Muscimole and Other Isoxazoles

It appears likely that mushrooms were the source of man's first psychedelic experiences. The "soma" praised in the Rig Veda of ancient India (about 2,000 BC) is now thought to be a mushroom (the fly agaric, Amanita muscaria, and possibly other Amanita species). Much of the Eurasian religion and mythology may have had its origin in the psychedelic properties of these mushrooms. Probably all members of the genus Amanita are psychedelic, but ingesting them is unwise since there is probably not a great difference between the psychedelic and lethal dose of mushroom for some species. Amanita muscaria itself is seldom lethal but be very careful if you eat it. It is said to be active if dried, powdered, and smoked. In the USA it fruits in early summer and again in late summer (in fall and winter on the Pacific coast). Since muscimole is very water soluble, it can be extracted from the dried, powdered mushroom (or perhaps even fresh ones) by soaking in water (hot or even boiling water may be OK) and straining ùù but drink cautiously. Most of the muscimole is contained in the red "skin" and the underlying yellow tissue. Some Russian peoples regarded thorough drying of Amanita as a necessary preliminary to ingestion. It has recently been suggested that there are significant chemical differences between New and Old World A. muscaria.

The main psychedelic component of these mushrooms appears to be muscimole (5-aminomethyl-3-OH-isoxazole), which is active in an oral dose of about 10 mg. See HCA 48,920(1965) for extraction of muscimole from the fungus.

The value of muscimole as a psychedelic is diminished by the dizziness and muscle twitching which seem to occur (at least in some people), but a small dose in conjunction with another psychedelic should be very interesting. Ibotenic acid also occurs in Amanita, and though not itself a desirable psychedelic, it can be converted to muscimole by dissolving in dimethylsulfoxide or refluxing in water.

Its synthesis is generally more difficult than that of muscimole (see CA 65,2266(1966) and CPB 15,1025(1967), 19,46(1971)). Several routes for the synthesis of muscimole (also called agarin or pantherine) follow. For a good review of this subject see Prog. Chem. Org. Nat. Prod. 27,262(1969). Muscimole is probably legal everywhere.

Muscimole Synthesis

Method 1 Rend. Ist. Lomb. 93A,150(1959), 94A, 735(1960), JCS 172(1968)

Dissolve 100 g 1-Cl-3-Butanone (JACS 75,5438(1953)) in 300 ml dimethylformamide; stir and add 77.5 g NaNO2 with cooling. Stir five hours and let stand twelve hours. Add 300 ml water; extract with ether and dry, evaporate in vacuum the extract (can distill residue 86/2) to get 50 g 1-N02-3-butanone (I). Dissolve 18 g (I) in 90 ml 48% HBr and reflux ten minutes. Add 100 ml water and steam distill. Dry, extract with ether and dry, evaporate in vacuum the extract (can distill 67-8/18) to get 4.8 g 3-Br-5-methyl-isoxazole (II). 32.4 g (II) and 85.5 g KOH in 220 ml methanol and reflux twenty four hours (under N2 if possible). Add 1 L water, extract with 4x250 ml ether and dry and evaporate the combined ether extracts to get 13 g liquid 3-methoxy-5-methyl-isoxazole (III). Add 100 ml n-butyllithium in hexane (2 moles) with stirring over twenty minutes below -65ø to 22.6 g (III) in 100 ml dry tetrahydrofuran. Stir at -75ø one hour and pour onto a stirred mixture of 1 kg dry ice (solid CO2) and 1 L ether. Let stand twelve hours or until CO2 evaporates and extract with 3x200 ml 2N NaOH. Acidify with dilute HCl and extract with 3x100 ml ether. Dry and evaporate in vacuum the extract to get 25 g 3-methoxy-isoxazole-5-acetic acid (1V) (re-crystallize-petroleum ether-ethyl acetate). Dissolve 5.2 g (IV) in 100 ml 3% HCl in methanol and reflux three hours. Evaporate in vacuum and recrystallize-petroleum ether (or dissolve residual oil in ether, wash with NaHCO3 and dry, evaporate in vacuum) to get 4.5 g of the methyl ester (V) (can distill 80/0.1 ). Add 6.5 g (V) in 20 ml methanol to 3 g hydrazine hydrate in 20 ml methanol and reflux four hours. Evaporate in vacuum and dry in exsiccator over concentrated sulfuric acid for twelve hours to get 6 g 3-methoxy-isoxazole-5-acetohydrazide (VI) (recrystallize-ethyl acetate). Dissolve 5.13 g (VI) in 50 ml dry tetrahydrofuran and treat with 50 ml 1.75 N HCl in tetrahydrofuran. Cool to -40ø and add over ten minutes with stirring 5 ml tertiary butyl nitrite in 20 ml tetrahydrofuran (temperature rises to about 10ø). Evaporate in vacuum and dissolve the yellow oil in 100 ml ethyl acetate. Wash with 50 ml NaCl containing 1% NaHCO3 and dry, evaporate in vacuum to get the azide. Dissolve the azide in 50 ml toluene and heat at 100ø until N2 evolution stops (about thirty minutes). Add 10 ml ethanol and reflux two hours. Evaporate (room temperature/15) to get 4 g 5-(ethoxy-carbonylamino)- methyl-3-methoxy-isoxazole (VII) (can distill 129/0.2). Test for psychedelic activity. Dissolve 4 g (VII) and 2.5 g KOH in 12 ml ethanol and reflux eight hours. Dissolve in 20 ml water, acidify with dilute HCl and evaporate to dryness. Dissolve residue in hot ethanol and evaporate to get 3 g 5-aminomethyl-3-methoxy-isoxazole (VIII). Test for activity. 1 g (VIII) in 10 ml glacial acetic acid and 4.5 g HBr and reflux one hour. Evaporate in vacuum to get muscimole.

Method 2 JACS 62,1147( 1940), TL 2077( 1963), CA 65,2267 (1966)

Pass chlorine gas into an ice cold, well-stirred solution of 5 ml acetylketene in 30 ml CCl4 until there is a 4.5 g increase in weight (solution is slightly yellow). Pour slowly into excess methanol or ethanol at 0ø and distill at 118/17 to get 6 ml methyl (or ethyl)-4-Cl-acetoacetate (I). To 2.7 ml methanol saturated with dry HCl at 0ø, add a mixture of 10 g (I), 20 g methyl orthoformate (trimethoxymethane) and 13 g methanol and reflux four hours. Pour hot into 200 ml ice water and adjust pH to 8 with 30% NaOH. Extract four times with ether and evaporate and distill to get methyl (or ethyl)-4-Cl-3,3-dimethoxy-butyrate (II). Dissolve 40 g (II) in 20 ml methanol and add hydroxylamine-HCl in methanol. After ninety-six hours at room temperature (under N2 if possible), evaporate in vacuum. Can purify the residue by dissolving in water and put on anionic column; wash column to neutrality and elute with 2N acetic acid; just before the acid elutes, the alkaline fraction giving a positive FeCl3 test appears; evaporate in vacuum this fraction below 40ø and dry at 40/0.5 for twelve hours. Dissolve 5 g product in 130 ml glacial acetic acid and saturate at room temperature, then at Oø with dry HCl. Let stand sixteen hours at room temperature and evaporate in vacuum at 40øC. Dilute with water and evaporate three times. Extract with 2x130 ml hot ether and filter, evaporate in vacuum to get 3-Cl-methyl-5-OH-isoxazole (III) (recrystallize in acetone). Heat (III) sixteen hours at 90ø in concentrated NH4OH in autoclave and evaporate to get muscimole.

Method 3 GCI 91,61 (1961), JCS 172(1968)

To 1 g 3-CI-5-methyl-isoxazole in 14 ml 1.4 N NaOH add with stirring 1.1 g KMnO4 in 20 ml water. Filter, decolorize with NaHSO3 and acidify with dilute sulfuric acid. Extract with ether and dry, evaporate in vacuum to get 0.8 g 3-Cl-isoxazole-5-COOH (I). To 2.5 g (I) in 7.5 ml water add 15 g KOH in 43 ml methanol and reflux four hours (under N2 if possible). Cool, acidify with concentrated HCl and extract with ether five times. Dry and evaporate in vacuum to get 2 g oil which precipitates to give 3-methoxy-isoxazole-5-COOH (11). Dissolve 5.2 g (ll) in 100 ml 3% HCl in methanol and reflux three hours. Evaporate in vacuum and recrystallize-petroleum ether (or dissolve residual oil in ether, wash with NaHCO3 and dry, evaporate in vacuum) to get the methyl ester (III). Add with stirring 0.8 g (III) to 40 ml aqueous NH3 (density about 0.88) and stir thirty minutes at room temperature. Filter, wash with cold water and dry to get 0.5 g 3-methoxy-isoxazole-5-carboxamide (IV). Dissolve 37.8 g NaBH4 in 100 ml diglyme (dimethyl ester of diethylene glycol) and 23 ml BF3 etherate in diglyme; add to 4.6 g (IV) in 100 ml tetrahydrofuran and reflux forty-eight hours. Add HCl, evaporate in vacuum and dissolve residue in water. Basify with 50% KOH and extract with ether. Dry, filter, evaporate in vacuum or treat with HCl to give (VIII) of method 1 which converts to muscimole as described.

Method 4 CA 65,2266(1966)

Dissolve 626 g 2-(2-nitrovinyl) furan in 3.13 L 48% HBr and 6.26 L glacial acetic acid and heat on steam bath nine hours. Evaporate in vacuum to 3 L; add 3 L water; boil; decolorize; cool and filter. Extract filtrate with 2L CHCl3 and evaporate in vacuum the extract. Add residue to precipitate. Filter out to get 3-Br-isoxazole-5-proprionic acid (I) (recrystallize-benzene-cyclohexane). Dissolve 44 g (I) in 440 ml concentrated sulfuric acid and add 80 g CrO3 in 80 ml water dropwise over three hours at 15-20ø. Pour onto 800 g ice and extract the aqueous solution with 3x500 ml ether. Evaporate in vacuum to get 3-Br-isoxazole-5-COOH (II) (recrystallize-benzene-toluene). Dissolve 30.7 g (II) and 27 g KOH in 500 ml methanol and stir two hours at 140ø. Cool, add 1.5 L water and extract with 3X 1.5 L ether. Can decolorize aqueous solution by boiling briefly with carbon, then filtering and acidifying with concentrated HCl. Filter and recrystallize-benzene to get 3-methoxy-isoxazole-5-COOH, which is the analog of (IV) in method 1 and is identical to (II) in method 3 and can be converted to muscimole by either method.

Method 5 Rend. Ist. Lomb. 94A,737(1960)

This method gives a somewhat lower yield than method 1. Dissolve 14.1 g 1-nitro-3-butanone in 30 ml glacial acetic acid and heat to 35ø; add slowly with stirring to a solution of 5.3 g bromine in 10 ml glacial acetic acid. Evaporate in vacuum, dissolve residue in ether and wash with water, NaHCO3 and water. Dry and evaporate in vacuum the ether (can distill 84/2) to get 6 g 1-nitro-4-Br-3- butanone (I). Add 4.4 g (I) to 25 ml 48% HBr and reflux three hours. Add 50 ml water and steam distill. Neutralize the distillate with K carbonate and extract with ether. Dry and evaporate the extract (can distill 128/20) to get 5.6 g 2-Br-5-Br-methyl-isoxazole (II). To a warm solution of 5.2 g urotropine (hexamethylenetetramine) in 45 ml CHCl3, add 8 g (II) in 10 ml CHCl3 and let stand four hours at room temperature. Filter, wash with CHCl3 and dry. Dissolve 2.3 g precipitate in 10 ml concentrated HCl and let stand two hours at room temperature. Add 30 ml water, basify with NaOH and extract with ether. Precipitate with dry HCl gas to get 3-Br-5-aminomethyl-isoxazole (III). Test for activity. Dissolve 8.8 g (III) and 5.6 g KOH in 60 ml methanol and reflux thirty hours. Dissolve in 100 ml water, acidify with dilute HCl; evaporate in vacuum and dissolve residue in hot methanol to get 3-methoxy-5-aminomethyl-isoxazole (IV). Test for activity. Convert (IV) to muscimole as described in method 1 or as follows: reflux in concentrated HCl four hours and evaporate in vacuum (recrystallize-methanol:tetrahydrofuran 1:1). Can add tetraethylamine to precipitate.

Method 6 (best yield) CPB 19,46(1971), JCS 121,1638(1922)

To prepare diethylacetone dicarboxylate (I) proceed as in method 3 for cocaine synthesis, substituting 720 ml ethanol for methanol. To 197 g (I), add rapidly with stirring and cooling 208 g PCl5, keeping temperature below 50ø. When HCl evolution stops, pour into water and add ice as necessary to cool. Extract the red oil with ether and dry, evaporate in vacuum. Boil residue 2 1/2 hours with 20% HCl, evaporate the water and dissolve the residue in ether. Dry and evaporate in vacuum to get 100 g 3-Cl-glutaconate (II). 100 g (II), 300 g ethanol, 50 ml sulfuric acid and reflux while passing in ethanol vapor until 1.5 L collects. Add water, extract with ether, wash with aqueous Na carbonate and dry, evaporate in vacuum to get 113 g diethyl-3-CI-glutaconate (Ill). Mix 28 g NaOH, 140 ml water, 10.4 g hydroxylamine-HCl, 280 ml ethanol and cool to -35ø in acetone-dry ice bath. Stir and add 30.3 g (III) in 50 ml ethanol over two minutes; stir one hour (can let stand twelve hours). Neutralize with 60 ml concentrated HCl while cooling in ice bath. Evaporate in vacuum and then add 62 ml dry ethanol, 130 ml benzene and 1/2 ml concentrated sulfuric acid to residue and reflux twenty-four hours to remove 80 ml of the azeotrope. Cool, pour into 60 ml ice water and separate the organic layer. Extract the aqueous layer with 30 ml benzene and add to organic layer. Wash organic layer with 2x15 ml NaHCO3 (2%), 2x30 ml water and dry, evaporate in vacuum to get 14 g ethyl-3-OH-5-isoxazoleacetate (IV) (recrystallize-ether). Add 3.36 g (IV) portionwise to 1.35 g hydrazine hydrate which has been heated at 94ø on water bath and heat 3 hours. Cool, filter and recrystallize in methanol to get 2.6 g of the hydrazide (V) (concentrate filtrate to get more). Dissolve 3.41 g (V) in 30 ml tetrahydrofuran and mix with 6.55 ml of a solution of dry HCl in tetrahydrofuran (9.94M). Cool to -50ø in dry ice-acetone bath and add dropwise 2.81 g isoamyl nitrite in 10 ml tetrahydrofuran over five minutes. Remove cooling bath, let stand fifteen minutes and stir fifteen minutes and filter. Evaporate in vacuum the liquid, dissolve the residue in 80 ml ethyl acetate; wash with 2x20 ml 1:9 mixture of saturated aqueous NaHCO3 and saturated aqueous NaCl and dry, evaporate in vacuum. Dissolve oily residue in 40 ml dry ethanol and reflux on oil bath forty-five minutes. Cool, evaporate in vacuum, filter and wash with benzene to get 1.2 g 3-OH-5-(carbeth-oxyamino)-methyl-isoxazole (VI) (recrystallize-benzene). 3.83 g (VI) in 80 ml 20% Ba(OH)2. Reflux fifteen hours and evaporate in vacuum (or cool and acidify with 10% sulfuric acid, filter, and evaporate in vacuum) to get muscimole (recrystallize-methanol).

Other References

The following papers describe the preparation of various muscimole analogs which may have activity: CJC 48,3753(1970); TL 21 O1 (1970); JOC 35,1806(1970); JMC 13,738(1970); BSC 2685(1970). For a simple two-step synthesis of 3-OH-5-methyl-isoxazole (which may substitute for (III) in method 1) see CA 74,76407(1971) (cf. CA 74,12552 I (1971)). The antibiotic Oxamycin (cycloserine) is often psychedelic (see Merck Index for synthesis).

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