1
SAR s udies on hyd open alene de i a i es – impo an co e uni s o
biologically ac i e e amic acid mac olac ams and p ychanolides
Vanessa Lu za, Fabian Mannchena, Michael K ebsa, Na ja Pa ka, Claudia K üge a, A una
Rajab, Flo enz Sasseb,*, Angelika Ba oa, Sabine Lascha a,*
a Ins i u ü O ganische Chemie de Uni e si ä S u ga , P a enwald ing 55, 70569 S u ga , Ge many
b Depa men o Chemical Biology, Helmhol z Cen e o In ec ion Resea ch, Inho ens . 7, 38124 B aun-
schweig, Ge many
ABSTRACT
S uc u ally di e se bicyclo[3.3.0]oc anes we e p epa ed and es ed o hei biological
ac i i y. Bo h he an ip oli e a i e ac i i y and he esul s o pheno ypic cha ac e iza ion
a ied wi h he subs i u ion pa e ns. Two de i a i es displayed high inhibi o y (IC50 3 µM)
ac i i y agains he L-929 cell line, bu di e ed in hei mode o ac ion. A clus e analysis
wi h impedance p o iling da a showed he wo compounds in ela ionship o mic o ubule
in e e ing compounds. In P K2 cells ea ed wi h bo h de i a i es a pe u bing e ec on he
mic o ubula ne wo k was obse ed, whe eas he ac in cy oskele on in incuba ed P K2 cells
was dis u bed only by one compound. The e ec s on ubulin and ac in polyme iza ion could
be con i med by in i o polyme iza ion expe imen s.
Keywo ds: Biological ac i i y, Click chemis y, Hyd open alene, S uc u e-ac i i y
ela ionships, Syn hesis
* Co esponding au ho s. Tel.: +49 711 685 64565, ax: +49 711 685 64285 (S.L.); el.: +49
531 6181 3429; ax: +49 531 6181 3499 (F.S.).
E-mail add esses: [email p o ec ed] (S. Lascha ),
[email p o ec ed] (F. Sasse).
2
1. In oduc ion
Highly subs i u ed bicyclo[3.3.0]oc anes (hyd open alenes) ep esen he co e uni o
se e al biologically ac i e na u al p oduc amilies such as e amic acid mac olac ams con-
aining, o example, cylind amide (1),1,2 al e amide A (2),3 geodin A (3),4,5 and abu a ubo-
lac am A (4),6,7 o p ychanolides 5, 68,9 (Figu e 1).
Figu e 1. Hyd open alene co e con aining na u al p oduc s 1–6.
The class o e amic acid lac ams displays a a ie y o biological ac i i ies. Cylind amide (1),
isola ed om he ma ine sponge Halichond ia cylind a a in 1989, o example, exhibi s p o-
nounced cy o oxici y agains B16 melanoma cells.1 Al e amide A (2)3 om a bac e ium
Al e omonas sp. associa ed wi h he sponge Halichond ia okadai is cy o oxic agains epide -
mal ca cinoma cell lines. Cylind amide's close ela i e geodin A (3) being isola ed as Mg2+
sal in 1999 om he Sou he n Aus alian ma ine sponge Geodia,4 di e s om 1 only in he
exocyclic me hylene g oup ins ead o me hyl. Biological s udies e ealed i s s ong nema o-
cidal ac i i y agains he pa asi ic nema ode Haemonchus con o us. Abu a ubolac am A (4)
isola ed in 1996 om he ma ine bac e ium S ep omyces sp. SCRC A-206 inhibi s he o -
ma ion o anionic supe oxides which a e ele an in bo h in lamma o y and degene a i e p o-
cesses and umo p omo ion.10 Whe eas na u al p oduc s 1–4 a e o ma ine o igin, a bicyclo-
[3.3.0]oc ane skele on has also been de ec ed in e es ial sou ces. Fo example, he sesqui-
e penoids p ychanolide 5 and 6 ha e been isola ed in 1981 om he li e wo P ychan hus
s ia us (Lehm. e Lindenb.) Nees.8
3
Besides some ini ial expe imen s on he biological ac i i y o hese na u al p oduc classes,
howe e , only limi ed sys ema ic s uc u e-ac i i y ela ionship (SAR) s udies ha e been ca -
ied ou . The in luence o he e amic acid moie y on cy o oxici y and an imic obial p ope -
ies o mac ocyclic acyl e ama es has been in es iga ed by Schobe .11 P e iously we aimed
owa ds elucida ion o he mode o ac ion o cylind amide (1).12 Fo his pu pose a se ies o
de i a i es o 1, including analogues whe e a simple cyclopen ane eplaced he unc ionalized
hyd open alene moie y, we e p epa ed. A b oad biological sc eening e ealed p omising an i-
p oli e a i e ac i i y agains se e al umo cell lines. The cy o oxici y o 1 was ound o be
calcium dependen and we obse ed acuolisa ion and esicle o ma ion in he endoplasmic
e iculum o P K2 (po o oo kidney) cells ha we e incuba ed wi h 1. A dec ease o ac i i y o
he cyclopen ane analogue o cylind amide (1) indica ed ha he ac i i y is s ongly co ela ed
o he in ac unc ionalized hyd open alene sys em.
SAR s udies o subs i u ed bicyclo[3.3.0]oc anes, howe e , a e a ely epo ed. Mos o
hem ocused on o ally ac i e PDE4 inhibi o s,13 ca bacycline de i a i es such as TXA2/
PGH2 ecep o an agonis s and p os aglandin analogues,14 o mammalian squalene syn hase
inhibi o s.15 The e o e, we we e mo i a ed o in es iga e he biological p ope ies o he
hyd open alene subuni in mo e de ail. Besides s udying analogues o he co e uni s o e a-
mic acid lac ams we we e in e es ed in a mo e gene al insigh in o he biological p ope ies o
a ious unc ionalized bicyclo[3.3.0]oc anes A–C. Ou esul s a e epo ed below.
2. Resul s and discussion
2.1. Chemis y
In o de o supply compounds o SAR s udies, a se ies o bicyclo[3.3.0]oc anes was p e-
pa ed om he easily accessible Weiss dike one (7a),16 i s dime hyl analogue 7b16 and he
known cylind amide co e p ecu so 82b (Figu e 2) leading o de i a i es o s uc u al
di e si y.
Figu e 2. S a ing ma e ials and posi ions o unc ionaliza ion leading o s uc u es A–C.
4
The hyd open alene de i a i es 11–18 a e based on Weiss dike one (7a) (Scheme 1). The
syn hesis o alkynes 11a and 11b which we e en isaged o "click chemis y" possibly allow-
ing isualiza ion o he cellula a ge , commenced wi h he educ ion o monoke al 9a17,18 o
gi e 10.18 The la e was ei he eac ed wi h p opiolic acid unde Mi sunobu condi ions o 11a
o con e ed in h ee s eps o 12 which was es e i ied analogously o 11b. Di ec es e i ica ion
o alcohol 10 wi h p opionyl chlo ide ga e de i a i e 11c. Alkenes 17 and 18, p o iding key
s uc u al mo i s o he bicyclo[3.3.0]oc ene co e o he na u al p oduc s 1–4, we e p epa ed
om monoke al 9a by a sequence o double dep o ona ion/elec ophilic apping ollowed by
swapping o p o ec ing g oups o yield compounds 13, 14. De i a i es 13b, 14b we e submi -
ed o Shapi o eac ion o gi e 15b, 16b, which we e subsequen ly unc ionalized o 17, 18.19
Scheme 1. Reagen s and condi ions: (a) 4.0 equi PPh3, 4.0 equi p opiolic acid, 4.0 equi
DEAD, 1.0 equi 10 o 12, E 2O, . ., 24 h, 40% o 11a, 37% o 11b; (b) 1.0 equi 5-{[ e -
bu yl(diphenyl)silyl]oxy}hexahyd open alen-2(1H)-one,18 3.0 equi NaBH4, MeOH, 0 °C, 3
h, quan .; (c) 1.0 equi 9a, 2.0 equi p opionyl chlo ide, E 3N, CH2Cl2, 0 °C, 14 h, 73%.
Numbe ing o NMR assignmen .
The syn hesis o hyd open alenes 21–23 based on bicyclo[3.3.0]oc ane-1,4-dione 82b is shown
in Scheme 2. Monoke al 19 was con e ed o silyl enol e he 20 which was submi ed o a Shi
epoxida ion.20 Reduc i e epoxide opening o he esul ing in e media e wi h BH3·THF p o ided
he ans con igu ed hyd open alenes 21a and 21b. Dienedione 222b yielded he alkyla ed bi-
cyclo[3.3.0]oc ene-1,4-dione 23a by Cu-ca alyzed G igna d eac ion wi h 3-bu enylmagnesium-
5
b omide in he p esence o TMSCl. Ke aliza ion o 23a wi h e hylene glycol ga e he monoke al
23b (Scheme 2).
Scheme 2. Reagen s and condi ions: (a) 1.0 equi KHMDS, 1.0 equi 19, THF, –78 °C, 20
min, 1.1 equi TBDPSCl, –78 °C o . ., 3 h; (b) aq. Na2B4O7·10H2O/Na2EDTA·2H2O, 0.3
equi Shi ca alys ,20 0.04 equi Bu4NHSO4, 1.4 equi oxone in aq. Na2EDTA·2H2O, 5.8
equi K2CO3, 3 h; (c) 2.5 equi BH3·THF, THF, 3 h, 15% o 21a, 38% o 21b; (d) 1.4 equi
CuCN, 1.0 equi TMEDA, 1.1 equi 3-bu enylmagnesiumb omide, THF, –78 °C, 1.2 equi
TMSCl, 1.0 equi 22, 30 min, 63%; (e) 2.0 equi e hylene glycol, 0.09 equi p-TsOH, e lux,
7 h, 60%. Only one dias e eome o 23a was de ec ed, i s con igu a ion was de e mined by
NOESY (see supplemen a y da a). Numbe ing o NMR assignmen .
Hyd open alenes o he p ychanolide se ies we e p epa ed om 3a,6a-dime hyl e ahyd o-
pen alene-2,5(1H,3H)-dione (7b)16 ollowing known p ocedu es17,18b,21 (Scheme 3).
Scheme 3. Reagen s and condi ions: (a) 2,2-dime hyl-1,3-p opanediol, p-TsOH in oluene,
e lux, 2 h;18b (b) 1. n-BuLi/bis[(R,R)-1-phenyle hyl]ammonium chlo ide, –100 °C; hen
–78 °C, ClSiE 3; 2. MeLi, MeI acco ding o e .;17 (c) 3 equi NaBH4, 1 equi 24, MeOH,
0 °C, 15 min; (d) 1 equi 25a,b in DMF, 1.2 equi TBDPSCl, 2.5 equi imidazole acco ding
o e .;18b (e) ca aly ic amoun s o p-TsOH acco ding o e .21
The a io o he dias e eome ic ke ones 24a and 24b depended on he empe a u e. A oom
empe a u e, 24a,b we e isola ed in 87% yield wi h d 39:61 whe eas he eac ion a –20 °C
p o ided 24a,b quan i a i ely wi h d 85:15. The ob ained enan ioselec i i y (10–12% ee)

6
could no be imp o ed by apping he in e media e enola e as silyl enol e he p io o
alkyla ion as p e iously epo ed.17 P esumably he s e ic bias o he axial me hyl g oups on
op o he con ex bicyclic oo in e e ed wi h he enan iodisc imina ion o he chi al li hium
amide base a he ke one moie y. Ne e heless, ke ones 24 we e educed o alcohols 25a,b
which we e p o ec ed o yield silyl e he s 25c,d. Final acidic ke al clea age p o ided ke ones
26a,b.
2.2. Biological s udies – p oli e a ion es s
The abili y o he se o nea ly 30 hyd open alene de i a i es o inhibi he p oli e a ion o
a ious ans o med cell lines including human umo cell lines was in es iga ed in an MTT
assay.22 The esul s a e summa ized in Tables 1 and 2.
Table 1
Cy o oxici y o bicyclo[3.3.0]oc anes agains di e en ans o med cell linesa
En y
Compounds
L-929 (mouse)
connec i e issue
IC50 (µM)
KB-3-1 (human)
ce ix ca cinoma
IC50 (µM)
1
11a
2.2
21.6
2
11b
9.3
–
3
11c
>142
>142
4
13a
7.4
15.2
5
13b
32.4
16.0
6
13c
17.8
6.9
7
13d
23.9
28.2
8
14a
14.8
7.4
9
14b
16.2
20.3
10
14c
5.1
16.2
11
14d
5.6
21.7
12
15b
20.0
13.3
13
16b
>67
12.5
14
17
20.4
>96
15
18
7.2
>96
16
21a
41.0
–
17
21b
38.8
–
18
22
3.0
6.0
19
23a
20.0
16.3
20
23b
25.6
32.0
a Fu he compounds in supplemen a y da a. IC50 alues a e means o wo assays in pa allel.
7
As shown in Table 1, he an ip oli e a i e ac i i ies o mos compounds a ied in a
mic omola ange. Excep ions we e he alkyne 11a and dienedione 22 showing p omising
po encies (IC50 3 ) agains L-929 mouse ib oblas s. The cy o oxici y o es e -subs i u ed
hyd open alenes 11 was ound o depend s ongly on he es e unc ion. In con as o bo h
p opiola es 11a and 11b, he co esponding p opiona e 11c was comple ely inac i e. We we e
wo ied ha he cy o oxici y o he p opiola e de i a i es 11a,b migh be due o a subsequen
hyd olysis p oduc gene a ed by es e ase ac i i y a he han caused by 11a and 11b
hemsel es. The e o e, he co esponding pen alene alcohol was s udied in compa ison
(supplemen a y da a), bu no cy o oxici y was obse ed o he alcohol, indica ing ha indeed
he in ac es e seemed o be he ac i e species. Bicyclo[3.3.0]oc enes 17 and 1819 displayed
cy o oxici ies (IC50 = 7.2–20.4 ) agains L-929 mouse ib oblas s bu poo inhibi o y (IC50
>96 ) ac i i y agains he KB-3-1 cell line (Table 1, En ies 14, 15). Unexpec edly, silyl-
p o ec ion had a signi ican e ec on he an ip oli e a i e ac i i y. The unp o ec ed analogues
o 13, 14 and 21 we e comple ely inac i e agains all es ed cell lines (supplemen a y da a).
A simila e ec was obse ed o unp o ec ed p ychanolide de i a i es 25a,b and hei
silyl-p o ec ed coun e pa s 25c,d (Table 2, En ies 2–5). In o de o s udy he in luence o he
silyl p o ec ing g oup, he cy o oxic e ec o O-silyla ed cyclopen anols23–25 o he wo cell
lines was in es iga ed. We obse ed an inc eased ac i i y in he o de o silyl p o ec ing
g oups TBS < TIPS < TBDPS (Table 2, En ies 8–11).
Table 2
Cy o oxici y o 3a,6a-dime hylhyd open alenes 24–26 and cyclopen anols agains di e en
ans o med cell linesa
En y
Compound
L-929
IC50 (µM)
KB-3-1
IC50 (µM)
1
24a
>150
–
2
25a
>150
–
3
25b
>150
–
4
25c
15.8
–
5
25d
11.8
–
6
26a
14.3
–
7
26b
35.7
–
8
-bu yl(cyclopen yloxy)dime hylsilane
>200
>200
9
(cyclopen yloxy)( iisop opyl)silane
111.4
123.7
10
-bu yl(cyclopen yloxy)diphenylsilane
61.6
–
a IC50 alues a e means o wo assays in pa allel.
8
2.3. Biological s udies – a ge iden i ica ion
Alkyne 11a and dihyd open alenedione 22 wi h he highes po ency we e selec ed o
u he biological s udies. In o de o ge hin s abou he mode o ac ion we employed a newly
de eloped impedance p o iling me hod which uses ime-dependen impedance cu es as a
inge p in o he mode o ac ion.26 In L-929 cell cul u es ha we e incuba ed wi h 11a and
22, impedance was moni o ed o e ime (Figu e 3).
Figu e 3. Impedance cu es o L-929 cells incuba ed wi h 11a (0.6 µg mL–1) and 22 (0.4 µg
mL–1). Tubulysin B (3 ng mL–1) was used as one o he e e ence compounds.
The ob ained cu es we e compa ed wi h hose o a se o e e ence compounds using s a is i-
cal me hods. Clus e analysis o he esul ing da a showed 11a and 22 in a g oup o e e ence
compounds such as nocodazole, ubulysin B and g iseo ul in ha in e e e wi h mic o ubule
polyme iza ion and spindle o ma ion (Figu e 4).
Figu e 4. Clus e analysis o da a ob ained by impedance moni o ing o incuba ed cell cul-
u es. De i a i es 11a (c9a) and 22 (c19) a e ela ed o compounds ha in e e e wi h ubulin
polyme iza ion and mi o ic spindle o ma ion.
9
Based on he esul s ob ained om he impedance p o iling, P K2 (po o oo kidney) cells
which showed in MTT assays an IC50 o app oxima ely 3 µM wi h 11a and 22, espec i ely,
we e incuba ed wi h bo h compounds and s ained o e ec s on mic o ubules (Figu e 5). Fo
bo h 11a and 22 a pe u bing e ec on he mic o ubula ne wo k in he cells and al e ed
mi o ic spindles oge he wi h an unusual dis ibu ion o ch omosomes was isible.
Figu e 5. E ec o 11a and 22 on he mic o ubule cy oskele on and mi o ic spindle. P K2
cells we e ea ed wi h 11a (4 µg mL–1; C) and 22 (4 µg mL–1; D) o e nigh and s ained o
mic o ubules (g een) and DNA (blue). Con ol cells we e incuba ed wi h me hanol only (A
and B).
To u he e i y he e ec o 11a and 22, ubulin polyme iza ion assays we e ca ied ou
in i o (Figu e 6). The e ec o 11a and 22 was compa ed o ha o me hanol and noco-
dazole, a known inhibi o o ubulin polyme iza ion.27 The basis o his assay is he inco po a-
ion o a luo escen epo e in o he de eloping mic o ubules. While nocodazole is only inhi-
bi ing he polyme iza ion p ocess, 11a and 22 induce a dec ease in luo escence. The e ec o
22 was much s onge han ha o 11a, which s ill allowed a delayed ubulin polyme iza ion.
A
D
C
B
16
o 2.5 h. A sa d NaHCO3 solu ion (4 mL) was added, he o ganic sol en emo ed and he
emaining aqueous laye ex ac ed wi h CH2Cl2 (3  30 mL). The combined ex ac s we e
d ied (MgSO4) and concen a ed. The esidue was pu i ied by lash ch oma og aphy on SiO2
(hexanes/E OAc, 10 : 1, R 0.91) o gi e 18 (67.0 mg, 85%, pu i y >95 % by 1H NMR) as a
yellowish oil.
20
D
]α[
–14.6 (c = 1.0, CH2Cl2). 1H NMR (500 MHz, CDCl3): δ 0.94 (d, J =
6.9 Hz, 3H, 4-CH3), 1.04 [s, 9H, SiC(CH3)3], 1.25 (d , J = 12.2, 8.6 Hz, 1H, 1-Ha), 1.61 (d , J
= 2.5, 8.9 Hz, 1H, 3a-H), 1.70 (dd , 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, 1’-Ha), 2.36–2.42 (m, 1H, 1’-Hb), 2.51–2.57 (m, 1H, 4-
H), 2.80–2.87 (m, 1H, 6a-H), 3.67 (d , J = 6.4, 8.6 Hz, 1H, 2-H), 4.90–4.98 (m, 2H, 3’-H),
5.47 (d , J = 5.5, 2.2 Hz, 1H, 5-H), 5.52 (d , J = 5.5, 2.0 Hz, 1H, 6-H), 5.71 (dddd, J = 14.5,
10.1, 7.7, 6.8 Hz, 1H, 2’-H), 7.34–7.38 (m, 4H, m-H, m’-H), 7.39–7.44 (m, 2H, p-H, p’-H),
7.64–7.69 (m, 4H, o-H, o’-H) ppm; 13C NMR (125 MHz, CDCl3): δ 19.2 [SiC(CH3)3], 21.5
(4-CH3), 27.0 [SiC(CH3)3], 37.0 (C-1’), 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-3’), 127.4, 127.5 (C-m, C-m’), 129.4, 129.5 (C-p, C-p’), 133.5 (C-
6), 134.2 (C-5), 134.3, 134.7 (C-i, C-i’), 136.0 (C-o, C-o’), 137.5 (C-2’) ppm; FT-IR (ATR):
ν
~
= 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 ( s), 701 ( s), 612 (m) cm–1; MS (EI):
m/z (%) = 401.2 (8) [M – Me]+, 359.2 (100) [M – -Bu]+, 281.1 (21), 199.1 (68); HRMS
(ESI): calcd. o C28H36OsiNa+ [M + Na]+ 439.2428; ound 439.2427.
4.7. e -Bu yl(diphenyl)[(3a’S,6a’S)-3’,3a’,6’,6a’- e ahyd o-2’H-spi o[1,3-dioxolane-
2,1’-pen alen]-4’-yloxy]silane (20)
A solu ion o 19 (72.0 mg, 0.22 mmol) in THF (2 mL) was slowly added d opwise o a
solu ion o KHMDS (64.0 mg, 0.22 mmol) in THF (3 mL) a –78 °C and he mix u e s i ed
o 20 min. A e addi ion o TBDPSCl (76.0 µL, 72.0 mg, 0.24 mmol), he eac ion mix u e
was allowed o wa m o oom empe a u e and s i ed o a u he 1 h. The sol en was e-
mo ed unde acuum, he esidue aken up wi h pen ane and il e ed h ough Celi e. The il-
a e was concen a ed unde acuum and he c ude p oduc pu i ied by ch oma og aphy on
SiO2 wi h hexanes/E OAc (10:1, R 0.75) o gi e 20 (72.0 mg, 0.17 mmol, 85%). 1H NMR
(500 MHz, CDCl3):

1.03 [s, 9H, SiC(CH3)3], 1.58–1.62 (m, 2H, 2’-H), 1.70–1.77 (m, 1H,
3’-Ha), 1.89–1.93 (m, 1H, 3’-Hb), 2.12–2.16 (m, 2H, 6’-H), 2.41–2.45 (m, 1H, 6a’-H), 3.01–
3.13 (m, 1H, 3a’-H), 3.75–3.80 (m, 1H, OCH2), 3.83–3.87 (m, 3H, OCH2), 4.07–4.09 (m, 1H,
5’-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):

19.9 [SiC(CH3)3], 26.4 (C-3’), 26.8 [SiC(CH3)3], 30.0 (C-6’),

17
32.9 (C-2’), 45.2 (C-6a’), 48.8 (C-3a’), 63.9 (OCH2), 65.2 (OCH2), 102.9 (C-5’), 119.8 (C-
1’), 128.1 (C-o) , 130.2 (C-p), 133.4 (C-i), 135.9 (C-m) ppm; FT-IR (ATR):
ν
~
= 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 ( s), 1030 (s),
1009 (m), 946 (s), 889 (w), 861 (m), 840 (s), 822 (s), 794 (m), 741 (m), 701 ( s), 651 (w), 613
(m), 574 (w) cm–1; MS (ESI): m/z = 443.20 [M + Na]+, 421.22 [M + H]+, 358.15, 304.26,
282.27, 239.11; HRMS (ESI): calcd. o C26H32O3Si [M + H]+ 420.2193; ound 420.2199.
4.8. (3a’S,4’R,5’R,6a’S)- and (3a’S,4’S,5’S,6a’S)-4’-{[ e -Bu yl(diphenyl)silyl]oxy}hexa-
hyd o-2’H-spi o[1,3-dioxolane-2,1’-pen alen]-5’-ol (21a) and (21b)
a) Silyl enol e he 20 (210 mg, 0.50 mmol) was added o a solu ion o MeCN (2.4 mL), di-
me hoxyme hane (4.80 mL) and s ock solu ion I (4.80 mL) [p epa ed om sodium e abo a e
decahyd a e (19.1 g, 50.0 mmol) and disodium e hylenediamine e aace a e dihyd a e (149
mg, 0.40 mmol) in H2O (1 L)] a 0 °C ollowed by addi ion o Shi ca alys (50.0 mg, 0.20
mmol) and Bu4NHSO4 (61.7 mg, 0.20 mmol). To his eac ion mix u e ice-cold solu ions o
oxone (427 mg, 0.70 mmol) in s ock solu ion II (3 mL) [p epa ed om disodium e hylenedi-
amine e aace a e dihyd a e (149 mg, 0.40 mmol) in H2O (1 L)] and K2CO3 (402 mg, 2.80
mmol) in H2O (3 mL) we e added successi ely o e 2 h. A e comple e addi ion, he wo-
phase mix u e was s i ed a 0 °C o a u he 3 h and dilu ed wi h H2O (30 mL) o dissol e
he p ecipi a ed sal s. The laye s we e sepa a ed and he aqueous laye was ex ac ed wi h
CH2Cl2 (3  30 mL). The combined o ganic laye s we e washed wi h b ine, d ied (MgSO4)
and concen a ed unde acuum o 5 mL. C ude e -bu yl[(1b’S,4a’S)-hexahyd o-1a’H-
spi o[1,3-dioxalane-2,4’-pen aleno[1,2-b]oxi en]-1a’-yloxy]diphenylsilane was used wi hou
u he pu i ica ion.
b) To a solu ion o c ude e -bu yl[(1a’R,1b’S,4a’S)-hexahyd o-1a’H-spi o[1,3-dioxolane-
2,4’-pen aleno[1,2-b]-1a’-yloxy]diphenylsilane (0.25 mmol) in THF (2 mL) a 0 °C was ad-
ded BH3·THF (0.64 mL, 0.64 mmol, 1.0 M in THF) and he eac ion mix u e s i ed o 1 h.
A e comple e con e sion (GC con ol), a 1 M is(hyd oxyme hyl)aminome hane hyd ochlo-
ide solu ion (5 mL) was added (gas o ma ion!). The bilaye sys em was wa med o oom
empe a u e and s i ed o 30 min. The laye s we e sepa a ed and he aqueous laye was ex-
ac ed wi h E OAc (3  10 mL). The combined o ganic laye s we e d ied (MgSO4) and he
sol en emo ed unde acuum. The esidue was pu i ied by ch oma og aphy on SiO2 (hex-
anes/E OAc, 4:13:12.5:1) o gi e 21a (14 mg, 15%), 21b (38 mg, 38%) and he espec-
18
i e cis-compound (19 mg, 19%). 21a: R 0.8 (hexanes/E OAc, 2:1). 1H NMR (500 MHz,
CDCl3):

1.10 (s, 9H, SiC(CH3)3), 1.53–1.59 (m, 1H, 3’-Ha), 1.59–1.62 (m, 1H, 2’-Ha), 1.68–
1.74 (m, 1H, 6’-Ha), 1.87–1.91 (m, 1H, 6’-Hb), 2.10–2.16 (m, 1H, 3’-Hb), 2.16–2.24 (m, 2H,
2’-Hb, 6a’-H), 2.41–2.47 (m, 1H, 3a’-H), 2.78–2.80 (d, J = 2.9 Hz, 1H, OH), 3.82–3.85 (m,
1H, 5’-H), 3.87–3.94 (m, 4H, OCH2CH2O), 4.05–4.08 (m, 1H, 4’-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):

19.4
(SiC(CH3)3), 22.3 (C-3’), 27.1 (SiC(CH3)3), 31.8 (C-6’), 33.6 (C-2’), 44.2 (C-3a’), 46.9 (C-
6a’), 64.0 (OCH2CH2O), 64.9 (OCH2CH2O), 74.0 (C-5’), 77.2 (C-4’), 119.4 (C-1’), 127.7 (C-
o), 127.8 (C-o), 129.87 (C-p), 129.94 (C-p), 133.4 (C-i), 133.6 (C-i), 135.6 (C-o), 135.7 (C-o)
ppm; FT-IR (ATR):
ν
~
= 2952 (m), 2889 (m), 2858 (m), 1472 (w), 1428 (w), 1362 (w), 1332
(w), 1213 (w), 1110 ( s), 1031 (m), 903 ( s), 854 (w), 822 (w), 729 ( s), 703 ( s), 649 (m),
614 (w), 537 (m) cm–1; MS (ESI): m/z = 461.21 [M + Na]+, 439.23 [M + H]+, 361.18 [M –
Ph]+, 331.14, 317.16, 283.14, 265.12, 239.11 [M – TBDPS]+, 211.08, 197.06, 165.09,
155.09, 121.06; HRMS (ESI): calcd. o C26H34O4SiNa+ [M + Na]+ 461.2119; ound
461.2117. 21b: R 0.70 (hexanes/E OAc, 2:1). 1H NMR (500 MHz, CDCl3):

1.10 (s, 9H,
SiC(CH3)3), 1.36–1.42 (m, 1H, 6’-Ha), 1.58–1.65 (m, 2H, 2’-Ha, 3’-Ha), 1.76–1.81 (m, 1H, 2’-
Hb), 1.89–1.98 (m, 2H, 6’-Hb, 3’-Hb), 2.29–2.34 (m, 1H, 6a’-H), 2.45–2.52 (m, 1H, 3a’-H),
3.86–3.92 (m, 4H, OCH2CH2O), 3.96–4.00 (m, 2H, 4’-H, 5’-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):

19.4
(SiC(CH3)3), 22.6 (C-3’), 27.1 (SiC(CH3)3), 30.4 (C-6’), 35.1 (C-2’), 42.8 (C-3a’), 44.7 (C-
6a’), 64.1 (OCH2CH2O), 65.0 (OCH2CH2O), 77.4 (C-4’/C-5’), 81.4 (C-4’/C-5’), 118.7 (C-1’),
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),
135.9 (C-o) ppm; FT-IR (ATR):
ν
~
= 2955 (m), 2889 (m), 2857 (m), 1472 (w), 1427 (w),
1362 (w), 1104 (m), 1041 (m), 942 (w), 906 ( s), 855 (m), 821 (m), 729 ( s), 701 ( s), 649
(m), 612 (m) cm–1; MS (ESI): m/z = 439.23 [M + H]+, 361.18 [M – Ph]+, 317.16, 299.15,
283.14, 239.11 [M – TBDPS]+, 235.11, 183.10, 165.09, 139.07, 121.06, 105.04; HRMS
(ESI): calcd. o C26H34O4SiNa+ [M + Na]+ 461.2119; ound 461.2125.
4.9. ac 3-Bu -3-enyl-2,3,3a,6a- e ahyd open alene-1,4-dione (23a)
To a suspension o CuCN (0.55 g, 6.16 mmol) in eshly dis illed THF was added TMEDA
(0.66 mL, 0.75 g, 4.40 mmol) and he eac ion mix u e cooled o –78 °C. A solu ion o 3-bu-
enylmagnesiumb omide, eshly p epa ed om Mg (0.43 g, 17.6 mmol) and 4-b omo-1-bu-
ene (0.49 mL, 0.65 g, 4.80 mmol), was slowly added d opwise and he eac ion mix u e
s i ed o 20 min a –78 °C. Then TMSCl (0.68 mL, 0.58 g, 5.28 mmol) was added ollowed
19
by a cold solu ion o 22 (0.59 g, 4.40 mmol) in THF (5 mL). A e s i ing o 30 min, he
eac ion mix u e was hyd olyzed wi h a mix u e om a sa d. NH4Cl solu ion/25%ic NH3
solu ion (10:1, 15 mL). The laye s we e sepa a ed and he aqueous laye was ex ac ed wi h
E 2O (3  10 mL). A 1 N HCl solu ion was added o he combined o ganic laye s, and he
mix u e s i ed o 30 min o hyd olyze he o med silyl enol e he . The laye s we e sepa a ed
and he o ganic laye was d ied (MgSO4) and concen a ed. The esidue was pu i ied by lash
ch oma og aphy (hexanes/ E OAc, 4:1) o gi e 23a (530 mg, 2.77 mmol, 63%) as a yellow
oil. R 0.5 (hexanes/E OAc, 2:1). 1H NMR (500 MHz, CDCl3): δ 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 (dd , J
= 17.8, 8.0, 0.8 Hz, 1H, 2-Hb), 2.77 (dd , 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): δ 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):
ν
~
= 3076 (w), 2926 (m), 1740 (s), 1703 ( s), 1640
(m), 1580 (m), 1452 (w), 1409 (w), 1334 (w), 1173 (m), 1073 (w), 995 (w), 912 (m), 788 (m)
cm–1; MS (EI, 70 eV): m/z (%) = 190.1 (2) [M]+, 162.1 (2), 148.1 (3), 135.0 (15) [M – C4H7],
108.1 (100) [C6H4O2], 91.1 (6), 81.1 (32), 67.1 (3), 55.0 (9); HRMS (EI): calcd. o C12H14O2
[M]+ 190.0994; ound 190.0989.
4.10. ac 3’-Bu -3-enyl-2’,3’,3a’,6a’- e ahyd o-4’H-spi o[1,3-dioxolane-2,1’-pen alen]-
4’-one (23b)
To a solu ion o oluene (5.00 mL), e hylene 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 he eac ion mix u e
hea ed a e lux o 7 h. A e cooling o oom empe a u e, a sa d. NaHCO3 solu ion (5 mL)
was added and he laye s we e sepa a ed. The o ganic laye was washed wi h b ine (4 mL)
and hen ex ac ed wi h E OAc (10 mL), d ied (MgSO4) and he sol en emo ed unde
educed p essu e. The esidue was pu i ied by p epa a i e HPLC on a K omasil column (250
 20 mm, 5 m po e size; MZ Analysen echnik GmbH) wi h hexanes/E OAc (4:1) o gi e
23b (100 mg, 0.44 mmol, 60%) as a yellow oil. R 0.6 (hexanes/E OAc, 1:1). 1H NMR (500
MHz, CDCl3): δ 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): δ
20
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), 138.5 (C-9), 162.7 (C-6), 211.7 (C-4)
ppm; FT-IR (ATR):
ν
~
= 3074 (w), 2923 (m), 1702 ( s), 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) cm–1; MS (EI, 70 eV): m/z (%) = 234.1 (33) [M]+, 179.1 (25) [M – C4H7], 153.1
(100), 125.1 (6), 107.0 (31), 99.0 (8), 86.0 (19), 79.0 (10); HRMS (EI): calcd. o C14H18O3
[M]+ 234.1256; ound: 234.1251.
4.11. Cy o oxici y assay
3-(4,5-Dime hyl hiazol-2-yl)-2,5-diphenyl e azolium b omide (MTT) was used o measu e
g ow h and iabili y o cells which a e capable o educing i o a iole o mazan p oduc . 60
µL o se ial dilu ions o he es compounds we e added o 120 µL aliquo s o a cell suspen-
sion (50 000 mL–1) in 96-well mic opla es. Blank and sol en con ols we e incuba ed unde
iden ical condi ions o 5 d. MTT in phospha e bu e ed saline (PBS) (20 µL) was added o a
inal concen a ion o 0.5 mg mL–1. A e 2 h, he p ecipi a e o o mazan c ys als was cen i-
uged, and he supe na an disca ded. The p ecipi a e was washed wi h PBS (100 µL) and
dissol ed in isop opanol con aining 0.4% hyd ochlo ic acid (100 µL). The mic opla es we e
gen ly shaken o 20 min o ensu e a comple e dissolu ion o he o mazan and inally
measu ed a 595 nm using an ELISA pla e eade . All expe imen s we e ca ied ou in wo
pa allel expe imen s. Ac i i y alues we e calcula ed as he mean wi h espec o he con ols
se o 100%.
4.12. Cell s aining
P K2 cells (ATCC CCL-56) we e g own in 750 µL medium in 4-well pla es (Nunc) on
glass co e slips, and incuba ed wi h he es compound o e nigh . Fo F-ac in s aining cells
we e ixed wi h o malin (3.7%) o 10 min, pe meabilized wi h 0.1% T i on
X-100 o 5 min, and hen incuba ed wi h Alexa Fluo 488 phalloidin (1:100; Molecula
P obes) o 1 h. Fo ER and α- ubulin s aining cells we e ixed wi h cold (–20°C) MeOH/ace-
one (1:1) o 10 min and la e incuba ed wi h a p ima y an ibody agains GRP-94 (1:1000;
A ini y Bio eagen s) and α- ubulin (1:100; Sigma), espec i ely, and hen wi h a seconda y
Alexa Fluo 488 goa an i- a IgG an ibody (1:200; Molecula P obes) and Alexa Fluo 488
goa an i-mouse IgG an ibody (1:200; Molecula P obes), espec i ely, and moun ed in
P oLong An i ade Gold (Molecula P obes), which included DAPI o s ain he nuclei.
21
4.13. Click chemis y
P K2 cells (ATCC CCL-56) we e g own in 750 µL medium in 4-well pla es (Nunc) on
glass co e slips, and incuba ed wi h 11a (3 µg mL–1) o e nigh . Cells we e ixed wi h
o malin (3.7%) o 10 min and pe meabilized wi h 0.1% T i on X-100 o 5 min. Cells we e
blocked wi h 3% FBS in PBS a 37 °C o 5 min. The Click-iT cock ail was p epa ed as pe
he manu ac u e ’s p o ocol jus p io i s usage and cells we e incuba ed o 30 min. Fo co-
localiza ion s udies, u he s aining was ca ied ou o F-ac in using Alexa Fluo 488
phalloidin (Ph488, 1:100 wi h 10% FBS, Molecula P obes), and moun ed in P oLong An i-
ade Gold (Molecula P obes) wi hou DAPI.
4.14. Impedance measu emen p o iling
The impedance o incuba ed cell cul u es was moni o ed on a RT-CES sys em
(xCelligence) om Acea Biosciences (Roche). The esul ing impedance cu es we e used o
a hie a chical clus e analysis o e e ence compounds oge he wi h compound o unknown
mode o ac ion. Co-clus e ing o he compound o unknown mode o ac ion wi h e e ence
compounds wi h known ac i i y class label is used o p edic he mode o ac ion. The me hod
was desc ibed p e iously.26
4.15. Tubulin polyme iza ion assay
This assay was ca ied ou acco ding o manu ac u e ’s p o ocol (Tubulin Polyme iza ion
Assay Ki , Ca alog #: BK0011P; Cy oskele on). Tubulin mas e mix was p epa ed which
included bu e , glyce ol, GTP s ock (100 mM) and po cine ubulin (10 mg mL–1) acco ding
o he manu ac u e ’s guidelines – all we e p o ided in he ki . 5 µL o he compounds we e
added o 50 µL o ubulin mix in a 96-well pla e which was immedia ely placed in a he mo-
egula ed luo ime e main ained a 37 °C. Fluo escence was measu ed o abou an hou a
460 nm (exci a ion: 340 nm).
4.16. Ac in polyme iza ion assay
This assay was ca ied ou acco ding o manu ac u e ’s p o ocol (Ac in Polyme iza ion
Biochem Ki , Ca alog #: BK003; Cy oskele on). Acco ding o he manu ac u e ’s guidelines
bu e and ac in we e p epa ed. The ac in s ock was added o a 96-well pla e and luo escence
was ead a 410 nm (exci a ion: 360 nm) o 3 min o a baseline eading. A e 3 min es

22
compounds (20 µL) we e added and ead o ano he 20 min. This was ollowed by addi ion
o 10X ac in polyme iza ion bu e and eadings we e aken o an hou .
Acknowledgemen s
Gene ous inancial suppo by he Deu sche Fo schungsgemeinscha , he Minis e ium ü
Wissenscha , Fo schung und Kuns des Landes Baden-Wü embe g (Landesg aduie en
ellowship o N.P.), he DAAD ( ellowship o N.P.) and he Fonds de Chemischen Indus-
ie and is g a e ully acknowledged.
Supplemen a y da a
Supplemen a y da a associa ed wi h his a icle can be ound, in he online e sion, a
h p://.
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