[CONTBIBUTION FILOY TBB DEPARTMENT OF PHYSIOLOQICAL CHEMISTRY, SCHOOL OB MEDICINE,UNIVERSITY OF PENNSYLVANIA]
CHEMICAL REACTIONS OF NITROGEN MUSTARD GASES.1 I. REACTIONS OF METHYL-BIS(8-CHLOR0ETHYL)AMINE WITH HEXAMETHYLENETETRAMINE
SAMUEL GURIN, ADELAIDE M. DELLUVA,
AND
DANA I. CRANDALL
Received A p ~ i l8, 1047
Previous work in this laboratory (1) has demonstrated that aqueous solutions of methyl-bis(8-chloroethy1)amine (M.B.A.) are rendered non-toxic by exposure to hexamethylenetetramine (H.M.T.). Furthermore, the administration of H.M.T. to animals will to a significant degree protect them against subsequent exposure to M.B.A., regardless of whether the toxic agent is administered subcutaneously as the hydrochloride, or whether it is applied to the skin as the free base. This report is concerned with a detailed study of the reaction between M.B.A. and H.M.T. undertaken with the hope that information might be obtained concerning the nature of thjs detoxication. Reaction of M.B.A. and H.M.T. in water. When aqueous solutions of M.B.A. and H.M.T. are mixed, the following events occur: 1. The resulting solution after standing less than 30 minutes is non-lethal when injected subcutaneously in four lethal doses. Although some toxic effects can be observed when large amounts of the solution are administered, these effects do not appear after the mixture has been allowed to stand for 1-2 hours. 2. After standing 30 minutes with H.M.T. the solution contains approximately equal amounts of ionic and covalent chloride. Chloride ions are liberated at about the same rate (at pH 7.5) irrespective of whether H.M.T. is present or not. 3. The color developed by M.B.A. with various reagents (which are specific for organic molecules containing covalent chlorine) fades at the same rate in the presence or absence of H.M.T. (1). 4. After standing 30 minutes, aqueous solutions containing M.B.A. and 2-3 moles of H.M.T. react to only a very slight extent when treated with thiosulfate for 10 minutes. T h s procedure which was developed by Golumbic, Fruton, and Rergmann (2) serves as a measure of ethylenimoniumions (11, Fig. 1) remaining in the solution. Fruton, Stein, Stahmann, and Golumbic (3) have utilized thiosulfate titration as a measure of the reactivity of test substances toward the nitrogen mustards. These authors were also able to show that H.M.T. reacts extremely rapidly with M.B.A. (3). Our results confirm the conclusions of these investigators, namely that there is very little of the ethylenimine form of M.B.A. left after exposure to H.M.T. for 30 minutes. These results have led to the following interpretation. Among the products of 1 This work was done in whole under Contract No. OEM-cmr-108between the University of Pennsylvania and the OEce of Scientific Research and Development, which assumes no responsibility for the accuracy of the statements contained herein. 606
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the reaction it should be possible to find substances containing carbon-bound chlorine. The covalent chlorine atom is not concerned with the rapid condensation occurring during the first 30 minutes. The neutralizing properties of H.M.T. cannot be ascribed to its effect upon the rate of hydrolysis of M.B.A. since chloride ion is liberated a t equal rates in the absence or presence of H.M.T. The rate of reaction as well as the mild conditions employed suggest the participation of a reactive chlorine in this reaction. The absence of the ethylenimonium form as indicated by the thiosulfate titration suggests that it is primarily the corresponding ethylenimonium form which reacts rapidly with H.M.T. Since formaldehyde, ammonia, and methylamine each react sluggishly or not at all with M.B.A., it appears that it is intact H.M.T. which participates in the reaction. All of the evidence is in accord with the belief that the ethylenimine form of M.B.A. reacts rapidly with H.M.T. t o form quaternary salts. The formation of other quaternary salts of M.B.A. has been described by Golumbic, Fruton, and Bergmann (4). When equimolar amounts of M.B.A. and H.M.T. were mixed and allowed to stand in Bo/,aqueous ethanol for 30 minutes, a variety of products was isolated. Among them were H.M.T., H.M.T. hydrochloride, a small amount of p-chloroethylmethylethanolamine (IIa) and considerable quantities of the hexamethylene tetraminium derivative of M.B.A. (111). Evidence was also obtained for the existence of small quantities of a quaternary salt of H.M.T. with a linear dimer of M.B.A. The quaternary salts are difficult to prepare in pure form. They are soluble in cold water, methanol, warm ethanol, less soluble in chloroform, and slightly soluble in acetone and ether. On warming, they decompose readily, causing brown amorphous products to be formed. Although the nature of these decomposition products has not been investigated, it is suggested that there is a ready loss of HCI, producing vinyl amines accompanied by cleavage of the hexamethylene tetramine portion as a result of the liberated acid. The analytical data indicate that these substances are monomeric and linear dimeric derivatives of M.B.A. with H.M.T. Peters and Ogston (5) have isolated a picrate to which they tentatively assign the structure of a linear dimer of M.B.A. Golumbic, Fruton, and Bergmann (4) have isolated from the hydrolysis products of 1-methyl-1-(P-chloroethy1)ethylenimoniumpicrylsufonate a linear dimer (X) which was characterized as a picrylsulfonate and as a Reineckate. Since the rate of reaction of M.B.A. with H.M.T. is much greater than is the rate of polymerization of M.B.A., it is suggested that the primary reaction is a condensation of H.M.T. with the ethylenimonium form of M.B.A., resulting in the formation of large amounts of product (111). This is supported by the isolation of considerable amounts of this substance. A41thoughthe analytical evidence for product YI is not satisfactory, fairly good evidence has been obtained for VII. While there is very little question concerning the mode of formation of 111,there are several routes by which the dimeric salts may be formed. The monomeric quaternary salt I11 may react with a second molecule of M.B.A. t o form either IV or V (Figure 1) which can
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hydrolyze further to VI, VII, VI11 and IX. Product I11 could, however, condense with @-chloroethylmethylethanolamine (IIa) yielding VI or VI11 directly. It is, of course, conceivable that such products may result from a preliminary dimerization of M.B.A. followed by condensation of the resulting linear dimer with H.M.T. Although this mechanism is unlikely, there is as yet not enough information available to make a decision concerning either the mode of formation or the structure of the resulting compounds. A small amount of impure product VI1 has been recovered from an aqueous solution of H.M.T. and M.B.A. which had been allowed to stand a t room temperature for 24 hours. The reaction has also been carried out in large volumes of water, thus avoiding the use of 50% aqueous alcohol as a solvent. After standing 30 minutes at room temperature, the solution was chilled and evaporated to dryness in the frozen state. From the residue, H.M.T., H.M.T. hydrochloride, @-chloroethylmethylethanolamine picrate (IIa) and I11 were isolated. No evidence was found for the presence of the dimer quaternary salts (VI-IX). It appears, therefore, that no appreciable linear dimerization occurs in aqueous solutions and that the primary action of H.M.T. upon M.B.A. is the formation of the nonomeric hexamethylene tetraminium derivative. EXPERIMENTAL
One fractionation procedure is reported here in detail for illustration. To 1 g. of M.B.A. was added 1 g. of H.M.T. and 1.5 ml. of water followed by 1.5 ml.of ethanol. The suspension cleared after stirring from 5 to 10 minutes at room temperature. After stirring for 30 minutes, 30 to 40 ml. of acetone was added and the solution chilled for several hours. A crystalline precipitate was removed by centrifugation, washed with acetone, and dried. The product, weighing 0.34.4 g., consisted mainly of H.M.T. which was recovered by extraction with chloroform. A small amount of insoluble material remained and was later identified as product 111. No cyclic dimer nf M.B.A. (XI) could be found in this fraction. The combined acetone mother liquors were evaporated to dryness at low temperature yieldinga colorless crystalline residue contaminatedwith asmall amount of oil. Extraction with ether removed the oil which proved t o be mainly 8-chloroethylmethylethsnolamine. This substance was recovered as the picrate from ether and the product recrystallized from hot water; yield, 0.21 g., m.p. 68-69'. Anal., C1,9.8; picrir acid, 62.3;theory: C1,9.7; picric acid, 62.5. The colorlese crystalline residue (insoluble in ether) was extracted several times with rhloroform t o remove any remaining H.M.T. Upon evaporation of the chloroform a small amount (0.1 9.) of product I11 was obtained; m.p. 146-149' dec. Recrystallization from cold methyl alcohol and ether raised the melting point to 152-154O. The remaining crystalline residue (insoluble in chloroform) was dissolved in anhydrous methyl alcohol and fractionally precipitated with ether. The first crop consisted of an oily precipitate which slowly crystallized. Upon recrystallization of this material from methyl alcohol and ether, two different fractions were obtained. The first product (needles) melted a t 140-143Owith decomposition. Anal., C1-, 15.0; total C1,23.0; theory for product VI, CY. 16.3; total C1,24.5. There was not enough product obtained for further purification. The second preparation which decomposed at 118-119' gave the following analyses; C1-, 16.5; total C1, 16.5; N, 19.9; theory for product VII: C1-, 17.1; total C1,17.1; N, 20.2. From the mother liquors the monomeric quaternary salt (product 111) was obtained by
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further addition of ether. A relatively large crop of colorless feathery needles was obtained after chilling for several days. After rectystallization from methyl alcohol-ether, the product melted a t 158-155" with decomposition; yield 0.7 g. AnaZ., C1-, 12.3; total c1, 23.6; N, 23.4; theory for product 111: C1-, 12.0; total C1,B.O; N, 23.65. The material was subjected to further analysis for ammonia and formaldehyde. This was accomplished by hydrolysis with mineral acid followed by distillation and estimation of the formaldehyde. Ammonia was estimated in the residue by alkalinization and distillation into standard acid. 14.2. The ratio CH~/"T--N Found: CHI, 27.5; NHa-Ii, 15.2; theory: CHz, 28.4; "1-N, was 1.8, whereas theoretically it should have been 2. For H.M.T. the ratio CH,/NHr--N is 1.66 (6CH2/4N) whereas in hexamethylene tetraminiurn compounds the ratio should be 2(6CII2/3N). For purposes of comparison, cetylhexamethylene tetraminiurn iodide wvas prepared and similarly analyzed. Found: CHI, 15.1; NHI-N, 8.48; CHZ/NHa-N, 1 3 , theory: CHZ, 17.0; h"a--N, 8.58; CHZ/NHs--N, 2.0. These analyses support the structure assigned to the compound (111). The substance is hygrosropic and unstable; it rapidly develops a tan color unless it is kept thoroughly dry. PHILADELPHIA, PA. REFERENCES (1) Unpublished data. (2) GOLCMBIC, FRUTOX, AND BERGMANN, J. Org. Chem., 11, 518 (1946). (3) FRUTON,STEIN,STAAMANN, AND GOLUMBXC, J . Org. Chem., 11,571 (1946). (4) GOLUMBIC, FRUTON, AND BERGMANN, J . Org. Chem., 11, 581 (1946). (5) PETER^ AND @%TON, Unpublished data in Great Britain.
