WO2012130906A1 - Derivatives useful for the treatment or prevention of bone tumors - Google Patents

Abstract

The invention relates to a compound of formula (I) or a pharmaceutically acceptable salt thereof, where: X is a halogen atom or an OR₃, SR₃, or NR₃R₄ group; A₁ is an X₁-A₃-X₂ group in which X₁ and X₂ independently are an -O-, -S-, -NR₅-, or -C=C- bond, and A₃ is a (C₁-C₁₀)alkyl chain optionally interrupted by one or more unit, preferably one unit, selected from among -O-, -S-, -NR₆-, -C(O)-, and -C=C-, or an optionally substituted aryl group; A₂ is a -COOH-, C(O)O-((C₁-C₆)alkyl), or -C(OH)(PO₃H₂)₂ group; and - R₁ is a hydrogen atom or a -C(O)-((C₁-C₆)alkyl) group. The invention also relates to the use of said compound as a medicament, in particular for the treatment and prevention of bone tumors, pharmaceutical compositions containing the same, and a method for producing the same.

Description

 DERIVATIVES USEFUL IN THE TREATMENT OR

 THE PREVENTION OF BONE TUMORS

The present invention relates to derivatives useful as a medicament, and more particularly for the treatment of bone tumors, as well as to a process for their preparation.

Two types of bone tumors are presently counted: primary bone tumors such as osteosarcomas, chondrosarcomas and Ewing's sarcomas; secondly, secondary tumors or bone metastases (particularly frequent in the evolution of cancerous pathologies) due to the dissemination of tumor cells that have a strong bone (osteophilic) tropism such as mammary or prostatic carcinoma cells.

 In pathological conditions, the balance between formation and bone resorption is broken, regardless of the origin of tumor cells (bone or not). Thus, bone remodeling, continuous throughout life, can be done either in favor of bone formation (osteoconductive tumors), or conversely, in favor of bone resorption (osteolytic tumors). But in most cases tumors are mixed, as in the case of osteosarcomas or bone metastases secondary to carcinoma. Osteosarcoma of osteoblastic origin is the most common of primary bone tumors, and is characterized by abundant bone formation associated with significant osteolytic lesions.

 The current standard treatment of primary bone tumors begins with a biopsy to establish the histological diagnosis. A poly-chemotherapy (OS 2006) is initiated which comprises 10 courses in total. Four neo-adjuvant induction courses combining ifosfamide and veside or methotrexate alone precede tumor exeresis before a new adjuvant poly-chemotherapy regimen of 6 courses. Depending on the response of the tumor to the first treatment, the same treatment is applied (good responder) or otherwise the combination adriamycin / cisplatin is used (in the case of poor responders to the first treatment).

Unfortunately, in many cases, a lack of response to anti-tumor drugs is observed leading to the development of metastases (mainly pulmonary) and then on the patient's death. Thus, the survival of patients is 56 to 83% at 5 years in the absence of pulmonary metastases at the time of diagnosis and 30% at 5 years when pulmonary metastases are detected at the time of diagnosis.

 There is therefore a real need to develop new molecules useful in the treatment and prevention of bone tumors.

The subject of the present invention is therefore a compound of formula (I) below:

or a pharmaceutically acceptable salt thereof,

for which :

X represents a halogen atom or a group OR ₃ , SR ₃ or R ₃ R ₄ ,

- Ai represents a group Xi-A ₃ -X ₂ for which:

^■ Xi and X ₂ are each independently of the other a bond, -O-, -S-, - R 5, -C (O) - or -C = C-, and

^■ A ₃ represents a chain (Ci-Cio) alkyl, preferably (Ci-Ce) alkyl optionally interrupted by one or more units, preferably one, selected from -O-, -S-, -NR _O -, - C (O) - and -C = C-; or an optionally substituted aryl, cycloalkyl or heteroaryl group,

- A ₂ represents a group -COOH, -C (O) O - ((C ₁ -C ₆ ) alkyl) or -C (OH) (P0 ₃ H ₂ ) ₂ ,

R 1 represents a hydrogen atom or a -C (O) - ((C ₁ -C ₆ ) alkyl) group,

Each of R ₃ and R ₄ represents, independently of one another, a hydrogen atom or a (C ₁ -C ₆ ) alkyl, aryl or aryl (C ₁ -C ₆ ) alkyl group, and

R ₅ and 5 each independently of one another represent a hydrogen atom or a (C ₁ -C ₆ ) alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, aryl (C 1 -C 6) alkyl group or acyl; especially a hydrogen atom or a (C ₁ -C ₆ ) alkyl, aryl or aryl (C ₁ -C ₆ ) alkyl group; preferably a hydrogen atom or a (C ₁ -C ₆ ) alkyl group,

 the cycloalkyl, heterocyclic, aryl and heteroaryl groups being optionally substituted; and preferably unsubstituted.

In the present invention, the term "pharmaceutically acceptable" is intended to mean that which is useful in the preparation of a pharmaceutical composition which is generally safe, non-toxic and neither biologically nor otherwise undesirable and which is acceptable for veterinary use as well as human pharmaceutical.

 The term "pharmaceutically acceptable salts" of a compound means salts which are pharmaceutically acceptable, as defined herein, and which possess the desired pharmacological activity of the parent compound. Such salts include:

 (1) hydrates and solvates,

 (2) pharmaceutically acceptable acid addition salts formed with pharmaceutically acceptable inorganic acids such as hydrochloric acid, hydrobromic acid, sulfuric acid, nitric acid, phosphoric acid and the like; or formed with pharmaceutically acceptable organic acids such as acetic acid, benzenesulfonic acid, benzoic acid, camphorsulfonic acid, citric acid, ethanesulfonic acid, fumaric acid, acid glucoheptonic acid, gluconic acid, glutamic acid, glycolic acid, hydroxynaphthoic acid, 2-hydroxyethanesulfonic acid, lactic acid, maleic acid, malic acid, mandelic acid, methanesulfonic acid, muconic acid, 2-naphthalenesulfonic acid, propionic acid, salicylic acid, succinic acid, dibenzoyl-L-tartaric acid, tartaric acid, p-acid, toluenesulphonic acid, trimethylacetic acid, trifluoroacetic acid and the like, or

(3) pharmaceutically acceptable base addition salts formed when an acidic proton present in the parent compound is either replaced by a metal ion, for example an alkali metal ion, an alkaline earth metal ion or a aluminum; is coordinated with a pharmaceutically acceptable organic or inorganic base. Acceptable organic bases include diethanolamine, ethanolamine, N-methylglucamine, triethanolamine, tromethamine and the like. Acceptable inorganic bases include aluminum hydroxide, calcium hydroxide, potassium hydroxide, sodium carbonate and sodium hydroxide.

 For the purposes of the present invention, the term "halogen atom" means the fluorine, chlorine, bromine and iodine atoms.

By "(C 1 -C 10) alkyl" or "(C ₁ -C ₆ ) alkyl" group, is meant, in the sense of the present invention, a saturated hydrocarbon chain, linear or branched, comprising 1 to 10, respectively 1 to 6, and preferably 1 to 4, carbon atoms. By way of example, mention may be made of methyl, ethyl, propyl, isopropyl, butyl, isobutyl, sec-butyl, tert-butyl, pentyl or hexyl groups.

 For the purposes of the present invention, the term "aryl" means an aromatic hydrocarbon group, preferably comprising from 6 to 10 carbon atoms, and comprising one or more contiguous rings, for example a phenyl or naphthyl group. Advantageously, it is phenyl.

By "aryl- (Ci-C ₆₎ alkyl" is understood within the meaning of the present invention, an aryl group, as defined above, bonded to the molecule via a chain (Ci-C ₆ ) alkyl, as defined above. By way of example, mention may be made of the benzyl group.

 By "heteroaryl" is meant, in the sense of the present invention, an aromatic group comprising one or more contiguous rings, 5 to 10 atoms forming the ring (s) including one or more heteroatoms, advantageously 1 to 4, and also more preferably 1 or 2, such as for example sulfur, nitrogen, oxygen, phosphorus or selenium atoms and preferably sulfur, nitrogen or oxygen, the other atoms of the cyclic or cyclic being carbon atoms. Examples of heteroaryl groups are furyl, thienyl, pyrrolyl, pyridinyl, pyrimidinyl, pyrazolyl, imidazolyl, triazolyl, tetrazolylindyl or even selenophenyl.

By "cycloalkyl" group is meant, in the sense of the present invention, a saturated mono- or poly-cyclic (and especially bicyclic or tricyclic) hydrocarbon chain, 3 to 10 carbon atoms forming the ring (s). In the case of a polycyclic group, the rings may be, two by two, contiguous, bridged or joined by a spiro-type ring junction. By way of example, mention may be made of cyclopropyl, cyclopentyl, cyclohexyl and cycloheptyl groups. By "heterocyclic" group is meant, in the sense of the present invention, a 5- to 10-membered ring, saturated or unsaturated, but not aromatic, and containing one or more, advantageously 1 to 4, still more advantageously 1 or 2, heteroatoms, such as, for example, sulfur, nitrogen or oxygen atoms. It may especially be pyrrolidinyl, piperidinyl, piperazinyl or morpholinyl group.

The aryl, cycloalkyl, heteroaryl and heterocyclic groups, when substituted, may be substituted with one or more groups selected from the group consisting of a halogen atom, -NO ₂ , -CN, -OH, -SH, - R ₇ R ₈ , -SO ₃ R ₉ , -COOR 10, -OP (O) (OR n) (ORi ₂ ), -P (O) (ORi ₃ ) (ORi 4), -C (O) Ri ₅ , (Ci-C) ₆ ) alkyl and (C ₁ -C ₆ ) alkoxy, with R ₇ to R ₁₅ representing, independently of each other, a hydrogen atom or a group (C ₁ -C ₆ ) alkyl.

By "acyl" group is meant, in the sense of the present invention, a -C (O) -R _{22 group} in which R ₂₂ represents a (C ₁ -C ₆ ) alkyl, aryl or aryl- (C ₁ -C ₆ ) group. ₆ ) alkyl.

According to one particular embodiment of the invention, the compounds according to the invention correspond to a compound of formula (I) for which:

X represents a halogen atom or a group OR ₃ , SR ₃ or R ₃ R ₄ ,

- Ai represents a group Xi-A ₃ -X ₂ for which:

Each of X ₁ and X ₂ independently of one another is a bond,

-O-, -S-, - R ₅ -, -C (O) - or -C = C-, and

A ₃ represents a (C 1 -C 10) alkyl chain, preferably (C ₁ -C ₆ ) alkyl chain, optionally interrupted by one or more units, preferably by one, selected from -O-, -S-, -NR _Ô- , -C (O) - and -C = C-; or an optionally substituted aryl, cycloalkyl or heteroaryl group,

- A ₂ represents a -COOH or-C (O) O - ((C ₁ -C ₆ ) alkyl) group,

 Ri represents a hydrogen atom or a -C (O) - ((C 1 -C 6) alkyl) group;

R ₃ and R ₄ each independently represent a hydrogen atom or a (C ₁ -C ₆ ) alkyl, aryl or aryl (C ₁ -C ₆ ) alkyl group,

R ₅ and 5 each independently of one another represent a hydrogen atom or a (C ₁ -C ₆ ) alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, aryl (C 1 -C 6) alkyl group or acyl; especially a hydrogen atom or a (C ₁ -C ₆ ) alkyl, aryl or aryl (C ₁ -C ₆ ) alkyl group; preferably a hydrogen atom or a (C ₁ -C ₆ ) alkyl group,

 the cycloalkyl, heterocycle, aryl and heteroaryl groups being optionally substituted.

According to another particular embodiment of the invention, the compounds according to the invention correspond to a compound of formula (I) for which:

X represents a halogen atom or a group OR ₃ , SR ₃ or R ₃ R ₄ ,

A 1 represents a (C 1 -C 10) alkyl group, preferably (C ₁ -C ₆ ) alkyl,

- A ₂ represents a group -COOH, -C (O) O - ((C ₁ -C ₆ ) alkyl) or -C (OH) (P0 ₃ H ₂ ) ₂ ,

R 1 represents a hydrogen atom or a -C (O) - ((C 1 -C 6) alkyl) group, and

R ₃ and R ₄ each independently represent a hydrogen atom or a (C ₁ -C ₆ ) alkyl, aryl or aryl (C ₁ -C ₆ ) alkyl group. Preferably, X will represent a halogen atom, such as a chlorine atom or optionally bromine.

Advantageously, Xi and X ₂ each represent a link.

Advantageously, A ₃ represent a chain (Ci-Cio) alkyl, preferably (C -C ₆₎ alkyl, optionally interrupted by a moiety selected from -O-, -S-, -NR _O -, -C (O) - and -C = C-; or an optionally substituted aryl group, and in particular an unsubstituted aryl group. Preferably, A ₃ represent a chain (Ci-Cio) alkyl, preferably (C -C ₆₎ alkyl, optionally interrupted by a moiety selected from -O-, -S-, and -NR _O -, in particular -O -; or an optionally substituted aryl group, and in particular an unsubstituted aryl group.

Advantageously, A _{2 will} represent a -COOH group.

 Preferably, R 1 will represent a hydrogen atom.

 According to a particularly advantageous embodiment, the compound according to the invention will be a compound of formula (I) for which:

 X represents a halogen atom such as Cl or Br,

- Ai represents a chain (Ci-Cio) alkyl, preferably (Ci-C ₆₎ alkyl, optionally interrupted by a moiety selected from -O-, -S-, and -NR _O -, and especially -O-; or an optionally substituted aryl group, and in particular an unsubstituted aryl group,

- A ₂ represents a group -COOH,

 Ri represents a hydrogen atom, and

- Re represents a hydrogen atom or a group (C ₁ -C ₆ ) alkyl, aryl or aryl- (C ₁ -C ₆ ) alkyl; preferably a hydrogen atom or a (C ₁ -C ₆ ) alkyl group. The compound of formula (I) may more particularly be one of the compounds

La présente invention a également pour objet un composé de formule (I) selon la présente invention pour son utilisation comme médicament, notamment dans le traitement ou la prévention d'une tumeur osseuse.

The subject of the present invention is also a compound of formula (I) according to the present invention for its use as a medicament, especially in the treatment or prevention of a bone tumor.

 The present invention also relates to the use of a compound of formula (I) according to the invention for the preparation of a medicament, in particular for treating or preventing a bone tumor.

 The present invention also relates to a method for treating or preventing a bone tumor, comprising administering to a person in need of an effective amount of at least one compound of formula (I) according to the invention.

 The bone tumor may be a primary bone tumor such as osteosarcoma, chondrosarcoma and Ewing's sarcoma; or a secondary bone tumor or bone metastases such as mammary or prostatic carcinoma cells. The present invention also relates to a pharmaceutical composition comprising at least one compound of formula (I) according to the invention and at least one pharmaceutically acceptable excipient.

 The pharmaceutical compositions according to the invention may be formulated for oral or parenteral administration such as intravenous, intended for mammals, including humans. The dosage varies according to the treatment and the condition in question.

 The active ingredient can be administered in unit dosage forms, in admixture with conventional pharmaceutical carriers, to animals or humans. Suitable unit dosage forms include oral forms such as tablets, capsules, powders, granules and oral solutions or suspensions, and parenteral administration forms such as intravenous.

When a solid composition in tablet form is prepared, the main active ingredient is mixed with a pharmaceutical carrier such as gelatin, starch, lactose, magnesium stearate, talc, gum arabic or the like. The tablets can be coated with sucrose or other suitable materials or they can be treated in such a way that they have prolonged or delayed activity and continuously release a predetermined amount of active ingredient. A preparation in capsules is obtained by mixing the active ingredient with a diluent and pouring the resulting mixture into soft or hard gelatin capsules.

 A syrup or elixir preparation may contain the active ingredient together with a sweetener, an antiseptic, as well as a flavoring agent and a suitable colorant.

 Water-dispersible powders or granules may contain the active ingredient in admixture with dispersing agents or wetting agents, or suspending agents, as well as with taste correctors or sweeteners.

 For parenteral, especially intravenous administration, aqueous suspensions, isotonic saline solutions or sterile and injectable solutions containing dispersing agents and / or pharmacologically compatible wetting agents are used.

 The active ingredient may also be formulated as microcapsules, optionally with one or more additive carriers.

 The compounds of the invention as active ingredients can be used at doses of between 0.01 mg and 1000 mg per day, given as a single dose once a day or administered in several doses throughout the day, for example twice a day in equal doses. The dose administered per day is advantageously between 5 mg and 500 mg, more advantageously between 10 mg and 200 mg. It may be necessary to use doses out of these ranges which the skilled person can realize himself.

 The pharmaceutical composition according to the invention may also contain another active ingredient such as an anticancer agent.

The present invention also relates to a process for preparing a compound of formula (I) according to the invention comprising the following successive stages: (a) opening of the epoxide and halogenation of the compound of formula (II) below:

for which A ₁ and A ₂ are as defined above,

 to give a compound of formula (Ia) below:

for which A ₁ and A ₂ are as defined above and Hal represents a halogen atom, the compounds of formula (Ia) corresponding to compounds of formula (I) according to the invention with X = Hal and Ri = H,

 optionally nucleophilic substitution of the OH or Hal function of the compound of formula (Ia) obtained in the preceding step to give a compound of formula (I) for which X ≠ Hal and / or Ri ≠ H, and

 optionally salifying the compound of formula (I) or (la) obtained in the preceding step to obtain a pharmaceutically acceptable salt thereof.

Step (a):

 This step will be carried out in the presence of a halogenation reagent under conditions well known to those skilled in the art.

 This reaction may especially be carried out in the presence of an acid such as acetic acid and a halide salt such as M-Hal with M selected from Li, K, Na,

Rb, Cs, or NH ₄ . M-Hal will represent in particular Li-Cl or Li-Br. In this case, the reaction may be carried out in a solvent such as tetrahydrofuran, especially at a temperature between 0 and 30 ° C. Any other halogenation reaction condition known to those skilled in the art may, however, be used.

 The compound of formula (II) can be obtained by coupling of the OH function of fumagillol of formula 2 below:

OH ₂

with the COOH function of the carboxylic acid of formula (III) below

HO. _^ A ^J , 1 -

O (III)

for which A ₁ and A ₂ are as defined above, A ₂ being optionally in a protected form which will be deprotected once the reaction is carried out or subsequently, or with the anhydride of formula (IV) below:

for which Ai is as defined above, to give in this case a compound of formula (II) with A ₂ = COOH.

 This coupling can be carried out by techniques well known to those skilled in the art and especially in the presence of dimethylaminopyridine (DMAP) in pyridine as a solvent in the case of a reaction with an anhydride of formula (IV). The reaction will be carried out especially at room temperature, that is to say at a temperature between 15 and 40 ° C, especially between 20 and 30 ° C.

 The fumagillol compound 2, for its part, can be obtained from fumagillin 1 according to a procedure described in the examples below.

Advantageously, the coupling will be carried out with a compound of formula (III) for which A ₂ = COOH optionally in protected form, or even more preferably with an anhydride of formula (IV). The COOH function can then be esterified subsequently to give access to a compound of formula (I) for which A ₂ = CO- ((C 1 -C 6) alkyl) under esterification conditions well known to those skilled in the art. Similarly, the function C OOH may be converted into a hydroxybisphosphonic acid function (-C (OH) (PO ₃ H ₂ ) ₂ ) under conditions well known to those skilled in the art such as those described in WO 2009/083613. .

 Step (b):

 This substitution step may be performed by techniques well known to those skilled in the art.

 If necessary, certain functionalities present on the molecule and sensitive to the conditions of the substitution reaction may be protected before being deprotected once the substitution is made.

 Step (c):

 This step may be carried out by techniques well known to those skilled in the art in the presence of a base or a pharmaceutically acceptable acid as defined above.

 In addition, the compound of formula (I) thus obtained may be separated from the reaction medium by methods well known to those skilled in the art, such as for example by extraction, evaporation of the solvent or by precipitation and filtration.

 The compound may be further purified if necessary by techniques well known to those skilled in the art, such as by recrystallization, by silica gel column chromatography or by high performance liquid chromatography (HPLC).

The present invention will be better understood in the light of the figures and non-limiting examples which follow.

FIGURES

Figure 1 represents the percentage of cell viability for the SaOS2 (a), MG63 (b), POS1 (c), OSRGa (d), HFF2 (e) and L929 (f) cell lines as a function of the compound concentration according to the administered invention, using a semi-logarithmic scale. Figure 2 represents the percentage of cell viability for the SaOS2 (a), MG63 (b), POS1 (c), OSRGa (d), HFF2 (e) and L929 (f) cell lines as a function of the administered concentration of the compound. 1-1 or TNP470, using a semi-logarithmic scale.

Figure 3 represents the weight follow-up (weight in grams) of the control mouse group and the group of mice that received the compound 1-1 according to the invention during the study (time in days).

 FIG. 4 represents the survival rate of the mice of the control mouse group and of the group of mice that received the compound 1-1 according to the invention as a function of time.

EXAMPLES

I. Preparation of the compounds according to the invention

 1.1. Preparation by extraction from a strain of Penicillium

 · Strain used

 The strain used is a saprotrophic micromycete of marine origin. It was isolated from a seawater sample taken in January 1997 at the La Prée site, Loire-Atlantique (Sallenave, 1999). It is kept in the marine mycotek of the laboratory MMS-EA2160 of the University of Nantes under the code MMS 351 as well as in the collection of the National Museum of Natural History (MNHN, Paris) under the reference LCP 99.43.43, and has has been identified morphologically as belonging to the species Penicillium waksmanii Zaleski.

 • Preparation of the culture medium

The culture medium used is the YES / seawater semisynthetic solid medium (Frisvad JC, 1981, Applied and Environmental Microbiology, 41 (3): 568-579, Filtenborg O. et al., 1990, In Modem Concepts. Penicillium and Aspergillus classification, Samson RA, Pitt JI eds, Plenum Press, New York, USA: 433-440) whose mass composition is as follows:

 Yeast extract: 2.0 g

 Sucrose (D (+) - sucrose): 15.0 g

MgSO ₄ , 7H ₂ O: 0.05 g

CuS0 ₄ , 5H ₂ 0: 0.0005 g ZnS0 ₄ .7H ₂ 0 0.001 g

 Bacteriological Agar E: 2.0 g

 Sterile seawater: 100 mL.

 • Inoculation and incubation

Cultures were incubated at 27 ° C under natural light after inoculation of sterile media at 50 mL with 250 mL Erlenmeyer flasks.

 • Fungal culture extraction protocol

 The cultures of strain MMS 35 1 were extracted after 11 days of incubation.

All cultures (biomass and agar) were contacted with 100 mL of ethyl acetate _T for 1 hour and then ground.

 The whole was placed on a shaker bench for 1 h at 150 rpm, and then subjected to the action of ultrasound for 30 min.

After successive filtrations (filter paper, then regenerated cellulose membrane with a porosity of 0.45 μιη) and adsorption of the residual water with anhydrous sodium sulphate, the solvent was evaporated on a rotary evaporator to dryness , resulting in obtaining a raw extract.

 • Separative methods of analysis and purification used and cytotoxicity tests carried out on cell lines

 - Low pressure liquid chromatography 1 (CLBP 1)

 The first stage of fractionation of the crude extract consisted of a low pressure liquid chromatography (CLBP) carried out on a glass column filled with 1.5 kg of porosity of 60 Å and particle size 35-70 μιη (Chromagel, SDS ). The dry deposit consisted of 33 g of crude extract adsorbed on 30 g of silica.

Elution was carried out by a discontinuous gradient of solvents of increasing polarities: hexane / EtOAc (0, 5, 10, 15, 20, 30, 40 and 50% of EtOAc by volume) then CH ₂ Cl ₂ / MeOH ( 0, 30 and 100% MeOH by volume); a volume of 4 L was used for each of the first 6 solvent mixtures, 8L for the hexane / EtOAc mixture 60: 40 (v / v) and the hexane / EtOAc mixture 50: 50 (v / v), 6L for the Pure CH ₂ C1 ₂ and 12L for 70: 30 (v / v) CH ₂ Cl ₂ / MeOH mixture and pure MeOH. The fractions obtained were evaporated to dryness. - Cytotoxicity tests

 The fractions were solubilized in absolute ethanol and then tested on two cell lines:

 a fibroblastic line of murine origin: L929 (non-tumor line);

 a line resulting from a mouse osteosarcoma: POS1 (tumor line).

To carry out the cytotoxicity tests, the various cell lines were seeded at T0 in 96-well plates and then incubated in a humid atmosphere at 37 ° C. delivering 5% of C0 ₂ .

 Depending on the cell types, seeding wells differed:

 - Mouse osteosarcoma cells (POS1): 2500 cells / well

 RPMI medium supplemented with 5% fetal calf serum (FCS)

 Mouse fibroblast cells (L929): 1000 cells / well

 RPMI medium supplemented with 5% FCS

At T = 24 h, the culture media were renewed and the different fractions were brought into contact with the cell lines at different concentrations.

 The percentage of ethanol was identical in each well for each concentration, ie 0.1% ethanol per well. Solvent control was carried out in parallel.

A cytotoxicity test (XTT-tetrazolium hydroxide, Cellproliferation kit II, Roche-Applied Sciences) was performed according to the manufacturer's specifications at T = 96h, after 72 hours of contacting the cells with the different extracts at different concentrations.

 - Low pressure liquid chromatography 2 (CLBP 2)

The second step of purification of the active fraction (eluted with hexane / EtOAc 60:40 v / v) consisted of a second low-pressure liquid chromatography, on a glass column filled with 20 g of 60 A porosity silica. and particle size 35-70 μιη (Chromagel, SDS). Dry deposition consisted of 272 mg of sample adsorbed on 600 mg of silica. Elution was carried out by a discontinuous gradient of solvents of increasing polarities (400 ml of each solvent): CH ₂ Cl ₂ / MeOH (0.5, 1, 2, 3, 5, 10, 20, 30, 40, 70 and 100% MeOH by volume). After evaporation of the solvent to dryness, the fractions were taken up in absolute ethanol and tested on lines POS 1 and L929 following the same protocol as that described previously. - High Performance Liquid Chromatography (HPLC)

 The third fractionation step consisted of high performance liquid chromatography. Fractions from CLBP 2 which showed an interesting activity on the tumor line were fractionated by HPLC equipped with a reverse phase diode array detector (Cl 8 grafted silica) with a MeOH / water gradient + 0.005% TFA ( trifluoroacetic acid) under the following conditions:

 - From t = 0 to t = 5min: MeOH / water + TFA 30: 70 (v / v)

 Increase in the percentage of MeOH until reaching 100% MeOH at t = 30 min

 Tray at 100% MeOH for 5 min (t = 30 min at t = 35 min)

 - Decrease of the percentage of MeOH from t = 35min to t = 53 min in order to return to the initial conditions, that is to say MeOH / water + TFA 30: 70 (v / v)

 Initial conditions MeOH / water + TFA 30: 70 (v / v) for 10 min (from t = 53 min to t = 63 min)

 This HPLC step yielded the molecule 1-1, called ligerine (tR ~ 28 min, 10 mg).

 The yield for obtaining this pure molecule, at this unoptimized stage, is therefore 0.03%) relative to the crude culture extract of the fungal strain.

 • Structural Elucidation

The analyzes performed on this molecule made it possible to determine the chemical structure of this compound as follows:

Nuclear magnetic resonance (NMR) data 1D (1 H and ¹³ C) of the compound 1-1 are shown in the table below (analysis performed on a 500MHz spectrometer with cryoprobe, Bruker):

The melting point of compound 1-1 was measured at 89.6 ° C on a Thermo Scientific 9300 Electrothermal block. High resolution mass spectrometry (HRMS) analysis made it possible to determine the crude formula of compound II as C20H31CIO7 (observation of sodium adduct [M + Na] ⁺ of m / z = 441, 16507 (C ₂ oH ₃ iC10 ₇ Na theoretical mass 441, 16560, Ammu = 0.53 = Appm = 1.2).

1.2. Synthesis by chemical way

 The compound I-1 according to the invention was also prepared by semisynthesis from fumarine according to the following reaction scheme:

Step a):

Fumagillin 1 (100 mg) was dissolved in NaOH (0.1N, 10 mL) and stirred at room temperature for 4 h. The final product was extracted with ether, the solution was filtered through a mixture NaHCO / NaiSC _A .and concentrated in vacuo. Fumagillol 2 was obtained in the form of a yellow oil (57 mg, yield = 93%). Step a) was carried out a second time to obtain 100 mg of additional fumagillol 2.

Step b):

Fumagillol 2 (145 mg, 0.5 mmol, 1 eq) was contacted with dimethylaminopyridine (DMAP) (80 mg, 0.655 mmol, 1.27 eq.) And succinic anhydride (200). mg, 2 mmol, 3.9 eq.) in 1 mL of anhydrous pyridine (Py). The whole was stirred for 16 h at room temperature. Silica gel chromatography was performed with a mobile phase consisting of a gradient of increasing polarity (from cyclohexane to a CH ₂ Cl ₂ / MeOH mixture 10: 1 v / v). Compound II-1 was obtained as a colorless oil (130 mg, yield = 66%).

Step c):

 Compound II-1 (65 mg, 0.17 mmol, 1 eq.) Was then contacted with acetic acid (50 μg, 54 mg, 0.9 mmol, 5.3 eq.), LiCl (30 mg, 0.715 mmol, 4.2 eq) in 0.5 mL of tetrahydrofuran (THF) at 0 ° C with stirring. The reaction mixture was gradually warmed to room temperature and then stirred for 24 h at this temperature. The mixture was then subjected to chromatography on silica gel with a gradient gradient from cyclohexane to a 10: 1 (v / v) CFLC / MeOH mixture as a mobile phase. The compound 1-1 according to the invention was obtained in the form of a colorless oil (44 mg, yield = 62%).

The nuclear magnetic resonance (NMR) spectra obtained correspond to those obtained from the molecule isolated from the strain MMS 351. The compounds 1-2 to 1-5 were also prepared by hemisynthesis according to the same process as that described above. above for the compound (1-1). Only the anhydride used for the esterification of step b) and the halide salt used in step c) have been modified. The compounds prepared and the reagents used are shown in the table below.

II. Evaluation de l'activité biologique des composés selon l'invention

II. Evaluation of the biological activity of the compounds according to the invention

II.1. Evaluation of the in vitro biological activity of compounds I-1 to 1-5

Compounds II to 1-5 were tested in vitro on four tumor lines (human osteosarcoma: MG63 and SaOS2, murine osteosarcoma: POS1 and rat osteosarcoma: OSRGα) and on two non-tumor lines (murine fibroblasts: L929 and human fibroblasts: HFF2).

 The results obtained are shown in FIG.

 All compounds thus have antiproliferative activity associated with a dose effect on all cell lines. On lines of the same origin (human or murine), the activities are weaker on non-tumor cells.

 Compound 1.1 exhibited a significantly higher activity on tumor cells compared to other compounds.

II 2. Evaluation of the in vitro biological activity of compound I-1 and TNP470

The compound TNP470 of the prior art has the following formula:

 Both compounds were tested on four tumor lines (human osteosarcoma: MG63 and SaOS2, murine osteosarcoma: POS 1 and rat osteosarcoma: OSRGa) and two non-tumor lines (murine fibroblasts: L929 and human fibroblasts: HFF2). The results obtained are shown in FIG.

 Compounds I-1 and T P470 exhibited antiproliferative activity associated with a dose effect on the 6 cell lines. In human tumor lines, the two compounds induce a maximum decrease in cell viability of 55% on the MG63 line and of respectively 52% and 59% for the TNP470 and the compound I-1 on the SaOS2 line.

Compound II, however, has a lower toxicity on healthy human cells HFF2 than TNP470 for a tested concentration of between 0, 125 ng / mL and 100 ng / mL. Thus, at 12.5 ng / mL, the decrease in viability of healthy cells HFF2 is only 15% for compound 1-1 while it is 43% for T P470.

 Thus, for a similar efficiency of the two molecules on human tumor cells, the toxicity observed on healthy human cells is much lower for compound 1-1 compared to T P470.

II.3. Evaluation of the in vivo biological activity of the compound 1-1

 The compound 1-1 was tested in vivo in an osteosarcoma model in the immunocompetent mouse according to the protocol described in Rousseau et al, Journal of Bone and Mineral Research, 2011, 26, 2452-2462.

 The model consists of the transplantation of murine tumor cells (POS1) near the shins of C3H / HeN mice. The tumor develops about 14 days after transplantation and progresses rapidly. In the context of osteosarcoma, the tumor cells used induce osteolysis.

The compound 1-1 has been used as a preventive treatment as soon as the tumors have been transplanted, that is to say that the injections have begun on the day of the transplantation. For this, the method of administration of the compound (in solution in 0.9% NaCl solution + 20%) EtOH) is as follows: 3 injections per week intraperitoneally at 30 mg / kg (10 ml / kg) spaced 24 hours each, for the duration of the study. A negative control group was formed with 0.9% NaCl injections (vehicle alone). At the start of the experiment (tumor fragment transplantation), the control group consisted of 12 animals and the group treated with compound 1-1 of 14. The tumor volume was calculated as follows: volume = (l ² xL) / 2, with 1 the short length and L the long length of the tumor.

The animals were euthanized when one of the following conditions was observed:

the volume of the tumor is greater than 2500 mm ³ ,

 one of the lengths of the tumor is greater than 25 mm, or

 - tumor necrosis.

 Thus, the survival of the animals, dependent on the size of the tumor, was evaluated.

Similarly, tumor-induced osteolysis was determined by analysis of X-ray images taken at different times during the experiments, namely at 0, 28 and 57. The weight of each animal was determined twice a week throughout the duration of the study. The results shown in Figure 3 indicate that there is no significant difference between the mice of the control group and those of the group administered the compound II. In both cases, the evolution of the weight corresponds to a physiological development.

 Animal survival is closely related to the rate of proliferation of tumors. The survival rate of the mice over time is thus represented in FIG. 4. This figure clearly shows that, whatever the stage of the study, the survival rate is higher for the group of mice that received the compound II. according to the invention compared to the group of control mice.

 In addition, at the end of the 54-day study, it can be noted that the survival rate is only 8.3% in the negative control group while it is 21.4% for the control group. mouse which has received the compound II according to the invention.

 Under the experimental conditions tested, the compound I-1 according to the invention therefore increases the survival rate of the mice compared to the mice of the control group.

Claims

1. Compound of formula (I) below:

or a pharmaceutically acceptable salt thereof, for which : X represents a halogen atom or a group OR ₃ , SR ₃ or R ₃ R ₄ , - Ai represents a group Xi-A ₃ -X ₂ for which: ^■ Xi and X ₂ are each independently of the other a bond, -O-, -S-, - R ₅ -, -C (O) - or -C = C-, and ^■ A ₃ represents a chain (Ci-Cio) alkyl, preferably (Ci-C ₆₎ alkyl, optionally interrupted by one or more units, preferably one, selected from -O-, -S-, -NR _Ô - , -C (O) - and -C = C-; or an optionally substituted aryl, cycloalkyl or heteroaryl group, - A ₂ represents a group -COOH, -C (O) O - ((C ₁ -C ₆ ) alkyl) or -C (OH) (P0 ₃ H ₂ ) ₂ , Ri represents a hydrogen atom or a -C (O) - ((C 1 -C 6) alkyl) group; Each of R ₃ and R ₄ represents, independently of one another, a hydrogen atom or a (C ₁ -C ₆ ) alkyl, aryl or aryl (C ₁ -C ₆ ) alkyl group, and R ₅ and 5 each independently of one another represent a hydrogen atom or a (C ₁ -C ₆ ) alkyl, cycloalkyl, heterocyclic, aryl, heteroaryl, aryl (C 1 -C 6) alkyl group or acyl;  the cycloalkyl, heterocyclic, aryl and heteroaryl groups being optionally substituted. 2. Compound according to claim 1, characterized in that X represents a halogen atom, such as a chlorine or bromine atom. 3. Compound according to any one of claims 1 and 2, characterized in that Xi and X ₂ each represent a bond. 4. Compound according to any one of claims 1 to 3, characterized in that A ₃ represents a (C 1 -C 10) alkyl chain, preferably (C ₁ -C ₆ ) alkyl, optionally interrupted by a unit selected from -O- , -S-, and -NR _O -, especially -O-; or an optionally substituted aryl group. 5. Compound according to any one of claims 1 to 4, characterized in that R1 represents a hydrogen atom. 6. Compound according to any one of claims 1 to 5, characterized in that A ₂ represents a COOH group. 7. Compound according to any one of claims 1 to 6, characterized in that R ₅ and 5 each represent, independently of one another, a hydrogen atom or a (C ₁ -C ₆ ) alkyl group, aryl or aryl (C ₁ -C ₆ ) alkyl; preferably a hydrogen atom or a (C ₁ -C ₆ ) alkyl group. 8. Compound according to any one of claims 1 to 7, characterized in that it is chosen from:

9. A compound according to any one of claims 1 to 8 for use as a medicament. 10. A compound according to any one of claims 1 to 8 for use in the treatment or prevention of a bone tumor. 11. A compound according to claim 10, characterized in that the bone tumor is a primary bone tumor such as osteosarcoma, chondro-sarcoma and Ewing's sarcoma; or a secondary bone tumor or bone metastases such as mammary or prostatic carcinoma cells. 12. Pharmaceutical composition comprising at least one compound of formula (I) according to any one of claims 1 to 8 and at least one pharmaceutically acceptable excipient. 13. Pharmaceutical composition according to claim 12, characterized in that it further comprises another active ingredient such as an anticancer agent. 14. Process for preparing a compound of formula (I) according to any one of claims 1 to 8, comprising the following successive steps:  (a) opening of the epoxide and halogenation of the compound of formula (II) below:

for which A ₁ and A ₂ are as defined in claim 1,  to give a compound of formula (Ia) below:

(the) for which A ₁ and A ₂ are as defined in claim 1 and Hal represents a halogen atom, the compounds of formula (Ia) corresponding to compounds of formula (I) according to claim 1 with X = Hal and Ri = H, optionally nucleophilic substitution of the OH or Hal function of the compound of formula (Ia) obtained in the preceding step to give a compound of formula (I) for which X ≠ Hal and / or Ri ≠ H, and  optionally salifying the compound of formula (I) or (la) obtained in the preceding step to obtain a pharmaceutically acceptable salt thereof. 15. Process according to claim 14, characterized in that the compound of formula (II) is obtained by coupling of the OH function of the fumagillol of formula 2 below:

OH ₂ with the COOH function of the carboxylic acid of formula (III) below HO. _^ A ^J , 1 - O (III) for which A ₁ and A ₂ are as defined in claim 1, A ₂ being optionally in protected form, or with the anhydride of formula (IV) below:

for which Ai is as defined in claim 1.