[CONTRIBUTION FROM
THE
LABORATORIES OF THEROCKEFELLER INSTITUTEFOR MEDICAL RESEABCH I
CHEMICAL REACTIONS OF THE NITROGEN MUSTARD GASES.' VII. MOKOSUBSTITUTION PRODUCTS OF ETHYL-BIS(8CHL0ROETHYL)AMINE AND METHYL-BIS (8CHLOROET1XYL)AMIXE CALVIN GOLUMBIC,1 JOSEPH S. FRUTOR-,' A N D MAX BERGRIANX-' Received March 22, 1946
Khen one of the chloroethyl groups of either ethyl-bis(8-chloroethy1)amine (EBA) or methyl-bis(8-chloroethy1)amine (MBX) reacts with a tertiary base or sulfide, it is to be expected that the chemical nature of the group which is introduced into the nitrogen mustard \Till influeRce the reactivity of the second chloroethyl group. IC order to secure information on this question, the preparation of moncsubstitution products of the nitrogen mustards was ucdertnken. T h e zsoldion of monosubstitution products of E B A and d f B A containing one chloroethyl group. When equimolar amounts of 1-ethyl-1-(8-ch1oroethyl)ethylenimoniurn picrylsulfonate (1)and pyridine are allowed to react in acetone solution (to minimize hydrolysis), the monopyridinium derivative of EBA [ethyl-& chloroethyl-8-pyridiniumethylamine(I)] is formed in good yield. The imonium picrylsulfonate also reacts with methyldiethanolamine under the same conditions to give r\'-ethyl-N-~-chloroethyl-N-8-[~'-methyl-h"-bis(8-hydroxyethyl)ammonium] ethylamine (11). C2H5
I
CICHZCHZNCH~CH~$C~H,
C2H6
I
CH2 CH2 OH
+/
C1 CH2 CHzNCHz CHZN,
I CHzCH2OH CH3
(1) (11) In the MBA series, l-methyl-l-(8-chloroethyl)ethylenimoniumpicrylsulfonate (2) has been found to undergo a similar type of condensation with pyridine to form methyl-j3-chloroethyl-8-pyridiniumethylamine(111). With nicotinic acid the analogous 3-carboxypyridinium derivative (IV) is formed. With thiodig lycol N-rnethyl-N-/3-chloroethyl-N-&[i";'-bis( 0 - hydroxyethy1)sulf onium] ethylamine (V) is obtained. The imonium compound also reacts with methyldiethanolamine t o form N-methyl-N-P-chloroethyl-r\'-P-[N'-methyl-n"-bis(~-hydroxyethyl)ammonium]ethylamine (VI), and with hexamethylene tetramine t o yield the hexamethylene tetraminium derivative (VII). Gurin and co-workers 1 Thin work was done in whole under Contract KO. OEMsr-313 between The Rockefeller Institute for Medical Research and the Office of Scientific Research and Development, which a ~ s u m e sno responsibility for the accuracy of the statements contained herein. The experiments were performed during the period June 1942-January 1944. 2 Present address, University of Pittsburgh, Pittsburgh, Pennsylvania. Present address, Yale University, New Haven, Connecticut. ' Cied, November 7, 1944.
581
682
QOLUMBIC, FRUTON, AND BERGXANN
(3) have reported the isolation of compound VI1 as a chloride. In the present work, all the monosubstitution products of EBA and MBA were isolated, in excellent yields, as dipicrylsulfonates. CHa CH2 CH2 OH +/ ClCH2 CH2kCH2 CH2S
\
CH2 CH2 OH
(V)
/* I "?I
CH2 CHS
N
,C&l
I
ClCH2 CHzNCH2 CH2NCHzN
\
CH2
N
CH2 (VW The properties of monosubstitutzon products of EBA and M B A . The reaction of the compounds I-VI1 with thiosulfate was studied by following the rate of disappearance of thiosulfate from the reaction mixtures. The results are given in Table I together with comparable data (1, 2) on the reaction with thiosulfate of the chlorohydrins (ethyl-P-chloroethyl-8-hydroxyethylamineand methyl-@chloroethyl-@-hydroxyethylamine)derived from EBA and MBA respectively. It will be noted that the monosubstitction products react with thiosulfate at a much slower rate than do the corresponding chlorohydrins. On the other hand, the speed of the reaction of each EBA derivative is considerably greater than that found for the corresponding compound of the MB,4 series. The parent mustards, EBA and MBA, are also known to show this difference in the rate of their reaction with thiosulfate (1, 2). When a monosubstitution product of MBA is subjected to hydrolysis at p H 8, it is found that the liberation of C1- from the chloroethyl group is slower than in the case of the chlorohydrin of MBA. Thus, in the hydrolysis of the nicotinic acid derivative about one hour is required for the release of the chlorine as C1-, whereas less than 20 minutes are necessary for a similar liberation of C1- from the chlorohydrin of MBA (2). When the dipicrylsulfonate of VI is hydrolyzed in the presence of bicarbonate, nearly one equivalent of HCI is liberated in 20 hours. From the hydrolysate, compound (VIII) was isolated as a dipicrylsulfonate. HOCHzCH2
\I
CHS
CHz
I
NCH2 CHzNCH2 CHZOH
/+
HO CH2 CH2
(VIII)
NITROGEN MUSTARD GAS.
583
VI1
Compound (VIII) was previously isolated as a product of the hydrolysis of 1methyl- 1-(8-chloroethyl)ethylenimoniumpicrylsulfonate in bicarbonate solution (2). THE REACTION
OF
TABLE I MONOSUBSTITUTION PRODUCTS
OF
ETHYL-BIS(&CHLOROETHYL)AMINE
(ElBA) AND METHYL-BIS(~-CHLOROETHYL)AMINE (MBA) WITH THIOSULFATE Concentration of reactants per cc.: 0.01 mM of EBA or MBA derivatives (employed as the dipicrylsulfonate) ; 0.025 mM of NazSzOa;0.04 mM of NaHC03. Temperature 25'. PARENT NITROGEN YUSTAED
EBA
Hydroxyl (chlorohydrin) Methyldiethanolamine Pyridine
MEA
Hydroxyl (chlorohydrin) Pyridine R'icotinic acid hlethyldiethanclamine Hexamethylenetetramine" Thiodiglycol
0
-
SU8STIIUENT
.-
10 min.
20 min.
60 min.
120 min.
0.93 * 47 .41
0.96 .72 .62
0.98 .92 .89
0.98 .98 .97
.81 .20 .22 .24 .08 .17
.97 .50 .40
.97 .72
1
.46
.18 .32
.62 .71
.34
.51
The titrations in this instance were carried out at 0' to retard fading of the endpoint.
TABLE I1 THETOXICITY TO MICE OF MONOSUBSTITUTION PRODUCTS OF ETHYL-BIS(&CHLOROETHYL)AMINE (EBA) AND ~~ETHYL-BIS(@-CHLOROETHYL)AMINE (MBA) The chlorohydrins were injected as their hydrochlorides, and the monosubstitution productti were injected as their chloride hydrochlorides.
--
COMPOUND
L D ~ (APPBOX.)5 D
mg.lk.
EBA chlorohydrin MBA chlorohydrin I1 VI I
10 30 200 350 75
6 Determined by intraperitoneal injection of a graded series of doses into sets of three mice.
The i!oxicity of monosubstitution products of EBA and MBA. For the toxicity studies, the dipicrylsulfonates of the EBA and MBA derivatives were converted into the corresponding chloride hydrochlorides by treatment with the dichloro cyclic dimer of MBA. This method of splitting picrylsulfonates has been described earlier (2). The approximate LDao obtained for each derivative upon intraperitoneal injection into mice is given in Table 11. It will be noted that the methyldiethanolammonium derivatives I1 and VI and the pyridinium derivatives I and I11 were less toxic than the corresponding chlorohydrins. Furthermore,
584
QOLUMBIC, FRUTON, AND BERGMANN
each compound of the EBA series is more toxic than its MBA analog. These data, coupled with the results given in Table I, suggest that there is a correlation between the chemical reactivity of the nitrogen mustard derivatives and their toxicity, the more reactive compounds being the more toxic. EXPERIMENTAL
Ethyl-6-chtoroethyl-6-pyridiniumethylamine(I). T o 1.70 g. (4.0 m M ) of I-ethyl-I(8-chloroethy1)ethylenimoniumpicrylsulfonate in 100 cc. of acetone was added 316 mg. (4.0 mM) of pyridine in acetone solution. The mixture was kept for one-half hour at room temperature, and then the acetone was evaporated off. The residue was taken up in 50 cc. of acetone and filtered from a slight amount of undissolved material. After addition of a n acetone solution of 1.46 g. (4.0 mM) of picrylsulfonic acid, the dipicrylsulfonate of I crystallized at once; yield 2.85 g. (91%); m.p. 202-205' with decomposition. Ana2. Calc'd for C1IHloC192.2CaH2X3O&:C, 34.6; H, 2.9; N, 14.0; C1, 4.4. Found: C, 34.6; H, 3.05; N, 14.0; C1,4.4. N-Ethyl-N-6-chloroethyl-N-6 - [N'-methyl-N'-his@ - hydroxyelhyl)ammonium] ethylamine (II). T o 1.7 g. (4.0 m M ) of I-ethyl-1-(6-chloroethy1)ethylenimoniumpicrylsulfonate in 100 cc. of acetone was added 475 mg. (4.0 mM) of methyldiethanolamine dissolved in 25 cc. of acetone. The remainder of the procedure was the same as that for compound I ; yield 2.9 g. (87%); m.p. 191-192'. H Z ~H, ~O 3.7; PS N: , 13.35; C1,4.2. Anal. Calc'd for C ~ ~ H ? , C ~ N Z O * . ~ CC,~32.9; Found: C, 33.2; H, 3.9; N, 13.2; C1,4.05. ilPethyZ-~-chloroethyl-fl-pyridiniumethylamine (IIZ). A solution of 413 mg. (1 m M ) of l-methyl-l-(p-chloroethyl)ethylenimonium picrylsulfonate in 100 cc. of acetone was treated with 80 nig. (1 mJI) of pyridine dissolved in 5 cc. of acetone. After standing one hour a t room temperature, the acetone was removed under reduced pressure. The residue was dissolved in 25 cc. of acetone and treated n i t h 293 mg. (1 mM) of anhydrous picrylsulfonic acid dissolved in 10 cc. of acetone. Tiny prismatic crystals appeared in a few min; utes. After cooling a t Oo, the crystals were filtered off and dried zn vacuo over P ~ O Syield 580mg. (74%). The substance was difficultly Eoluble in acetone but could be recrystallized from a large volume of this solvent by addition of petroleum ether. C, 33.7; II,2.7; N , 14.3; C1,4.5. Anal. Cnlc'd for CloH,rC1N~.2CsH1N3O~S: Found: C, 33.5; H, 3.0; E,14.2; C1,4.4. MethyE-P-chloroethyl-P-(S-car6oxy)pyridinzurriethylamine (Zv). To a solution of 2.672 g. (6.47 ndi') of l-~~ethyl-l-(~-chloroethyl)ethylenimonium picrylsulfonate in 500 cc. of acetone mas added 796 mg. (6.47 mM) of nicotinic acid dissolved in 400 cc. of acetone. The reaction mixture was concentrated under reduced pressure to about 150 cc., and after standing for an hour at room temperature, the remainder of the acetone was removed in vacuo. The residue was dissolved in 100 cc. of acetone and then 6.47 mM of picrylsulfonic acid dissolved in a little acetone uas added. T o this solution was added 150 cc. of petroleum ether. The dipicrylsulfonate of compound 1V precipitated as a n oil which crystallized upon scratching and cooling. The product was dried in vacuo over PzOS; yield 4.8 g . (90%). For analysis, the compound was recrystallized from acetone-ether solution; m.p. 163-165'. C, 33.3; 13, 2.6; N, 13.5; C1,4.3. Anal. Calc'd for C,lHlrClN202.2C611~?:30~S: Found: C, 33.4; E l , 2.7; N, 13.7; C1,4.5. N-F/lethyl-iV-p-chloroethyl-N-,T- [A"-bzs (8-hydroxyethyl)sulfoniuinIethylamine ( V ). A mixture of 1.652 g. (4.0 mM) of 1-methyl-I-(6-chloroethy1)ethylenimonium picrylsulfonate and 488 nig. (4.0 mM) of thiodiglycol was dissolved in 350 cc. of acetone and the solution was allowed to stand a t room temperature for one hour. The acetone was removed under reduced pressure. The residual oil W P S taken up in 50 cc. of acetone and filtered from a small amount of crystalline material (0.5 g.). T o the filtrate was added 4.0 m M of picrylsulfonic acid dissolved in a little acetone. The dipicrylsulfonzte of V crystallized out of the reaction misturc upon cooling at 4' overnight. I t was filtered off and dried in vacuo over P20s; yield 86%; m.p. 158-160".
NITROGEN MUSTARD GAS.
VI1
585
Anal. Calc'd for CpH&1NOzS.2C~H,N109S:C, 30.5; H, 3.2; N , 11.8; C1,4.3. Found: C, 30.7; HI 3.4; N, 11.7; C1, 4.1. N-Methyt-N-8-chloroethyl-N-fl[N'-methyl-N'-bis (8-hydroxyethyl) ammonium] ethylamins ( V I ) . Methyldiethanolamine (0.565 g., 4.75 m M ) was added to 2.065 g. (5.0 mM) of 1-methyl-l-(8-chloroethyl)ethylenimoniumpicrylsulfonate dissolved in 400 cc. of acetone. The solvent was removed under reduced pressure. The residual oil was dissolved in 60 cc. of acetone and a n acetone solution of 5.0 mMof picrylsulfonic acid was added. The dipicrylsulfonate of VI rapidly crystallized out of the reaction mixture in pure form. After cooling far a n hour at 4", the compound was filtered off and dried in vacuo over Pz06; yield 3.0 g. (77%); m.p. 213-215". Anal. Calc'd for CloH26C1~20*.2CsH1hT300S: C, 32.0; H, 3.5; N , 13.6; C1,4.3. Found: C, 32.1; H, 3.7; N , 13.6; C1,4.4. Methyl-8-chloroethyl-B-(hexamethylenetetraminium)ethylamine ( V I I ) . A solution of 2.065 g. (5.0 m M ) of I-methyl-I-(8-chloroethy1)ethylenimoniumpicrylsulfonate in 400 cc. of acetone was treated with 701 mg. (5.0 mM) of hexamethylenetetramine dissolved i n 250 cc. of acetone. The solvent was removed in vacuo. The residue was dissolved in 100 cc. of acetone and treated with 5.0 miM of picrylsulfonic acid dissolved in a little acetone. After standing overnight at 4 ', the crystalline dipicrylsulfonate of VI1 was filtered off and dried over PzOain vacuo; yield 3.65 g. (87%); m.p. 130-133' (decomp.). Anal. Calc'd for C11H2,C1S~~2CoHzTU'aOpS: C, 32.65; H, 3.3; N, 18.2; C1, 4.2. Found: C, 32.4; H, 3.4; N , 17.9; C1, 3.9. The hudrolysis of N-meth~l-N-~-chloroethyl-N-~-[N'-methyl-N'-bis(~-hydrozy~thyl)ammonium jeth~lamznedipicrylsulfonate. The dipicrylsulfonate of VI (865 mg., 1.05 mM) was added to 70 cc. of 0.08 N NaHCOI. The mixture was stirred a t room temperature for 20 hours. Ten-cc. aliquots of the reaction mixture were withdrawn for C1- and H+ determinations. Found, C1-, 0.015 mM per cc. and H+, 0.014 mM per cc. Theory, C1-, 0.015 mM per cc.; H+, 0.015 mM per cc. The remainder of the reaction mixture was cooled, acidified to Congo Red and filtered' The initial precipitate (0.35 g.) was separated from a small amount of high-melting substance (m.p. 250', possibly a cyclic dimer) by extraction with hot water. The dipicrylsulfonate of compound VI11 was obtained on cooling the filtrate; m.p. 196-200' with decomposition. It was again recrystallized from water and dried in uucuo over PlOa at room temperature. When dried at 115", the compound lost about one-half mole of water. C, 32.4; H, 3.8; H20, 1.2. Anal. Calc'd for CloH,,NzOj.2C,H,N30sS.tH*0: Found: C, 32.7; H, 3.9; H20, 1.5. A further quantity of VI11 was obtained when the mother liquor was concentrated to about 25 cc., cooled and filtered. The salt was dried in vacuo over P201; yield 0.30 g.; m.p. 202-205" with decomposition. For analysis i t was recrystallized from water; m.p. 204-206'. Anal. Calc'd for CloH20Nr03~2C6H2N30~S-~H20: C, 32.4; H, 3.8; N, 13.7; H20,1.2. Found: C, 32.2; H, 4.0; N, 13.7; H*0,1.5. The two samples of the salt described above, on admixture, melted at 201-205".
The anthors 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) FRUTON A N D BERGMANN, J. Org. Chem., (paper I11 thiR series). (2) GOLTJMBIC, FRUTON, A N D BERGMANN, J. Org. Chem., (paper I this series). (3) WILSON,VARS,GURIN,BROWN,CRANDALL, A N D DELLUVA (1943)." 0
Unpublished data obtained in the United States.
