[CONTRIBUTION FROM
THE
LABORATORIES OF THEROCKEFELLER INSTITUTE FOB MEDICAL RESEARCH 1
CHEMICAL REACTIONS OF THE NITROGEN MUSTARD GASES.1 VIII. T H E OXIDATION OF T H E NITROGEN MUSTARD GASES BY PERACIDS MARK A. STAHMANNZ
AND
MAX BERGMAN"
Received March $2, 1945
The rate and extent of oxidation by peracids. It has been observed that the nitrogen mustards are oxidized to the corresponding N-oxides by peracids in aqueous solution. This oxidation is rapid in weakly alkaline solution but is slow in acid solution. At p H 7.7, each of the three nitrogen mustards [methylbis(&chloroethyl)amine (MBA), ethyl-bis(&chloroethyl)amine (EBA), tris(0chloroethy1)amine (TBA)], as well as methyldiethanolamine, consumes about one equivalent of peracetic acid within 15 minutes. At pH 3.2,the peracetic acid consumption was 10% or less of the amount found at p H 7.7. The slower oxidation of the nitrogen mustards in acid solution might be due to ammonium salt formation. Peracids other than peracetic acid also oxidize the nitrogen mustards. When a bicarbonate buffered solution of MBA was shaken with a chloroform solution of perbenzoic acid or was treated with an aqueous solution of monoperphthalic acid, within 15 minutes 1.42and 0.85 equivalents of peracid respectively were consumed. The preparation of the N-oxides of the nitrogen mustards. The principal product of the peracid oxidation of each of the nitrogen mustards, the N-oxide, has been isolated as its hydrochloride from a bicarbonate buffered reaction mixture containing excess peracetic acid. The oxidation of MBA to methyl-bis(8-chloroethy1)amine oxide (I) and the formation of its hydrochloride (11) may be represented by the following equation : ClCHnCHz
+ peracid-
\ /NcHa
ClCH2 CH2
MBA
r
CICHz H2\ 'N'
I C 1CH2C Hz
OH
(11) 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. * Present address, University of Wisconsin, Madison, Wisconsin. *Died, November 7, 1944. 1
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NITROQEN MUSTARD QAS.
587
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Ethyl-lbis(j3-chloroethy1)amine oxide hydrochloride (111) and tris(j3-chloroethy1)amine oxide hydrochloride (IV) have also been prepared from EBA and TBA respectively by a procedure similar to that used for the preparation of 11.
(111) (IV) In all three cases, the amine oxide was isolated in a yield of 78435% of theory. This high yield indicates that oxidation of the nitrogen atom proceeds much more rapidly than does hydrolysis of the 8-chloroethyl groups. TABLE I THEREACTION OF METHYL-BIS(~-CHLOROETHYL)AMINE (MBA) OXIDE WITH WATER AND WITH THIOSULFATE Concentration of reactants per cc.: 0.02 mM of MBA oxide HCI; 0.02 m M of NaOH; 0.06 mM of NaHCOI. I n the experiment given in Column 4, 0.05 mM of NatSzOs per cc. was also present. pH 8; temperature 25".
--
TOTAL NAvSZO: UPTAKE PEB m M OF MBA OXIDE
--1 4 7
24 72
0.18 0.30 0.67 1.03 1.11
0.02 0.11 0.17 0.45 0.69
3-HOUR NAzSYO,
m. EQUIV.
UPTAKE^ PER mA! OF MBA OYIDE
(4)
(5)
0.25 1.12 1.21 1.79 1.79
Y. EQUIV.
0.84 0.82 0.20 0.00
0 This value was determined by adding, after the indicated time interval, excesa NazS20ato an aliquot of the hydrolysis mixture; after exactly 3 hours the unreacted thiosulfate was titrated.
The chemical reactions of M B A and TBA N-oxides. The stability of the ch1oroet:hyl groups of these N-oxides and their reaction with thiosulfate4 were studied 'by measuring the liberation of C1- and H+ and the consumption of thiosulfate in a bicarbonate solution. Table I shows that, under these condition, within 72 hours, I1 liberates 1.11equivalents of C1- and only 0.69 equivalents of H+. The smaller Hf liberation suggests that the over-all process is of a complex nature and warrants further investigation. Column 5 of Table I shows that the thiosulfate titer decreases as the hydrolysis proceeds and that the final hydralysis products show no thiosulfate consumption. The Cl- and H+ liberation, and thiosulfate consumption of IV in bicarbonate solution were measured in a manner similar to that employed for the study of 11. 4 The use of the reaction with thiosulfate as a n index of the presence of reactive ethylenimonium or p-chloroethyl groups has been discussed in previous papers of this series.
688
MARK A. STAHMANN AND MAX BERQMANN
The data in Table I1 show that within one hour in bicarbonate solution at 25”, 0.97 equivalents of C1- and 0.52 equivalents of H+ are liberated and 1.32 equivalents of Na2S203are consumed, In marked contrast, I1 liberates within the same time only 0.18 equivalents of C1-, 0.02 equivalents of H+ and reacts with only 0.25 equivalents of Na2S20s. It will be noted that in the hydrolysis of IV, the rate of C1- liberation is greater than is the rate of H+ liberation. The same observation was made with 11. Column 4 of Table I1 shows that I V is capable of reacting with approximately 2 equivalents of thiosulfate within 4 hours. Column 5 indicates that the 35-minute thiosulfate titer decreases as hydrolysis proceeds. Two reaction products have been isolated from a 48-hour old bicarbonate buffered solution of IV. The isolation of P-hydroxyethyl-bis(P-chloroethy1)amine TABLE I1
THE REACTIONOF TRIS(~-CHLOROETHYL)AMINE (“€3.4) OXIDEWITH WATERA N D
WITH
THIOSULFATE Concentration of reactants per cc.: 0.02 mil4 of TBA oxide HCl; 0.02 m M of NaOH; 0.08 mM of NaHCOa. In the experiment given in Column 4,0.07m M of Na&OI per cc. was also present. Temperature 25’; pH 8. TIXE, KIN.
(1)
15 30 60 120 240
CL- LIBEPATSD PER m Y OF TBA OXIDE K EQUIV.
(2)
1
HCLIBERATED
0.57 0.76 0.97 1.18 1.33
OF
PER
TB;)OXIDE
M.EQVIV.
0.81 0.96 1.32 1.73 2.04
0.09 0.29 0.52 0.79 0.91
1.08 1.08 1.07 0.90 0.53
~
0 These values were determined by removing, at the time indicated, an aliquot of the hydrolysis mixture, adding excess NanS1O1,and after exactly 35 minutes titrating the unreacted thiosulfate.
N-oxide (V) in a yield of 54% indicates that the major portion of I V is hydrolyzed under these conditions as follows : CICH2 CHn
\+
CICHzCHz-N-OH /
x-
ClCHs CH2 --+
\+
ClCHzCH2-N-OH
/
C I C HC ~ H~’
HOCH, CH2’
(IV)
(VI
x- + HCI
Triethanolamine also was isolated from the reaction mixture in a yield of 20%. It is possible that triethanolamine is not a normal product of the decomposition
of the N-oxide but was produced during the isolation procedure. Triethanolamine could arise as the result of decomposition of a portion of the N-oxide to yield TBA. Subsequent hydrolysis of the TBA would yield triethanolamine. A similar reaction was observed by Bamberger and Leyden (1)who found that the
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N-oxide of dimethylaniline decomposes on heating to form the original amine, dimethylaniline. The toxicity of the N-oxides of the nitrogen mustards. The toxicity of 11, 111, and IV was determined by intraperitoneal injection of graded doses into sets of three mice. The results indicate that the LD60 for I1 is 75-125 mg./kg.; for 111, 50-100 mg./kg.; and for IV, 2.5-5.0mg./kg. By this method of administration, the LD60 for MBA hydrochloride is 2.4 mg./kg. (2) and for EBA hydrochloride is 1.05 mg./kg. (3). It is of interest that oxidation of the nitrogen mustards to the corresponding N-oxides results in the formation of compounds which still possess appreciable toxicity. From the above data and those of Table 11,it will be noted that the greater toxicity of IV is associated with an increase in the rate of C1- liberation and thiosulfate consumption. EXPERIMENTAL
Preparation of peracids. Peracetic acid was prepared by stirring at 0" for 2 hours a mixture of 153 g. (1 mole) of sodium perborate and 71 cc. (0.75 mole) of acetic anhydride in 400 cc. of water. The mixture was filtered, the filtrate acidified to Congo Red with 5 N sulfuric acid and the peracetic acid was separated from the inorganic salts by extraction with ether or by distillation under reduced pressure. Monoperphthalic acid was prepared from sodium perborate and phthalic anhydride by a similar procedure. After filtration, the reaction mixture was extracted with ether and a n aqueous solution of monoperphthalic acid was obtained by shaking the ether extract with water. Perbeneoic acid was prepared by the method of Bergmann and Witte (4). Oxidation of the nitrogen mustards. The rate of oxidation of the nitrogen mustards by peracetic acid was measured by estimation of the peracid consumption in a reaction mixture containing per cc.: 0.05 mM of nitrogen mustard hydrochloride, 0.10 mM of peracetic acid, and 0.45 mM of sodium bicarbonate. The pH was 7.7 and the temperature was maintained a t 25". A control solution was made up without the nitrogen mustard. After 15 minutes, the unreacted peracetic acid was determined by the addition of an excess of sulfuric acid and potassium iodide followed by titration of the liberated iodine. The oxidations at pH 3.2 were carried out in a similar manner with a reaction mixture containing per cc. 0.09 mM of the nitrogen mustard hydrochloride and 0.18 mM of peracetic acid. The estimation of the extent of oxidation of MBA by monoperphthalic acid in NaHC03 solution was carried out as described above. The oxidation by perbenzoic acid was carried out at 25" by shaking a mixture of 5 cc. of a 0.2 .N chloroform solution of perbeneoic acid and 10 cc. of a solution containing 0.05 mM of MBA and 0.45 mM of NaHC03 per cc. After 15 minutes, the perbenzoic acid remaining in the mixture was determined. The isolation of methyl-bis(&chloroethyl)amine oxide hydrochloride (II). A solution of 25 g. of EdBA.HC1 (0.12 11.1) in 100 cc. of water was added over a period of 15 minutes with stirring t o 1500 cc. of 0.26 N peracetic acid (0.39 M) containing 98 g. of NaHC03 (1.16 M ) . The NaRC03was added to the peracid solution immediately before beginning the addition of the iVlBA.HC1. The mixture was stirred a t 25" for 15 minutes, and then acidified with HC1 to Congo Red. The acid solution was concentrated to dryness (bath temperature, 40") under reduced pressure. Anhydrous acetone (50 cc.) was added and the mixture was again concentrated to dryness. The operation was repeated three times. The residue was then extracted with three 150-cc. portions of anhydrous acetone. The combined acetone extracts were filtered and concentrated under reduced pressure t o a thin syrup. This was dissolved in 200 cc. of absolute ethyl alcohol, and 2 liters of anhydrous ether was then added with stirring. The amine oxide hydrochloride first separated as an oil which crystal-
590
MARK A. STAHMANS AKD MAX BERGMANN
lized when allowed to stand a t 4". The product was collected by filtration, washed with dry ether, and dried over PZOs. The yield was 21.3 g. (85%). After recrystallization from an anhydrous ethyl alcohol-ether mixture, the melting point was 109-110'. Anal. Calc'd for CLH1lC1lKO~HC1: C, 28.8; 11, 5.8; S , 6.7; C1, 51.1; C1-, 17.1. Found: C, 28.6; H , 5.6; N,6.7; C1, 50.6; C1-, 17.3. T h e isolation of ethyl-bis(p-ch1oroethyl)amine oxide hydrochloride ( I I I ) . The preparation of I11 was carried out in a manner similar t o that employed for 11. From 1.65 g. of EBA.HC1, 1.52 g. (85%) of the corresponding amine oxide hydrochloride was obtained. After recrystallization from an anhydrous alcohol-ether mixture the melting point was 8586". Anal. Calc'd for C&4C13KO: C, 32.4; H , 6.3; p1', 6.3; C1, 47.8; C1-, 15.9. Found: C, 32.1; H , 6.3; N , 6.3; C1, 47.4; C1-, 16.0. T h e isolation of tris(P-chloroethyl)amine oxide hydrochloride ( I V ) . The preparation of I V was performed in a manner similar to that employed for 11. From 1.94 g. of TBA.HC1, 1.79 g. (78'j?.0) of the corresponding amine oxide hydrochloride was obtained. After recrystallization from an anhydrous alcohol-ether mixture, the melting point was 91-92". Anal. Calc'd for CeH13C14SO: C, 28.0; H , 5 . 0 ; N, 5 . 5 ; C1, 55.2; C1-, 13.8. Found: C, 28.1; €1,5.1; K , 5.7; C1, 55.2; C1-, 13.8. T h e hydrolysis of tris(p-ch1oroethyl)amine oxide hydrochloride ( I V ). A reaction mixture containing 10.27 g. (40 mM) of IV, 13.44 g. (160 mill) of NaHC03, and 40 cc. (40 m M ) of N NaOH in 2 liters of water was allowed to stand for 48 hours a t 25" and mas then extracted with four 300-cc. portions of ether. The ether extracts were combined and then divided into t\To equal portions. To one portion of the combined ether extracts, 40 cc. of N HC1 was added and the ether was removed under reduced pressure until only an oily aqueous suspension remained. A solution of 7.06 g. of Reinecke salt in 60 cc. of absolute methyl alcohol was added with stirring to this suspension. The Reinecke salt of P-hydroxyethylbis(P-chloroethy1)amine oxide (V) separated. The mixture was allowed to stand a t 4" for 24 hours, filtered, and the product washed with about 50 cc. of dilute HCl. The yield was 6.16 g. (54y0). The Reineckate was recrystallized by dissolving in 50 cc. of absolute methyl alcohol followed by the addition of 100 cc. of dilute HCl; m.p. 146". c, 23.0; €1, 3.8; N,18.8; c1, 13.6; s, 24.6. Anal. Calc'd for C6H14Clz?r'z0.CIHpCr?u'sS4: Found: C, 23.0; H, 3.6; S , 18.4; C1, 13.7; S, 24.2. The aqueous phase remaining after the ether extraction was acidified with HCl to Congo Red and then concentrated under reduced pressure to dryness (bath temperature, 40"). Absolute alcohol (100 cc.) was added to the residue and the mixture again was concentrated to dryness. The addition of alcohol and concentration was repeated three times. The residue was then extracted with 150 cc. of boiling absolute alcohol. The hot alcoholic extract was filtered, cooled, 150 cc. of dry ether was added, and the mixture was allowed to stand a t 4" for 24 hours. The product was then collected by filtration and washed with dry ether. The yield of triethanolamine hydrochloride was 1.50 g. (20%). After two recrystallizations from absolute methyl alcohol, the melting point was 176-177". Knorr (5) reports the melting point of triethanolamine hydrochloride as 177". S o depression of the melting point was observed on admixture of the isolated material with an authentic sample of triethanolamine hydrochloride. Anal. Calc'd for C6H1~pr'o~"Cl: C, 38.8; H , 8 . 6 ; pr', 7.5; C1, 18.6. Found: C, 38.8; H, 8.7; N, 7.6; C1, 19.1.
The authors wish to acknowledge with thanks the helpful cooperation of Miss Rosalind E. Joseph, mho assisted in the conduct of these experiments, and of Rlr. Stephen M. Kagy, who performed the microanalyses reported in this paper. NEWYORK,N. Y.
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REFERENCES (1) BAJIBERGER .4SD LEYDEN, BET., 34, 12 (1901).
(2) University of Chicago Toxicity Laboratory (1942).a (3) ShlITH, JAGER, HOIE, ELLIS,GRAEF,BEYELANDER, SUMMERS, CRAWFORD, AND WING (1943).Q (4) BERGM.4NX AXD WITTE, Chem. Zentr., I, 1925 (1911). (5) KXORR,Ber., 30, 918 (1897). -0 Unpublished data obtained in the United States.
