Synthesis and biological evaluation of a class of 5-benzylidene-2-phenyl- thiazolinones as potent 5-lipoxygenase inhibitors

Article abstract of DOI:10.1016/j.bmc.2012.04.003

A class of 5-lipoxygenase (5-LO) inhibitors characterized by a central 5-benzylidene-2-phenyl-thiazolinone scaffold was synthesized as a new series of molecular modifications and extensions of a previously reported series. Compounds were tested in a cell-based and a cell-free assay and furthermore evaluated for their influence on cell viability. The presented substituted thiazolinone scaffold turned out to be essential for both the 5-LO inhibitory activity and the non-cytotoxic profile. With (Z)-5-(4-methoxybenzylidene)-2- (naphthalen-2-yl)-5H-thiazol-4-one (2k, ST1237), a potent, direct, non-cytotoxic 5-LO inhibitor with IC₅₀ of 0.08 μM and 0.12 μM (cell-free assay and intact cells), we present a promising lead optimization and development for further investigations as novel anti-inflammatory drug.

Full text of DOI:10.1016/j.bmc.2012.04.003

Bioorganic & Medicinal Chemistry 20 (2012) 3575–3583
Contents lists available at SciVerse ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Synthesis and biological evaluation of a class of 5-benzylidene-2-phenyl-thiaz-
olinones as potent 5-lipoxygenase inhibitors
Sebastian Barzen , Carmen B. Rödl , Andreas Lill, Dieter Steinhilber, Holger Stark ,à, Bettina Hofmann
⇑
⇑
,à
Institute of Pharmaceutical Chemistry, ZAFES/LiFF/OSF Goethe-University Frankfurt, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
A class of 5-lipoxygenase (5-LO) inhibitors characterized by a central 5-benzylidene-2-phenyl-thiazoli-
none scaffold was synthesized as a new series of molecular modifications and extensions of a previously
reported series. Compounds were tested in a cell-based and a cell-free assay and furthermore evaluated
for their influence on cell viability. The presented substituted thiazolinone scaffold turned out to be
essential for both the 5-LO inhibitory activity and the non-cytotoxic profile. With (Z)-5-(4-methoxyben-
zylidene)-2-(naphthalen-2-yl)-5H-thiazol-4-one (2k, ST1237), a potent, direct, non-cytotoxic 5-LO inhib-
Received 18 January 2012
Revised 28 March 2012
Accepted 2 April 2012
Available online 11 April 2012
Keywords:
5-Lipoxygenase
Asthma
Inflammation
itor with IC50 of 0.08
optimization and development for further investigations as novel anti-inflammatory drug.
Ó 2012 Elsevier Ltd. All rights reserved.
lM and 0.12 lM (cell-free assay and intact cells), we present a promising lead
Leukotrienes
Polymorphonuclear leukocytes
Structure–activity relationships
1. Introduction
activator for phagocytes.8 Furthermore, LTA4 can be conjugated
with reduced glutathione by LTC4 synthase, resulting in the cys-
teinyl-containing LTs C4, D4 and E4, which cause bronchoconstric-
tion and vascular permeability.4
Current studies showed that the ALOX5 gene is a critical regu-
lator for leukemia stem cells in BCR-ABL-induced chronic myeloid
leukemia (CML).9 This together with the multiple pathophysiolog-
ical actions of LTs explains the increasing interest to find an active
agent for anti-leukotriene therapy. To date, zileuton,10 which acts
by chelating the active site iron of the enzyme, is the only 5-LO
inhibitor that entered the market. Given that many 5-LO inhibitory
drug candidates lack sufficient selectivity or show mechanism-
based side effects11–14 there is a strong need for novel 5-LO inhib-
itors with a distinct mode of action.
Different inflammatory diseases like allergic rhinitis, asthma,
osteoporosis, cardiovascular diseases and cancer are linked to
leukotrienes (LTs).1–3 LTs are signalling lipids derived from polyun-
saturated fatty acids acting as important regulators in immunity
and inflammation.4 One key enzyme of LT biosynthesis is 5-lipoxy-
genase (5-LO): after arachidonic acid (AA) is released from the
membrane by cytosolic phospholipase A2 (cPLA2) and transferred
via the 5-lipoxygenase-activating-protein (FLAP) to 5-LO, this
non-heme iron containing enzyme catalyses the oxygenation of
AA to 5(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid
(5-HPETE). Afterwards, 5-HPETE can be reduced to the correspond-
ing alcohol 5-HETE or alternatively dehydrated to the unstable
5–7
epoxide LTA4.
LTA4 is converted by LTA4 hydrolase to LTB4,
which is a chemotactic, and chemokinetic agent as well as an
H3CO
H3CO
Cl
CH3
Abbreviations: 5-LO, 5-lipoxygenase; AA, arachidonic acid; CML, chronic mye-
loid leukemia; cPLA2, cytosolic phospholipase A2; FLAP, 5-LO-activating protein; 5-
H(P)ETE, 5(S)-hydro(pero)xy-6-trans-8,11,14-cis-eicosatetraenoic acid; LDH, lactate
dehydrogenase; LT, leukotriene; PBS, phosphate-buffered saline pH 7.4; PMNL,
polymorphonuclear leukocytes; S100, 100,000Âg supernatant; SAR, structure–
activity relationships; TLC, thin layer chromatography; TMS, tetramethylsilane.
S
S
N
N
O
O
ST1098
C06
⇑
Corresponding authors. Tel.: +49 69 798 29302; fax: +49 69 798 29258 (H.S.);
IC50 S100 = 0.08 µM
IC50 PMNL = 0.28 µM
IC50 S100 = 0.30 µM
IC50 PMNL = 0.66 µM
tel.: +49 69 798 29335; fax: +49 798 29323 (B.H.).
E-mail addresses: h.stark@pharmchem.uni-frankfurt.de (H. Stark), hofmann@
pharmchem.uni-frankfurt.de (B. Hofmann).
Scheme 1. Structures and 5-LO inhibitory activities of parent compounds ST1098
and C06.16
Both authors contributed equally to this work.
Share senior authorship.
à
0968-0896/$ - see front matter Ó 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.bmc.2012.04.003

3576
S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
A promising structure series based on a scaffold that was orig-
To get further insight into the SAR of this class of 5-LO inhibi-
tors, we developed compounds 1a–b, 2a–l, 3a–j, and 4a–f (Table
1), which were all synthesized via the ‘one-pot reaction’ mentioned
above (Scheme 2). Initially, we synthesized compounds 1a–b with
exchanged residues at R1 and R2 compared to C06 and ST1098. 1a–
b had a slightly higher inhibitory activity than their partners. The
electron donating p-methoxy group at R2 seems to be suitable at
this position as well. Next, we changed the substitution pattern
at R2 while keeping the p-methoxybenzylidene moiety at R1 con-
stant (2a–k). The position of the chloro-residue at R2 (ST1098,
2d, 2e) had only marginal influence on 5-LO inhibition as well as
the type of halogen introduced (ST1098, 2b, 2c). Only the introduc-
tion of a fluorine at R2 (2c) led to lesser 5-LO inhibition in the S100
assay, though a comparable potency in intact cells was observed.
All of these halogen derivatives (2b–e) were able to completely
abolish 5-LO product formation in both assays (Table 1). Derivati-
sation to p-propoxyphenyl or introduction of a benzophenone
group at R2 resulted in comparable potencies (2a, 2f), although
the p-propoxyphenyl derivative was not able to completely abolish
inally identified in a virtual screening campaign15 depict the thiaz-
olinone-based compounds exemplified by ST1098 (Scheme 1).16
Compound ST1098 is the most potent derivative of a series of
thiazolinones we previously prepared and tested to investigate
the influence of different substituents on the inhibitory activity
of this thiazolinone scaffold.16 One of the most promising com-
pounds derived from this series, C06 ((Z)-5-(4-methoxybenzyli-
dene)-2-(p-tolyl)-5H-thiazol-4-one, Scheme 1), was also
characterized by molecular biological and pharmacological ap-
proaches: it exhibits a selective, direct, allosteric, stimulus and
substrate independent, non-cytotoxic 5-LO inhibitory mode of ac-
tion different to other established 5-LO inhibitors.17 To extend our
knowledge of the structure–activity relationships (SAR) of this
class of 5-LO inhibitors, we here present an additional set of syn-
thesized derivatives with a wide range of variations, tested in a cell
based and a cell free assay. Furthermore, selected compounds were
tested for their influence on cell viability.
5-LO product formation (still ꢀ20% residual activity left at 10
lM,
2. Results and discussion
2.1. Chemistry
Table 1) in contrast to the benzophenone-derivative. The disubsti-
tuted derivative 2g, carrying a polar p-nitro and m-hydroxyl group,
showed the lowest inhibitory activity in this set of compounds. The
inhibitory activity decreased to IC50 values of 4.66 0.74
lM in the
Synthesis of the target compounds 1a–b and 2a–l and 3a–j and
4a–f is outlined in Scheme 2. The thiazolinones were prepared by a
‘one-pot domino reaction’ of thioglycolic acid, the corresponding
benzonitrile and the corresponding benzaldehyde according to
Zayed et al.18 Multicomponent reactions like these are an excellent
source for novel bioactive compounds.19
Since this domino reaction can be controlled at different steps it
can also be performed in two-step synthesis route as done with the
less reactive acetophenone derivatives resulting in the methyl-
branched compounds 5a–b. First thioglycolic acid and the corre-
sponding benzonitrile were refluxed for one hour in ethanol with
TEA to yield the thiazolinone core (Scheme 3).18 The intermediate
was then condensed with p-methoxy-acetophenone by heating it
with ammonium acetate in toluene for 90 min at 130 °C under
microwave irradiation (Scheme 3).20
S100 assay and to IC50 of 28.21 4.86 M in PMNL. The loss of
l
activity in intact cells might be contributed to hampered mem-
brane permeation of the potentially partially charged compound
under test and also physiological conditions.
Substitution at the R2 phenyl in meta position with a bulky ada-
mantly coupled via a carboxamide linker (2h) did not alter activity
at all (IC50 = 0.11 0.02 lM, S100). The adamantyl-free analogue
(2i) exhibited a five- to ten-fold loss of activity, whereas elongation
with a benzyl group (2j) even resulted in a 100-fold loss of inhib-
itory activity in the S100 assay (IC50 = 11.05 4.71
ingly, inhibition of 5-LO product formation in intact PMNL was
not hampered (IC50 = 0.26 0.04 M), suggesting interference with
lM). Interest-
l
FLAP or cPLA2 rather than 5-LO. Enlargement of the aromatic R2
phenyl group to naphthalene (2k) did not alter activity at all. Fur-
ther introduction of an electron donating m-hydroxyl group at the
R1 phenyl (2l) resulted in a six-fold loss of inhibitory potency in
PMNL compared to 2k yet without having any effects on the inhib-
itory activity in the S100 assay.
2.2. Biological evaluation
2.2.1. 5-LO activity assay
In the next set of derivatives the substitution at R1 was varied
while R2 remained p-chloro (3a–f). Regardless of which substituent
was introduced at position R1, the inhibitory potency in the S100
assay of the first five derivatives (3a–e) did not change. In the
PMNL assay, slight variations were observed, but compared to
the inhibitory activity of ST1098,16 no prominent effects of the
substitutions were detected, except for compound 3e. This deriva-
tive, which carries, together with 3f, a methoxy naphthalene
moiety as R1, showed a three- to six-fold decreased activity in
PMNL. All these halogen derivatives were either able to completely
abolish 5-LO product formation in both assays (3a–c, Table 1) or
All compounds were evaluated in two different assay systems
on 5-LO activity: (i) a cell-based test system using isolated human
polymorphonuclear leukocytes (PMNL), and (ii) utilizing the cell-
free supernatant of a 100,000Âg ultracentrifuge run of homoge-
nized PMNL, the S100 assay. The PMNL assay allows assessing
intracellular bioavailability of the compounds. The cell-free assay
(S100) was chosen to distinguish between direct inhibition of 5-
LO (comparable inhibition in both assays) and indirect effects
involving for example, FLAP or cPLA2 interaction (lower inhibitory
activity in the cell-free assay). The 5-LO inhibitor BWA4C,21
another well-established iron-ligand type 5-LO inhibitor, was ta-
resulted in only marginal residual 5-LO activity at 10 lM inhibitor
concentration in the S100 assay (3d–f).
ken as reference compound showing IC50 of 0.05 0.005
lM in
PMNL S100 and IC50 of 0.14 0.06
lM in intact PMNL (Table 1),
which is similar to that in literature.21
Next, the combination of a p-methoxy substitution at position
R2 and different bicyclic moieties as R1 (3h–j) led to ineffective
H
R1
SH
OH
R2
NC
(i)
O
R1
R2
S
O
N
O
Scheme 2. Synthesis of 1a–b, 2a–l, 3a–j, and 4a–f: (i) TEA, MeOH, reflux, 12 h.18

S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
3577
SH
OH
NC
(i)
S
N
R2
O
R2
O
H3CO
R2
H3CO
S
N
(ii)
S
R2
O
N
O
H3C
CH3
O
Scheme 3. Two-step reaction of the synthesis of methyl-branched compounds 5a–b: (i) TEA, EtOH, reflux, 1 h,18 (ii) NH4OAc, toluene, microwave, 130 °C, 45–90 min.20
inhibitors with 3i being the weakest derivative with an IC50 of
5.84 0.62 M in the S100 assay. Compound 3g within this set,
carrying a flexible p-propoxy substitution at R1, also exhibited low-
er inhibitory potency with IC50 of 2.14 0.84 M (S100). In this
This assay assesses mitochondrial metabolic activity after treat-
ment with increasing concentrations of test compounds for 48 h.
Inhibition of cell viability in this assay can be ascribed both to cyto-
toxic as well as anti-proliferative effects. The iron-ligand inhibitor
l
l
case, the p-methoxy substitution at position R2 seems to be detri-
mental as the inhibitory potency decreased up to 25-fold (S100)
compared to 3a, with the same R1 moiety, but a p-chloro substitu-
tion at R2. The same pattern was observed for the IC50 values in the
S100 assays for ST1098 and ST113016 and Table 1) and for 3f and
3h. These three compound pairs have exchanged the electron with-
drawing p-chloro substitution at R2 for an electron donating p-
methoxy, which resulted in lower inhibitory potencies in the
cell-free assay. Compound 3g is somehow a negative outlier within
these three compound pairs, as it also showed loss of activity in the
intact cell assay.
One of the last steps on the compound optimization were per-
formed on new combinations of bicyclic or biphenylic residues as
R1 and R2 (4a–f) for a potentially increased aromatic enzyme/li-
gand interaction in combination with improved cell permeation.
It resulted in a set of potent inhibitors with IC50 between 0.06–
BWA4C exhibited a non-cytotoxic profile with an IC50 >30
of the tested compounds (1a, 2h, 2k, 3c, 3f) showed no effect on
cell viability (IC50 >30 M) as well. Compound 3a showed a slight
effect with an IC50 value of 21.06 0.90 M. The derivative with
the methylated double-bond (5a) showed a high influence on cell
viability in the WST-1 assay (IC50 = 12.10 1.66 M). To elucidate
whether the decreased cell viability resulted from cytotoxicity like
necrosis or rather from anti-proliferative effects, loss of cell mem-
brane integrity of U937 cells was additionally measured. Cells were
lM. Five
l
l
l
treated with test compounds up to a concentration of 30 lM for
48 h. An increased LDH leakage points to necrotic events. The
known nonredox-type 5-LO inhibitor REV-590122 was used as
cytotoxic control.23 In this assay, BWA4C induced no LDH leakage
at a concentration of 30 lM. Compound 3a resulted in medium
LDH leakage, whereas 5a caused high LDH leakage. The latter is
in contrast to the results for the non-methylated analogue C06,
which showed no cytotoxic effects at all in both assays. The methyl
group seems to be detrimental for cell viability.
0.79 lM (S100). Only 4c, 4e and 4f showed a loss of activity in
the PMNL assay possibly due to the lipophilic and steric character-
istics of the substitutions that might hamper membrane perme-
ation in contrast to the initial drug design intentions, although
the latter does not hold for all derivatives of this subset.
3. Conclusions
Additionally, two compounds with a methylated double-bond
(5a–b) were synthesized (Scheme 3, Table 1). On the one hand, it
has not been clear if the Michael acceptor function is needed for
the enzyme inhibition and on the other hand, it is unclear if the re-
stricted steric access at the enzyme can be enlarged by methyl-
branching at the aromatic substituted methylene side-chain. Their
syntheses followed the fore cited two-step reaction procedure
(Scheme 3). In relation to their non-methylated analogues C06
and ST1131,16 the IC50 of 5a and 5b got worse (Table 1). Compound
5a, the methylated derivative of C06, showed a seven-fold loss of
activity in the S100 assay and a 1.5-fold loss of activity in intact
PMNL. This observation was confirmed for the methylated ana-
logue of ST113116, 5b, which exhibited an up to 10-fold loss of
inhibitory activity (Table 1). The Michael acceptor function is ex-
pected to have no effect in the physiological assay conditions,
therefore, the additional space-filling methyl moiety seems to dis-
turb the binding of the compound to the 5-LO. This fact highlights
that the original core 5-benzylidene-2-phenyl-thiazolinone scaf-
fold with an aromatic substituted methylene moiety is essential
for the inhibitory activity of this compound class.
Summarizing the results mentioned above, we gained further
insight into the SAR of this class of thiazolinone-based 5-LO inhib-
itors. With (Z)-5-(4-methoxybenzylidene)-2-(naphthalen-2-yl)-
5H-thiazol-4-one (2k, ST1237), we could identify a potent direct
5-LO inhibitor with IC50 of 0.08 0.03 lM and 0.12 0.02 lM in
the cell-free S100 assay and intact PMNL, respectively. Therewith,
it exhibits an about ten-fold higher inhibitory potency than zileu-
ton (IC50 = 0.5–1 l
M10). Furthermore ST1237 showed no cytotoxic
effects on treated cells. In line with the known non-cytotoxic ef-
fects of C06,17 several additional compounds tested showed no
influence on cell viability and the investigated SAR caused no neg-
ative influence on cytotoxicity except for the double-bond methyl-
ated derivative 5a, which showed high cytotoxic effects. This
additional methyl group also caused a decreased inhibitory activity
on 5-LO product formation. The core 5-benzylidene-2-phenyl-
thiazolinone scaffold is essential for the inhibitory activity of this
compound class. With ST1237, we presented a promising lead
optimization and development of a novel pharmacological tool
for further investigations as novel anti-inflammatory drug.
4. Experimental section
2.2.2. Cytotoxicity measurements
To determine possible negative influence on cell viability by the
most potent compounds of this series, we selected a subset of se-
ven derivatives with diverse scaffolds and wide variations of sub-
stituents (Table 2) and performed a WST-1 assay with U937 cells.
4.1. Compounds and chemistry
1H NMR and 13C NMR spectra were recorded on a Bruker AV 250
(1H: 250 MHz; 13C: 63 MHz), a Bruker AV 300 (1H: 300 MHz; 13C:

3578
S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
Table 1
Inhibition of 5-LO and 5-LO product formation by synthesized compoundsa
5-LO product formation IC50
(
lM)
Residual activity at 10 lM (%)
N°
R1
R2
S100
PMNL
S100
PMNL
—
1a
1b
2a
2b
2c
2d
2e
2f
0.21 0.02
0.28 0.04
0.24 0.08
0.50 0.13
0.18 0.05
0.20 0.02
0.18 0.04
0.51 0.08
0.44 0.04
28.21 4.86
0.15 0.01
1.84 0.63
0.26 0.04
20.9 2.0
0.09 0.03
0.21 0.02
0.09 0.01
0.55 0.13
0.10 0.005
0.18 0.06
0.15 0.02
4.66 0.74
0.14 0.02
0.76 0.14
11.05 4.71
30.1 3.9
27.6 3.8
—
18.5 3.8b
17.0 1.7
—
21.3 6.3
—
—
—
—
—
—
—
2g
2h
2i
28.6 7.4
16.2 2.4
20.1 1.3
52.0 7.8
61.8 4.3
—
25.8 5.6
14.8 1.8
2j

S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
3579
Table 1 (continued)
N°
5-LO product formation IC50
(lM)
Residual activity at 10 lM (%)
R1
R2
S100
PMNL
S100
PMNL
—
2kc
0.08 0.03
0.12 0.02
0.78 0.18
14.7 2.8
2l
0.08 0.01
0.08 0.01
—
—
—
—
3a
0.18 0.03
3b
0.09 0.01
0.32 0.08
—
—
3c
0.08 0.02
0.17 0.03
0.41 0.06
—
—
3d
0.08 0.003
12.3 1.7
26.6 3.1
3e
3f
0.07 0.009
0.13 0.02
2.14 0.84
1.24 0.61
0.64 0.10
>30
15.7 1.3
38.8 2.9
—
—
3g
43.3 1.7
60.8 3.8
3h
3i
4.31 1.24
5.84 0.62
0.57 0.14
0.90 0.14
3.55 1.62
0.67 0.12
46.8 1.4
38.3 5.0
21.4 1.1
28.9 7.8
43.3 3.7
20.5 4.8
3j
(continued on next page)

3580
S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
Table 1 (continued)
5-LO product formation IC50
(
lM)
Residual activity at 10 lM (%)
N°
R1
R2
S100
PMNL
S100
—
PMNL
4a
4b
0.13 0.04
0.52 0.09
0.65 0.34
26.9 0.9
0.09 0.02
0.14 0.01
0.06 0.003
0.17 0.03
0.79 0.42
14.5 1.5
14.6 2.4
—
29.4 2.3
42.3 2.2
23.7 5.2
37.4 2.2
45.8 3.3
4c
4d
4e
4f
2.15 0.28
0.48 0.15
1.99 0.50
5.78 2.12
15.3 0.5
31.5 3.7
ST1130d
ST1131d
4.00
0.63
0.32
1.93
38.8 2.4
21.4 3.4b
37.2 4.2b
72.9 3.5b
5a
2.08 0.44
1.02 0.08
12.4 2.1
—
5b
6.26 1.39
3.34 0.37
0.14 0.06
44.2 7.2
14.4 5.1
O
O
BWA4C
0.05 0.005
-
—
N
CH3
OH
a
b
c
Measured in cell-free S100 and intact PMNL, given as IC50
(lM) and residual 5-LO activity [percentage of control] as mean SE. ‘—’: residual activity <10%.
Residual activity at 1 M.
l
Laboratory code number is ST1237.
d
Recently published.16

S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
3581
Table 2
Cytotoxicity determination of selected compounds as IC50 for inhibition of cell viability in a WST-1 assay and
LDH release as percentage of the cytotoxic control Rev-5901a
N°
WST-1 assay IC50
(l
M)
LDH release at 30 lM (%)
1a
>30
>30
>30
21.06 0.90
>30
15.8 8.5
8.4 1.6
4.1 2.4
48.9 6.5
12.0 4.2
12.2 2.7
106.0 11.1
2.7 2.8
2h
2kb
3a
3c
3f
5a
C06
BWA4C
>30
12.10 1.66
>30c
>30
0.8 0.8
a
b
c
Given as IC50 (lM) and LDH release [percentage of cytotoxic control] as mean SE.
Laboratory code number is ST1237.
Recently published.17
75 MHz), or a Bruker AV 400 (1H: 400 MHz; 13C: 100 MHz) spec-
trometer (Bruker, Germany). Chemical shifts (d) are reported in
ppm relative to tetramethylsilane (TMS) as internal reference;
multiplicity: s, singlet; d, doublet; dd, double doublet; t, triplet;
m, multiplet; approximate coupling constants (J) are shown in
hertz (Hz); assignment: ada, adamantyl; ind, indol; naphth, naph-
thyl; ph, phenyl; thiaz, thiazol; ph, phenyl, where position 1 is the
substituent. Mass spectra were obtained on a VG Platform II (Fi-
sons Instruments, Great Britain) using electrospray ionization
(ESI). Data are listed as mass number ([M H+]) and relative inten-
sity (%). High resolution MS (HR-MS) were achieved on a LTQ Orbi-
trap XL (Thermo Fisher Scientific, USA). For final compounds the
analyses were within 5 ppm of the theoretical values. Elemental
analyses (C, H, N, S) were measured on a Vario MicroCube (Herae-
us, Germany) and were within 0.4% of the theoretical values for
all final compounds. Educts and all other reactants were commer-
cially obtained from Sigma–Aldrich, ABCR, Alfa Aesar, and Acros
Organics and were used without further purification. Analytical
TLC (thin layer chromatography) was performed with TLC plates
–CH@, naphth-3,5,8H), 7.53 (m, 4H, H3CO-ph-3,5H, naphth-6,7H),
6.94 (d, J = 8,8, 2H, CH3O-ph-2,6H), 3.81 (s, 3H, CH3O); 13C NMR
(63 MHz, CDCl3) d (ppm): 186.35 (thiaz-4C), 183.50 (thiaz-2C),
162.15 (CH3O-ph-1C), 138.47 (thiaz-5C), 136.67 (naphth-4C),
132.87 (CH3O-ph-3,5C), 132.66 (naphth-9C), 130.56 (–CH@),
129.73 (naphth-1C), 129.35 (naphth-3C), 129.08 (naphth-5C),
128.03 (naphth-8C), 127.38 (CH3O-ph-4C), 126.60 (naphth-2C),
124.14 (naphth-6,7C), 123.79 (naphth-10C), 115.00 (CH3O-ph-
2,6C), 55.61 (CH3O); C21H15NO2S, Mr = 345.4, ESI-MS (m/z): 346.3
[M+H+]; Anal. calcd: C (73.02%), H (4.38%), N (4.06%), S (9.28%);
found: C (73.14%), H (4.43%), N (3.96%), S (9.49%).
4.1.2. Preparation of (Z)-5-(1-(4-Methoxyphenyl)ethylidene)-2-
(p-tolyl)-5H-thiazol-4-one (5a)
0.52 g (5.64 mmol) 2-sulfanylacetic acid and 0.66 g (1.0 equiv)
p-tolunitrile were refluxed 1 h with 20 ml ethanol and 2.0 ml
(2.5 equiv) triethylamine. The reaction mixture was evaporated
under reduced pressure and the resulting solid was washed with
acetone and isopropanol.18 75 mg (0.39 mmol) of this intermediate
were heated 45 min with 177 mg (3.0 equiv) p-methoxyacetophe-
none and 91 mg (3.0 equiv) ammonium acetate in 3 mL absolute
toluene in the microwave at 130 °C.20 The reaction mixture was
evaporated under reduced pressure and the crude product was
purified by column chromatography eluting with hexane/ethyl
acetate (5:1). Yield: 56%; ocher solid; 1H NMR (250 MHz, DMSO-
d6) d (ppm): 7.96 (d, J = 8.3, 2H, CH3-ph-3,5H), 7.58 (d, J = 9.0, 2H,
CH3O-ph-3,5H), 7.42 (d, J = 8.0, 2H, CH3-ph-2,6H), 7.09 (d, J = 8.8,
2H, CH3O-ph-2,6H), 3.83 (s, 3H, CH3O), 2.78 (s, 3H, CH3); 2.41 (s,
3H, CH3-ph); 13C NMR (63 MHz, DMSO-d6) d (ppm): 184.88 (thi-
az-4C), 180.10 (thiaz-2C), 160.38 (CH3O-ph-1C), 155.43 (CH3-ph-
1C), 145.86 (CH3-C), 135.67 (thiaz-5C), 130.04 (CH3O-ph-3,5C),
128.99 (CH3-ph-3,5C), 128.82 (CH3-ph-4C), 127.89 (CH3-ph-2,6C),
126.10 (CH3O-ph-4C), 114.25 (CH3O-ph-2,6C), 55.40 (CH3O),
21.35 (CH3-ph), 21.01 (CH3-C); C19H17NO2S Â 0.25 H2O,
Mr = 323.4, ESI-MS (m/z): 324.5 [M+H+]; Anal. calcd: C (69.59%),
H (5.38%), N (4.27%), S (9.78%); found: C (69.70%), H (5.41%), N
(4.13%), S (9.55%).
F
254 (Merck, Germany) with detection using a UV-lamp. Flash chro-
matography separations were obtained on an IntelliFlash 310 Flash
Chromatography Workstation with Column Station (Agilent, USA).
The microwave syntheses were performed on a Biotage Initiator
2.0, 400 Watt (Biotage, Sweden).
4.1.1. General Procedure for preparation of compounds 1a–b,
2a-l, 3a–j, and 4a–f
A
solution of the corresponding aromatic aldehyde (0.8–
10.3 mmol), 2-sulfanylacetic acid (1.0–1.3 equiv) and the corre-
sponding aromatic nitrile (1.0–1.3 equiv) and triethylamine (1.5–
15.0 equiv) in methanol was refluxed over night. The reaction mix-
ture was evaporated under reduced pressure and the crude prod-
uct was recrystallized from ethanol and washed with acetone.18
The synthetic procedures are described representatively for 2k
for the one-pot reaction and in details for the two-step synthesis
for the methyl-branched derivatives 5a and 5b. Detailed synthesis
and analytical data of all synthesized compounds are provided in
the Supplementary data. For the Knoevenagel condensation, only
the Z-isomer was obtained.16
4.1.3. Preparation of (Z)-2-(4-Aminophenyl)-5-(1-(4-
methoxyphenyl)ethylidene)-5H-thiazol-4-one (5b)
4.1.1.1. (Z)-5-(4-Methoxybenzylidene)-2-(naphthalen-2-yl)-5H-
thiazol-4-one (2k, ST1237). 0.53 g (3.92 mmol) p-methoxybenz-
aldehyde and 0.36 g (1.0 equiv) 2-sulfanylacetic acid and 0.60 g
(1.0 equiv) 2-naphthonitrile were refluxed over night with 20 ml
methanol and 2.0 ml (14.2 equiv) triethylamine. The reaction mix-
ture was evaporated under reduced pressure and the pure product
was recrystallized from ethanol and washed with acetone. Yield:
39%; orange solid; 1H NMR (250 MHz, CDCl3) d (ppm): 8.65 (s,
1H, naphth-10H), 8.12 (dd, J = 8.6, 1H, naphth-2H), 7.86 (m, 4H,
3.68 g (40.00 mmol) 2-sulfanylacetic acid and 4.73 g (1.0 equiv)
p-aminobenzonitrile were refluxed one hour with 90 ml ethanol
and 9.0 ml (1.6 equiv) triethylamine. The reaction mixture was
evaporated under reduced pressure and the resulting solid was
washed with acetone and isopropanol.18 0.40 g (2.08 mmol) of this
intermediate were heated 90 min with 0.24 g (0.8 equiv) p-
methoxyacetophenone and 0.19 g (1.2 equiv) ammonium acetate
in 10.0 mL absolute toluene in the microwave at 130 °C.20 The
resulting precipitate was washed with methanol and purified with

3582
S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
a flash chromatography and dichloromethane/methanol as eluent.
Yield: 21%; red-brown solid; 1H NMR (300 MHz, CDCl3) d (ppm):
7.95 (d, J = 8.7, 2H, NH2-ph-3,5H), 7.47 (d, J = 8.9, 2H, CH3O-ph-
3,5H), 7.00 (d, J = 8.8, 2H, CH3O-ph-2,6H), 6.70 (d, J = 8.7, 2H,
NH2-ph-2,6H), 3.92 (s, 3H, CH3O), 2.88 (s, 3H, CH3); 13C NMR
(75 MHz, CDCl3) d (ppm): 184.67 (thiaz-4C), 181.60 (thiaz-2C),
160.38 (CH3O-ph-1C), 153.15 (NH2-ph-1C), 152.67 (CH3-C),
136.71 (thiaz-5C), 130.87 (NH2-ph-3,5C), 128.84 (CH3O-ph-3,5C),
127.38 (CH3O-ph-4C), 121.86 (NH2-ph-4C), 114.15 (CH3O-ph-
2,6C), 114.06 (NH2-ph-2,6C), 55.66 (CH3O), 21.57 (CH3-C);
4.3. Cytotoxicity measurements
4.3.1. Cell culture
The human leukemic monocyte cells U937 were maintained in
RPMI 1660 medium containing 10% FCS, 100 lg/mL streptomycin
and 100 U/mL penicillin. Cells were cultured at 37 °C in an atmo-
sphere containing 5% CO2.
4.3.2. In vitro cell viability assay
The WST-1 assay (Roche Diagnostic GmbH, Mannheim,
Germany) was used to determine cell viability after treatment with
test compounds. U937 cells were seeded in 96-well plates at a den-
sity of 104 cells/well and treated with increasing concentrations of
test compounds for 48 h in presence of 10% FCS. Cell viability was
assessed according to the distributor’s protocol using a microplate
reader (infinite M200, Tecan Group Ltd, Crailsheim, Germany). All
experiments were performed at least three times and the
mean SE were calculated.
C
18H16N2O2S, Mr = 324.4, ESI-MS (m/z): 325.0 [M+H+]; HR-MS
calcd: 325.10053; found: 325.10128 (À2.307 ppm).
4.2. 5-LO activity assay
4.2.1. Cell preparation
Human PMNL were freshly isolated from leukocyte concen-
trates obtained at Städtische Kliniken Frankfurt Höchst (Frankfurt,
Germany). In brief, venous blood was taken from healthy adult do-
nors with informed consent. Leukocyte concentrates were pre-
pared by centrifugation at 4,000g for 20 min at rt. PMNLs were
immediately isolated by dextran sedimentation, centrifugation on
Nycoprep cushions (PAA Laboratories, Linz, Austria), and hypotonic
lysis of erythrocytes as described.24 Cells were finally resuspended
in phosphate-buffered saline, pH 7.4 (PBS) containing 1 mg/mL
glucose (purity of >96–97%).
4.3.3. Lactate dehydrogenase (LDH) cytotoxicity assay
The LDH assay (cytotoxicity detection 1 kit; Roche Diagnostics
GmbH, Roche Applied Science, Mannheim, Germany) was used to
determine cell death after treatment of U937 cells with test com-
pounds. LDH leakage was measured as an index of loss of cell
membrane integrity. U937 cells were seeded in 96-well plates at
a density of 1.5 Â 104 cells/well and incubated with increasing con-
centrations of test compounds or vehicle (DMSO) for 48 h. Plates
were centrifuged (250Âg, 4 min) and an aliquot of the supernatant
was transferred to a clean microplate. Cytotoxicity was assessed
according to the distributor’s protocol using a microplate reader
(infinite M200, Tecan Group Ltd, Crailsheim, Germany). A control
detergent supplied by Sigma–Aldrich (Saint Louis, Mo, USA) was
used for maximum LDH release and set to 100%. Rev-590122
4.2.2. Determination of 5-LO product formation in intact cells
For whole-cell assay freshly isolated PMNL (5 Â 106) were
resuspended in 1 mL PBS, pH 7.4, containing 1 mg/mL glucose
and 1 mM CaCl2. After preincubation with the test compounds
for 15 min at 37 °C, 5-LO product formation was stimulated by
the addition of calcium ionophore A23187 (2.5
lM) and exogenous
(100 lM), a non-redox-type 5-LO inhibitor, was used as a cyto-
AA (20 M). Exogenous arachidonic acid was applied to overcome
l
toxic23 control and was set to 100%. All experiments were tested
three times and the mean SE were calculated.
any limitation of supply of endogenous substrate, mediated by
cPLA2. After 10 min at 37 °C, the reaction was stopped with the
addition of methanol (1 mL). HCl (30
lL, 1 N), prostaglandin B1
Acknowledgements
(200 ng) and PBS (500 L) were added and the 5-LO metabolites
l
were extracted and analyzed by HPLC as described in the litera-
ture.25 5-LO product formation was determined as nanograms of
5-LO products per 106 cells, which includes leukotriene B4 (LTB4)
and its all-trans isomers, and 5-H(P)ETE (5(S)-hydro(pero)xy-6-
trans-8,11,14-cis-eicosatetraenoic acid). Cysteinyl LTs C4, D4 and
E4 were not detected, and oxidation products of LTB4 were not
determined. Each compound was tested at least three times, and
the mean SE were calculated.
The study was supported by the EU (Grant LSHM-CT-2004-
0050333), the LOEWE Lipid Signaling Forschungszentrum Frank-
furt (LiFF), and LOEWE OSF. We thank Astrid Brüggerhoff for expert
technical assistance.
Supplementary data
Supplementary data associated with this article can be found, in
the online version, at http://dx.doi.org/10.1016/j.bmc.2012.04.003.
4.2.3. Determination of 5-LO product formation in cell-free
systems
For determination of the activity of 5-LO in S100 freshly isolated
PMNL cells were resuspended in 1 mL of PBS containing 1 mM
EDTA and the protease inhibitors soybean trypsin inhibitor
References and notes
1. Dahlén, S. E.; Hedqvist, P.; Hammarström, S.; Samuelsson, B. Nature 1980, 288,
484.
2. Peters-Golden, M.; Henderson, W. R., Jr. N. Engl. J. Med. 2007, 357, 1841.
3. Werz, O.; Steinhilber, D. Pharmacol. Ther. 2006, 112, 701.
4. Samuelsson, B.; Dahlén, S. E.; Lindgren, J. A.; Rouzer, C. A.; Serhan, C. N. Science
1987, 237, 1171.
5. Miller, D. K.; Gillard, J. W.; Vickers, P. J.; Sadowski, S.; Leveille, C.; Mancini, J. A.;
Charleson, P.; Dixon, R. A.; Ford-Hutchinson, A. W.; Fortin, R.; Gauthier, J. Y.;
Rodkey, J.; Rosen, R.; Rouzer, C.; Sigal, I. S.; Strader, C. D.; Evans, J. F. Nature
1990, 343, 278.
6. Woods, J. W.; Evans, J. F.; Ethier, D.; Scott, S.; Vickers, P. J.; Hearn, L.; Heibein, J.
A.; Charleson, S.; Singer, I. I. J. Exp. Med. 1993, 178, 1935.
7. Rådmark, O.; Werz, O.; Steinhilber, D.; Samuelsson, B. Trends Biochem. Sci. 2007,
32, 332.
(60
(10
l
g/mL), 1 mM phenylmethylsulfonyl fluoride, and leupeptine
lg/mL), cooled on ice for 10 min, and sonicated (3 Â 10 s) at
4 °C. The whole homogenate was then centrifuged (100,000g for
70 min at 4 °C) to obtain the S100. For determination of 5-LO activ-
ity, S100 corresponding to 7.5 Â 106 PMNL was added to 1 mL of a
5-LO reaction mix (PBS, pH 7.4, 1 mM EDTA, and 1 mM ATP). After
preincubation with the test compounds or vehicle (DMSO) for
15 min at 4 °C, the samples were prewarmed for 30 s at 37 °C,
and 20 lM AA and 2 mM CaCl2 were added to start the 5-LO reac-
tion. The reaction was stopped after 10 min at 37 °C by addition of
1 mL ice-cold methanol and the formed metabolites were analyzed
by HPLC as described for intact cells. Each compound was tested at
least three times, and the mean SE were calculated.
8. Ford-Hutchinson, A. W.; Bray, M. A.; Doig, M. V.; Shipley, M. E.; Smith, M. J.
Nature 1980, 286, 264.
9. Chen, Y.; Hu, Y.; Zhang, H.; Peng, C.; Li, S. Nat. Genet. 2009, 41, 783.
10. Carter, G. W.; Young, P. R.; Albert, D. H.; Bouska, J.; Dyer, R.; Bell, R. L.;
Summers, J. B.; Brooks, D. W. J. Pharmacol. Exp. Ther. 1991, 256, 929.

S. Barzen et al. / Bioorg. Med. Chem. 20 (2012) 3575–3583
3583
11. McMillan, R. M.; Walker, E. R. Trends Pharmacol. Sci. 1992, 13, 323. 18. Zayed, E. M.; Elbannany, A. A. A.; Ghozlan, S. A. S. Pharmazie 1985, 40, 194.
12. Steinhilber, D. Curr. Med. Chem. 1999, 6, 71.
19. Tietze, L. F.; Modi, A. Med. Res. Rev. 2000, 20, 304.
13. Nasser, S. M.; Bell, G. S.; Hawksworth, R. J.; Spruce, K. E.; MacMillan, R.;
Williams, A. J.; Lee, T. H.; Arm, J. P. Thorax 1994, 49, 743.
14. Turner, C. R.; Smith, W. B.; Andresen, C. J.; Eggler, J. F.; Watson, J. W. Inflamm.
Res. 1996, 45, 42.
20. Unangst, P. C.; Connor, D. T.; Cetenko, W. A.; Sorenson, R. J.; Kostlan, C. R.;
Sircar, J. C.; Wright, C. D.; Schrier, D. J.; Dyer, R. D. J. Med. Chem. 1994, 37, 322.
21. Tateson, J. E.; Randall, R. W.; Reynolds, C. H.; Jackson, W. P.; Bhattacherjee, P.;
Salmon, J. A.; Garland, L. G. Br. J. Pharmacol. 1988, 94, 528.
22. Ding, X. Z.; Iversen, P.; Cluck, M. W.; Knezetic, J. A.; Adrian, T. E. Biochem.
Biophys. Res. Commun. 1999, 261, 218.
23. Fischer, A. S.; Metzner, J.; Steinbrink, S. D.; Ulrich, S.; Angioni, C.; Geisslinger,
G.; Steinhilber, D.; Maier, T. J. Br. J. Pharmacol. 2010, 161, 936.
24. Werz, O.; Bürkert, E.; Samuelsson, B.; Rådmark, O.; Steinhilber, D. Blood 2002,
99, 1044.
15. Hofmann, B.; Franke, L.; Proschak, E.; Tanrikulu, Y.; Schneider, P.; Steinhilber,
D.; Schneider, G. ChemMedChem 2008, 3, 1535.
ˇ
´
16. Hofmann, B.; Barzen, S.; Rödl, C. B.; Kiehl, A.; Borig, J.; Zivkovic, A.; Stark, H.;
Schneider, G.; Steinhilber, D. J. Med. Chem. 2011, 54, 1943.
17. Hofmann, B.; Rödl, C.; Kahnt, A.; Maier, T.; Michel, A.; Hoffmann, M.; Rau, O.;
ˇ
´
Awwad, K.; Pellowska, M.; Wurglics, M.; Wacker, M.; Zivkovic, A.; Fleming, I.;
Schubert-Zsilavecz, M.; Stark, H.; Schneider, G.; Steinhilber, D. Br. J. Pharmacol.
2012, 165, 2304.
25. Brungs, M.; Rådmark, O.; Samuelsson, B.; Steinhilber, D. Proc. Natl. Acad. Sci.
U.S.A. 1995, 92, 107.