[CONTRIBUTION FROM
THE
LABORATORIES OF THE ROCKEFELLER INSTITUTE FOR MEDICAL RESEARCH ]
CHEMICAL REACTIONS OF THE NITROGEN MUSTARD GASES.' I. THE TRANSFORMATIONS OF METHYL-BIS(0-CHLOROETHYL)AMINE IN WATER CALVIN GOLUMBIC,* JOSEPH S. FRUTON3,
AND
MAX BERGMANN4
Received March 99, 1946
The work to be presented in this series of papers was undertaken in order to elucidate certain aspects of the chemistry of a new group of vesicant chemical warfare agents, the nitrogen mustards. These substances are bis(p-chloroethyl) substituted tertiary amines of the general formula RN(CH2CH2C1)2. One compound of this series, tris(p-chloroethyl)amine, was first described by Ward (1). The transformations undergone by the nitrogen mustards in water were chosen as the first point of attack since a detailed knowledge of these reactions seemed essential to an understanding of the general chemistry of the nitrogen mustards. Furthermore, the transformations of the nitrogen mustards in water are of interest from the biochemical point of view, since mater is a major constituent of all biological systems. Finally, the possibility that the nitrogen mustards might be employed in chemical warfare as water contaminants made it imperative to study exhaustively the chemical reactions undergone by these agents in water. The experimental results presented in this communication concern the sequence of chemical reactions undergone by methyl-bis(0-chloroethy1)amine (MBA) when this agent reacts with water in the presence or the absence of bicarbonate. On the basis of these results, a reaction mechanism (cf. Figure 1) has been proposed to explain the complex transformations of MBA in aqueous solutions. Two types of experimental evidence will be presented. The first involves an analytical study of the reactions undergone by MBA in aqueous bicarbonate solution buffered at pH 8 and in unbuffered aqueous solution [cf. also (2)]. The second type of evidence involves the isolation, in crystalline form, of the successive transformation products of MBA and a study of their properties. The transformations of methyl-bis(0-chloroethy1)aminein bicarbonate solution. When MBA (0.02 fM)reacts with water a t pH 8 (bicarbonate buffer), it goes into solution rapidly with the liberation of nearly one equivalent of C1- and the appearance of negligibly small amounts of H+ (Table I). It will be noted from Figure 1 that this is the result to be expected on formation of the l-methyl-l1 This work was done in whole under Contract No. OEMsr-313 between The Rockefeller Institute for Rledical Research and the Office of Scientific Research and Development, which amurnes no responsibility for the accuracy of the statements contained herein. The experiments were performed during the period June 1942-Ja,nuasry1944. 2 Present address, University of Pittsburgh, Pittsburgh, Pennsylvania. 3 Present address, Yale University, Kew Haven, Connecticut. Died, h-ovember 7 , 1944. 518
NITROGEN MUSTARD GAS.
519
I
(8-chloroethy1)ethylenimonium ion (I). Analytical evidence for the reaction was first presented by Hartley, Powell, and Rydon (3), and careful kinetio studies have been made by Bartlett (4)and Cohen ( 5 ) . The cyclization of MBA /CHzcH*cl CHsN
\
C HiCHzCl
11 CH3
V
CHzCIIZCI
1’ C Hz C H?OH
/
CHaN
\ C HzC HI C 1
ll
I1 CHzCHtOH
+/
‘CHZ
1
L
I11 C HzC HzO H
/ CHIN
\ C Ht CH2O H
I
I
IV
C H a NC HP C H20 H
VI1 FIG.1
is a special case of the conversion of chloroalkylamines into heterocyclic compounds, investigated by Freundlich (6). Additional evidence for the rapid and nearly quantitative conversion of MBA to the ethylenimonium form is provided by the data given in Table I on the thiosulfate consumption of the solution. The rapid reaction of the ethylenimonium forms of MBA with thiosulfate and the use of the thiosulfate titer as an
520
GOLUMBIC, FRUTON, AND BERGMANN
index of ethylenimonium formation is discussed in detail in a subsequent section. Conclusive evidence for the formation of I is furnished by its isolation as a crystalline salt of picrylsulfonic acid from solutions of MBA aged for 30 minutes at pH 8. As the hydrolysis in bicarbonate proceeds, there is observed a slow liberation of additional C1- and H+ in approximately equivalent amounts and a progressive fall in the thiosulfate titer. These observations are in accord with the reaction sequences presented in Figure 1. As will be noted from the Figure, the hydrolysis of ethylenimonium rings to form hydroxyethyl groups is accompanied by the liberation of H+ but not of C1-. On the other hand, when compounds containing quaternary nitrogen are formed, C1- is libeiated without the appearance of Hf. TABLE I THEHYDROLYSIS OF METHYL-BIS(~~-CHLOROETAYL)AMINE (MBA) IN BICARBONATE SOLUTION Concentration of reactants per cc.: 0.02 m M of MBAvHCI; 0.02 d of NsOH; 0.08 mM of KaHCOs. Tempere.turc 25"; pH 8. TIYE,
WIN.
L- LIBERATED FER 7liM OF
MBA Y . E Q U I V .
H+ LIBERATZD PER m M OF MBA M.EQUIV.
(CL-)-(H+) M.EQUIV.
NAn%&
CONSUW3D IN
3 XIN. PER m M OF MBA M.EQUIV.
_.____
a
20 60 120 240 420
0.945
1200 4320
1.83
1.20 1.31 1.50 1.65
0.085 .14 .25 .48 .65 .99
0.86 1.06 1.06 1.02 1.00 0.84
1.90
1.13 1.08
1.06 0.94 .82
.28
.06"
This value represents the thiosulfate consumed in 1 hour.
The steady liberation of greater amounts of C1- than of H+ during the hydrolysis in bicarbonate is evidence for the formation, during the reaction, of compounds containing quaternary nitrogen atoms. The difference between the values for the C1- and H+ liberated represents the amount of quaternary nitrogen present. It will be noted that the thiosulfate titer has decreased markedly after 20 hours without an equivalent diminution in the amount of quaternary nitrogen. This finding indicates that a portion of the intermediates having ethylenimonium rings and chloroethyl groups have been converted into quaternary nitrogen compounds which do not react with thiosulfate under these conditions. At the end of three days nearly the theoretical amount of C1- has been liberated. The low one-hour thiosulfate titer a t this time indicates the absence of any significant quantities of compounds containing chloroethyl groups or ethylenimonium rings. As will be shown later in this paper, the products isolated as picrylsulfonates from a solution aged in this manner were methyldiethanolamine
NITROQEN MUSTARD GAS. I
521
(IV) and the dihydroxy cyclic dimer IN, N’-dimethyl-N ,N’-bis(B-hydroxyethy1)piperazinium salt (VI)]. The latter compound also has been obtained (7) from the chlorohydrin [methyl-B-chloroethyl-,!?-hydroxyethylamine (II)] in alkaline solution. The dichloro cyclic dimer [N ,N’-dimethyl-N ,N’-bis(Bchloroethy1)piperazinium salt (V)] is not formed in appreciable amount in the presence of bicarbonat,e, but as will be shown later, is a major product of the reaction of MBA in unbuffered solution. It will. be noted from Figure 1that the formation of the dihydroxy cyclic dimer is thought to occur by combination of the chlorohydrin (11) and the l-methyl-l@-hydroxyethy1)ethylenimonium ion (111). It has been suggested (5) that these compounds condense to give a chlorinated intermediate (VIII) which subsequently cyclizes to the dihydroxy cyclic dimer VI.
I/
HO CH, CHJ!?CHI CHIIN +\
‘CH2 CHzCl c1-
(VIII) An alternative mechanism would involve the direct interaction of two molecules of I11 to give the cyclic compound. The latter mechanism appears unlikely, however; since, as will be shown later, the dihydroxy cyclic dimer (VI) results from the transformation of the chlorohydrin (11) a t pH 7-8 while, under similar conditions, I11 yields only the linear compound VI1 and methyldiethanolamine. These results suggest that compounds containing chloroethyl groups must participate in the reactions which lead to the formation of the dihydroxy cyclic dimer. It will be noted from Figure 1 that the dihydroxy cyclic dimer (VI), the linear compound VII, and methyldiethanolamine should result from the transformations in bicarbonate solution of both I and MBA itself. After aging I (0.02 M) in aqueous bicarbonate, compounds VI and VI1 were isolated. MBA (0.02M I , however, yields the dihydroxy cyclic dimer (VI) and methyldiethanolamine; the expected linear compound VI1 could not be isolated. No satisfactory explanation can be offered at present for this result. The results presented above, and in what follows, strongly support the reaction sequence given in Figure 1. It should be pointed out: however, that after hydrolysis of MBA, the relative amount
