Synthesis of the Carboline Disaccharide Domain of Shishijimicin A

Abstract

A synthetic route to the carboline disaccharide domain (2) of shishijimicin A (1) has been developed. The convergent synthesis relies on a novel application of the Reetz–Müller-Starke reaction to form the central, sulfur-bearing quaternary carbon center and addition of the carboline structural motif as a dianion to a disaccharide aldehyde fragment.

The enediyne class of natural products has captured the imagination of chemists, biologists and clinicians due to their novel molecular architectures, potent biological properties and medical potential. Thus, investigations of members of this family of compounds led to important advances in fundamental science and practical applications in cancer chemotherapy.¹

Isolated from the thin encrusting orange ascidian Didemnum proliferum, shishijimicin A (1, Figure 1) has joined its siblings (B and C)² and namenamicin³ as the only members of the enediyne class that originate from marine sources. All three shishijimicins carry the same enediyne core as the one found in namenamicin and calicheamicin γ₁^I (known as calicheamicinone).⁴ What sets the shishijimicins apart from all other enediynes, however, is their unique carboline structural motif. Indeed, β-carboline is known to intercalate into double-stranded DNA,⁵ and a number of β-carbolines have been shown to cleave DNA under photoirradiation conditions.⁶ Shishijimicin A (1) is the most potent of the shishijimicin family, exhibiting IC₅₀ values of 2.0, 1.8, and 0.47 pM cytotoxicities against 3Y1, HeLa, and P388 cell lines, respectively.² In light of these observations and the extreme scarcity of shishijimicin A, its total synthesis and that of its carboline disaccharide domain are deemed important. In this letter, we report the chemical synthesis of the carboline disaccharide domain 2 of shishijimicin A, whose features include a Reetz–Müller-Starke reaction to install the tetrasubstituted, sulfur-bearing carbon center and a carboline dianion-aldehyde coupling.

Figure 1.

Structures of shishijimicin A (1), carboline disaccharide domain 2, and retrosynthetic analysis of 2.

Retrosynthetic rupture of the targeted disaccharide fragment 2 as shown in Figure 1 revealed carboline derivative 3, methylthio sugar 4, and N-alloc aminoglycosyl fluoride 5 as the required building blocks.

Scheme 1 summarizes the developed expedient route to carboline derivative 3 starting with 5-methoxytryptamine 6. Thus, 6 was sequentially treated with triphosgene (60 – 70 °C) and 30% HBr in glacial acetic acid (100 °C) to afford the dihydrocarboline 7 in 72% overall yield. The fully aromatized carboline 8 was obtained from the latter compound through oxidation in the presence of 10% Pd/C in refluxing cumene (68% yield). Previous studies⁷ with carboline dianions employed bromide derivatives, however, in this case, the bromide was obtained in rather low yield (i.e. <40%), prompting us to target the corresponding iodide derivative, which was also expected to undergo a smoother halogen-metal exchange at the step of the anion-aldehyde coupling. Toward this end, and capitalizing on the reactivity of the hydroxypyridine moiety of compound 8, we transformed the latter, first to its triflate (Tf₂O, pyridine) and then to its iodide in the same pot by iodide displacement (NaI, TfOH) in 61% overall yield.⁸ Exchange of protecting group within the so-obtained iodo-β-carboline 9 (BBr₃ – demethylation; TBSOTf – silylation) led to the desired carboline block 3 in 55% overall yield.

Scheme 1.

Synthesis of Iodo-β-carboline 3

With the carboline coupling partner in hand, our attention turned to the synthesis of the central unit of the molecule, namely the methylthio sugar 4. Our successful construction of this intermediate commenced with differentially protected glycoside 10⁹ and proceeded as shown in Scheme 2. Cleavage of the benzylidene group under acidic conditions (TsOH) liberated the 1,3-diol system 11 (80% yield), whose tosylation (TsCl, Et₃N, DMAP cat.) furnished monotosylate 12 in 89% yield. Subsequent reduction of the tosylate 12 with LiAlH₄ led smoothly to 6-deoxymethylpyranoside 13 in 94% yield. The remaining hydroxyl group within 13 was then oxidized with NMO in the presence of TPAP (cat.), yielding ketone 14 (84% yield). Aiming for the eventual installment of the quaternary center bearing the methylthio and nitrile groups in the growing molecule, ketone 14 was treated with TMSSMe and TMSOTf in CH₂Cl₂ at −78 → 0 °C to furnish dithioketal 15 in 94% yield. Careful monitoring of the temperature for this reaction was important, as warming above 0 °C resulted in formation of by-products in which the anomeric methoxy group was replaced with a methylthio substituent.

Scheme 2.

Synthesis of Thiosugar 4

To complete the construction of the central carbon center bearing the required methylthio and nitrile groups, we called upon the Reetz–Müller-Starke reaction.¹⁰ This method had been successful previously on dithioketals but not demonstrated on carbohydrates such as 15, where the anomeric methoxy group may lead to complications. Despite these fears, we were pleased to observe that, under the conditions originally reported by Reetz and Müller-Starke (i.e. TMSCN, SnCl₄, CH₂Cl₂, 0 °C), dithioketal 15 reacted to afford the expected methylthio nitrile 16 as a ca. 2:1 diastereomeric mixture and in 75% combined yield. The structures of the chromatographically separated isomers 16 (major, desired) and 4-epi-16 (minor, undesired) were assigned by ¹H NMR spectroscopic analysis. Specifically, the desired isomer 16 exhibited a diagnostic nOe between the methylthio group protons and the C-2 proton, whereas the undesired epimer did not (see chair structures 16 and 4-epi-16 in Scheme 2). In support of the stereochemical assignment of 4-epi-16 were the nOe’s exhibited between its methylthio group protons and those of C-3 and C-5. All that remained to generate the targeted fragment 4 was the deallylation of intermediate 16, a reaction that proceeded smoothly, and in 77% yield, in the presence of Pd(PPh₃)₄.

The preparation of the remaining carbohydrate unit, glycosyl fluoride 5, was prepared from the known amino glycoside 17,¹¹ as summarized in Scheme 3. The alloc group was chosen as a protecting group for the secondary amine moiety instead of the Fmoc group employed in previous work,¹² due to the anticipated basic conditions of the carboline coupling. Thus, treatment of 17 with allyl chloroformate in the presence of K₂CO₃ and 18-crown-6 led to carbamate 18 (98% yield), whose exposure to AcOH:H₂O (5:1) at 95 °C liberated lactol 19 (70% yield; 90% based on recovered starting material). Finally, glycosyl fluoride 5 was formed from the latter compound through the action of DAST in 64% yield.

Scheme 3.

Synthesis of Glycosyl Fluoride 5

With all three required fragments available, their coupling and elaboration to the targeted carboline disaccharide domain 2 became the next task. Scheme 4 summarizes the accomplishment of this goal. Coupling of carbohydrate fragment 4 with glycosyl donor 5 proceeded smoothly following the Mukaiyama protocol.^11,13 Thus, in the presence of AgClO₄ and SnCl₂ under anhydrous conditions (4Å MS), the mixture afforded, stereoselectively, disaccharide 20 in 85% yield (based on 4). Direct addition of the dianion derived from carboline fragment 3 to disaccharide nitrile 20 gave a complex mixture of products, necessitating the intermediacy of aldehyde 21. The latter was produced from 20 by DIBAL-H reduction (88% yield). Reaction of this aldehyde with the dianion generated from iodocarboline derivative 3 by deprotonation (KHMDS, Et₂O, −20 °C), followed by halogen-metal exchange (t-BuLi, −78 °C) resulted in the formation of the corresponding coupling product as a mixture of diastereomeric alcohols (2′, epi-2′, not shown, dr ca. 1:1; 70% yield based on 50% recovered starting material 21).^7a This mixture was then oxidized with MnO₂ to afford the targeted ketone 2 in 74% yield.

Scheme 4.

Fragment Coupling and Completion of the Synthesis of Carboline Disaccharide 2

The described chemistry provides a highly convergent entry to the carboline disaccharide domain 2 of shishijmicin A (1), paving the way for an eventual total synthesis of the natural product itself, and for binding and biological evaluation studies with the synthesized domain 2 and its derivatives. This synthesis demonstrated the application of the Reetz–Müller-Starke reaction¹⁰ and of the carboline dianion coupling in complex situations.