[CONTRIBUTION FROM
THE LABORATORIES OF THE ROCKEFELLER h S T I T U T E FOR MEDICALRESEARCH]
CHEMICAL REACTIONS OF MUSTARD GAS AND /RELATED COMPOUNDS.’ I. THE TRANSFORMATIONS OF MUSTARD GAS I N WATER. FORMATION AND PROPERTIES O F SULFONIUM SALTS DERIVED FROM MUSTARD GAS WILLIAM H. STEIN, STANFORD MOORE,
AND
MAX BERGMANN*
Received March 22, 1946
The physiological effects of mustard gas [bis(P-chloroethyl)sulfide,to be designated hereafter by its U. S. Chemical Warfare symbol, HI are the consequence of chemical processes which the agent initiates by its reaction with body constituents. The experiments to be described in this and subsequent papers of this series were performed in order to gain insight into the mode of action of H upon proteins, amino acids, and other constituents of biological systems. Since water is a major component of biological systems, the transformations undergone by H in water are the subject of this first paper. The kinetics of the hydrolysis of H in nearly saturated aqueous solution (less than 0.1%) were studied in World War I (l),and these investigations have been greatly extended more recently (2, 3, 4, 5). The kinetic data show that in dilute aqueous solution H hydrolyzes according to Equation 1. CHaCHzCl
/ \
+
S
CH2 CHzC1
2H20
-
CH2CHzOH
/ \
+
S
2HC1
1.
C H2 CH2 OH
Davies and Oxford (6), on the other hand, demonstrated that when the ratio of water t o H is small (as in suspensions of H in water in the ratio 1:3), only a small quantity of thiodiglycol [bis(P-hydroxyethyl)sulfide, to be referred to hereafter by the symbol TG] and HC1 are formed, most of the H being converted to a mixture of sulfonium chlorides. The experiments of Davies and Oxford, however, were performed under conditions quite dissimilar to those obtaining when H reacts with water under physiological conditions. It was of interest t o find, therefore, that when H was shaken with 50 volumes of water at room temperature until a clear solution resulted, e.g. for 24 hoursa at 20°, the resulting solution contained only about 789;bof the theoretical amount of HC1 to be expected on complete hydrolysis of H according to Equation 1. ‘This work was done in whole under Contract KO.OEMsr-313 between The Rockefeller Institute for Medical Research and the Office of Scientific Research and Development, which assumes no responsibility for the accuracy of the statements contained herein. The experiments were performed during the period January 1942-August 1944. ZDied, November 7, 1944. 8At the end of this period free H is absent, as proved by the fact that nothing can be extracted from the solution by ether. 664
REACTIONS OF MUSTARD GAS.
I
665
On heating the neutralized solution a t 100" for 2 hours, however, the remainder of the theoretically possible HC1 was liberated. The properties of the H hydrolysate just described, coupled with the observations of Davies and Oxford (6), suggested the presence in the hydrolysate of sulfonium salts. Northrop (7) arrived a t similar conclusions. As will be shown later, the sulfonium salts derived from H under these conditions decompose on heating t o 100" in aqueous solution with the formation of one equivalent of acid for each sulfonium group present. Hence the amount of acid produced on heating an hydrolysate to 100" is an index of the extent of sulfonium salt formation. On this basis, about 22% of the chlorine of the original H is found as sulfonium chloride after hydrolysis of H with 50 volumes of water a t room temperature. If the ratio of water to H is increased to 200 volumes, the extent of sulfonium salt formation drops to about 16%, while if 1000 volumes of water is used, the extent of sulfonium salt formation falls to about 5%. From the hydrolysate resulting when 26 g. of H was shaken with 50 volumes of water a t room temperature, b o different sulfonium salts have been isolated. One of these sulfonium salts, bis-P-[bis(P-hydroxyethyl)sulfonium]ethylsulfide dichloride (compound IV in Figure 11, is formed by the reaction of one molecule of H with 2 molecules of TG. From the hydrolysate, 3.3 g. of this sulfonium dichloride was isolated, indicating that a t least 16% of the original H molecules had been transformed into this compound. This sulfonium salt was first described by Davies and Oxford (6), who prepared it by heating H with 3 volumes of water a t 100". The second sulfonium salt isolated from the H hydrolysate was p-hydroxyethyl-~-[bis(~-hydroxyethyl)sulfonium]ethylsulfide chloride (111). This compound might be formed by the reaction of one molecule of H chlorohydrin Ischloroethyl-P-hydroxyethylsulfide (I)] with one molecule of TG. From the hydrolysate, 10.3 g. of I11 was obtained as the picrylsulfonate, indicating that a t least 26% of the original H molecules had been transformed into this compound. The total amount of sulfonium compounds isolated represents about 50y0 OS the amount of sulfonium ion estimated above to be present in the hydrolysa.te. I n Figure 1 a mechanism is presented t o explain the formation of sulfonium salts when H is hydrolyzed in the presence of moderate quantities of water. It should be mentioned that Ogston independently has proposed the same scheme (8). The sequence of reactions proceeding from H, through H chlorohydrin (I), to TG, is the same as the simple hydrolysis given earlier in Equation 1. The existence of H chlorohydrin as an intermediate in this reaction sequence has been proved by the work of Ogston (9) and Kinsey and Grant (lo), who isolated the compound from H hydrolysates. In very dilute aqueous solutions simple hydrolysis occurs exclusively, since the concentration of TG is extremely low relative to that of water. As the ratio of water to H is decreased, however, some of the TG formed by hydrolysis of H undergoes further reaction t o give the sulfonium salts isolated. The formation of IV is envisaged in Figure 1to occur by the stepwise reaction
666
STEIN, MOORE, AND B E R G U N N
of one molecule of H with two molecules of TG. The intermediate, P-chloroethyl-~-[bis(/3-hydroxyethyl)sulfonium]ethylsulfide chloride (11), has not been isolated from H hydrolysates, but its existence as a precursor of IV is assumed on the basis of several lines of evidence. In the first place, it is improbable that two molecules of TG could react simultaneously with one molecule of
+ Ha0
/CHzcHac*
S
\
+ Ha0
S
\
CHzCHz C1
\
CHzCHzOH
CHXCHsOH
CHzCHiOH
+/
+/ CHiCHzS
\
CHZCHZOH
C 1-
C1-
/ S \
S/cHscHsoH
CHa CHzOH
+ H a O _ , / CH2CHaS\ S
\
CHz CHz Cl
CHzCHzOH
CHzCHzOH
\
(111)
(11)
GICHZCHzOH
+/ /cHzcH2s
\
CHzCHzOH
S
\
/CHzCH20H CHzCHzS
+\
CHzCH2OH
GI-
(IV) FIGURE 1.
H, since this would involve a trimolecular reaction. In addition, Rydon (11) has synthesized the sulfonium salt (11) by allowing equivalent molar quantities of H and TG t o react in ethanol. The sulfonium chloride was found by Rydon to be an unstable oil. In a later part of this paper, the preparation of this sulfonium salt as a stable crystalline picrylsulfonate is described, and compound I1 is shown t o be capable of reacting with TG in aqueous solution to form IV.
REACTIONS OF MUSTARD GAS.
I
667
According to Figure 1, there are two reaction sequences which may give rise t o the sulfonium salt 111. One mechanism involves a direct reaction between I and TG; the other involves the hydrolysis of the chloroethyl group of 11. The farmer mechanism is supported by the fact, to be demonstrated in a later paper of this series (12), that I readily reacts with T@ in aqueous solution to form a sulfonium salt. Hydrolysis of the chloroethyl group of 11, with the resultant formation of 111, will be demonstrated later in this paper. Although the ultimate products of the decomposition have not been isolated, it seems likely that, given sufficient time, the various sulfonium salts pictured in Figure 1 will break down to TG. The formation of sulfonium salts during the hydrolysis of H is of physiological as well as chemical interest, since, as will be shown below, these salts possess a relatively great toxicity. Accordingly, the chemical properties of the sulfonium salts were investigated in greater detail. The chemical properties of the sulfonium salt I V . The sulfonium dichloride IV was prepared by shaking H with an excess of TG in water. For example, when $5 cc. of H is shaken for 18 hours with 250 cc. of water containing 20 cc. of TG, only 10-13% of the H is hydrolyzed, the remainder combining with TG t o form the sulfonium dichloride, which may readily be isolated in good yield [cf. also (7)]. If the same amount of H is shaken with 33 cc. of TG and 250 oc. of water, 94% of the H combines with the TG. The reaction proceeds more readily in water than in non-aqueous solutions. No reaction was observed in chloroform or nitrobenzene, and lower yields were obtained in aqueous ethanol than in water alone. The sulfonium dichloride IV has an LD6o for mice of 50-100 mg./kg. If a, solution of the sulfonium salt is heated a t 100" for 1-2 hours, the toxicity is destroyed. At dosages of 250-1000 mg./kg. in mice, death occurs rapidly with flaccid paralysis and respiratory failure. When IV is administered at levels near the LDSO,death is delayed for a period of a week or more (13). It was also found that the sulfonium dichloride possesses a strong necrotizing action when injected intradermally into rabbits. The necrotizing action appeared to be roughly 1/10 as great as that of H itself (14). The chemical properties of IV present several points of interest. With respect to the stability of the salt, Davies and Oxford (6) observed that in dilute aqueous solution, it is 30% decomposed with the liberation of acid upon standing at 15-20' for three weeks. As was mentioned previously, the sulfonium salt is completely decomposed when heated a t 100" in dilute aqueous solution for one hour, two equivalents of acid being formed. I n 0.03 N NaOH at 4", the compound is fairly stable, only 25% of the theoretically possible acid being fiberatred in 24 hours. At pH 8.9 and 9.9 at 3", the sulfonium salt does not form any acid within this time period. As will be noted from Column 3 of Table I, when IV is incubated at 37' in NaHC03 solution a t pH 7.6, a slow liberation of acid occurs. If NazSz03 is also present in the reaction mixture, a reaction occurs which consumes thiosulfate (Column 2). This finding indicates that in the course of its decomposi-
668
STEIN, MOORE, AND BERGMANN
tion, IV liberates reactive alkylating groups capable of combining with thiosulfate. A comparison of the data in Columns 2 and 3 of Table I reveals that the speed of the reaction of the sulfonium salt with thiosulfate is only slightly greater than is the speed of its decomposition in aqueous NaHCOa in the absence of thiosulfate. Further evidence for the formation of reactive products during the decomposition of IV is provided by an experiment performed in the presence of cysteine. When cysteine and IV are allowed to remain in aqueous bicarbonate a t 37", crystals slowly separate from the solution. The material thus formed has the structure V indicated below, and must have resulted from the reaction shown in Equation 2.
c1-
+/ /HZ
\ CHlsS
S
\
CHz CH2 OH 2"
CHz CH2 OH CH2 CHzOH
+
I
2HSCHzCHCOOH
-
/ +\ CH2 CH2 OH c1-
CHzCHzS
(IV) 2"
/ S \
I
CH2 CHzSCHzCHCOOH
+ CHzCH2SCHtCHCOOH
I
/ 2s \
CHZ CH2 OH
+
2HCl
2.
CH2 CH2OH
"2
(VI The presence of TG in the reaction mixture has not been verified experimentally. Compound V has been isolated previously as a result of the reaction of H itself with two equivalents of cysteine (15). The formation of V coupled with the observations with thiosulfate reported above, make it appear that the sulfonium groups of IV possess, to a lesser degree, some of the reactive alkylating properties associated with the 8-chloroethyl groups of H. This observation makes it appear probable that the toxicity of some sulfonium salts may be a reflection of their ability to decompose under physiological conditions with the formation of reactive, toxic products. Further data on this point will be presented in a subsequent paper of this series (16). The chemical properties of the sulfonium salt II. Rydon first prepared the sulfonium salt I1 by allowing equivalent molar quantities of H and TG to react
REACTIONS OF MUSTARD GAS.
669
I
in alcohol a t room temperature for a week (11). The sulfonium compound waa isolated by Rydon as the chloride, which he found to be a hygroscopic oil, unstable a t room temperature. On standing for several days it decomposed with the formation of H and IV. If, however, picrylsulfonic acid is added t,o the week-old alcoholic reaction mixture described by Rydon, the pure picrylsulfonat,e of I1 separater, a t 0" in crystalline form: The picrylsulfonate of I1 is not hygroscopic and has shown no tendency to decompose after storage a t 0" for a period of month^.^ Neither has decomposition been observed after repeated short periods of exposure to room temperahre aggregating many hours. TABLE I REACTIONOF BI8-@-[BIS(~-HYDROXYETHYL)SULFONIUM]ETEYLSULFIDEDICHLORIDE (Iv)WITH SODIUM THIOSULFATE AND SODIUM BIC.4RBONATE Column 2: conc. of reactants per cc.: 0.05 m M of IV 0.12 m M of NaHCOs 0.10 m M of Na2S208 Temperature, 37'; pH 7.3. Column 3: conc. of reactants per cc.: 0.05 m M of IV 0.12 mM of NaHCOa Temperature, 37"; pH, 7.6.
THE
LIBERATED PEB mhf OF IV, M.EQUIV.
TIM& HOURS
THIOSULFATE UPTAKE PEP mhf OF IV, M.EQUIV.
1
2.
3.
0.08 0.37 0.80
0.08 0.34 0.68 0.90 1.06
2 17.5 41.5 65.5 89.5
1.10
1.40
The picrylsulfonate of I1 is soluble in about 10 volumes of acetone or methylcellosolve. It has a solubility of about 1% in water, and is very sparingly soluble in ethanol and methanol. The picrylsulfonate of I1 decomposes in aqueous methylcellosolve (1 part methylcellosolve plus 4 parts water) a t pH 7.5-8.0 and 25' with the liberation of one equivalent of chloride ion and nearly two equivalents of acid. As may be seen from Columns 2 and 3 of Table 11, the reaction proceeds in two stages. The first stage, in which one equivalent of acid and all of the chloride ion are liberated, has a half-time of about 2.5-3 hours and is complete in less than 24 hours; whereas the second stage, in which the second equivalent of acid is liberated, is much slower and has a half-time of about 2 days. Rydon (11) found that, in 0.01 molar aqueous solution a t 30" and pH 7.4, the chloride of I1 liberated acid, the half-time for the liberation of one equivalent of acid being about 3 hours. 'Compound IV also forms a n insoluble picrylsulfonate, the preparation of which is described in the experimental section. &Likemany polynitro compounds, the picrylsulfonates darken on prolonged exposure to light. The picrylsulfonate of I1 should, therefore, be stored in a dark place.
670
STEIN, MOORE, AND BERQMANN
I n order to elucidate the mechanism of the decomposition of 11, isolation of intermediate hydrolysis products was undertaken. For this purpose, the picrylsulfonate of I1 was allowed to hydrolyze in unbuffered aqueous methylcellosolve solution for 16 hours at 25". After this time 0.95 equivalents of C1- and 1.2 equivalents of H+ had been formed. (The liberation of C1- and the first equivalent of H+ is somewhat slower in unbuffered solution than it is a t pH 8.) The hydrolysate was found to contain a mixture of compounds. From it both the dipicrylsulfonate of IV, and the picrylsulfonate of the intermediate (111), have been isolated. The picrylsulfonate of IV was obtained in a yield of about 5% of the theory, whereas the picrylsulfonate of I11 was obtained in a yield of about 20% of the theory. TABLE I1
REACTION OF ~-CHLOROETHYL-~-[BIS(~-HYDROXYETHYL)SULFONIUM]ETHYLSULFIDE PICRYLSULFONATE WITH WATER A N D WITH THIOSULFATE Composition of reaction mixture per cc.: 0.0305 mM of sulfonium salt 0.09 mM of NaHCOs I n the experiment given in Column 4,O.075 m M of KazSzOsper cc. also was present. pH, 7.5-8.0; temperature, 25"; solvent, aqueous methylcellosolve ( 4 : l ) . LIBERATED PER ?ldf OF SULFONIUM SALT, M.EQUIV.
CITIME, HOURS
1.
1.75 3.75 4.0 6.25 7.0 23 47 71 95 167
H+ LIBERATED
PER mM OF SULFONIUM SALT, M.EQUIV.
2.
3.
0.38
0.33
0.63
0.62
0.70 1 .oo 1.01
0.85 1.31 1.51 1.64 1.72 1.84
THIOSULFATE CONSUMPTION PER mM OF s u b PONIUM SALT, M.EQUIV.
4.
0.46 0.77 0.93 1.31 1.51 1.59
The formation of I11 proves that hydrolysis of the chloroethyl group of I1 is a primary step in its decomposition (cf. Figure 1). The formation of IV probably arises as a result of the reaction of I1 with some TG which is formed during hydrolysis. It has been found that if I1 is allowed to hydrolyze in the presence of TG (2 equiv.), the H+ liberation is only half that observed in the absence of TG, whereas the C1- liberation is the same in both cases. This observation indicates that I1 reacts with TG. The reaction product, IV, has been isolated as a picrylsulfonate. The evidence presented above offers support for the mechanism of the hydrolysis of H in moderate quantities of water presented in Figure 1. It should be mentioned that Rydon (11) on the basis of his work with I1 has come to substantially the same conclusions relative to the hydrolysis of this compound. He also has obtained some evidence that the sulfonium chloride is capable of regenerating a small quantity of H during its decomposition in water.
REACTIONS OF MUSTARD GAS.
I
671
As may be seen from Column 4 of Table 11, the picrylsulfonate of I1 reacts with thiosulfate. This reaction also proceeds in two stages. The first stage, in which one equivalent of thiosulfate is consumed has a half-time of about 2 hours, and is 90% complete in about 6 hours. The rate of the consumption of the first equivalent of thiosulfate is somewhat faster than the rate of the liberation of chloride ion in the absence of thiosulfate. It would appear probable that it is the chloroethyl group which reacts with the first equivalent of thiosulfate. The second stage of the reaction is much slower; after 71 hours, 0.59 equivalents of additional thiosulfate are consumed. This second reaction proceeds a t a rate comparable to that of the liberation of the second equivalent of acid in the absence of thiosulfate. The second reaction probably involves the slow decomposition of the sulfonium group of the compound, and is analogous to the similar reactions discussed earlier for IV. The ,sulfoniumsalt I1 has a noteworthy toxicity. The LDw for this compound in mice is about 1.2 mg./kg., thus making it even more toxic to this species than is H itself. The pharmacological and toxicological properties of this substance are presented elsewhere (17). Although there is no direct evidence indicating that sulfonium salts are formed from H in vivo, the toxicity and mode of formation of the sulfonium salts described in this paper raise the possibility that these compounds may play a role in the physiological action of H. EXPERIMENTAL
The extent of sulfonium salt formation on hydrolysis of H . The mustard gas used in this work was a pure sample made by the thiodiglycol process. The H was shaken with the requisite amount of water (50,200, or IO00 volumes) a t room temperature until a clear solution resulted (18-24 hours). The solution was titrated to phenolphthalein with standard alkali, thus permitting a calculation of the acidity produced on hydrolysis. The neutralized solution was heated t o 100" for 1hour, and the amount of acid produced thereby again titrated. This figure furnished an index of the extent of sulfonium salt formation. Isolation of sulfonium salts f r o m H hydrolysates. H (20 cc.) was shaken at room temperature with 1 liter of water for 16 hours. The resulting clear solution was concentrated in vacuo to a sirup (bath temperature, 40-45'). Alcohol was added and the mixture again concentrated t o a sirup. This procedure was repeated twice more with alcohol and twice with acetone. The sirup was further dried by trituration with acetone, and 100 cc. of absolute dcohol was added. The resulting clear solution, on standing a t OD, deposited 5.3 g. of crystalline IV. After one crystallization from absolute alcohol this material melted at 97-102'; yield 3.3 g . A further recrystallization from alcohol yielded 2.5 g. of pure product, m.p. 101-103'. A pure sample of IV melts at 103" (see below). To the alcoholic solution from which IV had been removed, 3 g. of picrylsulfonic acid was added. On cooling to 0" an oil separated which gradually solidified on standing. It was obviously a mixture, and was not investigated further. The bulk of the solution was decanted from this material, and 7 g. of picrylsulfonic acid added to the solution. On cooling t o 0", 10.4 g. of the crystalline picrylsulfonate of I11 was obtained; m.p. 76-78'; mixed m.p. with a sample of the same compound prepared by hydrolysis of the picrylsulfonate of 11 (see below), 76-77'. Anal. Calc'd for C ~ H I ~ O & . C ~ H ~ NC, ~ O32.4; & ~ H, : 4.1; N, 8.1; S, 18.5. Found: C, 32.6; H, 3.9; N, 8.0; S, 18.7. Preparation of bis-8-[bis(@-hydroxyethyl)sulfonium]ethylsulfide dichloride ( I V ) . H (5 cc.) was shaken for 24 hours with 250 cc. of a n aqueous solution containing 20 cc. of TG. The resulting clear solution was concentrated in V ~ C U Ot o a sirup (bath temperature 40-45").
672
STEIN, MOORE, AND BERGUNN
The sirup was triturated repeatedly with acetone, and the acetone discarded. The sticky residue was taken up in about 150 cc. of boiling absolute ethanol, the solution filtered, and stored a t 0". The crystalline sulfonium salt was filtered, washed with cold ethanol, filtered, and dried; yield 10.5 g.; m.p. 101.5". This material is analytically pure, but the melting point may be raised t o 103" by one further crystallization from alcohol. Davies and Oxford (6) report the melting point 101.5-103". Anal. Calc'd for C1zHz&1n04Sa:C, 35.7; H, 7.0; C1, 17.6; S, 23.8. Found: C, 35.8; H, 7.1; C1, 17.4; S, 23.6. The sulfonium dichloride forms a chloroplatinate of the composition C12H280&*PtCle, which crystallizes as microscopic needles melting at 138". Preparation of bis-@-[bis(@-hydroxyethyl)sulfonium]ethylsulficledipicrylsulfonate. A solution of 2 g. of IV in 25 cc. of water was added to a solution of 3.65 g. of picrylsulfonic acid in 25 cc. of water. An oil separated which crystallized on scratching. After standing at 0" overnight, the dipicrylsulfonate was filtered off and washed with water and ethanol. For recrystallization the salt was dissolved in 45 cc. of acetone plus 15 cc. of water, filtered, and 150 cc. of ethanol added to the filtrate. An immediate crystallization occurred. After standing overnight at O", 3.6 g. of material was obtained; m.p. 138-139". A n a l . Calc'd for C ~ Z H Z E O * S ~ . ~ C ~ H C,~31.45; N ~ OH, ~ S3.5; : N, 9.2. Found: C, 31.4; H , 3.7; N, 9.3. The reaction of IV with sodium thiosuljale and sodium bicarbonate. The reaction mixtures were made up t o the concentrations indicated in Table I. COZwas bubbled through the NalS20s solutions (which contained a small amount of borax as a stabilizer) to bring them t o the desired pH. The thiosulfate consumption was determined by iodimetric titration in the usual manner. The extent of the liberation of hydrogen ions was determined in the following manner: T o a 10-cc. aliquot of the NaHCOa solution containing IV, 15 cc. of 0.1 N HC1 was added, the C 0 2 removed in vacuo, and the solution titrated t o phenolphthalein with 0.1 N NaOH. A control NaHC03 solution (containing no sulfonium salt) was treated in the same manner. The difference between the amounts of NaOH required by the two solutions is equal to the amount of hydrogenions liberated during the decomposition of IV. Controls were run t o prove the stability of the NaaSaOa and NaHCOs solutions under the conditions of these experiments. The reaction of IV with cysteine. Cysteine HC1 I788 mg. (5 mkf)], 1.0 g. (2.5 m M ) of IV, and 925 mg. (11 m M ) of NaHC03 were dissolved in 50 cc. of On-free water and kept at 37" under N2. Crystals appeared in the reaction mixture after 18 hours. After 2 days the crystalline material was filtered off, washed, dried, and analyzed; yield 0.26 g. The nitroprusside test in the presence of cyanide was negative. The material was soluble i n acids and alkalies, but insoluble in water. C, 36.6; H, 6.1; N, 8.5. Anal. Calc'd for CIGHZON~O$~J: Found: C, 36.3; H, 6.2; N, 8.3. Preparation of ~-chloroethyl-~-[bis(@-hydroxyethyl)sulfonium]ethylsulfide picrylsulfonate. H (40 g.) and T G (30.5 g.) were dissolved in 95% ethanol, made up to a volume of 1 liter with ethanol, and the mixture was allowed to stand at room temperature for a week. The solution was cooled t o 0", 30 g. of picrylsulfonic acid in 100 cc. of 95% ethanol was added, and the mixture was kept at 0" overnight. The picrylsulfonate crystallizes in large, heavy crystals, which are occasionally contaminated with small quantities of lighter crystals which can be removed by decantation. The salt was filtered, washed with alcohol, and dried over Pz06in vacuo at 0"; yield 16.4 g.; m.p. 89-91'. The mother liquor was allowed t o stand at room temperature for 4 days, 20 g. of picrylsulfonic acid added, and the mixture kept at 0" for 2-3 days. Twelve and two-tenths grams of sulfonium picrylsulfonate (m.p. 89-91') was obtained. A repetition of this procedure yielded another 8.6 g. of salt. For recrystallization, 10 g. of the picrylsulfonate was dissolved in 100 cc. of acetone, filtered, and 300 cc. of absolute ethanol was added. The clear solution was concentrated in vacuo (bath temperature, 40") until a faint turbidity appeared, and placed at 0" overnight.
REACTIONS OF MUSTARD GAS.
I
673
The crystallise product was filtered off, washed with ethanol, and dried as before; yield 8.5 g. The picrylsulfonate was stable on storage for at least 4 weeks a t room temperature, as evidenced by the fact that no change in melting point resulted after this treatment. The picrylsulfonate is practically pure as i t separates from the reaction mixture. The analyzed preparation had the melting point 91-92'. Anal. Calc'd for C~Hl&lO2S2.CsH2NsOeS: C, 31.3;H, 3.8; N, 7.8;S, 17.8;C1, 6.6. Found: C, 31.4;H , 3.8; N, 7.9; S, 18.2;C1, 6.6. Reactions of ~-chloroethyl-@-[bis(~-hydroxyethyl)sulfonium]ethylsulfide picrylsulfonate with water and with thiosulfate. I n the experiments recorded in Table 11, the powdered salt wa:3 dissolved in methylcellosolve, and an aqueous solution of the other reactants was added. I n the experiment employing Na2S203, the reaction mixture was brought t o p H 7.5 with Con. For the various analytical determinations, 10-cc. aliquots of the reaction mixture were withdrawn. For the determination of the chloride ion liberation, 0.02 N AgNOs solution was employed (dichlorofluorescein as indicator). Hydrogen ion liberation was determined in the manner already described for similar experiments with compound LV, except that 0.05N acid and alkali were employed. Thiosulfate uptake was determined by titration with 0.05 N IZsolution, the titration mixture being cooled in ice-water t o prevent, reaction of the IOwith the methylcellosolve. It should be mentioned that sodium picrylsulfonate reacts slowly with Na2S208. A control experiment indicated that the extent of this reaction was negligible for the first 7 hours. Hence, the thiosulfate consumption in the first stage of the reaction of the sulfonium picrylsulfonate with thiosulfate can all be attributed to reaction with the sulfonium salt. Iluring the second or slower stage, however, a control containing sodium picrylsulfonate and no sulfonium salt must be run simultaneously, and its thiosulfate consumption deducted from that of the reaction mixture containing the sulfonium salt. The initial thiosulfate concentration in this control, however, was 0.045 m M per cc., since the thiosulfate concentration in the reaction mixture containing the sulfonium salt a t the start of the second stage reaction had been reduced to this value as a result of the more rapid first stage reaction. Isolation of the products of the reaction of @-chloroethyl-~-[bis(j3-hydroxyethyl)sulfonium]ethylsulfide picrylsulfonate with water. The sulfonium picrylsulfonate (4.04 g.) was dissolved in 50 cc. of methylcellosolve and made up to a volume of 250 cc. with water. The mixture was kept at 25" for 16 hours, after which time a 10-cc. aliquot was withdrawn for the determination of Hf and C1- content. The results indicated that 1.2 equivalents of H+ and 0.95 equivalents of C1- had been formed. The bulk of the reaction mixture was concent>ratedin uacuo (bath temperature 40-15") to a sirup. Acetone was added and the mixture was concentrated again. This was repeated three times. The sirup was dissolved in acetone, and 200 mg. of crystalline material was filtered off; m.p. 133-135'; yield 5%. The crystalline precipitate was dissolved in aqueous acetone (1 part water and 3 parts acetone) and precipitated by addition of ethanol; m.p. 133-135". The dipicrylsulfonate of IV melts at 138-139". A mixed melting point of the isolated compound and an authentic sample of the dipicrylsulfonate of IV was 133-135'. The acetone solution obtained above was concentrated in uacuo to a sirup, and CHCls added. The mixture was cooled to 0", after which the CHCla could be decanted from the sticky oil. The oil was dissolved in acetone, filtered, and ether was added to the filtrate. After cooling to 0" and scratching, the oil which had formed soon crystallized; yield 2.6 g. The substance melted over a range beginning at 65". The crystalline material was treated with 100 cc. of hot absolute ethanol, and the solution decanted from a small oily residue. On cooling the solution to room temperature, a sticky solid deposited which was removed by filtration. Three volumes of ether was added to the filtrate. After cooling t o O", 750 mg. of the crystalline picrylsulfonate of I11 was obtained; m.p. 73-76" with preliminary softening. The yield corresponds to about 20% of theory. For analysis the compound was recrystallized from acetone with the addition of ether, the first fraction which precipitated being discarded; m.p. 76-78'.
674
STEIN, MOORE, A N D BERGbfANN
Anal. Calc'd for C ~ H l g O ~ S 2 - C ~ H ~ N aC, O g32.4; S : H, 4.1; N, 8.1; S, 18.5. Found: C, 32.2; H, 4.2; N, 8.1; S, 18.6. Reaction of ~-chloroethyl-~-[bis(~-hyd~oxyethyl)suZfonium]ethylsu~~de picrylsulfonate with thiodiglycol. The picrylsulfonate [970 mg. (1.8mM)] was dissolved in 12 cc. of methylcellosolve and 48 cc. of water containing 440 mg. (3.6m M ) of TG was added. After standing for 24 hours a t 25", the mixture was cooled to 0". Seven hundred milligrams of the dipicrylsulfonate of IV, m.p. 138-139', was obtained. This melting point is the same as that of an authentic sample of the salt. A mixed m.p. showed no depression. Anal. Calc'd for C12H2a04Sa.2CeH2NsOoS:C, 31.5;H, 3.5; N,9.2. Found: C,31.3; H, 3.6;N, 9.2.
The authors wish to acknowledge with thanks the helpful cooperation of Mr. Stephen M. Nagy, who performed the microanalyses reported in this paper. NEW YORIC, N. Y. REFERENCES (1) SARTORI,"The War Gases", New York, 1940. (2) OGSTON,(1941)." (3) CLARK,COREN,AND HARRIS,(1942).b (4) DOEBINCAND LINSTEAD,(1942).b (5) BARTLETT,(1943). (6) DAVIESAND OXFORD,J . Chem. SOC.,224 (1931). (7) NORTHROP, (1942). (8) OGSTON,(1943)." (9) OGSTON,(1941)." (10) GRANTAND KINSEY,(1943).b (11) RYDON,(1943)." J. Org. Chem. (paper IV this Benes). (12) STEINAND FRUTON, (13) SMITH,ADDIS,BEVERLANDER, CRAWFORD, GRAEF,HUBBARD, JAMES, AND G R N O F S K Y , (1942). (14) MCMASTER AND HOGEBOOM, (1942). (15) HELLERMAN, PORTER, AND PRESTA, (1942).' (16) STAHM.4NN, FRUTON, AND BERGMA", J. OTg. C h m . (paper VI this Series) (17) SMITH,JAOER, HOIE, ELLIS,GRAEF,BEVELANDER, SUMMERS, CRAWFORD, AND WINQ, (1943).
=Unpublished data obtained in Great Britain. Wnpublished data obtained in the United States.
