Small-molecule anticancer agents kill cancer cells by harnessing reactive oxygen species in an iron-dependent manner

Sara R. Fedorkaa, Kevin Soa, Ayad A. Al-Hamashia, Ibtissam Gadb, Ronit Shahb, Veronika Kholodovychb, Hanan D. Alqahtanib, William R. Taylor*b and L. M. Viranga Tillekeratne*a
aDepartment of Medicinal and Biological Chemistry, College of Pharmacy and Pharmaceutical Sciences, The University of Toledo, 2801, W. Bancroft Street, Toledo, Ohio 43606, USA. E-mail: ltillek@utoledo.edu; Tel: +1 419 530-1983
bDepartment of Biological Sciences, College of Natural Sciences and Mathematics, The University of Toledo, 2801, W. Bancroft Street, Toledo, Ohio 43606, USA. E-mail: william.taylor3@utoledo.edu; Tel: +1419 530-1966

Received 13th December 2017 , Accepted 30th January 2018

First published on 30th January 2018


In the course of generating a library of open-chain epothilones, we discovered a new class of small molecule anticancer agents that has no effect on tubulin but instead kills selected cancer cell lines by harnessing reactive oxygen species in an iron-dependent manner. Results of the preliminary studies are consistent with the recently described cell death mechanism ferroptosis. Studies are in progress to confirm ferroptosis as the cell death mechanism and to identify the specific molecular targets of these small molecule anticancer agents.


Introduction

Epothilones 1,2 (Fig. 1), first isolated from the myxobacterium Sorangium cellulosum in the 1980s,1 are potent cytotoxic agents2 that induce apoptosis through microtubule stabilization like the blockbuster drug paclitaxel.3,4 Since their isolation, they received the attention of the scientific community due to their potent cytotoxicity against a variety of cancer cell lines including breast, lung, and prostate cancers.2 Both paclitaxel and epothilone molecules bind to the α/β tubulin heterodimer subunits increasing tubulin aggregation to microtubules, while decreasing α/β heterodimer disassociation from microtubules.3
image file: c7ob03086j-f1.tif
Fig. 1 Natural epothilones 1, 2 and synthetic open-chain epothilones 3, 4.

We previously reported the synthesis and biological activity of a new class of structurally simplified open-chain epothilones 3 and 4 (Fig. 1)5 in which the C9–C15 sector of the epothilone molecules (1 and 2, Fig. 1)2–4 has been completely deleted, while retaining the two biologically relevant sectors C1–C8 and C16–C20 connected by a rigid cyclopentene molecular scaffold for conformational stability.

The diastereomer 3, with stereochemical assignments as in natural epothilones, had weak but selective activity against SNB-75 (GI50 = 21.9 μM) CNS cancer and OVCAR-4 (GI50 = 45.1 μM) ovarian cancer cell lines. A computational study performed by Rusinska-Roszak et al.6 suggested a high degree of conformational similarity between these open-chain molecules and natural epothilones and confirmed that conformational requirements for tubulin binding are being met with the open-chain analogues. In carrying out a structure–activity relationship study of these molecules, we focused on the substituent at the 2-positon of the aromatic ring as the rest of the structure embodied most of the important structural features important for activity. Thiazole was chosen as the aromatic moiety to be consistent with the thiazole of natural epothilones. Thus, we designed a library of open-chain epothilone analogues 5a–f (Scheme 1) with a variety of 2-substituted thiazole rings to investigate their effects on biological activity. One of the compounds showed strong cytotoxic activity on several cell lines in the NCI 60 human tumor cell line screen tested at 10−5 M. Follow up studies in our laboratory traced the observed activity to a part of the molecule which kills cells by a nonapoptotic mechanism of action. Preliminary studies show that these compounds do not target tubulin, but kills cells by a mechanism that shows the hallmarks of the recently described cell death mechanism “ferroptosis”,7 characterized by dependence on iron and ROS.


image file: c7ob03086j-s1.tif
Scheme 1 Retrosynthetic analysis of open-chain epothilones.

Results and discussion

Chemistry

Compounds 5a–f were derived by the esterification of the carboxylic acid 7 with 2-substituted thiazole cyclopentenols 6a–f as shown in the retrosynthetic analysis in Scheme 1.

We synthesized the carboxylic acid 7 as reported previously (Scheme 2),5 with the exception that the two diastereomers were separated efficiently by flash chromatography at the aldehyde stage. This was found to be easier and more efficient than separating the two carboxylic acids as reported earlier. The two aldehydes were then separately converted to the corresponding carboxylic acids 7 and 7a by Pinnick oxidation.8


image file: c7ob03086j-s2.tif
Scheme 2 Synthesis of carboxylic acids 7 and 7a.

The synthesis of cyclopentenols bearing aromatic moieties 6a–f began with dibromothiazole 12, which by nucleophilic substitution, metal-halogen exchange followed by alkylation or Sonogashira coupling, produced 2-substituted 4-bromothiazole derivatives 13–17 in good yields (Scheme 3).9–11 They were treated with n-butyllithium followed by trimethyltin chloride at −78 °C to produce the corresponding tin derivatives (Scheme 4a). t-Butyllithium proved more effective for the stannylation of the TMS-acetylene derivative 17. The tin derivatives were partially purified by passing through a plug of silica gel deactivated with 5% triethylamine in hexanes and were immediately subjected to Stille coupling with iodocyclopentenone 19 in the presence of palladium catalyst to obtain the aryl-substituted cyclopentenones 20a–e in good yields (Scheme 4).11 The TMS group of 20e was removed with K2CO3 in methanol to obtain 21 prior to reduction (Scheme 4b). Cyclopentenones 20a–d and 21 were selectively reduced using (R)-CBS catalyst to obtain cyclopentenols 6a–e.5 The stereochemistry of 6a–f was confirmed using the Mosher ester model.12,13 Enantioselective reduction of the piperonyl ketone 20c to alcohol 6c was not successful and the product was obtained as a racemic mixture, which was used in the next reaction without separation. Compound 21 was subjected to click chemistry with azidotrimethylsilane and copper iodide to obtain the triazole ketone 22 (Scheme 5).14


image file: c7ob03086j-s3.tif
Scheme 3 Synthesis of 2-substituted-bromothiazoles.

image file: c7ob03086j-s4.tif
Scheme 4 Synthesis of 2-substituted cyclopentenols 6a–e.

image file: c7ob03086j-s5.tif
Scheme 5 Synthesis of cyclopentenol 6f.

The triazole product 22 was insoluble in most organic solvents except for large quantities of methanol that precluded CBS reduction. It was methylated15 with MeI, and subjected to CBS reduction to obtain 6f.

We previously reported that desilylation of the TBS groups after esterification of 7 resulted in cleavage of the ester moiety.5 Therefore, as before the silyl groups were first removed with TFA and the desilylated carboxylic acid was esterified with cyclopentenols 6a–f using DCC and DMAP to yield open-chain epothilones 5a–f (Scheme 6). The purification of the final products was carried out on preparative TLC plates, which were first deactivated with 5% Et3N–hexanes to yield the final products 5a–f. Other purification methods including column chromatography on silica gel and reversed phase HPLC resulted in decomposition of the product and in most cases, the corresponding alcohol was isolated as a breakdown product.


image file: c7ob03086j-s6.tif
Scheme 6 Synthesis of target open-chain epothilone molecules.

Biological studies

The compounds were tested in the NCI 60 cell line one-dose screen at 10−5 M. The compounds with methyl (5a), thiomethoxy (5b), piperidinyl (5c) and substituted triazole (5f)-thiazole rings showed no activity on any of the cell lines. Interestingly, the two alkyne analogues with acetylene and phenyl acetylene substituted thiazole rings showed activity against some cell lines. The phenylacetylene derivative (5d) inhibited the growth of non-small cell lung cancer HOP-62 (35%), non-small cell lung cancer NCI-H226 (45%), melanoma UACC-62 (40%) and renal cancer A498 (78%) cell lines. The acetylene derivative (5e) showed 75% growth inhibition against non-small cell lung cancer cell line HOP-62 and 25% growth inhibitory activity against CNS cancer U251 cell line. What was more striking was its lethal effect on non-small cell lung cancer NCI-H522 (−62%), melanoma LOX IMVI (−23%), ovarian cancer IGROV1 (−32%) and renal cancer UO-31 (−36%). Intrigued by the remarkable lethality of the analogue 5e to NCI-H522 lung cancer cell line, we repeated this analysis. NCI-H522 cells were highly sensitive to analogue 5e (LC50 of ∼0.6 μM) whereas HOP-62 cells were less sensitive (LC50 of ∼30 μM) (ESI Fig. S1). These results show that analogue 5e with a linear terminal alkyne substituent group at the 2-position of the thiazole ring has a profound and selective lethal effect on NCI-H522 cells.

During their synthesis, esters 5a–f were found to be rather unstable and underwent ready hydrolysis in the presence of nucleophilic solvents like water and methanol. We therefore speculated that the observed cytotoxicity of compound 5e may in fact, be due to compound 6e formed by hydrolysis of the ester 5e in the assay media, possibly aided by cellular esterases. As speculated, compound 6e showed similar potency as 5e in the cellular assay and also had a similar selectivity profile (see below). Therefore, our subsequent studies focused on 6e. Comparison of several cancer cell lines exposed to 6e for three days indicated an LC50 of 1.5 μM for NCI-H522, and an LC50 of 3.6 μM for the fibrosarcoma cell line HT1080 (Fig. 2). Colon cancer cell line HCT116, lung cancer HOP-62 and breast cancer cell line MCF7 were relatively unaffected by compound 6e (Fig. 2). The large difference in sensitivities of these various cell lines suggests that 6e is not a general toxin, but relies on metabolic and/or genetic factors in certain tumor cells to allow sensitivity.


image file: c7ob03086j-f2.tif
Fig. 2 Survey of cytotoxicity. Cells were exposed to the indicated compounds at 10 μM and analyzed by methylene blue staining 3 days later. LC50 values of various cell lines determined 3 days post treatment. Cells were treated one day after plating. Cell viability was determined using methylene blue staining.

We then synthesized the (R)-enantiomer 23 and the racemic alcohol 24 (Fig. 3) using (S)-CBS catalyst and direct NaBH4 reduction, respectively. We also synthesized the corresponding oxime 25 by reaction of the ketone 21 with hydroxylamine hydrochloride and NaOAc in methanol (Fig. 3). The three compounds killed NCI-H522 cells with equal efficacy compared to 6e and had LC50 values of 2.6 μM, 2.5 μM and 2.3 μM, respectively. The ketone 21 was less lethal with an LC50 value of 8.0 μM (ESI Fig. S1). These results show that cytotoxic effect is independent of the stereochemistry of the alcohol function and that a H-bond donor is probably preferred over a H-bond acceptor at this position.


image file: c7ob03086j-f3.tif
Fig. 3 Other analogues synthesized.

Rapid, non-apoptotic cell death in response to compounds 5e and 6e

Initial time-lapse microscopy indicated that 50% of cells were killed by 5e in ∼10 h and 100% by 18 hours (Fig. 4A). The rapid mode of killing of NCI-H522 cells by 5e suggests that the mechanism of cell death is unlikely to be cell cycle-dependent. Similar rapid death was observed with the smaller 6e compound. Immunofluorescence analysis showed that compound 6e had no overt effects on tubulin in interphase or mitosis, suggesting that the compound does not mimic epothilone in mechanism of action (Fig. 4B). Furthermore, NCI-H522 cells were able to progress through mitosis in the presence of 6e (ESI Fig. S2). The rapid mode of cell death suggested that 6e might directly disrupt mitochondrial function; however, membrane potentials were intact until late in the death process (ESI Fig. S3). Additional time-lapse microscopy indicated that cell death was not associated with membrane blebbing (Fig. 4C). Cells lost contact with the substratum shortly before losing membrane integrity and pyknotic nuclei were not evident (Fig. 4D). Inhibiting caspases with ZVAD blocked killing by taxol but not compound 6e (Fig. 4E). These observations suggest that 6e does not induce apoptosis.
image file: c7ob03086j-f4.tif
Fig. 4 Rapid cell death induced by compound 5e and 6e. (A) NCI-H522 cells were analyzed by time-lapse microscopy to determine time of death. Imaging was started at the time of drug addition. (B) 6e does not affect tubulin. NCI-H522 were exposed to 10 μM 6e for 6 hours and then analyzed by confocal immunofluorescence with antibodies against alpha-tubulin and borealin (to detect centromeres). DNA was stained with Hoechst 33342. For comparison, cells were exposed to either epothilone B or taxol for 6 hours before analysis. (C) Morphological features of 6e-induced death. NCI-H522 cells exposed to compound 6e at 10 μM were analyzed by time-lapse microscopy. Cell death is characterized by loss of contact with the substratum followed by loss of membrane integrity. (D) Example of cytoplasmic shrinkage during cell death in the presence of 10 μM 6e. Immunofluorescence was performed as described in “B”, except cells were stained with phalloidin to detect actin. A wide-field image is shown. (E) Lack of protective effect of ZVAD on 6e-induced death. Cells were co-treated with ZVAD and either 10 μM compound 6e, or 10 μM taxol. Survival was assessed using methylene blue staining of fixed adherent cells.

Ferroptotic cell death in cells exposed to compound 6e

Next, we investigated potential non-apoptotic mechanisms of death in response to compound 6e. Additional studies suggest that these compounds kill cells by inducing ferroptosis, a novel iron-dependent, non-apoptotic form of cell death.7 Ferroptosis is a recently described mechanism of death characterized by a dependence on iron and ROS. Similarly to erastin, a prototypical inducer of ferroptosis, iron chelators ciclopirox olamine and hydroxyurea abolished killing of NCI-H522 cells by 6e (Fig. 5A). Sensitivity was enhanced by adding ferric citrate, suggesting that iron is the relevant cation (Fig. 5B). Death of NCI-H522 cells induced by 6e was also blocked by the free radical scavengers trolox and butylated hydroxyanisole (Fig. 5A). Thus, compound 6e relies on ROS for cytotoxic activity. ROS are under multiple levels of control in cells.16 Elevation of ROS, to the extent that they overwhelm antioxidant capacity, can induce damage to proteins, lipids, and nucleic acids causing cell death. Cellular sources of ROS include incomplete reduction of O2 during electron transport to form superoxide and direct generation of superoxide by the membrane bound NADPH oxidases.16 Inhibiting NADPH oxidases using diphenyleneiodonium chloride blocked killing by 6e, suggesting that these enzymes are important providers of ROS needed for killing (Fig. 5A). As predicted, exposure of NCI-H522 for 6 hours to compound 6e increased the level of intracellular ROS (Fig. 5C and D). ROS elevation by 6e was dependent on iron and NADPH oxidase and was blocked by trolox (Fig. 5D).
image file: c7ob03086j-f5.tif
Fig. 5 Compound 6e induces ferroptosis. (A) Effect of ferroptosis modulators on 6e toxicity. NCI-H522 were exposed to 10 μM 6e with or without the compounds indicated. Viability was analyzed 24 hours later using methylene blue. CPO: ciclopirox olamine; HU: hydroxyurea; BHA: butylated hydroxyanisole; DIC: diphenyleneiodonium chloride. All modulators eliminated 6e-induced killing (only 6e alone [+] versus DMSO [−] was significantly different using a student's t-test). (B) Elevating iron enhances 6e toxicity. NCI-H522 cells were exposed to 6e with or without 25 μM ferric citrate. Viability was determined by methylene blue staining 2 days later. (C) Elevation of ROS in NCI-H522 cells exposed to 10 μM compound 6e for 4 hours. Representative microscopic fields of H2DCFDA-stained cells are shown. Dye was extracted and quantified in (D).

Conclusions

During the course of our studies to develop structurally simplified epothilone analogues, we discovered a new class of small molecules that kill selected cancer cell lines by a novel mechanism of action. The cytotoxic activity is cell cycle-independent and does not involve apoptosis. The compounds had no effect on tubulin or mitochondrial function. Preliminary studies show that they harness reactive oxygen species to kill cancer cells in an iron-dependent manner showing the hallmarks of ferroptosis, a recently reported mechanism of action. Studies are in progress to further confirm the ferroptotic cell death mechanism and to determine the specific molecular targets of these promising “drug-like” small molecule anticancer agents.

Experimental section

General information

All reactions were carried out under nitrogen using anhydrous solvents unless otherwise noted. Tetrahydrofuran (THF) was distilled from Na and benzophenone under nitrogen prior to use. NMR spectra were recorded on Varian VXRS 400 MHz, Varian INOVA 600 MHz and Bruker Avance 600 MHz spectrometers and calibrated using residual undeuterated solvent as internal reference. Optical rotations were recorded on Rudolph AUTOPOL III and Rudolph AUTOPOL IV 589/586 polarimeters. High-resolution mass spectra (HRMS) were recorded on a (LCT Electrospray mass spectrometer at the Central Instrument Facility, Mass Spectrometry Laboratory, the Wayne State University, Detroit, Michigan) and on a Micromass Q-Tof II Electrospray mass spectrometer at the Mass Spectrometry and Proteomics Facility, the Ohio State University, Columbus, Ohio. Ozone was generated on a Welsbach Model T-408 commercial ozone generator. A Biotage Initiator was used in microwave synthesis. Crude products were purified by flash column chromatography on silica gel (standard grade, 40–63 μm, 230 × 400 mesh) purchased from Sorbent Technologies and using RediSep prepacked cartridges from Teledyne ISCO Inc. on a Combiflash Companion. 1000 μ Uniplates purchased from Analtech Inc. were used in preparative thin layer chromatography using commercial solvents as specified. HPLC analyses were performed on a Waters 1525 Binary Pump system with Waters 2487 Dual Wavelength Absorbance Detector on a Supleco C18 reverse phase column (5 μm, 15 cm × 4.6 mm) and on a Symmetry C18 reverse phase column (5 μ, 15 cm x 4.6 mm) using a linear gradient of 60–100% acetonitrile in water over 10–20 min; flow rate of 1 mL min−1 and UV detection at 254 nm. Structural integrity and purity of the test compounds were determined by the composite of 1H and 13C NMR, HRMS and HPLC and were found to be >95% pure.

Synthesis of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononoic acid (7) and the (3S,6S,7R) diastereomer (7a)

(3S,6R,7S)-3,7-bis(tert-Butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11) and (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal (11a). Ozone was passed through a solution of diastereomers (7S)-3,7-dihydroxy-2,4,6,6-tetramethyldec-9-en-5-one (10)8 (1.00 g, 2.12 mmol, 1 equiv.) in DCM (12 mL) at −78 °C until the reaction mixture reached a dark blue color. Nitrogen was bubbled through the solution for 15 min and triphenylphosphine (612 mg, 2.33 mmol, 1.1 equiv.) was added. The reaction mixture was stirred at room temperature overnight and concentrated in vacuo. The mixture of aldehydes was separated by flash column chromatography on silica gel in 10% DCM–hexanes, monitored by NMR, to obtain the (3S,6R,7S) diastereomer 11 (150 mg), the mixture of the two aldehydes (416 mg), followed by (3S,6S,7R) diastereomer 11a (245 mg) as colorless oils with an overall yield of 69%.

Data for the (3S,6R,7S) diastereomer 11: 1H NMR (400 MHz, CDCl3): δ 9.76 (s, 1H), 4.57 (t, J = 5.2 Hz, 1H), 3.70 (d, J = 7.6 Hz, 1H), 3.07–3.00 (m, 1H), 2.51–2.37 (m, 3H), 1.43–1.33 (m, 1H), 1.17 (m, 3H), 1.03 (m, 3H), 0.66 (m, 9H), 0.01 (m, 6H); 13C NMR (CDCl3, 100 MHz): δ 218.18, 201.55, 77.85, 70.42, 53.92, 49.24, 45.44, 33.25, 26.43, 26.20, 26.12, 22.71, 21.31, 19.44, 18.76, 18.31, 16, 16.035, −3.24, −3.51, −3.10, −4.22.

Data for the (3S,6S,7R) diastereomer 11a: 1H NMR (400 MHz, CDCl3): δ 9.71 (s, 1H), 4.45 (t, J = 4.8 Hz, 1H), 3.70 (d, J = 6.8 Hz, 1H), 3.05–3.01 (m, 1H), 2.48–2.32 (m, 3H), 1.44–1.38 (m, 1H), 1.14 (s, 3H), 0.98 (d, J = 8.0 Hz, 3H), 0.81 (m, 9H), 0.01 (m, 6H); 13C NMR (CDCl3, 100 MHz): δ 218.98, 201.47, 77.87, 71.25, 53.80, 49.79, 46.09, 46.05, 39.92, 32.89, 29.92, 26.46, 26.17, 26.10, 26.06, 24.12, 21.51, 18.791, 18.74, 18.32, 16.09, 15.86, −3.15, −3.46, −3.86, −4.28.

(3S,6R,7S)-3,7-bis(tert-Butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7). To a solution of aldehyde 11 (710 mg, 1.50 mmol, 1 equiv.) and 2-methyl-2-butene (7.6 mL) in t-butanol (32 mL) was added a solution of NaClO2 (1.244 g, 13.76 mmol, 9.1 equiv.) and NaH2PO4 (1.248 g, 10.38 mmol, 6.9 equiv.) in water (9 mL) dropwise. The reaction mixture was stirred for 1 h at RT and the reaction was quenched with saturated aqueous ammonium chloride (40 mL) and water (40 mL). The reaction mixture was extracted with EtOAc (3 × 15 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was taken directly to the next step without further purification. 1H NMR (400 MHz, CDCl3): δ 4.52–4.49 (m, 1H), 3.73 (dd, J = 8.0 Hz, 2.0 Hz, 1H), 3.09–3.01 (m, 1H), 2.47 (dd, J = 16.4 Hz, 3.6 Hz, 1H), 2.33 (dd, J = 16.4 Hz, 6.8 Hz, 1H), 1.46–1.38 (m, 1H), 1.16 (m, 3H), 1.01 (m, 3H), 0.98–0.82 (m, 9H), 0.03 (m, 6H).
(3S,6R,7S)-3,7-Dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid. A solution of the carboxylic acid 7 (350 mg, 0.72 mmol) in DCM (15 mL) was cooled to 0 °C. TFA (3.0 mL, 20% in DCM) was added and the reaction mixture was stirred at 4 °C for 25 h. Water was added (5 mL) and the reaction was evaporated to dryness under reduced pressure. The residue was dried azeotropically with toluene to obtain the desilylated (3S,6R,7S) carboxylic acid (168 mg, 90%) as a colorless oil: 1H NMR (400 MHz, CDCl3): δ 4.23 (dd, J = 10.0 Hz, 2.4 Hz, 1H), 3.33 (dd, J = 8.8 Hz, 1.6 Hz, 1H), 3.26–3.21 (m, 1H), 2.51–2.37 (m, 3H), 1.69–1.63 (m, 1H), 1.15 (d, J = 1.6 Hz, 6H), 1.03 (d, J = 6.8 Hz, 3H), 0.98 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.47, 177.25, 76.79, 72.47, 52.33, 41.079, 36.71, 30.66, 21.61, 19.54, 19.30, 19.17, 10.61.
(3S,6S,7R)-3,7-bis(tert-Butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanoic acid (7a). To a solution (3S,6S,7R)-3,7-bis(tert-butyldimethylsilyloxy)-4,4,6,8-tetramethyl-5-oxononanal 11 a (282 mg, 0.569 mmol, 1 equiv.) and 2-methyl-2-butene (3.0 mL) in t-butanol (12 mL) was added a solution of NaClO2 (497 mg, 5.50 mmol, 9.1 equiv.) and NaH2PO4 (497 mg, 4.15 mmol, 6.9 equiv.) in water (4 mL) dropwise. The reaction was stirred at RT for 1 h and the reaction was quenched with saturated aqueous ammonium chloride (15 mL) and water (15 mL). The crude reaction mixture was extracted with EtOAc (3 × 10 mL). The combined organic layers were dried over anhydrous sodium sulfate, filtered, and concentrated in vacuo. The crude product was passed through a silica gel plug to obtain the (3S,6S,7R) carboxylic acid 7a (241 mg, 87%) as a colorless oil: 1H NMR (400 MHz, CDCl3): δ 4.39 (m, 1H), 3.72 (dd, J = 8.0 Hz, 1.2 Hz, 1H), 3.08 (m, 1H), 2.46 (dd, J = 16.4 Hz, 2.8 Hz, 1H), 2.29 (dd, J = 16.4 Hz, 6.8 Hz, 1H), 1.45–1.42 (m, 1H), 1.17 (m, 3H), 1.08 (m, 3H), 0.88–0.73 (m, 9H), 0.03 (m, 6H); 13C (CDCl3, 100 MHz): δ 218.51, 178.65, 77.87, 73.40, 53.81, 46.07, 40.43, 32.94, 26.48, 26.22, 23.73, 21.53, 19.04, 18.80, 18.41, 16.18, 15.90, −3.18, −3.46, −4.05.
(3S,6S,7R)-3,7-Dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid. A solution the carboxylic acid 7a (882 mg, 1.81 mmol) in DCM (58 mL) was cooled to 0 °C and TFA (11.6 mL, 20% in DCM) was added. The reaction mixture was stirred at 4 °C for 25 h. Water (15 mL) was added and the reaction mixture was evaporated to dryness under reduced pressure. The residue was dried by azeotropically with toluene to obtain the desilylated (3S,6S,7R) carboxylic acid (450 mg, 95%) as a colorless oil: 1H NMR (400 MHz, CDCl3): δ 4.26 (dd, J = 10.0 Hz, 2.4 Hz, 1H), 3.30–3.22 (m, 2H), 2.53–2.40 (m, 2H), 1.70–1.63 (m, 1H), 1.20 (s, 3H), 1.15 (s, 3H), 1.06 (d, J = 7.2 Hz, 3H), 1.00 (d, J = 6.4 Hz, 3H), 0.86 (d, J = 7.2 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.61, 176.67, 76.58, 72.44, 52.16, 41.33, 36.38, 30.82, 21.60, 19.66, 19.21, 19.05, 10.54.

Synthesis of 2-substituted thiazole cyclopentenols (6a–f)

2-Methyl-4-bromothiazole (13). Synthesized using conditions described by Hofle et al.10 (0.765, 52%) reddish oil: TLC Rf = 0.68 (20% EtOAc–hexanes); 1H NMR (400 MHz, CDCl3): δ 7.04 (s, 1H), 2.71 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 167.20, 123.94, 116.30, 19.41.
2-(Methylthio)-4-bromothiazole (14). Synthesized using conditions described by Nicolaou et al.11 (1.415 g, 94%) yellow solid: TLC Rf = 0.48 (2% EtOAc–hexanes); 1H NMR (600 MHz, CDCl3): δ 7.05 (s, 1H), 2.68 (s, 3H); 13C NMR (CDCl3, 100 MHz): δ 124.44, 115.68, 16.85.
2-(Piperidin-1-yl)-4-bromothiazole (15). Synthesized using conditions described by Nicolaou et al.11 (1.30, 86%) yellow solid: TLC Rf = 0.43 (5% EtOAc–hexanes); 1H NMR (400 MHz, CDCl3): δ 6.35 (s, 1H), 3.42 (d, J = 5.6 Hz, 4H), 1.63 (s, 6H); 13C NMR (CDCl3, 100 MHz): δ 171.03, 121.70, 102.97, 49.26, 25.14, 24.08.
2-(Phenylethynyl)-4-bromothiazole (16). A mixture of 2,4-dibromothiazole 12 (2.92 g, 12.0 mmol, 1 equiv.), Pd(Ph3)4 (0.685 g, 0.60 mmol, 5 mol%), and CuI (0.228 g, 0.12 mmol, 10 mol%) was placed in a three-necked round bottom flask under nitrogen. Anhydrous THF (30 mL) was added, followed by N,N-diisopropylamine (2.55 mL, 18.02 mmol, 1.5 equiv.). A solution of phenylacetylene (2.0 g, 17.98 mmol, 1.5 equiv.) in anhydrous THF (6 mL) was slowly added to the reaction mixture via a syringe pump over 7 h. The reaction mixture was stirred for an additional 9 h. Water (50 mL) was added and the crude reaction mixture was extracted with ethyl acetate (3 × 20 mL). The combined organic extract was washed with brine, dried over anhydrous sodium sulfate, and concentrated in vacuo. The crude product was purified by flash column chromatography on silica gel in 5% EtOAc in hexanes and recrystallized from EtOAc–hexanes to obtain product 16 (3.174 g, 86%) as brown crystals: TLC Rf = 0.59 (10% EtOAc–hexanes); mp 79–80 °C; IR vmax: 3300, 3000, 2900, 2350 cm−1; 1H NMR (600 MHz, acetone-d6): δ 7.79 (s, 1H), 7.66 (m, 2H), 7.51 (m, 3H); 13C NMR (CDCl3, 100 MHz): δ 149.93, 132.249, 130.12, 128.78, 126.11, 121.10, 118.82, 95.69, 81.60; HRMS (m/z): [M + Na]+ calcd for C11H6NSBr, 285.9302; found, 285.9314.
2-((Trimethylsilyl)ethynyl)-4-bromothiazole (17). A mixture of 2,4-dibromothiazole 12 (3.000 g, 12.34 mmol, 1 equiv.), triphenylphosphine (486 mg, 1.851 mmol, 5 mol%), CuI (120 mg, 0.617 mmol, 5 mol%), and Pd(Ph3)2Cl2 (120 mg, 0.173 mmol, 1.4 mol%) was placed in a three-neck round bottom flask under nitrogen. Anhydrous toluene (42 mL) was added followed by anhydrous Et3N (2.2 mL, 15.04 mmol, 1.3 equiv.) and trimethylsilylacetylene (2.6 mL, 18.51 mmol, 1.5 equiv.). The reaction mixture was refluxed at 140 °C for 2 d. It was poured into water (50 mL) and extracted with EtOAc (3 × 20 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel in DCM–hexanes to obtain 17 (2.63 g, 81%) as a brownish/red solid: TLC Rf = 0.72 (10% EtOAc–hexanes); mp 37 °C; IR vmax: 3300, 2900, 2250 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.18 (s, 1H), 0.24 (s, 9H); 13C NMR (CDCl3, 100 MHz): δ 149.54, 125.87, 118.85, 103.03, 95.52, −0.41; HRMS (m/z): [M + H]+ calcd for C8H11NSSiBr, 259.9565; found, 259.9565.
2-Methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enone (20a). A solution of 2-methyl-4-bromothiazole 15 (351 mg, 1.97 mmol, 1 equiv.) in anhydrous ether (3 mL) was cooled to −78 °C and n-BuLi (1.30 mL of 2.5 M solution in THF, 3.35 mmol, 1.7 equiv.) was added dropwise. The reaction mixture was stirred at −78 °C for 2 h and a solution of trimethytin chloride (785 mg, 3.94 mmol, 2 equiv.) in anhydrous ether (2 mL) was added dropwise. The reaction mixture was stirred for an additional 1 h and allowed to warm to room temperature slowly. It was diluted with hexanes, passed through a silica gel plug deactivated with 5% Et3N–hexanes, and eluted with EtOAc. The partially purified product was concentrated in vacuo to obtain a reddish oil which was immediately taken to the next step. 1H NMR (600 MHz, CDCl3): δ 7.19 (s, 1H), 2.75 (s, 3H), 0.33 (t, J = 3.6 Hz, 9H). A mixture of 3-iodo-2-methylcyclopent-2-enone 10 (350 mg, 1.57 mmol, 0.8 equiv.) and Pd(Ph3)4 (455 mg, 0.394 mmol, 20 mol%) was placed in a μw vial under nitrogen. A solution of the tin derivative prepared above in anhydrous DMF (4.5 mL) was added via a syringe, and the vial was heated for 2 h 15 min at 145 °C in the microwave synthesizer. The reaction mixture was poured into water (15 mL) and extracted with EtOAc (3 × 5 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel in EtOAc–hexanes and recrystallized from EtOAc–hexanes to obtain 20a (336 mg, 88%) as tan crystals: TLC Rf = 0.39 (40% EtOAc–hexanes); mp 89–90 °C; 1H NMR (400 MHz, CDCl3): δ 7.45 (s, 1H), 2.95–2.92 (m, 2H), 2.73 (s, 3H), 2.49–2.47 (m, 2H), 2.09 (t, J = 2.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 210.24, 165.80, 158.67, 152.28, 136.60, 120.32, 33.89, 28.24, 19.56, 10.33; HRMS (m/z): [M + Na]+ calcd for C10H11NOS, 216.0459; found, 216.0461.
2-Methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enone (20b). A solution of 2-thiomethyl-4-bromothiazole 14 (300 mg, 1.43 mmol, 1 equiv.) in anhydrous ether (10 mL) was cooled to −78 °C. n-BuLi (1.80 mL, 1.6 M in hexanes, 2.86 mmol, 2 equiv.) was added dropwise and the reaction mixture was stirred at −78 °C for 2 h. A solution of trimethyltin chloride (708 mg, 3.57 mmol, 2.5 equiv.) in anhydrous ether (3 mL) was added dropwise. The reaction mixture was stirred at −78 °C for an additional 1 h and was allowed to warm slowly to room temperature. The reaction mixture was diluted with hexanes, passed through a silica gel plug deactivated with 5% Et3N–hexanes, and eluted with EtOAc. The partially purified product was concentrated in vacuo to obtain a reddish oil which was taken immediately to the next step. 1H NMR (400 MHz, CDCl3): δ 7.22 (s, 1H), 2.69 (s, 3H), 0.32 (t, J = 28.0 Hz, 9H). 3-Iodo-2-methylcyclopent-2-enone 10 (318 mg, 1.43 mmol, 1 equiv.) and Pd(Ph3)4 (330 mg, 0.286 mmol, 20 mol%) were placed in a μw vial under nitrogen. A solution of the tin derivative prepared above in anhydrous DMF (15 mL) was added via syringe, and the vial was heated for 2 h in the microwave synthesizer at 145 °C. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The product was purified by flash chromatography on silica gel in EtOAc–hexanes, and recrystallized from EtOAc–hexanes to obtain the product 20b (148 mg, 46%) as a pale yellow solid: TLC Rf = 0.60 (40% EtOAc–hexanes); mp 102 °C; 1H NMR (600 MHz, CDCl3): δ 7.46 (s, 1H), 2.92–2.90 (m, 2H), 2.73 (s, 3H), 2.52–2.50 (m, 2H), 2.14 (t, J = 2.0 Hz, 3H); 13C NMR (100 MHz, CDCl3): δ 210.27, 166.73, 157.83, 152.64, 137.03, 119.64, 33.93, 27.92, 16.71, 10.40; HRMS (m/z): [M + Na]+ calcd for C10H11NOS, 248.0180; found, 248.0189.
2-Methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enone (20c). A solution of 2-piperdin-1-yl-4-bromothiazole 15 (550 mg, 2.22 mmol, 1 equiv.) in anhydrous ether (19 mL) under nitrogen was cooled to −78 °C. n-BuLi (1.60 mL, 1.6 M in hexanes, 2.66 mmol, 1.2 equiv.) was added dropwise and the reaction mixture was stirred at −78 °C for 2 h. A solution of trimethyltin chloride (590 mg, 3.33 mmol, 1.5 equiv.) in anhydrous ether (7 mL) was added dropwise. The reaction mixture was stirred at −78 °C for an additional 1 h and was allowed to warm slowly to room temperature. The reaction mixture was diluted with hexanes, passed through a silica gel plug deactivated with 5% Et3N–hexanes, and eluted with EtOAc. The partially purified product was concentrated in vacuo to obtain a yellowish oil which was taken immediately to the next step. 1H NMR (400 MHz, CDCl3): δ 6.56 (s, 1H), 3.47 (m, 4H), 1.65 (m, 6H), 0.27 (t, J = 20 Hz, 9H). 3-Iodo-2-methylcyclopent-2-enone 10 (338 mg, 1.52 mmol, 1 equiv.) and Pd(Ph3)4 (352 mg, 0.304 mmol, 20 mol%) were placed in a μw vial under nitrogen. A solution of the tin derivative prepared above in anhydrous DMF (10 mL) was added via syringe. The vial was heated for 2 h in the microwave synthesizer at 140 °C. The reaction mixture was poured into water (20 mL) and extracted with EtOAc (3 × 8 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel in EtOAc–hexanes and recrystallized from EtOAc–hexanes to obtain 20c (341 mg, 58%) as yellow crystals: TLC Rf = 0.57 (40% EtOAc–hexanes); mp 108 °C; 1H NMR (600 MHz, CDCl3): δ 6.88 (s, 1H), 3.49 (m, 4H), 2.85 (m, 2H), 2.46 (m, 2H), 2.13 (t, J = 2.0 Hz, 3H), 1.66 (m, 6H); 13C NMR (CDCl3, 100 MHz): δ 221.25, 210.71, 170.84, 159.11, 149.68, 136.08, 109.85, 49.76, 33.93, 27.60, 25.33, 24.37, 10.39. HRMS (m/z): [M + Na]+ calcd for C14H18N2O, 285.1038; found, 285.1046.
2-Methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enone (20d). A solution of 2-(phenylethynyl)-4-bromothiazole 16 (1.000 g, 3.78 mmol, 1 equiv.) in anhydrous ether (15 mL) was cooled to −78 °C and n-BuLi (2.3 mL, 2.5 M in hexanes, 5.67 mmol, 1.5 equiv.) was added dropwise. The reaction mixture was stirred at −78 °C for 2 h and a solution of trimethyltin chloride (1.503 g, 7.56 mmol, 2 equiv.) in anhydrous ether (5 mL) was added dropwise. The reaction mixture was stirred for an additional 1 h at −78 °C before it was allowed to warm to room temperature slowly. The reaction mixture was diluted with hexanes, passed through a silica gel plug deactivated with 5% Et3N–hexanes, and eluted with EtOAc. The partially purified product was concentrated in vacuo to obtain a reddish oil which was used immediately in the next step. 1H NMR (400 MHz, CDCl3): δ 7.59 (m, 2H), 7.38 (m, 3H), 0.37 (t, J = 27.6 Hz, 9H). 3-Iodo-2-methylcyclopent-2-enone 10 (678 mg, 3.02 mmol, 0.8 equiv.) and Pd(Ph3)4 (873 mg, 0.756 mmol, 20 mol%) were placed in a μw vial under nitrogen. A solution of the tin derivative prepared above in anhydrous DMF (15 mL) was added via syringe. The vial was heated at for 2 h 15 min in the microwave synthesizer at 145 °C. The reaction mixture was poured into water (50 mL) and extracted with EtOAc (3 × 20 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel in EtOAc–hexanes and recrystallized from EtOAc–hexanes to obtain the product 20d (816 mg, 77%) as a brown solid: TLC Rf = 0.45 (40% EtOAc–hexanes); mp 117 °C; IR vmax: 3320, 3200, 2900, 2250, 1800, 1750 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.64 (s, 1H), 7.61 (m, 2H), 7.38 (m, 3H), 3.03 (m, 2H), 2.54 (m, 2H), 2.13 (t, J = 2.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 210.09, 157.99, 153.08, 148.76, 137.61, 132.27, 130.03, 128.76, 121.87, 121.24, 95.08, 82.16, 33.96, 28.40, 10.44; HRMS (m/z): [M + Na]+ calcd for C17H13NOS, 302.0616; found, 302.0620.
2-Methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone (20e). A solution of 2-((trimethylsilyl)ethynyl)-4-bromothiazole 17 (100 mg, 0.384 mmol, 1 equiv.) in anhydrous ether (3 mL) under nitrogen was cooled to −78 °C and t-BuLi (240 μL, 1.6 M, 0.786 mmol, 1 equiv.) was added dropwise. The reaction was stirred at −78 °C for 1 h and a solution of trimethyltin chloride (150 mg, 0.768 mmol, 2 equiv.) in anhydrous ether (2 mL) was added via syringe. The reaction mixture was stirred at −78 °C for an additional 1 h and was allowed to warm to room temperature slowly. The reaction mixture was diluted with hexanes, passed through a silica gel plug deactivated with 5% Et3N–hexanes, and eluted with EtOAc. It was concentrated in vacuo to obtain a yellow oil, which was immediately taken to the next step. 1H NMR (600 MHz, CDCl3): δ 7.33 (s, 1H), 0.34 (t, J = 14.8 Hz, 9H), 0.25 (m, 3H). 3-Iodo-2-methylcyclopent-2-enone 10 (83 mg, 3.84 mmol, 1 equiv.) and Pd(Ph3)4 (88 mg, 0.078 mmol, 20 mol%) were placed in a μw vial under nitrogen. A solution of tin derivative prepared above in anhydrous DMF (2 mL) was added via syringe. The μw vial was heated for 2 h 15 min in the microwave synthesizer at 145 °C. The reaction mixture was poured into water (30 mL) and extracted with EtOAc (3 × 10 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by flash chromatography on silica gel in EtOAc–hexanes and recrystallized from DCM–hexanes to obtain 20e as tan star shaped crystals (50 mg, 47%): TLC Rf = 0.63 (40% EtOAc–hexanes); mp 108–110 °C; IR vmax: 3300, 2900, 2200, 1750 cm−1; 1H NMR (600 MHz, CDCl3): δ 7.58 (s, 1H), 2.99 (t, J = 2.0 Hz, 2H), 2.52 (m, 2H), 2.10 (d, J = 2.0 Hz, 3H), 0.28 (s, 9H); 13C NMR (CDCl3, 100 MHz): δ 209.71, 157.66, 152.65, 148.22, 137.37, 121.79, 102.05, 96.15, 33.74, 28.18, 10.23, −0.46; HRMS (m/z): [M + Na]+ calcd for C14H17NOSSi, 298.0698; found, 298.0680.
3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enone (21). A solution of 2-methyl-3-(2-((trimethylsilyl)ethynyl)thiazol-4-yl)cyclopent-2-enone 20e (50 mg, 0.182 mmol, 1 equiv.) and K2CO3 (3 mg, 0.022 mmol, 12 mol%) in methanol (1 mL) was stirred for 5 min. The crude product was poured into water (8 mL) and extracted with EtOAc (3 × 3 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The product was recrystallized from DCM–hexanes to obtain 21 as tan needles: TLC Rf = 0.33 (40% EtOAc–hexanes); mp 138–139 °C; IR: 3000, 2900, 2250, 1750 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.62 (s, 1H), 3.52 (s, 3H), 2.99–2.96 (m, 2H), 2.53–2.51 (m, 2H), 2.11 (t, J = 2.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 209.98, 157.56, 153.08, 147.50, 137.84, 121.90, 83.24, 76.31, 33.92, 28.31, 10.38; HRMS (m/z): [M + Na]+ calcd for C11H9NOS, 226.0303; found, 226.0298.
3-(2-(1H-1,2,3-Triazol-4-yl)-2-methylcyclopent-2-enone) (22). 3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enone 21 (60 mg, 0.295 mmol, 1 equiv.) and CuI (3 mg, 0.015 mmol, 5 mol%) were placed in a μw vial under nitrogen. Anhydrous DMF (3.6 mL) and anhydrous MeOH (440 μL, 9[thin space (1/6-em)]:[thin space (1/6-em)]1 ratio DMF/MeOH) were added, followed by azidotrimethylsilane (58 μL, 0.443 mmol, 1.5 equiv.). The vial was heated for 2 h in the microwave synthesizer at 140 °C. The reaction mixture was poured into water (10 mL) and extracted with EtOAc (3 × 4 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude compound was purified by flash chromatography on silica gel in EtOAc–hexanes and recrystallized from methanol to obtain product 22 (51 mg, 70%) as a white solid: TLC Rf = 0.34 (50% EtOAc–hexanes); mp 241–243 °C; 1H NMR (400 MHz, CDCl3): δ 8.25 (S, 1H), 7.67 (s, 1H), 3.04–2.98 (m, 2H), 2.57–2.55 (m, 2H), 2.20 (d, J = 4.0 Hz, 3H); 13C NMR (DMSO-d6, 100 MHz): δ 208.87, 158.46, 157.43, 152.32, 141.26, 135.32, 141.26, 135.47, 122.25, 33.25, 27.46, 9.99; HRMS (m/z): [M + Na]+ calcd for C11H10N4OS, 269.0473; found, 269.0489.
2-Methyl-3-(2-(1-methyl-1H-triazol -4-yl)thiazol-4-yl)cyclopent-2-enone (20f). 3-(2-(1H-1,2,3-Triazol-4-yl)-2-methylcyclopent-2-enone) 22 (25 mg, 0.102 mmol, 1 equiv.) and NaOMe (5 mg, 0.100 mmol, 0.99 equiv.) were placed in a μw vial under nitrogen and anhydrous MeOH (2 mL) was added. The reaction mixture was heated for 7 min in the microwave synthesizer at 65 °C and methyl iodide (7 μL, 0.112 mmol, 1.1 equiv.) was added. The reaction mixture was reheated in the microwave synthesizer for 30 min at 90 °C. The insoluble starting material was filtered off using DCM. Purification by preparative thin-layer chromatography on silica gel in 60% EtOAc–hexanes gave the product 20f (9.6 mg, 36%) as a white solid: TLC Rf = 0.44 (50% EtOAc–hexanes); mp 154–156 °C; 1H NMR (400 MHz, CDCl3): δ 8.08 (s, 1H), 7.62 (s, 1H), 4.25 (s, 3H), 3.02–2.99 (m, 2H), 2.55–2.53 (m, 2H), 2.17 (t, J = 2.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 210.21, 158.19, 158.03, 153.40, 143.07, 137.36, 132.36, 120.48, 42.30, 33.93, 28.18, 10.41; HRMS (m/z): [M + Na]+ calcd for C12H12N4OS, found 283.0630; found, 283.0620.
(S)-2-Methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enol (6a). A solution of 2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enone 20a (100 mg, 0.517 mmol, 1 equiv.) and (R)-2-methyl-CBS-oxazaborolidine (29 mg, 0.103 mmol, 20 mol%) in anhydrous THF (2 mL) was cooled to 0 °C. A solution of borane-Me2S (260 μL of 2.0 M solution in THF, 0.517 mmol, 1 equiv.) was added dropwise. The reaction mixture was stirred at 0 °C for 1 h and the reaction was quenched by slow addition of water (5 mL) at 0 °C. The reaction mixture was extracted with EtOAc (3 × 2 mL), and the combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography on silica gel in 50% EtOAc–hexanes to obtain the product 6a (25 mg, 24%) as white crystals: TLC Rf = 0.34 (40% EtOAc–hexanes); mp 70–71 °C; [α]25D = −18.0° (c = 0.75, CHCl3); 1H NMR (400 MHz, CDCl3): δ 6.94 (s, 1H), 4.72 (d, J = 5.6 Hz, 1H), 2.86–2.77 (m, 1H), 2.70 (s, 3H), 2.65–2.57 (m, 1H), 2.41–2.32 (m, 1H), 2.13 (d, J = 0.4 Hz, 3H), 1.76–1.66 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 164.98, 153.10, 138.67, 131.66, 115.26, 82.38, 32.91, 32.72, 19.55, 13.36; HRMS (m/z): [M + Na]+ calcd for C10H11NOS, 218.0616; found, 218.0620.
(S)-2-Methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enol (6b). A solution of 2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enone 20b (100 mg, 0.444 mmol, 1 equiv.) and (R)-2-methyl-CBS-oxazaborolidine (25 mg, 0.089 mmol, 20 mol%) in anhydrous THF (2 mL) was cooled 0 °C. A solution of borane-Me2S (220 μL of 2.0 M solution in THF, 0.444 mmol, 1 equiv.) was added dropwise. The reaction mixture was stirred at 0 °C for 1 h and the reaction was quenched by slow addition of water (5 mL) at 0 °C. The reaction mixture was extracted with EtOAc (3 × 2 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography on silica gel in 60% EtOAc–hexanes to obtain 6b (64 mg, 63%) as a white solid: TLC Rf = 0.48 (40% EtOAc–hexanes); mp 46–49 °C; [α]25D = −5.8° (c = 1.40, CHCl3); 1H NMR (400 MHz, CDCl3): δ 6.95 (s, 1H), 4.71 (m, 1H), 2.83–2.76 (m, 1H), 2.70 (s, 3H), 2.61–2.55 (m, 1H), 2.42–2.34 (m, 1H), 2.18 (s, 3H), 1.77–1.69 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 165.25, 153.35, 139.23, 130.69, 114.73, 82.15, 32.52, 32.45, 16.69, 13.41; HRMS (m/z): [M + H]+ calcd for C10H13NOS2, 228.0517; found, 228.0525.
2-Methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enol (6c). A solution of 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enone 20c (100 mg, 0.380 mmol, 1 equiv.) and (R)-2-methyl-CBS-oxazaborolidine (21 mg, 0.076 mmol, 20 mol%) in anhydrous THF (4 mL) was cooled to 0 °C. A solution of borane-Me2S (190 μL of 2.0 M solution in THF, 0.380 mmol, 1 equiv.) was slowly added. The reaction was stirred at 0 °C for 1 h before the reaction was quenched by slow addition of water (8 mL). The reaction mixture was extracted with EtOAc (3 × 3 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography on silica gel in 50% EtOAc–hexanes to obtain racemic 6c yellow crystals (88 mg, 87%): TLC Rf = 0.53 (40% EtOAc–hexanes); mp 93–95 °C; 1H NMR (400 MHz, CDCl3): δ 6.35 (s, 1H), 4.67 (t, J = 5.2 Hz, 1H), 3.44 (m, 4H), 2.76–2.72 (m, 1H), 2.52–2.49 (m, 1H), 2.36–2.30 (m, 1H), 2.20 (s, 3H), 1.71–1.62 (m, 6H); 13C NMR (CDCl3, 100 MHz): δ 170.88, 150, 137.98, 131.63, 104.57, 82.52, 49.65, 32.63, 32.13, 25.31, 24.38, 13.35; HRMS (m/z): [M + H]+ calcd for C14H20N2OS, 265.1375; found, 265.1375.
(S)-2-Methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enol (6d). A solution of 2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enone 20d (100 mg, 0.358 mmol, 1 equiv.) and (R)-2-methyl-CBS-oxazaborolidine (20 mg, 0.072 mmol, 20 mol%) in anhydrous THF (2 mL) was cooled to 0 °C. A solution of borane-Me2S (179 μL of 2.0 M solution in THF, 0.358 mmol, 1 equiv.) was added dropwise. The reaction mixture was stirred at 0 °C for 5 min before being quenched by slow addition of water (4 mL) at 0 °C. The crude reaction mixture was extracted with EtOAc (3 × 1 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography on silica gel in 20% EtOAc–DCM to obtain product 6d (56 mg, 56%) as a red oil: TLC Rf = 0.55 (20% EtOAc–DCM); [α]25D = +3.5° (c = 0.650, CHCl3); IR vmax: 3350, 2900, 2300 cm−1; 1H NMR (600 MHz, CDCl3): δ 7.59 (m, 2H), 7.38 (m, 3H), 7.15 (s, 1H), 4.74 (d, J = 3.6 Hz, 1H), 2.89 (m, 1H), 2.69 (m, 1H), 2.41 (m, 1H), 2.15 (s, 3H), 1.76 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 154.17, 147.79, 139.98, 132.19, 131.14, 129.70, 128.68, 121.658, 117.38, 93.95, 82.78, 82.31, 32.99, 32.69, 13.51; HRMS (m/z): [M + H]+ calcd for C17H15NOS, 282.0953; found, 282.0953.
(S)-3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (6e). A solution of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone 20e (95 mg, 0.340, 1 equiv.) and (R)-2-methyl-CBS-oxazaborolidine (19 mg, 0.072 mmol, 20 mol%) in anhydrous THF (2 mL) was cooled to 0 °C. A solution of borane-Me2S (170 μL of 2.0 M solution in THF, 0.340 mmol, 1 equiv.) was added dropwise. The reaction mixture was stirred at 0 °C for 25 min and the reaction was quenched by slow addition of water (5 mL) at 0 °C. The reaction mixture was extracted with EtOAc (3 × 2 mL). The combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography on silica gel in 25% EtOAc–DCM to obtain the partial purified product 6e (72 mg, 75.1%) as a reddish/brown solid: TLC Rf = 0.58 (40% EtOAc–DCM); mp 69–71 °C; [α]25D = −12.8° (c = 1.50, CHCl3); IR vmax: 3300, 2900, 2300 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.13 (s, 1H), 4.73 (d, J = 4.4 Hz, 1H), 3.44 (s, 1H), 2.88–2.81 (m, 1H), 2.67–2.60 (m, 1H), 2.43–2.34 (m, 1H), 2.14 (d, J = 8.8 Hz, 3H), 1.78–1.70 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 154.15, 146.52, 140.24, 130.83, 117.48, 82.30, 82.14, 32.91, 32.67, 13.49; HRMS (m/z): [M + Na]+ calcd for C11H11NOS, 228.0458; found, 228.0459.
(S)-2-Methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enol (6f). A solution of 2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enone 20f (20 mg, 0.071 mmol, 1 equiv.) and (R)-2-methyl-CBS-oxazaborolidine (5 mg, 0.015 mmol, 20 mol%) in anhydrous THF (1.5 mL) was cooled to 0 °C. A solution of borane-Me2S (40 μL of 2.0 M solution in THF, 0.077 mmol, 1 equiv.) was added dropwise. The reaction mixture was stirred at 0 °C for 40 min and quenched by the slow addition of water (5 mL). The reaction mixture was extracted with EtOAc (3 × 2 mL), and the combined organic extract was dried over anhydrous sodium sulfate and concentrated in vacuo. The crude product was purified by preparative thin-layer chromatography on silica gel in 5% MeOH–DCM to obtain 6f (10 mg, 54%) as a white powder: TLC Rf = 0.51 (60% EtOAc–hexanes); mp 140–141 °C; [α]25D = −5.9° (c = 0.540, CHCl3); 1H NMR (600 MHz, CDCl3): δ 8.05 (s, 1H), 7.12 (s, 1H), 4.75 (d, J = 5.4 Hz, 1H), 4.24 (s, 3H), 2.87–2.85 (m, 1H), 2.67–2.65 (m, 1H), 2.43–2.40 (m, 1H), 2.22 (s, 3H), 1.78–1.74 (m, 1H); 13C NMR (CDCl3, 100 MHz): δ 157.26, 154.27, 143.57, 139.66, 132.20, 131.09, 115.85, 82.37, 42.21, 32.82, 32.71, 13.46; HRMS (m/z): [M + Na]+ calcd for C12H14N4OS, 285.0786; found, 285.0776.

Synthesis of open-chain epothilones (5a–f)

(3S,6R,7S)-((S)-2-Methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5a). A mixture of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid (20 mg, 0.077 mmol, 1equiv), (S)-2-methyl-3-(2-methylthiazol-4-yl)cyclopent-2-enol 6a (15 mg, 0.077 mmol, 1 equiv.), DCC (20 mg, 0.100 mmol, 1.3 equiv.), DMAP (1 mg, 0.008 mmol, 10 mol%), and CSA (4 mg, 0.015 mmol, 20 mol%) in anhydrous DCM (300 μL) was stirred under nitrogen overnight. The reaction mixture was filtered through a cotton plug and purified by preparative thin-layer chromatography on silica gel plates (deactivated with 5% Et3N–hexanes) in 25% EtOAc–hexanes to obtain 5a (5 mg, 23%) as a clear oil: TLC Rf = 0.44 (40% EtOAc–hexanes); [α]25D = +5.3° (c = 0.300, CHCl3); 1H NMR (400 MHz, CDCl3): δ 6.99 (s, 1H), 5.79 (s, 1H), 4.25–4.22 (m, 1H), 3.36–3.23 (m, 4H), 2.89–2.84 (m, 1H), 2.70 (s, 3H), 2.66 (m, 1H), 2.51–2.34 (m, 3H), 2.08 (s, 3H), 1.86–1.78 (m, 1H), 1.71–1.60 (m, 1H), 1.16 (s, 6H), 1.05 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.37, 173.29, 165.15, 152.50, 134.67, 134.12, 116.09, 85.53, 76.64, 72.78, 72.71, 52.40, 41.04, 37.10, 33.55, 30.79, 29.53, 21.69, 19.75, 19.61, 19.33, 19.28, 19.23, 13.69, 10.64; HRMS (m/z): [M + Na]+ calcd for C23H35NO5S, 460.2134; found 460.2144.
(3S,6R,7S)-((S)-2-Methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5b). A mixture of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid (66 mg, 0.253 mmol, 1 equiv.), (S)-2-methyl-3-(2-(methylthio)thiazol-4-yl)cyclopent-2-enol 6b (78 mg, 0.343 mmol, 1.4 equiv.), DCC (75 mg, 0.328 mmol, 1.3 equiv.), DMAP (32 mg, 0.253 mmol, 1 equiv.), and anhydrous Et3N (64 μL, 0.506 mmol, 2 equiv.) in anhydrous DCM (300 μL) was stirred under nitrogen for overnight. The reaction mixture was filtered through a cotton plug and purified by successive preparative thin-layer chromatography on silica gel (deactivated with 5% Et3N–hexanes) in 40% EtOAc–hexanes, 90% EtOAc–hexanes, 85% EtOAc–hexanes to obtain 5b (12 mg, 10%) as a clear oil: TLC Rf = 0.56 (40% EtOAc–hexanes); [α]25D = −4.6° (c = 0.600, CHCl3); 1H NMR (400 MHz, CDCl3): δ 6.99 (s, 1H), 5.80 (t, J = 3.2 Hz, 1H), 4.26–4.22 (m, 1H), 3.35–3.22 (m, 4H), 2.87–2.80 (m, 1H), 2.69 (s, 3H), 2.67–2.62 (m, 1H), 2.51–2.35 (m, 3H), 2.12 (s, 3H), 1.86–1.78 (m, 1H), 1.69–1.64 (m, 1H), 1.16 (s, 6H), 1.04 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.38, 173.24, 165.59, 152.76, 134.73, 133.90, 85.46, 76.62, 72.75, 72.62, 52.36, 41.02, 37.08, 37.05, 33.14, 30.74, 29.47, 21.67, 21.65, 19.68, 19.29, 19.24, 19.19, 16.73, 13.72, 13.70, 10.60; HRMS (m/z): [M + Na]+ calcd for C23H35NO5S2, 492.1854; found, 492.1858.
(3S,6R,7S)-((R)-2-Methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate compound with (3S,6R,7S)-((S)-2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5c). A mixture of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid (44 mg, 0.170 mmol, 1 equiv.), 2-methyl-3-(2-(piperidin-1-yl)thiazol-4-yl)cyclopent-2-enol 6c (45 mg, 0.170 mmol, 1 equiv.), DCC (46 mg, 0.221 mmol, 1.3 equiv.), DMAP (21 mg, 0.170, 1 equiv.), and anhydrous Et3N (47 μL, 0.340 mmol, 2 equiv.) in anhydrous DCM (400 μL) was stirred under nitrogen overnight. The reaction mixture was filtered through a cotton plug and purified by successive preparative thin-layer chromatography on silica gel (deactivated with 5% Et3N–hexanes) in 30% EtOAc–hexanes, 50% EtOAc–hexanes to obtain a diasteromeric mixture 5c (3 mg, 4%) as a yellow oil: TLC (20% EtOAc–hexanes); [α]25D = −1.2° (c = 0.650, CHCl3); 1H NMR (400 MHz, CDCl3): δ 6.39 (s, 1H), 5.77 (s, 1H), 4.21 (d, J = 10.2 Hz, 1H), 3.44 (m, 4H), 3.34–3.28 (m, 4H), 2.78 (m, 1H), 2.59 (m,1H), 2.50–2.36 (m, 3H), 2.13 (s, 3H), 1.82–1.79 (m, 1H), 1.63 (m, 6H), 1.15 (s, 6H), 1.04 (d, J = 8.0 Hz, 3H), 1.00 (d, J = 8.0 Hz, 3H), 0.85 (d, J = 8.0 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.31, 173.35, 173.32, 170.90, 149.47, 134.77, 134.72, 133.34, 133.28, 105.46, 105.42, 85.82, 76.60, 72.73, 72.66, 52.38, 52.34, 49.71, 40.10, 37.10, 37.02, 32.82, 30.74, 29.44, 25.388, 25.35, 24.42, 21.68, 21.64, 19.72, 19.22, 19.18, 13.66, 13.64, 10.61; HRMS (m/z): [M + Na]+ calcd for C27H42N2O5S, 529.2712; found, 529.2729.
(3S,6R,7S)-((S)-2-Methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5d). A mixture of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid (52 mg, 0.199 mmol, 1equiv), (S)-2-methyl-3-(2-(phenylethynyl)thiazol-4-yl)cyclopent-2-enol 6d (55 mg, 0.199 mmol, 1 equiv.), DCC (53 mg, 0.259 mmol, 1.3 equiv.), DMAP (25 mg, 0.199 mmol, 1 equiv.), and anhydrous Et3N (55 μL, 0.398 mmol, 2 equiv.) in anhydrous DCM (400 μL) was stirred under nitrogen overnight. The reaction mixture was filtered through a cotton plug and purified by successive preparative thin-layer chromatography on silica gel (deactivated with 5% Et3N–hexanes) in 40% EtOAc–hexanes, 5% EtOAc–hexanes, 25% EtOAc–hexanes to obtain 5d (10 mg, 10%) as a white semisolid: TLC Rf = 0.45 (40% EtOAc–hexanes); [α]25D = −13.5° (c = 0.275, CHCl3); IR vmax: 3500, 3200, 2900, 2100 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.57 (dd, J = 7.6 Hz, J = 5.6 Hz, 2H), 7.39–7.33 (m, 3H), 7.19 (s, 3H), 5.82 (s, 1H), 4.27–4.22 (m, 2H), 3.35–3.22 (m, 4H), 2.93–2.89 (m, 1H), 2.79–2.71 (m, 1H), 2.52–2.36 (m, 3H), 2.10 (s, 3H), 1.89–1.82 (m, 1H), 1.70–1.64 (m, 1H), 1.16 (s, 6H), 1.05 (d, J = 6.8 Hz, 3H), 1.01 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.34, 173.20, 153.50, 147.97, 135.49, 134.06, 132.20, 129.76, 128.70, 121.59, 118.06, 94.12, 85.30, 82.68, 76.62, 72.76, 72.69, 52.37, 41.03, 37.07, 33.61, 30.76, 29.49, 21.68, 19.70, 19.32, 19.27, 19.21, 13.79, 10.62; HRMS (m/z): [M + Na]+ calcd for C30H37NO5S, 546.2290; found 546.2285.
(3S,6R,7S)-((S)-3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5e). A mixture of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid (38 mg, 0.146 mmol, 1 equiv.), (S)-3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enol 6e (30 mg, 0.146 mmol, 1 equiv.), DCC (39 mg, 0.190 mmol, 1.3 equiv.), DMAP (4 mg, 0.029 mmol, 20 mol%), and CSA (2 mg, 0.007 mmol, 5 mol%) in anhydrous DCM (300 μL) was stirred under nitrogen overnight. The reaction mixture was filtered through a cotton plug and purified by successive preparative thin-layer chromatography on silica gel (deactivated with 5% Et3N–hexanes) in 10% AcCN–DCM, 50% DCM–hexanes, 10% acetone–hexanes to obtain 5e (10 mg, 15%) as a colorless oil: TLC Rf = 0.69 (50% acetone–hexanes); [α]25D = −6.3° (c = 0.600, CHCl3); IR vmax: 3300, 3050, 2900, 2100, 1750 cm−1; 1H NMR (400 MHz, CDCl3): δ 7.17 (s, 1H), 5.81 (s, 1H), 4.25–4.22 (m, 1H), 3.45 (s, 1H), 3.35–3.20 (m, 4H), 2.92–2.89 (m, 1H), 2.78–2.70 (m, 1H), 2.51–2.35 (m, 3H), 2.15 (s, 3H), 1.85–1.82 (m, 1H), 1.70–1.57 (m, 1H), 1.16 (s, 6H), 1.05 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.86 (d, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz) δ 222.37, 173.18, 153.48, 146.71, 135.78, 133.71, 118.20, 85.24, 82.31, 76.80, 76.62, 72.75, 72.68, 52.36, 41.03, 37.06, 33.51, 30.75, 29.46, 21.67, 19.70, 19.27, 19.20, 13.75, 10.61; HRMS (m/z): [M + Na]+ calcd for C24H33NO5S, 470.1977; found 470.1957.
(3S,6R,7S)-((S)-2-Methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enyl) 3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoate (5f). A mixture of (3S,6R,7S)-3,7-dihydroxy-4,4,6,8-tetramethyl-5-oxononanoic acid (55 mg, 0.201 mmol, 1 equiv.), (S)-2-methyl-3-(2-(1-methyl-1H-1,2,3-triazol-4-yl)thiazol-4-yl)cyclopent-2-enol 6f (52 mg, 0.201 mmol, 1 equiv.), DCC (54 mg, 0.261 mmol, 1.3 equiv.), DMAP (5 mg, 0.040 mmol, 20 mol%), and CSA (2 mg, 0.010 mmol, 5 mol%) in anhydrous DCM (400 μL) was stirred under nitrogen overnight. The reaction mixture was filtered through a cotton plug and purified by successive preparative thin-layer chromatography on silica gel (deactivated with 5% Et3N–hexanes) in 40% EtOAc–hexanes, 35% acetone–hexanes to obtain 5f (21 mg, 20%) as a colorless oil: TLC Rf = 0.44 (40% EtOAc–Hex); [α]25D = −4.5° (c = 1.17, CHCl3); 1H NMR (400 MHz, CDCl3): δ 8.04 (s, 1H), 7.16 (s, 1H), 5.82 (d, J = 2.4 Hz, 1H), 4.26 (d, J = 2.4 Hz, 1H), 4.18 (s, 3H), 3.35–3.23 (m, 4H), 2.94–2.88 (m, 1H), 2.75–2.71 (m, 1H), 2.52–2.34 (m, 3H), 2.15 (s, 3H), 1.89–1.81 (m, 1H), 1.71–1.60 (m, 1H), 1.16 (s, 1H), 1.04 (d, J = 6.8 Hz, 3H), 1.00 (d, J = 6.8 Hz, 3H), 0.85 (d, J = 6.8 Hz, 3H); 13C NMR (CDCl3, 100 MHz): δ 222.35, 173.22, 157.40, 153.59, 143.46, 135.08, 134.04, 132.19, 116.60, 85.40, 76.60, 72.74, 72.66, 52.36, 42.23, 41.01, 37.07, 33.43, 30.74, 29.50, 21.66, 19.69, 19.30, 19.25, 19.17, 13.74, 13.71, 10.61; HRMS (m/z): [M + Na]+ calcd for C25H36N4O5S, 527.2304; found, 527.2308.

Synthesis of alcohols (23) and (24) and oxime (25)

(R)-3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enol (23). A mixture of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone 21 (0.0258 g, 0.127 mmol, 1 equiv.), (S)-(−)-2-methyl-CBS-oxazaborolidine (0.008 g, 0.029 mmol, 22.7 mol%), and anhydrous THF (0.6 mL) was cooled to 0 °C. A solution of BH3-Me2S (0.063 mL of 2 M solution in THF, 0.126 mmol, 0.99 equiv.) was added dropwise via syringe and the reaction mixture was stirred at 0 °C for 25 min. The reaction was quenched by slow addition of water (∼2 mL) at 0 °C. The reaction mixture was extracted with EtOAc three times. The combined EtOAc extract was washed with brine and dried over anhydrous sodium sulfate and concentrated on a rotary evaporator to obtain a yellow oil. The crude product was purified by flash chromatography on silica gel in 15% acetone/hexanes. The product was further purified by preparative TLC in 25% acetone/hexanes to obtain analog 23 as a yellow oil (0.0237 g, 91%); 1H NMR (400 MHz, CDCl3) δ 7.18 (s, 1H), 4.80 (brs, 1H), 3.50 (s, 1H), 2.93 (m, 1H), 2.72 (m, 1H), 2.47 (m, 1H), 2.18 (s, 3H), 1.81 (s, 1H). 13C NMR (150 MHz, CDCl3) δ 13.45, 32.67, 32.92, 76.95, 82.10, 82.29, 117.44, 130.85, 140.29, 146.55, 154.22. HRMS (m/z): [M + Na]+ calcd for C11H11NOS, 228.0459; found 228.0452
2-Methyl-3-(2-((trimethylsilyl)ethynyl)thiazole-4-yl)cyclopent-2-enol (24). A mixture of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone 21 (0.0259 g, 0.127 mmol, 1 equiv.) and methanol (∼0.5 mL) was cooled to 0 °C with stirring. Sodium borohydride (0.0064 g, 0.169 mmol, 1.328 equiv.) was added and the reaction was monitored by TLC. An additional portion of sodium borohydride (0.0056 g, 0.148 mmol, 1.162 equiv.) was added to the reaction mixture and stirring was continued for another 40 min at 0 °C. Excess methanol was removed on a rotary evaporator. Water (∼2 mL) was added to the residue and extracted with EtOAc three times. The combined EtOAc extract was washed with brine, dried over anhydrous sodium sulfate and concentrated on a rotary evaporator. The crude product was purified by flash chromatography on silica gel in 25% EtOAc/hexanes to obtain analog 24 as a yellow solid (0.0161 g, 62%); mp 84–85 °C; 1H NMR (400 MHz, CDCl3) δ 7.17 (s, 1H), 4.80 (brs, 1H), 3.50 (s, 1H), 2.91 (m, 1H), 2.71 (m, 1H), 2.45 (m, 1H), 2.17 (s, 3H), 1.79 (m, 1H). 13C NMR (150 MHz, CDCl3) δ 13.45, 32.67, 32.92, 76.94, 82.12, 82.28, 117.44, 130.84, 140.30, 146.55, 154.22. HRMS (m/z): [M + Na]+ calcd for C11H11NOS, 228.0459; found 228.0452
3-(2-Ethynylthiazol-4-yl)-2-methylcyclopent-2-enone oxime (25). A mixture of 3-(2-ethynylthiazol-4-yl)-2-methylcyclopent-2-enone (4a) (0.0255 g, 0.126 mmol, 1 equiv.), sodium acetate (0.0248 g, 0.303 mmol, 2.409 equiv.), hydroxylamine hydrochloride (0.0102 g, 0.147 mmol, 1.17 equiv.), methanol (1 mL), and water (0.25 mL) was heated at 70 °C with stirring for 28 h. Methanol was removed on a rotary evaporator. Water (2 mL) was added to the residue and extracted with DCM three times. The combined DCM extract was washed with brine and dried over anhydrous sodium sulfate and concentrated on a rotary evaporator. The crude product was purified by flash chromatography on silica gel in 1% methanol/DCM to obtain analog 32 as a yellow solid (0.0179 g, 65%); mp 189–191 °C (decomp.); 1H NMR (600 MHz, acetone-d6) δ 9.86 (s, 1H), 7.74 (s, 1H), 4.35 (s, 1H), 2.88 (m, 2H), 2.73 (m, 2H), 2.20 (s, 3H). 13C NMR (150 MHz, acetone-d6) δ 12.05, 24.90, 32.19, 77.81, 84.83, 120.66, 135.94, 141.87, 147.92, 155.09, 168.54. HRMS (m/z): [M + Na]+ calcd for C11H10N2OS, 219.0592; found 219.0594

Biological studies

Materials and methods.
Cell lines and culture conditions. Cell lines were cultured in a humidified 37 °C atmosphere containing 10% CO2 in Dulbecco's Modified Eagle's Medium (Mediatech, Inc.) supplemented with 10% fetal bovine serum (Atlanta Biologicals) and 1000 U ml−1 of both Penicillin and Streptomycin (Mediatech). To determine viability 50[thin space (1/6-em)]000 cells were plated in each well of a 24-well plate. Compounds were added 1 day post plating and cells were stained after 3 days with a saturated solution of methylene blue in 50% ethanol. Plates were rinsed and retained dye quantified by absorbance at 630 nm. Results are representative of at least two independent experiments. Some experiments were repeated using MTS dye (CellTitre96, Promega) with similar results.
Microscopy. For immunofluorescence microscopy, cells were fixed with 2% formaldehyde in phosphate buffered saline (PBS) for 15 minutes, followed by permeabilization [150 mM NaCl, 10 mM Tris (pH 7.7), 0.1% Triton X-100, and 0.1% BSA] for 9 minutes. Fixed cells were blocked with PBS containing 0.1% BSA for 1 h at room temperature. Cells were then stained with antibodies to alpha-tubulin and borealin.17 Antibodies were visualized by incubating samples with Alexafluor-conjugated secondary antibodies (Invitrogen). DNA was visualized by staining with Hoechst 33342. Images were captured using a Leica SP8 confocal microscope. Time-lapse microscopy was carried out using an Olympus IX81 inverted microscope with an environmental chamber heated to 37 °C. Images were captured at defined intervals using an Photometrics Coolsnap HQ2 camera.

Conflicts of interest

LMVT, SRF and WRT are coinventors of a patent application that covers these anticancer agents.

Acknowledgements

We thank the Elsa U. Pardee Foundation and the deArce-Koch Memorial Endowment Fund for initial seed funding for the project and Michael Bekier for confocal imaging. This project was partially supported by grant NIH R15 GM100440-01 to WRT.

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Footnotes

Electronic supplementary information (ESI) available: 1H and 13C NMR spectra of all intermediates and final products, and materials and methods of biological studies. See DOI: 10.1039/c7ob03086j
These authors contributed equally to this work.

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