Bioinspired Diversification Approach Toward the Total Synthesis of Lycodine-Type Alkaloids

Nitrogen heterocycles (azacycles) are common structural motifs in numerous pharmaceuticals, agrochemicals, and natural products. Many powerful methods have been developed and continue to be advanced for the selective installation and modification of nitrogen heterocycles through C–H functionalization and C–C cleavage approaches, revealing new strategies for the synthesis of targets containing these structural entities. Here, we report the first total syntheses of the lycodine-type Lycopodium alkaloids casuarinine H, lycoplatyrine B, lycoplatyrine A, and lycopladine F as well as the total synthesis of 8,15-dihydrohuperzine A through bioinspired late-stage diversification of a readily accessible common precursor, N-desmethyl-β-obscurine. Key steps in the syntheses include oxidative C–C bond cleavage of a piperidine ring in the core structure of the obscurine intermediate and site-selective C–H borylation of a pyridine nucleus to enable cross-coupling reactions.


General Information
Reactions were performed under inert nitrogen atmosphere in oven-dried glassware unless otherwise noted. Room temperature (r.t.) refers to a standard 21 °C. All reactions were stirred with Teflon®-coated magnetic stir bars and were monitored by thin layer chromatography (TLC) performed on Macherey-Nagel TLC Silica gel 60 F254 precoated glass plates (0.25 mm thickness). Substances were visualized under UV light (λ = 254 nm) and/or staining with basic KMnO4 or p-anisaldehyde solutions. Chromatographic purification was performed on silica gel (SiliCycle, 40-63 μm mesh).
All chemicals were purchased from commercial suppliers and were used without further purification unless otherwise stated. THF, toluene, Et2O and Et3N were dried by passage over a column of activated alumina (JC Meyer Phoenix SDS Solvent System). CH2Cl2 and diisopropylamine (DIPA) were distilled over anhydrous CaH2. Anhydrous EtOH, CHCl3 and pyridine were obtained in sealed bottles from commercial suppliers. 1 H and 13 C-NMR spectra were recorded on Bruker spectrometers operating at 300, 400, 500, 600 MHz for 1

Synthesis of (5R)-5-methyl-2-(phenylthio)cyclohexan-1-one (S1)
Adapted from literature. 4 Thiophenol (23.8 mL, 25.5 g, 232 mmol, 2.05 equiv) was dissolved in anhydrous THF (500 mL) in a 1.0 L round-bottom flask with stir bar. Pieces of flattened sodium metal (5.17 g, 226 mmol, 2.0 equiv) are added and the developing colorless suspension was stirred at room temperature overnight. Pulegone epoxide 19 (2:1 mixture of aand b-isomers, 19.0 g, 113 mmol, 1.0 equiv) was dissolved in anhydrous THF (25 mL) in a 100 mL round bottom flask and was added dropwise via cannula to the PhSNa suspension over the course of 30 min. The mixture was heated to 85 °C and kept at reflux for 7 h after which TLC (hex/EtOAc 2:1, UV/p-anisaldehyde, Rf,prod = 0.71, Rf,sm = 0.46; diluted sample in a few µL MeOH) confirmed full conversion of the epoxide. The reaction was cooled to room temperature and water (200 mL) was added, followed by extraction with ethyl acetate (3 × 150 mL). The combined organic extracts were washed with sat. aq. K2CO3 (3 × 150 mL) and brine (200 mL), dried over MgSO4 and the solvent was evaporated under reduced pressure (40 °C) to give 25.8 g of S1 as a yellow-brownish oil, which slowly turns into to a waxy solid upon storage at 4 °C (104 % crude yield). The mixture of diastereomers was used in the next step without further purification.

Synthesis of (5R)-5-methyl-2-(phenylsulfinyl)cyclohexan-1-one (20)
Crude thioether S1 (25.0 g) was dissolved in acetic acid (glacial, 225 mL, 9 mL/g crude) in a 500 mL round bottom flask and heated to 40 °C. Sodium perborate monohydrate (12.4 g, 125 mmol, 1.1 equiv) was ground to a fine powder with a mortar before adding it to the dissolved thioether in ca. 2 g portions over the course of 5 min. The suspension was stirred at 40 °C for 45 min, after which the mixture clarified and TLC (hexanes/EtOAc 2:1, UV/p-anisaldehyde, Rf,prod = 0.25, brown) confirmed full conversion of the starting material. The reaction was cooled to 0 °C in an ice bath, and NH4OH (14.8 M, 265 mL) was added slowly (!) over 1.5 h with a dropping funnel whilst stirring to quench the acetic acid. The resulting colorless suspension was diluted with water (100 mL) and the pH adjusted to 7-8 by dropwise addition of aqueous ammonia solution (14.8 M). The aqueous mixture was extracted with CH2Cl2 (3 × 150 mL), the combined organic extracts washed with brine (150 mL), and dried over MgSO4. Evaporation of the solvent under reduced pressure (20 °C) gave 26.5 g of a thick, brownish oil (99% crude over 2 steps), which was purified by column chromatography using a gradient of hexanes/EtOAc 2:1-1:1-1:2. The title compound 20 was obtained as colorless oil, which solidifies upon standing at room temperature (17.3 g, 65% over two steps, mixture of diastereomers

Synthesis of (R)-3-(9-methyl-1,4-dioxaspiro[4.5]dec-6-en-6-yl)propan-1-amine (22)
Adapted from literature. 1 Ketal S2 (2.85 g, 13.8 mmol, 1.0 equiv) was dissolved in anhydrous Et2O (57.0 mL) and cooled to 0 °C. LiAlH4 (2.11 g, 55.6 mmol, 4.04 equiv) was added portion-wise over 12 minutes and the grey suspension was stirred for 4 h at 0 °C. Upon full consumption of starting material (TLC) the reaction was worked up according to the Fieser & Fieser protocol: The reaction mixture was diluted with Et2O (57 mL) and water (2.15 mL) was added dropwise (violent gas evolution) at 0 °C. 15% aq. NaOH solution (2.15 mL) was added and the mixture was stirred for 20 minutes at 0 °C. Water (6.30 mL) was added and the mixture was stirred for 20 minutes at room temperature. MgSO4 (5.7 g) was added and the mixture was stirred for another 20 minutes. The white suspension was filtered through a pad of celite and the filter was thoroughly washed with Et2O. Concentration under reduced pressure gave a slightly yellowish oil of amino ketal 22 (2.42 g, 11.51 mmol, 84%) which was used without further purification.

Synthesis of N-desmethyl-α-obscurine (5)
The crude material obtained by following a literature procedure 1 may be purified by a phase-transfer protocol if needed: The yellow residue was taken up in 1 M aq. HCl and washed with CH2Cl2 (2 × 50 mL). After adjusting the pH to 6 with sat. aq. Na2CO3, another three washes with CH2Cl2 followed. Further basification with sat. aq. Na2CO3 led to formation of a white precipitate. At pH 13-14, the mixture was extracted again with CH2Cl2 (3 × 50 mL). The combined organic extracts from the last step were dried over MgSO4 and concentrated under reduced pressure to afford N-desmethyl-αobscurine as yellowish/off-white solid. Spectral data is in agreement with literature. 1
phase was carefully adjusted to pH 7.0 with 0.5 M aq. HCl (9.5 mL). The aq. phase was extracted with CH2Cl2 (3 × 50 mL), the combined organic extracts were dried over MgSO4 and evaporated under reduced pressure afforded crude pyridonyl imide S4 as colorless amorphous solid (530 mg). The crude material was taken directly into the pyridone methylation.

Synthesis of 1-methoxy-9-oxo-N-Boc-lycodine (S5)
Ag2CO3 (749 mg, 2.72 mmol, 1.9 equiv) was added to a solution of crude pyridonyl imide S4 (530 mg, 1.43 mmol assumed from previous step, 1.0 equiv) in CHCl3 (10 mL) followed by a solution of MeI (111 µL, 1.78 mmol, 2.6 equiv) in CHCl3 (2.0 mL). The grey suspension was heated to reflux (75 °C) in the dark (wrap with tin foil) for 13 hours, after which TLC (n-hexane/EtOAc 1:1) showed full conversion of the starting material. After cooling to room temperature, the yellow-brown suspension was filtered through a pad of celite and the yellow oily residue obtained after evaporation was purified by silica gel chromatography (hexanes/EtOAc 2:1) to afford the title compound S5 as colorless foam (475 mg, 86% over two steps).

Synthesis of 1'-methyl-N-Boc-casuarinine H (27)
An oven-dried, tall tube with stir-bar and septum cap was loaded with methoxy acid 27 (100 mg, 247 µmol, 1.0 equiv), PdBr2 (2.0 mg, 7.4 µmol, 3 mol%) and DPE-Phos (12.0 mg, 22.3 µmol, 9 mol%) and subjected to three cycles of evacuation and backfilling with nitrogen. Anhydrous DMPU (1.2 mL) was added, followed by pivalic anhydride (105 µL, 519 µmol, 2.1 equiv) and anhydrous triethylamine (4.0 µL, 29.7 µmol, 12 mol%). The clear yellow solution was stirred at 130 °C for 4 h upon which the color changes to orange/red and then dark purple and TLC (n-hexane/EtOAc 2:1, p-anisaldehyde) confirmed full conversion of the starting material. The solution was cooled to room temperature, then cooled further to 0 °C, and 1 mL saturated aqueous NaHCO3 was added. The solution was allowed to warm to room temperature and stirred for 20 minutes. The mixture was diluted with EtOAc (30 mL) and washed with sat. aq.

Synthesis of (-)-casuarinine H (2)
Methoxy olefin 27 (10 mg, 0.028 mmol, 1.0 equiv) was concentrated in a 1 dram vial and introduced to a glovebox. In the glovebox, 27 was dissolved in anhydrous CHCl3 (0.5 mL) and trimethylsilyl iodide (TMSI) (40 µL, 0.28 mmol, 10 equiv) was then added dropwise. The vial was sealed, removed from the glovebox, and heated at 65 °C for 5 hours, resulting in a dark yellow solution. After cooling to room temperature, the reaction was transferred to a 2 dram vial with CH2Cl2 (2 × 1.0 mL). The solution was cooled to 0 °C and H2O, sat. aq. K2CO3, and sat. aq. Na2S2O3 (0.5 mL of each) were added while stirring. The pH of the resulting solution was confirmed to be >10 and the aqueous phase was extracted with CH2Cl2 (3 × 1 mL). The combined organic extracts were dried over MgSO4 and filtered. Concentration under reduced pressure afforded (-)-casuarinine H (2) as an off-white solid/foam (6.1 mg, 89%).

Synthesis of (-)-8,15-dihydrohuperzine A (3)
Methoxy internal olefin S6 (10.4 mg, 0.029 mmol, 1.0 equiv) was concentrated in a 1 dram vial and introduced to a glovebox. In the glovebox, S6 was dissolved in anhydrous CHCl3 (0.5 mL) and TMSI (42 µL, 0.29 mmol, 10.0 equiv) was then added dropwise. The vial was sealed, removed from the glovebox, and heated at 65 °C for 5 hours, resulting in a dark red/brown solution. After cooling to room temperature, the reaction was transferred to a 2 dram vial with CH2Cl2 (2 x 1.0 mL). The solution was cooled to 0 °C and H2O (1 mL

Synthesis of N-Boc lycodine boronic ester (S7)
The title compound was prepared from 30 according to a literature procedure 1 and was used without purification in the next step.

Synthesis of lycoplatyrine A (8)
Coupling product 33 (1.0 equiv., 9-13 µmol) and powdered anhydrous NaOH (   Spectral data of both isomers is in agreement with literature. 8 The natural product derived from deprotection of the coupling product with racemic 32 was obtained as a 1.5:1 mixture of (2'S) and (2'R) epimers, respectively. stirred solution (>1000 rpm). Upon complete addition the mixture was rapidly stirred at -78°C for 30 min, then the ice bath was removed and the mixture was allowed to warm to room temperature in ambient air before it was placed in a water bath at 21 °C and rapid stirring was continued for another 30 min. 31 (23.8 mg, 56.5 µmol, 0.95 equiv.) was provided in an oven-dried glass vial and dissolved in anhydrous MTBE (100 µL). The solution was transferred dropwise to the reaction using a cannula and the source vial is washed with another 100 µL MTBE. Then Pd(OAc)2 (0.7 mg, 3.0 µmol, 5 mol%) and t Bu3PHBF4 (1.1 mg, 3.6 µmol, 6 mol%) were added together as solids in a N2-stream. The resulting yellow, clear solution was placed in a preheated oil bath at 60 °C and the precipitation of Zn-salts was observed within the first 30 to 60 min. After 18 h, TLC (n-hexane/EtOAc 1:1, UV/KMnO4) and LC-MS confirmed full consumption of 31 and the beige suspension was cooled to room temperature. NH4OH (35%, 11.5 µL) was added and the beige suspension was stirred for 30 min at 21 °C before filtering through a short pad of celite. The reaction vial and plug were washed with MTBE (3 × 1.0 mL) and the filtrate was evaporated in vacuo to give a yellow oil, which was purified by column chromatography (EtOAc/n-hexane 3:1) or preparative TLC (EtOAc/n-hexane 1:1) to afford the coupling product (2'R)-36 as colorless foam (25.4 mg, 88%).

Synthesis of lycopladine F (9)
Coupling product 38 ( with sat. aq. K2CO3 and extracted with MTBE (3 × 1 mL). The organic extracts were dried over MgSO4, filtered, and concentrated in vacuo to afford the crude product S10 as a clear oil, which was analyzed by NMR spectroscopy and telescoped to the debenzylation without further purification. In a 1 dram vial, crude benzyl ester S10 (assuming 24 µmol) and palladium on activated charcoal (0.5 mg, 5% w/w) were taken up in anhydrous MeOH (500 µL), and trifluoroacetic acid (TFA) (2 µL, 24.4 µmol, 1.0 equiv) was added to the suspension. The vial, capped with a septa lid through which a large needle was inserted, was placed in a hydrogenation bomb at 500 psi hydrogen pressure. The suspension was stirred at 21 °C (900 rpm) for 3 h before the pressure was released and the reaction was filtered through a plug of Celite (moistened with MeOH). The plug was washed with 3 × 1.0 mL MeOH and the collected filtrate was evaporated under reduced pressure to yield a 1:1 epimeric mixture at C2' of lycopladine F (9) as a white solid (7.8 mg, 71% yield over two steps, calculated as the mono TFA salt) (1:1 mixture of 2'S and 2'R epimers). CDCl3 was deacidified by passing through a plug of basic alumina (Brockmann I) before dissolving the product for NMR analysis. Note: Omitting TFA from the debenzylation procedure yielded a product for which the NMR spectra did not match the spectra detailed in the isolation report.   Characterization data in agreement with material isolated from natural sources. 13 S32  Table S2. Screening of biocatalytic conditions towards a chemoselective piperidine C-N bond oxidation of N-desmethyl-aobscurine (5) and Boc-protected derivative (S3). Enzymes were selected based on their ability to oxidize C-heteroatom bonds in their substrate portfolio (references given).

Enzymatic kinetic resolution of the b-lactam coupling partner
All lipase-and esterase preparations used in kinetic resolution experiments were obtained from Sigma Aldrich and used as received: • Esterase from porcine liver (PLE, 20 U/mg and 69.3 U/mg, respectively) • Lipase A from Candida antarctica immobilized on immobead 150 (CalA, 1.59 U/mg, recombinant from A.