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A series of TAN-1511 analogues bearing a non-peptide spacer in place of the Gly-Gly-Gly sequence in the peptide moiety was synthesized, and the effects of these compounds on the proliferation of bone marrow cells in culture and experimental leukocytopenia in mice were examined. The structure-activity relationships obtained were as follows. As the substituent at the 2-position of the 4-thiaheptanoic acid framework, an amino group, methyl group or hydrogen was preferable; as a spacer in place of the Gly-Gly-Gly sequence, a 4-aminobenzoyl or 4-aminomethylbenzoyl group was suitable; and as the fatty acids bonded to the 6,7-dihydroxy groups, C 16 fatty acid was best. Compounds 12f, 30d and 30i potently promoted the proliferation of bone marrow cells in culture and the restoration of leukocyte counts in a murine leukocytopenia model.

The antitumor activity of recombinant human interleukin 2 (rIL-2) in combination with 5'-deoxy-5-fluorouridine (doxifluridine; 5'-DFUR) against marine colon carcinoma 26 (Colon 26) was studied. BALB/c mice were treated daily for 15 days with 5'-DFUR, rIL-2 or both, beginning on day 7 after subcutaneous transplantation of Colon 26. While mice treated with 5'-DFUR or rIL-2 alone died of tumor growth with pulmonary metastases within 9 weeks posttransplantation, the survival time was significantly prolonged in mice treated with both 5'-DFUR and rIL-2. Most of the combination-treated animals showed the regression of local tumors and the inhibition of pulmonary metastasis. Histopathologically, many tumor cells were degenerated and necrotized, with marked infiltration of mononuclear cells including large granular lymphocytes (LGLs) with periodic acid-Schiff-positive cytoplasmic granules. The cells were positive for CD3 epsilon, asialo GM1 and NK1.1. Spleen cells from the combination-treated mice showed high activities of natural killer (NK) cytotoxicity as well as growth inhibition of Colon 26 and Meth A fibrosarcoma in mice. The results suggest that the combination therapy of 5'-DFUR plus rIL-2 enhanced non-specific cytotoxicity of LGL/NK cells for Colon 26 in tumor-bearing mice and was effective in the inhibition of tumor growth.

The effects of lansoprazole given intravenously on indomethacin-induced gastric bleeding and mucosal lesions were investigated in rats in comparison with those of omeprazole, famotidine and ranitidine. Lansoprazole inhibited gastric bleeding induced by indomethacin with an ID50 value of 0.29 mg/kg. Omeprazole and famotidine significantly inhibited gastric bleeding, but ranitidine provided negligible inhibition. A correlation was found between the inhibitory action of lansoprazole on gastric bleeding, and acid secretion, and its inhibitory action on gastric bleeding was almost completely abolished by adding 50 mM-HCl to the gastric perfusate, suggesting that lansoprazole's inhibitory action on gastric bleeding was mainly due to its antisecretory action. Lansoprazole inhibited the development of gastric lesions induced by indomethacin with an ID50 value of 0.10 mg/kg, whereas histamine H2-receptor antagonists did not display a potent inhibitory effect. ID50 values for omeprazole, famotidine and ranitidine were 0.69, 2.58 and 24.6 mg/kg, respectively. These results indicate that lansoprazole has a potent inhibitory action on indomethacin-induced gastric bleeding and mucosal lesions and that it is useful in the treatment of acute gastric mucosal lesions.

Either the alpha- or gamma-carboxyl group of the glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5- yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and its related compound (1a) was replaced with a 1H-tetrazole ring, and the inhibitory effects of the resulting compounds on dihydrofolate reductase (DHFR) and the growth of murine fibrosarcoma Meth A cells were examined. The gamma-tetrazole analogs (2) were found to be much more potent DHFR inhibitors than TNP-351, and strongly inhibited the growth of Meth A cells. On the other hand, the alpha-tetrazole analogs (3) were much less active against Meth A cells, even though their DHFR-inhibitory activity was comparable to that of TNP-351. These findings suggest that the alpha-carboxyl group plays an important role in effective uptake via the reduced folate carrier, and a novel DHFR inhibitor could be obtained by chemically modifying the gamma-carboxyl moiety while leaving the alpha-carboxyl group intact.

TAN-1511 analogues were synthesized and their effects on the proliferation of bone marrow cells were examined. To exert potent activity the following conditions are necessary: the configuration of the 2-amino-6, 7-dihydroxy-4-thiaheptanoic acid moiety must be (2R, 6R), long chain acyl groups (C14 to C18) must be bound to both hydroxyl groups, the amino group must be free or acylated with the long chain fatty acid (ca. C14) and the pep tide moiety must have glutamic acid as a component. Among the synthesized compounds, trisodium (2R, 6R)-2-amino-6, 7-bis (hexadecanoyloxy)-4-thiaheptanoyl glycyl glutamyl glutamate, which has improved solubility, was effective in experimental leukocytopenia in mice.

Novel pyrrolo[2, 3-d]pyrimidine antifolates (1a, b and 2a, b) with a nitrogen atom in the bridge chain between the 2, 4-diaminopyrrolo[2, 3-d]pyrimidine and phenylene rings were designed and efficiently synthesized. These compounds exhibited more potent inhibitory activities than methotrexate (MTX) against the proliferation of human epidermoid carcinoma KB cells and human non-small cell lung carcinoma A549 cells despite their modest dihydrofolate reductase (DHFR)-inhibitory potency.

Novel pyrrolo[2, 3-d]pyrimidine antifolates (1a, b and 2a, b) with a nitrogen atom in the bridge chain between the 2, 4-diaminopyrrolo[2, 3-d]pyrimidine and phenylene rings were designed and efficiently synthesized. These compounds exhibited more potent inhibitory activities than methotrexate (MTX) against the proliferation of human epidermoid carcinoma KB cells and human non-small cell lung carcinoma A549 cells despite their modest dihydrofolate reductase (DHFR)-inhibitory potency.

A series of TAN-1511 analogues bearing a non-peptide spacer in place of the Gly-Gly-Gly sequence in the peptide moiety was synthesized, and the effects of these compounds on the proliferation of bone marrow cells in culture and experimental leukocytopenia in mice were examined. The structure-activity relationships obtained were as follows. As the substituent at the 2-position of the 4-thiaheptanoic acid framework, an amino group, methyl group or hydrogen was preferable; as a spacer in place of the Gly-Gly-Gly sequence, a 4-aminobenzoyl or 4-aminomethylbenzoyl group was suitable; and as the fatty acids bonded to the 6,7-dihydroxy groups, C 16 fatty acid was best. Compounds 12f, 30d and 30i potently promoted the proliferation of bone marrow cells in culture and the restoration of leukocyte counts in a murine leukocytopenia model.

A series of novel pyrrolo[2,3-d]pyrimidine derivatives was designed and synthesized as thymidylate synthase (TS) inhibitors. Molecular design was performed on the human TS complex model built on the basis of the reported structure of TS-deoxyuridinemonophosphate (dUMP)-CB3717 ternary complex. From a docking study, we expected that a one-carbon bridge between pyrrolo[2,3-d]pyrimidine and an aromatic ring was suitable. Moreover, we found that the bridge carbon could be replaced with an alkyl group to fill out the unoccupied space. Based on this design, we synthesized five pyrrolo[2,3-d]pyrimidine derivatives with one-carbon bridge and evaluated their TS inhibitory activities. All synthesized compounds inhibited TS more potently than compound 2 (LY231514), and the C8-ethyl analogue (7) showed a remarkable inhibitory activity against TS (IC50=0.017 μM).

A series of novel pyrrolo[2,3-d]pyrimidine derivatives was designed and synthesized as thymidylate synthase (TS) inhibitors. Molecular design was performed on the human TS complex model built on the basis of the reported structure of TS-deoxyuridinemonophosphate (dUMP)-CB3717 ternary complex. From a docking study, we expected that a one-carbon bridge between pyrrolo[2,3-d]pyrimidine and an aromatic ring was suitable. Moreover, we found that the bridge carbon could be replaced with an alkyl group to fill out the unoccupied space. Based on this design, we synthesized five pyrrolo[2,3-d]pyrimidine derivatives with one-carbon bridge and evaluated their TS inhibitory activities. All synthesized compounds inhibited TS more potently than compound 2 (LY231514), and the C8-ethyl analogue (7) showed a remarkable inhibitory activity against TS (IC50=0.017 microM).

The glutamic acid moiety of N-[4-[3-(2,4-diamino-7H-pyrrolo[2,3-d]pyrimidin-5-yl)propyl]benzoyl]-L-g lutamic acid (1b, TNP-351) and the related compound (1a), was replaced with various N(omega)-acyl-, sulfonyl-, carbamoyl- and aryl-2,omega-diaminoalkanoic acids, and the inhibitory effects of the resulting products (9, 11, 14, 18, 21, 23, 25, 30, 36) on dihydrofolate reductase (DHFR), the growth of murine fibrosarcoma Meth A cells, and methotrexate-resistant human CCRF-CEM cells, were examined. Compounds (9a-f) acylated with a hemiphthaloyl group were efficiently synthesized by coupling pyrrolo[2,3-d]pyrimidine carboxylic acids (7a,b) and N(omega)-phthaloyl 2,omega-diaminoalkanoic acid methyl esters (6a-c) and subsequent hydrolysis. The other N(omega)-acyl- and sulfonyl-ornithine analogs (21, 23, 25) were synthesized by acylation of free amino intermediates (19a,b) derived from tert-butoxycarbonyl-ornithine analogs (17a,b). A free ornithine analog (18) did not strongly inhibit Meth A cell growth, whereas all N(omega)-acyl-, sulfonyl-, carbamoyl- and aryl-ornithine analogs (9, 11, 21, 23, 25, 30, 36) exhibited much more potent inhibitory activities against both DHFR and Meth A cell growth. In particular, compounds 9c, 21k and 36a also showed remarkable growth-inhibitory activities against methotrexate-resistant CCRF-CEM cells. These results demonstrate that the potent inhibitory activities of N(omega)-masked ornithine analogs against the growth of Meth A cells and methotrexate-resistant CCRF-CEM cells, results from effective uptake via reduced folate carrier and their potent DHFR inhibition.

The glutamic acid moiety of N-[4-[3-(2, 4-diamino-7H-pyrrolo[2, 3-d]pyrimidin-5-yl)propyl]benzoyl]-L-glutamic acid (1b, TNP-351) and related compounds was replaced with some N5-substituted glutamines. Antifolates (4A-S) were effectively prepared by coupling pyrrolo[2, 3-d]pyrimidine carboxylic acids (11a, b) with some properly protected N5-substituted glutamine derivatives (10A-S), which were prepared by coupling Boc-Glu-OMe (7) with various amines (8A-S) using a suitable condensing reagent, followed by hydrolysis. The inhibitory effects of the resulting products on dihydrofolate reductase (DHFR), thymidylate synthetase (TS) and the growth of murine fibrosarcoma Meth A cells in culture were examined. All N5-substituted glutamine analogs (4A-S) inhibited DHFR much more strongly than TNP-351 and some analogs exhibited the same potent growth inhibition of Meth A cells as TNP-351. Some typical analogs (4Bb, 4Db, 4F, 4Oa) were also examined for inhibitory effects on the growth of methotrexate (MTX)-resistant human CCRF-CEM cells in culture and for in vivo antitumor activities against murine leukemia and solid tumors. MTX-resistant cells, with a defect in transport and decreased polyglutamylation activity, showed little cross resistance to the analog (4Oa) having a tetrazole moiety as a substituent of glutamine, which exhibited potent antitumor activities. These results demonstrate that the antifolate analogs (4) with N5-substituted glutamine in place of glutamic acid are novel potent DHFR inhibitors with activity against MTX-resistant tumors. The potent antitumor activity of these analogs (4) may result from their effective uptake via reduced folate carrier in combination with their potent inhibition of DHFR.

Electrophoretic isolation of a membrane-bound NADPH oxidase of guinea-pig polymorphonuclear leukocytes was attempted with the O2−-generating membranes of cells unstimulated or stimulated with C3b-zymosan or sodium dodecyl sulfate, and also with the phagosomes isolated from the phorbol myristate acetate-coated latex particle-phagocytosing cells. When these vesicles were subjected to discontinuous polyacrylamide gel electrophoresis in the presence of Triton X-100 and then assayed for NADPH-Nitroblue tetrazolium reducing activity, the activity was detected by the appearance of a single, blue band of the reduced dye on the gel, independent of the source of vesicles. In addition, the enzyme was able to generate O2− and its activity was significantly augmented with the homologous liver microsomal cytochrome b5. Its activity was heat-labile and inactivated by N-ethylmaleimide and p-chloromercuribenzene sulfonate. The enzyme, with an apparent molecular weight of 150 000, in the phagosomes was easily susceptible to limited proteolysis by trypsin and formed an active fragment with a molecular weight of 70000, accompanying the loss of O2−-generating activity of the vesicles.