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

Article abstract of DOI:10.1021/jm021070e

Recently we reported a novel series of hydroxamates, called 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamides (APHAs), acting as HDAC inhibitors (Massa, S.; et al. J. Med. Chem. 2001, 44, 2069-2072). Among them, 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide 1 was chosen as lead compound, and its binding mode into the modeled HDAC1 catalytic core together with its histone hyperacetylation, antiproliferative, and cytodifferentiating properties in cell-based assays were investigated (Mai, A.; et al. J. Med. Chem. 2002, 45, 1778-1784). Here we report the results of some chemical manipulations performed on (i) the aroyl portion at the C₄-pyrrole position, (ii) the N₁-pyrrole substituent, and (iii) the hydroxamate moiety of 1 to determine structure-activity relationships and to improve enzyme inhibitory activity of APHAs. In the 1 structure, pyrrole N₁-substitution with groups larger than methyl gave a reduction in HDAC inhibiting activity, and replacement of hydroxamate function with various non-hydroxamate, metal ion-complexing groups yielded poorly active or totally inactive compounds. On the contrary, proper substitution at the C₄-position favorably affected enzyme inhibiting potency, leading to 8 (IC₅₀ = 0.1 μM) and 9 (IC₅₀ = 1.0 μM) which were 38- and 3.8-fold more potent than 1 in in vitro anti-HD2 assay. Against mouse HDAC1, 8 showed an IC₅₀ = 0.5 μM (IC₅₀ of 1 = 4.9 μM), and also in cell-based assay, 8 was endowed with higher histone hyperacetylating activity than 1, although it was less potent than TSA and SAHA. Such enhancement of inhibitory activity can be explained by the higher flexibility of the pyrrole C₄-substituent of 8 which accounts for a considerably better fitting into the HDAC1 pocket and a more favorable enthalpy ligand receptor energy compared to 1. The enhanced fit allows a closer positioning of 8 hydroxamate moiety to the zinc ion. These findings were supported by extensive docking studies (SAD, DOCK, and Autodock) performed on both APHAs and reference drugs (TSA and SAHA).

Full text of DOI:10.1021/jm021070e

512
J . Med. Chem. 2003, 46, 512-524
3-(4-Ar oyl-1-m eth yl-1H-2-p yr r olyl)-N-h yd r oxy-2-a lk yla m id es a s a New Cla ss of
Syn th etic Histon e Dea cetyla se In h ibitor s. 1. Design , Syn th esis, Biologica l
Eva lu a tion , a n d Bin d in g Mod e Stu d ies P er for m ed th r ou gh Th r ee Differ en t
Dock in g P r oced u r es
Antonello Mai,*,§ Silvio Massa, Rino Ragno,*,# Ilaria Cerbara,§ Florian J esacher,‡ Peter Loidl,‡ and
Gerald Brosch*,‡
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
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 October 14, 2002
Recently we reported a novel series of hydroxamates, called 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-
2-propenamides (APHAs), acting as HDAC inhibitors (Massa, S.; et al. J . Med. Chem. 2001,
44, 2069-2072). Among them, 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-propenamide
1 was chosen as lead compound, and its binding mode into the modeled HDAC1 catalytic core
together with its histone hyperacetylation, antiproliferative, and cytodifferentiating properties
in cell-based assays were investigated (Mai, A.; et al. J . Med. Chem. 2002, 45, 1778-1784).
Here we report the results of some chemical manipulations performed on (i) the aroyl portion
at the C4-pyrrole position, (ii) the N1-pyrrole substituent, and (iii) the hydroxamate moiety of
1 to determine structure-activity relationships and to improve enzyme inhibitory activity of
APHAs. In the 1 structure, pyrrole N1-substitution with groups larger than methyl gave a
reduction in HDAC inhibiting activity, and replacement of hydroxamate function with various
non-hydroxamate, metal ion-complexing groups yielded poorly active or totally inactive
compounds. On the contrary, proper substitution at the C4-position favorably affected enzyme
inhibiting potency, leading to 8 (IC50 ) 0.1 µM) and 9 (IC50 ) 1.0 µM) which were 38- and
3.8-fold more potent than 1 in in vitro anti-HD2 assay. Against mouse HDAC1, 8 showed an
IC50 ) 0.5 µM (IC50 of 1 ) 4.9 µM), and also in cell-based assay, 8 was endowed with higher
histone hyperacetylating activity than 1, although it was less potent than TSA and SAHA.
Such enhancement of inhibitory activity can be explained by the higher flexibility of the pyrrole
C4-substituent of 8 which accounts for a considerably better fitting into the HDAC1 pocket
and a more favorable enthalpy ligand receptor energy compared to 1. The enhanced fit allows
a closer positioning of 8 hydroxamate moiety to the zinc ion. These findings were supported by
extensive docking studies (SAD, DOCK, and Autodock) performed on both APHAs and reference
drugs (TSA and SAHA).
In tr od u ction
One of the key steps in the regulation of expression of
target genes is the posttranslational modification of the
N-terminal tails of core histones by acetylation.4-6
Acetylation of Lys residues, predominantly in histones
H3 and H4, is catalyzed by enzymes with histone
acetyltranferase (HAT) activity. Such acetylation is
associated with relaxation of DNA wrapped around the
core histones and produces transcriptionally active
chromatin regions. On the other hand, histone deacety-
lase (HDAC) enzymes remove acetyl groups from ꢀ-N-
acetyl-lysine histone tails and restore a positive charge
to the Lys residues. Such chemical modifications result
in a tightening of nucleosome structure and gene
silencing.7-10
Chromatin is a nuclear macromolecular complex
containing DNA, histones, and nonhistone proteins.1
The basic functional unit of chromatin is the nucleo-
some, consisting of DNA wrapped around a histone
octamer of pairs of H2A, H2B, H3, and H4 proteins.2,3
Dynamic changes in nucleosomal packaging of DNA
define distinct levels of chromatin organization. Local
remodeling of higher-order chromatin structure plays
a key role in the regulation of gene expression affecting
proper cell function, differentiation, and proliferation.
* To whom correspondence should be addressed. A.M., Tel.: +396-
4991-3392; Fax: +396491491; e-mail: antonello.mai@uniroma1.it.
(Molecular Modeling) R.R., Tel.: +396-4991-3152. Fax: +396491491.
e-mail: rino.ragno@uniroma1.it. (Biology) G.B., Tel.: 0512-507-3608;
Fax: 0512-507-2866; e-mail: gerald.brosch@uibk.ac.at.
§ Dipartimento di Studi Farmaceutici, Universita` degli Studi di
Roma “La Sapienza”.
Aberrant acetylation of histone tails emerging from
HAT mutations or abnormal recruitment of HDACs has
been clearly linked to carcinogenesis. Rubinstein-Taybi
syndrome,11-14 acute promyelocytic leukemia (APL),15-18
acute myelogenous leukemia (AML) involving AML1-
ETO fusion protein,19-22 non-Hodgkin’s lymphoma with
Universita` degli Studi di Siena.
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze
#
Biologicamente Attive, Universita` degli Studi di Roma “La Sapienza”.
‡ University of Innsbruck.
10.1021/jm021070e CCC: $25.00 © 2003 American Chemical Society
Published on Web 01/21/2003

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 513
overexpression of the BCL-6 oncogene repressor,23 and
some colorectal and gastric carcinomas24 are examples
of cancer diseases associated with an upset of biological
HAT/HDAC balance. Although the molecular basis of
many neoplasias is still largely unexplored, it has been
widely established that inappropriate HDAC-mediated
transcriptional repression represents a common molec-
ular mechanism used by oncoproteins to produce alter-
ations in chromatin structure and blockage of normal
cell differentiation. From these data, compounds able
to inhibit HDAC activity are expected to reverse repres-
sion and to induce reexpression of differentiation-
inducing genes.25-29
than that observed for the corresponding HDLP/1
complex.53 In in vivo assays, 1 induced histone hyper-
acetylation on mouse A20 cells and was endowed with
antiproliferative and cellular differentiation activities
in murine erytroleukemic cells.53
Prompted by these results, in the chemical skeleton
of compound 1 we identified several structural features
to subject to chemical modifications for determining
structure-activity relationships (SARs) and for improv-
ing HDAC inhibitory activity of APHA analogues.
Particularly, (i) the aroyl portion at the C4-pyrrole
position, (ii) the N1-pyrrole substituent, and (iii) the
hydroxamate moiety of 1, were chosen for chemical
modifications. Novel pyrrole derivatives 1-29 bearing
several aroyl groups at C4-position, hydrogen, methyl,
isopropyl, or phenyl substituents at the N1-position, and
various hydroxamate or non-hydroxamate, metal ion-
complexing moieties on the unsaturated chain linked
at the C2-position of the pyrrole ring, were synthesized
and tested to determine their HDAC inhibitory activity
in vitro. 3-(1-Methyl-4-phenylacetyl-1H-2-pyrrolyl)-N-
hydroxy-2-propenamide 8, the most potent compound
resulting from such chemical manipulations, was fur-
ther investigated about its binding mode into the
HDAC1 catalytic core and its in vivo histone hyper-
acetylating activity on mouse A20 cells.
A number of natural or synthetic compounds have
been so far reported exhibiting HDAC inhibiting activ-
ity. Among them, trichostatin A (TSA),30 cyclic tetrapep-
tide trapoxin (TPX),31 HC toxin,32 apicidin,33 depsipep-
tide FK-228,34 and sodium butyrate35 are examples of
natural substances endowed with anti-HDAC activity.
Synthetic HDAC inhibitors can be represented by
sodium phenylbutyrate,36 sodium valproate,37,38 sub-
eroylanilide hydroxamic acid (SAHA),39 straight chain
TSA- and SAHA-like analogues,40-42 1,4-cyclohexylene-
and 1,4-phenylene-N-hydroxycarboxamides,43 scrip-
taid,44 oxamflatin45 and related compounds,46 cyclic
hydroxamic acid-containing peptides (CHAPs),47,48 and
the benzamide MS-275.49,50
Many of these compounds have been shown to have
potent antitumor effect in vivo in tumor-bearing ani-
mals, and some of them are currently in phase I or
phase I/II clinical trials.29 However, in some cases their
potential for clinical drug development could be limited
by low potency and lack of selectivity (butyrates and
analogues), cytotoxicity (TSA, CHAPs, MS-275), low
solubility in the aqueous vehicle (TSA), and/or low
stability in cell culture (TSA, trapoxin).51
Recently, we reported a novel series of hydroxamate
compounds, namely, 3-(4-aroyl-1H-2-pyrrolyl)-N-hydroxy-
2-propenamides (APHAs), acting as HDAC inhibi-
tors.52,53 The X-ray crystal structure of the catalytic core
of an archaebacterial HDAC homologue (histone deacety-
lase-like protein, HDLP), reported in 1999 by Finnin et
al.,54 revealed the mode by which the hydroxamic acid-
based HDAC inhibitors TSA and SAHA bind to the
pocket of the catalytic site of the enzyme. These data
prompted us to perform three-dimensional (3D) structure-
based design and molecular modeling studies on previ-
ously reported55,56 and newly synthesized aroyl-pyrrole-
hydroxyamides, with the aim to explore their capability
to bind the deacetylase catalytic core. An extended
VALIDATE QSAR/scoring function hybrid model57 pre-
dicted the pKi values of such derivatives complexed with
HDLP in the low (or sub-) micromolar range.52 Subse-
quent enzyme-based assay against maize histone deacety-
lase HD2 performed on pyrrole compounds revealed IC50
values at low micromolar concentrations in good
agreememt with the corresponding predicted pKi val-
ues.52 The binding mode of 3-(4-benzoyl-1-methyl-1H-
2-pyrrolyl)-N-hydroxy-2-propenamide 1, chosen as APHA
lead compound, into the HDAC1 (RPD3 homologue)
catalytic core was further investigated by modeling the
HDAC1 X-ray coordinates from those of HDLP.53 The
modeled HDAC1/1 complex, subjected to the VALIDATE
model, afforded lower absolute error of prediction (AEP)
Ch em istr y
Hydroxamic acids 1-12 (Scheme 1) were synthesized
from the corresponding pyrrolepropenoic acids 53-64
and hydroxylamine via ethoxycarbonyl anhydrides. O-
Methylhydroxamates (13, 17), hydrazides (14, 18), and
2′-hydroxyanilides (16, 20) were prepared in a similar
manner from the propenoic acids 53, 60 using O-
methylhydroxylamine, hydrazine, and 2-hydroxyaniline
instead of hydroxylamine. 2-Hydroxyethylamides (15,
19) were obtained by heating ethyl pyrrolepropenoates
41, 48 with 2-hydroxyethylamine. The propenoic acids
53-63 were readily obtained by a Wittig-Horner reac-
tion between 4-aroyl-1H-pyrrole-2-carboxaldehydes (30-
40) and triethyl phosphonoacetate in the presence of
potassium carbonate, followed by alkaline hydrolysis of
the resulting ethyl pyrrolepropenoates (41-51). 3-[4-
Benzoyl-1-(1-methylethyl)-1H-2-pyrrolyl]propenoic acid
64 was prepared by alkylation of ethyl N1-unsubstituted
pyrrolepropenoate 50 and subsequent alkaline hydroly-

514 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Mai et al.
Sch em e 1a
a
(a) (COCl)2, DMF, dichloroethane, 0 °C; (b) (1) R-Ph-X-COCl, AlCl3, rt; (2) NaOH 50%, rt; (c) (C2H5O)2OPCH2COOC2H5, K2CO3,
C2H5OH, 80 °C; (d) 2-iodopropane, K2CO3, DMF, 90 °C; (e) NH2CH2CH2OH, 140 °C; (f) KOH, C2H5OH, H2O, 70 °C; (g) (1) ClCOOC2H5,
(C2H5)3N, THF, 0 °C; (2) NH2OH, or NH2OCH3, or NHNH2, or NH2-(2′-OH)-Ph, rt.
Sch em e 2a
sis of the obtained ester 52. The pyrrolealdehydes 30-
40, key intermediates for the synthesis of title com-
pounds, were prepared by acylation of the Vilmeier-
Haack intermediates formed from pyrrole, 1-methylpyr-
role, or 1-phenylpyrrole, and a mixture of N,N-dimeth-
ylformamide and oxalyl chloride, with the proper aroyl
chloride under Friedel-Crafts conditions (Scheme 1).
Further compounds (Scheme 2) were prepared by
reacting the pyrrolealdehydes 30, 37 with tetraethyl
methylenediphosphonate or diethyl cyanomethylphos-
phonate under Wittig-Horner conditions. In the first
reaction two diethyl pyrroleethenylphosphonates (65,
66) were obtained, which were in turn transformed into
the corresponding monophosphonic acids 21, 22 by
alkaline hydrolysis. The latter reaction yielded the
pyrrolepropenonitriles 27, 28; among them, 27 was
converted into the related pyrrolepropenylamidine 29
with trimethylaluminum and ammonium chloride ac-
cording to the Garigipati procedure.58 Finally, conden-
a
(a) (C2H5O)2OPCH2PO(OC2H5)2, K2CO3, C2H5OH, 80 °C; (b)
sation of 30, 37 with barbituric and thiobarbituric acids
KOH, C2H5OH, H2O, 70 °C; (c) (thio)barbituric acid, H2O, C2H5OH,
rt; (d) (C2H5O)2OPCH2CN, K2CO3, C2H5OH, 80 °C; (e) (1) (CH3)3Al,
NH4Cl, n-hexane/toluene, from 0 to 80 °C; (2) CH3OH, 0 °C.
gave the pyrroleethenyl(thio)barbiturates 23-26 (Scheme
2).
Chemical and physical data of compounds 1-29 are
against three APHA compounds and SAHA and 3.5-fold
less sensitive to TSA.53 Additionally, two short chain
fatty acids (sodium butyrate and sodium valproate), two
hydroxamic acids (TSA and SAHA), and two cyclic
tetrapeptides (trapoxin and HC-toxin) were tested as
reference drugs.
listed in Table A (see Supporting Information). Chemical
and physical data of intermediate compounds 37-40,
48-52, 60-66 are listed in Table B (See Supporting
Information).
Biologica l Eva lu a tion a n d Str u ctu r e-Activity
Rela tion sh ip Stu d ies. In Vitr o En zym e In h ibition .
The pyrrole derivatives 1-29 were evaluated for their
ability to inhibit HDAC activity59 using maize histone
deacetylase HD2 as the enzyme source.60 HD2 was
characterized in detail by us61,63 and has an in vitro
enzyme activity comparable to those of HDACs from
other sources, such as fungi and vertebrates, using our
standard HDAC assay.59,64 In enzyme inhibition assays,
HD2 was 1.3-2.4-fold more sensitive than mouse HDAC1
The results, expressed as percent of enzyme inhibition
at fixed dose and IC50 (50% inhibitory concentration)
values, are reported in Table 1. The effects of some
chemical manipulations performed on (i) the aroyl
portion at the C4-pyrrole position, (ii) the N1-pyrrole
substituent, and (iii) the hydroxamate moiety of 1 on
the biological activity of the compounds are discussed
below.

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 515
Ta ble 1. HDAC Inhibitory Activity of Compounds 1-29a
a
b
Data represent mean values of at least three separate experiments. NI, not inhibition at starting concentration of 30 µM. c ND, not
determined.
Ar oyl Su bstitu tion a t th e C4-P yr r ole P osition . To
Tested as anti-HDAC agents, these derivatives showed
IC50 values between 1.9 and 3.9 µM, the 4′-chloro-, 4′-
methyl-, and 4′-dimethylaminobenzoylpyrrolepropenoic
hydroxamates 2, 5, 7 being only just 1.6-2-fold more
potent than the unsubstituted 1. Instead, an increase
of potency was obtained with the synthesis of 8 and 9,
develop SAR studies on APHA compounds, we prepared
a series of pyrrolepropenoic hydroxamates 1-7 showing
differently 4′-substituted (4′-H, 4′-chloro, 4′-fluoro, 4′-
nitro, 4′-methyl, 4′-methoxy, and 4′-dimethylamino)
benzoyl groups at the C4-position of the pyrrole ring.

516 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Mai et al.
Ta ble 2. Maize HD2 and Mouse HDAC1 Inhibitory Activity of
8
compd
HD2 IC50 ( SD (µM)
HDAC1 IC50 ( SD (µM)
8
0.1 ( 0.004
0.51 ( 0.02
1a
3.8 ( 0.15
4.9 ( 0.15
TSAa
SAHAa
0.007 ( 0.0003
0.05 ( 0.0015
0.002 ( 0.00006
0.112 ( 0.0045
Ta ble 3. Experimental vs VALIDATE and Autodock Predicted
HDAC1 Inhibitory Activities of TSA, SAHA, 1, and 8
expt pIC50
VALIDATE predicted pKi
Autodock
compd HDAC1 HD2 SAD DOCK Autodock predicted pKi
F igu r e 1. Schematic representation of hydroxamate-based
inhibitors-HDLP catalytic core interactions
TSA
SAHA
1
8
8.70
6.95
5.31
6.29
8.15 8.61a
7.30 6.69a
5.42 5.76a
7.00 6.30
7.26
6.79
4.76
6.98
7.92
7.15
7.31
6.37
7.41
6.21
5.65
5.98
in which the benzene ring of the 1 C4-benzoyl moiety is
spaced from the carbonyl group through a methylene
(8) or an ethenyl (9) link. Compounds 8 and 9 were 38-
and 3.8-fold more effective than 1 in inhibiting HDAC
enzyme, respectively.
a
See ref 53.
instead of the hydroxamate. Moreover, the monophos-
phonic acids 21, 22, the (thio)barbiturates 23-26, the
nitriles 27, 28, and the amidine 29 were synthesized
and tested as HDAC inhibitors because such chemical
functions are able to chelate metal ions.
In flu en ce of N1-Su bstitu en t a t th e P yr r ole Rin g.
To study the effect of pyrrole N1-substituent on enzyme
inhibition we prepared and tested N1-H, N1-isopropyl,
and N1-phenyl analogues of 1 (compounds 10-12). The
N1-H derivative 10 retained the anti-HDAC activity
showed by 1 resulting only 1.3-fold less potent than the
N1-methyl counterpart, while 11 and 12 carrying sub-
stituents bulkier than a methyl showed a decrease of
inhibitory activity of 14 and 29 times, respectively.
These data confirm our observations about the binding
mode of 1 into the modeled HDAC1 catalytic core,53 with
the hypothesis of a steric hindrance exerted by pyrrole
N1-substituent of APHA derivatives into the binding
pocket. However, the lack of increasing of activity from
N1-methyl compound 1 to N1-H counterpart 10 can be
explained through the display of unfavorable electronic
and hydrophilic effects exerted by the NH group into
the catalytic pocket. In fact, the calculated logP value
for 10 (1.54) is lower than that calculated for 1 (1.85),
and this difference accounts for an higher energy that
10 needs to passage by its solvated state to the receptor
bound state during the complex formation.
Rep la cem en t of Hyd r oxa m ic Acid Moiety. Crys-
tal structures of HDLP/TSA and HDLP/SAHA com-
plexes revealed that hydroxamic acid-based inhibitors
bind the deacetylase core by inserting their aliphatic
chains into the HDLP pocket and by making multiple
contacts to its tubelike hydrophobic portion.54 Particu-
larly, their hydroxamic acid group reaches the polar
bottom of the pocket, where it coordinates the zinc ion
in a bidentate fashion (through CO and OH groups) and
also contacts active-site residues (forming two hydrogen-
bonds between its NH and OH groups and the two
charge-relay systems His131/Asp166 and His132/Asp173,
and another one between its CO and the Tyr297
hydroxyl group) (Figure 1). Moreover, hydroxamic acid
function replaces the zinc-bound water molecule of the
active structure with its OH group.54
Unfortunately, all attempts to obtain non-hydroxam-
ate compounds active as anti-HDAC agents produced
only weakly potent (18, 25-27, 29) or totally inactive
(13-17, 19-24, 28) derivatives. In fact between 1
analogues, the nitrile 27 and the amidine 29 were 7-
and 6-fold less potent than their reference compound.
With regard to 8 analogues, the hydrazide 18 was 3-fold
less effective than 8 in inhibiting the enzyme at fixed
dose, and the (thio)barbiturates 25, 26 were 1000- and
850-fold less active than 8, respectively.
Dock in g a n d Bin d in g Mod e In sp ection of 1h in to
th e Mod eled HDAC1 Ca ta lytic Cor e. Chemical ma-
nipulations performed on APHA lead compound 1
highlighted that enzyme inhibiting potency of aroylpyr-
role-hydroxyalkylamides is strongly increased by con-
verting the C4-benzoyl into C4-phenylacetyl moiety at
the pyrrole ring, being 3-(1-methyl-4-phenylacetyl-1H-
2-pyrrolyl)-N-hydroxy-2-propenamide 8 38-fold more
active than 1 in in vitro HD2 inhibitory assay (Table
1).
Binding mode analysis of 8 was first investigated
using the modeled HDAC1 X-ray structure analogously
to that reported for 153 using a semiautomatic dock
(SAD) procedure. IC50 value of 8 against mouse HDAC1
enzyme was determined (Table 2), and its pKi was
predicted using the in house refined VALIDATE
model57,53 (Table 3). In parallel experiments, docking
studies into the HDAC1 catalytic core were also per-
formed on 1, 8, TSA, and SAHA structures by the mean
of the DOCK 4.0.265 and Autodock 3.0.566 programs.
Among the minimized ligand/HDAC1 complexes ob-
tained, the most stable ones were those proposed by
Autodock (see minimized complex steric energies in
Table 4). To compare the four docked molecules (1, 8,
TSA, and SAHA), in Figure 2 are reported their binding
conformations obtained from the Autodock run. Simi-
larly to that observed for the complexes, also the
Autodock conformations showed to be more stable than
those obtained with DOCK or SAD procedures (ligand
bound steric energies, Table 4).
This mechanism of action underlines the importance
of the presence of the CO+NH+OH (or similar, metal
ion-complexing structure) for the enzyme inhibiting
activity. From these data, we prepared some hydroxamic
acid-like derivatives 13-20 bearing an O-methylhy-
droxamate (13, 17), hydrazide (14, 18), 2-hydroxyethyl-
amide (15, 19), and 2′-hydroxyanilide (16, 20) moieties

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 517
Ta ble 4. Minimized Complex and Ligand Bound Steric
Energies (AMBER force field) Relative to 1, 8, SAHA, and TSA
Obtained by DOCK, Autodock, and SAD Procedures
enthalpy of binding that offsets the slightly more
unfavorable entropy (Table 4). Furthermore, this en-
hanced fitting allows a closer positioning of 8 hydrox-
amate moiety to the zinc ion (1: average CO‚‚‚Zn ) 4.0
Å, average OH‚‚‚Zn ) 4.7 Å; 8: average CO‚‚‚Zn ) 3.1
Å, average OH‚‚‚Zn ) 3.5 Å).
docking method
minimized complex steric energy (kJ /mol)
-7174.81 -9077.85 -7369.18 -8909.93
1
8
SAHA
TSA
DOCK
Autodock
SAD
-8913.90 -9405.68 -10363.13 -9976.13
In Figure 4 the SAD conformation of 8 into the
HDAC1 catalytic core is reported. Tyr297 hydroxyl
group is placed at hydrogen-bond distance from the 8
hydroxamate moiety (Tyr297-OH‚‚‚COhydroxamate ) 3.25
Å; Tyr297-OH‚‚‚OHhydroxamate ) 2.42 Å). Positive π-stack-
ing interactions can be observed between the pyrrolyl-
ethylene chain and the Phe141 and Phe198 residues of
the site. The pyrrole N1-methyl group, contrary to that
of 1,53 makes favorable interactions with the R-carbon
atom of Gly140 and partially with the Phe141 side
chain. This switched position of the 8 N1-methyl group
with respect to that of 153 allows the molecule to get
closer to the catalytic zinc ion. Finally, in the pyrrole
C4-phenylacetyl portion the ketone points toward the
Tyr91 hydroxyl group, probably making a hydrogen
bond by the mean of a water molecule (not shown), and
the benzene ring points to a hydrophobic region of the
enzyme (i.e., Phe200).
-7828.92 -9142.08
NDa
ND
Ligand Bound Steric Energy (kJ /mol)
DOCK
Autodock
SAD
-12.25
-25.11
-11.50
-49.03
-53.02
-54.90
-0.53
-13.38
ND
-48.20
-52.74
ND
a
ND, not determined.
In Vivo Histon e Hyp er a cetyla tion In d u ced by 8.
To examine whether in vitro HDAC inhibition by 8
correlates with increased acetylation level of core his-
tones in vivo, we treated exponentially growing A20 cells
with 8 (40 µM) together with TSA (200 nM) and SAHA
(11 µM) as reference drugs. After isolation of nuclei, the
effects on histone acetylation were analyzed by AUT-
PAGE (acid-urea-triton polyacrylamide gel electrophore-
sis) followed by staining with Coomasie brilliant blue
R-250. The effect of 1 (800 µM) on histone acetylation
on mouse A20 cells is also reported for a direct com-
parison with that of 8. AUT-PAGE allows separation
of individual subspecies of core histones with the
different extent of acetylation, because of slower migra-
tion rates of the acetylated species. Figure 5 shows that
8 induced the accumulation of highly acetylated histone
H4 subspecies (mono- to tetraacetylated) compared to
control cells where no toxin wax added, with decreases
in the most rapidly migrating bands of each histone
species.
The effect of 8 on acetylation patterns of histone H4
by an antibody against acetylated H4 peptide was also
studied. This antibody recognizes di-, tri-, and tet-
raacetylated H4, but not nonacetylated H4. In Western
Blot experiments isolated histones of Figure 5A were
subjected to SDS-PAGE and immunoblotting. Treat-
ment of cells with 8 as well as with 1, TSA and SAHA
led to a highly increased acetylation level of H4 with
significant immunoreaction, compared to control his-
tones which show only a faint signal (Figure 5B). J oined
to AUT-gel results, these data clearly demonstrate that
8 inhibits HDAC not only in vitro but also in the cells.
Moreover, 8 is endowed with higher histone hyperacety-
lating activity than the APHA prototype 1, still resulting
less potent than TSA and SAHA.
F igu r e 2. Superimposition of the Autodock docked conforma-
tions of 1 (green), 8 (red), TSA (yellow), and SAHA (blue). The
gray ball represents the mean position of the zinc ion. HDAC1
pocket is not shown for the sake of clarity
Interestingly, DOCK and Autodock results were in
good agreement each other and with those obtained by
either the SAD procedure (1 and 8) or experimental
values (TSA and SAHA) (see Figure 3, and Table C in
Supporting Information). The found different binding
conformations of 1 (Figure 3A) likely represent three
different bound states of the ligand, confirming the low
tightness of the HDAC1/1 complex. On the other hand,
for structures endowed with higher activities, the dock-
ing methodologies led to almost the same results,
reflecting that more stable and unique complexes are
achieved for 8, TSA, and SAHA (Figures 3B-D) (see
Experimental Section).
Inspection of 8 binding mode into the HDAC1 cata-
lytic core, compared with that described for 1,53 revealed
that the enhancement of inhibitory activity can be
mainly attributed to the higher flexibility of the pyrrole
C4-substituent of 8. Generally, an increase in flexibility
is unfavorable, due to the increase in the entropy of
binding. Nevertheless, the introduction of a methylene
connection between the phenyl and the carbonyl group
of the 1 benzoyl moiety disrupts the extended phenyl-
CO-pyrrole π-conjugation. As a consequence of this
flexibility, 8 fits considerably better than 1 into the
HDAC1 pocket, showing an higher average steric fit57
Con clu sion
We designed and performed structural modifications
on 3-(4-benzoyl-1-methyl-1H-2-pyrrolyl)-N-hydroxy-2-
propenamide 1, the representative member of aroyl-
(1average
) 1.7; 8average
) 2.0) (Table D in
steric fit
steric fit
Supporting Information) and providing a more favorable

518 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Mai et al.
F igu r e 3. Superimposition of the docked conformations of 1 (A), 8 (B), TSA (C), and SAHA (D) obtained from the three docking
procedures (see text). The minimized experimental (TSA ans SAHA) and the semiautomatic docked (SAD) (1 and 8) conformations
are in green. Autodock conformations are in red. DOCK conformations are in blue. The gray sphere indicates the Zn ion position
into the HDAC1.
F igu r e 4. Stereoview of the semiautomatic docked conformation of 8 (in white) into the HDAC1 catalytic core. Residues 4 Å
from the ligand are displayed in orange. In transparent cyan is the ligand available volume.
pyrrole-hydroxy-alkylamides (APHAs) recently de-
scribed by us as a new class of synthetic HDAC
inhibitors. In the 1 structure, pyrrole N1-substitution
with groups larger than methyl decreased HDAC in-
hibiting activity, and replacement of hydroxamate func-
tion with various non-hydroxamate, metal ion-complex-
ing groups yielded low active or totally inactive com-
pounds. On the contrary, proper substitution at the C4-
position of the pyrrole ring favorably affected enzyme
inhibiting potency, leading to 3-(1-methyl-4-phenylacetyl-
1H-2-pyrrolyl)-N-hydroxy-2-propenamide 8 and 3-[1-
methyl-4-(2-phenylethenyl)-1H-2-pyrrolyl]-N-hydroxy-
2-propenamide 9 which were 38- and 3.8-fold more
potent than 1 in in vitro anti-HD2 assay, respectively.
In comparison with reference drugs, in our enzyme
assay 8 showed a 1280-fold higher inhibitory potency
than sodium valproate; moreover, it was as active as
HC-toxin, and was 14-, 2-, and 10-fold less potent than

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 519
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.67 All
chemicals were purchased from Aldrich Chimica, Milan (Italy)
or Lancaster Synthesis GmbH, Milan (Italy) and were of the
highest purity.
Gen er a l P r oced u r e for th e Syn th esis of 4-Ar oyl-1H-
2-p yr r oleca r boxa ld eh yd es 30-40. Exa m p le: 4-Ben zoyl-
1H-p yr r ole-2-ca r boxa ld eh yd e (39). A 20 mL 1,2-dichloro-
ethane solution of oxalyl chloride (29.7 mmol, 2.6 mL) was
added to a cooled (0-5 °C) solution of N,N-dimethylformamide
(29.7 mmol, 2.3 mL) in 1,2-dichloroethane (20 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 1H-pyrrole (29.7 mmol, 2.0 g) in 1,2-
dichloroethane (20 mL). The mixture was stirred at room
temperature for 15 min and then treated with aluminum
trichloride (65.3 mmol, 8.7 g) and benzoyl chloride (29.7 mmol,
3.5 mL). After 3 h, the reaction mixture was poured onto
crushed ice (100 g) containing 50% NaOH (20 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 × 20 mL). The combined
organic solutions were washed with water, dried, and evapo-
rated to dryness. The residual oil was purified by column
chromatography on silica gel eluting with ethyl acetate:
chloroform 1:10. The solid obtained was recrystallized from
cyclohexane/benzene to give pure 39. IR 3220 (NH), 1677
F igu r e 5. Effects of 1, 8, TSA, and SAHA on histone
acetylation in mouse A20 cells. (A) Analysis of histone hyper-
acetylation by AUT-PAGE and Coomassie blue staining.
Positions of mono- to tetraacetylated H4 subspecies are
indicated. (1) control, (2) 1, (3) 8, (4) TSA, (5) SAHA, (6)
hyperacetylated MELC (murine erythroleukemia cells) his-
tones. (B) Analysis of H4-acetylation by immunoblotting.
Position of acetylated H4 is indicated. Data reported in lines
1, 2, 4-6 are the same used in ref 53.
(CHO), 1630 (CO) cm-1 1H NMR (CDCl3) δ 7.45 (m, 4 H,
.
pyrrole â-proton and benzene H-3,4,5), 7.69 (m, 1 H, pyrrole
R-proton), 7.81 (m, 2 H, benzene H-2,6), 9.58 (s, 1 H, CHO),
10.70 (s, 1 H, NH). Anal. (C12H9NO2) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Eth yl 3-(4-
Ar oyl-1H-2-p yr r olyl)p r op en oa tes 41-51. Exa m p le: Eth yl
3-(4-Ben zoyl-1-p h en yl-1H-2-p yr r olyl)p r op en oa te (51). A
suspension of 40 (5.7 mmol, 1.6 g) in absolute ethanol (20 mL)
was added in one portion to a mixture of triethyl phospho-
noacetate (6.8 mmol, 1.4 mL) and anhydrous potassium
carbonate (17.1 mmol, 2.4 g). After being stirred at 70 °C for
2 h, the reaction mixture was cooled to room temperature,
diluted with water (50 mL), and extracted with ethyl acetate
(3 × 30 mL). The organic layer was washed with water, dried,
and evaporated to dryness, and the solid residue was recrys-
tallized to furnish pure 51. IR 1705 (COOC2H5), 1633 (CO)
cm-1. 1H NMR (CDCl3) δ 1.24 (t, 3 H, CH3), 4.13 (q, 2 H, CH2),
6.16 (d, 1 H, CHdCHCO), 7.21 (s, 1 H, pyrrole â-proton), 7.31-
7.44 (m, 10 H, CH)CHCO, pyrrole R-proton, N-benzene H,
and CO-benzene H-3,4,5), 7.81 (m, 2 H, CO-benzene H-2,6).
Anal. (C22H19NO3) C, H, N.
TSA, SAHA, and trapoxin, respectively. Against mouse
HDAC1, 8 showed an IC50 ) 0.5 µM, 10 times more
efficient than 1 and 4.5- and 255- fold less potent than
SAHA and TSA, respectively. Also in cell-based assay
8 was endowed with higher histone hyperacetylating
activity than 1 on mouse A20 cells. Such enhancement
of inhibitory activity of 8 with respect to that of 1 can
be explained by the binding mode analysis of 8 into the
HDAC1 catalytic core. The higher flexibility of the
pyrrole C4-substituent of 8 accounts for a considerably
better fitting into the HDAC1 pocket compared to 1 and
allows a closer positioning of 8 hydroxamate moiety to
the zinc ion. These findings were supported by extensive
docking studies (SAD, DOCK, and Autodock) performed
on both APHAs and reference drugs (TSA and SAHA).
We are currently investigating the effect of chemical
modifications at the unsaturated chain connecting pyr-
role ring with the hydroxamate function in APHA
compounds and the capability of 8 to induce antiprolif-
erative effects and terminal cell differentiation.
E t h yl 3-[4-Ben zoyl-1-(1-m et h ylet h yl)-1H-2-p yr r olyl]-
p r op en oa te (52). A mixture of 50 (19.3 mmol, 5.3 g) and
2-iodopropane (38.6 mmol, 3.9 mL) in N,N-dimethylformamide
(10 mL) containing anhydrous potassium carbonate (38.6
mmol, 5.3 g) was heated at 90 °C while stirring overnight.
After being cooled, the mixture was poured onto water (300
mL) and extracted with ethyl acetate (3 × 50 mL). The organic
layer was washed, dried, and evaporated to dryness. The
residue was purified by column chromatography on silica gel
eluting with ethyl acetate:chloroform 1:10. IR 1705 (COOC2H5),
Exp er im en ta l Section
1627 (CO) cm-1 1H NMR (CDCl3) δ 1.34 (t, 3 H, CH2CH3),
.
1.53 (d, 6 H, CH(CH3)2), 4.29 (q, 2 H, CH2CH3), 4.56 (m, 1 H,
CH(CH3)2), 6.31 (d, 1 H, CHdCHCO), 7.09 (s, 1 H, pyrrole
â-proton), 7.55 (m, 4 H, pyrrole R-proton and benzene H-3-
5), 7.64 (d, 1 H, CHdCHCO), 7.84 (m, 2 H, benzene H-2,6).
Anal. (C19H21NO3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1H-
2-p yr r olyl)p r op en oic Acid s 53-64. Exa m p le: 3-[1-Meth -
yl-4-(2-ph en yleth en yl)-1H-2-pyr r olyl]pr open oic Acid (61).
A mixture of 49 (5.7 mmol, 1.8 g), 2 N KOH (22.9 mmol, 11.4
mL), and ethanol (15 mL) was heated at 70 °C for 3 h. After
being cooled, the solution was poured into water (50 mL) and
extracted with ethyl acetate (2 × 20 mL). To the aqueous layer
was added 2 N HCl until the pH was 5, and the precipitate
was filtered and recrystallized from benzene giving the pure
Ch em istr y. 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
1
instrument. H NMR spectra were recorded at 200 MHz on a
Bruker AC 200 spectrometer; chemical shifts are reported in
δ (ppm) units relative to the internal reference tetramethyl-
silane (Me4Si). All compounds were routinely checked by TLC
1
and H 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

520 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
compound 61. IR 3450 (OH), 1705 (COOH), 1640 (CO) cm-1
Mai et al.
.
(CDCl3) δ 1.65 (s, 1 H, OH), 3.58 (m, 2 H, CH2CH2OH), 3.71
(s, 3 H, NCH3), 3.84 (m, 2 H, CH2CH2OH), 4.02 (s, 2 H, PhCH2),
6.28 (m, 2 H, CHdCHCO and NH), 6.70 (d, 2 H, benzene
H-3,5), 7.01 (m, 1 H, pyrrole â-proton), 7.30 (m, 6 H, pyrrole
R-proton and benzene H), 7.53 (d, 1 H, CHdCHCO). Anal.
(C18H20N2O3) C, H, N.
1H NMR (DMSO-d6) δ 3.73 (s, 3 H, NCH3), 6.25 (d, 1 H, CHd
CHCOOH), 7.10-7.56 (m, 8 H, pyrrole â-proton, PhCHdCH,
pyrrole R-proton, and benzene H), 7.77 (m, 1H, PhCHdCH),
7.99 (m, 1 H, CHdCHCOOH), 12.10 (s, 1 H, OH). Anal. (C17H15
-
NO3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1-
m eth yl-1H-2-pyr r olyl)-N-h ydr oxypr open am ides 1-12. Ex-
a m p le: 3-(1-Meth yl-4-p h en yla cetyl-1H-2-p yr r olyl)-N-h y-
d r oxyp r op en a m id e (8). 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 60 (4.2 mmol, 1.1 g) in dry THF (10
mL), and the mixture was stirred for 10 min. The solid was
filtered off. The filtrate was added to a freshly prepared
solution of hydroxylamine, obtained by reaction between
hydroxylamine hydrochloride (6.2 mmol, 0.4 g) and KOH (6.2
mmol, 0.35 g), in methanol (10 mL). After being stirred at room
temperature for 15 min, the mixture was evaporated under
reduced pressure, and the residue was recrystallized from
benzene to give the pure compound 8. IR 3444-3215 (NHOH),
1640 (CONH), 1616 (CO) cm-1. 1H NMR (DMSO-d6) δ 3.74 (s,
3 H, NCH3), 4.01 (s, 2 H, CH2), 6.26 (d, 1 H, CHdCHCO), 6.92
(s, 1 H, pyrrole â-proton), 7.27 (m, 6 H, CH)CHCO, pyrrole
R-proton, and benzene H-2,3,5,6), 7.85 (m, 1 H, benzene H-4),
9.01 (s, 1 H, NH), 10.66 (s, 1 H, OH). Anal. (C16H16N2O3) C, H,
N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1-
m eth yl-1H-2-pyr r olyl)-2′-h ydr oxypr open ylan ilides 16, 20.
Exa m p le: 3-(1-Meth yl-4-p h en yla cetyl-1H-2-p yr r olyl)-2′-
h yd r oxyp r op en yla n ilid e (20). Triethylamine (2.9 mmol, 0.4
mL) and ethyl chloroformate (2.68 mmol, 0.26 mL) were added
to an ice-cooled solution of 60 (2.23 mmol, 0.6 g) in dry THF
(10 mL). After being stirred for 10 min, the obtained suspen-
sion was filtered, and 2-aminophenol (3.34 mmol, 0.36 g) was
added to the filtrate. The resulting mixture was stirred at room
temperature for 15 min and then was evaporated under
reduced pressure to give crude 20 which was purified by
crystallization (benzene/acetonitrile). IR 3120 (NH, OH), 1650
1
(CONH), 1590 (CO) cm-1. H NMR (DMSO-d6) δ 3.50 (s, 3 H,
NCH3), 3.96 (m, 2 H, CH2), 6.10 (m, 1 H, CHdCHCO), 6.67
(m, 1 H, pyrrole â-proton), 6.85 (m, 4 H, anilide H-3,4,5 and
NH), 7.08 (m, 1 H, anilide H-6), 7.25 (m, 5 H, pyrrole R-proton
and benzene H), 7.74 (m, 1 H, CHdCHCO), 9.2 (s, 1 H, OH).
Anal. (C22H21N2O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of Dieth yl 2-(4-
Ar oyl-1-m eth yl-1H-2-p yr r olyl)eth en ylp h osp h on a tes 65,
66. Exa m p le: Dieth yl 2-(4-Ben zoyl-1-m eth yl-1H-2-p yr -
r olyl)eth en ylp h osp h on a te (65). A suspension of 30 (4.7
mmol, 1.0 g) in absolute ethanol (20 mL) was added in one
portion to a mixture of tetraethyl methylenediphosphonate (5.6
mmol, 1.4 mL) and anhydrous potassium carbonate (14.1
mmol, 1.9 g). After being stirred at 70 °C for 2 h, the reaction
mixture was cooled to room temperature, poured into water
(50 mL), and extracted with ethyl acetate (3 × 25 mL). The
combined organic solution was washed with water, dried, and
evaporated to dryness. The residual oil was purified by column
chromatography on silica gel eluting with ethyl acetate:
chloroform 1:1 to furnish pure 65. IR 1635 (CO), 1240 (PdO),
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1-
m eth yl-1H-2-p yr r olyl)-N-m eth oxyp r op en a m id es 13, 17.
Exa m p le: 3-(1-Meth yl-4-p h en yla cetyl-1H-2-p yr r olyl)-N-
m eth oxyp r op en a m id e (17). To a cooled (0 °C) solution of
60 (1.11 mmol, 0.3 g) in dry THF (10 mL) were added
triethylamine (1.45 mmol, 0.21 mL) and ethyl chloroformate
(1.33 mmol, 0.13 mL) in succession. After being stirred for 10
min, the mixture was filtered and the filtrate was added to a
freshly prepared solution of O-methylhydroxylamine, obtained
by reaction between O-methylhydroxylamine hydrochloride
(1.66 mmol, 0.14 g) and KOH (1.66 mmol, 0.1 g), in methanol
(5 mL). The resulting mixture was stirred at room temperature
for 15 min and then was evaporated under reduced pressure,
and the residue was recrystallized from benzene to furnish
1025 (P(OC2H5)2) cm-1 1H NMR (CDCl3) δ 1.27 (t, 6 H,
.
CH2CH3), 3.68 (s, 3 H, NCH3), 4.04 (q, 4 H, CH2CH3), 5.97 (t,
1 H, CHdCHPO), 7.01 (s, 1 H, pyrrole â-proton), 7.42 (m, 5
H, CHdCHPO, pyrrole R-proton, and benzene H-3,4,5), 7.74
(m, 2 H, benzene H-2,6). Anal. (C18H22NO4P) C, H, N, P.
pure 17. IR 3190 (NH), 16550 (CONH), 1625 (CO) cm-1 1H
.
NMR (CDCl3) δ 3.71 (s, 3 H, NCH3), 3.82 (s, 3H, OCH3), 4.02
(s, 2 H, CH2), 6.31 (m, 1 H, CHdCHCO), 7.04 (s, 1 H, pyrrole
â-proton), 7.29 (m, 6 H, pyrrole R-proton and benzene H), 7.65
(d, 1 H, CHdCHCO), 9.16 (s, 1 H, NH). Anal. (C17H18N2O3) C,
H, N.
Gen er a l P r oced u r e for th e Syn th esis of Eth yl 2-(4-
Ar oyl-1-m eth yl-1H-2-p yr r olyl)eth en ylp h osp h on a tes 21,
22. E xa m p le: E t h yl 2-(1-Met h yl-4-p h en yla cet yl-1H-2-
p yr r olyl)eth en ylp h osp h on a te (22). A mixture of 66 (2.0
mmol, 0.8 g), 2 N KOH (16.1 mmol, 8.1 mL), and ethanol (15
mL) was heated at 70 °C for 3 h. After being cooled, the
solution was poured into water (50 mL) and extracted with
ethyl acetate (2 × 20 mL). To the aqueous layer was added 2
N HCl until the pH was 5. The precipitate was filtered and
recrystallized from benzene/acetonitrile to give pure 22. IR
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1-
m eth yl-1H-2-p yr r olyl)p r op en ylh yd r a zid es 14, 18. Ex-
a m p le: 3-(1-Met h yl-4-p h en yla cet yl-1H -2-p yr r olyl)p r o-
p en ylh yd r a zid e (18). A cooled (0 °C) solution of 60 (1.1 mmol,
0.3 g) in dry THF (10 mL) was treated with triethylamine (1.4
mmol, 0.21 mL) and ethyl chloroformate (1.3 mmol, 0.13 mL)
while stirring. After 10 min, the solid was filtered off, and
hydrazine hydrate (1.7 mmol, 0.08 mL) was added to the
filtrate. The resulting mixture was stirred at room tempera-
ture for further 15 min and then was evaporated, and the
residue was recrystallized to furnish pure 18. IR 3200 (NHNH2),
3130 (OH), 1626 (CO), 1366 (PdO), 960 (POC2H5) cm-1 1H
.
NMR (CDCl3) δ 1.34 (t, 3 H, CH2CH3), 3.58 (s, 3 H, NCH3),
4.00 (s, 2 H, PhCH2), 4.10 (q, 2 H, CH2CH3), 6.06 (t, 1 H, CHd
CHPO), 6.07 (s, 1 H, OH overlapped signal), 6.98 (s, 1 H,
pyrrole â-proton), 7.29 (m, 7 H, CHdCHPO, pyrrole R-proton,
and benzene H). Anal. (C18H20PNO5) C, H, N, P.
1
1650 (CONH), 1620 (CO) cm-1. H NMR (CDCl3) δ 3.73 (s, 3
H, NCH3), 4.03 (s, 2 H, CH2), 6.24 (d, 1 H, CHdCHCO), 7.02
(s, 1 H, pyrrole â-proton), 7.30 (m, 6 H, pyrrole R-proton and
benzene H), 7.51 (d, 1 H, CHdCHCO). Anal. (C16H17N3O2) C,
H, N.
Gen er a l P r oced u r e for th e Syn th esis of 5-[2-(4-Ar oyl-
1-m et h yl-1H -2-p yr r olyl)et h en yl]-1,2,3,4,5,6-h exa h yd r o-
2,4,6-tr ioxopyr im idin es 23, 25, an d 5-[2-(4-Ar oyl-1-m eth yl-
1H-2-p yr r olyl)eth en yl]-4,6-d ioxo-1,2,3,4,5,6-h exa h yd r o-2-
t h ioxop yr im id in es 24, 26. E xa m p le: 5-[2-(4-Ben zoyl-1-
m eth yl-1H-2-pyr r olyl)eth en yl]-1,2,3,4,5,6-h exah ydr o-2,4,6-
tr ioxop yr im id in e (23). A suspension of barbituric acid (4.4
mmol, 0.56 g) in 15 mL of water was added to a solution of 30
(4.4 mmol, 1.0 g) in ethanol (25 mL), and the mixture was
stirred at room temperature for 2 h. The resulting solid was
filtered under reduced pressure, collected, and recrystallized
to furnish pure 23. IR 3193 (NH), 1784 (CONHCO),1670
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1-
m e t h y l-1H -2-p y r r o ly l)-N -(2-h y d r o x y e t h y l)p r o p e n -
a m id es 15, 19. Exa m p le: 3-(1-Meth yl-4-p h en yla cetyl-1H-
2-p yr r olyl)-N-(2-h yd r oxyeth yl)p r op en a m id e (19). A solu-
tion of 2-ethanolamine (5.28 mmol, 0.32 mL) and ethyl 3-(1-
methyl-4-phenylacetyl-1H-2-pyrrolyl)propenoate 48 (1.32 mmol,
0.3 g) was stirred for 2 h a 140 °C. After being cooled, the
mixture was poured onto water (100 mL) and extracted with
ethyl acetate (3 × 50 mL). The organic layer was washed,
dried, and evaporated to dryness to furnish a solid residue (19)
which was purified by crystallization (benzene/acetonitrile).
(CONH), 1630 (CO) cm-1
NCH3), 7.48 (m, 5 H, pyrrole H and benzene H-3,4,5), 8.44 (d,
.
1H NMR (DMF-d7) δ 4.16 (s, 3 H,
IR 3280 (NH, OH), 1645 (CONH), 1605 (CO) cm-1 1H NMR
.

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 521
2 H, benzene H-2,6), 9.04 (s, 1 H, CH), 11.35 (s, 1 H, NH),
11.45 (s, 1 H, NH). Anal. (C17H13N3O4) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Ar oyl-1-
m eth yl-1H-2-p yr r olyl)p r op en on itr iles 27, 28. Exa m p le:
3-(4-Ben zoyl-1-m eth yl-1H-2-p yr r olyl)p r op en on itr ile (27).
A suspension of 30 (17.01 mmol, 4.47 g) in absolute ethanol
(20 mL) was added in one portion to a mixture of diethyl
cyanomethylphosphonate (20.41 mmol, 3.3 mL) and anhydrous
potassium carbonate (51.03 mmol, 7.05 g). After being stirred
at 70 °C for 2 h, the reaction mixture was cooled to room
temperature, diluted with water (50 mL), and extracted with
ethyl acetate (3 × 25 mL). The organic layer was washed with
water, dried, and evaporated to dryness. The residual oil was
purified by column chromatography on silica gel eluting with
ethyl acetate:chloroform 1:5. Compound 27 was obtained as a
solid which was then recrystallized from cyclohexane/benzene.
F igu r e 6. Semiautomatic docking procedure generating a
family of 748 conformations of 8. A represents the axis of
rotation passing through the center (x) of the benzene ring and
the hydroxamic carbon atom. τ1-τ5 indicate the five rotatable
bonds used for the grid searches.
1
IR 2200 (CN), 1635 (CO) cm-1. H NMR (CDCl3) δ 3.79 (s, 3
H, NCH3), 5.75 (d, 1 H, CHdCHCN), 7.15 (m, 1 H, pyrrole
â-proton), 7.31 (d, 1 H, CH)CHCN), 7.42 (m, 1 H, pyrrole
R-proton), 7.56 (m, 3 H, benzene H-3,4,5), 7.87 (m, 2 H, benzene
H-2,6). Anal. (C16H16N2O) C, H, N.
harvested by centrifugation. Isolation of nuclei, extraction of
histones, and analysis of histone hyperacetylation by AUT-
PAGE (acid-urea-triton polyacrylamide gel electrophoresis)
followed by staining with Coomasie brilliant blue R-250 were
performed according to standard procedures.62,69 Hyperacety-
lation was also investigated by immunoblotting. For this
purpose, equal amounts of isolated histones (estimated by laser
densitometry) were electrophoresed in precast 14% SDS-
polyacrylamide gels and blotted onto nitrocellulose membrane.
Membrane strips were incubated with an antibody against
acetylated peptide corresponding to the amino acids 2-19 of
Tetrahymena histone H4 (Upstate Biotechnology). This anti-
body recognizes highly acetylated histone H4 isoforms (di-, tri,
and tetraacetylated). Immunodetection was performed using
secondary antibody alkaline phosphatase conjugates.
Molecu la r Mod elin g Stu d ies. All molecular modeling
calculations and manipulations were performed using the
software packages Macromodel 7.1,70 MOPAC 2000,71,72 VALI-
DATE,57 GOLPE 4.5.12,73 XlogP 2,74 Autodock 3.0.5,65 DOCK
4.0.2,66 and MIDAS 2.175 running on Silicon Graphics O2
R10000, IBM-compatible Intel Pentium IV 1.4 GHz and AMD
Athlon 1.9 GHz workstations. For the conformational analysis
and for any minimization, the all-atom Amber force field76,77
was adopted as implemented in the Macromodel package. As
previously reported,53 the crystal structure of TSA extracted
from the HDLP/TSA complex filed in the Brookhaven Protein
Data Bank78 (entry code 1c3r) was used. The HDAC1 model
was constructed from HDLP by mutating all the residues
comprised in a shell of 12 Å from the cocrystallized TSA. Before
any docking studies the HDAC1 model was minimized in a
vacuum in the presence of TSA to relieve any steric contact
introduced by the mutated residue’s side chains.
Binding mode of 8 was extensively analyzed by the mean
of three different docking procedures. A semiautomatic docking
(SAD) was conducted similarly to that reported for 1. A family
of 748 conformations of 8 was generated using the following
procedure: the initial conformation of 8 obtained from the
reported bound conformation of 1 was arbitrarily rotated by a
step of 10° along an axis passing through the center of the
benzene ring and the hydroxamic acid carbon atom. Each of
the obtained new 36 HDAC1/8 complexes was submitted to a
grid search rotating the five dihedral angles by a step of 30°
(Figure 6). By filtering out all of the complexes showing a
molecular clashing between 8 and the enzyme pocket, the final
conformation family of 748 complexes was achieved. After
minimization (Figure 6), the chosen binding conformation of
8 was that associated with the most stable complex (global
minimum).
The 8 starting conformation for the docking studies was
obtained using molecular dynamics with simulated annealing
as implemented in Macromodel version 7.1 and conducted as
following: 8 was energy minimized to a low gradient. The
nonbonded cutoff distances were set to 20 Å for both van der
Waals and electrostatic interactions. An initial random velocity
to all atoms corresponding to 300 K was applied. Three
3-(4-Be n zoyl-1-m e t h yl-1H -2-p yr r olyl)p r op e n yla m i-
d in e (29). A solution of trimethylaluminum 2.0 M in n-hexane
(5.8 mmol, 2.9 mL) was slowly added to a 0 °C cooled
suspension of ammonium chloride (6.4 mmol, 0.34 g) in toluene
(30 mL). After being stirred at room temperature for 2 h, 3-(4-
benzoyl-1-methyl-1H-2-pyrrolyl)propenonitrile (27) (1.0 g, 3.5
mmol) was added, and the reaction mixture was stirred at 80
°C overnight. After the mixture was cooled at 0 °C, methanol
was added to the mixture, and the precipitate was filtered and
washed with methanol. The filtrate was evaporated and the
residue recrystallized from acetonitrile to afford pure 29. IR
1
3400 (NH, NH2), 1635 (CO) cm-1. H NMR (DMSO-d6) δ 3.84
(s, 3 H, NCH3), 6.21 (d, 1 H, CHdCHCdNH(NH2)), 7.79 (m, 7
H, pyrrole H and benzene H), 8.04 (m, 1 H, CHdCHCdNH-
(NH2)), 11.80 (s, 2 H, NH). Anal. (C16H19N3O) C, H, N.
Biologica l Assa ys. HD2 En zym e In h ibition . Radioac-
tively labeled chicken core histones were used as the enzyme
substrate according to established procedures.59 The enzyme
liberated tritiated acetic acid from the substrate which was
quantitated by scintillation counting. IC50 values are results
of triple determinations. Fifty microliters of maize enzyme (at
30 °C) was incubated (30 min) with 10 µL of total [3H]acetate-
prelabeled chicken reticulocyte histones (1 mg/mL). Reaction
was stopped by addition of 36 µL of 1 M HCl/0.4 M acetate
and 800 µL of ethyl acetate. After centrifugation (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 in a starting concentration of 40 µM,
and active substances were diluted further. Sodium butyrate,
sodium valproate, TSA, SAHA,67 trapoxin, and HC-toxin were
used as the reference compounds, and blank solvents were
used as negative controls.
Mou se HDAC1 En zym e Assa y. For inhibition assay,
partially purified HDAC1 from mouse A20 cells (ATCC: TIB-
208) (anion exchange chromatography, affinity chromatogra-
phy (J esacher and Loidl, unpublished results)) was used as
enzyme source. HDAC activity was determined as described68
using [3H]acetate-prelabeled chicken reticulocyte histones as
substrate. Fifty microliters of mouse HDAC1 was incubated
with different concentrations of compounds for 15 min on ice
and 10 µL of total [3H]acetate-prelabeled chicken reticulocyte
histones (4 mg/mL) were added, resulting in a concentration
of 41 µM. The mixture was incubated at 37 °C for 1 h. The
reaction was stopped by addition of 50 µL of 1 M HCl/0.4 M
acetylacetate and 1 mL ethyl acetate. After centrifugation at
10 000g for 5 min an aliquot of 600 µL of the upper phase was
counted for radioactivity in 3 mL of liquid scintillation cocktail.
In Vivo Histon e Hyp er a cetyla tion Assa y. Mouse A20
cells (ATCC: TIB-208) were maintained in RPMI 1640 me-
dium at 37 °C and 5% CO2. Exponentially growing cells were
incubated for 8 h with 8 at final concentration of 40 µM. As
references, cells were also treated with TSA (200 nM) and
SAHA (11 µM). After incubation, cells were washed and

522 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Mai et al.
subsequent molecular dynamics runs were then performed.
The first was carried out for 10 ps with a 1.5 fs time-step at a
constant temperature of 300 K for equilibration purposes. The
next molecular dynamic was carried out for 20 ps, during
which the system is coupled to a 150 °C thermal bath with a
time constant of 5 ps. The time constant represents ap-
proximately the half-life for equilibration with the bath;
consequently the second molecular dynamic command caused
the molecule to slowly cool to approximately 150 K. The third
and last dynamic cooled the molecule to 50 K over 20 ps. A
final energy minimization was then carried out for 250
iterations using conjugate gradient. The minimizations and
the molecular dynamics were in all cases performed in aqueous
solution. The atom charges automatically assigned by the
batchmin module were retained on 8 for the docking calcula-
tions.
To validate the use of the Autodock and DOCK programs,
analogue docking studies were performed on the reference
compounds SAHA and TSA. DOCK and Autodock successfully
reproduced the experimental binding conformations of the
reference drugs with acceptable root-mean-square deviation
(RMSD) of atom coordinates. In Table C
(see Supporting Information) are reported the RMSD values
of the most stable energy minimized complexes of TSA and
SAHA compared with the experimental bound structures
(SAHA PDB entry code: 1c3s).54
The VALIDATE procedure was used to estimate the binding
conformation of the docking studies as previously reported.
The partition coefficients were calculated using the program
XlogP.
Ack n ow led gm en t. The authors whish to thank Prof
G. Cruciani, University of Perugia, for providing the
GOLPE program. This work was supported by grants
of “Progetto Finalizzato Ministero della Salute 2002” (A.
M.) and the Austrian Science Foundation P13209 (G.
B.) and P13620 (P. L.).
For the second docking procedure the program Autodock
was used to explore the binding conformation of 8. For the
docking a grid spacing of 0.375 Å and 58 × 52 × 48 number of
points were used. The grid was centered on the mass center
of the experimental bound TSA coordinates. The GA-LS
method was adopted using the default settings. Amber united
atom were assigned to the protein using the program ADT
(Auto Dock Tools). Autodock generated 100 possible binding
conformations for 8 grouped in 32 clusters.
Su ppor tin g In for m ation Available: Chemical and physi-
cal data for compounds 1-29, 37-40, 48-52, 60-66, com-
parison of docking studies performed on 1, 8, TSA, and SAHA
(values are the root mean square deviations (RMSDs) for all
the atoms), and calculated steric fit values and hydroxamate/
Zn distances. This material is available free of charge via the
Internet at http://pubs.acs.org.
The third docking was performed by the mean of the
program DOCK. First, a Connolly surface of each receptor
active site was generated by using a 1.4 Å probe radius and
further used to generate a set of overlapping spheres that were
then clustered according to their spatial distribution. To
optimize docking accuracy, spheres located too far away from
the known ligand position were eliminated from the finally
selected cluster. To compute interaction energies, a 3-D grid
of 0.30 Å resolution was centered on TSA experimental bound
conformation. The size of the grid box was chosen to enclose
all selected spheres using an extra margin of 6 Å. The grid
had a size of about 36 × 31 × 33 Å and was composed of about
1 400 000 grid points. Energy scoring grids were obtained by
using an all-atom model and a distance-dependent dielectric
function (ꢀ ) 4r) with a 10 Å cutoff. Amber atomic charges
were assigned to all protein atoms. The ligand was then docked
into the protein active site by matching sphere centers with
ligand atoms. A flexible docking (peripheral search and torsion
drive) with subsequent minimization was performed as fol-
lows: (i) automatic selection and matching of an anchor
fragment within a maximum of 10 000 orientations, (ii) itera-
tive growing of the ligand using at least 30 conformations
(peripheral seeds) for seeding the next growing stage with
assignment of energy-favored torsion angles, (iii) simultaneous
relaxation of the base fragments as well as of all peripheral
segments and final relaxation of the entire molecule. Orienta-
tions/conformations were relaxed in 100 cycles of 100 simplex
minimization steps to a convergence of 0.1 kcal/mol. The top
50 solutions corresponding to the best Dock energy scores were
then stored in a single multi mol2 file.
Because of neither Autodock nor DOCK are able to perform
any energy minimization of the generated complexes, the
selection of the binding conformation for 8 was not straight-
forward by using the first Autodock/DOCK scored conforma-
tion. The selection of the binding conformation was performed
by an energy-based rescoring of the 32 Autodock cluster
representants and the 50 best contact scored DOCK conforma-
tions. The program Macromodel was used to minimize the
corresponding 32 Autodock and the 50 DOCK HDAC1/8
complexes. The ligand (8) and an 10 Å core of atoms of the
pocket were allowed to relax during the minimization. An
external fixed shell of 8 Å was also included for the long-range
interactions. Because of the presence of a metal Zn ion in the
HDAC1 catalytic core and the intrinsic molecular mechanic
electrostatic limitation of the AMBER force field, the minimi-
zations were performed applying AM1 charges calculated with
the program MOPAC 2000. Furthermore for comparison
purpose Autodock and DOCK calculations were also performed
on the starting lead compound 1.
Refer en ces
(1) Wu, J .; Grunstein, M. 25 Years after the nucleosome model:
chromatin modifications. Trends Biochem. Sci. 2000, 25, 619-
623.
(2) Urnov, F. D.; Wolffe, A. Chromatin organization and human
disease. Emerg. Ther. Targets 2000, 4, 665-685.
(3) Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.;
Richmond, T. J . Crystal structure of the nucleosome core particle
at 2.8 A resolution. Nature 1998, 389, 251-260.
(4) Davie, J . R. Covalent modifications of histones: expression from
chromatin templates. Curr. Opin. Genet. Dev. 1998, 8, 173-178.
(5) Kouzarides, T. Histone acetylases and deacetylases in cell
proliferation. Curr. Opin. Genet. Dev. 1999, 9, 40-48.
(6) Strahl, B. D.; Allis, C. D. The language of covalent histone
modifications. Nature 2000, 403, 41-45.
(7) Grunstein, M. Histone acetylation in chromatin structure and
transcription. Nature 1997, 389, 349-352.
(8) Pazin, M. J .; Kadonaga, J . T. What is up and down with histone
deacetylation and transcription? Cell 1997, 89, 325-328.
(9) Glass, C. K.; Rosenfeld, M. G. The coregulator exchange in
transcriptional functions of nuclear receptors. Genes Dev. 2000,
14, 121-141.
(10) Khochbin, S.; Verdel, A.; Lemercier, C.; Seigneurin-Berny, D.
Functional significance of histone deacetylase diversity. Curr.
Opin. Genet. Dev. 2001, 11, 162-166.
(11) Giles, R. H.; Peters, D. J .; Breuning, M. H. Conjunction dysfunc-
tion: CBP/p300 in human disease. Trends Genet. 1998, 14, 178-
183.
(12) Gayther, S. A.; Batley, S. J .; Linger, L.; Bannister, A.; Thorpe,
K.; Chin, S. F.; Daigo, Y.; Russell, P.; Wilson, A.; Sowter, H. M.;
Delhanty, J . D.; Ponder, B. A.; Kouzarides, T.; Caldas, C.
Mutations truncating the EP300 acetylase in human cancers.
Nature Genet. 2000, 24, 300-303.
(13) Goodman, R. H.; Smolik, S. CBP/p300 in cell growth, transfor-
mation, and development. Genes Dev. 2000, 14, 1553-1577.
(14) Grossman, S. R. p300/CBP/p53 Interactions and regulation of
the p53 response. Eur. J . Biochem. 2001, 268, 2773-2778.
(15) Lin, R. J .; Sternsdorf, T.; Tini, M.; Evans, R. M. Transcriptional
regulation in acute promyelocytic leukemia. Oncogene 2001, 20,
7204-7215.
(16) Zelent, A.; Guidez, F.; Melnick, A.; Waxman, S.; Licht, J . D.
Translocation of the RARR gene in acute promyelocytic leuke-
mia. Oncogene 2001, 20, 7186-7203.
(17) Pandolfi, P. P. Transcription therapy for cancer. Oncogene 2001,
20, 3116-3127.
(18) Grignani, F.; De Matteis, S.; Nervi, C.; Tomassoni, L.; Gelmetti,
V.; Cioce, M.; Fanelli, M.; Ruthardt, M.; Ferrara, F. F.; Zamir,
I.; Seiser, C.; Grignani, F.; Lazar, M. A.; Minucci, S.; Pelicci, P.
G. Fusion proteins of the retinoic acid receptor-R recruit histone
deacetylase in promyelocytic leukemia. Nature 1998, 391, 815-
818.

Histone Deacetylase Inhibitors
J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4 523
(19) Lutterbach, B.; Westendorf, J . J .; Linggi, B.; Patten, A.; Moniwa,
M.; Davie, J . R.; Huynh, K: D.; Bardwell, V. J .; Lavinsky, R.
M.; Rosenfeld, M. G.; Glass, C.; Seto, E.; Hiebert, S. W. ETO, a
target of t(8; 21) in acute leukemia, interacts with the N-CoR
and mSin3 corepressors. Mol. Cell. Biol. 1998, 18, 7176-7184.
(20) Gelmetti, V.; Zhang, J .; Fanelli, M.; Minucci, S.; Pelicci, P. G.;
Lazar, M. A. Aberrant recruitment of the nuclear receptor
corepressor-histone deacetylase complex by the acute myeloid
leukemia fusion partner ETO. Mol. Cell. Biol. 1998, 18, 7185-
7191.
(40) J ung, M.; Brosch, G.; Ko¨lle, D.; Scherf, H.; Gerha¨user, C.; Loidl,
P. Amide Analogues of Trichostatin A as Inhibitors of Histone
Deacetylase and Inducers of Terminal Cell Differentiation. J .
Med. Chem. 1999, 42, 4669-4679.
(41) Remiszewski, S. W.; Sambucetti, L. C.; Atadja, P.; Bair, K. W.;
Cornell, W. D.; Green, M. A.; Howell, K. L.; J ung, M.; Known,
P.; Trogani, N.; Walker, H. Inhibitors of Human Histone
Deacetylase: Synthesis and Enzyme and Cellular Activity of
Straight Chain Hydroxamates. J . Med. Chem. 2002, 45, 753-
757.
(21) Wang, J .; Hoshino, T.; Redner, R. L.; Kajigaya, S.; Liu, J . M.
ETO, fusion partner in t(8;21) acute myeloid leukemia, represses
transcription by interaction with the human N-CoR/mSin3/
HDAC1 complex. Proc. Natl. Acad. Sci. U.S.A. 1998, 95, 10860-
10865.
(22) Ferrara, F. F.; Fazi, F.; Bianchini, A.; Padula, F.; Gelmetti, V.;
Minucci, S.; Mancini, M.; Pelicci, P. G.; Lo Coco, F.; Nervi, C.
Histone-deacetylase targeted treatment restores retinoic acid
signaling and differentiation in acute myeloid leukemia. Cancer
Res. 2001, 61, 2-7.
(42) Woo, S. H.; Frechette, S.; Khalil, E. A.; Bouchain, G.; Vaisburg,
A.; Bernstein, N.; Moradei, O.; Leit, S.; Allan, M.; Fournel, M.;
Trachy-Bourget M.-C.; Li, Z.; Besterman, J . M.; Delorme, D.
Structurally Simple Trichostatin A-Like Straight Chain Hy-
droxamates as Potent Histone Deacetylase Inhibitors. J . Med.
Chem. 2002, 45, 2877-2885.
(43) Uesato, S.; Kitagawa, M.; Nagaoka, Y.; Maeda, T.; Kuwajima,
H.; Yamori, T. Novel Histone Deacetylase Inhibitors: N-Hy-
droxycarboxamides Possessing a Terminal Bicyclic Aryl Group.
Bioorg. Med. Chem. Lett. 2002, 12, 1347-1349.
(23) Huynh, K. D.; Bardwell, V. J . The BCL-6 POZ domain and other
POZ domains interact with the co-repressors N-CoR and SMRT.
Oncogene 1998, 17, 2473-2484.
(24) Muraoka, M.; Konishi, M.; Kikuchi-Yanoshita, R.; Tanaka, K.;
Shitara, N.; Chong, J . M.; Iwama, T.; Miyaki, M. p300 Gene
alterations in colorectal and gastric carcinomas. Oncogene 1996,
12, 1565-1569.
(25) Weidle, U. H.; Grossmann, A. Inhibition of Histone Deacety-
lases: a New Strategy to Target Epigenetic Modifications for
Anticamcer Treatment. Anticancer Res. 2000, 20, 1471-1486.
(26) Kramer, O. H.; Go¨ttlicher, M. G.; Heinzel, T. Histone deacetylase
as a therapeutic target. Trends Endocrinol. Metab. 2001, 12,
294-300.
(27) 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.
(28) J ung, M. Inhibitors of Histone Deacetylase as New Anticancer
Agents. Curr. Med. Chem. 2001, 8, 1505-1511.
(29) J ohnstone, R. W. Histone-deacetylase inhibitors: novel drugs
for the treatment of cancer. Nature Rev. Drug Discov. 2002, 1,
287-299.
(30) 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.
(44) Su, G. H.; Sohn, T. A.; Ryu, B.; Kern, S. E. A Novel Histone
Deacetylase Inhibitor Identified by High-Throughput Transcrip-
tional Screening of a Compound Library. Cancer Res. 2000, 60,
3137-3142.
(45) Kim, Y. B.; Lee, K. H., Sugita, K.; Yoshida, M.; Horinouchi, S.
Oxamflatin is a novel antitumor compound that inhibits mam-
malian histone deacetylase. Oncogene 1999, 18, 2461-2470.
(46) Lavoie, R.; Bouchain, G.; Frechette, S.; Woo, S. H.; Khalil, E.
A.; Leit, S.; Fournel, M.; Yan, P. T.; Trachy-Bourget M.-C.;
Beaulieu, C.; Li, Z.; Besterman, J . M.; Delorme, D. Design and
Synthesis of a Novel Class of Histone Deacetylase Inhibitors.
Bioorg. Med. Chem. Lett. 2001, 11, 2847-2850.
(47) Furumai, 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) Komatsu, Y.; Tomizaki, K.; Tsukamoto, M.; Kato, T.; Nishino,
N.; Sato, S.; Yamori, T.; Tsuruo, T.; Furumai, R.; Yoshida, M.;
Horinouchi, S.; Hayashi, H. Cyclic Hydroxamic-acid-containing
Peptide 31, a Potent Synthetic Histone Deacetylase Inhibitor
with Antitumor Activity. Cancer Res. 2001, 61, 4459-4466.
(49) Suzuki, T.; Ando, T.; Tsuchiya K.; Fukazawa, N.; Saito, A.;
Mariko, Y.; Yamashita, T.; Nakanishi, O. Synthesis and Histone
Deacetylase Inhibitory Activity of New Benzamide Derivatives.
J . Med. Chem. 1999, 42, 3001-3003.
(31) 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.
(32) Shute, R. E.; Dunlap, B.; Rich, D. H. Analogues of the Cytostatic
and Antimitogenic Agents Chlamydocin and HC-Toxin: Syn-
thesis and Biological Activity of Chloromethyl Ketone and
Diazomethyl Ketone Functionalized Cyclic Tetrapeptides. J .
Med. Chem. 1987, 30, 71-78.
(33) Han, J . W.; Ahn, S. H.; Park, S. H.; Wang, S. Y.; Bae, G. U.;
Seo, D. W.; Known, H. K.; Hong, S.; Lee, Y. W.; Lee, H. W.
Apicidin, a histone deacetylase inhibitor, inhibits proliferation
of tumor cells via induction of p21WAF1/Cip1 and gelsolin.
Cancer Res. 2000, 60, 6068-6074.
(34) Ueda, H.; Nakajima, H.; Hori, Y.; Fujita, T.; Nishimura, M.; Goto,
T.; Okuhara, M. FR901228, a novel antitumor bicyclic depsipep-
tide produced by Chromobacterium violaceum No. 968. I.
Taxonomy, fermentation, isolation, physicochemical and biologi-
cal properties, and antitumor activity. J . Antibiot. 1994, 47,
301-310.
(50) Saito, A.; Yamashita, T.; Mariko, Y.; Nosaka, Y.; Tsuchiya, K.;
Ando, T.; Suzuki, T.; Tsuruo, T.; Nakanishi, O. A synthetic
inhibitor of histone deacetylase, MS-27-275, with marked in
vivo antitumor activity against human tumors. Proc. Natl. Acad.
Sci. U.S.A. 1999, 96, 4592-4597.
(51) Vigushin, D. M.; Coombes, R. C. Histone deacetylase inhibitors
in cancer treatment. Anti-Cancer Drugs 2001, 13, 1-13.
(52) 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.
(53) Mai, A.; Massa, S.; Ragno, R.; Esposito, M.; Sbardella, G.; Nocca,
G.; Scatena, R.; J esacher, 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.
(54) Finnin, M. S.; Donigian, J . R.; Cohen, A.; Richon, V. M.; Rifkind,
R. A.; Marks, P. A.; Breslow, R.; Pavletich, N. P. Structures of
a histone deacetylase homologue bound to the TSA and SAHA
inhibitors. Nature 1999, 401, 188-193.
(35) Kruh, J . Effects of Sodium Butyrate, a New Pharmacological
Agent, on Cells in Culture. Mol. Cell. Biochem. 1982, 42, 65-
82.
(36) Gore, S. D.; Carducci, M. A.; Modifying histones to tame
cancer: clinical development of sodium phenylbutyrate and other
histone deacetylase inhibitors. Exp. Opin. Invest. Drugs 2000,
9, 2923-2934.
(55) Massa, S.; Artico, M.; Corelli, F.; Mai, A.; Di Santo, R.; Cortes,
S.; Marongiu, M. E.; Pani, A.; La Colla, P. Synthesis and
Antimicrobial and Cytotoxic Activities of Pyrrole-Containing
Analogues of Trichostatin A. J . Med. Chem. 1990, 33, 2845-
2849.
(37) Go¨ttlicher, M.; Minucci, S.; Zhu, P.; Kramer, 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.
(38) 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.
(39) 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.
(56) Corelli, F.; Massa, S.; Stefancich, G.; Mai, A.; Artico, M.; Panico,
S.; Simonetti, N. Ricerche su composti antibatterici ed anti-
fungini. Nota VIII - Sintesi ed attivita` antifungina di derivati
pirrolici correlati con la tricostatina A. (Researches on antibacte-
rial and antifungal agents. VIII. Synthesis and antifungal
activity of trichostatin A-related pyrrole derivatives.) Farm., Ed.
Sci. 1987, 42, 893-903.
(57) Head, R. D.; Smythe, M. L.; Oprea, T. I.; Waller, C. L.; Green,
S. M.; Marshall, G. R. VALIDATE: A New Method for the
Receptor-Based Prediction of Binding Affinities of Novel Ligands.
J . Am. Chem. Soc. 1996, 118, 3959-3969.
(58) Garigipati, R. S. An efficient conversion of nitriles to amidines.
Tetrahedron Lett. 1990, 31, 1969-1972.

524 J ournal of Medicinal Chemistry, 2003, Vol. 46, No. 4
Mai et al.
(59) 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.
(60) 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.60,61
(61) 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.
(62) Ko¨lle, D.; Brosch, G.; Lechner, T.; Lusser, A.; Loidl, P. Biochemi-
cal methods for analysis of histone deacetylases. Methods 1998,
15, 323-331.
(63) 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.
(64) 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.
(65) Oshiro, C. M.; Kuntz, I. D.; Dixon, J . S. Flexible ligand docking
using a genetic algorithm. J . Comput. Aided Mol. Des. 1995, 9,
113-130.
(66) Goodsell, D. S.; Morris, G. M.; Olson, A. J . Automated docking
of flexible ligands: applications of AutoDock. J . Mol Recognit.
1996, 9, 1-5.
(67) 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.
(68) Rowley, P. T.; Ohlsson-Wilhelm, B. M.; Farley, B. A.; La Bella,
S. Inducers of erythroid differentiation in K562 human leukemia
cells. Exp. Hemat. 1981, 9, 32-37.
(69) Alfageme, C. R.; Zweidler, A.; Mahowald, A.; Cohen, L. H.
Histones of Drosophila embryos. Electrophoretic isolation and
structural studies. J . Biol. Chem. 1974, 249, 3729-3736.
(70) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MACROMODEL - an integrated software system for model-
ing organic and bioorganic molecules using molecular mechanics.
J . Comput. Chem. 1990, 11, 440-467.
(71) Stewart, J . J . MOPAC:
a semiempirical molecular orbital
program. J . Comput. Aided Mol. Des. 1990, 4, 1-105.
(72) MOPAC 2000.00 Manual; Stewart, J . J . P., Ed; Fujitsu Lim-
ited: Tokyo, J apan, 1999.
(73) GOLPE. Multivariate Infometric Analysis Srl.: Viale dei Cast-
agni 16, Perugia, Italy, 1999.
(74) Wang, R.; Fu, Y.; Lai, L. A New Atom-Additive Method for
Calculating Partition Coefficients. J . Chem. Inf. Comput. Sci.
1997, 37, 615-621.
(75) Ferrin, T. E.; Huang, C. C.; J arvis, L. E.; Langridge, R. The
MIDAS Display System. J . Mol. Graphics 1988, 6, 13-27.
(76) Pearlman, D. A.; Case, D. A.; Caldwell, J . W.; Ross, W. S.;
Cheatham T. E., III.; Debolt, S.; Ferguson, D. M.; Seibel, G. L.;
Kollman, P. A. AMBER, a Package of Computer Programs for
Applying Molecular Mechanics, Normal-Mode Analysis, Molec-
ular Dynamics and Free Energy Calculations to Simulate the
Structural and Energetic Properties of Molecules. Comput. Phys.
Commun. 1995, 91, 1-41.
(77) Pearlman, D. A.; Case, D. A.; Caldwell, J . W.; Ross, W. S.;
Cheatham, T. E., III; Ferguson, D. M.; Seibel, G. L.; Singh, U.
C.; Weiner, P. K.; Kollman, P. A. AMBER 4.1; Department of
Pharmaceutical Chemistry, University of California: San Fran-
cisco, CA, 1995.
(78) Berman, H. M.; Westbrook, J .; Feng, Z.; Gilliland, G.; Bhat, G.
N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data
Bank. Nucl. Acids Res. 2000, 28, 235-242.
J M021070E