Reactivity of isothiazolones and isothiazolone-1-oxides in the inhibition of the PCAF histone acetyltransferase

Article abstract of DOI:10.1016/j.ejmech.2009.07.025

Development of small molecule inhibitors of the histone acetyltransferase p300/CBP associated factor (PCAF) is relevant for oncology. The inhibition of the enzyme PCAF and proliferation of the cancer cell line HEP G2 by a series of 5-chloroisothiazolones was compared to a series of 5-chloroisothiazolone-1-oxides. The PCAF inhibitory potency of 5-chloroisothiazolones and 5-chloroisothiazolone-1-oxides is influenced by substitution in the 4-position. A study on the reactivity of the HAT inhibitors towards thiols and thiolates indicates that 5-chloroisothiazolones reacted quickly with propane-1-thiolate to provide many products, whereas 5-chloroisothiazolone-1-oxides provide only one defined product. Growth inhibition studies indicate that 5-chloroisothiazolones inhibit proliferation of HEP G2 cells at concentrations between 8.6 and 24 μM, whereas 5-chloroisothiazolone-1-oxides required higher concentrations or showed no inhibition.

Full text of DOI:10.1016/j.ejmech.2009.07.025

European Journal of Medicinal Chemistry 44 (2009) 4855–4861
Contents lists available at ScienceDirect
European Journal of Medicinal Chemistry
journal homepage: http://www.elsevier.com/locate/ejmech
Original article
Reactivity of isothiazolones and isothiazolone-1-oxides in the inhibition of the
PCAF histone acetyltransferase
Massimo Ghizzoni, Hidde J. Haisma, Frank J. Dekker*
Department of Pharmaceutical Gene Modulation, Faculty of Mathematics and Natural Sciences, University of Groningen, Anthonius Deusinglaan 1, 9713 AV Groningen, The
Netherlands
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 13 May 2009
Received in revised form
Development of small molecule inhibitors of the histone acetyltransferase p300/CBP associated factor
(
PCAF) is relevant for oncology. The inhibition of the enzyme PCAF and proliferation of the cancer cell line
HEP G2 by a series of 5-chloroisothiazolones was compared to a series of 5-chloroisothiazolone-1-oxides.
The PCAF inhibitory potency of 5-chloroisothiazolones and 5-chloroisothiazolone-1-oxides is influenced
by substitution in the 4-position. A study on the reactivity of the HAT inhibitors towards thiols and
thiolates indicates that 5-chloroisothiazolones reacted quickly with propane-1-thiolate to provide many
products, whereas 5-chloroisothiazolone-1-oxides provide only one defined product. Growth inhibition
studies indicate that 5-chloroisothiazolones inhibit proliferation of HEP G2 cells at concentrations
6
July 2009
Accepted 28 July 2009
Available online 3 August 2009
Keywords:
Histone acetyltransferase
PCAF
between 8.6 and 24
m
M, whereas 5-chloroisothiazolone-1-oxides required higher concentrations or
Isothiazolone
Isothiazolone-1-oxide
Epigenetics
showed no inhibition.
Ó 2009 Elsevier Masson SAS. All rights reserved.
Antitumour
1
. Introduction
now. Potent and selective bisubstrate inhibitors for p300 and PCAF
have been reported but their lack of cell-permeability limits their
application [12]. The natural products curcumin [13], garcinol [14]
and anacardic acid [15] show HAT inhibitory activity, however
their potency is low. A promising class of inhibitors is the iso-
thiazolones that inhibit HATs with IC50’s in the micromolar range
[16–18].
The PCAF inhibition of isothiazolones is expected to derive from
their reactivity towards the enzyme’s active site thiol. The reaction of
isothiazolones with a thiolate involves nucleophilic attack at the
sulfur atom with concomitant cleavage of the S–N bond to give a ring
opened product that can react further [19]. For 5-chlor-
oisothiazolone-1-oxides the reactivity towards thiolates is unknown.
Previously, we have reported a significant difference in PCAF HAT
inhibition between N-aliphatic substituted 5-chloroisothiazolones
and isothiazolones [16]. The most potent derivative is denoted
isothiazolone A (Fig. 1). We also found that PCAF is inhibited by the
5-chloroisothiazolone-1-oxide denoted isothiazolone oxide A (Fig.1).
Furthermore, we have demonstrated that 5-chloroisothiazolones
inhibit the growth of cancer cell lines in the micromolar range,
Histone acetylation and deacetylation play a crucial role in
regulation of gene transcription in eukaryotes [1]. These histone
modifications are regulated by two classes of enzymes: histone
acetyltransferases (HATs) and histone deacetylases (HDACs), which
respectively catalyze the addition to and the removal of acetyl
groups on specific lysine residues [2]. Small molecule inhibitors are
useful tools to study the functions of these enzymes and have
potential therapeutic applications [3,4].
HATs are grouped into different families according to their
sequence similarity [5]. The GNAT (Gcn5 related N-acetyltransfer-
ase) family HATs include the closely related enzymes PCAF (p300/
CBP associated factor) and GCN5 (general control of amino-acid
synthesis 5) [6]. The members of this family play a key role in
endothelial growth factor (EGF) mediated gene transcription and in
cell cycle progression and have therefore been recognized as
potential anticancer targets [5,7–9]. Furthermore, deregulation of
the activities of GNAT and p300 family HATs plays an important role
in human immunodeficiency virus (HIV) and chronic obstructive
pulmonary disease (COPD) [10,11].
whereas isothiazolones showed no inhibition at 10 mM.
Despite significant efforts, very few small molecule and cell-
permeable inhibitors for GCN5 and PCAF have been described until
In this study we investigated synthetic modifications of
5-chloroisothiazolones and 5-chloroisothiazolone-1-oxides and
studied their reactivity to thiols and thiolates using HPLC and NMR.
We studied also their inhibition of the enzyme PCAF and inhibition
of cell proliferation. Chlorine and methyl substituents were
*
Corresponding author. Tel.: þ31 50 3638030; fax þ31 50 3637953.
E-mail address: f.j.dekker@rug.nl (F.J. Dekker).
0223-5234/$ – see front matter Ó 2009 Elsevier Masson SAS. All rights reserved.
doi:10.1016/j.ejmech.2009.07.025

4856
M. Ghizzoni et al. / European Journal of Medicinal Chemistry 44 (2009) 4855–4861
Fig. 1. Inhibitors of the HAT PCAF with an isothiazolone or isothiazolone-1-oxide core
structure.
introduced in the 4-position of the 5-chloroisothiazolone and
5
-chloroisothiazolone-1-oxide core in order to study the effect of
substituents on the inhibition of the enzyme PCAF. Finally, the
growth inhibition of HEP G2 liver cancer cells upon treatment with
5
-chloroisothiazolones and 5-chloroisothiazolone-1-oxides was
investigated to explore the toxicity of these molecules.
2
. Chemistry
Scheme 1. Reagents and conditions: (a) mCPBA, CH
(CH 11–SH, Et N, CH Cl
2 2 2 2 2 2 3
Cl , (b) SO Cl , CH Cl , (c) CH –
2.1. Synthesis of isothiazolones and isothiazolone-1-oxides
2
)
3
2
2
.
A series of 5-chloroisothiazolones and 5-chloroisothiazolone-1-
with dodecane-1-thiol in the presence of triethylamine in yields
oxides was synthesized in order to explore synthetic modifications
of the isothiazolone ring and their effects on inhibition of the
enzyme PCAF and cell proliferation [16,20]. Synthesis was per-
formed according to previously published procedures. Amines
around 60% after purification. Compound 12 was chlorinated in the
4-position using sulfuryl chloride as a reagent to give 13 in 58%
yield.
5-Chloro-4-methylisothiazolones 19 and 20 were synthesized
0
were reacted with 3,3 -dithiobispropionyl chloride to yield the
using the procedure shown in Scheme 2 [19,23]. Methacrylic acid
0
0
corresponding 3,3 -dithiobispropionic amides. The 3,3 -dithio-
bispropionic amides were treated with 3 equivalents sulfuryl
chloride in dichloromethane. This provided reaction mixtures in
which the 5-chloroisothiazolones were the main products
(
14) was subjected to Michael addition with thioacetic acid to give
the 3-(acetylthio)-2-methylpropanoic acid (15) as racemic
mixture in 50% yield. Deacetylation was achieved by treatment
a
with 6 N HCl, and the resulting thiol was oxidized using H
yield the 3,3 -dithiobis(2-methylpropanoic acid) (16) that was pure
enough to continue to the next step. The diacid 16 was converted to
2
O
2
to
(
40–60%) and the isothiazolones the minor product (10–20%).
0
According to the literature, 4,5-dichloroisothiazolones 7 and 8 can
0
be obtained in one step from 3,3 -dithiobispropionic amides by
the diacylchloride and coupled to different amines to give the crude
treatment with 5 equivalents of sulfuryl chloride in yields between
0
3
3
,3 -dithiobispropionic amides 17 and 18 in yields around 30% over
10% and 40% [20]. We hypothesized that chlorination of the purified
0
steps. The crude 3,3 -dithiobispropionic amides were treated
5
-chloroisothiazolones in the 4-position would provide the 4,5-
ꢁ
with 3 equivalents of sulfuryl chloride at 0 C in dichloromethane
dichloroisothiazolones with higher yields. Treatment of 1 and 3
with sulfuryl chloride gave the 4,5-dichloroisothiazolones 7 and 8
in yields of 55% and 90%, respectively (Scheme 1). Compound 2 was
synthesized according to the procedure described by Yue et al. [21].
However, product 2 was obtained with a yield of 10% instead of the
reported 30–70%. The lower yield may be explained by the steric
hindrance for formation of the S–N bond when the N is tert-butyl
substituted.
to give the 5-chloro-4-methylisothiazolones 19 and 20 in yields of
38% and 52%, respectively. Compounds 19 and 20 were oxidized by
mCPBA to the corresponding 1-oxide derivates 21 and 22 in yields
around 75%.
2.2. Reactivity of isothiazolones and isothiazolone-1-oxides
The 5-chloroisothiazolones were oxidized by mCPBA to yield the
corresponding 5-chloroisothiazolone-1-oxides 4–6 as racemic
mixtures in yields between 60% and 80% after purification. Combs
et al. demonstrated that comparable S-oxides can be resolved using
chiral HPLC and that the stereochemistry is stable [22]. However,
attempts to separate the enantiomers of 5-chloroisothiazolone-1-
oxides 6 using chiral HPLC failed.
The biological activity of isothiazolones and their derivatives
originates from both their binding to proteins and their reactivity
Attempts were made to chlorinate the 5-chloroisothiazolone-1-
oxides 4 and 6 in the 4-position using sulfuryl chloride. However,
overnight treatment with 3 equivalents of sulfuryl chloride at room
temperature did not show conversion of the starting materials into
the desired products 9 and 10. Apparently, it is not possible to
chlorinate 5-chloroisothiazolone-1-oxides in the 4-position under
these conditions. Compounds 9 and 10 were obtained via oxidation
of 7 and 8 using mCPBA in yields around 80%.
Thiol substitution on the 5-chloroisothiazolone-1-oxides 5 and
6
was studied using dodecane-1-thiol as a reagent. The thiol (–SH)
did not react with the 5-chloroisothiazolone-1-oxides, whereas
a very quick reaction (<30 min) with the thiolate (–S ) was
Scheme 2. Reagents and conditions: (a) thioacetic acid, cyclohexane, reflux, (b) 6 N
ꢀ
HCl in H
2 2 2 2 2 3 2 2
O, reflux (c) aqueous 35% H O , (d) SOCl , reflux, (e) R–NH , Et N, CH Cl , (f)
observed. Products 11 and 12 were obtained by reaction of 5 and 6
SO Cl , CH
2
2
2
Cl (g) mCPBA, CH Cl
2
2
2
.

M. Ghizzoni et al. / European Journal of Medicinal Chemistry 44 (2009) 4855–4861
4857
do not react with thiols, whereas they react quickly with thiolates
providing many products that could not be identified.
The thiol reactivity of 5-chloroisothiazolone-1-oxide 6 was
investigated. No reaction was observed between 6 and propane-1-
thiol after treatment overnight. On the contrary, 6 reacted quickly
with sodium propan-1-thiolate in acetonitrile to provide one new
product (HPLC chromatograms are shown in the Supporting infor-
mation). Treatment with 0.1 equivalent resulted in one new product
with a conversion of about 10%. Treatment with 0.5 equivalent
resulted in about 50% conversion into one new product. After treat-
ment with 1.0 equivalent, the starting material was completely con-
verted into one new product. NMR data showed that this product
derives from addition-elimination in the 5-position (NMR data can be
found in the Supporting information). Treatment of 5-chlor-
oisothiazolon-1-oxide with more than 1.0 equivalent of sodium
propan-1-thiolate provided substitution in the 5-position, followed
by substitution in the 4-position. These data show that 5-chlor-
oisothiazolone-1-oxides do not react with thiols, whereas they react
quickly with thiolates through addition-elimination inthe5-position.
Fig. 2. Reactivity of the isothiazolone core.
towards thiolates [24,25]. It has been described that isothiazolones
react with thiolates on the sulfur atom, with concomitant cleavage
of the S–N bond. A study on N-methylisothiazolone 25, with
N-Acetyl-Cys as thiolate, showed formation of intermediate 27
(Fig. 2) [19]. The reaction between 5-chloro-N-methylchlor-
3. Pharmacology
oisothiazolone 23 is expected to proceed through the same
mechanism, however intermediate 28 has not been identified. This
might be due to the increased reactivity of intermediate 28 due to
the presence of the chlorine atom. Another line of evidence for this
inhibitory mechanism has been provided by a study on inhibition of
3.1. Histone acetyltransferase inhibition
Inhibition of the HAT PCAF by 5-chloroisothiazolones and
5-chloroisothiazolone-1-oxides was investigated in order to
explore the binding properties of these compounds. The PCAF HAT
inhibition studies were performed using a fluorescent assay as
described previously [16,28]. 5-Chloroisothiazolones 1 and 3
lck
p56 tyrosine kinase by isothiazolones [25]. Morley et al. have
studied the reactivity of (5-chloro)isothiazolones towards
2
-methyl-2-propanethiol at pH 4 in aqueous environment. They
have found kinetic rate constants in the order 23 > 24 > 25 > 26
showed PCAF inhibition with IC50 values of 3.0 and 1.8 mM,
(
Fig. 2) [26].
respectively (Table 1). The effects of substitutions in the 5-chlor-
oisothiazolone 4-position on PCAF inhibition were studied. Intro-
duction of a chlorine in the 4-position provided compounds 7 and 8
The reactivity of 5-chloroisothiazolones and 5-chlor-
oisothiazolone-1-oxides towards thiols and thiolates was studied
using comparable methods as published previously [27]. HPLC
analysis showed that 3 did not react with propane-1-thiol in aceto-
nitrile as a solvent after 24 h treatment at room temperature (HPLC
chromatograms are shown in the Supporting information). In
contrast, treatment with sodium propane-1-thiolate in acetonitrile
resulted in a quick conversion into many new products. Treatment
with 0.1 equivalent thiolate resulted in 10% conversion of the starting
material. Treatment with 0.5 equivalents resulted in 50% conversion
and many new products were observed. After treatment with 1.0
equivalent thiolate the starting material disappeared and many new
peaks were observed. These resultsshow that 5-chloroisothiazolones
with an IC50 of 2.4 and 2.6
group in the 4-position provided compounds 19 and 20 with less
than 50% inhibition at 10 M.
mM, whereas introduction of a methyl
m
The inhibitory properties of these molecules derive both from
their binding to the enzyme active site and from their chemical
reactivity. Chloro- or methyl substitution in the 4-position of
5-chloroisothiazolones could change the binding configuration to
the active site of the enzyme and thus influence the inhibitory
properties.
Substitution of the 5-chloroisothiazolones in their 4-position
changes their reactivity. The methyl group is an electron donating
Table 1
Inhibition of HAT PCAF and proliferation of cell lines by 5-chloroisothiazolinone and isothiazolone-1-oxide.
Compound
R1
R2
R3
PCAF inhibition (IC50
m
M)a
HEP G2 (IC50 m
M)b
1
7
19
3
8
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Methyl
H
Cl
Ethyl
Ethyl
Ethyl
3.0 ꢂ 0.3
2.4 ꢂ 0.1
>10
1.8 ꢂ 0.2
2.6 ꢂ 0.6
>10
12 ꢂ 0.5
12 ꢂ 2
O
R2
R3
9.1 ꢂ 0.9
24 ꢂ 3
N
R1
S
–(CH
–CH
–(CH
2
)
2
CO
CO
2
Me
Me
2
)
2
2
16 ꢂ 3
20
Methyl
2
)
2
CO
2
Me
8.6 ꢂ 1
4
9
21
6
1
22
5
1
1
1
Cl
Cl
Cl
Cl
Cl
Cl
Cl
H
Cl
Methyl
H
Cl
Methyl
H
H
H
Cl
Ethyl
Ethyl
Ethyl
>10
>10
>10
>100
>100
>100
>100
>100
>100
27 ꢂ 9
>50
O
R2
–(CH
–(CH
–(CH
2
)
)
)
2
2
2
CO
CO
CO
2
2
2
Me
Me
Me
5.6 ꢂ 0.2
N
R3
0
2
2
>10
R1
S
>10
>10
>10
>10
O
tert-Butyl
tert-Butyl
1
2
3
–S(CH)11CH
–S(CH)11CH
–S(CH)11CH
3
3
3
–(CH
–(CH
2
)
)
2
CO
CO
2
Me
Me
>100
>100
2
2
2
>10
a
Inhibition concentration 50% (IC50) determination n ¼ 3.
Growth inhibition 50% determinations n ¼ 8.
b

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M. Ghizzoni et al. / European Journal of Medicinal Chemistry 44 (2009) 4855–4861
group (EDG) due to inductive effects, whereas the chlorine is a net
electron-withdrawing group (EWG) due to inductive effects
partially counteracted by an electron donating contribution
through resonance. Introduction of an EDG like a methyl group (20)
causes loss of potency, which suggests that an EDG in the 4-position
adds electron density to the isothiazolone ring and thus stabilizes it
for nucleophilic attack. On the contrary, the introduction of an EWG
like a chlorine atom (8) does not affect the inhibitory properties.
This datum suggests that the isothiazolone reactivity on the
reaction, multiple products were observed which hampers the
elucidation of the reaction mechanism. 5-Chloroisothiazolone-1-
oxides do not react with thiols, whereas they react quickly with
thiolates in the 5-position. The reaction occurs via an addition-
elimination mechanism to give 5-alkylthioisothiazolone-1-oxides.
The resulting 5-alkylthioisothiazolone-1-oxide was chlorinated in
the 4-position using sulfuryl chloride.
N-aliphatic substituted 5-chloroisothiazolones (1 and 3) showed
PCAF inhibition at micromolar concentrations (2–3 mM). Chlorine
1
-position towards thiolates is already at its maximum and cannot
substitution in the 4-position yielded products which retained PCAF
inhibition, whereas methyl substitution provided compounds that
be increased further by an EWG.
The effect of substitution in the 4-position of 5-chlor-
oisothiazolone-1-oxides on inhibition of PCAF activity was studied.
The compounds were tested as racemic mixtures. Compound 4,
with an N-ethyl substitution, showed less than 50% inhibition at
showed no inhibition at 10
with a methyl ester in the N-substitution showed an IC50 of 5.6
whereas 4showedanIC50 higherthan10 M. These dataindicatethat
the methyl ester contributes to PCAF inhibition. Methyl or chlorine
substitution in the 4-position and thiol substitution in the 5-position
of 5-chloroisothiazolone-1-oxides provided compounds with IC50
mM. 5-Chloroisothiazolone-1-oxide 6
mM
m
10
m
M. Introduction of either chlorine (9) or methyl group (21) in
the 4-position, provided compounds that did not show any
inhibition at 10 M. Compound 6 with a methyl ester in the
N-substitution showed PCAF inhibition with an IC50 value of
.6 M. Introduction of either a chlorine (10) or a methyl group (22)
provided compounds that showed less than 50% PCAF inhibition at
M. These differences in binding could derive from changes in
m
values higher than 10
growth of HEP G2 cancer cells in concentrations between 8.6 and
24 M, whereas little or no growth inhibition was observed for 5-
mM. 5-Chloroisothiazolones inhibited the
5
m
m
chloroisothiazolone-1-oxides.
5. Experimental protocols
5.1. General procedures
10 m
binding to the enzyme active site as well as from changes in
reactivity.
The 5-dodecylthioisothiazolone-1-oxides 11 and 12 and
5
-dodecylthio-4-chloroisothiazolone-1-oxide 13 showed no
detectable PCAF inhibition at 10 M. The lack of activity of these
compounds might derive both from their binding properties to the
enzyme active site as well as their reactivity towards thiolates.
m
Chemicals were obtained from commercial suppliers (Sigma–
Aldrich, Acros Organics) and in most cases used without further
purification. Dichloromethane was dried by distillation over CaH
2
before use. Aluminum sheets of Silica Gel 60 F254 were used for
Thin-layer chromatography (TLC). Spots were visualized under
3.2. Growth inhibition
4
ultraviolet light or stained with KMnO solution. MP Ecochrom
Growth inhibition of cancer cells was studied in order to explore
Silica Gel 32–63 60 Å was used for column chromatography.
Melting points were determined on an Electrothermal digital
melting point apparatus and are uncorrected. H and C NMR
spectra were recorded on a Varian Gemini-200 (50.32 MHz) spec-
trometer. Chemical shift values are reported relative to residual
how modifications of the isothiazolone core influence the inhibi-
tion of cell proliferation. The growth inhibition of the human cancer
cell line HEP G2 (liver cancer) was studied using a crystal violet
assay as described previously [16]. The 5-chloroisothiazolones
inhibited the growth of HEP G2 cells with IC50 values between 8.6
1
13
1
13
1
solvent peaks (CD
3
OD,
H
d
¼ 3.31,
C
d
¼ 49.00 or CHCl
3
, H
13
and 24
mM (Table 1). The differences in PCAF inhibitory potency are
d
¼ 7.26,
C
d
¼ 77.16). The coupling constants (J) are reported in
1
3
not reflected in the inhibition of cell proliferation. Other mecha-
nisms might be involved in the inhibition of cell proliferation by
these compounds like for example binding to other proteins or
reactions with cellular glutathione. Western blot on inhibition of
histone acetylation upon treatment of this cell line with the
inhibitors is required to investigate if the molecules inhibit histone
acetylation in cell-based assays.
Hertz (Hz). C spectra were recorded using the attached proton test
(APT) pulse sequence. HPLC analyses were performed with
a Waters 510 pump, equipped with an ISCO 2360 gradient
programmer and a Waters 486 UV detector (254 nm). The column
was a 25 cm ꢃ 4.6 mm (5
mm) Discovery C18, with a flow of 0.5 mL/
min. Electrospray ionization mass spectra (ESI-MS) were recorded
on an Applied Biosystems/SCIEX API3000-triple quadrupole mass
spectrometer. High-resolution mass spectra (HR-MS) were recor-
ded using a flow injection method on an LTQ-Orbitrap XL mass
spectrometer (Thermo Electron, Bremen, Germany) with a resolu-
tion of 60,000 at m/z 400. Internal recalibration in real time was
performed with protonated testosterone (lock mass m/
z ¼ 289.2162).
5
-Chloroisothiazolone-1-oxides showed less than 50% inhi-
bition of cell proliferation at 100 M, with the exception of
m
compound 5. In general, 5-chloroisothiazolone-1-oxides are less
lipophilic then 5-chloroisothiazolones, which might hamper
their cellular permeability. The partition coefficient (log P) for
isothiazolone-1-oxide 6 was determined to be 0.08, whereas for
isothiazolone 3 a log P value of 0.69 was measured. The low log P
value of isothiazolone-1-oxide 6 might hamper the inhibition of
cell proliferation.
5.2. Synthetic procedure 1
The starting material (1.0 mmol) was dissolved in dichloro-
4
. Conclusion
methane (10 mL), and m-chloroperbenzoic acid (70%) (1.2 mmol,
0.29 g) was added in several portions over 20 min. The mixture
A series of 5-chloroisothiazolones and 5-chloroisothiazolone-1-
was stirred for 3 h at room temperature. A few milligrams of
sodium metabisulfite were added to quench the excess mCPBA,
and the mixture was stirred for 5 min. The solvent was evapo-
rated under reduced pressure, and the residue was dissolved in
oxides with chlorine and methyl substitutions in the 4-position was
synthesized. 5-Chloroisothiazolones were readily chlorinated in
the 4-position by sulfuryl chloride, whereas the 5-chlor-
oisothiazolone-1-oxides could not be chlorinated in the 4-position
under the applied conditions. 5-Chloroisothiazolones do not react
with thiols whereas they react quickly with thiolates. Upon
EtOAc (15 mL), and extracted with saturated NaHCO
(3 ꢃ15 mL) and brine (1 ꢃ15 mL). The organic layer was dried
over Na SO , filtered and the solvent was evaporated under
3
solution
2
4

M. Ghizzoni et al. / European Journal of Medicinal Chemistry 44 (2009) 4855–4861
4859
reduced pressure. The crude product was purified by column
chromatography.
91%): R
f
¼ 0.40 (EtOAc/Hex 1:1); 1H NMR (200 MHz, CDCl
3
):
d
¼ 2.75 (t, J ¼ 6.1 Hz, 2H), 3.71 (s, 3H), 4.07 ppm (t, J ¼ 6.0, Hz 2H);
1
3
C NMR (50 MHz, CDCl
3
):
d
¼ 33.6, 41.0, 52.3, 114.5, 139.7, 162.1,
5.3. Synthetic procedure 2
171.7 ppm; HPLC: purity >98%, RT ¼ 10.5 min, mobile phase:
þ
H
2
O:CH
3
CN:TFA 50:50:0.1; HRMS: m/z [M þ H]
calcd for
3
5
The starting material (1.0 mmol) was dissolved in dichloro-
C
7
H
8
Cl
2 3
NO S: 255.9597, found: 255.9596.
methane (10 mL). Sulfuryl chloride (3.0 mmol, 0.24 mL) was added
slowly and the solution was stirred overnight at room temperature.
5.9. 4,5-Dichloro-2-ethylisothiazol-3(2H)-one-1-oxide (9)
H
2
O (1.0 mL) was added to the solution, and the mixture was stirred
for 5 min. The mixture was extracted with water (3 ꢃ10 mL) and
SO , and filtered. The solvent was
evaporated under reduced pressure, and the product was purified
by column chromatography.
The product was obtained using procedure 1 starting from 7 and
purified using column chromatography with EtOAc/Hexane 1:3 (v/
v) as eluent. The compound was obtained as a pale yellow solid
brine (1 ꢃ10 mL), dried over Na
2
4
1
(165 mg, 77%): R
CDCl ):
(50 MHz, CDCl d
f
¼ 0.58 (EtOAc/Hex, 1:2); H NMR (200 MHz,
13
3
d
¼ 1.32–1.40 (m, 3H), 3.72–3.91 (m, 2H) ppm. C NMR
):
¼ 14.7, 38.0, 131.0, 148.7, 159.8 ppm; HPLC:
5.4. Synthetic procedure 3
3
purity 98%, RT ¼ 12.2 min, mobile phase:
H
2
O:CH
3
CN:TFA
þ
35
The dithiobispropanamide (1.0 mmol) was dissolved in dry
50:50:0.1; HRMS: m/z [M þ H] calcd for C
5
H
6
Cl
2
NO S: 213.9491,
2
ꢁ
dichloromethane (5 mL) and cooled to 0 C. Sulfuryl chloride
found: 213.9491.
(
3.0 mmol, 0.24 mL) was added dropwise, and the mixture was
stirred for 2 h at room temperature. H O (1.0 mL) was added, and
the mixture was stirred for 5 min. Subsequently, the mixture was
2
5.10. Methyl 3-(4,5-dichloro-1-oxido-3-oxoisothiazol-2(3H)-
yl)propanoate (10)
extracted with water (3 ꢃ10 mL) and brine (1 ꢃ10 mL), dried over
Na
2
SO
4
, and filtered. The solvent was evaporated under reduced
The product was obtained using procedure 1 starting from 8 and
pressure, and the product was purified by column chromatography.
purified using column chromatography with EtOAc/Hex 1:2 (v/v) as
eluent. The compound was obtained as a yellow oil (212.2 mg, 78%):
1
5
.5. 5-Chloro-2-ethylisothiazol-3(2H)-one-1-oxide (4)
R
2
f
¼ 0.48 (EtOAc/Hex, 1:1); H NMR (200 MHz, CDCl
3
):
d
¼ 2.71–
13
.81 (m, 2H), 3.70 (s, 3H), 4.05 ppm (t, J ¼ 6.5 Hz, 2H); C NMR
The product was obtained using procedure 1 starting from 1 and
(50 MHz, CDCl
3
):
d
¼ 33.4, 38.2, 52.3, 130.6, 149.4, 160.3, 171.1 ppm;
purified using column chromatography with EtOAc/Hex 1:2 (v/v) as
eluent. The compound was obtained as a yellow oil (100 mg, 56%):
R
HPLC: purity 98%, RT ¼ 11.5 min, mobile phase: H
2
O:CH
3
CN:TFA
þ
35
50:50:0.1; HRMS: m/z [M þ H] calcd for C
7
H
8
Cl
2
NO S: 271.9546,
4
1
f
¼ 0.38 (EtOAc/Hex, 1:2); H NMR (200 MHz, CDCl
3
):
d
¼ 1.33 (t,
found: 271.9544.
13
J ¼ 7.1 Hz, 3H), 3.65–3.89 (m, 2H), 6.67 ppm (s, 1H); C NMR
50 MHz, CDCl ):
¼ 14.8, 36.8, 124.4, 156.9, 169.4 ppm; HPLC:
(
3
d
5.11. 2-Tert-butyl-5-(dodecylthio)isothiazol-
purity >98%, RT ¼ 9.6 min, mobile phase:
H
2
O:CH
3
CN:TFA
3(2H)-one-1-oxide (11)
þ
35
6
0:40:0.1; HRMS: m/z [M þ H] calcd for C
5
H
7
ClNO S: 179.9988,
2
found: 179.9876.
Dodecanethiol (1.2 mmol, 0.28 mL) was added to a solution of
2
-tert-butyl-5-chloroisothiazol-3(2H)-one-1-oxide (1.2 mmol,
5
.6. 2-Tert-butyl-5-chloroisothiazol-3(2H)-one 1-oxide (5)
0.25 mg) in dry dichloromethane at room temperature. Triethyl-
amine (1.2 mmol, 0.15 L) was added dropwise, and the mixture
was stirred for 3 h at room temperature. Dichloromethane
(50.0 mL) was added, and the organic layer was extracted with H
SO . The
m
The product was obtained using procedure 1 starting from 2
and purified using column chromatography with EtOAc/Hex 1:2 (v/
v) as eluent. The compound was obtained as a colorless oil (62 mg,
3
2
O
(3 ꢃ 50 mL) and brine (1 ꢃ50 mL), and dried with Na
2
4
1
0%): R
f
¼ 0.6 (EtOAc/Hex, 1:1); H NMR (200 MHz, CDCl
3
):
d
1.63
solvent was evaporated under reduced pressure, and the residue
was purified by column chromatography using EtOAc/Hex 1:25 (v/
v) as eluent. The compound was obtained as a white solid (241 mg,
13
(
s, 9H), 6.55 ppm (s, 1H); C NMR (50 MHz, CDCl
3
):
d
29.4, 59.7,
1
25.8, 136.3, 165.6 ppm; HPLC: purity 98%, RT ¼ 11.1 min, mobile
ꢁ
1
phase: H
2
O:CH
3
CN:TFA 50:50:0.1; MS (ES, 70 eV): m/z 207.9
57%): R
CDCl ):
.65–1.85 (m, 2H), 2.98 (t, J ¼ 7.3 Hz, 2H), 6.07 ppm (s, 1H). C NMR
(50 MHz, CDCl ):
¼ 14.3, 22.8, 28.3, 28.9, 29.1, 29.3, 29.5, 29.6,
9.7, 32.0, 33.6, 58.9, 118.0, 165.5, 167.2 ppm. MS (ESI): m/z 374.1
f
¼ 0.42 (EtOAc/Hex, 1:4); mp: 62 C; H NMR (200 MHz,
þ
[M þ H] .
3
d
¼ 0.87 (t, J ¼ 6.5 Hz, 3H), 1.20–1.50 (m, 18H), 1.62 (s, 9H),
13
1
5.7. 4,5-Dichloro-2-ethylisothiazol-3(2H)-one (7)
3
d
2
þ
The product was obtained using procedure 2 starting from 1 and
[M þ H] .
purified using column chromatography with EtOAc/Hex 1:1 (v/v) as
eluent. The compound was obtained as a yellow oil (109 mg, 55%):
5.12. Methyl 3-[5-(dodecylthio)-1-oxido-3-oxoisothiazol-2(3H)-yl]
propanoate (12)
1
R
f
¼ 0.51(EtOAc/Hex 1:2); H NMR (200 MHz, CDCl
3
):
d
¼ 1.32 (t,
13
J ¼ 7.2 Hz, 3H), 3.86 ppm (q, J ¼ 7.0 Hz, 2H); C NMR (50 MHz,
CDCl ):
¼ 14.7, 40.4, 138.4, 161.8, 168.0 ppm; HPLC: purity 99%,
3
d
The product was obtained using the same procedure as for
compound 11 starting from dodecanethiol and methyl 3-(5-chloro-
1-oxido-3-oxoisothiazol-2(3H)-yl)propanoate (6). The compound
was purified using column chromatography with EtOAc/Hex (v/v)
1:10 as eluent and was obtained as a white solid (335 mg, 69%):
RT ¼ 12.2 min, mobile phase: H
2
O:CH
3
CN:TFA 60:40:0.1; HRMS: m/
þ
35
z [M þ H] calcd for C
5 6 2
H Cl NOS: 197.9542, found: 197.9543.
5.8. Methyl 3-(4,5-dichloro-3-oxoisothiazol-2(3H)-yl)
ꢁ
1
propanoate (8)
R
d
f
¼ 0.39 (EtOAc/Hex, 1:2); mp: 55.7 C; H NMR (200 MHz, CDCl
3
):
¼ 0.86 (t, J ¼ 6.5 Hz, 3H), 1.20–1.50 (m, 18H), 1.65–1.85 (m, 2H),
The product was obtained using procedure 2 starting from 3 and
was purified using column chromatography with EtOAc/Hex 1:1 (v/
v) as eluent. The compound was obtained as a yellow oil (233 mg,
2.72 (dt, J ¼ 4.0, 7.1 Hz, 2H), 3.01 (t, J ¼ 7.3 Hz, 2H), 3.69 (s, 3H), 3.98
13
(t, J ¼ 6.8 Hz, 2H) 6.15 ppm (s, 1H). C NMR (50 MHz, CDCl
3
):
d
¼ 14.23, 22.8, 28.2, 28.8, 29.1, 29.4, 29.5, 29.6, 29.7, 32.0, 33.8, 33.9,

4860
M. Ghizzoni et al. / European Journal of Medicinal Chemistry 44 (2009) 4855–4861
þ
3
7.2, 52.1, 115.8, 166.4, 168.3, 171.4 ppm; HRMS: m/z [M þ H] calcd
149.8, 165.6, 171.2 ppm; HPLC: purity 97%, RT ¼ 11.2, mobile phase
for C19
H34NO
4
S
2
: 404.1924, found: 404.1925.
2
H O:CH
3
CN:TFA 60:40:0.1; HRMS: m/z [M þ H]þ calcd for
3
5
8 4
C H11ClNO S: 252.0092, found 252.0091.
5
2
.13. Methyl 3-[4-chloro-5-(dodecylthio)-1-oxido-3-oxoisothiazol-
(3H)-yl]propanoate (13)
5.18. Determination of the propane-1-thiol concentration
0
The product was obtained using procedure 2 starting from 12
The concentration of free thiol was quantified using 5,5 -Dithio-
bis(2-nitrobenzoic acid) (DTNB). A DTNB calibration curve was
determined by measurement of the UV extinction at 401 nm for
and was purified using column chromatography with EtOAc/Hex
:10 (v/v) as eluent. The compound was obtained as a colorless oil
254 mg, 58%) (the starting material 12 was isolated in a yield of
1
(
3
d
DTNB concentration between 1 mM and 10
(10 mM) propane-1-thiol was added. Subsequently, 100
a 1 mM propane-1-thiol solution was mixed with 100 L 20 mM
m
M to which an excess
1
1%): R
¼ 0.86 (t, J ¼ 6.5 Hz, 3H), 1.20-1.80 (m, 20H), 1.65–1.85 (m, 2H),
.65–2.80 (m, 2H), 2.85–3.00 (m, 2H), 3.69 (s, 3H), 3.95–4.10 ppm
):
¼ 14.4, 28.8, 29.0, 29.2, 29.3,
9.6, 29.7, 29.8, 30.9, 32.1, 33.2, 38.4, 39.9, 40.7, 52.4, 95.8, 167.8,
f
¼ 0.49 (EtOAc/Hex, 1:2); H NMR (200 MHz, CDCl
3
):
mL of
m
2
(
2
1
DTNB solution. The UV absorbance was measured and the –SH
content was calculated from the calibration curve. No significant
differences were observed between the calculated (0.50 mM) and
measured (0.48 mM) concentrations.
m, 2H); 13C NMR (50 MHz, CDCl
d
3
þ
35
69.5, 171.2 ppm; HRMS: m/z [M þ H] calcd for C19
H
33ClNO
4
S
2
:
4
38.1534, found: 438.1532.
5.19. Reaction of isothiazolone with propane-1-thiol
5.14. 5-Chloro-2-ethyl-4-methylisothiazol-3(2H)-one (19)
The isothiazolone(-1-oxide) (50
(2 mL). A 0.1 M solution of propane-1-thiol in CH
500 L) was added, and the progress of the reaction was followed
by HPLC analysis. The mobile phase contained H O:CH CN:TFA
m
mol) was dissolved in CH
3
CN
0
The product was obtained using procedure 3 starting from 3,3 -
3
CN (50 mol,
m
dithiobis(N-ethyl-2-methylpropanamide) and was purified using
column chromatography with EtOAc/Hex 1:6 (v/v) as eluent. The
m
2
3
compound was obtained as a yellow liquid (64 mg, 38%): R
f
¼ 0.34
¼ 1.26 (t, J ¼ 7.2, 3H),
):
10.9, 14.8, 39.0, 122.0, 138.4, 166.5 ppm; HPLC: purity 98%,
50:50:0.1 and the compounds were identified on the basis of their
retention times.
1
(
1
3
EtOAc/Hex, 1:2); H NMR (200 MHz, CDCl ): d
.97 (s, 3H), 3.78 ppm (q, J ¼ 7.3, 2H); C NMR (50 MHz, CDCl
13
3
d
5.20. Reaction of isothiazolone with sodium propane-1-thiolate
RT ¼ 9.2, mobile phase: H
2
O:CH
3
CN:TFA 60:40:0.1; HRMS: m/z
M þ H]þ calcd for C
H ClNOS: 178.0088, found: 178.0088.
35
A solution of 0.1 M sodium propane-1-thiolate in water (30 mL)
[
6 9
was prepared from propane-1-thiol (272
(108 mg, 2.7 mmol). The isothiazolone(-1-oxide) (50
dissolved in CH CN (2 mL). A 0.1 M solution of sodium propane-1-
mL, 3 mmol) and NaOH
5.15. Methyl 3-(5-chloro-4-methyl-3-oxoisothiazol-2(3H)-
mmol) was
yl)propanoate (20)
3
thiolate (0.1, 0.5 or 1.0 equiv) was added, and the progress of the
reaction was monitored by HPLC. The mobile phase contained
H O:CH CN:TFA 50:50:0.1 and the compounds were identified on
2 3
0
The product was obtained using procedure 3 starting from 3,3 -
dithiobis[N-(methyl 2-aminopropionate)-2-methylpropanamide]
and purified using column chromatography with EtOAc/Hex 1:4 (v/
the basis of their retention times.
v) as eluent. The compound was obtained as a yellow gum (122 mg,
1
5
d
2%): R
f
¼ 0.44 (EtOAc/Hex, 1:1); H NMR (200 MHz, CDCl
3
):
5.21. Log P measurement
¼ 1.98 (s, 3H), 2.71 (t, J ¼ 6.2 2H), 3.70 (s, 3H), 4.02 ppm (t, J ¼ 6.3
13
2
1
H
H); C NMR (50 MHz, CDCl
3
):
d
¼ 10.9, 33.8, 39.8, 52.2, 121.5,
The compound (4 mmol) was dissolved in 1-octanol (1 mL).
Water (1 mL) was added and the mixture was vigorously shaken
with a vortex mixer (3 ꢃ 5 min) and centrifuged (2000g, 10 s). The
two layers were separated and analyzed by HPLC The mobile phase
39.6, 166.9, 171.7; HPLC: purity 99%, RT ¼ 11.4 min, mobile phase:
2
O:CH
3
CN:TFA 60:40:0.1; HRMS: m/z [M þ H]þ calcd for
C8H1135ClNO3S: 236.0142, found: 236.0141.
2 3
contained H O:CH CN:TFA 50:50:0.1. The log P was calculated by
5.16. 5-Chloro-2-ethyl-4-methylisothiazol-3(2H)-one-1-oxide (21)
comparison of the peak area of the analyte in both layers.
The product was obtained using procedure 1 starting from 19
5.22. Enzyme inhibition studies
and purified using column chromatography with EtOAc/Hex 1:2 (v/
v) as eluent. The compound was obtained as a colorless liquid
A fluorescent histone acetyltransferase assay described by
Trievel et al. was used for enzyme inhibition studies [27]. Enzyme
inhibition was measured by determination of the residual
enzyme activity after 15 min incubation with the inhibitor. The
enzyme activity was measured by detection of CoA–SH by the
fluorescent dye 7-(diethylamino)-3-(40-maleimidylphenyl)-4-
methylcoumarin (CPM). The CoA–SH concentrations measured with
1
(
159 mg, 82%): R
CDCl ):
C NMR (50 MHz, CDCl
66.5 ppm; HPLC: 98%,
O:CH
f
¼ 0.72 (EtOAc/Hex, 1:1); H NMR (200 MHz,
3
d
¼ 1.32 (t, J ¼ 7.3, 3H), 2.05 (s, 3H), 3.69–3.87 ppm (m, 2H).
):
¼ 10.9, 14.8, 39.0, 122.0, 138.4,
1
3
3
d
1
H
RT ¼ 11.8 min,
mobile
phase:
CN:TFA 60:40:0.1; HRMS: m/z [M þ H]þ calcd for
2
3
3
5
6 9 2
C H ClNO S: 194.0037, found: 194.0032.
no inhibitor present were around 50
tions were maximal 10 M, so that inhibitory effects of more than
20% at 10 M inhibitor concentration cannot be explained by direct
reaction of the inhibitors with CoA–SH. Compounds that showed
more than 50% inhibition at 10
determination (n ¼ 3). The human recombinant histone acetyl-
transferase PCAF (p300/CREB-bindingprotein Associated Factor)
was obtained from Biomol International. The histone H3 peptide
(Ac-QTARKSTGGKAPRKQLATKNH2) was purchased from Pepscan
(Lelystad, NL).
mM. The inhibitor concentra-
5
.17. Methyl 3-(5-chloro-4-methyl-1-oxido-3-oxoisothiazol-2(3H)-
m
yl)propanoate (22)
m
The product was obtained using procedure 1 starting from 20
m
M (n ¼ 3) were subjected to IC50
and purified using column chromatography with EtOAc/Hex 1:4 (v/
v) as eluent. The compound was obtained as a colorless oil (181 mg,
1
72%): R
f
¼ 0.56 (EtOAc/Hex, 1:1); H NMR (200 MHz, CDCl
3
):
d
¼ 2.04 (s, 3H), 2.69–2.77 (m, 2H), 3.69 (s, 3H), 4.00 ppm (t, J ¼ 6.7,
13
2
H); C NMR (50 MHz, CDCl
3
):
d
¼ 10.8, 33.6, 37.4, 52.2, 133.6,

M. Ghizzoni et al. / European Journal of Medicinal Chemistry 44 (2009) 4855–4861
4861
5
.23. Cell growth inhibition
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All cell culture reagents were purchased from Invitrogen. The
human cancer cells HEP G2 (liver) were maintained in Dulbecco’s
modified Eagle’s Medium (DMEM) with 10% heat-inactivated fetal
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aspirated, and the cells were fixed with 50
mL 1% crystal violet in
7
0% ethanol for 30 min. The cells were washed with water and the
[
[
[
staining was solubilized by addition of 100 mL 1% SDS in water. The
plates were read at 550 nm. A blank extinction value in which no
cells were seeded was subtracted from all determinations and cell
growth with no inhibitor present was set to 100%. All concentra-
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[
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final products numbered 15–18 can be found in the supporting
information. HPLC chromatograms of the reaction of compounds 3
and 6 with thiols or thiolates and also the H and C NMR spectra for
reaction of 6 with propane-1-thiolate can be found in the supporting
information. Supplementary data associated with this article can be
found in the online version at doi:10.1016/j.ejmech.2009.07.025.
5
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