WO2011009899A2

WO2011009899A2 - Phenalenones as antitumoral agents

Info

Publication number: WO2011009899A2
Application number: PCT/EP2010/060588
Authority: WO
Inventors: José María SÁNCHEZ LÓPEZ; Marta MARTÍNEZ INSUA; María De Los Ángeles VINUESA-NAVARRO; Jesús Ángel DE LA FUENTE BLANCO; Antonio FERNÁNDEZ-MEDARDE
Original assignee: Instituto Biomar, S.A.
Priority date: 2009-07-22
Filing date: 2010-07-21
Publication date: 2011-01-27
Also published as: WO2011009899A3

Abstract

Compounds of general formula (Ia) or (Ib) or (Ic) or (Id), wherein the R groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, -CONH₂, alkali metal, and sugar, and their use for the treatment of cancers.

Description

PHENALENONES AS ANTITUMORAL AGENTS

TECHNICAL FIELD The present invention relates to phenalenone compounds, pharmaceutical compositions containing them and their use as antitumoral agents.

BACKGROUND OF THE INVENTION Cancer is a leading cause of death in animals and humans. Several efforts have been and are still being undertaken in order to obtain an active antitumoral agent for safe administration to patients suffering from a cancer. The problem to be solved by the present invention is to provide compounds that are useful in the treatment of cancer.

Some known phenalenes, as the amphilectenes showed in formulas i-iv (J. Nat. Prod. 1993, 56, 915-20; J. Nat. Prod. 2004, 67, 833-837) and the sinulobatins A-C (Tetrahedron 1997, 53, 4569-4578) have been disclosed to have only weak in vitro cytotoxic activities.

11 111 IV

Sinulobatin A (R=OAc) Sinulobatin C

Sinulobatin B (R=H)

SUMMARY OF THE INVENTION

The present invention is directed to compounds of general formula (Ia) or (Ib) or (Ic) or (Id) or pharmaceutically acceptable salts, derivatives, or stereoisomers thereof:

(Ia) (Ib)

(Ic) (Id) wherein the R groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, -CONH₂, alkali metal, and sugar.

In another aspect, the present invention is also directed to compounds of general formula (Ia) or (Ib) or (Ic) or (Id), pharmaceutically acceptable salts, derivatives, stereoisomers or mixtures thereof for use as a medicament, particularly a medicament for the treatment of cancer.

In another aspect, the present invention is also directed to the use of compounds of general formula (Ia) or (Ib) or (Ic) or (Id), pharmaceutically acceptable salts, derivatives, stereoisomers or mixtures thereof in the treatment of cancer, or in the preparation of a medicament for the treatment of cancer.

Other aspects of the invention are methods of treatment, and compounds for use in these methods. Therefore, the present invention further provides a method of treating cancer which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound of general formula (Ia) or (Ib) or (Ic) or (Id), or a pharmaceutically acceptable salt, derivative or stereoisomer thereof. Preferably, the patient in need of such treatment may be any mammal, notably a human.

The present invention also relates to the obtaining of compounds of general formula (Ia) or (Ib) or (Ic) or (Id) from a strain of a microorganism capable of producing them. In a preferred embodiment, the present invention relates to the obtaining of compounds of general formula (Ia) from a strain of a microorganism capable of producing them.

The preferred process comprises the steps of cultivating a strain of a microorganism capable of producing compounds of general formula (Ia) or (Ib) or (Ic) or (Id) in an aqueous nutrient medium with assimilable carbon and nitrogen sources and salts, under controlled submerged aerobic conditions, and then recovering and purifying the compounds according to the invention from the cultured broth.

The present invention also relates to the obtaintion of compounds of general formula (Ia) or (Ib) or (Ic) or (Id) by synthesis of derivatives from the natural compounds isolated from a strain of a microorganism capable of producing them.

The phenalenones of the present invention may be synthesised from commercially available starting materials using conventional procedures.

In another aspect, the present invention is directed to pharmaceutical compositions containing a compound of general formula (Ia) or (Ib) or (Ic) or (Id), or mixtures thereof, or pharmaceutically acceptable salts, derivatives, or stereoisomers thereof together with a pharmaceutically acceptable carrier. BRIEF DESCRIPTION OF THE DRAWINGS

Figure 1 represents an HPLC/UV chromatogram of purified Compound I, where the absorbance (measured in mAU) is plotted against time (measured in minutes).

Figure 2 represents an UV- Vis spectrum of purified Compound I, where the absorbance (measured in mAU) is plotted against wavelength (measured in nm).

Figure 3 represents an APCI-MS (APCI, Pos, Frag: 50) spectrum of purified

Compound I, where the % total chromatogram integration is plotted against m/z.

DETAILED DESCRIPTION OF THE INVENTION

(Ia) (Ib)

(Ic) (Id) wherein the R groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, -CONH₂, alkali metal, and sugar, and their use for the treatment of cancer.

The inventors have found that the above defined compounds show good antitumoral activity in in vitro assays and therefore are useful as anticancer agents. In particular, they have shown activity against solid cancer and also myeloid leukaemia, and are especially uselful for these types of cancer.

In the above definition of compounds of general formula (Ia) or (Ib) or (Ic) or (Id), the following terms have the meaning indicated:

An acyl group is of the form R¹CO-, wherein R¹ is an organic group.

Suitable acyl groups have from 2 to about 12 carbon atoms, more preferably from 2 to about 8 carbon atoms, still more preferably from 2 to about 6 carbon atoms, even more preferably 2 carbon atoms. In one embodiment the acyl group CH3CO- (represented as "Ac") is especially preferred.

Alkyl groups preferably have from 1 to about 20 carbon atoms, more preferably from 1 to about 12 carbon atoms, even more preferably from 1 to about 6. As used herein, the term alkyl, unless otherwise modified, refers to both cyclic and non- cyclic groups, although cyclic groups will comprise at least three carbon ring members. Non-cyclic alkyl refers to a straight-chain or branched alkyl group.

Preferred alkenyl and alkynyl groups in the compounds of the present invention have one or more unsaturated linkages and from 2 to about 20 carbon atoms. The terms alkenyl and alkynyl as used herein refer to both cyclic and non cyclic groups. Non-cyclic alkenyl or alkynyl refers to a straight-chain or branched alkenyl or alkynyl group.

The groups above mentioned may be substituted at one or more available positions by one or more suitable groups such as OR', =0, SR', SOR', SO₂R', NO₂, NHR', N(R )₂, =N-R\ NHCOR', N(COR )₂, NHSO₂R', CN, halogen, C(=O)R', CO₂R', OC(=O)R' wherein each of the R' groups is independently selected from the group consisting of H, OH, NO₂, NH₂, SH, CN, halogen, =0, C(=O)H, C(=O)CH₃, CO₂H, substituted or unsubstituted Ci-Ci₂ alkyl, substituted or unsubstituted C₂-Ci₂ alkenyl, substituted or unsubstituted C₂-Ci₂ alkynyl and substituted or unsubstituted aryl.

"Aryl" refers to single and multiple ring radicals, including multiple ring radicals that contain separate and/or fused aryl groups. Typical aryl groups contain from 1 to 3 separated or fused rings and from 6 to about 18 carbon ring atoms, such as phenyl, naphthyl, indenyl, fenanthryl or anthracyl radical. The aryl group in the compounds of the present invention may be substituted at one or more available positions by one or more suitable groups, e. g., halogen such as F, Cl, Br and I; cyano; hydroxyl; nitro; azido; acyl groups including those groups having 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; carboxamido; alkyl groups including those groups having 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms and more preferably 1 to 3 carbon atoms; alkenyl and alkynyl groups including groups having one or more unsaturated linkages and from 2 to about 12 carbon atoms or from 2 to about 6 carbon atoms; alkoxy groups having one or more oxygen linkages and from 1 to about 12 carbon atoms or 1 to about 6 carbon atoms; aryloxy such as phenoxy; alkylthio groups including those moieties having one or more thioether linkages and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; alkylsulfmyl groups including those moieties having one or more sulfmyl linkages and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; alkylsulfonyl groups including those moieties having one or more sulfonyl linkages and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; aminoalkyl groups such as groups having one or more N atoms and from 1 to about 12 carbon atoms or from 1 to about 6 carbon atoms; aralkyl such as benzyl. Unless otherwise indicated, an optionally substituted group may have a substituent at each substitutable position of the group, and each substitution is independent of the other.

Notable alkali metals include sodium or potassium.

"Sugar" refers to mono-, di- or tri- saccharides or saccharide derivatives, preferably mono- or di- saccharides. Pentose or hexose compounds are preferred. Derivatives include sugar glycosides, N-glycosylamines, O-acyl derivatives, O-methyl derivatives, sugar alcohols, sugar acids, and deoxy sugars.

The term "pharmaceutically acceptable salts" refers to any salt which, upon administration to the patient is capable of providing (directly or indirectly) a compound as described herein. 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 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, alcoholates, particularly methanolates) and it is intended that both forms are within the scope of the present invention. Methods of solvation are generally known within the art. The compounds of the invention may present different polymorphic forms, and it is intended that the invention encompasses all such forms.

Any compound that is a derivative of a compound of formula (Ia) or (Ib) or (Ic) or (Id) is within the scope and spirit of the invention. The term "derivative" is used in its broadest sense and encompasses those compounds that are converted in vivo to the compounds of the invention. Examples of derivatives include, but are not limited to, derivatives and metabolites of the compounds of formula I that include biohydrolyzable moieties such as biohydrolyzable amides, biohydrolyzable esters, biohydrolyzable carbamates, biohydrolyzable carbonates, biohydrolyzable ureides, and biohydrolyzable phosphate analogues. Preferably, derivatives of compounds with carboxyl functional groups are the lower alkyl esters of the carboxylic acid. The carboxylate esters are conveniently formed by esterifying any of the carboxylic acid moieties present on the molecule. Derivatives can typically be prepared using well-known methods, such as those described by Burger "Medicinal Chemistry and Drug Discovery 6th ed. (Donald J. Abraham ed., 2001, Wiley) and "Design and Applications of Prodrugs" (H. Bundgaard ed., 1985, Harwood Academic Publishers). Any compound referred to herein is intended to represent such specific compound as well as certain variations or forms. In particular, compounds referred to herein may have asymmetric centres and therefore exist in different enantiomeric or diastereomeric forms. Thus any given compound referred to herein is intended to represent any one of a racemate, one or more enantiomeric forms, one or more diastereomeric forms, and mixtures thereof. Likewise, stereoisomerism or geometric isomerism about the double bond is also possible, therefore in some cases the molecule could exist as (E)-isomer or (Z)-isomer (trans and cis isomers). If the molecule contains several double bonds, each double bond will have its own stereoisomerism, that could be the same or different than the stereoisomerism of the other double bonds of the molecule. All the stereoisomers including enantiomers, diastereoisomers and geometric isomers of the compounds referred to herein, and mixtures thereof, are considered within the scope of the present invention.

Unless otherwise stated, the compounds of the invention are also meant to include compounds which differ only in the presence of one or more isotopically enriched atoms. For example, compounds having the present structures except for the replacement of an hydrogen by a deuterium or tritium, or the replacement of a carbon by a ¹³C- or ¹⁴C-enriched carbon or ¹⁵N-enriched nitrogen are within the scope of this invention.

Pharmaceutical compositions useful for the method of the invention comprise a compound of formula (Ia) or (Ib) or (Ic) or (Id), or mixtures thereof, a pharmaceutically acceptable salt, derivative, or stereoisomer thereof together with a pharmaceutically acceptable carrier, for administration to a patient.

The term "carrier" refers to a diluent, adjuvant, excipient or vehicle with which the active ingredient is administered. Suitable pharmaceutical carriers are described in "Remington's Pharmaceutical Sciences" by E. W. Martin, 1995.

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.

In a preferred embodiment the pharmaceutical compositions are in oral form, either solid or liquid. Suitable dose forms for oral administration may be tablets, capsules, syrops or solutions and may contain conventional excipients known in the art such as binding agents, for example syrup, acacia, gelatin, sorbitol, tragacanth, or polyvinylpyrrolidone; fillers, for example lactose, sugar, maize starch, calcium phosphate, sorbitol or glycine; tabletting lubricants, for example magnesium stearate; disintegrants, for example starch, polyvinylpyrrolidone, sodium starch glycollate or microcrystalline cellulose; or pharmaceutically acceptable wetting agents such as sodium lauryl sulfate.

The solid oral compositions may be prepared by conventional methods of blending, filling or tabletting. Repeated blending operations may be used to distribute the active agent throughout those compositions employing large quantities of fillers. Such operations are conventional in the art. The tablets may for example be prepared by wet or dry granulation and optionally coated according to methods well known in normal pharmaceutical practice, in particular with an enteric coating.

The pharmaceutical compositions may also be adapted for parenteral administration, such as sterile solutions, suspensions or lyophilized products in the apropriate unit dosage form. Adequate excipients can be used, such as bulking agents, buffering agents or surfactants.

The mentioned formulations will be prepared using standard methods such as those described or referred to in the Spanish and US Pharmacopoeias and similar reference texts.

In view of their biological activity, the compounds of the invention are useful for the treatment of cancer.

Administration of the compounds of formula (Ia) or (Ib) or (Ic) or (Id) or compositions thereof may be by any suitable method, such as intravenous infusion, oral preparations, and intraperitoneal and intravenous administration. Oral administration is preferred because of the convenience for the patient and the chronic character of the diseases to be treated.

Generally an effective administered amount of a compound of formula (Ia) or (Ib) or (Ic) or (Id) will depend on the relative efficacy of the compound chosen, the severity of the disorder being treated and the weight of the sufferer. However, active compounds will typically be administered once or more times a day for example 1, 2, 3 or 4 times daily, with typical total daily doses in the range of from 0.1 to 1000 mg/kg/day. The compounds of formula (Ia) or (Ib) or (Ic) or (Id), and compositions thereof may be used with other drug(s) to provide a combination therapy. The other drug(s) may form part of the same composition, or be provided as a separate composition for administration at the same time or at different times.

A particularly preferred compound of the invention falling under the general formula (Ia) is the Compound I (Ia wherein R=H)

Compound I

Compound I is preferably obtained from a fungus, and more preferably from a terrestrial strain named HT-05-06-5008, being taxonomically classified as Periconia macrospinosa. A culture of this strain has been deposited under the Budapest Treaty in the Colecciόn Espanola de Cultivos Tipo at the University of Valencia, in Spain, under the accession number CECT 20556.

While the deposited strain is clearly preferred, the present invention is not restricted or limited to any particular strain or organism. It is the intention of the present invention to include other producing organisms of compounds of the general formula (Ia) or (Ib) or (Ic) or (Id), strains or mutants within the scope of this invention.

Periconia macrospinosa cultured under controlled conditions in a suitable medium produces the antitumoral Compound I. This strain is preferably grown in an aqueous nutrient medium, under aerobic and mesophilic conditions.

Taxonomic studies of the strain HT-05-06-5008 are summarized as follows: All cultures were incubated at 25°C and records of results were made weekly up to 30 days.

Culture characteristics: colonies reach 4 cm diameter in ten days at 25⁰C on potato dextrose agar in a culture chamber that maintains a humidity of 42%.

Colony characteristics: colonies growing moderately slow, white-yellow, hairy to cottony. The central part of the colony shows a darkest yellow colour. Microscopy: hyaline vegetative hyphae. Conidiophores erect, brown, bearing an apical head of abundant, dense branches. Conidiogenous cells produce short and sometimes branched chains of blastoconidia. Conidia globose, brown, rough-walled, formed in acropetal chains. Conidiophores up to 420 μm long and 7-12 μm wide at the base. Conidia 18-35 μm diameter, coarsely equinulate with spines 2-7 μm long.

The optimal temperature for growth on solid media is 24-28°C. The pH range for growth is between 5 to 7. Growth and sporulation were best with glucose, starch and sucrose. Other carbon sources such as flour, glycerol, and dextrose can also be used. In shacked cultures, the mycelium grows densely and sporulation is inhibited.

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

Compound I can be isolated from the mycelial cake by extraction with a suitable mixture of solvents.

Isolation and purification of Compound I 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 antitumoral activity of fractions.

On the basis of detailed analysis of their various spectral characteristics (Table 1), the pure Compound I can be identified as showed. The APCI-MS spectrum of

Compound I (Figure 3) displayed a (M+H)⁺ peak at 331.

Compound I

TABLE 1. ¹U NMR (400 MHz), ¹³C NMR (100 MHz), COSY, and HMBC spectral data for Compound I [δ (ppm and are referenced to either residual CHCI3 (7.26 ppm) or CDCl₃ (77.0 ppm) signals), J_HH (Hz); CDCl₃]

Position ¹³C (δ) ¹H (O) COSY HMBC

^„ 205Ϊ0 (Q H-Ϊ87HT^'H-2Ϊ 137.5 (C) H-18

146.5 (CH) 6.55 (s) H-18

68.0 (C) H-3, H-5

α 48.6 (CH₂) 1.76 (dd, 12.8,3.8) H-3, H-19

β 1.20 (dd, 12.8)

28.0 (CH) 2.00 (m) H-19 H-5, H-7, H-19

α 42.0 (CH₂) 1.93 (d, 11.2) H-7β, H-8 H-9, H-13, H-19

β 0.80 (dd, 24.0, 12.0) H-7α, H-8

32.0 (CH) 2.38 (brt, 11.2) H-7α, H-7β,H-13 H-7, H-9

125.6 (CH) 5.30 (brs) H-Il, H-20

0 134.0 (C) H-Il, H-20

1 59.3 (CH) 2.70 (s) H-9, H-13, H-15, H-20, H-212 48.3 (C) H-Il, H-13, H-21

3 44.0 (CH) 1.93 (d, 11.2) H-8 H-3, H-5, H-7, H-9, H-11, H-17, H-214 135.5 (C) H-Il, H-17

5 125.5 (CH) 5.25 (q, 6.3) H-16 H-Il, H-16, H-17

6 14.0 (CH₃) 1.53(d, 6.3) H-15 H-15

7 19.0 (CH₃) 1.53 (s) H-Il, H-15

8 62.0 (CH₂) 4.20 (S_AB, 6.3) H-3

9 22.2 (CH₃) 0.95 (d, 6.6) H-6

0 23.0 (CH₃) 1.53 (s) H-9, H-Il

1 23.4(CH₃) 1.25 (s) H-Il, H-13

Other phenalenones of the present invention, of general formula (Ia) or (Ib) or (Ic) or (Id) may be synthesised from Compound I using conventional procedures. For example, using standard organic synthetic reactions known by the skilled person.

Particularly preferred compounds of the invention falling under the general formula (Ia) or (Ib) or (Ic) or (Id) are also the Compounds II- VII.

Compound II Compound III Compound IV

Compound V Compound VI Compound VII The invention will be further illustrated by means of examples. These should be interpreted merely as illustrative of the invention.

EXAMPLES

Example 1: Production of Compound I

The following Example is presented only to illustrate this invention and it is not intended to limit its scope.

Stock culture: a spore suspension of a pure culture of Periconia macrospinosa, strain HT-05-06-5008 (CECT 20556), was kept frozen at -7O⁰C in 20% glycerol in H₂O.

Preparation of inoculum: a well grown agar culture was used to inoculate 50 ml of seed medium containing 2% oat meal, 2% malt extract, 0.01% KH₂PO₄, 0.005% MgSO₄ and tap water in 250 ml shake flasks and cultured at 25°C on a rotary shaker at 200 rpm. The flasks were incubated 48 hours, and used as a first stage inoculum.

Fermentation: 250 ml of the seed medium in 2 L erlenmeyer flasks were inoculated with 10% of the first stage inoculum. The fermentation was carried for 7 days at 25°C on a rotary shaker at 200 rpm.

Production of Compound I can be monitored by whole broth assay against A549 or Hl 16 or PSNl or T98G cell lines or any other sensitive cell or by HPLC or any other method with enough sensitivity.

Example 2: Isolation of Compound I

4 Litres of whole harvested broth were filtrated to separate the biomass and other solids. The mycelia cake was extracted twice with 2 L of EtOAc-MeOH (3: 1). The activity was concentrated in the upper layer. The organic solvent was concentrated and evaporated to dryness in vacuum to yield 9.1 g of crude extract.

The extract was applied to a silica gel VFC (Vacuum Flash

Chromatography) system, using mixtures of growing polarity solvents from n-hexane- EtOAc to EtOAc-MeOH as eluting solvents. The fractions containing Compound I (438 mg), with cytotoxic activity, were eluted with n-hexane-EtOAc (2:8) and EtOAc. The active fractions were chromatographied by column on silica gel, using CHCIs-MeOH mixtures as eluting solvent. The cytotoxic activity was detected in fractions eluted with CHCl₃-MeOH 97 : 3 ( 1 75 mg) . Further purification by C18 reversed phase chromatography afforded 34 mg of pure Compound I, eluted with MeOH-H₂O 95:5. HPLC (HP 1 100) analysis was performed at 20 ⁰C using an analytical column (3.9 x 150mm) Symmetry Cl 8 (5mm) and as a mobile phase a gradient from 50% MeOH/H₂O (0.04% TFA) to 100% in 20 minutes, a flow rate of 0.5 mL/min, injection volume: 3 μL, and plotted at 220 and 280 nm. Under these conditions Compound I retention time is 21.4 min as is shown in figure 1. Example 3: Synthesis of Compound II

A mixture of pyridine (2.4 μL), Ac₂O (2.4 μL) and CH₂Cl₂ (240 μL) was added, at O⁰C, to Compound I (8 mg, 24 μmol). After 90 min of reaction, a mixture of pyridine (2.4 μL), Ac₂O (2.4 μL) and CH₂Cl₂ (240 μL) was added to the reaction mixture. After 3 h at O⁰C, the temperature was left to warm to room temperature and a mixture of pyridine (2.4 μL), Ac₂O (2.4 μL) and CH₂Cl₂ (240 μL) was added. Finally, after 26 h, a mixture of pyridine (2.4 μL), Ac₂O (2.4 μL) and CH₂Cl₂ (240 μL) was added and the reaction mixture was stirred for 20 h more. The solvent was removed by evaporation in vacuum. Compound II was purified (6.2 mg, 17 mmol, 70%) by chromatography on silica gel (Hex: AcOEt, 1 :2 v/v). ¹H NMR (500 MHz) for Compound II: δ (J_HH Hz) 6.57 (s, IH), 5.30 (s, IH), 5.28 (q, 6.8, IH), 4.76 (d, 13.2, IH), 4.69 (d, 13.2, IH), 2.71 (s, IH), 2.37 (t_br, 11.3, IH), 2.07 (s, 3H), 2.03 (m, IH), 1.94 (d, 11.3, IH), 1.93 (d_br, 12.9, IH), 1.77 (dd, 13.6, 2.4, IH), 1.55 (s, 6H), 1.53 (d, 7.1, 3H), 1.30 (s, 3H), 1.23 (t, 12.8, IH), 0.98 (d, 6.5, 3H) and 0.80 (q, 12.2, IH) ppm. ¹³C NMR (125 MHz) for Compound II: δ 202.1, 170.5, 147.0, 134.7, 133.8 (x2), 125.1, 124.9, 68.0, 61.5, 59.2, 48.3, 47.6, 43.7, 41.3, 31.7, 27.4, 23.2, 22.7, 22.0, 20.9, 18.8 and 13.8 ppm. The APCI-MS spectrum of Compound II displayed a (M+H)⁺ peak at 373.

Example 4: Synthesis of Compound III

Trichloro acetyl isocyanate (13 μL, 107 μmol) was added dropwise to a solution of Compound I (6 mg, 18 μmol) in CH₂Cl₂ (900 μL). The resulting mixture was stirred for 4 h at 65⁰C in a sealed tube. Then, the mixture was deposited in a short column of neutral alumina that was pre-wetted with CH₂Cl₂. After 30 min on standing, the mixture was eluted with AcOEt. Compound III was purified (5.5 mg, 15 μmol, 86%) by chromatography on silica gel (Hex: AcOEt, 1 :1 v/v). ¹H NMR (500 MHz) for Compound III: δ (J_HH Hz) 6.93 (s, IH), 5.97 (s_br, IH), 5.36 (q, 7.7, IH), 5.33 (s, IH), 4.78 (s, 2H), 4.61(s_br, 2H), 2.77 (s, IH), 2.75 (m, IH), 2.56 (m, IH), 2.25 (t_br, 11.7, IH), 2.01 (dd, 12.4, 6.0, 2H), 1.70 (s, 3H), 1.58 (d, 6.2, 3H), 1.56 (s, 3H), 1.11 (d, 7.2, 3H) and 1.00 (s, 3H) ppm. ¹³C NMR (125 MHz) for Compound III: δ 201.6, 156.5, 142.7, 142.1, 136.0, 135.5, 132.9, 130.0, 124.5, 123.5, 62.3, 57.0, 48.3, 39.1, 37.4, 34.4, 33.3,

22.6, 22.1, 21.0, 19.0 and 13.8 ppm. The APCI-MS spectrum of Compound III displayed a (M+Na)⁺ peak at 378.

Example 5: Synthesis of Compound IV

A solution of trichloroacetyl isocyanate (18 μL, 150 μmol) in CH₂Cl₂ (750 μL) was added dropwise to a solution of Compound I (5 mg, 15 μmol) in CH₂Cl₂ (500 μL). The resulting mixture was stirred at room temperature for Ih. Then, the mixture was deposited in a short column of neutral alumina that was pre-wetted with CH₂Cl₂. After 30 min on standing, the mixture was eluted with AcOEt. Compound IV was purified (5.1 mg, 14 μmol, 93%) by chromatography on silica gel (Hex:AcOEt, 1 :1 v/v). ¹H NMR (500 MHz) for Compound IV: δ (J_HH Hz) 5.46 (s, IH), 5.44 (s_br, IH), 5.29 (s, IH), 5.25 (q, 6.5, IH), 5.19 (s, IH), 4.14 (s, IH), 2.63 (s, IH), 2.43 (t_br, 11.1, IH), 2.10 (d_br, 10.5, IH), 2.10 (m, IH), 1.94 (d_br, 12.8, IH), 1.89 (d, 11.3, IH), 1.52 (s, 3H), 1.51 (d, 6.5, 3H), 1.50 (s, 3H), 1.25 (t, 10.0, IH), 1.23 (s, 3H), 0.97 (d, 6.4, 3H) and 0.83 (q, 12.3, IH) ppm. ¹³C NMR (125 MHz) for Compound IV: δ 207.9, 156.7, 145.9, 133.5, 133.1, 126.1, 125.4, 122.2, 83.3, 62.5, 60.7, 49.2, 48.1, 42.9, 40.8, 32.8, 28.0, 22.3,

21.7, 20.6, 18.1 and 13.8 ppm. The APCI-MS spectrum of Compound IV displayed a (M+H)⁺ peak at 356.

Example 6: Synthesis of Compound V

A solution of trichloroacetyl isocyanate (1.5 μL, 12.4 μmol) in CH₂Cl₂ (250 μL) was added dropwise to a solution of Compound I (4.1 mg, 12.4 μmol) in CH₂Cl₂ (750 μL). The resulting mixture was stirred at room temperature for 30 min. Then, the mixture was deposited in a short column of neutral alumina that was pre-wetted with

CH₂Cl₂. After 30 min on standing, the mixture was eluted with AcOEt. Compound V was purified (2.1 mg, 5.6 μmol, 45%) by chromatography on silica gel (Hex: AcOEt, 3:1 v/v). ¹H NMR (500 MHz) for Compound V: δ (J_HH Hz) 6.61 (s, IH), 5.33 (s, IH), 5.31 (q, 6.4, IH), 4.76 (s, 2H), 4.61(s_br, 2H), 2.74 (s, IH), 2.40 (t_br, 11.1, IH), 2.03 (m, IH), 1.97 (d,l 1.4, IH), 1.95 (d_br, 13.0, IH), 1.79 (dd, 13.2, 2.3, IH), 1.58 (s, 3H), 1.57 (s, 3H), 1.56 (d, 6.5, 3H), 1.32 (s, 3H), 1.26 (t, 12.8, IH), 1.00 (d, 6.6, 3H) and 0.82 (q, 12.1, IH) ppm. ¹³C NMR (125 MHz) for Compound V: δ 203.9, 156.4, 147.0, 135.0, 134.3, 134.0, 125.4, 125.1, 68.2, 62.3, 59.4, 48.6, 47.9, 44.0, 41.6, 31.9, 27.7, 23.5, 23.0, 22.2, 19.0 and 14.1 ppm. The APCI-MS spectrum of Compound V displayed a (M+H)⁺ peak at 374.

Example 7: Synthesis of Compound VI

Sodium borohydride (700 μg, 18 μmol) was added to a solution of Compound IV (2.6 mg, 7.3 μmol) in MeOH (500 μL). After 2h of reaction another portion of sodium borohydride (700 μg, 18 μmol) was added and the reaction was stirred for 90 min more. The reaction was quenched by the addition of IN HCl (5 mL) and the aqueous phase was extracted with Et₂O (3 x 5 mL). Drying of the combined organic layers was followed by evaporation of the solvent under vacuum. Compound VI was purified (2.0 mg, 5.5 μmol, 75%) by chromatography on silica gel (Hex: AcOEt, 1 :1 v/v). ¹H NMR (500 MHz) for Compound VI: δ 5.35 (s, IH), 5.31 (q, 6.4, IH), 5.23 (s_br, IH), 3.73 (d, 4.0, IH), 2.54 (s, IH), 2.42 (qd, 6.7, 4.2, IH), 2.39 (t_br, 12.6, IH), 2.11 (m, IH), 2.07 (d, 11.3, IH), 2.06 (d_br ,12.0, IH), 1.96 (d_br, 11.3, IH), 1.51 (s, 3H), 1.60 (d, 6.7, 3H), 1.54 (s, 3H), 1.28 (t, 11.9, IH), 1.19 (s, 3H), 1.02 (d, 6.7, 3H), 0.99 (d, 6.5, 3H) and 0.85 (q, 12.3, IH) ppm. ¹³C NMR (125 MHz) for Compound VI: δ 214.6, 156.9, 134.0, 133.3, 126.3, 124.8, 82.9, 63.2, 61.3, 48.6, 48.0, 44.7, 42.4, 40.7, 32.9, 28.0, 22.3, 21.7, 19.5, 18.9, 13.9 and 10.8 ppm. The APCI-MS spectrum of Compound VI displayed a (M+H)⁺ peak at 358.

Example 8: Synthesis of Compound VII

250 μL of solution A (1.23 x 10^"4 M solution OfEt₃N in CH₂Cl₂) and 250 μL of solution B (1.27 x 10^"4 M solution OfHSiCl₃ in CH₂Cl₂) were added, successively, to a solution of Compound III (4.9 mg, 13.8 μmol) in CH₂Cl₂ (400 μL). After 5 h of reaction, another 250 μL of solution A and 250 μL of solution B were added. Finally, after 24 h, another 250 μL of solution A and 250 μL of solution B were added and the reaction was stirred for 2 h more. The mixture was the diluted with CH₂Cl₂ (10 mL) and washed with aqueous saturated solution Of NH₄Cl. The organic phase was dried over Na₂SO₄ and the solvent evaporated under vacuum. Compound VII was purified (1.2 mg, 3.2 μmol, 23%) by chromatography on silica gel (Hex:AcOEt, 1 :1 v/v). ¹H NMR (500 MHz) for Compound VII: δ 6.85 (s, IH), 5.95 (s_br, IH), 5.36 (q, 6.9, IH), 5.35 (s, IH), 4.31 (dd, 14.2, 6.7, IH), 4.25 (dd, 14.2, 7.3, IH), 2.78 (s, IH), 2.76 (m, IH), 2.57 (m, IH), 2.54 (t, 6.5, IH), 2.26 (t_br, 10.0, IH), 2.02 (m, 2H), 1.69 (s, 3H), 1.60 (s, 3H), 1.58 (d, 6.9, 3H), 1.11 (d, 7.1, 3H) and 1.03 (s, 3H) ppm. ¹³C NMR (125 MHz) for Compound VII: δ 203.9, 141.7, 141.5, 135.8, 135.3, 133.7, 132.9, 124.7, 123.7, 62.5, 58.9, 48.4, 39.2, 37.4, 34.4, 33.3, 22.7, 22.2, 21.0, 18.7 and 13.8 ppm. The APCI-MS spectrum of Compound VII displayed a (M+H)⁺ peak at 313.

Example 9: in vitro activity against solid cancer cell lines

Cell Culture

All the tumour cell lines were obtained from the ATCC. Human lung carcinoma A549, colon adenocarcinoma Hl 16, pancreatic adenocarcinoma PSNl, and human Caucasian glioblastoma T98G were cultured in RPMI medium containing glutamine, (2 mM) penicillin (50 IU/mL), streptomycin (50 μg/mL), supplemented with 5% FBS (A549 and Hl 16) or 10% FBS (PSNl and T98G).

Cell proliferation assay

The 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium (MTT; Sigma Chemical Co., St. Louis, MO) dye reduction assay in 96-well microplates was used, essentially as described in Mosmann, T. J. Immunol. Methods 1983, 65, 55. The assay is dependent on the reduction of MTT by mitochondrial dehydrogenases of viable cell to a blue formazan product, which can be measured spectophotometrically. Tumor cells (4xlO³ A-549 cells or 6xlO³ H-1 16 or 6xlO³ PSNl or 6xlO³ T98G cells in a total volume of 200 μL of complete medium) were seeded and serial dilutions in DMSO (10 μg/ml, 5 μg/ml, 1 μg/ml, 0.5 μg/ml, 0.1 μg/ml, 0.05 μg/ml, 0.01 μg/ml, and 0.005 μg/ml) of the tested compound were added to the wells. After 2 days of incubation (37 ⁰C, 5% CO₂ in a humid atmosphere), 50 μL of MTT (1 mg/mL in PBS) were added to each well and the plate was incubated for a further 2 h (37 ⁰C). The resulting formazan was dissolved in 100 μL DMSO and read at 490 nm. All determinations were carried out in triplicate. IC50 value was calculated as the concentration of drug yielding a 50% of cell survival. Table 2 illustrates data on the cytotoxic activity of the compounds of the present invention.

TABLE 2

Example 10: in vitro activity against myeloid leukaemia cell lines

Cell lines used

Cell line Type species

K-562 Chronic Myelogenous Leukemia Human

NB-4 Acute Promyelocytic Leukemia Human

HL-60 Acute Myeloid Leukemia with maturation Human

MV4-11 Acute Monocytic Leukemia Human

HEL Acute Erythroid Leukemia Human

KG-I Acute Erythroid Leukemia Human

Methods and materials

As in example 9, the method to assay proliferation/survival is based on the metabolic bromide reduction from 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazole (MTT).

Methodology

Cells are plated at 20,000 cells per well into the 96-well microtiter plates.

The cells are cultured for 24-48-72h (depending on the cell line) at 37 ⁰C, 5 % CO₂ to allow attachment to the surface of the well.

The compound to be assayed is prepared in DMSO 0.1%. Add 1 μL of the sample solution to the corresponding wells (assays are performed on quadruplicates).

Incubate for 24-48-72h in the incubator to allow the substance to act on all the cells.

Add to each well 22 μL of MTT (48-well microtiter plates) or 10 μL of MTT (96-well microtiter plates).

Incubate 2h to allow formazan crystal formation.

Add 220 μL of SDS plus HCl (48-well microtiter plates) / 100 μL (96-well microtiter plates).

Keep at room temperature until formazan crystals are dissolved. Read absorbance at 570 nm OD and use a reference wavelength of 650 nm.

Cell viability (percentage) is obtained as follows:% Viability = OD treated cells x 100/

OD control cells.

Each assay must be performed three times. Negative controls (solvent alone), positive controls (known cytotoxic substance), and untreated cells must be included.

Table 3 illustrates data on the in vitro cytotoxic activity against myeloid leukemias after 48 h of incubation.

TABLE 3

Claims

1.- A compound of general formula (Ia) or (Ib) or (Ic) or (Id)

(Ia) (Ib)

(Ic) (Id) wherein the R groups are each independently selected from the group consisting of hydrogen, acyl, alkyl, alkenyl, alkynyl, aryl, -CONH₂, alkali metal, and sugar, or a pharmaceutically acceptable salt, derivative, or stereoisomer thereof.

2.- A compound according to claim 1, wherein the compound of formula (Ia) is

3.- A compound according to claim 1, wherein the compound of formula (Ia) is

4.- A compound according to claim 1, wherein the compound of formula (Ia) is

5.- A compound according to claim 1, wherein the compound of formula (Ib) is

6.- A compound according to claim 1, wherein the compound of formula (Ib) is

7.- A compound according to claim 1, wherein the compound of formula (Ic) is

8.- A compound according to claim 1, wherein the compound of formula (Id) is

9.- 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.

10.- The process according to claim 10, which further comprises isolating and purifying the active compound from the cultured broth.

11.- A process according to any of claims 9 or 10, wherein the compound of formula (Ia) is

12.- A process according to any of claims 9 to 11, wherein the microorganism is a fungus.

13.- A process according to claim 12, wherein the microorganism is the substantially pure culture strain HT-05-06-5008, available under accession number CECT 20556, from the Colecciόn Espanola de Cultivos Tipo at the University of Valencia, Spain.

14.- A process for producing a compound as defined in claim 1 which comprises preparing a derivative of the compound as defined in claim 2.

15.- A process according to claim 14, wherein the compound as defined in claim 2 is treated with trichloro acetyl isocyanate.

16.- A pharmaceutical composition comprising a compound as defined in any of claims 1-8, or a pharmaceutically acceptable salt, derivative, or stereoisomer thereof, and a pharmaceutically acceptable carrier.

17.- A compound as defined in any of claims 1-8, or a pharmaceutically acceptable salt, derivative, or stereoisomer thereof, for use as a medicament.

18.- A compound as defined in any of claims 1-8, or a pharmaceutically acceptable salt, derivative, or stereoisomer thereof, for use as a medicament for the treatment of cancer.

19.- Use of a compound as defined in any of claims 1-8, or a pharmaceutically acceptable salt, derivative, or stereoisomer thereof, in the preparation of a medicament for the treatment of cancer.

20.- A method of treating cancer which comprises administering to a patient in need of such treatment a therapeutically effective amount of a compound as defined in any of claims 1-8, or a pharmaceutically acceptable salt, derivative or stereoisomer thereof.