Synthesis, structure-activity relationships, cocrystallization and cellular characterization of novel smHDAC8 inhibitors for the treatment of schistosomiasis

Article abstract of DOI:10.1016/j.ejmech.2021.113745

Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people worldwide and for which the control strategy consists of mass treatment with the only available drug, praziquantel. In this study, we chemically optimized our previously reported benzhydroxamate-based inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis provided insights into the inhibition mode of smHDAC8 activity by the highly potent inhibitor 5o. Structure-based optimization of the novel inhibitors was carried out using the available crystal structures as well as docking studies on smHDAC8. The compounds were evaluated in screens for inhibitory activity against schistosome and human HDACs (hHDAC). The in vitro and docking results were used for detailed structure activity relationships. The synthesized compounds were further investigated for their lethality against the schistosome larval stage using a fluorescence-based assay. The most promising inhibitor 5o showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying of adult worm pairs maintained in culture.

Full text of DOI:10.1016/j.ejmech.2021.113745

European Journal of Medicinal Chemistry 225 (2021) 113745
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Synthesis, structure-activity relationships, cocrystallization and
cellular characterization of novel smHDAC8 inhibitors for the
treatment of schistosomiasis
Ehab Ghazy a, Tino Heimburg a, Julien Lancelot b, Patrik Zeyen a, Karin Schmidtkunz c,
Anne Truhn a, Salma Darwish a, Conrad V. Simoben a, Tajith B. Shaik d, Frank Erdmann a,
Matthias Schmidt a, Dina Robaa a, Christophe Romier d, Manfred Jung c, Raymond Pierce b,
,
*
Wolfgang Sippl a
a Institute of Pharmacy, Martin-Luther University of Halle-Wittenberg, 06120, Halle/Saale, Germany
b
ꢀ
University of Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 - UMR 9017- CIIL - Centre d'Infection et d'Immunite de Lille, F-59000, Lille,
France
c Institute of Pharmaceutical Sciences, University of Freiburg, 79104, Freiburg, Germany
d
ꢀ
ꢀ ꢀ
ꢀ
ꢀ
Universite de Strasbourg, CNRS, INSERM, Institut de Genetique et de Biologie Moleculaire et Cellulaire (IGBMC), Departement de Biologie Structurale
ꢀ
Integrative, 67404, Illkirch Cedex, France
a r t i c l e i n f o
a b s t r a c t
Article history:
Schistosomiasis is a major neglected parasitic disease that affects more than 265 million people
worldwide and for which the control strategy consists of mass treatment with the only available drug,
praziquantel. In this study, we chemically optimized our previously reported benzhydroxamate-based
inhibitors of Schistosoma mansoni histone deacetylase 8 (smHDAC8). Crystallographic analysis pro-
vided insights into the inhibition mode of smHDAC8 activity by the highly potent inhibitor 5o. Structure-
based optimization of the novel inhibitors was carried out using the available crystal structures as well as
docking studies on smHDAC8. The compounds were evaluated in screens for inhibitory activity against
schistosome and human HDACs (hHDAC). The in vitro and docking results were used for detailed
structure activity relationships. The synthesized compounds were further investigated for their lethality
against the schistosome larval stage using a fluorescence-based assay. The most promising inhibitor 5o
showed significant dose-dependent killing of the schistosome larvae and markedly impaired egg laying
of adult worm pairs maintained in culture.
Received 17 June 2021
Received in revised form
21 July 2021
Accepted 26 July 2021
Available online 8 August 2021
© 2021 Elsevier Masson SAS. All rights reserved.
1. Introduction
of an effective vaccine, efforts to control Schistosomiasis rely almost
exclusively on preventive/curative chemotherapy through mass
Schistosomiasis is a neglected parasitic disease that affects
about 240 million people worldwide and is prevalent in poor
tropical and subtropical areas. It is estimated that the disease
causes more than 200 thousands deaths each year in sub-Saharan
Africa in addition to severe morbidity (anemia, growth retarda-
tion) and economic losses due to decreased ability of the infected
people to work [1]. The infection is caused by blood flukes from the
genus Schistosoma, mainly S. mansoni, S. haematobium and
S. japonicum. As there is currently no prospect of the development
drug administration of Praziquantel [2]. The WHO reports that
more than 66.5 million individuals received Praziquantel in 2015.
On one hand, this extensive use of Praziquantel has indeed ach-
ieved success given that the number of people requiring treatment
decreased from around 258.8 million in 2014 to 218.8 million in
2015 [1]. On the other hand, this mass treatment using a single drug
raises the concern of the development of drug resistance [3]. In
addition, Praziquantel lacks efficacy against immature juveniles
which can lead to treatment failure [3e6]. Therefore, it is impera-
tive to develop new therapeutic agents and identify novel drug-
gable targets as well as lead compounds to ensure the future of
schistosomiasis therapy.
* Corresponding author.
Research on schistosomiasis has recently identified several
E-mail address: wolfgang.sippl@pharmazie.uni-halle.de (W. Sippl).
https://doi.org/10.1016/j.ejmech.2021.113745
0223-5234/© 2021 Elsevier Masson SAS. All rights reserved.

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
promising chemical scaffolds that could provide novel therapies
[6]. Among these, epigenetic modulators have received great in-
terest. The complex lifecycle of schistosomes, characterized by
several morphologically distinct forms, among which the juvenile
and adult worms are the infective stages in humans, together with
intensive metabolic and reproductive activity, imply a tight control
of gene expression to control these changes [7,8]. Epigenetic
modulators of gene expression acting on histones are key to this
regulation and are therefore potential drug targets. In this regard,
the repurposing of already approved and well-established anti-
cancer drugs targeting histone modifications for antischistosomal
therapy seems very attractive [8e10]. One of the most investigated
posttranslational modification is histone acetylation, where
writers, readers and erasers work in concert to control the acety-
lation state of histone and consequently the activation level of
different genes. Of particular importance are the erasers, histone
deacetylases (HDACs), as they were found to be deregulated in
different human diseases, and inhibitors of human HDACs are
either already approved or in preclinical and clinical trials as anti-
cancer therapeutic agents [11e13]. HDAC inhibitors could be uti-
lized as potential antischistosomal agents, as well as against other
parasitic diseases [14,15].
The most promising compound obtained was XI (TH65, Fig. 1B)
showing nanomolar inhibition against smHDAC8 (IC50
293
35 nM) as well as dose dependent killing of the parasite
M). It also induced separation of the adult male
larvae (EC50 16.1
m
and female worm pairs with a corresponding reduction in egg
laying [34]. The solved crystal structure of smHDAC8 with different
selective HDAC8 inhibitors including TH65 allowed the identifica-
tion of key active site structural and functional determinants
important for potent inhibitors [35].
In the present work we wanted to further explore the SAR of
benzhydroxamates as smHDAC8 inhibitors as well as to develop
more potent compounds against the parasite. Therefore, we started
from the most promising inhibitor, TH65, and used structure-based
design strategies in combination with synthesis and in vitro testing
to develop further inhibitor series (Fig. 1B). We determined the
in vitro activity of the synthesized compounds against the recom-
binant parasitic and human enzymes as well as on parasites
maintained in culture. Finally, we solved the crystal structures of
one highly potent compound in complex with smHDAC8.
2. Results and discussion
Histone deacetylases are a group of enzymes that remove the
acyl group from lysine residues of histones and some non-histone
proteins. In the human, classical HDACs (class I, II and IV) depend
on zinc chelation for their catalytic activity, while sirtuins (class III)
utilize nicotinamide adenine dinucleotide (NADþ) for their action
[16,17]. In schistosomes, several orthologues of the human enzymes
have been characterized belonging to class I (smHDAC 1, 3 and 8),
class II (three HDACs, one related to human HDAC6 and two related
to the human clade composed of HDACs 4, 5, 7 and 9) and class III
(sirtuins 1, 2, 5, 6 and 7) [18,19]. While class I classical HDACs have
received the most interest, several studies also clarified some
physiological roles of schistosomal sirtuins, provided proofs of ev-
idence of the significance of their inhibition, and have recently
reported low micromolar inhibitors [19e21]. It was previously re-
ported that the HDAC inhibitor Trichostatin A (TSA) induces
hyperacetylation of schistosomal proteins and dose dependent
mortality of both schistosomula and adult worms [22] in addition
to blocking the transformation from the miracidia into the sporo-
cyst [23]. Class I smHDACs are expressed in all life cycles of the
parasite, with the smHDAC8 isoform being the most abundant,
whereas its human counterpart (hHDAC8) shows usually a lower
level of expression in human cells compared to HDAC1,2 and 3. This
suggests that this isoform has a specific function for the parasite
and could therefore be the perfect target for novel antischistosomal
therapy [8,24]. Indeed, RNA mediated down-regulation of
smHDAC8 decreased the ability of schistosomula to survive and
mature in infected mice and many selective smHDAC8 inhibitors
(Fig. 1A) were reported showing in vitro inhibition of enzymatic
activity accompanied by phenotypic effects such as impaired
viability of different life stages of the parasite, decreased egg pro-
duction and morphological alterations in the adult worms [25e29].
We have carried out several virtual screens for smHDAC8 in-
hibitors [30,31] and identified various hits that were further
confirmed through in vitro testing. We were able to obtain several
crystal structures for some of the hits that interestingly showed
different conformations of the binding pocket [32]. Based on the
crystal structure of IX (J1075), we recently reported an optimized
series of cinnamoyl hydroxamic acid derivatives (Fig. 1) having
nanomolar activity against smHDAC8 and also showing a dose
dependent mortality on the schistosomula and impaired egg laying
of adult worms [33]. Moreover, we previously utilized the crystal
structure of X (J1036) to guide our structure-based optimization of
novel benzhydroxamic acids as nanomolar smHDAC8 inhibitors.
Our previous studies have shown that the capping group of the
L-shaped m-substituted benzhydroxamates occupy the so-called
HDAC8-specific pocket; a hydrophobic pocket located between
the L6 loop, the L1 loop and Tyr341 of smHDAC8 [34,35]. Since this
pocket is not present in the other HDAC isoforms, it can be effec-
tively explored to develop potent and selective HDAC8 inhibitors.
As aforementioned, our lead compound TH65, which bears a
biphenyl capping group, was found to show high inhibitory activity
against smHDAC8. Structural data revealed that this extended
capping group can be well accommodated in the hydrophobic
HDAC8-specific pocket, displaying more extensive hydrophobic
contacts with Pro291 in addition to the commonly observed pi-pi
stacking interactions with Tyr341 [35]. This indicated that further
exploration of the hydrophobic HDAC8-specific side pocket by
bulky capping group is a promising strategy to achieve novel
smHDAC8 inhibitors with improved inhibitory activity and
enhanced selectivity over human HDAC isoforms (Fig. 2).
We started our campaign by synthesizing structurally related
derivatives of TH65 keeping firstly the biphenyl ring system, either
as such or substituted, as the capping group while changing the
substituents at position-4 of the benzhydroxamate moiety (5a-f).
These modifications resulted in the case of 5b, 5c and 5d in good
inhibition of the recombinant parasitic enzyme. Compounds with
substituted biphenyl ring system (5c-f) showed a decreased
inhibitory activity against the human isoform. Best selectivity for
smHDAC8 was observed for compound 5c with a methoxy sub-
stituent in o-position to the amide linker. The chloro-substituent at
the o-position of the capping group seems to be determinant for the
increased selectivity for smHDAC8 over hHDAC8 (Fig. 3-A). Docking
studies in smHDAC8 reveal that the chloro-substituent in 5c forces
the capping group to slightly tilt toward the L6 loop. Nevertheless,
the biphenyl capping group still undergoes various stabilizing in-
teractions with the residues lining the side pocket including hy-
drophobic interactions between the chloro group and Tyr341, pi-pi
stacking interactions between the phenyl group and His292, as well
as van-der-Waals interactions between the terminal phenyl group
and Pro291 (Fig. 3-A). In the human HDAC8 isoform, the predicted
binding mode reveals that the capping group does not protrude
into the HDAC8-specific pocket but is rather placed on top of the L6
loop. The interaction, which is usually observed between the
capping group of this class of compounds with Tyr306, cannot be
observed, and the capping group only shows van-der-Waals in-
teractions with the gatekeeper residue Met274 (Fig. 3-B). Apart
2

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Fig. 1. A) Chemical structures of some previously reported smHDAC8 inhibitors. B) Design of the smHDAC8 inhibitors of the current study.
from 5a, which showed potent inhibition of hHDAC6, the com-
pounds showed only micromolar inhibition against hHDAC1 and 6
(Table 1).
The next modification of the capping group was achieved by
changing the biphenyl ring system and introduction of an ether
linkage (19a and 19b). The compounds showed a less potent inhi-
bition against smHDAC8. Introduction of further chloro sub-
stituents at the terminal phenyl ring (19c) did not show a
significant impact on the schistosomal enzyme, but decreased the
activity on the human isoform.
We then tried a simplified capping group, namely one phenyl
ring with different substitution patterns. Substitutions by different
alkoxy groups at position-4 of the phenyl capping group (5g-i) led
to a decrease in the activity on smHDAC8, albeit the compounds
still showed submicromolar inhibitory potency. Interestingly, 2,4
dimethoxy substitution (5j) of the phenyl capping group led to a
sharp decrease in the activity. The combination of a more bulky
ethoxy substituent next to the amide linker and a 2,4-dichloro
substitution of the terminal aromatic ring (5k) restored the activ-
ity against both parasitic and hHDAC8, while showing good selec-
tivity against HDACs 1 and 6.
Finally, the introduction of an acetyl moiety in the capping
group led to the highly potent smHDAC8 inhibitor 5l. It was
therefore chosen for further derivatization to obtain more selective
derivatives.
Based on the crystal structure of smHDAC8/TH65, one suggested
modification of the capping group was to replace the biphenyl ring
system with polycyclic rings to address the hydrophobic HDAC8-
specific pocket (Table 2). Therefore, we used bicyclic rings (5m
and 5n) and observed that the nanomolar inhibition of smHDAC8
was retained, accompanied with a potent activity on hHDAC6 and 8.
Molecular docking studies of 5n revealed that the bicyclic capping
group occupies the hydrophobic side pocket of smHDAC8 showing
pi-pi stacking interactions with Tyr341 as well as hydrophobic
3

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Fig. 2. Crystal structure of smHDAC8 in complex with TH65 (PDB ID 6HTH). A) Surface depiction of smHDAC8 binding side. The capping group of TH65 (orange sticks) occupies the
HDAC8-specific pocket formed between L1 (green), L6 (yellow) and Tyr341 (blue). B) Observed binding mode of TH65 (orange sticks) in the catalytic pocket of smHDAC8 (white
cartoon). Side chains of the smHDAC8 residues lining the pocket are shown as white sticks and the zinc ion as a purple sphere. Metal coordination is depicted as cyan-dashed lines,
hydrogen bonds as yellow-dashed lines and cation-pi interactions as green-dashed lines. Water molecules are omitted.
Fig. 3. Docking poses of 5c in smHDAC8 and hHDAC8. A) Predicted binding mode of 5c (yellow sticks) in smHDAC8 (PDB ID 6HTH), B) Obtained docking pose of 5c (purple sticks) in
hsHDAC8 (PDB ID 2V5X). Side chains of the active site residues lining the pocket of smHDAC8 and hsHDAC8 are shown as white sticks. The zinc ion is displayed as a purple sphere.
Metal coordination is depicted as cyan-dashed lines, hydrogen bonds as yellow-dashed lines, cation-pi interactions as green-dashed lines and pi-pi stacking interactions as blue-
dashed lines. Water molecules are omitted.
interactions with Pro291 and His292. (Fig. 4-A). In human HDAC8,
docking studies showed that the compounds can adopt a similar
binding conformation as observed in smHDAC8. The bicyclic
capping group occupies the side pocket showing pi-pi stacking
interactions with Tyr306 as well as hydrophobic interactions with
Pro273 and Met274 (Fig. 4-B).
We next turned to tri-heterocyclic rings (5o) which showed a
comparable potent activity on both parasitic and human HDAC8. In
addition, we aimed to synthesize further analogues bearing
different bioisosteres of the dibenzofuran ring. 5p and 24 exhibited
comparable inhibitory activity to 5o against sm- and hHDAC8.
Similar to 5n, the tricyclic moiety of 5o was found to be embedded
in the hydrophobic side pocket of smHDAC8, displaying aromatic
and hydrophobic interactions with Tyr341, Pro 291 and His292
(Fig. 5-A). A similar binding mode was also observed for the anal-
ogous tricyclic derivatives 5p and 24 in smHDAC8 (Fig. 5-B).
Docking studies in the human HDAC8 isoform also revealed that the
tricyclic capping group can be well accommodated in the hydro-
phobic side pocket, where it is stabilized by interactions with
Tyr306, Pro273, and Met274 (Fig. 5-C). Meanwhile, in HDAC6 the
capping group resides above L1 and L6 loops, showing hydrophobic
interactions with Pro 501 and Leu 749 (Fig. 5-D). As previously
4

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Table 1
Chemical structures and in vitro results for benzhydroxamates.
ID
5a
Structure
IC50
m
M smHDAC8
IC50
m
M hHDAC8
IC50
m
M hHDAC1
IC50 mM hHDAC6
0.40 0.09
0.45 0.08
10.87 1.52
0.16 0.03
5b
5c
0.10 0.01
0.22 0.05
3.88 0.32
1.50 0.09
0.10 0.08
1.10 0.23
16 6% @ 10
m
m
M n.i. @ 1
m
M
46 7% @ 10 mM
15 5% @ 1 mM
5d
5e
0.11 0.01
0.32 0.02
0.78 0.19
48 5% @ 1
18 2% @ 10
M
2.4 0.3
7
2% @ 1 mM
m
M
n.d.
n.d.
5f
0.28 0.04
0.61 0.07
25 2% @ 1
59 3% @ 1
m
m
M
M
n.d.
n.d.
n.d.
n.d.
19a
19b
19c
5g
0.45 0.03
0.52 0.02
51 3% @ 0.5
60 4% @ 1
34 2% @ 1
n.d.
m
m
M
M
n.d.
n.d.
n.d.
n.d.
n.d.
n.d.
m
M
5h
5i
0.73 0.09
0.73 0.05
66 5% @ 1
58 5% @ 1
m
m
M
M
n.d.
n.d.
n.d.
n.d.
5j
2.08 0.27
0.10 0.01
63 4% @ 1
0.23 0.04
m
M
n.d.
n.d.
5k
21.1 2.3
7.74 0.57
5l
0.11 0.02
0.14 0.02
0.36 0.03
0.02 0.002
TH65
0.29 0.04
0.13 0.05
6.34 2.10
0.39 0.002
(continued on next page)
5

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Table 1 (continued)
ID
Structure
IC50
m
M smHDAC8
IC50
mM hHDAC8
IC50
mM hHDAC1
IC50 mM hHDAC6
SAHA
PCI34051
1.56 0.20
0.44 0.06
0.40 0.10
0.01 0.003
0.12 0.01
48 4% @ 100
0.04 0.01
41 4% @ 100 mM
m
M
Table 2
Chemical structures and in vitro results for benzhydroxamates containing bi- and tricyclic capping groups.
ID
Structure
IC50 mM smHDAC8
IC50 mM hHDAC8
IC50
n.d.
mM hHDAC1
IC50
n.d.
mM hHDAC6
5m
0.58 0.07
29 3% @ 1
mM
5n
0.33 0.05
0.18 0.04
n.d.
n.d.
5o
24
0.27 0.02
0.20 0.02
0.32 0.03
0.29 0.03
18.51 1.60
n.d.
0.29 0.07
n.d.
5p
8
0.32 0.03
0.45 0.09
0.15 0.03
0.86 0.13
n.d.
n.d.
n.d.
n.d.
Fig. 4. Binding modes of the bicyclic derivative 5n. A) Predicted binding modes of 5n (green sticks) in smHDAC8 (PDB ID 6HTH, white sticks); B) Predicted binding modes of 5n
(cyan sticks) in hHDAC8 (PDB ID 2V5X). The zinc ion is displayed as a purple sphere. Metal coordination is depicted as cyan-dashed lines, hydrogen bonds as yellow-dashed lines,
cation-pi interactions as green-dashed lines and pi-pi stacking interactions as blue-dashed lines.
observed for other m-substituted derivatives, 5o is not able to
properly chelate the catalytic zinc ion in HDAC1, which explains its
low HDAC1-inhibitory activity (Figure S2- Supplementary). More-
over, based on our previous work, where we reported that
hydroxamates with an amine linker are potent hHDAC8 inhibitors
[36], we synthesized a close analogue of 5o, where we replaced the
amide linker with an amine one to get 8. The compound showed
almost half the activity of its amide analogues against both sm- and
hHDAC8, but still in the nanomolar range.
We previously showed that inhibitors with inverted amide
6

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Fig. 5. Binding modes of the tricyclic derivatives. A) Docking pose of 5o (purple-blue sticks) in smHDAC8 (PDB ID 6HTH, white sticks) overplayed with the experimentally
determined binding mode of 5o (PDB ID 7P3S, orange sticks) in smHDAC8 (salmon sticks); B) Predicted binding modes of the 24 (yellow sticks) and 5p (green sticks) in smHDAC8
(PDB ID 6HTH); C) Predicted binding mode of 5o (purple-blue sticks) in hHDAC8 (PDB ID 2V5X); D) Predicted binding mode of 5o (purple-blue sticks) in hHDAC6 (PDB ID 5EDU).
The zinc ion is displayed as a purple sphere. Metal coordination is depicted as cyan-dashed lines, hydrogen bonds as yellow-dashed lines, and cation-pi interactions as green-dashed
lines.
linker are potent HDAC8 inhibitors [36]. We hypothesized then that
these “inverse amides” could also inhibit the parasitic enzyme. As a
proof of concept, we firstly tested our previously reported hHDAC8
inhibitors (13a-c) on smHDAC8, and the inhibitors indeed showed a
potent inhibition of the parasitic enzyme in the nanomolar range.
As mentioned above, compound 5l bearing the acetyl moiety
showed a potent inhibition against smHDAC8 as well as hHDACs1, 6
and 8. We then aimed to prepare another set of inhibitors (13d-j)
where we inverted the amide linker of 5l to benefit from the HDAC8
inhibitory activity of the inverse amides. In the cases of 13d and 13e
we observed potent inhibition of smHDAC8 and slight selectivity
against hHDAC8 (Table 3). With the need to improve the selectivity
of the compounds, we synthesized further inverse amides lacking
the acetyl group at the capping group (13k-n). Simple mono- and
disubstituted phenyl rings bearing methoxy and chloro sub-
stituents as well as a bicyclic benzothiophene ring were included.
Interestingly these inhibitors retained the nanomolar activity
against the parasitic and human HDAC8 while showing decreased
inhibition of hHDAC1 and 6.
A more detailed study describing all docking results, binding
free energy calculations, as well as the generation of quantitative
structure activity relationship (QSAR) models of the developed
smHDAC8 inhibitors that have been used in combination to the
chemical optimization has been published elsewhere [37].
3. Crystallographic studies
To confirm the docking results of the new inhibitors, the crystal
structure of smHDAC8 in complex with 5o (Fig. 5-A) was solved and
refined at 1.55 Å (Table S3 and Figure S4- Supplementary). The
crystal structure showed that 5o binds in the active site, where it
coordinates with the catalytic zinc ion, and the tricyclic capping
group was found to be embedded in the hydrophobic side pocket of
smHDAC8, displaying aromatic and hydrophobic interactions with
7

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Table 3
Chemical structures and in vitro results for benzhydroxamates with inverse amide linker.
ID
R
IC50 mM smHDAC8
IC50
m
M hHDAC8
IC50
n.d.
m
M hHDAC1
IC50
n.d.
mM hHDAC6
13a
0.31 0.07
0.04 0.002
13b
60 3% @ 1
mM
0.18 0.05
20 1% @ 1
m
M
94 4% @ 1 mM
13c
13d
0.33 0.04
0.16 0.02
0.11 0.01
0.59 0.09
3.37 0.63
0.60 0.04
7.7 1.5
0.12 0.016
13e
0.15 0.02
71 4% @ 1
m
M
M
0.36 0.25
0.02 0.002
13f
0.33 0.03
61 5% @ 1
m
n.d.
n.d.
13g
13h
0.29 0.03
0.09 0.01
n.d.
n.d.
0.14 0.02
0.22 0.04
18.1 6.2
0.11 0.02
13i
13j
0.18 0.03
0.09 0.01
n.d.
n.d.
n.d.
n.d.
0.21 0.02
0.07 0.004
8

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Table 3 (continued)
ID
R
IC50 mM smHDAC8
IC50
m
M hHDAC8
IC50
0
m
M hHDAC1
IC50 mM hHDAC6
13k
0.17 0.01
0.28 0.04
0.27 0.05
31 3% @ 1
1% @ 1
m
M
25 2% @ 1
18 2% @ 1
n.d.
m
M
13l
0.43 0.02
2
1% @ 1
m
M
m
M
13m
0.18 0.0
m
M
n.d.
13n
0.22 0.02
0.10 0.01
n.d.
n.d.
Tyr341, Pro 291 and His292, as predicted by the docking study. The
interaction is similar to our previously solved crystallographic
structures with other compounds [35].
used to synthesize the inverse amide hydroxamates (13a-n) as
illustrated in Scheme 3. In Scheme 4, Williamson ether synthesis
was utilized to build the ether bond with subsequent amide bond
formation and hydrolysis to yield intermediates (18a-c). Although
compound 24 is structurally related to compounds (5a-p), trials to
synthesize it in a similar sequence as in Scheme 1 didn't succeed
due to its high polarity that interfered with the purification of the
intermediates. Therefore a slightly modified synthetic sequence
was adopted e as shown in Scheme 5 e where the THP protected
intermediate 20 was first synthesized, coupled to carboxylic acid
23, and finally deprotected to give the final compound 24.
4. Chemistry
The compounds in the present work were synthesized through
five schemes. Apart from compound 24 (illustrated in Scheme 5),
the synthetic strategy aimed to get the carboxylic acid in-
termediates (4a-b, 7, 12a-n and 18a-c), converted then to the cor-
responding hydroxamates through the well-established method by
amide coupling with O-(Tetrahydro-2H-pyran-2-yl)hydroxylamine
followed by acidic deprotection [38]. Scheme 1 illustrates the
synthesis of compounds (5a-p), where amide coupling followed by
basic hydrolysis afforded the required carboxylic acid in-
termediates (4a-b). While carboxylic acids (4a-o) were smoothly
converted to the corresponding hydroxamates, 4-methoxy-3-(9-
oxo-9H-fluorene-1-carboxamido)benzoic acid 4p gave unex-
pected product N-[5-(hydroxycarbamoyl)-2-methoxyphenyl]-9-
(hydroxyimino)-9H-fluorene-1-carboxamide 5p instead of the
5. Phenotypic screening on schistosomula
We next analyzed the effect of the developed compounds on the
parasites maintained in culture. The compounds were initially
tested for their toxicity toward S. mansoni schistosomula using an
Alamar Blue-based viability assay (see Experimental Methods).
Initial testing was done at a concentration of 10
mM and selected
compounds were also tested at 20 M in order to determine dose-
m
originally
wanted
compound
N-[5-(hydroxycarbamoyl)-2-
dependency. Two biological replicates were carried out in triplicate
and the results are shown in Table 4. In addition to the compounds
developed during this study, the previously developed smHDAC8
inhibitors TH65, the reported HDAC8 selective inhibitor PCI-34051
and Praziquantel, the drug used for treating schistosomiasis, were
also included in the assay. Many of the tested compounds showed
toxic effects towards the parasite comparable to the reference
TH65. Nevertheless, some synthesized inhibitors were even more
potent, such as 19c, 5l, 13h and 13j. However, it was 5o which
provoked the most marked dose-dependent reductions in schis-
tosomula viability, killing almost 98% of the schistosomula. In this
methoxyphenyl]-9-oxo-9H-fluorene-1-carboxamide. This could be
explained e as shown in Fig. 6 e that the THP protected hydrox-
ylamine reacted not only with the activated acid (to produce the
THP protected hydroxamic acid), but also with the carbonyl group
in a condensation reaction to give the corresponding ketoxime.
Deprotection with HCl removed both THP groups yielding com-
pound 5p.
As shown in Scheme 2, reductive amination afforded directly
the key carboxylic acid intermediate 7, while again amide coupling
followed by alkaline hydrolysis yielded carboxylic acids (12a-n)
Scheme 1. Reagents and conditions: (a) i) thionyl chloride, reflux, ii) DIPEA, THF; (b) i) aqu. NaOH, Methanol, reflux, ii) dil. HCl; (c) i) PyBOP, DIPEA, H2NOTHP, THF, ii) dil. HCl, THF.
9

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Fig. 6. Predicted pathway for the synthesis of unexpected compound 5p.
Scheme 2. Reagents and conditions: (a) i) Na(AcO)3BH, acetic acid, THF; (b) i) PyBOP, DIPEA, H2NOTHP, THF, ii) dil. HCl, THF.
Scheme 3. Reagents and conditions: (a) i) oxalyl chloride, cat. DMF, dichloromethane, ii) DIPEA, dichloromethane; (b) i) aqu. NaOH, Methanol, reflux, ii) dil. HCl; (c) i) PyBOP, DIPEA,
H2NOTHP, THF, ii) dil. HCl, THF.
Scheme 4. Reagents and conditions: (a) K2CO3, DMF, RT; (b) i) aqu. NaOH, Methanol, reflux, ii) dil. HCl; (c) i) Thionyl chloride, reflux, ii) DIPEA, THF; (d) i) PyBOP, DIPEA, H2NOTHP,
THF, ii) dil. HCl, THF.
10

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Scheme 5. Reagents and conditions: (a) PyBOP, DIPEA, H2NOTHP, THF; (b) i) conc. HCl, Dioxane, reflux, ii) dil. NaOH; (c) dil. HCl, THF.
Table 4
Toxicity studies on S. mansoni schistosomula (Alamar blue assay) and cytotoxicity studies in HEK293 cells.
% viability S. mansoni 10
Alamar Blue
mM (n ¼ 2 ꢀ 3)
% viability S. mansoni 20
Alamar Blue
m
M (n ¼ 2 ꢀ 3)
% viability HEK293 50
Alamar Blue
m
M (n ¼ 3)
TH65
5a
5b
5c
5d
5e
5f
19a
19b
19c
5g
5h
5i
5j
5k
5l
5m
5n
5o
24
64.8 3.5
62.3 0.9
57.4 6.8
64.0 4.6
60.6 2.9
74.6 6.0
63.4 6.1
61.0 4.7
67.3 2.0
38.4 2.6
64.4 2.0
70.7 9.4
72.6 3.0
65.3 1.6
56.2 2.3
45.2 1.6
69.5 6.6
62.0 0.4
2.2 1.1
98.0 0.3
98.5 3.4
77.3 3.0
77.6 5.0
69.1 2.7
81.0 0.9
78.2 1.7
64.3 4.0
62.6 4.8
57.0 4.5
75.2 5.7
42.1 3.3
85.6 4.0
91.0 9.1
88.2 5.4
82.1 1.0
71.0 1.1
92.1 8.0
33.4 3.5
55.1 1.1
64.9 5.8
65.6 10.1
43.5 2.6
60.5 5.6
56.6 8.3
56.2 8.0
46.5 8.4
22.3 2.1
59.4 0.6
51.6 1.1
56.2 4.8
66.5 6.7
56.4 0.1
34.0 3.9
47.1 8.1
40.0 1.5
n.d.
100.0 0.5
96.0 5.5
59.2 3.2
62.3 1.7
50.2 12.2
67.3 0.6
66.2 1.7
56.7 1.3
32.3 6.3
33.1 4.9
69.5 5.0
37.0 1.6
82.8 4.0
83.3 1.4
83.4 4.0
77.1 9.8
62.3 4.1
88.9 6.4
12.3 0.9a
81.7 2.6
82.3 1.9
94.6 1.6
77.0 1.7
83.5 3.2
82.2 3.0
95.4 1.6
81.4 6.4
66.6 4.4
88.7 0.9
93.2 2.0
93.1 1.8
88.4 1.6
86.8 2.1
68.8 2.8
83.5 1.4
77.0 9.8
73.0 5.7
71.1 1.7
76.6 5.9
27.5 0.8
69.5 3.8
67.6 3.7
81.2 13.5
66.2 3.7
59.9 4.8
89.1 5.5
60.6 2.9
94.8 7.4
87.7 13.6
88.2 4.5
94.5 7.8
58.3 5.5
95.6 11.5
96.8 2.1
99.3 4.1
5p
13a
13b
13c
13d
13e
13f
13g
13h
13i
13j
13k
13l
13m
13n
PCI-34051
Praziquantel
a
IC50 value for TH65 was determined to be 199 mM [34].
11

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
assay PCI-34051 showed only very modest activity against schis-
tosomula and as expected, Praziquantel was inactive at the con-
centrations used. In view of the primary assay data and its
selectivity of inhibition with regard to human HDAC1, 5o was
chosen for further testing. We first showed that the EC50 value for
hHDACs1,6. The developed inhibitors were then screened for their
phenotypic activity against the schistosomula, and we have iden-
tified compound 5o as the most potent one with EC50-value of
3.5
mM. Due to the low solubility of 5o we synthesized a more
soluble bioisostere 24, which retained the inhibitory activity. Un-
fortunately, 24 lacked cellular activity. We could also solve the
crystal structures of 5o in complex with smHDAC8, gaining detailed
insights how this potent inhibitor, esp. the tricyclic capping group
interacts with the parasitic enzyme (Figure S4- Supplementary).
Finally, the developed compounds showed a good safety profile
against human HEK293 cells.
this compound using the Alamar Blue-based assay was 3.5
mM
(Fig. 7) on schistosomula after 72 h in culture. Taking into consid-
eration the potent EC50 value of 5o, we were then interested to test
this compound in vivo in mice infected with S. mansoni. However,
due to its lipophilicity and poor solubility, all our trials to prepare a
formulation to solubilize the amount required for the in vivo testing
were unsuccessful (data not shown). To overcome that, we
designed and synthesized its analogue 24 as a hydrochloride salt.
This derivative was indeed better soluble and showed almost the
same activity on smHDAC8, but unfortunately it showed a negli-
gible toxicity toward the schistosomula. A general observation from
our work on S. mansoni was that the more lipophilic inhibitors were
the most potent in the phenotypic screening, most likely due to
increased permeability into the parasite. A good balance between
lipophilicity and water solubility is therefore critical for these in-
hibitors to be advanced to in vivo testing.
8. Experimental protocols
8.1. Chemistry
8.1.1. General
All materials and reagents were purchased from Sigma-Aldrich
Co. Ltd. and abcr GmbH. All solvents were analytically pure and
dried before use. Thin layer chromatography was carried out on
aluminum sheets coated with silica gel 60 F254 (Merck, Darmstadt,
Germany). For column chromatography under normal pressure
silica gel 60 (0.036e0.200 mm) was used.
6. Cytotoxicity study
Final compounds were confirmed to be of >95% purity based on
HPLC. Purity was measured by UV absorbance at 254 nm. The HPLC
Parasite-specific HDAC inhibitors should have low toxicity to
mammalian cells. Therefore, we tested the potential cytotoxicity of
the synthesized inhibitors in a human epithelial kidney cell line
(HEK293). The cells were incubated for 48 h with the indicated
consists of an XTerra RP18 column (3.5
m
m, 3.9 mm ꢀ 100 mm)
from the manufacturer Waters (Milford, MA, USA) and two LC-
10AD pumps, a SPD-M10A VP PDA detector, and a SIL-HT auto-
sampler, all from the manufacturer Shimadzu (Kyoto, Japan). For
compounds at a concentration of 50
mM, and cell viability was
preparative tasks a XTerra RP18 column (7
m
m, 19 mm ꢀ 150 mm)
determined by Alamar Blue assay. The majority of the tested in-
hibitors showed only relatively low cytotoxicity in the human cell
system used and were all less toxic than TH65 (Table 4).
from the manufacturer Waters (Milford, MA, USA) and two LC-
20AD pumps were used. The mobile phase was in all cases a
gradient of methanol/water (starting at 95% water going to 5%
water).
7. Conclusion
Mass spectrometry analyses were performed with a Finnigan
MAT710C (Thermo Separation Products, San Jose, CA, USA) for the
ESIMS spectra and with a LTQ (linear ion trap) Orbitrap XL hybrid
mass spectrometer (Thermo Fisher Scientific, Bremen, Germany)
for the HRMS-ESI (high resolution mass spectrometry) spectra. For
the HRMS analyses the signal for the isotopes with the highest
prevalence was given and calculated (35Cl, 79Br).
In summary, we have designed, synthesized and tested in vitro
several new benzhydroxamates as smHDAC8 inhibitors to further
explore their SAR. In addition we performed docking and crystal-
lization studies to increase our understanding of their interaction
with the target enzyme as well as other human orthologues. Many
of the compounds developed in this work showed potent
smHDAC8, mainly accompanied with comparable hHDAC8, and -
apart from some exceptions - they showed decreased inhibition of
1H NMR and 13C NMR spectra were taken on a Varian Inova 500
using deuterated chloroform and deuterated DMSO as solvent.
Chemical shifts are referenced to the residual solvent signals. The
following abbreviations and formulas for solvents and reagents
were used: dimethylformamide (DMF), dimethylsulfoxide (DMSO),
tetrahydrofuran (THF), N,N-diisopropylethylamine (DIPEA) and
hydrochloric acid (HCl)
8.1.2. General synthetic methods
8.1.2.1. Method A, synthesis of amide bond using oxalyl chloride.
The appropriate carboxylic acid (1 eq.), DMF (one drop) and oxalyl
chloride (1.3 eq.) were stirred in dichloromethane at room tem-
perature for 3 h. The mixture was then added dropwise to a solu-
tion of the appropriate amine (0.9 eq.) and DIPEA (3 eq.) in
dichloromethane, and stirring was continued for another 2 h at
room temperature. The reaction mixture was washed with a satu-
rated aqueous solution of ammonium chloride and brine. The
organic layer was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified using column
chromatography (chloroform/methanol as eluents). The yields
were around 60e80%.
8.1.2.2. Method B, synthesis of amide bond using thionyl chloride.
The appropriate carboxylic acid (1 eq.) was cooled to 0 ꢁC and then
Fig. 7. The activity on schistosomula shown as EC50-value (3.5 mM) of compound 5o
using the Alamar Blue-based viability-assay.
12

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
thionyl chloride (2 eq.) was added dropwise. The mixture was then
heated under reflux for 2 h, and the excess of thionyl chloride was
evaporated under vacuum. The acid chloride was dissolved in dry
THF and added dropwise to a solution of the appropriate amine (0.9
eq.) and DIPEA (3 eq.) in THF. After completion, the reaction
mixture was diluted with ethyl acetate and washed with a satu-
rated aqueous solution of ammonium chloride and brine. The
organic layer was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified using column
chromatography (chloroform/methanol as eluents). The yields
were around 50e70%.
vacuum and the product was purified by column chromatography
(chloroform/methanol/triethylamine).
The obtained product was dissolved in THF and a catalytic
amount of conc. HCl was added and the reaction mixture was
stirred at room temperature and controlled by TLC. After that the
solvent was evaporated under vacuum and the hydroxamic acid
product was purified by column chromatography (chloroform/
methanol/formic acid). The yields were around 30e50%.
Other specific synthetic methods for some intermediates are
provided in (S1- supplementary).
8.1.3. Characterization data of the final compounds
8.1.2.3. Method C, synthesis of amide bond using PyBOP. To a
mixture of the appropriate carboxylic acid (1 eq.) and PyBOP (1.2
eq.) in DMF was added a solution of the appropriate amine (0.9 eq.)
and DIPEA (3 eq.) in DMF. The reaction mixture was stirred at room
temperature for 4e5 h, then diluted with ethyl acetate and washed
with a an aqueous solution of sodium hydroxide and brine. The
organic layer was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo. The residue was purified using column
chromatography (chloroform/methanol as eluents). The yields
were around 60e70%.
5a;
N-[5-(hydroxycarbamoyl)-2-chlorophenyl]-biphenyl-4-
carboxamide.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between biphenyl-4-carboxylic acid 1a
and methyl 3-amino-4-chlorobenzoate 2a using method B. The
resulting ester was hydrolyzed using method F to afford N-[5-
(hydroxycarbonyl)-2-chlorophenyl]-biphenyl-4-carboxamide 4a.
This acid was converted to the final compound using method G.
MS m/z: 365.10 [M ꢂ H]-
8.1.2.4. Method D, reductive amination. A mixture of the appro-
priate aldehyde (1 eq.) and amine (0.9 eq.) was dissolved in toluene,
and the reaction mixture was refluxed for 2 h using a dean stark
apparatus. The solvent was then evaporated under reduced pres-
sure, and the crude product was dissolved in THF and cooled to 0 ꢁC.
Sodium triacetoxyborohydride (3 eq.) was then added and the re-
action mixture was stirred overnight at room temperature. The
reaction was quenched by adding water and extracted with ethyl
acetate. The organic layer was dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuo. The residue was purified using
column chromatography (chloroform/methanol as eluents). The
yields were around 60e70%.
1H NMR (400 MHz, DMSO‑d6)
d 11.33 (s,1H),10.21 (s,1H), 9.12 (s,
1H), 8.11e8.06 (m, 2H), 8.01 (s, 1H), 7.87e7.82 (m, 2H), 7.78e7.73
(m, 2H), 7.68e7.62 (m, 2H), 7.54e7.47 (m, 2H), 7.45e7.39 (m, 1H).
13C NMR (101 MHz, DMSO‑d6)
d 165.56, 143.95, 139.49, 135.68,
132.95, 132.44, 130.08, 129.52, 128.86, 128.66, 127.39, 127.20, 126.01.
HRMS m/z: 365.0699 [M ꢂ H]-; calculated C20H14N2O3Clꢂ
365.0688.
HPLC: rt 12.01 min (98.51%)
5b; N-[5-(hydroxycarbamoyl)-2-methylthiophenyl]-biphenyl-
4-carboxamide.
8.1.2.5. Method E, synthesis of ether bond (Williamson ether syn-
thesis). To a suspension of the appropriate phenol (1 eq.) and po-
tassium carbonate (3 eq.) in DMF was added the appropriate alkyl
bromide (1.5 eq.), and the mixture was stirred at room temperature
overnight. The reaction was quenched by adding water and
extracted with ethyl acetate. The organic layer was dried over
anhydrous sodium sulfate, filtered, and concentrated in vacuo. The
residue was purified using column chromatography (n-hexane/
ethyl acetate as eluents). The yields were around 70e80%.
8.1.2.6. Method F, ester hydrolysis. The appropriate methyl ester (1
eq.) was dissolved in methanol followed by the addition of aqueous
solution of sodium hydroxide (10 eq.) and the mixture was stirred
under reflux for 2e4 h until complete hydrolysis of the ester.
Methanol was then evaporated and the reaction was neutralized
with aqueous solution of 1 M HCl until pH 6. The liberated free acid
was extracted with a mixture of ethyl acetate and THF, and the
organic layer was dried over anhydrous sodium sulfate, filtered, and
concentrated in vacuo to yield the product, which required no
further purification. Crude yields were around 80e90%.
We have previously described the synthesis and characteriza-
tion data of this compound [39].
5c;
3-(2-Chlor-4-phenylbenzamido)-N-hydroxy-4-
methoxybenzamide.
8.1.2.7. Method G, synthesis of hydroxamic acids. The appropriate
carboxylic acid (1 eq.) was dissolved in dry THF, followed by the
addition of PyBOP (1.2 eq.) and DIPEA (3 eq.). The mixture was
stirred for 15 min, then NH2OTHP (1.2 eq.) was added and the re-
action mixture was stirred at room temperature for 2e4 h. The
solvent was evaporated under vacuum and the mixture was dis-
solved in ethyl acetate (50 ml) and washed with 1 M sodium car-
bonate solution and brine. The organic layer was evaporated under
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 2-chloro-4-phenylbenzoic
acid 1b and methyl 3-amino-4-methoxybenzoate 2c using
method B. The resulting ester was hydrolyzed using method F to
afford 3-(2-chlor-4-phenylbenzamido)-4-methoxybenzoic acid 4c.
This acid was converted to the final compound using method G.
13

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
MS m/z: 395.22 [M ꢂ H]-
through the amide coupling between 4-methoxybenzoic acid 1d
and methyl 3-amino-4-ethoxybenzoate 2d using method B. The
resulting ester was hydrolyzed using method F to afford 3-(4-
methoxybenzamido)-4-ethoxybenzoic acid 4g. This acid was con-
verted to the final compound using method G.
1H NMR (400 MHz, DMSO‑d6)
d 11.10 (s, 1H), 9.75 (s, 1H), 8.90 (s,
1H), 8.36 (s, 1H), 7.82 (s, 1H), 7.76e7.70 (m, 3H), 7.65 (d, J ¼ 8.0 Hz,
1H), 7.58 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.53e7.47 (m, 2H), 7.42 (t,
J ¼ 7.2 Hz, 1H), 7.12 (d, J ¼ 8.5 Hz, 1H), 3.86 (s, 3H).
HRMS m/z: 397.0950 [MþH]þ; calculated C21H18ClN2Oþ4
397.0950.
MS m/z: 353.10 [MþNa]þ
1H NMR (400 MHz, DMSO‑d6)
d 11.11 (s, 1H), 9.29 (s, 1H), 8.97 (s,
HPLC: rt 11.74 min (99.99%)
1H), 8.18 (d, J ¼ 2.2 Hz, 1H), 7.94e7.86 (m, 2H), 7.54 (dd, J ¼ 8.6,
2.2 Hz, 1H), 7.08 (d, J ¼ 8.7 Hz, 1H), 7.06e7.00 (m, 2H), 4.11 (q,
J ¼ 6.9 Hz, 2H), 3.80 (s, 3H), 1.38e1.27 (m, 3H).
5d;
3-(2-Chlor-4-phenylbenzamido)-
4-chloro-N-hydrox-
ybenzamide.
13C NMR (101 MHz, DMSO‑d6)
d 164.65, 162.43, 153.24, 129.75,
127.39, 126.97, 125.08, 124.67, 123.64, 114.26, 112.05, 64.64, 55.88,
14.94.
HRMS m/z: 331.1285 [MþH]þ; calculated C17H19N2Oþ5 331.1289.
HPLC: rt 11.05 min (99.65%)
5h;
mide.
N-hydroxy-4-methoxy-3-(4-propoxybenzamido)benza-
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 2-chloro-4-phenylbenzoic
acid 1b and methyl 3-amino-4-chlorobenzoate 2a using method
B. The resulting ester was hydrolyzed using method F to afford 3-(2-
chlor-4-phenylbenzamido)-4-chlorobenzoic acid 4d. This acid was
converted to the final compound using method G.
MS m/z: 399.15 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.36 (s,1H),10.35 (s,1H), 9.11 (s,
1H), 8.09 (s, 1H), 7.85 (s, 1H), 7.81e7.67 (m, 4H), 7.63 (s, 2H),
7.53e7.47 (m, 2H), 7.43 (t, J ¼ 7.3 Hz, 1H).
HRMS m/z: 401.0461 [MþH]þ; calculated C20H15Cl2N2O3þ
401.0454.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 4-propoxybenzoic acid 1e
and methyl 3-amino-4-methoxybenzoate 2c using method B. The
resulting ester was hydrolyzed using method F to afford 4-
methoxy-3-(4-propoxybenzamido)benzoic acid 4h. This acid was
converted to the final compound using method G.
HPLC: rt 11.51 min (99.99%)
Yield: 65 mg; 0.16 mmol; 28%
5e;
3-[2-Chlor-4-(4-fluorphenyl)benzamido]-N-hydroxy-4-
MS m/z: 343.29 [M ꢂ H]-
methoxybenzamide.
1H NMR (400 MHz, DMSO‑d6)
d 11.07 (s, 1H), 9.34 (s, 1H), 8.88 (s,
1H), 8.16 (d, J ¼ 2.1 Hz, 1H), 7.92 (d, J ¼ 8.8 Hz, 2H), 7.58 (dd, J ¼ 8.6,
2.2 Hz,1H), 7.12 (d, J ¼ 8.7 Hz,1H), 7.03 (d, J ¼ 8.9 Hz, 2H), 4.05e3.95
(m, 2H), 3.86 (s, 3H), 1.82e1.67 (m, 2H), 0.98 (t, J ¼ 7.4 Hz, 3H).
HRMS m/z: 345.1446 [MþH]þ; calculated C18H21N2Oþ5 345.1445.
HPLC: rt 10.57 min (99.99%)
5i;
N-hydroxy-3-(4-isopropoxybenzamido)-4-
methoxybenzamide.
We have previously described the synthesis and characteriza-
tion data of this compound [39].
5f;
3-[2-Chlor-4-(4-fluorphenyl-)benzamido]-4-chlor-N-
hydroxybenzamide.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 4-isopropoxybenzoic acid 1f
and methyl 3-amino-4-methoxybenzoate 2c using method B. The
resulting ester was hydrolyzed using method F to afford 3-(4-
isopropoxybenzamido)-4-methoxybenzoic acid 4i. This acid was
converted to the final compound using method G.
We have previously described the synthesis and characteriza-
tion data of this compound [39].
5g; 4-Ethoxy-3-(4-methoxybenzamido)-N-hydroxybenzamide.
MS m/z: 343.25 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.07 (s, 1H), 9.32 (s, 1H), 8.88 (s,
1H), 8.17 (d, J ¼ 2.2 Hz, 1H), 7.94e7.87 (m, 2H), 7.58 (dd, J ¼ 8.6,
2.2 Hz,1H), 7.11 (d, J ¼ 8.7 Hz,1H), 7.04e6.97 (m, 2H), 4.79e4.64 (m,
1H), 3.86 (s, 3H), 1.28 (d, J ¼ 6.0 Hz, 6H).
HRMS m/z: 345.1445 [MþH]þ; calculated C18H21N2Oþ5 345.1445.
HPLC: rt 10.12 min (99.99%)
5j;
3-(2,4-Dimethoxybenzamido)-N-hydroxy-4-
This compound was synthesized e as shown in Scheme 1 e
methoxybenzamide.
14

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 1H-benzimidazole-2-
carboxylic acid 1j and methyl 3-amino-4-methoxybenzoate 2c
using method B. The resulting ester was hydrolyzed using method F
to afford N-[5-(hydroxycarbonyl)-2-methoxyphenyl]-1H-benzo[d]
imidazole-2-carboxamide 4m. This acid was converted to the final
compound using method G.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 2,4-dimethoxybenzoic acid
1g and methyl 3-amino-4-methoxybenzoate 2c using method B.
The resulting ester was hydrolyzed using method F to afford 3-(2,4-
dimethoxybenzamido)-4-methoxybenzamide 4j. This acid was
converted to the final compound using method G.
MS m/z: 369.1.31 [MþNa]þ
MS m/z: 325.39 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.04 (s, 1H), 10.53 (s, 1H),
1H NMR (500 MHz, DMSO‑d6)
d 13.55 (s,1H),11.13 (s,1H), 9.91 (s,
8.90e8.84 (m, 2H), 8.04 (d, J ¼ 8.7 Hz, 1H), 7.44 (dd, J ¼ 8.5, 2.1 Hz,
1H), 7.11 (d, J ¼ 8.6 Hz, 1H), 6.78e6.70 (m, 2H), 4.07 (s, 3H), 3.99 (s,
3H), 3.85 (s, 3H).
1H), 8.92 (s, 1H), 8.72 (d, J ¼ 1.7 Hz, 1H), 7.80 (d, J ¼ 7.3 Hz, 1H),
7.60e7.52 (m, 2H), 7.39e7.27 (m, 2H), 7.19 (d, J ¼ 8.1 Hz, 1H), 3.99 (s,
3H).
13C NMR (101 MHz, DMSO‑d6)
d 164.96, 164.15, 162.44, 159.10,
HRMS m/z: 327.1117 [MþH]þ; calculated C16H15N4Oþ4 327.1093.
150.45, 133.48, 128.36, 125.86, 122.52, 118.77, 114.09, 110.45, 107.11,
99.23, 57.00, 56.93, 56.10.
HPLC: rt 10.29 min (98.85%)
5n; N-(5-(hydroxycarbamoyl)-2-methoxyphenyl)quinoline-8-
carboxamide.
HRMS m/z: 347.1237 [MþH]þ; calculated C17H19N2Oþ6 347.1238.
HPLC: rt 10.85 min (92.03%)
5k; 3-(2,4-Dichlorbenzamido)-4-ethoxy-N-hydroxybenzamide.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 8-quinolinecarboxylic acid 1k
and methyl 3-amino-4-methoxybenzoate 2c using method B. The
resulting ester was hydrolyzed using method F to afford N-(5-
(hydroxycarbonyl)-2-methoxyphenyl)quinoline-8-carboxamide
4n. This acid was converted to the final compound using method G.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between 2,4-dichlorobenzoic acid 1h
and methyl 3-amino-4-ethoxybenzoate 2d using method B. The
resulting ester was hydrolyzed using method F to afford 3-(2,4-
dichlorobenzamido)-4-ethoxybenzoic acid 4k. This acid was con-
verted to the final compound using method G.
MS m/z: 360.10 [MþNa]þ1H NMR (400 MHz, DMSO‑d6)
d 11.07
(s, 1H), 9.18 (dd, J ¼ 4.2, 1.8 Hz, 1H), 9.03 (d, J ¼ 2.1 Hz, 1H), 8.88 (s,
1H), 8.78 (dd, J ¼ 7.4, 1.5 Hz, 1H), 8.65 (dd, J ¼ 8.4, 1.8 Hz, 1H), 8.29
(dd, J ¼ 8.1, 1.5 Hz, 1H), 7.86e7.82 (m, 1H), 7.76 (dd, J ¼ 8.3, 4.3 Hz,
1H), 7.49 (dd, J ¼ 8.5, 2.2 Hz, 1H), 7.16 (d, J ¼ 8.6 Hz, 1H), 4.08 (s, 3H).
HRMS m/z: 338.1130 [MþH]þ; calculated C18H16N3Oþ4 338.1140.
HPLC: rt 10.32 min (98.50%)
MS m/z: 367.67 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.10 (s, 1H), 9.74 (s, 1H), 8.93 (s,
1H), 8.25 (d, J ¼ 1.3 Hz, 1H), 7.71 (d, J ¼ 1.4 Hz, 1H), 7.63e7.49 (m,
3H), 7.09 (d, J ¼ 8.6 Hz, 1H), 4.11 (q, J ¼ 6.9 Hz, 2H), 1.32 (t, J ¼ 6.9 Hz,
5o;
N-[5-(hydroxycarbamoyl)-2-methoxyphenyl]-dibenzo-
3H).
furan-4-carboxamide.
HRMS m/z: 391.0217 [MþNa]þ; calculated C16H14N2O4Cl2Naþ
391.0223.
HPLC: rt 12.94 min (98.85%)
5l;
3-(4-Acetyl-2-propoxy-benzamido)-N-hydroxy-4-
methylbenzamide.
This compound was synthesized e as shown in Scheme 1 e
through the amide coupling between dibenzofuran-4-carboxylic
acid 1l and methyl 3-amino-4-methoxybenzoate 2c using method
B. The resulting ester was hydrolyzed using method F to afford N-
[5-(hydroxycarbonyl)-2-methoxyphenyl]-dibenzofuran-4-
carboxamide 4o. This acid was converted to the final compound
using method G.
MS m/z: 399.10 [MþNa]þ
We have previously described the synthesis and characteriza-
tion data of this compound [39].
5m; N-(5-(hydroxycarbamoyl)-2-methoxyphenyl)-1H-benzo[d]
imidazole-2-carboxamide.
1H NMR (400 MHz, DMSO‑d6)
d 11.12 (s, 1H), 10.20 (s, 1H),
9.07e8.74 (m, 2H), 8.40 (d, J ¼ 7.6 Hz, 1H), 8.24 (d, J ¼ 7.8 Hz, 1H),
8.14 (d, J ¼ 7.5 Hz, 1H), 7.83 (d, J ¼ 8.1 Hz, 1H), 7.64e7.44 (m, 4H),
7.21 (d, J ¼ 8.5 Hz, 1H), 4.10 (s, 3H).
15

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
HRMS m/z: 377.1132 [MþH]þ; calculated C21H17N2Oþ5 377.1137.
HPLC: rt 9.90 min (96.10%)
5p; N-[5-(hydroxycarbamoyl)-2-methoxyphenyl]-9-(hydrox-
yimino)-9H-fluorene-1-carboxamide.
We have previously described the synthesis and characteriza-
tion data of this compound [36].
13b;
4-Chloro-3-(4-chlorophenylcarbamoyl)-N-hydrox-
ybenzamide.
This compound was obtained as unexpected product instead of
the originally planned compound N-[5-(hydroxycarbamoyl)-2-
methoxyphenyl]-9-oxo-9H-fluorene-1-carboxamide.
The synthesis started e as shown in Scheme 1 e through the
amide coupling between 9-fluorenone-1-carboxylic acid 1m and
methyl 3-amino-4-methoxybenzoate 2c using method B. The
resulting ester was hydrolyzed using method F to afford 4-
methoxy-3-(9-oxo-9H-fluorene-1-carboxamido)benzoic acid 4p.
When method G was applied to this acid, it yielded the titled
compound.
We have previously described the synthesis and characteriza-
tion data of this compound [36].
13c;
3-(4-Chlorophenylcarbamoyl)-N-hydroxy-4-
MS m/z: 402.10 [M ꢂ H]-
methylbenzamide.
1H NMR (400 MHz, DMSO‑d6)
d 12.65 (s, 1H), 11.05 (s, 1H), 9.81
(s, 1H), 8.90 (s, 1H), 8.64 (s, 1H), 8.36 (d, J ¼ 7.6 Hz, 1H), 8.04e7.87
(m, 2H), 7.55e7.42 (m, 4H), 7.40 (t, J ¼ 7.5 Hz, 1H), 7.08 (d, J ¼ 8.6 Hz,
1H), 3.82 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 166.73, 164.84, 152.43, 151.89,
140.49, 139.86, 133.27, 131.31, 131.23, 130.04, 129.81, 129.21, 128.07,
127.83, 125.30, 123.67, 121.86, 120.74, 110.98, 56.48.
HRMS m/z: 426.1053 [MþNa]þ; calculated C22H17N3O5Naþ:
426.1060.
We have previously described the synthesis and characteriza-
tion data of this compound [36].
HPLC: rt 11.77 min (99.6%)
13d;
3-(4-Acetyl-2-ethoxy-phenylcarbamoyl)-N-hydrox-
8;
3-[N-(dibenzofuran-4-yl-methyl)amino]-N-hydroxy-4-
ybenzamide.
methoxybenzamide.
This compound was synthesized e as shown in Scheme 2 e
through
reductive
amination
between
dibenzofuran-4-
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 3-(methoxycarbonyl)benzoic
acid 10a and 1-(3-amino-4-ethoxyphenyl)ethanone 9b using
method A. The resulting ester was hydrolyzed using method F to
afford 3-[(5-acetyl-2-ethoxyphenyl)carbamoyl]benzoic acid 12d.
This acid was converted to the final compound using method G.
MS m/z: 341.35 [M ꢂ H]-
carbaldehyde 6 and 3-amino-4-methoxybenzoic acid 2c-CA using
method D. The resulting 3-[N-(dibenzofuran-4-yl-methyl)amino]-
4-methoxybenzoic acid 7 was converted to the final compound
using method G.
MS m/z: 361.27 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 10.84 (s, 1H), 8.67 (s, 1H), 8.13 (d,
J ¼ 7.5 Hz,1H), 8.00 (d, J ¼ 7.2 Hz, 1H), 7.75 (d, J ¼ 8.0 Hz,1H), 7.53 (t,
J ¼ 7.7 Hz, 1H), 7.47e7.25 (m, 3H), 7.01 (dd, J ¼ 8.2, 1.8 Hz, 1H), 6.96
(d, J ¼ 1.9 Hz,1H), 6.85 (d, J ¼ 8.3 Hz,1H), 5.71 (t, J ¼ 6.2 Hz,1H), 4.74
(d, J ¼ 6.0 Hz, 2H), 3.85 (s, 3H).
1H NMR (400 MHz, DMSO‑d6)
d 11.32 (s, 1H), 9.64 (s, 1H), 9.12 (s,
1H), 8.36e8.30 (m, 2H), 8.09e8.03 (m, 1H), 7.95e7.90 (m, 1H), 7.83
(dd, J ¼ 8.6, 2.2 Hz,1H), 7.61 (t, J ¼ 7.8 Hz,1H), 7.19 (d, J ¼ 8.7 Hz,1H),
4.20 (q, J ¼ 7.0 Hz, 2H), 2.52 (s, 3H), 1.36 (t, J ¼ 7.0 Hz, 3H).
13C NMR (101 MHz, DMSO‑d6)
d
155.89, 153.79, 149.21, 137.79,
13C NMR (101 MHz, DMSO‑d6)
d 196.63, 165.18, 164.04, 155.27,
127.98, 126.15, 125.79, 124.13, 123.82, 123.52, 121.65, 119.96, 115.82,
112.23, 109.53, 56.08.
135.09, 133.59, 130.38, 129.80, 129.18, 127.70, 127.23, 126.78, 124.72,
112.25, 64.85, 26.84, 14.86.
HRMS m/z: 363.1332 [MþH]þ; calculated C21H19N2Oþ4 363.1344.
HRMS m/z: 343.1315 [MþH]þ; calculated C18H19N2Oþ5 : 343.1293.
HPLC: rt 13.31 min (98.28%)
HPLC: rt 7.87 min (98.89%)
13a;
3-(4-Chlorophenylcarbamoyl)-N-hydroxy-4-
13e; 3-(4-Acetyl-2-methoxy-phenylcarbamoyl)-N-hydroxy-4-
methoxybenzamide.
methoxybenzamide.
16

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
(methoxycarbonyl)benzoic
methoxyphenyl)ethanone 9c using method A. The resulting ester
was hydrolyzed using method to afford 3-[(5-acetyl-2-
acid
10c
and
1-(3-amino-4-
F
methoxyphenyl)carbamoyl]-4-chlorobenzoic acid 12g. This acid
was converted to the final compound using method G.
MS m/z: 361.49 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.37 (s, 1H), 9.94 (s, 1H), 9.15 (s,
1H), 8.51 (d, J ¼ 1.8 Hz, 1H), 7.93 (d, J ¼ 1.7 Hz, 1H), 7.88e7.78 (m,
2H), 7.63 (d, J ¼ 8.4 Hz, 1H), 7.19 (d, J ¼ 8.7 Hz, 1H), 3.90 (s, 3H), 2.53
(s, 3H).
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 2-methoxy-5-(methox-
ycarbonyl)benzoic acid 10b and 1-(3-amino-4-methoxyphenyl)
ethanone 9c using method A. The resulting ester was hydrolyzed
using method F to afford 3-[(5-acetyl-2-methoxyphenyl)carba-
moyl]-4-methoxybenzoic acid 12e. This acid was converted to the
final compound using method G.
13C NMR (101 MHz, DMSO‑d6)
d 196.68, 165.15, 162.89, 155.14,
136.90, 133.32, 131.79, 130.28, 129.83, 127.79, 127.61e127.37 (m),
127.02, 123.29, 111.50, 56.64, 26.86.
HRMS m/z: 363.0734 [MþH]þ; calculated C17H16ClN2Oþ5 :
363.0747.
HPLC: rt 8.33 min (99.55%)
13h;
3-(4-Acetyl-2-ethoxy-phenylcarbamoyl)-4-chloro-N-
MS m/z: 357.52 [M ꢂ H]-
hydroxybenzamide.
1H NMR (400 MHz, DMSO‑d6)
d 11.27 (s, 1H), 10.59 (s, 1H),
9.18e8.88 (m, 2H), 8.59e8.47 (m, 1H), 7.96 (d, J ¼ 8.7 Hz, 1H), 7.77
(d, J ¼ 8.6 Hz,1H), 7.35 (d, J ¼ 8.6 Hz,1H), 7.20 (d, J ¼ 8.4 Hz,1H), 4.12
(s, 3H), 4.04 (s, 3H), 2.53 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 196.78, 163.67, 162.23, 159.50,
152.34, 132.55, 131.05, 130.29, 128.09, 126.11, 125.87, 121.14, 119.33,
113.06, 110.69, 57.38, 57.12, 26.82.
HRMS m/z: 359.1231 [MþH]þ; calculated C18H19N2Oþ6 : 359.1243.
HPLC: rt 10.74 min (99.72%)
13f;
3-(4-Acetyl-2-ethoxy-phenylcarbamoyl)-N-hydroxy-4-
methoxybenzamide.
We have previously described the synthesis and characteriza-
tion data of this compound [39].
13i; 3-(4-Acetyl-2-methoxy-phenylcarbamoyl)-N-hydroxy-4-
methylbenzamide.
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 2-methoxy-5-(methox-
ycarbonyl)benzoic acid 10b and 1-(3-amino-4-ethoxyphenyl)
ethanone 9b using method A. The resulting ester was hydrolyzed
using method F to afford 3-[(5-acetyl-2-ethoxyphenyl)carbamoyl]-
4-methoxybenzoic acid 12f. This acid was converted to the final
compound using method G.
MS m/z: 371.44 [M ꢂ H]-
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 5-(methoxycarbonyl)-2-
methylbenzoic acid 10d and 1-(3-amino-4-methoxyphenyl)etha-
none 9c using method A. The resulting ester was hydrolyzed using
method F to afford 3-[(5-acetyl-2-methoxyphenyl)carbamoyl]-4-
methylbenzoic acid 12i. This acid was converted to the final com-
pound using method G.
1H NMR (400 MHz, DMSO‑d6)
d 11.27 (s, 1H), 10.45 (s, 1H), 9.10
(d, J ¼ 2.1 Hz, 1H), 8.99 (s, 1H), 8.54 (d, J ¼ 2.3 Hz, 1H), 7.97 (dd,
J ¼ 8.7, 2.4 Hz, 1H), 7.75 (dd, J ¼ 8.6, 2.2 Hz, 1H), 7.36 (d, J ¼ 8.8 Hz,
1H), 7.19 (d, J ¼ 8.6 Hz, 1H), 4.27 (q, J ¼ 7.0 Hz, 2H), 4.11 (s, 3H), 2.53
(s, 3H), 1.49 (t, J ¼ 6.9 Hz, 3H).
HRMS m/z: 373.1384 [MþH]þ; calculated C19H21N2Oþ6 : 373.1399.
HPLC: rt 10.57 min (99.97%)
MS m/z: 341.57 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.22 (s, 1H), 9.59 (s, 1H), 9.03 (s,
13g;
3-(4-Acetyl-2-methoxy-phenylcarbamoyl)-4-chloro-N-
hydroxybenzamide.
1H), 8.41 (s, 1H), 7.90e7.80 (m, 2H), 7.75 (dd, J ¼ 7.9,1.5 Hz,1H), 7.36
(d, J ¼ 8.0 Hz, 1H), 7.19 (d, J ¼ 8.6 Hz, 1H), 3.90 (s, 3H), 2.53 (s, 3H),
2.43 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 196.70, 167.84, 163.94, 155.66,
139.35, 137.09, 131.10, 130.52, 129.93, 128.41, 127.62, 127.20, 126.34,
124.07, 111.49, 56.63, 26.87, 19.83.
HRMS m/z: 343.1281 [MþH]þ; calculated C18H19N2Oþ5 :
343.1293.
HPLC: rt 9.18 min (96.22%)
This compound was synthesized e as shown in Scheme 3 e
13j;
3-(4-Acetyl-2-ethoxy-phenylcarbamoyl)-N-hydroxy-4-
through
the
amide
coupling
between
2-chloro-5-
methylbenzamide.
17

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 2-methoxy-5-(methox-
ycarbonyl)benzoic acid 10b and 2,4-dichloroaniline 9f using
method A. The resulting ester was hydrolyzed using method F to
afford 3-[(2,4-dichlorophenyl)carbamoyl]-4-methoxybenzoic acid
12m. This acid was converted to the final compound using method
G.
We have previously described the synthesis and characteriza-
tion data of this compound [39].
13k;
3-(3-Chlorophenylcarbamoyl)-N-hydroxy-4-
methoxybenzamide.
MS m/z: 353.42 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.29 (s, 1H), 10.57 (s, 1H), 9.01
(s, 1H), 8.53 (d, J ¼ 1.9 Hz, 1H), 8.47 (d, J ¼ 2.1 Hz, 1H), 8.01e7.95 (m,
1H), 7.60 (d, J ¼ 8.6 Hz, 1H), 7.36 (d, J ¼ 8.6 Hz, 1H), 7.25 (dd, J ¼ 8.6,
2.4 Hz, 1H), 4.11 (s, 3H).
HRMS m/z: 355.0261 [MþH]þ; calculated C15H13Cl2N2Oþ4 :
355.0252.
HPLC: rt 11.51 min (99.67%)
13n;
3-(Benzo[b]thiophen-5-carbamoyl)-N-hydroxy-4-
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 2-methoxy-5-(methox-
ycarbonyl)benzoic acid 10b and 3-chloroaniline 9d using method A.
The resulting ester was hydrolyzed using method F to afford 3-[(3-
chlorophenyl)carbamoyl]-4-methoxybenzoic acid 12k. This acid
was converted to the final compound using method G.
MS m/z: 319.22 [M ꢂ H]-
methoxybenzamide.
1H NMR (400 MHz, DMSO‑d6)
d 11.18 (s,1H),10.32 (s,1H), 8.96 (s,
1H), 7.99 (d, J ¼ 2.3 Hz, 1H), 7.93e7.87 (m, 2H), 7.61 (d, J ¼ 9.1 Hz,
1H), 7.36 (t, J ¼ 8.1 Hz,1H), 7.23 (d, J ¼ 8.8 Hz, 1H), 7.16e7.12 (m, 1H),
3.91 (s, 3H).
This compound was synthesized e as shown in Scheme 3 e
through the amide coupling between 2-methoxy-5-(methox-
ycarbonyl)benzoic acid 10b and benzo[b]thiophen-5-amine 9g
using method A. The resulting ester was hydrolyzed using method F
to afford 3-(benzo[b]thiophen-5-ylcarbamoyl)-4-methoxybenzoic
acid 12n. This acid was converted to the final compound using
method G.
HRMS m/z: 321.0642 [MþH]þ; calculated C15H14ClN2Oþ4 :
321.0642.
HPLC: rt 11.14 min (98.18%)
13l;
N-Hydroxy-4-methoxy-3-(4-methoxyphenylcarbamoyl)
benzamide.
MS m/z: 341.32 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.19 (s,1H),10.26 (s,1H), 8.96 (s,
1H), 8.39 (d, J ¼ 1.9 Hz, 1H), 8.04 (d, J ¼ 2.3 Hz, 1H), 7.95e7.88 (m,
2H), 7.75 (d, J ¼ 5.4 Hz, 1H), 7.59 (dd, J ¼ 8.7, 1.9 Hz, 1H), 7.44 (d,
J ¼ 5.4 Hz, 1H), 7.24 (d, J ¼ 8.8 Hz, 1H), 3.93 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 164.51, 164.32, 158.96, 140.29,
136.31, 134.68, 131.03, 128.80, 125.51, 125.24, 124.54, 123.05, 118.19,
114.40, 112.28, 56.70.
þ
HRMS m/z: 343.0747 [MþH]þ; calculated C17H15N2O4S
:
This compound was synthesized e as shown in Scheme 3 e
343.0752.
through the amide coupling between 2-methoxy-5-(methox-
ycarbonyl)benzoic acid 10b and 4-methoxyaniline 9e using method
A. The resulting ester was hydrolyzed using method F to afford 4-
methoxy-3-[(4-methoxyphenyl)carbamoyl]benzoic acid 12l. This
acid was converted to the final compound using method G.
MS m/z: 315.38 [M ꢂ H]-
HPLC: rt 11.81 min (100%)
19a;
3-[3-[Benzyloxy]benzamido]-N-hydroxy-4-
methoxybenzamide.
1H NMR (400 MHz, DMSO‑d6)
d 11.17 (s, 1H), 9.99 (s, 1H), 8.94 (s,
1H), 8.01 (d, J ¼ 2.3 Hz, 1H), 7.88 (dd, J ¼ 8.7, 2.4 Hz, 1H), 7.65e7.59
(m, 2H), 7.21 (d, J ¼ 8.8 Hz, 1H), 6.93e6.87 (m, 2H), 3.91 (s, 3H), 3.73
(s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d
163.96, 163.80, 158.95, 155.93,
This compound was synthesized e as shown in Scheme 4 e
through Williamson ether synthesis between benzyl bromide 14a
and methyl 3-hydroxybenzoate 15 using method E. The resulting
ester was hydrolyzed using method F to afford 3-(benzyloxy)ben-
zoic acid 16a, which was coupled with methyl 3-amino-4-
methoxybenzoate 2c using method B. The resulted ester was
again hydrolyzed using method F to afford 3-[3-(benzyloxy)
132.56, 130.95, 128.95, 125.46, 125.17, 121.69, 114.28, 112.22, 56.66,
55.64.
HRMS m/z: 317.1128 [MþH]þ; calculated C16H17N2Oþ5 : 317.1137.
HPLC: rt 9.62 min (97.27%)
13m;
3-(2,4-Dichlorophenylcarbamoyl)-N-hydroxy-4-
methoxybenzamide.
18

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
benzamido]-4-methoxybenzoic acid 18a, which was then con-
verted to the final compound using method G.
MS m/z: 391.27 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.09 (s, 1H), 9.51 (s, 1H), 8.89 (s,
1H), 8.12 (d, J ¼ 2.2 Hz, 1H), 7.63e7.56 (m, 2H), 7.54 (d, J ¼ 7.8 Hz,
1H), 7.48e7.30 (m, 6H), 7.22 (dd, J ¼ 8.1, 2.0 Hz, 1H), 7.13 (d,
J ¼ 8.7 Hz, 1H), 5.18 (s, 2H), 3.86 (s, 3H).
HRMS m/z: 415.1285 [MþNa]þ; calculated C22H20N2O5Naþ
415.1269.
HPLC: rt 11.06 min (98.69%)
19b;
3-[3-[Benzyloxy]benzamido]-N-hydroxy-4-
This compound was synthesized e as shown in Scheme 5 e
through the amide coupling between 2-methyl-2,3,4,5-tetrahydro-
1H-pyrido [4,3-b]indole-8-carboxylic acid 23 and 3-amino-4-
methoxy-N-(tetrahydro-2H-pyran-2-yloxy)benzamide 20 using
method C. The THP protected product was then deprotected with
HCl - as described in method G. The product was directly purified
using preparative HPLC.
methylbenzamide.
MS m/z: 393.32 [M ꢂ H]-
1H NMR (400 MHz, DMSO-D6)
d 11.58 (s, 1H), 11.09 (s, 1H), 10.94
This compound was synthesized e as shown in Scheme 4 e
through Williamson ether synthesis between benzyl bromide 14a
and methyl 3-hydroxybenzoate 15 using method E. The resulting
ester was hydrolyzed using method F to afford 3-(benzyloxy)ben-
zoic acid 16a, which was coupled with methyl 3-amino-4-
methylbenzoate 2e using method B. The resulted ester was again
hydrolyzed using method F to afford 3-[3-(benzyloxy)benzamido]-
4-methylbenzoic acid 18b, which was then converted to the final
compound using method G.
(s, 1H), 10.01 (s, 1H), 9.30 (s, 1H), 8.25 (d, J ¼ 2.1 Hz, 1H), 8.12 (s, 1H),
7.74 (dd, J ¼ 8.6, 1.5 Hz, 1H), 7.58 (dd, J ¼ 8.6, 2.1 Hz, 1H), 7.44 (d,
J ¼ 8.5 Hz, 1H), 7.13 (d, J ¼ 8.7 Hz, 1H), 3.88 (s, 2H), 3.32 (s, 3H),
3.15e2.99 (m, 4H), 2.96 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
d 166.22, 153.82, 138.48, 132.34,
127.51, 125.76, 125.24, 124.79, 121.45, 118.30, 111.46, 111.21, 103.33,
56.33, 50.77, 45.88, 42.22, 20.74.
HRMS m/z: 395.1708 [MþH]þ; calculated C21H23N4Oþ4 395.1719.
HPLC: rt 6.97 min (97.62%).
MS m/z: 375.30 [M ꢂ H]-
1H NMR (400 MHz, DMSO‑d6)
d 11.17 (s, 1H), 9.95 (s, 1H), 8.97 (s,
8.2. In vitro HDAC inhibitory activity
1H), 7.74 (d, J ¼ 1.6 Hz, 1H), 7.62e7.60 (m, 1H), 7.58e7.53 (m, 2H),
7.49e7.36 (m, 5H), 7.35e7.30 (m, 2H), 7.25e7.21 (m, 1H), 5.18 (s,
2H), 2.24 (s, 3H).
The in vitro testing on recombinant HDACs were performed as
previously described [40].
Recombinant human HDAC1 and -6 were purchased from BPS
Biosciences. The enzyme inhibition was determined by using a
reported homogenous fluorescence assay [41]. The enzymes were
incubated for 90 min at 37 ꢁC, with the fluorogenic substrate ZMAL
HRMS m/z: 399.1357 [MþNa]þ; calculated C22H20N2O4Naþ
399.1320.
HPLC: rt 11.03 min (99.22%)
19c;
methylbenzamide.
3-[3-[2,6-Dichlorbenzyloxy]benzamido]-N-hydroxy-4-
(Z-(Ac)Lys-AMC) in a concentration of 10.5
concentrations of inhibitors with subsequent addition of 60
buffer containing trypsin (1 mg/ml) and TSA (2.75 M) and further
mM and increasing
mL of
m
incubation for 20 min at 37 ꢁC. Fluorescence intensity was
measured at an excitation wavelength of 390 nm and an emission
wavelength of 460 nm in a microtiter plate reader (BMG Polarstar).
Recombinant Sm- and hHDAC8 was produced by Romier et al. in
Strasbourg [32,35]. The HDAC8 activity assays were performed
according to the commercial HDAC8 Fluorometric Drug Discovery
Kit [Fluor de Lys(R)-HDAC8, BML-KI178] corresponding to the
manufacturer's instructions as described earlier [32]. As substrate a
tetrapeptide connected to aminomethylcoumarin (AMC) H2N-Arg-
His-Lys(Ac)-Lys(Ac)-AMC was synthesized as previously described
[40]. The enzyme was incubated for 90 min at 37 ꢁC, with a sub-
This compound was synthesized e as shown in Scheme 4 e
through Williamson ether synthesis between 2,6-dichlorobenzyl
bromide 14b and methyl 3-hydroxybenzoate 15 using method E.
The resulting ester was hydrolyzed using method F to afford 3-(2,6-
dichlorobenzyloxy)benzoic acid 16b, which was coupled with
methyl 3-amino-4-methylbenzoate 2e using method B. The resul-
ted ester was again hydrolyzed using method F to afford 3-[3-(2,6-
dichlorobenzyloxy)benzamido]-4-methylbenzoic acid 18c, which
was then converted to the final compound using method G.
MS m/z: 443.00 [M ꢂ H]-
strate concentration of 50
mM and increasing concentrations of
inhibitors. The stop-solution containing inhibitor, to stop the
hHDAC8 activity, and Trypsin, to release the AMC, was added. The
solution was incubated for 20 min at 37 ꢁC to develop the assay.
Fluorescence intensity was measured at an excitation wavelength
of 355 nm and an emission wavelength of 460 nm in a microtiter
plate reader (BMG Polarstar).
1H NMR (400 MHz, DMSO‑d6)
d
11.19 (s, 1H), 10.00 (s, 1H), 8.99
(s, 1H), 7.79e7.26 (m, 10H), 5.32 (s, 2H), 2.26 (s, 3H).
13C NMR (101 MHz, DMSO‑d6)
165.43, 164.17, 158.85, 137.80,
d
136.87, 136.53, 136.28, 132.03, 131.17, 130.75, 130.24, 129.26, 125.89,
124.73, 121.04, 118.48, 113.97, 65.60, 18.40.
HRMS m/z: 467.0533 [MþNa]þ; calculated C22H18N2O4Cl2Naþ:
467.0536.
8.3. Cytotoxicity
HPLC: rt 12.05 min (98.5%)
To determine the cytotoxicity of the developed compounds, a
human epithelial kidney cell line (HEK293) was used. HEK293 cells
(DSMZ Braunschweig, ACC305) were incubated at 37 ꢁC in a hu-
midified incubator with 5% CO2 in Dulbecco's Modified Eagle
24;
N-(5-(hydroxycarbamoyl)-2-methoxyphenyl)-2-methyl-
2,3,4,5-tetrahydro-1H-pyrido[4,3-b]indole-8-carboxamide hydro-
chloride.
19

E. Ghazy, T. Heimburg, J. Lancelot et al.
European Journal of Medicinal Chemistry 225 (2021) 113745
Medium (DMEM) supplemented with 10% fetal calf serum and
5 mM glutamine. Cells were seeded out at 1.5 ꢀ 103 cells per well in
a 96-well cell culture plate (TPP, Switzerland). The compounds to be
(TH65; PDB ID: 6HTH, chain A), hHDAC8 (PDB ID 2V5X), hDAC6
(PDB ID 5EDU) and hHDAC1 (PDB ID 4BKX) were downloaded from
the Protein Data Bank (PDB,; www.rcsb.org) [43]. Protein prepara-
tion was done using the protein preparation wizard implemented
tested were added immediately to the medium at 50 mM. After 24 h,
€
AlamarBlue reagent (Invitrogen, CA) was added according to the
manufacturer's instructions and incubated again for 21 h before
samples were analyzed. Detection of viable cells, which convert the
resazurine reagent into the highly fluorescent resorufin, was per-
formed by using a FLUOstarOPTIMA microplate reader (BMG Lab-
tec) with the following filter set: Ex 530 nm/Em 590 nm.
Measurements were performed in triplicate and data are means
with standard deviation < 14%. As a positive control daunorubicin
in Schrodinger version 2019.1 by adding hydrogen atoms, assigning
protonation states and minimizing the protein. Ligands structures
were generated in MOE [Molecular Operating Environment (MOE),
2020.01; Chemical Computing Group Inc., 1010 Sherbooke St. West,
Suite #910, Montreal, QC, Canada, H3A 2R7, 2018] in the hydrox-
amte form. The ligands were subsequently prepared for docking
€
using the LigPrep tool as implemented in Schrodinger's software
(version 2019.1) and energy minimized using the OPLS3e force
field. 25 conformers of all ligands were subsequently generated
with ConfGen. Docking of the generated conformers into the pre-
pared protein structures was performed using the program Glide
was used and an IC50 value of 12.55 0.07 mM was measured.
8.4. Phenotypic screening
€
(Schrodinger-release 2019.1) in the Standard Precision mode.
The effects of novel inhibitors targeting SmHDAC8 on the
viability of S. mansoni schistosomula was carried out as previously
described [34]. Briefly, newly transformed schistosomula (NTS)
were obtained in vitro by mechanical transformation of S. mansoni
cercariae as previously described [42]. An NTS suspension was
8.7. PAINS filter
All the herein described compounds were filtered for pan-assay
interference compounds (PAINS) [44]. For this purpose, PAINS1,
prepared at a concentration of 100 NTS per 100
199 (Invitrogen) supplemented with 10% fetal calf serum (Gibco),
penicillin (50 U.mL-1), streptomycin (50 g.mL-1) and rifampicin
(60
g.mL-1). Schistosomula were kept in culture 3 h at 37 ꢁC and
m
L using Medium
€
PAINS2 and PAINS3 filters, as implemented in Schrodinger's Canvas
program, were employed. None of the compounds was flagged as a
m
PAIN.
m
5% CO2 prior to use in screening. Stock solutions of 20 mM in DMSO
were used for each inhibitor. Mid-dilutions were performed in
Author contribution
100% DMSO and 1
black 96 well plates (Nunc, UK) with supplemented Medium 199
and 100 L of prepared NTS suspension (100 NTS/well). Live and
dead schistosomula (treated with 70% ethanol) were used as pos-
itive and negative controls. Experiments were carried out in trip-
licate wells in two biological replicates and the compounds were
mL added to 100 mL/well of M199 medium in
E.G. synthesized part of the compounds and wrote the paper.
T.H., A.T., S.D. synthesized part of the compounds. P.Z. carried out
the in vitro testing on HDAC8. K.S. carried out the in vitro testing on
HDAC1 and 6. J.L. and R.J.P. performed antiparasitic studies and
carried out data analysis. F.E. carried out the cellular testing on
HEK293 cells. C.V.S. and D.R. did the docking calculations and wrote
the manuscript. T.B.S. and C.R. solved the smHDAC8-inhibitor
crystal structures. W.S., M.S. and M.J. designed experiments,
analyzed data and wrote the paper. All authors have given approval
to the final version of the manuscript.
m
tested at final concentrations of 10 and 20
mM. After 48 h of drug
exposure, 20 L of Alamar Blue solution (AbdSerotec) were added
m
in each well. Finally, after a further 24 h of exposure, the fluores-
cence intensity of the red fluorescent resorufin product was
measured at 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
was determined for each drug dilution using wells containing only
DMSO as control. The EC50 concentration for compound 5o was
measured using the same assay with different concentrations of the
compound.
Declaration of competing interest
The authors declare that they have no known competing
financial interests or personal relationships that could have
appeared to influence the work reported in this paper.
Acknowledgment
8.5. Crystallization and X-ray data collection
E.G. appreciated the support of DAAD Egypt by GERLS scholar-
ship. This research was funded by the European Regional Devel-
opment Fund of the European Commission. CR is supported by
institutional funds from the Centre National de la Recherche Sci-
Diffraction-quality crystals of native smHDAC8 enzyme were
obtained at 17 ꢁC after three to four days by mixing equal volumes
of smHDAC8 (2.5 mg/ml) with a reservoir solution composed of 21%
PEG 3350 (Fluka) and 0.2 M Naþ/K þ
L-tartrate, and crystallized
ꢀ
entifique (CNRS), the Institut National de la Sante et de la Recherche
using the hanging-drop vapor diffusion technique. After 3 days,
grown crystals were soaked in mother liquor supplemented with
the inhibitor 5o (10 mM final concentration of the inhibitor) for
20 h. Crystals used for X-ray data collection were briefly transferred
in reservoir solution supplemented with 22% glycerol and flash-
frozen in liquid nitrogen. Crystallographic data obtained in this
project were collected at 100 K on ESRF beamline ID23. Atomic
coordinates and structure factors of the smHDAC8-5ꢁ complex
were deposited in the Protein Data Bank under the PDB ID code
7P3S.
ꢀ
ꢀ
Medicale (Inserm) and the Universite de Strasbourg.
Appendix A. Supplementary data
Supplementary data to this article can be found online at
https://doi.org/10.1016/j.ejmech.2021.113745.
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