Isophthalic Acid-Based HDAC Inhibitors as Potent Inhibitors of HDAC8 from Schistosoma mansoni

Article abstract of DOI:10.1002/ardp.201700096

Schistosoma mansoni histone deacetylase 8 (SmHDAC8) has been recently identified as a new potential target for the treatment of schistosomiasis. A series of newly designed and synthesized alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8. Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed rationalization of the observed selectivity profile.

Full text of DOI:10.1002/ardp.201700096

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Full Paper
Isophthalic Acid-Based HDAC Inhibitors as Potent Inhibitors of
HDAC8 from Schistosoma mansoni
Katharina Stenzel1, Alokta Chakrabarti2, Jelena Melesina3, Finn K. Hansen1,4, Julien Lancelot5,
1
1
6
6
5
€
Simon Herkenhohner , Beate Lungerich , Martin Marek , Christophe Romier , Raymond. J. Pierce ,
1
Wolfgang Sippl3, Manfred Jung2, and Thomas Kurz
1
€
Institute of Pharmaceutical and Medicinal Chemistry, Heinrich-Heine-University, Dusseldorf, Germany
Institute of Pharmaceutical Sciences, Albert-Ludwigs-University Freiburg, Freiburg, Germany
Institute of Pharmacy, Martin-Luther-University Halle-Wittenberg, Halle (Saale), Germany
Pharmaceutical/Medicinal Chemistry, Institute of Pharmacy, Leipzig University, Leipzig, Germany
Univ. Lille, CNRS, Inserm, CHU Lille, Institut Pasteur de Lille, U1019 UMR 8204CIIL – Centre d’Infection
2
3
4
5
ꢀ
et d’Immunite de Lille, Lille, France
6
́
IGBMC, Universite de Strasbourg, Illkirch, France
Schistosoma mansoni histone deacetylase 8 (SmHDAC8) has been recently identified as a new
potential target for the treatment of schistosomiasis. A series of newly designed and synthesized
alkoxyamide-based and hydrazide-based HDAC inhibitors were tested for inhibitory activity against
SmHDAC8 and human HDACs 1, 6, and 8. The front runner compounds showed submicromolar
activity against SmHDAC8 and modest preference for SmHDAC8 over its human orthologue hHDAC8.
Docking studies provided insights into the putative binding mode in SmHDAC8 and allowed
rationalization of the observed selectivity profile.
Keywords: Docking studies / HDAC8 / Histone deacetylase inhibitors / Schistosoma mansoni /
Schistosomiasis
Received: March 16, 2017; Revised: May 21, 2017; Accepted: May 22, 2017
DOI 10.1002/ardp.201700096
Additional supporting information may be found in the online version of this article at the publisher’s web-site.
:
symptoms is critical to the management of schistosomiasis.
Under risk are individuals having contact with freshwater
Introduction
The neglected parasitic disease schistosomiasis is endemic in
74 developing countries. The disease continues to spread to
new geographic areas despite comprehensive anthelmintic
drug therapy programs. There are two major forms of
schistosomiasis ꢀ intestinal and urogenital ꢀ caused by
Schistosoma mansoni and four further parasites of the genus
Schistosoma that infect humans. Chronic disease contributes
to major organ damage, and reducing the severity of
sources and in particular children under age 14. According to
World Health Organization (WHO) statistics at least 258
million people worldwide required preventive treatment and
62 million received treatment in 2014 [1, 2].
The life cycle of S. mansoni includes radical morphological
modifications and exhibits diverse phenotypes [3, 4]. The
complexity of the human endoparasite is reflected in a large
genome, variable transcriptome profiles, and a dynamic
epigenetic machinery in dependency of each life cycle stage
[5, 6]. There is currently no vaccine available for the treatment
of human schistosomiasis and no indication that a vaccine is
likely to become available soon [7].
€
Correspondence: Prof. Thomas Kurz, Institut fur Pharmazeutische
Since the anthelminthic drug praziquantel (PZQ) was
approved for treatment of schistosomiasis it has been widely
used for more than 30 years and remains to be the drug of
choice till now [8]. The WHO strategy for schistosomiasis
€
€
und Medizinische Chemie, Heinrich-Heine-Universitat Dusseldorf,
€
€
Universitatsstr. 1, 40225 Dusseldorf, Germany.
E-mail: thomas.kurz@uni-duesseldorf.de
Fax: þ49 211 81-13847
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control focuses on periodic large scale treatment with PZQ
(preventive chemotherapy) of affected populations.
However, the drug does not prevent reinfections and its
mechanism of action is not exactly known. The application of
PZQ is limited to adult worms, which has been shown in
in vitro tests and confirmed by clinical data [9, 10]. PZQ is safe,
effective and relatively cheap, but there is also a growing
concern regarding reports from patients not cured by
multiple doses [11, 12]. The development of resistance and
stable resistance after removal of drug pressure has been
demonstrated in the laboratory [13–15]. Moreover resistant
isolates have already been characterized in endemic areas and
the selection of field strains of schistosome that are resistant
to PZQ is more and more likely [9].
Like other human parasites schistosomes share some
properties with malignant tumors including intense meta-
bolic activity and uncontrolled cell division [16–18]. Histone
deacetylases (HDACs) are Zn2þ- or NADþ-dependent lysine
deacetylases that can modulate cell chromatin structure,
transcription, and gene expression. Consequently, histone
deacetylase inhibitors (HDACi) have emerged as a new class of
anticancer drugs. Currently, three HDACi are approved by the
US Food and Drug Administration (FDA) for the treatment of
T-cell lymphoma (vorinostat, romidepsin, and belinostat)
while panobinostat has been approved for combination
therapy use in certain cases of multiple myeloma. Further-
more, the potential therapeutic use of HDACi in other
diseases including inflammatory, immune, neurodegenera-
tive, cardiac, viral, and parasitic diseases is currently under
discussion [19–27].
against SmHDAC8 have been identified in recent years (see
Fig. 1 for selected examples) [32, 34]. Unfortunately, most
known SmHDAC8 inhibitors possess a higher activity against
human HDAC8. Thus, there is a strong need for new types of
HDACi with preferential activity for SmHDAC8. We herein
present the design, synthesis, and biological evaluation of a
novel series of SmHDAC8 inhibitors.
Results and discussion
Design and synthesis of target compounds
We used the 3-acylaminobenzohydraxamates of type I and II
(Fig. 1), a series of potent and in some cases preferential
SmHDAC8 inhibitors, as starting point for this project [34]. In a
previous study [21, 35] we identified an alkoxyamide group as
a novel connecting unit which presumably can enable charge-
assisted hydrogen bonds due to the additional polarization of
the N–H bond. We therefore decided to retain the meta-
substituted benzohydroxamate realized in I and to combine
this motif with an alkoxyamide connecting unit and various
cap groups. In addition, we designed compounds with
hydrazide-based connecting units in order to probe whether
these groups can serve as an alternative connecting unit.
The isophthalic acid-based target compounds 3a–i and 5a,b
were synthesized using a straightforward two-step protocol
as illustrated in Scheme 1. First, the 1,10-carbonyldiimidazole
(CDI)-mediated amide coupling reaction of mono-methyl
isophthalate with O-substituted hydroxylamines and hydra-
zine derivatives provided the alkoxyamide intermediates 2a–i
and hydrazides 4a,b (Scheme 1), respectively.
Using the “piggyback” strategy it has been shown that the
HDACi trichostatin A (TSA), vorinostat (SAHA), and valproic
The subsequent treatment of 2a–i and 4a,b with an excess
of hydroxylamine hydrochloride in the presence of meth-
anolic sodium methoxide afforded the desired target com-
pounds 3a–i and 5a,b. Using these synthetic methods allowed
us to efficiently modify the cap region of our target
compounds in order to potentially address the hydrophobic
side pocket of SmHDAC8.
acid (VPA) inhibited S. mansoni histone deacetylase
8
(SmHDAC8) activity at all life cycle stages and TSA and
VPA caused mortality of schistosomula and adult worms
[25, 28, 29]. Till now only class I S. mansoni HDAC1, -3, and -8
and class III S. mansoni Sirt1, -2, -5, -6, -7 HDACs have been
cloned and characterized [29, 30]. S. mansoni HDAC1, 3, and 8
mRNAs are expressed at all schistosome life cycle stages. In
particular, SmHDAC8 has been identified as a potential target
for antiparasitic therapy [31]. Transcripts of SmHDAC8 are
expressed at higher levels than SmHDAC1 and SmHDAC3
during all life cycle stages, pointing at specific and vital
functions in the parasite life cycle. At the same time human
HDAC8 shows the lowest level of expression of the four class I
HDACs in human and it has been reported that inhibition of
hHDAC8 shows only limited effects in many cell types [32].
Different HDACi have already been shown to inhibit
SmHDAC8 and to induce histone hyperacetylation and
apoptosis in S. mansoni [29, 31, 33].
Primary screening
All synthesized compounds were first tested in a primary
screen for inhibition of SmHDAC8 and representative human
HDAC isoforms (hHDAC1, hHDAC6, and hHDAC8) at
a
concentration of 1 mM (Table 1). The in vitro assays rely on
the use of fluorogenic substrates containing an e-acetyl lysine
linked to a fluorescent moiety at the C-terminus. Only upon
deacetylation the resulting peptide is a substrate for a
protease which upon cleavage of the fluorogenic moiety
releases the fluorophore for quantitation. For HDAC1 and 6
we used ZMAL (Z-Lys(Ac)-AMC) and trypsin as the protease.
The commercially available Fluor de Lys-HDAC8 substrate has
Arg-His-Lys(Ac)-Lys(Ac) as the substrate sequence. The exact
identity of fluorophore and developer are not revealed
[36, 37]. All compounds showed significant inhibition of
SmHDAC8 deacetylase activity, moderate inhibition of
hHDAC6 and only very low inhibition of hHDAC1. However,
Treatment of schistosomes with HDACi caused an accumu-
lation of acetylated cellular proteins and dose-dependent
mortality of schistosomula and adult worms [6, 28, 29]. Thus,
the development of small-molecule SmHDAC8 inhibitors
represents
a promising approach for the treatment of
schistosomiasis and several HDACi with confirmed activity
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Figure 1. Chemical structures of selected HDAC
inhibitors and IC50 values of SmHDAC8 and
hHDAC8 [22, 24].
most compounds inhibited hHDAC8 in similar fashion as
SmHDAC8. Interestingly, compounds 3d and 5a revealed a
somewhat stronger inhibition of SmHDAC8 in comparison to
hHDAC8 (Table 1). Based on this primary screening, we
decided to investigate the HDACi 3d, 5a and the unsubsti-
tuted prototype compound 3c in more detail.
compounds possess approximately 10-fold preference over
hHDAC6. The compound 3c exhibited a stronger inhibition of
hHDAC8 (IC50: 0.09 mM) compared with SmHDAC8 (IC50
0.33 mM) whereas 3d (hHDAC8 IC50: 0.63 mM vs. SmHDAC8
:
IC50: 0.40 mM) and 5a (hHDAC8IC50: 1.31 mM vs. SmHDAC8 IC50
:
0.75 mM) showed a very modest preference for SmHDAC8.
Docking study
Inhibition of hHDAC1, hHDAC6, hHDAC8, and
To rationalize the obtained biochemical data, notably to
understand the change of specificity between the schisto-
somal and human enzymes, the synthesized inhibitors were
docked to the available crystal structures of SmHDAC8,
hHDAC8, and hHDAC1. The applied docking method was
first successfully validated on the X-ray structures of hHDAC8
and SmHDAC8 (for details see the Experimental section).
Using this docking setup consistent binding models were
derived for both human and SmHDAC8. Comparison of the
available X-ray structures of hHDAC8 and SmHDAC8 showed a
SmHDAC8
In order to study whether 3c, 3d, and 5a can be considered as
preferential SmHDAC8 inhibitors, we determined IC50 values
against hHDAC1, hHDAC6, hHDAC8, and SmHDAC8. SAHA
(vorinostat, suberoylanilide hydroxamic acid) was used as a
reference pan-HDACi. The results are summarized in Table 2.
Compounds 3c, 3d, and 5a showed nanomolar activity against
SmHDAC8 with IC50 values in the range of 0.33–0.75 mM and
very good selectivity over hHDAC1. Furthermore, the
Scheme 1. Synthesis of alkoxyamide-based and
the hydrazide-based HDACi. Reagents and
conditions: a) CDI, CH2Cl2, R1-ONH2, or R1R2-
NNH2, r.t., 0.5 h, 12 h; b) 1) NaOMe, MeOH,
NH2OH.HCl, 70°C, 3–5 h, 2) NaOH, NH2OH,
MeOH, CH2Cl2, 0°C, r.t., 12 h.
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Table 1. % Inhibition of hHDAC1, hHDAC6, hHDAC8, and SmHDAC8 at 1 mM.
% Inhibition at 1 mM: Primary screen
R1
hHDAC1
hHDAC6
hHDAC8
SmHDAC8
3a
3b
3c
3d
3e
3f
3g
3h
3i
Ph-CH2-CH2
Ph-CH2
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
<10
65
43
31
20
52
41
50
51
41
26
49
81
63
79
56
76
73
88
78
72
38
70
87
84
89
86
90
78
90
84
82
64
87
Ph
2,4-Cl-Ph
2,3-Cl-Ph
3,5-Me-Ph
4-Me-Ph
1-naphthyl
3,4-F-Ph
5a
5b
high similarity especially within the binding pocket. The main
differences are the subsitution of Met274 in hHDAC8 to
His292 in SmHDAC8 and a flipped out conformation of
Phe151 in SmHDAC8 (for details see Fig. S1a and b in the
Supporting Information). Docking of inhibitors 3c, 3d, and 5a
to SmHDAC8 and hHDAC8 showed that the hydroxamate is
perfectly coordinating the zinc ion as observed in Sm/
hHDAC8-inhibitor crystal structures. In addition the hydrox-
amate group is making hydrogen bonds to conserved Tyr and
His residues nearby the catalytic zinc ion (see Fig. 2A–C for
details). Docking of 3c and 3d, having an alkoxyamide linker
between the two aromatic rings, showed in case of SmHDAC8
two additional hydrogen bonds to Lys20 and His292 (Fig. 2A,
B). These interactions are also observed in the crystal structure
of SmHDAC8 in complex with an amide containing inhibitor
(PDB ID 5FUE) [34]. Only compounds 3c and 3d are showing
these two hydrogen bonds, whereas the weaker SmHDAC8
inhibitor 5a is not able to form these hydrogen bonds but
interacts with Asp100. Additionally, the calculated binding
energies of 3c and 3d are more favourable compared to 5a
(see Supporting Information Table S1). The terminal aromatic
group of 3c and 3d is interacting with the residues of the so-
called side-pocket (His292, Pro293, Tyr306). The docking of 5a
showed that the piperazine ring is located nearby the acidic
residue Asp100 (hydrogen bond in case of SmHDAC8) whereas
no interaction with the residues of the side-pocket was
observed, which might explain the lower inhibitory activity.
In the hHDAC8 structure a methionine (Met274) is located
at the same position as His292 in SmHDAC8. The methionine is
not able to form hydrogen bonds with the docked inhibitors.
However, in the available crystal structures of hHDAC8 a
conserved water molecule bound to the zinc coordinating
histidine (His180 in hHDAC8) is observed which was found as a
hydrogen bonding partner with 3c and 3d (Fig. 2D,E). The
terminal aromatic ring of 3c makes favorable van der Waals
interaction with the side-pocket in hHDAC8 (Phe152, Pro273,
Met274, Tyr341, Fig. 2) and shows a perfect fit to this
hydrophobic subpocket (Fig. 3). This is reflected by
favourable docking score and binding energy calculated for
3c (Supporting Information Table S1). The docking of the
Table 2. IC50 profiling against human hHDAC1, hHDAC6, hHDAC8, and SmHDAC8.
IC50 (mM)
hHDAC1
hHDAC6
hHDAC8
SmHDAC8
0.33 ꢁ 0.04
3c
3d
136.1
2.95
0.09 ꢁ 0.12
47.5% @200 mM
15.3% @200 mM
0.32
5.12
7.11
0.11
0.63 ꢁ 0.14
1.31 ꢁ 0.13
0.91 ꢁ 0.26
0.40 ꢁ 0.078
0.75 ꢁ 0.22
1.38 ꢁ 0.70
5a
SAHA
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Figure 2. Docking poses calculated for inhibitor 3c (colored cyan), 3d (colored orange), and 5a (colored green) at SmHDAC8 (purple
ribbon) and at hHDAC8 (turquoise ribbon). Only surrounding amino acid residues are shown for clarity. Hydrogen bonds are shown
as orange dashed lines.
inhibitors to hHDAC1 showed that the hydroxamate is not
able to coordinate in a favorable manner to the catalytic zinc
ion due to a narrower binding pocket (Fig. 4).
was used as reference compound. The results are summa-
rized in Fig. 5. As expected, vorinostat exhibited strong
cytotoxicity against both HeK293T and HeLa cells. In
contrast, compounds 3d and 5a showed only relatively low
cytotoxicity (Fig. 5).
Cytotoxicity and phenotypic activity
Parasite-specific HDACi should possess low toxicity to
mammalian cells. We therefore tested 3d and 5a for
cytotoxicity against HeK293T and HeLa cells. Vorinostat
We next studied the phenotypic activity of 3d and 5a by
testingtheireffectsontheviabilityofthelarvae(schistosomula)
and the stability of adult worm pairs in culture exactly as
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Experimental
Chemistry
General procedure for the synthesis of 3a–i and 5a,b
Method A: Hydroxylamine hydrochloride (348 mg, 5.0 mmol,
10 eq) was added to a freshly prepared sodium methanolate
solution (175 mg, 7.5 mmol, 15 eq) in dry methanol (8 mL). The
mixture was stirred for 10 min before the respective ester 2a–i
or 4a (0.5 mmol, 1.0 eq) was added. The reaction mixture was
stirred in a high-pressure flask for 3–5 h at 70°C. The solvent
was removed under reduced pressure, water (15 mL) was
added, and the pH was adjusted to pH 7–8 using 4 M HCl. The
mixture was extracted with ethyl acetate (3 ꢂ 20 mL), the
combined organic layers were dried over anhydrous sodium
sulfate, filtered, and concentrated in vacuum. The crude
products were purified by flash column chromatography
using a linear dichloromethane/methanol gradient (pre-
packed silica cartridge, gradient: 97:3 to 91:9 in 20 min) to
yield the desired hydroxamic acids 3a–i and 5a–b (yield:
60–91%). Method B: The respective ester 4b (1 mmol, 1.0 eq)
was dissolved in dry dichlormethane/methanol (1:3) and
cooled down to 0°C. Hydroxylamine (50 wt% in water,
30 mmol, 30 eq) and NaOH (0.4 g, 10 mmol, 10 eq) were added
and stirred for 12 h at room temperature. The solvent was
removed under reduced pressure, water (15 mL) was added,
and the pH was adjusted to pH 7–8 using 4 M HCl. The mixture
was extracted with ethyl acetate (3 ꢂ 20 mL), the combined
organic layers were dried over anhydrous sodium sulfate,
filtered, and concentrated in vacuum (yield: 86%).
Figure 3. Molecular surface of the hHDAC8 binding pocket
colored according to the hydrophobicity (green ¼ hydrophobic,
magenta ¼ hydrophilic). The docking pose of inhibitor 3c is
shown in cyan, 3d is shown in orange, and 5a is shown in green.
previously described [34]. Using a fluorescence-based assay
3d showed moderate toxicity toward schistosomula at
10 mM, but this was not dose-dependent. 5a showed only
slight activity. Moreover, neither compound significantly
affected adult worm pairing during 5 days of culture in vitro
(Table 3). Of note, vorinostat is only moderately effective in
the same assays (not shown), whereas other hydroxamate-
based inhibitors are very active [29, 34]. The different
abilities of the compounds to pass through the parasite
tegument may provide a possible explanation for these
differences.
The InChI codes of the investigated compounds together
with some biological activity data are provided as Supporting
Information.
N1-Hydroxy-N3-(3-phenylpropoxy)isophthalamide (3a)
Colorless solid; yield: 84%; mp: 130°C; 1H NMR (600 MHz,
DMSO-d6) d 10.69 (s, 2H), 8.18 (s, 1H), 7.84–7.94 (m, 2H), 7.55
(t, J ¼ 7.1 Hz, 1H), 7.42–7.22 (m, 4H), 7.22–7.07 (m, 1H), 3.92
(s, 2H), 2.73 (s, 2H), 1.92 (s, 2H). 13C NMR (151 MHz, DMSO) d
162.95, 162.90, 141.01, 132.53, 132.07, 129.04, 128.96, 128.01,
127.79, 127.72, 125.32, 125.20, 73.96, 30.85, 29.05. tR:
11.68 min, purity: 97.2%; HRMS (ESI) Anal. calcd. for
Conclusion
In summary, we have designed and synthesized two new
types of isophthalic acid-based SmHDAC8 inhibitors. The
alkoxyamide-based HDACi 3d and the hydrazide-based
HDACi 5a were identified as potent inhibitors of SmHDAC8
with a high preference over hHDAC1 and good preference
over hHDAC6. Cytotoxicity studies revealed that
the compounds showed relatively low effects on the
proliferation of human cells. Molecular modeling and
docking studies allowed rationalization of the observed
biochemical data and suggest that two important hydrogen
bonds of the alkoxyamide connecting unit to Lys20 and
His292 contribute to the high activity of 3d against
SmHDAC8. Even though the preference over hHDAC8 and
the phenotypic activity need to be improved in the
future, we believe that 3d and 5a are valuable starting
points for the development of novel preferential SmHDAC8
inhibitors.
C
17H19N2O4 315.1345 [MþH]þ, Found 315.1342.
N1-Hydroxy-N3-phenethoxyisophthalamide (3b)
Colorless solid; yield: 78%; mp: 128°C; 1H NMR (500 MHz,
DMSO-d6) d 11.55 (s, 2H), 9.18 (s, 1H), 8.16 (s, 1H), 7.88
(t, J ¼ 8.1 Hz, 2H), 7.55 (t, J ¼ 7.7 Hz, 1H), 7.42–7.27 (m, 4H),
7.27–7.11 (m, 1H), 4.13 (t, J ¼ 6.9 Hz, 2H), 2.97 (t, J ¼ 6.9 Hz,
2H). 13C NMR (126 MHz, DMSO) d 163.43, 138.27, 133.00,
132.49, 129.58, 129.48, 128.78, 128.51, 128.20, 126.09, 125.86,
75.64, 33.93. tR: 10.65 min, purity: 97.5%; HRMS (ESI) Anal.
calcd. for C16H17N2O4 301.1188 [MþH]þ, Found 301.1182.
N1-(Benzyloxy)-N3-hydroxyisophthalamide (3c)
Colorless solid; yield: 60%; mp: 159°C; 1H NMR (500 MHz,
DMSO-d6) d 11.92 (s, 1H), 11.34 (s, 1H), 9.14 (s, 1H), 8.16 (s, 1H),
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Figure 4. Docking poses calculated for inhibitor 3c (colored cyan), 3d (colored orange), and 5a (colored green) at hHDAC1. Only
surrounding amino acid residues are shown for clarity. Hydrogen bonds are shown as orange dashed lines.
8.05–7.75 (m, 2H), 7.62–7.26 (m, 6H), 4.94 (s, 2H). 13C NMR
(126 MHz, DMSO) d 163.72, 163.46, 135.76, 132.98, 132.45,
129.62, 129.51, 128.82, 128.52, 128.23, 125.91, 76.93. tR:
9.62 min, purity: 97.2%; HRMS (ESI) Anal. calcd. for
N1-((2,4-Dichlorobenzyl)oxy)-N3-hydroxyisophthalamide (3d)
Colorless solid; yield 82%; mp: 130°C; 1H NMR (600 MHz,
DMSO-d6) d 11.51 (s, 2H), 9.43 (s, 1H), 8.14 (s, 1H), 7.88
(d, J ¼ 7.7 Hz, 1H), 7.84 (d, J ¼ 7.6 Hz, 1H), 7.67 (s, 1H), 7.64
(d, J ¼ 8.2 Hz, 1H), 7.53 (t, J ¼ 7.7 Hz, 1H), 7.49 (dd, J ¼ 8.3,
C
15H15N2O4 287.1032 [MþH]þ, Found 287.1024.
Figure 5. Comparisonofcellviabilityof3dand5a
in human cell lines. HeK293(A) and HeLa(B) cell
lines were treated with the indicated concen-
trations of inhibitors for 72 h. Cell viability was
measured using the 3-(4,5-dimethylthiazol-2-yl)-
5-(3-carboxymethoxyphenyl)-2-(4-sulfo-phenyl)-
2H-tetrazolium/phenazine methosulfate (MTS/
PMS) reagent. Data represent S.E. of the mean
(duplicates).
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16H17N2O4 301.1188 [MþH]þ, Found
Table 3. Toxicity studies on S. mansoni adult worms and
schistosomula.
Anal. calcd. for
301.1184.
C
Compound
% Pairing ꢁ SEM
% Viability ꢁ SD
N1-Hydroxy-N3-(naphthalen-1-ylmethoxy)isophthalamide
(3h)
3d
Colorless solid; yield: 81%; mp: 137°C; 1H NMR (600 MHz,
DMSO-d6) d 11.97 (s, 1H), 11.09 (s, 1H), 9.17 (s, 1H), 8.62
(d, J ¼ 8.0 Hz, 1H), 8.22 (s, 1H), 7.98 (d, J ¼ 8.1 Hz, 2H), 7.91
(t, J ¼ 6.5 Hz, 2H), 7.68–7.55 (m, 4H), 7.49–7.54 (m, 1H), 5.39
(s, 2H). 13C NMR (126 MHz, DMSO) d 164.78, 164.37, 134.21,
134.09, 133.47, 132.91, 132.33, 130.59, 130.45, 130.24, 129.47,
129.36, 129.12, 127.29, 126.87, 126.15, 125.75, 76.23. tR:
12.28 min, purity: 97.4%; HRMS (ESI) Anal. calcd. for
10 mM
20 mM
5a
10 mM
20 mM
95 ꢁ 5
85 ꢁ 5
76 ꢁ 6
75 ꢁ 4
95 ꢁ 5
95 ꢁ 5
90 ꢁ 9
79 ꢁ 3
Experimental conditions: Single dose at D ¼ 0, duration:
3 days data represent the means of three independent
experiments. Adult worm pairing assay: Number of
worms: 10/wells n ¼ 2 Alamar viability assay: Number of
schistosomula: 100/well n ¼ 2.
C
19H17N2O4 337.1188 [MþH]þ, Found 337.1185.
N1-((3,4-Difluorobenzyl)oxy)-N3-hydroxyisophthalamide
(3i)
2.2 Hz, 1H), 5.04 (s, 2H). 13C NMR (151 MHz, DMSO) d 163.12,
162.91, 133.63, 133.24, 132.47, 132.28, 132.03, 131.86, 129.11,
129.03, 128.20, 128.01, 126.83, 125.40, 72.46. tR: 12.40 min,
purity: 99.1%; HRMS (ESI) Anal. calcd. for C15H13Cl2N2O4
355.0252 [MþH]þ, Found 355.0244.
Colorless solid; yield 73%; mp: 120°C; 1H NMR (600 MHz,
DMSO-d6) d 11.57 (s, 2H), 9.28 (s, 1H), 8.18 (s, 1H), 7.88
(d, J ¼ 7.5 Hz, 1H), 7.91 (d, J ¼ 7.6 Hz, 1H), 7.64–7.50 (m, 2H),
7.49–7.42 (m, 1H), 7.33 (s, 1H), 4.95 (s, 2H). 13C NMR (151 MHz,
DMSO) d 163.02, 162.89, 148.71 (dd, J ¼ 245.4, 10.5 Hz), 148.61
(dd, J ¼ 245.2, 11.9 Hz), 133.46, 132.45, 131.82, 129.14, 129.02,
128.03, 125.37, 125.07 (dd, J ¼ 6.2, 3.0 Hz), 117.11 (d, J ¼ 17.3
Hz), 116.74 (d, J ¼ 17.1 Hz), 74.96. tR: 10.83 min, purity: 98.9%;
HRMS (ESI) Anal. calcd. for C15H13F2N2O4 323.0843 [MþH]þ,
Found 323.0835.
N1-((2,3-Dichlorobenzyl)oxy)-N3-hydroxyisophthalamide
(3e)
Colorless solid; yield 91%; mp: 116°C; 1H NMR (600 MHz,
DMSO-d6) d 11.75 (s, 1H), 11.47 (s, 1H), 9.13 (s, 1H), 8.13
(s, 1H), 7.89 (d, J ¼ 7.6 Hz, 1H), 7.84 (d, J ¼ 7.7 Hz, 1H), 7.67
(d, J ¼ 7.9 Hz, 1H), 7.60 (d, J ¼ 7.5 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz,
1H), 7.42 (t, J ¼ 7.8 Hz, 1H), 5.11 (s, 2H). 13C NMR (151 MHz,
DMSO) d 163.52, 162.93, 135.68, 135.63, 132.50, 131.76,
131.28, 130.60, 129.91, 129.15, 129.05, 128.03, 127.60,
125.42, 73.62. tR: 12.10 min, purity: 96.3%; HRMS (ESI)
Anal. calcd. for C15H13Cl2N2O4 355.0252 [MþH]þ, Found
355.0247.
N1-Hydroxy-N3-(4-methylpiperazin-1-yl)isophthalamide
(5a)
Colorless solid; yield: 65%; mp: 155°C; 1H NMR (600 MHz,
DMSO-d6) d 11.40 (s, 1H), 9.72 (s, 1H), 9.15 (s, 1H), 8.24 (s, 1H),
7.94–7.86 (m, 2H), 7.53 (t, J ¼ 7.5 Hz, 1H), 2.97 (s, 4H), 2.66
(s, 4H), 2.33 (s, 3H). 13C NMR (151 MHz, DMSO) d 163.05,
162.98, 133.43, 132.21, 129.35, 128.88, 127.81, 125.47, 53.20,
52.32, 43.97. tR: 4.79 min, purity: 98.1%; HRMS (ESI) Anal.
calcd. for C13H19N4O3 279.1457 [MþH]þ, Found 279.1453.
N1-((3,5-Dimethylbenzyl)oxy)-N3-hydroxyisophthalamide
(3f)
Colorless solid; yield 84%; mp: 105°C; 1H NMR (500 MHz,
DMSO-d6) d 11.86 (s, 1H), 11.33 (s, 1H), 9.14 (s, 1H), 8.16
(s, 1H), 7.94–7.83 (m, 2H), 7.56 (t, J ¼ 7.7 Hz, 1H), 7.07 (s, 2H),
7.00 (s, 1H), 4.86 (s, 2H), 2.29 (s, 6H). 13C NMR (126 MHz,
DMSO-d6) 13C NMR (151 MHz, DMSO) d 164.30, 164.06,
137.78, 136.13, 133.57, 133.12, 130.15, 129.10, 127.15,
126.51, 77.59, 21.32. tR: 12.03 min, purity: 97.5%; HRMS
(ESI) Anal. calcd. for C17H19N2O4 315.1345 [MþH]þ, Found
315.1343.
N-Hydroxy-3-(2-phenylhydrazine-1-carbonyl)benzamide
(5b)
Colorless solid; yield 86%; mp: 182°C; 1H NMR (600 MHz,
DMSO-d6) d 11.34 (s, 1H), 10.44 (s, 1H), 9.14 (s, 1H), 8.29 (s, 1H),
8.04 (d, J ¼ 7.7 Hz, 1H), 7.99–7.89 (m, 2H), 7.59 (t, J ¼ 7.7 Hz,
1H), 7.16 (t, J ¼ 7.9 Hz, 2H), 6.81 (d, J ¼ 7.8 Hz, 2H), 6.73
(t, J ¼ 7.3 Hz, 1H). 13C NMR (151 MHz, DMSO) d 165.33, 163.07,
148.78, 132.73, 132.56, 129.21, 129.15, 128.14, 128.06, 125.61,
118.08, 111.74. tR: 9.59 min, purity: 98.4% HRMS (ESI) Anal.
calcd. for C25H25N2O4 417.1804 [M þ H]þ, Found 417.1809.
HRMS (ESI) Anal. calcd. for C14H14N3O3 272.1035 [MþH]þ,
Found 272.1032.
N1-Hydroxy-N3-((4-methylbenzyl)oxy)isophthalamide (3g)
Colorless solid; yield 78%; mp: 169°C; 1H NMR (600 MHz,
DMSO-d6) d 11.56 (s, 2H), 9.15 (s, 1H), 8.15 (s, 1H), 7.89 (d,
J ¼ 7.7 Hz, 1H), 7.86 (d, J ¼ 7.8 Hz, 1H), 7.55 (t, J ¼ 7.7 Hz, 1H),
7.35 (d, J ¼ 7.6 Hz, 2H), 7.21 (d, J ¼ 7.6 Hz, 2H), 4.89 (s, 2H), 2.32
(s, 3H). 13C NMR (151 MHz, DMSO) d 163.16, 162.95, 137.02,
132.49, 132.28, 131.98, 129.04, 128.95, 128.43, 128.28, 128.01,
125.36, 76.24, 20.22. tR: 10.85 min, purity: 97.1%; HRMS (ESI)
Biological evaluation
Phenotypic screening of schistosomes
The viability of S. mansoni schistosomula in the presence of
SmHDAC8 inhibitors was measured with a fluorescence-based
assay using Alamar blue as previously described [24], as was
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Isophthalic Acid-Based HDAC Inhibitors from SmHDAC8
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Computational methods
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€
preparation was done using Schrodinger’s Protein Prepa-
ration Wizard [39] by adding hydrogen atoms, assigning
protonation states, and minimising the protein. Ligands
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Multiple low energy starting conformations were gener-
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Molecular docking was performed using program Glide
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In our previous study we found that rescoring the
docking poses by using a MM-GB/SA protocol resulted in
a significant correlation between calculated interaction
energies and in vitro inhibition data. Therefore, the same
protocol was applied to the compounds under study. To
calculate binding free energy, we used the AMBER12EHT
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2
̊
0.5 A (force constant (3/2) kT/(0.5) ).
The authors thank the COST action CM1406 (Epigenetic
Chemical Biology EPICHEMBIO) for support. This work and the
authors of this article received funding from the European
Union’s Seventh Framework Programme for Research,
Technological Development and Demonstration under Grant
Agreements 241865 (SEtTReND) and 602080 (A-ParaDDisE).
We thank Karin Schmidtkunz for technical assistance. The
work of J. L. and R. P. was also supported by institutional funds
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ꢀ
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