Dual inhibitors of thymidylate synthase and dihydrofolate reductase as antitumor agents: Design, synthesis, and biological evaluation of classical and nonclassical pyrrolo[2,3-d]pyrimidine antifolates

Journal of Medicinal Chemistry

Volumn 49, Issue 3, 2006, Pages 1055-1065

  Gangjee, Aleem ^a   Jain, Hiteshkumar D ^a   Phan, Jaclyn ^a   Lin, Xin ^a   Song, Xiaohong ^a,d   McGuire, John J ^b   Kisliuk, Roy L ^c  

^a DUQUESNE UNIVERSITY   (United States)

^b ROSWELL PARK CANCER INSTITUTE   (United States)

^c TUFTS UNIVERSITY SCHOOL OF MEDICINE   (United States)

^d ABBOTT LABORATORIES   (United States)

Author keywords

[No Author keywords available]

Indexed keywords

2 AMINO 6 ETHYL 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(1' NAPHTHYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(2' NAPHTHYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(2',6' DIMETHYLPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(3' BROMOPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(3' CHLOROPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(3' METHOXYPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(3',4' DICHLOROPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(3',5' TRIFLUOROMETHYLPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(4' BROMOPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(4' CHLOROPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(4' FLUOROPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(4' METHOXYPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; 2 AMINO 6 ETHYL 5 [(4' NITROPHENYL)SULFANYL] 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDINE; DIHYDROFOLATE REDUCTASE INHIBITOR; FOLINATE CALCIUM; METHOTREXATE; N [4 [(2 AMINO 6 ETHYL 3,4 DIHYDRO 4 OXO 7H PYRROLO[2,3 D]PYRIMIDIN 5 YL)THIO]BENZOYL]GLUTAMIC ACID; NOLATREXED; PEMETREXED; PLEVITREXED; PYRROLO[2,3 D]PYRIMIDINE DERIVATIVE; RALTITREXED; THYMIDYLATE SYNTHASE INHIBITOR; UNCLASSIFIED DRUG;

ADDITION REACTION; ANTINEOPLASTIC ACTIVITY; ARTICLE; DEPROTECTION REACTION; DRUG DESIGN; DRUG SYNTHESIS; HUMAN; HUMAN CELL; IC 50; NONHUMAN; OXIDATION; SAPONIFICATION;

ANTINEOPLASTIC AGENTS; BINDING SITES; CELL LINE, TUMOR; DRUG SCREENING ASSAYS, ANTITUMOR; ESCHERICHIA COLI; FOLIC ACID ANTAGONISTS; GLUTAMATES; HUMANS; MODELS, MOLECULAR; PYRIMIDINES; PYRIMIDINONES; PYRROLES; STRUCTURE-ACTIVITY RELATIONSHIP; TETRAHYDROFOLATE DEHYDROGENASE; THERMODYNAMICS; THYMIDYLATE SYNTHASE;

EID: 32344451388     PISSN: 00222623     EISSN: None     Source Type: Journal    
DOI: 10.1021/jm058276a     Document Type: Article

References (64)

1. Presented in part at the 227th American Chemical Society National Meeting, Anaheim, CA, March 28-April 1, 2004; Abstr. MEDI-76.
2. Kisliuk, R. L. The Biochemistry of Folates. In Folate Antagonists as Therapeutic Agents; Sirotnak, F. M., Burchall, J. J., Ensminger, W. D., Montgomery, J. A., Eds.; Academic Press: New York, 1984; pp 1-68.
3. Berman, E. M.; Werbel, L. M. The Renewed Potential for Folate Antagonists in Contemporary Cancer Chemotherapy. J. Med. Chem. 1991, 34, 479-485.
4. Carreras, C. W.; Santi, D. V. Catalytic Mechanism and Structure of Thymidylate Synthase. Ann. Rev. Biochem. 1995, 64, 721-762.
5. Blakley, R. L. Eukaryotic Dihydrofolate Reductase. Adv. Enzymol. Mol. Biol. 1995, 70, 23-102.
6. MacKenzie, R. E. Biogenesis and Interconversion of Substituted Tetrahydrofolates. In Folates and Pterins Chemistry and Biochemistry; Blakley, R. L., Benkovic, S. J., Eds.; Wiley: New York, 1984; Vol. 1, pp 255-306.
7. Takimoto, C. H. Antifolates in Clinical Development. Semin. Oncol. 1997, 24, S18-40-S18-51.
8. Jackman, A. L.; Taylor, G. A.; Gibson, W.; Kimbell, R.; Brown, M.; Calvert, A. H.; Judson, I. R.; Hughes, L. R. ICI D1694, A Quinazoline Antifolate Thymidylate Synthase Inhibitor That is a Potent Inhibitor of L1210 Tumour Cell Growth In Vitro and In Vivo: A New Agent for Clinical Study. Cancer Res. 1991, 51, 5579-5586.
9. Taylor, E. C.; Kuhnt, D.; Shih, C.; Rinzel, S. M.; Grindey, G. B.; Barredo, J.; Jannatipour, M.; Moran, R. A Dideazatetrahydrofolate Analogue Lacking a Chiral Center at C-6, N-[4-[2-(2-Amino-3,4-dihydro-4-oxo-7H-pyrrolo[2, 3-d]pyrimidin-5-yl)ethylbenzoyl]-L-glutamic Acid, Is an Inhibitor of Thymidylate Synthase. J. Med. Chem. 1992, 35, 4450-4454.
10. Bertino, J. R.; Kamen, B.; Romanini, A. Folate Antagonists. In Cancer Medicine; Holland, J. F., Frei, E., Bast, R. C., Kufe, D. W., Morton, D. L., Weichselbaum, R. R., Eds.; Williams & Wilkins; Baltimore, MD, 1997; Vol. 1, pp 907-921.
11. Rosowsky, A. Chemistry and Biological Activity of Antifolates. In Progress in Medicinal Chemistry; Ellis, G. P., West, G. B., Eds.; Elsevier Science: New York, 1989; Vol. 26, pp 1-252.
12. Gangjee, A.; Elzein, E.; Kothare, M.; Vasudevan, A. Classical and Nonclassical Antifolates as Potential Antitumor, Antipneumocystis and Antitoxoplasma Agents. Curr. Pharm. Des. 1996, 2, 263-280.
13. Mendelsohn, L. G.; Shih, C.; Chen, V. J.; Habeck, L. L.; Gates, S. B.; Shackelford, K. A. Enzyme Inhibition, Polyglutamation, and the Effect of LY231514 (MTA) on Purine Biosynthesis. Semin. Oncol. 1999, 26, 42-47.
14. Shih, C.; Chen, V. J.; Gossett, L. S.; Gates, S. B.; Mackellar, W. C.; Habeck, L. L.; Shackelford, K. A.; Mendelsohn, L. G.; Soose, D. J.; et al. LY231514, a Pyrrolo[2,3-d]pyrimidine-based Antifolate that Inhibits Multiple Folate-requiring Enzymes N-[4-[2-(2-amino-3,4-dihydro-4-oxo-7H-pyrrolo[2,3-d] pyrimidin-5-yl)-ethyl]-benzoyl]-L-glutamic Acid (LY231514) is a novel Pyrrolo[2,3-d]pyrimidine-based Antifolate Currently Undergoing Extensive Phase II Clinical Trials. Cancer Res. 1997, 57, 1116-1123.
15. Schultz, R. M.; Patel, V. F.; Worzalla, J. F.; Shih, C. Role of Thymidylate Synthase in the Antitumor Activity of the Multitargeted Antifolate, LY231514. Anticancer Res. 1999, 19, 437-443.
16. Gangjee, A.; Yu, J.; McGuire, J. J.; Cody, V.; Galitsky, N.; Kisliuk, R. L.; Queener, S. F. Design, Synthesis, and X-ray Crystal Structure of a Potent Dual Inhibitor of Thymidylate Synthase and Dihydrofolate Reductase as an Antitumor Agent. J. Med. Chem. 2000, 43, 3837-3851.
17. Kuyper, L. F.; Garvey, J. M.; Baccanari, D. P.; Champness, J. N.; Stammers, D. K.; Beddell, C. R. Pyrrolo[2,3-d]pyrimidines and Pyrido[2,3-d]pyrimidines as Conformationally Restricted Analogs of the Antibacterial Agent Trimethoprim. Bioorg. Med. Chem. 1996, 4, 593-602.
18. 10-Propargyl-5,8-dide-azafolic Acid (CB 3717) Polyglutamates in L1210 Cells. In Vitro. Biochem. Pharmacol. 1988, 37, 4047-4054.
19. 10-propargyl- 5,8-dideazafolic Acid (ICI 198583). Biochem. Pharmacol. 1991, 41, 1885-1895.
20. Nair, M. G.; Abraham, A.; McGuire, J. J.; Kisliuk, R. L.; Galivan, J. Polyglutamylation as a Determinant of Cytotoxicity of Classical Folate Analogue Inhibitors of Thymidylate Synthase and Glycinamide Ribonucleotide Formyltransferase. Cell. Pharmacol. 1994, 1, 245-249.
21. Bisset, G. M. F.; Pawelczak, K.; Jackman, A. L.; Calvert, A. H.; Hughes, L. R. Syntheses and Thymidylate Synthase Inhibitory Activity of the Poly-γ-glutamyl Conjugates of N-[5-[N-(3,4-Dihydro-2-methyl-4- oxoquinazolin-6-ylmethyl)-N-methylamino]-2-thienoyl]-L-glutamic Acid (ICI D1694) and Other Quinazoline Antifolates. J. Med. Ghent. 1992, 35, 859-866.
22. 10-propargyl-5,8-dideazafolic acid (ICI 198583). J. Med. Chem. 1994, 37, 3294-3302.
23. Jackman, A. L.; Kelland, L. R.; Kimbell, R.; Brown, M.; Gibson, W.; Aherne, W.; Hardcastle, A.; Boyle, F. T. Mechanisms of Acquired Resistance to the Quinazoline Thymidylate Synthase Inhibitor ZD1694 (Tomudex) in One Mouse and Three Human Cell Lines. Br. J. Cancer. 1995, 71, 914-924.
24. Barakat, R. R.; Li, W. W.; Lovelace, C.; Bertino, J. R. Intrinsic Resistance of Squamous Cell Carcinoma Cell Lines as a Result of Decreased Accumulation of Intracellular Methotrexate Polyglutamates. Gynecol. Oncol. 1993, 51, 54-60.
25. McCloskey, D. E.; McGuire, J. J.; Russell, C. A.; Rowan, B. G.; Bertino, J. R.; Pizzorno, G.; Mini, E. Decreased Folylpolyglutamate Synthetase Activity as a Mechanism of Methotrexate Resistance in CCRF-CEM Human Leukemia Sublines. J. Biol. Chem. 1991, 266, 6181-6187.
26. Braakhuis, B. J.; Jansen, G.; Noordhius, P.; Kegel, A.; Peters, G. J. Importance of Pharmacodynamics in the In Vitro Antiproliferative Activity of the Antifolates Methotrexate and 10-EDAM Against Head and Neck Squamous Cell Carcinoma. Biochem. Pharmacol. 1993, 46, 2155-2161.
27. Jackman, A. L.; Kimbell, R.; Aherne, G. W.; Brunton, L.; Jansen, G.; Stephens, T. C.; Smith, M. N.; Wardleworth, J. M.; Boyle, F. T. Cellular Pharmacology and In Vivo Activity of a New Anticancer Agent, ZD9331: A Water-soluble, Nonpolyglutamatable, Quinazoline-Based Inhibitor of Thymidylate Synthase. Clin. Cancer Res. 1997, 3, 911-921.
28. Benepal, T. S.; Judson, I. ZD9331: Discovery to Clinical Development. Anti-Cancer Drugs 2005, 16, 1-9.
29. Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. 5-Arylthio Substituted 2-Amino-4-oxo-6-methylpyrrolo[2,3-d]pyrimidine Antifolates as Thymidylate Synthase Inhibitors and Antitumor Agents. J. Med. Chem. 1995, 38, 4495-4502.
30. Tripos Inc., 1699 South Hanley Road, St. Louis, MO, 63144.
31. Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L.; Queener, S. F.; Barrows, L. R. Classical and Nonclassical Furo[2,3-d]pyrimidines as Novel Antifolates: Synthesis and Biological Activities. J. Med. Chem. 1994, 37, 1169-1176.
32. Gangjee, A.; Devraj, R.; McGuire, J. J.; Kisliuk, R. L. Effect of Bridge Region Variation on Antifolate and Antitumor Activity of Classical 5-substituted 2,4-diaminofuro[2,3-d]pyrimidines. J. Med. Chem. 1995, 38, 3798-3805.
33. Gangjee, A.; Lin, X.; McGuire, J. J.; Kisliuk, R. L. Synthesis of N-{4-[(2,4-diamino-5-methyl-4,7-dihydro-3H-pyrrolo[2,3-d]pyrimidin-6-yl)thio] benzoyl}-L-glutamic Acid and N-{4-[(2-amino-4-oxo-5-methyl-4,7-dihydro-3H- pyrrolo[2,3-d]pyrimidin-6-yl)thio]benzoyl}-L-glutamic Acid as Dual Inhibitors of Dihydrofolate Reductase and Thymidylate Synthase, and as Potential Antitumor Agents. J. Med. Chem. 2005, 48, 7215-7222.
34. Gangjee, A.; Jain, H. D.; McGuire, J. J.; Kisliuk, R. L. Benzoyl Ring Halogenated Classical 2-Amino-6-methyl-3,4-dihydro-4-oxo-5- substitutedthiobenzoyl-7H-pyrrolo[2,3-d]pyrimidine Antifolates as Inhibitors of Thymidylate Synthase and as Antitumor Agents. J. Med. Chem. 2004, 47, 6730-6739.
35. Cao, W.; Matherly, L. H. Structural Determinants of Folate and Antifolate Membrane Transport by the Reduced Folate Carrier. In Drug Metabolism and Transport; Lash, L. H., Ed.; Humana Press: Totowa, NJ, 2005; pp 291-318.
36. Liani, E.; Rothem, L.; Bunni, M. A.; Smith, C. A.; Jansen, G.; Assaraf, Y. G. Loss of Folylpoly-γ-glutamate Synthetase Activity is a Dominant Mechanism of Resistance to Polyglutamylation-dependent Novel Antifolates in Multiple Human Leukemia Sublines. Int. J. Cancer 2003, 103, 587-599.
37. Fry, D. W.; Jackson, R. C. Membrane Transport Alterations as a Mechanism of Resistance to Anticancer Agents. Cancer Surv. 1986, 5, 47-49.
38. Schornagel, J. H.; Pinard, M. F.; Westerhof, G. R.; Kathmann, I.; Molthoff, C. F. M.; Jolivet, J.; Jansen, G. Functional Aspects of Membrane Folate Receptors expressed in Human Breast Cancer Lines with Inherent and Acquired Transport-Related Resistance to Methotrexate. Proc. Am. Assoc. Cancer Res. 1994, 35, 302.
39. 10-propargyl-5,8-dideazafolate (DMPDDF) and Related Compounds. Proc. Am. Assoc. Cancer Res. 1989, 30, 476.
40. Jackman, A. L.; Gibson, W.; Brown, M.; Kimbell, R.; Boyle, F. T. The Role of the Reduced Folate Carrier and Metabolism to Intracellular Polyglutamates for the Activity of ICI D1694. Adv. Exp. Med. Biol. 1993, 339, 265-276.
41. Webber, S. E.; Bleckman, T. M.; Attard, J.; Deal, J. G.; Katherdekar, V.; Welsh, K. M.; Webber, S.; Janson, C. A.; Matthews, D. A.; Smith, W. W.; Freer, S. T.; Jordan, S. R.; Bacquet, R. J.; Howland, E. F.; Booth, C. J. L.; Ward, R. W.; Hermann, S. M.; White, J.; Morse, C. A.; Hilliard, J. A.; Bartlett, C. A. Design of Thymidylate Synthase Inhibitors Using Protein Crystal Structures: The Synthesis and Biological Evaluation of a Novel Class of 5-Substituted Quinazolines. J. Med. Chem. 1993, 36, 733-746.
42. Hughes, A.; Calvert, A. H. Preclinical and Clinical Studies with the Novel Thymidylate Synthase Inhibitor Nolatrexed Dihydrochloride (Thymitaq, AG337). In Antifolate Drugs in Cancer Therapy; Jackman, A. L., Ed.; Humana Press: Totowa, NJ, 1999; pp 229-241.
43. Gangjee A.; Mavandadi F.; Kisliuk R. L.; McGuire J. J.; Queener S. F. 2-Amino-4-oxo-5-substituted-pyrrolo[2,3-d]pyrimidines as Nonclassical Antifolate Inhibitors of Thymidylate Synthase. J. Med. Chem. 1996, 39, 4563-4568.
44. Gangjee, A.; Yang, J.; Ihnat, M. A.; Kamat, S. Antiangiogenic and antitumor agents. Design, synthesis, and evaluation of novel 2-amino-4-(3-bromoanilino)-6-benzyl substituted pyrrolo[2,3-d]pyrimidines as inhibitors of receptor tyrosine kinases. Bioorg. Med. Chem. 2003, 11, 5155-5170.
45. Kita, Y.; Takada, T.; Mihara, S.; Whelan, B. A.; Tohma, H. Novel and Direct Nucleophilic Sulfenylation and Thiocyanation of Phenol Ethers Using a Hypervalent Iodine(III) Reagent. J. Org. Chem. 1995, 60, 7144-7148.
46. Wahba, A. J.; Friedkin, M. The Enzymatic Synthesis of Thymidylate. Early Steps in the Purification of Thymidylate Synthetase of Escherichia coli. J. Biol. Chem. 1962, 237, 3794-3801.
47. Kisliuk, R. L.; Strumpf, D.; Gaumont, Y.; Leary, R. P.; Plante, L. Diastereoisomers of 5,10-Methylene-5,6,7,8-tetrahydropteroyl- D-glutamic Acid. J. Med. Chem. 1977, 20, 1531-1533.
48. Wong, S. C.; Zhang, L.; Witt, T. L.; Proefke, S. A.; Bhushan, A.; Matherly, L. H. Impaired Membrane Transport in Methotrexate-Resistant CCRF-CEM Cells Involves Early Translation Termination and Increased Turnover of a Mutant Reduced Folate Carrier. J. Biol. Chem. 1999, 274, 10388-10394
49. McGuire, J. J.; Magee, K. J.; Russell, C. A.; Canestrari, J. M. Thymidylate Synthase as a Target for Growth Inhibition in Methotrexate Sensitive and -Resistant Human Head and Neck Cancer and Human Leukemia Cell Lines. Oncol. Res. 1997, 9, 139-147.
50. Bertino, J. R. Rescue Techniques in Cancer Chemotherapy: Use of Leucovorin and Other Rescue Agents after Methotrexate Treatment. Semin. Oncol. 1977, 4, 203-216.
51. Hakala, M. T.; Taylor, E. The Ability of Purine and Thymine Derivatives and of Glycine to Support the Growth of Mammalian Cells in Culture. J. Biol. Chem. 1959, 234, 126-128.
52. Marsham, P. R.; Jackman, A. L.; Barker, A. J.; Boyle, F. T.; Pegg, S. J.; Wardleworth, J. M.; Kimbell, R.; O'Connor, B. M.; Calvert, A. H.; Hughes, L. R. Quinazoline Antifolate Thymidylate Synthase Inhibitors: Replacement of Glutamic Acid in the C2-Methyl Series. J. Med. Chem. 1995, 38, 994-1004.
53. Verdonk, M. L.; Cole, J. C.; Hartshorn, M. J.; Murray, C. W.; Taylor, R. D. Improved Protein-Ligand Docking Using GOLD. Proteins: Struct., Funct., Genet. 2003, 52, 609-623.
54. Jones, G.; Willett, P.; Glen, R. C.; Leach, A. R.; Taylor, R. Development and Validation of a Genetic Algorithm for Flexible Docking. J. Mol. Biol. 1997, 267, 727-748.
55. Jones, G.; Willett, P.; Glen, R. C. Molecular Recognition of a Receptor Sites Using a Genetic Algorithm with a Description of Desolvation. J. Mol. Biol. 1995, 245, 43-53.
56. Cody, V.; Luft, J. R.; Pangborn, W. Understanding the Role of Leu22 Variants in Methotrexate Resistance: Comparison of Wild-Type and Leu22Arg Variant Mouse and Human Dihydrofolate Reductase Ternary Crystal Complexes with Methotrexate and NADPH. Acta Crystallogr., Sect. D 2005, 61, 147-155.
57. Eldridge, M. D.; Murray, C. W.; Auton, T. R.; Paolini, G. V.; Mee, R. P. Empirical Scoring Functions: I. The Development of a Fast Empirical Scoring Function to Estimate the Binding Affinity of Ligands in Receptor Complexes. J. Comput.-Aided Mol. Des. 1997, 11, 425-445.
58. Blakley, R. L. The Biochemistry of Folic acid and Related Pleridines; Elsevier: Amsterdam, 1969; p 569.
59. Foley, G. F.; Lazarus, H.; Farber, S.; Uzman, B. G.; Boone, B. A.; McCarthy, R. E. Continuous Culture of Lymphoblasts from Peripheral Blood of a Child with Acute Leukemia. Cancer 1965, 18, 522-529.
60. Mini, E.; Srimatkandada, S.; Medina, W. D.; Moroson, B. A.; Carman, M. D.; Bertino, J. R. Molecular and Karyological Analysis of Methotrxate-Resistant and -Sensitive Human Leukemic CCRF-CEM Cells. Cancer Res. 1985, 45, 317-325.
61. Rosowsky, A.; Lazarus, H.; Yuan, G. C.; Beltz, W. R.; Mangini, L.; Abelson, H. T.; Modest, E. J.; Frei, E.; III Effects of Methotrexate Esters and Other Lipophilic Antifolates on Methotrexate-Resistant Human Leukemic Lymphoblasts. Biochem. Pharmacol. 1980, 29, 648-652.
62. Grindey, G. B.; Wang, M. C.; Kinahan, J. J. Thymidine Induced Perturbations in Ribonucleoside Triphosphate Pools in Human Leukemic CCRF-CEM Cells. Mol. Pharmacol. 1979, 16, 601-606
63. Gangjee, A.; Yu, J.; Kisliuk, R. L.; Haile, W. H.; Sobrero, G.; McGuire, J. J. Design, Synthesis, and Biological Activities of Classical N-{4-[2-(2-Amino-4-ethylpyrrolo[2,3-d]pyrimidin-5-yl)ethyl]benzoyl}-L-glutamic Acid and Its 6-Methyl Derivative as Potential Dual Inhibitors of Thymidylate Synthase and Dihydrofolate Reductase and as Potential Antitumor Agents. J. Med. Chem. 2003, 46, 591-600.
64. Sayre, P. H.; Finer-Moore, J. S.; Fritz, T. A.; Biermann, D.; Gates, S. B.; MacKellar, W. C.; Patel, V. F.; Stroud, R. M. Multi-targeted Antifolates Aimed at Avoiding Drug Resistance Form Covalent Closed Inhibitory Complexes with Human and Escherichia coli Thymidylate Synthases. J. Mol. Biol. 2001, 313, 813-829.