Identification of structural features of 2-alkylidene-1,3-dicarbonyl derivatives that induce inhibition and/or activation of histone acetyltransferases KAT3B/p300 and KAT2B/PCAF

Article abstract of DOI:10.1002/cmdc.201402371

Dysregulation of the activity of lysine acetyltransferases (KATs) is related to a variety of diseases and/or pathological cellular states; however, their role remains unclear. Therefore, the development of selective modulators of these enzymes is of paramount importance, because these molecules could be invaluable tools for assessing the importance of KATs in several pathologies. We recently found that diethyl pentadecylidenemalonate (SPV106) possesses a previously unobserved inhibitor/activator activity profile against protein acetyltransferases. Herein, we report that manipulation of the carbonyl functions of a series of analogues of SPV106 yielded different activity profiles against KAT2B and KAT3B (pure KAT2B activator, pan-inhibitor, or mixed KAT2B activator/KAT3B inhibitor). Among the novel compounds, a few derivatives may be useful chemical tools for studying the mechanism of lysine acetylation and its implications in physiological and/or pathological processes.

Full text of DOI:10.1002/cmdc.201402371

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DOI: 10.1002/cmdc.201402371
Identification of Structural Features of 2-Alkylidene-1,3-
Dicarbonyl Derivatives that Induce Inhibition and/or
Activation of Histone Acetyltransferases KAT3B/p300 and
KAT2B/PCAF
Sabrina Castellano,[a] Ciro Milite,[a] Alessandra Feoli,[a] Monica Viviano,[a] Antonello Mai,[b]
Ettore Novellino,[c] Alessandra Tosco,*[a] and Gianluca Sbardella*[a]
Dedicated to Prof. Marino Artico on the occasion of his 80th birthday.
Dysregulation of the activity of lysine acetyltransferases (KATs)
is related to a variety of diseases and/or pathological cellular
states; however, their role remains unclear. Therefore, the de-
velopment of selective modulators of these enzymes is of para-
mount importance, because these molecules could be invalua-
ble tools for assessing the importance of KATs in several path-
ologies. We recently found that diethyl pentadecylidenemalo-
nate (SPV106) possesses a previously unobserved inhibitor/acti-
vator activity profile against protein acetyltransferases. Herein,
we report that manipulation of the carbonyl functions of
a series of analogues of SPV106 yielded different activity pro-
files against KAT2B and KAT3B (pure KAT2B activator, pan-in-
hibitor, or mixed KAT2B activator/KAT3B inhibitor). Among the
novel compounds, a few derivatives may be useful chemical
tools for studying the mechanism of lysine acetylation and its
implications in physiological and/or pathological processes.
Introduction
Lysine acetylation was first identified nearly 50 years ago[1] and
has gained interest over the past two decades. Through cross-
talk with other prominent posttranslational modifications (e.g.,
iterative methylation),[2] lysine acetylation gives rise to a com-
plex network, named the “histone code”,[2b,3] that affects the
structure of nucleosomes and is crucial for the modulation of
chromatin-based transcriptional control and shaping inherita-
ble epigenetic programs.[2a,4] In fact, gene regulation is corre-
lated with the acetylation state of histones; transcriptionally re-
pressed genes are associated with hypoacetylated histones,
and transcriptionally active genes are associated with hyper-
acetylated histones. Similar crosstalk, named the “protein
modification code,”[3a,5] between acetylation and other post-
translational modifications occurs in many non-histone pro-
teins that have a diverse range of functions,[6] including tran-
scription factors (i.e., p53),[7] other nuclear regulators (i.e., a-tu-
bulin),[8] and cytoplasmic proteins.[5,9] As a result, lysine acetyla-
tion appears to have a crucial role not only in the nucleus but
also in the regulation of different cytoplasmic processes, in-
cluding cytoskeleton dynamics, energy metabolism, endocyto-
sis, autophagy, and signaling from the plasma membrane.[10]
Moreover, reversible lysine acetylation may alter enzymatic ac-
tivity to allow cells to respond to environmental changes in
metabolic demands, and it has been proposed to be an evolu-
tionarily conserved mechanism for the regulation of cellular
functions.[11]
The acetylation level of proteins is controlled by the action
of lysine acetyltransferases (KATs)[12] and deacetylases
(KDACs),[13] and its deregulation has been linked to several dis-
eases and disorders, including cancer, inflammation and meta-
bolic and neurodegenerative diseases.[14] A number of KDAC
inhibitors are currently undergoing clinical evaluation for effi-
cacy in the treatment of human tumors,[15] and two of these in-
hibitors, suberoylanilide hydroxamic acid (SAHA, vorinostat)[16]
and FK228 (FR901228, romidepsin),[17] have been recently ap-
proved by the US Food and Drug Administration (FDA) for clin-
ical use in refractory cutaneous T-cell lymphoma.
[a] Prof. Dr. S. Castellano, Dr. C. Milite, A. Feoli, Dr. M. Viviano,
Prof. Dr. A. Tosco, Prof. Dr. G. Sbardella
Dipartimento di Farmacia, Universitꢀ degli Studi di Salerno
Via Giovanni Paolo II 132, 84084 Fisciano (SA) (Italy)
E-mail: tosco@unisa.it
In contrast, KATs are less validated as chemotherapeutic tar-
gets. These enzymes can be organized into families (Figure 1)
based on primary structure homology within the catalytic
domain and the biochemical mechanism of acetyl transfer. Sev-
eral KAT families have been identified, but only four have been
extensively studied: the Gcn5-related N-acetyltransferase
(GNAT) family (KAT2), the E1A-associated 300 kDa protein
(p300)/CREB-binding protein (CBP) family (KAT3), the MYST
gsbardella@unisa.it
[b] Prof. Dr. A. Mai
Dipartimento di Chimica e Tecnologie del Farmaco
“Sapienza” Universitꢀ di Roma, P.le A. Moro 5, 00185 Rome (Italy)
[c] Prof. E. Novellino
Dipartimento di Farmacia, Universitꢀ di Napoli “Federico II”
Via D. Montesano 49, 80131 Naples (Italy)
Supporting information for this article is available on the WWW under
http://dx.doi.org/10.1002/cmdc.201402371.
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tors have been described
(Figure 2), with various degrees
of selectivity and cell permeabili-
ty. Mechanism-based peptide–
CoA conjugates (Lys–CoA and
H3–CoA-20) have been reported
as potent and selective bisub-
strate inhibitors for KAT3B (p300)
and KAT2B (PCAF). Unfortunately,
these conjugates are not meta-
bolically stable and have poor
cell permeability.[23a,25] Ethnome-
dicine has inspired the identifica-
tion of a few natural products,
including anacardic acid,[26] garci-
nol,[27] curcumin,[28] plumbagin,[29]
and guttiferone A,[30] and some
analogues[31] or semi-synthetic
derivatives,[20c,32] as inhibitors of
different classes of KATs.
Isothiazolones[33] and pyridoi-
sothiazolones[34] were reported
to be cell-permeable, potent in-
hibitors of KAT2B, but their
chemical reactivity (and limited
solubility in the case of pyridoi-
sothiazolones) limits their specif-
icity. Recently, a virtual screening
approach led to the identifica-
tion of pyrazolone derivatives as
selective KAT3B inhibitors.[24b,35]
Cell-permeable KAT2A (GCN5)-
selective inhibitors, such as g-bu-
tyrolactone MB-3[36] and a few
quinoline derivatives,[37] have
also been reported.
Figure 1. Phylogenetic tree of KAT enzymes and their relationships with diseases. The sizes of the colored circles
represent the number of articles (according to NCBI PubMed at the date of manuscript submission) associated
with a specific disease based on the presence of keywords in the abstract or MeSH terms of PubMed entries. This
tree was obtained from the Structural Genomic Consortium ChromoHub[21] and modified with Adobe Illustrator
CS5.
In addition to inhibitors, a few
KAT enzyme activators have also
family (KAT6), and the regulation of Ty1 transposition gene
product 109 (Rtt109) family (KAT11).[5,12,14c,18]
been reported. For example, anacardic acid-inspired N-(4-
chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-benza-
mide (CTPB, Figure 3)[26] and garcinol-related nemorosone[30]
were reported to selectively activate p300 HAT activity. Interest-
ingly, a series of CTPB analogues showed that the selectivity of
these compounds toward p300 and PCAF is dependent on the
length of the C6-aliphatic chain and the presence of terminal
polar groups.[38]
Dysregulation of the activity of KAT enzymes has been relat-
ed to a variety of diseases and/or pathological cellular states
(infographic in Figure 1).[14] In particular, the GCN5/PCAF and
CBP/p300 paralogue pairs have been pursued as potential
therapeutic targets in cancer,[19] inflammation,[20] viral dis-
eases,[18e,20c] cardiomyopathies,[22] and disturbances in neuronal
cell plasticity and differentiation.[23] However, at least in cancer,
their role cannot be simply generalized, because they can
function as either tumor suppressors or promoters depending
on the tumor type and development stage.[11a,14a] Therefore,
cell-permeable, selective modulators of these enzymes may be
important tools for reverse chemical genetics studies[24] and as-
sessing the implication of KATs in several pathologies. More-
over, these modulators can represent starting points for the
design of novel epigenetic drugs.
We recently reported a series of long-chain alkylidenemalo-
nates (LoCAMs), which were inspired by the structural simplifi-
cation of anacardic acid.[39] One of these compounds, diethyl
pentadecylidenemalonate 1 (SPV106, Figure 3), has a unique
activity profile. This compound exhibits inhibitory properties
against KAT3A/3B (CBP/p300) with a potency comparable to
that of anacardic acid, and it simultaneously enhances the ace-
tylating activity of KAT2B. Therefore, it is the first mixed activa-
tor/inhibitor of protein acetyltransferases.[39]
Different approaches have been used to identify KAT modu-
lators,[14c] but only a limited number of small molecule inhibi-
As a result of its peculiar activity profile, derivative 1 was
successfully used as a chemical probe[40] in a study that corre-
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tion of the extinction of condi-
tioned fear and neuronal plastici-
ty[23f] and the role of KAT2B ace-
tyltransferase activity in the reg-
ulation of nitroglycerin-depen-
dent arterial relaxation[22a] and
wound healing.[41] More recently,
treatment with compound 1 has
been shown to reverse altera-
tions in human cardiac mesen-
chymal cells obtained from dia-
betic patients and restore cellu-
lar function.[22c]
The uncommon biological
properties of derivative
1
prompted us to explore the
structure–activity relationships of
LoCAMs. First, we focused on
the alkyl chain and the flexibility
of the scaffold.[39a] Similar to
what was previously reported for
MB-3[36] and CTPB analogues,[38]
we found that variations in the
alkyl chain length influenced the
activity profile of KAT modula-
tors, whereas all other modifica-
tions, such as variations in flexi-
bility/rigidity of the core struc-
ture and the introduction of sub-
stituents, were detrimental.[39a]
Indeed, not only the selectivity
toward different KAT enzymes,
but also the inhibitory and acti-
vating properties, varied de-
pending on the substitution pat-
tern.[39a]
As a further exploration of the
structure–activity relationships of
this class of KAT modulators, and
with the aim of identifying po-
tential structural features that
differentially affect the activity of
acetyltransferases, we herein de-
scribe the synthesis of a number
of 1,3-dicarbonyl derivatives
(Figure 4), formally derived by
the replacement of one or both
ester functions with keto- or car-
boxylic acid groups while keep-
ing the alkyl chain length con-
stant. In addition, because we
previously found that the diketo
analogue of compound 1 (deriva-
tive 2d, Figure 4) retained inhibi-
tory activity against KAT3A,[39a] we also prepared a few shorter-
and longer-chain homologues of 2d.
Figure 2. Different classes of KAT inhibitors.
lated Duchenne cardiomyopathy with KAT2B-mediated lysine
acetylation levels of connexin 43.[22b] It was also examined
during investigations of the role of KAT enzymes in the regula-
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Results and Discussion
Chemistry
Novel alkylidene derivatives 2–5 were prepared in
a straightforward manner (Scheme 1) by Knoevenagel
condensation of a 1,3-dicarbonyl derivative with the
appropriate alkyl aldehydes,[39a] which were commer-
cially available or obtained from the corresponding
alcohol by oxidation with 2,2,6,6-tetramethylpiperi-
dine 1-oxyl (TEMPO), according to previously pub-
lished procedures.[43] Derivatives 2a–e,[44] 3a–d,[45]
and 4a,b were prepared by reacting, respectively,
pentane-2,4-dione, ethyl 3-oxobutanoate, tert-butyl
3-oxobutanoate, or malonic acid with the appropriate
aldehyde in dichloromethane using piperidine and
acetic acid as the catalysts (Scheme 1). The hydrolysis
of tert-butyl esters 3c,d with trifluoroacetic acid (TFA)
yielded the corresponding 2-alkylidene-3-oxobutano-
ic acids 5a,b.
Figure 3. KAT activators.
Notably, the Knoevenagel condensation of ethyl 3-
oxobutanoate and tert-butyl 3-oxobutanoate with
alkyl aldehydes furnished derivatives 3a,b and 3c,d,
respectively, as mixtures of E and Z isomers, which
were successfully separated by silica gel chromatog-
raphy. However, interconversion spontaneously oc-
curred, even at low temperatures. For this reason,
compounds 3a–d and the corresponding acids 5a,b
were biologically evaluated as the E/Z mixtures.
Copies of the NMR spectra of the separated isomers
of 3d are provided as representative examples in the
Supporting Information.
Alkylthiomethylidenepentane-2,4-diones 6a,b and
alkylaminomethylidenepentane-2,4-diones
8a–f[46]
were obtained in high yield (Scheme 2) by reacting,
respectively, the appropriate alkyl amine or alkyl thiol
with 3-(ethoxymethylene)-2,4-pentanedione 9 in THF
at reflux.[47] Alkyloxymethylene-2,4-pentanediones
7a,b were obtained in good yield (calculated by NMR
analysis of the crude reaction mixture) under the
same conditions from the corresponding alkyl alco-
hols. Unfortunately, compounds 7a,b were sensitive
to nucleophiles, including negligible amounts of
water. Therefore, any attempt to purify these deriva-
tives by chromatography was unsuccessful. Moreover,
because an aqueous medium is required for biologi-
cal screening, we abandoned any further purification
efforts.
Figure 4. 1,3-Dicarbonyl derivatives 2a–e, 3a–d, 4a,b, 5a,b, 6a,b, 7a,b, and 8a–f.
To avoid time-consuming, expensive assays, all compounds
were preliminarily screened using a surface plasmon reso-
nance-based binding assay, which we recently reported,[30,39a,42]
to study the interactions between small molecules and epige-
netic enzymes. The effects of selected derivatives on the cata-
lytic activity of the KAT2B and KAT3B enzymes was then deter-
mined, and the biological effects on different cell lines were
evaluated.
SPR-based binding assays
The most sensitive histone acetyltransferase assays typically in-
volve radioactivity. However, these methods tend to be costly,
particularly those involving radiolabeled acetyl-CoA, and re-
quire special handling precautions and radioactive disposal. In
contrast, non-radioactive spectrophotometric assays are rather
quick, safe, and inexpensive, but they are plagued by signifi-
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all compounds were injected on an immobilized
myoglobin. The binding of each compound was read
in real time as the change in mass at the sensor sur-
face. After injection, running buffer was allowed to
flow over the surface, and the dissociation of com-
pounds from the surface was observed (Figure 5).
Equilibrium dissociation constant (KD) values were de-
rived from the ratio between kinetic dissociation (kd)
and association (ka) constants, obtained by fitting
data from all injections at different concentrations of
each compound using the simple 1:1 Langmuir bind-
ing fit model of the BIAevaluation software.
Tetradecylidene- and pentadecylidene-substituted
pentane-2,4-diones, malonic acids, and 3-oxobutano-
ic acids (2c,d, 4a,b, and 5a,b, respectively) efficiently
interacted with the immobilized proteins as demon-
strated by the concentration-dependent responses
and the clearly evident exponential curves during
both the association and dissociation phases (the
sensorgrams for compounds 2d, 4b, and 5b are dis-
played in Figure 5). However, both 3-oxobutanoates
3a–d and derivatives 6–8, which are characterized by
the presence of a heteroatom in their aliphatic
chains, showed negligible interaction (not shown). In
addition, in agreement with what we previously re-
ported for LoCAMs,[39a] we found that variations in
the alkyl chain length influenced the binding profile
of the tested pentane-2,4-diones 2a–e. In fact,
superior (2e) and inferior (2a,b) homologues
also produced good sensorgrams but showed low
and/or concentration-independent responses (not
shown).
Scheme 1. Reagents and conditions: a) alkyl aldehyde, AcOH, piperidine, CH2Cl2, RT,
2–12 h; b) TFA/CH2Cl2 (5:95), RT, 30 min.
Scheme 2. Reagents and conditions: a) alkyl amine, thiol, or alcohol (n=11, 12), THF,
reflux, 2 h.
cantly lower sensitivity (enzyme-coupled assays), interference
from compound spectral density, autofluorescence, and/or re-
action of the enzyme-free sulfhydryl groups or substrates/
products with common coumarin reporters.[48]
Histone acetyltransferase IC50 profiling
After application of the SPR-based ligand filtering procedure,
the effect of the selected derivatives (SPR+) 2c,d, 4a,b, and
5a,b on the catalytic activity of the human recombinant acetyl-
transferase enzymes KAT3B and KAT2B was determined in Hot-
Spot HAT activity assays, which were performed by Reaction
Biology Corporation (Malvern, PA, USA) according to the com-
pany’s standard operating procedure, using curcumin[28] and
anacardic acid (AA),[26] respectively, as reference compounds.
Two negative controls (SPRꢀ, 6b and 8b) were also tested to
cross-validate the selection method.
Surface plasmon resonance (SPR) biosensor-based assays are
well suited to overcome many of the limitations of traditional
assays and are increasingly important in medicinal chemistry
for hit/lead identification and the optimization process.[49] Re-
cently, we established an SPR-based binding assay and suc-
cessfully employed it to study the real-time kinetic and ther-
modynamic parameters of ligand–protein interactions between
small-molecule derivatives and KAT3B[39] as well as other epige-
netic targets.[42]
As shown in Table 1, all of the SPR+ derivatives substantially
affected the activity of one or both of the HAT enzymes. In par-
ticular, pentane-2,4-diones 2c,d induced a marked dose-depen-
dent increase in the enzymatic activity of KAT2B (HAT activity
at 100 mm: 229% and 389%, respectively; Table 1), while they
did not affect the activity of KAT3B. In contrast, malonic acids
4a,b strongly inhibited both enzymes, with IC50 values in the
low micromolar range (1.3 and 1.1 mm, respectively, for KAT3B,
and 50.3 and 21.1 mm, respectively, for KAT2B; Table 1 and
Figure 6), which are similar to or more potent than the refer-
ence compounds (IC50: 6.5 mm and 33.9 mm for curcumin and
AA, respectively; see Table 1).
Therefore, we used this assay to select compounds capable
of binding to at least one of the acetyltransferase enzymes
(KAT2B and/or KAT3B). Thus, the human recombinant KAT3B
(aa 1284–1673) and KAT2B (aa 492–658) catalytic domains were
immobilized (up to ~10000 response units (RU)) on different
flow cells of the biosensor chip, and 1,3-dicarbonyl derivatives
2–8 were injected at various concentrations (from 25–100 mm)
over the protein surface. To decrease false positives from de-
tergent-sensitive, nonspecific aggregation-based binding,
0.005% NP20 was added to the running buffer in all experi-
ments. In addition, to evaluate potential nonspecific binding,
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Figure 5. Sensorgrams obtained from SPR interaction analysis of compounds 2d (left), 4b (middle), and 5b (right) binding to immobilized a) hKAT3B/p300
(catalytic domain, aa 1284–1673) and b) hKAT2B/PCAF (catalytic domain, aa 492–658). Each compound was injected at four different concentrations (25, 50,
75, and 100 mm). Equilibrium dissociation constants (KD) were derived from the ratio between kinetic dissociation (kd) and association (ka) constants obtained
by fitting data from all of the injections at various concentrations of each compound using the BIAevaluation software package (version 4.1) and the simple
1:1 Langmuir binding model.
Notably, 3-oxobutanoic acids 5a,b efficiently
Table 1. Effects of compounds 2c,d, 4a,b, 5a,b, 6b, and 8b on the activity of KAT2B and
inhibited KAT3B (IC50 values of 2.4 mm and
4.7 mm, respectively) but also caused strong
amplification of KAT2B enzymatic activity in
a dose-dependent manner (HAT activity at
100 mm: 346 and 497%, respectively; Table 1),
and this effect was even more significant than
that observed for pentane-2,4-diones 2c,d
(see above). In accordance with the SPR ex-
periments, the negative controls negligibly af-
fected the enzymatic activity of KAT2B and
KAT3B (see 6b and 8b in Table 1).
KAT3B.[a]
Compd
IC50 [mm][b] or Activity [%][c,d]
p300/KAT3B[e]
PCAF/KAT2B[e]
1
2c
2d
4a
4b
5a
5b
6b
18.8ꢁ1.2%
NI[f]
137ꢁ5%
229ꢁ7%
389ꢁ9%
50.3ꢁ0.4[b]
21.1ꢁ0.5[b]
346ꢁ9%
497ꢁ12%
NI[f]
NI[f]
1.3ꢁ0.1[b]
1.1ꢁ0.1[b]
2.4ꢁ0.1[b]
4.7ꢁ0.3[b]
NI[f]
Biological effects on various tumor cell lines
The aberrant regulation (or altered expression)
of histone-modifying enzymes may differen-
tially affect the level of histone modifications
in different cells, and this may influence both
normal biology and disease processes. Indeed,
different histone modification (comprising ace-
tylation) signatures have been recently de-
scribed for a number of human cancer cell
lines.[50] Therefore, we decided to examine the
effects of tested compounds on three different
cell lines, namely human leukemic monocyte
lymphoma U937 cells and human cervical car-
8b
NI[f]
NI[f]
curcumin[g]
AA[g]
6.5ꢁ0.2[b]
NT[h]
33.9ꢁ0.7[b]
NT[h]
[a] Compounds were tested in 10-dose IC50 mode with threefold serial dilutions starting at
100 mm. [b] Data were analyzed with GraphPad Prism software (version 6.0) for IC50 curve fits.
[c] Enzyme activity percentage determined at 100 mm with respect to DMSO. [d] Values are
the means ꢁSD determined for at least two separate experiments and are indicated in per-
centage points. [e] Histone H3 was used as substrate (5 mm), and [acetyl-3H]acetyl coenzyme A
(3.08 mm) was used as an acetyl donor. [f] Negligible inhibition or activation. [g] Control com-
pounds curcumin and anacardic acid were tested in a 10-dose IC50 mode with threefold serial
dilutions starting at 100 mm. [h] Not tested.
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the histone extracts were then immunoblotted with antibodies
to specific histone acetylation sites (Figure 8). Consistent with
the different patterns of acetylation recently described by
Garcia and co-workers,[50] we observed that the tested com-
pounds differentially affected the global H4 acetylation level
(Figure S2, Supporting Information) as well as the H4K5ac[53]
and H3K9ac levels in the three cell lines. In fact, the effect of
pentane-2,4-dione 2d on both markers was negligible in U937
cells (Figure 8, row c, left and middle panels), whereas a de-
crease was observed in both cervical carcinoma cell lines,
which was more evident for H4K5ac (Figure 8, rows a and b).
Both malonic acid 4b and 3-oxobutanoic acid 5b induced
a marked decrease in the acetylation of lysine H4K5 in C33A
cells (Figure 8, row a, left panel), whereas no appreciable effect
on the same marker was detected in HeLa cells, and an in-
crease was observed in U937 cells (Figure 8, rows b and c, re-
spectively).
In contrast, derivative 4b induced a moderate decrease in
the acetylation of H3K9 in C33A and HeLa cells (Figure 8,
rows a and a, respectively, middle panels), and there was no
appreciable variation in U937 cells (Figure 8, row c, middle
panel), whereas 5b induced a decrease in the acetylation of
H3K9 in C33A and HeLa cells (Figure 8, rows a and b, respec-
tively, middle panels) and a marked increase in the acetylation
level in U937 cells (Figure 8, row c, middle panel). As expected,
treatment with the reference compound SAHA reliably showed
a significant increase in the lysine acetylation level (Figure 8).
Figure 6. Dose–response inhibition of a) KAT3B/p300 and b) KAT2B/PCAF by
compounds 4a,b and 5a,b. Compounds were tested in 10-dose IC50 mode
with threefold serial dilutions starting at 100 mm. Data were analyzed with
GraphPad Prism software (version 6.0) for curve fits using a sigmoidal dose–
response with a variable slope equation.
cinoma C33A and HeLa cells. First, an MTT assay was per-
formed after treatment with SPR+ derivatives to assess the
maximum concentration of compounds that could be used
without significantly affecting cell viability. For solid C33A and
HeLa tumor cell lines, we observed no significant decrease in
the number of metabolically active cells after 24 h of treatment
with concentrations up to 200 mm for derivatives 2c,d and
4a,b and up to 50 mm for compounds 5a,b (data not shown).
On the other hand, in the case of the more sensitive U937 cell
line (Figure 7a), we registered a significant decrease in cell via-
bility starting from lower concentrations of tested compounds
(100 mm for 2c,d and 4a,b and 10 mm for 5a,b, respectively).
All of the SPR+ derivatives were then screened for their ef-
fects on the cell cycle using the U937 cell line. After 24 h of
treatment, compounds 4b, 5a, and 5b were able to arrest the
cell cycle in the G1 phase (Figure 7b). In the case of the latter
two compounds, this occurred at concentrations as low as
10 mm and induced an increase in the number of sub-G1 hypo-
diploid nuclei, as detected by the Nicoletti method (Figure S1,
Supporting Information).[51] This result is consistent with the
importance of acetylation for control of the G1/S transi-
tion.[19i,52] Under the same conditions, the other derivatives had
no significant effects on the cell cycle.
Conclusions
Lysine acetylation is a reversible process, resulting from the op-
posing actions of the KDAC and KAT enzymes, which is crucial
for the regulation of a number of cellular functions, including
chromatin remodeling, gene expression, cytoskeleton dynam-
ics, energy metabolism, endocytosis, autophagy, and even sig-
naling from the plasma membrane. Dysregulation of the activi-
ty of KAT enzymes has been related to a variety of diseases
and/or pathological cellular states including cancer, inflamma-
tion, viral diseases, cardiomyopathies, and disturbances in neu-
ronal cell plasticity and differentiation, yet their role is still un-
clear. Therefore, the development of selective modulators of
these enzymes is of paramount importance, because such mol-
ecules could be invaluable tools for assessing the implication
of KATs in several pathologies.
We recently identified diethyl pentadecylidenemalonate (1;
SPV106) as having a previously unobserved activity profile
against protein acetyltransferases (i.e., the ability to inhibit the
activity of KAT3A/3B and simultaneously enhance that of
KAT2B).[39] Because of this unusual activity profile, an SPV106
derivative was successfully used as a chemical probe to study
the role of KAT enzymes in cardiovascular, neurological, physio-
logical, and/or pathological pathways.[22,23f,41] With the aim of
further investigating structure–activity relationships, we pre-
pared a number of 1,3-dicarbonyl derivatives as analogues of
SPV106, replacing one or both of the ester functions with
keto- or carboxylic acid groups. Taking advantage of a binding
assay recently established by our group,[30,39a,42] we applied
We then examined the effects of compounds 2d, 4b, and
5b on the acetylation levels of specific lysine residues of core
histones H3 (K9) and H4 (K5) in the three cell lines. The effect
on the overall level of H4 acetylation was also observed. Cells
were incubated for 24 h with vehicle and tested with com-
pounds at the indicated concentrations or the reference com-
pound suberoylanilide hydroxamic acid (SAHA; 5 mm),[16] and
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the enzymatic activity of KAT2B and did not affect
the activity of KAT3B; malonic acids 4a,b strongly in-
hibited both enzymes with IC50 values in the low mi-
cromolar range, and 3-oxobutanoic acids 5a,b effi-
ciently inhibited KAT3B but also caused strong am-
plification of the KAT2B enzymatic activity in a dose-
dependent manner. Under the same conditions, SPR-
negative controls negligibly affected the enzymatic
activity of KAT2B and KAT3B. We then decided to
evaluate the effects of derivatives 2d, 4b, and 5b on
the acetylation of lysine residues H4K5 and H3K9 in
C33A, HeLa, and U937 cell lines. Consistent with the
distinct patterns of acetylation (and other histone
modifications) in the various cancer cell lines,[50] we
observed differential modulation of the same lysine
marker induced by the compounds in the different
cell lines. This observation is not surprising, consider-
ing that in the cellular context, acetyltransferases are
similar to other enzymes by not being isolated; they
participate in complex pathways and actively cross-
talk with each other and with other proteins. In par-
ticular, we found that malonic acid 4b and 3-oxobu-
tanoic acid 5b induce a marked decrease in H4K5ac
in C33A cells, whereas a significant increase in
H3K9ac was detected in U937 cells after treatment
with 3-oxobutanoic acid 5b.
The differences observed in the activity profile of
the structurally related compounds 2c,d, 4a,b, and
5a,b against the two acetyltransferases could have
a number of explanations, and the mechanisms un-
derlying their biological effects remain unclear. How-
ever, it has been previously reported that small modi-
fications in structurally related compounds lead to
dramatic differences in terms of selectivity between
KAT2B and KAT3B.[20c] Moreover, is worth mentioning
that the catalytic mechanisms of the two enzymes
are different, with KAT2B using a ternary complex
mechanism,[54] and a Theorell–Chance “hit and run”
model proposed for KAT3B[55] in which the histone
peptide binds weakly to the enzyme–cofactor com-
Figure 7. Cell viability and cell-cycle analysis in U937 cells by fluorescence-activated cell
sorting (FACS). a) Cell viability was assessed by measuring the mitochondrial-dependent
reduction of MTT to formazan. b) U937 cells were treated with compounds 2c,d, 4a,b,
and 5a,b at the indicated concentrations for 24 h, stained with propidium iodide, and
subjected to flow cytometric analysis to determine the distribution of cells in each phase
of the cell cycle. Data are reported as the mean ꢁSD of at least three independent ex-
periments. Statistical significance (relative to controls, Student’s t test): a) p<0.05 for 5a
and 5b at 1 mm and for 4a at 100 mm, p<0.005 for all other compounds and concentra-
tions; b) p<0.005 (G1 phase of cells treated with 4b, 5a, and 5b).
a SPR-based ligand filtering procedure to select compounds
capable of binding to the acetyltransferase enzymes KAT2B
and KAT3B. We found that the presence of a heteroatom in
the aliphatic chain of tested molecules resulted in negligible
interactions and that, similar to what was previously reported
for LoCAMs[39a] by us and other groups for analogues of
MB-3[36] and CTPB,[38] variations in the length of the alkyl chain
greatly influenced the binding profile of the tested derivatives;
tetradecylidene- and pentadecylidene-substituted compounds
interacted efficiently with both of the immobilized proteins,
whereas superior and inferior homologues had low and/or
concentration-independent responses. When tested in a bio-
chemical assay, all of the selected derivatives (SPR+) markedly
affected the catalytic activity of one or both of the KAT en-
zymes, and this effect was related to the nature of the carbon-
yl functions of the molecules. In fact, pentane-2,4-diones 2c,d
induced a marked and concentration-dependent increase in
plex, allowing enough time for the lysine side chain to snake
through the enzyme tunnel and receive the acetyl group.[56]
Furthermore, it has been recently reported that displacement
of the flexible auto-inhibitory loop (AIL) and inhibitory RING
domain enhance the activity of KAT3A/3B enzymes,[57] whereas
another report suggested that KAT2B is a dimer in its function-
al ATAC complex.[58] Ongoing experiments will shed more light
on this issue.
In conclusion, in this study, we obtained different activity
profiles against KAT2B and KAT3B (pure KAT2B activator, pan-
inhibitor, or mixed KAT2B activator/KAT3B inhibitor) by manip-
ulating the carbonyl functions of a series of analogues of
SPV106. Enzyme activators are rarely pursued in chemical biol-
ogy and drug discovery efforts, but they may be invaluable re-
agents alone or in combination with inhibitors.[42] Therefore,
a few of the compounds described, namely derivatives 2c,d
(EML317, EML76), 4a,b (EML341, EML264), and 5a,b (EML333,
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Figure 8. Western blot analyses performed with compounds 2d, 4b, and 5b at the indicated concentrations for 24 h on the acetylation of the specific lysine
residues H4K5 (left column) and H3K9 (middle column) in histone extracts from a) C33A and b) HeLa carcinoma cells, and c) U937 leukemic monocyte lym-
phoma cells. Acetylation was detected by immunoblotting with antibodies specific for histone acetylation sites as indicated. Total histone H3 was used to
check for equal loading. SAHA (5 mm) was used as a reference compound. Signals were detected with the ImageQuant LAS 4000 digital imaging system (GE
Healthcare, Waukesha, WI, USA) and quantified by ImageQuantTL software (version 8.1); total histone H3 levels were used for normalization. The results are
reported as the mean ꢁSD of three independent experiments with the control data set to 1; statistical significance: *p<0.05, **p<0.01, ***p<0.001 relative
to controls (Student’s t test).
TLC performed on aluminum-backed silica gel plates (Merck DC,
Alufolien Kieselgel 60 F254) with spots visualized by UV light (l=
254, 365 nm) or using a KMnO4 alkaline solution. Solvents were re-
EML334), may be useful chemical tools for mechanistic studies
of lysine acetylation and its implications in physiological and/
or pathological processes.
moved using a rotary evaporator operating at a reduced pressure
of ꢂ10 Torr. Organic solutions were dried over anhydrous Na2SO4.
Chromatographic separations were performed on silica gel (silica
gel 60, 0.015–0.040 mm; Merck DC) columns. Melting points were
determined on a Stuart SMP30 melting point apparatus in open ca-
pillary tubes and were uncorrected. Infrared (IR) spectra were re-
corded neat with a Shimadzu IR Affinity-1 FTIR fitted with a MIRacle
10 single reflection ATR accessory at room temperature. 1H and
13C NMR spectra were recorded at 300 MHz and 75 MHz, respec-
tively, with a Bruker Avance 300 spectrometer. Chemical shifts are
reported in d (ppm) relative to the internal reference tetramethylsi-
lane (TMS). Mass spectra were recorded with a Finnigan LCQ DECA
TermoQuest (San Jose, USA) mass spectrometer in electrospray-
Experimental Section
Chemistry
General directions: All chemicals were purchased from Aldrich
Chimica (Milan, Italy) and were of the highest purity. All solvents
were reagent grade and were purified and dried by standard meth-
ods when necessary. All reactions requiring anhydrous conditions
were conducted under a positive atmosphere of nitrogen in oven-
dried glassware. Standard syringe techniques were used for the an-
hydrous addition of liquids. Reactions were routinely monitored by
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positive and -negative ionization modes (ESI-MS). The purity of the
test compounds was established by combustion analysis, confirm-
ing purity ꢃ95%. Elemental analyses (C, H, N) were performed
with a PerkinElmer 2400 CHN elemental analyzer, and the analytical
results were within ꢁ0.4% of the theoretical values (Table S1, Sup-
porting Information).
a and b; 1H NMR (300 MHz, CDCl3): d=6.92 (t, J=7.7 Hz, 1H,
isomer a), 6.85 (t, J=7.7 Hz, 1H, isomer b), 4.31 (q, J=7.1 Hz, 2H,
isomer b), 4.25 (q, J=7.1 Hz, 2H, isomer a), 2.36–2.27 (m, 5H, iso-
mer b and 3H, isomer a), 2.25–2.21 (m, 2H, isomer a), 1.55–1.40 (m,
2H, isomer a and 2H isomer b), 1.30–1.21 (m, 23H, isomer a and
23H isomer b), 0.88 ppm (brt, J=6.3 Hz, 3H, isomer a and 3H, iso-
mer b); IR (neat): n˜ =2922, 2853, 1728, 1701, 1638, 1622 cmꢀ1; ESI-
MS m/z: 325 [M+H]+; Anal. calcd for C20H36O3: C 74.03, H 11.18,
found: C 73.92, H 11.20.
General procedure for the synthesis of derivatives 2a–d: A
stirred solution of the appropriate alkyl aldehyde (4.00 mmol) and
2,4-pentanedione (acetylacetone) (4.40 mmol) in anhydrous meth-
ylene chloride (3 mL) was treated with piperidine (0.08 mmol) and
acetic acid (0.08 mmol) at 08C. The reaction mixture was stirred at
room temperature for 1 h (TLC monitoring), diluted with methyl-
ene chloride (50 mL), washed with brine (3ꢁ10 mL), and dried
(Na2SO4). After removing the solvent, the crude residue was puri-
fied by column chromatography on silica gel (n-hexane/EtOAc) to
afford the title compounds.
Ethyl 2-acetylheptadec-2-enoate (3b, EML330): Low-melting-
point white solid (1056 mg, 78%): 1:1 mixture of geometric iso-
1
mers a and b; H NMR (300 MHz, CDCl3): d=6.91 (t, J=7.7 Hz, 1H,
isomer a), 6.83 (t, J=7.7 Hz, 1H, isomer b), 4.29 (q, J=7.1 Hz, 2H,
isomer b), 4.23 (q, J=7.1 Hz, 2H, isomer a), 2.36–2.27 (m, 5H, iso-
mer b and 3H, isomer a), 2.27–2.18 (m, 2H, isomer a), 1.54–1.40 (m,
2H, isomer a and 2H isomer b), 1.34–1.16 (m, 25H, isomer a and
25H isomer b), 0.86 ppm (brt, J=6.3 Hz, 3H, isomer a and 3H, iso-
mer b); 13C NMR (75 MHz, CDCl3): d=201.2, 195.2, 166.7, 164.8,
149.2, 148.8, 137.7, 137.2, 61.3, 32.0, 30.1, 29.8, 29.6, 29.5, 28.7,
28.5, 27.0, 22.8, 14.3, 14.2 ppm; IR (neat): n˜ =2913, 2851, 1717,
3-Pentylidenepentane-2,4-dione (2a, EML319): Colorless oil
(619 mg, 92%): 1H NMR (300 MHz, CDCl3): d=6.67 (t, J=7.7 Hz,
1H), 2.30 (s, 6H), 2.28–2.15 (m, 2H), 1.52–1.42 (m, 2H), 1.41–1.31
(m, 2H), 0.92 ppm (t, J=7.1 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
203.5, 197.2, 146.9, 145.4, 30.9, 29.5, 26.1, 22.5, 13.9 ppm; IR (neat):
n˜ =2959, 2932, 2862, 1705, 1667, 1628 cmꢀ1; ESI-MS m/z: 169
[M+H]+; Anal. calcd for C10H16O2: C 71.39, H 9.59, found: C 71.54, H
9.61.
1670, 1643 cmꢀ1
;
ESI-MS m/z: 339 [M+H]+; Anal. calcd for
C21H38O3: C 74.51, H 11.31, found: C 74.37, H 11.33.
tert-Butyl 2-acetylhexadec-2-enoate (3c, EML331): Low-melting-
point white solid (1072 mg, 76%): 1:1 mixture of geometric iso-
1
mers a and b; H NMR (300 MHz, CDCl3): d=6.80 (t, J=7.9 Hz, 1H,
3-Dodecylidenepentane-2,4-dione (2b, EML318): Colorless oil
(991 mg, 93%): 1H NMR (300 MHz, CDCl3): d=6.71 (t, J=7.7 Hz,
1H), 2.35 (s, 6H), 2.28–2.18 (m, 2H), 1.53–1.42 (m, 2H), 1.34–1.22
(m, 16H), 0.89 ppm (t, J=6.5 Hz, 3H); IR (neat): n˜ =2955, 2924,
2855, 1705, 1667, 1628 cmꢀ1; ESI-MS m/z: 267 [M+H]+; Anal. calcd
for C17H30O2: C 76.64, H 11.35, found: C 76.83, H 11.38.
isomer a), 6.75 (t, J=7.8 Hz, 1H, isomer b), 2.33–2.27 (m, 5H, iso-
mer b and 3H, isomer a), 2.25–2.14 (m, 2H, isomer a), 1.55–1.40 (m,
11H, isomer a and 11H isomer b), 1.30–1.21 (m, 20H, isomer a and
20H isomer b), 0.88 ppm (brt, J=6.5 Hz, 3H, isomer a and 3H, iso-
mer b); IR (neat): n˜ =2924, 2855, 1724, 1701, 1636, 1620 cmꢀ1; ESI-
MS m/z: 375 [M+Na]+; Anal. calcd for C22H40O3: C 74.95, H 11.44,
found: C 74.79, H 11.43.
3-Tetradecylidenepentane-2,4-dione (2c, EML317): Colorless oil
(1095 mg, 93%): 1H NMR (300 MHz, CDCl3): d=6.66 (t, J=7.7 Hz,
1H), 2.27 (s, 6H), 2.25–2.13 (m, 2H), 1.51–1.40 (m, 2H), 1.28–1.16
(m, 20H), 0.86 ppm (t, J=6.5 Hz, 3H); IR (neat): n˜ =2924, 2855,
1713, 1670, 1624 cmꢀ1; ESI-MS m/z: 295 [M+H]+; Anal. calcd for
C19H34O2: C 77.50, H 11.64, found: C 77.69, H 11.67.
tert-Butyl 2-acetylheptadec-2-enoate (3d, EML332): Low-melting-
point white solid (1056 mg, 72%): 1:1 mixture of geometric iso-
1
mers a and b; H NMR (300 MHz, CDCl3): d=6.80 (t, J=7.9 Hz, 1H,
isomer a), 6.75 (t, J=7.8 Hz, 1H, isomer b), 2.33–2.27 (m, 5H, iso-
mer b and 3H, isomer a), 2.25–2.14 (m, 2H, isomer a), 1.55–1.40 (m,
11H, isomer a and 11H isomer b), 1.30–1.21 (m, 22H, isomer a and
22H isomer b), 0.88 ppm (brt, J=6.5 Hz, 3H, isomer a and 3H, iso-
mer b); IR (neat): n˜ =2913, 2851, 1717, 1686, 1620 cmꢀ1; ESI-MS
m/z: 389 [M+Na]+; Anal. calcd for C23H42O3: C 75.36, H 11.55,
found: C 75.21, H 11.54.
3-Pentadecylidenepentane-2,4-dione (2d, EML76):[39a] Colorless
1
oil (1098 mg, 89%): H NMR (300 MHz, CDCl3): d=6.70 (t, J=7.7 Hz,
1H), 2.34 (s, 6H), 2.28–2.19 (m, 2H), 1.54–1.48 (m, 2H), 1.38–1.26
(m, 22H), 0.99 ppm (t, J=6.3 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=
203.7, 197.2, 147.1, 145.3, 32.1, 31.8, 29.8, 29.6, 29.5, 28.8, 26.2,
26.2, 22.8, 14.3 ppm; IR (neat): n˜ =2916, 2851, 1701, 1664,
1636 cmꢀ1; ESI-MS m/z: 309 [M+H]+; Anal. calcd for C20H36O2: C
77.87, H 11.76, found: C 78.05, H 11.79.
General procedure for the synthesis of derivatives 4a–b: Deriva-
tives 4a–b were prepared by reacting the appropriate alkyl alde-
hyde and malonic acid according to the procedure used for deriva-
tives 2a–e. The crude residue was purified by column chromatog-
raphy on silica gel (CH2Cl2/MeOH) to afford the title compounds.
3-Hexadecylidenepentane-2,4-dione (2e, EML320): Colorless oil
(1238 mg, 96%): 1H NMR (300 MHz, CDCl3): d=6.66 (t, J=7.7 Hz,
1H), 2.28 (s, 6H), 2.26–2.16 (m, 2H), 1.53–1.43 (m, 2H), 1.30–1.23
(m, 24H), 0.86 ppm (t, J=6.3 Hz, 3H); IR (neat): n˜ =2916, 2851,
1701, 1663, 1636 cmꢀ1; ESI-MS m/z: 323 [M+H]+; Anal. calcd for
C21H38O2: C 78.20, H 11.88, found: C 78.41, H 11.91.
2-Tetradecylidenemalonic acid (4a, EML341): White solid
(991 mg, 83%): mp: 69–708C; 1H NMR (300 MHz, [D6]DMSO): d=
6.99 (t, J=7.5 Hz, 1H), 2.42–2.29 (m, 2H), 1.44–1.33 (m, 2H), 1.32–
1.15 (m, 20H), 0.85 ppm (t, J=6.2 Hz, 3H); IR (neat): n˜ =2953,
2918, 2851, 1735, 1720, 1697, 1568 cmꢀ1; ESI-MS m/z: 297 [MꢀH]ꢀ;
Anal. calcd for C17H30O4: C 68.42, H 10.13, found: C 68.27, H 10.15.
General procedure for the synthesis of derivatives 3a–d: Deriva-
tives 3a–d were prepared by reacting the appropriate alkyl alde-
hyde and ethyl 3-oxobutanoate (for compounds 3a,b) or tert-butyl
3-oxobutanoate (for compounds 3c,d), according to the procedure
used for derivatives 2a–e. The crude residue was purified by
column chromatography on silica gel (n-hexane/EtOAc) to afford
the title compounds as a mixture of E and Z isomers.
2-Pentadecylidenemalonic acid (4b, EML264): White solid
(975 mg, 78%): mp: 73–758C; 1H NMR (300 MHz, [D6]DMSO): d=
6.88 (t, J=7.6 Hz, 1H), 2.41–2.27 (m, 2H), 1.49–1.38 (m, 2H), 1.34–
1.14 (m, 22H), 0.85 ppm (t, J=6.3 Hz, 3H); 13C NMR (75 MHz,
DMSO): d=169.8, 167.1, 147.3, 129.8, 31.3, 29.1, 28.8, 22.1,
13.8 ppm; IR (neat): n˜ =2954, 2916, 2848, 1735, 1710, 1697,
Ethyl 2-acetylhexadec-2-enoate (3a, EML329):[45] Low-melting-
point white solid (934 mg, 72%): 1:1 mixture of geometric isomers
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1568 cmꢀ1; ESI-MS m/z: 311 [MꢀH]ꢀ; Anal. calcd for C18H32O4: C
m/z: 327 [M+H]+; Anal. calcd for C19H34O2S: C 69.89, H 10.50, S
9.82, found: C 70.05, H 10.52, S 9.84.
69.19, H 10.32, found: C 69.05, H 10.34.
3-((Dodecylamino)methylene)pentane-2,4-dione (8a, EML321):[46]
Low-melting-point pale-yellow solid (287 mg, 97%): 1H NMR
(300 MHz, CDCl3): d=11.07 (brs, 1H), 7.71 (d, J=12.3 Hz, 1H),
3.38–3.31 (m, 2H), 2.48 (s, 3H), 2.26 (s, 3H), 1.67–1.56 (m, 2H),
1.41–1.19 (m, 18H), 0.88 ppm (t, J=6.4 Hz, 3H); IR (neat): n˜ =3202,
2916, 2847, 1651, 1605, 1582 cmꢀ1; ESI-MS m/z: 296 [M+H]+; Anal.
calcd for C18H33NO2: C 73.17, H 11.26, N 4.74, found: C 73.33, H
11.28, N 4.75.
General procedure for the synthesis of derivatives 5a–b: A mix-
ture of TFA and CH2Cl2 (1:3, 12 mL) was added to ester derivatives
3c or 3d (1.00 mmol). The reaction was stirred at room tempera-
ture for 30 min and then concentrated in vacuo to give the title
compounds as a mixture of E and Z isomers.
2-Acetylhexadec-2-enoic acid (5a, EML333): Low-melting-point
white solid (284 mg, 96%): 2:1 mixture of geometric isomers a and
1
b; H NMR (300 MHz, CDCl3): d=10.90 (brs, 1H, isomer a and 1H
3-((Tridecylamino)methylene)pentane-2,4-dione (8b, EML322):
Low-melting-point pale-yellow solid (303 mg, 98%): 1H NMR
(300 MHz, CDCl3): d=11.07 (brs, 1H), 7.72 (d, J=13.7 Hz, 1H),
3.41–3.29 (m, 2H), 2.48 (s, 3H), 2.24 (s, 3H), 1.68–1.58 (m, 2H),
1.34–1.19 (m, 20H), 0.88 ppm (t, J=6.5 Hz, 3H); 13C NMR (75 MHz,
CDCl3): d=200.4, 194.4, 160.0, 111.5, 50.6, 32.0, 30.7, 29.8, 29.6,
29.5, 29.3, 27.5, 26.6, 22.8, 14.2 ppm; IR (neat): n˜ =3202, 2916,
2851, 1651, 1605, 1585 cmꢀ1; ESI-MS m/z: 310 [M+H]+; Anal. calcd
for C19H35NO2: C 73.74, H 11.40, N 4.53, found: C 73.94, H 11.42, N
4.54.
isomer b), 7.41 (t, J=7.2 Hz, 1H, isomer a), 7.16 (t, J=7.8 Hz, 1H,
isomer b), 2.88–2.78 (m, 2H, isomer a), 2.46 (s, 3H, isomer a), 2.40
(s, 3H, isomer b), 2.35–2.25 (m, 2H, isomer b), 1.62–1.40 (m, 2H,
isomer a and 2H isomer b), 1.30–1.21 (m, 20H, isomer a and 20H
isomer b), 0.88 ppm (brt, J=6.3 Hz, 3H, isomer a and 3H, iso-
mer b); IR (neat): n˜ =2916, 2851, 1713, 1682, 1624 cmꢀ1; ESI-MS
m/z: 297 [M+H]+; Anal. calcd for C18H32O3: C 72.93, H 10.88,
found: C 72.80, H 10.90.
2-Acetylheptadec-2-enoic acid (5b, EML334): Low-melting-point
white solid (292 mg, 94%): 2:1 mixture of geometric isomers a and
1
3-((Dodecyl(ethyl)amino)methylene)pentane-2,4-dione
(8c,
b; H NMR (300 MHz, CDCl3): d=10.90 (brs, 1H, isomer a and 1H
EML323): Low-melting-point pale-yellow solid (301 mg, 93%):
1H NMR (300 MHz, CDCl3): d=7.36 (s, 1H), 3.32–3.20 (m, 4H), 2.32
(s, 6H), 1.58–1.42 (m, 2H), 1.33–1.20 (m, 18H), 1.19–1.03 (m, 3H),
0.87 ppm (t, J=6.3 Hz, 3H); IR (neat): n˜ =2924, 2855, 1655, 1620,
1578 cmꢀ1; ESI-MS m/z: 324 [M+H]+; Anal. calcd for C20H37NO2: C
74.25, H 11.53, N 4.33, found: C 74.47, H 11.55, N 4.34.
isomer b), 7.53 (t, J=7.1 Hz, 1H, isomer a), 7.38 (t, J=7.7 Hz, 1H,
isomer b), 3.00–2.91 (m, 2H, isomer a), 2.50 (s, 3H, isomer a), 2.46
(s, 3H, isomer b), 2.45–2.35 (m, 2H, isomer b), 1.65–1.47 (m, 2H,
isomer a and 2H isomer b), 1.40–1.15 (m, 22H, isomer a and 22H
isomer b), 0.88 ppm (brt, J=6.4 Hz, 3H, isomer a and 3H, iso-
mer b); 13C NMR (75 MHz, CDCl3): d=201.9, 201.1, 169.0, 165.5,
164.0, 153.7, 134.4, 131.0, 32.0, 31.4, 31.3, 30.0, 29.8, 29.6, 29.5,
28.7, 26.7, 22.8, 14.2 ppm; IR (neat): n˜ =2922, 2853, 1717, 1684,
1636 cmꢀ1; ESI-MS m/z: 311 [M+H]+; Anal. calcd for C19H34O3: C
73.50, H 11.04, found: C 73.35, H 11.06.
3-((Ethyl(tridecyl)amino)methylene)pentane-2,4-dione
(8d,
EML324): Low-melting-point pale-yellow solid (317 mg, 94%):
1H NMR (300 MHz, CDCl3): d=7.36 (s, 1H), 3.39–3.18 (m, 4H), 2.32
(s, 6H), 1.62–1.37 (m, 2H), 1.35–1.19 (m, 20H), 1.18–1.05 (m, 3H),
0.87 ppm (t, J=6.4 Hz, 3H); IR (neat): n˜ =2920, 2851, 1659, 1612,
1578 cmꢀ1; ESI-MS m/z: 338 [M+H]+; Anal. calcd for C21H39NO2: C
74.72, H 11.65, N 4.15, found: C 74.94, H 11.68, N 4.16.
General procedure for the synthesis of derivatives 6a,b and 8a–
f: To a solution of 3-(ethoxymethylene)-2,4-pentanedione 9[47]
(1.00 mmol) in anhydrous THF (3 mL), the appropriate alkylamine
(1.00 mmol) or alkylthiol (2.00 mmol) was added, and the reaction
mixture was stirred at reflux for 2 h (monitored by TLC). After cool-
ing at room temperature, the solvent was removed, and the crude
residue was purified by column chromatography on silica gel
(CH2Cl2/EtOAc) to afford the title compounds.
3-((Benzyl(dodecyl)amino)methylene)pentane-2,4-dione
(8e,
1
EML325): Pale-yellow solid (370 mg, 96%): mp: 53–568C; H NMR
(300 MHz, CDCl3): d=7.49 (s, 1H), 7.38–7.27 (m, 3H), 7.18–7.10 (m,
2H), 4.44 (s, 2H), 3.37–3.20 (m, 2H), 2.15 (s, 6H), 1.62–1.46 (m, 2H),
1.32–1.19 (m, 18H), 0.88 ppm (t, J=6.5 Hz, 3H); IR (neat): n˜ =2916,
2851, 1670, 1574 cmꢀ1; ESI-MS m/z: 386 [M+H]+; Anal. calcd for
C25H39NO2: C 77.87, H 10.20, N 3.63, found: C 78.10, H 10.22, N
3.64.
Dodecanethiol, dodecanamine, and tridecanamine used for the
preparation of derivatives 6a, 8a, and 8b, respectively, were com-
mercially available. Tridecanethiol used for the synthesis of 6b and
N-ethyldodecan-1-amine, N-ethyltridecan-1-amine, N-benzyldode-
can-1-amine, and N-benzyltridecan-1-amine used for the synthesis
of derivatives 8c, 8d, 8e, 8 f, respectively, were prepared by stan-
dard synthetic procedures (see Supporting Information for details).
3-((Benzyl(tridecyl)amino)methylene)pentane-2,4-dione
(8 f,
1
EML326): Pale-yellow solid (388 mg, 97%): mp: 61–648C; H NMR
(300 MHz, CDCl3): d=7.49 (s, 1H), 7.39–7.28 (m, 3H), 7.20–7.11 (m,
2H), 4.43 (s, 2H), 3.34–3.17 (m, 2H), 2.15 (s, 6H), 1.66–1.48 (m, 2H),
1.31–1.20 (m, 20H), 0.88 ppm (t, J=6.4 Hz, 3H); IR (neat): n˜ =2916,
2851, 1674, 1574 cmꢀ1; ESI-MS m/z: 400 [M+H]+; Anal. calcd for
C26H41NO2: C 78.15, H 10.34, N 3.51, found: C 78.38, H 10.36, N
3.52.
3-(Dodecylthiomethylene)pentane-2,4-dione (6a, EML327): Low-
1
melting-point white solid (288 mg, 92%): H NMR (300 MHz, CDCl3):
d=8.17 (s, 1H), 2.83 (t, J=7.4 Hz, 2H), 2.47 (s, 3H), 2.38 (s, 3H),
1.79–1.63 (m, 2H), 1.48–1.37 (m, 2H), 1.34–1.16 (m, 16H), 0.88 ppm
(t, J=6.5 Hz, 3H); IR (neat): n˜ =2951, 2920, 2847, 1659, 1643 cmꢀ1
;
ESI-MS m/z: 313 [M+H]+; Anal. calcd for C18H32O2S: C 69.18, H
Surface plasmon resonance
10.32, S 10.26, found: C 69.32, H 10.34, S 10.27.
3-(Tridecylthiomethylene)pentane-2,4-dione (6b, EML328): Low-
melting-point white solid (307 mg, 94%): H NMR (300 MHz, CDCl3):
SPR analyses were performed with a Biacore 3000 optical biosensor
equipped with research-grade CM5 sensor chips (Biacore AB). Re-
combinant p300/KAT3B (Enzo Life Sciences, cat.# BML-SE451; Gen-
Bank accession no. NM001429) and PCAF/KAT2B (Biovision, cat.#
1137–100; GenBank accession n. NM003884) HAT domains were
used in this analysis. Proteins (10 mgmLꢀ1 in 100 mm sodium ace-
tate, pH 4.5) were immobilized on individual flow cells of the
1
d=8.16 (s, 1H), 2.83 (t, J=7.4 Hz, 2H), 2.47 (s, 3H), 2.38 (s, 3H),
1.78–1.65 (m, 2H), 1.48–1.37 (m, 2H), 1.33–1.19 (m, 18H), 0.88 ppm
(t, J=6.5 Hz, 3H); 13C NMR (75 MHz, CDCl3): d=197.7, 194.7, 162.6,
134.2, 38.3, 32.1, 31.1, 30.2, 29.8, 29.6, 29.3, 28.6, 27.8, 22.8,
14.2 ppm; IR (neat): n˜ =2951, 2917, 2847, 1659, 1643 cmꢀ1; ESI-MS
ꢀ 2014 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 0000, 00, 1 – 15
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11
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These are not the final page numbers!

CHEMMEDCHEM
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sensor chip at a flow rate of 10 mLminꢀ1 using standard amine cou-
pling protocols to obtain densities of 8–9 kRU. Myoglobin was
used as a negative control, and one flow cell was left empty for
background subtraction. Compounds 2–8 were dissolved in DMSO
(100%) to obtain 50 mm solutions, and they were diluted in HBS-P
(10 mm HEPES pH 7.4, 0.15m NaCl, 0.005% surfactant P20) while
maintaining a final 0.2% DMSO concentration. Binding experi-
ments were performed at 258C using a flow rate of 30 mLminꢀ1
with 120 s association monitoring and 200 s dissociation monitor-
ing. Surface regeneration was performed when necessary by a 10 s
injection of 5 mm NaOH. The simple 1:1 Langmuir binding fit
model of BIAevaluation software (version 4.1) was used for deter-
mining equilibrium dissociation constants (KD) and kinetic dissocia-
tion (kd) and association (ka) constants using Equations (1) and (2),
where R represents the response unit, and C is the concentration
of the analyte.
Western blot analysis of acetyl-lysines: U937 (10 mL, 2.5ꢁ
105 cellsmLꢀ1), HeLa and C33A (sub-confluent) cells were seeded
and incubated with compounds 2d, 4b, and 5b at different con-
centrations, and after 24 h, the cells were harvested and washed
three times with 1ꢁPBS (Sigma) and then resuspended in lysis
buffer (10 mm Tris pH 8, 1 mm KCl, 1.5 mm MgCl2, and 1 mm DTT)
supplemented with a protease inhibitor cocktail (Sigma). All subse-
quent manipulations were performed at 48C. After incubation for
1 h on a rotator, nuclei were collected by centrifugation for 10 min
at 10000 g. The nuclear pellets were suspended in 0.4 N H2SO4,
and after incubation for 2 h, were centrifuged for 15 min at
12000 g. Histones contained in the supernatant were precipitated
by adding TCA to a final concentration of 33% and incubating
overnight at 48C. After centrifugation at 10000 g for 30 min, the
histone pellets were washed twice with acetone, air-dried, and fi-
nally redissolved in 50 mL of water. Protein concentrations were de-
termined using the Bradford assay, and 1 mg of histones from each
sample was loaded onto a 15% SDS-PAGE gel and transferred to
a nitrocellulose membrane. The following primary antibodies were
used: anti-Ac-histone H4 (Ser1/Lys5/Lys8/Lys12) (Santa Cruz cat.#
sc-34263); anti-histone H4 (acetyl K5) (Abcam cat.# ab61236); anti-
histone H3 (acetyl K9) (Abcam cat.# ab10812); anti-histone H3
(Abcam cat.# ab1791).
dR
ð1Þ
ð2Þ
¼ kaCðRmax ꢀ RÞ ꢀ kdR
dt
KD ¼ kd=ka
Biological methods
Histone acetyl transferase IC50 profiling: The effect of the test deriva-
tives on the catalytic activity of PCAF and p300 was determined Acknowledgements
with a HotSpot HAT activity assay by Reaction Biology Corporation
(Malvern, PA, USA) according to the company’s standard operating
This study was supported by grants from the Italian Ministero
dell’Istruzione, dell’Universitꢀ e della Ricerca (MIUR)–Progetti di
Ricerca di Interesse Nazionale (PRIN 2009PX2T2E,
procedure. Briefly, the recombinant catalytic domains of PCAF
(aa 492–658) or p300 (aa 1284–1673) were incubated with histone
H3 as
a
substrate (5 mm) and [acetyl-3H]-acetyl coenzyme A
PRIN 20103W4779, and PRIN 2012ZHN9YH), the Italian Ministero
dell’Istruzione, dell’Universitꢀ e della Ricerca (MIUR)–Futuro in Ri-
cerca (RBFR10ZJQT), the European Union’s Seventh Framework
Programme (FP7 BLUEPRINT/282510 and FP7 COST/TD0905), the
Universitꢀ di Salerno (Italy), and the Sapienza–Istituto Italiano di
Tecnologia (ITT) Project. C.M. and M.V. were supported by the
Universitꢀ di Salerno with postdoctoral research fellowships, and
A.F. by a predoctoral fellowship. The authors thank Dr. Kurumi
Horiuchi (Reaction Biology Corporation) for helpful discussions.
(3.08 mm) as an acetyl donor in reaction buffer (50 mm Tris-HCl
(pH 8.0), 50 mm NaCl, 0.1 mm EDTA, 1 mm DTT, 1 mm PMSF, 1%
DMSO) for 1 h at 308C in the presence or absence of a dose titra-
tion of the compounds. Histone H3 acetylation was assessed by
liquid scintillation. Anacardic acid or curcumin served as controls
that inhibit PCAF or p300 activity, respectively. Data were analyzed
using Excel and GraphPad Prism software (version 6.0, GraphPad
Software Inc., San Diego, CA, USA) for IC50 curve fits using sigmoi-
dal dose–response (variable slope) equations.
Cell viability assay: The U937 cell line (derived from a human histo-
cytic lymphoma) was cultured in RPMI-1640 medium (Sigma) sup-
plemented with 10% (v/v) fetal bovine serum (Sigma), 100 UmLꢀ1
penicillin, and 100 mgmLꢀ1 streptomycin (Sigma) at 378C in a 5%
CO2 atmosphere. U937 cell viability was measured via a 3-(4,5-di-
methylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. A
total of 100 mL of cells seeded in 96-well microtiter plates (2.5ꢁ
105 cellsmLꢀ1) were exposed for 24 h to different concentrations of
selected compounds, ranging from 1 to 200 mm, in media contain-
ing 0.4% DMSO. The mitochondrial-dependent reduction of MTT
to formazan was used to assess cell viability. Experiments were per-
formed in quadruplicate, and all values are expressed as the per-
centage of the control containing 0.4% DMSO.
Keywords: epigenetics
activators · structure–activity relationships · surface plasmon
resonance
· lysine acetyltransferases · PCAF
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FULL PAPERS
Ups and downs: Chemical manipulation
of the carbonyl functional groups of
a series of analogues of diethyl penta-
decylidenemalonate 1 (SPV106),
a mixed inhibitor/activator of lysine
acetyltransferases that we recently iden-
tified, yielded different activity profiles
against KAT2B and KAT3B (pure KAT2B
activator, pan-inhibitor, or mixed KAT2B
activator/KAT3B inhibitor).
S. Castellano, C. Milite, A. Feoli,
M. Viviano, A. Mai, E. Novellino, A. Tosco,*
G. Sbardella*
&& – &&
Identification of Structural Features of
2-Alkylidene-1,3-Dicarbonyl
Derivatives that Induce Inhibition and/
or Activation of Histone
Acetyltransferases KAT3B/p300 and
KAT2B/PCAF
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