[CONTRIBUTION FROW
THE
LABORATORIES OF THE ROCKEFELLER INSTITUTE FOR MEDICALRESEARCH]
CHEMICAL REACTIONS OF MUSTARD GAS AND RELATED COMPOUNDS.’ VII. THE CHEMISTRY OF BIS (P-CHLOR0ETHYL)SULFONE, DIVINYL SULFONE AND DIVINYL SULFOXIDE MARK A. STAHMA”,* AND
CALVIN GOLUMBIC? WILLIAM H. STEIN, JOSEPH S. FRUTON4
Received March 22, 1946
In the course of the past 25 years, much interest has centered on the chemistry of bis(P-chloroethy1)sulfone [mustard gas sulfone, to be referred to as H sulfone (I)]. The ease with which H sulfone reacts with sulfhydryl, phenolic hydroxyl, and amino groups was recognized by Helfrich and Reid (l), Cashmore and McCombie (2), and Lawson and Reid (3). In fact, the great chemical reactivity of H sulfone led Flury and Wieland (4)in 1921 to suggest that vesication by H mas due t o oxidation of H in the skin to the sulfone. No positive evidence has been adduced to support this hypothesis, and there appear to be theoretical grounds for questioning its validity ( 5 ) . Moreover, the recent work, which has demonstrated the great chemical reactivity of H itself, has rendered the hypothesis of Flury and Wieland unnecessary. Fevertheless, H sulfone continues to receive much attention because of its vesicancy and toxicity, and its close chemical relationship to H. Alexmder and McCombie (6) reported that H sulfone readily splits out HC1 to form divinyl sulfone (11) when treated with triethylamine in dry benzene. They found that divinyl sulfone was an extremely reactive substance, combining readily with sulfhydryl, phenolic hydroxyl, and amino groups to form P-substituted derivatives. Ford-Moore (7) studied further the reaction of H sulfone and divinyl sulfone with amino compounds and observed that the product of the reaction of these two sulfones with a given compound invariably was the same. Ford-Moore and Lidstone (8) found that divinyl sulfone was formed when an aqueous solution of H sulfone was heated with calcium carbonate, and Marshall and Williams (9) earlier had observed that H sulfone liberated C1- in aqueous phosphate solution (pH 7.3-7.7). Boursnell, Francis, and Wormall (10) noted that, on treatment of H sulfone with aqueous sodium bicarbonate, a product was formed which reacted more readily with the amino group of glycine than does H sulfone itself. They suggested that this intermediate product was divinyl sulfone. Price (11) showed by isolation that divinyl sulfone was formed from H sulfone in bicarbonate - buffered solution. Reaction o f H sulfone, H sulfoxide and divinyl sulfone with water. When H 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 %’ere performed during the period January 1942-August 1944. * Present address, University of Wisconsin, Madison, Wisconsin. * Present address, University of Pittsburgh, Pittsburgh, Pennsylvania. 4 Present address, Yale University, New Haven, Connecticut. 719
720
STAHMANN, GOLUMBIC, STEIIC, AND FRUTON
sulfone is dissolved in water, the pH falls slowly. After several hours, a pH of about 3.5-4.5 is attained by a saturated solution, and tests with silver nitrate then show a trace of C1-. As soon as the pH is raised by the addition of alkali, equivalent amounts of Hf and C1- are liberated. Table I shows that this elimination of HC1 is catalyzed by OH-. At pH 6.5-7.0, 0.35 m.equiv. of HCl is liberated within 30 minutes, whereas, at pH 7.5-7.8, 1.06 m.equiv. of HCl is liberated within 3 minutes and 1.47 m.equiv. within 30 minutes. The rate of HC1 liberation is greatly reduced by the addition of sodium bicarbonate. In the presence of one equivalent of bicarbonate a t p H 7.5-7.8, only about 0.37 m.equiv. of HC1 is liberated within 30 minutes. In marked contrast to H and H sulfone, the non-vesicant his(@-chloroethy1)sulfoxide (H sulfoxide) liberated HCl only very slowly a t physiological pH values. TABLE I IFFLWNCE OF pH AND BICARBONATE UPON THE REACTION OF H SULFONE WITH WATER The R sulfone (I)dissolved inmethyl cellosolve (1 m M of I i n 0.8 cc. of methyl cellosolve) was added to water. The initial concentration of I was 0.02 molar. The HC liberation was followed electrometrically by adding NaOH (0.5 N ) to maintain the pH as indicated. The C1- liberation was determined argentometrically on aliquots of the reaction mixture. Temperature, 25'. PH 7.5-1.8
fiH 6.5-7.0
No NaHCOa
TIYE,LUN.
I
No NaHCOa
H+liber,permddI, C1-liber.permH1 H + l i b e r . p e r d f I, C1-1iber.permH I, C1-liber.permM1, Y. EQUIV.
3 5 10
30 90 150 420
0.27 0.35 0.55
Y.EQUIV.
0.27 0.38 0.54
Y. EQUIV.
1.04 1.17 1.26 1.47 1.66 1.81
Y. EQUIv.
1.07 1.16 1.26 1.47 1.66 1.81
Y. EQUIV.
0.40 0.80 1.02 1.55
In the pH range 7-8, only about 0.02m.equiv. of HC1 is formed within 150 minl Utes. At higher pH values HC1 is liberated more rapidly, 0.23 m.equiv. of H C being formed a t pH 9.5within 150 minutes. It has also been found that the rate of HCl liberation is reduced by bicarbonate. Divinyl sulfone appears to be relatively stable in aqueous solution. Since, as will be shown below, divinyl sulfone reacts readily with thiosulfate, the disappearance of reactive vinyl groups on aging the sulfone in aqueous bicarbonate a t 25' and pH 8.4 was followed by determining the decrease in thiosulfate titer. It was found that the thiosulfate titer of aged divinyl sulfone solutions (0.05M ) decreased by about 15% in 22 hours, and by about 60% in 94 hours. Reaction of H sulfone and divinyl sulfone with sulfhydryl compounds. Both H sulfone and divinyl sulfone react readily with thiosulfate a t pH 7.5. The data in Table I1 show that the rate of the reaction of H sulfone is markedly decreased
REACTIONS OF MUSTARD GAS.
721
VI1
by bicarbonate. I n the absence of bicarbonate, 0.42 m.equiv. of thiosulfate was consumed per mM of H sulfone within 1 hour, while in the presence of 1 mole equivalent of sodium bicarbonate, only 0.20 m.equiv. of thiosulfate was consumed. In contrast, the rate of the reaction of divinyl sulfone with thiosulfate in not influenced by bicarbonate. Both in the presence and in the absence of bicarbonate, about 1.00 m.equiv. of sodium thiosulfate was consumed per mM of divinyl sulfone within 1hour. In this reaction OH- was liberated a t the %me rate as thiosulfate was consumed. In the initial stages of the reaction of H sulfone with thiosulfate HC1 accumulated, and 0.34 m.equiv. of NaOH was required during the first few minutes to TABLE I1 INFLUENCE OF BICARBONATE UPON THE REACTION OF H SULFONE (I) SULFONE (11) WITH THIOSULFATE
AND OF
DIVINYL
Concentration of reactants per cc. at start of reaction: Columns 2 and 4,0.02 mM of I; 0.05 mM of Na2S201. Columns 3 , 5 , and $0.02 mM of 11; 0.05 mM of NazS20s;0.02 mM of NaHCOs. Temperature, 25’; pH 7.4-7.6.
I TIYE.
mix
I
OR- PO-D
I No NaHCOs, Y. EQUIV.
1.
---
,5 10
30 60 120 180 240 360
-a
b
I
THIOSULFATE CONSTMED PEP m Y OF:
I1
+m.
NaHCOI, EQUIV.
No NaHCOq, Y. EQUIV.
2 .a
3.
4.b
0.11 .16 .27 .42 .67
0.05 .07 .12 .20 .39
1.04 1.27
.78 .99
0.20 .39 .73 1.01 1.28 1.43 1.56
PEP
mY
OF 11
NaHCOt
+ NaRCOr.
Y. EQUIV.
0.23 .37 .71 .99 1.29 1.54 1.62
0.20 .35 .66 1.01 1.33 1.54 1.68
+
0.34m.equiv. of NaOH per mM of I were added at the start to raise the pH t o 7.5. The pH was held as nearly as possible at pH 7.5. However, since the solution was not
buffered, the pH fluctuated between 4 and 10.
maintain the pH a t 7.5. The accumulation of HC1 shows that the reaction of H sulfone with groups such as thiosulfate must first involve the formation of a reactive vinyl group. The fact that bicarbonate inhibits the reaction of H sulfone with thiosulfate, but does not inhibit the reaction of divinyl sulfone with thiosulfate, is a clear indication that bicarbonate decreases the rate a t which vinyl groups are formed, but does not alter the reactivity of the vinyl groups once they are present. The reaction of H sulfone with thiosulfate may, therefore, be represented by the reactions portrayed in Figure 1. The “Bunte salt” (111)has been isolated from the reaction of divinyl sulfone nith thiosulfate. In alkaline solutions I11 slowly liberates substances which consume iodine. After 24 hours a t pH 8.7, the iodine consumption was 0.24
722
STARMANN, GOLUMBIC, STEIN, AND FRUTON
m.equiv., while after 48 hours, the iodine consumptionwas 0.32 m.equiv. Hence, it seems likely that the second reaction given in Figure 1 is reversed in strong11 alkaline solutions. Ford-Moore and Lidstone (8) report that the reaction of divinyl sulfone with thiophenol, which leads to the formation of bis(phenylthioethyl)sulfone, is catalyzed by nitrogenous bases. We have found that when divinyl sulfone and thiophenol were allowed to react in 55% aqueous methylcellosolve solution in the absence of a catalyst, the reaction was 73% complete within 90 minutes. However, the reaction was 99% complete within 7 minutes when a small amount (0.018 mole equivalent) of triethylamine was added. Aqueous sodium bicarbonate is equally as effective as catalyst as is triethylamine. When the triethylamine was previously neutralized to pH 7.0 with HC1, its catalytic effect was lost; the reaction was then only 77% complete within 90 minutes. Hence it would appear that this catalytic effect is not specific, but results from the fact that the reaction between thiophenol and divinyl sulfone is very sensitive to slight changes in pH. In aqueous solution, the rate of the reaction of divinyl sulfone with the SH group of both cysteine6and P-mercaptoethanolis also markedly dependent upon CHsCH2CI
CH=CH2
~~
CHzCH2SSOaNa
/ -+ so2
/ so2 \
+
\
CH2CH2CI
(1)
CH=CHz
2WeOH
CH2CH2SS03Na
(11)
(111)
FIG. 1
the pH, increasing rapidly as the pH rises. At pH 4.4, 1.18 m.equiv. of the SH groups of cysteine reacted per mM of divinyl sulfone within 20 minutes, while at pH 5.1, 1.24 m.equiv. reacted within 3 minutes, and a t pH 6.2,2.0m.equiv. reacted within 2 minutes. At pH 5.4, 1.00 m.equiv. of the SH groups of &mercaptoethanol reacted per mM of divinyl sulfone within 5 minutes, while a t p H 6.0 and 6.5, 1.46 and 1.96 m.equiv. respectively reacted within the same time. The product formed by the reaction of divinyl sulfone with P-mercaptoethanol, his[@-(P-hydroxyethylthio)ethyl]sulfone has been prepared. In contrast to the “Bunte salt” of divinyl sulfone, this product did not liberate reducing substances a t pH 8.5. Reaction of divinyl sulfone with nitrogenous bases. Divinyl sulfone reacts readily with pyridine to form the bis(@-pyridiniumethyl)sulfone derivative (IV) . Bis(p-pyridiniumethy1)sulfone dichloride was isolated from the reaction of divinyl sulfone with pyridine and pyridine hydrochloride in alcoholic solution. The rate and extent of the reaction of pyridine with divinyl sulfone were followed a t pH 7.5 by continuous electrometric titration of the OH- produced (Figure 2). The decomposition of the reaction product was followed under the same con6 Ford-Moore (7) has shown that divinyl sulfone reacts with two equivalents of cysteine t o form bis (cysteinylethyl)sulfone [SO2(CaH4SCH&H(“2) COOH)ZI.
REACTIONS OF MUSTARD GAS.
723
VI1
ditions by continuous titration of the H+ liberated when bis(p-pyridiniumethy1)sulfone dichloride was dissolved in water. The data in Table I11 show that the reaction of divinyl sulfone with pyridine at pH 7.5 starts at a rapid rate, 1.15 m.equiv. of OH- being formedwithin 5minutes. Subsequently, the reactionstops completely when 1.48 m.equiv. of OH- (74% of theory) have been formed. The reverse reaction, the decomposition of IV is also very rapid a t the start, 0.40 m.equiv. of H+ being formed within 1.5 minutes. The decomposition stops when 0.49 m.equiv. of H+ (25y0 of theory) are liberated. Since essentially the same equilibrium point was reached by the reverse reaction as obtained by the forward reaction, the addition of pyridine to divinyl sulfone must proceed aacording to the reversible reaction sequences given in Figure 2. Since OH- is formed in the reaction of divinyl sulfone with pyridine, the position of the equilibrium will be determined by the pH of the solution. High pH values favor decomposition of IV, while lower pH values favor its formation. At low pH values, the forward reaction does not proceed because pyridine is then almost exclusively in the ionized form. When the experiment described CH=CHz
/
+
so2
\
/
CHzCHzNC6Hs C6HsN
+
320 S
CH=CHz
/ SOz \
+
+
OH-
CH=CHz
CHzCHzNCsHa
+
OH-
FIQ. 2
in Column 2 of Table I11 was repeated, except that the pH was maintained a t 6.5, equilibrium was established when 1.72 m.equiv. of OH- had been liberated. At pH 7.5, this value was 1.48 m.equiv., and a t pH 8.6 and 9.7 equilibrium was established when 0.83 and 0.17 m.equiv. respectively of OH- had been liberated. It follows from the reversible nature of the reaction of pyridine with divinyl sulfone that, in aqueous solution, IV should give rise to reactive vinyl groups. The validity of this conclusion is supported by the observation that the pyridinium salt reacts with cysteine, thiosulfate, and alanine. When a bicarbonatebuffered solution of bis(P-pyridiniumethy1)sulfonedichloride was treated with cysteine, the SH groups slowly disappeared. After about 10 minutes, a distinct odor of pyridine was detectable and 0.52 m.equiv. of cysteine had reacted. After about 2 hours, 1.21 m.equiv. of cysteine had reacted and a crystalline precipitate appeared. This precipitate increased in amount during the next few hours until, a t the end of 20 hours, 2.04 m.equiv. of cysteine had reacted. The product was then filtered off and identified as the bis-cysteinyl derivative of divinyl sulfone (7).
724
STAFIMANN, GOLUMBIC, STEIN, AND FRUTON
For purposes of comparison, the reaction of bis(p-pyridiniumethy1)sulfide(12) with cysteine was investigated. In contrast to the sulfone, no reaction between the sulfide and cysteine occurred within 48 hours. It was also desirable to compare the stability of a p-pyridiniumethyl sulfonium salt. However, attempts to methylate bis(&pyridiniumethyl)sulfide with methyl iodide were unsuccessful, only the diiodide of bis(P-pyridiniumethy1)sulfide being obtained. However, the alkylating properties of p-pyridiniumethyl-1 ,4-dithiane sulfonium chloride have been pointed out in the previous paper of this series (13). TABLE I11 REACTION OF DIVINYL SULFONE (11) WITH PYRIDINE AND OF BIS(&PYRIDINIUMETHYL)SULFONE DICHLORIDE (IV) WITH WATER Concentration of reactants per cc. at start of reaction: 0.05 m M of I1 or IV; 0.20 mM of pyridine (Column 2) ;0.10 mM of pyridine (Column 3). The liberation of H+ or OH- was followed electrometrically by adding 0.5 N NaOH or HCl t o maintain the pH at about 7.5. In the experiment given in Column 3 sufficient water was added to the reaction mixture to make the final volume the same as in the experiment reported in Column 2. ' I = MINUTES ,
1.
0 .o 0.5 1 .o 1.5 2.0 2.5 3.0 4.0 5.0 6.0
7.0 8.0 9.0
10.0 11 .o 12.0 14.0 20.0 180.0
OH-
PRODUCED PEP m M
2.
1
or n, Y.EQUIV. H+PPODUCED PEP m K 3.
-
-
0.23 .38 .51
0.08
.68
.40 .49
.79 .92 1.06
1.15 1.25 1.34 1.38 1.41 1.44 1.46 1.47
.49 *
49
.49
1.48
1.48 1.48
.49
OF IV, Y. EQUIV.
REACTIONS OF MUSTARD GAS.
725
VI1
Compound IV was also isolated as a picrylsulfonate after a n aqueous bicarbonate-buffered reaction mixture containing pyridine and divinyl sulfone had stood for 2 hours. After 24 hours, P-pyridiniumethyl-8-hydroxyethylsulfone was isolated from a similar reaction mixture. Bis(P-pyridiniumethy1)sulfone dipicrylsulfonate slowly dissolves in aqueous thiosulfate solution with the consumption within 2 hours of 1.04 m.equiv., and within 24 hours of 1.78 m. equiv., of thiosulfate. A slight decrease in this thiosulfate consumption was noted after 48 hours, in agreement with the previously noted instability of the thiosulfate derivative of divinyl sulfone in alkaline solution. Similarly, 6-pyridiniumethyl-p-hydroxyethylsulfone picrylsulfonate consumes 0.96 m.equiv. of thiosulfate within 24 hours. The extent of the reaction of divinyl sulfone with nicotinic acid and nicotinamide was followed electrometrically by continuous titration with acid under the same experimental conditions employed in the case of pyridine. Divinyl sulfone reacts slowly with nicotinamide a t pH 7.5-8.0, the reaction ceasing after 14 minutes when only 0.079 m.equiv. of OH- has been liberated. Thus, i t would appear that equilibrium is established when only 4% of the theoretical quantity of OH- is produced. When nicotinic acid is allowed to react with divinyl sulfone a t pH 7.6-7.7, equilibrium is established when 1.14 m-equiv. of OH- (57% of theory) are liberated. Pyridine and its derivatives are not the only nitrogenous bases which react with divinyl sulfone. Attempts have been made to prepare the products of the reaction of divinyl sulfone with the following bases : ethyl-bis(P-chloroethy1)amine, methyl-bis(6-chloroethyl)amine, tris(P-chloroethyl)amine,ethyldiethanolamine, methyldiethanolamine, diethanolamine, quinoline, nicotine, brucine, and strychnine. The experimental conditions were the same as or similar to those employed in the synthesis of the pyridine derivative. With the P-chloroethylainines and ethyldiethanolamine, the only crystalline compounds that could be isolated were the corresponding hydrochlorides. No crystalline products were obtained from the reaction with methyldiethanolamine, quinoline, and nicotine. When brucine hydrochloride was treated with divinyl sulfone in alcohol, a crystalline derivative was obtained the elementary composition of which agreed with that of the expected structure (V). When strychnine hydrochloride was allowed to react with divinyl sulfone in aqueous methanol, a crystalline product (VI) was obtained.
c1-
c1CH2 C H2 &2 C21 H2a 0
/ so2 \
4
CHzCHzNz CsH2604 61(VI
CH2C ~ H , ~ C ~0I 2H ~ ,
/ so2 \
CH=CH,
(VI)
726
STAHMANN, GOLUMBIC, STEIN, AND FRUTON
The brucine and strychnine derivatives of divinyl sulfone both were found to consume thiosulfate. The brucine derivative consumed 1.2 m.equiv., and the strychnine compound 0.65 m.equiv. of thiosulfate within 22 hours. In the case of V, the consumption of thiosulfate must be attributed to decomposition of the compound with the liberation of alkylating groups. The strychnine derivative (VI), however, still retains one vinyl group which might react with thiosulfate. Diethanolamine hydrochloride reacts vigorously with divinyl sulfone in ethanol to form bis(P-hydroxyethy1)-1,4-thiazanium dioxide chloride (VII). The thiazanium chloride (VII) slowly liberates alkylating groups a t pH 7.5 when treated with cysteine or thiosulfate. Within 3 hours, 0.92 mM of cysteine SHgroups had reacted with VII, while within 44 hours, 1.36 m.equiv. of cysteine SH-groups and 0.79 m.equiv. of thiosulfate had disappeared. The dicysteinyl derivative of divinyl sulfone was formed in the reaction of VI1 with cysteine. When VI1 was treated with thionyl chloride, a product was obtained the elementary analysis of which agrees with the expected chloro-substituted cyclic structure (VIII). c1-
c1-
CHzCH2
/ so2 \-
CHzCH2OH
\+/
/ so2 \
N
/ \
CHzCH2
CH2CH2
\+/
N
/ \
CH2 CHP
CHzCH20H
(VII)
CHtCHzC1
CHaCHzCl
(VIII) CHI CH2
/ so2 \
c1-
CH2 CH2 C1
\ / NH+ \
CHaCH2 CH2 CH2 C1 (IX) On dissolving the chlorinated product (0.02 M ) in water, however, the pH falls to about 4.3, and when the solution is titrated in 80% alcohol to phenolphthalein, one equivalent of alkali is consumed. These findings suggest that the open chain isomer (IX) is present under these conditions. The product from the chlorination of VI1 will, therefore, be designated as /3-[bis(8-chloroethyl)amino]ethyl vinyl sulfone (IX), although its actual structure may be that represented either by VI11 or by IX. The rapid reversible formation of vinyl groups from other quaternary ammonium compounds derived from divinyl sulfone would suggest that VI11 would likewise readily be converted into IX. Compound IX is of some interest since it is not only a monosubstituted derivative of divinyl sulfone, but is also a nitrogen mustard by virtue of the two P-chloroethyl groups attached to a tertiary nitrogen atom. The chlorinated product is unstable in bicarbonate-buffered solution. The data in Table IV show that C1- is liberated a t a faster rate than H+, indicating that hydrolysis of the chloroethyl groups proceeds through the formation of intermediate un-
REACTIONS OF MUSTARD GAS.
727
VI1
TABLE IV
THEHYDROLYSIS OF /3-[BIS(/3-CHLOROETHYJi)AMINO~ETHYL VINYL SULFONE (Ix) Concentration of reactants per cc.: 0.01 m M of IX; 0.10 mM of NaHCOt. Temperature, 25'; pH 8.1. The liberation of H+ or C1- was followed in the manner H '
cl- LIBERATED
PEE tnY 01 I X , M.EQUIV.
TIME, MIN.
15 30 60 120 240 300 420 1440 2880
0.36 0.62 0.94 1.34 1.48 1.56 1.66 1.76
-
0.30 .21 .23 .I1 .I2 .I2 .I2 .03 .03 CH2 CH2 C1
I
CHz=CHSO*CHsCHpN
CHI CHZ C1
l/i"
- (H')
M.EQUIV.
CHzCHzCl
CH2=CHSOsCH~CH2N'
\
(C1-)
0.06 0.41 0.71 1.23 1.36 1.44 1.54 1.73 1.77
1.so
/
LIBERATED PEE d f OF IX, M.EQUIV.
'/I
CH2
+\
+
c1-
CHz
CH2CHzCl
CHz=CHS02 CHI CHzN
+\
+
/
Hz0 + CH,=CHSOzCHzCHzN
\
C H2
CHiCHzCl
+ Hi CHIC HZ0 H
CHzCH20H
I
CHe=CHSOaCHsCHaN
/ \
CHICHIOH
--+ CHZ=CHSOzCH2CH2N
I/$'
+\
CHz CHs C1
+
c1-
CHI
CHz CHZOH
!/T
CHz=CHS02 CH2 CHzN
+\
+
/CHzcBzoH H+
+
HzO + CHa=CHSOzCHzCH2N
CHz
\
CHzCHZOH
FIQ.3
stable ethylenimonium ions (Figure 3) (14). The data further indicate that the second ethylenimonium ion is more unstable than the first. Evidence for the
728
STAHMANN, QOLUMBIC, STEIN, AND FRUTON
presence of a vinyl group in the end products of hydrolysis was gainedthrouhg the observation that a solution of IX, after aging for 2 days a t pH 8.1, reacted with 0.46 m.equiv. of cysteine. The chlorinated product (IX) readily reacts with thiosulfate, 1.32 m.equiv. of thiosulfate being consumed within 1hour and 2.80 m.equiv. within 24 hours. The consumption of almost 3 equivalents of thiosulfate in 24 hours shows that the vinyl group and both chloroethyl groups have reacted with thiosulfate. Ford-Moore and Lidstone (8) have reported that divinyl sulfone reacts with proline to give the crystalline betaine (X). The betaine (X) liberates alkylating groups slowly a t pH 7.4, consuming 0.98 m.equiv. of cysteine SH-groups, and TABLE V THETOXICITY OF MICE TO DERIVATIVES OF DIVINYL SULFONE Intraperitoneal injection into sets of three mice. ~~
APPROXIMATE Isro
COm?OUND"
w.lk
I11 IV V VI VI1 IX
425 200
80 3.75 875 7.8
a Bis[p-(8-hydroxyethylthio)ethyl]sulfoneis non-toxic in doses of 1 g./kg. The betaine (X) is also non-toxic in doses of 1.5 g./kg. * A t doses below 50 mg./kg., death is delayed.
0.17 m.equiv. of thiosulfate, within 44 hours. The dicysteinyl derivative of divinyl sulfone was formed in the reaction with cysteine.
/
CH2 CH2
so2
CHzCHz
1
\+/
N
i / \ I CH, CH2 CHCHz I I
coo(X) Toxicity of derivatives of divinyl sulfone. The toxicity of several derivatives of divinyl sulfone was determined by intraperitoneal ijection of graded doses into sets of three mice. The results, presented in Table V, indicate that some of these compounds possess a noteworthy toxicity. This finding is not surprising in view of the ease withwhichmany of the substances decomposeunderphysiological conditions of pH and temperature t,o regenerate both the highly toxic divinyl sulfone, and the other component of the parent compound, which in some instances also possesses a high toxicity. It may be pointed out that chlorination of compound VI1 to yield I X results in over a 100-fold increase in toxicity.
REACTIONS OF MUSTARD GAS.
VI1
729
Chemical reactions of divinyl sulfoxide. In contrast to H sulfone and divinyl sulfone, divinyl sulfoxide does not exhibit vesicant action. It seemed of interest, therefore, to study more closely the chemical reactions of divinyl sulfoxide, and to compare its behavior with that of divinyl sulfone. Like divinyl sulfone, divinyl sulfoxide also reacts with the sulfhydryl group of cysteine and P-mercaptoethanol. However, these reactions are very much slower in the case of the sulfoxide than are the corresponding reactions of the sulfone. The rate of the reaction of the sulfoxide with SH groups is markedly dependent upon the pH of the solution. At pH 5.8, divinyl sulfoxide reacts within 40 minutes with only 0.43 m.equiv. of cysteine SH-groups, whereas, a t pH 7.1, it reacts within 40 minutes vi-ith 1.05 m.equiv. and within 24 hours with 2.02 m.equiv. of cysteine SH-groups. The product formed in this reaction is bis(@-cysteinylethyl)sulfoxide(XI). 2"
I
/
so
\
CH2 CH2 SCHz CHC 0OH
I
2"
(XI) Divinyl sulfoxide reacts with 0.06 m.equiv. of P-mercaptoethanol SH-groups within 1 hour a t pH 5.7 and with 0.34 m.equiv. a t pH 7.0; within 24 hours it reacts with 0.83 m.equiv. a t pH 5.7 and with 2.04 m.equiv. a t pH 7.0. The reaction of the sulfoxide with thiosulfate is also much slower than is that of the sulfone. Within 96 hours, divinyl sulfoxide reacts with only 0.39 m.equiv. of thiosulfate, whereas, under similar conditions, divinyl sulfone reacts with 1.90 m.equiv. of thiosulfate within 5 hours. The data presented in Table VI show that &vinyl sulfoxide also reacts with pyridine with the liberation of OH-. However, the rate and extent of the reaction of the sulfoxide is much less than was that of the sulfone. From Table VI it would appear that a t pH 7.5, only 0.277 m.equiv. of OH- are liberated within 480 minutes and that equilibrium conditions are not then attained. Under comparable conditions, divinyl sulfone liberated 1.48 m.equiv. of OH- and reached equilibrium within 14 minutes. At higher pH values, less OH- is liberated by both the sulfoxide and the sulfone. The data in Table VI suggest that the reaction of &vinyl sulfoxide with pyridine, like that of divinyl sulfone, involves the addition of pyridine to the double bonds, that the reaction is reversible, and that the position of the equilibrium is influenced by the pH of the reaction mixture. However, definite proof for all of these contentions must await the isolation and investigation of the reaction products. Attempts to prepare bis(P-pyridiniumethy1)sulfoxide dichloride by a procedure analogous to that used to prepare the corresponding
730
STAHMANN, GOLUMBIC, STEIN, AND FRUTON
sulfone were unsuccessful. From Table VI it would appear, however, that in the case of the sulfoxide, the equilibrium has been displaced rather far to the left, thus possibly explaining our failure to obtain the pyridinium sulfoxide. Discussion. The experiments reported in this communication, coupled with those given in the previous paper of this series (13), indicate a striking similarity in behavior between @-substitutedsulfones and p-substituted sulfonium salts. It has been demonstrated that two p-chloroethyl sulfonium salts and H sulfone all lose HC1 by elimination with the formation of reactive vinyl groups. This TABLE V I REACTIONOF DIVINYL SULFOXIDE WITH PYRIDINE Concentration of reactants per cc. at start of reaction: 0.05 m M of divinyl sulfoxide; 0.20m M of pyridine. Temperature, 25". The OH- formation was followed electrometrically by adding HCI (0.1 N in Column 2; 0.05 N in Columns 3 and 4) to maintain the pH a t the desired value.
INFLUENCE OF pH
ON THE
OH- PBODUCED PEE ndd
OF DIVINYL SULFOXIDE AT
TIME, MINUTES
PH 8.6, KEQUIV. 3.
1.
24 48 72 96 120 144 168 192 216 240 264 288 312 336 360 384 408 432 456 480
0.012 .033 .051 .070 .086
.lo3 .I18 .I33 .I50 .I63 .178 .187 .200 ,213 .224 .237 .247 .257 .266 .277
0.021 .041 ,056 .072 .OS5 .098 .IO9 .I19 .128 .136 .144 .152 .I58 .I64 .I71 .I75 .I79 .I83 .187
PH
9.7, M.EQUIV. 4.
0.011 .017 .021 .024 .025 .026 .026 .026 .026 ,026 .026 .026 .026
elimination reaction in the case of both the sulfonium salts and H sulfone is influenced in a similar manner by changes in pH and by the presence of salts. Thus, the rate of elimination of HC1 is markedly dependent upon pH, being very rapid at alkaline pH values, and slowing up as the pH falls; a t acid pH values HCl is not formed a t all. Moreover, the rate of elimination of HC1 is strongly inhibited by bicarbonate. The sulfonium salts and H sulfone appear to react readily with a number of substances to form p-substituted derivatives. It has been demonstrated, however, that chemical reaction is, in all cases, preceded by the elimination of HCl
REACTIONS OF MUSTARD GAS.
VI1
731
and the formation of reactive vinyl groups. There is, in addition, a striking similarity in the behavior of the vinyl sulfonium groups and vinyl sulfone groups thus formed. This similarity is exemplified by a comparison of the properties of vinyl-l,4-dithiane sulfonium chloride with those of divinyl sulfone. Both substances react readily with pyridine in aqueous solution a t pH 7.5 to form P-pyridinium derivatives. In both cases the reaction is reversible, and the speed of attainment of equilibrium, as well as the position of the equilibrium, is similarly influenced by pH. Furthermore, the pyridinium derivatives in both cases are unstable, and decompose with the formation of reactive alkylating groups which combine readily with SH groups or the amino group of alanine. A further similarity between the properties of vinyl-1 ,4-dithiane sulfonium chloride and divinyl sulfone resides in the behavior of the two substances towards thiosulfate. Both react with thiosulfate a t pH 6-8, the rate of the reaction increasing with rising pH and being unaffected by bicarbonate. At moderately alkaline pH values (pH 8.5-9.5), however, both thiosulfate derivatives decompose with the liberation of substances titratable with iodine. Finally, Bartlett (15) has shown that acetic acid is eliminated from diacetylthiodiglycol methylsulfonium picrylsulfonate and also from diacetylthiodiglycol sulfone by treatment with bicarbonate solution, and that, in both cases, the resulting products consume thiosulfate. The ready decomposition of sulfones (or sulfoniuin compounds) which contain a quaternary nitrogen atom in the @-positiont o the sulfur atom is of interest and may be compared with the usual decomposition of quaternary ammonium compounds described by Hofmann and familiarly known as the Hofmann degradation or exhaustive methylation. In the Hofmann degradation, a quaternary amrnonium base decomposes under the influence of heat with scission of one of the C-Sbonds and the resulting formation of a tertiary amine, an ethylenic group and water. On the other hand, the decomposition of the P-quaternary derivatives of sulfones (or sulfonium salts) occurs in aqueous solution a t pH values near neutrality and proceeds spontaneously a t room temperature with the formation of a tertiary amine, an ethylenic group and a hydrogen ion. In contrast to the Hofmann degradation, the decomposition of these sulfone (and sulfonium) derivatives is a reversible reaction and the extent of the decomposition is influenced by the pH. For the general problem of vesication, it is of some interest that derivatives of divinyl sulfone (and consequently of H sulfone as well) are far more unstable than are similar derivatives of H . It is also of interest that the sulfonecompounds decompose to yield reactive alkylating groups. It seems not unlikely, therefore, that in vivo the products of the reaction of divinyl sulfone with cellular constituents would undergo a similar decomposition. Should such a decomposition occur, it would become possible for one molecule of sulfone to react in succession with several functional groups in a cell. The sulfone residue might be handed on, so to speak, from one group t o another, until it finally either reacted with some tissue component with which it formed a stable compound or was removed by the circula,t,ion
732
STAHMANN, GOLUMBIC, STEIN, AND FRUTON
The question also may be raised as to whether the reactivity of oxidized H derivatives may play a role in the mechanism of the physiological action of H. In order to provide an experimental basis for this speculation, it is necessary to determine whether animal tissues contain enzyme systems capable of oxidizing the sulfide sulfur of H derivatives. Such an oxidation process might convert stable H residues attached to tissue constituents into unstable and reactive sulfoxide or sulfone residues. From a calculation of the energy necessary, Sugden ( 5 ) has concluded that the oxidation of H to H sulfone is unlikely to occur in vivo. No information has come to our attention concerning the energy required for the oxidation of H derivatives, however. The authors mould like t o express their thanks to Miss Jean Grantham who assisted in carrying out many of the experiments, and to Doctor Adalbert Elek who performed the numerous microanalyses reported in this paper. EXPERINMENTAL
Preparation of divinyl sulfone
