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

1 SAR studies on hydropentalene derivatives – important core units of biologically active tetramic acid macrolactams and ptychanolides Vanessa Lutza, Fabian Mannchena, Michael Krebsa, Natja Parka, Claudia Krügera, Aruna Rajab, Florenz Sasseb,*, Angelika Baroa, Sabine Laschata,* 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 ABSTRACT 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. Keywords: Biological activity, Click chemistry, Hydropentalene, Structure-activity relationships, Synthesis * Corresponding authors. Tel.: +49 711 685 64565, fax: +49 711 685 64285 (S.L.); tel.: +49 531 6181 3429; fax: +49 531 6181 3499 (F.S.). E-mail addresses: [email protected] (S. Laschat), [email protected] (F. Sasse). 2 1. Introduction Highly substituted bicyclo[3.3.0]octanes (hydropentalenes) represent the core unit of several biologically active natural product 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 (Figure 1). Figure 1. Hydropentalene core containing natural products 1–6. 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. Cylindramide'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 tumor promotion.10 Whereas natural products 1–4 are of marine origin, a bicyclo- [3.3.0]octane skeleton has also been detected in 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 3 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 reticulum of PtK2 (potoroo kidney) cells that were incubated with 1. A decrease of activity for the cyclopentane analogue of cylindramide (1) indicated that the activity is strongly correlated to the intact functionalized hydropentalene system. 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 biological properties of various functionalized bicyclo[3.3.0]octanes A–C. Our results are reported below. 2. Results and discussion 2.1. Chemistry 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 (Figure 2) leading to derivatives of structural diversity. Figure 2. Starting materials and positions for functionalization leading to structures A–C. 4 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 11c. Alkenes 17 and 18, providing key structural motifs of the bicyclo[3.3.0]octene core of the natural products 1–4, were prepared from monoketal 9a by a sequence of double deprotonation/electrophilic 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 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, r.t., 24 h, 40% for 11a, 37% for 11b; (b) 1.0 equiv 5-{[tertbutyl(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. The synthesis of hydropentalenes 21–23 based on bicyclo[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 BH3·THF provided the trans configured hydropentalenes 21a and 21b. Dienedione 222b yielded the alkylated bicyclo[3.3.0]octene-1,4-dione 23a by Cu-catalyzed Grignard reaction with 3-butenylmagnesium- 5 bromide in the presence of TMSCl. Ketalization of 23a with ethylene glycol gave the monoketal 23b (Scheme 2). 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 r.t., 3 h; (b) aq. Na2B4O7·10H2O/Na2EDTA·2H2O, 0.3 equiv Shi catalyst,20 0.04 equiv Bu4NHSO4, 1.4 equiv oxone in aq. Na2EDTA·2H2O, 5.8 equiv K2CO3, 3 h; (c) 2.5 equiv BH3·THF, THF, 3 h, 15% for 21a, 38% for 21b; (d) 1.4 equiv CuCN, 1.0 equiv TMEDA, 1.1 equiv 3-butenylmagnesiumbromide, 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. Hydropentalenes of the ptychanolide series were prepared from 3a,6a-dimethyltetrahydropentalene-2,5(1H,3H)-dione (7b)16 following known procedures17,18b,21 (Scheme 3). 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 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) 6 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 enantiodiscrimination 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 cleavage 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. Table 1 Cytotoxicity of bicyclo[3.3.0]octanes against different transformed cell linesa Entry Compounds L-929 (mouse) connective tissue IC50 (µM) KB-3-1 (human) cervix carcinoma 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 Further compounds in supplementary data. IC50 values are means of two assays in parallel. 7 As shown in Table 1, the antiproliferative activities of most compounds varied in a micromolar range. Exceptions were the alkyne 11a and dienedione 22 showing promising potencies (IC50 3 ) against L-929 mouse fibroblasts. The cytotoxicity of 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 displayed cytotoxicities (IC50 = 7.2–20.4 ) against L-929 mouse fibroblasts but poor inhibitory (IC50 >96 ) activity against the KB-3-1 cell line (Table 1, Entries 14, 15). Unexpectedly, silylprotection had a significant effect on the antiproliferative activity. The unprotected analogues of 13, 14 and 21 were completely inactive 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 cyclopentanols23–25 for the two cell lines was investigated. We observed an increased activity in the order of silyl protecting groups TBS < TIPS < TBDPS (Table 2, Entries 8–11). Table 2 Cytotoxicity of 3a,6a-dimethylhydropentalenes 24–26 and cyclopentanols against different transformed cell linesa Entry 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 t-butyl(cyclopentyloxy)dimethylsilane >200 >200 9 (cyclopentyloxy)(triisopropyl)silane 111.4 123.7 10 t-butyl(cyclopentyloxy)diphenylsilane 61.6 – a IC50 values are means of two assays in parallel. 8 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 developed 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 (Figure 3). Figure 3. Impedance curves of L-929 cells incubated with 11a (0.6 µg mL–1) and 22 (0.4 µg mL–1). Tubulysin B (3 ng mL–1) was used as one of the reference compounds. The obtained curves were compared with those of a set of reference compounds using statistical methods. Cluster analysis of the resulting data showed 11a and 22 in a group of reference compounds such as nocodazole, tubulysin B and griseofulvin that interfere with microtubule polymerization and spindle formation (Figure 4). 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. 9 Based on the results obtained from the impedance profiling, PtK2 (potoroo kidney) cells which showed in MTT assays an IC50 of approximately 3 µM with 11a and 22, respectively, were incubated with both compounds and stained for effects on microtubules (Figure 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. Figure 5. Effect of 11a and 22 on the microtubule cytoskeleton and mitotic spindle. PtK2 cells were treated with 11a (4 µg mL–1; C) and 22 (4 µg mL–1; D) overnight and stained for microtubules (green) and DNA (blue). Control cells were incubated with methanol only (A and B). To further verify the effect of 11a and 22, tubulin polymerization assays were carried out in vitro (Figure 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 developing microtubules. While nocodazole is only inhibiting the polymerization 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. A D C B 16 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  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) to give 18 (67.0 mg, 85%, purity >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 (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, 1’-Ha), 2.36–2.42 (m, 1H, 1’-Hb), 2.51–2.57 (m, 1H, 4H), 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, 3’-H), 5.47 (dt, J = 5.5, 2.2 Hz, 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, 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 (C6), 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 (vs), 701 (vs), 612 (m) cm–1; MS (EI): m/z (%) = 401.2 (8) [M – Me]+, 359.2 (100) [M – t-Bu]+, 281.1 (21), 199.1 (68); HRMS (ESI): calcd. for C28H36OsiNa+ [M + Na]+ 439.2428; found 439.2427. 4.7. tert-Butyl(diphenyl)[(3a’S,6a’S)-3’,3a’,6’,6a’-tetrahydro-2’H-spiro[1,3-dioxolane2,1’-pentalen]-4’-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 µL, 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 residue 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):  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 (C1’), 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 (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) 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. for C26H32O3Si [M + H]+ 420.2193; found 420.2199. 4.8. (3a’S,4’R,5’R,6a’S)- and (3a’S,4’S,5’S,6a’S)-4’-{[tert-Butyl(diphenyl)silyl]oxy}hexahydro-2’H-spiro[1,3-dioxolane-2,1’-pentalen]-5’-ol (21a) and (21b) a) Silyl enol ether 20 (210 mg, 0.50 mmol) was added to a solution of MeCN (2.4 mL), dimethoxymethane (4.80 mL) and stock solution I (4.80 mL) [prepared from sodium tetraborate decahydrate (19.1 g, 50.0 mmol) and disodium ethylenediaminetetraacetate 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 twophase mixture was stirred at 0 °C for a further 3 h and diluted with H2O (30 mL) to dissolve the precipitated salts. The layers were separated and the aqueous layer was extracted with CH2Cl2 (3  30 mL). The combined organic layers were washed with brine, dried (MgSO4) and concentrated under vacuum to 5 mL. Crude tert-butyl[(1b’S,4a’S)-hexahydro-1a’Hspiro[1,3-dioxalane-2,4’-pentaleno[1,2-b]oxiren]-1a’-yloxy]diphenylsilane was used without further purification. b) To a solution of crude tert-butyl[(1a’R,1b’S,4a’S)-hexahydro-1a’H-spiro[1,3-dioxolane2,4’-pentaleno[1,2-b]-1a’-yloxy]diphenylsilane (0.25 mmol) in THF (2 mL) at 0 °C was added BH3·THF (0.64 mL, 0.64 mmol, 1.0 M in THF) and the reaction mixture stirred 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 separated and the aqueous layer was extracted with EtOAc (3  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 respec- 18 tive cis-compound (19 mg, 19%). 21a: Rf 0.8 (hexanes/EtOAc, 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 (C6a’), 64.0 (OCH2CH2O), 64.9 (OCH2CH2O), 74.0 (C-5’), 77.2 (C-4’), 119.4 (C-1’), 127.7 (Co), 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 (vs), 1031 (m), 903 (vs), 854 (w), 822 (w), 729 (vs), 703 (vs), 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. for C26H34O4SiNa+ [M + Na]+ 461.2119; found 461.2117. 21b: Rf 0.70 (hexanes/EtOAc, 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 (C6a’), 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 (vs), 855 (m), 821 (m), 729 (vs), 701 (vs), 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. for C26H34O4SiNa+ [M + Na]+ 461.2119; found 461.2125. 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-butenylmagnesiumbromide, 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 19 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 solution (10:1, 15 mL). The layers were separated and the aqueous layer was extracted with Et2O (3  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 concentrated. The residue was purified by flash chromatography (hexanes/ 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): 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 (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): 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 (vs), 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. for C12H14O2 [M]+ 190.0994; found 190.0989. 4.10. rac 3’-But-3-enyl-2’,3’,3a’,6a’-tetrahydro-4’H-spiro[1,3-dioxolane-2,1’-pentalen]- 4’-one (23b) To a solution of toluene (5.00 mL), ethylene glycol (0.10 mL, 91 mg, 1.47 mmol) and pTsOH (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 Kromasil column (250  20 mm, 5 m pore size; MZ Analysentechnik GmbH) with hexanes/EtOAc (4:1) to give 23b (100 mg, 0.44 mmol, 60%) as a yellow oil. Rf 0.6 (hexanes/EtOAc, 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 (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) 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. for C14H18O3 [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 µL of serial dilutions of the test compounds were added to 120 µL aliquots of a cell suspension (50 000 mL–1) in 96-well microplates. Blank and solvent controls were incubated under identical conditions for 5 d. MTT in phosphate buffered saline (PBS) (20 µL) was added to a final concentration of 0.5 mg mL–1. After 2 h, the precipitate of formazan crystals was centrifuged, and the supernatant discarded. The precipitate was washed with PBS (100 µL) and dissolved in isopropanol containing 0.4% hydrochloric acid (100 µL). The microplates were gently shaken for 20 min to ensure a complete dissolution of the formazan and finally 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 µL 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 α-tubulin staining cells were fixed with cold (–20°C) MeOH/acetone (1:1) for 10 min and later incubated with a primary antibody against GRP-94 (1:1000; Affinity Bioreagents) and α-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. 21 4.13. Click chemistry PtK2 cells (ATCC CCL-56) were grown in 750 µL medium in 4-well plates (Nunc) on glass coverslips, and incubated with 11a (3 µg mL–1) overnight. Cells were fixed with formalin (3.7%) for 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 colocalization 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. 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 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 mL–1) according to the manufacturer’s guidelines – all were provided in the kit. 5 µL of the compounds were added to 50 µL of tubulin mix in a 96-well plate which was immediately placed in a thermoregulated fluorimeter maintained at 37 °C. Fluorescence was measured for about an hour at 460 nm (excitation: 340 nm). 4.16. Actin polymerization assay This assay was carried out according to manufacturer’s protocol (Actin Polymerization Biochem Kit, Catalog #: BK003; Cytoskeleton). 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