Total Synthesis of Aetokthonotoxin, the Cyanobacterial Neurotoxin Causing Vacuolar Myelinopathy

Abstract Aetokthonotoxin has recently been identified as the cyanobacterial neurotoxin causing Vacuolar Myelinopathy, a fatal neurologic disease, spreading through a trophic cascade and affecting birds of prey such as the bald eagle in the USA. Here, we describe the total synthesis of this specialized metabolite. The complex, highly brominated 1,2’‐biindole could be synthesized via a Somei‐type Michael reaction as key step. The optimised sequence yielded the natural product in five steps with an overall yield of 29 %.

used for high-resolution mass spectrometry, which was equipped with an ESI-DuoSpray-Ion-Source (operating in positive ion mode) and controlled by Analyst 1.7.1 TF software (Sciex).
The ESI source operation parameters were as follows: ion spray voltage: 5.500 V, nebulizing gas: 60 p.s.i., source temperature: 450 °C, drying gas: 70 p.s.i., curtain gas: 35 p.s.i. Data acquisition was performed in the MS1-ToF mode, scanned from 100 to 1500 Da with an accumulation time of 50 ms. Melting points were determined with a Leica DM LS2 microscope.

Supporting Information 3 of 36
Synthesis and characterization of building blocks

Synthesis of 2,4-dibromo-1-nitrosobenzene (7)
Following a reported protocol, 1 ammonium persulfate (4.6 g, 0.02 mol) and sulfuric acid (6.6 g, 0.067 mol) are stirred at room temperature for 1 h. This mixture is poured into 40 g of ice, after which 120 ml of water are added, followed by 2,4-dibromoaniline 7 (1.0 g, 0.04 mol). The mixture is allowed to reach room temperature and stirred for 4 h. The resulted precipitate is filtered off, washed with water, and dried in a desiccator to afford pure 2,4dibromo nitrosobenzene 8 (0.65 g, 61%) as a brown solid. 1

Synthesis of propiolonitrile (9)
Propiolamide (1.0 g, 14.5 mmol) and P 2 O 5 (8.2 g, 5.8 mmol) are carefully mixed and heated to 130 °C, as described in the literature. 2 Propiolonitrile 9 (0.41 g, 56%) is collected by distillation as a white solid at -78 °C. Caution: This product is a severe vesicant, it penetrates globes and causes painful burns and blistering! 1 H NMR (400 MHz, CDCl 3 ): δ = 2.52 (s, 1H). 13    Supporting Information 6 of 36 Attempts to "Penoni" indole synthesis with 2,4-dibromo-1-nitrosobenzene (7) The Penoni cycloaddition has been extensively explored for the construction of N-OMe indoles using various aryl alkynes in moderate to excellent yields. 3 The use of alkynes with electron withdrawing groups has been limited to methyl propiolate only. Therefore, we envisioned propiolonitrile 9 could be used for the cycloaddition with brominated nitrosobenzenes and generate in only one step the desired 5,7-dibromo-1-methoxy-1H-indole-3-carbonitrile 2. Thus, 2,4-dibromo-1-nitrosobenzene 8 (100 mg, 0.38 mmol) was reacted with propiolonitrile 9 (385 mg, 7.55 mmol) in the presence of K 2 CO 3 (312 mg, 2.3 mmol) and (CH 3 ) 2 SO 4 (191 µL, 2.3 mmol) according to the conditions described. 3 Unfortunately, a very complex mixture of products was obtained with no sign of the desired product. Changing the reaction conditions by using different solvents, higher temperatures or even microwave heating, did not show any improvement. While methyl propiolate or propiolamide have been reported to generate the corresponding indole derivative, 3 the nitrile functionality can be obtained by functional group transformations thereof, subsequently. However, none of these alkynes efficiently reacted with the 2,4-dibromo-1-nitrosobenzene 8. In the reaction with propiolamide, there was no sign of product formation. Only with methyl propiolate, the cycloaddition was achieved in 18% yield (NMR of the cycloaddition product detailed as follow). With these results, the present strategy was discarded. 1

Synthesis of 5,7-dibromo-1-methoxy-1H-indole (4)
The following procedure is an adaptation from a previous report. 4 Na 2 WO 4 ·2H 2 O (0.36 g, 1.1 mmol) dissolved in water (5.0 mL) is added under stirring to a solution of 2,4dibromoindoline 5 (1.5 g, 5.4 mmol) in MeOH/THF 3:1 (40 mL) at 5 °C. 30% H 2 O 2 (6.1 mL, 54 mmol) is added over 5 min to the previous solution, and the resulting homogeneous mixture is vigorously stirred at 0 °C for 15 min and then at 10 °C for additional 30 min. The reaction turns from violet to light yellow. By combination of TLC and ESI-MS, the formation of the corresponding N-hydroxylated derivative (Rf (toluene) = 0.29) is checked. Additional Na 2 WO 4 · 2 H 2 O (0.283, 0.84 mmol) and 30% H 2 O 2 (5 mL) are added, and the reaction is stirred at 10 °C for another 45 min. After ensuring complete consumption of the starting material, dimethyl sulfate (2.3 mL, 27 mmol) is added, followed by K 2 CO 3 (3.0 g, 21.7 mmol) and the reaction is stirred for 1 h, checking the reaction progress by TLC. In case of any sign of precipitation, further THF (5-10 mL) is added, until the reaction mixture gets homogeneous again. After this time, analysis by TLC shows no starting material and confirms the formation of the desired product Rf (n-hex/DCM 2:1) = 0.54). Then, brine (200 mL) is added, and the crude is extracted with CHCl 3 (3×40 mL). The combined organic phases are washed with brine, dried over Na 2 SO 4 , and evaporated under reduced pressure to leave a yellowish syrup when cooled below 30 °C. This crude is purified by column chromatography (n-hex/DCM 5:1) to give 5,7-dibromo-1-methoxy-1H-indole 4 (1.2 g, 72%) as a white amorphous solid. Rf

General procedure for Somei-Michael reaction
Indole derivative 3 (0.3 mmol) is dissolved in dry DMF (5 mL) and the solution is cooled at 0 °C. NaH (0.33 mmol, 1.1 equiv) is added in portions over 10 min and the mixture is stirred for 1 h. Maintaining the temperature at 0 °C, indole derivative 2 (0.36 mmol, 1.2 equiv), previously dissolved in DMF (2 mL) is slowly added and the reaction mixture is stirred for 12 h at room temperature. The reaction is quenched with sat. soln. of NH 4 Cl, diluted with AcOEt (100 mL), washed with brine, and dried over Na 2 SO 4 . After removing the solvent by evaporation at reduced pressure, the crude mixture obtained is triturated with MeOH to dissolve excess of starting material and byproducts to afford the insoluble biindole ~95% pure as a colorless solid in 50-70% yield.

Synthesis of the biindole 13b
Following the general procedure for the Somei coupling described above, indole 2 (100 mg, 0.30 mmol) and 3b (83 mg, 0.36 mmol) are combined with 60% NaH in mineral oil (

Failed attempts to synthesize AETX (1) by direct coupling of 2 and 3c
Following the general procedure for the Somei coupling described above, indole 2 (100 mg, 0.30 mmol) and 3c (83 mg, 0.36 mmol) are combined with 60% NaH in mineral oil (13.3 mg, 0.33 mmol) in DMF (5 ml). Unfortunately, the expected biindole was not obtained despite total consumption of the starting material 2. Instead, ESI-MS analysis of the reaction crude reveals the formation of the indole species 14. This byproduct corresponds to the auto decomposition of 2, when the methoxide anion, initially generated by a reductive cleavage of N-OMe with NaH, is the nucleophile engaging in the Michael addition. This side reaction is

Synthesis of biindole 16
Indoles 2 (200 mg, 0.60 mmol) and 3a (142 mg, 0.72 mmol) are combined with 60% NaH in mineral oil (26.6 mg, 0.66 mmol) in DMF (10 ml) according to the general procedure described above. However, instead of quenching the reaction with NH 4 Cl, SEM-Cl (0.26 mL, 1.5 mmol) was added, followed by Cs 2 CO 3 (0.39 g, 1.2 mmol), and the reaction is stirred for additional 12 h at room temperature. Then, the reaction is quenched with sat. soln. of NH 4 Cl, diluted with AcOEt (100 mL), washed with brine, and dried over Na 2 SO 4 . After removing the solvent by evaporation at reduced pressure, the crude is purified by column chromatography

Supporting Information 26 of 36
Bromination of biindoles

Bromination studies of 13a and 13b
Attempts to brominate simultaneously C-2 and C-3 in 13a or only C-2 in 13b were performed using different brominating agents in THF or DMF as solvents. The insolubility of these biindoles in nonpolar solvents such as CCl 4 , CHCl 3 , DCM, and DCE considerably limited the bromination studies. Test reactions (20 mg scale) were performed using protocols with the following reagents: NBS, 9 DBDMH, 10 PyHBr 3 , 11 and TMSBr-DMSO 12 . The progress of the reactions and, therefore, the consequent incorporation of bromine atoms was analyzed by ESI-MS in negative mode, due to the facile ionization of the NH group. As a result, it was concluded, that the mild brominating agent PyHBr 3 (3 equiv) in DMF efficiently transforms 13a to 13b. However, no further bromine atom is incorporated even when running the reaction for more than 24 h. Unfortunately, similar outcomes were observed when using TBSBr-DMSO (3 equiv each) in THF at room temperature. The use of DMBDH (3 equiv) in THF at room temperature also permits the formation of 13b, although additional side reactions beside bromination arise when leaving the reaction to run for a longer time.
Experiments using NBS are detailed as follows:

Bromination using NBS in THF
The method based on using NBS in THF at -78 ºC was initially considered the most promising attempt, based on the success in C-2,3 simultaneous dibromination of indole species achieved by Langer et al. 8 Moreover, the same procedure was used by Mathey et al.
for the dibromination of an also sterically hindered N-Aryl-Indole in 89% yield. 13 Accordingly, 13a (20 mg, 40 µmol) was treated with NBS (22 mg, 120 µmol, 3 equiv) reproducing the conditions reported. ESI-MS analysis after 1 h, 2 h, 3 h, and 4 h of reaction reveals that only one bromine atom is incorporated with the consequent formation of 13b. By warming the reaction up to 0 ºC and performing the reaction for longer time, it was evidenced by ESI-MS analysis that another bromo substitution took place. After 6 h at room temperature, the reaction was stopped because a hexabrominated biindole species was starting to emerge. The reaction was quenched with sat. soln. Na 2 S 2 O 3 , diluted with AcOEt (100 mL) and washed successively with sat. soln. of NaHCO 3 , and brine. Then, after drying the organic solution over Na 2 SO 4 , it is evaporated at reduced pressure, the crude is triturated with MeOH to afford the insoluble pentabrominated biindole (17 mg, 69% yield, 85% purity).
Unfortunately, the 1 H NMR of the compound obtained confirmed the formation of the biindole 15 instead of 1, due to an unexpected regioselective bromination of the position C-6' rather than C-2. The 1 H NMR proton signal at C-4' allows easy discrimination between 15 and 1. This signal resonates at the lowest field among other proton signals due to the anisotropy effect of the nitrile moiety. The appearance of a singlet in 15 confirmed the substitution at C-6', since for 13a and 13b this signal appears as a meta-coupled doublet (J ~ 2 Hz) as also observed for 1.