[CONTRIBUTION FROM
THE
LABORATORIES OF THE ROCKEFELLER INETITUTE FOR MEDICAL RESEARCH]
CHEMICAL REACTIONS OF T H E NITROGEN MUSTARD GASES.’ 111. THE TRANSFORMATIONS OF ETHYL-BIS(B-CHLOR0ETHYL)AMINE I N WATER JOSEPH S. FRUTON* AND MAX BERGMAN”
Received March 28, 1946
In the course of a systematic study of the chemical reactions of the nitrogen mustard gases (1,2), experiments were performed to determine the sequence of reactions undergone by ethyl-bis(p-chloroethy1)amine (EBA) in water. The methods employed in this series of experiments were analogous to those used in the case of the lower homolog, methyl-bis(Pchloroethy1)amine (MBA). ks may be expected from its close structural similarity to the latter compound, the behavior of EBA, in many respects, is similar to that of its homolog, MBA. The transformations of ethyl-bis(/3-chloroethyl)aminein bicarbonate solution. When EBB (0.02 M ) is shaken with aqueous bicarbonate (pH S), somewhat more than one equivalent of C1- is released during the first 15 minutes (Table I). :During this period very little H+ is liberated. The 2-hour thiosulfate titer of an aliquot is very high. From these three sets of data, it must be concluded that EBA has been transformed into ethylenimonium form (I) (cf. Figure 1). The use of the 2-hour thiosulfate titer represents a departure from the procedure employed in the case of MBA. As pointed out previously (l), it required 10 minutes for the theoretical uptake of one equivalent of thiosulfate per mole of MBA. On the other hand, EBA reacts with thiosulfate a t a faster rate than does MBA; thus, even when the time employed for its reaction with thiosulfate is reduced to 5 minutes, the thiosulfate test gives a value much higher than that theoretically possible for one equivalent of ethylenimonium group per mole of EBA. For this reason, in the present communication, the total thiosulfate consumption after 2 hours is used; when taken in conjunction with the data on C1- and H+ liberation, it provides a measure of the extent of ethylenimonium ion formation. When the reaction is allowed to go to completion, EBA like MBA. consumes 2 equivalents of thiosulfate to form the “Bunte salt” (cf. experimental section). As will be noted from Table I, during the later stages of the reaction of EBA in bicarbonate, there is released a second equivalent of C1-. This process is completed within 24 hours, and is accompanied by an appreciable liberation of H+. There also occurs a progressive decrease in the concentration of quaternary compounds as represented by the difference between the C1- and H+ values. 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 June 1942-January 1944. 2 Present address, Yale University, New Haven, Connecticut. * Died, November 7,1944.
543
544
JOSEPH S. FRUTON AND MAX BEROMANN
The H+ liberation continues after all the theoretically possible C1- has appeared, and after 96 hours reaches the value of 1.68 milliequivalent of H+ per mM of EBA. At this time, the thiosulfate titer has dropped to a low value which is not appreciably different from that for the concentration of quaternary
+/Y
CzHsN
\
CSHbNCHtCHlCl
\
C2H:N-
\
\
CHa
--f
-+
/CHIC&oH
CIH~N
\
CHtCHiCl
CHz
+/
-
CH2
/
CHICHZC1
CHtCHzOH
CXHpN
\
CHZC HzOH
CHiCHzOH
111
1v FIG.1
TABLE I THEHYDROLYSIS OF ETHYL-BIS(#-CHLOROETHYL)AMINE (EBA) IN BICARBONATE SOLUTION Concentration of reactants per cc.: 0.02 mM of EBAmHCI; 0.02 m M of NaOH; 0.03 mM of NaHCOt. Temperature 25'; pH 8. TIME. MIN.
15 60 120 300 600 1440 2880 5i60
CL-LIBERATED PEX OF EBA M.EQUIV.
1.11 1.19 1.27 1.47 1.70 2.00 2.02 (2.00)
H+LIBERATED OF
PER mM EBA I6.EQUIV.
0.08 0.18 0.22 0.43 0.65 1.10 1.43 1.68
(cL-)-m+)
LEQUIV.
1.03 1.01 1.05 1.05 1.05 0.90 0.59 (0.32)
NArSzOs CONSUbtED IN 2 HOUXS PER fl4M OF EBA Y.EQUIV.
1.87 1.80 1.68 1.46 1.27 0.76 0.44 0.27
nitrogen present. These data are best interpreted as indicating nearly complete hydrolysis of EBA to ethyldiethanolamine. This conclusion receives confirmation from the data presented in Table I11 which show that the EBA transformation products I1 and I11 are hydrolyzed rather completely to form ethyldiethanolamine. When the initial concentration of EBA is increased from 0.02 M to 0.13 M , compounds containing stable quaternary nitrogen are formed. The extent of
NITROGEN MUSTARD GAS.
545
111
the formation of these compounds is much less in the case of EBA, however, than had been observed previously for MBA under the same experimental conditions. The transformations of ethyl-his(@-chloroethy1)amine in unbuffered solution. When EBA (0.02 M ) is shaken with water in the absence of bicarbonate (Table 11),somewhat more than one equivalent of C1- is liberated rapidly (within 15 minutes) but no further C1- liberation is observed during the next 48 hours. Since, within 15 minutes, little H+ has been liberated and the 2-hour thiosulfate titer is very high, most of the EBA must have been transformed into the ethylenimonium form (I). As the reaction proceeds, Hf is liberated, but much more slowly than during the hydrolysis in aqueous bicarbonate. The acid liberation in the unbuffered solution gradually approaches one equivalent of H+ per mole of EBA. The thiosulfate titer drops much more slowly than in the experiment with bicarbonate. TABLE I1
THEHYDROLYSIS OF ETHYL-BIS@-CHLOROETHYL)AMINE (EBA) IN UNBUFFERED SOLUTION Concentration of reactants per cc.: 0.02 m M of EBAsHCl; 0.02 ntM of NaOH. Tem:perature 25"; pH ca. 7 (at start); pH ca. 5 (at end).
15 90 300 1440 2880
1.12 1.15 1.15 1.17 1.16
0.10 .25 .35 .75
.87
1.88 1.85 1.68 1.25 1.10
It is of interest that a t any time the SUMof the milliequivalents of thiosulfate consumed in 2 hours and the milliequivalents of H+ liberated is equal to two, within the limits of experimental error. This finding indicates that, under these experimental conditions, hydrolysis is the only reaction occurring in unbuffered solution. The presence in the solution of compounds containing stable quaternary nitrogen is, therefore, precluded. This conclusion is supported further by evidence to be presented below. It will be recalled that, under similar conditions, MBA gives rise to compounds containing stable quaternary nitrogen. It would appear, therefore, that, as was noted in bicarbonate solution, EBA has less of a tendency to form dimeric products in unbuffered solution than does MBA. The isolation of l-ethyl-l-(/3-chloroethyl)ethylenimonium picrylsulfonate. As shown above, EBA is rapidly converted into the ethylenimonium form (I). This irnonium compound has been isolated as its picrylsulfonate from a 30-minute old unbuffered solution of EBA (0.133 M ) . The procedure was essentially the same as that employed in the isolation of the corresponding ethylenimonium form of hlBA (1). The precipitate formed on addition of sodium picrylsulfonate to a 30-minute
546
JOSEPH 8. FRUTON AND MAX BERGMANN
old solution of EBA is pure I picrylsulfonate. In the case of MBA,the initial precipitate contained large amounts of the dipicrylsulfonate of the dichloro cyclic dimer of MBA [N ,N'-dimethyl-N ,N'-bis(p-chloroethy1)piperazinium dichloride]. It is of interest that under comparable conditions no appreciable quantity of dimeric products can be detected in the case of EBA. The isolation and hydrolysis of the chlorohydrin (11). After the hydrolysis of EBA in unbuffered solution has proceeded for 48 hours, one equivalent of C1and nearly one equivalent of H+ have been liberated, suggesting that the principal product present at this time is the chlorohydrin (11). This chlorohydrin TABLE I11 THEHYDROLYSIS OF THE TRANSFORMATION PRODUCTS OF ETHYL-BIS@-CHLOROETHYL)AMINI I N BICARBONATE SOLUTION Concentration of reactants per cc.:0.02 mM of picrylsulfonate of I1 or 111; 0.08 mM of
NaHCOa. Temperature 25". In the hydrolysis of I1 the pH was 7.4 a t the start, 8.2 after 20 hours, and 8.8 after 44 hours. In the hydrolysis of I11 the pH was 8.3 at the start and 8.6 after 24 hours. TIMZ, MIN.
CL- LIBEPATED PEP mM II M.EQUIV.
H+LIBEPATED 11 M.EQUfV.
30 60
180 300 480 1200 1440 2640
0.95 0.97 1.00
0.02 .05 .18
1
PEP
111'
mM Y.EQUIV.
1.05 0.10
1.00
.75
.25 .35 1.00
.47
1.01
.75
0.90
0.85 -60
-60
.95
.10
.30
0 It will be noted that the disappearance of 111 occurs more rapidly than when i t ie formed during the hydrolysis of 11. The higher initial pH during the hydrolysis of I11 may be the reason for this effect.
has been isolated as a salt of picrylsulfonic acid from such a 48-hour aged solution of EBA. The yield corresponded to 90% of the original EBA. As may be seen from Table 111, the picrylsulfonate of the chlorohydrin (11) dissolves readily in aqueous bicarbonate and, within 30 minutes at 25", is completely converted into the ethylenimonium form (111). The hydrolysis of this ethylenimonium compound to ethyldiethanolamine (IV) proceeds slowly. After 20 hours at 25", only about 40% is hydrolyzed, while after 44 hours, the extent of hydrolysis attains about 70%. The hydrolysis evidently is not complete even after 44 hours because, as will be noted from Table 111, there is an appreciable thiosulfate titer indicating that the solution still contains some 111. As shown below, I11 hydrolyzes to ethyldiethanolamine in bicarbonate solution. T h e isolation and hydrolysis of 1-ethyl-l-(8-hydroxyethyl)ethylenimonium picrylsulfonate. Compound I11 has been isolated in the form of a picrylsulfonate from
NITROGEN MUSTARD GAS.
547
I11
a solution of the chlorohydrin picrylsulfonate which had been aged a t pH 7-8 for 30 minutes at 25". The yield was 84%. It will be noted from Table I11 that during the hydrolysis of I11 in bicarbonate solution, H+ gradually appears and after 24 hours, the Hf liberation reaches a value of nearly one equivalent. The liberation of H+ is accompanied by a corresponding drop in the thiosulfate titer. These results indicate that in bicarbonate solution, I11 hydrolyzes to ethyldiethanolamine (IV). Under comparable conditions, the corresponding ethylenimonium compound derived from MBA is only partially hydrolyzed to methyldiethanolamine, the principal product being a linear compound formed by the reaction of the imonium compound with methyldiethanolamine. TABLE IV THETOXICITT TO MICEOF
THE
TRANSFORMATION PRODUCTS OF
ETHYL-BIS(&CHLOROETHYL)AMINE SUBSTAWE (EMPLOYED AS CHLORIDE OR EYDROCHLORIDE)
DOSAGE
EFFECT ON micE
NO. OB MlCF. INJECTED
mE./kC.
I1
I11
7.5 4.5
3 3
All dead within 15-63 min. 2 dead within 23-25 min. 1 dead within 2 days. 1 dead within 28 min. 2 dead within 2 4 days. 3 dead within 4 days. 3 alive after 5 dags. All alive after 5 days. All alive after 5 days.
19.5 13.5 8.5 5.5 2.75
3 3 3 3 3
All All All All All
55 40
3 3
20
3
10
6
dead within 18-26 min. dead within 17-19 min. dead within 2 9 4 5 min. alive after 7 days. alive after 7 days.
The toxicity o j the transformation products of ethyl-bis(&chloroethyl)amine. In order to study the toxicity of the transformation products of EBA, their picrylsulfonates were converted to the corresponding chlorides or hydrochlorides by double decomposition with the dichloro cyclic dimer of MBA as described previously (1). The approximate toxicities of these compounds upon intraperitoneal injection into mice are given in Table IV. The results are in good agreement with the LDao values of Smith et d.(3). These investigations reported that for mice the chlorohydrin (11) has an L D Bof~ less than 8 mg./kg. intraperitoneally or subcutaneously. The LD60 value for I11 was about 7.5 mg./kg. intraperitoneally, 5.5 mg. subcutaneously, and 5.0 mg./kg. intravenously. For I, the LD6o was less than 2 mg./kg. upon subcutaneous injection into mice. In contrast to the behavior of the corresponding ethylenimonium compound derived from MBA, compound I11 is no more toxic than its parent compound,
548
JOSEPH S. FRUTON AND MAX BERGMANN
the chlorohydrin (11). This might be explained by the fact that the cyclization of the chlorohydrin to I11 occurs much more rapidly than does the corresponding reaction in the MBA series. EXPERIMENTAL
Analytical methods. The mehods for determining the C1- and Hf liberation, and the thiosulfate consumption have been described previously (1,2). The isolation of 1-ethyl-1 -(P-chloroethy1)ethylenimonium picrylsulfonate. An aqueous solution (25 cc.) containing 20 mM of EBAeHCI was added to 203 cc. of 0.1 N NaOH. The mixture was shaken for 30 minutes, then chilled to 0" and acidified with HCl to Congo Red. A solution of 8 g. of sodium picrylsulfonate was then added. After 20 minutes, the crystalline precipitate was filtered and dried over P20s; yield 5.2 g. Anal. Calc'd for CsHIrClN.CsHzN300S: C, 33.8; H, 3.5; N, 13.1; C1,8.3. Found: C, 33.8; H, 3.6; N, 13.0; C1,8.4. The 2-hour thiosulfate titer, performed in the manner already described (2), was 2.02 equivalents. The isolation of the chlorohydrin (11)as a picrylsulfonate. To 403 cc. of water containing exactly 20 mM of NaOH were added 4.13 g. of EBA.HC1 (20 mM). The mixture was shaken for 15 minutes and the resulting clear solution was left at 25' for 48 hours. After chilling to 0', the solution was acidified with HC1 to Congo Red. Sodium picrylsulfonate (7.5 g. in 80 cc. of 0.5 N HCl) then was added. The solution was concentrated under reduced pressure (bath temperature 35-40') to a volume of 100 cc. and kept at 0" overnight. The yield 8.0 g. (90%). crystals which formed were filtered off and dried in vacuo over PzOs; The 2-hour thiosulfate consumption of this substance was 1.08 equivalents. The salt was recrystallized from acetone-petroleum ether. Its thiosulfate titer was now 1.02 equivalents; m.p. 110-111'. Anal. Calc'd for CEHtlClNO-CaHzOsN3S: C, 32.4; H, 3.6; N, 12.6; Cl, 8.0. Found: C, 32.6; H, 3.7; N, 12.4; C1,7.9. The isolation of 1 -ethyl-1 -(P-hydroxyethy2)ethylenimoniumpicrylsulfonate. The chlorohydrin picrylsulfonate (2.23 g., 5 mM) was stirred for 30 minutes a t 25' with 53 cc. of 0.1 N NaHC03. A slight amount of undissolved material was filtered off and the filtrate was chilled to 0" and acidified to Congo Red. Crystallization began quickly. After standing a t 0' overnight, the crystals were filtered off and dried in vacuo over P206.The filtrate was concentrated to 25 cc. under reduced pressure end a second crop of crystals was collected. The total yield was 1.7 g. (81%). The 2-hour thiosulfate titer of each fraction was 1.01 equivalents. On sodium fusion, the material gave a negative test for chloride. For analysis, the substance was recrystallized from acetone-petroleum ether. Anal. Calc'd for CeH,4N0.C6H2NaOoS: C, 35.3; H, 4.0; N, 13.7. Found: C, 35.4; H, 4.0; N, 13.5. Preparation of 'LBunteSalt" oj' E B A . Five cc. of a solution containing 4 mM of EBAHCl were added to 20 cc. of a solution containing 16 mM of NaZS203,16 mM of NaHCOs, and 4 m M of NaOH. The mixture was shaken for 30 minutes and left a t room temperature for 6 hours. The solution was evaporated to dryness under reduced pressure and the residue was extracted with three 100-cc.portions of hot absolute alcohol. On concentration of the alcohol extract under reduced pressure, the "Bunte Salt" was obtained in crystalline form; yield 2.2 g. The substance was recrystallized from alcohol-ether. For analysis, the material was dried to constant weight in air. Anal. Calc'd for C6H,,NP\TarOsS4.2H20: C, 17.8; H, 4.2; N, 3.5; S, 31.6; Na, 11.3; HnO, 8.9. Found: C, 17.6; H, 4.2; N, 3.4; S, 31.4; Na, 11.3; H 2 0 ,8.9.
NITROGEN MUSTARD GAS.
I11
549
The authors wish to acknowledge with thanks the helpful cooperation of Miss Rosalind E. Joseph, who assisted in the conduct of these experiments, and of Mr. Stephen M. Nagy, who performed the microanalyses reported in this paper. NEWYORK, N. Y. REFERENCES (1) GOLUIMBIC, FRUTON, AND BERGMANN, J. Org. Chen., (paper 1 this series). (2) GOLUMBIC AND BERGMANN, J. Org. Chem., (paper I1 this series).
(3)
SMITH, KARNOFfiKY
, ADDIS,JAMES, GRAEF,BEVELANDER, SUMMERS, AND
(1943).a 6
Unpublished data obtained in the United States.
CRAWFORD
