WO2010100297A1

WO2010100297A1 - Bisthiazole compounds that can be used to treat cancer

Info

Publication number: WO2010100297A1
Application number: PCT/ES2010/000087
Authority: WO
Inventors: Rodolfo LAVILLA GRÍFOLS; Joan Gil Santano; Fernando Albericio Palomera; Miriam Miguel Sala; Diana Maria GONZÁLEZ GIRONÈS; Anna ALCAIDE LÓPEZ
Original assignee: Universidad De Barcelona; Institut De Recerca Biomèdica De Barcelona; Institut D'investigació Biomèdica De Bellvitge
Priority date: 2009-03-03
Filing date: 2010-03-02
Publication date: 2010-09-10
Also published as: ES2344780B1; ES2344780A1

Abstract

The invention relates to compounds having formula (I), the pharmaceutically acceptable salts thereof, or the solvates of same, wherein: Ri is selected from phenyl, optionally mono-, di- or tri-substituted with a radical independently selected from F, Cl, Br, I, (C₁C₄)-alkyl, N,N-(C₁,C₄)-alkylamino, hydroxy, (C₁C₄)-alkoxy, phenoxy, methylenedioxy, trifluoromethoxy, trifluoromethyl, carboxyl, 4-phenoxyphenyl and (C₁C₄)-alkoxycarbonyl; 2-thiazolyl; 2-pyridinyl; 2,2'-bipyridinyl; 2,2'-bisthiazole; naphthyl; 4-phenoxyphenyl and isoquinolyl; R₂ is selected from the same group as R₁ and, in addition, is independent of H and I; and R₃ and R₄ are selected independently from H, Br and F. Said compounds inhibit cell proliferation of tumour cells independently of the p53 protein and can likewise induce apoptosis in different tumour cells independently of the p53 protein, making same suitable for use in the treatment of different types of cancer. Formula (I)

Description

Bistiazole compounds useful for cancer treatment

The present invention relates to new bistiazole compounds, pharmaceutical compositions containing them and their use for the treatment of cancer.

STATE OF THE PREVIOUS TECHNIQUE

Cancer is a heterogeneous disease characterized by the accumulation of tumor cells, which can cause the death of both animals and humans. Conventional methods for the treatment of cancer include surgical treatments, the administration of chemotherapeutic agents, and more recently treatments based on the immune response, which involve the administration of an antibody or an antibody fragment that can be conjugated to a unit. therapy. However, so far, such treatments have had limited success.

The development of antitumor therapies of general applicability is one of the main objectives in medical chemistry. Among the strategies planned to develop new antitumor treatments, inducing apoptosis is particularly attractive. In this context, the development of new compounds that can act as efficient inducers of apoptosis in the maximum number of types of cancer possible would have an enormous scientific, social and economic impact.

Although many drugs have been used in cancer therapies, today there is no curative therapy for most of them. Many of the drugs currently used in the treatment of cancer induce apoptosis of these cells at least partially, through the activation of the p53 protein pathway. When damage to deoxyribonucleic acid (DNA) occurs, p53 prevents the cell from replicating by stopping the cell cycle and induces apoptosis. The mechanisms of resistance to the drugs currently used include the inactivation of the p53 protein that is mutated in half of all the types of cancer analyzed, demonstrating the importance of the p53 pathway in the development of cancer. Chemotherapy works by killing rapidly dividing cells, one of the main properties of tumor cells. This means that it also damages cells that divide rapidly under normal conditions. Most chemotherapeutic regimens can cause depression of the immune system.

Various bioactive natural products maintain as a common feature the presence of thiazole rings in their structures, many of them have antitumor properties. There is a great structural diversity of these compounds such as cyclic peptides, polyheterocyclic alkaloids, or compounds of mixed biosynthetic origin that have structural motifs of bicar- and polycyanolic nature. The biosynthetic origin of these sub-structural units is related to the cyclodehydration procedures generated by the thiol of the cysteine side chain and the neighboring peptide bond. However, the synthesis of these natural products is usually complicated, both by solid phase peptide synthesis, and by condensation protocols (Hantszch synthesis) or through transition metal coupling reactions, from products that already contain its structures the thiazole ring (cf. eg N. Haginova et al., Bioorα. Med. Chem. 2004. vol. 12, pp. 5579-5586).

Likewise, there are few examples of symmetric bistiazole compounds in the literature and, more especially, by a 2.2 ^' bond. Some bistiazole compounds with the ability to inhibit cell proliferation have been described previously. Thus, WO 2004/016622 describes compounds with structure of type 4,4'-bipyridyl-2,2'-bisoxazole and 4,4'-bipyridyl-2,2'-bistiazole with antiproliferative activity, in particular, on cells HT-29 All the examples cited in said patent have a bridging bridge between the two thiazole nuclei.

In short, despite all the efforts invested so far, there is still a need to find new antitumor agents that are effective, and in particular, it would be of great interest to find antitumor therapies that act independently of p53. SUMMARY OF THE INVENTION

The inventors have found a new family of compounds with the bistiazole nucleus diversely substituted with a great variety of aromatic rings constituting constructions that contain in their structures up to three and four aromatic rings, with antitumor properties against several lines of cancer cells, and therefore , which are useful for cancer treatment. It has been found that these compounds inhibit the cellular proliferation of tumor cells independently of the p53 protein. Additionally, it has been seen that some compounds induce apoptosis in several tumor cells independently of the p53 protein.

The fact that the compounds of the present invention inhibit the proliferation of cells and even induce apoptosis of tumor cells, in both cases independently of the p53 protein, is of great importance since the resistance of the tumor cells to current treatments is partly due to its dependence on the p53 protein pathway. Therefore, the compounds of the present invention are advantageous because they reduce the problems that many antitumor agents have and are active against a wide variety of cancer cell lines.

Thus, one aspect of the present invention consists in providing a compound of general formula (I), or its pharmaceutically acceptable salts, or its solvates.

(D

where: Ri is selected from: phenyl optionally mono-, di-, or tri- substituted by a radical independently selected from the group consisting of: F, Cl, Br, I, (Ci-CvO-alkyl, N, N- ( Ci-C ₄ ) -alkylamino, hydroxyl,

(CrC ₄ ) -alkoxy, phenoxy, methylenedioxy, trifluoromethoxy, trifluoromethyl, carboxy, 2- (2- methoxyethoxy) ethoxy, and (CrC ₄ ) -alkoxycarbonyl; 2-thiazolyl; 2-pyridinyl; 2.2 ^' - bipyridinyl; 2,2'bistiazole;naphthyl; 4-phenoxyphenyl and isoquinolyl; R ₂ is selected from the same group of Ri and also includes independently H and I; and R ₃ and R ₄ are independently selected from H, Br and F; with the condition that the compound (I) is not one of the following compounds:

2,2'-bistazole, 5,5'-diphenyl (compound (I) with Ri = R ₂ = phenyl, R ₃ = R ₄ = H) having RN 109558-82-9;

2,2'-b¡stiazol, 5,5'-di-2-naphthalene (compound (I) with R ₁ = R ₂ = 2-naphthyl, R ₃ = R ₄ = H) having RN 1087742- 85-8;

2,2'-bistiazole, 5,5'-bis [4- (trifluoromethyl) phenyl] - (compound (I) with Ri = R ₂ = 4- trifluoromethylphenyl, R ₃ = R ₄ = H) having RN 869896- 77-5;

2,2'-bistiazole, 5,5'-bis (4-butoxyphenyl) - (compound (I) with Ri = R ₂ = 4- butoxyphenyl, R ₃ = R ₄ = H) having the RN 861958-17-0 ; or

2,2'-bistiazole, 5,5'-bis (4-ethoxyphenyl) - (compound (I) with R ₁ = R ₂ = 4-ethoxyphenyl, and R ₃ = R ₄ = H) having RN 72997-86- 5.

There is no knowledge of the use as anticancer agents of any of the aforementioned compounds.

The term "pharmaceutically acceptable salts" used herein encompasses any salt formed from both pharmaceutically acceptable inorganic and organic non-toxic acids or bases. Pharmaceutically acceptable salts may also be in the form of hydrates. There is no limitation with respect to salts, except that if they are used for therapeutic purposes, they must be pharmaceutically acceptable.

The compounds mentioned may have an asymmetry center, and therefore, exist in different enantiomeric forms. All optical isomers and individual stereoisomers of the compounds herein, and mixtures thereof, are considered within the scope of protection of the present invention. Thus, the compounds referred to herein are intended to represent any form of a racemic, one or more enantiomeric forms, one or more atropoisomeric forms, or a mix them.

In a preferred embodiment, the compounds of formula (I) are those where R ₁ is selected from phenyl, optionally mono-, di-, or tri-substituted by a radical independently selected from: F, Cl, Br, I,

(CrC ₄ ) -alkyl, N, N-dimethylamine, hydroxy, methoxy, ethoxy, isopropoxy, phenoxy, carboxy, and ethoxycarbonyl; 2-thiazolyl; 2-pyridinyl and ^2,2' -bipiridin¡l; and R ₂ is selected from the same group as Ri or is H.

In a more preferred embodiment, the compounds of formula (I) are those where Ri and R ₂ are independently selected from the group consisting of 3-isoproproxyphenyl, 3-chlorophenyl, 4-ethoxycarbonylphenyl, 4-ethylphenyl, 4-carboxyphenyl, 4 -methoxyphenyl, 3-ethoxyphenyl, 3,4-ethoxyphenyl, 4- (dimethylamino) phenyl, 3-isopropylphenyl, 3-chlorophenyl, 3,4,5-trimethoxyphenyl, 4-phenoxyphenyl, 3,4-dimethoxyphenyl, 2,4- difluorophenyl, 2-thiazolyl, ^2,2' -bipir¡dinil and 2-pyridinyl.

In an even more preferred embodiment, the compounds of formula (I) of the present invention are those where Ri is selected from the group consisting of 3-isoproproxyphenyl, 3-chlorophenyl, 4-ethylphenyl, 4-ethoxycarbonylphenyl, A-methoxyphenyl and 3 , 4-dimethoxyphenyl.

In another preferred embodiment, the compounds of formula (I) are those where Ri and R ₂ have the same meaning. In another preferred embodiment, the compounds of formula (I) are those where R ₂ is H.

In another preferred embodiment, the compounds of formula (I) are those where R ₃ and R ₄ are H. In another even more preferred embodiment, the compounds of formula (I) are those where R ₃ and R ₄ are F.

The most preferred compounds of formula (I) as defined above are those of the following Table 1:

The compounds of the present invention can be conveniently prepared from commercial reagents by a variety of procedures. They can be prepared simply and flexibly. The synthetic sequence is direct, allowing the synthesis of different structural analogues. The compounds of the present invention can be prepared by a method comprising Suzuki couplings. Scheme I illustrates a particular embodiment of the procedure.

Scheme I

Pd (II)

(D

According to the above scheme, the 2,2'-bistiazole of formula (I) where R ₃ and R4 are H can be obtained directly by the homocoupling of the 2-bromothiazole using a palladium catalyst, such as the Pd (OAc) ₂ , a base such as diisopropylethylamine and an appropriate solvent such as toluene. Other 2-halothiazoles can be used as starting products. ^2,2' -bistiazol can be selectively lithiated with lithium diisopropylamide ( "lithium diisopropylamide , " LDA) and the organometallic derivative obtained can be doubly halogenated with a source of iodine as molecular iodine to afford compound 5, 5 ^'- diiodo-2,2'-bisthiazol.

The preparation of the non-symmetrical compounds of formula (I) is carried out by sequentially performing two Suzuki couplings. The first coupling is made with a boronic acid of formula RiB (OH) 2 and then the second coupling is made with a boronic acid of formula R ₂ B (OH) ₂ . In the previous formula Ri and R ₂ have the same meaning as for the compounds of formula (I). As palladium catalysts, for example, Pd (OAc) ₂ , tetrakis (triphenylphosphine) -palladium (0) or tris (dibenzylidenacetone) dipaladium (0) can be used. Examples of suitable bases include potassium phosphate or sodium carbonate. In general, the reaction is heats to an approximate temperature of 80 ⁰ C.

The preparation of symmetric compounds of formula (I) can be carried out as illustrated in the above scheme, but by subjecting the iodinated derivative to a single Suzuki coupling, generally using two equivalents of the arylboronic acid of formula RiB (OH) ₂ .

In a preferred embodiment, the preparation of the symmetric compounds of formula (I) where Ri = R ₂ and R ₃ = R4 = H is carried out using as a ligand the 2-dicyclohexylphosphino-2 ¹ , 6'-dimethoxybiphenyl (S- Phos)

The non-symmetrical diarylbistiazole compounds of formula (I) where R ₂ = R ₃ = R ₄ = H can be prepared as indicated in scheme II:

Il scheme

(I)

According to the above scheme, 5-iodo-2,2'-bistiazole can be obtained by monolithing the corresponding diiodinated derivative and subsequent treatment with water. Then, 5-iodo-2,2'-bistiazole can be subjected to a Suzuki coupling with a boronic acid of formula R ₁ B (OH) ₂ , to obtain with good yields the compounds of formula (I) with R ₂ = R ₃ = R ₄ = H.

The compounds of the present invention can also be prepared by Stille couplings. A particular embodiment of the procedure is illustrated in the following Scheme III. Scheme

S NTT ((v

N / N CCHH ₃ g)) ₃₃ SSnnαQ I uUf VN Pd (O)

(i)

In the above scheme, X represents a halogen atom, preferably X is Br. According to the above scheme, both non-symmetric diarylbistiazoles and symmetric diarylbistiazoles in which Ri = R ₂ and R ₃ = R ₄ = H can be obtained.

Preparing unsymmetrical diarilbistiazoles of formula (I) includes two sequential Stille couplings from ^5,5' -b¡s (trimethylstannyl) -2,2 ^'- bisthiazol, first with a halogenated derivative of formula RIX and then with a second haloderivative of formula R ₂ X, where Ri and R ₂ can vary in the same way as they do in formula (I). Preferably, this coupling is performed in the presence of a palladium catalyst. A preferred catalyst is tetrakis (triphenylphosphine) palladium (0). Generally, to obtain derivatives ^5,5' ^{-bisaril-2,2'} -bistiazol 10% mole of the palladium catalyst is used. The coupling is carried out in a suitable solvent such as dimethylformamide. In general, the reaction is heated to a temperature of approximately 100 ⁰ C. The reaction can be carried out in the presence of silver oxide (I) as an additive.

The compounds of formula (I) where R ₂ = R ₃ = R ₄ = H can also be prepared by Stille couplings. A particular embodiment of this procedure is shown in the following Scheme IV. Scheme IV

(I)

According to the above scheme, 5-iodo-2,2'-bistazole is subjected to a StMIe coupling with a suitable trimethylstannilaryl to provide the compounds of formula (I) where R ₂ = R ₃ = R 4 = H with good yields .

The compounds of the present invention can also be prepared by a CH activation process. This procedure is useful for the preparation of compounds of formula (I) where R ₃ = R ₄ = H. Scheme V illustrates a particular embodiment of this process.

Scheme V

(I) According to the above scheme, 2,2'-bistiazole can be subjected to an oxidative coupling reaction with a suitable compound of formula RiI. This reaction is carried out in the presence of a suitable palladium catalyst. A preferred catalyst is tetrakis (triphephosphine) palladium (0). 10 mol% palladium catalyst is usually used. The coupling is usually carried out in a suitable solvent such as dimethylformamide and in the presence of a base such as cesium carbonate. In a particular embodiment, the reaction is carried out at a temperature of approximately 150 ⁰ C.

In another particular embodiment, triphenylphosphine (20 mol%) is used as a ligand. In another particular embodiment, copper (I) iodide is used as an additive.

The subsequent fluorination of the compound (I) in the thiazole ring with a fluorinating agent such as bis- (tetrafluoroborate) of 1-chloromethyl-4-fluoro-1, 4-diazononiabicyclo [2.2.2] octane (Selectfluor®), gives place compounds of formula (I) with R ₃ and / or R ₄ = F with good yields. The fluorination is carried out in a suitable solvent such as, for example, acetonitrile, generally at high temperatures, in particular, it is usually carried out at the reflux temperature of the solvent used.

The bromination of the compound (I) in the thiazole ring can be carried out with bromine, to give compounds of formula (I) with R ₃ and / or R ₄ = Br in good yields. The bromination is carried out in a suitable solvent such as acetonitrile, chloroform or in mixtures thereof.

The preparation procedures described above can be modified to obtain enantiopide compounds as well as mixtures of stereoisomers. It is possible to synthesize specific stereoisomeric compounds or specific mixtures by various methods, including the use of stereospecific reagents or by the introduction of chiral centers in the compounds during the synthesis. In addition, it is possible to separate the stereoisomers once the compound has been synthesized by standard resolution techniques well known to the person skilled in the art. The preparation of pharmaceutically acceptable salts can be carried out by methods known in the prior art. For example, they can be prepared from the primary compounds which contain a basic or acid reactive center, by conventional chemical methods. Generally, said salts are prepared by reacting the acid or the free base with the stoichiometric amount of a base or an acid in water, in an organic solvent or in a mixture thereof. The compounds of the present invention can be in crystalline form, both solvated solvent-free compounds and solvates (for example, hydrates) and it is understood that both forms are within the scope of protection of the present invention. Solvation methods are generally known in the literature.

An important characteristic of the compounds of the present invention is their bioactivity by inhibiting the cell growth of the tumor cell lines tested, and in particular their cytotoxic activity promoting apoptosis.

As illustrated in the Examples, the compounds of the present invention show antitumor properties (proapoptotic properties) against several cancer cell lines. The best results have been obtained for the compounds (Is) and (It), the (Is) having an IC ₅₀ of less than 1 to 6 μM. Both promote apoptosis of tumor cells independently of the p53 protein.

Thus, another aspect of the present invention is related to providing compounds of formula (I), or their pharmaceutically acceptable salts, or their solvates for use in the therapeutic treatment of cancer in a mammal, including humans,

(I) where: Ri is selected from the group consisting of: phenyl, optionally mono-, di-, or tri-substituted by an independently selected radical from the group consisting of: F, Cl, Br, I, (Ci-C ₄ ) -alkyl, N ₁ N- (CrC ₄ ) - alkylamino, hydroxy, (Ci-C ₄ ) -alkoxy, phenoxy, methylenedioxy, trifluoromethoxy, trifluoromethyl, carboxy, 2- (2-methoxyethoxy) ethoxy, and (dCO-alkoxycarbonyl; 2- thiazolyl, 2-pyridinyl, ^2,2' -bip¡rid¡n¡l; 2,2'-bisthiazol;naphthyl; 4-phenoxyphenyl and isoquinolyl; R ₂ is selected from the same group as Ri and also independent of H and I; and R ₃ and R ₄ are independently selected from H, Br and F.

Preferably, the types of cancer against which the compounds of the present invention are active are the following: leukemias, lymphomas, cervical carcinoma, colon cancer and neuroblastoma. More preferably, the cancer is leukemia or lymphomas. Even more preferably, the type of lymphoma or leukemia is B cell neoplasms.

This aspect can also be formulated as the use of the compounds of formula (I) as defined above, for the preparation of a medicament for the therapeutic treatment of cancer in a mammal, including humans.

The invention also relates to a method of treating cancer in a mammal, including humans, suffering from cancer, in particular to the aforementioned types of cancer, said method comprising administering to said patient a therapeutic therapeutically effective amount of a compound of formula (I), as defined above, together with pharmaceutically acceptable excipients or carriers.

The compounds of the present invention can be used in the same manner as other known chemotherapeutic agents. They can be used alone or in combination with other suitable bioactive compounds.

Another aspect of the present invention is related to a pharmaceutical composition containing a therapeutically effective amount of the compounds of the present invention, together with appropriate amounts of pharmaceutically acceptable excipients or carriers.

The chemotherapeutic treatment derived from the present invention is a new approach to cancer therapy and has the advantage of being useful for the treatment of various types of cancer. Throughout the description and the claims, the word "comprises" and its variants are not intended to exclude other technical characteristics, additives, components or steps.

For those skilled in the art, other objects, advantages and characteristics of the invention will emerge partly from the description and partly from the practice of the invention. The following examples and drawings are provided by way of illustration, and are not intended to be limiting of the present invention. In addition, the present invention covers all possible combinations of particular and preferred embodiments indicated herein.

BRIEF DESCRIPTION OF THE FIGURES

FIG. 1A shows the dose-response of the compound (l _t ) from 5 μM to 40 μM in Jurkat cells (T lymphocytes from a type T leukemia) at 24 h of incubation. Cell growth (CG) was measured with the MTT assay and is expressed as the percentage with respect to the control cells at the beginning of the treatment (100%). The results are shown as the mean ± the standard deviation (SD) of the triplicates.

FIG. 1 B shows the dose-response of the compound (l _t ) from 5 μM to 40 μM in Jurkat cells at 24 h of incubation. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells.

FIG. 2A shows the dose-response of compound (I ₅ ) from 5 μM to 40 μM in Jurkat cells at 24 h of incubation. Cell growth (CG) was measured with the MTT assay and is expressed as the percentage with respect to the control cells at the beginning of the treatment (100%). The results are shown as the mean ± SD of the triplicates.

FIG.2B shows the dose-response of compound (I ₅ ) from 5 μM to 40 μM in Jurkat cells at 24 h of incubation. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells. FIG. 3A shows the dose-response of compound (l _t ) from 5 μM to 40 μM in TK6 cells (B lymphoblasts from hereditary spherocytosis) at 24 h of incubation. Cell growth (CG) was measured with the MTT assay and is expressed as the percentage with respect to the control cells at the beginning of the treatment (100%). The results are shown as the mean ± SD of the triplicates.

FIG. 3B shows the effect of compound (l _t ) at 20 μM and 40 μM on TK6 cells at 24 h of incubation. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells.

FIG. 4A shows the effect of compound (l _t ) at 20 μM and 40 μM on Ramos cells (B lymphocytes from Burkitt lymphoma) at 24 h of incubation. Cell growth (CG) was measured with the MTT assay and is expressed as the percentage with respect to the control cells at the beginning of the treatment (100%). The results are shown as the mean ± SD of the triplicates.

FIG. 4B shows the dose response of the compound (l _t ) from 5 μM to 40 μM in the Ramos cells at 24 h of incubation. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells.

FIG. 5A shows the dose-response of the compound (l _t ) from 5 μM to 40 μM in cells from patients with chronic lymphocytic leukemia (CLL) at 24 h of incubation. A representative patient is shown. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells.

FIG. 5B shows the dose-response of compound (I ₅ ) from 5 μM to 40 μM in cells from patients with chronic lymphocytic leukemia (CLL) at 24 h of incubation. A representative patient is shown. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells. FIG. 5C shows the dose-response of compound (I ₁ ) from 5 μM to 40 μM in normal (non-tumor) B and T lymphocytes from healthy donors at 24 h of incubation. The results are shown as the mean ± SD. Viability (V) was measured by flow cytometry and is expressed as the percentage of non-apoptotic cells.

EXAMPLES

Unless otherwise indicated, all reactions were carried out under an argon atmosphere. Commercial reagents were used without further purification. The reaction temperatures were controlled by a modulator of the IKA temperature. Thin layer chromatography is performed on silica gel 60 F ₂₅₄ chromatopholics (Merck) and development is performed by visualization with UV light or with a KMnO ₄ solution. For the purification in flash chromatographic column, silica gel with a particle size of 35-70 μm is used as stationary phase. Symmetry C-iβ reverse phase columns of dimensions 4.6 mm * 150 mm, 5 μm (column A) of HPLC were used. The HPLC analyzes were performed in an apparatus composed of two solvent supply pumps, an automatic injector and a variable wavelength detector and a system controller (Breeze V3.20). The MALDI-TOF analyzes were performed using the ACH matrix. IR spectra are obtained with a Thermo Nicolet Nexus spectrophotometer and the absorption bands are indicated in cm ^{~ 1} . Melting points were made in a Büchi Melting Point B-540 apparatus.

Example 1: Preparation of 5.5'-bis (3-isopropoxyphenyl) -2.2'-bistazole (compound (Ic))

A glass flask (oven - dried) fitted with condenser and maintained under nitrogen was added ^5,5' ^{-d¡yodo-2,2'} -bit¡azol (190 mg, 0.45 mmol) and tetrakis (triphenylphosphine) palladium (0) (31.2 mg, 0.027 mmol). The flask was purged three times with nitrogen and then toluene (20 mL) was added. To the orange solution was added an aqueous solution of 2M Na ₂ CO ₃ (1.37 mL) and 3-isopropoxyphenylboronic acid (178 mg, 0.99 mmol). The reaction mixture was stirred at 80 ⁰ C for 12h. When the reaction was complete, the inorganic solids were filtered on a bed of diatomaceous earth (Celite ^® ) and the residue obtained was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2). Subsequently, a crystallization was carried out in toluene to yield the product 5, 5 ^'-bis (3-isopropoxyphenyl) -2,2'-bisthiazol as a yellow solid (95% yield). IR (NaCI), v (cm ^"1 ): 3073, 2969, 2924, 1581, 1255, 1116. MS (IE) m / z, (%): 437 (M ⁺ , 100). HRMS (ESI): Calculated for C ₂₄ H ₂₅ N ₂ O ₂ S ₂ : 437.1351; found: 437.1347. Melting point: 202.6-203.8 ⁰ C.

Example 2: Preparation of 4- ^{(5'-2,2'--vodo} bisthiazol-5-yl) -2-methoxyphenol (compound (Ij))

A glass flask (oven - dried) fitted with condenser and maintained under nitrogen was added ^5,5' ^-diyodo-2,2' -bitiazol (190 mg, 0.45 mmol) and tetrakis (triphenylphosphine) palladium (0) (15.6 mg, 0.013 mmol). The flask was purged three times with nitrogen and then toluene (20 ml_) was added. To the yellow solution was added an aqueous solution of Na ₂ Cθ ₃ 2M (680 μl_) and 4-hydroxy-3-methoxyphenylboronic acid pinacol ester (123mg, 0.49 mmol). The reaction mixture was stirred at 80 ⁰ C for 12h. When the reaction was completed, the inorganic solids were filtered over Celite ^® . The obtained residue was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2). Subsequently, a crystallization was carried out in toluene to yield the product 4- ^(5' - ^iodo-2,2' -bistiazol-5-yl) -2-methoxyphenol as a yellow solid (88% yield). IR (NaCI), v (cm ^"1 ): 3079, 2922, 2851, 1596, 1533, 1378, 1268, 917. MS (IE) m / z, (%): 416 (M ⁺ , 100). HRMS ( ESI): Calculated for Ci ₃ Hi ₀ N ₂ O ₂ S ₂ I: 416.9226; found: 416.9229. Melting point: 213.5-214.7 ⁰ C.

Example 3: Preparation of ethyl 4- [5 ^* (3,4-dimethoxyphenyl) -2.2 ^' -bistiazolyl-5-yl] -benzoate (compound (Iq))

A glass flask (oven - dried) fitted with a condenser and kept under nitrogen atmosphere, 4- ^(5' - iodo-2,2'-bisthiazol-5- l) -benzoic acid ethyl ester (162 mg , 0.37 mmol) and tetrakis (triphenylphosphine) palladium (0) (15.6 mg, 0.013 mmol). The flask was purged three times with nitrogen and then toluene (20 ml_) was added. The yellow solution is added an aqueous solution of 2M Na ₂ CO ₃ (680 μl_) and 3, 4-dimethoxyphenylboronic acid (74 mg, 0.40 mmol). The reaction mixture was stirred at 80 ⁰ C for 12h. When the reaction was completed, the inorganic solids were filtered over Celite ^® . The obtained residue was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2). Subsequently, a crystallization was carried out in toluene to yield the product 4- [5 ^' (3,4-dimethoxyphenyl) -2,2 ^' -bistiazolyl-5-yl] -benzoate as a beige solid (98% of performance). MS (IE) m / z, (%): 452 (M, 100). IR (NaCI), v (cm ^" ¹ ): 3045, 2942, 2893, 1705. HRMS (IE): Calculated for C ₂₃ H ₂₀ O ₂ N ₂ S ₂ : 452.0864; found: 452.0852. Melting point: 164.8- 165.2 ⁰ C.

Example 4: Preparation of 5- (3-fluoro-4-methoxyphenyl) -2.2'-bistiazole (compound (Ie))

A glass flask (oven - dried) fitted with a condenser and kept under nitrogen, was added ^5-iodo-2,2' -bistiazol (132 mg, 0.45 mmol) and tetrakis (triphenylphosphine) palladium (0) (15.6 mg, 0.013 mmol). The flask was purged three times with nitrogen and then toluene (20 ml_) was added. To the yellow solution was added an aqueous solution of 2M Na ₂ CO ₃ (680 μl_) and 3-fluoro-4-methoxyphenylboronic acid (84mg, 0.49 mmol). The reaction mixture was stirred at 80 ⁰ C for 12h. When the reaction was completed, the inorganic solids were filtered over Celite ^® . The obtained residue was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2). Subsequently, a crystallization was carried out in toluene to give the product 5- (3-fluoro-4-methoxyphenyl) -2,2 ^'-bistiazol as a yellow solid (42% yield). IR (NaCI), v (cnrf ¹ ): 3121, 2924, 1485, 1276. HRMS (ESI): Calculated for C ₁₃ H ₁₀ N ₂ OFS ₂ : 293.0213; Found: 293.0221. Melting point: 171.9-172.3 ⁰ C.

Example 5: Preparation of 4- ^{(5'-2,2'--vodo} bisthiazol-5-yl) -benzoic acid ethyl ester (compound (Ih))

Obtaining analogously to example 4 from 5,5'-diiodo-2,2'-bistiazole and 4-ethoxycarbonylphenylboronic acid (81% yield). IR (NaCI), v (cm ^'1 ): 3400, 3074, 2976, 2923, 2852, 1712, 1275, 1107, 912, 767, 692. MS (IE) m / z, (%): 442 (M ⁺ , 100). HRMS (ESI): Calculated for Ci ₅ H ₁₂ N ₂ O ₂ S ₂ I: 442.9379; Found: 442.9385. Melting point: 134.1-134.7 ⁰ C.

Example 6: Preparation of 5.5 ^'-bisr3.4- (metilend¡oxi) feniπ-2,2'-b¡stiazol

A glass flask (oven - dried) fitted with condenser and maintained under nitrogen was added ^5,5' ^-diyodo-2,2' -bistiazol (336mg, 0.8 mmol), S-Phos (197mg, 0.48 mmol), tris (dibenzyldenacetone) dipaladium (0) or Pd ₂ (dba) ₃ (146.5mg, 0.16 mmol),

K ₃ PO ₄ (1.05g, 4.97 mmol) and 3,4- (methylenedioxy) phenylboronic acid (531 mg, 3.2 mmol). The flask was purged three times with nitrogen and then anhydrous toluene (20 ml_) was added. The reaction mixture was stirred at 100 ⁰ C for 12h. When the reaction was completed, the inorganic solids were filtered over Celite ^® . The obtained residue was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2). Subsequently, a crystallization was carried out in toluene to yield the product 5, 5 ^'-bis (3-isopropoxyphenyl) -2,2'-bisthiazol as a yellow solid (67% yield). IR (NaCI), v (crτϊ ¹ ): 2922, 1469, 1444, 1249. MS (IE) m / z, (%): 408 (M, 100). HRMS (El): Calculated for C ₂₀ H ₁₂ N ₂ O ₄ S ₂ : 408.023893; Found: 408.023851. Melting point: 234.1-234.9 ⁰ C.

Example 7: Preparation of 5- (2-pyridinih-2.2'-bistiazole (compound (Im))

To a glass flask (oven dried) provided with a condenser and kept under a nitrogen atmosphere, 2-bromopyridine (58 μl_, 0.61 mmol), 2-trimethylstannylpyridine (0.30 mmol), and tetrakis (triphenylphosphine) - palladium ( 0) (116 mg, 0.1 mmol, 10 mol%). The flask was purged three times with nitrogen and then anhydrous dimethylformamide (5 ml_) was added. The reaction mixture was stirred at 80 ⁰ C for 12h. When the reaction was completed, the inorganic solids were filtered over Celite ^® . The obtained residue was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 9: 1, + 1% NEt ₃ ). Subsequently, crystallization was carried out in AcOEt-hexane to yield the product 5- (2-pyridinyl) -2,2'-bistiazole as a yellow solid (15% yield). IR (NaCI), v (crτϊ ¹ ): 3104, 3077, 1581, 1463. MS (IE) m / z, (%): 245 (M ⁺ , 100), 135 (61). HRMS (ESI): Calculated for CnH ₈ N ₃ S ₂ : 246.0154; Found: 246.0162. Calculated for CnH ₇ N ₃ S ₂ Na: 267.9974; Found: 267.9969. Melting point: 208.8-209.5 ⁰ C.

Example 8: Preparation of 5.5'-diphenyl-2.2'-bistiazole

To a glass flask (oven drying) provided with a condenser and kept under a nitrogen atmosphere, tetrakis (triphenylphosphine) palladium (0) (130 mg, 0.12 mmol, 20 mol%) and triphenylphosphine (63 mg, 0.24 mmol, 10 mol%). The flask was purged three times with nitrogen and then the anhydrous dimethylformamide (5 ml_) was added. After 10 minutes stirring at room temperature it was added slowly (about 15 minutes) copper iodide (I) (907 mg, 4.76 mmol), cesium carbonate (931 mg, 2.86 mmol), ^2,2' -bistiazol (200 mg, 1.19 mmol) and iodobenzene (318 μl_, 2.85 mmol). The reaction mixture was stirred at 150 ⁰ C for 12h. When the reaction was completed, the inorganic solids were filtered over Celite ^® . The obtained residue was washed several times with dichloromethane, then filtered and evaporated. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 9: 1).

Subsequently, a crystallization was carried out in toluene to yield the product 5, ^5' - diphenyl-2,2'-bisthiazol as orange solid (60% yield) yellow. IR (NaCI), v (cm ^"1 ): 3064, 1477, 1442, 1394, 1332. MS (IE) m / z, (%): 320 (M ⁺ , 100). HRMS (ESI): Calculated for C ₁₈ H ₁₃ N ₂ S ₂ : 321.0515; found: 321.0518. Melting point: 237.4-239.0 ⁰ C.

Example 9: Preparation of 4.4 ^' -difluoro-5.5'-diphenyl-2.2'-bistiazole (compound (Is))

To a solution of 5,5'-diphenyl-2,2'-bitiazole (125 mg, 0.39 mmol) in acetonitrile (10 ml_) was added Selectfluor ^® (276.8 mg, 0.78 mmol). The reaction was stirred for 12 h at the reflux temperature. The reaction was monitored by TLC (1: 4 ethyl acetate / hexane). Then, 15 mL of EtOAc was added and extracted with H ₂ O (3 x 15 mL) and a saturated aqueous solution of NaHCO ₃ (3 x 15 mL). The organic extracts were dried with Na ₂ SO ₄ , filtered and evaporated under reduced pressure. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2), obtaining the product ^4,4' -d¡fluoro-5,5'-diphenyl-2,2'-bisthiazol as (100% yield, 92% purity) as a yellow solid. MS (IE) m / z, (%): 356.9 (M ⁺ , 100). IR (NaCI) ₁ v (cm ^"1 ): 2924, 1530, 1450, 1370, 1210, 1154, 1125, 1021. HRMS (ESI): Calculated for Ci ₈ H ₁₀ N ₂ S ₂ F ₂ : 356.0253; found: 356.0252 Melting point: 119.5-119.9 ⁰ C. HPLC (Column A, R ₁ 16.553 min).

Example 10: Preparation of ^{4,4'-dibromo-2,2'-diphenyl} 5.5'-bisthiazol (compound (Iq))

To a solution of 5,5'-diphenyl-2,2'-bitiazole (60 mg, 0.18 mmol) in 1 ml of chloroform and 1.5 mL of acetonitrile was added, slowly, Br ₂ (40 μl, 0.75 mmol). The reaction was stirred for 12 h at room temperature. After this time, the mixture was filtered. On the filtrate a saturated aqueous solution of Na ₂ S ₂ O ₄ (10%) was added and the mixture was extracted with ether (3 x 25 mL). The organic extracts were dried with Na ₂ SO ₄ , filtered and evaporated under reduced pressure, obtaining a crystalline compound. The solid product was recrystallized from methylene chloride to yield the product ^4,4'-dibromo - ^5,5' ^{-d¡fenil-2,2'} -bistiazol as a yellow solid (99% yield). IR (NaCI), v (crτϊ ¹ ): 3369, 2923. MS (IE) m / z, (%): 478 (M, 100), 480 (55), 476 (49). HRMS (ESI): Calculated for Ci ₈ H ₁₁ N ₂ S ₂ Br ₂ : 478.8724; Found: 478.8715. Calculated for C ₁₈ H ₁₂ N ₂ S ₂ Br: 398.9619; Found: 398.9636. Melting point: 213.1-213.8 ⁰ C.

Example 11: Preparation of 5- (3,4-D-methoxyphenyl) -5 ^' -vodo-2.2'-biastiazole (compound (It))

Pd (PPh ₃ J ₄ (31.2 mg, 0.027 mmol), an aqueous solution of Na ₂ CO ₃ (2M, 1.37 mL) and 3,4-dimethoxyphenylboronic acid (179 mg, 0.99 mmol) were added to a solution 5,5'-diiodo-2,2'-bisthiazol (190 mg, 0.45 mmol) in anhydrous toluene (20 mL). The mixture of the reaction was stirred at 80 ⁰ C for 12h, then filtered over a thin layer of Celite ^® and the filtrates were evaporated under reduced pressure. The residue was purified by flash chromatographic column (SiO ₂ , hexane: AcOEt, 8: 2). Then, a crystallization was carried out in toluene to yield the desired product (165 mg, 95% yield) IR (NaCI): v 2989, 2928, 2829, 1598, 1513, 1476, 1443, 1382, 1258, 1144, 1024, 916, 836 cm ^'1. Melting point: 170.5-170.7 ° C. EM (IE): m / z (%): 431 (M ⁺ , 100). HRMS (ESI): Calculated for C ₁₄ H ₁₂ N ₂ O ₂ S ₂ I: 430.9376; Found: 430.9373.

Example 12: Biological tests for the detection of antitumor activity

An exploration of the antitumor activity of the compounds with formula (I) in Jurkat cells was carried out and a study of the most active compounds in Ramos cells, TK6 cells, cells from patients with chronic lymphocytic leukemia (LLC) was carried out ), and B and T lymphocytes from healthy donors.

Patients with CLL or healthy donors and cell isolation

Peripheral blood lymphocytes were obtained from patients with CLL or from healthy donors of the Hematology Unit at IDIBELL-Hospital de Bellvitge, L'Hospitalet de Llobregat, Barcelona, Spain. The CLL was diagnosed according to the standard clinical and laboratory criteria. Informed consent was obtained from all patients, according to the Bellvitge Hospital Ethics Committee. Purification of the mononuclear leukocytes was performed by a gradient of Ficoll-Hypaque (Seromed, Berlin, Germany). The purity of the LLC samples was evaluated by flow cytometry with allophycocyanin-conjugated anti-CD3 (allophycocyanin, APC) and phycoerythrin-conjugated anti-CD19 (phycoerythrin, PE) (Becton Dickinson, Frankiln Lakes, NJ, USA). The results were analyzed with a FACSCalibur and the analysis was carried out with the CelIQuest program (Becton Dickinson, Mountain View, CA, USA).

Cell culture

The human Jurkat cell lines (T lymphocytes from an acute T-cell leukemia), Ramos (B lymphocytes from a Burkitt lymphoma) and TK6 (B lymphoblasts from hereditary spherocytosis) were obtained from the European Cell Culture Collection.

All cell types were grown in RPMI 1640 medium containing 10% inactivated fetal serum (veal), 1% glutamine, and 1% penicillin. Streptomycin at 37 ⁰ C in humidified atmosphere with 5% carbon dioxide.

Reagents

3- (4,5-Dimethylthiazol-2-yl) 2,5-diphenyltetrazole (MTT) bromide and dimethyl sulfoxide (DMSO) were obtained from Sigma Chemicals Co. (St Louis, MO, USA). Annexin V-FITC and propidium iodide (propidium iodine, Pl) were obtained from Bender MedSystems (Vienna, Austria).

Analysis of the cell viability by the MTT assay

Cell viability was determined using the MTT assay. Cells (0.25-0.3 * 10 ⁵ cells / well) were incubated in a 96-well plate in the absence or in the presence of the compound of interest, in a final volume of 100 µl. 10 µl of MTT (3- (4,5-dimethyltiazol-2-yl) 2,5-diphenyltetrazole bromide) (Sigma Chemicals Co, St Louis, MO, USA) (5 mg / ml in phosphate buffer) was added saline, phosphate-buffered saline, PBS) to the control well at Oh incubation and all wells at 24h and an additional 4h was left. The blue MTT formazan precipitate was dissolved with 100 μl of isopropanol: 1 M HCI (24: 1) and the optical density (OD) values were measured at 550 nm in a multiwell plate reader.

A value of 100% cell growth (CG) has been given to the OD of the control well at the Oh of incubation. The percentage increase / decrease of the DO in the 24-hour wells was calculated by comparison with the control well of Oh. An increase in DO correlates with an increase in cell proliferation while a decrease in DO is indicative of induction of apoptosis.

The MTT test offers a convenient and quantitative method to evaluate the response of a cell population to external factors, which may be an increase in cell growth, no effect, or a decrease in growth due to necrosis or apoptosis. The yellow MTT (3- (4,5-dimethylthiazol-2-yl) 2,5-diphenyltetrazole bromide) is reduced to formazan blue in the mitochondria of living cells. A solubilization solution is added to dissolve the blue formazan insoluble product to a solution colored. The absorbance of this colored solution can be quantified by measuring it at a certain wavelength with a spectrophotometer. These reductions take place only when mitochondrial reductase enzymes are active, and therefore their conversion is frequently used as a measure of viable (live) cells. When the amount of formazan blue produced by the cells treated with the agent is compared with the amount produced by the control cells (untreated), the effectiveness of the agent in causing cell death is deduced through a dose-response curve. For each cell type, a linear relationship is established between the number of cells and the absorbance, allowing a direct and exact quantification of the changes in proliferation.

Analysis of apoptosis by flow cytometry

0.25-0.3 * 10 ⁶ cells were washed in phosphate buffered saline (PBS), resuspended in 100 µl of annexin binding buffer and incubated with 1 µl of Annexin V-Fluorescein-5-isothiocyanate (FITC ). After a 20 min incubation in the dark at room temperature, 100 µl of annexin binding buffer with 5 µl of propidium iodide (propidium iodide, Pl) (20 µg / ml) was added just before the analysis by cytometry of flow. The data were analyzed with the CeII Quest software (Becton Dickinson). In the case of the compound (l _t ), the cell viability was measured through the analysis of the exposure of phosphatidylserine and the entry of Pl, and is expressed as the percentage of annexin-V cells and double negative Pl. In the case of the compound (l _s ), apoptosis was quantified by a reduction in cell size ("cell forward scatter", FSC) and an increase in cell complexity ("cell side scatter", SSC).

Apoptosis, or programmed cell death, is a general mechanism of the immune system for the elimination of unwanted cells. It is characterized by the condensation of chromatin, a reduction in cell volume, and a cut of the DNA carried out by endonucleases that gives rise to fragments of oligonucleosomal length. Apoptosis is also accompanied by a loss of the asymmetry of the phospholipid membrane, resulting in the exposure of phosphatidylserine on the cell surface. The expression of phosphatidylserine on the cell surface plays an important role in the recognition and elimination of apoptotic cells that lead to out the macrophages. This is one of the earliest events of the apoptotic process. The method for the detection of apoptotic cells by means of flow cytometry uses the binding of annexin V linked to fluorescein isothiocyanate (or other fluorochromes) to phosphatidylserine.

In addition, the plasma membrane is disturbed during late apoptosis but also during necrosis, since it becomes permeable to substances such as Pl. The Pl is sandwiched between double-stranded nucleic acids and is a fluorescent molecule with a molecular weight of 668.4 It can be used to stain the DNA. Pl is excluded by viable cells but can penetrate the cell membranes of dying and dead cells.

Therefore, viable cells are annexin-V and Pl double negative, early apoptotics are annexin-V positive and Pl negative while late apoptotic cells are annexin-V and Pl double positive. These three populations are indicative of apoptosis. A fourth population of positive Pl cells correlates with necrotic cells.

Results

1. Exploratory test in Jurkat cells

An exploration of the effect of the compounds from (l _a ) to (l _v ) at 20 μM during a 24 h incubation was performed on Jurkat cells (T lymphocytes from an acute T-cell leukemia). Jurkat cells were chosen among other leukemic tumor cell lines because they have the mutated p53 protein.

The compounds from (l _a ) to (l _v ) were dissolved in the minimum amount of DMSO necessary to be completely dissolved. The cell viability was measured with the MTT methodology. It was verified that DMSO alone did not decrease cell viability. Thus, all the effects observed in the cell viability were due to the activity of these compounds.

The cell viability is expressed as the percentage with respect to the control cells at the beginning of the incubation (Oh). The results obtained are summarized in Table 2 and calculated with the formula:

(MTT compound (I) at 24 h - MTT Control at 0 h) / (MTT Control at 24 h - MTT Control at 0 h) x 100

The term MTT in the above formula means the OD of the sample minus the OD of the blank (culture medium).

A value of 100% is indicative of no effect, neither in cell proliferation nor apoptosis. Values between 100 and 0% are indicative of inhibition of cell proliferation while negative values indicate apoptosis.

The compounds (Ia) - (It) have the structure indicated in Table 1. The compound (Iu) is a compound of formula (I) where Ri = R ₂ = 2-naphthyl and R ₃ = R ₄ = H. compound (Iv) is a compound of formula (I) where Ri = R ₂ = phenyl and R ₃ = R ₄ = H.

In the table: "+" means normal activity; "++" means good activity and "+++" means very good activity.

Table 2:

2. Study of cell viability in Jurkat cells. Bouquets TK6 v of LLC

The most active compounds were subsequently studied in Jurkat cells and other leukemic B cell lines such as Ramos cells (with mutated p53) and TK6 cells (with wild, non-mutated p53). In addition, the effect of these compounds was tested in primary cells of patients with chronic lymphocytic leukemia (CLL).

These different cell types were incubated with these compounds at 20 μM for 24 hours and the cell viability was measured with the MTT assay and by flow cytometry.

The results of the MTT method are summarized in Table 3 and calculated as in Table 2.

The results of the flow cytometry are expressed as the effect of each compound at 20 μM with respect to the untreated cells (control). Cell viability is expressed as the percentage of double negative cells for annexin-V and Pl. In the case of compound (I ₅ ), cell viability is expressed as the percentage of high cell size (FSC) and low cell complexity ( SSC).

A MTT value of 100% is indicative of no effect, neither in cell proliferation nor apoptosis. MTT values between 100 and 0% are indicative of inhibition of cell proliferation while negative values indicate apoptosis.

Cytometry values below 100% are indicative of apoptosis.

In Table 3. citom. It means cytometry.

Therefore, the results show that these compounds induce inhibition of cell proliferation and that some of them induce apoptosis in these same cells.

3. Dose-response study of the compounds (Is) v (It)

The effect of the two proapoptotic compounds (Is) and (It) was determined on several tumor lines. The dose-response study was performed using cancer cells with wild p53, such as leukemic lines (TK6), cervical cancer (HeLa) and neuroblastoma (SH-SY-5Y). In addition, tumor cells with mutated p53 were also used, such as leukemic lines (Jurkat and Ramos) and colon cancer cells (HT-29). The study was completed using primary cells from CLL patients and cells from healthy donors.

Cell viability was measured with two methodologies:

- The MTT assay and is expressed as the percentage with respect to the control cells at the beginning of the incubation. - Flow cytometry and is expressed as the percentage of non-apoptotic cells with respect to the control cells.

Jurkat cells (which are T lymphocytes with mutated p53 were incubated with a dose range from 5 to 40 μM of both compounds for 24 hours. Compounds (It) and (Is) induced apoptosis in a dose-dependent manner measured by MTT (cf. FIG. 1 A and 2A respectively) and by flow cytometry (cf. FIG. 1B and 2B respectively).

TK6 cells (which are B lymphoblasts with wild p53) were incubated with a dose range from 5 to 40 μM of compound (It) for 24 hours. A flow cytometry test was performed with the doses of 20 and 40 μM while the entire dose-response curve was studied with the MTT test. The compound induced apoptosis in a dose-dependent manner measured by MTT (cf. FIG. 3A) and by flow cytometry (cf. FIG. 3B).

Ramos cells (which are B lymphocytes with mutated p53) were incubated with a dose range from 5 to 40 μM of compound (It) for 24 hours. An MTT test was carried out with the doses of 20 and 40 μM while the dose-response curve was studied by flow cytometry. Compound (It) induced apoptosis measured by MTT (cf. FIG. 4A) and by flow cytometry (cf. FIG. 4B), especially at high doses.

LLC cells (from 4 different patients) were incubated with a dose range from 5 to 40 μM of the compound (It) for 24 hours. Compound (Is) was tested in LLC cells (of a patient) using the same dose range. Compound (It) (cf. FIG. 5A) and compound (Is) (cf. FIG. 5B) induced apoptosis measured by flow cytometry.

B and T lymphocytes from healthy donors (n = 2) were incubated with a dose range of 5 to 40 μM of compound (It) for 24 hours. The compound induced apoptosis in B cells but not in T cells (cf. FIG. 5C). These results indicate that the B cells are much more sensitive than the T cells to the apoptosis induced by the compound (It). This differential effect is of great interest in B cell neoplasms. The IC ₅ or at 24 hours was calculated for the compound (Is) and (It) using the MTT method and by flow cytometry and the results are expressed in Table 4. ND indicates not determined. N indicates the number of experiments performed.

These results demonstrate that the apoptosis induced by these bistiazoles is independent of p53, an important difference with respect to the majority of drugs used in cancer therapy, which induce cell cycle arrest and apoptosis through the activation of p53.

Claims

1. Compound of formula (I) or a pharmaceutically acceptable salt thereof, or a solvate thereof,

(I where:

Ri is selected from the group consisting of: phenyl, optionally mono-, di-, or tri-substituted by a radical independently selected from the group consisting of: F, Cl, Br, I, (Ci-C ₄ ) -alkyl. lo, N, N- (Ci-C ₄ ) -alkylamine, hydroxyl, (Ci-Ci) -alkoxy, phenoxy, methylenedioxy, trifluoromethoxy, trifluoromethyl, carboxy, 2- (2-methoxyethoxy) ethoxy, and ( CrC ₄ ) -alkoxycarbonyl; 2-thiazolyl; 2-pyridinyl; 2,2 ^'- bipyridinyl; 2,2'-bistiazole;naphthyl; 4-phenoxyphenyl and isoquinolyl.

R ₂ is selected from the same group of Ri and also independent of H and I; Y

R ₃ and R ₄ are independently selected from the group consisting of H, Br and F;

with the condition that the compound (I) is not one of the following compounds:

Compound (I) with Ri = R ₂ = phenyl, and R ₃ = R ₄ = H; Compound (I) with R ₁ = R ₂ = 2-naphthyl, and R ₃ = R ₄ = H; Compound (I) with Ri = R ₂ = 4-trifluoromethylphenyl, and R ₃ = R ₄ = H; Compound (I) with Ri = R ₂ = 4-butoxyphenyl, and R ₃ = R ₄ = H; or Compound (I) with Ri = R ₂ = 4-ethoxyphenyl, and R ₃ = R ₄ = H;

2. Compound according to claim 1, wherein Ri is selected from the group consisting of: phenyl, optionally mono-, di-, or tri-substituted by a radical independently selected from the group consisting of: F, Cl, Br, I, (CrC-O-alkyl, NN-dimethylamino, methoxy, ethoxy, isopropoxy, phenoxy, methylenedioxy, carboxy, and ethoxycarbonyl; 2-thiazolyl, 2-pyridinyl, ^2,2' -bipiridin¡ly 2,2'-bistiazol; and R ₂ is selected from the same group of Ri or H.

3. A compound according to claim 2, wherein Ri and _R2 are independently selected from the group consisting of: 3-isoproproxifenil, 3- chlorophenyl, 4-ethoxycarbonylphenyl, 4-ethylphenyl, 4-carboxyphenyl, 4-methoxyphenyl, 3- ethoxyphenyl, 3,4-ethoxyphenyl, 4- (dimethylamino) phenyl, 3-isopropylphenyl, 3-chlorophenyl, 3,4,5-trimethoxyphenyl, 3,4- (methylenedioxy) phenyl, 4-phenoxyphenyl, 3,4-dimethoxyphenyl, 2,4-difluorophenyl, 2-thiazolyl, ^2,2' -bipiridinil, and 2-pyridinyl.

4. Compound according to claim 3, wherein Ri is selected from the group consisting of: 3-isoproproxyphenyl, 3-chlorophenyl, 4-ethylphenyl, 4-ethoxycarbonylphenyl, 4-methoxyphenyl and 3,4-dimethoxyphenyl.

5. Compound according to any of claims 1-4, wherein Ri and R ₂ are the same.

6. Compound according to any of claims 1-4, wherein R ₂ is H.

7. Compound according to any of claims 1-6, wherein R ₃ and R4 are H.

8. Compound according to any of claims 1-6, wherein R ₃ and R ₄ is F.

9. Compound according to claim 1, which is selected from the following table:

10. Compound of formula (I), or a pharmaceutically acceptable salt thereof, or a solvate thereof, for use in the therapeutic treatment of cancer in a mammal, including humans.

where:

Ri is selected from the group consisting of: phenyl, optionally mono-, di-, or tri-substituted by a radical independently selected from the group consisting of: F, Cl, Br, I, (CrC ₄ ) -alkyl, N, N- (CrC ₄ ) -alkylamino, (Ci-C ₄ ) -alkoxy, phenoxy, methylenedioxy, trifluoromethoxy, trifluoromethyl, hydroxy, carboxy, 4-phenoxyphenyl, and (CrC ₄ ) -alkoxycarbonyl; 2-thiazolyl; 2-pyridinyl; 2,2 ^'- bipyridinyl; 2,2'-bistiazole;naphthyl; 4-phenoxyphenyl and isoquinolyl; F * ₂ is selected from the same group as Ri and also independently of H and I; Y

R ₃ and R ₄ are independently selected from the group consisting of H, Br and F.

11. Compound according to claim 10, for use in the therapeutic treatment against a cancer selected from the group consisting of: leukemia, lymphoma, cervical carcinoma, colon cancer and neuroblastoma.

12. Compound according to claim 11, for use in the therapeutic treatment of leukemia or lymphoma.

13. Compounds according to claim 12, for use in the therapeutic treatment of leukemia or lymphoma, which are B cell neoplasms.

14. Use of the compound as defined in any of claims 1-9, for the preparation of a medicament for the therapeutic treatment of cancer in a mammal, including the human being.

15. Pharmaceutical composition comprising a therapeutically effective amount of the compound as defined in any of claims 1-9, together with appropriate amounts of pharmaceutically acceptable carriers or excipients.

16. Method of preparing a compound of formula (I) as defined in any of claims 1-9, comprising:

a) submit a compound of formula (II)

(II) to a Suzuki coupling with a compound of formula RiB (OH) 2, in the presence of a palladium catalyst to give a compound of formula (I) with Ri = R ₂ and R ₃ = R ₄ = H; or alternatively

b) subjecting a compound of formula (II) to a Suzuki coupling first with a compound of formula RiB (OH) ₂ , in the presence of a palladium catalyst, and subsequently with a compound of formula R ₂ B (OH) ₂ , in the presence of a palladium catalyst, to provide a compound of formula (I) with Ri different from R ₂ and R ₃ = R ₄ = H; or alternatively

c) subject to a compound of formula (III)

(Hl) to a Suzuki coupling with a compound of formula RiB (OH) ₂ , in the presence of a palladium catalyst, to give a compound of formula (I) with R ₂ = R ₃ = R ₄ = H; or alternatively

d) subject to a compound of formula (IV)

(IV) to a Stille coupling with a compound of formula RiX, in the presence of a palladium catalyst, to give a compound of formula (I) with R ₁ = R ₂ and R ₃ = R ₄ = H; or alternatively

e) subjecting a compound of formula (IV) to a Stille coupling first with a compound of formula RiX ₁ in the presence of a palladium catalyst, and subsequently with a compound of formula R ₂ X, in the presence of a palladium catalyst, to provide a compound of formula (I) with R ₁ other than R ₂ and R ₃ = R ₄ = H; or alternatively

f) subject to a compound of formula (V)

(V)

to a coupling of StNIe with a compound of formula RiSn (CH ₃ ) ₃ , in the presence of a palladium catalyst, to give a compound of formula (I) with R ₂ = R ₃ = R4 = H; or alternatively

g) subject to a compound of formula (Vl)

(Vl)

to an oxidative coupling with a compound of formula R ₁ I, in the presence of a palladium catalyst, to give a compound of formula (I) with R ₂ = R ₃ = R ₄ = H; or alternatively

h) subjecting a compound of formula (Vl) to an oxidative coupling first with a compound of formula R ₁ I, presence of a palladium catalyst, and subsequently with a compound of formula R ₂ I, in the presence of a catalyst of palladium, to provide a compound of formula (I) with R ₁ other than R ₂ and R ₃ = R ₄ = H; Y

i) optionally, subjecting the compounds obtained in any of the above procedures from a) to h) to a fluorination reaction with a fluorinating agent or a bromination reaction with a brominating agent to provide compounds of formula (I) where R ₃ and / or R ₄ are F or Br; Y j) optionally, converting the compounds obtained in any of the procedures a) to i) into a pharmaceutically acceptable salt by reacting the compound (I) with an appropriate pharmaceutically acceptable acid or with a pharmaceutically acceptable base appropriate to yield the corresponding pharmaceutically acceptable salt ;

where:

Ri, R2, R3, and R ₄ have the same meaning as in claim 1; Y

X is a halogen atom.