Phosphorofluoridate di-

In view of the high toxicity and very pronounced myotic effect of di-isopropyl phosphorofluoridate, di ( 1 Z dichlorowo propyl) phosphorofluoridate (XVI) was of special interest. It was prepared from l 3-dichlorohydrin and phosphorus trichloride. It did not have any appreciable myotic effect and the toxicity was of alow order. Di ( 1 -ethylpropyl) phosphorofluoridate (XVII),... 

We carried out a great deal of work on the relationship between the above physiological effects and chemical constitution, and it was shown conclusively that the more potent compounds were those derived from secondary alcohols.3 Thus, for example, di-isopropyl phosphorofluoridate is very much more potent than diethyl phosphorofluoridate or di-ra-propyl phosphorofluoridate and the toxicity of the dicycZohexyl ester is of a high order (L.C. 50 for mice, rats and rabbits was 0-11 mg./l.). Din-butyl phosphorofluoridate had low toxicity and produced only feeble... 

Further modifications were then made in this hydrogen phosphite method of preparing di-isopropyl phosphorofluoridate in order to put it on an industrial scale. 

After a large number of experiments, we found that the preparation could be run virtually as a one-stage process. The whole process consists simply in adding phosphorus trichloride to isopropyl alcohol, dissolved in a solvent such as carbon tetrachloride, without external cooling. The crude product (still in the solvent) is chlorinated and then heated with an inorganic fluoride, e.g. sodium fluoride. After filtration, the solvent is distilled off and the pure di-isopropyl phosphorofluoridate distilled. ... 

Di 2 fluoroethyl phosphorofluoridate (XVIII) was prepared with the idea of combining the toxic principles of the fluoro-aoetates and of the phosphorofluoridates. It was readily obtained by the action of phosphorus oxydichlorofluoride on... 

The individual stages having been established, the whole process was then re-examined with a view to preparing di-isopropyl phosphorofluoridate on a technical scale. As a result of many... 

Di-isopropyl phosphorofluoridate is a practically odourless, mobile liquid, b.p. 183°/760 mm. (by extrapolation), f.p. ca. -82°. This wide range of temperature over which the compound is liquid adds to its usefulness. A specimen of the pure liquid has remained unchanged in a glass vessel for several years. Whereas the phosphorochloridate was readily hydrolysed by water, hydrolysis of the phosphorofluoridate was slow and took 72 hr. for completion at 15° and then only in the presence of a large excess of water (1 per cent solution solubility 1-5 per cent) P(OPr<)2 of+hso - P(OPrf)2 oh + hf. 

Dimethyl and diethyl phosphorofluoridate were hydrolysed much more quickly, the order being Me>Et>Pr complete hydrolysis of the ethyl ester took about 4 hr. Small quantities of the di-isopropyl, but not the diethyl ester, could be steam-distilled. Bi-isopropyl phosphorochloridate can be readily identified by allowing it to react with aniline to give the crystalline phenylphosphoramidate ... 

Di-isopropyl phosphorofluoridate does not, like the phosphoro-chloridate, give the anilinophosphonate on treatment with aniline. 

Whereas diethyl phosphorofluoridate was hydrolysed in aqueous solution in about 4 hr., the di-isopropyl ester required 72 hr. for complete hydrolysis, and small quantities could be steam-distilled. DicycZohexyl phosphorofluoridate was very stable, and vigorous shaking with water did not produce any appreciable hydrolysis. It was hydrolysed by boiling water only after several hours. When stirred vigorously with 2 per cent sodium hydroxide solution at 28-5°, the time taken to bring about hydrolysis according to the equation... 

Di-isopropyl phosphorofluoridate. Phosphorus oxydichlorofluoride (50 g.) is dissolved in dry ether (100 c.c.), isopropyl alcohol (dried over calcium oxide 50 g., 15 per cent excess) in dry ether (100 c.c.) is added slowly with cooling, and the mixture kept for 1 hr. Dry ammonia is then passed through the liquid, with cooling. The liquid... 

Fig. 12. Progress curve of inhibition of horse-serum cholinesterase by eserine and by di -isopropyl phosphorofluoridate in the absence of a substrate at pH 7-4 and 20°. x---x, 5x 10 8 M eserine O—O.ca. 3 x 10-10 M di-isopropyl phosphorofluoridate.

Results.3 In preliminary experiments with di-isopropyl phosphorofluoridate, it was found that concentrations above 10 7 M completely inhibited cholinesterase almost instantaneously. 

With lower concentrations, the inhibition produced varied with the time of incubation. Fig. 12 shows the inhibition of cholinesterase by di-tsopropyl phosphorofluoridate, and by a comparable amount of eserine, after varying times. The action of eserine reaches a maximum within 5 min., while the inhibition by phosphorofluoridate is initially less rapid, but is progressive and ultimately more complete. The latter effect suggests an irreversible inactivation of the enzyme rather than an equilibrium. 

Under the conditions used, the enzyme was less sensitive to phosphorofluoridate than in the manometric experiments. With an acetylcholine concentration of 0-0045m, 50 per cent inhibition was produced by 2 x 10 7m eserine or 3-5 x 10 8m di-isopropyl phosphorofluoridate. When the substrate concentration was varied over the range 0-0004-0-06 m, the percentage inhibition by the phosphorofluoridate (compared with a standard having a similar substrate concentration but no inhibitor) remained more or less constant. On the other hand, the inhibition due to eserine decreased when the acetylcholine concentration was raised. The difference in the behaviour of... 

Fig. 13. Effect of substrate concentration on inhibition of horse-serum cholinesterase.1 Enzyme activity was estimated by titration with 0-01 n NaOH at pH 7-4 and 20°. — , control, no inhibitor x — x, 2x 10 7 m eserine 0—O, 5 x 10 8 m di-isopropyl phosphorofluoridate.

Fig. 13 shows the results obtained with 2 x 10 7m eserine and 5x 10 8m di-isopropyl phosphorofluoridate. Eserine behaves like a... 

True and pseudo-cholinesterase. The above serum preparations contained both the true and pseudo- cholinesterases of Mendel and Rudney.1 The effect of di-isopropyl phosphorofluoridate on these components was examined separately by means of the specific substrates described by Mendel, Mundel and Rudney,2 using the titration method described above. Phosphorofluoridate (5 x 10 8m) gave an inhibition of 57 per cent of the activity towards 00045m acetylcholine, 30 per cent of the activity towards 0-0005 m acetyl-/ methyl-choline, and 40 per cent of that towards 0-005 m benzoylcholine, after incubating the enzyme with the poison for 5 min. Thus in these experiments there appeared to be no appreciable difference in sensitivity of the true and pseudo-cholinesterases of horse serum to phosphorofluoridates. 

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