MXPA99009503A - Nucleophile substituted ecteinascidins and n-oxide ecteinascidins - Google Patents

Abstract

Five new nucleophile substituted ecteinascidin (Et) compounds have been isolated from extracts of Ecteinascidia turbinata. These compounds have been purified by chromatographic techniques and their structures and bioactivities have been determined. The five nucleophile substituted Et compounds have been designated herein as Et 802 (1), Et 788 (2), Et 760 (3), Et 858 (4) and Et (815) (5). Also obtained were three new N-oxide ecteinascidin compounds, which have been designated herein as Et 717 (6), Et 775 (7) and Et 789 (8). Some of these newly discovered Et compounds show exceedingly potent cytotoxicity against L1210.

Description

ECTEINASCI DI NAS AND N-OXI DO ECTEINASCI DIAS SUBSTITUTIONS WITH NUCLEOFI LOS BACKGROUND OF THE INVENTION Ecteinascidins (Ets), excessively potent antitumor agents, isolated first from the marine tunicate Ecteinascidia turbinata, especially Et 743, Et 729, Et 746 and Et 722 show significant efficacy in vivo against tumor cell lines including leukemia of murine P388, melanoma B16, Lewis lung carcinoma, and human tumor xenograft models in mice. Continuous studies are conducted by Rinehart et al in a variety of ways to provide adequate amounts of these compounds for clinical trials, study of the mechanism of antitumor action, and the determination of structure-activity relationships. In addition, the discovery of additional Et compounds, either minor natural components or precursor compounds, will not only provide evidence of their biosynthetic pathway, but would also be useful with respect to determining structure-activity relationships.

BRIEF DESCRIPTION OF THE INVENTION The present invention is directed to several newly discovered ecteinascidin (Et) compounds, all isolated from extracts of Ecteinascidia turbinata. For a detailed discussion of the previously discovered ecteinascidin compounds, as well as the methods used for their isolation and purification, see Sakai et al. , J.

Amer. Chem. Soc, 1 9-96, 1 18, 9017, the description of which is incorporated herein by reference. The structures of the new ectainascidin compounds reported herein are as follows: 1: ETT02, R1 = Ac, R2 = Me 2: ET760, R1 = H, R2 = Me 3: ET788, R1aAcf R2 = H 4: ET85B, R1 = Ac, R2 = e : ET815, R1 = Ac, R2 = Me 6: ET717, R1 = H, R2 = Me 7: ET776, R1 = Ac, R2 = H 8: ET789, R1 = Ac, R2 = Me (fragment ET757) 9: ET832, R1 = Ac, R2 = Me (fragment ET 813) BRIEF DESCRIPTION OF THE DRAWINGS Figure 1 schematically illustrates the chromatographic processes used to isolate Et 717, Et 81 5, Et 813, Et 729 and Et 731 from extracts of Ecteinascidia turbinata. Figure 2 illustrates schematically the chromatographic processes for isolating Et 729, Et 743, Et 788, Et 757, Et 789, Et 775, Et 745, Et 760, Et 802, Et 858 and Et 745 from extracts of Ecteinascidia turbinata. Figure 3 schematically illustrates the chromatographic processes used to isolate Et 771, Et 759A, Et 743 and Et 729 from extracts of Ecteinascidia turbinata. Figure 4 illustrates the MS / MS fragmentation of Et 802. Figure 5 illustrates the MS / MS fragmentation of Et 760. Figure 6 illustrates the MS / MS fragmentation of Et 788. Figure 7A illustrates the MS / MS fragmentation of Et. 858 and Figure 7B illustrates the MS / MS fragment fragment ion (m / z 800) thereof.

Figure 8 illustrates the MS / MS fragmentation of Et 717. Figure 9A illustrates the MS / MS fragmentation of Et 789 (8, R = CH3) and Et 775 (7, R = H) and Figure 9B shows the MS / MS fragmentation of the reaction product of Et 789 treated with oxalic acid.

DETAILED DESCRIPTION OF THE INVENTION The five new ecteinascidin compounds substituted with nucleophiles designated herein as Et 802 (1), Et 788 (2), Et 760 (3), Et 858 (4), Et 815 (5) and the three new N-oxide ecteinascidins, designated herein as Et 717 (6), Et 775 (7) and Et 789 (8) were isolated and purified from extracts of Ecteinascidia turbinata by RP, NP and CCC column chromatography and RP-C18 HPLC as described in Figures 1-3. The structures of the new Et compounds were assigned based on mass spectral data (HRFABMS, fragmentation MS / MS) and detailed data analysis of 1 D and 2D-NMR spectra. The Figures 4-9 illustrate MS / MS fragmentation for Et 802, Et 760, Et 788, Et 858, Et 717 and Et 789, respectively. The spectral data for some of the new ecteinascidin compounds include the following: Et 802 (1): HRFABMS: m / z 803.2962, M + H ion, C14H47N4 OnS,? = 3. 1 mDa; 1 H NMR, d 4.15 (d, 1, H-1), 3.45 (br.d, H-3), 4.50 (br, H-4), 4.19 (dd, 1, 2, H1 1), 3.09 (d, 12, H13), 2.98 (d, 1 5, H14a), 2.81 (dd, 12, 15, H 14b), 6.46 (s, H 1 5), 5.08 (dd, 2, 8, H21), 5.24 (d, 1 1, H22a), 4.01 (dd, 1, 1 1, H22b), 3.18 (ddd, H3'a), 2.78 (ddd, H3'b), 2.58 (ddd, H4'a), 2.30 (ddd, H4'b), 6.46 (s, H5 '), 6.36 (s, H8'), 2.24 ( d, 12, H 12'a), 1.93 (d, 12, H12'b), 6.09 (s, -OCH2O-), 6.03 (s, -OCH2O-), 3.52 (s, 7OMe), 3.66 (s, 17OMe), 2.25 (s, AcMe), 2.07 (s, NMe), 2.24 (s, 16Me), 1.96 (s, 6Me), 2.03 (s, NHCOMe). Et 788 (2): HRFABMS: m / z 789.2806, M + H ion, C ^ H ^ N ^ nS,? = 1. 0 mDa; 1H NMR, d 4.21 (d, 1, H-1), 3.45 (br.d, H-3), 4.50 (br, H-4), 4.24 (dd, 1.2, H11), 3.09 (d, 12, H13), 6.48 (a, H15), 5.11 (dd, 2, 8, H21), 5.30 (d, 11, H22a), 4.01 (dd, 1, 11, H22b), 6.48 (s, H5 ') , 6.39 (s, H8 '), 6.13 (s, -OCH2O-), 6.07 (s, -OCH2O-), 3.56 (s, 7'OMe), 3.67 (s, 17OMe), 2.25 (s, AcMe), 2.24 (s, 16Me), 1.96 (s, 6Me), 2.03 (s, NHCOMe). Et 760 (3). HRFABMS: m / z 761.2856, M + H ion, C39H45N4O10S,? = 0.2 mDa; 1H NMR, d 4.15 (d, 1, H-1), 3.56 (br.d, H-3), 4.50 (br, H-4), 4.32 (dd, 1.2, H11), 3.09 (d, 12, H13), 3.00 (d, 15, H14a), 2.86 (dd, 12, 15, H 14b), 6.52 (s, H15), 5.11 (dd, 2, 8, H21), 5.20 (d, 11, H22a), 4.01 (dd, 1, 11, H22b), 3.18 (ddd, H3'a), 2.78 (ddd, H3'b), 2.58 (ddd, H4'a), 2.30 (ddd, H4'b), 6.38 (s, H5 '), 6.34 (S, H8'), 2.24 (d, 12, H12'a), 2.03 (d, 12, H12'b), 5.98 (s, -OCH2O-), 5.85 (s) , -OCH2O-), 3.54 (s, 7'OMe), 3.74 (s, 17OMe), 2.13 (s, NMe), 2.29 (s, 16Me), 2.06 (s, 6Me), 2.12 (s, NHCOMe). Et 858 (4): HRFABMS- m / z 859.3192, M + H ion, C44H51N4O12S,? = 3.2 mDa; ion fragment m / z 800: m / z 800.2825, M + H ion,? = 2.8 mDa; Et 858, 1 H NMR, d 4.13 (br.s, H-1), 3.41 (br.d, H-3), 4.50 (br, H-4), 4.36 (d, 3, H11), 2.79 (d , 13, H13), 3.01 (d, 12, H14a), 2.88 (dd, 12, 13, H14b), 6.50 (s, H15), 5.10 (d, 2, H21), 5.28 (d, 11, H22a) , 4.09 (dd, 1.5, 11, H22b), 3.18 (ddd, H3'a), 2.62 (ddd, H3'b), 2.53 (ddd, H4'a), 2.45 (ddd, H4'b), 6.43 ( 5, H5 '), 6.38 (s, H8'), 2.25 (d, 12, H12'a), 2.14 (d, 12, H12'b), 6.08 (s, -OCH2O-), 5.98 (s, - OCH2O-), 3.57 (s, 7'OMe), 3.73 (s, 17OMe), 2.28 (s, AcMe), 2.27 (s, 16Me), 2.01 (s, 6Me), 2.15 (s, NHCOMe), 2.09 ( s, NHCOMe), 3.25 (NHCOMe), 2.64 (m, NHCOMe). Et 717 (6): HRFABMS: m / z 718.2435, M + H ion, C37H4oN3? 10S,? = -0.1 mDa-, 1H NMR, d 6.55 (H15), 6.44 (H8 '), 6.38 (H5'), 6.07, 5.92 (-OCH2O-), 5.78, 4.09 (H22a, b), 5.30 (H1), 5.19 (H21), 4.92 (H4), 4.57 (H11), 4.41 (H3), 3.64 (H13), 3.22, 2.91 (H14a, b), 3.00, 2.85 (H3'a, b), 2.61, 2.38 (H4) 'a, b), 3.74 (17-OCH3), 3.54 (7'-OCH3), 2.35 (6-CH3), 2.27 (16-CH3), 2.16 (N-CH3). Et 775 (7): HRFABMS: m / z 776,? = -0.0 mDa. Et 789 (8): HRFABMS: m / z 790, C40H44N3O12S,? = -0.2 mDa; 12N-CH3, d 2.65 (only) observed in 1H NMR.

Several of these new ecteinascidin compounds show excessively potent cytotoxicity against L1210 (see Table 1).

TABLE 1 Compound of Et IC50 ng / ml against L1210 Et 802 (1) 7 Et 788 (2) 0.5 Et 760 (3) 32 Et 858 (4) 0.4 Et 815 (5) 0.4 As shown above, the present invention is directs to bioactive compounds. These compounds have been prepared in a substantially pure form, that is, at a level of purity sufficient to allow the physical and biological characterization thereof. As described above, it has been found that these compounds possess specific antitumor activities and as such, will be useful as medicinal agents in mammals, particularly in humans. Thus, another aspect of the present invention concerns pharmaceutical compositions containing the active compounds identified herein and methods of treatment employing such pharmaceutical compositions. The active compounds of the present invention exhibit anti-tumor activity. Thus, the present invention also provides a method for treating any mammal afflicted by a malignant tumor sensitive to these compounds, which comprises administering to the affected individual a therapeutically effective amount of an active compound or mixture of compounds, or pharmaceutical compositions thereof. The present invention also relates to pharmaceutical preparations, which contain as active ingredient one or more of the compounds of this invention, as well as the processes for their preparation. Examples of the pharmaceutical compositions include any solid (tablets, pills, capsules, granules, etc.) or liquid (solutions, suspensions or emulsions) with suitable composition or oral, topical or parenteral administration, and may contain the pure compound or in combination with any carrier or other pharmacologically active compounds. These compositions may need to be sterile when they are to be administered parenterally. The correct dosage of a pharmaceutical composition comprising the compounds of this invention will vary according to the particular formulation, the mode of application and the particular situs, hosts and bacteria or tumors being treated. Other factors such as age, body weight, sex, diet, time of administration, rate of excretion, condition of the host, combinations of medications, reaction sensitivities and severity of the disease should be taken into account. The administration can be carried out continuously or periodically within the maximum tolerated dose. Several known ecteinascidin compounds were also isolated, including Et 743 and Et 749, as shown below in Table 2.

TABLE 2 Quantities of Et compounds isolated Fraction A (550 mg) Fraction B (261 mg) Fraction C (40 mg) Et 729 39.4 mg Et 729 31 .6 mg Et 743 10.2 mg Et 731 1 1 .1 mg Et 743 0.9 mg Et 771 5.8 mg Et 745 45.4 mg Et 745 6.0 mg Et 759A 3.5 mg Et 759B 46.4 mg Et 802 (1) 1.4 mg * Et 597 3.4 mg Et 788 (2) 0.2 mg * Et 717 (6) 2.5 mg * Et 760 (3 ) 1 .0 mg * Et 81 5 (5) 5.1 mg * Et 858 (4) 0.5 mg * Et 814 (9) 5.9 mg * Et 789 (8) 1.5 mg * Et 775 (7) 0.5 mg * * - New ecteinascidin compounds The present invention has been described in detail, including preferred embodiments thereof. However, it will be appreciated that those skilled in the art, upon consideration of the present disclosure, may make modifications and / or improvements in this invention, and will still be within the scope and spirit of this invention as set forth in the following claims.

Claims (27)

1. CLAIMS 1 . Ecteinascidin 717 substantially pure, free of cellular waste from Ecteinascidia turbinata.
2. 2. Ecteinascidin 815 substantially pure, free of cellular waste from Ecteinascidia turbinata.
3. 3. Ecteinascidin 814 substantially pure, free of cellular waste from Ecteinascidia turbinata.
4. 4. Ecteinascidina 802 substantially pure, free of cellular waste of Ecteinascidia turbinata.
5. 5. Ecteinascidin 788 substantially pure, free of cellular waste of Ecteinascidia turbinata.
6. 6. Ecteinascidina 760 substantially pure, free of cellular waste of Ecteinascidia turbinata.
7. 7. Ecteinascidina 858 substantially pure, free of cellular waste of Ecteinascidia turbinata.
8. 8. Ecteinascidin 789 substantially pure, free of cellular waste of Ecteinascidia turbinata.
9. 9. Ecteinascidina 775 substantially pure, free of cellular waste of Ecteinascidia turbinata.
10. 10. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 717 and a pharmaceutically acceptable carrier, diluent or excipient. eleven .
11. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 81 and a pharmaceutically acceptable carrier, diluent or excipient.
12. 12. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 814 and a pharmaceutically acceptable carrier, diluent or excipient.
13. 13. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 802 and a pharmaceutically acceptable carrier, diluent or excipient.
14. 14. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 788 and a pharmaceutically acceptable carrier, diluent or excipient.
15. 15. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 760 and a pharmaceutically acceptable carrier, diluent or excipient.
16. 16. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 858 and a pharmaceutically acceptable carrier, diluent or excipient.
17. 17. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 789 and a pharmaceutically acceptable carrier, diluent or excipient.
18. 18. A pharmaceutical or veterinary composition comprising an effective antitumor amount of the substantially pure compound designated herein as Et 775 and a pharmaceutically acceptable carrier, diluent or excipient.
19. 19. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 717 and a carrier, diluent or pharmaceutically acceptable excipient.
20. 20. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 815 and a carrier, diluent or pharmaceutically acceptable excipient. twenty-one .
21. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 814 and a carrier, diluent or excipient pharmaceutically acceptable.
22. 22. Use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 802 and a pharmaceutically acceptable carrier, diluent or excipient.
23. 23. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 788 and a carrier, diluent or pharmaceutically acceptable excipient.
24. 24. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 760 and a carrier, diluent or pharmaceutically acceptable excipient.
25. 25. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 858 and a carrier, diluent or pharmaceutically acceptable excipient.
26. 26. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 789 and a carrier, diluent or pharmaceutically acceptable excipient.
27. 27. The use for the manufacture of a medicament for the therapeutic or prophylactic treatment of a patient suffering from a mammalian tumor, of an effective antitumor amount of the substantially pure compound designated herein as Et 775 and a carrier, diluent or pharmaceutically acceptable excipient.