WO2004012756A1

WO2004012756A1 - New cytotoxic cyclopeptide and its use for the treatment of cancer

Info

Publication number: WO2004012756A1
Application number: PCT/GB2003/003433
Authority: WO
Inventors: Marta Trujillo; Maria Jesus Luna; Jose Luis Fernandez-Puentes; Julian Castro; Librada Maria Canedo; Gravalos Dolores Garcia
Original assignee: Instituto Biomar SA
Current assignee: Instituto Biomar SA
Priority date: 2002-08-06
Filing date: 2003-08-06
Publication date: 2004-02-12
Anticipated expiration: 2005-02-06
Also published as: AU2003249078A1

Abstract

A compound is provided with a cyclopeptide structure of formula (1) (IB01212) or pharmaceutically acceptable salts, derivatives, prodrugs or steroisomers thereof: the compound can be obtained by cultivating a microorganism such as the culture strain ESNA-A009, available under accession number CECT 20477, from the Collección Española de Cultivos Tipo at the University of Valencia, Spain.

Description

NEW CYTOTOXIC CYCLOPEPTIDE AND ITS USE FOR THE TREATMENT OF CANCER

FIELD OF THE INVENTION

The present invention relates to antitumoral compounds, in particular to a cyclopeptide, its pharmaceutical compositions and use thereof. It is also related to the obtention of the compound and the microorganism producing it.

BACKGROUND OF THE INVENTION

Several cyclic peptides obtained from marine organisms have been disclosed. Some of them have antitumoral properties, like Aplidine from Aplidium albicans that is the subject of WO 91 04985 and Kahalalide F from Elysia rτifescens that is the subject of EP 610 078.

Cancer is a leading cause of death in animals and humans. Several efforts have been and are still being undertaken in order to obtain antitumor agents active and safe to be administered to patients suffering from a cancer.

SUMMARY OF THE INVENTION

This invention provides a compound with a cyclopeptide structure of formula 1 (IB01212) or pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof:

We have found that compound 1 has significant antitumoral properties and therefore is useful as an antitumoral agent for the treatment of cancer.

The present invention also relates to the process of obtaining compound 1, and the preferred process comprises cultivating a strain of a microorganism capable of producing compound 1 in an aqueous nutrient medium with assimilable carbon and nitrogen sources and salts, under controlled submerged aerobic conditions. The compound 1 is recovered and purified from the cultured broth.

The present invention is also directed to a microorganism capable of producing compound 1. Preferably, the microorganism is a Chlonostachys sp., most preferably the substantially pure- culture strain ESNA-A009, available under accession number CECT 20477, from the Colecciόn Espaήola de Cultivos Tipo at the University of Valencia, Spain.

In another aspect, the present invention is directed to pharmaceutical compositions containing a compound of formula 1 or pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof, together with a pharmaceutically acceptable carrier or diluent. In another aspect, the present invention is also directed to the use of a compound of formula 1 or pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof in the treatment of cancer, or in the preparation of a medicament for the treatment of cancer.

The invention also provides a method of treatment using the compund of formula 1.

BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 is the HPLC/UV chromatogram and the UV spectrum of purified IB-01212 (compound 1)

Figure 2 is the IR spectrum of purified IB-01212 (compound 1) Figure 3 is the ^:H NMR spectrum of purified IB-01212 (compound 1) Figure 4 is the ¹³C NMR spectrum of purified IB-01212 (compound 1) Figure 5 is the DEPT spectrum of purified IB-01212 (compound 1) Figure 6 is the COSY 45 spectrum of purified IB-01212 (compound 1) Figure 7 is the HSQC spectrum of purified IB-01212 (compound 1) Figure 8 is the HMBC spectrum of purified IB-01212 (compound 1) Figure 9 is the HPLC/MS chromatogram and ESI-MS spectrum of IB- 01212 (compound 1)

DETAILED DESCRIPTION OF THE INVENTION

The present invention relates to compounds of general formula 1 as defined above. The new compounds of the invention are isolated from the fermentation broth of a microorganism, preferably a fungal strain Chlonostachys sp., ESNA-A009, a culture of which has been deposited by <depositor> on <date> in the Colecciόn Espaiiola de Cultivo Tipo at the Universidad de Valencia, Spain, under the accession number CECT 20477. This deposit has been made under the provisions of the Budapest Treaty and all restrictions on the availability thereof to the public will be irrevocably maintained upon the granting of a patent on this application.

The organism was isolated from an unidentified marine sponge collected in Japanese waters.

While the deposited strain is clearly preferred, the present invention is not restricted or limited to any particular strain or organisms. It is the intention of the present invention to include other producing organisms, strains or mutants within the scope of this invention.

Taxonomic studies of the strain ESNA-A009 are summarized as follows: All cultures were incubated at 24°C and records of results were made weekly up to 30 days.

Culture characteristics. Colonies are rather slow growing reaching 35 cm diameter in ten days at 24°C on oatmeal agar, potato dextrose agar, and malt agar. All media were -supplemented with 50% artificial seawater. Growth was recorded after 15 days at 24°C.

Colony characteristics. Colonies growing moderately slow, white, hairy to cottony. Pustulate due to formation of dark green sporodochia which appear after at least20 days incubation. Production of soluble pigments could not be observed on any of the media.

Microscopy. Phialoconidia in long chains are dry and subhyaline, mainly produced in sporodochia. Conidiophores aggregated, forming dark green sporodochia, simple or branched, with conidia in chains. Phialides not well differentiated. Conidia are cylindrical and are 7 to 10 μm in length and 4.5 to 5.5 μm in width.

The optimal temperature for growth on solid media is 24-28°C; the pH range for growth is between 3.0 to 9.0. The isolate was found to grow best on agar media supplemented with seawater. Growth and sporulation were best with glucose, starch and sucrose. Other carbon sources such as glycerol and dextrose can also be used. In shake cultures, the mycelium becomes transformed into a kind of blastospores.

Based on the preceding characteristics the culture has been determined as a member of the genus Chlonostachys.

Chlonostachys sp. ESNA-A009 cultured under controlled conditions in a suitable medium produces the antitumour compound 1. This strain is preferably grown in an aqueous nutrient medium, under aerobic and mesophilic conditions, preferably between 20 and 25°C at a pH 5.0 to 6.0.

A description of a process to obtain compound 1 is as follows:

Stock culture. A spore suspension of a pure culture of Chlonostachys sp. ESNA-A009 was kept frozen at -70°C in 20% glycerol.

Preparation of inoculum. A frozen culture or a well grown agar culture was used to inoculate 50 ml of a seed medium containing 0.4% glucose, 0.2%) beef extract, 0.2% yeast extract, 2% starch, 0.4% tryptone, 0.5% NaCl, 0.02% KC1, 0.3% MgCl₂, 0.2% and tap water in 250 ml shake flasks and cultured at 24°C on a rotary shaker at 150 rpm. The flasks were incubated 48 hours, and used as a first stage inoculum. Fermentation. 300 ml of the same medium in 2 L Erlenmeyer flasks were inoculated with 10% of the first stage inoculum. The fermentation was carried for 5 days at 24°C on a rotary shaker at 150 rpm.

Production of the compounds can be monitored by whole broth assay against HT-29 cell line or any other sensitive cell or by HPLC or any other method with enough sensitivity.

The antitumoral compound 1 can be isolated from the mycelial cake by extraction with a suitable mixture of solvent such as CHCl3:CH3θH:H∑O. Other solvents are contemplated within the scope of the invention. The activity is concentrated in the lower layer. The extracts from two repeated extraction can be combined and evaporated to dryness in vacuum.

Separation and purification of compound 1 from the crude active extract can be performed by the use of the proper combination of conventional chromatographic techniques.

Fractionation can be guided by the antitumor activity of fractions, or by TLC visualized with vanillin in cone. H2SO4, or analytical HPLC with photodiode-array and MS detectors. HPLC analysis is performed at room temperature using an analytical column Symmetry C18 (5DD with gradient elution from 40:60:0.1 to 85: 15:0.1 MeCN/H2O/TFA in 25 min, a flow rate of 0.5 ml/ min. and plotted at 220 nm, in this conditions Compound 1 IB-01212 retention time is 13.3 min as is shown in Fig. l.

On the basis of detailed analysis of their various spectral characteristics, the pure compound can be identified (see data reproduced in figures 1 to 9).

The U.V. spectrum shows absorption at 220, as reported in Fig. 1. The infrared absorption spectrum is shown in Fig. 2 of the accompanying drawings.

The iH, ¹³C and DEPT NMR. spectra of IB-01212 are reported in Fig.3, Fig.4 and Fig. 5 respectively.

The 2D NMR experiments COSY, HSQC and HMBC are reported in Fig. 6, Fig. 7 and Fig. 8 respectively.

The ES-MS spectrum of IB-01212 displayed a (M+H)⁺ peak at 1033 and (M+Na)⁺ 1055, is reported in Fig. 9.

The present invention is directed at compound 1 and its pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers.

The term "pharmaceutically acceptable salts, derivatives, prodrugs" refers to any pharmaceutically acceptable salt, ester, solvate, hydrate or any other compound which, upon administration to the recipient is capable of providing (directly or indirectly) a compound as described herein. However, it will be appreciated that non-pharmaceutically acceptable salts also fall within the scope of the invention since those may be useful in the preparation of pharmaceutically acceptable salts. The preparation of salts, prodrugs and derivatives can be carried out by methods known in the art.

For instance, pharmaceutically acceptable salts of compounds provided herein are synthesized from the parent compound which contains a basic or acidic moiety by conventional chemical methods. Generally, such salts are, for example, prepared by reacting the free acid or base forms of these compounds with a stoichiometric amount of the appropriate base or acid in water or in an organic solvent or in a mixture of the two. Generally, nonaqueous media like ether, ethyl acetate, ethanol, isopropanol or acetonitrile are preferred. Examples of the acid addition salts include mineral acid addition salts such as, for example, hydrochloride, hydrobromide, hydroiodide, sulphate, nitrate, phosphate, and organic acid addition salts such as, for example, acetate, maleate, fumarate, citrate, oxalate, succinate, tartrate, malate, mandelate, methanesulphonate and p-toluenesulphonate. Examples of the alkali addition salts include inorganic salts such as, for example, sodium, potassium, calcium and ammonium salts, and organic alkali salts such as, for example, ethylenediamine, ethanolamine, N,N- dialkylenethanolamine, triethanolamine and basic aminoacids salts.

The compounds of the invention may be in crystalline form either as free compounds or as solvates (e.g. hydrates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art.

Any compound that is a prodrug of a compound of formula 1 is within the scope of the invention. The term "prodrug" is used in its broadest sense and encompasses those derivatives that are converted in vivo to the compounds of the invention. Such derivatives would readily occur to those skilled in the art.

The compounds of the present invention represented by the above described formula 1 may include enantiomers depending on their asymmetry or diastereoisomers. The single isomers and mixtures of the isomers fall within the scope of the present invention.

An important feature of the above described compounds of formula 1 is their bioactivity and in particular their cytotoxic activity. With this invention we provide novel pharmaceutical compositions of compounds of formula 1 that possess cytotoxic activity, and their use as antitumor agents. Thus the present invention further provides pharmaceutical compositions comprising a compound of this invention, a pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof with a pharmaceutically acceptable carrier.

Examples of pharmaceutical compositions include any solid (tablets, pills, capsules, granules etc.) or liquid (solutions, suspensions or emulsions) composition for oral, topical or parenteral administration.

Administration of the compounds or compositions of the present invention may be by any suitable method, such as intravenous infusion, oral preparations, and intraperitoneal and intravenous administration. We prefer that infusion times of up to 24 hours are used, more preferably 2-12 hours, with 2-6 hours most preferred. Short infusion times which allow treatment to be carried out without an overnight stay in hospital are especially desirable. However, infusion may be 12 to 24 hours or even longer if required. Infusion may be carried out at suitable intervals of say 1 to 4 weeks. Pharmaceutical compositions containing compounds of the invention may be delivered by liposome or nanosphere encapsulation, in sustained release formulations or by other standard delivery means.

The correct dosage of the compounds will vary according to the particular formulation, the mode of application, and the particular situs, host and tumour 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.

The compounds and compositions of this invention may be used with other drugs to provide a combination therapy. The other drugs may form part of the same composition, or be provided as a separate composition for administration at the same time or at different time. The present invention also extends to the compounds of the invention for use in a method of treatment, and to the use of the compounds in the preparation of a composition for treatment of cancer.

Antitumoral activities of these compounds include leukaemias, lung cancer, colon cancer, kidney cancer, prostate cancer, ovarian cancer, breast cancer, pancreas cancer, cervix cancer, sarcomas and melanomas.

EXAMPLES

Example 1 :

Compound 1 was produced as described above.

2.5 liters of whole harvested broth were filtrated to separate the biomass and other solids. The mycelia cake was extracted twice with a mixture solvent (1.2 1) of CHC1₃: CH₃OH:H₂0 (2: 1: 1), the activity was concentrated in the lower layer. The organic solvent was concentrated and evaporated to dryness in vacuum to yield 1.3 g of crude extract.

The extract was applied to a silica gel VFC (vacuum flash chromatography) system, using a mixture of -hexane-EtOAc and EtOAc- MeOH as eluting solvents. The fractions containing compound 1 (IB- 01212) (120 mg) with antitumor activity were eluted with ethyl acetate/ methanol 1: 1, 1:3 and methanol. The active fractions were chromatographied by column on silica gel using CHC13-MeOH mixtures as eluting solvent. The cytotoxic activity was detected in fractions eluted with CHC13-MeOH 95:5 (28 mg). Further purification by C18 reversed phase chromatography afforded 20 mg of pure compound 1 (IB-01212) eluted with MeOH.

This compound has the following spectroscopic data. All the ¹H, ¹³C NMR data are summarized in Table 1 : Table 1. *H and ¹³C NMR data of IB-01212

Position 13 C (δ) 1H (δ) Position 13 C (δ) Η (δ)

N-MePhe Ser

N-CH₃ 30.4 2.82 (s) ΝH 7.73 (d, 8.0) αCH 61.5 4.59 (dd, 3.5, 11.9) αCH 47.7 5.16 (d, 8.0) βCH₂ 35.8 3.05 (dd, 11.9 ', 14.5) βCH₂ 66.4 3.95 (dd, 2.2, 11.6) 3.64 (dd, 3.5, 14.5) 4.52 (dd, 2.5, 11.4) l-C 136.1 CO 168.0

2,6-CH 129.4 7.29 (m)

3,5-CH 129.1 7.29 (m) N, 7V-DiMeLeu

4-CH 127.3 7.24 (m) N-CH₃ 42.4 2.26 (s)

CO 170.5 N-CH₃ 42.4 2.26 (s) αCH 67.2 2.94 (dd, 4.8, 8.6)

N-MeLeu βCH₂ 36.1 1.40 (m)

N-CH₃ 30.9 2.88 (s) 1.57 (m) αCH 52.6 4.65 (t, 6.9) γCH 26.1 1.65 (m) βCH₂ 38.2 1.08 (m) γCH₃ 22.3 0.90 (d, 6.4)

1.55 (m) γCH₃ 23.6 0.93 (d, 6.4) γCH 24.7 0.94 (m) CO 173.2 γCH₃ 22.9 0.55 (d, 6.6) γCH₃ 23.0 0.63 (d, 6.6)

CO 170.1

EXAMPLE 2: BIOLOGICAL ACTIVITY BIOASSAYS FOR ANTITUMOR SCREENING

The finality of these assays is to interrupt the growth of a "in vitro" tumor cell culture by means a continued exhibition of the cells to the sample to be testing.

CELL LINES

Name N° ATCC Species Tissue Characteristics

K-562 CCL-243 human leukemia erythroleukemia (pleural effusion)

A-549 CCL-185 human lung lung carcinoma "NSCL"

SK-MEL-28 HTB-72 human melanoma malignant melanoma

HT-29 HTB-38 human colon colon adenocarcinoma

LoVo CCL-229 human colon colon adenocarcinoma

LoNo-Dox human colon colon adenocarcinoma (MDR)

DU-145 HTB-81 human prostate prostate carcinoma, not androgen receptors

LΝCaP CRL-1740 human prostate prostate adenocarcinoma, with androgen receptors

SK-BR-3 HTB-30 human breast breast adenocarcinoma, Her2/neu+, (pleural effusion)

IGRON-1 human ovary ovary adenocarcinoma

IGRON-ET human ovary ovary adenocarcinoma, characterized as ET-743 resistant cells

SK-ON-3 HTB-77 human ovary ovary adenocarcinoma (malignant ascites)

HeLa CCL-2 human cervix cervix epitheloid carcinoma

HeLa-APL CCL-3 ^' human cervix cervix epitheloid carcinoma, characterized as aplidine resistant cells

PAΝC-1 CRL-1469 human pancreas pancreatic epitheloid carcinoma

HMEC1 human endothelium Inhibition of cells growth by colorimetric assay.

A colorimetric type of assay, using sulforhodamine B (SRB) reaction has been adapted for a quantitative measurement of cell growth and viability [following the technique described by Philip Skehan, et al. (1990), New colorimetric cytotoxicity assay for anticancer drug screening, J. Natl. Cancer Inst. , 82: 1107-1112]

This form of the assay employs 96 well cell culture microplates of 9 mm diameter (Faircloth, 1988; Mosmann, 1983). Most of the cell lines are obtained from American Type Culture Collection (ATCC) derived from different human cancer types.

Cells are maintained in RPMI 1640 10% FBS, supplemented with 0.1 g/1 penicillin and 0.1 g/1 streptomycin sulfate and then incubated at 37°C, 5% CO and 98% humidity. For the experiments, cells were harvested from subconfluent cultures using trypsin and resuspended in fresh medium before plating.

Cells are seeded in 96 well microtiter plates, at 5 x 10³ cells per well in aliquots of 195 μl medium, and they are allowed to attach to the plate surface by growing in drug free medium for 18 hours. Afterward, samples are added in aliquots of 5 μl in a ranging from 10 to 10"⁸ μg/ml, dissolved in DMSO/EtOH/PBS (0.5:0.5:99). After 48 hours exposure, the antitumor effect are measured by the SRB methodology: cells are fixed by adding 50 μl of cold 50% (wt/vol) trichloroacetic acid (TCA) and incubating for 60 minutes at 4°C. Plates are washed with deionized water and dried. One hundred μl of SRB solution (0.4% wt/vol in 1% acetic acid) is added to each microtiter well and incubated for 10 minutes at room temperature. Unbound SRB is removed by washing with 1% acetic acid. Plates are air dried and bound stain is 14 solubilized with Tris buffer. Optical densities are read on a automated spectrophotometric plate reader at a single wavelength of 490 nm.

The values for mean +/- SD of data from triplicate wells are calculated. Some parameters for cellular responses can be calculated: GI = growth inhibition, TGI = total growth inhibition (cytostatic effect) and LC = cell killing (cytotoxic effect).

Activity Data (Molar) of IB-01212.

Claims

CLAIMS:

1. A compound of formula 1

or pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof.

2. A process for preparing a compound as defined in claim 1 which comprises cultivating a strain of a microorganism capable of producing it in an aqueous nutrient medium.

3. A process according to claim 2 wherein the microorganism is a Chlonostachys sp.

4. A process according to claim 3 wherein the microorganism is is the substantially pure culture strain ESNA-A009, available under accession number CECT 20477, from the Colecciόn Espanola de Cultivos Tipo at the University of Valencia, Spain.

5. A substantially pure culture strain ESNA-A009 as isolated from marine invertebrates, and has been taxonomically classified as Chlonostachys sp.

6. A pharmaceutical composition comprising a compound according to claim 1 , pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof, together with a pharmaceutically acceptable carrier or diluent.

7. Use of a compound of formula 1 or pharmaceutically acceptable salts, derivatives, prodrugs or stereoisomers thereof in the treatment of cancer, or in the preparation of a medicament for the treatment of cancer.