Enantioselective Total Synthesis of Plectosphaeroic Acid B

Abstract

The first total synthesis of a member of the plectosphaeroic acid family of fungal natural products is reported. Key steps include the late-stage formation of the hindered N6–C9″ bond and stereoselective introduction of the two methylthio substituents.

In 2009, Mauk, Andersen and coworkers reported the isolation of plectosphaeroic acids A–C (1–3) from cultured extracts of the fungus Plectosphaerella cucumerina collected in Barkley Sound, British Columbia (Figure 1).¹ These highly functionalized, secondary metabolites are defined by the union of a tryp-tophan-derived epitrithiodioxopiperazine (or methylthio analog)² and 2-aminophenoxazin-3-one fragments,³ structural motifs commonly found in natural products but never before seen in conjunction. A number of epipolythiodioxopiperazine (ETP) alkaloids and their methylthio congeners (e.g., 5–11) share a polycyclic core that is homologous to the northern fragments of 1–3.^4,5 Naturally occurring cinnabarinic acid (4) comprises the southern fragment of 1–3. Molecules comprised of either structural motif display a broad spectrum of biological properties (e.g., antimicrobial, antiviral, antifungal, immunosuppressive, and anticancer activities),^2,3 with numerous studies focusing on chemotherapeutic applications.^6,7 In the isolation report, it was disclosed that 1–3 were equipotent (IC₅₀ = 2 μM) against indoleamine 2,3-dioxygenase (IDO), a recently identified molecular target for the potential treatment of cancer. Although it was found that only the phenoxazinone subunit is required for this activity,⁸ we were intrigued by the potential utility of molecules that contain both anticancer motifs,⁹ as well as the considerable synthetic challenges^10–12 posed by the complexity of 1–3. Herein, we describe our recent efforts that culminated in the first total synthesis of (+)-plectosphaeroic acid B (2).

Figure 1.

The plectosphaeroic acids and related metabolites.

Our plan for preparing plectosphaeroic acids A–C (1–3) is outlined in Scheme 1. We envisaged stereoselectively introducing the labile thioethers or bridging trisulfide functionalities of 1–3 after late-stage construction of the critical C9″–N6 bond between the indoline nitrogen atom of fragment 12 and a halogenated congener 13 of cinnabarinic acid. Although efficient transition metal-catalyzed methods are known, the hindered nature and structural complexity of these intermediates would make the successful joining of fragments 12 and 13 one of the most challenging applications of C–N cross-coupling to date.^13,14 Fragment 12 was seen arising from the simpler trioxopiperazine alkaloid (+)-gliocladin C (14)^4b,4e,10a,15 using a general sequence our group disclosed in 2011 for the total synthesis of the ETP natural product (+)-gliocladine C (5).¹⁰ Halide 13 was envisioned originating from the biomimetic, oxidative dimerization of 6-halo-3-hydroxyanthranilic acid 15.¹⁶

Scheme 1.

In order to assess the feasibility of—and identify conditions for—the critical C–N bond-forming step, we began our efforts by examining N-arylation of a structurally simplified indoline fragment (Scheme 2). The di-(tert-butoxycarbonyl) derivative 16 of (+)-gliocladin C, which is available on multi-gram scale by chemical synthesis,^10a was exposed to a catalytic amount of Sc(OTf)₃, resulting in the selective deprotection of the indoline nitrogen atom. After a considerable screening effort,¹⁷ joining of the two fragments was realized in 67% yield when indoline 17 was allowed to react with 2.3 equiv of iodide 18,¹⁸ 3.0 equiv of copper(I) thiophene-2-carboxylate (CuTC)¹⁹ and excess K₂CO₃ in toluene at 90 °C. Other Cu(I) salts and ligand combinations that were screened gave poor conversions to 19 (0–20% yields). Reducing the amount of CuTC also resulted in low yields of 19. Additionally, it was found that double protection of the 2-amino group of iodide 18 was critical,²⁰ and that N-arylation of the corresponding bromide was much less efficient (8% yield of 19). Although the excess of iodide 18 was recoverable, minor amounts of the by-product arising from undesired hydrodehalogenation of 18 were observed as well. Having discovered conditions to successfully unite the two fragments, we focused our attention on the synthesis of plectosphaeroic acid B (2).

Scheme 2.

The total synthesis of (+)-plectosphaeroic acid B (2) commenced with deprotection of the indoline nitrogen atoms of the individual epimers 20a and 20b, intermediates previously prepared en route to (+)-gliocladine C (5) (Scheme 3).¹⁰ Attempts to chemoselectively remove the Boc group of 20a or 20b with Lewis or protic acids proved challenging because of the other acid-labile functionalities that were present, including the C3- and C12-N,O-acetals. For this reason, a two-step (single-pot) procedure was developed. Thermolytic cleavage of both Boc groups of 20a or 20b, followed by selective reprotection of the indole nitrogen atom by reaction with 1 equiv of Boc₂O and a catalytic amount of DMAP afforded intermediates 21a and 21b in 60–80% yields. Complete inversion of the C12-stereocenter occurred during the thermolytic-deprotection step.²¹

Scheme 3.

The potential to form an N-acyliminium ion by loss of the oxygen substituents at C3 or C12 also complicated the ensuing copper-mediated C–N cross-coupling reaction. In preliminary experiments, treatment of epimer 21a with iodide 18, CuTC and K₂CO₃ resulted in inefficient conversion to the coupled product 22a (10–30% yields). In these reactions, some formation of the thiophene-2-carboxylate adduct 23 was observed. As in-situ activation of the angular N,O-acetal appeared unavoidable, we explored substituting CuTC with CuOAc²² in order to minimize the formation of by-products. After some optimization, exposure of 21a or 21b to 3.0 equiv of iodide 18 and 6.0 equiv of CuOAc in toluene at 90 °C delivered 22a and 22b in 50–58% yields.

We turned our attention to the stereoselective installation of the methylthio substituents of 2.²³ Activation of the N,O-acetals of 22a and 22b by exposure to excess BF₃·OEt₂ and MeSH in CH₂Cl₂ at −78 °C with slow warming to room temperature led to the generation of a 1.3:1.0 mixture of di(methylthio)ethers 24 and C3-epi-24 in high yield (79% from 22a, 92% from 22b). Alternatively, it was found that transforming 22a or 22b by reaction with H₂S and BF₃·OEt₂, then methylation with MeI and K₂CO₃, provided cis-di(methylthio)ether 24 in 80–90% yield as virtually a single stereoisomer. The difference in stereochemical outcomes for these sulfenylation procedures warrants further comment. Substantial precedent suggests that introduction of the sulfur nucleophile would occur first at C12, with high stereoselectivity from the concave face. ^2c,2e,10,11 The factors governing the facial selectivity of the subsequent addition of the sulfur nucleophiles at C3 are less certain. The greater stereoselectivity we observe in forming 2 by the two-step sequence could reflect the difference between directly forming configurationally stable thioether products or proceeding via configurationally less stable hemithioaminal intermediates, which could be equilibrating under the sulfenylation or methylation conditions to the more stable cis product.^24-26

Completion of the synthesis of (+)-plectosphaeroic acid B (2) required careful removal of the three remaining ester groups.²⁷ Methanolysis of the C11-acetate of 24 was achieved using excess La(OTf)₃ and 1 equiv of DMAP at 50 °C. Then, conversion of the methyl esters of the phenoxazinone subunit to carboxylic acids by the use of LiI in pyridine at 90 °C gave (+)-plectosphaeroic acid B (2) in 65% yield over two steps after HPLC purification. The optical rotation of synthetic 2, [α]_D²³ +228 (c 0.08, MeOH), was considerably higher than the reported value for the natural sample, [α]_D²³ +69.8 (c 0.27, MeOH). However, all other spectroscopic data, including CD spectra, compared well.

In conclusion, the first total synthesis of (+)-plectosphaeroic acid B (2) was achieved in seven steps from the known intermediates 20a and 20b. This total synthesis confirms the unique structure and absolute configuration of plectosphaeroic acid B, which had been assigned on the basis of NMR, MS and CD data.¹ Introduction of the highly congested, central C–N bond of 2 by a late-stage copper-mediated process provides one of the most demanding examples of C–N cross-coupling reported to date. The convergence of this synthesis strategy should enable the synthesis of the remaining plectosphaeroic acids and analogues, and allow the pharmacological evaluation of these and related molecules containing multiple anticancer motifs.