United States Patent (19) [11] Patent Number: 4,889,859 Taylor Et Al

United States Patent (19) [11] Patent Number: 4,889,859 Taylor et al. 45 Date of Patent: Dec. 26, 1989

(54) PYRIDO2,3-DPYRIMIDINEDERIVATIVES OTHER PUBLICATIONS (75) Inventors: Edward C. Taylor, Princeton, N.J.; Chuan Shih, Indianapolis, Ind. Merck Index (1983) pp. 599 & 1408. Primary Examiner-Mukund J. Shah (73) Assignee: The Trustees of Princeton University, Assistant Examiner-C. L. Cseh Princeton, N.J. Attorney, Agent, or Firm-Mathews, Woodbridge, 21 Appl. No.: 156,908 Goebel, Pugh & Collins 22) Filed: Feb. 5, 1988 57 ABSTRACT Derivatives of N-(2-(5,6,7,8-tetrahydropyrido-2,3- (51) Int. Cl* ...... A61K 31/505; C07D 471/04 dipyrimidin-6-yl)-alkylbenzoyl-L-glutamic acid are (52) U.S. C...... 514/258; 544/279 antineoplastic agents. A typical embodiment is N-(2- (58) Field of Search ...... 544/279; 514/258 fluoro-4-2-(2-amino-4-hydroxy-5,6,7,8-tetrahy References Cited dropyrido-2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- . (56 glutamic acid. U.S. PATENT DOCUMENTS 4,684,653 8/1987 Taylor et al...... 544/279 14 Claims, No Drawings 4,889,859 1 2 PYRIDO2,3-DPYRIMIDINEDERIVATIVES -continued TECHNICAL FIELD o The invention pertains to derivatives of N-(2-(5,6,7,8- R. H. H R5 R6 (IB)IB N1 tetrahydropyrido2,3-dipyrimidin-6yl)-alkylbenzoyl e CS ..H R2 L-glutamic acid, which are antineoplastic agents, and to N R 1s YCH-CH R4 their preparation and use. S. CH2 O H2N N N1 BACKGROUND ART h R R3 The folic acid antimetabolites aminopterin and ame thopterin (also known as 10-methylaminopterin or wherein R is hydroxy or amino; R2 is hydrogen, methotrexate) are antineoplastic agents. These com 15 methyl, or ethyl; and R3, R, R, R6, and R7 are se pounds inhibit enzymatic conversions involving meta lected such that (a) R3 is bolic derivatives of folic acid. Amethopterin, for exam ple, inhibits dihydrofolate reductase, an enzyme neces gH2CH2COOH sary for the regeneration of tetrahydrofolate from dihy 20 drofolate which is formed during the conversion of -conHo- (-COOH 2-deoxy-uridylate to thymidylate by the enzyme thy midylate synthetase. Other derivatives of folic acid and aminopterin have and each of R', R, R6, and R7 independently is hy been synthesized and tested as anti-metabolites. Among 25 drogen, chloro, or fluoro; or (b) Ris these are various "deaza' compounds in which a meth ylene or methylidene group occupies a position in the molecule normally occupied by an imino or nitrilo gH2CH2COOH group, respectively. These derivatives have varying 30 -conHo-C-COOH degrees of antimetabolic activity. 10-Deazaaminopterin H is highly active (Sirotak et al., Cancer Treat Rep., 1978, 62, 1047) whereas 10-deazafolic acid shows no signifi one member of R3, RS, R, and R7 is chloro or fluoro; cant activity (Struck et al., J. Med. Chem, 1971, 14, 35 and 693). 5-Deazafolic acid is only weakly cytotoxic the remaining members of R3, R5, R6, and R7 is hy whereas 5-deazaaminopterin has activity similar to that drogen, chloro, or fluoro; of amethopterin (Taylor et al., J. Org. Chen, 1983, 48, (ii) the pharmaceutically acceptable alkali metal, al 4852). 5,6,7,8-Tetrahydro-5-deazaaminopterin also is kaline earth, non-toxic metal, ammonium, and substi active (U.S. Pat. No. 4,684,653). 8, 10-Dideazafolic acid tuted ammonium salts thereof; and is only marginally effective as a dihydrofolate reductase (iii) diastereoisomeric mixtures of said tetrahy inhibitor (De Graw et al., "Chemistry and Biology of dropyrido(2,3-dlpyrimidine or said salts. Pteridines', Elsevier, 1979, 229) while 5,8,10 trideazafolic acid also shows only marginal activity 45 The invention also pertains to methods for the prepa against mouse L1210 leukemia (Oatis et al., J. Med. ration of such compounds, to intermediates useful in Chem., 1977, 20, 1393). 8,10-Dideazaaminopterin is re those preparations, and to methods and compositions ported to be active (U.S. Pat. No. 4,460,591) and 5,8,10 for the use of such compounds in combating neoplastic trideazaaminopterin exhibits activity against mouse 50 growth. L1210 leukemia (Yan et al., J. Heterocycl. Chem, 1979, MODES FOR CARRYING OUT THE 16, 541). INVENTION DISCLOSURE OF INVENTION 55 The compounds of the invention are derivatives of The invention pertains to the pyrido (2,3-dipyrimidine heterocyclic ring which is (i) a tetrahydropyrido2,3-dipyrimidine of the for numbered as follows: mula: 4. 5 R5 R6 (IA) r N6 Rl R2 ls 217 N N 1 "nHN. CH-CH R4 8 65 1.Sa l CH2I." The compounds of Formulas IA and IB exist in tauto H2N N N1 R R3 meric equilibrium with the corresponding 4-oxo and H 4-imino compounds: 4,889,859 3

OH O H-H H H-H N 21 n N1 - G N N n N1

H2N 1s N -CH2 <- H2N 1s N CH2 H H

NH2 NH

2- H-HN 1 H N H.--N 1

H2N IISa N 1 CH2 . .H2N Sa .N It1.CH2 H H

For convenience, the 3,4-dehydro-4-hydroxy and methyl or ethyl; or Z and R2 when taken together are 3,4-dehydro-4-amino forms are depicted, and the corre- 20 a carbon-carbon bond; sponding nomenclature issued, throughout this specifi in the presence of a palladium complex, to yield a com cation, it being understood that in each case such in pound of the formula: cludes the tautomeric 4(3H)-oxo and imino forms. The compounds can be prepared by allowing a com pound of the formula: 25 (IV) R5 R6 (II)

X R4' 30 in which R1, R2, R3, R4, R3, R6, R7, R10 and Z are as R R3' herein defined. The palladium complexes are those which have been wherein X is bromor or iodo; R3'is 35 employed previously in the reaction of aryl halides and allylic alcohols, as described for example by Melpoler et gH2CH2COOR9 al., J. Org. Chem, 41, No. 2, 1976, 265; Chalk et al., J. Org. Chem, 41, No. 7, 1976, 1206; Arai et al., J. Hetero -conHo- (-COOR8 cyclic Chem, 15, 351 (1978); Tamuru et al., Tetrahedron H 40 Papers, 10,919 (1978) 919; Tetrahedron, 35,329 (1979). Particularly useful are the palladium/trisubstituted phosphine complexes of Sakamoto, Synthesis, 1983, 312; one member of R', R5, R6, and R7 is hydrogen, e.g., a trisubstituted-phosphine such as a triarylphos chloro, or fluoro; or R'is phine, as for example triphenylphosphine, or a trialkyl 45 phosphine; a palladium salt such as palladium acetate or gH2CH2COOR9 a palladium halide such as palladium chloride; and a cuprous halide, such as cuprous iodide. -CONH--COOR8 The reaction preferably is conducted in the presence H of at least one molar equivalent of a secondary or ter 50 tiary amine which acts as an acid acceptor, as for exam one member of R3, R5, and R6 is chloro or fluoro; ple triethylamine, or diethylamine, and under and inert a second of the remaining members of R3, R5, and R6 atmosphere, optionally in the presence of an inert polar is hydrogen, chloro, or fluoro; and solvent such as acetonitrile, dimethylformamide, N the remaining member of R3, R, and R6is hydrogen; methylpyrrolidone and the like. Particularly preferred and 55 is the use of acetonitrile which serves as a solvent not each of R8 and R9 is a carboxylic acid protecting only for the reactants but also for the salt formed from group, with a compound of the formula: the acid acceptor and acid generated. Moderately ele vated temperatures, as for example from about 75 to Z R2 (III) 125 C., preferable at or below 100° C., generally are 60 advantageous. CC-H The amino and carboxylic acid protecting groups discussed herein are those conventionally employed, as described for example by Greene in "Protective Groups in Organic Synthesis', John Wiley & Sons, Inc., 1981, 65 and McOmie in "Protective Groups in organic Chemis wherein R10 is hydrogen or an amino protecting group; try', Plenum Press, 1983. Particularly preferred R10 Z when taken independently of R2 is hydrogen; and protecting groups are alkanoyl groups such as acetyl, R2 when taken independently of Z is hydrogen, propionyl, pivaloyl, and the like. 4,889,859 5 Catalytic hydrogenation of a compound of Formula tide bonds, such as activation of the carboxylic acid IV yields the corresponding 2-amino (or 2-protected through formation of the mixed anhydride, treatment amino)-6-substituted-5,6,7,8-tetrahydropyrido(2,3-d- with DCC, or use of diphenylchlorophosphonate. pyrimidine of the formula: Representative compounds of the present invention include: A. N-(3-2-(2amino-4-hydroxy-5,6,7,8-tetrahy 1 R5 R6 (V) dropyrido2,3-dlpyrimidin-6-yl)ethylbenzoyl)-L- R H H R2 glutamic acid. 2 CS B. N-(3-fluoro-5-[2-(2-amino4-hydroxy-5,6,7,8-tet N H-CH-CH R4 10 rahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- son'sSa N N1 CH2 R3' glutamic acid. C. N-(2-fluoro-3-2-(2-amino-4-hydroxy-5,6,7,8-tet rahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- glutamic acid. The compounds of Formula V are then subjected to 15 D. N-(2-fluoro-5-2-(2-amino-4-hydroxy-5,6,7,8-tet hydrolysis to remove the protecting groups R, R9 and rahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- R10. This is conducted at normal temperatures utilizing glutamic acid. ' aqueous acid or base, such as for example, an aqueous E. N-(4-fluoro-5-2-(2-amino-4-hydroxy-5,6,7,8-tet alkali metal hydroxide, optionally in the presence of a rahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- water miscible organic solvent such as methanol, etha 20 nol, tetrahydrofuran, dimethylformamide, and the like, glutamic acid or an acid, as for example trifluoroacetic acid. When F. N-(2-chloro-4-2-(2-amino-4-hydroxy-5,6,7,8-tet base is used, the cationic moiety of the salt is liberated rahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- and the product is formed as the dicationic glutamate glutamic acid. salt which can be readily precipitated by adjustment of 25 G. N-(3-chloro-4-2-(2-amino-4-hydroxy-5,6,7,8-tet pH, as through acidification with, for example, acetic rahydropyrido(2,3-dpyrimidin-6-yl)ethylbenzoyl)-L- acid. The resulting products generally are high melting glutamic acid. crystalline or microcrystalline solids. H. N-(2-fluoro-4-2-(2-amino-4-hydroxy-5,6,7,8-tet The absolute configuration about the chiral carbon in rahydropyrido(2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- the glutamic acid chain in R3 or Rin Formulas IA and 30 glutamic acid. IB is (S) or L, being the same absolute configuration as I. N-(3-fluoro-4-2-(2-amino-4-hydroxy-5,6,7,8-tet that about the corresponding alpha carbon atom in rahydropyrido(2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- L-alanine. In addition, the carbon atom in the 6-position glutamic acid. of the 5,6,7,8-tetrahydropyrido-2,3-dipyrimidine ring is J. N-(2,6-difluoro-4-2(2-amino-4-hydroxy-5,6,7,8-tet a chiral center, leading to the two (R,S) and (S,S) diaste 35 rahydropyridio2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- reomers shown in Formulas IA and IB. The mixture of glutamic acid. diastereomers can be utilized therapeutically, both serv K. N-(3,5-difluoro-4-2-(2-amino-4-hydroxy-5,6,7,8- ing as substrates for relevant folate enzymes. The diaste tetrahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)- reomers can also be separated so as to be in a form L-glutamic acid. substantially free of the other; i.e., in a form having an L. N-(2,3-difluoro-4-2-(2-amino-4-hyddroxy-5,6,7,8- optical purity of >95%. tetrahydropyrido2,3-dipyrimidin-6-yl)ethyl-benzoyl)- The diastereomers can be separated mechanically, as L-glutamic acid. by chromatography or a mixture of diastereomers can M. N-(2,3,5,6-tetrafluoro-4-2-(2-amino-4-hydroxy be treated with a chiral acid operable form a salt there 5,6,7,8-tetrahydropyrido2,3-dipyrimidin-6-yl)ethyl)- with. The resultant diastereoisomeric salts are then 45 benzoyl)-L-glutanic acid. separated through one or more fractional crystalliza The resultant compounds of Formulas IA and IB, tions and thereafter the free base of the cationic moiety including mixtures thereof and their pharmaceutically of at least one of the separated salts is liberated through acceptable alkali metal, alkaline earth metal, non-toxic treatment with a base and removal of the protecting metal, ammonium, and substituted ammonium salts, groups. The liberation of the cation of the salt can be 50 such as for example the sodium, potassium, lithium, performed as a discrete step before or after the removal calcium, magnesium, aluminum, zinc, ammonium, tri of the protecting groups, R, R9, and R0 or concomi methylammonium, triethylammonium, triethanolam tantly with the removal of such groups under basic monium, pyridinium, substituted pyridinium, and the hydrolysis. like, have an effect on one or more enzymes which Suitable chiral acids include the individual enantio 55 utilize folic acid, and in particular metabolic derivatives mers of 10-camphorsulfonic acid, camphoric acid, alpha offolic acid, as a substrate. The compounds can be used, bromocamphoric acid, menthoxyacetic acid, tartaric alone or in combination, to treat neoplasms including acid, diacetyltartaric acid, malic acid, pyrrolidine-5-car choriocarcinoma, leukemia, adenocarcinoma of the boxylic acid, and the like. female breast, epidermid cancers of the head and neck, The starting materials of Formula III can be prepared squamous or small-cell lung cancer, and various lym from the corresponding 6-halopyrido2,3-d-pyrimidine hosarcomas. The compounds can also be used to treat in the manner set forth in copending U.S. application fungoides, psoriasis and other autoimmune conditions Ser. No. 922,511. The hydrogenated starting materials such as rheumatoid arthritis which are responsive to of Formula II can be prepared by coupling the appro methotrexate. priate halobenzoic acid and a protected derivative of 65 The compounds may be administered either orally or L-glutamic acid in the manner described in PCT appli preferably parenterally, alone or in combination with cation WO 86/05181. The coupling reaction utilizes other anti-neoplastic agents, steroids, etc., to a mammal conventional condensation techniques for forming pep suffering from neoplasm and in need of treatment. Par 4,889,859 7 8 enteral routes of administration include intramuscular, (m, 1H), 7.68 (m, 1H), 8.26 (dd, 1H, J=2.0, 7.2 Hz), 8.37 intrathecal, intravenous or intraarterial. Dosage regi (s, 1H), 8.63 (d, 1H, J=2.2 Hz), 9.0 (d, 1H, J = 2.0 Hz). mens must be titrated to the particular neoplasm, the condition of the patient, and the response but generally EXAMPLE 2 doses will be from about 10 to about 100 mg/day for A sample of 0.115 g (0.202 mmol) of dimethyl N-(2- 5-10 days or single daily administration of 250-500 mg, fluoro-5-2-(2-pivaloylamino-4-hydroxypyrido-2,3- repeated periodically; e.g. every 14 days. Oral dosage dlpyrimidin-6-yl)ethynyl)-benzoyl)-L-glutamate and forms include tablets and capsules containing from 1-10 0.045g of platinum oxide is dissolved in 5 mL of glacial mg of drug per unit dosage. Isotonic saline solutions acetic acid. This mixture then is hydrogenated under containing 20-100 mg/ml can be used for parenteral O atmospheric hydrogen at room temperature for 5 hours. administration. The catalyst is removed by filtration, and the filtrate Representative IC50 values determined in whole cell concentrated in vacuo, and the crude solid flash chro human leukemia cell line (CCRF-CEM) are as follows: matographed on silica gel using 1:19 methanol: chloro form as the eluent to yield 0.76 g (66%) of dimethyl 5 N-(2-fluoro-5-2-2-pivaloylamino-4-hydroxy-5,6,7,8-tet Compound IC50 (mcg/mL) rahydropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- A. 0.015 glutamate as a gray-white solid. m.p. 165-170 C. (dec); D 0.00 R=0.33 (1:19 methanol:chloroform), Mass (FD)=573; F 0.027 IR (KBr, cm-1)=1161, 1205, 1223, 1232, 1439, 1460, H 0.004 20 1478, 1492, 1527, 1536, 1556, 1573, 1619, 1645, 1743, 3392, 3395; UV (EtOH) An-271 (epsilon=10200), Mice implanted with C3H mammary adenocarci 234 (epsilon=42900). noma were treated intraperitoneally for five days with Anal. Calcd. for C28H36NsO7F: C, 58.63; H, 6.33; N, compound H. At a dosage of 30 mg/kg, 72% inhibition 12.21. Found: C, 58.59; H, 6.38; N, 12.14. was observed. At a dosage of 100 mg/kg, 80% inhibi 25 INMR (300 MHz, CDCl3) delta 1.32 (s, 9H), 1.73 (m, tion was observed. No toxicity was observed at these dosages. 2H), 1.89 (m, 1H), 2.20 (m, 2H), 2.30-2.60 (m, 3H), The following inhibitory data were observed with 2.65-2.90 (m, 3H), 3.03 (m, 1H), 3.39 (m, 1H), 3.70 (s, Compound H in 6C3HED lymphosarcoma in mice: 3H), 3.83 (s, 3H), 4.66 (s, 1H), 4.91 (m, 1H), 7.09 (dd, 30 1H, J=8.4, 11.7 Hz), 7.36 (m, 2H), 7.75 (s, 1H), 7.91 (dd, 1H, J =2.2, 7.4 Hz). 6C3HED LYMPHOSARCOMA EXAMPLE 3 DOSE (MK/KG) 7% INHIBITION TOXIC/TOTAL 200 100 0/10 A sample of 0.024 g (0.042 mmol) of dimethyl N-(2- 100 00 0/10 35 fluoro-5-2-2-pivaloylamino-4-hydroxy-5,6,7,8-tetrahy 50 95 0/10 dropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L-gluta 25 94 0/9 mate is dissolved in 0.5 mL of 1N sodium hydroxide and stirred at room temperature for 5 days. The pale yellow The following examples will serve to further illus solution then is acidified with glacial acetic acid and the trate the invention. In the NMR data, 's' denotes sin solvent removed in vacuo. The residue is triturated with glet, "d” denotes doublet, "t' denotes triplet, “q' de water and the solid collected by suction filtration, notes quartet, and "m' denotes multiplet. washed with a small amount of water, and dried in vacuo at 100° C. to give 0.4 g (73%) of N-(2-fluoro-5-2- EXAMPLE 1. (2-amino-4-hydroxy-5,6,7,8-tetrahydropyrido2,3-d- A mixture of 0.016 g (0.093 mmol) of palladium chlo 45 pyrimidin-6-yl)ethylbenzoyl)-L-glutamic acid as a ride and 0.0486 g (0.185 mmol) of triphenylphosphine in white solid. m.p. 252-262 C. (dec); R4=0.04 (1:19 5 mL of acetonitrile is stirred under nitrogen at room methanol: chloroform), Mass (FAB)=462; IR (KBr, temperature for 30 minutes. To this mixture is added an cm-hu -1)=1230, 1248, 1263, 1307, 1349, 1379, 1398, additional 10 mL of acetonitrile and 0.80 g (1.89 mmol) 1462, 1442, 1544, 1616, 1650, 1705, 2928; UV (EtOH) of dimethyl N-(2-fluoro-5-iodobenzoyl)L-glutamate, 50 Amax=278 (epsilon=13300), 221 (epsilon=29800); 0.50 g (1.85 mmol) of 2-pivaloylamino-4-hydroxy-6- lNMR (300 MHz, Me2SO-d6) delta 1.57 (brs, 3H), ethynylpyrido(2,3-dipyrimidine, 0.0088 g (0.046 mmol) 1.70-2.10 (m, 4H), 2.34 (t, 2HJ=5.0 Hz), 2.60-2.85 (m, of copper iodide, and 0.54 mL (3.89 mmol) of triethyl 3H), 3.20 (m, 1H), 4.37 (m, 1H), 5.92 (s, 1H), 6.26 (s, amine. The mixture is then heated to reflux for 3 hours. 1H), 7.19 (t, 1H, J-9.2 Hz), (brs 1H), 7.45 (d, 1H, After cooled to room temperature, the solvent is re 55 J=6.0 Hz), 8.43 (d, 1H, J = 6.3 Hz), 9.70 (brs, 1H). moved in vacuo and the residue is flash chromato graphed on silica gel using 1:19 methanol:chloroform as EXAMPLE 4 the eluent to yield 0.92 g (88%) of dimethyl N-(2- By substituting a substantially molar equivalent fluoro-5-2-(2-pivaloylamino-4-hydroxypyrido2,3- amount of dimethyl N-(3-iodobenzoyl)-L-glutamate for dipyrimidin-6-yl)-ethynyl)-benzoyl)-L-glutamate as a 60 dimethyl N-(2-fluoro-5-iodobenzoyl)-L-glutamate in dark yellow solid m.p. 189-191 C. (dec); R = 0.48 the procedure of Example 1 there is obtained dimethyl (1:19 methanol:chloroform), Mass (FD)=565; IR (KBr, N-(3-2-(2-pivaloylamino-4-hydroxypyrido(2,3-d- cm)=1146, 1228, 1263, 1440, 1488, 1549, 1596, 1607, pyrimidin-6-yl)ethynyl)benzoyl)-L-glunate, m.p. 1622, 1673, 1742; UV (EtOH) Anay-319 (ep 161-166 C. silon=30600), 259 (epsilon=11200), 212 (ep 65 Anal. Calcd. for C28H29NsO7: C, 61.42; H, 5.38; N, silon=42100); NMR (300 MHz, CDCl3) delta 1.38 (s, 12.77. Found: C, 61.34; H, 5.23; N, 12.67. 9H), 2.19 (m, 1H), 2.3-2.6 (m, 3H), 3.72 (s, 3H), 3.85 (s, Upon hydrogenation of this material substantially in 3H), 4.95 (m, 1H), 7.21 (dd, 1H, J = 8.5, 11.2 Hz), 7.45 the manner described in Example 2 there then is ob

4,889,859 11 12 EXAMPLE 9 -continued The glutamate derivatives employed in the foregoing R5 R6 examples can be prepared as follows: 5 O gH2CH2COOH | : To 20 mL of benzene are added 2.0 g (0.00752 mol) of CNH-C-COOH 2-fluoro4-iodobenzoic acid, followed by 2.3 mL (320 mmol) of thionyl chloride and several drops of dimeth H ylformamide. The mixture is heated at reflux of 3.5 R R3 hours. The solventis then removed in vacuo and the 10 residue pumped dry. The resulting acid chloride is re wherein R is hydroxy or amino; dissolved in 10 ml of methylene chloride, and added R2 is hydrogen, methyl, or ethyl; dropwise to cooled mixture of 1.67 g (7.89 mmol) of one or two members of R3, R., R6, and R7 are se L-glutamic acid dimethyl ester hydrochloride, 5 mg of lected from the group consisting of chloro and 4-dimethylaminopyridine, and 2.2 mL (15.8 mmol) of 15 fluoro and the remaining members of R3, R5, R6, triethylamine in 30 mL of methylene chloride. After the and R7 are hydrogen; addition, the reaction stirred at room temperature for 18 (ii) the pharmaceutically acceptable salts thereof; and hours. The reaction mixture is then diluted with addi (iii) diastereoisomeric mixtures of said tetrahydro(2,3- tional methylene chloride, washed with 1N hydrochlo 20 dpyrimidines or their salts. ric acid, water and 5% sodium bicarbonate and dried 2. A compound according to claim 1 wherein R is over sodium sulfate. The solvent is removed in vacuo to hydroxy and R2 is hydrogen. give 2.34 g (74%) of dimethyl N-(2-fluoro-4-iodoben zoyl)-L-glutamate as a white solid m.p. 94-96 C.; IR 3. A compound according to claim 2 wherein one of (KBr, cm-1)= 1159, 1177, 1194, 1208, 1229, 1398, 1437, 25 R3, R5, R6, and R7 is chloro or fluoro and the remaining 1477, 1537, 1601, 1642, 1728, 1739, 1756, 3326. members of R3, R5, R6, and R7 are each hydrogen. Anal. Calcd. for C14H15NO5Fl: C, 39.74; H, 3.57; N, 4. A compound according to claim 3 which is N-(2- 3.31. Found: C, 39.98; H, 3.61; N, 3.27. chloro-4-2-(2-amino-4-hydroxy-5,6,7,8-tetrahy HNMR (300 MHz, CDCl3) delta 2.15 (m, 1H), 30 dropyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- 2.33-2.49 (m, 1H), 3.66 (s, 3H), 3.79 (s, 3H), 4.86 (m, glutamic acid. 1H), 7.55 (d, 1H, J = 11 Hz), 7.63 (d, 1H, J=8.0 Hz), 5. A compound according to claim 3 which is N-(2- 7.76 (t, 1H, J = 8.0 Hz). fluoro-4-2-(2-amino-4-hydroxy-5,6,7,8-tetrahydro Similarly prepared are dimethyl N-(2-fluoro-5- pyrido2,3-dipyrimidin-6-yl)ethylbenzoyl)-L-glutamic iodobenzoyl)-L-glutamate, m.p. 69-72 C., and di 35 methyl N-(2-chloro-4-bromobenzoyl)-L-glutamate, acid. mp. 77-80 C. 6. The method of inhibiting neoplastic growth in a The halobenzoic acid starting materials in turn can be mammal which growth is dependent on folic acid or a prepared according to the following procedure: metabolic derivative of folic acid as a substrate, which To 8.1 g (51.3 mmol) of potassium permangante in comprises administering to the mammal in a single or 150 mL of water are added 5.0 g (21.1 mmol) of 2 multiple dose regimen an effective amount of a com fluoro-5-iodotoluene. The reaction mixture is heated at pound according to claim 1. reflux for 4 hours. After being cooled to room tempera 7. A pharmaceutical composition for inhibiting neo ture, the reaction mixture is poured into 250 mL of 45 plastic growth in a mammal which growth is dependent methylene chloride of sodium bisulfate, and 1N hydro on folic acid or a metabolic derivative of folic acid as a chloric acid. The organic layer is separated, washed substrate, which comprises an amount of a compound with water and dried over sodium sulfate. The solvent is according to claim 1 which upon administration to the removed in vacuo and the solid triturated with hexane 50 mammal in a single or multiple dose regiment is effec to give 0.86 g (15%) of 2-fluoro-5-iodobenzoic acid as a tive to inhibit said growth, in combination with a phar white solid, m.p. 161-163' C. maceutically acceptable carrier. 2-Fluoro-4-iodobenzoic acid, m.p. 213-215 C. (dec) is similarly prepared from 2-fluoro-4-iodotoluene. 8. A compound selected from the group consisting of What is claimed is: 55 (i) a tetrahydro2,3-dipyrimidine the formula: 1. A compound selected from the group consisting of (i) a tetrahydro2,3-dipyrimidine the formula: RS R6 R1 R2 R2 60 "SH R1 H H N 2 N.C : CH2CH R4 N. CHCH NH CH2CH2COOH N 1 s ls -CH2 Y. H2N N N R NH b-g-COOH C H sn's N N1 65 H O H

O 4,889,859 13 14 -continued the remaining members of R, R5, R6, and R7 are each Rl R5 R6 hydrogen. HN-H 11. A compound according to claim 10 which is N-(3- 1 CS c.H (2-(2-amino-4-hydroxy-5,6,7,8-tetrahydropyrido-(2,3- YoHo H R4 5 dipyrimidin-6-yl)ethylbenzoyl)-L-glutamic acid. 1s -CH2 H 12. A compound according to claim 10 which is N-(2- H2N N N fluoro-5-2-(2-amino-4-hydroxy-5,6,7,8-tetrahy h R gNH --cooH dropyrido(2,3-dipyrimidin-6-yl)ethylbenzoyl)-L- glutamic acid. H 10 13. The method of inhibiting neoplastic growth in a mammal which growth is dependent on folic acid or a wherein R1 is hydroxy or amino; metabolic derivative of folic acid as a substrate, which R2 is hydrogen, methyl, or ethyl; comprises administering to the mammal in a single or none, one or two members of R, R, R, and R7 are multiple dose regimen an effective amount of a com selected from the group consisting of chloro and 15 pound according to claim 8. fluoro and the remaining members of R, R, R, 14. A pharmaceutical composition for inhibiting neo and R7 are hydrogen; plastic growth in a mammal which growth is dependent (ii) the pharmaceutically acceptable salts thereof; and on folic acid or a metabolic derivative of folic acid as a (iii) diastereoisomeric mixtures of said tetrahydro2,3- substrate, which comprises an amount of a compound dpyrimidines or their salts. 20 according to claim 8 which upon administration to the 9. A compound according to claim 8 wherein R is mammalin a single or multiple dose regimen is effective hydroxy and R2 is hydrogen. to inhibit said growth, in combination with a pharma 10. A compound according to claim 9 wherein one of ceutically acceptable carrier. R“, R5, R6, and R7 is hydrogen, chloro, or fluoro, and B. k it is at 25

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : 4, 889,859 Page l of 2 DATED December 26, 1989 INVENTOR(S) : Edward C. Taylor et al. It is certified that error appears in the above-identified patent and that said Letters Patent is hereby COrrected as shown below:

Claim 1 should read as follows:

1. A compound selected from the group consisting of

(i) a tetrahydro (2,3-d)pyrimidine the formula:

Or 2N-HH c n...H R? R52 .R6 CH2CH2COOH ls | Y CHcH Y)-(NH-6-cooh HN 1SN 1SN-"? ritium H H 3 R R wherein R1 is hydroxy or amino; R2 is hydrogen, methyl, or ethyl;

UNITED STATES PATENT AND TRADEMARK OFFICE CERTIFICATE OF CORRECTION PATENT NO. : A , 889,859 Page 2 of 2 DATED December 26, 1989 INVENTOR(S) : Edward C. Taylor et al. It is Certified that error appears in the above-identified patent and that said Letters Patent is hereby COrrected as shown below: - 2 - Claim 1 . . . (cont'd) one or two members of R3, R, R6, and R7 are selected from the group consisting of chloro and fluoro and the re maining members of R3, R5, R6, and R7 are hydrogen;

(ii) the pharmaceutically acceptable salts thereof; and

(iii) diastereoisomeric mixtures of said tetrahydro (2, 3 d)pyrimidines or their salts.

Signed and Sealed this Twenty-second Day of January, 1991

Attest:

HARRY F. MANBECK, JR.

Attesting Officer Commissioner of Patents and Trademarks

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTFCATE OF CORRECTION PATENT NO. : 4,889,859 Page 1 of 2 DATED : DECEMBER 26, 1989 INVENTOR(S) : EDWARD C. TAYLOR and CHUAN SHIH it is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below: Cols. 12, lines 54-66 and Col. 13, lines 1-10, Claim 8 chould read as follows: 8. A compound selected from the group consisting of (i) a tetrahydro2,3-dipyrimidine the formula:

R5 R6 1 R2 SC'CHCH R4 N YNH GHCHCOOH HN 1NNr N -H, R7 CNH-(-COOH h O H

R H R 6 C H H R2 N YCN. CHCH R4 2 1's-N-CH2 GH,CHCOOH R7 (5NH--COOH

UNITED STATES PATENT ANDTRADEMARK OFFICE CERTFCATE OF CORRECTION PATENT NO. : 4,889,859 Page 2 of 2 DATED : DECEMBER 26, 1989 INVENTOR(S) : EDWARD C. TAYLOR and CHUAN SHIH It is certified that error appears in the above-indentified patent and that said Letters Patent is hereby corrected as shown below: wherein R is hydroxy or amino; R’ is hydrogen, methyl, or ethyl; none, one or two members of R. R, R, and R' are selected from the group consisting of chloro and fluoro and the remaining members of R", R, R, and Rare hydrogen; (ii) the pharmaceutically acceptable salts thereof, and (iii) diastereoisomeric mixtures of said tetrahydro2,3-dipyrimidines or their salts.

Signed and Sealed this Twenty-first Day of October 1997 Attest: (a team

BRUCE LEHMAN Attesting Officer Connaissioner of Parents and Trade nic rks