Discovery of inhibitors of the wnt and hedgehog signaling pathways through the catalytic enantioselective synthesis of an iridoid-inspired compound collection

Article abstract of DOI:10.1002/anie.201306948

Cousins you can count on: An iridoid-inspired compound collection was synthesized efficiently by the resolution of cyclic enones in an asymmetric cycloaddition with azomethine ylides. The collection contained novel potent inhibitors of the Wnt and Hedgehog signaling pathways. Copyright

Full text of DOI:10.1002/anie.201306948

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Angewandte
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DOI: 10.1002/anie.201306948
Biology-Oriented Synthesis
Discovery of Inhibitors of the Wnt and Hedgehog Signaling Pathways
through the Catalytic Enantioselective Synthesis of an Iridoid-Inspired
Compound Collection**
Hiroshi Takayama, Zhi-Jun Jia, Lea Kremer, Jonathan O. Bauer, Carsten Strohmann,
Slava Ziegler, Andrey P. Antonchick,* and Herbert Waldmann*
In biology-oriented synthesis (BIOS), biological relevance is
employed as a key criterion to generate hypotheses for the
Iridoids are a large group of cyclopentano[c]pyran
monoterpene secondary metabolites of terrestrial and
[
1]
[8]
design and synthesis of focused compound libraries. In
particular, the underlying scaffolds of natural product (NP)
classes provide inspiration for BIOS since they define the
areas of chemical space explored by nature, and therefore
they can be regarded as “privileged”. The use of NPs and NP-
inspired compound collections has been particularly reward-
ing in the chemical-biological investigation of cellular signal-
marine flora and fauna. Structurally, they are predominantly
cis-fused bicycles decorated by various functional groups with
multiple stereogenic centers (Scheme 1). Iridoids constitute
[
1,2]
[3]
[4]
ing cascades.
For example, the Wnt and Hedgehog
signaling pathways are of major importance for the regulation
of differentiation, tissue regeneration, and stem-cell renewal,
and are major pathways with relevance to the establishment
of cancer. Modulators of Wnt-pathway signaling are efficient
tools for the dissection of signal progression through the
Scheme 1. Structural features of over 500 natural iridoids.
major components of traditional folk-medicinal plants and
exhibit a wide range of pharmacological and biological
[
2g,3,5]
pathway.
Aberrant activation of the Hedgehog pathway
[8,9]
is involved in particular in skin cancer (basal-cell carcinoma)
and brain tumors (medulloblastoma). Therapeutic strategies
aimed at targeting the Hedgehog pathway are in high
properties.
Given this pronounced bioactivity and biolog-
ical relevance, it is surprising that the synthesis of iridoid-
inspired compound collections has essentially remained
[
6]
[10]
demand. In light of the structural complexity and richness
in stereogenic centers of natural products, and consequently
of the compound collections inspired by their structure, the
development of efficient enantioselective synthetic methods
unexplored.
Herein, we describe the design and highly
enantioselective synthesis of a novel iridoid-inspired com-
pound collection and its investigation in cell-based assays for
modulation of the Wnt and Hedgehog signal-transduction
pathways.
[1,2,7]
is at the heart of BIOS.
For the synthesis of a compound collection that was
similar in structure and complexity to the iridoids, we
envisaged that a matching target scaffold might be accessed
by the kinetic resolution of racemic 2H-pyran-3(6H)-ones 2
by means of an asymmetric [2+3] cycloaddition with azome-
[
*] Dr. H. Takayama, Z.-J. Jia, L. Kremer, Dr. S. Ziegler,
Dr. A. P. Antonchick, Prof. Dr. H. Waldmann
Max-Planck-Institut fꢀr Molekulare Physiologie
Abteilung Chemische Biologie
Otto-Hahn-Strasse 11, 44227 Dortmund (Germany)
E-mail: andrey.antonchick@mpi-dortmund.mpg.de
herbert.waldmann@mpi-dortmund.mpg.de
thine ylides derived from amino acid ester imines
[11–13]
1
[Eq. (1)].
Such an endo-selective cycloaddition should
Z.-J. Jia, Dr. A. P. Antonchick, Prof. Dr. H. Waldmann
Technische Universitꢁt Dortmund
Fakultꢁt Chemie, Chemische Biologie
Otto-Hahn-Strasse 6, 44221 Dortmund (Germany)
J. O. Bauer, Prof. Dr. C. Strohmann
Technische Universitꢁt Dortmund
Fakultꢁt Chemie, Anorganische Chemie
Otto-Hahn-Strasse 6, 44221 Dortmund (Germany)
[**] We acknowledge funding from the European Research Council
through the Seventh Framework Programme of the European Union
provide bicyclic compounds 3 with cis-fused five- and six-
membered rings and five stereogenic centers. The target
compounds 3 can be regarded as aza analogues of natural
iridoids (Scheme 1). Racemic 2H-pyran-3(6H)-one deriva-
tives are readily accessible by the Achmatowicz oxidation of
furfuryl alcohols. Critical for the success of this strategy is the
(
FP7/2007–2013; ERC Grant No. 268309 to H.W.) J.O.B. thanks the
Fonds der Chemischen Industrie for a fellowship. The Compound
Management und Screening Center (COMAS), Dortmund is
acknowledged for carrying out the screening and data analysis.
Supporting information for this article is available on the WWW
under http://dx.doi.org/10.1002/anie.201306948.
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development of an efficient method for the resolution of
racemic derivatives 2 during the planned cycloaddition.
For the establishment of suitable reaction conditions, we
selected N-4-bromobenzylideneglycine methyl ester (1a) and
solvent, base, and catalyst loading (Table 1, entries 12–15; see
also the Supporting Information). An increase in the ee value
of 3a to 93% was observed when DBU was employed as the
base (Table 1, entry 12). The catalyst loading could be
reduced to 5 mol% without compromising the yield and
enantioselectivity for the formation of 3a (Table 1, entry 14).
Therefore, further cycloaddition reactions were carried out
5
-oxo-5,6-dihydro-2H-pyran-2-yl benzoate (2a) as substrates.
I
I
Initially, different salts of Ag and Cu were tested in the
presence of the chiral P,N ferrocene ligand 4 and triethyl-
amine as a base (Table 1, entries 1–5). Notably, the relative
configuration of bicycle 3a with its multiple stereocenters is
identical to the relative configuration of natural iridoids. The
highest enantioselectivity for the synthesis of 3a was observed
in the presence of [Cu(CH CN) ]BF . Subsequently, a variety
with [Cu(CH CN) ]BF (5 mol%), the chiral ligand
7
3
4
4
(6 mol%), and DBU (20 mol%) in dichloromethane (0.1m)
at ambient temperature.
We investigated the scope of the [2+3] cycloaddition for
the synthesis of an iridoid-inspired compound collection from
various glycine ester imines 1 and pyranone 2a under the
optimized reaction conditions (Table 2, entries 1–8). Regard-
less of the electronic properties of the substituents introduced
into the glycine ester imine, products 3 were obtained as
single diastereomers in 60–71% yield with 88–99% ee
3
4
4
of ligands were tested (Table 1, entries 6–11; see the Support-
ing Information for additional details). An increase in
enantioselectivity for 3a was observed when (R)-Fesulphos
(7) was employed as a ligand (Table 1, entry 11). For further
optimization of the reaction conditions, we focused on the
(
Table 2, entries 1–8). The use of ortho-, meta-, and para-
[
a]
Table 1: Optimization of the reaction conditions.
substituted benzylideneglycine methyl esters led to similar
results. Imines obtained from aliphatic aldehydes were not
[
a]
Table 2: Scope of the asymmetric [2+3] cycloaddition.
[
c]
Entry Catalyst
L
Solvent Base
CH Cl2 Et N
T
Yield 3a/3a’
ee
[%]
[
b]
[d]
[h] [%]
1
2
3
4
5
6
7
8
9
0
1
2
3
4
5
AgOTf
AgOAc
AgOCOCF3
CuPF6
CuBF4
CuPF6
CuBF4
CuPF6
CuBF4
CuPF6
4
4
4
4
4
5
5
6
6
7
7
7
7
7
7
4
4
4
4
4
76
68
63
61
51
>20:1
>20:1
>20:1
>20:1
>20:1
–
0
26
20
53
65
–
–
–
–
64
70
93
83
93
85
1
2
[b]
[c]
2
3
Entry
R
R
Product Yield [%]
ee [%]
CH Cl2 Et N
2
3
1
2
3
4
5
6
7
8
9
10
11
12
13
Ph
Bz
Bz
Bz
Bz
Bz
Bz
Bz
Bz
Piv
Piv
Piv
Piv
Piv
Piv
Piv
Ac
Ac
Ac
Ac
Ac
Ac
(+)-3b
(+)-3c
(+)-3d
(+)-3e
(+)-3 f
(+)-3g
(+)-3h
(+)-3i
(+)-3j
(+)-3k
(+)-3l
69
71
61
63
62
66
60
60
68
65
60
92
94
92
88
94
95
90
99
88
92
85
95
90
96
94
84
93
91
91
85
91
94
88
94
94
93
92
CH Cl2 Et N
2
3
p-MeOC H
p-MeC H
4
m-MeC H
o-MeC H
p-FC H
m-FC H
2-furanyl
p-BrC H
4
CH Cl2 Et N
6
4
2
3
CH Cl2 Et N
6
2
3
CH Cl2 Et N 24 trace
6
4
2
3
CH Cl2 Et N 24 trace
–
6
4
2
3
CH Cl2 Et N 24
–
–
6
4
2
3
CH Cl2 Et N 24
–
–
6
4
2
3
1
1
1
1
1
1
CH Cl2 Et N
4
4
4
4
4
4
55
62
65
65
69
65
>20:1
>20:1
>20:1
>20:1
>20:1
>20:1
2
2
3
3
CuBF
4
CH
Cl
2
Et
N
6
[
[
[
[
e]
Ph
CuBF4
CuBF4
CuBF4
CuBF4
CH Cl2 DBU
2
e]
p-MeOC H
p-FC H
4
m-FC H
o-FC H
2-naphthyl
p-BrC H
p-MeOC H
p-FC H
m-FC H
o-FC H
2-naphthyl
p-MeC H
p-FC H
m-FC H
2-naphthyl
p-BrC H
toluene DBU
6
4
e,f]
e,g]
(+)-3m 62
CH Cl2 DBU
6
2
(+)-3n
(+)-3o
(+)-3p
(+)-3q
(+)-3r
(+)-3s
(+)-3t
(+)-3u
(+)-3v
65
68
58
58
55
55
56
51
56
61
68
65
66
CH Cl2 DBU
6
4
2
14
6
4
[
a] Reaction conditions: 1a (1.0 equiv, 0.1 mmol), 2a (2 equiv), Et N
3
15
(
1 equiv), catalyst (10 mol%), chiral ligand (12 mol%), solvent (0.1m),
1
1
1
1
2
2
2
6
7
8
9
0
1
2
6
4
room temperature. [b] Yield of the isolated product after column
chromatography. [c] The diastereomeric ratio was determined by
6
4
6
4
1
H NMR spectroscopy. [d] The ee value was determined by HPLC
6
4
analysis on a chiral stationary phase. [e] DBU (20 mol%) was used.
6
4
[
f] The reaction was carried out with 5 mol% of the catalyst and 6 mol%
of 7. [g] The reaction was carried out with 2 mol% of the catalyst and
.4 mol% of 7. Bz=benzoyl, CuBF =Cu(CH CN) BF , CuPF =Cu-
methoxyacetyl (+)-3w
methoxyacetyl (+)-3x
methoxyacetyl (+)-3y
methoxyacetyl (+)-3z
4-pentenoyl
4-pentenoyl
6
4
2
4
3
4
4
6
23
24
6
4
(
CH CN) PF , DBU=1,8-diazabicycloundec-7-ene, Tf=trifluorometha-
3 4 6
6
4
nesulfonyl.
2
2
2
5
6
7
(+)-3aa 73
(+)-3ab 70
6
4
2-naphthyl
[a] Reaction conditions: imine 1 (1.0 equiv, 0.1 mmol), oxodihydropyran
2
(2 equiv), DBU (20 mol%), [Cu(CH CN) ]BF (5 mol%), 7 (6 mol%),
3
4
4
CH Cl (0.1m), room temperature, 4 h. [b] Yield of the isolated product
2
2
after column chromatography. [c] The ee value was determined by HPLC
analysis on a chiral stationary phase. Piv=pivaloyl.
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.
Angewandte
Communications
reactive under these reaction conditions. In subsequent
experiments, the scope of the [2+3] cycloaddition with
respect to the oxodihydropyran 2 was examined (Table 2,
entries 9–27). Various esters obtained from 6-hydroxy-2H-
pyran-3(6H)-one by acylation with aliphatic acids smoothly
underwent the cycloaddition. The desired products were
obtained with 84–96% ee and in 51–73% yield as single
diastereomers, regardless of the electronic and steric proper-
ties of the substituents on the substrates. In total, a collection
of 115 compounds was synthesized.
The absolute configuration of cycloadduct (+)-3j was
determined by X-ray crystal-structure analysis (see the
Supporting Information for details). The absolute configu-
ration of all other compounds was assigned by analogy on the
basis of this crystal structure. The high selectivity in the
kinetic resolution of racemic derivatives 2 arises from a steric
interaction in the 1,3-dipolar cycloaddition (Scheme 2). A
as transcriptional repressors by interaction with histone
deacetylases. In many epithelial cancers, the Wnt pathway is
constitutively active as a result of mutations in different
components of the pathway.
We used an HEK293 reporter cell line that is highly
sensitive to stimulation by the protein Wnt3a owing to the
presence of additional copies of the Frizzled receptor and the
resulting 10–20-fold induction of a luciferase reporter to
[
15]
screen the library at a 30 mm concentration. Compounds for
which cell viability remained above 80% with respect to
control experiments were used in a primary screening. Dose-
response analyses were carried out for hit compounds. To rule
out the inhibition of the luciferase protein by the compounds
or their interference with transcription or translation, we also
carried out dose-response analyses by using an HEK293 cell
line with constitutive luciferase expression. The results of the
assays are summarized in Table 3. Much to our delight, the
compound collection contained potent inhibitors of the Wnt
signaling pathway.
The delineation of a structure–activity relationship from
2
the screening results revealed that the R group plays an
important role in the bioactivity of the compounds (Table 3;
see the Supporting Information for details). The introduction
Table 3: Representative results of the evaluation of the compound
[
a]
collection for inhibition of the Wnt signaling pathway.
Scheme 2. Proposed model of kinetic resolution by 1,3-dipolar cyclo-
addition.
1
2
3
Entry
R
R
R
3
Wnt
IC50 [mm]
Luciferase
IC50 [mm]
[
b]
[c]
1
2
3
4
5
6
7
8
9
p-BrC H
Bz
Bz
Bz
Ac
Ac
Me 3a
Me 3e
Me 3h
Me 3q
Me 3ac
Me 3r
25.8Æ4.2 inactive
27.1Æ1.5 inactive
23.0Æ6.5 inactive
4.4Æ0.5 inactive
7.2Æ0.8 > 30
6
4
m-MeC
m-FC H
6
H
4
complex A is formed by coordination of the copper salt with
the chiral ligand 5e and the glycine ester imine 1. Deproto-
nation by DBU leads to the formation of an azomethine ylide,
which undergoes 1,3-dipolar cycloaddition with derivatives 2.
The cycloaddition occurs in such a way as to avoid unfavor-
6
4
p-BrC H
6
4
Ph
p-MeOC H Ac
13.0Æ1.9 inactive
6
4
p-MeC H
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Ac
Me 3ad 10.1Æ1.4 inactive
6
4
m-MeC H
Me 3ae
Me 3af
Me 3s
Me 3t
Me 3u
Me 3v
8.2Æ0.4 inactive
7.1Æ0.4 > 30
6
4
2
able interactions between the bulky R group and the
o-MeC H
6
4
azomethine ylide. The transition state C is preferred over B
owing to the occurrence of minimal steric interactions and
thus defines the absolute configuration of all stereogenic
centers of products 3.
10
p-FC
H
6
4
10.8Æ0.8 inactive
10.2Æ1.0 inactive
7.5Æ0.9 inactive
5.8Æ0.9 16.5Æ2.6
1
1
1
1
1
1
2
3
4
5
m-FC H
6
4
o-FC H
6
4
2-naphthyl
2-furanyl
p-MeC H
Me 3ag 14.8Æ4.3 inactive
To investigate whether the iridoid-inspired compound
collection contained members that modulate different bio-
logical processes, we subjected it to different cell-based
assays, including a Wnt-pathway reporter-gene assay. Upon
Wnt activation, b-catenin, the central player in the pathway,
accumulates in the cytoplasm and enters the nucleus. Nuclear
b-catenin associates with transcription factors of the TCF/
LEF family and recruits transcriptional coactivators and
chromatin remodeling complexes, which in concert drive the
MeOCH CO Me 3w 12.2Æ3.3 inactive
6
4
2
16
17
m-MeC H
MeOCH CO Me 3ah
8.0Æ0.5 inactive
11.0Æ1.7 inactive
10.2Æ1.0 inactive
3.1Æ0.6 inactive
8.7Æ2.6 inactive
6
4
2
p-FC
H
6
MeOCH
MeOCH CO Me 3y
CO Me 3x
2
4
18
19
20
m-FC H
6
4
2
2-naphthyl PhOCH CO Me 3ai
1-naphthyl
2
4-pentenoyl Bn 3aj
[a] See the Supporting Information for biological methods. [b] Mean
IC50 value for the inhibition of the Wnt pathway as determined in a Wnt
reporter-gene assay. [c] Mean IC50 value for the inhibition of luciferase
activity as determined in a recombinant luciferase assay. Compounds
referred to as “inactive” showed no inhibition of luciferase activity at
[14]
expression of target genes. In the absence of a Wnt signal,
the b-catenin level is low, and the TCF/LEF factors function
30 mm. Bn=benzyl.
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Angewandte
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of bulky carboxylic residues, such as benzoic, pivalic, and iso-
pentanoic acid, as the R group led to less active inhibitors.
Table 4: Representative results of the evaluation of the compound
collection for inhibition of the Hedgehog signaling pathway.
[
a]
2
Active compounds most frequently contained an acetic acid
2
ester (R = Ac). Nevertheless, aliphatic acid residues of
different chain lengths without substituents in the a or
b position were tolerated. Importantly, the use of more labile
compounds, such as mixed acetals, in which the carboxylic
acid residue was replaced with an alkyl group, did not lead to
1
2
3
[b]
[c]
Entry
R
R
R
3
IC50 [mm]
Viability
2
3
active inhibitors (R = Me, allyl). Methyl esters (R = Me)
scored well in the inhibition of the Wnt signaling pathway. An
increase in the size of the group at this position led to active
1
2
3
4
5
6
1-naphthyl 4-pentenoyl Me 3al 4.3Æ1.8
inactive
inactive
inactive
inactive
inactive
inactive
2-naphthyl MeOCH CO Me 3z 5.0Æ1.3
3ap 5.9Æ1.0
4-pentenoyl Me 3aq 6.0Æ2.0
4-pentenoyl Bn
3ar 7.0Æ1.2
Ac
allyl 3as 9.8Æ0.2
2
2-naphthyl MeOCH CO Bn
2
1
compounds in the luciferase control assay. Notably, R could
o-MeC H
6 4
2-furanyl
be varied widely without loss of activity; ortho-, meta-, and
para-substituted phenyl groups were tolerated at this position.
p-BrC H
6
4
1
However, bulky R groups, such as 2-naphthyl, may lead to
[a] See the Supporting Information for biological methods. [b] Mean
IC50 value for the inhibition of the Hedgehog signaling pathway.
c] Compounds referred to as “inactive” showed more than 80% cell
false-positive results owing to the inhibition of luciferase
activity. Finally, protection of the nitrogen atom of com-
pounds 3 with various carboxylic acid derivatives led to
inactive compounds.
[
viability at 10 mm.
Protein structures and natural product scaffold structures
are conserved in evolution, and the number of fold types and
ligand-sensing cores with similar 3D structures yet different
amino acid sequencers and precise ligand-recognition motifs
is fairly limited. Thus, ligand-binding pockets with similar
structures and sequences occur in various proteins with
differing biological functions. Therefore, NP-inspired com-
pound collections based on BIOS may contain members that
hit different targets and thereby modulate different biological
a novel class of inhibitors of the Wnt and Hedgehog signaling
pathways.
Received: August 7, 2013
Published online: October 2, 2013
Keywords: asymmetric catalysis · cycloaddition · inhibitors ·
.
iridoids · signaling pathways
[1,16,17]
phenomena.
On the basis of this insight, we investigated
the iridod-inspired compound collection for possible modu-
lation of the Hedgehog pathway.
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[
6a,18]
a highly luminescent product.
Inhibition of the Hedgehog
pathway results in a reduction in luminescence. Hits showed
reduction in the luminescence signal without alteration of the
cell viability. Dose-response analysis was performed for hit
compounds. Representative results of the assay are summar-
ized in Table 4 (see the Supporting Information for details).
To our delight, several iridoid-inspired compounds that were
not identified as valid hits in the Wnt assay selectively
inhibited the Hedgehog signaling pathway in the low micro-
molar range. This finding confirms that the application of NPs
and NP-inspired compound collections is a powerful method
for the identification of modulators and probes with diverse
bioactivity from one given compound collection.
In conclusion, we developed a novel asymmetric catalytic
method for the synthesis of an iridoid-inspired compound
collection by the resolution of 2H-pyran-3(6H)-one deriva-
tives. The desired products were formed efficiently and with
high diastereo- and enantioselectivity. Evaluation of the
obtained compound collection led to the discovery of
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