ES2381117T3 - Indolyl-alkylamine derivatives substituted as novel histone deacetylase inhibitors - Google Patents

Abstract

A compound of formula (I), N-oxide type forms, pharmaceutically acceptable addition salts and stereochemically isomeric forms thereof, where each n is an integer with a value of 0, 1 or 2 and, when n is 0, then it refers to a direct link; each m is an integer with a value of 1 or 2; each X is independently N or CH; each Y is independently O, S or NR4; where each R 4 is hydrogen, C 1-6 alkyl, (C 1-6 alkyloxy) (C 1-6 alkyl), C 3-6 cycloalkyl, (C 3-6 cycloalkyl) methyl, phenyl (C 1-6 alkyl), -C (= O ) -CHR5R6 or -S (= O) 2-N (CH3) 2; where each R5 and R6 is independently hydrogen, amino, C1-6 alkyl or C1-6 aminoalkyl; and when Y is NR4 and R2 is in position 7 of the indolyl, then R2 and R4 together can form the bivalent radical - (CH2) 2- (a-1),. (CH2) 3- (a-2); R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkylsulfonyl, (C 1-6 alkyl) carbonyl or mono- or di (C 1-6 alkyl) aminosulfonyl; R2 is hydrogen, hydroxy, amino, halo, C1-6 alkyl, cyano, C2-6 alkenyl, C1-6 polyhaloalkyl, nitro, phenyl, (C16 alkyl) carbonyl, hydroxycarbonyl, (C1-6 alkyl) carbonylamino, C1- alkyloxy 6, or mono- or di (C1-6 alkyl) amino; R3 is hydrogen, C1-6 alkyl or C1-6 alkyloxy; and when R2 and R3 are in adjacent carbon atoms, they can form the bivalent radical -O-CH2-O-.

Description

Indolyl-alkylamine derivatives substituted as novel histone deacetylase inhibitors

This invention relates to compounds that possess histone deacetylase inhibitory enzyme activity (HDAC). In addition, it refers to processes for its preparation, to compositions comprising them, as well as to its use, both in vitro and in vivo, to inhibit HDAC and as a medicament, for example, as a medicament to inhibit proliferative conditions such as Cancer or psoriasis

There is evidence that nuclear histones are dynamic and integral components of the machinery responsible for the regulation of genetic transcription and other processes shaped by DNA such as replication, repair, recombination and chromosomal segregation. They are subject to post-translational modifications, which include acetylation, phosphorylation, methylation, ubiquitination and ADP-ribosylation.

The histone deacetylase (s), referred to herein as "HDAC", are enzymes that catalyze the elimination of the acetyl modification in the lysine residues of the proteins, including the internal nucleosomal histones H2A, H2B, H3 and H4. Together with the histone acetyltransferase (s), referred to herein as "HAT", HDACs regulate the level of acetylation of histones. The balance of acetylation of nucleosomal histones plays an important role in the transcription of many genes. Hypoacetylation of histones is associated with the condensed structure of chromatin, which causes repression of genetic transcription, while acetylated histones are associated with a more open chromatin structure and activation of transcription.

Eleven structurally related HDACs have been described and are grouped into two classes. Class I HDACs consist of HDAC 1, 2, 3, 8 and 11, while Class II HDACs consist of HDAC 4, 5, 6, 7, 9 and

10. Members of a third class of HDAC are not structurally related to class I and class II HDACs. Class I / II HDACs operate through zinc-dependent mechanisms, while Class III HDACs depend on NAD.

In addition to histones, other proteins have also acted as substrates for acetylation, in particular, transcription factors such as p53, GATA-1 and E2F; nuclear receptors such as the glucocorticoid receptor, thyroid receptors, estrogen receptors; and cell cycle regulatory proteins such as pRb. Protein acetylation has been linked to protein stabilization such as p53 stabilization, cofactor recruitment and increased DNA binding. P53 is a tumor suppressor that can induce cell cycle arrest or apoptosis in response to a series of stress signals such as DNA lesions. The main target for cell cycle arrest induced by p53 appears to be the p21 gene. After activation by p53, p21 has been identified due to its association with cyclin complexes and cyclin-dependent kinase complexes, which cause the cell cycle to stop at phases G1 and G2, its increase during senescence and its interaction with the proliferating cell nuclear antigen.

The study of HDAC inhibitors indicates that they play an important role in cell cycle arrest, cell differentiation and the reversal of transformed phenotypes.

The tricostatin A (TSA) inhibitor, for example, causes the cell cycle to stop in phases G1 and G2, reverses the transformed phenotype of different cell lines and induces differentiation of Friend's leukemia cells, among others. It has been described that TSA (and SAHA suberoilanilide hydroxamic acid) inhibit cell growth, induce terminal differentiation and prevent tumor formation in mice (Finnin et al., Nature, 401: 188-193, 1999).

It has also been described that tricostatin A is useful in the treatment of fibrosis, e.g. eg, liver fibrosis and liver cirrhosis (Geerts et al., European patent application EP 0 827 742, published March 11, 1998).

The pharmacophore for HDAC inhibitors consists of a metal-binding domain that interacts with the active center that contains HDAC zinc, a linker domain and a surface recognition domain.

or restriction region, which interacts with the residues at the edge of the active center.

It has also been described that HDAC inhibitors induce p21 gene expression. The transcriptional activation of the p21 gene by these inhibitors is favored by chromatin remodeling, after acetylation of histones H3 and H4 in the region of the p21 promoter. This activation of p21 occurs independently of p53 and, therefore, HDAC inhibitors are operative in cells with mutated p53 genes, which represent a distinctive feature of numerous tumors.

In addition, HDAC inhibitors may have indirect activities such as increased host immune responses and inhibition of tumor angiogenesis and, therefore, can suppress the growth of primary tumors and prevent metastasis (Mai et al., Medicinal Research Reviews, 25: 261-309).

Given the above, HDAC inhibitors can have great potential in the treatment of cell proliferative diseases or conditions, including tumors with mutated p53 genes.

Patent application EP1472216, published on August 14, 2003, describes bicyclic hydroxamates as histone deacetylase inhibitors.

Patent applications EP1485099, EP1485348, EP1485353, EP1485354, EP1485364, EP1485365, EP1485370 and EP1485378, published on September 18, 2003, among others, describe substituted piperazinylpyrimidinylhydroxamic acids as histone deacetylase inhibitors; In addition, EP1485365 describes R306465.

Patent application EP1492534, published October 9, 2003, describes carbamic acids comprising a piperazine linker as HDAC inhibitors.

Patent application EP1495002, published October 23, 2003, describes substituted piperazinylphenylbenzamide compounds as histone deacetylase inhibitors.

Patent application WO04 / 009536, published on January 29, 2004, describes derivatives containing an alkyl linker between the aryl group and the hydroxamate as histone deacetylase inhibitors.

Patent application EP1525199, published on February 12, 2004, describes bicyclic hydroxamates substituted with (hetero) arylalkenyl as histone deacetylase inhibitors.

Patent application WO04 / 063146, published on July 29, 2004, describes N-hydroxybenzamide derivatives with anti-inflammatory and anti-tumor activity.

Patent application WO04 / 063169, published on July 29, 2004, describes substituted aryl hydroxamates as histone deacetylase inhibitors.

Patent application WO04 / 072047, published on August 26, 2004, describes indoles, benzimidazoles and naphh-imidazoles as histone deacetylase inhibitors.

Patent application WO04 / 082638, published on September 30, 2004, describes hydroxamates bound to non-aromatic heterocyclic ring systems as histone deacetylase inhibitors.

Patent application WO04 / 092115, published on October 28, 2004, describes hydroxamates as histone deacetylase inhibitors.

Patent application WO05 / 028447, published March 31, 2005, describes benzimidazoles as histone deacetylase inhibitors.

Patent applications WO05 / 030704 and WO05 / 030705, published on April 7, 2005, describe benzamides as inhibitors of histone deacetylase.

Patent application WO05 / 040101, published on May 6, 2005, describes hydroxamates connected to acylurea and connected to sulfonylurea as histone deacetylase inhibitors.

Patent application WO05 / 040161, also published on May 6, 2005, describes biaryl-linked hydroxamates as histone deacetylase inhibitors.

The compounds of the present invention differ from the prior art in their structure, in their pharmacological activity and / or pharmacological potency.

The problem that needs to be solved consists in obtaining histone deacetylase inhibitors with a high cellular and enzymatic activity that have a greater bioavailability and / or potency in vivo.

The novel compounds of the present invention solve the problem described above.

The compounds of the present invention exhibit excellent cellular and enzymatic activity inhibiting histone deacetylase. They have a great ability to activate the p21 gene, both at the cellular level and at the in vivo level. They have a desirable pharmacokinetic profile and a low affinity for P450 enzymes, which reduces the risk of adverse drug-drug interactions and also allows for a wider safety margin.

Some advantageous characteristics of the compounds herein are metabolic stability, solubility and / or the ability to induce the production of p21. More specifically, the compounds of the present invention have higher half-lives in rat hepatocytes, have greater solubility / stability in aqueous solutions and / or have a greater ability to induce p21 promoter production in vivo.

This invention relates to compounds of formula (I)

R3O

R1 YNHO N

N (CH2) n N (CH2) m

(I) H X R2

N-oxide type forms, pharmaceutically acceptable addition salts and stereochemically isomeric forms thereof, where 10 each n is an integer with a value of 0, 1 or 2 and, when n is 0, then refers to a direct link;

each m is an integer with a value of 1 or 2;

15 each X is independently N or CH;

each Y is independently O, S or NR4; where

each R4 is hydrogen, C1-6 alkyl, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl, (C3-6 cycloalkyl) methyl, phenyl (C1-6 alkyl), -C (= O ) -CHR5R6 or -S (= O) 2-N (CH3) 2; where

each R5 and R6 is independently hydrogen, amino, C1-6 alkyl or C1-6 aminoalkyl; Y

when Y is NR4 and R2 is in the 7 position of the indolyl, then R2 and R4 together can form the bivalent radical 25 - (CH2) 2- (a-1), or - (CH2) 3- (a-2);

R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkylsulfonyl, (C 1-6 alkyl) carbonyl or mono- or di (C 16 alkyl) aminosulfonyl;

R2 is hydrogen, hydroxy, amino, halo, C1-6 alkyl, cyano, C2-6 alkenyl, C1-6 polyhaloalkyl, nitro, phenyl, (C16 alkyl) carbonyl, hydroxycarbonyl, (C1-6 alkyl) carbonylamino, C1 alkyloxy -6, or mono- or di (C1-6 alkyl) amino;

R3 is hydrogen, C1-6 alkyl or C1-6 alkyloxy; and when R2 and R3 are in adjacent carbon atoms, they can form the bivalent radical -O-CH2-O-.

Lines drawn within the bicyclic ring systems relative to the substituents indicate that the bonds can be attached to any of the ring atoms that are suitable within the bicyclic ring system.

The expression "histone deacetylase inhibitor" or "histone deacetylase inhibitor" is used to identify a compound capable of interacting with a histone deacetylase and inhibit its activity, more specifically its enzymatic activity. Inhibition of the histone deacetylase enzyme activity refers to the reduction of histone deacetylase's ability to remove an acetyl group from a histone. Preferably, this

Inhibition is specific, that is, the histone deacetylase inhibitor reduces the ability of a histone deacetylase to remove an acetyl group from a histone with a concentration lower than the concentration of the inhibitor necessary to produce some other unrelated biological effect.

As used in the above definitions and hereinafter, "halo" is the generic term for fluoro, chloro, bromo and iodo; C1-2 alkyl defines saturated straight chain hydrocarbon radicals containing 1

or 2 carbon atoms such as, e.g. eg, methyl or ethyl; C1-6 alkyl defines C1-2 alkyl and saturated straight and branched chain hydrocarbon radicals containing 3 to 6 carbon atoms such as, e.g. e.g., propyl, butyl,

1-methyl ethyl, 2-methylpropyl, pentyl, 2-methylbutyl, hexyl, 2-methylpentyl and the like; and C1-6 polyhaloalkyl defines alkyl containing three identical or different halo substituents, for example, trifluoromethyl; C2-6 alkenyl defines

C1-6

Linear and branched chain hydrocarbon radicals containing a double bond and having 2 to 6 carbon atoms such as, for example, ethenyl, 2-propenyl, 3-butenyl, 2-pentenyl, 3-pentenyl, 3-methyl -2butenyl and the like; C3-6 cycloalkyl includes cyclic hydrocarbon groups containing from 3 to 6 carbons such

as cyclopropyl, cyclobutyl, cyclopentyl, cyclopentenyl, cyclohexyl, cyclohexenyl and the like.

Pharmaceutically acceptable addition salts encompass pharmaceutically acid addition salts. Acceptable and pharmaceutically acceptable base addition salts. It is intended that acid addition salts Pharmaceutically acceptable, as previously mentioned herein, understand the forms therapeutically active non-toxic acid addition salts that the compounds of formula (I) are capable of to form. Compounds of formula (I) that possess basic properties can be converted into their addition salts of pharmaceutically acceptable acid by treating said basic form with a suitable acid. Suitable acids they comprise, for example, inorganic acids such as halohydric acids, e.g. eg, hydrochloric or hydrobromic acid; sulfuric acid; nitric acid; phosphoric acid and similar acids; or organic acids such as, for example, acid acetic, trifluoroacetic, propanoic, hydroxyacetic, lactic, pyruvic, oxalic, malonic, succinic (i.e. acid butanedioic), maleic, fumaric, malic, tartaric, citric, methanesulfonic, ethanesulfonic, benzenesulfonic, ptoluenesulfonic, cyclamic, salicylic, p-aminosalicylic, pamoic and similar acids. Compounds of formula (I) that possess acidic properties can be converted into their base addition salts pharmaceutically acceptable by treating said acid form with a suitable organic or inorganic base. The Suitable basic salt forms comprise, for example, ammonium salts, alkali metal salts and alkaline earths, p. eg, salts of lithium, sodium, potassium, magnesium, calcium and the like, salts with organic bases, e.g. eg, benzathine, N-methyl-D-glucamine, hydrabamine salts, and salts with amino acids such as, for example, arginine, lysine and the like. The term "acid or base addition salts" also includes hydrates and forms of addition of solvent that the compounds of formula (I) are capable of forming. Examples of such forms are, e.g. eg hydrates, alcoholates and the like.

The expression "sterochemically isomeric forms of the compounds of formula (I)", as used in the present, defines all possible compounds constituted by the same atoms bonded by the same sequence of links but presenting different three-dimensional structures, which are not interchangeable, that the compounds of formula (I) may possess. Unless otherwise mentioned or indicated, the denomination A compound's chemistry encompasses the mixture of all possible stereochemically isomeric forms that said compound may possess. Said mixture may contain all diastereomers and / or enantiomers of the basic molecular structure of said compound. It is intended that all forms stereochemically isomeric compounds of formula (I), both in pure form and in a mixture of one another, remain encompassed within the scope of the present invention.

It is intended that the N-oxide forms of the compounds of formula (I) comprise the compounds of formula (I) in which one or several nitrogen atoms are oxidized to the so-called N-oxide, particularly the N-oxides in which one or more of the nitrogens of piperidine, piperazine or pyridazinyl are N-oxidized

Some of the compounds of formula (I) may also exist in their tautomeric forms. It is intended that such forms are included within the scope of the present invention, although they are not explicitly indicated in the above formula

Whenever used hereinafter, the term "compounds of formula (I)" is intended to include also pharmaceutically acceptable addition salts and all stereoisomeric forms.

As used herein, the terms "histone deacetylase" and "HDAC" are intended to refer to any of Enzyme families that remove acetyl groups from £ -amino groups of lysine residues at the N-terminus of a histone. Unless the context indicates otherwise, the term "histone" is intended to refer to any histone type protein, including H1, H2A, H2B, H3, H4 and H5, of any species. The products Genetic or human HDAC proteins include, without limitation, HDAC-1, HDAC-2, HDAC-3, HDAC-4, HDAC-5, HDAC-6, HDAC-7, HDAC-8, HDAC-9 HDAC-10 and HDAC-11. Histone deacetylase can also come from a fungal or protozoic source.

A first group of interesting compounds is constituted by the compounds of formula (I) where they are applied one or more of the following restrictions:

a) each R4 is hydrogen, C1-6 alkyl, C3-6 cycloalkyl, (C3-6 cycloalkyl) methyl, -C (= O) -CHR5R6 or -S (= O) 2-N (CH3) 2; b) R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkylsulfonyl, or mono- or di (C 1-6 alkyl) aminosulfonyl; or c) R3 is hydrogen.

A second group of interesting compounds is constituted by the compounds of formula (I) where they are applied one or more of the following restrictions:

a) each n is an integer with a value of 0 or 1; b) each X is independently N; c) each R4 is hydrogen or C1-6 alkyl; d) R 1 is hydrogen, C 1-6 alkyl or C 1-6 hydroxyalkyl; or e) R2 is hydrogen, halo, C1-6 alkyl or C1-6 alkyloxy.

A third group of interesting compounds is constituted by the compounds of formula (I) where a

or more of the following restrictions:

5 a) each n is an integer with a value of 0 or 1; b) each R4 is hydrogen, C1-6 alkyl, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl or phenyl (C1-6 alkyl); c) R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, (C 1-6 alkyl) carbonyl or (C 1-6 alkyl) sulfonyl; or d) R2 is hydrogen, halo, C1-6 alkyl, cyano, nitro or C1-6 alkyloxy.

A fourth group of interesting compounds consists of the compounds of formula (I) where one or more of the following restrictions apply:

a) each n is an integer with a value of 0 or 1; b) each m is an integer with a value of 1;

C) each R4 is hydrogen, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl or phenyl (C1-6 alkyl); d) R1 is hydrogen, C1-6 hydroxyalkyl, (C1-6 alkyl) carbonyl or (C1-6 alkyl) sulfonyl; e) R2 is hydrogen, halo, cyano, nitro or C1-6 alkyloxy; f) R3 is C1-6 alkyloxy; or g) when R2 and R3 are in adjacent carbon atoms, they can form the bivalent radical -O-CH2-O-.

A fifth group of interesting compounds consists of the compounds of formula (I) where one or more of the following restrictions apply:

a) each n is an integer with a value of 1;

25 b) each m is an integer with a value of 1; c) each X is independently N; d) each Y is independently NR4; e) each R4 is C1-6 alkyl; f) R1 is hydrogen;

30 g) R2 is hydrogen or halo; or h) R3 is hydrogen.

A group of preferred compounds is constituted by the compounds of formula (I) where n is an integer with a value of 0 or 1; each R4 is hydrogen, C1-6 alkyl, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl or phenyl (C1-6 alkyl); R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, (C 1-6 alkyl) carbonyl or (C 1-6 alkyl) sulfonyl; and R2 is hydrogen, halo, C1-6 alkyl, cyano, nitro, C1-6 polyhaloalkyl or C1-6 alkyloxy.

A group of more preferred compounds is constituted by the compounds of formula (I) where each n is an integer with a value of 1; each m is an integer with a value of 1; each X is independently N; 40 each Y is independently NR4; each R4 is C1-6 alkyl; R1 is hydrogen; R2 is hydrogen or halo; and R3 is hydrogen.

The most preferred compounds are compound No. 1a, compound No. 30, compound No. 39 and compound No. 50.

The compounds of formula (I) and (II), their pharmaceutically acceptable salts and N-oxides and the stereochemically isomeric forms thereof can be prepared in a conventional manner. The starting materials and some of the intermediates are known compounds and can be purchased from commercial suppliers or can be prepared according to conventional reaction procedures, which are commonly used in the

N HN NN N H N O HO

N H N N N N O N H HO F

C2HF3O2; Compound 1a

C2HF3O2 (1: 1); Compound 30

O H N NN H N HO N N

F N H NHO N H N N O N

C2HF3O2 (1: 1); Compound 39

 Compound 50

5 matter or which are described in patent applications EP1485099, EP1485348, EP1485353, EP1485354, EP1485364, EP1485365, EP1485370 and EP1485378. Some preparation methods will be described hereinafter in more detail. Other methods of obtaining the final compounds of formula (I) are described in the examples.

10 a) The hydroxamic acids of formula (I) can be prepared by reacting an intermediate of formula (II), where Q is tetrahydropyranyloxyaminocarbonyl, referred to herein as intermediate of formula (II-a), with a suitable acid such as, by example, trifluoroacetic acid. Said reaction is carried out in a suitable solvent such as, for example, methanol or dichloromethane.

Or R3

R1

Y

N

O o

N

N (CH2) n N (CH2) m

H

X

R2

(II-a)

R3O

R1CF3COOH Y

N

HO N

N (CH2) n N (CH2) mH

X

R2 (I)

B) Alternatively, the hydroxamic acids of formula (I) can be prepared by reacting an intermediate of formula (II), where Q is (C1-2 alkyloxy) carbonyl, referred to herein as intermediate of formula (II-c) , with hydroxylamine, in the presence of a base such as, for example, sodium hydroxide. This reaction is carried out.

20 in a suitable solvent such as, for example, methanol.

The compounds of formula (I) can also be converted into each other by reactions or transformations

25 functional groups commonly used in the field. Previously, a series of these transformations have already been described. Other examples consist of the hydrolysis of carboxylic esters to obtain the corresponding acid or alcohol; hydrolysis of amides to obtain the corresponding acids or amines; hydrolysis of nitriles to obtain the corresponding amides; the amino groups of the imidazole or the phenyl can be replaced by hydrogen by means of reactions of formation of diazonium salts commonly used in the field and later

Replacement of the diazo group with hydrogen; alcohols can be converted into esters and ethers; primary amines can be converted into secondary and tertiary amines; the double bonds can be hydrogenated to obtain the corresponding single link; an iodine radical in a phenyl group can be converted into an ester group by inserting carbon monoxide in the presence of a suitable palladium catalyst.

The present invention also relates to intermediates of formula (II)

R3 R1

And Q

N

N (CH2) n N (CH2) m

(II) X

R2

N-oxide type forms, pharmaceutically acceptable addition salts and stereochemically isomeric forms thereof, where 5 each n is an integer with a value of 0, 1 or 2 and, when n is 0, then refers to a direct link;

each m is an integer with a value of 1 or 2;

10 each X is independently N or CH;

each Y is independently O, S or NR4; where

each R4 is hydrogen, C1-6 alkyl, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl, (C3-6 cycloalkyl) methyl, phenyl (C1-6 alkyl), -C (= O ) -CHR5R6 or -S (= O) 2-N (CH3) 2; where

each R5 and R6 is independently hydrogen, amino, C1-6 alkyl or C1-6 aminoalkyl; Y

when Y is NR4 and R2 is in position 7 of the indolyl, then R2 and R4 together can form the bivalent radical 20 - (CH2) 2- (a-1), or - (CH2) 3- (a-2);

R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkylsulfonyl, (C 1-6 alkyl) carbonyl or mono- or di (C 16 alkyl) aminosulfonyl;

R2 is hydrogen, hydroxy, amino, halo, C1-6 alkyl, cyano, C2-6 alkenyl, C1-6 polyhaloalkyl, nitro, phenyl, (C16 alkyl) carbonyl, hydroxycarbonyl, (C1-6 alkyl) carbonylamino, C1 alkyloxy -6, or mono- or di (C1-6 alkyl) amino;

R3 is hydrogen, C1-6 alkyl or C1-6 alkyloxy; and 30 when R2 and R3 are in adjacent carbon atoms, they can form the bivalent radical -O-CH2-O-; Y

Q is (C1-2 alkyloxy) carbonyl, hydroxycarbonyl or tetrahydropyranyloxyaminocarbonyl.

The groups of interesting, preferred, most preferred and most preferred compounds can be defined for the compounds of formula (II) according to the groups defined for the compounds of formula (I).

a) The intermediates of formula (II-a) can be prepared by reacting an intermediate of formula (III) with an intermediate of formula (II), where Q is hydroxycarbonyl, referred to herein as intermediate of formula (II-b), in

The presence of suitable reagents such as N '- (ethylcarbonimidoyl) -N, N-dimethyl-1,3-propanediamine (EDC) monohydrochloride and 1-hydroxy-1 H -benzotriazole (HOBT). The reaction can be carried out in the presence of a base such as triethylamine, in a suitable solvent such as a mixture of dichloromethane and tetrahydrofuran.

Or R3

R1 YN

OO HO

NH2N (CH2) n N (CH2) m

+ X

R2

(III) (II-b)

R3O

R1 Y

N

OOEDC / HOBT N

N (CH2) n N (CH2) mH

X R2

(II-a)

B) The intermediates of formula (II-a) can also be prepared by reacting an intermediate of formula (VI) with the appropriate carboxaldehyde of formula (V), where t is an integer with a value of 0 or 1 and, when t is 0, then it refers to a direct bond, in the presence of a suitable reagent such as sodium borohydride, in a suitable solvent such as dichloromethane or methanol.

R3O

R1 YN OO

(CH2) tH

N

N (CH2) n NH +

X O

(VI) (V) R2

R3O

R1 Y

N

OO N

N (CH2) n N (CH2) mH

X

R2 (II-a)

c) The intermediates of formula (II-b) can be prepared by reacting an intermediate of formula (II), where Q is methyl- or ethyl-oxycarbonyl (C1-2 alkyl), referred to herein as the intermediate of formula (II-c) , with a solution

10 suitable acid, p. eg, hydrochloric acid, or a basic solution, e.g. eg, hydrogen bromide or sodium hydroxide, in a suitable solvent, e.g. eg, an alcohol such as ethanol or propanol.

D) The intermediates of formula (II-c) can be prepared by reacting the carboxylic acid ethyl ester of formula (IV) with the appropriate carboxaldehyde of formula (V), in the presence of a suitable reagent such as sodium borohydride , p. eg, sodium tetrahydroborate, in a suitable solvent such as an alcohol, e.g. eg, methanol.

e) Similarly, the intermediates of formula (II-c) can be prepared by reacting an intermediate of formula (XIV) with the appropriate intermediate of formula (XV), in the presence of a suitable reagent such as a sodium borohydride, p. eg, sodium tetrahydroborate, in a suitable solvent such as an alcohol, e.g. eg methanol

f) The intermediates of formula (IIc) can also be prepared by reacting an intermediate of formula (X) with an intermediate of formula (XI), where W is a suitable leaving group such as, for example, halo, p. eg, fluoro, chloro, bromo or iodo, or a sulfonyloxy radical such as methylsulfonyloxy, 4-methylphenylsulfonyloxy and the like. The reaction 10 can be carried out in a reaction inert solvent such as, for example, an alcohol, e.g. eg, methanol, ethanol, 2-methoxyethanol, propanol, butanol and the like; an ether, p. eg, 4,4-dioxane, 1,1'-oxybispropane and the like; a ketone,

p. eg, 4-methyl-2-pentanone; or N, N-dimethylformamide, nitrobenzene, acetonitrile and the like. A suitable base such as, for example, a carbonate or hydrogen carbonate of an alkali metal or alkaline earth metal, e.g. eg, triethylamine or sodium carbonate, to neutralize the acid that is released during the course of the reaction. Be

15 can add a small amount of a suitable metal iodide, e.g. eg, sodium or potassium iodide to promote the reaction. Stirring can increase the speed of the reaction. The reaction can be suitably carried out at a temperature between room temperature and the reflux temperature of the reaction mixture and, if desired, the reaction can be carried out under pressure.

g) Similarly, the intermediates of formula (II-c) can be prepared by reacting an intermediate of formula (XII) with an intermediate of formula (XIII), where W is a suitable leaving group as previously described.

The intermediates of formula (VI) can be prepared by reacting the intermediate of formula (VII) with piperidine in a suitable solvent, e.g. eg dichloromethane.

The intermediates of formula (VII) can be prepared by reacting an intermediate of formula (VIII) with an intermediate of formula (III), in the presence of suitable reagents such as N 'monohydrochloride

10 (ethylcarbonimidoyl) -N, N-dimethyl-1,3-propanediamine (EDC) and 1-hydroxy-1H-benzotriazole (HOBT). The reaction can be carried out in the presence of a base such as triethylamine, in a suitable solvent such as a mixture of dichloromethane and tetrahydrofuran.

OO N HO N (CH2) n NH

OR OO X

NH2 +

(VIII) (III)

OO NEDC / HOBT O o

N

N (CH2) n NH

OH

X

(VII)

The intermediates of formula (VIII) can be prepared by reacting an intermediate of formula (IX) with an intermediate of formula (IV), where R1 is hydrogen, referred to herein as intermediate of formula (IV-a), in the presence of sodium hydroxide, in a suitable solvent such as tetrahydrofuran, and then neutralizing with hydrochloric acid and adding sodium carbonate.

OR N

OO

ON

N (CH2) n NH2 +

H

X (VI-a) (IX)

OO N HO

N (CH2) n NH

Or X

(VIII)

The compounds of formula (I) and some of the intermediates may contain at least one stereogenic center in their structure. This stereogenic center may be present in an R or S configuration.

The compounds of formula (I) prepared according to the processes previously described herein are generally racemic mixtures of enantiomers, which can be separated from each other following resolution procedures commonly used in the art. The racemic compounds of formula (I) can be converted into the corresponding diastereomeric salt forms by reaction with a chiral acid

10 suitable. Subsequently, said diastereomeric salt forms are separated, for example, by fractional or selective crystallization and the enantiomers are released therefrom with base. An alternative way of separating the enantiomeric forms of the compounds of formula (I) involves the use of liquid chromatography using a chiral stationary phase. Said pure stereochemically isomeric forms may also come from the pure stereochemically isomeric forms of the appropriate starting materials, provided that the reaction takes place.

15 stereospecifically. Preferably, if a specific stereoisomer is desired, said compound will be synthesized by stereospecific methods of preparation. These methods will conveniently employ enantiomerically pure starting materials.

Compounds of formula (I), pharmaceutically acceptable acid addition salts and forms

20 stereoisomers of these possess valuable pharmacological properties, since they have a histone deacetylase (HDAC) inhibitory effect.

This invention provides a method for inhibiting the abnormal growth of cells, including transformed cells, by administering an effective amount of a compound of the invention. Growth

Abnormal cells refers to cell growth independent of normal regulatory mechanisms (eg, loss of contact inhibition). This includes the inhibition of tumor growth both directly, causing growth arrest, terminal differentiation and / or apoptosis of cancer cells, and indirectly, inhibiting the neovascularization of tumors.

This invention also provides a method for inhibiting tumor growth by administering an effective amount of a compound of the present invention to a subject, e.g. eg, a mammal (and more particularly a human) that needs such treatment. In particular, this invention provides a method for inhibiting tumor growth by administering an effective amount of the compounds of the present invention. Examples of tumors that can be inhibited include, but are not limited to, lung cancer (e.g.,

35 adenocarcinoma, including non-small cell lung cancer), pancreatic cancers (e.g., pancreatic carcinoma such as, for example, exocrine pancreatic carcinoma), colon cancers (e.g., colorectal carcinomas such as, for example, adenocarcinoma colon and colon adenoma), prostate cancer, including advanced disease, hematopoietic tumors of the lymphoid lineage (eg, acute lymphocytic leukemia, B-cell lymphoma, Burkitt lymphoma), myeloid leukemia (eg, myeloid leukemia acute (AML)), follicular thyroid cancer,

40 myelodysplastic syndrome (MDS), tumors of mesenchymal origin (e.g., fibrosarcomas and rhabdomyosarcomas), melanomas, teratocarcinomas, neuroblastomas, gliomas, benign skin tumors (e.g., keratoacamptoms), breast carcinoma (e.g. e.g., advanced breast cancer), kidney carcinoma, ovarian carcinoma, bladder carcinoma and epidermal carcinoma.

The compound according to the invention can be used for other therapeutic purposes, for example:

a) the sensitization of tumors to radiotherapy by administration of the compound according to the invention before, during or after irradiation of the tumor to treat cancer; b) the treatment of arthropathies and osteopathological conditions such as rheumatoid arthritis, osteoarthritis, arthritis juvenile, gout, polyarthritis, psoriatic arthritis, ankylosing spondylitis and systemic lupus erythematosus; c) the inhibition of proliferation of smooth muscle cells, including vascular proliferative disorders, atherosclerosis and restenosis; d) the treatment of inflammatory conditions and dermal conditions such as ulcerative colitis, disease of Crohn, allergic rhinitis, graft versus host disease, conjunctivitis, asthma, ARDS, Behcet's disease, transplant rejection, hives, allergic dermatitis, alopecia areata, scleroderma, rash, eczema, dermatomyositis, acne, diabetes, systemic lupus erythematosus, Kawasaki disease, multiple sclerosis, emphysema, cystic fibrosis and chronic bronchitis; e) the treatment of endometriosis, uterine fibroids, dysfunctional uterine bleeding and endometrial hyperplasia; f) the treatment of ocular vascularization, including vasculopathy affecting the choroidal and retinal vessels; g) the treatment of cardiac dysfunction; h) inhibition of immunosuppressive conditions, such as the treatment of HIV infections; i) the treatment of renal dysfunction; j) the suppression of endocrine disorders; k) inhibition of gluconeogenesis dysfunction; l) the treatment of a neuropathology, for example, Parkinson's disease or a neuropathology that causes a cognitive disorder, for example, Alzheimer's disease or related neuronal diseases with polyglutamine; m) the treatment of psychiatric disorders, for example, schizophrenia, bipolar disorder, depression, anxiety and psychosis; n) inhibition of a neuromuscular pathology, for example, amylootrophic lateral sclerosis; o) the treatment of spinal muscular atrophy; p) the treatment of other pathological conditions that can be treated by enhancing the expression of a gene; q) the improvement of genetic therapy; r) adipogenesis inhibition; s) the treatment of parasitosis such as malaria.

Therefore, the present invention describes the compounds of formula (I) for use as a medicine, as well as the use of these compounds of formula (I) for the production of a medicament to treat one or more of the conditions mentioned above.

Compounds of formula (I), pharmaceutically acceptable acid addition salts and forms These stereoisomers may have valuable diagnostic properties, since they can be used to detect or identify an HDAC in a biological sample, which includes detecting or evaluating the formation of a complex between a labeled compound and an HDAC.

The detection or identification methods may employ compounds labeled with labeling agents such such as radioisotopes, enzymes, fluorescent substances, luminous substances, etc. The examples of radioisotopes include 125I, 131I, 3H and 14C. Normally, enzymes are made detectable by conjugation with a substrate suitable, which in turn catalyzes a detectable reaction. Examples of these include, for example, betagalactosidase, beta-glucosidase, alkaline phosphatase, peroxidase and malate dehydrogenase, preferably the Horseradish peroxidase. Luminous substances include, for example, luminol, derivatives of luminol, Luciferin, aequorin and luciferase.

Biological samples can be defined as body tissue or body fluids. The examples of fluids Body are cerebrospinal fluid, blood, plasma, serum, urine, sputum, saliva and the like.

In view of their useful pharmacological properties, the compounds herein can be formulated in several pharmaceutical forms for administration purposes.

To prepare the pharmaceutical compositions of this invention, an effective amount of a particular compound, in the form of a base or in the form of an acid addition salt, which acts as the active substance is combined in a intimate mixing with a pharmaceutically acceptable carrier; said carrier may present a variety of forms, depending on the form of the preparation desired for administration. These pharmaceutical compositions they are presented, desirably, in a unit dosage form suitable, preferably, for oral, rectal, percutaneous or parenteral injection administration. For example, when preparing the compositions in a oral pharmaceutical form, any of the usual pharmaceutical means such as, by for example, water, glycols, oils, alcohols and the like, in the case of oral liquid preparations such as suspensions, syrups, elixirs and solutions; or solid carriers such as starches, sugars, kaolin, lubricants, binders, disintegrating agents and the like, in the case of powders, tablets, capsules and tablets

Due to their ease of administration, tablets and capsules represent the most advantageous oral unit dosage form, in which case solid pharmaceutical carriers are obviously employed. For parenteral compositions, the carrier will normally comprise sterilized water, at least in large part, although other ingredients may be included to increase solubility, for example. For example, injectable solutions can be prepared, in which the carrier comprises saline solution, glucose solution or a mixture of saline solution and glucose. Injectable suspensions can also be prepared, in which case liquid carriers, suspending agents and the like can be used. In compositions suitable for percutaneous administration, the carrier optionally comprises an agent that enhances penetration and / or a suitable wetting agent, optionally combined with suitable additives of any nature in minor proportions, wherein said additives do not cause any significant detrimental effect on the skin. . Such additives may facilitate administration to the skin and / or may be useful for preparing the desired compositions. These compositions can be administered in several ways, e.g. eg, as a transdermal patch, as a localized application or as an ointment.

It is especially advantageous to formulate the pharmaceutical compositions mentioned above in unit dosage forms to facilitate administration and dose uniformity. The term "unit dosage form", as used in the specification and the claims herein, refers to physically discrete units suitable as unit doses, each unit of which contains a predetermined amount of active ingredient, calculated to produce the desired therapeutic effect, associated with the required pharmaceutical carrier. Some examples of such unit dosage forms are tablets (including labeled or coated tablets), capsules, pills, packets of powders, wafers, injectable solutions or suspensions, small scoops, soup tablespoons and the like, and multiple segregated pharmaceutical forms thereof.

Those skilled in the art will be able to easily determine the effective amount from the results of the tests shown hereinafter. In general, a therapeutically effective amount is considered to be between 0.005 mg / kg and 100 mg / kg body weight, and in particular between 0.005 mg / kg and 10 mg / kg body weight. It may be appropriate to administer the necessary dose in the form of two, three, four or more sub-doses at appropriate intervals throughout the day. Such sub-doses can be formulated as unit dosage forms, for example, containing 0.5 to 500 mg, and in particular 10 mg to 500 mg of active ingredient per unit dosage form.

As another aspect of the present invention, a combination of an HDAC inhibitor with another anticancer agent is considered, especially for use as a medicine, more specifically in the treatment of cancer or related diseases.

For the treatment of the above conditions, the compounds of the invention can be advantageously used in combination with one or more different medicinal agents, more particularly, with other anti-cancer agents. Some examples of anticancer agents are:

-

coordination compounds of platinum, for example, cisplatin, carboplatin or oxaliplatin;

-

taxanes, for example, paclitaxel or docetaxel;

-

topoisomerase I inhibitors such as camptothecins, for example, irinotecan or topotecan;

-

topoisomerase II inhibitors such as the antitumor derivatives of podophyllotoxin, for example, etoposide or teniposide;

-

antitumor vinca alkaloids, for example, vinblastine, vincristine or vinorelbine;

-

anti-tumor nucleoside derivatives, for example, 5-fluorouracil, gemcitabine or capecitabine;

-

alkylating agents such as nitrogen mustard or nitrosourea, for example, cyclophosphamide, chlorambucil, carmustine or lomustine;

-

anthracycline antitumor derivatives, for example, daunorubicin, doxorubicin, idarubicin or mitoxantrone;

-

HER2 antibodies, for example, trastuzumab;

-

estrogen receptor antagonists or selective estrogen receptor modulators, for example, tamoxifen, toremifene, droloxifene, faslodex or raloxifene;

-

aromatase inhibitors such as exemestane, anastrozole, letrazole and vorozole;

-

differentiation agents such as retinoids, vitamin D and retinoic acid metabolism blocking agents (RAMBA), for example, acutane;

-

DNA methyltransferase inhibitors, for example, azacitidine;

-

kinase inhibitors, for example, flavoperidol, imatinib mesylate or gefitinib;

-

phamesyltransferase inhibitors; -other HDAC inhibitors;

-

ubiquitin-proteasome pathway inhibitors, for example, velcade; or

-

Yondelis

The term "platinum coordination compound" is used herein to mean any platinum coordination compound that inhibits the growth of tumor cells that provide platinum in the form of an ion.

The term "taxanes" indicates a class of compounds that contain the annular system of taxane and that are related to the extracts of certain species of yew trees (Taxus) or come from them.

The term "topoisomerase inhibitors" is used to indicate enzymes capable of altering the DNA topology in eukaryotic cells. They are crucial for the development of important cellular functions and for cell proliferation. There are two kinds of topoisomerases in eukaryotic cells, which are called type I and type II. Topoisomerase I is a monomeric enzyme with a molecular weight of approximately 100,000. The enzyme binds to the DNA and introduces a transient single chain break, undoes the double helix (or allows it to undo) and then reseals the break before to dissociate from the DNA chain. Topoisomerase II has a similar mechanism of action, which involves the induction of DNA chain breaks or the formation of free radicals.

The term "camptothecins" is used to indicate compounds related to or originating from the original camptothecin, which is a water-insoluble alkaloid that comes from the Chinese tree Camptothecin acuminata and the Indian tree Nothapodytes foetida.

The term "podophyllotoxins" is used to indicate compounds related to or originating from the original podophyllotoxin, which is extracted from the mandrake plant.

The expression "vinca alkaloids antitumor" is used to indicate compounds related to or derived from the vinca plant (Vinca rosea).

The term "alkylating agents" encompasses a diverse group of chemical agents that have the common characteristic that they possess the ability to provide, under physiological conditions, alkyl groups to biologically vital macromolecules, such as DNA. In most of the most important agents, such as nitrogen mustards and nitrosoureas, active alkylating moieties are generated in vivo after complex degradation reactions take place, some of which are enzymatic. The most important pharmacological actions of alkylating agents are those that affect the fundamental mechanisms involved in cell proliferation, in particular DNA synthesis and cell division. The ability of alkylating agents to interfere with DNA function and integrity in rapidly proliferating tissues, provides the basis for their therapeutic applications and for many of their toxic properties.

The term "anthracycline antitumor derivatives" includes antibiotics obtained from the Strep fungus. peuticus var. Caesius and its derivatives, which are characterized by having an annular structure of tetracycline with an unusual sugar, daunosamine, linked by a glycosidic bond.

It has been shown that amplification of the protein consisting of the human epidermal growth factor 2 receptor (HER 2) in primary breast carcinomas correlates with an unfavorable clinical prognosis in certain patients. Trastuzumab is a humanized monoclonal lgG1 kappa antibody derived from highly purified recombinant DNA that binds with high affinity and specificity to the extracellular domain of the HER 2 receptor.

Many breast cancers have estrogen receptors and the growth of these tumors can be stimulated by estrogen. The terms "estrogen receptor antagonists" and "selective estrogen receptor modulators" are used to indicate competitive estradiol inhibitors that bind to the estrogen receptor (ER). When selective estrogen receptor modulators bind to the ER, they induce a change in the three-dimensional shape of the receptor, which modulates their binding to the estrogen-responsive element (ERE) in the DNA.

In postmenopausal women, the main source of estrogen circulation is the conversion of adrenal and ovarian androgens (androstenedione and testosterone) into estrogens (estrone and estradiol) by the enzyme aromatase in peripheral tissues. Estrogen deprivation by inhibiting or deactivating aromatase is an effective and selective treatment for some postmenopausal patients suffering from hormone-dependent breast cancer.

The term "anti-estrogen agent" is used herein to include not only estrogen receptor antagonists and selective estrogen receptor modulators, but also aromatase inhibitors, as previously mentioned.

The term "differentiation agents" encompasses compounds that can inhibit cell proliferation and induce differentiation in several ways. There is evidence that vitamin D and retinoids play a key role in the regulation of growth and differentiation of a wide variety of malignant and normal cell types. Retinoic acid metabolism blocking agents (RAMBA) increase endogenous retinoic acid levels by inhibiting cytochrome P450-mediated catabolism of retinoic acids.

Changes due to DNA methylation are among the most common abnormalities in human malignancies. Hypermethylation in selected gene promoters is normally associated with the deactivation of the genes involved. The term "DNA methyltransferase inhibitors" is used to indicate compounds that act by pharmacological inhibition of DNA methyltransferase and reactivation of tumor suppressor gene expression.

The term "kinase inhibitors" comprises potent kinase inhibitors that are involved in cell cycle progression and programmed cell death (apoptosis).

The term "farnesyltransferase inhibitors" is used to indicate compounds that were designed to prevent farnesylation of Ras and other intracellular proteins. There is evidence that they exert some effect on the proliferation and survival of malignant cells.

The term "other HDAC inhibitors" includes, without limitation:

-

carboxylates, for example, butyrate, cinnamic acid, 4-phenylbutyrate or valproic acid;

-

hydroxamic acids, for example, suberoylanilide hydroxamic acid (SAHA), SAHA analogs containing piperazine, biaryl hydroxamate A-161906 and its analogs of carboxylic ether, tetrahydropyridine and tetralone, aryl-N-hydroxycarboxamides bicyclic, pyroxamide, CG 1521, PXD-101, sulfonamide hydroxamic acid, LAQ-824, LBH-589, tricostatin A (TSA), oxamflatin, scriptaid, tricyclic molecules related to scriptaid, m-carboxycinnamic acid bishydroxamic acid (CBHA), hydroxamic acid type CBHA, hydroxamic acid-trapoxin analog, R306465 and related heteroaryl and benzoyl hydroxamic acids, aminosuberates and malonyldiamides;

-

cyclic tetrapeptides, for example, trapoxin, apidicin, depsypeptide, spiruchostatin-related compounds, RedFK-228, cyclic tetrapeptides containing sulfydryl (SCOP), cyclic tetrapeptides containing hydroxamic acid (CHAP), TAN-174 and azumamides;

-

benzamides, for example, MS-275 or CI-994; or

-

depudecina

The term "ubiquitin-proteasome pathway inhibitors" is used to identify compounds that inhibit the strategic destruction of cellular proteins in the proteasome, including cell cycle regulatory proteins.

For the treatment of cancer, the compounds according to the present invention can be administered to a patient as described above, together with irradiation. Irradiation refers to ionizing radiation and, in particular, gamma radiation, especially that emitted by linear accelerators or by radionuclides commonly used today. The irradiation of the tumor with the radionuclides can be external or internal.

The present invention also relates to a combination according to the invention of an anticancer agent and an HDAC inhibitor according to the invention.

The present invention also relates to a combination according to the invention for use in medical therapy, for example, to inhibit the growth of tumor cells.

The present invention also relates to combinations according to the invention to inhibit the growth of tumor cells.

The present invention also relates to a method for inhibiting the growth of tumor cells in a human subject, which comprises administering to the subject an effective amount of a combination according to the invention.

This invention further provides a method for inhibiting the abnormal growth of cells, including transformed cells, by administering an effective amount of a combination according to the invention.

The other medicinal agent and the HDAC inhibitor can be administered simultaneously (e.g., in unit or separate compositions) or sequentially in any order. In the latter case, the two compounds will be administered over a period of time and in an amount and manner that are sufficient to ensure that a synergistic or advantageous effect is achieved. It will be understood that the preferred method and order of administration, as well as the amounts and regimens of the respective doses for each component of the combination will depend on the other medicinal agent and the particular HDAC inhibitor that are administered, of their route of administration, of the particular tumor to be treated and the particular host to be treated. Those skilled in the art will be able to easily determine the optimal method and order of administration, as well as the amounts and dose regime, using conventional methods and taking into account the information detailed herein.

The platinum coordination compound is conveniently administered in a dose of 1 to 500 mg per square meter (mg / m2) of body surface area, for example, 50 to 400 mg / m2, particularly for cisplatin a dose of approximately 75 mg / m2 and for carboplatin a dose of approximately 300 mg / m2 in each treatment.

The taxane is conveniently administered at a dose of 50 to 400 mg per square meter (mg / m2) of body surface area, for example, 75 to 250 mg / m2, particularly for paclitaxel a dose of approximately 175 to 250 is administered. mg / m2 and for docetaxel a dose of approximately 75 to 150 mg / m2 in each treatment.

Camptothecin is conveniently administered at a dose of 0.1 to 400 mg per square meter (mg / m2) of body surface area, for example, 1 to 300 mg / m2, particularly for irinotecan a dose of approximately 100 to 350 is administered mg / m2 and for the topotecan a dose of approximately 1 to 2 mg / m2 in each treatment.

The antitumor derivative of podophyllotoxin is conveniently administered in a dose of 30 to 300 mg per square meter (mg / m2) of body surface area, for example, 50 to 250 mg / m2, particularly for the etoposide a dose of approximately 35 to 100 mg / m2 and for teniposide a dose of approximately 50 to 250 mg / m2 in each treatment.

The vinca alkaloid antitumor is conveniently administered in a dose of 2 to 30 mg per square meter (mg / m2) of body surface area, particularly for vinblastine a dose of approximately 3 to 12 mg / m2 is administered, for vincristine a dose of approximately 1 to 2 mg / m2 and for vinorelbine a dose of approximately 10 to 30 mg / m2 in each treatment.

The antitumor nucleoside derivative is conveniently administered at a dose of 200 to 2500 mg per square meter (mg / m2) of body surface area, for example, 700 to 1500 mg / m2, particularly for 5-FU a dose of 200 is administered. at 500 mg / m2, for gemcitabine a dose of approximately 800 to 1200 mg / m2 and for capecitabine a dose of approximately 1000 to 2500 mg / m2 for each treatment.

Alkylating agents, such as nitrogen mustard or nitrosourea, are conveniently administered in a dose of 100 to 500 mg per square meter (mg / m2) of body surface area, for example, 120 to 200 mg / m2, particularly for Cyclophosphamide is administered a dose of 100 to 500 mg / m2, for chlorambucil a dose of approximately 0.1 to 0.2 mg / kg, for carmustine a dose of approximately 150 to 200 mg / m2 and for lomustine a dose of approximately 100 to 150 mg / m2 in each treatment.

The anti-tumor anthracycline derivative is conveniently administered in a dose of 10 to 75 mg per square meter (mg / m2) of body surface area, for example, 15 to 60 mg / m2, particularly for doxorubicin a dose of approximately 40 to 75 mg / m2, for daunorubicin a dose of approximately 25 to 45 mg / m2 and for idarubicin a dose of approximately 10 to 15 mg / m2 for each treatment.

Trastuzumab is conveniently administered at a dose of 1 to 5 mg per square meter (mg / m2) of body surface area, particularly 2 to 4 mg / m2 in each treatment.

The antiestrogen agent is conveniently administered at a dose of about 1 to 100 mg per day, depending on the particular agent and the condition to be treated. Tamoxifen is conveniently administered orally in a dose of 5 to 50 mg, preferably 10 to 20 mg twice daily, following therapy for a period of time sufficient to achieve and maintain a therapeutic effect. Toremifene is conveniently administered orally at a dose of approximately 60 mg once daily, following therapy for a sufficient period of time to achieve and maintain a therapeutic effect. Anastrozole is conveniently administered orally at a dose of approximately 1 mg once daily. Droloxifene is conveniently administered orally at a dose of approximately 20-100 mg once daily. Raloxifene is conveniently administered orally at a dose of approximately 60 mg once daily. Exemestran is conveniently administered orally at a dose of approximately 25 mg once daily.

These doses can be administered, for example, one, two or more times in each treatment, which can be repeated, for example, every 7,14, 21 or 28 days.

In view of their useful pharmacological properties, the compounds of the combinations according to the invention, that is, the other medicinal agent and the HDAC inhibitor, can be formulated in various pharmaceutical forms for administration purposes. The components may be formulated separately in individual pharmaceutical compositions or in a unit pharmaceutical composition containing both components.

Accordingly, the present invention also relates to a pharmaceutical composition comprising the other medicinal agent and the HDAC inhibitor, together with one or more pharmaceutical carriers.

The present invention also relates to a combination according to the invention in the form of a pharmaceutical composition comprising an anticancer agent and an HDAC inhibitor according to the invention, together with one or more pharmaceutical carriers.

The present invention further relates to the use of a combination according to the invention in the production of a pharmaceutical composition to inhibit the growth of tumor cells.

The present invention further relates to a product containing, as the first active ingredient, an HDAC inhibitor according to the invention and, as the second active ingredient, an anticancer agent, as a combined preparation for simultaneous, separate or separate use. sequential in the treatment of patients suffering from cancer.

Examples

Experimental part

The following examples are provided for illustrative purposes.

Hereinafter, "DCM" is defined as dichloromethane, "DIPE" is defined as diisopropyl ether, "DMA" is defined as N, N-dimethylacetamide, "DMSO" is defined as dimethyl sulfoxide, "EDC" is defined as N '- (ethylcarbonimidoyl) -N, N-dimethyl-1,3-propanediamine hydrochloride, "EtOAc" is defined as ethyl acetate, "EtOH" is defined as ethanol, "HOBt" is defined as 1-hydroxy -1H-benzotriazole, "MeOH" is defined as methanol, "TFA" is defined as trifluoroacetic acid and "THF" is defined as tetrahydrofuran.

A. Preparation of intermediate compounds

Example A1

a) Preparation of intermediate 1

N

H NN

N

O n

OR

A mixture of the ethyl ester of 2- [4- (aminomethyl) -1-piperidinyl] -5-pyrimidinecarboxylic acid (0.0114 mol), 1-methyl-1Hindole-3-carboxaldehyde (0.017 mol) and MgSO4 (0.5 g) in MeOH (80 ml) was stirred and heated at reflux for 15 hours, then cooled to room temperature. Sodium tetrahydroborate (0.018 mol) was added portionwise. The mixture was stirred at room temperature for 5 hours, poured into water and extracted with EtOAc. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (6.6 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 94/6 / 0.5). The pure fractions were collected and the solvent was evaporated, to obtain 4.3 g (90%) of intermediate 1.

b) Preparation of intermediate 2

N

H

NN

N

HO

N

OR

.Na

A mixture of intermediate 1 (0.0037 mol) and sodium hydroxide (0.0074 mol) in EtOH (60 ml) was stirred at 50 ° C for 15 hours, then cooled to room temperature and the solvent was evaporated to dryness, to obtain 1.5 g (100%) of intermediate 2.

c) Preparation of intermediate 3

N

H

NN

N

H

N

N OO

OR

EDC (0.0075 mol), then HOBt (0.0075 mol) and then O- (tetrahydro-2H-pyran-2il) hydroxylamine (0.015 mol) at room temperature were added to a mixture of intermediate 2 (0.005 mol) in DCM (100 ml) and THF (100 ml) with a flow of N2. The mixture was stirred at 40 ° C for 4 hours, poured into ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (4 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 94/6 / 0.5). The pure fractions were collected and the solvent was evaporated, to obtain 1 g (42%) of intermediate 3. A fraction

(0.051 g) was crystallized from DIPE. The precipitate was filtered off and dried, to obtain 0.03 g of intermediate 3; melting point: 70 ° C.

Example A2

a) Preparation of intermediate 4 OR

N

N

NO HO N

H

OR

A mixture of 2- [4- (aminomethyl) -1-piperidinyl] -5-pyrimidinecarboxylic acid ethyl ester (0.0072 mol) in THF (40 ml) and 1 N sodium hydroxide (40 ml) was stirred at room temperature for all night. 1 N hydrochloric acid (40 ml) was added. The mixture was stirred for 10 minutes. Sodium carbonate (0.0216 mol) was added. The mixture was stirred for 10 minutes. 1 - [[((9H-Fluoren-9-ylmethoxy) carbonyl] oxy] -2,5-pyrrolidinedione (0.0072 mol) was added portionwise. The mixture was stirred at room temperature for 6 hours, then cooled to 0 ° C and acidified with hydrochloric acid. The precipitate was filtered, washed with diethyl ether and dried, to obtain 4.1 g (100%) of intermediate 4.

b) Preparation of intermediate 5 OR

Triethylamine (0.02 mol), EDC (0.0082 mol) and HOBt (0.0082 mol) were added at room temperature to a mixture of intermediate 4 (0.0068 mol) in DCM / THF (200 ml) with a flow of N2. The mixture was stirred at room temperature for 15 minutes. O- (tetrahydro-2H-piran-2-yl) hydroxylamine (0.0082 mol) was added. The mixture was stirred at room temperature for 48 hours, poured into water and extracted with DCM. The organic phase was washed with 10% NaHCO3, dried (MgSO4), filtered and the solvent evaporated. The residue (4 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 98/2 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 3.4 g (89%) of intermediate 5.

c) Preparation of intermediate 6

N

NH2

NO

H

NO

N

OR

A mixture of intermediate 5 (0.0355 mol) and piperidine (0.089 mol) in DCM (400 ml) was stirred at 35 ° C for 72 hours. The solvent was evaporated. The residue was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 80/20/2). The pure fractions were collected and the solvent was evaporated, to obtain 6.7 g (56%). Part of the residue (0.79 g) was crystallized from diethyl ether. The precipitate was filtered off and dried, to obtain 0.62 g of intermediate 6; melting point: 129 ° C.

d) Preparation of intermediate 7 Cl

N

N

N

H

H OR N

OR

NN H

OR

A mixture of intermediate 6 (0.0009 mol) and 5-chloro-1H-indole-3-carboxaldehyde (0.0012 mol) in 1,2-dichloroethane (30 ml) was stirred at room temperature overnight. Sodium triacetoxyborohydride (0.0013 mol) was added portionwise. The mixture was stirred at room temperature for 4 hours, poured onto water / 3 N NaOH and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (0.5 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 93/7 / 0.5). Two fractions were collected and the solvent was evaporated, to obtain 0.07 g (16%) of intermediate 7.

a) Preparation of intermediate 8

A solution of 2- (methylsulfonyl) -5-pyrimidinecarboxylic acid ethyl ester (0.094 mol) in acetonitrile was added

(40 ml) at 10 ° C to a suspension of 4-piperidinemethanol (0.086 mol) and potassium carbonate (0.172 mol) in

5 acetonitrile (200 ml) with a flow of N2. The mixture was allowed to reach room temperature, then

stirred for 4 hours, poured into water and extracted with EtOAc. The organic phase was separated, dried (MgSO4),

filtered and the solvent was evaporated. The residue (23 g) was crystallized from acetonitrile / diethyl ether. The precipitate separated

by filtration and dried, to obtain 7.8 g (34%) of intermediate 8. The mother liquor was evaporated. The residue (17

g) purified by silica gel column chromatography (20-45 μm) (eluent: DCM / MeOH / NH4OH 10 97/3 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 4.6 g (20%) of intermediate 7;

melting point: 129 ° C.

b) Preparation of intermediate 9 O

Triethylamine (0.038 mol) and then methanesulfonyl chloride (0.025 mol) at 0 ° C was added to a solution of the

intermediate 8 (0.0189 mol) in DCM (80 ml) with a flow of N2. The mixture was stirred at 0 ° C for 2 hours and poured into ice water. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated, to obtain

6.5 g (100%) of intermediate 9.

c) Preparation of intermediate 10 H

A mixture of intermediate 9 (0.0189 mol), N-methyl-1H-indole-3-ethanamine (0.0172 mol) and potassium carbonate

20 (0.0344 mol) in acetonitrile (180 ml) was stirred and refluxed for 24 hours, poured onto water and extracted with EtOAc. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (8.5 g) was purified by silica gel column chromatography (70-200 μm) (eluent: DCM / MeOH / NH4OH from 98/2/0 to 97/3 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 1.25 g (20%) of intermediate 10.

d) Preparation of intermediate 11 H

HO

A mixture of intermediate 10 (0.003 mol) and sodium hydroxide (0.006 mol) in EtOH (80 ml) was stirred and refluxed overnight, then cooled to room temperature and the solvent evaporated to dryness, to obtain 1.3 g (100%) of intermediate 11; m.p. > 260 ° C

e) Preparation of intermediate 12

H

N NN

H

N

N OO O

EDC (0.0045 mol) and then HOBt (0.0045 mol) at room temperature were added to a mixture of intermediate 11 (0.003 mol) in THF (100 ml) and DCM (100 ml) with a flow of N2. The mixture was stirred at room temperature for 15 minutes. O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.012 mol) was added. The mixture was stirred at room temperature for 72 hours, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (3 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 94/6 / 0.1). The pure fractions were collected and the solvent was evaporated. Diethyl ether was added to the residue (0.82 g). The precipitate was filtered off and dried, to obtain

0.78 g of intermediate 12; melting point: 154 ° C.

10 Example A4

a) Preparation of intermediate 13 H

A mixture of 2- [4- (aminomethyl) -1-piperidinyl] -5-pyrimidinecarboxylic acid ethyl ester (0.0049 mol),

Methanesulfonate (ester) of 1H-indole-3-ethanol (0.0054 mol) and potassium carbonate (0.01 mol) in acetonitrile (20 ml) was stirred and refluxed overnight, then cooled, poured over ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (2.2 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 96/4 / 0.2). The pure fractions were collected and the solvent was evaporated, to obtain 0.442 g (22%) of the intermediate

20 13; melting point: 238 ° C.

b) Preparation of intermediate 14 H

A mixture of intermediate 13 (0.0025 mol), (2-bromoethoxy) (1,1-dimethylethyl) dimethylsilane (0.0034 mol) and N-ethyl-N- (1methylethyl) -2-propanamine (0.0038 mol) in DMSO (20 ml ) was stirred at 50 ° C for 15 hours, then cooled

25 to room temperature, it was poured onto ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1.7 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 98/2 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 0.76 g (54%) of intermediate 14.

c) Preparation of intermediate 15 H

HO

A mixture of intermediate 14 (0.0013 mol) and sodium hydroxide (0.0027 mol) in EtOH (40 ml) was stirred at 80 ° C overnight, then cooled to room temperature and the solvent evaporated, to obtain

0.75 g (100%) of intermediate 15.

d) Preparation of intermediate 16 HN

N

NN

H

N O oo

N

Yes

OR

EDC (0.002 mol) and then HOBt (0.002 mol) at room temperature were added to a mixture of intermediate 15 (0.0013 mol) in THF (80 ml) and DCM (80 ml) with a flow of N2. The mixture was stirred at room temperature for 15 minutes. O- (tetrahydro-2H-piran-2-yl) hydroxylamine (0.0068 mol) was added. The mixture was stirred at

At room temperature for 72 hours, it was poured onto water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1.3 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 0.38 g of intermediate 16.

e) Preparation of intermediate 17 NH

N

NN

H

N OH OO

NO

A mixture of intermediate 16 (0.0011 mol) and tetrabutylammonium fluoride (0.0032 mol) in THF (10 ml) was stirred at room temperature for 72 hours, poured into water and extracted with EtOAc. The organic phase was washed with water, dried (MgSO4), filtered and the solvent was evaporated, to obtain 0.5 g (88%) of intermediate 17.

15 Example A5

a) Preparation of intermediate 45 O

A solution of the 2-chloro-5-pyrimidinecarboxylic acid methyl ester (0.058 mol) in DMA (80 ml) was added dropwise to a solution of 4-piperidinemethanamine (0.116 mol) and N-ethyldiisopropylamine (0.145 mol) in DMA (150ml) with a flow of N2. The mixture was stirred at room temperature for 1 hour and 30 minutes, poured onto ice water.

20 and extracted with EtOAc and then with DCM. The organic phase was washed with water, dried (MgSO4), filtered and the solvent evaporated. The residue was crystallized from DIPE. The precipitate was filtered off and dried, to obtain 10 g (65%) of intermediate 45.

b) Preparation of intermediate 46 O OR-

N +

OR

N

OR

A mixture of intermediate 45 (0.0024 mol), 1-methyl-5-nitro-1H-indole-3-carboxaldehyde (0.0036 mol) and MgSO4 (0.25 g) in MeOH (80 ml) was stirred at 60 ° C throughout the night and then cooled to room temperature. Sodium tetrahydroborate (0.0041 mol) was added portionwise. The mixture was stirred at room temperature for 18 hours, poured into water and extracted with EtOAc. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1.1 g) was crystallized from acetonitrile. The precipitate was filtered off and dried,

30 to obtain 0.9 g (86%) of intermediate 46; melting point: 150 ° C.

N

H

NN

C)

Preparation of intermediate 47

HO

A mixture of intermediate 46 (0.002 mol) and sodium hydroxide (0.008 mol) in EtOH (60 ml) was stirred at 60 ° C overnight, then cooled to room temperature and evaporated. Diethyl ether was added to the residue. The precipitate was filtered off and dried, to obtain 0.6 g (67%) of intermediate 47.

d) Preparation of intermediate 48 O

O-N +

N

H NN

H

N

N

OO O

EDC (0.0019 mol) and HOBt (0.0019 mol) were added at room temperature to a solution of intermediate 47 (0.0013 mol) and triethylamine (0.0039 mol) in DCM / THF (50/50) (100 ml) with a flow of N2 . The mixture was stirred at room temperature for 15 minutes. O- (tetrahydro-2H-piran-2-yl) hydroxylamine (0.0026 mol) was added. The

The mixture was stirred at room temperature for 72 hours, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1 g) was purified by kromasil column chromatography (5 μm) (eluent: DCM / MeOH / NH4OH from 98/2 / 0.2 to 92/8 / 0.2). The pure fractions were collected and the solvent was evaporated, to obtain 0.101 g (15%) of intermediate 48.

15 Example A6

a) Preparation of intermediate 49 O

A solution of the 2-chloro-5-pyrimidinecarboxylic acid methyl ester (0.033 mol) in DCM (80 ml) was added at room temperature to a solution of 4-piperidinemethanol (0.066 mol) and N-ethyldiisopropylamine (0.083 mol) in DCM

20 (100 ml) with a flow of N2. The mixture was stirred at room temperature for 3 hours and 30 minutes, poured into ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. Pentane was added to the residue. The precipitate was filtered off and dried, to obtain 7.88 g (95%) of intermediate 49.

b) Preparation of intermediate 50 O

DMSO (0.058 mol) was added dropwise at -78 ° C to a solution of ethanedioyl dichloride (0.0278 mol) in DCM (50 ml) with a flow of N2. The mixture was stirred for 15 minutes. A solution of intermediate 49 (0.023 mol) in DCM (200 ml) was added dropwise. The mixture was stirred at -78 ° C for 1 hour and 30 minutes. Triethylamine (0.118 mol) was added dropwise. The mixture was stirred at -78 ° C for 1 hour, poured into water and extracted with DCM. The

The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue was crystallized from diethyl ether. The precipitate was filtered off and dried, to obtain 3.06 g (54%) of intermediate 50.

c) Preparation of intermediate 51 O

Intermediate 50 (0.0122 mol) was added at 5 ° C to a solution of 1-methyl-1H-indole-3-ethanamine (0.0122 mol) in MeOH (270 ml) with a flow of N2. The mixture was stirred a few minutes. Sodium cyanoborohydride (0.0183 mol) and acetic acid (0.0183 mol) were added. The mixture was stirred at room temperature for 48 hours, 5 was poured onto 10% potassium carbonate and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (4.9 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 97/3 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain

1.2 g (24%) of intermediate 51.

d) Preparation of intermediate 52 O

HO

A mixture of intermediate 51 (0.0009 mol) and sodium hydroxide (0.0039 mol) in EtOH (60 ml) was stirred and refluxed for 15 hours, then evaporated to dryness, to obtain intermediate 52. This intermediate It was used directly in the next reaction step.

e) Preparation of intermediate 53 OR OO

N

N

H

NN

H N

N

HOBt (0.0019 mol) and then EDC (0.0019 mol) at room temperature were added to a solution of intermediate 52 (0.0009 mol) and O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.0019 mol) in DCM / THF (130 ml). The mixture was stirred at room temperature for 48 hours, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (0.93 g) was purified by chromatography

20 on a silica gel column (15-40 μm) (eluent: DCM / MeOH / NH4OH 97/3 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 0.155 g (33%) of intermediate 53.

Example A7

a) Preparation of intermediate 54

A mixture of 2- [4- (aminomethyl) -1-piperidinyl] -5-pyrimidinecarboxylic acid ethyl ester (0.0038 mol), 1-ethyl-1Hindole-3-carboxyaldehyde (0.0049 mol) and 10% Pd / C (0.5 g) in MeOH (20 ml) containing 1 ml of a 10% thiophene solution in EtOH, was hydrogenated at room temperature for 24 hours with a pressure of 3 bar, then filtered through celite. The solvent was evaporated to dryness. The residue (1.8 g) was purified by

30 silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH from 95/5 / 0.2 to 93/7 / 0.5). The pure fractions were collected and the solvent was evaporated, to obtain 0.7 g (44%) of intermediate 54.

b) Preparation of intermediate 55 O

60% sodium hydride (0.009 mol) at 0 ° C was added to a solution of intermediate 54 (0.0045 mol) in THF (50 ml) with a flow of N2. The mixture was stirred at room temperature for 1 hour. A solution of iodoethane (0.0062 mol) in THF (10 ml) was added dropwise. The mixture was stirred at room temperature overnight, it was

5 poured over water and extracted with EtOAc. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (0.6 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 0.16 g (8%) of intermediate 55.

c) Preparation of intermediate 56 O

HO

A mixture of intermediate 55 (0.0003 mol) and sodium hydroxide (0.03 g) in EtOH (15 ml) was stirred at 80 ° C for 6 hours and then evaporated to dryness, to obtain 0.16 g (100%) of the intermediate 56.

d) Preparation of intermediate 57 OR OO

N

N

H

NN N N

15 EDC (0.0005 mol) and HOBt (0.0005 mol) were added at room temperature to a mixture of intermediate 56 (0.0003 mol) in DCM (20 ml) and THF (20 ml) with a flow of N2. The mixture was stirred for 15 minutes. O (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.0007 mol) was added. The mixture was stirred at room temperature for 72 hours, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (0.3 g) was purified by kromasil column chromatography (5 μm) (eluent:

20 DCM / MeOH / NH4OH 93/7/0. 35). The pure fractions were collected and the solvent was evaporated, to obtain 0.03 g (16%) of intermediate 57.

Example A8

a) Preparation of intermediate 58 O

Sodium hydroxide (0.011 mol) at 5 ° C was added to a solution of intermediate 13 (0.0037 mol) in THF (30 ml) with a flow of N2. The mixture was stirred for 30 minutes. A solution of iodomethane (0.0081 mol) in THF (10 ml) was added dropwise. The mixture was stirred at 10 ° C for 2 hours, then allowed to reach room temperature for 1 hour and 30 minutes, poured into ice water and extracted with DCM. The phase

The organic was separated, dried (MgSO4), filtered and the solvent was evaporated. The residue (1.7 g) was purified by kromasil column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 98/2 / 0.1). Two fractions were collected and the solvent was evaporated, to obtain 0.265 g of intermediate 58 and 0.57 g (17%) of intermediate 10.

b) Preparation of intermediate 59 OR

HO

N

NN

N

N

.Na

A mixture of intermediate 58 (0.0006 mol) and sodium hydroxide (0.0012 mol) in EtOH (30 ml) was stirred at 80 ° C overnight, then cooled to room temperature and the solvent was evaporated, to obtain

0.26 g (100%) of intermediate 59.

c) Preparation of intermediate 60 OR OO

N

N

H

NN N

N

EDC (0.0009 mol) and HOBt (0.0009 mol) were added at room temperature to a solution of intermediate 59 (0.0006 mol) in THF (30 ml) and DCM (30 ml) with a flow of N2. The mixture was stirred at room temperature for 15 minutes. O- (tetrahydro-2H-piran-2-yl) hydroxylamine (0.0012 mol) was added. The mixture was stirred at room temperature for 24 hours, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (0.6 g) was purified by kromasil column chromatography (5 μm) (eluent: DCM / MeOH / NH4OH 99/1 / 0.05 and 94/6 / 0.3). The pure fractions were collected and the solvent was evaporated, to obtain 0.1 g (33%) of intermediate 60.

Example A9

a) Preparation of intermediate 61 OR

O n

NN OR

DMSO (0.127 mol) was added at -78 ° C to a solution of ethanedioyl dichloride (0.061 mol) in DCM (110 ml) with a flow of N2. The mixture was stirred for 15 minutes. A solution of intermediate 8 (0.051 mol) in DCM (200 ml) was added. The mixture was stirred at -78 ° C for 1 hour and 30 minutes. Triethylamine (0.26 mol) was added dropwise. The mixture was stirred at -78 ° C for 15 minutes and then allowed to reach room temperature for 2 hours and 30 minutes. Water was added. The mixture was extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (14 g) was purified by silica gel column chromatography (20-45 μm) (eluent: cyclohexane / EtOAc 70/30). The pure fractions were collected and the solvent was evaporated, to obtain 7.6 g (57%) of intermediate 61.

b) Preparation of intermediate 62 OR

O N Cl

NN

H

N

Sodium cyanoborohydride (0.049 mol) and acetic acid (0.034 ml) were added at room temperature to a solution of 5-chloro-1-methyl-1H-indole-3-ethanamine (0.031 mol) and intermediate 61 (0.034 mol) in MeOH (700 ml) with a flow of N2. The mixture was stirred at room temperature for 24 hours, poured onto 10% potassium carbonate and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (14.8 g) was purified by silica gel column chromatography (20-45 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.2). The pure fractions were collected and the solvent was evaporated, to obtain 4.52 g (32%) of intermediate 62.

c) Preparation of intermediate 63 O o

N Cl

OR

Or NN

S N N

Methanesulfonyl chloride (0.0049 mol) was added at 5 ° C to a solution of intermediate 62 (0.004 mol) and triethylamine

(0.008 mol) in DCM (150 ml) with a flow of N2. The mixture was stirred at room temperature for 24 hours, poured into ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and evaporated.

5 the solvent. DIPE was added to the residue (2.39 g). The precipitate was filtered off and dried, to obtain 1.78 g (84%) of intermediate 63; melting point: 162 ° C.

d) Preparation of intermediate 64 OR

HO N Cl

OR

O NN S N N

.Na

A mixture of intermediate 63 (0.0032 mol) and sodium hydroxide (0.0128 mol) in EtOH (150 ml) was stirred and heated

10 at reflux for 5 hours, then cooled to room temperature and diethyl ether was added. The precipitate was filtered off and dried, to obtain 1.57 g (99%) of intermediate 64; melting point> 260 ° C.

e) Preparation of intermediate 65 OR OO

N N ClH O

ONN S

N N

EDC (0.0064 mol) and HOBt (0.0064 mol) were added at room temperature to a solution of intermediate 64

15 (0.0032 mol) in THF (160 ml) and DCM (160 ml) with a flow of N2. The mixture was stirred at room temperature for 30 minutes. O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.0064 mol) was added. The mixture was stirred at room temperature for 3 days, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (2.77 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 97/3 / 0.1). The pure fractions were collected and

20 evaporated the solvent. The residue (0.385 g) was crystallized from CH3CN / diethyl ether. The precipitate was filtered off and dried, to obtain 0.084 g of intermediate 65; melting point: 179 ° C.

Example A10

a) Preparation of intermediate 66 H

A solution of 2- (methylsulfonyl) -5-pyrimidinecarboxylic acid ethyl ester (0.094 mol) in acetonitrile (240 ml) was added at room temperature to a solution of 1,1-dimethyl ethyl ester of 4-piperidinylcarbamic acid (0.078 mol ) and potassium carbonate (0.156 mol) in acetonitrile (120 ml) with a flow of N2. The mixture was stirred at room temperature overnight, poured into ice water and extracted with EtOAc. The organic phase was washed with

30 water, dried (MgSO4), filtered and the solvent evaporated. The residue was crystallized from diethyl ether. The precipitate was filtered off and dried, to obtain 14.4 g (53%) of intermediate 66; melting point: 160 ° C.

b) Preparation of intermediate 67

TFA (20 ml) was added at room temperature to a solution of intermediate 66 (0.0225 mol) in DCM (110 ml). The mixture was stirred at room temperature for 15 hours, poured into water and basified with potassium carbonate. The mixture was extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the evaporated.

5 solvent, to obtain 5.5 g (98%) of intermediate 67.

Example A11

a) Preparation of intermediate 68

N

OR

OR

10 60% sodium hydride in oil (0.0069 mol) at 0 ° C was added to a solution of the 2-methyl-1-indole-3-acetic acid ethyl ester (0.0046 mol) in THF (10 ml) with a flow of N2 The mixture was stirred at room temperature for 1 hour. Iodoethane (0.006 mol) was added. The mixture was stirred at room temperature for 18 hours and poured onto EtOAc and saturated NaCl. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated to dryness. The residue (1.1 g) was purified by silica gel column chromatography (15-40 μm) (eluent:

15 cyclohexane / EtOAc 80/20). The pure fractions were collected and the solvent was evaporated, to obtain 0.73 g (65%) of intermediate 68.

b) Preparation of intermediate 69

A solution of diisobutylaluminum hydride in toluene (0.0045 mol) was added dropwise at -78 ° C to a solution

20 of intermediate 68 (0.003 mol) in DCM (15 ml) and 1,2-dimethoxyethane (15 ml) (molecular sieves: 3 angstrom) with a flow of N2. The mixture was stirred at -78 ° C for 3 hours, then stopped with 3 N HCl and extracted with DCM. The organic phase was washed with water, dried (MgSO4), filtered and the solvent evaporated to dryness, to obtain 0.7 g (> 100%) of intermediate 69.

c) Preparation of intermediate 70

25 Titanium (IV) ethoxide (0.0023 mol) was added to a mixture of intermediate 67 (0.0021 mol) and intermediate 69 (0.0021 mol) in 1,2-dichloroethane (25 ml). The mixture was stirred at room temperature for 30 minutes. Sodium triacetoxyborohydride (0.0023 mol) was added portionwise. The mixture was stirred at room temperature for 18 hours, then stopped with NaHCO3 and extracted with DCM. The organic phase was separated, dried (MgSO4),

30 was filtered and the solvent was evaporated to dryness. The residue (1.2 g) was purified by silica gel column chromatography (5 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 0.2 g (21%) of intermediate 70.

d) Preparation of intermediate 71 O

HO

A mixture of intermediate 70 (0.0004 mol) and sodium hydroxide (0.0009 mol) in EtOH (30 ml) was stirred at 60 ° C overnight, then cooled to room temperature and evaporated, to obtain 0.2 g ( 100%) of intermediate 71.

5 e) Preparation of intermediate 72 OR OO

N N

H NNN

N

H

EDC (0.0007 mol) and HOBt (0.1 g) were added at room temperature to a solution of intermediate 71 (0.0004 mol) and triethylamine (0.0009 mol) in DCM / THF (40 ml) with a flow of N2. The mixture was stirred at room temperature for 15 minutes. O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.0009 mol) was added. The mixture was stirred at

At room temperature for 72 hours, it was poured onto water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (0.4 g) was purified by kromasil column chromatography (5 μm) (eluent: DCM / MeOH / NH4OH of 98/2 / 0.1 at 90/10/1). The pure fractions were collected and the solvent was evaporated, to obtain 0.087 g (37%) of intermediate 72.

15 Example A12

a) Preparation of intermediate 73 O

Intermediate 50 (0.0046 mol) was added at 5 ° C to a solution of 6-methoxy-1-methyl-1H-indole-3-ethanamine (0.0046 mol) in MeOH (100 ml) with a flow of N2. The mixture was stirred for 30 minutes. Sodium cyanoborohydride (0.0068 mol) and then acetic acid (0.0046 mol) were added portionwise. The mixture was stirred at room temperature for 48 hours, poured onto 10% potassium carbonate and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (4 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 96/4 / 0.2). The pure fractions were collected and the solvent was evaporated. A part (0.7 g) of the residue (2.2 g) was crystallized from acetonitrile. The precipitate separated

25 by filtration and dried, to obtain 0.43 g (61%) of intermediate 73; melting point: 122 ° C.

b) Preparation of intermediate 74 O

HO

A mixture of intermediate 73 (0.0015 mol) and sodium hydroxide (0.006 mol) in EtOH (90 ml) was stirred and refluxed for 8 hours, then evaporated to dryness, to obtain intermediate 74.

c) Preparation of intermediate 75 OR OO

N

N

H

NN H

N N

OR

HOBt (0.003 mol) and then EDC (0.003 mol) were added at room temperature to a solution of intermediate 74 (0.0015 mol) and O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.003 mol) in THF / DCM (200 ml) with a flow of N2. The mixture was stirred at room temperature for 48 hours, poured into water and extracted with DCM. The

The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1.1 g) was purified by silica gel column chromatography (10 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.5). The pure fractions were collected and the solvent was evaporated. The residue (0.24 g) was purified by silica gel column chromatography (10 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.5). The pure fractions were collected and the solvent was evaporated, to obtain 0.17 g (21%) of intermediate 75.

10 Example A13

a) Preparation of intermediate 76

A mixture of 6-methoxy-1H-indole-3-ethanamine (0.053 mol) and 1,3-isobenzofurandione (0.058 mol) in toluene (130 ml)

It was stirred and heated at reflux for 48 hours, then filtered. The filtrate was evaporated, to obtain 5.4 g (32%) of intermediate 76.

b) Preparation of intermediate 77

A solution of intermediate 76 (0.017 mol) in DMF (19 ml) was added dropwise at room temperature at

20 suspension of sodium hydride (0.034 mol) in DMF (11 ml) with a flow of N2. The mixture was stirred at room temperature for 1 hour and 30 minutes. Iodopropane (0.034 mol) was added. The mixture was stirred at room temperature for 1 hour and 15 minutes. Saturated NaCl was added. The mixture was extracted with EtOAc. The organic phase was washed with water, dried (MgSO4), filtered and the solvent evaporated, to obtain 4.9 g of intermediate 77. This product was used directly in the next reaction step.

25 c) Preparation of intermediate 78

A mixture of intermediate 77 (0.068 mol) and hydrazine monohydrate (0.068 mol) in EtOH (60 ml) was stirred and refluxed for 1 hour, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent was evaporated, to obtain 3.53 g of intermediate 78. This product was used.

30 directly in the next reaction step.

d) Preparation of intermediate 79

Sodium cyanoborohydride (0.024 mol) and acetic acid (0.0167 mol) were added at room temperature to a solution of intermediate 61 (0.0167 mol) and intermediate 78 (0.015 mol) in MeOH (380 ml) with a flow of N2. The mixture was stirred at room temperature for 30 minutes, poured onto 10% potassium carbonate and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (8.84 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 96/4 / 0.2). The pure fractions were collected and the solvent was evaporated, to obtain 2.85 g (40%) of the intermediate

79.

e) Preparation of intermediate 80

A mixture of intermediate 79 (0.0028 mol), iodoethane (0.0056 mol) and triethylamine (0.0085 mol) in DMF (60 ml) was stirred at 50 ° C for 7 hours, poured into ice water and extracted with EtOAc. The organic phase was washed with water, dried (MgSO4), filtered and the solvent evaporated. The residue (1.8 g) was purified by kromasil column chromatography (5 μm) (eluent: DCM / MeOH from 100/0 to 95/5). The pure fractions were collected and

15 evaporated the solvent, to obtain 1.1 g (78%) of intermediate 80.

f) Preparation of intermediate 81

HO

A mixture of intermediate 80 (0.0022 mol) and sodium hydroxide (0.0088 mol) in EtOH (100 ml) was stirred and refluxed for 6 hours, then stirred at room temperature overnight and evaporated at 20 dryness. Diethyl ether was added to the residue. The precipitate was filtered off and dried, to obtain 0.965 g (88%) of intermediate 81; melting point> 260 ° C.

g) Preparation of intermediate 82

O o

O n

N N

H

OR NN N

EDC (0.0038 mol) and HOBt (0.0038 mol) were added at room temperature to a solution of intermediate 81

25 (0.0019 mol) in THF (100 ml) and DCM (100 ml) with a flow of N2. The mixture was stirred at room temperature for 30 minutes. O- (tetrahydro-2H-piran-2-yl) hydroxylamine (0.0038 mol) was added. The mixture was stirred at room temperature for 48 hours, poured into water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1.2 g) was purified by kromasil column chromatography (5 μm) (eluent: DCM / MeOH / NH4OH from 99/1 / 0.05 to 93/7 / 0.35). The pure fractions were collected and

30 evaporated the solvent, to obtain 0.114 g of intermediate 82.

Example A14

a) Preparation of intermediate 83 OR

HO

N

Cl

NN

H

N

.Na

A mixture of intermediate 62 (0.0034 mol) and sodium hydroxide (0.0134 mol) in EtOH (150 ml) was stirred at 80 ° C for 3 hours, then cooled to room temperature and evaporated to dryness. Diethyl ether was added to the residue. The precipitate was filtered off and dried, to obtain 1.18 g (78%) of intermediate 83; melting point> 260 ° C.

b) Preparation of intermediate 84 O oo

N

N Cl

H

NN

H

N

EDC (0.0052 mol) and HOBt (0.0052 mol) were added at room temperature to a solution of intermediate 83 (0.0026 mol) in THF (120 ml) and DCM (120 ml) with a flow of N2. The mixture was stirred at room temperature for 30 minutes. O- (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.0052 mol) was added. The mixture was stirred at room temperature for 6 days, poured into ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (2 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 96/4 / 0.5). The pure fractions were collected and the solvent was evaporated. The residue (0.75 g, 55%) was purified by kromasil column chromatography (10 μm) (eluent: DCM / MeOH / NH4OH 96/4 / 0.5). The pure fractions were collected and the solvent was evaporated, to obtain 0.625 g of intermediate 84. This product was used directly in the next reaction step.

Example A15

Preparation of intermediate 85

NH2

NN

O n

OR

4-Piperidinemethanamine (0.65 mol) and potassium carbonate (96 g) in acetonitrile (1000 ml) were stirred and then a solution of the ethyl ester of 2- (methylsulfonyl) -5-pyrimidinecarboxylic acid was added dropwise

(0.37 mol) in acetonitrile (500 ml) at room temperature for 1 hour. The reaction mixture was stirred overnight at room temperature and the solvent was evaporated. The residue was stirred in water and the mixture was extracted with DCM (2 x 500 ml). The organic phase was separated, washed with water, dried (MgSO4), filtered and the solvent evaporated. The residue was purified by silica gel column chromatography (eluent 1: EtOAc / Hexane 1/1; eluent 2: MeOH + a little NH4OH). The product fractions were collected, stirred with a suspension of potassium carbonate and the mixture was extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent was evaporated, to obtain 31 g (32%) of intermediate 85.

Example A16

a) Preparation of intermediate 86 OR

Cl

N

Sodium hydride (0.0095 mol) was added at 5 ° C to a solution of 5-chloro-1H-indole-3-carboxaldehyde (0.0056 mol) in THF (52 ml) with a flow of N2. The mixture was stirred at 0 ° C for 1 hour. Iodopropane (0.0067 mol) was added. The mixture was stirred at room temperature for 2 days, poured into water and extracted with EtOAc. The organic phase was separated, dried (MgSO4), filtered and the solvent was evaporated, to obtain 1.5 g of intermediate 86. This product was used directly in the next reaction step.

b) Preparation of intermediate 87

5 Sodium cyanoborohydride (0.0068 mol) and acetic acid (0.0046 mol) were added at room temperature to a solution of intermediate 85 (0.0042 mol) and intermediate 86 (0.0051 mol) in MeOH (120 ml) with a flow of N2. The mixture was stirred and heated at reflux for 2 days, then cooled to room temperature, poured onto 10% potassium carbonate and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (2.42 g) was purified by silica gel column chromatography (15-40 μm)

10 (eluent: DCM / MeOH / NH4OH 95/5 / 0.2). The pure fractions were collected and the solvent was evaporated, to obtain

1.2 g (60%) of intermediate 87.

c) Preparation of intermediate 88

HO

A mixture of intermediate 87 (0.0025 mol) and sodium hydroxide (0.01 mol) in EtOH (120 ml) was stirred and heated to

15 reflux for 4 hours, then evaporated to dryness. Diethyl ether was added to the residue. The precipitate was filtered off and dried, to obtain 0.845 g (72%) of intermediate 88; melting point> 260 ° C.

Cl N

d) Preparation of intermediate 89

H

NN N

H

N N

OO

OR

EDC (0.0036 mol) and HOBt (0.0036 mol) were added at room temperature to a solution of intermediate 88

20 (0.0018 mol) in THF (90 ml) and DCM (90 ml) with a flow of N2. The mixture was stirred for 30 minutes. O (tetrahydro-2H-pyran-2-yl) hydroxylamine (0.0036 mol) was added. The mixture was stirred at room temperature for 3 days, poured into ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the solvent evaporated. The residue (1.3 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 92/8 / 0.5). The pure fractions were collected and the solvent was evaporated, to obtain 0.54 g

25 (56%) of intermediate 89.

Example A17

a) Preparation of intermediate 90 O

30 Methanesulfonyl chloride (0.004 mol) was added at 10 ° C to a solution of 1,2-dimethyl-1H-indole-3-ethanol (0.0026 mol) and triethylamine (0.008 mol) in DCM (10 ml) with a flow of N2. The mixture was stirred at 10 ° C for 4 hours. The solvent was evaporated to dryness, to obtain intermediate 90. This product was used directly in the next reaction step.

b) Preparation of intermediate 91

A mixture of intermediate 85 (0.0054 mol), intermediate 90 (0.0075 mol) and potassium carbonate (0.021 mol) in acetonitrile (150 ml) was stirred and refluxed for 2 days, then cooled to room temperature, It was poured onto ice water and extracted with EtOAc. The organic phase was separated, dried (MgSO4),

5 filtered and the solvent was evaporated. The residue (1.88 g) was purified by silica gel column chromatography (15-40 μm) (eluent: DCM / MeOH / NH4OH 97/3 / 0.1). The pure fractions were collected and the solvent was evaporated, to obtain 0.15 g (7%) of intermediate 91.

c) Preparation of intermediate 92

HO

OR

.Na

A mixture of intermediate 91 (0.0003 mol) in sodium hydroxide (0.0014 mol) and EtOH (20 ml) was stirred and refluxed for one day, then cooled to room temperature and evaporated to dryness. Diethyl ether was added to the residue. The precipitate was filtered off and dried, to obtain 0.12 g (82%) of intermediate 92; melting point> 260 ° C.

d) Preparation of intermediate 93

N

N

H NN

H N

N

OO O

15 EDC (0.0005 mol) and HOBt (0.0005 mol) were added at room temperature to a solution of intermediate 92 (0.0002 mol) in THF (15 ml) and DCM (15 ml). The mixture was stirred for 15 minutes. O- (tetrahydro-2Hpiran-2-yl) hydroxylamine (0.0005 mol) was added. The mixture was stirred at room temperature for 4 days, poured into ice water and extracted with DCM. The organic phase was separated, dried (MgSO4), filtered and the evaporated.

20 solvent. The residue (0.14 g) was purified by silica gel column chromatography (10 μm) (eluent: DCM / MeOH / NH4OH 95/5 / 0.3). The pure fractions were collected and the solvent was evaporated, to obtain 0.035 g (25%) of intermediate 93.

Table F-1 lists the intermediates prepared according to one of the previous examples. 25 Table F-1 (intermediate)

N N HN N NO O N N HN N NHO O

 Interm. one; Ex [A1]

Interm. 2; Ex [A1]; m.p. > 260 ° C

OR

N HN NN N H N O O NH NO N H N O N H N O Cl

 Interm. 3; Ex [A1]; m.p. 70 ° C

 Interm. 7; Ex [A2]

H N

H N

N

N

N

N

N

N

OR

N HO N

OR

Interm. 10; Ex [A3] H N   O. Na; Interm. eleven; Ex. [A3]; m.p. > 260 ° C H N  

N

N

N

N

N

N H

OR

H NO N OR N

Or interm. 12; Ex. [A3]; m.p. 154 ° C H N

  Or interm. 13; Ex. [A4]; m.p. 238 ° C H N  

N

N

N

N

N

N

OR

N Or if HO N Or if

OR

Interm. 14; Ex [A4] H N    OR .Na; Interm. fifteen; Ex [A4] H N  

N

N

OR

H NO N N N Or if  OR H NO N N N OH

OR

O. Na; Interm. 16; Ex. [A4] N N N N H H N O O H N Interm. 18; Ex [A1]; m.p. 109 ° C    Or interm. 17; Ex. [A4] O H N O NN N Interm. 19; Ex [A1] N    

H N

N  H N N

HO

NN N OR H N O NN N

OR

 .Na; Interm. twenty; Ex. [A1] H N H N   Or interm. twenty-one; Ex [A1]; m.p. 70 ° C H N H N  

N

N

N

N

OR

N HO N

Or interm. 22; Ex [A1]; m.p. 159 ° C

  O. Na; Interm. 2. 3; Ex [A1]  

H N

OR

H NO NN N H N O o NN N N H N H  

Or interm. 24; Ex [A1]; m.p. 100 ° C

  Interm. 25; Ex [A1]; m.p. 120 ° C

HO

NN N N H N H OR H NO N N N N H N H

OR

Interm. 26; Ex [A1]   Or interm. 27; Ex [A1]; m.p. 84 ° C  

NH

NH

N

N

N

N

OR

N S HO N S

OInterm. 28; Ex [A1]; m.p. 83 ° C

  O. Na; Interm. 29; Ex [A1]  

N

N

NH N N HNO

OR

O H N N S OR N

Or interm. 30; Ex [A1]

  OInterm. 31; Ex [A1]; m.p. 85 ° C  

N N HNO

N N HNO

HO

N OR O H N N

OR

 .Na; Interm. 32; Ex [A1]   Or interm. 33; Ex [A1]  

N

N

N H  N N  N N H

N

HO

N OR H NO N

OR

O. Na; Interm. 3. 4; Ex. [A1] O O N H N H N N N H N      OR Or interm. 35; Ex. [A1] O H N N NN N H O H N  F

OR

Interm. 36; Ex. [A2] O H N N NN N H O H N   OR  Interm. 37; Ex. [A2] N H NO N H N N O N H Br

OR

 Interm. 38; Ex. [A2]; m.p. 173 ° C N N H N N H N H NO OR OR  Interm. 39; Ex [A2] N N HN N NH Cl

Or interm. 40; Ex [A2]; m.p. 110 ° C

  Or interm. 41; Ex. [A3]; m.p. 240 ° C  

NHNH NN

HNN NN H

ClH ClHO N NNOO

O o

.Na; Interm. 42; Ex. [A3] Interm. 43; Ex. [A3]; m.p. 88 ° C

N

H

NN N

O n

OR

 Interm. 44; Ex [A1]

O O O-O-N + N +

N

N

N H  N  N  N N H

N

OR

N HO N

OR

 OR

 Interm. 46; Ex [A5]; m.p. 150 ° C

 .Na; Interm. 47; Ex [A5]

OR

OR

O-N + O

N NN

N HH NNN

NH

NNOO O

 Interm. 48; Ex. [A5] Interm. 51; Ex [A6]

OO

OOHO N N N

HNN NNH H

N N

N N

 .Na; Interm. 52; Ex. [A6] Interm. 53; Ex [A6]

OR

O O N

HO

N NN NN

NN

N N

 Interm. 55; Ex. [A7] .Na; Interm. 56; Ex [A7]

OO

OO N N O N

H NN NN

N N N N

 Interm. 57; Ex. [A7] Interm. 58; Ex [A8]

OOOO N N

HO N

HNNNN NN NN

  .Na; Interm. 59; Ex. [A8] Interm. 60; Ex [A8]

OR

OR

OR

N N N S OO Cl HO N N N S OO Cl

N

N

N

N

 Interm. 63; Ex. [A9]; m.p. 162 ° C

.Na; Interm. 64; Ex. [A9]; m.p. > 260 ° C

OR

N

OR

N H O N N N S OO Cl

N

HN  N  N OR

N

N

OR

 Interm. 65; Ex. [A9]; m.p. 179 ° C

 Interm. 70; Ex [A11]

OR

OR

HO

N OR N H O N

NN

N N N N

N H

N H

 .Na; Interm. 71; Ex [A11]

 Interm. 72; Ex [A11]

OR

OR

OR

N HO N

N

N

H N  N  N H N

N

N

OO

 Interm. 73; Ex. [A12]; m.p. 122 ° C. Na; Interm. 74; Ex [A12]

O oo

N N

Or NN

H O

NH

NN N

NN ON

OR

 Interm. 75; Ex [A12] Interm. 80; Ex [A13]

OR

OR

OR

OR

N

N

HO N

N

H

N

NN

OR

NN

N

N

.Na; Interm. 81; Ex [A13]; m.p. > 260 ° C

 Interm. 82; Ex. [A13]

OR

OR

O n

HO

N

Cl

Cl

NN

NN

H

H

N

N

N

N

 Interm. 62; Ex [A9]

 .Na; Interm. 83; Ex. [A14]; m.p. > 260 ° C

OR

Cl

OO

N

N

N

H

Cl

H

NN

NN

N

H

OR

N

N

OR

N

 Interm. 84; Ex [A14]

 Interm. 87; Ex [A16]

Cl

N

N

H

NN

H

NN

N

H

N

N

N

HO N

OO

OR

OR

 .Na; Interm. 88; Ex [A16]; m.p. > 260 ° C

 Interm. 89; Ex [A16]

N

N

N

N

H

H

NN

NN

O n

HO

N

OR

OR

 Interm. 91; Ex. [A17]

 .Na; Interm. 92; Ex [A17]; m.p. > 260 ° C

N

H

N

N

N

N

H

NN

OR

N

N

H

H

N

N

OR

OO O

 Interm. 93; Ex. [A17]

 Interm. 94; Ex [A1]

H

H

N

N

N

N

N

N

H

HO

ON

OR

N

N

N

N

H

H

OR

OR

.Na; Interm. 95; Ex [A1]

 Interm. 96; Ex [A1]

OR

OR

O n

HO N

F

F

N

N

NN

NN

H

H

N

N

Interm. 97; Ex [A1]

 .Na; Interm. 98; Ex [A1]

OR

N OO

NN

F

N N

H

H NN

NN H N

OR

NO

  Interm. 99; Ex [A1]

 Interm. 100; Ex [A1]

N N

N NH HNN

NN H

HO N NN

OO OR

OR

  .Na; Interm. 101; Ex [A1]; m.p. > 260 ° C

 Interm. 102; Ex [A1]

N

N Cl

Cl

N N H HNN

NN OR

HO

N OR

NO

 Interm. 103; Ex [A1]

  .Na; Interm. 104; Ex [A1]; m.p. > 260 ° C

OCl

N N

N

H H

NN NNNOO

N N

H NO OR

OR

   Interm. 105; Ex [A1]; m.p. 80 ° C

 Interm. 106; Ex [A1]

N

NO

OR

N N

H HNN NNO HHO N NN

OR OR

OR

  .Na; Interm. 107; Ex [A1]; m.p. > 260 ° C

Interm. 108; Ex [A1]; m.p. 80 ° C

N

HNN

N HO

NO

  Interm. 109; Ex [A1]; m.p. ° C

 .Na; Interm. 110; Ex [A1]; m.p. 260 ° C

N

HN NNH NNN NOH O N NNO O

OR

  Interm. 111; Ex [A1]

Interm. 112; Ex [A1]

OR

O H N N NN N H NH OR H N O O N N N N H N

Or interm. 113; Ex [A1]

    Interm. 114; Ex [A2]

OR

N N H N O O N HN Interm. 115; Ex [A2] OR N    OR N N N H N O O N H Interm. 116; Ex [A2] OR N  

OR

N H O N N N H N N  OR N H O N N N H N N

Interm. 117; Ex [A2]

F    Interm. 118; Ex [A2]  

OR

H NO OR N N H N N Interm. 119; Ex [A2] N Cl  OR NH NO N H N N O Interm. 120; Ex [A2] N  

OR

OR

NH N N H N N O Interm. 121; Ex [A2] N  OR OR  OR NH N N H N N O Interm. 122; Ex [A2] N  

OR

OR

H N O N N N N H N  OR OR H NO N N N N H N

 Interm. 123; Ex [A2]

  Or interm. 124; Ex [A2]  

OR

O H N  OR N H N NN Interm. 125; Ex [A2] N OR   OR NN H N O O N H N Interm. 126; Ex [A2] N  

OR

NN H N O O N H N Interm. 127; Ex [A2] N    OR N H N NN H N O O Interm. 128; Ex [A2] N  

OR

H NO OR N NN N H N F  OR H NO OR N NN N H N F

Interm. 129; Ex [A2]

 Interm. 130; Ex [A2]

OR

H NO N N N N H N  OR OR H N O N N N N H N

OR

Interm. 131; Ex [A2]

 Interm. 132; Ex [A2]

N

Cl

OR

O H N N NN N H N OR H NO N N N N H N

OR

 OR

Interm. 133; Ex [A2]

 Interm. 134; Ex [A2]

OR

H N O O NN N N H N   OR OR H N O N N N N H N Cl

Interm. 135; Ex [A2]

 Interm. 136; Ex [A2]

OR

OR

N H O O N NN H N N O o   OR O N H NN N   H N N O o

Interm. 137; Ex [A2]

 Interm. 138; Ex [A2]

OR

N

NN

N H N N N N H

OR

H NO N  OR H N O N

OR

 OR

Interm. 139; Ex [A2]

 Interm. 140; Ex [A2]

OR

OR

N H O N NN N N N H NH

HN

OR O H N N

OR

HN

Interm. 141; Ex [A2]

Interm. 142; Ex [A2]

OR

OR

OR

N Cl HO N Cl

N

N

N

N

N

N

N

N

Interm. 143; Ex [A7]

 .Na; Interm. 144; Ex. [A7]; m.p. > 260 ° C

OR

OR

OR

N H O N Cl  OR N

N

N

N

N

N

N

N

N

Interm. 145; Ex [A7]

 Interm. 146; Ex [A7]

OR

OR

HO

N OR N H O N

N

N

N

N

N

N

N

N

 .Na; Interm. 147; Ex. [A7]; m.p. > 260 ° C

Interm. 148; Ex [A7]

OR

OR

OR

N HO N

N

N  N N

N

N

N

N

Interm. 149; Ex [A7]

 .Na; Interm. 150; Ex [A7]

OR

OR

N H O N NN NN N N

OR

N

N

N

OR  

 Interm. 151; Ex [A7]

 Interm. 152; Ex [A7]

N

N

HO

NN N N OR O H N N NN N

OR

  OR  

 .Na; Interm. 153; Ex [A7]

 Interm. 154; Ex [A7]

OR

OR

OR

N N N OR Cl HO N N N OR Cl

N

N

N

N

Interm. 155; Ex [A9]

 .Na; Interm. 156; Ex. [A9]; m.p. > 260 ° C

OR

OR

OR

N H O N N N  OR Cl  OR N N N OR

N

N

N

N

Interm. 157; Ex [A9]

 Interm. 158; Ex [A9]

HO O

N N N NO N OR N H O O N N N NO N

OR

 .Na; Interm. 159; Ex [A9]    OR  Interm. 160; Ex [A9]  

OR

N N N NO N HO N N N NO N

OR

Interm. 161; Ex. [A9] N N N N H O O N O  OR  N O. Na; Interm. 162; Ex. [A9]; m.p. > 260 ° C N N N N NO O

OR

HO O O

Interm. 163; Ex. [A9] N N N N N O. Na; Interm. 165; Ex [A9] OR    Interm. 164; Ex. [A9] O N N N N H N O OO Interm. 166; Ex. [A9] OR N  

OR

N HO N

N

N

N

N

N

N

N

N

OR

 Interm. 167; Ex. [A12] N N N N N H O O OR  N O. Na; Interm. 168; Ex. [A12]; m.p. > 260 ° C O H N NO N N O

Or interm. 169; Ex. [A12]; m.p. 80 ° C

  Interm. 170; Ex [A13] N  

O H N NHO N N N O

O NN H N O H N N N O O

 .Na; Interm. 171; Ex. [A13] N H ON NN NO O  Interm. 172; Ex. [A13] N H ON NN NHO O

 Interm. 173; Ex [A13]; m.p. 173 ° C

.Na; Interm. 174; Ex [A13]; m.p. > 260 ° C

OR

N NN N H N O O O N H N N HN NH NO O

 Interm. 175; Ex [A13]

 Interm. 176; Ex. [A17]

N N HN NH NHO O

N O N HN NH N H N O O

 .Na; Interm. 177; Ex [A17]; m.p. > 260 ° C

Interm. 178; Ex. [A17]

B. Preparation of the final compounds

Example B1 5 Preparation of compound 1a

N

H

NN

NH

N

N

HO O

.C2HF3O2

TFA (2 ml) was added to a mixture of intermediate 3 (0.0006 mol) in MeOH (40 ml). The mixture was stirred at room temperature for 30 hours. The solvent was evaporated. The residue was crystallized from EtOAc / diethyl ether. The precipitate was filtered off and dried, to obtain 0.31 g (86%) of compound 1a; melting point: 130 ° C.

10 Alternative synthesis of compound 1

Preparation of compound 1b

N

H

NN

NH

N

N

HO O

.2HCl

Hydroxylamine (50% in water, 7.5 ml) and then 1 N NaOH (15 ml) was added at 10 ° C to a mixture of intermediate 1 (0.0098 mol) in MeOH (10 ml). The mixture was stirred at room temperature for 24 hours. The mixture was acidified to a pH of 5-6 by adding a solution of 1 N HCl. The precipitate was filtered off, washed with diethyl ether and dried. The residue (4.5 g) was purified by silica gel column chromatography LiChroprep® NH2 (25-40 μm) (eluent: DCM / MeOH / H2O 90/10/1). The pure fractions were collected and the solvent was evaporated, to obtain 3.1 g (80%). The hydrochloric salt was prepared with a fraction (0.5 g) in EtOH and the

The precipitate was filtered off, washed with diethyl ether and dried, to obtain 0.43 g of compound 1b; melting point: 220 ° C.

Example B2

Preparation of compound 2 HO

10 TFA (0.5 ml) was added to a mixture of intermediate 7 (0.0001 mol) in MeOH (10 ml) and the mixture was stirred at room temperature for 24 hours. The solvent was evaporated. The residue was crystallized from acetonitrile / diethyl ether. The precipitate was filtered off and dried, to obtain 0.036 g (50%) of compound 2; melting point: 205 ° C.

15 Example B3

Preparation of compound 3

HO

TFA (5 ml) was added at room temperature to a mixture of intermediate 12 (0.0014 mol) in MeOH (100 ml). The

The mixture was stirred at room temperature for 48 hours. The solvent was evaporated to dryness. The residue was crystallized from EtOAc / diethyl ether. The precipitate was filtered off and dried, to obtain 0.545 g (74%) of compound 3; melting point: 121 ° C.

Example B4

25 Preparation of compound 4 H

HO

TFA (0.5 ml) was added to a mixture of intermediate 17 (0.0009 mol) in MeOH (80 ml). The mixture was stirred at room temperature for 4 days. The solvent was evaporated. The residue was crystallized from diethyl ether. The precipitate was filtered off and dried, to obtain 0.19 g (32%) of compound 4; melting point: 103 ° C.

30 Example B5

Preparation of compound 18 O OR-

N +

N

H

NN

H

N

NHO

Or .C2HF3O2

A mixture of intermediate 48 (0.0002 mol) in TFA (0.75 ml) and MeOH (15 ml) was stirred at room temperature for 24 hours. The solvent was evaporated. The residue was crystallized from diethyl ether. The precipitate was filtered off and dried, to obtain 0.071 g (59%) of compound 18.

5 Example B6

Preparation of compound 19 O HO

N H

A mixture of intermediate 53 (0.0003 mol) in TFA (1 ml) and MeOH (20 ml) was stirred at room temperature for 24 hours. The solvent was evaporated to dryness. The residue was crystallized from MeOH / CH3CN / diethyl ether. The precipitate

10 was filtered off and dried, to obtain 0.133 g (80%) of compound 19; melting point: 174 ° C.

Example B7

Preparation of compound 20 O H

A mixture of intermediate 57 (0.00005 mol) in TFA (0.25 ml) and MeOH (10 ml) was stirred at room temperature for 24 hours. The solvent was evaporated. The residue (0.04 g) was crystallized from CH3CN / diethyl ether. The precipitate was filtered off and dried. The residue (0.04 g) was purified by column chromatography on silica gel LiChroprep® NH2 (25-40 μm) (eluent: DCM / MeOH / H2O 80/20/2). The pure fractions were collected and the solvent was evaporated, to obtain 0.02 g (80%) of compound 20; melting point: 90 ° C.

20 Example B8

Preparation of compound 21 O HO

A mixture of intermediate 60 (0.0001 mol) in TFA (0.5 ml) and MeOH (10 ml) was stirred at room temperature

25 throughout the night. The solvent was evaporated. The residue (0.15 g) was purified by silica gel column chromatography LiChroprep® NH2 (25-40 μm) (eluent: DCM / MeOH / H2O 80/20/2). The pure fractions were collected and the solvent was evaporated, to obtain 0.064 g (78%) of compound 21; melting point: 83 ° C.

Example B9 30 Preparation of compound 22 Or HO

N N ClH O

O NN S

N N

A mixture of intermediate 65 (0.002 mol) in TFA (6 ml) and MeOH (120 ml) was stirred at room temperature for 24 hours. The solvent was evaporated to dryness. The residue was crystallized from CH3CN / MeOH / diethyl ether. The precipitate was filtered off and dried, to obtain 0.9 g (87%) of compound 22; melting point: 183 ° C.

Preparation of compound 23 O HO

N

H

A mixture of intermediate 72 (0.0001 mol) in TFA (0.5 ml) and MeOH (10 ml) was stirred at room temperature for 48 hours. The solvent was evaporated. The residue was crystallized from diethyl ether. The precipitate was filtered off and dried, to obtain 0.059 g (59%) of compound 23; melting point: 182 ° C.

10 Example B11

Preparation of compound 24 OR HO

N

N

H

NN

H

N N

OR

.C2HF3O2

A mixture of intermediate 75 (0.0003 mol) in TFA (1 ml) and MeOH (20 ml) was stirred at room temperature for 24 hours. The solvent was evaporated to dryness. The residue was crystallized from MeOH / CH3CN / diethyl ether. The precipitate was filtered off and dried, to obtain 0.147 g (78%) of compound 24; melting point: 160 ° C.

Example B12

Preparation of compound 25

OR

HO

N

N

N H

NN OR

N

.C2HF3O2

A mixture of intermediate 82 (0.0009 mol) in TFA (2.5 ml) and MeOH (56 ml) was stirred at room temperature for 24 hours. The solvent was evaporated to dryness. The residue was purified by silica gel column chromatography (25-40 μm) (eluent: DCM / MeOH / H2O 90/10/1). The pure fractions were collected and the solvent was evaporated. The residue was crystallized from DIPE. The precipitate was filtered off and dried, to obtain 0.286 g.

25 (53%) of compound 25; melting point: 80 ° C.

Example B13

Preparation of compound 26 O HO

N

H

A mixture of intermediate 84 (0.0012 mol) in TFA (3 ml) and MeOH (60 ml) was stirred at room temperature for 24 hours. The solvent was evaporated to dryness. The residue was crystallized from DCM / MeOH. The precipitate was filtered off, washed with diethyl ether and dried, to obtain 0.322 g (50%) of compound 26; melting point: 188 ° C.

Cl N

Preparation of compound 27

H

NN NH N

N

HO O

.C2HF3O2

A mixture of intermediate 89 (0.001 mol) in TFA (2.5 ml) and MeOH (50 ml) was stirred at room temperature for 24 hours, then evaporated to dryness. The residue was crystallized from MeOH / CH3CN / diethyl ether. The precipitate was filtered off, washed with water and dried, to obtain 0.33 g (55%) of compound 27; melting point: 171 ° C.

Example B15

A mixture of intermediate 93 (0.00007 mol) in TFA (0.2 ml) and MeOH (4 ml) was stirred at room temperature for 3 days, then evaporated to dryness, to obtain 0.041 g (100%) of compound 28; melting point: 80 ° C.

15 Table F-2 lists the compounds prepared according to one of the previous examples. The following abbreviations were used in the tables: .C2HF3O2 refers to trifluoroacetic salt and m.p. refers to the melting point.

Table F-2 (final compounds)

N HN NN N H N O HO

N HN NN N H N O HO

C2HF3O2; Co. No. 1a; Ex. [B1]; m.p. 163 ° C

.HCl; Co. No. 1b; Ex. [B1]; m.p. 220 ° C

N NHHO HNN N H N O Cl

NN N H N O HO N H N

C2HF3O2; Co. No. 2; Ex [B2]; m.p. 205 ° C

Co. No. 3; Ex [B3]; m.p. 121 ° C

N N N H N N O OHH NHO

O H NHO N N N N H H N

.C2HF3O2; Co. No. 4; Ex. [B4]; m.p. 103 ° C

.C2HF3O2; Co. No. 5; Ex. [B1]; m.p. 120 ° C

H

N

N HH NN

NN H

NN H NNN N

HO OR

HOO

.C2HF3O2; Co. No. 6; Ex. [B1]; m.p. 132 ° C C2HF3O2; Co. No. 7; Ex. [B1]; m.p. 136 ° C

NH

H NNNN NH HH NSN NNHO

HO OR

OR

C2HF3O2; Co. No. 8; Ex. [B1]; m.p. 110 ° C C2HF3O2; Co. No. 9; Ex. [B1]; m.p. 217 ° C

N N H HN

NO

NN N H

H

N NNNHO

HO OR

OR

C2HF3O2; Co. No. 10; Ex. [B1]; m.p. 192 ° C C2HF3O2; Co. No. 11; Ex. [B1]; m.p. 186 ° C

OR HO

O HO N NN N HHHH NN NNNN HH NN F

C2HF3O2. H2O; Co. No. 12; Ex [B2]; m.p. 192 ° C C2HF3O2; Co. No. 13; Ex [B2]; m.p. 202 ° C

BrN NH HNN NNN NH H HHNN NNHO

HO OR

OR

C2HF3O2; Co. No. 14; Ex [B2]; m.p. 145 ° C C2HF3O2; Co. No. 15; Ex [B2]; m.p. > 260 ° C

NH

OR

N

H NNN HN NNH HN N H ClHO NNHOO

OR

2 C2HF3O2 .1 / 2 H2O; Co. No. 16; Ex [B2]; m.p. 180 ° C. C4H10O. C2HF3O2; Co. No. 17; Ex [B3]; m.p. 138 ° C

O OO-N + HO N

N H

NNN HH NNN N NH NN

HO OR

.C2HF3O2 (1: 1); Co. No. 18; Ex. [B5] .C2HF3O2 (1: 1); Co. No. 19; Ex [B6]; m.p. 174 ° C

OR

OR OR

HON N NHN HHNN NN NN

N

N

Co. No. 20; Ex [B7]; m.p. 90 ° C Co. No. 21; Ex [B8]; m.p. 83 ° C

OR

HO N N ClH

OR

O NN S

N N

 Co. No. 22; Ex [B9]; m.p. 183 ° C

OR

HO N N H NN H

N N

OR

 .C2HF3O2 (1: 1); Co. No. 24; Ex. [B11]; m.p. 160 ° C

OR

HO N N ClH NN H

N N

 .C2HF3O2 (1: 1); Co. No. 26; Ex. [B13]; m.p. 188 ° C

N

N

HNN

H N

NHO O

 .C2HF3O2 (1: 1); Co. No. 28; Ex. [B15]; m.p. 80 ° C

OR

HO

NN FH N

NN H

N

.C2HF3O2 (1: 1); Co. No. 30; Ex. [B1]; m.p. 197 ° C

N Cl

N

H

NN

H N

NHOO

 .C2HF3O2 (1: 1); Co. No. 32; Ex. [B1]; m.p. 186 ° C

N

HNN

N H

NNHOO

 Co. No. 34; Ex. [B1]; m.p. 287 ° C

OR

HON N

H N

NN N

H

.C2HF3O2 (1: 1); Co. No. 23; Ex. [B10]; m.p. 182 ° C

OR

HON N NH NN O

N

.C2HF3O2 (1: 1); Co. No. 25; Ex. [B12]; m.p. 80 ° C

Cl N

H

NN NH NNHOO

.C2HF3O2 (1: 1); Co. No. 27; Ex [B14]; m.p. 171 ° C

H

N N

N N

H

HO N N

HO

.C2HF3O2 (1: 1); Co. No. 29; Ex. [B1]; m.p. 173 ° C

N

N

H NN

H N

N HO O

 .C2HF3O2 (1: 1); Co. No. 31; Ex. [B1]; m.p. 160 ° C

NO

N

HNN

H N

NHO O

 .C2HF3O2 (1: 1); Co. No. 33; Ex. [B1]; m.p. 196 ° C

N

HNN

NH H

NNHO O

 Co. No. 35; Ex [B1]

N

H

NN N

H N

NHO O

 .C2HF3O2 (1: 1); Co. No. 36; Ex [B2]; m.p. > 300 ° C

N

HNN NH NNHO O

 .C2HF3O2 (1: 1); Co. No. 38; Ex [B2]; m.p. 188 ° C

OR

HO N NH

N HNN N

 .C2HF3O2 (1: 1); Co. No. 40; Ex [B2]; m.p. 183 ° C

NN

N

H H

NHO N

NO

.C2HF3O2 (1: 1); Co. No. 42; Ex [B2]; m.p. 134 ° C

NN

H NH

NHO

N NO

.C2HF3O2 (1: 1); Co. No. 44; Ex [B2]; m.p. 124 ° C

N

HNN

N H

NNHOO

 .C2HF3O2 (1: 1); Co. No. 46; Ex [B2]; m.p. 183 ° C

N

NH NH

N NHO O

C2HF3O2 (1: 1); Co. No. 48; Ex [B2]; m.p. 181 ° C

NN

H

NN H

N NHO O

 Co. No. 50; Ex [B2]; m.p. 114 ° C

N

HNN N

H N

NHO O

 .C2HF3O2 (1: 1); Co. No. 37; Ex [B2]; m.p. 189 ° C

OR

HO N N

H N NN H

N F

 .C2HF3O2 (1: 1); Co. No. 39; Ex [B2]; m.p. 239 ° C

NN Cl

N N

H H

HO

NN O

 .C2HF3O2 (1: 1); Co. No. 41; Ex [B2]; m.p. 148 ° C

NN

N

H H N

HO N

NO

 .C2HF3O2 (1: 1); Co. No. 43; Ex [B2]; m.p. 143 ° C

OR

NN O

N N

H H

HON

NO

 .C2HF3O2 (1: 1); Co. No. 45; Ex [B2]; m.p. 116 ° C

N

N NN O H

H NN

HO OR

.C2HF3O2 (1: 1); Co. No. 47; Ex [B2]; m.p. 161 ° C

NN

H

NN H

N NHO O

 Co. No. 49; Ex [B2]; m.p. 104 ° C

F

N

H NN

N H

NN HO O

 C2HF3O2 (1: 1); Co. No. 51; Ex [B2]; m.p. 140 ° C

F

N

H NN

N H NN

HO

OR

 C2HF3O2 (1: 1); Co. No. 52; Ex [B2]; m.p. 114 ° C

N

N NN H

H

N NHO

OR

 C2HF3O2 (1: 1); Co. No. 54; Ex [B2]; m.p. 159 ° C

Cl

N

H NN N

H

N HO

O n

 Co. No. 56; Ex [B2]; m.p. 145 ° C

Clnn

N

H H N

HO N

NO

 C2HF3O2 (1: 1); Co. No. 58; Ex [B2]; m.p. 130 ° C

OR

HO N N

H NO

NN H

NO

Co. No. 60; Ex [B2]; m.p. 105 ° C

N

H

NN NH

N NHO O

C2HF3O2 (1: 1); Co. No. 62; Ex [B2]; m.p. 162 ° C

NHN

H

NN

H N

N

HO

OR

C2HF3O2 (1: 1); Co. No. 64; Ex [B2]; m.p. 135 ° C

OR

HO N N

H

NN

N N

 Co. No. 66; Ex [B7]; m.p. 88 ° C

N

HNN NH NNHOO

 C2HF3O2 (1: 1); Co. No. 53; Ex [B2]; m.p. 178 ° C

N

N

H

NN N

H N

NHO O

 C2HF3O2 (1: 1); Co. No. 55; Ex [B2]; m.p. 144 ° C

N

H

NN NH

N HO

O n

Co. No. 57; Ex [B2]; m.p. 100 ° C

OR

HON N H N ONN H

NO

 Co. No. 59; Ex [B2]; m.p. 122 ° C

OR

N

HNN

N H

NNHO O

 Co. No. 61; Ex [B2]; m.p. 117 ° C

OR

HO N N

H NN HN HN

 .C2HF3O2 (1: 1); Co. No. 63; Ex [B2]; m.p. 110 ° C

OR

HO N N ClH NN N N

 .C2HF3O2 (1: 1); Co. No. 65; Ex [B7]; m.p. 111 ° C

OR

HO N N

H NN NN

.C2HF3O2 (1: 1); Co. No. 67; Ex [B7]; m.p. 119 ° C

N

NN N

H NNHO O

Co. No. 68; Ex [B7]; m.p. 210 ° C

OR

HO N N

H ONN

N N

Co. No. 70; Ex [B9]; m.p. 80 ° C

N

NN ONH NNHO O

Co. No. 72; Ex [B9]; m.p. 219 ° C

OR

HON N OH NN

H N N

Co. No. 74; Ex. [B12]; m.p. 149 ° C

NH

N

HNN

H N

N

HOO

 .C2HF3O2 (1: 1); Co. No. 76; Ex. [B15]; m.p. 164 ° C

I

N

H NN N

H

N HO

O n

 .C2HF3O2 (1: 1.3); Co. No. 78; Ex [B2]; m.p. 114 ° C

ON N HONH HN

N

OR

HON N ClH O NN N N

 Co. No. 69; Ex [B9]; m.p. 110 ° C

OR

HO N N

H O

NN

N N

OR

 Co. No. 71; Ex [B9]; m.p. 114 ° C

OR

HO

N N H NN

N N

OR

 Co. No. 73; Ex. [B11]; m.p. 98 ° C

NO

N

HNN

H NNHOO

 .C2HF3O2 (1: 1); Co. No. 75; Ex. [B12]; m.p. 184 ° C

CF3

N

H NN N

H N

N HO

OR

 .C2HF3O2 (1: 1.4); Co. No. 77; Ex [B5]; m.p. 190 ° C

N

H

NN NH NNHO O

.C2HF3O2 (1: 1.27); Co. No. 79; Ex. [B1]; m.p. 189 ° C

F

N

HNN

N H

NNHO O

C. Pharmacological example:

The in vitro assay for the inhibition of histone deacetylase (see example C.1) evaluates the inhibition of the enzymatic activity of the HDAC obtained with the compounds of formula (I).

The cellular activity of the compounds of formula (I) was determined in A2780 tumor cells, using a colorimetric assay for cell toxicity or survival (Mosmann Tim, Journal of Immunological Methods 65: 55-63, 1983) (refer to the example C.2).

The solubility of a compound determines the ability of the compound to remain in solution.

In a first method, the ability of a compound to remain in aqueous solution when diluted is determined (refer to example C.3.a). Standard solutions are diluted in DMSO with a single aqueous buffer solvent in 3 consecutive steps. For each dilution, turbidity is measured with a nephelometer.

In a second method, the solubility of a compound at different pHs can be determined using a chemiluminescence nitrogen detector (see example C.3.b).

The permeability of a drug expresses its ability to move from one medium to another. Specifically, its ability to move through the intestinal membrane into the bloodstream and / or from the bloodstream to the target. Permeability (refer to example C.4) can be determined by the formation of a phospholipid bilayer that acts as an artificial membrane immobilized in a filter. In the test of the artificial membrane immobilized in a filter, a "sandwich" is formed with a 96-well microtiter plate and a 96-well filtration plate, so that each composite well is divided into two chambers, with a solution donor at the bottom and an acceptor solution at the top, separated by a 125 μm microfiltration disc (pores of 0.45 μm), coated with a 2% (w / v) dodecane solution of dioleoylphosphatidylcholine, under conditions where those that form multilamellar bilayers within the filter channels when the system comes into contact with an aqueous buffer solution. The permeability of the compounds through this artificial membrane is measured in cm / s. The objective is to observe the permeability of drugs through an artificial membrane parallel to 2 different pHs: 4.0 and 7.4. The detection of the compound is carried out with UV spectrometry at an optimum wavelength between 250 and 500 nm.

Drug metabolism refers to the fact that a fat-soluble endobiotic or xenobiotic compound is enzymatically transformed into a polar metabolite (s), water soluble (s) and excretable (s). The main organ where drug metabolism takes place is the liver. Metabolic products are usually less active than the original drug or inactive. However, some metabolites may have increased activity or toxic effects. Therefore, drug metabolism can include both "detoxification" and "toxification" processes. One of the main enzymatic systems that determines the ability of an organism to deal with drugs and chemical agents is represented by the cytochrome P450 monooxygenases, which are enzymes that depend on NADPH. The metabolic stability of the compounds can be determined in vitro using subcellular human tissue (see example C.5.a). Here, the metabolic stability of the compounds is expressed as the% of the metabolized drug after incubating these compounds with microsomes for 15 minutes. The quantification of the compounds was carried out by LC-MS analysis. The metabolic stability of the compounds can also be determined by calculating the half-life of the compounds in rat liver cells (see example C.5.b).

A wide range of antitumor agents have been shown to activate p21 protein, including DNA damaging agents and histone deacetylase inhibitors. Agents that damage DNA activate the p21 gene through the p53 tumor suppressor, while histone deacetylase inhibitors transcriptionally activate the p21 gene by transcription of the Sp1 factor. Thus, agents that damage DNA activate the p21 promoter through the response element to p53, while histone deacetylase inhibitors activate the p21 promoter through sp1 sites (located in the region of –60 bp to +40 bp relative to the TATA box), and both cause greater expression of the p21 protein. When the p21 promoter in a cell is constituted by a fragment of the 1300 bp p21 promoter that does not comprise the response elements to p53, it will therefore not respond to the DNA damaging agents.

The ability of the compounds to induce the production of p21 can be determined in several ways. A first method consists in treating the tumor cells with the compound of interest and, after lysis of the cells, detecting the production of p21 with the p21 enzyme linked immunosorbent assay (WAF1 oncogenes ELISA). The p21 assay consists of a "sandwich" enzyme immunoassay that uses mouse monoclonal antibodies and rabbit polyclonal antibodies. A rabbit polyclonal antibody, specific for human p21 protein, has been immobilized on the surface of the plastic wells provided in the kit. The p21 present in the sample to be analyzed will bind to the capture antibody. The biotinylated monoclonal detection antibody also recognizes human p21 protein and will bind to any p21 that has been retained by the capture antibody. The detection antibody, in turn, is bound by streptavidin conjugated to horseradish peroxidase. Radish peroxidase catalyzes the conversion of the tetragenic methylbenzidine chromogenic substrate from a colorless solution to a blue solution (or yellow, after the addition of the stop reagent), whose intensity is proportional to the amount of p21 protein bound to the plate. The colored reaction product is quantified using a spectrophotometer. Quantification is carried out by constructing a standard curve using known concentrations of p21 (which are provided lyophilized). This assay can evaluate the induction of p21 production as a result of DNA damage or as a consequence of histone deacetylase inhibition (refer to example C.6.a).

Another method determines the ability of the compounds to induce the production of p21 as a consequence of the inhibition of HDAC at the cellular level. The cells can be stably transfected with an expression vector that contains a fragment of the p21 promoter of 1300 bp and that does not comprise the response elements to p53, where an increase in the expression of the marker gene, compared to levels control, determines that the compound has the capacity to induce the production of p21. The marker gene is a fluorescent protein and the expression of the marker gene is determined as the amount of fluorescent light emitted (refer to example C.6.b).

The last method is an in vivo method in which mice are used to perform a systematic detection of the pharmaceutical activity of a compound. The stably transformed tumor cells described above can be administered to mice in an amount sufficient to produce a tumor. After the tumor cells have had sufficient time to form a tumor, a potentially active compound can be administered to the animals and the effect of said compound on the tumor cells is evaluated by determining the expression of the marker gene. Incubation with active pharmaceutical compounds will cause an increase in expression of the marker gene compared to control levels (see example C.6.c).

Specific HDAC inhibitors should not inhibit other enzymes, such as abundant CYP P450 proteins. CYP P450 proteins (expressed in E. coli) 3A4, 2D6 and 2C9 convert their specific substrates into a fluorescent molecule. The CYP3A4 protein converts 7-benzyloxytrifluoromethylcoumarin (BFC) into 7-hydroxytrifluoromethylcoumarin. CYP2D6 protein converts 3- [2- (N, N-diethyl-N-methylamino) ethyl] -7-methoxy-4-methylcoumarin (AMMC) into 3- [2- (N, N-diethylamino) ethyl hydrochloride] -7 -hydroxy-4-methylcoumarin and the CYP2C9 protein converts 7-methoxy-4-trifluoromethylcoumarin (MFC) into 7-hydroxytrifluoromethylcumarine. Compounds that inhibit the enzymatic reaction will cause a reduction in the fluorescent signal (see example C.7).

Example C.1: In vitro assay for histone deacetylase inhibition

Example C.1.a: In vitro assay with a substrate labeled with [3H]

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg / ml with 75 μM of the substrate. As a substrate to determine HDAC activity, a synthetic peptide was used, that is, amino acids 14-21 of histone H4. The substrate was biotinylated in the NH2-terminated part with a 6-aminohexanoic acid spacer, protected in the COOH-terminated part with an amide group and an [3H] acetyl group was introduced into lysine 16. The substrate, biotinylated (6-aminohexanoic) Gly-Ala - ([3 H] -acetyl-Lys-Arg-His-Arg-Lys-Val-NH2), was added in a buffer containing 25 mM Hepes, 1 M sucrose, 0.1 mg / ml of BSA and 0.01% Triton X-100 at pH 7.4. After 30 min, the deacetylation reaction was stopped by adding HCl and acetic acid (final concentration of 0.035 mM and 3.8 mM, respectively). After stopping the reaction, the free 3H-acetate was extracted with ethyl acetate. After mixing and centrifuging, the radioactivity was counted in an aliquot of the upper (organic) phase with a � counter.

For each experiment, controls (containing HeLa and DMSO nuclear extract without compound), an incubation blank (containing DMSO, but did not contain HeLa nuclear extract or compound) and samples (containing the compound dissolved in DMSO and HeLa nuclear extract). First, the compounds were evaluated at a concentration of 10-5 M. When the compounds exhibited activity at 10-5 M, a concentration-response curve was performed, in which the compounds were evaluated at concentrations between 10- 5 M and 10-12 M. In each test, the blank value was subtracted from the control and sample values. The control sample represented 100% deacetylation of the substrate. For each sample, the radioactivity was expressed as a percentage of the average value of the controls. When necessary, the IC50 values (concentration of the drug necessary to reduce the amount of metabolites up to 50% of the control) were computed using probit analysis for stratified data. The effects of the test compounds are expressed herein as pCI50 (the negative log value of the IC50 value) (refer to Table F-3).

Example C.1.b: In vitro assay with a fluorescent labeled substrate

The HDAC fluorescent activity test / Biomol drug discovery kit (Cat. No .: AK-500-0001) was used. The HDAC fluorescent activity assay is based on the combination of the developer and the Fluor de Lys substrate (Fluorogenic Histone de Acetylase Lysyl; Fluorogenic histone deacetylase lysyl). The Fluor de Lys substrate comprises an acetylated lysine side chain. De-substrateization sensitizes the substrate, so that, in a second step, treatment with the Fluor de Lys developer produces a fluorophore.

HeLa nuclear extracts (supplier: Biomol) were incubated at 60 μg / ml with 75 μM of the substrate. The Fluor de Lys substrate was added in a buffer containing 25 mM Tris, 137 mM NaCl, 2.7 mM KCl and 1 mM MgCl2.6H2O at pH 7.4. After 30 min, 1 developer volume was added. The fluorophore was excited with 355 nm light and the emitted light (450 nm) was detected with a fluorometer to read plates.

For each experiment, the controls (containing HeLa nuclear extract and buffer), an incubation blank (containing buffer, but not containing HeLa nuclear extract) and samples (containing the compound dissolved in DMSO and further diluted further) were determined in parallel in buffer, and nuclear extract HeLa). First, the compounds were evaluated at a concentration of 10-5 M. When the compounds exhibited activity at 10-5 M, a concentration-response curve was performed, in which the compounds were evaluated at concentrations between 10- 5 M and 10-9 M. All samples were evaluated 4 times. In each test, the blank value was subtracted from the control and sample values. The control sample represented 100% deacetylation of the substrate. For each sample, the fluorescence was expressed as a percentage of the average value of the controls. When necessary, the IC50 values (concentration of the drug necessary to reduce the amount of metabolites up to 50% of the control) were computed using probit analysis for stratified data. The effects of the test compounds are hereby expressed as pCI50 (the negative log value of the IC50 value) (refer to Table F3).

Example C.2: Determination of antiproliferative activity in A2780 cells

All the compounds evaluated were dissolved in DMSO and additional dilutions were made in the culture medium. The final concentrations of DMSO never exceeded 0.1% (v / v) in cell proliferation assays. The controls contained A2780 and DMSO cells without compound, and the targets contained DMSO but did not contain cells. MTT was dissolved with a concentration of 5 mg / ml in PBS. A glycine buffer comprising 0.1 M glycine and 0.1 M NaCl, buffered at a pH of 10.5 with NaOH (1 N) was prepared (all reagents were from Merck).

Human A2780 ovarian carcinoma cells (a generous donation from Dr. TC Hamilton [Fox Chase Cancer Center, Pennsylvania, USA)) were grown in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μg / ml gentamicin and 10% fetal bovine serum. The cells were routinely maintained as monolayer cultures at 37 ° C in a humidified 5% CO2 atmosphere. The cells were inoculated once a week using a trypsin / EDTA solution with a 1:40 split rate. All media and accessories were purchased from Life Technologies. The cells were free of Mycoplasma contamination, as determined using the Mycoplasma tissue culture kit from Gen-Probe (supplier: BioMérieux).

The cells were grown in NUNCTM 96-well culture plates (supplier: Life Technologies) and allowed to adhere to the plastic overnight. The densities used in the plates were 1500 cells per well in a total volume of 200 μl of medium. After the cells adhered to the plates, the medium was changed and the drugs and / or solvents were added to a final volume of 200 µl. After four days of incubation, the medium was replaced with 200 µl of fresh medium and the density and viability of the cells were determined using an MTT-based assay. To each well, 25 µl of MTT solution was added and the cells were further incubated for 2 hours at 37 ° C. Then, the medium was carefully aspirated and the blue formazan-MTT product was dissolved by adding 25 µl of glycine buffer and then 100 µl of DMSO. The microassay plates were shaken for 10 min on a microplate shaker and the absorbance at 540 nm was measured using an Emax 96-well spectrophotometer (supplier: Sopachem).

In one experiment, the results for each experimental condition are the average of a triplicate of wells. In order to perform an initial screening, the compounds were evaluated with a single fixed concentration of 10-6 M. For the active compounds, the experiments were repeated to establish complete concentration-response curves. For each experiment, controls (which did not contain drug) and an incubation blank (which did not contain cells or drug) were performed in parallel. The blank value was subtracted from the control and sample values. For each sample, the average value for cell growth (in absorbance units) was expressed as a percentage of the average value for cell growth of the control. When necessary, the IC50 values (concentration of the drug necessary to reduce cell growth up to 50% of the control) were computed using probit analysis for stratified data (Finney, DJ, Probit Analyzes, 2nd edition, chapter 10, Graded Responses, Cambridge University Press, Cambridge 1962). The effects of the test compounds are expressed herein as pCI50 (the negative log value of the IC50 value) (refer to Table F-3).

Example C.3: Solubility / Stability

C.3.1. Solubility in aqueous medium

In the first dilution step, 10 µl of a concentrated standard solution of the active compound, dissolved in DMSO (5 mM), was added to 100 µl of a pH 7.4 phosphate citrate buffer and mixed. In the second dilution step, an aliquot (20 µl) of the first dilution step to 100 µl of the phosphate citrate buffer pH 7.4 was added and mixed. Finally, in the third dilution step, a sample (20 µl) of the second dilution step was further diluted in 100 µl of the pH 7.4 phosphate citrate buffer and mixed. All dilutions were made in 96-well plates. Immediately after the last dilution step, the turbidity of the three consecutive dilution steps was determined with a nephelometer. Dilution was performed in triplicate for each compound in order to exclude occasional errors. Based on turbidity measurements, a classification was made in 3 groups. Compounds with a high solubility obtained a score of 3 and, for these compounds, the first dilution is transparent. Compounds with a medium solubility obtained a score of 2. For these compounds, the first dilution is not transparent and the second dilution is transparent. Compounds with a low solubility obtained a score of 1 and, for these compounds, both the first and the second dilution are not transparent (refer to Table F-3).

C.3.b. Solubility / stability at different pH

The solubility of a compound at different pHs can also be determined using a chemiluminescence nitrogen detector (see Table F-3).

Example C.4: Analysis of permeability through a parallel artificial membrane

The standard samples (10 μl aliquots of a 5 mM standard solution in 100% DMSO) were diluted in a deep well or premix plate containing 2 ml of an aqueous buffer system of pH 4 or pH 7.4 (PSR4 System Solution Concentrate (pION)). Before adding the samples to the reference plate, 150 μl of buffer was added to the wells and a UV blank measurement was made. Next, the buffer was removed and the plate was used as the reference plate. All measurements were made on UV resistant plates (supplier: Costar or Greiner).

After measurement of the blank for the reference plate, 150 μl of the diluted samples were added to the reference plate and 200 μl of the diluted samples were added to the donor plate 1. An acceptor filtration plate 1 was coated (supplier : Millipore, type: MAIP N45) with 4 μl of an artificial membrane forming solution (1,2-dioleoyl-sn-glycer-3-phosphocholine in dodecane containing 0.1% 2,6-di-tert-butyl- 4-methylphenol) and placed on donor plate 1 to form a "sandwich." Buffer (200 µl) was added on top of the acceptor wells. The sandwich was covered with a lid and stored for 18 h at room temperature in the dark.

A blank measurement was made for the acceptor plate 2 by adding 150 µl of buffer to the wells, and then a UV measurement was performed. After measuring the blank for the acceptor plate 2, the buffer was removed and 150 µl of the acceptor solution from the acceptor filtration plate 1 was transferred to the acceptor plate 2. Next, the acceptor filtration plate 1 was removed from the sandwich. After measurement of the target for donor plate 2 (see above), 150 μl of the donor solution of the donor plate 1 was transferred to the donor plate 2. The UV spectra of the wells of the donor plate 2, acceptor plate 2 and reference plate were scanned (with a SpectraMAX 190). All spectra were processed to calculate permeability with the PSR4p command software. All compounds were evaluated in triplicate. In each experiment, carbamazepine, griseofulvin, acicloguanisin, atenolol, furosemide and chlorothiazide were used as standards. The compounds were classified into 3 categories, according to whether they had low permeability (mean effect <0.5 x 10-6 cm / s; score 1), average permeability (1 x 10-6 cm / s> average effect � 0.5 x 10 -6 cm / s; score 2) or high permeability (� 1 x 10-6 cm / s; score 3).

Example C.5: Metabolic Stability

Example C.5.a

Subcellular tissue preparations were obtained according to Gorrod et al. (Xenobiotica 5: 453-462, 1975) by centrifugal separation after mechanical homogenization of the tissue.

The liver tissue was rinsed with 0.1 M Tris-HCl buffer (pH 7.4) cooled with ice to wash off excess blood. Next, the tissue was dried with absorbent material, weighed and cut into large pieces using surgical scissors. The tissue pieces were homogenized in 3 volumes of 0.1 M phosphate buffer (pH 7.4) cooled in ice, using a Potter-S homogenizer (Braun, Italy) equipped with a Teflon mortar hand or an Omni-Mix Sorvall homogenizer, during 7 x 10 s. In both cases, the container was kept in / on ice during the homogenization process.

Tissue homogenates were centrifuged at 9000 x g for 20 minutes at 4 oC using a Sorvall centrifuge or a Beckman ultracentrifuge. The resulting supernatant was stored at -80 oC and was designated "S9".

Fraction S9 can be further centrifuged at 100,000 x g for 60 minutes (4 oC) using a Beckman ultracentrifuge. The resulting supernatant was carefully aspirated, an aliquot was taken and called "cytosol." The pellet was resuspended in 0.1 M phosphate buffer (pH 7.4) in a final volume of 1 ml per 0.5 g of original tissue weight and was called "microsomes".

Aliquots of all subcellular fractions were taken, immediately frozen in liquid nitrogen and stored at -80 ° C until use.

To evaluate the samples, the incubation mixture contained PBS (0.1 M), compound (5 μM), microsomes (1 mg / ml) and a NADPH generating system (0.8 mM glucose-6-phosphate, 0.8 mM magnesium chloride and 0.8 glucose-6-phosphate dehydrogenase units). Control samples contained the same material, but the microsomes were replaced by microsomes deactivated by heating (10 min at 95 degrees Celsius). The recovery of the compounds in the control samples was always 100%.

The mixtures were previously incubated for 5 min at 37 degrees Celsius. The reaction was started at zero time (t = 0) with the addition of 0.8 mM NADP and the samples were incubated for 15 min (t = 15). The reaction was terminated with the addition of 2 volumes of DMSO. The samples were then centrifuged for 10 min at 900 x g and the supernatants were stored at room temperature for no more than 24 h before analysis. All incubations were performed in duplicate. The analysis of the supernatants was performed by LC-MS analysis. Elution of the samples was performed on an Xterra MS C18 column (50 x 4.6 mm, 5 μm, Waters, USA). An HPLC Alliance 2790 system (provider: Waters, USA) was used. Elution was carried out with buffer A (25 mM ammonium acetate (pH 5.2) in H2O / acetonitrile (95/5)), solvent B being acetonitrile and solvent C methanol, with a flow rate of 2.4 ml / min The gradient used consisted of increasing the concentration of the organic phase from 0%, going through 50% of B and 50% of C in 5 min, up to 100% of B in 1 min in a linear way, and the concentration of the organic phase remained stationary for 1.5 min more. The total injection volume of the samples was 25 μl.

A triple quadrupole mass spectrometer (supplier: Micromass, Manchester, United Kingdom) equipped with an ESI source was used as a detector. The source temperature and the desolvation temperature were set at 120 and 350 ° C, respectively, and nitrogen was used as a nebulizer and drying gas. Data were recorded in a positive scan mode (single ion reaction). The cone voltage was set at 10 V and the residence time was 1 s.

Metabolic stability was expressed as the% metabolism of the compound after 15 min incubation (E (act)) (% metabolism = 100%

. Compounds with a metabolism percentage lower than one

twenty%

 They were identified as very metabolically stable. Compounds with a metabolism between 20 and 70% were defined as intermediate stability and compounds that presented a percentage of metabolism greater than 70% were defined as poor metabolically stable. Whenever a metabolic stability screen was performed, three reference compounds were included. Verapamil was included as a compound with low metabolic stability (% metabolism = 73%). Cisapride was included as a compound with a medium metabolic stability (% of metabolism = 45%) and propanol was included as a compound with a metabolic stability of intermediate to high (25% metabolism). These reference compounds were used to validate the metabolic stability test.

C.5.b: Metabolic stability with a rat hepatocyte cell culture

Hepatocytes from rats were isolated from Sprague Dowley rats. The compounds were dissolved to obtain a 5 mM standard solution in 100% DMSO and incubated with a final concentration of 5 μM for 0, 15, 30, 60 and 120 min with rat hepatocyte cell cultures (0.5 million viable cells /0.5 ml) using 24-well plates.

Samples were prepared for LC-MS by adding two volumes of DMSO. The samples were shaken well and subsequently centrifuged at 900 g for 10 min (room temperature). All experiments were performed in triplicate. Of the resulting supernatant, 50 μl were analyzed by LC-MS.

In LC-MS, elution of the samples was carried out on a Hypersil BDS C18 column (50 x 4.6 mm, 5 μm, Thermohypersil, United Kingdom). The HPLC system comprised a Surveyor supply system (Surveyor Inc., San Jose, USA) equipped with a Surveyor self-sampling device. Elution was carried out using buffer A (10 mM ammonium acetate (pH 6.9) in H2O / acetonitrile (95: 5)) and solvent B (acetonitrile), with a flow rate of 1.2 ml / min. The gradient used consisted of 0.5 min of solvent A as the initial condition and then the concentration of the organic phase was increased from 0% B to 95% B for 2 min linearly. This phase remained stationary for 2 more minutes and was reduced again to 0% B in

0.5 min

The total injection volume of the samples was 50 μl. The temperature of the column oven was maintained at 40 ° C. The LC flow was divided for the detection of MS and 0.1 ml was allowed to enter the source. For detection, a TSQ Quantum triple quadrupole mass spectrometer (Thermofinnigan, LaJolla, USA) equipped with an ESI source was used. The source voltage was set at 3800 volts and the capillary temperature at 300 ° C. The mass spectrometer was programmed with a positive ion mode in a SIM adjusted for the mass of M + H with a scanning width of 1 Da, to carry out the quantification. The control of the instrument, as well as the acquisition and processing of the data were carried out using the Xcalibur software (ThermoFinnigan, San José, CA, USA). The metabolic stability of the compounds in rat hepatocytes was expressed as half-lives in vitro.

As a reference, compound R306465 (WO03 / 76422) (half-life in vitro: 8 min) was used. Compound 1 and compound 5 were evaluated and presented an in vitro half-life of 81 min and 60 min, respectively.

Example C.6: Ability to induce the production of p21

Example C.6.a: Enzyme-linked immunosorbent assay p21

To determine the level of p21 protein expression in human A2780 ovarian carcinoma cells, the following protocol has been applied. A2780 cells (20,000 cells / 180 μl) were cultured in 96-well plates in RPMI 1640 medium supplemented with 2 mM L-glutamine, 50 μg / ml gentamicin and 10% fetal bovine serum. The compounds were added with final concentrations of 10-5, 10-6, 10-7 and 10-8 M, 24 hours before lysis of the cells. All the compounds evaluated were dissolved in DMSO and additional dilutions were made in the culture medium. Supernatants were removed from the cells 24 hours after the compound was added. The cells were washed with 200 µl of ice-cold PBS. The wells were aspirated and 30 µl of the lysis buffer (50 mM Tris.HCl (pH 7.6), 150 mM NaCl, 1% Nonidet p40 and 10% glycerol) were added. The plates were incubated overnight at -70 ° C.

An appropriate number of microtiter plates were removed from the aluminum bag and placed in an empty well holder. A working solution (1x) of the wash buffer (20x plate wash concentrate: 100 ml of 20 times concentrated solution of PBS and surfactant was prepared; it contains 2% chloroacetamide). The lyophilized p21WAF standard was reconstituted with distilled H2O and was further diluted with sample diluent (supplied with the kit).

Samples were prepared by diluting them 1: 4 in sample diluent. Samples (100 μl) and p21WAF1 standards (100 μl) were pipetted into the appropriate wells and incubated at room temperature for 2 hours. The wells were washed 3 times with 1x wash buffer and then 100 µl of the detection antibody reagent (a biotinylated monoclonal p21WAF1 antibody solution) was pipetted into each well. These wells were incubated at room temperature for 1 hour and then washed three times with 1x wash buffer. The x400 conjugate (peroxidase-streptavidin conjugate: 400 times concentrated solution) was diluted and 100 µl of the x1 solution was added to the wells. The wells were incubated at room temperature for 30 min and then washed 3 times with 1x wash buffer and 1 time with distilled H2O. The substrate solution (chromogenic substrate) (100 µl) was added to the wells and the wells were incubated for 30 minutes in the dark at room temperature. Stop solution was added to each well in the same order as the previously added substrate solution. The absorbance in each well was determined using a spectrophotometric plate reader with dual wavelengths of 450/595 nm.

For each experiment, controls (which contained no drug) and an incubation blank (which did not contain cells or drugs) were performed in parallel. The blank value was subtracted from all control and sample values. For each sample, the value for the induction of the production of p21WAF1 (in absorbance units) was expressed as a percentage of the value for p21WAF2 present in the control. An induction percentage greater than 130% was defined as a significant induction. Nine compounds were evaluated and all showed a significant induction at 10-6 M.

Example C.6.b .: Cellular method

A2780 cells (ATCC) were grown in RPMI 1640 medium supplemented with 10% FCS, gentamicin and 2 mM Lglutamine at 37 ° C in a humidified incubator with 5% CO2. All cell culture solutions were purchased from Gibco-BRL (Gaithersburg, MD). Other materials were purchased from Nunc.

Genomic DNA was extracted from growing A2780 cells and used as a template for the isolation of nested PCR from the p21 promoter. The first amplification was performed for 20 cycles at a hybridization temperature of 55 ° C using the oligonucleotide pair GAGGGCGCGGTGCTTGG and TGCCGCCGCTCTCTCACC with the genomic DNA as template. The resulting 4.5 kb fragment containing the –4551 to +88 fragment relative to the TATA box was amplified again with the oligonucleotides TCGGGTACCGAGGGCGCGGTGCTTGG and ATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycles with hybridization at 88 ° C, to subsequently obtain a 4.5 kb fragment, and subsequently with the pair of oligonucleotides TCGGGTACCGGTAGATGGGAGCGGATAGACACATC and ATACTCGAGTGCCGCCGCTCTCTCACC for 20 cycles with hybridization at 88 ° C, to obtain a 1.3 kb fragment containing the –1300 to +88 fragment relative to the TATA box. The Xhol and Kpnl restriction sites present in the oligonucleotides (underlined sequence) were used to subclone.

The luciferase marker was removed from PGL3-basic and replaced by the ZsGreen marker (from plasmid pZsGreen1-N1) at restriction sites KpnI and XbaI. PGL3-basic-ZsGreen-1300 was constructed by inserting the aforementioned 1.3 kb fragment of the human p21 promoter region into the XhoI and KpnI sites of pGL3-basic-ZsGreen. All restriction enzymes were purchased from Boehringer Manheim (Germany).

A2780 cells were added to a 6-well plate with a density of 2x105 cells, incubated for 24 hours and transfected with 2 μg of pGL3-basic-ZsGreen-1300 and 0.2 μg of the pSV2neo vector using Lipofectamine 2000 (Invitrogen, Brussels, Belgium) as described by the manufacturer. Transfected cells were selected for 10 days with G418 (Gibco-BRL, Gaithersburg, MD) and individual cell suspensions were cultured. After three weeks, individual clones were obtained.

Selected clones of A2780 were expanded and seeded at 10,000 cells per well in 96-well plates. 24 hours after seeding, the cells were treated for a further 24 hours with the compounds (which affect sp1 sites in the region of the proximal p21 promoter). Subsequently, the cells were fixed with 4% PFA for 30 ’and stained with Hoechst dye. The activation of the p21 promoter that leads to the production of ZsGreen and, therefore, fluorescence production, was monitored with the Ascent Fluoroskan (Thermo Labsystems, Brussels, Belgium).

For each experiment, controls (which contained no drug) and an incubation blank (which did not contain cells or drugs) were performed in parallel. The blank value was subtracted from all control and sample values. For each sample, the value for the induction of p21 production was expressed as a percentage of the value for p21 present in the control. An induction percentage greater than 130% was defined as a significant induction.

Seventy-one compounds were evaluated and all showed a significant induction at 10-6 M.

Example C.6.c: In vivo method

A selected clone was injected subcutaneously (107 cells / 200 µl) into the slit of mice without fur and after 12 days a tumor was obtained that could be measured with a caliber. From day 12 onwards, doses were administered to the animals, orally or intravenously, daily for 6 days of solvent and 20-40 mpk of compound (4-10 animals for each). Tumors were evaluated for fluorescence using the internally developed automated full-body imaging system (an Olympus® SZX12 type fluorescent stereomicroscope equipped with a GFP filter and coupled to a controlled JAI® CV-M90 type CCD camera for a software package based on the National Instruments® IMAQ Vision software). Compound R306465 (WO03 / 76422) was used as reference. The compounds were classified as inactive (no fluorescence was detected), weaker, identical or better than R306465. Compound 1 was evaluated and was found to be better than R306465.

Example C.7: Ability to inhibit P450

All compounds were dissolved in DMSO (5 mM) and an additional dilution was made up to 5 10-4 M in acetonitrile. Additional dilutions were made in the assay buffer (0.1 M NaK phosphate buffer pH 7.4) and the final solvent concentration was never greater than 2%.

The CYP3A4 protein assay comprises, per well, 15 pmol of P450 / mg protein (in 0.01M NaK phosphate buffer + 1.15% KCl), a NADPH generating system (3.3 mM glucose-6-phosphate, 0.4 U / ml of glucose-6-phosphate dehydrogenase, 1.3 mM NADP and 3.3 mM MgCl2.6H2O in assay buffer) and the compound in a total assay volume of 100 μl. After a previous incubation of 5 min at 37 ° C, the enzymatic reaction was initiated with the addition of 150 µM of the substrate of the BFC fluorescent probe in assay buffer. After a 30 minute incubation at room temperature, the reaction was terminated after the addition of 2 volumes of acetonitrile. Fluorescence determinations were carried out at an excitation wavelength of 405 nm and an emission wavelength of 535 nm. In this experiment, Ketoconazole (IC50 value = 3 X 10-8 M) was included as the reference compound.

The CYP2D6 protein assay comprises, per well, 6 pmol of P450 / mg protein (in 0.01M NaK phosphate buffer + 1.15% KCl), a NADPH generating system (0.41 mM glucose-6-phosphate, 0.4 U / ml of glucose-6 phosphate dehydrogenase, 0.0082 mM NADP and 0.41 mM MgCl2.6H2O in assay buffer) and the compound in a total assay volume of 100 μl. After a previous incubation of 5 min at 37 ° C, the enzymatic reaction was initiated with the addition of 3 µM of the substrate of the AMMC fluorescent probe in assay buffer. After a 45 minute incubation at room temperature, the reaction was terminated after the addition of 2 volumes of acetonitrile. Fluorescence determinations were carried out at an excitation wavelength of 405 nm and an emission wavelength of 460 nm. In this experiment, quinidine (IC50 value <5 X 10-8 M) was included as the reference compound.

The CYP2C9 protein assay comprises, per well, 15 pmol of P450 / mg protein (in 0.01M NaK phosphate buffer + 1.15% KCl), a NADPH generating system (3.3 mM glucose-6-phosphate, 0.4 U / ml of glucose-6-phosphate dehydrogenase, 1.3 mM NADP and 3.3 mM MgCl2.6H2O in assay buffer) and the compound in a total assay volume of 100 μl. After a previous incubation of 5 min at 37 ° C, the enzymatic reaction began with the

5 addition of 200 μM of the MFC fluorescent probe substrate in assay buffer. After a 30 minute incubation at room temperature, the reaction was terminated after the addition of 2 volumes of acetonitrile. Fluorescence determinations were carried out at an excitation wavelength of 405 nm and an emission wavelength of 535 nm. In this experiment, sulfafenazole (IC50 value = 6.8 X 10-7 M) was included as the reference compound.

10 To perform an initial screening, the compounds were evaluated at a single fixed concentration of 1 X 10-5 M. For the active compounds, the experiments were repeated to establish complete concentration-response curves. For each experiment, the controls (which did not contain drug) and an incubation blank (which did not contain enzyme or drugs) were performed in parallel. All compounds were analyzed in quadruplicate. The value

15 of the blank was subtracted from all control and sample values. For each sample, the average value for the P450 activity of the sample (in relative fluorescence units) was expressed as a percentage of the average value for the P450 activity of the control. The percent inhibition was expressed as 100% minus the average value for the P450 activity of the sample. When necessary, the IC50 values (concentration of the drug necessary to reduce the activity of P450 up to 50% of the control) were calculated.

20 Table F-3: List the results of the compounds evaluated according to examples C.1.a, C.1.b, C.2,

C.3.a and C.3.b.

Compound number

Enzymatic activity pCI50 C.1.a Enzymatic activity pCI50 C.1.b PCI50 C.2 cell activity Solubility C.3.a. Solubility C.3.b pH = 2.3 (mg / ml)

5

8.6 6.5 2.0

1st

 8.8 9.2 8.2 3 1.4

6

8.2 6.3

7

8.5 6.1

3

8.5 6.7

4

8.7 7.0 3

17

7.8 6.8

8

8.3 7.1 2.4

9

8.3 7.1

10

8.1 7.5  3 3.7

16

8.6 7.1  3

eleven

8.2 7.5  3 2.8

2

8.4 7.5  3

fifteen

8.5 7.5

14

8.4 7.4 1.7

13

6.0 7.5  3

12

8.2 7.1  3

35

8.2 6.7

68

> 9.0 7.2  2.5

63

> 9.0 7.5

67

8.3 7.5  1.9

76

7.8 6.7

72

7.9 6.8

80

7.8 7.1

3. 4

8.4 7.5

33

10.0 7.5  1.6

32

8.0 8.4  1.5

twenty-one

8.5 7.6  1.9

49

8.7 7.5 3.3

Compound number

Enzymatic activity pCI50 C.1.a Enzymatic activity pCI50 C.1.b PCI50 C.2 cell activity Solubility C.3.a. Solubility C.3.b pH = 2.3 (mg / ml)

53

10.0 8.0

twenty

9.5 8.1  2.7

64

8.9 7.8

62

9.5 8.0

28

8.1 7.1

61

8.9 7.6

60

8.7 6.9  3.1

59

9.0 7.7

58

10.0 5.8

19

8.0 7.1

24

7.6 6.5  1.6

57

10.2 7.6  2.8

56

9.2 7.6

55

10.1 7.8  2.0

54

10.2 7.9

52

10.3 8.1

51

9.8 8.0

fifty

8.6 7.6  2.4

48

> 9.0 8.2

66

> 9.0 7.4  1.7

73

7.9 7.1

47

8.8 8.0  4.0

74

8.2 7.1

46

9.0 8.0

31

8.2 7.4

75

7.3 7.1

27

> 9.0 8.0

Four. Five

9.0 6.7  1.7

44

8.2 7.4  1.8

43

8.5 7.8  2.1

29

7.9 6.9

42

8.3 7.5

71

7.5 6.5

70

8.5 6.8

69

7.6 6.7

25

7.6 7.0

41

8.4 5.8

40

8.5 7.5

2. 3

8.4 7.5

22

9.1 7.2

26

> 9.0 7.5

65

> 9.0 7.5  1.6

39

9.0 7.5  2.0

30

9.0 7.5  2.1

38

> 9.0 7.5

37

8.6 7.5

36

8.7 8.6  2.5

Compound number

Enzymatic activity pCI50 C.1.a Enzymatic activity pCI50 C.1.b PCI50 C.2 cell activity Solubility C.3.a. Solubility C.3.b pH = 2.3 (mg / ml)

79

8.4 8.1  2.7

18

8.3 8.0

81

8.9 7.5

D. Composition example: Film-coated tablets

Tablet core preparation

A mixture of 100 g of a compound of formula (I), 570 g of lactose and 200 g of starch is properly mixed and then moistened with a solution of 5 g of sodium dodecyl sulfate and 10 g of polyvinyl pyrrolidone in approximately 200 ml of Water. The wet powder mixture is screened, dried and screened again. Next, 100 mg of microcrystalline cellulose and 15 g of hydrogenated vegetable oil are added. It mixes

10 properly the entire mass and is compressed into tablets, to provide 10,000 tablets, each of which comprises 10 mg of a compound of formula (I).

Covering

To a solution of 10 g of methylcellulose in 75 ml of denatured ethanol, a solution of 5 g of ethylcellulose in 150 ml of dichloromethane is added. Next, 75 ml of dichloromethane and 2.5 ml of 1,2,3propanotriol are added. 10 g of polyethylene glycol are ground and dissolved in 75 ml of dichloromethane. This last solution is added to the previous one and then 2.5 g of magnesium octadecanoate, 5 g of polyvinyl pyrrolidone and 30 ml of a concentrated color suspension are added, and the entire mass is homogenized. The cores of the tablets are

20 cover with the mixture obtained in this way in a coating apparatus.

Claims (12)

1. A compound of formula (I), R3O R1 YNHO N N (CH2) n N (CH2) m (I) H X R2 5 N-oxide type forms, pharmaceutically acceptable addition salts and stereochemically isomeric forms thereof, where each n is an integer with a value of 0, 1 or 2 and, when n is 0, then refers to a direct link; each m is an integer with a value of 1 or 2; each X is independently N or CH; 15 each Y is independently O, S or NR4; where each R 4 is hydrogen, C 1-6 alkyl, (C 1-6 alkyloxy) (C 1-6 alkyl), C 3-6 cycloalkyl, (C 3-6 cycloalkyl) methyl, phenyl (C 1-6 alkyl), -C (= O ) -CHR5R6 or -S (= O) 2-N (CH3) 2; where each R5 and R6 is independently hydrogen, amino, C1-6 alkyl or C1-6 aminoalkyl; and when Y is NR4 and R2 is in position 7 of the indolyl, then R2 and R4 together can form the bivalent radical - (CH2) 2- (a-1), or 25 - (CH2) 3- (a-2) ; R1  is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkylsulfonyl, (C 1-6 alkyl) carbonyl or mono- or di (C 1-6 alkyl) 6) aminosulfonyl; R2 is hydrogen, hydroxy, amino, halo, C1-6 alkyl, cyano, C2-6 alkenyl, C1-6 polyhaloalkyl, nitro, phenyl, (C16 alkyl) carbonyl, hydroxycarbonyl, (C1-6 alkyl) carbonylamino, C1- alkyloxy 6, or mono- or di (C1-6 alkyl) amino; R3 is hydrogen, C1-6 alkyl or C1-6 alkyloxy; and when R2 and R3 are in adjacent carbon atoms, they can form the bivalent radical -O-CH2-O-.

2.

A compound according to claim 1, wherein

each n is an integer with a value of 0 or 1; each R4 is hydrogen, C1-6 alkyl, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl or phenyl (C1-6 alkyl); R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, (C 1-6 alkyl) carbonyl or (C 16 alkyl) sulfonyl; and R2 is hydrogen, halo, C1-6 alkyl, cyano, nitro, C1-6 polyhaloalkyl or C1-6 alkyloxy.

3.

A compound according to claim 1 and 2, wherein

45 each n is an integer with a value of 1; each m is an integer with a value of 1; each X is independently N; Each Y is independently NR4; each R4 is C1-6 alkyl; R1 is hydrogen; R2 is hydrogen or halo; and R3 is hydrogen.

Four.

A compound as claimed in claim 1, 2 and 3, wherein said compound is compound No. 1a, compound No. 30, compound No. 39 and compound No. 50.

5.

A pharmaceutical composition comprising pharmaceutically acceptable carriers and, as active ingredient, a therapeutically effective amount of a compound as claimed in claims 1 to

N HN NN N H N O HO

N H N N N N O N H HO F

C2HF3O2; Compound 1st

C2HF3O2 (1: 1); Compound 30

O H N NN H N HO N N

F H NHO O N N H N N N

C2HF3O2 (1: 1); Compound 39

Compound 50

Four. 5 6. A process for preparing a pharmaceutical composition as claimed in claim 5, wherein the pharmaceutically acceptable carriers and a compound as claimed in claims 1 to 4 are intimately mixed. A compound as claimed in any one of claims 1 to 4 for use as a medicine. 8. The use of a compound as claimed in any one of claims 1 to 4 for production of a medicine for the treatment of proliferative diseases. fifteen 9. A combination of an anticancer agent and an HDAC inhibitor as claimed in any one of claims 1 to 4. 10. A process for preparing a compound as claimed in claim 1, characterized in that a) an intermediate of formula (II) is reacted, where Q is tetrahydropyranyloxyaminocarbonyl, referred to herein as intermediate of formula (II-a), with a suitable acid such as, for example, trifluoroacetic acid, to obtain a hydroxamic acid of formula (I). Or R3 R1 Y N O o N N (CH2) n N (CH2) m H X R2 (II-a) R3 OR R1 CF3COOH Y N HO N N (CH2) n N (CH2) mH X R2 (I) b) an intermediate of formula (II) is reacted, where Q is (C1-2 alkyloxy) carbonyl, referred to herein as formula (II-c), with hydroxylamine, in the presence of a base and in a suitable solvent , to produce a hydroxamic acid of formula (I). 11. A compound of formula (II), N-oxide type forms, pharmaceutically acceptable addition salts and stereochemically forms isomeric of this where each n is an integer with a value of 0, 1 or 2 and, when n is 0, then it refers to a direct link; each m is an integer with a value of 1 or 2; each X is independently N or CH; each Y is independently O, S or NR4; where each R4 is hydrogen, C1-6 alkyl, (C1-6 alkyloxy) (C1-6 alkyl), C3-6 cycloalkyl, (C3-6 cycloalkyl) methyl, phenyl (alkyl C1-6), -C (= O) -CHR5R6 or -S (= O) 2-N (CH3) 2; where each R5 and R6 is independently hydrogen, amino, C1-6 alkyl or C1-6 aminoalkyl; and when Y is NR4 and R2 is in position 7 of the indolyl, then R2 and R4 together can form the bivalent radical - (CH2) 2- (a-1), or - (CH2) 3- (a-2); R 1 is hydrogen, C 1-6 alkyl, C 1-6 hydroxyalkyl, C 1-6 alkylsulfonyl, (C 1-6 alkyl) carbonyl or mono- or di (C 16 alkyl) aminosulfonyl; R2 is hydrogen, hydroxy, amino, halo, C1-6 alkyl, cyano, C2-6 alkenyl, C1-6 polyhaloalkyl, nitro, phenyl, (C1 alkyl 6) carbonyl, hydroxycarbonyl, (C1-6 alkyl) carbonylamino, C1-6 alkyloxy, or mono- or di (C1-6 alkyl) amino; R3 is hydrogen, C1-6 alkyl or C1-6 alkyloxy; when R2 and R3 are in adjacent carbon atoms, they can form the bivalent radical -O-CH2-O-; and Q is (C1-2 alkyloxy) carbonyl, hydroxycarbonyl or tetrahydropyranyloxyaminocarbonyl. 12. A process for preparing a compound as claimed in claim 11, characterized in that a) a compound of formula (II) is reacted, where Q is hydroxycarbonyl, referred to in the present compound of formula (II-b), with an intermediate of formula (III) in the presence of suitable reagents such as N monohydrochloride '- (ethylcarbonimidoyl) -N, N-dimethyl-1,3-propanediamine (EDC) and 1-hydroxy-1H-benzotriazole (HOBT), to form a compound of formula (II-a), Or R3 R1 YN OO HO NH2N (CH2) n N (CH2) m + X R2 (III) (II-b) R3O R1 Y N OOEDC / HOBT N N (CH2) n N (CH2) mH X R2 (II-a) b) an intermediate of formula (VI) is reacted with the appropriate carboxaldehyde of formula (V), where t is an integer with a value of 0 or 1 and, when t is 0, then it refers to a direct bond, in the presence of a suitable reagent, to form a compound of formula (II-a), R3O R1 YN OO (CH2) tH N N (CH2) n NH + X O (VI) (V) R2 R3O R1 Y N OO N N (CH2) n N (CH2) mH X R2 (II-a) c) a compound of formula (II) is reacted, where Q is methyl- or ethyloxycarbonyl (C1-2 alkyl), referred to as The present compound of formula (II-c), with an acid solution or a suitable basic solution, to form a compound of formula (II-b), D) the carboxylic acid ethyl ester of formula (IV) is reacted with the appropriate carboxaldehyde of formula (V), in the presence of a suitable reagent, to form a compound of formula (II-c), e) an intermediate of formula (XIV) is reacted with the appropriate intermediate of formula (XV), in the presence of a suitable reagent in a suitable solvent, to form a compound of formula (II-c), f) an intermediate of formula (X) is reacted with an intermediate of formula (XI), where W is a suitable leaving group such as, for example, halo, p. eg, fluoro, chloro, bromo or iodo, or a sulfonyloxy radical such as methylsulfonyloxy, to form a compound of formula (II-c), or g) an intermediate of formula (XII) is reacted with an intermediate of formula (XIII), where W is a suitable leaving group 15 as previously described, to form a compound of formula (II-c).