Biology-oriented synthesis of a natural-product inspired oxepane collection yields a small-molecule activator of the Wnt-pathway

Article abstract of DOI:10.1073/pnas.1015269108

In Biology Oriented Synthesis the scaffolds of biologically relevant compound classes inspire the synthesis of focused compound collections enriched in bioactivity. This criterion is met by the structurally complex scaffolds of natural products (NPs) selected in evolution. The synthesis of NP-inspired compound collections approaching the complexity of NPs calls for the development of efficient synthetic methods.We have developed a one pot 4-7 step synthesis of mono-, bi-, and tricyclic oxepanes that resemble the core scaffolds of numerous NPs with diverse bioactivities. This sequence entails a ring-closing ene-yne metathesis reaction as key step and makes productive use of polymer-immobilized scavenger reagents. Biological profiling of a corresponding focused compound collection in a reporter gene assay monitoring for Wntsignaling modulation revealed active Wntepanes. This unique class of small-molecule activators of the Wnt pathway modulates the van-Gogh-like receptor proteins (Vangl), which were previously identified in noncanonical Wnt signaling, and acts in synergy with the canonical activator protein (Wnt-3a).

Full text of DOI:10.1073/pnas.1015269108

Biology-oriented synthesis of a natural-product
inspired oxepane collection yields a small-
molecule activator of the Wnt-pathway
Sudipta Basua,b,1,2, Bernhard Ellingera,b,1, Stefano Rizzoa, Céline Deraevea,3, Markus Schürmannb,4, Hans Preutb,4
Hans-Dieter Arndta,b, and Herbert Waldmanna,b,5
,
aDepartment of Chemical Biology, Max-Planck-Institute of Molecular Physiology, Otto-Hahn-Strasse 11, 44227 Dortmund, Germany; and bFaculty of
Chemistry, Technische Universität Dortmund, Otto-Hahn-Strasse 6, 44221 Dortmund, Germany
Edited by Stuart L. Schreiber, Broad Institute, Cambridge, MA, and approved February 7, 2011 (received for review October 27, 2010)
In Biology Oriented Synthesis the scaffolds of biologically relevant
compound classes inspire the synthesis of focused compound
collections enriched in bioactivity. This criterion is met by the struc-
turally complex scaffolds of natural products (NPs) selected in evo-
lution. The synthesis of NP-inspired compound collections
approaching the complexity of NPs calls for the development of
efficient synthetic methods. We have developed a one pot 4–7 step
synthesis of mono-, bi-, and tricyclic oxepanes that resemble the
core scaffolds of numerous NPs with diverse bioactivities. This
sequence entails a ring-closing ene-yne metathesis reaction as key
step and makes productive use of polymer-immobilized scavenger
reagents. Biological profiling of a corresponding focused com-
pound collection in a reporter gene assay monitoring for Wnt-
signaling modulation revealed active Wntepanes. This unique class
of small-molecule activators of the Wnt pathway modulates the
van-Gogh-like receptor proteins (Vangl), which were previously
identified in noncanonical Wnt signaling, and acts in synergy with
the canonical activator protein (Wnt-3a).
of efficient synthesis methods which give rapid access to NP scaf-
folds and allow for the flexible assembly of structural diversity.
Sequential multistep reaction sequences which can be carried
out as one-pot syntheses, e.g., with the help of polymer-immobi-
lized scavenging reagents may provide advantageous solutions to
the problem (24). Here we report on the development of a solu-
tion phase one-pot, 4–7 step reaction sequence that efficiently
gives access to a collection of mono-, bi-, and tricyclic oxepanes.
NPs with multiple bioactivities (Fig. 1) like heliannuol B and C
(allelopathic and phytotoxic) (25), sodwanone S (antitumor) (26),
and zoapatanol (contraceptive) (27) served as an inspiration.
From this privileged library an activator of Wnt signaling was
identified, which acts synergistically with the Wnt-3a protein
and binds to the van-Gogh-like 1 transmembrane receptor (28).
Results
Retrosynthetic Analysis. Oxepanes have been synthesized by means
of different strategies (29), including ruthenium-catalyzed ring-
closing metathesis of unsaturated ethers (30). For the synthesis of
an NP-inspired oxepane collection a ring-closing ene-yne metath-
esis (RC-eneyne-M) of unsaturated linear ene-yne ethers 2 was
chosen as robust key transformation with wide scope. This syn-
thetic approach gives rise to a seven-membered oxacyclic ether
embodying a diene unit with one olefin incorporated into the se-
ven-membered ring (Scheme 1) and allows to flexibly introduce
diversity at multiple intermediate steps. Homoallylic alcohols 2
should be readily accessible by means of asymmetric Brown ally-
lation, which should proceed with predictable stereocontrol (31).
The resulting secondary alcohol would remain within the ring-
closing metathesis (RCM) products and allow for further functio-
nalization. Coupling of selected propargyl alcohols (PA1-4) with
α-bromo ethyl acetate derivatives (BEA1-3) would yield inter-
mediate esters 5 and provide an opportunity for introduction
of diversity. Notably, this synthesis sequence would allow for
chemical biology ∣ compound libraries
n Biology Oriented Synthesis (BIOS) biological relevance and
I
prevalidation are employed as key criteria in the selection of
compound classes for the synthesis of focused compound collec-
tions (1–4). The underlying scaffolds of natural product (NP)
classes define the areas of chemical space explored by nature
in evolution. NPs bind to multiple proteins in the course of their
biosynthesis and when exerting their biological function. There-
fore, NP scaffolds define “privileged” and biologically relevant
molecular frameworks in chemical space (5). Consequently, com-
pound collections based on and inspired by NP structure are
expected to be enriched in bioactivity and to yield modulators
of multiple biological processes. The use of NPs and analogues
thereof has been particularly rewarding in the study of biological
signal transduction events and pathways, often related to the
establishment of disease (6). Thus, the synthesis of NP inspired
compound collections and their evaluation in cell-based assays
monitoring biological signal transduction cascades is a highly
promising strategy for the identification of unique biologically
relevant compound classes and probes, as well as unique candi-
date molecules inspiring drug discovery programs. For instance,
the canonical and the noncanonical Wnt-signaling pathways play
decisive roles in differentiation, patterning, and tissue regenera-
tion (7–10). Modulators of Wnt signaling are efficient tools for
the study of these processes and have been intensively searched
for in drug discovery programs aimed at cancer therapy (11–14).
Wnt signaling also holds promise in the development of unique
methods to chemically induce stem cell differentiation and
wound healing (15–20). However, Wnt-pathway activators which
are required for this purpose have been identified in only very few
cases (21–23).
Author contributions: M.S., H.-D.A., and H.W. designed research; S.B., B.E., S.R., C.D., and
H.P. performed research; S.B., B.E., S.R., C.D., H.-D.A., and H.P. analyzed data; and S.B.,
B.E., S.R., H.-D.A., and H.W. wrote the paper.
The authors declare no conflict of interest.
This article is a PNAS Direct Submission.
Data deposition: The atomic coordinates have been deposited in the Cambridge Structural
Database, Cambridge Crystallographic Data Centre, Cambridge CB2 1EZ, United Kingdom
(CSD reference nos. 803785 and 803786).
1S.B. and B.E. contributed equally to this work.
2Present address: Department of Medicine, Harvard-MIT Division of Health Science and
Technology, Brigham and Women's Hospital, Cambridge, MA 02139.
3Present address: Laboratoire de Chimie de Coordination du CNRS, 205 Route de
Narbonne, 31077 Toulouse, Cedex 4, France.
4X-ray crystal structure analysis.
5To whom correspondence should be addressed. E-mail: herbert.waldmann@
mpi-dortmund.mpg.de.
The structural complexity of NPs, and, consequently also of
NP-inspired compound collections, calls for the development
This article contains supporting information online at www.pnas.org/lookup/suppl/
doi:10.1073/pnas.1015269108/-/DCSupplemental.
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were obtained as inseparable mixtures of two isomers in 20–
25% yield after 6–7 steps (SI Appendix: Table S1, entries 17–20,
26, 60–72). Cross metathesis reaction of 1 with methyl acrylate
in the presence of second generation Grubbs catalyst followed
by either esterification or carbamoylation of the secondary alco-
hol on the oxepane ring yielded 15 compounds (subcollection 2)
in 25–75% overall yield after 5 or 6 steps. The cross metathesis
products (SI Appendix: Table S2, entries 78–88) were formed
exclusively E- configured based on the coupling constants
recorded for the olefinic protons (JHa−Hb ¼ 16.0 Hz).
In order to create a small collection of keto-oxepane deriva-
tives (subcollection 3), the crude alcohols 1 were oxidized to the
corresponding ketones using pyridinium chlorochromate (PCC)
and then submitted to Diels-Alder cycloaddition with selected
dienophiles (D1-5) to give keto cycloaducts as single isomers in
10–25% overall yield after 5 steps (SI Appendix: Table S3,
entries 89–95). When treated with either O-methyl hydroxyla-
mine- or O-benzyl hydroxylamine hydrochloride, a selected num-
ber of ketones were converted into oximes (1∶1 E∕Z mixtures;
SI Appendix: Table S3, entries 96–100) in 10–15% overall yield
from intermediate 1.
Fig. 1. Structures of representative bioactive NPs embodying the oxepane
scaffold.
the use of solid-supported scavengers and extractive work-up
procedures and thereby open up an opportunity to develop a
one-pot approach.
Primary Screening for Wnt-Signaling Modulators in a Reporter Gene
Assay. A HEK293 reporter cell line highly sensitive for stimula-
tion by Wnt3a due to additional copies of the Frizzled receptor
resulting in a 20- to 30-fold induction of a luciferase reporter (33)
was used for screening of the library at 20 μM concentration. For
50 compounds the screen indicated activation of the Wnt-respon-
sive reporter construct, resulting in an increase of the luciferase
signal up to >250% relative activity (Fig. 2A). Notably, the Wnt-
pathway activators acted synergistically with the Wnt3a protein.
In the absence of Wnt3a (Fig. 2A) or upon stimulation with
the GSK-3β inhibitors lithium chloride or (2’Z, 3’E)-6-bromoin-
dirubine-3’-oxime (BIO) activation (34) by the oxepanes was
not observed (SI Appendix: Fig. S4). Luciferase activity was not
affected by the compounds, as checked by independent control
experiments.
One-Pot Synthesis of an Oxepane Collection. Propargyl alcohols
PA1-4 were converted to aldehydes 4 by alkylation with α-bromo
ethyl acetate derivatives BEA1-3 and subsequent reduction of the
ester groups with diisobutylaluminium hydride (DIBAL-H)
(Scheme 2A, SI Appendix: Fig. S1). Crude aldehydes 4 were ally-
lated using allylmagnesium chloride and (þ)- or (−)-diisopino-
campheylboron chloride (DIPCl) as chiral auxiliary. Enantio-
and/or diastereomerically enriched homoallyl alcohols 2 were
obtained after scavenging of excess allyl Grignard reagent with
polymer-bound sulfonic acid resin S1. As expected, matched
and mismatched cases were encountered. During library synthesis
we sometimes observed an unexpectedly variable stereoselectivity
(8∶1 to 2∶1). This variation was obviously correlated to comple-
teness of formation and quality of the allylation reagent. On dedi-
cated resynthesis of hit compounds with freshly prepared reagent
(see below), diastereomeric ratios of 12∶1 were regularly
achieved.
In order to develop a swift one-pot synthesis, diastereomers
were not separated but directly subjected to the decisive ring-
closing metathesis reaction using first generation Grubbs’ catalyst
(20 mol%) without purification. Upon completion of the trans-
formation, the ruthenium catalyst was scavenged with the poly-
mer-supported resin S2 (32) to afford oxepene-containing
intermediates 1. According to NMR-spectroscopic analysis, the
enantio- and diastereomeric ratio of the precursors was preserved
after ring closure, as expected.
Establishment of a Structure Activity Relationship. A structure activ-
ity relationship delineated from the screening results (Fig. 2B,
SI Appendix: Table S5) revealed that for high activity two methyl
substituents at positions R1 and R2 are beneficial. If instead of a
Intermediates 1 were then employed for introduction of
structural diversity and synthesis of subcollections by means of
chemical transformations involving the diene and the secondary
alcohol (Scheme 2B). Thus, O-acylation or O-carbamoylation and
subsequent Diels-Alder reaction yielded 65 compounds (subcol-
lection 1) in 15–70% overall yield after 4–7 steps. Chromatogra-
phy delivered all compounds either as single isomers or as
inseparable mixtures of two isomers. p-Benzoquinone (D1, see
SI Appendix) yielded hydroquinones (SI Appendix: Table S1,
entries 10, 14–16, 46, 48, 49, and 51) due to aromatization of the
initial Diels-Alder adducts. A nuclear Overhauser effect (NOE)
study performed for compound 47 (SI Appendix: Fig. S2), indi-
cated endo-selectivity for the Diels-Alder reaction. This finding
was later confirmed by crystal structure determination of racemic
compound 109 (SI Appendix: Fig. S3). The configuration of the
other library members was assigned by analogy.
Further introduction of structural diversity to the oxepane
scaffold was achieved through Diels-Alder cycloaddition with
Scheme 1. Retrosynthetic analysis and strategic disconnection of oxepane
maleic anhydride (D5) followed by hydrolysis to diacids, which candidates.
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Scheme 2. (A) General synthesis of the oxepane core. Diversification sites are highlighted (arrows). Building blocks, solid-supported scavengers, reagents, and
conditions: (A) NaH, THF, 0 °C to room temperature (r.t.), 10 h; (B) DIBAL-H, Et2O, 78 °C, 20 min. then aqueous HCl (1 M), −78 °C to r.t.; (C) allylmagnesium
chloride (2 M in THF), (þ)-DIPCl or (−)-DIPCl, THF, −78 ° > C to r.t., 4 h, then solid-supported scavenger S1; (D) First generation Grubbs catalyst (10% mol), CH2Cl2,
reflux, 18 h, then S2. (B) General synthesis of the oxepane collections. Building blocks, reagents, and conditions: (A) dienophile (D2), toluene, 70 °C, 3 h; (B) acyl
chloride (AC1-8), pyridine, 6 h, r.t., then S3; (C) isocyanate (I1-4), pyridine, 6 h, r.t., then S3; (D) carbonyldiimidazole, CH2Cl2, r.t. overnight; (E) amine (A1-12),
K2CO3, dimethylformamide (DMF), r.t. overnight, then S1; (F) dienophile (D1-5), toluene, 70 °C, 3 h; (G) 20% H2O in THF, 10 h, r.t.; (H) methyl acrylate, second
generation Grubbs catalyst (15% mol), CH2Cl2, 18 h, reflux, then S2; (I) PCC, CH2Cl2, 10 h, r.t.; (J) dienophile (D1-5), toluene, 70 °C, 3 h; (K) hydroxylamine
(HA1,2), EtOH∕H2O (2∶1), r.t., 10 h; and (L) 20% H2O in THF, 10 h, r.t.
geminal dimethyl group or a cyclohexyl group a di-n-pentyl group
was installed, toxicity was found to increase. Maleic acid imide
coupling products (R3 ¼ H) and the N-phenyl analogues at
the R3-position activate the Wnt signalling pathway to a higher
extent than the corresponding dicarboxylic acids or oxepanes 1
and oxepane collection 3, which embody a 1,4-diene substructure.
Notably, R4 could be varied widely without loss of activity
(SI Appendix: Table S5) including esters and carbamates, but
introducing a methyl substituent R5 already reduced the activity.
Based on these results tricyclic oxepane 109 (Fig. 3) was chosen
as most promising candidate for further investigation and for
tracking the cellular target.
four stereoisomers 109–112 of the most active Wnt activator were
synthesized separately (see SI Appendix) and investigated inde-
pendently in the reporter gene assay which revealed that 109
was most active (ED50 ¼ 1.8 ꢀ 0.9 μM) (Fig. 2). To identify a
suitable site for linker attachment compounds 113–116 carrying
an additional para-methoxy group in the benzyl carbamate
moiety were synthesized (see SI Appendix) and investigated as
well. Activity was identical for compound 113, which has the same
absolute configuration as 109. The other isomers 114–116 were
inactive (up to 20 μM, Fig. 2). Therefore, the para position at
the benzyl carbamate moiety was selected to attach a biotinylated
linker. Probe molecule 121 was synthesized from 113, and stereo-
isomer 122 was prepared as control (see SI Appendix). In addi-
tion, compound 125 was synthesized as alternative probe having
Synthesis of a Bait Probe for Target Identification. For target identi-
fication by means of a chemical proteomics approach (see below)
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Fig. 3. Oxepanes used in the biological investigations. For active compounds
the ED50 values based on their activity in the reporter gene assay are given.
Inactive compounds were measured up to 20 μM final concentration.
the linker directly attached to the oxepane core, and 123 was
used as additional control substance.
Identification of Target Proteins by a Chemical Proteomics Approach.
To gain initial insight into the biological mode of action of Wnt-
pathway activation, oxepane 35 (Fig. 3) was exposed to SW480
cancer cells (SI Appendix: Fig. S6). These cells harbor a mutation
in the tumor suppressor gene APC (adenomatous polyposis coli)
and hence show constitutively activated Wnt signaling. Treatment
of SW480 cells with oxepane 35 did not additionally activate
Wnt signaling suggesting that the compound must act upstream
of APC. In the light of this finding and the results that 35 and
109 act synergistically with Wnt-3a but not with two glycogen
synthase kinase 3 beta (GSK3-β) inhibitors (SI Appendix:
Fig. S4), the cellular target was therefore assumed to be closely
related to the Wnt receptor complex.
For target identification, HEK293 cell lysate was treated with
affinity probes 125 or 121 immobilized on streptavidin-coated
magnetic beads. Bound proteins were then released either by
heating to 95 °C or by elution with increasing concentrations
of nonbiotinylated ligand 109 and identified by means of PAGE,
in-gel digestion with trypsin, and mass-spectrometric identifica-
tion of the resulting peptides (see SI Appendix). Control pull
down experiments were performed with 123 and with 122
(Fig. 3C). Proteins that were found at least three times in five
independent pull down experiments with active probe 125 and
could be confirmed in additional pull down experiments with
probe 121 but not with the control probes 123 and 122 were con-
sidered further, if a connection to Wnt signaling had been docu-
mented in the literature. These criteria led to the proteins CDC2
(35), Pescadillo-1 (36), and van-Gogh-like receptor protein 1
(37–39) as plausible candidates. While CDC2 and Pescadillo-1
could not be eluted from the affinity beads by nonimmobilized
ligand 109, van-Gogh-like receptor protein 1 (Vangl1) was speci-
fically released with increasing concentration of compound 109
(Fig. 2D). This finding indicates that Wnt activating oxepane
109 binds reversibly to Vangl1. Because Vangl1 is a multipass
hydrophobic integral membrane protein (see below), we were
not surprised to find that it bound to a minor extent to the triethy-
lene glycol control resin from which it could be released by heat-
ing and identified by means of Western blotting.
Fig. 2. Biological analysis of the oxepane libraries. (A) Wnt pathway mod-
ulating activity of compounds 75, 86, and 97 (see Fig. 3 for structures). Data
are normalized to DMSO as control. The compounds do not activate the Wnt
pathway if used in combination with cell growth media but activate if used in
combination with Wnt-3a preactivated cells. (B) Structure activity relation-
ship of the oxepanes. (C) Structures of the affinity reagents used for proteo-
mics approaches. Compound 123 and 125 were used for proteomics and
competitive Western blotting. Compound 121 and 122 represent a second
generation of affinity purification reagents used for proteomics. (D) Compe-
titive Western blot of Vangl1. The Western blot was done after affinity
purification with either compound 125 (lane 1, 3–5) or compound 123 (con-
trol, lane 2) as control. Lanes 3–5 show the amount of Vangl1 protein bound
on the affinity resign after elution of reversibly bound protein with the
indicated concentration of compound 109.
Discussion
The Wnt signal transduction pathway is of major importance in
the regulation of cell differentiation and patterning as well as
tissue regeneration, wound healing, and stem cell differentiation
(15–20). Small-molecule modulators of Wnt signaling are consid-
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ered efficient tools for the study of these processes and unique
sources of inspiration for drug discovery programs aimed at e.g.,
cancer therapy (11–14). Notably, small-molecule activators of
Wnt signaling which might for instance be invaluable as reagents
for directed stem cell differentiation have been identified in only
very few cases (21–23).
with the lack of synergism of Wntepane 1 with Liþ ions, which
inhibit GSK3, and its lack of activity in SW480 cells.
Conclusion
Overall, our results demonstrate that NP inspired compound
collections with an oxepane scaffold can efficiently be synthesized
and that they may be rich sources for unique modulators of
biological systems. The identification of the Wntepanes as ligands
for the Vangl1 is notable, because small molecules targeting
this protein have not been described before. In the future, Wnte-
pane1 may serve as an advantageous tool for the study of
biological processes mediated by Vangl1.
For the development of unique small-molecule collections
enriched in bioactivity we have forwarded BIOS as a principle
that builds on the selection of biologically relevant compound
classes in evolution. In BIOS the underlying scaffolds of biologi-
cally relevant compound classes, in particular NPs inspire the
synthesis of focussed compound collections. In order to approach
NP-like diversity and structural complexity, for the synthesis of
BIOS-based compound collections efficient reliable and flexible
multistep synthesis sequences are required. The use of multistep,
one-pot solution phase sequences is particularly promising be-
cause it minimizes synthesis-, isolation-, and purification efforts.
For an oxepane collection, we have developed a 4–7 step synth-
esis sequence that gives efficient access to mono-, di-, and tricyclic
NP-inspired oxepanes. The synthesis proceeds in one pot and
without isolation of intermediates and employs a ring-closing
ene-yne metathesis as robust central step. This strategy enabled
the assembly of a focussed compound collection that resembles
the core structures of NPs with a mono- or polycyclic oxepane-
based structural framework. The synthesis is operationally conve-
nient and does not require elaborate instrumentation. By virtue
of the use of polymer-immobilized scavenging reagents this ap-
proach yields crude products that are readily purified by means
of conventional chromatography methods.
Materials and Methods
Luciferase Based Screening of Wnt-Signalling Modulators. Reporter gene carry-
ing cells (3,500) were seeded in a white 96 well plate (Corning) and incubated
for 4 h at 37 °C and 5% CO2 in a tissue culture incubator. The medium was
replaced by 100 μL of 20% Wnt-3a enriched medium supplemented with the
appropriate amount of test compound. The cells were incubated for 14 h in
the presence of compound and subsequently lysed. The medium was re-
moved and 30 μL lysis buffer [25 mM Tris-HCl (pH ¼ 7.5), 150 mM NaCl,
5 mM MgCl2, 1% NP-40, 1 mM DDT, 5% glycerol] was added and cells were
incubated for 10 min. 100 μL luciferase reagent (25 mM Tris-HCl pH ¼ 7.8,
0.5 mM coenzymeA, 0.5 mM EDTA, 0.5 mM ATP, 10 mM MgCl2, 40 mM tricine,
0.5 mM luciferine, 10 mM DTT) was added, and measurements were started
immediately. An Infinite 200 M (Tecan) plate reader was used, implementing
shaking at 120 rpm for 5 s before data acquisition and an integration of the
signal for 1 s per well. Every concentration was measured in quadruplicate
and normalized against data from DMSO-treated samples. Assessment
of the assay in 96 well format using DMSO and PKF118-310 (SI Appendix:
Fig. S6) as control confirmed the robustness of the experimental set-up
and readout and generally resulted in Z’-factors >0.7 (42). Six of the most
active oxepanes (35, 36, 104, rac-105, rac-106, and 109) were tested at higher
concentrations and showed no cytotoxicity to the liver cell line HepG2
and the endothelial cell lines HeLa and Hek293 up to a concentration of
160 μM (SI Appendix: Fig. S5).
The oxepane NPs inspiring this synthesis effort display multi-
ple biological activities and most likely target multiple proteins
involved in various phenomena. Thus, we expected that the use
of the oxepane collection in several cell-based assays monitoring
different biological processes should yield modulators of complex
biological systems at significantly enhanced frequency.
Solution Phase Synthesis of Oxepane 1 Using Polymer-Supported Scavenging
Reagents. To a cooled suspension (0 °C) of sodium hydride [1.5 equivalent
(equiv.) , 95% dispersion in mineral oil] in 50 mL of THF, a solution of a
selected building block PA1-4 (1 equiv.) in THF (20 mL) was added drop wise
over 20 min. The mixture was warmed to 25 °C and stirred for 15 min. After
cooling to 0 °C, a solution of selected building block BEA1-3 (1.5 equiv.) in
THF (10 mL) was added drop wise over 30 min and the resulting mixture
was warmed to 25 °C and stirred for 6 h. Water was added (20 mL, then
100 mL), followed by Et2O (100 mL) and the layers were separated. The
aqueous layer was extracted with Et2O (2 × 200 mL). The combined ether
extracts were washed with brine (2 × 20 mL), dried with Na2SO4, filtered,
and concentrated. Purification of the residue by silica gel chromatography
(cyclohexane/ethyl acetate 9∶1) furnished propargyl ethers 5 (70–80% yield).
To a solution of the ester 5 in diethyl ether at −78 °C, diisobutylaluminium
hydride (1 M in hexane, 1.5 equiv.) was added slowly by a syringe pump
over 30 min. The mixture was stirred at −78 °C for 20 min. HCl solution
was added (1 M) and the mixture was stirred for 1 h. The mixture was
extracted with diethyl ether (1 × 50 mL, 2 × 20 mL). The combined extracts
were washed with water (2 × 10 mL) and brine (2 × 10 mL), dried with
Na2SO4, filtered, and concentrated under reduced pressure to afford alde-
hydes 4 (90%-quantitative yield).
(þ) or (−)-DIPCl was dissolved in THF in a two-neck round-bottom flask
and cooled to −78 °C. Allylmagnesium chloride (2 M in THF, 1.5 equiv.)
was added drop wise and the mixture was stirred at −78 °C for 1 h. The reac-
tion mixture was warmed to 20 °C and stirred for 1 h. After cooling to −78 °C,
crude aldehyde 4 (dissolved in THF) was added drop wise and the mixture
was stirred at −78 °C for 1 h. The reaction mixture was allowed to warm
to room temperature, stirred for 1 h, and then diluted with methanol
(10 mL). Sulfonic acid resin (S1) was added and the mixture was shaken at
room temperature for 6 h. The resin was drained, washed with methanol
and CH2Cl2 and the solvents were evaporated to afford the crude homoallylic
alcohols 2.
The oxepanes such derived were subjected to cell-based assays
that monitor signaling through the Ras- or the Wnt-pathway,
neurite outgrowth, and cell cycle progression. Much to our
delight the most promising hits were activators of Wnt signaling
that act synergistically with the Wnt3a protein and appeared to
target the Wnt receptor complex. Employing a chemical proteo-
mics approach we found that the most potent oxepane 109 binds
to and most likely exerts its biological activity through the Vangl1.
Due to this Wnt-signaling enhancing activity we would like
to term the compound class “Wntepanes” (Wnt activating oxe-
panes) with compound 109 being Wntepane 1. The finding that
the Vangl1 is a target of the Wntepane is in accordance with the
activity profile of this molecule and with reports on Vangl1 func-
tion in the literature. Vangl1 is a multipass hydrophobic integral
membrane protein and serves as receptor in the noncanonical
Wnt-signaling pathway. This protein can directly bind to and
thereby antagonize the Wnt target protein Dishevelled (Dsh).
Thus, van-Gogh-like receptors are modulators of Wnt signaling
in vivo (40) and mediate cross talk between canonical and non-
canonical Wnt signaling (40, 41). The antagonistic activity of
van-Gogh-like receptors towards Wnt signaling had been demon-
strated by knockdown with antisense oligonucleotides which led
to an increase of Wnt signaling (41).
It is therefore likely that Wntepane 1 interacts with Vangl1
(and possibly Vangl2 as well) and thereby at least partially liber-
ates and restores the signal transducing activity of Dsh. This
hypothesis is supported by the observed synergistic mode of
action with Wnt, because only in the case of Wnt mediated
activation will the pool of Dsh be sensitive to modulation by
Vangl. Modulation by a small molecule should then lead to an
increase of signal. This mechanism would also be in accordance
In a two-neck round-bottom flask with attached refluxing condenser,
alcohol 2 was dissolved in CH2Cl2 (0.002 M). Ar was bubbled through
the solution by a needle for 30 min. First generation Grubbs catalyst
(20 mol%) was added and the reaction mixture was heated to reflux. After
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the reaction was complete [thin layer chromatography (TLC) control], poly-
mer-bound ruthenium scavenger resin S2 (20 equiv. relative to the catalyst
added) was added and the mixture was shaken at room temperature for
10 h. The resin was filtered off over a pad of silica gel, washed with dichlor-
omethane, and the solvents were evaporated to obtain the crude product 1.
ACKNOWLEDGMENTS. We are grateful to Petra Janning and Andreas Brock-
meyer for support in performing and analysis of the proteomics experiments.
This research was supported by the Max-Planck-Gesellschaft, the state of
Nord-Rhein-Westfalen [Zentrum für Angewandte Chemische Genomik
(ZACG) Dortmund], the European Commission (FP6, RTN Endocyte), and
the European Research Council (FP7, ERC Grant agreement n° 268309).
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