Class II (IIa)-selective histone deacetylase inhibitors. 1. Synthesis and biological evaluation of novel (aryloxopropenyl)pyrrolyl hydroxyamides

Article abstract of DOI:10.1021/jm049002a

Chemical manipulations performed on aroyl-pyrrolyl-hydroxyamides (APHAs) led to (aryloxopropenyl)pyrrolyl hydroxamates 2a-w, and their inhibition against maize HDACs and their class I or class II HDAC selectivity were determined. In particular, from these

Full text of DOI:10.1021/jm049002a

3344
J. Med. Chem. 2005, 48, 3344-3353
Class II (IIa)-Selective Histone Deacetylase Inhibitors. 1. Synthesis and
Biological Evaluation of Novel (Aryloxopropenyl)pyrrolyl Hydroxyamides
Antonello Mai,*,§ Silvio Massa,# Riccardo Pezzi,§ Silvia Simeoni,§ Dante Rotili,§ Angela Nebbioso,
,‡
Annamaria Scognamiglio, Lucia Altucci,*, Peter Loidl,| and Gerald Brosch*,|
Istituto Pasteur - Fondazione Cenci Bolognetti, Dipartimento di Studi Farmaceutici, Universita` degli Studi di Roma “La
Sapienza”, P.le A. Moro 5, 00185 Roma, Italy, Dipartimento Farmaco Chimico Tecnologico, Universita` degli Studi di Siena,
via A. Moro, 53100 Siena, Italy, Dipartimento di Patologia Generale, Seconda Universita` degli Studi di Napoli,
vico L. De Crecchio 7, 80138 Napoli, Italy, Centro di Oncogenomica AIRC, CEINGE Biotecnologia avanzata, Napoli, and
Department of Molecular Biology, Innsbruck Medical University, Peter-Mayr-Strasse 4b, 6020 Innsbruck, Austria
Received December 7, 2004
Chemical manipulations performed on aroyl-pyrrolyl-hydroxyamides (APHAs) led to (aryloxo-
propenyl)pyrrolyl hydroxamates 2a-w, and their inhibition against maize HDACs and their
class I or class II HDAC selectivity were determined. In particular, from these studies some
benzene meta-substituted compounds emerged as highly class II (IIa)-selective HDAC inhibitors,
the most selective being the 3-chloro- and 3-fluoro-substituted compounds 2c (SI ) 71.4) and
2f (SI ) 176.4). The replacement of benzene with a 1-naphthyl ring afforded 2s, highly active
against the class II homologue HD1-A (IC50 ) 10 nM) but less class II-selective than 2c,f.
When tested against human HDAC1 and HDAC4, 2f showed no inhibitory activity against
HDAC1 but was able to inhibit HDAC4. Moreover, in human U937 acute myeloid leukaemia
cells 2f did not produce any effect on apoptosis, granulocytic differentiation, and the cell cycle,
whereas 2s (that retain class I HDAC inhibitory activity) was 2-fold less potent than SAHA
used as reference.
Introduction
acid-containing peptides (CHAPs), depsipeptide FK-228,
and MS-275) can de-repress these genes, resulting in
antiproliferative effects in vitro and antitumor effects
in vivo.17-25
Histone acetylation and deacetylation play an es-
sential role in modifying chromatin structure and
regulating gene expression in eukaryotic cells. Hyper-
acetylated histones are generally found in transcription-
ally active genes and hypoacetylated histones in tran-
scriptionally silent regions of the genome.1-4 Key
enzymes, which modify histone proteins and thereby
regulate gene expression, are histone acetyltransferases
(HATs) and histone deacetylases (HDACs).5-7 In mam-
mals, both these acetylating/deacetylating enzymes are
components of multiprotein complexes containing other
proteins known to function in transcriptional activation/
repression.8-10 Such multiprotein complexes are re-
cruited to specific regions in the mammalian genome
and generate an unique spectrum of expressed and
silenced genes. Known repressor multiprotein complexes
contain DNA binding proteins (such as Rb and Rb-like
proteins, N-CoR, SMRT, MEF, MeCP2, sin3A, etc.) that
can recruit HDACs to repress transcription and to block
the function of some tumor suppressor genes.11-16
Compounds able to inhibit HDAC activity (HDAC
inhibitors, such as trichostatin A (TSA), trapoxin (TPX),
suberoylanilide hydroxamic acid (SAHA), sodium bu-
tyrate (NaB), sodium valproate (VPA), cyclic hydroxamic
To date, eighteen distinct human HDACs have been
reported, grouped into three classes (I, II, and III)
depending on their primary homology to three Saccha-
romyces cerevisiae HDACs (RPD3, HDA1, and SIR2,
respectively).26 Class I and II HDACs show some degree
of homology in their catalytic domain, whereas class III
HDACs (SIRT1-7, sirtuins) show no homology to class
I/II enzymes. Class I HDACs, comprise HDAC1-3 and
8, are predominantly nuclear enzymes, and are ubiqui-
tously expressed. HDAC11 is most closely related to the
class I HDACs, but its overall sequence similarity is too
low27 for classifying it into class I, and a class IV of
human HDACs (containing the sole HDAC11 until now)
has been proposed.28 Class II HDACs comprise HDAC4-
7, 9, and 10 and are divided into two subclasses, IIa
(HDAC4-7 and 9) and IIb (HDAC6 and 10, containing
as a unique feature two deacetylase domains), according
to their sequence homology and domain organization.29-31
Class IIa HDACs shuttle between the nucleus and the
cytoplasm, depending on their CaMK-mediated phos-
phorylation extent and subsequent binding of 14-3-3
proteins. In particular, phosphorylation-dependent bind-
ing of 14-3-3 proteins to the N-termini of class IIa
HDACs masks the nuclear import signal and prevents
nuclear import. Class IIb HDACs are mainly cytoplas-
mic, but show significant nuclear amounts in several
cell lines. Differently from class I HDACs that are
ubiquitarian, class II HDACs are expressed in a re-
stricted number of cell types. Most class IIa HDACs
(HDAC4, 5, and 9) are abundant in heart, skeletal
* To whom correspondence should be addressed: A.M., Tel.: +396-
4991-3392; Fax:+396-491491; e-mail: antonello.mai@uniroma1.it.
Biology: L.A., Tel.: +3981-566-7569; Fax: +3981450-169; e-mail:
lucia.altucci@unina2.it. Biochemistry: G. B., Tel.: 0512-507-3608;
Fax: 0512-507-9880; e-mail: gerald.brosch@uibk.ac.at.
§ Universita` degli Studi di Roma “La Sapienza”.
# Universita` degli Studi di Siena.
Seconda Universita` degli Studi di Napoli.
‡ Centro di Oncogenomica AIRC, CEINGE Biotecnologia avanzata,
Napoli.
| Innsbruck Medical University.
10.1021/jm049002a CCC: $30.25 © 2005 American Chemical Society
Published on Web 04/07/2005

Class II (IIa)-Selective Histone Deacetylase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3345
Chart 1. Known Unselective and Selective HDAC
Inhibitors
Chart 2. From Aroyl-pyrrolyl-hydroxamides (APHAs)
to (Aryloxopropenyl)pyrrolyl Hydroxamates 2a-w
Further development of new small molecules with the
specificity to selectively perturb only a (sub)class of the
HDAC family is a very attractive goal to pursue, because
such as compounds could be useful tools to dissect the
role of a given HDAC in different protein complexes.53
Since 2001 a new class of HDAC inhibitors, namely
aroyl-pyrrolyl-hydroxy-amides (APHAs), have been de-
scribed by us.54-60 In early studies, APHAs have been
tested again HD2,61 a maize deacetylase different from
mammalian HDACs but being a good predictive model
for the behavior of class I HDACs with various series
of HDAC inhibitors.55,56,58,62 Recently, we have under-
taken a broad strategy59,63,64 by testing the new deriva-
tives against HD1-B65,66 and HD1-A enzymes,67,68 two
maize deacetylases that are homologous of mammalian
class I and class II (IIa) HDACs, respectively, to explore
the potential selectivity of our compounds. Our maize
system offers the advantage of completely separated
enzyme forms, while human HDACs act as multipro-
tein- and, in some cases, multi-HDAC-containing com-
plexes in their active forms.69,70 First, anti-HD1-B/
HD1-A data59,63 showed that in APHA compounds just
one type of chemical manipulation performed on the
lead compound 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-
N-hydroxy-2-propenamide 1 (Chart 2) was efficient to
yield highly selective derivatives, that is the insertion
of an alkyl/alkenyl spacer between the benzoyl moiety
and the carbon atom at the pyrrole-C4 position in the 1
structure.63
Here we report in full details the synthesis and
biochemical characterization of novel (aryloxopropenyl)-
pyrrolyl hydroxyamides 2a-w, structurally related to
APHAs and highly selective against the class II (class
IIa) histone deacetylase homologue HD1-A. Moreover,
on selected compounds the inhibitory activities against
human HDAC1 and HDAC4 as well as the cellular
effects on apoptosis, granulocytic differentiation, and
cell cycle phases in U937 acute myeloid leukemia (AML)
cells are described.
Chemistry. Vilsmeier-Haack formylation of ethyl
3-(1-methyl-1H-pyrrol-2-yl)-2-propenoate71 with oxalyl
chloride/N,N-dimethylformamide afforded the ethyl 3-(4-
formyl-1-methyl-1H-pyrrol-2-yl)-2-propenoate 3 as a sole
product in high yield. Further condensation of 3 with
the properly substituted alkyl aryl ketone in alkaline
medium furnished the 3-[4-(3-aryl-3-oxopropen-1-yl)-1-
methyl-1H-pyrrol-2-yl]-2-propenoic acids 4a-w, key
intermediate for the synthesis of the title compounds.
By reaction of 4a-w with ethyl chloroformate, followed
by addition of O-(2-methoxy-2-propyl)hydroxylamine72
and acidic treatment in the presence of the Amberlyst
15 ion-exchange resin, the desired 3-[4-(3-aryl-3-oxo-
propen-1-yl)-1-methyl-1H-pyrrol-2-yl]-N-hydroxy-2-pro-
penamides 2a-w have been obtained (Scheme 1).
muscle, and brain,32-36 and HDAC7 is highly expressed
in CD4/CD8 double-positive thymocytes.37 Class IIb
HDAC6 is predominantly expressed in testis,32 and
HDAC10 is expressed in liver, spleen, and kidney.38,39
Class I HDACs are well-known transcriptional co-
repressors, acting through the block of the expression
of some tumor suppressor genes. Class IIa HDACs have
been reported to interact with one or more DNA-binding
transcription factors, as well as with transcriptional co-
repressors.29 Among these interactions, the class IIa
HDACs-MEF2 interactions are the most studied.40
MEF2 plays a critical role in cardiac and skeletal
myogenesis, in negative selection of developing thy-
mocytes, and in the transcriptional regulation of Ep-
stein-Barr virus (EBV). Moreover, MEF2 has a role in
neuronal resistance to excitotoxicity. By binding to
MEF2 several promoters, class IIa HDACs can act as
transcriptional repressors in a variety of biological
functions, from myogenesis41 to EBV latency.42 Differ-
ently, class IIb HDAC6 takes part, together with the
class III HDAC SIRT2, in the microtubule network and
acts as specific R-tubulin deacetylase in vitro and in
vivo.43,44
Most of the HDAC inhibitors described so far inhibit
to the same extent class I as well as class II members
of the HDAC family. The only exceptions are VPA, 5-fold
more potent against HDAC1 than HDAC5 and HDAC6,45
and FK-228, a class I-selective prodrug.46 Class IIb
HDAC6 and HDAC10 have been found to be resistant
to the inhibitors TPX-A and -B, CHAPs, and NaB47,48
and are selectively inhibited by tubacin, a 1,3-dioxane-
containing hydroxamate discovered through a multidi-
mensional, chemical genetic screen of 7392 molecules
as a specific R-tubulin deacetylation inhibitor in mam-
malian cells (Chart 1).49-52

3346 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Mai et al.
Scheme 1a
a (a) (COCl)2, DMF, dichloroethane, room temp; (b) aryl alkyl ketone, KOH, C2H5OH, H2O, room temp; (c) ClCOOC2H5, (C2H5)3N, THF,
0 °C; (d) NH2OC(CH3)2OCH3; (e) Amberlyst 15, MeOH, 45 °C.
Table 1. Chemical and Physical Data for Compounds 2a-w
compd
R
R1
mp, °C
recrystn solvent
% yield
formula
anal.a
2a
2b
2c
2d
2e
2f
2g
2h
2i
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
205-207
224-226
198-200
>260
220-222
212-215
>260
168-170
253-255
250-252
222-224
215-217
246-248
165-166
194-196
>260
207-209
>260
184-185
205-207
184-185
174-175
190-192
CH3CN/benzene
CH3CN/MeOH
MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
MeOH
54
58
72
80
65
62
53
61
56
61
45
67
61
69
65
52
46
45
76
68
69
61
63
C17H16N2O3
C, H, N
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-F-Ph
C17H15ClN2O3
C, H, N, Cl
C, H, N, Cl
C, H, N, Cl
C, H, N, F
C, H, N, F
C, H, N, F
C, H, N, Br
C, H, N, Br
C, H, N, Br
C, H, N
C17H15ClN2O3
C17H15ClN2O3
C17H15FN2O3
C17H15FN2O3
C17H15FN2O3
C17H15BrN2O3
3-F-Ph
4-F-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
3-CF3-Ph
3-CF3O-Ph
1-naphthyl
2-naphthyl
4-biphenyl
Ph
C
17H15BrN2O3
17H15BrN2O3
2j
2k
2l
MeOH
CH3CN
C
C18H18N2O3
C18H18N2O3
C18H18N2O3
CH3CN/MeOH
CH3CN/MeOH
MeOH
C, H, N
2m
2n
2o
2p
2q
2r
2s
2t
C, H, N
C18H18N2O4
C18H18N2O4
C18H18N2O4
C, H, N
MeOH
C, H, N
MeOH/DMF
THF/MeOH
MeOH/DMF
CH3CN/MeOH
MeOH
C, H, N
C18H15F3N2O3
C17H15FN2O3
C21H18N2O3
C, H, N, F
C, H, N, F
C, H, N
C
21H18N2O3
23H20N2O3
C, H, N
2u
2v
2w
MeOH
CH3CN/benzene
CH3CN/benzene
C
C, H, N
C18H18N2O3
C19H20N2O3
C, H, N
Ph
C, H, N
a Analytical results were within (0.4% of the theoretical values.
Chemical and physical data of compounds 2a-w are
listed in Table 1. Chemical and physical data of the
intermediate compounds 3 and 4a-w are listed in Table
2.
IC50-HD1-A ratio) have been assessed. TSA, SAHA, and
tubacin have been also tested for comparison purpose
(Table 4).
HD2 Inhibitory Activity and Structure-Activity
Relationships (SARs). (Aryloxopropenyl)pyrrolyl hy-
droxyamides 2a-w have been first evaluated against
maize HD2, in comparison with NaB, VPA, TSA, SAHA,
TPX, HC-toxin, and tubacin (Table 3). From inhibitory
data, it is clear that the type and the position of the
substituent inserted at the benzene ring of the unsub-
stituted 3-[4-(3-phenyl-3-oxopropen-1-yl)-1-methyl-1H-
pyrrol-2-yl]-N-hydroxy-2-propenamide 2a play a crucial
role in determining the inhibitory activity of the tested
derivatives. Compound 2a inhibited HD2 at submicro-
molar concentrations (IC50 ) 0.28 µM). The introduction
of a halogen atom (chlorine, fluorine, or bromine) at each
of the three positions of the 2a benzene ring generally
decreased or abated the potency of derivatives, the only
exception being the 2-bromo analogue 2h that retained
almost the same activity as 2a. The 2-chloro- and
2-fluoro derivatives 2b and 2e were 18- and 25-fold less
active than 2a, respectively; when the halogen was
Results and Discussion
The novel (aryloxopropenyl)pyrrolyl hydroxyamides
2a-w have been evaluated for their ability to inhibit
maize HD2.61,73-76 Two short-chain fatty acids (NaB77
and VPA37,78), two hydroxamic acids (TSA79 and SA-
HA80), two cyclic tetrapeptides (TPX81 and HC-toxin82),
and tubacin,50-52 the sole class II (class IIb)-selective
HDAC inhibitor reported to date, have been used as
reference drugs. The results, expressed as the percent
of inhibition at a fixed dose and IC50 (50% inhibitory
concentration) values, are reported in Table 3. Com-
pounds 2a-w have been also tested against maize HD1-
B65,66 and HD1-A,67,68 two mammalian class I and class
II (IIa) HDAC homologues, respectively, and the result-
ing fold selectivity values (for class I HDACs: IC50-HD1-A
/
/
IC50-HD1-B ratio; for class II HDACs: IC50-HD1-B

Class II (IIa)-Selective Histone Deacetylase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3347
Table 2. Chemical and Physical Data for Compounds 3 and 4a-w
compd
R
R1
mp, °C
recrystn solvent
% yield
formula
anal.a
3
102-104
205-207
224-226
215-217
>260
220-222
210-212
>260
255-256
230-232
258-260
258-260
>260
246-248
205-207
193-195
203-204
232-234
234-236
230-232
261-263
232-234
174-176
190-192
cyclohexane
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN/MeOH
CH3CN
CH3CN/MeOH
CH3CN
CH3CN/MeOH
CH3CN/MeOH
MeOH
74
52
57
57
75
56
51
66
57
58
65
41
47
45
50
66
74
86
84
71
69
70
42
37
C10H13NO2
C, H, N
4a
4b
4c
4d
4e
4f
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
C17H15NO3
C, H, N
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-F-Ph
C17H14ClNO3
C17H14ClNO3
C17H14ClNO3
C17H14FNO3
C17H14FNO3
C17H14FNO3
C17H14BrNO3
C17H14BrNO3
C17H14BrNO3
C18H17NO3
C, H, N, Cl
C, H, N, Cl
C, H, N, Cl
C, H, N, F
C, H, N, F
C, H, N, F
C, H, N, Br
C, H, N, Br
C, H, N, Br
C, H, N
3-F-Ph
4-F-Ph
4g
4h
4i
4j
4k
4l
4m
4n
4o
4p
4q
4r
4s
4t
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
3-CF3-Ph
3-OCF3-Ph
1-naphthyl
2-naphthyl
4-biphenyl
Ph
C18H17NO3
C, H, N
C18H17NO3
C, H, N
C18H17NO4
C, H, N
C18H17NO4
C, H, N
C18H17NO4
C, H, N
THF/MeOH
MeOH
C18H14F3NO3
C17H13FNO3
C21H17NO3
C21H17NO3
C, H, N, F
C, H, N, F
C, H, N
CH3CN
CH3CN
C, H, N
4u
4v
4w
MeOH
C23H19NO3
C18H17NO3
C19H19NO3
C, H, N
benzene
C, H, N
Ph
benzene
C, H, N
a Analytical results were within (0.4% of the theoretical values.
Table 3. Maize HD2 Inhibitory Activity of Compounds 2a-wa
less (2o,p) active. Differently, 3-trifluoromethyl- and
3-trifluoromethoxy-substituted compounds 2q and 2r as
well as 4-biphenyl derivative 2u were substantially
inactive. Replacement of the 2a benzene ring with the
bulky 1-naphthyl group led to 2s, that was 1.4-fold more
potent than 2a, while compound 2t bearing a 2-naphthyl
moiety at the 3-propenyl position showed 6.4 times
lower activity than 2a. Finally, an alkyl (methyl or
ethyl) substituent at the C2 position of the 3-oxopropenyl
chain was tolerated for HD2 inhibition, 2v and 2w being
2.4- and 1.5-fold less active than the unsubstituted 2a,
respectively.
HD1-B and HD1-A Inhibitory Assays: Assess-
ment of Class Selectivity. The novel (aryloxoprope-
nyl)pyrrolyl hydroxyamides 2a-w have been tested
against maize HD1-B and HD1-A, two mammalian class
I and class II (IIa) HDAC homologues, and their class
selectivity has been calculated. The unselective inhibi-
tors TSA and SAHA and the class IIb-selective tubacin
have been also tested as reference drugs. Data sum-
marized in Table 4 show that the inhibitory trend of
2a-w against HD1-B as well as against HD1-A is the
same as that observed for 2a-w against HD2, the main
difference lying in the diverse degree of susceptibility
of HD1-B and HD1-A to (aryloxopropenyl)pyrrolyl hy-
droxamate inhibitors. The inhibitory activity of the
unsubstituted 2a was at submicromolar concentrations
(IC50-HD1-B ) 0.26 µM; IC50-HD1-A ) 0.19 µM), and the
potency of substituted compounds against both the two
maize enzymes varied according to the order of ortho >
meta > para-substituted analogues, the last being in
general the lowest active (or inactive) derivatives, while
the ortho-substituted analogues were among the most
potent compounds of the series. In anti-HD1-B assay,
the chloro- and fluoro-substitution at the benzene C2
position led to 2b and 2e, that were 6- and 13-fold less
active than 2a, respectively, while the corresponding C3-
and C4-substituted 2c,d,f,g were substantially inactive.
Bromine and methyl substitutions generated more
active compounds, the C2-substituted analogues being
% inhbtn
compd
R
R1 (at a fixed dose, µM) IC50 ( SD (µM)
2a
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
92.3 (26)
79.1 (23.3)
49 (23.3)
0.28 ( 0.01
5.2 ( 0.16
24.2 ( 1.21
25.2 ( 1.26
7.1 ( 0.35
22.0 ( 1.32
35.3 ( 1.06
0.36 ( 0.02
2.1 ( 0.13
28.5 ( 1.71
0.27 ( 0.008
7.9 ( 0.24
10.2 ( 0.41
0.25 ( 0.01
0.54 ( 0.02
0.57 ( 0.03
49.0 ( 2.0
176.0 ( 5.3
0.20 ( 0.006
1.8 ( 0.09
26.5 ( 1.06
0.68 ( 0.02
0.42 ( 0.02
NDb
128.0 ( 3.8
0.007 ( 0.0002
0.05 ( 0.001
0.01 ( 0.0003
0.11 ( 0.004
2.0 ( 0.1
2b
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-F-Ph
2c
2d
45.5 (23.3)
73.9 (24.5)
56.5 (24.5)
33.2 (24.5)
92.4 (20.5)
76.8 (20.5)
41.7 (20.5)
96 (24.8)
2e
2f
3-F-Ph
4-F-Ph
2g
2h
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
3-CF3-Ph
3-CF3O-Ph H
1-naphthyl H
2-naphthyl H
4-biphenyl
Ph
Ph
2i
2j
2k
2l
72 (24.8)
65 (24.8)
2m
2n
95.7 (23.6)
78.2 (23.6)
74.2 (23.6)
46.1 (21.1)
19.5 (20.2)
92 (22.2)
62.9 (25.2)
34 (20.6)
89.9 (24.8)
93 (23.7)
35 (5000)
2o
2p
2q
2r
2s
2t
2u
H
Me
Et
2v
2w
NaB
VPA
TSA
SAHA
TPX
HC-toxin
tubacin
92.9 (40)
a
Data represent mean values of at least three separate
b
experiments. ND, not determined.
introduced at benzene C3 as well as C4 position, only
substantially inactive compounds have been obtained,
with the exception of the 3-bromo derivative 2i (IC50
)
2.1 µM), that was 7.5 times less potent than 2a. Methyl
substitution at the benzene C2 position furnished a
compound (2k) showing the same activity as 2a, while
the 3- and 4-methyl counterparts (2l and 2m) were from
28 to 36 times less potent than 2a in inhibiting HD2.
In comparison with the unsubstituted 2a, the three
methoxy derivatives 2n-p were equally (2n) or slightly

3348 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Mai et al.
Table 4. Maize HD1-B and HD1-A Inhibitory Activities of Compounds 2a-wa
IC50 ( SD (µM)
SIb
compd
R
R1
HD1-B
HD1-A
class I
class II (IIa)
2a
Ph
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Me
Et
0.26 ( 0.02
1.6 ( 0.08
31.4 ( 1.26
29.4 ( 1.18
3.4 ( 0.14
38.8 ( 1.16
24.9 ( 1.24
0.18 ( 0.007
1.1 ( 0.04
0.19 ( 0.01
0.05 ( 0.003
0.44 ( 0.02
18.6 ( 1.12
0.10 ( 0.004
0.22 ( 0.01
15.7 ( 0.94
0.21 ( 0.008
1.6 ( 0.06
2b
2-Cl-Ph
3-Cl-Ph
4-Cl-Ph
2-F-Ph
32
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
71.4
34
176.4
3-F-Ph
4-F-Ph
2-Br-Ph
3-Br-Ph
4-Br-Ph
2-Me-Ph
3-Me-Ph
4-Me-Ph
2-MeO-Ph
3-MeO-Ph
4-MeO-Ph
3-CF3-Ph
3-CF3O-Ph
1-naphthyl
2-naphthyl
4-biphenyl
Ph
24.3 ( 0.97
0.39 ( 0.02
2.5 ( 0.08
20.3 ( 0.81
0.06 ( 0.002
0.17 ( 0.007
0.50 ( 0.02
0.16 ( 0.01
0.39 ( 0.02
0.79 ( 0.02
9.3 ( 0.46
6.5
14.7
9.6
4.8 ( 0.29
0.17 ( 0.008
0.60 ( 0.03
0.80 ( 0.05
159.0 ( 6.4
298.0 ( 14.9
0.33 ( 0.02
1.8 ( 0.07
2p
2q
17.1
3.3
33
45
14.4
2r
90.0 ( 4.5
2s
0.01 ( 0.0007
0.04 ( 0.002
2.2 ( 0.07
2t
2u
2v
31.8 ( 1.27
0.57 ( 0.02
4.5 ( 0.22
0.0004 ( 0.00001
0.03 ( 0.001
0.98 ( 0.04
0.78 ( 0.02
0.22 ( 0.01
0.0008 ( 0.00003
0.18 ( 0.009
0.45 ( 0.02
2w
TSA
SAHA
tubacin
Ph
20.4
2
6
2.2
a Data represent mean values of at least three separate experiments. bSI, Selectivity Index.
more potent (2h) than or as active (2k) as 2a, and the
C3-substituted 2i and 2l were 4- and 10-fold less potent
than 2a. 2-Methoxy-substituted 2n showed higher
activity than 2a, and the corresponding 3- and 4-isomers
2o and 2p were 2-4 times less active than 2a in
inhibiting HD1-B. As it has been observed in the anti-
HD2 assay and also against HD1-B, the 3-trifluoro-
methyl (2q) and 3-trifluoromethoxy (2r) as well as the
4-biphenyl (2u) derivatives failed in inhibiting the
enzyme. Compound 2s, in which the 2a benzene ring
has been replaced with 1-naphthyl moiety, retained
almost the same activity as 2a, while the 2-naphthyl
isomer 2t was 7-fold less potent. At last, 2v and 2w
carrying methyl and ethyl substituent at the C2 position
of the 3-oxopropenyl chain showed from 2 to 17 times
lower activity than 2a in inhibiting HD1-B.
In anti-HD1-A assay the order of potency of substi-
tuted compounds (ortho > meta > para) in comparison
with the unsubstituted 2a was fully maintained, but the
susceptibility of the enzyme to the (aryloxopropenyl)-
pyrrolyl hydroxamate inhibitors was very high. The
2-chloro- and 2-fluoro-substituted 2b and 2e were 4- and
2-fold more active than 2a in inhibiting HD1-A, and the
3-substituted analogues 2c and 2f showed the same
activity (2f) as or 2-fold lower potency (2c) than 2a.
Among bromo-substituted derivatives, the benzene-C2
substituted 2h retained the same activity than 2a, the
C3 isomer 2i being 8 times less potent than the unsub-
stituted reference. Methyl and methoxy substitutions
gave highly active compounds, methyl- being more
potent than the corresponding methoxy-containing de-
rivatives. In particular, 2-methyl-, 3-methyl-, and 2-meth-
oxyphenyloxopropenyl-pyrrolyl hydroxamates 2k,l,n were
up to 3 times more potent than 2a in inhibiting HD1-
A. Differently, the insertion of trifluoromethyl or tri-
fluoromethoxy substituent at the C3 position of the
benzene ring led to inactive compounds (2q and 2r).
Both the naphthyl analogues 2s and 2t were more
efficient (19- and 5-fold, respectively) than the phenyl
counterpart in inhibiting HD1-A, and the 4-biphenyl
compound 2u, inactive against HD2 as well as HD1-B
enzymes, showed IC50 ) 2.2 µM against HD1-A. Finally,
the insertion of a methyl or ethyl substituent at the
oxopropenyl chain was well tolerated in anti-HD1-A
assay, as phenyloxobutenyl and phenyloxopentenyl
derivatives 2v and 2w showed slightly lower activity
than the phenyloxopropenyl 2a.
To determine the class selectivity for (aryloxopro-
penyl)pyrrolyl hydroxamates 2a-w, the IC50-HD1-A
IC50-HD1-B ratio for class I-selectivity and the IC50-HD1-B
/
/
IC50-HD1-A ratio for class II (IIa)-selectivity have been
calculated. Only selectivity values g2 have been re-
ported. Table 4 shows that compounds substituted at
C2 or better at C3 position of the benzene ring with
chloro- (2b,c), fluoro- (2e,f), or methyl (2k,l) groups, as
well as the naphthyl derivatives 2s,t, were highly
selective for the class II (IIa) HDAC homologue HD1-
A, 3-[4-(3-(3-chlorophenyl)- and 3-[4-(3-(3-fluorophenyl)-
3-oxopropen-1-yl)-1-methyl-1H-pyrrol-2-yl]-N-hydroxy-
2-propenamides 2c and 2f being the most class II (IIa)-
selective compounds, with a selectivity ratio of 71.4 and
176.4, respectively. Among the reference drugs, TSA and
SAHA showed to be slightly selective (2 and 6 times)
toward the class I HDAC homologue HD1-B. Tubacin,
a highly “in cell” selective class IIb HDAC inhibitor, was
2.2 times more potent against HD1-A (class II homo-
logue) than HD1-B (class I homologue). This result is
as expected, because the tubacin selectivity is truly
manifest in “in cell” functional assay, by comparing the
induction of acetylated histone (EC50 ) 217 ( 96 µM)
and acetylated tubulin (EC50 ) 2.9 ( 0.9 µM; selectivity
ratio ) 75) in mammalian cells,50 while in enzyme
(HDAC1, HDAC4, and HDAC6) assays tubacin showed

Class II (IIa)-Selective Histone Deacetylase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3349
Table 5. Human HDAC1 and HDAC4 Inhibitory Activities of
2f and SAHAa
(48h) apoptosis, 73% CD11c positive cells, and cell cycle
arrest in G2/M phase. In the same assays, 2f at 5 µM
showed % values (apoptosis, granulocytic differentiation,
cell cycle phases) similar to those of controls.
% inhibitory activity at 5 µM
assay
2f
SAHA
HDAC1
HDAC4
0
54.9
52.9
37.2
Conclusion
A novel series of (aryloxopropenyl)pyrrolyl hydrox-
amates 2a-w have been described as HDAC inhibitors,
and their selectivity for maize HD1-A, a mammalian
class II (IIa) HDAC homologue, has been determined.
In general, the introduction of a substituent (from
halogen atoms to methyl or methoxy groups) at the
benzene ring of the (phenyloxopropenyl)pyrrolyl skel-
eton of the molecules did not improve the inhibitory
activity, the order of potency of compounds (ortho- >
meta- > para-substituted) being the same for all the
three tested enzymes (HD2, HD1-B, and HD1-A). Nev-
ertheless, in the anti-HD1-A assay some highly active
derivatives have been obtained, in particular whit
chloro-, fluoro-, methyl- and methoxy-substitutions at
the benzene C2 and, to a lesser extent, C3 position.
Moreover, since such derivatives (mainly the C3-
substituted compounds) showed little or no activity
against HD1-B, a mammalian class I HDAC homologue,
they were highly class II (IIa)-selective, the most selec-
tive being the 3-chloro- and 3-fluoro-substituted com-
pounds 2c and 2f, with selectivity ratios of 71.4 (2c) and
176.4 (2f). Also the replacement of the benzene ring with
the bulkier 1- and 2-naphthyl moieties furnished highly
active derivatives against HD1-A (2s: IC50 ) 10 nM;
2t: IC50 ) 40 nM), although less selective than 2c,f. In
comparison with TSA and SAHA, the inhibitory con-
centrations of (aryloxopropenyl)pyrrolyl hydroxyamides
against HD1-A are substantially higher than that of
TSA and in the range of SAHA. About selectivity,
properly substituted (aryloxopropenyl)pyrrolyl hydroxy-
amides were class II (IIa) HDAC selective inhibitors,
while TSA and SAHA showed a little selectivity against
class I HDACs.
When tested against immunoprecipitated human
HDAC1 and HDAC4, 2f showed no inhibitory activity
against HDAC1, while was able to inhibit HDAC4 (%
inhibition at 5 µM ) 54.9%). Moreover, in human U937
cancer cells 2f did not produce any effect on apoptosis,
granulocytic differentiation, and the cell cycle (class II
HDAC-selective inhibitor behavior), whereas 2s which
retains class I HDAC inhibitory activity was 2-fold less
potent than SAHA, used as reference to induce apoptosis
and granulocytic differentiation, and blocked the cell
cycle in G1 phase.
(Aryloxopropenyl)pyrrolyl hydroxamates 2a-w are
the first reported examples of class II (IIa)-selective
compounds and together with the “in cell” class IIb-
selective tubacin can be useful tools to probe the biology
and to elucidate the functions, largely still unknown,
of HDACs. Molecular modeling studies are in progress
to rationalize the different behaviors of our class II (IIa)-
selective molecules against HD1-B and HD1-A enzymes.
a Data represent mean values of at least three separate experi-
ments.
about 4-fold selectivity for HDAC6 (J. C. Wong, personal
communication).
Human HDAC1 and HDAC4 Inhibitory Assays.
The most selective compound in the maize HD1-B/
HD1-A system, i.e. the 3-[4-(3-(3-fluorophenyl)-3-oxo-
propen-1-yl)-1-methyl-1H-pyrrol-2-yl]-N-hydroxy-2-pro-
penamide (2f) (class II fold selectivity ) 176.4), was
evaluated against human HDAC1 and HDAC4 in com-
parison with SAHA as reference drug. Human breast
cancer ZR-75.1 cell lysates were immunoprecipitated
with antibodies against HDAC1 and HDAC4, and
inhibitory assays were performed on such immunopre-
cipitates (IPs) with 2f (5 µM) and SAHA (5 µM). Data
reported in Table 5 clearly show that 2f lacked any
inhibitory activity against human HDAC1 but was
effective in inhibiting human HDAC4 enzyme.
Cellular Activities Evaluation: Apoptosis, Granu-
locytic Differentiation, and Cell Cycle Analysis. To
investigate on cellular activities of selected (aryloxo-
propenyl)pyrrolyl hydroxamides, the effects of 2f and
2s on apoptosis, cytodifferentiation, and cell cycle phases
in human U937 cell line were determined in comparison
with SAHA.
It is well-known that class I HDACs induce the
expression of some tumor suppressor genes, such as the
cyclin-dependent kinase inhibitor p21WAF1/Cip1 17,83
while
,
class II HDACs interact with one or more DNA-binding
transcription factors as well as with transcriptional co-
repressors, such as MEF2.29 A detailed study using
small interfering double stranded RNA (siRNA) to
selectively knockdown HDACs 1, 3, 4, and 7 in HeLa
S3 cells showed that knockdowns of HDACs 1 and 3
produced a significant morphological phenotype similar
to that observed with HDAC inhibitor treatment of
these cells, giving an antiproliferative effect, whereas
knockdowns of HDACs 4 and 7 produced no changes in
cell morphology.69,84 These studies established the criti-
cal role of class I HDACs in tumor cell proliferation/
survival, while class II HDACs may not be involved in
the process that controls proliferation and apoptosis in
cancer cells, their involvement being important in
diverse biological processes such as cardiac and skeletal
myogenesis, negative selection of developing thy-
mocytes, and other unknown processes.29
On these bases, the treatment of U937 AML cells with
2s, that retain class I HDAC activity (IC50-HD1-B ) 0.33
µM; IC50-HD1-A ) 0.01 µM), was expected to induce
apoptosis, cell differentiation, and cell cycle arrest, while
treatment of U937 AML cells with 2f, the selective class
II (IIa)-inhibitor (IC50-HD1-B ) 38.8 µM; IC50-HD1-A
)
Experimental Section
0.22 µM), was expected to have no effect. Indeed, as
depicted in Figure 1, 2s at 5 µM induced 4% (24 h) and
8% (48 h) apoptosis, 56% CD11c positive cells, and
arrest of cell cycle in G1 phase in U937 cells. SAHA,
used as reference, at 5 µM produced 9% (24 h) and 22%
Chemistry. Melting points were determined on a Bu¨chi 530
melting point apparatus and are uncorrected. Infrared (IR)
spectra (KBr) were recorded on a Perkin-Elmer Spectrum One
instrument. 1H NMR spectra were recorded at 200 MHz on a
Bruker AC 200 spectrometer; chemical shifts are reported in

3350 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Mai et al.
Figure 1. Effects of 2s and 2f on apoptosis, granulocytic differentiation, and cell cycle phases (U937 AML cell line) in comparison
with SAHA.
δ (ppm) units relative to the internal reference tetramethyl-
silane (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 standards meth-
ods. Concentration of solutions after reactions and extractions
involved the use of a rotary evaporator operating at a reduced
pressure of ca. 20 Torr. Organic solutions were dried over
anhydrous sodium sulfate. Analytical results are within
(0.40% of the theoretical values. A SAHA sample for biological
assays was prepared as previously reported by us.85 Tubacin
was kindly provided by Jason C. Wong (Harvard University,
Cambridge, MA). All chemicals were purchased from Aldrich
Chimica, Milan (Italy) or Lancaster Synthesis GmbH, Milan
(Italy) and were of the highest purity.
Ethyl 3-(4-Formyl-1-methyl-1H-pyrrol-2-yl)-2-propen-
oate (3). A 50 mL 1,2-dichloroethane solution of oxalyl chloride
(0.06 mol, 5.2 mL) was added to a cooled (0-5 °C) solution of
N,N-dimethylformamide (0.06 mol, 4.6 mL) in 1,2-dichloro-
ethane (50 mL) over a period of 5-10 min. After being stirred
at room temperature for 15 min, the suspension was cooled
(0-5 °C) again and treated with a solution of ethyl 3-(1-methyl-
1H-pyrrol-2-yl)-2-propenoate71 (0.06 mol, 10.7 g) in 1,2-dichlo-
roethane (50 mL). The mixture was stirred at room temper-
ature for 1 h and then was poured onto crushed ice (200 g)
containing 50% NaOH (50 mL) and stirred for 10 min. The
pH of the solution was adjusted to 4 with 37% HCl, the organic
layer was separated, and the aqueous one was extracted with
chloroform (2 × 50 mL). The combined organic solutions were
washed with water, dried, and evaporated to dryness. The
residual solid was purified by recrystallization. 1H NMR
(DMSO-d6) δ 1.24 (t, 3 H, CH2CH3), 3.96 (s, 3 H, NCH3), 4.20
(q, 2 H, CH2CH3), 6.60 (d, 1 H, CHdCHCO), 6.96 (d, 1 H,
pyrrole â-proton), 7.03 (d, 1 H, pyrrole R-proton), 7.60 (d, 1 H,
CH)CHCO), 9.58 (s, 1 H, CHO). Anal. (C10H13NO2) C, H, N.
General Procedure for the Synthesis of 3-[4-(3-Aryl-
3-oxopropen-1-yl)-1-methyl-1H-pyrrol-2-yl]-2-propeno-
ic Acids 4a-w. Example: 3-[4-(3-(4-Chloro)phenyl-3-oxo-
1-propen-1-yl)-1-methyl-1H-pyrrol-2-yl]-2-propenoic Acid
4d. A mixture of ethyl 3-(4-formyl-1-methyl-1H-pyrrol-2-yl)-
2-propenoate 3 (6.0 mmol, 1.07 g), 4-chloroacetophenone (6.0
mmol, 0.93 g), and 2 N KOH (24.0 mmol, 12.4 mL) in ethanol
(15 mL)/water (15 mL) was stirred at room temperature for
24 h. Afterward, the solution was poured into water (100 mL)
and was made acid with 2 N HCl. The obtained precipitate
was filtered and recrystallized to give the pure acid 4d. 1H
NMR (DMSO-d6) δ 3.75 (s, 3 H, NCH3), 6.34 (d, 1 H, CHd
CHCOOH), 6.96 (d, 1 H, pyrrole â-proton), 7.21 (d, 1 H, pyrrole
R-proton), 7.54 (d, 1 H, CH)CHCOOH), 7.65 (d, 2 H, benzene
H-2,6), 7.73 (d, 1 H, COCH)CH), 7.77 (d, 1 H, COCHdCH),
8.12 (d, 2 H, benzene H-3,5), 12.22 (s, 1 H, OH). Anal. (C17H14-
ClNO3) C, H, N, Cl.
propen-1-yl)-1-methyl-1H-pyrrol-2-yl]-N-hydroxy-2-
propenamide (2a). Ethyl chloroformate (5.0 mmol, 0.5 mL)
and triethylamine (5.4 mmol, 0.8 mL) were added to a cooled
(0 °C) solution of 3-[4-(3-phenyl-3-oxopropen-1-yl)-1-methyl-
1H-pyrrol-2-yl]-2-propenoic acid 4a (4.2 mmol, 1.18 g) in dry
THF (10 mL), and the mixture was stirred for 10 min. The
solid was filtered off, and O-(2-methoxy-2-propyl)hydroxyl-
amine (12.6 mmol, 1.1 mL)72 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
methanol (30 mL). Amberlyst 15 ion-exchange resin (0.6 g) was
added to the solution of the O-protected hydroxamate, and the
mixture was stirred at 45 °C for 1 h. Afterward, the reaction
was filtered and the filtrate was concentrated in vacuo to give
1
the crude 4a, which was purified by crystallization. H NMR
(DMSO-d6) δ 3.73 (s, 3 H, NCH3), 6.30 (d, 1 H, CHdCH-
CONHOH), 6.67 (s, 1 H, pyrrole â-proton), 7.17 (s, 1 H, pyrrole
R-proton), 7.32 (d, 1 H, PhCOCHdCH), 7.53 (m, 3 H, benzene
H-3,4,5), 7.62 m, 2 H, benzene H-2,6), 8.08 (d, 2 H, CHdCH-
CONHOH and PhCOCHdCH). Anal. (C17H16N2O3) C, H, N.
In Vitro Maize HD2, HD1-B, and HD1-A Enzyme
Inhibition. Radioactively labeled chicken core histones were
used as the enzyme substrate according to established
procedures.73-76 The enzyme liberated tritiated acetic acid from
the substrate, which was quantified by scintillation counting.
IC50 values are results of triple determinations. A 50 µL sample
of maize enzyme (at 30 °C) was incubated (30 min) with 10
µL of total [3H]acetate-prelabeled chicken reticulocyte histones
(2 mg/mL). Reaction was stopped by addition of 50 µL of 1 M
HCl/0.4 M acetate and 800 µL of ethyl acetate. After centrifu-
gation (10 000g, 5 min), an aliquot of 600 µL of the upper phase
was counted for radioactivity in 3 mL of liquid scintillation
cocktail. The compounds were tested at a starting concentra-
tion of 40 µM, and active substances were diluted further. NaB,
VPA, TSA, SAHA,85 TPX, HC-toxin, and tubacin were used
as the reference compounds, and blank solvents were used as
negative controls.
Human HDAC1 and HDAC4 Inhibition. Cell Culture
and Ligands. Human breast cancer ZR-75.1 cells were
propagated in DMEM medium supplemented with 10% FBS
and antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin,
and 250 ng/mL amphotericin-B). Before use, cells were plated
at 60% confluence and treated the day after with 5 µM SAHA85
or 2f. All compounds were resuspended in DMSO (Sigma-
Aldrich).
HDAC1 and HDAC4 Assays. A 1000 µg amount of total
protein extracts (RIPA buffer for HDAC1 immunoprecipitation
and NP-40 buffer for HDAC4 immunoprecipitation) was im-
munoprecipitated with 10 µg of HDAC1 antibody (Alexis) or
HDAC4 antibody (Santa Cruz) overnight at 4 °C in slow
rotation. As negative control the same amount of protein
extracts was immunoprecipitated with purified IgG (Santa
Cruz). One day later, the samples were incubated with 40 µL
of protein A/G plus agarose (Santa Cruz) at 4 °C in slow
rotation for 2-4 h. The samples were than washed six times
with RIPA buffer or NP40-buffer respectively and twice in PBS
General Procedure for the Synthesis of 3-[4-(3-Aryl-
3-oxopropen-1-yl)-1-methyl-1H-pyrrol-2-yl]-N-hydroxy-2-
propenamides 2a-w. Example: 3-[4-(3-Phenyl-3-oxo-1-

Class II (IIa)-Selective Histone Deacetylase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3351
(6) Grunstein, M. Histone acetylation in chromatin structure and
transcription. Nature 1997, 389, 349-352.
(7) Kouzarides, T. Histone acetylases and deacetylases in cell
proliferation. Curr. Opin. Genet. Dev. 1999, 9, 40-48.
(8) Roth, S.; Denu, J. M.; Allis, C. D. Histone acetyltransferases.
Annu. Rev. Biochem. 2001, 70, 81-120.
and resuspended in 20 µL of sterile PBS. The HDAC assays
were carried out according to suppliers instructions (Upstate).
Briefly, all samples immunoprecipitated with the HDAC1,
HDAC4, or purified IgG were pooled respectively to homog-
enize all samples. A 10 µL amount of each HDAC IP was
incubated with a previously labeled 3H-Histone H4 peptide
linked with streptavidin agarose beads (Upstate). In detail,
100.000 cpm of the H4-3H-acetyl-peptide was used for each
tube and incubated in 1X HDAC buffer with 10 µL of the
sample in the presence or absence of the HDAC inhibitor with
a final volume of 200 µL. Those samples were incubated
overnight at 37 °C in slow rotation. One day later, 50 µL of a
quenching solution was added and 100 µL of the samples was
counted in duplicate after a brief centrifugation in a scintil-
lation counter.
(9) Grant, P. A.; Berger, S. L. Histone acetyltransferase complexes.
Semin. Cell Dev. Biol. 1999, 10, 169-177.
(10) Cress, W. D.; Seto, E. Histone deacetylases, transcriptional
control, and cancer. J. Cell Physiol. 2000, 184, 1-16.
(11) Ng, H. H.; Bird, A. Histone deacetylases: silencers for hire.
Trends Biochem. Sci. 2000, 25, 121-126.
(12) Ferreira, R.; Magnaghi-Jaulin, L.; Robin, P.; Harel-Bellan, A.;
Trouche, D. The three members of the pocket proteins family
share the ability to repress E2F activity through recruitment of
a histone deacetylase. Proc. Natl. Acad. Sci. U.S.A. 1998, 95,
10493-10498.
(13) Stiegler, P.; De Luca, A.; Bagella, L.; Giordano, A. The COOH-
terminal region of pRb2/p130 binds to histone deacetylase 1
(HDAC1), enhancing transcriptional repression of the E2F-
dependent cyclin A promoter. Cancer Res. 1998, 58, 5049-5052.
(14) Nagy, L.; Kao, H.-Y.; Chakravarti, D.; Lin, R. J.; Hassig, C. A.;
Ayer, D. E.; Schreiber, S. L.; Evans, R. M. Nuclear receptor
repression mediated by a complex containing SMRT, mSin3A,
and histone deacetylase. Cell 1997, 89, 373-380.
(15) Alland, L.; Muhle, R.; Hou, H.; Potes, J.; Chin, L.; Schreiber-
Agus, N.; DePinho, R. A. Role for N-CoR and histone deacety-
lase in Sin3-mediated transcriptional repression. Nature 1997,
387, 49-55.
(16) Heinzel, T.; Lavinsky, R. M.; Mullen, T.-M.; Soderstrom, M.;
Laherty, C. D.; Torchia, J.; Yang, W.-M.; Brard, G.; Ngo, S. D.;
Davie, J. R.; Seto, E.; Eisenman, R. N.; Rose, D. W.; Glass, C.
K.; Rosenfeld, M. G. A complex containing N-CoR, mSin3 and
histone deacetylase mediates transcriptionalrepression. Nature
1997, 387, 43-48.
(17) Weidle, U. H.; Grossmann, A. Inhibition of Histone Deacety-
lases: a New Strategy to Target Epigenetic Modifications for
Anticancer Treatment. Anticancer Res. 2000, 20, 1471-1486.
(18) Kramer, O. H.; Go¨ttlicher, M. G.; Heinzel, T. Histone deacetylase
as a therapeutic target. Trends Endocrinol. Metab. 2001, 12,
294-300.
(19) Marks, P. A.; Richon, V. M.; Breslow, R.; Rifkind, R. A. Histone
deacetylase inhibitors as new cancer drugs. Curr. Opin. Oncol.
2001, 13, 477-483.
(20) Marks, P. A.; Rifkind, R. A.; Richon, V. M.; Breslow, R.; Miller,
T.; Kelly, W. K. Histone deacetylases and cancer: causes and
therapies. Nat. Rev. Cancer 2001, 1, 194-202.
(21) Jung, M. Inhibitors of Histone Deacetylase as New Anticancer
Agents. Curr. Med. Chem. 2001, 8, 1505-1511.
(22) Vigushin, D. M.; Coombes, R. C. Histone deacetylase inhibitors
in cancer treatment. Anti-Cancer Drugs 2002, 13, 1-13.
(23) Johnstone, R. W. Histone deacetylase inhibitors: novel drugs
for the treatment of cancer. Nat. Rev. Drug Discovery 2002, 1,
287-299.
(24) Kelly, W. K.; O′Connor, O. A.; Marks, P. A. Histone deacetylase
inhibitors: from target to clinical trials. Expert Opin. Investig.
Drugs 2002, 11, 1695-1713.
(25) Miller, T. A.; Witter, D. J.; Belvedere, S. Histone deacetylase
inhibitors. J. Med. Chem. 2003, 46, 5097-5116.
(26) Grozinger, C. M.; Schreiber, S. L. Deacetylase Enzymes: Biologi-
cal Functions and the Use of Small-Molecule Inhibitors. Chem.
Biol. 2002, 9, 3-16.
(27) Gao, L.; Cueto, M. A.; Asselbergs, F.; Atadja, P. Cloning and
functional characterization of HDAC11, a novel member of the
human histone deacetylase family. J. Biol. Chem. 2002, 277,
25748-25755.
Determination of Apoptosis, Cell Differentiation, and
Cell Cycle Effect on U937 AML Cells. Cell Lines and
Cultures. U937 cell line was cultured in RPMI with 10% fetal
calf serum, 100 U/mL penicillin, 100 mg/mL streptomycin, 10
mM HEPES, and 2 mM glutamine.
Ligands. SAHA85 was dissolved in DMSO and used at 5
µM. Compounds 2f and 2s were dissolved in DMSO and used
at 5 µM.
Granulocytic Differentiation. U937 cells were harvested
and resuspended in 10 µL of phycoerythrine-conjugated CD11c
(CD11c-PE). Control samples were incubated with 10 µL of
PE conjugated mouse IgG1, incubated for 30 min at 4 °C in
the dark, washed in PBS, and resuspended in 500 µL of PBS-
containing propidium iodide (0.25 µg/mL). Samples were
analyzed by FACS with Cell Quest technology (Becton Dick-
inson) as previously reported.86 Propidium iodide positive
samples have been excluded from the analysis.
Cell Cycle Analysis. 2.5 × 105 Cells were collected and
resuspended in 500 µL of an hypotonic buffer (0.1% Triton
X-100, 0.1% sodium citrate, 50 µg/mL propidium iodide, RNAse
A). Cells were incubated in the dark for 30 min. Samples were
acquired on a FACS-Calibur flow cytometer using the Cell
Quest software (Becton Dickinson) and analyzed with standard
procedures using the Cell Quest software (Becton Dickinson)
and the ModFit LT version 3 Software (Verity) as previously
reported.86 All the experiments were performed 3 times.
FACS Analysis of Apoptosis. Apoptosis was measured
with Annexin V/Propidium iodide double staining detection
(Roche) as recommended by the supplier; samples were
analyzed by FACS with Cell Quest technology (Becton Dick-
inson) as previously reported.87
Acknowledgment. The authors thank Dr. Jason C.
Wong (Harvard University, Cambridge, MA) for the
kind gift of tubacin. This work was partially supported
by grants from Progetto Finalizzato Ministero della
Salute 2002 (A.M.), AIRC 2003 (A.M.), PRIN 2004
(A.M.), European Union (QLG1-CT2000-01935 and
QLK3-CT2002-02029), Ministero della Salute R. F. 02/
184 (L.A.), PRIN 2004 (L.A.), and the Austrian Science
Foundation Grant P13620 (P.L.).
(28) Gregoretti, I. V.; Lee, Y.-M.; Goodson, H. V. Molecular Evolution
of the Histone Deacetylase Family: Functional Implications of
Phylogenetic Analysis. J. Mol. Biol. 2004, 338, 17-31.
(29) Verdin, E.; Dequiedt, F.; Kasler, H. G. Class II histone deacety-
lases: versatile regulators. Trends Genet. 2003, 19, 286-293.
(30) Bertos, N. R.; Wang, A. H.; Yang, X.-J. Class II histone
deacetylases: Structure, function, and regulation. Biochem. Cell
Biol. 2001, 79, 243-252.
Supporting Information Available: Elemental analyses.
This material is available free of charge via the Internet at
http://pubs.acs.org.
References
(31) Fischle, W.; Kiermer, V.; Dequiedt, F.; Verdin, E. The emergency
role of class II histone deacetylases. Biochem. Cell Biol. 2001,
79, 337-348.
(32) Verdel, A.; Khochbin, S. Identification and characterization of
a new family of higher eukaryotic histone deacetylases. Coor-
dinate expression of differentiation-dependent chromatin modi-
fiers. J. Biol. Chem. 1999, 274, 2440-2445.
(33) Fischle, W.; Emiliani, S.; Hendzel, M. J.; Nagase, T.; Nomura,
N.; Voelter, W.; Verdin, E. A new family of human histone
deacetylases related to Saccharomyces cerevisiae HDA1p. J.
Biol. Chem. 1999, 274, 11713-11720.
(34) Grozinger, C. M.; Hassig, C. A.; Schreiber, S. L. Three proteins
define a class of human histone deacetylases related to yeast
Hda1p. Proc. Natl. Acad. Sci. U.S.A. 1999, 96, 4868-4873.
(1) Hassig, C. A.; Schreiber, S. L. Nuclear histone acetylases and
deacetylases and transcriptional regulation: HATs off to HDACs.
Curr. Opin. Chem. Biol. 1997, 1, 300-308.
(2) Strahl, B. D.; Allis, C. D. The language of covalent histone
modifications. Nature 2000, 403, 41-45.
(3) Wu, J.; Grunstein, M. 25 Years after the nucleosome model:
chromatin modifications. Trends Biochem. Sci. 2000, 25, 619-
623.
(4) Wolffe, A. P.; Guschin, D. Chromatin structural features and
targets that regulate transcription. J. Struct. Biol. 2000, 129,
102-122.
(5) Cheung, W. L.; Briggs, S. D.; Allis, C. D. Acetylation and
chromosomal functions. Curr. Opin. Cell Biol. 2000, 12, 326-
333.

3352 Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9
Mai et al.
(35) De Ruijter, A. J. M.; van Gennip, A. H.; Caron, H. N.; Kemp, S.;
van Kuilenburg A. B. P. Histone deacetylases (HDACs): char-
acterization of the classical HDAC family. Biochem. J. 2003, 370,
737-749.
(36) Zhang, C. L.; McKinsey, T. A.; Chang, S.; Antos, C. L.; Hill, J.
A.; Olson, E. N. Class II histone deacetylases act as signal-
responsive repressors of cardiac hypertrophy. Cell 2002, 110,
479-488.
(37) Dequiedt, F.; Kasler, H. G.; Fischle, W.; Kiermer, V.; Weinstein,
M.; Herndier, B. G.; Verdin, E. HDAC7, a thymus-specific Class
II histone deacetylase, regulates Nur77 transcription and TCR-
mediated apoptosis. Immunity 2003, 18, 687-698.
(38) Fischer, D. D.; Cai, R.; Bhatia, U.; Asselbergs, F. A.; Song, C.;
Terry, R.; Trogani, N.; Widmer, R.; Atadja, P.; Cohen, D.
Isolation and characterization of a novel class II histone deacety-
lase, HDAC10. J. Biol. Chem. 2002, 277, 6656-6666.
(39) Kao, H. Y.; Lee, C. H.; Komarov, A.; Han, C. C.; Evans, R. M.
Isolation and characterization of mammalian HDAC10, a novel
histone deacetylase. J. Biol. Chem. 2002, 277, 187-193.
(40) McKinsey, T. A.; Zhang, C. L.; Olson, E. N. MEF2: a calcium-
dependent regulator of cell division, differentiation and death.
Trends Biochem. Sci. 2002, 27, 40-47.
(41) McKinsey, T. A.; Zhang, C. L.; Olson, E. N. Control of muscle
development by dueling HATs and HDACs. Curr. Opin. Genet.
Dev. 2001, 11, 497-504.
(42) Gruffat, H.; Manet, E.; Sergeant, A. MEF2-mediated recruitment
of class II HDAC at the EBV immediate early gene BZLF1 links
latency and chromatin remodeling. EMBO Rep. 2002, 3, 141-
146.
(43) Hubbert, C.; Guardiola, A.; Shao, R.; Kawaguchi, Y.; Ito, A.;
Nixon, A.; Yoshida, M.; Wang, X. F.; Yao, T. P. HDAC6 is a
microtubule-associated deacetylase. Nature 2002, 417, 455-458.
(44) Matsuyama, A.; Shimazu, T.; Sumida, Y.; Saito, A.; Yoshimatsu,
Y.; Seigneurin-Berny, D.; Osada, H.; Komatsu, Y.; Nishino, N.;
Khochbin, S.; Horinouchi, S.; Yoshida, M. In vivo destabilization
of dynamic microtubules by HDAC6-mediated deacetylation.
EMBO J. 2002, 21, 6820-6831.
(45) Go¨ttlicher, M.; Minucci, S.; Zhu, P.; Kra¨mer, O. H.; Schimpf, A.;
Giavara, S.; Sleeman, J. P.; Lo Coco, F.; Nervi, C.; Pelicci, P. G.;
Heinzel, T. Valproic acid defines a novel class of HDAC inhibitors
inducing differentiation of transformed cells. EMBO J. 2001, 20,
6969-6978.
(46) Fumurai, R.; Matsuyama, A.; Kobashi, N.; Lee, K.-H.; Nish-
iyama, M.; Nakajima, H.; Tanaka, A.; Komatsu, Y.; Nishino, N.;
Yoshida, M.; Horinouchi, S. FK228 (Depsipeptide) as a Natural
Prodrug That Inhibits Class I Histone Deacetylases. Cancer Res.
2002, 62, 4916-4921.
(47) Fumurai, R.; Komatsu, Y.; Nishino, N.; Khochbin, S.; Yoshida,
M.; Horinouchi, S. Potent histone deacetylase inhibitors built
from trichostatin A and cyclic tetrapeptide antibiotics including
trapoxin. Proc. Natl. Acad. Sci. U.S.A. 2001, 98, 87-92.
(48) Guardiola, A. R.; Yao, T.-P. Molecular Cloning and Characteriza-
tion of a Novel Histone Deacetylase HDAC10. J. Biol. Chem.
2002, 277, 3350-3356.
(49) Sternson, S. M.; Wong, J. C.; Grozinger, C. M.; Schreiber, S. L.
Synthesis of 7200 Small Molecules Based on a Substructural
Analysis of the Histone Deacetylase Inhibitors Trichostatin and
Trapoxin. Org. Lett. 2001, 3, 4239-4242.
(50) Wong, J. C.; Hong, R.; Schreiber, S. L. Structural Biasing
Elements for In-Cell Histone Deacetylase Paralog Selectivity.
J. Am. Chem. Soc. 2003, 125, 5586-5587.
(51) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Butcher, R. A.;
Schreiber, S. L. Multidimensional Chemical Genetic Analysis
of Diversity-Oriented Synthesis-Derived Deacetylase Inhibitors
Using Cell-Based Assays. Chem. Biol. 2003, 10, 383-396.
(52) Haggarty, S. J.; Koeller, K. M.; Wong, J. C.; Grozinger, C. M.;
Schreiber, S. L. Domain-selective small-molecule inhibitor of
histone deacetylase 6 (HDAC6)-mediated tubulin deacetylation.
Proc. Natl. Acad. Sci. U.S.A. 2003, 100, 4389-4394.
(53) Tong, J. K. Dissecting Histone Deacetylase Function. Chem. Biol.
2002, 9, 668-670.
(54) Massa, S.; Mai, A.; Sbardella, G.; Esposito, M.; Ragno, R.; Loidl,
P.; Brosch, G. 3-(4-Aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propena-
mides, a New Class of Synthetic Histone Deacetylase Inhibitors.
J. Med. Chem. 2001, 44, 2069-2072.
(55) Mai, A.; Massa, S.; Ragno, R.; Esposito, M.; Sbardella, G.; Nocca,
G.; Scatena, R.; Jesacher, F.; Loidl, P.; Brosch, G. Binding Mode
Analysis of 3-(4-Benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-
propenamide: A New Synthetic Histone Deacetylase Inhibitor
Inducing Histone Hyperacetylation, Growth Inhibition, and
Terminal Cell Differentiation. J. Med. Chem. 2002, 45, 1778-
1784.
(56) Mai, A.; Massa, S.; Ragno, R.; Cerbara, I.; Jesacher, F.; Loidl,
P.; Brosch, G. 3-(4-Aroyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-
alkylamides as a New Class of Synthetic Histone Deacetylase
Inhibitors. 1. Design, Synthesis, Biological Evaluation, and
Binding Mode Studies Performed Through Three Different
Docking Procedures. J. Med. Chem. 2003, 46, 512-524.
(57) Mai, A.; Massa, S.; Cerbara, I.; Valente, S.; Ragno, R.; Bottoni,
P.; Scatena, R.; Loidl, P.; Brosch, G. 3-(4-Aroyl-1-methyl-1H-2-
pyrrolyl)-N-hydroxy-2-propenamides as a New Class of Synthetic
Histone Deacetylase Inhibitors. 2. Effect of Pyrrole C2 and/or
C4 Substitutions on Biological Activity. J. Med. Chem. 2004, 47,
1098-1109.
(58) Ragno, R.; Mai, A.; Massa, S.; Cerbara, I.; Valente, S.; Bottoni,
P.; Scatena, R.; Jesacher, F.; Loidl, P.; Brosch, G. 3-(4-Aroyl-1-
methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides as
a New
Class of Synthetic Histone Deacetylase Inhibitors. 3. Discovery
of Novel Lead Compounds through Structure-Based Drug Design
and Docking Studies. J. Med. Chem. 2004, 47, 1351-1359.
(59) Mai, A.; Massa, S.; Rotili, D.; Cerbara, I.; Valente, S.; Pezzi, R.;
Simeoni, S.; Ragno, R. Histone Deacetylation in Epigenetics: an
Attractive Target for Anticancer Therapy. Med. Res. Rev. 2004,
DOI 10.1002/med.20024.
(60) Mai, A.; Cerbara, I.; Valente, S.; Massa, S.; Walker, L. A.;
Tekwani, B. L. Antimalarial and Antileishmanial Activities of
Aroyl-Pyrrolyl-Hydroxyamides, a New Class of Histone Deacety-
lase Inhibitors. Antimicrob. Agents Chemother. 2004, 48, 1435-
1436.
(61) Lusser, A.; Brosch, G.; Loidl, A.; Haas, H.; Loidl, P. Identification
of maize histone deacetylase HD2 as an acidic nucleolar phos-
phoprotein. Science 1997, 277, 88-91.
(62) Wittich, S.; Scherf, H.; Xie, C.; Brosch, G.; Loidl, P.; Gerha¨user,
C.; Jung, M. Structure-Activity Relationships on Phenylalanine-
Containing Inhibitors of Histone Deacetylase: In Vitro Enzyme
Inhibition, Induction of Differentiation, and Inhibition of Pro-
liferation in Friend Leukemic Cells. J. Med. Chem. 2002, 45,
3296-3309.
(63) Mai, A.; Massa, S.; Pezzi, R.; Rotili, D.; Loidl, P.; Brosch, G.
Discovery of (Aryloxopropenyl)pyrrolyl Hydroxamides as Selec-
tive Inhibitors of Class IIa Histone Deacetylase Homologue HD1-
A. J. Med. Chem. 2003, 46, 4826-4829.
(64) Mai, A.; Massa, S.; Pezzi, R.; Valente, S.; Loidl, P.; Brosch, G.
Synthesis and Biological Evaluation of 2-, 3-, and 4-Acylamino-
cinnamyl-N-hydroxyamides as Novel Synthetic HDAC Inhibi-
tors. Med. Chem. 2005, 25, 261-309.
(65) Ko¨lle, D.; Brosch, G.; Lechner, T.; Pipal, A.; Helliger, W.; Taplick,
J.; Loidl, P. Different types of maize histone deacetylases are
distinguished by a highly complex substrate and site specificity.
Biochemistry 1999, 38, 6769-6773.
(66) Lechner, T.; Lusser, A.; Pipal, A.; Brosch, G.; Loidl, A.; Goralik-
Schramel, M.; Sendra, R.; Wegener, S.; Walton, J. D.; Loidl, P.
RPD3-Type histone deacetylases in maize embryos. Biochemistry
2000, 39, 1683-1692.
(67) Brosch, G.; Goralik-Schramel, M.; Loidl, P. Purification of histone
deacetylase HD1-A of germinating maize embryos. FEBS Lett.
1996, 393, 287-291.
(68) Brosch, G.; Georgieva, E.; Lopez-Rodas, G.; Lindner, H.; Loidl,
P. Specificity of zea mays histone deacetylase is regulated by
phosphorylation. J. Biol. Chem. 1992, 267, 20561-20564.
(69) Curtin, M.; Glaser, K. Histone Deacetylase Inhibitors: The
Abbott Experience. Curr. Med. Chem. 2003, 10, 2373-2392.
(70) Li, J.; Staver, M. J.; Curtin, M. L.; Holms, J. H.; Frey, R. R.;
Edalji, R.; Smith, R.; Michaelides, M. R.; Davidsen, S. K.; Glaser,
K. B. Expression and functional characterization of recombinant
human HDAC1 and HDAC3. Life Sci. 2004, 74, 2693-2705.
(71) Sinisterra, J. V.; Mouloungui, Z.; Delmas, M.; Gaset, A. Barium
hydroxide as catalyst in organic reactions. V. Application in the
Horner reaction under solid-liquid phase-transfer conditions.
Synthesis 1985, 1097-1100.
(72) Mori, K.; Koseki, K. Synthesis of trichostatin A, a potent
differentiation inducer of Friend leukemic cells, and its antipode.
Tetrahedron 1988, 44, 6013-6020.
(73) HD2-activity was extensively purified by anion exchange chro-
matography (Q-Sepharose), affinity chromatography (Heparin-
Sepharose, Histone-Agarose), and size exclusion chromatography
(Superdex S200) as described elsewhere.74-76
(74) Lechner, T.; Lusser, A.; Brosch, G.; Eberharter, A.; Goralik-
Schramel, M.; Loidl, P. A comparative study of histone deacety-
lases of plant, fungal and vertebrate cells. Biochim. Biophys.
Acta 1996, 1296, 181-188.
(75) Brosch, G.; Lusser, A.; Goralik-Schramel, M.; Loidl, P. Purifica-
tion and characterization of a high molecular weight histone
deacetylase complex (HD2) of maize embryos. Biochemistry 1996,
35, 15907-15914.
(76) Ko¨lle, D.; Brosch, G.; Lechner, T.; Lusser, A.; Loidl, P. Biochemi-
cal methods for analysis of histone deacetylases. Methods 1998,
15, 323-331.
(77) Kruh, J. Effects of Sodium Butyrate, a New Pharmacological
Agent, on Cells in Culture. Mol. Cell. Biochem. 1982, 42, 65-
82.
(78) Phiel, C. J.; Zhang, F.; Huang, E. Y.; Guenther, M. G.; Lazar,
M. A.; Klein, P. S. Histone deacetylase is a direct target of
valproic acid, a potent anticonvulsant, mood stabilizer, and
teratogen. J. Biol. Chem. 2001, 76, 36734-36741.

Class II (IIa)-Selective Histone Deacetylase Inhibitors
Journal of Medicinal Chemistry, 2005, Vol. 48, No. 9 3353
(79) Yoshida, M.; Kijima, M.; Akita, M.; Beppu, T. Potent and Specific
Inhibition of Mammalian Histone Deacetylase both In Vivo and
In Vitro by Trichostatin A. J. Biol. Chem. 1990, 265, 17174-
17179.
(80) Richon, V. M.; Emiliani, S.; Verdin, E.; Webb, Y.; Breslow, R.;
Rifkind, R. A.; Marks, P. A. A class of hybrid polar inducers of
transformed cell differentiation inhibits histone deacetylases.
Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 3003-3007.
(84) Glaser, K. B.; Li, J.; Staver, M. J.; Wei, R. Q.; Albert, D. H.;
Davidsen, S. K. Role of class I and class II histone deacetylases
in carcinoma cells using siRNA. Biochem. Biophys. Res. Com-
mun. 2003, 310, 529-536.
(85) Mai, A.; Esposito, M.; Sbardella, G.; Massa, S. A new facile and
expeditious synthesis of N-hydroxy-N′-phenyloctanediamide, a
potent inducer of terminal cytodifferentiation. Org. Prep. Proced.
Int. 2001, 33, 391-394.
(81) Kijima, M.; Yoshida, M.; Suguta, K.; Horinouchi, S.; Beppu, T.
Trapoxin, an Antitumor Cyclic Tetrapeptide, Is an Irreversible
Inhibitor of Mammalian Histone Deacetylase. J. Biol. Chem.
1993, 268, 22429-22435.
(82) Brosch, G.; Ransom, R.; Lechner, T.; Walton, J.; Loidl, P.
Inhibition of maize histone deacetylases by HC toxin, the host-
selective toxin of Cochliobolus carbonum. Plant Cell 1995, 33,
1941-1950.
(83) Sambucetti, L. C.; Fischer, D. D.; Zabludoff, S.; Kwon, P. O.;
Chamberlin, H.; Trogani, N.; Xu, H.; Cohen, D. Histone deacety-
lase inhibition selectively alters the activity and expression of
cell cycle proteins leading to specific chromatin acetylation and
antiproliferative effects. J. Biol. Chem. 1999, 274, 34940-34947.
(86) Nebbioso, A.; Clarke, N.; Voltz, E.; Germain, E.; Ambrosino, C.;
Bontempo, P.; Alvarez, R.; Schiavone, E. M.; Ferrara, F.;
Bresciani, F.; Weisz, A.; de Lera, A. R.; Gronemeyer, H.; Altucci,
L. Tumor-selective action of HDAC inhibitors involves TRAIL
induction in acute myeloid leukemia cells. Nat. Med. 2005, 11,
77-84.
(87) Altucci, L.; Rossin, A.; Raffelsberger, W.; Reitmair, A.; Chomi-
enne, C.; Gronemeyer, H. Retinoic acid-induced apoptosis in
leukemia cells is mediated by paracrine action of tumor-selective
death ligand TRAIL. Nat. Med. 2001, 7, 680-686.
JM049002A