Evaluation of Improved Glycogen Synthase Kinase-3α Inhibitors in Models of Acute Myeloid Leukemia

Article abstract of DOI:10.1021/acs.jmedchem.5b01200

The challenge for glycogen synthase kinase-3 (GSK-3) inhibitor design lies in achieving high selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid leukemia (AML), may require α-isoform specific targeting. The s

Full text of DOI:10.1021/acs.jmedchem.5b01200

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Article
Evaluation of Improved Glycogen Synthase Kinase-3#
Inhibitors in Models of Acute Myeloid Leukemia
Theresa Neumann, Lina Benajiba, Stefan Göring, Kimberly Stegmaier, and Boris Schmidt
J. Med. Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jmedchem.5b01200 • Publication Date (Web): 23 Oct 2015
Downloaded from http://pubs.acs.org on November 1, 2015
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Evaluation of Improved Glycogen Synthase Kinaseꢀ3α Inhibitors in
Models of Acute Myeloid Leukemia
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Theresa Neumann1, Lina Benajiba2, Stefan Göring1, Kimberly Stegmaier2 and Boris Schmidt1†.
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1Clemens Schöpf-Institute of Organic Chemistry and Biochemistry, Technische Universität Darmstadt,
64287 Darmstadt, Germany.
2Department of Pediatric Oncology, Dana-Farber Cancer Institute, Harvard Medical School, Boston,
MA, USA.
† Corresponding author: Tel.: +49 6151 163075; Fax: +49 6151 163278
E-mail: Schmidt_boris@t-online.de
Keywords: Acute myeloid leukemia, drug discovery, glycogen synthase kinaseꢀ3, inhibitors,
zebrafish.
Abstract
The challenge for Glycogen Synthase Kinaseꢀ3 (GSKꢀ3) inhibitor design lies in achieving high
selectivity for one isoform over the other. The therapy of certain diseases, such as acute myeloid
leukemia (AML) may require αꢀisoform specific targeting. The scorpion shaped GSKꢀ3 inhibitors
developed by our group achieved the highest GSKꢀ3α selectivity reported so far, but suffered from
insufficient aqueous solubility. This work presents the solubilityꢀdriven optimization of our isoformꢀ
selective inhibitors using a scorpion shaped lead. Among 15 novel compounds, compound 27 showed
high activity against GSKꢀ3α/β with the highest GSKꢀ3α selectivity reported to date. Compound 27
was profiled for bioavailability and toxicity in a zebrafish embryo phenotype assay. Selective GSKꢀ3α
targeting in AML cell lines was achieved with compound 27, resulting in a strong differentiation
phenotype and colony formation impairment, confirming the potential of GSKꢀ3α inhibition in AML
therapy.
Introduction
Glycogen Synthase Kinaseꢀ3 (GSKꢀ3) is a constitutively active, ubiquitous serine/threonine kinase,
which takes part in a number of physiological processes ranging from glycogen metabolism to
apoptosis.1ꢀ3 GSKꢀ3 is a key mediator of various signalling pathways, such as the Wnt and the
Insulin/AKT signalling pathways.1 Therefore, dysregulation of GSKꢀ3 has been linked to various
human diseases, such as cancer, diabetes and neurodegenerative diseases.1ꢀ2, 4ꢀ7 Two related isoforms
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of GSKꢀ3 exist in mammals, GSKꢀ3α and β, which share a sequence identity within their catalytic
domains of 98%.2ꢀ3, 8 Beyond the catalytic domains they show significant differences.2, 8 Although
these isoforms are structurally related, they are not functionally equivalent, and one cannot
compensate for loss of the other.6 The debate on the respective contributions of the isoforms GSKꢀ3α
and GSKꢀ3β on the pathogenesis of different diseases is ongoing.9 Various studies indicate that the
therapies of certain diseases benefit from specific targeting of GSKꢀ3α and GSKꢀ3β.6, 10ꢀ14 GSKꢀ3α
was recently identified as a differentiation target in acute myeloid leukemia (AML).10 AML is a
haematopoietic malignancy defined by uncontrolled proliferation and disrupted myeloid
differentiation.15ꢀ16 AML is the second most common form of leukaemia in adults.16 The current
treatment of AML with conventional chemotherapy is very aggressive yet ineffective for the majority
of patients with the disease.15 Thus, alternative targeted treatment approaches for AML are highly
desirable.10, 15 GSKꢀ3α recently emerged as a potential target in this disease.10
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Many molecular approaches for the inhibition of GSKꢀ3 have been reported. However, most of these
inhibitors lack selectivity for GSKꢀ3 over other related kinases. This selectivity problem can challenge
the clinical utility of these inhibitors leading to doseꢀlimiting toxicities. Moreover, due to the structural
similarity between the two ATP binding pockets of GSKꢀ3α and GSKꢀ3β most published inhibitors do
not differ effectively between the GSKꢀ3 isoforms. Therefore, our research focuses on the
development of isoformꢀselective, ATPꢀcompetitive inhibitors using a scorpion shaped lead
(Figure 1A).9 Compound 1 (Figure 1) shows high kinase inhibitory activity and an impressive
selectivity for GSKꢀ3α.9 However, compound 1 could not be tested in vivo due to insufficient
solubility. Therefore, it was chosen as lead structure for the design of improved GSKꢀ3 inhibitors.
Here we report the synthesis and optimization of scorpion shaped GSKꢀ3 inhibitors with improved
solubility. The most promising compounds were evaluated in a wildꢀtype zebrafish embryo assay and
in AML cell lines.
Optimization Strategy
Optimization of compound 1 was performed to improve its pharmacokinetics and cell penetration. In
addition, our intention was to increase the selectivity towards GSKꢀ3α. Unfortunately, the structure of
GSKꢀ3α has not been solved. Therefore, targetꢀoriented synthesis of isoformꢀspecific inhibitors can
inform the mechanism behind αꢀselectivity of our scorpion shaped GSKꢀ3 inhibitors. All structures
share the oxadiazole moiety, as it provides a high inhibitory activity and isoformꢀselectivity.9 Different
substitution patterns at the biphenylic scaffold were explored in order to enhance GSKꢀ3α selectivity
and to concurrently improve solubility.
We next designed 140 leadꢀlike compounds by the systematic modification of scaffold elements: the
heteroaromatic head group (Figure 1A: blue), the spacer between the oxadiazole and the biphenylic
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scaffold (Figure 1A: yellow), the first aromatic ring of the biphenylic scaffold (Figure 1A: green) and
the terminal aromatic ring (Figure 1A: red). To find promising compounds all leadꢀlike structures were
evaluated by molecular docking. Initially, the essential ligandꢀreceptor interactions of compound 1
with GSKꢀ3β had to be determined to make it possible to compare the candidates with the lead
structure and the respective docking hypothesis. Docking of compound 1 into the GSKꢀ3β active site
(PDB: 3F88) was done by the software MOE 2014.09.17 Afterwards the docking poses were rescored
by the DSX rescoring function18 with the aim to identify the best binding mode of compound 1
(Figure 1C). The resulting conformations agree with already published findings from our group.9 The
head group (dihydrobenzodioxine and oxadiazole) of compound 1 is oriented to the hinge region
where it forms hydrophobic interactions with Tyr134. The oxadiazole ring is located between Val70
and Cys199. The biphenylic tail group establishes πꢀπꢀstacking interaction with Phe67 and
Hꢀπꢀinteractions with Gln185. In addition, the cyano moiety builds an Hꢀbond to Thr138. From this
model a phamacophore was generated. It consists of three aromatic features fulfilled by the oxadiazole
ring and two phenyl rings (Figure 1B: orange), one hydrophobic centroid covered by one phenyl ring
(Figure 1B: yellow) and one Hꢀbond acceptor pharmacophore feature fulfilled by one nitrogen atom of
the oxadiazole ring (Figure 1B: blue). With the intention to identify the best candidates of 140
leadꢀlike compounds, docking was performed with phamacophore placement. Then the resulted
docking poses were again rescored by the DSX rescoring function.18 Thereby the best rated docking
poses reproduce the overall orientation of the lead structure compound 1 (Figure 1D).
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C
D
Figure 1: A) Compound 1 was used as lead structure for systematic modification. B) Docking study of compound
1 using MOE 2014.09 and the known GSKꢀ3β crystal structure (PDB: 3F88). C) From the docking study (B)
Resulted pharmacophore hypothesis is used for screening. The pharmacophore is represented by an Hꢀbond
acceptor (blue), aromatic rings (orange) and hydrophobic centroids (yellow). D) Selection of compounds that
match the docking model of 1.17
Based on the results of the docking simulations the best candidates were evaluated further. The next
filtering was set by limitation of the ClogP value to be less than or equal to 4.5. This ensured that the
number of hits were limited to more polar compounds than 1. This filtering step reduced the number of
hits to 94. The analysis of possible ways for derivatization and chemical accessibility led to 15
promising candidates, which were synthesized in the next steps as outlined below.
Chemistry
The reference Compound 1 and some of the screening hits were synthesized according to the
published procedure.9 The building blocks of the head group and the tail group were prepared in a
converging synthesis route.9
The synthesis of the head group building blocks 4a-b was accomplished starting from the carboxylic
acids 2a-b. The carboxylic acids 2a-b were esterified to the methyl esters 3a-b. The oxadiazole
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derivates 4a-b resulted from 3a-b after reaction with hydrazine monohydrate followed by cyclization
of the resulting hydrazides with carbondisulfide under basic conditions (Scheme 1).9
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.
Scheme 1: Reagents and conditions: (a) MeOH, SOCl2, 0 – 50 °C, 96 – 99%; (b) NH2NH2 H2O, EtOH, reflux, then
CS2, Et3N, EtOH, reflux, 11 ꢀ 45%.
The synthesis of the biphenylic scaffold starts either with a Suzuki coupling of the commercially
available boronic acid 6 with various bromides 5a-d,g or (hydroxyl)boronic acid 11 with
bromines 10e,f,h (Scheme 2). For the cases of intermediates 7a-d,g a radical bromination with NBS
and AIBN was performed to obtain bromides 8a-d,g. The resulting benzylic alcohols 9e,f,h, however,
were treated with phosphorous tribromide, to convert them into the benzylic bromides 8e,f,h
(Scheme 2). Coupling of the biphenylic bromides 8aꢀh to the mercaptanes 4a-b gave the final
compounds 12a, 13b-h.
Scheme 2: Reagents and conditions: (a) Pd(PPh3)4, 2N aq. Na2CO3, toluene/EtOH 1:1, 80 °C, 27 – 99%;
(b) NBS, AIBN, CCl4, reflux, 52 – 64%; (c) PBr3, toluene, reflux, (d) 4aꢀb, 4N aq. NaOH, DMF, rt, 12 – 66%.
Suzuki coupling of the fluoroboronic acids 15a-c with bromines 14a-c followed by radical
bromination gave the benzylic bromides 17a-c, which were coupled under basic conditions to the
mercaptane 4b to yield the final compounds 18a-c (Scheme 3).
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Scheme 3: Reagents and conditions: (a) Pd(PPh3)4, 2N aq. Na2CO3, toluene/EtOH 1:1, 80 °C, 87 – 99%;
(b) NBS, AIBN, CCl4, reflux, 22 – 45%; (c) 4b, 4N aq. NaOH, DMF, rt, 31 – 37%.
The orthoꢀsubstituted product 21 was obtained by a nucleophilic aromatic substitution of
2,5ꢀdifluorobenzonitrile (20) and methylpiperidineꢀ4ꢀcarboxylate (19) with cesium carbonate
(Scheme 4). The reduction of the methoxy ester 21 by lithium borohydride provided the primary
alcohol 22. Appel reaction of 22 gave the bromide 23, which was coupled under basic conditions to
the mercaptane 4b to yield the final compound 24.
Scheme 4: Reagents and conditions: (a) Cs2CO3, DMSO, 100 °C, 42%; (b) LiBH4, THF, reflux, 98%; (c) CBr4,
PPh3, acetonitrile, rt, 35%; (d) 4b, K2CO3, DMF, 80 °C, 71%.
The sulfur atom of the previously synthesized Compound 1 was oxidized by mCPBA to the final
compounds (rac)-25 and 26 (Scheme 5). The final compound 27 was obtained by the partial
hydrolysis of the cyano group of 1 under acidic conditions (Scheme 5).
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Scheme 5: Reagents and conditions: (a) mCPBA, DCM, 0° C – RT, 79%; (b) mCPBA, DCM, 0 – 30 °C, 68%;
(c) H2SO4, TFA, reflux, 78%.
In vitro Phamacology and StructureꢀActivity Relationship
The obtained compounds were tested for their inhibitory activity against GSKꢀ3α and GSKꢀ3β in a
commercial in vitro assay.19ꢀ20 The assay was performed at an inhibitor concentration of 1 µM
(100 nM for 26), using isolated GSKꢀ3α and GSKꢀ3β from porcine brain and staurosporine as
control.19ꢀ20 The results are expressed as percentage of control specific activity.19ꢀ20 The structureꢀ
activity relationship was analyzed by systematic modification of scaffold moieties.
Initially, the hinge binding dihydrobenzodioxine moiety of the lead structure 1 was replaced by a
benzimidazole group as potential hinge binder. This lead to a massive decreased inhibitory activity
against both isoforms as compared to the lead structure 1 (Table 1). Consequently, the benzimidazole
scaffold was abandoned. For all further modification steps the dihydrobenzodioxine scaffold was
unmodified.
In the next step, the impact of the spacer between the oxadiazole and the biphenylic scaffold was
investigated. The oxidation of the thioether 1 to the racemic sulfoxide (rac)-25 resulted in a decrease
of inhibitory activity, especially against GSKꢀ3α (Table 1). Due to the heavy loss of isoform
selectivity the racemic product was not separated into the pure enantiomers, as neither of the
enantiomeric sulfoxides is as active as the sulfide. The further oxidation of 1 to the sulfone 26 led to
an even stronger decrease of inhibitory activity (Table 1). The introduction of oxygen atoms on the
linking sulfur apparently results in repulsive interactions, presumably as the biphenylic scaffold is
displaced and the important πꢀstacking interaction with Phe67 or with Gln185 cannot be formed.
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Table 1: Inhibitory activity against GSKꢀ3α and GSKꢀ3β.
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GSKꢀ3α
Inhibition
[%]b
GSKꢀ3β
Inhibition
[%]b
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Compound
Het
Y
ClogPa
tPSAa
1 ꢁM of compound
1
S
4.60
3.98
76.2
82.1
80
36
48
45
0
12a
S
(rac)ꢀ25
SO
3.40
3.11
93.3
42
0
0
26
SO2
110.3
(100 nM)
(100 nM)
a Calculated by ChemBioDraw Ultra (version 13.0.2).21
b
The results are expressed as a percent of control specific activity = 100 – ((measured specific
activity/control specific activity)*100), at a concentration of 1 µM (100 nM for 26) of the test
compound. The measurements were performed in duplicates.
The influence of the biphenylic scaffold was investigated next keeping the hinge binding moiety and
the spacer constant. The introduction of a nonꢀaromatic piperidine in 24 resulted in an immense drop
of inhibitory potency (Table 2), because the πꢀstacking interaction with Phe67 or with Gln185 can no
longer be formed.
The impact of the cyano substituent on the activity and αꢀselectivity of 1 was addressed next (Table 2).
The complete removal of the cyano substituent in 13b (Table 2) resulted in a substantial loss of
inhibitory activity.
However, the exchange of the cyano substituent for the amide substituent in Compound 27 resulted in
the most potent compound (Table 2). Compound 27 inhibits GSKꢀ3α and GSKꢀ3β at a concentration
of 1 µM at 100 percent. The substitution by 3ꢀfluoropyridine in 13c and 2ꢀfluoropyridine in 13f led to
an improved inhibitory potency against GSKꢀ3β (Table 2). The inhibitory potency against GSKꢀ3α
increased slightly. However, the increase of the inhibitory potency against the αꢀisoform is stronger for
13f than for 13c. Consequently, the pyridine in 13f was changed back to a phenyl with a cyano
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substituent in ortho position to the fluorine. Due to the chemical accessibility the first phenyl ring of
the biphenylic scaffold had to be changed simultaneously to a 2ꢀpyridine in 18b and a 3ꢀpyridine in
18a. In both cases the modifications led to an immense drop of inhibitory potency (Table 2).
Moreover, a total loss of activity against GSKꢀ3β was observed for 18a. We assumed that the
orthoꢀcyano substituent in 18b and 18a was responsible for the sharp drop of activity. This hypothesis
was confirmed by the introduction of a second 2ꢀpyridine (18c) into 13f, where 18c shows the same
activity like 13f (Table 2). In the last step the impact of the fluoro substituent within the pyridines of
13f and 13c on the activity and αꢀselectivity was explored. The introduction of electronꢀdonating
methyl substituents in 13e and 13h leads to a slight improvement of inhibitory potency against
GSKꢀ3β (Table 2). However, the inhibitory potency against GSKꢀ3α was not affected. Consequently,
the differentiation between the isoforms was reduced. The introduction of an electronꢀwithdrawing
cyanoꢀsubstituent in 13d and 13g led to a decrease of inhibitory potency against GSKꢀ3α (Table 2).
However, both compounds showed the best discrimination between the isoforms with αꢀselectivity
towards GSKꢀ3α. In the case of the inhibition of the βꢀisoform both compounds behave differently.
13d showed the same inhibitory activity against GSKꢀ3β as 1. However, in the case of 13g a striking
decrease of inhibitory activity was observed against the GSKꢀ3β. The docking of 13d and 13g into the
GSKꢀ3β active site suggested that both cyano substituents contribute to the inhibitor activity by
establishing a Hꢀbond to the amino acid Arg144. The different activities between both compounds can
be explained by the rotation of the biphenylic residue. The biphenylic residue of 13g is rotated by
about 60 degree compared to 13d. Consequently, 13g can not form a πꢀstacking interaction with
Gln185. Due to the πꢀstacking interaction with Gln185, 13d interacts better with the active site.
Accordingly, 13d shows a higher activity against GSKꢀ3β than 13g.
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Table 2: Inhibitory activity against GSKꢀ3α and GSKꢀ3β.
GSKꢀ3α
Inhibition
[%]b
GSKꢀ3β
Inhibition
[%]b
Compound
R
ClogPa
tPSAa
1 ꢁM of compound
1
4.60
76.2
80
45
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13b
13c
13d
13e
13f
13g
13h
18a
18b
18c
24
5.17
3.97
3.37
4.24
3.76
3.36
4.03
3.15
3.36
2.55
3.88
3.39
52.4
64.8
88.6
64.8
64.8
88.6
64.8
88.6
88.6
77.1
79.4
95.5
13
86
86
86
95
58
99
21
49
93
20
100
6
84
44
88
78
21
89
0
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75
10
97
27
a Calculated by ChemBioDraw Ultra (version 13.0.2).21
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b
The results are expressed as a percent of control specific activity = 100 – ((measured specific
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6
activity/control specific activity)*100), at a concentration of 1 µM of the test compound. The
measurements were performed in duplicates.
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The IC50 values of the lead compound 1 and the most potent compound 27 were determined for
detailed analysis (Table 3). With our novel inhibitor 27 the activity and selectivity against GSKꢀ3α/β
could be increased further. With an IC50 value of 42 nM for GSKꢀ3α and 140 nM for GSKꢀ3β,
compound 27 inhibits GSKꢀ3α/β stronger than the lead compound 1.
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Table 3: Inhibitory activity against GSKꢀ3α and GSKꢀ3β IC50 [ꢁM].
Compound IC50 GSKꢀ3α [nM]a IC50 GSKꢀ3β [nM]a
1
230
42
>1000
140
27
a
The evaluation of the activity in GSKꢀ3α (h)19 and GSKꢀ3β (h)20 was performed by the company
Cerep using a published procedure by Greengard et al. (Supporting Info).22 The IC50 values were
determined by measurement of eight concentrations (0.03 nM, 0.3 nM, 1 nM, 3 nM, 10 nM, 30 nM,
100 nM, 1 µM).
With the intention to elucidate the difference between both isoforms of GSKꢀ3 a sequence alignment
of GSKꢀ3α (UniProtKB: P49840) on the 3Dꢀstructure of GSKꢀ3β (PDB: 3F88) was performed. The
sequence identity between GSKꢀ3β and GSKꢀ3α was calculated to be 86% by MOE 2014.09.17
However, all differences between the two isoforms are located away from the ATPꢀbinding site. This
results in a similarity of 98% within the active site. The SAR study of 13d is best suited to illustrate
the reasons for isoformꢀselectivity (Figure 2A). It shows that the cyano substituent contributes to the
inhibitor selectivity by establishing an Hꢀbond to the amino acid Arg144 (GSKꢀ3β) and within the
αꢀisoform to Arg207 (GSKꢀ3α). Arg144 and Arg207 are located in the Argꢀrich loop. However, the
neighboring amino acids within the Argꢀrich loop differ between the isoforms. Within GSKꢀ3β there is
Arg148 (green) while within GSKꢀ3α there is Lys211 (pink). Within GSKꢀ3β Arg144 interacts with
the next located amino acid Arg148 over a coordinated water molecule (Figure 2A). However, within
GSKꢀ3α the adjacent amino acids, Arg207 and Lys211, do not interact with each other (Figure 2A).
We suggest that the waterꢀmediated Hꢀbond between Arg144 and Arg148 is responsible for the
isoform selectivity of 13d. The amino acids within the Argꢀrich loop are likely solvent exposed and
quite flexible. But the waterꢀmediated Hꢀbond between Arg144 and Arg148 may restrict the motion of
the side chains, which would cause a conformational entropic penalty.23 This could cancel out any gain
in binding energy from the interaction between ligand and protein.23 Consequently, 13d inhibits
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GSKꢀ3β less potent than GSKꢀ3α (Table 2). We therefore propose that the isoform selectivity of our
scorpion shaped GSKꢀ3 inhibitors is caused by the two different amino acids, GSKꢀ3β: Arg148
(green), GSKꢀ3α: Lys211 (pink), within the Argꢀrich loop. Consequently, the paraꢀsubstituent of the
biphenylic moiety of our scorpion shaped GSKꢀ3 inhibitors plays a key role in the selectivity of the
inhibitor (Figure 2B). Obviously, this is a speculative hypothesis, which has to be confirmed by more
advanced theoretical and experimental work as well as Xꢀray studies of GSKꢀ3α.
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A
B
Figure 2: A) Docking study of compound 13d using MOE 2014.09 and the aligned structure of GSKꢀ3α
(UniProtKB: P49840) and GSKꢀ3β (PDB: 3F88).17 Differing amino acids are highlighted, GSKꢀ3β (green) and
(GSKꢀ3α) pink. B) Schematic overview of the selectivity hypothesis of our scorpion shaped GSKꢀ3 inhibitors.
Aqueous solubility of selected compounds
The aqueous solubility of chemical agents affects the uptake of the substances into cells and their oral
bioavailability. Consequently, a good waterꢀsolubility is essential for sufficient bioavailability, which
must be considered for the performance of the compound within the organism. Solubilityꢀdriven
optimization of 1 was the main intention of this work. The solubility values of the most active
compounds 1, 13c-h, 18c and 27 are shown in Table 4. Some of the compounds (1, 13e, 13h, 18c)
showed a poor water solubility of below 1 µg/mL, which makes them unsuitable for further
development. 13d and 27 are not only the most potent compounds, but also the most soluble. The
highest water solubility of 78 µg/mL (181 µM) was observed for 13d. The most potent compound 27
also has good aqueous solubility of 34 µg/mL (73 µM).
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Table 4: Aqueous solubility of selected compounds.
Compound Solubility [ꢁg/mL]a Solubility [ꢁM]a
6
7
8
9
1
< 1
dec
78
1
dec
181
1
13c
13d
13e
13f
13g
13h
18c
27
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< 1
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< 1
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< 1
< 1
34
73
a
Related to the in vivo assay conditions all diluted samples contain water and 2% DMSO. The
determination was done by the “ShakeꢀFlask” method24 and HPLC measurements.
In vivo evaluation
The most soluble and most active candidates 13d and 27 were further profiled for in vivo activity in
wildꢀtype zebrafish (Danio rerio) embryos. The zebrafish embryo is a useful model to assess doseꢀ
dependent bioavailability and toxicity of compounds. The embryos were collected and maintained in
E3 medium at ~26°C. The compounds were added 24 hours post fertilization (hpf) and the phenotypes
compared at 96 and 120 hpf. Both compounds showed no lethality in our concentration range
(< 60µM). Compound 13d showed no effects on the wildꢀtype zebrafish embryos. At lower
concentrations of compound 27 from 1 to 15 µM no abnormalities were observed (Figure 3). At
concentrations of 27 higher than 30 µM all zebrafish embryos showed a stunted and crooked tailed
phenotype (Figure 3). These observations support the absorption and cell penetration of compound 27.
Furthermore, this zebrafish embryo assay demonstrates that compound 27 disturbs the zebrafish
development. For embryonic development of the zebrafish the Wnt/βꢀcateninꢀsignalling pathway plays
an important role.25 The Wntꢀsignalling pathway involves the complex interplay of multiple proteins,
including GSKꢀ3β in this signalling network.26 The observed phenotype is consistent with activation
of βꢀcatenin, which is associated with inhibition of GSKꢀ3β.25, 27 Compound 27 exhibits IC50 values of
42 nM for GSKꢀ3α and 14 nM for GSKꢀ3β inhibition. The concentrations in this experiment are too
high to achieve isoformꢀselectivity. Consequently, compound 27 is no longer selective to GSKꢀ3α at
concentrations higher than 30 µM. It is likely that with this gross experiment isoformꢀselectivity
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cannot be detected. Therefore, we tested compound 27 in a cell assay at lower concentrations to
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investigate the effects on GSKꢀ3α versus GSKꢀ3β.
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Control
1ꢁM
15 ꢁM
30 ꢁM
45 ꢁM
60 ꢁM
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Figure 3: In vivo effects on wt zebrafish embryos by compound 27 (AꢀF).
Compound 27 was added 24 hours postꢀfertilization (hpf) to five embryos in
duplicates, and the phenotypes were compared after 96 hpf. (A) Control embryo in
2% DMSO. Zebrafish embryos treated with compound 27 at increasing
concentrations of (B) 1 ꢁM, (C) 15 ꢁM, (D) 30 ꢁM, (E) 45 ꢁM, (F) 60 ꢁM.
In vitro effects in AML cell lines
To further investigate the activity of compound 27 in a diseaseꢀbased model in vitro, we treated two
AML cell lines (HLꢀ60 and NB4) with increasing concentrations of compound 27. Compound 27
shows a specific onꢀtarget effect with a concentrationꢀdependent decrease in Tyr279 GSKꢀ3α
phosphorylation without any relevant effect on Tyr216 GSKꢀ3β phosphorylation up to 20 µM
(Figure 4A). GSKꢀ3α inhibition with compound 27 induces morphological (Figure 4B) and surface
marker changes (Figure 4C) consistent with AML differentiation. Selective GSKꢀ3α inhibition results
in methylcellulose colony formation impairment (Figure 4D) in NB4 and HLꢀ60 cell lines.
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Figure 4: In vitro effects of compound 27 in AML cell lines (HLꢀ60 and NB4). Selective GSKꢀ3α inhibition after 24h
treatment with increasing concentrations of compound 27 in NB4. Histograms show a quantification of pꢀGSKꢀ3α
tyr279 and pꢀGSKꢀ3β tyr216 relative to total GSKꢀ3α and β respectively (A). Morphological changes (B:
decreased nuclear to cytoplasmic ratio and increased vacuolization) and surface markers expression (CD11b,
CD11c and CD14) consistent with differentiation after 6 days of treatment, histograms and means of fluorescence
of 10 000 independent events are represented (C). Concentrationꢀdependent colony formation impairment.
*p<0,05 relative to corresponding DMSO. Error bars represent means +/ꢀ SEM (D).
Conclusion
Starting from the poorly soluble lead compound 1 a series of improved GSKꢀ3 inhibitors was obtained
by rational design. Optimization, primarily of the biphenylic scaffold of 1 led to the potent and αꢀ
selective GSKꢀ3 inhibitor 27 with improved solubility. Compound 27 exhibits IC50 values of 42 nM
for GSKꢀ3α and 140 nM for GSKꢀ3β. The homology model of GSKꢀ3α/β and the in vitro activities of
the isoformꢀselective compounds lead us to the hypothesis, that interactions to Arg148 (GSKꢀ3β) and
Lys211 (GSKꢀ3α) within the Argꢀrich loop are significant contributors for isoformꢀselectivity.
Our best compounds 13d and 27 were further profiled for efficacy and toxicity in the wt zebrafish
embryo assay. Compound 27 showed reported phenotypes without lethality in vivo and proof of
efficacy in vitro in two AML cell lines. A strong dose dependent differentiation phenotype and colony
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formation impairment through selective GSKꢀ3
α
inhibition was observed in AML cell lines. The
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properties of 27 make it an excellent tool compound to study the different pathogenesis contributions
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of the isoforms GSKꢀ3α and GSKꢀ3β and a promising therapeutic candidate in AML.
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Experimental Section
General Information
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All chemicals were purchased as reagent grade from commercial suppliers and used without further
purification. All reactions under anhydrous conditions were carried out under argon atmosphere with
dry solvents.
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The 1HꢀNMR spectra were recorded on a Bruker DRX 300 spectrometer at 300 MHz and on a Bruker
DRX 500 spectrometer at 500 MHz. The 13Cꢀspectra were recorded on a Bruker DRX 300 spectrometer
at 75 MHz and on a Bruker DRX 500 spectrometer at 125 MHz. Chemical shifts are expressed in parts
per million (ppm) and calibrated from the used solvent: CHCl3
(δCDCl3 = 7.26 ppm,
δCDCl = 77.16 ppm), DMSOꢀd6 ( (CHD )SO(CD ) = 2.50 ppm, (CD )SO(CD ) = 39.52 ppm). The respective
δ
δ
3
2
3
3
3
measuring frequency, the used solvent and the measuring temperature are added in brackets to the
spectroscopic data. Fine structures of proton signals are labeled with the following abbreviations: s for
singlet, d for doublet, t for triplet, q for quartet and m for multiplet. Coupling constants (J values) are
given in hertz (Hz).
Mass spectrometry was performed on a MAT 95 double focusing sector field EIꢀMS. Detected ion
masses (m/z) are expressed in u.
Analytical high performance liquid chromatographies (HPLC) were carried out on an Agilent 1100
(column: reversed phase, Synergi 4u Polar-RP 80A, 4.6x150 mm, Variable Wavelength Detector
λ = 254 nm). As a standard method a solvent gradient of acetonitrile (B) and 0.1% trifluoroacetic acid
in water (A) (70% A; 30% B (0 to 1 min) up to 90% B) with a flow rate of 1 mL/min and an elapsed
time of 12 min was used.
Flash column chromatography was carried out using silica gel 60 (15 – 40 µm, Merck). The automated
flash chromatography was carried out on a Combi Flash Rf from Teledyne Isco using a reversed phase
C18 high performance column (RediSep) and a mixture of MeCN/H2O. Thinꢀlayer chromatography
was carried out using aluminium sheets precoated with silica gel 60 F254 (0.2 mm, Merck). UV light
with a wavelength of 254 nm and 360 nm was used for detection.
All compounds that were evaluated in biological assays had >95% purity using HPLC method
described above.
Methyl 1Hꢀbenzo[d]imidazoleꢀ5ꢀcarboxylate (3a). 3a was synthesized in a similar manner to
that described for 4b in the literature procedure by Schmidt et al.9 HPLC: 83%, tR = 0.83 min.1H-
NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 3.87 (s, 3H), 7.68 (d, J = 8.3 Hz, 1H), 7.84 (dd,
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J = 1.4 Hz, J = 8.3 Hz, 1H), 8.23 (s, 1H), 8.40 (s, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
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δ (ppm) = 51.9, 112.1, 123.0, 123.3, 125.7, 137.9, 142.1, 144.6, 166.8.
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Methyl 2,3ꢀdihydrobenzo[
the literature procedure by Schmidt et al.9
b][1,4]dioxineꢀ6ꢀcarboxylate (3b). 3b was synthesized according to
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5ꢀ(1HꢀBenzo[d]imidazolꢀ5ꢀyl)ꢀ1,3,4ꢀoxadiazoleꢀ2ꢀthiol (4a). 4a was synthesized in a similar
manner to that described for 4b in the literature procedure by Schmidt et al.9 HPLC: 90%,
tR = 0.92 min.1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 7.75 (dd, J = 1.6 Hz, J = 8.4 Hz, 1H),
7.79 (d, J = 8.4 Hz, 1H), 8.10 (d, J = 0.8 Hz, 1H), 8.50 (s, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
δ (ppm) = 113.9, 116.2, 120.1, 123.2, 137.9, 140.0, 144.5, 163.3, 177.3.
5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazoleꢀ2ꢀthiol (4b). 4b was synthesized
according to the literature procedure by Schmidt et al.9
General Procedure A: Suzuki coupling reaction (7aꢀd,g; 9e,f,h; 16aꢀc).9
Under an argon atmosphere the aryl bromide (2.50 mmol, 1.00 eq) was dissolved in 7 mL of
toluene/EtOH (1/1). To the solution Pd(PPh3)4 (0.07 mmol, 0.03 eq) and 3.75 mL of 2 N aqueous
Na2CO3 were added and the twoꢀphase system was stirred for 15 min. Then the boronic acid
(3.00 mmol, 1.20 eq) was added and the reaction mixture was refluxed at 80 °C for 2 d. After
confirming the completion of the reaction by HPLC the mixture was diluted with water and extracted
with EtOAc. The organic layers were dried over MgSO4. The solvent was evaporated under reduced
pressure and the crude product was purified by column chromatography using a mixture of
cyclohexane/EtOAc.
4ꢀFluoroꢀ4'ꢀmethylꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile (7a). 7a was synthesized according to the
literature procedure by Schmidt et al.9
4ꢀFluoroꢀ4'ꢀmethylꢀ1,1'ꢀbiphenyl (7b). Yield 99%, colorless solid. HPLC: 97%,
tR = 8.12 min.1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 2.34 (s, 3H), 7.26 (m, 4H), 7.53 (d,
J = 8.07 Hz, 2H), 7.66 (m, 2H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 20.6, 115.6 (d,
JC-F = 19 Hz, 2C), 126.4 (2C), 128.3 (d, JC-F = 8 Hz, 2C), 129.5 (2C), 136.2, 136.5 (d, JC-F = 3 Hz),
136.6, 161.6, (d, JC-F = 242 Hz).
5ꢀFluoroꢀ2ꢀ(p
ꢀtolyl)pyridine (7c). Yield 67%, beige solid. HPLC: 98%, tR = 6.17 min.1H-NMR
(DMSO, 500 MHz, 300 K): δ (ppm) = 2.35 (s, 3H), 7.29 (d, J = 7.9 Hz, 2H), 7.78 (td, J = 8.8 Hz,
J = 3.0 Hz, 1H), 7.94 (d, J = 8.2 Hz, 2H), 7.99 (dd, J = 8.9 Hz, J = 4.4 Hz, 1H), 8.62 (d, J = 3.0 Hz,
1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 20.7, 121.2 (d, JC-F = 4 Hz), 124.0 (d,
JC-F = 18 Hz), 126.3 (2C), 129.3 (2C), 135.0, 137.2 (d, JC-F = 24 Hz), 138.4, 152.7 (d, JC-F = 3 Hz),
158.4 (d, JC-F = 254 Hz).
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6ꢀ(
p
ꢀTolyl)nicotinonitrile (7d). Yield 85%, beige solid. HPLC: 96%, tR = 6.77 min.1H-NMR
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8
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(DMSO, 500 MHz, 300 K): δ (ppm) = 2.38 (s, 3H), 7.35 (d, J = 7.9 Hz, 2H), 8.08 (m, 2H), 8.16 (dd,
J = 8.4 Hz, J = 0.8 Hz, 1H), 8.34 (dd, J = 8.4 Hz, J = 2.2 Hz, 1H), 9.06 (dd, J = 2.2 Hz, J = 0.8 Hz,
1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 20.9, 107.0, 117.3, 119.7, 127.1 (2C), 129.6
(2C), 134.2, 140.5, 140.8, 152.4, 159.1.
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1
5ꢀ(pꢀTolyl)picolinonitrile (7g). Yield 75%, colorless solid. HPLC: 96%, tR = 6.71 min. H-NMR
(DMSO, 500 MHz, 300 K): δ (ppm) = 2.37 (s, 3H), 7.35 (d, J = 7.9 Hz, 2H), 7.73 (d, J = 8.2 Hz, 2H),
8.08 (d, J = 8.2 Hz, 1H), 8.31 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H), 9.07 (d, J = 2.3 Hz, 1H). 13C-NMR
(DMSO, 125 MHz, 300 K): δ (ppm) = 20.8, 117.7, 127.2 (2C), 129.1, 129.9 (2C), 130.8, 132.3,
134.9, 138.9, 139.2, 149.0.
(4ꢀ(5ꢀMethylpyridinꢀ2ꢀyl)phenyl)methanol (9e). Yield 62%, colorless solid. HPLC: 99%,
tR = 0.83 min. 1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 2.33 (s, 3H), 4.55 (d, J = 5.7 Hz, 2H),
5.21 (t, J = 5.7 Hz, OH), 7.41 (d, J = 8.0 Hz, 2H), 7.67 (m, 1H), 7.83 (d, J = 8.1 Hz, 1H), 8.01 (d,
J = 8.1 Hz, 2H), 8.48 (s, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 17.6, 62.6, 119.4,
125.9 (2C), 126.7 (2C), 131.5, 137.1, 137.4, 1143.1, 149.7, 153.3.
(4ꢀ(6ꢀFluoropyridinꢀ3ꢀyl)phenyl)methanol (9f). Yield 27%, colorless solid. HPLC: 99%,
1
tR = 3.55 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.61 (s, 2H), 6.96 (dd, J = 8.5 Hz,
J = 3.0Hz, 1H), 7.40 (d, J = 8.3 Hz, 2H), 7.45 (d, J = 8.2 Hz, 2H), 7.93 (ddd, J = 8.5 Hz, J = 2.6 Hz,
J = 1.3 Hz, 1H), 8.31 (d, J = 2.3 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 63.0,
108.9 (d, JC-F = 36 Hz), 126.2 (2C), 126.9 (2C), 134.1 (d, JC-F = 4 Hz), 134.4, 139.1 (d, JC-F = 7 Hz),
141.7, 144.8 (d, JC-F = 15 Hz), 162.4 (d, JC-F = 239 Hz).
(4ꢀ(6ꢀMethylpyridinꢀ3ꢀyl)phenyl)methanol (9h). Yield 52%, colorless solid. HPLC: 93%,
tR = 0.81 min. 1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 2.50 (s, 3H), 4.54 (d, J = 5.7 Hz, 2H),
5.23 (t, J = 5.7 Hz, OH), 7.32 (d, J = 8.0 Hz, 1H), 7.42 (d, J = 8.0 Hz, 2H), 7.65 (d, J = 8.1 Hz, 2H),
7.94 (dd, J = 8.1 Hz, J = 2.3 Hz, 1H), 8.73 (d, J = 2.3 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
δ (ppm) = 23.6, 62.6, 123.1, 126.3 (2C), 127.1 (2C), 132.6, 134.2, 135.5, 142.2, 146.7, 156.7.
2ꢀFluoroꢀ5ꢀ(6ꢀmethylpyridinꢀ3ꢀyl)benzonitrile (16a). Yield 87%, colorless solid. HPLC: 99%,
1
tR = 1.75 min. H-NMR (CDCl3, 500 MHz, 300 K): δ (ppm) = 2.75 (2, 3H), 7.36 (t, J = 8.4 Hz, 1H),
7.41 (d, J = 7.9 Hz, 1H), 7.81 (dd, J = 11.9 Hz, J = 5.6 Hz, 2H), 7.91 (d, J = 7.9 Hz, 1H), 8.73 (s, 1H).
13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 23.0, 102.9 (d, JC-F = 17 Hz), 113.5, 117.7 (d,
JC-F = 23 Hz), 124.9, 132.0, 132.1, 133.7 (d, JC-F = 8 Hz), 134.2, 137.0, 145.0, 157.4, 163.3 (d,
JC-F = 263 Hz).
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2ꢀFluoroꢀ5ꢀ(5ꢀmethylpyridinꢀ2ꢀyl)benzonitrile (16b). Yield 91%, colorless solid. HPLC: 91%,
tR = 2.61 min. 1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 2.34 (s, 3H), 7.61 (t, J = 9.0 Hz, 1H),
7.74 (dd, J = 8.6 Hz, 2.1 Hz, 1H), 7.96 (d, J = 8.1 Hz, 1H), 8.46 (ddd, J = 8.8 Hz, 5.3 Hz, 2.4 Hz, 1H),
8.52 (d, J = 2.1 Hz, 1H), 8.54 (dd, J = 6.3 Hz, 2.4 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
δ (ppm) = 17.6, 100.6 (d, JC-F = 16 Hz), 113.9, 116.9 (d, JC-F = 21 Hz), 119.9, 131.3, 132.9, 133.6 (d,
JC-F = 8 Hz), 136.1 (d, JC-F = 4Hz), 137.8, 149.9, 150.3, 162.6 (d, JC-F = 257 Hz).
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6'ꢀFluoroꢀ5ꢀmethylꢀ2,3'ꢀbipyridine (16c). Yield 99%, orange solid. HPLC: 92%, tR = 1.31 min.
1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 2.35 (s, 3H), 7.28 (dd, J = 8.8 Hz, 2.7 Hz, 1H), 7.74
(ddd, J = 8.1 Hz, 2.2 Hz, 0.7 Hz, 1H), 7.94 (d, J = 8.1 Hz, 1H), 8.53 (dd, J = 1.5 Hz, 0.7 Hz, 1H), 8.59
(m, 1H), 8.88 (d, J = 2.5 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 17.5, 109.5 (d,
JC-F = 36 Hz), 119.5, 128.7 (d, JC-F = 10 Hz), 131.4 (d, JC-F = 16 Hz), 132.7, 132.8 (d, JC-F = 4 H),
137.7, 139.8 (d, JC-F = 8 Hz), 145.5 (d, JC-F = 16 Hz), 150.0, 163.2 (d, JC-F = 237 Hz).
General Procedure B: Radical bromination (8aꢀd,g; 17aꢀc).9
The substituted toluene (1.00 eq) was diluted in 20 mL of CCl4 and NBS (0.95 eq) and AIBN (5 mg
per mmol) was added in portions over a period of 1 h. The reaction mixture was heated to reflux for
16 h. After cooling to rt the mixture was diluted with water and CCl4 was evaporated. The residue was
extracted with EtOAc and washed with brine. The organic layers were dried over MgSO4 and
concentrated under reduced pressure. The crude product was purified by column chromatography
using a mixture of cyclohexane/EtOAc.
4'ꢀ(Bromomethyl)ꢀ4ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile (8a). 8a was synthesized according
to the literature procedure by Schmidt et al.9
4ꢀ(Bromomethyl)ꢀ4'ꢀfluoroꢀ1,1'ꢀbiphenyl (8b). Yield 52%, colorless solid. HPLC: 87%,
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tR = 8.39 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.76 (s, 2H), 7.29 (m, 2H), 7.53 (m,
2H), 7.64 (m, 2H), 7.72 (m, 2H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 34.2, 115.6, 115.8,
126.9 (2C), 128.6, 128.7, 129.9 (2C), 136.0 (d, JC-F = 2 Hz), 137.1, 139.0, 162.0 (d, JC-F = 245 Hz).
2ꢀ(4ꢀ(Bromomethyl)phenyl)ꢀ5ꢀfluoropyridine (8c). Yield 65%, colorless solid. HPLC: 61%,
tR = 7.15 min.1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.67 (s, 2H), 7.55 (d, J = 8.7 Hz, 2H),
7.82 (dd, J = 8.0 Hz, J = 3.0 Hz, 1H), 8.03 (d, J = 8.7 Hz, 2H), 8.03 (d, J = 8.0 Hz, 1H), 8.66 (d,
J = 3.0 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 34.5, 122.2, 124.6 (d,
JC-F = 24 Hz), 127.2, 127.8, 130.2, 132.7, 138.0 (d, JC-F = 24 Hz), 139.2, 152.6, 159.1 (d,
JC-F = 245 Hz).
6ꢀ(4ꢀ(Bromomethyl)phenyl)nicotinonitrile (8d). Yield 89%, pale yellow solid. HPLC: 73%,
tR = 7.26 min.1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.78 (s, 2H), 7.61 (d, J = 8.4 Hz, 2H),
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7.78 (d, J = 8.4 Hz, 2H), 8.20 (m, 1H), 9.11 (m, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
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δ (ppm) = 34.2, 108.0, 117.6, 121.0, 128.2, 130.4, 132.9, 141.5, 144.5, 153.2, 158.4.
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5ꢀ(4ꢀ(Bromomethyl)phenyl)picolinonitrile (8g). Yield 64%, colorless solid. HPLC: 96%,
tR = 7.17 min. 1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.78 (s, 2H), 7.62 (d, J = 8.3 Hz, 2H),
7.84 (m, 2H), 8.12 (d, J = 8.1 Hz, 1H), 8.36 (dd, J = 8.2 Hz, 2.3 Hz, 1H), 9.11 (d, J = 2.3 Hz, 1H). 13C-
NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 33.7, 117.6, 127.7 (2C), 129.8, 130.2 (2C), 131.3, 135.1,
135.4, 138.4, 139.3, 149.3.
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5ꢀ(6ꢀ(Bromomethyl)pyridinꢀ3ꢀyl)ꢀ2ꢀfluorobenzonitrile (17a). Yield 22%, pale yellow solid.
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HPLC: 85%, tR = 6.77 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.70 (d, J = 63.6 Hz,
2H), 7.36 (t, J = 8.4 Hz, 1H), 7.61 (dd, J = 20.0 Hz, J = 8.4 Hz, 1H), 7.85 (m, 3H), 8.75 (s, 1H). 13C-
NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 29.9, 113.5, 117.7 (d, JC-F = 21 Hz), 124.1, 132.2, 133.3,
133.8 (d, JC-F = 8 Hz),134.6 (d, JC-F = 4 Hz), 136.0, 147.4, 156.8, 163.3 (d, JC-F = 251 Hz).
5ꢀ(5ꢀ(Bromomethyl)pyridinꢀ2ꢀyl)ꢀ2ꢀfluorobenzonitrile (17b). Yield 39%, beige solid. HPLC:
85%, tR = 6.77 min. 1H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.80 (s, 2H), 7.65 (t, J = 9.0 Hz,
1H), 8.00 (dd, J = 8.2 Hz, 2.3 Hz, 1H), 8.08 (d, J = 8.2 Hz, 1H), 8.50 (ddd, J = 8.7 Hz, 5.3 Hz, 2.4 Hz,
1H), 8.59 (dd, J = 6.3 Hz, 2.4 Hz, 1H), 8.76 (d, J = 2.1 Hz, 1H). 13C-NMR (DMSO, 125 MHz,
300 K): δ (ppm) = 30.6, 100.8 (d, JC-F = 25 Hz), 113.9, 117.1 (d, JC-F = 15 Hz), 120.5, 131.9, 133.8,
134.1 (d, JC-F = 15 Hz), 135.5, 138.3, 150.1, 152.7, 162.9 (d, JC-F = 256 Hz).
5ꢀ(Bromomethyl)ꢀ6'ꢀfluoroꢀ2,3'ꢀbipyridine (17c). Yield 45%, orange solid. HPLC: 76%,
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tR = 4.92 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.80 (s, 2H), 7.31 (dd, J = 8.2 Hz,
J = 2.9 Hz, 1H), 8.00 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H), 8.05 (dd, J = 8.2 Hz, J = 0.7 Hz, 1H), 8.63 (m,
1H), 8.76 (d, J = 1.9 Hz, 1H), 8.92 (d, J = 2.5 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
δ (ppm) = 30.6, 109.7, 120.5, 132.8, 133.6, 138.2, 140.3, 146.1, 149.9, 150.1, 163.5 (d, JC-F = 245 Hz).
General Procedure C: Bromination of the benzylic alcohols (8e,f,g).
To a solution of the benzylic alcohol 9e,f,g (2.50 eq) in toluene (10 mL per mmol) was added
phosphorous tribromide (1.00 eq). The reaction mixture was refluxed at 110 °C for 3 h. After cooling
to rt the solvent was evaporated under a reduced pressure. The crude product was used for the next
reaction without any purification.
General Procedure D: Preparation of thioethers by nucleophilic substitution (1;
12a;13bꢀh; 18aꢀc).
To a solution of the mercaptane (1.00 eq) in DMF (10 mL per mmol) was added 4N NaOH. The
mixture was stirred at rt for 15 min. Afterwards the benzylic bromide (1.25 eq) was added and the
reaction mixture was stirred at rt for another 6 h. The formed precipitate is filtered off, washed once
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with a small amount of DMF and then thoroughly with EtOH. The product was dried under reduced
pressure.
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Purification of compounds, which did not precipitate in DMF was performed as follows. The mixture
was diluted with EtOAc. The separated organic layer was washed with brine several times, dried over
MgSO4 and dried under reduced pressure. The crude product was purified by column chromatography
using silica gel and a mixture of cyclohexane/EtOAc or by automated flash chromatography using a
reversed phase C18 high performance column (RediSep) and a mixture of MeCN/H2O.
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4'ꢀ(((5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl)thio)methyl)ꢀ4ꢀfluoroꢀ
[1,1'ꢀbiphenyl]ꢀ2ꢀcarbonitrile (1). 1 was synthesized according to the literature procedure by
Schmidt et al.9
4'ꢀ(((5ꢀ(1
HꢀBenzo[
d
]imidazolꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl)thio)methyl)ꢀ4ꢀfluoroꢀ[1,1'ꢀ
1
biphenyl]ꢀ2ꢀcarbonitrile (12a). Yield 13%, colorless solid. HPLC: 95%, tR = 5.53 min. H-NMR
(DMSO, 500 MHz, 300 K): δ (ppm) = 4.68 (s, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.66 (m, 4H), 7.77 (d,
J = 8.4 Hz, 1H), 7.83 (dd, J = 8.4 Hz, J = 1.4 Hz, 1H), 7.95 (m, 1H), 8.19 (s, 1H), 8.42 (s, 1H). 13C-
NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.5, 111.6 (d, JC-F = 10 Hz), 116.6, 117.4 (d,
JC-F = 27 Hz), 120.3 (d, JC-F = 7 Hz), 120.5, 121.0 (d, JC-F = 21 Hz), 122.6 (d, JC-F = 23 Hz), 125.5,
129.0 (2C), 129.4 (2C), 132.4 (d, JC-F = 8 Hz), 136.2, 137.5, 139.6, 140.8 (d, JC-F = 3 Hz), 144.3,
144.5, 160.8 (d, JC-F = 246 Hz), 162.6, 166.2. EI-MS: m/z (%): 427 (20, [M+]), 428 (5, [M+ + H]).
2ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ5ꢀ(((4'ꢀfluoroꢀ[1,1'ꢀbiphenyl]ꢀ4ꢀyl)methyl)thio)ꢀ
1,3,4ꢀoxadiazole (13b). Yield 48%, colorless solid. HPLC: 99%, tR = 8.72 min. 1H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = 4.32 (m, 4H), 4.60 (s, 2H), 7.04 (d, J = 8.4, 1H), 7.27 (td, J = 6.7 Hz,
3.4 Hz, 2H), 7.39 (d, J = 2.1 Hz, 1H), 7.44 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 7.54 (d, J = 8.3 Hz, 2H), 7.62
(d, J = 8.3 Hz, 2H), 7.69 (m, 2H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.6, 64.0, 64.4,
115.0, 115.6, 115.7, 115.9, 118.1, 119.9, 126.8 (2C), 128.5, 128.6, 129.6 (2C), 135.9, 136.0, 138.5,
143.8, 146.7, 161.9 (d, JC-F = 245 Hz, 1C), 162.5, 164.9. EI-MS: m/z (%): 420 (16, [M+]), 421 (5,
[M+ + H]).
2ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ5ꢀ(4ꢀ(5ꢀfluoropyridinꢀ2ꢀyl)benzylthio)ꢀ1,3,4ꢀoxaꢀ
diazole (13c). Yield 66%, colorless solid. HPLC: 96%, tR = 8.09 min. 1H-NMR (DMSO, 500 MHz,
300 K): δ (ppm) = 4.31 (dd, J = 12.6 Hz, J = 5.0 Hz 4H), 4.61 (s, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.43
(m, 2H), 7.57 (d, J = 8.2 Hz, 2H), 7.80 (td, J = 8.7 Hz, J = 2.9 Hz, 1H), 8.05 (m,3H), 8.63 (d,
J = 2.9 Hz, 1H).13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.6, 64.1, 64.4, 115.1, 115.9,
118.1, 119.9, 121.7 (d, JC-F = 5 Hz), 124.1 (d, JC-F = 18 Hz), 126.6 (2C), 129.5 (2C), 137.1, 137.3,
137.5 (d, JC-F = 6 Hz), 143.8, 146.7, 152.2 (d, JC-F = 4 Hz), 158.6 (d, JC-F = 254 Hz), 162.5, 165.0. EI-
MS: m/z (%): 421 (20, [M+]), 422 (4, [M+ + H]).
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6ꢀ(4ꢀ((5ꢀ(2,3ꢀDihydrobenzo[
b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀylthio)methyl)phenyl)ꢀ
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nicotinonitrile (13d). Yield 51%, colorless solid. HPLC: 95%, tR = 7.89 min. H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = 4.31 (dtd, J = 2.1 Hz, J = 3.5 Hz, 4.3 Hz, 4H), 4.63 (s, 2H), 7.04 (d,
J = 8.5 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.43 (dd, J = 2.1 Hz, J = 8.4 Hz, 1H), 7.63 (d, J = 8.4 Hz,
2H), 8.14 (d, J = 8.4 Hz, 2H), 8.18 (dd, J = 0.6 Hz, J = 8.4 Hz, 1H), 8.37 (dd, J = 2.2 Hz, J = 8.4 Hz,
1H), 9.07 (dd, J = 0.6 Hz, J = 2.1 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.6,
64.0, 64.4, 107.4, 115.0, 115.9, 117.2, 118.1, 119.9, 120.2, 127.4 (2C), 127.9 (2C), 136.3, 139.4,
140.9, 143.8, 146.7, 152.5, 158.6, 162.4, 164.6. EI-MS: m/z (%): 428 (38, [M+]).
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2ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ5ꢀ(4ꢀ(5ꢀmethylpyridinꢀ2ꢀyl)benzylthio)ꢀ1,3,4ꢀ
1
oxadiazole (13e). Yield 27%, colorless solid. HPLC: 98%, tR = 5.26 min. H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = 2.33 (s, 3H), 4.32 (ddd, J = 5.2 Hz, J = 4.5 Hz, J = 1.6 Hz, 4H), 4.61 (s,
2H), 7.04 (d, J = 8.4 Hz, 1H), 7.40 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 8.4 Hz, 2.1 Hz, 1H), 7.56 (d,
J = 8.3 Hz, 2H), 7.68 (dd, J = 8.2 Hz, 1.7 Hz, 1H), 7.84 (d, J = 8.1 Hz, 1H), 8.02 (d, J = 8.3 Hz, 2H),
8.49 (d, J = 1.2 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 17.6, 35.8, 64.0, 64.4,
115.0, 115.9, 118.1, 119.6, 119.9, 126.4 (2C), 129.4 (2C), 131.9, 137.1, 137.5, 138.1, 143.8, 146.7,
149.7, 152.8, 162.5, 164.9. EI-MS: m/z (%): 417 (32, [M+]).
2ꢀ(2,3ꢀDihydrobenzo[
b
][1,4]dioxinꢀ6ꢀyl)ꢀ5ꢀ((4ꢀ(6ꢀfluoropyridinꢀ3ꢀyl)benzyl)thio)ꢀ1,3,4ꢀ
1
oxadiazole (13f). Yield 58%, colorless solid. HPLC: 98%, tR = 8.10 min. H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = 4.32 (m, 4H),4.61 (s, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.27 (dd, J = 8.5 Hz,
J = 2.8 Hz, 1H), 7.38 (d, J = 2.0 Hz, 1H), 7.44 (dd, J = 8.4 Hz, 2.0 Hz, 1H), 7.59 (d, J = 8.3 Hz, 2H),
7.70 (d, J = 8.3 Hz, 2H), 8.27 (td, J = 8.2 Hz, J = 2.6 Hz, 1H), 8.53 (d, J = 2.6 Hz, 1H). 13C-NMR
(DMSO, 125 MHz, 300 K): δ (ppm) = 35.6, 64.1, 64.4, 109.6 (d, JC-F = 37 Hz), 115.1, 115.9, 118.1,
119.9, 127.0 (2C), 129.8 (2C), 133.6 (d, JC-F = 4 Hz), 135.2 (d, JC-F = 4 Hz), 136.9, 140.3 (d,
JC-F = 9 Hz), 143.8, 145.4 (d, JC-F = 16 Hz), 146.7, 162.5,, 162.7 (d, JC-F = 263 Hz), 165.0. EI-MS: m/z
(%): 421 (100, [M+]), 422 (26, [M+ + H]), 423 (8, [M+ + 2H]).
5ꢀ(4ꢀ(((5ꢀ(2,3ꢀDihydrobenzo[
b
][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl)thio)methyl)phenyl)
1
picolinonitrile (13g). Yield 58%, colorless solid. HPLC: 99%, tR = 8.02 min. H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = 4.32 (dtd, J = 7.3 Hz, J = 3.5 Hz, J = 2.1 Hz, 4H), 4.63 (s, 2H), 7.04 (d,
J = 8.5 Hz, 1H), 7.38 (d, J = 2.1 Hz, 1H), 7.43 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 7.64 (d, J = 8.3 Hz,
2H), 7.82 (d, J = 8.3 Hz, 2H), 8.11 (d, J = 8.2 Hz, 1H), 8.33 (dd, J = 8.2 Hz, J = 2.3 Hz, 1H), 9.09 (d,
J = 1.7 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.5, 64.1, 64.4, 115.1, 155.9,
117.6, 118.2, 119.9, 127.5 (2C), 129.1, 130.0 (2C), 131.2, 134.6, 135.3, 138.3, 138.4, 143.8, 146.7,
149.2, 162.5, 165.0. EI-MS: m/z (%): 428 (100, [M+]), 429 (28, [M+ + H]), 430 (8, [M+ + 2H]).
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2ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ5ꢀ(4ꢀ(6ꢀmethylpyridinꢀ3ꢀyl)benzylthio)ꢀ1,3,4ꢀ
4
5
1
oxadiazole (13h). Yield 12%, colorless solid. HPLC: 97%, tR = 5.17 min. H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = CH3 fehlt, 4.32 (m, 4H), 4.61 (s, 2H), 7.04 (d, J = 8.4 Hz, 1H), 7.33 (d,
J = 8.1 Hz, 1H), 7.39 (d, J = 2.1 Hz, 1H), 7.44 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 7.57 (d, J = 8.3 Hz,
2H), 7.66 – 7.69 (m, 2H), 7.95 (dd, J = 8.1 Hz, J = 2.5 Hz, 1H), 8.73 (d, J = 2.2 Hz, 1H). 13C-NMR
(DMSO, 125 MHz, 300 K): δ (ppm) = 24.1, 36.1, 64.6, 64.9, 114.9, 115.6, 116.4, 118.6, 120.4, 123.6,
127.2, 130.2, 132.6, 134.8, 136.9, 137.0, 144.3, 147.2, 157.4, 163.1, 165.5. EI-MS: m/z (%): 417 (100,
[M+]), 418 (26, [M+ + H], 419 (8, [M+ + 2H]).
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2ꢀ(6ꢀ((5ꢀ(2,3ꢀDihydrobenzo[
b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀylthio)methyl)pyridinꢀ3ꢀ
1
yl)ꢀ5ꢀfluorobenzonitrile (18a). Yield 33%, colorless solid. HPLC: 98%, tR = 7.32 min. H-NMR
(CDCl3, 500 MHz, 300 K): δ (ppm) = 4.31 (dddd, J = 6.8 Hz, J = 5.0 Hz, 3.4 Hz, 1.6 Hz, 4H), 5.03 (s,
2H), 6.95 (m, 1H), 7.46 (dd, J = 2.0 Hz, J = 6.9 Hz, 3H), 7.85 (ddd, J = 12.3 Hz, J = 4.7 Hz,
J = 2.1 Hz, 2H), 8.32 (m, 2H), 8.87 (s, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 29.9,
64.4, 64.8, 102.5 (d, JC-F = 6 Hz), 114.4, 116.1, 116.1, 117.7 (d, JC-F = 29 Hz), 118.3, 120.6, 122.9,
128.4, 131.2, 132.4 (d, JC-F = 7 Hz), 133.8 (d, JC-F = 8 Hz), 134.0, 138.4, 144.1, 144.4, 149.8, 151.8,
155.4, 157.8 (d, JC-F = 254 Hz), 165.0. EI-MS: m/z = 463 (100, [M+]), 464 (26, [M+ + H]), 465 (7,
[M+ + 2H].
5ꢀ(5ꢀ(((5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀyl)thio)methyl)pyridinꢀ
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2ꢀyl)ꢀ2ꢀfluorobenzonitrile (18b). Yield 37%, colorless solid. HPLC: 99%, tR = 7.58 min. H-
NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.32 (m, 4H), 4.64 (s, 2H), 7.04 (d, J = 8.5 Hz, 1H),
7.37 (d, J = 2.1 Hz, 1H), 7.43 (dd, J = 8.5 Hz, 2.1 Hz, 1H), 7.65 (t, J = 9.1 Hz, 1H), 8.03 (dd,
J = 8.3 Hz, 2.2 Hz, 1H), 8.07 (dd, J = 8.2 Hz, 0.5 Hz, 1H), 8.48 (ddd, J = 8.9 Hz, 5.3 Hz, 2.4 Hz, 1H),
8.58 (dd, J = 6.2 Hz, 2.3 Hz, 1H), 8.77 (d, J = 1.7 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K):
δ (ppm) = 32.8, 64.1, 64.4, 95.2, 113.9, 115.1, 115.8, 117.0, 117.1, 118.1, 119.9, 120.3, 131.8, 132.8,
134.0 (d, JC-F = 8 Hz), 136.1, 138.0, 143.8, 146.7, 150.0, 152.2, 156.7, 163.6 (d, JC-F = 356 Hz). EI-
MS: m/z (%): 446 (100, [M+]), 447 (27, [M+ + H]), 448 (8, [M+ + 2H]).2ꢀ(2,3ꢀ
Dihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ5ꢀ(((6'ꢀfluoroꢀ[2,3'ꢀbipyridin]ꢀ5ꢀyl)methyl)thio)ꢀ1,3,4ꢀ
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oxadiazole (18c). Yield 31%, pale yellow solid. HPLC: 95%, tR = 6.69 min. H-NMR (DMSO,
500 MHz, 300 K): δ (ppm) = 4.31 (dtd, J = 7.3 Hz, J = 3.6 Hz, 2.1 Hz, 4H), 4.64(s, 2H), 7.04 (d,
J = 2.1 Hz, 1H), 7.30 (dd, J = 8.6 Hz, J = 2.6 Hz, 1H), 7.37 (d, J = 2.1 Hz, 1H), 7.43 (dd, J = 8.5 Hz,
J = 2.1 Hz, 1H), 8.01 – 8.05 (m, 2H), 8.61 (td, J = 8.3 Hz, J = 2.6 Hz, 1H), 8.77 (d, J = 1.7 Hz, 1H),
8.90 (d, J = 2.5 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 32.8, 64.0, 64.4, 109.6 (d,
JC-F = 39 Hz), 115.1, 115.8, 118.1, 119.9, 120.3, 132.4 (d, JC-F = 7 Hz), 132.5, 137.9, 140.2 (d,
JC-F = 9 Hz), 143.8, 145.8, 145.9, 146.7, 150.1, 151.9, 162.4, 163.6 (d, JC-F = 356 Hz). EI-MS: m/z
(%): 422 (100, [M+]), 423 (36, [M+ + H]), 424 (11, [M+ + 2H]).
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Methylꢀ1ꢀ(2ꢀcyanoꢀ4ꢀfluorophenyl)piperidineꢀ4ꢀcarboxylate (21). Methyl piperidineꢀ4ꢀ
carboxylate (0.62 g, 4.32 mmol, 1.20 eq) and 2,5ꢀdifluorobenzonitrile (0.50 g, 3.60 mmol, 1.00 eq)
were dissolved in 10 mL DMSO. Cesium carbonate (1.41 g, 4.32 mmol, 1.2 eq) was added and the
reaction mixture was stirred at 100°C for 18 h. After cooling to rt, the reaction mixture was diluted
with water and extracted with EtOAc. The organic layer was washed with saturated NaHCO3 solution
and brine, dried over MgSO4 and concentrated under reduced pressure. Purification by column
chromatography (cyclohexane/EtOAc 4:1) gave (0.39 g, 42%) of compound 20 as a colorless solid..
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HPLC: 98%, tR = 6.93 min. H-NMR (CDCl3, 500 MHz, 300 K): δ (ppm) = 2.03 (m, 4H), 2.48 (m,
1H), 2.86 (m, 2H), 3.43 (ddd, J = 3.16 Hz, J = 6.95 Hz, J = 8.38 Hz, 2H), 3.71 (s, 3H), 7.00 (dd, J =
4.61 Hz, J = 9.02 Hz, 1H), 7.20 (ddd, J = 3.05 Hz, J = 7.79 Hz, J = 9.02 Hz, 1H), 7.26 (m, 1H). 13C-
NMR (CDCl3, 125 MHz, 300 K): δ (ppm) = 28.5(2C), 40.5, 51.9(2C), 52.1, 56.7, 107.9 (d,
JC-F = 9 Hz), 112.7 (d, JC-F = 8 Hz), 117.1, 120.5 (d, JC-F = 25 Hz), 120.8 (d, JC-F = 8 Hz), 121.1 (d,
JC-F = 23 Hz), 153.1, 157.1 (d, JC-F = 243 Hz), 175.0.
5ꢀFluoroꢀ2ꢀ(4ꢀ(hydroxymethyl)piperidinꢀ1ꢀyl)benzonitrile (22). To a solution of 21 (0.34 g,
1.30 mmol, 1.00 eq) in anhydrous THF (15 mL) was added lithium borohydride (1.9 mL, 3.38 mmol,
2.60 eq). The mixture was heated to reflux for 16 h under argon atmosphere. After cooling to rt, the
reaction mixture was diluted carefully with water and extracted with EtOAc. The combined organic
layer was washed with brine and dried over MgSO4. After removing the solvent compound 22 (0.30 g,
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98%) was obtained as a pale yellow solid. HPLC: 99%, tR = 4.94 min. H-NMR (CDCl3, 500 MHz,
300 K): δ (ppm) = 1.51 (ddd, J = 3.91 Hz, J = 12.42 Hz, J = 24.3 Hz, 2H), 1.60 – 1.69 (m, 1H), 1.87
(dd, J = 1.62 Hz, J = 12.42 Hz, 2H), 2.76 (td, J = 1.62 Hz, J = 12.42 Hz, 2H), 3.49 (d, J = 11.95 Hz,
2H), 3.56 (d, J = 6.47 Hz, 2H), 6.98 (dd, J = 4.43 Hz, J = 9.19 Hz, 1H), 7.18 (ddd, J = 2.95 Hz,
J = 7.99 Hz, J = 9.19 Hz, 1H), 7.24 (dd, J = 2.95 Hz, J = 7.99 Hz, 1H). 13C-NMR (CDCl3, 125 MHz,
300 K): δ (ppm) = 29.2 (2C), 38.3, 52.8 (2C), 67.8, 107.6 (d, JC-F = 9 Hz), 117.3 (d, JC-F = 2 Hz),
120.3 (d, JC-F = 26 Hz), 120.7 (d, JC-F = 8 Hz, 1C), 121.1 (d, JC-F = 23 Hz), 153.5, 156.9 (d,
JC-F = 243 Hz).
2ꢀ(4ꢀ(Bromomethyl)piperidinꢀ1ꢀyl)ꢀ5ꢀfluorbenzonitrile (23). Anhydrous conditions and inert
gas are essential for this reaction! 22 (0.18 g, 0.77 mmol, 1.00 eq), tetrabromomethane (0.38 g,
1.15 mmol, 1.50 eq) and triphenylphosphine (0.40 g, 1.55 mmol, 2.00 eq) was added in a dried vessel
and purged for 15 min with argon. Afterwards the educts were dissolved in dry acetonitrile. The
reaction mixture was stirred at rt. The progression of the reaction was controlled by HPLC. After no
further conversion was observed, the reaction was stopped by addition of 15% NaOH solution. The
reaction mixture was extracted with DCM. The combined organic layer was washed with brine and
dried over MgSO4. The solvent was evaporated under reduced pressure and the crude product was
purified by column chromatography (cyclohexane/EtOAc 9:1) to give 23 (0.08 g, 35%) as a brown oil.
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HPLC: 97%, tR = 8.41 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 1.43 (qd, J = 3.80 Hz,
J = 11.94 Hz, 2H), 1.73 – 1.81 (m, 1H), 1.89 (d, J = 12.25 Hz, 2H), 2.77 (td, J = 2.09 Hz,
J = 11.94 Hz, 2H), 3.41 (d, J = 12.25 Hz, 2H), 3.54 (d, J = 6.13 Hz, 2H), 7.20 (dd, J = 4.73 Hz,
J = 8.99 Hz, 1H), 7.47 (ddd, J = 0.66 Hz, J = 3.11 Hz, J = 8.73 Hz, J = 8.99 Hz, 1H), 7.68 (dd,
J = 3.11 Hz, J = 8.32 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm) =30.5 (2C), 37.2, 39.9,
51.7 (2C), 106.3 (d, JC-F = 11 Hz), 116.9 (d, JC-F = 2 Hz), 120.2 (d, JC-F = 26 Hz), 121.3 (d,
JC-F = 8 Hz), 121.4 (d, JC-F = 23 Hz), 152.9 (d, JC-F = 3 Hz), 156.2 (d, JC-F = 240 Hz).
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2ꢀ(4ꢀ((5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀylꢀthio)methyl)piperidinꢀ
1ꢀyl)ꢀ5ꢀfluorbenzonitrile (24). A mixture of 4b (0.03 g, 0.14 mmol, 1.00 eq) and potassium
carbonate (0.02 g, 0.17 mmol, 1.20 eq) was stirred in dry DMF (3.6 mL) at rt for 15 min. 23 (0.02 g,
0.17 mmol, 1.50 eq) was added to the orange solution. Afterwards the reaction mixture was stirred for
3 h at 80°C under an argon atmosphere. The resolution mixture was diluted with water and EtOAc.
The aqueous layer was extracted with EtOAc. The organic layer was washed with bine, successively
dried over MgSO4 and concentrated under reduced pressure. The obtained residue was purified by
column chromatography (cyclohexane/EtOAc 2:1) to give (0.05 g, 71%) of compound 24 as a
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colorless solid. HPLC: 99%, tR = 8.86 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) =1.66
(ddd, J = 3.86 Hz, J = 12.18 Hz, J = 15.67 Hz, 2H), 2.01 (m, 1H), 2.07 (d, J = 13.17 Hz, 2H), 2.84 (td,
J = 1.75 Hz, J = 11.82 Hz, 2H), 3.28 (d, J = 6.82 Hz, 2H), 3.51 (d, J = 12.05 Hz, 2H), 4.29 ꢀ 4.34 (m,
4H), 6.96 (d, J = 8.32 Hz, 1H), 7.09 (dd, J = 4.60 Hz, J = 8.97 Hz, 1H), 7.21 (ddd, J = 2.99 Hz,
J = 7.84 Hz, J = 8.97 Hz, 1H), 7.26 ꢀ 7.28 (m, 1H), 7.49 ꢀ 7.52 (m, 2H). 13C-NMR (DMSO, 125 MHz,
300 K): δ (ppm) =31.7 (2C), 35.4, 38.6, 52.8 (2C), 64.4, 64.8, 107.9 (d, JC-F = 9 Hz), 116.1, 117.1,
118.2, 120.5, 120.6 (d, JC-F = 25 Hz), 121.1, 121.2 (d, JC-F = 12 Hz), 121.3, 144.0, 146.9, 151.8, 158.3,
164.8 (d, JC-F = 232 Hz), 167.9. EI-MS: m/z (%): 452 (100, [M+]), 453 (28, [M+ + H]).
4'ꢀ((5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀylꢀsulfinyl)methyl)ꢀ4ꢀ
fluorbiphenylꢀ2ꢀcarbonitrile ((rac)ꢀ25). Under an argon atmosphere 1 (0.05 g, 0.11 mmol,
1.00 eq) was dissolved in 8 mL of DCM. The solution was cooled to 0°C and mCPBA (0.03 g,
0.11 mmol, 1.00 eq) was added. The reaction mixture was allowed to warm to rt and was stirred for
2 d. After confirming the completion of the reaction with HPLC, saturated sodium sulphite solution
was added to the mixture. The organic layer was washed with saturated NaHCO3 solution and dried
over MgSO4. Purification was performed by column chromatography (cyclohexane/EtOAc 1:1) to
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give (0.04 g, 79%) of compound (rac)-25 as a colorless solid. HPLC: 99%, tR = 7.71 min. H-NMR
(DMSO, 500 MHz, 300 K): δ (ppm) = 4.31 – 4.33 (m, 2H), 4.35 – 4.36 (m, 2H), 4.92 (q,
J = 12.57 Hz, 2H), 7.09 (d, J = 8.56 Hz, 1H), 7.48 – 7.50 (m, 3H), 7.53 (dd, J = 2.11 Hz, J = 8.41 Hz,
1H), 7.56 – 7.57 (m, 1H), 7.57 – 7.58 (m, 1H), 7.62 (dd, J = 5.40 Hz, J = 8.56 Hz, 1H), 7.68 (td,
J = 2.67 Hz, J = 8.56 Hz, 1H), 7.96 (dd, J = 2.67 Hz, J = 8.56 Hz, 1H). 13C-NMR (DMSO, 125 MHz,
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300 K): δ (ppm) = 58.2, 64.2, 64.7, 111.8, 115.3, 115.9, 117.4, 118.5, 120.6 (d, JC-F = 24 Hz), 120.9,
121.2 (d, JC-F = 23 Hz), 129.2 (2C), 130.1, 131.0 (2C), 132.6 (d, JC-F = 8 Hz), 137.2, 140.6, 144.1,
147.6, 165.0 (d, JC-F = 243 Hz), 165.5. EI-MS: m/z (%): 461 (100, [M+]), 445 (43, [M+ ꢀ O], 462 (25,
[M+ + H], 463 (6, [M+ + 2H]).
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4'ꢀ((5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀylꢀsulfonyl)methyl)ꢀ4ꢀ
fluorbiphenylꢀ2ꢀcarbonitrile (26). Under argon atmosphere 2 (0.03 g, 0.07 mmol, 1.00 eq) was
dissolved in 6 mL of DCM. The solution was cooled to 0°C and mCPBA (0.04 g, 0.20 mmol, 3.00 eq)
was added. The reaction mixture warmed to 30°C and was stirred for 3 d. In the case of full
implementation the reaction was stopped by adding saturated NaSO3 solution. The organic layer was
washed with saturated NaHCO3 solution and dried over MgSO4. Purification was performed by
column chromatography (cyclohexane/EtOAc 1:1) to give (0.02 g, 68%) of compound 26 as a
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colorless solid. HPLC: 97%, tR = 8.07 min. H-NMR (DMSO, 500 MHz, 300 K): δ (ppm) = 4.34
(ddd, J = 2.7 Hz, 5.9 Hz, 7:8 Hz, 4H), 5.33 (s, 2H), 7.09 (d, J = 8.4 Hz, 1H), 7.44 (d, J = 2.1 Hz, 1H),
7.50 (dd, J = 2.1 Hz, J = 8.5 Hz, 1H), 7.54 (d, J = 8.2 Hz, 2H), 7.63 (m, 3H), 7.69 (td, J = 2.7 Hz,
J = 8.5 Hz, 1H), 7.98 (dd, J = 2.7 Hz, J = 8.7 Hz, 1H). 13C-NMR (DMSO, 125 MHz, 300 K): δ (ppm)
= 60.2, 64.1, 64.6, 111.7 (d, JC-F = 10 Hz), 114.6, 116.0, 117.2, 118.4, 120.5 (d, JC-F = 25 Hz), 121.1,
121.1 (d, JC-F = 23 Hz), 126.8, 129.1 (2C), 131.7 (2C), 132.4 (d, JC-F = 8 Hz), 137.7, 140.4, 143.9,
147.9, 160.7, 160.9 (d, JC-F = 227 Hz), 165.9. EI-MS: m/z (%): 477 (100, [M+]), 478 (28, [M+ + H]),
479 (8, [M+ + 2H]).
4'ꢀ((5ꢀ(2,3ꢀDihydrobenzo[b][1,4]dioxinꢀ6ꢀyl)ꢀ1,3,4ꢀoxadiazolꢀ2ꢀylꢀthio)ꢀmethyl)ꢀ4ꢀ
fluorbiphenylꢀ2ꢀcarboxamide (27). Under an argon atmosphere 2 (0.05 g, 0.11 mmol, 1.00 Äq.)
was added to a dried vessel. Trifluoroacetic acid (0.8 mL) and concentrated sulphoric acid (0.2 mL)
were added and the red mixture was stirred 6h at 70°C. Subsequently the reaction is poured into a
water/iceꢀmixture (50 mL), extracted with CH2Cl2, washed with saturated NaHCO3 solution and dried
over MgSO4. Purification by column chromatography (cyclohexane/EtOAc 1:2) gave (0.04 g, 78%) of
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compound 27 as a colorless solid. HPLC: 96%, tR = 6.93 min. H-NMR (DMSO-
d
6, 500 MHz,
300 K): δ (ppm) = 4.32 (td, J = 5.2 Hz, J = 3.7 Hz, 4H), 4.60 (s, 2H), 7.05 (d, J = 8.4 Hz, 1H), 7.25
(dd, J = 9.1 Hz, J = 2.7 Hz, 1H), 7.31 (td, J = 8.6 Hz, J = 2.8 Hz, 1H), 7.38 (m, 3H), 7.41 (d,
J = 2.0 Hz, 1H), 7.45 (dd, J = 8.4 Hz, J = 2.1 Hz, 1H), 7.49 (d, J = 8.2 Hz, 2H), 7.73 (s, 1H). 13C-
NMR (DMSO, 125 MHz, 300 K): δ (ppm) = 35.6, 64.1, 64.4, 114.3 (d, JC-F = 21 Hz), 115.0, 115.9
(d, JC-F = 21 Hz), 115.9, 118.1, 120.0, 128.6 (2C), 128.8 (2C), 132.0 (d, JC-F = 8 Hz), 134.8, 135.5,
138.9, 139.0 (d, JC-F = 7 Hz), 143.8, 146.7, 160.9 (d, JC-F = 247 Hz), 162.7, 164.9, 169.5. EI-MS: m/z
= 463 (100, [M+]), 464 (26, [M+ + H]), 465 (7, [M+ + 2H].
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Supporting Information
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Molecular docking, determination of aqueous solubility, in vitro activity in GSKꢀ3α (h) and GSKꢀ3β
(h), in vitro activity in AML cell lines, in vivo on golden zebrafish embryo. This material is available
free of charge via the Internet at http://pubs.acs.org.
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*Phone: +496151ꢀ163075. Eꢀmail: schmidt_boris@tꢀonline.de (B.S.), theresa_neumann@web.de
(T.N.).
Acknowledgments
The authors thank the students Martin Brodrecht, Anne Schieferdecker and David Fiebig (Technische
Universität Darmstadt), who supported this work. The authors thank Dennis Bensinger (Technische
Universität Darmstadt) for help with scripting. We thank the Hans und Ilse Breuer-Stiftung for
financial support. K.S. is a Leukemia and Lymphoma Society Scholar, and this work was supported in
part by R01 CA140292.
References and notes
(1)
Cohen, P.; Frame, S., The renaissance of GSK3. Nat. Rev. Mol. Cell Biol. 2001, 2 (10), 769ꢀ
776.
(2)
Frame, S.; Cohen, P., GSK3 takes centre stage more than 20 years after its discovery.
Biochem. J. 2001, 359 (1), 1ꢀ16.
(3) Dajani, R.; Fraser, E.; Roe, S. M.; Young, N.; Good, V.; Dale, T. C.; Pearl, L. H., Crystal
Structure of Glycogen Synthase Kinase 3β: Structural Basis for PhosphateꢀPrimed Substrate
Specificity and Autoinhibition. Cell 2001, 105 (6), 721ꢀ732.
(4)
Biochem. Sci. 2004, 29 (2), 95ꢀ102.
(5) Lei, P.; Ayton, S.; Bush, A. I.; Adlard, P. A., GSKꢀ3 in Neurodegenerative Diseases. Int. J.
Alzheimers Dis. 2011, 2011, 189246.
(6) McCubrey, J. A.; Steelman, L. S.; Bertrand, F. E.; Davis, N. M.; Sokolosky, M.; Abrams, S.
Jope, R. S.; Johnson, G. V. W., The glamour and gloom of glycogen synthase kinaseꢀ3. Trends
L.; Montalto, G.; D’Assoro, A. B.; Libra, M.; Nicoletti, F.; Maestro, R.; Basecke, J.; Rakus, D.; Gizak,
A.; Demidenko, Z.; Cocco, L.; Martelli, A. M.; Cervello, M., GSKꢀ3 as potential target for therapeutic
intervention in cancer. Oncotarget 2014, 5 (10), 2881ꢀ2911.
(7)
Bhat, R. V.; Budd Haeberlein, S. L.; Lindquist, J. M., Inhibition of GSKꢀ3 as Therapeutic
Strategy in Disease: Efficacy of ARꢀA014418. In Glycogen Synthase Kinase 3 (GSK-3) and Its
Inhibitors, John Wiley & Sons, Inc.: 2006; pp 243ꢀ255.
(8)
Woodgett, J. R., Molecular cloning and expression of glycogen synthase kinaseꢀ3/factor A.
EMBO J. 1990, 9, 2431ꢀ8.
(9)
Lo Monte, F.; Kramer, T.; Gu, J.; Anumala, U. R.; Marinelli, L.; La Pietra, V.; Novellino, E.;
Franco, B.; Demedts, D.; Van Leuven, F.; Fuertes, A.; Dominguez, J. M.; Plotkin, B.; Eldarꢀ
Finkelman, H.; Schmidt, B., Identification of Glycogen Synthase Kinaseꢀ3 Inhibitors with a Selective
Sting for Glycogen Synthase Kinaseꢀ3α. J. Med. Chem. 2012, 55 (9), 4407ꢀ4424.
(10)
Banerji, V.; Frumm, S. M.; Ross, K. N.; Li, L. S.; Schinzel, A. C.; Hahn, C. K.; Kakoza, R.
M.; Chow, K. T.; Ross, L.; Alexe, G.; Tolliday, N.; Inguilizian, H.; Galinsky, I.; Stone, R. M.;
DeAngelo, D. J.; Roti, G.; Aster, J. C.; Hahn, W. C.; Kung, A. L.; Stegmaier, K., The intersection of
genetic and chemical genomic screens identifies GSKꢀ3α as a target in human acute myeloid
leukemia. J. Clin. Invest. 2012, 122 (3), 935ꢀ947.
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KaidanovichꢀBeilin, O.; Woodgett, J. R., GSKꢀ3: functional insights from cell biology and
animal models. Front. Mol. Neurosci. 2011, 4, 1ꢀ40.
(12) MacAulay, K.; Doble, B. W.; Patel, S.; Hansotia, T.; Sinclair, E. M.; Drucker, D. J.; Nagy, A.;
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Woodgett, J. R., Glycogen Synthase Kinase 3αꢀSpecific Regulation of Murine Hepatic Glycogen
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Metabolism. Cell Metab. 2007, 6 (4), 329ꢀ337.
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(13)
KaidanovichꢀBeilin, O.; Lipina, T.; Takao, K.; van Eede, M.; Hattori, S.; Laliberté, C.; Khan,
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