[CONTRIBUTION FROM TE~E LABORATORIES OF TBE ROCKEFELLEB INSTITUTE FOR MEDICAL RESEARCH]

CHEMICAL REACTIONS OF MUSTARD GAS AND RELATED COMPOUNDS.' V. THE CHEMICAL REACTIONS OF

1,2-BIS (a-CHLOROETHYLTHI0)ETHANE WILLIAM H. STEIN, JOSEPH S. FRUTONa, AND MAX BERGMANNa

Received March 98,1946

The experiments outlined in this communication were undertaken as part of a general investigation of the chemical reactions of the vesicant war gases. An attempt has been made to study the reactions of 1,2-bis(/3-chloroethylthio)- ethane (I) with water and with various substances of biological interest.

CHzSCHz CH2 C1

CH2 SCHz CHa C1 I

Interest in the chemical reactions of I arises from its structural relationship to mustard gas ~is(~-chloroethyl)sulfide] and from the fact that, like the mustard gas, I is a potent vesicant when applied to the skin. These vesicants differ markedly, however, in their physical properties. I is a solid melting at 54" while mustard gas is a liquid at room temperature. In addition, I is much less soluble in water than is mustard gas. T h hydrolysis of 1,6-bis(8-chloroethylthio)ethune ( I ) . When a suspension

of I is shaken with 50 volumes of water a t room temperature, all of the carbon- bound chlorine is hydrolyzed to C1- in 48 to 72 hours? On the other hand, the H+ liberated after 72 hours' shaking equals only 8045% of the amount to be expected on complete hydrolysis of I to yield two equivalents of HC1. The extent of H+ liberation does not surpass 85% of theory after an additional 24 hours' standing at room temperature. This indicates that about 15% of the C1- is not neutralized by H+, but by sulfonium ions. The extent of H+ liberation may be raised to 95% of the theory if the 72-hour hydrolysate of I is neutralized and then heated at 100" for 2 hours.

The rate of the liberation of C1- and H+ when I is shaken with 50 volumes of water is shown in Table I. It will be noted that in the course of the hydroly- sis C1- is formed faster than is H+, and that H+ continues to be formed after all the C1- has been liberated. This observation makes it probable that some of the sulfonium salts formed during the hydrolysis of I are unstable. Thus, after 28 to 54 hours' shaking, about 30% of all the C1- present in solution is

1 This work was done in whole under Contract No. 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.

2 Present address, Yale University, New Haven, Connecticut. a Died, November 7,1944. 4 Since I is extremely insoluble in water, the rate and extent of hydrolysis must depend

upon the method of agitation and the particle size of the I employed. In these experiments no attempt wasmade to control these factors.

692

REACTIONS OF MUSTARD GAS. V 693

0.17 0.52 1.57

1.98 2.00

neutralized by sulfonium ion, while after 72 hours only about 15% is neutralized by sulfonium ion.

The hydrolysis of I on shaking with an aqueous NaHC08 solution (pH 8) appears to be somewhat slower than the hydrolysis of I shaken with water (cf. Table 1). The amount of sulfonium ion at pH 8 is, at any given time, less than that found in the absence of bicarbonate.

After I has been shaken for 72 hours with either water or aqueous NaHCOa, a precipitate is present in the reaction mixture. From 10 g. of I shaken with 50 volumes of water, 2.5 g. of insoluble material was obtained. This material, after two crystallizations from absolute ethanol, melted at 99-101" and contained no chlorine. The elementary analysis of the substance corresponded well with

0.13 0.04 0.14 0.32 0.20 0.33 1.06 0.51

1.16 1.41 0.57 1.74 0.26 1.99

TABLE I HYDROLYSIS OF I IN WATER AWD AQUEOUS NaHCOs

TDIZ. xnms

--- 2.5 6.5 28 30.5 54 72

WATER (50 VOLUXES) I NaHCOi (0.3 N; 50 vnmums)

Cl- liberated H+ liberated (cr) - !H+), Ci- liberated

m.equiv. m .equiv . m.equiv. per m+ I, per 0% 1, m.equ,v. per + 1, H+ liberated

m .equiv . per + 1, 0.13 0.25

0.93

1.91

(ClWH+), m.equiv.

0.01 0.08

0.23

0.08

the empirical formula CIOHBO~S~. It was suspected that this product was 1,2-bis[2-(2-hydroxyethylthio)ethylthio]ethane (11), which may also be desig- nated pentaethylene tetraaulfide-o , o'-diol.

For purposes of comparison with the isolated material, the tetrasulfide diol was synthesized by reaction of I with two equivalents of P-mercaptoethanol (111) in the following manner: CH2SCH2CH2 C1 CH2SCH2 CH2SCHzCH2 OH

CH2SCHzCH2SCH2C;HzOH CH2SCH2CH2 C1 + 2HSCH2CH20H -+ I I

The product obtained from the above reaction of I with 6-mercaptoethanol was found to have the elementary composition required for compound 11, and to have the melting point 108-111°. As was mentioned above, the product isolated from the hydrolysate of I had the same elementary composition, but the lower melting point 99-101'. Further recrystallization of this isolated

694 STEIN, FRUTON, AND BERGMANN

material [from water: ethanol (2:l) instead of from absolute ethanol] raised the melting point slightly to 102-104°. The mixed melting point of the two substances was 101-109". In view of the discrepancy in melting points, proof of the identity of the isolated and synthesized products required the prepara- tion of derivatives. For this purpose the diacetate and dibenzoate of both sub- stances were prepared. The melting point of the derivative of the compound isolated from the hydrolysate of I was, in each case, the same as that of the corre- sponding derivative of the substance synthesized from I and 0-mercaptoethanol. The elementary analyses were in good agreement with the theory, and the mixed melting points of the derivatives of the isolated and synthesized products showed no depression. The derivatives of the product isolated from the hydrolysate

X CH&HzSCHzCHnSCHzCHzOH + ClCHzCHzSCHzCHzSCHz CHzX

I c1- + CH;I

X C H Z C H ~ ~ C H , CHzSCHaCH,S CHZCHnSCHnCHzX I I

CHzOH

(Iv)

i X CHZCH~SCH~CHISCH~CHPSCH,CHZSCH~CH~X + HOCHzCHzOH + HCl

(VI (X = either C1 or OH)

FIGURE 1

of I were more difficult to purify and were obtained in lower yield than were the corresponding derivatives of the synthetic product. It appears, therefore, that the substance isolated from the hydrolysate of I consisted largely of pentaethylene tetrasulfide-w ,w'-diol, but contained as impurities some sub- stances of similar properties which were difficult to remove.

The mechanism by which the tetrasulfide diol is formed on hydrolysis of I, and the probable nature of the impurities mentioned above, are discussed in what follows.

The tetrasulfide diol is probably formed as a result of the reaction of I chloro- hydrin (or I) with I glycol (or I chlorohydrin) and subsequent decomposition in the manner indicated in Figure 1.

It cannot be stated at this time whether sulfonium salt formation and sub-

REACTIONS OF MUSTARD GAS. V 695

sequent elimination of ethylene glycol occurs prior to hydrolysis of the two chlorine atoms indicated in Formulas IV and V by an X. It seems highly probable, however, that the sulfur atom which reacts to become a sulfonium sulfur bears a hydroxyethyl and not a chloroethyl group. It is known that the tendency to form sulfonium salts is much greater with the group HOCHZCHZS- than it is with the group C1CH2CH2S-. It also seems probable that ethylene glycol, not ethylene chlorohydrin, is eliminated from compound IV. The chlorine of ethylene chlorohydrin is relatively resistant to hydrolysis in water, whereas, as was shown earlier, all the chlorine of I is liberated as chloride ion.

If the tetrasulfide diol actually is formed by a mechanism such as that por- trayed in Figure 1, it is easy to see how similar reactions might lead to the for- mation of other sulfides of analogous structure, and thus account for the presence in the tetrasulfide diol of the impurities referred to above. The intermediate sulfonium salt (IV) was assumed, in Figure 1, to decompose by fission of a carbon- sulfur bond, thus yielding the tetrasulfide diol (11). It also seems possible, however, that the sulfonium salt IV might decompose in a different manner with th.e resulting formation of a tetraethylene trisulfide diol.

Furthermore, by analogy with mustard gas, I might give rise to a sulfonium salt formed from one molecule of I and two molecules of I-glycol. This sul- fonium salt could decompose in several ways to yield a variety of products. Depending upon the particular carbon-sulfur bond broken, there would result the tetrasulfide diol (II), a hexaethylene pentasulfide diol, or a heptaethylene hexasulfide diol.

It should be pointed out that elementary analysis would scarcely distinguish between these various compounds. The carbon and hydrogen content is prac- tically the same for all of the substances. The sulfur content is 39.7% for the trisulfide diol, 42.4% for the tetrasulfide diol, 44.2% for the pentasulfide diol, and 45.5% for the hexasulfide diol. The various compounds would also un- doubtedly have similar properties and would be difficult to separate from one another. The presence of such a mixture of substances in hydrolysates of I would readily explain the difficulties encountered in purifying the tetrasulfide diol. It should be emphasized that much of the foregoing is speculative and would prove difficult to establish in detail. No attempt was made to isolate all the various homologs of the tetrasulfide diol which might be present in a hydrolysate of I. It should be mentioned that, in addition to the tetrasulfide diol, I-glycol (VI) has also been isolated from a hydrolysate of I in a yield of 20-25y7 of the theory. This substance would result, of course, from the hy- drolysis of the two carbon-bound chlorine atoms of I.

CHZSCHZCHZOH I CHzSCH2CHzOH

As will be noted from Formula VII, p-chloroethyl-l,4-dithiane sulfonium It seemed possible, therefore, that VI1 might arise chloride is isomeric with I.

696 STEIN, FRUTON, AND BERQMA"

during the hydrolysis of I. Attempts to demonstrate the formation of VI1 during the hydrolysis of I, however, have been unsucces3ful.

CHz CHz c1-, \

ClCHz CHZ S+' 'S /

CHz CH2 \

After shaking I with water for 48 hours and removal of the precipitate, the a t ra te was neutralized and boiled under reflux for 4 hours. Under these con- ditions, 1,4-dithiane sulfonium compounds decompose readily to yield 1,4di- thiane which sublimes into the reflux condenser (2). However, in the case of the hydrolysate of I no dithiane formation was observed on boiling. Similarly, when I was boiled under reflux with 50 volumes of water for 24 hours, no evidence for the formation of 1,4-dithiane was obtained.

The reaction of I With thiodiglycol and pgridine. It has been shown that mus- tard gas reacts with thiodiglycol (TG) in aqueous solution at room temperature to form a sulfonium salt derived from one molecule of mustard gas and two molecules of TG (1). This compound was isolated as a chloride and as a pic- rylsulfonate (VIII). It seemed of interest to determine, therefore, whether I behaves similarly. Accordingly, I was shaken with an aqueous solution of TG for 4 days. At the end of this period, the amount of C1- and H+ present in the solution waa determined.

HO CHz CHz CHz CH2 OH

(VIII)

HO CH2 CHa' 'CH~ C H ~ OH P P

P- = picrylsulfonate ion

It was found that only about 1570 of the C1- present in the reaction mixture could be accounted for as HC1. The remainder of the C1-, therefore, must have been neutralized by sulfonium ion. It waa shown in the preceding sec- tion of this paper that when I was shaken with water alone, 80-85 per cent of the theoretical H+ was formed. It follows, therefore, that in the presence of excess TG, I forms a sulfonium salt. When the reaction mixture containing the sul- fonium salt is heated at 100' for 2 hours, acid is formed. The extent of H+ liberation is about 90% of that to be expected from complete hydrolysis of the I employed. Thus the sulfonium salt formed from I and TG is unstable a t 100' in aqueous solution. This behavior is similar to that exhibited by the sulfonium salts formed from mustard gas and TG.

The product of the reaction between I and TG has been isolated as its crystal- line dipicrylsulfonate (IX).

REACTIONS OF MUSTARD GAS. V 697

HO CH2 CH2 CH2 CH2 OH \+ /

S CH2

HO CH2 CH2' P- P-

P- = picrylsulfonate ion

As shown in Paper I of this series, the corresponding sulfonium salt derived from mustard gas was shown to decompose with the liberation of acid on stand- ing in aqueous NaHC03 a t 37", and to react slowly with both NazSaOs and the

TABLE I1 THE CHEMICAL REACTIVITY OF THE SULFONIUM SALTS VI11 AND IX

Concentration of reactants per cc.: 0.005 mM of sulfonium salt in all experiments. 0.025 mM of NaHCOs in Columns 2, 3, 4 , and 5. 0.025 mM of Na$%Os in Columns 4 and 5.

Solvent, acetone-water (1: l ) ; temperatui ---

TME, EOUBS

1.

:3 1'7 16.5 21 47 71 74 92 98

138

H+ LIBERATED IN NaHCO: SOLUTION PEE mM OF

IX, m.equiv. 2.

0.75

1.24 1.52

1.72

1.73

VIII, m.equiv. 3.

0.16

0.32 0.40

0.52

0.70

25".

Na& 01 CONSLTMED PER mM OF

IX, m.equiv. 4.

0.60

1.36 1.68

1.76

1.90

VIII, m.equiv. 5.

0.20

0.68 0.92

1.00

1.30

H* LIBERATED IN UNBUFFERED SOLU-

TION PEB mM OF

M, m.equiv. 6 .

0.16

0.75 1.12

1.52

1.68

SH group of cysteine at pH 8 and 37". The appreciable chemical reactivity of this sulfonium salt (VIII) is of interest in view of the marked toxicity of the compound. It seemed worth while, therefore, to study the properties of the sulfonium salt IX for purposes of comparison.

As can be seen from Columns 2 and 6 of Table 11, compound IX decomposes slowly in acetone-water solution with the liberation of acid, both in the presence and absence of NaHCOs. The rate of the decomposition of IX at pH 8 (Column 2) is much greater than is that of VI11 (Column 3) studied under the same condit,ions. As will be noted from the Table, IX reacts with thiosulfate (Column 4), the rate of this reaction also being far greater than that observed for VI11 (Column 5) . It would appear, therefore, that the sulfonium salt IX is markedly more reactive than is VIII.

When I is shaken with an aqueous solution of pyridine, only about 743%

698 STEIN, FRUTON, AND BERQUNN

SUBSTANCE

of the C1- present is bound as HCI. It would appear, therefore, that I reacts with pyridine to form a strong base (a pyridinium compound) which neutralizes the remaining C1-.

The extent of the decrease in amino nitrogen when amino acids are treated with I at weakly alkaline p H values is given in Table 111. The reactions were carried out by shaking I at room temperature for 5 days with an aqueous methyl cel- losolve solution of the amino acids. At the start of the reaction the vesicant was not completely in solution. The disappearance of amino nitrogen was followed by the Van Slyke nitrous acid method.

The reaction of I with the amino groups of amino acids and peptides.

PH

TABLE I11 REACTION OF I WITH THE AMINO GROUPS OF AMINO ACIDS AND PEPTIDES

Concentration of reactants per cc.: 0.205 mM of "2-N.

0.205 m M of NaHCOa. 0.0523 m M of I.

Solvent, 23% methyl cellosolve in water. Temperature, 20-25"; reaction time, 5 days.

ECBEASE IN "rb PES CC., M.EQUIV.

0.03% .0238 .0354 .0238 .0277 .0308 . (3438 ,0631 .0785 .M62 .0769 .0208

E C B E A S E IN "1-N PEB OF I,

M.EQU1V.

0.68 .46 .68 .46 .53 .58 .84

1.21 1.50 0.88 1.47 0.40

a One equivalent of NaOH also added.

It Rill be noted from Table I11 that, although I reacts at p H 8 with the amino groups of all the amino acids tested, the various amino groups differ in their reactivity towards I. Thus the amino groups of glycine, histidine, lysine, glutamic acid, and serine are more reactive than are those of alanine or arginine. I, therefore, appears to be more selectfive in its action on amino groups than are the nitrogen mustards, for it was found that the latter vesicants react to the same extent with the amino groups of all of these amino acids, with the exception of histidine and methionine (3). Sufficiently extensive data on mustard gas are not available for comparison.

From the data in Table I11 it would appear that the imidazole group of histi- dine is not competing very effectively with the amino group in the same molecule for reaction with I. It will be recalled that the imidazole ring reacts readily

REACTIONS OF MUSTARD GAS. V 699

with inustard gas (4) and with all the nitrogen mustards (5 ) . On the basis of the evidence given here and further data to be presented in the next section of this paper, it cannot be stated with certainty whether or not the imidazole group of histidine reacts with I.

As can be seen from Table 111, the amino group of glycylglycine, the only peptide investigated, reacts to a greater extent with I than do the amino groups of any of the amino acids. A similar observation was made with mustard gas and with the nitrogen mustards.

It will be noted that I, like mustard gas and the nitrogen mustards, reacts to a far greater extent with amino groups at p H 9.5 than it does at p H 8. At the higher p H the difference between the reactivities of glycine and alanine is accentuated. The behavior of phenylalanine resembles that of glycine rather than that of alanine. The amino group of methionine, on the other hand, reach to only a slight extent a t p H 9.5. It appears probable, therefore, that the --SCH3 group of methionine reacts with I to form a sulfonium salt, and that this reaction removes part of the I which would otherwise be available for reaction with the amino group. Mustard gas also reacts with the 4 C H S group of methionine to form a sulfonium salt (6).

The competitive e$ect of various substances on Ihe reaction of I with the amino group of glycine. As shown in the previous section, I reacts with the amino group of glycine and other amino acids. In order to study the reaction of various substances with I, their competitive effect on the extent of the reaction between I and glycine was determined. The competitive effect of such substances on the extent of the disappearance of the glycine NH2-N can be taken as an indication of the reactivity of these compounds with I.

The data in Table IV show that imidazole has an appreciable competitive effect on the reaction of I with glycine. The data presented in the previous section suggested that the imidazole ring of histidine did not compete appreciably with the amino group of this amino acid for reaction with I. The explanation for the difference between the effect of free imidazole and that of combined imidazole is not clear at present.

Both nicotinic acid and the corresponding amide react very readily with I, presumably with the formation of pyridinium derivatives. In this respect I resembles mustard gas (7) and the nitrogen mustards (5) which have been shown to react with pyridine nitrogen. In contrast to the appreciable reactivity observed with the aromatic tertiary nitrogen compounds such as pyridine, I reacts to a much lesser extent with aliphatic tertiary amines such as triethyl- amine or triethanolamine.

I reacts appreciably with organic phosphate compounds such as glycero- phosphate and with inorganic phosphate. This behavior is also shown by mus- tard gas (8) and the nitrogen mustards (5 ) . Like mustard gas, I reacts with the carboxyl groups of acetic acid or carbobenzoxyglutamic acid. Thiodiglycol also shows an appreciable competitive effect on the reaction of I with glycine. The product of the reaction between I and thiodiglycol has been isolated and was described earlier in this paper. I reacts with proline; however, the com-

700 STEIN, FRUTON, AND BERGMANN

0.70 -42 .20 .09 * 33 .31 .44 .33 .52 -56 .66 .37

petition data do not permit of a decision as to whether the imino group or the carboxyl group of the amino acid is involved. The reaction of I with thiosulfate and with cysteine. On shaking I for 72 hours

with thiosulfate, a clear solution resulted. Iodometric titration indicated that 1.92 equivalents of thiosulfate had been consumed. The product of the re- action of I with NazS203, the "Bunte salt" (X), has been isolated and found to have the expected composition.

0.28 .50 .61 .37 .39 .26 .37 .18 .14 .04 .33

TABLE IV COMPETITIVE EFFECT OF VARIOUS SUBSTANCES ON THE REACTION OF I WITH THE AMINO

GROUP OF GLYCINE Concentration of reactants per cc.: 0.205 m M of glycine.

0.205 m M of competing substance. 0.205 m M of NaHCOs. 0.056 m M of I.

Solvent, 23% methyl cellosolve in water. Temperature, 20-25"; pH 7.6-8.0; reaction time, 3 days.

CO-ETING SUBSTANCE

. . . . . . . . . . . . . . . . . Imidazole. . . . . . . . Nicotinic acid.. . . . . . . . . . . . . Nicotinamide. ...................... Na2HPOA. . . . . . . . . . . . . . . . . . .

Sodium acetate.. .. . . . . . . . . . . . . . . . . Carbobeneoxyglutamic acid.. . . 1-Proline . . . . . . . . Triethylamine . . . . . . . . . . . . . . . . . Triethanolamine . . . . . . . . . . . . . .

%

40 71 87 53 56 37 53 26 20 6

47

I also reacts readily with the sulfhydryl group of cysteine to yield the com- pound (XI).

-CH2SCH2 CHzSCHz CHCOOH

"2 I 1, (XI)

[ CH2SCH2CH2S20gNa

CH2SCH2 CH2S20sNa I

(X> This compound was prepared by a method similar to that employed by Hellerman et al. (9) for the synthesis of the corresponding derivative of mustard gas.

EXPERIMENTAL

Hydrolysis af 1 ,P-bis(j3-chloroethyIthio)ethane ( I ) . I was hydrolyzed by shaking with 50 volumes of water or 50 volumes of 0.3 N NaHCOI at room temperature. After filtering the mixture, the filtrate was made up to 100 cc., and C1- and H+ determinations were per- formed in the manner described for mustard gas (1) and the nitrogen mustard gases (10).

REACTIONS OF MUSTARD GAS. V 701

Isolation of pentaethylene tetrasuljtde-w,w'-diol from hydrolysate of I . I (10 g.) was shaken at room temperature for 5 days with 500 CC. of water. The precipitate which had formed was filtered off and dried; yield 2.1 g. This material should be handled cautiously since i t frequently is contaminated by traces of unchanged I. After removal of the crude tetrasulfide diol, the filtrate was kept at 0" overnight. The precipitate was iiltered off and recrystallized from ethanol, yielding a n additional 0.4 g. of tetrasulfide diol. The crude tetrasulfide diol was recrystallized twice from absolute ethanol and dried over PZOS in vacuo at room temperature; yield 1.4 g. of microscopic needles; m.p. 99-101' with pre- liminary softening a t 96". The compound contains no chlorine.

Anal . Calc'd for CldfZaO&: C, 39.7; H, 7.3; S, 42.4. Found: C, 39.7; H, 7.25; S, 42.3.

Further recrystallization of this product from water-ethanol (2: 1) raised the melting point slightly t o 102-104".

Synthesis of pentaethylene tetrasulfide-w, w'-diol f rom I and 6-mercaptoethanol. T o 2.2 g. (0.01 M ) of I in 40 cc. of ethanol were added, 3.1 g. (0.04 M ) of 8-mercaptoethanol in 5 cc. of water, and 2.6 g. (0.04 M ) of KOH (86%) in 20 cc. of ethanol. Nitrogen was bubbled through the mixture for 2 hours, and the mixture was allowed t o stand under NS for 48 hours. The precipitate which had formed was removed by filtration and dried; yield 2.6 g. After one crystallization from absolute ethanol, 2.0 g. of material melting at 102- 108" was obtained. After three more crystallizations from absolute ethanol the melting point was 108-111". The mixed melting point with the tetrasulfide diol isolated from a I hydrolysate was 101-109". The substance contained no chlorine.

Anal . Calc'd for C ~ I Z Z O ~ S ~ : C, 39.7; H, 7.3; S, 42.4. Found: C, 39.4; H, 7.15; S, 42.1.

Pentaethylene tetrasulfide-w , w'-diol diacetate. The pentaethylene tetrasulfide-w,w'-diol (600 mg.) obtained from the reaction of I with 8-mercaptoethanol was dissolved in 25 cc. of anhydrous pyridine and 0.8 cc. of acetic anhydride was added. The mixture was allowed t o remain at room temperature for 24 hours, and the pyridine removed in vacuo. The residue was crystallized twice from absolute ethanol and once from acetone and petroleum ether; yield 400 mg.; m.p. 85-87"with softening a t 83".

Anal . Calc'd for C14H2804S4: C, 43.5; H, 6.8; S, 33.2. Found: C, 43.3; H, 6.7; S, 33.0.

When the diol isolated from a hydrolysate of I was subjected t o the same procedure, 300 mg. of diacetate was obtained after two crystallizations from alcohol and two from acetone and petroleum ether; m.p. 81-83"; mixed m.p. 81-85".

Anal . Found: C, 42.9; H, 6.5. Further recrystallization of this product did not raise the melting point or change the

The mother liquors fromwhich this product was obtained yielded elemen1,ary composition. 75 mg. of the pure diacetate; m.p. 86-88', mixed m.p. 85-88'.

Anal . Found: C, 43.6; H, 6.8; S, 33.2. Pentaethylene tetrasuZficle-w,w'-diol dibenzoate. The pentaethylene tetrasulfide diol

(600 mg.) obtained from the reaction of I with p-mercaptoethanol was dissolved in 25 cc. of anhydrous pyridine and 1 cc. of benzoyl chloride was added. The mixture was allowed t o stand for 24 hours at room temperature, and the pyridine was removed in vacuo. The residue was crystallized twice from absolute ethanol, once from acetone and petroleum ether, and once from ether and petroleum ether; yield 300 mg.; m.p. 60-63" with softening at 58".

Anal . Calc'd for Cz4Haa04S4: C, 56.4; H, 5.9; S, 25.1. Found: C, 56.4; H, 5.8; S, 25.2.

The diol isolated from a hydrolysate of I was subjected t o the same procedure. Several recrystallizations from the above solvents were required before 175 mg. of the pure di- benzoate was obtained; m.p. 59-63"; mixed m.p. 59-63'.

Anal . Found: C, 56.4; H, 5.9; S, 25.1.

702 STEIN, FRUTON, AND BERGMANN

An additional 100 mg. of material, m.p. 58-62", was obtained from the mother liquors. Isolation of I-glycol f r o m a hydrolysate of I . I (10 8.) was shaken at room temperature

for 5 days with 500 cc. of water. After removal of the crude tetrasulfide diol from the hydrolysate of I by filtration, the filtrate was kept at 0" overnight. The precipitate which separated was iiltered off. The clear aqueous filtrate was concentrated t o a sirup in vacuo, alcohol was added and the mixture again was concentrated. This was repeated twice more. The sirup which remained was triturated with acetone, which dissolved about half of the sirup. The acetone solution was concentrated t o a sirup in vacuo, and the residue shaken with about 100 cc. of ether. A small amount of material did not dissolve and was discarded. Petroleum ether was added to the ether solution, and the mixture cooled t o 0". Crude I glycol (2 g.) (m.p. 35-46'} was thus obtained. After three recrystallizations from ether- petroleum ether the glycol melted a t 59-62"with preliminary softening at 57".

Anal . Calc'd for CeHlrO&: C, 39.5; H, 7.7; S, 35.2. Found: C, 39.3; H, 7.4; S, 35.1.

Rosen and Reid (11) give the m.p. 114-117" for I-glycol. Their product, however, con- tained 37.4% sulfur, and could not have been pure. Bennet and Whincop (12) give m.p. 64' for this substance. Their product gave the correct elementary analysis for carbon and hydrogen. A sulfur analysis was not reported.

Preparation of the sulfonium salt I X . Thiodiglycol (5 9.) was shaken with 2 g. of I i n 100 cc. of methyl cellosolve-water (1:3) for 4 days. The reaction mixture was filtered, and con- centrated in vacuo (bath temperature, 45") t o a sirup. Alcohol was added and removed in vacuo. This procedure was repeated twice more, the residue triturated with acetone, and the acetone decanted. Since the residual sirup could not be induced to crystallize, i t was dissolved in water and an aqeuous solution of 8 g. of picrylsulfonic acid added slowly with stirring. After the first 500 mg. of picrylsulfonic acid had been added, the solution was fil- tered, and the precipitate discarded. After all the picrylsulfonic acid had been added, the mixture was left at 0" overnight, the precipitate filtered off, and dried over PZOS in vacuo, at room temperature; yield 4.8 g.; m.p. 125-127'.

Anal . Calc'd for C ~ ~ H J Z O I S , . ~ C ~ H B N ~ O ~ S : C, 32.0; H, 3.7; N, 8.6; S, 19.7. Found: C, 32.2; H, 3.8; N, 8.6; S, 19.6.

The chemical reactivity of the sulfonium salts VI I I and I X . For the study of the hydroly- sis in unbuffered solution, IX was suspended in acetone, and water added slowly until the concentration reached about 10-15%. At this concentration the salt dissolved slowly, and after i t was in solution, sufficient water was added to make the ratio of acetone to water 1: 1. At the times indicated in Table 11, aliquots were withdrawn and titrated t o bromthymol blue with 0.1 N NaOH.

For the study of the rate of hydrolysis in NaHCOs solution, the solutions of both sul- fonium salts were prepared as above, the requisite amount of h'aHCOs being added to the clear solution. On standing, the reaction mixtures became red. At the indicated times, aliquots were withdrawn, acidified with a known quantity of 0.1 N HC1, and the solution concentrated in vacuo (bath temperature, 35") for about 10 minutes t o remove acetone and COZ. The mixture was titrated to bromthymol blue with 0.1 N NaOH. Because of the red color of the solution, the end point is not sharp, the change being from clear red to a muddy purple. The acid generated is represented by the additional quantity of 0.1 N NaOH re- quired t o neutralize the sulfonium salt solution over that required t o neutralize a con- trol solution treated in the same manner but containing no sulfonium salt.

For the reaction with thiosulfate, the solutions were made up as above, the requisite amount of NazSz0, being added. At the times indicated in Table 11, aliquots were with- drawn, Concentrated in VQCUO to remove acetone, and titrated with 0.1 N 12 solution. The solutions become highly colored on standing a t 25", thus making i t somewhat difficult t o observe the end point in the iodometric titration. The development of this color also in- dicates that the picrylsulfonate ion is undergoing some chemical reaction in the medium. It would seem unwise, therefore, to place too much reliance on the absolute values presented in Table 11. The fact that IX is much more reactive than VI11 is, however, not subject t o this uncertainty.

REACTIONS OF MUSTARD GAS. V 703

"Bunte salt" of I . I (1.1 g.) was shaken for 72 hours at room temperature with 2.5 g. of NaZS2O3.5H20 in 50 cc. of water. The solution was filtered and concentrated to dryness under reduced pressure (bath temperature, 40"). The residue was extracted with boiling ethanol, and the extract filtered. The filtrate was concentrated under reduced pressure t o a small volume, and the crystals which separated were filtered off, washed with ether, and dried in air; yield of crude product, 1.5 g. The product was recrystallized 3 times from hot ethanol.

And. Calc'd for C&2Na20&: C, 17.2; H, 2.9; S, 45.9; Na, 11.0. Found: C, 17.4; H, 3.1; S, 45.6; Na, 11.0.

Cysteine derivative of I ( X I ) . To 1.8 g. of I suspended in 10 cc. of ethanol, was added a solution of 2.6 g. of cysteine-HC1 in 5 cc. of water and 25 cc. of ethanol. Nitrogen was bub- bled through the reaction mixture and a solution of 2.17 g. of KOH (85%) in 15 cc. of ethanol was added. After 48 hours under nitrogen, 50 cc. of water was added. The mixture was filtered and the pH of the filtrate was adjusted to 6.5 with acetic acid. The mixture was chilled for 12 hours and the precipitate was filtered. The product was recrystallized twice by dissolving i t in concentrated ",OH and allowing the ammonia to evaporate a t room temperature; yield 1.2 g. (38% of theory).

Anal. Calc'd for CIZHZ~NZOL~,: C, 37.0; H, 6.2; N, 7.2; S, 33.0. Found: C, 36.7; H, 6.2; N, 7.0; S, 32.8.

The authors wish to acknowledge with thanks the helpful cooperation of Miss Rosalind E. Joseph and Miss Jean Grantham, who assisted in the conduct of these experiments, and of Mr. Stephen M. Nagy, who performed the micro- analyses reported in this paper.

Nnw YOBK, N. Y.

REFERENCES (1) STEIN, MOORE, AND BERGMANN, J. Org. Chem., (paper I, this series): (2) STAHMANN, FRUTON, AND BERGMANN, J. Org. Chem., (paper VI, this series):

(4) DU VIGNEAUD, MCKENNIS, MELVILLE, STEVENS, AND WOOD, (1942,1943)." (5) FRUTON, STEIN, STAHMANN, AND GOLUMBIC, J . Org. Chem., 11,571 (1946). (6) STEIN AND MOORE, J . Org. Chem., (paper 111, this series). (7) MOORE, STEIN, AND FRUTON, J. Org. Chem., (paper 11, this series). (8) OILMAN, GOODMAN, AND PHILIPS, (1943) .a

(9) HELLERMAN, PORTER, AND PRESTA, (1942)."

(3) ~PRUTON,STEIN,ANDBERGMAN~,~. erg. Chem., 11,559 (1946).

(10) GOLUMBIC, FRUTON, AND BERGMANN, J. Org. Chem., 11, 518 (1946). (11) ROSEN AND REID, J . Am. Chem. SOC., 44,634 (1922) (12) HENNET AND WHINCOP, J . Chem. SOC., 119, 1860 (1920).

0 Unpublished data obtained i n the United States.