ES2410704A1 - Fluorinated indolenines for use in the treatment of cancer - Google Patents

Abstract

The compounds of formula (I), or the pharmaceutically acceptable salts thereof or the stereoisomers or mixture of stereoisomers thereof, in which: R1, R2, R4, R5 and R7 are independently selected from the group consisting of H, Cl, C(=O)O(C1-C4)alkyl, O(C1-C4)alkyl and (C1-C4)alkyl R3 is selected from the group consisting of H1, Cl, C(=O)O(C1-C4)alkyl, O(C1-C4)alkyl, (C1-C4)alkyl, pyrrolidinyl-1-carbonyl, and morpholin-1-carbonyl R6 is selected from the group consisting of H and (C1-C4)alkyl and R8 is selected from the group consisting of H, F, Br, (C1-C4)alkyl, and phenyl said compounds inhibit the cellular proliferation of tumour cells independently of the p53 protein and are also able to induce apoptosis in various tumour cells independently of the p53 protein, which makes them useful for the treatment of various types of cancer.

Description

Fluorinated Indolenins useful for cancer treatment

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

STATE OF THE TECHNIQUE

Cancer is a heterogeneous disease characterized by the accumulation of tumor cells, which can cause death in 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.

Chemotherapy, despite all its limitations, is still today one of the most widespread methods for the treatment of different types of cancer. Therefore, the development of new antitumor therapies of general applicability is one of the main objectives of medical chemistry.

The inability of chemical agents to distinguish between normal rapidly dividing cells and tumor cells can lead to a depression of the patient's immune system. This has been considered one of the main problems associated with chemotherapy, as well as the resistance mechanisms developed by cancer cells, which include the inactivation of the p53 pathway. Although the majority of drugs currently used in therapy induce apoptosis in these cells, at least partially, through the activation of the p53 pathway, the p53 gene is mutated in half of the tumors analyzed, demonstrating its importance in development of cancer

Great efforts are being made to improve antitumor treatments, trying to get active and selective compounds that can act independently of the p53 pathway, to administer to patients with incipient and recurrent cancer or with metastases.

However, despite the efforts made so far, there is currently no curative therapy for most tumors, so there is still a need to find effective antitumor agents. In particular, it would be of great interest to find antitumor therapies that can act independently of p53.

EXPLANATION OF THE INVENTION

The inventors have found a new family of compounds with the indolenin nucleus substituted by fluorine atoms that have antitumor properties (apoptotic properties) against different cancer cell lines. Therefore, these compounds are useful for the treatment and / or prevention of cancer.

In particular, the inventors have found that the compounds of the present invention promote apoptosis in several tumor cell lines independently of the p53 protein. Under normal conditions, when detecting DNA damage, the p53 protein prevents cell replication by stopping the cell cycle in the G1 phase, or interface, allowing DNA repair. However, it induces apoptosis if cell damage is very extensive and repair attempts fail. Any interruption in the regulation of the p53 pathway, in its functioning or in the target genes increases the possibility of tumor formation.

The fact that the compounds of the present invention promote apoptosis of tumor cells independently of the p53 protein is of great importance, since the resistance of the tumor cells to current treatments is partially due to their dependence on the p53 pathway. . Therefore, the compounds of the present invention are advantageous because they reduce the chemoresistance problems associated with many antitumor agents and are active against some carcinogenic cell lines.

Thus, one aspect of the present invention is related to providing compounds of general formula (I), or their pharmaceutically acceptable salts, or their stereoisomers or mixture of stereoisomers,

(I)

where: R1, R2, R4, R5, and R7, are independently selected from the group consisting of H, Cl, C (= O) O (C1C4) alkyl, O (C1-C4) alkyl; and (C1-C4) alkyl; R3 is selected from the group consisting of H, Cl, C (= O) O (C1-C4) alkyl, O (C1-C4) alkyl, (C1-C4) alkyl, pyrrolidinyl-1-carbonyl, and morpholin-1 -carbonyl; R6 is selected from the group consisting of H and (C1-C4) alkyl; and R8 is selected from the group consisting of H, F, Br, (C1-C4) alkyl, and phenyl; with the proviso that the compound of formula (I) is different from the compound of formula (I) where R1-R8 = H and with the proviso that the compound of formula (I) is different from the compound of formula (I) where R1 and R3-R8 are H and R2 is methoxy.

The compound of formula (I) where R1-R8 = H and the compound of formula (I) where R1 and R3-R8 is H and R2 is methoxy are described in Riyuan Lin et al., Organic Letters 2011, vol. 13, No. 17, pp. 4998-4501. However, nothing is said in this document regarding its use in the treatment of cancer.

The term "pharmaceutically acceptable salts" used herein comprises any salt formed from pharmaceutically acceptable non-toxic acids or bases including inorganic or organic acids or bases. There is no restriction on salts, except that if they are used for therapeutic purposes they must be pharmaceutically acceptable.

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

Preferably, the compounds of formula (I) are those in which R1, R4 and R5 are H.

In a preferred embodiment, the compounds of formula (I) are those in which R 6 is H.

In a more preferred embodiment, the compounds of formula (I) are those in which R2 and R7 are independently selected from H and Cl.

In an even more preferred embodiment, the compounds of formula (I) are those in which R3 is independently selected from the group consisting of H, Cl, and (C1-C4) alkoxycarbonyl. Preferably, the (C1-C4) alkoxycarbonyl is ethoxycarbonyl.

In another preferred embodiment, the compounds of the formula (I) according to the above definition are those in which R 8 is H or methyl.

The most preferred compounds of formula (I) are those of Table 1:

Table 1:

Comp. (I)

R1  R2  R3  R4  R5  R6  R7  R8

Ia

H H Cl H H H H H

Ib

H H Methoxy H H H H H

Ic

H H Ethoxycarbonyl H H H H H

Id

H H Cl H H H Cl H

Ie

Cl H H H H H H H

If

H H Ethyl H H H H H

Ig

H Cl H H H H H H

Ih

 H H H H H H H Met ilo

Ii

 H Methoxy Methoxy H H H H H

Ij

 H H H H H Ethyl H H

Ik

 H H 4-pyrrolidin-1carbonyl H H H H H

Il

 H H 4-morpholin-1carbonyl H H H H H

Im

 H H Ethoxycarbonyl H H H Cl H

In

 H H Methoxycarbonyl H H H H Fen ilo

The most preferred compounds of formula (I) are those selected from the following table: Table 2:

Comp (I)

R1  R2  R3  R4  R5  R6  R7  R8

Ia

 H H Cl H H H H H

Ic

 H H Ethoxycarbonyl H H H H H

Id

 H H Cl H H H Cl H

Ig

 H Cl H H H H H H

Im

 H H Ethoxycarbonyl H H H Cl H

In a particular preferred embodiment, the compound of formula (I) is that where R1-R2, R4-R8 is H and R3 is ethoxycarbonyl. In another particular preferred embodiment, the compound of formula (I) is that where R1-R2, R4-R6, and R8 are H and R3 and R7 are chlorine.

The compounds of the present invention can be prepared simply and flexibly from commercial reagents by a variety of procedures. For example, they can be prepared by the procedure illustrated in Scheme I.

Scheme I F where R9 is

F R8 R8 R8

R9B (OH) 2 fluorination R9

R9

30

N N NR7 Pd (II) R7 R7H

H R6R6 R6

(III) (II) (I)

In the above scheme, R6-R8 have the same meaning as mentioned above for the compound of formula (I).

In the above scheme R9 is a radical selected from the group consisting of: phenyl, phenyl substituted with at least one radical selected from the group consisting of Cl, (C1-C4) -alkoxy, (C1-C4) -alkyl, (C1 -C4) alkoxycarbonyl, 4-pyrrolidin-1carbonyl, and 4-morpholin-1-carbonyl.

Preferably, R9 is selected from the group consisting of phenyl, 4-chlorophenyl, 4-methoxyphenyl, 4 ethoxycarbonylphenyl, 4 ethylphenyl, 3-chlorophenyl, 3,4-dimethoxyphenyl, 4-phenyl-1-pyrrolidine carbonyl, 4 morpholin-1- carbonylphenyl.

According to the above scheme, 3,3-difluoro-3H-indole of formula (I) can be obtained by the oxidative coupling of Suzuki of a compound of formula (III) with a boronic acid of formula R9-B (OH) 2 using a palladium catalyst such as Pd (OAc) 2, an oxidizing agent such as TEMPO, a base such as KF and an appropriate solvent such as propionic acid.

Generally, the rection is carried out at room temperature (20-25 ° C). The subsequent fluorination of the indoline ring of compound (II) obtained with a fluorinating agent such as bis-(tetrafluoroborate) of 1-chloromethyl-4-fluoro1,4-diazononiabicyclo [2.2.2] octane (Selectfluor®), results in to compounds of formula (I). The fluorination is carried out in a suitable solvent such as acetonitrile, generally at room temperature (20-25 ° C).

The compounds of formula (I) where R 8 is Br can be prepared by a process comprising direct arylation followed by a fluorination reaction. In this particular embodiment, the compound of formula (III) wherein R8 = H is subjected to an activation reaction CH with a compound of formula R8-I, in the presence of a palladium (II) catalyst such as palladium acetate, and a base such as cesium acetate and an appropriate solvent such as N, N-dimethylacetamide (DMA). The reaction can be carried out at a temperature between 110 and 130 ° C. The subsequent fluorination of the indolenin compound of formula (II) can be carried out as mentioned above.

The preparation procedures described above can be modified to obtain enantiopide compounds as well as mixtures of stereoisomers. It is possible to prepare specific stereoisomers or specific mixtures by various procedures, including the use of stereospecific reagents or by introducing chiral centers in the compounds during their preparation process. Furthermore, it is possible to separate the stereoisomers once the compound has been prepared by standard resolution techniques well known to the person skilled in the art.

The preparation of pharmaceutically acceptable salts of the compounds of formula (I) can be carried out by methods known in the state of the 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, for example, by reacting the free acid or base forms of those compounds with the stoichiometric amount of a pharmaceutically acceptable base or acid in water or in an organic solvent or in a mixture thereof.

The compounds of the present invention may be in crystalline form, both as solvent-free compounds or as 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 within the art.

An important feature of the compounds of the present invention is their ability to inhibit cell growth in tested tumor lines, and in particular their ability to induce cytotoxicity by promoting apoptosis. As shown in the Examples, the compounds of the present invention have antitumor properties on two cancer cell lines.

Thus, another aspect of the present invention is related to the preparation of compounds of general formula (I), or their pharmaceutically acceptable salts or their stereoisomers or mixture of stereoisomers, including the compound of formula (I) where R1-R8 is H and the compound of formula (I) where R1 and R3-R8 are H and R2 is methoxy, for use as a medicament.

Another aspect of the present invention is related to the preparation of compounds of formula (I), or their pharmaceutically acceptable salts, or their stereoisomers or mixture of stereoisomers, including the compounds of formula (I) where R1-R8 are H and the compound of formula (I) where R1 and R3-R8 are H and R2 is methoxy, for use in the treatment and / or prevention of cancer, since they are active against all types of cancer that have been tested.

In a particular embodiment, compounds of formula (I), or their pharmaceutically acceptable salts, or their stereoisomers or mixture of stereoisomers for use in the treatment and / or prevention of tumors with mutated p53 are provided.

Preferably, the compounds of the present invention are especially active against acute T-cell leukemia and cervical cancer.

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

The invention also relates to a method of treating cancer in a mammal, including the human being, who suffers or is susceptible to cancer, in particular to the aforementioned types of cancer, said method comprising the administration to said patient of a therapeutically effective amount of a compound of formula (I), as defined above, together with pharmaceutically acceptable carriers or excipients.

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, of a compound of formula (I) where R1-R8 is H or the compound of formula (I) wherein R1 and R3-R8 are H and R2 is methoxy, together with appropriate amounts of pharmaceutically acceptable excipients or carriers. Preferably, the compound of formula (I) is as mentioned above without including the compound of formula (I) where R1-R8 = H, or a compound of formula (I) where R1 and R3-R8 are H and R2 is methoxy.

The term "therapeutically effective amount" as used herein, refers to the amount of a compound that, when administered, is sufficient to prevent the development of, or relieve to some degree, one or more of the symptoms of the disease a The one that goes. The particular dose of compound administered according to this invention will of course be determined by the particular conditions surrounding the case, including the compound administered, the route of administration, the particular condition being treated, and similar considerations.

The term "pharmaceutically acceptable excipients or carriers" refers to pharmaceutically acceptable materials, components or vehicles. Each component must be pharmaceutically acceptable in the sense of being compatible with the other ingredients of the pharmaceutical composition. It should also be suitable for use in contact with tissues or organs of humans and animals without too much toxicity, irritation, allergic response, immunogenicity or other problems or complications in accordance with a reasonable benefit / risk ratio.

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 features of the invention will be derived 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 DRAWINGS

FIG. 1 shows the dose-response of compound Ic from 2 µM to 40 µM in the Jurkat cell line (T lymphocytes from an acute type T leukemia, with mutated p53) at 24 hours of incubation. Viability was measured by flow cytometry and expressed as the percentage of non-apoptotic cells (annexin V APC negative).

FIG. 2 shows the dose-response of compound Ic from 5 µM to 40 µM in the HeLa cell line (cervical adenocarcinoma epithelial cell line with p53 inactivated) at 48 hours of incubation. Viability was measured by flow cytometry and expressed as the percentage of non-apoptotic cells (annexin V APC negative).

FIG. 3 shows the dose-response of compound Id from 2 µM to 40 µM in the Jurkat cell line (T lymphocytes from an acute type T leukemia, with mutated p53) at 24 hours of incubation. Viability was measured by flow cytometry and expressed as the percentage of non-apoptotic cells (annexin V APC negative).

FIG. 4 shows the dose-response of compound Id from 5 µM to 40 µM in the HeLa cell line (cervical adenocarcinoma epithelial cell line with p53 inactivated) at 48 hours of incubation. Viability was measured by flow cytometry and expressed as the percentage of non-apoptotic cells (annexin V APC negative).

EXAMPLES

Unless otherwise indicated, all reactions were carried out under an atmosphere of argon and dry glass material. Commercial reagents were used without further purification. Reaction temperatures were controlled by an IKA temperature modulator. Thin layer chromatography was performed on silica gel chromatofolios 60 F254 (Merck) and developed by visualization with UV light or with a solution of KMnO4. Silica gel (particle size 35-70 µm) was used for flash chromatographic column purification. Symmetry C18 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 length detector.

variable wave and a system controller (Breeze V3.20). MALDI-TOF analyzes were performed using the ACH matrix. IR spectra were 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 2- (4-chlorophenyl) -1H-indole

Indole (240 mg, 2.00 mmol), (2,2,6,6-tetramethyl-piperidin-1-yl) oxyl (TEMPO) (319 mg, were dissolved in a closed tube

2.00 mmol), KF (116 mg, 2.00 mmol), p-chlorophenylboronic acid (329 mg, 2.00 mmol), palladium acetate (11.3 mg, 0.05 mmol) in propionic acid (2 mL). The mixture was stirred at room temperature for 2 h. After completion of the reaction, it was diluted with a saturated solution of Na2CO3 (25 mL) and extractions were made with dichloromethane (DCM) (3 x 10 mL). The organic extracts were dried over Na2SO4, filtered and evaporated under reduced pressure. The residue was purified by flash chromatographic column (instantaneous) (SiO2, hexane: AcOEt, 9: 1), obtaining 79 mg of 2- (4-chlorophenyl) -1H-indole as a white solid (37% yield) . 1 H NMR (CDCl 3, 400 MHz): 8.28 (s, 1 H),

7.63 (d, J = 7.8 Hz, 1H), 7.61 - 7.57 (m, 2H), 7.44 - 7.39 (m, 3H), 7.24 - 7.18 (m, 1H), 7.15 - 7.10 (m, 1H), 6.81 ( d, J =

1.3 Hz, 1H) ppm. HRMS (ESI): calculated for (M + H +) C14H10ClN: 228.0502; Found: 228.0578.

Example 2: Preparation of 2- (4-methoxyphenyl) -1H-indole

The title compound was prepared analogously to Example 1 from indole (240 mg, 2.00 mmol) and pmethoxyphenylboronic acid (304 mg, 2.00 mmol) for 24 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 7: 3), obtaining 203.7 mg of 2- (4-methoxyphenyl) -1H-indole as a white solid (46% yield). 1H NMR (CDCl3, 400 MHz): 5 8.24 (s, 1H), 7.59 (d, J = 8.8 Hz, 3H), 7.40 - 7.36 (m, 1H), 7.20 - 7.08 (m, 2H), 6.98 (d, J = 8.8 Hz, 2H), 6.71 (dd, J = 2.1, 0.7 Hz, 1H), 3.86 (s, 3H) ppm.

Example 3: Preparation of 2- (4-ethoxycarbonylphenyl) -1H-indole

The title compound was prepared analogously to Example 1 from indole (240 mg, 2.00 mmol) and petoxycarbonylphenylboronic acid (388 mg, 2.00 mmol) for 24 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 9: 1), obtaining 104.6 mg of 2- (4-ethoxycarbonylphenyl) -1H-indole as a white solid (27% yield). 1 H NMR (CDCl 3, 400 MHz): 5 8.12 (d, J = 5.7 Hz, 2H), 7.72 (m, 2H), 7.65 (d, J = 7.8 Hz, 1H), 7.42 (d, J = 6.8 Hz, 1H ), 7.23 - 7.20 (m, 1H), 7.14 (t, J = 7.5 Hz, 1H), 6.97 (s, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.0 Hz , 3H) ppm. HRMS (ESI): calculated for (M + H +) C17H15NO2: 266.1103; Found: 266.1178.

Example 4: Preparation of 6-chloro-2- (4-chlorophenyl) -1H-indole

The title compound was prepared analogously to Example 1 from 6-chloroindole (310 mg, 2.00 mmol) and p-chlorophenylboronic acid (388 mg, 2.00 mmol) for 24 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 9: 1), obtaining 149.2 mg of 6-chloro-2- (4-chlorophenyl) -1H-indole as a white solid (33% yield ). 1 H NMR (CDCl 3, 400 MHz): 5 8.27 (s, 1 H), 7.59 - 7.55 (m, 2 H), 7.54 - 7.50 (m, 1 H), 7.44 - 7.41 (m, 2 H), 7.39 (s, 1 H), 7.10 (dd, J = 8.4, 1.8 Hz, 1H), 6.78 (d, J = 1.5 Hz, 1H) ppm. HRMS (ESI): calculated for (M + H +) C14H9Cl2N: 262.0112; Found: 262.0214.

Example 5: Preparation of 2- (4-ethylphenyl) -1H-indole

The title compound was prepared analogously to Example 1 from indole (200 mg, 1.67 mmol) and ethylphenylboronic acid (317 mg, 2.00 mmol) for 24 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 9: 1), yielding 58.7 mg of 2- (4-ethylphenyl) -1H-indole as a white solid (16% yield). 1H NMR (CDCl3, 400 MHz): 5 8.31 (s, 1H), 7.63 - 7.58 (m, 3H), 7.39 (dd, J = 8.0, 0.8 Hz, 1H), 7.28 (d, J = 8.4 Hz, 2H) , 7.20 - 7.16 (m, 1H), 7.13 - 7.09 (m, 1H), 6.81 - 6.77 (m, 1H), 2.69 (q, J = 7.6 Hz, 2H), 1.28 (t, J = 7.6 Hz, 3H ) ppm. HRMS (ESI): calculated for (M + H +) C16H15N: 222.1204; Found: 222.1303.

Example 6: Preparation of 2- (3-chlorophenyl) -1H-indole

The title compound was prepared analogously to Example 1 from indole (200 mg, 1.71 mmol) and michlorophenylboronic acid (304.3 mg, 2.04 mmol) for 48 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 7: 3), obtaining 41 mg of 2- (3-chlorophenyl) -1H-indole as a white solid (11% yield). 1 H NMR (CDCl 3, 400 MHz): 5 7.64 (dd, J = 6.6, 4.9 Hz, 2H), 7.56 - 7.53 (m, 1H), 7.42 -

7.39 (m, 1H), 7.36 (d, J = 7.8 Hz, 1H), 7.29 (ddd, J = 8.0, 1.9, 1.0 Hz, 1H), 7.24 - 7.20 (m, 1H), 7.16 - 7.11 (m, 1H), 6.85 (d, J = 1.3 Hz, 1H) ppm.

Example 7: Preparation of 2- (2-chlorophenyl) -1H-indole

The title compound was prepared analogously to Example 1 from indole (200 mg, 1.65 mmol) and ochlorophenylboronic acid (310.8 mg, 2.00 mmol) for 48 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 7: 3), obtaining 39 mg of 2- (2-chlorophenyl) -1H-indole as a white solid (10% yield). 1 H NMR (CDCl 3, 400 MHz): 5 8.78 (s, 1 H), 7.70 - 7.65 (m, 2 H), 7.49 (dd, J = 7.9, 1.4 Hz, 1 H), 7.43 (dd, J = 8.1, 0.8 Hz, 1H), 7.35 (td, J = 7.6, 1.4 Hz, 1H), 7.30 - 7.26 (m, 1H), 7.25 - 7.21 (m, 1H), 7.14 (td, J = 7.6, 1.0 Hz, 1H), 6.87 (dd, J = 2.1, 0.8 Hz, 1H) ppm.

Example 8: Preparation of 2- (3,4-dimethoxyphenyl) -1H-indole

The title compound was prepared analogously to Example 1 from indole (240 mg, 2.00 mmol) and 3,4-dimethoxyphenylboronic acid (364 mg, 2.00 mmol) for 24 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 6: 4), giving 244 mg of 2- (3,4-dimethoxyphenyl) -1H-indole as a white solid (48% yield).

Example 9: Preparation of 6-chloro-2- (4-ethoxycarbonylphenyl) -1H-indole

The title compound was prepared analogously to Example 1 from 6-chloroindole (200 mg, 1.30 mmol) and p-ethoxycarbonylphenylboronic acid (320 mg, 1.56 mmol) for 24 h at room temperature. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 9: 1), yielding 64.5 mg of 6-chloro-2- (4-ethoxycarbonylphenyl) -1H-indole as a white solid (17% yield). 1 H NMR (CDCl 3, 400 MHz): 5 8.44 (s, 1 H), 8.14

- 8.08 (m, 2H), 7.72 - 7.68 (m, 2H), 7.54 (t, J = 7.6 Hz, 1H), 7.40 (d, J = 0.8 Hz, 1H), 7.11 (dd, J = 8.4, 1.8 Hz, 1H), 6.91 (dd, J = 2.1, 0.8 Hz, 1H), 4.41 (q, J = 7.1 Hz, 2H), 1.42 (t, J = 7.1 Hz, 3H) ppm.

Example 10: Preparation of 5-bromo-2-phenyl-1H-indole

In a closed tube 5-bromoindole (200 mg, 1.02 mmol) and CsOAc (777 mg, 4.13 mmol) were added. The tube was purged 3 times with nitrogen and then iodobenzene (240 μL, 2.14 mmol) and 300 μL of a solution of palladium aacetate in DMA [11 mg of Pd (OAc) 2 in 1 ml of DMA] was added. The mixture was stirred under Ar atmosphere for 72 h at 125 ° C. When the reaction was complete, AcOEt was added and stirred for a few minutes. The solution was then passed through SiO2, washing the residue several times with AcOEt. It was then filtered and evaporated under reduced pressure. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt 95: 5), obtaining 53 mg of 5-bromo-2-phenyl-1H-indole as a white solid (19% yield). 1H NMR (CDCl3, 400 MHz): 7.80 (d, J = 1.9 Hz, 1H), 7.55 - 7.49 (m, 2H), 7.46 (dt, J = 8.2, 1.8 Hz, 2H), 7.42 - 7.37 (m, 2H ), 7.33 (d, J = 3.3 Hz, 1H), 7.29 (dd, J = 8.8, 1.9 Hz, 1H), 6.61 (dd, J = 3.2, 0.6 Hz, 1H) ppm. HRMS (ESI): calculated for (M + H +) C14H10BrN: 271.9997; Found: 272.007

Example 11: Preparation of 5-methyl-2-phenyl-1H-indole

The title compound was prepared analogously to Example 10 from 5-methylindole (140 mg, 1.06 mmol) and iodobenzene (250 µL, 2.22 mmol) for 72 h at 125 ° C. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 95: 5), yielding 53 mg of 5-methyl-2-phenyl-1H-indole as a white solid (24% yield). 1H NMR (CDCl3, 400 MHz): 5 8.23 (s, 1H), 7.65 (d, J = 8.5 Hz, 2H), 7.42 (d, J = 8.0 Hz, 2H), 7.32 (d, J = 7.4 Hz, 1H ), 7.29 (d, J = 8.3 Hz, 2H), 7.02 (d, J = 8.2 Hz, 1H), 6.75 (d, J = 1.6 Hz, 1H), 2.45 (s, 3H) ppm.

Example 12: Preparation of 7-ethyl-2-phenyl-1H-indole

The title compound was prepared analogously to Example 10 from 7-ethylindole (145 µg, 1.03 mmol) and iodobenzene (160 µL, 1.40 mmol) for 72 h at 125 ° C. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 95: 5), yielding 82 mg of 7-ethyl-2-phenyl-1H-indole as a white solid (36% yield). 1 H NMR (CDCl 3, 400 MHz): 5 8.22 (s, 1 H), 7.71 - 7.66 (m, 2 H), 7.53 - 7.41 (m, 3 H), 7.36 - 7.29 (m, 1 H),

7.13 - 7.01 (m, 2H), 6.84 (dd, J = 4.4, 2.0 Hz, 1H), 2.92 (qd, J = 7.5, 2.4 Hz, 2H), 1.41 (ddd, J = 9.5, 6.7, 3.0 Hz, 3H) ppm.

Example 13: Preparation of 5-phenyl-2- (4-methoxycarbonylphenyl) -1H-indole

The title compound was prepared analogously to Example 10 from 5-phenylindole (105 mg, 0.52 mmol) and methyl 4iodobenzoate (199 mg, 0.72 mmol) for 72 h at 125 ° C. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 95: 5), obtaining 24 mg of 5-phenyl-2- (4-ethoxycarbonylphenyl) -1H-indole as a white solid (14% yield ). 1H NMR (CDCl3, 400 MHz): 8.45 (s, 1H), 8.12 (d, J = 8.3 Hz, 2H), 7.86 (s, 1H), 7.75 (d, J = 8.3 Hz, 2H), 7.66 (dd, J = 8.2, 1.0 Hz, 2H), 7.49 (d, J = 1.5 Hz, 2H), 7.45 (t, J = 7.7 Hz, 2H), 7.33 (t, J = 7.4 Hz, 1H), 7.00 (d, J = 2.1 Hz, 1H), 3.95 (s, 3H) ppm.

Example 14: Preparation of 2- (4-pyrrolidine-1-carbonylphenyl) -1H-indole

The title compound was prepared analogously to Example 10 from indole (100 mg, 0.85 mmol) and 4iodophenyl (pyrrolidin-1-yl) methanone (360 mg, 1.19 mmol) for 48 h at 125 ° C. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 30:70), yielding 75 mg of 2- (4-pyrrolidine-1-carbonylphenyl) -1 Hindol as a white solid (30% yield). 1H NMR (CDCl3, 400 MHz): 5 8.53 (s, 1H), 7.69 (dd, J = 8.3, 6.5 Hz, 2H), 7.61 (t, J = 8.5 Hz, 3H), 7.43 (dd, J = 11.5, 6.0 Hz, 1H), 7.24 - 7.19 (m, 1H), 7.16 - 7.11 (m, 1H), 6.87 (s, 1H), 3.66 (s, 5H), 1.94 (s, 5H) ppm.

Example 15: Preparation of 2- (4-morpholine-1-carbonylphenyl) -1H-indole

The title compound was prepared analogously to Example 10 from indole (40.8 mg, 0.34 mmol) and 4iodophenyl (morpholin-1-yl) methanone (151.4 mg, 0.48 mmol) for 48 h at 125 ° C. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 30:70), obtaining 47 mg of 2- (4-morpholine-1-carbonylphenyl) -1 Hindol as a white solid (45% yield). 1 H NMR (CDCl 3, 400 MHz): 5 9.07 (s, 1 H), 7.67 - 7.61 (m, 3 H), 7.40 (t, J = 8.8 Hz, 3 H), 7.20 (d, J = 1.0 Hz, 1 H), 7.12 (s, 1H), 6.83 (d, J = 1.4 Hz, 1H), 3.72 (s, 8H) ppm.

Example 16: Preparation of 4-iodophenyl (piperidin-1-yl) methanone

To a solution of 4-iodobenzoic acid (400 mg, 1.58 mmol) in anhydrous dichloromethane (10 mL) and catalytic DMF (48 µL) was added oxalyl chloride (1.6 mL, 19 mmol). The mixture was stirred at reflux temperature (65 ° C) for 12 h. After this time, the excess oxalyl chloride was distilled to obtain a yellow solid. Then, at a temperature of 0 ° C, a solution of pyrrolidine (660 μL, 7.9 mmol) in anhydrous dichloromethane (10 mL) was added, stirring the mixture for 1 h. The mixture was then concentrated to dryness. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 40:60), obtaining 445 mg of 4-iodophenyl (piperidin-1il) methanone as a yellowish solid (94% yield). 1 H NMR (CDCl 3, 400 MHz): 5 7.79 - 7.71 (m, 2H), 7.28 -

7.24 (m, 2H), 3.63 (t, J = 6.9 Hz, 2H), 3.41 (t, J = 6.6 Hz, 2H), 2.01 - 1.84 (m, 4H) ppm.

Example 17: Preparation of (4-iodophenyl) (morpholino) methanone

To a solution of 4-iodobenzoic acid (400 mg, 1.58 mmol) in

Anhydrous dichloromethane (10 mL) and catalytic DMF (48 µL) oxalyl chloride (1.6 mL, 19 mmol) was added. The mixture was stirred at reflux temperature (65 ° C) for 12 h. After this time, the excess oxalyl chloride was distilled to obtain a yellow solid. Then at the temperature of 0 ° C a solution of morpholine (695 μL,

7.9 mmol) in anhydrous dichloromethane (10 mL), stirring the mixture for 1 h. The mixture was then concentrated to dryness. The residue was purified by flash chromatographic column (SiO2, hexane: AcOEt, 40:60), obtaining 446 mg of (4-iodophenyl) (morpholino) methanone as a yellowish solid (89% yield). 1 H NMR (CDCl 3, 400 MHz): 5 7.80 - 7.74 (m, 2H), 7.19 - 7.13 (m, 2H), 3.58 (m, 8H) ppm.

Example 18: Preparation of 3,3-Difluoro-2-phenyl-3H-indole

In a flask, 2-phenylindole (1.5 g, 7.37 mmol) was dissolved in anhydrous ACN (80 mL). Next Na2CO3 was added

(s) (15 g) and the Selectfluor® fluorinating agent (5.74 g, 16.2 mmol). The mixture was stirred at room temperature for 5 h. After completion of the reaction, diethyl ether (100 mL) was added and H2O extractions (5 x 30 mL) were made from the organic phases. The organic extracts were dried over Na2SO4, filtered and evaporated under reduced pressure, yielding 1.74 g of 3,3-difluoro-2-phenyl-3H-indole as an orange solid (99% yield). 1 H NMR (CDCl 3, 400 MHz): 5 8.21 (d, J = 7.2 Hz, 2H), 7.62-7.46 (m, 6H), 7.31 (t, J = 7.9 Hz, 1H) ppm. NMR19F (CDCl3, 400 MHz): 5

116.84 (s) ppm. NMR 13C (CDCl3, 100 MHz): 5 169.15 (t, J = 24.8 Hz), 152.54 (t, J = 9.7 Hz), 132.26, 132.48, 129.08,

128.74 (m), 128.7 (t, J = 37.1 Hz), 128.54, 127.47, 123.07 (t, J = 256.3 Hz), 123.02, 121.94 ppm. HRMS (ESI): calculated for (M + H +) C14H9F2N: 230.0703; Found: 230.0784.

Example 19: Preparation of 2- (4-chlorophenyl) -3,3-difluoro-3H-indole

The title compound was prepared analogously to Example 18 from the compound of Example 1 (59 mg, 0.25 mmol) in anhydrous ACN (7 mL), Na2CO3 (s) (500 mg) and the Selectfluor® fluorinating agent (227.3 mg, 0.61 mmol). The mixture was stirred at room temperature for 2 h, obtaining 50.8 mg of 2- (4-chlorophenyl) -3,3-difluoro-3H-indole as an orange solid (77% yield). 1 H NMR (CDCl3, 400 MHz): 5 8.13 (d, J = 8.5 Hz, 2H), 7.57 (d, J = 7.3 Hz, 1H), 7.54 - 7.49 (m, 4H), 7.32 (td, J = 6.8, 2.1 Hz, 1H) ppm. NMR19F (CDCl3, 400 MHz): 5-117.11 ppm. NMR 13C (CDCl3, 100 MHz): 5 168.39 (t, J = 25.0 Hz), 152.63 (t, J = 9.6 Hz), 139.08 (s), 133.61 (s), 129.99 (s), 129.52 (s), 128.96 (t, J = 24.0 Hz), 127.92 (s, J = 7.1 Hz), 127.55 (t, J = 2.8 Hz), 123.35 (s), 123.14 (t, J = 256.2 Hz), 122.29 (s) ppm. HRMS (ESI): calculated for (M + H +) C14H8ClF2N: 264.0313; Found: 264.0391.

Example 20: Preparation of ethyl 4- (3,3-difluoro-3H-indole-2-yl) benzoate

The title compound was prepared analogously to Example 18 from the compound of Example 3 (70 mg, 0.264 mmol) in anhydrous ACN (7 mL), Na2CO3 (s) (600 mg) and the Selectfluor® fluorinating agent (236.1 mg, 0.63 mmol). The mixture was stirred at room temperature for 3 h, obtaining 58.6 mg of ethyl 4- (3,3-difluoro-3H-indole-2-yl) benzoate as an orange solid (74% yield). NMR1H (CDCl3, 400 MHz): 5 8.26 (d, J = 8.3 Hz, 2H), 8.20 -

8.17 (m, 2H), 7.61 - 7.51 (m, 3H), 7.37 - 7.31 (m, 1H), 4.43 (q, J = 7.1 Hz, 2H), 1.43 (t, J = 7.1 Hz, 3H) ppm. NMR19F (CDCl3, 400 MHz): -117.71 ppm. NMR13C (CDCl3, 100 MHz): 5 168.61 (t, J = 25.0 Hz), 166.20 (d, J = 31.9 Hz), 152.50 (t, J = 9.5 Hz), 133.80 (s), 133.63 (s), 132.76 (t, J = 2.7 Hz), 130.15 (s), 130.74 - 129.73 (m), 129.32 - 128.77 (m),

128.58 (s), 128.28 (s), 123.38 (s), 123.06 (s), 122.59 (s), 61.62 (s, J = 36.8 Hz), 14.52 (s) ppm. HRMS (ESI): calculated for (M + H +) C17H13F2NO2: 302.0914; Found: 302.0993.

Example 21: Preparation of 6-chloro-2- (4-chlorophenyl) -3,3-difluoro-3H-indole (Ie),

The title compound was prepared analogously to Example 18 from the compound of Example 4 (80 mg, 0.34 mmol) in anhydrous ACN (7.5 mL), Na2CO3 (s) (340 mg) and the Selectfluor® fluorinating agent (282.5 mg, 0.76 mmol). The mixture was stirred at room temperature for 2 h, obtaining 71 mg of 6-chloro-2- (4-chlorophenyl) -3,3-difluoro-3 Hindole as an orange solid (70% yield). RMN1H (CDCl3, 400 MHz): 5 8.12 (d, J = 8.6 Hz, 2H), 7.55 - 7.46 (m, 4H), 7.30 (dd, J = 7.8, 1.7 Hz, 1H) ppm. NMR19F (CDCl3, 400 MHz): 5-116.33 ppm. NMR13C (CDCl3, 100 MHz): 5

169.74 (t, J = 25.0 Hz), 153.99 (t, J = 9.7 Hz), 139.61 (s, J = 10.5 Hz), 139.50 (s), 130.18 (s), 129.61 (s), 127.72 (s, J =

18.9 Hz), 127.24 (dd, J = 26.3, 22.6 Hz), 127.19 (s), 124.09 (s), 123.02 (s), 122.53 (t, J = 256.6 Hz) ppm. HRMS (ESI): calculated for (M + H +) C14H7Cl2F2N: 297.9924; Found: 298.0009.

Example 22: Preparation of 2- (3-chlorophenyl) -3,3-difluoro-3H-indole (Ih)

The title compound was prepared analogously to Example 18 from the compound of Example 6 (10 mg, 0.044 mmol) in anhydrous ACN (1.5 mL), Na2CO3 (s) (100 mg) and the Selectfluor® fluorinating agent (34 mg, 0.09 mmol). The mixture was stirred at room temperature for 2 h, obtaining 7.3 mg of 2- (3-chlorophenyl) -3,3-difluoro-3H-indole as an orange solid (63% yield). 1H NMR (CDCl3, 400 MHz): 5 8.20 (s, 1H), 8.06 (d, J = 7.7 Hz, 1H), 7.61 - 7.52 (m, 7H), 7.47 (t, J = 7.9 Hz, 2H), 7.33 (td, J = 7.1, 1.6 Hz, 2H) ppm. NMR19F (CDCl3, 400 MHz): 5-117.49 ppm. HRMS (ESI): calculated for (M + H +) C14H8ClF2N: 264.0313; Found: 264.039.

Example 23: Preparation of ethyl 4- (6-chloro-3,3-difluoro-3H-indole-2-yl) benzoate

The title compound was prepared analogously to Example 18 from the compound of Example 9 (49 mg, 0.16 mmol) in anhydrous ACN (3.2 mL), Na2CO3 (s) (160 mg) and the Selectfluor ® fluorinating agent (134.1 mg, 0.36 mmol). The mixture was stirred at room temperature for 5 h, obtaining 49 mg of ethyl 4- (6-chloro-3,3-difluoro-3H-indole-2-yl) benzoate as an orange solid (86% yield). 1 H NMR (CDCl3, 400 MHz): 5 8.25 (d, J = 8.4 Hz, 2H), 8.20 - 8.17 (m, 2H), 7.57 (s, 1H), 7.53 - 7.49 (m, 1H), 7.33 (dd, J = 7.8, 1.7 Hz, 1H), 4.42 (t, J = 7.1 Hz, 2H), 1.44 (d, J = 7.1 Hz, 3H) ppm. NMR19F (CDCl3, 400 MHz): 5-116.94 ppm HRMS (ESI): calculated for (M + H +) C17H12ClF2NO2: 336.0525; Found: 336.0596.

Example 24: Biological assays for the detection of antitumor activity

An exploration of the antitumor activity of the compounds of formula (I) in Jurkat cells was carried out and a more detailed study of the compounds with better activity in Jurkat and HeLa cells was performed.

Cell culture

Jurkat human cell lines (T lymphocytes from acute T-cell leukemia) and HeLa (cervical adenocarcinoma epithelial cell line) were obtained from the European Cell Culture Collection.

The Jurkat cell line grew in RPMI-1640 medium and the HeLa cell line in DMEM. Both culture media were supplemented with 10% inactivated fetal serum (veal), 1% glutamine, and 1% penicillin-streptomycin and were grown at 37 ° C in an atmosphere dampened with 5% carbon dioxide.

Reagents

Dimethyl sulfoxide (DMSO) was obtained from Sigma Chemicals Co. (St Louis, MO, USA). Propidium iodide ("propidium iodide", PI) was obtained from Bender MedSystems (Vienna, Austria). Annexin V-APC was obtained from eBioscience (St Diego, USA).

Analysis of apoptosis by flow cytometry

0.25-0.3x106 cells were washed in phosphate buffered saline (PBS), resuspended in 100 µl of annexin binding buffer and incubated with 2-5 µl of Annexin V-Allophycocyanin (APC ). After a 15 min incubation in the dark at room temperature, 100 µl of annexin binding buffer with 5 µl of propidium iodide ("propidium iodide", PI) (20 µg / ml) was added just before of flow cytometric analysis. Data were analyzed with the appropriate program. Cell viability was measured by the analysis of phosphatidylserine outsourcing and the incorporation of PI, and is expressed as the percentage of annexin-V cells and double negative PI.

Apoptosis, or programmed cell death, is a general physiological mechanism for the removal of unwanted cells. It is characterized by chromatin condensation, a reduction in cell volume, and DNA cutting carried out by endonucleases that results in fragments of oligonucleosomal length. Apoptosis is also accompanied by a loss of 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 carried out by macrophages. This is one of the earliest events of the apoptotic process. The method for the detection of apoptotic cells by flow cytometry uses the binding of annexin V labeled with a fluorochrome to phosphatidylserine.

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

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

Results

1. Exploratory test of cell viability in Jurkat cells

An exploration of the effect of some compounds of formula (I) on the Jurkat cell line (T lymphocytes from an acute T-cell leukemia) was performed at a single maximum dose of 40 µM during a 24-hour incubation. The structure of the compounds tested is indicated in Table 1. The compound of formula (I) where R1-R8 is H (compound Io) has also been tested.

Jurkat cells were chosen among other leukemic tumor cell lines because they have the mutated p53 protein.

All the compounds mentioned were dissolved in the minimum amount of DMSO needed to be completely dissolved. Cell viability was measured by flow cytometry. It was verified that DMSO alone did not decrease cell viability. Thus, all the effects observed in cell viability were due to the activity of these compounds.

The results are summarized in Table 3: "+" means low activity; "++" means good activity and "+++" means very good activity; "-" means that it is not active.

Table 3:

Compound

Biological activity

Ia

 +++

Ib

 ++

Ic

 +++

Id

 +++

Ie

 ++

If

 ++

Ig

 +++

Ih

 ++

Ii

 ++

Ij

 ++

Ik

 ++

Il

 +

Im

 +++

In

 ++

Io

 +++

2. Study of the dose-response of compounds Ic and Id

A dose-response analysis of the compounds Ic and Id was performed in Jurkat cells with mutated p53 and in HeLa cells with inactivated p53.

Cell viability was measured by flow cytometry and expressed as a percentage of non-apoptotic cells (annexin V negative) with respect to untreated cells at 24 and 48 hours of incubation. Therefore, values below 100% are indicative of apoptosis or loss of cell viability.

Jurkat cells (which are T lymphocytes from an acute type T leukemia, with mutated p53) were incubated with a dose range from 2 µM to 40 µM for each compound for 24 and 48 hours. All compounds induced apoptosis in a dose-dependent manner. Viability was measured by flow cytometry (cf. FIG. 1 and 3).

HeLa cells (cervical adenocarcinoma epithelial cell line with inactivated p53) were incubated with a dose range from 5 µM to 40 µM for each compound for 24 and 48 hours. All compounds induced apoptosis in a dose-dependent manner. Viability was measured by flow cytometry (cf. FIG. 2 and 4).

The IC50 at 24 hours was calculated for each compound using the viability values obtained by flow cytometric analysis. The results are expressed in Table 4 as the percentage of non-apoptotic cells (annexin V negative) with respect to untreated cells at 24 and 48 hours.

Table 4:

Compound

IC50 (μM) 24 hours IC50 (μM) 48 hours

Jurkat

HeLa  Jurkat HeLa

Ic

 16 twenty 11.5 eleven

Id

 8 9 7.5 7

Io

25 39 31 40

These results demonstrate that these compounds have antitumor activity. In addition, the apoptosis induced by these indolenin compounds is independent of the p53 protein, an important difference from the majority of drugs currently used in cancer therapy, which induce cell cycle arrest and apoptosis through the activation of p53.

Claims (14)

1. Compound of formula (I) or a pharmaceutically acceptable salt thereof, or a stereoisomer thereof, or a mixture of stereoisomers,      (I) where: R1, R2, R4, R5, and R7, are independently selected from the group consisting of H, Cl, C (= O) O (C1-C4) alkyl, O (C1 C4) alkyl; and (C1-C4) alkyl; R3 is selected from the group consisting of H, Cl, C (= O) O (C1-C4) alkyl, O (C1-C4) alkyl, (C1-C4) alkyl, pyrrolidinyl-1carbonyl, and morpholin-1-carbonyl ; R6 is selected from the group consisting of H and (C1-C4) alkyl; Y R8 is selected from the group consisting of H, F, Br, (C1-C4) alkyl, and phenyl; 20 with the proviso that the compound of formula (I) is different from the compound of formula (I) where R1-R8 = H, and with the proviso that the compound of formula (I) is different from the compound of formula (I) where R1 and R3-R8 are H and R2 is methoxy.

2.

 Compound of formula (I) according to claim 1, wherein R1, R4 and R5 are H.

3.

 Compound according to claim 2, wherein R6 is H.

4. A compound according to any of claims 1-3, wherein R2 and R7 are independently selected from the group consisting of H and Cl. 5. Compound according to any of claims 1-3, wherein R3 is independently selected from the group consisting of H, Cl, and (C1-C4) alkoxycarbonyl. 6. Compound according to claim 5, wherein the (C1-C4) alkoxycarbonyl is ethoxycarbonyl. Compound according to any of claims 1-6, wherein R8 is H or methyl.

8.

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

9.

 Compound according to claim 8, which is selected from the following table:

10.

 Compound according to claim 9, which is selected from: compound (I) wherein R1-R2, R4-R8 are H and R3 is ethoxycarbonyl, and compound (I) wherein R1-R2, R4-R6 and R8 are H and R3 and R7 are chlorine.

eleven.

Compound of formula (I), as defined in any of claims 1-9, including a compound of formula (I) wherein R1-R8 = H or a compound of formula (I) wherein R1 and R3-R8 are H and R2 is methoxy, for use as a medicine.

12.

 Use of the compound of the compound of formula (I), as defined in any of claims 1-9, including a compound of formula (I) wherein R1-R8 = H or a compound of formula (I) wherein R1 and R3- R8 are H and R2 is methoxy, for the preparation of a medicament for the treatment and / or prevention of cancer in a mammal, including humans.

13.

 Use according to claim 12, wherein the cancer is selected from the group consisting of leukemia and cervical cancer.

14.

 Pharmaceutical composition comprising an effective therapeutic amount of the compound of formula (I), defined in any of claims 1-9, or a compound of formula (I) wherein R1-R8 = H or a compound of formula (I) wherein R1 and R3-R8 are H and R2 is methoxy, together with sufficient amounts of pharmaceutically acceptable excipients or carriers.

fifteen.

 Pharmaceutical composition according to claim 14, wherein the compound of formula (I) is as defined in any of claims 1-9.

Comp. (I)

R1  R2  R3  R4  R5  R6  R7  R8

Ia

H H Cl H H H H H

Ib

H H Methoxy H H H H H

Ic

H H Ethoxycarbonyl H H H H H

Id

H H Cl H H H Cl H

Ie

Cl H H H H H H H

If

H H Ethyl H H H H H

Ig

H Cl H H H H H H

Ih

H H H H H H H Met ilo

Ii

H Methoxy Methoxy H H H H H

Ij

H H H H H Ethyl H H

Ik

 H H 4-pyrrolidin-1carbonyl H H H H H

Il

 H H 4-morpholin-1carbonyl H H H H H

Im

H H Ethoxycarbonyl H H H Cl H

In

H H Methoxycarbonyl H H H H Fen ilo

Comp. (I)

R1  R2  R3  R4  R5  R6  R7  R8

Ia

H H Cl H H H H H

Ic

H H Ethoxycarbonyl H H H H H

Id

H H Cl H H H Cl H

Ig

H Cl H H H H H H

Im

H H Ethoxycarbonyl H H H Cl H

Ic FIG. 1: Jurkat Ic FIG. 2: HeLa fifteen Id FIG. 3: Jurkat FIG. 4: HeLa 16 SPANISH OFFICE OF PATENTS AND BRANDS SPAIN REPORT ON THE STATE OF THE TECHNIQUE 51 Int. Cl.: C07D209 / 30 (2006.01) A61K31 / 404 (2006.01) twenty-one N.O. application: 2011 31915 22 Date of submission of the application: 28.11.2011 32 Date of priority: RELEVANT DOCUMENTS

Category

56 Documents cited Claims Affected

x

R LIN et al., Organic Letters 2011, vol 13, # 17, pages 4498-4501. "An efficient eleven

difluorohydroxylation of indoles using Selectfluor as a fluorinating reagent ", table 2, compounds 13

and 14.

TO

   1-10

TO

WO 2009037308 A1 (JANSSEN PHARMACEUTICA) 26.03.2009, 12-15

claims 1-14.

Category of the documents cited x: of particular relevance Y: of particular relevance combined with other / s of the same category A: reflects the state of the art O: refers to unwritten disclosure P: published between the priority date and the date of priority submission of the application E: previous document, but published after the date of submission of the application

This report has been produced • for all claims • for claims No:

Date of realization of the report 19.02.2013

Examiner M. P. Fernandez Fernandez Page 1/4

TECHNICAL STATUS REPORT Application NO .: 201131915 Minimum documentation sought (classification system followed by classification symbols) C07D, A61K Electronic databases consulted during the search (name of the database and, if possible, terms of search used) INVENES, EPODOC, WPI, CAS, REGISTRY, EMBASE State of the Art Report Page 2/4  WRITTEN OPINION Application NO .: 201131915 Date of Written Opinion: 19.02.2013 Statement

Novelty (Art. 6.1 LP 11/1986)

Claims 1-10,12-15 Claims 11 IF NOT

Inventive activity (Art. 8.1 LP11 / 1986)

Claims 1-10,12-15 Claims 11 IF NOT

The application is considered to comply with the industrial application requirement. This requirement was evaluated during the formal and technical examination phase of the application (Article 31.2 Law 11/1986).  Opinion Base.- This opinion has been made on the basis of the patent application as published. State of the Art Report Page 3/4  WRITTEN OPINION Application NO .: 201131915  1. Documents considered.- To continuation, the documents pertaining to the technique study taken in consideration for the realization of this opinion are listed.

Document

Publication or Identification Number publication date

D01

R LIN et al., Organic Letters 2011, vol 13, # 17, pages 4498-4501. "An efficient difluorohydroxylation of indoles using Selectfluor as a fluorinating reagent", table 2, compounds 13 and 14. 09.2011

 2. Statement motivated according to articles 29.6 and 29.7 of the Regulations for the execution of Law 11/1986, of March 20, on Patents on novelty and inventive activity; quotes and explanations in support of this statement The request is effected at the 3,3-difluoro-3 (H) indoles of formula (I) of claim 1, (claims 1-7), with the exception of compounds in which R1 to R8 are H and R1, R3 to R8 are H and R2 is OCH3, more specifically to the compounds described in claims 8 to 10, to the pharmaceutical composition (claims 14 and 15) comprising a compound according to claims 1-9 and to its use for the treatment of cancer (claims 12 and 13). Claim 11 refers to the compounds excluded from the formula (I) of claim 1. Document D1 is considered the closest to the state of the art. It discloses compounds (see table 2 of D1) of formula similar to those of claim 1 of the same, are not yet found previously disclosed by him or that are considered to be new; however, in claim 11 of the application two compounds disclosed in D1 are specifically claimed, then this claim cannot be considered new. On the other hand, claims 1-10 are considered inventive as it is necessary to vary the substituents of the base formula to study which confer upon the molecule the desired pharmacological properties. The pharmaceutical composition comprising compounds of the formula (I) of the application or the use of these compounds for the treatment of cancer has not been disclosed, and claims 12-15 are considered new. Therefore, it is concluded that claims 1-10 and 12-15 of the application are new and inventive and claim 11 is novel, as set forth in Art. 6.1 and 8.1 of Patent Law 11/1986. State of the Art Report Page 4/4