SAR studies on hydropentalene derivatives - Important core units of biologically active tetramic acid macrolactams and ptychanolides

Article abstract of DOI:10.1016/j.bmc.2014.04.063

Structurally diverse bicyclo[3.3.0]octanes were prepared and tested for their biological activity. Both the antiproliferative activity and the results of phenotypic characterization varied with the substitution patterns. Two derivatives displayed high inhibitory (IC₅₀ ≤3 μM) activity against the L-929 cell line, but differed in their mode of action. A cluster analysis with impedance profiling data showed the two compounds in relationship to microtubule interfering compounds. In PtK₂ cells treated with both derivatives a perturbing effect on the microtubular network was observed, whereas the actin cytoskeleton in incubated PtK₂ cells was disturbed only by one compound. The effects on tubulin and actin polymerization could be confirmed by in vitro polymerization experiments.

Full text of DOI:10.1016/j.bmc.2014.04.063

Bioorganic & Medicinal Chemistry xxx (2014) xxx–xxx
Contents lists available at ScienceDirect
Bioorganic & Medicinal Chemistry
journal homepage: www.elsevier.com/locate/bmc
SAR studies on hydropentalene derivatives—Important core units of
biologically active tetramic acid macrolactams and ptychanolides
Vanessa Lutz a, Fabian Mannchen a, Michael Krebs a, Natja Park a, Claudia Krüger a, Aruna Raja b,
Florenz Sasse b, , Angelika Baro a, Sabine Laschat a,
⇑
⇑
a Institut für Organische Chemie der Universität Stuttgart, Pfaffenwaldring 55, 70569 Stuttgart, Germany
b Department of Chemical Biology, Helmholtz Centre for Infection Research, Inhoffenstr. 7, 38124 Braunschweig, Germany
a r t i c l e i n f o
a b s t r a c t
Article history:
Structurally diverse bicyclo[3.3.0]octanes were prepared and tested for their biological activity. Both the
antiproliferative activity and the results of phenotypic characterization varied with the substitution
patterns. Two derivatives displayed high inhibitory (IC50 63 lM) activity against the L-929 cell line,
but differed in their mode of action. A cluster analysis with impedance profiling data showed the two
compounds in relationship to microtubule interfering compounds. In PtK2 cells treated with both deriv-
atives a perturbing effect on the microtubular network was observed, whereas the actin cytoskeleton in
incubated PtK2 cells was disturbed only by one compound. The effects on tubulin and actin polymeriza-
tion could be confirmed by in vitro polymerization experiments.
Received 6 February 2014
Revised 28 April 2014
Accepted 29 April 2014
Available online xxxx
Keywords:
Biological activity
Click chemistry
Hydropentalene
Structure–activity relationships
Synthesis
Ó 2014 Elsevier Ltd. All rights reserved.
O
O
1. Introduction
H
H
HN
HO
H
NH
Highly substituted bicyclo[3.3.0]octanes (hydropentalenes)
represent the core unit of several biologically active natural prod-
uct families such as tetramic acid macrolactams containing, for
example, cylindramide (1),1,2 alteramide A (2),3 geodin A (3),4,5
and aburatubolactam A (4),6,7 or ptychanolides 5, 68,9 (Fig. 1).
The class of tetramic acid lactams displays a variety of biological
activities. Cylindramide (1), isolated from the marine sponge
Halichondria cylindrata in 1989, for example, exhibits pronounced
cytotoxicity against B16 melanoma cells.1 Alteramide A (2)3 from a
bacterium Alteromonas sp. associated with the sponge Halichondria
okadai is cytotoxic against epidermal carcinoma cell lines. Cylindra-
mide’s close relative geodin A (3) being isolated as Mg2+ salt in 1999
from the Southern Australian marine sponge Geodia,4 differs from 1
only in the exocyclic methylene group instead of methyl. Biological
studies revealed its strong nematocidal activity against the
parasitic nematode Haemonchus contortus. Aburatubolactam A (4)
isolated in 1996 from the marine bacterium Streptomyces sp. SCRC
A-206 inhibits the formation of anionic superoxides which are
relevant in both inflammatory and degenerative processes and
OH
H
H
O
H
O
HO
NH
NH
O
O
1
3
OH
2
4
OH
H
N
O
H
OH
O
HN
HO
H
N
H
H
HO
O
O
NH
O
O
OH
O
O
O
O
O
O
5
6
Figure 1. Hydropentalene core containing natural products 1–6.
⇑
Corresponding authors. Tel.: +49 531 6181 3429; fax: +49 531 6181 3499 (F.S.);
tel.: +49 711 685 64565; fax: +49 711 685 64285 (S.L.).
E-mail addresses: florenz.sasse@helmholtz-hzi.de (F. Sasse), sabine.laschat@oc.
uni-stuttgart.de (S. Laschat).
tumor promotion.10 Whereas natural products 1–4 are of marine
origin, a bicyclo[3.3.0]octane skeleton has also been detected in
http://dx.doi.org/10.1016/j.bmc.2014.04.063
0968-0896/Ó 2014 Elsevier Ltd. All rights reserved.
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2
V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
O
reticulum of PtK2 (potoroo kidney) cells that were incubated with
H
6a
Me
H
6a
1. A decrease of activity for the cyclopentane analogue of cylindra-
mide (1) indicated that the activity is strongly correlated to the
intact functionalized hydropentalene system.
6
4
1
3
6
4
1
2
5
2
5
O
O
O
O
3a
3a
3
H
7a
Me
7b
H
8
O
SAR studies of substituted bicyclo[3.3.0]octanes, however, are
rarely reported. Most of them focused on orally active PDE4
inhibitors,13 carbacycline derivatives such as TXA2/PGH2 receptor
antagonists and prostaglandin analogues,14 or mammalian squalene
synthase inhibitors.15 Therefore, we were motivated to investigate
the biological properties of the hydropentalene subunit in more
detail. Besides studying analogues of the core units of tetramic acid
lactams we were interested in a more general insight into the biolog-
ical properties of various functionalized bicyclo[3.3.0]octanes A–C.
Our results are reported below.
R3
R1
R4
R4
R4
R6
H
Me
H
R2
R5 R2
R5
R5
H
A
Me
B
H
C
R1
Figure 2. Starting materials and positions for functionalization leading to struc-
tures A–C.
2. Results and discussion
2.1. Chemistry
terrestrial sources. For example, the sesquiterpenoids ptychanolide
5 and 6 have been isolated in 1981 from the liverwort Ptychanthus
striatus (Lehm. et Lindenb.) Nees.8
Besides some initial experiments on the biological activity of
these natural product classes, however, only limited systematic
structure–activity relationship (SAR) studies have been carried
out. The influence of the tetramic acid moiety on cytotoxicity
and antimicrobial properties of macrocyclic acyltetramates has
been investigated by Schobert.11 Previously we aimed towards
elucidation of the mode of action of cylindramide (1).12 For this
purpose a series of derivatives of 1, including analogues where a
simple cyclopentane replaced the functionalized hydropentalene
moiety, were prepared. A broad biological screening revealed
promising antiproliferative activity against several tumor cell lines.
The cytotoxicity of 1 was found to be calcium dependent and we
observed vacuolisation and vesicle formation in the endoplasmic
In order to supply compounds for SAR studies, a series of
bicyclo[3.3.0]octanes was prepared from the easily accessible
Weiss diketone (7a),16 its dimethyl analogue 7b16 and the known
cylindramide core precursor 82b (Fig. 2) leading to derivatives of
structural diversity.
The hydropentalene derivatives 11–18 are based on Weiss
diketone (7a) (Scheme 1). The synthesis of alkynes 11a and 11b
which were envisaged for ‘click chemistry’ possibly allowing
visualization of the cellular target, commenced with the reduction
of monoketal 9a17,18 to give 10.18 The latter was either reacted with
propiolic acid under Mitsunobu conditions to 11a or converted in
three steps to 12 which was esterified analogously to 11b. Direct
esterification of alcohol 10 with propionyl chloride gave derivative
O
O
O
H
H
3'
4'
5'
O
O
3a'
2'
1'
RO
RO
O
6a'
6'
H
H
11b
11a
a
a
O
O
H
H
H
O
O
O
O
b
c
OH
OH
H
12
H
10
H
11c
ref.18
H
O
O
ref.17,18
13 16 R1
R2
Me Me
b allyl Me
7a
−
O
a
H
R = TBDPS
9a
ref.19
c
Me allyl
d Me prenyl
R1
R2
R1
H
H
H
H
ref.19
ref.19
15b
ref.19
Z
OR
+
Z
OR
16b
ref.19
R2
13 Z = O
14 Z = O
H
H
OR
OR
15b 16b Z = N-NHTs
,
H
17
H
18
Scheme 1. Reagents and conditions: (a) 4.0 equiv PPh3, 4.0 equiv propiolic acid, 4.0 equiv DEAD, 1.0 equiv 10 or 12, Et2O, rt, 24 h, 40% for 11a, 37% for 11b; (b) 1.0 equiv 5-
{[tert-butyl(diphenyl)silyl]oxy}hexahydropentalen-2(1H)-one,18 3.0 equiv NaBH4, MeOH, 0 °C, 3 h, quant.; (c) 1.0 equiv 9a, 2.0 equiv propionyl chloride, Et3N, CH2Cl2, 0 °C,
14 h, 73%. Numbering for NMR assignment.
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V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
3
11c. Alkenes 17 and 18, providing key structural motifs of the bicy-
clo[3.3.0]octene core of the natural products 1–4, were prepared
from monoketal 9a by a sequence of double deprotonation/electro-
philic trapping followed by swapping of protecting groups to yield
compounds 13, 14. Derivatives 13b, 14b were submitted to Shapiro
reaction to give 15b, 16b, which were subsequently functionalized
to 17, 18.19
The synthesis of hydropentalenes 21–23 based on bicy-
clo[3.3.0]octane-1,4-dione 82b is shown in Scheme 2. Monoketal
19 was converted to silyl enol ether 20 which was submitted to
a Shi epoxidation.20 Reductive epoxide opening of the resulting
intermediate with BH3ꢀTHF provided the trans configured hydro-
pentalenes 21a and 21b. Dienedione 222b yielded the alkylated
bicyclo[3.3.0]octene-1,4-dione 23a by Cu-catalyzed Grignard reac-
tion with 3-butenylmagnesiumbromide in the presence of TMSCl.
Ketalization of 23a with ethylene glycol gave the monoketal 23b
(Scheme 2).
The ratio of the diastereomeric ketones 24a and 24b depended
on the temperature. At room temperature, 24a,b were isolated in
87% yield with dr 39:61 whereas the reaction at ꢁ20 °C provided
24a,b quantitatively with dr 85:15. The obtained enantioselectivity
(10–12% ee) could not be improved by trapping the intermediate
enolate as silyl enol ether prior to alkylation as previously
reported.17 Presumably the steric bias of the axial methyl groups
on top of the convex bicyclic roof interfered with the enantiodis-
crimination of the chiral lithium amide base at the ketone moiety.
Nevertheless, ketones 24 were reduced to alcohols 25a,b which
were protected to yield silyl ethers 25c,d. Final acidic ketal cleav-
age provided ketones 26a,b.
2.2. Biological studies–proliferation tests
The ability of the set of nearly 30 hydropentalene derivatives to
inhibit the proliferation of various transformed cell lines including
human tumor cell lines was investigated in an MTT assay.22 The
results are summarized in Tables 1 and 2.
As shown in Table 1, the antiproliferative activities of most
compounds varied in a micromolar range. Exceptions were the
Hydropentalenes of the ptychanolide series were prepared from
3a,6a-dimethyltetrahydropentalene-2,5(1H,3H)-dione (7b)16 fol-
lowing known procedures17,18b,21 (Scheme 3).
O
OTBDPS
b
OTBDPS
O
H
H
H
H
H
H
Table 1
ref.2b
a
Cytotoxicity of bicyclo[3.3.0]octanes against different transformed cell linesa
8
O
O
O
Entry Compounds L-929 (mouse)
connective tissue IC50
M)
KB-3-1 (human) cervix
carcinoma IC50 (lM)
O
O
O
c
19
20
(
l
OTBDPS
OTBDPS
OH
H
H
1
2
3
4
5
6
7
8
11a
11b
11c
13a
13b
13c
13d
14a
14b
14c
14d
15b
16b
17
2.2
9.3
>142
7.4
32.4
17.8
23.9
14.8
16.2
5.1
21.6
—
>142
15.2
16.0
6.9
3'
4'
5'
3a'
2'
OH
+
6a'
1'
O
O
6'
H
21a
H
21b
O
O
1'
2'
28.2
7.4
O
O
O
H
H
H
H
6
ref.2b
O
d
1
e
9
20.3
16.2
21.7
13.3
12.5
>96
>96
—
6a
3a
5
2
8
10
11
12
13
14
15
16
17
18
19
20
4
3
10
5.6
9
H
22
H
23a
O
O
O
7
20.0
>67
20.4
7.2
41.0
38.8
3.0
8
23b
Scheme 2. Reagents and conditions: (a) 1.0 equiv KHMDS, 1.0 equiv 19, THF,
ꢁ78 °C, 20 min, 1.1 equiv TBDPSCl, ꢁ78 °C to rt, 3 h; (b) aq Na2B4O7ꢀ10H2O/
Na2EDTAꢀ2H2O, 0.3 equiv Shi catalyst,20 0.04 equiv Bu4NHSO4, 1.4 equiv oxone in aq
Na2EDTAꢀ2H2O, 5.8 equiv K2CO3, 3 h; (c) 2.5 equiv BH3ꢀTHF, THF, 3 h, 15% for 21a,
38% for 21b; (d) 1.4 equiv CuCN, 1.0 equiv TMEDA, 1.1 equiv 3-but-
enylmagnesiumbromide, THF, ꢁ78 °C, 1.2 equiv TMSCl, 1.0 equiv 22, 30 min, 63%;
(e) 2.0 equiv ethylene glycol, 0.09 equiv p-TsOH, reflux, 7 h, 60%. Only one
diastereomer of 23a was detected, its configuration was determined by NOESY
(see Supplementary data). Numbering for NMR assignment.
18
21a
21b
22
—
6.0
23a
23b
20.0
25.6
16.3
32.0
a
Further compounds in Supplementary data. IC50 values are means of two assays
in parallel.
O
O
O
O
a,b
R
S
Table 2
O
O
O
O +
c
O
Cytotoxicity of 3a,6a-dimethylhydropentalenes 24–26 and cyclopentanols against
different transformed cell linesa
7b
24a
24b
Entry
Compound
L-929 IC50
M)
KB-3-1 IC50
M)
(l
(l
O
e
1
2
3
4
5
6
7
8
9
10
24a
25a
25b
25c
25d
26a
26b
>150
>150
>150
15.8
11.8
14.3
35.7
>200
111.4
61.6
—
—
—
—
—
—
—
>200
123.7
—
OTBDPS
(R)-26a,(S)-26b
OR
O
R = H
R = TBDPS
(R)-25a,(S)-25b
(R)-25c,(S)-25d
d
Scheme 3. Reagents and conditions: (a) 2,2-dimethyl-1,3-propanediol, p-TsOH in
toluene, reflux, 2 h;18b (b) (1) n-BuLi/bis[(R,R)-1-phenylethyl]ammonium chloride,
ꢁ100 °C; then ꢁ78 °C, ClSiEt3, (2) MeLi, MeI according to Ref. 17; (c) 3 equiv NaBH4,
1 equiv 24, MeOH, 0 °C, 15 min; (d) 1 equiv 25a,b in DMF, 1.2 equiv TBDPSCl,
2.5 equiv imidazole according to Ref. 18b; (e) catalytic amounts of p-TsOH
according to Ref. 21.
t-Butyl(cyclopentyloxy)dimethylsilane
(Cyclopentyloxy)(triisopropyl)silane
t-Butyl(cyclopentyloxy)diphenylsilane
a
IC50 values are means of two assays in parallel.
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V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
alkyne 11a and dienedione 22 showing promising potencies (IC50
63 M) against L-929 mouse fibroblasts. The cytotoxicity of
increased activity in the order of silyl protecting groups
TBS < TIPS < TBDPS (Table 2, entries 8–11).
l
ester-substituted hydropentalenes 11 was found to depend
strongly on the ester function. In contrast to both propiolates
11a and 11b, the corresponding propionate 11c was completely
inactive. We were worried that the cytotoxicity of the propiolate
derivatives 11a,b might be due to a subsequent hydrolysis product
generated by esterase activity rather than caused by 11a and 11b
themselves. Therefore, the corresponding pentalene alcohol was
studied in comparison (Supplementary data), but no cytotoxicity
was observed for the alcohol, indicating that indeed the intact ester
seemed to be the active species. Bicyclo[3.3.0]octenes 17 and 1819
2.3. Biological studies–target identification
Alkyne 11a and dihydropentalenedione 22 with the highest
potency were selected for further biological studies. In order to
get hints about the mode of action we employed a newly devel-
oped impedance profiling method which uses time-dependent
impedance curves as a fingerprint for the mode of action.26 In L-
929 cell cultures that were incubated with 11a and 22, impedance
was monitored over time (Fig. 3).
displayed cytotoxicities (IC50 = 7.2–20.4
fibroblasts but poor inhibitory (IC50 >96
l
l
M) against L-929 mouse
M) activity against the
The obtained curves were compared with those of a set of refer-
ence compounds using statistical methods. Cluster analysis of the
resultingdata showed11a and 22 in a groupof reference compounds
such as nocodazole, tubulysin B and griseofulvin that interfere with
microtubule polymerization and spindle formation (Fig. 4).
Based on the results obtained from the impedance profiling,
PtK2 (potoroo kidney) cells which showed in MTT assays an IC50
KB-3-1 cell line (Table 1, entries 14 and 15). Unexpectedly, silyl-
protection had a significant effect on the antiproliferative activity.
The unprotected analogues of 13, 14 and 21 were completely inac-
tive against all tested cell lines (Supplementary data).
A similar effect was observed for unprotected ptychanolide
derivatives 25a,b and their silyl-protected counterparts 25c,d
(Table 2, entries 2–5). In order to study the influence of the silyl
protecting group, the cytotoxic effect of O-silylated cyclopenta-
nols23–25 for the two cell lines was investigated. We observed an
of approximately 3 lM with 11a and 22, respectively, were incu-
bated with both compounds and stained for effects on microtu-
bules (Fig. 5). For both 11a and 22 a perturbing effect on the
microtubular network in the cells and altered mitotic spindles
together with an unusual distribution of chromosomes was visible.
To further verify the effect of 11a and 22, tubulin polymeriza-
tion assays were carried out in vitro (Fig. 6). The effect of 11a
and 22 was compared to that of methanol and nocodazole, a
known inhibitor of tubulin polymerization.27 The basis of this
assay is the incorporation of a fluorescent reporter into the devel-
oping microtubules. While nocodazole is only inhibiting the poly-
merization process, 11a and 22 induce a decrease in fluorescence.
The effect of 22 was much stronger than that of 11a, which still
allowed a delayed tubulin polymerization.
We also checked for the influence of 11a and 22 on the F-actin
cytoskeleton, the microfilaments (Fig. 7). Whereas compound 22
did not show a perturbing influence on the actin cytoskeleton
(Fig. 7D), this was indeed the case with 11a (Fig. 7C).
For 11a, further studies were carried out that should possibly
show co-localization with F-actin. We used a ‘click chemistry’
approach, which is based on a copper(I)-catalyzed azide-alkyne
cycloaddition (CuAAC) reaction.28,29 PtK2 cells were treated with
terminal alkyne 11a (3 l
g mLꢁ1) for 12 h and then fixed. The fixed
cells were permeabilized with Triton X-100 and incubated with a
click reagent mixture for 30 min. Further staining for F-actin was
carried out using fluorescently labeled phalloidin which binds
Figure 3. Impedance curves of L-929 cells incubated with 11a (0.6
(0.4 was used as one of the reference
g mLꢁ1). Tubulysin (3 ng mLꢁ1
compounds.
l
g mLꢁ1) and 22
l
B
)
Figure 4. Cluster analysis of data obtained by impedance monitoring of incubated cell cultures. Derivatives 11a (c9a) and 22 (c19) are related to compounds that interfere
with tubulin polymerization and mitotic spindle formation.
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V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
5
Figure 5. Effect of 11a and 22 on the microtubule cytoskeleton and mitotic spindle. PtK2 cells were treated with 11a (4
l
g mLꢁ1; C) and 22 (4
l
g mLꢁ1; D) overnight and
stained for microtubules (green) and DNA (blue). Control cells were incubated with methanol only (A and B).
3. Conclusions
We have synthesized a series of hydropentalene derivatives
starting from hexahydropentalene-1,4-dione (8), cis-tetrahydro-
pentalene-2,5(1H,3H)-dione 7a (Weiss diketone) or its 3a,6a-
dimethyl analogue 7b. Hydropentalenes are not only biologically
important parts of natural macrolactams but show pharmaco-
phoric properties themselves as was demonstrated by biological
studies. Their antitumor activities on mammalian cell lines varied
with the substitution patterns. In the series of 1,4-dione 8 derived
compounds 21–23 the presence of unsaturated rings seemed to
play a key role. Consequently, the cytotoxicity against the tested
L-929 cell line increased in the order saturated 21 (IC50 ca.
40
l
M) < single unsaturated 23 (IC50 ca 26
lM) < double unsatu-
rated 22 (IC50
3
lM). The latter was also most potent within all
investigated compounds. In the Weiss diketone 7a based series,
this structural element may be negligible although derivatives 17
and 18 displayed antiproliferative activities against L-929 cells
comparable to 23. The most active compound in this group, how-
ever, was the alkyne 11a. A hierarchical cluster analysis of imped-
ance monitoring data measured with 11a and 22 suggested a mode
of action that might be similar to microtubule affecting com-
pounds. During further investigations with 11a, tubulin and also
actin could be identified as the probable target proteins. Both actin
filaments and tubulin network in PtK2 cells were disturbed when
treated with 11a, and in vitro experiments also showed that the
polymerization of tubulin and of actin is impaired. With actin we
measured an enhancing effect, with tubulin an inhibition of poly-
merization. An incubation of PtK2 cells with 22 only induced a per-
turbing effect on the microtubules, not on the microfilaments.
Tubulin polymerization assays showed a strong inhibitory effect
in vitro also. Future work should reveal whether pentalene-con-
taining tetramic acid macrolactams 1–4 are indeed binding to
the same target proteins or whether the tetramic acid moiety
results in binding to a completely different target.
Figure 6. In vitro tubulin polymerization assay (37 °C). Compound 11a and 22
(500
g mLꢁ1 each) induced a decrease in fluorescence while the rate of tubulin
polymerization progressed in the methanol control. While 11a still allowed some
delayed polymerization, this process was completely blocked by 22. Fluorescence
was monitored at 460 nm (excitation at 340 nm).
l
selectively to F-actin. A blue fluorescence was observed that was
co-localized with the green phalloidin label depicting the
microfilaments.
In order to check the effect of 11a on actin in vitro, a polymer-
ization assay with pyrene muscle actin was carried out, wherein
the environmentally sensitive fluorophore pyrene indicates the
polymerization state of actin. Thus, an increase in fluorescence is
directly proportional to an increase in F-actin. As shown in Figure 8,
an increase of the polymerization rate was measured with increas-
ing concentration of 11a.
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6
V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
Figure 7. Effect of 11a and 22 on the F-actin cytoskeleton. PtK2 cells were incubated with 11a (2 l l
g mLꢁ1; C) and 22 (3 g mLꢁ1; D) overnight and stained for F-actin (green)
and DNA (blue). Control cells were treated with methanol only (A and B).
Flash chromatography was performed on silica gel, grain size 40–
63 m (Fluka). Moisture-sensitive reactions were performed under
l
nitrogen atmosphere in oven-dried glassware. All reagents were
used as purchased unless otherwise noted. Solvents used for chro-
matography were distilled. THF was distilled from sodium/benzo-
phenone, CH2Cl2 and toluene from CaH2. The reactions were
monitored by TLC (Merck 60 F254 plates).
Cell lines were obtained from the German Collection of Micro-
organisms and Cell Cultures (DSMZ) or American Type Culture Col-
lection (ATCC) and cultivated in the media recommended by the
supplier at 37 °C and 10% CO2.
4.2. (3a0R,6a0S)-5,5-Dimethylhexahydro-10H-spiro[1,3-dioxane-2,
20-pentalen]-50-yl propiolate (11a)
A suspension of propiolic acid (248 mg, 3.52 mmol) and PPh3
(923 mg, 3.52 mmol) in abs Et2O (10 mL) under N2 atmosphere
was stirred for 15 min. Then DEAD (0.56 mL, 3.52 mmol) was
added followed by a solution of 1018 (200 mg, 0.88 mmol) in abs
Et2O (5 mL). The reaction mixture was stirred for 24 h, filtered
off, and the precipitate was washed with Et2O (15 mL). The filtrate
was concentrated and the residue purified by flash chromatogra-
phy on SiO2 (hexanes/EtOAc, 20:1) to give 11a (98 mg, 0.35 mmol,
40%) as a white solid. Rf 0.40 (hexanes/EtOAc, 10:1). 1H NMR
(500 MHz, CDCl3): d 0.96 [s, 6H, C(CH3)2], 1.67–1.79 (m, 4H, 10-
Ha, 30-Ha, 40-Ha, 60-Ha), 1.98–2.07 (m, 2H, 40-Hb, 60-Hb), 2.17 (dd,
J = 13.5, 8.9 Hz, 2H, 10-Hb, 30-Hb), 2.67–2.77 (m, 2H, 3a0-H, 6a0-H),
2.83 (s, 1H, CCH), 3.46 (s, 2H, OCH2), 3.47 (s, 2H, OCH2), 5.31–
5.38 (m, 1H, 50-H) ppm; 13C NMR (125 MHz, CDCl3): d 22.5
[C(CH3)2], 30.1 [C(CH3)2], 38.2 (C-3a0, C-6a0), 39.0 (C-40, C-60), 39.8
(C-10, C-30), 72.0, 72.1 (OCH2), 74.0 (CCH), 75.1 (CCH), 80.9 (C-50),
~
Figure 8. In vitro actin polymerization assay. Compound 11a induced an increased
polymerization rate of pyrene actin in vitro. The actin fluorescence baseline was
first read without any drug addition for 3 min. Then methanol (control) and 11a (at
different concentrations) were added and fluorescence read for a further 20 min to
test if the drug itself has an effect before the polymerization reaction was started by
addition of 10X actin polymerization buffer. Fluorescence was monitored at 420 nm
(excitation 360 nm).
4. Experimental
4.1. General
NMR spectra were recorded on a Bruker Avance 500 spectrom-
eter in CDCl3 with TMS as an internal standard. IR spectra were
recorded on a Bruker FT-IR-spectrometer Vektor 22 with MKII
golden gate single reflection Diamant ATR-system. Mass spectra
were recorded on a Varian MAT 711 (EI, 70 eV) and a Bruker Dal-
tonics micrOTOF_Q (ESI) with nitrogen as carrier gas. Optical rota-
tions were measured with a Perkin-Elmer 241 polarimeter at 20 °C.
110.0 (C-2), 152.4 [(CO)O] ppm; FT-IR (ATR):
m = 3246 (w), 2954
(m), 2865 (w), 2359 (w), 2112 (w), 1706 (s), 1472 (w), 1434 (w),
1395 (w), 1352 (w), 1334 (w), 1299 (w), 1233 (s), 1220 (vs),
1121 (s), 1105 (s), 1053 (m), 1011 (m), 993 (m), 966 (w), 943
Please cite this article in press as: Lutz, V.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.063

V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
7
(w), 926 (w), 905 (w), 877 (w), 792 (w), 758 (m), 731 (w), 694 (w),
594 (w), 561 (w), 528 (w) cmꢁ1; MS (EI): m/z (%) = 278.2 (61) [M]+,
263.1 (2), 235.1 (10), 225.1 (8), 209.2 (56), 193.1 (26), 181.1 (7),
168.1 (72), 155.1 (19), 141.1 (9), 123.1 (36), 105.1 (4), 95.1 (23),
81.1 (17), 69.1 (100), 53.0 (30), 41.0 (44); HRMS (ESI): calcd for
(3aS,4R,5R,6aR)-5-{[tert-butyl(diphenyl)silyl]oxy}-4-methylhexa-
hydropentalen-2(1H)-one (266 mg, 0.68 mmol) and prenyl bro-
mide (0.18 mL, 269 mg, 1.60 mmol). Compound 13d: colorless oil,
20
yield: 113 mg (36%), Rf 0.46 (hexanes/EtOAc, 15:1), [
a]
ꢁ52.7
D
(c = 5.1, CH2Cl2). 1H NMR (500 MHz, CDCl3): d 0.86 (d, J = 6.8 Hz,
3H, 4-CH3), 1.05 [s, 9H, SiC(CH3)3], 1.51 (ddd, J = 13.6, 7.0, 4.7 Hz,
1H, 6-Ha), 1.60 (q, J = 1.2 Hz, 3H, 40-H or 50-H), 1.65 (q,
J = 1.2 Hz, 3H, 40-H or 50-H), 1.71 (ddq, J = 9.2, 7.0, 6.8 Hz, 1H,
4-H), 1.96 (dq, J = 9.1, 3.9 Hz, 1H, 3a-H), 2.00–2.07 (m, 1H, 10-Ha),
2.03 (ddd, J = 13.6, 8.5, 7.0 Hz, 1H, 6-Hb), 2.15 (dq, J = 8.5, 4.7 Hz,
1H, 6a-H), 2.18–2.24 (m, 1H, 1-H), 2.28 (ddd, J = 19.1, 3.9, 1.6 Hz,
1H, 3-Ha), 2.31–2.38 (m, 1H, 10-Hb), 2.36 (dd, J = 19.1, 9.3 Hz, 1H,
3-Hb), 3.80 (q, J = 7.0 Hz, 1H, 5-H), 4.98–5.04 (m, 1H, 20-H),
7.35–7.39 (m, 4H, m-H, m0-H), 7.41–7.45 (m, 2H, p-H, p0-H), 7.63–
7.68 (m, 4H, o-H, o0-H) ppm; 13C NMR (125 MHz, CDCl3): d 17.0
(4-CH3), 17.8 (C-40 or C-50), 19.2 [SiC(CH3)3], 25.8 (C-40 or C-50),
27.0 [SiC(CH3)3], 28.5 (C-10), 41.4 (C-6), 42.4 (C-6a), 42.6 (C-3,
C-3a), 49.2 (C-4), 56.3 (C-1), 81.6 (C-5), 121.1 (C-20), 127.5, 127.6
(C-m, C-m0), 129.61, 129.62 (C-p, C-p0), 133.3 (C-30), 134.1, 134.4
(C-i, C-i0), 135.90 (C-o, C-o0), 221.4 (C-2) ppm; FT-IR (ATR):
~
C
16H22O4Na+ [M+Na]+ 301.1410; found 301.1421.
4.3. (3aR,6aS)-5-{[tert-Butyl(diphenyl)silyl]oxy}octahydropenta-
len-2-ol (12)
NaBH4 (90.0 mg, 2.38 mmol) was added portionswise to a solu-
tion of (3aR,6aS)-5-{[tert-butyl(diphenyl)silyl]oxy}hexahydropent-
alen-2(1H)-one18 (300 mg, 0.79 mmol) in dry MeOH (40 mL) at
0 °C and the reaction mixture stirred at 0 °C for 2 h. Then the reac-
tion mixture was hydrolyzed with H2O (15 mL), warmed to room
temperature, and extracted with Et2O (3 ꢂ 20 mL). The combined
organic layers were washed with brine (20 mL) and dried (MgSO4).
After removal of the solvent under reduced pressure, 12 (300 mg,
0.79 mmol, quant.) was obtained as a colorless oil. 1H NMR
(500 MHz, CDCl3): d 0.96 [s, 6H, C(CH3)2], 1.73–1.90 (m, 4H,
10-Ha, 30-Ha, 40-Ha, 60-Ha), 2.15–2.33 (m, 4H, 10-Hb, 30-Hb, 40-Hb,
60-Hb), 2.45–2.62 (m, 2H, 3a0-H, 6a0-H), 2.99 (s, 3H, SO2-CH3), 3.48
[s, 4H, (OCH2)2], 5.02 (tt, J = 6.7, 5.9 Hz, 1H, 50-H) ppm; 13C NMR
(75 MHz, CDCl3): d 22.5 [C(CH3)], 22.6 [C(CH3)], 30.1 (C-3a0,
C-6a0), 30.5 [C(CH3)2], 38.2 (OCH3), 40.2 (C-40), 40.3 (C-60), 48.3
m
= 3071 (w), 2958 (s), 2929 (s), 2858 (m), 1737 (vs), 1589 (w),
1460 (m), 1428 (m), 1378 (w), 1110 (vs), 1033 (m), 874 (m), 822
(m), 740 (m), 702 (s), 613 (m), 548 (w), 526 (w) cmꢁ1; MS (ESI):
m/z = 483.3 [M+Na]+, 461.3, 383.2 [M+NaꢁC6H6]+, 205.2, 187.2,
86.1; HRMS (ESI): calcd for C30H40O2SiNa+ [M+Na]+ 483.2692;
0
0
~
m
(C-1 , C-3 ), 109.8 [(OCH2)2] ppm; FT-IR (ATR):
= 3071 (w), 2957
found 483.2692. 14d: colorless oil, yield: 64 mg (20%), Rf 0.46 (hex-
ꢁ4.0 (c = 4.1, CH2Cl2). 1H NMR (500 MHz,
20
(m), 2929 (m), 2855 (m), 1590 (w), 1487 (w), 1471 (w), 1461
(w), 1427 (m), 1390 (w), 1360 (w), 1262 (w), 1188 (w), 1108
(vs), 1039 (m), 999 (m), 975 (w), 938 (w), 898 (w), 821 (m), 801
(w), 740 (m), 701 (vs), 610 (m) cmꢁ1; MS (ESI): m/z = 403.21
[M+Na]+, 381.22 [M]+, 365.19, 248.09, 226.95; HRMS (ESI): calcd
for C24H32O2SiNa+ [M+Na]+ 403.2064; found 403.2074.
anes/EtOAc, 15:1), [
a]
D
CDCl3): d 0.82 (d, J = 6.6 Hz, 3H, 6-CH3), 1.04 [s, 9H, SiC(CH3)3], 1.50
(dq, J = 13.3, 6.6 Hz, 1H, 4-Ha), 1.58 (q, J = 1.1 Hz, 3H, 40-H or 50-H),
1.67 (q, J = 1.1 Hz, 3H, 40-H or 50-H), 1.77–1.86 (m, 2H, 6a-H, 6-H),
2.04–2.11 (m, 2H, 10-Ha, 4-H), 2.20–2.35 (m, 3H, 1-H, 10-Hb, 3-Ha),
2.43–2.49 (m, 2H, 3a-H, 3-Hb), 3.80 (q, J = 6.4 Hz, 1H, 5-H), 5.02–
5.07 (m, 1H, 20-H), 7.35–7.39 (m, 4H, m-H, m0-H), 7.41–7.45 (m,
2H, p-H, p0-H), 7.62–7.68 (m, 4H, o-H, o0-H) ppm; 13C NMR
4.4. (3aR,6aS)-5-{[tert-Butyl(diphenyl)silyl]oxy}octahydropent-
alen-2-yl propiolate (11b)
(125 MHz, CDCl3): d
17.6 (6-CH3), 17.8 (C-40 or C-50), 19.1
[SiC(CH3)3], 25.8 (C-40 or C-50), 27.0 [SiC(CH3)3], 29.4 (C-10), 33.9
(C-3a), 42.0 (C-4), 45.2 (C-3), 49.0 (C-6), 51.0 (C-6a), 54.4 (C-1),
81.8 (C-5), 121.3 (C-20), 127.53, 127.54 (C-m, C-m0), 129.59,
129.61 (C-p, C-p0), 133.4 (C-30), 134.1, 134.3 (C-i, C-i0), 135.87,
~
As described for 11a, from propiolic acid (130 lL, 2.10 mmol),
PPh3 (551 mg, 2.10 mmol) in abs Et2O (10 mL), DEAD (0.33 mL,
2.10 mmol) and 12 (200 mg, 0.53 mmol) in abs Et2O (5 mL), flash
chromatography on SiO2 (hexanes/EtOAc, 100:1, Rf 0.37), colorless
needles, yield: 84 mg (0.35 mmol, 37%). 1H NMR (500 MHz, CDCl3):
d 1.03 [s, 9H, C(CH3)3], 1.45–1.52 (m, 2H, 10-Ha, 30-Ha), 1.79–1.89
(m, 2H, 10-Hb, 30-Hb), 1.92–2.07 (m, 4H, 40-Ha, 60-Ha, 40-Hb, 60-Hb),
2.49–2.59 (m, 2H, 3a0-H, 6a0-H), 2.81 (s, 1H, CCH), 4.23 (dt,
J = 10.7, 5.4 Hz, 1H, 20-H), 5.40–5.45 (m, 1H, 50H), 7.34–7.39 (m,
4H, m-H), 7.39–7.45 (m, 2H, p-H), 7.62–7.67 (m, 4H, o-H) ppm;
13C NMR (125 MHz, CDCl3): d 19.0 [C(CH3)3], 26.9 [C(CH3)3], 39.4
(C-3a0, C-6a0), 39.7 (C-40, C-60), 41.8 (C-10, C-30), 73.9 (CCH), 75.2
(CCH), 81.5 (C-50), 127.6 (C-m), 129.6 (C-p), 134.2 [(CO)O], 135.8
~
135.91 (C-o, C-o0), 222.0 (C-2) ppm; FT-IR (ATR):
m = 3070 (w),
2956 (s), 2930 (s), 2860 (m), 1736 (vs), 1454 (w), 1429 (w), 1380
(w), 1110 (vs), 867 (w), 821 (w), 740 (m), 703 (s), 613 (m),
504 (m) cmꢁ1
;
MS (ESI): m/z = 483.3 [M+Na]+, 461.3, 383.2
[M+NaꢁC6H6]+, 205.2, 187.2, 86.1; HRMS (ESI): calcd for
C
30H40O2SiNa+ [M+Na]+ 483.2692; found 483.2683.
4.6. {[(2R,3R,3aS,4R,6aR)-3-Allyl-4-methyl-1,2,3,3a,4,6a-hexahy-
dropentalen-2-yl]oxy}(tert-butyl)diphenylsilane (18)
(C-o) ppm; FT-IR (ATR):
m
= 3268 (w), 3071 (w), 2962 (m), 2929
In analogy to Ref. 19 a 1.6 M solution of n-BuLi in hexane
(w), 2856 (w), 2114 (w), 1711 (s), 1589 (w), 1472 (w), 1461 (w),
1427 (m), 1391 (w), 1373 (w), 1360 (w), 1304 (w), 1262 (vs),
1260 (s), 1175 (w), 1106 (vs), 1044 (s), 1019 (vs), 1001 (s), 940
(w), 902 (w), 861 (w), 820 (s), 798 (s), 758 (m), 740 (m),
701 (vs), 611 (m) cmꢁ1; MS (ESI): m/z = 455.20 [M+Na]+, 248.09.
HRMS (ESI): calcd for C27H32O3SiNa+ [M+Na]+ 455.2013; found
455.2007.
(0.29 mL, 460
16b (115 mg, 190
l
mol) was slowly added dropwise to a solution of
mol) in abs THF (5 mL) and TMEDA (0.5 mL)
l
at ꢁ78 °C. Then the reaction mixture was warmed to room temper-
ature and stirred for 2.5 h. A satd NaHCO3 solution (4 mL) was
added, the organic solvent removed and the remaining aqueous
layer extracted with CH2Cl2 (3 ꢂ 30 mL). The combined extracts
were dried (MgSO4) and concentrated. The residue was purified
by flash chromatography on SiO2 (hexanes/EtOAc, 10:1, Rf 0.91)
4.5. (1R,3aS,4R,5R,6aS)-5-{[tert-Butyl(diphenyl)silyl]oxy}-4-
methyl-1-(3-methylbut-2-enyl)hexahydropentalen-2(1H)-one
(13d) and (1S,3aS,5R,6R,6aS)-5-{[tert-butyl(diphenyl)silyl]oxy}-
6-methyl-1-(3-methylbut-2-enyl)hexahydropentalen-2(1H)-
one (14d)
to give 18 (67.0 mg, 85%, purity >95% by 1H NMR) as a yellowish
20
oil. [
a]
ꢁ14.6 (c = 1.0, CH2Cl2). 1H NMR (500 MHz, CDCl3): d
D
0.94 (d, J = 6.9 Hz, 3H, 4-CH3), 1.04 [s, 9H, SiC(CH3)3], 1.25 (dt,
J = 12.2, 8.6 Hz, 1H, 1-Ha), 1.61 (dt, J = 2.5, 8.9 Hz, 1H, 3a-H), 1.70
(ddt, J = 12.9, 8.6, 4.4 Hz, 1H, 3-H), 1.84 (ddd, J = 12.2, 8.6, 6.4 Hz,
1H, 1-Hb), 1.87–1.91 (m, 1H, 10-Ha), 2.36–2.42 (m, 1H, 10-Hb),
2.51–2.57 (m, 1H, 4-H), 2.80–2.87 (m, 1H, 6a-H), 3.67 (dt, J = 6.4,
8.6 Hz, 1H, 2-H), 4.90–4.98 (m, 2H, 30-H), 5.47 (dt, J = 5.5, 2.2 Hz,
According to GP1 in Ref. 19 from bis(1-phenylethyl)ammonium
chloride (213 mg, 0.81 mmol), BuLi (0.99 mL, 1.59 mmol),
Please cite this article in press as: Lutz, V.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.063

8
V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
1H, 5-H), 5.52 (dt, J = 5.5, 2.0 Hz, 1H, 6-H), 5.71 (dddd, J = 14.5, 10.1,
7.7, 6.8 Hz, 1H, 20-H), 7.34–7.38 (m, 4H, m-H, m0-H), 7.39–7.44 (m,
2H, p-H, p0-H), 7.64–7.69 (m, 4H, o-H, o0-H) ppm; 13C NMR
2,40-pentaleno[1,2-b]oxiren]-1a0-yloxy]diphenylsilane was used
without further purification.
(b) To a solution of crude tert-butyl[(1a0R,1b0S,4a0S)-hexahydro-
1a0H-spiro[1,3-dioxolane-2,40-pentaleno[1,2-b]-1a0-yloxy]diphe-
nylsilane (0.25 mmol) in THF (2 mL) at 0 °C was added BH3ꢀTHF
(0.64 mL, 0.64 mmol, 1.0 M in THF) and the reaction mixture stir-
(125 MHz, CDCl3):
d
19.2 [SiC(CH3)3], 21.5 (4-CH3), 27.0
[SiC(CH3)3], 37.0 (C-10), 39.8 (C-1), 45.5 (C-6a), 46.8 (C-4), 51.4
(C-3a), 53.7 (C-3), 78.3 (C-2), 115.5 (C-30), 127.4, 127.5 (C-m, C-
m0), 129.4, 129.5 (C-p, C-p0), 133.5 (C-6), 134.2 (C-5), 134.3, 134.7
(C-i, C-i0), 136.0 (C-o, C-o0), 137.5 (C-20) ppm; FT-IR (ATR):
~
red for 1 h. After complete conversion (GC control),
a 1 M
tris(hydroxymethyl)aminomethane hydrochloride solution (5 mL)
was added (gas formation!). The bilayer system was warmed to
room temperature and stirred for 30 min. The layers were sepa-
rated and the aqueous layer was extracted with EtOAc
(3 ꢂ 10 mL). The combined organic layers were dried (MgSO4)
and the solvent removed under vacuum. The residue was purified
by chromatography on SiO2 (hexanes/EtOAc, 4:1 ? 3:1 ? 2.5:1) to
give 21a (14 mg, 15%), 21b (38 mg, 38%) and the respective cis-
compound (19 mg, 19%). 21a: Rf 0.8 (hexanes/EtOAc, 2:1). 1H
NMR (500 MHz, CDCl3): d 1.10 (s, 9H, SiC(CH3)3), 1.53–1.59 (m,
1H, 30-Ha), 1.59–1.62 (m, 1H, 20-Ha), 1.68–1.74 (m, 1H, 60-Ha),
1.87–1.91 (m, 1H, 60-Hb), 2.10–2.16 (m, 1H, 30-Hb), 2.16–2.24 (m,
2H, 20-Hb, 6a0-H), 2.41–2.47 (m, 1H, 3a0-H), 2.78–2.80 (d,
J = 2.9 Hz, 1H, OH), 3.82–3.85 (m, 1H, 50-H), 3.87–3.94 (m, 4H,
OCH2CH2O), 4.05–4.08 (m, 1H, 40-H), 7.36–7.49 (m, 4H, o-H),
7.42–7.46 (m, 2H, p-H), 7.66–7.71 (m, 4H, m-H) ppm; 13C NMR
(125 MHz, CDCl3): d 19.4 (SiC(CH3)3), 22.3 (C-30), 27.1 (SiC(CH3)3),
31.8 (C-60), 33.6 (C-20), 44.2 (C-3a0), 46.9 (C-6a0), 64.0 (OCH2CH2O),
64.9 (OCH2CH2O), 74.0 (C-50), 77.2 (C-40), 119.4 (C-10), 127.7 (C-o),
127.8 (C-o), 129.87 (C-p), 129.94 (C-p), 133.4 (C-i), 133.6 (C-i),
~
m
= 3050 (w), 2953 (m), 2928 (m), 2859 (m), 2359 (w), 1640 (w),
1427(w), 1373 (w), 1264 (s), 1109 (s), 997 (w), 909 (m), 866 (w),
822 (w), 734 (vs), 701 (vs), 612 (m) cmꢁ1
; MS (EI): m/z
(%) = 401.2 (8) [MꢁMe]+, 359.2 (100) [Mꢁt-Bu]+, 281.1 (21),
199.1 (68); HRMS (ESI): calcd for
439.2428; found 439.2427.
C
28H36OSiNa+ [M+Na]+
4.7. tert-Butyl(diphenyl)[(3a0S,6a0S)-30,3a0,60,6a0-tetrahydro-20H-
spiro[1,3-dioxolane-2,10-pentalen]-40-yloxy]silane (20)
A solution of 19 (72.0 mg, 0.22 mmol) in THF (2 mL) was slowly
added dropwise to a solution of KHMDS (64.0 mg, 0.22 mmol) in
THF (3 mL) at ꢁ78 °C and the mixture stirred for 20 min. After
addition of TBDPSCl (76.0 lL, 72.0 mg, 0.24 mmol), the reaction
mixture was allowed to warm to room temperature and stirred
for a further 1 h. The solvent was removed under vacuum, the res-
idue taken up with pentane and filtered through Celite. The filtrate
was concentrated under vacuum and the crude product purified by
chromatography on SiO2 with hexanes/EtOAc (10:1, Rf 0.75) to give
20 (72.0 mg, 0.17 mmol, 85%). 1H NMR (500 MHz, CDCl3): d 1.03 [s,
9H, SiC(CH3)3], 1.58–1.62 (m, 2H, 20-H), 1.70–1.77 (m, 1H, 30-Ha),
1.89–1.93 (m, 1H, 30-Hb), 2.12–2.16 (m, 2H, 60-H), 2.41–2.45 (m,
1H, 6a0-H), 3.01–3.13 (m, 1H, 3a0-H), 3.75–3.80 (m, 1H, OCH2),
3.83–3.87 (m, 3H, OCH2), 4.07–4.09 (m, 1H, 50-H), 7.36–7.41 (m,
4H, o-H), 7.42–7.45 (m, 2H, p-H), 7.68–7.71 (m, 4H, m-H) ppm;
13C NMR (125 MHz, CDCl3): d 19.9 [SiC(CH3)3], 26.4 (C-30), 26.8
[SiC(CH3)3], 30.0 (C-60), 32.9 (C-20), 45.2 (C-6a0), 48.8 (C-3a0), 63.9
(OCH2), 65.2 (OCH2), 102.9 (C-50), 119.8 (C-10), 128.1 (C-o), 130.2
~
135.6 (C-o), 135.7 (C-o) ppm; FT-IR (ATR):
m = 2952 (m), 2889
(m), 2858 (m), 1472 (w), 1428 (w), 1362 (w), 1332 (w), 1213
(w), 1110 (vs), 1031 (m), 903 (vs), 854 (w), 822 (w), 729 (vs),
703 (vs), 649 (m), 614 (w), 537 (m) cmꢁ1; MS (ESI): m/z = 461.21
[M+Na]+, 439.23 [M+H]+, 361.18 [MꢁPh]+, 331.14, 317.16, 283.14,
265.12, 239.11 [MꢁTBDPS]+, 211.08, 197.06, 165.09, 155.09,
121.06; HRMS (ESI): calcd for C26H34O4SiNa+ [M+Na]+ 461.2119;
found 461.2117. 21b: Rf 0.70 (hexanes/EtOAc, 2:1). 1H NMR
(500 MHz, CDCl3): d 1.10 (s, 9H, SiC(CH3)3), 1.36–1.42 (m, 1H, 60-
Ha), 1.58–1.65 (m, 2H, 20-Ha, 30-Ha), 1.76–1.81 (m, 1H, 20-Hb),
1.89–1.98 (m, 2H, 60-Hb, 30-Hb), 2.29–2.34 (m, 1H, 6a0-H), 2.45–
2.52 (m, 1H, 3a0-H), 3.86–3.92 (m, 4H, OCH2CH2O), 3.96–4.00 (m,
2H, 40-H, 50-H), 7.36–7.40 (m, 4H, o-H), 7.41–7.44 (m, 2H, p-H),
7.66–7.72 (m, 4H, m-H) ppm; 13C NMR (125 MHz, CDCl3): d 19.4
(SiC(CH3)3), 22.6 (C-30), 27.1 (SiC(CH3)3), 30.4 (C-60), 35.1 (C-20),
42.8 (C-3a0), 44.7 (C-6a0), 64.1 (OCH2CH2O), 65.0 (OCH2CH2O),
77.4 (C-40/C-50), 81.4 (C-40/C-50), 118.7 (C-10), 127.7 (C-o), 127.8
(C-o), 129.8 (C-p), 129.9 (C-p), 133.7 (C-i), 134.5 (C-i), 135.8 (C-o),
~
(C-p), 133.4 (C-i), 135.9 (C-m) ppm; FT-IR (ATR):
m = 3071 (w),
2955 (s), 2941 (s), 2889 (m), 2858 (s), 2357 (w), 1650 (s), 1588
(w), 1472 (m), 1428 (m), 1391 (w), 1348 (s), 1302 (m), 1269 (m),
1239 (m), 1202 (s), 1183 (m), 1109 (vs), 1030 (s), 1009 (m), 946
(s), 889 (w), 861 (m), 840 (s), 822 (s), 794 (m), 741 (m), 701 (vs),
651 (w), 613 (m), 574 (w) cmꢁ1; MS (ESI): m/z = 443.20 [M+Na]+,
421.22 [M+H]+, 358.15, 304.26, 282.27, 239.11; HRMS (ESI): calcd
for C26H32O3Si [M+H]+ 420.2193; found 420.2199.
4.8. (3a0S,40R,50R,6a0S)- and (3a0S,40S,50S,6a0S)-40-{[tert-
Butyl(diphenyl)silyl]oxy}hexahydro-20H-spiro[1,3-dioxolane-
2,10-pentalen]-50-ol (21a) and (21b)
135.9 (C-o) ppm; FT-IR (ATR):
m = 2955 (m), 2889 (m), 2857 (m),
1472 (w), 1427 (w), 1362 (w), 1104 (m), 1041 (m), 942 (w), 906
(vs), 855 (m), 821 (m), 729 (vs), 701 (vs), 649 (m), 612 (m) cmꢁ1
;
MS (ESI): m/z = 439.23 [M+H]+, 361.18 [MꢁPh]+, 317.16, 299.15,
283.14, 239.11 [MꢁTBDPS]+, 235.11, 183.10, 165.09, 139.07,
121.06, 105.04; HRMS (ESI): calcd for C26H34O4SiNa+ [M+Na]+
461.2119; found 461.2125.
(a) Silyl enol ether 20 (210 mg, 0.50 mmol) was added to a solu-
tion of MeCN (2.4 mL), dimethoxymethane (4.80 mL) and stock
solution I (4.80 mL) [prepared from sodium tetraborate decahy-
drate (19.1 g, 50.0 mmol) and disodium ethylenediaminetetraace-
tate dihydrate (149 mg, 0.40 mmol) in H2O (1 L)] at 0 °C followed
by addition of Shi catalyst (50.0 mg, 0.20 mmol) and Bu4NHSO4
(61.7 mg, 0.20 mmol). To this reaction mixture ice-cold solutions
of oxone (427 mg, 0.70 mmol) in stock solution II (3 mL) [prepared
from disodium ethylenediaminetetraacetate dihydrate (149 mg,
0.40 mmol) in H2O (1 L)] and K2CO3 (402 mg, 2.80 mmol) in H2O
(3 mL) were added successively over 2 h. After complete addition,
the two-phase mixture was stirred at 0 °C for a further 3 h and
diluted with H2O (30 mL) to dissolve the precipitated salts. The lay-
ers were separated and the aqueous layer was extracted with CH2
Cl2 (3 ꢂ 30 mL). The combined organic layers were washed with
brine, dried (MgSO4) and concentrated under vacuum to 5 mL.
Crude tert-butyl[(1b0S,4a0S)-hexahydro-1a0H-spiro[1,3-dioxolane-
4.9. rac 3-But-3-enyl-2,3,3a,6a-tetrahydropentalene-1,4-dione
(23a)
To a suspension of CuCN (0.55 g, 6.16 mmol) in freshly distilled
THF was added TMEDA (0.66 mL, 0.75 g, 4.40 mmol) and the
reaction mixture cooled to ꢁ78 °C.
A solution of 3-but-
enylmagnesiumbromide, freshly prepared from Mg (0.43 g,
17.6 mmol) and 4-bromo-1-butene (0.49 mL, 0.65 g, 4.80 mmol),
was slowly added dropwise and the reaction mixture stirred for
20 min at ꢁ78 °C. Then TMSCl (0.68 mL, 0.58 g, 5.28 mmol) was
added followed by a cold solution of 22 (0.59 g, 4.40 mmol) in
THF (5 mL). After stirring for 30 min, the reaction mixture was
hydrolyzed with a mixture from a satd NH4Cl solution/25%ic NH3
Please cite this article in press as: Lutz, V.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.063

V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9
solution (10:1, 15 mL). The layers were separated and the aqueous
layer was extracted with Et2O (3 ꢂ 10 mL). A 1 N HCl solution was
added to the combined organic layers, and the mixture stirred for
30 min to hydrolyze the formed silyl enol ether. The layers were
separated and the organic layer was dried (MgSO4) and concen-
trated. The residue was purified by flash chromatography (hex-
anes/EtOAc, 4:1) to give 23a (530 mg, 2.77 mmol, 63%) as a
yellow oil. Rf 0.5 (hexanes/EtOAc, 2:1). 1H NMR (500 MHz, CDCl3):
d 1.45–1.54 (m, 1H, 7-Ha), 1.69–1.78 (m, 1H, 7-Hb), 2.05–2.20 (m,
3H, 2-Ha, 8-H), 2.30–2.38 (m, 1H, 3-H), 2.53 (ddt, J = 17.8, 8.0,
0.8 Hz, 1H, 2-Hb), 2.77 (ddt, J = 6.4, 3.6, 0.8 Hz, 1H, 3a-H), 3.59–
3.63 (m, 1H, 6a-H), 4.95–5.05 (m, 2H, 10-H), 5.72–5.82 (m, 1H, 9-
H), 6.20–6.23 (m, 1H, 5-H), 7.56–7.58 (m, 1H, 6-H) ppm; 13C
NMR (125 MHz, CDCl3): d 31.6 (C-8), 35.6 (C-7), 35.8 (C-3), 43.2
(C-2), 52.6 (C-3a), 55.7 (C-6a), 115.6 (C-10), 134.9 (C-5), 137.6
(C-9), 159.5 (C-6), 210.0 (C-4), 212.2 (C-1) ppm; FT-IR (ATR):
~
(100
acid (100
ensure
l
L) and dissolved in isopropanol containing 0.4% hydrochloric
L). The microplates were gently shaken for 20 min to
complete dissolution of the formazan and finally
l
a
measured at 595 nm using an ELISA plate reader. All experiments
were carried out in two parallel experiments. Activity values were
calculated as the mean with respect to the controls set to 100%.
4.12. Cell staining
PtK2 cells (ATCC CCL-56) were grown in 750 lL medium in
4-well plates (Nunc) on glass coverslips, and incubated with the
test compound overnight. For F-actin staining cells were fixed with
formalin (3.7%) for 10 min, permeabilized with 0.1% Triton X-100
for 5 min, and then incubated with Alexa Fluor 488 phalloidin
(1:100; Molecular Probes) for 1 h. For ER and
a-tubulin staining
cells were fixed with cold (ꢁ20 °C) MeOH/acetone (1:1) for
m
= 3076 (w), 2926 (m), 1740 (s), 1703 (vs), 1640 (m), 1580 (m),
10 min and later incubated with a primary antibody against GRP-
1452 (w), 1409 (w), 1334 (w), 1173 (m), 1073 (w), 995 (w), 912
(m), 788 (m) cmꢁ1; MS (EI, 70 eV): m/z (%) = 190.1 (2) [M]+, 162.1
(2), 148.1 (3), 135.0 (15) [MꢁC4H7], 108.1 (100) [C6H4O2], 91.1
(6), 81.1 (32), 67.1 (3), 55.0 (9); HRMS (EI): calcd for C12H14O2
[M]+ 190.0994; found 190.0989.
94 (1:1000; Affinity Bioreagents) and a-tubulin (1:100; Sigma),
respectively, and then with a secondary Alexa Fluor 488 goat
anti-rat IgG antibody (1:200; Molecular Probes) and Alexa Fluor
488 goat anti-mouse IgG antibody (1:200; Molecular Probes),
respectively, and mounted in ProLong Antifade Gold (Molecular
Probes), which included DAPI to stain the nuclei.
4.10. rac 30-But-3-enyl-20,30,3a0,6a0-tetrahydro-40H-spiro[1,3-
dioxolane-2,10-pentalen]-40-one (23b)
4.13. Click chemistry
To a solution of toluene (5.00 mL), ethylene glycol (0.10 mL,
91 mg, 1.47 mmol) and p-TsOH (13 mg, 0.07 mmol) was added
23a (140 mg, 0.74 mmol) and the reaction mixture heated at reflux
for 7 h. After cooling to room temperature, a satd NaHCO3 solution
(5 mL) was added and the layers were separated. The organic layer
was washed with brine (4 mL) and then extracted with EtOAc
(10 mL), dried (MgSO4) and the solvent removed under reduced
pressure. The residue was purified by preparative HPLC on a Kro-
PtK2 cells (ATCC CCL-56) were grown in 750
4-well plates (Nunc) on glass coverslips, and incubated with 11a
(3
g mLꢁ1) overnight. Cells were fixed with formalin (3.7%) for
lL medium in
l
10 min and permeabilized with 0.1% Triton X-100 for 5 min. Cells
were blocked with 3% FBS in PBS at 37 °C for 5 min. The Click-iT
cocktail was prepared as per the manufacturer’s protocol just prior
its usage and cells were incubated for 30 min. For co-localization
studies, further staining was carried out for F-actin using Alexa
Fluor 488 phalloidin (Ph488, 1:100 with 10% FBS, Molecular
Probes), and mounted in ProLong Antifade Gold (Molecular Probes)
without DAPI.
masil column (250 ꢂ 20 mm, 5
GmbH) with hexanes/EtOAc (4:1) to give 23b (100 mg, 0.44 mmol,
60%) as
yellow oil. Rf 0.6 (hexanes/EtOAc, 1:1). 1H NMR
lm pore size; MZ Analysentechnik
a
(500 MHz, CDCl3): d 1.54–1.65 (m, 2H, 2-Ha, 7-Ha), 1.75–1.83 (m,
1H, 7-Hb), 1.93 (dd, J = 13.3, 7.4 Hz, 1H, 2-Hb), 2.00–2.07 (m, 1H,
3-H), 2.12 (q, J = 7.4 Hz, 2H, 8-H), 2.51 (dd, J = 6.5, 4.3 Hz, 1H, 3a-
H), 3.36–3.40 (m, 1H, 6a-H), 3.86–4.05 (m, 4H, OCH2CH2O), 4.94–
4.97 (m, 1H, 10-Ha), 5.01–5.06 (m, 1H, 10-Hb), 5.78–5.87 (m, 1H,
9-H), 6.14 (ddd, J = 5.9, 2.2, 0.7 Hz, 1H, 5-H), 7.57 (ddd, J = 5.6,
2.9, 0.7 Hz, 1H, 6-H) ppm; 13C NMR (125 MHz, CDCl3): d 32.1 (C-
8), 35.0 (C-7), 38.5 (C-3), 40.7 (C-2), 54.0 (C-3a), 54.6 (C-6a),
64.7, 64.8 (OCH2CH2O), 114.9 (C-10), 116.1 (C-1), 134.2 (C-5),
~
4.14. Impedance measurement profiling
The impedance of incubated cell cultures was monitored on a
RT-CES system (xCelligence) from Acea Biosciences (Roche). The
resulting impedance curves were used for a hierarchical cluster
analysis of reference compounds together with compound of
unknown mode of action. Co-clustering of the compound of
unknown mode of action with reference compounds with known
activity class label is used to predict the mode of action. The
method was described previously.26
138.5 (C-9), 162.7 (C-6), 211.7 (C-4) ppm; FT-IR (ATR):
m = 3074
(w), 2923 (m), 1702 (vs), 1640 (m), 1585 (m), 1437 (w), 1341
(m), 1175 (m), 1113 (m), 1066 (m), 1018 (m), 947 (m), 911 (m),
884 (w), 842 (w), 798 (w), 769 (w) cmꢁ1; MS (EI, 70 eV): m/z
(%) = 234.1 (33) [M]+, 179.1 (25) [MꢁC4H7], 153.1 (100), 125.1
(6), 107.0 (31), 99.0 (8), 86.0 (19), 79.0 (10); HRMS (EI): calcd for
4.15. Tubulin polymerization assay
This assay was carried out according to manufacturer’s
protocol (Tubulin Polymerization Assay Kit, Catalog #: BK0011P;
Cytoskeleton). Tubulin master mix was prepared which included
buffer, glycerol, GTP stock (100 mM) and porcine tubulin
(10 mg mLꢁ1) according to the manufacturer’s guidelines–all were
C
14H18O3 [M]+ 234.1256; found: 234.1251.
4.11. Cytotoxicity assay
3-(4,5-Dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide
(MTT) was used to measure growth and viability of cells which
are capable of reducing it to a violet formazan product. 60 lL of
provided in the kit. 5 lL of the compounds were added to 50 lL of
tubulin mix in a 96-well plate which was immediately placed in a
thermo-regulated fluorimeter maintained at 37 °C. Fluorescence
was measured for about an hour at 460 nm (excitation: 340 nm).
serial dilutions of the test compounds were added to 120 lL
aliquots of a cell suspension (50,000 mLꢁ1) in 96-well microplates.
Blank and solvent controls were incubated under identical
conditions for 5 d. MTT in phosphate buffered saline (PBS)
4.16. Actin polymerization assay
(20
l
L) was added to a final concentration of 0.5 mg mLꢁ1. After
This assay was carried out according to manufacturer’s
protocol (Actin Polymerization Biochem Kit, Catalog #: BK003;
Cytoskeleton). According to the manufacturer’s guidelines buffer
2 h, the precipitate of formazan crystals was centrifuged, and the
supernatant discarded. The precipitate was washed with PBS
Please cite this article in press as: Lutz, V.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.063

10
V. Lutz et al. / Bioorg. Med. Chem. xxx (2014) xxx–xxx
9. (a) Huguet, J.; Karpf, M.; Dreiding, A. S. Tetrahedron Lett. 1983, 24, 4177; (b)
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Acknowledgements
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Generous financial support by the Deutsche Forschungsge-
meinschaft, the Ministerium für Wissenschaft, Forschung und
Kunst des Landes Baden-Württemberg (Landesgraduierten fellow-
ship for N.P.), the DAAD (fellowship for N.P.) and the Fonds der
Chemischen Industrie is gratefully acknowledged.
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Supplementary data
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Please cite this article in press as: Lutz, V.; et al. Bioorg. Med. Chem. (2014), http://dx.doi.org/10.1016/j.bmc.2014.04.063