The Phosphorofluoridates

In Chapter i a brief description was given of D.F.P. and some related compounds, and in this chapter a more detailed account is given of the work initiated and carried out on the toxic phos phorofluoridates during World War II at Cambridge by an extramural Ministry of Supply research team working with the author. 

For security reasons, during the war the work was not published at the time, though secret reports (which were also made available to American workers almost from the inception of the investigations) were regularly submitted to the Ministry of Supply. 

Until this work began in Cambridge at the begiiming of the war, the alkyl phosphorofluoridates had received practical no attention. Lange had given a tedious and laborious method for preparing dimethyl and diethyl phosphorofluoridates in very poor yield as follows. 

Death took place rapidly (for example, a concentration of 1 10,000 of the di-isopropyl ester killed 6/6 rats, 10/10 mice and 2/3 rabbits within 25 min. from the beginning of exposure of 10 min.). Such rapid effect and quick knock-out action was ihown by few other gases or vapours. 

These initial observations encouraged us to search for new and simple methods of preparation. It is convenient therefore to defer a more detailed discussion of the physiological action of these compounds until we have indicated the methods of preparation. 

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Concurrently with experiments on animals, the action of the phosphorofluoridates on enzymes was investigated in Cambridge.3 It was shown in 1942 that esters of phosphorofluoridic acid inhibit4 the action of the enzyme cholinesterase, which is present in tissue fluids and hydrolyses acetylcholine to the much less active choline. 

Many of these compounds were toxic for example, tetra-methyl phosphorodiamidic fluoride (dimethylaminofluorophos-phine oxide (VII)) had a l.c. 50 of 0-1 mg./l. Unlike the phosphorofluoridate esters, however, they were devoid of myotic action. 

As a matter of interest, we obtained a compound, diethyl phosphorofluoridite (IX), of a lower state of oxidation than the corresponding phosphorofluoridate by the action of phosphorus dichlorofluoride on ethyl alcohol. The new compound, unlike the phosphorofluoridate, was readily hydrolysed by water, was relatively non-toxic and did not produce myosis.1... 

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... 

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. 

Hydrolysis of D.F.P. by alkali.1 When D.F.P. was heated under reflux with an excess of n/2 sodium hydroxide solution for 30 min., and then back-titrated with n/2 sulphuric acid (phenolphthalein aa indicator), it was found that 1 mol. of the phosphorofluoridate required 2 0 mol. of sodium hydroxide. 

Hydrolysis by n/2 sodium hydroxide at room temperature, (a) Diisopropyl phosphorofluoridate (2-0532g.) was shaken with 100 ml. of 0-49 n sodium hydroxide at 17°. The oily drops disappeared after about 5 min. The shaking was continued for a total of 30 min., and then 25 ml. were withdrawn and required 13-35 ml. of 0-5n sulphuric acid for neutralization (phenolphthalein). Therefore 1 mol. of the phosphorofluoridate had reacted with 1-994 mol. of alkali. 

The reaction depended upon the marked difference in reactivity between the chlorine atoms and the fluorine atom in phosphorus oxydichlorofluoride. In general, the reaction with alcohols waa clear-cut, and most of the phosphorofluoridic esters were obtained in excellent yield and uncontaminated with the phosphoric triester. For example, when ethyl alcohol and phosphorus oxydichlorofluoride were allowed to react in the odd, diethyl phosphorofluoridate (VII, R = Et) was obtained in 93 per cent yield. No tertiary base was necessary to remove the hydrogen chloride produced in the reaction. The general process was patented during the war.1... 

The cholinesterase-inhibiting activity of the phosphorofluoridates was compared quantitatively with that of eserine sulphate thus. To 0-2 ml. of heparinized human plasma was added 05 ml. of a solution containing either eserine or the phosphorofluoridate in varying concentrations then the mixture was kept at room temperature for 10 min. before 1 /tg. of acetylcholine in 1 c.c. saline solution was added. After 5 min. at room temperature, the mixture was made up to 10 ml. with frog saline containing eserine 1/100,000, which at once stopped the action of any cholinesterase not yet inactivated. The solution was then assayed for acetylcholine on the frog rectus-muscle preparation. 

Poisons. These were the phosphorofluoridates prepared by the Cambridge team of chemists (pp. 2-7), and eserine. Since the phosphorofluoridates slowly hydrolyse in water, stock solutions of these and of eserine were prepared in ethylene glycol monoethyl ether, and... 

Reversibility. It is known that the effect of eserine on cholinesterase can be completely reversed by prolonged dialysis against water. On the other hand, it proved impossible to obtain any reversal of the poisoning by the phosphorofluoridate esters (see table below). The enzyme solution (5 ml.) was treated with the inhibitor for 15 min. at 38° 1 ml. was used at once for activity estimation, and the remainder dialysed against running water for 24 hr. in the case of the eserine experiment, 36 hr. in the others. It was clear that the combination between the phosphorofluoridate esters and the enzyme is much firmer than that between eserine and the enzyme. 

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... 

The above proof of the extremely high affinity for cholinesterase of the phosphorofluoridates, especially those with branched-chain alkyl groups, suggested that the toxic and myotic power was due to inhibition of cholinesterase in vivo. Bloch1 showed... 

The active trichloride was converted into di-isopropyl hydrogen phosphite and thence through the phosphorochloridate into the phosphorofluoridate essentially according to the scheme... 

It was suggested,1 on the basis of kinetic measurements, that the phosphorofluoridates inhibit esterases by virtue of a highly specific affinity for the active centres of this group of enzymes. Preliminary experiments by Boursnell and Webb2 with diisopropyl phosphorofluoridate containing 32P gave results which were in accordance with this view. 

The reactions were carried out in each case with a 0-1 per cent protein solution in phosphate buffer (pH 6 8), to which the radioactive phosphorofluoridate was added as a concentrated solution in dry ethanol. At the end of the reaction time, the product was dialysed for 20 hr. against running water, and precipitated at 0° by addition of two volumes of acetone. The precipitate was spun off and washed at —5° with ethanol and ether, and dried in air or over sulphuric acid. Samples of 25-50 mg. of dry powder were used for radioactivity determinations, and compared with a standard prepared by hydrolysing a weighed amount (ca. 1 mg.) of the phosphorofluoridate in n sodium hydroxide, neutralizing and drying. 

Stoichiometric deductions from these results are difficult, owing to the inhomogeneity of theenzyme preparations but if the figure of 80 per cent for the purity of the esterase is used, the results showthat 1 g. molecule of the phosphorofluoridate combines with 96,000 g. esterase under conditions which produce complete in-aotivation. This low figure is consistent with the value obtained by Jansen, Nutting and Balls1 for crystalline chymotrypsin.2... 

Ethyl phosphorodifluoridate (XXI) was obtained by the action of sodium fluoride on the corresponding phosphoro-dichloridate unlike diethyl phosphorofluoridate, it was rapidly attacked by cold water. Alcohol converted (XXI) into the phosphorofluoridate ... 

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 was obviously of interest to determine whether other esters of fluoroacetic acid would prove to be more or less toxic than the methyl ester. In the phosphorofluoridate series, for example, we found that esters of secondary alcohols were far more potent than those of primary alcohols for instance, di-isopropyl fluorophosphonate (I) was a compound of considerable activity. Accordingly ethyl, ra-propyl and isopropyl fluoroacetates were prepared by heating the corresponding esters of chloroacetic acid in the rotating autoclave with potassium fluoride. The toxicity figures of these esters were very similar to those of methyl fluoroacetate. 

In the phosphorofluoridate series, we found that the diphenyl ester (p. 53) was relatively non-toxic. Phenyl fluoroacetate, however, was toxic with an l.d. 50 of 6-10 mg./kg. for subcutaneous injection into mice. The symptoms were similar to those displayed by methyl fluoroacetate. 

The two types of fluorine compounds are (a) compounds containing the P—F link, e.g. the phosphorofluoridates (b) compounds containing the C—F link and known collectively as fluoroaoetates. 

In each class the problem may be resolved into two essential parts (i) the breakdown of the organic compound under appropriate conditions to give a quantitative yield of fluoride ions in aqueous solution, and (ii) the determination of the concentration of these fluoride ions. Methods of breaking down the organic compounds were examined and the procedure adopted for the phosphorofluoridate was different from that used for the fluoroacetate series. From both, however, sodium fluoride was obtained as the breakdown product containing all the fluorine present. After numerous preliminary experiments we came to the conclusion that on the macro-scale a very convenient method of determining the quantity of fluoride ions in the products was by precipitation as lead chlorofluoride,2 PbCIF, which was then dissolved in dilute nitric acid and the chloride was determined by the Volhard method and calculated to the equivalent amount of fluorine. We determined carefully the conditions for the quantitative precipitation of lead chlorofluoride. 

These conditions were realized by treating the phosphorofluoridate ester, dissolved in alcohol, with five times the quantity of sodium required by the equation... 

Here again the fluorine was determined ultimately by precipitation as lead chlorofluoride, but the breakdown of the organic compound is more difficult than with the phosphorofluoridates. The two2 methods recommended are... 

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