Evaluation of a large library of (thiazol-2-yl)hydrazones and analogues as histone acetyltransferase inhibitors: Enzyme and cellular studies

Article abstract of DOI:10.1016/j.ejmech.2014.04.042

Recently we described some (thiazol-2-yl)hydrazones as antiprotozoal, antifungal and anti-MAO agents as well as Gcn5 HAT inhibitors. Among these last compounds, CPTH2 and CPTH6 showed HAT inhibition in cells and broad anticancer properties. With the aim to identify HAT inhibitors more potent than the two prototypes, we synthesized several new (thiazol-2-yl)hydrazones including some related thiazolidines and pyrimidin-4(3H)-ones, and we tested the whole library existing in our lab against human p300 and PCAF HAT enzymes. Some compounds (1x, 1c', 1d', 1i' and 2m) were more efficient than CPTH2 and CPTH6 in inhibiting the p300 HAT enzyme. When tested in human leukemia U937 and colon carcinoma HCT116 cells (100 μM, 30 h), 1x, 1i' and 2m gave higher (U937 cells) or similar (HCT116 cells) apoptosis than CPTH6, and were more potent than CPTH6 in inducing cytodifferentiation (U937 cells).

Full text of DOI:10.1016/j.ejmech.2014.04.042

European Journal of Medicinal Chemistry 80 (2014) 569e578
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Evaluation of a large library of (thiazol-2-yl)hydrazones and analogues
as histone acetyltransferase inhibitors: Enzyme and cellular studies
,
,
Simone Carradori a 1, Dante Rotili a 1, Celeste De Monte a, Alessia Lenoci a,
Melissa D’Ascenzio a, Veronica Rodriguez a, Patrizia Filetici b, Marco Miceli c,
Angela Nebbioso c, Lucia Altucci c d, Daniela Secci a , Antonello Mai
,
,
a,e,
*
*
a Department of Drug Chemistry and Technologies, Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy
b Institute of Molecular Biology and Pathology (IBPM), CNR National Research Council of Italy, c/o Sapienza Università di Roma, 00185 Rome, Italy
c Department of Biochemistry, Biophysics and General Pathology, Second University of Naples, vico L. De Crecchio 7, 80138 Naples, Italy
d CNR-IGB, Institute of Genetics and Biophysics, via P. Castellino, 80100 Naples, Italy
e Pasteur Institute, Cenci Bolognetti Foundation, 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:
Recently we described some (thiazol-2-yl)hydrazones as antiprotozoal, antifungal and anti-MAO agents
as well as Gcn5 HAT inhibitors. Among these last compounds, CPTH2 and CPTH6 showed HAT inhibition
in cells and broad anticancer properties. With the aim to identify HAT inhibitors more potent than the
two prototypes, we synthesized several new (thiazol-2-yl)hydrazones including some related thiazoli-
dines and pyrimidin-4(3H)-ones, and we tested the whole library existing in our lab against human p300
and PCAF HAT enzymes. Some compounds (1x, 1c’, 1d’, 1i’ and 2m) were more efficient than CPTH2 and
CPTH6 in inhibiting the p300 HAT enzyme. When tested in human leukemia U937 and colon carcinoma
Received 3 February 2014
Received in revised form
7 April 2014
Accepted 12 April 2014
Available online 15 April 2014
Keywords:
Apoptosis
Epigenetics
HAT inhibitor
Hydrazone
Thiazole
HCT116 cells (100 mM, 30 h), 1x, 1i’ and 2m gave higher (U937 cells) or similar (HCT116 cells) apoptosis
than CPTH6, and were more potent than CPTH6 in inducing cytodifferentiation (U937 cells).
Ó 2014 Elsevier Masson SAS. All rights reserved.
1. Introduction
functions [3]. Nuclear histone lysine acetyltransferases (type A
HATs or KATs) are grouped into five distinct evolutionary conserved
Nε-Lysine acetylation was the first discovered post-translational
modification (PTM) of canonical and variant histones, and it is
considered a direct regulator of chromatin structure and function.
Histone acetyltransferases (HATs) and histone deacetylases
(HDACs) are the enzymes involved in this equilibrium, acting as
concerted gene repressors or activators [1]. Histones can be also
remodelled by other PTMs including methylation, phosphorylation,
sumoylation, poly-ADP-ribosylation, and ubiquitilation. These
changes alter dynamically and reversibly the chromatin structure
recruiting specific effector proteins and thereby influencing gene
expression, DNA replication and repair, and chromosome conden-
sation and segregation [2]. Recent studies also investigated lysine
acetylation levels of non-histone proteins, and highlighted the
numerous roles that HATs and HDACs play as multifunctional fac-
tors in a variety of other cellular processes and vital physiological
major families (based on structural homology in the primary
sequence and biochemical mechanism of acetyl transfer): general
control non-derepressible 5 (Gcn5)-related N-acetyltransferases
(GNATs) (Gcn5p, PCAF, Elp3, Hat1, Hpa2, and Nut1), MYST (Esa1,
Morf, Ybp2, Sas2, Sas3, Tip60, and Hbo1), p300 (adenoviral E1A-
associated protein of 300 kDa)/CBP (CREB-binding protein), gen-
eral transcription factors HATs, and nuclear hormone receptor-
related HATs [4].
In view of the increasing evidence that associates HAT function
with cancer development and progression, these enzymes are
appealing as drug targets for the development of small molecule
inhibitors [5]. It may seem paradoxical to seek inhibitors of both
HATs and HDACs when these enzymes have opposing catalytic re-
actions. However, the biology of HATs and HDACs is complex and it
is unlikely that a simple ‘‘on-off’’ acetylation model will apply to
gene transcription and cancer development [6]. The correlation
between epigenetic aberrations and cancer underscores the
importance of epigenetic mechanisms which may be the result of
some pleiotropic effects. This evidence may indicate that: (i) a
disequilibrium based on both genetic and epigenetic disregulations
* Corresponding authors. Department of Drug Chemistry and Technologies,
Sapienza University of Rome, P.le A. Moro 5, 00185 Rome, Italy.
E-mail addresses: daniela.secci@uniroma1.it, antonello.mai@uniroma1.it (A. Mai).
Equal contribution.
1
http://dx.doi.org/10.1016/j.ejmech.2014.04.042
0223-5234/Ó 2014 Elsevier Masson SAS. All rights reserved.

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S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
brings to tumorigenesis; (ii) the deregulation of selective epi-
enzymes might be crucial for cancer thus emphasizing the rele-
vance of tissue-selective disregulations. In addition, multiple epi-
enzyme-containing complexes might be altered in tumorigenesis,
thus repressing or activating both HDAC- or HAT-dependent targets
into chromatin [7].
More in detail, mutations in several HAT genes have been
observed in solid tumors [8]; furthermore, biallelic mutations of
EP300 have been identified in epithelial tumors [9]. In hemato-
logical malignancies, and less commonly in solid tumors, chromo-
somal translocations involving HAT genes such as EP300, CREBBP,
MYST3 and MYST4 (hematological malignancies) or MYST4 and
NCOA1 (solid tumors) have been reported [10]. The translocations
involved in leukemogenesis seem to be due to aberrant acetylation
caused by mistargeting of HATs. The HAT NCOA3 gene is frequently
amplified and overexpressed in human breast cancer and behaves
as a classical oncogene [11].
A large number of HDAC inhibitors have already entered into the
clinical arena, alone or in combination with other therapeutics, and
two of them, vorinostat and romidepsin, have been approved by US
FDA for the treatment of refractory cutaneous T-cell lymphoma
[12]. Differently, only a few HAT inhibitors (HATi) have been
described so far, and a limited number of them showed efficacy in
preclinical cancer models, mainly because they suffer from absence
of a recognized pharmacophore structure for chemical optimiza-
tion, lack of specificity, and/or low cell-permeability (Fig. 1) [13].
Thus, the urgency to find new selective HATi as pharmacological
tools has emerged as a priority especially in the clinic. Overall, the
understanding of structureeactivity relationship of newly devel-
oped molecules as well as the knowledge of their biological
mechanisms of action applied to the medical field will pave the way
for novel epigenetic approaches to fight human cancer [4].
The chemistry of thiazole-containing compounds is particularly
interesting because of its capability to easily furnish valuable che-
motherapeutics such as anticancer, antibacterial, antifungal, and
antiprotozoal agents as well as monoamine oxidase inhibitors [14].
Recently, we reported some (thiazol-2-yl)hydrazones screened by a
chemo-genomic approach in S. cerevisiae yeast cultures, demon-
strating that some of them were endowed with selective inhibitory
Fig. 2. Structural modifications applied on the reported lead compounds CPTH2 and
CPTH6.
activity against the Gcn5 HAT subfamily by means of a fluorescent
read-out, and showed a moderate inhibitory activity against U87-
glioblastoma, and BE-neuroblastoma [15]. Successively, some of
the most potent derivatives (CPTH2, now also provided by com-
mercial suppliers as selective Gcn5 inhibitor, and CPTH6, Fig. 2)
were used as pharmacological tools to unravel the superoxide-
generating system in leukocytes [16] and as new therapeutic op-
tions for the treatment of a panel of leukemia cell lines, confirming
the predominant inhibition of Gcn5 HAT activity (inhibition of H3/
H4 histones and
a-tubulin acetylation, concentration- and time-
dependent inhibition of cell viability paralleled by accumulation
of cells in the G0/G1 phase, depletion from the S/G2M phases, in-
duction of apoptosis via mitochondrial pathway, and involvement
of Bcl-2 and Bcl-xL proteins) [17]. Solid tumor cell lines from several
origins were shown to be differently sensitive to CPTH6 treatment
in terms of cell viability, and a correlation between the inhibitory
efficacy on H3/H4 histones acetylation and cytotoxicity was found
[17]. In addition, differentiating effect on leukemia and neuro-
blastoma cell lines was also induced by CPTH6 exposure [17]. We
also found that CPTH6 had an effect on autophagy developed
through the Atg-7-mediated elongation of autophagosomal mem-
branes and the blockage of autophagic cargo degradation indicating
a putative role of
a-tubulin acetylation in CPTH6-induced alteration
in autophagy [18].
Fig. 1. Known HAT inhibitors.

S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
571
In view of the above findings and our interest in designing new
HAT inhibitors [19], we focused our attention on the (thiazol-2-yl)
hydrazone scaffold and, having available a large in-house library of
such compounds mostly assayed against other biological targets,
[14cef, hej, 15], we decided to test them against human HAT p300
and PCAF enzymes (Fig. 2) using a radiochemical assay and, for
selected derivatives, against human U937 leukemia and HCT116
cells to determine their effects in cell cycle perturbation, apoptosis
induction and cytodifferentiation.
In this library, first we kept constant the 4-(4-chlorophenyl)-2-
thiazolylhydrazone moiety of CPTH2 and CPTH6 replacing the
cyclopentane portion with shorter and larger residues, either
(cyclo)aliphatic or (hetero)aromatic, through processes of ring
opening/chain shortening and chain lengthening/ring complication
(1aej’) (Fig. 3, section A).
Successively, we studied the influence of different substitutions
at the C4-phenyl ring of the thiazole, or its replacement with
smaller (methyl, ethoxycarbonyl) or larger (2-naphthyl, 3-
chromenyl) groups, leaving the iso-propyl, (substituted)cyclo-
pentyl, (substituted)cyclohexyl, or cycloheptyl hydrazone function
at the thiazole C2 position (2aei’) (Fig. 3, section B). Also the effect
of methyl substitution at the C5 position of the thiazole ring has
been explored (3aec) (Fig. 3, section C). Lastly, some compounds
have been designed and prepared by either deleting the hydrazone
part (4aec) (Fig. 3, section D) or replacing the thiazole nucleus with
a thiazolidinone (5a,b) (Fig. 3, section E) or a 4-oxopyrimidin-2-yl
nucleus (6ael) (Fig. 3, section F).
Scheme 1. Synthesis of compounds 1e5. Reagents and conditions: a) (for R ¼ NH2)
R1COR2, CH3COOH, EtOH, MW, 103 ꢀC, 5 min, 300 W; b) BrCH(R4)COR3, MeOH, MW,
90 ꢀC, 10 min, 300 W; c) BrCH2COPh(Y), CH3COOH, EtOH, 12 h, rt; d) BrCH2COOEt,
CH3COONa, MeOH, 2e6 h, rt.
developed in our laboratory as outlined in Scheme 1. Carbonylic
compounds reacted directly with thiosemicarbazide in ethanol
with catalytic amounts of acetic acid under MW irradiation (103 ꢀC,
5 min, 300 W), and the obtained thiosemicarbazones 7aej’ were
subsequently converted into (thiazol-2-yl)hydrazones by Hantzsch
reaction with
a-bromo-substituted acetophenones/ketones/
ketoesters in methanol under MW irradiation (90 ꢀC, 10 min,
300 W). The 2-hydrazino- and 2-aminothiazoles 4aec [14a,b] were
prepared by reaction of thiosemicarbazide or thiourea with the
appropriate a-bromo-acetophenone in ethanol/acetic acid (Scheme
2. Results and discussion
1). Thiazolidine compounds (5) were obtained by the same versa-
tile thiosemicarbazone intermediates 7 by cyclization with ethyl
bromoacetate and sodium acetate in methanol (Scheme 1).
The synthetic route followed for the preparation of 6 is depicted
in Scheme 2. As reported previously [20], substituted ethyl
2.1. Chemistry
(Thiazol-2-yl)hydrazone derivatives (1e3) were synthesized in
high yields according to an improved MW-assisted protocol
Fig. 3. Library of (thiazol-2-yl)hydrazones and analogues described in this paper.

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S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
benzoylacetates and thiourea were added to an ethanolic solution
of sodium, the resulting mixture was stirred at reflux and subse-
quent ring closure afforded the intermediates (8), which were then
converted, in the presence of hydrazine monohydrate, to the cor-
responding (un)substituted 2-hydrazinyl-6-phenylpyrimidin-
4(3H)-ones (9). The final compounds 6 were obtained by conden-
sation between the intermediates (9) and the appropriate
commercially available ketones in a refluxing solution of dry
ethanol with glacial acetic acid as a catalyst.
2.2. Enzyme assays
Fig. 4. Human p300 inhibitory activity of compounds 1e6. Color code for inhibition:
red, 60e80%; orange, 40e60%; yellow, 20e40%; green, <20% inhibition. (For inter-
pretation of the references to colour in this figure legend, the reader is referred to the
web version of this article.)
Compounds 1e6 were tested at 100 mM against human p300
and PCAF (catalytic domains) using histone H3 as a substrate and
[acetyl-3H]-acetyl coenzyme A as an acetyl donor (HotSpot HAT
activity assays). In these conditions, compounds 1e6 displayed very
poor (if any) PCAF inhibiting activity (Tables S1eS5 in
Supplementary Material). The percentages of p300 inhibitory ac-
at C4. The insertion of an additional methyl substituent at the
thiazole-C5 position (compounds 3, section C) did not improve the
potency of the (thiazol-2-yl)hydrazones, as well as the removal of
the hydrazine moiety (compounds 4, section D) or the replacement
of the thiazole with the thiazolidine ring (compounds 5, section E).
Among the 2-hydrazonopyrimidin-4(3H)-ones 6 (section F), com-
pound 6k, bearing the same substituents of CPTH6, displayed
improved p300 inhibitory activity respect to the prototype.
Selected 1, 2, and 6 derivatives were also tested at 200 and
tivities for 1e6 tested at 100 mM are summarized in Fig. 4. Two
compounds, 1x and 1i’, showed the highest p300 inhibition (>60%),
and six further compounds (i.e., 1c’, 1d’, 2i, 2o, 2i’ and 6k) exhibited
higher potency (>40%) than the two prototypes CPTH2 and CPTH6.
In the 1 series (section A) carrying the 4-chlorophenyl substit-
uent at the C4-thiazole position, the replacement at the hydrazone
function of the (3-methyl)cyclopentyl moiety of CPTH2 and -6 with
smaller or larger, straight or branched, saturated or unsaturated
(cyclo)alkyl portions (compounds 1aer) did not improve the p300
inhibitory activity of the derivatives. The introduction of (hetero)
aryl substituents at this level was in general also ineffective, with
some important exceptions. In particular, the insertion of a thiazol-
2-yl or pyrid-2-yl ring at the hydrazone function yielded com-
pounds (1c’ and 1d’) more potent than CPTHs in inhibiting p300.
Among the bicyclic rings, the heteroaromatic (1H)-indol-3-yl and
2(2H)-chromenon-3-yl furnished the most potent derivatives (1x
and 1i’), while 2-naphthyl, benzo[d][1,3]dioxol-5-yl and 2-carboxy-
9(9H)-fluorenyl substituents led to less potent analogues.
400
mM against human p300 (catalytic domain) to assess their
percentages of inhibition. Data in Table 1 indicate that 1x, 1d’ and
1i’ displayed the highest p300 inhibitory potency, with IC50 values
of 77.6 ꢁ 21.4 (1x), 99.3 ꢁ 20.8 (1d’) and 78.3 ꢁ 6.6 (1i’)
mM,
highlighting the crucial role of the 4-chlorophenyl substituent at
the C4 position of the thiazole ring. Nevertheless, the high inhibi-
tion registered with 2h and 2m suggested the introduction of a 3-
nitro- or 4-fluorophenyl at C4 as a valid alternative moiety. As
regards to the hydrazone function, together with the iso-propyl and
cyclopentyl residues also heterocycles such as 3(1H)-indolyl (1x),
2-pyridyl (1d’) and 2(2H)-chromenon-3-yl (1i’) furnished high
inhibition.
When the 4-chlorophenyl ring at the thiazole C4 was replaced
with other (substituted)phenyl or (hetero)aromatic rings (com-
pounds 2, section B), an increase of p300 inhibitory activity respect
to CPTHs was obtained with the introduction of a 3-nitrophenyl
(2i), 4-bromophenyl (2o), or 2(2H)-chromenon-3-yl (2i’) portion
2.3. Cellular studies
Selected compounds 1x, 1c’, 1d’, 1i’, 2h, 2i, 2m, 2i’, 6g and 6k
were tested in human leukemia U937 and in human colon
Table 1
Human p300 inhibitory activities for the most active derivatives.a
Compound
% Inhibition
100
m
M
200
mM
400 mM
1a
1x
1c’
1d’
1i’
2g
2h
2i
2k
2m
2o
2q
2h’
2i’
6g
6k
CPTH2
31 ꢁ 1.7
61 ꢁ 3.4
46 ꢁ 4.5
51 ꢁ 1.6
62 ꢁ 0.9
37 ꢁ 0.2
33 ꢁ 2.4
44 ꢁ 1.9
31 ꢁ 1.6
34 ꢁ 0.9
42 ꢁ 2.1
33 ꢁ 0.6
32 ꢁ 0.3
40 ꢁ 0.02
31 ꢁ 0.6
41 ꢁ 0.7
34 ꢁ 4.4
45 ꢁ 0.2
79 ꢁ 1.5
55 ꢁ 1.4
69 ꢁ 1.8
86 ꢁ 5.6
56 ꢁ 2.2
54 ꢁ 1.6
51 ꢁ 1.7
51 ꢁ 1.2
62 ꢁ 2.4
53 ꢁ 2.3
47 ꢁ 2.0
46 ꢁ 1.1
52 ꢁ 1.8
46 ꢁ 1.8
55 ꢁ 2.9
47 ꢁ 2.5
60 ꢁ 2.2
97 ꢁ 0.4
67 ꢁ 4.0
87 ꢁ 3.4
97 ꢁ 1.3
54 ꢁ 1.2
76 ꢁ 0.3
60 ꢁ 2.7
62 ꢁ 2.5
91 ꢁ 1.5
63 ꢁ 1.2
57 ꢁ 2.6
56 ꢁ 2.1
69 ꢁ 1.0
54 ꢁ 2.5
64 ꢁ 2.4
61 ꢁ 2.0
Scheme 2. Synthesis of compounds 6. Reagents and conditions: a) thiourea, Na, dry
EtOH, reflux; b) hydrazine monohydrate, reflux; c) R1COR2, dry EtOH, cat. CH3COOH.
a
The assays were performed in duplicate.

S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
573
carcinoma HCT116 cells at 100
m
M for 30 h, to determine their ef-
activation/deactivation. Aberrant HAT activity and mutation in
several HAT genes are strictly related to tumorigenesis. Few HAT
inhibitors have been characterized so far, and the development of
new pharmacological agents is required.
fects on cell cycle progression and induction of apoptosis. In U937
cells, granulocytic cytodifferentiation at 30 h has been also evalu-
ated. CPTH6, already known for its anticancer properties [17,18],
was used as a reference drug.
By the use of thiazole chemistry, we prepared some (thiazol-2-
yl)hydrazones as antimicrobial or anti-MAO agents [14cef, hej].
Other analogues were identified as Gcn5 HAT inhibitors in yeast,
including CPTH2 and CPTH6 after recognized as valuable anti-
tumor agents [15e18]. Since all these derivatives carried the same
pharmacophoric core e the thiazole ring e linked to structurally
diverse substituents at the thiazole C4 position and/or at the
hydrazone moiety, we decided to increase our in-house thiazole
library through the synthesis of further derivatives
2b,f,h,l,n,o,z,c’,d’ including some thiazolidine (5a,b) and pyr-
imidin-4(3H)-one (6ael) analogues, and to screen the whole li-
brary against human p300 and PCAF HAT enzymes, with the aim
to identify compounds more potent than CPTH2 and CPTH6. From
this screening, 1x, 1c’, 1d’, 1i’ and 2m were more efficient than the
two prototypes in enzyme assays, highlighting the possibility to
replace the (3-methyl)cyclopentyl group at the hydrazone moiety
of CPTH2 and CPTH6 with a 3(1H)-indolyl or 2-pyridyl or 2(2H)-
chromenon-3-yl moiety, and to insert at the thiazole-C4 position a
4-fluorophenyl instead of the 4-chlorophenyl ring typical of the
two prototypes. When tested in human leukemia U937 cells at
In U937 cells, the treatment with 1d’ and 2m among the tested
derivatives, together with CPTH6, gave an arrest of cell cycle in the
G1 phase, with lower percentages of cells in S phase respect to the
control (Fig. 5A). For compounds 1x and 1i’, it was no possible to
determine the changes in cell cycle due to the intense apoptosis.
The pre-G1 peak in cell cycle was taken as an index of apoptosis
induction. As shown in Fig. 5B, in U937 cells 1x, 1i’ and 2m elicited
huge apoptosis (from 44 to 60%), they being 1.5/2-fold more potent
than CPTH6 (% apoptosis: 29).
To confirm that the observed apoptotic effects of 1x, 1i’ and 2m
in U937 cells were not due to off-target effects, such compounds
were used in combination with CPTH6 (all at 100 mM for 30 h) in
U937 cells, and the percentages of apoptosis were determined
(Fig. 5C). Neither synergic nor additive effects were observed, thus
confirming an on-target phenotype with these compounds.
On the basis of the relevance of histone acetylation changes on
regulation of cell differentiation [25], we also investigated whether
the selected compounds were able to induce granulocytic differ-
entiation in U937 cells. The superficial antigen CD11c was taken as a
marker of differentiation. After treatment of U937 cells with 1x,1d’,
100
much more differentiation than CPTH6, used as reference drug. In
colon carcinoma HCT116 cells, treated at 100 M for 30 h, 1x, 1i’,
mM for 30 h, 1x, 1i’ and 2m induced higher apoptosis and
1i’ and 2m (100 mM, 30 h), a significant increase of the CD11c-
positive cells was observed when compared with the control, after
subtraction of the dead cells (propidium iodide (PI) positive cells)
(Fig. 5D).
When tested in HCT116 cells, 2m, 2i’ and, to a lower extent, 2h
gave a block in G1 phase of the cell cycle (Fig. 6A). As apoptotic
inducers, 1x, 1i’, 2h and 2m were slightly less potent than CPTH6
displaying nearly 10% of apoptosis (CPTH6: 11.5%) (Fig. 6B).
The reversible acetylation of a broad range of histone and non-
histone proteins is an epigenetic regulator in eukaryotic gene
m
2h and 2m gave similar percentage of apoptosis as CPTH6. Further
studies will be performed on such thiazole derivatives to assess
their anticancer utility.
3. Conclusion
With the aim to identify HAT inhibitors more potent than our
two promising prototypes (CPTH2 and CPTH6), we synthesized
Fig. 5. Effects of selected derivatives 1x, 1c’, 1d’, 1i’, 2h, 2i, 2m, 2i’, 6g and 6k on cell cycle progression (A), apoptosis induction (B) and cytodifferentiation (D) in human leukemia
U937 cells when tested at 100 M for 30 h (C), percentages of apoptosis using 1x, 1i’ and 2m in combination with CPTH6 in U937 cells treatment at 100 M for 30 h.
m
m

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S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
by UV light. Preparative flash column chromatography was carried
out on silica gel (230e400 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 of ca. 20 Torr.
4.1.1. General procedure for the synthesis of thiosemicarbazone
intermediates (7)
Carbonylic compound (3.4 mmol) and the corresponding cata-
lyst (glacial acetic acid) were added to a suspension of thio-
semicarbazide (0.30 g, 3.4 mmol) in 2 mL of absolute ethanol in a
5 mL vessel suitable for microwave reactor (2.45 GHz high-
frequency microwaves, power range 0e300 W). The vessel was
sealed, the mixture pre-stirred for 30 s and then heated by mi-
crowave irradiation for 5 min at fixed temperatures (103 ꢀC). If not
set, the irradiation power reaches its maximum at the beginning of
reaction and it subsequently decreases to lower and constant
values. The vial internal temperature was controlled by an equip-
ped IR sensor. After cooling in a stream of pressurized air the re-
action mixture was filtered and the obtained solid washed with
petroleum ether, n-hexane, and diethyl ether. The crude mixture
was purified by column chromatography (SiO2, ethyl acetate/n-
hexane).
4.2. Characterization data for new compounds
4.2.1. General procedure for the synthesis of (thiazol-2-yl)
hydrazone derivatives 1e3
The appropriate thiosemicarbazone (7) (1.5 mmol) was added to
a solution of the proper a-bromo-acetophenone in 2 mL of meth-
anol. The mixture was pre-stirred in a sealed vessel for 1 min and
then heated up by microwave irradiation for 10 min at fixed tem-
peratures (90 ꢀC). The reaction mixture was cooled down with
pressurized air, filtered, and the obtained solid washed with n-
hexane and diethyl ether. The crude mixture was purified by col-
umn chromatography (SiO2, ethyl acetate/n-hexane) to give all
compounds in high yields.
Fig. 6. Effects of selected derivatives 1x, 1c’, 1d’, 1i’, 2h, 2i, 2m, 2i’, 6g and 6k on cell
cycle progression (A) and apoptosis induction (B) in human colon carcinoma HCT116
cells when tested at 100 mM for 30 h.
several new (thiazol-2-yl)hydrazones including some related
thiazolidines and pyrimidin-4(3H)-ones to extrapolate broader
structureeactivity relationships within this scaffold. Moreover, we
tested them, along with our in-house library [15], against human
p300 and PCAF HAT enzymes in vitro with a radiochemical assay.
Compounds 1x, 1c’, 1d’, 1i’ and 2m were potent p300 inhibitors in
4.2.1.1. 1-Cyclopentylidene-2-(4-methylthiazol-2-yl)hydrazine (2b).
White powder, yield 79%, mp 157e158 ꢀC; 1H NMR (CDCl3)
d 1.90e
1.94 (m, 2H, CH2, cyclopentyl), 1.95e2.00 (m, 2H, CH2, cyclopentyl),
2.35 (s, 3H, CH3), 2.50e2.54 (m, 2H, CH2, cyclopentyl), 2.61e2.64
(m, 2H, CH2, cyclopentyl), 6.15 (s, 1H, C5H-thiazole), 12.50 (bs, 1H,
NH, D2O exch.). Anal. Calcd for C9H13N3S: C, 55.35%; H, 6.71%; N,
21.52%. Found C, 55.60%; H, 6.44%; N, 21.87%.
the micromolar range. In U937 and HCT116 cells at 100 mM, 1x, 1i’
and 2m determined remarkable apoptosis and cytodifferentiation.
4. Experimental section
4.2.1.2. 1-(4-Phenylthiazol-2-yl)-2-(propan-2-ylidene)hydrazine (2f).
White solid, yield 77%, mp 220e223 ꢀC; 1H NMR (CDCl3)
d 2.14 (s,
4.1. Chemistry
3H, CH3), 2.24 (s, 3H, CH3), 6.71 (s, 1H, C5H-thiazole), 7.49e7.51 (m,
3H, Ar), 7.73e7.75 (m, 2H, Ar), 12.47 (bs, 1H, NH, D2O exch.). Anal.
Calcd for C12H13N3S: C, 62.31; H, 5.66; N, 18.17. Found: C, 62.03; H,
5.89; N, 17.91.
Starting materials and reagents were obtained from commercial
suppliers (Aldrich, Milan (Italy) and Lancaster Synthesis GmbH,
Milan (Italy)) and were used without further purification.
Microwave-assisted reactions were performed in a Biotage Ini-
tiatorÔ 2.0 (Uppsala, Sweden). Melting points (mp) were deter-
mined by the capillary method on an FP62 apparatus (Mettler-
Toledo) and are uncorrected. 1H NMR spectra were recorded at
400 MHz on a Bruker AC 400 spectrometer using DMSO-d6 or CDCl3
4.2.1.3. 1-(4-(3-Nitrophenyl)thiazol-2-yl)-2-(propan-2-ylidene)hy-
drazine (2h). Light yellow solid, yield 78%, mp 215e216 ꢀC; 1H NMR
(CDCl3)
d 2.08 (s, 3H, CH3), 2.11 (s, 3H, CH3), 6.97 (s, 1H, C5H-thia-
zole), 7.62e7.66 (m,1H, Ar), 8.12e8.14 (m,1H, Ar), 8.19e8.22 (m,1H,
Ar), 8.56e8.57 (m, 1H, Ar), 12.42 (bs, 1H, NH, D2O exch). Anal. Calcd
for C12H12N4O2S: C, 52.16; H, 4.38; N, 20.28. Found: C, 52.30; H,
4.22; N, 20.16.
as solvent. 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 PerkineElmer 240
B microanalyzer and the analytical results were ꢂ95% purity for all
compounds. All reactions were monitored by TLC performed on
0.2 mm thick silica gel plates (60 F254 Merck) with spots visualized
4.2.1.4. 1-(4-(4-Fluorophenyl)thiazol-2-yl)-2-(propan-2-ylidene)hy-
drazine (2l). Light yellow solid, yield 72%, mp 185e187 ꢀC; 1H NMR
(CDCl3)
d 2.06 (s, 3H, CH3), 2.22 (s, 3H, CH3), 6.65 (s, 1H, C5H-

S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
575
thiazole), 7.21e7.23 (d, J ¼ 7.0 Hz, 2H, Ar), 7.72e7.74 (d, J ¼ 7.0 Hz,
4.4. General procedure for the synthesis of thiazolidine derivatives
(5)
2H, Ar), 12.64 (br s, 1H, NH, D2O exch.). Anal. Calcd. for C12H12FN3S:
C, 57.81; H, 4.85; N, 16.85. Found: C, 57.65; H, 4.79; N, 16.64.
Equimolar amounts of the prepared thiosemicarbazone (7,
5 mmol) and ethyl bromoacetate (5 mmol), both suspended in
50 mL of methanol, were reacted at room temperature under
magnetic stirring for 2e6 h in the presence of 5 mmol of
CH3COONa. The obtained solution was poured into ice and
extracted with CHCl3 (3 ꢃ 50 mL), dried over anhydrous sodium
sulphate, and evaporated. The crude product has been purified by
column chromatography (SiO2, ethyl acetate/n-hexane).
4 . 2 .1. 5 . 1 - ( 4 - ( 4 - F l u o r o p h e n y l ) t h i a z o l - 2 - y l ) - 2 - ( 3 -
methylcyclopentylidene)hydrazine (2n). Yellow powder, yield 81%,
mp 225e227 ꢀC; 1H NMR (CDCl3)
d 1.13 (s, 3H, CH3), 1.38e1.40 (m,
1H, cyclopentyl), 1.50e1.51 (m, 1H, cyclopentyl), 2.01e2.03 (m, 1H,
cyclopentyl), 2.17e2.19 (m, 1H, cyclopentyl), 2.23e2.25 (m, 1H,
cyclopentyl), 2.57e2.58 (m, 1H, cyclopentyl), 2.71e2.73 (m, 1H,
cyclopentyl), 6.63 (s, 1H, C5H-thiazole), 7.18e7.20 (d, J ¼ 7.7 Hz, 2H,
Ar), 7.70e7.72 (d, J ¼ 7.7 Hz, 1H, Ar), 12.23 (bs, 1H, NH, D2O exch.).
Anal. Calcd for C15H16FN3S: C, 62.26%; H, 5.57%; N, 14.52%. Found C,
62.41%; H, 5.33%; N, 14.87%.
4.5. 2-(2-(Propan-2-ylidene)hydrazono)thiazolidin-4-one (5a)
White powder, yield 89%, mp 170e174 ꢀC; 1H NMR (CDCl3)
d 1.97
(s, 3H, CH3), 2.00 (s, 3H, CH3), 3.70 (s, 2H, CH2, thiazolidinone), 11.91
(bs, 1H, NH, D2O exch.). Anal. Calcd for C6H9N3OS: C, 42.09%; H,
5.30%; N, 24.54%. Found C, 41.82%; H, 5.12%; N, 24.79%.
4.2.1.6. 1-(4-(4-Bromophenyl)thiazol-2-yl)-2-(propan-2-ylidene)hy-
drazine (2o). Light brown solid, yield 73%, mp 240e243 ꢀC; 1H NMR
(DMSO-d6)
d 1.94 (s, 3H, CH3), 1.96 (s, 3H, CH3), 7.32 (s, 1H, C5H-
thiazole), 7.60e7.63 (d, J ¼ 10.0 Hz, 2H, Ar), 7.79e7.81 (d,
4.6. 2-(2-Cyclopentylidenehydrazono)thiazolidin-4-one (5b)
White powder, yield 90%, mp 207e210 ꢀC; 1H NMR (400 MHz,
J ¼ 10.0 Hz, 2H, Ar), 10.78 (br s, 1H, NH, D2O exch.). Anal. Calc. for
C
12H12BrN3S: C 46.46; H, 3.90; N, 13.55. Found: C 46.59; H, 4.06; N,
13.32.
CDCl3):
d 1.79e1.82 (m, 4H, cyclopentyl), 2.50e2.53 (m, 4H, cyclo-
pentyl), 3.79 (s, 2H, CH2-thiazolidinone), 11.77 (bs, 1H, NH, D2O
exch.). Anal. Calcd for C8H11N3OS: C, 48.71%; H, 5.62%; N, 21.30%.
Found C, 48.99%; H, 5.34%; N, 21.67%.
4 . 2 . 1. 7 . 1 - ( 4 - ( 4 - c y a n o p h e n y l ) t h i a z o l - 2 - y l ) - 2 - ( 3 -
methylcyclopentylidene)hydrazine (2z). Yellow powder, yield 89%;
mp 180e182 ꢀC; 1H NMR (CDCl3)
d 1.28 (s, 3H, CH3), 1.54e1.55 (m,
1H, cyclopentyl), 1.56e1.58 (m, 1H, cyclopentyl), 1.59e1.61 (m, 1H,
cyclopentyl), 2.36e2.38 (m, 1H, cyclopentyl), 2.52e2.54 (m, 1H,
cyclopentyl), 2.66e2.68 (m, 1H, cyclopentyl), 2.83e2.85 (m, 1H,
cyclopentyl), 6.87 (s, 1H, C5H-thiazole), 7.77e7.78 (d, J ¼ 8.2 Hz, 2H,
Ar), 7.84e7.87 (d, J ¼ 8.2 Hz, 2H, Ar), 12.15 (bs, 1H, (E)-NH, D2O
exch.), 14.21 (bs, 1H, (Z)-NH, D2O exch.). Anal. Calcd for C16H16N4S:
C, 64.84%; H, 5.44%; N, 18.90%. Found C, 65.08%; H, 5.26%; N, 19.11%.
4.7. General procedure for the preparation of 6-(substituted)
phenyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-ones (8)
4.7.1. Example: 6-(4-bromophenyl)-2-thioxo-2,3-
dihydropyrimidin-4(1H)-one (8d)
Sodium metal (1.3 g, 56.1 mg-formula) was dissolved in 30 mL of
absolute ethanol, then thiourea (2.9 g, 38.2 mmol) and 4-
bromobenzoylacetate (6.9 g, 25.5 mmol) were added in sequence
to the clear solution. The mixture was heated at reflux for 12 h. The
resulting mixture was cooled at room temperature, the solvent was
distilled in vacuo at 40e50 ꢀC, and the residue was dissolved in a
little amount of water to obtain a basic solution which was
extracted with diethyl ether (2 ꢃ 50 mL). The basic aqueous solu-
tion was acidified while cooling with ice bath to pH ¼ 2 to give a
precipitate that was filtered and washed with water. The resulting
compound was a white solid used in the next step without further
4. 2.1. 8 . 1- (4 -(2, 4-Di fluorophenyl)thiaz ol-2-yl )-2-(3-
methylcyclopentylidene)hydrazine (2c’). Light yellow powder, yield
83%, mp 131e134 ꢀC; 1H NMR (CDCl3)
d 1.11 (s, 3H, CH3), 1.42e1.45
(m, 1H, cyclopentyl), 1.47e1.51 (m, 1H, cyclopentyl), 2.43e2.46 (m,
1H, cyclopentyl), 2.61e2.64 (m, 1H, cyclopentyl), 2.67e2.69 (m, 1H,
cyclopentyl), 2.78e2.80 (m, 1H, cyclopentyl), 2.84e2.2.86 (m, 1H,
cyclopentyl), 6.91 (s, 1H, C5H-thiazole), 6.94e6.99 (m, 1H, Ar) 7.07e
7.11 (m, 1H, Ar), 7.88e7.91 (m, 1H, Ar), 12.36 (bs, 1H, (E)-NH, D2O
exch.), 14.40 (bs, 1H, (Z)-NH, D2O exch.). Anal. Calcd for
purification. 1H NMR (400 MHz, DMSO-d6)
d ppm 6.11 (s, 1H, CH-
C
15H15F2N3S: C, 58.62%; H, 4.92%; N, 13.67%. Found C, 58.40%; H,
pyrimidine ring), 7.64e7.66 (d, 2H, Ar), 7.69e7.71 (d, 2H, Ar),
12.56 (s, 2H, NH).
5.17%; N, 13.92%.
4.2.1.9. 1-(4-(Biphenyl)thiazol-2-yl)-2-(propan-2-ylidene)hydrazine
6-phenyl-2-thioxo-2,3-dihydropyrimidin-4(1H)-one (8a). See
ref. [20a].
(2d’). Brown solid, yield 69%, mp 241e243 ꢀC; 1H NMR (CDCl3)
d
2.16 (s, 3H, CH3), 2.26 (s, 3H, CH3), 6.73 (s, 1H, C5H-thiazole), 7.25e
7.52 (m, 9H, Ar), 12.40 (bs, 1H, NH, D2O exch.). Anal. Calcd for
18H17N3S: C, 70.33; H, 5.57; N, 13.67. Found: C, 70.64; H, 5.84; N,
6-(3-nitrophenyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
ꢀ
(8b). White powder, yield 65%, mp > 250 C.
C
6-(4-chlorophenyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
(8c). See ref. [20b].
13.90.
6-(4-bromophenyl)-2-thioxo-2,3-dihydropyrimidin-4(1H)-one
ꢀ
4.3. General procedure for the synthesis of derivatives 4
(8d). White powder, yield 86%, mp > 250 C.
Thiourea or thiosemicarbazide (1.5 mmol) was added to a so-
5. General procedure for the preparation of 2-hydrazinyl-6-
lution of the correspondent
a-bromo-acetophenone in 2 mL of
(substituted)phenylpyrimidin-4(3H)-ones (9)
methanol. The mixture was pre-stirred in a sealed vessel for 1 min
and then heated up by microwave irradiation for 10 min at fixed
temperatures (90 ꢀC). The reaction mixture was cooled down with
pressurized air, filtered, and the obtained solid washed with n-
hexane and diethyl ether. The crude mixture was purified by col-
umn chromatography (SiO2, ethyl acetate/n-hexane) to give all
compounds in high yields.
5.1. Example: 6-(4-chlorophenyl)-2-hydrazinylpyrimidin-4(3H)-
one (9c)
A mixture of 6-(4-chlorophenyl)-3,4-dihydro-2-thiopyrimidin-
4(3H)-one 8b (500 mg, 2.1 mmol) and hydrazine monohydrate
(7.15 mL, 7.36 g, 0.15 mol) was stirred at reflux for 4 h. Afterwards,

576
S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
hydrazine was evaporated under reduced pressure. The residue was
then triturated with water for 30 min. The obtained precipitate was
filtered off and subsequently washed with water to give a white
powder used in the next step without further purification. 1H NMR
(400 MHz, DMSO-d6) d ppm 6.11 (s, 1H, CH-pyrimidine ring), 7.49e
7.51 (d, 2H, Ar), 7.98e8.01 (d, 2H, Ar), 8.57 (s, 1H, NH).
Anal. Calcd for C13H13BrN4O: C, 48.62%; H, 4.08%; N,17.44%. Found C,
48.36%; H, 3.89%; N, 17.76%.
6.5. 2-(2-Cyclopentylidenehydrazinyl)-6-phenylpyrimidin-4(3H)-
one (6e)
White powder, yield 80%, mp 231e233 ꢀC; 1H NMR (DMSO-d6)
ppm 1.70e1.72 (m, 2H, cyclopentyl ring), 1.78e1.80 (m, 2H,
cyclopentyl ring), 2.42 (m, 4H, cyclopentyl ring), 6.23 (s, 1H, CH-
pyrimidine), 7.45e7.47 (m, 3H, Ar), 7.99e8.01 (m, 2H, Ar), 10.39
(s, 2H, NH). Anal. Calcd for C15H16N4O: C, 67.15%; H, 6.01%; N,
20.88%. Found C, 67.44%; H, 6.19%; N, 20.61%.
2-hydrazinyl-6-phenylpyrimꢀidin-4(3H)-one (9a). White pow-
der, yield 51%, mp 221e223 C
d
2-hydrazinyl-6-(3-nitrophenyl)pyrimidin-4(3H)-one
White powder, yield 62%, mp 227e229 C
(9b).
(9c).
(9d).
ꢀ
6-(4-chlorophenyl)-2-hydrazinylpyrimidin-4(3H)-one
White powder, yield 59%, mp 243e245 ꢀC
6-(4-bromophenyl)-2-hydrazinylpyrimidin-4(3H)-one
6.6. 2-(2-Cyclopentylidenehydrazinyl)-6-(3-nitrophenyl)
pyrimidin-4(3H)-one (6f)
ꢀ
White powder, yield 56%, mp > 250 C
6. General procedure for the preparation of 2-(2-
White powder, yield 67%, mp 230e232 ꢀC; 1H NMR (DMSO-d6)
ppm 1.69e1.81 (m, 4H, cyclopentyl ring), 2.41 (m, 4H, cyclopentyl
ring), 6.31 (s, 1H, s, CH-pyrimidine), 7.29e7.32 (m, 1H, Ar), 7.47e
7.53 (m, 1H, Ar), 7.82e7.87 (m, 2H, Ar), 10.38e10.49 (s, 2H, NH).
Anal. Calcd for C15H15N5O3: C, 57.50%; H, 4.83%; N, 22.35%. Found C,
57.23%; H, 4.79%; N, 22.68%.
alkylidenehydrazinyl)-6-(substituted)phenylpyrimidin-4(3H)-
ones (6). Example: 2-(2-cyclopentylidenehydrazinyl)-6-(3-
nitrophenyl)pyrimidin-4(3H)-one (6f)
d
A mixture of 2-hydrazinyl-6-(3-nitrophenyl)pyrimidin-4(3H)-
one 9b (80 mg, 0.32 mmol), cyclopentanone (0.14 mL, 0.14 g,
1.6 mmol) and a catalytic amount of glacial acetic acid in dry
ethanol (2 mL) was heated at reflux for 2 h. After cooling at room
temperature, from the reaction mixture a white solid separated,
which was collected by filtration, washed with ethanol and pe-
troleum ether to provide the desired compound as a white solid
finally purified by recrystallization from ethanol.
6.7. 6-(4-Chlorophenyl)-2-(2-cyclopentylidenehydrazinyl)
pyrimidin-4(3H)-one (6g)
White powder, yield 72%, mp 238e240 ꢀC; 1H NMR (DMSO-d6)
ppm 1.69e1.74 (m, 2H, cyclopentyl ring), 1.76e1.81 (m, 2H,
d
cyclopentyl ring), 2.39e2.43 (m, 4H, cyclopentyl ring), 6.26 (s, 1H,
CH-pyrimidine), 7.52 (d, 2H, Ar), 8.03 (d, 2H, Ar), 10.41 (s, 2H, NH).
Anal. Calcd for C15H15ClN4O: C, 59.51%; H, 4.99%; N, 18.51%. Found C,
59.76%; H, 5.12%; N, 18.24%.
6.1. 6-Phenyl-2-(2-(propan-2-ylidene)hydrazinyl)pyrimidin-4(3H)-
one (6a)
White powder, yield 78%, mp 232e234 ꢀC; 1H NMR (DMSO-d6)
6.8. 6-(4-Bromophenyl)-2-(2-cyclopentylidenehydrazinyl)
d
ppm 1.97 (s, 3H, CH3), 2.02 (s, 3H, CH3), 6.24 (s, 1H, CH-
pyrimidin-4(3H)-one (6h)
pyrimidine), 7.45e7.47 (m, 3H, Ar), 7.99e8.01 (m, 2H, Ar), 10.49
(s, 2H, NH). Anal. Calcd for C13H14N4O: C, 64.45%; H, 5.82%; N,
23.13%. Found C, 64.78%; H, 5.96%; N, 22.81%.
White powder, yield 88%, mp 237e240 ꢀC; 1H NMR (DMSO-d6)
d
ppm 1.67e1.81 (m, 4H, cyclopentyl ring), 2.40e2.41 (m, 4H,
cyclopentyl ring), 6.26 (s, 1H, CH-pyrimidine), 7.66 (m, 2H, Ar), 7.96
(m, 2H, Ar), 10.44 (s, 2H, NH). Anal. Calcd for C15H15BrN4O: C,
51.89%; H, 4.35%; N, 16.14%. Found C, 52.17%; H, 4.46%; N, 15.97%.
6.2. 6-(3-Nitrophenyl)-2-(2-(propan-2-ylidene)hydrazinyl)
pyrimidin-4(3H)-one (6b)
White powder, yield 75%, mp 217e219 ꢀC; 1H NMR (DMSO-d6)
d
ppm 1.97 (s, 3H, CH3), 2.02 (s, 3H, CH3), 6.32 (s, 1H, CH-
6.9. 2-(2-(3-Methylcyclopentylidene)hydrazinyl)-6-
pyrimidine), 7.29e7.33 (m, 1H, Ar), 7.47e7.53 (m, 1H, Ar), 7.82e
7.87 (m, 2H, Ar), 10.47e10.54 (s, 2H, NH). Anal. Calcd for
phenylpyrimidin-4(3H)-one (6i)
White powder, yield 73%, mp 202e204 ꢀC; 1H NMR (DMSO-d6)
C
13H13N5O3: C, 54.35%; H, 4.56%; N, 24.38%. Found C, 54.11%; H,
4.39%; N, 24.61%.
d
ppm 1.03 (m, 3H, CH3), 1.24e1.29 (m, 1H, cyclopentyl ring), 1.93e
2.14 (m, 3H, cyclopentyl ring), 2.28e2.38 (m, 1H, cyclopentyl ring),
2.51e2.69 (m, 2H, cyclopentyl ring), 6.22 (s,1H, CH-pyrimidine),
7.46 (m, 3H, Ar), 8.00 (m, 2H, Ar), 10.39 (s, 2H, NH). Anal. Calcd
for C16H18N4O: C, 68.06%; H, 6.43%; N, 19.84%. Found C, 68.29%; H,
6.54%; N, 19.62%.
6.3. 6-(4-Chlorophenyl)-2-(2-(propan-2-ylidene)hydrazinyl)
pyrimidin-4(3H)-one (6c)
White powder, yield 81%, mp 237e239 ꢀC; 1H NMR (DMSO-d6)
d
ppm 1.96 (s, 3H, CH3), 2.01 (s, 3H, CH3), 6.26 (s, 1H, CH-
pyrimidine), 7.51e7.54 (d, 2H, Ar), 8.02e8.05 (d, 2H, Ar), 10.50e
10.62 (s, 2H, NH). Anal. Calcd for C13H13ClN4O: C, 56.42%; H, 4.74%;
N, 20.25%. Found C, 56.74%; H, 4.89%; N, 20.02%.
6.10. 2-(2-(3-Methylcyclopentylidene)hydrazinyl)-6-(3-
nitrophenyl)pyrimidin-4(3H)-one (6j)
White powder, yield 69%, mp 210e212 ꢀC; 1H NMR (DMSO-d6)
6.4. 6-(4-Bromophenyl)-2-(2-(propan-2-ylidene)hydrazinyl)
d ppm 1.02e1.06 (m, 3H, CH3), 1.27e1.41 (m, 1H, cyclopentyl ring),
pyrimidin-4(3H)-one (6d)
1.91e2.07 (m, 3H, cyclopentyl ring), 2.31e2.59 (m, 3H, cyclopentyl
ring), 6.31 (s, 1H, CH-pyrimidine), 7.29e7.32 (m, 1H, Ar), 7.47e7.51
(m, 1H, Ar), 7.81e7.86 (m, 2H, Ar), 10.35e10.47 (s, 2H, NH). Anal.
Calcd for C16H17N5O3: C, 58.71%; H, 5.23%; N, 21.39%. Found C,
58.47%; H, 5.11%; N, 21.64%.
White powder, yield 69%, mp 238e240 ꢀC; 1H NMR (DMSO-d6)
d
ppm 1.96 (s, 3H, CH3), 2.01 (s, 3H, CH3), 6.26 (s, 1H, CH-
pyrimidine), 7.66 (d, 2H, Ar), 7.96 (d, 2H, Ar), 10.53 (s, 2H, NH).

S. Carradori et al. / European Journal of Medicinal Chemistry 80 (2014) 569e578
577
6.11. 6-(4-chlorophenyl)-2-(2-(3-methylcyclopentylidene)
8.4. Differentiation
hydrazinyl)pyrimidin-4(3H)-one (6k)
The cells after centrifugation were resuspended in 10
m
L of
White powder, yield 79%, mp 210e212 ꢀC; 1H NMR (DMSO-d6)
ppm 1.16 (m, 3H, CH3), 1.47e1.52 (m, 1H, cyclopentyl ring), 1.91
(m,1H, cyclopentyl ring), 2.01e2.19 (m, 2H, cyclopentyl ring), 2.36e
2.40 (m, 1H, cyclopentyl ring), 2.57e2.70 (m, 2H, cyclopentyl ring),
6.26 (s, 1H, CH-pyrimidine), 7.52 (d, 2H, Ar), 8.03 (d, 2H, Ar), 10.34e
10.41 (s, 2H, NH). Anal. Calcd for C16H17ClN4O: C, 60.66%; H, 5.41%;
N, 17.69%. Found C, 60.94%; H, 5.57%; N, 17.33%.
conjugate phycoerythrine CD11c (CD11c-PE, PharMingen). Control
samples were incubated with 10 L of mouse IgG1 PE conjugated;
after incubation for 1 h at 4 ꢀC in the dark, the cells were washed in
PBS and resuspended in 500 L PBS containing propidium iodide
(0.25 g/mL). The samples were then analyzed according to stan-
d
m
m
m
dard flow cytometry (FACSCalibur, BD).
Acknowledgements
6.12. 6-(4-bromophenyl)-2-(2-(3-methylcyclopentylidene)
hydrazinyl)pyrimidin-4(3H)-one (6l)
This work was supported by FIRB RBFR10ZJQT, Sapienza Ateneo
Project 2012, IIT-Sapienza Project, AIRC nꢀ 11812, FP7 Project
BLUEPRINT/282510 and COST/TD0905.
White powder, yield 70%, mp 215e217 ꢀC; 1H NMR (DMSO-d6)
d
ppm 1.04 (d, 3H, CH3), 1.35e1.40 (m, 1H, cyclopentyl ring), 1.96e
2.06 (m, 3H, cyclopentyl ring), 2.30e2.37 (m, 1H, cyclopentyl ring),
2.53e2.58 (m, 2H, cyclopentyl ring), 6.26 (s, 1H, CH-pyrimidine),
7.66 (d, 2H, Ar), 7.96 (d, 2H, Ar), 10.36e10.48 (s, 2H, NH). Anal.
Calcd for C16H17BrN4O: C, 53.20%; H, 4.74%; N, 15.51%. Found C,
53.58%; H, 4.87%; N, 15.13%.
Appendix A. Supplementary data
Supplementary data related to this article can be found at http://
dx.doi.org/10.1016/j.ejmech.2014.04.042.
References
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mM) and [Ace-
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mM) as an acetyl donor in reaction
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containing 5% CO2 in RPMI-1640 medium (SIGMA) supplemented
with 10% heat-inactivated fetal bovine serum (HYCLONE), penicillin
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