Total Synthesis and Selective Activity of a NewClass of Conformationally Restrained Epothilones

Abstract

Stereoselective total syntheses of two novel conformationally restrained epothilone analogues are described. Evans asymmetric alkylation, Brown allylation, and a diastereoselective aldol reaction served as the key steps in the stereoselective synthesis of one of the two key fragments of the convergent synthetic approach.Enzyme resolution was employed to obtain the second fragment as a single enantiomer. The molecules were assembled by esterification, followed by ring-closing metathesis. In preliminary cytotoxicity studies, one of the analogues showed strong and selective growth inhibitory activity against two leukemia cell lines over solid human tumor cell lines. The precise biological mechanism of action and high degree of selectivity of this analogue remain to be examined.

Introduction

The clinically used anticancer drug paclitaxel (taxol) exerts its cytotoxic activity by a mechanism invoking the stabilization of microtubules.[1,2] However, the susceptibility of the drug to multiple drug resistance has stimulated extensive research on molecules with a paclitaxel-like mechanism of action.[3,4] The most extensively investigated of the new molecular entities discovered are the epothilones, a class of macrolide natural products initially isolated from a soil bacterium.[3,5,6] Some epothilones exhibit more potent anticancer activity than paclitaxel and have favorable attributes that may improve their pharmacological profile.[7,8]

Replacing the thiazole ring in epothilone B by methylpyridine rings, in which the nitrogen atom is ortho to the pyridine-vinyl linker attachment, produced analogues with higher potencies against drug-resistant tumor cells.[9] The role of the N atom at an ortho-position in the heterocycle has been attributed to a hydrogen-bond interaction between the thiazole N atom and a protonated His residue in tubulin.[10–13] The 16-Me and 19-H adopt a syn orientation, liberating the thiazole N atom from the steric bulk of the 16-Me, making it more accessible for potential hydrogen bonding. Rigidification of the aromatic side chain through incorporation of the C-16–C-17 olefinic double bond into a fused heteroaromatic ring system such as benzothiazole resulted in analogues more potent than parent epothilone B and D.[14,15] Interestingly, in contrast to the pyridine analogues, there was no dependence of the tubulin-polymerizing activity of these rigidified analogues on the position of the nitrogen atom in the heterocycles.[16] Antiproliferative activity, on the other hand, showed a strong dependence on the position of the nitrogen atom, as in the pyridine analogues. Attempts to rigidify the southern C-1–C-8 sector of the molecule have resulted in loss of biological activity.[17,18] Analogues made by rigidification of some parts of the northern C-9–C-13 sector have retained biological activity.[19–21] A series of conformationally rigidified analogues bridging the northern and southern sectors across the macrocycle were prepared recently. However, their biological activities have not been reported.[22]

The extensive database on SAR for epothilone has led to the discovery of potent epothilones.[23,24] At least five analogues are currently in different stages of clinical development.[7] Most of the current potent epothilone analogues bear close structural resemblance to the parent compounds. While largely uninvestigated, compounds with more stringent structural modifications of the epothilone scaffold may produce new lead structures with altered pharmacological profiles for drug discovery.[25,26]

Results and Discussion

We designed a new class of conformationally restrained analogues of epothilones (compounds 1a and 1b) by restricting the mobility of the aromatic side chain. In doing so, we retained the C-1–C-8 sector unchanged, in view of its seemingly crucial role in biological activity. A single methylene bridge was introduced between C-14 and C-17. The resulting cyclopentene moiety, which incorporated the C-16–C-17 double bond, was designed to rigidify the side chain while still permitting sufficient mobility of the pyridine ring to allow for the preferred N-atom orientation for a hydrogen bonding interaction with the receptor.[11]

The choice of a S configuration at the new chiral center at C-14 in the designed analogues was based on molecular modeling studies, and is consistent with the observations of Taylor et al.,[27, 28] whose extensive studies on the bioactive conformation of epothilones have shown that the (S)-C-14-Me analogue of epothilone D is far more active than the corresponding R epimer. We performed energy minimizations for 1b (C-14-S) as well as for its C-14-(R) epimer and for epothilone D by using Gaussian 03[29] at the B3LYP/6—311++G(d,p) level (Figure 1). Compound 1b was found to be more stable than the C-14-(R) epimer by 8.8 kcalmol⁻¹. The main ring configuration in the minimized structure of epothilone D remained very similar to the X-ray crystal structure of 14-(S)-methyl-epothilone D (Figure 1).[27] In Figure 1, the energy-minimized structures of 1b and its C-14-(R) epimer are aligned with the minimum-energy structure of epothilone D by using the heavy atoms in the large ring and the root-mean-square deviations were found to be 0.287 and 0.283 Å for 1b and its C-14-(R) epimer, respectively. In comparison, the root mean deviation of the heavy atom alignment for epothilone D and the X-ray crystal structure of 14-(S) -methyl-epothilone D was found to be 0.186 Å. As can be seen from Figure 1, the side chain heteroaromatic ring segment in 1b, compared to its C-14-(R) epimer, can adopt an overall configuration close to those of epothilone D and 14-(S)-methyl-epothilone D. The conformational flexibility of the heteroaromatic ring-containing side chains of these analogues permit the N atoms in the heteroaromatic rings to come within 1 Å of each other. In contrast, the heteroaromatic ring-containing side chain of the C-14-(R) epimer of 1b is virtually perpendicular to the plane of the macrocycle.

Figure 1.

Energy-minimized epothilone D (gray); X-ray crystal structure of 14-(S)-methyl-epothilone D (cyan); compound 1b (green); and C-14-(R)-1b (magenta). The nitrogen atoms are shown in blue, the oxygen atoms are in red, and the sulfur atoms are in yellow. Main-ring heavy atoms were used in the alignment and the energy-minimized epothilone D was used as the reference structure. The figure was created by using Sybyl 7.3 (Tripos, St. Louis, MO).

A convergent synthetic approach with ring-closing metathesis (RCM) of 2a and 2b in the final step was adapted (Scheme 1). Retrosynthetic disconnection of 2a and 2b led to the three key synthons 3, 4, and either 5a or 5b. The stereoselective aldol coupling product between aldehyde 3 and ketone 4 would be converted to the corresponding carboxylic acid and then esterified with alcohols 5a and 5b to give 2a and 2b. This intermediate would then undergo RCM to yield the desired macrolide skeleton.

Scheme 1.

Retrosynthetic analysis of the designed epothilones.

Aldehyde 3 was made by using the Evans asymmetric alkylation protocol (Scheme 2).[30] Wittig olefination of the keto acid 6 gave the alkene 7. It was converted to the aldehyde 3 via the imide 9, following Schinzer’s procedure.[31]

Scheme 2.

a) MeP⁺(Ph)₃Br⁻, nBuLi, DMSO/THF, RT, 48 h, 78%; b) (COCl)₂, benzene; c) (S)-4-isopropyl-2-oxazolidonone, nBuLi, THF, −78°C.

The bis-silyl ether ketone 4 was made as shown in Scheme 3. Compound 10 was synthesized as reported earlier.[32] Selective reduction of the aldehyde group of 10 with NaBH₄ gave the primary alcohol 11 as a mixture with its hemiacetal 12, and was converted to the bis-silyl ether 4 with TBSCl and imidazole.

Scheme 3.

a) NaBH₄, CH₂Cl₂, EtOH, −78°C; b) TBSCl, imidazole, CH₂Cl₂, 0°C. TBSCl = tert-butyldimethylsilyl chloride.

A highly diastereoselective aldol reaction between the aldehyde 3 and ketone 4 under kinetic control generated aldol 13 (Scheme 4). The desired syn aldol 13 was formed together with the unwanted syn diastereomer (10:1) without any detectable formation of the anti product.[33] The syn diastereomers were separated by column chromatography. The S stereochemistry at C-7 in 13 was confirmed by Mosher’s ester analysis.[34] The hydroxyl function was protected with a Troc group to give compound 14. The use of the Troc group[35] in this context was based on an earlier failed sequence. We had initially approached the synthesis of 1a by employing Suzuki coupling for making the C-12–C-13 double bond and Yamaguchi macrolactonization for final ring closure and with a TBS (and later TES) protecting group at this position instead of Troc. However, desilylation of these groups in the final step proved problematic. Milder desilylating agents were ineffective and harsher conditions gave decomposition products. Thus, the present protocol was adopted with olefin metathesis for final ring closure. Selective desilylation of 14, followed by sequential DMP and Pinnick’s oxidations gave the carboxylic acid 17.

Scheme 4.

a) LDA, THF, −78°C, 83%; b) TrocCl, pyridine, CH₂Cl₂, 0°C, 1 h, 93%; c) CSA, CH₂Cl₂/MeOH, 0°C, 7 h, 87%; d) DMP, CH₂Cl₂, RT, 15 min; e) NaClO₂, NaH₂PO₄, H₂O/tBuOH, 2-methyl-2-butene, RT, 1 h, 90% (2 steps). TrocCl = 2,2,2-trichloroethyl chloroformate, CSA = (1S)-(+)−10-camphorsulfonic acid, LDA = lithium diisopropylamide, DMP = Dess–Martin periodinane.

The synthesis of enantiomerically pure β-ketoalcohols 22a and 22b by means of diastereoselective reduction of the corresponding β-ketoesters 21a and 21b seemed a logical and convenient approach to this moiety as the racemate of these β-ketoesters could be very conveniently prepared by aldol cyclization of the corresponding γ-aryl-substituted β-ketoesters 20a and 20b (Scheme 5). We investigated the conversion of 21a and 21b to 22a and 22b in high enantiomeric purity.

Scheme 5.

a) NaH, THF, 0°C, 82% (20a), 76% (20b); b) NaOH, anhydrous EtOH, 78% (21a), 73% (21b).

The diketoesters 20a and 20b were synthesized from ethyl propionylacetate 18 and α-bromoacetoaromatic derivatives 19a and 19b (Scheme 5). Direct intramolecular aldol condensation led to the cyclopentenones 21a and 21b. The 2-bromoketone 19b was prepared as shown in Scheme 6.[36] Bromination of 25 to 19b was carried out conveniently and reproducibly by using commercially available polymer-supported tribromide (Amberlyst A26-Br₃⁻).[37]

Scheme 6.

a) nBuLi, Et₂O, −78°C; b) N,N-dimethyl-acetamide, Et₂O, −78°C, 72% (2 steps); c) polymer-supported Amberlyst A26-Br₃⁻, THF, 91%.

After a number of unsuccessful attempts at stereoselective reduction of the ketone 21a,[38,39] including reduction with high catalyst loading of Corey’s oxazaborolidine CBS reagent,[40] the desired trans product 22a and its enantiomer ent-22a were finally obtained in equal amounts by reduction under chelation control by using Zn(BH₄)₂ (Scheme 7).[41]

Scheme 7.

a) Zn(BH₄)₂/ether, 4°C, 75%; b) PS-D lipase, vinyl acetate, 4 Å MS, pentane, RT, 4 d, 48% (ent-22a), 49% (26); c) anhydrous K₂CO₃, anhydrous EtOH, RT, 12h, 92%. MS = molecular sieves.

The trans racemate mixture 22a/ent-22a was efficiently separated by enzymatic resolution by using Amano PS-D Lipase enzyme (Scheme 7). Alcohol 22a was acetylated to form 26, whilst ent-22a remained unchanged. They were separated by column chromatography (49% 26 and 48% ent-22a). The stereochemistry at the secondary hydroxyl carbon atom of the ent-22a isomer and its enantiomeric purity were determined by Mosher’s ester analysis (R, 99% ee).[34] Ethanolysis of 26 gave the desired alcohol 22a (S, 98% ee by Mosher’s ester analysis).[34] The trans configuration of 22a was confirmed by NOE experiment (vide infra).

Compound 21b was found to be much more resistant to reducing agents, including Zn(BH₄)₂. Indeed, using even an excess of NaBH₄ resulted in only partial reduction. We speculated that altering electronic effects exerted by the basic nitrogen atom could render the molecule susceptible to reduction. Gratifyingly, prior conversion of 21b to the corresponding trifluoroacetate salt by treatment with two equivalents of trifluoroacetic acid (TFA) allowed rapid NaBH₄ reduction producing a mixture of all four diastereomers (Scheme 8). The product mixture was separated into the cis and trans enantiomeric pairs by column chromatography (trans/cis 2:1 determined by ¹H NMR spectroscopy). The fraction containing the pair of trans enantiomers (22b and ent-22b) was resolved by using Amano PS-D Lipase enzyme as described earlier for the phenyl analogue.

Scheme 8.

a) i) TFA, CH₂Cl₂, solvent evaporated, ii) residue in MeOH, NaBH₄, 0°C, 30 min; b) PS-D lipase, vinyl acetate, 4 Å MS, pentane, RT, 3d, 48% (ent-22b), 49% (28); c) anhydrous K₂CO₃, anhydrous EtOH, RT, 12h, 94 %. TFA = trifluoroacetic acid.

As with the phenyl analogue the desired enantiomer 22b was acetylated to 28 while the enantiomer ent-22b remained unchanged. They were separated by column chromatography and 28 was subjected to ethanolysis as before to obtain 22b (S, 95% ee by Mosher’s ester analysis).[34] NOE experiments confirmed the trans configuration of the molecule. The cis isomers 27 and ent-27 isolated by a similar enzymemediated resolution protocol showed a strong NOE between the two protons at the two stereogenic centers, and no such NOE was observed between the two corresponding protons in 22b or ent-22b.

Reduction of the esters 29a and 29b with DIBAL-H gave the corresponding aldehydes 30a and 30b, which were subjected to Wittig olefination to obtain the olefins 31a and 31b (Scheme 9). Desilylation with TBAF gave the desired alcohols 5a and 5b. At this stage, we confirmed the relative trans stereochemistry of the protons H-1 and H-5 in the cy-clopentene moiety 5a and the absolute stereochemistry of the molecule as shown based on NOE correlations (Figure 2) in conjunction with the already-established S configuration of the secondary hydroxyl carbon atom.[34]

Scheme 9.

a) TESCl, imidazole, CH₂Cl₂, 0°C, 2 h, 93% (29a), 92% (29b); b) DIBAL-H, toluene, −78°C, 1 h; c) MeP⁺(Ph)₃Br⁻, nBuLi, THF, 0°C, 30 min, 71% (31a), 73% (31b); d) TBAF, THF, 0°C, 30 min, 80% (5a) 83% (5b). TESCl = triethylsilyl chloride DIBAL-H = diisobutylaluminum hydride TBAF = tetrabutylammonium fluoride.

Figure 2.

NOE correlations of (1S,5S)-2-methyl-3-phenyl-5-vinylcyclopent-2-enol (5a).

The carboxylic acid 17 was esterified to 32a with alcohol 5a by using DCC/DMAP (Scheme 10). The Troc and TBS protecting groups were sequentially removed with zinc dust/ ammonium chloride in dry ethanol and TAS-F,[42] respectively, to give intermediate 2a. Finally, RCM of 2a by using second-generation Grubbs catalyst gave the desired Z-alkene 1a, accompanied by what appeared to be the E isomer as a minor product. A strong NOE correlation between the C-12-methyl protons and the C-13 olefinic proton confirmed the Z stereochemistry of the double bond of 1a.

Scheme 10.

a) DCC, DMAP, CH₂Cl₂, 0°C (15 min), RT (16 h), 64%; b) Zn, NH₄Cl, anhydrous EtOH, RT, 45 min; c) TAS-F, DMF, 2 d, 62% (2steps); d) CH₂Cl₂, 50°C, 16 h, 50% (Z+E). DCC=1,3-dicyclohexylcarbodiimide, DMAP=4-dimethylaminopyridine, TAS-F=tris(dimethylamino)sulfur (trimethylsilyl)difluoride.

A similar approach to synthesize the pyridine analogue 1b by starting from the carboxylic acid 17 and the alcohol 5b was complicated by an unexpected retroaldol decoupling of the alcohol obtained by the removal of the Troc protecting group upon treatment with TAS-F. Gratifyingly, this was easily overcome by changing the order of removal of the two protecting groups (Scheme 11). Thus, the TBS protecting group was removed first with TAS-F[42] to give 34 which was then treated directly with zinc dust and ammonium chloride in dry ethanol to give intermediate 2b. Finally, RCM of 2b by using second-generation Grubbs catalyst gave the desired product 1b. The Z configuration of the double bond was confirmed by NOESY. The phenyl analogue 35 and a dimerlike product from 2b were isolated as byproducts. A large coupling constant between the two olefinic protons and the absence of NOE correlations established the E configuration of the nonterminal double bond of 35. Formation of 35 can be attributed to high catalyst loading required to overcome the sluggishness of the RCM reaction.

Scheme 11.

a) DCC, DMAP, CH₂Cl₂, 0°C (15 min), RT (16 h), 85%; b) TAS-F, DMF, 2 d; c) Zn, NH₄Cl, anhydrous EtOH, RT, 45 min, 62% (2steps); d) CH₂Cl₂, 50°C, 16 h, 55%.

Cytotoxic activity

In preliminary in vitro cytotoxicity studies in the NCI-60 cell line human tumor screen, compound 1b showed strong growth inhibitory activity on CCRF-CEM and SR leukemia cell lines with GI₅₀ values of 2.7 and 2.9 nm, respectively (Table 1 and Figure 3 A). Surprisingly, it showed no significant activity on any of the other cell lines, including those derived from breast (MCF-7) and ovarian (SK-OV-3) cancers (Table 1 and Figure 3B,C). Although activity data of natural epothilone D against the NCI-60 cell lines is not available, Danishefsky et al. reported a potent and nonselective cytotoxic activity of epothilone D against these cell lines (Table 1).[43] It is also significant to note that the mechanistically analogous cancer drug paclitaxel (taxol) is slightly less effective (GI₅₀ of 12.6 nm for CCRF-CEM and 15.8 nm for SR) and less selective against these cell lines (NCI-60 cell line screen data). The reason for the highly selective inhibition of compound 1b against leukemia cell growth over solid tumor cell lines is still under investigation. We also tested the open-chain analogue 35, which was isolated as a byproduct of the RCM reaction along with 1b, and it showed no significant activity on any of the NCI-60 cell lines (Table 1). This is in accordance with our previous data on acyclic epothilone analogues which showed weak cytotoxic activity.[44] So far we have subjected analogue 1a only to a preliminary growth inhibition assay on MCF-7 breast cancer cells, in which it showed no activity. We are currently generating more material for testing in the NCI-60 cell line tumor screen.

Table 1.

In vitro cytotoxicities (GI₅₀ and IC₅₀) against human tumor cell lines.^[a]

			GI50 [µm]		IC50 [µm]
Cell line	1b	1a	35	Paclitaxel	Epothilone D[b]
CCRF-CEM	0.0027	nd	>12.5	0.0126	0.0095
SR	0.0029	nd	> 12.5	0.0158	nd
MCF-7	>15	192	>12.5	0.0100	0.0029
SK-OV-3	>15	nd	>12.5	0.0251	0.0069

^aIn vitro cell growth inhibition was measured by using the NCI-60 cell line screen (SRB assay). CCRF-CEM and SR are leukemia cell lines, MCF-7 is breast cancer cell line, and SK-OV-3 is ovarian cancer cell line.

^bData from reference [43] (measured by SRB assay). nd: Not Determined.

Figure 3.

NCI in vitro 60 cell line human tumor screen (dose response curves for A) leukemia (◆: CCRF-CEM, : HL-60(TB), ▲: K-562, : RPMI-8226, : SR) B) breast cancer (◆: MCF7, : NCI/ADR-RES, : MDA-MB-231/, : HS 578T, : MDA-MB-435, : BT-549), and C) ovarian cancer (◆: IGROV1, : OVCAR-3, : OVCAR-4, : OVCAR-5, : OVCAR-8, : SK-OV-3.

Conclusion

Two novel conformationally restrained epothilones 1a and 1b were synthesized. The strategy developed should be applicable to the synthesis of other important analogues of the series. The strong and selective growth inhibitory effect exhibited by analogue 1b on two leukemia cell lines, while not suppressing the proliferation of breast cancer and ovarian cancer cells which are very sensitive to natural epothilones, indicates that it may be possible to also develop new lead molecules of varying pharmacological profile by substantial modification of the epothilone scaffold. Our future efforts will focus on a more detailed investigation of this new class of conformationally restrained epothilone analogues to elucidate the biological mechanism in relation to selective activity.

Experimental Section

General methods

NMR spectra were recorded on Varian INOVA 600, Varian VXRS-400, Bruker AC-F 300 MHz, or Nicolet NM-500 MHz (modified with a Tecmag Libra interface) instruments and calibrated using residual undeuterated solvent as internal reference. Optical rotations were recorded on an AUTOPOL III 589/546 polarimeter. High-resolution mass spectra (HRMS) were recorded on a Micromass LCT Electrospray mass spectrometer performed at the Mass Spectrometry and Proteomics Facility, The Ohio State University.

(3S,6R,7S,8S)-1,3-Bis(tert-butyldimethylsilanyloxy)-7-hydroxy-4,4,6,8,12-pentamethyltridec-12-en-5-one (13)

A solution of ketone 4 (1.8 g, 4.48 mmol, 2.3 equiv) in THF (5 mL) was added dropwise to a solution of freshly prepared LDA in THF (prepared by adding nBuLi (2.92 mL of 1.6mM solution in hexanes, 4.67 mmol) to diisopropylamine (4.67 mmol, 0.655 mL) in THF (5 mL) at −78°C, hen warming the solution to 0°C for 20 min, and finally cooling back to −78°C). The reaction mixture was stirred at −78°C for 1 h and at −40°C for 30 min and was then cooled back to −78°C. A precooled (−78°C) solution of aldehyde 3 (0.272 g, 1.95 mmol, 1 equiv) in THF (10 mL) was added by the use of a cannula to the mixture over 2min. The reaction mixture was stirred at −78°C for 15 min before it was quenched rapidly by injection of a solution of acetic acid (0.55 mL) in THF (1.64 mL). The mixture was stirred at −78°C for 5 min and brought to room temperature. Saturated aqueous ammonium chloride (20 mL) and Et₂O (25 mL) were added and the layers were separated. The aqueous layer was extracted with Et₂O (3 × 25 mL) and the organic extracts were combined, dried over anhydrous sodium sulfate, and concentrated in vacuo. Flash column chromatography (4–20 % Et₂O/hexanes) gave recovered ketone 4 (0.78 g) followed by syn aldol 13 (0.87 g, 83 %) as the pure diastereomer along with the other syn aldol diastereomer (74 mg, 7 %) as colorless oils.

Data for 13: ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-39.6$ (c=0.7 in CHCl₃); R_f=0.57 (silica gel, 20% Et₂O/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=4.66 (s, 1H), 4.65 (s, 1H), 3.88 (dd, J=2.4, 7.8 Hz, 1H), 3.67–3.63 (m, 1H), 3.60–3.55 (m, 1H), 3.30–3.27 (m, 2H), 2.05–1.95 (m, 2H), 1.69 (s, 3H), 1.76–1.26 (m, 7H), 1.19 (s, 3H), 1.07 (s, 3H), 1.01 (d, ³J=6.6Hz, 3H), 0.88 (s, 9 H), 0.87 (s, 9H), 0.82(d, J=7.2Hz, 3H), 0.09 (s, 3H), 0.06 (s, 3H), 0.02ppm (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ=222.7, 146.5, 109.9, 75.1, 74.3, 60.7, 54.2, 41.5, 38.4, 35.7, 32.8, 26.4, 26.3, 26.2, 25.0, 23.1, 22.6, 20.7, 18.6, 18.5, 15.6, 9.8, −3.5, −3.8, −5.0 ppm; HRMS (ESI): m/z: calcd for C₃₀H₆₂O₄Si₂+Na⁺: 565.4084 [M+Na⁺]; found: 565.4067.

(3S,6R,7S,8S)-Carbonic acid-1-[4,6-bis(tert-butyldimethylsilanyloxy)-1,3,3-trimethyl-2-oxohexyl]-2,6-dimethyl-1-hept-6-enyl ester 2,2,2-trichloroethyl ester (14)

To a solution of aldol 13 (0.80 g, 1.48 mmol) in methylene chloride (30 mL) at 0 °C was added pyridine (0.96 mL, 11.84 mmol, 8 equiv) followed by 2,2,2-trichloroethyl chloroformate (0.8 mL, 5.92mmol, 4 equiv), and the reaction mixture was stirred at 0 °C for 1 h. Saturated aqueous sodium bicarbonate (50 mL) was added and the organic layer was separated. The aqueous layer was extracted with methylene chloride (3×50 mL), and the combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography (2% EtOAc/hexanes) afforded protected aldol 14 (0.98 g, 93%) as a colorless oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-51.5$ (c=1.6 in CHCl₃); R_f=0.72(silica gel, 17% EtOAc/hexanes); ¹H NMR (600MHz, CDCl₃): δ=4.85 (d, J=12.0 Hz, 1H), 4.78 (dd, J=4.2, 7.8 Hz, 1H), 4.66 (d, J=12.0Hz, 1H), 4.66 (s, 1H), 4.62(s, 1H), 3.72(dd, J=2.4, 7.8 Hz, 1H), 3.63–3.59 (m, 1H), 3.58–3.53 (m, 1H), 3.50–3.45 (m, 1H), 1.98–1.91 (m, 2H), 1.72–1.61 (m, 2H), 1.67 (s, 3H), 1.51–1.42 (m, 2H), 1.34 (s, 3H), 1.31–1.24 (m, 3H), 1.04 (d, J=6.6Hz, 3H), 0.99 (s, 3H), 0.94 (d, J=6.6Hz, 3H), 0.88 (s, 9H), 0.85 (s, 9H), 0.082ppm (s, 6H), 0.001 (s, 6H); ¹³C NMR (100 MHz, CDCl₃): δ = 215.8, 154.5, 145.8, 110.3, 95.0, 83.1, 76.8, 75.8, 60.5, 53.8, 42.7, 38.2, 34.9, 31.5, 26.4, 26.1, 24.9, 23.7, 22.6, 21.2, 18.6, 16.3, 11.3, −3.3, −4.1, −5.0, −5.1 ppm; HRMS (ESI): m/z: calcd for C₃₃H₆₃Cl₃O₆Si₂+Na⁺ 739.3126; found: 739.3163 [M+Na⁺].

(3S,6R,7S,8S)-Carbonic acid-1-[4-(tert-butyldimethylsilanyloxy)-6-hy-droxy-1,3,3-trimethyl-2-oxohexyl]-2,6-dimethyl-1-hept-6-enyl ester 2,2,2- trichloroethyl ester (15)

A solution of bis-silyl compound 14 (0.8 g, 1.11 mmol, 1 equiv) in methylene chloride (30 mL) and methanol (20 mL) was cooled to 0°C. A solution of (1S)-(+)-10-camphorsulfonic acid (77 mg, 0.33 mmol, 0.3 equiv) in methanol (10 mL) was added to this solution. The reaction mixture was stirred at 0°C for 7 h before it was quenched with saturated aqueous sodium bicarbonate (10 mL). The solid precipitate was filtered and the filtrate was concentrated in vacuo. The residue was diluted with Et₂O (100 mL) and washed with brine. The organic layer was separated and the aqueous layer was extracted with Et₂O (3×20mL). The combined organic phases were dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography (10% EtOAc/hexanes) afforded the unstable alcohol 15 (0.583 g, 87%). It was used directly in the next step. R_f=0.57 (silica gel, 33% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=4.87–4.80 (m, 2H), 4.69–4.61 (m, 3H), 3.92 (dd, J= 3.0Hz, 7.8 Hz, 1H), 3.67–3.59 (m, 2H), 3.45–3.40 (m, 1H), 1.98–1.90 (m, 2H), 1.66 (s, 3H), 1.70–1.42 (m, 8H), 1.26 (s, 3H), 1.07–1.06 (m, 6H), 0.95 (d, J=6.6Hz, 3H), 0.89 (s, 9H), 0.10 (s, 3H), 0.08 ppm (s, 3H); HRMS (ESI): m/z: calcd for C₂₇H₄₉Cl₃O₆Si+Na⁺: 625.2262 [M+Na⁺]; found: 625.2260.

(3S,6R,7S,8S)-3-(tert-Butyldimethylsilanyloxy)-4,4,6,8,12-pentamethyl-5-oxo-7-(2,2,2-trichloroethoxycarbonyloxy)tridec-12-enoic acid (17)

Dess–Martin periodinane (0.545 g, 1.28 mmol, 1.4 equiv) was added to a solution of alcohol 15 (0.550 g, 0.914 mmol) in methylene chloride (4 mL). The mixture was stirred at room temperature for 15 min. An additional amount of Dess–Martin reagent (0.23 g, 0.6 equiv) was added and the reaction mixture was stirred for 15 min and was then subjected to flash column chromatography (10% EtOAc/hexanes) to furnish crude aldehyde 16 which was used directly in the next step. A solution of sodium dihydrogenphosphate (270 mg, 2.25 mmol, 2.46 equiv) and sodium chlorite (270 mg, 3 mmol, 3.27 equiv) in distilled water (5 mL) was added to the crude aldehyde 16 (ca. 0.914 mmol) in tert-butanol (25 mL) and 2-methyl-2-butene (6 mL). The mixture was stirred at room temperature for 1 h and quenched by the addition of saturated aqueous ammonium chloride (50 mL) and water (50 mL). The mixture was extracted with ethyl acetate (3×60 mL) and the combined organic extracts were dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography (10% EtOAc/hexanes) afforded the pure carboxylic acid 17 (0.505 g, 90% over two steps) as a colorless oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-53.7$ (c=0.8 in CHCl₃); R_f=0.58 (silica gel, 40% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=4.85 (d, J=12.0 Hz, 1H), 4.76 (dd, J=4.2, 7.2 Hz, 1H), 4.66 (d, J=12.0 Hz, 1H), 4.66 (s, 1H), 4.62 (s, 1H), 4.22 (dd, J=3.6, 6.6 Hz, 1H), 3.47–3.41 (m, 1H), 2.60 (dd, J=3.6, 17.4 Hz, 1H), 2.21 (dd, J=6.6, 16.8 Hz, 1H), 1.96–1.91 (m, 2H), 1.74 –1.68 (m, 1H), 1.66 (s, 3H), 1.52–1.42 (m, 2H), 1.31 (s, 3H), 1.28 (d, J=16.8Hz, 3H), 1.06 (s, 6H), 0.94 (d, J=7.2Hz, 3H), 0.86 (s, 9H), 0.1 (s, 3H), 0.03 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=215.5, 177.7, 154.5, 145.8, 110.3, 94.9, 82.7, 76.9, 75.0, 53.8, 42.4, 39.9, 38.2, 34.9, 31.5, 26.3, 26.2, 24.8, 23.0, 22.6, 20.3, 18.4, 16.3, 11.5, −4.3 ppm; HRMS (ESI): m/z: calcd for C₂₇H₄₇Cl₃O₇Si+Na⁺: 639.2054 [M+Na⁺]; found: 639.2079.

3-Oxo-2-(2-oxo-2-phenylethyl)pentanoic acid ethyl ester (20a)

Ethyl propionylacetate 18 (10 g, 69.4 mmol) was added slowly to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 3.33 g, 83.3 mmol, 1.2equiv) in THF (100 mL) at 0°C and the mixture was stirred for 30 min. 2-Bromoacetophenone 19a (15.2g, 76.34 mmol, 1.1 equiv) in THF (10 mL) was added dropwise and the reaction mixture was stirred at room temperature for 16 h. Saturated aqueous ammonium chloride (60 mL) was added and the mixture was subsequently extracted with Et₂O (3×70 mL). The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give a dark-yellow oil that was purified by flash column chromatography (10% EtOAc/hexane) to give the diketoester 20a (14.8 g, 82%) as a yellow oil. R_f=0.50 (silica gel, 25% EtOAc/hexanes); ¹H NMR (400MHz, CDCl₃): δ=7.98 (d, J=8.4Hz, 2H), 7.58 (m, 1H), 7.46 (m, 2H), 4.22 (q, J=7.2Hz, 3H), 3.74 (dd, J=8.4, 18.4 Hz, 1H), 3.53 (dd, J=5.2, 18.4Hz, 1H), 2.91–2.70 (m, 2H), 1.28 (t, J=7.2Hz, 3H), 1.12ppm (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ=205.4, 197.4, 169.3, 136.3, 133.6, 128.8, 128.3, 61.9, 53.2, 37.7, 36.6, 14.2, 7.8 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₈O₄+Na⁺ 285.1103; found: 285.1093 [M+Na⁺].

3-Methyl-2-oxo-4-phenylcyclopent-3-enecarboxylic acid ethyl ester (21 a)

A solution of the diketoester 20a (12g, 45.8 mmol) in dry ethanol (150 mL) was added dropwise to a solution of sodium hydroxide (1.83 g, 45.8 mmol) in dry ethanol (75 mL) with vigorous stirring. The solution was heated to 50°C and stirred overnight at that temperature. Et₂O (1.5 L) was added and the organic phase was washed with HCl (2n, 3×300 mL) and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give an oil that was purified by flash column chromatography (6% EtOAc/hexane) to give the cyclic β-ketoester 21a (8.7 g, 78 %) as a yellow oil. R_f=0.40 (silica gel, 25 % EtOAc/hexane); ¹H NMR (400 MHz, CDCl₃): δ=7.56–7.52(m, 2H), 7.50–7.42(m, 3H), 4.26 (q, J=7.2Hz, 2H), 3.58 (dd, J=3.2, 7.6 Hz, 1H), 3.38–3.31 (m, 1H), 3.15–3.07 (m, 1H), 1.99 (s, 3H), 1.32ppm (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ=202.6, 169.6, 166.5, 135.8, 134.9, 130.2, 128.9, 127.9, 61.9, 51.3, 33.7, 14.5, 10.5 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₆O₃+Na⁺: 267.0997 [M+Na⁺]; found: 267.0974.

1-(5-Methylpyridin-2-yl)ethanone (25)

nBuLi (6.25 mL of 1.6m solution in hexanes, 10 mmol, 1 equiv) was added dropwise to a solution of 2-bromo-5-methyl pyridine 23 (1.73 g, 10 mmol) in dry Et₂O (20 mL), cooled to −78°C. The reaction mixture was allowed to warm to −40°C for 15 min, then cooled back to −78°C again. N,N-Dimethylacetamide (1.023 mL, 11 mmol, 1.1 equiv) was added dropwise and the mixture was stirred at −78°C for 2 h. Saturated aqueous ammonium chloride (10 mL) was added and the organic layer was separated. The aqueous layer was extracted with Et₂O (3×10 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo to give an oily residue that was subjected to flash column chromatography by using (5% methanol/methylene chloride) to give compound 25 (0.977 g, 72%) as a yellow oil. R_f=0.48 (silica gel, 25% EtOAc/hexanes); 1H NMR (400 MHz, CDCl₃): δ=8.50–8.48 (brs, 1H), 7.94 (d, J=8.0Hz, 1H), 7.63–7.60 (m, 1H), 2.70 (s, 3H), 2.41 ppm (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ=200.2, 151.7, 149.7, 137.8, 137.4, 121.7, 26.0, 18.9 ppm; HRMS (ESI): m/z: calcd for C₈H₉NO+Na⁺: 158.0582 [M+Na⁺]; found: 158.0580.

2-Bromo-1-(5-methylpyridin-2-yl)ethanone (19b)

Amberlyst A26-Br₃⁻ (Aldrich, 1.26 mmol Br₃ per g; 4.76 g, 6 mmol, 0.9 equiv) was added in one portion to a solution of compound 25 (0.9 g, 6.67 mmol) in THF (25 mL). The mixture was stirred at 50°C for 10 h and the decolored resin was filtered off and washed with ethyl acetate. The organic solution was washed with water, dried over anhydrous sodium sulfate, and concentrated in vacuo to give an oily residue that was chromatographed on silica gel with (10–30% methylene chloride/hexanes) to give compound 19b (1.29 g, 91%) as a yellow oil. R_f=0.62(silica gel, 20% EtOAc/ hexane); ¹H NMR (400 MHz, CDCl₃): δ=8.50 (brs, 1H), 8.01 (d, J=8.0 Hz, 1H), 7.66 (dd, J=1.6, 8.0 Hz, 1H), 4.84 (s, 2H), 2.44 ppm (s, 3H); ¹³C NMR (75 MHz, CDCl₃): δ=192.5, 149.8, 149.3, 138.7, 137.6, 122.6, 32.5, 18.9 ppm; HRMS (ESI): m/z: calcd for C₈H₈BrNO+Na⁺: 235.9687 [M+Na⁺]; found: 235.9676.

3-Oxo-2-(2-oxo-2-pyridin-2-ylethyl)pentanoic acid ethyl ester (20b)

Ethyl propionylacetate 18 (5 g, 34.7 mmol) was added slowly to a stirred suspension of sodium hydride (60% dispersion in mineral oil, 1.665 g, 41.65 mmol, 1.2equiv) in THF (50 mL) at 0 °C and the mixture was stirred for 30 min. Compound 19b (8.092g, 38.17 mmol, 1.1 equiv) in THF (5 mL) was added dropwise and the reaction mixture was stirred at room temperature for 16 h. Saturated aqueous ammonium chloride (30 mL) was added and the mixture was subsequently extracted with Et₂O (3×30mL). The combined organic layers were washed with brine and dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give a dark-yellow oil that was purified by flash column chromatography (10% EtOAc/hexanes) to give the diketoester 20b (7.3 g, 76%) as a yellow oil: TLC R_f=0.33 (silica gel, 20% EtOAc/hexane); ¹H NMR (400 MHz, CDCl₃): δ=8.50 (brs, 1H), 7.89 (d, J=8.0Hz, 1H), 7.66 (dd, J=1.6, 8.0 Hz, 1H), 4.20 (q, J=7.2Hz, 2H), 4.15 (dd, J=6.0, 8.0 Hz, 1H), 3.92(dd, J=8.4, 18.8 Hz, 1H), 3.74 (dd, J=6.0, 18.8 Hz, 1H), 2.85–2.66 (m, 2H), 2.41 (s, 3H), 1.27 (t, J=7.2Hz, 3H), 1.10 ppm (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ=205.5, 198.9, 169.6, 150.7, 149.8, 138.1, 137.3, 121.7, 61.8, 53.5, 37.1, 36.2, 18.9, 14.2, 7.9 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₉NO₄+Na⁺: 300.1212; found: 300.1200 [M+Na⁺].

3-Methyl-2-oxo-4-pyridin-2-ylcyclopent-3-ene carboxylic acid ethyl ester (21 b)

A solution of the diketoester 20b (3.1 g, 11.19 mmol) in dry ethanol (35 mL) was added dropwise to a solution of sodium hydroxide (0.447 g, 11.19 mmol) in dry ethanol (15 mL) with vigorous stirring. The solution was stirred at room temperature overnight. Et₂O (200 mL) was added and the organic phase was washed with HCl (2N, 3×100 mL). The aqueous layer was cooled to 0°C and made slightly basic by the addition of sodium bicarbonate. The aqueous layer was then extracted with ethyl acetate (3×300 mL) and the organic phase was dried over anhydrous sodium sulfate. The solvent was removed in vacuo to give an oil that was purified by flash column chromatography (10% EtOAc/hexanes) to give the cyclic β-ketoester 21b (2.115 g, 73%) as a yellow oil. R_f=0.35 (silica gel, 33% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃): δ=8.59 (s, 1H), 7.62–7.60 (m, 1H), 7.54–7.52 (m, 1H), 4.24 (q, J=7.2Hz, 2H), 3.56 (dd, J=2.8, 7.2 Hz, 1H), 3.42–3.25 (m, 2H), 2.41 (s, 3H), 2.12 (s, 3H), 1.31 ppm (t, J=7.2Hz, 3H); ¹³C NMR (75 MHz, CDCl₃): δ=203.2, 169.5, 164.4, 151.4, 150.6, 136.8, 136.5, 134.2, 123.3, 61.7, 51.1, 32.5, 18.6, 14.3, 10.6 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₇NO₃+Na⁺: 282.1106 [M+Na⁺]; found: 282.1109.

(1R*,2S*,3E)-2-Hydroxy-3-methyl-4-phenylcyclopent-3-enecarboxylic acid ethyl ester (22a) and (ent-22a)

A solution of zinc borohydride (150 mL of 0.3m solution in Et₂O, 45 mmol, 4 equiv) was added dropwise at 0°C to a stirred solution of β-ketoester (+/−)-21a (2.75 g, 11.25 mmol) in THF (5 mL). The mixture was stirred overnight at 4°C, quenched by slow addition of water and stirred for an additional 1 h. Anhydrous sodium sulfate was added and the resulting suspension was filtered and the filtrate was concentrated. The residue was dissolved in methylene chloride and filtered again and dried. Purification by flash column chromatography (8% EtOAc/hexanes) gave recovered starting material (0.27 g, 10%) and racemic β-hydroxyesters 22a and ent-22a (2.1 g, 75%) as a yellow oil. R_f=0.17 (silica gel, 25% EtOAc/hexanes); ¹H NMR (400MHz, CDCl₃): δ=7.37–7.24 (m, 5H), 4.98 (s, 1H), 4.22 (q, J=7.2 Hz, 2H), 3.03–2.94 (m, 3H), 2.32 (d, J=5.6Hz, 1H), 1.90 (s, 3H), 1.31 ppm (t, J=7.2Hz, 3H); ¹³C NMR (100MHz, CDCl₃): δ=174.9, 137.2, 135.7, 135.1, 128.4, 127.9, 127.3, 83.9, 61.1, 51.8, 37.1, 14.5, 12.8 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₈O₃+Na⁺: 269.1154 [M+Na⁺]; found: 269.1143.

(1R,2S,3E)-2-Acetoxy-3-methyl-4-phenylcyclopent-3-ene carboxylic acid ethyl ester (26) and (1R,2R,3E)-2-hydroxy-3-methyl-4-phenyl-cyclopent-3-ene carboxylic acid ethyl ester (ent-22a)

Vinyl acetate (7.5 mL, 81.4 mmol) was added to a solution of 22a and ent-22a (2g, 8.13 mmol) in anhydrous pentane (30 mL). Amano PS-D lipase (2.0 g) and 4 Å MS (2.0 g) were added and the suspension was stirred at room temperature. The reaction was monitored by TLC and ¹H NMR spectroscopy and after 4 d, 48–50% conversion of 22a to the acetate was achieved. The sieves and lipase were filtered and washed with Et₂O. The solvent was removed and crude product was purified by flash column chromatography (20% Et₂O/hexanes) to give 26 (1.15 g, 49%) as a slightly yellow oil and ent-22a (0.96 g, 48%, 98% ee).

Data for 26: ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-50.5$ (c=0.6 in CHCl₃); R_f=0.38 (silica gel, 25% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=7.40–7.25 (m, 5H), 6.06 (s, 1H), 4.20 (q, J=7.2Hz, 2H), 3.08–2.95 (m, 3H), 2.11 (s, 3H), 1.80 (s, 3H), 1.28 ppm (t, J=7.2Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=174.2, 170.9, 138.4, 136.7, 131.8, 128.5, 128.0, 127.7, 85.9, 61.2, 48.7, 38.6, 21.3, 14.4, 12.9 ppm; HRMS (ESI): m/z: calcd for C₁₇H₂₀O₄+Na⁺: 311.1259 [M+Na⁺]; found: 311.1240.

Data for ent-22a: ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-15.2$ (c=0.6 in CHCl₃); see 22a below for ¹H and ¹³C NMR spectroscopic data.

(1R,2S,3E)-2-Hydroxy-3-methyl-4-phenylcyclopent-3-ene carboxylic acid ethyl ester (22a)

Anhydrous potassium carbonate (240 mg, 1.74 mmol) was added at 0°C to a solution of 26 (500 mg, 1.74 mmol) in dry ethanol (15 mL) and the solution was stirred at room temperature for 12h. Ethanol was removed under reduced pressure and the residue was dissolved in methylene chloride and washed with a saturated aqueous solution of ammonium chloride. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (25 % EtOAc/hexane) to give compound 22a (393 mg, 92%, 99 % ee) as a slightly yellow oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=+14.8$ (c=0.8 in CHCl₃); R_f=0.17 (silica gel, 25% EtOAc/hexane); ¹H NMR (400 MHz, CDCl₃): δ=7.37–7.25 (m, 5H), 4.99 (s, 1H), 4.23 (q, J=7.2Hz, 2H), 3.05–2.94 (m, 3H), 2.33 (d, J=5.6Hz, 1H), 1.90 (s, 3H), 1.32 ppm (t, J=7.2Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=174.9, 137.2, 135.8, 135.1, 128.4, 127.9, 127.3, 84.0, 61.1, 51.7, 37.1, 14.5, 12.9 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₈O₃+Na⁺ 269.1154 [M+Na⁺]; found: 269.1148.

(1R*,2S*,3E)-2-Hydroxy-3-methyl-4-pyridin-2-ylcyclopent-3-ene carboxylic acid ethyl ester (22b/ent-22b) and (27/ent-27)

Trifluoroacetic acid (0.297 mL, 3.86 mmol) was added to a stirred solution of compound 21b (0.5 g, 1.93 mmol) in methylene chloride (6 mL). The solvent was removed in vacuo and the resulting salt was dissolved in methanol (7 mL) and cooled to 0°C. Sodium borohydride (0.73 g, 19.3 mmol) was added rapidly at once to this solution at 0°C. After stirring at the same temperature for 30 min, chloroform was added and the mixture was washed with saturated aqueous sodium bicarbonate solution and brine. The organic layer was dried over anhydrous sodium sulfate and concentrated. The residue (0.459 g, 91%, cis/trans 1:2, determined by ¹H NMR spectroscopy) was separated by using silica-gel chromatography (16–25 % EtOAc/hexane) to obtain 22b/ent-22b (0.306 g, 67%) and 27/ent-27 (0.153 g, 33%).

Data for 27/ent-27: R_f=0.29 (silica gel, 20% EtOAc/hexane); ¹H NMR (C₆D₆, 600 MHz): δ=8.38 (s, 1H), 6.88–6.87 (m, 2H), 4.61 (s, 1H), 3.99–3.89 (m, 2H), 3.54–3.49 (m, 1H), 3.05–3.01 (m, 1H), 2.88–2.84 (m, 1H), 2.33 (d, J=7.8 Hz, 1H), 2.13 (s, 3H), 1.77 (s, 3H), 0.90ppm (t, J=14.4, 3H); ¹³C NMR (C₆D₆, 400 MHz): δ=172.9, 153.5, 149.9, 138.6, 136.2, 135.9, 130.7, 121.9, 82.0, 60.4, 46.5, 36.2, 17.8, 14.1, 13.8 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₉NO₃+Na⁺: 284.1263 [M+Na⁺]; found: 284.1248.

(1R,2S,3E)-2-Acetoxy-3-methyl-4-pyridin-2-ylcyclopent-3-ene carboxylic acid ethyl ester (28) and (1R,2R,3E)-2-hydroxy-3-methyl-4-pyridin-2-ylcyclopent-3-ene carboxylic acid ethyl ester (ent-22b)

Vinyl acetate (0.95 mL, 10.3 mmol) was added to a solution of 22b/ent-22b (0.27 g, 1.03 mmol) in anhydrous pentane (3 mL). Amano PS-D lipase (0.27 g) and 4 Å MS (0.27 g) were added and the suspension was stirred at room temperature. The reaction was monitored by TLC and ¹H NMR spectroscopy and after 3 d, 49–50% conversion of 22b/ent-22b to acetate 28 was achieved. The sieves and lipase were filtered off and washed with Et₂O. The solvent was removed and the crude product was purified by column chromatography (17% EtOAc/hexane) to give 28 (0.154 g, 49%) as a pale-yellow oil and ent-22b (0.130 g, 48%).

Data for 28: ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-40.6$ (c=1.0 in CHCl₃); R_f=0.26 (silica gel, 75% EtOAc/hexanes); ¹H NMR (CDCl₃, 600 MHz): δ=8.44 (s, 1H), 7.47 (d, J=7.8 Hz, 1H), 7.20 (d, J=7.8 Hz, 1H), 6.07 (s, 1H), 4.19 (q, J=14.4 Hz, 2H), 3.22–3.16 (m, 1H), 3.05–3.01 (m, 2H), 2.32 (s, 3H), 2.09 (s, 3H), 1.94 (s, 3H), 1.26ppm (t, J=7.2Hz, 3H); ¹³C NMR (CDCl₃, 400 MHz): δ=174.1, 170.9, 152.5, 150.0, 137.2, 136.7, 135.0, 137.8, 122.5, 85.9, 61.1, 48.5, 37.5, 21.3, 18.5, 14.4, 13.2 ppm; HRMS (ESI): m/z: calcd for C₁₇H₂₁NO₄+Na⁺: 326.1368 [M+Na⁺]; found: 326.1354.

Data for ent-22b: ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=+7.8$ (c=1.0 in CHCl₃). See 22b below for ¹H and ¹³C NMR spectroscopic data.

(1R,2S,3E)-2-Hydroxy-3-methyl-4-pyridin-2-ylcyclopent-3-ene carboxylic acid (22b)

Anhydrous potassium carbonate (45.6 mg, 0.33 mmol) was added at 0 °C to a solution of 28 (100 mg, 0.33 mmol) in dry ethanol (2.8 mL) and the solution was stirred at room temperature for 12 h. Ethanol was removed under reduced pressure and the residue was dissolved in methylene chloride and washed with a saturated aqueous solution of ammonium chloride. The organic phase was dried over anhydrous sodium sulfate and the solvent was removed in vacuo. The residue was purified by flash column chromatography (25 % EtOAc/hexane) to give compound 22b (81 mg, 94%, 95% ee) as a pale-yellow oil. ${\left[\alpha \right]}_{\text{D}}^{\text{20}}=-8.1$ (c=0.26 in CHCl₃); TLC R_f=0.27 (silica gel, 75% EtOAc/hexanes); ¹H NMR (C₆D₆, 600 MHz): δ=8.48 (s, 1H), 6.97–6.92(m, 2H), 5.03 (s, 1H), 4.04–4.00 (m, 2H), 3.22–3.21 (m, 2H), 3.02–2.98 (m, 1H), 2.18 (s, 3H), 1.87 (s, 3H), 0.98ppm (t, J=14.4Hz, 3H); ¹³C NMR (CDCl₃, 400 MHz): δ=174.6, 153.1, 149.9, 138.4, 136.7, 134.9, 131.4, 122.4, 84.1, 61.0, 51.7, 35.9, 18.5, 14.5, 13.1 ppm; HRMS (ESI): m/z: calcd for C₁₅H₁₉NO₃+Na⁺: 284.1263 [M+Na⁺]; found: 284.1279.

(1R,2S,3E)-3-Methyl-4-phenyl-2-triethylsilanyloxycyclopent-3-ene car-boxylic acid ethyl ester (29 a)

Triethylsilyl chloride (876 µL, 5.22 mmol, 1.5 equiv) was added dropwise to a solution of alcohol 22a (856 mg, 3.48 mmol) and imidazole (711 mg, 10.44 mmol, 3 equiv) in methylene chloride (25 mL) at 0°C. After stirring at 0°C for 2 h, the reaction was quenched with water (15 mL) and extracted with ethyl acetate (3×30 mL). The organic extracts were washed with brine (20 mL) and dried over anhydrous sodium sulfate. Filtration and evaporation of the solvents in vacuo furnished an oily crude product which was purified by flash column chromatography (2% EtOAc/hexanes) to give TES ether 29a (1.16g, 93%) as a yellow oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-34.2$ (c=0.6 in CHCl₃); R_f=0.53 (silica gel, 10% EtOAc/hexanes); ¹H NMR (400 MHz, CDCl₃): δ=7.36–7.22 (m, 5H), 5.11 (s, 1H), 4.21 (q, J=7.2Hz, 2H), 3.08–2.82 (m, 3H), 1.83 (s, 3H), 1.32(t, J=7.2Hz, 3H), 1.00 (t, J=8.0Hz, 9H), 0.68 ppm (q, J=8.0Hz, 6H); ¹³C NMR (75 MHz, CDCl₃): δ=175.5, 137.5, 135.7, 134.9, 128.3, 128.0, 127.2, 84.5, 60.9, 51.8, 38.6, 14.5, 12.9, 7.1, 5.1, 0.2ppm; HRMS (ESI): m/z: calcd for C₂₁H₃₂O₃Si+Na⁺: 383.2018 [M+Na⁺]; found: 383.2019.

(1R,2S,3E)-3-Methyl-4-phenyl-2-triethylsilanyloxycyclopent-3-enecarb-aldehyde (30 a)

A solution of DIBAL-H (2.2 mL, 1.0m in hexane, 2.2 mmol, 1.1 equiv) was added dropwise to a solution of silyl ether 29 a (720 mg, 2 mmol) in toluene (10 mL) at −78°C. The reaction was stirred at that temperature for 1 h and quenched by dropwise addition of saturated NH₄Cl (1.0 mL). The reaction was allowed to reach room temperature and a saturated aqueous solution of Rochelle salt (3.0 mL) and brine (2.0 mL) were added. The mixture was extracted with ethyl acetate (3×7 mL) and the combined organic extracts were dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo to give crude aldehyde 30a as a colorless liquid which was used in the next step without further purification; ¹H NMR (400 MHz, CDCl₃): δ=9.86 (d, J=2.0Hz, 1H), 7.38–7.25 (m, 5H), 5.08 (s, 1H), 3.12–2.98 (m, 2H), 2.90–2.84 (m, 1H), 1.84 (s, 3H), 1.00 (t, J=8.0Hz, 9H), 0.68 ppm (q, J=8.0Hz, 6H); ¹³C NMR (75 MHz, CDCl₃): δ=200.2, 135.5, 133.9, 133.7, 126.6, 126.3, 125.6, 79.6, 57.4, 33.1, 11.3, 5.3, 3.4 ppm.

(1S,2E,5S)-Triethyl-(2-methyl-3-phenyl-5-vinylcyclopent-2-enyloxy)silane (31 a)

nBuLi (0.775 mL of 1.6m solution in hexanes, 1.24 mmol, 1.95 equiv) was added dropwise to a precooled (0 °C) solution of methyltriphenylphosphonium bromide (455 mg, 1.27 mmol, 2 equiv) in THF (5 mL). The reaction mixture was stirred at 0°C for 30 min. A solution of aldehyde 30 a (0.2g, 0.633 mmol) in THF (2mL) was added and the mixture was stirred at 0 °C for 30 min and quenched with saturated aqueous ammonium chloride (5 mL). The solvent was removed under reduced pressure and the aqueous residue was extracted with ethyl acetate (3×5 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography (5 % Et₂O/hexanes) afforded pure olefin 31a (0.14 g, 71 %) as a colorless oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-14.3$ (c=0.4 in CHCl₃); R_f=0.73 (silica gel, 10% Et₂O/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=7.34–7.21 (m, 5H), 5.94–5.88 (m, 1H), 5.12 (d, J=16.8 Hz, 1H), 5.04 (dd, J=1.8, 10.2Hz, 1H), 4.52 (d, J=4.8 Hz, 1H), 2.88–2.82 (m, 1H), 2.74–2.68 (m, 1H), 2.52–2.47 (m, 1H), 1.82 (s, 3H), 0.98 (t, J=7.8Hz, 9H), 0.66 ppm (q, J=7.8 Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ=141.1, 138.1, 136.3, 135.7, 128.3, 127.9, 126.9, 115.2, 87.0, 52.1, 40.4, 13.1, 7.2, 5.5 ppm; HRMS (ESI): m/z: calcd for C₂₀H₃₀OSi+Na⁺: 337.1964 [M+Na⁺]; found: 337.1970.

(1S,2E,5S)-2-Methyl-3-phenyl-5-vinyl-cyclopent-2-enol (5a)

Tetrabutylammonium fluoride (1.53 mL of 1m solution in THF, 1.53 mmol) was added dropwise to a solution of compound 31a (0.48 g, 1.53 mmol) in THF (7 mL) at 0 °C. The reaction mixture was stirred at 0°C for 30 min and then water (6 mL) and ethyl acetate (10 mL) were added. The layers were separated and the aqueous layer was extracted with ethyl acetate (2×10 m). The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography (10% EtOAc/hexanes) afforded pure alcohol 5a (0.245 g, 80%) as a white solid. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-14.17$ (c=1.2 in CHCl₃); R_f=0.45 (silica gel, 33% EtOAc/hexanes); ¹H NMR (600MHz, CDCl₃): δ=7.36–7.23 (m, 5H), 6.00–5.94 (m, 1H), 5.18 (dd, J=1.2, 16.8 Hz, 1H), 5.08 (dd, J=0.6, 10.2Hz, 1H), 4.48 (d, J=6.6Hz, 1H), 2.85–2.81 (m, 1H), 2.71–2.65 (m, 1H), 2.59–2.54 (m, 1H), 1.89 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=140.4, 137.8, 136.3, 135.9, 128.4, 127.9, 127.2, 115.4, 86.4, 52.4, 39.8, 12.9 ppm; HRMS (ESI): m/z: calcd for C₁₄H₁₆O+Na⁺: 223.1099 [M+Na⁺]; found: 223.1100.

(1R,2S)-3-Methyl-4-(5-methylpyridin-2-yl)-2-triethylsilanyloxycyclopent-3-ene carboxylic acid ethyl ester (29 b)

Triethylsilyl chloride (145 µL, 0.863 mmol, 1.5 equiv) was added dropwise to a solution of alcohol 22b (150 mg, 0.575 mmol) and imidazole (17 mg, 1.72 mmol, 3 equiv) in methylene chloride (4 mL) at 0°C. After stirring at 0°C for 2h, the reaction was quenched with water (2.5 mL) and extracted with ethyl acetate (3×5 mL). The organic extracts were washed with brine (3.5 mL) and dried over anhydrous sodium sulfate. Filtration and evaporation of the solvents in vacuo furnished an oily crude product which was purified by flash column chromatography on silica (2–7% EtOAc/hexanes) to give 29b (198 mg, 92%) as a yellow oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-51.04$ (c=0.7 in CHCl₃); R_f=0.29 (silica gel, 25% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=8.41 (s, 1H), 7.44 (dd, J=1.8, 8.4 Hz, 1H), 7.19 (d, J=7.8Hz, 1H), 5.12 (d, J=4.8Hz, 1H), 4.18 (dd, J=7.2, 14.4 Hz, 2H), 3.17–3.12 (m, 1H), 3.00–2.89 (m, 2H), 2.30 (s, 3H), 1.97 (s, 3H), 1.28 (t, J=14.4Hz, 3H), 0.96 (t, J=7.8Hz, 9H), 0.65 ppm (q, J=7.8Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ=175.3, 153.3, 149.9, 139.1, 136.5, 134.1, 131.1, 122.4, 84.5, 60.8, 51.6, 37.4, 18.4, 14.5, 13.3, 7.0, 5.2 ppm; HRMS (ESI): m/z: calcd for C₂₁H₃₃NO₃Si+H⁺: 376.2308 [M+H⁺]; found: 376.2302.

(3S,4S)-5-Methyl-2-(2-methyl-3-triethylsilanyloxy-4-vinylcyclopent-1-enyl)pyridine (31 b)

A solution of DIBAL-H (0.44 mL, 1.0 m in hexane, 0.44 mmol, 1.1 equiv) was added dropwise to a solution of silyl ether 29b (150 mg, 0.4 mmol) in toluene (2 mL) at −78°C. The reaction mixture was stirred at that temperature for 1 h and quenched by dropwise addition of saturated aqueous ammonium chloride solution (0.2 mL). The reaction mixture was allowed to reach room temperature and a saturated aqueous solution of Rochelle salt (1 mL) and brine (0.4 mL) were added. The mixture was extracted with ethyl acetate (3×2 mL) and the combined organic extracts were dried over anhydrous sodium sulfate, filtered, and evaporated in vacuo to give crude aldehyde 30b as a yellow liquid which was used in the next step without further purification. nBuLi (0.49 mL of 1.6 m solution in hexanes, 0.78 mmol, 1.95 equiv) was added dropwise to a precooled (0 °C) solution of methyltriphenylphosphonium bromide (286 mg, 0.8 mmol, 2 equiv) in THF (3 mL). The reaction mixture was stirred at 0°C for 30 min. A solution of crude aldehyde 30b (ca. 0.4 mmol) in THF (1.5 mL) was added and the mixture was stirred at 0°C for 30 min and then quenched with saturated aqueous ammonium chloride solution (3 mL). The solvent was removed under reduced pressure and the aqueous residue was extracted with ethyl acetate (3×3 mL). The combined organic extracts were dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography on silica (7% EtOAc/hexanes) afforded pure olefin 31b (96 mg, 73%) as a yellowish oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-21.29$ (c=0.7 in CHCl₃); R_f=0.66 (silica gel, 33% EtOAc/hexanes); ¹H NMR (600MHz, CDCl₃): δ=8.42 (s, 1H), 7.44 (dd, J=1.8, 7.8 Hz, 1H), 7.18 (d, J=7.8 Hz, 1H), 5.9–55.88 (m, 1H), 5.12 (d, J=16.8 Hz, 1H), 5.04 (d, J=10.8 Hz, 1H), 4.54 (d, J=5.4Hz, 1H), 2.97–2.93 (m, 1H), 2.74–2.68 (m, 1H), 2.60–2.56 (m, 1H), 2.30 (s, 3H), 1.96 (s, 3H), 0.98 (t, J=7.8Hz, 9H), 0.64ppm (q, J=7.8Hz, 6H); ¹³C NMR (100 MHz, CDCl₃): δ=153.9, 149.8, 140.9, 139.8, 136.5, 135.0, 130.9, 122.4, 115.2, 87.2, 51.8, 39.2, 18.4, 13.4, 7.2, 5.5 ppm; HRMS (ESI): m/z: calcd for C₂₀H₃₁NOSi+H⁺: 330.2253 [M+H⁺]; found: 330.2258.

(1S,5S)-2-Methyl-3-(5-methylpyridin-2-yl)-5-vinylcyclopent-2-enol (5 b)

Tetrabutylammonium fluoride (0.152mL of 1m solution in THF, 0.152 mmol) was added dropwise to a solution of compound 31b (50 mg, 0.152 mmol) in THF (1 mL) at 0°C. The reaction mixture was stirred at 0°C for 30 min and then water (1 mL) and ethyl acetate (2mL) were added. The layers were separated and the aqueous layer was extracted with ethyl acetate (2×2 mL). The combined organic layers were dried over anhydrous sodium sulfate and concentrated in vacuo. Purification by flash column chromatography on silica (16–50% EtOAc/hexanes) afforded pure alcohol 5b (28 mg, 83%) as a white solid. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-26.0$ (c=0.3 in CHCl₃); R_f=0.25 (silica gel, 50% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=8.43 (s, 1H), 7.46 (dd, J=1.8Hz, 7.8 Hz, 1H), 7.18 (d, J=7.8 Hz, 1H), 6.0–5.95 (m, 1H), 5.14 (d, J=16.8 Hz, 1H), 5.08 (d, J=10.2 Hz, 1H), 4.48 (s, 1H), 2.96–2.91 (m, 1H), 2.69–2.60 (m, 2H), 2.32 (s, 3H), 2.03 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=153.6, 149.9, 140.2, 139.4, 136.7, 135.6, 131.2, 122.3, 115.4, 86.4, 52.3, 38.5, 18.5, 13.2ppm; HRMS (ESI): m/z: calcd for C₁₄H₁₇NO+Na⁺: 238.1208 [M+Na⁺]; found: 238.1191.

(3S,6R,7S,8S)-3-(tert-Butyldimethylsilanyloxy)-4,4,6,8,12-pentamethyl-5-oxo-7-(2,2,2-trichloroethoxycarbonyloxy)tridec-12-enoic acid (1S,2E,5S)-2-methyl-3-phenyl-5-vinylcyclopent-2-enyl ester (32 a)

DCC (0.027 mL of 1m solution in CH₂Cl₂, 0.027 mmol, 1.3 equiv) was added dropwise to a solution of acid 17 (13 mg, 0.021 mmol), alcohol 5a (4.6 mg, 0.023 mmol, 1.1 equiv), and DMAP (1 mg, 0.008 mmol, 0.4 equiv) in methylene chloride (0.5 mL) at 0 °C. The reaction mixture was stirred for 15 min at 0°C and for 16 h at room temperature. The solid precipitate was filtered off and the filtrate was concentrated in vacuo. Purification by flash column chromatography (5% Et₂O/hexanes) afforded ester 32 a (11 mg, 64%) as a colorless oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-53.2$ (c=0.85 in CHCl₃); TLC R_f=0.67 (silica gel, 25% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=7.36–7.25 (m, 5H), 5.98–5.91 (m, 1H), 5.70 (d, J=4.2Hz, 1H), 5.10 (d, J=17.4 Hz, 1H), 5.03 (d, J=10.2Hz, 1H), 4.88–4.85 (m, 1H), 4.70 (dd, J=3.0, 8.4 Hz, 1H), 4.66–4.60 (m, 3H), 4.29 (t, J=4.2Hz, 1H), 3.50–3.46 (m, 1H), 2.96–2.92 (m, 1H), 2.86–2.81 (m, 1H), 2.68 (dd, J=3.6, 17.4 Hz, 1H), 2.59–2.55 (m, 1H), 2.23 (dd, J=5.4, 17.4 Hz, 1H), 1.95–1.87 (m, 2H), 1.75 (s, 3H), 1.66 (s, 3H), 1.54–1.53 (m, 6H), 1.33–1.27 (m, 5H), 1.06–1.04 (m, 3H), 0.96 (d, J=6.6Hz, 3H), 0.87 (s, 9H), 0.13 (s, 3H), 0.05 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=215.4, 172.3, 154.4, 145.8, 139.3, 137.3, 132.4, 128.4, 127.9, 127.4, 115.3, 110.3, 95.0, 88.2, 82.4, 76.9, 75.0, 56.0, 54.0, 47.6, 42.2, 40.5, 40.3, 38.2, 35.2, 34.9, 31.8, 31.2, 26.2, 25.7, 24.9, 24.8, 22.7, 22.6, 20.6, 18.4, 16.1, 13.1, 11.1, −4.1, −4.5 ppm; HRMS (ESI): m/z: calcd for C₄₁H₆₁Cl₃O₇Si+Na⁺: 821.3150 [M+Na⁺]; found: 821.3178.

(3S,6R,7S,8S)-3,7-Dihydroxy-4,4,6,8,12-pentamethyl-5-oxotridec-12-enoic acid (1S,2E,5S)-2-methyl-3-phenyl-5-vinylcyclopent-2-enyl ester (2 a)

Anhydrous ammonium chloride (75 mg) followed by zinc dust (75 mg) was added to a solution of ester 32a (12mg, 0.015 mmol) in dry ethanol (1.5 mL). The reaction mixture was stirred at room temperature for 45 min before it was diluted with ethyl acetate (5 mL) and filtered though a plug of Celite. The solution was concentrated and passed though a small plug of silica gel to give compound 33 which was used in the next step without further purification. To the crude solution of compound 33 was added a solution of tris(dimethylamino)sulfur (trimethylsilyl)difluoride (TAS-F) (5 mg, 0.187 mmol, 1 equiv) in N,N-dimethylformamide (0.2mL). After 24 h, another 5 mg of TAS-F were added and the mixture was stirred for an additional 24 h after which it was diluted with ethyl acetate (5 mL) and washed with phosphate buffer pH 7 (5 mL). The aqueous layer was extracted with ethyl acetate (3×5 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by silica gel chromatography (10% EtOAc/hexanes) to give pure compound 2a (4.7 mg, 62% over two steps) as a colorless material. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-77.5$ (c=0.2in CHCl₃); R_f=0.74 (silica gel, 50% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=7.37–7.25 (m, 5H), 6.0–5.92 (m, 1H), 5.76 (d, J=3.6 Hz, 1H), 5.13 (d, J=16.8 Hz, 1H), 5.06 (d, J=10.2 Hz, 1H), 4.66 (d, J=10.2 Hz, 2H), 4.29–4.26 (m, 1H), 3.39–3.35 (m, 2H), 3.27–3.23 (m, 2H), 2.99–2.93 (m, 1H), 2.88–2.83 (m, 1H), 2.61–2.57 (m, 1H), 2.52 (dd, J=1.8, 16.2 Hz, 1H), 2.44 (dd, J=10.2, 16.2 Hz, 1H), 2.04–1.96 (m, 2H), 1.79 (s, 3H), 1.75–1.72 (m, 1H), 1.69 (s, 3H), 1.54–1.52(m, 1H), 1.36–1.30 (m, 1H), 1.20–1.18 (s overlapping with m, 4H), 1.15 (s, 3H), 1.06 (d, J=7.2 Hz, 3H), 0.84 ppm (d, J=7.2Hz, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=222.5, 173.2, 146.4, 139.8, 139.4, 137.1, 131.8, 128.5, 127.9, 127.6, 115.5, 109.9, 88.6, 75.0, 72.7, 52.3, 47.8, 41.1, 40.4, 38.4, 36.9, 35.7, 32.7, 25.0, 22.6, 21.5, 19.2, 15.7, 13.1, 10.2ppm; HRMS (ESI): m/z: calcd for C₃₂H₄₆O5+Na⁺: 533.3243 [M+Na⁺]; found: 533.3213.

2E,4S,7S,10R,11S,12S,16Z,18S-7,11-Dihydroxy-3,8,8,10,12,16-hexamethyl-2-phenyl-3 a,7,8,10,11,12,13,14,15,17 a-decahydro-1H,6H-4-oxacyclopentacyclohexadecene-5,9-dione (1 a)

A solution of the second-generation Grubbs catalyst (1.5 mg, 0.0018 mmol; weighed under argon) in methylene chloride (1.5 mL) was added to a solution of compound 2a (1.5 mg, 0.0032 mmol) in methylene chloride (0.5 mL). The reaction mixture was heated at 50 °C for 16 h and applied directly to a preparative TLC plate (25% EtOAc/hexanes) to give the target (Z)-1a and slightly impure (E)-1a (0.2mg) separately (≈50% overall yield). The Z isomer was purified by a second preparative TLC by using (3% methanol/methylene chloride) to remove the last traces of the catalyst and to furnish the desired target molecule (Z)-1a (0.5 mg). ¹H NMR (600 MHz, CDCl₃): δ=7.36–7.33 (m, 2H), 7.27–7.24 (m, 3H), 5.82 (d, J=7.2Hz, 1H), 5.24 (d, J=10.2Hz, 1H), 4.2–4.17 (m, 1H), 3.69–3.66 (m, 1H), 3.22–3.15 (m, 2H), 2.81 (dd, J=8.4, 15.6 Hz, 1H), 2.66 (d, J=4.2 Hz, 1H), 2.58 (dd, J=10.8, 16.8 Hz, 2H), 2.52–2.47 (m, 1H), 2.39–2.31 (m, 2H), 1.80–1.75 (s overlapping with m, 4H), 1.68 (s, 3H), 1.66–1.62 (m, 3H), 1.36 (s, 3H), 1.18 (d, J=6.6 Hz, 3H), 1.07 (s, 3H), 0.98 ppm (d, J=7.2 Hz, 3H); HRMS (ESI): m/z: calcd for C₃₀H₄₂O₅+Na⁺: 505.2930 [M+Na⁺]; found: 505.2893.

(3S,6R,7S,8S,1′S,5′S)-3-(tert-Butyldimethylsilanyloxy)-4,4,6,8,12-pentamethyl-5-oxo-7-(2,2,2-trichloroethoxycarbonyloxy)tridec-12-enoic acid 2-methyl-3-(5-methylpyridin-2-yl)-5-vinylcyclopent-2-enyl ester (32 b)

DCC (0.017 mL of 1 m solution in CH₂Cl₂, 0.017 mmol, 1.3 equiv) was added dropwise to a solution of acid 17 (8.0 mg, 0.013 mmol), alcohol 5b (3.0 mg, 0.014 mmol, 1.1 equiv), and DMAP (1 mg, 0.008 mmol, 0.6 equiv) in methylene chloride (0.5 mL) at 0°C. The reaction mixture was stirred for 15 min at 0°C and for 16 h at room temperature. After this time, the solid precipitate was filtered off and the filtrate was concentrated in vacuo. Purification by flash column chromatography on silica (5% EtOAc/hexanes) afforded ester 32b (9 mg, 85%) as a colorless oil. ${\left[\alpha \right]}_{\text{D}}^{\text{22}}=-29.8$ (c=1 in CHCl₃); R_f=0.48 (silica gel, 25% EtOAc/hexanes); ¹H NMR (600 MHz, CDCl₃): δ=8.43 (s, 1H), 7.46 (dd, J=1.8, 7.8 Hz, 1H), 7.18 (d, J=7.8 Hz, 1H), 5.98%5.91 (m, 1H), 5.73 (d, J=4.8 Hz, 1H), 5.11 (d, J=16.8 Hz, 1H), 5.02 (d, J=10.2 Hz, 1H), 4.86 (d, J=12.0 Hz, 1H), 4.73–4.70 (m, 1H), 4.67–4.61 (m, 3H), 4.30–4.28 (m, 1H), 3.50–3.45 (m, 1H), 3.06–3.02 (m, 1H), 2.88–2.83 (m, 1H), 2.70–2.65 (m, 1H), 2.33 (s, 3H), 2.23 (dd, J=5.4, 17.4 Hz, 1H), 1.98–1.91 (m, 2H), 1.95 (s, 3H), 1.76–1.74 (m, 1H), 1.66 (s, 3H), 1.50–1.22 (m, 5H), 1.10–1.04 (m. 6H), 0.96 (d, J=7.2 Hz, 3H), 0.87 (m, 12H), 0.13 (s, 3H), 0.05 ppm (s, 3H); ¹³C NMR (100 MHz, CDCl₃): δ=215.4, 172.3, 154.4, 153.1, 149.9, 145.8, 139.5, 138.3, 136.7, 135.7, 131.5, 125.5, 122.5, 115.4, 110.3, 95.0, 88.2, 82.4, 75.0, 54.0. 47.5, 42.2, 40.2, 39.2, 38.2, 34.9, 31.8, 29.9, 26.3, 24.8, 22.6, 22.5, 20.7, 18.5, 18.4, 16.1, 13.4, 11.1, −4.1, −4.5 ppm; HRMS (ESI): m/z: calcd for C₄₁H₆₂Cl₃NO₇Si+H⁺: 814.3434 [M+H⁺]; found: 814.3481.

(3S,6R,7S,8S,1′S,5′S)-3,7-Dihydroxy-4,4,6,8,12-pentamethyl-5-oxotridec-12-enoic acid 2-methyl-3-(5-methyl-pyridin-2-yl)-5-vinylcyclopent-2-enyl ester (2 b)

A solution of tris(dimethylamino)sulfur (trimethylsilyl)difluoride (TAS-F) (1.7 mg, 0.008 mmol, 1 equiv) in N,N-dimethylformamide (0.2mL) was added to a solution of ester 32b (5 mg, 0.006 mmol) in DMF (0.2mL). After 24 h, another 1.7 mg of TAS-F were added and the mixture was diluted with ethyl acetate (3 mL) and washed with phosphate buffer pH 7 (5 mL). The aqueous layer was extracted with ethyl acetate (3×5 mL) and the combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude oil was purified by silica-gel chromatography (10% EtOAc/hexanes) to give compound 34 which was dissolved in dry ethanol (1.2mL) and treated with anhydrous ammonium chloride (62mg) followed by zinc dust (62mg). The reaction mixture was stirred at room temperature for 45 min before it was diluted with ethyl acetate (3 mL) and filtered though a plug of Celite. The solution was concentrated and purified by silica-gel chromatography (20% EtOAc/hexanes) to give pure compound 2b (2mg, 62% over two steps) as a colorless material. R_f=0.36 (silica gel, 33% EtOAc/hexanes); ¹H NMR (500 MHz, CDCl₃): δ=8.47 (s, 1H), 7.50 (d, J=8.0 Hz, 1H), 7.21 (d, J=8.5 Hz, 1H), 6.02–5.95 (m, 1H), 5.81 (d, J=4.5 Hz, 1H), 5.16 (d, J=17.5 Hz, 1H), 5.07 (d, J=10.5 Hz, 1H), 4.68 (d, J=9.0 Hz, 2H), 4.30 (d, J=10.5 Hz, 1H), 3.41–3.37 (m, 2H), 3.30–3.27 (m, 1H), 3.25 (d, J=3.5 Hz, 1H), 3.10–3.3.06 (m, 1H), 2.91–2.88 (m, 1H), 2.71–2.69 (m, 1H), 2.54 (dd, J=2.5, 16.5 Hz, 1H), 2.46 (dd, J=10.5, 16.5 Hz, 1H), 2.35 (s, 3H), 2.06–2.02 (m, 2H), 1.98 (s, 3H), 1.78–1.76 (m, 1H), 1.72(s, 3H), 1.33–1.31 (m, 2H), 1.22 (s, 3H), 1.17 (s, 3H), 1.08 (d, J=6.5 Hz, 3H), 0.87 ppm (d, J=7.0 Hz, 3H); ¹³C NMR (125 MHz, CDCl₃): δ=222.1, 172.9, 152.8, 149.8, 146.2, 139.1, 136.5, 135.0, 131.4, 122.2, 115.3, 109.7, 88.3, 74.9, 72.5, 52.1, 47.5, 41.0, 38.9, 38.2, 36.8, 35.5, 32.5, 29.7, 24.9, 22.4, 21.3, 19.0, 18.3, 15.6, 13.2, 10.0 ppm; HRMS (ESI): m/z: calcd for C₃₂H₄₇NO₅+Na⁺: 548.3352[M+Na⁺]; found: 548.3331.

(3S,6R,7S,8S,1′S,5′S)-7,11-Dihydroxy-3,8,8,10,12,16-hexamethyl-2-(5-methylpyridin-2-yl)-3a,7,8,10,11,12,13,14,15,17a-decahydro-1H,6H-4-oxacyclopentacyclohexadecene-5,9-dione (1 b)

A solution of the second-generation Grubbs catalyst (2.5 mg, 0.003 mmol; weighed under argon) in methylene chloride (1.5 mL) was added to a solution of compound 2b (2.5 mg, 0.0052 mmol) in methylene chloride (0.5 mL). The reaction mixture was heated at 50 °C for 16 h and applied directly to a preparative TLC plate and developed (25% EtOAc/hexanes) to give the target (Z)-1b (0.5 mg) along with phenyl analogue 35 and the dimer 36 (≈0.5 mg each; ≈ 55 overall yield).

Data for compound 1b: R_f=0.23 (silica gel, 50% EtOAc/hexanes); ¹H NMR (500 MHz, CDCl₃): δ=8.60 (s, 1H), 8.16 (d, J=8.0 Hz, 1H), 7.54 (d, J=8.0 Hz, 1H), 5.84 (d, J=7.0 Hz, 1H), 5.25 (d, J=10.0 Hz, 1H), 4.53 (d, J=10.0 Hz, 1H), 3.64–3.62 (m, 1H), 3.47–3.40 (m, 2H), 3.26–3.21 (m, 2H), 2.81 (dd, J=2.5, 16.5 Hz, 1H), 2.65 (dd, J=10.5, 16.5 Hz, 1H), 2.59 (s, 3H), 2.38–2.31 (m, 1H), 2.00 (s overlapping with m, 5H), 1.84–1.79 (m, 2H), 1.72 (s, 3H), 1.42 (s, 3H), 1.35–1.31 (m, 2H), 1.16 (d, J=6.5 Hz, 3H), 1.06 (s, 3H), 1.02ppm (d, J= 7.0 Hz, 3H); HRMS (ESI): m/z: calcd for C₃₀H₄₃NO₅+H⁺: 498.3219 [M+H⁺]; found: 498.3231.

3,7-Dihydroxy-4,4,6,8,12-pentamethyl-5-oxotridec-12-enoic acid 2-methyl-3-(5-methylpyridin-2-yl)-5-styrylcyclopent-2-enyl ester (35)

R_f=0.61 (silica gel, 50% EtOAc/hexanes); ¹H NMR (500 MHz, CDCl₃): δ=8.48 (d, J=2.0 Hz, 1H), 7.51 (dd, J=2.0, 8.0 Hz, 1H), 7.37–7.30 (m, 4H), 7.25–7.21 (m, 2H), 6.52 (d, J=16.0 Hz, 1H), 6.34 (dd, J=8.0, 16.0 Hz, 1H), 5.87 (d, J=4.5 Hz, 1H), 4.68 (d, J=9.0 Hz, 2H), 4.31–4.28 (m, 1H), 3.42–3.36 (m, 2H), 3.30–3.26 (m, 1H), 3.24 (d, J=4.0 Hz, 1H), 3.19–3.04 (m, 2H), 2.81–2.76 (m, 1H), 2.55 (dd, J=2.5, 16.0 Hz, 1H), 2.46 (dd, J=10.5, 16.0 Hz, 1H), 2.36 (s, 3H), 2.01 (s overlapping with m, 5H), 1.72 (m, 2H), 1.71 (s, 3H), 1.35 (m, 2H), 1.22 (s, 3H), 1.17 (s, 3H), 1.07 (d, J=7.0 Hz, 3H), 0.86 (d, J=7.0 Hz, 3H); HRMS (ESI): m/z: calcd for C₃₈H₅₁NO₅+Na⁺: 624.3665 [M+Na⁺]; found: 624.3713.

Dimer 36: R_f=0.37 (silica gel, 50% EtOAc/hexanes); ¹H NMR (500 MHz, CDCl₃): δ=8.45 (d, J=2.0 Hz, 1H), 7.48 (dd, J=2.0, 8.0 Hz, 1H), 7.17 (d, J=8.0 Hz, 1H), 5.81–5.78 (m, 1H), 5.66 (dd, J=2.5, 5.5 Hz, 1H), 4.66 (d, J=7.5 Hz, 2H), 4.45–4.41 (m, 1H), 4.20–4.17 (m, 1H), 3.55 (s, 1 H), 3.40 (d, J=9.0 Hz, 1 H), 3.35 (dd, J=6.5, 13.5 Hz, 1H), 2.98–2.93 (m, 1H), 2.87–2.83 (m, 1H), 2.65–2.60 (m, 1H), 2.54–2.51 (m, 2H), 2.34 (s, 3H), 2.05–1.94 (m, 3H), 1.91 (s, 3H), 1.70 (s, 3H), 1.26 (s overlapping with m, 5H), 1.18 (s, 3H), 1.09 (d, J=7.0 Hz, 3H), 0.87 ppm (d, J=7.0 Hz, 3H); HRMS (ESI): m/z: calcd for C₆₂H₉₀N₂O₁₀+Na⁺: 1045.6493 [M+Na⁺]; found: 1045.6522.