On the origin of the thiopyrone CTP-431 “unexpectedly” isolated from the marine sponge Cacospongia mycofijiensis

An intriguing hypothesis that latrunculin A, a well-known natural product, might have undergone transformation into the unprecedented thiopyrone CTP-431 upon long-term storage in methanol is advanced. Thus opening of the hemiacetal of latrunculin A, followed by E1CB elimination, and dehydration would give a polyene that could undergo intramolecular Diels-Alder reaction, followed by methanolysis of the thiazolidinone ring and ring closure by intramolecular thiol addition to an enone. Experimental evidence that the novel thiazolidinone to thiopyrone rearrangement can occur is presented.

2 mycothiazole, 4,5 and the macrolide latrunculins ( Figure 1). 6 The thiazolidinone-containing latruculins are of mixed polyketide synthesis (PKS) and non-ribosomal peptide synthesis (NRPS) origin, and latrunculin A 1 disrupts microfilament assembly to such an extent that it is the most widely used chemical tool to study actin binding. In the course of their seminal studies on the biology and chemistry of metabolites extracted from C. mycofijiensis, in 2008 the Crews laboratory reported the "unexpected" isolation of a new compound, CTP-431 7, containing an unprecedented thiopyrone ring fused to a hydrindane, the structure being confirmed by X-ray crystallographic analysis. 1  product of methanolysis of the thiazolidinone ring would be a methyl carbamate resembling the one found in CTP-431, providing support for our hypothesis.
We therefore propose that a possible mechanism (Scheme 1) for this interconversion would proceed from the hemiacetal of latrunculin A 1 to its open-chain isomer 2, followed by E1CB elimination of the lactone and subsequent dehydration of alcohol 3 to give polyene 4; the structures of congeners such as latrunculeic acid ( Figure 1) provide some evidence that this carboxylate elimination can occur readily. 7 Intramolecular Diels-Alder (IMDA) reaction of 4, followed by isomerization of the double bond to restore conjugation, would give the hydrindane 5. Methanolysis of the thiazolidinone would lead to rapid 6-endo-trig conjugate addition of the thiol to give thiopyranone 6, and autoxidation of the tricyclic system would give the final product, CTP-431 7 (Scheme 1).

Scheme 1. Proposed conversion of latrunculin A into CTP-131.
The fascinating possibility of such an extended sequence of spontaneous reactions occurring over a prolonged period prompted us to embark upon a model study to investigate the feasibility of such a transformation. There are two pivotal steps in the proposed sequence of events outlined in Scheme subsequent methanolysis of the thiazolidinone ring followed by reclosure by intramolecular thiol addition to the enone (5 to 6, Scheme 1). Of these, the former is well precedented with several examples of IMDA reactions of trienes leading to hydrindane derivatives, including many with a similar substitution pattern on diene and dienophile fragments, [8][9][10][11][12] although the conditions usually required to effect IMDA reactions vary from those used to store the natural sample. Nevertheless, we took the view that such an IMDA step was feasible, and thus our experimental focus was on the key thiazolidinone ring-opening step.
The ring opening of thiazolidinones is reported to occur under aqueous alkaline conditions, followed by decarboxylation to give cysteamine derivatives. 13,14 The present case necessitates a methanolysis initiated ring-rearrangement reaction of the thiazolidinone to a tetrahydrothiopyranone, and therefore as a model for the CTP-431 system, we selected the  According to our initial hypothesis, the ring rearrangement requires the attack of methanol on the thiazolidinone carbonyl, followed by elimination and conjugate addition of the thiol to the enone functionality via a 6-endo-trig process, a known but rare process. [16][17][18] Although the ring opening of thiazolidinones is known under aqueous conditions (vide supra), alcoholysis reactions are rare.
Mellor et al. have reported the conversion of 4-vinyl thiazolidinones to methyl carbamates using potassium hydroxide in methanol, 19 but these conditions gave no identifiable products in the case of our cyclohexenyl derivative 12. The use of sodium or potassium methoxide in methanol at various temperatures similarly led only to decomposition of the starting material. With basic conditions having failed and Brønsted acids such as TsOH giving no reaction at all, a variety of metal salts were employed as Lewis acid catalysts (see Table S1, Supporting Information), in the hope that coordination to the sulfur atom might increase the reactivity of the thiazolidinone towards methanol.
Silver(I) acetate, lead(II) chloride, gold(I) chloride, zinc(II) chloride and scandium(III) triflate all gave no reaction, while other silver salts such as AgBF 4 and AgPF 6 resulted in decomposition of the thiazolidinone 12. An attempt to use mercury(II) acetate in methanol at room temperature gave no isolable product, but when these conditions were followed by addition of excess 1,3-propanedithiol to sequester the mercury away from the thiol, the products 13, 14 and 15 were isolated in 7%, 12% and 51% yield respectively (Scheme 3). While we were pleased to identify the desired product of the thiol conjugate addition reaction 13, it was in a disappointing yield, with most of the material converted to the side-products 14 and 15. The stereochemistry of 14 was suggested by NOESY spectroscopy, but is inconsequential since compound 14 represents a dead end in our proposed reaction pathway (Scheme 4). Attempts to improve on this result using other mercury sources proved fruitless, but the use of a less polar solvent mixture -19:1 toluene:methanol -disfavoured the hemiacetal formation, giving none of the side-product 14 and a slightly improved yield (12%) of the desired product 13. Applying the1,3-propanedithiol work-up to reactions with other metal catalysts did not improve the outcomes of those reactions, and attempts to convert the cyclic carbamate 15 into the methyl carbamate 13 under acidic or basic conditions were unsuccessful. 7 Scheme 3. Ring opening rearrangement of thiazolidinone 12 into thiopyranone derivatives.
The formation of the major product 15, identified by X-ray crystallography (Figure 3), initially suggested the possibility that the thiazolidinone had undergone attack not by methanol, but by the hemiacetal of the pendant ketone. However, the fact that conjugate addition had also occurred indicated that the thiol must have been liberated while the enone functionality was still intact, leading us to propose instead that an elimination reaction occurs first, producing a thiol and an isocyanate 16 (Scheme 4); the thiol is rapidly trapped by the enone, and the ketone oxygen is well placed to undergo reaction with the isocyanate as its hemiacetal. Further support for this mechanism came from the failed ring-rearrangement of the PMB-protected thiazolidinone 11 under the same reaction conditions. When the hydrolysis was carried out under aqueous conditions, followed by treatment of the product with an excess of methyl chloroformate, the intermediate thiol 16 was trapped, the isocyanate intercepted by the ketone oxygen in its enol form to give, after further reaction at nitrogen with methyl chloroformate, the adduct 17 (Scheme 5), the structure of which was also confirmed by X-ray crystallography ( Figure 3).  In summary, we have investigated some of the key steps required for the conversion of latrunculin A 1 to its congener CTP-431 7 and demonstrated experimentally that the critical ring rearrangement sequence of the thiazolidinone to the corresponding tetrahydrothiopyranone is viable. Although the conditions required for both this key step and the proposed IMDA reaction vary significantly from those used to store the natural sample, given the crude nature of the sponge extract it is impossible to discount the possibility of some form of catalysis in the latter case. Hence we believe that these are feasible steps along the pathway from latrunculin A to CTP-431, and we hope that this will stimulate further work on the origins of this remarkable natural, or possibly unnatural, product.

General experimental details
Commercially available reagents were used throughout without purification unless otherwise stated.
All anhydrous solvents were used as supplied, except tetrahydrofuran and dichloromethane that were freshly distilled according to standard procedures. Reactions were routinely carried out under an argon atmosphere unless otherwise stated, and all glassware was flame-dried before use. Light petroleum refers to the fraction with bp 40-60 °C. Ether refers to diethyl ether. 10 Analytical thin layer chromatography was carried out on aluminum-backed plates coated with silica gel, and visualized under UV light at 254 and/or 360 nm and/or by chemical staining. Flash chromatography was carried out using silica gel, with the eluent specified.
Infrared spectra were recorded using an FT-IR spectrometer over the range 4000-600 cm −1 . NMR spectra were recorded at 400 or 500 MHz (1H frequency, 100 or 125 MHz 13C frequency).
Chemical shifts are quoted in parts per million (ppm), and are referenced to residual H in the deuterated solvent as the internal standard. Coupling constants, J, are quoted in Hz. In the 13C NMR spectra, signals corresponding to CH, CH 2 , or CH 3 groups are assigned from the DEPT spectra. Mass spectra were recorded on a time-of-flight (TOF) mass spectrometer using electrospray ionization (ESI).

Ethyl (R)-2-oxothiazolidine-4-carboxylate
1,1-Carbonyldiimidazole (8.11 g, 50 mmol) was added in portions over 5 min to a stirred suspension of (L)-cysteine ethyl ester hydrochloride (9.28 g, 50 mmol) in THF (150 mL). The mixture was stirred at room temperature for 16 h, filtered through a pad of Celite ® and washed with THF (150 mL). The filtrate was concentrated in vacuo and purified by column chromatography eluting with light petroleum and ethyl acetate (1:1) to give the title compound as a colorless oil (
The residue was dissolved in THF (2 mL), and cooled to 0 °C, then triethylamine (0.04 mL, 0.24 mmol) and methyl chloroformate (0.08 mL, 1.0 mmol) were added sequentially. The mixture was stirred at room temperature for 5 min, diluted with hydrochloric acid (1 M; 2 mL) and extracted with ethyl acetate (5 mL). The organic phases was washed with hydrochloric acid (1 M; 3 x 1 mL) and brine (2 mL

Supporting Information
The Supporting Information is available free of charge on the ACS Publications website at DOI: xxxxxx.

AUTHOR INFORMATION
Corresponding Author