3-(4-Aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides, a new class of synthetic histone deacetylase inhibitors

Article abstract of DOI:10.1021/jm015515v

Novel 3-(4-aroyl-2-pyrrolyl)-N-hydroxy-2-propenamides are disclosed as a new class of histone deacetylase (HDAC) inhibitors. Three-dimensional structure-based drug design and conformational analyses into the histone deacetylase-like protein (HDLP) catalyt

Full text of DOI:10.1021/jm015515v

J . Med. Chem. 2001, 44, 2069-2072
2069
been shown to have potent antitumor effect in vivo in
tumor-bearing animals.22,23
3-(4-Ar oyl-1H-p yr r ol-2-yl)-N-h yd r oxy-2-
p r op en a m id es, a New Cla ss of Syn th etic
Histon e Dea cetyla se In h ibitor s
Crystal structures of a bacterial HDAC homologue
(histone deacetylase-like protein, HDLP) bound to 2 and
3 have been reported.24 On the basis of the X-ray
coordinates of these complexes, the design of new
compounds able to bind the deacetylase core could be
made by computational procedures.
Silvio Massa,*,† Antonello Mai,*,‡
Gianluca Sbardella,‡ Monica Esposito,‡ Rino Ragno,§
Peter Loidl,| and Gerald Brosch*,|
Previously we described the synthesis and biological
evaluation of some 3-(4-aroyl-1-methyl-1H-pyrrol-2-yl)-
N-hydroxy-2-propenamides 7a -e as antifungal, anti-
bacterial, and antiviral agents.25,26 Compounds 7a -e
are characterized by an aroylpyrrole moiety connected
to a hydroxamic acid group by an unsaturated chain.
These chemical features, resembling some structural
elements exhibited by 2 and/or 3, prompted us to
investigate new compounds of general formula 7 as
HDAC inhibitors. The present communication reports
preliminary data on 3D structure-based drug design,
synthesis, biological evaluation, and structure-activity
relationships (SARs) of compounds 7a -j.
Dipartimento Farmaco Chimico Tecnologico,
Universita` degli Studi di Siena, via Aldo Moro,
53100 Siena, Italy, Dipartimento di Studi Farmaceutici,
Universita` degli Studi di Roma “La Sapienza”,
P.le A. Moro 5, 00185 Roma, Italy, Dipartimento di Studi di
Chimica e Tecnologia delle Sostanze Biologicamente Attive,
Universita` degli Studi di Roma “La Sapienza”,
P.le A. Moro 5, 00185 Roma, Italy, and Department of
Microbiology, University of Innsbruck, Medical School,
Fritz-Pregl-Strasse 3, 6020 Innsbruck, Austria
Received March 9, 2001
Abstr a ct: Novel 3-(4-aroyl-2-pyrrolyl)-N-hydroxy-2-propen-
amides are disclosed as a new class of histone deacetylase
(HDAC) inhibitors. Three-dimensional structure-based drug
design and conformational analyses into the histone deacet-
ylase-like protein (HDLP) catalytic core suggested the syn-
thesis and biological evaluation of compounds 7a -h . Experi-
mental pKi values are in good agreement with VALIDATE
predicted pKi values of new derivatives. All compounds 7a -h
show HDAC inhibitory activity in the micromolar range, with
7e as the most potent derivative (IC50 ) 1.9 µM). The influence
of the 4′-substituent in the aroyl moiety is not significant for
the inhibitory activity, as all compounds 7a -g show IC50
values between 1.9 and 3.9 µM. Otherwise, the unsaturated
chain linking the pyrrole ring to the hydroxamic acid group is
clearly important for the anti-HDAC activity, the saturated
analogue 7h being 10-fold less active than the unsaturated
counterpart 7a .
In tr od u ction . Acetylation of nuclear histones plays
a crucial role in gene expression, since histone hyper-
acetylation or hypoacetylation has been found to be
clearly associated with transcriptionally activated or
inactive genes.1-4 Enzymatic complexes such as histone
acetyltransferases (HATs) and histone deacetylases
(HDACs) have been identified as transcriptional coac-
tivators and transcriptional corepressors, respectively.5-8
Recently, a link between oncogene-mediated suppres-
sion of transcription and recruitment of HDAC into a
nuclear complex has been established.9-12 On the other
hand, compounds acting as HDAC inhibitors such as
sodium butyrate 1,13 trichostatin A (TSA) 2,14 suberoyl-
anilide hydroxamic acid (SAHA) 3,15 the cyclic tetrapep-
tides trapoxin 4,16 HC-toxin 5,17 and apicidin 618 (Chart
1) and synthetic compounds19-21 have been reported to
induce differentiation of several cancer cell lines and
suppress cell proliferation. Moreover, some of them have
Th r ee-Dim en sion a l Str u ctu r e-Ba sed Dr u g De-
sign a n d Molecu la r Mod elin g Stu d ies. The crystal
structure of 2 extracted from the HDLP/2 complex filed
in the Brookhaven Protein Data Bank27 (entry code
1c3r) was used as a template to build the 3D structures
of 7a -e. Following replacement of 2 with the new
modeled compounds in the HDLP catalytic core, five
new complexes were furnished which were then refined
by geometry optimization (MACROMODEL 6.5, all
atoms Amber force field)28-30 and conformational searches
(MCMM routine). Figure 1 shows the global minimum
obtained from the calculations on the HDLP/7a complex.
The binding conformation of the ligand shows a “SAHA-
like” disposition of the ketone group (Figure 2), different
from that shown by its starting conformation modeled
directly from 2 (“TSA-like” disposition).
A 3D QSAR model using the VALIDATE paradigm31-34
was then applied to predict the inhibitory activities
(expressed as pKi values) of compounds 7a -e. A previ-
ous VALIDATE model without the HDLP/2 complex was
used, and the pKi values of 2 and 3 were also recalcu-
lated to assess the general applicability of such QSAR
methodology to the HDLP coordinates (Table 1).
* To whom correspondence should be sent. E-mail addresses: S.M.,
massa@unisi.it; A.M., antonello.mai@uniroma1.it; G.B., gerald.brosch@
uibk.ac.at.
† Universita` degli Studi di Siena.
‡ Dipartimento di Studi Farmaceutici, Universita` degli Studi di
Roma “La Sapienza”.
§ Dipartimento di Studi di Chimica e Tecnologia delle Sostanze
Biologicamente Attive, Universita` degli Studi di Roma “La Sapienza”.
| University of Innsbruck.
10.1021/jm015515v CCC: $20.00 © 2001 American Chemical Society
Published on Web 05/26/2001

2070 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
Ch a r t 1. Structures of Known Histone Deacetylase Inhibitors
Letters
Sch em e 1a
a
a: ClCOOEt, Et3N, THF, 0 °C. b: NH2OH, NH2NH2, or
NH2CH2CH2OH, MeOH, rt. c: H2, Pd/C, rt.
various substituents in the benzene ring (7f,g) or a
saturated N-hydroxypropanamide in the place of the
N-hydroxy-2-propenamide chain (7h ). Predicted pKi
data of 7f-h complexed with HDLP, again in the
submicromolar range, suggested that we prepare the
new derivatives and test them as HDAC inhibitors.
Moreover, the hydrazido and 2-hydroxyethylamido ana-
logues of 7a (7i and 7j, respectively) have been pre-
pared.
F igu r e 1. Model of 7a docked into the HDLP catalytic core.
Ta ble 1. VALIDATEa Predicted and Experimental Anti-HDAC
Activities of 2, 3, and 7a -h
pKi
compd
7a
7b
7c
7d
7e
7f
7g
R
X
Y
predicted exprmtb
Ch em istr y. The synthesis of the new derivatives
7f-j was accomplished by a one-step reaction, under
neutral conditions, of 3-(4-aroyl-1-methyl-1H-pyrrol-2-
yl)-2-propenoic acids 8 with hydroxylamine, hydrazine,
and 2-hydroxyethylamine to give the hydroxamides 7a -
g, the hydrazide 7i, and the 2-hydroxyethylamide 7j,
respectively, via ethoxycarbonyl anhydrides 9 (Scheme
1). The saturated hydroxamide 7h has been prepared
by catalytic reduction of 7a with hydrogen and Pd/C
(Scheme 1).
H
Cl
F
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
OH
OH
OH
OH
OH
OH
OH
6.52
7.06
6.75
6.02
7.02
6.92
6.89
6.62
8.66
6.27
5.64
5.84
5.64
5.63
5.95
5.76
5.84
8.36
8.36
6.88
NO2
CH3
OCH3
N(CH3)2 CHdCH
7h
H
CH2-CH2 OH
2 (TSA)
3 (SAHA)
a
A modified procedure with an extended training set of 86
b
complexes was used. Experimental pKi values of 7a -h were
obtained by the corresponding IC50 values (Table 2) in comparison
with the pKi and IC50 values of TSA (2) (see ref 41).
The pyrrolylpropenoic acids 8 were easily prepared
by a Wittig-Horner reaction between 4-aroyl-1-methyl-
1H-pyrrole-2-carboxaldehydes (10) and triethyl phospho-
noacetate in the presence of potassium carbonate,
followed by alkaline hydrolysis of the resulting ethyl
pyrroleacrylates (11). The aldehydes 10 were obtained
by acylating the Vilsmeier-Haack intermediate, formed
from 1-methylpyrrole, DMF, and oxalyl chloride, under
Since the predicted pKi values of 7a -e complexed
with HDLP were in the low (or sub-) micromolar range,
we have designed and modeled into the HDLP catalytic
core new aroylpyrrole-N-hydroxypropenamides 7f-h
exhibiting, when compared to the lead compound 7a ,

Letters
J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13 2071
F igu r e 2. Proposed binding conformation of 7a (green) overlapped with TSA (2, yellow) and SAHA (3, purple).
Sch em e 2a
Ta ble 2. Anti-HDAC Activity of Compounds 7a -j
%
IC50 ( SD
(µM)
compd
R
X
Y
inhbtnb
7a
7b
7c
7d
7e
7f
7g
7h
7i
H
Cl
F
NO2
CH3
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
CHdCH
OH
OH
OH
OH
OH
OH
OH
86
87
84
78
86.4
75.2
88
45
0.2
0.9
3.8 ( 0.1
2.4 ( 0.07
3.8 ( 0.1
3.9 ( 0.2
1.9 ( 0.06
2.9 ( 0.06
2.4 ( 0.07
36.8 ( 1.1
OCH3
N(CH3)2 CHdCH
H
H
H
CH2-CH2 OH
CHdCH
CHdCH
NH2
CH2CH2-
OH
a
a: (COCl)2, DMF, dichloroethane, 0 °C. b: (1) R-Ph-COCl,
7j
AlCl3, rt; (2) NaOH 50%, rt. c: (EtO)2POCH2COOEt, K2CO3,
EtOH, 80 °C. d: KOH, EtOH, H2O, 70 °C.
sodium
butyrate (1)
35c
TSA (2)
0.0072 ( 0.0003
0.05 ( 0.0015
0.01 ( 0.0003
0.11 ( 0.0044
normal Friedel-Crafts conditions with the proper aroyl
chloride (Scheme 2).25
SAHA (3)
trapoxin (4)
HC-toxin (5)
Resu lts a n d Discu ssion . The pyrrole derivatives
7a -j have been evaluated for their ability to inhibit
HDAC as published previously35 in comparison with
sodium butyrate (1), TSA (2), SAHA (3), trapoxin (4),
and HC-toxin (5) tested as reference drugs.
a
Data represent mean values of at least three separate experi-
ments. Percent of HDAC activity inhibition at 30 µM. c Percent
of HDAC activity inhibition at 5 mM.
b
TSA (2), SAHA (3), trapoxin (4), and HC-toxin (5), 7e
exibits a 264-, 38-, 190-, and 17-fold lower potency,
respectively. The influence of the 4′-substituent in the
benzoyl portion is not significant for the biochemical
effect, as all compounds 7a -g show IC50 values between
1.9 and 3.9 µM. Otherwise, the unsaturated chain
linking the pyrrole ring to the hydroxamic acid residue
is clearly important for the anti-HDAC activity, the
saturated analogue 7h being 10-fold less active than the
unsaturated counterpart 7a . Replacement of the hy-
droxamide function with a hydrazide or 2-hydroxyethy-
lamide moiety (compounds 7i and 7j, respectively)
produces a loss of inhibitory activity.
The maize histone deacetylase HD2 was used as the
enzyme source.36 HD2, which was characterized in
detail,37,39 was shown to have an in vitro enzyme activity
comparable to those of HDACs from other sources, such
as fungi and vertebrates, using our HDAC assay.35,40
The results, expressed as percent of inhibition at fixed
dose and IC50 (50% inhibitory concentration) values, are
summarized in Table 2.
The pKi values of compounds 7a -h , calculated from
the corresponding IC50 values in comparison with the
pKi/IC50 ratio of 2 (Ki ) 0.0043 µM, IC50 ) 0.0072 µM),41
are in good agreement with the VALIDATE predicted
pKi values.42 Inhibitory activities of 7a -g are only 2-
to 12-fold weaker than their predicted values (Table 1).
All compounds 7a -h are endowed with HDAC inhibi-
tory activity in the micromolar range, confirming that
the hydroxamic acid group is crucial for an anti-HDAC
effect.43 Compound 7e is the most active derivative
(IC50 ) 1.9 µM), showing a 500-fold higher inhibitory
potency than sodium butyrate (1). In comparison with
Ack n ow led gm en t. The authors are grateful to Prof.
Marino Artico for the helpful critical discussion. This
work was supported in part by the Austrian Academy
of Sciences (to G.B.).
1
Su p p or tin g In for m a tion Ava ila ble: Melting points, H
NMR spectra, and IR spectra for compounds 7f-j are reported.

2072 J ournal of Medicinal Chemistry, 2001, Vol. 44, No. 13
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