The Fluoroacetates

The work described in this section was carried out in Cambridge during the war, and was originally submitted by us to the Ministry of Supply in communications entitled Fluoroacetates and related compounds . These communications were made available to American workers from the inception of the work. The present section is concerned mainly with a description of methyl fluoroacetate, CHaF-COgMe, and of certain otha derivatives of fluoroacetic acid. 

In Chapter iv we have described toxic fluorine compounds containing the POF grouping. Such compounds possessed quick knock-out action, and many of them were powerful myotics. Compounds of the fluoroacetate series are characterized by the CH2F group. Many of them are highly toxic wilh delayed action, but are completely devoid of myotic activity. The action is, broadly speaking, that of a convulsant poison (but see p. 136). 

Methyl fluoroacetate was first prepared by Swarts in small yield by the action of silver or mercurous fluoride on meth iodoacetate. The method is impracticable for large-scale work and therefore the preparation was reinvestigated in detail. Methyl chloroacetate was used in place of the expensive iodoacetate, and a variety of fluorinating agents was tried. It was found that fluorination could be effected by heating method chloroacetate in a rotating autoclave with potassium fluoride at 220° for 4 hr. Sodium fluoride, on the other hand, was almost without action. 

Other methods are available for the preparation of M.F.A. without the use of an autoclave, but it is doubtful whether they possess advantages over the autoclave method having regard to quality of product and yield. 

Methyl fluoroacetate (M.F.A.) is a liquid of b.p. 104 and f.p. ca. — 32° and is almost odourless. During a 10 min. exposure to a lethal concentration of the vapour, small animals did not appear to be affected in any way. After exposure, no very obvious s3miptoms developed until some 30-60 min. later (depending upon the concentration). The symptoms then shown depended to some extent upon the species, but all animals suffered convulsions, from which a partial recovery was sometimes made. Finally, however, a recurrence of the convulsions would cause death. 

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Relationship between physiological action and chemical constitution in the fluoroacetate series 133... 

Dry barium fluoroacetate (58 g., 0-2 mol.) is slowly added to 100 per cent sulphuric acid (122-5 g., 1 25 mol.). On distillation under reduced pressure, using a wide air-condenser, the fluoroacetic acid comes over between 83 and 100°/17 mm., and crystallizes immediately. It can be redistilled at atmospheric pressure and comes over at 167-168-5° yield 29-5 g. (94-2 per cent) colourless needles, m.p. 31-32°. (Found F, 24-3. Calc, for C2H302F F, 24-4 per cent.) These yields are considerably higher than those obtainable by Swarts s original method. 

The group Cl CH2 CH2 N< occurs in the nitrogen mustards which are powerful vesicants, e.g. CH3,N(CH2,CH2C1)2. It was decided therefore to introduce this group into the fluoroacetamide molecule in the hope of combining vesicant properties with the delayed convulsant action of the fluoroacetates. For this purpose N-2 hydroxyethylfluoroacetamide (IV) was prepared by the action of monoethanolamine on methyl fluoroacetate and was readily converted into N-2-chloroethylfluoroacetamide (V) by the action of thionyl chloride ... 

It was to be expected that the imino ether hydrochlorides would be hydrolysed in the animal body to give the corresponding fluoroacetate and ammonium chloride, and the toxicities should be roughly the same as those of the fluoroacetates. The results show this to be the case. The compound (VIII iZ = CH2,CH2F) was expectedly more toxic than the other compounds, as this would be hydrolysed to 2-fluoroethyl fluoroacetate which is known to be twice as toxic as methyl or ethyl fluoroacetate,1 as indicated below. 

Relationship between Physiological Action and Chemical Constitution in the Fluoroacetate Series1... 

For purposes of comparison the magnitude of the toxicity of fluoroacetic acid is represented as B A indicates higher toxicity (up to a factor of 2) and C indicates a lower toxicity (down to about 1/4 of that of fluoroacetic acid) D represents very low or negligible toxicity of the fluoroacetate type. 

Fluoroethanol, in contrast to ethanol, is only weakly oxidized by purified alcohol dehydrogenases, the rate being one-tenth to one-twentieth. Nevertheless, this rate appears sufficient to produce a typical fluoroacetate poisoning. A fairly long lag period in the development of the fluoroacetate symptoms possibly masks the time required for oxidation of fluoroethanol. 

From a study of the fluoroacetates so far mentioned, it appears that any compound which can give rise to fluoroacetic acid (or the fluoroacetate ion), either by hydrolysis or by oxidation (or both), is toxic. The toxic grouping is thus F-CH2-CO, and any substitution in this radical destroys the toxicity as far as relatively simple compounds are concerned. We had reached this conclusion by May 1943.1 We subsequently showed that esters of / -fluoropropionic acid were non-toxic, whereas esters of y-fluorobutyric acid were shown by American workers to be toxic. In 19442 we reported the synthesis of ethyl 5-fluoro-pentanecarboxylate, F,[CH2]g C02Et (I). This is a stable, colourless liquid and we showed that it possessed very potent toxic properties of the fluoroacetate type. By subcutaneous injection of the propylene glycol solution into mice the l.d. 50 was 4 mg./kg. Methyl fluoroacetate (II) may be taken as a convenient standard (p. 115) and has a l.d. 50 of about 6 mg./kg. for saline solutions, and 15 mg./kg. for propylene glycol solution.3 Therefore ethyl 5-fluoropentanecarboxylate was about 7 times as toxic as methyl fluoroacetate (molecule for molecule).4... 

The very high toxicity of ethyl 5-fluoropentanecarboxylate and its derivatives and the fluoroacetate-like symptoms produced seemed to us to be of particular interest, since by a process of /9-oxidation in the animal body 5-fluoropentanecarboxylic acid would readily give rise to the toxic fluoroacetic acid. Similar remarks apply to y-fluorobutyric acid and its derivatives prepared independently by American workers. The non-toxicity of /9-fluoropropionic acid and its derivatives may, on the other hand, be due to the inability of this acid to give the toxic fluoroacetic acid by a process of /9-oxidation. 

We see from the above that there is a striking alternation in the physiological properties of w-fluorocarboxylic esters of the general formula of F- [CH2]w-C02.R. Thus when n is an odd number the compound is highly toxic to animals, whereas when n is even the compound is non-toxic. All the toxic compounds are powerful convulsant poisons and showed symptoms of the fluoroacetate type. 

Chemically they are extremely inert, being much more un-reactive even than the fluoroacetates. The inertness of the fluorocarbons and their nearly perfect physical properties arise from the strength of the F—C linkage and from their compact structure. The effective atomic radius of covalently bound fluorine is 0-64 A., which although greater than hydrogen (0-30) is smaller than other elements, e.g. Cl 0-99 A., Br 1-14 A. 

Similarly with regard to the fluoroacetate story, other factors in addition to the jamming of the Krebs cycle may be looked for. One profitable line may well be the examination of the mobility, by chemical and enzyme methods, of the firmly... 

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. 

The toxicity of fluoroacetic acid and of its derivatives has played an historical decisive role at the conceptual level. Indeed, it demonstrates that a fluorinated analogue of a natural substrate could have an activity profile that is far different from that of the nonfluorinated parent compound. The toxicity of fluoroacetic acid is due to its ability to block the citric acid cycle (Krebs cycle), which is an essential process of the respiratory chain. The fluoroacetate is transformed in vivo into 2-fluorocitrate by the citrate synthase. It is generally admitted that aconitase (the enzyme that performs the following step of the Krebs cycle) is inhibited by 2-fluorocitrate the formation of aconitate through elimination of the water molecule is a priori impossible from this substrate analogue (Figure 7.1). 

Lewandowicz A, Sicinska D, Rudzinski J, Ichiyama S, Kurihara T, Esaki N, Paneth P (2001) Chlorine Kinetic Isotope Effect on the Fluoroacetate Dehalogenase Reaction. J Am Chem Soc 123 9192... 

There are two Krebs cycle inhibitors that are worth mentioning. Malonate inhibits succinate dehydrogenase because of its very similar structure. Fluoro-acetate inhibits cis-aconitase, which is an Fe-S enzyme. The fluoroacetate replaces acetate as a substrate in the citrate synthase reaction when this combines with cis-aconitase, however, no further reaction becomes possible. 

Fluoroacetate is rapidly absorbed by the gastrointestinal tract but not well absorbed dermally. Fluoroacetate is converted to the ultimate toxicant, fluorocitrate. Fluoroacetate is distributed to lipid-rich organs, such as the liver, brain, and kidneys. Fluoroacetate is primarily eliminated through urine. Up to 50% of the fluoroacetate is excreted unchanged in the urine by 72 h following administration. The kinetic half-life for sodium fluoroacetate is species dependent. Reported half-lives in rabbits, goats, and sheep are 1.1, 4-7, and 13.3 h, respectively. 

MFA. Examined as a possible CW agent but humans are less susceptible than laboratory animals. The fluoroacetates inhibit the mitochondrial enzyme aconitase and disrupt the citric acid cycle. The first signs of poisoning are nausea and apprehension, followed by convulsions. Death is due to ventricular fibrillation. 

Etherification of lower organic acids improves their chromatographic properties and enhances the detection sensitivity. To determine sodium fluoroacetate, Stahr [135] devised a method based on preliminary formation of methyl fluoroacetate. The fluoroacetate was extracted from the sample with methanol, and the water was removed by heating after adding a certain amount of an alkali in order to prevent volatilization of the fluoroacetate. The dehydrated sample was etlierifled in a sulphuric acid—methanol solution, yielding 85% of ethyl ether. The latter was analysed by GC. The detection limit was 1 10" %. 

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