Di isopropyl phosphorofluoridate

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. ... 

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. 

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

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. 

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. 

Fig. 14. Inhibition of horse-serum cholinesterase by various compounds. Incubated for 15 min. at 20° before addition of 2 mg. of acetylcholine chloride. A, di-isopropyl phosphorofluoridate B, di ec.-butyl phosphorofluoridate C, eserine D, diphenyl phosphorofluoridate E, dithioethyl phosphorofluoridate F, tetramethylphosphorodiamidic fluoride O, diethyl ALmethylphosphor-amidate.

It is considered that the direct method of preparing radioactive di-isopropyl phosphorofluoridate possesses advantages compared to the method of B. Witten and J. I. Miller, which starts with radioactive potassium dihydrogen phosphate.5... 

Results. Phosphorus (1 g.) and phosphorus trichloride (1 ml.) were used. The weight of di-isopropyl phosphorofluoridate obtained was 5-5 g. (62 per cent). The purity, determined by fluorine analysis1 on 50 mg. of the active product, was 98-5 per cent. 

N.B. With inefficient stirring the product contained less di-isopropyl phosphorofluoridate.)... 

The radioactive phosphorus used had an activity of 28,000 counts/min./mg. (counter efficiency, ca. 1 per cent, i.e. specific activity ca. 1 mc./g.), whilst the radioactive di-isopropyl phosphorofluoridate had an activity of 2200 counts/min./mg. (corrected to zero time). 

We found that the toxicity and myotic activity of di-isopropyl phosphorofluoridate (XI) were far greater than that of di- propyl phosphorofluoridate. In Report no. 6 on fluoro-phosphonates to the Ministry of Supply3 we described the preparation of di aec.-butyl phosphorofluoridate (XII) by the hydrogen phosphite method (p. 6). The compound was found to be very toxic and to produce severe myosis in man and animals. The symptoms displayed during and after exposure were identical with those produced by di-isopropyl phosphorofluoridate. The L.c. 50 for di-sec.-butyl phosphorofluoridate for mice for deaths within 2 hr. was 0-6 mg./l., and that for deaths within 48 hr. was 0-54 mg./l. 

Negligible sensory irritation was caused by di-isopropyl phosphorofluoridate at a concentration of 1 part in 10. This, coupled with the fact that the odour was practically undetectable, means that sufficient warning is not usually given at this concentration to suggest the use of respirators. Exposures at this concentration cause severe myosis which persists for several days and causes considerable incapacitation (Report no. 12 by McCombie and Saunders to Ministry of Supply, 4 August 1943). 

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),... 

Nevertheless, there is some similarity of structure between compounds (II) and (IV). It is known that with grem.-diethyl groups in the phosphorofluoridate molecule, e.g. di-(l-ethyl- -propyl) phosphorofluoridate (V), the toxicity is less than with <7em.-dimethyl groups (di-isopropyl phosphorofluoridate (IV)). We found that similarly tetraethylphosphorodiamidic fluoride... 

It has been reported4 that cholinesterase inhibitors (such as di-isopropyl phosphorofluoridate) increase the permeability of squid giant axons towards sodium and potassium. There is also an indication that the erythrocyte requires, among other factors, an adequate acetylcholine-cholinesterase system to prevent a gain of sodium or a loss of potassium.5 The conclusion that permeability is dependent on cholinesterase activity, however, seems to be contested by Strickland and Thompson.6... 

The above method (of which details are given in the example below) requires at most 45 min. heating with pure di-isopropyl phosphorofluoridate 5 min. heating was found sufficient. 

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