Neutral nitrogen concentrate

The cells were incubated with 8 x 10 M of pyrazofurin for one hour at 37 C. This concentration is about 10 times that needed for the inhibition of 90% of the activity of orotidylate decarboxylase in L1210 cells (15). After incubation, cells were washed twice with 0.15 M NaCl to remove free pyrazofurin. Next 5 volumes of 1 N perchloric acid was added to the cell pellets and centrifuged for 15 min to remove the precipitate. The acid-soluble portion of the extract was neutralized with concentrated KOH and centrifuged to remove the precipitated potassium perchloride. The resulting extract was evaporated to total dryness under nitrogen stream (dry, room temperature), and the resulting residue (somewhat brownish in appearance, probably due to some charring) was derivatized as described later. 

A total of 3 g (0.13 moles) of sodium hydride is added to a solution consisting of 10 g of 17 -hydroxy-5a-androstan-3-one (36 mmoles) in 200 ml of benzene and 10 ml of ethyl formate. The reaction mixture is allowed to stand under nitrogen for 3 days followed by dropwise addition of 10 ml of methanol to decompose the excess of sodium hydride. The solution is then diluted with 300 ml water and the layers are separated. The basic aqueous solution is extracted with ether to remove neutral material. The aqueous layer is acidified with 80 ml of 3 A hydrochloric acid and the hydroxymethylene steroid is extracted with benzene and ether. The combined organic extracts are washed with water and saturated sodium chloride solution and then dried over magnesium sulfate and concentrated. The residue, a reddish-yellow oil, crystallized from 10 ml of ether to yield 9.12 g (83%) of 17 -hydroxy-2-hydroxymethylene-5a-androstan-3-one mp 162-162.5°. Recrystallization from chloroform-ether gives an analytical sample mp 165-165.5° [a]o 53° (ethanol) 2 ° 252 mjj. (g 11,500), 307 m u (e 5,800). 

A mixture of 50 g of betamethasone, 50 cc of dimethylformamide, 50 cc of methyl orthobenzoate and 1.5 g of p-toluenesulfonicacid Is heated for 24 hours on oil bath at 105°C while a slow stream of nitrogen is passed through the mixture and the methanol produced as a byproduct of the reaction is distilled off. After addition of 2 cc of pyridine to neutralize the acid catalyst the solvent and the excess of methyl orthobenzoate are almost completely eliminated under vacuum at moderate temperature. The residue Is chromatographed on a column of 1,500 g of neutral aluminum oxide. By elution with ether-petroleum ether 30 g of a crystalline mixture are obtained consisting of the epimeric mixture of 170 ,21 -methyl orthobenzoates. This mixture is dissolved without further purification, in 600 cc of methanol and 240 cc of methanol and 240 cc of aqueous 2 N oxalic acid are added to the solution. The reaction mixture is heated at 40°-50°C on water bath, then concentrated under vacuum. The residue, crystallized from acetone-ether, gives betamethasone 17-benzoate, MP 225°-231°C. 

The organic phase is dried over sodium sulfate and then concentrated to dryness. The thus obtained crude 6-chloro-1a-chloromethyl-A -pregnadiene-17o-ol-3,20-dione-17-acetate is heated to boiling in 20 cc of collidine for 20 minutes under nitrogen. After dilution with ether it is washed with 4N hydrochloric acid and washed with water until neutral. 

The reaction velocity is comparatively slow, but increases with increasing concentration of acid. The addition of three drops of a neutral 20 per cent ammonium molybdate solution renders the reaction almost instantaneous, but as it also accelerates the atmospheric oxidation of the hydriodic acid, the titration is best conducted in an inert atmosphere (nitrogen or carbon dioxide). 

A solution containing 447 mg (1.38 mmol) of 3-(4-mcthylphenylsulfonyl)-1-nonene, 928 mg (3.19 mmol) of tributyltin hydride, and 10 mg of AIBN. in dry benzene is heated under reflux under an atmosphere of nitrogen for 2 h. Concentration under reduced pressure and chromatography on neutral alumina using benzene as eluant gives the product yield 443 mg (65%) further purification by distillation, bp 136-142X/0.003 Torr. 

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