Synthesis and biological testing of novel pyridoisothiazolones as histone acetyltransferase inhibitors

Article abstract of DOI:10.1016/j.bmc.2011.01.063

We present a combination of database screening, synthesis and in vitro testing to identify novel histone acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors. Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for new anticancer agents directed against HATs as new targets have been identified.

Full text of DOI:10.1016/j.bmc.2011.01.063

Bioorganic & Medicinal Chemistry 19 (2011) 3678–3689
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological testing of novel pyridoisothiazolones as histone
acetyltransferase inhibitors
Silviya D. Furdas a, Suhaib Shekfeh b, Elisabeth-Maria Bissinger a, Julia M. Wagner a, Sonja Schlimme b,
Vassil Valkov a, Michael Hendzel c, Manfred Jung a, Wolfgang Sippl b,
⇑
a Institute of Pharmaceutical Sciences, Albert-Ludwigs-University of Freiburg, Germany
b Department of Pharmaceutical Chemistry, Martin-Luther-Universität Halle-Wittenberg, Wolfgang-Langenbeckstr. 4, 06120 Halle(Saale), Germany
c Department of Oncology, Cross Cancer Institute, University of Alberta, Edmonton, Alberta, Canada
a r t i c l e i n f o
a b s t r a c t
Article history:
We present a combination of database screening, synthesis and in vitro testing to identify novel histone
acetyltransferase (HAT) inhibitors. The National Cancer Institute compound collection (NCI) and several
commercial databases were filtered by similarity-based virtual screening to find new HAT inhibitors.
Employing the recombinant HAT p300/CBP-associated factor (PCAF) and two different histone substrates
for screening, pyridoisothiazolones were identified as inhibitors of human PCAF. Due to the limited
solubility of the initial hits, we synthesized and tested them on PCAF. The compounds inhibit the
proliferation of cancer cells. In summary, valuable chemical tools and potential lead candidates for
new anticancer agents directed against HATs as new targets have been identified.
Received 14 October 2010
Revised 27 January 2011
Accepted 27 January 2011
Available online 2 February 2011
Keywords:
Histone acetyltransferase
PCAF
Database screening
Epigenetics
Ó 2011 Elsevier Ltd. All rights reserved.
Pyridoisothiazolones
1. Introduction
They possess various degrees of selectivity and cell permeability.
Peptide-CoA conjugates (Lys-CoA and H3-CoA-20)5 as well as a
Acetylation and deacetylation of histones have emerged as key
mechanisms regulating transcriptional activity. Histone acetyl-
transferases (HATs) catalyze the transfer of acetyl groups to lysine
residues in histones, which results in a more open conformation of
nucleosomes and increased accessibility of regulatory proteins to
DNA.1 The acetylation status of several non-histone proteins
including p53, ataxia-telangiectasia mutant (ATM), heat shock pro-
few natural small molecules such as anacardic acid6 and ana-
logues,7 garcinol,8 and curcumin9 and the
c-butyrolactone MB-3
were described as potent p300 or PCAF inhibitors.10 Moreover,
the selective inhibition of p300 HAT by semi-synthetic derivatives
of garcinol (i.e., LTK-14)11 was described. Recently, Aherne and
co-workers used high throughput screening to identify isothiazo-
lone derivatives as irreversible PCAF inhibitors.16 The compounds
were shown to decrease the cellular acetylation level in a time-
and concentration dependent manner and they inhibited the
growth of a panel of human tumor cell lines. It was also shown
that the preservative agent Kathon CG, which also contains an
isothiazolone structure, inhibits HAT and cancer cell growth.12
In order to identify new inhibitors of HATs, we applied a stra-
tegy that combined computational screening methods with a robust
biochemical assay that has been successfully applied to several tar-
gets recently.13–15 Virtual database filtering was used to conduct
virtual screening with the National Cancer Institute (NCI) com-
pound collection and several commercial compound libraries. As
search query we used the isothiazolone and isothiazolidinone ring
system which was derived from recently identified isothiazolone
HAT inhibitors. Subsequently, the initial hits were docked into
the active site of the HAT PCAF. Thus, 21 compounds were identi-
fied as inhibitor candidates and these were then tested in a PCAF
in vitro assay. Due to the limited solubility of some of the identified
hits and to obtain further structure-activity relationships, a series
tein 90, and
a-tubulin, is intimately related to their functions.
Reversible acetylation of
a-tubulin marks stabilized microtubule
structures and may contribute to regulating microtubule dynam-
ics.2 Acetylation of p53 tumor suppressor protein by two HAT
subtypes (p300/cAMP-responsive element binding protein (CBP)
and p300/CBP-associated factor (PCAF)) was linked to its trans-
activation potential and ability to regulate cell cycle arrest and
apoptosis.3 Furthermore, several HATs were found to be genetically
altered in a variety of cancer types.4 In view of the increasing
evidence that associates HAT function with cancer formation and
progression, these enzymes are appealing as drug targets for the
development of small-molecule inhibitors.
Whereas an increasing number of HDAC inhibitors is available
nowadays, only a few HAT inhibitors have been described so far.
⇑
Corresponding author. Tel.: +49 345 5525040; fax: +49 345 5527355.
E-mail address: wolfgang.sippl@pharmazie.uni-halle.de (W. Sippl).
0968-0896/$ - see front matter Ó 2011 Elsevier Ltd. All rights reserved.
doi:10.1016/j.bmc.2011.01.063

S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
3679
Table 1
of further analogs were synthesized and biologically tested. We
show for the first time that pyridoisothiazolones inhibit PCAF in
the submicromolar range and are promising anticancer lead struc-
tures with good cellular activity.
Chemical structures of pyridoisothiazolones from the NCI compound repository and
synthesized compounds 4a-g and 8a-h according to the scaffold in Figure 1
Compound
n
R1
R2
R3
NSC 694614
NSC 694615
NSC 694616
NSC 694617
NSC 694618
NSC 694619
NSC 694620
NSC 694621
NSC 694622
NSC 694623
NSC 698599
NSC 698600
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
0
1
1
1
1
1
1
1
1
CH3
H
NO2
H
NO2
CH3
NO2
H
NO2
H
CH3
CH3
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
Cl
H
F
H
OCH3
H
H
Cl
H
F
H
OCH3
H
Br
Br
Br
NO2
NO2
OC6H5 (OPhe)
OC6H5 (OPhe)
OCH3
OCH3
C4H9
NO2
OCH3
C4H9
H
Cl
Cl
F
F
CH3
H
H
Cl
Cl
F
F
CH3
H
CF3
2. Results and discussion
We chose to computationally screen the National Cancer
Institute (NCI)’s 3D database since it contains structurally diverse
synthetic compounds collected from many laboratories around
the world as well as natural products. The recently identified iso-
thiazolone inhibitor V,16 which acts as an irreversible inhibitor by
covalently binding to Cys574 at the PCAF active site, was used to
derive a search query for the in silico screening. The crystal struc-
ture of PCAF complexed with the cofactor Acetyl-CoA (pdb code
1CMO) was used for docking studies. We identified 51 compounds
with an isothiazolone or isothiazolidinone substructure that were
subsequently docked into the PCAF substrate binding site to test
whether they are able to bind at the catalytic site. 32 compounds
were identified that contain a reactive S–N bond which was located
in close proximity to the active site Cys574 in the model (S–S
distance below 4.5 Å). Among these, 15 compounds (Fig. 1 and
Table 1) could be obtained from the NCI. Using the same search
query five other pyridoisothiazolones were identified and
purchased from commercial suppliers (Ambinter, ChemDiverse,
Enamine, see Fig. 2).
NSC 700864
C6H5 (Phe)
4a
4b
4c
4d
4e
4f
4g
8a
8b
8c
8d
8e
8f
H
H
H
H
H
H
H
H
H
H
H
H
H
H
H
8g
8h
The 21 selected compounds were tested for in vitro inhibition of
histone acetyltransferase activity using recombinant human PCAF.
Biotinylated oligopeptide sequences from both histones H31–21 and
H42–24 were used as the substrates. The conversion was detected
with a primary antibody against acetylated histone H3 and H4
and quantification was achieved by using a secondary Europium
labelled antibody with a final measurement of time resolved fluo-
rescence. This assay had been used before for the determination of
cellular hypoacetylation caused by isothiazolones.16 We had previ-
ously used the same approach for the determination of histone
methyltransferase activity in vitro and in cancer cells17,18 and also
use it here for the characterization of in vitro HAT inhibition. The
published isothiazolone V16 was synthesized and used as reference
inhibitor.
The most active pyridoisothiazolones showed PCAF inhibition
in the submicromolar range. In most cases a higher potency was
observed on the H4 substrate. Thus, the binding of the histone sub-
strate seems to influence the structure or reactivity (or both) of the
acetyltransferase with regard to inhibitor responsiveness.
The amino-substituted isothiazolone NSC 145097 (Fig. 1, Table
1) as well as the isothiazolone-thione NSC 279225 (Fig. 1, Table 1)
did not show significant HAT inhibition. Isothiazolones substituted
with an aminocarbonylmethyl linker (A4033-0171899, K783-
6791, and K783-6798 (Fig. 2)) showed only moderate inhibition
on PCAF inhibition. The highest potency was observed for the
pyridoisothiazolones NSC 694614 and NSC 694622 (Fig. 1, Table 1).
Generally, the nitro substituted compounds from the NCI reposi-
tory suffered from severe solubility problems and this made deter-
mination of the cellular activity impossible. In some cases,
compounds precipitated in the cell culture medium and no growth
inhibition could be observed. For that reason and in order to obtain
further structure–activity relationships a series of analogs was
synthesized on the basis of the derived initial hits.
N-Substituted pyridoisothiazolones were synthesized by two
various routes. Starting out from 2-mercaptonicotinic acid, N-
phenyl and N-benzyl derivatives (Fig. 1, Table 1) were prepared in
order to determine structure–activity relationships. After protec-
tion of the reactive thiol group of the starting material, amide forma-
tion with aniline derivatives and following oxidative cyclisation
provided N-aryl substituted derivatives (Scheme 1, compounds
4a–g).19 N-Benzyl-derivatives (compounds 8a–h) were obtained
by nucleophilic substitution using the parent pyridoisothiazolone
structure (compound 7, Scheme 2) and benzyl bromides.20–22
For the most potent compound from the class of N-benzyl
pyridoisothiazolones (8c), a two-step sulphur oxidation was carried
out in order to investigate the influence of the oxidation state on
the inhibitory activity. Treatment with KHSO5 leads to the racemic
sulphoxide 9 and the subsequent phase-transfer oxidation with
hypochloride provides the sulphone 10 (Scheme 3).23 For the oxi-
dized analogues the reduced inhibitory properties in vitro against
PCAF/H31–21 (Table 2) can be correlated with the increasing sul-
phur oxidation state. With each oxidation step a huge reduction
of the IC50 value was observed. Mono- and dioxidized pyrido-
isothiazolone derivatives are probably much less reactive. The
electron withdrawal effect of the oxygen on the sulphur hampers
the S–N bond cleavage.24 Due to the sterically more demanding
sulphoxide and sulphone group it cannot be excluded that the
low activity is resulting from unfavourable steric effects.25
Molecular modelling studies were carried out to analyze the
binding mode of the identified pyridoisothiazolones. The PCAF
crystal structure complexed with the cosubstrate CoA was used
to dock the inhibitors into the substrate and cosubstrate binding
pocket. The docking studies (e.g., for compound 4d, Fig. 3) showed
a preferred binding mode in which the isothiazolone S–N bond is
located nearby the active site Cys574. A T-shaped aromatic pi–pi
stacking interaction is observed between the pyridine ring of the
pyridoisothiazolones and Tyr616 (distance pyridine-N/aromatic
plane 3.8 Å). The binding of the compounds is further stabilized
O
R2
R1
N
CH2
R3
n
S
N
Figure 1. Pyridoisothiazolones scaffold for compounds from the NCI compound
repository and synthesized compounds 4a-g and 8a-h.

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S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
NO2
O
O
O
O
S
Cl
H
O
CH3
CH2
N
N
O
O
N
S
N
S
S
O
H3C
V
NSC 145097
NSC 279225
O
O
O
N
N
CH3
N
S
H
N
N
S
S
F
F
S
O
N
S
A4033-0171899
T0504-9123
A3737-0158368
CH3
O
O
CH3
O
N
N
S
N
H
H
N
CH3
S
N
N
H3C
O2N
S
N
H3C
O
O
K783-6791
Ambinter 455691
K783-6798
F
Cl
Figure 2. Chemical structures of further isothiazolones.
O
R2
COOH
COOH
S
i)
N
R3
N
SH
N
1
S
N
4a-g
R2
v)
ii) or
iii)
vi)
H2N
R3
R2
R2
R3
R3
O
O
S
N
H
iv)
N
H
N
S
N
3
2a-g
O
Scheme 1. Synthesis routes for compounds with N-phenylpyridoisothiazolones scaffold (compounds 4a–g). Reagents and conditions: (i) BnCl, KOH, isopropyl alcohol, reflux,
30 min; (ii) BOP–Cl, DIPEA, CH2Cl2, rt, overnight; (iii) IBCF, Et3N, CH2Cl2, rt, overnight; (iv) m-CPBA, CH2Cl2, 0 °C, 15 min rt, overnight; (v) TCAA, CH2Cl2, 0–25 °C, 4 h; (vi)
SO2Cl2, Cl(CH2)2Cl, reflux, 45 min; (rt: room temperature).
by van der Waals interactions with aliphatic and aromatic residues
of the pocket (Leu526, Met 529, Ala613 and Phe617). The observed
binding mode of the potent inhibitors NSC 694614-NSC 694623
(Fig. 1, Table 1) as well as the novel compounds 4a–g (Fig. 1, Table
1) is very similar and no correlation between the docking scores
(Goldscore, Chemscore) and the PCAF inhibition data could be de-
rived. Introducing a methylene group between the aromatic substi-
tuent and the isothiazole core (e.g., compound 8h) results in
increasing the distance Cys574/S–N bond and is therefore
unfavourable.
To show that the pyridoisothiazolones bind irreversibly to PCAF,
potentially through a reactive S–N bond, we analyzed the role of
thiol interactions in the inhibitory activity of the compounds
toward PCAF. We determined the effect of a thiol reagent on the
PCAF inhibition by NSC 694622 (Fig. 1, Table 1). Without DTT, an
tack of a nucleophile (e.g., cysteine-SH) at the sulfur atom of the
heterocycle. This proceeds with concomitant ring-opening. The
authors showed for example that the presence of a 5-Cl substituent
at the isothiazolone ring which is increasing the reactivity of the
S–N bond by the higher electronegativity is also increasing the
HAT activity. Therefore, we calculated the electronic properties of
the tested isothiazolones on the basis of semi-empirical AM1 cal-
culations (AM1 bcc1 partial charge on the S and N atom of the iso-
thiazolone, HOMO and LUMO energies) and tried to correlate them
with the observed PCAF inhibition data. No significant correlation
between any of the calculated descriptors and the HAT in vitro
inhibition was observed indicating that the reactivity of the S–N
bond alone is not able to explain the biological activity of the
tested compounds. Additionally, the appropriate interaction of
the isothiazolone system with Cys574 might be sterically
influenced by unfavourable substituents (e.g., NSC 700864).
The new pyridoisothiazolones 4d and 8h, as well as NSC 694616
(Fig. 1, Table 1) were then tested for their growth inhibitory
properties using SK-N-SH neuroblastoma and MCF7 breast cancer
cells (Table 3). The more soluble fluorophenyl isothiazolone
IC50 value of 3.42 0.35 lM was determined. In the presence of
1 mmol/L DTT, which alone had no significant effect on enzyme
activity, HAT-inhibitory activity was significantly reduced (Fig. 4).
It was reported by Gorsuch et al.26 that the observed in vitro
HAT inhibitory results for isothiazolones are consistent with an at-

S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
3681
O
4. Experimental section
O
O
OH
v)
NH2
ii) iii) iv)
4.1. Computational methods
NH
N
SH
S
N
6
SH
N
7
All calculations were performed on a Pentium IV 2.2 GHz based
Linux cluster (20 CPUs). The GOLD software (Cambridge Crystallo-
graphic Data Centre) was used for docking, whereas the calculation
of all molecular descriptors and the analysis of the docking results
were carried out in MOE2008.1028 (Chemical Computing Group).
Br
i)
R2
R3
vi)
O
N
OH
N
4.2. Ligand docking
S
S
N
N
5
R2
8a-h
The crystal structure of human PCAF in complex with Acetyl-
CoA (pdb code 1CM0, chain B) resolved at 2.30 Å was taken from
the Protein Data Bank. The cofactor and the water molecules were
removed, hydrogen atoms were added and the protein was mini-
mized using the AMBER force field29 and the conjugate gradient
minimization (MOE 2008.10) until the gradient of 0.1 kcal/mol
was reached. Docking of the ligands was carried out using the
GOLD 4.0 program with default settings. A sphere of 14 Å around
the S-atom of Cys574 was defined for ligand docking. Details about
the GOLD program can be found elsewhere.30 To test the applicability
of the docking tool, a control docking was carried out with the
cofactor acetyl-CoA. Using GoldScore as scoring function an RMSD
value of 1.45 was derived for the top-ranked conformation of Acet-
yl-CoA (data not shown). For all compounds under study the Gold-
Score as well as the ChemScore was calculated and analyzed.
R3
Scheme 2. Synthesis routes for compounds with N-benzylpyridoisothiazolones
scaffold (compounds 8a–h). Reagents and conditions: (i) DPPA, Et3N, pyridine, rt,
1 h; (ii) SOCl2, BOP–Cl, toluene, reflux, 3 h; (iii) NH4OH, NH4Cl, rt, 18 h; (iv) NaBH4,
rt, 1 h; (v) conc. H2SO4, reflux, 3 h; (vi) DIPEA, methanol, rt, overnight; (rt: room
temperature).
showed good inhibition especially on the neuroblastoma cell line.
Surprisingly, the cellular activity of the benzyl congener was higher,
particularly on the SK-N-SH cells, and it was also higher than its own
in vitro inhibition. This indicates the involvement of additional
mechanisms which indeed have been described for isothiazolones
in the literature, for example, inhibition of farnesyltransferases.26
For compound 4d, clear time dependence was shown for the
antiproliferative action (Fig. 5).
4.3. Database screening
The 260,000 3D structures of the National Cancer Institute (NCI)
database, generated with the program CORINA, were obtained
from the NCI homepage (http://dtp.nci.nih.gov/docs/3d_database/
dis3d.html) and were imported as Mol2 files into the MOE program.
The 3D structures of the Ambinter, ChemDiverse, Chemical Block
and Enamine were retrieved from the ZINC database (http://
zinc.docking.org). Using an isothiazolone/isothiazolidinone ring
as search query we identified 51 compounds from the NCI database
that were subsequently docked into the PCAF protein structure
using the GOLD program as described above. 32 molecules were suc-
cessfully docked into the CoA binding pocket showing a distance
<5 Å between the S–N bond and the thiol group of Cys574 (distance
S to S). From the 32 compounds selected, 15 could be obtained
from the NCI. By using the same isothiazolone search query six fur-
ther compounds were identified and purchased from Ambinter,
ChemDiverse, Chemical Block, and Enamine.
3. Conclusions
Isothiazolones had been presented as new lead structures for
HAT inhibition in the literature but suffer from general high reac-
tivity. We hoped that compounds with decreased reactivity, for
example, obtained by annellation, would still retain HAT inhibition
but might be more selective towards that target and could be
useful for cellular and eventual further preclinical evaluation as
potential anticancer agents. A covalent attachment to thiol groups
does not necessarily rule out clinical application as can be shown,
for example, for the antiulcer drug omeprazole. Indeed, pyrido-
thiazolones could be identified via virtual screening and in vitro
enzyme testing as new PCAF inhibitors. Initial chemical variations
of the lead structures led to improved cellular activity as compared
to the hits from the in-silico screening. During the course of this
work, the group of Aherne just recently published similar
compounds and presented them as lead structures with increased
potency27 but no cellular activity was disclosed. We applied a
combination of virtual and biological screening approach to potent
PCAF inhibitors and show that these pyridoisothiazolones show
promising anticancer activity in cellular models. Other mecha-
nisms of action cannot be ruled out at the moment but these initial
results merit further structure–activity relationships in this class of
compounds.
4.4. Calculation of electronic properties
Geometrey optimization and calculation of partial charges
was carried out at the semi-empirical level using the AM1
paramterization (Dewar et al., 1985). ESP partial charges were
calculated on the basis of the AM1 optimized geometries.
Energies for the highest occupiet molecular orbital (HOMO) and
the lowest unoccupied molecular orbital (LUMO) were calculated.
O
O
O
i)
ii)
N
N
N
S
N
S
S
*
N
N
O
O
Cl
Cl
Cl
O
10
8c
9
Cl
Cl
Cl
Scheme 3. Synthesis routes for sulphur oxidation of compound 8c. Reagents and conditions: (i) KHSO5 (oxoneÒ), methanol, rt, 1 h; (ii) NaClO, TBAB, ethyl acetate, rt, 1 h;
(rt: room temperature). ⁄Racemic.

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S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
Table 2
100%
Inhibition of PCAF histone acetyltransferase activity for histone substrates H3 (amino
acid residues 1–21) and H4 (amino acid residues 2–24): IC50 value [ M] standard
error [ M] or enzyme inhibition [%] at the specified concentration
l
80%
60%
40%
20%
0%
l
Compound
PCAF, H3 (1–21)
PCAF, H4 (2–24)
NSC 694614
NSC 694615
NSC 694616
NSC 694617
NSC 694618
NSC 694619
2.99 0.27
4.43 0.20
4.91 0.39
4.10 0.60
11.8 1.83
12.9 0.95
n.t.
n.t.
0.86 0.08
2.99 0.32
15.8 5.76
n.t.
NSC 694620
48% @ 10
lM
n.t.
H
T1
T2
C
NSC 694621
NSC 694622
NSC 694623
NSC 698599
NSC 698600
NSC 700864
4a
4b
4c
4d
4e
4f
4g
5
8a
8b
8c
8d
8e
8f
8g
8h
9
10
5.71 0.30
3.42 0.35
15.9 2.20
15.3 6.21
6.51 0.49
n.t.
1.83 0.47
n.t.
2.72 0.60
n.t.
n.t.
88% @ 50
87% @ 50
91% @ 50
0.72 0.05
n.t.
75% @ 50
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
25.5 2.85
n.t.
n.t.
n.t.
20% @ 200
n.t.
51.3 8.83
n.t.
n.t.
n.t.
-20%
Figure 4. Reversibility of HAT inhibition by thiols (compound NCS 694622). DTT
Influence on HAT inhibition by pyridoisothiazolones; Column H: 10
(0.01 mg/ml), NSC 694622 (5), 100 AcCoA; Column T1: 10
(0.01 mg/ml), 5
100 M AcCoA; Column T2: 10
added after preincubation step, 100
l
ll
l
PCAF
PCAF
5
l
l
M
l
M
49% @ 25
l
M
M NSC 694622 (5), 50 mM DTT added at preincubation step,
l PCAF (0.01 mg/ml), NSC 694622 (5), 50 mM DTT
M AcCoA; Column C: 10 l PCAF (0.01 mg/ml),
7.85 0.46
3.53 0.07
4.57 0.23
1.64 0.11
5.90 0.85
4.63 0.24
5.03 0.14
l
l
l
M
M
M
l
l
l
l
50 mM DTT, 100 lM AcCoA.
l
M
Table 3
17% @ 50
l
M
Antiproliferative effects in cancer cells (GI50 value [lM] standard error [lM] or
growth inhibition [%] at the specified concentration; SK-N-SH: human neuroblastoma
cell line, HCT116: colorectal carcinoma cell line, MCF-7: breast adenocarcinoma cell
line)
22.96 2.06
17.5 1.27
6.94 0.57
86.3 3.40
101 4.92
27.9 4.50
22.2 1.97
130 8.49
Compound
SK-N-SH
HCT116
n.i. @ 25
MCF-7
NSC 694614
NSC 694615
NSC 694616
NSC 694617
NSC 694619
NSC 694620
NSC 694621
NSC 694623
21.3 4.17
15.2 2.53
53.5 2.23
15% @ 50
32% @ 25
l
M
n.t.
n.t.
67% @ 25
lM
n.t.
n.t.
44.25 3.55
n.t.
11% @ 50
6% @ 50
lM
l
l
l
M
M
M
l
M
29% @ 25
<10% @ 25
lM
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
n.t.
V
4.80 0.20
48% @ 300
n.i. @ 25 lM
7.66 1.29
n.i. @ 25
l
M
NSC 145097
NSC 279225
lM
lM
19.2 1.22
8.93 0.20
12.2 0.20
55% @ 25
11.2 1.88
16.1 1.88
29% @ 25
21% @ 25
40% @ 25
33% @ 25
lM
l
M
M
M
A 455691 (Ambinter)
A3737-0158368 (Chemical Block)
A4033-0171899 (Chemical Block)
T0504-9123 (Enamine)
K783-6791 (ChemDiverse)
K783-6798 (ChemDiverse)
NSC 698600
NSC 700864
l
l
3.69 0.17
l
M
21% @ 50
46% @ 50
48% @ 50
38% @ 50
l
M
4a
4b
4c
4d
4f
4g
n.i. @ 25
23% @ 25
56% @ 20
28% @ 25
20% @ 25
24% @ 25
l
M
l
l
l
M
M
M
l
l
l
l
l
M
M
M
M
M
n.t.
n.t.
37% @ 20
l
M
n.t.
10.3 1.24
6.20 0.07
6.99 1.09
37.2 0.32
n.t.
n.t.: not tested; n.i.: no enzyme inhibition (<5%) at the specified assay
concentration.
n.t.
8a
8b
8c
8f
8g
8h
V
18% @ 10
20% @ 10
9.53 0.57
31% @ 10
26% @ 10
0.48 0.02
23.2 1.52
35% @ 100
l
M
100% @ 25
l
M
M
n.t.
n.t.
n.t.
n.t.
n.t.
30.2 1.79
n.t.
n.t.
n.t.
lM
97% @ 25
15.8 1.67
79% @ 25
l
lM
l
M
M
M
lM
42% @ 25
38% @ 25
n.t.
l
l
A3737-0158368
A4033-0171899
K783-6791
K783-6798
l
M
M
49% @ 100
l
M
n.i. @ 40
l
n.i. @ 40
l
M
25.7 5.52
22.1 0.71
22.6 1.59
35.8 1.48
n.t.
n.t.
n.t.: not tested; n.i.: no growth inhibition (<5%) at the specified assay concentration.
4.5. Enzyme preparation (PCAF)
The plasmid construct pQE80_P/CAF1.16 (5.2 kb, PCAF residues
493–658 (a gift of Professor John M. Denu, University of Wisconsin,
Madison) was transformed for propagation and maintenance in
competent Escherichia coli (TG1). The recombinant HAT domain
of PCAF (residues 157–657) was overexpressed as N-terminal
His-tagged fusion protein in ampicillin-sensitive Escherichia coli
(BL21 D3) and 1 L LB medium. After IPTG induction at 18 °C over-
night,31–33 bacterial cells were centrifuged for 15 min (8000 rcf,
4 °C) and the supernatant was removed. The cell pellet was resus-
pended in 40 mL of lysis buffer pH 8.0 (1 mg/mL lysozyme, 0.1 mM
phenylmethanesulfonyl fluoride, 1 mM b-mercaptoethanol,
300 mM NaCl, 50 mM Tris). After 30 min incubation and following
Figure 3. GOLD docking solution for pyridoisothiazolone 4d (colored magenta) at
the PCAF binding site. The sulphur of the isothiazolone inhibitor is in close
proximity to the active site Cys574 thus enabling the formation of a covalent bond
between the enzyme and the isothiazolone.

S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
3683
performed for 10 min at 30 °C. Controls are treated in the same
manner, using HAT-buffer instead of the inhibitor solution. Then,
each mixture is transferred to the plate. To start the enzymatic
reaction, 10
added to each well (final assay concentration 100
CoA). The amount of the turnover is detected by a primary rabbit
IgG antibody against the modification. 100 L/well of anti-acetyl-
l
L/well of a 400
lM acetyl-CoA solution in water is
lM acetyl-
l
histone H3 (Millipore, #06–599) or anti-acetyl-lysine (Abcam,
#ab21623) respectively is added in appropriate dilutions in
Tris-buffer pH 7.5 (0.1% (v/v) Tween 20, 0.5% (w/v) BSA (protease-
free), 150 mM NaCl, 20 mM Tris base, 80 mM Tris–HCl). In the
next step incubation with 5 pmol/well of a N1-Eu-labelled anti-
rabbit secondary antibody (Perkin-Elmer, #AD0105), diluted in
the same Tris-buffer as the first antibody, is followed. The euro-
pium label is cleaved off by adding of 100 lL/well of enhancement
solution (Perkin-Elmer, #1244–105). After incubation for 10 min at
room temperature, time-resolved fluorescence measurement at
kex/kem: 340/615 nm (BMG Polarstar) is performed for the quantifi-
cation. Readout data were plotted as relative fluorescence units
against logarithm of compound concentration using GraphPad
Prism 4.00 Software in order to obtain IC50 values.
Figure 5. Time dependence of growth inhibition (compound 4d). Growth inhibition
of SK-N-SH neuroblastoma cells caused by different concentrations of compound 4d
was time-dependent up to 72 h of incubation time (dotted bars—4d 25
bars—4d 10 M, striped bars—4d 5 M).
lM, grey
l
l
4.7. Cell lines
sonification (5-fold for 15 s, 60% output), the cell debris were
removed by centrifugation (9000 rcf, 4 °C) in order to obtain clear
supernatant. Protein purification was performed using Ni-NTA
Superflow resin (Qiagen) according to the supplier’s instructions.
Prewashing of the beads with lysis buffer pH 8.0 (300 mM NaCl,
50 mM Tris) was repeated twice and subsequently the collected
supernatant was placed on the resin. After washing with 40 mL
of wash buffer pH 8.0 (1 mM b-mercaptoethanol, 10 mM imidaz-
ole, 300 mM NaCl, 50 mM Tris), the enzyme elution was performed
according to a replacement from the resin with elution buffer pH
8.0 (1 mM b-mercaptoethanol, 500 mM imidazole, 300 mM NaCl,
50 mM Tris). Sephadex™ G-25 PD-10 Desalting column (Amershem
Biosciences) was used according to the technical instructions for
buffer exchange against storage buffer pH 6.0 (1 mg/mL BSA, 10%
(v/v) glycerol, 20 mM trisodium citrate, 250 mM NaCl). In order
to verify the purity and the presence of a 6xHis-tagged protein,
SDS–PAGE and immunodetection with HRP mouse anti-6xHis anti-
body (BD Pharmingen, #552565), respectively, were performed
under standard conditions. Protein quantification (12.6 mg enzyme
from 1 L LB medium) was determined using PierceÒ BCA Protein
Assay Kit (Pierce Biotechnology). Samples with high specific his-
tone acetyltransferase activity were stored in aliquots at ꢀ80 °C
and used for the enzyme inhibition assay described below.
SK-N-SH neuroblastoma cells were grown in DMEM low glucose
(PAA) supplemented with 10% fetal bovine serum (PAA), 2 mM
L-glutamine (Gibco Invitrogen) and 1% penicillin-streptomycin
(Gibco Invitrogen). HCT116 colorectal cancer cells were grown in
DMEM high glucose (PAA) supplemented with 10% fetal bovine
serum (PAA), 2 mM L-glutamine (PAA), 1% non essential amino
acids (PAA) and 1% penicillin-streptomycin (PAA), MCF-7 human
breast adenocarcinoma cells were grown in RPMI-1640 (PAA)
supplemented with 10% fetal bovine serum (PAA), 2 mM
L-glutamine (Gibco Invitrogen) and 1% penicillin-streptomycin
(Gibco Invitrogen).
4.8. Cell proliferation assay
Cells were seeded in 96-well tissue culture plates at a density of
5000 per well for SK-N-SH and MCF-7 and 4000 per well for
HCT116. After 24 h of incubation, diluted compounds or DMSO
vehicle were added to each well to a total volume of 100 lL; three
replicates per concentration were used. Growth inhibition was
determined using the CellTiter 96Ò AQueous Non-Radioactive Cell
Proliferation Assay according to the manufacturer’s instructions.
Data was plotted as absorbance unit against logarithm of com-
pound concentration using GraphPad Prism 4.02. 50% Growth inhi-
bition (GI50) was determined as compound concentration required
to reduce the number of metabolic active cells by 50% compared to
DMSO control.
4.6. In vitro time-resolved fluorescence immunosorbent
acetyltransferase assay for PCAF
The heterogeneous assay is performed in streptavidin-coated
96-well plates (Nunc). After each incubation step (1 h at 30 °C),
six washing steps as well as a prewash step are necessary to re-
move the non bound fraction using wash buffer pH 7.5 (0.1% (v/
v) Tween 20, 150 mM NaCl, 20 mM Tris base, 80 mM Tris–HCl)
4.9. Inhibitors
Standard chemicals were purchased from Acros, Sigma Aldrich,
or Fluka. Solvents were dried before use according to standard pro-
cedures. Merck silica gel 60 was used for flash chromatography
with the given eluents. Melting points were obtained after mea-
surement with SMP2 (Stuart Scientific) and are uncorrected.
NMR spectra were recorded on an Avance DRX 400 MHz spectro-
meter (Bruker) or an Unity 300 MHz spectrometer (Varian) respec-
tively. Chemical shifts are reported in parts per million (d)
relative to residual solvent peaks for 1H and 13C NMR spectra
((CD3)2SO: 1H d = 2.50, 13C d = 39.92; CD3OD: 1H d = 3.34, 13C
d = 40.01; CDCl3: 1H d = 7.29, 13C d = 76.98) or in negative parts
per million (d) relative to CFCl3 for 19F NMR spectra respectively.
The coupling constants (J) are reported in Hertz. EI- and CI-mass
with 300 lL/step in an overflow mode (Tecan Columbus Plate
washer). In the first incubation step, 20 pmol/well biotinylated his-
tone peptide residues 1–21 of human histone H3 or residues 2–24
of human histone H4, respectively (Millipore, #12–403 respec-
tively #12–372), diluted in the same buffer as mentioned above
is bound to the wells. In the second step, the bound substrate is
turned over in an enzymatic reaction. For this, preincubation of
10
v) BSA (protease-free), 0.8% (v/v) Triton X-100, 100 mM HEPES),
of inhibitor DMSO-solution 18 of the HAT-buffer is
lL/well enzyme PCAF, diluted in HAT-buffer pH 7.5 (0.1% (w/
2
l
L
lL

3684
S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
spectra were measured with a TSQ700 mass spectrometer (Ther-
moelectron), ESI- and PCI-mass spectra were recorded with a
LCQ-Advantage mass spectrometer. Microanalysis were performed
with Vario-EL (Elementaranalysensysteme). HPLC data for purity
verification were obtained with Alliance Equipment (Waters,
2695 Separations Module, 2487 Dual k Absorbance Detector).
Quantification was performed by determination of the peak area
using the Millenium Software (Waters). HPLC analysis was per-
formed under following conditions: LiChrospherÒ 60 RP—select B,
and recrystallized from ethanol to give white crystals of 1 (2-(ben-
zylthio)nicotinic acid) (yield: 26.7 mmol, 81%). Mp 194 °C; 1H NMR
(400 MHz, (CD3)2SO): d = 13.26 (bs 1H, COOH), 8.66 (dd, 3J = 4.7 Hz,
4J = 1.7 Hz, 1Hpy, 6-H), 8.22 (dd, 3J = 7.8 Hz, 4J = 1.7 Hz, 1Hpy, 4-H),
7.42–7.40 (m, 2Hbn
,
20/60-H), 7.32–7.23 (m, 4Hbn/py
,
30/40/50/5-H), 4.37 (s, 2H, CH2); 13C NMR (100 MHz, (CD3)2SO):
d = 166.76 (COOH), 161.05 (Cq, 2-Cpy), 152.35 (CHar, 6-Cpy),
139.47 (CHar, 4-Cpy), 138.38 (Cq, 10-Cbn), 129.62 (2 ꢁ CHar, Cbn),
128.74 (2 ꢁ CHar, Cbn), 127.28 (CHar, 40-Cbn), 123.58 (Cq, 3-Cpy),
119.51 (CHar, 5-Cpy), 34.22 (CH2); MS (CI NH3 direct mode): m/z
(%) 241.6 [M+H]+ (100).
5
l
m, 60Å, 250 ꢁ 4 mm (Merck); gradient mode elution (CH3CN/
H2O:0.05% TFA); flow rate: 1 mL/min; detection mode: UV absorp-
tion: k = 254 nm). The samples for the HPLC analysis were solved in
suitable solvent (acetonitrile or methanol, concentration: 1 mg/
mL).
4.12.2. 2-(Benzylthio)-N-phenylnicotinamide (2a)
Starting out from 1 (2-(benzylthio)nicotinic acid) (10 mmol,
2.5 g) and aniline (11 mmol, 1.0 g, 1.0 mL) according to synthetic
procedure 1, 2a (2-(benzylthio)-N-phenylnicotinamide) was ob-
tained as white crystals (yield: 4.9 mmol, 49%). Mp 171 °C; 1H
NMR (400 MHz, (CD3)2SO): d = 10.39 (s, 1H, NH), 8.61 (dd,
3J = 4.8 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 7.92 (dd, 3J = 7.6 Hz, 4J = 1.7 Hz,
4.10. Synthetic procedure 1: amide preparation
Compound 1 (2-(benzylthio)nicotinic acid) (10 mmol, 2.5 g)
was suspended in 50 mL of absolute dichloromethane and an equi-
molar amount of N,N-diisopropylethylamine (10 mmol, 1.3 g,
1.7 ml) was added. After stirring at room temperature for 5 min
bis(2-oxo-3-oxazolidinyl)phosphonic chloride (10 mmol, 2.6 g)
was added and the batch was stirred for further 5 min. Subse-
quently appropriate substituted aniline derivative (11 mmol) and
N,N-diisopropylethylamine (20 mmol, 2.6 g, 3.3 ml) were added.
The mixture was stirred at room temperature overnight. After sol-
vent removal in vacuum, the residue was solved in ethyl acetate
and washed consecutive three times with 2 M hydrochloric acid,
water, 5% (w/v) sodium bicarbonate solution, water and brine.
The organic phase was dried over sodium sulphate and evaporated
to dryness. The crude product was recrystallized from ethyl acetate
with petroleum ether (80–110 °C).
3
1Hpy, 4-H), 7.69 (d, J = 8.2, 2Har, 200/600-H), 7.41–7.37 (m, 2Hbn, 20/
60-H), 7.36–7.32 (m, 2Har, 300/500-H), 7.31–7.26 (m, 3Hbn/py, 30/50/5-
H), 7.24–7.20 (m, 1Har, 400-H), 7.13–7.09 (m, 1Hbn, 40-H), 4.43 (s,
2H, CH2); 13C NMR (100 MHz, (CD3)2SO): d = 165.19 (C@O),
157.04 (Cq, 2-Cpy), 150.54 (CHar, 6-Cpy), 139.25 (Cq, 10-Cbn),
138.51 (Cq, 100-C), 136.12 (CHar, 4-Cpy), 130.63 (Cq, 3-Cpy), 129.48
(2 ꢁ CHar, 300/500-C), 129.16 (2 ꢁ CHar, Cbn), 128.74 (2 ꢁ CHar, Cbn),
127.32 (CHar, 40-Cbn), 124.33 (CHar, 400-C), 120.19 (2 ꢁ CHar, 200/600-
C), 119.64 (CHar, 5-Cpy), 33.84 (CH2); MS (CI NH3 direct mode):
m/z (%) 321.0 [M+H]+ (100).
4.12.3. 2-(Benzylthio)-N-(4-chlorophenyl)nicotinamide (2b)
Starting out from 1 (2-(benzylthio)nicotinic acid) (10 mmol,
2.5 g) and 4-chloroaniline (11 mmol, 1.4 g) according to synthetic
procedure 1, 2b (2-(benzylthio)-N-(4-chlorophenyl)nicotinamide)
was obtained as white crystals (yield: 5.4 mmol, 54%). Mp
155 °C; 1H NMR (400 MHz, (CD3)2SO): d = 10.60 (s, 1H, NH), 8.62
4.11. Synthetic procedure 2: phenylpyridoisothiazolones (4a–f)
Appropriate substituted amide (3 mmol) was dissolved in
30 mL dichloroethane and heated to 70 °C. Sulfuryl chloride
(11.9 mmol) was added dropwise as 1 M solution in dichloroethane.
After stirring for 45 min at 70 °C, the preparation was cooled to
room temperature and washed then with 25 mL saturated solution
of sodium bicarbonate. The organic layer was separated and the
solvent was evaporated in vacuum. The products were purified
using silica gel column chromatography with the reported eluents.
(dd, 3J = 4.8 Hz, 4J = 1.7 Hz, 1Hpy
,
6-H), 7.95 (dd, 3J = 7.9 Hz,
3
4J = 1.7 Hz, 1Hpy, 4-H), 7.72 (d, J = 8.9 Hz, 2Har, 200/600-H), 7.42–
7.39 (m, 4Har/bn, 20/60/300/500-H), 7.31–7.27 (m, 3Hbn/py, 30/50/5-H),
7.24–7.22 (m, 1Hbn, 40-H), 4.42 (s, 2H, CH2); 13C NMR (100 MHz,
(CD3)2SO): d = 165.27 (C@O), 157.11 (Cq, 2-Cpy), 150.70 (CHar, 6-
C
py), 138.46 (Cq, 10-Cbn), 138.19 (Cq, 100-C), 136.20 (CHar, 4-Cpy),
130.31 (Cq, 3-Cpy), 129.48 (2 ꢁ CHar, 300/500-C), 129.10 (2 ꢁ CHar,
C
bn), 128.74 (2 ꢁ CHar, Cbn), 127.94 (Cq, 400-C), 127.34 (CHar, 40-
4.12. Synthetic procedure 3: benzylpyridoisothiazolones (8a–h)
Cbn), 121.74 (2 ꢁ CHar, 200/600-C), 119.64 (CHar, 5-Cpy), 33.85 (CH2);
MS (CI NH3 direct mode): m/z (%) 355.0 [M35Cl]+ (100).
Compound 7 (isothiazolo[5,4-b]pyridin-3(2H)-one) (10 mmol,
1.5 g) was dissolved in 40 mL of absolute methanol. After addition
of 13 mmol of appropriate substituted benzyl bromide derivative
and N,N-diisopropylethylamine (11 mmol, 1.4 g, 1.9 ml), the
preparation was stirred at room temperature overnight. The
reaction mixture was poured onto 150 mL 1 M hydrochloric acid
and extracted three times with ethyl acetate. The combined
organic layers were washed three times with water, once with
brine and then dried over sodium sulphate and evaporated to
dryness. The products were purified using silica gel column
chromatography with ethyl acetate/cyclohexane 1:1 (v/v) as eluent.
4.12.4. 2-(Benzylthio)-N-(3,4-dichlorophenyl)nicotinamide (2c)
Starting out from 1 (2-(benzylthio)nicotinic acid) (10 mmol,
2.5 g) and 3,4-dichloroaniline (11 mmol, 1.8 g) according to syn-
thetic procedure 1, 2c (2-(benzylthio)-N-(3,4-dichlorophenyl)nico-
tinamide) was obtained as white crystals (yield: 4.7 mmol, 47%).
Mp 172 °C; 1H NMR (400 MHz, (CD3)2SO): d = 10.75 (s, 1H, NH),
8.64 (dd, 3J = 4.9 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.07 (t, J = 1.3 Hz,
1Har, 200-H), 7.97 (dd, 3J = 7.6 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.62 (d,
J = 1.3 Hz, 2Hbn, 20/60-H), 7.41–7.39 (m, 2Har, 500/600-H), 7.32–7.27
(m, 3Hbn/py, 30/50/5-H), 7.24–7.22 (m, 1Hbn, 40-H), 4.42 (s, 2H,
CH2); 13C NMR (100 MHz, (CD3)2SO): d = 165.51 (C@O), 157.28
(Cq, 2-Cpy), 150.93 (CHar, 6-Cpy), 139.31 (Cq, 10-Cbn), 138.40 (Cq,
100-C), 136.33 (Car, 4-Cpy), 131.44 (Cq, 300-C), 131.16 (CHar, 500-C),
129.82 (Cq, 3-Cpy), 129.49 (2 ꢁ CHar, Cbn), 128.75 (2 ꢁ CHar, Cbn),
127.36 (CHar, 40-Cbn), 125.85 (Cq, 400-C), 121.34 (CHar, 200-C),
120.24 (CHar, 600-C), 119.64 (CHar, 5-Cpy), 33.88 (CH2); MS (CI NH3
direct mode): m/z (%) 389.0 [M35Cl35Cl]+ (100).
4.12.1. 2-(Benzylthio)nicotinic acid (1)
2-Mercaptonicotinic acid (32 mmol, 5.0 g) was dissolved to-
gether with potassium hydroxide (65 mmol, 3.6 g) in 40 mL of iso-
propyl alcohol and 8 mL of water. Benzyl chloride was added
dropwise and the mixture was refluxed for 30 min. After cooling,
the reaction mixture was concentrated to approximately 30 mL.
The residue was treated with 30 mL of water and slightly acidified
with acetic acid. The precipitate was filtered, washed with water

S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
3685
4.12.5. 2-(Benzylthio)-N-(4-fluorophenyl)nicotinamide (2d)
Starting out from 1 (2-(benzylthio)nicotinic acid) (10 mmol,
2.5 g) and 4-fluoroaniline (11 mmol, 0.82 g, 1.0 mL) according to
synthetic procedure 1, 2d (2-(benzylthio)-N-(4-fluorophenyl)nico-
tinamide) was obtained as white crystals (yield: 6.0 mmol, 60%).
Mp 162 °C; 1H NMR (400 MHz, (CD3)2SO): d = 10.52 (s, 1H, NH),
8.62 (dd, 3J = 4.8 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 7.94 (dd, 3J = 7.6 Hz,
4J = 1.7 Hz, 1Hpy, 4-H), 7.73–7.69 (m, 2Har, 200/600-H), 7.41–7.39
(m, 2Hbn, 20/60-H), 7.31–7.27 (m, 3Hbn/py, 30/50/5-H), 7.24–7.17 (m,
1.4 g, 1.4 mL) was followed and the mixture was heated to 45 °C
for 1 h. After cooling at room temperature, 3-methoxyaniline
(12 mmol, 1.4 g, 1.3 mL) was added and the preparation was stir-
red overnight. The mixture was then treated with 50 mL of 1 M so-
dium hydroxide solution and the remaining aqueous phase was
washed three times with dichloromethane. The combined organic
layers were washed with brine, dried over sodium sulphate and
evaporated in vacuum. The crude product was recrystallized from
ethyl acetate with petroleum ether (80–110 °C) to give 2g (2-(ben-
zylthio)-N-(3-methoxyphenyl)nicotinamide) as white crystals
(yield: 3.4 mmol, 33%). Mp 134 °C; 1H NMR (400 MHz, (CD3)2SO):
d = 10.44 (s, 1H, NH), 8.61 (dd, 3J = 4.8 Hz, 4J = 1.7 Hz, 1Hpy, 6-H),
3Har/bn
,
40/300/500-H), 4.42 (s, 2H, CH2); 13C NMR (100 MHz,
1
(CD3)2SO): d = 165.08 (C@O), 158.87 (d, JC–F = -238.9 Hz, Cq, 400-
C), 157.10 (Cq, 2-Cpy), 150.61 (CHar, 6-Cpy), 138.49 (Cq, 10-Cbn),
4
136.13 (CHar, 4-Cpy), 135.63 (d, JC–F = -2.5 Hz, Cq, 100-C), 130.42
7.92 (dd, 3J = 7.6 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.41–7.39 (m, 3Har/bn
,
(Cq, 3-Cpy), 129.49 (2 ꢁ CHar, Cbn), 128.74 (2 ꢁ CHar, Cbn), 127.33
20/60/600-H), 7.31–7.22 (m, 6Har/bn/py, 30/40/50/200/500/5-H), 6.71–6.68
(m, 1Har, 400-H), 4.42 (s, 2H, CH2), 3.34 (s, 3H, CH3); 13C NMR
(100 MHz, (CD3)2SO): d = 165.22 (C@O), 159.89 (Cq, 2-Cpy), 157.07
(Cq, 300-C), 150.56 (CHar, 6-Cpy), 140.43 (Cq, 10-Cbn), 138.50 (Cq, 100-
C), 136.13 (CHar, 4-Cpy), 130.57 (Cq, 3-Cpy), 129.97 (CHar, 500-C),
129.49 (2 ꢁ CHar, Cbn), 128.74 (2 ꢁ CHar, Cbn), 127.33 (CHar, 40-
3
(CHar, 40-Cbn), 122.00 (d, JC–F = -7.7 Hz, 2 ꢁ CHar, 200/600-C), 119.64
2
(CHar, 5-Cpy), 115.78 (d, JC–F = -22.1 Hz, 2 ꢁ CHar, 300/500-C), 33.85
(CH2); 19F NMR (376 MHz, (CD3)2SO): d = 118.38–118.45 (m, 1F,
400-F); MS (CI NH3 direct mode): m/z (%) 339.2 [M+H]+ (100).
C
bn), 119.62 (CHar, 5-Cpy), 112.45 (CHar, 600-C), 109.81 (CHar, 400-C),
4.12.6. 2-(Benzylthio)-N-(3,4-difluorophenyl)nicotinamide (2e)
Starting out from 1 (2-(benzylthio)nicotinic acid) (10 mmol,
2.55 g) and 3,4-difluoroaniline (11 mmol, 1.4 g, 1.1 mL) according
to synthetic procedure 1, 2e (2-(benzylthio)-N-(3.4-diflorophe-
nyl)nicotinamide) was obtained as white crystals (yield: 6.9 mmol,
69%). Mp 149 °C; 1H NMR (400 MHz, (CD3)2SO): d = 10.69 (s, 1H,
NH), 8.63 (dd, System, 3J = 4.8 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 7.96 (dd,
3J = 7.6 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.78–7.82 (m, 1Har, 200-H), 7.45–
7.39 (m, 4Har/bn/py, 20/60/600/5-H), 7.32–7.27 (m, 3Hbn, 30/40/50-H),
7.24–7.20 (m, 1Har, 500-H), 4.42 (s, 2H, CH2); 13C NMR (100 MHz,
(CD3)2SO): d = 165.32 (C@O), 157.20 (Cq, 2-Cpy), 150.83 (CHar, 6-
105.92 (CHar, 200-C), 55.44 (CH3), 33.85 (CH2); MS (CI NH3 direct
mode): m/z (%) 351.2 [M+H]+ (100).
4.12.9. 2-(Benzylsulfinyl)-N-(3-methoxyphenyl)nicotinamide 3
Compound 2g (2-(benzylthio)-N-(3-methoxyphenyl)nicotin-
amide) (2.9 mmol, 1.0 g) was dissolved in 25 mL of absolute
dichloromethane and cooled to 0 °C. After addition of 70% m-chloro-
perbenzoic acid, the batch was stirred for 15 min at 0 °C. Then the
reaction was quenched with 100 mL of 2 M sodium hydroxide
solution and stirred intensively. The preparation was filtrated, the
organic solvent was removed in vacuum and the residue after
evaporation was combined with the precipitate to give compound
3 (2-(benzylsulfinyl)-N-(3-methoxyphenyl)nicotinamide) as white
crystals (yield: 2.4 mmol, 83%). Mp 193 °C; 1H NMR (400 MHz,
(CD3)2SO): d = 10.73 (bs 1H, NH), 8.88 (dd, 3J = 4.7 Hz, 4J = 1.5 Hz,
1Hpy, 6-H), 8.29 (dd, 3J = 7.7 Hz, 4J = 1.3 Hz, 1Hpy, 4-H), 7.73 (dd,
3J = 7.7 Hz, 3J = 4.7 Hz, 1Hpy, 5-H), 7.38–7.33 (m, 4H, 30/50/200/600-
C
py), 149.90 (dd, 1JC–F = -241.9 Hz, 2JC–F = -13.1 Hz, Cq, 300-C), 146.06
2
(dd, 1JC–F = -188.7 Hz, JC–F = -12.8 Hz, Cq, 400-C), 138.43 (Cq, 10-Cbn),
136.30–136.19 (m, Cq, 100-C), 129.99 (CHar, 4-Cpy), 129.49 (2 ꢁ CHar,
C
bn), 128.75 (2 ꢁ CHar, Cbn), 127.35 (CHar, 40-Cbn), 127.45 (Cq, 3-Cpy),
119.64 (CHar, 5-Cpy), 117.88 (d, 2JC–F = -17.6 Hz, CHar, 500-C), 116.57
3
4
2
(dd, JC–F = -5.9 Hz, JC–F = -3.4 Hz, CHar, 600-C), 109.14 (d, JC–F
=
-21.5 Hz, CHar, 200-C), 33.86 (CH2); 19F NMR (376 MHz, (CD3)2SO):
d = 136.97–137.16 (m, 1F, 400-F), 143.79–143.91 (m, 1F, 300-F); MS
(CI NH3 direct mode): m/z (%) 357.1 [M+H]+ (100).
H
ar/bn), 7.31–7.26 (m, 4Har/bn, 20/40/60/500-H), 6.75–6.71 (m, 1Har,
400-H), 4.49 (d, 2J = 12.5 Hz, 1H, CH2), 4.15 (d, 2J = 12.5 Hz, 1H, CH2),
3.77 (s, 3H, CH3); 13C NMR (100 MHz, (CD3)2SO): d = 162.23 (C@O),
164.12 (Cq, 2-Cpy), 159.91 (Cq, 300-C), 151.72 (CHar, 6-Cpy), 137.48
(CHar, 4-Cpy), 132.48 (Cq, 100-C), 131.89 (Cq, 10-Cbn), 130.66 (2 ꢁ CHar,
4.12.7. 2-(Benzylthio)-N-(4-methylphenyl)nicotinamide (2f)
Starting out from 1 (2-(benzylthio)nicotinic acid) (10 mmol,
2.5 g) and and p-toluidine (11 mmol, 1.2 g) according to synthetic
procedure 1, 2f (2-(benzylthio)-N-(4-methylphenyl)nicotinamide)
was obtained as white powder (yield: 4.8 mmol, 48%). Mp
159 °C; 1H NMR (400 MHz, (CD3)2SO): d = 10.37 (s, 1H, NH), 8.60
C
bn), 130.04 (CHar, 500-C) 128.92 (2 ꢁ CHar, Cbn), 128.32 (CHar, 5-Cpy),
125.72 (CHar, 40-Cbn), 123.70 (Cq, 3-Cpy), 112.91 (CHar, 600-C), 109.93
(CHar, 400-C), 106.46 (CHar, 200-C), 60.99 (CH2), 55.49 (CH3); MS (CI
NH3 direct mode): m/z (%) 367.2 [M+H]+ (100).
,
(dd, 3J = 4.8 Hz, 4J = 1.7 Hz, 1Hpy 6-H), 7.92 (dd, 3J = 7.6 Hz,
4J = 1.7 Hz, 1Hpy, 4-H), 7.57 (d, 3J = 8.3 Hz, 2Hbn, 20/60-H), 7.41–
7.38 (m, 2Har, 200/600-H), 7.31–7.26 (m, 3Har/py, 300/500/5-H), 7.22–
7.20 (m, 1Hbn, 40-H), 7.15 (d, 3J = 8.3 Hz, 2Hbn, 30/50-H), 4.42 (s,
2H, CH2), 2.27 (s, 3H, CH3); 13C NMR (100 MHz, (CD3)2SO):
d = 164.97 (C@O), 157.06 (Cq, 2-Cpy), 150.47 (CHar, 6-Cpy), 138.52
(Cq, 10-Cbn), 136.75 (Cq, 400-C), 136.06 (CHar, 4-Cpy), 133.32 (Cq, 100-
C), 130.68 (Cq, 3-Cpy), 129.52 (2 ꢁ CHar, 300/500-C), 129.49 (2 ꢁ CHar,
4.12.10. 2-Phenylisothiazolo[5,4-b]pyridin-3(2H)-one (4a)
Starting out from 2a (2-(benzylthio)-N-phenylnicotinamide)
(3 mmol, 1 g) using synthetic procedure 2, 4a (2-phenylisothiazol-
o[5,4-b]pyridin-3(2H)-one) was obtained as white crystals after
purification through silica gel column chromatography and cyclo-
hexane/ethyl acetate 7:3 (v/v) (yield: 0.3 mmol, 10%). Mp 159 °C;
1H NMR (400 MHz, (CDCl3): d = 8.83–8.82 (m, 1Hpy, 6-H), 8.36 (d,
J = 7.8 Hz, 1Hpy, 4-H), 7.72 (d, J = 7.7 Hz, 2Har, 20/60-H), 7.52–7.49
(m, 2Har, 30/50-H), 7.44–7.41 (m, 1Hpy, 5-H), 7.38–7.35 (m, 1Har,
40-H); 13C NMR (100 MHz, (CDCl3): d = 162.44 (C@O), 161.86 (Cq,
2-Cpy), 154.05 (CHar, 6-Cpy), 136.59 (Cq, 10-C), 135.25 (CHar, 4-
C
bn), 128.74 (2 ꢁ CHar, Cbn), 127.32 (CHar, 40-Cbn), 120.19 (2 ꢁ CHar,
200/600-C), 119.62 (CHar, 5-Cpy), 33.85 (CH2), 20.92 (CH3); MS (CI NH3
direct mode): m/z (%) 335.1 [M+H]+ (100).
4.12.8. 2-(Benzylthio)-N-(3-methoxyphenyl)nicotinamide (2g)
Compound 1 (2-(benzylthio)nicotinic acid) (10. mmol, 2.5 g)
was suspended in 100 mL of absolute dichloromethane and trieth-
ylamine (12 mmol, 1.2 g, 1.6 mL) was added to the stirred suspen-
sion. After 5 min, addition of isobutylchloroformiate (10 mmol,
C
py), 129.45 (2 ꢁ CHar, 30/50-C), 127.42 (CHar, 40-C), 124.80
(2 ꢁ CHar, 20/60-C), 120.97 (CHar, 5-Cpy), 119.75 (Cq, 3-Cpy); MS (EI
direct mode): m/z (%) 228.1 [M]+ (100); microanalysis [C12H8N2OS]:
found/calculated C (63.19/63.14), H (2.99/3.53), N (11.86/12.27),
S (14.09/14.05).

3686
S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
4.12.11. 2-(4-Chlorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-one
(4b)
3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy
, 4-H), 7.73–7.68 (m, 1Hpy, 5-H),
7.46–7.41 (m, 2H, 20/60-H), 7.33–7.26 (m, 1H, 50-H); 13C NMR
Starting out from 2b (2-(benzylthio)-N-(4-chlorophenyl)nico-
tinamide) (3 mmol, 1 g) using synthetic procedure 2, 4b (2-(4-
chlorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as white-yellowish crystals after purification through silica gel
column chromatography and dichloromethane/methanol 100:6.25
(v/v) (yield: 0.5 mmol, 15%). Mp 196 °C; 1H NMR (400 MHz,
(CDCl3): d = 8.84 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.37 (dd,
3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.69 (dt, 3J = 5.2 Hz, 4J = 3.0 Hz,
5J = 2.2 Hz, 2H, 20/60-H), 7.49 (dt, 3J = 5.2 Hz, 4J = 3.0 Hz, 5J = 2.2 Hz,
2H, 30/50-H), 7.45–7.43 (m, 1Hpy, 5-H); 13C NMR (100 MHz, (CDCl3):
d = 162.46 (C@O), 161.65 (Cq, 2-Cpy), 154.25 (CHar, 6-Cpy), 135.32
(CHar, 4-Cpy), 135.14 (Cq, 10-C), 132.96 (Cq, 40-C), 129.58 (2 ꢁ CHar,
30/50-C), 125.89 (2 ꢁ CHar, 20/60-C), 121.13 (CHar, 5-Cpy), 119.53
(Cq, 3-Cpy); MS (EI direct mode): m/z (%) 261.8 [M35Cl]+ (100);
microanalysis [C12H7ClN2OS]: found/calculated C (54.86/54.68), H
(2.90/2.69), N (10.33/10.60), S (11.03/12.21).
(100 MHz, (CDCl3): d = 162.48 (C@O), 161.54 (Cq, 2-Cpy), 154.40
(CHar, 6-Cpy), 150.21 (dd, JC–F = -249.0 Hz, JC–F = -13.6 Hz, Cq,
1
2
1
2
30-C), 149.16 (dd, JC–F = -248.5 Hz, JC–F = -12.5 Hz, Cq, 40-C),
3
4
135.39 (CHar, 4-Cpy), 132.76 (dd, JC–F = -8.0 Hz, JC–F = -3.9 Hz, Cq,
3
4
10-C), 121.24 (CHar, 5-Cpy), 120.75 (dd, JC–F = -6.4 Hz, JC–F = -
2
3.7 Hz, CHar, 60-C), 119.33 (Cq, 3-Cpy), 117.83 (d, JC–F = -18.2 Hz,
CHar, 50-C), 114.62 (d, JC–F = -18.2 Hz, CHar, 20-C); 19F NMR
2
(376 MHz, (CDCl3): d = 134.00–134.11 (m, 1F, 40-F), 137.52–
137.64 (m, 1F, 30-F); MS (EI direct mode): m/z (%) 264.0 [M]+
(100); HPLC purity analysis: 97.0%, retention time 19.51 min.
4.12.15. 2-(4-Methylphenyl)isothiazolo[5,4-b]pyridin-3(2H)-one
(4f)
Starting out from 2f (2-(benzylthio)-N-(4-methylphenyl)nico-
tinamide) (3 mmol, 1 g) using synthetic procedure 2, 4f (2-(4-
methylphenyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as white-yellowish powder after purification through silica gel
column chromatography and dichloromethane/methanol 100:6.25
(v/v) (yield: 1.8 mmol, 59%). Mp 143 °C; 1H NMR (400 MHz, (CDCl3):
d = 8.82 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.37 (dd, 3J = 7.9 Hz,
4.12.12. 2-(3,4-Dichlorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-
one (4c)
Starting out from 2c (2-(benzylthio)-N-(3,4-dichlorophenyl)nic-
otinamide) (3 mmol, 1 g) using synthetic procedure 2, 4c (2-(3,4-
dichlorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as fawn powder after purification through silica gel column chro-
matography and dichloromethane/methanol 100:6.25 (v/v) (yield:
1.6 mmol, 52%). Mp 252 °C; 1H NMR (400 MHz, (CDCl3): d = 8.85
(dd, 3J = 4.7, 4J = 1.7, 1Hpy, 6-H), 8.36 (dd, 3J = 7.9, 4J = 1.7, 1Hpy, 4-
4J = 1.7 Hz, 1Hpy 4-H), 7.59–7.56 (m, 2H, 20/60-H), 7.42 (dd,
,
3J = 7.9 Hz, 3J = 4.7 Hz, 1Hpy, 5-H), 7.32–7.28 (m, 2H, 30/50-H), 2.42
(s, 3H, CH3); 13C NMR (100 MHz, (CDCl3): d = 162.49 (C@O),
161.91 (Cq, 2-Cpy), 153.93 (CHar, 6-Cpy), 137.66 (Cq, 40-C), 135.19
(CHar, 4-Cpy), 133.85 (Cq, 10-C), 130.02 (2 ꢁ CHar, 30/50-C), 124.94
(2 ꢁ CHar, 20/60-C), 120.90 (CHar, 5-Cpy), 119.73 (Cq, 3-Cpy), 21.09
(CH3); MS (EI direct mode): m/z (%) 241.8 [M]+ (100); HPLC purity
analysis: 98.9%, retention time 19.77 min.
4
4
H), 7.94 (d, J = 2.5, 1H, 20-H), 7.62 (dd, 3J = 8.7, J = 2.5, 1H, 50-H),
7.57 (d, J = 8.7, 1H, 60-H), 7.44 (dd, 3J = 7.9, 3J = 4.7, 1Hpy, 5-H);
3
13C NMR (100 MHz, (CDCl3): d = 162.44 (C@O), 161.54 (Cq, 2-Cpy),
154.44 (CHar, 6-Cpy), 136.11 (Cq, 10-C), 135.33 (CHar, 4-Cpy) 133.44
(Cq, 30-C), 131.16 (Cq, 40-C), 130.91 (CHar, 50-C), 126.12 (CHar, 20-
C), 123.42 (CHar, 60-C), 121.19 (CHar, 5-Cpy), 119.37 (Cq, 3-Cpy);
MS (EI direct mode): m/z (%) 295.9 [M35Cl35Cl]+ (100); HPLC purity
analysis: 97.2%, retention time 25.71 min.
4.12.16. 2-(3-Methoxzphenyl)isothiazolo[5,4-b]pyridin-3(2H)-
one (4g)
A suspension of 3 (2-(benzylsulfunyl)-N-(3-methoxyphenyl)
nicotinamide) (2 mmol, 0.7 g) in 50 mL dichloromethane was
cooled to 0 °C and treated dropwise with trichloroacetic anhydride
(2.3 mmol, 0.69 g, 0.41 mL). The preparation was stirred for 4 h
while the temperature was rising to 25 °C. The reaction mixture
was then quenched with 75 mL of 2 M sodium hydroxide solution
and extracted. The aqueous phase was washed three times with
dichloromethane. The combined organic layers were dried over
sodium sulphate and the solvent was removed in vacuum. The
residue was purified using silica gel column chromatography
and dichloromethane/methanol 100:6.25 (v/v) as eluent to give
4g (2-(3-methoxzphenyl)isothiazolo[5,4-b]pyridin-3(2H)-one) as
white crystals (yield: 0.9 mmol, 43%). Mp 114 °C; 1H NMR
(400 MHz, (CDCl3): d = 8.83 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H),
8.37 (dd, 3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.45–7.26 (m, 4H, 20/
4.12.13. 2-(4-Fluorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-one
(4d)
Starting out from 2d (2-(benzylthio)-N-(4-fluorophenyl)nico-
tinamide) (3 mmol, 1 g) using synthetic procedure 2, 4d (2-(4-
fluorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as white-yellowish crystals after purification through silica gel col-
umn chromatography and cyclohexane/ethyl acetate 7:3 (v/v)
(yield: 1.1 mmol, 38%). Mp 204 °C; 1H NMR (400 MHz, (CDCl3):
d = 8.84 (dd, 3J = 4.7 Hz, 4J = 1.6 Hz, 1Hpy
,
6-H), 8.37 (dd,
3J = 8.1 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.69–7.65 (m, 2H, 20/60-H), 7.44
(dd, 3J = 7.8 Hz, 3J = 4.8 Hz, 1Hpy, 5-H), 7.23–7.18 (m, 2H, 30/50-H);
13C NMR (100 MHz, (CDCl3): d = 162.65 (C@O), 161.37 (d, JC–F
=
50/60/5-H), 6.92 (ddd, 3J = 8.3 Hz, 4J = 2.5 Hz, J = 2.4 Hz, 1H, 40-H),
1
4
-239.0 Hz, Cq, 40-C), 161.79 (Cq, 2-Cpy), 154.16 (CHar, 6-Cpy),
3.88 (s, 3H, CH3); 13C NMR (100 MHz, (CDCl3): d = 162.45 (C@O),
161.86 (Cq, 2-Cpy), 160.26 (Cq, 30-C), 154.08 (CHar, 6-Cpy), 137.64
(Cq, 10-C), 135.22 (CHar, 4-Cpy), 130.14 (CHar, 50-C), 120.97 (CHar,
5-Cpy), 119.83 (Cq, 3-Cpy), 116.76 (CHar, 60-C), 113.40 (CHar, 40-C),
110.44 (CHar, 20-C), 55.48 (CH3); MS (EI direct mode): m/z
(%) 258.2 [M]+ (100); microanalysis [C13H10N2O2S]: found/calcu-
lated (%) C (60.64/60.45), H (4.10/3.90), N (10.69/10.85), S (12.13/
12.41).
4
135.30 (CHar, 4-Cpy), 132.32 (d, JC–F = -3.0 Hz, Cq, 10-C), 127.09 (d,
3JC–F = -8.5 Hz, 2 ꢁ CHar, 20/60-C), 121.08 (CHar, 5-Cpy), 119.40
2
(Cq,3-Cpy), 116.42 (d, JC–F = -22.8 Hz, 2 ꢁ CHar, 30/50-C); 19F NMR
(376 MHz, (CDCl3): d = 112.99–113.06 (m, 1F, 40-F); MS (EI direct
mode): m/z (%) 246.0 [M]+ (100); HPLC purity analysis: 99.8%,
retention time 19.77 min.
4.12.14. 2-(3,4-Difluorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-
one (4e)
4.12.17. Isothiazolo[5,4-b]pyridin-3-ol (5)
Starting out from 2e (2-(benzylthio)-N-(3,4-difluorophenyl)nic-
otinamid) (3 mmol, 1 g) using synthetic procedure 2, 4e (2-(3,4-
difluorophenyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as white-yellowish crystals after purification through silica gel
column chromatography and cyclohexane/ethyl acetate 7:3
(v/v) (yield: 0.4 mmol, 13%). Mp 219 °C; 1H NMR (400 MHz,
(CDCl3): d = 8.85 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.36 (dd,
To obtain 5 (isothiazolo[5,4-b]pyridin-3-ol), diphenyl phos-
phoryl azide (20 mmol, 5.5 g, 4.3 mL) was cooled to 0 °C in
triethylamine (33 mmol, 3.3 g, 4.5 mL). Then, a suspension of
2-mercaptonicotinic (20 mmol, 3.10 g) acid in 10 mL pyridine
was added dropwise and the mixture was stirred overnight
at room temperature. The crude product was absorbed on
Al2O3(basic, activate I) and washed first with methanol and

S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
3687
subsequently with 10% acetic acid methanol solution. The acid
direct mode): m/z (%)242.0 [M]+ (100); microanalysis [C13H10
-
phase was neutralized with 5% (w/v) solution of sodium bicarbon-
ate and extracted three times with dichlorometane. The combined
organic layers were washed with diluted hydrochloric acid, treated
with brine and dried over sodium sulphate. After solvent removal
in vacuum, 5 (isothiazolo[5,4-b]pyridin-3-ol), was obtained as a
yellow powder (yield: 4.2 mmol, 41%). Mp 243 °C; 1H NMR
(400 MHz, (CD3)2SO): d = 11.81 (bs 1H, OH), 8.82 (dd, 3J = 4.6 Hz,
4J = 1.6 Hz, 1Hpy, 6-H), 8.31 (dd, 3J = 8.0 Hz, 4J = 1.6 Hz, 1Hpy, 4-H),
7.51 (dd, 3J = 8.0 Hz, 3J = 4.6 Hz, 1Hpy, 5-H); 13C NMR (100 MHz,
(CD3)2SO): d = 168.18 (C-OH), 163.51 (Cq, 2-Cpy), 152.95 (CHar,
6-Cpy), 133.55 (CHar, 4-Cpy), 120.77 (CHar, 5-Cpy), 118.92 (Cq,
3-Cpy); MS (EI direct mode): m/z (%) 152.0 [M]+ (100); HPLC purity
analysis: 98.8%, retention time 9.35 min.
N2OS]: found/calculated (%) C (64.26/64.44), H (4.26/4.16), N
(11.50/11.56), S (13.19/13.23).
4.12.21. 2-(4-Chlorobenzyl)isothiazolo[5,4-b]pyridin-3(2H)-one
(8b)
Starting out from
7 (isothiazolo[5,4-b]pyridin-3(2H)-one)
(10 mmol, 1.5 g) and 4-chlorobenzyl bromide (13 mmol, 2.7 g,)
according to synthetic procedure 3, 8b (2-(4-chlorobenzyl)iso-
thiazolo[5,4-b]pyridin-3(2H)-one) was obtained as white crystals
(yield: 3.0 mmol, 30%). Mp 162 °C; 1H NMR (400 MHz, (CDCl3):
d = 8.77 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy
,
6-H), 8.32 (dd,
3J = 8.0 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.41–7.32 (m, 5H, 20/30/50/60/5-
H), 5.06 (s, 2H, CH2); 13C NMR (100 MHz, (CDCl3): d = 163.65
(C@O), 162.28 (Cq, 2-Cpy), 153.73 (CHar, 6-Cpy), 134.92 (CHar,
4-Cpy), 134.41 (Cq, 10-C), 134.21 (Cq, 40-C), 129.78 (2 ꢁ CHar),
129.12 (2 ꢁ CHar), 120.79 (CHar, 5-Cpy), 119.13 (Cq, 3-Cpy), 46.73
(CH2); MS (CI NH3 direct mode): m/z (%) 222.0 [M35Cl+H]+ (100);
4.12.18. 2-Mercaptonicotinamide (6)
A suspension of 2-mercaptonicotinic acid (77.3 mmol, 12.0 g) in
120 mL toluene and thionyl chloride (500 mmol, 58.9 g, 36.0 mL)
was heated at reflux for 3 h. The preparation was cooled to room
temperature and then concentrated in vacuum. The residue was
resuspended in 80 mL toluene and concentrated again in vacuum.
The obtained acid chloride was subsequently solved in a mixture
of ammonium chloride (260 mmol, 14.0 g), concentrated ammonia
(2.6 mmol, 100 mL) and 34 mL of water and stirred at room temper-
ature overnight. After treatment with sodium borohydride
(40 mmol, 1.51 g) for 1 h, the mixture was acidified with 3 M hydro-
chloric acid. The precipitate of 6 (2-mercaptonicotinamide) was ob-
tained as brown powder (yield: 58.0 mmol, 75%). Mp 246 °C; 1H
NMR (400 MHz, (CD3)2SO): d = 14.00 (bs, 1H, SH), 10.07 (bs 1H,
microanalysis [C13H9ClN2OS]: found/calculated (%)
56.42), H (3.33/3.28), N (10.10/10.12), S (11.13/11.59).
C (56.26/
4.12.22. 2-(3,4-Dichlorobenzyl)isothiazolo[5,4-b]pyridin-3(2H)-
one (8c)
Starting out from
7 (isothiazolo[5,4-b]pyridin-3(2H)-one)
(10 mmol, 1.5 g) and 3,4-dichlorobenzyl bromide (13 mmol, 3.1 g,
1.9 mL) according to synthetic procedure 3, 8c (2-(3,4-dichloroben-
zyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained as white
crystals (yield: 3.7 mmol, 37%). Mp 136 °C; 1H NMR (400 MHz,
(CDCl3): d = 8.79 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.33 (dd,
NH2), 8.49 (dd, 3J = 7.6 Hz, 4J = 1.9 Hz, 1Hpy
,
6-H), 7.95 (dd,
3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.48 (d, J = 2.1 Hz, 1H, 20-H),
4
3J = 5.9 Hz, 4J = 1.8 Hz, 2H, 4-Hpy/1H-NH2), 7.01 (dd, 3J = 7.6 Hz,
3J = 5.9 Hz, 1Hpy, 5-H); 13C NMR (100 MHz, (CD3)2SO): d = 174.49
(C@O), 165.52 (Cq, 2-Cpy), 142.62 (CHar, 6-Cpy), 141.70 (CHar,
4-Cpy), 133.84 (Cq, 3-Cpy), 114.05 (CHar, 5-Cpy); MS (EI direct mode):
m/z (%) 154.1 [M]+ (100).
7.45 (d, 3J = 8.2 Hz, 1H, 50-H), 7.41 (dd, 4J = 7.9 Hz, 4J = 4.7 Hz, 1Hpy
,
5-H), 7.23 (dd, 3J = 8.2 Hz, J = 2.1 Hz, 1H, 60-H), 5.05 (s, 2H, CH2);
13C NMR (100 MHz, (CDCl3): d = 163.69 (C@O), 162.22 (Cq, 2-Cpy),
153.90 (CHar, 6-Cpy), 135.91 (Cq, 10-C), 135.02 (CHar, 4-Cpy), 133.08
(Cq, 30-C), 132.68 (Cq, 40-C), 130.90 (CHar, 50-C), 130.21 (CHar, 20-C),
127.59 (CHar, 60-C), 120.90 (CHar, 5-Cpy), 118.94 (Cq, 3-Cpy), 46.22
(CH2); MS (CI NH3 direct mode): m/z (%) 311.0 [M35Cl35Cl+H]+
4
4.12.19. Isothiazolo[5,4-b]pyridin-3(2H)-one (7)
A mixture of 6 (2-mercaptonicotinamide) (20 mmol, 3.1 g) with
20 mL concentrated sulphuric acid was refluxed at 100 °C until the
amide was completely dissolved. The preparation was then cooled
to room temperature, poured over 50 g ice and made alkaline (pH
11) with concentrated ammonia solution. Thereby, a small amount
of precipitate was separated by filtration and the filtrate was
heated to boiling. The hot aqueous phase was acidified with acidic
acid (pH 4) and allowed to cool. The precipitate was isolated by fil-
tration and washed with water to give 7 (isothiazolo[5,4-b]pyridin-
3(2H)-on) as fawn-yellow powder (yield: 16.2 mmol, 81%). Mp
225 °C; 1H NMR (400 MHz, CD3OD, -16 °C): d = 8.82 (dd,
3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.37 (dd, 3J = 8.0 Hz, 4J = 1.7 Hz,
1Hpy, 4-H), 7.52 (dd, 3J = 8.0 Hz, 3J = 4.7 Hz, 1Hpy, 5-H); 13C NMR
(100 MHz, CD3OD, -16 °C): d = 168.30 (C@O), 165.52 (Cq, 2-Cpy),
152.76 (CHar, 6-Cpy), 133.70 (CHar, 4-Cpy), 120. (CHar, 5-Cpy),
118.52 (Cq, 3-Cpy); MS (EI direct mode): m/z (%) 152.0 [M]+ (100).
(100); microanalysis [C13H8Cl2N2OS]: found/calculated (%)
(50.33/50.18), H (2.44/2.59), N (9.00/9.00), S (9.63/10.30).
C
4.12.23. 2-(4-Fluorobenzyl)isothiazolo[5,4-b]pyridin-3(2H)-one
(8d)
Starting out from
7 (isothiazolo[5,4-b]pyridin-3(2H)-one)
(10 mmol, 1.5 g) and 4-fluorobenzyl bromide (13 mmol, 2.5 g,
1.6 mL) according to synthetic procedure 3, 8d (2-(4-fluoroben-
zyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained as white
crystals (yield: 2.0 mmol, 20%). Mp 152 °C; 1H NMR (400 MHz,
(CDCl3): d = 8.76 (dd, 3J = 4.7 Hz, 4J = 1.7 v, 1Hpy, 6-H), 8.32 (dd,
3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.40–7.36 (m, 3H, 30/50/5-H),
7.10–7.06 (m, 2H, 20/60-H), 5.07 (s, 2H, CH2); 13C NMR (100 MHz,
1
(CDCl3): d = 163.60 (C@O), 163.11 (d, JC–F = -163.6 Hz, Cq, 40-C),
162.29 (Cq, 2-Cpy), 153.70 (CHar, 6-Cpy), 134.89 (CHar, 4-Cpy),
4
3
131.56 (d, JC–F = -3.5 Hz, Cq, 10-C), 130.31 (d, JC–F = -8.3 Hz,
2 ꢁ CHar, 20/60-C), 120.75 (CHar, 5-Cpy), 119.23 (Cq, 3-Cpy), 115.88
2
4.12.20. 2-Benzylisothiazolo[5,4-b]pyridin-3(2H)-one (8a)
(d, JC–F = -21.7 Hz, 2 ꢁ CHar, 30/50-C), 46.72 (CH2); 19F NMR
Starting out from
7
(isothiazolo[5,4-b]pyridin-3(2H)-one)
(376 MHz, (CDCl3): d = 113.14–113.21 (m, 1F, 40-F); MS (CI NH3 di-
rect mode): m/z (%) 261.1 [M+H]+ (100); HPLC purity analysis:
98.9%, retention time 18.87 min.
(10 mmol, 1.5 g) and benzyl bromide (13 mmol, 2.2 g, 1.5 mL)
according to synthetic procedure 3, 8a (2-benzylisothiazolo[5,4-
b]pyridin-3(2H)-one) was obtained as white crystals (yield:
1.7 mmol, 17%). Mp 79 °C; 1H NMR (400 MHz, (CDCl3): d = 8.74
4.12.24. 2-(3,4-Difluorobenzyl)isothiazolo[5,4-b]pyridin-3(2H)-
one (8e)
(dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy
,
6-H), 8.31 (dd, 3J = 7.9 Hz,
4J = 1.7 Hz, 1Hpy, 4-H), 7.39–7.35 (m, 6H, 20/30/40/50/60/5-H), 5.09
(s, 2H, CH2); 13C NMR (100 MHz, (CDCl3): d = 163.62 (C@O),
162.37 (Cq, 2-Cpy), 153.60 (CHar, 6-Cpy), 135.71 (Cq, 10-C), 134.84
(CHar, 4-Cpy), 128.91 (2 ꢁ CHar), 128.46 (2 ꢁ CHar), 128.44 (CHar,
40-C), 120.66 (CHar, 5-Cpy), 119.28 (Cq, 3-Cpy), 47.44 (CH2); MS (EI
Starting out from
7 (isothiazolo[5,4-b]pyridin-3(2H)-one)
(10 mmol, 1.5 g) and 3,4-difluorobenzyl bromide (13 mmol, 2.7 g,
1.7 mL) according to synthetic procedure 3, 8e (2-(3,4-difluoroben-
zyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained as white
crystals (yield: 3.5 mmol, 35%). Mp 125 °C; 1H NMR (400 MHz,

3688
S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
2
(CDCl3): d = 8.78 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.33 (dd,
3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.40 (dd, 3J = 7.9 Hz, 3J = 4.7 Hz,
1Hpy, 5-H), 7.24–7.13 (m, 3H, 20/50/60-H), 5.04 (s, 2H, CH2); 13C
NMR (100 MHz, (CDCl3): d = 163.65 (C@O), 162.22 (Cq, 2-Cpy),
4-Cpy), 130.62 (d, JC–F = -32.4 Hz, Cq, 40-C), 128.55 (2 ꢁ CHar,
3
3
20/60-C), 125.90 (dd, JC–F = -7.5 Hz, JC–F = -3.7 Hz, 2 ꢁ CHar,
1
30/50-C), 123.85 (d, JC–F = -270.6 Hz, Cq, CF3), 120.88 (CHar, 5-Cpy),
118.95 (Cq, 3-Cpy), 46.82 (CH2); 19F NMR (376 MHz, (CDCl3):
d = 62.67 (2, 3F, CF3); MS (EI direct mode): m/z (%) 310.1 [M]+
(74), 159.0 [C8H6F3]+ (100); HPLC purity analysis: 98.4%, retention
time 20.98 min.
1
2
153.86 (CHar, 6-Cpy), 150.39 (dpt, JC–F = -248.3 Hz, JC–F
=
2
-12.8 Hz, JC–F = -12.4 Hz, 2 ꢁ Cq, 30/40-C), 134.98 (CHar, 4-Cpy),
3
4
132.71 (dd, JC–F = -5.2 Hz, JC–F = -4.0 Hz, Cq, 10-C), 124.48 (dd,
3JC–F = -6.3 Hz, JC–F = -3.7 Hz, CHar, 60-C), 120.87 (CHar, 5-Cpy),
4
2
119.00 (Cq, 3-Cpy), 117.74 (d, JC–F = -17.4 Hz, CHar, 50-C), 117.47
4.12.28. 2-(3,4-Dichlorobenzyl)-1-oxoisothiazolo[5,4-b]pyridin-
3(2H)-one (9)
(d, JC–F = -17.6 Hz, CHar, 20-C), 46.38 (CH2); 19F NMR (376 MHz,
2
(CDCl3): d = 136.21–136.31 (m, 1F, 40-F), 137.61–137.73 (m, 1F,
30-F); MS (CI NH3 direct mode): m/z (%) 279.1 [M+H]+ (100); HPLC
purity analysis: 99.3%, retention time 19.44 min.
Compound 8c (2-(3,4-dichlorobenzyl)isothiazolo[5,4-b]pyridin-
3(2H)-one) (13 mmol, 4.0 g) was solved at room temperature in
50% (v/v) methanol (21 mL). Potassium peroxomonosulphate (ox-
oneÒ, 19.3 mmol, 11.9 g) was added in small portions to the stirred
mixture. After consumption of the reactants (1 h), the batch was
poured into 21 mL of water and extracted three times with dichlo-
romethane. The collected organic layers were dried over sodium
sulphate and evaporated. The residue was recrystallized from ethyl
4.12.25. 2-(4-Methylbenzyl)isothiazolo[5,4-b]pyridin-3(2H)-one
(8f)
Starting out from
(10 mmol, 1.5 g) and 4-methylbenzyl bromide (
7
(isothiazolo[5,4-b]pyridin-3(2H)-one)
-bromo-p-xylene,
a
13 mmol, 2.1 g,) according to synthetic procedure 3, 8f (2-(4-
methylbenzyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as white crystals (yield: 3.2 mmol, 32%). Mp 116 °C; 1H NMR
(400 MHz, (CDCl3): d = 8.75 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H),
8.32 (dd, 3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.37 (dd, 4J = 7.9 Hz,
acetate to give 9 (2-(3,4-dichlorobenzyl)-1-oxoisothiazolo[5,4-
b]pyridin-3(2H)-one) as white crystals (yield: 8.3 mmol, 64%). Mp
150 °C; 1H NMR (400 MHz, (CDCl3): d = 8.98 (dd, 3J = 4.8 Hz,
4J = 1.6 Hz, 1Hpy, 6-H), 8.34 (dd, 3J = 7.8 Hz, 4J = 1.6 Hz, 1Hpy, 4-H),
7.74 (dd, 4J = 7.8 Hz, 4J = 4.8 Hz, 1Hpy, 5-H), 7.56 (d, 4J = 2.0 Hz,
3
3
4J = 4.7 Hz, 1Hpy, 5-H), 7.29 (d, J = 8.0 Hz, 2H, 30/50-H), 7.20 (d,
1H, 20-H), 7.46 (d, J = 8.2 Hz, 1H, 50-H), 7.31 (dd, 3J = 8.2 Hz,
3J = 7.2 Hz, 2H, 20/60-H), 5.06 (s, 2H, CH2), 2.37 (s, 3H, CH3); 13C
NMR (100 MHz, (CDCl3): d = 163.87 (C@O), 162.40 (Cq, 2-Cpy),
153.54 (CHar, 6-Cpy), 138.37 (Cq, 10-C), 134.81 (CHar, 4-Cpy),
132.67 (Cq, 40-C), 129.59 (2 ꢁ CHar), 128.53 (2 ꢁ CHar), 120.62
(CHar, 5-Cpy), 119.41 (Cq, 3-Cpy), 47.25 (CH2), 21.17 (CH3); MS (EI
direct mode): m/z (%) 256.1 [M]+ (96), 105.1 [C8H9]+ (100); micro-
analysis [C14H12N2OS]: found/calculated (%) C (65.45/65.60), H
(4.81/4.72), N (10.94/10.93), S (12.57/12.51).
4J = 2.1 Hz, 1H, 60-H), 5.22 (d, 2J = 15.5 Hz, 1H, CH2), 4.80 (d, 2J =
15.6 Hz, 1H, CH2); 13C NMR (100 MHz, (CDCl3): d = 165.24 (C@O),
163.96 (Cq, 2-Cpy), 155.17 (CHar, 6-Cpy), 135.61 (Cq, 10-C), 134.81
(CHar, 4-Cpy), 133.08 (Cq, 30-C), 132.69 (Cq, 40-C), 130.91 (CHar, 50-
C), 130.58 (CHar, 20-C), 127.98 (CHar, 60-C), 127.44 (CHar, 5-Cpy),
122.55 (Cq, 3-Cpy), 46.61 (CH2); MS (CI NH3 direct mode): m/z (%)
344.0 [M35Cl35Cl+NH4]+ (100); microanalysis [C13H8Cl2N2O2S]:
found/calculated C (47.58/47.72), H (2.40/2.46), N (8.53/8.56), S
(9.89/9.80).
4.12.26. 2-(3-Methoxybenzyl)isothiazolo[5,4-b]pyridin-3(2H)-
one (8g)
4.12.29. 2-(3,4-Dichlorobenzyl)-1,1-dioxoisothiazolo[5,4-b]pyr-
idin-3(2H)-one (10)
Starting out from
7
(isothiazolo[5,4-b]pyridin-3(2H)-one)
(10 mmol, 1.5 g) and 3-methoxybenzyl bromide (13 mmol, 2.6 g,
1.8 mL) according to synthetic procedure 3, 8g (2-(3-methoxyben-
zyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained as white
crystals (yield: 2.7 mmol, 27%). Mp 107 °C; 1H NMR (400 MHz,
(CDCl3): d =8.76 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy, 6-H), 8.32 (dd,
3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.38 (dd, 4J = 7.9 Hz, 4J = 4.7 Hz,
1Hpy, 5-H), 7.33–7.29 (m, 1H, 50-H), 6.98–6.96 (m, 1H, 60-H),
6.92–6.88 (m, 2H, 20/40-H), 5.07 (s, 2H, CH2), 3.82 (s, 3H, CH3);
13C NMR (100 MHz, (CDCl3): d = 163.63 (C@O), 162.41 (Cq, 2-Cpy),
159.98 (Cq, 30-C), 153.62 (CHar, 6-Cpy), 137.17 (Cq, 10-C), 134.86
(CHar, 4-Cpy), 129.96 (CHar, 50-C), 120.66 (2 ꢁ CHar, 5py/60-C),
119.27 (Cq, 3-Cpy), 113.97 (CHar, 40-C), 113.94 (CHar, 20-C), 55.25
(CH3), 47.39 (CH2); MS (EI direct mode): m/z (%) 272.1 [M]+ (98),
121.1 [C8H9O]+ (100); microanalysis [C14H12N2O2S]: found/calcu-
lated (%) C (61.36/61.75), H (4.52/4.44), N (10.16/10.29), S (11.66/
11.77).
A mixture of 9 (2-(3,4-dichlorobenzyl)-1-oxoisothiazolo[5,4-
b]pyridin-3(2H)-one) (6.0 mmol, 2.0 g) and tetrabutylammonium
bromide (0.24 mmol, 0.77 g) in 40 mL ethyl acetate was treated with
5% (w/v) sodium hypochlorite solution (24 mmol, 36 g). The prepa-
ration was stirred intensively at room temperature and the reaction
progress was monitored by thin layer chromatography. When the
reaction had been completed (1 h), water (100 mL) was added to
the mixture. The ethyl acetate phase was separated and washed
twice with water. The organic layer was dried over sodium sulphate
and evaporated. The crude product was purified using silica gel col-
umn chromatography and ethyl acetate/cyclohexane 2:1 (v/v) as
eluent to obtain 10 (2-(3,4-dichlorobenzyl)-1,1-dioxoisothiazol-
o[5,4-b]pyridin-3(2H)-one) as white crystals (yield: 2.2 mmol,
36%). Mp 175 °C; 1H NMR (400 MHz, (CDCl3): d = 9.04 (dd, 3J =
4.9 Hz, 4J = 1.6 Hz, 1Hpy, 6-H), 8.41 (dd, 3J = 7.9 Hz, 4J = 1.6 Hz,
1Hpy, 4-H), 7.81 (dd, 4J = 7.9 Hz, 4J = 4.9 Hz, 1Hpy, 5-H), 7.64 (d, 4J =
2.1 Hz, 1H, 20-H), 7.46 (d, 3J = 8.2 Hz, 1H, 50-H), 7.38 (dd, 3J = 8.2 Hz,
4J = 2.1 Hz, 1H, 60-H), 4.90 (s, 2H, CH2); 13C NMR (100 MHz, (CDCl3):
d = 156.85 (C@O), 155.93 (Cq, 2-Cpy), 155.65 (CHar, 6-Cpy), 134.19 (Cq,
10-C), 133.86 (CHar, 4-Cpy), 132.88 (Cq, 30-C), 132.80 (Cq, 40-C), 130.78
(CHar, 50-C), 130.74 (CHar, 20-C), 128.39 (CHar, 60-C), 128.16 (CHar, 5-
4.12.27. 2-(4-(Trifluoromethyl)benzyl)isothiazolo[5,4-b]pyridin-
3(2H)-one (8h)
Starting out from
7
(isothiazolo[5,4-b]pyridin-3(2H)-one)
(10 mmol, 1.5 g) and 4-(trifluoromethyl)benzyl bromide (13 mmol,
2.4 g) according to synthetic procedure 3, 8 h (2-(4-(trifluoro-
methyl)benzyl)isothiazolo[5,4-b]pyridin-3(2H)-one) was obtained
as white-yellowish crystals (yield: 4.6 mmol, 46%). Mp 117 °C; 1H
C
py), 122.23 (Cq, 3-Cpy), 41.48 (CH2); MS (CI NH3 direct mode): m/z
(%) 360.0 [M35Cl35Cl+NH4]+ (100); microanalysis [C13H8Cl2N2O3S]:
found/calculated (%) C (45.53/45.50), H (2.31/2.35), N (8.11/8.16),
S (9.28/9.34).
NMR (400 MHz, (CDCl3): d = 8.78 (dd, 3J = 4.7 Hz, 4J = 1.7 Hz, 1Hpy
,
6-H), 8.34 (dd, 3J = 7.9 Hz, 4J = 1.7 Hz, 1Hpy, 4-H), 7.65 (d, 3J = 8.2
v, 2H, 30/50-H), 7.50 (d, 3J = 8.1 Hz, 2H, 20/60-H), 7.41 (dd,
3J = 7.9 Hz, 3J = 4.7 Hz, 1Hpy, 5-H), 5.15 (s, 2H, CH2); 13C NMR
(100 MHz, (CDCl3): d = 163.75 (C@O), 162.23 (Cq, 2-Cpy), 153.88
Acknowledgments
Funding by the Wilhelm Sander Foundation is gratefully
acknowledged. We thank Ursula Predoiu for technical assistance.
5
(CHar, 6-Cpy), 139.66 (d, JC-F = -1.0 Hz, Cq, 10-C), 135.00 (CHar,

S. D. Furdas et al. / Bioorg. Med. Chem. 19 (2011) 3678–3689
3689
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