AU2003204339B2 - Cytotoxic Alkaloid Derivatives Including Asmarine A and B Isolated from a Sponge - Google Patents

Description

S&FRef: 513700D1

AUSTRALIA

PATENTS ACT 1990 COMPLETE SPECIFICATION FOR A STANDARD PATENT Name and Address of Applicant: Actual Inventor(s): Address for Service: Invention Title: Instituto Biomar S.A.

Polig. Industrial, Edificio CEI Mod. 2.02 y 2.03 E-24231 Onzonilla Spain Yoel Kashman Amira Rudi Tesfamariam Yosief Dolores G-Gravalos Spruson Ferguson St Martins Tower Level 31 Market Street Sydney NSW 2000 (CCN 3710000177) Cytotoxic Alkaloid Derivatives Including Asmarine A and B Isolated from a Sponge The following statement is a full description of this invention, including the best method of performing it known to me/us:- 5845c 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 A family of these metabolites is the diterpene-alkaloid family; in 1984 (Reference 2) it was reported the structure of four Agelasines: Agelasine A Agelasine B

H

Agelasine D Agelasine C We have isolated from the sponge Raspailia sp. new cytotoxic diterpene-alkaloids related to this agelasine family.

Summary of the Invention The present invention provides an extracted and isolated compound having either s the formula or the formula (II):

R

4 N R 4

X-

-s

RX-N

N NR N y S^ N

/N

'OR' OR R

R

3 R2O

(II)

wherein R' represents hydrogen or lower alkyl or lower alkanoyl; R 2 represents hydrogen or lower alkyl; R 3 is an alkyl group containing one or more isoprene units; R 4 and R represent hydrogen or lower alkyl; R 6 represents lower alkyl; X represents F or Cl or Br to or I, and the lower alkyl and the lower alkyl moiety of the lower alkanoyl mean a straightchain 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.

In one aspect of the invention there is provided a substantially purified compound having either the formula or the formula (II): N4

R

6 R N R 4 1 X-N

N

N H R 5 N R

R

3

R

2 R RR 2

OR

1

(II)

wherein R' represents hydrogen or lower alkyl or lower alkanoyl; R 2 represents methyl; R is

H

R

4 and R 5 represent hydrogen; R 6 represents hydrogen, lower alkyl or lower alkanoyl; X represents F or Cl or Br or I, and 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 2a 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 exhibit 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 or the formula which comprises administering a therapeutically effective amount of the compound with either the formula or the formula or a pharmaceutical composition thereof.

The present invention further provides pharmaceutical compositions which contain as active ingredient a compound with either the formula or the formula 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.

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 either the formula or the formula 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.

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 (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 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 lxl04 cells per well in 1 ml aliquots of MEMcontaining 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% C02 in a 98% humid atmosphere, an approximately ICso 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 2x10 4 cells per well in 1 ml aliquots of MEM 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% CO2 in a 98% humid atmosphere, the wells were stained with 0.1% Crystal Violet. An approximately IC5o 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: ICso (lM) P-388 A-549 HT-29 MEL-28 Asmarine A 1.18 1.18 1.18 1.18 Asmarine B 0.24 0.12 0.12 0.24 Extraction and Isolation Low-resolution mass spectra were recorded on a EIMS mass spectrometer. IH and 13

C-

NMR spectra were recorded on a Bruker ARX-500 spectrometer. All chemical shifts are reported with respect to TMS (8=0 ppm).

Raspailia sp. (Rob Van 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 CHCI3 and CC4 fractions were similar. These two fractions were combined and chromatographed on a sephadex LH-20 column eluting with MeOH:CHCl3 giving five fractions. Fractions were further chromatographed repeatedly on a sephadex LH-20 with the same solvent system yielding Asmarine A (100 mg) as a solid, mp= 232 m/z+ 423 (C 2 5

H

37 NsO), (100%) 188 (CsHNsO+), [a] 20 +55* CHC 3 IR 3400, 2928, 1600, 1553, 1451, 1400, 1388, 900 cm 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 (C2sH 37 NsO), (100%) 188 [a]D 20 .+60r 0.5, CHC 3 IR 3400, 2927, 1606, 1553, 1451, 1404, 1388, 900 cm''.

For NMR data of Asmarine A and B see next Table: in all cases the solvent is CDC 3 and all chemical shifts are reported with respect to TMS (8=0 ppm).

OH

17 C No. Asmarin A Asmarine B Asmarine A Asmarine B 21.82 t 28.56 t 33.20 t 160.59 s 40.05 s IIf\ I 4* tL 27.36 t 36.68 d 39.26 s 48.62 d 31.23 t 33.01 t 64.20 s 36.68 t 42.30 t 21.17t 24.13 t 31.65 t 153.65 s 39.35 s 38.12t 27.22 t 38.12 d 40.54 s 46.56 d 31.05 t 31.55 t 64.95 s 36.40 t 42.33 t 1.70 d, 1H 1.45 m, 1H 1.85 brd, 1H 1.21 m, 1H 2.25 dt, 1H 2.05 dd, 1H 1.50 m,2H 1.45 m, 2H 1.37 m, 1H 1.05 d, 1H 1.55 dt, 1H 1.25 m, 1H 1.95 dt, 1H 1.43 m, 1H 2.50 dt, 1H 2.15 dd, 1H 4.25 dt, 1H 1.82 m, 2H 1.75 m, 1H 1.60 m, 1H 2.45 m, 1H 2.12 m, IH 2.10 t, 1H 1.20 t, 1H 1.54 m, 1H 1.20 m, 1H 1.35 m, 1H 1.39 m, 1H 1.59 m, 1H 1.30 t, 1H 1.95 dt, 1H 1.55 m, 1H 2.55 m, 1H 2.25 m, 1H 4.30 t, 2H 21.75 q 15.93 q 102.45 t 20.08 q 18.28 q 151.68 d 149.00 s 109.31 s 158.70 s 143.10 d 23.09 q 15.84 q 105.69 t 32.87 q 19.85 q 151.64 d 149.57 s 109.32 s 158.38 s 143.32 d 4.20 dd, 1H 1.44 s, 3H 0.70 d, 3H 4.60 s, 2H 1.00 s, 3H 0.65 s, 3H 8.50 s, 1H 7.95 s, 1H 1.49 s, 3H 0.74 d, 3H 4.70 d, 2H 1.11 s,3H 0.82 s, 3H 8.50 d, 1H 7.95 s, 1H References 1. Faulkner, D. Nat.ProdRep. 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., Gaiy T. Elliot and Carl W. Porter. Antineoplastic and antiherpetic activity of spermidine catecholamide iron chelators. Biochem. Bioph. Res. Comm. 1984, 121, 848-854.

Claims (6)

1. A substantially purified compound having either the formula or the formula (II): R 6 X- wherein R' represents hydrogen or lower alkyl or lower alkanoyl; R 2 represents methyl; R 3 is R 4 and R 5 represent hydrogen; R 6 represents hydrogen, lower alkyl or lower alkanoyl; X represents F or C1 or Br or I, and 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.

2. A substantially purified compound, Asmarine A, having the formula: RALIBXX513 700dl speci.doc:aak 11

3. A substantially purified compound, Asmarine B, having the formula: 7, ;9 6 and having the following NMR data in CDC1 3 [R:\LEBXX1S I3700d Ispeci.doc:aak ~I _8 (No. Asmarine.13 1 ~21.17 t 2 ~24.13 t A smarine B 1.82 m, 2H 1.75 ni, 1H 1.60 m, IH 2.45 m, IH 2.12 m, IH

31.65t

153.65 s 39.35Ss 38.12 t 27.22t 38.12 d 40.54 s 46.56 d 31.05 t 31.55t 64.95 s 36.40 t 42.33 t 23.09 q 15.84 q 105.69 t 32.87 q 19.85 q 151.64 d 149.57 s 109.32s

158.38 s 143.32 d 2.l 1.20, 1.54 m 1.20 m 1.35 m 1.39 m, I1H 1.59 m, IH 1.30 t, 1H 1.95 dt, I1H 1.55m IH 2.55m 1H 2 .25m1 IH 4.30 t, 2H 1.49 s, 3H 0.74 d, 3H 4.70 d, 2H 1.11 s, 3H 0.82 s, 3H 8.50 d, I H 7.95 s, I H 4. Use of a compound with either the formula or the formula (II) as defined in claim 1, in the manufacture of a medicament for the treatment of malignant tumors. Use of Asmarine A (as defined in claim 2) or Asmarine B (as defined in claim in the manufacture of a medicament for the treatment of malignant tumors. 6. A pharmaceutical preparation which contains as active ingredient a compound with either the formula or the formula as defined in claim 1. 7. A pharmaceutical preparation which contains as active ingredient Asmarine A (as defined in claim 2) or Asmarine B (as defined in claim 3). 8. Use of a compound with either the formula or the formula as defined in claim 1, together with one or more other antitumoral compounds, in the manufacture of a medicament for the treatment of malignant tumors. 9. Use of Asmarine A (as defined in claim 2) or Asmarine B (as defined in claim together with one or more other antitumoral compounds, in the manufacture of a medicament for the treatment of malignant tumors. 10. A method of treating a mammal affected by a malignant tumour sensitive to a compound having either the formula or the formula as defined in claim 1, which comprises administering to the affected individual a therapeutically effective amount of the compound or a pharmaceutical composition thereof. 11. A method of treating a mammal affected by a malignant tumour which comprises administering to the affected individual a therapeutically effective amount of a compound having either the formula or the formula as defined in claim 1, together with one or more other antitumoral compounds. 12. A compound of claim 1, when used to treat a mammal affected by a malignant tumour sensitive to a compound having either the formula or the formula as defined in claim 1. 13. A pharmaceutical preparation as claimed in claim 6 when used to treat a mammal affected by a malignant tumour sensitive to a compound having either the formula or the formula as defined in claim 1. 14. The compound of any one of claims 1 to 3 said compound being substantially purified by the process substantially as herein described with reference to the section headed Extraction and Isolation. Dated 30 January, 2006 Instituto Biomar S.A. Patent Attorneys for the Applicant/Nominated Person SPRUSON FERGUSON [R:\LIBXX1513700dspeci.doc:aak