tert-Butylcarbamate-Containing Histone Deacetylase Inhibitors: Apoptosis Induction, Cytodifferentiation, and Antiproliferative Activities in Cancer Cells

Article abstract of DOI:10.1002/cmdc.201300005

Herein we report novel pyrrole- and benzene-based hydroxamates (8, 10) and 2′-aminoanilides (9, 11) bearing the tert-butylcarbamate group at the CAP moiety as histone deacetylase (HDAC) inhibitors. Compounds 8b and 10c selectively inhibited HDAC6 at the nanomolar level, whereas the other hydroxamates effected an increase in acetyl-α-tubulin levels in human acute myeloid leukemia U937 cells. In the same cell line, compounds 8b and 10c elicited 18.4 and 21.4% apoptosis, respectively (SAHA: 16.9%), and the pyrrole anilide 9c displayed the highest cytodifferentiating effect (90.9%). In tests against a wide range of various cancer cell lines to determine its antiproliferative effects, compound 10c exhibited growth inhibition from sub-micromolar (neuroblastoma LAN-5 and SH-SY5Y cells, chronic myeloid leukemia K562 cells) to low-micromolar (lung H1299 and A549, colon HCT116 and HT29 cancer cells) concentrations. In HT29 cells, 10c increased histone H3 acetylation, and decreased the colony-forming potential of the cancer cells by up to 60%.

Full text of DOI:10.1002/cmdc.201300005

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DOI: 10.1002/cmdc.201300005
tert-Butylcarbamate-Containing Histone Deacetylase
Inhibitors: Apoptosis Induction, Cytodifferentiation, and
Antiproliferative Activities in Cancer Cells
Sergio Valente,[a] Daniela Trisciuoglio,[b] Maria Tardugno,[a] Rosaria Benedetti,[c]
Donatella Labella,[a] Daniela Secci,[a] Ciro Mercurio,[d] Roberto Boggio,[e, f] Stefano Tomassi,[g]
Salvatore Di Maro,[g] Ettore Novellino,[g] Lucia Altucci,*[c, h] Donatella Del Bufalo,*[b]
Antonello Mai,*[a] and Sandro Cosconati*[i]
Herein we report novel pyrrole- and benzene-based hydroxa-
mates (8, 10) and 2’-aminoanilides (9, 11) bearing the tert-bu-
tylcarbamate group at the CAP moiety as histone deacetylase
(HDAC) inhibitors. Compounds 8b and 10c selectively inhibit-
ed HDAC6 at the nanomolar level, whereas the other hydroxa-
mates effected an increase in acetyl-a-tubulin levels in human
acute myeloid leukemia U937 cells. In the same cell line, com-
pounds 8b and 10c elicited 18.4 and 21.4% apoptosis, respec-
tively (SAHA: 16.9%), and the pyrrole anilide 9c displayed the
highest cytodifferentiating effect (90.9%). In tests against
a wide range of various cancer cell lines to determine its anti-
proliferative effects, compound 10c exhibited growth inhibi-
tion from sub-micromolar (neuroblastoma LAN-5 and SH-SY5Y
cells, chronic myeloid leukemia K562 cells) to low-micromolar
(lung H1299 and A549, colon HCT116 and HT29 cancer cells)
concentrations. In HT29 cells, 10c increased histone H3 acety-
lation, and decreased the colony-forming potential of the
cancer cells by up to 60%.
Introduction
[a] Dr. S. Valente, Dr. M. Tardugno,+ Dr. D. Labella, Prof. D. Secci, Prof. A. Mai
Istituto Pasteur—Fondazione Cenci Bolognetti
The acetylation and deacetylation of histones have emerged as
critical components of an epigenetic indexing system, demar-
cating transcriptionally active chromatin domains or gene si-
lencing. This dynamic balance is regulated by two families of
enzymes: histone acetyltransferases (HATs) and histone deace-
tylases (HDACs).[1] Acetylation of lysine residues, catalyzed by
HATs, neutralizes the positive charges of e-amino groups on
lysine residues, relaxes chromatin structure, and increases ac-
cessibility for the transcription machinery. Conversely, removal
of acetyl groups from histones and other nuclear proteins by
HDACs induces chromatin condensation and transcriptional re-
pression.[2]
Dipartimento di Chimica e Tecnologie del Farmaco
“Sapienza” Universitꢀ di Roma, P.le A. Moro 5, 00185 Roma (Italy)
E-mail: antonello.mai@uniroma1.it
[b] Dr. D. Trisciuoglio,+ Dr. D. Del Bufalo
Regina Elena National Cancer Institute
Laboratorio di Chemioterapia Sperimentale Preclinica
Via delle Messi d’Oro 156, 00158 Roma (Italy)
E-mail: delbufalo@ifo.it
[c] Dr. R. Benedetti, Prof. L. Altucci
Dipartimento di Patologia Generale
Seconda Universitꢀ degli Studi di Napoli
vico L. De Crecchio 7, 80138 Napoli (Italy)
E-mail: lucia.altucci@unina2.it
[d] Dr. C. Mercurio
HDACs are expressed in all eukaryotic cells, and HDAC activi-
ty is essential for cell proliferation, differentiation, and homeo-
stasis. Eighteen HDACs have been identified in humans and
are classified on the basis of their homology to yeast HDACs.
Eleven of these HDACs contain highly conserved deacetylase
domains, are zinc-dependent enzymes, and are divided into
class I (HDAC1, -2, -3, and -8, homologues of yeast RPD3), clas-
s IIa (HDAC4, -5, -7, and -9 homologues of yeast HDA1), class I-
Ib (HDAC6 and -10, having two catalytic domains), and class IV
HDACs (HDAC11, the only member of this class, HDAC11 has
less sequence similarity with both class I and II deacetylases).
Overexpression of specific HDACs has been observed in many
types of cancer, and often correlates with poor prognosis.[3]
HDAC inhibition induced by HDAC inhibitors (HDACi) may
regulate gene expression, both directly and indirectly, by hy-
peracetylation of histones and/or transcription factors such as
NF-kB, STAT1, STAT3, YY1, E2F, and Rb. In cancer cells, HDACi
Genextra Group, DAC s.r.l., Via Adamello 16, 20139 Milano (Italy)
[e] Dr. R. Boggio
Genextra Group, Congenia s.r.l., Via Adamello 16, 20139 Milano (Italy)
[f] Dr. R. Boggio
Present address: Nerviano Medical Sciences
Via Pasteur 10, 20014 Nerviano (Italy)
[g] Dr. S. Tomassi, Dr. S. Di Maro, Prof. E. Novellino
Dipartimento di Chimica Farmaceutica e Tossicologica
Universitꢀ degli Studi di Napoli “Federico II”
Via D. Montesano 49, 80131 Napoli (Italy)
[h] Prof. L. Altucci
IGB-CNR, Via P. Castellino Napoli (Italy)
[i] Dr. S. Cosconati
DiSTABiF, Seconda Universitꢀ di Napoli
Via G. Vivaldi 43, 81100 Caserta (Italy)
E-mail: sandro.cosconati@unina2.it
[+] These authors contributed equally to this work.
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201300005.
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induce cell-cycle arrest by up-regulation of p21 and/
or down-regulation of cyclins, suppress angiogenesis
by decreased expression of pro-angiogenic factors
(VEGF, HIF-1a), and modulate the release of cytokines
(TNF-a, interleukin-1).[4] Thus, HDACi and their devel-
opment have become the subject of intense interest
by both academic and industrial scientists, and many
of them have now entered the clinical arena.[3a]
Multiple classes of HDACi have been developed,
and members of these classes differ substantially in
their potency and target specificity. Among them,
vorinostat (suberoylanilide hydroxamic acid, SAHA)
and romidepsin (FK-228) were approved by the US
Food and Drug Administration (FDA) in 2006 and
2009, respectively, for the treatment of refractory cu-
taneous T-cell lymphoma (CTCL).[5] In addition, valpro-
ic acid (VPA), panobinostat (LBH589), belinostat
(PDX101), givinostat (ITF2357), resminostat (4SC-201),
entinostat (MS-275), and mocetinostat (MGCD0103)
(Figure 1) are examples of HDACi entered into pha-
se II/III clinical trials, alone or in combination with
other chemotherapeutics for the therapy of hemato-
logical disorders and/or solid tumors.[3a]
According to the well-known pharmacophore
model for HDACi,[6] a deacetylase inhibitor should
bear a CAP group to interact with the rim of the cat-
alytic tunnel of the enzyme,
Figure 1. HDACi approved by the FDA and/or in clinical trials.
a connection unit, linking the
CAP to a hydrophobic spacer
(HS) and lying into the tunnel,
and an enzyme inhibiting group
(EIG), able to complex the zinc
ion, crucial for the catalysis at
the bottom of the tunnel. From
2001, our research group has de-
scribed various chemically differ-
ent series of HDACi [aroylpyrro-
lylhydroxamates (APHAs) and
(aryloxopropenyl)pyrrolylhydrox-
amates 1,[7] aroyl/arylamino-,
(phenyloxopropenyl)-, and (ami-
dopropenyl)cinnamyl and -pyri-
dinylpropenoic
2,[8] and uracil-based hydroxa-
mates and 2-aminoanilides
(UBHAs) 3],[9] fitting with this
hydroxamates
Figure 2. Design of novel HDAC inhibitors.
pharmacophore
(Figure 2).
model
Some analogues of 1, namely
MC1575 and MC1568, displayed class-II-selective HDAC inhibi-
tic inhibition of HCT116 cancer cell growth when used in com-
bination with paclitaxel,[11] inhibition of the estrogen-stimulat-
ed growth of human breast cancer MCF7 cells,[11] and block the
proliferation of a panel of pancreatic cancer cell lines.[12] Thus,
prompted by the interest aroused by the insertion of a Boc-
amino moiety at the CAP group, we designed and synthesized
a series of analogues of 1 and 2 containing this chemical fea-
ture at the CAP benzene ring, and carrying as EIG either the
tory activity, and have been used as valuable tools to dissect
the role of class II HDACs in a number of different biological
contexts.[10] Some derivatives of 2 and 3 showed high anticanc-
er effects. Recently, several authors highlighted structurally dif-
ferent HDACi bearing the N-tert-butyloxycarbonyl (Boc)-amino
group at the CAP moiety (compounds 4–7; Figure 2). Such
compounds showed anti-HDAC6 specific activity and synergis-
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hydroxamate (8, 10) or the 2-
aminoanilide (9, 11) moiety
(Figure 2).
All the new derivatives 8–11
were tested at 5 mm against
human recombinant (hr) HDAC1
and -4, and for selected com-
pounds IC50 values (compound
dose required to inhibit enzyme
activity by 50%) were deter-
mined against all the HDAC1–11
isoforms. Compounds 8–11 were
also assayed in human leukemia
U937 cells to determine their ef-
fects on cell cycle, apoptosis in-
duction, and granulocytic differ-
entiation after treatment for
30 h. The most pro-apoptotic de-
rivatives were then tested in
three different cancer cell lines
to assess their antiproliferative
activities, and the most potent
compound was further charac-
terized (antiproliferative activity
against a wider panel of cancer
cells, histone H3 acetylation, clo-
nogenic assay) as a valuable an-
ticancer agent.
Scheme 1. Reagents and conditions: a) C2H5ONa, anhyd. C2H5OH, RT, 2 h; b) LiOH, THF/H2O, overnight;
c) 1. ClCOOC2H5, (C2H5)3N, anhyd. THF, 08C, 30 min, 2. CH3O(CH3)2ONH2, anhyd. THF, 08C, 1 h, 3. Amberlyst 15,
CH3OH, RT, 1.5 h; d) 1. (C2H5)3N, BOP reagent, anhyd. DMF, N2(g), RT, 2. o-phenylendiamine, anhyd. DMF, N2(g), RT,
1 h.
Results and Discussion
Chemistry
Human recombinant HDAC1 and HDAC4 assays
Aldol condensation of commer-
Compounds 8–11 were tested at 5 mm against hrHDAC1 (sub-
cially available ethyl 4-formyl-1-methyl-1H-pyrrole-2-carboxyl-
ate 12a or ethyl 3-(4-formyl-1-methyl-1H-pyrrol-2-yl)acrylate
12b[7e] with 3- or 4-tert-butoxycarbonylaminoacetophenones
13a,b was carried out in dry ethanol in the presence of
sodium ethylate and afforded the pyrrole-containing ethyl
esters 14a–d (Scheme 1). The same reaction performed start-
ing from ethyl 4-formylbenzoate or ethyl-(4-formylphenyl)acry-
late 15a,b furnished the ethyl benzoates/cinnamates 16a–d.
The esters 14 and 16 were then hydrolyzed with lithium hy-
droxide in tetrahydrofuran/water to afford the corresponding
carboxylic/acrylic acids 17 and 18, key intermediates for the
synthesis of the related hydroxamates (reaction with ethyl
chloroformate/triethylamine followed by addition of O-(2-me-
thoxy-2-propyl)hydroxylamine and final acidic treatment with
the Amberlyst 15) 8, 10 or 2’-aminoanilides [treatment with
benzotriazole-1-yloxytris(dimethylamino)phosphonium hexa-
fluorophosphate (BOP reagent, Castro’s reagent), ortho-phenyl-
endiamine, and (C2H5)3N in dry N,N-dimethylformamide] 9, 11.
Chemical and physical data of compounds 8–11 are listed in
Table 1. Chemical and physical data of the intermediate com-
pounds 14, 16–18 are listed in table S1 in the Supporting In-
formation.
strate: histone H3) and hrHDAC4 (substrate: the non-histone
trifluoroacetyllysine derivative),[13] and the results are summar-
ized in Table 2. Among the hydroxamates 8 and 10, the ben-
zene compounds 10 showed, in general, similar or better po-
tency against HDAC1 than the corresponding pyrrole ana-
logues (with the exception of 10d) but lower than SAHA and,
with some exceptions (10c and 8c), than MS-275, used as ref-
erence drugs.
Typically, 3-NH-Boc-substituted compounds were more effi-
cient than their 4-NH-Boc counterparts, and the arylcarbonyl
hydroxamates were less potent than the arylacrylic analogues,
at least in the pyrrole series 8. However, 10a–c and the pyrrole
8a displayed higher potency than SAHA against HDAC4. Re-
garding the 2’-aminoanilides 9 and 11, both the pyrrole and
benzene derivatives showed low inhibitory potency against
HDAC1 if compared with the related hydroxamates, but
against HDAC4 the pyrrole compounds 9 (with the exception
of 9a) were more potent than the corresponding hydroxa-
mates, thus being quite HDAC4-selective. Against HDAC4 the
benzene anilides 11, despite a drop of potency when com-
pared with the related hydroxamates, maintained 36–41% in-
hibition at 5 mm. In relation particularly to the pyrrole series 9,
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Effect of compounds 8–11 on
a-tubulin acetylation in human
leukemia U937 cells as an indi-
cation of HDAC6 inhibition
Table 1. Chemical and physical properties of compounds 8–11.
Western blot analyses in human
leukemia U937 cells were per-
formed on 8–11 (5 mm, 24 h) to
detect their effects on the acety-
lation
level
of
a-tubulin,
Compd
NH-Boc
position
n
mp [8C]
Recrystallization
solvent
Yield [%]
a marker of HDAC6 inhibition.
MS-275 and SAHA (5 mm, 24 h)
were used as reference drugs.
Data depicted in Figure 3 show
that both pyrrole (8) and ben-
zene (10) hydroxamates were
able to strongly increase a-tubu-
lin acetylation, with 10 more effi-
cient than 8. In contrast, the 2’-
aminoanilides 9 and 11 (includ-
ing the reference MS-275) yield-
ed a signal similar or slightly
higher than the control (DMSO).
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
11 a
11 b
11 c
11 d
3
4
3
4
3
4
3
4
3
4
3
4
3
4
3
4
0
0
1
1
0
0
1
1
0
0
1
1
0
0
1
1
121–123
102–104
120–122
122–124
185–187
119–121
162–164
220–222
125–127
190–192
162–164
180–182
210–212
194–196
205–207
123–125
toluene
toluene
toluene
toluene
77
75
73
75
69
88
72
87
82
75
78
81
67
74
74
59
acetonitrile
toluene
acetonitrile
methanol
toluene
acetonitrile
acetonitrile
acetonitrile
methanol
acetonitrile
methanol
toluene
Table 2. hrHDAC1 and hrHDAC4 inhibitory activity of compounds 8–11.
Compd
Inhibition [%][a]
HDAC1
HDAC4
8a
8b
8c
8d
9a
9b
9c
9d
10a
10b
10c
10d
11 a
11 b
11 c
11 d
SAHA
MS-275
37Æ0.8
24Æ1
71Æ4
21Æ2
65Æ3
60Æ3
59Æ2
51Æ2
22Æ2
43Æ2
11 Æ1
32Æ2
31Æ1.2
25Æ2.1
43Æ1.8
37Æ1.1
65Æ2.4
35Æ1.8
14Æ0.9
10Æ0.3
9Æ0.2
64Æ3
Figure 3. Effects of compounds 8–11, MS-275 (MS), and SAHA (5 mm, 24 h)
on a-tubulin acetylation (AcTub) in human U937 leukemia cells. Western
blot analyses were performed with specific antibodies. ERK proteins were
used for equal loading. Blots representative of two independent experi-
ments with similar results are shown.
66Æ2.2
83Æ4.4
83Æ5.2
76Æ3.2
63Æ2.5
38Æ1.4
36Æ2.4
41Æ1.4
40Æ1.3
63Æ1.3
10Æ1.4
Profiling of selected compounds 8b and 10c on HDAC1-11
isoforms
11 Æ0.9
87Æ2.2
54Æ2.1
Two selected compounds, 8b and 10c, chosen because of
their effect on tubulin acetylation and their high pro-apoptotic
properties in U937 cells (see below), were tested against all the
HDAC1–11 isoforms in ten-dose IC50 mode with threefold serial
dilution starting from 50 mm solutions (Table 3). From these
data, the tert-butyl-(3-(3-(4-(3-(hydroxyamino)-3-oxoprop-1-en-
1-yl)phenyl)acryloyl)phenyl)carbamate 10c was generally more
potent than the tert-butyl (4-(3-(5-(hydroxycarbamoyl)-1-
methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate 8b against
HDACs (with the exception of HDAC5), and was less efficient
than SAHA, used as reference drug. Compound 10c displayed
single-digit micromolar inhibition of HDAC1, -2, -4, -8, and -10,
and nanomolar activity against HDAC6 (IC50 =10.2 nm). Com-
[a] Percent inhibition at 5 mm; data represent mean values ÆSD of at
least three separate experiments.
the introduction of the Boc-amino substituent at the 3-position
of the benzene ring seemed to be preferred, and the insertion
of a double bond between the carbonyl function and the 2’-
aminoanilide moiety led to more potent compounds (9c,d).
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Table 3. Inhibition of HDAC1–11 isoforms by compounds 8b and 10c
and SAHA.
HDAC
IC50 [mm]
8b
10c
SAHA
1
2
3
4
5
6
7
8
9
54.8
28%[a]
82.2
74.4
70.6
2.9
2.1
10.8
3.2
101.0
0.01
34%[a]
1.2
68.8
5.1
0.26
0.92
0.35
0.49
0.38
0.03
0.34
0.24
0.32
0.46
0.36
0.03
30%[a]
19.8
99.9
10
11
NA[b]
16%[a]
12.0
[a] Percent inhibition at 50 mm. [b] Not active.
pound 8b was also highly potent against HDAC6, with an IC50
value similar to SAHA (IC50 8b=27.9 nm; IC50 SAHA=28.6 nm),
confirming the western blot data on acetyl-a-tubulin, and be-
cause of its low activities against the other HDAC isoforms, it is
extremely HDAC6-selective.
Theoretical binding pose of 10c in the HDAC6 binding site
Figure 4. Predicted binding pose of 10c in the HDAC6 homology model.
The ligand is represented as dark-gray sticks, whereas the enzyme is repre-
sented as white sticks. Hydrogen bonds are displayed as black dashed lines.
To better rationalize the enhanced inhibitory activity against
HDAC6 of the newly discovered compounds, molecular model-
ing studies were undertaken. As the X-ray structure of HDAC6
has not been solved, a homology-based model of its major
functional domain (second catalytic subunit CDII, G482–
G801)[14] was constructed. In this step, multiple HDAC X-ray
templates and subsequent multiple-threading alignments were
employed following the I-TASSER methodology.[15] Interestingly,
the same methodology has been already applied by Kozikow-
ski and co-workers to propose viable ligand–HDAC6 theoretical
complexes.[16]
10d (figure S1 in the Supporting Information) revealed that
the ligand is unable to form the aforementioned hydrogen
bond interaction with the enzyme while the hydrophobic con-
tacts with M682 are maintained.
According to these calculations, the Boc-unsubstituted N-hy-
droxy-3-(4-(3-oxo-3-phenylprop-1-en-1-yl)phenyl)acrylamide
would be unable to establish the same interaction pattern con-
tacts with the outer portion of the enzyme. These data would
explain why 10c (IC50 0.01 mm) is a more potent HDAC6i than
its close analogue 10d or the Boc-unsubstituted prototype
(IC50 =0.04 mm and 0.5 mm, respectively).
The resulting HDAC6 model was then used to dock com-
pound 10c, which is among the most active and selective
HDAC6 inhibitors described herein. Analysis of the modeled
enzyme binding cavity revealed that its outer portion is lined
by five loops that could rearrange upon ligand binding. There-
fore, to allow for a certain amount of protein plasticity during
the docking calculation an induced-fit docking approach (IFD)
was used (see the Experimental Section for a full description).
In the best ranking binding pose predicted by the IFD
(Figure 4), 10c is well inserted in the enzyme binding cavity
with its hydroxamate group coordinating the zinc ion. More-
over, the rigid aromatic spacer is embedded in an aromatic
cage formed by H500, F620, and F680 engaging favorable
charge–transfer interactions and hydrophobic contacts with
P501. On the other hand, the tert-butylcarbamate group at po-
sition 3 of the terminal phenyl ring extends toward the outer
portion of the binding gorge contacting one of the loops
lining the rim of the catalytic tunnel (residues G677–G686). In
this position the latter group hydrogen bonds with the F199
backbone CO group and engages in hydrophobic contacts
with the M682 side chain. Analysis of the results obtained for
Effect of compounds 8–11 on cell-cycle distribution, apopto-
sis induction, and granulocytic differentiation in human
U937 leukemia cells
The described compounds 8–11 were tested at 5 mm for 30 h
in U937 cells to determine their effects on cell cycle, apoptosis
induction, and granulocytic differentiation (Figure 5). SAHA
and MS-275 (5 mm) were used as reference drugs. Among the
hydroxamates 8, 10, the compounds 8a, 8b, and 10c dis-
played an arrest in the G1 phase similar to (10c) or higher than
(8a, 8b) MS-275. Compound 8c blocked the cycle at the S
phase, similar to SAHA. In relation to the 2’-aminoanilides, the
benzene compounds 11 a,b showed a block at the G1 phase
similar to MS-275, whereas the remaining derivatives produced
a weaker effect (Figure 5a). Taking into account the pre-G1
peak as an index of pro-apoptotic properties of the com-
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Antiproliferative activities of selected compounds in a panel
of different cancer cell lines
Selected pro-apoptotic compounds (8a, 8b, 10c, and 10d)
were first tested in three different human cancer cell lines
(colon carcinoma HCT116, non-small-cell lung adenocarcinoma
A549, and chronic myeloid leukemia K562), using the CellTiter-
Glo luminescent cell viability assay, to determine their antiproli-
ferative effects after 72 h of treatment (Table 4). The benzene
Table 4. In vitro antiproliferative effects of selected compounds against
cancer cell lines.
Compd
IC50 [mm][a]
HCT116
A549
K562
8a
8b
10c
10d
3.6
9.7
1.0
1.3
12.2
18.8
4.1
1.5
3.1
0.6
1.6
3.1
[a] Concentration of test compound required for 50% inhibition of cell vi-
ability. Determined by CellTiter-Glo assays (72 h), performed in replicate
(nꢀ2); values are shown are the mean (SD<30% of the mean). HCT116:
human colon carcinoma, A549: human non-small-cell lung adenocarcino-
ma, K562: chronic myeloid leukemia.
hydroxamates 10c and 10d showed the highest antiprolifera-
tive activities in these assays, with IC50 values ranging from 0.6
to 4.1 mm. Thus, we selected the tert-butyl-3-(3-(4-(3-(hydroxya-
mino)-3-oxoprop-1-en-1-yl)phenyl)acryloyl)phenylcarbamate
10c to extend the characterization against a panel of different
cancer cells (colon carcinoma HT29, melanoma M14, non-
small-cell lung adenocarcinoma H1299, breast adenocarcinoma
MCF7, ovarian carcinoma HEY, glioblastoma U87, neuroblasto-
ma LAN-5 and SH-SY5Y, pancreatic carcinoma PANC-1, prostate
carcinoma PC3, and ovarian carcinoma SKOV3 cancer cells)
after 48 h treatment, using the 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyltetrazolium bromide (MTT) method (Table 5).
Figure 5. a) Cell-cycle analysis, b) apoptosis induction, and c) granulocytic
differentiation in human U937 leukemia cells treated with compounds 8–11,
SAHA, and MS-275 at 5 mm for 30 h.
Table 5. In vitro antiproliferative activity of 10c against a panel of cancer
cell lines.
pounds, Figure 5b shows that the hydroxamates 8a–c and
10c,d exhibited 15– 21% apoptotic induction in this assay,
with 8b and 10c being more potent than SAHA [percent pre-
G1 peak: 18.4 (8b), 21.4 (10c), and 16.9% (SAHA)].
Cell line
IC50 [mm][a]
colon carcinoma HT29
melanoma M14
3.2
21
2.2
21
20
40
non-small-cell lung adenocarcinoma H1299
breast carcinoma MCF7
ovarian carcinoma HEY
ovarian carcinoma SKOV3
glioblastoma U87
The expression of the surface antigen CD11c was deter-
mined as a marker of granulocytic differentiation in U937 leu-
kemia cells. The cells were treated with 8–11 at 5 mm for 30 h,
and then the percentage values of CD11c-positive/propidium
iodide (PI)-negative cells were determined. In this assay, both
the pyrrole hydroxamates (8c,d) and 2-aminoanilides (9c,d)
were able to induce cytodifferentiation effects more potently
than MS-275, one of the most efficient cytodifferentiating
agents among the HDAC inhibitors described in literature.[9e,17]
In particular, 9c and 9d displayed respectively 90.9 and 76.1%
of CD11c+/PIÀ cells (Figure 5c).
13
neuroblastoma LAN-5
0.4
0.6
28
neuroblastoma SH-SY5Y
pancreatic carcinoma PANC-1
prostate carcinoma PC3
24.5
[a] Concentration of 10c required for 50% inhibition of cell viability. De-
termined by MTT assay; cells were treated with 10c ranging from 0.1 to
100 mm for 48 h. The results are reported as the average of two or three
independent experiments (SD<30% of the mean).
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In these assays, the two neuroblastoma LAN-5 and SH-SY5Y
cell lines were the most sensitive, with 10c displaying sub-mi-
cromolar antiproliferative activities, followed by H1229 and
HT29 carcinoma cells (10c active at single-digit micromolar
range), whereas the other screened cell lines were less suscep-
tible to the treatment.
Effects of 10c in HT29 cells: histone H3 acetylation, prolifer-
ation, and colony-forming ability in HT29 cells
Choosing colon HT29 as a representative cancer cell line, the
effect of 10c on acetylation level of histone H3 (10 mm, 24 h)
was detected by western blot analysis. SAHA was used at
50 mm as reference drug. As depicted in Figure 6a, an in-
creased acetylation at Lys 9/14 of histone H3 was observed
after exposure to 10c when compared with untreated cells.
To further characterize the antiproliferative effect of 10c in
HT29 cells, experiments were performed by treating cells for
24 h with the compound (0.5, 1, and 5 mm) and assessing cell
proliferation both at the end of treatment and from 24 to 96 h
after the end of treatment (Figure 6b). As reported in Fig-
ure 6b, 10c induced a dose-dependent decrease of cell surviv-
al up to 16% (5 mm, 96 h after the end of treatment). SAHA,
tested at the same conditions, was less effective (see figure S2
in Supporting Information).
Finally, we assessed the ability of HT29 cells to form colonies
after treatment with 10c (0.5, 1.0, and 5.0 mm, for 24 h). As re-
ported in Figure 6c, a dose-dependent decrease in colony-
forming potential was evident in HT29 cells after 10c treat-
ment. In particular, an approximate 60% reduction was ob-
served after exposure to 5 mm 10c for 24 h as compared with
control untreated cells.
Conclusions
Using HDAC6-selective inhibitors 4–7, which bear the NH-Boc
group at the CAP moiety and show growth inhibition of colon
HCT116, estrogen-stimulated breast MCF7, and a panel of pan-
creatic carcinoma cell lines as a structural basis, we designed,
new pyrrole- and benzene-containing hydroxamates (8, 10)
and 2’-aminoanilides (9, 11). These compounds (8, 10, 9, 11)
are substituted with a NH-Boc group either at the 3- or 4-posi-
tion of the benzoyl group and are therefore analogues of com-
pounds 1, 2 previously described by us and endowed with an-
ticancer properties.[7e,h,8b,d,f,9b,c,e,17,18] When tested against
HDAC1 and -4, the 3-NH-Boc-substituted compounds seemed
to be more potent than the corresponding 4-NH-Boc counter-
parts. The benzene hydroxamates 10 displayed higher inhibi-
tion than the related pyrrole analogues 8, and exhibited
a great decrease in activity when changed into 2’-aminoani-
lides (11). In the pyrrole series a similar decrease of potency
from hydroxamates 8 to anilides 9 was not registered. Both
the pyrrole (8) and benzene (10) hydroxamates increased the
acetylation level of a-tubulin in human leukemia U937 cells,
a marker of HDAC6 inhibition, whereas both the anilides (9,
11) did not. Two compounds, 8b and 10c, were tested on all
the HDAC1–11 isoforms, and in agreement with previous data,
Figure 6. Effects of 10c in human colon HT29 cancer cells: a) Western blot
analysis of acetylated histone H3 on Lys 9/14 (Ac-H3) in protein extracts ob-
tained after treatment of HT29 cells with 10c (10 mm) and SAHA (50 mm) for
24 h. b-Actin is shown as loading and transferring control. A blot representa-
tive of two independent experiments with similar results is shown. b) Per-
centage of cell survival after treatment with 10c at doses ranging from 0.5
to 5 mm for 24 h. Analysis was performed at the end of treatment (24 h), and
24 (24R), 48 (48R), 72 (72R), and 96 h (96R) after the end of treatment (R=re-
covery). The results are reported as (viability of treated cells)/(viability of
control cells)ꢁ100, and represent the average ÆSD of three independent
experiments. c) In vitro clonogenic ability of HT29 cells after treatment with
10c at doses ranging from 0.5 to 5 mm for 24 h.
10c was more potent than 8b in 10 of the 11 tested isoforms,
and both the compounds showed HDAC6 inhibition at a nano-
molar level, 8b being threefold less potent than 10c but ex-
tremely more selective because of its low potency against the
other enzymes. In comparison with other known HDAC6-selec-
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6.51 (d, 1H, CH=CHCOOEt), 6.76 (bs, 1H, (CH3)3CONH), 7.50–7.71
(m, 8H, benzene protons, CH=CHCOOEt and PhCOCH=CH), 7.77–
7.81 (d, 1H, PhCOCH=CH), 8.00–8.02 ppm (d, 2H, benzene pro-
tons); 13C NMR (CDCl3): d=28.5 (3C), 79.5, 118.9, 121.4, 122.1 (2C),
126.3 (4C), 130.1 (2C), 133.5, 134.4 (2C), 141.7, 144.0, 145.2, 153.9,
161.6, 189.7 ppm; MS (EI) calcd for C25H27NO5 m/z [M]+: 421.1889,
found: 421.1885.
tive inhibitors—such as the Boc-containing compounds 4–
7,[11,12] the tetrahydropyrido[4,3-b]indole derivative tubastati-
n A,[16] or the macrocyclic hydroxamates (S)-9, (S)-34, and (S)-
35[19]—compounds 8b and 10c displayed similar potency and/
or selectivity, despite some chemical differences shown mainly
at the CAP and HS level in the structures.
In U937 cells, 8b and 10c (5 mm, 30 h) induced greater
apoptosis (18.4 and 21.4%) than SAHA (16.9%), and the pyr-
role 2’-aminoanilides 9c and 9d (5 mm, 30 h) showed the high-
est cytodifferentiating effect (76.1 and 90.9%). When tested
against a wide panel of different cancer cell lines, 10c showed
low or single-digit micromolar antiproliferative effect, probably
related to its ability to inhibit HDAC1 and -2, largely overex-
pressed in cancer.[3c,d] Remarkably, against neuroblastoma LAN-
5 and SH-SY5Y cell lines 10c exerted growth inhibition at
nanomolar level, suggesting an additional role for HDAC6 in-
hibition in neurologic diseases.[20] In colon HT29 cell line,
chosen as a representative cell line, 10c increased the level of
histone H3 acetylation (10 mm, 24 h), and decreased up to 60%
the colony-forming potential in a dose-dependent manner.
Further studies with 10c in vivo are required to assess its po-
tential use as anticancer agent.
General procedure for the synthesis of the acids 17 and 18. Ex-
ample: Synthesis of 3-(4-(3-(4-((tert-butoxycarbonyl)amino)phen-
yl)-3-oxoprop-1-enyl)-1-methyl-1H-pyrrol-2-yl)acrylic acid (17d):
A solution of ethyl 3-(4-(3-(4-((tert-butoxycarbonyl)amino)phenyl)-3-
oxoprop-1-enyl)-1-methyl-1H-pyrrol-2-yl)acrylate 14d (0.82 mmol,
0.35 g) and 2N KOH (1.64 mmol, 0.092 g) in C2H5OH (15 mL) was
stirred overnight at room temperature. Then the solvent was
evaporated and 2n HCl was slowly added till the aqueous phase
reached pH 5.0. The colorless solid was filtered and washed first
with H2O and then with a mixture of (C2H5)2O/petroleum ether 1:1
1
to afford pure 17d. H NMR ([D6]DMSO, 400 MHz): d=1.46 (s, 9H,
C(CH3)3), 3.87 (s, 3H, CH3), 6.34 (d, 1H, CH=CHCOOH), 7.11 (s, 1H,
pyrrole-b proton), 7.26–7.37 (m, 3H, benzene protons and
PhCOCH=CH), 7.57–7.63 (m, 4H, pyrrole-a proton, benzene pro-
tons, PhCOCH=CH and CH=CHCOOH), 8.08–8.09 (m, 1H, benzene
proton), 9.52 (bs, 1H, (CH3)3CONH), 13.0 ppm (bs, 1H, COOH);
13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 34.0, 79.5, 104.8,
108.0, 121.6, 122.1 (2C), 123.2, 127.4, 130.1 (2C), 130.6, 133.5, 138.8,
141.7, 145.2, 153.9, 170.6, 189.7 ppm; MS (EI) calcd for C22H24N2O5
m/z [M]+: 396.1685, found: 396.1690.
Experimental Section
General procedure for the synthesis of the hydroxamic acids 8,
10. Example: Synthesis of tert-butyl-(3-(3-(5-(hydroxycarbamo-
Chemistry
yl)-1-methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate
(8a):
Melting points were determined on a Bꢂchi 530 melting point ap-
1
paratus and are uncorrected. H NMR and 13C NMR spectra were re-
(C2H5)3N (0.12 mmol, 0.017 mL) and ClCOOC2H5 (0.11 mmol,
0.015 mL) were slowly added to a cooled (08C) solution of 4-(3-(3-
((tert-butoxycarbonyl)amino)phenyl)-3-oxoprop-1-enyl)-1-methyl-
1H-pyrrole-2-carboxylic acid 17a (0.092 mmol, 0.034 g) in anhy-
drous THF (5 mL), and the mixture was stirred for 45 min at room
temperature under N2 atmosphere. Afterward, the solid was fil-
tered, washed with anhydrous THF (3ꢁ5 mL) and then O-(2-me-
thoxy-2-propyl)hydroxylamine (0.27 mmol, 0.02 mL) was added,
and the resulting mixture was stirred for 15 min at room tempera-
ture. After this time the solvent was removed under vacuum, the
residue was diluted in CH3OH (10 mL), and Amberlyst 15 ion-ex-
change resin (0.36 g) was added. The mixture was stirred at room
temperature for 1 h. After that, the resin was filtered and the fil-
trate was concentrated under reduced pressure to give crude 8a,
which was purified by recrystallization. 1H NMR ([D6]DMSO,
400 MHz): d=1.50 (s, 9H, C(CH3)3), 3.94 (s, 3H, CH3), 7.04 (s, 1H,
pyrrole-b proton), 7.26–7.67 (m, 6H, benzene proton, PhCOCH=CH
and PhCOCH=CH), 8.09 (s, 1H, pyrrole-a proton), 8.99 (bs, 1H,
(CH3)3CONH), 9.56 (bs, 1H, CONHOH), 10.95 ppm (bs, 1H,
CONHOH); 13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 32.9, 79.5,
111.0, 118.7, 122.1 (2C), 126.1, 127.4, 130.1 (2C), 132.0, 133.5, 141.7,
145.2, 153.9, 163.0, 189.7 ppm; MS (EI) calcd for C20H23N3O5 m/z
[M]+: 385.1638, found: 385.1632.
corded at 400 MHz on a Bruker AC 400 spectrometer; chemical
shifts (d) are reported in ppm relative to the internal reference tet-
ramethylsilane. EIMS data were recorded with a Fisons Trio 1000
spectrometer; only molecular ions [M]+ and base peaks are given.
All compounds were routinely checked by TLC, 1H NMR and
13C NMR spectra. TLC was performed on aluminum-backed silica
gel plates (Merck DC, Alufolien Kieselgel 60 F254) with spots visual-
ized by UV light. All solvents were reagent grade and, when neces-
sary, were purified and dried by standard methods. Concentration
of solutions after reactions and extractions involved the use of
a rotary evaporator operating at reduced pressure of ~20 Torr. Or-
ganic solutions were dried over anhydrous Na2SO4. Elemental anal-
ysis was used to determine purity of the described compounds,
that is >95%. Analytical results are within Æ0.40% of the theoreti-
cal values. All chemicals were purchased from Aldrich Chimica,
Milan (Italy) or from Alfa Aesar, Milan (Italy), and were of the high-
est purity.
General procedure for the synthesis of the esters 14 and 16. Ex-
ample: Synthesis of ethyl 3-(4-(3-(4-((tert-butoxycarbonyl)ami-
no)phenyl)-3-oxoprop-1-enyl)phenyl)acrylate (16d): tert-Butyl 4-
acetylphenylcarbamate 13b (2.12 mmol, 0.5 g) and ethyl 3-(4-for-
mylphenyl)acrylate 15b (2.12 mmol, 0.433 g) were added to a solu-
tion of Na0 (2.54 mmol, 0.05 g) in C2H5OH (5 mL). The resulting mix-
ture was stirred at room temperature for 4 h. The reaction was
quenched with H2O (30 mL) and extracted with EtOAc (3ꢁ50 mL).
The combined organic extracts were washed with saturated NaCl
solution (3ꢁ50 mL), dried with Na2SO4, and concentrated to obtain
a residue that was purified by column chromatography (SiO2 elut-
ing with EtOAc/n-hexane 1:2) to give the compound 16d as
General procedure for the synthesis of the 2’-aminoanilides 9,
11. Example: Synthesis of tert-butyl-(3-(3-(5-((2-aminophenyl)car-
bamoyl)-1-methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate (9a):
(C2H5)3N (1.6 mmol, 0.22 mL) and benzotriazole-1-yloxytris(dimethy-
lamino)phosphonium
hexafluorophosphate
(BOP
reagent)
(0.48 mmol, 0.21 g) were added to a solution of 4-(3-(3-((tert-butox-
ycarbonyl)amino)phenyl)-3-oxoprop-1-enyl)-1-methyl-1H-pyrrole-2-
carboxylic acid 17a (0.4 mmol, 0.32 g) in anhydrous DMF (5 mL)
under N2 atmosphere. The resulting mixture was stirred for 30 min
1
a pale-yellow solid. H NMR (CDCl3, 400 MHz): d=1.33–1.37 (t, 3H,
OCH2CH3), 1.54 (s, 9H, C(CH3)3), 4.25–4.31 (q, 2H, OCH2CH3), 6.47–
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at room temperature; afterward 1,2-phenylenediamine (0.44 mmol,
0.05 g) was added under N2 atmosphere and the reaction was
stirred overnight. The reaction was quenched with H2O (50 mL)
and extracted with EtOAc (3ꢁ30 mL). The combined organic ex-
tracts were dried and the residue obtained upon evaporation of
solvent was purified by column chromatography (SiO2 eluting with
EtOAc/n-hexane 1:1) to give pure 9a. 1H NMR ([D6]DMSO,
400 MHz): d=1.48 (s, 9H, C(CH3)3), 3.87 (s, 3H, CH3), 4.89 (bs, 2H,
NH2), 6.59–6.61 (m, 1H, benzene proton), 6.75–6.77 (d, 1H, ben-
zene proton), 6.96–6.98 (m, 1H, benzene proton), 7.14–7.16 (d, 1H,
benzene proton), 7.26–7.66 (m, 7H, benzene protons, pyrrole-
a and -b protons, PhCOCH=CH and PhCOCH=CH), 8.09 (s, 1H, ben-
zene proton), 9.35 (bs, 1H, (CH3)3CONH), 9.52 ppm (bs, 1H, CONH);
13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 32.9, 79.5, 111.0, 116.5,
118.7, 119.0, 119.6, 122.4, 122.9, 125.2, 125.5, 126.1, 127.4 (2C),
129.5, 132.0, 136.4, 138.1, 141.8, 145.2, 153.9, 162.7, 189.7 ppm; MS
(EI) calcd for C26H28N4O4 m/z [M]+: 460.2111, found: 460.2118.
COCH=CH), 9.10 (bs, 1H, (CH3)3CONH), 9.84 (bs, 1H, CONHOH),
11.39 ppm (bs, 1H, CONHOH); 13C NMR ([D6]DMSO, 400 MHz): d=
28.5 (3C), 79.5, 121.4, 122.1 (2C), 126.5 (2C), 127.4 (2C), 130.1 (2C),
133.4, 133.5, 138.6, 141.7, 145.2, 153.9, 163.0, 189.7 ppm; MS (EI)
calcd for C21H22N2O5 m/z [M]+: 382.1529, found: 382.1535.
tert-Butyl-(3-(3-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl) phenyl)-
acryloyl)phenyl)carbamate (10c): 1H NMR ([D6]DMSO, 400 MHz):
d=1.48 (s, 9H, C(CH3)3), 6.52–6.56 (d, 1H, CH=CHCONH), 7.47–7.50
(d, 1H, PhCOCH=CH), 7.63–7.96 (m, 10H, benzene protons, CH=
CHCONH and PhCOCH=CH), 9.12 (bs, 1H, (CH3)3CONH), 9.55 (bs,
1H, CONHOH), 10.79 ppm (bs, 1H, CONHOH); 13C NMR ([D6]DMSO,
400 MHz): d=28.5 (3C), 79.5, 118.9, 119.6, 121.4, 125.5, 126.3 (4C),
127.4, 129.5, 134.4 (2C), 136.4, 138.1, 144.0, 145.2, 153.9, 161.6,
189.7 ppm; MS (EI) calcd for C23H24N2O5 m/z [M]+: 408.1685, found:
408.1691.
tert-Butyl-(4-(3-(4-(3-(hydroxyamino)-3-oxoprop-1-enyl)phenyl)a-
cryloyl)phenyl)carbamate (10d): 1H NMR ([D6]DMSO, 400 MHz):
d=1.47 (s, 9H, C(CH3)3), 6.52–6.55 (d, 1H, CH=CHCONH), 7.45–7.47
(d, 1H, PhCOCH=CH), 7.49–8.09 (m, 10H, benzene protons, CH=
CHCONH and PhCOCH=CH), 9.10 (bs, 1H, (CH3)3CONH), 9.80 (bs,
1H, CONHOH), 10.78 ppm (bs, 1H, CONHOH); C13 NMR ([D6]DMSO,
400 MHz): d=28.5 (3C), 79.5, 118.9, 121.4, 122.1 (2C), 126.3 (4C),
130.1 (2C), 133.5, 134.4 (2C), 141.7, 144.0, 145.2, 153.9, 161.6,
189.7 ppm; MS (EI) calcd for C23H24N2O5 m/z [M]+: 408.1685, found:
408.1691.
tert-Butyl-(4-(3-(5-(hydroxycarbamoyl)-1-methyl-1H-pyrrol-3-yl)a-
1
cryloyl)phenyl)carbamate (8b): H NMR ([D6]DMSO, 400 MHz): d=
1.50 (s, 9H, C(CH3)3), 3.94 (s, 3H, CH3), 7.04 (s, 1H, pyrrole-
b proton), 7.26–7.67 (m, 6H, benzene protons, PhCOCH=CH and
PhCOCH=CH), 8.09 (s, 1H, pyrrole-a proton), 8.99 (bs, 1H,
(CH3)3CONH), 9.56 (bs, 1H, CONHOH), 10.95 ppm (bs, 1H,
CONHOH); 13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 32.9, 79.5,
111.0, 118.7, 122.1 (2C), 126.1, 127.4, 130.1 (2C), 132.0, 133.5, 141.7,
145.2, 153.9, 163.0, 189.7 ppm; MS (EI) calcd for C20H23N3O5 m/z
[M]+: 385.1638, found: 385.1632.
tert-Butyl-(4-(3-(5-((2-aminophenyl)carbamoyl)-1-methyl-1H-
pyrrol-3-yl)acryloyl)phenyl)carbamate (9b): 1H NMR ([D6]DMSO,
400 MHz): d=1.49 (s, 9H, C(CH3)3), 3.91 (s, 3H, CH3), 4.89 (bs, 2H,
NH2), 6.49–6.51 (m, 1H, benzene proton), 6.56–6.63 (d, 1H, ben-
zene proton), 6.75–6.81 (m, 1H, benzene proton), 6.92–6.94 (d, 1H,
benzene proton), 7.31–7.64 (m, 7H, benzene protons, pyrrole-
a and -b protons, PhCOCH=CH and PhCOCH=CH), 8.08–8.09 (d,
1H, benzene proton), 9.35 (bs, 1H, (CH3)3CONH), 9.52 ppm (bs, 1H,
CONH); 13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 32.9, 79.5,
111.0, 116.5, 118.7, 119.0, 122.1 (2C), 122.4, 122.9, 125.2, 126.1,
127.4, 130.1 (2C), 132.0, 133.5, 141.7, 141.8, 145.2, 153.9, 162.7,
189.7 ppm; MS (EI) calcd for C26H28N4O4 m/z [M]+: 460.2111, found:
460.2118.
tert-Butyl-(3-(3-(5-(3-(hydroxyamino)-3-oxoprop-1-enyl)-1-
methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate (8c): 1H NMR
([D6]DMSO, 400 MHz): d=1.50 (s, 9H, C(CH3)3), 3.94 (s, 3H, CH3),
7.04 (s, 1H, pyrrole-b proton), 7.26–7.67 (m, 7H, benzene protons,
PhCOCH=CH and PhCOCH=CH, CH=CHCONHOH), 8.09–8.3 (m, 2H,
pyrrole-a proton, CH=CHCONHOH), 8.99(bs, 1H, (CH3)3CONH), 9.56
(bs, 1H, CONHOH), 10.95 ppm (bs, 1H, CONHOH); 13C NMR
([D6]DMSO, 400 MHz): d=28.5 (3C), 34.0, 79.5, 104.8, 108.0, 119.6,
123.2, 124.9, 125.5, 127.4 (2C), 129.5, 130.6, 134.8, 136.4, 138.1,
145.2, 153.9, 161.6, 189.7 ppm; MS (EI) calcd for C22H25N3O5 m/z
[M]+: 411.1794, found: 411.1788.
tert-Butyl-(4-(3-(5-(3-(hydroxyamino)-3-oxoprop-1-enyl)-1-
methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate (8d): 1H NMR
([D6]DMSO, 400 MHz): d=1.48 (s, 9H, C(CH3)3), 3.8 (s, 3H, CH3), 6.98
(s, 1H, pyrrole-b proton), 7.16–7.7 (m, 7H, benzene protons,
PhCOCH=CH and PhCOCH=CH, CH=CHCONHOH), 8.09–8.3 (m, 2H,
pyrrole-a proton, CH=CHCONHOH), 9.02 (bs, 1H, (CH3)3CONH), 9.43
(bs, 1H, CONHOH), 10.97 ppm (bs, 1H, CONHOH); 13C NMR
([D6]DMSO, 400 MHz): d=28.5 (3C), 34.0, 79.5, 104.8, 108.0, 122.1
(2C), 123.2, 124.9, 127.4, 130.1 (2C), 130.6, 133.5, 134.8, 141.7,
145.2, 153.9, 161.6, 189.7 ppm; MS (EI) calcd for C22H25N3O5 m/z
[M]+: 411.1794, found: 411.1788.
tert-Butyl-(3-(3-(5-(3-(2-aminophenylamino)-3-oxoprop-1-enyl)-1-
methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate (9c): 1H NMR
([D6]DMSO, 400 MHz): d=1.48 (s, 9H, C(CH3)3), 3.79 (s, 3H, CH3),
4.89 (bs, 2H, NH2), 6.57–6.59 (m, 1H, benzene proton), 6.72–6.75
(d, 3H, CH=CHCONH, benzene protons), 6.88–6.90 (m, 1H, benzene
proton), 7.15 (s, 1H, pyrrole-a proton), 7.33–7.35 (m, 1H, benzene
proton) 7.42–7.72 (m, 6H, benzene protons, pyrrole-b proton, CH=
CHCONH, PhCOCH=CH, PhCOCH=CH), 8.12 (s, 1H, benzene
proton), 9.34 (bs, 1H, (CH3)3CONH), 9.52 ppm (bs, 1H, CONH);
13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 34.0, 79.5, 104.8,
108.0, 116.5, 119.0, 119.6, 122.4, 122.9, 123.2, 124.9, 125.2, 125.5,
127.4 (2C), 129.5, 130.6, 134.8, 136.4, 138.1, 141.8, 145.2, 153.9,
166.7, 189.7 ppm; MS (EI) calcd for C28H30N4O4 m/z [M]+: 486.2267,
found: 486.2261.
tert-Butyl-(3-(3-(4-(hydroxycarbamoyl)phenyl)acryloyl) phenyl)-
carbamate (10a): 1H NMR ([D6]DMSO, 400 MHz): d=1.47 (s, 9H,
C(CH3)3), 7.44–7.48 (m, 1H, COCH=CH), 7.50–8.03 (m, 8H, benzene
protons, PhCOCH=CH), 8.10–8.14 (m, 1H, benzene proton), 9.09
(bs, 1H, (CH3)3CONH), 9.53 (bs, 1H, CONHOH), 11.30 ppm (bs, 1H,
CONHOH); 13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 79.5, 119.6,
121.4, 125.5, 126.5 (2C), 127.4 (3C), 129.5, 133.4, 136.4, 138.1, 138.6,
145.2, 153.9, 163.0, 189.7 ppm; MS (EI) calcd for C21H22N2O5 m/z
[M]+: 382.1529, found: 382.1535.
tert-Butyl-(4-(3-(5-(3-(2-aminophenylamino)-3-oxoprop-1-enyl)-1-
methyl-1H-pyrrol-3-yl)acryloyl)phenyl)carbamate (9d): 1H NMR
([D6]DMSO, 400 MHz): d=1.49 (s, 9H, C(CH3)3), 3.78 (s, 3H, CH3),
4.93 (bs, 2H, NH2), 6.55–6.59 (m, 1H, benzene proton), 6.71–6.75
(d, 3H, CH=CHCONH, benzene protons), 6.88–6.91 (m, 1H, benzene
proton), 7.17 (s, 1H, pyrrole-a proton), 7.34–7.36 (m, 1H, benzene
proton) 7.52–7.74 (m, 5H, benzene proton, pyrrole-b proton, CH=
CHCONH, PhCOCH=CH, PhCOCH=CH), 8.04–8.06 (d, 2H, benzene
protons), 9.34 (bs, 1H, (CH3)3CONH), 9.52 ppm (bs, 1H, CONH);
tert-Butyl-(4-(3-(4-(hydroxycarbamoyl)phenyl)acryloyl) phenyl)-
carbamate (10b): 1H NMR ([D6]DMSO, 400 MHz): d=1.49 (s, 9H,
C(CH3)3), 6.65–8.11 (m, 10H, benzene protons, PhCOCH=CH and
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13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 34.0, 79.5, 104.8,
108.0, 116.5, 119.0, 122.1 (2C), 122.4, 122.9, 123.2, 124.9, 125.2,
127.4, 130.1 (2C), 130.6, 133.5, 134.8, 141.7, 141.8, 145.2, 153.9,
166.7, 189.7 ppm; MS (EI) calcd for C28H30N4O4 m/z [M]+: 486.2267,
found: 486.2261.
de Lys substrate, which comprises an acetylated lysine side chain.
For the HDAC4 assay, the HDAC4-selective, non-histone substrate
reported by Lahm et al.[13] was used. Full-length HDAC1 and
HDAC4 with C-terminal His tags were expressed using baculovirus
systems. Deacetylation sensitizes the substrates that, in the second
step, treated with the developer produce a fluorophore. Fluores-
cence was with a TECAN Infinite M200 station.
tert-Butyl-(3-(3-(4-((2-aminophenyl)carbamoyl)phenyl) acryloyl)-
1
phenyl)carbamate (11a): H NMR ([D6]DMSO, 400 MHz): d=1.49 (s,
9H, C(CH3)3), 4.93 (bs, 2H, NH2), 6.58–6.61 (m, 1H, benzene proton),
6.77–6.79 (d, 1H, benzene proton), 6.96–6.99 (m, 1H, benzene
proton), 7.16–7.18 (d, 1H, benzene proton), 7.63–7.65 (d, 2H, ben-
zene protons), 7.69–7.74 (d, 1H, PhCOCH=CH), 7.96–8.23 (m, 5H,
benzene protons and PhCOCH=CH), 8.15–8.23 (d, 2H, benzene pro-
tons), 9.75 (bs, 1H, (CH3)3CONH), 9.83 ppm (bs, 1H, CONH);
13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 79.5, 116.5, 119.0,
119.6, 121.4, 122.4, 122.9, 125.2, 125.5, 126.5 (2C), 127.4 (3C), 129.5,
133.4, 136.4, 138.1, 138.6, 141.8, 145.2, 153.9, 164.8, 189.7 ppm; MS
(EI) calcd for C27H27N3O4 m/z [M]+: 457.2002, found: 457.2008.
Molecular modeling studies: The human HDAC6 homology model
was obtained using the I-TASSER approach (http://zhanglab.ccmb.
med.umich.edu/I-TASSER/ [accessed February 27, 2013]). In particu-
lar the sequence of the second catalytic subunit of HDAC6
(Q9UBN7), retrieved from the Uniprot server (http://www.uniprot.
org [accessed February 27, 2013]), was used as the input for the I-
TASSER method. The position of the zinc ion and the conformation
of the chelating residues were extracted from the resolved struc-
ture of human HDAC7 complexed with SAHA (PDB ID: 3C0Z).[21]
The constructed model was then processed using the Protein Prep-
aration Wizard panel of the Schrçdinger 2012 molecular modeling
package (Schrçdinger Suite 2012, Schrçdinger LLC, New York, NY,
USA). In particular, using the preprocess and analyze structure tool,
the bond orders were assigned, all the hydrogen atoms were
added, and the disulfide bonds were assigned. Using Epik 2.0 a pre-
diction of the heterogroups ionization and tautomeric states was
performed. An optimization of the hydrogen bonding network was
performed using the H-bond assignment tool. Finally, using the
impref utility, the positions of the hydrogen atoms were optimized
by keeping all the heavy atoms in place.
tert-Butyl-(4-(3-(4-((2-aminophenyl)carbamoyl)phenyl) acryloyl)-
phenyl)carbamate (11b): 1H NMR ([D6]DMSO, 400 MHz): d=1.49 (s,
9H, C(CH3)3), 4.93 (bs, 2H, NH2), 6.58–6.61 (m, 1H, benzene proton),
6.77–6.79 (m, 1H, benzene proton), 6.96–6.99 (m, 1H, benzene
proton), 7.16–7.18 (m, 1H, benzene proton), 7.63–7.65 (d, 2H, ben-
zene protons), 7.69–7.74 (d, 1H, PhCOCH=CH), 7.96–8.23 (m, 5H,
benzene protons and PhCOCH=CH), 8.15–8.23 (d, 2H, benzene pro-
tons), 9.75 (bs, 1H, (CH3)3CONH), 9.83 ppm (bs, 1H, CONH);
13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 79.5, 116.5, 119.0,
121.4, 122.1 (2C), 122.4, 122.9, 125.2, 126.5 (2C), 127.4 (2C), 130.1
(2C), 133.4, 133.5, 138.6, 141.7, 141.8, 145.2, 153.9, 164.8,
189.7 ppm; MS (EI) calcd for C27H27N3O4 m/z [M]+: 457.2002, found:
457.2008.
Ligand files (10c and 10d) were prepared employing the Builder
tool and generated with the Ligprep module within Schrçdinger
2012 molecular modeling package (Schrçdinger Suite 2012, Schrç-
dinger LLC, New York, NY, USA). Partial ligand charges were calcu-
lated by means of the Jaguar suite (Jaguar 4.1; Schrçdinger, Inc.,
Portland, OR, USA) within the same package. The electrostatic po-
tential was computed using 6-31G* basis set.
tert-Butyl-(3-(3-(4-(3-(2-aminophenylamino)-3-oxoprop-1-enyl)-
phenyl)acryloyl)phenyl)carbamate (11c): 1H NMR ([D6]DMSO,
400 MHz): d=1.49 (s, 9H, C(CH3)3), 4.95 (bs, 2H, NH2), 6.59–6.63 (m,
1H, benzene proton), 6.73–6.75 (m, 1H, benzene proton), 6.83–
6.85 (d, 1H, PhCOCH=CH), 6.97–6.99 (m, 1H, benzene proton),
7.18–7.20 (m, 1H, benzene proton), 7.33–7.35 (d, 1H, PhCOCH=
CH), 7.44–7.47 (t, 1H, benzene proton), 7.73–8.19 (m, 8H, benzene
protons, PhCOCH=CH and PhCOCH=CH), 8.30–8.31 (m, 1H benzene
proton), 9.57 (bs, 1H, (CH3)3CONH), 9.75 ppm (bs, 1H, CONH);
13C NMR ([D6]DMSO, 400 MHz): d=28.5 (3C), 79.5, 116.5, 118.9,
119.0, 119.6, 121.4, 122.4, 122.9, 125.2, 125.5, 126.3 (4C), 127.4,
129.5, 134.4 (2C), 136.4, 138.1, 141.8, 144.0, 145.2, 153.9, 166.7,
189.7 ppm; MS (EI) calcd for C29H29N3O4 m/z [M]+: 483.2158, found:
483.2165.
Ligands were submitted as starting geometries to IFD calculations
(Schrçdinger Suite 2012, Schrçdinger LLC, New York, NY, USA)
using Glide (Glide 5.7; Schrçdinger Suite 2012, Schrçdinger LLC,
New York, NY, USA) In the first IFD stage, a softened-potential
docking was performed using GlideSP (Standard Precision) mode
generating 20 initial poses. For each one, a full circle of protein re-
finement was performed using Prime (Prime 3.0; Schrçdinger Suite
2011, Schrçdinger LLC, New York, NY, USA). Residues of the protein
within 5.0 ꢃ of ligand poses were refined, and side chains were
conformationally changed and subsequently minimized. Ligands
were re-docked with GlideXP included in IFD to generate poses. All
ligand–protein complex pictures were rendered employing the
UCSF Chimera software.[22]
tert-Butyl-(4-(3-(4-(3-(2-aminophenylamino)-3-oxoprop-1-enyl)-
phenyl)acryloyl)phenyl)carbamate (11d): 1H NMR ([D6]DMSO,
400 MHz): d=1.47 (s, 9H, C(CH3)3), 4.55 (bs, 2H, NH2), 6.56–6.91 (m,
5H, CH=CHCONH, benzene protons), 7.33–8.10 (m, 11H, PhCOCH=
CH, CH=CHCONH, PhCOCH=CH and benzene protons), 9.30 (bs,
1H, (CH3)3CONH), 9.80 ppm (bs, 1H, CONH); 13C NMR ([D6]DMSO,
400 MHz): d=28.5 (3C), 79.5, 116.5, 118.9, 119.0, 121.4, 122.1 (2C),
122.4, 122.9, 125.2, 126.3 (4C), 130.1 (2C), 133.5, 134.4 (2C), 141.7,
141.8, 144.0, 145.2, 153.9, 166.7, 189.7 ppm; MS (EI) calcd for
C29H29N3O4 m/z [M]+: 483.2158, found: 483.2165.
Cell cultures: U937 human acute leukemia cells were cultured in
RPMI with 10% fetal calf serum, 100 UmLÀ1 penicillin, 100 mgmLÀ1
streptomycin, 250 ngmLÀ1 amphotericin-B, 10 mm HEPES, and
2 mm glutamine. U937 cells were kept at a constant concentration
of 200000 cells per mL culture medium. HT29 (colon carcinoma
cancer), M14 (melanoma), H1299 (non-small-cell lung adenocarci-
noma), MCF7 (breast adenocarcinoma), HEY (ovarian carcinoma),
U87 (glioblastoma), LAN-5 (neuroblastoma), SH-SY5Y (neuroblasto-
ma), PANC-1 (pancreatic carcinoma), PC3 (prostate carcinoma),
SKOV3 (ovarian carcinoma), K562 (chronic myeloid leukemia), A549
(non-small-cell lung adenocarcinoma), and HCT116 (colon carcino-
ma) cells were cultured in RPMI (Invitrogen, Carlsbad, CA, USA)
with 10% fetal calf serum (HyClone, Thermo Scientific, South
Logan, UT, USA), and 100 UmLÀ1 penicillin.
Fluorimetric hrHDAC1 and hrHDAC4 assays: The HDAC Fluorescent
Activity Assay for HDAC1 and HDAC4 is based on the Fluor de Lys
Substrate and Developer combination (BioMol), and has been car-
ried out according to the supplier’s instructions and as previously
reported.[8d] First, the inhibitors and purified recombinant HDAC1
or HDAC4 enzymes have been pre-incubated at room temperature
for 15 min before the addition of the substrate which is the Fluor
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Western blot analysis for a-tubulin and histone H3 acetylation:
For determination of the acetylation levels of a-tubulin in U937
cells and histone H3 in HT29 cells, 25 mg of total protein extracted
from the cells were separated on 10 and 13.5% polyacrylamide
gels, respectively, and blotted as previously described.[18,23] Immu-
nodetection was performed using antibodies directed to acetylated
a-tubulin (Sigma–Aldrich, St. Louis, MO, USA), total ERKs (Santa
Cruz Biotechnology, Santa Cruz, CA, USA), acetyl (Lys 9/14)-histone
H3 (Ac-H3, Millipore, Billerica, MA, USA), and b-actin (Sigma–Al-
drich). Anti-mouse or anti-rabbit immunoglobulin G (IgG)–horse-
radish peroxidase (HRP)-conjugated antibodies (Amersham Biosci-
ences, Freiburg, Germany) were used as secondary antibodies at
1:10000 dilution. Antibody binding was visualized by enhanced
chemiluminescence according to manufacturer’s specification and
recorded on autoradiography film (Amersham Biosciences).
Aldrich). Briefly, exponentially growing tumor cells were seeded
(3ꢁ103 cells per well) in sextuplicate in 96-well culture plates
(Nunc, Milan, Italy), and 24 h later cells were treated with 10c at
concentrations ranging from 0.1 to 100 mm for 24 or 48 h. Cells
were also treated with 1% DMSO, as control. Recovery experiments
were performed by treating cells for 24 h with the compound and
assessing cell proliferation both at the end of treatment and from
24 to 96 h after the washout of the drug. Cell viability was evaluat-
ed by adding MTT to each well at a final concentration of
0.5 mgmLÀ1 and after 4 h of incubation at 378C, the formazan salt
was dissolved with 200 mL isopropyl alcohol. The absorbance of
each well was measured with an ELISA reader (DASIT, Milan, Italy)
at l 570 nm and the viability was calculated for each concentration
of 10c used as (ODtreated cells)/(ODcontrol cells)ꢁ100. The concentration
of 10c that causes a 50% of cell viability inhibition (IC50) was also
calculated.
HDAC1–11 isoforms inhibition assay: Individual IC50 values for
each HDAC isozyme were measured with the homogeneous fluo-
rescence release HDAC assay. Purified recombinant enzymes were
incubated with serial diluted inhibitors at the indicated concentra-
tion. The deacetylase activities of HDACs 1, 2, 3, 6, and 10 were
measured by assaying enzyme activity using AMC-K(Ac)GL sub-
strate and AMC-K(TFA)GL substrate for HDACs 4, 5, 7, 8, 9, and 11
as previously described.[13,24] Deacetylated AMC-KGL was sensitive
toward 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 was analyzed on a plate to plate
basis in relationship to the control and imported into analytical
software (GraphPad Prism).
In vitro colony-forming assay: Suspensions from treated (0.5–
5 mm 10c, 24 h) and untreated HT29 cells were seeded into 60 mm
Petri dishes (Nunc, Mascia Brunelli, Milano, Italy) for 10 days. Colo-
nies were stained with 2% methylene blue in 95% ethanol and
counted (1 colony >50 cells). The surviving fractions were calculat-
ed as the ratio of number of colonies per number of plated cells
ꢁ100.
Acknowledgements
This work was supported by PRIN 2009PX2T2E, FIRB RBFR10ZJQT,
EPIGEN (MIUR-CNR), the Italian Association for Cancer Research
(AIRC no. 11812 and MFAG no. 11502), and FP7 Project BLUE-
PRINT/282510.
Cell-cycle analysis of U937 cells: 2.5ꢁ105 treated and untreated
cells were collected, fixed, and resuspended in 500 mL of a hypoton-
ic buffer (0.1% Triton X-100, 0.1% sodium citrate, 50 mgmLÀ1 propi-
dium iodide (PI), RNase A). Cells were incubated in the dark for
30 min. Samples were acquired on a FACS-Calibur flow cytometer
using Cell Quest software (Becton Dickinson, Franklin Lakes, NJ,
USA) and analyzed with standard procedures using Cell Quest soft-
ware (Becton Dickinson) and ModFit LT version 3 Software (Verity)
as previously reported.[18] All the experiments were performed
three times.
Keywords: anilides · apoptosis · hydroxamates · pyrroles · tert-
butylcarbamates
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Received: January 3, 2013
Revised: February 16, 2013
Published online on March 25, 2013
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ChemMedChem 2013, 8, 800 – 811 811