A modular synthesis of tetracyclic meroterpenoid antibiotics

Stachyflin, aureol, smenoqualone, strongylin A, and cyclosmenospongine belong to a family of tetracyclic meroterpenoids, which, by nature of their unique molecular structures and various biological properties, have attracted synthetic and medicinal chemists alike. Despite their obvious biosynthetic relationship, only scattered reports on the synthesis and biological investigation of individual meroterpenoids have appeared so far. Herein, we report a highly modular synthetic strategy that enabled the synthesis of each of these natural products and 15 non-natural derivatives. The route employs an auxiliary-controlled Diels–Alder reaction to enable the enantioselective construction of the decalin subunit, which is connected to variously substituted arenes by either carbonyl addition chemistry or sterically demanding sp2–sp3 cross-coupling reactions. The selective installation of either the cis- or trans-decalin stereochemistry is accomplished by an acid-mediated cyclization/isomerization reaction. Biological profiling reveals that strongylin A and a simplified derivative thereof have potent antibiotic activity against methicillin-resistant Staphylococcus aureus.


Mass spectrometry
All mass spectra were measured by the analytic section of the Department of Chemistry, Ludwig-Maximilians-Universität München. Mass spectra were recorded on the following spectrometers (ionisation mode in brackets): MAT 95 (EI) and MAT 90 (ESI) from Thermo Finnigan GmbH. Mass spectra were recorded in highresolution. The method used is reported at the relevant section of the experimental section.

IR spectroscopy
IR spectra were recorded on a PerkinElmer Spectrum BX II FT-IR system. If required, substances were dissolved in CH2Cl2 prior to direct application on the ATR unit. Data are represented as follows: frequency of absorption (cm −1 ).

Optical rotation
Optical rotation values were recorded on a PerkinElmer 241 or Anton Paar MCP 200 polarimeter. The specific rotation is calculated according to Supplementary Equation 1.
Thereby, the wave length λ is reported in nm and the measuring temperature ϕ in °C. α represents the recorded optical rotation at the apparatus, c the concentration of the analyte in 10 mg/mL and d the length of the cuvette in dm. Thus, the specific rotation is given in 10 −1 ·deg·cm 2 ·g −1 . Usage of the sodium D line (λ = 589 nm) is indicated by D instead of the wavelength in nm. The respective concentration as well as the solvent is reported at the relevant section of the experimental section.

Melting Points
Melting points were determined on a B-450 melting point apparatus from BÜCHI Labortechnik AG. The values are uncorrected.

X-Ray Crystallographic Data
The data collections were performed either on an Oxford Diffraction Xcalibur diffractometer, on a Bruker D8Quest diffractometer or on a Bruker D8Venture at 100 K or at 173 K using MoKα-radiation (λ = 0.71073 Å, graphite monochromator). The CrysAlisPro software (version 1.171.33.41)[S8] was applied for the integration, scaling and multi-scan absorption correction of the data. The structures were solved by direct methods with SIR97 3 and refined by least-squares methods against F2 with SHELXL-97. 4 All non-hydrogen atoms were refined anisotropically. The hydrogen atoms were placed in ideal geometry riding on their parent atoms. Further details are summarized in the tables at the different sections.

Antibacterial assays
Overnight cultures of the bacteria were grown aerobically at 37 °C in Müller Hinton broth with added 1% glucose and pH 7.2 for Gram-negative strains, or with Trypticase soy yeast extract medium (TSY-30 g/l trypticase soy broth, 3 g/L yeast extract, pH 7.2) for Gram-positive strains. The cultures were adjusted to an OD600nm of 0.001, which resulted in a final start OD600nm of 0.0005 in the test. 25 μL of test culture was added to 25 μL of a serial dilution of the test compounds in the appropriate medium for the different strains in accordance with standardized procedures in 384 well plates (DIN 58940-7: Medical microbiologysusceptibility testing of microbial pathogens to antimicrobial agentsdetermination of the minimum bactericidal concentration (MBC) with the method of micro boullion dilution; text in German and English). Test compounds from stock solutions in DMSO were used at final concentrations of 100, 50, 25, 12.5, 6,25, 3.125, 1.56, 0.78, 0.39, 0.2 µM. As positive control compounds, Linezolid (both MRSA strains), Ciprofloxacin (E. faecium, E. coli, A. baumannii, K. pneumonia), Amikacin (P. aeruginosa) and Amphotericin (C. albicans) were applied. The highest DMSO concentration in the assay was 1%, which had no apparent effect on the growth of the bacteria. After an incubation time of 18

Antiproliferative assays
The effect of compounds on cell viability was probed with a WST-1 test using the procedure of Ishiyama et al. 5 as modified by Sasse et al. 6 The following cell lines were used: mouse fibroblast cell line L929 ( 195 mmol, 1.10 equiv) was added and the reaction mixture was allowed to warm to 23 °C. After 90 min, the reaction mixture was filtered and the filtrate was concentrated. The residue was dissolved in pentane, the soobtained mixture was filtered through a plug of Celite ® and the solvent was removed under reduced pressure.
This process was repeated twice, yielding 10 (50.3 g, 99%) as a pale yellow oil. The obtained characterization data were in full agreement with the values previously reported. 7

Dimethyl-3,5-dihydroxyphthalate 11
A mixture of dimethyl acetylenedicarboxylate (S02) (12.6 g, 88.4 mmol, 1 equiv) and diene 10 (50.3 g, 177 mmol, 2.00 equiv) was heated to 120 °C. After 17 h, the reaction mixture was diluted with a mixture of ethyl acetate-hexanes (2:3, 400 mL) and the resulting suspension was filtered through a plug of Celite ® . The filtrate was concentrated and the residue was purified by flash-column chromatography on silica gel (30% ethyl acetate in hexanes initially, grading to 50% ethyl acetate in hexanes). The obtained yellowish solid was dissolved in a minimum amount of hot dichloromethane and precipitated by the addition of hexanes to give 11 (17.2 g, 86%) as a white powder.
The organic layer was washed with aqueous hydrochloric acid solution (2 M, 2 × 200 mL) and saturated aqueous sodium chloride solution (200 mL). The washed organic extract was dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated to give the phthalamide (10.6 g) as a yellow solid.
The crude phthalamide (10.6 g) was placed in a bulb-to-bulb distillation apparatus. To a suspension of phenol 13 (5.05 g, 14.7 mmol, 1 equiv) and iodine (2.24 g, 8.83 mmol, 0.60 equiv) in ethanol (60 mL) was added a solution of periodic acid (671 mg, 2.94 mmol, 0.20 equiv) in water (3.5 mL) and the reaction mixture was heated to 40 °C. After
The obtained analytical data were in full agreement with those values reported in literature. 9

Grabowski. 11
A solution of (R)-(+)-diphenyl-2-pyrrolidinemethanol (S10) (5.25 g, 20.7 mmol, 1 equiv) and butylboronic acid (2.10 g, 20.7 mmol, 1 equiv) in toluene (250 mL) in a two-necked, round-bottomed flask equipped with an additional funnel (containing a cotton plug and 100 g of 4 Å molecular sieves) was heated to 125 °C. After 16 h, the reaction mixture was allowed to cool to 23 °C and the solvent was removed to give S11 (6.80 g, 98%) as a colorless oil. The obtained analytical data were in full agreement with those values reported in literature. 11

Mosher ester S13
The enantiomeric excess of alcohol 17 was determined as 83% by 1 H-NMR analysis of its corresponding-MTPA ester S13 according to the procedure described by E. J. Corey. 12 To a solution of alcohol 17 (10.0 mg, 70.0 µmol, 1 equiv) in dichloromethane (1 mL) were added 4dimethylaminopyridine (34.4 mg, 28.0 µmol, 4.00 equiv) and (R)-(−)-α-methoxy-α-(trifluoromethyl)phenylacetyl chloride (S12) (20.0 µL, 110 µmol, 1.50 equiv) at 23 °C. After 1 h, water (2 mL) and dichloromethane (3 mL) were added and the layers were separated. The aqueous layer was extracted with dichloromethane (2 × 3 mL) and the combined organic extracts were dried over magnesium sulfate. The dried solution was filtered and the filtrate was concentrated to give S13 as a yellow oil. The obtained analytical data were in full agreement with those values reported in literature.

Diene 20
Note: benzene was degassed via freeze-pump-thaw (three cycles) prior to use.

Alcohol 24
Lithium aluminium hydride (1.96 g, 51.7 mmol, 5.00 equiv) was added portionwise to a solution of thioester 23 (4.00 g, 10.3 mmol, 1 equiv) at 0 °C. After complete addition, the reaction mixture was heated to 45 °C. After 3 h, the reaction mixture was cooled to 0 °C, diluted with diethyl ether (100 mL) and saturated aqueous potassium sodium tartrate solution (400 mL) was carefully added. After stirring vigorously for 1 h, the layers were separated and the aqueous layer was extracted with diethyl ether (3 × 150 mL). The combined organic extracts were dried over magnesium sulfate, the dried solution was filtered and the filtrate was concentrated. [ ] = +50.9° (c = 0.89, CH2Cl2).
After 30 min, the orange, turbid reaction mixture was allowed to warm to 23 °C. After 3 h, dichloromethane (50 mL) and pH 7 phosphate buffer (100 mL) were added. The layers were separated and the aqueous layer was extracted with dichloromethane (2 × 60 mL). The combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to give mesylate S15 as an orange oil.
The so-obtained mesylate was directly used in the following reaction without further purification.

Pentacycle S17 and S18
A solution of hydrochloric acid (~1.25 M in methanol, 10 mL) was added to a solution of 26 (290 mg, 424 µmol, 1 equiv) in dichloromethane (5 mL) and the reaction mixture was heated to 40 °C. After 1 h, the reaction mixture was diluted with dichloromethane (30 mL) and saturated aqueous sodium bicarbonate solution (70 mL) was added. The layers were separated, the aqueous layer was extracted with dichloromethane (3 × 60 mL) and the combined organic extracts were dried over magnesium sulfate. The dried solution was filtered and the filtrate was concentrated to yield S17 as a yellow foam that was directly used in the following step without further purification.
A solution of boron trifluoride diethyl etherate (48% in diethyl ether, 1.11 mL, 4.24 mmol, 10.0 equiv) was added dropwise to a solution of the crude phenol S17 (271 mg, 424 µmol, 1 equiv) in dichloromethane (40 mL) at -40 °C and the reaction mixture was allowed to slowly warm to -15 °C over a period of 1 h. After 5 h, saturated aqueous sodium bicarbonate solution (50 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 50 mL). The combined organic extracts were dried over magnesium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was filtered through a short plug of silica to yield a mixture of S18 (83%) and rearranged S19 (17%) as a colorless foam that was used in the following step without further purification.
The layers were separated and the aqueous layer was extracted with diethyl ether (3 × 150 mL). The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated.
The residue was purified by flash-column chromatography on silica gel (20% diethyl ether in pentane) to yield S22 (7.67 g, 48%) as a colorless oil. The obtained analytical data were in full agreement with those previously reported. 16

2,2-Dimethyl-1-vinylcyclohexan-1-ol (S23)
A solution of S22 (23.0 g, 182 mmol, 1 equiv) in tetrahydrofuran (90 mL) was added dropwise to a solution of vinylmagnesium bromide (1 M in tetrahydroduran, 219 mL, 219 mmol, 1.20 equiv) over a period of 45 min at 0 °C. After 1.5 h, saturated aqueous ammonium chloride solution (500 mL) was added and the layers were separated. The aqueous layer was extracted with diethyl ether (2 × 300 mL) and the combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to yield crude S23 as a yellow oil. The residue was used without further purification in the next step.

Diene 31
Note: CuSO4•5H2O was dried over night in a 140 °C oven.
To a solution of crude S23 (28.1 g, 182 mmol, 1 equiv) in benzene (400 mL) was added anhydrous copper(II) sulfate (63.9 g, 400 mmol, 2.20 equiv) and the reaction mixture was heated to 90 °C under Dean-Stark conditions. After 16 h, the reaction mixture was allowed to cool to 23 °C, filtered through a pad of Celite ® and was washed thoroughly with n-pentane. The filtrate was carefully concentrated (>220 mbar, 30 °C) and the residue was purified by flash-column chromatography on silica gel (n-pentane) to yield 31 as a yellow oil (17.7 g, 71% over 2 steps). The obtained analytical data were in full agreement with those previously reported. 17  was added over a period of 15 min to the reaction mixture. After complete addition, the reaction mixture was allowed to warm to 23 °C. After 36 h, aqueous hydrogen chloride solution (1 M, 100 mL) was carefully added, the layers were separated and the aqueous layer was extracted with dichloromethane (3 × 100 mL). The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (14% diethyl ether in n-pentane) to yield S24 as a yellow highly viscous oil (9.02 g, 61%). The obtained analytical data were in full agreement with those previously reported.

Thioester 32
A solution of n-butyllithium (2.40 M in hexanes 50.8 mL, 122 mmol, 4.70 equiv) was added dropwise to a solution of ethanethiol (11.3 mL, 153 mmol, 5.90 equiv) in tetrahydrofuran (250 mL) at 0 °C. After complete addition, the reaction mixture was slowly allowed to warm to 23 °C. After 30 min, a solution of S24 (9.02 g, 26.0 mmol, 1 equiv) in tetrahydrofuran (70 mL) was added. After 7 h, diethyl ether (100 mL) and saturated aqueous ammonium chloride solution (100 mL) were added. The layers were separated, the aqueous layer was extracted with diethyl ether (3 × 100 mL) and the combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated. The residue was purified by flashcolumn chromatography on silica gel (2% diethyl ether in n-pentane) to yield 32 as a yellow oil (7.10 g, 98%).
The obtained analytical data were in full agreement with those previously reported. 18

Aldehyde 33
Triethylsilane (1.07 g, 9.20 mmol, 1.20 equiv) was added to a solution of thioester 32 (2.15 g, 7.67 mmol, 1 equiv) and palladium(II) acetate (103 mg, 460 µmol, 0.060 equiv) in acetone (60 mL). After 2.5 h, the dark brown solution was filtered through a plug of silica, the filtrate was concentrated and the residue was purified by flash-column chromatography on silica gel (2% ethyl acetate in hexanes) to provide 33 (1.43 g, 85%) as a colorless solid. The obtained analytical data were in full agreement with those previously reported. 18 Recrystallization from diethyl ether gave crystals suitable for single-crystal X-ray diffraction.

(+)-Aureol (2)
A solution of hydrochloric acid (~1.25 M in methanol, 9 mL) was added to a solution of 35 (300 mg, 744 µmol, 1 equiv) in dichloromethane (3 mL) and the resulting solution was heated to 30 °C. After 5 h, the reaction mixture was diluted with dichloromethane (25 mL) and saturated aqueous sodium bicarbonate solution (25 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 20 mL).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated to yield S26 as a colorless foam that was directly used in the following step.    Synthesis of (+)-Strongylin A (4), 5-epi-Strongylin A (45), 46, 47, 3-

(+)-Strongylin A (4)
A solution of hydrochloric acid (~1.25 M in methanol, 4 mL) was added to a solution of S30 (33.0 mg, 76.0 µmol, 1 equiv) in dichloromethane (2 mL) and the resulting solution was heated to 30 °C. After 6.5 h, the reaction mixture was diluted with dichloromethane (5 mL) and saturated aqueous sodium bicarbonate solution (10 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 10 mL).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated to yield S31 as a colorless solid that was directly used in the following step.

5-epi-strongylin A (45)
A solution of hydrochloric acid (~1.25 M in methanol, 9 mL) was added to a solution of S30 (117 mg, 270 µmol, 1 equiv) in dichloromethane (7 mL) at 23 °C and the mixture was heated to 30 °C. After 6 h, the reaction mixture was diluted with dichloromethane (30 mL) and saturated aqueous sodium bicarbonate solution (20 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 20 mL).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated to yield S31 as a colorless solid that was directly used in the following reaction.
A solution of hydroiodic acid (57 wt.% in water, 357 µL, 2.70 mmol, 10.0 equiv) was added to a solution of the crude hydroquinone S31 (93.2 mg, 270 µmol, 1 equiv) in benzene (10 mL) in an Ace ® pressure tube. The tube was sealed and the reaction mixture was heated to 90 °C. After 16 h, the reaction mixture was cooled to 23 °C and saturated aqueous sodium bicarbonate solution (15 mL) and dichloromethane (10 mL) were added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 15 mL). The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated.  [ ] = -8.7° (c = 0.21, CH2Cl2).
After 13 h, aqueous hydrochloric acid solution (2 M, 100 mL) and dichloromethane (60 mL) were added. The layers were separated and the aqueous layer was extracted with dichloromethane (2 × 100 mL). The combined organic extracts were dried over magnesium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (20% ethyl acetate in hexanes) to yield aldehyde S33 (1.72 g, 70%) as a colorless solid. The obtained analytical data were in full agreement with those previously reported.

Phenol S39
A solution of hydrochloric acid (~1.25 M in methanol, 12 mL) was added to a solution of S37 (145 mg, 269 µmol, 1 equiv) in dichloromethane (6 mL) and the resulting solution was heated to 35 °C. After 1 h, the reaction mixture was diluted with dichloromethane (25 mL) and saturated aqueous sodium bicarbonate solution (25 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 30 mL).
The combined organic extracts were dried over magnesium sulfate, the dried solution was filtered and the filtrate was concentrated to yield S38 as a yellow oil that was directly used in the following reaction without further purification. [ ] = +73.6° (c = 1.48, CH2Cl2).

epi-Smenoqualone (50) Bromide S42
Benzaldehyde S40 24,25 (1.48 g, 5.38 mmol, 1 equiv) was added to a solution of selenium dioxide (47.8 mg, 430 µmol, 8.0 mol%) and hydrogen peroxide (30% in water, 1.21 mL, 11.8 mmol, 2.20 equiv) in dichloromethane (30 mL). After 14 h, saturated aqueous ammonium chloride solution (40 mL) was added, the layers were separated and the aqueous layer was extracted with dichloromethane (3 × 30 mL). The combined organic extracts were dried over magnesium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was dissolved in methanol (40 mL) and an aqueous solution of potassium carbonate (14%, 10 mL) was added. After 1 h, the reaction mixture was extracted with dichloromethane (3 × 30 mL), the combined organic extracts were dried over magnesium sulfate, the dried solution was filtered and the filtrate was concentrated to give crude S41 (924 mg) as a yellow oil that was used without further purification.
To a solution of crude S41 (924 mg, 3.51 mmol, 1 equiv) in N,N-dimethylformamide (12 mL) was added sodium hydride (60% mineral oil dispersion, 211 g, 5.27 mmol, 1.50 equiv) at 0 °C. After 1 h, bromomethyl methyl ether (344 µL, 4.21 mmol, 1.20 equiv) was added and the reaction mixture was allowed to warm to 23 °C. After 1.5 h, water (20 mL) was added and the mixture was extracted with diethyl ether (3 × 20 mL). The combined organic extracts were washed with saturated aqueous sodium chloride solution (40 mL). The washed solution was dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (20 ethyl acetate in hexanes) to provide S42 (510 mg, 48% over

Arene S43
A mixture of aryl bromide S42 (503 mg, 1.64 mmol, 1 equiv), sodium methoxide (177 mg, 3.28 mmol, 2.00 equiv), copper(I) chloride (6.49 mg, 65.5 µmol, 0.04 equiv) and formic acid methyl ester (40.6 µL, 65.5 µmol, 0.40 equiv) in methanol (1 mL) was heated to 120 °C in a pressure tube. After 13 h, the reaction mixture was cooled to 23 °C, dichloromethane (10 mL) and saturated aqueous ammonium chloride solution (10 mL) were added. The layers were separated and the aqueous layer was extracted with dichloromethane (2 × 10 mL). The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was purified by flash-column chromatography on silica gel (20% ethyl acetate in hexanes initially, grading to 50% ethyl acetate in hexanes) to provide S43 (350 mg, 83%) as a colorless oil. were added, the layers were separated and the aqueous layer was extracted with ethyl acetate (3 × 30 mL).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was filtered through a short plug of silica and the obtained crude xanthogenate was used without further purification.

Phenol S47
A solution of hydrochloric acid (~1.25 M in methanol, 3 mL) was added to a solution of S45 (24.4 mg, 52.7 µmol, 1 equiv) in dichloromethane (1 mL) and the resulting solution was heated to 30 °C. After 4 h, the reaction mixture was diluted with dichloromethane (5 mL) and saturated aqueous sodium bicarbonate solution (3 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 5 mL).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated to yield S46 as a yellow foam that was directly used in the following step.
The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 5 mL). The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was  [ ] = +45.8° (c = 1.46, CH2Cl2).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated. [ ] = +492.3° (c = 0.16, CH2Cl2).
The layers were separated and the aqueous layer was extracted with dichloromethane (2 × 20 mL). The combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to yield S46 as a yellow oil that was directly used in the following reaction. [ ] = +54.9° (c = 0.12, CH2Cl2).
The combined organic extracts were dried over sodium sulfate, the dried solution was filtered and the filtrate was concentrated. The residue was filtered through a short plug of silica and the obtained residue was used without further purification.
Note: benzene was degassed via freeze-pump-thaw (three cycles) prior to use.

(-)-Mamanuthaquinone (6)
A solution of hydrochloric acid (~1.25 M in methanol, 3 mL) was added to a solution of 37 (33.0 mg, 76.3 µmol, 1 equiv) in dichloromethane (1 mL) and the resulting solution was heated to 30 °C. After 5 h, the reaction mixture was diluted with dichloromethane (5 mL) and saturated aqueous sodium bicarbonate solution (5 mL) was added. The layers were separated and the aqueous layer was extracted with dichloromethane (3 × 5 mL).
The combined organic extracts were dried over sodium sulfate. The dried solution was filtered and the filtrate was concentrated to yield S60 as a brown foam that was directly used in the following reaction.