Design, synthesis and evaluation of 2-aminothiazole derivatives as sphingosine kinase inhibitors

Article abstract of DOI:10.1016/j.bmc.2014.07.044

Sphingosine kinases (SphK1, SphK2) are main regulators of sphingosine-1-phosphate (S1P), which is a pleiotropic lipid mediator involved in numerous physiological and pathophysiological functions. SphKs are targets for novel anti-cancer and anti-inflammatory agents that can promote cell apoptosis and modulate autoimmune diseases. Herein, we describe the design, synthesis and evaluation of an aminothiazole class of SphK inhibitors. Potent inhibitors have been discovered through a series of modifications using the known SKI-II scaffold to define structure-activity relationships. We identified N-(4-methylthiazol-2-yl)-(2,4′-bithiazol)-2′-amine (24, ST-1803; IC₅₀values: 7.3 μM (SphK1), 6.5 μM (SphK2)) as a promising candidate for further in vivo investigations and structural development.

Full text of DOI:10.1016/j.bmc.2014.07.044

Bioorganic & Medicinal Chemistry 22 (2014) 5354–5367
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
Design, synthesis and evaluation of 2-aminothiazole derivatives
as sphingosine kinase inhibitors
Dominik Vogt a, Julia Weber a, Katja Ihlefeld a, Astrid Brüggerhoff a, Ewgenij Proschak a, Holger Stark b,
⇑
a Institute of Pharmaceutical Chemistry, Goethe University, Max-von-Laue-Str. 9, D-60438 Frankfurt am Main, Germany
b Institute of Medicinal and Pharmaceutical Chemistry, Heinrich Heine University, Universitätsstr. 1, D-40225 Düsseldorf, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Sphingosine kinases (SphK1, SphK2) are main regulators of sphingosine-1-phosphate (S1P), which is a
pleiotropic lipid mediator involved in numerous physiological and pathophysiological functions. SphKs
are targets for novel anti-cancer and anti-inflammatory agents that can promote cell apoptosis and mod-
ulate autoimmune diseases. Herein, we describe the design, synthesis and evaluation of an aminothiazole
class of SphK inhibitors. Potent inhibitors have been discovered through a series of modifications using
the known SKI-II scaffold to define structure–activity relationships. We identified N-(4-methylthiazol-
Received 16 April 2014
Revised 23 July 2014
Accepted 28 July 2014
Available online 6 August 2014
Keywords:
2-yl)-(2,40-bithiazol)-20-amine (24, ST-1803; IC50 values: 7.3
candidate for further in vivo investigations and structural development.
lM (SphK1), 6.5 lM (SphK2)) as a promising
Sphingosine-1-phosphate
Sphingolipids
Sphingosine
Ó 2014 Elsevier Ltd. All rights reserved.
ST-1803
Enzyme inhibitor
1. Introduction
cancer, asthma, atherosclerosis, sepsis, inflammatory bowel dis-
ease, rheumatic arthritis and multiple sclerosis.3,4
Sphingolipids represent a major class of lipids that are ubiqui-
tous localized and essential constituents of eukaryotic cells.
Besides structural roles in membrane formation, they act as mod-
ulators in cell signaling processes. Sphingolipid metabolism dis-
plays a complex network of enzymatically controlled reactions
with ceramide (Cer) in the center of biosynthesis and catabolism.1
Most important sphingolipid mediators are sphingosine-1-phos-
phate (S1P), sphingosine (Sph) and Cer.2 The balance between cel-
lular concentrations of Cer and S1P (called ‘sphingolipid rheostat’)
has been proposed to determine the physiological fate of cells. Cer
and S1P elicit opposing cellular fates, for example, growth arrest
and apoptosis versus proliferation and survival. Changes of
intracellular S1P levels also affect the levels of Cer and Sph. The
bioactive S1P is a potent mitogenic and migratory signaling lipid.
It has been linked to the development and progression of numer-
ous hyperproliferative and inflammatory diseases including
Therapeutic opportunities targeting the S1P/Cer rheostat are
manifold. The major objective of drug discovery has focused on
molecules that are capable of agonizing or antagonizing S1P recep-
tors. S1P receptor modulator Fingolimod (FTY720), a Sph analogue,
is the first oral therapeutic approved for the treatment of multiple
sclerosis.5 Another approach is to reduce the bioavailability of S1P
at its receptors using S1P neutralizing antibodies. The anti-S1P
monoclonal antibody Sonepcizumab (LT1009) is currently in clini-
cal trials phase I for age-related macular degeneration (iSONEP™)
and for advanced solid tumors (ASONEP™).6 Sphingosine kinase
(SphK) inhibitors are another therapeutic option.7
Sph is phosphorylated by SphK to form S1P. SphK exists in two
isoforms, SphK1 and SphK2, which differ in their substrate prefer-
ences, subcellular localizations and tissue distributions, suggesting
that they perform different physiological roles.8 Studies using iso-
form-specific siRNA and knock-out mice have indicated that SphK1
and SphK2 have distinct and non-redundant functions involved in
(patho)physiology. This promoted the search for isoform-specific
inhibitors of SphK1 and SphK2.9 The actions of SphK1 and S1P
are complex and far away from being fully understood, especially
with regard to the involvement in inflammation and cancer. Can-
cers of stomach, lung, brain, colon, kidney and breast as well as
non-Hodgkin’s lymphoma have increased SphK1 expression.10
Up-regulation of SphK1 increases S1P production and correlates
Abbreviations: ATP, adenosine triphosphate; ADP, adenosine diphosphate; Cer,
ceramide; ERK-1/2, extra-cellular signal-regulated kinase 1/2; HDAC, histone
deacetylase; LDH, lactate dehydrogenase; S1P, sphingosine-1-phosphate; SAR,
structure–activity relationship; Sph, sphingosine; SphK1, sphingosine kinase 1;
SphK2, sphingosine kinase 2; TRAF2, TNF
a receptor associated factor 2; TNFa,
tumor necrosis factor alpha; TPSA, topological polar surface area.
⇑
Corresponding author. Tel.: +49 211 81 10478; fax: +49 211 81 13359.
E-mail address: stark@hhu.de (H. Stark).
http://dx.doi.org/10.1016/j.bmc.2014.07.044
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5355
with poor cancer prognosis.11 The activity and expression of SphK1
is increased in response to growth factors and pro-inflammatory
effects in part on its ability to induce the proteasomal24 or lyso-
somal25 degradation of SphK1. PF-543 (42) is the first nanomolar
SphK1-selective inhibitor that rapidly reduces S1P in cells
(Fig. 1).26 It appears to be a useful tool for inhibiting SphK1
in vitro, but its in vivo effects were not described so far.
We focused on SKI-II as promising lead compound for the
development of a small library of 2-aminothiazole derivatives as
SphK inhibitors with different selectivity ratios. 2-Aminothiazoles
and derivatives provide a wide spectrum of biological activities27
and are seen in many bioactive scaffolds as ‘privileged
structures’.28,29
cytokines.8 TNF
a-induced interaction of SphK1 with TNFa receptor
associated factor 2 (TRAF2) is required for the extracellular signal-
regulated kinase (ERK-1/2)-mediated phosphorylation of SphK1
and subsequent translocation of SphK1 to the plasma membrane,
where it interacts with phosphatidylserine.12 The physiological
and pathophysiological relevance of this translocation remains to
be determined. It might be required for the so-called ‘inside-out’
signaling of S1P.13
Less is known about the role and regulation of SphK2. Some
studies have supported a role for SphK2 in survival and migra-
tion.14 By contrast, other studies have demonstrated the suppres-
sion of cell cycle arrest and apoptosis.15 The ambiguous nature
might be determined by the subcellular localization of SphK2
and resulting intracellular S1P pools.16 Recently, histone deacetyl-
ase (HDAC) has been claimed as an intracellular target for nuclear
localized SphK2-derived S1P. S1P binds to and inhibits HDAC1 and
HDAC2 within the repressor complexes that are enriched at the
promoters of genes encoding the transcriptional regulator c-fos
and cyclin-dependent kinase inhibitor p21. S1P promotes their
expression by inhibiting HDACs and increasing histone acetylation
(epigenetic S1P effect).17
2. Chemistry
Compounds 1–37 were prepared as shown in Scheme 1. We
started with the bromination of the corresponding ketone deriva-
tives (A) that gave a-bromoketones (B). The substituted thioureas
(E) that were not commercially available were synthesized by con-
densation of the appropriate aniline or amine derivative (C) with
benzoyl chloride in the presence of ammonium thiocyanate, fol-
lowed by saponification of the resultant N-aryl-N0-benzoylthioure-
as (D) to remove the benzoyl group.30,31 In the final step,
microwave-assisted Hantzsch thiazole synthesis, the condensation
There is a need for specific inhibitors of SphK1 and/or SphK2 to
determine whether strategies targeting the isoenzymes alone or in
combination offer the best therapeutic option for the diseases
mentioned above. A major difficulty in the development of isoen-
zyme-selective inhibitors has been the lack of structural informa-
tion of substrate recognition and catalysis. The recent elucidation
of the X-ray crystal structure of SphK1 in 201318 will lead to a bet-
ter understanding of the enzyme functionality. The crystal struc-
ture of SphK2 remains to be solved and would accelerate the
development of selective inhibitors for therapeutic uses.
of
a-bromoketones (B) and N-substituted thioureas (E), provided
the desired 2-aminothiazole derivatives (1–37).32
For the synthesis of compounds 38–41 we used different amide
bond formation procedures to couple 2-aminothiazole to activated
cinnamic acid derivatives.33 Acyl chlorides (for 38) or carboxylic
acids after previous activation by DIC/HOBt (39) or EDC/HOBt
(40, 41) were coupled to the aromatic amine.34 Reference com-
pound PF-543 (42) was synthesized as described by Schnute
et al. (cf. Supplementary material).26
To date, most SphK inhibitors exhibit Ki or IC50 values in the
micromolar range.7 The roles of SphKs and S1P in different pathol-
ogies were first elucidated by the discovery of the non-selective
SphK inhibitor N,N-dimethylsphingosine (DMS)19 (Fig. 1). Due to
its high structural similarity to the endogenous Sph, the therapeu-
tic potential of this substrate analogue is limited. A milestone in
the development of more ‘drug like’ molecules was the synthesis
and evaluation of non-selective SphK inhibitor SKI-II (4-((4-(4-
chlorophenyl)-2-thiazolyl)amino)phenol, compound 1)20 (Fig. 1).
SKI-II has been used for in vitro and in vivo studies.21,22 It reduces
intracellular S1P, inhibits proliferation and induces apoptosis in
various cancer cell lines.23 SKI-II is orally bioavailable.22 Its
described potency may depend beside several non-Sph-related
3. Results and discussion
3.1. Biological evaluation
Compounds were screened for SphK inhibitory activity at
10 lM (Table 1). An ADP-detecting fluorescence assay has been
used as ADP and S1P are equimolar products of the enzymatic
phosphorylation reaction. Inhibition of SphK1 or SphK2 was deter-
mined by incubating SphK1 or SphK2 in the presence of sphingo-
sine, ATP and inhibitor or control (DMSO). The inhibitory
potential of the lead structure SKI-II (1) has been shown to be only
moderate under these assays conditions (15–25% inhibition),
whereas PF-543 (42) was able to inhibit SphK1 almost quantita-
tively at this concentration (94%).
The initial round of lead compound SKI-II (1) modifications
aimed at improving its inhibitory potential at SphK1 and/or SphK2
via structural variations at R3. Small functional groups were
attached at different positions of the aromatic ring to affect the
hydrogen-bond donor/acceptor ability of the moiety, thereby play-
ing a role in the interactions with the target binding site.
The hydroxy group in 4-position of the phenyl ring seems to be
crucial for inhibitory potential, as both its shift to the 2- and 3-
position (3, 4) and methylation (6) led to loss of inhibition. The thi-
oether derivative 12 nevertheless was able to selectively inhibit
SphK1. The insertion of a methylene spacer (5) between hydroxyl
moiety and aromatic ring produced an inactive compound. Inter-
estingly, the methoxy-group in 2-position of the phenyl ring (7)
showed a slight tendency towards SphK1-selective inhibition. Ana-
logues containing alkyloxy-substituents in 4- and 3-position as
dimethoxy (8) as well as methylene or ethylene linked diethers
(10, 11) lost their ability to inhibit SphKs. By introducing a third
methoxy group in 5-position (9, ST-1780), a substantial increase
Figure 1. Representative SphK inhibitors and screening hit ST-1803.

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D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
Scheme 1. General procedure for the synthesis of 2-aminothiazole derivatives 1–37. Reagents and conditions: (i) Br2, chloroform, rt, 2 h; (ii) benzoyl chloride, NH4SCN,
acetone, 60 °C, 45 min; (iii) NaOH (2 M), H2O/THF, 100 °C, 1 h; (iv) ethanol, microwave irradiation, 80 °C, 30 min.
in potency and isoenzyme preference could be achieved. Trifluo-
romethylation at different positions or different functionalities
led to inactive compounds as well as isopropylation in 4-position
(13–16). In contrast, the 4-dimethylamino group with 17 re-estab-
lished inhibitory potential at SphK1.
molecule. Oral efficacy of the drugs can be extended and more lipo-
philic interactions in the active site of an enzyme are provided as
shown on other compound classes.37 Adamantyl-containing com-
pounds have already been successfully established as SphK inhib-
itors. Aryladamantane compound ABC294640 (3-(4-chlor
Since the variations on the phenyl substitution pattern for R3
led to a potent compound only for 7, 9, 12, we focused in the next
step on the 5-methylation of the central thiazole core ring. It is
described that 2-aminothiazoles without substituents at C-5 that
are able to inhibit metabolism can undergo oxidation in vivo to
generate potentially toxic reactive epoxide metabolites.35 Recently,
2-aminothiazole derivatives were evaluated for microsomal stabil-
ity and the regions of the scaffold predestined for phase I metabo-
lism were determined. The observed thiazole ring modifications
were either the 4,5-epoxide or the 5-hydroxythiazole whose tauto-
mer is a thiazolone. Ring opening of the thiazolone via hydrolytic
opening was determined as well. The introduction of a methyl-
group at C-5 of the thiazole ring avoids this metabolic pathway.36
Therefore, the presence of a substituent at the C-5 might be useful
in terms of improved pharmacokinetics and toxicological aspects.
Compound 18, the methylated derivative of SKI-II (1), and com-
pound 21 did not show any inhibition. In contrast, the methyl-
group in compound 20 maintained inhibitory potential compared
to compound 9 (ST-1780). Surprisingly, the methyl group in com-
pound 19 provided selective inhibitory potential for SphK1 com-
ophenyl)adamantane-1-carboxylic
amide) (Fig. 2) selectively inhibits SphK2 in vitro, competitive to
Sph with a Ki of 9.8 M. It attenuated S1P formation, suppressed
acid
(pyridin-4-ylmethyl)
l
cell proliferation in several cancer cell lines and promoted autoph-
agy.38 ABC294640 has a good oral bioavailability and it showed
promising inhibitory potency in tumor growth in vivo in several
xenograft rodent models, such as different breast, kidney and pan-
creatic cancer models.38,39 ABC294640 is currently in a phase I
clinical trial for patients with advanced solid tumors (ClinicalTri-
als.gov identifier, NCT01488513). In addition, this compound was
successfully evaluated in animal models of ulcerative colitis40
and Crohn’s disease41, were it decreased S1P levels and the levels
of inflammatory cytokines. ABC294735 (3-(4-chlorophenyl)ada-
mantane-1-carboxylic acid 3,4-dihydroxybenzylamide) (Fig. 2) is
a non-selective SphK inhibitor. It is Sph-competitive with Ki values
for SphK1 and SphK2 of 3.1 and 4.2 l
M, respectively.42 Both,
ABC294640 and ABC294735, have been demonstrated to cause a
comparable delay in tumor growth, an effect that was potentiated
by the co-administration of Sorafenib.39
Taken together, these results indicate that adamantyl is a struc-
tural element in SphK inhibitors that is worth to be further
investigated.
Within the adamantyl series, we also explored the previously
successful optimization by methylation at C-5 of the 2-aminothia-
zole. The promising substitution patterns of compounds 7, 9 (ST-
1780), 20, and 22 were confirmed. In combination with the ada-
mantyl-moiety at C-4 of the aminothiazole, the trimethoxy-substi-
tution of the phenyl ring resulted in an increase in potency, but
SphK1 selectivity was lost (compounds 27 and 36). Introduction
of only one methoxy group in 2-position of the phenyl ring (com-
pounds 28 and 35) led to a significant increase in activity com-
pared to that of compound 7. SphK1 selectivity was only
observed for compound 25. Compound 29, the thioether analogue
of compound 25 was comparable potent at both isoenzymes.
SphK1 selectivity was restored by additional introduction of the
methyl group at C-5 of the aminothiazole ring (compound 37).
Dimethoxy-substituted derivative 26 is completely inactive like
pared to the inactive compound
6
whereas the same
modification in 12 compared to 21 led to an inactive derivative.
With structural modifications on the substituent at C-4 of the 2-
aminothiazole we examined whether the 4-chloro-phenyl moiety
could be replaced by other functionalities that are able to maintain
or to improve selectivity and potency of the inhibitor. The slight
modification of SKI-II (1) by introducing 4-bromo-phenyl (2) led
to a comparable inhibition of SphK2 but a reduced inhibition of
SphK1. The exchange into a thiazol-2-yl ring (23) resulted in selec-
tive SphK1 inhibition. Taking into consideration the hydrophobic
properties that are supposed to be responsible for the binding of
sphingosine in the active site, we prepared a series of adamantyl-
containing derivatives (compounds 25–37). The introduction of
an adamantyl group provides a rigid scaffold and a larger volume
compared to the aromatic phenyl ring. It confers higher lipophilic-
ity to the entire molecule. This may completely change the absorp-
tion, distribution, metabolism or excretion (ADME) properties of a

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5357
Table 1
Human SphK1 and SphK2 screening for compounds 1–42a
No.
% Inhibitiona SphK1 @10
lM
% Inhibitiona SphK2 @10
lM
R1
R2
R3
Ph-4-OH
1b
2
3
4-Cl-Ph
H
25.0 6.1⁄⁄⁄
15.5 4.2⁄⁄
13.6 7.0
—
4-Br-Ph
4-Cl-Ph
H
H
Ph-4-OH
Ph-3-OH
10.3 4.7
g
—
4
4-Cl-Ph
H
Ph-2-OH
—
—
5
4-Cl-Ph
H
Ph-4-CH2-OH
—
—
6
4-Cl-Ph
H
Ph-4-OCH3
—
—
7
8
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
4-Cl-Ph
Thiazol-2-yl
Thiazol-2-yl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
Adamantyl
4-Cl-Ph
H
H
H
H
H
H
H
H
H
H
H
CH3
CH3
CH3
CH3
CH3
H
H
H
H
H
H
H
H
H
Ph-2-OCH3
19.1 8.6
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
—
Ph-3,4-(OCH3)2
Ph-3,4,5-(OCH3)3
Ph-3-O-CH2-O-4
Ph-3-O-C2H4-O-4
Ph-4-SCH3
Ph-4-OCF3
Ph-4-CF3
Ph-3,5-(CF3)2
Ph-4-CH(CH3)2
Ph-4-N(CH3)2
Ph-4-OH
Ph-4-OCH3
Ph-3,4,5-(OCH3)3
Ph-4-SCH3
Thiazol-2-yl-4-CH3
Ph-4-OH
Thiazol-2-yl-4-CH3
Ph-4-OCH3
Ph-3,4-(OCH3)2
Ph-3,4,5-(OCH3)3
Ph-2-OCH3
Ph-4-SCH3
Ph-4-CH(CH3)2CH
Ph-4-N(CH3)2
Ph-3-O-CH2-O-4
Ph-3-O-C2H4-O-4
Thiazol-2-yl-4-CH3
Ph-2-OCH3
Ph-3,4,5-(OCH3)3
Ph-4-SCH3
C(O)CH@CHAPh-3,4-(OCH3)2
C(O)CH@CHAPh-3,4,5-(OCH3)3
C(O)CH@CHAPh-3,4-(OCH3)2
C(O)CH@CHAPh-3,4,5-(OCH3)3
9d
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24e
25
26
27
28
29
30
31
32
33
34
35
36
37
38
39f
40
41
42c
30.7 9.1⁄
—
—
29.8 14.7
—
—
—
—
15.3 7.3
—
21.8 6.0
31.4 15.8
—
26.7 11.1
29.5 8. 6⁄
59.4 3.3⁄⁄⁄
20.3 12.6
—
43.1 6.0
27.3 5.8⁄⁄
16.7 8.6
—
19.2 5.2⁄
-
50.0 9.9⁄⁄
—
—
20.8 12.0
19.0 5.4⁄
18.6 7.6
—
21.9 12.7
20.8 10.8
—
—
H
H
41.3 17.1
31.7 18.6
20.9 5.9⁄
41.6 10.1⁄
21.5 8.4
—
CH3
CH3
CH3
CH3
H
H
H
H
38.9 10.9⁄
33.9 5.9⁄⁄
36.1 3.8⁄⁄⁄
19.2 8.0
—
31.7 7.9⁄⁄
32.0 8.1⁄⁄
26.5 3.3
26.6 4.5⁄⁄
14.7 4.0⁄⁄
4-Cl-Ph
H
H
—
14.3 8.3
17.7 8.8
94.1 3.2⁄⁄⁄
See Figure 1
a
Compounds were screened at 10 lM. Results are presented as % inhibition of SphK1 and SphK2 compared to control (DMSO). Results were calculated as mean SEM of
five independent experiments and were analyzed using one sample t-test. Significant inhibition is illustrated as follows: ⁄p 6 0.05, ⁄⁄p 6 0.01 and ⁄⁄⁄p 6 0.0001. Two-tailed
Student’s t-tests were performed (c.f. Supplementary material): for PF-453 selectivity profile towards SphK1 was highly significant (p 6 0.05).
b
Reference compound: SKI-II.
Reference compound: PF-543.
Reference compound: ST-1780.
Reference compound: ST-1803.
Reference compound: ST-1577.
(—) no significant inhibition at the concentration tested.
c
d
e
f
g
isopropyl- by N,N-dimethyl moieties (compounds 16 and 17) was
confirmed by compounds 30 and 31.
Based on the modelling results discussed below we decided to
evaluate the role of an amide linkage between the central thiazole
and the aromatic system, wherein the carbonyl provides a further
hydrogen bond acceptor. The amide group is additionally part of a
Figure 2. Adamantyl-containing SphK inhibitors ABC294640 and ABC294735.
substituted acrylamide as electrophile. Acrylamide-based com-
pounds, also known as Michael acceptors, are targeting cysteine
residues in the enzyme that display nucleophilic thiol groups. This
potentially covalent and irreversible inhibition is a powerful
approach for enhancing pharmacological potency and selectivity.
It may have advantages for the extent and duration of the thera-
peutic effect, since restoration of pharmacological activity requires
re-synthesis of the protein target.43
compound 8, but interestingly compound 33 with dioxin partial
structure and adamantyl moiety instead of 4-chloro-phenyl
showed preferred SphK2 inhibition, compared to non-selective
dioxole-containing non-selective compound 32. The positive
trend towards selective SphK1 inhibition via replacement of

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D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
We discovered four candidates as potential covalent com-
ide-containing compounds, 38 and 39 (ST-1577) emerged as
SphK2-selective inhibitors. To assess the importance of the 4-
chloro-phenyl moiety, we tested compounds 40 and 41. Both lost
their isoenzyme preference, thereby indicating that a further sub-
stitution at C-4 of the aminothiazole is crucial for SphK2 selectiv-
pounds through our screening in the biological assay. The proof
of concept—the investigation of the covalent molecular mecha-
nism—has to be elucidated in future experiments beyond this
structure–activity relationships evaluation. Among the acrylam-
Scheme 2. Summary of molecular profiling of 2-aminothiazole derivatives as SphK inhibitors and development of SphK1-selective ST-1780, non-selective ST-1803 and
SphK2-selective ST-1577 as promising new lead compounds.
Scheme 3. Structure–activity relationships of non-selective SphK inhibitors.

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5359
Scheme 4. Structure–activity relationships of inhibitors with preference for SphK1.
ity. Taken together, the results of the four amide-containing deriv-
atives demonstrate that the amino moiety could also be embodied
into an amide bond without loss of inhibitory activity at SphKs.
To further define functionalities tolerated by SphK1 and SphK2,
we replaced the characteristic 4-hydroxy-phenyl and/or the 4-
chloro-phenyl substituent of SKI-II (1) by thiazole and methyl-thi-
azole moieties (compounds 22, 23, 24 and 34). This bioisosteric
replacement was successful as all four derivatives were active as
SphK inhibitors. Whereas non-selective compound 22 showed
comparable inhibition to that of SKI-II (1), compound 34 had a
2-fold higher inhibitory potency at SphK1. With compound 23,
we identified a SphK1-selective inhibitor. A summary of the molec-
ular profiling and the observed structure activity relationships of
2-aminothiazole derivatives as SphK inhibitors is shown in
Schemes 2–5. Tri-thiazole compound 24 (ST-1803) was found to
be the most active compound among all screened 2-aminothiazole
derivatives with 59.4 3.3% inhibition of SphK1 and 50.0 9.9%
10.8
l
M for SphK1 and 4.5 to 9.4
l
M for SphK2). ST-1803 fulfills
Lipinski’s Rules of Five for oral bioavailability,44–46 which generally
simplifies the administration and drug formulation systems. A cal-
culated log P value of 2.9 and a calculated TPSA value of 38.7 Å2 for
ST-1803 suggest a good permeation of cell membranes and even
the blood brain barrier (c log P <5, TPSA <60 Å2).47 ST-1803 was
also tested to assess its cytotoxicity. We measured the leakage of
LDH which points to a loss of cell membrane integrity. U937 cells
were treated with ST-1803 up to a concentration of 30
ST-1803 showed no signs of cell toxicity with an LDH release of
0.69 0.12% at 30 M.
lM for 24 h.
l
3.2. Molecular modelling
We identified the distinction between the binding sites of SphK1
and SphK2 by building a homology model of non-crystallized
SphK2, based on the sequences of both molecules and the crystal
structure of SphK1 (28.1% sequence identity). We identified three
amino acid substitutions (Ille174 ? Val340, Met272 ? Leu272,
Phe288 ? Cys589) within a radius of 4.5 Å to the reference ligand
SKI-II (1). It is conspicuous that all amino acids substituted in
SphK2 are of a smaller size, than the corresponding amino acids
in SphK1. These findings indicate that the binding pocket of SphK2
is larger as compared to that of SphK1. Furthermore, the lipophilic-
ity seems not to be affected by the substitutions (see Fig. 3). Molec-
ular docking studies suggest that the lacking activity for SphK1 of
compounds 38 and 39 (ST-1577) arises from steric hindrance as
the molecules are too big to fit into the binding site. The measured
inhibition of compounds 38 and 39 (ST-1577) on SphK2 is a second
indicator for a bigger binding site. It is apparent that the SphK2
selective compounds 38 and 39 both have an acrylamide function-
inhibition of SphK2 at 10
determination of IC50 values for 24 (ST-1803) at SphK1 and SphK2
of 7.3 M and 6.5 M, respectively (95% confidence interval 4.9 to
lM. Extensive screening allowed the
l
l
Scheme 5. Structure–activity relationships of inhibitors with preference for SphK2.
Figure 3. Binding site comparison of SphK1 (a) and SphK2 homology model (b). The homology model was built on the basis of the co-crystallized structure of SphK1 and SKI-
II (PDB code: 3VZD). SKI-II (blue) is shown within both binding sites as well as the substituted amino acids (Ille174 ? Val340, Met272 ? Leu272, Phe288 ? Cys569)
determined by the sequence alignment and the shape of the binding pocket colored by lipophilicity (green: lipophilic; magenta: hydrophilic; white: neutral).

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D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
investigation of S1P metabolism, but also in a clinical setting. Cur-
rent efforts are aimed at modifying several regions of this lead
compound to achieve selectivity and increased potency.
Overall, our study supports the continued and future design of
SphK inhibitors. Ongoing development will enhance treatment
options for diseases possessing S1P-dependent components. As
these molecular targets are distinct from those of conventional
drugs in the clinical management of cancer, inflammatory disor-
ders and autoimmune diseases, they are a further step towards
progress in therapy.
5. Experimental section
5.1. Chemistry
Figure 4. Reference SphK inhibitor SKI-II (grey) and docking pose of ST-1803
(blue). Close interactions (4.5 Å distance cutoff) between ST-1803 and the corre-
sponding amino acids are shown as dashed lines.
All commercial chemicals were reagent grade and used as pur-
chased by Sigma–Aldrich (Steinheim/Germany), ABCR (Karlsruhe/
Germany), Acros Organics (Geel/Belgium), Alfa-Aesar (Karlsruhe/
Germany) and Fluorochem (Derbyshire/UK) unless otherwise indi-
cated. Only dried solvents were used. Reactions were monitored by
thin-layer chromatography using silica gel 60 F254 aluminum-
backed plate from Merck (Darmstadt/Germany) with detection
using a UV lamp and ninhydrin. Column chromatography was per-
ality. A hypothetical mode of action could be the acrylamide form-
ing a covalent bond as Michael acceptor with the Cys569 residue
which is only present within the binding site of SphK2. Additional
studies to investigate covalent binding potential of the SphK2 selec-
tive inhibitors are projected. The docking pose of the most potent
compound 24 (ST-1803) is shown in Figure 4. The compound forms
formed on silica gel (SiO2, 40–63
lM). Microwave-assisted synthe-
sis was performed using Biotage Initiator 2.0, 400 Watt
a
mostly hydrophobic interactions and p-stacking which should not
microwave synthesizer (Biotage, Uppsala/Sweden). All products
were characterized by 1H NMR and 13C NMR. NMR spectra were
recorded on a Bruker AV 250 (1H: 250 MHz; 13C: 63 MHz) spec-
trometer (Bruker, Karlsruhe/Germany). Chemical shifts (d) are
reported in ppm relative to tetramethylsilane (TMS) as internal
standard. Multiplicity: s = singlet, d = doublet, t = triplet, q = quar-
tet, m = multiplet; coupling constants (J) are shown in hertz (Hz);
number and assignment of protons. Mass spectra were obtained
on a VG Platform II (Fisons Instruments, Ipswich/UK) using electro-
spray ionization (ESI). Data are listed as mass number [M+H]+ or
[MꢀH]ꢀ. High resolution MS (HR-MS) were achieved on a LTQ
Orbitrap XL (Thermo Fisher Scientific, Waltham/USA). For final
compounds the analyses were within 5 ppm of the theoretical
values. The purity of the compounds was determined by elemental
analysis (C, H, N, S) on a MicroCube instrument (Elementar, Hanau/
Germany) and was within 0.4% of the theoretical values for all
final compounds, which corresponds to P95% purity. Melting
points were recorded using a Buchi B-540 melting point instru-
ment (Buchi, Flawil/Switzerland) and are uncorrected.
be affected by the given amino acid substitutions and therefore
explains the nonselective behavior of the compound. A common
problem with kinase inhibitors is their tendency towards non-
selective and undesired inhibition of other ATP-dependent
enzymes because of an interaction with the highly conserved nucle-
otide binding site. Binding mode of SphK inhibitor SKI-II turned out
to be non-competitive with ATP.18,20 As we could show in the dock-
ing pose compound 24 (ST-1803) binds congruent with SKI-II to
SphKs (Fig. 4). The structural similarity suggests a similar non-com-
petitive binding mechanism.
4. Conclusion
The aim of the present study was to identify inhibitors of SphK1
and/or SphK2 with an improved inhibition profile compared to
non-selective SphK inhibitor SKI-II. Described is the optimization
and SAR of 2-aminothiazole-based inhibitors with low micromolar
activity. Modifications led to a variable range of inhibitory activi-
ties and allowed us to construct a plausible SAR as described above.
Microwave-assisted Hantzsch thiazole synthesis demonstrates a
fast and facile synthesis of 2-aminothiazole derivatives (Scheme 1).
The 3,4,5-trimethoxyphenyl moiety is an auspicious substitu-
tion pattern for inhibitory activity. Isoenzyme preference or selec-
tivity depend on further functionalities that are part of the 2-
aminothiazole scaffold. From this series, SphK1-selective inhibitors
9 (ST-1780) and 20 are candidates for continuative structural mod-
ifications and in vivo experiments, same for non-selective SphK
inhibitors 27 and 36. Compounds 38 and 39 (ST-1577), character-
ized by the substituted acrylamide were identified as selective
SphK2 inhibitors. Finally, we successfully introduced the di- and
tri-thiazole pattern as completely novel functionality for the
design of potent SphK inhibitors (compounds 18, 23, 24 and 34).
Most promising screening hit 24 (ST-1803, Fig. 1) showed an
5.1.1. General procedure for the synthesis of
(B, Scheme 1)
a-bromoketones
To a solution of the appropriate ketone (A, 1 equiv) in chloro-
form, bromine (1 equiv) in chloroform was added dropwise at
0 °C. The mixture was stirred at room temperature for 2 h and
was washed with H2O (3 ꢁ 50 ml) and saturated Na2S2O3 solution
(2 ꢁ 50 ml). The organic phase was dried over Na2SO4, filtered and
the solvent was removed in vacuum. The crude
was recrystallized from petrolether.48
a-bromoketone (B)
5.1.2. General procedure for the synthesis of N-aryl-N0-
benzoylthioureas (D, Scheme 1)
Benzoyl chloride (1 equiv) was added over 5 min to a freshly
prepared solution of NH4SCN (1.1 equiv) in acetone and the mix-
ture was heated to reflux for 15 min. The in situ generated benzoyl
isothiocyanate reacted with added amine/aniline derivatives (C,
1 equiv) in acetone. The mixture was heated to reflux for 30 min,
and then poured on ice with vigorous stirring. The resulting solid
(D) was collected and washed with H2O, followed by cold
acetone.30
encouraging inhibition of over 50% (at 10
with an IC50 of 7.3 M for SphK1 and 6.5
tively. Moreover, ST-1803 is completely devoid of apparent cyto-
toxicity (LDH assay: 0.69 0.12% LDH release at 30 M). These
lM) for both isoenzymes,
l
l
M for SphK2, respec-
l
favorable properties qualify ST-1803 for further physicochemical
investigations. If the inhibitory effect of this potent drug candidate
proves to be reproducible in vivo, it may be useful not only in the

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5361
5.1.3. General procedure for the synthesis of N-substituted
thioureas (E, Scheme 1)
5.1.4.4.
(4).
2-(4-(4-Chlorophenyl)thiazol-2-ylamino)phenol
General procedure 5.1.4 was used to couple 2-bromo-1-
A solution of N-aryl-N0-benzoylthiourea (D) in aqueous sodium
hydroxide (2 M)/THF (1:1) was heated to 100 °C for 1 h. The precip-
itating solid N-substituted thiourea (E) was collected and washed
with H2O.30
(4-chlorophenyl)ethanone (0.55 g, 2.38 mmol) and 1-(2-hydroxy-
phenyl)thiourea (0.40 g, 2.38 mmol) in 4 ml ethanol to yield the
title compound. Beige powdered solid, yield: 498 mg (69%). 1H
NMR (250 MHz, DMSO-d6) d = 9.89 (s, 1H, –OH), 9.50 (s, 1H, –
NH–), 8.26 (t, J = 4.4 Hz, 1H, 4H-Ph-OH), 7.90 (d, J = 8.6 Hz, 2H,
2H,6H-Ph-Cl), 7.48 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.34 (s, 1H,
5H-thiazole), 6.88–6.84 (m, 3H, 3H,5H,6H-Ph-OH); 13C NMR
(63 MHz, DMSO-d6) d = 164.2, 148.2, 146.3, 133.4, 131.7, 129.1,
128.5, 127.1, 122.4, 119.2, 119.0, 114.9, 103.9; ESI-MS (m/
z) = 303.1 (100.0%), 305.1 (79.0%) [M+H]+; Anal. Calcd for C15H11
ClN2OS: C (59.50) H (3.66) N (9.25) S (10.59); found: C (59.17) H
(3.72) N (8.85) S (10.63); mp = 198.9 °C.
5.1.4. General procedure for the synthesis of 2-aminothiazole
derivatives (F, Scheme 1, compounds 1–37)
2-Aminothiazole derivatives 1–37 were prepared by Hantzsch
thiazole synthesis: The cyclic condensation of an
a-bromoketone
(1 equiv) and N-substituted thiourea (1 equiv) in anhydrous etha-
nol, stirring under microwave irradiation at 80 °C for 30 min. The
precipitating hydrobromide salts were collected and washed with
H2O and cold ethanol. If needed, further purification was done after
neutralization with saturated NH4OH solution, stirring at room
temperature, filtration of the precipitate and washing with cold
ethanol. Colum chromatography on silica gel (eluent: hexane/eth-
ylacetate 2:1 or dichloromethane/methanol 9:1) afforded the title
compound.30
5.1.4.5. (4-(4-(4-Chlorophenyl)thiazol-2-ylamino)phenyl)meth-
anol (5).
General procedure 5.1.4 was used to couple 2-
bromo-1-(4-chlorophenyl)ethanone (0.26 g, 1.10 mmol) and 1-(4-
(hydroxymethyl)phenyl)thiourea (0.20 g, 1.10 mmol) in 4 ml etha-
nol to yield the title compound. Pale brown solid, yield: 226 mg
(65%). 1H NMR (250 MHz, DMSO-d6) d = 10.28 (s, 1H, –NH), 7.93
(d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.65 (d, J = 8.5 Hz, 2H, 2H,6H-Ph-
CH2OH), 7.49 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.39 (s, 1H, 5H-thia-
zole), 7.28 (d, J = 8.5 Hz, 2H, 3H,5H-Ph-CH2OH), 4.79 (s, 1H, –OH–),
4.44 (s, 2H, -CH2-); 13C NMR (63 MHz, DMSO-d6) d = 160.6, 150.2,
139.4, 134.3, 131.2, 131.1, 129.3, 129.2, 128.9, 120.6, 105.0, 64.7;
ESI-MS (m/z) = 317.3 (100.0%), 319.3 (43.0%) [M+H]+; Anal. Calcd
for C16H13ClN2OS: C (60.66) H (4.14) N (8.84) S (10.12); found: C
(60.65) H (4.27) N (8.58) S (9.97); mp = 167.0 °C.
5.1.4.1. 4-(4-(4-Chlorophenyl)thiazol-2-ylamino)phenol hydro-
bromide (1, SKI-II).
General procedure 5.1.4 was used to cou-
ple 2-bromo-1-(4-chlorophenyl)-ethanone (1.39 g, 5.95 mmol) and
1-(4-hydroxyphenyl)thiourea (1.00 g, 5.94 mmol) in 30 ml ethanol
to yield the title compound. White solid, yield: 1.65 g (92%). 1H
NMR (250 MHz, DMSO-d6) d = 9.99 (s, 1H, –NH–), 9.20 (s, 1H, Ph-
OH), 7.97 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.56 (d, J = 8.9 Hz, 2H,
3H,5H-Ph-OH), 7.50 (d, J = 8.5 Hz, 2H, 2H,6H-Ph-Cl), 7.35 (s, 1H,
5H-thiazole), 6.81 (d, J = 8.9 Hz, 2H, 2H,6H-Ph-OH); 13C NMR
(63 MHz, DMSO-d6) d = 164.39, 152.40, 148.99, 133.52, 133.17,
131.81, 128.57, 127.27, 119.32, 115.43, 102.63; ESI-MS (m/
z) = 303.0 [M+H]+; Anal. Calcd for C15H11ClN2OSꢂHBr: C (46.95) H
(3.15) N (7.30) S (8.36); found: C (46.70) H (2.96) N (7.28) S
(8.09); mp = 171.0 °C.
5.1.4.6. 4-(4-Chlorophenyl)-N-(4-methoxyphenyl)thiazol-2-
amine (6).
General procedure 5.1.4 was used to couple 2-
bromo-1-(4-chlorophenyl)ethanone (0.64 g, 2.74 mmol) and 1-(4-
methoxyphenyl)thiourea (0.50 g, 2.74 mmol) in 5 ml ethanol to
yield the title compound. Grey crystalline solid, yield: 396 mg
(46%). 1H NMR (250 MHz, DMSO-d6) d = 10.09 (s, 1H, –NH), 7.92
(d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.62 (d, J = 9.0 Hz, 2H, 2H,6H-Ph-
OCH3), 7.47 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.33 (s, 1H, 5H-thia-
zole), 6.94 (d, J = 9.0 Hz, 2H, 3H,5H-Ph-OCH3), 3.74 (s, 3H, –
OCH3); 13C NMR (63 MHz, DMSO-d6) d = 163.8, 154.1, 148.7,
134.5, 133.3, 131.7, 128.5, 127.2, 118.6, 114.2, 102.9, 55.1; ESI-
MS (m/z) = 317.2 (100.0%), 319.3 (38.5%) [M+H]+; Anal. Calcd for
C16H13ClN2OS: C (60.66) H (4.14) N (8.84) S (10.12); found: C
(60.43) H (4.08) N (8.56) S (10.27); mp = 193.0 °C.
5.1.4.2.
(2).
4-(4-(4-Bromophenyl)thiazol-2-ylamino)phenol
General procedure 5.1.4 was used to couple 2-bromo-1-
(4-bromophenyl)ethanone (0.30 g, 1.08 mmol) and 1-(4-hydroxy-
phenyl)thiourea (0.18 g, 1.08 mmol) in 4 ml ethanol to yield the
title compound. White solid, yield: 299 mg (80%). 1H NMR
(250 MHz, DMSO-d6) d = 9.91 (s, 1H, –NH–), 9.11 (s, 1H, Ph-OH),
7.84 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Br), 7.60 (d, J = 8.6 Hz, 2H,
3H,5H-Ph-Br), 7.46 (d, J = 8.9 Hz, 2H, 2H,6H-Ph-OH), 7.29 (s, 1H,
5H-thiazole), 6.75 (d, J = 8.8 Hz, 2H, 3H,5H-Ph-OH); 13C NMR
(63 MHz, DMSO-d6) d = 164.3, 152.3, 148.7, 133.7, 133.1, 131.4,
127.5, 120.3, 119.2, 115.4, 102.6; ESI-MS (m/z) = 347.0 (100.0%),
349.0 (95.0%) [M+H]+; Anal. Calcd for C15H11BrN2OS: C (51.89) H
(3.19) N (8.07) S (9.42); found: C (51.70) H (3.23) N (7.97) S
(9.42); mp = 190.3 °C.
5.1.4.7. 4-(4-Chlorophenyl)-N-(2-methoxyphenyl)thiazol-2-amine
hydrobromide (7).
General procedure 5.1.4 was used to cou-
ple 2-bromo-1-(4-chlorophenyl)ethanone (1.28 g, 5.49 mmol) and
1-(2-methoxyphenyl)thiourea (1.00 g, 5.49 mmol) in 5 ml ethanol
to yield the title compound. Pale yellow solid, yield: 1.767 g
(81%). 1H NMR (250 MHz, DMSO-d6) d = 9.70 (s, 1H, –NH), 8.42
(m, 1H, 3H-Ph-OCH3), 7.90 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.48
(d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.37 (s, 1H, 5H-thiazole), 7.03–
6.97 (m, 3H, 4H,5H,6H-Ph-OCH3), 3.86 (s, 3H, –OCH3); 13C NMR
(63 MHz, DMSO-d6) d = 162.5, 148.2, 145.3, 133.0, 132.6, 132.3,
129.4, 128.3, 127.2, 122.6, 120.5, 118.5, 110.9, 104.3, 91.4, 55.6;
ESI-MS (m/z) = 317.3 (100.0%), 319.3 (38.4%) [M+H]+; Anal. Calcd
for C16H13ClN2OSꢂHBr: C (48.32) H (3.55) N (7.04) S (8.06); found:
C (48.13) H (3.41) N (7.07) S (8.09); mp = 214.0 °C.
5.1.4.3. 3-(4-(4-Chlorophenyl)thiazol-2-ylamino)phenol hydro-
bromide (3).
General procedure 5.1.4 was used to couple 2-
bromo-1-(4-chlorophenyl)ethanone (0.42 g, 1.78 mmol) and 1-(3-
hydroxyphenyl)thiourea (0.30 g, 1.78 mmol) in 4 ml ethanol to
yield the title compound. White solid, yield: 519 mg (76%). 1H
NMR (250 MHz, DMSO-d6) d = 10.16 (s, 1H, –NH–), 9.42 (s, 1H,
Ph-OH), 7.96 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.49 (d, J = 8.6 Hz,
2H, 3H,5H-Ph-Cl), 7.39 (s, 1H, 5H-thiazole), 7.31 (s, 1H, 2H-Ph-
OH), 7.10 (t, 1H, 5H-Ph-OH), 7.02 (d, J = 8.7 Hz, 1H, 4H-Ph-OH),
6.38 (d, J = 8.9 Hz, 1H, 6H-Ph-OH); 13C NMR (63 MHz, DMSO-d6)
d = 163.2, 157.9, 154.2, 148.8, 142.0, 133.3, 131.9, 128.5, 127.3,
108.6, 107.9, 104.1, 100.4; ESI-MS (m/z) = 303.3 (100.0%), 305.3
(35.0%) [M+H]+; Anal. Calcd for C15H11ClN2OSꢂHBr: C (46.95) H
(3.15) N (7.30) S (8.36); found: C (46.98) H (3.17) N (7.20) S
(8.61); mp = 278.9 °C.
5.1.4.8. 4-(4-Chlorophenyl)-N-(3,4-dimethoxyphenyl)thiazol-2-
amine (8).
General procedure 5.1.4 was used to couple 2-
bromo-1-(4-chlorophenyl)ethanone (0.20 g, 0.86 mmol) and 1-
(3,4-dimethoxyphenyl)thiourea (0.18 g, 0.86 mmol) in 3 ml etha-
nol to yield the title compound. Yellow powdered solid, yield:
253 mg (85%). 1H NMR (250 MHz, DMSO-d6) d = 10.12 (s, 1H, –

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D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
NH–), 7.92 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.50–7.46 (m, 3H,
2H,6H-Ph-Cl+5H-thiazole), 7.35 (s, 1H, 2H-Ph-(OCH3)2), 7.15 (dd,
1H, 4J = 2.4 Hz, 3J = 8.6 Hz, 6H-Ph-(OCH3)2), 6.94 (d, 1H, 5H-Ph-
(OCH3)2), 3.80 (s, 3H, 3-(OCH3)-Ph-), 3.73 (s, 3H, 4-(OCH3)-Ph);
13C NMR (63 MHz, DMSO-d6) d = 163.7, 148.9, 148.6, 143.6, 135.0,
133.4, 131.8, 128.5, 127.2, 112.7, 108.8, 103.0, 102.7, 55.9, 55.3;
ESI-MS (m/z) = 347.2 (100.0%), 348.7 (37.9%) [M+H]+; Anal. Calcd
for C17H15ClN2O2S: C (58.87) H (4.36) N (8.08) S (9.25); found: C
(58.58) H (4.28) N (7.94) S (9.30); mp = 155.0 °C.
7.94 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.69 (d, J = 8.8 Hz, 2H, 3H,5H-
Ph-SCH3), 7.49 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.40 (s, 1H, 5H-thi-
azole), 7.29 (d, J = 8.7 Hz, 2H, 2H,6H-Ph-SCH3), 2.45 (s, 3H, –SCH3);
13C NMR (63 MHz, DMSO-d6) d = 163.0, 148.7, 138.8, 133.2, 131.9,
129.2, 128.5, 128.0, 127.2, 117.5, 103.6, 16.0; ESI-MS (m/
z) = 333.2 (100.0%), 335.3 (43.8%) [M+H]+; Anal. Calcd for C16H13-
ClN2S2: C (57.73) H (3.94) N (8.42) S (19.27); found: C (57.89) H
(3.94) N (8.32) S (19.28); mp = 180.0 °C.
5.1.4.13.
thiazol-2-amine (13).
4-(4-Chlorophenyl)-N-(4-(trifluoromethoxy)phenyl)
General procedure 5.1.4 was used to
5.1.4.9. 4-(4-Chlorophenyl)-N-(3,4,5-trimethoxyphenyl)thiazol-
2-amine hydrobromide (9, ST-1780).
General procedure
couple 2-bromo-1-(4-chlorophenyl)ethanone (0.18 g, 0.85 mmol)
and 1-(4-(trifluoromethoxy)phenyl)thiourea (0.20 g, 0.85 mmol)
in 2 ml ethanol to yield the title compound. Pale yellow solid,
yield: 188 mg (60%). 1H NMR (250 MHz, DMSO-d6) d = 10.52 (s,
1H, –NH–), 7.95 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.84 (d,
J = 9.1 Hz, 2H,6H-Ph-OCF3), 7.50 (s, 1H, 5H-thiazole), 7.46 (d,
J = 8.5 Hz, 2H, 3H,5H-Ph-Cl), 7.35 (d, J = 8.4 Hz, 2H, 3H,5H-Ph-
OCF3); 13C NMR (63 MHz, DMSO-d6) d = 162.8, 148.7, 141.9,
140.2, 133.2, 132.0, 128.6, 127.3, 121.9, 117.8, 104.2; ESI-MS (m/
z) = 371.6 (100.0%), 371.6 (70.0%) [M+H]+; Anal. Calcd for C16H10-
ClF3N2OS: C (51.83) H (2.72) N (7.56) S (8.65); found: C (51.50) H
(2.66) N (7.42) S (8.81); mp = 134.1 °C.
5.1.4 was used to couple 2-bromo-1-(4-chlorophenyl)ethanone
(0.19 g, 0.83 mmol) and 1-(3,4,5-trimethoxyphenyl)thiourea
(0.20 g, 0.83 mmol) in 3 ml ethanol to yield the title compound.
White solid, yield: 378 mg (78%). 1H NMR (250 MHz, DMSO-d6)
d = 10.29 (s, 1H, –NH–), 7.92 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.47
(d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 7.40 (s, 1H, 5H-thiazole), 7.12 (s,
2H, 6H-Ph-(OCH3)3), 3.80 (s, 6H, 3-OCH3, 5-OCH3), 3.62 (s, 3H, 4-
OCH3); 13C NMR (63 MHz, DMSO-d6) d = 163.2, 161.9, 152.9,
148.5, 145.5, 137.1, 129.3, 128.3, 127.1, 103.4, 95.2, 60.0, 55.6;
ESI-MS (m/z) = 377.2 (100.0%), 379.2 (62.5%) [M+H]+; Anal. Calcd
for C18H17ClN2O3SꢂHBr: C (47.23) H (3.96) N (6.12) S (7.00); found:
C (47.22) H (3.98) N (6.19) S (7.18); mp = 209.0 °C.
5.1.4.14. 4-(4-Chlorophenyl)-N-(4-(trifluoromethyl)phenyl)thia-
5.1.4.10. N-(Benzo[d][1,3]dioxol-5-yl)-4-(4-chlorophenyl)thia-
zol-2-amine (14).
General procedure 5.1.4 was used to couple
zol-2-amine hydrobromide (10).
General procedure 5.1.4
2-bromo-1-(4-chlorophenyl)ethanone (0.21 g, 0.91 mmol) and 1-
(4-(trifluoromethyl)phenyl)thiourea (0.20 g, 0.91 mmol) in 2 ml
ethanol to yield the title compound. Yellow powdered solid, yield:
236 mg (73%). 1H NMR (250 MHz, DMSO-d6) d = 10.73 (s, 1H, –
NH–), 7.98 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.91 (d, J = 8.6 Hz, 2H,
2H,6H-Ph-CF3), 7.69 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-CF3), 7.52 (s, 1H,
5H-thiazole), 7.49 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl); 13C NMR
(63 MHz, DMSO-d6) d = 162.4, 148.9, 144.2, 133.1, 132.1, 128.6,
127.4, 126.2, 121.2, 120.7, 116.5, 104.9; ESI-MS (m/z) = 355.6
(100.0%), 357.6 (70.0%) [M+H]+; Anal. Calcd for C16H10ClF3N2S: C
(54.17) H (2.84) N (7.90) S (9.04); found: C (53.79) H (2.98) N
(7.63) S (9.34); mp = 123.4 °C.
was used to couple 2-bromo-1-(4-chlorophenyl)ethanone (0.15 g,
0.64 mmol) and 1-(benzo[d][1,3]dioxol-5-yl)thiourea (0.13 g,
0.64 mmol) in 2.5 ml ethanol to yield the title compound. White
solid, yield: 182 mg (69%). 1H NMR (250 MHz, DMSO-d6)
d = 10.17 (s, 1H, –NH–), 7.90 (d, J = 8.5, 2H 2H,6H-Ph-Cl), 7.50–
7.46 (m, 3H, 3H,5H-Ph-Cl + 5H-thiazole), 7.36 (s, 1H, 4H-
benzo[d][1,3]dioxol), 7.05 (dd, 1H, 4J = 2.3 Hz, 3J = 8.5 Hz, 6H-
benzo[d][1,3]dioxol), 6.89 (d, 1H, J = 8.4 Hz, 7H-benzo[d][1,3]diox-
ol), 5.99 (s, 2H, –CH2–); 13C NMR (63 MHz, DMSO-d6) d = 163.6,
148.6, 147.3, 141.6, 135.8, 133.3, 131.9, 128.6, 127.2, 109.6,
108.3, 103.3, 100.8, 99.5; ESI-MS (m/z) = 321.2 (100.0%), 333.5
(31.2%) [M+H]+; Anal. Calcd for C16H11ClN2O2SꢂHBr: C (46.68) H
(2.94) N (6.80) S (7.79); found: C (46.63), H (3.02), N (6.87), S
(7.75); mp = 251.0 °C.
5.1.4.15. N-(3,5-Di(trifluoromethyl)phenyl)-4-(4-chlorophenyl)
thiazol-2-amine (15).
General procedure 5.1.4 was used to
couple 2-bromo-1-(4-chlorophenyl)ethanone (0.24 g, 1.04 mmol)
and 1-(3,5-di(trifluoromethyl)phenyl)thiourea (0.30 g, 1.04 mmol)
in 3 ml ethanol to yield the title compound. Pale yellow powdered
solid, yield: 313 mg (71%). 1H NMR (250 MHz, DMSO-d6) d = 11.12
(s, 1H, –NH–), 8.43 (s, 2H, 2H,6H-Ph-(CF3)2), 7.90 (d, J = 8.6 Hz, 2H,
2H,6H-Ph-Cl), 7.60 (s, 1H, 4H-Ph-(CF3)2), 7.58 (s, 1H, 5H-thiazole),
7.51 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl); 13C NMR (63 MHz, DMSO-
d6) d = 162.2, 148.7, 142.4, 132.9, 132.3, 130.6, 128.7, 127.0,
125.4, 121.1, 116.2, 105.5; ESI-MS (m/z) = 423.1[M+H]+; Anal.
Calcd for C17H9ClF6N2S: C (48.30) H (2.15) N (6.63) S (7.58); found:
C (47.91) H (2.23) N (6.47) S (7.75); mp = 139.5 °C.
5.1.4.11.
4-(4-Chlorophenyl)-N-(2,3-dihydrobenzo[b][1,4]
General
dioxin-6-yl)thiazol-2-amine hydrobromide (11).
procedure 5.1.4 was used to couple 2-bromo-1-(4-chloro-
phenyl)ethanone (0.22 g, 0.95 mmol) and 1-(2,3-dihydro-
benzo[b][1,4]dioxin-6-yl)thiourea (0.20 g, 0.95 mmol) in 3 ml
ethanol to yield the title compound. Grey powdered solid, yield:
263 mg (65%). 1H NMR (250 MHz, DMSO-d6) d = 10.09 (s, 1H, –
NH–), 7.89 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.49 (d, J = 8.6 Hz, 2H,
3H,5H-Ph-Cl),
7.40
(d,
J = 2.5 Hz,
1H,
7H-2,3-dihydro-
benzo[b][1,4]dioxin), 7.35 (s, 1H, 5H-thiazole), 7.01 (dd,
4J = 2.5 Hz, 3J = 8.8 Hz, 1H, 8H-2,3-dihydrobenzo[b][1,4]dioxin),
6.82 (s, 1H, 5H-2,3-dihydrobenzo[b][1,4]dioxin), 4.24–4.22 (m,
4H, –CH2–CH2–); 13C NMR (63 MHz, DMSO-d6) d = 163.6, 148.6,
143.1, 138.0, 135.0, 133.3, 131.9, 138.6, 127.2, 117.1, 110.4,
106.1, 103.1, 64.1; ESI-MS (m/z) = 344.7 (100.0%) [M+H]+; Anal.
Calcd for C17H13ClN2O2SꢂHBr: C (47.96) H (3.31) N (6.58) S (7.53);
found: C (47.84) H (3.29) N (6.51) S (7.43); mp = 247.0 °C.
5.1.4.16.
amine hydrobromide (16).
to couple 2-bromo-1-(4-chlorophenyl)ethanone
1.29 mmol) and 1-(4-isopropylphenyl)thiourea
4-(4-Chlorophenyl)-N-(4-isopropylphenyl)thiazol-2-
General procedure 5.1.4 was used
(0.30 g,
(0.25 g,
1.29 mmol) in 3 ml ethanol to yield the title compound. White
solid, yield: 410 mg (78%). 1H NMR (250 MHz, DMSO-d6)
d = 10.23 (s, 1H, –NH–), 7.94 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.62
(d, J = 8.6 Hz, 2H, 2H,6H-Ph-CH(CH3)2), 7.49 (d, J = 8.6 Hz, 2H,
3H,5H-Ph-Cl), 7.38 (s, 1H, 5H-thiazole), 7.22 (d, J = 8.5 Hz, 2H,
3H,5H-Ph-CH(CH3)2), 2.86 (septet, 1H, –CH(CH3)2), 1.20 (d,
J = 6.9 Hz, 6H, –CH(CH3)2); 13C NMR (63 MHz, DMSO-d6)
d = 163.4, 162.1, 148.6, 142.0, 138.2, 132.2, 129.5, 128.3, 126.7,
117.4, 103.3, 32.6, 23.6; ESI-MS (m/z) = 329.3 (100.0%), 331.4
5.1.4.12. 4-(4-Chlorophenyl)-N-(4-(methylthio)phenyl)thiazol-
2-amine (12).
2-bromo-1-(4-chlorophenyl)ethanone (0.41 g, 1.77 mmol) and 1-
(4-(methylthio)phenyl)thiourea (0.35 g, 1.77 mmol) in 5 ml etha-
nol to yield the title compound. Pale yellow crystalline solid, yield:
335 mg (57%). 1H NMR (250 MHz, DMSO-d6) d = 10.32 (s, 1H, –NH),
General procedure 5.1.4 was used to couple

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5363
(35.0%) [M+H]+; Anal. Calcd for C18H17ClN2SꢂHBr: C (52.76) H (4.43)
5.1.4.21.
4-(4-Chlorophenyl)-5-methyl-N-(4-(methyl-
N (6.84) S (7.83); found: C (53.07) H (4.45) N (6.62) S (7.95);
thio)phenyl)thiazol-2-amine hydrobromide (21).
General
mp = 214.0 °C.
procedure 5.1.4 was used to couple 2-bromo-1-(4-chloro-
phenyl)propan-1-one (0.35 g, 1.77 mmol) and 1-(4-(methyl-
thio)phenyl)thiourea (0.44 g, 1.77 mmol) in 5 ml ethanol to yield
the title compound. White crystalline solid, yield: 580 mg (77%).
1H NMR (250 MHz, DMSO-d6) d = 10.33 (s, 1H, –NH–), 7.67 (d,
J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.60 (d, J = 8.7 Hz, 2H, 3H,5H-Ph-Cl),
7.51 (d, J = 8.6 Hz, 2H, 4H-Ph-SCH3), 7.24 (d, J = 8.8 Hz, 2H,
2H,6H-Ph-SCH3), 2.43 (s, 3H, 5H-thiazole), 2.39 (s, 3H, Ph-SCH3);
13C NMR (63 MHz, DMSO-d6) d = 179.3, 142.5, 131.9, 130.7, 129.7,
128.8, 128.6, 128.3, 128.0, 126.5, 125.9, 118.3, 118.2, 117.0, 16.0,
14.5, 11.8; ESI-MS (m/z) = 347.4 (100.0%), 349.2 (42.5%) [M+H]+;
Anal. Calcd for C17H15ClN2S2ꢂHBr: C (47.73) H (3.77) N (6.55) S
(14.99); found: C (47.68) H (3.80) N (6.54) S (15.17); mp = 238.0 °C.
5.1.4.17. N1-(4-(4-Chlorophenyl)thiazol-2-yl)-N4,N4-dimethyl-
benzene-1,4-diamine
hydrobromide
(17).
General
procedure 5.1.4 was used to couple 2-bromo-1-(4-chlorophenyl)-
ethanone (0.36 g, 1.54 mmol) and 1-(4-(dimethylamino)
phenyl)-2-thiourea (0.30 g, 1.54 mmol) in 3 ml ethanol to yield the
title compound. White solid, yield: 515 mg (81%). 1H NMR
(250 MHz, DMSO-d6) d = 11.58 (s, 1H, –NH–), 7.95 (d, J = 8.6 Hz,
2H, 2H,6H-Ph-Cl), 7.95 (m, 2H, 3H,5H-Ph-N(CH3)2), 7.61 (m, 2H,
2H,6H-Ph-N(CH3)2), 7.50–7.48 (m, 3H, 5H-thiazole, 3H,5H-Ph-Cl),
3.15 (s, 6H, –N(CH3)2); 13C NMR (63 MHz, DMSO-d6) d = 164.1,
155.7, 153.8, 150.3, 141.6, 135.9, 133.1, 131.9, 129.5, 127.2, 118.5,
45.3; ESI-MS (m/z) = 330.9 (100.0%), 332.8 (38.0%) [M+H]+; Anal.
Calcd for C17H16ClN3SꢂHBr: C (49.71) H (4.17) N (10.23) S (7.81);
found: C (49.67) H (4.23) N (10.05) S (7.83); mp = 213.0 °C.
5.1.4.22.
yl)thiazol-2-amine (22).
to couple 2-bromo-1-(4-chlorophenyl)propan-1-one (0.36 g,
1.44 mmol) and 1-(4-methylthiazol-2-yl)thiourea (0.25 g,
4-(4-Chlorophenyl)-5-methyl-N-(4-methylthiazol-2-
General procedure 5.1.4 was used
5.1.4.18.
4-(4-(4-Chlorophenyl)-5-methylthiazol-2-ylami-
1.44 mmol) in 4 ml ethanol to yield the title compound. White
crystalline solid, yield: 289 mg (62%). 1H NMR (250 MHz, DMSO-
d6) d = 12.00 (s, 1H, –NH–), 7.74 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl),
7.51 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 6.54 (s, 1H, 5H-thiazole),
2.47 (s, 3H, 5-CH3-thiazole), 2.22 (s, 3H, 4-CH3-thiazole; 13C NMR
(63 MHz, DMSO-d6) d = 162.3, 159.8, 152.4, 150.0, 133.6, 131.5,
129.3, 128.3, 105.6, 16.8, 12.0; ESI-MS (m/z) = 322.2 (10.0%)
[M+H]+, 344.2 (100.0%) [M+Na]+; Anal. Calcd for C14H12ClN3S2: C
(52.25) H (3.76) N (13.06) S (19.93); found: C (52.30) H (3.87) N
(13.06) S (20.24); mp = 256.0 °C.
no)phenol (18).
General procedure 5.1.4 was used to couple
2-bromo-1-(4-chlorophenyl)propan-1-one (0.25 g, 1.01 mmol)
and 1-(4-hydroxyphenyl)thiourea (0.17 g, 1.01 mmol) in 3 ml eth-
anol to yield the title compound. Brown solid, yield: 286 mg (89%).
1H NMR (250 MHz, DMSO-d6) d = 9.70 (s, 1H, –NH–), 9.10 (s, 1H,
OH), 7.67 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.49 (d, J = 8.6 Hz, 2H,
3H,5H-Ph-Cl), 7.40 (d, J = 8.9 Hz, 2H, 2H,6H-Ph-OH), 6.71 (d,
J = 8.9 Hz, 2H, 3H,5H-Ph-OH), 2.38 (s, 3H, –CH3); 13C NMR
(63 MHz, DMSO-d6) d = 160.4, 152.1, 143.8, 134.0, 133.2, 131.3,
129.4, 128.2, 119.0, 115.8, 115.3, 11.9; ESI-MS (m/z) = 317.3
(100.0%), 319.3 (38.0%) [M+H]+; Anal. Calcd for C16H13ClN2OS: C
(60.66) H (4.14) N (8.84) S (10.12); found: C (60.35) H (3.96) N
(8.57) S (9.71); mp = 163.0 °C.
5.1.4.23. 4-(2,40-Bithiazol-20-ylamino)phenol (23).
General
procedure 5.1.4 was used to couple 2-(bromoacetyl)-1,3-thiazole
(0.15 g, 0.73 mmol) and 1-(4-hydroxyphenyl)thiourea (0.12 g,
0.73 mmol) in 2 ml ethanol to yield the title compound. Pale brown
solid, yield: 130 mg (65%). 1H NMR (250 MHz, DMSO-d6) d = 10.08
(s, 1H, –NH–), 9.19 (s, 1H, –OH), 7.85 (d, J = 3.2 Hz, 1H, 4H-thia-
zole), 7.70 (d, J = 3.2 Hz, 1H, 5H-thiazole), 7.41 (d, J = 8.8 Hz, 2H,
2H,6H-Ph), 7.36 (s, 1H, 5H-aminothiazole), 6.76 (d, J = 8.8 Hz, 2H,
3H,5H-Ph); 13C NMR (63 MHz, DMSO-d6) d = 164.9, 162.5, 152.7,
144.2, 143.6, 132.7, 119.9, 119.8, 115.4, 104.2; ESI-MS (m/
5.1.4.19. 4-(4-Chlorophenyl)-N-(4-methoxyphenyl)-5-methyl-
thiazol-2-amine hydrobromide (19).
General procedure
5.1.4 was used to couple 2-bromo-1-(4-chlorophenyl)propan-1-
one (0.30 g, 1.21 mmol) and 1-(4-methoxyphenyl)thiourea
(0.22 g, 1.21 mmol) in 3 ml ethanol to yield the title compound.
White crystalline solid, yield: 425 mg (85%). 1H NMR (250 MHz,
DMSO-d6) d = 10.21 (s, 1H, –NH–), 7.65 (d, J = 8.6 Hz, 2H, 2H,6H-
Ph-Cl), 7.54–7.50 (m, (2+2)H, 3H,5H-Ph-Cl, 2H,6H-Ph-OCH3), 6.94
(d, J = 9.0 Hz, 2H, 3H,5H-Ph-OCH3), 3.74 (s, 3H, Ph-OCH3), 2.36 (s,
3H, 5-CH3-thiazole); 13C NMR (63 MHz, DMSO-d6) d = 159.6,
153.8, 147.9, 142.4, 134.6, 131.9, 129.6, 128.8, 121.7, 117.6,
114.7, 55.5, 15.8; ESI-MS (m/z) = 331.3 (100.0%), 333.3 (37.6%)
[M+H]+; Anal. Calcd for C17H15ClN2OSꢂHBr: C (49.59) H (3.92) N
(6.80) S (7.79); found: C (49.86) H (3.88) N (6.65) S (7.47);
mp = 238.0 °C.
z) = 274.0 (100.0%) [MꢀH]ꢀ; Anal. Calcd for C12H9N3OS2:
C
(52.34) H (3.29) N (15.26) S (23.29); found: C (52.14) H (3.44) N
(15.04) S (23.66); mp = 204.0 °C.
5.1.4.24.
hydrobromide (24).
N-(4-Methylthiazol-2-yl)-2,40-bithiazol-20-amine
General procedure 5.1.4 was used to
couple 2-(bromoacetyl)-1,3-thiazole (0.30 g, 1.46 mmol) and 1-
(4-methylthiazol-2-yl)thiourea (0.25 g, 1.46 mmol) in 3 ml ethanol
to yield the title compound. Pale brown powdered solid, yield:
387 mg (73%). 1H NMR (250 MHz, DMSO-d6) d = 7.89 (d,
J = 3.2 Hz, 1H, 4H-thiazol-2-yl), 7.75 (d, J = 3.2 Hz, 1H, 5H-thiazol-
2-yl), 7.63 (s, 1H, 5H-aminothiazole), 7.58 (s, 1H, –NH–), 6.62 (s,
1H, 5H-(4-methylthiazol-2-yl)), 2.24 (s, 3H, -CH3); 13C NMR
(63 MHz, DMSO-d6) d = 162.3, 161.9, 158.9, 143.6, 143.3, 120.4,
120.2, 108.6, 108.2, 14.6; ESI-MS (m/z) = 281.1 (100.0%) [M+H]+;
Anal. Calcd for C10H8N4S3ꢂHBr: C (33.24) H (2.51) N (15.51) S
5.1.4.20.
4-(4-Chlorophenyl)-5-methyl-N-(3,4,5-trimethoxy-
General proce-
phenyl)thiazol-2-amine hydrobromide (20).
dure 5.1.4 was used to couple 2-bromo-1-(4-chlorophenyl)
propan-1-one (0.20 g, 0.83 mmol) and 1-(3,4,5-trimethoxy-
phenyl)thiourea (0.20 g, 0.83 mmol) in 2 ml ethanol to yield the
title compound. White crystalline solid, yield: 290 mg (75%). 1H
NMR (250 MHz, DMSO-d6) d = 10.34 (s, 1H, –NH–), 7.70 (d,
J = 8.4 Hz, 2H, 2H,6H-PhCl), 7.51 (d, J = 8.6 Hz, 2H, 3H,5H-Ph-Cl),
7.04 (s, 2H, 2H,6H-Ph(OCH3)3), 3.76 (s, 6H, 3-OCH3, 5-OCH3),
3.61 (s, 3H, 4-OCH3); 13C NMR (63 MHz, DMSO-d6) d = 168.7,
161.7, 152.8, 150.5, 146.5, 131.9, 129.6, 128.7, 128.3, 118.0,
95.2, 60.7, 55.9, 11.7; ESI-MS (m/z) = 391.9 (100.0%), 393.9
(40.1%) [M+H]+; Anal. Calcd for C19H19ClN2O3SꢂHBr: C (48.37) H
(4.27) N (5.94) S (6.80); found: C (48.53) H (4.23) N (6.02) S
(7.18); mp = 206.0 °C.
(26.62); found:
C (33.47) H (2.87) N (15.42) S (26.48);
mp = 261.0 °C.
5.1.4.25.
hydrobromide (25).
4-Adamantyl-N-(4-methoxyphenyl)thiazol-2-amine
General procedure 5.1.4 was used to
couple 1-adamantylbromomethylketone (0.30 g, 1.17 mmol) and
1-(4-methoxyphenyl)thiourea (0.21 g, 1.17 mmol) in 3 ml ethanol
to yield the title compound. White crystalline solid, yield:
378 mg (77%). 1H NMR (250 MHz, DMSO-d6) d = 10.45 (s, 1H, –

5364
D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
NH–), 7.46 (d, J = 8.9 Hz, 2H, 2H,6H-Ph-Cl), 7.00 (d, J = 8.9 Hz, 2H,
3H,5H-Ph-Cl), 6.44 (s, 1H, 5H-thiazole), 3.78 (s, 3H, –OCH3), 2.03
(m, 3H, –CH–(adamantyl)), 1.87 (m, 6H, –CH2–(adamantyl)), 1.71
(m, 6H, –CH2–(adamantyl)); 13C NMR (63 MHz, DMSO-d6)
d = 161.3, 154.6, 134.2, 118.6, 114.1, 99.1, 85.0, 55.2, 40.6, 37.6,
36.0, 28.0; ESI-MS (m/z) = 341.4 (100.0%) [M+H]+; Anal. Calcd for
27.8, 15.9; ESI-MS (m/z) = 358.1 (100.0%) [M+H]+; Anal. Calcd for
C20H24N2S2ꢂHBr: C (54.91) H (5.76) N (6.40) S (14.66); found: C
(54.79) H (5.59) N (6.41) S (15.16); mp = 264.0 °C.
5.1.4.30.
4-Adamantyl-N-(4-isopropylphenyl)thiazol-2-amine
General procedure 5.1.4 was used to
hydrobromide (30).
C
20H24N2OSꢂHBr: C (57.00) H (5.98) N (6.65) S (7.61); found: C
couple 1-adamantylbromomethylketone (0.33 g, 1.29 mmol) and
1-(4-isopropylphenyl)-2-thiourea (0.25 g, 1.29 mmol) in 3 ml eth-
anol to yield the title compound. White solid, yield: 370 mg
(66%). 1H NMR (250 MHz, DMSO-d6) d = 10.45 (s, 1H, –NH–), 7.45
(d, J = 8.5 Hz, 2H, 2H,6H-Ph), 7.26 (d, J = 8.5 Hz, 2H, 3H,5H-Ph),
6.44 (s, 1H, 5H-thiazole), 2.88 (septett, 1H, –CH(CH3)2), 2.03 (m,
3H, -CH–(adamantyl)), 1.89 (m, 6H, –CH2–(adamantyl)), 1.71 (m,
6H, –CH2–(adamantyl)), 1.19 (d, J = 6.9 Hz, 6H, –CH(CH3)2); 13C
NMR (63 MHz, DMSO-d6) d = 160.9, 155.1, 141.5, 138.7, 127.0,
118.7, 99.5, 41.1, 37.7, 36.1, 32.8, 28.4, 23.8; ESI-MS (m/z) = 353.9
(100.0%) [M+H]+; Anal. Calcd for C22H28N2SꢂHBr: C (60.96) H
(6.74) N (6.46) S (7.40); found: C (61.04) H (6.43) N (6.39) S
(7.65); mp = 276.0 °C.
(57.22) H (5.62) N (6.43) S (7.28); mp = 255.0 °C.
5.1.4.26.
amine (26).
4-Adamantyl-N-(3,4-dimethoxyphenyl)thiazol-2-
General procedure 5.1.4 was used to couple 1-
adamantylbromomethylketone (0.50 g, 1.94 mmol) and 1-(3,4-
dimethoxyphenyl)thiourea (0.41 g, 1.94 mmol) in 8 ml ethanol to
yield the title compound. Beige solid, yield: 502 mg (70%). 1H
NMR (250 MHz, DMSO-d6) d = 9.87 (s, 1H, –NH–), 7.66 (d,
J = 2.2 Hz, 1H, 6H-Ph), 6.94 (dd, 4J = 2.2 Hz, 3J = 8.7 Hz, 1H, 5H-Ph),
6.88 (s, 1H, 2H-Ph), 6.27 (s, 1H, 5H-thiazole), 3.77 (s, 3H, 3-
OCH3), 3.70 (s, 3H, 4-OCH3), 2.02 (m, 3H, –CH–(adamantyl)), 1.89
(m, 6H, –CH2–(adamantyl)), 1.71 (m, 6H, –CH2–(adamantyl)); 13C
NMR (63 MHz, DMSO-d6) d = 162.9, 161.7, 148.8, 143.0, 135.6,
112.8, 108.2, 102.4, 98.2, 55.9, 55.0, 41.5, 36.4, 36.7, 27.9; ESI-MS
(m/z) = 371.3 (100.0%) [M+H]+; Anal. Calcd for C21H26N2O2S: C
(68.08) H (7.07) N (7.56) S (8.65); found: C (67.88) H (7.05) N
(7.38) S (8.84); mp = 195.0 °C.
5.1.4.31.
zene-1,4-diamine hydrobromide (31).
N1-(4-Adamantyl-thiazol-2-yl)-N4,N4-dimethylben-
General procedure
5.1.4 was used to couple 1-adamantylbromomethylketone
(0.40 g, 1.54 mmol) and 1-(4-(dimethylamino)phenyl)-2-thiourea
(0.30 g, 1.54 mmol) in 4 ml ethanol to yield the title compound.
Yellow solid, yield: 530 mg (79%). 1H NMR (250 MHz, DMSO-d6)
d = 10.35 (s, 1H, –NH–), 7.74–7.52 (m, (2+2)H, 2H,6H-Ph, 3H,5H-
Ph), 6.43 (s, 1H, 5H-thiazole), 3.12 (m, 6H, –N(CH3)2), 2.03 (m,
3H, –CH2–(adamantyl)), 1.90 (m, 6H, –CH2–(adamantyl)), 1.72
(m, 6H, –CH2–(adamantyl)); 13C NMR (63 MHz, DMSO-d6)
d = 166.3, 157.4, 140.9, 124.0, 118.0, 115.6, 99.4, 41.3, 40.4, 36.2,
35.9, 27.8; ESI-MS (m/z) = 354.9 (100.0%) [M+H]+; Anal. Calcd for
5.1.4.27.
amine (27).
4-Adamantyl-N-(3,4,5-trimethoxyphenyl)thiazol-2-
General procedure 5.1.4 was used to couple 1-
adamantylbromomethylketone (0.30 g, 1.17 mmol) and 1-(3,4,5-
trimethoxyphenyl)thiourea (0.28 g, 1.17 mmol) in 3 ml ethanol to
yield the title compound. Pale yellow powdered solid, yield:
276 mg (59%). 1H NMR (250 MHz, DMSO-d6) d = 10.00 (s, 1H, –
NH–), 7.11 (s, 2H, 2H,6H-Ph), 6.31 (s, 1H, 5H-thiazole), 3.77 (s,
3H, 3-OCH3, 5-OCH3), 3.60 (s, 3H, 4-OCH3), 2.01 (m, 3H, –CH–(ada-
mantyl)), 1.90 (m, 6H, –CH2–(adamantyl)), 1.70 (m, 6H, –CH2–(ada-
mantyl)); 13C NMR (63 MHz, DMSO-d6) d = 162.4, 161.6, 152.7,
137.4, 131.4, 96.7, 94.4, 60.0, 55.3, 41.5, 36.4, 36.0, 27.8; ESI-MS
(m/z) = 401.3 (100.0%) [M+H]+; Anal. Calcd for C22H28N2O3S: C
(65.97) H (7.05) N (6.99) S (8.01); found: C (65.82) H (6.58) N
(6.57) S (7.81); mp = 214.0 °C.
C
21H27N3SꢂHBr: C (58.06) H (6.50) N (9.67) S (7.38); found: C
(58.15) H (6.44) N (9.60) S (7.58); mp = 256.0 °C.
5.1.4.32.
4-Adamantyl-N-(benzo[d][1,3]dioxol-5-yl)thiazol-2-
amine (32).
General procedure 5.1.4 was used to couple 1-
adamantylbromomethylketone (0.15 g, 0.58 mmol) and 1-
(benzo[d][1,3]dioxol-5-yl)thiourea (0.11 g, 0.58 mmol) in 2.5 ml
ethanol to yield the title compound. White solid, yield: 141 mg
(68%). 1H NMR (250 MHz, DMSO-d6) d = 9.92 (s, 1H, –NH–), 7.42
(d, J = 2.0 Hz, 6H-benzo[d][1,3]dioxol), 6.93 (dd, 4J = 2.0 Hz,
3J = 8.4 Hz, 1H, 7H-benzo[d][1,3]dioxol), 6.84 (d, J = 8.4 Hz, 1H,
4H-benzo[d][1,3]dioxol), 6.29 (s, 1H, 5H-thiazole), 5.96 (s, 2H, –
OCH2O–), 2.02 (m, 3H, –CH2–(adamantyl)), 1.88 (m, 6H, –CH2–
(adamantyl)), 1.72 (m, 6H, –CH2–(adamantyl)); 13C NMR
(63 MHz, DMSO-d6) d = 162.8, 161.9, 147.2, 141.1, 136.3, 109.0,
108.1, 100.6, 99.2, 98.4, 41.5, 36.3, 35.9, 27.8; ESI-MS (m/
5.1.4.28.
(28).
4-Adamantyl-N-(2-methoxyphenyl)thiazol-2-amine
General procedure 5.1.4 was used to couple 1-adaman-
tylbromomethylketone (0.30 g, 1.17 mmol) and 1-(2-methoxy-
phenyl)thiourea (0.21 g, 1.17 mmol) in 3 ml ethanol to yield the
title compound. Pale yellow powdered solid, yield: 241 mg (61%).
1H NMR (250 MHz, DMSO-d6) d = 9.25 (s, 1H, –NH–), 8.36–8.32
(m, 1H, 3H-Ph), 7.02–6.90 (m, 3H, 4H,5H,6H-Ph), 6.30 (s, 1H, 5H-
thiazole) 3.84 (s, 3H, –OCH3), 2.02 (m, 3H, –CH2–(adamantyl)),
1.89 (m, 6H, –CH2–(adamantyl)), 1.72 (m, 6H, –CH2–(adamantyl));
13C NMR (63 MHz, DMSO-d6) d = 162.9, 161.5, 147.7, 130.5, 121.4,
120.5, 117.6, 110.7, 99.5, 55.5, 41.5, 36.3, 35.9, 27.8; ESI-MS (m/
z) = 341.3 (100.0%) [M+H]+; Anal. Calcd for C20H24N2OS: C (70.55)
H (7.10) N (8.23) S (9.42); found: C (70.26) H (6.70) N (7.61) S
(9.62); mp = 107.0 °C.
z) = 354.8 (100.0%) [M+H]+; Anal. Calcd for C20H22N2O2S:
C
(67.77) H (6.26) N (7.90) S (9.05); found: C (67.85) H (6.25) N
(7.86) S (9.18); mp = 173.0 °C.
5.1.4.33.
yl)thiazol-2-amine (33).
to couple 1-adamantylbromomethylketone (0.25 g, 0.95 mmol)
and 1-(2,3-dihydrobenzo[b][1,4]dioxin-6-yl)thiourea (0.20 g,
4-Adamantyl-N-(2,3-dihydrobenzo[b][1,4]dioxin-6-
General procedure 5.1.4 was used
5.1.4.29.
amine hydrobromide (29).
4-Adamantyl-N-(4-(methylthio)phenyl)thiazol-2-
General procedure 5.1.4 was used
0.95 mmol) in 3 ml ethanol to yield the title compound. Pale brown
solid, yield: 132 mg (38%). 1H NMR (250 MHz, DMSO-d6) d = 9.83
(s, 1H, –NH–), 7.36 (s, 1H, 5H-(2,3-dihydrobenzo[b][1,4]dioxin)),
to couple 1-adamantylbromomethylketone (0.30 g, 1.17 mmol)
and 1-(2-methoxyphenyl)thiourea (0.23 g, 1.17 mmol) in 3 ml eth-
anol to yield the title compound. White crystalline solid, yield:
469 mg (92%). 1H NMR (250 MHz, DMSO-d6) d = 10.33 (s, 1H, –
NH–), 7.54 (d, J = 8.7 Hz, 2H, 3H,5H-Ph), 7.28 (d, J = 8.7 Hz, 2H,
2H,6H-Ph), 6.41 (s, 1H, 5H-thiazole), 2.45 (s, 3H, –SCH3), 2.03 (m,
3H, –CH2–(adamantyl)), 1.89 (m, 6H, –CH2–(adamantyl)), 1.72
(m, 6H, –CH2–(adamantyl)); 13C NMR (63 MHz, DMSO-d6)
d = 160.5, 155.0, 138.5, 127.9, 118.2, 99.3, 86.2, 41.2, 36.2, 36.0,
6.88
(dd,
4J = 2.5 Hz,
3J = 8.8 Hz,
1H,
7H-(2,3-dihydro-
benzo[b][1,4]dioxin)), 6.77 (d, J = 8.7 Hz, 1H, 8H-(2,3-dihydro-
benzo[b][1,4]dioxin)), 6.27 (s, 1H, 5H-thiazole), 4.20 (m, 4H,
2H,3H-(2,3-dihydrobenzo[b][1,4]dioxin)), 2.02 (m, 3H, –CH2–(ada-
mantyl)), 1.87 (m, 6H, –CH2–(adamantyl)), 1.72 (m, 6H, –CH2–(ada-
mantyl)); 13C NMR (63 MHz, DMSO-d6) d = 162.8, 161.9, 143.0,
137.6, 135.5, 116.9, 110.0, 105.7, 98.2, 64.1, 41.5, 36.3, 35.9, 27.8;

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5365
ESI-MS (m/z) = 369.6 (100.0%) [M+H]+; Anal. Calcd for C21H24N2O2-
S: C (68.45) H (6.56) N (7.60) S (8.70); found: C (68.15) H (6.49) N
(7.53) S (8.49); mp = 151.0 °C.
5.1.4.38.
thoxyphenyl)acrylamide
(E)-N-(4-(4-Chlorophenyl)thiazol-2-yl)-3-(3,4-dime-
(38)33,34
4-(4-Chlorphenyl)thia-
.
zol-2-amine (0.25 g, 0.12 mmol) was solved together with (E)-3-
(3,4-dimethoxyphenyl)acryloyl chloride (0.27 g, 0.12 mmol) in
3 ml pyridine. The resulting mixture was heated to 60 °C under
microwave irradiation for 30 min. The crude product was purified
by column chromatography on silica gel (eluent: dichloromethane/
methanol 9:1 (v/v)) that afforded the title compound. Brown solid,
yield: 160 mg (33%). 1H NMR (250 MHz, DMSO-d6) d = 12.38 (s, 1H,
–NH–), 7.93 (d, J = 8.3 Hz, 2H, 2H,6H-Ph-Cl), 7.71 (s, 1H, 5H-thia-
zole), 7.66 (d, J = 15.7 Hz, 1H, ANHACOACH@CHA), 7.50 (d,
J = 8.3 Hz, 2H, 3H,5H-Ph-Cl), 7.22 (m, 2H, 2H,6H-Ph-(OCH3)2),
7.04 (m, 1H, 5H-Ph-(OCH3)2), 6.82 (d, J = 15.7 Hz, 1H,
ANHACOACH@CHA), 3.82 (m, 6H, 3-OCH3, 4-OCH3); 13C NMR
(63 MHz, DMSO-d6) d = 163.6, 158.2, 150.8, 148.8, 147.7, 142.5,
133.1, 132.1, 128.6, 127.3, 127.0, 121.9, 117.0, 111.8, 110.5,
109.0, 55.5; ESI-MS (m/z) = 401.1 (100.0%) [M+H]+; HR-MS (C20H17-
ClN2O3S) calcd: 401.07212; found: 401.07192; mp = 184.2 °C.
5.1.4.34. 4-Adamantyl-5-methyl-N-(4-methylthiazol-2-yl)thia-
zol-2-amine hydrobromide (34).
General procedure 5.1.4
was used to couple 1-(1-adamantyl)-2-bromopropan-1-on
(0.24 g, 0.87 mmol) and 1-(4-methylthiazol-2-yl)thiourea (0.15 g,
0.87 mmol) in 2 ml ethanol to yield the title compound. White
solid, yield: 214 mg (58%). 1H NMR (250 MHz, DMSO-d6)
d = 12.98 (s, 1H, –NH–), 6.87 (s, 1H, 5H-(4-methylthiazol-2-yl)),
2.48 (s, 3H, 5-CH3-thiazole), 2.28 (s, 3H, 4-CH3-(4-methylthiazol-
yl), 2.06 (m, (3+6)H, -CH–(adamantyl), –CH2–(adamantyl)), 1.72
(m, 6H, –CH2–(adamantyl)); 13C NMR (63 MHz, DMSO-d6)
d = 156.8, 155.3, 147.8, 146.8, 115.2, 104.7, 38.4, 37.5, 35.7, 27.7,
14.2, 12.9; ESI-MS (m/z) = 346.2 (100.0%) [M+H]+; Anal. Calcd for
C18H23N3S2ꢂHBr: C (50.70) H (5.67) N (9.85) S (15.04); found: C
(50.46) H (5.68) N (9.91) S (15.40); mp = 300.0 °C (sublimation).
5.1.4.35. 4-Adamantyl-5-methyl-N-(2-methoxyphenyl)thiazol-
5.1.4.39. (E)-N-(4-(4-Chlorophenyl)thiazol-2-yl)-3-(3,4,5-trime-
2-amine hydrobromide (35).
General procedure 5.1.4 was
thoxyphenyl)acrylamide (39, ST-1577)33
.
(E)-3-(3,4,5-Tri-
used to couple 1-(1-adamantyl)-2-bromopropan-1-on (0.25 g,
0.92 mmol) and 1-(2-methoxyphenyl)thiourea (0.17 g, 0.92 mmol)
in 2 ml ethanol to yield the title compound. White crystalline solid,
yield: 212 mg (53%). 1H NMR (250 MHz, DMSO-d6) d = 10.04 (s, 1H,
–NH–), 7.66 (m, 1H, 3H-Ph), 7.27–7.15 (m, 1H, 4H,5H-Ph), 7.06–
7.00 (m, 1H, 6H-Ph), 3.86 (s, 3H, –OCH3), 2.36 (s, 3H, –CH3-thia-
zole), 2.03 (m, (3+6)H, –CH–(adamantyl), –CH2–(adamantyl)),
1.73 (m, 6H, –CH2–(adamantyl)); 13C NMR (63 MHz, DMSO-d6)
d = 163.7, 152.1, 150.7, 130.5, 127.1, 126.8, 126.7, 112.8, 112.1,
55.8, 40.4, 37.0, 35.6, 27.7, 12.2; ESI-MS (m/z) = 355.3 (100.0%)
[M+H]+; Anal. Calcd for C21H26N2OSꢂHBr: C (57.93) H (6.25) N
(6.43) S (7.36); found: C (57.79) H (6.14) N (6.29) S (7.31);
mp = 222.0 °C.
methoxyphenyl)acrylic acid (0.30 g, 1.26 mmol) was solved in
2 ml DMF. N,N0-Diisopropylcarbodiimide (DIC, 0.15 g, 1.26 mmol)
and 1-hydroxybenzotriazol (HOBt, 0.17 g, 1.26 mmol) were added
and the solution was stirred for 10 min at room temperature. In
a
second reaction batch, 4-(4-chlorphenyl)thiazol-2-amine
(0.24 g, 1.14 mmol) was suspended in 4 ml dichloromethane.
N,N-Diisopropylethylamine (DIPEA, 0.22 g, 1.72 mmol) was added.
Both reaction batches were combined and stirred under inert argon
atmosphere at room temperature for 48 h. The crude product was
purified by column chromatography on silica gel (eluent: hexane/
EtOAc 1:2 (v/v)) that afforded the title compound. Yellow solid,
yield: 147 mg (30%). 1H NMR (250 MHz, DMSO-d6) d = 12.42 (s,
1H, –NH–), 7.94 (d, J = 8.6 Hz, 2H, 2H,6H-Ph-Cl), 7.73 (s, 1H, 5H-thi-
azole), 7.66 (d, J = 16.0 Hz, 1H, ANHACOACH@CHA), 7.51 (d,
J = 8.6 Hz, 2H, 3H,5H-Ph-Cl), 6.99 (s, 2H, 2H,6H-Ph(OCH3)3), 6.91
(d, J = 15.8 Hz, 1H, ANHACOACH@CHA), 3.85 (s, 6H, 3-OCH3, 5-
OCH3), 3.72 (s, 3H, 4-OCH3); 13C NMR (63 MHz, DMSO-d6)
d = 163.4, 158.0, 153.0, 147.7, 142.4, 139.4, 133.0, 132.1, 129.8,
128.7, 127.3, 118.8, 109.1, 105.4, 60.0, 55.8; ESI-MS (m/z) = 431.3
(100.0%) [M+H]+; HR-MS (C21H19ClN2O4S) calcd: 431.08268;
found: 431.08214; mp = 210.0 °C.
5.1.4.36.
phenyl)thiazol-2-amine (36).
4-Adamantyl-5-methyl-N-(3,4,5-trimethoxy-
General procedure 5.1.4 was
used to couple 1-(1-adamantyl)-2-bromopropan-1-on (0.25 g,
0.92 mmol) and 1-(3,4,5-trimethoxyphenyl)thiourea (0.22 g,
0.92 mmol) in 3 ml ethanol to yield the title compound. Pale brown
solid, yield: 194 mg (51%). 1H NMR (250 MHz, DMSO-d6) d = 9.73
(s, 1H, –NH–), 7.07 (s, 2H, 2H,6H-Ph), 3.75 (s, 6H, 3-OCH3, 5-
OCH3), 3.59 (s, 3H, 4-OCH3), 2.33 (s, 3H, –CH3-thiazole), 2.05–
2.00 (m, (3+6)H, –CH–(adamantyl), –CH2–(adamantyl)), 1.71 (m,
6H, –CH2–(adamantyl)); 13C NMR (63 MHz, DMSO-d6) d = 157.2,
152.8, 137.8, 131.2, 112.5, 99.5, 94.1, 60.1, 55.4, 41.7, 38.5, 36.4,
28.2, 11.7; ESI-MS (m/z) = 416.1 (100.0%) [M+H]+; Anal. Calcd for
5.1.4.40. (E)-3-(3,4-Dimethoxyphenyl)-N-(thiazol-2-yl)acrylam-
ide (40)33
.
(E)-3-(3,4-dimethoxyphenyl)acrylic acid (2.08 g,
0.99 mmol) was solved in 10 ml acetone. 1-Ethyl-3-(3-dimethyl-
aminopropyl)carbodiimide (EDC, 1.71 g, 1.10 mmol) and 1-hydrox-
ybenzotriazol (HOBt, 1.49 g, 1.10 mmol) were added and the
mixture was stirred at room temperature for 30 min. 2-Aminothi-
azole (1.00 g, 0.99 mmol) was added and the mixture was heated
to 60 °C under microwave irradiation for 10 min. The crude prod-
uct was purified by column chromatography on silica gel (eluent:
dichloromethane/methanol 98:2 (v/v)) that afforded the title com-
pound. White solid, yield: 1.31 g (45%). 1H NMR (250 MHz, DMSO-
d6) d = 12.19 (s, 1H, –NH–), 7.66 (d, J = 15.8 Hz, 1H,
ANHACOACH@CHA), 7.50 (d, J = 3.5 Hz, 1H, 4H-thiazole), 7.24–
7.21 (m, (1–2)H, 5H-thiazole, 2H,6H-thiazole), 7.03 (d, J = 8.8 Hz,
1H, 5H-Ph), 6.80 (d, J = 15.8 Hz, 1H, ANHACOACH@CHA), 3.81
(d, 6H, 3-OCH3, 4-OCH3); 13C NMR (63 MHz, DMSO-d6) d = 163.3,
158.0, 150.7, 148.8, 142.2, 137.7, 127.0, 121.9, 117.1, 113.5,
111.7, 110.4, 55.5; ESI-MS (m/z) = 289.1 (100.0%) [MꢀH]ꢀ; Anal.
Calcd for C14H14N2O3S: C (57.92) H (4.86) N (9.65) S (11.04); found:
C (57.58) H (4.88) N (9.57) S (11.07); mp = 225.0 °C.
C23H30N2O3S: C (66.64) H (7.29) N (6.76) S (7.73); found: C
(66.77) H (7.36) N (6.41) S (7.34); mp = 221.0 °C.
5.1.4.37. 4-Adamantyl-5-methyl-N-(4-(methylthio)phenyl)thia-
zol-2-amine (37).
General procedure 5.1.4 was used to cou-
ple 1-(1-adamantyl)-2-bromopropan-1-on (0.25 g, 0.92 mmol)
and 1-(2-methoxyphenyl)thiourea (0.18 g, 0.92 mmol) in 3 ml eth-
anol to yield the title compound. Pale yellow powdered solid, yield:
180 mg (53%). 1H NMR (250 MHz, DMSO-d6) d = 9.78 (s, 1H, –NH–),
7.54 (d, J = 8.7 Hz, 2H, 3H,5H-Ph), 7.22 (d, J = 8.7 Hz, 2H, 2H,6H-Ph),
2.42 (s, 3H, –SCH3), 2.34 (s, 3H, 5-CH3-thiazole), 2.03 (m, (3+6)H, –
CH–(adamantyl), CH2–(adamantyl)), 1.72 (m, 6H, –CH2–(adaman-
tyl)); 13C NMR (63 MHz, DMSO-d6) d = 157.0, 153.0, 139.5, 128.2,
127.9, 116.9, 112.6, 41.5, 37.6, 36.2, 28.1, 16.3, 11.8; ESI-MS (m/
z) = 371.8 (100.0%) [M+H]+; Anal. Calcd for C21H26N2S2: C (68.06)
H (7.07) N (7.56) S (17.31); found: C (68.34) H (6.94) N (7.60) S
(17.19); mp = 137.0 °C.

5366
D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5.1.4.41. (E)-3-(3,4,5-Trimethoxyphenyl)-N-(thiazol-2-yl)acryl-
amide (41)33
(E)-3-(3,4,5-trimethoxyphenyl)acrylic acid
5.2.1.2. Assay.
itive fluorescence intensity assay based on the detection of ADP,
flexible with regard to the ATP concentration (0.1–100 M ATP).
The TranscreenerÒ ADP2 FI assay is a compet-
.
(2.38 g, 0.99 mmol) was solved in 10 ml acetone. 1-Ethyl-3-(3-
dimethylaminopropyl)carbodiimide (EDC, 1.71 g, 1.10 mmol) and
1-hydroxybenzotriazol (HOBt, 1.49 g, 1.10 mmol) were added and
the mixture was stirred at room temperature for 30 min. 2-Amino-
thiazole (1.00 g, 0.99 mmol) was added and the mixture was
heated to 60 °C under microwave irradiation for 10 min. The crude
product was purified by column chromatography on silica gel (elu-
ent: dichloromethane/methanol 99:1 (v/v)) that afforded the title
compound. Beige crystalline solid, yield: 1.68 g (53%). 1H NMR
(250 MHz, DMSO-d6) d = 12.22 (s, 1H, –NH–), 7.66 (d, J = 15.7 Hz,
1H, ANHACOACH@CHA), 7.51 (d, J = 3.6 Hz, 1H, 4H-thiazole),
7.24 (d, J = 3.6 Hz, 1H, 5H-thiazole), 6.97 (s, 2H, 2H,6H-Ph), 6.88
(d, J = 15.8 Hz, 1H, ANHACOACH@CHA), 3.84 (s, 6H, 3-OCH3, 5-
OCH3), 3.70 (s, 3H, 4-OCH3); 13C NMR (63 MHz, DMSO-d6)
d = 163.1, 157.9, 153.0, 142.2, 139.3, 137.8, 129.8, 118.9, 113.6,
105.4, 60.0, 55.8; ESI-MS (m/z) = 321.0 (100.0%) [MꢀH]ꢀ; Anal.
Calcd for C15H16N2O4S: C (56.24) H (5.03) N (8.74) S (10.01); found:
C (56.62) H (5.27) N (8.37) S (9.68); mp = 216.0 °C.
l
It is a one-step detection assay that provides signal evaluation
at low substrate conversion. SphK1 and SphK2 use sphingosine
and ATP to form S1P and ADP. We performed the assay according
to the suppliers’ protocol. We performed the experiments under
conditions that ensured a 10–15% substrate conversion as pro-
posed. We also considered the different activities of the isoen-
zymes that were relevant for incubation times to ensure that
the reactions were linear over the time course of reactions at
the concentrations of the enzymes used in the assay. Using this
protocol and standard assay conditions, we generated an ATP/
ADP standard curve and screened our test compounds including
SKI-II (1) and PF-543 (42) as reference compounds and compared
to control (DMSO). Compounds and enzymes were pre-incubated
for 15 min at room temperature. SphK1 reaction was incubated at
room temperature for 30 min, the SphK2 reaction for 60 min.
Readout of the assay is a percentage inhibition. An IC50 value
could be determined for the most promising compound ST-
1803 (24). For the calculation of the IC50 value of ST-1803 we
measured eight increasing concentrations of the inhibitor (0.03–
5.1.4.42. (R)-(1-(4-((3-Methyl-5-(phenylsulfonylmethyl)phen-
oxy)methyl)benzyl)pyrrolidin-2-yl)methanol
543). Reference substance PF-543 (compound 42) was syn-
(PF-
100
lated with GraphPad Prism 5.0 using a non-linear regression
(95% confidence interval 4.89 to 10.81 M for SphK1 and 4.45
to 9.42 for SphK2). For c log P calculation of ST-1803 we used
ChemOffice UltraÒ 10.0 (CambridgeSoft/USA). TPSA was calcu-
lated with Molinspiration Property Engine v2013.09 (Molinspira-
tion Cheminformatics/Slovakia).
lM) in 3 independent experiments. IC50 values were calcu-
thesized according to Schnute et al.26 (cf. Suppl. mat.). 1H NMR
(250 MHz, DMSO-d6) d = 7.74–7.68 (m, 3H, 2H,6H-Ph, 4H-Ph),
7.61–7.55 (m, 2H, 3H,5H-Ph), 7.30 (s, 4H, 2H,6H-Ph-CH2-prolinol,
3H,5H-Ph-CH2-prolinol), 6.77 (s, 1H, 6H-Ph-CH3), 6.54 (s, 1H, 2H-
Ph-CH3), 6.51 (s, 1H, 4H-Ph-CH3), 4.88 (s, 2H, –SO2–CH2–), 4.55
(s, 2H, –O–CH2–), 4.41 (br s, 1H, –OH), 4.04 (d, J = 13.3 Hz, 1H, –
CH-prolinol), 3.50–3.40 (m, 1H, prolinol), 3.36–3.22 (m, 2H, prolin-
ol), 2.83–2.69 (m, 1H, prolinol), 2.63–2.54 (m, 1H, prolinol), 2.17 (s,
3H, –CH3), 2.14–2.04 (m, 1H, prolinol), 1.92–1.75 (m, 1H, prolinol),
1.67–1.48 (m, 3H prolinol); 13H NMR (75 MHz, DMSO-d6) d = 158.0,
138.7, 138.4, 133.7, 129.6, 129.0, 128.7, 128.0, 127.4, 124.1, 115.2,
114.6, 68.9, 65.0, 60.6, 58.3, 58.2, 58.2, 53.9, 27.8, 22.3, 20.9; ESI-
MS (m/z) = 466.2 (100.0%) [M+H]+; Anal. Calcd For C27H31NO4S2ꢂ0.5
H2O: C (68.33) H (6.80) N (2.95) S (6.76); found: C (68.35) H (6.84)
N (2.71) S (6.55).
l
5.3. 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-
pound ST-1803 (24). 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 ꢁ 104 cells/well and incubated with
increasing concentrations of the test compound or vehicle (DMSO)
for 24 h. Plates were centrifuged (250 ꢁ g, 4 min) and an aliquot of
the supernatant was transferred to a clean microplate. Cell toxicity
was assessed according to the distributor’s protocol using a micro-
plate 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%. All exper-
iments were done three times and mean SE were calculated.
5.2. Biology
5.2.1. Sphingosine kinase assay
5.2.1.1. Materials.
All general reagents were ordered from
Sigma–Aldrich (Steinheim/Germany) and AppliChem (Darmstadt/
Germany). Human recombinant SphK1 and SphK2 were supplied
from Echelon (Salt Lake City/USA). SphK1 was formulated in
40 mM Tris–HCl, pH 8.0, 110 mM NaCl, 2.2 mM KCl, 200 mM
imidazole, 3 mM DTT and 20% glycerol (enzyme concentration:
1.39 mg/mL). SphK2 was formulated in 45 mM Tris–HCl, pH 8.0,
124 mM NaCl, 2.4 mM KCl, 225 mM Imidazole, 3 mM DTT and
5.4. Molecular modelling
The software package Molecular Operating Environment (MOE,
2013.08; Chemical Computing Group, Montreal/Canada) was used
for all computational tasks carried out for this work. The structure
of SphK1 co-crystallized with SKI-II (PDB code: 3VZD)16 was used
as a template for docking as well as for the homology modelling of
SphK2. The receptor was arranged by means of the functions Pro-
tonate 3D and Energy Minimization (Amber 12:EHT force field).
The Pharmacophore Query Editor was used to define the common
pharmacophore of the docked structures and the template
molecule SKI-II. Pharmacophore was furthermore used as docking
placement algorithm which generated a maximum of 1000 dock-
ing poses for each molecule (time-out: 300 s). The 30 best poses
where kept and energy minimized using Amber 12:EHT force field.
For rescoring and final pose ranking the scoring function GBVI/WSA
dG was applied. The sequence of human SphK2 was taken from
Protein Knowledge Base UniProtKB49 (ID: Q9NRA0) and
aligned to the template sequence of SphK1 (PDB code: 3VZD.B).
10% glycerol (enzyme concentration: 0.5 mg/mL).
D-Erythro-
sphingosine was supplied by Cayman/Biomol (Hamburg/Ger-
many). Sphingosine kinase reaction buffer, DTT and BellBrook
TranscreenerÒ ADP2 FI assay kit were provided by Echelon (Salt
Lake City/USA) within the sphingosine kinase inhibitor screen
assay kit K-4400. BellBrook TranscreenerÒ ADP2 FI Assay Kit con-
tains Alexa FluorÒ 594 labeled tracer (800 nM in 2 mM HEPES pH
7.5, 0.01% Brij-35), IRDyeÒ-QC-1quencher-labeled monoclonal
antibody (1.3 mg/mL in 100 mM K2HPO4 pH 8.5), stop and detect
buffer (200 mM HEPES, 0.2% Brij-35, 400 mM EDTA, pH 7.5), ADP
(5 mM in deionized water, pH 7.0) and ATP (5 mM in deionized
water, pH 7.0). For fluorescence measurement, we used Infinite
M200 plate reader from Tecan (Crailsheim/Germany). We utilized
384-well black low volume round bottom plates from Corning
(Tewksbury/USA).

D. Vogt et al. / Bioorg. Med. Chem. 22 (2014) 5354–5367
5367
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6. Authorship
23. French, K. J.; Upson, J. J.; Keller, S. N.; Zhuang, Y.; Yun, J. K.; Smith, C. D. J.
Pharmacol. Exp. Ther. 2006, 318(2), 596.
Conceived and designed the experiments: D.V., H.S. Performed
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putational modelling: J.W., E.P. Wrote the paper: D.V., J.W., H.S.
24. Loveridge, C.; Tonelli, F.; Leclercq, T.; Lim, K. G.; Long, J. S.; Berdyshev, E.; Tate,
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Broadus, R.; Ogawa, S.; Cukyne, K.; Zawadzke, L. E.; Peterkin, V.; Iyanar, K.;
Scholten, J. A.; Wendling, J.; Fujiwara, H.; Nemirovskiy, O.; Wittwer, A. J.;
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Acknowledgement
This work was supported by grants from Else-Kröner-Fresenius-
Foundation and the Hessian excellence initiative in science and
economy (LOEWE) Fh-TNP.
Supplementary data
30. Rasmussen, C. R.; Villani, F. J.; Weaner, L. E.; Reynolds, B. E.; Hood, A. R.; Hecker,
L. R.; Nortey, S. O.; Hanslin, A.; Costanzo, M. J.; Powell, E. T.; Molinari, A. J.
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