New anacardic acid-inspired benzamides: Histone lysine acetyltransferase activators

Article abstract of DOI:10.1002/cmdc.201000158

A series of N-(4-cyano-3-trifluoromethyl-phenyl)-2-ethoxy-6-alkyl (and alkenyl) benzamides related to the anacardic acid derivative CTPB have been prepared from 2,6-dihydroxybenzoic acid with a Suzuki coupling and addition of the anion of 4-cyano-3-trifluoromethylphenylamine to a benzodioxinone as the key steps. In U937 cells, these analogues, in particular 7c, 7d, 7 f and 7j, induced cell-cycle arrest in the G1 phase, caused apoptosis in about 20% of the cells, and increased the acetylation levels of H3. These activities correlate with the enzymatic activation of histone lysine acetyltransferases (KATs): CBP and PCAF.

Full text of DOI:10.1002/cmdc.201000158

MED
DOI: 10.1002/cmdc.201000158
New Anacardic Acid-Inspired Benzamides: Histone Lysine
Acetyltransferase Activators
Josꢀ A. Souto,[a] Rosaria Benedetti,[b, d, e] Katharina Otto,[a] Marco Miceli,[b] Rosana ꢁlvarez,*[a]
Lucia Altucci,*[b, c] and Angel R. de Lera*[a]
A
series of N-(4-cyano-3-trifluoromethyl-phenyl)-2-ethoxy-6-
7d, 7 f and 7j, induced cell-cycle arrest in the G1 phase,
caused apoptosis in about 20% of the cells, and increased the
acetylation levels of H3. These activities correlate with the en-
zymatic activation of histone lysine acetyltransferases (KATs):
CBP and PCAF.
alkyl (and alkenyl) benzamides related to the anacardic acid
derivative CTPB have been prepared from 2,6-dihydroxybenzo-
ic acid with a Suzuki coupling and addition of the anion of 4-
cyano-3-trifluoromethylphenylamine to a benzodioxinone as
the key steps. In U937 cells, these analogues, in particular 7c,
Introduction
Data accumulated over the past decade clearly link cancer
onset and tumour progression to the deregulation of the enzy-
matic machinery responsible for epigenetic modifications of
both DNA and histone tails within the nucleosomes—the basic
units of chromatin.[1–5] The “epigenetic marks” on chromatin in-
clude methylation of DNA at CpG islands, as well as a variety
of covalent modifications (notably methylation, acetylation,
ADP-ribosylation, phosphorylation, sumoylation and ubiquityla-
tion) of basic amino acid residues located primarily at the tails
of histones H3 and H4.[6] These alterations become docking
sites for additional proteins that trigger the assembly of supra-
molecular structures,[7,8] which among other cellular processes
regulate chromatin remodelling, cell cycle, splicing, nuclear
transport and actin nucleation.[9–11] The reversible nature of
most of the epigenetic modifications[12] has been exploited for
the development of novel approaches to cancer[11,13–15] and
therapies for other diseases as well.[16]
Histone lysine acetyltransferases1 (KATs)[17,18] are responsible
for the transfer of acetyl groups to lysine residues, whereas his-
tone deacetylases (HDACs) catalyse the reversal of this cova-
lent modification and remove acetyl substituents. Acetylation
(but not methylation) weakens the interactions of histone tails
with the negatively charged phosphate groups of DNA in the
nucleosome, converting the nontranscribed heterochromatin
to the more open euchromatin state, which is now accessible
to the transcriptional machinery.[19]
number of transcription factor complexes;[21] TAF250, which is
part of the basic transcription complex TFIID that binds to the
TATA box; and finally SRC-1 and ACTR, which are coactivators
for the ligand-activated nuclear receptors, and their KAT activi-
ties are controversial.[21–23]
Although all KAT enzymes require acetyl-CoA as a cofactor,
the precise mechanism of acetyl transfer to the lysine residue
by the KAT enzyme[20,24] might vary with isoform. A ternary
complex, a “ping-pong” and a “hit and run” mechanism have
been proposed.[16] Regardless of the mechanistic details, dys-
function of acetyltransferase enzymatic activity has been asso-
ciated to several diseases, including cancer, Huntington’s dis-
ease, inflammatory disease, diabetes mellitus and AIDS.[17] Re-
storing the balance between acetylation and deacetylation via
small-molecule modulation might correct aberrant cell growth
and differentiation.[25] In fact, inhibitors of HDACs display anti-
cancer actions,[4,26,27] and two drugs—SAHA (also known as
vorinostat or Zolinzaꢂ) and FK228 (also known as romidepsin
or Istodaxꢂ)—are in the clinic for the treatment of cutaneous
[a] Dr. J. A. Souto, K. Otto, Prof. Dr. R. ꢀlvarez, Prof. Dr. A. R. de Lera
Departamento de Quꢁmica Orgꢂnica, Facultade de Quꢁmica, Universidade
de Vigo, Campus As Lagoas-Marcosende, 36310 Vigo (Spain)
Fax: (+34)986-811-940
E-mail: rar@uvigo.es
qolera@uvigo.es
[b] R. Benedetti, M. Miceli, Prof. Dr. L. Altucci
Dipartimento di Patologia Generale, Seconda Universitꢃ degli Studi di
Napoli, Vico L. de Crecchio 7 80138 Napoli (Italy)
Fax: (+39)081-450-169
KATs have been recently grouped into seven families[17,18] al-
though only four of them have intrinsic KAT activities:[20] Gcn5
and PCAF, which are related to the yeast KAT; the cyclic adeno-
sine monophosphate response element-binding protein (CREB)
binding protein (CBP) and p300, which act as coactivators for a
E-mail: lucia.altucci@unina2.it
[c] Prof. Dr. L. Altucci
IGB-CNR, Via Pietro Castellino, 80100 Napoli (Italy)
[d] R. Benedetti
1
Dipartimento di Fisica, Universitꢃ di Napoli Federico II, Napoli (Italy)
Traditionally histone acetyltransferases were abbreviated as HATs, however, to
reflect their general activity beyond that on histone, the preferred abbrevia-
tion for lysine (K) acetyltransferases is now KATs. Further details on this
change in nomenclature are given in Reference [17].
[e] R. Benedetti
Dipartimento di Chimica Organica e Biochimica, Universitꢃ di Napoli Feder-
ico II, Napoli (Italy)
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Anacardic Acid-Derived Histone Lysine Acetyltransferase Activators
T-cell lymphomas. Modulators of KATs (inhibitors or activators)
also have the potential to become new generation therapeu-
tics.[16]
benzamide) 6 was reported as a potent activator of p300/CBP,
and moreover, devoid of HDAC inhibitory activity.[32,34] Howev-
er, the related benzamides 7a,b (Figure 2) with a shorter satu-
rated side chain were characterised as p300 inhibitors with a
profile similar to the parent 5.[35] A series of novel derivatives
of CTPB, most notably 4-pyridylamides (8a,b; Figure 2) were
shown to inhibit p300/CBP in the micromolar range.[36]
KAT modulators of different structural classes, both of natu-
ral and synthetic origin, have been described in the literature
(Figure 1).[16] Bisubstrate analogues, such as LysCoA (1) and H3-
CoA-20 (2) act as inhibitors of p300 and PCAF/MYST Esa1/
Tip60, respectively.[28] Inhibitors discovered by virtual ligand
Figure 2. Anacardic acid-inspired amides.
Since the strongest KAT inhibitors (7a, with an octyl group
being the most potent) had the shortest hydrophobic alkyl
chains of the series of CTPB analogues,[35] we decided to fur-
ther reduce the size of the chain and incorporate terminal
polar and/or unsaturated groups in the same position of the
C6-salicylic amide substituent, while preserving the ethoxy[32]
and the 4-cyano-3-trifluoromethylbenzamide functionali-
ties.[32,35]
Figure 1. Selected histone acetyltransferase modulators.
Results and Discussion
Synthesis
screening or rational drug design include isothiazolones, alkyli-
denemalonates, thiazol-2-yl hydrazones, quinolines, the curcu-
minoid multiple epigenetic ligands, or the methylenebutyrolac-
tone MB-3, among others.[16] Recently, C646 (3) was described
as a selective, linear, competitive inhibitor of p300 (86% inhibi-
tion of p300 at 10 mm compared with 10% inhibition for six
other KATs) versus acetyl-CoA with a Ki value of 400 nm.[29] It is
the most potent KAT inhibitor reported, about 12-fold more
potent than LTK14 (4),[30] a synthetic phloroglucinol structurally
related to garcinol.[31]
Following the synthetic approach to the CTPB analogues that
uses a Suzuki cross-coupling reaction[37] between an aryl triflate
and different trialkylboranes,[38] known triflate 9[39,35] containing
a 1,3-benzodioxinone group[40–42] was coupled to the trialkyl-
borane obtained by hydroboration of the terminal alkene with
9-BBN in the presence of PdCl2(dppf), MeONa and KBr.[42] Five
alkenes (1-heptene, 10c; 1-hexene, 10d; 4-penten-1-ol, 10e;
3-buten-1-ol, 10 f; 2-propen-1-ol, 10g) were selected as precur-
sors of the organoboranes. The combined yields for the hydro-
boration/coupling step ranged from 39 to 66%.
6-Pentadecylsalicylic acid (anacardic acid, AA; 5), the main
component of the cashew nut-shell oil, was the first in vitro
noncompetitive inhibitor of both p300 and PCAF reported
(IC50 =8.5 and 0.5 mm, respectively).[32] It also inhibits the KAT
activity of recombinant pGcn5 from Plasmodium falciparum.[33]
Synthetic modifications of anacardic acid 1 have yielded com-
pounds with contrasting epigenetic profiles. In particular, CTPB
(N-(4-chloro-3-trifluoromethyl-phenyl)-2-ethoxy-6-pentadecyl-
The acyl transfer/dioxinone deprotection step required the
treatment of the corresponding aniline 12 with nBuLi in DMPU
and heating with 11 at 808C, as reported for ester formation
starting from similar substrates.[43] Formation of the ethyl ether
from salicylamides 13 provided the final benzamides 7c,d and
the protected primary alcohols 14e–g, which were deprotect-
ed to afford 7e–g upon treatment with tetra-n-butylammoni-
um fluoride (TBAF) in THF (Scheme 1; Table 1).
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Scheme 1. Reagents and conditions: a) 10c–g, 9-BBN, MeONa, PdCl2(dppf),
KBr, 708C, THF; b) Aniline 12, nBuLi, DMPU, 808C, 2 h; c) Et2SO4, K2CO3,
acetone; d) TBAF, THF. The yield for each step is given in Table 1.
Table 1. Yields for the steps a–d of the synthesis (Scheme 1).[a]
R1
Step a
Step b
Step c
Step d
[%]
P
[%]
P
[%]
P
[%]
P
Scheme 2. Reagents and conditions: a) EtI, K2CO3, acetone, 258C, 12 h, 91%;
b) 12, nBuLi, DMPU, 808C, 2 h, 88%; c) TFAA, pyridine, 258C, 87%; d) 20a,b,
21 or 22, Pd(PPh3)4, K2CO3, dioxane, microwave, 858C, 15 min (7h, 43%; 7i,
27%; 7j, 80%; 7k, 92%); e) 2-(Dichloromethyl)pinacolboronate, CrCl2, LiI,
THF, 258C, 16 h (20a, 81%; 20b, 91%).
C5H11
C4H9
(CH2)2CH2OTBS
CH2CH2OTBS
CH2OTBS
11 c
11 d
11 e
11 f
11 g
39
41
32
66
32
13c
13d
13e
13 f
13g
76
65
35
50
37
7c
7d
76
46
–
–
–
–
–
7e
7 f
7g
61[b]
51[b]
97[b]
[a] The product (P) and associated yield (%) is given for each step. R1 in-
troduced using compounds 10 (see Scheme 1). [b] Combined yield for
steps c and d.
Biological evaluation
To evaluate whether these novel CTPB-inspired benzamides
are able to have an effect on KAT activity, we tested the com-
pounds in enzymatic and whole-cell assays. In particular, cell-
based assays were performed in U937 leukaemia cells to deter-
mine the antiproliferative potential and the ability of the syn-
thetic compounds to alter the cell cycle. Compared with the
vehicle-treated cells (negative control) and to the cells treated
with the pan-HDAC inhibitor SAHA, used as a positive control
for its known actions on cell cycle and apoptosis,[50] at 5 mm
the test compounds did not significantly alter the cell cycle. In
contrast to SAHA, which accumulates the cell population in
the S phase after 24 h of treatment, at the same concentration
the synthetic salicylamides showed only a modest effect
(~10% increase of U937 cells in the S phase; Figure 3a). How-
ever, after 24 h of induction, compounds 7c–e and 7k at
50 mm induced a time-dependent accumulation of U937 cells
in the G1 phase that reached 70% (Figure 3b), doubling the
effect of the controls SAHA and AA (5). The parent compound
CTPB (6) had no effect on the cell cycle at the tested concen-
trations of 5 and 50 mm. The failure of 5 to influence the cell
cycle regardless of its concentration (see Figure 3a and 3b) is
consistent with reports on its inability to pass through the cell
membrane.[51] In contrast, some of the salicylamides altered
the cell cycle, entering cells most likely due to the presence of
functionalities that provide greater membrane permeability. In
addition, the series of compounds induced a ~20% apoptosis
in U937 cells (Figure 3).
A reversal of the strategic key steps is also possible, and this
strategy was employed for the synthesis of the C6-unsaturated
salicylamides 7h–k. The 1,3-benzodioxinone 15 derived from
2,6-dihydroxybenzoic acid[40–42] (the precursor to triflate 9) was
converted into the ethyl ether 16 as described above (91%
yield). Addition of the anion derived from aniline 12 to 16 ef-
fected the acyl transfer/deprotection[43] in 88% yield. Triflate
18 was obtained in 87% yield upon treating 17 with trifluoro-
acetic anhydride (TFAA) in pyridine at 258C (Scheme 2).
The alkenylboron reagents required for the completion of
the series of C6-unsaturated salicylamides were acquired fol-
lowing complementary methods. (E)-Alkenyl boronates 20a,b
were obtained in high yield and excellent stereoselectivity
from commercially available benzaldehydes 19a,b using the
Takai–Utimoto condensation reaction,[44] after activation of 2-
(dichloromethyl)pinacolboronate with chromium(II) in the pres-
ence of lithium iodide.[45] Two previously described alkenylbor-
on reagents 21[46] and 22[47] were obtained by the hydrobora-
tion of the precursor alkynes and hydrolysis for 22.
The Suzuki coupling of triflate 18 and organoboranes 20–22
was complete after about 15 min in concentrated solutions
(0.2m) using microwave irradiation.[48,49] Unfortunately, the in-
stability of pinacolboronates 20 under the reaction conditions
decreased the yield of the corresponding products 7h and 7i.
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Anacardic Acid-Derived Histone Lysine Acetyltransferase Activators
low cell permeability, CTPB 6 is able to induce an increase in
the acetylation of H3 lysine tails due to its action on KAT en-
zymes (Figure 4a). Interestingly, a similar effect was observed
with most of the salicylamides at 50 mm (Figure 4a). This effect
can most likely be ascribed to the increase of KAT activity and
the up-regulation of the acetylation reaction. Compounds 7c,
7e, 7 f and 7k, which showed induction of KAT activity, also
caused significant cell-cycle arrest in the G1 phase (see Fig-
ure 3a and 3b, and Figure 4a). The insensitivity of the intracel-
lular acetylation status to the presence of AA, as deduced from
Western blot analysis, further confirms its well-known inability
to enter the cells.
To evaluate the effective induction of KAT activity, two enzy-
matic assays were performed: a PCAF and a CBP radioactive
assay. Figure 4 shows the correlation between the enzymatic
and whole-cell assay results. The CTPB analogues, in particular
7e, 7 f, 7h, 7k, enhanced the CBP acetyltransferase activity by
30–40% relative to the control (Figure 4b). In our previous
report, we determined that benzamides 7a and 7b, with an
n-octyl and n-decyl chains at C6, respectively, behaved as
modest inhibitors of p300. Indeed, the shorter homologue 7c
is a rather weak inhibitor of CBP, but the n-hexyl derivative 7d
is inactive, thus signalling the lower limit for modifications at
that position with saturated groups.[35]
Likewise, all the compounds in the series induced activation
of PCAF in the radioactive enzymatic assay. Indeed these com-
pounds enhanced the PCAF activity by at least 150%, and in
particular 7e, 7 f, 7g and 7k induced a 200–250-fold activation
relative to the control (Figure 4c). In both assays, AA behaves
as a very potent enzymatic inhibitor,[32,51] whereas benzamide
CTPB 6 is a selective activator of p300/CBP (Figure 4b, 4c and
4d) as previously described.[32,34] On the contrary, benzamides
7 activate KATs and, moreover, exhibit a preference for the
PCAF family. These experiments confirm that the analogues are
able to modulate the acetylation balance inside the cell by the
direct activation of KAT enzymes.
Conclusions
The development of chemical probes and modulators of KATs
has provided valuable insights into the catalytic features of
this enzyme class and revealed their roles in various cellular
pathways.[16] The great majority of these modulators are KAT
inhibitors with various degrees of potency, selectivity and cell
permeability. As far as we know, only the AA-derived benz-
amide CTPB is an activator of the p300/CBP KAT. However, no
drugs have been described that selectively distinguish be-
tween the subtypes p300 and CBP or PCAF and GCN5. As a
follow-up of our previous studies on the activities of AA-de-
rived CTPB analogues,[35] we have designed and synthesised a
new series of compounds, which carry polar terminal groups
to improve the permeability and enhance their activity. From
the analysis of the biological readouts, we conclude that these
amides act specifically on KAT enzymes both in enzymatic and
whole-cell assays. The increase in KAT activity was ~30% for
CBP and ~200% for PCAF when the compounds, in particular
those with a primary alcohol on the side chain 7e and 7k,
Figure 3. a) Cell-cycle analysis of U937 cells treated with the indicated com-
pounds at 5 mm for 24 h. b) Cell-cycle analysis of U937 cells treated with the
indicated compounds at 50 mm for 24 h. Cell-cycle phases (G1, S and S2) and
apoptosis (Apo) are shown. The data represent the mean of three independ-
ent experiments.
To confirm the general involvement of these CTPB analogues
in the modulation of KAT activity; we analysed the acetylation
status of the representative histone protein H3, which is de-
pendent upon the opposing activities of HDAC and KAT en-
zymes (Figure 4a). The activities of these AA-derived benza-
mides were compared to those of AA (50 mm), CTPB (50 mm),
and the HDAC inhibitors SAHA (5 mm) and MS-275 (5 mm). The
inhibition of HDACs is demonstrated by an increase in the
global level of acetyl groups on H3 lysine tails after treatment
of U937 cells for 24 h (Figure 4a), in comparison to the control
experiments using SAHA and MS-275. Despite the reported
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2) Further reduction in the size
of the alkyl chain (7c, 7d)
was inconsequential, thus
demonstrating a lower limit
for modifications at that posi-
tion.
3) The incorporation of hydroxy
groups at the terminus of
the C6-group (7e, 7 f, 7g,
7k) modified the KAT inhibi-
tory activities of the alkylben-
zamide analogues 7a, 7b
and provided activators of
both
p300
and
PCAF
(Figure 4).
4) In contrast to CTPB (6), these
compounds (most notably 7 f
and 7g) exhibit a preference
for the activation of PCAF
over p300, thus modifying
the selectivity of parent 6.
An examination of the biologi-
cal results indicates the lack of a
linear correlation between the
enzymatic and whole-cell data
for every compound. In fact,
some of the CTPB analogues
show a strong G1 phase block-
ade but not a direct activation
of PCAF and/or CBP. The exis-
tence of ancillary mechanisms
and additive pathways by which
the balance of acetylated/deace-
tylated histones within a cell is
maintained, in addition to the
chemical structure of the com-
pound itself, might partially ex-
plain some of the differences.
Figure 4. a) Western Blot analysis of histone H3 acetylation carried out in U937 cells after 24 h induction with
compounds at 50 mm. b) CBP radioactive assay performed with 1 mg of recombinant CBP enzyme. The compounds
were tested at 50 mm and the CBP activity (%) was compared in each case to anacardic acid (AA; 5) and CTPB (6)
at the same concentration. c) PCAF radioactive assay performed with 200 ng of recombinant PCAF enzyme. The
compounds were tested at 50 mm and the inhibition value was reported as a percentage of residual activity in
comparison to the control, to anacardic acid (AA; 5) and to CTPB (6). The data represent the mean of three inde-
pendent experiments. d) PCAF dose–response assay with the selected compounds (5 and 6 as reference com-
pounds) at three different concentrations (50 mm, 10 mm and 1 mm).
Given that the compounds
showing a well-defined KAT acti-
vation profile (7e, 7 f, 7g, 7k)
act at a quite high concentration
(50 mm), additional chemical
modifications of the general
scaffold might provide more
potent activators that could
were used at 50 mm. At the same concentration they also
showed cell-cycle arrest in the G1 phase and a strong induc-
tion of acetylation of H3 tails.
become valuable tools for understanding the correlation be-
tween the acetylation status of histones and nonhistone pro-
teins, and transcriptional activation.
From the limited number of CTPB-related compounds re-
ported,[32,34–36] the modifications at the C6-position have
proved most informative:
Experimental Section
Chemistry
1) Shorter saturated side chain benzamides 7a,7b (Figure 2)
revert the modulatory profile of the parent CTPB (6)[32,34] and
behave as p300 inhibitors similar to anacardic acid (5).[35]
General Procedures: Solvents were dried according to published
methods and distilled before use. All other reagents were commer-
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Anacardic Acid-Derived Histone Lysine Acetyltransferase Activators
cial compounds of the highest purity available. All reactions were
carried out under argon atmosphere, and those not involving
aqueous reagents were carried out in oven-dried glassware. Analyt-
ical thin-layer chromatography (TLC) was performed on aluminium
plates with Merck kieselgel 60 F254 and visualised by UV irradiation
(254 nm) or by staining with solution of phosphomolibdic acid.
Flash column chromatography was carried out using Merck kiesel-
gel 60 (230–400 mesh) under pressure. UV/VIS spectra were record-
ed on a Cary 100 bio-spectrophotometer. Infrared (IR) spectra were
obtained on a JASCO FTIR 4200 spectrophotometer from a thin
film deposited onto a NaCl glass. Mass Spectra (MS) were obtained
on a Hewlett–Packard HP59970 instrument operating at 70 eV by
electron ionisation. High-resolution mass spectra (HRMS) were
taken either on a VG Autospec instrument, a Micromass GC-TOF or
a Bruker FT-MS apex-Qe. 1H NMR spectra were recorded in CDCl3
and (CD3)2CO at room temperature on a Bruker AMX-400 spec-
trometer at 400 MHz with residual protic solvent as the internal ref-
erence (CDCl3, dH =7.26 ppm; (CD3)2CO, dH =2.05 ppm); chemical
shifts (d) are given in parts per million (ppm), and coupling con-
stants (J) are given in Hertz (Hz). The proton spectra are reported
as follows: d (multiplicity, coupling constant J, number of protons).
13C NMR spectra were recorded in CDCl3 and (CD3)2CO at room
temperature on the same spectrometer at 100 MHz, with the cen-
tral peak of CDCl3 (dC =77.0 ppm) or (CD3)2CO (dC =30.8 ppm) as
the internal reference. Although DEPT 135 was used to aid the as-
signment of signals in the 13C NMR spectra, the multiplicity is only
shown for fluorine–carbon bonds, with the JCꢀF values measured.
separated and the aqueous layer was extracted with EtOAc (ꢄ3).
The combined organic layers were washed with 10% aq HCl (ꢄ1),
water (ꢄ2) and brine (ꢄ1), dried (Na2SO4) and the solvent was
evaporated. The residue was purified by column chromatography
(85:15, hexane/EtOAc) to afford 13c as a yellow oil (133 mg, 76%);
mp: 1478C (hexane/acetone); 1H NMR (400 MHz, (CD3)2CO): d=
10.08 (s, 1H), 8.83 (s, 1H), 8.54 (s, 1H), 8.30 (d, J=8.4 Hz, 1H), 8.07
(d, J=8.4 Hz, 1H), 7.21 (t, J=7.9 Hz, 1H), 6.82 (d, J=7.6 Hz, 1H),
6.81 (d, J=8.2 Hz, 1H), 2.68 (t, J=8.2 Hz, 2H), 1.7–1.5 (m, 2H), 1.3–
1.2 (m, 8H), 0.82 ppm (t, J=6.4 Hz, 3H); 13C NMR (100 MHz,
(CD3)2CO): d=167.3, 154.0, 144.0, 142.0, 136.3, 132.7 (2JCꢀF
=
32.1 Hz), 130.4, 124.6, 122.7 (1JCꢀF =273.1 Hz), 121.9, 120.7, 116.7
(3JCꢀF =5.6 Hz), 115.5, 113.3, 102.8, 32.9, 31.5, 31.3, 29.2, 28.8, 22.4,
˜
13.4 ppm; IR (NaCl): n=3600–3000 (br, NꢀH and OꢀH), 2929 (m,
CꢀH), 2855 (m, CꢀH), 1651 (m, C=O), 1588 (s, C=C), 1535 (s, C=C),
1426 (s), 1188 cmꢀ1 (s); MS (ESI+): m/z (%): 404 [M]+ (8), 273 (11),
220 (15), 219 (100), 218 (21), 186 (37), 134 (20), 108 (21), 107 (24);
HRMS (ESI+): m/z [M]+ calcd for C22H23F3N2O2: 404.1712, found:
404.1731; Anal. calcd for C22H23F3N2O2: C, 65.34; H, 5.73; found: C,
64.79; H, 5.74.
N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-heptylbenz-
amide (7c): General procedure for the alkylation of phenols. A solu-
tion of 13c (0.1 g, 0.25 mmol) in acetone (10 mL) was treated se-
quentially with K2CO3 (0.085 g, 0.617 mmol) and Et2SO4 (0.071 mL,
0.54 mmol). After stirring for 3 h, saturated aq NH4Cl was added
(1.5 mL) and the aqueous layer was extracted with Et2O (ꢄ3). The
combined organic layers were washed with brine (ꢄ2), dried
(Na2SO4) and the solvent was evaporated. The residue was purified
by column chromatography (90:10, hexane/EtOAc) to afford 7c as
a white solid (82 mg, 76%); mp: 808C (hexane/EtOAc); 1H NMR
(400 MHz, (CD3)2CO): d=10.04 (s, 1H), 8.51 (s, 1H), 8.26 (d, J=
8.5 Hz, 1H), 8.06 (d, J=8.5 Hz, 1H), 7.32 (dd, J=8.3, 7.6 Hz, 1H),
6.90 (d, J=8.3 Hz, 1H), 6.89 (d, J=7.6 Hz, 1H), 4.07 (q, J=6.9 Hz,
2H), 2.66 (t, J=7.7 Hz, 2H), 1.7–1.6 (m, 2H), 1.4–1.2 (m, 11H),
0.81 ppm (t, J=6.8 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO): d=
168.9, 157.6, 145.8, 143.8, 138.2, 134.7 (2JCꢀF =31.8 Hz), 132.3, 128.2,
124.7 (1JCꢀF =272.7 Hz), 123.6, 123.5, 118.5 (3JCꢀF =5.7 Hz), 117.4,
111.5, 104.8, 65.9, 34.7 (2C), 33.4, 33.1, 31.4, 24.2, 16.0, 15.3 ppm; IR
2,2-Dimethyl-5-heptyl-4H-1,3-benzodioxin-4-one (11c): General
procedure for the alkyl Suzuki coupling reaction (Method A). A solu-
tion of 10c (0.09 mL, 0.65 mmol) in THF (0.6 mL) was treated with
9-BBN (1.3 mL, 0.5m in THF, 0.65 mmol) and the reaction was
stirred at 258C for 24 h. The mixture was transferred to a flask con-
taining MeONa (0.035 g, 0.65 mmol) and the solution was stirred
for 2 h. A mixture of dichloro-1,1’-bis-(diphenylphosphino)ferrocene
palladium (0.006 g, 0.018 mmol), KBr (0.087 g, 0.728 mmol) and 9
(0.2 g, 0.61 mmol) in THF (4 mL) was added and the mixture was
stirred at 708C for 4 h. Hexane (1 mL), aq NaOH (1 mL, 2m) and
H2O2 (1 mL, 30% w/w) were then added and the mixture was
stirred at 258C. The aqueous layer was extracted with Et2O (ꢄ3)
and the combined organic layers were washed with a saturated aq
NaHCO3 (ꢄ3) and dried (Na2SO4), and the solvent was evaporated.
The residue was purified by column chromatography (90:10,
˜
(NaCl): n=3300–3100 (br, NꢀH), 2928 (s, CꢀH), 2856 (m, CꢀH), 1668
(m, C=O), 1587 (s, C=C), 1528 (s, C=C), 1326 (s), 1269 (s), 1179 (s),
1140 cmꢀ1 (s); MS (ESI+): m/z (%): 432 [M]+ (3), 248 (60), 247 (100),
147 (19), 145 (16), 135 (19), 133 (15), 107 (14), 105 (11); HRMS
(ESI+): m/z [M]+ calcd for C24H27F3N2O2: 432.2025, found:
432.2024; Anal. calcd for C24H37F3N2O2: C, 66.65; H, 6.29; found: C,
66.81; H, 6.66.
1
hexane/EtOAc) to afford 11 c as a yellow oil (70 mg, 39%): H NMR
(400 MHz, CDCl3): d=7.37 (t, J=7.9 Hz, 1H), 6.91 (d, J=7.6 Hz, 1H),
6.78 (d, J=8.2 Hz, 1H), 3.07 (t, J=7.7 Hz, 2H), 1.67 (s, 6H, 2CH3),
1.6–1.5 (m, 2H), 1.5–1.2 (m, 8H), 0.85 ppm (t, J=6.5 Hz, 3H);
13C NMR (100 MHz, CDCl3): d=160.2, 157.1, 148.5, 135.0, 125.1,
115.0, 112.1, 104.9, 34.4, 31.8, 31.2, 29.6, 29.1, 25.6 (2C), 22.6,
2,2-Dimethyl-5-hexyl-4H-1,3-benzodioxin-4-one (11d): Following
the general procedure described for the Suzuki cross coupling
(Method A), the reaction of 9 (0.25 g, 0.07 mmol), 10d (0.06,
0.65 mmol), 9-BBN (1.3 mL, 0.65 mmol), MeONa (0.04 g, 0.65 mmol),
PdCl2(dppf) (6 mg, 0.02 mmol) and KBr (0.09 g, 0.73 mmol) in THF
(5.4 mL) afforded, after purification by column chromatography
(90:10, hexane/EtOAc), 11 d as a colourless oil (71 mg, 41%);
1H NMR (400 MHz, CDCl3): d=7.40 (t, J=7.9 Hz, 1H), 6.94 (d, J=
7.6 Hz, 1H), 6.80 (d, J=8.2 Hz, 1H), 3.09 (t, J=7.9 Hz, 2H), 1.70 (s,
6H, 2CH3), 1.6–1.5 (m, 2H), 1.4–1.3 (m, 6H), 0.89 ppm (t, J=6.2 Hz,
3H); 13C NMR (100 MHz, CDCl3): d=160.2, 157.1, 148.5, 135.0,
125.0, 115.0, 112.0, 104.9, 34.3, 31.6, 31.1, 29.3, 25.6 (2C), 22.6,
˜
14.1 ppm; IR (NaCl): n=2998 (w, CꢀH), 2954 (m, CꢀH), 2926 (s, Cꢀ
H), 2856 (m, CꢀH), 1738 (s, C=O), 1605 (m, C=C), 1582 cmꢀ1 (C=C);
MS (ESI+): m/z (%): 276 [M]+ (7), 219 (12), 218 (100), 176 (14), 162
(38), 161 (20), 148 (13), 147 (43), 134 (77), 133 (11), 105 (34), 91
(11), 77 (11); HRMS (ESI+): m/z [M]+ calcd for C17H24O3: 276.1725,
found: 276.1733.
N-(4-Cyano-3-trifluoromethylphenyl)-6-heptyl-2-hydroxybenz-
amide (13c): General procedure for the amidation of dioxinones. A
cooled (08C) solution of 12 (0.4 g, 2.15 mmol) and 1,3-dimethyl-
3,4,5,6-tetrahydro-2(1H)-pyrimidinone (DMPU; 0.38 mL, 3.14 mmol)
in THF (8.3 mL) at 08C was treated with nBuLi (1.5 mL, 1.40m in
hexane, 2.15 mmol). After stirring for 30 min at 258C a solution of
11 c (0.12 g, 0.43 mmol) in THF (8.3 mL) was added and the reac-
tion was stirred at 808C for 2 h. Water was added, the layers were
˜
14.0 ppm; IR (NaCl): n=2998 (w, CꢀH), 2925 (s, CꢀH), 2855 (m, Cꢀ
H), 1739 (s, C=O), 1605 (m, C=C), 1582 (m, C=C), 1312 (m), 1213
(m), 1042 cmꢀ1 (m); MS (ESI+): m/z (%): 262 [M]+ (8), 204 (100),
162 (32), 147 (22), 134 (57), 105 (25); HRMS (ESI+): m/z [M]+ calcd
for C16H22O3: 262.1569, found: 262.1572.
ChemMedChem 2010, 5, 1530 – 1540
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1535

A. R. de Lera, L. Altucci, R. ꢀlvarez et al.
MED
N-(4-Cyano-3-trifluoromethylphenyl)-6-hexyl-2-hydroxybenz-
amide (13d): Following the general procedure described for
amide formation, the reaction of 11 d (0.06 g, 0.23 mmol), 12
(0.21 g, 1.15 mmol), nBuLi (0.82 mL, 1.40m in hexane, 1.15 mmol)
and DMPU (0.2 mL, 1.68 mmol) in THF (8.8 mL) afforded, after pu-
rification by column chromatography (80:20, hexane/EtOAc), 13d
as a white solid (58 mg, 65%); mp: 1618C (hexane/acetone);
1H NMR (400 MHz, (CD3)2CO): d=10.07 (br, 1H), 8.54 (s, 1H), 8.29
(d, J=8.4 Hz, 1H), 8.07 (d, J=8.5 Hz, 1H), 7.21 (t, J=7.9 Hz, 1H),
6.82 (d, J=7.7 Hz, 1H), 6.81 (d, J=8.1 Hz, 1H), 2.85 (br, 1H), 2.68 (t,
J=7.9 Hz, 2H), 1.7–1.6 (m, 2H), 1.4–1.3 (m, 2H), 1.3–1.2 (m, 4H),
0.82 ppm (t, J=6.7 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO): d=
167.2, 154.0, 143.9, 142.0, 136.3, 132.7 (2JCꢀF =31.3 Hz), 130.4, 124.5,
123.0 (1JCꢀF =273.0 Hz), 121.9, 120.7, 116.7 (3JCꢀF =5.7 Hz), 115.5,
(0.08 g, 0.21 mmol), 12 (0.20 g, 1.05 mmol), nBuLi (0.75 mL, 1.40m
in hexane, 1.05 mmol) and DMPU (0.19 mL, 1.54 mmol) in THF
(8.1 mL) afforded, after purification by column chromatography
(80:20, hexane/EtOAc), 13e as a colourless oil (37 mg, 35%);
1H NMR (400 MHz, (CD3)2CO): d=10.00 (br, 1H), 8.86 (s, 1H), 8.54 (s,
1H), 8.30 (d, J=8.2 Hz, 1H), 8.07 (d, J=8.3 Hz, 1H), 7.21 (t, J=
8.0 Hz, 1H), 6.82 (d, J=7.6 Hz, 1H), 6.81 (d, J=8.2 Hz, 1H), 3.59 (t,
J=6.2 Hz, 2H), 2.69 (t, J=7.9 Hz, 2H), 1.7–1.4 (m, 6H), 0.86 (s, 9H,
SiC(CH3)3), 0.01 ppm (s, 6H, 2SiCH3); 13C NMR (100 MHz, (CD3)2CO):
d=167.2, 154.1, 144.0, 141.9, 136.3, 132.8 (2JCꢀF =32.1 Hz), 130.4,
124.5, 122.8 (1JCꢀF =273.0 Hz), 121.9, 120.7, 116.7 (3JCꢀF =4.9 Hz),
115.5, 113.3, 102.9, 62.5, 33.0, 32.4, 31.1, 25.5, 25.4 (3C), 17.9,
˜
ꢀ6.1 ppm (2C); IR (NaCl): n=3500–3000 (br, NꢀH and OꢀH), 2931
(s, CꢀH), 2858 (m, CꢀH), 1656 (m, C=O), 1588 (s, C=C), 1533 (s, C=
C), 1426 (s), 1330 cmꢀ1 (s); MS (ESI+): 507 [M+H]+ (24), 371 (56),
197 (100); HRMS (ESI+): m/z [M+H]+ calcd for C26H34F3N2O3Si:
507.2285, found: 507.2298.
˜
113.3, 102.9, 32.9, 31.4, 31.2, 28.9, 22.2, 13.3 ppm; IR (NaCl): n=
3600–3000 (br, NꢀH and OꢀH), 3020 (m, CꢀH), 2931 (m, CꢀH),
2857 (w, CꢀH), 1654 (m, C=O), 1588 (s, C=C), 1534 (s, C=C), 1426
(s), 1331 (s), 1217 cmꢀ1 (s); MS (ESI+): m/z (%): 390 [M]+ (7), 212
(29), 205 (100), 186 (54), 108 (31), 107 (34), 77 (18); HRMS (ESI+):
m/z [M]+ calcd for C21H21F3N2O2: 390.1555, found: 390.1552.
N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-(5-hydroxypent-
1-yl)benzamide (7e): Following the general procedure described
for the alkylation of phenols, the reaction of 13e (0.03 g,
0.05 mmol), Et2SO4 (0.02 mL, 0.12 mmol), K2CO3 (0.02 g, 0.13 mmol)
in acetone (2.1 mL) afforded, after purification by column chroma-
tography (90:10, hexane/EtOAc), a colourless oil (16 mg) that was
used in the next step without further purification. General proce-
dure for the deprotection of silyl ethers. A solution of the residue ob-
tained above (0.02 g, 0.03 mmol) in THF (0.5 mL) at 08C was treat-
ed with TBAF (0.05 mL, 1m in THF, 0.055 mmol) and stirred at RT
for 5 h. The mixture was diluted with EtOAc and washed with satu-
rated aq NaHCO3 (ꢄ3). The aqueous layer was extracted with
EtOAc (ꢄ3) and the combined organic layers were washed with
brine (ꢄ3), dried (Na2SO4) and the solvent was evaporated. The res-
idue was purified by column chromatography (50:50, hexane/
EtOAc) to afford 7e as a colourless oil (8 mg, 61% combined
N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-hexylbenz-
amide (7d): Following the general procedure described for the al-
kylation of phenols, the reaction of 13d (0.03 g, 0.08 mmol), Et2SO4
(0.02 mL, 0.17 mmol) and K2CO3 (0.03 g, 0.19 mmol) in acetone
(3.2 mL) afforded, after purification by column chromatography
(90:10, hexane/EtOAc), 7d as a white solid (15 mg, 46%); mp:
1098C (hexane/EtOAc); 1H NMR (400 MHz, CDCl3): d=8.1–8.0 (m,
3H), 7.81 (d, J=8.2 Hz, 1H), 7.31 (t, J=8.0 Hz, 1H), 6.89 (d, J=
7.7 Hz, 1H), 6.80 (d, J=8.3 Hz, 1H), 4.08 (q, J=6.9 Hz, 2H), 2.71 (t,
J=7.7 Hz, 2H), 1.6–1.5 (m, 2H), 1.4–1.2 (m, 9H), 0.85 ppm (t, J=
6.5 Hz, 3H); 13C NMR (100 MHz, CDCl3): d=166.5, 155.5, 143.6,
142.4, 136.0, 134.1 (2JCꢀF =33.0 Hz), 131.2, 124.6, 122.6, 122.2
(1JCꢀF =274.0 Hz), 121.7, 117.0 (3JCꢀF =4.8 Hz), 115.6, 109.7, 104.1,
˜
64.5, 33.4, 31.6, 31.5, 29.2, 22.5, 14.8, 14.0 ppm; IR (NaCl): n=3500–
1
yield); H NMR (400 MHz, CDCl3): d=8.27 (s, 1H), 8.06 (s, 1H), 8.04
3100 (br, NꢀH), 2929 (s, CꢀH), 2857 (m, CꢀH), 1668 (m, C=O), 1587
(s, C=C), 1528 (s, C=C), 1327 (s), 1179 (s), 1140 cmꢀ1 (s); MS (ESI+):
m/z (%): 418 [M]+ (6), 234 (99), 233 (100), 205 (22), 147 (47), 145
(63), 135 (58), 134 (29), 133 (52), 107 (52), 105 (35), 91 (29), 77 (28);
HRMS (ESI+): m/z [M]+ calcd for C23H25F3N2O2: 418.1868, found:
418.1870; Anal. calcd for C23H25F3N2O2: C, 66.02; H, 6.02; found: C,
66.21; H, 6.07.
(d, J=8.1 Hz, 1H), 7.82 (d, J=8.2 Hz, 1H), 7.32 (t, J=8.0 Hz, 1H),
6.90 (d, J=7.7 Hz, 1H), 6.81 (d, J=8.3 Hz, 1H), 4.09 (q, J=6.6 Hz,
2H), 3.60 (t, J=6.3 Hz, 2H), 2.74 (t, J=7.6 Hz, 2H), 1.7–1.4 ppm (m,
9H); 13C NMR (100 MHz, CDCl3): d=166.6, 155.6, 143.0, 142.4,
136.0, 134.1 (2JCꢀF =33.0 Hz), 131.2, 124.8, 122.5, 122.2 (1JCꢀF
=
273.1 Hz), 121.7, 117.1 (3JCꢀF =4.8 Hz), 115.6, 109.8, 104.1, 64.5, 62.7,
˜
33.2, 32.3, 30.9, 25.4, 14.8 ppm; IR (NaCl): n=3500–3000 (br, NꢀH
2,2-Dimethyl-5-(tert-butyldimethylsilyloxypent-1-yl)-4H-1,3-ben-
zodioxin-4-one (11e): Following the general procedure described
for the Suzuki cross coupling (Method A), the reaction of 9 (0.25 g,
0.07 mmol), 10e (0.13 g, 0.65 mmol), 9-BBN (1.3 mL, 0.5m in THF,
0.65 mmol), MeONa (0.04 g, 0.65 mmol), PdCl2(dppf) (6 mg,
0.02 mmol) and KBr (0.09 g, 0.73 mmol) in THF (5.4 mL) afforded,
after purification by column chromatography (90:10, hexane/
EtOAc), 11 e as a colourless oil (80 mg, 32%); 1H NMR (400 MHz,
CDCl3): d=7.40 (t, J=7.9 Hz, 1H), 6.93 (d, J=7.6 Hz, 1H), 6.81 (d,
J=8.2 Hz, 1H), 3.61 (t, J=6.5 Hz, 2H), 3.09 (t, J=7.7 Hz, 2H), 1.70
(s, 6H, 2CH3), 1.6–1.4 (m, 6H), 0.89 (s, 9H, SiC(CH3)3, 0.04 ppm (s,
6H, 2SiCH3); 13C NMR (100 MHz, CDCl3): d=160.2, 157.1, 148.3,
135.1, 125.1, 115.1, 112.0, 104.9, 63.1, 34.3, 32.7, 30.9, 26.0 (3C),
and OꢀH), 2932 (s, CꢀH), 2859 (m, CꢀH), 1679 (m, C=O), 1588 (s,
C=C), 1529 (s, C=C), 1327 (s), 1138 cmꢀ1 (s); MS (ESI+): 443
[M+Na]+ (33), 421 [M+H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd
for C22H24F3N2O3: 421.1734, found: 421.1736.
2,2-Dimethyl-5-(tert-butyldimethylsilyloxybut-1-yl)-4H-1,3-ben-
zodioxin-4-one (11 f): Following the general procedure described
for the Suzuki cross coupling (Method A), the reaction of 9 (0.25 g,
0.07 mmol), 10 f (0.12 g, 0.65 mmol), 9-BBN (1.3 mL, 0.5m in THF,
0.65 mmol), MeONa (0.04 g, 0.65 mmol), PdCl2(dppf) (6 mg,
0.02 mmol) and KBr (0.09 g, 0.73 mmol) in THF (5.4 mL) afforded,
after purification by column chromatography (90:10, hexane/
1
EtOAc), 11 f as a colourless oil (160 mg, 66%); H NMR (400 MHz,
˜
25.8, 25.6 (2C), 18.4, ꢀ5.2 ppm (2C); IR (NaCl): n=2929 (m, CꢀH),
CDCl3): d=7.39 (t, J=7.9 Hz, 1H), 6.93 (d, J=7.6 Hz, 1H), 6.80 (d,
J=8.2 Hz, 1H), 3.63 (t, J=5.8 Hz, 2H), 3.11 (t, J=7.0 Hz, 2H), 1.69
(s, 6H, 2CH3), 1.7–1.6 (m, 4H), 0.88 (s, 9H, SiC(CH3)3), 0.03 ppm (s,
6H, 2SiCH3); 13C NMR (100 MHz, CDCl3): d=160.2, 157.1, 148.1,
135.1, 125.1, 115.2, 112.1, 104.9, 63.1, 34.0, 32.7, 27.3 , 26.0 (3C),
2857 (m, CꢀH), 1740 (s, C=O), 1606 (m, C=C), 1582 (s, C=C),
1476 cmꢀ1 (s, C=C); MS (ESI+) 401 [M+Na]+ (100), 379 [M+H]+
(76), 287 (9), 201 (20); HRMS (ESI+): m/z [M+H]+ calcd for
C21H35O4Si: 379.2299, found: 379.2300.
˜
N-(4-Cyano-3-trifluoromethylphenyl)-6-(5-tert-butyldimethylsilyl-
oxypent-1-yl)-2-hydroxybenzamide (13e): Following the general
procedure described for amide formation, the reaction of 11 e
25.6 (2C), 18.3, ꢀ5.2 ppm (2C); IR (NaCl): n=2929 (s, CꢀH), 2857
(m, CꢀH), 1739 (s, C=O), 1605 (m, C=C), 1582 (m, C=C), 1312 (m),
1217 cmꢀ1 (m); MS (ESI+): 387 [M+Na]+ (42), 365 [M+H]+ (100);
1536
www.chemmedchem.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1530 – 1540

Anacardic Acid-Derived Histone Lysine Acetyltransferase Activators
HRMS (ESI+): m/z [M+H]+ calcd for C20H33O4Si: 365.2143, found:
365.2142.
(ESI+): 373 [M+Na]+ (71), 351 [M+H]+ (100); HRMS (ESI+): m/z
[M+H]+ calcd for C19H31O4Si: 351.1986, found: 351.1989.
N-(4-Cyano-3-(trifluoromethyl)phenyl)-6-(4-tert-butyldimethylsi-
lyloxybut-1-yl)-2-hydroxybenzamide (13 f): Following the general
procedure described for amide formation, the reaction of 11 f
(0.16 g, 0.44 mmol), 12 (0.41 g, 2.20 mmol), nBuLi (1.57 mL, 1.40m
in hexane, 2.20 mmol) and DMPU (0.39 mL, 3.20 mmol) in THF
(17 mL) afforded, after purification by column chromatography
(80:20, hexane/EtOAc), 13 f as a colourless oil (109 mg, 50%);
1H NMR (400 MHz, (CD3)2CO): d=10.06 (br, 1H), 8.54 (s, 1H), 8.30
(d, J=8.5 Hz, 1H), 8.06 (d, J=8.6 Hz, 1H), 7.21 (t, J=7.9 Hz, 1H),
6.82 (d, J=7.6 Hz, 1H), 6.81 (d, J=7.6 Hz, 1H), 3.61 (t, J=6.3 Hz,
2H), 2.71 (t, J=7.4 Hz, 2H), 1.7–1.4 (m, 4H), 0.84 (s, 9H, SiC(CH3)3),
ꢀ0.01 ppm (s, 6H, 2SiCH3); 13C NMR (100 MHz, (CD3)2CO): d=167.3,
154.1, 144.0, 141.9, 136.3, 132.8 (2JCꢀF =32.1 Hz), 130.4, 124.6, 122.8
(1JCꢀF =273.0 Hz), 121.9, 120.6, 116.7 (3JCꢀF =4.1 Hz), 115.6, 113.4,
102.8, 62.5, 32.7, 32.5, 27.6, 25.4 (3C), 17.9, ꢀ6.1 ppm (2C); IR
N-(4-Cyano-3-(trifluoromethyl)phenyl)-6-(3-tert-butyldimethylsi-
lyloxyprop-1-yl)-2-hydroxybenzamide (13g): Following the gener-
al procedure described for amide formation, the reaction of 11 g
(0.11 g, 0.17 mmol), 12 (0.16 g, 0.85 mmol), nBuLi (0.57 mL, 1.50m
in hexane, 0.85 mmol) and DMPU (0.15 mL, 1.24 mmol) in THF
(6.5 mL) afforded, after purification by column chromatography
(80:20, hexane/EtOAc), 13g as a colourless oil (30 mg, 37%);
1H NMR (400 MHz, (CD3)2CO): d=10.07 (br, 1H), 8.57 (s, 1H), 8.27
(d, J=8.5 Hz, 1H), 8.06 (d, J=8.5 Hz, 1H), 7.22 (t, J=7.9 Hz, 1H),
6.9–6.8 (m, 2H), 3.65 (t, J=6.1 Hz, 2H), 2.75 (t, J=7.8 Hz, 2H), 1.9–
1.8 (m, 2H), 0.83 (s, 9H, SiC(CH3)3), 0.00 ppm (s, 6H, 2SiCH3);
13C NMR (100 MHz, (CD3)2CO): d=167.2, 154.1, 144.0, 141.5, 136.2,
132.7 (2JCꢀF =34.5 Hz), 130.4, 124.7, 122.7 (1JCꢀF =272.5 Hz), 121.9,
120.7, 116.7 (3JCꢀF =5.6 Hz), 115.6, 113.3, 102.8, 62.3, 34.5, 29.5, 25.3
˜
(3C), 17.8, ꢀ6.1 ppm (2C); IR (NaCl): n=3500–3000 (br, NꢀH and
˜
(NaCl): n=3500–3000 (br, NꢀH and OꢀH), 2953 (s, CꢀH), 2931 (s,
OꢀH), 2928 (m, CꢀH), 2858 (m, CꢀH), 1652 (m, C=O), 1588 (s, C=C),
1534 (s, C=C), 1427 (s), 1330 cmꢀ1 (s); MS (ESI+): 501 [M+Na]+
(100), 479 [M+H]+ (14); HRMS (ESI+): m/z [M+H]+ calcd for
C24H30F3N2O3Si: 479.1972, found: 479.1984.
CꢀH), 2857 (m, CꢀH), 1656 (m, C=O), 1588 (s, C=C), 1533 (s, C=C),
1426 (s), 1329 (s), 1187 cmꢀ1 (m); MS (ESI+): 515 [M+Na]+ (38),
493 [M+H]+ (100); HRMS (ESI+): m/z [M+H]+ calcd for
C25H32F3N2O3Si: 493.2129, found: 493.2128.
N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-(3-hydroxyprop-
1-yl)benzamide (7g): Following the general procedure described
for the alkylation of phenols, the reaction of 13g (0.03 g,
0.07 mmol), Et2SO4 (0.02 mL, 0.16 mmol) and K2CO3 (0.03 g,
0.18 mmol) in acetone (2.8 mL) afforded, after purification by
column chromatography (80:20, hexane/EtOAc), a colourless oil
(25 mg) that was used in the next step without further purification.
Following the general procedure described for the deprotection of
silyl ethers, the reaction of the residue obtained above (24 mg,
0.047 mmol) and TBAF (0.071 mL, 1m in THF, 0.071 mmol) in THF
(0.8 mL) afforded, after purification by column chromatography
(60:40, hexane/EtOAc), 7g as a colourless oil (18 mg, 97%);
1H NMR (400 MHz, (CD3)2CO): d=10.08 (s, 1H), 8.49 (s, 1H), 8.25 (d,
J=8.4 Hz, 1H), 8.05 (d, J=8.5 Hz, 1H), 7.33 (t, J=8.0 Hz, 1H), 6.9–
6.8 (m, 2H), 4.07 (q, J=7.0 Hz, 2H), 3.6–3.5 (m, 2H), 2.74 (t, J=
8.5 Hz, 2H), 1.9–1.8 (m, 2H), 1.29 ppm (t, J=6.9 Hz, 3H); 13C NMR
(100 MHz, (CD3)2CO): d=169.0, 157.7, 145.8, 143.3, 138.2, 134.7
(2JCꢀF =31.0 Hz), 132.4, 128.4, 125.1 (1JCꢀF =270.6 Hz), 123.9, 123.5,
118.7 (3JCꢀF =4.9 Hz), 117.4, 111.6, 104.8, 65.9, 62.7, 36.2, 31.1,
N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-(4-hydroxybut-
1-yl)benzamide (7 f): Following the general procedure described
for the alkylation of phenols, the reaction of 13 f (0.08 g,
0.16 mmol), Et2SO4 (0.05 mL, 0.36 mmol), K2CO3 (0.06 g, 0.41 mmol)
in acetone (6.5 mL) afforded, after purification by column chroma-
tography (80:20, hexane/EtOAc), a colourless oil (67 mg) that was
used in the next step without further purification. Following the
general procedure described for the deprotection of silyl ethers,
the reaction of the residue obtained above (67 mg, 0.129 mmol)
and TBAF (0.19 mL, 1m in THF, 0.19 mmol) in THF (2.2 mL) afford-
ed, after purification by column chromatography (60:40, hexane/
EtOAc), 7 f as a colourless oil (27 mg, 51%); 1H NMR (400 MHz,
(CD3)2CO): d=10.07 (br, 1H), 8.51 (s, 1H), 8.26 (d, J=7.1 Hz, 1H),
8.07 (d, J=8.5 Hz, 1H), 7.33 (t, J=8.0 Hz, 1H), 6.92 (d, J=7.4 Hz,
2H), 4.08 (q, J=7.0 Hz, 2H), 3.50 (t, J=6.0 Hz, 2H), 3.39 (t, J=
5.2 Hz, 1H), 2.70 (t, J=7.8 Hz, 2H), 1.7–1.5 (m, 4H), 1.30 ppm (t, J=
6.9 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO): d=167.0, 155.8, 143.9,
141.7, 136.3, 132.8 (2JCꢀF =31.3 Hz), 130.4, 126.3, 122.7 (1JCꢀF
=
273.9 Hz), 121.9, 121.6, 116.7 (3JCꢀF =4.8 Hz), 115.5, 109.6, 102.9,
˜
16.0 ppm; IR (NaCl): n=3500–3200 (br, NꢀH and OꢀH), 2928 (m,
˜
63.9, 61.3, 32.6, 32.5, 27.6, 14.1 ppm; IR (NaCl): n=3500–3000 (br,
CꢀH), 1671 (m, C=O), 1589 (s, C=C), 1526 (s, C=C), 1327 (s),
1220 cmꢀ1 (s); MS (ESI+): 415 [M+Na]+ (100), 393 [M+H]+ (65);
HRMS (ESI+): m/z [M+H]+ calcd for C20H20F3N2O3: 393.1421, found:
393.1419.
NꢀH and OꢀH), 2933 (m, CꢀH), 1678 (m, C=O), 1588 (s, C=C), 1529
(s, C=C), 1327 cmꢀ1 (s); MS (ESI+): 429 [M+Na]+ (23), 407 [M+H]+
(100); HRMS (ESI+): m/z [M+H]+ calcd for C21H22F3N2O3: 407.1577,
found: 407.1585.
2,2-Dimethyl-5-(3-tert-butyldimethylsilyloxyprop-1-yl)-4H-1,3-
benzodioxin-4-one (11g): Following the general procedure de-
scribed for the Suzuki cross coupling (Method A), 9 (0.25 g,
0.07 mmol), 10g (0.11 g, 0.65 mmol), 9-BBN (1.3 mL, 0.5m in THF,
0.65 mmol), MeONa (0.04 g, 0.65 mmol), PdCl2(dppf) (6 mg,
0.02 mmol) and KBr (0.09 g, 0.73 mmol) in THF (5.4 mL) afforded,
after purification by column chromatography (90:10, hexane/
EtOAc), 11 g as a colourless oil (74 mg, 32%); 1H NMR (400 MHz,
CDCl3): d=7.41 (t, J=7.9 Hz, 1H), 6.96 (d, J=7.6 Hz, 1H), 6.81 (d,
J=8.2 Hz, 1H), 3.69 (t, J=6.4 Hz, 2H), 3.14 (t, J=7.7 Hz, 2H), 1.84
(t, J=6.6 Hz, 2H), 1.70 (s, 6H, 2CH3), 0.91 (s, 9H, SiC(CH3)3),
0.07 ppm (s, 6H, 2SiCH3); 13C NMR (100 MHz, CDCl3): d=160.2,
157.1, 147.8, 135.1, 125.2, 115.2, 112.1, 105.0, 62.8, 34.0, 30.7, 26.0
5-Ethoxy-2,2-dimethyl-4H-1,3-benzodioxin-4-one (16): Following
the general procedure described for the alkylation of phenols, the
reaction of 15 (0.10 g, 0.51 mmol), K2CO3 (0.18 g, 1.28 mmol) and
ethyl iodide (0.09 mL, 1.13 mmol) in acetone (16 mL), afforded,
after purification by column chromatography (90:10, hexane/
EtOAc), 16 as a white solid (0.106 g, 91%); mp: 1018C (hexane/ace-
1
tone); H NMR (400 MHz, CDCl3): d=7.42 (t, J=8.4 Hz, 1H), 6.62 (d,
J=8.4 Hz, 1H), 6.55 (d, J=8.4 Hz, 1H), 4.19 (q, J=6.8 Hz, 2H), 1.72
(s, 6H, 2CH3), 1.53 ppm (t, J=6.8 Hz, 3H); 13C NMR (100 MHz,
CDCl3): d=160.7, 158.0, 157.6, 136.2, 108.8, 106.3, 105.0, 103.3,
˜
64.8, 25.5 (2C), 14.4 ppm; IR (NaCl): n=2988 (w, CꢀH), 2938 (w, Cꢀ
H), 2888 (w, CꢀH), 1736 (s, C=O), 1582 (m, C=C), 1461 (s), 1254 (s),
1081 cmꢀ1 (s); MS (ESI+): 245 [M+Na]+ (100), 223 [M+H]+ (46);
HRMS (ESI+): m/z [M+Na]+ calcd for C12H14NaO4: 245.0784; found
245.0779; Anal. calcd for C12H14O4: C, 64.85; H, 6.35; found: C,
64.76; H, 6.38.
˜
(3C), 25.6 (2C), 18.3, ꢀ5.3 ppm (2C); IR (NaCl): n=2952 (m, CꢀH),
2928 (m, CꢀH), 2887 (w, CꢀH), 2857 (m, CꢀH), 1739 (s, C=O), 1606
(m, C=C), 1582 (m, C=C), 1476 (s), 1313 (s), 1271 cmꢀ1 (s); MS
ChemMedChem 2010, 5, 1530 – 1540
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1537

A. R. de Lera, L. Altucci, R. ꢀlvarez et al.
MED
N-(4-Cyano-3-trifluoromethylphenyl)-6-ethoxy-2-hydroxybenz-
amide (17): Following the general procedure described for amide
formation, the reaction of 16 (0.34 g, 1.51 mmol), 12 (0.703 g,
3.78 mmol), nBuLi (3.10 mL, 1.22m in hexane, 3.78 mmol) and
DMPU (4.15 mL, 34.50 mmol) in THF (56 mL) afforded, after purifi-
cation by column chromatography (60:10:30, hexane/EtOAc/
CH2Cl2), 17 as a white solid (0.47 g, 88%); mp: 1678C (hexane/ace-
(42 mL) afforded, after purification by column chromatography
(95:5, hexane/EtOAc), 20b as a yellow oil (0.78 g, 91%); 1H NMR
(400 MHz, CDCl3): d=7.63 (t, J=1.7 Hz, 1H), 7.42 (td, J=8.0, 1.6 Hz,
2H), 7.32 (d, J=18.4 Hz, 1H), 7.23 (t, J=7.6 Hz, 1H), 6.18 (d, J=
18.4 Hz, 1H), 1.33 ppm (s, 12H); 13C NMR (100 MHz, CDCl3): d=
147.5, 139.5, 131.5, 129.9 (2C), 129.7, 125.4, 122.6, 83.3 (2C),
24.7 ppm (4C); MS (ESI+): 429 [M+Na]+ (23), 407 [M+H]+ (100);
1
˜
tone); H NMR (400 MHz, (CD3)2CO): d=11.06 (s, 1H), 8.40 (s, 1H),
IR (NaCl): n=2979 (m, CꢀH), 1626 (m, C=O), 1563 (w, C=C), 1471
8.1–8.0 (m, 2H), 7.43 (t, J=8.4 Hz, 1H), 6.67 (d, J=8.4 Hz, 1H), 6.61
(dd, J=8.4, 0.9 Hz, 1H), 4.38 (q, J=7.0 Hz, 2H), 1.66 ppm (t, J=
7.0 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO): d=171.3, 166.4, 160.0,
(w), 1345 (s), 1210 (m), 1145 cmꢀ1 (s); MS (ESI+): 333 [M+Na]+
(51), 331 [M+Na]+ (62), 311 [M+H]+ (100), 309 [M+H]+ (100);
HRMS (ESI+): m/z [M+H]+ calcd for C14H19B79BrO2: 309.0659,
found: 309.0667.
144.1, 138.1, 137.0, 134.7 (2JCꢀF =32.5 Hz), 125.6, 124.5 (1JCꢀF
=
273.4 Hz), 120.2 (3JCꢀF =5.6 Hz), 117.2, 113.2, 106.0 (4JCꢀF =2.6 Hz),
(E)-N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-phenyl-
ethen-1-yl Benzamide (7j): General procedure for the Suzuki cross
coupling (Method B). 21 (41.9 mg, 0.19 mmol), 18 (0.05 g,
0.11 mmol), Pd(PPh3)4 (6 mg, 5.2 mmol) and K3PO4 (0.04 g,
0.19 mmol) were suspended in dioxane (0.55 mL). The suspension
was irradiated in a microwave reactor for 15 min at 1008C (90 W).
Benzene (1 mL), aq NaOH (100 mL, 3m) and H2O2 (30%, 100 mL)
were added and the mixture was stirred at RT for 1 h. Water was
added and the aqueous layer was extracted with EtOAc (ꢄ3). The
combined organic layers were dried (Na2SO4) and the solvent was
evaporated. The residue was purified by column chromatography
(-NH2 silica gel; 90:10, hexane/EtOAc) to afford 7j as a white solid
(36.8 mg, 80%); mp: 1828C (hexane/acetone); 1H NMR (400 MHz,
(CD3)2CO): d=10.16 (s, 1H), 8.50 (s, 1H), 8.29 (dd, J=8.5, 1.4 Hz,
1H), 8.07 (d, J=8.5 Hz, 1H), 7.6–7.4 (m, 4H), 7.4–7.2 (m, 5H), 7.03
(dd, J=7.7, 1.3 Hz, 1H), 4.12 (q, J=7.0 Hz, 2H), 1.31 ppm (t, J=
7.0 Hz, 3H); 13C NMR (100 MHz, (CD3)2CO): d=168.4, 158.0, 145.8,
139.0, 138.3, 138.2, 134.7 (2JCꢀF =34.6 Hz), 133.5, 132.6, 130.6 (2C),
129.9, 128.6 (2C), 127.7, 126.7, 124.6 (1JCꢀF =272.0 Hz), 123.9, 119.3,
118.7 (3JCꢀF =5.6 Hz), 117.4, 113.1, 105.0, 66.1, 16.0 ppm; IR (NaCl):
˜
105.7, 104.6, 67.8, 15.8 ppm; IR (NaCl): n=3600–3000 (br, NꢀH and
OꢀH), 1648 (m, C=O), 1585 (s, C=C), 1544 (s, C=C), 1451 (m), 1430
(m), 1326 (s), 1234 (s), 1136 cmꢀ1 (s); MS (ESI+): 373 [M+Na]+ (20),
351 [M+H]+ (92), 209 (100); HRMS (ESI+): m/z [M+H]+ calcd for
C17H14F3N2O3: 351.0951; found 351.0965; Anal. calcd for
C17H13F3N2O3: C, 58.29; H, 3.74; found: C, 58.25; H, 3.76.
2-(4-Cyano-3-trifluoromethylphenylcarbamoyl)-3-ethoxyphenyl
trifluoromethanesulfonate (18): Following the general procedure
described for the synthesis of triflates, the reaction of 17 (0.15 g,
0.43 mmol) and trifluoromethanesulfonic anhydride (0.07 mL,
0.43 mmol) in pyridine (0.75 mL) afforded, after purification by
column chromatography (70:30, hexane/EtOAc), 18 as a white
1
solid (0.18 g, 87%); mp: 1488C (hexane/EtOAc); H NMR (400 MHz,
CDCl3): d=8.52 (s, 1H), 8.02 (d, J=9.2 Hz, 1H), 7.96 (s, 1H), 7.79 (d,
J=8.4 Hz, 1H), 7.49 (t, J=8.4 Hz, 1H), 7.03 (d, J=8.8 Hz, 1H), 6.98
(d, J=8.4 Hz, 1H), 4.19 (q, J=7.2 Hz, 2H), 1.46 ppm (t, J=7.2 Hz,
3H); 13C NMR (100 MHz, CDCl3): d=161.0, 157.0, 147.6, 141.7,
136.0, 134.1 (2JCꢀF =33.2 Hz), 132.8, 122.2, 122.1 (1JCꢀF =274.0 Hz),
118.6, 118.5 (1JCꢀF =320.6 Hz), 117.3 (3JCꢀF =4.9 Hz), 115.5, 114.9,
˜
˜
112.5, 104.8, 65.7, 14.6 ppm; IR (NaCl): n=3286 (m, NꢀH), 2979 (w,
n=3292 (br, NꢀH), 2964 (s, CꢀH), 1668 (s, C=O), 1587 (s, C=C),
CꢀH), 1650 (m, C=O), 1582 (s, C=C), 1545 (s, C=C), 1431 (s, C=C),
1322 cmꢀ1 (s); MS (ESI+): 505 [M+Na]+ (91), 483 [M+H]+ (100);
HRMS (ESI+): m/z [M+H]+ calcd for C18H13F6N2O5S: 483.0444,
found: 483.0446; Anal. calcd for C18H12F6N2O5S: C, 44.82; H, 2.51;
found: C; 44.63; H, 1.98.
1530 (s, C=C), 1426 (m), 1260 (s), 1137 cmꢀ1 (s); MS (ESI+): 459
[M+Na]+ (38), 437 [M+H]+ (100), 391 (38); HRMS (ESI+): m/z
[M+H]+ calcd for C25H20F3N2O2: 437.1471, found: 437.1468.
(E)-N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-(4-trifluoro-
methylphenyl-ethen-1-yl benzamide (7h): Following the general
procedure described for the Suzuki cross coupling (Method B), the
reaction of 20a (0.06 g, 0.19 mmol), 18 (0.05 g, 0.11 mmol),
Pd(PPh3)4 (6 mg, 5.19 mmol) and K3PO4 (0.04 g, 0.19 mmol) in diox-
ane (0.55 mL) afforded, after purification by column chromatogra-
phy (-NH2 silica gel; 90:10!85:15, hexane/EtOAc), 7h as a white
solid (22.7 mg, 43%); mp: 1808C (hexane/acetone); 1H NMR
(400 MHz, (CD3)2CO): d=10.19 (s, 1H), 8.50 (s, 1H), 8.28 (dd, J=8.5,
1.0 Hz, 1H), 8.07 (d, J=8.5 Hz, 1H), 7.71 (d, J=8.4 Hz, 2H), 7.67 (d,
J=8.4 Hz, 2H), 7.5–7.4 (m, 3H), 7.36 (d, J=16.3 Hz, 1H), 7.08 (d, J=
7.9 Hz, 1H), 4.13 (q, J=7.0 Hz, 2H), 1.32 ppm (t, J=7.0 Hz, 3H);
13C NMR (100 MHz, (CD3)2CO): d=168.2, 158.1, 145.7, 142.9, 138.3,
137.8, 134.7 (2JCꢀF =32.5 Hz), 132.8, 131.9, 130.7 (2JCꢀF =32.5 Hz),
(E)-4,4,5,5-Tetramethyl-2-(4-trifluoromethylphenyl-vin-1-yl)-1,3,2-
dioxaborolane (20a): General procedure for the Takai–Utimoto reac-
tion. A suspension of CrCl3 (2.76 g, 22.43 mmol) in THF (30 mL) was
treated with 19a (0.38 mL, 2.80 mmol) and 3-(dichloromethyl)-
4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.18 g, 5.60 mmol) in THF
(6 mL). Then LiI (1.50 g, 11.20 mmol) in THF (6 mL) was added and
the suspension was stirred for 16 h at RT. The mixture was poured
into water, extracted with EtOAc (ꢄ3), and the solvent was evapo-
rated. The residue was purified by column chromatography (95:5,
hexane/EtOAc) to afford 20a as a white solid (0.68 g, 81%); mp:
608C (hexane/EtOAc); 1H NMR (400 MHz, CDCl3): d=7.6–7.5 (m,
4H), 7.39 (d, J=18.4 Hz, 1H), 6.25 (d, J=18.4 Hz, 1H), 1.28 ppm (s,
12H, 4CH3); 13C NMR (100 MHz, CDCl3): d=147.6, 140.8, 130.4
129.7, 129.1 (2C), 127.9, 127.5 (3JCꢀF =3.9 Hz, 2C), 126.3 (1JCꢀF
=
(2JCꢀF =32.5 Hz), 127.1 (2C), 125.5 (3JCꢀF =4.2 Hz, 2C), 124.1 (1JCꢀF
=
271.7 Hz), 124.6 (1JCꢀF =273.8 Hz), 124.0, 119.6, 118.7 (3JCꢀF =4.9 Hz),
˜
˜
274.7 Hz), 119.5, 83.5 (2C), 24.7 ppm (4C); IR (NaCl): n=2923 (s, Cꢀ
H), 2853 (m, CꢀH), 1623 cmꢀ1 (w, C=C); MS (ESI+): 321 [M+Na]+
(72), 299 [M+H]+ (100), 279 (20); HRMS (ESI+): m/z [M+H]+ calcd
for C15H19BF3O2: 299.1427, found: 299.1438; Anal. calcd for
C15H18BF3O2: C, 60.43; H, 6.09; found: C, 60.49; H, 6.09.
117.4, 113.8, 105.1, 66.2, 16.0 ppm; IR (NaCl): n=3500–3200 (br, Nꢀ
H), 2963 (m, CꢀH), 2931 (m, CꢀH), 1666 (m, C=O), 1588 (s, C=C),
1531 (s, C=C), 1326 (s), 1121 cmꢀ1 (s); MS (ESI+): 527 [M+Na]+
(48), 505 [M+H]+ (71), 391 (100); HRMS (ESI+): m/z [M+H]+ calcd
for C26H19F6N2O2: 505.1345, found: 505.1343.
(E)-4,4,5,5-Tetramethyl-2-(3-bromophenyl)ethen-1-yl-1,3,2-dioxa-
borolane (20b): Following the general procedure described for the
Takai–Utimoto reaction, 19b (0.33 mL, 2.80 mmol), 3-(dichloro-
methyl)-4,4,5,5-tetramethyl-1,3,2-dioxaborolane (1.18 g, 5.60 mmol),
CrCl3 (2.76 g, 22.43 mmol) and LiI (1.50 g, 11.20 mmol) in THF
(E)-N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-(3-bromo-
phenyl)ethen-1-yl benzamide (7i): Following the general proce-
dure described for the Suzuki cross coupling (Method B), the reac-
tion of 20b (58.4 mg, 0.19 mmol), 18 (50 mg, 0.11 mmol), Pd(PPh3)4
(6 mg, 5.19 mmol) and K3PO4 (40.1 mg, 0.19 mmol) in dioxane
1538
www.chemmedchem.org
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1530 – 1540

Anacardic Acid-Derived Histone Lysine Acetyltransferase Activators
(0.55 mL) afforded, after purification by column chromatography
(-NH2 silica gel; 90:10!85:15, hexane/EtOAc), 7i as a white solid
(15 mg, 27%); mp: 1448C (hexane/acetone); 1H NMR (400 MHz,
(CD3)2CO): d=10.17 (s, 1H), 8.49 (d, J=1.6 Hz, 1H), 8.28 (dd, J=
8.4, 1.6 Hz, 1H), 8.07 (d, J=8.6 Hz, 1H), 7.68 (t, J=1.7 Hz, 1H), 7.52
(d, J=7.8 Hz, 1H), 7.5–7.4 (m, 3H), 7.39 (d, J=16.3 Hz, 1H), 7.30 (t,
J=7.9 Hz, 1H), 7.25 (d, J=16.3 Hz, 1H), 7.06 (dd, J=6.7, 2.4 Hz,
1H), 4.12 (q, J=7.0 Hz, 2H), 1.31 ppm (t, J=7.0 Hz, 3H); 13C NMR
(100 MHz, (CD3)2CO): d=168.2, 158.0, 145.7, 141.5, 138.3, 138.0,
134.6 (2JCꢀF =31.2 Hz), 132.7, 132.6, 132.5, 131.9, 131.4, 128.6, 127.8,
peak”), monitored by FACS and analysed by CellQuest technology,
was used as an indicator of apoptosis.
Histone Extraction Protocol: Cells were harvested and washed twice
with ice-cold phosphate-buffered saline (PBS) and lysed in Triton
extraction buffer (TEB; PBS containing 0.5% Triton X-100 (v/v),
2 mm phenylmethylsulfonyl fluoride (PMSF), 0.02% (w/v) NaN3) at a
cellular density of 107 cells per mL for 10 min on ice, with gentle
stirring. After brief centrifugation at 2000 rpm at 48C, the superna-
tant was removed and the pellet was washed in half the volume of
TEB and centrifuged as before. The pellet was resuspended in
0.2m HCl at a cell density of 4ꢄ107 cells per mL, and acid extrac-
tion was left to proceed overnight at 48C on a rolling table. Next,
the samples were centrifuged at 2000 rpm for 10 min at 48C, the
supernatant was removed and the protein content was determined
using the Bradford assay.
127.3, 124.6 (1JCꢀF =272.7 Hz), 124.3, 124.0, 119.6, 118.7 (3JCꢀF
=
˜
4.9 Hz), 117.4, 113.6, 105.0, 66.1, 16.0 ppm; IR (NaCl): n=3500–3200
(br, NꢀH), 2980 (w, CꢀH), 2937 (w, CꢀH), 2891 (w), 1654 (s, C=O),
1584 (s, C=C), 1510 (s, C=C), 1424 (s), 1271 (s), 1178 (s), 1133 cmꢀ1
(s); MS (ESI+): 539 [M+Na]+ (28), 537 [M+Na]+ (28), 515 [M+H]+
(31), 515 [M+H]+ (31), 38 (100); HRMS (ESI+): m/z [M+H]+ calcd
for C25H19BrF3N2O2: 515.0577, found: 515.0585; Anal. calcd for
C25H18BrF3N2O2: C, 58.27; H, 3.52; found: C, 58.03; H, 3.62.
Western Blot analyses: Western Blot analyses were performed ac-
cording to standard procedures following the suggestions of the
antibody suppliers.
(E)-N-(4-Cyano-3-trifluoromethylphenyl)-2-ethoxy-6-(6-hydroxy-
hex-1-enyl)benzamide (7k): Following the general procedure de-
scribed for the Suzuki cross coupling (Method B), the reaction of
22 (27.2 mg, 0.19 mmol), 18 (50 mg, 0.11 mmol), Pd(PPh3)4 (6 mg,
5.19 mmol) and K3PO4 (40.1 mg, 0.19 mmol) in dioxane (0.55 mL) af-
forded, after purification by column chromatography (-NH2 silica
gel; 90:10!55:45, hexane/EtOAc), 7k as a colourless oil (43.5 mg,
Determination of Histone H3 Specific Acetylation: For histone H3
acetylation in U937 cells, 5 mg of histone extract were separated on
15% polyacrylamide gels and blotted. Western blots were shown
for pan-acetylated histone H3 (Upstate Biotechnology; Milan, Italy).
Human recombinant CBP assay: The recombinant cyclic adenosine
monophosphate response element-binding protein (CREB) binding
protein (CBP) was prepared in E. coli BL21 and purified by affinity
chromatography. The recombinant CBP fraction corresponded to
amino acids 1098–1877. CBP was incubated in KAT buffer x 5
(250 mm TRIS base pH 8.0, 50% glycerol, 0.5 mm EDTA, 5 mm DTT)
with 10 mg of histone H4 peptide (corresponding to amino acids
2–24) and 20 mm acetyl-CoA containing 0.5 mCimLꢀ1 [3H]acetyl-CoA
in the presence of inhibitors and putative KAT activators. After 2 h
a 378C, 5 mL of samples were spotted onto Whatman P81 paper (in
triplicate). The paper squares were washed in 5% TCA (ꢄ3) and
100% acetone (ꢄ1) and then placed into scintillation vials contain-
ing scintillation fluid to allow the disintegrations per minute (dpm)
reading. The dpm value of enzyme samples was compared with
the dpm value of negative and positive controls. Data have been
expressed as the percentage of activity considering the control
without treatment as 100%.
1
92%); H NMR (400 MHz, (CD3)2CO): d=10.09 (s, 1H), 8.50 (s, 1H),
8.25 (d, J=8.5 Hz, 1H), 8.05 (d, J=8.5 Hz, 1H), 7.34 (t, J=8.1 Hz,
1H), 7.22 (d, J=7.9 Hz, 1H), 6.94 (d, J=8.2 Hz, 1H), 6.53 (d, J=
15.7 Hz, 1H), 6.32 (td, J=15.6, 7.0 Hz, 1H), 4.08 (q, J=6.9 Hz, 2H),
3.49 (q, J=5.7 Hz, 2H), 3.4–3.3 (m, 1H), 2.18 (q, J=6.6 Hz, 2H), 1.6–
1.5 (m, 4H), 1.28 ppm (t, J=6.9 Hz, 3H); 13C NMR (100 MHz,
(CD3)2CO): d=168.6, 157.8, 145.8, 138.7, 138.2, 136.1, 134.6 (2JCꢀF
=
32.5 Hz), 132.3, 128.2, 127.1, 125.0 (1JCꢀF =272.7 Hz), 123.8, 119.4,
118.6 (3JCꢀF =4.9 Hz), 117.4, 112.4, 104.8, 66.0, 63.1, 34.6, 34.1, 27.3,
˜
16.0 ppm; IR (NaCl): n=3500–3200 (br, NꢀH and OꢀH), 2935 (m,
CꢀH), 1675 (m, C=O), 1591 (s, C=C), 1532 (s, C=C), 1329 (s), 1266
(s), 1177 cmꢀ1 (s); MS (ESI+): 455 [M+Na]+ (61), 433 [M+H]+ (100);
HRMS (ESI+): m/z [M]+ calcd for C23H24F3N2O3: 433.1734, found:
433.1731.
Biology
PCAF radioactive assay: 200 ng of human PCAF were incubated in
KAT buffer (Upstate Biotechnology) with 10 mg of histone H4 pep-
tide substrate (corresponding to amino acids 2–24) and 20 mm
acetyl-CoA containing 0.5 mCimLꢀ1 [3H]acetyl-CoA. The acetylation
reaction was performed in a volume of 25 mL in the presence of
test compounds at the desired final concentration. After 2 h a
378C, 5 mL of samples were spotted onto chromatographic What-
man P81 paper (in triplicate). After a washing session (3ꢄ5% TCA;
1ꢄ100% acetone), the paper squares were placed into scintillation
vials containing scintillation fluid to allow the dpm reading. The
dpm value of enzyme samples was compared to the dpm value of
the negative and positive controls and reported as the percentage
of activity considering the untreated control as 100%.
Ligands and materials: SAHA (Merck; Rome, Italy), MS-275 (Bayer
Schering AG; Berlin, Germany), and anacardic acid and CTPB
(Alexis–Enzo Life Sciences; New York, USA) were dissolved in
DMSO and used at 5ꢄ10ꢀ6 m. All other compounds described
were dissolved in DMSO (Sigma–Aldrich; Milan, Italy) and used at
5 mm and 50 mm.
Cell Culture: Human U937 and HL60 leukaemia cell lines were
propagated in RPMI medium supplemented with 10% fetal bovine
serum (FBS; Hyclone, Milan, Italy) and antibiotics (100 UmLꢀ1 peni-
cillin, 100 mgmLꢀ1 streptomycin and 250 ngmLꢀ1 amphotericin-B).
Cells were kept at the constant concentration of 200000 cells per
mL of culture medium.
Cell Cycle Analysis: 2.5ꢄ105 U937 cells were collected and resus-
pended in 500 mL of hypotonic buffer (0.1% Triton X-100, 0.1%
sodium citrate, 50 mgmLꢀ1 propidium iodide, RNAse A). Cells were
incubated in the dark for 30 min. Data were acquired on a FACS-
Calibur flow cytometer using the CellQuest software (Becton, Dick-
inson & Co.) and analysed with standard procedures also using
CellQuest and the ModFit LT v3 software (Verity) as previously re-
ported.[27] Nuclear fragmentation (the so-called “sub-G1 DNA
Acknowledgements
This work was partially supported by grants from the European
Union (EPITRON, LSHC-CT2005-518417; ATLAS, 221952), AIRC,
MICINN-Spain (SAF2007-63880-FEDER) and Xunta de Galicia-
Spain (Inbiomed).
ChemMedChem 2010, 5, 1530 – 1540
ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
www.chemmedchem.org
1539

A. R. de Lera, L. Altucci, R. ꢀlvarez et al.
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Published online on August 3, 2010
1540
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ꢃ 2010 Wiley-VCH Verlag GmbH & Co. KGaA, Weinheim
ChemMedChem 2010, 5, 1530 – 1540