CYTOTOXIC ALKALOID DERIVATIVES INCLUDING ASMARINE A AND B ISOLATED FROM A SPONGE

Abstract: Asmarine A and B, novel cytotoxic diterpene-alkaloids, have been isolated from the sponge Raspailia sp. The structure of these compounds have been established on the basis of NMR data and confirmed by X-ray analysis. Also claimed are compounds (I) or (II), wherein R<1> represents hydrogen or lower alkyl or lower alkanoyl; R<2> represents hydrogen or lower alkyl; R<3> is either an alkyl or a cycloalkyl group containing one or more isopreneunits, or a monoterpene or a sesquiterpene or a sesquiterpene or a diterpene group; R<4> or R<5> represent hydrogen or lower alkyl; R<6> represents lower alkyl; X represents F or Cl or Br or (I).

Description: CYTOTOXIC ALKALOID DERIVATIVES INCLUDING ASMARINE A AND B ISOLATED FROM A SPONGE The present invention relates to new cytotoxic alkaloids, Asmarine A and B, isolated from the sponge Raspailia sp. 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). A family of these metabolites is the diteipene-alkaloid family; in 1984 (Reference 2) it was reported the structure of four Agelasines: Agelasine A Agelasine B We have isolated from the sponge Raspailia sp. new cytotoxic diteφene-alkaloids related to mis agelasine famly. Summary of the Invention The present invention provides new diteφene-alkaloid having either the foπnula (I) or the formula (II): (D cπ) wherein R 1 represents hydrogen or lower allcyl or lower alkanoyl; R 2 represents hydrogen or lower alkyl; R 3 is either an alkyl or a cycloallcyl group containig one or more isoprene units, or a monoteφene or a sesquiteφene or a diteφene group; R 4 and R 5 represent hydrogen or lower alkyl; R 6 represents lower alkyl; X represents F or Cl or Br or I. In the definitions of the groups in foπnulas (I) and (II), the lower alkyl and the lower alkyl moiety of the lower alk.anoyl 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 Asmarine A and Asmarine B, extracted and isolated from the sponge Raspailia sp. The structures of these compounds are the following: Asmarine A Asmarine B The stereochemistry showed is for a relative configuration in both cases. Asmarine A and Asmarine B exWbit antitumor activity. .In particular, Asmarine A and Asmarine B 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, it is active against other tumor cell lines, like leukemia and lymphoma. The present invention also provides a method of treating a mammal affected by a malignant tumor sensitive to a compound with either the formula (I) or the formula (II), which comprises administering a therapeutically effective amount of the compound with either the formula (I) or the formula (IT), or a pharmaceutical composition thereof. The present invention further provides pharmaceutical compositions which contain as active ingredient a compound with either the formula (I) or the formula (II), as well as a process for its preparation. A further aspect of the invention is a method for preparing the compounds Asmarine A and Asmarine B, which comprises extraction and isolation from the sponge Raspailia sp. Ex.amples 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 phaπnaceutical composition comprising compounds with either the formula (I) or the formula (II), 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 m.aximum tolerated dose. Antitumour 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 (ElVDEM/neaa); supplemented with 10% Fetal Calf Senim (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 foi of the method described by Bergeron et al. (Reference 3). The antitumor cells employed were P-388 (suspension culture of a lymphoid neoplasm from DB.A/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 5FCS 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. it 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 M . EM 10FCS 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, the wells were stained with 0.1% Crystal Niolet. An approximately ICs 0 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 Asmarine A and Asmarine B with the cell lines P-388, A-549, HT-29 and MEL-28 are given in the following table: IC 50 (μM) P-388 A-549 HT-29 MEL-28 Asmarine A 1J8 1J8 1J8 1J8 Asmarine B 0.24 0J2 0J2 0.24 Extraction and Isolation Low-resolution mass spectra were recorded on a EIMS mass spectrometer. 1H and 13 C- NMR spectra were recorded on a Bruker .ARX-500 spectrometer. All chemical shifts are reported with respect to TMS (δ=0 pp ). Raspailia sp. (Rob Nan Soest), (Class Demorspongia, Order poecilosclerida, Family Raspailiidea) was collected in Dahlak .Archipelago, Eritrea by SCUBA diving to a depth of 23.5 m in May 1997. A reference sample is deposited in Tel Aviv University (ET- 338). The sponge was frozen on site. The freeze dried sponge (20 gr) was extracted with ethyl acetate 2x to give a brown gum 1.2 g after evaporation. This brown gum was fractionated by Kupchon into four fractions: hexane, carbon tetrachloride, chloroform and water. The CHC1 3 and CC fractions were similar. These two fractions were combined .and chromatographed on a sephadex LH-20 column eluting with MeOH:CHCl 3 (1 :1) giving five fractions. Fractions (3-5) were further chromatographed repeatedly on a sephadex LH-20 with the same solvent system yielding Asmarine A (100 mg) as a solid, mp= 232 °C, m/z+ [α] D 20 +55° (c=0.5, CHC1 3 ), IR 3400, 2928, 1600, 1553, 1451, 1400, 1388, 900 cm "1 , the structure of this Asmarine A was confirmed by X-ray analysis. With the same solvent was also isolated Asmarine B (120 mg) as an oil, m/z + = 423 (C 25 H 37 N 5 O), (100%) 188 [α] D 20 .+60° (c= 0.5, CHC1 3 ), IR 3400, 2927, 1606, 1553, 1451, 1404, 1388, 900 cm- 1 . For NMR data of Asmarine A and B see next Table: in all cases the solvent is CDC1 3 and all chemical shifts are reported with respect to TMS (δ=0 ppm). -: >c δπ C No. Asmarin A Asmarine B Asmarine A Asmarine B 1 21.821 21.17 t 1.70 d, IH 1.82 m, 2H 1.45 m, IH 2 28.56 1 24.13 1 1.85 brd, IH 1.75 m, IH 1.21 m, IH 1.60 m, IH 3 33.201 31.651 2.25 dt, IH 2.45 m, IH 2.05 dd, IH 2J2 m, IH 4 160.59 s 153.65 s 5 40.05 s 39.35 s 6 37.241 38.121 1.50 m, 2H 2J0 t, IH 1.201, IH 7 27.361 27.221 1.45 m, 2H 1.54 m, IH 1.20 m, IH 8 36.68 d 38.12 d 1.37 m, IH 1.35 m, IH 9 39.26 s 40.54 s 10 48.62 d 46.56 d 1.05 d, IH 1.39 m, IH 11 31.23 t 31.05 1 1.55 dt, IH 1.59 m, IH 1.25 m, IH 1.301, IH 12 33.01 1 31.55 1 1.95 dt, IH 1.95 dt, IH 1.43 m, IH 1.55 m, IH 13 64.20 s 64.95 s 14 36.68 1 36.401 2.50 dt, IH 2.55 m, IH 2.15 dd, IH 2.25 m, IH 15 42.301 42.33 1 4.25 dt, IH 4.301, 2H 1.49 s,3H 0.74 d, 3H 4.70 d, 2H l.Hs,3H 0.82 s, 3H 8.50 d, IH 7.95 s, IH References 1. Faulkner, D. Nat.Prod.Rep. 1997, 14, 259-302 and references therein. 2. Nakamura, H. et al. Tetrahedron Lett. 1984, 25, 2989-2992 3. Raymond J. Bergeron, Paul F. Cavanaugh, Jr., Steven J. Kline, Robert G. Hughes, Jr., Gary T. Elliot and Carl W. Porter. Antineoplastic and antiheφetic activity of spermidine catecholamide iron chelators. Biochem. Bioph. Res. Comm. 1984, 121, 848-854.

Claims: CLAIMS: 1. A compound having the either the formula (I) or the formula (II): (I) (D) wherein R represents hydrogen or lower alkyl or lower alkanoyl; R represents hydrogen or lower alkyl; R 3 is either an alkyl or a cycloalkyl group containig one or more isopreneunits, or a monoteφene or a sesquiteφene or a diteφene group; R 4 and R 5 represent hydrogen or lower alkyl; R 6 represents lower alkyl; X represents F or Cl or Br or I. In the definitions of the groups in formulas (I) and (II), the lower alkyl and the lower alkyl moiety of the lower alkanoyl 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. . A compound, Asmarine A, according to claim 1 , having the following formula: Asmarine A A compound, Asmarine B, according to claim 1, having the following formula: Asmarine B 4. A method of treating a mammal affected by a malignant tumor sensitive to a compound with either the formula (I) or the formula (H), as defined in claim 1 , wliich 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 Asmarine A (as defined in claim 2) or Asmarine B (as defined in claim 3), wliich comprises administering to the affected individual a therapeutically effective amount of Asmarine A or Asmarine B or a pharmaceutical composition thereof. 6. A pharmaceutical preparation which contains as active ingredient a compound with either the formula (I) or the formula (II), as defined in claim 1. 7. A pharmaceutical preparation which contains as active ingredient Asmaiine A (as de.fined in claim 2) or Asmarine B (as defined in claim 3). 8. 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 either the formula (I) or the formula (II), as defined in claim 1, together with other or others antitumoral compounds. 9. A method of treating a mammal affected by a malignant tumor wliich comprises administering to the affected individual a therapeutically effective .amount of Asmarine (as defined in claim 2) or Asmarine B (as defined in claim 3), together with other or others antitumoral compounds. 10. A method for prepaiϊng Asmaiine A (as defined in claim 2) or Asmarine B (as defined in claim 3), wliich comprises extraction and isolation from the sponge Raspailia sp.

Inventors: GRAVALOS DOLORES G, KASHMAN YOEL, RUDI AMIRA, YOSIEF TESFAMARIAM, GRAVALOS, DOLORES, G., KASHMAN, YOEL, RUDI, AMIRA, YOSIEF, TESFAMARIAM
Applicants: GRAVALOS DOLORES G, INST BIOMAR SA, KASHMAN YOEL, RUDI AMIRA, RUFFLES GRAHAM KEITH, YOSIEF TESFAMARIAM, GRAVALOS, DOLORES, G., INSTITUTO BIOMAR S.A., KASHMAN, YOEL, RUDI, AMIRA, RUFFLES, GRAHAM, KEITH, YOSIEF, TESFAMARIAM
Application year: 1998
Main classification: C07D487
IPC codes: A61K31, A61P35, C07D487