Novel Pyridine-Based Hydroxamates and 2′-Aminoanilides as Histone Deacetylase Inhibitors: Biochemical Profile and Anticancer Activity

Article abstract of DOI:10.1002/cmdc.202000854

Starting from the N-hydroxy-3-(4-(2-phenylbutanoyl)amino)phenyl)acrylamide (5 b) previously described by us as a HDAC inhibitor, we prepared four aza-analogues, 6–8, 9 b, as regioisomers containing the pyridine nucleus. Preliminary screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-phenylbutanoyl)amino)pyridyl)acrylamide (9 b) as the most potent inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-based hydroxamates (9 a, 9 c–f, and 11 a–f) and 2′-aminoanilides (10 a–f and 12 a–f), related to 9 b, to be tested against HDACs. Among them, the nicotinic hydroxamate 11 d displayed sub-nanomolar potency (IC₅₀: 0.5 nM) and selectivity up to 34 000 times that of HDAC4 and from 100 to 1300 times that of all the other tested HDAC isoforms. The 2′-aminoanilides were class I-selective HDAC inhibitors, generally more potent against HDAC3, with the nicotinic anilide 12 d being the most effective (IC₅₀^HDAC3=0.113 μM). When tested in U937 leukemia cells, the hydroxamates 9 e, 11 c, and 11 d blocked over 80 % of cells in G2/M phase, whereas the anilides did not alter cell-cycle progress. In the same cell line, the hydroxamate 11 c and the anilide 10 b induced about 30 % apoptosis, and the anilide 12 c displayed about 40 % cytodifferentiation. Finally, the most potent compounds in leukemia cells 9 b, 11 c, 10 b, 10 e, and 12 c were also tested in K562, HCT116, and A549 cancer cells, displaying antiproliferative IC₅₀ values at single-digit to sub-micromolar level.

Full text of DOI:10.1002/cmdc.202000854

Accepted Article
Title: Novel Pyridine-Based Hydroxamates and 2'-Aminoanilides
as Histone Deacetylase Inhibitors: Biochemical Profile and
Anticancer Activity
Authors: Clemens Zwergel, Elisabetta Di Bello, Rossella Fioravanti,
Mariarosaria Conte, Angela Nebbioso, Roberta Mazzone,
Gerald Brosch, Ciro Mercurio, Mario Varasi, Lucia Altucci,
Sergio Valente, and Antonello Mai
This manuscript has been accepted after peer review and appears as an
Accepted Article online prior to editing, proofing, and formal publication
of the final Version of Record (VoR). This work is currently citable by
using the Digital Object Identifier (DOI) given below. The VoR will be
published online in Early View as soon as possible and may be different
to this Accepted Article as a result of editing. Readers should obtain
the VoR from the journal website shown below when it is published
to ensure accuracy of information. The authors are responsible for the
content of this Accepted Article.
To be cited as: ChemMedChem 10.1002/cmdc.202000854
Link to VoR: https://doi.org/10.1002/cmdc.202000854
01/2020

10.1002/cmdc.202000854
ChemMedChem
FULL PAPER
Novel Pyridine-Based Hydroxamates and 2'-Aminoanilides as
Histone Deacetylase Inhibitors: Biochemical Profile and
Anticancer Activity
Clemens Zwergel+,[a] Elisabetta Di Bello+,[a] Rossella Fioravanti,[a] Mariarosaria Conte,[b] Angela
Nebbioso, [b] Roberta Mazzone,[a] Gerald Brosch,[c] Ciro Mercurio,[d] Mario Varasi,[d] Lucia Altucci,[b]
Sergio Valente,*[a] and Antonello Mai[a]
[a]
Dr. C. Zwergel, Dr. E. Di Bello, Dr. R. Fioravanti, Dr. R. Mazzone, Prof. S. Valente, Prof. A. Mai
Department of Drug Chemistry and Technologies, Sapienza University of Rome, P. le A. Moro 500185 Rome (Italy)
E-mail: sergio.valente@uniroma1.it.
[b]
[c]
[d]
Dr. M. Conte, Prof. A. Nebbioso, Prof. L. Altucci
Department of Precision Medicine, Università degli Studi della Campania Luigi Vanvitelli, Vico L. De Crecchio, 7-80138, Naples (Italy)
Prof. G. Brosch
Institute of Molecular Biology, Biocenter, Medical University of Innsbruck, 6020 Innsbruck, Austria.
Dr. M. Varasi, Dr. C. Mercurio
Department of Experimental Oncology, Academic Drug Discovery, European Institute of Oncology IRCCS, Via Adamello 16, 20139 Milan, Italy
[+] These authors contributed equally to this work
Supporting information for this article is given via a link at the end of the document.
Abstract:
Starting
from
the
N-hydroxy-3-(4-(2-
enzymes are also involved in neurological diseases, inflammation,
parasite infections, and other human disorders [4]
phenylbutanoyl)amino)phenyl)acrylamide 5b previously described by
us as HDAC inhibitor, we prepared four aza-analogues of 5b (6-8, 9b)
as regioisomers containing the pyridine nucleus. A preliminary
screening against mHDAC1 highlighted the N-hydroxy-5-(2-(2-
phenylbutanoyl)amino)pyridyl)acrylamide 9b as the most potent
inhibitor. Thus, we further developed both pyridylacrylic- and nicotinic-
based hydroxamates (9a, 9c-f, and 11a-f) and 2'-aminoanilides (10a-
f and 12a-f), related to 9b, to be tested against HDACs. Among them,
the nicotinic hydroxamate 11d displayed subnanomolar potency (IC50:
0.5 nM) and selectivity up to 34000-fold over HDAC4 and from 100-
to 1300-fold over all the other tested HDAC isoforms. The 2'-
aminoanilides were class I-selective HDAC inhibitors, generally more
potent against HDAC3, with the nicotinic anilide 12d being the most
effective (IC50HDAC3 = 0.113 μM). When tested in U937 leukemia cells,
the hydroxamates 9e, 11c, and 11d blocked over 80% cells in G2/M
phase, whereas the anilides did not alter the cell cycle progress. In
the same cell line, the hydroxamate 11c and the anilide 10b induced
about 30% apoptosis, and the anilide 12c displayed about 40%
cytodifferentiation. Finally, the most potent compounds in leukemia
cells 9b, 11c, 10b, 10e, and 12c were also tested in K562, HCT116,
and A549 cancer cells, displaying antiproliferative IC50 values at
single-digit to sub-micromolar level.
.
HDAC inhibitors (HDACi) are capable to activate several
antitumor pathways, such as extrinsic (death receptors and
ligands upregulation) or intrinsic (downregulation of anti-apoptotic
factors, upregulation of pro-apoptotic ones) apoptosis induction.
Furthermore, HDACi either target directly or modulate indirectly
the (over)expression of non-histone proteins. They influence a
whole bunch of inflammation/immune response mediators,
transcription factors and regulators, chaperones, and structural
proteins as well as DNA repair enzymes [2-3, 5]. These enzymes
can also induce senescence, growth arrest via p21 upregulation
or downregulation of cyclins, lead to mitotic and autophagic cell
death, and possess anti-angiogenic effects via HIF-1α function or
VEGF downregulation [6]
.
A large number of HDACi are involved in preclinical as well as
clinical settings [6c, 7]. HDACi are mainly used in blood cancers
such as leukemias and are poorly successful in solid tumors as
single agents [8]. Inhibitors designed for HDACs have in common
a well-validated pharmacophore model composed of a zinc-
binding group (ZBG) attached to a linker chain (hydrophobic
spacer, HS) mimicking the lysine side chain terminated by a
functional "cap" group. A connecting unit (CU) between the cap
and the linker chain can also be modulated to improve interactions
[6c]
.
In detail, most HDACi typically possess either an hydroxamate or
a 2-aminoanilide function as ZBG. Numerous examples are
described in the recent literature and have been summarized in
Introduction
In eukaryotic biological processes, epigenetic alterations resulting
in the gain or loss of function of regulatory proteins contribute
fundamentally to human diseases' onset and progression [1]. One
of the most studied epigenetic targets in the last three decades
various reviews [2, 4c, 6c]
.
are histone deacetylases (HDACs) [2]
.
Human HDACs have been investigated as key components of the
epigenetic machinery regulating gene expression and often
behave as oncogenes in cancer except for HDAC2 [3]. Besides
HDAC deregulation and/or overexpression in cancer, these
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1

10.1002/cmdc.202000854
ChemMedChem
FULL PAPER
O
A
Our research group acquired extensive experience in the design
of HDACi in the last two decades [6c, 13, 16]. In 2001, we described
a new class of selective HDACi, 3-(4-aroyl-1H-2-pyrrolyl)-N-
hydroxy-2-propenamides (APHAs, 1, Figure 2) as agents capable
of inhibiting HDAC activity in the micromolar range, characterized
by an aroyl moiety as CAP+CU, a pyrrylacrylic moiety as HS, and
the hydroxamate group as ZBG [16e, 16g, 17]. Extensive structure-
activity relationship studies performed on the 1 model led to
analogues with improved HDAC inhibitory potency and good
O
O
O
HN
O
H
N
OH
N
H
NH
NH
O
S
S
O
Vorinostat
O
Romidepsin
O
H
O
O
N
OH
S
OH
O
H
N
N
N
H
O
H
HN
Panobinostat
Belinostat
anticancer properties in mouse A20 cells[16a]
.
N
NH2
H
H
Later, through a structure-based drug design, two isomers of the
APHA scaffold, the 3-(2-aroyl-1-methyl-1H-pyrrol-4-yl)-N-
hydroxy-2-propenamide and the 3-(2-aroyl-1-methyl-1H-pyrrol-5-
yl)-N-hydroxy-2-propenamide (iso-APHAs 2, Figure 2), were
designed and prepared. When tested in human leukemia U937
cell line, the (E)-3-(5-([1,1'-biphenyl]-4-carbonyl)-1-methyl-1H-
pyrrol-3-yl)-N-hydroxy-2-propenamide impaired cell cycle while
inducing apoptosis and cytodifferentiation [16e]. A vinylogy study
applied on the 2 model allowed us to discover a new series of
compounds, the (aryloxopropenyl)pyrrolyl hydroxyamides (3), as
selective inhibitors of class II HDACs. The hit compound, MC1568
(Ar2 = 3-fluorophenyl, Figure 2), has been extensively used as a
tool for dissecting the HDAC biological functions in different cell
contexts, including cancers, type 2 diabetes, hepatic and renal
fibrosis, amyotrophic lateral sclerosis (ALS), influenza A virus and
N
N
O
Tucidinostat
F
B
O
N
O
O
N
NH2
N
N
NH2
H
N
H
H
H
N
N
N
O
O
Entinostat
Mocetinostat
O
OH
O
N
H
O
N
HN
Trichostatin A
N
N
O
NH2
H
N
N
H
NH2
HIV [16j, 16l, 18]
Replacement of the pyrrole with benzene or pyridine ring in the
scaffold afforded the N-hydroxy-3-(4-(3-oxo-3-phenylprop-
.
O
N
N
compound 40
N
O
compound 7i
S
O
3
1enyl)phenyl/pyridin)acrylamides (4) (Figure 2) [16i, 16j], acting as
anticancer agents through HDAC inhibition. A further change of
the oxopropenyl with an amide moiety led to the N-hydroxy-3-
(acylaminophenyl)acrylamides (5) (Figure 2) [16c] as an additional
class of HDACi potent up to nanomolar level [16c]. Most of the
tested compounds showed a preference for HDAC1 inhibition and
Figure 1. (A) Chemical structures of HDAC inhibitors approved by FDA and
CFDA. (B) Chemical structures of HDAC inhibitors in clinical trials and active in
preclinical settings
So far, only five HDACi have reached the market: the pan-HDAC
inhibitor vorinostat (SAHA) [9] and the HDAC1,2 selective inhibitor
romidepsin (FK-228) [10] were both approved for the treatment of
refractory cutaneous T-cell-lymphoma (CTCL), and the latter also
for peripheral T-cell lymphoma (PTCL); moreover, the pan-HDAC
were able to arrest the U937 cell cycle in the G2/M phase [16h]
.
[11]
inhibitors belinostat (PDX101) and panobinostat (LBH589)
have been applied for the treatment of PTCL and
[12]
multiple
myeloma (MM), respectively. All drugs mentioned above have
been approved by the FDA, whereas tucidinostat (chidamide),
active against HDAC1, -2, -3, -10 was approved for PTCL only in
China in 2015 [6c] (Figure 1A).
Entinostat (MS-275) (Figure 1B) is a HDAC1-3 selective inhibitor
in clinical trials that can be used alone or in combination with 5-
azacytidine or ganciclovir for the treatment of myelodysplastic
syndrome (MDS), chronic myeloid leukemia (CML), acute
myelogenous leukemia (AML) and cancer-related viral infections
such as Epstein-Barr virus (EBV) [13]. Mocetinostat, a HDAC1,2
selective inhibitor, is currently evaluated in numerous cancer
types, especially in leukemia and lymphoma. Trichostatin A (TSA),
a pan-HDAC inhibitor, is currently evaluated in many types of
diseases, including pancreatic adenocarcinoma, HIV infection,
[14]
CML, BCR/ABL positive breast cancer
(Figure 1B). In 2007,
Figure 2. Several pyrrolylacrylic (1-3) and phenyl/pyridylacrylic (4, 5) scaffolds
developed by our research group for HDACi design.
Hamblett et al. explored around the nicotinamide core, describing
several compounds displaying good selectivity against HDAC1
and high capability to induce cell cycle arrest, apoptosis, and
cytodifferentiation in human leukemia U937 cells. Furthermore,
extensive exploration around of these compounds led to the
discovery of the class I selective HDACi 7i and compound 40
(Figure 1B) with a good in vivo efficacy in a HCT116 xenograft
Results and Discussion
[16b, 16h-j]
Following our previous investigations mentioned above
,
and taking into account that pyridine-based HDACi were active in
preclinical settings [15], we selected the N-(4-(3-(hydroxyamino)-3-
model [15]
.
2
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oxoprop-1-en-1-yl)phenyl)-2-phenylbutanamide 5b as one of the
most potent compounds among the 5 series, and designed and
prepared four corresponding pyridine-based regioisomers, 6-8
and 9b, to be tested against mouse HDAC1 (mHDAC1). The
results clearly highlighted that the N-(5-(3-(hydroxyamino)-3-
oxoprop-1-en-1-yl)pyridin-2-yl)-2-phenylbutanamide 9b displayed
the highest mHDAC1 inhibition (IC50: 0.075 μM), being 4-fold
more potent than the reference 5b. Hence, we selected 9b for
further structural optimization. Two series of 2-acylamino-5-(3-
oxoprop-1-en-1-yl)pyridine hydroxamates (9a,c-f) and 2'-
aminoanilides (10a-f), as well as the corresponding nicotinic
analogs 11a-f (hydroxamates) and 12a-f (2'-aminoanilides), were
designed and synthesized to be tested in enzyme and in cellular
assays. At the acyl moiety, the derivatives differ in the presence
of phenylacetyl groups with growing substituents at the methylene
unit (ethyl, iso-propyl, benzyl), or of the bulkier 1- and 2-
naphthylacetyl groups (Figure 3).
Scheme 1. Reagents and conditions: a) i) SOCl2, 80°C; ii) Et3N, CH2Cl2; b):
Method 1: Pd(OAc)2, Et3N , n-butyl acrylate, anhydrous DMA, 100° C, sealed
tube, 8h; Method 2. P(Ph)3, Pd(OAc)2, Et3N , n-butyl acrylate, NaHCO3,
anhydrous DMF, 100° C, sealed tube, 2h; Method 3: P(Ph)3, Pd(OAc)2, n-BuN4I,
NaOAc x 3 H2O, H2O, butyl acrylate, 140° C, anhydrous DMF, overnight; c) KOH
o LiOH, H2O, EtOH; d) i) ClCOOC2H5, Et3N, THF, 0° C; ii) NH2OC(CH3)2OCH3;
iii) Amberlist 15, CH3OH; e) Et3N, BOP reagent, 1,2-phenylendiamine, DMF, N2.
For the synthesis of the lead compounds, the appropriate
commercial carboxylic acids were treated with thionyl chloride
and then with 2-amino-5-bromopyridine, or with methyl 6-
aminonicotinate, in the presence of triethylamine to afford the
amides 13-15 and 16a-f or 25a-f, respectively. The
bromopyridylamides 13-15 and 16a-f underwent Heck reaction
(three different methods) to obtain the pyridylacrylate
intermediates 17-19 and 20a-f. The esters 17-19, 20a-f, and 25a-
f were hydrolyzed in basic medium to the corresponding
carboxylic acids 21-23, 24a-f, and 26a-f, respectively. Next, these
compounds were activated with ethyl chloroformate and
triethylamine, and subsequently treated with O-(2-methoxy-2-
propyl)hydroxylamine and then Amberlyst 15 ion-exchange resin
in methanol to yield the hydroxamates 6-8, 9a-f, and 11a-f.
Alternative treatment of the acids 24a-f and 26a-f with
benzotriazole-1-yloxytris(dimethylamino)-phosphonium
hexafluorophosphate (BOP reagent), triethylamine and 1,2-
phenylenediamine afforded the corresponding 2'-aminoanilides
10a-f and 12a-f, respectively (Scheme 1). Chemical and physical
data of the final compounds 6-12 and the intermediates 13-26 as
well as elemental analyses for the final compounds are reported
in tables S1–S10 in Supporting Information.
Figure 3. Design of (A) novel pyridine hydroxamates isomers 6-8 and 9b, and
(B) 2-acylamino-5-(3-oxoprop-1-en-1-yl)pyridine hydroxamates 9a-f and 2'-
aminoanilides 10a-f, as well as the corresponding nicotinic analogs 11a-f
(hydroxamates) and 12a-f (2'-aminoanilides).
Biochemical activities of 9a-f, 11a-f, 10a-f, and 12a-f against
human recombinant (hr) HDAC isoforms
The new hydroxamates 9a-f, 11a-f, and 2-aminoanilides 10a-f,
12a-f were screened in 10-dose IC50 mode with 3-fold serial
dilution starting from 100 μM solutions, against selected hrHDAC1,
-3, -4, -6, and -8, representatives of class I (HDAC1,3,8), class IIa
(HDAC4) and class IIb (HDAC6) HDACs, to assess their inhibitory
capability. For this purpose, either the fluorogenic peptide from
p53 residues 379-382 (RHKK(Ac)AMC) substrate (for HDAC1, -3,
-6), or the fluorogenic class IIa (Boc-Lys(trifluoroacetyl)-AMC)
substrate (for HDAC4), or the diacetylated peptide from p53
residues 379-382 (RHK(Ac)K(Ac)AMC) substrate (for HDAC8)
has been used, and SAHA was employed as reference compound
and positive control.
Synthetic routes of novel pyridine-based hydroxamates 6-8, 9a-f, and 11a-f, and
2'-aminoanilides 10a-f and 12a-f.
The biochemical data (Table 1) highlighted that both the
pyridylacrylic (9a-f) and the nicotinic (11a-f) hydroxamates
potently and selectively inhibited HDAC6. In detail, the first group
showed IC50 values ranging from submicromolar (9a, IC50= 164
nM) to single-digit nanomolar (9e, IC50: 7 nM) level. In particular,
3
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(28.6)
(655)
9e, bearing the 1-naphthylacetyl moiety at the amide function,
displayed up to 1000-fold selectivity for HDAC6 over HDAC4 and
more than 38- or 50-fold selectivity over all the other tested HDAC
isoforms. The nicotinic hydroxamates 11a-f were even more
10b
10c
10d
10e
10f
3.43
(8)
0.426
0.681
0.406
0.187
0.694
NA
NA
NA
NA
NA
98.3
(231)
86.8
(204)
5.77
(8.5)
115.8
(170)
98.5
(145)
potent against HDAC6 than 9a-f, reaching subnanomolar
HDAC6
inhibition with 11d (IC50
= 0.5 nM), characterized by a 2,3-
diphenylpropanamide moiety at the pyridine C2 position, whereas
all the other analogs (11a-c,e-f) provided dual- to single-digit
nanomolar inhibition. Noteworthy, the nicotinic series displayed
very high selectivity for HDAC6: indeed, 11d displayed up to
34000-fold selectivity over HDAC4 and from 100- to 1300-fold
over all other HDAC isoforms. Moreover, in both pyridylacrylic and
nicotinic hydroxamates, the insertion of a bulky or branched
arylacetamide group at the pyridine C2 position increases the
inhibition potency and selectivity towards HDAC6, when
compared to the phenylacetyl derivatives 9a and 11a.
Regarding the 2'-aminoanilide series 10a-f and 12a-f, both the
pyridylacrylic and nicotinic derivatives displayed, as predictable,
HDAC1 and -3 selectivity, with a preference for HDAC3 over
HDAC1. More in detail, both series displayed single-digit
micromolar to submicromolar inhibition potency against HDAC1
and -3, with 10e among the pyridylacrylic anilides, bearing the 1-
naphthylacetamide moiety at C2, and 12d among the nicotinic
anilides, carrying a 3-methyl-2-phenylbutanamide portion at C2,
being the most potent against HDAC3 (IC50 values: 0.187 and
0.113 μM, respectively).
3.18
(7.8)
NA
NA
181
(446)
0.366
(2)
140
(749)
2.57
(3.7)
309
(445)
NA
11a
11b
11c
11d
11e
11f
2.42
(142)
2.71
(159)
26.0
(1529)
0.017
0.006
0.006
0.0005
0.007
0.019
NA
0.386
(22.7)
1.12
(187)
0.917
(153)
28.8
(4800)
0.277
(46.2)
1.0
(167)
0.665
(111)
46.6
(7767)
0.357
(59.5)
0.117
(234)
0.056
(112)
17.2
(34400)
0.643
(1286)
1.03
(147)
0.765
(109)
14.4
(2057)
0.40
(57.1)
0.784
(41.3)
0.301
(15.8)
6.0
(316)
1.0
(52.6)
Cell-cycle analyses, apoptosis induction and granulocytic
differentiation in human U937 leukemia cells
12a
12b
12c
12d
12e
12f
1.41
(7)
0.202
0.176
0.185
0.113
0.389
0.752
0.35
NA
NA
NA
NA
NA
NA
0.49
NA
Using FACS analysis, compounds 9a-f, 10a-f, 11a-f, and 12a-f
were tested at 5 µM for 30 h in human U937 acute myeloid
leukemia cell line to evaluate their effects on cell cycle, apoptosis,
and granulocytic differentiation. SAHA and MS-275 were used as
reference and positive controls. In cell cycle assays (Figure 4A),
the nicotinic hydroxamates led to an arrest in G2/M phase (see
11c and 11d, 50% and 80% blockage, respectively), while the
pyridinacrylic hydroxamates displayed a blockage of the cell cycle
either at the S phase, with 9c resulting in more than 60% blockage,
or G2/M phase, with 9b, 9d, 9e, the latter two being the most
potent with about 80% blockage.
1.13
(6.4)
NA
103
(585)
1.27
(6.9)
NA
136
(735)
0.379
(3.3)
NA
NA
1.42
(3.6)
NA
78
(200)
2.36
(3.1)
NA
96
(128)
Table 1. Biochemical inhibition (IC50, μM) of 9a-f, 10a-f, 11a-f and 12a-f towards
hrHDAC 1, -3, -4, -6 and -8 isoforms. The HDAC6- (for 9, 11) or HDAC3- (for
10, 12) selectivity index is given in round brackets.
SAHA
0.26
0.03
0.24
[a] NA, not active, impossible to calculate the IC50 even by extrapolation.
compound
HDAC1
HDAC3
HDAC4
HDAC6
0.164
HDAC8
In addition, 11c was unique to induce over 30% of the pre-G1
fraction, indicating a cell death effect, more potent than SAHA
(about 15% of pre-G1 phase) in these assay conditions. None of
the tested hydroxamates 9 and 11 were able to induce
cytodifferentiation, which was evaluated through the expression
of the surface antigen CD11c marker in propidium iodide (PI)-
negative cells.
9a
7.08
(43.2)
6.30
(38.4)
155
(945)
2.94
(17.9)
9b
9c
1.56
(130)
1.23
(102)
31.7
(2642)
0.012
0.016
1.15
(95.8)
2.26
(141)
2.19
(137)
39.4
(2462)
1.45
(90.6)
Concerning the 2'-aminoanilides 10a-f and 12a-f, none of both
series displayed cell cycle arrest/death induction, with the only
exception of 10b, giving almost 30% of cell death and being more
potent than SAHA in this assay. Moreover, 10b, 10e, and 12c
provided the most potent cytodifferentiating effects (40% CD11c-
positive/PI-negative cells with 12c), although less effective than
MS-275 (about 70% CD11c-positive/PI negative cells) in these
assay conditions.
9d
9e
2.02
(91.8)
0.781
(35.5)
27.3
(1241)
0.022
0.007
0.013
NA
1.41
(64.1)
0.271
(38.7)
0.351
(50.1)
7.05
(1007)
0.352
(50.3)
9f
0.425
(32.7)
0.581
(44.6)
10.7
(823)
0.552
(42.3)
10a
5.89
0.206
NA[a]
135
4
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leukemia U937 cells to evaluate the effects on the acetylation
levels of histone H3 and α-tubulin by the treatment with three of
the most effective compounds 10e, 11c, and 12c, tested at 5 μM
for 24 h. SAHA and MS-275 were used as positive controls. As
highlighted by Figure 5, the nicotinic hydroxamate 11c confirmed
to be a potent HDAC6 inhibitor since it induced a strong α-tubulin
acetylation, even better than SAHA, whereas none of the nicotinic
2'-aminoanilides 10e and 12c provided any effect on acetyl-
tubulin, as expected. In histone H3 acetylation assay, 10e, 11c
and 12c increased the acetyl-H3 level respect to the control, with
10e and 11c displaying a level comparable to those of SAHA and
MS-275, tested in the same conditions.
G1
S
G2/M
100
90
A
80
70
60
50
40
30
20
10
0
5
A
9f
1f
7
9a
9c
9e
1a
1c
1e
9b
9d
1b
1d
1
1
H
1
1
1
1
MSO
SA
D
MS-2
G1
S
G2/M
100
90
80
70
60
50
40
30
20
10
0
B
C
5
A
7
H
10f
12f
10a
10c
10e
12a
12c
12e
10b
10d
12b
12d
MSO
SA
D
MS-2
50
40
30
20
10
0
5
A
9f
1f
7
9a
9c
9e
1a
1c
1e
1
9b
9d
1b
1d
1
1
H
1
1
1
MSO
SA
D
MS-2
Figure 5. Effects on acetylation level of histone H3 (up) and a-tubulin (down) in
U937 cells treated with the indicated compounds at 5 μM of compounds for 24
h. Histone H4 and GAPDH were used as loading controls.
40
30
20
10
0
D
E
According to the best profile proved in U937 leukemia cells,
selected hydroxamates 9b, 11c, and the 2'-aminoanilides 10b,
10e and 12c were screened against three different cancer cell
lines, as human chronic myelogenous leukemia K562, colorectal
carcinoma HCT116, lung adenocarcinoma A549, to assess their
antiproliferative activities (IC50 values, Table 2). In general, both
the hydroxamates and the 2'-aminoanilides displayed IC50 values
ranging from single-digit to sub-micromolar levels against all the
three tested cancer cell lines, with HCT116 and K562 being more
responsive than A549 (Table 2) to the selected compounds
treatment.
5
5
5
A
7
H
H
H
10f
12f
10a
10c
10e
12a
12c
12e
10b
10d
12b
12d
MSO
MSO
MSO
SA
SA
SA
D
D
D
MS-2
80
70
60
50
40
30
20
10
0
A
9f
1f
1
7
9a
9c
9e
1a
1
1c
1
1e
1
9b
9d
1b
1
1d
1
Table 2. Antiproliferative activity (IC50, μM ± SD)a,b of selected compounds in a
panel of human cancer cell lines
MS-2
80
70
60
50
40
30
20
10
0
F
compound 1[a]
Colorectal
carcinoma
HCT116
Lung
adenocarcinoma
A549
Chronic
myelogenous
leukemia K562
9b
0.88 (±0.23)
0.92 (±0.07)
1.63 (±0.20)
2.67 (±0.12)
2.06 (±0.14)
5.72 (±0.86)
0.92 (±0.19)
1.04 (±0.15)
2.76 (±0.61)
11c
A
7
10f
12f
10a
10c
10e
12a
12c
12e
10b
10d
12b
12d
10b
10e
12c
MS-2
Figure 4. (A, B, C, D) Effects on cell cycle phases (E, F), and cytodifferentiation
of hydroxamates 9a-f, 11a-f, and 2'-aminoanilides 10a-f, 12a-f in human U937
leukemia cells treated at 5 μM of compounds for 30 h.
0.84 (±0.19)
3.15 (±0.09)
4.26 (±1.11)
8.51 (±2.10)
0.60 (±0.0007)
3.11 (±0.64)
aCells were treated for 72 h with various inhibitor concentrations; bStandard
deviation (SD) is reported in round brackets.
Effects on acetylation level of histone (histone H3) and non-
histone (α-tubulin) substrates
Next, to assess the in-cell target engagement, western blot
analyses with specific antibodies were performed in human
5
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10.1002/cmdc.202000854
ChemMedChem
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rotary evaporator operating at reduced pressure of ca. 20 Torr. Organic
solutions were dried over anhydrous sodium sulfate. Elemental analysis
has been used to determine the purity of the described unknown final
compounds, which is >95%. Analytical results are within ±0.40% of the
theoretical values (Table S1 in Supporting Information). All chemicals were
purchased from Sigma Aldrich s.r.l, Milan (Italy), or TCI Europe N.V.,
Zwijndrecht (Belgium), and were of the highest purity.
Conclusion
In conclusion, in the present work we discuss first a regioisomeric
study of novel acylaminopyridylacrylic hydroxamates 6-8 and 9b
as azaisosters of our previously reported cinnamyl hydroxamate
5b. This preliminary screening led to the identification of the N-(5-
(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)pyridin-2-yl)-2-
General procedure for the synthesis of nicotinates 13-15, 16a-f, and
25a-f. Example: methyl 6-(3-methyl-2-phenylbutanamido)nicotinate
(25c).
3-methyl-2-phenyl-butyryl chloride (6.1 mmol, 1.0 g) and triethylamine
were added to a solution of 6-amino-methylnicotinate (6.1 mmol, 1.39 g)
in DCM dry (20 mL) at 0 °C. After stirring at room temperature for two hours,
the reaction was quenched with water (50 mL) and extracted with
dichloromethane (3 x 50 mL). The organic phase was then washed with
brine (100 mL), dried over sodium sulfate, and evaporated under reduced
pressure. The residual crude was finally purified by column
chromatography on silica gel eluting with a mixture ethyl acetate:n-hexane
1:5.
Mp: 92-94 °C; yield: 78.4%; rec.solv.: cyclohexane/benzene 1H NMR
(CDCl3) δ 0.72 (d, 3H, CH(CH3)2, J = 6.7 Hz), 1.08 (d, 3H, CH(CH3)2, J =
6.5 Hz), 2.45-2.55 (m, 1H, CH(CH3)2), 3.05 (d, 1H, PhCHCO, J = 10.2 Hz),
3.89 (s, 3H, COCH3), 7.30-7.32 (m, 5H, benzene protons), 8.25 (d, 1H,
pyridine proton, J = 2.1 Hz), 8.28 (s, 1H, pyridine proton), 8.45 (bs, 1H,
CONH), 8.82 (d, 1H, pyridine proton, J = 1.4 Hz).
phenylbutanamide 9b as the most potent HDAC inhibitor. By the
following optimization of this scaffold, new 9b analogs, both as
pyridylacrylic and nicotinic hydroxamates 9a, 9c-f, and 11a-f, and
the corresponding 2'-aminoanilides 10a-f and 12a-f were
designed and prepared. When tested against hrHDAC1, -3, -4, -
6, and -8 isoforms, 9e and 11d proved to be most potent and
selective against HDAC6 among the pyridylacrylic and nicotinic
hydroxamates, respectively. In detail, 9e displayed IC50 = 7 nM
against HDAC6 and selectivity over the other HDAC isoforms
ranging from 40- to 1000-fold, whereas 11d displayed
subnanomolar potency (IC50 = 0.5 nM) against HDAC6 and
selectivity from 112- to 34000-fold over the other isoforms. The 2'-
aminoanilides 10a-f and 12a-f preferentially inhibited class I
HDACs, in particular HDAC3 with submicromolar potency. Among
them, 10e and 12d were most potent against HDAC3, showing
IC50 values of 0.187 and 0.113 μM, respectively. When tested in
U937 leukemia cells to assess their capability to induce cell cycle
arrest, apoptosis induction, and cytodifferentiation, the
hydroxamates 9e, 11c, and 11d blocked the cell cycle in G2/M
phase (over 80% blockage), whereas the anilides did not alter cell
cycle progression significantly. Among the tested compounds, the
nicotinic hydroxamate 11c and the pyridylacrylic 2'-aminoanilide
10b were the sole to induce about 30% of proapoptotic effect. In
cytodifferentiation assays, the hydroxamates had no effect,
whereas the nicotinic 2'-aminoanilide 12c exhibited about 40% of
CD11c-positive/PI-negative cells.
General Procedure for the synthesis of (E)-n-butyl 3-(6-
(arylacetamido)pyridinyl)acrylates 17-19, 20a-f.
Method 1. (17): Palladium acetate (0.95 mmol, 0.21 g) was added under
nitrogen atmosphere to
a
solution of N-(6-bromopyridin-2-yl)2-
phenylbutanamide 13 (6.8 mmol, 0.22 g), triethylamine (16.73 mmol, 2.32
mL) and n-butyl acrylate (20.57 mmol, 2.95 mL) in anhydrous N,N-
Dimethylacetamide (8 mL) and in a sealed tube. The resulting mixture was
stirred at 100°C for 8h. The reaction was quenched by water (30 mL) and
extracted with ethyl acetate (3 x 50 mL). The collected organic phases
were washed with 2N HCl (3 x 50 mL) and saturated sodium chloride
solution (3 x 50 mL), dried and concentrated to obtain an oily residue that
was purified by silica gel chromatography eluting with ethyl acetate/n-
hexane 1/3 to provide pure 17 as a pale yellow solid.
Method 2. (18, 20a-f). Example: Synthesis of (E)-n-butyl 3-(6-(2-
(phenyl-1-yl)butanamido)pyridin-3-yl)acrylate (20b).
Triphenylphosphine (0.22 mmol, 0.06 g) and palladium acetate (0.081
mmol, 0.02 g) were added under nitrogen atmosphere to a solution of N-
(5-bromopyridin-2-yl)-2-(phenyl-1-yl) butanamide 16b (4.40 mmol, 1.50 g),
sodium bicarbonate (8,79 mmol, 0.74 g), triethylamine (18.90 mmol, 2.6
mL) and n-butyl acrylate (5.28 mmol, 0.76 mL) in anhydrous N,N-
dimethylformamide (5 mL) and in a sealed tube. The resulting mixture was
stirred at 100°C for 2h. The reaction was quenched by water (30 mL) and
extracted with ethyl acetate (3 x 50 mL). The collected organic phases
were washed with 2N HCl (3 x 50 mL) and saturated sodium chloride
solution (3 x 50 mL), dried and concentrated to obtain an oily residue that
was purified by silica gel chromatography eluting with ethyl acetate/n-
hexane 1/3 to provide pure 20b as a pale yellow solid.
Mp: 102-104 °C; yield: 88.2%; rec solv: cyclohexane/benzene 1H-NMR
(CDCl3) δ 0.91-0.94 (t, 3H, OCH2CH2CH2CH3, J = 7.4 Hz), 0.95-0.98 (t,
3H, CH2CH3, J = 7.3 Hz), 1.42-1.44 (m, 2H, OCH2CH2CH2CH3), 1.66-1.70
(m, 2H, OCH2CH2CH2CH3), 1.85-1.87 (m, 2H, CH2CH3), 3.42 (t, 1H,
PhCHCO, J = 6.5 Hz), 4.19-4.22 (t, 2H, OCH2CH2CH2CH3, J = 6.7 Hz),
6.40 (d, 1H, CH2=CH2COOBu, J = 16.0 Hz), 7.27-7.36 (m, 5H, benzene
protons), 7.59 (d, 1H, CH2=CH2COOBu, J = 16.0 Hz), 7.87 (d, 1H, pyridine
proton, J = 2.3 Hz), 8.19 (bs, 1H, CONH), 8.27 (s, 1H, pyridine proton),
8.30 (d, 1H, pyridine proton, J = 2.5 Hz).
Among the most effective compounds, the hydroxamate 11c
potently induced α-tubulin acetylation, proving HDAC6-
engagement in U937, and both the 2'-aminoanilides 10e and 12c
together with 11c led to a strong histone H3 acetylation signal in
the same cells.
The most efficient compounds in leukemia, i.e., 9b, 11c, 10b, 10e,
and 12c, were also screened against three further cancer cell
lines, including K562 leukemia, HCT116 colon cancer, and A549
lung cancer cells, where they displayed antiproliferative activity at
single-digit to sub-micromolar concentrations. Further studies are
needed to assess the anticancer potential of such derivatives.
Experimental Section
Synthesis and characterization
General
Melting points were determined on a Büchi 530 melting point apparatus
and are uncorrected. 1H-NMR spectra were recorded at 400 MHz on a
Bruker AC 400 spectrometer; chemical shifts are reported in δ (ppm) units
relative to the internal reference tetramethylsilane (Me4Si). All compounds
were routinely checked by TLC and 1H-NMR. TLC was performed on
aluminum-backed silica gel plates (Merck DC, Alufolien Kieselgel 60 F254)
with spots visualized by UV light. All solvents were reagent grade and,
when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved using a
Method 3. (19): butyl (E)-3-(5-(2-phenylbutanamido)pyridin-2-
yl)acrylate
Triphenylphosphine (0.37 mmol, 0.10 g) and palladium acetate (0.18 mmol,
0.04 g) were added under nitrogen atmosphere to a solution of N-(6-
bromopyridin-3-yl)-2-phenylbutanamide 15 (4.40 mmol, 1.50 g), sodium
acetate trihydrate (11.43 mmol, 1.55 g), water (0.5 mL) and n-butyl acrylate
(8.79 mmol, 1.26 mL) in anhydrous N,N-dimethylformamide (8.5 mL) and
in a sealed tube. The resulting mixture was stirred at 140°C overnight. The
6
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10.1002/cmdc.202000854
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FULL PAPER
reaction was quenched by water (50 mL) and extracted with ethyl acetate
(3 x 50 mL). The collected organic phases were washed with saturated
sodium chloride solution (100 mL), dried, and concentrated to obtain an
oily residue that was purified by silica gel chromatography eluting with ethyl
acetate/n-hexane 1/3 to provide pure 19 as a pale yellow solid.
General Procedure for the Synthesis of acids 21-23, 24a-f, and 26a-f.
Example: 6-(2-phenylbutanamido)nicotinic acid (26c).
PhCHCH2CH3CO), 2.05-2.12 (m, 1H, PhCHCH2CH3CO), 3.64 (t, 1H,
PhCHCO, J = 7.2 Hz), 6.56 (d, 1H, PyrCH=CHCO, J = 16 Hz), 7.13-7.43
(m, 6H, PyrCH=CHCO and benzene protons), 8.48 (d, 2H, pyridine proton,
J = 5 Hz), 8.62 (s, 1H, pyridine proton), 9.03 (bs, 1H, CONHOH), 10.66 (s,
1H, CONHOH), 10.85 (s, 1H, CONHPyr).
(8)
(E)-N-(6-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)pyridin-3-yl)-2-
A mixture of methyl 6-(2-phenylbutanamido)nicotinate 25c (0.57 mmol,
0.20g), THF (15 mL), and 2 N KOH (1.14 mmol, 0.48 g) was stirred at room
temperature overnight. Afterward, THF was removed under reduced
pressure, and the residue was poured into water (50 mL) and extracted
with ethyl acetate (3 x 30 mL). The aqueous layer was acidified by adding
2 N HCl at 0 °C; the obtained precipitate was filtered and dried to give
compound 26c.
Mp: 254-256 °C; yield: 95.7%; rec solv: methanol 1H-NMR (DMSO) δ 0.61
(d, 3H, CH(CH3)2, J = 6.6 Hz), 0.97 (d, 3H, CH(CH3)2, J = 6.4 Hz), 2.35-
2.32 (m, 1H, CH(CH3)2), 3.5 (d, 1H, PhCHCO, J = 10.7 Hz), 7.21 (t, 1H,
benzene proton, J = 7.0 Hz), 7.29 (t, 2H, benzene protons, J = 7.3 Hz),
7.37 (d, 2H, benzene protons, J = 7.8 Hz), 8.14-8.17 (m, 2H, pyridine
protons), 8.75 (s, 1H, pyridine protons), 11.02 (s, 1H, CONH), 13.21 (s, 1H,
COOH).
phenylbutanamide
Mp: 69-71 °C; yield: 59.8%; rec solv: cyclohexane/benzene 1H NMR
(DMSO) δ 0.87 (t, 3H, PhCHCH2CH3CO, J = 7.5 Hz), 1.70-1.77 (m, 1H,
PhCHCH2CH3CO), 2.03-2.08 (m, 1H, PhCHCH2CH3CO), 3.62 (t, 1H,
PhCHCO, J = 8 Hz), 6.80 (d, 1H, PyrCH=CHCO, J = 16 Hz), 7.23-7.42 (m,
6H, PyrCH=CHCO and benzene protons), 7.53 (d, 1H, pyridine proton, J
= 8 Hz), 8.08 (d, 1H, pyridine proton, J = 8.3 Hz), 8.77 (s, 1H, pyridine
proton), 9.31 (bs, 1H, CONHOH), 10.54 (s, 1H, CONHOH), 10.86 (s, 1H,
CONHPyr).
(9a) (E)-N-hydroxy-3-(6-(2-phenylacetamido)pyridin-3-yl)acrylamide
Mp: 195-197 °C; yield: 67.3%; rec solv: acetonitrile 1H NMR (DMSO) δ
3.70 (s, 2H, PhCH2CO), 6.42 (d, 1H, PyrCH=CHCO, J = 15.4 Hz), 7.21-
7.30 (m, 5H, benzene protons), 7.40 (d, 1H, PyrCH=CHCO, J = 15.5 Hz),
7.94 (d, 1H, pyridine proton, J = 8.4 Hz), 8.06 (d, 1H, pyridine proton, J =
8.5 Hz), 8.46 (s, 1H, pyridine proton), 9.00 (bs, 1H, CONHOH), 10.71 (s,
1H, CONHOH), 10.84 (s, 1H, CONHPyr).
General procedure for the synthesis of acrylamides 6-8, 9a-f and 11a-
f.
Example:
(E)-N-hydroxy-3-(6-(2-(naphthalen-2-
yl)acetamido)pyridin-3-yl)acrylamide (9f).
Ethyl chloroformate (1.26 mmol, 0.12 mL) and Et3N (1.37 mmol, 0.19 mL)
were added to
a
cooled (0 °C) solution of (E)-N-hydroxy-3-(6-(2-
(9b)
(E)-N-hydroxy-3-(6-(2-phenylpropanamido)pyridin-3-
(naphthalen-2-yl)acetamido)pyridin-3-yl)acrylic acid 24f (1.05 mmol, 0.42
g) in THF dry (10 mL) and the mixture was stirred for 10 min. The solid
was filtered off, and O-(2-methoxy-2-propyl)hydroxylamine (3.15 mmol,
0.23 mL) was added to the filtrate. The solution was stirred for 15 min at
0 °C, then was evaporated under reduced pressure, and the residue was
diluted in MeOH (10 mL). Amberlyst 15 ion-exchange resin (0.42 mg) was
added to the solution of the O-protected hydroxamate, and the mixture was
stirred at RT for 1 h. Afterward, the mixture was filtered, and the filtrate
was concentrated in vacuo to give the crude 9f, which was purified by
crystallization.
yl)acrylamide
Mp: 149-151; yield: 87.0%; rec solv: benzene 1H NMR (DMSO) δ 0.84 (t,
3H, PhCHCH2CH3CO, J = 7.3 Hz), 1.67-1.71 (m, 1H, PhCHCH2CH3CO),
2.01-2.04 (m, 1H, PhCHCH2CH3CO), 3.78 (t, 1H, PhCHCO, J = 8.5 Hz),
6.44 (d, 1H, PyrCH=CHCO, J = 15.9 Hz), 7.21-7.40 (m, 6H, PyrCH=CHCO
and benzene protons), 7.95 (d, 1H, pyridine proton, J = 8 Hz), 8.12 (d, 1H,
pyridine proton, J = 8.3 Hz), 8.45 (s, 1H, pyridine proton), 9.02 (bs, 1H,
CONHOH), 10.73 (s, 1H, CONHOH), 10.82 (s, 1H, CONHPyr).
(9c) (E)-N-(5-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)pyridin-2-yl)-3-
General procedure for the synthesis of nicotinamides 10a-f and 12a-
methyl-2 phenylbutanamide
Mp: 178-180; yield: 59.8%; rec solv: acetonitrile H NMR (DMSO) δ 0.61
1
f.
Example:
(E)-N-(2-aminophenyl)-3-(6-(2-(naphthalen-1-
yl)acetamido)pyridin-3-yl)acrylamide (10e).
(bs, 3H, CH3CHCH3), 0.96 (t, 3H, CH3CHCH3), 2.32 (m, 1H, CH3CHCH3),
3.05 (m, 1H, PhCHCO), 6.41 (d, 1H, PyrCH=CHCO, J = 0.6 Hz), 7.19-7.37
(m, 6H, PyrCH=CHCO and benzene protons), 7.93 (d, 1H, pyridine proton,
J = 1.5 Hz), 8.08 (s, 1H, pyridine proton), 8.41 (s, 1H, pyridine proton), 9.01
(bs, 1H, CONHOH), 10.70 (s, 1H, CONHOH), 10.83 (s, 1H, CONHPyr).
Et3N (2.24 mmol, 0.31 mL) and BOP reagent (0.67 mmol, 0.30 g) were
added under N2 atmosphere to a solution of (E)-3-(6-(2-(naphthalen-1-
yl)acetamido)pyridin-3-yl)acrylic acid 24e in DMF dry (3 mL), and the
resulting mixture was stirred for 30 min at room temperature. After this time,
1,2-phenylendiamine (0.56 mmol, 0.06 g) was added. After the completion
of the reaction, the mixture was quenched with water (30 mL) and
extracted with ethyl acetate (3 x 30 mL). The combined organic layers were
washed with brine, dried over anhydrous sodium sulfate, and concentrated
under reduced pressure. The obtained residue was purified by
chromatography on silica gel eluting with EtOAc/CHCl3 1:1 to give
compound 10e as a white solid.
(9d)
(E)-3-(6-(2,3-diphenylpropanamido)pyridin-3-yl)-N-
hydroxyacrylamide
Mp: 201-203 °C; yield: 78.5%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 2.90-2.95 (m, 1H, CHCH2Ph), 3.36-3.42 (m, 1H, CHCH2Ph),
4.25-4.27 (m, 1H, PhCHCO), 6.40 (d, 1H, PyrCH=CHCO, J = 16.0 Hz),
7.10-7.43 (m, 11H, benzene protons and PyrCH=CHCO), 7.92 (d, 1H,
pyridine proton, J = 8.0 Hz), 8.04 (d, 1H, pyridine proton, J = 8.0 Hz), 8.40
(s, 1H, pyridine proton), 9.00 (bs, 1H, CONHOH), 10.67 (s, 1H, CONHOH),
10.78 (s, 1H, CONHPyr).
Chemical and physical data of compounds 6-8, 9a-f, 10a-f, 11a-f, 12a-
f, 13-15, 16a-f, 17-19, 20a-f, 21-23, 24a-f, 25a-f, 26a-f are in Supporting
info (Table S1-9).
1H NMR spectral data of compounds 6-8, 9a-f, 10a-f, 11a-f, 12a-f.
(9e)
(E)-N-hydroxy-3-(6-(2-(naphthalen-1-yl)acetamido)pyridin-3-
yl)acrylamide
(6)
(E)-N-(6-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)pyridin-2-yl)-2-
Mp: 212-214°C; yield: 68.4%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 4.22 (s, 2H, NaphtCH2CO), 6.42 (d, 1H, PyrCH=CHCO, J = 8.0
Hz), 7.40-7.52 (m, 6H, PyrCH=CHCO and aromatic protons), 7.82 (d, 1H,
aromatic proton, J = 4.0 Hz), 7.89-7.94 (m, 1H, aromatic protons), 8.03 (d,
1H, pyridine proton, J = 4 Hz), 8.09 (d, 1H, pyridine proton, J = 8.0 Hz),
8.49 (s, 1H, pyridine proton), 9.01 (bs, 1H, CONHOH), 10.71 (s, 1H,
CONHOH), 10.99 (s, 1H, CONHPyr).
phenylbutanamide
Mp: 143-145 °C; yield: 67.3%; rec solv: benzene 1H NMR (DMSO) δ 0.82
(t, 3H, PhCHCH2CH3CO, J = 8 Hz), 1.63-1.67 (m, 1H, PhCHCH2CH3CO),
2.01-2.05 (m, 1H, PhCHCH2CH3CO), 3.80 (t, 1H, PhCHCO, J = 8 Hz), 6.76
(d, 1H, PyrCH=CHCO, J = 16 Hz), 7.18-7.37 (m, 7H, PyrCH=CHCO and
benzene protons), 7.75 (t, 1H, pyridine proton, J = 8 Hz), 8.01 (d, 1H,
pyridine proton, J = 8 Hz), 9.02 (bs, 1H, CONHOH), 10.59 (s, 1H,
CONHOH), 10.82 (s, 1H, CONHPyr).
(9f)
(E)-N-hydroxy-3-(6-(2-(naphthalen-2-yl)acetamido)pyridin-3-
yl)acrylamide
(7)
(E)-N-(5-(3-(hydroxyamino)-3-oxoprop-1-en-1-yl)pyridin-3-yl)-2-
Mp: 167-169°C; yield: 65.7%; rec solv: benzene/acetonitrile 1H NMR
(DMSO) δ 3.89 (s, 2H, NaphtCH2CO), 6.44 (d, 1H, PyrCH=CHCO, J = 16.0
Hz), 7.38-7.50 (m, 4H, PyrCH=CHCO and aromatic protons), 7.85-7.94 (m,
4H, aromatic proton), 7.95 (d, 1H, pyridine proton, J = 4.0 Hz), 8.07 (d, 1H,
phenylbutanamide
Mp: 190-192 °C; yield: 87.0%; rec solv: acetonitrile 1H NMR (DMSO) δ
0.88 (t, 3H, PhCHCH2CH3CO,
J = 7.5 Hz), 1.71-1.78 (m, 1H,
7
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10.1002/cmdc.202000854
ChemMedChem
FULL PAPER
pyridine proton, J = 4.0 Hz), 8.47 (s, 1H, pyridine proton), 10.03 (bs,
(d, 1H, pyridine proton, J = 4.0 Hz), 8.53 (s, 1H, pyridine proton), 9.34 (s,
1H,CONHOH ), 10.85 (s, 1H, CONHOH ), 10.97 (s, 1H, CONHPyr).
1H, CONHPyr), 10.97 (s, 1H, CONHaniline).
(10a)
yl)acrylamide
(E)-N-(2-aminophenyl)-3-(6-(2-phenylacetamido)pyridin-3-
(11a) N-hydroxy-6-(2-phenylacetamido)nicotinamide
Mp: 199-201°C; yield: 67.3%; rec solv: acetonitrile 1H NMR (DMSO) δ 3.71
(s, 2H, PhCH2CO), 7.21-7.30 (m, 5H, benzene protons), 8.06 (s, 2H,
pyridine proton), 8.64 (s, 1H, pyridine proton), 9.07 (bs, 1H, NHOH), 10.94
(s, 1H, NHOH), 11.22 (s, 1H, CONHPyr).
Mp: 215-217°C; yield: 61.3%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 3.71-3.74 (m, 2H, PhCH2CO), 4.97 (bs, 2H, PhNH2), 6.54 (d,
1H, PyrCH=CHCO, J = 8.0 Hz), 6.72 (d, 1H, PyrCH=CHCO, J = 8.0 Hz),
6.90 (m, 1H, aniline proton), 7.21-7.24 (m, 5H, benzene protons), 7.31-
7.32 (m, 1H, aromaric proton), 7.34-7-35 (m, 1H, aromatic proton), 7.52-
7.53 (m, 3H, aromatic proton), 8.79 (s, 1H, pyridine proton), 9.34 (s, 1H,
CONHPyr), 10.90 (s, 1H, CONHaniline).
(11b) N-hydroxy-6-(2-phenylpropanamido)nicotinamide
Mp: 169-171°C; yield: 87.0%; rec solv: benzene/acetonitrile 1H NMR
(DMSO) δ 0.81 (t, 3H, PhCHCH2CH3, J = 16.0 Hz), 1.65-1.68 (m, 1H,
PhCHCH2CH3CO), 2.01-2.04 (m, 1H, PhCHCH2CH3CO), 3.76 (t, 1H,
PhCHCO, J = 16.0 Hz), 7.20-7.37 (m, 5H, benzene protons), 8.05 (d, 1H,
pyridine proton, J = 12.0 Hz), 8.10 (d, 1H, pyridine proton, J = 12.0 Hz),
8.62 (s, 1H, pyridine proton), 9.05 (bs, 1H, NHOH), 10.90 (s, 1H, NHOH),
11.21 (s, 1H, CONHPyr).
(10b) (E)- N-(2-aminophenyl)-3-(6-(2-phenylpropanamido)pyridin-3-
yl)acrylamide
Mp: 172-174°C; yield: 77.4%; rec solv: acetonitrile 1H NMR (DMSO) δ 0.85
(t, 3H, PhCHCH2CH3CO, 7.2 Hz), 1.69-1.72 (m, 1H, PhCHCH2CH3CO),
2.05-2.09 (m, 1H, PhCHCH2CH3CO), 3.81 (t, 1H, PhCHCO, J = 4.0 Hz),
4.93 (bs, 2H, PhNH2), 6.57 (t, 1H, aniline proton, J = 16.0 Hz), 6.74 (d, 1H,
aniline proton, J = 8.0 Hz), 6.87-6.93 (m, 2H, PyrCH=CHCO and aniline
proton), 7.22-7.25 (m, 1H, aniline proton), 7.30-7.54 (m, 5H, benzene
protons + 1H PyrCH=CHCO), 8.01 (d, 1H, pyridine proton, J = 8.0 Hz),
8.17 (d, 1H, pyridine róton, J = 12.0 Hz), 8.52 (s, 1H, pyridine proton), 9.36
(s, 1H, CONHPyr), 10.87 (s, 1H, CONH aniline).
(11c) N-hydroxy-6-(3-methyl-2-phenylbutanamido)nicotinamide
Mp: 132-134°C; yield: 59.8%; rec solv: benzene 1H NMR (DMSO) δ 0.62
(d, 3H, CH3CHCH3, J = 4.0 Hz), 0.97 (d, 3H, CH3CHCH3, J = 4.0 Hz), 2.35-
2.37 (m, 1H, CH3CHCH3), 3.47 (d, 1H, PhCHCO, J = 8.0 Hz), 7.20-7.38
(m, 5H, benzene protons), 8.04 (d, 1H, pyridine proton, J = 8.0 Hz), 8.09
(d, 1H, pyridine proton, J = 8.0 Hz), 8.62 (s, 1H, pyridine proton), 9.10 (bs,
1H, NHOH), 10.91 (s, 1H, NHOH), 11.15 (s, 1H,CONHPyr).
(10c)
(E)-N-(5-(3-((2-aminophenyl)amino)-3-oxoprop-1-en-1-
yl)pyridin-2-yl)-3-methyl-2-phenylbutanamide
(11d) 6-(2,3-diphenylpropanamido)-N-hydroxynicotinamide
Mp: 195-197°C; yield: 78.5%; rec solv: acetonitrile 1H NMR (DMSO) δ
2.91-2.95 (m, 1H, CHCH2Ph), 3.37-3.41 (m, 1H, CHCH2Ph), 4.24-4.26 (m,
1H, PhCHCO), 7.10-7.43 (m, 10H, benzene protons), 8.04 (d, 1H, pyridine
proton, J = 4.0 Hz), 8.09 (d, 1H, pyridine proton, J = 8.0 Hz), 8.58 (s, 1H,
pyridine proton), 9.10 (bs, 1H,NHOH), 10.87 (s, 1H, NHOH), 11.20 (s,
1H,CONHPyr).
Mp: 204-206°C; yield: 59.2%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 0.62 (d, 3H, CH3CHCH3, J = 8.0 Hz), 0.98 (d, 3H, CH3CHCH3,
J = 4.0 Hz), 2.30-2.33 (m, 1H, CH3CHCH3), 3.48 (d, 1H, PhCHCO, J = 12
Hz), 4.91 (bs, 2H, PhNH2), 6.54 (t, 1H, aniline proton, J = 12.0 Hz), 6.71
(d, 1H, aniline proton, J = 8.0 Hz), 6.83-6.89 (m, 2H, PyrCH=CHCO and
aniline proton), 7.20-7.22 (m, 1H, aniline proton), 7.27-7.39 (m, 5H,
benzene protons), 7.49 (d, 1H, PyrCH=CHCO, J = 16.0 Hz), 7.97 (d, 1H,
pyridine proton, J = 8.0 Hz), 8.14 (d, 1H, pyridine proton, J = 12.0 Hz), 8.49
(s, 1H, pyridine proton), 9.32 (s, 1H, CONHPyr), 10.86 (bs, 1H,
CONHaniline).
(11e) N-hydroxy-6-(2-(naphthalen-1-yl)acetamido)nicotinamide
Mp: 221-223°C; yield: 68.4%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 4.24 (s, 2H, NaphtCH2CO), 7.49-8.09 (m, 7H, aromatic protons),
8.17 (d, 1H, pyridine proton, J = 4.0 Hz), 8.67 (d, 1H, pyridine proton, J =
4.0 Hz), 8.82 (s, 1H, pyridine proton), 9.08 (bs, 1H, NHOH), 11.08 (s,
1H,NHOH), 11.22 (s, 1H,CONHPyr ).
(10d) (E)-N-(2-aminophenyl)-3-(6-(2,3-diphenylpropanamido)pyridin-
3-yl)acrylamide
Mp: 180-182°C; yield: 78.5%; rec solv: acetonitrile 1H NMR (DMSO) δ
2.90-2.95 (m, 1H, CHCH2Ph), 3.37-3.41 (m, 1H, CHCH2Ph), 4.27-4.29 (m,
1H, PhCHCO), 4.90 (bs, 2H, PhNH2), 6.53 (t, 1H, aniline proton, J = 8.0
Hz), 6.71 (d, 1H, aniline proton, J = 8.0 Hz), 6.82-6.87 (m, 2H,
PyrCH=CHCO and aniline proton), 7.11-7.12 (m, 1H, aniline proton), 7.19-
7.22 (m, 5H, benzene protons), 7.27-7.31 (m, 3H, benzene protons), 7.42-
7.48 (m, 3H, PyrCH=CHCO and benzene protons), 7.96 (d, 1H, pyridine
proton, J = 8.0 Hz), 8.09 (d, 1H, pyridine proton, J = 8.0 Hz), 8.45 (s, 1H,
pyridine proton), 9.32 (s, 1H, CONHPyr), 10.82 (s, 1H, CONHaniline).
(11f) N-hydroxy-6-(2-(naphthalen-2-yl)acetamido)nicotinamide
Mp: 217-219°C; yield: 65.7%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 3.92 (s, 2H, NaphtCH2CO), 7.46-8.01 (m, 7H, aromatic protons),
8.07 (d, 1H, pyridine proton, J = 4.0 Hz), 8.11 (d, 1H, pyridine proton, J =
4.0 Hz), 8.65 (s, 1H, pyridine proton), 9.08 (bs, 1H, NHOH), 11.02 (s, 1H,
NHOH), 11.22 (s, 1H, CONHPyr).
(12a) N-(2-aminophenyl)-6-(2-phenylacetamido)nicotinamide
Mp: 238-240°C; yield: 61.3%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 3.73 (s, 2H, PhCH2CO), 4.91 (bs, 2H, PhNH2), 6.55 (t, 1H,
aniline proton, J = 4.0 Hz), 6.73 (d, 1H, aniline proton, J = 8.0 Hz), 6.94 (t,
1H, aniline proton, J = 8.0 Hz), 7.22 (d, 1H, aniline proton, J = 8.0 Hz),
7.28-7.32 (m, 5H, benzene protons), 8.12 (d, 1H, pyridine proton, J = 8.0
Hz), 8.28 (d, 1H, pyridine proton, J = 8.0 Hz), 8.89 (s, 1H, pyridine proton),
9.67 (s, 1H, CONHPyr), 11.00 (s, 1H, CONHaniline).
(10e)
(E)-N-(2-aminophenyl)-3-(6-(2-(naphthalen-1-
yl)acetamido)pyridin-3-yl)acrylamide
Mp: 155-157°C; yield: 58.3%; rec solv: benzene/acetonitrile 1H NMR
(DMSO) δ 4.23 (s, 2H, NaphtCH2CO), 4.90 (bs, 2H, PhNH2), 6.53 (t, 1H,
aniline proton, J = 8.0 Hz), 6.71 (d, 1H, aniline proton, J = 8.0 Hz), 6.84-
6.87 (m, 2H, aniline protons), 7.30 (d, 1H, PyrCH=CHCO, J = 8.0 Hz),
7.43-7.55 (m, 5H, aromatic protons), 7.82 (d, 1H, PyrCH=CHCO, J = 8.0
Hz), 7.91 (d, 1H, aromatic proton, J = 8.0 Hz), 7.98 (d, 1H, aromatic proton,
J = 4.0 Hz), 8.06-8.12 (m, 2H, pyridine protons), 8.54 (s, 1H, pyridine
proton), 9.33 (s, 1H, CONHPyr), 11.02 (s, 1H, CONHaniline).
(12b) N-(2-aminophenyl)-6-(2-phenylpropanamido)nicotinamide
Mp: 98-100°C; yield: 77.4%; rec solv: cyclohexane/benzene 1H NMR
(DMSO) δ 0.82 (t, 3H, PhCHCH2CH3CO, J = 16.0 Hz), 1.68-1.70 (m, 1H,
PhCHCH2CH3CO), 2.05-2.07 (m, 1H, PhCHCH2CH3CO), 3.79 (t, 1H,
PhCHCO, J = 16.0 Hz), 4.90 (bs, 2H, PhNH2), 6.56 (t, 1H, aniline proton,
J = 8.0 Hz), 6.73 (d, 1H, aniline proton, J = 8.0 Hz), 6.93 (t, 1H, aniline
proton, J = 4.0 Hz), 7.11 (d, 1H, aniline proton, J = 8.0 Hz), 7.21-7.39 (m,
5H, benzene protons), 8.15 (d, 1H, pyridine proton, J = 8.0 Hz), 8.29 (d,
1H, pyridine proton, J = 12.0 Hz), 8.87 (s, 1H, pyridine proton), 9.67 (s, 1H,
CONHPyr), 10.96 (s, 1H, CONHaniline).
(10f)
(E)-N-(2-aminophenyl)-3-(6-(2-(naphthalen-2-
yl)acetamido)pyridin-3-yl)acrylamide
Mp: 238-240°C; yield: 72.8%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 3.90 (s, 2H, NaphtCH2CO), 4.91 (bs, 2H, PhNH2), 6.54 (t, 1H,
aniline proton, J = 4.0 Hz), 6.71 (d, 1H, aniline proton, J = 8.0 Hz), 6.84-
6.88 (m, 2H, PyrCH=CHCO and aniline proton), 7.30-7.31 (m, 1H, aniline
proton), 7.48-7.53 (m, 4H, PyrCH=CHCO and aromatic protons), 7.82-7.86
(m, 4H, aromatic protons), 8.00 (d, 1H, pyridine proton, J = 4.0 Hz), 8.10
(12c)
N-(2-aminophenyl)-6-(3-methyl-2-
phenylbutanamido)nicotinamide
8
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000854
ChemMedChem
FULL PAPER
Mp: 110-112°C; yield: 59.2%; rec solv: cyclohexane/benzene 1H NMR
(DMSO) δ 0.71 (d, 3H, CH3CHCH3, J = 8.0 Hz), 1.07 (d, 3H, CH3CHCH3,
J = 8.0 Hz), 2.45-2.47 (m, 1H, CH3CHCH3), 3.03 (d, 1H, PhCHCO, J = 4.0
Hz), 4.90 (bs, 2H, PhNH2), 6.78-6.80 (m, 2H, aniline protons), 7.03-7.06
(m, 1H, aniline proton), 7.24-7.33 (m, 6H, benzene protons and aniline
proton), 8.01 (d, 1H, pyridine proton, J = 4.0 Hz), 8.22 (d, 1H, pyridine
proton, J = 4.0 Hz), 8.37 (s, 1H, pyridine proton), 8.70 (s, 1H, CONHPyr),
9.92 (bs, 1H, CONH aniline).
software. IC50 values were calculated using the GraphPad Prism 4
program based on a sigmoidal dose-response equation.
Cellular assays
[16h]
Cell lines and cultures: U937 were grown as described in
whereas
K562, A549, and HCT116 cells were maintained as reported in [16j]
Cell-cycle analysis using U937 cells: 2.5x105 cells were harvested and
resuspended in 500 mL hypotonic buffer (0.1 % Triton X-100, 0.1 %
sodium citrate, 50 mg mL-1 propidium iodide (PI), RNAse A). Cells were
incubated in the dark for 30 min.. Cell death was measured as percentage
of cells in pre-G1 phase of the cell cycle. Samples were measured on a
FACS-Calibur flow cytometer using the Cell Quest software (Becton
Dickinson) and processed with the Cell Quest software (Becton Dickinson)
and ModFit LT ver. 3 software (Verity) as previously reported [16h, 21]. All
experiments were performed three times.
(12d) N-(2-aminophenyl)-6-(2,3-diphenylpropanamido)nicotinamide
Mp: 186-188°C; yield: 78.5%; rec solv: acetonitrile 1H NMR (DMSO) δ
2.93-2.98 (m, 1H, CHCH2Ph), 3.38-3.43 (m, 1H, CHCH2Ph), 4.26-4.30 (m,
1H, PhCHCO), 4.89 (bs, 2H, PhNH2), 6.54 (t, 1H, aniline proton, J = 8.0
Hz), 6.73 (d, 1H, aniline proton, J = 8.0 Hz), 6.92 (t, 1H, aniline proton, J =
8.0 Hz), 7.11 (d, 2H, aniline proton and benzene proton, J = 12.0 Hz), 7.19-
7.23 (m, 5H, benzene protons), 7.28-7.32 (m, 2H, benzene protons), 7.43-
7.45 (m, 2H, benzene protons), 8.11 (d, 1H, pyridine proton, J = 8.0 Hz),
8.26 (d, 1H, pyridine proton, J = 8.0 Hz), 8.83 (s, 1H, pyridine proton), 9.64
(s, 1H, CONHPyr), 10.93 (s, 1H, CONHaniline).
Granulocytic differentiation in U937 cells: Granulocytic differentiation was
[16h,
carried out as previously described
22]. Briefly, U937 cells were
(12e)
N-(2-aminophenyl)-6-(2-(naphthalen-1-
harvested and resuspended in 10mL phycoerythrin-conjugated CD11c
(CD11c-PE). Control samples were incubated with 10 mL PE-conjugated
mouse IgG1 for 30 min at 4°C in the dark, washed in PBS, and
resuspended in 500 mL PBS containing PI (0.25 μg mL-1). Samples were
analyzed by FACS with Cell Quest technology (Becton Dickinson). PI-
positive cells were excluded from the analysis.
yl)acetamido)nicotinamide
Mp: 218-220°C; yield: 58.3%; rec solv: acetonitrile/methanol 1H NMR
(DMSO) δ 4.25 (s, 2H, NaphtCH2CO), 4.91 (bs, 2H, PhNH2), 6.55 (t, 1H,
aniline proton, J = 16.0 Hz), 6.74 (d, 1H, aniline proton, J = 12.0 Hz), 6.94
(t, 1H, aniline proton, J = 16.0 Hz), 7.11 (d, 1H, aniline proton, J = 8.0 Hz),
7.44-7.53 (m, 4H, aromatic protons), 7.83 (d, 1H, aromatic protons, J = 8.0
Hz), 7.91 (d, 1H, aromatic protons, J = 8.0 Hz), 8.07-8.12 (m, 2H, aromatic
proton and pyridine proton), 8.27 (d, 1H, pyridine proton, J = 8.0 Hz), 8.53
(s, 1H, pyridine proton), 8.91 (s, 1H, CONHPyr), 11.16 (s, 1H,
CONHaniline).
Antiproliferative activity: The antiproliferative effect of the HDAC inhibitors
on cell proliferation was evaluated against K562 (chronic myeloid
carcinoma), A549 (non-small-cell lung cancer), and HCT-116 (human
colon cancer) tumor cell lines using the CellTiter-Glo luminescent cell
viability assay (Promega, Madison, WI) according to the manufacturer's
instructions. K562, A549, and HCT-116 cells, in exponential growth, were
incubated for 72 h at different concentrations of the inhibitors. Then an
equivalent of the CellTiter-Glo reagent was added, the solution was mixed
for 2 min in order to induce cell lysis, and the luminescence was recorded
after a further 10 min. The IC50 was calculated using GraphPad software
(12f)
N-(2-aminophenyl)-6-(2-(naphthalen-2-
yl)acetamido)nicotinamide
Mp: >250°C; yield: 72.8%; rec solv: methanol 1H NMR (DMSO) δ 3.92 (s,
2H, NaphtCH2CO), 4.90 (bs, 2H, PhNH2), 6.55 (t, 1H, aniline proton, J =
16.0 Hz), 6.74 (d, 1H, aniline proton, J = 12.0 Hz), 6.94 (t, 1H, aniline
proton, J = 12.0 Hz), 7.11 (d, 1H, aniline proton, J = 8.0 Hz), 7.46-7.50 (m,
3H, aromatic protons), 7.83-7.86 (m, 4H, aromatic protons), 8.13 (d, 1H,
pyridine proton, J = 8.0 Hz), 8.28 (d, 1H, pyridine proton, J= 8.0 Hz), 8.90
(s, 1H, pyridine proton), 9.66 (s, 1H, CONHPyr), 11.08 (s, 1H,
CONHaniline).
[16j]
.
Histone Extraction: After stimulation with compounds, U937 cells were
collected by centrifugation and washed two times with PBS. Then the
samples were resuspended in Triton extraction buffer [TEB; PBS
containing 0.5% Triton X-100 (v/v), 2 mM PMSF, and 0.02% (w/v) NaN3)],
and the lysis was performed for 10 min at 4 °C. Next, samples were
centrifugated at 2000 rpm for 10 min at 4 °C, and the pellets were washed
in TEB (half the volume). After a new centrifugation under the same
conditions, the samples were resuspended in 0.2 N HCl and the acidic
histone extraction was carried out overnight at 4 °C.
Mouse HDAC1 Enzyme Assay: For inhibition assay, partially purified
HDAC1 from mouse A20 cells (ATCC: TIB208) (anion exchange
chromatography, affinity chromatography) was used as enzyme source.
HDAC activity was determined as described
prelabeled chicken reticulocyte histones as substrate. 50 µL of mouse
HDAC1 enzyme fractions were incubated with different concentrations of
compounds for 15 min on ice, and 10 µL of total [3H] acetate-prelabeled
chicken reticulocyte histones (4 mg/mL) were added, resulting in a
concentration of 41 µM. The mixture was incubated at 37 °C for 1 h. The
reaction was stopped by the addition of 50 µL of 1 M HCl/0.4 M
acetylacetate and 1 mL ethyl acetate. After centrifugation at 10000g for 5
min, an aliquot of 600 µL of the upper phase was counted for radioactivity
in 3 mL of liquid scintillation cocktail.
[19]
using [3H] acetate-
Total proteins extraction: U937 cells were harvested and washed once with
cold PBS and lysed in a lysis buffer containing 50mM Tris-HCl pH 7.4,
150mM NaCl, 1%NP40, 10mM NaF, 1mM PMSF (phenyl-
methylsulphonylfluoride) and protease inhibitor cocktail (Roche). The
lysates were centrifuged at 13000 rpm for 30 min at 4°C. Protein
concentrations were estimated by Bradford assay (Bio-Rad).
Western Blot Analysis: 30 μg total protein and 2 μg histone extracts were
denatured and boiled in buffer (0.25 M Tris-HCl, pH 6.8, 8% SDS, 40%
glycerol, 5% 2-mercaptoethanol, and 0.05% bromophenol blue) for 3 min
before electrophoresis. Proteins were subjected to sodium dodecyl sulfate-
polyacrylamide gel electrophoresis in Tris-glycine–SDS buffer (25 mM Tris,
192 mM glycine 0.1% SDS). After electrophoresis, total proteins and
histones were transferred to nitrocellulose membranes (Bio-Rad mini-
protean gel and Transblot Turbo transfer system, Bio-Rad).
The employed antibodies were acetylated histone H3 (Millipore, 06-599)
and Acetyl-tubulin (SIGMA, T7451). Total H4 (Cell Signaling, 2935S) was
used to normalize for equal loading of histone extracts, while GAPDH
(Santa Cruz, sc-47724) was used to normalize for equal loading of total
protein extraction
HDAC1, -3, -4, -6, -8 isoform inhibition assay: The described
compounds were tested against purified hrHDAC1, -3, -4, -6, and -8 in 10-
dose IC50 mode with 3-fold serial dilution starting from 100 μM solutions.
Three fluorogenic peptides, from p53 residues 379-382 (RHKK(Ac)AMC)
(for HDAC1, -3, -6), or the fluorogenic class IIa (Boc-Lys(trifluoroacetyl)-
[20]
AMC) substrate (for HDAC4)
the diacetylated p53 residues 379-382
(RHK(Ac)K(Ac)AMC) (for HDAC8) were used as substrates, and SAHA
was employed as reference compound and positive control. Deacetylated
peptides were sensitive towards lysine peptidase, and free fluorogenic 4-
methylcoumarin-7-amide (MCA) was generated, which can be excited at
355 nm and observed at 460 nm. The data were analyzed on a plate to
plate basis in relationship to the control and imported into analytical
9
This article is protected by copyright. All rights reserved.

10.1002/cmdc.202000854
ChemMedChem
FULL PAPER
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Acknowledgments
This work was supported by Ricerca Finalizzata 2013 PE-2013-
02355271, AIRC 2016 (n. 19162), Vanvitelli per la Ricerca
“AdipCare”
(ID
263), Campania
Regional
Government
Technology Platform Lotta alle Patologie Oncologiche: iCURE
(B21C17000030007), Campania Regional Government FASE2:
IDEAL (B63D18000560007), MIUR, Proof of Concept
POC01_00043, VALERE 2020 “NETWINS” funds.
Keywords: chromatin • histone deacetylase inhibitors • cancer•
apoptosis • cell differentiation
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10.1002/cmdc.202000854
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Entry for the Table of Contents
O
O
NHOH
N
N
H
11d
Highest and specific HDAC6
inhibition, IC50: 0.5 nM
Highest apoptosis induction
Highest cytodifferentiation
A novel pyridine-based hydroxamates and 2'-aminoanilides as HDAC inhibitors have been described. Among them, the nicotinic
hydroxamates, in particular 11d, proved very high potent inhibition against HDAC6 (IC50: 0.5 nM) with a selectivity index ranging from
100- to 30000-fold over all other HDACs. 11c displayed the highest apoptosis induction whereas the 2'-aminoanilide 12c was the
best to induce cytodifferentiation in leukemia U937 cells.
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