Development and evaluation of ST-1829 based on 5-benzylidene-2-phenylthiazolones as promising agent for anti-leukotriene therapy

Article abstract of DOI:10.1016/j.ejmech.2014.10.054

Different inflammatory diseases and allergic reactions are mediated by leukotrienes, which arise from the oxygenation of arachidonic acid catalyzed by 5-lipoxygenase (5-LO). One promising approach for an effective anti-leukotriene therapy is the inhibition of this key enzyme. This study presents the synthesis and development of a potent and direct 5-LO inhibitor based on the well characterized 5-benzylidene-2-phenylthiazolone C06, whose further pharmacological investigation was precluded due to its low solubility. Through optimization of C06, evaluation of structure-activity relationships including profound assessment of the thiazolone core and consideration of the solubility, the 5-benzyl-2-phenyl-4-hydroxythiazoles represented by 46 (ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol) were developed. Compound 46 showed an improved 5-LO inhibitory activity in cell-based (ICinf50/inf values 0.141/4M) and cell-free assays (ICinf50/inf values 0.03 1/4M) as well as a prominent enhanced solubility. Furthermore, it kept its promising inhibitory potency in the presence of blood serum, excluding excessive binding to serum proteins. These facts combined with the non-cytotoxic profile mark a major step towards an effective anti-inflammatory therapy.

Full text of DOI:10.1016/j.ejmech.2014.10.054

Accepted Manuscript
Development and evaluation of ST-1829 based on 5-Benzylidene-2-
phenylthiazolones as promising agent for anti-leukotriene therapy
Andreas P. Lill, Carmen B. Rödl, Dieter Steinhilber, Holger Stark, Prof. Bettina
Hofmann, Dr.
PII:
S0223-5234(14)00982-9
10.1016/j.ejmech.2014.10.054
EJMECH 7462
DOI:
Reference:
To appear in: European Journal of Medicinal Chemistry
Received Date: 25 June 2014
Revised Date: 14 October 2014
Accepted Date: 17 October 2014
Please cite this article as: A.P. Lill, C.B. Rödl, D. Steinhilber, H. Stark, B. Hofmann, Development
and evaluation of ST-1829 based on 5-Benzylidene-2-phenylthiazolones as promising agent
for anti-leukotriene therapy, European Journal of Medicinal Chemistry (2014), doi: 10.1016/
j.ejmech.2014.10.054.
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ACCEPTED MANUSCRIPT

ACCEPTED MANUSCRIPT

5-LO and leukotrienes are associated with inflammatory diseases as asthma and
cancer.



Optimization of thiazolone-based C06 revealed soluble potent 5-LO inhibitors.
SAR evaluation identified essential 5-LO inhibitory elements of di-aryl-thiazoles.
Evolution towards 5-arylmethyl-2-aryl-4-hydroxythiazoles as highly potent
derivatives.

ST-1829 exhibits a promising pharmacological profile with enhanced solubility.

ACCEPTED MANUSCRIPT
Development and Evaluation of ST-1829 Based on
5
-Benzylidene-2-phenylthiazolones as Promising
Agent for Anti-leukotriene Therapy
#,‡
#,‡
#
#,§*
Andreas P. Lill , Carmen B. Rödl , Dieter Steinhilber , Holger Stark , Bettina
#
*
Hofmann
#
[
] Institute of Pharmaceutical Chemistry, Goethe-University Frankfurt, Max-von-Laue-Str. 9,
D-60438 Frankfurt am Main, Germany.
§
[
] Institute of Pharmaceutical and Medicinal Chemistry, Heinrich Heine University
Düsseldorf, Universitätsstr. 1, D-40225 Düsseldorf, Germany.
‡
These authors contributed equally to this work.
*
These authors contributed equally as senior authors.
*
Authors to whom correspondence should be addressed: Dr. Bettina Hofmann, Institute of
Pharmaceutical Chemistry, Goethe University Frankfurt, Max-von-Laue-Str. 9, D-60438
Frankfurt, Germany. Phone: ++49-69-798-29335; Fax: ++49-798-29323; e-mail:
hofmann@pharmchem.uni-frankfurt.de; Prof. Dr. Holger Stark, Institute of Pharmaceutical
and Medicinal Chemistry, Heinrich Heine University Düsseldorf, Universitätsstr. 1, D-40225
Düsseldorf, Germany. Phone: ++49-211-8110478; Fax: ++49-211-8113359; e-mail:
stark@hhu.de
1

ACCEPTED MANUSCRIPT
ABSTRACT
Different inflammatory diseases and allergic reactions are mediated by leukotrienes, which
arise from the oxygenation of arachidonic acid catalyzed by 5-lipoxygenase (5-LO). One
promising approach for an effective anti-leukotriene therapy is the inhibition of this key
enzyme. This study presents the synthesis and development of a potent and direct 5-LO
inhibitor based on the well characterized 5-benzylidene-2-phenylthiazolone C06, whose
further pharmacological investigation was precluded due to its low solubility. Through
optimization of C06, evaluation of structure-activity relationships including profound
assessment of the thiazolone core and consideration of the solubility, the 5-benzyl-2-phenyl-
4
-hydroxythiazoles represented by 46 (ST-1829, 5-(4-chlorobenzyl)-2-p-tolylthiazol-4-ol)
were developed. Compound 46 showed an improved 5-LO inhibitory activity in cell-based
IC values 0.14 µM) and cell-free assays (IC values 0.03 µM) as well as a prominent
(
5
0
50
enhanced solubility. Furthermore, it kept its promising inhibitory potency in the presence of
blood serum, excluding excessive binding to serum proteins. These facts combined with the
non-cytotoxic profile mark a major step towards an effective anti-inflammatory therapy.
2

ACCEPTED MANUSCRIPT
KEYWORDS
Anti-leukotriene therapy, Hydroxythiazoles, Inflammation, 5-Lipoxygenase, Solubility,
Structure-activity relationship
ABBREVIATIONS USED
aa: amino acids; AA: arachidonic acid; abs: absolute; AIBN: azobisisobutyronitrile; aq.:
aqueous; conc: concentrated; cPLA2: cytosolic phospholipase A2; DBU: 1,8-
diazabicyclo[5.4.0]undec-7-ene; DIPEA: N,N-diisopropylethylamine; DME: dimethoxy
ethane; DMF: dimethyl formamide; DMSO: dimethyl sulfoxide; FCS: fetal calf serum;
FLAP: 5-LO-activating protein; 5-HPETE: 5(S)-hydroperoxy-6-trans-8,11,14-cis-
eicosatetraenoic acid; LDH: lactate dehydrogenase; 5-LO: 5-lipoxygenase; LOOH:
hydroperoxide; LT: leukotriene; NBS: N-Bromosuccinimide; PBS: phosphate-buffered saline;
PGC: PBS buffer containing 1 mg/mL glucose and 1 mM CaCl ; PMNL: polymorphonuclear
2
leukocytes; py: pyridine; RT: room temperature; S100: 100,000g supernatant; SAR: structure-
activity relationships; µw: microwave.
3

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1
. INTRODUCTION
5
-Lipoxygenase (5-LO) is one of six known lipoxygenases in the human body, playing a
key role in leukotriene (LT) biosynthesis, which are small lipid mediators in immunity and
inflammation. [1] Recently, the crystal structure of human 5-LO was solved in 2.4 angstrom
resolution. [2] The enzyme can structurally be separated in two distinct domains: the N-
terminal regulatory C2-like domain (~120 amino acids (aa)) and the large C-terminal catalytic
domain (~553aa) with the active site containing a non-heme iron. Inactive, the iron has the
2
+
3+
ferrous state (Fe ) and the enzyme is not able to start the catalytic cycle. Oxidation to Fe by
lipid hydroperoxides (LOOH) activates the enzyme. [3] After activation, 5-LO translocates to
the nuclear membrane where the cytosolic phospholipase A (cPLA ) releases arachidonic
2
2
acid (AA) out of membrane phospholipids. AA is transferred to 5-LO supported by 5-
lipoxygenase activating protein (FLAP). [4] 5-LO first oxidizes AA at carbon 5 resulting in
5
(S)-hydroperoxy-6-trans-8,11,14-cis-eicosatetraenoic acid (5-HPETE); further dehydration
leads to the instable epoxide LTA . [5,6] LTA is further hydrolysed into LTB , a chemotactic
4
4
4
and chemokinetic agent activating phagocytosis. [7] Moreover, LTA is conjugated to the
4
cysteinyl-containing LTs (LTC , -D and -E ) causing bronchoconstriction and vascular
4
4
4
permeability. [8] LTs with their pro-inflammatory properties are linked to several chronic
diseases like bronchial asthma, atherosclerosis, cardiovascular diseases, rheumatoid arthritis
[
9] and different types of cancer. [1,10,11]
Concerning the multiple pathophysiological properties of LTs, it is important to develop an
anti-leukotriene therapy without harmful drawbacks. Most drug candidates lack selectivity
and lead to severe side effects or lose their activity under chronic inflammatory conditions, so
that to date only one direct 5-LO inhibitor, zileuton, [12] was able to enter the market for the
treatment of asthma. This fact encouraged us to further investigate a substance class we
identified by virtual screening previously. [13] One of the most promising compounds in this
class was C06 (Fig. 1), which was molecular pharmacologically characterized. [14] It showed
4

ACCEPTED MANUSCRIPT
a selective, direct and non-cytotoxic 5-LO inhibitory mode of action with an IC value of
5
0
0
.66 µM for inhibition of 5-LO product formation in intact cells (polymorphonuclear
leukocytes (PMNL)).
Supported by the promising characteristics of this compound class, we further investigated
this series. Within the following structure activity relationship (SAR) studies [15,16] we have
demonstrated that different ethers, amides, halides and larger, lipophilic systems are generally
well tolerated by 5-LO, although the variations were only applied to position 2 of the
thiazolone ring (eastern part) of C06. The most potent lead structures in this series are shown
in Figure 1 (IC values in a cell-free assay (S100) of 0.08 µM for both compounds and 0.28
5
0
µM and 0.12 µM in intact PMNL, respectively). [15,16] This SAR confirms large, lipophilic
moieties on position 2 of the thiazolone ring as beneficial for 5-LO inhibition. However, no
data about the relevance of the thiazolone core is available so far. Furthermore, beside the
auspicious activity of C06 and the previous hits, they only possess a low solubility in aqueous
buffer and DMSO (cp. Figure 1). The poor solubility and a lack of activity in presence of
blood serum have hampered their further development and in vivo studies of C06 [14].
Therefore, we aimed at optimizing this compound towards an effective, more soluble, non-
cytotoxic 5-LO inhibitor together with completing our knowledge of the SAR of this
substance class by synthesizing compounds with different polar structures, morpholine
containing derivatives and heteroaromatic systems as well as variations of the thiazolone ring
(Figure 1).
[
FIG1]
2
. RESULTS AND DISCUSSION
.1 Chemistry
2
5

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2
.1.1 Synthesis of the 5-arylidene-2-arylthiazol-4(5H)-ones: Most of the thiazolone-
based final compounds were synthesized as outlined in Scheme 1 either in a three component
one-pot-reaction (marked with * in Tables 1 and 4) or by a related two step procedure
(marked with ** in Tables 1, 2, 4 and 6). These known synthetic routes [15,16,17] were
improved by applying complete microwave conducted synthesis that resulted in shorter
reaction times, less side product formation and therefore easier isolation of the products.
Some aldehydes for the aldol condensation with 2-p-tolylthiazol-4(5H)-one, 3, in the two step
procedure had to be synthesized as shown in the Supporting Information (marked with # in
Tables 1 and 6).
[
SC01]
Scheme 2 shows the synthesis of the ylidene substituted derivatives 4 and 5. The
appropriate ketone was condensed with 3 under ammonium acetate catalysis [18] in toluene
using microwave irradiation.
[
SC02]
2
.1.2 Synthesis of the none-thiazol-4(5H)-one compounds: The none-thiazol-4(5H)-one
compounds were prepared by distinct synthetic routes. For the synthesis of the furan analog 8
the corresponding γ-keto acid 6 was cyclized [19] to 7 with acetyl chloride and 7 was further
condensed with 4-methoxybenzaldehyde at 0 °C (8). By treatment of 8 with ammonia [20]
compound 9 could be obtained in good yields (Scheme 3).
[
SC03]
The oxazol-5-one- and imidazol-4-one 12 and 13 were prepared from 4-methylbenzoic acid
1
0, which was amidated with glycine. [21] Intermediate 11 was in situ cyclized and
condensed with 4-methoxybenzaldehyde to give 12. [22] The oxazol-5-one was converted
into the imidazol-4-one 13 by treatment with aqueous ammonia (Scheme 4). [23]
6

ACCEPTED MANUSCRIPT
[
SC04]
For the synthesis of the thia to oxa exchange in C06 (20), 4-methylbenzoic acid, 10, was
converted into the amide 14 (Scheme 5). [24] Intermediate 14 was treated with chloroacetyl
chloride to give 15 that was further converted into the Wittig agent 16. [25] The Wittig-
Horner-reaction was conducted using DBU (1,8-diazabicyclo[5.4.0]undec-7-ene) as base to
yield 17 in (77%). [25] The double bond of 17 was then brominated to 18, which was
subsequently treated with triethylamine. The Z-isomer, 19a, could be separated from the E-
isomer by crystallization. Compound 19a was then cyclized to the oxazol-4(5H)-one 20 using
sodium hydride (adapted from Iyengar et al. [26]).
[
SC05]
Tolunitrile (2) was treated with ammonium sulfide under microwave conditions to give the
thioamide 21, [27] that was converted into the dione 22 with oxalyl chloride. [28,29]
Decarbonylation of 22 and subsequent addition of 4-methoxyisocyanate gave the imino
derivative (23) of C06 in good yield. [28,29] Compound 24 was obtained by addition of
methanol to the imine moiety of 23 (Scheme 6).
[
SC06]
For the synthesis of compound 28, which is an equilibrium of its amino and imino tautomer,
rhodanine (25), was condensed to the 2-thioxothiazolidin-4-one 26 in high yield (adapted
from Abell et al. [30]). The thioxo structure of 26 was then converted into the corresponding
thioether 27, which was aminated with p-toluidine under microwave irradiation to give 28
(
Scheme 7). [31]
SC07]
[
7

ACCEPTED MANUSCRIPT
The thioxothiazolidinone core was synthesized by cyclization of methyl 2-sulfanylacetic
acid (31) and the isothiocyanate 30. [32] 30 was obtained from p-toluidine hydrochloride,
2
9xHCl, and carbon disulfide catalyzed by cyanuric chloride (Scheme 8). [33] The C-H acidic
compound 32 was condensed with 4-methoxybenzaldehyd under microwave irradiation using
sodium acetate as a base to give 33. [32]
[
SC08]
4
-Methylbenzaldehyde (34) was cyclized with methylamine and methyl 2-sulfanylacetic
acid (31) to give the N-methylated thiazolidinone 35 (Scheme 9). For the following
condensation with 4-methoxybenzaldehyde under microwave irradiation sodium hydroxide
had to be used as a base to yield 36 – under the regular conditions with piperidine as a base no
conversion could be detected.
[
SC09]
2
.1.3 Synthesis of the thiazole derivatives: The (cyclohexylidene)methyl-
hydroxythiazoles have also been prepared by the two step procedure involving aldol
condensation. As the aliphatic aldehydes 37 and 38 (for synthesis of 38 see Supporting
Information) possess acidic protons, the intermediates dehydrate to the aromatic, more stable
4
-hydroxythiazoles 39 and 40 (Scheme 10).
[
SC10]
Since the direct synthesis of benzylhydroxythiazoles by reduction of the double bond in
benzylidenethiazolones (e.g. transition metal catalyzed hydrogenation) is not possible, the
ring closure to 45 and 46 was performed with the thiobenzamides 21 and 44, respectively, and
an α-bromomethylester 43 as generally described by Metzger. [34] The bromomethyl ester 43
was obtained by Meerwein arylation of the diazonium bromide of 41 and methyl acrylate (42)
in hydrobromic acid (Scheme 11). [35]
8

ACCEPTED MANUSCRIPT
[
SC11]
The 5-aryl substituted 4-hydroxythiazoles 52-54 were prepared by microwave assisted
cyclization of 2-mercapto-2-(4-methoxyphenyl)acetic acid (51) and the appropriated aromatic
nitrile (modified from Metzger [34]). The mercapto acid 51 was prepared from the
corresponding acetic acid 47 that was esterified [36] to 48, brominated [37] (49), treated with
sodium thioacetic acid to give 50 and finally cleaved with potassium hydroxide (Scheme 12).
[
[
38]
SC12]
Hantzsch thiazole synthesis was applied to obtain the 4-methylthiazole 57 (Scheme 13).
Therefore, the α-bromoketone 56, which was obtained by bromination [39] of 55, was reacted
with 4-methylbenzothioamide (21) under microwave irradiation.
[
SC13]
The benzoic acid 63 (Table 1) was prepared by ester cleavage of 62 with acetic and
hydrochloric acid. The morpholino derivatives 67 and 69 (Table 2) were synthesized from the
phenol 58 by alkylation with N-(2-chloroethyl)morpholine and 2-chloro-1-morpholin-
ethanone, [40] respectively. For compound 79 methyl 2-sulfamylacetate was used instead of
the free acid. The methylpyridinium iodide 80 (Table 4) was obtained by treatment of the
corresponding pyridine 77 with methyl iodide.
2
.2 5-LO inhibitory activity: All final compounds were tested in a cell-free and a whole-
cell assay. The cell-based assay was performed with freshly isolated human
polymorphonuclear leukocytes (PMNL), whereas the cell-free assay, the S100 assay, was
conducted with the supernatant of a 100,000  g ultracentrifuge run of homogenized PMNL.
9

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The PMNL assay assesses intracellular bioavailability of the compounds whereas the cell-
free (S100) assay was performed to study direct inhibition of 5-LO. Comparing the inhibitory
potential of the compounds in both assay systems will point out, if the potent inhibition of 5-
LO product formation results from indirect interaction with upstream enzymes like FLAP or
cPLA (lower inhibitory activity in the cell-free assay) or a direct inhibition of 5-LO. As
2
control compound BWA4C was applied, a well-established direct 5-LO inhibitor. BWA4C
was able to inhibit 5-LO product formation to a residual activity of 58.36 ± 4.54% in intact
PMNL and to 45.80 ± 1.94% in cell-free S100 at a concentration of 80 nM, which confirms to
the literature. [16]
To complete our knowledge about the SAR for 5-LO inhibition by the 5-arylidene-2-aryl-
thiazolones and to improve the compounds solubility in DMSO as well as in aqueous buffer
for trouble-free screening conditions, we started with variations concerning the 5-position on
the thiazolone ring of C06. By synthesizing several polar compounds with different electronic
structures and H-bond-formation capabilities (Table 1) we increased the polarity and therefore
tried to improve the compounds solubility.
[
TAB1]
The hydroxy analog 58 with the additional H-bond donor as well as the two electron-
deficient derivatives 59 and 60 showed similar inhibitory potency as C06 resulting in IC50
values between 0.16 to 0.33 µM and 0.58 to 1.13 µM in S100 and PMNL, respectively.
Compared to 60 the additional double bound in 61 did not alter the inhibitory potency in S100
but showed only a weak inhibitory activity in PMNL which might be caused by cross-
reactivity of the Michael acceptor system. Compound 62, which will most likely be
deprotonated under assay conditions, showed a clearly decreased inhibitory potency in terms
of both assays. The more electron rich aniline derivative 63 with its two H-bond-donors had a
slightly higher IC value compared to C06. The two sulfonyl derivatives 64 and 65 resulted
5
0
1
0

ACCEPTED MANUSCRIPT
in similar inhibitory activities. Their IC values in S100 are comparable to those of C06,
5
0
however, the inhibitory potency in PMNL is decreased to IC values of 1.43 and 5.29 µM,
5
0
respectively. The poor inhibitory activity on 5-LO product formation in PMNL by compound
1 to 65 may be due to a decreased ability to cross the cell membrane and a poor
6
bioavailability in the cell.
To further study the inhibitory potential of better soluble compounds, we additionally
synthesized a series of morpholine containing derivatives (Table 2).
[
[
TAB2]
TAB3]
Since C06 is characterized by poor solubility in DMSO and aqueous buffer therewith
limiting further investigations, we analyzed the solubility of selected C06 derivatives (Table
). Surprisingly, the 4-morpholinophenyl-C06-derivative 66 showed a decreased solubility in
3
buffer and a three times lower 5-LO inhibitory potency under cell-free conditions than C06.
Compound 67, the ethoxy analog of 66, showed the same solubility as C06 without any
improvement in IC values despite its higher calculated logS-value (Supporting Information).
5
0
The N-(oxyacetyl)morpholine derivative 68 had an increased solubility in both DMSO and
aqueous buffer, but possessed a lower inhibitory potential of 5-LO product formation.
Compound 69 showed almost the same inhibitory potential as 58 and possessed a higher
solubility in buffer solution (cp. Table 3). Derivative 69 can be considered as a precursor of
5
8 and the partial hydrolysis of the carbamate might contribute to the related IC values.
50
Compound 64 showed an improved solubility in both DMSO and aqueous buffer, whereas the
clogS-values are comparable (Supporting Information). The inhibitory activity in S100 is
comparable to C06, and the activity in PMNL only slightly decreased to an IC of 1.44 µM.
5
0
However, no pronounced improvement of solubility combined with a superior inhibitory
potential could be achieved with the derivatives in Table 1 and 2. Interestingly, the clogS-
1
1

ACCEPTED MANUSCRIPT
values did not correlate with experimental solubility in DMSO and buffer. Again, the different
substituents of the tested compounds caused only slight changes in 5-LO inhibitory activity -
according to the continuous SAR observed in our previous studies. [15,16] To leave the
typical diphenyl scaffold of C06 and further explore the SAR, we synthesized a series of
heterocyclic-substituted derivatives to increase polarity along with solubility (Table 4).
[
TAB4]
The pyridine compounds 70 and 71, both being electron deficient and bearing a hydrogen-
bond acceptor, showed similar activities under cell-free conditions wherein the position of the
nitrogen only caused marginal changes. However, the IC50 values in intact cells were
considerably higher. The pyrrole and indole analogs 72 and 73, both having a hydrogen-bond
donor and no acceptor, only showed poor inhibitory activity in the S100 assay, but exhibit
moderate activity in intact PMNL. The increased activity in the whole-cell assay might be due
to indirect inhibition of other upstream enzymes like FLAP. Comparing 72 with 74, the
necessity of an H-bond acceptor for direct interaction with the 5-LO enzyme becomes
obvious: The thiophene bearing derivative 74 possessed an IC value of 0.86 µM whereas 72
5
0
with its heteroaromatic H-bond donor exhibited an IC value of 8.02 µM in the cell-free
5
0
assay. The inhibitory activity in intact cells resulted in comparable values for both
compounds. The inhibitory potential in S100 of the imidazole derivatives 75 and 76 was
found between those of 72 and 74, which is in line with the fact that both bear a hydrogen-
bond acceptor and donor that might flip to the required geometry, at least for 75. The
additional methyl group of 76 is unfavorable and resulted in a further decreased inhibitory
potency, possibly due to steric hindrance of the ´H-bond donor/acceptor rotation´.
Compounds 77 to 79, all bearing a nitrogen aromatic heterocycle on 2-position of the 5-
arylidene-2-aryl-thiazolone parent structure, exhibited inhibitory activities comparable to
1
2

ACCEPTED MANUSCRIPT
those of 70 and 71 in both assay systems. Under cell-free conditions, the nitrogen at 4-
position seemed to be favored, since the IC value doubled for 77 compared to 78.
5
0
The pyrazine 79 had a similar inhibitory activity as C06. The charged methylpyridium
compound 80 was inactive in the intact cell assay (most probably due to poor membrane
penetration) and showed only weak inhibitory activity in the cell-free system.
As seen with the polar aryl substituents (58-69), the aromatic heterocycles in the 5-
arylidene-2-aryl-thiazolones 70-80 also possessed a continuous SAR but did not show
improved solubility properties (data not shown). Since we did not succeed in optimizing the
aryl substituents on the thiazolone core of C06 towards improved solubility and effective
inhibition of 5-LO product formation, we subsequently investigated the importance of the
individual substructures of the 5-arylidene-2-aryl-thiazolone scaffold starting at the thiazolone
core itself. Therefore we synthesized a series of different heterocyclic five-membered core
derivatives (Table 5).
[
TAB5]
Replacement of the thiazolone by a furanone or an oxazol-5-one core structure (8, 12) while
keeping the 2- and 5-aryl substituents of the thiazolone ring of C06 constant led to a complete
loss of inhibitory activity of 5-LO product formation with IC values greater than 30 µM.
5
0
This might be caused by enzymatic instability of the lacton moiety against esterases or
hydrolases - though both derivatives are also inactive in a purified enzyme assays (data not
shown). However, the nitrogen derivatives of 8 and 12, the pyrrolone 9 and imidazolone 13,
showed a partly restored inhibitory activity. The H-bond-acceptor at 1-position of the core
ring (13) seemed to be necessary for 5-LO activity, since 9 lacking it, exhibits a ten times
higher IC value in PMNL assay. The replacement of sulfur in C06 by oxygen (20) caused a
5
0
decreased inhibitory activity in both assay systems. The oxazol-4-one 20 exhibited IC values
5
0
1
3

ACCEPTED MANUSCRIPT
of 1.86 µM and 10.51 µM in S100 and PMNL, respectively. Obviously, the larger sulfur atom
radius in the core ring and therefore a distinct geometry seems to be essential for a potent
inhibition of 5-LO product formation. To study the influence of more flexibility concerning
the ring at the ylidene moiety we replaced the benzylidene carbon by nitrogen (23). This
modification was not advantageous for the inhibitory potential which is reduced to IC values
5
0
about 10 µM. The activity is completely lost when the imine and therefore the whole aromatic
system along with the geometry is destroyed by addition of methanol (24). In 28, additional
nitrogen is inserted between the thiazolone ring and the 2-aryl residue. Beside the
conformational changes and flexibility that come with the equilibrium between the amine and
imine tautomeres, it resulted in a total loss of direct 5-LO inhibitory activity while retaining
moderate indirect, cellular inhibition (IC50 2.90 µM). The switch from a 2,5-substituted
thiazolone core to a 3,5-substituted 2-thioxothiazolidinone (33) led to a total loss of inhibitory
activity in terms of both assays, which could primarily be attributed to the changed
constitution. The N-methylated C06 derivative 36 which lacks the overall conjugation seemed
to be a potent indirect inhibitor of 5-LO product formation (IC of 0.94 µM in the whole-cell
5
0
assay). Besides the loss of overall aromaticity, the thiazole carbon-2 and the nitrogen in 36 are
3
sp hybridized. Therefore, the tolyl residue and the nitrogen lone pair have another orientation
that may be responsible for the loss of direct inhibition.
These core variations suggest, that the 2-arylthiazole structure is highly beneficial for a
potent inhibition of 5-LO activity. Hence, further compounds with variations of the arylidene
substructure were synthesized and evaluated for their 5-LO inhibitory potential (Table 6).
[
TAB6]
The space filling methyl- (4) or methoxyphenyl-group (5) at the ylidene position impaired
the binding to 5-LO resulting in decreased inhibitory potencies of 5-LO product formation
IC50 S100: 1.97 µM and 6.15 µM; PMNL: 1.04 µM and 8.55 µM, respectively). If the
(
1
4

ACCEPTED MANUSCRIPT
aromatic ring at the ylidene moiety is displaced by an additional double bond (81), the IC50
values were comparable to C06 in terms of both assays. To investigate, if the ylidene aromatic
ring is needed for 5-LO activity, it was replaced by an adamantyl moiety (82). The unaffected
inhibitory potential of these compounds showed the variability of this moiety. To prove this
observation, 39 with a cyclohexyl residue was synthesized. Due to the tertiary hydrogen in the
cyclohexyl ring, 39 exist as cyclohexylidenmethyl-4-hydroxythiazole. This novel structure
exhibited good inhibitory potential with IC values of 0.35 µM in S100 and 0.79 µM in the
5
0
PMNL assay. For comparability towards C06, the methoxy analog 40 was also synthesized.
This derivative showed a slight decreased inhibitory activity in terms of both assays.
Examination of the solubility of these two 4-hydroxythiazole derivatives revealed improved
solubility in DMSO and aqueous buffer (DMSO: 50 mM (both), aqueous buffer: 100 µM (39)
and 80 µM (40)).
C06 contains an - unsaturated carbonyl group and it can be questioned if this acts as a
Michael acceptor in this scaffold resulting in unspecific, reactive binding. Derivatives 39 and
4
0 lack this functionality but maintain their inhibitory potential, therewith contradicting this
hypothesis. Furthermore, for C06 the reduction of the arylidene double bond was not possible
even under optimized synthesis conditions; it also shows enhanced chemical stability to
different nucleophiles. In addition, C06 is characterized by a reversible (cp. Supporting
Information), highly selective, non-cytotoxic inhibitory mode of action with no unusual high
Hill slopes of the dose-response curves [14], all characteristics for a specific inhibitory
mechanism, not supporting the „frequent hitter‟ hypothesis for related classes of compounds.
To further clarify the role of the ylidene moiety as substructure of the 5-arylidene-2-aryl-
thiazolone and because of the great potential of the 4-hydroxythiazoles in terms of improved
1
5

ACCEPTED MANUSCRIPT
solubility along with retained potency, we synthesized and tested other hydroxythiazoles
(
Table 7.1).
TAB7.1]
[
The C06 analog with a reduced double bond and therefore a hydroxythiazole structure was
synthesized (45).Compound 45 exhibited a promising inhibitory potency with an IC value of
5
0
0
.20 µM in the S100 assay, and 0.72 µM in PMNL. The potency of 45 and the fact that the
arylidene structure shows great chemical stability against nucleophiles (reducing agents and
methyl lithium with or without lewis acid catalysis) further refutes the thesis, that the double
bond functions as a Michael acceptor (and that this functionality is needed for affinity).
To examine the relevance of the reduced double bond and to simplify the scaffold, we
deleted the benzylic methylene group in 52 which resulted only in a slight change in
inhibitory potency with IC50 values of 0.17 µM and 0.43 µM in S100 and PMNL,
respectively. This last SAR step led us to a compound closely related to a
diarylhydroxythiazole that has been described as 5-LO inhibitor before [41,42] (Fig. 2). 4-
HT_2 is the most similar derivative, among a class of different 4-hydroxythiazoles (4-HTs),
exhibiting an IC value of 0.5 µM in a 20,000g supernatant assay with RBL-1 (rat basophilic
5
0
leukemia) cells [41].
Exchange of the 4-hydroxy by a methyl group (57) merely inhibited 5-LO product
formation indirectly in intact cells, but showed no direct 5-LO inhibition.
[
FIG2]
One limiting factor of C06 was the poor solubility in DMSO and in aqueous buffer. With
compound 45 and 52 we were able to improve the solubility for both conditions (cp. Table
.2) while maintaining the 5-LO inhibitory potential. The 2,5-diarylthiazol-4-ole 52 is soluble
up to 50 µM in buffer and 50 mM in DMSO. Compound 45 with its rotatable bond shows an
7
1
6

ACCEPTED MANUSCRIPT
even higher solubility of 100 mM in DMSO and 80 µM in buffer, which might also contribute
to its increased solubility compared to C06 having a more rigid double bond.
With 45 and 52 as more soluble lead structures, we further examined the group of 2,5-
diarylthiazol-4-oles and 2-aryl-5-arylmethylthiazol-4-oles as 5-LO inhibitors. In our previous
work, we have experienced an enhancement in inhibitory activity by exchange of the
substituent on the aryl residue on 2-position of the thiazolone from p-methyl to p-chloro (cp.
Fig. 1) and from p-fluoro to p-chloro. [15,16] This might be attributed to a sigma hole
interaction [43] resulting in an improvement of the IC value from 0.55 µM (p-F) over 0.3
5
0
µM (p-CH ) to 0.08 µM (p-Cl) (cp. [15,16]). In line with this, the chloro derivatives 46 (ST-
3
1
829) and 53 (ST-1748) exhibited excellent inhibitory potentials with IC50 values in the
nanomolar concentration range (0.03 µM and 0.05 µM in the cell-free assay and 0.14 µM and
.01 µM in intact PMNL, respectively (Fig. 3A)). Again, they both keep the enhanced
solubility of their p-methyl analogs (cp. Table 7.2).
0
[
TAB7.2]
Compared to the known 4-hydroxythiazoles (Fig. 2) the substitution pattern presented
herein (p-chlorine at 2-position and p-methoxy at 5-position) improved the 5-LO inhibitory
activity about 10 – 50 fold (for compound 53 (ST-1748)) and 5 – 20 fold (46 (ST-1829)). The
known derivative 2,5-diphenyl-thiazol-4-ol (4-HT_1, Fig. 2) exhibits in vivo inhibitory
activity in a rat anaphylaxis model after i.p. administration [41]. However, a lack of oral
bioavailability possible due to low absorption and solubility was stated [41]. It can be
speculated whether the here presented substitution pattern in particular together with the
improved potency and the high solubility may confer oral bioavailability to the 4-
hydroxythiazole lead structure: The chloro substitution at the 2-phenyl ring might show
advantageous in terms of metabolic stability of the phenyl ring and the methoxy substitution
1
7

ACCEPTED MANUSCRIPT
at the 5-phenyl yet together with the flexible linker in compound 46 (ST-1829) is beneficial
for solubility. Further studies are needed to evaluate the pharmacokinetic profile of this class.
With regard to the optimization of C06, the 5-LO inhibitory potential was improved by
more than tenfold for compound 46 (ST-1829) and over sixfold for 53 (ST-1748) in the S100
assay. Furthermore, both optimized compounds proved to be selective with regard to the other
two main lipoxygenases, 12-LO and 15-LO1: 46 (ST-1829) inhibits 12-LO and 15-LO1
product formation with IC values > 10 µM (data not shown). As for C06 we have observed
50
a loss of activity in presence of blood serum (Fig. 3B) - what points among others to high
serum protein binding of C06. It is known, that serum albumin has high, tight binding affinity
to several established drugs [44,45] and these drugs lose inhibitory potency in the presence of
blood serum, therefore higher doses are needed. We further investigated the inhibitory
potency of 46 (ST-1829) and 53 (ST-1748) and incubated the compounds in the absence and
presence of 10% fetal calf serum (FCS) (Fig. 3B) to explore their inhibitory activity in
presence of serum. C06 showed a decreased inhibitory activity of 5-LO product formation if
the cells are incubated with 10% FCS, what discourages further in vivo studies next to its poor
solubility. Compound 46 (ST-1829) showed a decreased activity in the presence of 10% FCS
at a concentration of 1 µM but this effect is abolished at a concentration of 10 µM. At this
concentration C06 resulted in a sevenfold decrease of activity, however for compound 46
(
ST-1829) only a threefold loss of activity could be observed. The inhibitory potency of 53
ST-1748) is not affected by the presence of blood serum regardless of inhibitor
(
concentration.
FIG3]
[
2
.3 Cytotoxicity: To further characterize these promising candidates, we studied their
influence on cell viability of U937 cells, with two different assay systems. Impaired cell
viability measured with a WST-1 assay can be ascribed to a reduced number of viable cells
1
8

ACCEPTED MANUSCRIPT
caused by anti-proliferative as well as cytotoxic properties of test compounds. To distinguish
between these two effects, the loss of cell membrane integrity was additional measured in
terms of LDH leakage since an increased LDH release points to cytotoxic effects like
necrosis. For both assays, U937 cells were treated with 5-LO inhibitors or vehicle for 48 h up
to a concentration of 30 µM. For comparison, the only marked direct 5-LO inhibitor zileuton
(100 µM) exhibits a non-cytotoxic profile, whereas Rev-5901 (100 µM) served as cytotoxic
control. The promising non-cytotoxic profile from C06 [15,16] was not affected by the
different modifications leading to the new candidates 46 (ST-1829) and 53 (ST-1748) (Table
8
). Both lead structures showed no influence on cell viability in terms of both assays.
[
TAB8]
3
. CONCLUSIONS
With the diverse polar and heteroaromatic C06 analogs (Table 1,2,4), we further explored
and confirmed the continuous SAR of the 5-aryliden-2-aryl-thiazoles. The heteroaryl
derivatives pointed out that a hydrogen bond-acceptor on the 5-arylring is favorable for 5-LO
activity (cp. 72 with 74). However, most of the inhibitors with an improved aqueous solubility
exhibited a reduced 5-LO inhibitory potential in the whole-cell assay, e.g. 64. Therefore the
thiazolone core itself was investigated (Table 5). With the core variation series we were able
to prove the importance of the 2-arylthiazole structure for the 5-LO inhibitory potential. Slight
variations on the thiazolone core, such as the exchange of the sulfur atom, diminished the 5-
LO activity or led to inactive compounds. Further synthetic evaluation of the arylidene
substructure of the 2-arylthiazole (Table 6 and 7) showed that aliphatic residues are also well
tolerated and that corresponding hydroxythiazoles posses an increased solubility. This
optimization step led to the development of the benzyl-aryl- (45, 46 (ST-1829)) and aryl-aryl-
hydroxythiazoles (52, 53 (ST-1748)) that also lack the arylidene-thiazolone substructure
denominated as potential “frequent hitter” substructure in others compounds before. [46] With
1
9

ACCEPTED MANUSCRIPT
4
6 (ST-1829) and 53 (ST-1748), we developed two highly potent compounds with a
promising molecular pharmacological, non-cytotoxic profile. Both compounds showed an
excellent direct inhibitory potential of 5-LO product formation with IC values of 0.03 and
5
0
0
.14 µM (46) and 0.05 and 0.01 µM (53) in S100 and PMNL, respectively.
With the development of the novel benzyl-aryl-hydroxythiazole scaffold we were able to
improve the solubility in DMSO by tenfold compared to C06 (46 (ST-1829) 100 mM vs. 10
mM for C06) and almost doubled the solubility in aqueous buffer. Unlike C06, compound 46
(ST-1829) showed only a slightly decreased inhibitory potential at a concentration of 10 µM
in the presence of blood serum (10% FCS). This together with the greatly enhanced solubility
felicitates the investigation of this novel class of 5-LO inhibitors in in vivo inflammation
models. The development of these benzyl-aryl-hydroxythiazoles represented by ST-1829
together with its promising molecular pharmacological profile represents an important step
towards an effective anti-leukotriene therapy.
4
4
. EXPERIMENTAL SECTION
.1 Chemistry
General. All chemicals were purchased from Sigma-Aldrich, Alfa Aesar, and Acros
Organics were used without further purification. Analytical TLC (thin layer chromatography)
was performed with TLC plates F254 (Merck, Germany) with detection using a UV-lamp. A
1
Biotage Initiator 2.0 microwave reactor has been used. H-NMR were recorded on a Bruker
1
1
1
AV 250 ( H: 250 MHz), a Bruker AV 300 ( H: 300 MHz), or a Bruker AV 400 ( H: 400
1
3
13
MHz) spectrometer (Bruker, Germany). C-NMR were recorded on a Bruker AV 250 ( C:
1
3
13
6
3 MHz), a Bruker AV 300 ( C: 75 MHz), or a Bruker AV 400 ( C: 100 MHz) spectrometer
(Bruker, Germany). Chemical shifts (δ) are reported in ppm relative to tetramethylsilane
(TMS) as reference; multiplicity: s, singlet; d, doublet; dd, double doublet; t, triplet; m,
multiplet; pq, pseudoquartett; approximate coupling constants (J) are shown in hertz (Hz).
2
0

ACCEPTED MANUSCRIPT
Melting points were determined on a Büchi Melting Point M-560. Mass spectra were obtained
on a VG Platform II (Fisons Instruments, Great Britain) using electrospray ionization (ESI).
Data are listed as mass number ([M±H+]). High resolution MS (HRMS) were achieved on a
LTQ Orbitrap XL (Thermo Fisher Scientific, USA). Elemental analyses (C, H, N) were
measured on a Vario MicroCube (Heraeus, Germany) and were within ± 0.4% of the
#
theoretical values for all final compounds, which corresponds to ≥ 95% purity. For synthesis
of aldehydes used in two step procedure see Supporting Information.
4
.1.1 2-p-Tolylthiazol-4(5H)-one (3): To a solution of 843 g of p-tolunitrile, 2, (7.05
mmol, 1.0 eq) in 1.0 mL of triethylamine (7 mmol, 1.0 eq) were added 0.5 mL of 2-
mercaptoacetic acid, 1, (7.05 mmol, 1.0 eq) and the mixture was headed for 15 min to 100 °C
by microwave irradiation. The solvent was removed under reduced pressure, the residue was
dissolved in 2 mL of methanol and cooled at -5 °C for 3-4 days. The yellow precipitate was
collected and washed with methanol. The mother liquor was concentrated and after 7 days in
the refrigerator additional product can be obtained. This can be repeated several times to yield
1
4
56 mg (34%) of foliated yellow solid. Modification to [17]: H-NMR (400 MHz, DMSO-d ):
6
3
δ = 11.00 (s, 1H, -OH), 9.54 (s, 1H, -C=CH-), 7.66 (d, 2H, J = 8.2 Hz, 3H,5H-tolylketone),
3
3
7
2
.40 (d, 2H, J = 8.3 Hz, 2H,6H-tolyl
), 7.25 (d, 2H, J = 8.0 Hz, 3H,5H-zolylenol), 7.19 (d,
ketone
3
3
3
H, J = 8.0 Hz, 2H,6H-zolyl ), 3.78 (d, 1H, J = 15.3 Hz, -CH -), 3.65 (d, 1H, J = 15.3 Hz,
enol
2
+
-
CH -), 2.33 (s, 3H, -CH ), 2.31 (s, 3H, -CH ); ESI-MS: m/z = 191.6 [M+H]
2 3 3
4
4
.1.2 Condensation under ammonium acetate catalysis:
.1.2.1 (Z)-5-(1-(4-Methoxyphenyl) ethylidene)-2-p-tolylthiazol-4(5H)-one (4): A
suspension of 75 mg of 2-p-tolylthiazol-4(5H)-one, 3, (0.39 mmol, 1.0 eq), 177 mg of 4-
methoxyacetophenone (1.18 mmol, 3.0 eq) and 91 mg of ammonium acetate (1.18 mmol, 3.0
eq) in 3 mL of abs. toluene was heated for 45 min to 130 °C by microwave irradiation. The
solvent was removed under reduced pressure and the residue was purified by column
1
chromatography (hexane:ethyl acetate 5:1) to yield 71 mg (56%) of a ocher solid. H-NMR
2
1

ACCEPTED MANUSCRIPT
3
3
(
250 MHz, CDCl ): δ = 7.96 (d, J = 8.3 Hz, 2H, 3,5H-tolyl), 7.58 (d, J = 9.0 Hz, 2H, 3,5H-
3
3
3
PhOMe), 7.42 (d, J = 8.0 Hz, 2H, 2,6H-tolyl), 7.09 (d, J = 8.8 Hz, 2H, 2,6H-PhOMe), 3.83 (s,
1
3
3
1
2
H, -OCH ), 2.78 (s, 3H, -CH ), 2.41 (s, 3H, tolyl-CH ); C-NMR (63 MHz, CDCl ): δ =
3 3
3
3
84.9, 180.1, 160.4, 155.4, 145.9, 135.7, 130.0, 129.0, 128.8, 127.9, 126.1, 114.3, 55.4, 21.4,
+
1.01; mp = 161 °C; ESI-MS: m/z = 324.5 [M+H] ; MALDI-HRMS anal. calcd. for
+
+
C H NO S: m/z = 324.10528, [M+H] , found: m/z = 324.10556 [M+H] ; Anal. calcd. for
19
17
2
.
C H NO S 0.25H O: C (69.59), H (5.38), N (4.37), S (9.78); found: C (69.70), H (5.41), N
19
17
2
2
(4.13), S (9.55).
4
.1.2.2 5-(bis(4-Methoxyphenyl)methylene)-2-p-tolylthiazol-4(5H)-one
(5):
A
suspension of 105 mg of 2-p-tolylthiazol-4(5H)-one, 3, (0.55 mmol, 1.0 eq), 274 mg of bis(4-
methoxyphenyl)methanone (1.10 mmol, 2.0 eq) and 127 mg of ammonium acetate (1.65
mmol, 2.0 eq) in 3 mL of abs. toluene was heated for 45 min to 130 °C by microwave
irradiation. The solvent was removed under reduced pressure. The residue was taken up in 25
mL of water and extracted with ethyl acetate (3 x 30 mL). The combined organic phases were
dried over Na SO , filtered and the solvent was removed under reduced pressure. The crude
2
4
product was purified by column chromatography (hexane:ethyl acetate 4:1) to yield 49 mg
1
3
(
22%) of a ocher solid. H-NMR (250 MHz, CDCl ): δ = 7.99 (d, 2H, J = 8.2 Hz, 3H,5H-
3
toyl), 7.35-7.27 (m, 6H, 2H,6H-toyl + 2 x 2H,6H-PhOMe), 6.94-6.87 (m, 4H, 2 x 3H,5H-
1
3
PhOMe), 3.86 (pd, 6H, 2x OCH ), 2.44 (s, 3H, -CH ). C-NMR (63 MHz, CDCl ): δ = 166.7,
3
3
3
1
2
65.5, 161.2, 155.5, 132.6, 131.9, 129.7, 128.2, 123.9, 118.6, 115.6, 113.9, 113.5, 55.5, 55.3,
+
+
1.9; mp = 208.6 °C; ESI-MS: m/z = 416.7 [M+H] , 438.6 [M+Na] ; Anal. calcd. for
C H NO S: C (72.27), H (5.09), N (3.37), S (7.72), found: C (72.00), H (5.38), N (3.20), S
25
21
3
(7.57).
4
.1.3 General Procedure I: One-pot three component reaction. A mixture of the
corresponding arylnitrile (1 eq), arylaldehyde (1 eq), 2-mercaptoacetic acid (1 eq) and
2
2

ACCEPTED MANUSCRIPT
triethylamine (0.7-1.0eq) was heated in the microwave at 100 °C for 30 min. The product was
either obtained by suspension in methanol and collection of the precipitate by vacuum
filtration or the solvent was removed under reduced pressure and the residue was
recrystallized.
4
.1.3.1 (Z)-5-(4-Hydroxybenzyliden)-2-p-tolylthiazol-4(5H)-one (58): 1.00 g p-
Tolunitrile (8.62 mmol), 1.05 g 4-hydroxybenzaldehyde (8.62 mmol), 0.6 mL 2-
mercaptoacetic acid, 1, (8.62 mmol, 1 mL triethylamine (0.84 eq). Yield: 832 mg (33%)
orange solid. 58 can also be obtained by ether cleavage C06 with boron tribromide [47]
1
3
(
quant. yield). H-NMR (250 MHz, DMSO-d ): δ = 10.50 (s, 1H -OH), 8.10 (d, 2H, J = 8.2
6
3
3
Hz, 3H,5H-tolyl), 7.94 (s, 1H, -CH-), 7.68 (d, 2H, J = 8.7 Hz, 2H,6H-PhOH), 7.48 (d, 2H, J
3
13
=
8.0 Hz, 2H,6H-tolyl), 6.96 (d, 2H, J = 8.6 Hz, 3H,5H-PhOH), 2.45 (s, 3H, -CH ); C-
3
NMR (63 MHz, CDCl ): δ = 182.3, 160.9, 146.4, 138.1, 133.2, 130.2, 128.7, 128.4, 124.4,
3
-
1
22.0, 116.5, 104.5, 21.4; mp = 280.2 °C; ESI-MS: m/z = 294.1 [M-H] ; MALDI-HRMS
+
+
anal. calcd. for C H NO : m/z = 296.07398 [M+H] , found: m/z = 296.07417 [M+H] ;
1
7
13
2
.
Anal. calcd. for C H NO 1.0H O: C (65.16), H (4.82), N (4.47), S (10.23), found: C
1
7
13
2
2
(65.18), H (4.67), N (4.10), S (10.07).
4
.1.3.2 (Z)-5-(Pyridin-3-ylmethylene)-2-p-tolylthiazol-4(5H)-one (71): 500 mg p-
Tolunitrile (4.27 mmol), 0.4 mL freshly distilled 3-pyridine carbaldehyde (4.27 mmol), 0.3
mL 2-mercaptoacetic acid, 1, (4.27 mmol), 0.5 mL triethylamine (0.85 eq). Recrystallization
from acetone/diethyl ether and column chromatography (dichloromethane:methanol 98:2).
1
4
Yield: 159 mg (13%) light yellow solid. H-NMR (250 MHz, CDCl ): δ = 8.92 (d, 1H, J =
3
4
3
3
2
.0 Hz, 2H-Py), 8.67 (dd, 1H, J = 1.2 Hz, J = 4.7 Hz, 6H-Py), 8.11 (d, 2H, J = 8.3 Hz,
3
3
3
H,5H-tolyl), 8.00-7.96 (m, 2H, -CH- + 4H-Py), 7.46 (dd, 2H, J = 4.7 Hz, J = 7.9 Hz, 5H-
3
13
Py), 7.38 (d, 2H, J = 8.1 Hz, 2H,6H-tolyl), 2.49 (s, 3H, -CH ); C-NMR (63 MHz, CDCl ):
3
3
δ = 186.9, 182.8, 151.7, 151.0, 147.2, 136.5, 133.7, 130.1, 130.0, 129.1, 129.0, 129.0, 124.0,
2
3

ACCEPTED MANUSCRIPT
+
2
2.0; mp = 214.6 °C; ESI-MS: m/z = 281.16 [M+H] ; MALDI-HRMS anal. calcd. for
+
+
C H N OS: m/z = 281.07431 [M+H] , found: m/z = 281.07546 [M+H] .
16
12
2
4
.1.3.3 (Z)-5-((1H-Indol-2-yl)methylene)-2-p-tolylthiazol-4(5H)-one (73): 842 mg p-
Tolunitrile (7.19 mmol), 1044 mg 2-indole carbaldehyde (7.19 mmol), 0.5 mL 2-
mercaptoacetic acid, 1, (7.19 mmol), 1.0 mL triethylamine (1 eq). Reaction time 15 min.
Recrystallization from water/methanol and ethyl acetate. Yield: 155 mg (7%) dark red solid.
1
H-NMR (250 MHz, CDCl ): δ = 12.41 (br s, 1H, NH), 8.28 (s, 1H, 3H-indole), 8.11-8.07 (m,
3
3
H, 3H,5H-tolyl + -CH-), 8.00-7.97 (m, 1H, 4H-indole), 7.56-7.53 (m, 1H, 7H-indole), 7.48
3
13
(
d, 2H, J = 8.0 Hz, 2H,6H-toyl), 7.32-7.21 (m, 2H, 5H,6H-indole), 2.45 (s, 3H, -CH ); C-
3
NMR (63 MHz, CDCl ): δ = 182.9, 181.9, 145.7, 136.6, 130.5, 130.1, 130.0, 128.9, 128.1,
3
+
1
2
26.7, 123.3, 121.5, 119.4, 118.5, 112.5, 111.8, 21.3; ESI-MS: m/z = 319.4 [M+H] ; mp =
+
86.1 °C; MALDI-HRMS: calcd.: m/z = 319.08996 [M+H] , found: m/z = 319.09048
+
.
[
M+H] ; Anal. calcd. for C H N OS 0.2H O: C (70.78), H (4.51), N (8.70), S (9.96), found
19 14 2 2
C (70.94), H (4.19), N (8.69), S (10.51).
.1.3.4 (Z)-5-(Thiophene-2-ylmethylene)-2-p-tolylthiazol-4(5H)-one (74): 313 mg p-
4
Tolunitrile (2.68 mmol), 1044 mg thiophen-2-carbaldehyde (2.68 mmol), 0.19 mL 2-
mercaptoacetic, 1, acid (2.68 mmol), 0.37 mL triethylamine (1 eq). Reaction time 15 min.
1
Recrystallization from acetone/water. Yield: 130 mg (17%) yellow solid. H-NMR (250 MHz,
CDCl ): δ = 12.41 (br s, 1H, NH), 8.28 (s, 1H, 3H-indole), 8.11-8.07 (m, 3H, 3H,5H-tolyl + -
3
3
CH-), 8.00-7.97 (m, 1H, 4H-indole), 7.56-7.53 (m, 1H, 7H-indole), 7.48 (d, 2H, J = 8.0 Hz,
1
3
2
H,6H-toyl), 7.32-7.21 (m, 2H, 5H,6H-indole), 2.45 (s, 3H, -CH3); C-NMR (63 MHz,
CDCl ): δ = 182.9, 181.9, 145.7, 136.6, 130.5, 130.1, 130.0, 128.9, 128.1, 126.7, 123.3,
3
+
1
21.5, 119.4, 118.5, 112.5, 111.8, 21.3; ESI-MS: m/z = 319.4 [M+H] ; mp = 209 °C
+
(
sublimation); MALDI-HRMS: calcd.: m/z = 319.08996 [M+H] , found: m/z = 319.09048
+
.
2
[
M+H] ; Anal. calcd. for C H NOS 0.2H O: C (70.78), H (4.51), N (8.70), S (9.96), found
15
11
2
C (70.94), H (4.19), N (8.69), S (10.51).
2
4

ACCEPTED MANUSCRIPT
4
.1.3.5 (Z)-5-(4-Methoxybenzylidene)-2-(pyridin-3-yl)thiazol-4(5H)-one (77): 525 mg 3-
Cyanopyridine (5.04 mmol), 0.5 mL 4-methoxybenzaldehyde (5.04 mmol), 0.35 mL 2-
mercaptoacetic, 1, acid (5.04 mmol), 0.7 mL triethylamine (1 eq). Recrystallization from
1
methanol. Yield: 796 mg (53%) yellow solid. H-NMR (250 MHz, CDCl ): δ = 9.37 (d, 1H,
3
4
4
3
J = 1.6 Hz, 2H-Py), 8.88 (dd, 1H, J = 1.4 Hz, J = 4.8 Hz, 6H-Py), 8.53-8.48 (m, 1H, 4H-
3
Py), 8.08 (s, 1H, -CH-), 7.66 (d, 2H, J = 8.6 Hz, 2H,6H-PhOMe), 7.56-7.51 (m, 1H, 5H-Py),
3
13
7
.05 (d, 2H, J = 8.8 Hz, 3H,5H-PhOMe), 3.90 (s, 3H, -OCH ); C-NMR (63 MHz, CDCl ):
3 3
δ = 183.8, 182.6, 162.5, 154.6, 154.6, 149.1, 140.0, 135.9, 133.0, 126.2, 122.7, 115.0, 105.0,
+
5
5.6; mp = 237.7 °C; ESI-MS: m/z = 297.1 [M+H] ; Anal. calcd. for C H N O S: C (64.85),
1
6
12
2
2
H (4.08), N (9.45), S (10.82), found: C (64.87), H (4.03), N (9.35), S (10.87).
.1.3.6 (Z)-5-(4-Methoxybenzylidene)-2-(pyridin-4-yl)thiazol-4(5H)-one (78): 525 mg 4-
4
Pyridinnitrile (5.04 mmol), 0.6 mL 4-methoxybenzaldehyde (5.04 mmol), 0.35 mL 2-
mercaptoacetic, 1, acid (5.04 mmol), 0.5 mL triethylamine (0.7 eq). Recrystallization from
1
ethanol/diethyl ether, ethanol and chloroform/hexane. Yield: 86 mg (6%) orange solid. H-
3
NMR (250 MHz, CDCl ): δ = 8.89 (d, 2H, J = 6.2 Hz, 2H,6H-Py), 8.11 (s, 1H, -CH-), 7.99
3
3
3
3
(
d, 2H, J = 6.1 Hz, 3H,5H-Py), 7.67 (d, 2H, J = 8.8 Hz, 2H,6H-PHOMe), 7.05 (d, 2H, J =
1
3
8
1
.8 Hz, 3H,5H-PhOMe), 3.91 (s, 3H, -OCH ); C-NMR (63 MHz, CDCl ): δ = 187.3, 182.6,
3 3
62.7, 150.5, 141.0, 139.3, 133.2, 126.2, 122.6, 121.5, 115.1, 55.6; mp = 237.8 °C; ESI-MS:
+
m/z = 297.80 [M+H] ; MALDI-HRMS anal. calcd. for C H N O S: m/z = 297.06922
1
6
12
2
2
+
+
[
M+H] , found: m/z = 297.06936 [M+H] .
.1.3.7 (Z)-5-(4-Methoxybenzylidene)-2-(pyrazin-2-yl)thiazol-4(5H)-one (79): A mixture
of 866 mg pyrazine-2-carbonitrile (8.24 mmol, 1.0 eq), 1122 mg of 4-methoxybenzaldehyde
8.24 mmol, 1.0 eq), 1.1 mL of triethylamine (8.24 mmol, 1.0 eq) in 5 mL of abs. methanol
4
(
and 875 mg of methyl 2-sulfanylacetic, 31, (8.24 mmol, 1.0 eq) was heated for 5 min at 90 °C
by microwave irradiation. The crude product was diluted with 70 mL DCM , filtered through
a plug of silica gel and eluted with 150 mL dichloromethane. The solvent was removed under
2
5

ACCEPTED MANUSCRIPT
reduced pressure and the residue was recrystallized from EtOH, suspended in diethyl ether,
1
dissolved in dichloromethane and filtered to give 120 mg (5%) orange-red solid. H-NMR
4
3
(
250 MHz, CDCl ): δ = 9.46 (d, 1H, J = 1.4 Hz, 3H-pyrazine), 9.04 (d, 1H, J = 2.4 Hz, 6H-
3
4
3
pyrazine), 8.96 (dd, 1H, J = 1.5 Hz, J = 2.3 Hz, 5H- pyrazine), 8.08 (s, 1H, -CH-), 7.84 (d,
3
3
2
H, , J = 8.8 Hz, 2H,6H-PhOMe), 7.16 (d, 2H, J = 8.8 Hz, 3H,5H-PhOMe), 3.87 (s, 3H, -
13
OCH ); C-NMR (63 MHz, CDCl ): δ = 186.0, 182.2, 162.1, 149.7, 145.2, 143.5, 143.3,
3
3
+
1
3
39.7, 133.2, 125.8, 123.0, 115.1, 55.9, 18.4; mp = 216 °C; ESI-MS: m/z = 298.3 [M+H] ,
+
2+
20.3 [M+Na] , 617.2 [M+Na] ; Anal. calcd. for C H N O S: C (60.59), H (3.73), N
1
5
11
3
2
(
14.13), S (10.78), found: C (60.49), H (3.87), N (13.79), S (10.68).
.1.4 General Procedure II: Condensation of 2-p-tolylthiazol-4(5H)-one with a
4
corresponding aldehyde. A suspension of 2-p-tolylthiazol-4(5H)-one, 3, (1 eq), aldehyde (1
eq), 1-2 drops piperidine in methanol was heated in the microwave to 90-100 °C for 3 to 20
min. The product was either directly isolated by collection of the precipitate or the solvent
was removed under reduced pressure and the residue was recrystallized.
4
.1.4.1 (Z)-5-(4-Acetylbenzylidene)-2-p-tolylthiazol-4(5H)-one (59): 150 mg 2-p-
Tolylthiazol-4(5H)-one, 3, (0.78 mmol), 116 mg 4-acetylbenzaldehyde (0.78 mmol) in 2 mL
1
methanol (3 min at 90 °C). Yield: 186 mg (74%) yellow solid. H-NMR (250 MHz, CDCl ): δ
3
3
3
=
8.12 (d, 2H, J = 8.3 Hz, 3H,5H-tolyl), 8.07 (d, 2H, J = 8.4 Hz, 2H,6H-PhAc), 8.04 (s, 1H,
3
3
-
CH-), 7.75 (d, 2H, J = 8.3 Hz, 3H,5H-PhAc), 7.39 (d, 2H, J = 8.0 Hz, 2H,6H-tolyl), 2.65 (s,
1
3
3
1
H, -C(O)CH ), 2.49 (s, 3H, -CH ); C-NMR (63 MHz, CDCl ): δ = 197.0, 187.1, 183.0,
3 3 3
47.1, 138.1, 138.1, 135.9, 130.5, 130.1, 129.2, 129.1, 129.0, 129.0, 26.7, 22.0; mp = 234 °C;
+
+
ESI-MS: m/z = 322.19 [M+H] , 344.18 [M+Na] ; MALDI-HRMS anal. calcd. for
+
+
C H NO S: m/z = 322.08963 [M+H] , found: m/z = 322.09062 [M+H] ; Anal. calcd. for
19
15
2
.
C H NO S 0.1H O: C (70.61), H (4.74), N (4.33), S (9.92), found C (70.50), H (4.60), N
19
15
2
2
(4.29), S (10.11).
2
6

ACCEPTED MANUSCRIPT
4
.1.4.2 Methyl (Z)-4-((4-oxo-2-p-tolylthiazol-5(4H)-ylidene)methyl)benzoate (60): 150
mg 2-p-Tolylthiazol-4(5H)-one, 3, (0.78 mmol), 116 mg methyl 4-formylbenzoate (0.78
1
mmol) in 2 mL methanol (10 min at 100 °C). Yield: 194 mg (73%) pale yellow solid. H-
NMR (250 MHz, DMSO-d ): δ = 8.15-8.07 (m, 4H, 3H,5H-tolyl + 2H,6H-PhCOOCH ), 8.07
6
3
3
3
(
s, 1H, -CH-), 7.94 (d, 2H, J = 8.2 Hz, 3H,5H-PhCOOCH ), 7.52 (d, 2H, J = 8.0 Hz, 2H,6H-
3
1
3
tolyl), 3.90 (s, 3H, -COOCH ), 2.47 (s, 3H, -CH ); C-NMR (75 MHz, DMSO-d ): δ = 186.6,
3
3
6
1
2
82.0, 165.5, 147.2, 137.8, 135.6, 131.1, 130.6, 130.3, 129.9, 128.8, 128.8, 128.8, 128.4, 52.4,
+
1.5; mp = 215 °C; ESI-MS: m/z = 338.0 [M+H] ; Anal. calcd. for C H NO S: C (67.64), H
1
9
15
2
(
4.48), N (4.15), S (9.50), found: C (67.59), H (4.47), N (3.99), S (9.50).
.1.4.3 Methyl (E)-3-(4-((Z)-(4-oxo-2-p-tolylthiazol-5(4H)-
4
ylidene)methyl)phenyl)acrylate (61): 75 mg 2-p-Tolylthiazol-4(5H)-one, 3, (0.39 mmol), 75
#
mg methyl (E)-3-(4-formylphenyl)acrylate (0.39 mmol) in 1.5 mL methanol (10 min at 100
1
3
°
C). Yield: 186 mg (82%) yellow solid. H-NMR (250 MHz, DMSO-d ): δ = 8.15 (d, 2H, J =
6
3
8
.2 Hz, 3H,5H-tolyl), 8.04 (s, 1H, -CH-), 7.94 (d, 2H, J = 8.4 Hz, 2H,6H-
3
3
PhCH=CHCOOCH ), 7.85 (d, 2H, J = 8.4 Hz, 3H,5H- PhCH=CHCOOCH ), 7.73 (d, 1H, J
3
3
3
3
=
16.1 Hz, Ph-CH=CH-), 7.51 (d, 2H, J = 8.2 Hz, 2H,6H-tolyl), 6.80 (d, 1H, J = 16.1 Hz,
13
Ph-CH=CH-), 3.75 (s, 3H, -COOCH ), 2.46 (s, 3H, -CH ); C-NMR (75 MHz, DMSO-d ): δ
3
3
6
=
186.2, 182.1, 166.4, 147.0, 136.4, 136.3, 136.3, 135.1, 131.0, 130.3, 129.2, 128.8, 128.5,
+
+
1
27.2, 119.9, 51.6, 21.5; mp = 223 °C; ESI-MS: m/z = 364.22 [M+H] , 386.23 [M+Na] ;
Anal. calcd. for C H NO S: C (69.40), H (4.71), N (3.85), S (8.82), found: C (69.08), H
2
1
17
3
(4.73), N (3.72), S (8.76).
4
.1.4.4 (Z)-5-(4-Aminobenzylidene)-2-p-tolylthiazol-4(5H)-one (63): 110 mg 2-p-
s
Tolylthiazol-4(5H)-one, 3, (0.58 mmol), 70 mg 4-aminobenzaldehyde (0.58 mmol) in 2.5 mL
1
methanol (20 min at 100 °C). Yield: 124 mg (73%) dark red solid. H-NMR (250 MHz,
3
3
DMSO-d ): δ = 8.07 (d, 2H, J = 8.0 Hz, 3H,5H-tolyl), 7.82 (s, 1H, -CH-), 7.53 (d, 2H, J =
6
3
3
8
.5 Hz, 2H,6H-PhNH ), 7.47 (d, 2H, J = 8.0 Hz, 2H,6H-tolyl), 6.72 (d, 2H, J = 8.4 Hz,
2
2
7