Small-molecule inhibitors of histone acetyltransferase activity: Identification and biological properties

Article abstract of DOI:10.1021/jm060601m

Starting from a yeast phenotypic screening performed on 21 compounds, we described the identification of two small molecules (9 and 18) able to significantly reduce the S. cerevisiae cell growth, thus miming the effect of GCN5 deletion mutant. Tested on a GCN5-dependent gene transcription assay, compounds 9 and 18 gave a high reduction of the reporter activity. In S. cerevisiae histone H3 terminal tails assay, the H3 acetylation levels were highly reduced by treatment with 0.6-1 mM 9, while 18 was effective only at 1.5 mM. In human leukemia U937 cell line, at 1 mM 9 and 18 showed effects on cell cycle (arrest in G1 phase, 9), apoptosis (9), and granulocytic differentiation (18). When tested on U937 cell nuclear extracts to evaluate their histone acetyltransferase (HAT) inhibitory action, both compounds were able to reduce the enzyme activity when used at 500 μM. Another quinoline, compound 22, was synthesized with the aim to improve the activity observed with 9 and 18. Tested in the HAT assay, 22 was able to reduce the HAT catalytic action at 50 and 25 μM, thereby being comparable to anacardic acid, curcumin, and MB-3 used as references. Finally, in U937 cells, compounds 9 and 18 used at 2.5 mM were able to reduce the extent of the acetylation levels of histone H3 (9) and α-tubulin (9 and 18). In the same assay, 22 at lower concentration (100 μM) showed the same hypoacetylating effects with both histone and non-histone substrates.

Full text of DOI:10.1021/jm060601m

J. Med. Chem. 2006, 49, 6897-6907
6897
Small-Molecule Inhibitors of Histone Acetyltransferase Activity: Identification and Biological
Properties
Antonello Mai,*,§ Dante Rotili,§ Domenico Tarantino,§ Prisca Ornaghi,# Federica Tosi,# Caterina Vicidomini,†
Gianluca Sbardella,† Angela Nebbioso,‡ Marco Miceli,‡ Lucia Altucci,*,‡,| and Patrizia Filetici*,
Dipartimento di Studi Farmaceutici, Istituto PasteursFondazione Cenci Bolognetti, UniVersita` degli Studi di Roma “La Sapienza”, P.le A.
Moro 5, 00185 Roma, Italy, Dipartimento di Genetica e Biologia Molecolare, UniVersita` degli Studi di Roma “La Sapienza”, P.le A. Moro 5,
00185 Roma, Italy, Dipartimento di Scienze Farmaceutiche, UniVersita` degli Studi di Salerno, Via Ponte Don Melillo, 84084 Fisciano (SA),
Italy, Dipartimento di Patologia Generale, Seconda UniVersita` degli Studi di Napoli, Vico L. De Crecchio 7, 80138 Napoli, Italy, Centro di
Oncogenomica AIRC, CEINGE Biotecnologia AVanzata, Napoli, Italy, and Dipartimento di Genetica e Biologia Molecolare, Istituto di Biologia
e Patologia Molecolari CNR, UniVersita´ degli Studi di Roma “La Sapienza”, P.le A. Moro 5, 00185 Roma, Italy
ReceiVed May 20, 2006
Starting from a yeast phenotypic screening performed on 21 compounds, we described the identification of
two small molecules (9 and 18) able to significantly reduce the S. cereVisiae cell growth, thus miming the
effect of GCN5 deletion mutant. Tested on a GCN5-dependent gene transcription assay, compounds 9 and
18 gave a high reduction of the reporter activity. In S. cereVisiae histone H3 terminal tails assay, the H3
acetylation levels were highly reduced by treatment with 0.6-1 mM 9, while 18 was effective only at 1.5
mM. In human leukemia U937 cell line, at 1 mM 9 and 18 showed effects on cell cycle (arrest in G1 phase,
9), apoptosis (9), and granulocytic differentiation (18). When tested on U937 cell nuclear extracts to evaluate
their histone acetyltransferase (HAT) inhibitory action, both compounds were able to reduce the enzyme
activity when used at 500 µM. Another quinoline, compound 22, was synthesized with the aim to improve
the activity observed with 9 and 18. Tested in the HAT assay, 22 was able to reduce the HAT catalytic
action at 50 and 25 µM, thereby being comparable to anacardic acid, curcumin, and MB-3 used as references.
Finally, in U937 cells, compounds 9 and 18 used at 2.5 mM were able to reduce the extent of the acetylation
levels of histone H3 (9) and R-tubulin (9 and 18). In the same assay, 22 at lower concentration (100 µM)
showed the same hypoacetylating effects with both histone and non-histone substrates.
Introduction
Similar to HDACs, GCN5/PCAF HATs are typically found in
cells in large multiprotein complexes such as SAGA and ADA,
yet unlike HDACs they exhibit robust catalytic activity as
purified proteins, with high selectivity for Lys-14 in histone
H3.13-16 However, their substrate specificity in protein com-
plexes appears to be broader, and in addition to histones, GCN5/
PCAF HATs target non-histone protein substrates, such as
transcription factors (i.e., p53)17 and structural proteins (i.e.,
R-tubulin).18
The anticancer effects of HDAC inhibitors are well-known,
and a number of them are in clinical trials,19,20 while the
chemotherapeutic potential of HAT targets has been less
validated so far. Missense and deletion mutations in the p300
gene have been found in colorectal, gastric, and epithelial
cancers, and the loss of heterozygosity of p300 gene has been
related to glioblastoma.21,22 Amplification and overexpression
of AIB-1 (amplified in breast cancer-1), a steroid receptor
coactivator with HAT activity, in breast, ovarian, and gastric
cancers furnish an example of deregulated HATs in oncogen-
esis.23,24 In acute myeloid leukemia (AML), the gene for CBP
is translocated and fused to MYST family HATs such as MOZ
and MORF.25,26 p300 and PCAF have been reported to play an
important role in MyoD dependent cell cycle arrest,27 and
deregulation of GCN5/PCAF in genetic diseases and cancer has
led to the supposition that selective inhibitors of these HATs
may exert therapeutic applications.28,29
The reversible process of histone acetylation occurring at the
ꢀ-amino group of lysine residues in the N-terminal tails of core
histones mediates conformational changes in nucleosomes. Two
superfamilies of enzymes are involved in such a process: histone
acetyltransferases (HATs) and histone deacetylases (HDACs),
which catalyze respectively the addition to and the removal of
acetyl units to histones. These modifications affect the acces-
sibility of transcription factors to DNA and regulate gene
expression.1-5 Indeed, hyperacetylation of histones has been
associated with relaxed chromatin structure and active gene
transcription, while hypoacetylated histones lead to transcrip-
tional repression by condensing the structure of chromatin and
restricting the access of transcription factors.6-9
The first nuclear HAT to be discovered, general control of
nuclear-5 (GCN5), and its paralogue p300/CBP accessory factor
(PCAF) are conserved in organisms from yeast to humans, and
GCN5/PCAF HATs have served as paradigms for the biochemi-
cal analysis of HAT structure, mechanism, and function.10-12
* To whom correspondence should be addressed. For A.M., phone,
+3906-4991-3392; fax, +3906-491491; e-mail, antonello.mai@uniroma1.it.
For L.A., phone, +39081-566-7569; fax, +39081-450-169; e-mail,
lucia.altucci@unina2.it. For P.F., phone, +3906-4991-2241; fax, +3906-
4440-812; e-mail, patrizia.filetici@uniroma1.it.
§ Dipartimento di Studi Farmaceutici, Universita` degli Studi di Roma
“La Sapienza”.
# Dipartimento di Genetica e Biologia Molecolare, Universita` degli Studi
di Roma “La Sapienza”.
To date, a small number of HAT inhibitors have been
reported, the first being the bisubstrate analogues Lys-CoA and
H3-CoA-20, respectively selective for p300 and PCAF.30 As
the second step, a few natural small molecules such as anacardic
acid,31 garcinol,32 and curcumin33 were described as potent p300
† Universita` degli Studi di Salerno.
‡ Seconda Universita` degli Studi di Napoli.
| CEINGE Biotecnologia Avanzata.
Istituto di Biologia e Patologia Molecolari CNR, Universita` degli Studi
di Roma “La Sapienza”.
10.1021/jm060601m CCC: $33.50 © 2006 American Chemical Society
Published on Web 10/17/2006

6898 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Mai et al.
Figure 1. Known HAT inhibitors.
and PCAF inhibitors. Recently, the γ-butyrolactone MB-3 has
been discovered as a small, cell-permeable GCN5 inhibitor,34
and some isothiazolones were disclosed as inhibitors of p300
and PCAF HAT activity35 (Figure 1).
HDACi and HAT activators could induce similar phenotypes
in a cell-based assay.
Among the first tested compounds, 1-14, only the 3-carbe-
thoxy-2-methylquinoline 952 was found to be active in inhibiting
yeast cell growth by resembling a GCN5 loss-of-function
mutation. Thus, a series of analogues of compound 9 (com-
pounds 15-21) were prepared and tested in Saccharomyces
cereVisiae as GCN5 inhibitors. Among them, only compound
18 was able to inhibit the yeast growth as well as compound 9.
Since compounds 9 and 18, which have been characterized with
a wide range of cellular activities, were active at (sub)millimolar
concentrations, other functional targets may be involved and
may explain the observed effects and phenotype. Thus, we tried
to combine the structural features shown by compounds 9 and
18 (i.e., the C3-substituted quinoline moiety) with those typical
of AA31 and MB-334 (i.e., the salicylic acid or the 4-carboxy-
γ-butyrolactone function together with the presence of a long
alkyl chain), obtaining a new series of quinolines of which the
4-hydroxy-2-pentylquinoline-3-carboxylic acid 22 (Figure 3) can
be considered a prototype.
Pursuing our searches on design, synthesis, and biological
evaluation of small molecules as epigenetic tools for regulating
gene expression and transcription,36-46 we performed a yeast
phenotypic screening on a set of both newly synthesized and
commercially available molecules (1-21) (Figure 2) to find
among them one or more modulators of GCN5 activity. Indeed,
GCN5 is involved in maximal expression of certain amino acid
biosynthesis genes,47 and loss of GCN5 leads to poor yeast
growth under amino acid starvation. Small-molecule inhibitors
of GCN5 activity in wild-type yeast, as well as gcn5∆ mutants,
are expected to cause clear gcn5- phenotypes.
Among tested compounds, derivatives 1-4 were designed
after a molecular pruning/isosterism approach on the transition-
state analogue Lys-CoA. Anacardic acid (AA) 531 was used as
a template for the design and synthesis of analogues 6-10, in
which the AA benzene ring was replaced by a pyrimidine (6,
7) or quinoline (8) moiety, including the two simpler quinolines
9 and 10. all-trans-Retinoic acid (ATRA) 11,48 a well-known
transcriptional modulator, and the corresponding hydroxamate
12 also show some structural analogies with AA and were thus
included in the assay. Apicidin 1349 and HC-toxin 14,50 two
HDAC inhibitors belonging to the cyclic tetrapeptide family,
were also tested in the cell-based assay to investigate an eventual
positive modulation of GCN5-mediated phenotypic effects. In
fact, an amide analogue of AA, (N-(4-chloro-3-trifluorometh-
ylphenyl)-2-ethoxy-6-pentadecylbenzamide, CTPB),31 was re-
ported as a p300 activator, and we consequently expected that
Chemistry
The LysCoA-based compounds 1 and 2 were prepared by
acylation of NR-acetyl-L-lysine methyl ester hydrochloride with
2-methylthioacetic acid or thiophene-2-carboxylic acid in the
presence of N,N′-carbonyldiimidazole (Scheme 1). For the
synthesis of 3 and 4, the same acylation reactions were
performed on the 2-[4-(4,5-dihydrooxazol-2-yl)phenoxy]ethy-
lamine 23, prepared by Mitsunobu reaction between tert-
butoxycarbonyl- (BOC-) protected 2-aminoethanol and the
4-(4,5-dihydrooxazol-2-yl)phenol 2451 in the presence of diiso-

Inhibitors of Histone Acetyltransferase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6899
Figure 2. Compounds 1-21 subjected to the cell-based screening.
Figure 3. 4-Hydroxy-2-pentylquinoline-3-carboxylic acid 22.
acetoacetate (for 15) or ethyl 3-oxooctanoate (for 28) with isatoic
anhydride in the presence of sodium hydroxide as catalyst.
Alkaline hydrolysis of 28 with 4 N KOH furnished the hydroxy
acid 22 (Scheme 3). The synthesis of 9 was accomplished by a
single-step variant of the Friedla¨nder synthesis involving
o-nitrobenzaldehyde and ethyl acetoacetate in the presence of
SnCl2 and ZnCl2,52 and the corresponding carboxylic acid 1053
was obtained from 9 by standard procedure (Scheme 3).
The N-hydroxyretinamide 1254 was prepared by reaction of
the commercial all-trans-retinoic acid 11 with ethyl chlorofor-
mate, followed by addition of O-(2-methoxy-2-propyl)hydroxy-
lamine55 and subsequent acidic treatment in the presence of
Amberlyst 15 ion-exchange resin (Scheme 4).
propyl azodicarboxylate (DIAD)/triphenylphosphine, followed
by acidic removal of the protecting tert-butoxycarbonyl group
(Scheme 1).
Acylation of potassium ethyl malonate with pentadecyl
imidazolide in the presence of magnesium dichloride and
triethylamine furnished the corresponding â-oxoester 25, which
was in turn condensed with thiourea and sodium ethoxide in
ethanol to give the 2-thiouracil 6. Following treatment of 6 with
methyl iodide afforded the 2-methylthiopyrimidin-4(3H)-one 26,
which was converted into the corresponding 5-bromoderivative
27 with N-bromosuccinimide (NBS). Further reaction of 27 with
n-butyllithium and carbon dioxide at -78 °C furnished the
4-hydroxy-2-methylthio-6-tetradecylpyrimidine-5-carboxylic acid
7 (Scheme 2).
The commercial 3-quinolinecarboxylic acid 20 was the
starting material for the preparation of both the corresponding
ethyl ester 1856 (by standard method) and the carboxyamide
2157 (by a two-step, one-pot reaction with ethyl chloroformate/
0.5 M ammonia solution in dioxane). Finally, the ethyl 2-naph-
thoate 19 was prepared from 2-naphthoyl chloride by standard
reaction.58
Chemical and physical data for compounds 1-4, 6-8, 12,
15, 21-23, and 25-28 are reported as Supporting Information.
The 3-carbethoxy-4-hydroxyquinolines 8, 15, and 28 were
obtained by reaction of ethyl 3-oxooctadecanoate (for 8) or ethyl

6900 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Mai et al.
Scheme 1a
well as the replacement of the quinoline ring of 9 with a pyridine
(compound 16) or the deletion of the C3-carbethoxy function
(compound 17) abolished the GCN5 inhibiting activity. In
contrast, compound 18, lacking the C2-methyl substituent,
retained the GCN5 inhibitory activity, but again the replacement
of its quinoline moiety (with naphthalene, 19) or the modifica-
tion of the 3-carbethoxy group at the quinoline C3 position into
a carboxyl (20) or carboxamide (21) function yielded inactive
products.
To ascertain the existence of a direct correlation between the
activities of compounds 9 and 18 and the HAT catalytic activity
of GCN5, we tested the effects of 9 and 18 (0.6 mM) on cell
growth in the mutant F221A and E173H yeast strains, and these
effects were compared with those obtained with the respective
isogenic wild-type MK839 strain. As negative control, the
ineffective compound 16 (0.6 mM) was also tested. F221A
mutant strain contains the F221A point mutation in the HAT
minimal catalytic domain of GCN5, this causing a major defect
in cell growth.60 In the E173H mutant strain the glutamic acid
173 playing an essential role in the HAT catalytic mechanism
and function of GCN5 is replaced by a histidine residue, unable
to give the deprotonation of the histone substrate essential for
the HAT catalytic activity.61 In comparison with the effects
exerted by compounds 9 and 18 on wild-type MK839, the
reduction of the cell growth on F221A and E173H mutant strains
was lower, thus providing the evidence that the inhibition
observed with 9 and 18 is exerted on the catalytic activity of
GCN5 but not on the whole protein (Figure 6). Finally, we tested
compounds 9 and 18 on spt20∆, a S. cereVisiae mutant strain
containing null mutation in the SPT20 gene that selectively
disrupts the SAGA but not the ADA complex among the two
high-molecular-mass native HAT complexes recruiting GCN5
as catalytic subunit for acetylation of chromosomal histones.15,62
In S. cereVisiae, the mutation of SPT20 causes poor cell growth
and a set of severe transcriptional defects.63 Interestingly, the
yeast spt20∆ mutant strain was more susceptible than the wild-
type MK839 strain toward compounds 9 and 18 inhibition
(Figure 6). Compound 16 did not exert any effect on WT
MK839, F221A, E173H, and spt20∆ yeast strains.
Effect on Transcriptional Activation. The HAT activity of
GCN5 is required for transcriptional activation of specific target
genes in vivo, through nucleosomal acetylation in the gene
promoter regions.59 In yeast, GCN5 was first described as a
transcriptional coactivator of amino acid biosynthetic genes,
HIS3 being one of the most affected.47 The 3-carbethoxyquino-
line 18 and the 3-carbethoxy-2-methylpyridine 16 (as negative
control) (0.6 mM) were tested in a transcription assay using
the reporter HIS3-LacZ in comparison with compound 9 (0.6
mM), to evaluate the functional inhibition of GCN5-dependent
gene transcription. The test was performed in basal and in
activated conditions, in which yeast strain was starved for amino
acids including histidine to induce HIS3 expression, by applying
3-aminotriazole (3-AT), an amino acid analogue, to the medium.
The levels of â-galactosidase (â-Gal) activity produced were
used to measure the transcriptional activity. In this test, the
reporter activity was highly reduced in the presence of 9 and
18 both in basal (minimal SD medium; see Experimental
Section) and, more drastically, in activated (SD + 3AT)
conditions, while the treatment with 16 was ineffective (Figure
7A). To check the effects of our compounds toward global
transcription, a promoter (GAL10-CYC1) not responding to
GCN5 fused to a Lac-Z reporter was used. In this case activated
transcription was induced by addition of galactose to the
medium, and the same reporter assay was repeated for com-
a Reagents and conditions: (a) 2-methylthioacetic acid or 2-thiophen-
ecarboxylic acid; (b) N,N′-carbonyldiimidazole, triethylamine, dichlo-
romethane, room temp; (c) di-tert-butyl dicarbonate, dioxane, room temp;
(d) DIAD/PPh3, THF, room temp; (e) CF3COOH, dichloromethane, room
temp.
Scheme 2a
a Reagents and conditions: (a) MgCl2, Et3N, acetonitrile, room temp;
(b) 13% HCl, room temp; (c) thiourea, EtONa, EtOH, reflux; (d) MeI, DMF,
room temp; (e) NBS, benzoyl peroxide, carbon tetrachloride, reflux; (f)
2.5 M n-butyllithium, diethyl ether, -70 °C; (g) CO2, room temp.
Synthetic procedures for known compounds have been reported
only if different from those already published.
Results and Discussion
Yeast Phenotypic Screening. Compounds 1-21 at doses
ranging from 0.2 to 1 mM were tested on S. cereVisiae to
evaluate the effects on cell growth inhibition (Figure 4; only
the effects of compounds at 0.6 mM is reported). Because yeast
strains lacking GCN5 (gcn5∆) showed poor growth under amino
acid starvation and alteration in cell cycle phases,59 we expected
that compounds able to reduce yeast cell growth could mimic
the effects produced by the deletion of GCN5. Among the first
tested compounds, 1-14, only 9 gave a significant reduction
of cell growth.52 Tested on gcn5∆ yeast strain, compound 9
had little effect on cell growth.52 Prompted by these results, we
prepared and tested in our cell-based phenotypic screen seven
novel analogues of compound 9 (compounds 15-21) to acquire
SAR information about the inhibiting activity. Among com-
pounds 15-21, only the 3-carbethoxyquinoline 18 was able to
reduce the growth of wild-type S. cereVisiae, being less effective
in inhibiting the gcn5∆ strain growth (Figure 5). The insertion
of a hydroxyl group at the C4 position of 9 (compound 15) as

Inhibitors of Histone Acetyltransferase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6901
Scheme 3a
a Reagents and conditions: (a) NaOH, dioxane, reflux; (b) 4 N KOH, EtOH, reflux; (c) SnCl2, ZnCl2, EtOH, 80 °C; (d) 10% NaHCO3, room temp; (e)
KOH, EtOH, 80 °C.
Scheme 4a
showing that the inhibitory effect of 18 on transcription is not
fully GCN5-specific such as that observed with 9.
Yeast Acetylation Level of Histone H3. To confirm that
the acetylation reaction is the primary target for the inhibiting
activity of compounds 9 and 18, an in vivo assay was performed
by measuring the global acetylation level of histone H3
N-terminal tails in protein extracts from wild-type S. cereVisiae
cells, grown for 16 h in YPD medium (see Experimental
Section) alone and in the presence of compounds 9 and 18 used
at 0.6, 1, and 1.5 mM (Figure 8). In parallel, the assay was
performed on the gcn5∆ strain for comparison purposes. To
determine the acetylation levels, histone-specific antibodies anti-
acetyl H3 were used. In these conditions, 9 already inhibited
H3 histone tail acetylation at 0.6-1 mM, while 18 showed a
low inhibitory activity only at 1.5 mM.
a Reagents and conditions: (a) ClCOOEt, Et3N, THF, room temp; (b)
H2NOC(CH3)2OCH3, room temp; (c) Amberlyst 15, MeOH, room temp.
Cell Cycle, Apoptosis, and Cytodifferentiating Effect on
Human Leukemia U937 Cells. Compounds 9 and 18 were also
tested for their effects on cell cycle, apoptosis induction, and
granulocytic differentiation in human leukaemia U937 cell line.
After 24 h of treatment at 1 mM, compound 9 prompted arrest
of the cell cycle in the G1 phase and was able to induce 27%
of apoptosis, whereas compound 18 had very weak effects on
cell cycle and apoptosis induction at the same conditions (Figure
9A). To test differentiation, the granulocytic maturation of U937
cells was determined by CD11c expression levels upon 30 h of
stimulation with compounds 9 and 18 (both used at 1 mM). As
indicated in Figure 9B, whereas compound 9 was similar to
the control (8.8% vs 8.2%), 18 was able to induce a weak
increase in the CD11c expression levels (14% of CD11c positive
cells). Note that propidium iodide (PI) positive cells (dead cells)
have been excluded from the analysis.
Figure 4. Inhibition of yeast cell growth observed with compounds
1-21 (0.6 mM). The numbers of yeast cells were evaluated as arbitrary
units by optical density.
HAT Inhibitory Assay. To ascertain that compounds 9 and
18 were biologically active as HAT inhibitors, we performed a
HAT assay using AA,31 curcumin,33,64,65 and MB-334 (all at 50
µM) as references. As shown in Figure 10, both 9 and 18 at
500 µM were able to reduce the HAT enzymatic activity in
U937 cell nuclear extracts (32% (for 9) and 25% (for 18) of
inhibition of HAT activity). Given that 9 and 18, active at the
submillimolar level, were 1 order of magnitude less potent than
the references, other functional targets may be involved and
may explain the observed cellular effects and phenotypes. Thus,
by combining some structural features shown by 9 and 18 (i.e.,
the C3-substituted quinoline moiety) with those typical of AA31
and MB-334 (i.e., the salicylic acid or the 4-carboxy-γ-lactone
function together with the presence of a long alkyl chain), we
designed a new series of quinolines of which the 4-hydroxy-
2-pentylquinoline-3-carboxylic acid 22 can be considered a
prototype. Tested in the same assay, compound 22 showed HAT
Figure 5. (A) Effects on yeast (WT) cell growth exerted by 18 and
by the ineffective compound 16 (both at 0.6 mM). (B) Inhibitory effects
of 18 (0.6 mM) on WT and gcn5∆ yeast strains. Ten-fold serial dilutions
of the liquid yeast cell culture were spotted on solid medium.
parison. Figure 7B clearly shows that 9 and 16 were completely
ineffective in inhibiting the GCN5-independent GAL10-CYC1-
LacZ transcription in both basal and activated conditions, while
18 displayed little inhibitory effect in activated conditions, thus

6902 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Mai et al.
Figure 6. Cell growth inhibitory effects of 9, 18, and 16 on a panel of WT and mutant yeast strains. Ten-fold serial dilutions of the liquid yeast
cell culture were spotted on solid medium.
Figure 7. (A) Effects of 9, 18, and 16 (0.6 mM) on GCN5-dependent
(HIS3-LacZ) transcription. (B) Effects of the same compounds on
GCN5-independent (GAL10-CYC1-LacZ) transcription.
Figure 8. Effect of 9 and 18 on acetylation levels of yeast H3 histones.
Figure 9. (A) Cell cycle analysis and apoptosis induction for
compounds 9 and 18 in U937 cells after 24 h of stimulation. (B)
Cytodifferentiating effect of compounds 9 and 18 in U937 cells after
30 h of treatment.
inhibitory activity at 50 µM (30%) and 25 µM (24%) concentra-
tions, thereby being more potent than AA (15% of HAT
inhibiting activity at 50 µM) and curcumin (17.5% of inhibition
at 50 µM) and just a bit weaker than MB-3 (44% of inhibition
at 50 µM) in this assay (Figure 10).
These results suggest that, mainly for compound 18, the potential
activity of HAT inhibition may have non-histone molecular
targets.
Effects on Histone H3 and r-Tubulin Acetylation. The
capability of compounds 9 and 18 to reduce the acetylation
levels of H3 terminal tails as well as the non-histone substrate
R-tubulin was assessed in the human U937 leukemia cell line.
To this aim, the U937 cell line was initially treated for 2 h with
5 µM suberoylanilide hydroxamic acid (SAHA),66 a well-known
HDAC inhibitor, to increase H3 as well as R-tubulin acetylation
levels. As second step, cells were washed and incubated with
vehicle or compounds 9 and 18 (at 2.5 mM) for an additional
4 h. As shown in Figure 11A by Western blot analysis,
compound 9 was highly efficient in reduction of the H3
acetylation level whereas 18 was much less effective in the same
conditions. Tested on R-tubulin, both compounds 9 and 18 were
able to decrease the acetylation level of the non-histone
substrate, 18 being slightly more potent than 9 (Figure 11B).
The same assay was repeated with compound 22 at 100 µM,
using AA, curcumin, and MB-3 (all at 100 µM) as reference
drugs. As shown in Figure 12, the acetylation levels of R-tubulin
(lane 1) as well as histone H3 (lane 2) increased with the 2 h
pretreatment with SAHA (5 µM). After washing out and
incubation for a further 4 h with several compounds, the addition
of SAHA (5 µM) to the medium produced higher acetylation
levels of both tubulin and histone H3, whereas the incubation
with curcumin, AA, MB-3 as well as with 22 (all at 100 µM)
clearly reduced the acetylation extents of the histone (lane 2)
and the non-histone (lane 1) substrates, 22 being more efficient
than AA and slightly less active than curcumin and MB-3 with
the histone H3 as substrate. The same assay repeated without
the 2 h pretreatment with SAHA (lane 4 for histone H3 and

Inhibitors of Histone Acetyltransferase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6903
first approach, we performed a screening of various molecules
(1-14) ranging from transition-state mimics, anacardic acid
analogues, to HDAC inhibitors to evaluate the modulating effect
on the cell growth of the wild-type S. cereVisiae strain in
comparison with that of the gcn5∆ mutant. Among compounds
1-14, only compound 9 was able to significantly reduce the
yeast cell growth, thus miming the effect of GCN5 deletion
mutant. On the basis of these findings, we synthesized and tested
in a cell-based assay some analogues of 9 (derivatives 15-21)
to acquire SAR information. Among compounds 15-21, only
the 9-desmethyl derivative 18 was active in inhibiting the yeast
cell growth. Tested on the HIS3-LacZ transcription assay,
compounds 9 and 18 gave a high reduction of the GCN5-
dependent gene transcription both in basal and in activated
(amino acid starvation) conditions while the 3-carbethoxy-2-
methylpyridine 16 was ineffective. Nevertheless, when tested
on the GCN5-independent GAL-10-CYC1-LacZ reporter, 9 was
completely inactive to inhibit transcription, whereas 18 showed
a little inhibiting activity, thus indicating that activity profiles
of the two derivatives are not fully superimposable. To
determine the in vivo effects of 9 and 18 on the GCN5
acetylation activity, the acetylation of histone H3 N-terminal
tails in the presence of the two compounds was determined in
S. cereVisiae. Under steady-state conditions, the H3 acetylation
levels were highly reduced by treatment with 0.6 mM 9, while
18 was effective only at 1.5 mM.
Figure 10. HAT assay performed with 9, 18, and 22 in U937 cell
nuclear extracts. Anacardic acid (AA), curcumin, and MB-3 were used
as reference drugs. The concentrations used are indicated.
In the human leukemia U937 cell line, at 1 mM, compound
9 showed effects on cell cycle (arrest in G1 phase) and apoptosis
induction while 18 did not alter cell cycle and apoptosis.
Moreover, both compounds at higher concentrations were able
to induce apoptosis, as determined by Annexin V/PI double
staining and by caspase3 cleavage, with compound 9 being more
powerful than 18 in both cases (data not shown). When the
granulocytic cytodifferentiating effect in U937 cells was tested,
compound 18 displayed a higher number of CD11c positive PI
negative (14%) cells while 9 was ineffective. When the HAT
inhibitory activity was tested in U937 cell nuclear extracts, both
9 and 18 used at 500 µM were able to reduce the enzymatic
activity (32% (for 9) and 25% (for 18) of HAT activity
inhibition). With the aim of looking for small molecules active
at lower concentrations such as the compounds used as reference
drugs (AA, curcumin, and MB-3), we designed a new series of
quinolines with the 4-hydroxy-2-pentylquinoline-3-carboxylic
acid 22 as a prototype. In the HAT assay, compound 22 was
able to reduce the HAT activity at 50 µM (30%) and 25 µM
(24%). Finally, in human U937 cells, compound 9 (2.5 mM)
was able to reduce the acetylation level of H3 terminal tails
whereas 18 (2.5 mM) showed just a weak, if any, effect. On
the other hand, both compounds showed high hypoacetylating
properties on the non-histone substrate R-tubulin, 18 being
slightly more potent than 9. In the same assay, at the lower
concentration of 100 µM, compound 22 showed a clear
reduction of the acetylation extents of both histone H3 and
R-tubulin comparable to that obtained with curcumin, AA, and
MB-3 (all at 100 µM).
Figure 11. (A) Effects exerted by 9 and 18 (2.5 mM) on H3
acetylation. (B) Effects exerted by 9 and 18 (2.5 mM) on R-tubulin
acetylation.
Further studies on compound 22 and analogues will be
performed to further increase their HAT inhibiting properties,
substrate specificities, and modulating effects on HAT-related
phenomena.
Figure 12. Histone H3 and R-tubulin hypoacetylating effects exerted
by AA, curcumin, MB-3, and 22 (all at 100 µM).
lane 5 for tubulin) produced no observed effects (with the
exception of the SAHA-dependent hyperacetylation).
Experimental Section
Chemistry. Melting points were determined on a Buchi 530
melting point apparatus and are uncorrected. Infrared (IR) spectra
(KBr) were recorded on a Perkin-Elmer Spectrum One instrument.
1H NMR spectra were recorded at 400 MHz on a Bruker AC 400
Conclusion
Starting from a yeast phenotypic screening, we described the
discovery of two small molecules as GCN5 inhibitors. As the

6904 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Mai et al.
spectrometer; chemical shifts are reported in δ (ppm) units relative
to the internal reference tetramethylsilane (Me4Si). Electronic impact
mass spectrometry (EI-MS) was performed on a Finnigan LCQ
DECA TermoQuest (San Jose, CA) mass spectrometer. All
O), 2.37 (s, 3H, CH3-S), 3.45 (s, 2H, CH2-S), 3.51-3.55 (t, 2H,
CH2-NH), 3.98 (s, 3H, CH3-O), 4.80-4.83 (t, 1H, CH), 6.58 (s,
broad, NH), 7.50 (s, broad, NH).
Ethyl 3-Oxoheptadecanoate (25). Triethylamine (1.83 mL,
13.20 mmol) and magnesium chloride (0.98 g, 10.31 mmol) were
added to a stirred suspension of potassium ethyl 2-methylmalonate
(1.47 g, 8.66 mmol) in dry acetonitrile (28 mL), and stirring was
continued at room temperature for 2 h. Then a previously prepared
mixture of pentadecanoic acid (1.00 g, 4.12 mmol) and N,N′-
carbonyldiimidazole (0.73 g, 4.53 mmol) in dry acetonitrile (15
mL) was added, and the resulting slurry was stirred overnight. After
completion (TLC monitoring, SiO2/CHCl3), the mixture was
cautiously acidified with 13% HCl while keeping the temperature
below 25 °C, and the resulting mixture was stirred for a further 15
min. The organic layer was separated and evaporated, and the
residue was treated with ethyl acetate (20 mL). The aqueous layer
was extracted with ethyl acetate (2 × 20 mL), and the organic
phases were combined, washed with saturated sodium bicarbonate
solution (2 × 30 mL) and brine (3 × 30 mL), dried, and
concentrated to give 25 (96%) as a white solid purified by column
chromatography (SiO2/CHCl3). MS (EI, 70 eV) m/z: 312. 1H NMR
(CDCl3) δ 1.07-1.11 (t, 3H, CH3-aliphatic chain), 1.39-1.42 (m,
22H, aliphatic chain), 1.63-1.65 (m, 2H, CH2CH2CdO), 2.54-
2.58 (t, 2H, CH2CH2CdO), 3.45 (s, 2H, CH2), 4.13-4.15 (m, 2H,
1
compounds were routinely checked by TLC and H NMR. TLC
was performed on aluminum-backed silica gel plates (Merck DC,
Alufolien Kieselgel 60 F254) with spots visualized by UV light or
using a KMnO4 alkaline solution. All solvents were reagent grade
and, when necessary, were purified and dried by standard methods.
Concentration of solutions after reactions and extractions involved
the use of a rotary evaporator operating at a reduced pressure of
∼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.67 All chemicals were purchased from Aldrich
Chimica (Milan, Italy) or from Lancaster Synthesis GmbH (Milan,
Italy) and were of the highest purity.
As a rule, samples prepared for physical and biological studies
were dried in high vacuum over P2O5 for 20 h at temperatures
ranging from 25 to 110 °C, depending on the sample melting point.
Synthetic procedures for known compounds have been reported
only if different from those already published.
2-[4-(4,5-Dihydrooxazol-2-yl)phenoxy]ethylamine (23). To a
stirred solution of 2-ethanolamine (8.19 mmol, 0.50 g) in anhydrous
dioxane (30 mL) at 0 °C a solution of di-tert-butyl dicarbonate
(6.96 mmol, 0.15 g) in dioxane (10 mL) was added dropwise. The
resulting mixture was stirred at room temperature for an additional
4 h. After completion (TLC monitoring, CHCl3 on silica gel plate),
the solvent was distilled in vacuo at 40-50 °C until dry and the
residue was dissolved in water (30 mL) and extracted with
dichloromethane (3 × 20 mL). The organic layers were collected,
washed with brine (3 × 20 mL), dried overnight, then evaporated
to furnish a TLC pure colorless oily residue (4.17 mmol, 0.76 g,
60%), which was redissolved in anhydrous tetrahydrofuran (20 mL)
and mixed with 4-(4,5-dihydrooxazol-2-yl)phenol 2451 (4.17 mmol,
0.68 g) and anhydrous triphenylphosphine (4.17 mmol, 1.09 g).
To the magnetically stirred resulting mixture, a solution of
diisopropyl azodicarboxylate (4.17 mmol, 0.84 g) in anhydrous
tetrahydrofuran (10 mL) was added dropwise. The resulting mixture
was stirred at room temperature for 12 h and then filtered, and the
solvent was removed under reduced pressure. The residue was taken
up with water (50 mL) and extracted with dichloromethane (3 ×
30 mL). The organic layer was washed with 2 N KOH (2 × 20
mL) and then with brine (3 × 30 mL), dried, and evaporated.
Column chromatography on silica gel (ethyl acetate as eluent)
provided a pure colorless oil (0.70 g, 55%), which was dissolved
in dichloromethane (10 mL) and treated with trifluoroacetic acid
(3 mL). The resulting mixture was stirred at room temperature
overnight. Afterward, the solvent was removed under reduced
pressure to give TLC pure 23 as a white solid, which was further
CH2O). IR: 1725, 1700 cm-1
.
3,4-Dihydro-2-thioxo-6-tetradecylpyrimidin-4(3H)-one (6). So-
dium metal (88.3 mg, 3.84 mmol) was dissolved in 20 mL of
absolute ethanol, and thiourea (0.15 g, 1.92 mmol) and 25 (0.50 g,
1.60 mmol) were added to the clear solution. The mixture was
heated at reflux for 5 h. After the mixture was cooled, the solvent
was distilled in vacuo at 40-50 °C until dry and the residue was
dissolved in a little water (20 mL) and made acidic with 0.5 N
acetic acid. The resulting precipitate was filtered under reduced
pressure, washed with water and then with diethyl ether, and
vacuum-dried at 80 °C for 12 h to give title compound as a pure
solid, which was further purified by crystallization. MS (EI, 70
1
eV) m/z: 324. H NMR (DMSO-d6): δ 1.06-1.08 (t, 3H, CH3),
1.45-148 (m, 24H, CH2), 1.73-1.74 (d, 2H, CH2-Ar), 5.90 (s, 1H,
CH), 12.51 (d broad, 2H, NH, exchangeable with D2O).
3,4-Dihydro-2-methylthio-6-tetradecylpyrimidin-4(3H)-one (26).
A mixture of 3,4-dihydro-2-thioxo-6-tridecylpyrimidin-4(3H)-one
6 (0.40 g, 1.23 mmol), methyl iodide (0.15 mL, 2.46 mmol), and
2 mL of anhydrous N,N-dimethylformamide was stirred at room
temperature for 2 h. The reaction content was poured on cold water
(80 mL) and extracted with chloroform (3 × 30 mL). The organic
layers were collected, washed with brine (3 × 50 mL), dried
overnight, then evaporated to furnish crude 26 as a white solid,
which was purified by crystallization. MS (EI, 70 eV) m/z: 338.
1H NMR (DMSO-d6) δ 1.07-1.12 (t, 3H, CH3-aliphatic chain),
1.30-1.37 (m, 22H, aliphatic chain), 1.66-1.73 (m, 2H, CH2-
CH2Cd), 2.63-2.68 (t, 2H, CH2CH2Cd), 2.72 (s, 3H, CH3), 6.12
(s, 1H, CH), 11.02 (s, 1H, NH, exchangeable with D2O). IR: 2900,
1
purified by crystallization. MS (EI, 70 eV) m/z: 206. H NMR
(CDCl3) δ 1.62 (s, 2H, NH2), 3.62-3.66 (t, 2H, CH2NH2), 4.01-
4.15 (t, 2H, CH2N), 4.40-4.46 (m, 4H, OCH2CH2NH2 and OCH2
oxazoline ring, overlapped signals), 6.89-6.92 (d, 2H, H2,6 benzene
ring), 7.87-7.90 (d, 2H, H3,5 benzene ring).
1640 cm-1
.
5-Bromo-3,4-dihydro-2-methylthio-6-tetradecylpyrimidin-
4(3H)-one (27). To a stirred supension of 26 (0.59 mmol, 0.20 g)
and anhydrous carbon tetrachloride (50 mL) were added N-
bromosuccinimide (0.59 mmol, 0.11 g) and catalytic benzoyl
peroxide (10 mg), and the resulting mixture was refluxed for 2.5
h. After completion, the mixture was diluted with water (30 mL),
the two phases were separated, and the aqueous layer was extracted
with CHCl3 (3 × 20 mL). The organic phases were combined,
washed with saturated sodium bicarbonate solution (2 × 30 mL)
and brine (3 × 30 mL), dried, and concentrated to give TLC pure
27 as a white solid, which was further purified by crystallization.
General Procedure for the Preparation of Derivatives 1-4.
Example: Synthesis of N2-Acetyl-N6-methylthioacetyl-L-lysine
Methyl Ester (1). A mixture of NR-acetyl-L-lysine methyl ester
hydrochloride (0.42 mmol, 100.0 mg) and triethylamine (1.26 mmol,
0.18 mL) in anhydrous dichloromethane (5 mL) was treated with
a previously prepared solution of methylthioacetic acid (0.42 mmol,
36.5 µL) and N,N′-carbonyldiimidazole (0.46 mmol, 74.9 mg) in
anhydrous dichloromethane (5 mL). The resulting mixture was
magnetically stirred at room temperature until completion (TLC
monitoring, 9:1 CHCl3/MeOH on silica gel plate), then diluted to
30 mL with dichloromethane and washed with water (3 × 10 mL)
and brine (3 × 10 mL). The organic layer was dried and evaporated
under reduced pressure to give compound 1 as a pure solid, which
was further purified by crystallization. MS (EI, 70 eV) m/z: 290.
1H NMR (CDCl3) δ 1.59-1.62 (m, 2H, ø-CH2), 1.63-1.65 (m,
2H, δ-CH2), 1.78-1.81 (m, 2H, â-CH2), 2.29 (s, 3H, CH3-Cd
1
MS (EI, 70 eV) m/z: 417. H NMR (DMSO-d6) δ 1.05-1.08 (t,
3H, CH3-aliphatic chain), 1.28-1.35 (m, 22H, aliphatic chain),
1.66-1.73 (m, 2H, CH2CH2Cd), 2.66 (s, 3H, CH3), 2.73-2.78 (t,
2H, CH2CH2Cd).
3,4-Dihydro-2-methylthio-4-oxo-6-tetradecylpyrimidin-5-car-
boxylic Acid (7). To a solution of 27 (0.48 mmol, 0.20 g) in
anhydrous diethyl ether (20 mL), magnetically stirred at -70 °C,

Inhibitors of Histone Acetyltransferase
Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23 6905
a 2.5 M solution of n-BuLi in hexanes (1.2 mmol, 0.48 mL) was
added dropwise. The resulting mixture was stirred for an additional
30 min, then poured on anhydrous diethyl ether (20 mL) saturated
with dry ice, and kept stirring until warmed to room temperature.
The mixture was then poured on cold water (80 mL). The aqueous
layer was acidified with 10% HCl and extracted with ethyl acetate
(3 × 25 mL). The combined organic extracts were washed with
brine (3 × 20 mL), dried, and concentrated to give TLC pure 7,
which was further purified by crystallization. MS (EI, 70 eV) m/z:
382. 1H NMR (DMSO-d6) δ 1.06-1.10 (t, 3H, CH3-aliphatic chain),
1.39-1.49 (m, 22H, aliphatic chain), 1.83-1.87 (m, 2H, CH2-
CH2Cd), 2.72 (s, 3H, CH3), 2.80-2.86 (t, 2H, CH2CH2Cd), 11.2
(s broad, 1H, NH), 11.3 (s, 1H, COOH, exchangeable with D2O).
General Procedure for the Synthesis of Derivatives 8, 15, 28.
Example: 3-Carbethoxy-4-hydroxy-2-pentadecilquinoline (8).
To a stirred solution of isatoic anhydride (1.50 g, 9.2 mmol) and
ethyl 2-oxostearate (4.50 g, 13.8 mmol) in dry dioxane (12 mL)
was added NaOH pearl (55 mg, 1.4 mmol). The mixture was heated
at reflux for 36 h. After the completion of reaction, the cooled
mixture was poured on water (36 mL) and extracted with ethyl
acetate (3 × 40 mL). The organic layers were collected, washed
with brine (3 × 40 mL), dried, and evaporated to afford a residue,
which was purified by chromatography on silica gel column (eluent,
solution in dioxane (30 mL). The resulting mixture was then stirred
at room temperature for 1 h. After the completion of the reaction,
the solvents were removed in vacuum and the residue was poured
into water (30 mL) and extracted with ethyl acetate (3 × 30 mL).
Organic phases were combined, washed with 0.5 N HCl (3 × 30
mL) and brine (3 × 30 mL), then dried and evaporated to afford
1
21, which was purified by crystallization. H NMR (DMSO-d6) δ
7.67 (m, 1H, H C6), 7.84 (m, 1H, H C7), 8.06 (m, 2H, H C5 and H
C8), 8.32 (s, 2H, NH2), 8.84 (s, 1H, H C4), 9.31 (s, 1H, H C2).
Cell-Based Screening. Yeast Strains and Growth. All yeast
strains used in this study are listed in Table C (Supporting
Information). The yPO4 and yPO13 strains were produced by gene
disruption using a polymerase chain reaction (PCR) cassette carrying
kanMX468 or the HIS3 gene. Cells were grown at 28 °C in YPD-
rich medium (1% yeast extract, 2% bactopeptone, 2% glucose), in
SD minimal medium (0.67% yeast nitrogen base (YNB), 2%
glucose), or in SG minimal medium (0.67% YNB, 2% galactose),
the last two supplemented with 0.01% of requested amino acids.
For amino acid deprivation experiments, the yPO14 strain was
inoculated into 20 mL of SD and incubated at 28 °C for 16 h. Then
25 mM 3-aminotriazole (3-AT) was added for 4 h at 28 °C with
shaking. Cells were harvested and used for â-galactosidase
determinations. For galactose induction experiments, the yPO1569
strain was inoculated into 20 mL of SD and incubated at 28 °C for
16 h. The cells were then washed and grown in SG for 4 h at 28
°C. Microscopic observations were performed using a fluorescence
microscope (Axioskop Zeiss) equipped with oil immersion ocular
objectives at 100× and 10×.
1
3:1 ethyl acetate/hexane) to furnish 8 as a white solid. H NMR
(DMSO-d6) δ 0.81 (t, 3H, C15H3 alkyl chain), 1.22 (m, 27H,
OCH2CH3 and C(3-14)H2 alkyl chain), 1.63 (m, 2H, C2H2), 2.61
(m, 2H, C1H2 alkyl chain), 4.21 (q, 2H, OCH2CH3), 7.32 (m, 1H,
H C6 quinoline ring), 7.55 (m, 1H, H C7 quinoline ring), 7.65 (m,
1H, H C8 quinoline ring), 8.04 (m, 1H, H C5 quinoline ring), 11.78
(s, 1H, OH).
4-Hydroxy-2-pentil-3-quinolinecarboxylic Acid (22). To a
stirred solution of 3-carbethoxy-4-hydroxy-2-pentilquinoline 28
(0.44 g, 1.5 mmol) in ethanol (8 mL) was added 4 N KOH solution
(15 mL). The mixture was then heated at 70 °C for 1 week. After
the completion of reaction, the cooled mixture was poured on water
(36 mL) and extracted with ethyl acetate (3 × 20 mL). Then 2 N
HCl was added to the aqueous layer until the pH was 2. The
precipitate was filtered and recrystallized from MeOH to yield the
title compound as a TLC pure white solid. 1H NMR (DMSO-d6) δ
0.85 (t, 3H, CH3), 1.33 (m, 4H, CH2CH2CH3), 1.63 (m, 2H, CH2-
CH2CH2CH3), 3.27 (m, 2H, CH2CH2CH2CH2CH3), 7.51 (m, 1H,
H C6 quinoline ring), 7.73 (m, 1H, H C7 quinoline ring), 7.83 (m,
1H, H C8 quinoline ring), 8.20 (m, 1H, H C5 quinoline ring), 12.42
(s, 1H, OH).
Determination of â-Gal Activity. Protein extracts were prepared
from 0.8 OD600 harvested yeast cultures. Cells were washed twice
with H2O and once with extraction buffer (100 mM Tris-HCl, 1
mM DTT, 20% glycerol). Soluble extracts were prepared by
resuspending whole cells in extraction buffer and grinding them
with glass beads in a Vortex mixer. Protein concentration was
determined by the Bradford method.70 â-Gal activities were
determined as previously described.71 One unit of â-galactosidase
corresponds to 1 µmol of o-nitrophenol produced per minute.
Protein Extraction and Western Blot Analysis. Protein extrac-
tion from yeast was performed using an alkaline protocol.72
Extracted proteins were run on 15% SDS-PAGE gels and blotted
onto Hybond membranes (Amersham). Histones H3 and H3-Ac
were detected using primary histone antibodies (Upstate Biotech-
nology) and HRP-labeled IgG secondary antibody, diluted 1:10000.
Detected proteins signals were visualized using an enhanced
chemiluminescence (ECL) system.
Determination of Cell Cycle Effect, Cell Differentiation, and
Apoptosis in U937 AML Cells. Cell Culture and Ligands.
Human leukemia cell line U937 was propagated in RPMI medium
supplemented with 10% FBS (fetal bovine serum, Hyclone) and
antibiotics (100 U/mL penicillin, 100 µg/mL streptomycin, and 250
ng/mL amphotericin-B). Cells were kept at a constant concentration
of 200 000 cells per milliliter of culture medium. SAHA67 was used
at 5 µM. All compounds described were resuspended in DMSO
(Sigma-Aldrich).
Cell Cycle Analysis. The 2.5 × 105 cells were collected and
resuspended in 500 µL of an hypotonic buffer (0.1% Triton X-100,
0.1% sodium citrate, 50 µg/mL propidium iodide, RNAse A). Cells
were incubated in the dark for 30 min. Samples were acquired on
a FACS-Calibur flow cytometer using the Cell Quest software
(Becton Dickinson) and analyzed with standard procedures using
the Cell Quest software (Becton Dickinson) and the ModFit LT
version 3 software (Verity). All the experiments were performed
three times.
N-Hydroxyretinamide (12). To a 0 °C cooled solution of 11
(0.50 g, 1.67 mmol) in dry tetrahydrofuran (10 mL) were added
ethyl chloroformate (0.19 mL, 2.0 mmol) and triethylamine (0.30
mL, 2.17 mmol), and the mixture was stirred for 10 min. The solid
was filtered off, and to the filtrate was added O-(2-methoxy-2-
propyl)hydroxylamine55 (0.37 mL, 5.0 mmol). The resulting mixture
was stirred at room temperature for 1 h. Then the solvent was
evaporated under reduced pressure and the residue was diluted in
MeOH (6 mL). Amberlyst 15 ion-exchange resin (0.17 g) was added
to the solution of the O-protected hydroxamate, and the mixture
was stirred at room temperature for 1 h. Afterward, the mixture
was filtered and the filtrate was concentrated in vacuum to give a
1
residue, which was purified by crystallization. H NMR (DMSO-
d6) δ 0.99 (s, 6H, two CH3 at C6 cyclohexene ring), 1.42 (m, 2H,
CH2 position 5 cyclohexene ring), 1.54 (m, 2H, CH2 position 4
cyclohexene ring), 1.66 (s, 3H, CH3 at C7 position alkenylic chain),
1.96 (s, 3H, CH3 at C2 cyclohexene ring), 1.98 (m, 2H, CH2 position
3 cyclohexene ring), 2.25 (s, 3H, CH3 at C3 position alkenylic
chain), 5.75 (s, 1H, CH position 2 alkenylic chain), 6.19 (m, 3H,
CH positions 4, 6, 8 alkenylic chain), 6.37 (m, 1H, CH position 9
alkenylic chain), 6.99 (m, 1H, CH position 5 alkenylic chain), 10.55
(s, 1H, NHOH), 11.06 (s, 1H, NHOH).
3-Carboxamidoquinoline (21). To a 0 °C cooled solution of
20 (0.50 g, 2.9 mmol) in dry tetrahydrofuran (25 mL) were added
ethyl chloroformate (0.66 mL, 6.9 mmol) and triethylamine (1.05
mL, 7.5 mmol), and the mixture was stirred for 10 min. The solid
was filtered off, and to the filtrate was added 0.5 M ammonia
FACS Analysis of Apoptosis. Apoptosis was measured with
Annexin V/propidium iodide double-staining detection (Roche and
Sigma-Aldrich, respectively), as recommended by the suppliers.
Samples were analyzed by FACS with Cell Quest technology
(Becton Dickinson). As second assays, the caspase 3 detection (B-
Bridge) was performed and quantified by FACS (data not shown,
Becton Dickinson).

6906 Journal of Medicinal Chemistry, 2006, Vol. 49, No. 23
Mai et al.
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Granulocytic Differentiation. Granulocytic differentiation was
carried out according to Altucci . et al.73 Briefly, U937 cells were
harvested and resuspended in 10 µL of phycoerythrine-conjugated
CD11c (CD11c-PE). Control samples were incubated with 10 µL
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gels and blotted.74 Western blots were shown for pan-acetylated
histone H3 (Upstate), and total tubulin (Sigma) was used to
normalize for equal loading.
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Acknowledgment. This work was partially supported by
Grants AIRC 2005 (A.M.), PRIN 2004030405_005 (A.M.),
PRIN 2004055579_03 (L.A.), EU EPITRON Contract No.
518417 (L.A.), and Regione Campania Grant 2003 LR 5/02
(G.S.). A.N. and M.M. were supported by EU fundings. P.F.
was partially supported by RTL, CNR Project.
Supporting Information Available: Characterization and
elemental analysis data for compounds 1-4, 6-8, 12, 15, 21, 22,
24-26 and table of yeast strains used in this study. This material
is available free of charge via the Internet at http://pubs.acs.org.
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