Inhibition of the PCAF histone acetyl transferase and cell proliferation by isothiazolones

Article abstract of DOI:10.1016/j.bmc.2008.12.008

Small molecule HAT inhibitors are useful tools to unravel the role of histone acetyl transferases (HATs) in the cell and have relevance for oncology. We present a systematic investigation of the inhibition of the HAT p300/CBP Associated Factor (PCAF) by i

Full text of DOI:10.1016/j.bmc.2008.12.008

Bioorganic & Medicinal Chemistry 17 (2009) 460–466
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Inhibition of the PCAF histone acetyl transferase and cell proliferation
by isothiazolones
*
Frank J. Dekker , Massimo Ghizzoni, Nanette van der Meer, Rosalina Wisastra, Hidde J. Haisma
Department of Therapeutic 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:
Small molecule HAT inhibitors are useful tools to unravel the role of histone acetyl transferases (HATs) in
the cell and have relevance for oncology. We present a systematic investigation of the inhibition of the
HAT p300/CBP Associated Factor (PCAF) by isothiazolones with different substitutions. 5-chloro-
isothiazolones proved to be the most potent inhibitors of PCAF. The growth inhibition of 4 different cell
lines was studied and the growth of two cell lines (A2780 and HEK 293) was inhibited at micromolar con-
Received 12 September 2008
Revised 3 December 2008
Accepted 4 December 2008
Available online 13 December 2008
TM
centrations by 5-chloroisothiazolones. Furthermore, the 5-chloroisothiazolone preservative Kathon CG
that is used in cosmetics inhibited PCAF and the growth of cell lines A2780 and HEK 293, which indicates
that this preservative should be applied with care.
Keywords:
PCAF
Histone acetyl transferase
Acetylation
Ó 2008 Elsevier Ltd. All rights reserved.
Isothiazolones
1. Introduction
p300 and PCAF by coupling the histone H3 peptide to CoA.15 How-
ever, this provided compounds with poor membrane permeability.
Posttranslational modifications of histone proteins play a cru-
cial role in gene-specific transcription regulation in eukaryotes.1
These histone modifications occur in distinct patterns that mediate
specific interactions with multiprotein complexes, which initiate
or inhibit gene transcription via the so-called ‘histone-code’.2–4
There is increasing evidence that the histone code plays a crucial
role in normal and aberrant cell function and differentiation. Small
molecule modulators of histone modifying enzymes are useful
tools to unravel the functions of these enzymes and might ulti-
mately lead to therapeutic applications.
The histone acetyl transferases (HATs) form a disparate group of
enzymes that mediate acetyl transfer to histones or other pro-
teins.5,6 The GNAT (Gcn5 related N-acetyltransferase) family HATs
include the closely related enzymes PCAF (p300/CBP associated
factor) and GCN5 (general control of amino-acid synthesis 5).7 PCAF
acetylates histone H3 on lysine 14 and less efficiently histone H4 on
lysine 8.8 The GNAT family HATs have been recognized as potential
anticancer and antiviral targets.6,9 The HAT GCN5 plays a key role in
EGF mediated gene transcription, which is relevant for cancer ther-
apy.10 Furthermore, GCN5 is crucial for cell cycle progression.11
Deregulation of the activity of GNAT and p300 family HATs plays
an important role in a number of human cancers.12–14
The natural products Curcumin,16 Garcinol17 and Anacardic acid18
show HAT inhibitory activity, however their potency is low. GCN5
and PCAF are inhibited by
a-methylene-c
-butyrolactones19 and
isothiazolones,20 that covalently capture the active site thiol of
these enzymes.
The isothiazolones provide an interesting starting point for
structure based design of PCAF and GCN5 inhibitors. The isothiazo-
lone functionality is readily available via organic synthesis21 and
can easily be decorated with diverse substitutions to enhance
binding to the enzyme active site. The crystal structure and the cat-
alytic mechanism of GCN522 and PCAF23 provide inspiration for
inhibitor design.24,25 The crystal structure of the enzyme GCN5 in
complex with a bisubstrate inhibitor26 shows that the pyrophos-
phate and the pantothenic acid arm make extensive hydrogen
bonding interactions with the enzyme. The same is observed in
the crystal structure of the enzyme PCAF in complex with CoA
(PDB entry 1CM0).23 This suggests that hydrogen bonding interac-
tions are relevant for binding to the enzyme active site. Thus,
inhibitors with hydrogen bond donor/acceptors may show im-
proved binding to the enzyme active site.
In this study we investigated structure activity relationships for
inhibition of the enzyme PCAF by isothiazolones. The (5-
chloro)isothiazolone scaffold was used to target the active site
thiol and the substitution was explored to enhance binding by pro-
viding specific interactions with the enzyme. A series of 19 com-
pounds was synthesized and tested for inhibition of PCAF
activity. 5-Chloroisothiazolones showed the most potent inhibition
of PCAF. 5-Chloroisothiazolone 2e showed a slightly increased
Despite the potential therapeutic relevance of the GNAT family
HATs, very few small molecule inhibitors for GCN5 and PCAF have
been described so far. Lau et al. described bisubstrate inhibitors for
* Corresponding author. Tel.: +31 50 3638030; fax: +31 50 3637953.
E-mail address: f.j.dekker@rug.nl (F.J. Dekker).
0968-0896/$ - see front matter Ó 2008 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2008.12.008

F. J. Dekker et al. / Bioorg. Med. Chem. 17 (2009) 460–466
461
Table 1
potency compared to the other 5-chloroisothiazolones. Cell lines
A2780 and HEK 293 showed growth inhibition upon treatment
with micromolar concentrations N-aliphatic substituted 5-chloro-
isothiazolones, whereas the cell lines WiDr and HEP G2 were much
less affected.
Inhibition of the HAT PCAF and proliferation of cell lines by (5-chloro)isothiazolones
PCAF inhibition A2780 GI50 WiDr GI50 HEPG2 GI50 HEK 293
IC50
(lM)
(l
M)
(l
M)
(lM)
GI50 (lM)
2a/3a 3:1 3.0 0.6
2.0 0.2
>10
2.3 0.2
>10
3.3 0.3
3.0 0.4
5.2 0.1
7.0 0.8
3.0 0.1
>10
5.0 0.4
>10
>10
>10
7.5 0.3
>10
>10
5.6 0.5
>10
>10
>10
6.0 1.2
>10
6.4 0.2
>10
1.4 0.1
5.6 0.4
1.7 0.1
>10
2.8 0.3
1.5 0.6
0.7 0.1
2.9 0.8
>10
2b
2c
2d
2e
2f
2g
2h
2i
3.0 0.3
2.9 0.3
2.9 0.7
1.8 0.2
2.5 0.3
2.8 0.3
3.2 0.2
2.0 0.2
2.5 0.1
2.6 0.1
>10
2. Results and discussion
2.1. Chemistry
10
1
7.0 0.8
8.0 0.4
9.4 0.3
>10
>10
>10
A collection of N-functionalized isothiazolones was synthesized
using procedures shown in Scheme 1.21,27–29 Different amines were
reacted with 3,30-dithiodipropanoyl chloride 1 to give the dithiodi-
propionic amides in moderate to high yields. The dithiodipropionic
amides were treated with sulfuryl chloride (3 equiv) at 0 oC in
dichloromethane to give the 5-chloroisothiazolones 2 and isot-
hiazolones 3 in ratios between 3:1 and 2:1, which were readily sep-
arated using column chromatography. 5-Chloroisothiazolones were
obtained in yields between 50% and 70% and isothiazolones were ob-
tained in yields around 20% from the same reaction mixture. Surpris-
ingly, 2d yielded only 23% of the 5-chloroisothiazolone and the
corresponding isothiazolone could not be isolated from the same
reaction mixture. 5-Chloroisothiazolone 2e was oxidized by mCPBA
to yield 5-chloroisothiazolone-1-oxide 4e.28 Treatment of 5-chloro-
isothiazolone 2e with POCl3 and ammonia provided 5-chloro-
isothiazol-3-amine 5e.21 4-acylamino-isothiazolone 7e was
obtained using a modified procedure published by Nádel et al.29
2j
>10
3.0 0.4
>10
2k
3b
3c
3e
3f
3h
3j
4e
5e
7e
8.1 0.7
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
>10
4.2 0.6
5.6 0.2
>10
>10
>10
>10
5.8 0.6
>10
>10
8.6 1.0
>10
>10
>10
2.0
>10
>10
>10
4.7 0.4
10
0.3
4.9 0.9
Inhibition concentration 50% (IC50) determination n = 3, growth inhibition 50%
(GI50) determinations n = 8, standard deviations from the non-linear curve fitting
are reported.
direct reaction of the inhibitors with CoA-SH, but result from inhi-
bition of the enzyme. Compounds that showed more than 50%
inhibition at 10 lM (n = 3) were subjected to IC50 determination
Cbz protected L-Cystin 6 was coupled to an amine HCl-salt using
DCC, HOBt and triethylamine as a base followed by treatment with
sulfuryl chloride to yield the 4-carbamoyl-5-chloroisothiazolone 7e.
(n = 3). A representative example is shown in Figure 1A.
The compounds were designed to evaluate the PCAF inhibition
of 5-chloroisothiazolones and isothiazolones with different N-sub-
stitutions. The 5-chloroisothiazolone core with only an N-ethyl
substitution 2b shows a potent PCAF inhibition. The PCAF inhibi-
tion of 2b is equal to the commercially available preservative
2.2. Structure–activity relationships for PCAF inhibition
Structure–activity relationships for inhibition of the HAT PCAF
by a series of (5-chloro)isothiazolones were investigated in order
to explore the binding properties of these compounds (Table 1).
Binding studies were performed using a procedure published by
Trievel et al.22 The enzyme activity was measured by detection of
CoA-SH by the fluorescent dye 7-(diethylamino)-3-(40-maleimidyl-
phenyl)-4-methylcoumarin (CPM). The CoA-SH concentrations
measured with no inhibitor present were around 50 lM. Enzyme
inhibition was measured by determination of the residual enzyme
activity after 15 min incubation with the inhibitor. The inhibitor
TM
Kathon CG 2a/3a, which is a mixture of N-methyl-5-chloro-
isothiazolone and N-methylisothiazolone 3:1. An N-pentyl substi-
tution 2c did not improve binding compared to 2b. A series of N-
substitutions with a methyl ester at different numbers of carbon
atoms distance to the (5-chloro-)isothiazolone 2d–h was studied.
Compound 2e showed a slightly increased potency compared to
the other 5-chloroisothiazolones, which suggests that the N-sub-
stitution provides additional interaction with PCAF. Based on com-
pound 2e a molecular modeling was performed to suggest a
binding pose to aid future optimizations of the PCAF inhibition (
Fig. 2).
concentrations were maximal 10
lM, so inhibitory effects of more
than 20% at 10 M inhibitor concentration cannot be explained by
l
There is a pronounced difference in potency between N-ali-
phatic substituted 5-chloroisothiazolones 2 and isothiazolones 3.
5-Chloroisothiazolones 2b–i,k inhibit PCAF with IC50 values
around 2–3
than 50% inhibition at 10
l
M, whereas isothiazolones 3b,c,e,f,h, showed less
M. The difference in potency for the
l
N-aromatic substituted 5-chloroisothiazolone 2j and isothiazolone
3j is much less pronounced. The same in observed in the data pro-
vided by Stimson et al.20 in which the difference in PCAF inhibitory
potency between N-aromatic 5-chloroisothiazolones and iso-
thiazolones is a factor 2 or less.
Compounds with modifications directly on the isothiazolone
scaffold were synthesized and the consequences for PCAF inhibi-
tion were studied. 5-Chloroisothiazolone-1-oxide 4e inhibited the
PCAF activity at micromolar concentrations. The 5-chloro-
isothiazol-3-amine 5e did not inhibit PCAF. Substitution of the 5-
chloroisothiazolone scaffold in the 4-position with an amino acyl
substituent 7e reduced the PCAF inhibition compared to com-
pound 2e. Nevertheless, compound 7e provides an interesting
starting point for further optimization of the PCAF inhibition by
variation of the 4-amino acyl substituent.
Scheme 1. Synthesis of a focused compound collection. Reagents and conditions:
(a) R-NH2, Et3N, CH2Cl2, (b) SO2Cl2, CH2Cl2, (c) mCPBA, CH2Cl2, (d) POCl3, (e) NH3,
CH3CN, (f) DCC, HOBt, Et3N, R-NHþClꢀ, CH2Cl2.
3

462
F. J. Dekker et al. / Bioorg. Med. Chem. 17 (2009) 460–466
Figure 1. Panel A shows the concentration dependence for inhibition of the PCAF enzyme activity by 5-chloroisothiazolone 2e (n = 3, standard deviation shown) and panel B
shows the concentration dependence for growth inhibition of the cell line A2780 by 2e (n = 8, standard deviation shown).
used for docking studies.23 The molecular modeling study sug-
gested a binding pose in which the methyl ester is hydrogen
bonded to a pocket in which the pyrophosphate is bound in the
PCAF crystal structure 1CMO (Fig. 2).23 The pyrophosphate binding
pocket is formed by the backbone of amides of Glutamine 581, Va-
line 582, Lysine 583, Glycine 584 and Tyrosine 585. The carbonyl
from the 5-chloroisothiazolone is hydrogen bonded to Glutamine
581 and Valine 576. Docking of 5-chloroisothiazolone-1-oxide 4e
provided a similar binding pose as observed for 2e. Docking of 7e
provided also a binding pose comparable to 2e. These results sug-
gests that the PCAF inhibitory potency of 2e, 4e and 7e might be
improved by variations in the substitution in the 4-position of
the isothiazolone core. In conclusion, compounds 2e, 4e and 7e
provide starting points to design inhibitors with an improved PCAF
inhibitory potency.
2.4. Growth inhibition of cell lines
Inhibition of cancer cell lines was studied in order to explore the
relevance of (5-chloro)isothiazolones as research tools to study the
biological mechanisms underlying cell proliferation.31 The growth
inhibition of the human cancer cell lines A2780 (ovarian), WiDr
(colon) and HEP G2 (liver) and the human embryonic cell line
293 (kidney) was studied. Growth inhibition was determined using
Figure 2. Binding pose for 2e in the PCAF active site (PDB entry 1CM0) that was
proposed by a molecular modeling study. Cys 574 is the active site cysteine.
Hydrogen bonds are formed to valine 576 and Glutamine 581 and to the
pyrophosphate binding pocket formed by the backbone amides of Glutamine 581,
Valine 582, Lysine 583, Glycine 584 and Tyrosine 585.
Reports on the reactivity of N-methylisothiazolones and N-
methyl-5-chloroisothiazolones indicate a high reactivity of 5-chlo-
roisothiazolones. 5-chloroisothiazolones are prone to nucleophilic
substitution in the 5-position and reductive cleavage of the N–S
bond.30 In contrast, for isothiazolones only reductive cleavage of
the N–S bond by sulfides is observed and no nucleophilic addition
in the 5-position.30 This study supports the idea that the potent
inhibition of 5-chloroisothiazolones is due to their reactivity. Inter-
estingly, isothiazolone 3j with a N-aromatic substitution shows an
a crystal violet assay. The compounds were screened at 10 lM
inhibitor concentration for growth inhibition (n = 8) and com-
pounds with more than 50% growth inhibition were subjected to
growth inhibition 50% determination (GI50) (n = 8) (Table 1). A rep-
resentative example is shown in Figure 1B. The N-aliphatic substi-
tuted 5-chloroisothiazolones are the most potent compounds for
growth inhibition of cell lines A2780 and HEK 293. The growth of
cell lines WiDr and HEP G2 were not inhibited or required higher
concentrations for inhibition compared to cell lines A2780 and
HEK 293, which might be due to differences in the growth rate
or differences in cellular biochemistry. Western blots on histone
acetylation in cell lines that are treated with optimized inhibitors
are required to proof a connection between the PCAF HAT inhibi-
tion and the cellular activity.
IC50 of 4.2 lM for PCAF inhibition. This indicates that N-aromatic
isothiazolones are either more prone to nucleophilic addition in
the 5-position of the isothiazolone or that they are more prone
to reductive cleavage of the N–S bond, compared to N-aliphatic
substituted isothiazolones.
It is remarkable that compounds 2j and 3j do not inhibit the
growth of the four cell lines that were studied. This indicates that
N-aromatic isothiazolones like 2j are less potent inhibitors of cell
proliferation. Compound 5e with a 5-chloroisothiazol-3-amine
scaffold showed no inhibitory effect on PCAF but shows a potent
inhibition of WiDr, HEP G2 and HEK 293 cell lines. This is the first
2.3. Molecular modeling
Modeling studies were performed to propose a binding pose for
inhibitor 2e to aid future optimization of the inhibitors. The crystal
structure of PCAF complexed with CoA (PDB entry code 1CM0) was

F. J. Dekker et al. / Bioorg. Med. Chem. 17 (2009) 460–466
463
time that a biological effect is described for a compound with an 5-
chloroisothiazol-3-amine scaffold.
the solution, and the mixture was stirred at 0 °C for 2 h. Subse-
quently, the mixture was washed with water (2 times 200 mL)
and brine (1 time 200 mL), dried with Na2SO4 and filtered. The
solution was concentrated under reduced pressure and purified
using column chromatography.
TM
The commercially available preservative Kathon CG 2a/3a
showed a potent PCAF inhibition and a potent growth inhibition
of the four cell lines studied (Table 1). This is remarkable because
the mixture has been used as a preservative in rinse-off cosmetics
such as shampoos and conditioners for more than 25 years.
4.2.1. 5-Chloro-2-ethylisothiazol-3(2H)-one (2b)
TM
Kathon CG is claimed to be safe and effective, however skin sensi-
The product was obtained using procedure 1 starting from 10b.
Purification was performed using column chromatography with
hexane/EtOAc 3:1 (v/v) as eluent. Yield 46%. Brown gum.
Rf = 0.44 (CH2Cl2/MeOH 9:1). 1H NMR (200 MHz, CDCl3): d 1.32
(t, J = 7.2 Hz, 3H), 3.81 (q, J = 7.2 Hz, 2H), 6.36 (s, 1H). 13C NMR
(50 MHz, CDCl3): d 15.0, 39.4, 114.8, 146.3, 167.0. HPLC: purity
>99%, retention time 6.6 min (column C8 mobile phase H2O/
CH3CN/TFA 60:40:1). HRMS (m/z) 163.9933 [M+H]+, calcd
163.9937 C5H7ClNOS.
tization have been reported.30,32 The growth inhibition of cell lines
TM
shows that Kathon CG 2a/3a is cell-permeable and acts on intra-
cellular targets. The inhibition of PCAF indicates that this preserva-
tive might modify the epigentics regulation of gene transcription.
TM
Taking these data together we conclude that Kathon CG should
be applied with care.
3. Conclusion
4.2.2. 2-Ethylisothiazol-3(2H)-one (3b)
In conclusion, N-aliphatic substituted 5-chloroisothiazolones
After elution of 2b product 3b was eluted from the column
using EtOAc eluent. Yield 10%. Brown gum. Rf = 0.38 (CH2Cl2/MeOH
9:1). 1H NMR (200 MHz, CDCl3) d 1.60 (m, 3H), 3.88 (m, 2H), 6.36
(m, 1H) 8.20 (m, 1H). 13C NMR (50 MHz, CDCl3) d 15.3, 39.8,
114.9, 140.4, 168.9. GC–MS purity >99%, retention time 2.69 min,
(EI) [M+] m/z 129. HRMS (m/z) 130.0323 [M+H]+, calcd 130.03266
C5H8NOS.
inhibit the enzyme PCAF with an IC50 around 2–3
N-aliphatic substituted isothiazolones show less than 50% inhibi-
tion at 10 M. The difference in PCAF inhibitory potency between
lM, whereas
l
N-aromatic substituted 5-chloroisothiazolones and isothiazolones
is much less pronounced. N-Aromatic and N-aliphatic substituted
5-chloroisothiazolones showed a comparable potency for PCAF
inhibition. N-Aliphatic substituted 5-chloroisothiazolones inhib-
ited the growth of the cell lines A2780 and HEK 293 in the low
micromolar range, whereas growth inhibition of WiDr and HEP
G2 required higher concentrations or was not observed. N-Aro-
matic substituted 5-choroisothiazolones, N-aromatic and N-ali-
phatic substituted isothiazolones showed less than 50% inhibition
4.2.3. 5-Chloro-2-pentylisothiazol-3(2H)-one (2c)
The product was obtained using procedure 1 starting from 10c.
Purification was performed using column chromatography with
hexane/EtOAc 8:1 (v/v) as eluent. An extra column chromatogra-
phy step was required to obtain the pure product. Yield 55%. Or-
ange oil. Rf = 0.53 (CH2Cl2/MeOH 94:6). 1H NMR (200 MHz,
CDCl3) d 0.88 (t, J = 6.8 Hz, 3H), 1.24-1.38 (m, 4H), 1.59-1.73 (m,
2H), 3.71 (t, J = 7.3 Hz, 2H), 6.25 (s, 1H). 13C NMR (50 MHz, CDCl3)
d 14.1, 22.4, 28.8, 29.6, 44.0, 115.0, 145.8, 167.1. GC-MS RT 5.2 min,
purity >99%, MS(EI) (m/z) 205, calcd 205. HRMS (m/z) 206.0402
[M+H]+, calcd 206.04064 C8H13ClNOS.
of the cell proliferation at 10 lM. Finally, it has been shown that
TM
the preservative Kathon CG inhibits the HAT PCAF and inhibits
the cell growth of A2780, WiDr, HEP G2 and HEK 293 cell lines.
4. Experimental
4.1. Organic synthesis
4.2.4. 2-pentylisothiazol-3(2H)-one (3c)
General: Chemicals were obtained from commercial sources
(Sigma–Aldrich, Acros Organics) and used without previous purifi-
cation, except dichloromethane that was distilled over CaH2 before
use. Thin-layer chromatography (TLC) was performed on alumi-
num sheets of Silica Gel 60 F254. Spots were visualized under
ultraviolet light, with I2 vapour, KMnO4 solution or ninhydrin solu-
tion. Column chromatography was performed with MP Ecochrom
Silica Gel 32–63, 60 Å. 1H and 13C NMR spectra were recorded on
a Varian Gemini-200 (50.32 MHz). Chemical shift values are re-
ported as part per million (d) relative to residual solvent peaks
(CHCl3, 1H = 7.26, 13C d = 77.16 or CD3OD, 1H = 3.31, 13C
d = 49.00). The coupling constants (J) are reported in Hertz (Hz).
13C spectra were recorded using the attached proton test (APT)
pulse sequence. Electrospray ionization mass spectra (ESI-MS)
were recorded on a Applied Biosystems/SCIEX API3000-triple
quadrupole mass spectrometer. High-resolution mass spectra
(HR-MS) were recorded using a flow injection method on a
LTQ-Orbitrap XL mass spectrometer (Thermo Electron, Bremen, Ger-
many) with a resolution of 60,000 at m/z 400. Protonated testoster-
one (lock mass m/z = 289.2162) was used for internal recalibration
in real time. Melting points were determined on an Electrothermal
digital melting point apparatus and are uncorrected.
After elution of 2c product 3c was eluted from the column using
hexane/EtOAc 1:1 (v/v) as eluent. An extra column chromatogra-
phy step was required to obtain the pure product. Yield 25%. Brown
gum. Rf = 0.29 (CH2Cl2/MeOH 94:6). 1H NMR (200 MHz, CDCl3) d
0.89 (t, J = 6.5 Hz, 3H), 1.24-1.37 (m, 4H), 1.51-1.79 (m, 2H), 3.79
(t, J = 7.3 Hz, 2H), 6.32 (d, J = 6.2 Hz, 1H), 8.1 (d, J = 6.2 Hz, 1H). 13C
NMR (50 MHz, CDCl3) d 14.2, 22.6, 28.8, 29.7, 44.5, 114.7, 139.7,
169.1. GC-MS RT 4.8 min, purity >95%, MS(EI) (m/z) 171, calcd
171. HRMS (m/z) 172.0792 [M+H]+, calcd 172.0796 C8H14NOS.
4.2.5. Methyl 2-(5-chloro-3-oxoisothiazol-2(3H)-yl)ethanoate
(2d)
The product was obtained using procedure 1 starting from the
10d. Purification was performed using column chromatography
with hexane/EtOAc 3:2 (v/v) as eluent. Yield 23%. Dark brown
gum. Rf = 0.76 (CH2Cl2/MeOH 9:1). 1H NMR (200 MHz, CDCl3) d
3.78 (s, 3H), 4.48 (s, 2H), 6.32 (s, 1H). 13C NMR (50 MHz, CDCl3) d
44.1, 53.0, 112.9, 148.2, 167.3, 168.0. GC–MS rt 4.88 min, MS (EI)
gave anomalous results. MS (ESI) (m/z) 208.0 [M+H]+, calcd
208.0. HRMS (m/z) 207.9831 [M+H]+, calcd 207.9835 C6H7ClNO3S.
4.2.6. Methyl 3-(5-chloro-3-oxoisothiazol-2(3H)-yl)propanoate
(2e)
4.2. General procedure 1
The product was obtained using procedure 4 starting from 10e.
Purification was performed using column chromatography with
hexane/EtOAc 1:2 (v/v) as eluent. Yield 49%. Orange oil. Rf = 0.58
(CH2Cl2/MeOH 10:1). 1H NMR (200 MHz, CDCl3) d 2.67 (t, J = 6.2
The dithiobispropanamide (8 mmol) was dissolved in dry
CH2Cl2 (50 mL) at 0 °C. SO2Cl2 (24 mmol) was added dropwise to

464
F. J. Dekker et al. / Bioorg. Med. Chem. 17 (2009) 460–466
Hz, 2H), 3.66 (s, 3H), 3.95 (t, J = 6.2 Hz, 2H), 6.19 (s, 1H). 13C NMR
(50 MHz, CDCl3) d 33.9, 39.8, 52.3, 114.3, 147.0, 167.1, 171.8. GC–
MS rt 5.97 min, purity >99%, MS(EI) (m/z) 221 [M], calcd 221.
HRMS (m/z) 221.9987 [M+H]+, calcd 221.9992 C7H9ClNO3S, (m/z)
243.9805 [M+Na]+, calcd 243.9811 C7H8ClNNaO3S. HPLC purity
>99%%, rt 5.0 min, (column C8 mobile phase H20/CH3CN/TFA
60:40:1).
hexane/EtOAc 3:1 (v/v) as eluent. Yield 61%. Yellow oil. Rf = 0.56
(EtOAc/hexane 6:1). 1H NMR (200 MHz, CDCl3) d 1.30–1.42 (m,
2H), 1.56–1.71 (m, 4H), 2.29 (t, J = 7.2 Hz, 2H), 3.63 (s, 3H), 3.71
(t, J = 7.2 Hz, 2H), 6.24 (s, 1H). 13C NMR (50 MHz, CDCl3) d 24.4,
26.0, 29.4, 33.8, 43.5, 51.6, 114.8, 145.7, 166.9, 173.9. GC-MS RT
8.36 min, purity >99%, MS (EI) (m/z) 263, calcd 263. HRMS (m/z)
264.0456 [M+H]+, calcd 264.04612 C10H15ClNO3S.
4.2.7. Methyl 3-(3-oxoisothiazol-2(3H)-yl)propanoate (3e)
After elution of 2e product 3e was eluted from the column using
hexane/EtOAc 1:20 (v/v) as eluent. Yield 23%. Brown oil. Rf = 0.49
(CH2Cl2/MeOH 10:1). 1H NMR (200 MHz, CDCl3) d 2.76 (t, J = 6.5
Hz, 2H), 3.70 (s, 3H), 4.09 (t, J = 6.5 Hz, 2H), 6.31 (d, J = 6.4 Hz,
1H), 8.10 (d, J = 6.2 Hz, 1H). 13C NMR (50 MHz, CDCl3) d 33.9,
40.2, 52.2, 114.2, 140.4, 169.1, 171.6. GC–MS rt 6.36 min, purity
>99%, MS (EI) (m/z) 187 [M], calcd 187. HRMS (m/z) 188.0378
[M+H]+, calcd 188.0381 C7H9ClNO3S, (m/z) 210.01961 [M+Na]+,
calcd 210.0201 C7H9NNaO3S.
4.2.13. Methyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate
(3h)
After elution of 2h product 3h was eluted from the column
using hexane/EtOAc 1:5 (v/v) as eluent. Yield 25%. Yellow oil.
Rf = 0.30 (EtOAc/hexane 6:1). 1H NMR (200 MHz, CDCl3) d 1.30–
1.74 (m, 6H), 2.29 (t, J = 7.2 Hz, 2H), 3.63 (s, 3H), 3.76 (t, J = 7.2
Hz, 2H), 6.23 (d, J = 6.5 Hz, 1H), 8.04 (d, J = 6.5 Hz, 1H). 13C NMR
(50 MHz, CDCl3) d 24.5, 26.1, 29.6, 33.9, 43.8, 51.7, 114.8, 139.0,
169.0, 174.0. GC–MS rt 7.98 min, purity >99%, MS (EI) (m/z) 229,
calcd 229. HRMS (m/z) 230.0846 [M+H]+, calcd 230.08509
C10H16NO3S.
4.2.8. Methyl 4-(5-chloro-3-oxoisothiazol-2(3H)-yl)butanoate
(2f)
4.2.14. Ethyl 3-(5-chloro-3-oxoisothiazol-2(3H)-yl)propanoate
(2i)
The product was obtained using procedure 1 starting from 10f.
Purification was performed using column chromatography with
hexane/EtOAc 1:1 (v/v) as eluent. Yield 67%. Brown solid.
Mp = 47.9 °C. Rf = 0.83 (CH2Cl2/MeOH 9:1). 1H NMR (200 MHz,
CDCl3) d 1.92–2.06 (m, 2H), 2.38 (t, J = 7.2 Hz, 2H), 3.66 (s, 3H),
3.78 (t, J = 7.1 Hz, 2H), 6.19 (s, 1H). 13C NMR (50 MHz, CDCl3): d
24.9, 30.7, 42.9, 51.9, 114.8, 146.1, 167.1, 173.0. GC–MS rt
6.69 min, purity >99%, MS(EI) (m/z) 235 [M]+, calcd 235. HRMS
(m/z) 236.0143 [M+H]+, calcd 236.0148 C8H11ClNO3S, (m/z)
257.9962 [M+Na]+, calcd 257.9968 C8H10ClNNaO3S.
The product was obtained using procedure 1 starting from 10i.
Purification was performed using column chromatography with
hexane/EtOAc 2:1 (v/v) as eluent. Yield 51%. Brown solid.
Mp = 73.2 °C. Rf = 0.67 (CH2Cl2/MeOH 9:1). 1H NMR (200 MHz,
CDCl3) d 1.26 (t, J = 7.2 Hz, 3H), 2.70 (t, J = 6.2 Hz, 2H), 4.00 (t,
J = 6.2 Hz, 2H), 4.16 (q, J = 7.1, 7.3 Hz, 2H), 6.24 (s, 1H). 13C NMR
(50 MHz, CDCl3) d 14.4, 34.2, 39.9, 61.4, 114.3, 147.1, 167.2,
171.4. GC–MS rt 6.43 min, purity >99%, MS (EI) (m/z) 235, calcd
235. HRMS (m/z) 236.0143 [M+H]+, calcd 236.0148 C8H11ClNO3S.
4.2.9. Methyl 4-(3-oxoisothiazol-2(3H)-yl)butanoate (3f)
After elution of 2f product 3f was eluted from the column using
hexane/EtOAc 1:7 (v/v) as eluent. Yield 9%. Brown oil. Rf = 0.78
(CH2Cl2/MeOH 9:1). 1H NMR (200 MHz, CDCl3) d 2.02 (t, J = 7.1
Hz, 2H), 2.37 (t, J = 7.2 Hz, 2H), 3.66 (s, 3H), 3.87 (t, J = 7.1 Hz,
1H), 6.28 (d, J = 6.2 Hz, 1H), 8.1 (d, J = 6.2 Hz, 1H). 13C NMR (50
MHz, CDCl3) d 25.1, 30.8, 43.1, 51.9, 114.8, 139.2, 173.2, 180.3.
GC–MS rt time 6.36 min, purity >99%, MS (EI) (m/z) 201 [M]+, calcd
201. HRMS (m/z) 202.0534 [M+H]+, calcd 202.0538 C8H12NO3S, (m/
z) 224.0352 [M+Na]+, calcd 224.03573 C8H11NNaO3S.
4.2.15. 5-Chloro-2-(3-chloro-4-fluorophenyl)isothiazol-3(2H)-
one (2j)
The product was obtained using procedure 1 starting from 10j.
Purification was performed using column chromatography with
ethyl acetate/hexanes 1:10 (v/v) as eluent. Yield 62%. White solid,
Mp = 116.9 °C; Rf = 0.66 (ethyl acetate/cyclohexane 1:1). 1H NMR
(200 MHz, CDCl3) d 6.36 (s, 1H), 7.10–7.21 (m, 1H), 7.43–7.36 (m,
1H), 7.63 (dd, J = 2.6, 6.5 Hz, 1H). 13C NMR (50 MHz, CDCl3) d
114.8, 117.2, 117.6, 124.9, 125.0, 127.6, 146.5, 154.8, 159.8,
165.4. GC–MS purity >99%, retention time 7.8 min, MS (EI) (m/z)
263, calcd 263. HRMS (m/z) 263.9448 [M+H]+, calcd 263.9453
C9H5Cl2FNOS.
4.2.10. Methyl 4-(5-chloro-3-oxoisothiazol-2(3H)-
yl)pentanoate (2g)
The product was obtained using procedure 1 starting from 10g.
Purification was performed using column chromatography with
hexane/EtOAc 3:1 (v/v) as eluent. Yield 62%. Brown oil. Rf = 0.52
(CH2Cl2/MeOH 94:6). 1H NMR (200 MHz, CDCl3) d 1.60–1.90 (m,
4H), 2.35 (t, J = 7.2 Hz, 2H), 3.65 (s, 3H), 3.75 (t, J = 7.1 Hz, 2H),
6.32 (s, 1H). 13C NMR (50 MHz, CDCl3) d 21.9, 29.2, 33.5, 43.7,
51.9, 114.9, 146.4, 167.3, 173.6. GC–MS rt 7.58 min, purity >99%,
MS (EI) (m/z) 249, calcd 249. HRMS (m/z) 250.0300 [M+H]+, calcd
250.0305 C9H13ClNO3S, (m/z) 272.0119 [M+Na]+, calcd 272.0124
C9H12ClNNaO3S.
4.2.16. 2-(3-Chloro-4-fluorophenyl)isothiazol-3(2H)-one (3j)
After elution of 2j product 3j was eluted from the column using
ethyl acetate/hexanes 1:2 as eluent. The product was obtained
using. Yield 23%. Orange oil, Rf = 0.18 (ethyl acetate/cyclohexane
1:1). 1H NMR (200 MHz, CDCl3) d 6.33 (d, J = 6.3 Hz, 1H), 7.10–
7.21 (m, 1H), 7.47–7.41 (m, 1H), 7.68 (dd, J = 2.6/6.5 Hz, 1H), 8.19
(d, J = 6.3 Hz, 1H). 13C NMR (50 MHz, CDCl3) d 114.8, 117.0,
117.5, 125.0, 127.4, 140.1, 154.7, 159.7, 167.7. GC–MS purity
>95%, retention time 7.7 min, MS (EI) (m/z) 229, calcd 229. HRMS
(m/z) 229.9838 [M+H]+, calcd 229.98427 C9H6ClFNOS.
4.2.11. Methyl 4-(3-oxoisothiazol-2(3H)-yl)pentanoate
4.2.17. Benzyl [2-(5-chloro-3-oxoisothiazol-2(3H)-
After elution of 2g the product was eluted from the column using
hexane/EtOAc 1:8 (v/v) as eluent. Yield 12%. Brown oil. Rf = 0.39
(CH2Cl2/MeOH 94:6). The compound was not pure enough for bio-
chemical characterization and was thus excluded from the study.
yl)ethyl]carbamate (2k)
The product was obtained using procedure 1 starting from 10k.
Purification was performed using column chromatography with
ethyl acetate/hexanes 3:1 (v/v) as eluent. Yield 29%. Yellow gum,
Rf = 0.35 (ethyl acetate/hexane 3:1), 1H NMR (200 MHz, CDCl3) d
3.45–3.47 (m, 2H), 3.86 (t, J = 6 Hz, 2H), 5.01 (s, 2H), 6.25 (s, 1H),
7.35 (m, 5H). 13C NMR (50 MHz, CDCl3) d 41.1, 43.5, 66.9, 114.3,
128.1, 128.2, 128.6, 136.4, 152.4, 156.9, 160.8. HPLC purity
>94%%, RT 12.3 min, (column C8 mobile phase H20/CH3CN/TFA
4.2.12. Methyl 6-(5-chloro-3-oxoisothiazol-2(3H)-yl)hexanoate
(2h)
The product was obtained using procedure 1 starting from 10h.
Purification was performed using column chromatography with

F. J. Dekker et al. / Bioorg. Med. Chem. 17 (2009) 460–466
465
60/40/1). HRMS (m/z) 313.0414 [M+H]+, calcd 313.0408
C13H14ClN2O3S. (m/z) 335.0233 [M+Na]+, calcd 335.0228
C13H13ClN2NaO3S.
prepared for docking using the default settings in MVD.33 The 5-
chloroisothiazolones, isothiazolones and 5-chloro-isothiazolones-
1-oxide were docked with a distance constraint of 6 Å between
the Cys 574 thiol and the carbon atom 5 in the isothiazolone scaf-
fold. Docking solutions were calculated within a sphere of 15 Å
from the Cys 574 thiol. Docking solutions were selected based on
4.2.18. Methyl 3-(5-chloro-1-oxido-3-oxoisothiazol-2(3H)-
yl)propanoate (4e)
33
Compound 2e (1 mmol) was dissolved in CH2Cl2 (15 mL) and m-
chloroperbenzoic acid was added in several portions. The mixture
was stirred at room temperature for 16 h. Several crystals Na2-
S2O3ꢁ5H2O and H2O (5 mL) were added and the mixture was stirred
for 10 min. A saturated solution of NaHCO3 (50 mL) was added, this
mixture was stirred for 5 min and the phases were separated. The
organic layer was washed with saturated NaHCO3 (50 mL), brine
(50 mL) and dried with Na2SO4 and the solvent was evaporated un-
der reduced pressure. The residue was purified by column chroma-
tography with ethyl acetate/hexanes 1:2 as eluent. Yield 50%,
yellow oil, Rf = 0.72 (ethylacetate/hexane 10:1). 1H NMR (200
MHz, CD3OD) d 2.70–2.77 (m, 2H), 3.68 (s, 3H), 3.99 (t, J = 6.8 Hz,
2H), 6.98 (s, 1H). 13C NMR (50 MHz, CDCL3) d 34.5, 38.3, 52.4,
125.4, 158.6, 166.5, 173.7. GC–MS rt 6.1 min, purity >99%, MS
(ESI) (m/z) 206, calcd 206 (MꢀOMe). HRMS (m/z) 237.9936
[M+H]+, calcd 237.9941 C7H9ClNO4S.
the MOLDOCKSCORE and the docking solutions were evaluated man-
ually and the amino acid side chain in a radius of 10 Å from the li-
gand were energy minimized, followed by energy minimization of
the ligand.
4.4. Enzyme inhibition studies
A fluorescent histone acetyl transferase assay described by Trie-
vel et al.22 was used for enzyme inhibition studies. The human re-
combinant histone acetyl transferases PCAF (p300/CREB-binding
protein Associated Factor) was purchased from Biomol Interna-
tional. The histone H3 peptide (Ac-QTARKSTGGKAPRKQLATK-
NH2) was purchased from Pepscan (Lelystad, NL) (purity >98%,
m/z 1034.9 [M+2H]2+ calcd 1035.2).The other chemicals were pur-
chased from Sigma–Aldrich. Detail on the enzyme inhibition stud-
ies can be found in the Supplementary data.
4.2.19. Methyl 3-[(5-chloroisothiazol-3-yl)amino]propanoate
(5e)
4.5. Cell growth inhibition
Compound 2e (0.74 mmol) was dissolved in POCl3 (1.5 mL) and
stirred overnight at room temperature. The next morning an or-
ange-brown precipitate was observed. Isopropylether (20 mL)
was added and the precipitate intensified. The suspension was cen-
trifuged (4000 rpm, 10 min), the supernatant was discarded and
the residue washed with isopropylether. Subsequently, the residue
was suspended in CH3CN (10 mL), cooled to 0 oC and NH3 gas was
bubbled through the solution for 30 min. The red-white suspension
turned into a brown solution with a white precipitate. The suspen-
sion was filtered and the filtrate was concentrated under reduced
pressure and purified using column chromatography ethyl ace-
tate/hexanes 1:6 as eluent. Yield 44% Yellow solid, Mp = 73.2 °C,
Rf = 0.33 (ethylacetate/hexane 3:1), 1H NMR (200 MHz, CDCl3) d
2.64 (t, J = 6.2 Hz, 2H), 3.61 (t, J = 5.9 Hz, 2H), 3.68 (s, 3H), 6.34
(s, 1H). 13C NMR (50 MHz, CDCl3) d 33.9, 38.5, 51.9, 112.3, 151.8
163.6, 173.2. GC–MS rt 5.9 min, purity >99%, MS (EI) (m/z) 220,
calcd 220. HRMS (m/z) 221.0146 [M+H]+, calcd 221.0152
C7H10ClN2O2S.
4.5.1. Cell culture
The growth inhibition of the human cancer cell lines A2780
(ovarian), WiDr (colon) and HEP G2 (liver) and the human embry-
onic cell line 293 (kidney) was studied. The cells were maintained
in Dulbecco’s modified Eagles Medium with 10% heat-inactivated
fetal calf serum, 50 IU/mL penicillin and 50 mg/mL streptomycin
at 37 °C in a humidified 5% CO2 incubator. All cell culture reagents
were purchased from invitrogen.
4.5.2. Cell proliferation
Cell proliferation was measured by a Crystal Violet assay. For
this assay, cells were seeded at 5000 cells per well into 96-well
plates, grown for 24 h, and treated for an additional 48 h with
the different inhibitors. After this, the medium was aspirated and
the cells were fixed with 50
for 30 min. Subsequently, the cells were washed with water and
the staining was solubilized by addition of 100 L 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 concen-
trations were tested in 8-fold on one plate and the GI50 values of
most potent inhibitors were measured again on a new plate.
lL 1% crystal violet in 70% ethanol
l
4.2.20. Methyl 3-[4-{[(benzyloxy)carbonyl]amino}-5-chloro-3-
oxoisothiazol-2(3H)-yl]propanoate (7e)
The product was obtained using procedure 1 starting from 9.
Purification was performed using column chromatography with
ethyl acetate/hexanes 1:1 (v/v) as eluent. Yield 70%. Off-white
gum. Rf = 0.38 (ethyl acetate/cyclohexane 2:1). 1H NMR (200
MHz, CDCl3) d 2.67 (m, 2H), 3.70 (s, 3H), 3.96 (m, 2H), 5.15 (s,
2H), 7.34 (m, 5H). 13C NMR (50 MHz, CDCl3) d 33.6, 40.5, 52.2,
67.9, 119.1, 128.3, 128.4, 128.6, 135.7 153.2, 163.0 171.6. HPLC
purity >99%. HRMS (m/z) 371.0463 [M+H]+, calcd 371.0469
C15H16ClN2O5S.
Acknowledgment
We gratefully acknowledge the University of Groningen and the
European Union (FP7-PEOPLE-2007-2-2-ERG_DEKKER40) for
financial support.
Supplementary data
4.3. Modeling
The synthesis of the precursors of the final products and their
characterizations can be found in the supporting information num-
bered 8b–f, 9, 10b–k. Details on the enzyme inhibition studies can
also be found. Supplementary data associated with this article can
be found, in the online version, at doi:10.1016/j.bmc.2008.12.008.
Molecular dockings were performed using the program Molegro
Virtural Docker 2007 (MVD) from Molegro ApS, Aarhus, Denmark.
The crystal structure from PCAF (PDB entry 1CM0) chain B,23 was
prepared for dockings using the default settings in MVD. The water
molecules were removed before docking. The ligands were geom-
etry optimized using HYPERCHEM 7.5 Professional (Hypercube, Inc)
using molecular mechanics with the Amber force field and Polar-
Ribiere (conjugate gradient) algorithm. The optimized ligands were
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