Synthesis of a novel series of thiazole-based histone acetyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.01.022

Acetylation, which targets a broad range of histone and non-histone proteins, is a reversible mechanism and plays a critical role in eukaryotic genes activation/deactivation. Acetyltransferases are very well conserved through evolution. This allows the use of a simple model organism, such as budding yeast, for the study of their related processes and to discover specific inhibitors. Following a simple yeast-based chemogenetic approach, we have identified a novel HAT (histone acetyltransferase) inhibitor active both in vitro and in vivo. This new synthetic compound, 1-(4-(4-chlorophenyl)thiazol-2- yl)-2-(propan-2-ylidene)hydrazine, named BF1, showed substrate selectivity for histone H3 acetylation and inhibitory activity in vitro on recombinant HAT Gcn5 and p300. Finally, we tested BF1 on human cells, HeLa as control and two aggressive cancer cell lines: a neuroblastoma from neuronal tissue and glioblastoma from brain tumour. Both global acetylation of histone H3 and specific acetylation at lysine 18 (H3AcK18) were lowered by BF1 treatment. Collectively, our results show the efficacy of this novel HAT inhibitor and propose the utilization of BF1 as a new, promising tool for future pharmacological studies.

Full text of DOI:10.1016/j.bmc.2014.01.022

Bioorganic & Medicinal Chemistry 22 (2014) 1680–1689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis of a novel series of thiazole-based histone acetyltransferase
inhibitors
Daniela Secci c, Simone Carradori c, Bruna Bizzarri c, Adriana Bolasco c, Paola Ballario b, Zoi Patramani a,
Paola Fragapane a, Stefano Vernarecci b, Claudia Canzonetta b, Patrizia Filetici a,
⇑
a Institute of Molecular Biology and Pathology, CNR, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Institute Pasteur-Fondazione Cenci Bolognetti, Department of Biology and Biotechnologies C. Darwin, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
c Department of Drug Chemistry and Technologies, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
a r t i c l e i n f o
a b s t r a c t
Article history:
Acetylation, which targets a broad range of histone and non-histone proteins, is a reversible mechanism
and plays a critical role in eukaryotic genes activation/deactivation. Acetyltransferases are very well con-
served through evolution. This allows the use of a simple model organism, such as budding yeast, for the
study of their related processes and to discover specific inhibitors. Following a simple yeast-based
chemogenetic approach, we have identified a novel HAT (histone acetyltransferase) inhibitor active both
in vitro and in vivo. This new synthetic compound, 1-(4-(4-chlorophenyl)thiazol-2-yl)-2-(propan-2-yli-
dene)hydrazine, named BF1, showed substrate selectivity for histone H3 acetylation and inhibitory activ-
ity in vitro on recombinant HAT Gcn5 and p300. Finally, we tested BF1 on human cells, HeLa as control
and two aggressive cancer cell lines: a neuroblastoma from neuronal tissue and glioblastoma from brain
tumour. Both global acetylation of histone H3 and specific acetylation at lysine 18 (H3AcK18) were low-
ered by BF1 treatment. Collectively, our results show the efficacy of this novel HAT inhibitor and propose
the utilization of BF1 as a new, promising tool for future pharmacological studies.
Received 20 September 2013
Revised 20 December 2013
Accepted 16 January 2014
Available online 26 January 2014
Keywords:
Histone acetyltransferase
Inhibitor
Acetylation
Thiazole
Gcn5
p300
Ó 2014 Elsevier Ltd. All rights reserved.
1. Introduction
arrest, redifferentiation and apoptosis.3–5 Histone acetylation is
catalyzed by Lys-acetyltransferases (KATs) grouped in five classes
The genome architecture is a dynamic structure, which provides
an actively regulated platform for gene expression influenced by
different degree of compaction. Waves of chromatin remodeling al-
low transcription in response to external stimuli and environmen-
tal conditions. Centrally involved in this regulation is the
chromatin structure whose accessibility is determined by nucleo-
some positioning which is affected by post-translational modifica-
tions (PTMs) such as acetylation, methylation, phosphorylation,
ADP-ribosylation, and ubiquitination.1 Chromatin modifiers depos-
it epigenetic PTMs on histone N-terminal tails or non-histone sub-
strates and represent central pleiotropic regulators determining
cell fate and differentiation.2 Among other modifications, histone
acetylation is a positive mark associated with active transcription
that plays an important role in carcinogenesis and cancer therapy.
Histone acetylation levels are finely regulated through a balanced
competing activity of histone acetyltransferases (HATs) and his-
tone deacetylases (HDACs). Interestingly, in tumor cells, either
increasing or decreasing histone acetylation results in cell cycle
of enzymes conserved throughout evolution.6 HATs and HDACs
are centrally implicated in tumorigenesis by acting on oncogenes
or oncosuppressors2 and, accordingly, mutated forms have been
found in several types of cancer. A growing number of examples
where acetylation is involved in tumour etiology could be re-
ported. It has been established that the pro-inflammatory NF-jB
is involved in cellular transformation and it is well known the role
of p300 as its coactivator; in addition recent studies revealed that
NF-
onstrated that curcumin—an acetyltransferase inhibitor of natural
origin—is able to inhibit NF- B and therefore to suppress prolifer-
ation.7 Moreover, dysregulated Notch signaling, mainly upregu-
lated by acetylation, is frequently observed in variety of
jB is also substrate of p300-mediated acetylation. It was dem-
j
a
malignancies and consistently, HAT inhibitors are promising mole-
cules for the pharmacological inhibition of this target.8 Similarly,
increased acetylation of STAT3 is found in tumors, such as breast
cancer, therefore these observations provide a strong rationale
for targeting HAT as a potential anticancer strategy.9
Along with the variety of pathological cellular states linked to
the activity of certain members of the HAT family, we can include
the Rubinstein–Taybi syndrome that is caused by specific
⇑
Corresponding author. Tel.: +39 06 49912235.
E-mail address: patrizia.filetici@uniroma1.it (P. Filetici).
http://dx.doi.org/10.1016/j.bmc.2014.01.022
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
1681
chromosomal alterations corresponding to an area encoding for
CBP. Other fusion proteins with aberrant histone acetyltransferase
activity have been described as the main contributors to leukemo-
genesis. Moreover, increased p300 expression upon androgen star-
vation is crucial for prostate cancer cell proliferation,10 clearly
indicating the importance of targeting HATs in drug design.
Nevertheless, among the inhibitory molecules effective on chro-
matin modifiers, acetyltransferase inhibitors (HATi) described are
fewer than the HDAC inhibitors.11–13 Therefore, there is an active
ongoing effort to identify specific modulators (activator/inhibitor)
of HAT activity. Some already known active molecules are natural
compounds extracted from plants such as curcumin,14 anacardic
acid,15 garcinol and its synthetic derivatives,16 epigallocatechin-
3-gallate,17 and plumbagin;18 conversely, new and selective p300
inhibitors have been recently discovered by an extensive high-
throughput screening program (i.e., L002, C646).19
In addition, several authors have undertaken a series of studies
involving the biological validation of new synthetic HATs inhibi-
tors, with various degrees of selectivity and cell permeability.
These can be divided into two different classes: the synthetic
bisubstrate inhibitors (Lys-CoA, specific for p300, and H3-CoA-20,
specific for PCAF)20 and small molecules. Examples of this latest
class are Gcn5 inhibitor butyrolactone MB-3,21 a group of isot-
hiazolones acting on p300/PCAF with anticancer properties in the
micromolar range,22–25 and different quinoline derivatives.26,27
In a previous study,28 our lab identified the compound CPTH2
(Fig. 1A), from a series of cycloaliphatic thiazole-based derivatives,
as a modulator of the HAT Gcn5 network. Accordingly, CPTH2
inhibited acetylation of recombinant Gcn5 in vitro and in vivo
decreasing histone H3 global acetylation. Moreover, CPTH2 was
used as an epigenetic modulator of essential functions in
leukocytes.29
A
RING OPENING
PHARMACOPHORE
Cl
5
2
3
4
1
N
7
9
6
8
NH
S
N
CPTH2
11
RING MODIFICATION
12
10
RING AROMATIZATION AND COMPLICATION
N
N
O
O
S
S
S
N
H
H
H
13
14
15
16
17
18
19
20
O
O
N
N
NH
N
H
H
H
H
21
22
23
24
25
26
HOOC
O
O
Fe
27
28
30
29
Cl
B
S
O
S
i
N
ii
H2N
Br
O
N
HN
N
N
NH2
N
H
NH2
S
H
Cl
BF1-BF30
Figure 1. Lead modifications and general synthetic pattern. (A) Structural changes of the cyclopentane substituent on the hydrazonic nitrogen of the pharmacophore. (B)
General synthetic pattern of BF1–BF30 derivatives. Reagents and conditions: (i) acetic acid (cat.), EtOH; (ii) EtOH.

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D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
With the aim to identify compounds with specific HAT inhibi-
Rtt10932,33—was further characterized. The yeast methodology,
with a simple and straightforward approach, allows the identifica-
tion of synthetic molecules able to modulate key epigenetic regu-
latory hubs and to target genes involved in the functional
response of a query gene, in this case HAT Gcn5. In addition, this
assay may be employed in a variety of query deleted genes belong-
ing to the same functional subgroup that is, acetyltransferases,
methyltransferases, kinases etc. Finally, BF1 was then tested
in vitro for its activity on the catalytic Lys-acetylation reaction
and its inhibitory activity was also confirmed in vivo against two
aggressive tumour derived cell lines, glioblastoma-U87 and neuro-
blastoma-BE.
tory activity, we developed novel molecules starting from this lead
compound as shown in Figure 1. On the basis of HATs evolutionary
conservation, we choose to screen in yeast Saccharomyces cerevisiae
active compounds for their ability to inhibit growth of a strain de-
leted in GCN5. We based our screening on the genetic principle
that deletion of two genes, whose function is connected, will pro-
duce a double mutant showing a sicker phenotype with respect to
each single one. In line with this, we screened for compounds able
to block a gene whose function is connected to our query gene, in
this case GCN5 (Fig. 2). For this reason, we explored the effects of
the chemical substitutions on the hydrazonic nitrogen with respect
to the biological activity, by modifying the cyclopentane ring of the
lead compound taking advantage of molecular simplification (ring
opening, compounds BF1–BF9), cycle modification (BF10–BF12),
and molecular complication (cycle aromatization, compounds
BF13–BF30), without affecting the 4-(4-chlorophenyl)thiazol-2-
ylhydrazine pharmacophore (Fig. 1A). Here we report on the
synthesis of a large array of derivatives and the identification of
a novel active compound, 1-(4-(4-chlorophenyl)thiazol-2-yl)-2-
(propan-2-ylidene)hydrazine (BF1), derived from the lead com-
pound CPTH2. The synthesized molecules were screened in yeast
and compound BF1—which resulted active and selective on Gcn5
but not on other yeast HATs, such as Elp3,30 Sas2,31 and
2. Materials and methods
2.1. Chemistry
Starting materials and reagents were obtained from commercial
suppliers and were used without further purification. Melting
points (mp) were determined by the capillary method on an
FP62 apparatus (Mettler-Toledo) and are uncorrected. 1H NMR
spectra were recorded at 400 MHz on a Bruker spectrometer using
DMSO-d6 or CDCl3 as solvents. Chemical shifts are expressed as d
units (ppm) relative to TMS. Coupling constants J are expressed
in hertz (Hz). Elemental analyses for C, H, and N were determined
with a Perkin-Elmer 240 B microanalyzer and the analytical results
were P95% purity for all compounds. All reactions were monitored
by TLC on 0.2 mm thick silica gel plates (60 F254 Merck). Prepara-
tive flash column chromatography was carried out on silica gel
(230–400 mesh, G60 Merck). Organic solutions were dried over
anhydrous sodium sulfate. Concentration and evaporation of the
solvent after reaction or extraction was carried out on a rotary
evaporator (Büchi Rotavapor) operating at reduced pressure.
[4-(4-Chlorophenyl)thiazol-2-yl]hydrazine derivatives (BF1–
BF30) were synthesized in high yields (83–99%) as reported in
our previous studies.34 Selected carbonyl compounds reacted di-
rectly with thiosemicarbazide in ethanol with catalytic amounts
of acetic acid, and the obtained thiosemicarbazones were subse-
quently converted into (4-substituted-thiazol-2-yl)hydrazines by
reaction with a,4-dichloroacetophenone in the same solvent (Han-
tzsch reaction) (Fig. 1B). All synthesized products were purified by
washing with petroleum ether, hexane, and diethyl ether and by
column chromatography (SiO2, ethyl acetate/n-hexane) before
characterization by 1H NMR and elemental analysis.
Moreover, the presence of a C@N double bond can give rise to
isomeric geometry E/Z. In some examples the 1H NMR spectra anal-
ysis revealed that the E isomer was more favoured and stable than
the Z-configuration by comparison of peaks area integration. Our
choice, as reaction medium, of a polar alcoholic solvent appeared
to be preferred to obtain the E-configuration and limit the inter-
conversion according to the results of our previous theoretical
and chromatographic study for similar compounds.35
2.2. General procedure for the synthesis of derivatives BF1–BF30
The appropriate carbonyl compound, aldehyde or ketone, (1.0 g,
50 mmol) was dissolved/suspended in 100 mL of ethanol and stir-
red with an equimolar quantity of thiosemicarbazide for 12–24 h
at room temperature with catalytic amounts of acetic acid. Within
30 min the starting reaction mixture turned into a suspension and
the precipitated thiosemicarbazone was filtered, washed with suit-
able solvent (petroleum ether, n-hexane, diethyl ether), and dried
under vacuum. The structures of the synthesized intermediates
were confirmed by 1H NMR and TLC. Equimolar amounts of the
Figure 2. Screening in yeast S. cerevisiae. (A) Schematic representation of chemo-
genetic interaction in which small compounds can target a gene belonging to the
functional network of the query gene. The result is a synthetic sick phenotype that
resembles the phenotype produced by double deletions of interconnected genes. (B)
Yeast growth spot assay. Liquid cell cultures were serially diluted (1/5), spotted on
Petri dishes (solid medium) and grown for 48 h at 28 °C in presence of the
compounds to be tested. The panel shows wild type (WT) and GCN5 deleted
(gcn5D) strains grown in presence of the reported compounds, each dissolved in
YPD/DMSO 1%. Presence of 1% DMSO itself is not toxic as shown in the first line of
the panel.
prepared thiosemicarbazone (25 mmol) and
a,4-dichloroacetophe-
none both suspended in ethanol, were reacted at room tempera-

D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
1683
ture under magnetic stirring for 12–24 h. The suspension was fil-
tered on Gooch filter, washed with adequate solvents (diethyl
ether, petroleum ether, hexane), and dried under vacuum. All prod-
ucts have been purified by column chromatography (SiO2, ethyl
acetate/n-hexane) to give compounds BF1–BF30 in high yield.
CH2), 1.48–1.51 (m, 2H, CH2), 2.45–2.48 (m, 2H, CH2), 2.53–2.56
(m, 2H, CH2), 6.68 (s, 1H, C5H-thiazole), 7.45–7.47 (d, J = 7.9 Hz,
2H, Ar), 7.67–7.69 (d, J = 7.9 Hz, 2H, Ar), 12.41 (br s, 1H, NH, D2O
exch.). Anal. Calcd for C16H20ClN3S: C, 59.70; H, 6.26; N, 13.06.
Found C, 59.56; H, 5.96; N, 13.44.
2.3. Characterization data for new compounds
2.3.8. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(heptan-2-
ylidene)hydrazine (BF8)
2.3.1. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(propan-2-
ylidene)hydrazine (BF1)
Grey powder, 89% yield; mp 158–160 °C; 1H NMR (DMSO-d6) d
0.90–0.94 (m, 3H, CH3), 1.33–1.35 (m, 4H, 2 Â CH2), 1.57–1.60 (m,
2H, CH2), 2.10 (s, 3H, CH3), 2.35–2.39 (m, 2H, CH2), 6.60 (s, 1H, C5H-
thiazole), 7.46–7.48 (d, J = 8.0 Hz, 2H, Ar), 7.64–7.66 (d, J = 8.0 Hz,
2H, Ar), 12.48 (br s, 1H, NH, D2O exch.). Anal. Calcd for C16H20ClN3-
S: C, 59.70; H, 6.26; N, 13.06. Found C, 59.66; H, 5.99; N, 13.41.
White powder, 98% yield; mp 208–210 °C; 1H NMR (DMSO-d6) d
2.10 (s, 3H, CH3), 2.17 (s, 3H, CH3), 6.72 (s, 1H, C5H-thiazole), 7.50–
7.52 (m, 2H, Ar), 7.70–7.72 (m, 2H, Ar), 12.35 (br s, 1H, NH, D2O
exch.). Anal. Calcd for C12H12ClN3S: C, 54.23; H, 4.55; N, 15.81.
Found C, 54.39; H, 4.69; N, 15.99.
2.3.9. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(octan-2-
ylidene)hydrazine (BF9)
2.3.2. 1-(Butan-2-ylidene)-2-(4-(4-chlorophenyl)thiazol-2-
yl)hydrazine (BF2)
Grey powder, 97% yield; mp 163–165 °C; 1H NMR (DMSO-d6) d
0.89–0.91 (m, 3H, CH3), 1.25–1.29 (m, 4H, 2 Â CH2), 1.31–1.35 (m,
2H, CH2), 1.57–1.59 (m, 2H, CH2), 2.18 (s, 3H, CH3), 2.36–2.39 (m,
2H, CH2), 6.69 (s, 1H, C5H-thiazole), 7.46–7.48 (d, J = 8.0 Hz, 2H,
Ar), 7.64–7.66 (d, J = 8.0 Hz, 2H, Ar), 12.45 (br s, 1H, NH, D2O exch.).
Anal. Calcd for C17H22ClN3S: C, 60.79; H, 6.60; N, 12.51. Found C,
61.12; H, 6.99; N, 12.89.
White powder, 89% yield; mp 203–205 °C; 1H NMR (DMSO-d6) d
1.16–1.18 (m, 3H, CH3), 2.20 (s, 3H, CH3), 2.27–2.32 (m, 2H, CH2),
6.70 (s, 1H, C5H-thiazole), 7.26–7.27 (d, J = 8.1 Hz, 2H, Ar), 7.47–
7.48 (d, J = 8.1 Hz, 2H, Ar), 12.38 (br s, 1H, NH, D2O exch.). Anal.
Calcd for C13H14ClN3S: C, 55.81H, 5.04; N, 15.02. Found C, 55.50;
H, 4.83; N, 15.30.
2.3.3. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(pentan-3-
ylidene)hydrazine (BF3)
2.3.10. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(4,4-
dimethylcyclohex-2-enylidene)hydrazine (BF10)
Grey powder, 85% yield; mp 177–179 °C; 1H NMR (DMSO-d6) d
1,14–1,18 (m, 3H, CH3), 1.23–1.27 (m, 3H, CH3), 2.41–2.44 (m, 2H,
CH2), 2.54–2.57 (m, 2H, CH2), 6.70 (s, 1H, C5H-thiazole), 7.45–7.47
(d, J = 8.0 Hz, 2H, Ar), 7.66–7.68 (d, J = 8.0 Hz, 2H, Ar), 12.46 (br s,
1H, NH, D2O exch.). Anal. Calcd for C14H16ClN3S: C, 57.23; H,
5.49; N, 14.30. Found C, 57.01; H, 5.20; N, 14.59.
Light yellow powder, 97% yield; mp 230–233 °C; 1H NMR
(DMSO-d6) d 1.02 (s, 6H, 2 Â CH3), 1.58–1.59 (m, 2H, CH2), 2.50–
2.51 (m, 2H, CH2), 5.96–5.99 (m, 2H, 2 Â CH@), 7.33 (s, 1H, C5H-thi-
azole), 7.43–7.45 (d, J = 7.9 Hz, 2H, Ar), 7.84–7.86 (d, J = 7.9 Hz, 2H,
Ar), 12.41 (br s, 1H, NH, D2O exch.). Anal. Calcd for C17H18ClN3S: C,
61.53; H, 5.47; N, 12.66. Found C, 61.19; H, 5.88; N, 12.88.
2.3.4. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(pentan-2-
ylidene)hydrazine (BF4)
2.3.11. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-
cyclooctylidenehydrazine (BF11)
Grey powder, 83% yield; mp 207–209 °C; 1H NMR (DMSO-d6) d
0.95–0.98 (m, 3H, CH3), 1.60–1.64 (m, 4H, 2 Â CH2), 2.18 (s, 3H,
CH3), 7.27 (s, 1H, C5H-thiazole), 7.46–7.48 (d, J = 7.9 Hz, 2H, Ar),
7.66–7.68 (d, J = 7.9 Hz, 2H, Ar), 12.45 (br s, 1H, NH, D2O exch.).
Anal. Calcd for C14H16ClN3S: C, 57.23; H, 5.49; N, 14.30. Found C,
57.03; H, 5.17; N, 14.63.
Grey powder, 99% yield; mp 197–198 °C; 1H NMR (DMSO-d6) d
1.44–1.46 (m, 2H, CH2), 1.68–1.72 (m, 2H, CH2), 1.89–1.95 (m, 3H,
CH2), 2.01–2.06 (m, 2H, CH2), 2.46–2.51 (m, 3H, CH2), 2.67–2.77
(m, 2H, CH2), 6.69 (s, 1H, C5H-thiazole), 7.45–7.48 (m, 2H, Ar),
7.66–7.69 (m, 2H, Ar), 12.39 (br s, 1H, NH, D2O exch.). Anal. Calcd
for C17H20ClN3S: C, 61.15; H, 6.04; N, 12.59. Found C, 61.49; H,
5.88; N, 12.88.
2.3.5. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(4-methylpentan-2-
ylidene)hydrazine (BF5)
2.3.12. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-
Grey powder, 93% yield; mp 190–191 °C; 1H NMR (DMSO-d6) d
0.95–0.96 (m, 6H, 2 Â CH3), 1.98–2.00 (m, 1H, CH), 2.18 (s, 3H,
CH3), 2.24–2.26 (m, 2H, CH2), 6.69 (s, 1H, C5H-thiazole), 7.46–
7.48 (d, J = 7.8 Hz, 2H, Ar), 7.66–7.68 (d, J = 7.8 Hz, 2H, Ar), 12.39
(br s, 1H, NH, D2O exch.). Anal. Calcd for C15H18ClN3S: C, 58.52;
H, 5.89; N, 13.65. Found C, 58.76; H, 5.60; N, 13.86.
cyclohexylethylidene)hydrazine (BF12)
Grey powder, 97% yield; mp 209–211 °C; 1H NMR (DMSO-d6) d
1.30–1.35 (m, 6H, 3 Â CH2), 1.61–1.63 (m, 2H, CH2), 1.70–1.72 (m,
1H, CH), 2.13–2.16 (m, 2H, CH2), 2.18 (s, 3H, CH3), 6.69 (s, 1H, C5H-
thiazole), 7.46–7.48 (d, J = 8.1 Hz, 2H, Ar), 7.66–7.68 (d, J = 8.1 Hz,
2H, Ar), 12.27 (br s, 1H, NH, D2O exch.). Anal. Calcd for C17H20ClN3-
S: C, 61.15; H, 6.04; N, 12.59. Found C, 61.50; H, 6.32; N, 12.27.
2.3.6. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(hex-5-en-2-
ylidene)hydrazine (BF6)
2.3.13. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(furan-2-
ylmethylene)hydrazine (BF13)
Green powder, 98% yield; mp 180–181 °C; 1H NMR (DMSO-d6) d
2.19 (s, 3H, CH3), 2.40–2.44 (m, 2H, CH2), 2.50–2.55 (m, 2H, CH2),
5.08–5.12 (m, 2H, @CH2), 5.83–5.88 (m, 1H, @CH), 6.76 (s, 1H,
C5H-thiazole), 7.46–7.48 (d, J = 8.0 Hz, 2H, Ar), 7.66–7.68 (d,
J = 8.0 Hz, 2H, Ar), 12.39 (br s, 1H, NH, D2O exch.). Anal. Calcd for
C15H16ClN3S: C, 58.91; H, 5.27; N, 13.74. Found C, 59.23; H, 5.02;
N, 14.02.
Light brown powder, 98% yield; mp 225–227 °C; 1H NMR
(DMSO-d6) d 6.60–6.64 (m, 1H, furan), 6.80–6.82 (m, 1H, furan),
7.38 (s, 1H, C5H-thiazole), 7.44–7.46 (d, J = 8.0 Hz, 2H, Ar), 7.79–
7.81 (m, 1H, furan), 7.84–7.86 (d, J = 8.0 Hz, 2H, Ar), 7.91 (s, 1H,
@CH), 12,24 (br s, 1H, NH, D2O exch.). Anal. Calcd for C14H10ClN3-
OS: C, 55.35; H, 3.32; N, 13.83. Found C, 55.54; H, 3.08; N, 13.48.
2.3.7. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(heptan-3-
ylidene)hydrazine (BF7)
2.3.14. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(furan-2-
yl)ethylidene)hydrazine (BF14)
Grey powder, 86% yield; mp 171–173 °C; 1H NMR (DMSO-d6) d
0.95–0.97 (m, 3H, CH3), 1.15–1.18 (m, 3H, CH3), 1.25–1.28 (m, 2H,
Yellow powder, 93% yield; mp 238–240 °C; 1H NMR (DMSO-d6)
d 2.22 (s, 3H, CH3), 6.47–6.50 (m, 1H, furan), 6.87–6.89 (m, 1H,

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D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
furan), 7.45–7.47 (d, J = 7.9 Hz, 2H, Ar), 7.56 (s, 1H, C5H-thiazole),
7.72 (m, 1H, furan), 7.87–7.89 (d, J = 7.9 Hz, 2H, Ar), 11.26 (br s,
1H, NH, D2O exch.). Anal. Calcd for C15H12ClN3OS: C, 56.69; H,
3.81; N, 13.22. Found C, 56.31; H, 3.49; N, 13.00.
9.02 (s, 1H, pyridine), 11.65 (br s, 1H, NH, D2O exch). Anal. Calcd
for C16H13ClN4S: C, 58.44; H, 3.98; N, 17.04. Found C, 58.05; H,
4.26; N, 17.37.
2.3.22. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(pyridin-4-
ylmethylene)hydrazine (BF22)
2.3.15. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(thiophen-2-
ylmethylene)hydrazine (BF15)
Orange powder, 94% yield; mp 278–280 °C; 1H NMR (DMSO-d6)
d 7.47–7.49 (d, J = 8.0 Hz, 2H, Ar), 7.58 (s, 1H, C5H-thiazole), 7.86–
7.88 (d, J = 8.0 Hz, 2H, Ar), 8.05–8.07 (m, 2H, pyridine), 8.12 (s, 1H,
@CH), 8.79–8.81 (m, 2H, pyridine), 13.38 (br s, 1H, NH, D2O exch.).
Anal. Calcd for C15H11ClN4S: C, 57.23%; H, 3.52; N, 17.80. Found C,
57.61; H, 3.12; N, 18.03.
Grey powder, 97% yield; mp 230–231 °C; 1H NMR (DMSO-d6) d
7.11–7.12 (m, 1H, tiophene), 7.35–7.36 (m, 1H, tiophene), 7.39–
7.40 (m, 1H, tiophene), 7.44–7.46 (d, J = 8.0 Hz, 2H, Ar), 7.57 (s,
1H, C5H-thiazole), 7.84–7.86 (d, J = 8.0 Hz, 2H, Ar), 8.22 (s, 1H,
@CH), 12.44 (br s, 1H, NH, D2O exch.). Anal. Calcd for C14H10ClN3S2:
C, 52.57; H, 3.15; N, 13.14. Found C, 52.30; H, 3.40; N, 13.47.
2.3.23. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(pyridin-4-
yl)ethylidene)hydrazine (BF23)
2.3.16. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(thiophen-2-
yl)ethylidene)hydrazine (BF16)
Red powder, 86% yield; mp >280 °C; 1H NMR (DMSO-d6) d 2.38
(s, 3H, CH3), 7.48–7.50 (m, 2H, Ar), 7.56 (s, 1H, C5H-thiazole), 7.89–
7.91 (m, 2H, Ar), 8.14–8.16 (m, 2H, pyridine), 8.81–8.83 (m, 2H,
pyridine), 12.04 (br s, 1H, NH, D2O exch). Anal. Calcd for C16H13-
ClN4S: C, 58.44; H, 3.98; N, 17.04. Found C, 58.69; H, 4.19; N, 17.36.
Yellow powder, 88% yield; mp 245–247 °C; 1H NMR (DMSO-d6)
d 2.43 (s, 3H, CH3), 7.07 (s, 1H, thiophene), 7.38 (s, 1H, thiophene),
7.44–7.46 (d, J = 8.0 Hz, 2H, Ar), 7.53 (s, 1H, C5H-thiazole), 7.80–
7.82 (d, J = 8.0 Hz, 2H, Ar), 7.85–7.88 (m, 1H, thiophene), 11.36
(br s, 1H, NH, D2O exch.). Anal. Calcd for C15H12ClN3S2: C, 53.96;
H, 3.62; N, 12.59. Found C, 54.19; H, 4.00; N, 12.28.
2.3.24. 1-((1H-Indol-3-yl)methylene)-2-(4-(4-
chlorophenyl)thiazol-2-yl)hydrazine (BF24)
2.3.17. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(thiazol-2-
yl)ethylidene)hydrazine (BF17)
Grey powder, 98% yield; mp 222–224 °C; 1H NMR (DMSO-d6) d
7.18–7.21 (m, 2H, indole), 7.33 (s, 1H, C5H-thiazole), 7.43–7.46 (m,
2H, indole), 7.76 (s, 1H, @CH), 7.86–7.88 (d, J = 8.0 Hz, 2H, Ar), 8.21
(s, 1H, indole), 8.23–8.25 (d, J = 8.0 Hz, 2H, Ar), 11.50 (br s, 1H, NH-
indole, D2O exch.), 11.89 (br s, 1H, NH, D2O exch.). Anal. Calcd for
Light red powder, 86% yield; mp 230–231 °C; 1H NMR (DMSO-
d6) d 2.41 (s, 1H, CH3), 7.45–7.47 (m, 2H, Ar), 7.57 (s, 1H, C5H–thi-
azole), 7.68–7.70 (m, 2H, Ar), 7.73–7.75 (m, 1H, thiazole), 7.89 (m,
1H, thiazole), 11.33 (br s, 1H, NH, D2O exch.). Anal. Calcd for C14-
H11ClN4S2: C, 50.22; H, 3.31; N, 16.73. Found C, 50.55; H, 3.02; N,
16.39.
C18H13ClN4S: C, 61.27; H, 3.71; N, 15.88. Found C, 61.59; H, 3.45; N,
16.09.
2.3.25. 1-(Benzodioxol-5-ylmethylene)-2-(4-(4-
2.3.18. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-
phenylethylidene)hydrazine (BF18)
chlorophenyl)thiazol-2-yl)hydrazine (BF25)
Yellow powder, 83% yield; mp 240–242 °C; 1H NMR (DMSO-d6)
d 6.06 (s, 2H, CH2, benzodioxole), 6.97–6.98 (m, 1H, benzodioxole),
7.21–7.22 (m, 1H, benzodioxole), 7.37 (s, 1H, C5H-thiazole), 7.44 (s,
1H, @CH), 7.45–7.47 (d, J = 7.8 Hz, 2H, Ar), 7.85–7.87 (d, J = 7.8 Hz,
2H, Ar), 7.94–7.95 (m, 1H, benzodioxole), 12.65 (br s, 1H, NH, D2O
exch.). Anal. Calcd for C17H12ClN3O2S: C, 57.06; H, 3.38; N, 11.74.
Found C, 57.39; H, 3.65; N, 11.50.
Light brown powder, 97% yield; mp 173–175 °C; 1H NMR
(DMSO-d6) d 2.34 (s, 3H, CH3), 7.40–7.42 (m, 2H, Ar), 7.44–7.46
(d, J = 7.9 Hz, 2H, Ar), 7.53–7.55 (m, 2H, Ar), 7.58 (s, 1H, C5H-thia-
zole), 7.63–7.65 (m, 1H, Ar), 7.87–7.89 (d, J = 7.9 Hz, 2H, Ar), 11.35
(br s, 1H, NH, D2O exch.). Anal. Calcd for C17H14ClN3S: C, 62.28; H,
4.30; N, 12.82. Found C, 62.01; H, 4.56; N, 13.07.
2.3.19. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(pyridin-2-
yl)ethylidene)hydrazine (BF19)
2.3.26. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(naphthalen-1-
ylmethylene)hydrazine (BF26)
Dark yellow powder, 83% yield; mp 214–216 °C; 1H NMR
(DMSO-d6) d 2.40 (s, 3H, CH3), 7.46 (s, 1H, C5H-thiazole), 7.48–
7.50 (m, 2H, Ar), 7.75–7.76 (m, 1H, pyridine), 7.86–7.88 (m, 2H,
Ar), 8.10–8.12 (m, 1H, pyridine), 8.17–8.19 (m, 1H, pyridine),
8.89–8.91 (m, 1H, pyridine), 11.69 (br s, 1H, NH, D2O exch). Anal.
Calcd for C16H13ClN4S: C, 58.44; H, 3.98; N, 17.04. Found C,
58.10; H, 4.29; N, 17.31.
Light red powder, 97% yield; mp 235–236 °C; 1H NMR (DMSO-
d6) d 7.43 (s, 1H, C5H-thiazole), 7.46–7.48 (m, 2H, Ar), 7.59–7.61
(m, 3H, naphthalene), 7.86–7.89 (m, 2H, Ar), 7.90–7.91 (m, 1H,
naphthalene), 7.97–8.01 (m, 2H, naphthalene), 8.67 (s, 1H, @CH),
8.74–8.76 (m, 1H, naphthalene), 12.12 (br s, 1H, NH, D2O exch.).
Anal. Calcd for C20H14ClN3S: C, 66.02; H, 3.88; N, 11.55. Found C,
66.38; H, 3.52; N, 11.79.
2.3.20. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(pyridin-3-
ylmethylene)hydrazine (BF20)
2.3.27. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(naphthalen-2-
yl)ethylidene)hydrazine (BF27)
Yellow powder, 96% yield; mp 245–249 °C; 1H NMR (DMSO-d6)
d 7.46–7.48 (m, 2H, Ar), 7.49 (s, 1H, C5H-thiazole), 7.86–7.88 (m,
2H, Ar), 7.90 (s, 1H, @CH), 8.13–8.15 (m, 1H, pyridine), 8.50–8.56
(m, 1H, pyridine), 8.60–8.63 (m, 1H, pyridine), 9.00 (s, 1H, pyri-
dine), 12.59 (br s, 1H, NH, D2O exch.). Anal. Calcd for C15H11ClN4S:
C, 57.23; H, 3.52; N, 17.80. Found C, 57.54; H, 3.09; N, 17.49.
Light yellow powder, 98% yield; mp 235–237 °C; 1H NMR
(DMSO-d6) d 2.45 (s, 3H, CH3), 7.47 (s, 1H, C5H-thiazole), 7.47–
7.49 (m, 2H, Ar), 7.53–7.56 (m, 2H, naphthalene), 7.88–7.90 (m,
2H, Ar) 7.91–7.95 (m, 3H, naphthalene), 8.17–8.19 (m, 1H, naph-
thalene), 8.29–8.34 (m, 1H, naphthalene), 11.27 (br s, 1H, NH,
D2O exch.). Anal. Calcd for C21H16ClN3S: C, 66.75%; H, 4.27%; N,
11.12%. Found C, 66.31; H, 4.59; N, 11.39.
2.3.21. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(pyridin-3-
yl)ethylidene)hydrazine (BF21)
2.3.28. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(2H-chromen-2-
oxo-3-yl)ethylidene)hydrazine (BF28)
Yellow powder, 85% yield; mp 235–236 °C; 1H NMR (DMSO-d6)
d 2.38 (s, 3H, CH3), 7.46 (s, 1H, C5H-thiazole), 7.48–7.50 (d, J = 8.7,
2H, Ar), 7.77–7.79 (m, 1H, pyridine), 7.88–7.90 (d, J = 8.7 Hz, 2H,
Ar), 8.51–8.53 (m, 1H, pyridine), 8.70–8.71 (m, 1H, pyridine),
Dark yellow powder, 98% yield; mp 243–244 °C; 1H NMR
(DMSO-d6) d 2.26 (s, 3H, CH3), 7.42–7.45 (m, 3H, chromene),
7.46–7.48 (m, 2H, Ar), 7.53 (s, 1H, C5H-thiazole), 7.82–7.85 (m,

D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
1685
1H, chromene), 7.87–7.89 (m, 2H, Ar), 8.16 (m, 1H, chromene),
11.75 (br s, 1H, NH, D2O exch.). Anal. Calcd for C20H14ClN3O2S: C,
60.68; H, 3.56; N, 10.61. Found C, 60.99; H, 3.78; N, 10.98.
main (80 ng, p300, Gcn5 or PCAF), 0.5 mM Acetyl-CoA and histone
H3 (50 ng) were mixed and incubated at room temperature for
30 min, Developer and STOP solution were finally added. The assay
is based on the response of a thiol-reactive fluorescent dye to the
Co-A-SH generated by the histone acetyltransferase activity to give
a fluorescent read-out.
2.3.29. 9-(2-(4-(4-Chlorophenyl)thiazol-2-yl)hydrazono)-9H-
fluorene-2-carboxylic acid (BF29)
Yellow powder, 93% yield; mp 190–191 °C; 1H NMR (DMSO-d6)
d 7.33 (s, 1H, C5H-thiazole), 7.36–7.41 (m, 3H, fluorene), 7.48–7.50
(m, 2H, Ar), 7.74–7.79 (m, 2H, fluorene), 7.87–7.89 (m, 2H, Ar),
8.06–8.10 (m, 1H, fluorene), 8.33–8.38 (m, 1H, fluorene), 11.03 (s,
1H, NH, D2O exch), 12.45 (br s, 1H, COOH, D2O exch). Anal. Calcd
for C23H14ClN3O2S: C, 63.96; H, 3.27; N, 9.73. Found C, 63.70; H,
3.50; N, 9.99.
2.7. Assessment of cell viability
BF1 was added at a final concentration of 50 and 100 lM, cells
were collected and analyzed after incubation of 48/72 h at 37 °C.
Cell viability of HeLa, U87 glioblastoma and BE-SK-N-BE(2) from
ATCC (CRL2271) neuroblastoma cell lines was evaluated by trypan
blue exclusion assay with respect to untreated controls.
2.3.30. 1-(4-(4-Chlorophenyl)thiazol-2-yl)-2-(1-(ferrocen-2-
yl)ethylidene)hydrazine (BF30)
3. Results
Brown powder, 89% yield; mp 220–221 °C; 1H NMR (DMSO-d6)
d 2.21 (s, 3H, CH3), 4.17–4.21 (m, 5H, ferrocene), 4.38–4.40 (m, 2H,
ferrocene), 4.62–4.65 (m, 2H, ferrocene), 7.33 (s, 1H, C5H-thiazole),
7.44–7.46 (d, J = 8.0 Hz, 2H, Ar), 7.85–7.87 (d, J = 8.0 Hz, 2H, Ar),
11.08 (br s, 1H, NH, D2O exch). Anal. Calcd for C21H20ClFeN3S: C,
57.62; H, 4.60; N, 9.60. Found C, 57.99; H, 4.84; N, 9.98.
3.1. Yeast based screening for selective HAT inhibitors
In this study, the lead compound CPTH2 was used as the start-
ing point for the targeted design and synthesis of a novel set of thi-
azole derivatives shown in Figure 1. In budding yeast, a strain
carrying the deletion of two genes, which interact at the functional
level, results in a sick phenotype and poor growth on plate. Simi-
larly, a chemical compound may block the product of a gene that
interacts with the query gene resulting in a sick phenotype
2.4. Yeast strains and growth
All yeast strains used in this study are listed in Table 1. The
gcn5D
, elp3
D
, and sas2D strains were produced in wild type
(Fig. 2A). Overall, deletion of a non-essential query gene (D), that
S288c, whereas rtt109
D
in BY4742 genetic backgrounds. Yeast cells
works together with the drug-target, causes hypersensitivity to
sublethal drug concentrations and decreased growth. Therefore,
compounds of BF series were tested in a yeast growth assay on
wild type (WT) and an isogenic strain deleted in the HAT GCN5
were grown at 28 °C in YPD medium (1% yeast extract, 2% bacto-
peptone, 2% glucose) liquid or solid (plus 2% agar). YPD was supple-
mented with solvent DMSO (1%). To test the sensitivity of the
derivatives, 1 OD600/mL of exponentially growing yeast cells were
serially diluted (1/5) and spotted on solid medium containing each
compound. All compounds were dissolved in DMSO and added to
the medium at the indicated concentrations.
(gcn5
of the gcn5
(S288c) and gcn5
D
) in order to identify molecules able to decrease the growth
strain on plate. Liquid cell cultures of wild type
strains were serially diluted, spotted on agar so-
D
D
lid medium (YPD–DMSO 1%), and grown for 48 hours at 28 °C. The
most active compounds BF1, BF2, and BF3 (0.2 mM), identified in a
previous screening, markedly reduced the growth of the gcn5
2.5. Histone bulk preparations and analysis
D
strain compared to CPTH2 (0.5 mM) and BF19 (1 mM) chosen as
an example of less active thiazole-based derivatives (Fig. 2B). The
selectivity of compounds was then tested on other yeast strains de-
Yeast and human cell lines were lysed and total protein extracts
were resuspended in 2Â Laemli Buffer, boiled 10 min, loaded on
15% SDS–PAGE and blotted onto Hybond-C extra membranes
(Amersham). Histones were detected with primary antibodies anti
Ac-H3 (Millipore-06599), Ac-H4 (Millipore-06866), H3-AcK18 (Ab-
cam-AB1191) and unmodified H3 (Millipore-06755). Signals were
normalized with anti-Ada2 (Santa-Cruz SC6651) and anti-GAPDH
leted in different HATs such as Elp3 (elp3D), Sas2 (sas2D), and
Rtt109 (rtt109 ). The strains were spotted on plate and their
D
growth was compared with the respective isogenic wild type:
S288c and BY4742 (Fig. 3).
Previously characterized HAT inhibitors CPTH2 (0.5 mM) and
curcumin (0.75 mM) were used as reference inhibitors (Fig. 3A).
(Glyceraldehyde
3-phosphate
dehydrogenase,
Santa-Cruz-
SC32233). Proteins were detected by Enhanced Chemio-Lumines-
cence (ECL) (Amersham) and visualized by film and ChemiDoc™
MP Imaging System (Biorad). Signals were quantified by Optiquant
analysis software.
Results show that CPTH2 and curcumin inhibited gcn5
growth in both genetic backgrounds; they were instead ineffective
on elp3 , sas2 , and rtt109 strains. The assay carried out with the
synthesized derivatives demonstrated that among BF1, BF2 and
BF3, BF1 was the most selective compound, able to inhibit almost
D strain
D
D
D
2.6. In vitro HAT assay
exclusively the growth of gcn5D strain (Fig. 3B). Therefore, BF1 was
tested at different concentrations in budding yeast and it showed a
dose-dependent inhibition with a significative effect at 0.1 mM
(Fig. 3C). On the basis of these results, we focused on BF1 com-
pound for further analysis.
In vitro HAT assay was performed using ‘HAT Assay Kit’ (Active
Motif). Briefly, HAT Buffer, specific recombinant HAT catalytic do-
Table 1
S. cerevisiae strains used in this work28
3.2. BF1 lowers global histone H3 acetylation in vivo and in vitro
Strain
Genotype
S288c
MAT alpha, SUC2, gal2, mal, mel, flo1 flo8-1, hap1
S288c background. Gcn5::KanMX
S288c background. Sas2::KanMX
S288c background. Elp3::KanMX
MATa, his3-D1, leu2D, met15D, ura3D
We tested the ability of BF1 to decrease global histone acetyla-
tion in the cell. Total protein extracts were analyzed and the global
acetylation of histone H3 and H4 was tested by immunoblot
(Fig. 4). Yeast cells treated with BF1 (0.6 and 0.8 mM) and CPTH2
(0.8 mM), used as a control, were lysed and total extracts fraction-
ated on PAGE.
gcn5
sas2
elp3
BY4742
gcn5
rtt109
D
D
D
D
BY4742 background, Gcn5::KanMX
BY4742 background, Rtt109::KanMX
D

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D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
Figure 3. Growth inhibition tested on different HAT deleted strains. (A) Yeast growth assay was carried out on WT (S288c background) and isogenic strains deleted in HATs
GCN5 (gcn5 ), ELP3 (elp3 ), SAS2 (sas2 ), and on WT (BY4742 background) and isogenic GCN5 deleted (gcn5 ) and RTT109 deleted (rtt109 ) strains. Cells were serially
diluted and spotted on YPD–DMSO 1%, CPTH2 (0.5 mM) and curcumin (0.75 mM) used as reference inhibitors. (B) Compounds BF1, BF2, and BF3 (0.2 mM) tested on indicated
HAT deleted strains. (C) Gcn5 selective inhibitor BF1 tested at increasing concentrations (0.05, 0.1 and 0.2 mM) showed to preferentially inhibit the gcn5 strain.
D
D
D
D
D
D
Immunoblot was probed for acetylated histone H3 (Ac-H3) and
H4 (Ac-H4), unmodified histone H3 (H3) and constitutively ex-
pressed ADA2 protein (Fig. 4A). Results were quantified, normal-
ized and levels of histone AcH3 and AcH4 with respect to
untreated controls (C) were reported (Fig. 4B). BF1 treated cells
showed a 50% decrease in histone H3 acetylation, whereas the level
of histone H4 acetylation was not affected, indicating histone H3
substrate selectivity. Based on these results, we tested if BF1 was
also able to inhibit the catalytic HAT reaction: this was evaluated
in order to assess the specificity of BF1 and avoid more indirect ef-
fects. Fluorescent HAT in vitro assay was used and human recom-
binant p300, Gcn5 and PCAF were tested on histone H3. Untreated
samples were taken as control (C) and HATs inhibition was mea-
sured by determination of residual enzyme activity in presence
of the inhibitor. PCAF was tested but its catalytic activity was not
inhibited by BF1 treatment (data not shown). The results were dia-
grammed in Figure 5. The reaction catalyzed by Gcn5 (Fig. 5A) was
no significatively reduced, while that catalyzed by p300 (Fig. 5B)
was almost abolished by BF1 (0.6 mM) with respect to untreated
control and is fully comparable to anacardic acid, used as reference
inhibitor. Overall, the measured inhibitory activity revealed that
BF1 not only had effect on functional interacting partners of
Gcn5, but showed a preferential effect on p300 in vitro. These re-
sults suggest that the yeast approach may represent a preliminary
convenient step to screen a large number of compounds, although
further in vitro inhibitory activity characterization of the most ac-
tive derivatives is required.
3.3. BF1 affects histone H3 acetylation in human cancer cell
lines
Next we wanted to evaluate the effect of BF1 on human cell
lines. HeLa cells were used as a widely accepted control line.
Two different cell lines derived from aggressive tumours: U87,

D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
1687
Figure 4. BF1 inhibits acetylation of histone H3 at global level. (A) Acetylation of
bulk histones H3 and H4 was investigated by Western blot. Wild type yeast cells
(S288c) were grown in rich medium YPD–DMSO 1% (C), supplemented with BF1
(0.6 and 0.8 mM) or with lead compound CPTH2 (0.8 mM). Total proteins extracts
were run on PAGE and hybridized with antibody against panacetylated histones
(Ac-H3 and Ac-H4), unmodified histone H3 and constitutively expressed ADA2
protein, both used as internal loading standards. (B) Histograms report global
histone H3 (black bar) and H4 (grey bar) acetylation percentage observed in
absence (C) or in presence of BF1 (0.6 and 0.8 mM) and CPTH2 (0.8 mM). Each value
was normalized to the internal amount of constitutively expressed ADA2. The error
bars represent the standard deviation from experiments performed in triplicate.
Figure 5. BF1 inhibition of in vitro histone H3 acetylation by recombinant Gcn5 and
p300. (A) Recombinant Gcn5 was assayed in the presence of acetyl-CoA and histone
H3 substrate. Reaction was performed without inhibitor (C), in the presence of
anacardic acid, AA (15 lM) as reference inhibitor and at increasing BF1 concentra-
tions (0.15, 0.3 and 0.6 mM). (B) Recombinant p300 was tested in the same
conditions of Gcn5. Each reaction was repeated three times. Standard errors are
shown and p = 0.0003.
glioblastoma and BE, neuroblastoma were tested in parallel. Cells
were incubated for 24 (not shown), 48 and 72 h in presence of
BF1 at two different concentrations, 50 and 100 lM. BF1 concen-
neuroblastoma. The cell viability was not much affected during
BF1 treatment indicating a low toxicity of BF1 on human cells.
trations used on cell cultures were sensibly lower than in yeast
where the cell wall requires higher concentrations of compounds
to obtain an efficient cellular uptake. The decrease of both, bulk
acetylation of histone H3 (Ac-H3) and acetylation of lysine K18
(H3-AcK18), which has been indicated as a promising prognostic
mark for a number of tumours,36 was evaluated. Cells were lysed
and total protein extracts analyzed. Figure 6 (Panel A) shows wes-
tern blot analysis of total protein extracts of respectively HeLa, U87
glioblastoma, and BE neuroblastoma cell lines. Levels of respec-
tively, panacetylated Ac-H3 and H3-AcK18 were normalized to
GAPDH, used as uniformly expressed protein internal loading con-
trol. Collected results are diagrammed in the histograms of Figure 6
(Panels B and C). Although the drug response is variable, all the
tested cell lines showed a concentration-dependent decrease of
4. Discussion
A major challenge in drug discovery is to identify the proteins
and cellular pathways affected by a new drug. On the basis of
the biological activity recorded for a large series of derivatives of
previously described HAT inhibitor CPTH2,28 we can describe the
general features of the structural requirements that lead to a novel
group of HAT inhibitors named BF. Our analysis indicates that the
4-chlorophenyl moiety in the C4 position of the thiazole nucleus
seems to be preferred. Furthermore, chemical changes of the
hydrazonic nitrogen in C2 suggested that both, the cyclopentane
ring modification (compounds BF10–BF12) and/or the aromatiza-
tion/complication (compounds BF13–BF30) reduce the biological
activity (i.e., BF19 in Fig. 2B). On the opposite, better results were
obtained by ring opening and chain shortening, thus correlating
the inhibitory strength to a limited steric hindrance in this portion
of the pharmacophore (compounds BF1–BF9). The best data were
registered with the smallest derivatives of the series (BF1–BF3),
where the cyclopentane skeleton of the lead compound CPTH2
was simplified. Our collected results indicate that BF1 represents
a novel HAT inhibitor and, by means of yeast based genetic assay,
this test compound is shown to interfere with a functional counter-
part of Gcn5. In fact, similarly to the double deletion genetically
obtained of two interacting genes, BF1 is able to affect one compo-
nent required by Gcn5 at functional level.
acetylation and after 72 h incubation with BF1 at 100 lM, both
AcH3 and H3-AcK18 were drastically and almost completely re-
duced. However, in this figure, 72 h treatment was not statistically
analyzed in detail because of unwanted side effects due to pro-
longed drug treatment. Noteworthy, BF1 inhibited histone H3 acet-
ylation without affecting the expression of constitutive GAPDH.
Having corroborated the inhibitory effect of BF1 on histone H3
acetylation, we evaluated the viability of cells exposed to the same
BF1 treatment (Fig. 6 Panel D). As shown, cell viability in HeLa and
glioblastoma U87 cell lines was only slightly affected by BF1
administration. In neuroblastoma BE cells, a decrease (ꢀ30%) in
cell viability, compared to the control, was observed after 72 h
treatment (BF1 at 100 lM). Collectively, these results show
that BF1 is able to inhibit histone H3 acetylation in human cell
lines and, in particular, in derived tumours glioblastoma and
This pre-screening offers a simple approach and represents a
convenient tool for the biological evaluation of new compounds.37

1688
D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
Figure 6. BF1 is a p300/Gcn5 inhibitor and reduces acetylation of histone H3 and H3K18 in human cell lines. Panel (A) Western blot analysis of histone H3, panacetylated
(AcH3), acetylated at Lys-18 (H3AcK18) and GAPDH as internal loading control. HeLa, U87-glioblastoma, and BE-neuroblastoma cell lines were grown for 48 and 72 h without
(C) or in presence of BF1 (50 and 100
lM). Histograms show the percentage of AcH3 (Panel B) and H3AcK18 (Panel C) normalized to the constitutively expressed GAPDH in
HeLa (dark grey bar), U87 (white bar), and BE (light grey bar) cells. Each experiment represents the average of three different biological replicates, unless otherwise stated.
Standard errors are shown and asterisks are as follows: ⁄⁄p <0.001 and ⁄p <0.05. (Panel D) viability of indicated cell lines treated with BF1 (50 and 100
lM) for 48 and 72 h. On
the average, 1.5 Â 105 cells were counted for each point. Viability was expressed as the mean percentage of viable/dead cells with respect to the untreated control.
On the basis of highly conserved HAT catalytic domain in evolu-
tion, the identification of an inhibitory activity selective for GCN5
established in yeast may further justify a similar biological effect
on other HATs such as p300 working in human cells. In addition,
it was recently reported that a single inhibitor may show activity
towards multiple HATs such as p300, CBP and GCN5.19b Similarly,
CPTH2, the lead compound of BF1, firstly identified in yeast,28
was successfully employed as potent inhibitor of GCN5 and PCAF,
confirming its role on epigenetic regulation.29
Here we report the identification of a biologically active mole-
cule, BF1, that was further validated in vivo, in yeast and human
cell cultures, and in vitro.
A selective inhibition of histone H3 acetylation as well as an
increased level of AcH4 were observed by BF1 treatment: this re-
sult fits to a previous report showing that bulk hyperacetylation
of histone H4 is simultaneously found with H3 hypoacetylation.38
BF1 inhibitory activity was next tested by in vitro HAT assay
(with Gcn5, p300 and PCAF): this experiment was performed to
rule out an indirect effect of this proposed lead compound. BF1
showed to be able to lower the catalytic activity of recombinant
Gcn5 and mainly p300, thus providing direct evidence for its effi-
cacy on the acetylation reaction itself. In vitro, the inhibition on
p300 was higher than the inhibition on Gcn5, whereas no effect
on PCAF was observed, highlighting the selective inhibition of
BF1 toward different HATs. The higher activity found on p300 is
not surprising since we used the yeast approach only to narrow
the search for active compounds. In budding yeast p300 is not
present; therefore it is likely that BF1 may show a higher selectiv-
ity for p300. To gain insights on BF1 efficacy on human cell lines,
we decided to analyze the histone H3 acetylation in two aggres-
sive tumour derived cell lines, glioblastoma-U87 and neuroblas-
toma-BE. HeLa was used as a bona fide reference cell line, not
derived from brain. We measured the acetylation status of both
global histone H3 and its specific lysine 18 (H3AcK18) because
it was demonstrated to be a valuable prognostic indicator in sev-
eral human cancers.36 BF1 showed to be active on human cell
lines and after 72 h of treatment, the global level of AcH3 and
H3AcK18 was drastically reduced.

D. Secci et al. / Bioorg. Med. Chem. 22 (2014) 1680–1689
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Collectively, our results describe the synthesis, identification,
and characterization of a novel HAT inhibitor, BF1, which was
shown in vitro to be mainly active on p300. Furthermore, BF1
proved to lower in vivo the global acetylation of histone H3
(AcH3 and H3AcK18) in yeast and in human cell lines. Moreover,
it is known that acetylation targets not only histones but also
non-histone factors, such as transcription activators. Accordingly,
the induction of oncogenes or aberrant gene activation like in car-
diomyocyte hypertrophy39 or on HIV replication cycle40 may be
profitably investigated with selective HAT inhibitors, which are
far less studied with respect to the widely described HDACs inhib-
itors. For all these reasons, we believe that the identification and
characterization of novel HAT inhibitors will open the way to novel
applications in basic research and in therapy.
Acknowledgments
This work was supported by PRIN 2009 (Prot. 200973ST5Y_
002), FIRB; Fondazione Cenci-Bolognetti, and Filas (ASR2, Regione
Lazio, Italy). We thank contribution and technical skill of M.
Marchioni, IBPM-CNR, for experiments on human cell cultures.
26. Mai, A.; Rotili, D.; Tarantino, D.; Nebbioso, A.; Castellano, S.; Sbardella, G.; Tini,
M.; Altucci, L. Bioorg. Med. Chem. Lett. 2009, 19, 1132.
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G.; Nebbioso, A.; Miceli, M.; Altucci, L.; Filetici, P. J. Med. Chem. 2006, 49, 6897.
28. Chimenti, F.; Bizzarri, B.; Maccioni, E.; Secci, D.; Bolasco, A.; Chimenti, P.;
Fioravanti, R.; Granese, A.; Carradori, S.; Tosi, F.; Ballario, P.; Vernarecci, S.;
Filetici, P. J. Med. Chem. 2009, 52, 530.
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