II. CHEMISTRY 111 



This observation suggested that CF might be a formyl derivative of PGA in 

 view of the simihir behavior of OF in counteracting PGA antagonists. This 

 supposition was confirmed by the finding that catalytic hydrogenation of 

 formyl PGA produced a mixture of compounds with high growth-promoting 

 activity for CF."-" 



b. Synthesis and Purification 



The synthesis of leucovorin (XII) appears to consist in two essential 

 steps (cf. Fig. 7): 



1. Reduction of either PGA (I) or 10-formyl PGA (XV) in 87 to 100% 

 formic acid at 0° to 30° over a platinum catalyst, whereby 2 moles of hy- 

 drogen are absorbed to form 10-formyl-5,(3,7,8-tetrahydro PGA (XIV). 



2. Rearrangement of the neutralized reduction mixture under anaerobic 

 conditions by long standing at room temperature or by heating the alkaline 

 solution at pH 10 to 12. This treatment results in the rearrangement of 

 10-formyl-5,6,7,8-tetrahydro PGA to leucovorin in a 40 to 50% over-all 

 yield from either PGA or 10-formyl PGA. During this process the formyl 

 group shifts from the N'" to the N^ position of the molecule. The leucovorin 

 is isolated by chromatographic adsorption and purification of the neutral 

 calcium or barium salt. The free acid of leucovorin is crj^stallized as the 

 free acid after acidification of the calcium salt to pH 3.5. 



c. Stage of Reduction and Location of Hydrogen A toms 



Elemental analyses of crystalline leucovorin (XII) are in agreement with 

 the formula C20H23N7O7 , and the presence of one formyl group has been 

 demonstrated. Thus from this analytical data and the method of prepara- 

 tion described above, the conclusion seemed justified that leucovorin is a 

 tetrahydroformyl PGA. 



Allen and coworkers^^ studied the stage of reduction of pteridines by 

 means of the potential at the dropping mercury electrode in the polaro- 

 graph. They demonstrated that it is possible to distinguish between PGA, 

 10-formyl PGA, and dihydro and tetrahydro PGA by means of the potential 

 at pH 9.0. Thus, with PGA (and pteridines in general) the polarograph 

 gives three inflection points in the curve corresponding to the tetrahydro, 

 dihydro, and aromatic forms; the ({uantity of a particular reduction state 



75 W. Shive, T. J. Bardos, T. J. Bond, and L. L. Rogers, J. Am. Chem. Soc. 72, 2S17 



(1950). 

 ^« J. A. Brockman, Jr., B. Roth, H. P. Broquist, M. E. Hultquist, J. M. Smith, Jr., 



M. J. Fahrenbach, D. B. Cosulich, R. P. Parker, E. L. R. Stokstad, and T. H. 



Jukes, J. Am. Chem. Soc. 72, 4325 (1950). 

 " E. IT. Flynn, T. J. Bond, T. J. Bardos, and W. Shive, ./. .1///. Chen,. Soc. 73, 1979 



(1951). 

 " W. Allen, R. L. Pasternak, and W. Seaman, ./. Am. ('hem. Soc. 74, .3264 (1952). 



