Synthesis of aminophenylhydroxamate and aminobenzylhydroxamate derivatives and in vitro screening for antiparasitic and histone deacetylase inhibitory activity

Article abstract of DOI:10.1016/j.ijpddr.2018.01.002

A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthesized and screened for their antiparasitic activity against Leishmania, Trypanosoma, and Toxoplasma. Their anti-histone deacetylase (HDAC) potency was determined. Moderate to no an

Full text of DOI:10.1016/j.ijpddr.2018.01.002

IJP:DrugsandDrugResistance8(2018)59–66
Contents lists available at ScienceDirect
IJP: Drugs and Drug Resistance
journal homepage: www.elsevier.com/locate/ijpddr
Synthesis of aminophenylhydroxamate and aminobenzylhydroxamate
derivatives and in vitro screening for antiparasitic and histone deacetylase
inhibitory activity
C. Loeuilleta,d, B. Touquetb, B. Ouryc,d, N. Eddaikrae,f, J.L. Ponsg, J.F. Guichoug,1, G. Labesseg,1
,
D. Serenoc,d,∗,1
a
Univ. Grenoble Alpes, CNRS, CHU Grenoble Alpes, Grenoble INP, TIMC-IMAG, F-38000 Grenoble, France
b
Institute for Advanced Biosciences (IAB), Team Host-Pathogen Interactions & Immunity to Infection, INSERM U 1209, CNRS UMR 5309, Université Grenoble Alpes,
Grenoble, France
c
IRD, Univ Montpellier, InterTryp, Montpellier, France
IRD, Univ Montpellier, MiVegec, Montpellier, France
Laboratoire d’Eco-épidemiologie Parasitaire et Génétique des Populations, Institut Pasteur d’Alger, Route du Petit Staoueli, Dely Brahim, Alger, Algeria
Laboratoire de Biochimie Analytique et Biotechnologies, Université Mouloud Mammeri de Tizi Ouzou, Algeria
d
e
f
g
Centre de Biochimie Structurale (CBS), INSERM, CNRS, Université de Montpellier, France
A B S T R A C T
A series of aminophenylhydroxamates and aminobenzylhydroxamates were synthesized and screened for their
antiparasitic activity against Leishmania, Trypanosoma, and Toxoplasma. Their anti-histone deacetylase (HDAC)
potency was determined. Moderate to no antileishmanial or antitrypanosomal activity was found
(IC50 > 10 μM) that contrast with the highly efficient anti-Toxoplasma activity (IC50 < 1.0 μM) of these com-
pounds. The antiparasitic activity of the synthetized compounds correlates well with their HDAC inhibitory
activity. The best-performing compound (named 363) express a high anti-HDAC6 inhibitory activity (IC50 of
0.045
0.015 μM) a moderate cytotoxicity and a high anti-Toxoplasma activity in the range of known anti-
Toxoplasma compounds (IC50 of 0.35–2.25 μM). The calculated selectivity index (10–300 using different human
cell lines) of the compound 363 makes it a lead compound for the future development of anti-Toxoplasma
molecules.
1. Introduction
endemic areas (Lira et al., 1999; Sereno et al., 2012; Seblova et al.,
2014; Uliana et al., 2016). Altogether, for all these neglected tropical
Parasitic diseases cause significant morbidity and mortality in-
fecting hundreds of millions of people particularly in developing
countries (Lozano et al., 2012). Human African trypanosomiasis (HAT)
or sleeping sickness is caused by two Trypanosoma brucei (T.b.) sub-
species transmitted through the bites of tsetse flies (Glossina). Seventy
million people risk to contract HAT (Franco et al., 2014). Trypanosoma
cruzi, the agent of Chagas disease, is mainly transmitted by triatomines,
but transmission may occur orally and congenitally, by transfusion or
transplantation (Rassi et al., 2010). About 6–7 million people world-
wide, mostly in Latin America, are infected. Leishmaniasis is a world-
wide threatening problem (Akhoundi et al., 2017). New antileishmanial
compounds are currently needed, because of the decreasing efficiency
of classical drugs (antimonial containing drugs) reported in various
diseases, drugs currently available are few, limited in efficacy display a
wide range of side effects and are challenged by drug resistant organ-
isms (see Chellan et al., 2017; Sereno et al., 2012; Stein et al., 2014;
Zingales, 2017). All these diseases belong to Neglected tropical Dis-
eases, that affect the poorest individual (Molyneux et al., 2017). Tox-
oplasma gondii is transmitted through meat containing T. gondii cysts or
through water containing oocysts from feline feces. During the acute
phase of infection in immunocompetent patients, the disease is largely
asymptomatic but can present as a “flu-like syndrome” in 20% of pa-
tients. Severe toxoplasmosis can be observed especially in fetuses and
immunocompromised patients, such as transplant and AIDS patients, in
whom the infection can lead to encephalitis, chorioretinitis, heart and
lung lesions (Tenter et al., 2000).
∗
Corresponding author. IRD, Office 164, 34032 Montpellier, France.
E-mail address: denis.sereno@ird.fr (D. Sereno).
Contributed equally.
1
https://doi.org/10.1016/j.ijpddr.2018.01.002
Received 6 November 2017; Received in revised form 10 January 2018; Accepted 16 January 2018
2211-3207/©2018TheAuthors.PublishedbyElsevierLtdonbehalfofAustralianSocietyforParasitology.ThisisanopenaccessarticleundertheCCBYlicense
(http://creativecommons.org/licenses/BY/4.0/).

C. Loeuillet et al.
IJP:DrugsandDrugResistance8(2018)59–66
The discovery of novel drug targets and new chemotherapies with
products were used such as in the next step. Amide or sulfonamide
derivatives (1 eq.) were dissolved in THF (3 ml). A solution of LiOH (3
eq.) in 3 ml of water was added and the reaction mixture was heated at
40 °C overnight. The reaction mixture was concentrated. The residue
was taken up with 30 ml of water and the aqueous phase was washed
with 20 ml of EtOAc, then the aqueous phase was acidified to pH 2 with
a solution of 1 M HCl. The aqueous phase was extracted three times
with 20 ml of EtOAc. The organic phases were combined and dried over
Na2SO4, filtrated and concentrated to afford the acid derivatives. The
products were used such as in the next step. Acids derivatives (1 eq.)
were dissolved in dry DMF (5 ml). Ethyl chloroformate (1.2 eq.) and N-
methyl-morpholine (1.3 eq.) were added successively at 0 °C. After 10
mn, a solution of hydroxylamine (2 eq.) in MeOH (10 ml) was added
and the reaction mixture was warm up to room temperature and let
overnight. The reaction mixture was concentrated. The residue was
taken up with EtOAc and the organic phase was washed with a satu-
rated solution of NaHCO3 and brine, dried over Na2SO4, filtrated and
concentrated. The crude products were purified by flash chromato-
graphy to afford the hydroxamate. The characterization data of com-
pound ST3 was reported in a previous work (Reddy et al., 2010), and
the characterization data of compounds 345, 349–351, 360–363 were
given a follows and NMR spectra can be found in Supplementary data
(S1).
novel mechanisms of action that kill intracellular parasites of humans,
are high priorities. Small molecules that act on epigenetic regulatory
proteins are of increasing interest as chemical tools for dissecting fun-
damental mechanisms of parasite growth and as new drug leads.
Histone deacetylases (HDACs) play key roles in diverse intracellular
processes and epigenetic regulation through the modification of histone
and nonhistone proteins to repress transcription. In human cells, 18
HDACs have been identified (Mariadason, 2008) and classified ac-
cording to their dependency on either zinc or NAD+as the co-factor
and to their sequence homology to yeast proteins (Xu et al., 2007). The
first evidence of the potential of HDACs as targets for antiparasitic drug
development came with the discovery of apicidin, a fungal metabolite
that exhibits nanomolar HDAC inhibitor (HDACi) activity and high
anti-Plasmodium falciparum activity (Darkin-Rattray et al., 1996). Ac-
cumulating evidence has indicated that zinc- or NAD-dependent HDACs
are promising drug targets in a wide variety of parasitic diseases, in-
cluding Schistosomiasis, malaria, leishmaniasis, trypanosomiasis and
toxoplasmosis (Mai et al., 2004; Saksouk et al., 2005; Sereno et al.,
2005; Vergnes et al., 2005; Strobl et al., 2007; Andrews et al., 2012;
Soares et al., 2012; Stolfa et al., 2014; Carrillo et al., 2015; Engel et al.,
2015; Campo, 2016; Chua et al., 2016) and reviewed by Hailu et al.
(2017).
In this study, we synthesized and ascertained the antiparasitic ac-
tivity, cytotoxicity and HDACi activity of a series of aminophenylhy-
droxamates and aminobenzylhydroxamates derived from an initial
alpha-hydroxyacetone compound (ST3) that have demonstrated an in
vitro antileishmanial activity associated with a moderate cytoxicity
(Sereno et al., 2008).
2.1.1. 4-{[(3-fluorophenyl)formamido]methyl}-N-hydroxybenzamide
(345)
1H NMR (DMSO-d6) δ 11.17 (s, 1H), 9.19 (t, J = 4.0 Hz, 1H), 9.00
(s, 1H), 7.70 (m, 4H), 7.56 (m, 1H), 7.39 (m, 3H), 4.51 (d, J = 4.0 Hz,
2H). HPLC purity 99%. MS ESI+H+ calc. 289.28 exp. 289.10.
2.1.2. 4-(3-fluorobenzamido)-N-hydroxybenzamide (349)
1H NMR (DMSO-d6) δ 10.40 (s br, 1H), 7.75 (m, 6H), 7.58 (m, 1H),
7.45 (m, 1H).
2. Materials and methods
2.1. General procedures for the synthesis of aminophenylhydroxamate and
aminobenzylhydroxamate derivatives
HPLC purity 98%. MS ESI+H+ calc. 275.25 exp. 275.10.
2.1.3. 4-(3-fluorobenzenesulfonamidomethyl)-N-hydroxybenzamide (350)
1H NMR (DMSO-d6) δ 11.17 (s, 1H), 9.02 (s, 1H), 8.37 (s, 1H), 7.60
(m, 4H), 7.50 (m, 2H), 7.29 (d, J = 5.6 Hz, 2H), 4.07 (s, 2H). HPLC
purity 97%. MS ESI+H+ calc. 325.30 exp. 325.10.
A series of aminophenylhydroxamates and aminobenzylhydrox-
amates were designed from an initial benzohydroxamate compound
(ST3) (Sereno et al., 2008, 2014). Synthesis of compound ST3, amino-
phenylhydroxamate, and aminobenzylhydroxamate derivatives was
performed as previously described (Reddy et al., 2010) and a synthetic
view is given in Fig. 1. Amine derivative (1 eq.) was dissolved in dry
DCM (0.2 M). Triethylamine (3 eq.) and the acid chloride or sulfonyl
chloride (1 eq.) were added successively and the reaction mixture was
heated at 40 °C. After the reaction was complete (TLC control), the
reaction mixture was concentrated. The residue was taken up with
EtOAc and the organic phase was washed with a solution of 1 M HCl, a
saturated solution of NaHCO3 and brine, dried over Na2SO4, filtrated
and concentrated to afford the amide or sulfonamide derivatives. The
2.1.4. 4-(3-fluorobenzenesulfonamido)-N-hydroxybenzamide (351)
1H NMR (DMSO-d6) δ 11.07 (s, 1H), 10.75 (s, 1H), 8.95 (s, 1H),
7.55 (m, 5H), 7.48 (m, 1H), 7.15 (d, J = 5.8 Hz, 2H). HPLC purity 99%.
MS ESI+H+ calc. 311.31 exp. 311.10.
2.1.5. N-{[4-hydroxycarbamoyl)phenyl]methyl}-4-(2-methyl-1,3-thiazol-
4-yl)benzamide (360)
1H NMR (DMSO-d6) δ 11.18 (s, 1H), 9.13 (t, J = 4.0 Hz, 1H), 9.01
Fig. 1. Synthetic routes of aminophenylhydroxamate and aminobenzylhydroxamate derivatives. Reagents and conditions: (a) R-Y-Cl, NEt3, DCM; (b) LiOH, THF/H2O 40 °C; (c) (i)
Cl-CO2Et, NMM, DMF, (ii) NH2OH, MeOH.
60

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IJP:DrugsandDrugResistance8(2018)59–66
(s, 1H), 8.09 (s, 1H), 7.98 (d, J = Hz, 2H), 7.95 (d, J = Hz, 2H), 7.70 (d,
J = 5.8 Hz, 2H), 7.38 (d, J = 5.8 Hz, 2H), 4.52 (d, J = 4.0 Hz, 2H),
2.73 (s, 3H). HPLC purity 96%. MS ESI+H+ calc. 368.43 exp. 368.10.
medium supplemented with 10% FCS, 2 mM GlutaMAX, 100 U/ml
penicillin, and 100 μg/ml streptomycin. HFF cells were cultivated in
Dulbecco's modified Eagle's medium (DMEM) with Earle's salts con-
taining 10% FCS, 10 mM HEPES, 1 mM sodium pyruvate, 2 mM
GlutaMAX, 100 U/ml penicillin, and 100 μg/ml streptomycin. The THP-
1 and HFF cells were maintained in a 5% CO2 incubator at 37 °C.
Cytotoxicity was evaluated via the MTT assay, and the IC50 values were
calculated using Prism software (Prism 4 for Mac OS X, version 5.0b,
December, 2008).
2.1.6. N-[4-hydroxycarbamoyl)phenyl]-4-(2-methyl-1,3-thiazol-4-yl)
benzamide (361)
1H NMR (DMSO-d6) δ 11.13 (s, 1H), 10.46 (s, 1H), 8.95 (s, 1H),
8.14 (s, 1H), 8.10 (d, J = 4.2 Hz, 2H), 8.03 (d, J = 4.2 Hz, 2H), 7.87 (d,
J = 4.4 Hz, 2H), 7.76 (d, J = 4.4 Hz, 2H), 2.75 (s, 3H).
HPLC purity 99%. MS ESI+H+ calc. 354.40 exp. 354.10.
2.3. HDAC assays
2.1.7. N-hydroxy-4-{[2-(3-methoxyphenyl)acetamido]methyl}benzamide
(362)
The HDACi effect against HeLa nuclear extracts and recombinant
proteins were measured using a fluorometric HDAC assay kit (Active
Motif, Belgium) according to the manufacturer's instructions. Briefly,
30 μL of HeLa nuclear extract (Active Motif, Belgium) or various HDAC
recombinant proteins (Active Motif, Belgium) was mixed with 5 μL of a
10 × compound and 10 μL of assay buffer. A negative control con-
taining trichostatin A at a final concentration of 2 μM was analyzed for
each independent experiment. A fluorogenic substrate (10 μL) was
added, and the reaction was allowed to proceed for 30 min at room
temperature and then stopped by the addition of a developer containing
trichostatin A. The fluorescence was monitored after 30 min at excita-
tion and emission wavelengths of 360 and 460 nm, respectively.
HDAC activity was determined from total protein extracted from T.
gondii. Confluent cell cultures (T-175 cm2) were infected with 5 × 107
tachyzoïtes in DMEM supplemented with 1% (v/v) FCS. Tachyzoïtes
were harvested after 72 h, resuspended in RIPA buffer (0.1 M PBS, pH
7.2, 0.5% deoxycholate, 0.1% SDS, and 0.5% Nonidet P-40) and in-
cubated for 30 min at 4 °C. After centrifugation at 13000 rpm for
30 min at 4 °C, the supernatant was collected, adjusted to a total protein
concentration of 312 μg/ml and then used for determining the HDAC
activity as described above.
1H NMR (DMSO-d6) δ 11.17 (s, 1H), 8.99 (s, 1H), 8.58 (t,
J = 4.0 Hz, 1H), 7.67 (d, J = 5.4 Hz, 2H), 7.29 (d, J = 5.4 Hz, 2H), 7.19
(t, J = 5.8 Hz, 1H), 6.80 (m, 3H), 4.30 (d, J = 4.0 Hz, 2H), 3.73 (s, 3H),
3.45 (s, 2H). HPLC purity 99%. MS ESI+H+ calc. 315.34 exp. 315.10.
2.1.8. N-hydroxy-4-[2-(3-methoxyphenyl)acetamido]benzamide (363)
1H NMR (DMSO-d6) δ 11.09 (s, 1H), 10.35 (s, 1H), 8.93 (s, 1H),
7.70 (d, J = 5.8 Hz, 2H), 7.64 (d, J = 5.8 Hz, 2H), 7.22 (t, J = 5.4 Hz,
1H), 6.89 (m, 2H), 6.82 (d, J = 4.8 Hz, 1H), 3.74 (s, 3H), 3.63 (s, 2H).
HPLC purity 99%. MS ESI+H+ calc. 301.32 exp. 301.10.
2.2. Antiparasitic activity and cytotoxicity
The antitrypanosomatid activity of the compounds was ascertained
as follows. Leishmania parasites were cultured at 26 °C in SDM-79
medium supplemented with 10% fetal calf serum (FCS), and the antil-
eishmanial activity was determined according to a previously published
protocol for LUC-expressing and wild-type Leishmania (Sereno et al.,
2001; Abbassi et al., 2008). Trypanosoma brucei gambiense were grown
at 26 °C in Cunningham's medium supplemented with 10% FCS, 2 mM
GlutaMAX-1 (Gibco, USA), 100 U/ml penicillin, 100 μg/ml strepto-
mycin and 20 mg/ml bovine hemin. The epimastigote form of Trypa-
nosoma cruzi (strain Y cl7 scl2, lineage Tc2) (kindly provided by Phi-
lippe Truc and Christian Barnabé, UMR 177 IRD-CIRAD InterTryp,
Montpellier, France) were grown at 26 °C in liver infusion tryptose (LIT)
medium, supplemented with 10% FCS, 2 mM GlutaMAX™-I, 100 U/ml
penicillin, 100 μg/ml streptomycin and bovine hemin (20 mg/l)
(Gibco). The antitrypanosomatids activity was determined using a
protocol that was initialy set up for wild-type Leishmania (Abbassi et al.,
2008). To assess the drug activity on Toxoplasma gondii, confluent
human foreskin fibroblast (HFF) monolayers were infected with YFP-
type I Rh kindly provided by B. Striepen, see (Striepen et al., 1998) or
Tomato-type II Pru kindly provided by N. Blanchard (Toulouse), ex-
pressing parasites at 5 × 104 per well, which were then centrifuged for
30 s at 1300 rpm and incubated for 30 min in a water bath at 37 °C to
allow invasion. The wells were then washed three times with PBS to
eliminate extracellular parasites, and the drugs were added at various
concentrations ranging from 1 to 50 μM. After incubation for 24 h at
37 °C in a humidified atmosphere containing 5% CO2, the cells were
fixed in 2.5% methanol-free formaldehyde (Tebu-bio)/PBS for
30 min at room temperature. Nuclei were stained with Hoechst 33258
(2 μg/ml, Molecular Probes) for 20 min and then washed three times
with water for 10 min each time. The number of infected cells, i.e., cells
harboring a parasitophorous vacuole, and the number of parasites per
vacuole were determined using an Olympus ScanR microscope ( × 20
objective) and ScanR software. The parasitic index in % compared to
the control was calculated as follows: (PI=((number of parasite/
100 cells in treated wells) x (% of infected macrophage in treated well)/
(number of parasite/100 cells in untreated wells) x (% of infected
macrophage in untreated wells))*100). Then, the 50% inhibitory con-
centration (IC50) was calculated using Prism software (Prism 4 for Mac
OS X, version 5.0b, December, 2008).
2.4. Comparative modeling and ligand docking
Protein sequences were recovered from the appropriate databases
for two toxoplasmal strains GT1 and Me49 as representatives of type I
and type II T. gondii (http://toxodb.org/) and from UNIPROT for the
leishmanial and human enzymes. Unfortunately the leishmanial en-
zymes provided poor sequence-structure alignment precluding further
analysis. Meanwhile, the sequences from both strains, GT1 and ME49
revealed similar trends, so the docking study was performed on three
GT1 sequences only. The best results were obtained for three sequences
from T. gondii (A0A125YPH2, S7UUG9 and S7W8W1, T. gondii GT1) but
not for two other sequences (S7UW66 and S7UZC5). The latter showed
only partial alignment and significant sequence divergence with known
crystal structures of HDACs. For those two sequences, the truncated
structural alignment did not allow complete modeling of the active site,
which prevented further ligand docking studies. Otherwise, for the
three other sequences, active site boundaries in each structural model
was deduced as the vicinity of the co-crystallized ligand (or hydro-
xamate compounds transferred from related HDACs by protein-protein
superposition) using @TOME-2 comparative option. The same chemical
entities served, in addition, to define a shape restraint to guide the
docking, in absence of the zinc atom, in the automatically computed
models.
All the results for the HDACs from T. gondii (GT1 and Me49 strains)
as well as for Leishmania infantum and human HDACs, can be found on
the following web pages (the links therein):
http://atome3.cbs.cnrs.fr/AT23/DB/HDAC_TOXGG.html
http://atome3.cbs.cnrs.fr/AT23/DB/HDAC_TOXGM.html
http://atome3.cbs.cnrs.fr/AT23/DB/HDAC_LEIIN.html
http://atome3.cbs.cnrs.fr/AT23/DB/HDAC_HUMAN.html
The human monocytic cell line THP-1 was cultivated in RPMI
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IJP:DrugsandDrugResistance8(2018)59–66
Fig. 2. HDACi screen and antiparasitic and cytotoxicity activities correlation. Screen for anti-HDAC activity (A). HDAC activity of HeLa nuclear extracts and of recombinant HDAC
1, 3, 6, and 8 proteins was a certained in the presence of 1.0 μM of each of the 8 synthesized compounds, as well as of SAHA. The experiments were carried out in triplicate, and the results
are expressed as the percentage of deacetylase activity compared to the untreated control. Inhibition of T. gondii deacetylase activity by SAHA and the compound 363 (B). The total
protein extract of T. gondii tachyzoites were prepared as described in the Materials and Methods section. The deacetylase activity was determined after the addition of 0.125 μM of
compound 363 or of SAHA. The results are expressed as the percentage of deacetylase activity compared to untreated T. gondii protein extracts. The results are the mean of 4 experiments,
carried out in triplicate. Correlation between HDACi and anti T. gondii activity or cytotoxicity potency (C).
3. Results and discussion
amine and acid chloride or sulfonyl chloride in the presence of tri-
methylamine afforded the amide or sulfonamide intermediates, which
subsequently reacted with lithium hydroxide to obtain corresponding
acids intermediates, then the acid are activated by reaction with ethyl
The synthetic routes of aminophenylhydroxamate and amino-
benzylhydroxamate derivatives are summarized in Fig. 1. Reaction of
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IJP:DrugsandDrugResistance8(2018)59–66
Table 2
data, we used a well-known HDACi, vorinostat (SAHA). We obtained an
IC50 for vorinostat in the micromolar range (2 μM) (see Table 1), which
agrees with the literature data on this molecule (1.3 μM) (Jiao et al.,
2009). For all the other compounds, the determined IC50 ranges from
more than 1.8 μM for the less active compounds (ST3, 350 and 361) to
0.085 μM for the more active compound (363). This latter inhibitory
concentration is lower than the IC50 that we determined for SAHA and
more than 20-fold lower than those of well-known HDACis. To further
characterize the HDACi effect of our series of compounds, we first as-
certained the inhibitory effect of all the compounds at a single con-
centration of 1.0 μM against HeLa nuclear extract and recombinant
HDAC1, 3, 6, and 8. As shown in Fig. 2A, the sole compound that ex-
hibited a strong HDACi effect at 1.0 μM is compound 363 (90% in-
hibition) (Fig. 2A). We further investigated the specificity of 363
against a set of recombinant HDAC enzymes (HDAC1, 3, 6, and 8)
(Table 2) and found that 363 is more potent for HDAC6 (Class II HDAC)
as compared to the other HDACs, that belong to the class I. We then
assessed the effect of 363 on T. gondii HDAC activity. As shown in
Fig. 2B, at 0.125 μM, compound 363 exerted a strong inhibitory effect
on the deacetylase activity measured in the total protein extract of T.
gondii. Compound 363 inhibited 44% of the T. gondii deacetylase ac-
tivity, whereas SAHA inhibited only 22% of this activity (Fig. 2B). The
compound 363 is structurally related to the HDAC6 inhibitor Nex-
turastat or BRD73954 (Wagner et al., 2013; Vreese and D’hooge, 2017)
and it will be interesting to ascertain Nexutarastat's anti T. gondii
pontency in the future.
Among the 8 compounds, those with the strongest anti-Toxoplasma
activity, i.e IC50 < 50 μM (345, 350, 360, 363), are among the best
HDACis of the series. A high correlation between HDACi and T. gondii
activity is recorded (R2 = 0.9950). In addition, their cytotoxic activity
against HFF and THP-1 correlates also well with their HDACi
(R2 = 0.9000 and R2 = 0.9933) respectively (Fig. 2C). But the corre-
lations appear not to be obvious when compounds with low anti T.
gondii activity IC50 > 50 μM (ST3, 349, 351, 352) were included in the
analysis, R2 = 0.3758, 0.4437 and 0.6667 for T. gondii, HFF and THP-1
respectively (see Supplementary data S2). Therefore, improved anti-T.
gondii potency of the series is largely dependent on potent HDACi ac-
tivity. However, since three compounds exhibit improved HDACi ac-
tivity that does not lead to improved anti-T. gondii activity it is likely
that other factors are also involved in the modulation of their cyto-
toxicity and anti T. gondii potency. Among them, the capacity of each
compound to reach their intracellular targets and/or to readily inhibit
HDAC activity might play a role. The THP-1 cell line, which was derived
from the peripheral blood of a 1-year-old human male with acute
monocytic leukemia, is known to be highly susceptible towards hy-
droxamate derivatives (Sung et al., 2010), and HDACis are known to
exhibit relatively selective cytotoxicity against growing malignant cells
compared to healthy ones (Popovic and Licht, 2012). Non-malignant
cells such as HFFs are less susceptible to the synthesized compounds.
HDAC inhibitory effect of the compound 363 as compared to SAHA.
rHDAC
363
SAHA
Fold
IC50 μM
IC50 μM
HDAC 1
HDAC 3
HDAC 6
HDAC 8
0.257
0.160
0.045
0.863
0.050
0.052
0.015
0.045
0.627
0.170
1.573
0.230
0.039
0.014
0.800
0.130
2.43
1.06
34.9
0.26
chloroformate in the presence of N-methyl-morpholine, the inter-
mediates react with hydroxylamine to afforded corresponding target
compounds 345, 349–351, 360–363.
The compound ST3, a benzohydroxamate, exert antileishmanial
activity against promastigote forms of L. infantum and L. braziliensis in
the range of 20 μM but is less potent against L. tropica (Table 1). In
addition, this compound is also active against T. brucei but do not dis-
play anti-T. cruzi activity (IC50 > 100 μM). It is moderately cytotoxic
for THP-1 cell line or for HFF, with a determined cytotoxicity above
250 μM (Table 1). Nevertheless when tested against the intracellular
form of Leishmania it discloses a low antiparasitic activity (IC50 of
140 μM and 111 μM against L. braziliensis and L. infantum respectively).
As the calculated index of selectivity for ST3 is of about 5.4, this small
compound (MW: 138) was considered as an interesting lead from which
a series of derivative targeting trypanosomatid parasites was developed.
Unexpectedly, almost all the aminophenylhydroxamates and amino-
benzylhydroxamates synthetized exert low or no antitrypanosomatid
activity (Table 1), irrespective of the parasite tested (T. brucei or
Leishmania spp.). The sole exception is the compound named 345,
which exhibited moderate antileishmanial activity in the range of
15–50 μM, L. braziliensis being the most susceptible Leishmania species.
Nevertheless, this compound is highly cytotoxic to THP-1 and HFF cells.
We further evaluated the capacity of ST3 and all aminopheylhy-
droxamate and aminobenzylhydroxamates synthetized to act against
Toxoplasma gondii, an intracellular parasite belonging to Phyllum
Apicomplexa. For T. gondii, the IC50 of ST3 is above 50 μM. The 8
compounds synthesized express IC50 values ranging from more than
60 μM to less than 1 μM (Table 1). Of the 8 derivatives synthesized, 3
strongly inhibited the proliferation of a type I T. gondii strain at con-
centrations below 10 μM. These 3 compounds were 345, 360 and 363,
with IC50 values of 5.0 μM, 4.0 μM, and 0.35 μM, respectively (Table 1).
The capacity of 363 to inhibit a type II strain of T. gondii was found to
be of 2.25 μM (see Table 1#). Among all the aminophenylhydroxamates
and aminobenzylhydroxamates synthetized, the compound 363 ex-
hibited a high selectivity for the intracellular proliferative stage of T.
gondii (Table 1), with calculated selectivity indexes of 300 against HFF
and of 10 for THP-1 cells.
In a HeLa cell nuclear extract, HDAC 1, 2, 6, and 8 are the pre-
dominant HDACs. To compare our results with previously reported
Fig. 3. Docking of two inhibitors in the models of two
TgHDACs. Model of S7W8W1 from T. gondii GT1 in
complex with ligand 363 (A). Model of A0A125YPH2
from T. gondii GT1 in complex with ligand 350 (B). Both
models were deduced automatically using the server
@TOME-2 and ligands were docked using PLANTS and a
shape restraint extracted from a co-crystallized ligand. The
putative binding site of the zinc atom is indicated by the
symbol Zn. The chelating sidechains are shown as wire-
frame. Similarly the two phenylalanine and the aspartate
residues interacting with the benzohydroxamate inhibitors
are shown (discussed in the text). Only one hydrogen bond
(black dashed line) is predicted and shown for each com-
pound beside the interactions involving the hydroxamate
group which are not shown for clarity. The zinc atom is
shown as a grey sphere. The figure was drawn using pymol
(www.pymol.org).
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IJP:DrugsandDrugResistance8(2018)59–66
Nevertheless, to gather more information on the selectivity of 363, we
investigated its cytotoxicity in the HEPG2 cell line, which is considered
a model system for studies on liver metabolism and xenobiotic toxicity.
Compound 363 is mildly cytotoxic for HEPG2 cells, with an IC50 of
greater than 15 μM. The calculated index of selectivity of 42 confirms
the high selectivity of this compound for T. gondii tachyzoites.
Among the compounds synthesized, those expressing strong anti-
Toxoplasma activity are among the best HDACis of the series, particu-
larly compound 363, the most effective HDACi with high selectivity for
Toxoplasma. The IC50 of 363 is equivalent, in terms of in vitro efficiency,
to the IC50 pyrimethamine the classical anti-Toxoplasma drug (IC50 of
0.35 for 363 and 0.4 μM for pyrimethamine) (Butler et al., 2013). We
propose that compound 363 be considered as a drug-like compound,
whose capacity to act against T. gondii infection in vivo must now be
probed.
The currently identified HDACi-based anti-T. gondii compounds
target class I HDACs. Among the 5 HDAC-encoding genes in the genome
of the GT1 strain of T. gondii (www.toxodb.org/), 3 are class I HDACs,
one is a class IV HDAC, and 1 is a class II HDAC. Fold recognition using
the server @TOME-2 (Pons and Labesse, 2009) was used to yield se-
quence-structure alignments with well-characterized HDACs and to
derive models of the parasite enzymes. First, it highlighted strong si-
milarities for two Toxoplasma gondii TgHDACs with Homo sapiens
HsHDAC2 (with 56% and 64% of sequence identity over ∼360 re-
sidues). Two other TgHDACs showed weaker similarities with bacterial
HDACs (40% identity), being far more distant to the Homo sapiens
HsHDADC6 (∼20% identity). The last one appeared to encode only a
partial HDAC fold and might correspond to a pseudogene. Taking ad-
vantage of the comparative docking implemented in @TOME-2, active
site delimitations and pharmacophoric shape restraints could be de-
fined to allow focused screening performed using PLANTS (Korb et al.,
2007). The docking of the lead compound ST3 was rather straightfor-
ward. It perfectly matches the binding mode of the phenyl-hydroxamate
moiety of Nexturastat as observed in HDAC6 from Danio rerio
(PDB5G0J) (Miyake et al., 2016). Satisfactory docking poses for most
the other benzohydroxamates were also obtained for the three targets
we could model. In all cases, the common aromatic phenyl moiety
showed favorable interactions with two aromatic side-chains (mainly
phenylalanines) lying at the entrance of the active site (Fig. 3). A
neighboring aspartate which is also conserved at the edge of the active
site is pointing toward the amide/sulfonamide groups present in the
chemical series derived from ST3, and accordingly it may (or may not)
form a favorable hydrogen bond with the incoming ligand. This could
explained the observed change in affinity depending on the position of
the amide/sulfonamide relative to the phenyl-hydroxamate moiety. In
the case of the leishmanial HDACs, significantly lower similarities were
detected (25–40% sequence identity) with more important insertion/
deletions. This more pronounced divergence precluded proper mod-
eling of the active site entrance and ligand docking. But this divergence
could potentially explain the detrimental impact of the substitutions we
designed on our lead compound. All these targets deserve further
characterization to guide the design of more potent compounds.
For the first time, we have demonstrated that a potent inhibitor of
the mammalian class IIb of HDACs (HDAC6) is effective as an anti-T.
gondii compound. The molecular target now needs to be further char-
acterized in T. gondii. Unexpectedly, among the 8 derivatives synthe-
sized there was minimal efficacy against Trypanasomes and only one
compound with demonstrated antileishmanial activity (345), but this
compound also exhibited high in vitro cytotoxicity. In order to address
whether these HDAC inhibitors can be developed to target intracellular
parasites more broadly, we will synthesize new compounds from the
ST3 and or the 345 molecules in order to improve their efficacy against
Leishmania parasites and decrease their cytotoxicity.
Acknowledgements
We thank the SCIMI Microbiology-Imaging-Microscopy Facility of
the TIMC IMAG University Grenoble Alpes for access to instruments and
technical advices. This work was supported by the IRD (821849H) and
Univ. Grenoble (Idex innovation grant Detox) as well as by a grant
(ANR-10-BINF-0003). We thank Mme Garcia Deborah (IRD) and Nelly
Boeckler for their technical assistance.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://dx.
doi.org/10.1016/j.ijpddr.2018.01.002.
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