Heteroareno-Annelated Estranes by Triene Cyclization

DOI: 10.2478/s11532-006-0016-2 Research article CEJC 4(3) 2006 375–402

Heteroareno-annelated estranes by triene cyclization

Masataka Watanabe1, Taisuke Matsumoto2, Shuntaro Mataka2, Thies Thiemann2∗ 1 Interdisciplinary Graduate School of Engineering Sciences, Kyushu University, Kasuga-shi 816-8580, Japan 2 Institute of Materials Chemistry and Engineering, Kyushu University, Kasuga-shi 816-8580, Japan

Received 23 June 2005; accepted 8 March 2006

Abstract: 17-Thienyl- and 17-benzo[b]thienyl-3-hydroxyestra-1,3,5(10),16-tetraenes were synthesized by either sequential or one-pot Suzuki cross-coupling and Wittig oleﬁnation reactions. At 160 ◦C, heteroaryl substituted steroids were observed to undergo thermal intramolecular cyclization to produce E-ring heteroareno-annelated estranes. c Versita Warsaw and Springer-Verlag Berlin Heidelberg. All rights reserved.

Keywords: Steroids, annelation, cyclization, trienes

1 Introduction

Areno- and heteroareno-annelated steroids were proposed as compounds with potential antiﬂammatory, antibacterial and antimicrobial activity. Recently, bioactive benzoan- nelated pentacyclic and hexacyclic terpenoids were isolated from natural sources. A number of adociasulfates, such as I which was isolated from the Palauan sponge Haliclona (aka Adocia)[1–3], were determined to inhibit kinesin motor proteins [2]. Haliclotriol B, II, isolated from the Indo-Paciﬁc sponge Haliclona [4], was shown to possess microbial activity against Bacillus subtilis and Staphylococcus aureus. The hexacyclic disidein, III, [5] was isolated from the marine sponge Disidea pallescens. Of these, the adociasulfate 1, I, was synthesized by polyene cyclization (Fig. 1)[6]. Compounds arising from the areno-annelation of the estrane framework occurred less

∗ E-mail: [email protected] 376 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

Fig. 1 Naturally occuring Terpenoid Structures featuring an annelated areno moiety: adociasulfate 1 (I) from the Paluan sponge Haliclona (aka Adocia) [1–3], haliclotriol B (II) from the Indo-Pacific sponge Haliclona [4], and disidein (III) from the sponge Disidea pallescens [5]. frequently and thus far no natural products of this type were isolated. Research was conducted, however, to develop hybrids of estrane molecules with bioactive moieties [7, 8]. In connection with our studies of novel estrane-derived systems with potential affinity to the estrogen receptor, the authors became interested in the preparation of areno- and heteroareno-annelated estranes [7, 9, 10]. Syntheses of areno-annelated estranes by a tandem nitro-olefination/Heck reaction/cyclisation/aromatisation protocol [9]andby Ru- and Pt-catalysed cyclization of estrane-diene-ynes [10] were reported. The structures were also prepared by Robinson annelation with subsequent aromatisation [11], by Diels reaction using a steroidal diene [7], and use of steroidal 17-oxo-16-ketene acetals [12]. Because of the steric demand of the steroid, it is advantageous to conduct the annelation step intramolecularly rather than intermolecularly. Typically, a cyclization step would be involved. In this research, the construction of 17-thieno- and 17-benzothienoestra- 1,3,5(10),16-tetraenes having a substituted vinyl group at C16 is detailed. The thermal cyclization of such trienes to thiaindeno- and thiafluorenoestra-1,3,5(10),16-tetraenes is described.

2 Results and discussion

Six-membered ring construction via triene electrocyclization, while not used frequently, is a powerful tool, especially in natural product synthesis [13]. Potentially, one or more double bonds in the triene system can become part of a heterocycle. Recently, the authors developed a new approach to the synthesis of thienyldienes via a Suzuki coupling–Wittig olefination sequence [14, 15]. Therefore, it was expedient to utilize this technique in combination with a subsequent thermal triene cyclization for rapid construction of thieno- annelated estranes. The reaction strategy, involving synthesis from the corresponding bromoenaldehydes (Arnold-Vilsmeier adducts), was first developed using less expensive model compounds, based on dihydronaphthalene [15]. The preparative strategy for the construction of the triene began with a cycloalkanone, M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 377 which was transformed to the bromocyclohexenal using the Arnold-Vilsmeier reaction [16]. The bromo functional group was utilized for C-C coupling reactions; the carbaldehyde functional group for an olefination reaction. For the synthesis of 17-thienyl- and 17-benzo[b]thienyl-16-vinylestra-1,3,5(10),16-tetraenes, the Suzuki coupling was selected as the C-C forming reaction at C-17. The Wittig olefination was used for the formation of the substituted vinyl moiety at C-16. Initially, the phenolic function at C-3 of commercially available estrone (1) was protected and 1 was transformed to the 3-methoxy, 3-benzyloxy, and 3-benzoyloxy derivatives 2a – 2c by published procedures (Scheme 1)[17, 18]. All three derivatives were subjected successfully to the Arnold-Vilsmeier reaction to generate the corresponding 3-O-protected 17-bromo-3-hydroxyestra-1,3,5(10),16-tetraene-16-carbaldehydes 3a–3c (Scheme 1)[19]. The order of the reactions for the final preparation of the steroidal trienes 7 was in- terchangeable: a) Suzuki coupling followed by Wittig olefination; b) Wittig olefination followed by Suzuki coupling; or c) one pot Suzuki coupling–Wittig olefination.

Scheme 1 Preparation of three protected 17-bromo-3-hydroxyestra-1,3,5(10),16-tetraene- 16-carbaldehydes (3).

2.1 Suzuki coupling with subsequent Wittig oleﬁnation

17-Bromoestra-1,3,5(10),16-tetraen-16-carbaldehydes 3a–3c were subjected to the Suzuki cross coupling reaction with thienylboronic acids 6a,c and benzo[b]thienylboronic acids 6b,d. A two phase system was used in the reactions with DME as the organic solvent and aq. Na2CO3 as the aqueous medium. In the experiments, bistriphenylphosphinopalladium (II) dichloride [(PPh3)2PdCl2][20] was utilized as the pre-catalyst for the Suzuki cross- coupling reactions, where triphenylphosphine (PPh3) was added as a ligand. The desired products were obtained in good yield (Scheme 2). When 3a was reacted with benzo[b]thien-3-ylboronic acid (4d), a mixture of two products was obtained in a ratio approximately 1:1, as observed from the 1H NMR spectrum by integration of the C18-methyl group and the carbaldehyde proton. The mixture was subjected to repeated reactions under the same conditions, but the ratio did not change. At this time, it was highly unlikely that the mixture still contained starting material. However, the possibility existed that the mixture contained reduced material as observed in Pd(0) catalysed C-C coupling reactions. The spectroscopic data clearly demonstrated 378 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

Scheme 2 Preparation of 3-O-protected 17-heteroaryl-3-hydroxyestra-1,3,5(10),16- tetraene-16-carbaldehydes (5). that both components of the mixture possessed the benzo[b]thienyl moiety. Furthermore, the same reaction with bromo-formyldihydronaphthalene as substrate posed no problems. Migration of the substituent of the benzo[b]thiophene, that would lead to a mixture of benzo[b]thien-2-yl and benzo[b]thien-3-yl substituted steroids, was not predicted. Lastly, single crystals (see also below) were prepared from a solution of the mixture in hexane/ether/chloroform (1:1:1). Dissolution of the crystals in deuterated chloroform led to the 1H NMR pattern observed for the previous mixture. From this, it was deduced that the solution of 5d consisted of two rotamers. MM2-modeling of 5d demonstrated that the C16-(steroidal)-C3’-(benzo[b]thienyl) bond cannot rotate freely, therefore, in the NMR time scale, the two rotamers occurred as separate entities. X-ray crystal structural analyses of a single crystal of 5d grown in hexane/ether/CHCl3 (1:1:1) revealed the presence of only one of the rotamers (Fig. 2), which corresponded to the rotamer determined by MM2 calculations to have the lower energy. Only one of the rotamers may pack well enough to form the single crystal. The dihedral angle between the benzo[b]thienyl group at C17 and the average plane of ring C is 50.17(9)◦ in the crystal. Normally, it can be expected that the formyl group at C16 and the benzothienyl group at C17 will occur in the same plane because of possible conjugation of the π-systems. A planar conformation is diﬃcult to achieve as the benzo[b]thienyl group interacts sterically with the formyl group at C16 and the methyl group at C18. The results gathered from the X-ray crystal structure resembled those obtained from MM2 calculation of the molecule. From the X-ray crystal structural analysis, the torsion angle C16-C17-C22-C21 is 128.9(3)◦,from MM2 calculations, it is 120.0◦. M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 379

Fig. 2 Aviewof5d with the atom-numbering scheme. Displacement ellipsoids are drawn at 50 % probability level and H atoms are shown as small spheres of arbitrary radii.

Fig. 3 The unit cell of 5d viewed down the b axis. The ﬁgure also shows the CH-p interaction between molecules.

The occurrence of rotamers was also evidenced with 7i, the 17-benzo[b]thien-3-yl substituted compound. Partial and total hydrolysis of 7i to 7i-OH and 7i-(OH)2, respectively, was conducted to better observe the 1H NMR pattern of the compounds. These compounds each demonstrated the NMR signals for two rotamers. The compounds of type 5 were converted by Wittig-oleﬁnation to the 16-vinyl-17- heteroarylestra-1,3,5(10),16-tetraenes of type 7. Mixtures of type 5 compounds and type 6 phosphoranes were dissolved in a minimal amount of solvent to solubilize and mix the 380 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 compounds homogeneously and were heated in an oven at 100◦C (Scheme 3). Reac- tions with ethoxycarbonylmethylidenetriphenylphosphorane (6a) were completed within 30 min., while reactions with the less reactive acetylmethylidenetriphenylphosphorane (6b) and benzoylmethylidenetriphenylphosphorane (6c) were allowed to react for 2h.

Scheme 3 Wittig oleﬁnation as the ﬁnal step for the preparation of estrane trienes of general type 7.

2.2 Wittig oleﬁnation followed by Suzuki coupling

Compounds of type 3 were also subjected directly to Wittig olefination reactions. A number of conditions were utilized to perform the reactions: a.) heating of a mixture of compounds of type 3 and the phosphorane in benzene in the presence of benzoic acid [19]; b.) ultrasonication of a mixture of compounds of type 3 and phosphoranes in a biphasic (water/hexane-ether) medium [21]; and c.) reaction of compounds of type 3 with the phosphorane in a minimal amount of solvent (CHCl3)inanelectricoven [22]. Variant c is often the method of choice because of the simplicity of handling, the reduction in solvents and additives used, and the frequently increased yields of the products. Therefore, 8a and 8d were prepared by Wittig olefination in which the reaction of 3a and 3c with phosphorane 6a in a minimal amount of solvent within 20 min., was conducted in an electric oven at 100 ◦C. The transformations demonstrated the benefits of a virtually solvent-free Wittig reaction. The authors reported [19] earlier the use of benzene as the solvent with a reaction time of 3 h and a yield of 75 %. In the reaction developed in this research, the reaction time was shortened to 20 min, the workup was simplified, and the yield was increased to 96 %. As observed previously in Suzuki coupling with subsequent Wittig olefination, acetylmethylenetriphenylphosphorane (6b) and benzoylmethylidenetriphenylphosphorane (6c) were much less reactive and reaction M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 381 times of 90 min to 2h were necessary (Scheme 4)

Scheme 4 Wittig oleﬁnation of estrane carbaldehydes of type 3 with stabilized phosphoranes using minimal amounts of solvent.

Wittig products of type 8 were transformed by Suzuki cross-coupling reaction to the steroids of type 7 under the reaction conditions discussed above [Pd(PPh3)2Cl2, PPh3, aq. Na2CO3, DME] (Scheme 5). Double Suzuki coupling reactions were possible with Wittig products of type 8 that were prepared by the reaction of compounds of type 3 with halobenzoylmethylidenetriphenylphosphoranes, such as for 8e. The reactions lead to additional, appended heteroaromatic substituents in the steroidal trienes as produced in 7j (Scheme 6).

2.3 One-pot Suzuki coupling–Wittig oleﬁnation

Alternatively, compounds of type 7 were prepared using a one-pot Suzuki cross coupling– Wittig oleﬁnation protocol. The reaction was possible because the conjugated phosphoranes were stable under the reaction conditions utilized in Suzuki cross coupling transformations (Scheme 7)

2.4 Intramolecular Thermal Triene Cyclization

The products produced using the Wittig oleﬁnation–Suzuki cross coupling protocols (vari- ants a – c) were subsequently cyclized thermally. A number of solvents were used previously in triene electrocyclization reactions [23], especially when the reactions were conducted at temperatures below 150 ◦C. Desimoni et al. [23] performed the reaction of (1Z,3Z,5E)-1,2,6-triphenylhexa-1,3,5-triene to cis-1,5,6-triphenylcyclohexa-1,3-diene as a typical triene-electrocyclization reaction in 15 diﬀerent solvents and concluded that 382 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

Scheme 5 Suzuki cross-coupling reaction as the ﬁnal step in the preparation of estrane trienes of type 7.

Scheme 6 Double Suzuki cross-coupling reaction. the reaction did not demonstrate any signiﬁcant solvent eﬀect. As it was necessary to conduct the cyclization reactions at higher temperatures in this research, diphenyl ether was used as the solvent, which was not included in the list of solvents investigated by Desimoni et al. [23]. Diphenyl ether exhibited a number of advantages over decaline, which was normally used for electrocyclic reactions at high temperatures. Diphenyl ether solubilized substrates better than decaline. Improved solubilization generated less poly- merization of the starting materials, which often occurred in decaline, where the starting M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 383

Scheme 7 One-pot Suzuki cross-coupling–Wittig olefination in a direct preparation of estrane trienes of type 7 from compounds of type 3. material resided in areas of high concentration before it was solubilized completely at higher temperatures. Although diphenyl ether had a greater polarity than decaline, it can be separated from the reaction mixture by column filtration with hexane as eluant. As long as the desired product had a functional group, it was not eluted with diphenyl ether. Additionally, diphenyl ether had the advantage that its elution through the column was monitored spectroscopically in the UV range. The thermal triene cyclizations of the steroids of type 7 yielded the desired products of type 9 (Scheme 8). High temperatures were required for the cyclization. Thus, 7m produced no cyclization product when heated at 120 ◦C for 15h. When compounds of type 7 were heated in diphenyl ether at 160 ◦C, thermal cyclization occurred with subsequent aromatisation of the cyclohexadienes initially formed. The yields of the products at this temperature were fair. For certain compounds, the acetyl group was eliminated in the products under the thermal conditions used, leading to non-substituted thiafluoreno- and thiaindeno-annelated estranes such as 9g and 9n. A major difference was observed in the cyclization behavior of the thien-2-yl and thien-3-yl substituted steroids (eg.,of 7a vs. 7e). While the former reacted sluggishly, the cyclization reactions of the thien- 3-yl substituted steroids were complete within 12h at 160 ◦C. Thien-2-yl substituted steroids generated low yields in the cyclization reaction. The benzothien-3-yl substituted product 7i was not cyclized at all, probably resulting from the steric problems posed by an intermediate having all three double bonds of the triene system in 7i in one plane. This result contrasted sharply with that found for the benzothien-3-yl substituted dihydronaphthalenes, which did not possess a steric constraint and underwent easily thermal cyclizations at 160 ◦C. As the cyclohexadienes primarily formed undergo aromatisation under the stated conditions, the problem of the formation of double bond isomeric mixtures can be circumvented. The formation of isomeric mixtures occurred in some electrocyclization reactions due to a 1,3-hydride shift. 384 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

Scheme 8 Thermal cyclization of estrane trienes of type 7. M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 385

Scheme 8 (continued) Thermal cyclization of estrane trienes of type 7.

In general, the coupling procedure–Wittig oleﬁnation/Suzuki C-C bond formation– in combination with a triene cyclization complemented nicely the annelation methods developed by Voigt et al. [24]a and Zezschwitz et al. [24]b which relied on the double- Heck or Heck/Stille coupling reactions with a subsequent cyclization reported by Gilchrist and Summersell [25] which proceeded via sequential Wittig oleﬁnation, coupling with organozinc halides, and cyclization. While the benzoyl protective group was cleaved with ease (cf., see 9c), reduction of the benzyl protective group in the ring-annelated estranes by hydrogenation was also possible. The ring-annelated steroids synthesized in this research are currently being tested for potential anti-cancer and antimicrobial activities.

3 Experimental

3.1 General

2-Thienylboronic acid (4a), benzo[b]thien-2-ylboronic acid (4b), 3-thienylboronic acid (4c), and benzo[b]thien-3-ylboronic acid (4d) were acquired commercially (Aldrich). The starting material estrone (1) (Wako Pure Chemical Industries, Ltd.) was used as pur- chased. 3-Methoxyestra-1,3,5(10)-trien-3-one (2a) (KOH, MeI, DMSO), 3-Benzyloxyestra- 1,3,5(10)-trien-3-one (2b) (BnBr, NaH, dry DMF), O-protected 17-bromo-3-hydroxyestra- 386 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

1,3,5(10),16-tetraen-16-carbaldehydes (3a-c) [19] ethoxy- and methoxycarbonylmethyli- denetriphenylphosphorane (6a)[26]and(6d), acetylmethylidenetriphenylphosphorane (6b)[27] and benzoylmethylidenetriphenylphosphorane (6c)[27] were synthesized according to procedures published in the literature. 8d is a known compound [19]. Melting points were measured on a Yanaco microscopic hotstage and were uncor- rected. IR spectra were measured with JASCO IR-700 and Nippon Denshi JIR-AQ2OM instruments. 1Hand13C NMR spectra were recorded with a JEOL EX-270 (1H at 270 MHz and 13C at 67.8 MHz) and JEOL Lambda 400 spectrometer (1H at 395 MHz and 13 C at 99.45 MHz). The chemical shifts were reported relative to TMS (solvent CDCl3) unless otherwise noted. Mass spectra were measured with a JMS-01-SG-2 spectrometer [electron impact mode (EI), 70 eV or fast atom bombardment (FAB)]. Column chromatography was performed on Wakogel 300. All reactions were conducted under an inert atmosphere.

3.1.1 3-Methoxy-17-(thien-3’-yl)estra-1,3,5(10),16-tetraene-16-carbaldehyde (5c)

Asolutionof3a (262 mg, 0.7 mmol), 4c (180 mg, 1.4 mmol), Pd(PPh3)2Cl2 (25 mg, . −5 . −5 3.6 10 mol) and PPh3 (25 mg, 9.5 10 mol) in a mixture of DME (4 mL) and 2M ◦ aq. Na2CO3 (3 mL) was allowed to react at 65 C for 9 h. Column chromatography of the crude residue on silica gel (hexane/ether/CHCl3 3:1:1) yielded 5c (206 mg, 78 %) as colorless needles, mp. 201 ◦C; IR (KBr) ν 3096, 2930, 2868, 1652, 1612, 1572, 1498, 1456, 1279, 1254, 1228, 1151, 1124, 1035, 1020, 853, 813, 792, 699 cm−1; 1H NMR (270 2 3 MHz, CDCl3) δ 1.06 (s, 3H, CH3), 1.45 – 2.48 (m, 10H), 2.75 (dd, 1H, J 15.2 Hz, J 4 3 6.3 Hz), 2.91 (m, 2H), 3.78 (s, 3H, OCH3), 6.65 (d, 1H, J 2.7 Hz), 6.71 (dd, 1H, J 8.4 Hz, 4J 2.7 Hz), 7.14 (dd, 1H, 3J 4.9 Hz, 4J 1.2Hz),7.19(d,1H,3J 8.4 Hz), 7.33 (dd, 1H, 3J 3.0 Hz, 4J 1.2 Hz), 7.42 (dd, 1H, 3J 4.9 Hz, 3J 3.0Hz),9.80(s,1H,CHO); 13 C NMR (67.8 MHz, CDCl3) δ 16.52, 26.30, 27.54, 29.02, 29.64, 34.53, 37.41, 43.95, 51.19, 54.12, 55.21, 111.55, 113.83, 125.88, 125.94, 126.12, 127.87, 132.22, 133.59, 137.99, 140.04, 157.57, 167.65, 190.87; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 379 (MH+, 24), + + 378 (M , 17). HRMS Found: 379.1729. Calcd. for C24H27O2S: 379.1732 (MH ,FAB).

3.1.2 17-(Benzothien-3’-yl)-3-methoxyestra-1,3,5(10),16-tetraene-16-carbaldehyde (5d) A mixture of 3a (750 mg, 2.0 mmol), benzothien-3-ylboronic acid (4d) (534 mg, 3.0 . −2 . −2 mmol), Pd(PPh3)2Cl2 (35 mg, 5 10 mmol), triphenylphosphine (20 mg, 7.6 10 mmol) ◦ in DME (5 mL) and a 1.5 M aq. Na2CO3 solution was kept at 65 C for 7 h. The cooled mixture was diluted with water (20 mL) and extracted with chloroform (3 x 20 mL). The organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. Column chromatography of the residue on silica gel (hexane / ether / CHCl3 5:1:1)yielded5d (668 mg, 78 %) as a yellow solid (two rotamers), mp. 207 ◦C; IR (KBr) ν 3112, 2924, 2846, 1664, 1605, 1498, 1281, 1256, 1229, 1037, 822, 760, 733 cm−1; 1H NMR (270 MHz, CDCl3) δ 0.96 (s, 3H, CH3 [1 rotamer], 1.27 (s, 3H, CH3 [one rotamer]), 1.54 – 2.99 (m, 13H [for both rotamers]), 3.78 (s, 3H, OCH3 [both rotamers]), 6.67 – 6.72 (m, 2H [for both rotamers]), 7.13 – 7.90 (m, 5H [for both rotamers]), 9.48 (s, 1H, CHO [1 rotamer]), M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 387

9.51 (s, 1H, CHO [1 rotamer]); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 429 (MH+, 9,9). HRMS Found: 429.1880. Calcd. for C28H29O2S: 429.1888.

3.1.3 3-Benzyloxy-(E)-16-ethoxycarbonylethenyl-17-(thien-2’-yl)estra-1,3,5(10),16-tetraene (7a) General Procedure A. - A mixture of 5a (105 mg, 0.23 mmol) and 6a (324 mg, 1.0 mmol) in chloroform (0.5 mL) was heated at 100 ◦Cinanelectricovenfor30min.Erlen- r meyer ﬂasks with their outlet covered with Saran Wrap were used for these reactions. Typically, a small amount of chloroform evaporated during the reaction to yield a ho- mogenous melt of the reactants. Column chromatography of the cooled mixture on silica gel (hexane/ether/CHCl3 5:1:1) yielded 7a (111 mg, 92 %) as an oﬀ-white solid; mp. 172 ◦C; IR (KBr) ν 3028, 2980, 2924, 2850, 1710, 1610, 1496, 1280, 1244, 1229, 1173, 1150, −1 1 1032, 1019, 847, 699 cm ; H NMR (270 MHz, CDCl3) δ 1.04 (s, 3H, CH3), 1.29 (t, 3 3 3H, J 7.3 Hz, CH3), 1.47 – 2.56 (m, 11H), 2.91 (m, 2H), 4.21 (q, 2H, J 7.3 Hz), 5.04 (s,2H),5.94(d,1H,3J 16.7 Hz), 6.73 (d, 1H, 4J 2.7Hz),6.78(dd,1H,3J 8.4 Hz, 4J 2.7 Hz), 7.01 (dd, 1H, 3J3.5 Hz, 4J1.1Hz),7.09(dd,1H,3J 5.1 Hz, 3J 3.5 Hz), 7.18 (d, 1H, 3J 8.4 Hz), 7.31 – 7.44 (m, 6H), 7.78 (d, 1H, 3J 16.7 Hz); 13C NMR (67.8 MHz, CDCl3) δ 14.33, 16.60, 26.46, 27.63, 29.65, 31.64, 35.09, 37.30, 44.05, 50.52, 54.07, 60.21, 112.29, 114.89, 119.31, 126.00, 126.37, 127.17, 127.43, 127.84, 128.03, 128.53, 132.78, 135.97, 136.25, 137.29, 137.89, 140.09, 153.54, 156.81, 167.50; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 524 (M+, 27), 479 (3.3), 441 (5.1). HRMS Found: 524.2388. Calcd. for C34H36O3S: 524.2385.

3.1.4 (E)-16-Acetylethenyl-3-benzyloxy-17-(thien-2’-yl)estra-1,3,5(10),16-tetraene (7b) 5a (100 mg, 0.22 mmol) and 6b (122 mg, 0.385 mmol) in chloroform (1 mL) were allowed to react according to Procedure A (100 ◦C, 90 min.) Column chromatography on silica gel (hexane/ether/CHCl3 7:1:1) yielded 7b (78 mg, 72 %) as a colorless solid, mp. 90 ◦ −1 1 C; IR (KBr) ν 2926, 1664, 1605, 1499, 1253, 696 cm ; H NMR (270 MHz, CDCl3) δ 2 1.05 (s, 3H, CH3), 1.48 – 2.40 (m, 10H), 2.33 (s, 3H, COCH3), 2.61 (d, 1H, J 13.1 Hz, 3J 6.5 Hz), 2.92 (m, 2H), 5.04 (s, 2H), 6.21 (d, 1H, 3J 15.9 Hz), 6.74 – 6.80 (m, 2H), 3 13 7.04 – 7.46 (m, 9H), 7.65 (d, 1H, J 15.9 Hz); C NMR (67.8 MHz, CDCl3) δ 16.67, 26.48, 27.14, 27.65, 29.67, 31.66, 35.11, 37.34, 44.07, 50.78, 54.11, 70.00, 112.36, 114.92, 126.02, 126.67, 127.32, 127.46, 127.88, 127.93, 128.56, 128.73, 132.73, 135.80, 136.67, 137.31, 138.10, 139.17, 154.90, 157.00, 199.05; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) + 495 (MH , 8.7). HRMS Found: 495.2364. Calcd. for C33H35O2S: 495.2358.

3.1.5 3- Benzyloxy-(E)-16-ethoxycarbonylethenyl-17-(benzo[b]thien-2’-yl)estra- 1,3,5(10), 16-tetraene (7c) 5b (368 mg, 0.73 mmol) and 6a (380 mg, 1.09 mmol) in chloroform (1 mL) were allowed to react according to Procedure A (100 ◦C, 20 min). Column chromatography on silica gel ◦ (hexane/ether/CHCl3 2:1:1) yielded 7c (400 mg, 95 %) as a colorless solid, mp. 105 C; IR (KBr) ν 3054, 2924, 1705, 1612, 1499, 1455, 1304, 1280, 1250, 1158, 1039, 857, 745, 388 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

−1 1 3 730 cm ; H NMR (270 MHz, CDCl3) δ 1.10 (s, 3H, CH3), 1.28 (t, 3H, J 7.3 Hz, CH3), 3 1.50 – 2.69 (m, 11H), 2.92 (m, 2H), 4.20 (q, 2H, J 7.3 Hz), 5.04 (s, 2H, OCH2Ph), 5.98 (d, 1H, 3J 15.7 Hz), 6.76 (d, 1H, 4J 2.7 Hz), 6.79 (dd, 1H, 3J 8.6 Hz, 4J 2.7 13 Hz), 7.17 – 7.80 (m, 11H); C NMR (67.8 MHz, CDCl3) δ 14.33, 16.64, 22.65, 26.45, 27.65, 29.66, 31.59, 31.78, 35.09, 37.32, 44.08, 50.73, 54.22, 60.28, 70.03, 112.33, 114.93, 120.07, 121.95, 121.96, 123.66, 124.42, 124.54, 124.64, 126.64, 127.45, 127.85, 128.54, 132.74, 136.45, 137.30, 137.88, 137.92, 139.56, 140.38, 153.66, 156.85, 167.36; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 575 (MH+, 10), 574 (M+, 11). HRMS Found: 574.2539. Calcd. for C38H38O3S: 574.2542.

3.1.6 (E)-16-Acetylethenyl-17-(benzo[b]thien-2’-yl)-3-benzyloxyestra-1,3,5(10), 16-tetraene (7d)

5b (135 mg, 0.27 mmol) and 6b in CHCl3 (0.5 mL) were allowed to react according to Pro- ◦ cedure A (100 C, 90 min). Column chromatography on silica gel (hexane/ether/CHCl3 2:1:1) yielded 7d (113 mg, 77 %) as a colorless solid, mp. 88 ◦C; IR (KBr) ν 2924, 1665, −1 1 1603, 1497, 1455, 1253, 973, 746, 695 cm ; H NMR (67.8 MHz, CDCl3) δ 1.11 (s, 3H, CH3), 1.47 – 2.73 (m, 11H), 2.27 (s, 3H, COCH3), 2.93 (m, 2H), 5.04 (s, 2H, OCH2Ph), 6.24 (d, 1H, 3J 15.9 Hz), 6.73 (d, 1H, 4J 2.7 Hz), 6.78 (dd, 1H, 3J 8.5 Hz, 4J 2.7 Hz), 7.19 (d, 1H, 3J 8.5 Hz), 7.26 – 7.86 (m, 10H), 7.68 (d, 1H, 3J 15.9 Hz); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 545 (MH+, 14), 544 (M+, 7). HRMS Found: 545.2510. + Calcd. for C37H37O2S: 544.2514 (FAB, MH ).

3.1.7 3-Benzoyloxy-(E)-16-ethoxycarbonylethenyl-17-(thien-3’-yl)estra-1,3,5(10), 16-tetraene (7e)

General Procedure B - A mixture of 8a (220 mg, 0.41 mmol), 3-thienylboronic acid (4c) . −2 (79 mg, 0.62 mmol), Pd(PPh3)2Cl2 (35 mg, 5 10 mmol) and triphenylphosphine (26 mg, 0.1 mmol) in DME (6 mL) and 1.5 M aq. Na2CO3 solution (4 mL) was heated at 65 ◦C for 8 h. The cooled solution was extracted with chloroform (3 x 20 mL) and the organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography on silica gel (hexane/ether/CHCl3 5:1:1) and yielded 7e (122 mg, 55 %) as a colorless solid, mp. 219◦C; IR (KBr) ν 2928, 1733, 1713, −1 1 1615, 1277, 1213, 1150, 1062, 1024, 851, 712 cm ; H NMR (270 MHz, CDCl3) δ 1.02 3 (s, 3H, CH3), 1.28 (t, 3H, J 7.3 Hz, CH3), 1.49 – 2.43 (m, 11H), 2.97 (m, 2H), 4.20 (q, 3 3 3 2H, J 7.3 Hz, OCH2), 5.90 (d, 1H, J 15.7 Hz), 7.00 – 7.66 (m, 10H), 8.20 (d, 2H, J 13 7.0 Hz); C NMR (67.8 MHz, CDCl3) δ 14.33, 16.71, 26.23, 27.54, 29.47, 31.44, 35.14, 37.03, 44.22, 50.19, 54.25, 60.18, 118.61, 118.73, 121.67, 124.17, 125.45, 126.12, 128.06, 128.52, 129.72, 130.14, 133.50, 135.37, 137.94, 138.22, 140.31, 148.80, 149.59, 156.55, 165.47, 167.50; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 539 (MH+, 16), 538 (M+, 13). + HRMS Found: 539.2258. Calcd. for C30H35O4S: 539.2256 (MH , FAB). Found: C, 75.41; H, 6.36 %. Calcd. for C34H34O4S: C, 75.81; H, 6.36 %. M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 389

3.1.8 (E)-16-(Acetylethenyl)-3-benzoyloxy-17-(thien-3’-yl)estra-1,3,5(10), 16-tetraene (7f)

8b (625 mg, 1.24 mmol), 3-thienylboronic acid (4c) (238 mg, 1.86 mmol), (PPh3)2PdCl2 (35 mg, 5.2 . 10−2 mmol) and triphenylphosphine (20 mg, 7.6 . 10−5 mol) in DME (5 mL) and a 1.5 M aq. Na2CO3 solution (4 mL) were allowed to react and subjected to processing according to Procedure B (65 ◦C, 8 h). Column chromatography on silica gel ◦ (hexane/ether/CHCl3 4:1:1) yielded 7f (493 mg, 78 %) as colorless needles, mp. 226 C; IR (KBr) ν 2928, 2846, 1732, 1680, 1587, 1493, 1263, 1212, 1171, 1061, 1024, 982, 710 cm-1; 1H NMR (270 MHz, CDCl3) δ 1.03 (s, 3H, CH3), 2.28 (s, 3H, COCH3), 1.49 – 2.42 (m, 10H), 2.59 (dd, 1H, 2J 14.6 Hz, 3J 6.2 Hz), 2.97 (m, 2H), 6.17 (d, 1H, 3J 15.7 Hz), 6.96 – 7.01 (m, 2H), 7.09 (dd, 1H, 3J 4.9 Hz, 4J 1.3Hz),7.21(dd,1H,4J 3.0 Hz, 4J 1.3 Hz), 7.32 (d, 1H, 3J 8.4Hz),7.38(dd,1H,3J 4.9 Hz, 4J 3.0 Hz), 7.41 – 7.63 (m, 3H), 3 13 8.20 (d, 2H, J 8.2 Hz); C NMR (67.8 MHz, CDCl3) δ 16.25, 25.77, 26.70, 27.02, 28.94, 30.88, 34.64, 36.56, 43.80, 49.87, 53.79, 118.27, 121.22, 123.71, 125.20, 125.63, 127.53, 127.57, 128.04, 129.23, 129.66, 133.01, 134.90, 135.28, 137.38, 137.72, 138.77, 148.36, 156.98, 165.06, 198.56; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 509 (MH+, 32), 105 (100). HRMS Found: 509.2148. Calcd. for C33H33O3S: 509.2150 (MH;, FAB).

3.1.9 3-Benzoyloxy-(E)-16-(phenacylethenyl)-17-(thien-3’-yl)estra-1,3,5(10),16-tetraene (7g)

8c (249 mg, 0.44 mmol), 3-thienylboronic acid (4c) (112 mg, 0.87 mmol), Pd(PPh3)2Cl2 (35 mg, 5.10−2 mmol), triphenylphosphine (20 mg, 7.6.10−2 mmol) in DME (5 mL) and a 1.5 M aq. Na2CO3 solution were allowed to react and subjected to processing according ◦ to Procedure B (65 C, 7 h). Column chromatography on silica gel (hexane/ether/CHCl3 5:1:1) yielded 7g (221 mg, 88 %) as pale yellow solid, mp. 236 ◦C; IR (KBr) ν 3058, 2926, 1735, 1635, 1579, 1493, 1450, 1330, 1263, 1216, 1173, 1152, 1081, 1066, 1036, 1020, −1 1 978, 849, 778, 705 cm ; H NMR (270 MHz, CDCl3) δ 1.06 (s, 3H, CH3), 1.54 – 2.10 (m, 7H), 2.38 – 2.43 (m, 3H), 2.73 (dd, 1H, 2J 14.3 Hz, 3J 6.3 Hz), 2.98 (m, 2H), 6.96 (d, 1H, 4J 2.4 Hz), 6.97 (d, 1H, 3J 15.4 Hz), 7.01 (dd, 1H, 3J 8.4 Hz, 4J 2.4 Hz), 7.12 (dd, 1H, 3J 5.1 Hz, 4J 1.1Hz),7.23(dd,1H,4J 3.0 Hz, 4J 1.1 Hz), 7.34 (d, 1H, 3J 8.4Hz),7.37(dd,1H,3J 5.1 Hz, 4J 3.0 Hz), 7.44 – 7.67 (m, 6H), 7.81 (d, 1H, 3J 15.4 Hz), 7.95 (dd, 1H, 3J 7.8 Hz, 4J 1.9Hz),8.21(dd,1H,3J 7.3 Hz, 4J 1.9 Hz); 13CNMR (67.8 MHz, CDCl3) δ 16.80, 26.43, 27.59, 29.46, 31.63, 35.16, 37.11, 44.30, 50.39, 54.30, 118.77, 121.70,. 122.60, 124.52, 125.59, 126.16, 126.69, 128.06, 128.34, 128.50, 128.54, 130.16, 132.47, 133.51, 135.34, 136.11, 137.94, 138.19, 138.55, 140.65, 148.76, 158.25, 165.42, 190.79; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 571 (MH+, 10). HRMS Found: 571.2310. Calcd. for C38H35O3S: 571.2307.

3.1.10 17-(Benzo[b]thien-3’-yl)-3-benzoyloxy-(E)-16-(ethoxycarbonylethenyl)estra-1,3, 5(10),16-tetraene (7i)

. −2 8a (230 mg, 0.44 mmol), 4d (137 mg, 0.77 mmol), Pd(PPh3)2Cl2 (18 mg, 2.6 10 mmol), . −2 and PPh3 (20 mg, 7.6 10 mmol) in DME (4 mL) and 1.5 M aq. Na2CO3 (2.5 mL) 390 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 were allowed to react and subjected to processing according to Procedure B at (65 ◦C, 8h). Column chromatography on silica gel (hexane/ether/CHCl3 5:1:1) yielded 7i (two rotamers) (200 mg, 77 %) as a pale yellow solid; mp. 208 ◦C; IR (KBr) ν 3056, 2976, 2918, 1736, 1708, 1621, 1496, 1300, 1282, 1261, 1220, 1163, 1063, 1024, 758, 734, 705 −1 1 cm ; H NMR (270 MHz, CDCl3) δ 0.91 (s, 3H, CH3 [one rotamer]), 1.11 (s, 3H, CH3 [one rotamer]), 1.21 (m, 3H, CH3 [both rotamers]), 1.55 - 2.55 (m, 10H [both rotamers]), 2.63 (m, 1H [both rotamers]), 2.97 – 3.01 (m, 2H [both rotamers]), 4.10 (m, 2H, OCH2 [for both rotamers]), 5.89 (d, 1H, 3J 15.7 Hz [both rotamer]), 6.98 (m, 2H, [both rotamers]), 7.17 – 7.63 (m, 9H [for both rotamers]), 7.88 (m, 1H [both rotamers]), 8.19 (dd, 2H, 3J 8.6 Hz, 4J 1.6 Hz [both rotamer]); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 589 (MH+, + 6.5), 588 (M , 6.0). HRMS Found: 588.2329. Calcd. for C38H36O4S: 588.2334.

3.1.11 17-(Benzothien-3’-yl)-(E)-16-(carboxyethenyl)-3-hydroxyestra-1,3,5(10),16-tetraene [7i-(OH2)]

A suspension of 7i (25 mg, 0.042 mmol) and NaOMe (100 mg, 1.8 mmol) in a mixture of ether (10 mL) and ethanol (10 mL) was stirred at rt for 5 h. The solution was concentrated in vacuo and the residue was diluted with water (10 mL). The mixture was acidiﬁed with conc. aq. HCl and was extracted with chloroform (3 X 10 mL). The combined organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. Column chromatography of the crude mixture (hexane/ether/CHCl3 2:1:1) yielded 17-(benzothien-3’-yl)- (E)-16-(ethoxycarbonylethenyl)-3-hydroxyestra-1,3,5(10),16-tetraene (7i-OH) (7 mg, 34 %) as a colorless solid, mp. 133 ◦C; IR (KBr) ν 3418 (bs, OH), 2924, 1686, 1618, 1499, −1 1 1453, 1366, 1283, 1175, 759, 735 cm ; H NMR (270 MHz, CDCl3) δ 0.88 (s, 3H, CH3 [one rotamer]), 1.09 (s, 3H, CH3 [one rotamer]), 1.26 (m, 3H, CH3 [both rotamers]), 1.50 – 2.68 (m, 11H [both rotamers]), 2.90 (m, 2H [both rotamers]), 4.11 (m, 2H, OCH2 [both rotamers]), 5.89 (d, 1H, 3J 15.7 Hz [both rotamers]), 6.59 – 6.62 (m, 2H [both rotamers]), 7.11 – 8.13 (m, 7H [both rotamers]), MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 484 + (M , 10); HRMS Found: 484.2075; calcd. for C31H32O3S: 484.2072; further elution with ether yielded 17-(benzothien-3’-yl)-(E)-16-(carboxyethenyl)-3-hydroxyestra-1,3,5(10),16- ◦ tetraene [7i-(OH2)] (10 mg, 52 %) as a colorless solid, mp. 197 C; IR (KBr) ν 3390 (bs, OH), 2926, 1683, 1608, 1500, 1453, 1280, 1217, 785, 761, 735 cm−1; 1H NMR (270 MHz, CDCl3) δ 0.88 (s, 3H, CH3 [one rotamer]), 1.09 (s, 3H, CH3 [one rotamer]), 1.50 – 2.68 (m, 11H [for both rotamers]), 2.90 (m, 2H [both rotamers]), 5.84 (d, 1H, 3J 15.4 Hz), 6.59 – 6.63 (m, 2H [for both rotamers]), 7.10 – 7.88 (m, 7H); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 457 (MH+, 1.4[5]), 456 (M+, 1.8). HRMS Found: 456.1765. Calcd. for C29H28O3S: 456.1759.

3.1.12 3-Methoxy-(E)-16-(p-[thien-3’-yl]phenylethenyl)-17-(thien-3”-yl)estra-1,3,5(10), 16-tetraene (7j)

. −2 8e (250 mg, 0.45 mmol), 4c (230 mg, 1.8 mmol), Pd(PPh3)2Cl2 (35 mg, 5 10 mmol) and triphenylphosphine (26 mg, 0.1 mmol) in DME (6 mL) and 1.5 M aq. Na2CO3 solution (4 mL) were allowed to react and subjected to processing according to Procedure B (65 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 391

◦ C, 12 h). Column chromatography on silica gel (hexane/ether/CHCl3 5:1:1) yielded 7j (215 mg, 85 %) as a pale yellow solid, mp. 163 ◦C; IR (KBr) ν 2930, 1653, 1606, 1575, −1 1 1281, 1185, 786, 733 cm ; H NMR (270 MHz, CDCl3) δ 1.05 (s, 3H, CH3), 1.55 – 2.75 4 3 (m, 11H), 2.95 (m, 2H), 3.79 (s, 3H, OCH3), 6.67 (d, 1H, J 2.4Hz),6.74(dd,1H, J 8.1 Hz, 4J 2.4 Hz), 7.02 (d, 1H, 3J 15.1 Hz), 7.11 (d, 1H, 3J 4.9 Hz), 7.19 – 7.44 (m, 6H), 7.58 (d, 1H, 4J 1.6 Hz), 7.70 (d, 1H, 3J 8.4Hz),7.83(d,1H,3J 15.1 Hz), 8.01 (d, 2H, 3J 13 8.4 Hz); C NMR (67.8 MHz, CDCl3) δ 16.81, 23.30, 27.79, 29.70, 31.58, 31.63, 35.18, 44.10, 50.45, 54.28, 55.22, 111.47, 113.92, 121.86, 122.40, 124.50, 125.55, 126.02, 126.20, 126.35, 126.69, 128.07, 129.08, 132.55, 135.40, 136.14, 137.30, 137.85, 139.66, 140.55, 141.28, 157.56, 158.37, 192.10; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 563 (MH+,4). HRMS Found: 563.2075. Calcd. for C36H35O2S2: 563.2078.

3.2 Wittig Oleﬁnation–Suzuki Coupling in one pot

3.2.1 3-Methoxy-(E)-16-(methoxycarbonylethenyl)-17-(thien-3-yl)estra-1,3,5(10),16-tetraene (7k)

A mixture of 3a (375 mg, 1.0 mmol), 3-thienylboronic acid (4c) (192 mg, 1.5 mmol), . −2 phosphorane 6d (500 mg, 1.5 mmol), Pd(PPh3)2Cl2 (28 mg, 4.0 10 mmol), PPh3 (30 ◦ mg, 0.11 mmol) in DME (5 mL) and 1.5 M aq. Na2CO3 (3 mL) was kept at 65 C for 8 h. The cooled mixture was extracted with chloroform (3 X 20 ml). The organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography on silica gel (hexane/ether/CHCl3 2:1:1) to yield 7k (374 mg, 86 %) as a colorless solid; mp. 106 ◦C; IR (KBr) ν 2924, 1714, 1613, 1501, 1280, 1257, −1 1 1162, 1040, 853, 788 cm ; H NMR (270 MHz, CDCl3) δ 0.86 – 2.41 (m, 10H), 1.00 (s, 2 3 3H, CH3), 2.57 (dd, 1H, J 14.7 Hz, J 6.9 Hz), 2.92 (m, 2H), 3.73 (s, 3H, OCH3), 3.78 3 4 3 (s, 3H, OCH3), 5.89 (d, 1H, J 15.4 Hz), 6.65 (d, 1H, J 2.7 Hz), 6.71 (dd, 1H, J 8.4 Hz, 4J 2.7Hz),7.07(dd,1H,3J 4.9 Hz, 4J 1.1 Hz), 7.18 (m, 2H), 7.36 (dd, 1H, 3J 4.9 Hz, 3 3 13 J 3.1Hz),7.61(d,1H, J 15.4 Hz); C NMR (67.8 MHz, CDCl3) δ 16.71, 26.39, 27.69, 29.68, 31.36, 35.13, 37.37, 44.08, 50.24, 51.46, 54.21, 55.20, 111.46, 113.84, 118.06, 124.17, 125.45 (2C), 125.99, 128.05, 132.52, 135.33, 137.86, 140.55, 156.46, 157.51, 167.99; MS + (EI, 70 eV) m/z (%) 434 (M , 100). HRMS Found: 434.1916. Calcd. for C27H30O3S: 434.1916.

3.2.2 3-Methoxy-(E)-16-phenacylethenyl-17-(thien-3-yl)estra-1,3,5(10),16-tetraene (7m)

A mixture of 3a (375 mg, 1.0 mmol), 3-thienylboronic acid (4c) (192 mg, 1.5 mmol), . −2 phosphorane 6c (570 mg, 1.5 mmol), Pd(PPh3)2Cl2 (28 mg, 4.0 10 mmol), PPh3 (32 ◦ mg, 0.12 mmol) in DME (5 mL) and 1.5 M aq. Na2CO3 (3 mL) was kept at 65 C for 3.5 h. Thereafter, 4c (192 mg, 1.5 mmol) was added and the reaction mixture was kept at 65 ◦C for an additional 5 h. The cooled mixture was extracted with chloroform (3 X 20 ml). The organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. The residue was subjected to column chromatography on silica gel (hexane/ether/CHCl3 392 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

2:1:1) to yield 7m (250 mg, 52 %) as pale yellow needles, mp. 180 ◦C; IR (KBr) ν 2924, −1 1 1641, 1573, 1495, 1278, 1257, 779, 706 cm ; H NMR (270 MHz, CDCl3) δ 1.04 (s, 3H, 2 3 CH3), 1.50 – 2.42 (m, 10H), 2.71 (dd, 1H, J 14.5 Hz, J 6.4 Hz), 2.96 (m, 2H), 3.79 4 3 4 (s, 3H, OCH3), 6.67 (d, 1H, J 2.5Hz),6.73(dd,1H, J 8.4 Hz, J 2.5Hz),6.98(d, 1H, 3J 15.1 Hz), 7.11 (dd, 1H, 3J 5.1 Hz, 4J 1.5 Hz), 7.22 (d, 1H, 3J 8.4Hz),7.23(dd, 1H, 3J 2.7 Hz, 4J 1.5 Hz), 7.37 (dd, 1H, 3J 5.1 Hz, 3J 2.7 Hz), 7.43 – 7.57 (m, 3H), 3 13 7.89 (d, 1H, J 15.1 Hz), 7.95 (m, 2H); C NMR (67.8 MHz, CDCl3) δ 16.79, 26.41, 27.77, 29.69, 30.93, 31.58, 35.15, 37.43, 44.07, 50.43, 54.24, 55.21, 111.45, 113.89, 122.52, 124.48, 125.53, 126.01, 128.04, 128.31, 128.48, 132.45, 132.52, 136.09, 137.84, 138.53, 140.69, 157.53, 158.38, 190.77; MS (EI, 70 eV) m/z (%) 480 (30), 252 (100). HRMS (Found): 480.2126. Calcd. for C32H32O2S: 480.2123.

3.2.3 3-Benzoyloxy-17-bromo-(E)-16-(ethoxycarbonylethenyl)estra-1,3,5(10),16-tetraene (8a)

A mixture of 3c (210 mg, 0.45 mmol) and 6a (220 mg, 0.675 mmol) in chloroform (0.5 mL) was heated in an electric oven at 100 ◦C for 20 min. The cooled solution was subjected to column chromatography on silica gel (hexane/ether/CHCl3 7:1:1) to yield 8a (232 mg, 96 %) as a colorless solid; mp. 180 ◦C; IR (KBr) ν 3072, 2976, 2932, 1735, 1715, 1622, 1493, 1452, 1300, 1268, 1216, 1171, 1061, 996, 981, 896, 713 cm−1; 1H NMR (270 MHz, 3 CDCl3) δ 0.92 (s, 3H, CH3), 1.32 (t, 3H, J 7.1 Hz, CH3), 1.46 – 1.85 (m, 6H), 1.94 – 2.01 (m, 2H), 2.12 – 2.21 (m, 1H), 2.33 – 2.52 (m, 2H), 2.95 (t, ), 4.24 (q, 2H, 3J 7.1 Hz), 13C NMR (67.8 MHz, CDCl3) δ 14.32, 15.51, 26.02, 27.03, 29.33, 29.70, 31.03, 33.57, 44.42, 50.85, 53.35, 60.47, 118.85, 120.48, 121.71, 126.13, 128.55, 129.67, 130.15, 133.53, 135.90, 137.61, 138.02, 138.42, 143.79, 148.86, 165.46, 167.01; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 537 ([81Br]MH+, 14), 535 ([79Br]MH+, 15). HRMS Found: 535.1480. Calcd. for 79 79 + C30H32O4 Br: 535.1484 ([ Br]MH ,FAB).

3.2.4 3-O-Benzoyloxy-17-bromo-(E)-16-(acetylethenyl)estra-1,3,5(10),16-tetraene (8b)

A mixture of acetylmethylenetriphenylphosphorane (6b) (400 mg, 1.26 mmol) and 3c (210 mg, 0.45 mmol) in chloroform (0.5 mL) was heated in an electric oven at 100 ◦C for 45 min. The cooled solution was subjected to column chromatography on silica gel ◦ (hexane/ether/CHCl3 6:1:1) to yield 8b (220 mg, 96 %) as a colorless solid, mp. 173 C; IR (KBr) ν 2930, 1734, 1692, 1666, 1595, 1493, 1265, 1217, 1174, 1063, 711 cm−1; 1H NMR (270 MHz, CDCl3) δ 0.93 (s, 3H, CH3), 1.45 – 2.49 (m, 11H), 2.43 (s, 3H, COCH3), 2.95 (m, 2H), 6.15 (d, 1H, 3J 15.9 Hz), 6.96 – 7.00 (m, 2H), 7.26 – 7.63 (m, 5H), 8.19 3 13 (d, 2H, J 8.1 Hz); C NMR (67.8 MHz, CDCl3) δg5.52, 25.99, 26.98 (2C), 29.30, 31.01, 34.64, 37.11, 44.39, 51.03, 55.34, 118.34, 121.71, 126.10, 128.53, 129.51, 129.52, 130.13, 133.38, 133.52, 136.40, 137.53, 137.99, 145.09, 148.66, 199.04; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 507 ([81Br]MH+, 4.7), 505 ([79Br]MH+, 5.0). HRMS Found: 505.1378. 79 Calcd. for C29H30O3 Br: 505.1378. Found: C, 68.92; H, 5.77%. Calcd. for C29H29BrO3: C, 68.91; H, 5.78%. M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 393

3.2.5 3-O-Benzoyloxy-17-bromo-(E)-16-(phenacylethenyl)estra-1,3,5(10),16-tetraene (8c)

A mixture of 3c (210 mg, 0.45 mmol) and benzoylmethylenetriphenylphosphorane (6c) (340 mg, 0.89 mmol) was heated in an electric oven at 100 ◦C for 75 min. Thereafter, the cooled solution was subjected to column chromatography on silica gel (hexane/ether/ ◦ CHCl3 5:1:1) to yield 8c (196 mg, 77 %) as a colorless solid, mp. 194 C; IR (KBr) ν 2928, 1732, 1658, 1586, 1493, 1306, 1265, 1214, 1173, 1061, 1020, 702 cm−1; 1H NMR (270 MHz, CDCl3) δ 0.95 (s, 3H, CH3), 1.54 – 2.60 (m, 11H), 2.97 (m, 2H), 6.94 – 7.02 (m, 3H), 7.32 – 7.64 (m, 7H), 7.73 (d, 1H, 3J 15.4 Hz), 7.97 (d, 2H, 3J 7.0 Hz), 8.21 (d, 2H, 3 13 J 7.3 Hz); C NMR (67.8 MHz, CDCl3) δ 15.56, 26.00, 27.10, 29.33, 31.23, 34.68, 37.18, 44.40, 51.07, 53.39, 118.85, 121.71, 124.31, 126.15, 128.46, 128.54, 128.57, 129.67, 130.14, 132.69, 133.51, 136.55, 137.59, 137.97, 138.17, 138.63, 145.79, 148.86, 165.44, 190.83; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 569 ([81Br]MH+, 11), 567 ([79Br]MH+, 12). HRMS 79 79 + Found: 567.1530. Calcd. for C34H32O3 Br: 567.1535 ([ Br]MH ,FAB).

3.2.6 Ethyl 3’-benzyloxyestra-1’,3’,5’(10’)-tetraeno[17’,16’-g]-1-thiaindene-4-carboxylate (9a) thermolysis of 7a

Asolutionof7a (67 mg, 0.13 mmol) in diphenyl ether (1.1 mL) was kept at 160 ◦C for 24 h. Thereafter, the cooled solution was subjected to column chromatography on silica gel. Initially, hexane was used to elute the diphenyl ether; subsequently, hexane/ether/CHCl3 (7:1:1) was used to elute 9a (14 mg, 21 %) as a colorless solid; mp. 153 ◦C; IR (KBr) ν 2924, 2858, 1708, 1612, 1496, 1448, 1282, 1257, 1192, 1115, 1042, 797, 730 cm−1; 1HNMR 3 (270 MHz, CDCl3) δ 1.10 (s, 3H, CH3), 1.42 – 3.00 (m, 13H), 1.45 (t, 3H, J 7.0 Hz, CH3), 3 4.44 (q, 2H, J 7.0 Hz, OCH2), 5.05 (s, 2H), 6.77 – 6.83 (m, 2H), 7.24 – 7.45 (m, 6H), 7.54 (d, 1H, 3J 5.7 Hz), 8.06 (s, 1H), 8.27 (d, 1H, 3J 5.7 Hz); MS (FAB, 3-nitrobenzyl alcohol) + m/z (%) 522 (M , 10). HRMS Found: 522.2224. Calcd. for C34H34O3S: 522.2229.

3.2.7 4-Acetyl-3’-benzyloxyestra-1’,3’,5’(10’),16’-tetraeno[17’,16’-g]-1-thiaindene (9b)

Thermal cyclization of 7b - a solution of 7b (78 mg, 0.16 mmol) in diphenyl ether (1.1 mL) was heated at 160 ◦C for 14 h. Thereafter, the cooled mixture was subjected to column chromatography on silica gel. Initially, hexane was used to elute the diphenyl ether; subsequently, hexane/CHCl3/ether (7:1:1) was used to elute 9b (12 mg, 15%) as a slowly solidifying oil; IR (neat) ν 2926, 1672, 1499, 1253, 785, 733 cm−1; 1H NMR (270 MHz, CDCl3) δ 0.86 – 3.01 (m, 13H), 1.11 (s, 3H, CH3), 2.70 (s, 3H, COCH3), 5.06 (s, 2H), 6.77 – 6.83 (m, 2H), 7.26 – 7.45 (m, 6H), 7.57 (d, 1H, 3J 5.7 Hz), 7.87 (s, 1H), 8.41 (d, 1H, 3J 5.7 Hz); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 493 (MH+, 7.8). HRMS + Found: 493.2197. Calcd. for C33H33O2S: 493.2201 (MH ,FAB). 394 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

3.2.8 3’-Benzoyloxy-7-ethoxycarbonyl-estra-1’,3’,5’(10’),16’-tetraeno[16’,17’-e]-1-thiaindene (9c) To generate thermal cyclization of 7e.–7e, 70 mg (0.13 mmol) in diphenyl ether (1.1 mL) was heated at 160 ◦C for 12 h. Column chromatography of the cooled mixture on silica gel (initially hexane, followed by hexane/ether/CHCl3 5:1:1) yielded 9c (31 mg, 45 %) as a colorless solid; mp. 136 ◦C; IR (KBr) ν 2928, 1735, 1702, 1494, 1452, 1373, 1263, −1 1 1209, 1173, 1079, 1062, 1024, 707 cm ; H NMR (270 MHz, CDCl3) δ 1.13 (s, 3H, CH3), 3 1.28 (t, 3H, J 7.0 Hz, CH3), 1.44 – 3.00 (m, 10H), 2.97 – 3.05 (m, 3H), 4.48 (q, 2H, 3 4 3 4 J 7.0 Hz, OCH2), 6.99 (d, 1H, J 2.4 Hz), 7.02 (dd, 1H, J 8.4 Hz, J 2.4 Hz), 7.39 (d, 1H, 3J 8.4 Hz), 7.49 – 7.64 (m, 3H), 7.57 (s, 2H), 8.19 (s, 1H), 8.21 (d, 2H, 3J 7.3 Hz); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 537 (MH+, 18), 105 (100). HRMS Found: + 537.2102. Calcd. for C34H33O4S: 537.2100 (MH ,FAB).

3.2.9 3’-Hydroxyestra-1,’3’,5’(10’),16’-tetraeno[16’,17’-e]thiaindene-6-carboxylic acid (9c-OH2)

. −5 t A mixture of 9c (30 mg, 5.5 10 mol), KOBu (400 mg, 3.6 mmol), and H2O (about 0.1 mL) in diethylether (6 mL) was stirred for 10 h at rt. Thereafter, the reaction mixture was diluted with water (6 mL) and extracted with ether (10 mL), followed by acidiﬁcation with conc. aq. HCl. The mixture was extracted with chloroform (3 x 10 mL); the organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. Column chromatography of the residue on silica gel (initially ether/hexane 1:2) yielded ethyl 3’-hydroxyestra-1,’3’,5’(10’),16’-tetraeno[16’,17’-e]thiaindene-6-carboxylate (9c-OH)(10 mg, 41%) as a colorless solid; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 433 (MH+,2.7), + 405 (M , 4.2); HRMS Found: 433.1840; calcd. for C27H29O3S: 433.1837; subsequent elution with ether and recrystallisation from ether/hexane 1:1 yielded 3’-hydroxyestra- 1,’3’,5’(10’),16’-tetraeno[16’,17’-e]thiaindene-6-carboxylic acid (9c-OH2) (12 mg, 53%) as a colorless solid; mp. > 250 ◦C; IR (KBr) ν 3428, 2924, 1674, 1501, 1282, 1222, 1092 −1 1 cm ; H NMR (270 MHz, CDCl3) δ 1.02 (s, 3H, CH3), 1.52 – 3.03 (m, 13H), 6.61 (d, 1H, 4J 2.4Hz),6.67(dd,1H,3J 8.6 Hz, 4J 2.4Hz),7.21(d,1H,3J 8.6 Hz), 7.59 (d, 1H, 3J 6.4 Hz), 7.61 (d, 1H, 3J 6.4 Hz), 8.13 (s, 1H); MS (FAB, 3-nitrobenzyl alcohol) m/z + + (%) 405 (MH , 3.1), 404 (M , 3.0). HRMS Found: 404.1450. Calcd. for C25H24O3S: 404.1446 (M+,FAB).

3.2.10 Ethyl 3’-methoxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-e]1-thiaindene-7-carboxylate (9d) Thermal cyclization of 7h -amixtureof7h (75 mg, 0.17 mmol) in diphenylether (2.0 mL) was kept at 160 ◦C for 24 h. Column chromatography of the cooled solution on silica gel (ﬁrst hexane, thereafter hexane/ether/CHCl3 5:1:1) yielded 9d (36 mg, 48 %) as a colorless solid; mp. 152 ◦C; IR (KBr) ν 2924, 1699, 1611, 1501, 1372, 1280, 1209, −1 1 1037, 764, 711 cm ; H NMR (270 MHz, CDCl3) δ 1.03 (s, 3H, CH3), 1.38 (t, 3H, 3 3 J 7.0 Hz, CH3), 1.51 – 2.96 (m, 13H), 3.72 (s, 3H, OCH3), 4.41 (q, 2H, J 7.0 Hz, 4 3 4 3 OCH2), 6.60 (d, 1H, J 2.7 Hz), 6.67 (dd, 1H, J 8.4 Hz, J 2.7Hz),7.18(d,1H, J 8.4 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 395

13 Hz),7.50 (vs, 2H), 7.98 (s, 1H); C NMR (67.8 MHz, CDCl3) δ 14.47, 17.99, 26.74, 27.79, 29.75, 31.97, 36.18, 37.64, 44.04, 47.84, 55.23, 56.73, 61.16, 111.59, 113.91, 120.04, 122.21, 124.19, 126.11, 129.43, 132.46, 135.71, 137.88, 138.57, 140.01, 153.50, 157.61, 166.53; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 447 (MH+, 76), 401 (22). HRMS Found: 447.1998. Calcd. for C28H31O3S: 447.1994.

3.2.11 Methyl 3’-methoxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-e]1-thiaindene-7- carboxylate (9e)

Thermal cyclization of 7k - a solution of 7k (319 mg, 0.73 mmol) in diphenyl ether (2.5 mL) was kept at 160 ◦C for 15h. The cooled reaction mixture was separated by column chromatography on silica gel (hexane, followed by hexane/ether/CHCl3 4:1:1) to yield 9e (158 mg, 50 %) as a colorless solid, mp. 162 ◦C; IR (KBr) ν 2932, 1708, 1607, 1500, 1436, −1 1 1365, 1280, 1211, 1032, 764, 714 cm ; H NMR (270 MHz, CDCl3) δ 1.11 (s, 3H, CH3), 1.50 – 2.78 (m, 10H), 2.94 – 3.02 (m, 3H), 3.80 (s, 3H, OCH3), 4.00 (s, 3H, COOCH3), 6.67 (d, 1H, 4J 2.7 Hz), 6.75 (dd, 1H, 3J 8.6 Hz, 4J 2.7 Hz), 7.24 (d, 1H, 3J 8.6 Hz), 7.58 13 (bs, 2H), 8.04 (s, 1H); C NMR (67.8 MHz, CDCl3) δ 17.97, 26.71, 27.77, 29.72, 31.94, 36.13, 37.60, 44.00, 47.83, 52.14, 55.21, 56.67, 111.55, 113.88, 120.04, 121.79, 124.20, 126.08, 129.42, 132.42, 135.70, 137.86, 138.58, 140.03, 153.62, 157.56, 166.97; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 433 (MH+, 9), 432 (M+, 8). HRMS Found: 432.1759. Calcd. for C27H28O3S: 432.1759.

3.2.12 3’-Benzoyloxyestra-1’,3’,5’(10’),16’-tetraeno[17’,16’-e]-1-thiaindene (9g) and 3’- benzoyloxy-estra-1’,3’,5’(10’),16’-tetraeno[17’,16’-e]-7-acetyl-1-thiaindene (9f)

Thermal Cyclization of 7f - a solution of 7f (100 mg, 0.20 mmol) was kept at 160 ◦C for 24 h. Thereafter, the cooled solution was subjected to column chromatography on silica gel (hexane, followed by hexane/ether/CHCl3 5:1:1) to produce 3’-benzoyloxy-estra- 1’,3’,5’(10’),16’-tetraeno[17’,16’-e]-1-thiaindene (9g) (10 mg, 11%) as a colorless solid, mp. 194 ◦C; IR (KBr) ν 2928, 1736, 1493, 1452, 1262, 1215, 1173, 1147, 1060, 1021, −1 1 894, 708 cm ; H NMR (270 MHz, CDCl3) δ 1.11 (s, 3H, CH3), 1.52 – 3.05 (m, 13H), 6.97 (d, 1H, 4J 2.4Hz),7.03(dd,1H,3J 8.4 Hz, 4J 2.4 Hz), 7.28 (d, 1H, 3J 8.1 Hz), 13 8.20 (m, 2H); C NMR (67.8 MHz, CDCl3) δ 18.02, 26.64, 27.66, 29.56, 32.20, 36.39, 37.26, 44.37, 47.37, 56.81, 118.76, 120.12, 121.70, 121.83, 126.25, 126.34, 126.44, 128.00, 128.54, 130.17, 133.49, 136.10, 138.09, 138.21, 138.30, 139.00, 147.34, 148.50, 166.60; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 465 (MH+, 2.6). HRMS Found: 465.1888. Calcd. for C31H29O2S: 465.1888 and 3’-benzoyloxy-estra-1’,3’,5’(10’),16’- tetraeno[17’,16’-e]-7- acetyl-1-thiaindene (9f) (50 mg, 50%) as a pale yellow solid, mp. 269 ◦C; IR (KBr) ν 2928, 1735, 1657, 1602, 1544, 1495, 1372, 1253, 1219, 1160, 1080, 1062, 1027, 893, 785, 713 −1 1 cm ; H NMR (270 MHz, CDCl3) δ 1.15 (s, 3H, CH3), 1.62 – 3.09 (m, 13H), 2.75 (s, 3H, 4 3 4 3 COCH3), 6.99 (d, 1H, J 2.7 Hz), 7.02 (dd, 1H, J 8.4 Hz, J 2.7 Hz), 7.39 (d, 1H, J8.4 Hz), 7.47 – 7.67 (m, 3H), 7.58 (d, 1H, 3J 5.7Hz),7.66(d,1H,3J 5.7 Hz), 7.97 (s, 1H), 8.21 13 (m, 2H); C NMR (67.8 MHz, CDCl3) δ 17.99, 26.29, 26.54, 27.60, 29.48, 36.12, 37.27, 44.24, 47.85, 56.77, 118.83, 119.48, 121.74, 124.72, 126.24, 128.63, 129.07, 129.73, 130.13, 396 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402

130.15, 131.11, 135.94, 136.30, 137.82, 138.15, 138.38, 148.88, 154.07, 165.56, 197.10; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 507 (MH+, 2.7). HRMS Found: 507.1995. Calcd. for C33H31O3S: 507.1994.

3.2.13 3’-Benzyloxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-e]-7-phenacyl-1-thiaindene (9h) Thermal cyclization of 7g - 7g (115 mg, 0.20 mmol) in diphenyl ether (1.0 mL) was kept at 160 ◦C for 11 h. Thereafter, the cooled solution was subjected directly to column chromatography on silica gel (initially hexane followed by hexane/ether/CHCl3 4:1:1) to yield 9h (71 mg, 62 %) as a colorless solid; mp. 168 ◦C (unidentiﬁed mesophase from 145◦C); IR (neat) ν 3058, 2924, 2854, 1737, 1641, 1494, 1263, 1215, 1175, 1061, 1023, −1 1 705 cm ; H NMR (270 MHz, CDCl3) δ 1.15 – 3.02 (m, 13H), 1.17 (s, 3H, CH3), 6.97 (d, 1H, 4J 2.7Hz),7.02(dd,1H,3J 8.1 Hz, 4J 2.7 Hz), 7.39 (d, 1H, 3J 8.1 Hz), 7.49 – 13 7.79 (m, 11H), 8.20 (m, 2H); C NMR (67.8 MHz, CDCl3) δ 18.00, 26.57, 27.58, 29.48, 30.91, 36.18, 37.28, 44.26, 47.88, 56.77, 118.84, 119.79, 121.74, 126.24, 127.53, 128.23, 128.54, 129.55, 129.70, 130.15, 130.66, 131.45, 133.51, 136.09, 137.77, 138.05, 138.16, 138.82, 139.79, 148.85, 153.81, 165.44, 195.76; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) + 569 (MH , 61). HRMS Found: 569.2158. Calcd. for C38H33O3S: 569.2150.

3.2.14 3’-Hydroxyestra-1’,3’,5’(10’),16’-tetraeno[16,17-e]-7-phenacyl-1-thiaindene (9h- OH) Asolutionof9h (25 mg, 4.4.10−5 mol) and NaOMe (100 mg, 1.8 mmol) in a mixture of diethylether (5 mL) and methanol (10 mL) was stirred for 10h. Thereafter, the solution was concentrated in vacuo, the residue diluted with water (30 mL), and the resulting mixture acidiﬁed with conc. HCl. The mixture was extracted with chloroform (3 x 20 mL); the combined organic phase was dried over anhydrous MgSO4 and concentrated in vacuo. Column chromatography on silica gel (ether) yielded 9h-OH (13 mg, 65 %) as a colorless solid, mp. 170 ◦C; IR (KBr) ν 3434 (bs, OH), 2926, 1638 (bs, s), 1499, 1282, −1 1 1219, 1057, 730 cm ; H NMR (270 MHz, CDCl3) δ 1.15 (s, 3H, CH3), 1.51 – 2.95 (m, 13H), 4.55 (bs, OH), 6.61 (d, 1H, 4J 2.7 Hz), 6.67 (dd, 1H, 3J 8.6 Hz, 4J 2.7 Hz), 7.18 – 7.26 (m, 2H), 7.48 – 7.79 (m, 7H), 7.72 (s, 1H); MS (FAB, 3-nitrobenzyl alcohol) m/z + (%) 465 (MH , 10). HRMS Found: 465.1890. Calcd. for C31H29O2S: 465.1888.

3.2.15 3’-Methoxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-e]-7-phenacyl-1-thiaindene (9i) Thermal cyclization of 7m - a solution of 7m (250 mg, 0.52 mmol) in diphenyl ether (2.5 mL) was heated at 160 ◦C for 13 h. Elution of the cooled crude mixture on silica gel (hexane followed by hexane/ether/CHCl3 4:1:1) gave 9i (144 mg, 58 %) as a colorless solid, mp. 132 ◦C; IR (KBr) ν 2926, 1639, 1500, 1280, 1256, 1219, 1056, 841, 727, 701 −1 1 cm ; H NMR (270 MHz, CDCl3) δ 1.15 (s, 3H, CH3), 1.52 – 2.78 (m, 11H), 2.95 (m, 4 3 4 2H), 3.80 (s, 3H, OCH3), 6.67 (d, 1H, J 2.6 Hz), 6.75 (dd, 1H, J 8.6 Hz, J 2.6 Hz), 7.25 (d, 1H, 3J 8.6 Hz), 7.48 – 7.78 (m, 5H), 7.63 (d, 1H, 3J 5.8 Hz), 7.70 (d, 1H, 3J 13 5.8Hz),7.72(s,1H); C NMR (67.8 MHz, CDCl3) δ 17.99, 26.71, 27.75, 29.71, 32.02, M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 397

36.17, 37.59, 44.03, 47.91, 55.21, 56.72, 111.56, 113.89, 119.80, 126.09, 127.54, 128.21 (2C), 129.53 (3C), 130.59, 131.43, 132.35, 136.07, 137.81, 138.09, 138.79, 139.75, 153.95, 157.57, 195.89; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 479 (MH+, 14). HRMS Found: + 479.2044. Calcd. for C32H31O2S: 479.2045 (MH ,FAB).

3.2.16 3’-Methoxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-e]-7-(4’-[thien-3”-yl]phenacyl)-1- thiaindene (9j)

Thermal Cyclization of 7j – 7j (75 mg, 0.13 mmol) in diphenyl ether (1.0 mL) was heated at 160 ◦C for 13 h. Column chromatography of the cooled mixture on silica gel (hexane/ether/CHCl3) 4:1:1) gave 9j (48 mg, 65 %) as an oﬀ-white solid; mp. > 300 ◦C; IR (KBr) ν 2924, 1634, 1603, 1497, 1360, 1280, 1255, 1219, 763 cm−1; 1H NMR (270 MHz, CDCl3) δ 1.16 (s, 3H, CH3), 1.45 – 3.01 (m, 13H), 3.80 (s, 3H, OCH3), 6.68 (d, 1H, 4J 2.7Hz),6.75(dd,1H,3J 8.6 Hz, 4J 2.7Hz9,7.27(d,1H,3J 8.6Hz),7.45(dd,1H, 3J 4.8 Hz, 4J 2.7 Hz), 7.48 (dd, 1H, 3J 4.8 Hz, 4J 1.6Hz),7.60(dd,1H,4J 2.7 Hz, 4J 1.6 Hz), 7.63 (d, 1H, 3J 5.9 Hz), 7.70 (d, 1H, 3J 5,9 Hz), 7.75 (d, 2H, 3J 8.6 Hz), 7.77 (s, 1H), 7.84 (d, 2H, 3J 8.6 Hz); MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 561 (MH+,0.6). HRMS Found: 561.1915. Calcd. for C36H33O2S2: 561.1922.

3.2.17 Ethyl 3’-benzyloxyestra-1’,3’,5’(10’),16’-tetraeno[16,17-a]-9-thiaﬂuorene-7- carboxylate (9k)

Thermal cyclization of 7c.–7c was generated by heating 103 mg (0.18 mmol) in diphenyl ether (2 mL) at 160 ◦C for 24 h. The cooled reaction mixture was subjected to column chromatography on silica gel (ﬁrst hexane to elute the diphenyl ether followed by hexane/ether 4:1) to yield 9k (46 mg, 45 %) as colorless needles, mp. 179 ◦C; IR (KBr) ν 2926, 2860, 1713, 1611, 1497, 1438, 1370, 1283, 1255, 1220, 1193, 1113, 1024, 761, 730 −1 1 3 cm ; H NMR (270 MHz, CDCl3) δ 1.10 (s, 3H, CH3), 1.46 (t, 3H, J 7.0 Hz, CH3), 1.47 3 4 – 2.98 (m, 13H), 4.54 (q, 2H, J 7.0 Hz, OCH2), 5.05 (s, 2H), 6.76 (d, 1H, J 2.7 Hz), 6.81 (dd, 1H, 3J 8.6 Hz, 4J 2.7 Hz), 7.24 – 7.46 (m, 8H), 7.61 (s, 1H), 7.85 (dd, 1H, 3J 4 3 4 13 8.1 Hz, J 1.6 Hz), 8.33 (dd, J 7.6 Hz, J 1.6 Hz); C NMR (67.8 MHz, CDCl3) δ 14.30, 16.08, 26.55, 27.80, 29.70, 32.42, 35.04, 37.53, 44.16, 47.56, 56.68, 61.58, 70.01, 112.39, 114.95, 122.63, 123.26, 124.16, 124.88, 126.15, 126.67, 127.08, 127.46, 127.88, 128.56, 132.00, 132.73, 133.95, 134.59, 137.32, 137.88, 139.87, 140.25, 150.07, 156.89, 169.58; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 572 (M+, 22), 307 (37), 154 (100). HRMS Found: 572.2384. Calcd. for C38H36O3S: 572.2385. Found: C, 79.85; H, 6.31%. Calcd. for C38H36O3S: C, 79.69; H, 6.34 %.

3.2.18 4-Acetyl-3’-benzyloxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-a]-9-thiaﬂuorene (9m) and 3’-benzyloxy-estra-1’,3’,5’(10’),16’-tetraeno[16’,17’-a]-9-thiaﬂuorene (9n)

Thermal cyclization of 7d.–7d was produced by heating 38 mg (6.9.10−5 mol) in diphenyl ether (1.5 mL) at 160 ◦C for 24 h. The cooled reaction mixture was subjected to column chromatography on silica gel (first hexane to elute diphenyl ether fol- 398 M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 lowed by hexane/ether 4:1) to yield 3’-benzyloxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-a]- 9-thiafluorene (9n) (10 mg, 28%) as a colorless solid; IR (KBr) ν 2924, 1612, 1495, 1434, −1 1 1232, 1107, 1018, 815, 762, 743 cm ; H NMR (270 MHz, CDCl3) δ 1.11 (s, 3H, CH3), 1.54 – 2.98 (m, 13H), 5.06 (s, 2H), 6.76 (d, 1H, 3J 2.1 Hz), 6.82 (dd, 1H, 3J 8.4 Hz, 4J 2.1 Hz), 7.26 – 7.47 (m, 9H), 7.85 (m, 1H), 7.96 (d, 1H, 3J 8.1 Hz), 8.13 (m, 1H); 13C NMR (67.8 MHz, CDCl3) δ 16.28, 26.57, 27.84, 29.75, 32.67, 35.14, 37.57, 44.30, 46.95, 56.79, 69.99, 112.34, 114.94, 119.37, 121.29, 121.90, 122.77, 124.23, 126.14, 126.24, 127.45, 127.85, 128.55, 132.93, 135.66, 137.94, 141.32, 147.15, 156.95; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 501 (MH+, 1.2), 500 (M+, 1.5); HRMS Found: 500.2166; calcd. for C35H32OS: 500.2174, and 4-acetyl-3’-benzyloxyestra-1’,3’,5’(10’),16’-tetraeno[16’,17’-a]-9- thiafluorene (9m) (20 mg, 52 %) as colorless needles, mp.:175 ◦C, 1H NMR (270 MHz, CDCl3) δ 1.11 (s, 3H, CH3), 1.55 – 3.02 (m, 13H), 2.76 (s, 3H, COCH3), 5.06 (s, 2H), 6.77 (d, 1H, 4J 2.4 Hz), 6.82 (dd, 1H, 3J 8.4 Hz, 4J 2.4 Hz), 7.25 – 7.46 (m, 9H), 7.86 (dd, 1H, 3J 7.6 Hz, 4J 1.6Hz),8.07(dd,1H,3J 7.3 Hz, 4J1.6 Hz); 13C NMR (67.8 MHz, CDCl3) δ 16.13, 26.56, 27.83, 29.71, 31.26, 32.52, 35.07, 37.54, 44.20, 47.51, 56.73, 70.02, 112.41, 114.98, 120.77, 122.81, 124.33, 124.49, 126.15, 126.64, 127.47 (2C), 127.89, 128.57 (2C), 130.66, 132.71, 133.87, 134.74, 136.45, 137.32, 137.88, 139.84, 140.31, 149.50, 156.91, 204.91; MS (FAB, 3-nitrobenzyl alcohol) m/z (%) 543 (MH+, 5.1). HRMS Found: 543.2360. Calcd. for C37H35O2S: 543.2358.

3.3 X-ray crystal structural analysis of 5d 3.3.1 Analysis data

C28H28O2S, Mr 428.59, monoclinic, space group P21 (No. 4), a = 7.054(2) A,˚ b = 7.601(2) ◦ 3 3 A,˚ c = 20.531(6) A,˚ β = 95.652(1) , V = 1095.4(6) A˚ , Z =2,Dcalc = 1.30 g/cm , μ(MoKα)1.71cm−1, F(000) = 456, λ = 0.71070 A.˚ Data were collected using a crystal of 0.15 X 0.05 X 0.05 mm3 on a Rigaku RAXIS-RAPID diffractometer. A total number of 8962 reflections were collected for 3.2◦<θ<27.5◦and -9 ≤ h ≤ 7, -7 ≤ k ≤ 9, -26 ≤ l ≤ 26. In the refinement, 4120 independent reflections and 3353 reflections with I > 2σ(I) were used. The structure was solved by direct methods [28] and refined using CRYSTALS [29]. All H atoms were refined as riding on their parent atoms, with Uiso(H) values set at 1.2 Ueq of the parent O and C atoms. ψ Scan absorption correction was applied with Tmin = 0.990 and Tmax = 0.991. CrystalStructure 3.6.0 [30]wasused for the preparation of the material for publication. The final R indices were [I>2σ(I)] 2 R1 0.042 and (all data), R1 = 0.055, wR2 = 0.119. The goodness-of-fit on F was 1.004 and the largest peak-and-hole difference was 0.42 and -0.44 e A˚−3. The crystallographic data were deposited with the Cambridge Crystallographic Data Centre (CCDC 275437).

3.3.2 Conformational analysis of 5d Ring A demonstrated a little distortion from planarity as was also evident in other estrane derivatives for which X-ray crystal structural analyses were conducted. Ring D had an envelope conformation (Q = 0.367(3) A,˚ φ = 213.6(5)◦; for a perfect envelope conforma- M. Watanabe et al. / Central European Journal of Chemistry 4(3) 2006 375–402 399 tion: φ =kx36◦)[31, 32], with atom C14 as the ﬂap and a pseudorotation angle Δ ◦ ◦ = 15.6(3) , and a minimum torsion angle[33] φm = 36.7(2) for the atom sequence C13, C14,...,C17. Ring B had a half-chair conformation (Q = 0.503(3) A,˚ θg 45.6(4)◦, φ = 158.38(5)◦; for a perfect half-chair conformation: θg 50.8◦, φ =kx60+30◦)[31, 32]. Ring C, with trans-fusion to rings B and D, was ﬁxed in a chair conformation (Q = 0.595(3) A,˚ θg8.7(3)◦, φ = 293(12)◦; for a perfect chair conformation: θg0◦)[31, 32].

3.3.3 Description of the packing of 5d in the crystal

In the crystal, 5d packed in columns of pairs of molecules, where the interaction between the molecules in the pair was a CH-pi interaction [34] (C25 – H25. . . Cg1 [H25. . . Cg1: 2.84 A;˚ symmetry code: 1-x, y-1/2, -z]). Close contact between molecules facing in the same direction existed (interpair but intracolumn): (C18-H(C18). . . Cg1 [H(C18). . . Cg1: 2.87 A,˚ where Cg1 was an atom of the thiophene ring, symmetry code: x + 1, y, z]). Between columns,inthedirectionoftheb-axis,a shortcontactexistedatS1...O1(3.239(2) A;˚ symmetry code: x, y + 1, z).

Acknowledgment

The authors thank Ms. Yasuko Tanaka for mass and high resolution mass measurements. Elemental analyses were conducted at the Center for Instrumental Analysis, Hakozaki Campus, Kyushu University.

References

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