540
ANALYTICAL CHEMISTRY
been confirmed. The reaction proceeds quantitatively in equimolecular concentrations according to the equation:
o=c--, I 1 HO-6
1
CHzCO
A ?I
+
HOH-A-J
'
i
I
\
S.Br
/
CH&O
H0-L-H
i
the titration process. The end point is thus easily observed by the appearance of a blue color in the ascorbic acid solution or extract. The presence of other reducing substances does not interfere with the titration with ,V-bromosuccinimide, since iodine is selectively liberated from potassium iodide before reducing substances present, other than ascorbic acid, are oxidized by X-bromosuccinimide, and consequently t,he end point is easily determined in the presence of starch. The end point is definitely blue in the case of pure solutions and pharmaceuticals, but violet in the case of biologicals and fruits.
CHzOH
LITERATURE CITED
H - L I
HO-~--H
I
CHiOH The fact that ascorbic acid reacts very rapidly with N-bromosuccinimide, whereas many of the interfering substances react more slowly or even do not react a t all, provides a reliable titrimetric method for the determination of ascorbic acid. N-Bromosuccinimide is an oxidizing agent and thus can liberate iodine from potassium iodide in aqueous acetic acid medium, but it oxidizes ascorbic acid to dehydroascorbic acid preferentially. Until all the ascorbic acid present in the solution is oxidized, no iodine is liberated from potassium iodide. The slightest excess of N-bromosuccinimide added, after all the ascorbic acid content has been oxidized, Fill liberate iodine from potassium iodide, n-hich is easily detected by the blue color developed with a few drops of starch solution added a t the beginning of
(1) Barakat, h l . Z., Badran, S . ,and Shehab, S. K., J. Pharm. and Pharmacol., 4 , 46 (1952). (2) Barakat, R l . Z., and Mousa, G. XI,,Ibid., 4, 115 (1952). (3) Basu, K. P., and Nath, XI. C., J . I n d i a n Chem. Soc., 15, 133 (1938). (4) Bessey, 0. A., J . Am. M e d . Assoc., 111, 1290 (1938). ( 5 ) Bessey, 0. A , J . Bid. Chem.. 126, 771 (1938). (6) Bessey, 0. A., and King, C. G., Ibid., 103, 687 (1933). (7) Fieser, L. F., and Rajagopalan, S., J . Am. Chem. Soc., 71, 3935, 3938 (1949). (8) Ibid., 72, 5530 (1950). (9) G I , Imre, AVafure,138, 799 (November 1936). (10) Gawron, O., and Berg, R., IND. EX. CHEY.,A h - a ~ED., . 16, 757 (1 944). (11) Hochberg, A I . , XIelnick, D., and Oser, B. L., I b i d . , 15, 182 (1943). (12) King, C. G., Ibid., 13, 225 (1941). (13) Penney, J. R., and Zilva, S. S.,Biochem. J . , 39, 392 (1945). (14) Roe, J. H., and Kuether, C., J . B i d . Chem., 147, 399 (1943). (15) Roe, J. H.. Mills, R l . B., Oesterling, XI. J., and Damron, C. M., Ibid.. 174, 201 (1948). (16) Roe, J. H., and Oesterling, RI. J., Ibid., 152, 511 (1944). (17) Sherman, H. C.. La Mer. V. K., and Campbell, H. L., J . A m . Chem. SOC., 44, 165 (1922). (18) Snow, G. A . , and Zilva, S.S.,Biochem. J . , 38, 458 (1944). (19) Tauber, H., and Kleiner, I. S., J . Bid. Chem., 108, 563 (1935). (20) Tillmans, J. Z., 2. L'ntersuch. Lebensm., 54, 33 (1927).
RECEIVED for reriew February 3,
1954.
Accepted November 22, 1954.
Method -of Assay for Ethylenimine Derivatives .L
EUGENE ALLEN and WILLIAM SEAMAN Research Division, American Cyanamid Co., Bound Brook,
A method was devised for the determination of ethylenimine derivatives, which are being studied experimentally for use in the treatment of cancer. It is based on a rapid reaction between the ethylenimino group and thiosulfate ion at pH 4. This reaction consumes 1 mole of acid for each ethylenimino group. A large excess of thiosulfate is needed to suppress competing reactions. -4s this excess is difficult to measure with precision, the acid consumption is measured instead, by adding standard acid and thiosulfate and titrating the excess acid with standard alkali. The method has been studied for the assay of the following ethylenimine derivatives and homologs: 2,4,6-tris(laziridy1)-s-triazine (triethylenemelamine), 2-amino4,6- bis(1- aziridyl) s - triazine (diethylenemelamine), N , N ',N " triethylenephosphoramide, iV,N ', N triethylenetbiophosphoramide, N ( 3 oxapentamethylene"N',N''-diethylenephosphoramide, N,N',N"-tris(1methylethylene) phosphoramide, iV,N',N"-tris(1,l-dimethylethylene) phosphoramide, p-toluenesulfonN-ethyleneamide, and iV,N'-diethylenebenzene-1,3disulfonamide. The standard deviation of these determinations, where applicable, is in the neighborhood of
-
*O.l%.
-
- -
"-
N. 1,
S
EVERAL derivatives of ethylenimine have recently been
studied as possible agents for the treatment of certain types of cancer. These compounds have physiological effects similar to those of the nitrogen mustards, which have been used for the palliative treatment of cancer ( 3 ) . However, the ethylenimines are reported to be easier to administer and to bring about unpleasant side effects less often ( 2 ) . When a nitrogen mustard is dissolved in water, it is transformed to an ethylenimonium ion ( 1 ) . Equation I shows such a reaction for methylbis (6-chloroethyl)amine, a typical nitrogen mustard:
The ethylenimonium ion is thought to be the active chemotherapeutic agent. The ethylenimino group has a structure similar to that of the ethylenimonium ion, except that the nitrogen atom does not have a positive charge. 2,4,6-Tris( 1-arizidy1)-s-triazine (triethylenemelamine) (I), also known as TEM, is the ethylenimine derivative which has been most thoroughly studied clinically. Another typical ethylenimine derivative is X,X', N"-triethylene phosphoramide (11).
V O L U M E 27, NO. 4, A P R I L 1 9 5 5
541 mine, but the reaction could be speeded by lowering the pH. Furthermore, when the reaction was conducted a t p H 4 (found to be the optimum pH), about 4 equivalents of acid per mole of triethylenemelamine were consumed. -4partial explanation of the acid consumption may be the reaction of Equation 4. This would account for 3 equivalents of acid per mole of triethylenemelamine. Monobasicity of the reaction product would account for the additional equivalent.
--S--C.12CH&03A7a I
f SaOH
(4)
k The basicity of the reaction product was verified by running a potentiometric titration curve on the reaction mixture. This' curve showed two breaks, a t pH 4 and 8. The difference between the breaks corresponded t o l equivalent of acid per mole of triethylenemelamine The phosphoramides so far investigated give reaction products with thiosulfate Fhich are not quite so basic, and consume less than 1 equivalent of acid per mole between p H 4 and 8. The method finally chosen is to use a large excess of thiosulfate solution in high concentration. This suppresses side reactions. The thiosulfate consumption is not measured because of the large excess involved, but the acid consumption is measured instead. The effect of the basicity of the reaction product is cancelled by back-titrating the solution to the phenolphthalein end point after the reaction has proceeded a t p H 4 for 0.5 hour. A similar method in which the titration is made directly with acid to the phenolphthalein end point, in a refluxing acetonewater solution, is used by Ross ( 4 ) .
\
N- CHz '&HZ I1
.4 method for the assay of the ethylenimines was needed both for the regulation of dosages and for synthetic research. THEORY OF METHOD
Golumbic, Fruton, and Bergmann (1) have shown t h a t the ethylenimoniuni ion can be determined in aqueous solution by reaction with thiosulfate according t o Equation 2.
Fz+
)S'+-CHz
Sa2S203
>
+.
N-CHsCHzS2O3Xa
+ Na+ (2)
Since each molecule of methylbis (p-chloroethy1)amine has two p-chloroethyl groups, it reacts with 2 molecules of thiosulfate. The titration is conducted by treating t h e solution of the nitrogen mustard with excess standard thiosulfate solution and then backtitrating with standard iodine solution. The data presented by these authors show that the results are not quantitative. The reason is probably t h e occurrence of side reactions mentioned by these authors. For example, the imonium ion can undergo hydrolysis as shown in Equation 3.
DESCRIPTION OF METHOD
T o an accurately weighed portion of about 3 meq. of sample (1 equivalent corresponds to 1 ethylenimino group) add 50 ml. of sodium thiosulfate solution (20 grams of Sa2S203.5Hz0per 100 ml.), followed by 1 drop of methyl orange indicator solution. Titrate the solution immediately with 0.LV hydrochloric acid t o a p H of 4, as indicated by the methyl orange color. (The proper color may be judged by comparison with a p H 4 buffer solution containing methyl orange.) If the end point does not persist for a t least 10 seconds, continue the titration until it does. Allow the solution to stand for 0.5 hour. Add several drops of phenolphthalein indicator solution and titrate with 0.1S sodium hydroxide t o the phenolphthalein end point. Run a blank in the same manner on 50 ml. of the sodium thiosulfate solution without the sample. The acid consumption minus the consumption of alkali, corrected for the blank. is equivalent to the ethylenimino groups present.
Another possible side reaction is dimerization, which produces a piperazine ring. Preliminary work showed t h a t low results were also obtained when the thiosulfate reaction was applied t o triethylenemelaCY.-
so00
xloo
2000
1300
IS00
100
I
1100
DISCUSSION OF RESULTS 1000
SW
w
mo
750
IOQ
I
PURE TRIETHYLENEMELAMINE
Y
11
I
Y
0 2
3
4
5
6
7
8
9
IO
ii
17
MICRONS
Figure 1.
Spectrum of pure triethylenemelamine
I3
.I
!5
Assay of T r i e t h y l e n e melamine. Table I shows assay values of several samples of t r i e t h y l e n e m e l a mine. The standard deviation, in the neighborhood of =!=0.2%, indicates that the method has adequate precision for the purpose for which it is intended. I n general, the samples which are known to be crude or which have been aged give lower values, m is to be expected. However, t h e aged samples
542
ANALYTICAL CHEMISTRY
only a minor effect in the assay value. It is possible Kx)r 12 DAY 55'C POLYMER that these samples are only partially polymerized. They w U contain appreciable insoluz 4 ble matter, but a quantitative determination of soluv) I 60. bility was not made and z the amount of insoluble 4 1 c K may be small. However, I- 40. t h e sample which w a i h e a t e d f o r 18 h o u r s a t K 100' C. gave a low assay Y m. n value. Polymerization in this case was probably fair15 L %J 0 complete. 9 M li iz I3 i4 15 I n d e p e n d e n t evidence for the relation betwren the polymers and the pure compound was o b t a i n e d from infrared spectra. Figures 1, 2, and 3 show xxx) quo 21500 lsoQ 1100 epo ea, 7% XLO 6 spectra of pure triethyl100 18 HOUR 10OoC.POLYMER enemelamine and the two polymers prepared by heating triethylenemelamine in the dry state a t 55' C. for 12 days and a t 100' C. foi 18 hours. The spectra of the pure material and of the 55" C. polymer are very similar, whereas that of the 100" C. polymer is different Assay of Other Ethylenimines. Table I11 shows assay values obtained on o t h e r ethylenimines a n d 9 io ii 14 I5 k I3 4 5 6 7 8 e t h y 1en i m i n e homologs. MICRONS The homolog with 1 methyl Figure 3. Spectrum of polymer prepared by heating triethrlenemelamine in the dry group on each ethylenimine state at 100' C. for 18 hours ring yields almost quantitative values, whereas the homolog with 2 methyl groups on each ethylenimine ring is on11 partially titrated. Steric hindrance may be responsible. The Table I. Assay of Triethylenemelamine Samples procedure is not applicable to the two sulfonamide derivative* Description Values, % 9Q.7,9Q.G, QQ.G Purified shown in the table. Q9.7,99.8,Q9.8 Stability Studies on Ethylenimines. When clinical trial3 80.Q,82.5 Crude 1 95.5,95.5 were started on the ethylenimines, it was important to know 98.2,98.2 98.9.98.9 in which form the compound would be most stable-in the solid 5mo
w
2000
I500
IMO
llpo
lope*
1%
800
m
f \v--
E
rj;
i
6
One year old Purified (slightly turbid in water) Purified (decomposition point slightly low)
1"'
97.3.97.1,97.4 98.Q,QQ.4 99.7,99.7 Q9.8,99.7 QQ .5,QQ.5
Table 11. Assay of Triethylenemelamine Polymers Description $&,:ed
were not aged under ideal conditions, as no effort was made to keep them absolutely dry. Assay of Triethylenemelamine Polymers. Triethylenemelamine shows a tendency to polymerize. Samples of triethylenemelamine which have been aged always show the presence of polymer by forming a milky solution when dissolved in water. Polymerization of triethylenemelamine can be induced by heating the sample, either with water or in the dry state. Table I1 shows assay values for some of the triethylenemelamine polymers. The first polymer, which was simply isolated from the crude sample by filtration, has a low assay value. The second polymer, prepared by heating triethylenemelamine with water, likewise shows low purity. The three last polymers in the table were prepared by heating triethylenemelamine in the dry state. The table shows that heating at 55' C. for up to 12 days produced
from crude sample
Values, % 3 1,3 0
In 4 1 1 1
Heated Heated Heated Heated
with water on steam bath 1 hour 24 hours a t 55' C. 18 hours a t 100' C. 12 days a t 55' C.
0 7,o
97.5
n
0.2
91 3
Table 111. .issay of Other Ethylenimines and IIomologs Designation 2-Amino-4.6-bis(l-aairidyl)-s-triaaine (diethylenemelamine) N N ' K"-Triethylenephosphoramidea N:.~~f:NU-Triethylenethiophosphora,~idea h'-("Oxapentamethylene)-~',K"~diethylenephosphoramide N V' Y"-Tris(1-methylethylenejphosphoramide ~V~:Vt~:V"-Tris(l ,1-diniethylethylenejphosphoramide
Values, % 99 7, 99 7 99 A , 99 3 99 9,100 0 99 3 98 6 , ?8 4 53 8 , 02 fi
p-Toluenesulfon-.V-ethyleneamide b iV,N'-Diethylenebenzene-l,3-disulfonaniide Weighings conducted under stream of dried nitrogen because of hygroscopicity of comppund. b Reaction too slow t o be practical.
V O L U M E 2 7 , NO. 4, A P R I L 1 9 5 5 state, or dissolved in physiological saline solution, phosphate buffer solution, or sesame oil. Accordingly, stability studies in these solvents and in the dry state were carried out on iV,N',N"triethylenephosphoramide and N,N',N"-triethylenethiophosphoramide. The analytical procedure for these determinations, except for the solutions in sesame oil, was the same as the proredure already described. (With the phosphate buffer solution it was necessary to run careful blanks to allow for the effect of the buffer in consuming alkali.) For the solution of the thiophosphoramide in sesame oil a modification was used in which (Lath addition of the acid or alkali was accompanied by vigorous -haking. The purpose was to extract the compound from the *?same oil layer into the water layer. But with phosphoramide dissolved in sesame oil, this extraction into the aqueous phase was much too slow. Therefore, boiling thiosulfate solution was added to the sample, followed immediately by a measured volume of boiling standard hydrochloric acid solution. The titration mas then completed a t the boiling point with standard hydrochloric acid and sodium hydroxide solutions. The procedure gave results of relatively poor precision, but served to indicate the stability in a satisfactory manner. Results of these determinations show t h a t the aqueous solutions, both physiological Qalineand phosphate buffer, were unstable. However, the compound in the solid state and in the sesame oil solution showed little, if any, loss in strength over the period studied.
543 ACKNOWLEDGMENT
The authors are greatly indebted to Frederick S. Philipe, Sloan-Kettering Institute for Cancer Research, New York, N. Y., who suggested a t the outset of the study that the thiosulfate titration method used for the nitrogen mustards ( 1 ) might be applied to the assay of the ethylenimines. Philips also obtained some preliminary data which indicated that the reaction of triethylenemelamine with thiosulfate was accompanied by a release of acid. The authors also Tl-ish to thank Erwin Kuh and Doris Seeger for furnishing the samples of the phosphoramides, P. F Dreisbach and C. M. Hofmann for furnishing the other samples, D. S . Kendall for the infrared work, and Z. F. Smith for obtaining some of the results in the stability studies. LITERATURE CITED (1) Golumbic, C . , Fruton, J.
S.,and Bergmann, XI., J . Ore. Chem., 11,518(1946). ( 2 ) Karnofsky, D. .4.,Burchenal, J. H., Armistead, G. C., Jr., Southam, C . >I., Bernstein, J. L., Craver, L. F., and Rhoads, C. P., Arch. Internal M e d . , 87, 477 (1951). ( 3 ) Philips. F. S., and Thiersch, J. B., J . Pharmacol. E r p t l . Therap., 100,398(1950). (4) Ross, W. C. J., J . Cham. SOC.,1950,2257.
RECEIVEDfor review June 17, 1954. Accepted December 1, 1954, Presented a t the Meeting-in-Miniature of the North Jersey Section, AMERICAN CHEMICAL SOCIETY, Newark, N. J., January 26, 1953.
Volumetric Determination of Nitrocellulose and Nitroguanidine By Transnitration of Salicylic Acid HARRY STALCUP and RICHARD W. WILLIAMS U. S. Naval Powder Factory, lndian Head, Md. This investigation was undertaken because of the need for a direct chemical method for the determination of per cent nitrogen in nitrocellulose for specialized applications where the nitrometer was either inapplicable or inaccurate. Nitrogen in nitrocellulose samples of various levels of nitration and nitramine nitrogen in nitroguanidine were determined volumetrically by the transnitration of salic?-licacid in sulfuric acid solution, the sample being used as the nitrating agent, followed by reduction of the resulting nitrosalicylic acid with standard titanous chloride. Determinations on samples of known nitrogen content showed a standard deviation of 0.02% and were in good agreement with nitrometer values. The method offers a new approach to the determination of nitramine-type ingredients in propellant-powder formulations.
T
H E determination of nitrate nitrogen in nitrocellulose and nitramine nitrogen in nitroguanidine is of importance in the production of gun and propellant powders. A nitrometer is ordinarily used, but because of their low solubility in cold sulfuric acid, some nitrocellulose fractions prepared in this laboratory under various experimental conditions failrd to yield their true nitrogen values in the nitrometer. For this reason a rapid and accurate titration procedure was sought. A procedure has been developed, based in part on the Moore (4)modification of the Forster ( 2 ) salicylsulfonic acid method for nitrate nitrogen, in which the nitrate compound is used as the agent for the nitration of salicylic acid in a sulfuric acid medium. The resulting nitro compound is then reduced with standard titanous chloride by the Knrcht-Hibbert method ( 3 ) . Compounds containing
both nitrate and nitramine nitrogen-such as b-nitroxyethylnitroguanidine-have been analyzed, and nitroguanidine in gun propellant compositions has been determined. The rompound produced in the salicylic acid-nitrate reaction has been identified as 5-nitrosalicylic acid. APPARATUS
Cylinder of carbon dioxide or nitrogen gas. Heat source, Variac-controlled hot plates or heating mantle to fit 250-ml. refluxing flask. Refluxing flask, 250 ml. Soxhlet unit, 20-ml. capacity siphoning cup. Reflux condenser, water-cooled to fit Soxhlet unit. Reflux condenser. water-cooled with ground-glass standardtaper tip. Glass bottle of 2-liter capacity, equipped with an automatic buret for storage and use of the titanous chloride solution. The siphon tube and buret must be connected in such a way that only carbon dioxide or nitrogen gas, supplied from a tank, will be drawn into the reagent bottle as the solution is used. The bottle should be covered with black paint or black paper to exclude light. Refluxing flask, 500-ml. capacity, with a round bottom and two necks. One of the necks should be of ground glass with a standard taper to fit the reflux condenser. REAGENTS
TITANOUS CHLORIDESOLUTION (0.3N). For each liter of titanous chloride solution, 225 ml. of 20% titanium trichloride is mixed with 100 ml. of 38% hydrochloric acid. The mixing operation should take place before diluting, and in these operations the solution should be protected from the air as much as possible by means of carbon dioxide. The solution should then be thoroughly mixed by means of a current of carbon dioxide and stored in reagent bottle. An alternative procedure for making titanous chloride from titanous hydride is as follows:
