Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D401/00—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom
  • C07D401/14—Heterocyclic compounds containing two or more hetero rings, having nitrogen atoms as the only ring hetero atoms, at least one ring being a six-membered ring with only one nitrogen atom containing three or more hetero rings
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P35/00—Antineoplastic agents
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/38—Nitrogen atoms
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D215/00—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems
  • C07D215/02—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom
  • C07D215/16—Heterocyclic compounds containing quinoline or hydrogenated quinoline ring systems having no bond between the ring nitrogen atom and a non-ring member or having only hydrogen atoms or carbon atoms directly attached to the ring nitrogen atom with hetero atoms or with carbon atoms having three bonds to hetero atoms with at the most one bond to halogen, e.g. ester or nitrile radicals, directly attached to ring carbon atoms
  • C07D215/38—Nitrogen atoms
  • C07D215/42—Nitrogen atoms attached in position 4
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07D—HETEROCYCLIC COMPOUNDS
  • C07D409/00—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms
  • C07D409/14—Heterocyclic compounds containing two or more hetero rings, at least one ring having sulfur atoms as the only ring hetero atoms containing three or more hetero rings

Definitions

• the present invention relates to cancer therapy and relates to new 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 new non-nucleotide chemical compounds which interact with specific structures of deoxyribonucleic acid (DNA), or ribonucleic acid (RNA). These new compounds consist of a distributing agent linked to two aminoaromatic groups. These new compounds are useful in the treatment of cancers and act in particular as telomerase inhibiting 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 stopping of cell mitosis and the death of rapidly dividing cells such as cancer cells and possibly the induction of cell senescence cancerous.
• telomerase makes it possible to add repeated DNA sequences of the TTAGGG type, called telomeric sequences, at the end of the telomer, during cell division.
• telomerase makes the cell immortal. In fact, in the absence of this enzymatic activity, the cell loses 100 to 150 bases each division, which makes it rapidly sinking. When rapidly dividing cancer cells appeared, these cells appeared to have telomeres maintained at a stable length during cell division.
• telomere was strongly activated and that it allowed the addition of repeating motifs of telomeric sequences at the end of the telomer and therefore allowed the conservation of the length of the telomer in cancer cells. It has appeared for some time that more than 85% of cancer cells have positive tests for the presence of telomerase while somatic cells do not have this feature.
• telomerase is a very renowned 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), 2635-2639).
• nucleotide structures which have been used in the prior art, mention may be made of diaminoanthraquinones (Sun et al. J. Med. Chem. 40 (14), 2113-6) or diethyloxadicarbocyanines (Wheelhouse RT Et al. J. Am. Chem. Soc. 1998 (120) 3261-2).
• WO 99/40087 describes the use of compounds which interact with G-quadruplex structures which are perylenes and carbocyanines containing at least seven rings including two heterocycles.
• the compounds of the present invention which meet the objective aimed at, that is to say which fix the G-quadruplex structure of DNA or RNA and in particular the G-quadruplex structure of telomeres and therefore exhibit activity telomerase inhibitors have the following general formula:
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or - a short-chain alkyl or alkoxy group in C1
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or
• a phenyl ring optionally substituted by a halogen group, C1-C4 alkoxy, cyano, carbonylamino optionally substituted by one or more C1-C4 alkyl groups, guanyl, alkylthio in
• a mono or bi or tricyclic aromatic or non-aromatic heterocyclic ring comprising 0 to 2 heteroatoms per cycle provided that at least one heteroatom is present in at least one ring optionally substituted by one or more C1-C4 alkyl groups or by C1-C4 alkylene or C2-C4 alkenylene groups • R3 and R'3, identical or different, independently of one another represent hydrogen or a C1-C4 alkyl radical
• a triazine group optionally substituted by one or more radicals chosen from halogen atoms, alkyl radicals having 1 to 4 carbon atoms and thio, oxy or amino radicals themselves optionally substituted by one or more short-chain alkyl chains containing 1 to 4 carbon atoms or 0 a heterocyclic radical containing 5 to 6 links containing a sulfur, oxygen or nitrogen atom
• the subject of the present invention is therefore in particular the products which correspond to the formula gen ral following: nitrogen-containing aromatic ring NR 3 - (CO) n splitter - (CO) m NR '3 - aromatic ring or nonaromatic with n and m are identical or different represent the integer 0 or 1, wherein
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or
• a phenyl ring optionally substituted by a halogen group, C1-C4 alkoxy, cyano, carbonylamino optionally substituted by one or more C1-C4 alkyl groups, guanyl, C1-C4 alkylthio, amino, C1-C4 alkylamino, dialkylamino in C1-C4 for each alkyl, nitro, C1-C4 alkyleneamino or C2-C4 alkenyleneamino or 0 a mono or bi or tricyclic aromatic or non-aromatic heterocyclic ring comprising 0 to 2 heteroatoms per cycle provided that at least a heteroatom is present in at least one ring optionally substituted by one or more C1-C4 alkyl groups or by C1-C4 alkylene or C2-C4 alkenylene groups
• R3 and R'3, identical or different, independently of one another represent hydrogen or a C1-C4 alkyl radical
• the distributor represents: 0 a triazine group optionally substituted by one or more radicals chosen from halogen atoms, alkyl radicals having 1 to 4 carbon atoms and the thio, oxy or amino radicals themselves optionally substituted by one or more short chain alkyl chains containing 1 to 4 carbon atoms or 0 a heterocyclic radical containing 5 to 6 links containing a sulfur, oxygen or nitrogen atom
• a phenyl radical -NH-phenyl-NH-, -NH-phenyl-CH2- NH-, -NH-CH2-phenyl-CH2-NH- or 0 a diazine group, heterocyclic radicals, phenyl, -NH-phenyl -NH-, -NH- phenyl-CH2-NH-, -NH-CH2-phenyl-CH2-NH- and diazine being optionally substituted by the same groups as triazine, it being understood that when the distributor represents phenyl optionally substituted by NH2, that n and m represent 1 and R3 and R3 'represent hydrogen then the nitrogen aromatic cycle and the aromatic cycle do not both represent a quinoline unsubstituted or substituted on its nitrogen atom by an alkyl radical containing 1 to 6 carbon atoms , or one of its salts.
• nitrogen aromatic ring means 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 such as a guanidino or guanyl chain.
• Preferred among all of the above compounds are those comprising a distributor chosen from heterocyclic groups such as, for example, thienyl and pyridyl, a phenyl radical, -NH-phenyl-NH-, -NH-phenyl-CH2-NH-, -NH-CH2-phenyl-CH2-NH- and diazine as defined above and optionally substituted as indicated above.
• a distributor chosen from heterocyclic groups such as, for example, thienyl and pyridyl, a phenyl radical, -NH-phenyl-NH-, -NH-phenyl-CH2-NH-, -NH-CH2-phenyl-CH2-NH- and diazine as defined above and optionally substituted as indicated above.
• diazine groups it is preferred to use pyrimidines.
• the compounds defined above are particularly preferred, characterized in that p and q represent the integer 1.
• the present invention relates particularly to the compounds defined above, characterized in that they correspond to the formula (IA) below:
• n, m, p and q identical or different represent the integer 0 or 1 and in which:
• R3 and R'3 identical or different, independently of one another represent hydrogen or a C1-C4 alkyl group
• a and Ar 2 identical or different represent when A and Ar 2 are identical: • a quinoline motif optionally substituted with at least
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or
• Ar represents one of the possibilities above and Ar 2 represents
• a phenyl ring optionally substituted by a halogen group, C1-C4 alkoxy, cyano, carbonylamino optionally substituted by one or more C1-C4 alkyl groups, guanyl, C1-C4 alkylthio, amino, C1-C4 alkylamino, dialkylamino in C1-C4 for each alkyl, nitro, C1-C4 alkyleneamino or C2- alkenyleneamino
• a heterocyclic ring, aromatic or non-aromatic, mono or bi or tricyclic comprising 0 to 2 heteroatoms per cycle provided that at least one heteroatom is present in at least one ring optionally substituted by one or more C1-C4 alkyl groups or by C1-C4 alkylene or C2-C4 alkenylene groups, it being understood that when A represents optionally substituted phenyl by NH2, that n, m, p and q represent 1 and R3 and R3 'represent hydrogen then the aromatic nitrogen cycle and the aromatic cycle do not both represent a quinoline unsubstituted or substituted on its nitrogen atom by an alkyl radical containing 1 to 6 carbon atoms or one of its salts and when A represents a triazine and p and q both represent the whole 1 then n and m do not both represent the whole 0.
• n and m identical or different represent the integer 0 or 1 and in which:
• • - A represents: 0 a heterocyclic radical containing 5 to 6 links containing a sulfur, oxygen or nitrogen atom,
• a phenyl radical -NH-phenyl-NH-, -NH-phenyl-CH2- NH-, -NH-CH2-phenyl-CH2-NH- or 0 a diazine group
• the heterocyclic radicals phenyl, -NH-phenyl-NH-, -NH-phenyl-CH2- NH-, -NH-CH2-phenyl-CH2-NH- and diazine that may represent A, optionally substituted by one or more chosen radicals among the halogen atoms, the alkyl radicals having 1 to 4 carbon atoms and the thio, oxy or amino radicals themselves optionally substituted by one or more short chain alkyl chains containing 1 to 4 carbon atoms,
• R3 and R'3 identical or different, represent independently of one another hydrogen or a C1-C4 alkyl group - An, and Ar 2 identical or different represent
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or
• a and Ar 2 both represent one of the possibilities mentioned above for A and Ar 2 or
• Ar 2 represents * a phenyl nucleus optionally substituted by a halogen group, C1-C4 alkoxy, cyano, carbonylamino optionally substituted by one or more C1-C4 alkyl groups, guanyl, C1-C4 alkylthio, amino, C1-C4 alkylamino, C1-C4 dialkylamino for each alkyl, nitro group, C1-C4 alkyleneamino or C2-C4 alkenyleneamino
• heterocyclic ring, aromatic or non-aromatic, mono or bi or tricyclic comprising 0 to 2 heteroatoms per cycle provided that at least one heteroatom is present in at least one ring optionally substituted by one or more C1-C4 alkyl groups or by C1-C4 alkylene or C2-C4 alkenylene groups, it being understood that when A represents phenyl optionally substituted by NH2, that n and m represent 1 and R3 and R3 'represent hydrogen, then the nitrogen aromatic ring and the aromatic ring do not represent not both a quinoline unsubstituted or substituted on its nitrogen atom by an alkyl radical containing 1 to 6 carbon atoms, or one of its salts.
• quinoline units can be substituted by any other group which does not intervene 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.
• the diazine groups which A can represent are preferably pyrimidines.
• Ra and Rb represent hydrogen or a C1-C4 alkyl radical or - a short-chain alkyl or alkoxy group containing 1 to 4 carbon atoms or
• a and Ar 2 represent a group chosen from the following groups: 4-amino- or 4-methylamino-, 4-dimethylamino- or 4-alkoxyquinolyl or quinolinium, the quinolinium nucleus of which is optionally substituted by a methyl group.
• A is optionally substituted by one or more radicals chosen from halogen atoms and thioalkyl, amino, alkylamino or dialkylamino radicals, radicals in which the alkyl groups have 1 to 4 carbon atoms and very particularly the compounds characterized in that A is optionally substituted by a methylthio group and optionally by a halogen atom.
• the present invention relates in particular to the compounds of formula (I) as defined above in which: n, m, p and q, identical or different, represent the whole 0 or 1 • - A represents:
• a pyrimidyl radical optionally substituted by one or more radicals chosen from halogen atoms and alkylthio radicals having 1 to 4 carbon atoms,
• R3 and R'3 identical or different, independently of one another represent a hydrogen atom or a C1-C4 alkyl group
• Ra and Rb which are identical or different represent hydrogen or a C1-C4 alkyl radical or
• a and Ar 2 both represent one of the possibilities mentioned above for A and Ar 2 or
• heterocyclic ring aromatic or non-aromatic, mono or bi or tricyclic comprising 0 to 2 heteroatoms per cycle provided that at least one heteroatom is present in at least one ring optionally substituted by one or more C1-C4 alkyl groups or by C1-C4 alkylene or C2-C4 alkenylene or a salt thereof.
• the present invention relates in particular to the compounds of formula (I) as defined above in which:
• n and m identical or different represent the integer 0 or 1
• p and q represent the integer 1
• • - A represents: 0 a thienyl or pyridyl radical
• a phenyl radical -NH-phenyl-NH-, -NH-phenyl-CH2- NH-, -NH-CH2-phenyl-CH2-NH- or
• a pyrimidyl radical optionally substituted by one or more radicals chosen from halogen atoms and alkylthio radicals having 1 to 4 carbon atoms,
• R3 and R'3 identical or different, independently of one another represent a hydrogen atom or a C1-C4 alkyl group
• Ra and Rb which are identical or different, represent hydrogen or a C1-C4 alkyl radical or - a short chain alkyl or alkoxy group containing
• Ar when Ar., And Ar 2 are different • Ar, and Ar 2 both represent one of the possibilities mentioned above for Ar, and Ar 2 or
• Ar represents one of the possibilities above and Ar 2 represents
• the present invention thus particularly relates to the compounds defined above, characterized in that Ar, and Ar 2, which are identical or different, represent a group chosen from the groups 4-amino- or 4-methylamino- or 4-dimethylamino-, or 4-alkoxy -quinolyl or -quinolinium, the quinolinium nucleus of which is optionally substituted by one or two methyl group (s).
• Ra and Rb identical or different represent hydrogen or a C1-C4 alkyl radical or
• a pyridyl ring * a quinoline, benzimidazole, indole, benzothiophene, benzofuran, benzothiazole, benzoxazole, carbazole, carbazole, quinazoline, quinoxaline, piperidyl, piperazinyl, morpholino, azepine, diazaazepine ring, optionally substituted by one or more C1-C4 alkyl groups or by C1-C4 alkylene or C2-C4 alkenylene or a salt thereof.
• R represents a methoxy, amino or dimethylamino group and A represents an aromatic system.
• Another object of the present invention relates to the use of the compounds of formula (I) as a pharmaceutical product for human use.
• the products of formula (IA) as defined above can be prepared as indicated below for the products of formula (I).
• the compounds of general formula (I) can be obtained in particular by condensation of diacids and quinaldines using method A or B which are described below and which are illustrated in the preparation of the examples of the present application below. These methods are not limiting and other methods of activating mono- or di-acids to form the corresponding amide derivatives can also be used. We can refer in this to Richard's 'Comprehensive Organic Transformation' C. Larock.
• the quinaldines can in particular be prepared as indicated in the following references:
• the products of general formula (I) can be prepared after activation of the diacid with bromotripyrrolidino-phosphonium hexafluorophosphate, taking inspiration from the conditions described in Bioorg. Med. Chem. Lett. 7 (1997) 1903-1908.
• the products of general formula (I) can also be prepared using 4- (4,6-dimethoxy-1, 3,5-triazine-2-yl) -4-methylmorpholinium chloride as coupling agent. using the conditions described in Tetrahedron 2001, 57, 1551-1558.
• the group Ar is a substituted or unsubstituted aromatic or heteroaromatic derivative.
• the substituent X can be a halogen atom or an activated group such as a triflate group (-OSO 2 CF 3 ).
• the substituents R1 and R2 optionally represent at least the substituents "nitrogen aromatic ring and R 3 " or "aromatic or non-aromatic ring and R ' 3 ".
• This reaction can be carried out with or without a catalyst (Pd or Cu for example), with or without an organic or mineral base.
• the amino compound (I) can optionally be activated by transforming it into amide.
• the compounds of general formula (I) can be obtained in the form of libraries, by applying the methods A, B or C described above in parallel and / or combinatorial chemistry in the liquid phase or in the solid phase, it being understood that, when working in the solid phase, any of the reactants is previously fixed on a solid support, chosen as a function of the chemical reaction involved, and that said chemical reaction is followed by an operation of cleavage of the product of the reaction of the solid support.
• the present invention also relates to the therapeutic compositions containing a compound according to the invention, in association with a pharmaceutically acceptable support according to the mode of administration chosen.
• the pharmaceutical composition can be in solid, liquid or liposome form.
• solid compositions mention may be made of powders, capsules, tablets.
• oral forms it is also possible to include the solid forms protected with respect to the acid medium of the stomach.
• the supports used for the solid forms consist in particular of mineral supports such as phosphates, carbonates or of organic supports such as lactose, celluloses, starch or polymers.
• Liquid forms are made up 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 according to the route of administration of the patient and the state of the latter.
• the compounds of the present invention can be administered alone or in admixture with other anticancer agents.
• anticancer agents include anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents, anticancer agents
• alkylating agents and in particular cyclophosphamide, melphalan, ifosfamide, chlorambucil, busulfan, thiotepa, prednimustine, carmustine, lomustine, semustine, steptozotocine, decarbazine, temozolomide, procarbazine and l '' hexamethylmelamine
• platinum derivatives such as cisplatin, carboplatin or oxaliplatin
• antibiotic agents such as bleomycin, mitomycin, dactinomycin
• antimicrotubule agents such as vinblastine, vincristine, vindesine, vinorelbine, taxoides (paclitaxel and docetaxel)
• anthracyclines such as doxorubicin, daunorubicin, idarubicin, epirubicin, mitoxantrone, losoxantrone
• topoisomerases such as etoposide, teniposide, amsacrine, irinotecan, topotecan and tomudex
• fluoropyrimidines such as 5-fluorouracil, UFT, floxuridine
• cytidine analogues such as 5-azacytidine, cytarabine, gemcitabine, 6-mercaptomurine, 6-thioguanine
• adenosine analogs such as pentostatin, cytarabine or fludarabine phosphate • methotrexate and folinic acid
• enzymes and various compounds such as L-asparaginase, hydroxyurea, trans-retinoic acid, suramin, dexrazoxane, amifostine, herceptin as well as the oestrogenic and androgenic hormones
• anti-vascular agents such as combretastatin or colchicine derivatives and their prodrug.
• 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.
• the oligonucleotide FAM + DABCYL carries the catalog reference, OL-0371-0802. It has the sequence: 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 by spectrophotometry, recording the absorbance spectrum between 220 and 700 nm and using the molar extinction coefficient provided by the supplier.
• a stock solution of oligonucleotide at the strand concentration of 0.2 ⁇ M in a 0.1 M LiCl 10 mM cacodylate buffer pH 7.6 is previously prepared, briefly heated to 90 ° C and slowly cooled to 20 ° C, then distributed in aliquots of 600 ⁇ l in fluorescence cells. 3 ⁇ l of water (for control) or 3 ⁇ l of the product to be tested (stock at 200 ⁇ M, 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.
• 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 G-quadruplex stabilizing compound induces an increase in Tm. This increase is considered significant if it is greater than 3 °.
• the biological anti -lomerase activity is determined by the following experimental protocol:
• 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
• telomerase activity is determined by a protocol for extending the oligonucleotide TS ' AATCGTTCGAGCAGAGTTTT ' ), in the presence of a cell extract enriched in telomerase activity and of compounds which are added at different concentrations (10, 1, 0.1 and 0.01 ⁇ M).
• the extension reaction is followed by a PCR amplification of the extension products using the oligonucleotides TS and CXext ' GTGCCCTTACCCTTACCCTTACCCTAA 3' ).
• the reaction medium is prepared according to the following composition:
• Bovine albumin serum 0.1 mg / ml
• the oligonucleotides are obtained from Eurogentec (Belgium) and are stored at -20 ° C at a stock concentration of 1 mg / ml in distilled water.
• 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.
• 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.
• the acrylamide gels are then dried on a sheet of Whatmann paper 3 mm at 80 ° C for 1 hour.
• the concentration of compound inducing a 50% inhibition of the telomerase reaction 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 active as an antelomerase 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:
• A549 human cell lines originate from ATCC
• the A549 cells are cultured in a layer in a culture flask in RPMI 1640 medium,
• KB cells are cultured in a layer in a culture flask in medium
• the cells in the exponential growth phase are trypsinized, washed in PBS 1X and are seeded in 96-well microplates (Costar) at the rate of 4x10 4 cells / ml for A549 and 1.5 ⁇ 10 4 cells / ml (0.2 ml / well) then incubated for 96 hours in the presence of variable concentrations of product to be studied (10, 1, 0.1 and 0.01 ⁇ M, each point in quadruplicate). 16 hours before the end of the incubation, 0.02% final neutral red is added to each well. At the end of the incubation, the cells are washed with 1 ⁇ PBS and lysed with 1% of sodium lauryl sulfate. The cellular incorporation of the dye, which reflects cell growth, is evaluated by spectrophotometry at a wavelength of 540 nm for each sample using a Dynatech MR5000 reading device.
• a compound is considered to be active as a cytotoxic agent if the inhibitory concentration of 50% of the growth of the tumor cells tested is notably less than 10 ⁇ M.
• the reaction mixture is concentrated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C.
• the residue obtained is taken up in 5 cm 3 of acetonitrile, filtered through a sintered glass and then washed with 3 cm 3 d diisopropyl ether.
• the brown powder thus obtained is purified by FLASH chromatography on a BOND-ELUT cartridge (27 mm in diameter) packed with 20 g of silica (15-35 ⁇ m) conditioned and then eluted with a mixture (dichloromethane / 2M ammoniacal methanol) (75-25 in volumes).
• the fractions containing the sought product are combined and concentrated to dryness under reduced pressure (2.7 kPa) at 40 ° C.
• the reaction mixture is taken up successively with 2 cm 3 of acetonitrile and 2 cm 3 of diisopropyl ether, the precipitate thus obtained is filtered slowly on a 6 cm 3 BOND-ELUT cartridge fitted with a frit.
• the insoluble material obtained is washed with diisopropyl ether and then dried by circulation of argon.
• a solution of 50 mg of phenylene-1, 3-diisocyanate and 235.1 mg of 6-amino-4-methoxy-2-methyl-quinoline in 1 cm 3 of dimethylformamide is stirred at a temperature in the region of 20 ° C for 3 hours.
• the reaction mixture is filtered through a frit, and the solid residue is rinsed with 1 cm 3 of dimethylformamide.
• the filtrate thus obtained is diluted with 2 cm 3 of dimethylformamide, then 333 mg of polystyrene-isocyanate resin (Argonaut, 1.49 mMol / g) and 419 mg of polystyrene-trisamine resin (Argonaut, 3.75 mMol / g) are added. ).
• the suspension obtained is stirred at a temperature in the region of 20 ° C for 19 hours, filtered through a frit, then the solid residue is washed with 20 cm 3 of a dichloromethane-methanol mixture (90-10 by volume).
• the filtrate obtained is concentrated under reduced pressure (2.7 kPa) at a temperature in the region of 50 ° C. 0.404 g of a brown-purple suspension is thus obtained.
• To this reaction mixture are added 7 cm 3 of dichloromethane, 2 cm 3 of dimethylformamide and 333 mg of polystsyrene isocyanate resin (Argonaut, 1.49 mMol / g).
• the suspension obtained is stirred at a temperature in the region of 60 ° C.
• the suspension obtained is stirred at a temperature in the region of 20 ° C for about 20 hours, filtered through a frit, then the solid residue is washed with 10 cm 3 of a dichloromethane-methanol mixture (90-10 by volume).
• the filtrate obtained is concentrated under reduced pressure (2.7 kPa) at a temperature in the region of 50 ° C, and the residue obtained is coevaporated under the same conditions as above successively with toluene, water, dichloromethane, and methanol. 113 mg of a yellow solid are thus obtained.
• This solid is taken up with 2 cm 3 of dimethyl sulfoxide, filtered through a frit, filtered through a Celite cartridge.
• the insoluble residue is washed with 1 cm 3 of dimethyl sulfoxide and 1 cm 3 of methanol.
• the filtrate obtained is centrifuged (5 minutes at 3000 rpm), and the supernatant liquid is purified by HPLC in 7 injections (column: C18 Waters, 5 M; eluent: elution gradient water-acetonitrile-TFA (0.07 %) from 95-5 to 5-95 in 25 minutes).
• the fractions containing the desired product are combined and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 50 ° C. 25.8 mg of 1- (4-dimethylamino-2-methyl-quinolin-) is obtained.
• the suspension obtained is stirred at a temperature in the region of 20 ° C for approximately 23 hours, filtered through a frit, then the solid residue is washed with 4 times 2 cm 3 of a dichloromethane-methanol mixture (90-10 by volume).
• the filtrate obtained is concentrated under air flow at a temperature in the region of 40 ° C, then reconcentrated under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C.
• the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 6-chloro-2-methylsulfanyl-pyrimidine-4-carboxylic acid can be prepared by operating as follows:
• the residue obtained is dissolved in 20 cm 3 of dimethyl sulfoxide and purified by HPLC (column: C18 Waters, 5M, 50x19 mm; eluent: gradient of elution water-acetonitrile-TFA (0.07%) from 95-5 to 5-95 in 30 minutes).
• HPLC column: C18 Waters, 5M, 50x19 mm; eluent: gradient of elution water-acetonitrile-TFA (0.07%) from 95-5 to 5-95 in 30 minutes).
• the fractions containing the desired product are combined and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C.
• Example 13 The amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 6- (4-methoxy-2-methyl-quinolin-6-ylamino) -2-methylsulfanyl-pyrimidine-4 acid carboxylic
• the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 6- (4-methoxy-2-methyl-quinolin-6-ylamino) -2-methylsulfanyl-pyrimidine-4-carboxylic acid can be prepared by operating as for the preparation of the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of the acid 6- (4-dimethylamino-2-methyl-quinolin-6-ylamino) -2 -methylsulfanyl-pyrimidine-4-carboxylic:
• Amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) 6- (4-amino-) acid 2-methyl-quinolin-6-ylamino) -2-methylsulfanyl-pyrimidine-4-carboxylic can be prepared by operating as for the preparation of the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) '6- (4-dimethylamino-2-methyl-quinolin-6-ylamino) -2-methylsulfanyl-pyrimidine-4-carboxylic acid: From 19 mg of 6-amino-4-amino-2-methyl-quinoline, 50 mg of amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 6-chloro-2-methylsulfanyl-pyrimidine-4-carboxylic acid, 32 mg of sodium carbonate and 3 cm 3 of dimethylformamide, 12 mg of the amide (4-dimethylamino-2-methyl-quino
• the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 2-methylsulfanyl-6- (pyridin-4-ylamino) -pyrimidine-4-carboxylic acid can be prepared by operating as for the preparation amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) 6- (4-dimethylamino-2-methyl-quinolin-6-ylamino) -2-methyl-Isulfanyl-pyrimidine-4-carboxylic acid : From 9.4 mg of 4-amino-pyridine, 50 mg of the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 6-chloro-2-methylsulfanyl-pyrimidine- acid 4- carboxylic, 32 mg of sodium carbonate and 3 cm 3 of dimethylformamide, 9 mg of the amide (4-dimethylamino-2-methyl-quinolin-6-ylamino) of 2-methylsulfany
• the filtrate is concentrated under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C, then the residue obtained is purified by HPLC (column: C18 Waters, 5 M, 50x19 mm; eluent: water-elution gradient- acetonitrile-TFA (0.07%) from 95-5 to 5-95 in 30 minutes).
• the fractions containing the desired product are combined and concentrated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C. 12 mg of N, N'-bis (4-amino-2-methyl- 6-quinolyl) -2,4-diamino-6-chloro-5-methylsulfanyl-pyrimidine in the form of an ecru solid.
• N N- (4-amino-2-methyl-6-quinolyl) -2-amino-4,6-dichloro-5-methylsuIfanyl-pyrimidine can be prepared by operating as follows: To a solution of 1 g of 2,4,6-trichloro-5-methylsulfanyl-pyrimidine 6 cm 3 of 2-butanone, at a temperature in the region of 0 ° C., 1.1 g of 4.6- are added in portions. diamino-2-methyl-quinoline, then at a temperature in the region of 20 ° C, 0.434 cm 3 of 30% sodium hydroxide.
• 2,4,6-Trichloro-5-methylsulfanyl-pyrimidine can be prepared as described in: Mattioda, Georges; Obellianne, Pierre; Gauthier, Henri; Loiseau, whatsoever; Millischer, René; Donadieu, Anne M.; Mestre, Michel. Synthesis and pharmacological properties of 4-piperazino-5-methylthiopyrimidines. Selection of new antiemetic agents. J. Med. Chem. (1975), 18 (6), 553-9.
• the crude product thus obtained is dissolved in a mixture (dichloromethane / 2M ammoniacal methanol) (90-10 by volume) then deposited on a 20 ⁇ 20 MERCK preparative plate of reference 1.05744 with a thickness of 0.5 mm. After elution in the mixture (dichloromethane / 2M ammoniacal methanol) (90-10 by volume) the product is isolated, taking up the silica with a mixture (dichloromethane / methanol (80-20 by volume).
• reaction mixture is taken up in 2 cm 3 of diisopropyl ether, filtered through sintered glass and then washed with 2 x 2 cm 3 of diisopropyl ether.
• the insoluble material obtained is dried under vacuum at a temperature in the region of 20 ° C for 2 hours. 157 mg of 2,5-pyridine dicarboxylic acid bis - [(4-dimethylamino-2-methyl-quinolin-6-yl) are thus obtained. ) -amide] in the form of a yellow solid, the characteristics of which are as follows:
• Example 21 Preparation of N, N'-Bis- (4-dimethylamino-2-methyl-quinolin-6-yl) -1, 4-phenylenediacetamide.
• the reaction mixture is concentrated to dryness under reduced pressure (2.7 kPa) at 40 ° C.
• the residue obtained is taken up in 5 cm 3 of toluene and then concentrated to dryness under reduced pressure (2.7 kPa) at 40 ° C.
• the yellowish solid thus obtained is purified by FLASH chromatography on a BOND-ELUT cartridge (27 mm in diameter) packed with 25 g of silica (15-35 ⁇ m) conditioned and then eluted with a mixture (dichloromethane / 2M ammoniacal methanol) (90-10 in volumes) at a clearance of 10 cm 3 per minute.
• the fractions between 180 and 250 cm 3 are combined and concentrated to dryness under reduced pressure (2.7 kPa) at 40 ° C.
• the mixture obtained is stirred at a temperature in the region of 20 ° C for approximately 15 hours.
• the reaction mixture is deposited on a BOND-ELUT VARIAN cartridge of reference 1225-6054 containing 3 g of SCX phase conditioned with dichloromethane.
• the cartridge is washed successively with dichloromethane (10 cm 3 ) and methanol (10 cm 3 ) before being eluted with 2M ammoniacal methanol.
• the ammoniacal fractions are concentrated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C. 179 mg of crude are thus obtained.
• the reaction mixture is deposited on a MEGA BOND ELUT VARIAN cartridge of reference 1225-6065 containing 20 g of SCX phase conditioned with dimethylformamide.
• the cartridge is washed successively with dimethylformamide (30 cm 3 ) and methanol (30 cm 3 ) before being eluted with 2M ammoniacal methanol.
• the ammoniacal fractions are concentrated to dryness under reduced pressure (2.7 kPa) at a temperature in the region of 40 ° C. 400 mg of 5- (methoxycarbonyl) -pyridine-2-carboxylic acid - [(4-dimethylamino-2-methyl-quinolin-6-yl) -amide] are thus obtained in the form of a brown insoluble matter.
• the reaction mixture is taken up successively with 2 cm 3 of acetonitrile and 2 cm 3 of diisopropyl ether, the precipitate thus obtained is filtered slowly on a 6 cm 3 BOND-ELUT cartridge fitted with a frit.
• the insoluble material obtained is washed with 1 cm 3 of ethanol, taken up in 2 cm 3 of diisopropyl ether and then dried under vacuum at a temperature in the region of 20 ° C for 2 hours.
• 47 mg of the hydrochloride of -2,6-pyridine dicarboxylic acid bis - [(4-amino-2-methyl-quinolin-6-yl) - amide] are thus obtained in the form of a gray powder, the characteristics of which are the following :
• Example 25 The G-quartet, antitelomerase and cytotoxic activities of the various compounds exemplified are determined according to the operating protocols described above.

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