Separation and washing

Phenyl-3-oxopropanoic acid (25 mmol) and EtjN (87.5 mmol) were dissolved in THF (150 ml) and cooled to —40°C. Ethyl chloroformate (27.5 mmol) was added dropwise to this solution and then the reaction mixture was stirred for 30 min at —20°C. Di-n-hexylamine (27.5 mmol) was added to the suspension and it was stirred at room temperature for an additional hour. The reaction mixture was diluted with water (100 ml) and extracted with ether (400 ml). The extract was washed with aq. 5% HCl (100 ml) and brine (2 X 100 ml) and dried over NajSO. The crude amide was obtained by removal of the solvent in vacuo and phenylhydrazine (25 mmol) was added. The mixture was heated to 100°C for 30 min. The residue was held in vacuo to remove the water formed and then powdered ZnCl2 (125 mmol) was added. The mixture was heated at 170"C with manual stirring for 5 min. The cooled residue was dissolved in acetone (100 ml) and diluted with ether (500 ml). Water (100 ml) was added. The organic layer was separated and washed successively with 5% aq. HCl (100 ml) and brine (2 x 100 ml) and dried over NajSO. The solvent was removed in vacuo, and the residue was recrystallized from EtOAc-hexane. The yield was 79%. 

Such a reaction is controlled by the rate of addition of the acid. The two-phase system is stirred throughout the reaction the heavy product layer is separated and washed thoroughly with water and alkaU before distillation (Fig. 3). The alkaU treatment is particularly important and serves not just to remove residual acidity but, more importantiy, to remove chemically any addition compounds that may have formed. The washwater must be maintained alkaline during this procedure. With the introduction of more than one bromine atom, this alkaU wash becomes more critical as there is a greater tendency for addition by-products to form in such reactions. Distillation of material containing residual addition compounds is ha2ardous, because traces of acid become self-catalytic, causing decomposition of the stiU contents and much acid gas evolution. Bromination of alkylthiophenes follows a similar pattern. 

Bone cleaning is the second stage of en2ymatic extraction. The soHd bone fraction from the first separation is mixed 1 1 with hot water (65—75°C) and treated with alkaline-type proteases. After a reaction time of one hour, the bones are separated and washed with water. The cleaned bones make an excellent raw material for the production of gelatin. 

After the addition of 2 1. of water, the mixture is steam-distilled as long as any oil comes over. The crude, heavy, yellow oil is separated and washed with two 200-cc. portions of 10 per cent sodium hydroxide, once with 100 cc. of water, twice with 150-cc. portions of concentrated sulfuric acid, and finally with 100 cc. of 5 per cent, sodium carbonate solution. It is dried with about 5 g. of calcium chloride, filtered through glass wool, and distilled using a long air condenser. Most of the product boils at i8o-i83°/75o mm. The yield of pure colorless material, b.p. i83°/76o mm., is 125-135 g. (36-39 per cent of the theoretical amount, based on the amount of -toluidine originally used, or 54-59 per cent based on the amount of 3-bromo-4-amino-toluene). 

B. (l-Azido-3,3-dimethoxy-l-propenyl)benzene. In a 2-1., one-necked, round-bottomed flask equipped with a magnetic stirrer and powder funnel are placed 156 g. (0.45 mole) of the iodoazide from Part A and 1500 ml. of anhydrous ether. The solution is stirred and cooled in an ice-salt bath (— 5° to 0°), and 62 g. (0.55 mole) of potassium <-butoxide (Note 6) is added. The powder funnel is then replaced by a calcium chloride drying tube and the mixture is stirred for 4 5 hours at 0°. At the end of this time 350 ml. of water is added while the mixture is still cold. The ethereal layer is then separated and washed with three 350-ml. portions of water and dried over magnesium sulfate. The solvent is removed with a rotary evaporator without heating, leaving 67-75 g. (68-76%) of (l-azido-3,3-dimethoxy-l-propenyl)-benzene as a dark oily liquid (Note 7). This material can be used without further purification for Part C (Note 8). 

The benzene solution is separated and washed twice with 100 cc. of water, once with 200 cc. of 5 per cent potassium hydroxide, and twice with water, and in case the solution is not colorless it is clarified with i g. of Norite. The benzene is removed by distillation and the resulting syrup is dissolved in 300 cc. of alcohol by heating. The solution on cooling deposits cholestanone as well-formed needles. The yield of collected, washed, and air-dried material, m.p. 129-130°, is 41.5-42 g. (83-84 per cent of the theoretical amount). The addition of 80 cc. of water to the filtrate gives about 2 g. of material melting at 125-126°. 

The mixture is cooled to room temperature, then filtered. The solvent is removed under reduced pressure, leaving the tribromide (47) as a foam. The foam is mixed with sodium iodide (9.55 g, 0.064 mole) and acetone (74 ml) and heated under reflux in a nitrogen atmosphere for 3.5 hr. The acetone is removed under reduced pressure and the residue is treated with chloroform and aqueous sodium thiosulfate solution. The chloroform layer is separated and washed with sodium thiosulfate solution until it is free from iodine, then dried over magnesium sulfate, filtered and evaporated to dryness under reduced pressure. The crude product (48) is obtained as a brown sohd (4.85 g) which is chromatographed over alumina (122 g, Merck acid-washed). The column is developed with hexane, benzene and ethyl acetate mixtures. The product (3.43 g) is eluted by benzene and benzene-ethyl acetate (10 1). Recrystallization from acetone yields purified 3jS-acetoxy-pregna-5,14,16-trien-20-one (48), 3.25 g, mp 158-159° 309 m/ (e 10,700). 

The enol lactone (12.7 g) is added to 157 ml of 0.5 M perbenzoic acid in benzene and allowed to stand at 25° for 140 hr. The solution is cooled to 15° and 15% sodium bisulfite solution is added to neutralize the excess peracid. The organic layer is separated and washed with saturated sodium bicarbonate solution and water. The benzene solution is dried over anhydrous sodium sulfate, filtered and concentrated to 30 ml. The product is crystallized by adding 80 ml of petroleum ether, filtered and washed with petroleum ether to yield 12.8 g (98%) of 3a,20,23-trihydroxy-16a-methyl-17(20)-oxido-ll-oxo-21-norchol-22-enoic acid-24(20)-lactone 3,23-diacetate mp 225-227°. 

After an additional 10 min, a 1 % solution of hydrochloric acid (100 ml) is slowly added to the stirred reaction mixture and the resultant mixture is transferred to a separatory funnel. The ether layer is separated and washed sequentially with water, 5 % sodium bicarbonate solution, water and saturated salt solution. The washed ether solution is dried over anhydrous sodium sulfate, filtered, and evaporated to give an oily residue (0.45 g). Chromatography of the crude product on silica gel (50 g) followed by crystallization of the solid thus obtained (0.18 g) from ethanol gives 3 -hydroxy-B-homo-cholest-5-en-7a-one acetate (67 0.14 g) mp 90-91° [a]o 99° (CHCI3). 

The trialkylborane is oxidized by the addition to the stirred reaction mixture of 32 ml of a 3 solution of sodium hydroxide, followed by the dropwise addition of 32 ml of 30 % hydrogen peroxide at a temperature of 30-32° (water bath). The reaction mixture is saturated with sodium chloride and the tetrahydrofuran layer formed is separated and washed with saturated sodium chloride solution. The organic solution is dried over anhydrous magnesium sulfate and the THF is removed. Distillation affords 24.5 g (80%) of 4-methyl-1-pentanol, bp I51-153°/735 mm. 

Aldehydes are usually most easily separated from the essential oils in which they occur, by means of acid sodium sulphite. The oil—or the suitable fraction thereof— is well shaken for a time varying according to the nature of the aldehyde, with an equal volume of a saturated solution of sodium bisulphite, with a little ether added, in order to hinder the non-aldehydic portion of the oil from becoming occluded in the crystals of the bisulphite compound of the aldehyde. These crystals are separated and washed well with ether They are then decomposed by warming with a solution of sodium carbonate, and the regenerated aldehyde is extracted by means of ether. 

Paraformaldehyde (7.5 g) (0.25 mol) and 18.3 g (0.25 mol) of diethylamine are mixed in 25 cc of alcohol and warmed until a clear solution Is obtained. The solution is cooled and mixed with 26.6 g (0.10 mol) of 3,3 -diallyl-4,4 -biphenol in 25 cc of alcohol. After standing several hours, the solution is warmed for one hour on the steam bath, allowing the alcohol to boil off. The residue is then taken up in ether and water, the ether layer separated and washed with 2% sodium hydroxide solution and finally with water. The washed ether solution is dried over solid potassium carbonate, and filtered. After acidifying with alcoholic hydrogen chloride, the ether is distilled off and the alcoholic residue diluted with an equal volume of acetone. The crystalline hydrochloride is filtered off, triturated with alcohol, diluted with several volumes of acetone, filtered and dried MP 209°-210°C. 

The mixture was filtered, the ethyl acetate layer separated and washed with three 100 ml portions of water, dried over Na2S04, filtered and treated with 30 ml of sodium 2-ethyl-hexanoate in n-butanol (34 ml = 0.1 mol). The oil which settled out was scratched to induce crystallization. After stirring for 20 minutes the product, sodium 7-(a-bromoacet-amido)cephalosporanate, was scraped from the sides of the flask and collected. The filter cake was washed with several portions of acetone, air dried, and dried in vacuo over P Os. The yield was 22.5 g and decomposed at 193°C. 

To a solution of 35.3 parts of phenylacetonitrile and 47.6 parts of 2-bromopyridine in 175 parts of dry toluene is added 53.4 parts of sodamide slowly with stirring over a period of 45 minutes. The resultant mixture is stirred at 100 C for 2 hours before it is cooled and the excess sodamide is decomposed by the addition of water. The toluene layer is separated and washed with water to remove excess alkali. The toluene solution is extracted with 6N hydrochloric acid and the acid extract is made alkaline and then extracted with toluene. The toluene solution is dried over sodium sulfate and the solvent is evaporated. Recrystal-lization of the residue from alcohol-hexane gives a-phenvl-2-pyridineacetonitrile melting at about 87°-88°C. 

The toluene layer is separated and washed with water and extracted with 6N hydrochloric acid. The acid extract Is made alkaline and extracted with toluene. The toluene solution is washed with water and dried and the solvent is evaporated. Distillation of the residue gives 4-diisopropvlamino-2-phenvl-2-(2-pvridyl)-butvronitrile boiling at about 145 -160°C at 0.3 mm pressure. 

The reaction mixture is allowed to stand at room temperature for 60 to 70 hours under the exclusion of air humidity. Then the excess of the chloroformyl compound is hydrolyzed with crushed ice. Ethyl acetate is added and after shaking, the ethyl acetate solution is separated and washed with water, dried over sodium sulfate and evaporated in vacuo to dryness. 

The lower glycol layer was separated and washed with benzene. The benzene layer and the benzene washings were combined and the combined solution was divided into two equal portions, one of which was used for the isolation of 17a-hydroxyprogesterone 3,20-bis-(ethylene ketal) as follows. The benzene solution was washed with 5% sodium carbonate solution, water and saturated sodium chloride solution. After being dried over anhydrous magnesium sulfate the solution was concentrated to dryness at reduced pressure. 

To a slurry of sodamide in 200 cc of toluene representing 6.7 g of sodium was added at 30° to 40°C, 32.3 g (0.31 mol) of 2-aminopyridine. The mixture was heated to reflux temperature and was refluxed for VA hours. To the resulting mixture was added over a period of approximately one hour a solution of 32 g of freshly distilled N,N-dimethyl-i3-chloroethylamine in 40 to 50 cc of dry toluene. The reaction mixture was then haated for 2 hours at reflux temperature. Thereafter, 200 cc of water was added and the toluene layer was separated and washed with water. The toluene was stripped from the mixture by distillation and the residue was distilled under reduced pressure. The distillate was refractionated and the portion distilled at 93° to 103°C/1 mm was recovered. Yield of N-(2-pvridvl)-N, N -dlmethyl-ethvlenediamine, 60%. 

The aqueous layer Is separated and washed with ether, the ether extracted with 5% hydrochloric acid, the acidic solution is made basic with 10% sodium hydroxide and again extracted with ether. Since some spontaneous crystallization occurs in the ether, the solvent is removed without drying under vacuum and the residue is recrystallized from 35 ml of ethanol giving 18.0 g of 1-cyclopropylmethyl-5-phenyl-7-chloro-1 H-1,4-benzodiazepine-2(3H)-one, MP 145° to 146°C (65% yield), according to U.S. Patent 3,192,199. 

The mixture is cooled and the excess of lithium aluminum hydride is decomposed with cracked ice. The water layer is separated and washed with diethyl ether. The combined ether extracts are dried over anhydrous magnesium sulfate and the solvent is removed by distillation under reduced pressure. Yield, 8.8 g boiling point, 160°C to 165°C/0.1 mm Hg. 

CH2C12 (500 mL). The CH2C]2 phase was separated and washed withH2Q, dried (Na2S04)and evaporated under reduced pressure, The residue was dissolved in benzene (200 mL), absorbed on silica gel (10 g) (0.05 0.2 mm) and the silica gel was then washed with portions of benzene (100 mL). The benzene washings were combined and evaporated to give the product, which was dissolved in hexane, and the solution cooled to 0 C, whereupon the pure dibenz[ft,/]azcpinc crystallized. 

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