118 PTEROYLGLUTAMIC ACID 



of lO-iiilroKo PC5A by treatment of PGA with nitrous acid in cold mineral 

 acid has been reported.^^ This is evidence that the nitrogen atom in the 10 

 position of PGA is capable of reacting as a secondary aromatic amine. 

 Under similar conditions, 10-formyl PGA (XV) does not react with nitrous 



H 



HOOC 



XXVIII 



{2-Amino-4-hydro.\y-5-formyl-10-methyl-5,6,7,8-tetrahydro PGA) 



acid, which is reasonable proof that the formyl group is in the 10 position 

 in this compound. Furthermore, immediate and rapid addition of 1 mole of 

 nitrous acid to leucovorin in cold dilute hydrochloric acid results in the 

 formation of the 10-nitroso derivative. ^^ This shows that the 10 position, 

 as in PGA, is free to act as a secondary amine, and thus the formyl group 

 in leucovorin must be attached only at the 5 position. However, when leu- 

 covorin is allowed to stand in dilute mineral acid for a short time at room 

 temperature, there is no reaction with nitrous acid in the cold. This indi- 

 cates that both the 5 and 10 positions had become blocked. 



The above experiments suggest that under mild acid conditions the 

 formyl group of leucovorin forms a new ring linking the 5 and 10 positions. 

 This assumption was proved by the subsequent isolation of an imidazolin- 

 ium salt, isoleucovorin chloride (XIII), ^"^^ ^^ obtained by treatment of 

 leucovorin with hydrochloric acid at pH 1.3 or below. Isoleucovorin chlo- 

 ride does not react with nitrous acid; furthermore, it contains ionic chlorine 

 and a potential tormyl group, since 1 mole of formic acid is formed during 

 the drastic acid hydrolysis employed in the usual formyl analysis. 



Leucovorin can be considered a formyl derivative of a N,N'-aromatic 

 disubstituted ethylene diamine. As a model of such a system, the mono- 

 formyl derivative of N,N'-diphenylethylenediamine, N-(2-anilinoethyl)- 

 formanilide (XXIX), was prepared''^ in order to compare its behavior to 



95 D. B. Cosulich, B. Roth, J. M. Smith, Jr., M. E. Hukquist, and K. V. Parker, 



/. Am. Chem. Soc. 73, 5006 (1951). 

 98 M. May, T. J. Bardos, F. L. Harger, M. I.ansford, J. M. Ravel, G. L. Sutherhiiid, 



and W. Shive, J. Am. Chem. Soc. 73, 3067 (1951). 



