl3): δ [ppm] = 52.5 (1C, CO2CH3), 120.9 (2C, C-
2'phenyl, C-6'phenyl), 125.7 (1C, C-5triazole), 129.7 (1C, C-4'phenyl), 130.1 (2C,
C-3'phenyl, C-5'phenyl), 136.4 (1C, C-1'phenyl), 140.7 (1C, C-4triazole), 161.2 (1C,
CO2CH3); IR (neat): ꢀꢁ [cm-1] = 3152, 1728, 1593, 1528, 1497, 1369, 1227,
1150, 1038, 768, 694; HRMS (m/z): [M+H]+ calcd for C10H10N3O2,
204.0768; found, 204.0764; HPLC (method 1): tR = 15.2 min, purity 95.1%.
carboxylate (5j): Tetrabutylammonium fluoride trihydrate (315 mg, 1.0
mmol) was added to a solution of 7 (250 mg, 0.84 mmol) in dichlromethane
(15 mL) and the reaction mixture was stirred at ambient temperature for
30 min. Then the solution was diluted with water and extracted with
dichloromethane (3×). The combined organic phases were dried with
Na2SO4, filtered and the solvent was removed in vacuo. The crude product
was dissolved in mixture of pyridine and MeOH (1:1, 4 mL). Then
phenylacetylene (0.46 mL, 4.2 mmol), copper(II) acetate (309 mg, 1.7
mmol) were added and the mixture was stirred overnight at ambient
temperature. Then the reaction mixture was diluted with water and
extracted with ethyl acetate (3×). The combined organic phases were dried
with Na2SO4, filtered and the solvent was removed in vacuo. The residue
was purified by flash column chromatography (Ø = 2 cm, h = 15 cm, V =
10 mL, cyclohexane/ethyl acetate = 3:1, Rf = 0.28) to give 5j as colorless
crystalline solid (169 mg, 0.52 mmol, 62% yield). m.p. = 198 °C; 1H NMR
(CDCl3): δ [ppm] = 4.00 (s, 3H, CO2CH3), 7.33 – 7.42 (m, 3H, 3''-Hphenyl
,
4''-Hphenyl, 5''-Hphenyl), 7.53 – 7.56 (m, 2H, 2''-Hphenyl, 6''-Hphenyl), 7.68 – 7.72
(m, 2H, 3'-H4-(4-phenylbuta-1,3-diynyl)phenyl, 5'-H4-(4-phenylbuta-1,3-diynyl)phenyl), 7.75 –
7.79 (m, 2H, 2'-H4-(4-phenylbuta-1,3-diynyl)phenyl, 6'-H4-(4-phenylbuta-1,3-diynyl)phenyl), 8.53
(s, 5-Htriazole); 13C NMR (CDCl3): δ [ppm] = 52.6 (1C, CO2CH3), 73.6 (1C,
C≡C), 76.4 (1C, C≡C), 79.7 (1C, C≡C), 83.1 (1C, C≡C), 120.7 (2C, C-2'4-
(4-phenylbuta-1,3-diynyl)phenyl, C-6'4-(4-phenylbuta-1,3-diynyl)phenyl), 121.5 (1C, C-1''phenyl),
123.6 (1C, C-1'4-(4-phenylbuta-1,3-diynyl)phenyl), 125.4 (1C, C-5triazole), 128.7 (2C,
C-3''phenyl, C-5''phenyl), 129.7 (1C, C-4''phenyl), 132.7 (2C, C-2''phenyl, C-6''phenyl),
134.2 (2C, C-3'4-(4-phenylbuta-1,3-diynyl)phenyl, C-5'4-(4-phenylbuta-1,3-diynyl)phenyl), 136.3
(1C, C-4'4-(4-phenylbuta-1,3-diynyl)phenyl), 140.9 (1C, C-4triazole), 161.0 (1C,
CO2CH3); IR (neat): ꢀꢁ [cm-1] = 2986, 1728, 1520, 1439, 1369, 1231, 1153,
1042, 833, 756, 691; HRMS (m/z): [M+H]+ calcd for C20H14N3O2, 328.1081;
found, 328.1095; HPLC (method 1): tR = 21.8 min, purity 98.0%.
Methyl 1-[4-(phenylethynyl)phenyl)]-1H-1,2,3-triazole-4-carboxylate
(5h): Under N2 atmosphere tetrakis(triphenylphosphine)palladium(0) (12
mg, 0.01 mmol) and copper(I) iodide (4 mg, 0.02 mmol) were added to a
solution of 5a (329 mg, 1.0 mmol) in a 1:2 mixture of triethylamine and
DMF (15 mL). Then phenylacetylene (0.13 mL, 1.2 mmol) was added
slowly and the mixture was stirred overnight at 70 °C. Then the reaction
mixture was diluted with water and extracted with ethyl acetate (3×). The
combined organic phases were dried with Na2SO4, filtered and the solvent
was removed in vacuo. The residue was purified by flash column
chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, cyclohexane/ethyl
acetate = 3:1, Rf = 0.28) to give 5h as colorless crystalline solid (291 mg,
0.96 mmol, 96% yield). m.p. = 189 °C; 1H NMR (CDCl3): δ [ppm] = 4.00 (s,
3H, CO2CH3), 7.35 – 7.40 (m, 3H, Harom.), 7.53 – 7.57 (m, 2H, Harom.), 7.68
– 7.73 (m, 2H, Harom.), 7.75 – 7.79 (m, 2H, Harom.), 8.54 (s, 5-Htriazole); 13C
NMR (CDCl3): δ [ppm] = 52.6 (1C, CO2CH3), 87.9 (1C, C≡C), 91.9 (1C,
C≡C), 120.7 (2C, Carom.), 122.7 (1C, Carom.), 125.0 (1C, Carom.), 125.5 (1C,
C-5triazole), 128.6 (2C, Carom.), 129.0 (1C, Carom.), 131.8 (2C, Carom.), 133.2
(2C, Carom.), 135.7 (1C, Carom.), 140.8 (1C, C-4triazole), 161.1 (1C, CO2CH3);
IR (neat): ꢀꢁ [cm-1] = 3132, 2951, 1701, 1543, 1520, 1435, 1350, 1265,
1157, 1034, 760, 694; HRMS (m/z): [M+H]+ calcd for C18H14N3O2,
304.1081; found, 304.1066; HPLC (method 1): tR = 21.2 min, purity 98.1%.
N-Hydroxy-1-phenyl-1H-1,2,3-triazole-4-carboxamide
(2b):
Hydroxylamine hydrochloride (139 mg, 2.0 mmol) and a 2.0 M solution of
sodium methoxide in methanol (1.5 mL, 3.0 mmol) were added to a
solution of 5b (203 mg, 1.0 mmol) in dry methanol (8 mL). The reaction
mixture was stirred at ambient temperature for 20 h until TLC showed
complete conversion of the ester. The reaction mixture was acidified with
1.0 M HCl to pH 5-6. Then the mixture was extracted with ethyl acetate
(3×). The combined organic phases were dried with Na2SO4, filtered and
the solvent was dried in vacuo. The crude mixture was purified by flash
column chromatography (Ø
= 2 cm, h = 15 cm, V = 10 mL,
dichloromethane/methanol= 10:1, Rf = 0.37) to give 2b as colorless
crystalline solid (135 mg, 0.66 mmol, 66%). m.p. = 171 °C; 1H NMR
(CD3OD): δ [ppm] = 7.50 – 7.55 (m, 1H, H-4'phenyl), 7.58 – 7.63 (m, 2H, 3'-
Hphenyl, 5'-Hphenyl), 7.86 – 7.90 (m, 2H, 2'-Hphenyl, 6'-Hphenyl), 8.91 (s, 5-
Htriazole); 13C NMR (CD3OD): δ [ppm] = 121.8 (2C, C-2'phenyl, C-6'phenyl),
125.3 (1C, C-5triazole), 130.5 (1C, C-4'phenyl), 131.0 (2C, C-3'phenyl, C-5'phenyl),
138.0 (1C, C-1'phenyl), 143.0 (1C, C-4triazole), 160.3 (1C, CONHOH); IR
(neat): ꢀꢁ [cm-1] = 3325, 3136, 1628, 1566, 1493, 1412, 1254, 1177, 1030,
876, 760, 683; HRMS (m/z): [M+H]+ calcd for C9H9N4O2, 205.0720; found,
205.0712; HPLC (method 2): tR = 12.5 min, purity 99.3%.
Methyl
carboxylate
1-{4-[2-(trimethylsilyl)ethynyl]phenyl}-1H-1,2,3-triazole-4-
(7): Under N2 atmosphere
tetrakis(triphenylphosphine)palladium(0) (12 mg, 0.01 mmol), copper(I)
iodide (4 mg, 0.02 mmol) and triethylamine (0.70 mL, 5.0 mmol) were
added to a solution of 5a (329 mg, 1.0 mmol) in dry acetonitrile (15 mL).
Then trimethylsilylacetylene (0.28 mL, 2.0 mmol) was added slowly. After
stirring the reaction mixture at ambient temperature for 3 h, water was
added and the mixture was extracted with ethyl acetate (3×). The
combined organic phases were dried with Na2SO4, filtered and the solvent
removed in vacuo. The residue was purified by flash column
chromatography (Ø = 2 cm, h = 15 cm, V = 10 mL, cyclohexane/ethyl
acetate = 4:1, Rf = 0.29) to give 7 as colorless crystalline solid (290 mg,
0.97 mmol, 97% yield). m.p. = 188 °C; 1H NMR (CDCl3): δ [ppm] = 0.27 (s,
9H, Si(CH3)3), 4.00 (s, 3H, CO2CH3), 7.61 – 7.65 (m, 2H, 3'-H4-[2-
(trimethylsilyl)ethynyl]phenyl, 5'-H4-[2-(trimethylsilyl)ethynyl]phenyl), 7.70 – 7.75 (m, 2H, 2'-H4-
N-Hydroxy-1-[4-(2-phenylethynyl)phenyl]-1H-1,2,3-triazole-4-
carboxamide (2h): Hydroxylamine hydrochloride (195 mg, 2.8 mmol) and
a 2.0 M solution of sodium methoxide in methanol (1.4 mL, 2.8 mmol) were
added to a solution of 5h (170 mg, 0.56 mmol) in dry methanol (10 mL).
The reaction mixture was stirred at ambient temperature for 20 h until TLC
showed complete conversion of the ester. Then reaction mixture was
acidified with a 1.0 M solution of HCl until pH 5-6 was reached. The
precipitate was washed with dichloromethane, water and ethyl acetate and
dried in vacuo for 4 h to give 2h as colorless solid (156 mg, 0.51 mmol,
91% yield). TLC (CH2Cl2:methanol = 10:1): Rf = 0.35; m.p. = 219 °C; 1H
NMR (DMSO-d6): δ [ppm] = 7.44 – 7.48 (m, 3H, 3''-Hphenyl, 4''-Hphenyl, 5''-
Hphenyl), 7.58 – 7.62 (m, 2H, 2''-Hphenyl, 6''-Hphenyl), 7.77 – 7.81 (m, 2H, 3'-
[2-(trimethylsilyl)ethynyl]phenyl, 6'-H4-[2-(trimethylsilyl)ethynyl]phenyl),8.58 (s br, 5-Htriazole); 13
NMR (CDCl3): δ [ppm] = 0.0 (3C, Si(CH3)3), 52.6 (1C, CO2CH3), 97.3 (1C,
C≡CSi(CH3)3), 103.3 (1C, C≡CSi(CH3)3), 120.6 (2C, C-2'4-[2-
C
,
C-6'4-[2-(trimethylsilyl)ethynyl]phenyl), 124.8 (1C, C-1'4-[2-
(trimethylsilyl)ethynyl]phenyl
(trimethylsilyl)ethynyl]phenyl), 133.7 (2C, C-3'4-[2-(trimethylsilyl)ethynyl]phenyl C-5'4-[2-
(trimethylsilyl)ethynyl]phenyl), 136.0 (1C, C-4'4-[2-(trimethylsilyl)ethynyl]phenyl), 161.1 (1C,
CO2CH3), the signals for the carbon atoms of the triazole ring could not be
,
9
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10.1002/cmdc.201900583
ChemMedChem
FULL PAPER
H4-(2-phenylethynyl)phenyl, 5'-H4-(2-phenylethynyl)phenyl), 8.00 – 8.06 (m, 2H, 2'-H4-(2-
phenylethynyl)phenyl, 6'-H4-(2-phenylethynyl)phenyl), 9.18 (s br, 1H, NHOH), 9.33 (s, 5-
Htriazole), 11.45 (s br, 1H, NHOH); 13C NMR (DMSO-d6): δ [ppm] = 88.2 (1C,
by automatic flash column chromatography (100 % H2O → 100 % CH3CN,
Biotage SNAP KP-C18-HS 12 g) to give 13 as colorless solid (43 mg, 0.17
mmol, 43% yield). m.p. = 220 °C; TLC (dichloromethane/methanol, 10/1
V/V): Rf = 0.36; 1H NMR (DMSO-d6): δ [ppm] = 7.12 – 7.16 (m, 1H, 3'-
Hphenyl), 7.41 – 7.46 (m, 1H, 4'-Hphenyl), 7.71 – 7.74 (m, 1H, 6'-Hphenyl), 8.86
(s, 1H, 5-Htriazole), 9.16 (s br, 1H, CONHOH), 11.05 (s br, 1H, OH), 11.40
(s br, 1H, CONHOH); 13C NMR (DMSO-d6): δ [ppm] = 118.6 (1C, C-3'phenyl),
122.6 (1C, C-5'phenyl), 124.8 (1C, C-1'phenyl), 124.9 (1C, C-6'phenyl), 127.5
(1C, C-5triazole), 130.4 (1C, C-4'phenyl), 141.1 (1C, C-4triazole), 149.1 (1C, C-
2'phenyl), 157.4 (1C, CONHOH); IR (neat): ꢀꢁ [cm-1] = 3267, 3156, 1670,
1601, 1558, 1408, 1296, 1254, 1200, 1042, 883, 814, 737, 660; HRMS
(m/z): [M+H]+ calcd for C9H8ClN4O3, 255.0279; found, 255.0305; HPLC
(method 2): tR = 14.0 min, purity 97.2 %.
C≡C), 90.9 (1C, C≡C), 120.5 (2C, C-2'4-(2-phenylethynyl)phenyl
, C-6'4-(2-
phenylethynyl)phenyl), 121.9 (1C, C-1''phenyl), 122.9 (1C, C-4'4-(2-phenylethynyl)phenyl),
124.5 (1C, C-5triazole), 128.8 (2C, C-3''phenyl, C-5''phenyl), 129.1 (1C, C-
4''phenyl), 131.5 (2C, C-2''phenyl, C-6''phenyl), 132.9 (2C, C-3'4-(2-phenylethynyl)phenyl
,
C-5'4-(2-phenylethynyl)phenyl), 135.9 (1C, C-1'4-(2-phenylethynyl)phenyl), 142.4 (1C, C-
4triazole), 157.1 (1C, CONHOH); IR (neat): ꢀꢁ [cm-1] = 3310, 3140, 2986,
1632, 1566, 1516, 1497, 1439, 1373, 1258, 1177, 1030, 880, 841, 752,
691; HRMS (m/z): [M+H]+ calcd for C17H13N4O2, 305.1033; found,
305.1035; HPLC (method 2): tR = 16.6 min, purity 96.1%.
N-Hydroxy-1-[4-(4-phenylbuta-1,3-diynyl)phenyl]-1H-1,2,3-triazole-4-
carboxamide (2j): Hydroxylamine hydrochloride (192 mg, 2.8 mmol) and
a 2.0 M solution of sodium methoxide in methanol (1.4 mL, 2.8 mmol) were
added to a solution of 5j (150 mg, 0.46 mmol) in dry methanol (10 mL).
The reaction mixture was stirred at ambient temperature for 20 h until TLC
showed complete conversion of the ester. The reaction mixture was
acidified with a 1.0 M solution of HCl until pH 5-6 was reached. The
precipitate was washed with dichloromethane, water and ethyl acetate and
dried in vacuo for 4 h to give 2j as colorless solid (102 mg, 0.31 mmol,
68% yield). TLC (CH2Cl2:methanol = 9:1): Rf = 0.30; m.p. = 188 °C
(decomposition); 1H NMR (DMSO-d6): δ [ppm] = 7.43 – 7.54 (m, 3H, 3''-
Methyl 1-{5-chloro-2-[(4-iodobenzyl)oxy]phenyl}-1H-1,2,3-triazole-4-
carboxylate (15): 4-Iodobenzyl bromide (490 mg, 1.7 mmol) was added
to a stirring suspension of 11 (350 mg, 1.4 mmol) and cesium carbonate
(900 mg, 2.8 mmol) in N,N-dimethylformamide (6.5 mL). The reaction
mixture was heated to 90 °C for 100 min. After cooling to room temperature,
water was added and the mixture was extracted with ethyl acetate (3×).
The combined organic layers were dried (Na2SO4), filtered and the solvent
was removed in vacuo. The residue was purified by flash column
chromatography (Ø = 3 cm, h = 15 cm, cyclohexane/ethyl acetate = 9/1 →
0/1, V = 15 mL) to give 15 (380 mg, 0.82 mmol, 59%) as colorless solid.
m.p. = 186 °C; TLC (cyclohexane/ethyl acetate, 2/1 V/V): Rf = 0.58; 1H
NMR (DMSO-d6): δ [ppm] = 3.88 (s, 3H, CO2CH3), 5.21 (s, 2H, OCH2Ar),
7.14 – 7.17 (m, 2H, 2''-H4-iodophenyl, 6''-H4-iodophenyl), 7.39 – 7.42 (m, 1H, 3'-
H5-chlorophenyl), 7.62 – 7.66 (m, 1H, 4'-H5-chlorophenyl), 7.70 – 7.74 (m, 2H, 3''-
H4-iodophenyl, 5''-H4-iodophenyl), 7.82 – 7.84 (m, 1H, 6'-H5-chlorophenyl), 9.14 (s, 1H,
5-Htriazole); 13C NMR (DMSO-d6): δ [ppm] = 52.0 (1C, CO2CH3), 70.0 (1C,
OCH2Ar), 94.3 (1C, C-4''4-iodophenyl), 116.1 (1C, C-3'5-chlorophenyl), 124.7 (1C,
Carom.), 126.0 (1C, C-6'5-chlorophenyl), 126.2 (1C, Carom.), 129.6 (2C, C-2''4-
iodophenyl, C-6''4-iodophenyl), 131.0 (1C, C-4'5-chlorophenyl), 131.1 (1C, C-5triazole),
135.7 (1C, C-1''4-iodophenyl), 137.2 (2C, C-3''4-iodophenyl, C-5''4-iodophenyl), 138.5
(1C, C-4triazole), 149.9 (1C, C-2'5-chlorophenyl), 160.5 (1C, CO2CH3); IR (neat):
ꢀꢁ [cm-1] = 3657, 3063, 2978, 1744, 1501, 1462, 1369, 1285, 1246, 1211,
1134, 1038, 999, 949, 826, 795, 772; HRMS (m/z): [M+H]+ calcd for
C17H14ClIN3O3, 469.9763; found, 469.9786; HPLC (method 1): tR = 23.3
min, purity 96.7 %.
H
phenyl, 4''-Hphenyl, 5''-Hphenyl), 7.61 – 7.65 (m, 2H, 2''-Hphenyl, 6''-Hphenyl), 7.83
– 7.87 (m, 2H, 3'-H4-(4-phenylbuta-1,3-diynyl)phenyl, 5'-H4-(4-phenylbuta-1,3-diynyl)phenyl),
8.04 8.08 (m, 2H, 2'-H4-(4-phenylbuta-1,3-diynyl)phenyl 6'-H4-(4-phenylbuta-1,3-
–
,
diynyl)phenyl), 9.21 (s br, 1H, NHOH), 9.35 (s, 5-Htriazole), 11.46 (s br, 1H,
NHOH); 13C NMR (DMSO-d6): δ [ppm] = 73.3 (1C, C≡C), 74.9 (1C, C≡C),
80.6 (1C, C≡C), 82.7 (1C, C≡C), 120.2 (1C, C-1''phenyl), 120.6 (2C, C-2'4-(4-
,
C-6'4-(4-phenylbuta-1,3-diynyl)phenyl), 121.0 (1C, C-1'4-(4-
phenylbuta-1,3-diynyl)phenyl
phenylbuta-1,3-diynyl)phenyl), 124.6 (1C, C-5triazole), 129.0 (2C, C-3''phenyl, C-5''phenyl),
130.2 (1C, C-4''phenyl), 132.5 (2C, C-2''phenyl, C-6''phenyl), 134.1 (2C, C-3'4-(4-
,
C-5'4-(4-phenylbuta-1,3-diynyl)phenyl), 136.7 (1C, C-4'4-(4-
phenylbuta-1,3-diynyl)phenyl
phenylbuta-1,3-diynyl)phenyl), 142.5 (1C, C-4triazole), 157.1 (1C, CONHOH); IR
(neat): ꢀꢁ [cm-1] = 3333, 3129, 2920, 2851, 1655, 1570, 1485, 1377, 1250,
1184, 1038, 833, 748, 683; HRMS (m/z): [M+Na]+ calcd for C19H12N4O2Na,
351.0852; found, 351.0867; HPLC (method 2): tR = 18.1 min, purity 93.3%.
Methyl 1-(5-chloro-2-hydroxyphenyl)-1H-1,2,3-triazole-4-carboxylate
(11): Methyl propiolate (0.36 mL, 336 mg, 4.0 mmol) was added to a
stirring solution of 9 (507 mg, 3.0 mmol) in a 1:1 mixture of water and tert-
butyl alcohol (15 mL). Then sodium ascorbate (40 mg, 0.20 mmol) and
copper(II) sulfate pentahydrate (10 mg, 0.04 mmol) were added and the
mixture was stirred for 18 h at room temperature. Then water was added
and the mixture was extracted with ethyl acetate (3×). The combined
organic layers were dried (Na2SO4), filtered and the solvent was removed
in vacuo. The residue was purified by flash column chromatography (Ø =
3 cm, h = 15 cm, cyclohexane/ethyl acetate = 2/1, V = 15 mL, Rf = 0.41) to
give 11 (675 mg, 2.7 mmol, 89%) as pale brown solid. m.p. = 130 °C; 1H
NMR (DMSO-d6): δ [ppm] = 3.88 (s, 3H, CO2CH3), 7.12 – 7.16 (m, 1H, 3-
H'phenyl), 7.43 – 7.47 (m, 1H, 4-H'phenyl), 7.72 – 7.74 (m, 1H, 6-H'phenyl), 9.07
(s, 1H, 5-Htriazole), 11.03 (s br, 1H, OH); 13C NMR (DMSO-d6): δ [ppm] =
52.0 (1C, CO2CH3), 118.6 (1C, C-3'phenyl), 122.6 (1C, C-5'phenyl), 124.5 (1C,
C-1'phenyl), 125.1 (1C, C-6'phenyl), 130.5 (1C, C-5triazole), 130.6 (1C, C-4'phenyl),
138.5 (1C, C-4triazole), 149.2 (1C, C-2'phenyl), 160.6 (1C, CO2CH3); IR (neat):
ꢀꢁ [cm-1] = 3183, 3144, 1748, 1532, 1508, 1439, 1292, 1157, 1157, 1130,
1049, 818, 733, 698, 656; HRMS (m/z): [M+H]+ calcd for C10H9ClN3O3,
254.0327; found, 254.0329; HPLC (method 1): tR = 17.7 min, purity 99.6 %.
1-{5-Chloro-2-[(4-iodobenzyl)oxy]phenyl}-N-hydroxy-1H-1,2,3-
triazole-4-carboxamide (17): A 5.4 M solution of sodium methoxide in
methanol (0.3 mL, 1.6 mmol) was added to a solution of 15 (110 mg, 0.23
mmol) and hydroxylamine hydrochloride (94 mg, 1.4 mmol) in dry
methanol (5 mL). The mixture was stirred at ambient temperature
overnight. Then the solvent was removed in vacuo and the residue was
purified by automatic flash column chromatography using a Biotage
purification apparatus (5% → 100% ACN in H2O, Biotage® SNAP KP-C18-
HS 30 g). Fractions containing the desired product were combined, dried
from acetonitrile under reduced pressure and then subjected to
lyophilization to give 17 (55 mg, 0.12 mmol, 52%) as colorless solid. m.p.
= 168-170 °C; 1H NMR (DMSO-d6): δ [ppm] = 5.22 (s, 2H, OCH2Ar), 7.15
– 7.19 (m, 2H, 2''-H4-iodophenyl, 6''-H4-iodophenyl), 7.37 – 7.40 (m, 1H, 3'-H5-
chlorophenyl), 7.59 – 7.62 (m, 1H, 4'-H5-chlorophenyl), 7.69 – 7.73 (m, 2H, 3''-H4-
iodophenyl, 5''-H4-iodophenyl), 7.79 – 7.81 (m, 1H, 6'-H5-chlorophenyl), 8.82 (s, 1H, 5-
Htriazole); 13C NMR (DMSO-d6): δ [ppm] = 69.8 (1C, OCH2Ar), 94.3 (1C, C-
4''4-iodophenyl), 116.1 (1C, C-3'5-chlorophenyl), 124.7 (1C, C-5'5-chlorophenyl), 125.8
(1C, C-6'5-chlorophenyl), 126.5 (1C, C-1'5-chlorophenyl), 127.5 (1C, C-5triazole),
129.6 (2C, C-2''4-iodophenyl, C-6''4-iodophenyl), 130.6 (1C, C-4'5-chlorophenyl), 135.8
(1C, C-1''4-iodophenyl), 137.2 (2C, C-3''4-iodophenyl, C-5''4-iodophenyl), 141.9 (1C, C-
4triazole), 149.7 (1C, C-2'5-chlorophenyl), 157.3 (1C, CONHOH); IR (neat): ꢀꢁ [cm-
1] = 3159, 2978, 2886, 1647, 1620, 1578, 1504, 1462, 1404, 1373, 1285,
1250, 1177, 1130, 1096, 1007, 880, 799, 745, 660; HRMS (m/z): [M+H]+
calcd for C16H13ClIN4O3, 470.9715; found, 470.9706; HPLC (method 2): tR
= 16.6 min, purity 96.8 %.
1-(5-Chloro-2-hydroxyphenyl)-N-hydroxy-1H-1,2,3-triazole-4-
carboxamide (13): Hydroxylamine hydrochloride (165 mg, 2.4 mmol) and
a 2 M solution of sodium methoxide in methanol (1.5 mL, 3.0 mmol) were
added to a solution of 11 (101 mg, 0.40 mmol) in dry methanol (10 mL)
and the mixture was stirred at ambient temperature for 20 h. Then water
was added. The mixture was acidified with 1 M HCl to pH 5-6 and extracted
with ethyl acetate (3×). The combined organic layers were dried (Na2SO4),
filtered and the solvent was removed in vacuo. The residue was purified
Methyl
1-[5-chloro-2-(4-fluorophenoxy)phenyl]-1H-1,2,3-triazole-4-
carboxylate (19): A 25 mL round-bottom flask was charged with 11 (150
10
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10.1002/cmdc.201900583
ChemMedChem
FULL PAPER
mg, 0.59 mmol), Cu(OAc)2 (107 mg, 0.59 mmol), 4-fluorophenylboronic
acid (99 mg, 0.71 mmol), and powdered 4 Å molecular sieves. Then
dichloromethane (4.5 mL) was added. After the addition of triethylamine
(0.41 mL, 2.9 mmol), the reaction mixture was stirred at ambient
temperature overnight. Then the suspension was filtered. The filtrate
diluted with water and extracted with ethyl acetate (3×). The combined
organic layers were dried (Na2SO4), filtered and the solvent was removed
in vacuo. The residue was purified by flash column chromatography (Ø =
2 cm, h = 15 cm, cyclohexane/ethyl acetate = 9/1 → 3/1, V = 10 mL) to
give 19 (27 mg, 0.08 mmol, 13%) as colorless solid. m.p. = 163-164 °C;
TLC (cyclohexane/ethyl acetate, 2/1 V/V): Rf = 0.68; 1H NMR (DMSO-d6):
δ [ppm] = 3.86 (s, 3H, CO2CH3), 7.03 – 7.07 (m, 1H, 3'-H5-chlorophenyl), 7.16
– 7.22 (m, 2H, 2''-H4-fluorophenyl, 6''-H4-fluorophenyl), 7.23 – 7.29 (m, 2H, 3''-H4-
fluorophenyl, 5''-H4-fluorophenyl), 7.61 – 7.65 (m, 1H, 4'-H5-chlorophenyl), 7.97 – 7.98
(m, 1H, 6'-H5-chlorophenyl), 9.25 (s, 1H, 5-Htriazole); 13C NMR (DMSO-d6): δ
[ppm] = 52.0 (1C, CO2CH3), 116.9 (d, J = 23.6 Hz, 2C, C-3''4-fluorophenyl, C-
5''4-fluorophenyl), 119.8 (1C, C-3'5-chlorophenyl), 121.5 (d, J = 8.6 Hz, 2C, C-2''4-
fluorophenyl, C-6''4-fluorophenyl), 126.7 (1C, C-6'5-chlorophenyl), 127.2 (1C, Carom.),
127.5 (1C, Carom.), 131.0 (1C, C-5triazole), 131.5 (1C, C-4'5-chlorophenyl), 138.7
(1C, C-4triazole), 149.4 (1C, C-2'5-chlorophenyl), 150.9 (d, J = 2.6 Hz, 1C, C-1''4-
fluorophenyl), 159.0 (d, J = 241 Hz, 1C, C-4''4-fluorophenyl), 160.4 (1C, CO2CH3);
IR (neat): ꢀꢁ [cm-1] = 3175, 3063, 2924, 1728, 1524, 1504, 1489, 1458,
1435, 1369, 1254, 1211, 1184, 1150, 1123, 1034, 991, 853, 814, 775, 687;
HRMS (m/z): [M+H]+ calcd for C16H12ClFN3O3, 348.0546; found, 348.0543;
HPLC (method 1): tR = 22.3 min, purity 97.6 %.
(1C, C-62-azido-4-chlorophenyl), 123.6 (1C, C-4'phenyl), 126.0 (1C, C-52-azido-4-
chlorophenyl), 128.8 (1C, C-42-azido-4-chlorophenyl), 130.1 (2C, C-3'phenyl, C-5'phenyl),
132.6 (1C, C-22-azido-4-chlorophenyl), 146.4 (1C, C-12-azido-4-chlorophenyl), 156.7 (1C,
C-1'phenyl); IR (neat): ꢀꢁ [cm-1] = 3063, 2106, 1585, 1481, 1400, 1296, 1227,
1196, 1161, 1142, 1107, 833, 748, 691; HRMS (m/z): [M+H]+ calcd for
C12H9ClN3O, 246.0429; found, 246.0442; HPLC (method 1): tR = 24.5 min,
purity 99.8 %.
Methyl 1-(5-chloro-2-phenoxyphenyl)-1H-1,2,3-triazole-4-carboxylate
(27): Methyl propiolate (0.13 mL, 140 mg, 1.6 mmol) was added to a
stirring solution of 25 (380 mg, 1.6 mmol) in a 1:1 mixture of water and tert-
butyl alcohol (10 mL). Then sodium ascorbate (30 mg, 0.15 mmol) and
copper(II) sulfate pentahydrate (9 mg, 0.04 mmol) were added and the
mixture was stirred for 14 h at room temperature. Then water was added
and the mixture was extracted with ethyl acetate (3×). The combined
organic layers were dried (Na2SO4), filtered and the solvent was removed
in vacuo. The residue was purified by flash column chromatography (Ø =
4 cm, h = 15 cm, cyclohexane/ethyl acetate = 9/1 → 3/1, V = 30 mL) to
give 27 (350 mg, 1.1 mmol, 69%) as colorless solid. m.p. = 135-136 °C;
TLC (cyclohexane/ethyl acetate, 2/1 V/V): Rf = 0.71; 1H NMR (DMSO-d6):
δ [ppm] = 3.86 (s, 3H, CO2CH3), 7.07 – 7.12 (m, 3H, 2''-Hphenyl, 6''-Hphenyl
,
3'-H5-chloro-2-phenoxyphenyl), 7.19 – 7.23 (m, 1H, 4''-Hphenyl), 7.39 – 7.44 (m, 2H,
3''-Hphenyl, 5''-Hphenyl), 7.63 – 7.66 (m, 1H, 4'-H5-chloro-2-phenoxyphenyl), 7.97 –
7.99 (m, 1H, 6'-H5-chloro-2-phenoxyphenyl), 9.22 (s, 1H, 5-Htriazole); 13C NMR
(DMSO-d6): δ [ppm] = 52.0 (1C, CO2CH3), 119.2 (2C, C-2''phenyl, C-6''phenyl),
120.5 (1C, C-3'5-chloro-2-phenoxyphenyl), 124.9 (1C, C-4''phenyl), 126.7 (1C, C-6'5-
chloro-2-phenoxyphenyl), 127.3 (1C, Carom.), 127.9 (1C, Carom.), 130.3 (2C, C-
3''phenyl, C-5''phenyl), 130.9 (1C, C-5triazole), 131.5 (1C, C-4'5-chloro-2-phenoxyphenyl),
138.7 (1C, C-4triazole), 149.0 (1C, C-2'5-chloro-2-phenoxyphenyl), 155.0 (1C, C-
1''phenyl), 160.4 (1C, CO2CH3); IR (neat): ꢀꢁ [cm-1] = 2978, 1744, 1585, 1528,
1485, 1462, 1435, 1369, 1238, 1215, 1153, 1123, 1034, 995, 945, 868,
814, 772, 691; HRMS (m/z): [M+H]+ calcd for C16H13ClN3O3, 330.0640;
found, 330.0634; HPLC (method 1): tR = 22.3 min, purity 99.7 %.
1-[5-Chloro-2-(4-fluorophenoxy)phenyl]-N-hydroxy-1H-1,2,3-triazole-
4-carboxamide (21): A 5.4 M solution of sodium methoxide in methanol
(0.2 mL, 1.1 mmol) was added to a solution of 19 (39 mg, 0.11 mmol) and
hydroxylamine hydrochloride (38 mg, 0.55 mmol) in dry methanol (3 mL).
The mixture was stirred at ambient temperature overnight. Then the
solvent was removed in vacuo and the residue was purified by automatic
flash column chromatography using a Biotage purification apparatus (5%
→ 50% ACN in H2O, Biotage® SNAP KP-C18-HS 12 g). Fractions
containing the desired product were combined, dried from acetonitrile
under reduced pressure and then subjected to lyophilization to give 21 (37
mg, 0.11 mmol, 95%) as yellowish solid. m.p. = 168 °C (decomposition);
1H NMR (DMSO-d6): δ [ppm] = 7.02 – 7.06 (m, 1H, 3'-H5-chlorophenyl), 7.15 –
7.20 (m, 2H, 2''-H4-fluorophenyl, 6''-H4-fluorophenyl), 7.22 – 7.28 (m, 2H, 3''-H4-
fluorophenyl, 5''-H4-fluorophenyl), 7.53 – 7.57 (m, 1H, 4'-H5-chlorophenyl), 7.87 – 7.90
(m, 1H, 6'-H5-chlorophenyl), 8.31 (s, 1H, 5-Htriazole); 13C NMR (DMSO-d6): δ
[ppm] = 116.9 (d, J = 23.7 Hz, 2C, C-3''4-fluorophenyl, C-5''4-fluorophenyl), 120.4
(1C, C-3'5-chlorophenyl), 121.1 (d, J = 8.7 Hz, 2C, C-2''4-fluorophenyl, C-6''4-
fluorophenyl), 123.1 (1C, C-5triazole), 125.7 (1C, C-6'5-chlorophenyl), 127.4 (1C, C-
5'5-chlorophenyl), 128.8 (1C, C-1'5-chlorophenyl), 130.2 (1C, C-4'5-chlorophenyl), 147.1
(1C, C-4triazole), 148.4 (1C, C-2'5-chlorophenyl), 151.3 (d, J = 2.4 Hz, 1C, C-1''4-
fluorophenyl), 158.3 (1C, CONHOH), 158.8 (d, J = 241 Hz, 1C, C-4''4-fluorophenyl);
IR (neat): ꢀꢁ [cm-1] = 3175, 2978, 2886, 1605, 1493, 1454, 1393, 1250,
1219, 1184, 1130, 1092, 1030, 876, 849, 822, 772, 683; HRMS (m/z):
[M+H]+ calcd for C15H11ClFN4O3, 349.0498; found, 349.0522; HPLC
(method 2): tR = 16.3 min, purity 92.4 %.
1-(5-Chloro-2-phenoxyphenyl)-N-hydroxy-1H-1,2,3-triazole-4-
carboxamide (29): A 5.4 M solution of sodium methoxide in methanol (0.6
mL, 3.2 mmol) was added to a solution of 27 (300 mg, 0.91 mmol) and
hydroxylamine hydrochloride (250 mg, 3.6 mmol) in dry methanol (12 mL).
The mixture was stirred at ambient temperature overnight. Then the
solvent was removed in vacuo and the residue was purified by automatic
flash column chromatography using a Biotage purification apparatus (5%
→ 80% ACN in H2O, Biotage® SNAP KP-C18-HS 30 g). Fractions
containing the desired product were combined, dried from acetonitrile
under reduced pressure and then subjected to lyophilization to give 29
(210 mg, 0.64 mmol, 70%) as colorless solid. m.p. = 116-118 °C; 1H NMR
(DMSO-d6): δ [ppm] = 7.07 – 7.12 (m, 3H, 2''-Hphenyl, 6''-Hphenyl, 3'-H5-chloro-
2-phenoxyphenyl), 7.19 – 7.23 (m, 1H, 4''-Hphenyl), 7.39 – 7.43 (m, 2H, 3''-Hphenyl
,
5''-Hphenyl), 7.61 – 7.63 (m, 1H, 4'-H5-chloro-2-phenoxyphenyl), 7.95 – 7.97 (m, 1H,
6'-H5-chloro-2-phenoxyphenyl), 8.92 (s, 1H, 5-Htriazole); 13C NMR (DMSO-d6): δ
[ppm] = 119.1 (2C, C-2''phenyl, C-6''phenyl), 120.7 (1C, C-3'5-chloro-2-phenoxyphenyl),
124.8 (1C, C-4''phenyl), 126.3 (1C, C-6'5-chloro-2-phenoxyphenyl), 127.4 (1C, C-5'5-
chloro-2-phenoxyphenyl), 127.6 (1C, C-5triazole), 128.3 (1C, C-1'5-chloro-2-phenoxyphenyl),
130.3 (2C, C-3''phenyl, C-5''phenyl), 131.1 (1C, C-4'5-chloro-2-phenoxyphenyl), 141.7
(1C, C-4triazole), 148.6 (1C, C-2'5-chloro-2-phenoxyphenyl), 155.1 (1C, C-1''phenyl),
157.2 (1C, CONHOH); IR (neat): ꢀꢁ [cm-1] = 3152, 2978, 2886, 1647, 1578,
1485, 1458, 1381, 1265, 1238, 1177, 1126, 1030, 876, 768, 691; HRMS
(m/z): [M+H]+ calcd for C15H12ClN4O3, 331.0592; found, 331.0624; HPLC
(method 2): tR = 16.2 min, purity 99.3 %.
2-Azido-4-chloro-1-phenoxybenzene (25): 2-Amino-4-chlorophenyl
phenyl ether (650 mg, 3.0 mmol) was dissolved in 2 M HCl (10 mL) and
the solution was stirred in an ice bath. Then an ice-cold solution of sodium
nitrite (301 mg, 4.4 mmol) in water (1.5 mL) was added dropwise over a
period of 5 min. After additional 5 min, urea (27 mg) was added to destroy
the excess of nitrous acid. Then an ice-cold solution of sodium azide (384
mg, 5.9 mmol) and sodium acetate (1.7 mg, 0.02 mmol) in water (8 mL)
was added. The mixture was stirred in an ice bath for 2 h. Then it was
extracted with ethyl acetate (3×). The combined organic layers were dried
(Na2SO4), filtered and the solvent was removed in vacuo. The residue was
purified by flash column chromatography (Ø = 4 cm, h = 15 cm,
cyclohexane/ethyl acetate = 9/1, V = 30 mL) to give 25 (459 mg, 1.9 mmol,
63%) as yellowish oil. 1H NMR (DMSO-d6): δ [ppm] = 6.95 – 7.00 (m, 2H,
2'-Hphenyl, 6'-Hphenyl), 7.02 – 7.05 (m, 1H, 6-H2-azido-4-chlorophenyl), 7.12 – 7.17
(m, 1H, 4'-Hphenyl), 7.23 – 7.26 (m, 1H, 5-H2-azido-4-chlorophenyl), 7.36 – 7.42 (m,
3H, 3-H2-azido-4-chlorophenyl, 3'-Hphenyl, 5'-Hphenyl); 13C NMR (DMSO-d6): δ [ppm]
= 117.2 (2C, C-2'phenyl, C-6'phenyl), 121.3 (1C, C-32-azido-4-chlorophenyl), 122.5
Methyl
1-(2-benzoylphenyl)-1H-1,2,3-triazole-4-carboxylate
(31):
Methyl propiolate (0.15 mL, 140 mg, 1.7 mmol) was added to a stirring
solution of 30 (390 mg, 1.7 mmol) in a 1:1 mixture of water and tert-butyl
alcohol (10 mL). Then sodium ascorbate (34 mg, 0.17 mmol) and
copper(II) sulfate pentahydrate (9 mg, 0.04 mmol) were added and the
mixture was stirred for 79 h at room temperature. Then water was added
and the mixture was extracted with ethyl acetate (3×). The combined
organic layers were dried (Na2SO4), filtered and the solvent was removed
in vacuo. The residue was purified by flash column chromatography (Ø =
11
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900583
ChemMedChem
FULL PAPER
3 cm, h = 15 cm, cyclohexane/ethyl acetate = 4/1 → 1/1, V = 20 mL) to
give 31 (225 mg, 0.73 mmol, 42%) as colorless solid. m.p. = 147-148 °C;
TLC (cyclohexane/ethyl acetate, 2/1 V/V): Rf = 0.33; 1H NMR (DMSO-d6):
δ [ppm] = 3.82 (s, 3H, CO2CH3), 7.41 – 7.46 (m, 2H, 3''-Hphenyl, 5''-Hphenyl),
7.57 – 7.62 (m, 3H, 2''-Hphenyl, 4''-Hphenyl, 6''-Hphenyl), 7.70 – 7.73 (m, 1H, 3'-
H2-benzoylphenyl), 7.75 – 7.79 (m, 1H, 4'-H2-benzoylphenyl), 7.83 – 7.89 (m, 2H, 5'-
H2-benzoylphenyl, 6'-H2-benzoylphenyl), 9.28 (s, 1H, 5-Htriazole); 13C NMR (DMSO-
d6): δ [ppm] = 52.0 (1C, CO2CH3), 125.1 (1C, C-6'2-benzoylphenyl), 128.6 (2C,
C-3''phenyl, C-5''phenyl), 129.0 (2C, C-2''phenyl, C-6''phenyl), 129.8 (1C, C-5triazole),
129.9 (1C, C-3'2-benzoylphenyl), 130.2 (1C, C-4'2-benzoylphenyl), 132.0 (1C, C-5'2-
benzoylphenyl), 133.6 (1C, C-4''phenyl), 133.7 (1C, C-1'2-benzoylphenyl), 134.0 (1C,
C-2'2-benzoylphenyl), 135.8 (1C, C-1''phenyl), 139.1 (1C, C-4triazole), 160.3 (1C,
CO2CH3), 193.7 (1C, C=O); IR (neat): ꢀꢁ [cm-1] = 3113, 3048, 2963, 1721,
1663, 1597, 1531, 1504, 1453, 1369, 1296, 1269, 1231, 1150, 1034, 930,
775, 752, 710, 671, 637; HRMS (m/z): [M+H]+ calcd for C17H14N3O3,
308.1030; found, 308.1050; HPLC (method 1): tR = 19.0 min, purity 99.8 %.
with Phenix.[55] Crystallographic statistics are provided in the Supporting
Information, Table S1. The smHDAC8/2b structure has been deposited in
the PDB under the PDB code 6TLD.
Docking: To prepare protein structures for docking studies, available
crystal structures were taken: in-house crystal structure of smHDAC8 with
triazole derivative 2b (PDB ID 6TLD) and human HDAC isoform crystal
structures
downloaded
from
the
Protein
Data
Bank
(http://www.rscb.org)[56] (hsHDAC8 PDB ID 2V5X, hsHDAC6 PDB ID
5EDU, hsHDAC1 PDB ID 4BKX). All protein structures were prepared with
Protein Preparation Wizard from Schrödinger Suite[57] in several steps.
First, hydrogen atoms and missing side chains were added. Second,
solvent molecules were removed. Only one water molecule bound to the
conserved catalytic zinc ion coordinating histidine and observed in multiple
HDAC crystal structures was considered. Third, structure was optimized
by automatic assignment of protonation states and tautomers of the amino
acid residues with PROPKA tool at pH 7.0. Finally, optimized structure was
minimized using default settings: 0.3 Å restraint of the atom displacement,
OPLS 2005 force field.[58]
1-(2-Benzoylphenyl)-N-hydroxy-1H-1,2,3-triazole-4-carboxamide (32):
A 5.4 M solution of sodium methoxide in methanol (0.6 mL, 3.2 mmol) was
added to a solution of 31 (200 mg, 0.64 mmol) and hydroxylamine
hydrochloride (178 mg, 2.6 mmol) in dry methanol (10 mL). The mixture
was stirred at ambient temperature overnight. Then the solvent was
removed in vacuo and the residue was purified by automatic flash column
chromatography using a Biotage purification apparatus (5% → 60% ACN
in H2O, Biotage® SNAP KP-C18-HS 30 g). Fractions containing the desired
product were combined, dried from acetonitrile under reduced pressure
and then subjected to lyophilization to give 32 (58 mg, 0.19 mmol, 29%)
as colorless solid. m.p. = 114-116 °C; 1H NMR (DMSO-d6): δ [ppm] = 7.39
Ligand preparation was carried out with MOE software.[59] A random
starting conformation for each ligand was generated from SMILES and
energy minimized with default settings: Amber10:EHT force field.[60-61]
Molecular docking studies were performed with our well established
protocol[3] using Glide docking program from Schrödinger Suite. Grids
were generated with default settings (centroid on ligand), except the grid
size for smHDAC8 was specified as 30 Å instead of automatic size
determination by the ligand size, because the co-crystal triazole 2b is
rather small and default grid size was not enough to accommodate large
ligands 2i-2j. For docking run default standard precision mode was chosen
with flexible ligand sampling and enhanced planarity of conjugated π-
systems. No constraints were used. One water molecule described
previously was toggled. Twenty poses were submitted for post-docking
energy minimization and ten final poses per each ligand were output and
ranked with Glide SP score.
– 7.45 (m, 2H, 3''-Hphenyl, 5''-Hphenyl), 7.55 – 7.61 (m, 3H, 2''-Hphenyl, 4''-Hphenyl
6''-Hphenyl), 7.68 – 7.71 (m, 1H, 3'-H2-benzoylphenyl), 7.72 – 7.77 (m, 1H, 4'-H2-
benzoylphenyl), 7.81 – 7.89 (m, 2H, 5'-H2-benzoylphenyl, 6'-H2-benzoylphenyl), 8.98 (s,
1H, 5-Htriazole); 13C NMR (DMSO-d6): δ [ppm] = 124.6 (1C, C-6'2-benzoylphenyl),
,
126.7 (1C, C-5triazole), 128.6 (2C, C-3''phenyl, C-5''phenyl), 129.0 (2C, C-2''phenyl
,
C-6''phenyl), 129.7 (1C, C-3'2-benzoylphenyl), 129.9 (1C, C-4'2-benzoylphenyl), 131.9
(1C, C-5'2-benzoylphenyl), 133.5 (1C, C-4''phenyl), 133.9 (2C, C-1'2-benzoylphenyl, C-
2'2-benzoylphenyl), 135.8 (1C, C-1''phenyl), 142.0 (1C, C-4triazole), 156.9 (1C,
CONHOH), 193.8 (1C, C=O); IR (neat): ꢀꢁ [cm-1] = 3144, 2978, 2886, 1655,
1597, 1574, 1497, 1450, 1315, 1273, 1180, 1153, 1030, 930, 876, 768,
702, 633; HRMS (m/z): [M+H]+ calcd for C16H13N4O3, 309.0982; found,
309.0992; HPLC (method 2): tR = 14.4 min, purity 99.5 %.
Performance of the docking protocol was evaluated by calculation of
RMSD value between co-crystal ligands binding modes and their docking
poses. Low values were observed despite using non-native protein
conformations in some cases: SAHA (1), 2b and 21 in smHDAC8 were
showing RMSD of 1.4 Å, 0.9 Å and 1.1 Å respectively.
Biological evaluation
X-ray crystal structures: Crystallization, X-ray data collection, structure
determination, model building, and refinement were performed as
described earlier.[28] Briefly, smHDAC8 expression was carried out in
BL21(DE3) cells in 2xLB medium. Cultures were grown and induced at
37 °C with 0.7 mM IPTG in the presence of 100 µM ZnCl2. After overnight
incubation at 37 °C, cells were harvested and resuspended in a lysis buffer
composed of 150 mM NaCl and 50 mM Tris pH 8.0. Lysis was done by
sonication, the lysate was clarified by centrifugation. The supernatant was
loaded onto Talon Superflow Metal Affinity Resin (Clontech) pre-
equilibrated with the lysis buffer. The his-tagged protein was released from
the Talon resin by thrombin protease treatment in a buffer composed of 50
mM KCl and 10 mM Tris pH 8.0 and subsequently loaded onto a 16/60
Superdex 200 gel filtration column (GE Healthcare) pre-equilibrated with a
buffer composed of 50 mM KCl, 10 mM Tris-HCl pH 8.0 and 2 mM DTT.
Peak fractions were concentrated with an Amicon Ultra centrifugal filter
unit. Diffraction-quality crystals of the native smHDAC8 enzyme were
obtained at 207 °C after 3 days by mixing equal volumes of smHDAC8 (2.5
mg/mL) with reservoir solution composed of 21% PEG 3350 (Fluka) and
0.05 M Na+/K+ L-tartrate, and crystallized using the sitting-drop vapor
diffusion technique. After 3 days, grown crystals were soaked for 20 h in
mother liquor supplemented with inhibitor 2b at 10 mM. Crystals used for
X-ray data collection were briefly transferred in reservoir solution
supplemented with 22% glycerol and flash-frozen in liquid nitrogen. The
crystallographic data were processed and scaled using XDS.[53] Phases
for smHDAC8/inhibitor complexes were obtained by molecular
replacement followed by rigid body refinement against smHDAC8 native
structure as a model (4BZ5). The initial models were refined through
several cycles of manual building using Coot[54] and automated refinement
Enzymes and in vitro inhibition assays: Recombinant human HDAC1
and 6 were purchased from BPS biosciences. Recombinant human
HDAC8 was produced as previously described.[5] Recombinant smHDAC8
enzyme was overproduced in E. coli cells and purified by a method
previously described.[5] Inhibition assays of smHDAC8 and human HDACs
were performed as described earlier.[3, 5] Briefly, the commercial Fluor de
Lys drug discovery kit (BML-KI178) was used for testing inhibition of
smHDAC8 and human HDAC8. Test compounds, Fluor de Lys-HDAC8
substrate (50 µM) and enzyme were incubated for 90 min at 37 °C with
subsequent addition of 50 µL Developer II (BML-KI176) and further
incubation for 45 min at 30 °C. Fluorescence was measured in a plate
reader (BMG Polarstar) with excitation at λ = 390 nm and emission at λ =
460 nm. Inhibition tests of human HDAC1 and 6 were conducted using
ZMAL (Cbz-(Ac)Lys-AMC) as substrate and trypsin as a developer. After
incubation of test compounds, ZMAL (10.5 µM) and enzyme for 90 min at
37 °C, 60 µL of trypsin was added and further incubated for 20 min at 37 °C.
Trichostatin A (2 µM) was used in both assays to stop the reaction.
Fluorescence was measured similarly as mentioned above. IC50 values
were determined with OriginPro (version 9.0.0, Northampton,
Massachusetts). Values represent mean ± S.E.M.
Schistosome viability testing: The resazurin-based assay to determine
the effects of novel inhibitors targeting smHDAC8 on the viability of S.
mansoni schistosomula was carried out exactly as previously described.[27,
62]
Briefly, newly transformed schistosomula (NTS) were obtained in vitro
as previously described[63] by mechanical transformation of S. mansoni
cercaria. A suspension of NTS was prepared at a concentration of 100 per
12
This article is protected by copyright. All rights reserved.

10.1002/cmdc.201900583
ChemMedChem
FULL PAPER
Oliveira, W. Sippl, M. Jung, J. Cavarelli, R. J. Pierce, C. Romier, PLOS
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100 µL using Medium 199 (Invitrogen) supplemented with 10% fetal calf
serum (Gibco), penicillin (50 U × mL-1), streptomycin (50 µg × mL-1) and
rifampicin (60 µg × mL-1). Schistosomula were kept in culture for 3 h at
37 °C and 5% CO2 prior to use in screening. Drug stock solutions of 20
mM in DMSO were used. Mid-dilutions were performed in 100% DMSO
and 1 µL added to 100 µL/well of M199 medium in black 96 well plates
(Nunc, UK) with supplemented Medium 199 and 100 µL of the prepared
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added to each well. Finally, after 72 h of exposure, the fluorescence
intensity of the highly red fluorescent resorufin product was measured
using an excitation wavelength of 530 nm and an emission wavelength of
590 nm in an Infinite M200 Pro microplate reader (TECAN). Background
fluorescence of the drug containing medium were determined for each
drug dilution using wells containing only DMSO as control.
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WS, MJ, and CR received funding from the European Union's
Seventh Framework Program for research, technological
development and demonstration under grant agreement no.
602080. WS was supported by the European Regional
Development Fund of the European Commission. Testing on
human subtypes was in part supported by the Deutsche
Forschungsgemeinschaft (DFG, MJ: Ju 295/13-1, WS: Si 846/13-
1). RJP and JL were supported by institutional funds from the
Centre National de la Recherche Scientifique (CNRS), the Institut
National de la Santé et de la Recherche Médicale (INSERM), the
Institut Pasteur de Lille and the Université de Lille. RH was
supported by the German Center for Infection Research (DZIF),
DVK by the Cells-in-Motion Cluster of Excellence (EXC 1003 -
CiM), and SKJ by the NRW International Graduate School of
Chemistry, which is gratefully acknowledged. We thank Paul
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Conflict of interest
[28] M. Marek, T. B. Shaik, T. Heimburg, A. Chakrabarti, J. Lancelot, E.
Ramos-Morales, C. Da Veiga, D. Kalinin, J. Melesina, D. Robaa, K.
Schmidtkunz, T. Suzuki, R. Holl, E. Ennifar, R. J. Pierce, M. Jung, W.
Sippl, C. Romier, Journal of Medicinal Chemistry 2018, 61(22), 10000-
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The authors declare no conflict of interest.
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Sippl, M. Jung, T. Kurz, Archiv der Pharmazie (Weinheim) 2017,
350(8).
Keywords: Schistosoma mansoni • histone deacetylases •
triazole derivatives • crystal structures • molecular docking
studies
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Entry for the Table of Contents
Triazole-based smHDAC8 inhibitors exhibiting an improved selectivity profile over human HDACs were synthesized, evaluated in in
vitro inhibition assays, and tested for their effects on the viability of Schistosoma mansoni larvae. Crystallographic as well as molecular
docking studies revealed key interactions between smHDAC8 and the developed triazole derivatives, thus explaining their unique
selectivity profile.
15
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