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

Article abstract of DOI:10.1021/jm030990+

Previous SAR studies (Part 1: Mai, A.; et al. J. Med. Chem. 2003, 46, 512-524) performed on some portions (pyrrole-C₄, pyrrole-N ₁, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamide (1a) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl (1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the present paper, we investigated the effect on anti-HD2 activity of chemical substitutions performed on the pyrrole-C₂ ethene chains of 1a-c, which were replaced with methylene, ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl moiety into the 1a chemical structure (IC₅₀(2f) = 0.77 μM; IC ₅₀(1a) = 3.8 μM). IC₅₀ values of compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups at the pyrrole-C₄ position a hydrocarbon spacer length ranging from two to five methylenes is well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes) are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives. Particularly, 3a (IC₅₀ = 0.043 μM) showed the same potency as SAHA in inhibiting HD2 in vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was 4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information potentially useful for a future 3D-QSAR study, showed the same (4a,b) or higher (4c,d) HD2 inhibiting activities in comparison with those of the reference drugs. Molecular modeling and docking calculations on the designed compounds performed in parallel with the chemistry work fully supported the synthetic effort and gave insights into the binding mode of the more flexible APHA derivatives (i.e., 3a). Despite the difference of potency between 1b and 3a in the enzyme assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the differentiation assay only at the highest tested dose (48 μM).

Full text of DOI:10.1021/jm030990+

1098
J . Med. Chem. 2004, 47, 1098-1109
3-(4-Ar oyl-1-m eth yl-1H-2-p yr r olyl)-N-h yd r oxy-2-p r op en a m id es a s a New Cla ss of
Syn th etic Histon e Dea cetyla se In h ibitor s. 2. Effect of P yr r ole-C2 a n d /or -C4
Su bstitu tion s on Biologica l Activity†
Antonello Mai,*,§ Silvio Massa, Ilaria Cerbara,§ Sergio Valente,§ Rino Ragno,# Patrizia Bottoni,|
Roberto Scatena,| 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, Istituto di Biochimica e Biochimica Clinica, Universita` Cattolica del Sacro Cuore,
L. go F. Vito 1, 00168 Roma, Italy, and Department of Molecular Biology, University of Innsbruck, Peter-Mayr-Strasse 4b,
6020 Innsbruck, Austria
Received August 6, 2003
Previous SAR studies (Part 1: Mai, A.; et al. J . Med. Chem. 2003, 46, 512-524) performed on
some portions (pyrrole-C4, pyrrole-N1, and hydroxamate group) of 3-(4-benzoyl-1-methyl-1H-
pyrrol-2-yl)-N-hydroxy-2-propenamide (1a ) highlighted its 4-phenylacetyl (1b) and 4-cynnamoyl
(1c) analogues as more potent compounds in inhibiting maize HD2 activity in vitro. In the
present paper, we investigated the effect on anti-HD2 activity of chemical substitutions
performed on the pyrrole-C2 ethene chains of 1a -c, which were replaced with methylene,
ethylene, substituted ethene, and 1,3-butadiene chains (compounds 2). Biological results clearly
indicated the unsubstituted ethene chain as the best structural motif to get the highest HDAC
inhibitory activity, the sole exception to this rule being the introduction of the 1,3-butadienyl
moiety into the 1a chemical structure (IC50(2f) ) 0.77 µM; IC50(1a ) ) 3.8 µM). IC50 values of
compounds 3, prepared as 1b homologues, revealed that between benzene and carbonyl groups
at the pyrrole-C4 position a hydrocarbon spacer length ranging from two to five methylenes is
well accepted by the APHA template, being that 3a (two methylenes) and 3d (five methylenes)
are more potent (2.3- and 1.4-fold, respectively) than 1b, while the introduction of a higher
number of methylene units (see 3e,f) decreased the inhibitory activities of the derivatives.
Particularly, 3a (IC50 ) 0.043 µM) showed the same potency as SAHA in inhibiting HD2 in
vitro, and it was 3000- and 2.6-fold more potent than sodium valproate and HC-toxin and was
4.3- and 6-fold less potent than trapoxin and TSA, respectively. Finally, conformationally
constrained forms of 1b,c (compounds 4), prepared with the aim to obtain some information
potentially useful for a future 3D-QSAR study, showed the same (4a ,b) or higher (4c,d ) HD2
inhibiting activities in comparison with those of the reference drugs. Molecular modeling and
docking calculations on the designed compounds performed in parallel with the chemistry work
fully supported the synthetic effort and gave insights into the binding mode of the more flexible
APHA derivatives (i.e., 3a ). Despite the difference of potency between 1b and 3a in the enzyme
assay, the two APHA derivatives showed similar antiproliferative and cytodifferentiating
activities in vivo on Friends MEL cells, being that 3a is more potent than 1b in the
differentiation assay only at the highest tested dose (48 µM).
In tr od u ction
core histones mediates conformational changes in nu-
cleosomes. Such modifications affect the accessibility of
transcription factors to DNA and regulate gene ex-
pression.1-5 Two classes of enzymes are involved in
determining the state of acetylation of histones: histone
acetyltransferases (HATs), which catalyze the acetyla-
tion of histones acting as transcriptional coactivators,
and histone deacetylases (HDACs), which are recruited
to the promoter regions by transcriptional repressors
and corepressors such as Sin3, SMRT, and N-CoR,
leading to hypoacetylated histones.6-10 Aberrant re-
cruitment of HDACs by oncogenic proteins or a pertur-
bation of the balance between HAT and HDAC activities
in normal cells is implicated in malignant diseases.11-15
HDAC inhibitors (Chart 1), such as the natural products
trichostatin A (TSA),16 trapoxin (TPX),17 and depsipep-
Reversible histone acetylation occurring at the ꢀ-ami-
no group of lysine residues at the N-terminal tails of
† Part 1: Mai, A.; Massa, S.; Ragno, R.; Cerbara, I.; J esacher, 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 Inhibi-
tors. 1. Design, Synthesis, Biological Evaluation, and Binding Mode
Studies Performed through Three Different Docking Procedures. J .
Med. Chem. 2003, 46, 512-524.
* To whom correspondence should be addressed. For A.M.: (phone)
+396-4991-3392; (fax) +396491491; (e-mail) antonello.mai@uniroma1.it.
For G.B.: (phone) 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”.
Universita` degli Studi di Siena.
Dipartimento di Studi di Chimica e Tecnologia delle Sostanze
#
Biologicamente Attive, Universita` degli Studi di Roma “La Sapienza”.
| Universita` Cattolica del Sacro Cuore, Roma.
‡ University of Innsbruck.
10.1021/jm030990+ CCC: $27.50 © 2004 American Chemical Society
Published on Web 01/21/2004

Propenamides as Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1099
Ch a r t 1. Structures of Histone Deacetylase Inhibitors
tide FK-228,18 the short-chain fatty acids sodium bu-
tyrate,19 phenylbutyrate,20 and valproate,21,22 the hy-
droxamates suberoylanilide hydroxamic acid (SAHA),23
pyroxamide, scriptaid,24 oxamflatin,25 and cyclic hy-
droxamic acid containing peptides (CHAPs),26,27 together
with the benzamide MS-27528,29 are potent inducers of
growth arrest, differentiation, or apoptosis in a variety
of transformed cells in culture and in animal models.30-34
Among them, sodium phenylbutyrate (alone or in as-
sociation with retinoic acid, azacytidine, fluorouracil +
indomethacin, or dexamethasone), depsipeptide, SAHA,
pyroxamide, and MS-275 are in phase I and/or phase
II clinical trials for the treatment of many different
cancer diseases.35 Nevertheless, the clinical benefit of
some of them seems to be limited by problems of toxicity
(TSA, CHAPs, MS-275), low stability (TSA, trapoxin)
and/or solubility (TSA), and low potency and lack of
selectivity (butyrates and analogues).34
tested in the enzyme assay against maize histone
deacetylase HD2,43 we chose compound 1a as the lead
compound of the APHA series. Investigation of the 1a
binding mode into the modeled HDAC1 catalytic core
was performed, and its histone hyperacetylation activity
on mouse A20 cells was determined.37 Furthermore, 1a
proved to be effective in growth inhibition (45% and 85%
cell growth inhibition at 40 and 80 µM, respectively) and
cellular differentiation (18% and 21% of benzidine
positive cells at the same concentrations) in murine
erythroleukemia (MEL) cells.37
Prompted by these findings, we designed chemical
modifications on the 1a structure to determine struc-
ture-activity relationships and to improve the HDAC
inhibitory effect of APHAs (Figure 1).38 Replacement of
the pyrrole N1-methyl group either with bulkier sub-
stituents (isopropyl, phenyl) or with a hydrogen atom
led to less potent compounds. Introduction of non-
hydroxamate, metal ion complexing moieties at the end
of the unsaturated chain linked at the pyrrole-C2
position again furnished weakly potent or totally inac-
Recently, a new class of synthetic HDAC inhibitors,
3-(4-aroyl-1H-pyrrol-2-yl)-N-hydroxy-2-propenamides
(APHAs), have been described by us.36-38 The pKi values
of modeled complexes between some aroylpyrrolehy-
droxyamides previously reported by us as antimicrobial
agents39,40 and archaebacterial HDAC homologues (his-
tone-deacetylase-like protein, HDLP)41 were predicted
in the low (or sub-) micromolar range by an extended
VALIDATE 3D QSAR model.42 Because the inhibiting
potencies of other lead HDAC inhibitors were in the
submillimolar (sodium valproate),21,22 micromolar (MS-
275),28,29 or submicromolar (SAHA)23 range and because
of the good agreement between predicted and experi-
mental inhibiting activities of our pyrrole derivatives

1100 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mai et al.
Compounds endowed with the best enzyme inhibiting
activities have then been evaluated in cell-based assays
on antiproliferative and cytodifferentiating effects on
Friend MEL cells.
Ch em istr y
4-Benzoyl-, 4-phenylacetyl-, and 4-cynnammoyl-1-
methyl-1H-2-pyrrolecarboxaldehydes 5a -c,38,40 starting
materials for the synthesis of title compounds, were
treated under Wittig-Horner conditions with the ap-
propriate triethylphosphono esters to give the ethyl
2-substituted 3-pyrrolepropenoates 6d-f,j-o. Such com-
pounds were converted into the corresponding N-hy-
droxy-2-substituted-3-pyrrolepropenamides 2d -f,l-
n ,q-s by reaction between the 2-substituted 3-pyrrole-
propenoic acids 7d -f,j-o, obtained from 6d -f,j-o by
alkaline hydrolysis, and hydroxylamine via ethoxycar-
bonyl anhydrides (Scheme 1). N-Hydroxy-3-pyrrolepro-
panamides 2c,k were prepared by catalytic hydrogena-
tion of known 3-(4-benzoyl- and 3-(4-phenylacetyl-1-
methyl-1H-2-pyrrolyl)-2-propenoic acids38,40 followed by
reaction with hydroxylamine. Base-catalyzed condensa-
tion of pyrrolealdehydes 5a ,b with malonic acid fur-
nished the pyrroledicarboxylic acids 2g,o, promptly
transformed into the corresponding bis-hydroxamides
2h ,p (Scheme 1). Friedel-Crafts reaction of methyl
2-pyrroleacetate with benzoyl and phenylacetyl chloride
yielded a mixture of isomers, the 2,4-, 2,5-, and 2,3-
disubstituted pyrroles (6a -c and 6g-i) being the reac-
tion products (Scheme 2). Among them, acylpyrrole-
acetates 6a ,b,g,h were subjected to alkaline hydrolysis
and in turn converted into the corresponding hydrox-
amates 2a ,b,i,j (Scheme 2). Finally the synthesis of
compounds 3a -f and 4a -d , which failed using the one-
pot Vilsmeier-Haack/Friedel-Crafts method previously
reported by us,36,38-40 was easily accomplished by acy-
lation of ethyl (1-methyl-1H-pyrrol-2-yl)propenoate46
with the appropriate acyl chloride and aluminum trichlo-
ride under Friedel-Crafts conditions (Scheme 3). The
obtained ethyl 3-(4-acyl-1-methyl-1H-pyrrol-2-yl)-2-pro-
penoates 6p -y were then hydrolyzed in alkaline me-
dium and treated with hydroxylamine to afford the
desired hydroxamates 3 and 4 (Scheme 3). Chemical and
F igu r e 1. Chemical manipulations38 performed on lead
compound 1a .
tive derivatives, whereas appropriate C4-aroyl substitu-
tion yielded an increase of inhibitory potency.
Particularly, 3-(1-methyl-4-phenylacetyl-1H-pyrrol-2-
yl)-N-hydroxy-2-propenamide 1b and 3-[1-methyl-4-(3-
phenyl-2-propenoyl)-1H-pyrrol-2-yl]-N-hydroxy-2-pro-
penamide 1c were 38- and 3.8-fold more potent than
1a in the in vitro anti-HD2 assay, respectively.38
In the present paper we explore the effect on HDAC
inhibiting activity of the replacement of the pyrrole-C2
ethene moieties of 1a -c with different alkyl/alkenyl
chains (compounds 2a -s, Chart 2). Moreover, 1b ho-
mologues with various hydrocarbon linker lengths
between the terminal phenyl ring and the carbonyl
adjacent to pyrrole ring (compounds 3a -f, Chart 2) have
been synthesized and tested as anti-HD2 agents. Fi-
nally, conformationally constrained 1b,c analogues
(compounds 4a -d , Chart 2) have been synthesized by
appropriate C4-acyl substitutions, with the aim to study
the effect of the introduction of conformational restric-
tions on the APHAs’ deacetylase inhibiting activity and
to obtain some information potentially useful for a
future 3D-QSAR study.
In parallel with the chemistry work, docking studies
(Autodock 3.0.5 program)44,45 on the designed APHA
derivatives 2-4 into the modeled HDAC1 catalytic
binding pocket were performed with the aim of assess-
ing the reliability of the synthetic effort.
Ch a r t 2. New Designed APHA Derivatives

Propenamides as Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1101
Sch em e 1a
a
(a) (C2H5O)2OPCHRCOOC2H5 (R ) CH3, C2H5, CHdCH), K2CO3, C2H5OH, 80 °C; (b) KOH, C2H5OH, H2O, 70 °C; (c) (1) ClCOOC2H5,
(C2H5)3N, THF, 0 °C; (2) NH2OH; (d) malonic acid, aniline, C2H5OH, room temp.
Sch em e 2a
a
(a) Ph-X-COCl, AlCl3, dichloroethane, room temp; (b) KOH, C2H5OH, H2O, 70 °C; (c) (1) ClCOOC2H5, (C2H5)3N, THF, 0 °C; (2)
NH2OH.
Sch em e 3a
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 together with pyrrole compounds as
reference drugs. The results, expressed as the percent
of enzyme inhibition at a fixed dose and IC50 (50%
inhibitory concentration) values, are reported in Table
1. The effects on biological activity of chemical substitu-
tions performed on (i) the ethene chains of 1a -c
connecting the aroylpyrrole with the hydroxamate
group, (ii) the spacer between benzene and carbonyl
groups at the pyrrole-C4 of 1b, and (iii) the C4-aroyl
moieties of 1b,c with the production of constrained
forms of such compounds are discussed below.
a
(a) W-COCl (W ) Ph-(CH2)n, 1-(1-Ph)cyclopropyl, 1-naphthyl,
1-(2-Ph)cyclopropyl, 2-naphthyl; n ) 2-7), AlCl3, dichloroethane,
room temp; (b) KOH, C2H5OH, H2O, 70 °C; (c) (1) ClCOOC2H5,
(C2H5)3N, THF, 0 °C; (2) NH2OH.
Rep la cem en t of 1a -c Eth en e Moieties a t P yr -
r ole-C2 P osition w ith Oth er (Un )sa tu r a ted Ch a in s.
In the previous paper, 3-(4-benzoyl-1-methyl-1H-pyrrol-
2-yl)-N-hydroxy-2-propenamide 1a , chosen as the APHA
lead compound, was subjected to various chemical
manipulations at the pyrrole-C4 or -N1 position, as well
as to the replacement of hydroxamate moiety with other
metal ion complexing groups, with the aim to study the
effect of such substitutions on HDAC inhibiting activ-
ity.38 The sole C4-aroyl substitution of the benzoyl (1a )
with phenylacetyl (1b) or cynnamoyl (1c) moieties was
successful in improving the anti-HDAC activity, 1b and
1c being 38 and 3.8 times more potent than 1a in the
in vitro assay, respectively.38 In an effort to complete
the SAR data on APHA compounds, we replaced the
1a -c ethene chains connecting the pyrrole ring (C2
position) with the hydroxamate moiety with other
physical data of compounds 2-4 are listed in Table A
(see Supporting Information). Chemical and physical
data of intermediate compounds 6 and 7 are listed in
Table B (see Supporting Information).
In Vitr o En zym e In h ibition a n d
Str u ctu r e-Activity Rela tion sh ip s (SARs)
The pyrrole derivatives 2-4 were evaluated for their
ability to inhibit HDAC activity47 using maize histone
deacetylase HD2 as the enzyme source.48 Maize HD249,51
has an in vitro enzyme activity comparable to those of
HDACs from other sources, such as fungi and verte-
brates, using our standard HDAC assay.47,52 Moreover,
maize HD2 has been shown to be a good predictive
model of mammalian HDACs with various series of
HDAC inhibitors.36-38,53,54

1102 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Ta ble 1. HDAC (Maize HD2) Inhibiting Activity of Compounds 2-4a
Mai et al.
% inhibition
(at fixed dose (µM))
IC50 ( SD
(µM)
compd
X
n
R
Y
2a
2b
2c
2d
2e
2f
2g
2h
2i
none
none
none
none
none
none
none
none
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CH2
CHdCH
CHdCH
CHdCH
CH2
45 (29.8)
61.3 (29.8)
45 (33.4)
83 (27)
37 (25.8)
94 (28)
34.7 ( 1.0
20.5 ( 0.8
36.8 ( 1.1
2.7 ( 0.08
ND
CH2CH2
CHdC(CH3)
CHdC(C2H5)
CHdCH-CHdCH
0.77 ( 0.04
NI
CHdC(CONHOH)
CH2
22.7 (23.3)
62.8 (28.2)
64.2 (28.2)
96 (27)
95 (26)
NI
89.8 (24.8)
NI
91 (22.4)
96 (25)
66 ( 1.3
10.8 ( 0.5
17.1 ( 0.7
1.2 ( 0.04
1.4 ( 0.03
2j
2k
2l
CH2CH2
CHdC(CH3)
CHdC(C2H5)
CHdCH-CHdCH
2m
2n
2o
2p
2q
2r
2s
3a
3b
3c
3d
3e
3f
4a
4b
4c
4d
1a
1b
1c
1.48 ( 0.06
CHdC(CONHOH)
CHdC(CH3)
CHdC(C2H5)
0.2 ( 0.006
1.3 ( 0.05
NI
CHdCH-CHdCH
66 (23.9)
98 (25.7)
94 (24.6)
94 (23.3)
97.2 (22.6)
95 (21.7)
96 (20.8)
94.2 (21.3)
93.6 (24)
97.6 (24.8)
93.8 (24)
86 (33)
9.9 ( 0.50
2
3
4
5
6
7
0.043 ( 0.003
0.13 ( 0.003
0.11 ( 0.005
0.071 ( 0.003
0.266 ( 0.013
0.209 ( 0.008
0.101 ( 0.005
0.144 ( 0.004
0.31 ( 0.01
0.115 ( 0.006
3.8 ( 0.15
1-Ph-c-Pr
1-naphthyl
2-Ph-c-Pr
2-naphthyl
96 (29)
95 (28)
35 (5000)
0.1 ( 0.004
1 ( 0.03
ND
sodium butyrate
sodium valproate
TSA
SAHA
trapoxin
128 ( 3.8
0.0072 ( 0.0002
0.05 ( 0.0015
0.01 ( 0.0003
0.11 ( 0.0044
HC-toxin
a
Data points each represent the mean value of at least three separate experiments. NI: no inhibition at starting concentration of
30 µM. ND: not determined.
(un)saturated alkyl chains and we evaluated the effect
of such substitutions on enzyme inhibitory activity.
N-Hydroxypyrroleacetamides 2a ,b (1a analogues) and
2i,j (1b analogues) was from 5 to 170 times less potent
than the reference compounds in inhibiting HDAC, and
N-hydroxypyrrolepropanamides 2c,k showed IC50 val-
ues 10 times lower than 1a and 1b, respectively. The
introduction of a methyl substituent at the C2 position
of the 1a -c propenoic chains was well tolerated in the
cases of 2d and 2q (1a and 1c analogues) [IC50(2d ) )
2.7 µM; IC50(2q ) ) 1.3 µM; IC50(1a ) ) 3.8 µM;
IC50(1c) ) 1.0 µM], while 2l (1b analogue) was 10 times
less active than the unsubstituted counterpart 1b. Ethyl
substitution at the C2-propenoic chain ruined the anti-
HDAC effect of APHA derivatives (see 2e,m ,r ). Replace-
ment of ethene (1a -c) with 1,3-butadiene (2f,n ,s)
moieties yielded a significant increase of enzyme inhib-
iting activity only in the benzoyl series [IC50(2f) )
0.77 µM; 5 times more potent than 1a in in vitro assay],
while the phenylacetyl and cynnammoyl derivatives 2n
and 2s were 10 times less potent than the corresponding
references 1b and 1c. Pyrrolemethylidenmalonic acids
2g,o failed in inhibiting HDAC activity, while among
pyrrole bis-hydroxamates (2h ,p ), compound 2p showed
an interesting anti-HDAC activity at submicromolar
concentrations, even if lower than that of the monohy-
droxamate counterpart 1b.
1b Hom ologu es w ith Va r iou s Hyd r oca r bon Sp a -
cer Len gth s. As previously described, the replacement
of the C4 benzoyl moiety of 1a with a phenylacetyl group
led to 1b, a compound 38-fold more potent than 1a in
HDAC inhibiting activity.38 On this basis, we explored
the effect on biological activity of the insertion of a
growing number of methylene units between the ter-
minal phenyl ring and the carbonyl adjacent to the
pyrrole ring. Thus, various 1b homologues (3a -f) bear-
ing from two to seven methylene units in such positions
have been synthesized and tested against HD2. N-Hy-
droxy-3-[1-methyl-4-(3-phenylpropionyl)-1H-pyrrol-2-yl]-
2-propenamide 3a was endowed with excellent anti-
HDAC activity in the in vitro assay (IC50 ) 0.043 µM),
being 2.3-fold more potent than 1b and showing the
same potency of SAHA in inhibiting the maize HD2
enzyme. In our assay, 3a was 3000- and 2.6-fold more
potent than sodium valproate and HC-toxin and was
4.3- and 6-fold less potent than trapoxin and TSA,
respectively. The HD2 inhibitory potency of 1b homo-
logues with three to five methylene units was well
maintained (with 3b,c as active as 1b and with 3d 1.4-
fold more potent than 1b), while that shown by six- or
seven-methylene-substituted compounds was decreased
(see 3e and 3f, respectively 2.7- and 2-fold less potent
than 1b).

Propenamides as Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1103
Ta ble 2. Experimental Anti-HD2 pIC50 and Autodock
Predicted (BE Method) Anti-HDAC1 pKi Values for
Compounds 1-4
Con for m a tion a l Con str a in ed An a logu es of 1b,c.
Some constrained 1b analogues (compounds 4a ,b) bear-
ing at the pyrrole-C4 position 1-phenylcyclopropylcar-
bonyl or 1-naphthoyl moiety as conformationally re-
stricted forms of phenylacetyl, and 1c analogues
(compounds 4c,d ) bearing at the pyrrole-C4 position
2-phenylcyclopropylcarbonyl or 2-naphthoyl moiety as
rigid cynnamoyl analogues, have been synthesized and
their anti-HDAC activities have been assessed. IC50
values showed that enzyme inhibiting activity was well
maintained by 4a ,b, which showed the same activity
as 1b, and it was improved by 4c and 4d , which were 3
and 9 times more potent than 1c in the in vitro assay.
a
b
exptl pIC50
(mol/L)
predicted pKi BE
compd
1a
1b
1c
2a
2b
2c
2d
2e
2f
2g
2h
2i
2j
2k
2l
2m
2n
2o
2p
2q
2r
2s
3a
3b
3c
3d
3e
3f
4a
4b
4c
(mol/L)
5.42
7.00
6.00
4.46
4.69
4.43
5.57
NAc
6.11
NAc
4.18
4.97
4.77
5.92
5.85
NAc
5.83
NAc
6.70
5.89
NAc
5.00
7.37
6.89
6.96
7.15
6.58
6.68
7.00
6.84
6.51
6.94
7.37
4.18
6.65
5.98
7.08
5.68
5.61
5.55
5.87
5.15
3.77
4.79
3.46
5.33
4.79
6.19
5.96
6.21
6.76
5.09
3.62
6.64
4.20
4.32
6.79
6.50
5.39
6.68
4.69
4.27
6.69
7.01
5.95
6.82
7.08
3.46
0.91
Molecu la r Mod elin g a n d Dock in g Stu d ies
Docking studies were performed on all the newly
designed APHA derivatives 2-4 using our previously
reported HDAC1 coordinates modeled from the HDLP
structure (PDB entry code 1c3r).37
The molecules were drawn by the Macromodel graphi-
cal interface Maestro 3.0 using as a template the model
of 1a taken from previous calculations. Initial docking
conformations of 2-4 were obtained from a simulated
annealing run performed in water (GBSA).55
From docking studies, three different binding confor-
mations were selected for each docked APHA deriva-
tive: (a) the first Autodock scored conformation, herein
named “best docked” (BD) conformation; (b) the repre-
sentative conformation of the most populated cluster,
herein named “best cluster” (BC) conformation; (c) the
conformation extracted from the lowest energy complex
obtained after a single-point minimization of all pro-
posed HDAC1/APHAs complexes by mean of the Mac-
romodel 7.1 program,56 herein named “best energy” (BE)
conformation.
Autodock pKi values associated with the 2-4 selected
BE binding conformations are reported in Table 2. The
pKi values obtained by BD and BC conformations are
listed in Table C in Supporting Information. For com-
parison purpose, the same calculations were performed
on lead compounds 1a -c.
4d
max value
min value
AAEP
a
pIC50 ) -log(IC50). b BE: best energy conformation, the lowest
energy from a single-point minimization of the Autodock results
(see text). c NA: not available.
Data in Table 2 showed a good agreement between
predicted pKi and experimental pIC50 values of com-
pounds 2-4, although it is not possible to make a direct
correlation between HD2 and HDAC1 inhibitory activi-
ties. Nevertheless, considering that the HD2 pIC50 value
is a close estimation of the HDAC1 pKi value,36-38,53,54
the average absolute error of prediction (AAEP) ranged
from 0.87 to 1.07, displaying an absolute error of
estimation under 1.0 pKi unit for 54% (BD set), 67% (BC
set), and 71% (BE set) of occurrences.
Conformations selected by the BE method are those
showing the lowest error of predictions (AAEPBE ) 0.91).
The plot in Figure 2 shows the Autodock pKi predictions
associated with such BE conformations. BD and BC
method predictions (AAEPBD ) 1.07, AAEPBC ) 0.87)
together with mean predictions (AAEP ) 0.77) are also
reported in Figure A (see Supporting Information).
BE conformations for compounds 1-4 in the HDAC1
catalytic pocket are depicted in Figure B (see Supporting
Information). Inspection of the APHA’s binding mode
reveals that the substructure pyrrole-(un)saturated-
chain-hydroxamate shows in all molecules almost a
unique binding conformation (part A of Figure B), while
F igu r e 2. Plot of the Autodock HDAC1 pKi predictions (BE
method) versus the experimental HD2 pIC50 values. The black
line represents the perfect correlation where Y ) X.
the pyrrole-C4 acyl moieties are almost randomly posi-
tioned because of the fact that these portions are no
longer buried in the enzyme pocket. Interestingly, the
orientation of the N-methylpyrrole moiety is highly
influenced by the chemical nature of the acyl group.
Indeed, for APHA derivatives belonging to the aroylpyr-
rolyl (1a , 2a -h ), cynnamoylpyrrolyl (1c, 2q-s), phen-

1104 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mai et al.
F igu r e 3. Orthographic view of 1b (yellow) and 3a (cyan) binding modes into the HDAC1 catalytic core: (left) shaded in white,
the N-methylpyrrole binding subpocket; (right) shaded in orange, the pyrrole C4-acyl subsite. The green sphere indicates the Zn
ion position in the HDAC1. Hydrogen atoms are not reported for the sake of clarity.
ylpolimethyleneacylpyrrolyl (3a -f), and conformation-
ally constrained acylpyrrolyl (4a -d ) series (parts B, D,
E, and F of Figure B), for 16 molecules out of 23 the
N-methyl group lies in the same protein cavity, fitting
the same area occupied by the C4-methyl of TSA (not
shown). In contrast, with phenylacetylpyrrolyl deriva-
tives 2i-p (part C of Figure B), it is not possible to
describe any common binding mode for the N-meth-
ylpyrrole moiety. In the former group, a further inspec-
tion of the seven molecules that do not place the
N-methyl in the common binding subsite (2a ,d ,g,h ,r ,s
and 3e) reveals that for five cases out of seven (2a ,g,h ,r ,
and 3e) they correspond to inactive or low-active deriva-
tives. About the remaining two compounds, 2d (light-
green in part B of Figure B) and 2s (red in part E of
Figure B), they showed similar inhibitory activities in
comparison with those of the reference compounds 1a
and 1c. The latter observation agrees with our previous
report38 in which we demonstrated that low-active
inhibitors bind in a loose mode, while compounds with
higher pIC50 display a tighter common binding mode.
An orthogonal view of the 3a binding mode into the
HDAC1 catalytic core is depicted in Figure 3. A com-
parison between the 3a binding mode and those de-
scribed for 1a 37 and 1b38 revealed that the enhancement
of inhibitory activity can be mainly attributed to the
higher flexibility of the pyrrole-C4 substituent of 3a . As
previously reported,38 an increase in flexibility of the
chemical structure is unfavorable for binding because
of the increase in the entropy. Nevertheless, the intro-
duction of a methylene (1b)/ethylene(3a ) connection
between the phenyl and the carbonyl group of the 1a
benzoyl moiety disrupts the extended phenyl-CO-
pyrrole π-conjugation. As a consequence of this flex-
ibility, 3a and to a lesser extent 1b were found to fit
better than 1a into HDAC1 (3a , CO‚‚‚Zn ) 2.87 Å, OH‚‚‚
Zn ) 3.01 Å; 1b, CO‚‚‚Zn ) 3.01 Å, OH‚‚‚Zn ) 3,09 Å;
1a , CO‚‚‚Zn ) 2.83 Å, OH‚‚‚Zn ) 4.37 Å).
bond distance from the NH hydroxamate moieties (1b,
Gly140-CO‚‚‚HNhydroxamate ) 2.65 Å; 3a , Gly140-CO‚‚‚
HNhydroxamate ) 2.77 Å). Positive π-stacking interactions
can be observed for both 1b and 3a between the
pyrrolylethylene chain and the Phe141 and Phe198
residues of the site. Pyrrole N1-methyl groups of either
1b or 3a make favorable interactions with the R-carbon
atom of Gly140 and partially with the Phe198 side chain
(white area of Figure 3). Finally, the greater difference
between 1b and 3a is due to the placement of their
pyrrole C4-acyl substituents. The introduction of a
further methylene in 3a enhances the flexibility of this
tail, allowing its placement into a hydrophobic cleft
made by portions of the side chains of Pro22, Tyr91,
Phe141, and Leu265 (orange area of Figure 3), while
the carbonyl group points outside the HDAC1 binding
pocket, free to make favorable hydrogen bonds with the
water environment.
An tip r olifer a tive a n d Cytod iffer en tia tin g Ef-
fects of 1b a n d 3a on F r ien d MEL Cells. From this
study, N-hydroxy-3-(1-methyl-4-(3-phenylpropionyl)-1H-
pyrrol-2-yl)-2-propenamide 3a emerged as the most
potent compound belonging to the APHA series in the
in vitro HDAC (HD2) inhibitory assay, with an IC50
value of 0.043 µM. This derivative is a homologue (at
pyrrole-C4) of N-hydroxy-3-(1-methyl-4-phenylacetyl-
1H-pyrrol-2-yl)-2-propenamide 1b, the highly active
HDAC inhibitor (IC50 ) 0.1 µM) chosen as a new lead
compound of APHAs.38 In addition to the in vitro
enzyme inhibition assay, the capability of 1b and 3a to
induce the antiproliferative effect and cell differentiation
in Friend MEL cells in vivo has been investigated.
Figure 4A shows the effect of the two compounds (1b,
blue; 3a , red) on the growth of MEL cells, cultured for
48 h. The compounds showed similar, significant dose-
dependent inhibitory effects on the growth rate of the
cell line (p < 0.01). Interestingly, after 48 h, the
inhibitors were not cytotoxic at the tested concentrations
(data not shown).
An inspection of 1b and 3a binding modes shows
that the Gly140 carbonyl group is placed at hydrogen-

Propenamides as Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1105
F igu r e 5. Structural modifications performed on APHA
compounds and associated with best HDAC inhibitory activity.
ethylene, substituted ethene, and 1,3-butadiene chains
(compounds 2). Biological results clearly indicated the
unsubstituted ethene chain linked at the pyrrole-C2
position as the best structural motif to get the highest
HDAC inhibitory activity, and it was reported with
other classes of HDAC inhibitors.57 The sole exception
to this rule is the introduction of the 1,3-butadiene
moiety into the 1a chemical structure, 2f being 5 times
more potent than 1a in inhibiting HD2 activity. Nev-
ertheless, the same substitution performed on 1b and
1c (giving 2n and 2s) failed in increasing the anti-HD2
activities of the derivatives, maybe because 2n and 2s
are too long and too rigid for obtaining suitable accom-
modation in the enzyme catalytic pocket (Figure 5).
Because the insertion of a methylene between the
phenyl and carbonyl groups of 1a led to 1b, which is
38-fold more potent, some homologues of 1b (compounds
3) bearing a polimethylene spacer at the pyrrole-C4 acyl
portion were synthesized and tested as anti-HDAC
agents. IC50 values of compounds 3 revealed that a
hydrocarbon spacer length ranging from two to five
methylene groups is well accepted by the APHA tem-
plate, being that 3a (two methylenes) and 3d (five
methylenes) are more potent (2.3- and 1.4-fold, respec-
tively) than 1b. Introduction of a larger number of
methylenes (see 3e and 3f, with six and seven methyl-
enes) decreased the inhibitory activities of the deriva-
tives. Finally, conformationally constrained forms of
1b,c (compounds 4), prepared with the aim to obtain
some information potentially useful for a future 3D-
QSAR study, showed the same (4a ,b) or higher (4c,d )
HD2 inhibiting activities in comparison with those of
the reference drugs 1b,c (Figure 5).
F igu r e 4. Antiproliferative and cytodifferentiating effects of
1b and 3a on MEL cell line. (A) Effects of 1b (blue) and 3a
(red) on cell growth of MEL cells, cultured for 48 h. Data are
expressed as area under the curve (AUC) (mean ( SEM, n )
4): (//) p < 0.01. (B) Effects of 1b (blue) and 3a (red) on
differentiation of MEL cells. The cells were cultured with
various concentrations of drugs for 48 h. Each point is the
mean ( SEM (n ) 4): (/) p < 0.05; (//) p < 0.01.
In MEL cells, the hemoglobin accumulation, revealed
by benzidine staining, is linked to activation of the
differentiation process. The results (Figure 4B) clearly
indicate a dose-dependent increase of hemoglobin syn-
thesis for both 1b and 3a at the tested doses, 3a being
more potent than 1b as a cytodifferentiating agent at
the highest tested concentration (48 µM).
Con clu sion
Molecular modeling and docking calculations, per-
formed on the designed APHAs in parallel with the
chemical work, fully supported the synthesis and bio-
logical evaluation of novel derivatives 2-4. Indeed, more
than 75% of these compounds were predicted to have
submicromolar activity.
Previous SAR studies performed on some portions
(pyrrole-C4, pyrrole-N1, and hydroxamate group) of the
3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-N-hydroxy-2-pro-
penamide (1a ) skeleton highlighted the 4-phenylacetyl
(1b) and 4-cynnamoyl (1c) analogues of 1a as more
potent compounds than 1a in inhibiting maize HD2
activity in vitro. Docking and binding mode studies
performed on 1b into the modeled HDAC1 catalytic core
allow us to explain, in part, the increase of HDAC
inhibiting activity from 1a to 1b.38
Despite the difference in potency between 1b (IC50
)
0.1 µM) and 3a (IC50 ) 0.043 µM) in the enzyme assay,
the two APHA derivatives showed similar antiprolif-
erative and cytodifferentiating activities in vivo on
Friends MEL cells, 3a being more potent than 1b in
the differentiation assay only at the highest tested dose
(48 µM).
In the present paper, we investigated the effect of
chemical substitutions performed on the 1a -c pyrrole-
C2 ethene chains, which were replaced with methylene,

1106 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mai et al.
being stirred at 70 °C for 2 h, the reaction mixture was cooled
to room temperature and diluted with water (50 mL). The
obtained precipitate was filtered and recrystallized from
benzene to give pure 6n . 1H NMR (CDCl3) δ 1.13 (t, 3 H, CHd
C-CH2CH3), 1.27 (t, 3 H, OCH2CH3), 2.58 (q, 2 H, CHdC-
CH2CH3), 3.67 (s, 3 H, NCH3), 4.22 (q, 2 H, OCH2CH3), 6.98
(d, 1 H, pyrrole â-proton), 7.20 (d, 1 H, pyrrole R-proton), 7.33
(m, 5 H, Ph-CHdCH-CO and benzene H-3,4,5), 7.43 (m,
2 H, benzene H-2,6), 7.75 (d, 1 H, pyrrole-CHdCHCO). Anal.
(C21H23NO3) C, H, N.
From such findings, a 3D QSAR study on APHA
derivatives will be performed to acquire useful data and
structural information for structure-based and ligand-
based drug design. Moreover, new synthetic efforts will
be made to prepare 1b and 3a analogues by introducing
a variety of substituents at ortho, meta, or para posi-
tions of the phenyl ring as well as by replacing the
phenyl ring with cycloaliphatic moieties.
Gen er a l P r oced u r e for th e Syn th esis of Eth yl 3-(4-
Acyl-1-m eth yl-1H-p yr r ol-2-yl)-2-p r op en oa tes 6p -y. Ex-
a m p le: Eth yl 3-[1-Meth yl-4-(4-p h en ylbu tyr yl)-1H-p yr r ol-
2-yl]-2-p r op en oa te (6r ). Aluminum trichloride (22.3 mmol,
3.00 g) was slowly added to a cooled (0-5 °C) solution of ethyl
3-(1-methyl-1H-pyrrol-2-yl)-2-propenoate46 (11.2 mmol, 2.0 g)
and 4-phenylbutyryl chloride (previously prepared by heating
the corresponding acid (22.3 mmol, 3.7 g) with SOCl2 for 1 h
at 50 °C) in 1,2-dichloroethane (100 mL). After being stirred
at room temperature for 30 min, the reaction mixture was
poured onto crushed ice (100 g) and 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
(3 × 50 mL). The combined organic solution was washed with
water (100 mL), dried, and evaporated to dryness. The residual
oil was purified by column chromatography on silica gel by
eluting with a 1:20 mixture of ethyl acetate and chloroform.
Exp er im en ta l Section
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
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.58 All
chemicals were purchased from Aldrich Chimica, Milan (Italy),
or from 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 Meth yl Acyl-
(1-m et h yl-1H-p yr r ol-2-yl)a cet a t es 6a -c,g-i. E xa m p le:
Meth yl (4-Ben zoyl-1-m eth yl-1H-p yr r ol-2-yl)a ceta te (2a ),
Meth yl (5-Ben zoyl-1-m eth yl-1H-p yr r ol-2-yl)a ceta te (2b),
a n d Meth yl (3-Ben zoyl-1-m eth yl-1H-p yr r ol-2-yl)a ceta te
(2c). Aluminum trichloride (47 mmol, 6.3 g) was slowly added
to a cooled (0-5 °C) solution of methyl (1-methyl-1H-pyrrol-
2-yl)acetate (39.2 mmol, 5.6 mL) and benzoyl chloride (47
mmol, 5.5 mL) in 1,2-dichloroethane (100 mL). After being
stirred at room temperature for 30 min, the reaction mixture
was poured onto crushed ice (100 g) and 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
(3 × 50 mL). The combined organic extracts were washed with
water (100 mL), dried, and evaporated to dryness. The residual
brown oil was purified by column chromatography on silica
gel by eluting with a 1:10 mixture of ethyl acetate and
chloroform. Three structural isomers have been obtained: the
2,4 isomer (2a ), which was recrystallized from cyclohexane/
benzene; the 2,5 isomer (2b), recrystallized from cyclohexane;
and the 2,3 isomer (2c), recrystallized from cyclohexane/
benzene.
1
Compound 6r was obtained as a pure oil. H NMR (CDCl3) δ
1.37 (t, 3 H, OCH2CH3), 2.07 (m, 2 H, COCH2CH2CH2Ph), 2.71
(m, 4 H, COCH2CH2CH2Ph), 3.76 (s, 3 H, NCH3), 4.38 (q, 2 H,
OCH2CH3), 6.27 (d, 1H, CHdCHCO), 7.01 (m, 1 H, pyrrole
â-proton), 7.28 (m, 6 H, pyrrole R-proton and benzene H), 7.55
(d, 1 H, CH)CHCO). Anal. (C20H25NO3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of (4-Acyl-1-
m eth yl-1H-p yr r ol-2-yl)a lk a n oic a n d -a lk en oic Acid s 7a-
y. Exa m p le: 5-(1-Meth yl-4-p h en yla cetyl-1H-p yr r ol-2-yl)-
2,4-p en ta d ien oic Acid (7l). A mixture of ethyl 5-(1-methyl-
4-phenylacetyl-1H-pyrrol-2-yl)-2,4-pentadienoate (6l) (4.3 mmol,
1.3 g), 2 N KOH (17.0 mmol, 8.3 mL), and ethanol (15 mL)
was heated at 70 °C for 3 h while being stirred. After cooling,
the solution was poured into water (50 mL) and extracted with
ethyl acetate (3 × 50 mL). A sample of 2 N HCl was added to
the aqueous layer until the pH was 5, and the precipitate was
filtered and recrystallized from acetonitrile to yield pure 7l.
1H NMR (DMSO-d6) δ 3.67 (s, 3 H, NCH3), 3.97 (s, 2 H,
PhCH2), 6.00 (m, 1 H, CHdCH-CHdCH-CO), 6.70 (d, 1H,
CHdCH-CHdCH-CO), 7.00 (m, 1 H, pyrrole â-proton), 7.28
(m, 6 H, pyrrole R-proton and benzene H), 7.85 (m, 2 H, CHd
CH-CHdCH-CO), 12.15 (bs, 1 H, OH). Anal. (C18H17NO3) C,
H, N.
Gen er a l P r oced u r e for th e Syn th esis of 3-(4-Acyl-1-
m eth yl-1H-p yr r ol-2-yl)p r op a n oic Acid s 7c,i. Exa m p le:
3-(4-Ben zoyl-1-m eth yl-1H-pyr r ol-2-yl)pr opan oic Acid (7c).
Palladium on active carbon (10%, 0.2 g) was added to a solution
of 3-(4-benzoyl-1-methyl-1H-pyrrol-2-yl)-2-propenoic acid40 (2.2
mmol, 0.6 g) in 20 mL of 95% ethanol. The mixture was stirred
under H2 (45 psi) for 3 h and then was filtered to eliminate
the catalyst. After evaporation of the solvent, the residual oil
was purified by column chromatography on silica gel by eluting
with a 1:9 mixture of methanol and chloroform to obtain 0.5 g
of a pure solid that was recrystallized from benzene. 1H NMR
(CDCl3) δ 2.75 (m, 2 H, CH2CH2CO), 2.87 (m, 2 H, CH2CH2-
CO), 3.59 (s, 3 H, NCH3), 6.46 (s, 1 H, pyrrole â-proton), 7.10
(s, 1 H, pyrrole R-proton), 7.44 (m, 3 H, benzene H-3,4,5), 7.77
2a : 1H NMR (CDCl3) δ 3.65 (s, 3 H, NCH3), 3.67 (s, 2 H,
CH2 overlapped signal), 3.75 (s, 3 H, OCH3), 6.62 (m, 1 H,
pyrrole â-proton), 7.18 (d, 1 H, pyrrole R-proton), 7.48 (m, 3
H, benzene H-3,4,5), 7.82 (m, 2 H, benzene H-2,6). Anal.
(C15H15NO3) C, H, N.
2b: 1H NMR (CDCl3) δ 3.68 (s, 2 H, CH2), 3.70 (s, 3 H, NCH3
overlapped signal), 3.92 (s, 3 H, OCH3), 6.06 (d, 1 H, pyrrole
H-3), 6.63 (d, 1 H, pyrrole H-4), 7.42 (m, 3 H, benzene H-3,4,5),
7.76 (m, 2 H, benzene H-2,6). Anal. (C15H15NO3) C, H, N.
2c: 1H NMR (CDCl3) δ 3.65 (s, 3 H, NCH3), 3.76 (s, 3 H,
OCH3), 4.19 (s, 2 H, CH2), 6.40 (m, 1 H, pyrrole â-proton), 6.59
(d, 1 H, pyrrole R-proton), 7.48 (m, 3 H, benzene H-3,4,5), 7.82
(m, 2 H, benzene H-2,6). Anal. (C15H15NO3) C, H, N.
(m, 2 H, benzene H-2,6), 12.10 (bs, 1 H, OH). Anal. (C15H15
NO3) C, H, N.
-
Gen er a l P r oced u r e for th e Syn th esis of Eth yl (4-Acyl-
1-m eth yl-1H-p yr r ol-2-yl)a lk en oa tes 6d -f,j-o. Exa m p le:
Eth yl 3-[4-(3-P h en yl-2-p r op en oyl)-1-m eth yl-1H-p yr r ol-2-
yl]-2-eth yl-2-p r op en oa te (6n ). A suspension of 4-(3-phenyl-
2-propenoyl)-1-methyl-1H-pyrrole-2-carboxaldehyde38 (4.2 mmol,
1.0 g) in absolute ethanol (20 mL) was added in one portion to
a mixture of triethyl 2-phosphonobutyrate (5.0 mmol, 1.2 mL)
and anhydrous potassium carbonate (12.5 mmol, 1.7 g). After
Gen er a l P r oced u r e for th e Syn th esis of (4-Acyl-1-
m eth yl-1H-p yr r ol-2-yl)-N-h yd r oxya lk a n a m id es a n d -a l-
k en a m id es 2a -f,i-n ,q-s, 3a -f, a n d 4a -d . Exa m p le:
N-Hydr oxy-3-[1-m eth yl-4-(5-ph en ylpen tan oyl)-1H-pyr r ol-
2-yl]p r op en a m id e (3d ). Ethyl chloroformate (2.9 mmol,
0.3 mL) and triethylamine (3.1 mmol, 0.4 mL) were added to
a cooled 0 °C solution of 7s (2.4 mmol, 0.8 g) in dry THF
(10 mL), and the mixture was stirred for 10 min. Afterward,

Propenamides as Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1107
the solid was filtered off and the filtrate was added to a freshly
prepared solution of hydroxylamine in methanol, which was
obtained from the filtration of the mixture from the reaction
between hydroxylamine hydrochloride (3.6 mmol, 0.3 g) and
KOH (3.6 mmol, 0.2 g). The resulting mixture was stirred at
room temperature for 15 min, then it was evaporated, and the
residue was recrystallized from benzene/acetonitrile to afford
pure 3d . 1H NMR (DMSO-d6) δ 1.58 (m, 4 H, CH2CH2CH2-
CH2CO), 2.55 (m, 2 H, CH2CH2CH2CH2CO), 2.68 (m, 2 H, CH2-
CH2CH2CH2CO), 3.72 (s, 3 H, NCH3), 6.25 (d, 1 H, CHd
CHCO), 6.85 (m, 1 H, pyrrole â-proton), 7.24 (m, 6 H, pyrrole
R-proton and benzene H), 7.70 (m, 1 H, CHdCHCO), 8.95
(s, 1 H, NH), 10.65 (s, 1 H, OH). Anal. (C19H22N2O3) C, H, N.
Gen er a l P r oced u r e for th e Syn th esis of 2-(4-Acyl-1-
m eth yl-1H-p yr r ol-2-yl)m eth ylid en m a lon ic Acid s 2g,o.
E xa m p le: 2-(4-Ben zoyl-1-m et h yl-1H -p yr r ol-2-yl)m et h -
ylid en m a lon ic Acid (2g). Malonic acid (16.9 mmol, 1.8 g)
and aniline (30.9 mmol, 2.9 mL) were added to a solution of
4-benzoyl-1-methyl-1H-pyrrole-2-carboxaldehyde40 (14.1 mmol,
3.0 g) in 20 mL of ethanol. The reaction mixture was stirred
at room temperature for 2 h. Afterward, the solution was
poured into water (50 mL) and extracted with ethyl acetate
(3 × 50 mL). A sample of 2 N HCl was added to the aqueous
layer until the pH was 5, and the precipitate was filtered and
recrystallized from acetonitrile/ethanol to give pure 2g. 1H
NMR (DMSO-d6) δ 3.80 (s, 3 H, NCH3), 7.10 (s, 1 H, pyrrole
â-proton), 7.50 (m, 4 H, pyrrole R-proton and benzene H-3,4,5),
7.75 (m, 3 H, benzene H-2,6 and CHdC(COOH)2), 13.05 (bs,
2 H, OH). Anal. (C16H13NO5) C, H, N.
Protein Data Bank63 (entry code 1c3r) was used. The HDAC1
model was constructed from HDLP by mutating all the
residues comprising 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.
The binding modes of APHAs 1-4 were analyzed by a
docking procedure using the program Autodock. 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
atoms were assigned to the protein using the program ADT
(Auto Dock Tools). Autodock generated 100 possible binding
conformations grouped in clusters.
The 1-4 starting conformations for the docking studies were
obtained using a molecular dynamic run with simulated
annealing procedure as implemented in Macromodel version
7.1 and conducted as follows. Each molecule 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 corre-
sponding to 300 K was applied. Three 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 K thermal bath with a time constant of 5 ps. The
time constant represents approximately the half-life for equili-
bration 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 a conjugate gradient.
The minimizations and the molecular dynamics were in all
cases performed in aqueous solution.
Because of Autodock is not able to perform any energy
minimization of the generated complexes, the selection of the
binding conformations was not straightforward by using the
first Autodock scored conformation. Three binding conforma-
tion for each APHA were selected: (a) the first scored Autodock
conformation; (b) the most populated cluster representative
structure; (c) the lowest energy APHA/HDAC1 complex upon
energy-based rescoring of the Autodock cluster representants.
The program Macromodel was used to minimize the corre-
sponding Autodock HDAC1/APHA complexes. The ligand and
a 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 minimizations were performed by
applying AM1 charges calculated with the program MOPAC
2000.
Gr ow t h In h ib it ion a n d Cell Differ en t ia t ion Assa y.
Cell Cu ltu r e a n d Rea gen ts. Murine erythroleukemia (MEL)
cells were obtained from Interlab Cell Line Collection (CBA)
(Genoa, Italy). Cells were maintained at 37 °C under a
humidified atmosphere of 5% CO2 in RPMI 1640 Hepes
modified medium supplemented with 10% (v/v) heat inacti-
vated fetal calf serum, 2 mmol/L glutamine, 100 IU/mL
penicillin, and 100 µg/mL streptomycin. Unless indicated, all
chemicals and reagents (cell culture grade) were obtained from
Sigma Chemical Co., Milan, Italy.
Cell Via bility a n d Gr ow th In h ibition Assa y. Cell num-
ber was determined using a Neubauer hemocytometer, and
viability was assessed by their ability to exclude trypan blue.
The stock solutions were prepared immediately before use.
Compounds 1b and 3a were dissolved in DMSO. MEL expo-
nentially growing cells (1 × 105 cells/mL) were set at day 0 in
media containing various concentrations of drugs for 48 h. The
final concentrations of the drugs were 2.4, 4.8, 24, and 48 µM.
The final concentration of DMSO, used as a vehicle, was the
same (0.1% v/v) in all samples during the experiments. Data
Gen er a l P r oced u r e for th e Syn th esis of 2-(4-Acyl-1-
m et h yl-1H -p yr r ol-2-yl)m et h ylid en -N,N′-d ih yd r oxym a -
lon a m id es 2h ,p . Exa m p le: 2-(1-Meth yl-4-p h en yla cetyl-
1H-p yr r ol-2-yl)m eth ylid en -N,N′-d ih yd r oxym a lon a m id e
(2p ). Ethyl chloroformate (22.2 mmol, 2.2 mL) and triethyl-
amine (24.1 mmol, 3.4 mL) were added to a cooled 0 °C solution
of 2o (6.3 mmol, 2.9 g) in dry THF (10 mL), and the mixture
was stirred for 10 min. Afterward, the solid was filtered off
and the filtrate was added to a freshly prepared solution of
hydroxylamine in methanol (obtained from the reaction be-
tween hydroxylamine hydrochloride (27.8 mmol, 1.9 g) and
KOH (27.8 mmol, 1.6 g)). The resulting mixture was stirred
at room temperature for 15 min and was evaporated, and the
1
residue was recrystallized from benzene to yield pure 2p . H
NMR (DMSO-d6) δ 3.71 (s, 3 H, NCH3), 3.94 (s, 2 H, Ph-CH2),
6.95 (d, 1 H, pyrrole â-proton), 7.21 (m, 5 H, benzene H), 7.33
(d, 1 H, pyrrole R-proton), 7.96 (d, 1 H, CHdC(CONHOH)2),
9.00 (bs, 2 H, NH), 11.05 (bs, 2 H, OH). Anal. (C17H15NO5)
C, H, N.
In Vitr o HD2 En zym e In h ibition . Radioactively labeled
chicken core histones were used as the enzyme substrate
according to established procedures.47 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 (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 (10000g,
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 concentration of 40 µM,
and active substances were diluted further. Sodium butyrate,
sodium valproate, TSA, SAHA,58 trapoxin, and HC-toxin were
used as the reference compounds, and blank solvents were
used as negative controls.
Molecu la r Mod elin g a n d Dock in g Stu d ies. All molec-
ular modeling calculations and manipulations were performed
using the software packages Macromodel 7.1,56 MOPAC
2000,59,60 and Autodock 3.0.544,45 running on IBM compatible
AMD Athlon 2.4 GHz workstations. For the conformational
analysis and for any minimization, the all-atom Amber force
field61,62 was adopted as implemented in the Macromodel
package. As previously reported37 the crystal structure of TSA
extracted from the HDLP/TSA complex filed in the Brookhaven

1108 J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5
Mai et al.
(18) 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, physico-chemical and biologi-
cal properties, and antitumor activity. J . Antibiot. 1994, 47,
301-310.
(19) Kruh, J . Effects of Sodium Butyrate, a New Pharmacological
Agent, on Cells in Culture. Mol. Cell. Biochem. 1982, 42, 65-
82.
(20) Gore, S. D.; Carducci, M. A.; Modifying histones to tame
cancer: clinical development of sodium phenylbutyrate and other
histone deacetylase inhibitors. Expert Opin. Invest. Drugs 2000,
9, 2923-2934.
are graphically reported in Figure 4A as area under the curve
(AUC) values.
Cytom or p h ologica l Assa y for MEL Cell Differ en tia -
tion . The most widely used method for scoring erythroid
differentiation is benzidine staining, which reveals the produc-
tion of hemoglobin.64 Benzidine dihydrochloride (2 mg/mL) was
prepared in 3% acetic acid. Hydrogen peroxide (1%) was added
immediately before use. The MEL cell suspensions were mixed
with the benzidine solution in a 1:1 ratio and counted in a
hemocytometer after 5 min. Blue cells were considered to be
positive for hemoglobin.
(21) 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.
(22) 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.
(23) 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.
(24) 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.
(25) 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.
(26) 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.
(27) 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.
(28) 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.
(29) 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.
(30) 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.
(31) Kramer, O. H.; Go¨ttlicher, M. G.; Heinzel, T. Histone deacetylase
as a therapeutic target. Trends Endocrinol. Metab. 2001, 12,
294-300.
(32) 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.
(33) J ung, M. Inhibitors of Histone Deacetylase as New Anticancer
Agents. Curr. Med. Chem. 2001, 8, 1505-1511.
(34) Vigushin, D. M.; Coombes, R. C. Histone deacetylase inhibitors
in cancer treatment. Anti-Cancer Drugs 2001, 13, 1-13.
(35) J ohnstone, R. W. Histone-deacetylase inhibitors: novel drugs
for the treatment of cancer. Nat. Rev. Drug Discovery 2002, 1,
287-299.
(36) 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.
(37) 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.
(38) Mai, A.; Massa, S.; Ragno, R.; Cerbara, I.; J esacher, 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.
(39) 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
Sta tistica l An a lysis. All results are expressed as the mean
( SEM. The group mean values were compared by analysis
of variance (ANOVA) followed by a multiple comparison of
means by the Dunnet test. p < 0.05 was considered significant.
Ack n ow led gm en t. This work was supported by
grants from “Progetto Finalizzato Ministero della Salute
2002” (A.M.), “AIRC Proposal 2003” (A.M.), and the
Austrian Science Foundation (Grants P13209 (G.B.) and
P13620 (P.L.)).
Su ppor tin g In for m ation Available: Chemical and physi-
cal data for compounds 2-4, 6, and 7 (Tables A and B) and
additional molecular modeling data (Table C, Figures A and
B). This material is available free of charge via the Internet
at http://pubs.acs.org.
Refer en ces
(1) Davie, J . R. Covalent modifications of histones: expression from
chromatin templates. Curr. Opin. Genet. Dev. 1998, 8, 173-178.
(2) Kouzarides, T. Histone acetylases and deacetylases in cell
proliferation. Curr. Opin. Genet. Dev. 1999, 9, 40-48.
(3) Strahl, B. D.; Allis, C. D. The language of covalent histone
modifications. Nature 2000, 403, 41-45.
(4) Pazin, M. J .; Kadonaga, J . T. What’s up and down with histone
deacetylation and transcription? Cell 1997, 89, 325-328.
(5) Glass, C. K.; Rosenfeld, M. G. The coregulator exchange in
transcriptional functions of nuclear receptors. Genes Dev. 2000,
14, 121-141.
(6) Wu, J .; Grunstein, M. 25 Years after the nucleosome model:
chromatin modifications. Trends Biochem. Sci. 2000, 25, 619-
623.
(7) Urnov, F. D.; Wolffe, A. Chromatin organization and human
disease. Emerging Ther. Targets 2000, 4, 665-685.
(8) Luger, K.; Mader, A. W.; Richmond, R. K.; Sargent, D. F.;
Richmond, T. J . Crystal structure of the nucleosome core particle
at 2.8 Å resolution. Nature 1998, 389, 251-260.
(9) Grunstein, M. Histone acetylation in chromatin structure and
transcription. Nature 1997, 389, 349-352.
(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) Lin, R. J .; Sternsdorf, T.; Tini, M.; Evans, R. M. Transcriptional
regulation in acute promyelocytic leukemia. Oncogene 2001, 20,
7204-7215.
(12) 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.
(13) Pandolfi, P. P. Transcription therapy for cancer. Oncogene 2001,
20, 3116-3127.
(14) 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.
(15) 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.
(16) 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.
(17) 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.

Propenamides as Inhibitors
J ournal of Medicinal Chemistry, 2004, Vol. 47, No. 5 1109
pirrolici correlati con la tricostatina A. (Research on antibacterial
and antifungal agents. VIII. Synthesis and antifungal activity
of trichostatin A-related pyrrole derivatives.) Farmaco, Ed. Sci.
1987, 42, 893-903.
(53) 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.
(54) Wittich, S.; Scherf, H.; Xie, C.; Brosch, G.; Loidl, P.; Gerha¨user,
C.; J umg, M. Structure-Activity Relationships on Phenylala-
nine-Containing Inhibitors of Histone Deacetylase: In Vitro
Enzyme Inhibition, Induction of Differentiation, and Inhibition
of Proliferation in Friend Leukemic Cells. J . Med. Chem. 2002,
45, 3296-3309.
(55) Still, W. C.; Tempczyk, A.; Hawley, R. C.; Hendrickson, T.
Semianalytical Treatment of Solvation for Molecular Mechanics
and Dynamics. J . Am. Chem. Soc. 1990, 112, 6127-6129.
(56) Mohamadi, F.; Richards, N. G. J .; Guida, W. C.; Liskamp, R.;
Lipton, M.; Caufield, C.; Chang, G.; Hendrickson, T.; Still, W.
C. MACROMODELsan integrated software system for modeling
organic and bioorganic molecules using molecular mechanics.
J . Comput. Chem. 1990, 11, 440-467.
(57) 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.
(40) 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.
(41) 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.
(42) 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.
(43) 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.
(44) Goodsell, D. S.; Morris, G. M.; Olson, A. J . Automated docking
of flexible ligands: applications of AutoDock. J . Mol Recognit.
1996, 9, 1-5.
(45) 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.
(58) 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.
(46) 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.
(59) Stewart, J . J . MOPAC:
a semiempirical molecular orbital
program. J . Comput.-Aided Mol. Des. 1990, 4, 1-105.
(60) MOPAC 2000.00 Manual; Stewart, J . J . P., Ed; Fujitsu Lim-
ited: Tokyo, J apan 1999.
(61) 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.
(62) 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.
(63) Berman, H. M.; Westbrook, J .; Feng, Z.; Gilliland, G.; Bhat, G.
N.; Weissig, H.; Shindyalov, I. N.; Bourne, P. E. The Protein Data
Bank. Nucleic Acids Res. 2000, 28, 235-242.
(47) 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.
(48) 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.49,50
(49) 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.
(50) Ko¨lle, D.; Brosch, G.; Lechner, T.; Lusser, A.; Loidl, P. Biochemi-
cal methods for analysis of histone deacetylases. Methods 1998,
15, 323-331.
(51) 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.
(52) 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.
(64) Rowley, P. T.; Ohlsson-Wilhelm, B. M.; Farley, B. A.; La Bella,
S. Inducers of erythroid differentiation in K562 human leukemia
cells. Exp. Hematol. 1981, 9, 32-37.
J M030990+