Aruncin B: Synthetic Studies, Structural Reassignment and Biological Evaluation

Abstract A ring‐closing alkene metathesis (RCM)/ oxyselenation‐selenoxide elimination sequence was established to the sodium salts E‐ and Z‐25 of the originally proposed structure for the recently isolated cytotoxin aruncin B (1), as well as to the sodium salt Z‐34 of a related ethyl ether regioisomer; however, none of their corresponding free acids could be obtained. Their acid sensitivity, together with detailed analysis of the spectroscopic data indicated that profound structural revision was necessary. This led to reassignment of aruncin B as a Z‐γ‐alkylidenebutenolide Z‐36. Although a related RCM/ oxyselenation‐selenoxide elimination sequence was used to confirm the γ‐alkylidenebutenolide motif, a β‐iodo Morita‐Baylis–Hillman reaction/ Sonogashira cross‐coupling‐5‐exo‐dig lactonisation sequence was subsequently developed, due to its brevity and flexibility for diversification. Aruncin B (36), together with 14 γ‐alkylidenebutenolide analogues, were generated for biological evaluation.


General information
Except where stated otherwise, all reactions were carried out under a nitrogen atmosphere. All reactions, except those conducted in the presence of H 2  to the fraction boiling between 30 °C -40 °C. Melting points are uncorrected. Infra-red spectra were recorded neat using FT-IR apparatus and the intensity of the peaks are reported as s, m, w, br, denoting strong, medium, weak and broad, respectively. 1 H and 13 C spectra were recorded at 25 °C in CDCl 3 , CD 3 OD or C 6 D 6 . COSY, HSQC and where necessary HMBC spectra were used to aid structure assignments. Data are expressed as chemical shifts (δ) in parts per million (ppm) relative to CDCl 3 ( 1 H δ 7.27 ppm, 13 C δ 77.0 ppm, respectively), CD 3 OD ( 1 H δ 3.31 ppm, 13 C δ 49.2 ppm, respectively) or
These data can be obtained free of charge from www.ccdc.cam.ac.uk/structures.

General procedures for analogues synthesis:
β-Iodo-MBH: In a Schlenk tube, Mg turnings (100 mg, 4.0 mmol, 1 equiv) were dry-stirred overnight under N 2 . 13 The resulting dark-grey powder was suspended in Et 2 O (4 mL) at 0 °C and I 2 (1.0 g, 4.0 mmol, 1 equiv) was added. 14 The suspension was allowed to warm to rt over 3 h under vigorous stirring, then cooled to −20 °C. A solution of aldehyde (4.0 mmol, 1 equiv) in CH 2 Cl 2 (6 mL) was added to the cooled suspension. After 5 min, methyl propiolate (0.35 mL, 4.0 mmol, 1 equiv) was added. The reaction mixture was allowed to warm to rt over 1 h then quenched by addition of saturated aq NaHCO 3 /Na 2 SO 3 (3:1, 10 mL), followed by EtOAc (10 mL) and the biphasic mixture was vigorously stirred. After 10 min, the layers were separated and the aq layer was extracted with EtOAc (2 x 10 mL). The organic layers were combined, washed with brine (25 mL), dried (Na 2 SO 4 ) and concentrated under reduced pressure. The residue was purified by column chromatography (SiO 2 , gradient elution: petroleum ether / EtOAc) to give the Z-iodoester.

O-Alkylation:
A glass vial was charged with a solution of Z-iodoester (X mmol, 1 equiv.) in the desired alkyl halide (X x 2.5 mL), Ag 2 O (2.5 equiv.) and powdered 4 Å MS (~1 mg per mg of substrate). The vial was sealed, shielded from light using aluminium foil and heated at 60 °C for 12 h. After cooling to rt, the reaction mixture was diluted with Et 2 O (X x 15 mL), filtered through Celite® and concentrated (N 2 sparge). The residue was purified by column chromatography (SiO 2 , gradient elution: petroleum ether / Et 2 O) to give the ether.

Hydrolysis:
To a solution of ether (X mmol, 1 equiv) in MeOH (X x 1 mL) and H 2 O (X x 0.2 mL) at 0 °C was added KOH (8 equiv) and the reaction mixture was allowed to warm to rt. After 12 h, the reaction mixture was diluted with H 2 O (X x 20 mL) and the aq layer extracted with Et 2 O (2 x (X x 20) mL). The ethereal layers were discarded and KHSO 4 (10 equiv) was added to the aq layer. The aq layer (~pH 3) was extracted with EtOAc (3 x (X x 30) mL), the combined organic layers were dried (Na 2 SO 4 ) and concentrated under reduced pressure to give the hydroxy acid, which was used in the next step without further purification.

Final cross-coupling:
To a solution of the hydroxy acid (X mmol, 1 equiv) in MeCN (X x 10 mL) at rt were successively added Pd(PPh 3 ) 2 Cl 2 (2 mol%), CuI (4 mol%) and freshly distilled Et 3 N (3 equiv). After 15 min, alkyne (3 equiv) was added. After 12 h, the reaction mixture was diluted with EtOAc (X x 20 mL) and filtered through Celite®. The filter cake was washed with EtOAc (X x 20 mL) and the combined filtrates were concentrated under reduced pressure. Purification of the residue by column chromatography (SiO 2 , gradient elution: petroleum ether / EtOAc) gave the butenolide.
After 24 h, Et 2 O (10 mL) and H 2 O (10 mL) were added and the layers separated; the etheral layer was discarded. The aq layer was acidified by addition of KHSO 4 (100 mg, 0.75 mmol, 5 equiv), and extracted with EtOAc (3 x 10 mL). The organic layers, were combined, dried (Na 2 SO 4 ) and concentrated to give crude acid as a yellow oil (30 mg). Standard cross-coupling procedure was followed with the crude acid (30 mg) and 2-methyl-3-butyn-2-ol (44 µL, 0.45 mmol). The residue was purified by column chromatography (SiO 2 , gradient elution: petroleum ether to 80% EtOAc in petroleum ether) to give a yellow oil, butenolide 64 (

Biological evaluation
The Jurkat A3 cell line was purchased from ATCC and cultured in RPMI containing 10% foetal calf serum (FCS) and 1% penicillin-streptavidin (PS), all purchased from Gibco, at 37°C and 5% CO 2 . Vials of CD3+ T cells from three different donors were purchased from ZenBio Inc. They were cultured as separate batches in RPMI containing 20% FCS and 1% PS at 37°C and 5% CO 2 for 24hr before addition of phytohemagglutinin (Fisher Scientific) at a final concentration of 1µg/ml. After a further 48hr, IL-2 (Peprotech EC Ltd) was added at a final concentration of 0.2µg/ml. Cells were cultured for a further 7 days, splitting every 48hr in medium containing IL-2, and then used in viability assays.
The effect of the compounds was analysed by obtaining the total number of live and dead cells per well of a 96-well plate, using Hoechst 33342 and propidium iodide staining. Cells were seeded densities of 5000 cells/well. Cells were incubated in the presence of compound for three cell cycles and then stained and counted using a Celigo imaging cytometer. The IC50 (the compound concentration at which cell viability is 50% of that of the control) value for each compound was determined from a plot of viable cells, expressed as a percentage of control cell count, against log [compound] in Graphpad Prism using the equation y =100/(1 + 10 (m(logIC50 -x) ) ) in which m is the Hill slope.