Discovery of Inhibitor of Wnt Production 2 (IWP-2) and Related Compounds As Selective ATP-Competitive Inhibitors of Casein Kinase 1 (CK1) δ/?

Article abstract of DOI:10.1021/acs.jmedchem.8b00095

Inhibitors of Wnt production (IWPs) are known antagonists of the Wnt pathway, targeting the membrane-bound O-acyltransferase porcupine (Porcn) and thus preventing a crucial Wnt ligand palmitoylation. Since IWPs show structural similarities to benzimidazol

Full text of DOI:10.1021/acs.jmedchem.8b00095

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Discovery of Inhibitor of Wnt Production 2 (IWP-2) and related compounds
as selective ATP-competitive inhibitors of Casein Kinase 1 (CK1) #/#
Balbina García-Reyes, Lydia Witt, Björn Jansen, Ebru Karasu, Tanja Gehring, Johann
Leban, Doris Henne-Bruns, Christian Pichlo, Elena Brunstein, Ulrich Baumann, Fabian
Wesseler, Bernd Rathmer, Dennis Schade, Christian Peifer, and Uwe Knippschild
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.8b00095 • Publication Date (Web): 09 Apr 2018
Downloaded from http://pubs.acs.org on April 9, 2018
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Discovery of Inhibitor of Wnt Production 2 (IWPꢀ2)
and related compounds as selective ATPꢀcompetitive
inhibitors of Casein Kinase 1 (CK1) δ/ε
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,‡
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Balbina García-Reyes , Lydia Witt , Björn Jansen , Ebru Karasu , Tanja Gehring , Johann
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Leban , Doris Henne-Bruns , Christian Pichlo , Elena Brunstein , Ulrich Baumann , Fabian
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2 #
1 #
Wesseler , Bernd Rathmer , Dennis Schade , Christian Peifer * , and Uwe Knippschild *
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Department of General and Visceral Surgery, Ulm University Hospital, AlbertꢀEinsteinꢀAllee
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3, Dꢀ89081 Ulm, Germany
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Institute of Pharmacy, ChristianꢀAlbrechtsꢀUniversity of Kiel, Gutenbergstraße 76, Dꢀ24116
Kiel, Germany
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Oncotyrol GmbH, KarlꢀKapfererꢀStraße 5, 6020 Innsbruck, Austria
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Department for Chemistry, University of Cologne, Zülpicher Str. 47B, Dꢀ50674 Cologne,
Germany
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Department of Chemistry and Chemical Biology, TU Dortmund University, OttoꢀHahnꢀStr. 6,
Dꢀ44227 Dortmund
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Institute of Pharmacy, ErnstꢀMoritzꢀArndtꢀUniversity of Greifswald, FelixꢀHausdorffꢀStr. 1, Dꢀ
17489 Greifswald
‡
#
These authors contributed equally, *corresponding authors, shared senior authorship
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Abstract
Inhibitors of Wnt Production (IWPs) are known antagonists of the Wnt pathway, targeting the
membraneꢀbound Oꢀacyltransferase Porcupine (Porcn) and thus preventing a crucial Wnt ligand
palmitoylation. Since IWPs show structural similarities to benzimidazoleꢀbased CK1 inhibitors,
we hypothesized that IWPs could also inhibit CK1 isoforms. Molecular modeling revealed a
plausible binding mode of IWPꢀ2 in the ATP binding pocket of CK1δ which was confirmed by
Xꢀray analysis. In vitro kinase assays demonstrated IWPs to be ATPꢀcompetitive inhibitors of
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CK1δ. IWPs also strongly inhibited the gatekeeper mutant
CK1δ. When profiled in a panel
of 320 kinases IWPꢀ2 specifically inhibited CK1δ. IWPꢀ2 and IWPꢀ4 also inhibited the viability
of various cancer cell lines. By a medicinal chemistry approach, we developed improved IWPꢀ
derived CK1 inhibitors. Our results suggest that the effects of IWPs are not limited to Porcn, but
also might influence CK1δ/εꢀrelated pathways.
Introduction
Casein Kinase 1 (CK1) is an ubiquitously expressed serine/threonine kinase family in mammals,
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known to phosphorylate a broad range of proteins. Accordingly, CK1 isoforms play essential
regulatory roles in diverse cellular processes including proliferation, DNA repair, apoptosis, cell
1,2,3
differentiation, and circadian rhythm.
The CK1 family consists of different isoforms (α, γ1,
4
γ2, γ3, δ, and ε) in humans, and their various alternative splice variants. CK1 isoforms possess a
highly conserved kinase domain but differ significantly in length and composition of their Nꢀ and
5,6
Cꢀterminal sequences. Since deregulation of CK1 expression and activity contributes to the
development of disorders such as cancer (CK1α/γ/δ/ε), neurodegenerative diseases (CK1δ), and
inflammation (CK1α/δ/ε), there is an increased interest to develop CK1ꢀspecific inhibitors to be
1
used as pharmacological tools, and for new therapeutic approaches.
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One of the most prominent cellular processes involving regulation by CK1 is Wnt signaling, a
rather complex group of signal transduction pathways controlling several processes including
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embryonic development and tissue homeostasis. CK1 isoforms are involved in both, negative
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and positive regulation of canonical as well as nonꢀcanonical Wnt pathways.
For example,
CK1α is part of the destruction complex that targets βꢀcatenin for ubiquitination and subsequent
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degradation in the absence of the Wnt ligand. Following binding of the Wnt ligand to Frizzled
(Fzd), the Wnt coꢀreceptor LRP5/6 is phosphorylated either by membraneꢀbound CK1γ (positive
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regulation), or by CK1ε (negative regulation). Furthermore, CK1δ and CK1ε phosphorylate
axin and dishevelled (DVL), thereby causing a conformational change in the βꢀcatenin
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destruction complex which prevents βꢀcatenin from being degraded.
In addition, Wntꢀ
activation promotes recruitment of DDX3 to CK1ε, and their interaction directly stimulates CK1ε
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kinase activity, which in turn contributes to stabilization of βꢀcatenin.
Targeting Wnt signaling for therapeutic purposes is challenging due to its complexity, and its role
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in a variety of developmental and homeostatic processes. Currently, there are several compounds
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at different drug development stages able to modulate Wnt signaling. Among these
pharmacologically active substances used in preclinical settings, small molecule inhibitors termed
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IWPs (Inhibitors of Wnt Production) have been reported. IWP compounds, originally
identified by screening of a synthetic chemical library, were able to block Wnt signaling by
inhibiting Porcupine (Porcn), a member of the membraneꢀbound Oꢀacyltransferases (MBOAT)
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7,18
protein family. Palmitoylation of Wnt by Porcn is essential for Wnt secretion and signaling.
Consequently, inactivation of Porcn captures Wnt3A in the endoplasmic reticulum, and thus,
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leads to inhibition of Wnt signaling. However, when used at higher concentrations, additionally
to their inhibition of Porcn, IWPs have been shown to modify further events related to Wnt
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signaling. These include inhibition of phosphorylation of Lrp6 and Dvl2, suggesting IWPs
targeting related protein kinase activity.
Since IWPs show structural similarities to known CK1ꢀspecific benzimidazole inhibitors (e.g., 2ꢀ
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benzamidoꢀNꢀ(1Hꢀbenzo[d]imidazolꢀ2ꢀyl)thiazoleꢀ4ꢀcarboxamide , (Bischofꢀ5; Figure 1AꢀC),
we hypothesized that IWP compounds could also inhibit CK1 isoforms. Thus, we performed
docking studies of IWPs in the ATP binding pocket of CK1δ which revealed plausible binding
modes (Figure 1DꢀE). To further prove this hypothesis, we performed in vitro kinase assays with
different CK1 isoforms in the presence of commercially available IWP derivatives. Moreover,
IWPꢀ2 was tested at a concentration of 1 µM in a panel of 320 kinases to determine a selectivity
profile. Additionally, we could show that IWPꢀ2 and IWPꢀ4 were able to inhibit the proliferation
of various CK1 dependent cancer cell lines indicating their potential in cancer research,
additionally to their use in stem cell applications.
Results
IWPꢀderivatives inhibit the activity of CK1δ
IWP small molecule compounds have been reported to target Porcn by inhibition of palmitoyl
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transfer to Wnt proteins, a crucial modification for Wnt secretion. Therefore, IWP compounds
in particular IWPꢀ2 and more recently WntꢀC59) have been routinely used as pharmacological
(
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tools to inhibit the Wnt pathway in various settings. Since IWPs exhibit structural similarities
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to CK1ꢀspecific smallꢀmolecule inhibitors,
we became interested in analyzing their ability to
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inhibit CK1 isoforms. For this purpose, IWPꢀ2, IWPꢀ2ꢀV2, IWPꢀ3, and IWPꢀ4 (Figure 2)
were initially screened for their biological activities against different CK1 isoforms. Whereas
fullꢀlength CK1δ, the Cꢀterminal truncated form of CK1δ (CK1δKD) and CK1ε were inhibited
by IWPꢀ2 and IWPꢀ4, CK1α and CK1γ were only weakly affected. IWPꢀ2 inhibited fullꢀlength
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CK1δ to a 23% of residual activity, CK1δ KD to 19% and CK1ε to 35%. IWPꢀ4 inhibited fullꢀ
length CK1δ to a 25% of residual activity, CK1δ KD to 22% and CK1ε to 43%. In addition,
IWPꢀ2ꢀV2 and IWPꢀ3 did not inhibit CK1α, while inhibiting CK1γ3 and CK1ε only moderately.
In contrast, the activity of both, fullꢀlength CK1δ and CK1δKD were significantly reduced in
these assays. Thus, our initial results suggested that the direct effects of IWP compounds on the
Wnt pathway reported so far are not limited to the inhibition of Porcn, but may also affect CK1δ
and/or CK1ε activity, respectively. We therefore aimed to analyze the biological impact of IWPs
towards CK1δ and ε in more detail.
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Next, IC50 values against rat GSTꢀ wildꢀtype CK1δ ( CK1δ), rat CK1δ KD, rat GSTꢀCK1δ
M82F
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M82F (
CK1δ, a typical “gatekeeper” mutation), and human CK1ε were determined by in
vitro kinase assays using αꢀcasein as substrate, CK1 isoforms as enzymes, and different
concentrations of either IWPꢀ2, IWPꢀ2ꢀV2, IWPꢀ3 or IWPꢀ4 (Table 1, IC50 curves in
wt
Supplementary Figures 1 and 2). Herein, IC values of all four IWP derivatives against CK1δ
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were 4 to 7ꢀfold lower than those against CK1ε. This CK1δ preference increased up to 17ꢀfold
when IC values against CK1δKD were compared to those against CK1ε (Table 1). The high
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degree of phosphorylation of CK1δ within its Cꢀterminal domain, which is missing in
CK1δKD, explains the 4ꢀfold reduced IC value of CK1δKD and is in line with previous reports
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on the influence of the Cꢀterminal domain on CK1 kinase activity.
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Interestingly, the gatekeeper mutant
CK1δ was strongly blocked by all tested IWPs with
IC values in the nanomolar range (Figure 3), suggesting a significant impact of the mutated
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phenylalanine residue towards ligand binding. Strikingly, IWPꢀ2ꢀV2 showed an almost 28ꢀfold
M82F
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increased inhibition of
CK1δ when compared to CK1δ. To analyze the role of
CK1δ in
more detail, we docked IWPꢀ2ꢀV2 both into the ATPꢀbinding sites of a protein structure of
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CK1δ (PDB 4TW9) , and its gatekeeper
CK1δ mutant structure generated by homology
modeling (Figure 4, A/B). While the general binding poses in both kinases turned out to be quite
similar, a focused analysis of ligandꢀprotein interactions regarding gatekeeper residue 82 revealed
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significant lipophilic πꢀstacking interactions towards 82F in comparison to 82M (Figure 4, C/D).
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Thus, the high affinity of IWP compounds towards
CK1δ correlates with the modeled pose,
further indicating a valid calculated binding mode.
IWP compounds inhibit CK1δ in an ATPꢀcompetitive manner
To investigate if IWP compounds were ATPꢀcompetitive inhibitors, IWPꢀ2 and its derivative
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IWPꢀ2ꢀV2 were analyzed for their ability to inhibit GSTꢀ CK1δ in the presence of different
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ATP concentrations (Figure 4 E/F). While ATP concentrations increased, incorporation of P
labeled phosphate into αꢀcasein decreased indicating that both compounds act as ATPꢀ
competitive inhibitors.
Selectivity profiling of IWPꢀ2 in a panel of 320 kinases reveals CK1δ selectivity
To characterize the selectivity of IWPꢀ2, its inhibitor profile was measured in a panel of 320
protein kinases at concentrations of 0.1 µM and 1 µM (see SI Table 1 and SI Figure 3A). Herein,
at a concentration of 1 µM, only three kinases were identified to be moderately inhibited: CK1δ
(55% of residual activity), TLK2 (59%), and ZAP70 (54%). Interestingly, besides CK1δ, no
further CK1 family members or their paralogs were significantly inhibited (see Supplementary
Figure 3B). To confirm the inhibitory effect of IWPꢀ2 as suggested by the profiling, we carried
out inꢀhouse enzymatic kinase assays using TLK2 and ZAP70 as enzymes. In contrast to the
panel data, we could not confirm the inhibitory effects of IWPꢀ2 on TLK2 and ZAP70 in the
tested range of IWPꢀ2 concentrations (see Supplementary Figure 3EꢀF). Thus, these results
indicate that IWPꢀ2 seems to be specific for CK1δ kinase inhibition.
Binding mode of IWPꢀ2 in CK1δ – parameters triggering CK1 isoform specificity
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Based on the high CK1δ specificity as indicated by the profiling we became interested in further
proving the modeled molecular binding mode of IWPꢀ2 in the active site of CK1δ (see Figure 1).
Hence, we aimed to elucidate a structure of IWPꢀ2/CK1δ ligandꢀprotein complex by Xꢀray
crystallography. Crystals were obtained by coꢀcrystallization of a truncated version of CK1δ
(tCK1δ) with IWPꢀ2, which diffracted to a resolution of 2.1Å. The asymmetric unit contains four
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molecules of CK1δ, which are all structurally very similar (RMSD of backbone alignment: 0.438
ꢀ 0.991 Å for at least 272 residues). In line with kinetic data and docking results shown above, in
the crystal structure we identified the ATP binding pocket as a binding site for IWPꢀ2 (Figure 5,
and SI Table 2 and SI Figure 4). As predicted by the modeling, the ligand is hydrogen bonded
involving its amide group and the benzothiazole moiety to the main chain of the hinge region. In
more detail, binding of IWPꢀ2 in CK1δ is further stabilized by numerous vanꢀderꢀWaals
interactions between the benzothiazole moiety and side chain residues Ile23 and Leu85. More
importantly, the benzothiazole moiety also interacts with the gatekeeper residue Met82 resulting
in a significant rearrangement of the Met82 side chain compared to inhibitorꢀfree CK1δ. As a
consequence, the side chain of Ile68 is rotated by 180° which in turn may probably be one
important factor for the isoform selectivity of IWPꢀ2. Rotation of Ile68 is mainly restricted by
steric clashes with the side chain of Met80 in CK1δ. In comparison, in CK1α the rotation of the
corresponding Ile76 is typically limited by steric hindrance with the side chain residue of Leu88.
The diastereotopic methyl groups of Leu88 hold the deltaꢀmethyl group of Ile76 in its position
and might prevent rotation of Ile76 to a greater extent compared to Met80 in CK1δ upon IWPꢀ2
binding. Furthermore, CK1α exhibits a tyrosine (Tyr79) in proximity to Ile76 whereas CK1δ
exhibits only a cysteine (Cys71) at this position, thus giving Met80 more conformational freedom
compared to Leu88. As a consequence, it is likely that Ile68 is able to rotate with lower energy
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barrier in CK1δ compared to Ile76 in CK1α, thus favoring IWPꢀ2 binding in CK1δ. Mutation of
the Met82 to phenylalanine may not only lead to tighter interactions between IWPꢀ2 and CK1δ
(as described above) but also might remove the residual steric clashes arising from the
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conformational changes of Met82.
Based on the data obtained for IWPꢀ2/CK1δ ligandꢀprotein complex, we speculate that the Cꢀ
terminal extensions of CK1δ compared to CK1ɛ isoform may contribute to IWPꢀ2 binding since
wt
wt
there is a remarkable difference in the susceptibility of CK1δ KD and GSTꢀ CK1δ towards
IWPꢀ2 inhibition (as shown in Table 1). Noteworthy, when reꢀdocking IWPꢀ2 into the newly
generated CK1 structure, the binding pose determined by xꢀray analysis could be reproduced.
IWPs inhibit the proliferation of cancer cell lines
We next performed cell viability assays to determine EC values of IWP compounds for eight
50
selected established tumor cell lines (Table 2, and Supplementary Figures 5ꢀ7). Herein, IWPꢀ2,
IWPꢀ2ꢀV2, and IWPꢀ3 inhibit the proliferation of the investigated cell lines within the single
digit µM range. In contrast, IWPꢀ4 consistently inhibited cell proliferation in all tested cell lines
with EC values even in the subꢀµM range. For example, while the EC value obtained for
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IWPꢀ2 on MiaPaCa2 cells was 1.90 µM, IWPꢀ4 was approximately 10ꢀfold more effective (EC50
value 0.23 µM).
Effect of IWPꢀ2 on kinase activity in Pancꢀ1 cells
In order to analyze whether IWPꢀ2 has an impact on intracellular CK1 kinase activity, we
26
selected CK1 dependent Pancꢀ1 cells for further experiments. Pancꢀ1 cells were either untreated
or treated with 2.33 µM IWPꢀ2 for 48 h. Following incubation, cells were lysed, and lysates were
subsequently fractionated via an anion exchange column (as described in Material and Methods).
1–64
Herein, CK1δ kinase activity was determined using GSTꢀp53
(FP267) as a substrate, and
fractions were used as enzyme sources for in vitro kinase assays. In IWPꢀ2 treated cells the
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CK1δ kinase peak activity was reduced to approximately 66% residual activity compared to the
activity in untreated cells, respectively (Figure 6A). Furthermore, in vitro kinase assays in the
27a
27b
presence and absence of control CK1δ inhibitors, IC261
and PF670462 , revealed a
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reduction of kinase activity in the kinase peak fraction by approximately 40%, providing further
evidence for the presence of CK1δ in the kinase peak fraction (Figure 6 B). Thus, our results
suggest that IWPꢀ2 reduces the activity of CK1δ in Panc1 cells. In a more general context,
inhibition of Wnt signaling by IWP compounds used at µM concentrations may not be
exclusively affecting their known target Porcn but could additionally result in CK1δ inhibition.
These findings have substantial implications on IWPꢀmediated inhibition in various studies of
proliferation of tumor cells, and within stem cell biology, respectively. IWP derivatives are
commonly used in modern stem cell protocols for the generation of human induced pluripotent
28ꢀ30
stem cell (hiPSC)ꢀderived cardiomyocytes,
or in the conversion of mouse embryonic stem
3
1
cells (ES) to epiblastꢀlike stem cells at concentrations of 5 µM, a concentration corresponding
to the EC values of IWPs for effective cellular CK1δ inhibition.
50
Design and synthesis of novel IWP derived CK1δ inhibitors
Based on the data obtained so far, we set out to optimize IWP hit compounds towards potent,
specific, and effective CK1δ/ε inhibitors. We aimed to establish a flexible synthetic route to
produce modified IWP derived compounds for the generation of initial CK1ꢀtargeted structureꢀ
activity relationships (SAR). Thus, the original synthesis of IWP compounds was adapted
32
(Scheme 1). Herein, a convergent strategy was followed coupling a benzothiazole building
16
block (16) with substituted tetrahydrothienoꢀpyrimidinones (9ꢀ15) to yield test compounds 17ꢀ
2
3.
In order to enhance ligandꢀprotein interactions towards the hydrophobic pocket I, and accordingly
3
to the substitution pattern in benzimidazole CK1δ inhibitor Bischofꢀ5 (see Figure 1) and further
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related CK1 inhibitors, we introduced a trifluoromethyl group to the IWP benzothiazole
scaffold. Besides this slight modification, we decided to initially keep the 2ꢀamidoꢀbenzothiazole
core as it was shown to mediate bidentate Hinge binding and, at the same time, quite optimally fit
into the hydrophobic pocket I of CK1δ. In contrast to maintaining the 2ꢀamidoꢀbenzothiazole
scaffold, we focused on variations of the phenylꢀ and benzyl moiety attached to the
tetrahydrothienoꢀpyrimidinone core, respectively. In line with our modeling and Xꢀray analysis of
IWPꢀ2 in CK1δ (Figure 5), this part of the inhibitors was found to address the solventꢀexposed
hydrophobic region II. Thus, we designed a small set of compounds with variability at the aryl
system attached to the tetrahydrothienoꢀpyrimidinone core (17ꢀ23, Scheme 1). Modeling of these
compounds in the active site of CK1δ indicated plausible binding modes. However, within this
preliminary set, it was not possible to prioritize any compound since all ligands were calculated
to have very similar docking scores. Thus, we decided to prepare this small set of compounds for
subsequent initial determination of SAR. Synthetically, modifications were readily introduced by
using substituted isothiocyanate precursors (2, 4, 5, 6, and 7, Scheme 1 and SI) during the
pyrimidinone ring closure reaction.
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wt
wt
Next, we determined IC values of these new test compounds against rat CK1δ KD, rat GSTꢀ
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wt
CK1δ, rat GSTꢀ
CK1δ, and human CK1ε by in vitro kinase assays using αꢀcasein as
substrate (Table 3, and Supplementary Figures 9ꢀ11). According to the design concept regarding
lipophilic interactions towards hydrophobic region II, the compounds were shown in general to
have modestly increased binding affinity to their targets. Given only minor variations in
substitution patterns for all compounds, the gain in affinity turned out to be within a similar
range. When compared to IWPꢀ2, compound 19 was determined to have approximately 2ꢀfold
higher affinity for CK1δ (IC = 0.41 µM). To further characterize CK1 isoform specificity of
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CK1γ3. Herein, all compounds showed no significant inhibition indicating selectively towards
CK1 isoforms δ and ε (see Supplementary Figure 12).
To judge the structural impact of these modifications towards kinase selectivity we selected
compound 19 for further profiling in a panel of 320 kinases (see Supplementary Figure 3AꢀD and
Supplementary Table 1). By this screen, compound 19 was determined to be selective for CK1δ
at a concentration of 1 µM which is comparable to the selectivity profile of IWPꢀ2. However,
selectivity profiling of 19 at 10 µM revealed a wealth of other inhibited kinases (see SI Table 1
and Figure 3AꢀB). Since the CK1 inhibition of 19 (IC50 = 0.41 µM) was slightly improved
compared to that of the original hit compound IWPꢀ2 (IC = 0.93 µM), we next determined their
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effects on Wnt signaling in cellular settings also using relevant subꢀµM concentrations.
Novel IWP derivatives exhibit distinct profiles on Wnt/βꢀcatenin signaling
Functional perturbation of canonical Wnt signaling by IWPꢀ2 and its derivative 19 was probed in
two specific cellꢀbased assay setups to discriminate between a Porcupineꢀdependent and
4
c
Porcupineꢀindependent modeꢀofꢀaction. HEK293T cells were transiently transfected with a 7ꢀ
tandemꢀrepeat of TCF/LEF response elements (i.e., SuperTOPFlash vector) and Wnt signaling
was activated by exogenous addition of Wnt3Aꢀcontaining conditioned medium from mouse Lꢀ
cells (i.e., paracrine pathway activation) or by autologous expression of Wnt3A from a coꢀ
33,34
transfected Wnt3A expression vector (i.e., autocrine and paracrine pathway activation).
The
latter assay setup strongly depends on the cells' capacity to postꢀtranslationally process Wnt3A by
the action of Porcupine to secrete mature ligands for functional pathway activation. The data
showed that all IWPꢀtype Wnt inhibitors inhibited Porcupineꢀdependent canonical Wnt signaling
3
5
in this setup (Figure 7A). WntꢀC59 is among the most potent currently reported Porcupine
36
inhibitors and served as a positive control (IC < 1 nM), while IWPꢀ2 and 19 had IC ’s of
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.157 µM and 0.71 µM, respectively.
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Strikingly, both IWPꢀ2 and WntꢀC59 did not show doseꢀdependent inhibition when the signaling
cascade was activated by exogenous Wnt3A (Figure 7B), whereas 19 appeared to synergize with
Wnt3A and thus further activated canonical signaling. At first, this finding was somewhat
surprising and required further validation concerning possible assay artifacts. For instance, it is
wellꢀknown that 2ꢀaminobenzothiazoles, such as IWPꢀ2 and probably 19, too, can potentially
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show unspecific reporter gene activation through inhibitorꢀbased stabilization of luciferases.
Thus, we considered this possibility by coꢀtransfection of a constitutively active Renilla
luciferase reporter in all experiments. Indeed, a characteristic lowꢀlevel reporter activation was
observed up to 1.3ꢀfold and 1.5ꢀfold of the baseline Wnt activity in the autocrine/paracrine and
paracrine assay setup, respectively (see Figure 7). However, this basal unspecific activity is taken
into account by normalization of Firefly by Renilla luciferase activity. It should also be noted that
compound/scaffoldꢀdependent effects on distinct proliferation rates are considered by this
method. Moreover, compound exposure in a timeꢀdependent manner (6h and 22h) in cell viability
assays complemented this data (see Supplementary Figures 13 and 14).
The offꢀtarget activity of the herein reported IWP derivative 19 on CK1 isoforms results in a
unique profile on canonical Wnt signaling. It is wellꢀknown that different CK1 isoforms fulfill
diverse roles within the signaling cascade, depending on the temporal and tissueꢀspecific
9
regulation of the actual pathway. In fact, pathway synergy or stimulation can be explained by an
underlying inhibitory activity on CK1δ isoforms. It has been shown that CK1δ phosphorylates
the Wntꢀdependent transcription factor Lef1, thereby inhibiting its binding interaction with βꢀ
catenin. Thus, inhibition of this inhibitory effect on canonical signaling results in pathway
3
8
stimulation. In this context, IWPꢀ2’s lack of activity in the paracrine assay setup is likely due to
its ca. 10ꢀfold less potent CK1δ inhibition compared to 19 (Tables 1 and 3), while Porcupine
inhibition cannot be effective at all. On the other hand, 19’s Wnt3A synergy appears to be
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overridden by still effective Porcupine inhibition in the autocrine/paracrine assay setup, thus
explaining the modest inhibitory activity of 19 (IC = 0.71 µM) in this setup compared to the
50
parent IWPꢀ2 (IC50 = 0.157 µM). Notably, profiling of two different chemotypes of CK1δ/ε
inhibitors, i.e. SRꢀ3029 and PFꢀ670462, raises questions whether the observed activity of 19 is
solely due to a unique inhibition profile of CK1 isoforms or a consequence of an additional offꢀ
target (see Figure S14). SRꢀ3029 showed doseꢀdependent Wnt/βꢀcatenin inhibition up to 0.1 µM
in both assay setups while being toxic at higher concentrations. Another explanation could be
differences in the subcellular distribution and cellular target engagement of these three unrelated
chemotypes of CK1δ/ε inhibitors. Additional studies are required to decipher compound 19’s
activity as a Wnt signaling modulator.
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Taken together, this data underlines the utility of the herein presented IWP derivatives as novel
pharmacological tools for CK1δꢀdependent Wnt/βꢀcatenin perturbation in different cellular,
tissue and disease context. However, for future projects, it would be desirable to eliminate any
remaining activity on Porcupine while keeping (and improving) CK1δ/ε inhibition of these
compounds.
Discussion and Conclusions
1
7
IWPꢀ2 and its derivatives have been initially described to be potent Wnt pathway inhibitors , a
discovery that has led to extensive use of IWPꢀ2 in biological research applications studying this
highly complex signaling process. IWPs effectively block the secretion of Wnt ligands by
inhibiting Porcn, a membraneꢀbound Oꢀacyltransferase catalyzing the palmitoylation of Wnt.
However, in our current project, we have found that IWP compounds also inhibit CK1δ/ε in vitro
and cell culture, adding a new layer of complexity to the effects of these small molecules on the
Wnt pathway.
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Initial screenings were performed using different IWP derivatives at 10 µM on the isoforms of
CK1 α, γ3, δ (in its full form and kinaseꢀdomainꢀonly form) and CK1ε (Figure 1). CK1δ was
consistently inhibited at various extents by the IWP derivatives, while CK1ε was more weakly
blocked despite its structural similarity to CK1δ. In contrast, the tested IWPs showed low
affinities to CK1γ3 and CK1α which were thus mostly unaffected by any of the treatments
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(Figure 1 and Supplementary Data). Additionally, we provide structural evidence by determining
a complex of IWPꢀ2 in CK1δ (xꢀray crystallography). Furthermore, the selectivity profiling of
IWPꢀ2 in a panel of 320 kinases demonstrated its preference for CK1δ. Moreover, IWPꢀ2 was
M82F
shown to inhibit the
CKδ gatekeeper mutant with an IC50 even in the nanomolar range
(Figure 3). Typically, smallꢀmolecule kinase inhibitors show a higher capability to inhibit wildꢀ
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type kinases, rather than mutant forms. To the best of our knowledge, this is the first report of
IWPs inhibiting protein kinases as targets. Finally, we started to reveal preliminary SAR to
optimize the IWP scaffold towards isoformꢀspecific CK1 inhibitors.
In summary, we characterized IWP derivatives as small molecule CK1 isoformꢀspecific agents
suggesting that some effects of prior IWP applications as Wnt inhibitors may be not be limited to
their known target Porcn. The presented data renders IWPs as interesting hits for the
development of novel potent, specific, and effective CK1δ/ε inhibitors. Compound 19 will serve
as starting point towards further optimized derivatives as novel pharmacological tools to study
CK1ꢀdependent processes in cancer and stem cell research.
Experimental Section
Commercially available chemical compounds and enzymes
Inhibitors of Wnt Production IWPꢀ2, IWPꢀ2ꢀV2, IWPꢀ3 and IWPꢀ4 were purchased from
Cayman Chemical (Ann Arbor, MI, USA) and were diluted in DMSO (GIBCO, Karlsruhe,
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Germany). αꢀcasein and poly(Lꢀglutamic acidꢀLꢀtyrosine) were acquired from SigmaꢀAldrich (St.
Louis, MO, USA), rat recombinant CK1δ kinase domain (KD) from New England Biolabs,
(NEB, Frankfurt am Main, Germany), and human CK1ε from Invitrogen (Karlsruhe, Germany).
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TLK2 and ZAP70 were obtained from ProQinase (Freiburg, Germany).
Plasmids
For the expression of wildꢀtype (wt) bovine CK1α the plasmid pGEXꢀ2TꢀCK1α (FP296) was
4
0
used. Human CK1γ3 was expressed using the plasmid pGEXꢀ2TꢀCK1γ3 (FP1054). Expression
wt
of wildꢀtype rat CK1δ and its gatekeeper mutant was carried out using pGEXꢀ2Tꢀ CK1δ
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M82F
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1ꢀ64
(
FP449) and pGEXꢀ2Tꢀ
expressed using pGEXꢀ2Tꢀp53
GSTꢀfusion proteins were carried out previously described. A Cꢀterminally truncated construct
tCK1δ) encoding amino acids 1 to 294 in a codonꢀoptimized version for expression in E.coli was
CK1δ (FP1153), respectively. The GSTꢀp53 fusion protein was
1
ꢀ64
(FP267), to use as substrate. Expression and purification of the
4
2
(
ordered from Geneart (Thermofisher, Germany) and cloned into the NdeI and XhoI sites of the
pET28a vector, resulting in an Nꢀterminally hexaꢀhistidine tagged protein with a thrombin site to
cleave off the tag. Wnt/βꢀCatenin reporter assays were performed with the following
commercially available vectors: M50 Super 8x TOPFlash (Addgene Plasmid 12456) coding for
firefly luciferase under control of TCF/LEF binding region (7 repeats), TKꢀdriven pRL Renilla
Luciferase control reporter vector (Promega Cat#E2241) and Wnt3a cDNA in pUSEamp (Upstate
Biotech, Cat#21ꢀ124).
In vitro kinase assays
In vitro kinase assays were performed with different CK1 isoforms and IWP derivatives at an
43 44
ATP concentration of 10 µM and using DMSO controls as described previously. , Where
indicated, higher ATP concentrations (50, 100, 250, and 500 µM) were used. Bovine GSTꢀCK1α
wt
(
FP296), rat recombinant CK1δ kinase domain (CK1δKD), rat GSTꢀ CK1δ (FP449), rat GSTꢀ
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CK1δ (FP1153), recombinant human CK1ε, TLK2, and ZAP70 were used as sources of
enzyme. Phosphorylated proteins were separated by SDSꢀPAGE and stained with Coomassie. αꢀ
casein served as a substrate for most kinase assay reactions. Kinase assays performed with
ZAP70 were done using poly(Lꢀglutamic acidꢀLꢀtyrosine) as substrate. Phosphate incorporation
was detected by autoradiography of dried gels. The phosphorylated protein bands were cut out
and quantified by Cherenkov counting. Doseꢀresponse analyses were carried out using GraphPad
Prism 6 (GraphPad Software, Inc., La Jolla CA, USA) statistical software.
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HighꢀThroughput Kinase Profiling
The residual activity of 320 eukaryotic kinases was measured by ProQinase GmbH (Freiburg,
Germany) in the presence of compounds IWPꢀ2 and 19 (1 µM). Dendrograms illustrating the
phylogenetic relations of the kinases was generated using TREEspotTM Software Tool Image
®
and reprinted with permission from KINOMEscan , a division of DiscoveRx Corp., ©
DISCOVERX CORPORATION 2010.
Cell lines
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The pancreatic cancer cell lines MiaPaCa, Panc1, the human embryonic kidney cell line
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HEK293 were grown in Dulbecco’s modified Eagle’s medium (DMEM). The human colon
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adenocarcinoma cell line HT29 was grown in McCoy's 5A medium. The pancreatic cancer cell
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line Panc89 was grown in DMEM:RPMI (1:1) medium. The cell line Capan1, also of
pancreatic origin, was grown in RPMI medium. The colorectal adenocarcinoma cell line
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SW620 was grown in Leibovitz Lꢀ15 medium. All media were supplemented with 10 % fetal
calf serum (FCS; Biochrom, Berlin, Germany), 100 units/ml penicillin, 100 µg/ml streptomycin
(Gibco, Karlsruhe, Germany) and 2 mM glutamine. All cells were cultured at 37°C in a
humidified 5 % carbon dioxide atmosphere. HEK293T cell line was kindly provided by the Max
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Planck Institute of Molecular Physiology (Dortmund, Germany). These cells cultured in DMEM
supplemented with 10% FCS.
Cell viability assay
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Cells were seeded at a concentration of 5 x 10 cells/ml in 96ꢀwell cell culture plates and allowed
to attach overnight at 37°C and 5% CO . To investigate the effects of compounds on cancer cell
2
proliferation, cells were treated with various concentrations (ranging from 0.313 µM to 10 µM)
of inhibitor, with untreated and DMSOꢀtreated cells serving as a control. After an incubation
period of 48h at 37°C, 10 µl of
a
MTT (3ꢀ(4,5ꢀdimethylthiazolꢀ2ꢀyl)ꢀ2,5ꢀ
diphenyltetrazoliumbromide) 12 mM solution in PBS) were added, followed by further
incubation for 4 h at 37°C. Media containing MTT was then removed carefully and 100 µl of
0
.04 N HCl in isopropanol were added. To dissolve the formazan crystals, the plates were placed
for 30 min on an orbital shaker. The resulting purple solution was spectrophotometrically
measured at 570 nm. Experiments were repeated at least three times with four replicates per
assay.
Fractionation of cell extracts by FPLC
IWPꢀ2ꢀtreated (EC = 2,33 µM) and DMSOꢀtreated Panc1 cells were lysed in sucrose lysis
5
0
buffer. Total protein extract (1,4 mg) was diluted in preꢀfiltered FPLC buffer A (50 mM Tris–
HCl pH 7.5, 1 mM EDTA, 1 mM EGTA, 5 % (v/v) glycerol, 0.03 % (v/v) Brijꢀ35, 1 mM
benzamidine, 25 µg/ml aprotinin, 0.1 % (v/v) βꢀmercaptoethanol). Cell lysates were then passed
through a 0.45 µm filter and injected into an anion exchange column (Resource Q; GE Healthcare
UK) attached to an EttanLC FPLC system (GE Healthcare, UK). Proteins bound to the cationic
surfaces of the column were eluted with a linear ascending NaCl gradient by gradually increasing
percentage of FPLC buffer B (equal to buffer A plus 1 M NaCl). Fractions of 250 µl volume
were collected. 3 µl from selected protein fractions were used for in vitro kinase assays, as
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described above, to determine CK1ꢀspecific kinase activity. To confirm CK1 in the peak fraction,
kinase assays were repeated in the presence of given concentrations of CK1δꢀspecific inhibitors
1
ꢀ64
IC261 and PF670462. DMSO was used as a control and GSTꢀp53 as substrate.
0
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Dual Luciferase Reporter Gene Assay
10b,33
The assay was performed as described previously.
Briefly, compounds were tested in
HEK293T cells in a 96ꢀwell plate format. For transient transfection, Lipofectamine®2000
(Thermo Fisher) and plasmids were preꢀincubated in OptiꢀMEM® medium for 15 min at room
6
temperature. For the autocrine/paracrine assay setup, 3 × 10 cells (for one 96ꢀwell plate) were
transfected with the Wnt3Aꢀexpressing vector and the Super(8x)TOPflash reporter vector
together with the TKꢀRenilla luciferase control vector for internal luminescence normalization
purposes, followed by incubation for 8 h. Transfected cells were harvested and seeded on 96ꢀ
wellꢀplates in 110 µL media at 25,000 cells per well and allowed to adhere for 1 h. Cells were
treated either with 10 µL of compound dilution (final concentrations 5, 2.5, 1, 0.5, 0.1, 0.05, 0.01,
0
.001, 0.001 µM with 0.5% DMSO) or DMSO (0.5%) as the vehicle controls. For paracrine
pathway activation, cells were stimulated by addition of Wnt3Aꢀconditioned medium that was
5
2
freshly harvested from mouse Lꢀcells overexpressing the Wnt protein (LꢀWnt3A). Control cells
were treated with Lꢀcell medium. Transfection of cells was done with the same vectors as
described above, except for the Wnt3Aꢀexpressing vector. Cells were seeded in 80 µL and 25,000
cells/well 12 h posttransfection before stimulation with 30 µL of Wnt3Aꢀconditioned medium.
Compound treatment was conducted described as above. After 22 h incubation, the medium was
carefully aspirated and both luciferase activities measured using the DualꢀGlo®Luciferase Assay
System (Promega) according to the manufacturer’s protocol on a TecanꢀInfinite 200 Plate
Reader. Data was processed by normalizing Firefly with Renilla luciferase signals from each
well. Each condition was repeated in technical triplicates with at least two independent biological
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replicates. EC ꢀ and IC ꢀvalues were calculated by nonꢀlinear regression analysis using the
5
0
50
GraphPad Prism 5 software (version 5.03).
Molecular Modeling
1
1
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Molecular modeling was performed on a DELL 4 core system. For visualization Maestro, version
1
0.3, Schrödinger, LLC, (New York, NY, USA, 2014) was used. Protein crystal structures were
prepared prior to docking by the Protein Preparation Wizard using default settings. The Xꢀray
crystal structure refinement process included the addition of hydrogen atoms, optimization of
hydrogen bonds, and removal of atomic clashes. Missing side chains and loops were filled in.
Furthermore, selenomethionines were converted to methionines, and water molecules were
deleted.
Small molecule ligands were prepared to create energetically minimized 3D geometries and
assign proper bond orders (MacroModel). Accessible tautomer and ionization states were
calculated prior to screening (LigPrep). To generate bioactive conformers, a conformational
search method was used (ConfGen). Receptor grid generation was performed by Glide. For
ligand docking, the Glide SP workflow was used (default settings). Energetically minimized
ligand conformations were docked into the active site of the protein; possible binding poses were
determined and subsequently ranked based on their calculated docking score.
Chemistry
All chemical reagents were commercially available and were used without further purification
from abcr GmbH, Karlsruhe, Germany; SigmaꢀAldrich Chemie GmbH, Merck Group, Munich,
Germany; Merck Millipore, Darmstadt, Germany; Acros Organics, Thermo Fisher Scientific,
Geel, Belgium. Infrared spectra (IR) were recorded on a Shimadzu IRAffinityꢀ1S FTIRꢀ
spectrometer. NMR spectra were recorded on a Bruker Avance III 300 spectrometer (300 MHz
1
13
H frequency), (75 MHz C NMR frequency). Chemical shifts are reported in ppm, multiplicity
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and coupling constant J (Hz). Spectra were referenced to internal DMSOꢀd or internal CDCl .
6
3
Whenever appropriate, signal assignments were deduced from DEPT, COSY, and CH
CORRELATION NMR experiments. LCꢀMS was performed using an Agilent 1100 HPLC
system over an Agilent Eclipse XDBꢀC8 column. Mass spectra were recorded on a Bruker
Esquire ~LC ion trap mass spectrometer (ESI). Column chromatography was performed using a
LaFlash system (VWR) with MachereyꢀNagel silica gel 60 (63ꢀ200 µm) for precolumns and preꢀ
packed Interchim PuriFlashꢀ30SIHP silica gel columns (30 µm, 40 g) using mixtures of
petroleum ether (PE) and ethyl acetate (EA). Where necessary, reactions were carried out in a
nitrogen atmosphere using dry solvents. HPLC analysis was performed on a HewlettꢀPackard
1050 Series using a ZORBAX® Eclipse XDBꢀC8 column or an STAGROMA YMCꢀCꢀ18
column. All test compounds were proved by HPLC to have ≥ 95% purity.
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General methods for the synthesis of isothiocyanate precursors
Method A
1
ꢀ(Bromomethyl)ꢀbenzene derivatives
To a solution of the appropriate benzyl alcohol in dichloromethane, phosphorus tribromide was
added at 0°C. The reaction mixture was allowed to warm to room temperature and stirred for an
additional hour. The reaction was quenched with a saturated solution of sodium hydrogen
carbonate and extracted with diethyl ether. The organic layer was washed with a saturated
solution of sodium thiosulfate and brine, dried over sodium sulfate and concentrated under
vacuum. The obtained products were used without further purification.
1
ꢀ(Isothiocyanatomethyl)ꢀbenzene derivatives
1
ꢀ(Bromomethyl)ꢀbenzene derivatives were heated with potassium thiocyanate and sodium iodide
in anhydrous dimethylformamide (DMF) at 90°C for 7 h. The reaction mixture was poured into
water and extracted with diethyl ether. The organic layer was washed three times with water,
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dried over sodium sulfate and concentrated under vacuum. The crude product was purified by
silica gel column flash chromatography (5 % EA/PE).
Method B
1
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1
ꢀ(Isothiocyanatomethyl)ꢀbenzene derivatives
The appropriate amine dissolved in dichloromethane or tetrahydrofuran was stirred at 0°C with
trimethylamine for 10 minutes. Thiophosgene was added dropwise at 0°C and the mixture was
stirred for additional 20 minutes, then allowed to reach room temperature and stirred for 3 h. The
solution was acidified with aqueous HCl and extracted with dichloromethane. The combined
organic phases were dried over sodium sulfate and the solvent was removed under reduced
pressure. The crude product was purified by silica gel column flash chromatography (5 %
EA/PE).
1
1
5
ꢀ(Bromomethyl)ꢀ4ꢀ(methoxy)benzene (1)
ꢀ(Bromomethyl)ꢀ4ꢀ(methoxy)benzene (1) was obtained from 4ꢀmethoxybenzyl alcohol (696 mg,
.04 mmol) with phosphorus tribromide (352 µL, 3.75 mmol) in dichloromethane (20 mL)
1
according to method A. Yield: 913 mg (90 %); C H BrO (M 201.06); H NMR (CDCl ): δ =
8
9
r
3
2
3
13
7
.25 (m , 2 H, C H ), 6.79 (m , 2 H, C H ), 4.43 (s, 2 H, CH ), 3.74 (s, 3 H, CH ) ppm; C
C ar C ar 2 3
4
2
1
3
NMR (CDCl ): δ = 159.7 (C ), 130.4 (C H ), 130.0 (C ), 114.2 (C H ), 55.3 (CH ), 24.0
3
ar
ar
ar
ar
3
(
CH ) ppm; MS (ESI, 70 eV) m/z = not detected.
2
1
ꢀ(Isothiocyanatomethyl)ꢀ4ꢀ(methoxy)benzene (2)
1ꢀ(Isothiocyanatomethyl)ꢀ4ꢀ(methoxy)benzene (2) was obtained from 1 (549 mg, 2.73 mmol)
with potassium thiocyanate (480 mg, 4.94 mmol) and sodium iodide (60.0 mg, 0.40 mmol) in
1
DMF (5 mL) according to method A. Yield: 325 mg (66 %); C H NOS (M 179.24); H NMR
9
9
r
2
3
(
CDCl ): δ = 7.19ꢀ7.14 (m, 2 H, C H ), 6.84 (m , 2 H, C H ), 4.56 (s, 2 H, CH ), 3.74 (s, 3 H,
3 ar C ar 2
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2
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1
3
4
1
2
CH ) ppm; C NMR (CDCl ): δ = 159.6 (C ), 131.9 (C ), 128.4 (C H ), 126.3 (N=C=S),
3
3
ar
ar
ar
3
+
1
1
1
14.2 (C H ), 55.3 (CH ), 48.3 (CH ) ppm; MS (ESI, 70 eV) m/z = 356.9 [MM] .
ar 3 2
ꢀ(Bromomethyl)ꢀ2,3ꢀ(dimethoxy)benzene (3)
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ꢀ(Bromomethyl)ꢀ2,3ꢀ(dimethoxy)benzene (3) was obtained from 2,3ꢀdimethoxybenzyl alcohol
(
842 mg, 5.01 mmol) with phosphorus tribromide (352 µL, 3.75 mmol) in dichloromethane (20
1
mL) according to method A. Yield: 845 mg (73 %); C H BrO (M 231.09); H NMR (CDCl ):
9
11
2
r
3
3
6
3
4
5
δ = 7.04 (q, J = 8.1 Hz, 1 H, C H ), 6.98 (dd, J = 7.8 Hz, J = 1.9 Hz, 1 H, C H ), 6.90 (dd,
ar
ar
3
4
4
2
J = 7.9 Hz, J = 1.8 Hz, 1 H, C Har), 4.59 (s, 2 H, CH ), 3.98 (s, 3 H, C OꢀCH ), 3.89 (s, 3 H,
2
3
3
13
2
3
1
6
C OꢀCH ) ppm; C NMR (CDCl ): δ = 152.8 (C ), 147.4 (C ), 131.9 (C ), 124.2 (C H ),
3
3
ar
ar
ar
ar
5
4
2
3
122.5 (C H ), 113.0 (C H ), 60.8 (C OꢀCH ), 55.8 (C OꢀCH ), 28.1 (CH ) ppm; MS (ESI,
ar
ar
3
3
2
7
1
1
0 eV) m/z = not detected.
ꢀ(Isothiocyanatomethyl)ꢀ2,3ꢀ(dimethoxy)benzene (4)
ꢀ(Isothiocyanatomethyl)ꢀ2,3ꢀ(dimethoxy)benzene (4) was obtained from 3 (586 mg, 2.53 mmol)
with potassium thiocyanate (449 mg, 4.62 mmol) and sodium iodide (58.0 mg, 0.387 mmol) in
1
DMF (4 mL) according to method A. Yield: 214 mg (41 %); C H NO S (M 209.26); H NMR
1
0
11
2
r
3
6
4
5
(
CDCl ): δ = 7.08 (t, J = 7.9 Hz, 1 H, C H ), 6.93 (m , 2 H, C H C H ), 4.73 (s, 2 H, CH ),
3 ar C ar, ar 2
2
3
13
2
3.89 (s, 3 H, C OꢀCH ), 3.88 (s, 3 H, C OꢀCH ) ppm; C NMR (CDCl ): δ = 152.8 (C ), 146.6
3
3
3
ar
3
1
6
5
4
2
(
C ), 131.6 (N=C=S), 128.1 (C ), 124.4 (C H ), 120.4 (C H ), 113.0 (C H ), 61.1 (C OꢀCH ),
ar
ar
ar
ar
ar
3
3
+
5
6.0 (C OꢀCH ), 44.1 (CH ) ppm; MS (ESI, 70 eV) m/z = 417.0 [MM] .
3 2
1ꢀ(Isothiocyanatomethyl)ꢀ3ꢀ(trifluoromethyl)benzene (5)
ꢀ(Isothiocyanatomethyl)ꢀ3ꢀtrifluoromethylbenzene (5) was obtained from 3ꢀtrifluoroꢀmethyl
benzylamine (220 µL, 1.53 mmol) with thiophosgene (120 µL, 1.57 mmol) and trimethylamine
840 µL, 6.06 mmol) in dichloromethane (10 mL) according to method B. Yield: 90.0 mg (28 %);
1
(
1
2
C H F NS (M 217.21); H NMR (CDCl ): δ = 7.63ꢀ7.53 (m, 1 H, C H ), 7.57ꢀ7.53 (m, 3 H,
9
6
3
r
3
ar
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C H , C H , C H ), 4.80 (s, 2 H, CH ) ppm; C NMR (CDCl ): δ = 135.3 (C ), 133.9
ar
ar
ar
2
3
ar
2
3
4
5
6
(
N=C=S), 131.4 (d, J = 32.7 Hz, C ), 130.1 (d, J = 1.7 Hz. C H ), 129.6 (C H ), 125.3 (q,
CF ar CF ar ar
3
2
1
3
4
J = 3.8 Hz, C H ), 123.8 (d, J = 272.5 Hz, CF ), 123.7 (q, J = 3.8 Hz, C H ), 48.3 (CH )
CF
ar
CF
3
CF
ar
2
+
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
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5
5
5
5
5
5
5
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0
1
2
3
4
5
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7
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ppm; MS (ESI, 70 eV) m/z = 432.9 [MM] .
1
ꢀ(Isothiocyanatomethyl)ꢀ4ꢀ(trifluoromethyl)benzene (6)
ꢀ(Isothiocyanatomethyl)ꢀ4ꢀtrifluoromethylbenzene (6) was obtained from 4ꢀtrifluoroꢀmethyl
1
benzylamine (715 µL, 5.02 mmol) with thiophosgene (410 µL, 5.35 mmol) and trimethylamine
(
2.80 mL, 20.2 mmol) in dichloromethane (27 mL) according to method B. Yield: 625 mg
1
3
3
(
56 %); C H F NS (M 217.21); H NMR (CDCl ): δ = 7.68 (d, J = 8.0 Hz, 2 H, C H ), 7.47 (dt,
9 6 3 r 3 ar
3
4
2
13
J = 8.0 Hz, J = 0.7 Hz, 2 H, C H ), 4.82 (s, 2 H, CH ) ppm; C NMR (CDCl ): δ = 138.2
ar
2
3
1
2
4
2
3
(
C ), 130.7 (d, J = 32.8 Hz, C ), 129.9 (N=C=S), 127.1 (C H ), 125.0 (q, J = 3.8 Hz,
ar CF ar ar CF
3
1
C H ), 123.8 (d, J = 271.8 Hz, CF ), 48.2 (CH ) ppm; MS (ESI, 70 eV) m/z = not detected.
ar
CF
3
2
1
ꢀ(Isothiocyanatomethyl)ꢀ4ꢀ(trifluoromethoxy)benzene (7)
1
ꢀ(Isothiocyanatomethyl)ꢀ4ꢀ(trifluoromethoxy)benzene (7) was obtained from 4ꢀtrifluoroꢀ
methoxy benzylamine (382 µL, 2.50 mmol) with thiophosgene (200 µL, 2.61 mmol) and
trimethylamine (1.39 mL, 10.0 mmol) in tetrahydrofuran (13 mL) according to method B. Yield:
1
2
360 mg (62 %); C H F NOS (M 233.21); H NMR (CDCl ): δ = 7.40ꢀ7.36 (m, 2 H, C H ), 7.27
9
6
3
r
3
ar
3
3
13
(
d, J = 8.4 Hz, 2 H, C H ), 4.75 (s, 2 H, CH ) ppm; C NMR (CDCl ): δ = 149.1 (d,
ar 2 3
3
4
2
1
ar
1
J = 1.8 Hz, C ), 133.0 (C H ), 128.4 (C ), 125.5 (N=C=S), 120.4 (d, J = 257.7 Hz, CF ),
CF
ar
ar
CF
3
3
+
121.5 (C H ), 48.2 (CH ) ppm; MS (ESI, 70 eV) m/z = 464.9 [MM] .
ar
2
Methyl 3ꢀaminoꢀ4,5ꢀdihydrothiopheneꢀ2ꢀcarboxylate (8)
To a solution of 1,8ꢀdiazabicyclo[5.4.0]undecꢀ7ꢀene (DBU, 10.0 mL, 67.0 mmol) in anhydrous
methanol (25 mL) under nitrogen atmosphere was added methyl thioglycolate (4.20 mL, 46.7
mmol) at 0°C. Acrylonitrile (3.30 mL, 49.8 mmol) was added dropwise, and the solution was
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2
2
2
2
2
2
2
2
3
3
3
3
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3
3
3
3
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stirred at 0°C for 5 h and at 80°C overnight. After cooling to room temperature, the solvent was
evaporated and quenched with a saturated solution of ammonium chloride. The aqueous phase
was extracted with ethyl acetate (3 x 100 mL), the organic layer was dried over sodium sulfate,
and the solvent was removed under reduced pressure. The crude product was purified by silica
gel column chromatography (30 % EA/PE) to obtain (8) as yellow solid. Yield: 3.20 g (43 %);
0
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C H NO S (M 159.21); H NMR (DMSOꢀd ): δ = 7.08 (s, 2 H, NH ), 3.57 (s, 3 H, CH ), 2.96ꢀ
6
9
2
r
6
2
3
13
2.91 (m, 2 H, SCH CH ), 2.85ꢀ2.78 (m, 2 H, SCH CH ) ppm; C NMR (DMSOꢀd ): δ = 165.1
2
2
2
2
6
(
C=O), 159.2 (C ꢀNH ), 85.9 (SC C=O), 50.3 (CH ), 38.4 (SCH CH ), 26.9 (SCH CH ) ppm;
q 2 q 3 2 2 2 2
+
+
MS (ESI, 70 eV) m/z = 316.9 [MM] , 159.8 [M+H] .
General method for the synthesis of tetrahydrothienoꢀpyrimidinone derivatives
The appropriate isothiocyanate was heated with methyl 3ꢀaminoꢀ4,5ꢀdihydrothiopheneꢀ2ꢀ
carboxylate in anhydrous pyridine at 110°C under nitrogen atmosphere for 24 h. After cooling of
the solution the solvent was evaporated and the residue was purified by silica gel column flash
chromatography (gradient EE/PE).
3
ꢀPhenylꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (9)
3
ꢀPhenylꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (9) was obtained from
commercially available phenyl isothiocyanate (420 µL, 3.51 mmol) with 8 (461 mg, 2.90 mmol)
in anhydrous pyridine (9 mL). After silica gel column flash chromatography (gradient EE/PE
starting with 30 % EE) the obtained product was washed three times with ethyl acetate. Yield:
1
113 mg (15 %); C H N OS (M 262.35); H NMR (CDCl ): δ = 13.23 (s, 1 H, NH), 7.49ꢀ7.35
12
10
2
2
r
3
2
4
3
(
m, 3 H, C H
, C H ), 7.19ꢀ7.16 (m, 2 H, C H ), 3.41ꢀ3.36 (m, 2 H, SCH CH ), 3.28ꢀ3.22
phen phen phen 2 2
13
(
m, 2 H, SCH CH ) ppm; C NMR (CDCl ): δ = 175.3 (C=S), 156.9 (C=O), 149.5 (C NHR),
2 2 3 q
1
3
2
4
1
39.1 (C
), 129.0 (C H ), 128.6 (C H ), 128.2 (C H ), 114.4 (SC C=O), 34.6
phen phen phen phen q
+
(
SCH CH ), 29.0 (SCH CH ) ppm; MS (ESI, 70 eV) m/z = 262.8 [MM] .
2 2 2 2
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ꢀBenzylꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidineꢀ4(1H)ꢀone (10)
3
ꢀBenzylꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidineꢀ4(1H)ꢀone (10) was obtained from
commercially available benzyl isothiocyanate (560 µL, 4.22 mmol) with 8 (596 mg, 3.74 mmol)
in anhydrous pyridine (12 mL).
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
After silica gel column flash chromatography (gradient EE/PE starting with 30 % EE) the
obtained product was washed three times with ethyl acetate. Yield: 207 mg (20 %); C H N OS
1
3
12
2
2
1
2
3
(M 276.38); H NMR (DMSOꢀd ): δ = 13.24 (s, 1 H, NH), 7.30ꢀ7.21 (m, 5 H, C H
, C H
benz
,
r
6
benz
4
C H ), 5.53 (s, 2 H, NꢀCH ), 3.38ꢀ3.32 (m, 2 H, SCH CH ), 3.24ꢀ3.18 (m, 2 H, SCH CH )
benz
2
2
2
2
2
1
3
1
ppm; C NMR (DMSOꢀd ): δ = 174.5 (C=S), 156.6 (C=O), 149.3 (C NHR), 136.2 (C benz),
6
q
3
2
4
128.2 (C H ), 127.2 (C H ), 127.0 (C H ), 114.0 (SC C=O), 48.7 (NꢀCH ), 34.5
benz
benz
benz
q
2
+
(SCH CH ), 28.9 (SCH CH ) ppm; MS (ESI, 70 eV) m/z = 276.7 [M+H] .
2 2 2 2
3
ꢀ(4ꢀMethoxybenzyl)ꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (11)
ꢀ(4ꢀMethoxybenzyl)ꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (11) was
3
obtained from 2 (313 mg, 1.75 mmol) with 8 (269 mg, 1.69 mmol) in anhydrous pyridine (5 mL).
The crude product was purified by silica gel column flash chromatography (gradient EE/PE
1
starting with 20 % EE). Yield: 120 mg (23 %); C H N O S (M 306.40); H NMR (DMSOꢀd ):
1
4
14
2
2
2
r
6
3
2
3
3
δ = 13.19 (s, 1 H, NH), 7.29 (d, J = 8.8 Hz, 2 H, C H ), 6.85 (d, J = 8.8 Hz, 2 H, C Hbenz),
benz
3
5
.45 (s, 2 H, NꢀCH ), 3.71 (s, 3 H, CH ), 3.33 (t, J = 8.0 Hz, 2 H, SCH CH ), 3.20 (t,
2
3
2
2
3
13
J = 8.0 Hz, 2 H, SCH CH ) ppm; C NMR (DMSOꢀd ): δ = 174.4 (C=S), 158.4 (C=O), 156.6
2
2
6
4
2
1
3
(C
), 149.2 (C NHR), 129.1 (C Hbenz), 128.2 (C
), 114.1 (SC C=O), 113.5 (C Hbenz), 55.0
benz q
benz
q
(CH ), 48.1 (NꢀCH ), 34.5 (SCH CH ), 28.9 (SCH CH ) ppm; MS (ESI, 70 eV) m/z = 306.9
3 2 2 2 2 2
+
[
M+H] .
3
ꢀ(2,3ꢀDimethoxybenzyl)ꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (12)
2
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9
1
1
1
1
1
1
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1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
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4
5
5
5
5
5
5
5
5
5
5
6
3
ꢀ(2,3ꢀDimethoxybenzyl)ꢀ2ꢀthioxoꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (12) was
obtained from 4 (495 mg, 1.75 mmol) with 8 (432 mg, 2.71 mmol) in anhydrous pyridine (8 mL).
After silica gel column flash chromatography (gradient EE/PE starting with 20 % EE) the
obtained product was washed three times with ethyl acetate. Yield: 226 mg (28 %);
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
C H N O S (M 336.43); H NMR (DMSOꢀd ): δ = 13.25 (s, 1 H, NH), 6.92 (m, 2 H, C Hbenz,
15
16
2
3
2
r
6
5
6
C H ), 6.35ꢀ6.32 (m, 1 H, C H ), 5.52 (s, 2 H, NꢀCH ), 3.80 (s, 6 H, CH ), 3.36 (t,
benz
benz
2
3
3
3
13
J = 8.0 Hz, 2 H, SCH CH ), 3.24 (t, J = 7.7 Hz, 2 H, SCH CH ) ppm; C NMR (DMSOꢀd ):
2
2
2
2
6
2
3
1
δ = 174.6 (C=S), 156.5 (C=O), 152.3 (C
), 149.3 (C NHR), 145.8 (C benz), 129.3 (C benz),
q
benz
5
6
4
2
1
23.7 (C H ), 117.0 (C H ), 113.8 (SC C=O), 111.5 (C Hbenz), 59.6 (C
ꢀOCH ), 55.6
benz 3
benz
benz
q
3
(C
ꢀOCH ), 44.5 (NꢀCH ), 34.5 (SCH CH ), 28.9 (SCH CH ) ppm; MS (ESI, 70 eV)
benz 3 2 2 2 2 2
+
m/z = 336.9 [M+H] .
ꢀThioxoꢀ3ꢀ(3ꢀ(trifluoromethyl)benzyl)ꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone
13)
ꢀThioxoꢀ3ꢀ(3ꢀ(trifluoromethyl)benzyl)ꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (13)
2
(
2
was obtained from 5 (598 mg, 2.75 mmol) with 8 (366 mg, 2.30 mmol) in anhydrous pyridine (7
mL). After evaporating the solvent, the residue was washed with dichloromethane and the
1
product was obtained as beige solid. Yield: 250 mg (32 %); C H F N OS (M 344.38); H
1
4
11
3
2
2
r
2
4
NMR (DMSOꢀd ): δ = 13.30 (s, 1 H, NH), 7.70 (bs, 1 H, C H ), 7.63ꢀ7.52 (m, 3 H, C Hbenz,
6
benz
5
6
3
3
C H , C H ), 5.60 (s, 2 H, NꢀCH ), 3.35 (t, J = 8.0 Hz, 2 H, SCH CH ), 3.22 (t, J = 7.9 Hz,
benz
benz
2
2
2
13
2 H, SCH CH ) ppm; C NMR (DMSOꢀd ): δ = 174.4 (C=S), 156.7 (C=O), 149.6 (C NHR),
2
2
6
q
1
6
5
2
3
1
37.7 (C
), 131.3 (C H ) 129.3 (C H ), 128.9 (d, J = 31.4 Hz, C benz), 124.1 (d,
benz benz , benz CF
1
3
2
3
4
J = 272.3 Hz, CF ), 124.1 (d, J = 3.8 Hz, C H ), 123.9 (d, J = 3.7 Hz, C Hbenz), 114.0
CF
3
CF
benz
CF
(SC C=O), 48.5 (NꢀCH ), 34.5 (SCH CH ), 28.9 (SCH CH ) ppm; MS (ESI, 70 eV) m/z = 344.9
q 2 2 2 2 2
+
[
M+H] .
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ꢀThioxoꢀ3ꢀ(4ꢀ(trifluoromethyl)benzyl)ꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone
(14)
2
ꢀThioxoꢀ3ꢀ(4ꢀ(trifluoromethyl)benzyl)ꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone (14)
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
was obtained from 6 (550 mg, 2.53 mmol) with 8 (358 mg, 2.25 mmol) in anhydrous pyridine (7
mL). After evaporating the solvent, the residue was washed with dichloromethane and the
1
product was obtained as beige solid. Yield: 373 mg (48 %); C H F N OS (M 344.38); H
1
4
11
3
2
2
r
3
3
NMR (DMSOꢀd ): δ = 13.32 (s, 1 H, NH), 7.67 (d, J = 8.2 Hz, 2 H, C Hbenz), 7.47 (d,
6
3
2
J = 8.2 Hz, 2 H, C H ), 5.60 (s, 2 H, NꢀCH ), 3.39ꢀ3.33 (m, 2 H, SCH CH ), 3.26ꢀ3.20 (m,
benz
2
2
2
13
2
H, SCH CH ) ppm; C NMR (DMSOꢀd ): δ = 174.5 (C=S), 156.6 (C=O), 149.6 (C NHR),
2
2
6
q
1
2
2
4
3
3
141.1 (C
), 127.6 (C H ) 127.6 (d, J = 32.2 Hz, C
), 125.1 (q, J = 3.9 Hz, C Hbenz),
benz CF
benz
benz ,
CF
1
1
24.3 (d, J = 271.8 Hz, CF ), 114.0 (SC C=O), 48.6 (NꢀCH ), 34.5 (SCH CH ), 29.0
CF
3
q
2
2
2
+
(SCH CH ) ppm; MS (ESI, 70 eV) m/z = 344.9 [M+H] .
2 2
2
ꢀThioxoꢀ3ꢀ(4ꢀ(trifluoromethoxy)benzyl)ꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀ
one (15)
ꢀThioxoꢀ3ꢀ(4ꢀ(trifluoromethoxy)benzyl)ꢀ2,3,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ4(1H)ꢀone
15) was obtained from 7 (294 mg, 1.26 mmol) with 8 (181 mg, 1.14 mmol) in anhydrous
pyridine (3.5 mL). After silica gel column flash chromatography (gradient EE/PE starting with 10
EE) the obtained product was obtained as beige solid. Yield: 112 mg (27 %); C H F N O S
2 2
2
(
%
14
11
3
2
1
3
3
(M 360.37); H NMR (DMSOꢀd ): δ = 13.28 (s, 1 H, NH), 7.43 (d, J = 8.4 Hz, 2 H, C Hbenz),
r
6
3
2
3
7.30 (d, J = 8.3 Hz, 2 H, C H ), 5.54 (s, 2 H, NꢀCH ), 3.34 (t, J = 7.5 Hz, 2 H, SCH CH ),
benz
2
2
2
3
13
3
.22 (t, J = 7.7 Hz, 2 H, SCH CH ) ppm; C NMR (DMSOꢀd ): δ = 174.4 (C=S), 156.6 (C=O),
2 2 6
4
1
2
3
1
49.5 (C NHR), 147.3 (C benz), 135.7 (C
) 129.2 (C H ), 120.8 (C Hbenz), 120.1 (d,
benz , benz
q
1
J = 255.9 Hz, CF ), 114.1 (SC C=O), 48.2 (NꢀCH ), 34.5 (SCH CH ), 29.0 (SCH CH ) ppm;
CF
3
q
2
2
2
2
2
+
MS (ESI, 70 eV) m/z = 360.9 [M+H] .
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2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
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5
5
5
5
5
5
5
6
2
ꢀChloroꢀNꢀ(6ꢀ(trifluoromethyl)benzo[d]thiazolꢀ2ꢀyl)acetamide (16)
A mixture of 5ꢀ(trifluoromethyl)benzo[d]thiazolꢀ2ꢀamine (596 mg, 2.73 mmol) and
trimethylamine (453 µL, 3.27 mmol) in dichloromethane (6 mL) was added over a period of 10
minutes to a solution of 2ꢀchloracetyl chloride (239 µL, 3.00 mmol) in dichloromethane (4 mL).
The reaction mixture was kept under stirring for 24 h and then concentrated in vacuum. The
obtained product was washed with water and dried in vacuum. Yield: 596 g (74 %);
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
4
C H ClF N OS (M 294,68); H NMR (DMSOꢀd ): δ = 12.96 (s, 1 H, NH), 8.52 (d, J = 1.6 Hz,
10
6
3
2
r
6
7
3
4
3
4
1
H, C H
), 7.94 (d, J = 8.5 Hz, 1 H, C H
), 7.75 (dd, J = 8.5 Hz, J = 1.6 Hz, 1 H,
benzth
benzth
5
13
2
C H
), 4.50 (s, 2 H, CH ꢀCl) ppm; C NMR (DMSOꢀd ): δ = 166.4 (C=O), 160.8 (C benzth),
benzth
2
6
3
a
7a
benzth ,
1
2
151.2 (C benzth), 132.0 (C
) 124.5 (d, J = 271.8 Hz, CF ), 123.9 (d, J = 31.9 Hz,
CF 3 CF
6
3
5
4
3
7
C
), 123.0 (d, J = 3.6 Hz, C H
), 121.2 (C H
), 120.0 (d, J = 4.0 Hz, C Hbenzth),
CF
benzth
CF
benzth
benzth
+
4
2.5 (CH ꢀCl) ppm; MS (ESI, 70 eV) m/z = 294.8 [M+H] .
2
General method for the synthesis of IWP derived compounds
Appropriate acetamide and tetrahydrothienoꢀpyrimidinone derivative was dissolved in DMF
under a positive pressure of nitrogen. Triethylamine was added and the mixture was stirred at
8
0°C for 2 h. The reaction mixture was cooled to room temperature and quenched with water.
The organic layer was separated and the aqueous layer was extracted with ethyl ether (3 times).
The combined organic phases were washed three times each with water and brine, dried over
sodium sulfate and the solvent was reduced in vacuum. The crude product was purified by silica
gel column flash chromatography (gradient EE/PE).
2
ꢀ((4ꢀOxoꢀ3ꢀphenylꢀ3,4,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ2ꢀyl)thio)ꢀNꢀ(6ꢀ
trifluoromethyl)benzo[d]thiazolꢀ2ꢀyl)acetamide (17)
ꢀ((4ꢀOxoꢀ3ꢀphenylꢀ3,4,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ2ꢀyl)thio)ꢀNꢀ(6ꢀ
trifluoromethyl)benzo[d]thiazolꢀ2ꢀyl)acetamide (17) was obtained from 16 (220 mg,
(
2
(
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.747 mmol), 9 (186 mg, 0.708 mmol) and triethylamine (300 µL, 2.16 mmol) in DMF (7 mL).
The crude product was purified by silica gel column chromatography (gradient EE/PE starting
1
with 20 % EE). Yield: 95 mg (26 %); C H F N O S (M 520.57); HPLC (purity): 97 %; H
22 15
3
4
2
3
r
4
5
7
1
1
1
1
1
1
1
1
1
1
2
2
2
2
2
2
2
2
2
2
3
3
3
3
3
3
3
3
3
3
4
4
4
4
4
4
4
4
4
4
5
5
5
5
5
5
5
5
5
5
6
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
6
7
8
9
0
NMR (DMSOꢀd ): δ = 12.85 (s, 1 H, NH), 8.50 (dd, J = 1.6 Hz, J = 0.6 Hz, 1 H, C Hbenzth), 7.92
6
3
4
5
3
(d, J = 8.5 Hz, 1 H, C H
), 7.76ꢀ7.73 (m, 1 H, C H
), 7.62ꢀ7.57 (m, 3 H, C Hphen,
benzth
benzth
4
2
3
C H ), 7.46ꢀ7.42 (m, 2 H, C Hphen), 4.16 (s, 2 H, CH ꢀC=O), 3.30 (t, J = 8.1 Hz, 2 H,
phen
2
3
13
SCH CH ), 3.10 (t, J = 8.1 Hz, 2 H, SCH CH ) ppm; C NMR (DMSOꢀd ): δ = 167.6 (CH ꢀ
2
2
2
2
6
2
2
3a
C=O), 161.0 (C
), 160.5 (C N=CꢀS), 158.5 (N=C ꢀS), 156.9 (NRR’ꢀC=O), 151.3 (C benzth),
q q
benzth
1
7a
3
4
2
1
35.4 (C phen), 132.0 (C
), 130.2 (C H ), 129.7 (C H ), 128.8 (C Hphen), 124.5 (d,
benzth phen phen
1
2
6
3
5
J = 271.7 Hz, CF ), 123.8 (d, J = 31.5 Hz, C
), 122.9 (d, J = 3.2 Hz, C Hbenzth),
CF
CF
3
CF
benzth
4
7
1
21.0 (C H
), 119.9 (SC C=O), 119.8 (C H
), 37.1 (SCH CH ), 36.1 (CH ꢀC=O), 28.4
benzth 2 2 2
benzth
q
+
(SCH CH ) ppm; MS (ESI, 70 eV) m/z = 520.8 [M+H] ; IR: 3173, 3073, 1707, 1647, 1545,
2 2
ꢀ1
1
2
308, 1244, 1121, 756, 698 cm .
ꢀ((3ꢀBenzylꢀ4ꢀoxoꢀ3,4,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ2ꢀyl)thio)ꢀNꢀ(6ꢀ
(
trifluoromethyl)benzo[d]thiazolꢀ2ꢀyl)acetamide (18)
ꢀ((3ꢀbenzylꢀ4ꢀoxoꢀ3,4,6,7ꢀtetrahydrothieno[3,2ꢀd]pyrimidinꢀ2ꢀyl)thio)ꢀNꢀ(6ꢀ
(trifluoromethyl)benzo[d]thiazolꢀ2ꢀyl)acetamide (18) was obtained from 16 (235 mg,
.797 mmol), 10 (196 mg, 0.709 mmol) and triethylamine (300 µL, 2.16 mmol) in DMF (7 mL).
2
0
The crude product was purified by silica gel column flash chromatography (gradient EE/PE
starting with 20 % EE). Yield: 278 mg (73 %); C H F N O S (M 534.60); HPLC (purity): 100
2
3
17
3
4
2
3
r
1
4
5
%
; H NMR (DMSOꢀd ): δ = 12.92 (s, 1 H, NH), 8.49 (dd, J = 1.3 Hz, J = 0.6 Hz, 1 H,
6
7
3
4
3
4
5
C H
), 7.92 (d, J = 8.5 Hz, 1 H, C H
), 7.74 (dd, J = 8.6 Hz, J = 1.5 Hz, 1 H, C Hbenzth),
benzth
benzth
2
3
4
7
.40ꢀ7.25 (m, 5 H, C H , C H , C H ), 5.27 (s, 2 H, NꢀCH ), 4.27 (s, 2 H, CH ꢀC=O), 3.27
phen phen phen 2 2
3
3
13
(t, J = 8.5 Hz, 2 H, SCH CH ), 3.04 (t, J = 8.5 Hz, 2 H, SCH CH ) ppm; C NMR (DMSOꢀd ):
2
2
2
2
6
29
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