CHEMICAL REACTIONS OF MUSTARD GAS AND RELATED

[CONTRIBUTION FROM T

~ LABORATORIES E OF THE ROCKEFELLER INSTITUTEFOR

MEDICAL RESEARCH]

CHEMICAL REACTIONS OF MUSTARD GAS AND RELATED COMPOUNDS.' VI. THE CHEMISTRY OF SULFONIUM SALTS RELATED TO MUSTARD GAS MARK A. STAHhlANX,2 JOSEPH S. FRC'TON,'

AKD MAX

BERGMANN4

Received March 22, 1946

In a previous paper of this series (I) i t was shown that, on hydrolysis with moderate quantities of water, mustard gas (H) gives rise to several different sulfonium salts. The interesting chemical and toxicological properties of these sulfonium derivatives prompted an investigation of the chemical and physiological properties of other sulfonium compounds of this type. Preparation and properties of tris(p-chloroethy1)sulfonium chloride. Tris(/3-chloroethyl)sulfonium chloride (11) was prepared by treatment of the corresponding hydroxy compound (I) with thionyl chloride according to the method of Ettel and Kohlik (2). HOCH2CHz Cl-

ClCHzCH,

\+

HO CH2 CHZ

+ CHz CH2 OH

(1)

3SOCl2

* ClCHz CHZ

C1-

\+ S / \

1.

CHzHzC1

(11)

So far as we are aware, compound I1 and its derivative (111) are unique in that they are the only compounds known in which the sulfur of bis(@-chloroethy1)sulfide is alkylated. In fact, there are several reports in the literature (2, 3, 4) which attest to the resistance to alkylation of the sulfur of 8-chloroethylsulfides. When I1 is dissolved in water, the pH falls rapidly to about 2.8. When the pH is raised by the addition of alkali, H+ and Cl- are formed in equivalent amounts, and a t a rate which is markedly dependent upon pH (Table I). Thus, a t pH 3.0, the half-time for the liberation of the first equivalent of HC1 is about 3 minutes, and that for the second equivalent of HC1 is about 25 minutes. The third equivalent of HC1 is not liberated a t this pH. As the pH is raised, both the speed and extent of HC1 formation are increased, so that a t pH 9, three equivalents of HC1 are liberated in 10 minutes. The data in Table I1 show that, a t pH 7.5, the rate of liberation of HC1 from I1 is reduced greatly by borate or bicarbonate and slightly by acetate or sulfate. 'This work was done in whole under Contract No. OEMsr-313 between The Rockefeller Inst,it,ute 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. *Present address, University of Wisconsin, Madison, Wisconsin. 'Present address, Yale University, New Haven, Connecticut. 'Died, November 7, 1944. 704

705

REACTIONS O F MUSTARD GAS. VI

TABLE I REACTION OF TRIS(~~-CHLOROETHYL)SULFONIUM CHLORID~ (11)WITH WATER The H+ liberation was followed electrometrically by adding NaOH (1.0 N ) to maintain the pH a t the deeired value. The C1- liberation wa8 determined argentometrically on aliquots of the reaction mixture. Temperature, 25". Concentration of 11, 0.05 M .

INFLUENCE OF pH

UPON TRE

pH 3.0 TIhCE, MIN.

H+liber m M of

\r

M.EQUIV.

1 3 5 10' 15 20 30 45 60 90 150

270

--

pH 9.0

#H 7.5

C1- liber

m M of

.F I

0.73

0.79

1.25

1.30

1.62

1.59

1.87 1.94 2 .OO 2.00

1.84 1.91 1.98 2.02

mM o f ' I r

Cl- liber. m M of I r

H+ fiber. er m M of IF,

M.EQUIV.

M.EQUIV.

M.EQUIV.

2.00 2.39 2.45 2.57

2.30 2.54

2.00 2.89 2.97 2.99

2.92 3.00

2.64 2.73 2.80 2.84 2.88 2.94

2.64 2.70 2.76 2.78 2.85 2.91

3.00

3.00

XI+ liber

M.EQUIV.

C1- liber. er mM.EQUIV. M of If

TABLE I1 INFLUXNCE OF VARIOUS ACIDSUPON ELIMINATION OF HCL FROM TRIS(@-CELOROETHYL)SULFONIUM CHLORIDE (11) Concentration of reactants per cc. a t start of reaction: 0.05 mM of 11; 0.05 mM of acid. Temperature, 25". Unless otherwise noted, the acid solution was first neutralized to pH 7.5 with NaOH and I1 was then added. The H+ liberation was followed electrometrically by adding NaOH (1.0 N ) as required to maintain the pH about 7.5. The C1- liberation was also followed by titration of an aliquot a t intervals with AgNO,. H+ and C1- were liberated at the same rate. MILLIEEQUIVALENTSOF HCL LIBERATED PER m Y OF II WITHIN THE FOLLOWING TIYE (IN MINUTES)

-

ACID

5

None. ............................. HsBOs. ............................ H&OjO. ........................... HiCO b . .......................... HC1. .............................. CH8COOH. ........................ H80,c.. . . . . . . . . . . . . . . . . . . . . . . . . . . . $0,

" 0 8

..............................

Added as NaHCOj. b Concentration 0.005 mM per cc. c Concentration 0.025 mM per cc.

0

2.38 0.46 0.82 2.02 1.90 2.18 2.26 2.46

I

10

2.56 0.75 1.11 2.21 2.06 2.32 2.44 2.62

1

20

I

30

2.64 2.72 1.16 1.44 1.47 1.67 2.34 2.43 2.23 2.33 2.46 2.57 2.55 2.62 2.72 2.79

45

60

90

150

--__.

2.78 1.70 1.84 2.50 2.38 2.64 2.65 2.85

-

2.81 1.84 1.96 2.54 2.46 2.70 2.67 2.90

2.87 2.93 1.93 2.22 2.14 2.42 2.64 2.73 2.58 2.65 2.76

706

STAHMANN, FRUTON, AND BERQMANN

It will be noted that, on addition of one mole equivalent of borate, the time required for the liberation of two equivalents of HC1 was increased from a period of less than 1 minute to about 97 minutes. In the presence of one equivalent of bicarbonate, this time was about 65 minutes, whereas in the presence of onetenth equivalent of bicarbonate only 4 minutes were required. On the other hand, one equivalent of acetate increased the time required for the liberation of 2.5 equivalents of HC1 from 8 minutes to 23 minutes; one equivalent of sulfate increased this time to 14 minutes; but under similar conditions, nitrate did not change the rate of the reaction. Thus it would appear tha,t the salts of very weak acids, such as boric or carbonic, which are only slightly dissociated a t this pH, act as strong inhibitors of the reaction, while the salts of stronger acids, such c1-

c1-

C1CHI CHa

-HCl

\+ SCH, CHZ C 1 C1CH2 CHz

C1CHz CH,

\+

+

SCH-CHa

/

/

ClCHiCHz

(11)

(111)

I

I

c1CHz=CH

-HC1

\+

t-

CH1=CH

\+ /scH=CH3

CH= CH

CH%=CH

-HC1 c1-

CICHZCH2

(Iv)

(V)

FIG: 1

as acetic, sulfuric or nitric, which are more completely dissociated, are weak inhibitors or shorn no inhibition a t all. The mechanism of the hydrolysis of tris(8-chloroethy1)sulfonium chloride is given in Figure 1. The sequence of reactions given in the figure is supported by several lines of evidence. In the first place, the intermediate bis(8-chloroethy1)vinylsulfonium compound (111) was isolated as a picrylsulfonate from a bicarbonate-buffered reaction mixture aged for 10 minutes. Furthermore, the final product, the trivinylsulfonium salt (V), was isolated as a picrylsulfonate from a reaction mixture aged for 96 hours. Additional support for the reaction sequence given in Figure 1 was provided by a study of the reaction of I1 with thiosulfate. It was found that I1 reacts with thiosulfate in bicarbonate solution. However, the extent of thiosulfate consumption did not decrease concomitantly

REACTIONS OF MUSTARD GAS.

707

VI

with the liberation of HCl, and aged solutions of I1 still reacted with thiosulfate. These observations are accounted for by the fact that the final product of the reaction contains vinyl groups, and, therefore, also reacts with thiosulfate. In fact, trivinylsulfonium picrylsulfonate consumes almost two equivalents of thiosulfate within 16 hours. Three additional reactions of tris(p-chloroethy1)sulfonium chloride are of interest. In the presence of an excess of cysteine, three equivalents of SH groups disappear, but bis(cysteinylethy1)sulfide is formed. This compound has also been prepared by the reaction of H ( 5 ) , or bis-8-[bis(P-hydroxyethy1)sulfonium]ethylsulfide dichloride (1) with cysteine. When only two equivalents of cysteine are present, this product is not obtained. It would appear, therefore, that substitution of the three chlorine atoms of I1 by cysteine leads to the formation ofan unstable sulfonium salt which decomposes to yield the sulfide. A somewhat analogous reaction must occur in the presence of an excess of pyridine, for here again a sulfide, bis(8-pyridiumethy1)sulfide) is formed. However, when treated with an alcoholic solution of KaOH, I1 yields tris(P-ethoxyethy1)sulfonium chloride. Preparation and properties of 8-chloroethyl-1 ,d-dithiane sulfonium chloride and vinyl-1 ,4-dithiane sulfonium chloride. Since @-chloroethyl-14-dithiane sulfonium chloride (VI) contains only one 8-chloroethyl group, it is well suited to a study of the properties of P-chloroethyl sulfonium compounds. It was prepared by chlorinating P-hydroxyethyl-l,4-dithianesulfonium chloride with thionyl chloride. The behavior of VI in aqueous solution is in many respects similar to that of tris(j3-chloroethy1)sulfonium chloride. Thus when VI is dissolved in mater, there is an initial fall in pH. If the pH is raised, HC1 is liberated a t a rate which is markedly dependent upon pH. It will be noted from Table I11 that at pH 5.0, 0.34 m. equiv. of HC1 is liberated within 120 minutes, while a t pH 7.5, 0.78 m. equiv. is liberated within 5 minutes. Furthermore, it will be noted from Table I11 that in the presence of one equivalent of bicarbonate, the rate of the formation of HC1 is greatly decreased. )

c1-

(VI) (VII) The product of the reaction of VI with water, the vinyl-l,4-dithiane sulfonium compound (VII), has been isolated as a picrylsulfonate from a bicarbonatebuffered reaction mixture. As will be described later, the vinyl compound (VII) was also obtained, as a chloride, by treatment of the zinc chloride double salt of S ,S’-endoethylene-l,4-dithiane sulfonium dichloride with silver carbonate. The 8-chloroethyl and vinylsulfonium salts VI and VI1 both react with thiosulfate t o form the @-thiosulfonatoethyl-l,4dithianesulfonium inner salt

708

STAHMANN, FRUTON, AND BERQMANN

(VIII) (Figure 2). The rate of the reaction of VI and VI1 with thiosulfate in aqueous solution a t pH 7.5 is given in Columns 2 and 4 respectively of Table IV. TABLE I11 INFLUENCE OF pH AND BICARBONATE UPON THE REACTION O F ~-CHLOROETHYL-1,4-DITHIANE

SULFONIUM CHLORIDE] (VI) WITH WATER The procedure was similar to that given in Table I. Temperature, 25'; concentration of VI, 0.05 M. pH 5.0

pH 1.5

no NaHCO:

T D 5 , YIN.

H;;bc;:f\~f'

df of vpe,

Y.EQUIV.

X.EQUIV.

0.11 .16

0.16

2 5 10 15 30 60 120 180

.20 .23 .28 .33

.21 -25 .28

\+

\

SCHzCHSC1

/

vy

C1- liber df of

Y.EQUIV.

0.74 .79 .82 .85

LEQUIV.

X.EQIJIV.

Y.EQUIV.

0.77

0.01 .03 .08

0.04 .10

.25 .42 .53 .55

.25 .46 .54 .56

.80 .83 -88

.90 .93

.!?4

.34

c1-

S

H c;~:;

+ NaHCO: (0.05 li)

I

no NaHCOt

Cl- liber

-

CHnCHp

c1-

\+

S

\

SCH=CHI

/

+ HCl

CHgCHn

(VI)

(VI11

c1-

S/CH2CH2 \+

\

/

SCH-CHa

+ Na&3*Ol+ Hp0

CHnCHt

(VI111

FIG.2

/3-Hydroxyethyl-l,4dithianesulfonium chloride, on the other hand, does not react with thiosulfate under these conditions.


709

VI

Data presented above indicated that the rate of the elimination of HC1 from VI is markedly influenced by bicarbonate. The results presented in Column 3 of Table IV show that the rate of the reaction of VI with thiosulfate is also reduced by bicarbonate. In the absence of bicarbonate; 0.39 m. equiv. of thiosulfate was consumed per mM of VI within l hour; while in the presence of one equivalent of bicarbonate, only 0.10 m. equiv. of thiosulfate was consumed. In marked contrast, the rate of the reaction of VI1 with thiosulfate is not changed by the addition of bicarbonate (Columns 4 and 5 of Table IV). It will also be noted from Table IV that the rate of reaction of the vinyl group with thiosulfate is considerably faster than that of the P-chloroethyl group. Furthermore, when the vinyl group reacts with thiosulfate, OH- is liberated at the same rate as thiosulfate is consumed. A comparison of the data of Table I V with TABLE IV BICARBONATE UPON THE REACTION O F @-CHLOROETHYL-1,4-DITHIANE SULFONIUM CHLORIDE (VI) AND O F VINYL-I,4-DITHIANE SULFONIUhl CHLORIDE(VII) WITH THIOSULFATE Concentration of reactants per cc. a t start of reaction: Columns 2 and 4,0.05mM of Sulfonium Chloride (VI or VII); 0.10 mM of Xa2S203. Columns 3, 5, and 6,0.05 mM of Sulfonium Chloride (VI or VII); 0.10 mM of Na2S203; 0.05m M of XaHCOs. Temperature, 25'. The OH- liberation (Column 6) was followed by continuous electrometric titration with HC1. The pH was maintained at about 7.5. INFLUENCE O F

THIOSULFATE CONSUMED PEP m Y OF VI

THIOSULFATE CONSubIED P E P VIM OF VI1

OH- FOBYED P E P nY OF VI1

I

M.EQUIV.

M.EQUN.

5.

6.

0.23 .49 .74 1 .oo

0.23 .49 .74 1.02

TIACE, MIN. Y.EQUIV.

1 ,,

1.0 30 60 1\50 300 1380 a

4.

0.04 .18 .39 .64 .73 1.01

0.01 .04 .10 .30 .51 1 .oo

0.24 .48 .73 1.02

0.35m.equiv. of NaOH per mM of V I were added at start t o raise the pH t o 7.7.

those of Table I shows that the rate of thiosulfate consumption by either the j3-chloroethyl or vinyl group is much slower than the elimination of HC1 from the chloroethyl group under similar conditions. This result indicates that loss of HCI. with the formation of a reactive vinyl group is the first step in the reaction of /3-chloroethyl sulfonium compounds with such groups as thiosulfate. Additional evidence for the formation of a vinyl group during the first stages of the reaction with thiosulfate lies in the fact that the pH of a reaction mixture containing VI and thiosulfate rapidly fell from 6.5 to about 3.5 as soon as the sulfonium salt was added. To raise the pH to 7.7,0.35 m. equiv. of NaOH per mM of sulfonium salt was added, after which the pH again slowly fell to about 6.5. A pH of about 6.5 t o 7.0 was maintained by the reaction mixture for at least 300 minutes, and then the pH rose to about 9.0 as the thiosulfate was consumed,

710

STAHMANN, FRUTON, AND BERGMA"

The fact that NaHCOBinhibits the reaction of 8-chloroethyl-1,4dithiane sulfonium chloride with thiosulfate, but does not inhibit the reaction of the vinyl compound with thiosulfate, is explained by the results presented above. Since a vinyl group must be formed prior to reaction with thiosulfate, the effect of NaHC03is to reduce the rate of the formation of vinyl groups, but not the rate of reaction of vinyl groups once they are formed. The over-all effect, therefore, is a retardation of thiosulfate consumption. The mechanism of the reaction of VI and VI1 with thiosulfate is given in Figure 2. It will be noted from Table IV that at pH 7.5 the reaction of VI1 with thiosulfate proceeds to completion. It has been found, however, that at a higher pH (8.5-9.0) the reaction appears t o stop 15% short of completion. Moreover, when solutions of the isolated inner salt VI11 are maintained at alkaline pHvalues, gradual decomposition occurs with the liberation of groups titratable with iodine. Thus after exposure of VI11for 100 hours t o a pH of 8.0,0.22 m. equiv. of iodine was consumed. After 100 hours at pH 8.5,0.38 m. equiv. of iodine was consumed, while if the pH of exposure was raised to 9.0,0.94 m. equiv. of iodine was consumed. The fLchloroethy1and vinyl sulfonium salts (VI) and (VII) both react readily with pyridine to form the p-pyridiniumethyl-l,4dithianesulfonium salt (IX) which has been obtained as a dichloride and as a dipicrylsulfonate. The reaction may be represented by Equation 2.

/

CH2 CH2

\+

S

\

CH2 CH2

/

SCH = CH2 -I- C&N

\

4- HzO

/

$

CH2 CH2 (IX) As can be seen from Equation 2, one equivalent of OH- is liberated for each vinyl group which combines with pyridine. The rate and extent of the reaction were therefore followed by continuous electrometric titration with acid. The results given in Column 2 of Table V, show that the reaction starts a t a rapid rate, 0.20 m. equiv. of OH- being formed within 9 minutes. Subsequently, the reaction stops completely when only 28% of the theoretically possible amount of OH- has been formed. On the basis of these data, it was suspected that the reaction between VI1 and pyridine waa reversible, and that equilibrium had been attained when the forward reaction had gone 28% to completion. In order to prove the reversible nature of the reaction, the decomposition of IX was studied. The pyridinium derivative (IX) was synthesized by treating a mixture of pyridine and pyridine hydrochloride with VI1 in ethanol-acetone.


711

VI

The same product was also obtained from pyridine and VI in methanol. It was found that I X decomposes rapidly in aqueous solution, liberating within 10 minutes 66% of the theoretical quantity of H+ (Column 3 of Table V). The equilibrium reached by the reverse reaction is, therefore, almost the same as that attained by the forward reaction, a strong ind.ication that the reaction is reversible. It follows from the foregoing that IX, in the course of its decomposition, should give rise t o a reactive group (probably a vinyl group) and, therefore, should act as an alkylating agent. Indeed, it has been found that I X consumes thiosulfate at pH 7.5 and 25". During this reaction, a small amount of crystalTABLE V VINYL-1,4-DITHIANE SULFONIUM CHLORIDE (VII) WITH PYRIDINE AND OP ,8-PYRIDINIUMETHYL-1,4-DITHIANE SULFONIUM DICHLORIDE (Ix)WITH WATER

REACT'ION OF

Concentration of reactants per cc. at start of reaction: Column 2, 0.10 mM of VII; 0.20 m,M of pyridine. Column 3, 0.105 m M of IX; 0.105 mM of pyridine. Temperature, 25'. The OH--or Hf formation was followed electrometrically by adding HC1 or NaOH (0.5 N) to majntain the pH at 7.3 to 7.5. The initial concentration of reactants for the experiment given in Column 3 was chosen so that the final volume was the same as that in the experiment recorded in Column 2. TIME, MIN.

OH-

LIBERATED PER M.EQUIV.

mM

OF

vu,

H+ LIBERATED PER mM

2.

3.

1 2 3 4 5 6 8 10 12 14 16 20 30 60

0.05

0.32 .55 .62 .64 .65

.06 .10 .12 .14 .16 .19 .22 .24 .25 .27 .28 .28 I28

OF IX,

M.EQUIV.

1.

.66

.66 .66

.66

line solid began to separate from the reaction mixture after about 4 hours. This product was identified as the inner salt (VIII) described above. It has also been found that the rate of the transformation of IX into the vinyl compound is much more rapid than is the rate of the reaction of the sulfonium dichloride with thiosulfate. It is clear, therefore, that the major part of the thiosulfate consumption is attributable to a reaction involving the intermediate vinyl compound. Additional evidence for the fact that I X can act as an alkylating agent is furnished by a study of the reaction of this substance with alanine. It has been found that the pyridinium compound reacts slowly with alanine, 0.99 m.

712

STAHiMANN, FRUTON, AND BERGMANN

equiv. of amino nitrogen disappearing per mM of pyridinium salt within 48 hours. During the reaction with alanine, a small amount of dithiane separated after 24 hours, indicating partial decomposition of one of the sulfonium salts present in the reaction mixture. Preparation and properties of S ,8'-endoethylene-1 ,&dithiane disulfonium dichloride. The synthesis of S ,S'-endoethylene-1 ,4-dithiane disulfonium dichloride (X) involved the reaction of thiodiglycol with concentrated hydroc1CHI CHZOH

+/

CH, CHzOH

/ S \

-+SHCI CHsCHzOH

-1

/ S \

CHzCHoS

s

/ \

STG

\

CHzCHzCl

c1-

\

CHZCHZOH

CHzCHnC1

GI-

CHnCHz

\+

\+ SCHgCHsOH +HCI+

\

/

CHzCHz

ClCHpCHzOH

c1-

b/

/sCHzcHsCi

CHzCHp

/

c1-

CHnCHz

+/

S-CH2CH2-S

\

CH2 CHz

\+

/

(XI FIG. 3

chloric acid a t 100'. The compound wzts isolated and analyzed as a double salt of zinc chloride and has also been analyzed as a dipicrylsulfonate. Figure 3 is intended to indicate the general types of reaction which might lead to the formation of X. Possible variations of this general reaction sequence are obvious. Davies and Oxford ( 6 ) have described the isolation of &hydroxyethyl-l,4dithiane sulfonium chloride from the reaction of H with 2 to 6 volumes of boiling water. Upon treatment with silver carbonate, X is transformed into MI. The re-


713

VI

action is analogous to a Hofmann degradation, only one sulfonium group being degraded under these conditions. The relationship between chemical reactivity and toxicity o j sulfonium salts. As a result of the chemical investigations detailed in this and a previous paper of this series (l), it has become possible to correlate the chemical reactivity of several sulfonium salts with their toxicity. The toxicity of a group of sulfonium salts is given in Table VI. It can be seen from Table VI that those sulfonium salts which contain a reactive side chain capable of undergoing alkylation reactions are, in general, toxic. The most toxic compound in the table is XI, which is the only substance TABLE VI THETOXICITY OF SEVERAL SULFONIUM SALTS The approximate LDso was determined in each case by intraperitoneal injection of graded doses into sets of three mice. ~

SUBSTANCE

--

APPPOXndATE LD'o

m.lkE.

p - Chloroethyl - B - [his(@ - hydroxyethyl)sulfoniumlethylsulfide chloride ( X I ) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Bis - p - [bis(p - hydroxyethyl)sulfonium]ethylsulfide dichloridc (XII) . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . p - Hydroxyethyl - B - [bis(p - hydroxyethyl)sulfoniumlethylsul fide chloride (XIIa).. . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . . Tris(p-chloroethy1)sulfonium chloride (11). ..................... @-Chloroethyl-l14-dithiane sulfonium chloride (VI). . . . . . . . . . . . . Vinyl-l14-dithiane sulfonium chloride (VII) .................... fi-Pyridiniumethyl-1,4-dithiane sulfonium dichloride (IX). . , , , . p-Hydroxyethyl-l,4-dithianesulfonium chloride ( X I I I ) . . . . . . . . . p-Thiosulfonatoethyl-1,4-dithiane sulfonium inner salt (VIII) . ,

Methyl-bis(&hydroxyethyl)sulfonium chloride (XIV) . . . . . . . . . . . Methionine methyl sulfonium iodide (XV). .................... Tris(8-hydroxyethy1)sulfonium chloride (I).....................

1.2a 50-1oob

86" 80 75

75 125 175 460 1700 2000 2000

Reported by Smith, et al. (8)

* Reported previously

(1).

possessing a 0-chloroethylsulfide moiety. It will be recalled that XI is a potent alkylating agent (1). Compounds 11,VI, and VII, all of which are good alkylating agents, also possess marked toxicity. In general, it would appear that the substitution of a reactive 6-chloroethyl (or vinyl) group for an unreactive j3hydroxyethyl group results in an increase in toxicity. Thus compound XI1 is less toxic than compound XI, compound I is much less toxic than compound 11, and compound XI11 is markedly less toxic than are compounds VI or VII. Similarly, 0-pyridiniumethyl-1,4-dithiane sulfonium chloride (IX) is slightly more toxic than is the corresponding hydroxyethyl compound (XIII). It will be recalled that compound I X decomposes at pH 7.5 with the liberation of an

714

STAHMANN, FRUTON, AND BERGMANN

active alkylating group (probably a vinyl group). Compound VIII, on the other hand, which is relatively non-toxic compared to the substances listed above, appears to be relatively stable a t pH's below 8. In comparing the toxicity and chemical reactivity of various sulfonium salts, the presence of a reactive side chain (0-chloroethyl or vinyl group) is not the only factor to be considered. Attention must also be directed towards the stability of the sulfonium sulfur atom. The ability of the sulfonium group to react with thiosulfate and/or to liberate acid when heated in aqueous solution may be taken as an index of stability or chemical reactivity. Of the sulfonium salts which do not possess reactive side chains, compounds XI1 and XIIa are the most toxic. It has already been shown, however, that these substances react with thiosulfate and decompose readily when heated in aqueous solution. Compound XIII, which possesses moderate toxicity, does not react with thiosulfate, but decomposes readily on heating in alkaline solution to give 1,4-dithiane,which can be isolated from the reaction mixture. Compounds I, XIV, and XV are by far the least toxic, and are also the most stable. Compounds I and XIV do not liberate appreciable quantities of HC1 on heating in aqueous solution a t 100" for 1 hour. Compound XV liberates only 17% of the theoretically possible quantity of acid under these conditions. It may be mentioned that the stability of XV is in marked contrast to the lability of the sulfonium salt formed from H and methionine. It will be recalled that-the latter compound decomposes readily on heating in aqueous solution (7). Considering the data in Table VI as a whole, two facts emerge. The more innocuous sulfonium salts all possess a relatively stable sulfonium sulfur atom, and do not possess any reactive, alkylating side chains. The more toxic sulfonium salts, on the other hand, each contain either a reactive side chain, or a relatively unstable sulfonium sulfur atom, or both. EXPERIMENTAL

Tris(@-ch1oroethyZ)sulfonium chloride (ZZ). Tris(@-hydroxyethy1)sulfonium chloride was prepared by the method of Davies and Oxford (6). It was chlorinated in a manner similar to that described by Ettel and Kohlik (2) except that the chlorination was carried out a t 25". The yield of I1 was 50% of theory. The compound was recrystallized from the minimum amount of absolute ethyl alcohol by the addition of anhydrous ether; m.p. 108-109". Anal. Calc'd for CsHlpClaS: C, 27.9; H, 4.7; S, 12.4; C1, 55.0. Found: C, 27.9; H, 4.8; S, 12.4; C1, 55.2. Tris(p-ch1oroethyE)sulfoniumpicrylsulfonate. To a solution containing 1.28 g. (5 m M ) of tris(6-chloroethy1)sulfonium chloride in 50 cc. of water, was added slowly 50 cc. of a solution containing 1.76 g. (5 m M ) of sodium picrylsulfonate. The solid product which separated immediately was collected and washed with water; yield 2.40 g., corresponding t o 9401, of theory. The salt was recrystallized by dissolving i t in 50 cc. of dry acetone and adding 60 cc. of anhydrous ether; m.p. 154". C, 28.0; H, 2.7; N, 8.2; S, 12.5; C1, 20.7. Anal. Calc'd for C~Hl2Cl3S.COH2N80&3: Found: C, 27.8; H, 2.7; N, 8.2; S, 12.7; C1, 21.0. Bis(@-chloroethy1)vinylsulfoniumpicrylsulfonate. A reaction mixture containing 2.56 g. (10 m M ) of 11,4.2 g. (50m M )of NaHC03, and 0.40 g. (10 m M ) of NaOH in 200 cc. of water was allowed to stand 10 minutes at 25". Titration of an aliquot indicated that 1.20m.equiv.


VI

715

of C1- had been liberated per m M of sulfonium salt. The remaining portion (180 cc.) was immediately acidified with 45 cc. of N HCl and a solution of 3.16 g. (9 m M ) of sodium picrylsulfonate in 50 cc. of water was then added. The reaction mixture was concentrated to 50 cc. under reduced pressure (bath temperature, 40°), allowed to stand at 4' for 4 hours, and filtered. The product was washed with cold water and dried; yield 3.49 g., corresponding to 73% of theory. Itwas recrystallized four times by the addition of 50 cc. of anhydrous ether to a solution of the substance in 50 cc. of dry acetone; m.p. 134". Anal. Calc'd for C6H~~C1zS.C6H2N30sS: C, 30.1; H, 2.7; N, 8.8; S, 13.4; C1, 14.8. Found: C , 30.0; H, 2.9; N, 8.8; S, 13.4; C1, 14.9. Trivinylsulfonium picrylsulfonate. A reaction mixture containing 6.38 g. (25 m M ) of 11,10.5 g. (125 mM) of NaHC03, and 1.0 g. (25 m M ) of NaOH in 500 cc. of water was allowed 1 o stand for 96 hours at 25". Titration of an aliquot showed that 2.87 m.equiv. of C1- had been liberated per mM of sulfonium salt. The remaining portion was acidified with 48 cc. of 2.7 ATHCI and a solution of 7.56 g. of sodium picrylsulfonate in 50 cc. of water waa added. The reaction mixture was concentrated to 50 cc. under reduced pressure (bath temperature, a"), allowed to stand at 4" for 4 hours, and filtered. The product was washed with cold water and dried; yield 7.4 g., corresponding to 74% of theory. It was twice recrystallized from methyl alcohol; m.p. 157". Anal. Calc'd for C&S.C6H,N~o&: c, 35.5; H, 2.7; N, 10.4; S,15.8. Found: C, 35.5; H, 2.8; N, 10.4; S, 15.6. Reaction of tris(&chloroethyl)sulfonium chloride (11)with cysteine. A reaction mixture was made up to contain 1.28 g. (5 m M ) of 11, 2.35 g. (15 m M ) of cysteine hydrochloride, 0.8 g. (20 m M ) of NaOH, and 1.26 g. (15 m M ) of NaHC03 in 50 cc. of Oe-free water. The mixture was kept at 25" under Ne. A solid began to separate after 3 hours. After 24 hours, the bis(cysteinylethy1)sulfide was collected, washed with water, and dried; yield 1.10 g., corresponding to 61% of theory. The filtrate gave a negative nitroprusside test. The crude product was recrystallized by dissolving i t in 30 cc. of concentrated ammonium hydroxide and allowing the ammonia to evaporate slowly. Anal. Calc'd for C10H20N201S3: C, 36.6; H, 6.1; N, 8.6; S, 29.3. Found: C, 36.4; H, 6.1; N, 8.6; S, 29.4. Reaction of tris(8-chloroethy1)sulfoniumchloride (ZZ)with pyridine. A reaction mixture was made up t o contain 2.56 g. (10 m M ) of I1 and 8.05 cc. (50 m M ) of pyridine in 25 cc. of absolute ethyl alcohol. One drop of triethylamine was added and the reaction mixture was allowed to stand 4 days a t room temperature. Anhydrous ether (60 cc.) was then added t o the reaction mixture to induce crystallization. The crude product was collected after 4 hours at 4'. The yield of crude bis(8-pyridiniumethy1)sulfide dichloride was 1.40 g . corresponding to 50% of theory. Since the dichloride was not analytically pure, i t was purified by conversion t o the dipicrylsulfonate. The crude dichloride (1.04 9.) waa dissolved in 25 cc. of water, and 2.0 cc. of a solution containing 3.00 g. of picrylsulfonic acid in 25 cc. of water was added; the reaction was filtered, and the rest of the picrylsulfonic acid solution was added. The picrylsulfonate was collected and recrystallized from SO'% methyl cellosolve. The melting point of the bis(P-pyridiniumethy1)sulfide dipicrylaiulfonate was 216-218". C, 37.6; H, 2.7; N, 13.5; S, 11.6. AnaE. Calc'd for CI~H18nT*S.2C*H2NsO(S: Found: C, 37.7; H, 2.7; h', 13.6; S, 11.7. Tris(fi-ethoxyethyl)sulfonium picrylsulfonate. A solution containing 1.28 g. (29.5 m M ) of sodium hydroxide in 75 cc. of absolute ethyl alcohol was added with stirring t o 25 cc. of an alcoholic solution containing 2.58 g. (10 m M ) of 11. After 20 minutes, the sodium chloride was removed and the filtrate concentrated under reduced pressure at 40" to a thin sirup. Anhydrous ether (100 cc.) was slowly added to the sirup. After 48 hours, a small amount of additional sodium chloride was removed and the filtrate added t o 50 cc. of an aqueous solution containing 3.65 g. (10 mM) of picrylsulfonic acid. An oil separated which crystallized as the ether was removed under reduced pressure. The yield of tris(8ethoxyethy1)sulfonium picrylsulfonate was 4.06 g. corresponding t o 76% of theory. The

716


product was recrystallized from 15 cc. of methyl alcohol by the addition of 50 cc. of water m.p. 62-64". Anal. Calc'd for C I Z H W O & ~ . C ~ H ~ NC,~ O 39.8; ~ S :H, 5.4; N, 7.8; S, 11.8. Found: C, 39.9; H, 5.4; N, 7.8; S, 11.6. 8-Chloroethyl-i ,4-dithiane sulfonium chloride ( V I ) . 8-Hydroxyethyl-l , 4-dithiane sulfonium chloride (6) (20.0 g., 0.1 M ) was treated at 25" with 35.7 g. (0.3 mole) of thionyl chloride for 2 hours. The reaction mixture was kept at 50" for 1 hour. The excess thionyl chloride was removed by maintaining the mixture under reduced pressure at 40" for 3 hours. The crude product solidified as the thionyl chloride was removed. The product was suspended in 50 cc. of dry ether, collected by filtration, and washed with dry ether; yield 20.7 g., corresponding to 95% of theory. The crude product was recrystallized by dissolving i t in 120 cc. of boiling absolute ethyl alcohol, cooling, and adding 100 cc. of dry ether; m.p. 144". Anal. Calc'd for CaH1&12Se: C, 32.9; H, 5.5; C1, 32.3. Found: C , 33.1; H, 5.5; C1, 32.3. Vinyl-I ,d-dithiane sulfonium picrylsulfonate. 0-Chloroethyl-l , 4-dithiane sulfonium chloride (2.19 g., 10mM) was dissolved in 200 cc. of a solution containing0.4 g .(10mM) of NaOH and 1.68 g. (20 m M ) of NaHCQ3. The reaction mixture was allowed to stand at 25' for 24 hours, and 3.63 g (10 mM) of picrylsulfonic acid dissolved in 50 cc. of water was added. The mixture was concentrated under reduced pressure t o 40 cc. Crystallization occurred during the concentration. The concentrate was cooled to 4" for 2 hours, filtered, and the product washed with water. The yield of vinyl-l,4-dithiane sulfonium picrylsulfonate was 3.92 g., corresponding to 85% of theory. The product was recrystallized by suspending i t in 50 cc. of boiling methyl cellosolve, filtering, and adding 50 cc. of ether t o the filtrate. The melting point was 154-155' and no depression was observed on admixture with a sample prepared from S , S'-endoethylene-1 ,4-dithiane sulfonium dichloride. Anal. Calc'd for CsH11Sz.CaH2N30pS:C, 32.8; H, 3.0; N, 9.6; S, 21.9. Found: C , 32.9; H, 3.1; N, 9.5; S, 21.8. 8-Thiosulfonatoethyl-1,J-dithiane sulfonium inner salt (VZZZ). Vinyl-1 ,4-dithiane sulfonium chloride (3.66 g., 20 m M ) , 10.0 g. (40 mM) of sodium thiosulfate, and 3.4 g. (40 mM) of sodium bicarbonate were dissolved in 100 cc. of water and the solution was allowed to stand at room temperature for 24 hours. Crystals of the thiosulfate salt appeared after 15 minutes and gradually increased in amount. The reaction mixture was cooled at 0", filtered, and the crystalline product was dried in vacuo over PgOa; yield 3.9 g. (75% of theory) of nearly pure product (C, 28.0; H, 4.7). It was recrystallized from hot water, washed with alcohol and ether, and dried as before. The melting point was 151-153"with decomposition. Anal. Calc'd for C6HuO3Sa: C, 27.7; H, 4.6; S, 49.2. Found: C, 27.7; H, 4.6; S. 49.2. The same product was obtained in a similar manner from a reaction mixture containing 2.19 g. (10 mM) of @-chloroethyl-l,4-dithianesulfonium chloride, 5.0 g. (20 mM) of sodium thiosulfate, and 1.7 g. (20 mM) of sodium bicarbonate dissolved in 50 cc. of water. The yield was 2.03 g. corresponding to 78% of theory. The melting point showed no depression when this product was mixed with that prepared from the vinyl sulfonium salt. Anal. Calc'd for C6H1203S4:C, 27.7; H, 4.6; S, 49.2. Found: C, 27.7; H, 4.7; S, 49.2. Compound VI11 also crystallized from a reaction mixture containing 1.63 g. (5 mM) of IX, 2.5 g. (10 mM) of sodium thiosulfate, and 1.7 g. (20 mM) of sodium bicarbonate in 50 CC. of water. Anal. Calc'd for CsH120aS4:C , 27.7; H, 4.6; S, 49.2. Found: C, 27.6; H , 4.5; S, 49.1. ~-Pyridiniumethyl-l,.&dithiane sulfonium dichloride ( Z X ). Vinyl-1 ,4-dithiane sulfonium chloride (3.65 g., 20 mM) was dissolved in 20 cc. of absolute alcohol and t o this solution were added 2.64 g. (23 mM) of pyridine hydrochloride dissolved in 5 cc. of absolute alcohol and 1.6 cc. (20 mM) of pyridine. After standing at room temperature for 24 hours, the


VI

717

reaction mixture contained a mass of crystalline material which was collected (1.52 g.) and recrystallized from absolute ethyl alcohol-ether; m .p. 150-152". The reaction mixture, after standing for 10 more days at room temperature, deposited an additional 1.40 g. of IX. The total yield corresponds t o 49% of theory. The substance w w dried for analysis at 78" over P z Oi~n vwuo. A d . Calc'd for CIIH1,CIZNSz: C, 44.3; H, 5.7; N, 4.7; S, 21.5. Found: C, 44.4; H, 5.7; N, 4.8; S, 21.5. The anhydrous substance, when exposed t o air at 23" and 32% humidity, showed an increase in weight of 8.3%. Calculated for 1.5 moles of H20, 8.3%. The same product was obtained from fl-chloroethyl-1,4-dithianesulfonium chloride and pyridine. The sulfonium chloride (10.9 g., 50 m M ) was dissolved in 35 cc. of absolute methyl alcohol and 12.1 cc. (150 m M ) of pyridine was added. The reaction mixture was allowed to stand a t room temperature for 48 hours; 25 cc. of dry ether then was added to induce crystallization. After standing a t 4" for 4 hours, the product was collected and washed with dry ether; yield 9.24 g. After recrystallization from absolute ethyl alcohol and ether, i t melted at 150-152", no depression in mixture with a sample of the substance prepared from vinyl-1,4-dithiane sulfonium chloride. After standing for 5 more days at room temperature, the mother liquors deposited an additional 3.84 g. of IX. The product was dried over P206 at 78" i n vacuo for analysis. The total yield corresponds t o 88% of theory Anal. Calc'd for CuHI,C12NSz: C, 44.3; H, 5.7; N, 4.7. Found: C, 44.1; H, 5.6; N, 4.9. 8,s'-Endoethylene-1,d-dithiune disulfonium dichloride ( X ) . A mixture of 200 g. of thiodiglycol (Kromfax), 225 g. of ZnCIZ,and 700 cc. of HC1 was boiled under reflux for 24 hours. During the reaction, a crystalline precipitate separated, which w&s collected and recryst,allized from hot water. The filtrate was refluxed for 48 hours longer; on cooling, a further crop of crystals was obtained; total yield of recrystallized material, 26 g. For analysis, the material was recrystallized once more from hot water. Anal. Calc'd for CsHd32S2.ZnC12: C, 20.3; H, 3.4; S, 18.1; Zn, 18.4; C1, 39.8. Found: C, 20.3; H, 3.5; S, 17.8; Zn, 18.4; C1, 39.9. For the preparation of the dipicrylsulfonate, 0.9 g. of the ZnCla double salt was dissolved in 50 cc. of water and a solution of 4 g. of picrylsulfonic acid in a mixture of 50 cc. of water and 15 cc. of N HCl was added. The dipicrylsulfonate (1.8 9.) crystallized out in the form of yellow leaflets. C, 29.5; H, 2.2; N, 11.4; S, 17.5. Anal. Calc'd for C~HIZSYPC~HSN~OQS: Found: C, 29.95; H, 2.5; N, 11.1; S, 17.4. Vinyl-I,4-dithiune sulfonium chloride ( V I Z ) . The double salt X (7 g.) was dissolved in 100 cc. of water and stirred at room temperature with 25 g. of Ag2COa. After 90 minutes, the reaction mixture was filtered and the filtrate (which was free of C1-) was acidified with HCl t o Congo Red. On evaporation i n vacw,a colorless crystalline chloride was obtained. For purification i t was repeatedly dissolved in cold absolute ethanol and reprecipitated by the addition of anhydrous ether. Anal. Calc'd for CeH11CISZ: C, 39.45; H, 6.0; Q 1 9 . 4 . Found: C, 39.1; H, 5.9; C1, 19.2. The chloride (7.3 g.) was dissolved in 200 cc. of water and 15 g. of picrylsulfonic acid was added. The picrylsulfonate crystallized a t once. The yield was 17 g., corresponding t o 957" of the theory. Anal. Calc'd for CeHl1Sz.CeHzNpOpS: C, 32.8; H, 3.0; N, 9.6; S, 21.9. Found: C, 32.7; H, 3.0; E,9.5; S, 21.8. Methyl-bis(,8-hydrox~ethyl)sulfonium salts. The iodide was obtained by heating 6.1 g. of thiodiglycol with 35.5 g. of methyl iodide (5 equivalents) in a mixture of 50 cc. of methanol and 10 cc. of water for 20 hours. On evaporation i n vucuo, the iodide was obtained as a yellowish brown liquid (Davies and Oxford, 6). The sulfonium compound was isolated in crystalline form as a salt of flavianic acid. For this purpose the iodide was dissolved in

718


u)cc. of water and a solution of 15.7 g. of flavianic acid in 20 cc. of water and 1 cc. of 3 N HCI was added. The methyl-bis(j3-hydroxyethy1)sulfonium flavianate crystallized a t once as microscopic needles; yield 19.6 g. or 83% of the theory. I t was thoroughly washed with ether and recrystallized from water containing some HCI; yield 18.25 g. Anal. Calc'd for C6&,O2S~CloH,X2OgS: C, 40.0; H , 4.0; N, 6.2; S, 14.2. Found: C, 39.9; H, 4.1; N, 6.3; S, 14.4. The flavianate was converted to the chloride by treatment, in aqueous solution, with one equivalent of arginine monohydrochloride. The arginine flavianate was removed a t 0" and the filtrate, which contained traces of flavianic acid, was employed for toxicity and stability tests. Methionine methyl sulfonium iodide. This compound was prepared by the procedure of Toennies (9). The salt was recrystallized twice from the minimum amount of water with the addition of acetone; m.p. 168-169". Anal. Calc'd for CeH14INOZS: C, 24.7; H , 4.8; N, 4.8; S, 11.0. Found: C, 24.6; H, 4.8; N, 4.8; S, 11.1.

The authors wish to acknowledge with thanks the helpful cooperation of Miss Jean Grantham and Miss Rosalind E. Joseph, who assisted in the conduct of these experiments, and of Dr. Adalbert Elek and Mr. Stephen M. Nagy, who performed the microanalyses reported in this paper. NEWYORK,N. Y . REFERENCES STEIN,MOORE,AND BERGMANN, J . Org. Chem. (paper I this series). ETTELAND KORLIK,Coll. Czechoslov. Chem. Communications,3, 585 (1931). NENITZESCU AND SCARLETESCU, Bey., 67, 1142 (1943). HURD(1944)." HELLERMAN, PORTER,AND PRESTA(1942)." DAVIES AND OXFORD, J . Chem. SOC.,224 (1931). STEINAND MOORE, J . Org. Chem. (paper I11 this series). (8) SMITH,JAGER,HOIE, ELLIS,GRAEF,BEVELANDER, SUMMERS, CRAWFOBD, AND WING (1943)." (9) TOENNIES, J . Bioi. (?hem.,132, 455 (1940).

(1) (2) (3) (4) (5) (6) (7)

oUnpublished data obtained in the United States.

CHEMICAL REACTIONS OF MUSTARD GAS AND RELATED

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