FR2801588A1 - Diarylamino diazine and triazine derivatives are telomerase inhibitors and G-quadruplex stabilizers, useful in the treatment of cancers - Google Patents

Abstract

Arylamine derivatives (I) and their salts, especially (I'), which are capable of fixing the G-quadruplex structure of telomers. Arylamine derivatives of formula N-containing aromatic ring - NR<3> - dividing group - NR'<3> - aromatic ring (I) and their salts, which are capable of fixing the G-quadruplex structure of telomers. N-containing aromatic ring = quinoline optionally substituted by N(Ra)(Rb), or 1-4C alkoxy, quinolinium, benzamidine, or pyridine; Ra and Rb = H or 1-4C alkyl or alkoxy; aromatic ring = N-containing aromatic ring as defined above, phenyl optionally substituted by a halo, 1-4C alkoxy, CN, carbonylamino optionally substituted by one or two 1-4C alkyl groups, guanyl, 1-4C alkylthio, amino, 1-4C mono- or dialkyl amino, NO2, 1-4C alkylene amino, or 2-4C alkenylene amino, or a mono- di- or tri-cyclic heterocyclic ring having 0-2 hetero atoms per ring so at least one hetero atom is present, optionally substituted by one or more 1-4C alkyl or alkylene groups or 2-4C alkenylene groups; R<3> and R'<3> = H or 1-4C alkyl; dividing group = triazine optionally substituted by one 1-4C alkyl group, thio, oxy, amino, which may themselves be substituted by one or more 1-4C alkyl groups or halo, carbonyl, -C(=NH)-NH-C(=NH)- 3-7 alkyl diyl, or diazine optionally substituted by the same groups as for the triazine An Independent claim is also included for an arylamine derivative of formula (I') with the exception of 2-amino-bis-4,6-((4'-amino-6'-quinaldinyl)amino)triazine dihydrochloride and 2-amino-bis-4,6-(p-amidino anilino) triazine. A = NR<1>R<2>, OR<1>, SR<1>, 1-4C alkyl, CF3, H, F, Cl, Br, or I; R<1>, R<2> = H or 1-4C alkyl; R<3> and R'<3> = H or 1-4C alkyl; Ar<1> and Ar<2> when they are identical = quinoline optionally substituted by N(Ra)(Rb), or 1-4C alkoxy, quinolinium, benzamidine, or pyridine attached through the 4-position or fused with an aryl or heteroaryl group and optionally substituted by 1-4C alkyl; Ar1, Ar2, when they are different = a group as defined above, or Ar2 may be phenyl optionally substituted by one halo, 1-4C alkoxy, CN, carbonylamino optionally substituted by one or two 1-4C alkyl groups, guanyl, 1-4C alkylthio or alkylamino, amino, di-(1-4C alkyl)amino, NO2, 1-4C alkylene amino or 2-4C alkenylene amino, or a mono- di- or tri-cyclic heterocyclic ring as defined above.

Description

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CHEMICAL DERIVATIVES AND THEIR APPLICATION AS AN AGENT
ANTITELOMERASE
The present invention relates to cancer therapy and relates to novel anticancer agents having a very specific mechanism of action. It also relates to new chemical compounds as well as their therapeutic application in humans.

The present invention relates to the use of novel non-nucleotide chemical compounds which interact with specific structures of deoxyribonucleic acid (DNA). These new compounds consist of a distribution agent linked to two amino aromatic groups.

These new compounds are useful in the treatment of cancers and in particular act as telomerase inhibitory agents. They are particularly useful for stabilizing DNA in a G-quadruplex structure (guanine tetrads). The therapeutic application of telomerase inhibition via the stabilization of these G-quadruplexes is the arrest of cell mitosis and the death of rapidly dividing cells such as cancer cells and possibly the induction of cell senescence. cancerous.

The compounds of the present invention have the therapeutic advantage of blocking telomerase. From a biological point of view, telomerase allows the addition of repeated DNA sequences of the T T A G G G type, called telomeric sequences, at the end of the telomere, during cell division. By this action telomerase makes the cell immortal. Indeed, in the absence of this enzymatic activity, the cell loses with each division 100 to 150 bases, which quickly makes it senescent. When rapidly dividing cancer cells appeared, these cells appeared to have telomeres maintained at a stable length during cell division. In these cancer cells it appeared that telomerase was strongly activated and that it allowed the addition of repeated units of telomeric sequences at the end of the telomere and therefore allowed the conservation of the length of the telomere in cancer cells. It has been found for some time that more than 85% of cancer cells test positive for the presence of

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telomerase while somatic cells do not exhibit this characteristic.

Thus telomerase is a highly coveted target for treating cancer cells. The first obvious approach to block telomerase was the use of nucleotide structures (Chen et al., Proc.

Natl. Acad. Sci. USA 93 (7), Among the non-nucleotide compounds which have been used in the prior art, mention may be made of diaminoanthraquinones (Sun et al. J. Med. Chem. 40 (14), or diethyloxadicarbocyanines (Wheelhouse RT et al. J. Am. Chem. Soc.

1998 (120) 3261-2).

Patent WO 99/40087 describes the use of compounds which interact with G-quadruplex structures which are perylene compounds and carbocyanines containing at least seven cycles including two heterocycles.

It appeared, quite surprisingly, that simple structures made it possible to obtain an at least equivalent result with structures which were much less complicated from the chemical point of view. The compounds of the present invention which meet the intended objective, that is to say which fix the G-quadruplex structure and thereby exhibit telomerase inhibitory activity correspond to the following general formula: nitrogenous aromatic ring - NH - distributor - NH - nitrogenous aromatic ring in which # the nitrogenous aromatic rings, identical or different, represent:
0 a quinoline substituted by at least one group
N (Ra) (Rb) in which Ra and Rb, identical or different, represent hydrogen or a short-chain alkyl radical in
C1-C4 and / or
0 a quinoline having a nitrogen atom in quaternary form or
0 a benzamidine # the distributor represents:

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0 a triazine group optionally substituted by an alkyl radical having 1 to 4 carbon atoms, an oxy or thio or amino radical themselves optionally substituted by one or more alkyl chains containing 1 to 4 carbon atoms or a halogen atom or
0 a carbonyl group or
0 a C (= NH) -NH-C (= NH) group or 0 an alkylediyl group containing 3 to 7 carbon atoms or
0 a diazine group such as a pyrimidine or tetrahydrodiazine group optionally substituted with the same groups as the triazine or one of its salts.

Within the meaning of the above formula, by nitrogenous aromatic ring is meant a heterocycle comprising at least one nitrogen atom or an aromatic group not comprising a heteroatom in the ring but containing at least one nitrogen atom in a hydrocarbon chain linked to the cycle, for example a guanidino or guanyl chain.

dans laquelle : - A représente # un groupe amino de formule NR1 R2 dans lequel R1 et
R2 identiques ou différents représentent l'hydrogène ou un groupe alkyle droit ou ramifié contenant 1 à 4 atomes de carbone ou It is preferred, among all of the compounds included above, to use those comprising a triazine or diazine group as a distributor. Among the diazine groups, the use of pyrimidines is preferred. Among the triazines, the compounds corresponding to formula (I) below are preferred:

in which: - A represents # an amino group of formula NR1 R2 in which R1 and
Identical or different R2 represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or

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# an OR1 or SR1 group in which R1 has the same meaning as above or # a short chain alkyl group containing 1 to 4 carbon atoms or the trifluoromethyl radical or # a hydrogen atom or # a halogen atom chosen from fluorine, chlorine, bromine or iodine - Ar, and Ar2, which are identical or different, represent # a quinoline unit substituted by at least one group
NRaRb in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl radical containing 1 to 4 carbon atoms and / or # a quinoline having a nitrogen atom in quaternary form or # a benzamidine or one of its salts.

It is obvious that the quinoline units can be substituted by any other group not involved in the intended application, thus groups acridines or isoquinolines or quinazolines or quinoxalines or phthalazines or benzothiazines or benzoxazines or phenoxazines or phenothiazines are included in the definition of quinoline groups.

Preference is given, among the compounds of formula (1) above, to those which comprise two heterocycles chosen from 4-aminoquinolyl, 4-aminoquinolinium or quinolinium groups, the quinolinium nucleus of which is optionally substituted by a methyl group.

As regards the groups R1 and R2, they preferably represent the methylthio, amino, alkylamino or dialkylamino radicals in which the alkyl groups have 1 to 4 carbon atoms.

As representative compounds of formula (I), mention may be made of the following compounds:

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- le diodure de N',N5-bis(7-chloro-1-méthyl-4-quinoléinio)pentane-1,5- diamine - le trichlorhydrate pentahydrate de bis-2,4-[(4'-amino-6'quinaldinyl)amino]pyrimidine - le trichlorhydrate dihydrate de 1,5-(4'-amino-6'quinaldinyl)biguanide - 2-amino-bis-4,6 dichloride - [(1'-methyl-4'-amino-6'-quinaldinio) amino] -triazine - 2-amino-bis-4,6- [dichloride (1'-ethyl-4'-amino-6'-quinaldinio) amino] -triazine - 2-dimethylamino-bis-4,6 dichloride - [(1'-methyl-4'-amino-6'quinaldinio) amino] -triazine - 2-methylamino-bis-4,6 trihydrochloride - [(4'-amino-6'quinaldinyl) amino] -triazine - 2-amino-bis-4,6 dichloride - [(1 '-methyl-6'-quinolinio) amino] triazine - 2-methylamino-bis-4,6 dichloride trihydrochloride - [(4'methylamino-6'-quinaldinyl) amino] -triazine - 2-dichloride hydrochloride -amino-bis-4,6 - [(9'-amino-10'methyl-2'-acridinio) amino] -triazine - 2-amino-bis-4,6 trihydrochloride - [(4'-amino- 6'-Quinaldinyl) amino] -triazine - 2-amino-bis-4,6- (p-amidinoanilino) -triazine trichloride, - 2-methylthio-bis-4,6 dichloride - [(1'- methyl-4'-amino-6'quinaldinio) amino] -triazine - 2-chloro-bis-4,6 dihydrate dihydrate - [(4'-dimethylamino-6'-quinaldinyl) amino] -triazine - hydrate of 2-methylthio-bis-4,6 - [(4'-dimethylamino-6'-quinaldinyl) amino] -triazine - N, N '- (4-amino-6-quinaldinyl) urea dihydrochloride

- N ', N5-bis (7-chloro-1-methyl-4-quinolinio) pentane-1,5-diamine diodide - bis-2,4-pentahydrate trihydrochloride - [(4'-amino-6' quinaldinyl) amino] pyrimidine - 1,5- (4'-amino-6'quinaldinyl) biguanide trihydrochloride dihydrate

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dans laquelle : - A représente # un groupe amino de formule NR1 R2 dans lequel R1 et
R2 identiques ou différents représentent l'hydrogène ou un groupe alkyle droit ou ramifié contenant 1 à 4 atomes de carbone ou # un groupe OR1 ou SR1 dans lequel R1 a la même signification que précédemment ou # un groupe alkyle contenant 1 à 4 atomes de carbone ou un groupe trifluorométhyle ou # un atome d'hydrogène ou # un atome d'halogène choisi parmi le fluor, le chlore, le brome ou l'iode -Ar1 et Ar2identiques ou différents représentent # un motif quinoléine substitué par au moins un groupe
N (Ra)(Rb) dans lequel Ra et Rb identiques ou différents représentent l'hydrogène ou un radical alkyle à chaîne courte contenant 1 à 4 atome de carbone et/ou # une quinoléine possédant un atome d'azote sous forme quaternaire ou # une benzamidine sauf dans le cas où A représente la diéthylamine, l'hydrogène ou un groupe amine Another object of the present invention relates to the compounds of formula (I) as new chemicals. It therefore concerns new products corresponding to the following formula (I):

in which: - A represents # an amino group of formula NR1 R2 in which R1 and
Identical or different R2 represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or # an OR1 or SR1 group in which R1 has the same meaning as above or # an alkyl group containing 1 to 4 carbon atoms or a trifluoromethyl group or # a hydrogen atom or # a halogen atom selected from fluorine, chlorine, bromine or iodine -Ar1 and Ar2 identical or different represent # a quinoline unit substituted by at least one group
N (Ra) (Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl radical containing 1 to 4 carbon atoms and / or # a quinoline having a nitrogen atom in quaternary form or # a benzamidine except where A represents diethylamine, hydrogen or an amine group

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or a salt thereof excluding 2-amino-bis-4,6- [(4'-amino-6'-quinaldinyl) amino] -triazine trihydrochloride and 2-amino-bis-4 trihydrochloride, 6- (p-amidinoanilino) -triazine.

Indeed the first of these two compounds is described in a publication published under the reference Indian Journal of Animal Sciences 43 (4), pages 226-29 as an antitrypanosome agent for animals and in no case as an antitelomerase agent and the second is also described as an antitrypanosome agent in J. Chem. Soc., 1960, 4525
The compounds of formula (I) which are preferred are those for which Ar1 and Ar2 represent a group chosen from the following units: 4-amino- or 4-methylamino- or 4-dimethylamino-quinolyl or quinolynium in which the quinolinium nucleus is optionally substituted. by a methyl group.

The compounds of general formula (I) which are preferred are those for which A represents an amino or dimethylamino group or more preferably methylthio.

Another object of the present invention relates to the use of the compounds of formula (1) as a pharmaceutical product for human use.

sont décrits ci-après. Processes for the preparation of the compounds of formula (I)

are described below.

General method 1
According to a first process for the preparation of compounds of general formula (I) in which Ar1 and Ar2 are identical and defined as above and R represents a halogen atom such as chlorine or fluorine, an amino, alkylamino or dialkylamino function of which the straight or branched alkyl parts contain 1 to 4 carbon atoms, alkyloxy or alkylthio in which the straight or branched alkyl parts contain 1 to 4

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carbon atoms, can be obtained by amination of a dihalogenotriazine, very generally a dichloro-s-triazine, of general formula (B) in which A is defined as above by an aromatic or heteroaromatic amine of general formula (C) in which Ar is defined as previously by operating according to scheme 1:

Diagram 1
In the case where A represents a halogen atom, it is useful to react the corresponding 2,4,6-trihalo-s-triazine of general formula (B) with the aromatic or heteroaromatic amine ArNH2 of general formula (C ).

The operation is generally carried out by condensing one mole of dihalo-striazine, or trihalo-s-triazine, with 2 moles of aromatic or heteroaromatic amine. The reaction takes place in an inert medium under the reaction conditions. Among the inert solvents, mention may be made of acetone, which may be aqueous, or an alcohol which may be aqueous, such as ethanol, or a halogenated solvent such as dichloromethane, or an ether such as diethyl ether or dioxane, or a polar aprotic solvent. such as DMF, DMSO or NMP. The operation is preferably carried out at a temperature between 20 ° C. and reflux, in the presence in particular of an organic base, such as triethylamine, or inorganic base, such as sodium hydroxide or sodium or potassium carbonate. It is also possible not to use a base during the amination reaction, and to isolate a hydrochloride of the product of general formula (A), the base of which can then be released.

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The dihalo or trihalo-s-triazines of general formula (B) are either commercial or known, and can be obtained under the conditions described in the literature.

The aromatic or heteroaromatic amines of general formula (C) are either known or can be easily prepared by known methods for the synthesis of aromatic or heteroaromatic amines.

In the case where Ar and Ar2 are different, the triazine of general formula (A) can be obtained by sequential displacement of the halogen atoms, very generally of the chlorine atoms, of the products of general formula (B) by the amines Ar, then Ar2 of general formula (C) according to scheme 2:

Diagram 2
Generally, the operation is carried out with 1 mole of dihalo-s-triazine, or trihalo-s-triazine, and 1 mole of amine Ar1. It is preferred to operate in an inert solvent such as acetone, optionally aqueous or an alcohol optionally aqueous, such as ethanol, or a halogenated solvent, such as

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dichloromethane, or an ether such as diethyl ether or dioxane, or a polar aprotic solvent such as DMF, DMSO or NMP. According to a better way of carrying out the invention, the operation is carried out at a temperature of between 20 ° C. and 50 ° C. Then 1 mole of amine Ar2 is added to the product of general formula (D), which can optionally be isolated. The operation is carried out in particular at a temperature between 50 ° C. and reflux.

Advantageously, it is possible to operate under the conditions described in J. Fluor. Chem., 1988, 39 (1), 117-123.

General method 2
According to a second method, the products of general formula (A) in which Ar are defined as above and R represents a group NR1 R2 or OR1 or SR1 can also be prepared by nucleophilic displacement of a halogen atom, generally an atom of chlorine, of a product of general formula (A) in which R represents a halogen atom according to scheme 3:

R = CI (or F or Br or 1) R = NR1R2 or OR1 or SR1
Diagram 3
The operation is generally carried out by condensing 1 mole of product of general formula (A) in which R represents a halogen atom, preferably a chlorine atom, with 1 mole of amine R1 R2NH or alcoholate R1O- or thioalcoholate R1S. The reaction takes place in an inert medium under the reaction conditions. Mention may be made, among the inert solvents, of acetone, optionally aqueous, or an optionally aqueous alcohol such as ethanol, or a halogenated solvent such as dichloromethane, or an ether such as diethyl ether or dioxane, or an aprotic solvent.

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polar such as DMF, DMSO or NMP. When the entering group represents an R1 R2NH group, the operation is preferably carried out at a temperature of between 20 ° C. and reflux, in the presence in particular of an organic base, such as triethylamine, or inorganic base, such as sodium hydroxide or carbonate. sodium or potassium. It is also possible not to use a base during the amination reaction, and to isolate a hydrochloride of the product of general formula (A), the base of which can then be released.

When the entering group represents a group R10 'or R1S - the operation is preferably carried out with an alkaline or alkaline earth alcoholate or thioalcoholate, such as a sodium or potassium or lithium or ammonium or cesium or barium salt, in a polar aprotic solvent such as DMF or DMSO or NMP, at a temperature between 50 and reflux.

General method 3
According to a third preparation process, the compounds for which R represents a hydrogen atom or an alkyl group, straight or branched containing from 1 to 4 carbon atoms, can also be prepared by condensation of a bisguanide of general formula (E) , in which Ar, and Ar2 are identical or different, with an acid derivative, preferably an acid chloride or a methyl ester of general formula (F) according to scheme 4:

Diagram 4
The condensation between the bisguanide of general formula (E) and the acid derivative of general formula (F) is generally carried out in a

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alcohol such as methanol or ethanol. It is preferred to operate at a temperature between 0 ° C. and the reflux temperature.

The symmetric or asymmetric bisguanides of general formula (E) can be obtained by operating under the conditions described in the literature and in particular according to J. P. 94-4993.

General method 4
The products of general formula (A), in which Ar1 and Ar2 are identical, defined as above and represented by Ar, and where R represents an alkyl group, straight or branched containing from 1 to 4 carbon atoms, can also be prepared by condensation of a cyanoguanidine of general formula (G), in which Ar is defined as above, with a nitrile of general formula (H) according to scheme 4:

Diagram 5
The condensation of the cyanoguanidine of general formula (G) with the nitrile of general formula (H) is carried out in particular by operating at reflux of a polar solvent with a high boiling point such as 2-methoxyethanol or 1, 2-dimethoxyethane.

The cyanoguanidines of general formula (G) can be prepared under the conditions described in the literature.

It is understood that the s-triazines of general formula can be obtained in the form of libraries, by applying the methods described in schemes 1, 2, 3, 4 or 5 in parallel and / or combinatorial chemistry in liquid phase or in solid phase. , it being understood that, when working in the solid phase, any one of the reagents is fixed beforehand on a solid support, chosen according to the chemical reaction involved, and that said

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chemical reaction is followed by an operation of cleavage of the reaction product from the solid support.

The present invention also relates to therapeutic compositions containing a compound according to the invention, in association with a pharmaceutically acceptable carrier according to the chosen mode of administration. The pharmaceutical composition can be in solid, liquid or liposome form.

Among the solid compositions, mention may be made of powders, capsules and tablets. Among the oral forms one can also include the solid forms protected vis-à-vis the acidic medium of the stomach. The supports used for the solid forms consist in particular of mineral supports such as phosphates, carbonates or organic supports such as lactose, celluloses, starch or polymers. Liquid forms consist of solutions of suspensions or dispersions. They contain as dispersive support either water or an organic solvent (ethanol, NMP or others) or mixtures of surfactants and solvents or complexing agents and solvents.

The administered dose of the compounds of the invention will be adapted by the practitioner depending on the route of administration of the patient and the condition of the latter.

The compounds of the present invention can be administered alone or in admixture with other anticancer drugs. Among the possible combinations, we can cite # alkylating agents and in particular cyclophosphamide, melphalan, ifosfamide, chlorambucil, busulfan, thiotepa, prednimustine, carmustine, lomustine, semustine, steptozotocin, decarbazine, temozolomide, procarbazine and hexamethylmelamine # platinum derivatives such as in particular cisplatin, carboplatin or oxaliplatin # antibiotic agents such as in particular bleomycin, mitomycin, dactinomycin,

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# antimicrotubule agents such as vinblastine, vincristine, vindesine, vinoreibine, taxoids (paclitaxel and docetaxel) # anthracyclines such as doxorubicin, daunorubicin, idarubicin, epirubicin, losoxantrone # group @ and II topoisomerases such as etoposide, teniposide, amsacrine, irinotecan, topotecan and tomudex, # fluoropyrimidines such as 5-fluorouracil, UFT, floxuridine, # cytidine analogs such as 5-azacytidine, cytarabine, gemcitabine, 6-mercaptomurine, 6-thioguanine # adenosine analogs such as pentostatin, cytarabine or fludarabine phosphate # methotrexate and acid folinic acid # enzymes and various compounds such as
L-asparaginase, hydroxyurea, trans-retinoic acid, suramin, dexrazoxane, amifostine, herceptin as well as oetrogenic and androgenic hormones.

It is also possible to combine the compounds of the present invention with a radiation treatment. These treatments can be administered simultaneously, separately, sequentially. The treatment will be adapted to the patient to be treated by the practitioner.

The stabilization activity of the G-quadruplexes can be determined by a method using the formation of a complex with fluorescein, the experimental protocol of which is described below.

Oligonucleotides
All the olignucleotides, modified or not, were synthesized by Eurogentec SA, Seraing, Belgium. The FAM + DABCYL oligonucleotide bears the catalog reference, OL-0371-0802. It has the sequence:

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GGGTTAGGGTTAGGGTTAGGG corresponding to 3.5 repetitions of the human telomeric motif (strand rich in G). Fluorescein is attached to the 5 'end, DABCYL to the 3' end, by the chemical arms described by Eurogentec. The concentration of the samples is verified spectrophotometrically, recording the absorbance spectrum between 220 and 700 nm and using the molar extinction coefficient provided by the supplier.

Buffers
All the experiments were carried out in a 10 mM sodium cacodylate buffer, pH 7.6, containing 0.1 M of lithium chloride (or of sodium chloride). The absence of fluorescent contamination in the buffer was previously checked. The fluorescent oligonucleotide is added to the final concentration of 0.2 M.

Fluorescence study
All the fluorescence measurements were carried out on a Spex Fluorolog DM1 B apparatus, using an excitation linewidth of 1.8 nm and an emission linewidth of 4.5 nm. The samples are placed in a micro quartz cuvette of 0.2 x 1 cm. The temperature of the sample is controlled by an external water bath. The oligonucleotide alone was analyzed at 20.30, 40.50, 60.70 and 80 C. The emission spectra are recorded using an excitation wavelength of 470 nm. The excitation spectra are recorded using either 515 nm or 588 nm as the emission wavelength. The spectra are corrected for the response of the instrument by reference curves. A significant extinction (80-90%) of fluorescein fluorescence at room temperature is observed, in agreement with an intramolecular fold of the oligonucleotide at 20 C in the form of a G-quadruplex, which induces a juxtaposition of its 5 'and 3' ends, respectively linked to fluorescein and DABCYL.

This juxtaposition leads to a phenomenon already described of fluorescence quenching, used for the “Molecular Beacons”.

Tm in fluorescence:

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A stock solution of oligonucleotide at a strand concentration of 0.2 M in a 0.1 M buffer 10 mM LiCl cacodylate pH 7.6 is prepared beforehand, heated briefly to 90 ° C. and slowly cooled to 20 ° C., then distributed in aliquots of 600 l in the fluorescence cells.

3 l of water (for the control) or 3 l of the product to be tested (stock at 200 μM, final concentration 1 M) are then added and mixed. The samples are then left to incubate for at least 1 hour at 20 ° C. before each measurement. The use of longer incubation times (up to 24 hours) has no influence on the result obtained.

Each experiment only allows the measurement of a single sample.

This is first incubated at an initial temperature of 20 ° C., brought to 80 ° C. in 38 minutes, left for 5 minutes at 80 ° C., then cooled to 20 ° C. in 62 minutes.

During this time, fluorescence is measured simultaneously at two emission wavelengths (515 nm and 588 nm) using 470 nm as the excitation wavelength. A measurement is taken every 30 seconds. The temperature of the water bath is recorded in parallel, and the fluorescence profile as a function of temperature is reconstructed from these values. The fluorescence profiles are then normalized between 20 C and 80 C, and the temperature for which the emission intensity at 515 nm is the average of those at high and low temperature is called Tm.

Under these conditions, the Tm of the reference sample without addition of product is 44 ° C. in a lithium chloride buffer. This temperature is brought to more than 55 ° C. in a sodium chloride buffer. The addition of a compound stabilizing G-quadruplex induces an increase in Tm.

This increase is considered significant if it is greater than 3.

The biological antitelomerase activity is determined by the following experimental protocol:
Preparation of the extract enriched in human telomerase activity
The HL60 leukemia line is obtained from the ATCC (Americam Type Culture Collection, Rockville USA). The cells are cultured in suspension in RPMI 1640 medium containing, L-Glutamine at 2 mM, Penicillin 200 U / ml, streptomycin 200 g / ml, gentamycin 50 g / ml and supplemented with 10% of fetal calf serum inactivated by the heat.

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An aliquot of 105 cells is centrifuged at 3000xG and the supernatant discarded. The cell pellet is resuspended by several successive pipettings in 200 μl of lysis buffer containing 0.5% CHAPS, 10 mM Tris-HCl pH 7.5, 1 mM MgCl2, 1 mM EGTA, 5 mM ss-mercaptoethanol, PMSF 0 1 mM and 10% glycerol and kept in ice for 30 minutes. The lysate is centrifuged at 16 OOOOxG for 20 minutes at 4 ° C. and 160 l of the supernatant is recovered. The protein content of the extract is assayed by the Bradford method. The extract is stored at -80 C.

Determination of telomerase activity
Inhibition of telomerase activity is determined by a protocol for extending the oligonucleotide TS (5'AATCGTTCGAGCAGAGTT3 '), in the presence of a cell extract enriched in telomerase activity and compounds which are added at different concentrations (10 , 1, 0. 1 and 0.01 g / ml). The extension reaction is followed by PCR amplification of the extension products using the TS and CXext oligonucleotides (5'GTGCCCTTACCCTTACCCTTACCCTAA3 ').

The reaction medium is prepared according to the following composition:
Tris HCI pH 8.3 20 mM MgCl2 1.5 mM
Tween 20 0.005% (W / V)
EGTA 1 mM dATP 50 uM dGTP 50 uM dCTP 50 uM dTTP 50 uM
Oligonucleotide TS 2 g / ml
CXext Oligonucleotide 2 g / ml
Serum Bovine albumin 0.1 mg / ml
Taq DNA polymerase 1 U / ml alpha 32P dCTP (3000 Ci / mmol) 0.5 l

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Telomerase extract 200 ng in a volume of 10 l
Product to be tested or solvent in a volume of 5 l
QS bi-distilled water ................... 50 l
The oligonucleotides are obtained from Eurogentec (Belgium) and are stored at -20 C at a stock concentration of 1 mg / ml in distilled water.

The reaction samples are assembled in 0.2 ml PCR tubes and a drop of paraffin oil is placed on each of the reactions of the experiment before the tubes are closed.

The reaction samples are then incubated in a Cetus 4800 type PCR apparatus under the following temperature conditions:
15 minutes at 30 C,
1 minute at 90 C, followed by 30 cycles of,
30 seconds at 94 C,
30 seconds at 50 C, and 1 minute 30 seconds at 72 C, followed by a final cycle of 2 minutes at 72 C.

For each of the samples, a 10 L aliquot is pipetted under the oil layer and mixed with 5 L of a deposition buffer containing:
TBE 3X glycerol 32% (W / V)
Bromophenol blue 0.03%
Xylene cyanol 0.03%
The samples are then analyzed by 12% acrylamide gel electrophoresis in 1X TBE buffer for 1 hour at a voltage of 200 volts, using a Novex electrophoresis system.

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The acrylamide gels are then dried on a sheet of whatmann 3MM paper at 80 ° C. for 1 hour.

The analysis and quantification of the reaction products are carried out using an Instantlmager device (Pacard).

For each concentration of compound tested, the results are expressed as a percentage inhibition of the reaction and calculated from the untreated enzymatic control and the sample without enzyme (blank) according to the following formula: (Compound value - blank value / Enzymatic control value - blank value) x 100.

The concentration of compound inducing a 50% inhibition of the telomerase reaction (IC50) is determined using a semi-logarithmic graphical representation of the inhibition values obtained as a function of each of the concentrations of compound tested.

A compound is considered to be active as an antitelomerase agent when the amount inhibiting 50% of the telomerase reaction is in particular less than 5 m.

The cytotoxic biological activity on human tumor lines is determined according to the following experimental protocol:
The KB and A549 human cell lines originate from the ATCC (Americam Type Culture Collection, Rockville USA). The A549 cells are cultured in a layer in a culture flask in RPMI 1640 medium, 2 mM L-glutamine, 200 U / ml penicillin, 200 g / ml streptomycin and supplemented with 10% of heat-inactivated fetal calf serum. The KB cells are cultured in a layer in a culture flask in Dulbelco's medium containing, L-Glutamine at 2 mM, Penicillin 200 U / ml, streptomycin 200 g / ml and supplemented with 10% of heat-inactivated fetal calf serum. .

The cells in exponential growth phase are trypsinized, washed in 1X PBS and are seeded in 96-well microplates (Costar) at a rate of 4x104 cells / ml for A549 and 1.5x104 cells / ml (0.2 ml / well). then incubated for 96 hours in the presence of concentrations

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product variables to be studied (10.1, 0. 1 and 0.01 g / ml, each point in quadruplicate). 16 hours before the end of the incubation, final 0.02% of neutral red is added to each well. At the end of the incubation, the cells are washed with 1 × PBS and lysed with 1% sodium lauryl sulfate.

Cellular incorporation of the dye, which reflects cell growth, is assessed spectrophotometrically at a wavelength of 540 nm for each sample using a Dynatech MR5000 reader.

For each concentration of compound tested, the results are expressed as a percentage of inhibition of cell growth and calculated from the untreated control and the culture medium without cells (blank) according to the following formula: (Compound value - blank value / Value cell control - blank value) x 100.

The concentration of compound inducing a 50% inhibition of growth (IC50) is determined using a semi-logarithmic graphical representation of the inhibition values obtained as a function of each of the concentrations of compound tested.

A compound is considered to be active as a cytotoxic agent if the 50% inhibitory concentration of the growth of the tumor cells tested is in particular less than 10 M.

The following non-limiting examples are given to illustrate the invention.

Préparation du dichlorure de 2-amino-bis-4.6-[1'-métharl-4'-amino-6'- quinaldinio) amino]-triazine
Dans un tricol de 2 dm3, on introduit 200 cm3 d'eau distillée et on charge sous agitation 41,6 g (0,16 mol) de chlorhydrate du chlorure de 1-méthyl-4,6-diaminoquinaldinium, qui peut-être obtenu selon J. Chem. Soc., 1953,50. On obtient une solution limpide jaune foncée où l'on coule 800 cm3 d'éthanol, provoquant un abondant précipité. On porte à 45 C pour dissoudre, puis on ajoute 13,2 g (0,08 mol) de 2-amino-4,6-dichloro-triazine, qui peut être préparé selon J. Amer. Chem. Soc., 1945,67, 662. Après Example 1

Preparation of 2-amino-bis-4.6- [1'-metharl-4'-amino-6'- quinaldinio) amino] -triazine dichloride
200 cm3 of distilled water are introduced into a three-necked flask of 2 dm3 and 41.6 g (0.16 mol) of 1-methyl-4,6-diaminoquinaldinium chloride hydrochloride are charged with stirring, which can be obtained. according to J. Chem. Soc., 1953.50. A clear dark yellow solution is obtained into which 800 cm3 of ethanol are poured, causing an abundant precipitate. The mixture is brought to 45 ° C. to dissolve, then 13.2 g (0.08 mol) of 2-amino-4,6-dichloro-triazine, which can be prepared according to J. Amer. Chem. Soc., 1945,67, 662. After

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a few minutes, a yellow precipitate appears, and the mixture is refluxed for 1 hour.

Cool and leave overnight in a cooler. The precipitate obtained is filtered off and washed with four times 100 cm3 of 80% aqueous ethanol and then dried at 45 ° C.

47 g (100%) of 2-amino-bis-4,6 - [(1'-methyl-4'-amino-6'-quinaldinio) amino] -triazine dichloride monohydrochloride are thus obtained.

Liberation and purification of the base form
In a three-necked flask of 2 dm3, 1.2 dm3 of distilled water are added then 47 g of the monohydrochloride obtained above, the mixture is brought to 55 C and 30 cm3 of concentrated ammonia (d = 0.925) is poured in, then the mixture is heated at 85 C to promote solubilization. An insoluble material is filtered hot through 40 g of Supercel and washed with three times 50 cm3 of boiling water. After concentrating the filtrate halfway, and again filtering through 10 g of supercel, 1.2 dm3 of ethanol are added and the mixture is stirred for 5 minutes, then left to stand overnight in a cooler. In the morning, filtered, washed with three times 50 cm3 of 66% ethanol and twice with 50 cm3 of ethanol, dried under vacuum at 45 C, and 34.2 g (79%) are obtained. of crude 2-amino-bis-4,6 - [(1'-methyl-4'-amino-6'-quinaldinio) amino]-triazine dichloride. 600 cm3 of distilled water and 34.2 g of crude base are introduced into a three-necked flask of 2 dm3, with stirring the mixture is brought to 50 ° C. until almost complete dissolution, and the insoluble material is filtered off. The resulting filtrate is loaded into a 3 dm3 three-necked flask, with stirring rapidly pouring 1.4 dm3 of ethanol. The gelatinous whitish precipitate obtained is filtered, washed with three times 100 cm3 of ethanol, dried under vacuum at 45 ° C., and 30.7 g (71%) of 2-amino-bis-4.6 dichloride are obtained. - [(1'-methyl-4'-amino-6'-quinaldinio) amino] -triazine, in the form of a white solid, the characteristics of which are as follows: - melting point = 334 C.

- 1 H NMR spectrum (300 MHz, (CD3) 2S0 d6, d in ppm): 2.72 (s: 6H); 4.01 (s: 6H); (s: 2H); (mf: 2H); 8.11 (d, J = 10Hz: 2H); 8.21 (dd, J = 10 and 2Hz: 8.40-8.75 (broad mf: 4H); 9.01 (broad s: 2H); 9.83 (mf: 2H).

Example 2
Preparation of 2-amino-bis-4,6 - [(1'-ethyl-4'-amino-6'-quinaldinio) amino] -triazine dichloride

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75 cm3 of distilled water are introduced into a three-necked flask of 1 dm3 and 16.45 g (0.06 mol) of 1-ethyl-4,6-diamino-quinaldinium chloride hydrochloride are charged with stirring, which can be obtained according to the US patent

2,585,905, then 300 cm3 of ethanol are poured in. The mixture is brought to 55 ° C., then 4.95 g (0.03 mol) 2-amino-4,6-dichloro-triazine are added and the mixture is refluxed for 2 and a half hours. Cool and leave overnight in a cooler. The precipitate obtained is filtered off, washed with 100 cm3 of 80% ethanol and then dried at 45 ° C. 16.47 g (91%) of 2-amino-bis-4,6 - [(( 1'-ethyl-4'-amino-6'-quinaldinio) amino] -triazine.

Liberation and purification of the base form
In a 250 cm3 flask containing 200 cm3 of distilled water, with stirring, the 16.47 g of monohydrochloride obtained above are charged and 8 cm3 of concentrated ammonia (d = 0.925) are poured, the mixture is brought to reflux to promote solubilization . A light insoluble material is filtered hot on Supercel and washed with twice 10 cm3 of boiling water. After concentrating the filtrate to half, 350 cm3 of ethanol are added with stirring, causing an abundant white precipitate, left overnight in a cooler. The precipitate is filtered off, washed with five times 10 cm3 of 80% ethanol, dried under vacuum at 55 ° C., and 12.87 g (76%) of 2-amino-bis-4.6 dichloride are obtained. - [(1'-ethyl-4'-amino-6'-quinaldinio) amino] -triazine, in the form of a hygroscopic white powder with the following characteristics: - melting point = 302 C - 1 H NMR spectrum (300 MHz, (CD3) 2 SO d6, d in ppm): 1.42 (t, J = 7 Hz: 6H); 2.74 (s: 6H); 4.57 (q, J = 7Hz: 6.80 (s: 2H); 7.09 (mf: 2H); 8.13 (d, J = 10Hz: 2H); 8.21 (dd, J = 10 and 2 Hz: 8.40-8.75 (broad mf: 4H); 9.01 (broad s: 2H); 9.83 (mf: 2H).

Préparation du dichlorure de 2-diméth5ilamino-bis-4.6-[(1'-méthll-4'- amino-6'-quinaldinio) amino]-triazine
Dans un tricol de 1 dm3, on introduit 60 cm3 d'eau distillée et on charge sous agitation 13,01 g (0,05 mol) du chlorhydrate du chlorure de 1-méthyl-4,6-diamino-quinaldinium, qui peut être obtenu selon J. Chem. Example 3

Preparation of 2-dimeth5ilamino-bis-4.6 - [(1'-methll-4'-amino-6'-quinaldinio) amino] -triazine dichloride
60 cm3 of distilled water are introduced into a three-necked flask of 1 dm3 and 13.01 g (0.05 mol) of 1-methyl-4,6-diamino-quinaldinium chloride hydrochloride are charged with stirring, which can be obtained according to J. Chem.

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Soc., 1953, 50. A yellow solution is obtained into which 240 cm3 of ethanol are poured, causing an abundant yellow precipitate. After dissolution by heating at 50 ° C., 4.83 g (0.025 mol) of 2-dimethylamino-4,6-dichloro-triazine are added, which can be prepared according to J. Amer. Chem. Soc., 1948,70, 3726. After a few minutes, a precipitate appears and the mixture is refluxed for 1 hour and a half.

The mixture is cooled for 1 hour in an ice bath, then the precipitate obtained is filtered, which is washed with four times 30 cm3 of ethanol and dried. 12.92 g (86%) of 2-dimethylamino-bis-4,6 - [(1'-methyl-4'-amino-6'-quinaldinio) amino] -triazine dichloride are obtained in the form of a hygroscopic cream powder, the characteristics of which are as follows: - melting point = 345 C - 1 H NMR spectrum (300 MHz, (CD3) 2S0 d6, d in ppm): 2.72 (s: 6H); 3.18 (s: 6H); (s: 6H); (s: 2H); (d, J = 9.5Hz: 2H); 8.22 (mt: 2H); 8.40-8.65 (broad mf: 4H); 8.79 (broad s: 2H); 9.83 (mf: 2H).

Example 4
Preparation of 2-Methylamino-bis-4.6 - [(4'-amino-6'-quinaldinyl) amino] -triazine trihydrochloride
34 cm3 of distilled water and 126 cm3 of normal hydrochloric acid are introduced into a three-necked flask of 2 dm3, and 21.82 g (0.1 mol) of 4,6-diaminoquinaldine, which can be obtained, are charged with stirring. according to J. Chem. Soc., 1953, 50. To the orange solution obtained, 600 cm3 of ethanol are poured, brought to 65 ° C., then 10.74 g (0.06 mol) of 2-methylamino-4,6-dichloro are added. - triazine, which can be prepared according to Chem. Berichte, 1899, 32, 700, and 40 cm3 of ethanol are poured. A yellow precipitate appears which thickens rapidly on heating at reflux for 2 hours. After cooling overnight in a cooler, the precipitate obtained is filtered, washed with three times 120 cm3 of 80% ethanol, dried and 27.3 g (81%) of 2-methylamino-bis-4,6 trihydrochloride are obtained. - [(4'-amino-6'-quinaldinyl) amino] -triazine, in the form of a hygroscopic white powder, the characteristics of which are as follows: - melting point = 340 C - NMR spectrum. 1H (300 MHz, (CD3) 2S0 d6, d in ppm): (broad s: 6H); 2.94 (br s: 3H); 6.64 (br s: 2H); from 7.60 to 7.80 (mf

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spread: 1H); 7.89 (broad d, J = 9.5Hz: 2H); 8.10 (broad d, J = 9.5 Hz: from 8.35 to 8.65 (mf spread: 4H); from 8.70 to 8.95 (mf spread: 2H); from 9.80 to 10 0.00 (broad mf: 2H); 13.76 (mf: 2H).

Example 5
Preparation of 2-Amino-bis-4,6- [1'-methyl-6'-quinolinio) amino] -triazine dichloride
225 cm3 of distilled water are introduced into a three-necked flask of 2 dm3 and 41.5 g (0.18 mol) of 1-methyl-6-aminoquinolinium chloride hydrochloride are charged with stirring, which can be obtained according to Zh. Org.Khim. ; 1993,29 (10), 2018.

A yellow solution is obtained into which 900 cm3 of ethanol are poured, causing an abundant precipitate. After dissolution by heating at 50 ° C., 14.8 g (0.09 mol) of 2-amino-4,6-dichloro-triazine are added, and the mixture is refluxed for 1 hour, a yellow precipitate rapidly appears. The mixture is cooled overnight in the cooler, then the precipitate obtained is filtered, which is washed with twice 50 cm3 of ethanol, dried and 36.6 g (79%) of 2-amino-bis-4 dichloride monohydrochloride are obtained. , 6 - [(1'-methyl-6'-quinolinio) amino] -triazine.

Liberation and purification of the base form
In a three-necked flask of 1 dm3, 200 cm3 of distilled water are added, then the 36.6 g of monohydrochloride obtained above are charged with stirring, the mixture is brought to 80 ° C. and 10 cm3 of concentrated ammonia are poured (d = 0.925) , and an insoluble material is filtered through Supercel. In a three-necked flask of 6 dm3, containing 3 dm3 of ethanol, the previous filtrate is poured with stirring over 5 minutes, a fine bright yellow precipitate appears, which is left for 2 days in a cooler, then filtered, washed with twice 50 cm3 d 'ethanol, dried and 19.1 g (44%) of 2-amino-bis-4,6 - [(1'-methyl-6'-quinolinio) amino] -triazine dichloride are obtained as a hygroscopic yellow powder, the characteristics of which are as follows: - melting point = 296 C - 1 H NMR spectrum (300 MHz, (CD3) 2S0 d6, d in ppm): 4.64 (s: 6H); 7.11 (mf: 2H); 8.10 (dd, J = 8.5 & 6Hz: 2H); 8.46 (d, J = 10 Hz:

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2H); 8.56 (dd, J = 10 and 2Hz: 9.17 (broad d, J = 8.5Hz: 2H); 9.30 (mt: 4H); 10.26 (mf: 2H).

4.6-[(4'-méthvlamino-6'-quinaldinynamino1-triazine
Etape A : Préparation de la 4-méthylamino-6-amino-quinaldine
Dans un tricol de 2 dm3, on introduit 240 cm3 d'acide acétique et on charge sous agitation 57,4 g (0,25 mol) de 6-acétamido-4-méthoxyquinaldine préparée selon J. Amer. Chem. Soc., 1948,70, 4065. On fait barbotter sous agitation de la méthylamine jusqu'à saturation, puis on porte à reflux 2 heures. On refroidit, et fait de nouveau l'opération précédente, on refroidit, et on coule sous agitation la solution obtenue dans un tricol de 2 dm3, contenant 300 cm3 d'eau distillée et 470 cm3 d'acide chlorhydrique normal. On chauffe alors à 100 C pendant 11 heures puis on laisse une nuit en glacière. Le produit cristallisé est filtré, donnant 25 g de chlorhydrate, les liqueurs mères sont concentrées, laissées une nuit en glacière puis filtrées pour donner de nouveau 150 g de chlorhydrate. Les 175 g de chlorhydrate sont repris par 300 cm3 d'eau distillée et dissous par chauffage à 50 C, traités par 1 g de noir et filtrés sur Supercel. On porte le filtrat à 90 C et on alcalinise par addition de 54 cm3 de soude concentrée. Le précipité obtenu par refroidissement la nuit en glacière est lavé par quatre fois 100 cm3 d'eau distillée, séché et on obtient 33 g (70 %) du 4-méthylamino-6-aminoquinaldine
Etape B
Dans un tricol de 2 dm3, on introduit 25 cm3 d'eau distillée et 101 cm3 d'acide chlorhydrique normal, et on charge 18.9 g (0,101 mol) de 4-méthylamino-6-amino quinaldine, la solution orangée obtenue est portée à 75 C, puis on coule en 2 minutes 500 cm3d'éthanol, et on ajoute d'un coup les 8,59 g (0,048 mol) 2-méthylamino-4,6-dichloro-triazine, préparée selon l'exemple 4. Après 5 minutes, un précipité apparaît et on porte à reflux 2 heures, on laisse refroidir sous agitation 3 heures et abandonner 8 jours en glacière. Le précipité obtenu est filtré, lavé par deux fois 100 cm3 d'éthanol à Example 6
Preparation of 2-methylamino-bis- dichloride trihydrochloride

4.6 - [(4'-methvlamino-6'-quinaldinynamino1-triazine
Step A: Preparation of 4-methylamino-6-amino-quinaldine
240 cm3 of acetic acid are introduced into a three-necked flask of 2 dm3 and 57.4 g (0.25 mol) of 6-acetamido-4-methoxyquinaldine prepared according to J. Amer are charged with stirring. Chem. Soc., 1948,70, 4065. Methylamine is bubbled with stirring until saturation, then refluxed for 2 hours. Cooled, and repeat the previous operation, cooled, and the solution obtained is poured with stirring into a three-necked flask of 2 dm3, containing 300 cm3 of distilled water and 470 cm3 of normal hydrochloric acid. Then heated at 100 C for 11 hours and then left overnight in a cooler. The crystallized product is filtered, giving 25 g of hydrochloride, the mother liquors are concentrated, left overnight in a cooler and then filtered to again give 150 g of hydrochloride. The 175 g of hydrochloride are taken up in 300 cm3 of distilled water and dissolved by heating at 50 ° C., treated with 1 g of charcoal and filtered through Supercel. The filtrate is brought to 90 ° C. and basified by adding 54 cm3 of concentrated sodium hydroxide. The precipitate obtained by cooling overnight in a cooler is washed with four times 100 cm3 of distilled water, dried and 33 g (70%) of 4-methylamino-6-aminoquinaldine are obtained.
Step B
25 cm3 of distilled water and 101 cm3 of normal hydrochloric acid are introduced into a three-necked flask of 2 dm3, and 18.9 g (0.101 mol) of 4-methylamino-6-amino quinaldine are charged, the orange solution obtained is brought to 75 C, then poured in 500 cm3 of ethanol in 2 minutes, and the 8.59 g (0.048 mol) 2-methylamino-4,6-dichloro-triazine, prepared according to Example 4, are added all at once. 5 minutes, a precipitate appears and the mixture is refluxed for 2 hours, the mixture is left to cool with stirring for 3 hours and leave for 8 days in an icebox. The precipitate obtained is filtered off, washed with twice 100 cm3 of ethanol at

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80% and three times 100 cm3 of ethanol, dried to give 21.7 g (77%) of the trihydrochloride of 2-methylamino-bis-4,6 - [(4'-methylamino-6'-quinaldinyl) amino] -triazine, in the form of a hygroscopic cream powder, the characteristics of which are as follows: - melting point = 355 C - NMR spectrum. 1H (300 MHz, (CD3) 2S0 d6, d in ppm): 2.68 (s: 6H); 2.93 (s: 3H); 3.04 (mf: 6H); 6.59 (s: 2H); 7.40-7.70 (broad mf: 1H); 7.88 (d, J = 9Hz: 2H); 8.08 (broad d, J = 9Hz: 8.50-8.95 (mf spread: 2H); 8.75-8.95 (mf: 2H); 9.70-10.10 (mf broad: 2H); 13.79 (mf: 2H).

amino-10'-méthyl-2'-acridiniolamino]'-triazine
Etape A : Préparation de la 2-acétamido-9-amino-acridine
Dans un tricol de 250 cm3, on introduit 72 cm3 d'acide acétique et on charge sous agitation 12 g (0,0575 mol) de 2,9-diaminoacridine, préparée selon J. Chem. Soc., 1949,1148, puis on coule 4,1 cm3 (0,0575 mol) de chlorure d'acétyle. La température monte à 50 C et la solution se prend en masse. On maintient à 60 C pendant 1 heure, on refroidit et on dilue par 200 cm3 d'oxyde de diéthyle. Par filtration, on obtient 15,2 g de chlorhydrate. Example 7
Preparation of 2-amino-bis-4.6-f (9'-

amino-10'-methyl-2'-acridiniolamino] '- triazine
Step A: Preparation of 2-acetamido-9-amino-acridine
72 cm3 of acetic acid are introduced into a 250 cm3 three-necked flask and 12 g (0.0575 mol) of 2,9-diaminoacridine, prepared according to J. Chem. Soc., 1949, 1148, then poured 4.1 cm3 (0.0575 mol) of acetyl chloride. The temperature rises to 50 C and the solution solidifies. Maintained at 60 C for 1 hour, cooled and diluted with 200 cm3 of diethyl ether. By filtration, 15.2 g of hydrochloride are obtained.

2 dm3 of distilled water are introduced into a 4 dm3 flask and the 15.2 g of hydrochloride are charged with stirring. It is brought to reflux, filtered and basified with ammonia. The base crystallizes, is filtered and dried. 11.2 g (78%) of 2-acetamido-9-amino-acridine are obtained.

StepB: Preparation of 2-acetamido-9-amino-10-methyl-acridinium sulfate
300 cm3 of nitrobenzene are introduced into a three-necked flask of 1 dm3 and then successively with stirring 11.2 g (0.0446 mol) of 2-acetamido-9-amino acridine and 11 cm3 (0.116 mol) of dimethyl sulfate. It is then heated to 140 ° C. for 20 minutes. After cooling, the precipitate obtained is filtered off, which is washed with 20 cm3 of nitrobenzene and six times 20 cm3 of sodium oxide.

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diethyl, dried in air and 14.35 g (85%) of 2-acetamido-9-amino-10-methyl-acridinium sulfate are obtained.

Step C: Preparation of 2-acetamido-9-amino-1 0-methyl-acridinium chloride hydrochloride.

600 cm3 of distilled water are introduced into a 2 dm3 flask and 13 g of 2-acetamido-9-amino-10-methylacridinium sulfate (0.0344 mol) are charged with stirring, the mixture is brought to reflux until almost complete dissolution and the insoluble material is filtered. After cooling, 900 cm3 of an aqueous 35% sodium chloride are poured in, the mixture is left to precipitate and filtered. 20 cm3 of concentrated hydrochloric acid (d = 1.18) are introduced into a 100 cm3 flask and the preceding compound is charged, which is brought to reflux for 5 minutes, diluted with 67 cm3 of ethanol and left crystallize in ice. The crystals formed are filtered off, washed with twice 5 cm3 of ethanol and with three times 10 cm3 of diethyl ether, dried and 5.1 g (50%) of 2-acetamido-9-amino chloride hydrochloride are obtained. -10-methyl-acridinium.

Step D
5 cm3 of distilled water and 37 cm3 of ethanol are introduced into a 100 cm3 three-necked flask and 1 g (0.00338 mol) of 2-acetamido-9-amino-10-methyl chloride hydrochloride is charged with stirring. -acridinium, the mixture is brought to 80 ° C. and 0.28 g (0.0017 mol) of 2-amino-4,6-dichloro-triazine is added. After 5 minutes, a precipitate appears and the mixture is refluxed for 2 hours, then allowed to cool, filtered, washed with three times 3 cm3 of ethanol and with 5 cm3 of ether, dried under vacuum to give 0, 8 g (73%) of 2-amino-bis-4,6 - [(9'-amino-10'-methyl-2'-acridinio) amino] -triazine dichloride hydrochloride, as an ocher powder hygroscopic yellow with the following characteristic: - melting point = 310 C Example 8
Preparation of 2-amino-bis-4.6 - [(4'-amino-6'- quinaldinyl) amino] -triazine trihydrochloride (the corresponding base is also called SURFENE C)

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5.25 dm3 of distilled water are introduced into a three-necked flask of 10 dm3 and 368.4 g (2.127 mol) of 4,6-diaminoquinaldine, prepared according to Example 4, are added with stirring, then 117 g ( 0.709 mol) 2-amino-4,6dichloro-triazine, prepared according to Example 1. A yellow precipitate forms immediately, the mixture is refluxed for 2 hours. After cooling and acidification to pH = 3.4 by adding 1.3 dm3 of normal hydrochloric acid, the mixture is refluxed for 15 minutes, 20 g of charcoal is added and the reflux is maintained for 10 minutes, then filtered. The previous solution is introduced into a 10 dm3 three-necked flask and brought to 80 ° C., then 852 cm3 of hydrochloric acid (d = 1.19) are poured in 30 minutes. At the end of the casting, a pale yellow precipitate appears which, after cooling in an ice bath, is filtered, washed four times with 250 cm3 of a hydrochloric acid solution, composed of 250 cm3 of hydrochloric acid (d = 1, 19) and 2 dm3 of distilled water, then dried under vacuum at 60 C to give 385.4 g (99%) trihydrochloride. In a three-necked flask of 4 dm3, 1.9 dm3 of ethanol are introduced and the previous 385.4 g are charged with stirring for 5 hours at room temperature in the dark, filtered and then washed twice with 250 cm3 d ethanol and dry; 346 g (89%) of 2-amino-bis-4,6 - [(4'-amino-6'quinaldinyl) amino] -triazine trihydrochloride are obtained in the form of a hygroscopic cream powder, the characteristic of which is following: - melting point = 345 C Example 9
2-Amino-bis-4.6- (p-amidinoanilino) -triazine hydrochloride
This product can be obtained according to J. Chem. Soc., 1960,4525 in the form of a hygroscopic cream powder.

Example 10
Preparation of 2-methylthio-bis-4,6 - [(1'-methyl-4'-amino-6'-quinaldinio) amino] -triazine dichloride
60 cm3 of distilled water are introduced into a three-necked flask of 1 dm3 and 13.01 g (0.05 mol) of 1-methyl-4,6-diaminoquinaldinium chloride hydrochloride are charged with stirring, which can be obtained according to J. Chem. Soc., 1953, 50. A yellow solution is obtained into which 240 cm3 is poured

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of ethanol, causing an abundant yellow precipitate. After dissolution by heating at 50 ° C., 4.90 g (0.025 mol) of 2-methylthio-4,6-dichlorotriazine are added, which can be prepared according to Ang. Chem. Int Ed., 1966.5, 960.

diméthvlamino-6'-quinaldinynamino"l-triazine
Etape A : Préparation de la 4-diméthylamino-6-amino-quinaldine
On opère comme à l'étape A de l'exemple 6, mais à partir de 57,4 g (0,25 mol) de 6-acétamido-4-méthoxy-quinaldine, préparée selon J. Amer. Chem. Soc., 1948,70, 4065, et de 100 cm3 d'une solution aqueuse à 40 % de diméthylamine dans 250 cm3 d'acide acétique chauffés à 100 C pendant 2 heures dans un autoclave de 1 dm3. On obtient alors, après purification acide-base en opérant comme à l'étape A de l'exemple 6, 38,71 g (77 %) de 4-diméthylamino-6-amino-quinaldine. After a few minutes, a precipitate appears and the mixture is refluxed for an hour and a half. The mixture is cooled overnight in an icebox, then the precipitate obtained is filtered, which is washed with four times 30 cm3 of ethanol and dried. 10.70 g (75%) of 2-methylthio-bis-4,6 - [(1'-methyl-4'-amino-6'quinaldinio) amino] -triazine dichloride are obtained in the form of a powder. hygroscopic cream, the characteristics of which are as follows: - melting point = 320 C Example 11
Preparation of 2-chloro-bis-4,6 - [(4'-

dimethvlamino-6'-quinaldinynamino "l-triazine
Step A: Preparation of 4-dimethylamino-6-amino-quinaldine
The procedure is as in step A of Example 6, but starting from 57.4 g (0.25 mol) of 6-acetamido-4-methoxy-quinaldine, prepared according to J. Amer. Chem. Soc., 1948,70, 4065, and 100 cm3 of a 40% aqueous solution of dimethylamine in 250 cm3 of acetic acid heated at 100 C for 2 hours in a 1 dm3 autoclave. Then, after acid-base purification by operating as in step A of Example 6, 38.71 g (77%) of 4-dimethylamino-6-amino-quinaldine are obtained.

Step B
In a 25 cm3 three-necked flask, 402 mg (2 mmol) of 4-dimethylamino-6-amino-quinaldine are dissolved in 7 cm3 of acetic acid, then 184 mg (1 mmol) of cyanuryl chloride are added over 2 minutes, then it is heated at 90 ° C. for 3 hours. After cooling, the crystals formed are drained, washed with 2.5 cm3 of acetic acid and dried under vacuum at 100 C. This gives 588 mg (94%) of 2-chloro-bis-4,6- dihydrochloride dihydrate. [(4'-dimethylamino-6'-quinaldinyl) amino] -triazine, in the form of pale yellow crystals, the characteristics of which are as follows: - melting point = 350 C

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- elemental analysis:% C = 51.75 (shim = 52.06); H = 5.67 (wedge = 5.50); N = 19.98 (shim = 20.23) ~ Example 12
Preparation of 2-methylthio-bis-4,6 - [(4'-dimethylamino- hydrate
6'-quinaldinyl) amino] -triazine
Step A: Preparation of 4-dimethylamino-6-amino-quinaldine
The procedure is as in step A of Example 6, but starting from 57.4 g (0.25 mol) of 6-acetamido-4-methoxy-quinaldine, prepared according to J. Amer.

Chem. Soc., 1948,70, 4065, and 100 cm3 of a 40% aqueous solution of dimethylamine in 250 cm3 of acetic acid heated at 100 C for 2 hours in a 1 dm3 autoclave. 6.38.71 g (77%) of 4-dimethylamino-6-amino-quinaldine are then obtained after acid-base purification by operating as in step A of Example.

Step B
100 cm3 of 90% aqueous ethanol are introduced into a 250 cm3 three-necked flask and 4.02 g (0.02 mol) of 4-dimethylamino-6-amino-quinaldine and 1.96 g (0 , 01 mol) of 2-methylthio-4,6-dichloro-triazine, which can be prepared according to Ang. Chem. Int Ed., 1966.5, 960. After a few minutes, a precipitate appears and the mixture is refluxed for 1 hour and a half.

The mixture is cooled overnight in an icebox, then the precipitate obtained is filtered, which is washed with four times 30 cm3 of ethanol and dried. 5.26 g (88%) of crude hydrochloride are obtained.

75 cm3 of 90% aqueous ethanol are added to a 250 cm3 three-necked flask, then the 5.26 g of hydrochloride obtained above are charged with stirring, the mixture is brought to 80 ° C. and 5 cm3 of concentrated ammonia (d = 0.925), and left to crystallize for 2 days in a cooler. It is filtered, washed with twice 5 cm3 of 90% aqueous ethanol, dried and 3.20 g (59.5%) of 2-methylthio-bis-4,6- hydrate are obtained [ (4'-dimethylamino-6'-quinaldinyl) amino] -triazine, in the form of a pale yellow solid, the characteristics of which are as follows: - melting point = 280-3 C

<Desc / Clms Page number 31>

- elemental analysis:% C = 62.06 (shim = 62.48); % H = 5.81 (wedge = 62.48); N = 23.80 (wedge = 23.42).

Example 13
Preparation of N, N '- (4-amino-6-quinaldinyl) urea dihydrochloride (the corresponding base, also called SURFENE can be prepared according to Ang. Chem 1939, 891)
400 cm3 of distilled water are introduced into a three-necked flask of 500 cm3 and 30 g (0.2 mol) of 4,6-diaminoquinaldine are charged with stirring, which can be prepared according to J. Chem. Soc., 1953, 50, then 8.4 g (0.06 mol) of sodium acetate trihydrate are added and the mixture is heated to 95-96 C. A stream of phosgene is then passed until saturation (5 to 10 minutes), then maintained at 95-96 C for 1 hour. After cooling and acidification by adding 200 cm3 of 6N hydrochloric acid, the precipitate formed is filtered off, washed with 200 cm3 of N hydrochloric acid and dried under vacuum at 60 ° C., in this way 30 g of crude hydrochloride is obtained, which is recrystallized from 300 cm3 of distilled water and 30 cm3 of concentrated hydrochloric acid in the presence of black. After cooling, the crystals formed are drained, washed with N hydrochloric acid, then with acetone and dried under vacuum at 40 C. In this way 27 g (60.5%) of N, N ′ - dihydrochloride are obtained ( 4-amino-6-quinaldinyl) urea, in the form of a hygroscopic cream powder, the characteristic of which is as follows: - melting point = 329-40 C Example 14
Preparation of N1, N5-bis (7-chloro-1-methyl-4-quinolinio) pentane-1.5-diamine diodide
3 g (7 mmol) of N ', N5-bis (7-chloroquinolin-4-yl) pentan-1,5-diamine, which can be prepared according to J. Med. Chem. 1992, 35, 2129, in 60 cc of butan-2-one. 3 g (21 mmol) of methyl iodide are added and the mixture is refluxed for 5 hours. The crystals formed are filtered off, washed with butan-2-one and then with diethyl ether and dried under vacuum. This gives 3 g (60%) of N1, N5- diodide.

<Desc / Clms Page number 32>

bis (7-chloro-1-methyl-4-quinolinio) pentan-1,5-diamine, in the form of beige crystals, the characteristic of which is as follows: - melting point = 277-78 C.

Example 15
Preparation of bis-2.4 - [(4'-amino-6'-quinaldinyl) amino] pyrimidine trihydrochloride pentahydrate
1.73 g (10 mmol) of 4,6-diamino-1-methyl-quinoline, which can be obtained according to J. Chem. Soc., 1953,50, and 0.74 g of 2,4-dichloropyrimidine in 75 cm3 of ethanol and 5 cm3 of water for 5 hours. The reaction medium is concentrated by half, then left to crystallize in a cooler overnight. The precipitate formed is filtered off, washed with ethanol and then with diethyl ether and dried under vacuum. The precipitate obtained is stirred for 6 hours in 10 cm3 of 0.2N hydrochloric acid, then drained, washed with water and dried under vacuum at 80 C. This gives 0.98 g (46.5%) of trihydrochloride. bis-2,4 - [(4'-amino-6'- quinaldinyl) amino] pyrimidine pentahydrate, in the form of a yellow solid with the following characteristics: - melting point = 310-20 C - elemental analysis :% C = 47.37 (calc = 47.46); H = 5.51 (calc = 5.67); N = 18.41 (calc = 18.45); % CI = 15.84 (calc = 15.76) Example 16
Preparation of 1.5- (4'-amino-6'- quinaldinyl) biguanide trihydrochloride dihydrate
In a 25 cm3 three-necked flask, 5 cm3 of water, 3 cm3 of 5N hydrochloric acid, 1.3 g (7.5 mmoles) of 4,6-diamino-quinaldine are added, which can be obtained according to J. Chem. . Soc., 1953.50, and 0.34 g (3.75 mmol) of sodium dicyanamide and brought to 50-55 C overnight. After cooling, the precipitate formed is filtered off, washed with ice-cold water and dried under vacuum at 70 ° C. In this way 0.47 g (22.5%) of 1,5- (4'-amino- trihydrochloride dihydrate is obtained. 6'-quinaldinyl) biguanide, in the form of yellow crystals with the following characteristics:

<Desc / Clms Page number 33>

- melting point = 262-66 C - elemental analysis:% C = 47.85 (shim = 47.56); H = 5.67 (wedge = 5.38); N = 22.96 (wedge = 22.69); % CI = 18.79 (wedge = 19.14).

Example 17
The G-quartet, antitelomerase and cytotoxic activities of the various exemplified compounds are determined according to the operating protocols described above.

<tb>
<tb>

EXAMPLE <SEP> FLUORESCENCE <SEP> TRAP <SEP> Cytotox. <SEP> A549
<tb> Tm <SEP> (C) <SEP> IC50 (M) <SEP> IC50 <SEP>(<SEP> M)
<tb> 1- <SEP> 0.25 <SEP> 0.59 / 1.9 *
<tb> 2 <SEP> 48 <SEP> 0.056 <SEP> 4.7
<tb> 3 <SEP> 52 <SEP> 0.22-
<tb> 4 <SEP> 48 <SEP> 0.51 <SEP> 3.1
<tb> 5 <SEP> 57 <SEP> 0.13 <SEP> 0.56 / 1.8 *
<tb> 6 <SEP> 44 <SEP> 0.3 <SEP> 1.9
<tb> 7 <SEP> 55 <SEP> 0.89 <SEP> 4.9
<tb> 8- <SEP> 0.051 <SEP> 9.1
<tb> 9- <SEP> 0.74 <SEP> 0.53
<tb> 10- <SEP> 0.24 <SEP> 3.6
<tb> 11 <SEP> 57 <SEP> 3 <SEP> 5.14
<tb> 12 <SEP> 70 <SEP> 0.041 <SEP> 0.44 / 1.1
<tb> 13- <SEP> 0.72-
<tb> 14- <SEP> 1.4 / 4.9 * <SEP> 6.5
<tb> 15 <SEP> 53 <SEP> 0.49 <SEP> 8.7
<tb> 16 <SEP> 52 <SEP> 2 / 4.5 * <SEP> 5.8
<tb> * <SEP>: <SEP> results <SEP> of <SEP> two independent <SEP><SEP> experiments
<tb>

Claims (19)

1- Compounds fixing the G-quadruplex structure of telomeres characterized in that they correspond to the following general formula: nitrogenous aromatic ring - NH - distributor - NH - nitrogenous aromatic ring in which # the nitrogenous aromatic rings, identical or different represent: 0 a quinoline substituted by at least one group N (Ra) (Rb) in which Ra and Rb, identical or different, represent hydrogen or a short-chain alkyl radical in C1-C4 and / or 0 a quinoline having a nitrogen atom in quaternary form or 0 a benzamidine # the distributor represents: 0 a triazine group optionally substituted by an alkyl radical having 1 to 4 carbon atoms, a thio, oxy or amino radical themselves optionally substituted by one or more short chain alkyl chains containing 1 to 4 carbon atoms or an atom of halogen or 0 a carbonyl group or 0 a C (= NH) -NH-C (= NH) group or 0 an alkylediyl group containing 3 to 7 carbon atoms or 0 a diazine group optionally substituted with the same groups as triazine or a salt thereof. 2 - Compounds according to claim 1 characterized in that the distributor is chosen from triazine or diazine groups. <Desc / Clms Page number 35> 3 - Compounds according to claim 2 characterized in that the diazine groups are pyrimidines. 4 - Compounds according to claim 1 characterized in that they correspond to formula (I) below:

in which: - A represents # an amino group of formula NR1 R2 in which R1 and Identical or different R2 represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or # an OR1 or SR1 group in which R1 has the same meaning as above or # an alkyl group containing 1 to 4 carbon atoms or or a trifluoromethyl group or * a hydrogen atom or # a halogen atom selected from fluorine, chlorine, bromine or iodine -Ar1 and Ar2 identical or different represent # a quinoline unit substituted by at least one group N (Ra) (Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl radical containing 1 to 4 carbon atoms and / or # a quinoline having a nitrogen atom in quaternary form or # a benzamidine or a salt thereof. <Desc / Clms Page number 36> 5 - Compounds according to claim 3 characterized in that Ar1 and Ar2 represent a group selected from the following groups: 4-amino- or 4-methylamino- or 4-dimethylamino-quinolyl or quinolinium in which the quinolinium nucleus is optionally substituted by a methyl group . 6 - Compounds according to claim 1 characterized in that the groups R1 and R2 represent the thiomethyl, amino, alkylamino or dialkylamino radicals in which the alkyl groups have 1 to 4 carbon atoms. 7 - Compounds according to claim 2 characterized in that A represents a methylthio group. 8 - Compounds of claim 1 characterized in that they have telomerase inhibitory activity. 9 - Compounds according to any one of the preceding claims, characterized in that they have anticancer activity. 10 - New compounds corresponding to the following formula (I):

in which: - A represents # an amino group of formula NR1 R2 in which R1 and Identical or different R2 represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or # an OR1 or SR1 group in which R1 has the same meaning as above or # an alkyl group containing 1 to 4 carbon atoms or a trifluoromethyl group or <Desc / Clms Page number 37> N (Ra) (Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a short-chain alkyl radical containing 1 to 4 carbon atoms and / or # a quinoline having a nitrogen atom in quaternary form or # a benzamidine except in the case where A represents diethylamine, hydrogen or an amine group or a salt thereof excluding the dihydrochloride of 2-amino-bis-4,6 - [(4'amino-6 ' -quinaldinyl) amino] -triazine and 2-amino-bis-4,6- (p-amidinoanilino) -triazine. # a hydrogen atom or # a halogen atom chosen from fluorine, chlorine, bromine or iodine - Ar1 and Ar2, which are identical or different, represent # a quinoline unit substituted by at least one group 11 - Compounds according to claim 10, characterized in that Ar and Ar2 represent a group chosen from 4-amino- or 4-methylamino- or 4-dimethylaminoquinolyl or quinolinium groups, the quinolinium nucleus of which is optionally substituted by a methyl group . 12 - Compounds according to claim 10 characterized in that R1 and R2 represent hydrogen. 13 - Compounds according to claim 10 characterized in that A represents a methylthio group. 14 - Compounds according to claim 10 chosen from: - 2-amino-bis-4,6 dichloride - [(1'-methyl-4'-amino-6'-quinaldinio) amino] -triazine - 2-amino dichloride -amino-bis-4,6 - [(1'-ethyl-4'-amino-6'-quinaldinio) amino] -triazine <Desc / Clms Page number 38> - N ', NS-bis (7-chloro-1-methyl-4-quinolinio) pentane-1,5-diamine diodide - bis-2,4-pentahydrate trihydrochloride - [(4'-amino-6' quinaldinyl) amino] pyrimidine - 1,5- (4'-amino-6'-quinaldinyl) biguanide trihydrate dihydrate

- 2-dimethylamino-bis-4,6 dichloride - [(1'-methyl-4'-amino-6'quinaldinio) amino] -triazine - 2-methylamino-bis-4,6 trihydrochloride - [( 4'-amino-6'-quinaldinyl) amino] -triazine - 2-amino-bis-4,6 dichloride - [(1'-methyl-6'-quinolinio) amino] triazine - 2-dichloride trihydrochloride -methylamino-bis-4,6 - [(4'methylamino-6'-quinaldinyl) amino] -triazine - 2-amino-bis-4,6 - [(9'-amino-10'methyl dichloride hydrochloride -2'-acridinio) amino] -triazine - 2-methylthio-bis-4,6 dichloride - [(1'-methyl-4'-amino-6'quinaldinio) amino] -triazine - 2-hydrochloride dihydrate -chloro-bis-4,6 - [(4'-dimethylamino-6'-quinaldinyl) amino] -triazine - hydrate of 2-methylthio-bis-4,6 - [(4'-dimethylamino-6'- quinaldinyl) amino] -triazine - N, N '- (4-amino-6-quinaldinyl) urea dihydrochloride 15 - Use of the compounds of claim 10 as a pharmaceutical product for human use. 16 - Therapeutic combinations consisting of a compound according to claim 1 and another anticancer compound. <Desc / Clms Page number 39> 17 - Combinations according to claim 16 characterized in that the anticancer compound is chosen from alkylating agents, platinum derivatives, antibiotic agents, antimicrotubule agents, anthracyclines, topoisomerases of groups 1 and II, fluoropyrimidines, analogues cytidine, adenosine analogues, enzymes and various compounds such as L-asparaginase, hydroxyurea, transretinoic acid, suramin, irinotecan, topotecan, dexrazoxane, amifostine, herceptin as well as oetrogenic, androgenic hormones. 18 - Therapeutic combination consisting of a compound according to claim 1 and radiation. 19 - Combinations according to any one of claims 16 to 18 characterized in that each of the compounds or treatments is administered simultaneously, separately or sequentially.