Hydroxamic Acid-Based Histone Deacetylase (HDAC) inhibitors bearing a pyrazole scaffold and a cinnamoyl linker

Article abstract of DOI:10.3390/ijms20040945

Genetic abnormalities have been conventionally considered as hallmarks of cancer. However, recent studies have demonstrated that epigenetic mechanisms are also implicated in the insurgence and development of cancer. Patterns of the epigenetic component in

Full text of DOI:10.3390/ijms20040945

International Journal of
Molecular Sciences
Article
Hydroxamic Acid-Based Histone Deacetylase (HDAC)
Inhibitors Bearing a Pyrazole Scaffold and a
Cinnamoyl Linker
Chiara Zagni 1, Andrea Citarella 2, Mahjoub Oussama 3, Antonio Rescifina 1,4
,
Alessandro Maugeri 2, Michele Navarra 2 , Angela Scala 2 , Anna Piperno 2 and
Nicola Micale 2,
*
1
Department of Drug Sciences, University of Catania, Viale A. Doria 6, 95125 Catania, Italy;
chiarazagni@gmail.com (C.Z.); arescifina@unict.it (A.R.)
2
Department of Chemical, Biological, Pharmaceutical, and Environmental Sciences, University of Messina,
Viale F. Stagno D’Alcontres 31, 98166 Messina, Italy; acitarella@unime.it (A.C.); amaugeri@unime.it (A.M.);
mnavarra@unime.it (M.N.); ascala@unime.it (A.S.); apiperno@unime.it (A.P.)
Higher Institute of Applied Sciences and Technology of Mahdia, University of Monastir, Sidi Massa-oud,
Hiboun Mahdia Tunisian 5100, Tunisia; oussamamahjoub1993@gmail.com
3
4
Consorzio Interuniversitario Nazionale di ricerca in Metodologie e Processi Innovativi di
Sintesi (C.I.N.M.P.S.), Via E. Orabona, 4, 70125 Bari, Italy
*
Correspondence: nmicale@unime.it; Tel.: +39-090-676-6419
ꢀꢁꢂꢀꢃꢄꢅꢆꢇ
ꢀꢁꢂꢃꢄꢅꢆ
Received: 31 January 2019; Accepted: 18 February 2019; Published: 21 February 2019
Abstract: Genetic abnormalities have been conventionally considered as hallmarks of cancer.
However, recent studies have demonstrated that epigenetic mechanisms are also implicated
in the insurgence and development of cancer. Patterns of the epigenetic component include
DNA methylation and histone modifications. Acetylation of histones is controlled by histone
acetyltransferases (HATs) and histone deacetylases (HDACs). Imbalance of these two enzymatic
systems is known to be a key factor in tumor progression. Because HDACs have been found to
function incorrectly in cancer, various HDAC inhibitors (HDACIs) are being investigated to act as
cancer chemotherapeutics. Herein, we report the synthesis, docking studies and biological activity of
a series of hydroxamic acid-based HDACIs bearing an N1-aryl or N1-H pyrazole nucleus as surface
recognition motif and a cinnamoyl group as a linker to the hydroxamic acid zinc-binding group (ZBG).
Some of the tested compounds exhibited inhibitory properties towards HDACs and antiproliferative
activity against neuroblastoma SH-SY5Y tumor cell line both at micromolar concentrations.
Keywords:
HDAC inhibitors; hydroxamic acid;
N1-aryl-pyrazole;
N1-H-pyrazole;
antiproliferative activity
1. Introduction
The incidence of malignancies has dramatically risen in recent years. According to the recent
report (2018) of the World Health Organization, cancer is currently the second leading cause of
death worldwide. Therefore, the identification and improvement of new therapeutic strategies are
urgently needed [1]. In this regard, it is well known the role of various histone deacetylase (HDAC)
isoforms which act as epigenetic modulators of gene transcription and whose inhibition is crucial
to identify novel anticancer agents. The epigenetic process is in large part coordinated through the
control of the chromatin structure, and it is highly regulated by post-translational modifications of
histones, around which nuclear DNA is wrapped, and the DNA itself. In particular, it has been shown
that hyperacetylation of histones leads to transcriptional activation of suppressed genes, whereas
Int. J. Mol. Sci. 2019, 20, 945; doi:10.3390/ijms20040945
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hypoacetylation is correlated with reduced gene expression [
2]. Imbalance of the activity of both
histone acetyltransferase (HAT) and HDAC is strictly related to the insurgency and progression of
tumors. Therefore, these epigenetic markers have been the focus of drug discovery during the last
decade. In particular, histone deacetylase inhibitors (HDACIs) represent a promising new class of
compounds for the treatment of cancer [3–5].
However, there are no selective HDACIs approved for clinical use in oncology, albeit some of them
reached Phase I or early Phase II trials [6]. The US FDA has approved only a few pan-HDACIs, and
most of them are hydroxamic acid-based compounds which retrace the classic pharmacophoric model
of these type of enzyme inhibitors consisting in: (i) a terminal metal-binding moiety (i.e., hydroxamic
acid; ZBG) that coordinates the cation Zn2+ within the HDAC active site; (ii) a hydrophobic capping
group that interacts with the external domain of the enzyme and regulates the interaction between
the HDAC and substrate; (iii) a linker (generally a chain of six methylene groups) that sets the
metal-binding moiety and the capping group for interactions within the binding site [7]. (Figure 1).
Figure 1. Pharmacophoric model of the designed histone deacetylases inhibitors (HDACIs).
On the basis of these fruitful features, we report herein the synthesis of two sets of new potential
HDACIs in which the hydroxamic acid moiety was maintained as a steady point for the zinc-binding.
The cinnamoyl group was used to resemble the classical six methylene groups linker, and a pyrazole
scaffold (joined to the linker throught an amide connection unit) was elected as capping group to
achieve potency and possibly selectivity for the different HDAC isoforms. The cinnamoyl hydroxamic
acid is a well-known and fruitfully exploited chemical fragment for HDACs inhibition [8–13]. Indeed,
some cinnamoyl hydroxamates such as Belinostat (peripheral T-cell lymphoma) and Panobinostat
(multiple myeloma) have been already approved by the FDA for the anticancer combination therapy.
On the other hand, the pyrazole scaffold has been recently reassessed as a valid surface recognition
motif [14,15].
2. Results and Discussion
2.1. Chemistry
The first synthetic strategy (Figure 2 and Scheme S1) was intended to obtain an
N1-aryl-pyrazole capping group and entails the condensation of different aryl-hydrazines with ethyl
2,4-dioxovalerate [16]. The resulting N1-aryl-pyrazole scaffolds bearing an ester moiety at C3 or C5
(two isomers were isolated because the condensation is not regiospecific. See Supporting Information
for details) were conjugated after hydrolysis with the cinnamoyl linker using an amide moiety under
standard coupling conditions. Finally, the terminal ester moiety of each adduct capping group/linker
was converted into the corresponding hydroxamic acid moiety by a three-step procedure (Figure 2 and
Scheme S1) [17].

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Figure 2. Design of the first set of HDACIs (N1-aryl-pyrazole derivatives). For synthetic details, see Scheme S1.
The second synthetic strategy (Figure 3 and Schemes S2 and S3) was designed to obtain the
N1-H pyrazole derivatives substituted at (C4)C5 with an aryl group, and entails the formation
of α,γ-diketoesters by Claisen condensation of various aryl methyl ketones with diethyl malonate
followed by cyclization with hydrazine [18]. The desired new pyrazole capping groups were then
transformed into the final compounds according to the previous synthetic methodology. The structures
of new final compounds, as well as of each intermediate, were determined by analytical and
spectroscopic data (see Supplementary Materials).
Figure 3. Design of the second set of HDACIs (aryl-substituted N1-H-pyrazole derivatives).
For synthetic details, see Schemes S2 and S3.
2.2. Biological Evaluation
The N1-phenylpyrazole derivatives 1a (the isomer with the connection unit at C5) and 1b (the
isomer with the connection unit at C3) were evaluated for their ability to inhibit the HDAC activity to
have a preliminary insight about the SAR of this set of compounds. The commercially available assay
kit HDAC Inhibitor Drug Screening Kit (Biovision) was employed for this test and Trichostatin A (TSA),
which is a natural pan-HDACI, was used as positive control. As can be seen from the data reported in
Table 1, only the derivative 1a turned out to be active (IC50 = 7.1 µM). Therefore, the development of

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the other two derivatives of this class, i.e., the p-CN-phenyl derivative
2
and the p-Br-phenyl derivative
3
, was focused on the single isomer C5-substituted. As for 1a, the detected inhibitory activity of 2
and 3 was in the low micromolar range (6.3 µM and 1.6 µM, respectively; Table 1). Regarding the set
of N1-H pyrazole derivatives, only the indane derivative 6 displayed fair HDAC inhibitory activity
(IC50 = 46.5 µM), whereas the p-Br-phenyl derivative 5 turned out to be inactive (IC50 > 500
µM) and
the phenyl-derivative 4 showed only a modest inhibition on the intended target (IC50 = 155.2 µM).
Table 1. HDAC inhibitory activity of compounds
1–6, Trichostatin A (TSA) and suberoylanilide
hydroxamic acid (SAHA) and calculated ∆Gbind and the corresponding Ki.
IC50 ±SD b
(µM)
Inhibition at 10 µM
Inhibition at 500 µM
Calcd Ki
(µM)
c
Compd.
Calcd ∆Gbind
(%) a
(%) a
1a
1b
2
3
4
5
—
6
86
30
88
88
71
15
87
—
—
7.1 ± 0.5
−7.96
−6.04
−8.08
−8.79
−6.40
−5.53
−7.27
−11.13
−10.03
1.45
37.19
1.19
>500
51
74
13
—
29
—
—
6.3 ± 0.3
1.6 ± 0.2
155.2 ± 2.8
>500
46.5 ± 1.5
3.4 ± 0.2
—
0.36
20.25
87.99
4.69
0.007
0.044
6
TSA d
SAHA d
a
Standard deviations (SD) for the percent inhibition (mean of 3 independent measurements) were always <10%.
b
c
d
SD values. kcal/mol. Experimental Ki = 45 and 480 nM for TSA and SAHA, respectively [19].
Finally, the compounds which turned out to be the most active in the enzyme assay,
i.e., the N1-aryl-pyrazole derivatives 1a
and , were also tested in vitro by the MTT
[3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide] test to evaluate their antiproliferative
activity against the neuroblastoma SH-SY5Y tumor cell line [20 21]. The treatment of the SH-SY5Y
cells with growing concentrations (1–50 M) of 1a (Figure 4A) and (Figure 4C) significantly reduced
cell proliferation, both reaching more than 75% of inhibition at 25 M after 72 h (**** p < 0.0001 vs.
CTRL). Compounds 1a and showed an IC50 of 5.34 and 5.61 M, respectively. None of the tested
concentrations of 2a, instead, turned out to be effective (Figure 4B).
,
2
3
,
µ
3
µ
3
µ
Figure 4. Cont.

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Figure 4. Compounds 1a
increasing concentration of synthesized compounds for 24, 48, and 72 h. No antiproliferative effect was
observed for compound 2a ). Each value is the mean S.E.M. of three experiments performed eight
(A) and 3 (C) exerted antiproliferative effect on SH-SY5Y cells exposed to
(
B
±
times. **** p < 0.0001, *** p < 0.001, ** p < 0.01, * p < 0.05 vs. CTRL.
2.3. Molecular Docking
In order to explain the different ability of the compounds 1–6 to inhibit HDAC enzymes, we
performed a docking simulation using the active site of the three-dimensional structure of the HDAC8
enzyme. In fact, except for HDAC8, functional HDACIs were found as high-molecular-weight
multiprotein complexes, and most purified recombinant HDACIs are enzymatically inactive [22].
Therefore, HDAC8 is the best model among mammalian HDACIs if we look at it from a structural
biology perspective. Thus, looking at the pose of TSA in the co-crystallized structure with the human
HDAC8 (PDB ID: 1T64) and considering that hydroxamic acid group chelates the zinc ion in a bidentate
fashion and forms hydrogen bonds with Tyr306 and Asp178 (Figure 5) we docked compounds
starting from this pose.
1–6
The molecular docking simulations of all new compounds showed that the trend of activity is
maintained along the series but with about a 10-fold underestimated value [23 25]. However, the
–
same order is maintained for the TSA and suberoylanilide hydroxamic acid (SAHA), the two most
famous HDACIs, chosen as reference compounds (Table 1) and used to validate the docking procedure.
The 3D and 2D sketch of the docked pose for the best-scored compound
3 (Figure 6) highlight that this
compound chelates the Zn2+ bidentately, as expected, whereas a hydrogen bond is established between
hydroxamic NH hydrogen and His143, while another with OH hydrogen and His142. Moreover, a
series of
π–π stacked interactions occur between the aromatic rings and Phe152, Phe208, His180, and
Tyr100; furthermore, a π-anion interaction involves the pyrazole ring and Asp101 (Figure 6).

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Figure 5. Interactions of TSA in the co-crystallized structure with the human HDAC8 (PDB ID: 1T64)
and chelation with Zn2+
.
Figure 6. 3D and 2D view of the interactions for 3 inside the binding pocket of HDAC8 (PDB ID: 1T64);
lengths in Å. The 3D plot was generated by UCSF ChimeraX (release 0.8, Resource for Biocomputing,
Visualization, and Informatics, San Francisco, CA, USA) [26]; hydrogen bonds are shown as green
dotted lines, while coordination bonds are in yellow. The 2D plot was generated by LigPlot+ (2.1,
EMBL-EBI, Wellcome Genome Campus, Hinxton, Cambridgeshire, United Kingdom) [27]; hydrogen
bonds are shown as green dotted lines, while the spoked arcs represent residues making nonbonded
contacts with the ligand.
3. Materials and Methods
3.1. Chemistry
3.1.1. General Experimental Information
All reagents used for the development of the HDACIs, including N-(3-dimethylaminopropyl)-
N0-ethyl carbodiimide hydrochloride (EDCI), 1-hydroxybenzotriazole hydrate (HOBt) and various
hydrazines, as well as all synthesis solvents and deuterated solvents employed for NMR analysis,
were purchased from Sigma-Aldrich/Fluka (Milano, Italia) and used without further purification.
NMR spectra were recorded by means of a Varian Gemini 500 MHz (1H) e 125 MHz (13C)
instrument or, alternatively, a Brüker Avance III 400 MHz (1H), using CDCl3, Acetone-d6, CD3OD, D2O
e DMSO-d6 as solvents depending on the solubility of the compounds; the chemical shifts (
δ) were
provided in ppm using TMS as internal standard and coupling constants (J) in Hertz (Hz). The patterns

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of splitting were described as singlet (s), doublet (d), double doublet (dd), triplet (t), quartet (q),
multiplet (m), and broad singlet (bs). NMR data of each intermediate of synthesis are reported only
within the Supporting Information.
Melting points of the various synthetic intermediates and final compounds were determined
using a “BUCHI Melting Point B-545 Apparatus” and are incorrect. Elemental analysis of the final
compounds was performed using a “Carlo Erba Model 1106” instrument (Elemental Analyzer for C,
H, and N) and the obtained results were in the range of ±0.4% compared with the theoretical values.
Thin layer chromatography (TLC) was performed on Merck 60 F254 plates (Merck KGaA,
Darmstadt, Germany). For the column chromatography Macherey-Nagel 60 M (0.040–0.063 mm)
silica gel was used.
3.1.2. General Procedure for the Synthesis of the N1-phenyl-pyrazole Scaffold
In a round bottom flask, 400 mg of ethyl 2,4-dioxovalerate (0.35 mL, 2.53 mmol) were dissolved in
ethanol (~40 mL) and reacted under reflux for ~3h with 1 equivalent of phenyl-hydrazine (0.25 mL,
2.53 mmol) in the presence of a catalytic amount of HClconc.
.
Then, the solvent was removed under reduced pressure, and the resulting crude product was
dissolved with brine and extracted thrice with EtOAc. The collected organic phases were dried over
anhydrous Na2SO4, filtered and evaporated under vacuum conditions to afford 655 mg of crude oil.
The latter was purified by column chromatography using a mixture of petroleum ether/ethyl acetate
8:2 as eluent to afford isomers 11a and 11b in a 1.2:1 molar ratio due to the fact that the cyclization of
the starting α,γ-diketoester with phenylhydrazine is not regiospecific.
1
From the mere H NMR analysis, it turned out to be difficult to ascribe with certainty the
stereochemistry of the two isomers; therefore, further experiments of bidimensional NMR spectroscopy
(2D NOESY) were performed before proceeding separately with the design and synthesis of the
N1-aryl-pyrazole HDACIs.
From these experiments (see Supporting Information) emerged that the only difference between
the two isomers is the detection of a long-range coupling between the –CH3 group at 2.31 ppm and the
aromatic hydrogens in the range 7.45–7.39 ppm of the phenyl ring observed for the isomer 11b, which
clearly indicates the proximity of these groups and the subsequent ascription therein reported.
3.1.3. General Procedure for the Hydrolysis of the N1-phenyl-pyrazole Scaffold
To free the carboxy group of the pyrazole derivative in such a way to allow its subsequent coupling
with the selected cinnamoyl linker, 245 mg (1.06 mmol) of 11a were dissolved in EtOH (~30 mL) and
placed in an ice bath under vigorous stirring. Then, a solution of LiOH 1N was added dropwise to
the reaction mixture, which was left to react at room temperature and continuously monitored by
TLC (EP/EtOAc 8:2). After ~5 h, the chromatographic check indicated the completion of the reaction.
Thus, the solvent was removed under reduced pressure, and the residue was solubilized in H2O and
extracted with a small amount of ethyl ether to eliminate the residual organic impurities. The aqueous
phase containing the desired compound (as Li+ salt) was acidified with HCl and re-extracted several
times with ethyl acetate. The combined organic phases were dried over anhydrous Na2SO4, filtered,
and evaporated to afford the product as yellow-brownish solid which was purified by trituration with
a mixture of ethyl ether/petroleum ether. With the same procedure was obtained the isomer 14b.
3.1.4. General Procedure for the Coupling between the N1-phenyl-pyrazole Scaffold (Cap) and the
Cinnamoyl Linker
The acid pyrazole derivative 14a (202 mg, 1.0 mmol) was placed in a flask and solubilized
in CH2Cl2 (~20 mL). The reaction mixture was cooled with an ice bath and left under vigorous
stirring for ~20 min. Then, HOBt (203 mg, 1.5 mmol), EDCI (288 mg, 1.5 mmol) and the linker (ethyl
4-amino-cinnamate, 191 mg, 1.0 mmol) were added in sequence, and the reaction mixture was left at
room temperature overnight. Work-up: The synthesis mixture was diluted with CH2Cl2 and washed

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in sequence with a solution of citric acid (10%), NaHCO3 (5%) and brine. The organic phase was dried
over anhydrous Na2SO4, filtered and removed in vacuo. The resulting crude was purified by column
chromatography (eluting solution EP/EtOAc 7:3) to afford the desired product 17a. The coupling of
the isomer 14b was performed with the same procedure, starting from 167 mg (0.83 mmol).
3.1.5. General Procedure for the Hydrolysis of the Adduct N1-phenyl-pyrazole (Cap)/
Cinnamoyl Linker
251 mg (0.67 mmol) of 17a were dissolved in THF (~30 mL) and the resulting solution was cooled
with an ice bath before adding LiOH 1N dropwise. The progress of the reaction was monitored by TLC
(disappearance of the starting material) and turned out to be completed in ~6h. Afterward, the reaction
solution was concentrated with the rotary evaporator and the residue was diluted with H2O, acidified
with HCl and extracted several times with ethyl acetate. The combined organic phases were dried
over anhydrous Na2SO4, filtered and removed under vacuum to afford a crude product which was
purified by column chromatography using petroleum ether/ethyl acetate 1:1 + 2% of formic acid as
eluent to obtain the desired product. In the same way, was performed the hydrolysis of the CAP-linker
17b starting from 195 mg (0.52 mmol).
3.1.6. General Procedure for the Synthesis of the Final Cinnamoyl Hydroxamic Acid with CAP
N1-phenyl-pyrazole (1a,1b)
In order to obtain the final hydroxamic zinc-binding domain (ZBD), the terminal group –COOH
of the adduct CAP-linker 20a (202 mg, 0.58 mmol) was joined with 1 equivalent of an O-protected
derivative of the hydroxylamine [O-(tert-butyldimethylsilyl)hydroxylamine] (86 mg, 0.58 mmol) easily
to be de-protected in mild conditions, using EDCI (167 mg, 0.87 mmol) as a coupling reagent in CH2Cl2.
The formation of the desired new adduct was detected by the TLC [Rf = 0.35 (TLC: Hexane/EtOAc 1:1)]
and quickly checked by NMR (data not shown). After the removal of the solvent, the resulting crude
product was employed as such in the next synthetic step. Thus, the crude product was reacted with a
solution of TFA (25% in CH2Cl2) at 0 ◦C for ~5h to free the hydroxamic acid moiety and eventually
the solvent was removed under vacuum. After these two consecutive synthetic steps, the pure final
product (1a) was obtained by trituration of the resulting crude product with ethyl ether.
(E)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-3-methyl-1-phenyl-1H-pyrazole-5-
carboxamide (1a); 1H NMR (500 MHz, CD3OD):
δ
ppm 7.63–7.40 (m, 10H, 9ArH + Ar–CH=), 6.79 (s,
1H, H-4), 6.40 (d, 1H, J = 15.9 Hz, =CHCONHOH), 2.36 (s, 3H, Pyr–CH3).
13C NMR (125 MHz, CD3OD):
ppm 165.0 (–CONHOH), 158.9 (–CONH), 149.1 (C5), 139.9
δ
(ArCH=), 139.4, 137.9, 131.1, 128.6, 128.0, 127.8, 126.1, 124.5, 120.2, 116.2 (C4), 108.6 (=CHCO), 13.2
(Pyr–CH3). Rf = 0.51 (TLC: EtOAc/MeOH 8:2); yellowish powder. M.p. = 103–107 ◦C. Yield: 101 mg
(48%). Anal. Calcd for C20H18N4O3: C, 66.29; H, 5.01; N, 15.46. Found: C, 66.35; H, 5.03; N, 15.50.
HRMS-EI (m/z) [M]+ calcd for C20H18N4O3 362.1379, found 362.1381.
With the same procedure was obtained the final product 1b starting from 123 mg (0.35 mmol) of
the related acid adduct 20b, 52 mg (0.35 mmol) of TBDMSiO-NH2 and 102 mg (0.53 mmol) of EDCI.
(E)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-5-methyl-1-phenyl-1H-pyrazole-3-
carboxamide (1b); 1H NMR (500 MHz, CD3OD):
δ ppm 7.81 (d, 2H, J = 8.4 Hz, ArH), 7.72 (d, 2H,
J = 8.4 Hz, ArH), 7.65–7.54 (m, 5H, ArH), 7.53 (d, 1H, J = 13.8 Hz, Ar–CH=), 6.80 (s, 1H, H-4), 6.30 (d,
1H, J = 13.8 Hz, =CHCONHOH), 2.36 (s, 3H, Pyr–CH3).
13C NMR (125 MHz, CD3OD):
δ ppm 165.2 (–CONHOH), 159.8 (–CONH), 148.1 (C3), 143.4
(ArCH=), 140.7, 139.6, 129.9, 129.3, 129.1, 128.4, 126.8, 125.4, 118.8, 117.1 (C4), 107.5 (=CHCO), 11.9
(Pyr-CH3). Rf = 0.18 (TLC: EtOAc/MeOH 8:2); yellowish powder. M.p. = 103–107 ◦C. Yield: 45 mg
(42%). Anal. Calcd for C20H18N4O3: C, 66.29; H, 5.01; N, 15.46. Found: C, 66.26; H, 4.99; N, 15.49.
HRMS-EI (m/z) [M]+ calcd for C20H18N4O3 362.1379, found 362.1382.

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3.1.7. Synthesis of the Other Hydroxamic Acids with CAP N1-aryl-pyrazole (2,3)
Regarding the other N1-aryl-pyrazole derivatives (i.e., p-cyanophenyl
the design and synthesis were carried out only on the related pyrazole scaffolds of the isomer 12a
and 13a in accordance with the preliminary biological data obtained with the N1-phenyl analogs 1a
,b.
2 and p-bromophenyl 3),
Analogous synthetic procedures were used, and the experimental data of each intermediate as well as
of the final compounds are reported hereinafter.
(E)-1-(4-cyanophenyl)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-3-methyl-1H-
pyrazole-5-carboxamide (2); 1H NMR (500 MHz, CD3OD):
δ
ppm 8.00–7.38 (m, 9H, 8ArH +
Ar–CH=), 6.90 (s, 1H, H-4), 6.83 (d, 1H, J = 15.9 Hz, =CHCONHOH), 2.35 (s, 3H, Pyr–CH3).
13C NMR (125 MHz, CD3OD):
ppm 164.9 (–CONHOH), 158.7 (–CONH), 150.3 (C5), 143.3
δ
(ArCH=), 132.7, 129.2, 128.9, 128.1, 126.4, 124.9, 124.1 120.3 (–CN), 117.8 (C4), 115.1 (C-CN), 110.6
(=CHCO), 11.2 (Pyr–CH3). Yellowish powder. Rf = 0.12 (TLC: EtOAc/MeOH 8:2); M.p. = 110–114 ◦C.
Anal. Calcd for C21H17N5O3: C, 65.11; H, 4.42; N, 18.08. Found: C, 65.18; H, 4.45; N, 18.13.
HRMS-EI (m/z) [M]+ calcd for C21H17N5O3 387.1331, found 387.1333.
(E)-1-(4-bromophenyl)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-3-methyl-1H-
pyrazole-5-carboxamide (3); 1H NMR (300 MHz, CD3OD):
δ
ppm 7.83–7.22 (m, 9H, 8ArH + Ar–CH=),
6.83 (s, 1H, H-4), 6.40 (d, 1H, J = 15.9 Hz, =CHCONHOH), 2.35 (s, 3H, Pyr–CH3).
13C NMR (75 MHz, CDCl3):
ppm 166.1 (–CONHOH), 158.7 (–CONH), 148.9 (C5), 143.4 (ArCH=),
δ
140.7, 139.7, 137.8, 131.6, 130.5, 128.7, 126.1, 122.0 (C-Br), 119.8, 117.0 (C4), 109.2 (–CHCO), 13.0
(Pyr–CH3). Rf = 0.13 (TLC: EtOAc/MeOH 8:2); M.p. = 103–107 ◦C. Pale pink solid. Anal. Calcd for
C20H17BrN4O3: C, 54.44; H, 3.88; N, 12.70. Found: C, 54.52; H, 3.89; N, 12.73.
HRMS-EI (m/z) [M]+ calcd for C20H17BrN4O3 440.0484, found 440.0486.
3.1.8. Synthesis of the α,γ-Diketoester Intermediate 26 and 27
The synthesis of the
condensation between acetophenones 23 and 24 and diethyl oxalate 25 in the presence of a strong base.
Specifically, 750 mg of acetophenone 23 (6.24 mmol, 730 L) were solubilized in ethanol (~50 mL) and
α,γ-diketoester intermediate 26 and 27 was carried out by Claisen
µ
then EtONa (850 mg, 12.5 mmol,) was added to the resulting solution. The temperature of reaction
mixture was raised to ~50 ◦C for a few minutes, and afterward 1.83 g of diethyl oxalate (12.5 mmol,
1.6 mL) were added dropwise. The reaction was brought to reflux for ~3h and then dried under
vacuum. The residue was solubilized in H2O acidified with HCl. The aqueous phase was extracted
several times with ethyl acetate, and the combined organic phases were dried over anhydrous Na2SO4,
filtered and concentrated under reduced pressure to afford a crude oil. The product (obtained in the
enol form only) was purified by column chromatography using a mixture of petroleum ether/ethyl
acetate 8:2 as mobile phase.
3.1.9. Synthesis of N1H-pyrazole Scaffold C5-phenyl-substituted
The starting
on a plate in an oil bath. Then, an excess (3 equivalent) of hydrazine hydrate (506 mg, 15.9 mmol,
499 L) and a catalytic amount of HClconc. were added to the solution and the reaction mixture was
α,γ-diketoester 26 (1.17 g, 5.3 mmol) was dissolved in ethanol (~50 mL) and placed
µ
refluxed for ~3 h. Afterward, it was cooled, dried with a rotary evaporator, collected with brine, and
extracted several times with ethyl ether. The combined organic phases were dried over anhydrous
Na2SO4 and filtered. Then, the solvent was removed under reduced pressure, and the resulting crude
oil was triturated with ethyl ether to provide a solid product which by NMR analysis turned out to be
the hydrazide derivative of the initially designed compound. The filtered obtained by trituration with
ethyl ether was purified by column chromatography using a mixture of n-hexane/ethyl acetate 7:3 as
eluent to afford the expected product 28. Beside of the expected ester 28, the corresponding hydrazide
derivative was recovered.

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3.1.10. Hydrolysis of the N1H-pyrazole Scaffolds C5-phenyl-substituted
As for the N1-aryl-pyrazole derivatives, the ester and the hydrazide moiety of the two
N1H-pyrazole scaffolds must be hydrolyzed to –COOH group to carry out the coupling with the
cinnamoyl linker.
Route a: 437 mg (2.0 mmol) of the ester derivative were dissolved in EtOH (~20 mL), placed in
an ice bath under vigorous stirring and added dropwise with a solution of LiOH 1N. The reaction
mixture was left to room temperature and monitored by TLC (n-Hexane/EtOAc 7:3). After ~5 h,
the chromatographic check indicated the completion of the reaction (disappearance of the starting
material). The reaction solvent was removed under vacuum, and the residue was collected with brine,
acidified with HCl and extracted several times with ethyl acetate. The combined organic phases were
dried over anhydrous Na2SO4 and filtered. Then, the solvent was removed under reduced pressure to
afford the desired product as a whitish solid, which was purified by trituration with a mixture of ethyl
ether/petroleum ether.
Route b: to a 538 mg (2.7 mmol) of the hydrazide derivative placed in a flask was added a mixture
of HCl 6N/CH3COOHgl. (1:1; ~40 mL). The reaction was brought to reflux and left overnight under
vigorous stirring. Work-up: The reaction solution was diluted with H2O and extracted several times
with ethyl acetate. Then, the combined organic phases were dried over anhydrous Na2SO4 and filtered.
The organic solvent was removed in vacuum and also in this case the desired product was obtained in
a pure form by simple trituration of the residue with a mixture of ethyl ether/petroleum ether.
3.1.11. Coupling Reaction between N1H-5-phenyl-pyrazole Scaffold (CAP) and Cinnamoyl Linker
The coupling reaction between the N1H-pyrazole CAP and the cinnamoyl linker and the
pertaining workup were carried out with the same procedures used to obtain the N1-aryl-pyrazole
derivatives 1–3, starting from 350 mg of acid 30 (1.86 mmol), 356 mg of linker (1.86 mmol), 377 mg
of HOBt (2.79 mmol) and 535 mg of EDCI (2.79 mmol). Due to solubility issues, in some cases, a
few drops of DMF were added to the reaction mixture. The desired pure adduct CAP-linker 32 was
obtained by column chromatography using a mixture of CH2Cl2/EtOAc (8:2) as mobile phase.
3.1.12. Hydrolysis of the Adduct N1H-pyrazole CAP/Cinnamoyl Linker
The hydrolysis of the ester moiety of the adduct N1H-pyrazole CAP/cinnamoyl linker 32 was
performed in basic conditions using a solution of LiOH 1N in ethanol, starting from 383 mg (1.06 mmol)
of product 34. In addition, the work-up of the synthesis was performed with the same procedure.
3.1.13. Synthesis of the Final Hydroxamic Acid with CAP N1H-5-phenyl-pyrazole (4)
The final conversion of the carboxylic acid moiety to hydroxamic acid moiety was carried out
analogously to the N1-aryl-pyrazole derivatives
1–3 by means of a two steps synthetic procedure
which entails the conjugation of a silyl derivative of the hydroxylamine (i.e., TBDMSiO–NH2) and
the subsequent removal of the protecting silyl group by a solution of TFA (25%) in CH2Cl2, starting
from 219 mg of acid 34 (0.66 mmol), 97 mg hydroxylamine derivative (0.66 mmol), 134 mg of HOBt
(0.99 mmol) and 190 mg of EDCI (0.99 mmol).
(E)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-5-phenyl-1H-pyrazole-3-carboxamide
(4); 1H NMR (500 MHz, DMSO-d6):
δ ppm 13.89 (bs, 1H, PyrNH), 9.64 (bs, 1H, –CONH), 7.84 (d, 2H,
J = 8.8 Hz, ArH), 7.75 (d, 2H, J = 7.9 Hz, ArH), 7.73 (d, 1H, J = 16.1 Hz, ArCH=), 7.64 (d, 2H, J = 8.8
Hz, ArH), 7.47 (t, 2H, J = 7.9 Hz, ArH), 7.38 (t, 1H, J = 7.9 Hz, ArH), 7.20 (s, 1H, PyrH-4), 6.62 (d, 1H,
J = 16.1 Hz, =CHCO).
13C NMR (125 MHz, DMSO-d6):
δ ppm 165.5 (–CONHOH), 161.0 (–CONH), 158.9 (ArCH=), 147.2
(C3), 141.1 (C5), 137.9, 131.3, 130.0, 129.5, 129.0, 125.9, 120.9, (=CHCO), 103.7 (C4). Rf = 0.14 (TLC:
2% HCOOH in EtOAc/MeOH 8:2). Yellowish powder; M.p. = 254–258 ◦C. Yield: 87 mg (38%). Anal.
Calcd for C19H16N4O3: C, 65.51; H, 4.63; N, 16.08. Found: C, 65.59; H, 4.60; N, 16.12.

Int. J. Mol. Sci. 2019, 20, 945
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HRMS-EI (m/z) [M]+ calcd for C19H15N4O3 347.1144, found 347.1146.
3.1.14. Synthesis of the Other Hydroxamic Acids with CAP N1H-aryl-substituted-pyrazole (5, 6)
Regarding this second group of derivatives, the other compounds, which were designed and
synthesized with the same procedure employed for the phenyl-derivative 4, are the p-bromophenyl (
5)
and the 1-indanone derivative ( ), whose experimental data of the various intermediates as well as of
6
the final compounds are hereinafter reported.
(E)-5-(4-bromophenyl)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-1H-pyrazole-3-
carboxamide (5); 1H NMR (500 MHz, DMSO-d6):
δ ppm 10.56 (bs, 1H, –CONH), 7.89 (d, 2H,
J = 8.8 Hz, ArH), 7.80 (d, 2H, J = 8.3 Hz, ArH), 7.77 (d, 2H, J = 8.8 Hz, ArH), 7.64 (d, 2H, J = 8.3 Hz,
ArH), 7.50 (d, 1H, J = 15.9 Hz, ArCH=), 7.19 (s, 1H, PyrH-4), 6.43 (d, 1H, J = 15.9 Hz, =CHCO).
13C NMR (125 MHz, DMSO-d6):
δ ppm 168.4 (–CONHOH), 164.1 (–CONH), 148.0 (C3), 143.6
(ArCH=), 141.0 (C5), 132.1, 129.8, 129.4, 128.0, 125.1, 121.7 (C–Br), 120.5, 118.1, 107.1 (=C◦HCO), 104.2
(C4). Rf = 0.17 (TLC: 2% HCOOH in EtOAc/CH3OH 8:2). Beige powder; M.p. = 222–224 C. %). Anal.
Calcd for C19H15BrN4O3: C, 53.41; H, 3.54; N, 13.11. Found: C, 53.47; H, 3.51; N, 13.15.
HRMS-EI (m/z) [M]+ calcd for C19H14BrN4O3 425.0249, found 425.0248.
(E)-N-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)-1,4-dihydroindeno[1,2-c]pyrazole-3-
carboxamide (6); 1H NMR (500 MHz, DMSO-d6):
δ ppm 13.88 (bs, 1H, PyrNH), 9.98 (bs, 1H, –CONH),
8.00–7.80 (m, 1H, ArH), 7.67 (d, 2H, J = 8.8 Hz, ArH), 7.65 - 7.53 (m, 1H, ArH), 7.50 (d, 1H, J = 16.1 Hz,
ArCH=), 7.36 (t, 1H, J = 7.9 Hz, ArH), 7.32 (d, 2H, J = 8.8 Hz, ArH), 7.29 (t, 1H, J = 7.9 Hz, ArH), 6.40 (d,
1H, J = 16.1 Hz, =CHCO), 3.69 (s, 2H, Ind–CH2).
13C NMR (125 MHz, DMSO-d6):
δ ppm 169.5 (–CONHOH), 165.1 (–CONH), 155.0, 149.1, 143.9
(ArCH=), 141.5, 138.5, 130.8, 129.9, 129.4, 127.2, 126.9, 120.6, 119.4, 117.8 (=CHCO), 29.6 (Ind–CH2).
Rf = 0.58 (TLC: 2% HCOOH in EtOAc/CH3OH 8:2). Rusty powder; M.p. > 250 ◦C. Anal. Calcd for
C20H16N4O3: C, 66.66; H, 4.48; N, 15.55. Found: C, 66.70; H, 4.46; N, 15.58.
HRMS-EI (m/z) [M]+ calcd for C21H19N4O3 375.1457, found 375.1456.
3.2. Biological Activity
3.2.1. HDAC Inhibitor Drug Screening Kit
HDAC-inhibitory activity was evaluated using a fluorometric HDAC assay kit (K340-100,
Biovision, (Milpitas, CA, USA). The compounds, assay buffer, and HDAC fluorometric substrate
were added to HeLa nuclear extract in a 96-well plate and incubated at 37 ◦C for 60 min. The reacti◦on
was stopped by adding a lysine developer, and the mixture was incubated for another 30 min at 37 C.
TSA was used as a positive control. Plates were read with excitation at 360 nm and emission at 450 nm.
The data analysis was performed using GraphPad Prism 6.0.
3.2.2. Cell Cultures and Treatment
Human neuroblastoma cell line SH-SY5Y was originally obtained from ATCC (Rockville, MD,
USA). Cells were cultured as previously reported [20]. Briefly, we employed RPMI 1640 medium
with 10% heat-inactivated fetal bovine serum (FBS), 2 ◦mM L-glutamine, 1 mM sodium pyruvate,
100 IU/mL penicillin and 100 µg/mL streptomycin at 37 C in a humidified atmosphere with 5% CO2.
All the reagents for cell culture were from Carlo Erba (Milan, Italy). For biological investigations,
compounds 1a and were solubilized in dimethyl sulfoxide (DMSO) to obtain stock solutions at
a final concentration of 50 mM that were stored at
20 ◦C. Just prior use, aliquots were diluted in
medium to the final concentrations ranging from 1 to 50 M. The highest DMSO concentration here
employed (0.1%) did not show any appreciable effect on cell proliferation.
,
2
3
−
µ

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3.2.3. Antiproliferative Activity
The abovementioned compounds were evaluated by the MTT assay to assess their
anti-proliferative activity, as reported by Ferlazzo et al. [21] Cells were seeded at a density of 5
×
103 cell/well in 96-well plates and incubated overnight at 37 ◦C. In treated cells, the medium was
then replaced with a fresh one in which the compounds were diluted from 1 to 50 µM. Untreated
cells together with ones exposed to DMSO 0.1% served as control and solvent control, respectively.
After incubation for 24, 48, and 72 h, plates were firstly centrifuged, then the medium was removed,
and a solution of 3-(4,5-dimethylthiazole-2-yl)-2,5-diphenyltetrazolium bromide (MTT; Sigma-Aldrich,
Milan, Italy) at a concentration of 0.5 mg/mL in fresh medium (w/o phenol red) was added. Plates
were incubated for further 4h. Afterward, MTT solution was removed, and the obtained formazan
crystals were solubilized in 100
µL of a lysis buffer consisting of HCl/isopropanol 0.1 N and regularly
shaken. Optical density was measured employing a microplate reader (iMark
™, Bio-Rad Laboratories,
Milan, Italy) at 595 nm. All experiments were carried out in eight replicates for three times. Results are
expressed as the percentage of viable cells compared to untreated cells.
3.2.4. Statistical Analysis
Data were expressed as mean
analysis of variance (ANOVA), followed by Tukey–Kramer multiple comparisons test (GraphPad
Prism Software for Science).
±
SEM and statistically evaluated for differences using one-way
3.3. Docking Studies
Docking Protocol
Macromolecule and ligands were prepared with Vega ZZ (3.1.1, Drug Design Laboratory, Milano,
Italy) [23] assigning Gasteiger charges to the protein and AM1BCC ones to the ligand. Fine docking
was performed by applying the Lamarckian genetic algorithm (LGA) implemented in AutoDock
4.2.6, The Scripps Research Institute, San Diego, California Jupiter, FL, US) [24] in combination with a
specialized potential describing the interactions of zinc-coordinating ligands, named AutoDock4Zn
(The Scripps Research Institute, San Diego, California Jupiter, FL, US) [25]. The ligand-centered maps
were generated by the program AutoGrid (4.2.6, The Scripps Research Institute, San Diego, California
Jupiter, FL, US) with a spacing of 0.375 Å and dimensions that encompass all atoms extending 5 Å
from the surface of the ligand. All of the parameters were inserted at their default settings. In the
docking tab, the macromolecule and ligand are selected, and GA parameters are set as ga_runs =
100, ga_pop_size = 150, ga_num_evals = 25,000,000, ga_num_generations = 27,000, ga_elitism = 1,
ga_mutation_rate = 0.02, ga_crossover_rate = 0.8, ga_crossover_mode = two points, ga_cauchy_alpha
= 0.0, ga_cauchy_beta = 1.0, number of generations for picking worst individual = 10.
Because no water molecule is directly involved in complex stabilization, they were not considered
in the docking process. All protein amino acid residues were kept rigid whereas all single bonds of
ligands were treated as fully flexible.
4. Conclusions
To sum up, two sets of new hydroxamic acid-based derivatives endowed with a pyrazole capping
group and a cinnamoyl linker were designed, synthesized, and evaluated as HDACIs. All compounds
having the N1-aryl-pyrazole scaffold as the capping group connected through the C5 at the cinnamoyl
linker (i.e., 1a
assay, whereas similar compounds having the N1-H-pyrazole scaffold (i.e.,
group turned out to be much less active ( and ) or even inactive ( ). Docking studies performed
,
2
, and
3) showed promising inhibitory activity (IC50 range 1.3–6.3
µM) in the enzyme
4
,
5, and
6
) as the capping
4
6
5
on these compounds using a simulation model of TSA co-crystallized with human HDAC8 isoform
confirmed the effective insertion of the synthesized molecules within the tubular hydrophobic pocket
of the target and the chelation of the terminal hydroxamic acid moiety with the Zn2+ enzyme cofactor.

Int. J. Mol. Sci. 2019, 20, 945
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Overall, these studies also confirmed the trend observed in the enzyme assay. Besides, the results
obtained from the evaluation of the antiproliferative activity of the N1-aryl-pyrazole derivatives 1a
and on neuroblastoma SH-SY5Y cells was in agreement with the enzyme inhibitory activity, except
for the p-CN-phenyl derivative , which turned out to be inactive. The other two compounds, instead
(i.e., the phenyl derivative 1a and the p-Br–phenyl derivative ), reduced the cell proliferation with a
dose- and time-dependent fashion, showing comparable IC50 values in the micromolar range.
, 2,
3
2
3
Given these combined preliminary results on this type of derivatives, compound
considered as the lead structure for further developments.
3 can be
Supplementary Materials: Schematic procedures of synthesis (Schemes S1–S3), 1H and 13C NMR spectra, and
other analytic data can be found at http://www.mdpi.com/1422-0067/20/4/945/s1.
Author Contributions: Conceptualization, N.M.; methodology, C.Z., A.C., M.O. and A.M.; software, A.R.;
validation, A.R., M.N., A.P. and A.S.; formal analysis, C.Z., A.C. and A.M.; investigation, N.M.; resources, N.M.
and M.N.; data curation, C.Z., A.R., M.N. and N.M.; writing—original draft preparation, N.M.; writing—review
and editing, A.R., A.P., A.S. and M.N.; visualization, A.P., A.S. and M.N.; supervision, N.M.; project administration,
N.M.; funding acquisition, N.M.
Funding: This research was partially aided by the grant FFABR2017_MICALE_NICOLA_RI.
Conflicts of Interest: The authors declare no conflict of interest.
Abbreviations
0
EDCI
HATs
N-(3-dimethylaminopropyl)-N -ethylcarbodiimide hydrochloride
histone acetyltransferases
HDACIs
HDACs
HOBt
histone deacetylase inhibitors
histone deacetylases
hydroxybenzotriazole
MTT
PDB
3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
protein data bank
SAHA
TBDMSiO-NH2
TSA
suberoylanilide hydroxamic acid
O-(tert-butyldimethylsilyl)hydroxylamine
Trichostatin A
ZBD
zinc-binding domain
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2019 by the authors. Licensee MDPI, Basel, Switzerland. This article is an open access
article distributed under the terms and conditions of the Creative Commons Attribution
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