AU2002229859B2 - Chemical derivatives and their use as anti-telomerase agent - Google Patents

Description

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

The present invention relates to the use of novel nonnucleotide chemical compounds which interact with specific structures of deoxyribonucleic acid (DNA). These novel compounds consist of a distribution agent linked to an aminoaromatic group. These novel compounds are useful in the treatment of cancers and act in particular as telomerase-inhibiting agents. They are particularly useful for stabilizing DNA in G-quadruplex structure (guanine tetrads). The therapeutic application of the inhibition of telomerase via the stabilization of these G-quadruplexes is the termination of cellular mitosis and the death of rapidly-dividing cells such as cancer cells and possibly the induction of the senescence of cancer cells.

The compounds of the present invention have the advantage, from the therapeutic point of view, of blocking telomerase. From a biological point of view, telomerase allows the addition of repetitive DNA sequences of the T T A G G G type, termed telomeric sequences, at the end of the telomer, during cell division. Through this action, telomerase renders the cell immortal. Indeed, in the absence of this enzymatic activity, the cell loses, at each division, 100 to 150 bases, which rapidly renders it senescent. During the appearance of rapidly-dividing cancer cells, it appeared that these cells possessed telomers which were maintained at a stable length during cell division. In these cancer cells, it appeared that telomerase was highly activated and that it allowed the addition of repetitive motifs of telomeric sequences at the end of the telomer and therefore allowed conservation of the length of the telomer in the cancer cells. It appeared during the past few years that more than 85% of cancer cells showed positive tests for the presence of telomerase whereas somatic cells do not show this characteristic.

Thus, telomerase is a highly coveted target for treating cancer cells. The first obvious approach for blocking telomerase was the use of nucleotide structures (Chen et al., Proc. Natl. Acad. Sci. USA 93(7), 2635-2639). Among the nonnucleotide compounds which have been used in the prior art, there may be mentioned the diaminoanthraquinones (Sun et al., J.

Med. Chem. 40(14), 2113-6) or the diethyloxadicarbocyanins (Wheelhouse R.T. et al., J. Am. Chem. Soc.

1998(120) 3261-2).

27-Feb-2008 13:06 Watermark +61298887600 15/26 00 3 0 SPatent WO 99/40087 describes the use of compounds which interact with .C the G-quadruplex structures which are perylene compounds and carbocyanins containing at least seven rings including two heterocycles.

It appeared, quite surprisingly, that simple structures made it possible to obtain a result which is at least equivalent with structures which are a lot less complicated from a chemical point of view. The compounds of the present 00 invention which meet the intended objective, that is to say which bind the Gquadruplex structure and thereby exhibit a telomerase-inhibiting activity, ccorrespond to the following general formula: O 10 nitrogen-containing aromatic ring NR3 distribution agent NR' 3 nonaromatic Cl hydrocarbon chain, nonaromatic hydrocarbon chain being selected from a linear or branched alkyl (CI-C 4 a linear or branched alkenyl (C 2

-C

4 chain, a cycloalkyl (C3-.C 18 chain, a cycloalkenyl (C3-C18) chain and a heterocycloalkyl (C 3 -C1a) chain which optionally includes the nitrogen atom, wherein 1) the nitrogen-containing aromatic ring represents: a) a quinoline optionally substituted with at least i) a group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a C1-C 4 alkyl radical or ii) a group ORa in which Ra is as defined above b) a quinoline possessing a nitrogen atom in quaternary form optionally substituted with a group selected from: 4-amino-, 4-methylamino- and 4-dimethylamino-, attached at the 4 position or c) a benzamidine or d) a pyridine, attached at the 4 position, 2) R 3 and R' 3 which are identical or different, represent independently of each other, hydrogen or a C1-C4 alkyl radical 3) the distribution agent represents: a triazine group, a triazine group substituted with i) a alkyl radical having 1 to 4 carbon atoms, ii) a thiol radical, iii) an hydroxy radical, or iv) an amino radical, COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:07 Watermark +61298887600 16/26 00 4 0 0 v) wherein the thiol, hydroxy or amino radicals are unsubstituted or .0 substituted with one or more short-chain alkyl groups containing 1 to

C)

[L 4 carbon atoms and wherein the alkyl is unsubstituted or substituted with a halogen atom or a salt thereof; wherein the nonaromatic hydrocarbon chain is optionally substituted with one or more atoms or radicals chosen from halogen atoms, hydroxyl, aryl, heteroaryl, Salkyloxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, C dialkylamino, diarylamino, amidino, guanidine, alkylcarbonylamino, o 10 arylcarbonylamino, carboxyl, alkyloxycarbonyl or aryloxycarbonyl, aminocarbonyl, l alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, alkycarbonyl arylcarbonyl, cyano and trifluoromethyl.

The alkyl chains of the optional substituents of the hydrocarbon chain preferably contain 1 to 4 carbon atoms and the aryl groups of the optional substituents of the hydrocarbon chain preferably contain 5 to 18 carbon atoms.

COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 PAGES 5 TO 7 HAVE BEEN LEFT BLANK INTENTIONALLY 8 It is evident that the quinoline motifs may be substituted by any other group not involved in the intended application; thus, acridine or isoquinoline or quinazoline or quinoxaline or phthalazine or benzothiazine or benzoxazine or phenoxazine or phenothiazine groups are included in the definition of the quinoline groups.

Among the above compounds of formula there are preferred those comprising a heterocycle chosen from the 4-aminoquinolyl, 4-alkyl- or 4-dialkylaminoquinolyl, 4-aminoquinolinium or quinolinium groups in which the quinolinium ring is optionally substituted with a methyl group.

As regards the groups A, they preferably represent the methylthio, amino, alkylamino or dialkylamino radical, in which radicals the alkyl groups possess 1 to 4 carbon atoms.

As regards the nonaromatic hydrocarbon chain, it preferably represents a 2-(dialkylamino)ethyl, 3-(dialkylamino)propyl, 2-(N-alkyl-N-arylamino)ethyl or 3-(N-alkyl-N-arylamino)propyl chain in which the alkyl groups preferably contain 1 to 4 carbon atoms, or more preferably still 1 to 2 carbon atoms and the aryl groups preferably contain 5 to 18 carbon atoms, more preferably still 6 carbon atoms.

Another subject of the present invention relates to the compounds of formula as novel 27-Feb-2008 13:07 Watermark +61298887600 17/26 00 9 0 Schemical products. It therefore relates to the novel products corresponding to the .C following formula

A

C N N RN N NR' 3 Ar, Alk 00 in which: C 5 1) A represents o a) an amino group of formula NRIR2, in which R1 and R2, which are 0 identical or different, represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or b) a group OR1 or SR1 in which R1 has the same meaning as above or c) an alky] group containing 1 to 4 carbon atoms or a trifluoromethyl group or d) a hydrogen atom or e) a halogen atom chosen from fluorine, chlorine, bromine and iodine 2) R3 and R'3, which are identical or different, represent independently of each other hydrogen or a C1-C4 alkyl group 3) Arl represents a nitrogen-containing aromatic ring representing: a) a quinoline optionally substituted with at least i) a group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a C1-C4 alkyl radical or ii) a group ORa in which Ra is as defined above b) a quinoline possessing a nitrogen atom in quaternary form optionally substituted with a group selected from: 4-amino-, 4-methylamino- and 4-dimethylamino-, attached at the 4 position or c) a benzamidine or d) a pyridine attached at the 4-position or fused with an aryl or heteroaryl group optionally substituted with a C1-C4 alkyl group 4) Alk represents a nonaromatic, optionally substituted, hydrocarbon chain as hereinbefore defined, chosen from alkyl (C1-C4), alkenyl (C2-C4), wherein the alkyl and alkenyl chain are linear or branched, cycloalkyl (C3-C18), COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:08 Watermark +61298887600 18/26 00 0 cycloalkenyl (C3-C18), heterocycloalkyl (C3-C18), and heterocycloalkyl (C3- CC18) including the nitrogen atom of the NR'3 group

C)

(1)or a salt thereof; wherein the nonaromatic hydrocarbon chain is optionally substituted with one or more atoms or radicals chosen from halogen atoms, hydroxyl, aryl, heteroaryl, alkyloxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino andlor 00 arylamino, dialkylamino, diarylamino, amidino, guanidine, alkylcarbonlamino, arylcarbonlamino, carboxyl, alkyloxycarbonyl, aryloxycarbonyl, aminocarbonyl, N alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, alkylcarbonyl, o 10 arycarbonyl, cyano and trifluoromethyl.

0 Ci The preferred compounds of formula are those for which AR 1 represents a group chosen from the COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 11 following units: 4-amino- or 4-methylamino- or 4dimethylaminoquinolyl or quinolynium whose quinolinium nucleus is optionally substituted with a methyl group.

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

The compounds of general formula which are preferred are those for which the nonaromatic hydrocarbon chain represents a 2-(dialkylamino)ethyl, 3-(dialkylamino)propyl, 2-(N-alkyl-N-arylamino)ethyl or 3-(N-alkyl-N-arylamino)propyl chain in which the alkyl groups contain 1 to 4 carbon atoms, in particular 1 to 2 carbon atoms and the aryl groups contain 5 to 18 carbon atoms, in particular 6 carbon atoms.

The compounds of general formula for which the nonaromatic hydrocarbon chain represents a 2-(N-alkyl-N-arylamino)ethyl chain such as the 2-(N-m.tolyl-N-ethylamino)ethyl chain are most particularly preferred.

Another subject of the present invention relates to the use of the compounds of formula as pharmaceutical product for human use.

The methods of preparing the compounds of formula (I)

A

N" N Ar I Alk NR N

NR'

3

(I)

are described below.

In the case where Arl and Alk are present, the triazine of general formula (A),may be obtained by sequential displacement of the halogen atoms, most generally of chlorine atoms, from the products of general formula by the amines Arl and then Alk of general formula according to scheme 1: R

R

NI rNN N X N N II 1- X N X R 3 NR N X X =Cl (or F or Br or I)

(D)

Alk-NH (C)

R'

3

R

N Alk

NR

3 N NR'

(A)

Scheme 1 Generally, the procedure is carried out with 1 mole of dihalo-s-triazine, or trihalo-s-triazine, and 1 mole of amine Arl. The procedure is preferably carried out in an inert solvent such as acetone which is optionally aqueous or an alcohol which is optionally 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. According to a better way of carrying out the invention, the procedure is carried out at a temperature of between 20 0 C and 50°C. Next, 1 mole of 13 amine Alk is added to the product of general formula which may be optionally isolated. The procedure is carried out in particular at a temperature of between 0 C and the reflux temperature.

Advantageously, it is possible to carry out the procedure 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 in which Ar are as defined above and R represents a group NR1R2 or OR1 or SR1 may also be prepared by nucleophilic displacement of a halogen atom, generally a chlorine atom, from a product of general formula in which R represents a halogen atom according to scheme 2:

R

N N RR 2 NH or RO- or R 1

S-

Ar N /A kAlk SNR NR ArA N N /N A lk

NR

3 N NRNR' N NR

(A)

R CI (or F or Br or I) R= NRR 2 or OR 1 or SR, Scheme 2 The procedure is generally carried out by condensing 1 mole of product of general formula in which R represents a halogen atom, preferably a chlorine atom, with 1 mole of amine R1R2NH or alcoholate R10- or thioalcoholate R1S. The reaction 14 takes place in an inert medium under the reaction conditions. There may be mentioned among the inert solvents acetone which is optionally aqueous or an alcohol which is optionally 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. When the entering group represents a group R1R2NH, the procedure is preferably carried out at a temperature of between 20 0 C and the reflux temperature, in the presence in particular of an organic base such as triethylamine, or an 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 the base of which can then be released. When the entering group represents a group R10- or R1S-, the procedure is preferably carried out with an alkali metal or alkaline-earth metal 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 of between 50 0 C and the reflux temperature.

General method 3 According to a third method of preparing the compounds for which R represents a hydrogen atom or a straight or branched alkyl group containing from 1 to 4 carbon atoms, may also be prepared by condensation of a bisguanide of general formula with an acid derivative, preferably an acid chloride or a methyl ester of general formula according to scheme 3: 0

R

NH NH R CI Ar N Alk or N N Ak NRN N NR'\ Alk H Aro0 1

NR

3 N NR' 3 R O

(A)

Scheme 3 The condensation between the bisguanide of general formula and the acid derivative of general formula is generally carried out in an alcohol such as methanol or ethanol. The procedure is preferably carried out at a temperature of between 0°C and the reflux temperature.

The symmetric or asymmetric bisguanides of general formula may be obtained by carrying out the procedure under the conditions described in the literature and in particular according to Patent J.P. 94-4993.

General method 4 It is understood that the s-triazines of general formula may be obtained in the form of libraries, by applying the methods described in schemes 1, 2, 3 in parallel and/or combinatorial chemistry in liquid phase or in solid phase, it being understood that when the work is carried out in solid phase, any 16 one of the reagents is attached beforehand onto a solid support, chosen according to the chemical reaction involved, and that said chemical reaction is followed by an operation of cleaving the product of the reaction from the solid support.

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

Among the solid compositions, there may be mentioned powders, gelatin capsules, tablets. Among the oral forms, it is also possible to include the solid forms which are protected from the acidic medium of the stomach. The carriers used for the solid forms consist in particular of inorganic carriers such as phosphates, carbonates or organic carriers such as lactose, celluloses, starch or polymers. The liquid forms consist of solutions, suspensions or dispersions. They contain, as dispersive carrier, either water or an organic solvent (ethanol, NMP and the like) or mixtures of surfactants and solvents or of complexing agents and solvents.

The administered dose of the compounds of the invention will be adjusted by the practitioner 17 according to the route of administration to the patient and the condition of the latter.

The compounds of the present invention may be administered alone or mixed with other anticancer agents. Among the possible combinations, there may be mentioned 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, antimicrotubule agents such as in particular vinblastine, vincristine, vindesine, vinorelbine, taxoids (paclitaxel and docetaxel) anthracyclines such as in particular doxorubicin, daunorubicin, idarubicin, epirubicin, mitoxantrone, losoxantrone 18 group I and II topoisomerases such as etoposide, teniposide, amsacrine, irinotecan, topotecan and tomudex, fluoropyrimidines such as 5-fluorouracil, UFT, floxuridine, cytidine analogs such as 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, suramine, dexrazoxane, amifostine, herceptin as well as etrogenic and androgenic hormones.

It is also possible to combine a radiation treatment with the compounds of the present invention.

These treatments may be administered simultaneously, separately or sequentially. The treatment will be adapted to the patient to be treated by the practitioner.

The G-quadruplex stabilizing activity may be determined by a method using the formation of a complex with fluorescein of which the experimental protocol is described below.

Oligonucleotides All the oligonucleotides, modified or otherwise, were synthesized by Eurogentec SA, Seraing, Belgium. The oligonucleotide FAM DABCYL carries the catalog reference OL-0371-0802. It has the sequence: GGGTTAGGGTTAGGGTTAGGG corresponding to 3.5 repeats of the human telomeric motif (strand rich in The fluorescein is attached to the 5' end, the DABCYL to the 3' end, by the chemical arms described by Eurogentec. The concentration of the samples is checked by spectrophotometry, 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 mM sodium cacodylate buffer pH 7.6 containing 0.1 M Lithium Chloride (or Sodium Chloride). The absence of fluorescent contamination in the buffer was checked beforehand. The fluorescent oligonucleotide is added at the final concentration of 0.2 pM.

Study of fluorescence All the measurements of fluorescence were carried out on a Spex Fluorolog DM1B apparatus, using an excitation line width of 1.8 nm and an emission line width of 4.5 nm. The samples are placed in a microquartz 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, 70 and 800C. 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 emission wavelength. The spectra are corrected for the response of the instrument by reference curves. A high extinction (80-90%) of the fluorescence of fluorescein at room temperature is observed, in agreement with an intramolecular folding of the oligonucleotide at 20 0 C in the form of a G-quadruplex, which induces juxtaposition of its 5' and 3' ends which are respectively linked to fluorescein and to DABCYL. This juxtaposition causes an alreadydescribed phenomenon of extinction of fluorescence which is used for "Molecular Beacons".

Fluorescence Tm: An oligonucleotide stock solution at the strand concentration of 0.2 gM in 0.1 M LiCI, 10 mM cacodylate buffer, pH 7.6, is prepared beforehand, heated briefly at 900C and slowly cooled to 20 0 C, and then distributed in aliquots of 600 il in the fluorescence cuvettes. 3 4~ of water (for the control) or 3 41 of test product (stock at 200 AM, final concentration 1 gM) are then added and mixed. The samples are then allowed to incubate for at least 1 hour at 200C before each measurement. The use of longer incubation times (up to 24 hours) has no influence on the result obtained.

21 Each experiment allows the measurement of only one sample. The latter is first incubated at an initial temperature of 200C, heated to 80 0 C over 38 minutes, left for 5 minutes at 800C and then cooled to 20 0 C over 62 minutes. During this time, the fluorescence is measured simultaneously at two emission wavelengths (515 nm and 588 nm) using 470 nm as excitation wavelength. A measurement is carried out every 30 seconds. The temperature of the water bath is recorded in parallel, and the fluorescence profile as a function of the temperature is reconstituted from these values. The fluorescence profiles are then normalized between 200C and 80 0 C, and the temperature for which the intensity of emission at 515 nm is the mean of those at high and low temperature is called Tm. Under these conditions, the Tm of the reference sample without addition of product is 440C in a Lithium Chloride buffer. This temperature is increased to more than 550C in a Sodium Chloride buffer. The addition of a G-quadruplex stabilizing compound induces an increase in the Tm. This increase is judged to be significant if it is greater than The antitelomerase biological activity is determined by the following experimental protocol: Preparation of the extract enriched in human telomerase activity The leukemia line HL60 is obtained from ATCC (American Type Culture Collection, Rockville USA). The 22 cells are cultured in suspension in RPMI 1640 medium containing L-Glutamine at 2 mM, Penicillin 200 U/ml, streptomycin 200 gg/ml, gentamycin 50 ig/ml and supplemented with 10% heat-inactivated fetal calf serum.

An aliquot of 105 cells is centrifuged at 3000xG and the supernatant discarded. The cell pellet is resuspended by several successive pipettings in 200 gi of lysis buffer containing 0.5% CHAPS, 10 mM Tris-HCl pH 7.5, 1 mM MgC1 2 1 mM EGTA, 5 mM P-mercaptoethanol, 0.1 mM PMSF and 10% glycerol and is stored in ice for 30 minutes. The lysate is centrifuged at 16 0000xG for 20 minutes at 4 0 C and 160 pl of supernatant is recovered. The proteins in the extract are assayed by the Bradford method. The extract is stored at -80 0

C.

Assay of the telomerase activity The inhibition of the telomerase activity is determined by a protocol for extension of the oligonucleotide TS 5

AATCGTTCGAGCAGAGTT

3 in the presence of a cellular extract enriched in telomerase activity and compounds which are added at various concentrations (10, 1, 0.1 and 0.01 ig/ml). The extension reaction is followed by a PCR amplification of the extension products with the aid of the oligonucleotides TS and CXext

GTGCCCTTACCCTTACCCTTACCCTAA

3 23 The reaction medium is prepared based on the following composition: Tris HC1 pH 8.3 20 mM MgC12 1.5 mM Tween 20 0.005% (W/V) EGTA 1 mM DATP 50 IM DGTP 50 AM DCTP 50 AM DTTP 50 ttM Oligonucleotide TS 2 pg/ml Oligonucleotide CXext 2 gg/ml Bovine serum albumin 0.1 mg/ml Taq DNA polymerase 1 U/ml alpha 32P dCTP (3000 0.5 l Ci/mmol) Telomerase extract 200 ng in a volume of 10 il Test product or solvent in a volume of 5 pl Double distilled water QS 50 tl The oligonucleotides are obtained from Eurogentec (Belgium) and are stored at -20 0 C at a stock concentration of 1 mg/ml in distilled water.

The reaction samples are assembled in 0.2 ml PCR tubes and one drop of paraffin oil is deposited on each of the reactions of the experiment before closing the tubes.

24 The reaction samples are then incubated in a Cetus 4800-type PCR apparatus under the following temperature conditions: minutes at 300C, 1 minute at 90 0

C,

followed by 30 cycle of, seconds at 940C, seconds at and 1 minute 30 seconds at 720C, followed by a final cycle of 2 minutes at 720C.

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

The acrylamide gels are then dried on a sheet of whatmann 3MM paper at 800C for 1 hour.

The analysis and the quantification of the reaction products are carried out with the aid of an InstantImager apparatus (Pacard).

For each compound concentration tested, the results are expressed as percentage inhibition of the reaction and calculated from the untreated enzymatic control and from the enzyme-free sample (blank) according to the following formula: (Compound Value blank value/enzymatic control value blank value) x 100.

The concentration of compound inducing a inhibition of the telomerase reaction (IC50) is determined with the aid of a semilogarithmic graphical representation of the inhibition values obtained as a function of each of the compound concentrations tested.

A compound is considered to be active as an antitelomerase agent when the quantity inhibiting of the telomerase reaction is in particular less than gM.

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

26 The cells at the exponential growth phase are trypsinized, washed in IX PBS and are inoculated in 96-well microplates (Costar) in an amount of 4x10 4 cells/ml for A549 and of 1.5x10 4 cells/ml (0.2 ml/well) and then incubated for 96 hours in the presence of variable concentrations of product to be studied (10, 1, 0.1 and 0.01 gg/ml, each point in quadruplicate). 16 hours before the end of the incubation, 0.02% final of neutral red is added to each well. At the end of the incubation, the cells are washed with IX PBS and lysed with 1% sodium lauryl sulfate. The cellular incorporation of the dye, which reflects cellular growth, is evaluated by spectrophotometry at a wavelength of 540 nm for each sample with the aid of a Dynatech MR5000 reading apparatus.

For each compound concentration tested, the results are expressed as percentage inhibition of cellular growth and calculated from the untreated control and the culture medium free of cells (blank) according to the following formula: (Compound Value blank value/cell control value blank value) x 100.

The concentration of compound inducing a inhibition of growth (IC50) is determined with the aid of a semilogarithmic graphical representation of the inhibition values obtained as a function of each of the compound concentrations tested.

27 A compound is considered to be active as cytotoxic agent if the concentration inhibiting the growth of the tumor cells tested by 50% is in particular less than 10 gM.

The following and nonlimiting examples are given to illustrate the invention.

Example 1: Parallel synthesis of substituted derivatives of N6-[6-amino-4-methylsulfanyl-[1,3,5]triazin-2-yl]-2-methylquinoline-4,6-diamine

S-

N N

NH

2 SN N N -3

N

Preparation of N6-(6-chloro-4-methylsulfanyl- [1,3,5]triazin-2-yl)-2-methylquinoline-4,6-diamine 4.4 g (25 mmol) of 2-methylquinoline- 4,6-diamine, which may be prepared according to J. Med.

Chem. 1992, 35, 252, and 2.8 g (25 mmol) of sodium carbonate are successively added, in a 1 litre threenecked flask, to a solution of 5 g (25 mmol) of 2,6-dichloro-6-methylsulfanyl-[1,3,5]triazine, which may be prepared according to J. Amer. Chem. Soc., 1945, 67, 662, in 400 ml of tetrahydrofuran. The reaction mixture is heated under reflux for 16 hours. After evaporation of the tetrahydrofuran, the residue is 28 taken up in 400 ml of a mixture of water and dichloromethane (50-50 by volume). The organic phase is separated after settling out, dried over sodium sulfate and concentrated to dryness under reduced pressure.

7.5 g of N6-(6-chloro-4-methylsulfanyltriazin-2yl)-2-methylquinoline-4,6-diamine are then obtained, in the form of a pale yellow solid whose characteristics are the following: melting point 294 0

C

H NMR spectrum (300 MHz, (CD 3 2 SO d6, 6 in ppm) 2.43 3H); 2.52 3H); 6.47 1H); 6.61 (unresolved complex: 2H); 7.62 (broad d, J 9 Hz: 1H); 7.69 J 9 Hz: 1H); 8.32 (unresolved complex: 1H); 10.80 (unresolved complex: 1H).

Parallel synthesis of N6-[6-(2-dimethylaminoethylamino)-4-methylsulfanyl-[1,3,5]triazin-2-yl]- 2-methylquinoline-4,6-diamine (Example 1-1) mg (0.15 mmol) of N6-(6-amino-4-methylsulfanyl-[1,3,5]triazin-2-yl)-2-methylquinoline- 4,6-diamine are introduced into a heating magnetic reactor with a Zymark condenser, of the STEM RS2050 type, containing 25 wells in parallel each provided with a 50 ml glass tube. 5 ml of dioxane, 16 mg (0.15 mmol) of sodium carbonate, 23 mg (0.15 mmol) of sodium iodide and 27 mg (0.3 mmol) of 2-dimethylaminoethylamine are successively added to the first tube (Example The reaction medium is heated by reflux and under argon for 24 hours. After cooling, the 29 content of the tube is evaporated under reduced pressure, taken up in 5 ml of water and 5 ml of ethyl acetate and filtered. The organic phase is separated by settling out, dried and concentrated under reduced pressure. The crude product obtained is then purified by LC/MS using a Waters Xterra 3.5 gM C18 silica column 3 mm in diameter and 50 mm in length, eluting with a linear elution gradient consisting, at the starting time (to 0 min), of water containing 0.05% trifluoroacetic acid and, at the final time (tf 4 min), of acetonitrile containing 0.05% trifluoroacetic acid. 58 mg of N6-[(6-(methylquinolin- 6-ylamino)-4-methylthiotriazin-2-yl]quinaldine- 4,6-diamine trifluoroacetate are thus obtained, after purification, whose characteristics are the following: mass spectrum (DAD-TIC) 454 (MH retention time 2.69 min (the retention times are obtained on a hypersil C 18 5 pm column 50 mm diameter 4.6 mm trade mark Purity Elite, eluting with a mixture of solvents A (H20/TFA 0.05%) and B (ACN/TFA 0.05%) with a linear gradient ranging from 95% A/5% B (t 0 min) to 10% A/90% B at t 3.5 min, then step 2 min).

Examples 1-1 to 1-26 were obtained by carrying out the procedure as above in a Zymark STEM RS2050 reactor. The structures, the various operating conditions used and the characteristics of Examples 1-1 to 1-26 are summarized in the table below: Example StutReaction conditions Characteristics No. of Mass Retention Ak('-Solven Heating mmol Of MH* time amine (min) 11dioxane 17 h.i 00* 0.3 384 2.69 1-2 dioxane 17 h.1100' 0.3 410 2.91 1-3 dioxane 17 h./100* 0.15 411 2.86 HO- N trans racemic 1-4 Ndioxane 3d/100 0.45 422 2.85 3-RSj (Ndioxane 17 h./iCC' 0.15 396 2.84

N~N

M2-S

NH

1-6 dioxane 17 h.1100' 0.15 424 2.79 '-NH 2-RS 1-7 H2 N dioxane 17 h.1100 0.15 410 2.72 HN-' NH trans racemic 1-8 HN dioxane 2d./100* 0.3 452 2.81 H,N NH 1-9 /dioxanelO 24 h./100* 0.3 425 2.43 HN mI/DMF1 -NH 1-10 dioxanelO 24 h.1100* 0.3 410 2.51 HN Nml/DMF1 NH 1 1-11 -N N dioxanelO 24 h./1 DO. 0.3 424 2.50 mlDMF1 V12 N za 2: 0.3 398 24 dioxanelO 24 h.lDO 0.3 398 2.46 mlIDMF1 1-14 dioxanelO 24h./lD 0.3 412 2.47 mL'DMF1 1-15 H dioxanelO 24 h./100' 0.3 384 2.48 NN mIDMF1 NH 1-16H doaeD 24 hJ1100' 0.3 396 2.49 KN- NH -i/DMF1

MVMF

1.17 dioxanelO 24h.100* 0.3 511 2.38 mUDMF1 1-18 N dioxanelO 24 h./iC' 0.3 459 2.62 N N/ mUDMF1 1-19 dioxanelO 24 n.1100' 0.3 410 2.44 C N mlDMFI 1-20 dioxanelO 24 h.1100C 0.3 410 2.52 mVDMF1 1-21 dioxanelO 24 h.1100* 0.3 422 2.55 m11DMF1 C NH 3-S 1-22 H dioxanel0 24 h./100' 0.3 400 2.36 HO N '-eNh mLDMF1 1-23 H dioxanel1 24 h./100' 0.3 384 2.40 N2 mltDMF1 1-24 dioxanel0 24 h.1100' 0.3 440 2.58 N m V D M F 1 1-25 dioxanelO 24 h./1001 0.3 410 2.48 N- mVDMF1 1-26 dioxanel0 24 h.1100' 0.3 474 2.86 mI/DMF 1 i~s~NH Example 2: Parallel synthesis of substituted derivatives of NG-[6-amino-4-diethylamino-[1,3,5]triazin-2-yl]-2-methylquinoline-4, 6-diamine

N

N NN

NH

2 1-

H

K'3 33 Preparation of N6-(6-chloro-4-diethylamino- [1,3,5]triazin-2-yl)-2-methylquinoline-4,6-diamine 3.91 g (22.5 mmol) of 2-methylquinoline- 4,6-diamine, which may be prepared according to J. Med.

Chem. 1992, 35, 252, and 2.4 g (22.5 mmol) of sodium carbonate are successively added, in a 1 litre threenecked flask, to a solution of 5 g (22.5 mmol) of commercial 2,6-dichloro-4-diethylamino-[1,3,5]triazine in 300 ml of tetrahydrofuran. The reaction mixture is heated to reflux for 20 hours. After evaporation of the tetrahydrofuran, the residue is taken up in 400 ml of a mixture of water and dichloromethane (50-50 by volume).

The organic phase is separated after settling out, dried over sodium sulfate and concentrated to dryness under reduced pressure. 7.4 g of N6-(6-chloro-4diethylaminotriazin-2-yl)-2-methylquinoline-4,6-diamine are thus obtained in the form of a yellow solid whose characteristics are the following: melting point 120 0

C

H NMR spectrum (300 MHz, (CD 3 2 SO d6, 8 in ppm): 1.14 (mt: 6H); 2.42 3H); from 3.50 to 3.70 (mt: 4H); 6.47 (s and unresolved complex: 3H in total); 7.54 (broad d, J 9 Hz: 1H); 7.67 (dd, J 9 and 2 Hz: 1H); 8.27 (unresolved complex: 1H); 10.09 (unresolved complex: 1H).

Parallel synthesis of N6-[(6-(3-dimethylaminopropylamino)-4-diethylamino-[1,3,5]triazin-3-yl]-2-methylquinoline-4,6-diamine (Example 2-1) 34 mg (0.13 mmol) of N6-(6-chloro-4-diethylamino-[1,3,5]triazin-2-yl)-2-methylquinoline- 4,6-diamine are introduced into a heating magnetic reactor with a Zymark condenser, of the STEM RS2050 type, containing 25 wells in parallel each provided with a 50 ml glass tube. 5 ml of DMF, 19 mg (0.14 mmol) of potassium carbonate, 21 mg (0.14 mmol) of sodium iodide and 14 mg (0.14 mmol) of 3-dimethylaminopropylamine are successively added to the first tube (Example The reaction medium is heated at 1200C under argon for 16 hours. After cooling, the content of the tube is evaporated under reduced pressure and taken up in 5 ml of water, filtered and washed with diethyl ether. The crude product obtained is then purified by LC/MS using a Waters Xterra 3.5 gM C18 silica column 3 mm in diameter and 50 mm in length, eluting with a linear elution gradient consisting, at the starting time (to 0 min), of water containing 0.05% trifluoroacetic acid and, at the final time (tf 4 min), of acetonitrile containing 0.05% trifluoroacetic acid. 12 mg of N6-[(6-(3-dimethylaminopropylamino)-4-diethylamino-[1,3,5]triazin-2-yl]- 2-methylquinoline-4,6-diamine are thus obtained, after purification, whose characteristics are the following: mass spectrum (DAD-TIC) 423 (MH retention time 0.79 min (the retention times are obtained on a hypersil C 18 5 pm column 50 mm diameter 4.6 mm trade mark Purity Elite, eluting with a mixture of solvents A (H20/TFA 0.05%) and B (ACN/TFA 0.05%) with a linear gradient ranging from 95% A/5% B (t 0 min) to 10% A/90% B at t 3.5 min, then step 2 min).

Examples 2-1 to 2-2 were obtained by carrying out the procedure as above in a Zymark STEM RS2050 reactor. The structures, the various operating conditions used and the characteristics of Examples 2-1 to 2-2 are summarized in the table below: Example Structure Reaction conditions Characteristics AlkN(R'3)- No. of Mass Retention Solvent Heating mmol of MH' time amine (min) 2-1 DMF 16 h./120' 0.14 423 0.79 -,IN 2-2 DMF 16 h./120' 0.14 421 0.79 -N N 36 Table of biological results Example TRAP G-4 Cytotoxicity telomerase ATm A549 g.M 0 C IC50 M 1-1 0.79 6 1-2 0.5 5.6 1-3 4.4 3.1 1-4 0.1 5.6 1.6 2.8 1-6 1.36 1-7 0.47 1-8 0.98 1-9 1.64 7 1-10 0.94 7 1-11 1.1 1-12 3.1 1-13 1-14 3.2 1-15 4.6 1-16 1.29 1-17 1.6 1-19 1 1-20 3.1 1-21 0.7 1-22 1-23 3.8 1-24 1-25 1.5 1-26 0.86 33 3 2-1 0.90 7.9 2-2 5.4

Claims (3)

1. 27-Feb-2008 13:09 Watermark +61298887600 19/26 00 38 0 0 THE CLAIMS DEFINING THE INVENTION ARE AS FOLLOWS: 1. A compound which binds the G-quadruplex structure of a telomere, characterized in that it has the following general formula: nitrogen-containing aromatic ring NR 3 distribution agent NR' 3 nonaromatic hydrocarbon chain, nonaromatic hydrocarbon chain being selected from a linear 0 or branched alkyl (C1-C4), a linear or branched alkenyl (C2-C4) chain, a cycloalkyl (C3-Cio) chain, a cycloalkenyl (C3-C18) chain and a heterocycloalkyl (C 3 -C 8 a) chain Cl which optionally includes the nitrogen atom, O wherein 0 C 10 1) the nitrogen-containing aromatic ring represents: b) a quinoline optionally substituted with at least i) a group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a Cl-C4 alkyl radical or ii) a group ORa in which Ra is as defined above c) a quinoline possessing a nitrogen atom in quaternary form optionally substituted with a group selected from: 4-amino-, 4-methylamino- and 4-dimethylamino-, attached at the 4 position or d) a benzamidine or e) a pyridine, attached at the 4 position, 2) R 3 and R' 3 which are identical or different, represent independently of each other, hydrogen or a C1-C4 alkyl radical 3) the distribution agent represents: a triazine group, a triazine group substituted with i. a alkyl radical having 1 to 4 carbon atoms, ii. a thiol radical, iii. an hydroxy radical, or iv. an amino radical, v. wherein the thiol, hydroxy or amino radicals are unsubstituted or substituted with one or more short-chain alkyl groups containing 1 to 4 carbon atoms and wherein the alkyl is unsubstituted or substituted with a halogen atom or a salt thereof; COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:09 Watermark +61298887600 20/26 00 39 0 0 wherein the nonaromatic hydrocarbon chain is optionally substituted with one or .O more atoms or radicals chosen from halogen atoms, hydroxyl, aryl, heteroaryl, alkyloxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino, arylamino, dialkylamino, diarylamino, amidino, guanidine, alkylcarbonylamino, arylcarbonylamino, carboxyl, alkyloxycarbonyl or aryloxycarbonyl, aminocarbonyl, alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, alkycarbonyl 0 arylcarbonyl, cyano and trifluoromethyl. S2. The compound according to claim 1, wherein the distribution agent is a Striazine group. 3. The compound according to claim 1, wherein the nonaromatic hydrocarbon chain is chosen from the following: i) alkyl (C1-C4), alkenyl (C2-C4), wherein the alkyl and alkenyl are linear or branched, ii) cycloalkyl (C3-C18) iii) cycloalkenyl (C3-C18) iv) heterocycloalkyl (C3-Cia) and v) heterocycloakyl (C3-Cia) including the nitrogen atom of the NR' 3 group. 4. A compound according to claim 1, wherein the alkyl chains of the optional substituents of the hydrocarbon chain contain 1 to 4 carbon atoms and the aryl groups of the optional substituents of the hydrocarbon chain contain 5 to 18 carbon atoms. A compound according to claim 3, wherein the alkyl chains contain 2 to 3 carbon atoms, and the heterocycloalkyl or cycloalkyl chains contain 5 to 7 carbon atoms. 6. A compound according to claim 1, wherein it corresponds to formula (I) below: COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:10 Watermark +61298887600 21/26 00 0 A N N A RN N NR'3 Ci Ar, Alk in which: IO 1) A represents 00 (0 a) an amino group of formula NR1R 2 in which Ri and Rz, which are identical or different, represent hydrogen or a straight or branched alkyl Sgroup containing 1 to 4 carbon atoms or o b) a group OR 1 or SR 1 in which R, has the same meaning as above or c) an alkyl group containing 1 to 4 carbon atoms or a trifluoromethyl group or d) a hydrogen atom 2) R 3 and R'3, which are identical or different, represent independently of each other hydrogen or a CI-C4 alkyl group 3) Arl represents a nitrogen-containing aromatic ring representing: a) a quinoline optionally substituted with at least i) a group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a CI-C4 alkyl radical or ii) a group ORa in which Ra is as defined above b) a quinoline possessing a nitrogen atom in quaternary form optionally substituted with a group selected from: 4-amino-, 4-methylamino- and 4- dimethylamino-, attached at the 4 position or c) a benzamidine or d) a pyridine attached at the 4-position 4) Alk represents a nonaromatic, optionally substituted, hydrocarbon chain as hereinbefore defined, chosen from alkyl (C1-C4), alkenyl (C2-C4), wherein the alkyl and alkenyl chain are linear or branched, cycloalkyl (C3-Ce), cycloalkenyl (C 3 -C 1 8 heterocycloalkyl (C 3 -C 18 and heterocycloalkyl (C3-C 18 including the nitrogen atom of the NR'3 group or a salt thereof. COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:11 Watermark +61298887600 22/26 00 41 0 o 7. A compound according to claim 6, wherein Arj is chosen from: 4-amino-, 4- Smethylamino-, 4-dimethylamino- quinolyl, and quinolinium. CD) 8. A compound according to claim 6, wherein A is chosen from: a thiomethyl, amino, alkylamino and dialkylamino, in which the alkyl groups posssess 1 to 4 carbon atoms. O 0 9. A compound according to claim 6, wherein A represents a methylthio Cgroup. 0 0 10. A compound according to claim 6, wherein Alk represents an alkyl containing 2 to 3 linear or branched carbon atoms, wherein the alkyl is substituted with at least one of the following: i) an amino, alkylamino, arylamino, dialkylamino or diarylamino, or ii) an alkenyl unit containing 2 to 3 carbon atoms, which is substituted with at least one substituent selected from an amino, alkylamino, arylamino, dialkylamine, diarylamino and heterocyclyl, or iii) cycloalkyl unit containing from 4 to 7 carbon atoms. 11. A compound according to claim 6, wherein Alk represents a 2- (dialkylamino)ethyl, 3-(dialkylamino)propyl, 2-(N-alkyl-N-arylamino)ethyl or 3-(N- alkyl-N-arylamino)propyl in which the alkyl groups contain 1 to 4 carbon atoms and the aryl groups contain 5 to 18 carbon atoms. 12. A compound according to claim 6, characterized in that alk represents 2- (N-m-tolyl-N-ethylamino)ethyl. 13. A compound of claim 1, for use as a telomerase-inhibiting agent. 14. A compound according to any one of the preceding claims, for anticancer use. 15. A compound corresponding to the following formula COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:12 Watermark +61298887600 23/26 00 42 0 0 A "RN N NR' Ar., alk 00 in which: N 1) A represents Sa) an amino group of formula NR 1 R 2 in which R 1 and R 2 which are S 5 identical or different, represent hydrogen or a straight or branched alkyl group containing 1 to 4 carbon atoms or b) a group OR 1 or SR, in which R 1 has the same meaning as above or c) an alkyl group containing 1 to 4 carbon atoms or a trifluoromethyl group or d) a hydrogen atom or e) a halogen atom chosen from fluorine, chlorine, bromine and iodine 2) R 3 and R' 3 which are identical or different, represent independently of each other a hydrogen or a C1-C4 alkyl group 3) Ar represents a nitrogen containing aromatic ring representing: a) a quinoline optionally substituted with at least i) a group N(Ra)(Rb) in which Ra and Rb, which are identical or different, represent hydrogen or a C1-C4 alkyl radical or ii) a group ORa in which Ra is as defined above b) a quinoline possessing a nitrogen atom in quaternary form optionally substituted with a group selected from: 4-amino-, 4-methylamino- and 4- dimethylamino-, attached at the 4 position or c) a benzamidine or d) a pyridine attached at the 4-position or fused with an aryl or heteroaryl group optionally substituted with a C1-C4 alkyl group 4) alk represents a nonaromatic, optionally substituted, hydrocarbon chain as hereinbefore defined, chosen from alkyl (C1-C4), alkenyl (C2-C4) wherein the alkyl and alkenyl chain are linear or branched, cycloalkyl (C3-CIa), cycloalkenyl (C3-C 18 or heterocycloalkyl (C3-C8) chain optionally including the nitrogen COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:12 Watermark +61298887600 24/26 00 43 0 0 atom of the NR'3 group Sor a salt thereof C) wherein the nonaromatic hydrocarbon chain is optionally substituted with one or more atoms or radicals chosen from halogen atoms, hydroxyl, aryl, heteroaryl, alkyloxy, aryloxy, thio, alkylthio, arylthio, amino, alkylamino and/or arylamino, VB dialkylamino, diarylamino, amidino, guanidine, alkylcarbonlamino, 00 0arylcarbonlamino, carboxyl, alkyloxycarbonyl, aryloxycarbonyl, aminocarbonyl, Ci alkylaminocarbonyl, arylaminocarbonyl, dialkylaminocarbonyl, alkylcarbonyl, 0 arycarbonyl, cyano and trifluoromethyl. 16. A compound according to claim 15, wherein Arl represents a group chosen from: 4-amino-, 4-methylamino-, 4-dimethylamino-quinolyl and quinolinium. 17. A compound according to claim 15, wherein group A represents a thiomethyl, amino, alkylamino or dialylamino, in which the alkyl groups possess 1 to 4 carbon atoms. 18. A compound according to claim 15, wherein R 1 and R2 represent hydrogen. 19. A compound according to claim 17, wherein A represents a methylthio group. A compound according to claim 15, wherein alk represents: i) an alkyl unit containing 2 to 3 linear or branched carbon atoms which is substituted with at least one substituent selected from: an amino, alkylamino, arylamino, dialkylamino and diarylamino. ii) an alkenyl unit containing 2 to 3 carbon atoms, which is substituted with at least one substituent selected from: an amino, alkylamino, arylamino, dialkylamino and diarylamino, or iii) a heterocyclyl unit containing from 4 to 7 carbon atoms. 21. A compound according to claim 15, wherein alk represents 2- COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 13:13 Watermark +61298887600 25/26 00 44 0 0 (dialkylamino)ethyl, 3-(dialylamino)propyl, 2-(N-alkyl-N-arylamino)ethyl or 3-(N- alkyl-N-arylamino)propyl, in which the alkyl groups contain 1 to 4 carbon atoms C) and the aryl groups contain 5 to 18 carbon atoms. 22. A compound according to claim 15, wherein alk represents a 2-(N-m-tolyl- N-ethylamino)ethyl chain. 00 23. Use of the compound of claim 15, as pharmaceutical product for human Ci use. 0 O 24. A therapeutic combination consisting of a compound according to claim 1 and one or more other anticancer compounds. 25. A combination according to claim 24, wherein the one or more other anticancer compounds are chosen from: alkylating agents, platinum derivatives, antibiotic agents, antimicrotubule agents, anthracyclines, group I and II topoisomerases, fluoropyrimidines, cytidine analogues, adenosine analogues, L- asparaginase, hydroxyurea, trans-retinoic acid, suramine, irinotecan, topotecan, dexrazoxane, amifostine, herceptin, estrogenic and androgenic hormones. 26. Therapeutic combination consisting of a compound according to claim 1 and of radiation. 27. A combination according to claim 25 or claim 26, wherein each of the compounds or treatments is administered simultaneously, separately or sequentially.
2. 28. A method of inhibiting telomerase activity, the method including administering to a patient a therapeutically effective amount of at least one compounds of any one of claims 1 to 22, wherein the level of telomerase activity in the patient following the administration is reduced relative to the level of telomerase activity existing prior to the administration. COMS ID No: ARCS-180904 Received by IP Australia: Time 13:18 Date 2008-02-27 27-Feb-2008 16:34 Watermark +61298887600 3/4 00 0 0
3. 29. A method of treating a cancer, the method including administering to a patient a therapeutically effective amount of at least one compound of any one of 00 claims 1 to 22, wherein the level of telomerase activity following the administration is reduced relative to the level of telomerase activity existing prior to the administration. 00 AVENTIS PHARMA S.A. C WATERMARK PATENT TRADE MARK ATTORNEYS O 0 P22968AU00 ci COMS ID No: ARCS-180965 Received by IP Australia: Time 16:38 Date 2008-02-27