Novel uracil-based 2-aminoanilide and 2-aminoanilide-like derivatives: Histone deacetylase inhibition and in-cell activities

Article abstract of DOI:10.1016/j.bmcl.2008.03.055

A novel series of non-hydroxamate HDAC inhibitors (HDACi) showing a uracil group at the left and a 2-aminoanilide/2-aminoanilide-like portion at the right head have been reported. In particular, the new compounds incorporating a 2-aminoanilide moiety behaved as class I-selective HDACi. Compound 8, the most potent and class I-selective, showed weak apoptosis (higher than MS-275) joined to cytodifferentiating activity on U937 cells. Surprisingly, the highest differentiation was observed with 13, through an effect that seems to be unrelated to HDAC inhibition.

Full text of DOI:10.1016/j.bmcl.2008.03.055

Available online at www.sciencedirect.com
Bioorganic & Medicinal Chemistry Letters 18 (2008) 2530–2535
Novel uracil-based 2-aminoanilide and 2-aminoanilide-like
derivatives: Histone deacetylase inhibition and in-cell activities
Antonello Mai,a,* Andrea Perrone,a Angela Nebbioso,b Dante Rotili,a Sergio Valente,a
Maria Tardugno,a Silvio Massa,c Floriana De Bellisb,d and Lucia Altuccib,*
aIstituto Pasteur—Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici,
`
Sapienza Universita di Roma, P.le A. Moro 5, 00185 Roma, Italy
`
b
Dipartimento di Patologia Generale, Seconda Universita degli Studi di Napoli, vico L. De Crecchio 7, 80138 Napoli, Italy
c
`
Dipartimento Farmaco Chimico Tecnologico, Universita degli Studi di Siena, via A. Moro, 53100 Siena, Italy
dDepartment of Cancer Biology, IGBCM, Strasbourg, France
Received 4 February 2008; revised 18 March 2008; accepted 18 March 2008
Available online 22 March 2008
Abstract—A novel series of non-hydroxamate HDAC inhibitors (HDACi) showing a uracil group at the left and a 2-aminoanilide/2-
aminoanilide-like portion at the right head have been reported. In particular, the new compounds incorporating a 2-aminoanilide
moiety behaved as class I-selective HDACi. Compound 8, the most potent and class I-selective, showed weak apoptosis (higher than
MS-275) joined to cytodifferentiating activity on U937 cells. Surprisingly, the highest differentiation was observed with 13, through
an effect that seems to be unrelated to HDAC inhibition.
Ó 2008 Elsevier Ltd. All rights reserved.
Chromatin remodelling is involved in the epigenetic reg-
ulation of gene expression.1,2 This mechanism includes a
growing number of families of enzymes that catalyse
several covalent modifications (acetylation/deacetyla-
tion, methylation/demethylation, phophorylation, ubiq-
uitilation, ADP-ribosylation, etc.) at the histone tails of
nucleosomal histones H3 and H4.3,4 In particular, his-
tone deacetylases (HDACs) catalyse the removal of the
N-acetyl lysine residues from the histone tails, thus
changing the accessibility of transcription factors to
DNA and switching the chromatin in its heterochroma-
tin form, transcriptionally silent.5–7 The inhibition of
HDACs restores the recruitment of transcription factors
to DNA and silences the growth of cancer cells by
inducing pathways that lead to growth arrest, terminal
differentiation and apoptosis, both in in vitro and in
in vivo assays.8–11 In humans, 18 HDACs have been
identified and grouped into four classes according to
their homology with the yeast deacetylases. Class I, II
and IV HDACs are zinc-binding enzymes, whereas class
III HDACs (sirtuins) have a NAD+-dependent mecha-
nism of deacetylation, do not share any homology with
class I, II, IV HDACs, are not sensitive to class I, II, IV
HDAC inhibitors (HDACi), and do not have histones
as primary targets. Class I (HDAC1–3, 8) and class II
(HDAC4–7, 9, 10) HDACs also deacetylate a growing
number of non-histone proteins in addition to histones
such as transcription factors and proteins related to cell
proliferation, differentiation, migration and death.8–11
Class I and II HDACs are generally considered valuable
therapeutic targets for the treatment of leukemia and so-
lid tumors,8,11 and suberoylanilide hydroxamic acid
(SAHA) (Fig. 1) is the first class I/II HDACi approved
O
O
H
O
N
H
NH2
N
H
N
NHOH
N
O
O
SAHA
PAOA
MS-275
NH2
OH
H
H
N
H
N
H
N
N
O
O
O
O
MC1863
Keywords: Chromatin remodelling; Histone deacetylase; Aminoanilide;
Non-hydroxamate; Apoptosis; Cytodifferentiation.
Figure 1. HDACi belonging to the hydroxamate (SAHA) or 2-
aminoanilide/2-aminoanilide-like (MS-275, PAOA and MC1863)
series.
*
Corresponding authors. Tel.: +39 0649913392; fax: +39 06491491;
e-mail: antonello.mai@uniroma1.it
0960-894X/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmcl.2008.03.055

A. Mai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2530–2535
2531
by the FDA in October 2006 for the treatment of cuta-
neous T-cell lymphoma (CTCL), and it is currently in
clinical trial of Phase II or III (alone or in combination)
for the treatment of a great number of tumors.12,13 Class
I HDACs are ubiquitary, primarily with nuclear locali-
sation, and often act as transcriptional co-repressors,
whereas class II HDACs have a tissue-specific expression
and can shuttle between nucleus and cytoplasm. Mount-
ing evidences suggest distinct roles for different classes of
HDACs. Class I HDAC overexpression has been re-
ported for human gastric, prostate (HDAC1) and colon
tumors (HDAC3),14–16 whereas the knockdown of
HDAC1 and HDAC3 (but not HDAC4 and HDAC7)
with siRNA in HeLa cells deeply inhibited proliferation
and induced apoptosis.17 Moreover, by combining the
use of class-selective HDACi with the siRNA technique,
Cohen et al. reported that inhibition of HDAC1 and
HDAC2 but not HDAC3, HDAC6 and HDAC8 are pri-
marily responsible for sensitisation to TRAIL-induced
apoptosis in Jurkat, K562, U937, DU145 and CLL cell
lines.18 On the other hand, Altucci and co-workers clearly
demonstrated that HDAC1 and HDAC2 reside on the
TRAIL promoter in leukemia cells thus contributing
with its aberrant silencing in tumors.19,20 Finally, siR-
NA-mediated knockdown of HDAC1 or HDAC3 but
not HDAC2 (unless the concomitant ablation of both
HDAC1 and HDAC2 was made) led to an inhibition of
cell proliferation in U2OS, MCF7 and MCF10A cancer
cell lines.21 Thus, the development of class I-selective
HDACi seems to be a tool to evaluate if anticancer agents
with improved efficacy and weaker toxicity profile com-
pared to pan-HDACi could be obtained.22
In our hands, when tested in the U937 cells, PAOA
and MC1863 at 5 lM shared a inhibiting behaviour sim-
ilar to MS-275: they were active against HDAC1 [%
inhibition: 28.9 (PAOA), 31.7 (MC1863), 47.8 (SAHA),
43.7 (MS-275)] but not against HDAC4 [% inhibition: 0
(PAOA), 7.9 (MC1863), 31.4 (SAHA), 0 (MS-275)], they
both induced histone H3 and H4 (PAOA only) but not
a-tubulin hyperacetylation (Fig. 2), and they were highly
efficient cytodifferentiating agents [% CD11c positive
cells: 18.3 (PAOA), 36.6 (MC1863), 6.6 (SAHA), 30.8
(MS-275)], but very weak if at all pro-apoptotic agents
after 30 h of treatment (Fig. S1 in Supplementary
material).
Recently we reported a new series of uracil-based
hydroxamates (UBHAs) as novel HDACi.25,31–34 Two
of them (MC1641 and MC1751, 1d and 1j in Ref. 25,
respectively) were active against HDAC1 but not
against HDAC4, and were able to highly increase the
levels of both acetyl-H3 and acetyl-a-tubulin. In cellular
assays (U937 cells), they displayed some differentiation
activity and low apoptosis.25
Following our researches on HDACi,25,26,31–41 with the
aim to increase the class-I selectivity of our uracil deriv-
atives to obtain more efficient compounds in the cellular
tests, we designed and synthesised a new series of uracil-
based 2-aminoanilide and 2-aminoanilide-like deriva-
tives 1–13 (Fig. 3), and we determined, in addition to
the anti-HDAC activity, their effects on histone H3
and a-tubulin acetylation in the human leukemia U937
cell line. Moreover, p21WAF1/CIP1 induction as well as
the activities of the compounds 1–13 on cell cycle, apop-
tosis induction, and granulocytic differentiation in the
U937 cells have been assayed. The 2-aminoanilide and
2-aminoanilide-like derivatives 1–13 were synthesised
from the uracil-containing carboxylic acids 24–29 by
treatment with benzotriazole-1-yl-oxy-tris-(dimethyl-
MS-275 (Fig. 1) is a class I-selective HDACi from the
Bayer Schering AG Pharma group, that inhibits tumor
growth in vivo and promotes differentiation or apopto-
sis following the induction of the cyclin-dependent ki-
nase inhibitor p21WAF1/CIP1, the apoptotic TRAIL and
the generation of ROS species.23,24 In the U937 cell line,
MS-275 showed an enzymatic inhibition profile deeply
different from that of SAHA (Table 1).25,26
The main chemical difference between the two HDACi is
the zinc-binding group,27 which is the hydroxamate in
SAHA28 and probably the 2-aminoanilide portion in
MS-275.29 Thus, we prepared (see Supplementary
material for chemical and physical data) and tested in
both enzyme and cellular assays the pimeloylanilide
orthoaminoanilide (PAOA) (Fig. 1), described by Wong
et al.30 as class-I selective HDACi, and its 2-hydroxyani-
lide analogue MC1863 (Fig. 1), as 2-aminoanilide/2-
aminoanilide-like analogues of a SAHA homologue.
Figure 2. Effects of PAOA and MC1863 on histone H3/H4 and a-
tubulin acetylation and on p21 induction.
Table 1. Biological properties of SAHA and MS-275 in the human leukemia U937 cell linea
Compound
HDAC1 inhbtnb
HDAC4 inhbtnb
Ac-H3
Ac-a-tubulin
p21 induct
Apoptosis inductc
Granuloc differentd
SAHA
MS-275
+++
++
++
+++
++
+++
—
+++
+++
+++
+
+
+++
a See Refs. 25 and 26.
b Assay performed on immunoprecipitate (IP).
c Measured by the Annexin V/PI method.
d Measured by the CD11c expression.

2532
A. Mai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2530–2535
O
N
1
3
6
, R = H; Ar =
2
4
7
, R = H; Ar =
, R = H; Ar =
, R = Ph; Ar =
NH
O
NH2
Ar
OH
N
S
N
H
N
N
R
1-7
, R = H; Ar =
, R = Ph; Ar =
NH2
NH2
5
, R = H; A =
H2N
NH2
H2N
O
NH
8
9
, R = H
N
S
NH2
, R = Ph
H
N
R
O
8,9
10, R = H; Ar =
12, R = Ph; Ar =
11, R = H; Ar =
O
NH2
NH2
H2N
NH
O
Ar
N
N
H
13, R = Ph; Ar =
H2N
R
10-13
Figure 3. Novel uracil-based 2-aminoanilide and 2-aminoanilide-like derivatives 1–13.
amino)-phosphonium hexafluorophosphate (Bop-re-
agent) and the appropriate aromatic amine in the pres-
ence of triethylamine. Alkaline hydrolysis of the ethyl
5-(6-oxo-1,6-dihydropyrimidin-2-ylthio)pentanoates 2025
and 21, and of the ethyl 4-((6-oxo-1,6-dihydropyrimi-
din-2-ylthio)methyl)benzoates 22 and 23 yielded the
acids 24,25 25, 26 and 27, respectively. Condensation
of the 3-amino-3-phenylacrylamides 18 and 19, readily
obtained by the reaction of b-oxoesters 14 25 and 15 with
ammonium hydroxide, with diethyl pimelate in the pres-
ence of sodium ethoxide furnished the 6-(6-oxo-1,6-
dihydropyrimidin-2-yl)hexanoic acids 28 and 29. The
ethyl esters 20–23 were prepared by reaction the of ethyl
x-bromovalerate or ethyl 4-(bromomethyl)benzoate and
the 6-substituted-2-thiouracils 1625 and 17, in turn ob-
tained by the condensation of the appropriate b–oxoest-
ers 14 and 15 with thiourea (Scheme 1).42
Compounds 1–13 were tested at 5 lM against human re-
combinant (hr) HDAC1 and HDAC4 enzymes, in com-
parison with MS-275 and SAHA at 5 lM (Table 2). By
the analysis of the data reported in Table 2, we can draw
the following structure-activity relationships (SAR): (i)
amongst the aromatic amines linked at the right head
of the uracil-based molecules, only the 1,2-phenylenedi-
amine and 2-hydroxyaniline were able to elicit the
HDAC inhibiting effect of the compounds (1, 2, 6, 8–
10, 12), whereas the introduction at the same position
of 2,3-diaminopyridine, 4,5-diaminopyrimidine, or
naphthalene-1,8-diamine was not effective (see com-
pounds 3–5, 7, 11 and 13). In addition, (ii) the replace-
ment of the phenyl group at the C6 position of the
uracil ring with a 4-biphenyl moiety did not influence
(compare 1 with 6 and 8 with 9) or gave a little decrease
(compare 10 and 12) of the HDAC1 inhibiting capacity,
O
O
N
O
NH
NH
O
NH
O
g
b
Ar
N
H
S
S
OX
N
S
N
H
20,21
X = C2H5
24,25
X = H
R
R
R
R
R
1-7
d
16,17
c
O
N
a
O
N
NH
O
O
NH
S
OC2H5
g
S
NH2
H
N
OX
14,15
22,23
26,27
R
R
X = C2H5
X = H
8,9
d
O
O
O
O
e
O
N
O
NH
NH2
O
NH
f
Ar
g
N
H
NH2
N
OH
10-13
R
18,19
R
28,29
Scheme 1. Reagents and conditions; (a) EtONa, thiourea, EtOH, reflux, 5–6 h, 70–80%; (b) Ethyl x-bromovalerate, K2CO3, DMF, rt, 1 h, 50%; (c)
Ethyl 4-(bromomethyl)benzoate, K2CO3, DMF, rt, 1 h, 51–55%; (d) 2 N KOH, EtOH, H2O, rt, 18 h, 90–94%; (e) NH4OH 28%, 120 °C, 2–3 h, 66–
70%; (f) diethyl pimelate, EtONa, EtOH, reflux, 6 h, 90–95%; (g) i—BOP-reagent, Et3N, dry DMF, N2, rt, 0.5 h; ii—aromatic amine, rt, 0.5–1 h, 40–
60%.

A. Mai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2530–2535
2533
Table 2. Human recombinant HDAC1 and HDAC4 inhibitory
activities of the new uracil-based compounds 1–13 at 5 lMa
tive and class I-selective derivative amongst those
prepared in this study was the N-(2-aminophenyl)-4-
((6-oxo-4-phenyl-1,6-dihydropyrimidin-2-ylthio)methyl)-
benzamide 8, which showed an activity/selectivity simi-
lar to MS-275 [IC50s (lM) against HDAC1: 0.2 (8)
and 0.3 (MS-275); IC50s (lM) against HDAC4: 125 (8)
and 142 (MS-275)].
Compound
% Residual activity
HDAC1
HDAC4
1
47.2
74.3
99.3
96.7
84.7
44.3
89.2
16.0
12.3
17.7
91.1
32.6
92.1
6.7
97.0
50.7
99.4
110.7
97.0
57.5
92.5
88.8
33.8
59.2
100.8
48.5
96.5
61.7
87.6
2
3
4
In the U937 cells, we have determined the effects of 1–13
(at 5 lM for 24 h) on histone H3 and a-tubulin acetyla-
tion levels taken as markers of class I HDACs and
HDAC6 inhibiting activities, respectively (Fig. 4). The
induction of p21WAF1/CIP1 for the tested compounds
was assessed too (Fig. 4). SAHA and MS-275 have been
taken as reference compounds. In the acetylation assays,
the majority of the tested compounds showed high acet-
yl-H3 levels and no a-tubulin acetylation, with the
exceptions of 2 and 9 which gave a modest signal on
H3 acetylation, and 6 and 12 which weakly increased
a-tubulin acetylation. Some of the tested compounds
(1, 5, 8, 10 and 12) showed high p21 induction. Com-
pared with the hydroxamates MC1641 and MC1751,
which behaved as class I/IIb HDACi,25 the new 2-
aminoanilide-like derivatives displayed a higher level
of class I selectivity, the majority of them being unable
to hyperacetylate a-tubulin.
5
6
7
8
9
10
11
12
13
SAHA
MS-275
7.3
a Data represent mean values of at least three separate experiments.
but significantly increased the HDAC4 inhibiting action
of the derivatives (the effect is particularly clear with
1 ! 6 and 8 ! 9, and less evident with 10 ! 12), thus
abating the class I-selectivity of the compounds. Finally,
the use of the 4-methylbenzamide moiety (compounds 8
and 9) instead of the pentanamide chain (compounds 1
and 6) as a spacer connecting the uracil portion with
the 2-aminoaniline group highly increased the anti-
HDAC1 activity of the derivatives with no or little influ-
ence on HDAC4, whereas the replacement of the sul-
phur atom (in 1 and 6) with the isoster methylene unit
(in 10 and 12) improved the HDAC1 as well as the
HDAC4 inhibiting action of the compounds, thus low-
ering the class I-selectivity. In conclusion, the most ac-
Compounds 1–13 were then tested in the U937 cell line
(at 5 lM for 30 h) to evaluate effects on cell cycle, apop-
tosis induction, and granulocytic differentiation. SAHA
and MS-275 (5 lM) were used as reference drugs. At the
tested conditions, 1, 3, 4, and 5 showed a weak arrest of
the cell cycle in the S phase, and 8 gave a block of the
cycle at the G2 phase (Fig. S2 in Supplementary mate-
rial). Apoptosis induction was measured with the An-
Figure 4. Western blot analyses on U937 cells performed on 1–13 (at 5 lM) to determine their effects on histone H3 and a-tubulin acetylation, and on
p21 induction. SAHA and MS-275 (5 lM) were used for a comparison.

2534
A. Mai et al. / Bioorg. Med. Chem. Lett. 18 (2008) 2530–2535
`
(L.A.), and Regione Campania, L.5 annualita 2005
(L.A.).
Supplementary data
Supplementary data associated with this article can be
found, in the online version, at doi:10.1016/j.bmcl.
2008.03.055.
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in this test, compound 8 and, to a lower extent, 10 and
12 displayed 4.8% to 8.4% of apoptosis induction, they
being less active than SAHA (16.8%) and more potent
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stimulation with 1–13 at 5 lM. Amongst the tested com-
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as cytodifferentiating agent (Fig. 5).
In conclusion, we reported here a new series of uracil-
based HDACi 1–13 bearing a 2-aminoanilide or 2-
aminoanilide-like moiety at the right head of the mole-
cules. Such compounds were in general more efficient
in inhibiting hrHDAC1 than hrHDAC4, and were able
to increase the H3 but not a-tubulin acetylation levels in
the U937 cells. The highest class I-selectivity was dis-
played by the uracils bearing at C6 a phenyl ring, at
C2 a sulphur atom, and showing a 4-methylbenzamide
moiety as a spacer between the uracil ring and the 2-
aminoanilide group. Compound 8, the most potent
and class I-selective HDACi for this series, yielded to-
gether with 12 a high level of CD11c positive/PI negative
cells in the cytodifferentiation assay (U937 cells, 5 lM,
30 h). Surprisingly, compound 13, which failed in inhib-
iting HDACs, showed in the same assay a cytodifferen-
tiating effect higher than MS-275. Studies are in progress
to probe this issue.
Acknowledgement
This work was partially supported by grants from AIRC
2007 (A.M.), PRIN 2006 (A.M.), European Union
(CancerDip Project No. 200620) (L.A.), PRIN 2006

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42. Chemical and physical data of compounds 1–29 are
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