NEW CYTOTOXIC TRIS(OXAZOLE)-CONTAINING MACROLIDES

Abstract: HA-1 and TH-2, two novel cytotoxic tris(oxazole)-containing macrolides, have been shown. These compounds have been obtained by chemical modification of marine natural products. The structure of these compounds has been established on the basis of NMR data.

Description: NEW CYTOTOXIC TRIS(OXAZOLE)-CONTAINING MACROLIDES The present invention relates to new cytotoxic tris(oxazole)-containing macrolides obtained by chemical modification of marine natural products. Background of the Invention Marine organisms, especially soft corals, sponges and tunicates, provide many secondary metabolites and exhibit a varying degree of biological activity (Reference 1). An important family of these metabolites is the cytotoxic tris(oxazole)-containing macrolide family; in 1986 it was reported the structure of the three first tris(ox--zole)-containing macrolides: Ulapualide A and B (Reference 2) and Kabiramide C (Reference 3). OHC-^ OH OMe Since then, over 30 additional tris(oxazole)-containing macrolides have been published (Reference 4). Summary of the Invention The present invention relates to new cytotoxic tris(oxazole)-containing macrolides having the formula (I): OHC R όa R όb R 8 (I) wherein X represents a lower alkyl; R 1 and R 2 represent hydrogen or lower alkyl; R 3 represents hydrogen, and R represents or OMe or R 3a and R 3b together represent oxygen; R 4 represents hydrogen, lower alkoxy or lower alkyl; R 5 represents hydrogen, carbamoyl, or lower alkanoyl; R 6a represents hydrogen, and R 6b represents hydroxy, or R 6a and R 6 together represent oxygen; R 7 represents hydrogen, lower alkyl, or hydroxymethyl; R represents lower alkoxy or lower alkyl; Y represents hydrogen or methyl, and y represents hydrogen, or Y and Y together represent a double bond. In the definitions of the groups in formula (I), the lower alkyl and the lower alkyl moiety of the lower alkanoyl or of the lower alkoxy mean a straight-chain or branched alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, pentyl, neopentyl and hexyl. More particularly, the present invention relates to HA-1 and TH-2, with structural formulas: OHC OMe HA-1 OHC OMe TH-2 HA-1 and TH-2 were obtained by chemical modification of the known Kabiramide B and C respectively. HA-1 and TH-2 exhibit antitumor activity. In particular, HA-1 and TH-2 exhibit antitumor activity against cell lines derived from human solid tumors, such as human lung carcinoma, human colon carcinoma and human melanoma, and, the like, they are active against other tumor cell lines, like leukemia and lymphoma. These compounds, HA-1 and TH-2, have in vitro antitumor selectivity for solid tumors. The present invention also relates to a method for treating a mammal affected by a malignant tumor sensitive to a compound with formula (I), which comprises administering to the affected individual a therapeutically effective amount of the compound with formula (I) or a pharmaceutical composition thereof. The present invention further provides pharmaceutical compositions which contain as active ingredient a compound with formula (I), as well as a process for its preparation. A further aspect of the invention is a method for preparing the compounds HA- land TH-2, which comprises chemical modification of Kabiramide B and C respectively. Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable formulation of oral, topical or parenteral administration, and they may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when administered parenterally. The correct dosage of a pharmaceutical composition comprising compounds with formula (I), will vary according to the pharmaceutical formulation, the mode of application, and the particular situs, host and tumor being treated. Other factors like age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, drug combinations, reaction sensitivities and severity of the disease shall be taken into account. Administration can be carried out continuously or periodically within the maximum tolerated dose. Antitu our Activity Cells were maintained in logarithmic phase of growth in Eagle's Minimum Essential Medium, with Earle's Balanced Salts, with 2.0 mM L-glutamine, with non-essential amino acids, without sodium bicarbonate (EMEM/neaa); supplemented with 10% Fetal Calf Serum (FCS), 10 "2 M sodium bicarbonate and 0,1 g/1 penicillin-G + streptomycin sulfate. A screening procedure has been carried out to determine and compare the antitumor activity of these compounds, using an adapted form of the method described by Bergeron et al. (Reference 5). The antitumor cells employed were P-388 (suspension culture of a lymphoid neoplasm from DBA/2 mouse), A-549 (monolayer culture of a human lung carcinoma), HT-29 (monolayer culture of a human colon carcinoma) and MEL-28 (monolayer culture of a human melanoma). P-388 cells were seeded into 16 mm wells at lxl 0 4 cells per well in 1 ml aliquots of MEM 5 FCS containing the indicated concentration of drug. A separate set of cultures without drug was seeded as control growth to ensure that cells remained in exponential phase of growth. All determinations were carried out in duplicate. After three days of incubation at 37°C, 10% CO 2 in a 98% humid atmosphere, an approximately IC 50 was determined by comparing the growth in wells with drug to the growth in wells control. A-549, HT-29 and MEL-28 cells were seeded into 16 mm Wells at 2xl0 4 cells per well in 1 ml aliquots of MEM 1 OFCS containing the indicated concentration of drug. A separate set of cultures without drug was seeded as control growth to ensure that cells remained in exponential phase of growth. All determinations were carried out in duplicate. After three days of incubation at 37°C, 10% CO in a 98% humid atmosphere, the wells were stained with 0.1% Crystal Violet. An approximately IC 50 was determined by comparing the growth in wells with drug to the growth in wells control. The results of the in vitro cytotoxic assays for HA-1 and TΗ-2 with the celular lines P- 388, A-549, HT-29 and MEL-28 are given in the following table: IC 50 (uM) Compound P-388 A-549 HT-29 MEL-28 HA-1 0.010 0.001 0.001 0.001 TH-2 0.011 0.002 0.002 0.002 It is showed that these compounds, HA-1 and TH-2, with a terminal aldehyde methyl group have in vitro antitumor selectivity for solid timors like A-549, HT-29,and MEL- 28. Isolation of the known Kabiramide B and C A sample of an unidentified black sponge (wet, 13.2 kg) was collected at Sichang Island, May 1997. Its CH 2 C1 2 portion (67 g) was separated first by silica vacuum flash chromatography (VFC, stepwise elution), then by Sephadex (CH 2 Cl 2 -MeOH, 1- 1), and finally by reversed phase HPLC (MeOH-H 2 O-EtOAc, 3-2-1) to give both Kabiramide B (Reference 6) (24.0 mg) and Kabiramide C (Reference 3) (35.0 mg). Synthesis of HA-1 Compound HA-1 was preparated by treating Kabiramide B (24.0 mg) in 10 mL of aqueous acidic dioxane (0.5 N HCl-dioxane, 2-3) for 2 hr at 50°C. The crude product was separated on reversed phase HPLC to give 6.4 mg (28 ) of compound HA-1 as a glass, and 7.1 mg (30 %) of recovered Kabiramide B. The NMR data for HA-1 are: All chemical shifts are reported with respect to TMS (δ=0 ppm). Η NMR (CDClj): δ 9.75 (IH, t, J = 2.5 Hz), 8.10 (1H, s), 8.04 (1H, s), 7.59 (1H, s), 7.29 (IH, m), 6.33 (IH, d, J = 16.0 Hz), 5.18 (IH, dt, J = 2.0, 10.0 Hz), 5.02 (IH, dd, J = 8.0, 12.0 Hz), 4.85 (IH, s), 4.15 (IH, m), 3.84 (IH, m), 3.45 (3H, s), 3.37 (3H, s), 3.30 (3H, s), 3.27 (IH, dd, J = 4.0, 7.5 Hz), 3.07 (IH, m), 2.73 (IH, m), 2.62 (IH, m), 2.57 (IH, m), 2.53 (2H, m), 2.45 (IH, m), 2.43 (2H, m), 2.36 (IH, dd, J = 2.0, 8.0 Hz), 2.25 (IH, m), 2.09 (IH, m), 1.85 (3H, m), 1.75 (IH, m), 1.72 (3H, m), 1.46 (2H, m), 1.35 (IH, m), 1.05 (3H, d, J = 7.0 Hz), 1.02 (3H, d, J = 7.0 Hz), 1.01 (3H, d, J = 7.0 Hz), 0.97 (3H, d, J = 7.0 Hz), 0.93 (3H, d, J = 7.0 Hz), 0.83 (3H, d, J = 7.0 Hz). 13 C NMR (CDC1 3 ): δ 213.3 s, 201.8 d, 172.0 s, 163.9 s, 157.7 s, 156.5 s, 155.4 s, 145.5 d, 141.7 s, 137.2 d, 136.9 d, 135.7 d, 131.1 s, 129.8 s, 115.1 d, 87.5 d, 82.2 d, 78.2 d, 74.4 d, 73.5 d, 70.4 d, 68.2 d, 60.6 q, 57.9 q, 57.6 q, 48.7 t, 46.1 t, 45.3 t, 43.4 t, 42.8 t, 42.7 d, 41.7 t, 37.8 t, 37.3 d, 34.7 d, 34.6 t, 31.0 d, 25.3 t, 25.1 d, 18.3 q (2C), 15.6 q, 13.4 q, 10.6 q, 9.2 q. IR (CHCI 3 ) 3470, 3015, 1720, 1650, 1460, 1315, 1215, 915, 665 cm "1 . Synthesis of TH-2 A mixture of Kabiramide C (35.0 mg) in 10 mL of 0.5 N HCl-dioxane (2-3) was stirred at 50°C for 2 h. The mixture was partitioned between EtOAc and water, and the organic layer was concentrated The crude product was separated on reversed phase HPLC (MeCN-H 2 0-EtOAc, 6-4-1) to give 11.0 mg (33 %) of compound TH-2 as a glass, and 18.3 mg (52 %) of recovered Kabiramide C. The NMR data for TH-2 are: All chemical shifts are reported with respect to TMS (δ=0 ppm). 1H NMR (CDCI 3 ) δ 9.75 (IH, t, J = 2.3 Hz), 8.09 (IH, s), 8.03 (IH, s), 7.58 (IH, d, J = 1.0 Hz), 7.46 (IH, ddd, J = 16.0, 9.5, 5.0 Hz), 6.29 (IH, d, J = 16.0 Hz), 5.32 (IH, m), 5.17 (IH, t, J = 10.5 Hz), 4.80 (IH, s), 3.84 (IH, m), 3.67 (IH, m), 3.45 (3H, s), 3.43 (3H, s), 3.33 (3H, s), 3.30 (3H, s), 3.27 (IH, m), 3.01 (IH, m), 2.81 (IH, m), 2.72 (IH, t, J = 8.0 Hz), 2.59 (IH, dd. J = 14.3, 9.7 Hz), 2.52 (IH, m), 2.44 (IH, m), 2.41 (IH, m), 2.36 (IH, m), 2.32 (IH, m), 2.15 (IH, m), 1.85 (1H, m), 1.46 (IH, m), 1.34 (IH, m), 1.04 (3H, d, J = 6.1 Hz), 1.01 (3H, d, J = 7.0 Hz), 0.99 (3H, d, J = 7.0 Hz), 0.94 (3H, d, J = 7.0 Hz), 0.87 (3H, d, J = 6.4 Hz), 0.83 (3H, d, J = 7.0 Hz). 13 C NMR (CDC1 3 ) δ 213.4 s, 201.8 d, 171.6 s, 163.2 s, 157.3 s, 156.4 s, 155.4 s, 142.0 d, 141.4 s, 137.1 d, 136.8 d, 135.6 d, 131.1 s, 129.9 s, 115.5 d, 87.4 d, 82.0 d, 79.2 d, 78.3 d, 74.0 d, 73.3 d, 69.3 d, 60.6 q, 57.9 q, 57.6 q, 57.5 q, 48.6 t, 46.0 t, 45.0 t, 43.6 t, 42.9 t, 41,7 t, 40.4 d, 37.3 d, 34.5 d, 33.9 t, 32.9 t, 30.9 d, 25.0 d (2C), 18.2 q (2C), 15.5 q, 13.3 q, 10.7 q, 8.4 q. 1 0 References 1. Faulkner, D. Nat.Prod.Rep. 1997, 14, 259-302 and references therein. 2. Scheuer, P.J. et al. J.Am.Chem.Soc. 1986, 108, 846-847. 3. Fusetani, N. et al. J.Am.Chem.Soc. 1986, 70S, 847-849. 4. Marine Biotechnology, Vol.l, Ed. by David H. Attaway and Oskar R. Zaborsky, Plenum Press, New York 1993, pp. 211-213, and references therein. 5. Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline, Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter. Antineoplastic and antiherpetic activity of spermidine catecholamide iron chelators. Biochem. Bioph. Res. Comm. 1984, 121, 848-854. 6. Fusetani, N. et al. J. Org. Chem. 1989, 54, 1360-1363. 1 1

Claims: CLAIMS: 1. A compound having formula (I): R όa R 6b R 8 (I) wherein X represents a lower alkyl; R 1 and R represent hydrogen or lower alkyl; R 3a represents hydrogen, and R 3b , represents o ~0 ' OMe OMe or o OMe or R and R together represent oxygen; R represents hydrogen, lower alkoxy or lower alkyl; R represents hydrogen, carbamoyl, or lower alkanoyl; R 6a represents hydrogen, and 1 2 R 6b represents hydroxy, or R 6a and R 6b together represent oxygen; R represents hydrogen, lower alkyl, or hydroxymethyl; R represents lower alkoxy or lower alkyl; Y represents hydrogen or methyl, and y represents hydrogen, or Y and Y together represent a double bond. In the definitions of the groups in formula (I), the lower alkyl and the lower alkyl moiety of the lower alkanoyl or of the lower alkoxy mean a straight-chain or branched alkyl group having 1 to 6 carbon atoms such as methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec- butyl, tert-butyl, pentyl, neopentyl and hexyl. 2. A compound, HA-1 , according to claim 1, having the following formula: OHC OMe HA-1 1 3 3. A compound, TH-2, according to claim 1, having the following formula: OHC OMe TH-2 4. A method of treating a mammal affected by a malignant tumor sensitive to a compound with formula (I), as defined in claim 1 , which comprises administering to the affected individual a therapeutically effective amount of the compound or a pharmaceutical composition thereof. 5. A method of treating a mammal affected by a malignant tumor sensitive to HA- 1, as defined in claim 2, which comprises administering to the affected individual a therapeutically effective amount of HA-1 or a pharmaceutical composition thereof. 6. A method of treating a mammal affected by a malignant tumor sensitive to TH-2, as defined in claim 3, which comprises administering to the affected individual a therapeutically effective amount of TH-2 or a pharmaceutical composition thereof. 7. A pharmaceutical preparation which contains as active ingredient a compound with formula (I), as defined in claim 1. m 8. A pharmaceutical preparation which contains as active ingredient HA-1 (as defined in claim 2), or TH-2 (as defined in claim 3). 9. A method of treating a mammal affected by a malignant tumor which comprises administering to the affected individual a therapeutically effective amount of a compound with formula (I), as defined in claim 1, together with other or others antitumoral compounds. 10. A method for preparing HA-1 (as defined in claim 2) or TH-2 (as defined in claim 3), which consists of chemical modification of the known Kabiramide B an C respectively. 1 5

Inventors: FERNANDEZ-PUENTES JOSE LUIS, GARCIA GRAVALOS DOLORES, HIGA TATSUO, FERNANDEZ-PUENTES, JOSE LUIS, GARCIA GRAVALOS, DOLORES, HIGA, TATSUO
Applicants: FERNANDEZ PUENTES JOSE LUIS, GARCIA GRAVALOS DOLORES, HIGA TATSUO, INST BIOMAR SA, RUFFLES GRAHAM KEITH, FERNANDEZ-PUENTES, JOSE LUIS, GARCIA GRAVALOS, DOLORES, HIGA, TATSUO, INSTITUTO BIOMAR S.A., RUFFLES, GRAHAM, KEITH
Application year: 1999
Main classification: C07D498
IPC codes: A61K31, A61P35, C07D498