There is disclosed a novel intermediate useful in the preparation of pyrido[2,3-d]pyrimidines, which includes N-[4- [(2-amino-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-glut amic acid (5-deazafolic acid), N-[4-[[(2-amino-4(3H)-oxopyrido[2,3-d]-pyrimidin-6-yl)methyl]methylamino]b enzoyl]-L-glutamic acid (5-deaza-N.sup.10 -methylfolic acid), N-[4-[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L-gluta mic acid (5-deazaaminopterin), and N-[4-[[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methyl]methylamino]benzoyl] -L-glutamic acid (5-deazamethotrexate). This intermediate is the compound 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde.

BACKGROUND OF THE INVENTION 
This invention relates to an intermediate useful in the preparation of 
pyrido[2,3-d]pyrimidines, which includes 
N-[4-[(2-amino-4(3H)-oxopyrido[2,3-d]pyrimidin-6-yl)methylamino]benzoyl]-L 
-glutamic acid (5-deazafolic acid), 
N-[4-[[(2-amino-4(3H)-oxopyrido-[2,3-d]-pyrimidin-6-yl)methyl]methylamino] 
benzoyl]-L-glutamic acid (5-deaza-N.sup.10 -methylfolic acid), 
N-[4-[(2,4-diaminopyrido[2,3-d]-pyrimidin-6-yl)methylamino]benzoyl]-L-glut 
amic acid (5-deazaaminopterin), and 
N-[[(2,4-diaminopyrido[2,3-d]pyrimidin-6-yl)methyl]-methylamino]benzoyl]-L 
-glutamic acid (5-deazamethotrexate). This intermediate is the compound 
2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde. This invention also 
relates to a process for using said intermediate; to the novel compounds 
5-deazamethotrexate and 5-deaza-N.sup.10 -methylfolic acid; and to methods 
for preparing such novel compounds. 
Powerful dihydrofolate reductase inhibitors such as aminopterin and 
methotrexate are known folic acid antagonists useful in the suppression 
and treatment of actue leukemia and related conditions. They have as their 
principal mechanism of action a competitive inhibition of the enzyme 
dihydrofolate reductase. Folic acid and its 7,8-dihydro derivative must be 
reduced to tetrahydrofolic acid by this enzyme in the process of DNA 
synthesis and cellular reproduction. Compounds having antifolate activity 
such as aminopterin and methotrexate inhibit the reduction of both folic 
acid and 7,8-dihydrofolic acid and interfere with tissue-cell 
reproduction. 
Several types of quinazolinyl (5,8-dideazapteridinyl) analogs of folic 
acid, aminopterin, and methotrexate were reported to be inhibitors both of 
dihydrofolate reductase and thymidylate synthetase [A. H. Calvert, T. R. 
Jones, P. J. Dady, B. Grzelakowska, R. M. Paine, G. A. Taylor and K. R. 
Harrap, Europ. J. Cancer, 16, 713 (1980); K. J. Scanlon, B. A. Moroson, J. 
R. Bertino and J. B. Hynes, Mol. Pharmacol., 16, 261 (1979); O. D. Bird, 
J. W. Vaitkus and J. Clarke, Mol. Pharmacol., 6, 573 (1970)]. Recently, 
N-[4-[N-[(2-amino-4-hydroxy-6-quinazolinyl)methyl]prop-2-ynylamino]benzoyl 
]-L-glutamic acid (5,8-dideaza-10-propargylfolic acid) was identified as a 
potent inhibitor of thymidylate synthetase [T. R. Jones, A. H. Calvert, A. 
L. Jackman, S. J. Brown, M. Jones and K. R. Harrap, Europ. J. Cancer, 17, 
11 (1981)]. This enzyme catalyzes the de novo synthesis of thymidine 
nucleotidase, which are required for DNA synthesis. 
The synthesis of derivatives of the pyrido[2,3-d]pyrimidine ring system has 
been reviewed by W. J. Irwin and D. G. Wibberley, Advan. Heterocycl. 
Chem., 10, 149 (1969), which covers the literature to the beginning of 
1968. Although many methods are reported in this review, major routes to 
this ring system include the cyclization of the functional derivatives of 
2-aminonicotinic acids with various reagents [e.g., R. K. Robins and G. H. 
Hitchings, J. Am. Chem. Soc., 77, 2256 (1955)], and the reaction of 
derivatives of 4-aminopyrimidine with 1,3-dicarbonyl comounds or their 
masked derivatives [e.g., B. S. Hurlbert and B. F. Valenti, J. Med. Chem., 
11, 708 (1968)]. The condensations of 4-aminopyrimidines with 
malondialdehyde derivatives to give pyrido[2,3-d]pyrimidines are reported 
by R. Bernetti, F. Mancini and C. C. Price, J. Org. Chem., 27, 2863 
(1962), and B. S. Hurlbert and B. F. Valenti, J. Med. Chem., 11, 708 
(1968). A procedure for the preparation of 
5-oxo-(8H)-pyrido[2,3-d]pyrimidines was reported by B. H. Rizkalla and A. 
D. Broom, J. Org. Chem., 37, 3980 (1972). This reference discloses the 
following compound. 
##STR1## 
The development of procedures for the conversion of the above compound to 
N-[4-[(2,4-diamino-5-oxo(8H)-pyrido[2,3-d]pyrimidin-6-yl)-methylamino]benz 
oyl]-L-glutamic acid (5-deaza-5-oxoaminopterin), i.e., a compound having 
the formula: 
##STR2## 
was reported by A. Srinivasan and A. D. Broom, J. Org. Chem., 45, 3746 
(1980). In addition, 
N-[4-[(2-amino-4(3H)-oxo-10-formylpyrido[2,3-d]-pyrimidin-6-yl)methylamino 
]benzoyl]-L-glutamic acid (5-deaza-10-formylfolic acid), characterized only 
by spectral data, was reported to be formed from 5-deazafolate and formic 
acid by G. K. Smith, W. T. Mueller, P. A. Benkovic and S. J. Benkovic, 
Biochemistry, 20, 1241 (1981). A method for preparing 5-deazafolate is not 
disclosed. 
The inhibition of bacterial dihydrofolate reductase by 
pyrido[2,3-d]pyrimidines has been summarized in the Advan. Heterocycl. 
Chem. reference. Recently, a pyrido[2,3-d]pyrimidine derivative was 
reported to be a potent lipid-soluble inhibitor of mammalian dihydrofolate 
reductase by E. M. Grivsky, S. Lee, C. W. Sigel, D. S. Duch and C. A. 
Nichol, J. Med. Chem., 23, 327 (1980). This reference discloses the 
compound: 
##STR3## 
Other derivatives of this ring system have been evaluated for 
antihypertensive activity. Thus, L. R. Bennett et al, J. Med. Chem., 24, 
382 (1981) reported that the following compound lowered blood pressure in 
the hypertensive rat: 
##STR4## 
The synthesis of 5-deazafolic acid has been reported by D. T. Hurst, "An 
Introduction to the Chemistry and Biochemistry of Pyrimidines, Purines, 
and Pteridines," John Wiley and Sons, Ltd., 231 (1980). The synthesis of 
this compound using as an intermediate 
2-amino-6-formyl-5-deazapteridine-4(3H)-one, i.e., a compound having the 
formula: 
##STR5## 
has been proposed by C. Temple, Jr. and J. A. Montgomery, "Synthesis of 
Potential Anticancer Agents," Cancer Chemotherapy National Service Center, 
Southern Research Institute Report 85, pages 1 and 2 (1966) and Report 86, 
pages 8 and 10 (1967). The synthesis of 5-deazafolic acid via a 
condensation reaction involving triformylmethane has been reported by C. 
P. Tseng, Dissertation Abstracts Int. B, 40, 3752 (1980). The thesis upon 
which this abstract was based, C. P. Tseng, Studies in Heterocyclic 
Chemistry, 171-185 (1979) also describes unsuccessful work on the 
preparation of 5-deaza-2,4-diaminopteridine-6-carboxaldehyde dimethyl 
acetal, i.e., a compound having the formula: 
##STR6## 
the synthesis of 5-deaza-6-formylpterin; and the conversion of this 
compound to 5-deazafolic acid via acetylated 5-deaza-6-formylpterin. The 
preparation of 5-deazaaminopterin via a long sequence of reactions 
involving the elaboration of a pyrimidine intermediate has been described 
by E. F. Elslager and J. Davoll, "Lectures in Heterocyclic Chemistry," 2, 
S-97, S-119-S-121 (1974). 
SUMMARY OF THE INVENTION 
The 5-deaza analogs of folic acid, N.sup.10 -methylfolic acid, aminopterin, 
methotrexate and the diethyl ester of aminopterin inhibit the growth of 
human epidermoid carcinoma cells No. 2 and are active against leukemia in 
laboratory animals. The 5-deaza analogs of folic acid, N.sup.10 
-methylfolic acid, aminopterin and methotrexate have the following 
structures: 
##STR7## 
wherein R is either hydrogen or methyl. 
A novel intermediate has now been found which is useful in the preparation 
of the compounds of Formulas I and II. This intermediate has the 
structure: 
##STR8## 
This compound is prepared by reaction of a compound having the structure: 
##STR9## 
with the quaternary salt of triformylmethane (or its hydrolyzed 
derivatives ) having the structure: 
EQU CH[CH.dbd.N.sup.+ (CH.sub.3).sub.2 ].sub.3 3X.sup.- V 
wherein X is a halogen atom, preferably chlorine. 
Reductive alkylation of dialkyl-p-aminobenzoyl-L-glutamate having the 
structure: 
##STR10## 
with the compounds of Formula III afforded a compound having the 
structure: 
##STR11## 
wherein R.sub.1 is a lower alkyl group, i.e., a group containing up to six 
carbon atoms. 
The compound of Formula VII was converted to the compound of Formula II 
(R.dbd.H) by saponification. In addition, the compound of Formula II 
(R.dbd.H) was treated under more drastic conditions with base to hydrolyze 
the 4-amino group to give the compound of Formula I (R.dbd.H). Also, 
methylation of II (R.dbd.H) with formaldehyde in the presence of sodium 
cyanoborohydride provided the compound of Formula II (R.dbd.CH.sub.3). In 
addition, the 4-amino group of the compound of Formula II (R.dbd.CH.sub.3) 
was hydrolyzed with base to give the compound of Formula I 
(R.dbd.CH.sub.3). The compound of Formula I (R.dbd.CH.sub.3) was also 
prepared by methylation of the compound of Formula I (R.dbd.H) with the 
formaldehyde-sodium cyanoborohydride combination. 
DETAILED DESCRIPTION OF THE INVENTION 
The synthesis of triformylmethane was reported by Z. Arnold and J. 
Zemlicka, Coll. Czech. Chem. Commun., 25, 1318 (1960) and Z. Arnold, Coll. 
Czech. Chem. Commun., 26, 3051 (1961). Thus, in one method, reaction of 
bromoacetic acid with the complex [(CH.sub.3).sub.2 N.dbd.CHCl].sup.+ 
Cl.sup.- resulting from treatment of N,N-dimethylformamide with phosphorus 
oxychloride gave a quaternary salt, probably V, which was treated with 
aqueous potassium carbonate to give triformylmethane. The isolation and 
purification of the latter is difficult, and in the procedure described 
herein, the intermediate quaternary salt or its hydrolyzed derivatives is 
used. 
The condensation of V with 2,4,6-triaminopyrimidine (IV) in water at reflux 
gave 2,4-diaminopyrido[2,3-d]pyrimidine-6-carboxaldehyde (III). The 
structure of III was confirmed as described hereinafter in Example 7B by 
the alkaline potassium permanganate oxidation of the formyl group and 
hydrolysis of the 4-amino group to give the known 
2-amino-4(3H)oxopyrido[2,3-d]pyrimidine-6-carboxylic acid (VIII) [R. 
Bernetti, F. Mancini and C. C. Price, J. Org. Chem., 27, 2863 (1962); D. 
M. Mulvery, S. G. Cottis and H. Tieckelmann, J. Org. Chem., 29, 2903 
(1964)]. An authentic sample of VIII was prepared as described hereinafter 
in Example 7A by alkaline potassium permanganate oxidation of 
2-amino-6-methyl-4(3H)oxopyrido[2,3-d]-pyrimidine, which was synthesized 
by the method of E. Stark and E. Breitmaier, Tetrahedron, 29, 2209 (1973). 
It has been established that in the 2,4-diaminopyrido[2,3-d]pyrimidine 
ring system, the 4-amino function undergoes alkaline hydrolysis readily 
[R. Tratner, G. Elion, G. Hitchings, and D. Sharefkin, J. Org. Chem., 29, 
2674 (1964)]. 
Although the mechanism of the condensation reaction is unknown, two of the 
formyl groups or potential formyl groups of V must react with the enamine 
moiety of the 4-aminopyrimidine with the elimination of either water or 
dimethylamine. The initial reaction involves the electrophilic attack of 
one formyl group or derivative either with the 5-position of the 
pyrimidine ring or with the 4-amino group to give a Schiff base followed 
by cyclization of the resulting monocyclic intermediate to give the 
desired bicyclic ring system. In the J. Org. Chem. reference above, Price 
et al. observed that pyrido[2,3-d]pyrimidines were readily formed under 
mild conditions from 4-aminopyrimidines and malondialdehydes containing 
electron-withdrawing groups. Compound V can be considered a 
malondialdehyde derivative substituted with an electro-withdrawing group. 
Reductive alkyation of diethyl p-aminobenzoyl-L-glutamate with III and 
hydrogen in 70% acetic acid containing Raney nickel gave a 32% yield of 
5-deazaaminopterin diethyl ester. Saponification of the ester groups in a 
mixture of dimethyl sulfoxide-water at ambient temperature gave an 87% 
yield of 5-deazaaminopterin (II, R.dbd.H). Methylation of the latter 
compound was accomplished by treatment of II (R.dbd.H) with formaldehyde 
and sodium cyanoborohydride in aqueous solution at pH 6.4 to give an 85% 
yield of 5-deazamethotrexate (II, R.dbd.CH.sub.3). The structure of II 
(R.dbd.CH.sub.3) was established as described hereinafter in Example 7C by 
oxidation with alkaline potassium permanganate to give the previously 
prepared 2-amino-4(3H)-oxopyrido[2,3-d]pyrimidine-6-carboxylic acid 
(VIII), which indicated that methylation had occurred either on the 4- or 
10-amino group. Methylation of the 4-amino group was eliminated from 
consideration by alkaline hydrolysis of the 4-amino group to give 
5-deaza-10-methylfolic acid (I, R.dbd.CH.sub.3). 
The preferred route for the preparation of I (R.dbd.H) involved the 
hydrolysis of 5-deazaaminopterin diethyl ester in aqueous sodium hydroxide 
at reflux temperature, which resulted in replacement of the 4-amino group 
as well as hydrolysis of the ester functions to give a 79% yield of 
5-deazafolic acid (I, R.dbd.H). Methylation of the compound of Formula I 
(R.dbd.H) with formaldehyde and sodium cyanoborohydride gave an 84% yield 
of 5-deaza-10-methylfolic acid (I, R.dbd.CH.sub.3), which was identical to 
that prepared by the alkaline hydrolysis of the compound of Formula II 
(R.dbd.CH.sub.3). The structures of I (R.dbd.H and CH.sub.3), II (R.dbd.H 
and CH.sub.3), and 5-deazaaminopterin diethyl ester were confirmed by 
elemental analyses, .sup.1 H-NMR and mass spectral data.