The synthesis of disteroidal macrocyclic molecular rotors by an RCM approach

Tetrahedron 70 (2014) 9427e9435

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The synthesis of disteroidal macrocyclic molecular rotors by an RCM approach Dorota Czajkowska-Szczykowska a, *, Izabella Jastrzebska a, Rosa Santillan b, Jacek W. Morzycki a a b

Institute of Chemistry, University of Białystok, Piłsudskiego 11/4, 15-443 Białystok, Poland n y de Estudios Avanzados del IPN, Apdo. Postal 14-740, M Departamento de Química, Centro de Investigacio exico D.F. 07000, Mexico

a r t i c l e i n f o

a b s t r a c t

Article history: Received 21 May 2014 Received in revised form 22 September 2014 Accepted 6 October 2014 Available online 17 October 2014

The syntheses of four macrocyclic molecular rotors are described. The starting oxosteroids, 5b-androsterone, and 23-oxosarsasapogenin, were subjected to Grignard ethynylation followed by Sonogashira coupling with 1,4-diiodobenzene. The obtained acyclic dimers were further transformed into the corresponding 3-esters with terminal double bonds. These compounds were converted into the final products via an RCM protocol. Ó 2014 Elsevier Ltd. All rights reserved.

Keywords: Steroids Molecular rotors Macrocyclization Sonogashira coupling Metathesis

1. Introduction In recent years, nanotechnology has become one of the most fascinating and prosperous fields of study, mainly because its achievements can be applied in materials science.1 Noteworthy, biological systems are the main inspiration for the study of molecular machines. In myosin, the common example of a motor protein, chemical energy released during ATP hydrolysis is converted into mechanical energy when this protein moves along linear bundles of the actin (typically, a linear motor can generate forces of up to w10 pN).2 This molecular motion of myosin is responsible for the lengthening and shortening of filaments that are intensified and changed into useful macroscale motion. The fundamental biological functions, e.g., muscle contraction, transport of membrane vesicles, cell division, and locomotion, can be achieved by this means.3 The concept of molecular machinery, followed by the chemical synthesis resulted in the growth of the number of compounds that emulate the structure and dynamics of macroscopic objects. Some examples of macromolecular systems working this way include catenanes, rotaxanes or pseudorotaxanes.4 Our interests, however, have focused on designing molecules with shapes inspired by

* Corresponding author. Tel.: þ48 857457604; fax: þ48 857457595; e-mail address: [email protected] (D. Czajkowska-Szczykowska). http://dx.doi.org/10.1016/j.tet.2014.10.022 0040-4020/Ó 2014 Elsevier Ltd. All rights reserved.

modern technologydcreated forms are composed of analogous parts to macroscopic gyroscopes. The fundamental elements of this kind of structure are: a static component, placed to support the dynamic parts of the system, and a rotating component. The first is defined as the stator, whereas the second is the rotator. These terms are implicitly related to amphidynamic crystalsdassemblage of autonomous systems that undergo rapid molecular motion within the close-packed environment.5 The essential approach to provide dynamically operating crystals is based on the accurate control of the rotator and the stator alignment associated with the source of energy. If those demands are fulfilled, proper functions of the materials depending on the purpose are attained. Ultimately, they can find application in electronics or optoelectronics,6 i.e., techniques that take advantage of a specific property of light for data detection (photosensors), data storage (CD or DVD), data transmission (optical fibers), data processing, and presentation (LCD, PDP). This kind of molecular rotor can be attached to various types of surface, e.g., plastics or polymers,7 dissolved in solution,8 or constitute self-assembled crystalline solids.8a,9 The rotors are also used in medicine, for example, as viscosity sensors in living cells.10 It has been established that there is a significant correlation between structure and dynamics of molecular gyroscopes in the crystal lattice. The stator should provide an encapsulating frame to shield the rotator from steric contacts with adjacent molecules in the crystal. This can be achieved in many ways, e.g., by suitable

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prevent disadvantageous interdigitation, and to form a local cavity that improves dynamics of the molecular rotors. A cis-linkage between steroid rings A and B is important, as it facilitates closure of the macrocyclic dimer through a metathesis protocol. The essential transformation in the synthetic routes to the rotors was introduction of the rotating component (rotator) between the carbon atoms of rings D of two molecules of 5b-androsterone 3 or between the positions 23 of two molecules of 23-oxosarsasapogenin 11. In the first synthesis, the 3a-hydroxyl group of 3 was protected as silyl ether 4 by reacting with TBS-Cl in DMF using imidazole as a base (Scheme 1). It was next reacted with ethynylmagnesium chloride in dry THF at 0  C to afford the 17a-ethynylsubstituted product 5 in 61% yield. It is well-known that the addition of the Grignard reagent to 17-oxosteroids is stereocontrolled by the angular methyl group at C13.20 For this reason, the 17a-ethynyl-17b-ol derivative was exclusively formed. The structure of product 5 was confirmed by the appearance of the one-proton signal of the free alkyne at d¼2.62 (s, 1H) in the 1H NMR spectrum and of the two peaks at d¼74.1 and 87.5 of acetylenic carbon atoms in the 13C NMR spectrum with simultaneous disappearance of the C-17 signal at d¼221.4 corresponding to the carbonyl group of 4. To the terminal alkyne carbon atom, the phenylene group was attached by the Sonogashira cross-coupling reaction with 1,4-diiodobenzene. This transformation proved to be more demanding, because the desired dimer 6 was not the only product of the reaction. The employed conditionsdPd(PPh3)2Cl2/CuI/DIPEA, caused the side reactions: Glaser acetylenic coupling21 and partial coupling concomitant with reduction. As a result, compound 6 (33% yield) was produced, accompanied by the diacetylenic dimer 7 (18% yield) and the phenylacetylene compound 8 (19% yield) (Scheme 1). The dimeric structure of 6 was evidenced by the appearance of the phenylene four-proton signal at d¼7.47 as required. Deprotection of the 3a-OH groups in 6 with TBAF, followed by Steglich esterification22 with 3-butenoic acid afforded diester 9 in 98% yield (Scheme 2). The tertiary hydroxyl group at C-17 remained intact under these conditions. The length of the acid chain for this reaction was chosen by inspection of Dreiding models, and confirmed by molecular modeling using MMþ force-field (HyperChem),23 so as to obtain in the next step the smallest low-strain macrocycle. The structure 9 was proved by the 1H NMR spectrum, in which three characteristic multiplets at d¼5.14 (2H), 5.18 (2H), and 5.93 (2H) of the 3-butenoate olefinic protons appeared. In the following step the ring-closing metathesis was performed. The macrocyclization was carried out in the presence of the HoveydaeGrubbs second-generation catalyst in toluene. A mixture of two (E/Z) isomers of 1 in the ratio of 2 to 1 was obtained in 67% yield. After HPLC separation, the diastereoisomers were

spatial shape of the stator and/or bridging chains introduced between them. The rotator may be any axially symmetric group with its center of mass aligned along a single bond that supplies both the rotary axis and the point of attachment to the rigid frame of the stator.11 Following the studies on molecular gyroscopes there is an opportunity to contribute to future developments in this topic, especially when the comparison with already synthesized molecular rotors was performed. Having in mind numerous examples of rotors achieved by Garcia-Garibay5a,12,13 and Gladysz,14 we have carried out a detailed analysis of the kind of molecules that provide the most promising designs in the field of molecular machines and materials, based on dynamically functional crystals. This analysis prompted us into the synthesis of molecular rotors based on steroidal units. Steroids, apart from regulation of different biological processes and being drugs for the treatment of a large number of diseases,15 frequently have been used as significant building blocks in the formation of macrocyclic structures.16 We have decided to synthesize a series of prospective steroidal molecular rotors that may lead to the preparation of new rotary dielectrics and novel molecular compasses, especially since several examples of their utility have been already published.17

2. Results and discussion 2.1. Syntheses of macrocycles Here we report two syntheses of rotors based on a steroidal framework built from 5b-androsterone (3)18 and 23-oxosarsasapogenin (11).19 In these rotors the 1,4-diethynylphenylene group acts as a rotator (Fig. 1). Additionally, one meridional (northesouth) bridging chain was introduced between the two steroidal units to OH O

O

O

O O

O

O

O

O

OH

O

OH

O

OH

O

1E, 1Z

2E, 2Z

Fig. 1. Molecular rotors based on 1,4-diethynylphenylene rotator prepared in this work aimed to emulate the macroscopic gyroscope.

O

HO

OH

O TBS-Cl imidazole DMF 98%

H

HC C Mg Cl TBSO

THF 61%

H

3

TBSO

H

5

4

OH I

OH

I

Pd(PPh ) Cl CuI, DIPEA

OH

OTBS

+

THF

OTBS

+

OTBS

TBSO

OTBS

H

OH OH

7 (18%)

6 (33%) Scheme 1. Synthetic route to the compound 6 from 5b-androsterone 3.

8 (19%)

D. Czajkowska-Szczykowska et al. / Tetrahedron 70 (2014) 9427e9435

OH

6

2.

O

toluene 67%

O

OH

O

Hoveyda-Grubbs II (25 mol%)

O

O

DCC/DMAP CH Cl 98%

1E

OH

O 1. TBAF/THF; 86%

9429

O

HPLC separation O

O

OH

OH

9

1Z

1 (E/Z = 2:1)

Scheme 2. Synthetic route to macrocycles 1E and 1Z from 6.

distinguished by a difference in the 1H NMR spectradthe signal of the olefinic protons of 1E appeared at d¼5.72 (m, 2H), whereas that of 1Z was observed at d¼5.80 (m, 2H). More information was provided by HSQC experiment. Based on these spectra we could properly identify the E and Z isomers, because the chemical shift values of the carbon atoms a to the double bond allow to assign the configuration. For compound 1E, the allylic CH2 group peak appeared at d¼39.3 due to downfield shift,24 whereas for the compound 1Z at d¼33.4. Small differences have been also noticed in the infrared spectra, where stretching bands for the ester groups occurred at 1711 and 1713 cm1 for the isomers E and Z, respectively. HRMS analysis showed an m/z peak at 837.5086 for the 1E and 1Z compounds corresponding to the expected molecular ions clustered with a sodium ion. The synthesis of the second cyclic rotor started from sarsasapogenin acetate (10),25 that was converted to 23oxosarsasapogenin (11) in a classical manner, using an improved Barton procedure.26 The 3b-hydroxyl group of 23-oxo compound 11 was then protected as a silyl ether 12 under standard conditions (Scheme 3). Further treatment of 23-ketone 12 with ethynylmagnesium chloride in dry THF at 0  C provided stereoselectively the 23b-hydroxy-23a-ethynyl derivative 13. The attack of the Grignard compound from the a-side of 23-carbonyl group is a consequence of steric hindrance from the axial methyl group at C25 position. The product 13 was obtained in a moderate yield 53%. The structure 13 was established by 1H NMR spectroscopy showing a signal at d¼2.41 (s, 1H) coming from the alkyne proton. Sonogashira coupling was a key reaction, which allowed us to obtain dimer 14. In this step a palladium(0) complexdPd(PPh3)427 was employed, instead of Pd(PPh3)2Cl2 used in the previous synthesis. This modification of reaction conditions was successful, because the desired dimer 14 was produced in higher yield (52%), accompanied by only one minor productdthe iodo-compound 15 (20%

2.2. Single crystal X-ray diffraction studies Single crystals of molecular rotor 2E suitable for X-ray diffraction studies were grown by slow evaporation of a saturated solution in chloroform. The ORTEP diagrams derived from data acquired at 173(2) K are illustrated in Fig. 2. The structure 2E was solved in the orthorhombic space group P212121 with one molecule per

1. BF . Et O NaNO AcOH

O O AcO

yield). The structure of dimer 14 was confirmed by the appearance of aromatic protons signal at d¼7.33 (s, 4H) in 1H NMR and the absence of the alkyne proton signal. The tert-butyldimethylsilyl ether group was then deprotected with BF3$Et2O in dichloromethane at 0  C with excellent yield.28 After esterification29 with 5-hexenoic acid in the presence of DCC as a promoter and DMAP, acyclic dimer 17 was prepared being a substrate for RCM. Because of steric hindrance, the esterification of the tertiary 23-hydroxyl group was not observed. Ester 17 was characterized by the presence of signals of terminal olefinic protons at d¼5.78 (m, 2H) and 5.01 (m, 4H). The described synthesis is shown in Scheme 4. Ring-closing metathesis of compound 17 afforded a mixture of diastereoisomeric compounds 2E and 2Z (Scheme 5) with the ratio dependent on catalyst used. Results of the RCM reactions are presented in Table 1. HPLC showed two well-resolved peaks coming from the distinct isomeric dimers. Unfortunately, irrelevant differences in their 1H NMR spectra were observed. The olefinic protons of 2E and 2Z were present at d¼5.41 in both cases. The only difference was observed for 18- and 19-protons. In 2Z the singlet coming from methyl protons appeared at d¼1.000 while in 2E compounddat d¼1.013. The methyl proton singlet in 2Z was observed at d¼0.973 while in 2E dimerdat d¼0.964. The structure of rotor 2E was unequivocally proved by an X-ray experiment (Fig. 2).

O O O

2. NaHCO MeOH 46%

H

HO

TBSO

H

11

12

O

TBSO OH O C Mg Cl O

THF 53%

OH

O

I

I

TBSO

O O

DMF 73%

H

10

HC

O

TBS-Cl imidazole

OOH

Pd(PPh ) CuI, DIPEA

+

THF

H

O

13

TBSO

OH O

14 (52%) Scheme 3. Synthetic route to dimer 14.

O TBSO

H

I

15 (20%)

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BF3.Et2O CH2Cl2 99%

CH2Cl2 60% O

OH

O CH2=CH(CH2)3COOH DCC, DMAP

HO

O

O

OH

O

14

O

O

HO

O

OH O

O

16

O

OH O

17

Scheme 4. Synthetic route to acyclic dimer 17.

O

O

O

OH

O

17

2E

RCM

HPLC separation

toluene O O

O

OH O

2Z

2

Scheme 5. Completion of the synthesis of macrocycles 2E and 2Z.

asymmetric unit and four molecules in the unit cell. This compound also contained one chloroform molecule located outside of the macrocyclic cavity, but with no noticeable disorder.32 The molecular structures of the macrocyclic rotors revealed that the 1,4-diethynylphenylene fragment is close to linearity (2E¼176.12 ) and relatively distant from the bridging chain. At the supramolecular level, there are close contacts between the bridge and the 1,4-diethynylphenylene fragment. Also, in the molecular rotor, the chain was only in one position, contrary to what is commonly observed in flexible fragments containing alkene functionalities (of course the ‘invisibility’ of this motion has been recognized and although we do not discard it, no further variable temperature X-ray diffraction studies were conducted to properly prove or disprove the motion).33

Table 1 Results of metathesis reaction of acyclic dimer 17

PCy3 Cl Ru Cl PCy3 Ph

N

Mes Mes Cl Ru Cl PCy3 Ph

A Temperature ( C) Time of reaction (min) Yield (%) Ratio of isomers (E/Z)

80 40 85 64:36

N

N N Mes Mes Cl Ru Cl O i Pr

B 100 50 68 87:13

Fig. 2. ORTEP diagrams of compound 2E with the chloroform molecule. The ellipsoids are drawn at 50% probability.

N N Mes Mes Cl Ru Cl O Me

C 100 60 75 83:17

30

D 90 55 59 81:19

N N Mes Mes Cl Ru Cl O

OMe OMe

E31

Me

100 55 57 81:19

The trans substitution in the isomer 2E causes the bridge to acquire a conformation similar to a distorted ‘S’, which places the two steroids in a staggered position relative to the diethynyl molecular axis. The C3eC30 distance between the edges of the steroid fragments in 2E is 9.65  A (see Supplementary data). The macrocyclic molecular rotor 2E presents a very distinctive packing array in its corresponding crystal. The packing arrangement can be described as a combination of normal OH/O and ‘weak’ CH/O hydrogen bonds that hold the molecules together. Rotor 2E (space group P212121) packs in perpendicular interpenetrating layers with the rotator fragment facing towards the next coplanar neighboring bridge (Fig. 3). The packing array of 2E is very similar to the one observed in the Z isomer of the molecular rotor derived from 17a-ethynyl-5a-androstan-17b-ol17c where the phenylene rotator undergoes motion in the fast exchange regime at 273 K with a rotational activation barrier of 7.19 kcal/mol. In accordance with literature reports, the double bond proximity to the neighboring rotator plays a major role defining the internal rotation. Similarly, the hydroxyl groups at C17 of neighboring rotors interact with each other with DeA distance of 2.78 and :DeA of 172.9 . Additional distances and angles of the observed

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CDCl3 (77.0 ppm), respectively, unless otherwise noted. NMR resonance multiplicities are reported using the following abbreviations: br¼broad, s¼singlet, d¼doublet, t¼triplet, q¼quartet, and m¼multiplet; and coupling constants J are reported in hertz (Hz) (only selected signals in the 1H NMR spectra are reported). IR spectral data were obtained using Magna IR 550 Nicolet spectrometer (taken in CHCl3 solutions or KBr pellets) and are reported € etius in cm1. Melting points were determined by Kofler bench (Bo type) melting point apparatus. HRMS were obtained on an electrospray ionization-time of flight (ESI-TOF) or electron ionization (EI) Micromass spectrometer. The HPLC separations of the isomeric rotors were performed, using a LabAlliance instrument equipped with UVevis detector, 525 model (wave range 190e800 nm) and reversed phase semipreparative columns LC 18 (Supelco). The HPLC grade solvents (CH3CN and CH2Cl2) were purchased from JT Baker.

Fig. 3. Crystal packing of compound 2E with an interpenetrated array. The chloroform molecules contained in the structure were removed for clarity purposes. CCDC no.1030165.

intermolecular interactions in compound 2E can be found in Supplementary data. The obtained new molecular rotors will be tested in order to determine a relationship between structure and dynamics for each isomer. 3. Conclusion The promising macrocyclic molecular rotors 1E, 1Z, 2E, and 2Z were successfully synthesized and characterized. The two isomers of compound 1 were prepared quite efficiently via a six-step synthesis starting from 5b-androsterone (3), while the E and Z isomers of compound 2dthrough an eight-step synthesis starting from sarsasapogenin acetate (10). In general, the syntheses consisted of similar transformations, including palladium-catalyzed coupling reaction, Steglich esterification and RCM reaction. It was also demonstrated that the replacement of the catalyst Pd(PPh3)2Cl2 by Pd(PPh3)4 in the Sonogashira coupling improves the yield of the desired product and prevents formation of by-products. Analysis of the crystal structure of 2E revealed that the 1,4-diethynylphenylene units are close to linear and comparatively distant from the bridging chain. However, the double bond proximity to the neighboring rotator is crucial defining the internal rotation and is expected to be more restricted in the E isomers regardless the steroidal frameworks. Further physicochemical study will show if constructing macrocyclic rotors from steroids with cis-fused A/B rings provides prospective materials for modern technology. 4. Experimental section 4.1. General All reagents were purchased from SigmaeAldrich and used as received. The solvents were dried prior to use by distillation over the following drying agents: DMF (4  A molecular sieves), THF (Na/ benzophenone), toluene, and methylene chloride (CaH2). Flash column chromatography and dry flash chromatography were performed with JT Baker silica gel, pore size 40  A (70e230 mesh), unless otherwise stated. Reactions were monitored by TLC on silica gel plates 60 F254 (Merck) and spots were visualized either by UV lamp (UV Emita VP 60, 180 W) and/or by charring with H2SO4/ vanillin in ethanol. All reactions were carried out under argon atmosphere using standard Schlenk techniques. 1H and 13C NMR data for all previously uncharacterized compounds were recorded at ambient temperature using Bruker AC 200F or Bruker Ultrashield Plus 400 spectrometers and are referenced to TMS (0.0 ppm) and

4.2. 3a-tert-Butyldimethylsilyloxy-5b-androstan-17-one (4) To 5b-androsterone (3) (320 mg, 1.1 mmol) dissolved in dry DMF (5 mL) in a 25 mL round-bottomed flask, imidazole (225 mg, 3.3 mmol, 3 equiv) and tert-butyldimethylsilyl chloride (200 mg, 1.33 mmol, 1.2 equiv) were added. The reaction mixture was stirred at ambient temperature for 1 h. Then, the reaction was quenched by water and extracted three times with benzene (100 mL each). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Dry flash chromatography (gradient, 99:1 to 97:3 hexane/AcOEt) afforded product 4 (437 mg, 98% yield) as a colorless oil: Rf¼0.62 (hexane/ AcOEtd8:2). nmax (CHCl3): 2931, 2859, 1732, 1471, 1374, 1255, 1053, 837 cm1. dH (ppm, CDCl3): 3.56e3.63 (m, 1H, TBSOCH), 2.44 (dt, J¼8.2, 0.4 Hz, 1H, C(O)CHaHb), 2.08 (dt, J¼19.2, 9.3 Hz, 1H, C(O) CHaHb), 0.94 (s, 3H, Me), 0.89 (s, 9H, t-BuSi), 0.85 (s, 3H, Me), 0.06 (s, 6H, Me2Si); dC (ppm, CDCl3): 221.4 (C), 72.6 (CH), 51.4 (CH), 47.9 (C), 42.2 (CH), 40.5 (CH), 36.9 (CH2), 35.9 (CH2), 35.5 (CH2), 35.4 (CH), 34.8 (C), 31.7 (CH2), 31.0 (CH2), 27.0 (CH2), 25.9 (3CH3), 25.3 (CH2), 23.3 (CH3), 21.8 (CH2), 20.1 (CH2), 18.3 (C), 13.8 (CH3), 4.6 (2CH3). HRMS (ESI) calculated for [C25H44O2SiNa]þ requires m/z 427.3008, found m/z 427.3019. 4.3. 3a-tert-Butyldimethylosilyloxy-17a-ethynyl-5b-androstan-17-ol (5) A solution of 3a-tert-butyldimethylsilyloxy-5b-androstan-17one (4) (400 mg, 0.99 mmol) in dry THF (20 mL) was placed in a flame-dried 50 mL round-bottomed flask. Then ethynylmagnesium chloride (2.4 mL, 0.5 M in THF, 1.19 mmol, 1.2 equiv) was added to the reaction mixture at ambient temperature. After 3 h, the reaction was quenched by slow addition of saturated NH4Cl (50 mL). The phases were separated, and the aqueous phase was extracted twice with Et2O (100 mL each). The combined organic layers were washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Flash column chromatography (gradient, 98:2 to 97:3 hexane/AcOEt) afforded product 5 (260 mg, 61% yield) as a white solid. Mp 131e133  C (hexane/AcOEt). Rf¼0.54 (hexane/AcOEtd8:2). nmax (CHCl3): 3598, 3304, 2930, 2859, 1471, 1254, 1055, 837 cm1. dH (ppm, CDCl3): 3.56e3.63 (m, 1H, TBSOCH), 2.62 (s, 1H, C^CH), 2.23e2.33 (m, 1H, HOC(C^CH)eCHaHb), 1.92e2.01 (m, 1H, HOC(C^CH)eCHaHb), 0.93 (s, 3H, Me), 0.91 (s, 9H, t-BuSi), 0.83 (s, 3H, Me), 0.08 (s, 6H, Me2Si); dC (ppm, CDCl3): 87.5 (C), 79.9 (C), 74.1 (CH), 72.9 (CH), 50.3 (CH), 46.9 (C), 42.3 (CH), 40.0 (CH), 38.9 (CH2), 36.9 (CH2), 36.5 (CH), 35.6 (CH2), 34.7 (C), 32.8 (CH2), 31.0 (CH2), 27.1 (CH2), 26.9 (3CH3), 26.0 (CH2), 23.3 (CH3), 23.1 (CH2), 20.4 (CH2), 18.4 (C), 12.7 (CH3), 4.7 (2CH3). HRMS (EI) calculated for [C27H46O2SiNa]þ requires m/z 453.3165, found m/z 453.3181.

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4.4. 1,4-Bis[3a-[(tert-butydimethyl)silyl]-17b-hydroxy-5b-androstan-17a-yl-ethynyl]benzene (6) A solution of bis(triphenylphosphine)palladium(II) dichloride (9 mg, 0.013 mmol, 10 mol %) and 1,4-diiodobenzene (22 mg, 0.67 mmol, 0.52 equiv) in dry THF (10 mL) was placed in a flamedried 50 mL round-bottomed flask. To the resulting homogeneous solution, compound 5 (55 mg, 0.13 mmol) was added and the reaction mixture was heated to 42  C. After 10 min, copper(I) iodide (2.5 mg, 0.013 mmol, 10 mol %) and N,N-diisopropylethylamine (0.2 mL, 8.61 mmol, 5.9 equiv) were added. After stirring at 42  C for 22 h, the reaction mixture was cooled to room temperature, filtered through a Celite pad in the fritted funnel, and washed by Et2O (100 mL). Then, the mixture of solvents was washed with brine, dried over Na2SO4, and concentrated under reduced pressure. Flash column chromatography afforded desired product 6 (20 mg, 33% yield) as a white solid, dimer 7 (10 mg, 18% yield) as an amorphous solid, and product 8 (12 mg, 19% yield) as an amorphous solid (gradient from 94:6 to 92:8, 96:4 to 94:6 and 98:2 to 96:4 hexane/AcOEt, respectively). 4.4.1. Compound 6. Mp 140e142  C (hexane/AcOEt). Rf¼0.65 (hexane/AcOEtd7:3). nmax (CHCl3): 3596, 2931, 2858, 1254, 1069, 838 cm1. dH (ppm, CDCl3): 7.47 (s, 4H, Ph), 3.55e3.64 (m, 2H, TBSOCH), 2.33e2.42 (m, 2H, HOC(C^C)eCHaHb), 2.01e2.11 (m, 2H, HOC(C^C)eCHaHb), 0.94 (s, 6H, Me), 0.90 (s, 18H, t-BuSi), 0.88 (s, 6H, Me), 0.07 (s, 12H, Me2Si); dC (ppm, CDCl3): 131.7 (4CH), 122.8 (2C), 94.4 (2C), 85.9 (2C), 80.5 (2C), 72.9 (2CH), 50.6 (2CH), 47.5 (2C), 42.4 (2CH), 40.0 (2CH), 39.3 (2CH2), 37.0 (2CH2), 36.7 (2CH), 35.7 (2CH2), 34.8 (2C), 33.2 (2CH2), 31.0 (2CH2), 27.3 (2CH2), 26.1 (2CH2), 26.0 (6CH3), 23.4 (2CH2), 23.4 (2CH3), 20.5 (2CH2), 18.4 (2C), 12.9 (2CH3), 4.6 (4CH3). HRMS (ESI) calculated for C60H94O4Si2Naþ requires m/z 957.6588, found m/z 957.6601. 4.4.2. Compound 7. Rf¼0.67 (hexane/AcOEtd7:3). dH (ppm, CDCl3): 3.56e3.66 (m, 2H, TBSOCH), 2.34e2.44 (m, 2H, HOC(C^C)eCHaHb), 1.98e2.06 (m, 2H, HOC(C^C)eCHaHb), 0.93 (s, 6H, Me), 0.91 (s, 18H, t-BuSi), 0.83 (s, 6H, Me), 0.08 (s, 12H, Me2Si). HRMS (ESI) calculated for [C54H90O4Si2Na]þ requires m/z 881.6275, found m/z 881.6289. 4.4.3. Compound 8. Rf¼0.68 (hexane/AcOEtd7:3). dH (ppm, CDCl3): 7.50e7.72 (m, 2H, Ph), 7.33e7.36 (m, 3H, Ph), 3.55e3.64 (m, 1H, TBSOCH), 2.33e2.42 (m, 1H, HOC(C^C)eCHaHb), 1.99e2.11 (m, 1H, HOC(C^C)eCHaHb), 0.94 (s, 3H, Me), 0.90 (s, 9H, t-BuSi), 0.88 (s, 3H, Me), 0.08 (s, 6H, Me2Si); dC (ppm, CDCl3): 131.7 (2CH), 128.2 (3CH), 123.0 (C), 92.7 (C), 86.0 (C), 80.4 (C), 72.9 (CH), 50.5 (CH), 47.4 (C), 42.4 (CH), 40.0 (CH), 39.3 (CH2), 36.9 (CH2), 36.7 (CH), 35.7 (CH2), 34.8 (C), 33.1 (CH2), 31.0 (CH2), 27.3 (CH2), 26.0 (CH2), 26.0 (3CH3), 23.4 (CH3), 23.4 (CH2), 20.5 (CH2), 18.4 (C), 12.9 (CH3), 4.6 (2CH3). HRMS (ESI) calculated for [C33H50NaO2SiNa]þ requires m/z 529.3478, found m/z 529.3488. 4.5. 1,4-Bis[3a-(but-30 -enoyloxy)-17b-hydroxy-5b-androstan17a-yl-ethynyl]benzene (9) A solution of dimer 6 (68 mg, 0.07 mmol) in dry THF (3 mL) was placed in a 10 mL round-bottomed flask, to which tetra-n-butylammonium fluoride (0.7 mL, 1 M in THF, 0.7 mmol, 10 equiv) was dropwise added. After stirring at room temperature for 2 h, the reaction mixture was poured into water and extracted three times with Et2O (50 mL each) and twice with AcOEt (50 mL each). The combined organic layers were dried over Na2SO4, and evaporated in vacuo. Flash column chromatography (gradient, 6:4 to 1:1 hexane/ AcOEt) afforded deprotected 6 (44 mg, 86% yield) as a white solid. Mp 187  C (hexane/AcOEt). Rf¼0.31 (hexane/AcOEtd1:1). nmax

(CHCl3): 3601, 3407, 2929, 1222, 1034 cm1. dH (ppm, CDCl3): 7.41 (s, 4H, Ph), 3.56e3.64 (m, 2H, TBSOCH), 2.32e2.41 (m, 2H, HOC(C^C)eCHaHb), 1.98e2.08 (m, 2H, HOC(C^C)eCHaHb), 0.93 (s, 6H, Me), 0.87 (s, 6H, Me). dC (ppm, CDCl3): 131.6 (4CH), 122.8 (2C), 94.6 (2C), 85.5 (2C), 80.3 (2C), 71.7 (2CH), 50.7 (2CH), 47.6 (2C), 42.1 (2CH), 40.2 (2CH), 39.1 (2CH2), 36.5 (2CH), 36.2 (2CH2), 35.3 (2CH2), 34.6 (2C), 33.1 (2CH2), 30.3 (2CH2), 27.0 (2CH2), 26.0 (2CH2), 23.3 (2CH3), 23.3 (2CH2), 20.5 (2CH2), 12.9 (2CH3). HRMS (ESI) calculated for [C48H66O4Na]þ requires m/z 729.4859, found m/z 729.4871. The linear dimeric tetraol (deprotected 6) (30 mg, 0.04 mmol), N,N0 -dicyclohexylcarbodiimide (DCC) (25 mg, 0.12 mmol, 3 equiv), and 4-dimethylaminopyridine (DMAP) (1 mg, 0.008 mmol, 0.2 equiv) were placed together in a 20 mL round-bottomed flask and dissolved in methylene chloride (10 mL). After stirring at 26  C for 5 min, 3-butenoic acid (0.008 mL, 8 mg, 0.09 mmol, 2.3 equiv) was dropwise added. The reaction mixture was stirred at the same temperature for 48 h, then the mixture was cooled to room temperature, filtered through Na2SO4 pad in the fritted funnel, and washed with CH2Cl2 (50 mL). Finally, the filtrate was concentrated under reduced pressure. Flash column chromatography (gradient from 84:16 to 82:18 hexane/AcOEt) afforded diester 9 (35 mg, 98% yield) as a colorless oil: Rf¼0.51 (hexane/AcOEtd7:4). nmax (CHCl3): 3596, 2928, 2858, 1721, 1180, 1025 cm1. dH (ppm, CDCl3): 7.46 (s, 4H, Ph), 5.87e5.98 (m, 2H, CH2]CHeCH2), 5.17e5.19 (m, 2H, CHaHb]CHeCH2), 5.13e5.15 (m, 2H, CHaHb]CHeCH2), 4.72e4.81 (m, 2H, C(O)OCH), 3.06 (dt, J¼6.9, 1.4 Hz, 4H, CH2]CHeCH2), 2.33e2.42 (m, 2H, HOC(C^C)eCHaHb), 2.01e2.11 (m, 2H, HOC(C^C)eCHaHb), 0.97 (s, 6H, Me), 0.89 (s, 6H, Me). dC (ppm, CDCl3): 171.1 (2C), 131.7 (4CH), 130.5 (2C), 122.9 (2CH), 118.3 (2CH2), 94.7 (2C), 85.6 (2C), 80.5 (2C), 74.7 (2CH), 50.7 (2CH), 47.5 (2C), 42.0 (2CH), 40.2 (2CH), 39.5 (2CH2), 39.3 (2CH2), 36.6 (2CH), 35.1 (2CH2), 34.8 (2C), 33.2 (2CH2), 32.3 (2CH2), 27.0 (2CH2), 26.7 (2CH2), 26.0 (2CH2), 23.4 (2CH2), 23.4 (2CH3), 20.5 (2CH2), 12.9 (2CH3). HRMS (ESI) calculated for [C56H74O6Na]þ requires m/z 865.5383, found m/z 865.5399. 4.6. Ring-closing metathesis: synthesis of macrocycles 1E and 1Z A solution of diester 9 (14 mg, 0.017 mmol) in dry toluene (150 mL) was placed under argon atmosphere in a flame-dried 250 mL round-bottomed flask, then the reaction mixture was heated to 80  C. After 5 min, HoveydaeGrubbs second-generation catalyst (2.7 mg, 0.0043 mmol, 25 mol %) was added. The reaction mixture was stirred for 23 h at 80  C, then cooled to room temperature, and quenched with ethylvinyl ether (0.5 mL). The solvents were removed under reduced pressure. The separation of the products (isomers 1E and 1Z) from the catalyst and/or its decomposition products was performed by flash column chromatography (gradient from 84:16 to 82:18 hexane/AcOEt using 230e400 mesh silica gel). The mixture of E/Z isomeric rotors 1E and 1Z was obtained (9 mg, 67% yield; E/Z ratiod2:1, determined by 1H NMR analysis). The separation of the isomers was achieved by HPLC (see: General information) with a solvent mixture CH3CN/CH2Cl2 (95:5) using a reversed phase column. 4.6.1. Compound 1E. The retention time (tR)¼13.15 min. Isomer 1E is a white solid. Mp 272e274  C (CH3CN/CH2Cl2). Rf¼0.49 (hexane/ AcOEtd6:4). nmax (CHCl3): 3596, 2932, 2856, 1711, 1179 cm1. dH (ppm, CDCl3): 7.47 (s, 4H, Ph), 5.68e5.78 (m, 2H, C]CH), 4.69e4.79 (m, 2H, C(O)OCH), 3.04e3.06 (m, 4H, C]CHeCH2), 2.33e2.44 (m, 2H, HOC(C^C)eCHaHb), 2.01e2.11 (m, 2H, HOC(C^C)eCHaHb), 0.97 (s, 6H, Me), 0.89 (s, 6H, Me). dC (100 MHz, CDCl3): 170.8 (2C), 131.7 (4CH), 126.2 (2CH), 122.9 (2C), 94.6 (2C), 85.4 (2C), 80.4 (2C), 74.3 (2CH), 50.8 (2CH), 47.5 (2C), 41.9 (2CH), 40.3

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(2CH), 39.3 (2CH2), 39.3 (2CH2), 36.5 (2CH), 35.0 (2CH2), 34.7 (2C), 33.3 (2CH2), 32.1 (2CH2), 26.9 (2CH2), 26.6 (2CH2), 26.0 (2CH2), 23.3 (2CH2), 23.3 (2CH3), 20.5 (2CH2), 12.9 (2CH3). HRMS (ESI) calculated for [C54H70O6Na]þ requires m/ z 837.5070, found m/z 837.5086. 4.6.2. Compound 1Z. The retention time (tR)¼14.36 min. Isomer 1E is a white solid. Mp 283e285  C (CH3CN/CH2Cl2). Rf¼0.49 (hexane/ AcOEtd6:4). nmax (CHCl3): 3594, 2929, 2855, 1713, 1178 cm1. dH (ppm, CDCl3): 7.46 (s, 4H, Ph), 5.78e5.83 (m, 2H, C]CH), 4.69e4.78 (m, 2H, C(O)OCH), 3.05e3.13 (m, 4H, C]CHeCH2), 2.35e2.43 (m, 2H, HOC(C^C)eCHaHb), 2.01e2.11 (m, 2H, HOC(C^C)eCHaHb), 0.97 (s, 6H, Me), 0.89 (s, 6H, Me). dC (ppm, CDCl3): 170.8 (2C), 131.6 (4CH), 124.3 (2CH), 122.9 (2C), 94.6 (2C), 85.4 (2C), 80.4 (2C), 74.5 (2CH), 50.8 (2CH), 47.5 (2C), 41.9 (2CH), 40.3 (2CH), 39.2 (2CH2), 36.5 (2CH), 35.0 (2CH2), 34.7 (2C), 33.4 (2CH2), 33.3 (2CH2), 32.2 (2CH2), 26.9 (2CH2), 26.6 (2CH2), 25.9 (2CH2), 23.3 (2CH2), 23.3 (2CH3), 20.5 (2CH2), 12.9 (2CH3). HRMS (ESI) calculated for [C54H70O6Na]þ requires m/z 837.5070, found m/z 837.5086. 23-Oxosarsasapogenin acetate was prepared by an improved Barton procedure.25 4.7. 3b-tert-Butyldimethylosilyloxy-23-oxosarsasapogenin (12) 23-Oxosarsasapogenin (11, 0.6 g, 1.4 mmol) was dissolved in dry DMF (100 mL), TBSeCl (0.42 g, 2 equiv), imidazole (0.19 g, 2 equiv), and DMAP (cat.) were added. The reaction mixture was stirred at room temperature for 3 days. The solution was poured into water and extracted with benzene. The organic layer was dried over Na2SO4 and evaporated in vacuo. Silica gel column chromatography afforded pure ether 12 (0.55 g, 73%) eluted with benzene/hexane 1:1 as a white solid. Mp 223e225  C. Rf¼0.73 (hexane/AcOEt 17:3). nmax (CHCl3): 2857, 1729, 1471, 1381, 1164, 1059 cm1. dH (ppm, CDCl3): 4.62 (m, 1H, CH2CHO), 4.29 (dd, J1¼2.9 Hz, J2¼11.2 Hz, 1H, OCHaHb), 4.03 (br s, 1H, TBSOCH), 3.42 (dt, J1¼2.1 Hz, J2¼11.2 Hz, 1H, OCHaHb), 2.91 (m, 2H, CH2C]O), 2.38 (m, 1H), 1.08 (d, J¼7.1 Hz, 3H, Me), 0.96 (d, J¼6.7 Hz, 3H, Me), 0.95 (s, 3H, Me), 0.89 (s, 9H, t-BuSi), 0.78 (s, 3H, Me), 0.02 (s, 6H, Me2Si). dC (ppm, CDCl3): 202.4 (C), 110.8 (C), 83.6 (CH), 67.4 (CH), 64.6 (CH2), 61.8 (CH), 56.7 (CH), 43.9 (CH2), 41.2 (C), 40.2 (CH2), 40.1 (CH), 36.5 (CH), 35.3 (CH), 35.2 (CH), 35.1 (C), 34.5 (CH2), 33.7 (CH), 31.7 (CH2), 30.0 (CH2), 28.6 (CH2), 26.8 (CH2), 26.7 (CH2), 25.8 (3CH3), 24.0 (CH3), 20.1 (CH2), 18.1 (C), 17.7 (CH3), 16.2 (CH3), 14.2 (CH3), 4.8 (CH3), 4.9 (CH3). Anal. Calcd for C33H56O4Si: C, 72.74; H, 10.36; Si, 5.15. Found: C, 72.67; H, 10.46; Si, 5.21. 4.8. 3b-tert-Butyldimethylosilyloxy-23a-ethynyl-23b-hydroxysarsasapogenin (13) Ether 12 (0.6 g, 1.1 mmol) was dissolved in dry THF (60 mL) and ethynylmagnesium chloride (0.5 M solution in THF, 13.25 mL, 6 equiv) at 0  C was added under argon. The reaction mixture was stirred at room temperature for 24 h. The solution was poured into aqueous solution of ammonium chloride and extracted with ethyl acetate. The organic layer was dried over Na2SO4 and evaporated in vacuo. Silica gel column chromatography afforded pure 23a-ethynyl-23b-hydroxysarsasapogenin 13 (0.55 g, 30%) eluted with ethyl acetate/hexane 1:99 as a white solid. Mp 190e192  C. Rf¼0.47 (hexane/AcOEt 17:3). nmax (CHCl3): 3598, 3307, 2857, 1471, 1380, 1058 cm1. dH (ppm, CDCl3): 4.45 (m, 1H, CH2CHO), 4.07 (dd, J1¼3.5 Hz, J2¼11.2 Hz, 1H, OCHaHb), 4.03 (br s, 1H, TBSOCH), 3.38 (d, J¼11.2 Hz, 1H, OCHaHb), 2.69 (m, 1H, HOC(C^C)CH), 2.41 (s, 1H, C^CH), 1.27 (d, J¼7.4 Hz, 3H, Me), 1.17 (d, J¼6.9 Hz, 3H, Me), 0.96 (s, 3H, Me), 0.92 (s, 3H, Me), 0.89 (s, 9H, t-BuSi), 0.02 (s, 6H, Me2Si); dC

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(ppm, CDCl3): 109.3 (C), 86.0 (C), 81.7 (CH), 72.3 (CH), 71.3 (C), 67.4 (CH), 64.1 (CH2), 63.4 (CH), 56.5 (CH), 41.9 (C), 40.5 (CH2), 40.1 (CH2), 40.0 (CH), 39.0 (CH), 36.5 (CH), 35.3 (CH), 35.2 (C), 34.5 (CH2), 31.7 (CH2), 30.0 (CH2), 28.3 (CH2), 26.8 (2CH2), 26.3 (CH), 25.8 (3CH3), 23.9 (CH3), 20.9 (CH2), 20.3 (CH3), 18.1 (C), 16.6 (CH3), 16.5 (CH3), 4.8 (CH3), 4.9 (CH3). MS (ES) m/z: 593 (MNaþ, 100). Anal. Calcd for C35H58O4Si: C, 73.63; H, 10.24; Si, 4.92. Found: C, 73.76; H, 10.29; Si, 4.86. 4.9. 1,4-Bis[3b-[(tert-butydimethyl)silyloxy]-23b-hydroxysarsasapogenin-23a-yl-ethynyl]benzene (14) 23a-Ethynyl-23b-hydroxysarsasapogenin 13 (0.35 g, 0.6 mmol) was dissolved in dry THF (100 mL) then 1,4-diodobenzene (0.1 g, 0.3 mmol), Pd(PPh3)4 (35 mg, 5% mol), Cu2I2 (24 mg, 10% mol), and DIPEA (2.5 mL) were added. The reaction mixture was stirred at reflux for 4 h. The solution was poured into aqueous solution of ammonium chloride and extracted with dichloromethane. The organic layer was dried over Na2SO4 and evaporated in vacuo. Silica gel column chromatography afforded pure dimer 14 (0.195 g, 52%) as a white solid and iodo-compound 15 (0.095 g, 20%) as a brown oil eluted with ethyl acetate/hexane 4:96 and 1:99, respectively. 4.9.1. Compound 14. Mp 324e325  C. Rf¼0.28 (hexane/AcOEt 17:3). nmax (CHCl3): 3595, 2856, 1725, 1463, 1380, 1165, 1135, 1058, 838 cm1. dH (ppm, CDCl3): 7.33 (s, 4H, Ph), 4.47 (m, 2H, CH2CHO), 4.10 (dd, J1¼3.6 Hz, J2¼11.2 Hz, 2H, OCHaHb), 4.03 (br s, 2H, TBSOCH), 3.41 (d, J¼11.2 Hz, 2H, OCHaHb), 2.74 (m, 2H, HOC(C^C) CH), 2.45 (dd, J1¼6.1 Hz, J2¼14.4 Hz, 2H, CH2CH(CH3)CH2O), 0.95 (s, 6H, Me), 0.92 (s, 6H, Me), 0.89 (s, 18H, t-BuSi), 0.02 (s, 12H, Me2Si); dH (ppm, CDCl3): 131.5 (CH2), 122.7 (C), 109.6 (C), 93.2 (C), 83.7 (C), 81.6 (CH), 71.8 (C), 67.4 (CH), 64.1 (CH2), 63.5 (CH), 56.6 (CH), 41.7 (C), 40.4 (CH2), 40.1 (CH), 40.1 (CH2), 39.1 (CH), 36.5 (CH), 35.3 (C), 35.1 (CH), 34.4 (CH2), 31.8 (CH2), 30.0 (CH2), 28.6 (CH2), 26.8 (CH2), 26.7 (CH2), 26.4 (CH), 25.8 (3CH3), 24.1 (CH3), 20.9 (CH2), 20.4 (CH3), 18.1 (C), 16.7 (CH3), 16.6 (CH3), 4.8 (CH3), 4.9 (CH3). MS (photospray) m/z: 459 (MþC48H69O5Si, 7.5), 754 (MþC28H49O3Si, 100), 1216 (MHþ, 25). Anal. Calcd for C76H118O8Si2: C, 75.07; H, 9.78; Si, 4.62. Found: C, 75.13; H, 9.85; Si, 4.58. 4.9.2. Compound 15. Mp 252e254  C. Rf¼0.41 (hexane/AcOEt 17:3). nmax (CHCl3): 3520, 3437, 1463, 1255, 1006, 816 cm1. dH (ppm, CDCl3): 7.65 (d, J¼8.5 Hz, 2H, Ph), 7.12 (d, J¼8.5 Hz, 2H, Ph), 4.48 (m, 1H, CH2CHO), 4.10 (dd, J1¼3.9 Hz, J2¼10.8 Hz, 1H, OCHaHb), 4.03 (br s, 1H, TBSOCH), 3.41 (d, J¼11 Hz, 1H, OCHaHb), 2.73 (m, 1H, HOC(C^C)CH), 2.45 (dd, J1¼6.2 Hz, J2¼14.4 Hz, 1H, CH2CH(CH3) CH2O), 2.36 (s, 1H, OH), 1.30 (d, J¼7.4 Hz, 6H, Me), 1.20 (d, J¼6.9 Hz, 6H, Me), 0.95 (s, 3H, Me), 0.90 (s, 3H, Me), 0.89 (s, 9H, t-BuSi), 0.02 (s, 6H, Me2Si); dC (ppm, CDCl3): 137.4 (CH2), 133.2 (CH2), 122.4 (C), 109.5 (C), 94.1 (C), 92.8 (C), 83.1 (C), 81.6 (CH), 71.8 (C), 67.4 (CH), 64.1 (CH2), 63.5 (CH), 56.6 (CH), 41.7 (C), 40.4 (CH2), 40.1 (CH), 39.1 (CH), 36.5 (CH), 35.3 (C), 35.1 (C), 34.4 (CH2), 31.8 (CH2), 30.0 (CH2), 29.7(CH2), 28.6 (CH2), 26.8 (CH2), 26.7 (CH2), 26.4 (CH), 25.8 (3CH3), 24.1 (CH3), 20.9 (CH2), 20.3 (CH3), 18.1 (C), 16.7 (CH3), 16.5 (CH3), 4.8 (CH3), 4.9 (CH3). MS (ESI) m/z: 795 (MNaþ, 100). Anal. Calcd for C41H61IO4Si: C, 63.71; H, 7,95; I, 16.42; Si, 3.63. Found: C, 63.79; H, 7.85; I, 16.51; Si, 3.51. 4.10. 1,4-Bis[3b-hydroxy-23b-hydroxysarsasapogenin-23a-ylethynyl]benzene (16) Dimer 14 (0.35 g, 0.3 mmol) was dissolved in dry dichloromethane (75 mL) and BF3$Et2O (0.11 mL, 3 equiv) was added. The reaction mixture was stirred at 0  C for 4 h. The solution was poured into water and extracted with dichloromethane. The organic layer was dried over Na2SO4 and evaporated in vacuo. Silica gel column

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chromatography afforded pure compound 16 (0.28 g, 99%) eluted with ethyl acetate/hexane 35:65 as a white solid: Rf¼0.2 (hexane/ AcOEt 17:3). nmax (CHCl3): 3627, 1167, 1101, 1058, 1001, 836 cm1. dH (ppm, CDCl3): 7.34 (s, 4H, Ph), 4.47 (m, 2H, CH2CHO), 4.12 (br s, 2H, OH), 4.10 (dd, J1¼3.1 Hz, J2¼11.0 Hz, 2H, OCHaHb), 3.41 (d, J¼11.1 Hz, 1H, OCHaHb), 2.74 (m, 2H, HOC(C^C)CH), 2.45 (dd, J1¼6.1 Hz, J2¼14.4 Hz, 2H, CH2CH(CH3)CH2O), 1.30 (d, J¼7.4 Hz, 6H, Me), 1.20 (d, J¼6.9 Hz, 6H, Me), 0.98 (s, 6H, Me), 0.92 (s, 6H, Me); dC (ppm, CDCl3): 131.5 (CH2), 122.7 (C), 109.6 (C), 93.3 (C), 83.7 (C), 81.6 (CH), 71.8 (C), 67.1 (CH), 64.1 (CH2), 63.6 (CH), 56.6 (CH), 41.7 (C), 40.4 (CH2), 40.2 (CH2), 39.9 (CH), 39.1 (CH), 36.5 (CH), 35.3 (C), 35.2 (CH), 33.6 (CH2), 31.8 (CH2), 30.0 (CH2), 29.7 (CH2), 27.9 (CH2), 26.5 (CH2), 26.4 (CH), 24.0 (CH3), 20.9 (CH2), 20.4 (CH3), 16.7 (CH3), 16.6 (CH3). MS (EI) m/z: 1010 (MNaþ, 100). Anal. Calcd for C64H90O8: C, 77.85; H, 9.19. Found: C, 77.97; H, 9.08. 4.11. 1,4-Bis[3b-(hex-50 -enoyloxy)-23b-hydroxysarsasapogenin23a-yl-ethynyl]benzene (17) To compound 16 (0.28 g, 0.28 mmol) dissolved in dry dichloromethane (75 mL), DCC (0.29 g, 5 equiv), 5-hexenoic acid (0.17 mL, 5 equiv), and DMAP (catalytic amount) were added. The reaction mixture was stirred at 0  C for 45 h. The solution was filtered then poured into water, washed three times by diluted acetic acid and by solution of sodium hydrogen carbonate, and extracted with dichloromethane. The organic layer was dried over Na2SO4 and evaporated in vacuo. Silica gel column chromatography afforded pure compound 17 (0.2 g, 60%) eluted with ethyl acetate/hexane 4:6 as a white solid. 4.11.1. Compound 17. Mp 280e283  C. Rf¼0.53 (hexane/AcOEt 4:1). nmax (CHCl3): 1719, 1154, 1102, 1025, 998 cm1. dH (ppm, CDCl3): 7.32 (s, 4H, Ph), 5.78 (m, 2H, CH2]CH), 5.08 (br s, 2H, HCO(C]O)), 5.01 (m, 4H, CH2]CH), 4.46 (m, 2H, CH2CHO), 4.08 (dd, J1¼3.4 Hz, J2¼11.2 Hz, 2H, OCHaHb), 3.40 (d, J¼11.2 Hz, 2H, OCHaHb), 2.73 (m, 2H, HOC(C^C)CH), 2.44 (dd, J1¼6.2 Hz, J2¼14.4 Hz, 2H, CH2CH(CH3) CH2O), 1.29 (d, J¼7.3 Hz, 6H, Me), 1.19 (d, J¼6.8 Hz, 6H, Me), 0.97 (s, 6H, Me), 0.91 (s, 6H, Me); dC (ppm, CDCl3): 173.1 (C), 137.7 (CH), 131.4 (CH2), 122.6 (C), 115.2 (CH2), 109.5 (C), 93.2 (C), 83.6 (C), 81.5 (CH), 71.7 (C), 70.4 (CH), 64.0 (CH2), 63.4 (CH), 56.4 (CH), 41.6 (C), 40.2 (CH2), 40.1 (CH2), 40.0 (CH), 39.1 (CH), 37.3 (CH), 35.1 (C), 34.9 (CH), 34.0 (2CH2), 33.1 (CH2), 31.7 (CH2), 30.8 (CH2), 30.6 (CH2), 26.4 (CH), 26.3 (CH2), 25.0 (CH2), 24.2 (CH2), 23.9 (CH3), 20.8 (CH2), 20.3 (CH3), 16.7 (CH3), 16.5 (CH3). MS (EI) m/z: 1202 (MNaþ, 100). Anal. Calcd for C76H108O10: C, 77.38; H, 9.06. Found: C, 77.46; H, 9.01. 4.12. Ring-closing metathesis: synthesis of macrocycles 2E and 2Z To diester 17 (0.03 g, 0.025 mmol) in dry toluene (160 mL), an olefin metathesis catalyst (see Table 1) (40 mol %) was added. The reaction mixture was stirred at 80 or 100  C for about 1 h. The consumption of the starting material was monitored by TLC. After reaction the solvent was removed. Silica gel column chromatography afforded a mixture of compounds 2 (E and Z) eluted with ethyl acetate/hexane 1:9. The mixture was separated by HPLC (elution with CH2Cl2/CH3CN 85:15). 4.12.1. Compound 2Z. Mp 267e269  C. Rf¼0.37 (hexane/AcOEt 5:1). nmax (CHCl3): 3448, 1717, 1449, 1032, 1001 cm1. dH (ppm, CDCl3): 7.32 (s, 4H, Ph), 5.41 (m, 2H, HC]C), 5.09 (br s, 2H, HCO(C]O)), 4.48 (m, 2H, CH2CHO), 4.09 (dd, J1¼3.5 Hz, J2¼11.2 Hz, 2H, OCHaHb), 3.41 (d, J¼11.2 Hz, 2H, OCHaHb), 2.74 (m, 2H, HOC(C^C)CH), 2.45 (dd, J1¼3.1 Hz, J2¼14.4 Hz, 2H, CH2CH(CH3)CH2O), 1.30 (d, J¼7.4 Hz, 6H, Me), 1.20 (d, J¼6.9 Hz, 6H, Me), 1.01 (s, 6H, Me), 0.96 (s, 6H, Me); dC (ppm, CDCl3): 173.1 (C), 131.4 (CH2), 130.1(CH), 122.7 (C), 109.4 (C),

93.1 (C), 83.7 (C), 81.5 (CH), 71.8 (C), 70.5 (CH), 64.0 (CH2), 63.5 (CH), 56.5 (CH), 41.7 (C), 40.2 (CH2), 40.1 (CH2), 40.0 (CH), 39.1 (CH), 37.5 (CH), 35.2 (CH), 35.0 (CH2), 34.8 (C), 32.1 (CH2), 31.7 (CH2), 30.9 (CH2), 30.6 (CH2), 26.4 (CH), 26.3 (CH2), 25.0 (CH2), 24.9 (CH2), 24.2 (CH3), 22.7 (CH2), 20.8 (CH2), 20.4 (CH3), 16.8 (CH3), 16.7 (CH3). MS (EI) m/z: 1173 (MNaþ, 100). Anal. Calcd for C75H106O10: C, 77.15; H, 9.15. Found: C, 77.23; H, 9.06. 4.12.2. Compound 2E. Mp 322e324  C. Rf¼0.37 (hexane/AcOEt 5:1). nmax (CHCl3): 3490, 1727, 1449, 1025, 1000 cm1. dH (ppm, CDCl3): 7.32 (s, 2H, Ph), 5.41 (m, 1H, HC]C), 5.10 (br s, 1H, HCO(C] O)), 4.48 (m, 1H, CH2CHO), 4.10 (dd, J1¼3.5 Hz, J2¼11.0 Hz, 1H, OCHaHb), 3.41 (d, J¼11.2 Hz, 1H, OCHaHb), 2.75 (m, 1H, HOC(C^C) CH), 2.46 (dd, J1¼6.1 Hz, J2¼14.3 Hz, 1H, CH2CH(CH3)CH2O), 1.30 (d, J¼7.4 Hz, 6H, Me), 1.20 (d, J¼6.8 Hz, 6H, Me), 1.00 (s, 6H, Me), 0.96 (s, 6H, Me); dC (ppm, CDCl3): 173.1 (C), 131.4 (CH2), 130.1 (CH), 122.7 (C), 109.4 (C), 93.1 (C), 83.7 (C), 81.5 (CH), 71.8 (C), 70.5 (CH), 64.0 (CH2), 63.6 (CH), 56.5 (CH), 41.7 (C), 40.2 (CH2), 40.1 (CH2), 40.0 (CH), 39.1 (CH), 37.5 (CH), 35.2 (C), 35.0 (CH), 34.8 (CH2), 32.1 (CH2), 31.7 (CH2), 30.9 (CH2), 30.6 (CH2), 26.4 (CH), 26.4 (CH2), 25.0 (CH2), 25.0 (CH3), 24.2 (CH2), 22.7 (CH2), 20.8 (CH2), 20.4 (CH3), 16.8 (CH3), 16.7 (CH3). MS (EI) m/z: 1173 (MNaþ, 100). Anal. Calcd for C75H106O10: C, 77.15; H, 9.15. Found: C, 77.26; H, 9.03. Acknowledgements The authors thank the Polish National Science Centre for the grant support (DEC-2011/02/A/ST5/00459). Supplementary data Supplementary data associated with this article can be found in the online version, at http://dx.doi.org/10.1016/j.tet.2014.10.022. References and notes 1. (a) Horinek, D.; Michl, J. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 14175; (b) Shirai, Y.; Morin, J.-F.; Sasaki, T.; Guerrero, J. M.; Tour, J. M. Chem. Soc. Rev. 2006, 35, 1043; (c) Browne, W. R.; Feringa, B. L. Nat. Nanotechnol. 2006, 1, 25; (d) Akutagawa, T.; Koshinaka, H.; Sato, D.; Takeda, S.; Noro, S. I.; Takahashi, H.; Kumai, R.; Tokura, Y.; Nakamura, T. Nat. Mater. 2009, 8, 342. 2. van den Heuvel, M. G. L.; Dekker, C. Science 2007, 317, 333. 3. Berg, J. S.; Powell, B. C.; Cheney, R. E. Mol. Biol. Cell 2001, 12, 780. 4. (a) Raymo, F. M.; Stoddart, F. Chem. Rev. 1999, 99, 1643; (b) Schalley, C. A.; Beizai, € gtle, F. Acc. Chem. Res. 2001, 34, 465; (c) Aucagne, V.; Leigh, D. A.; Lock, J. K.; Vo S.; Thomson, A. R. J. Am. Chem. Soc. 2006, 128, 1784; (d) Movsisyan, L. D.; Kondratuk, D. V.; Franz, M.; Thompson, A. L.; Tykwinski, R. R.; Anderson, H. L. Org. Lett. 2012, 14, 3424. ~ ez, J. E.; Godinez, C. E.; Garcia-Garibay, M. A. Acc. Chem. 5. (a) Khuong, T.-A. V.; Nun Res. 2006, 39, 413; (b) Vogelsberg, C. S.; Garcia-Garibay, M. A. Chem. Soc. Rev. 2012, 41, 1892. 6. Kottas, G. S.; Clarke, L. I.; Horinek, D.; Michl, J. Chem. Rev. 2005, 105, 1281. 7. (a) Deleuze, M. J. Am. Chem. Soc. 2000, 122, 1130; (b) Zheng, X.; Mulcahy, M.-E.; Horinek, D.; Galeotti, F.; Magnera, T. F.; Michl, J. J. Am. Chem. Soc. 2004, 126, 4540; (c) Das, B.; Sebastian, K. L. Chem. Phys. Lett. 2002, 357, 25; (d) Das, B.; Sebastian, K. L. Chem. Phys. Lett. 2000, 330, 433. 8. (a) Hughs, M.; Jimenez, M.; Khan, S.; Garcia-Garibay, M. A. J. Org. Chem. 2013, 78, 5293; (b) Leigh, D. A.; Wong, J. K. Y.; Dehez, F.; Zerbetto, F. Nature 2003, 424, 174; (c) Koumura, N.; Zijlstra, R. W.; van Delden, R. A.; Harada, N.; Feringa, B. L. Nature 1999, 401, 152. 9. (a) Commins, P.; Garcia-Garibay, M. A. J. Org. Chem. 2014, 79, 1611; (b) Rodriguez-Molina, B.; Ochoa, M. E.; Farf an, N.; Santillan, R.; Garcia-Garibay, M. A. J. Org. Chem. 2009, 74, 8554; (c) Karlen, S. D.; Reyes, H.; Taylor, R. E.; Khan, S. I.; Hawthorne, M. F.; Garcia-Garibay, M. A. Proc. Natl. Acad. Sci. U.S.A. 2010, 107, 14973; (d) O’Brien, Z. J.; Karlen, S. D.; Khan, S.; Garcia-Garibay, M. A. J. Org. Chem. 2010, 75, 2482; (e) Dominguez, Z.; Khuong, T.-A. V.; Dang, H.; Sanrame, ~ ez, J. E.; Garcia-Garibay, M. A. J. Am. Chem. Soc. 2003, 125, 8827; (f) C. N.; Nun Dominguez, Z.; Dang, H.; Strouse, M. J.; Garcia-Garibay, M. A. J. Am. Chem. Soc. 2002, 124, 2398. 10. (a) Michl, J.; Sykes, C. H. ACS Nano 2009, 3, 1042; (b) Sutharsan, J.; Lichlyter, D.; Wright, N. E.; Dakanali, M.; Haidekker, M. A.; Theodorakis, E. A. Tetrahedron 2010, 66, 2582. 11. Karlen, S. D.; Garcia-Garibay, M. A. Top. Curr. Chem. 2005, 262, 179. 12. Garcia-Garibay, M. A. Proc. Natl. Acad. Sci. U.S.A. 2005, 102, 10771.

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