[CONTRIBUTION FROM THE DEPARTMENT OF PHYSIOLOGICAL CHEMISTRY, SCHOOL OF MEDICINE, UNIVERSITY OF PENNSYLVANIA]
CHEMICAL REACTIONS OF NITROGEN MUSTARD GASES.* TI. REACTIONS WITH SECONDARY AND TERTIARY AMINES SAMUEL GURIN, DANA I. CRANDALL,
AND
ADELAIDE M. DELLW.4
Received April 8, ls4r
It has been shown in an earlier report from this laboratory (1) that hexamethylenetetramine reacts with methyl-bis(6-chloroethy1)amine (M.B.A.) to form quaternary salts. Fruton and co-workers (2, 3, 4) have made an extensive study of the relative reactivities of a number of cyclic nitrogen compounds and amino acids when mixed with M.B.A. This report is concerned with a similar study of a number of aliphatic and cyclic amines, which was undertaken in order to deteimine, if possible, the relationship of molecular structure to the reactivity of these amines toward tfhenitrogen mmtards. The methods wed were based on the thiosulfate titration procedure devised by Ogston ( 5 ) and further developed by Golumbic, Fruton, and Bergmann ( 6 ) . According to these authors, the relative amounts of ethylenimine form of M.B.A. remaining, after a reaction with another reagent has occurred, can be measured by titration with sodium thiosulfate. The experiments were carried out in the following manner: t o 1 ml. of M.B.A. hydrochloride (0.078 mM) in water were added 3 ml. of 0.FM sodium bicarbonate and 1 ml. of an aqueous solution containing 0.29 millimoles of the test amine. In all cases the pH was maintained between 7.5 and 8.0. When necessary, the amine solutions were adjusted with acetic acid to p H 7.5-8.0prior to mixing. The mixed solution was allowed to stand for exactly 30 minutes at room temperature and then treated with 10 ml. of 0.009 M sodium thiosulfate. After 10 minutes the solutions were acidified and the remaining thiosulfate titrated with a standard iodine solution. Control solutions containing M.B.A. without added test substance were simultaneously run with each serie.. of determinations. The amount of thiosulfate which reacted during the 10-minute period furnished a measure of the ethylenimine form of M.B.A. remaining at the end of the 30 minute reaction period. This value subtracted from the control value gave the amount of M.B.A. which had reacted with the test substance. This quantity divided by the control value and multiplied by 100, gave the percentage of M.B.A. which had reacted with the test substance under these standardized conditions. The results obtained with several cyclic amines (Table I) are in substantial agreement with those reported by Bergmann and Fruton. The competition rates of some of these substances are sufficiently high to suggest that those present in the living body would react very rapidly upon exposure t o M.B.A. It 1 This work waa done in whole under Contract No. OEM-cmr-108between the University of Pennsylvania and the Office of Scientific Research and Development, which assumes no responsibility for the accuracy of the statements contained herein.
612
REACTIOSS OF NITROGEN MUSTARD GASES.
613
I1
is noteworthy that tertiary amines are in general more reactive than secondary amines. For example, N-methylpiperidine and anserine were more reactive than piperidine and carnosine respectively. When the aliphatic series (Table 11)was studied, it became apparent that the introduction of alkyl groups larger than methyl seriously interferes with the reaction (compare triethylamine with trimethylamine). TABLE I REACTION OF MBA WITH CYCLICAbfIXEs In each experiment 0.078 millimoles MBA, 0.29 millimoles test substance and 1.5 mitlimoles NaHCOa, in a total volume of 5 ml. was employed. The reaction time was 30 minutes.
--
TEST SUBSTANCE
0 8 - CONSU?dED
MBA
BY COXTROL
RElwxwG
-Hexamethylenetetramine. . . . Tetramethylmethylenediamin Diethylenetetramethylenetetramine (9). . . . . . . . . . . . . . . . . . . . . . . . . . . Trimethyltrimethylenetriamine (9) Triethyltrimethylenetriamine. . . . . . . . Triisopropyltrimethylenetriamine. . , .
D A COKBINED WlTH TEST
SUBSTANCE
millimoles
millimoles
0.otK.l ,067
0.010 .006
0.050
.OM .065 .OB5 .065
.001
.065 .044 .035 .019 .027 .021
.021 .030 .046 .037
.or14 Nicotinamide. . . . . . . . . . . . . . . . . . . . . . . .
.OM .OM
.043
. Otj4 Pyridoxine . . . . . . . . . . . . . . . . . . . . . . . . . . ...............
N -Methyl pi peridine . . . . . . . . . . . . . . . . . Piperidine. . . . . . . . . ........... Anserine . . . . . . . . . . . . . . . . . . . . . . . . . . . . Carnosine. . . . . . . . .......... N-Methylmorpholine . . . . . . . . . . . . . . . . Morpholine . . . . . . . .......... Adenylic acid. . . . . . . . . . . . . . . . . . . . . . .
,004 .OB6
.004 .OM .065 .065 .Oti4 ,064 .006
.056 .057 .062 .063 .053 .060
.049 .056 .025 .029 .062
%
REACTED
millimoles
.061
,008 .007 f002 .003 .011 .004 ,016 .009 .039 .035 .004
83 91 99 68 54 29 42 33 13 11 3 5 17 6 25
14 61 55 6
Similar results were obtained when two methyl groups in triemethylamine were replaced by ethanol side chains (compare trimethylamine and methyldiethanolamine 11).
I (CH~)~NCH~CHZOHIV (C2H&NCH&H20H I1 CHsN(CH&HtOH)2
V (CHa)&CH&OOH
I11 C2HbN(CH2CH20H)t
VI CH3NHCH2COOH
Compounds I11 and IV were still less reactive. The introduction of one ethanol group in place of a methyl did not affect the reaction with M.B.A. Thus dimethylaminoethanol I was fully as effective as trimethylamine. Furthermore, the replacement of one ethanol or methyl group by an acetic acid residue
614
S. GURIN, D . I. CRANDALL, AND A . M. DELLUVA
did not inhibit the reaction. Dimethylglycine (V) proved to be as reactive as dimethylaminoethanol (I) or trimethylamine. That tertiary nitrogen is more reactive than secondary nitrogen, was again indicated by the more complete reaction obtained with dimethylglycine (V) as compared with sarcosine (VI). This information suggested the synthesis and study of a number of other compounds which it was hoped would prove to be more reactive. Among them were tetramethylmethylenediamine (VII), trimethyltrimethylenetriamine (VIII), and diethylenetetramethylenetetrsmine, which was prepared from ethylenediamine and formaldehyde (7), and t o which has been assigned the tentative structure IX.
VI1 (CH3)2NCH2N(CHa)2
CH3 N
/-\
CH2 I C&r;r
CH2
I
rjC%
\ /
CH2 VI11
CHz-N
' /
N-CH2
CH2 CH2-N
CH2
I
\ /
N-CHz
1
CHs IX
TABLE I1 REACTION OF MBA WITH ACYCLIC AMINES All concentrations and exwrimental conditions were maintained ae deecribed in Table I. TEST SUBSTANCE -.
Triethylamine. . . . . . . . . . . . . . . . . . . . . . Dimethylamine. . . . . . . . . . . . . . . . . . . . . .
millimoles
millimoles
millimoles
.062 ,064
,057 .OB
.005
8
.011
17
RE.lCTIOSS OF NITROGEN MUSTA 11D G.ISES.
Gl5
I1
significantly more effective than its corresponding ethyl and isopropyl homologs (triethyl- and triisopropyl-trimethylenetriamine). Several of these subtances were tested similarly with other nitrogen mustards, namely ethyl-bis(p-chloroethy1)amine(EBA)and tris(p-chloroethy1)amine (TBA) and were found t o react at about the same rate under similar conditions (Table 111). The data indicate that amines will react rapidly with the nitrogen mustards when the following conditions are fulfilled: 1. The nitrogen must be tertiary. 2. In the aliphatic series, the replacement of methyl groups t y ethyl or larger alkyl radicals diminishes the reactivity. 3. Althougb one ethanol or acetic acid side chain may be substituted for a TABLE: I11 REACTIVITY OF TRI~(@-CHLOROETHYL)AMINE; AND ETHYL-BIS(@-CHLOROETHYL)AMINE All concentrations and experimental conditions were maintained as described in Table I. TEST SUBSTANCE
I 1 1 1
N-MSISTA~D co:i$&D N-A~USTAPD COMBINED % c o ~ ~ I p pXEMAINING oL WITH TEST REACTED
N-YUS,TARD EMPLOYED
BY
SUBSTANCE
Hexamethylenetetramine
TBA
Trimet hyltrimet hylenetriamine
TB.4 EBA
Diethylenetetramethylenetetramine
TB.4 EBB
1 1
j
I ~
1
0.074
.070 .076
!, .023 1 .020
0.017 ,017
.076
1 I
.077 I .077 1
.014 .001
0.057 .059
77 78
.047
67 74 82
methyl group, further introduction of larger groups impairs the activity. 4. The nitrogen in cyclic amines appears to be somewhat more reactive than that of aliphatic amines. Substances containing; two or more ethyl groups (or larger radicals) attached to nitrogen will react efkiently only if the substance is cyclic. 5. The most effective reagents appear to be those substances containing two AF
mnrn tnrtiorv nitrnann atnmr
I'HILADELPHIA,
aonaratorl
hv mothvlono
ui m i n a
YA.
REFERENCES (1) GURIK,DELLUVA, AND CRANDALL, J. Org. Chem., 12, 606 (1947). (2) FRUTON, STEIN,AND BERGMANN, J. Org. (!hem., 11, 559 (1946). (3) FRUTON, STEIN,STAHMANN, AND GOLUMBCC, J. Org. Chem., 11, 571 (1946). (4) GOLUMBIC, FRUTON, AND BERGMANN, J . Org. Chem., 11, 581 (1946). (5) OGSTON(1942);Unpublished data in GreiLt Britain. (6) GOLUMBIC, FRUTOK, AND BERQMANN, J . O r g . Chem., 11, 518 (1946). (7) BISCHOFF,Ber., 31, 3254 (1898). (8) HENRY, Bull. Acad. Roy. Belg., 26,203 (1912);28, 368 (1914);Ber. (Ref.) 26,934 (1893);
28, 852 (1895). (9) HENRY,Bull. Acad. Roy. Belg., 28, 359 (1Si14). (10)MICHAELISAND SCHUBERT, J. Biol. Chem., 115, 221 (1936). (11) LOB,Bioch. Z.,51, I22 (1913).
