FIELD OF THE INVENTION

The present disclosure relates to a novel compounds for use in treatment or prevention of cancer, in particular to use of said compounds as an inhibitors of interactions between TRF1-TIN2 or TRF2-TIN2 telomeric proteins.

Background of the invention

Telomeres are nucleoprotein structures at the end of eukaryotic chromosomes. These complexes are composed of non-coding fragments of DNA with the six proteins collectively called shelterin [1-3]. Telomeric DNA of the vertebrae consists of many repeats of the six nucleotides (TTAGGG), and its length varies in 5-15 kbp in humans and up to 100 kbp in rodents [4], Main function of the telomeres is protection of the terminal fragments of the linear chromosome. Length of the telomere is decreasing during every cell division. Critical shortening of the telomeres leads to arrest of the proliferation of cells and eventually to their senescence or apoptosis. As a result, after the fixed number of population doublings (PDs) cells stop proliferation and number of cells in the culture reach a plateau. Estimated maximum number of PDs to reach cellular senescence was defined as a Hayflick limit [5]. In most somatic cells of the adult organism telomerase becomes dormant. In cancerous cells telomerase is reactivated in order to achieve the replicative immortality of cells [6, 7],

Due to the fact that telomerase is reactivated in most cancerous cells it became a promising target for anticancer chemotherapy [6, 8, 9]. However, despite the fact that telomeres of cancerous cells are usually shorter than telomeres of surrounding cells, decreasing of telomere lengthening by inhibition of telomerase would not cause the immediate cytotoxic effect [10]. Studies in mouse models and in human clinical trials have shown some benefits of telomerase inhibitors (especially Imetelstat) in myeloid malignancies but limitations concerning solid tumors [11-15]. In consequence still there are no clinically approved strategies exploiting this cancer target [15].

Therefore, other proteins from shelterin complex have been considered as potential targets for anticancer therapy. Shelterin complex is composed of six proteins: telomeric repeat binding factor 1 (TRF1 ), and 2 (TRF2), repressor/activator protein (RAP1 ), protection of telomeres protein (POT1), TRF1 -interacting nuclear protein 2 (TIN2), and TIN2- and POT1 -interacting protein (TPP1 ) [16, 17]. This shelterin complex binds specifically to telomeric DNA [16, 18, 19]. These capping structures have the crucial function of maintaining genome stability by protecting the chromosome end from being recognized as DNA double-strand breaks (DSBs) [18]. They also represent challenging structures for the replication machinery, which is associated with telomere fragile sites [20-22],

TRF1 is a key member of the shelterin complex. TRF1 and TRF2 proteins directly bind DNA TTAGGG telomere repeats and assemble the remaining shelterin proteins therefore TRFs are critical determinants of telomere’s protection [16, 23]. TRF1 comprising 439 amino acids possesses a specific conserved domain (TRFH) which assists in the formation of a stable homodimeric TRF1-TRF1 structure. TRFTs myb- domains (two per homodimer) create dimer stable interaction with the duplex DNA at the telomere. TRF1 plays a key role in the assembly of the shelterin complex by recruiting/binding to Telomere Repeat Binding Factor2 (TRF2) via TRF-1 Interacting Nuclear Protein-2 (TIN2) [19, 24, 25]. TIN2 protein is the central hub of the shelterin complex. TIN2 directly binds to and consequently stabilizes the TRF1 through two distinct mechanisms. First, TIN2 protects TRF1 from tankyrase 1 -mediated poly(ADP- ribosyl)ation, which in turn ensures TRFTs association with telomeres [26]. Second, TIN2 competes with SCFFBX4 for binding to TRF1 , thus preventing TRF1 from ubiquitin-dependent proteolysis [18]. Because of the important functions of TRF1 and TRF1 -recruited TIN2 protein in telomere maintenance, the generation of small molecular compounds which bind to TRF1 and interfere with its coupling to TIN2 offer a potential tool to dissect the molecular mechanism of TRF-1 -TIN2 interactions and may become a tool for destabilizing the whole shelterin complex, thus breaching cancer’s survival strategy. This strategy has started to be explored also by other groups showing that small molecules may modulate or inhibit TRF1/2 functions [20, 27-29].

To this end it is a need to search or design novel TRF1/2 inhibitors which can impair function of these proteins in cancer cells. The present invention meets these needs.

SUMMARY OF THE INVENTION

To this end it is a need to search or design novel TRF1/2 inhibitors which can impair function of these proteins in cancer cells. The present invention meets these needs. The present invention is defined in the appended claims. Embodiments and examples not falling under these claims are for reference purposes only.

The first aspect of the present invention relates to a compound of Formula I:

Formula I or compound of Formula II:

Formula II wherein: n is integer between 1 and 3,

Het-Ar is heteroaryl, possibly substituted, selected among of the following groups: wherein: X means S, O or NH, and R means CH3, C2H5, OCH3, Cl or Br

R ¹ is one of the following groups: or a pharmaceutically acceptable salt or a solvate thereof as an inhibitors of interactions between TRF1-TIN2 or TRF2-TIN2 telomeric proteins for use in the treatment or the prevention of cancer.

Preferably, said compound has been selected among of following compounds: The second aspect of subject invention relates to compound of of Formula I:

Formula I or compound of Formula II:

Formula II wherein: n is integer between 1 and 3,

Het-Ar is heteroaryl, possibly substituted, selected among of the following groups: wherein: X means S, O or NH, and R means CH3, C2H5, OCH3, Cl or Br

R ¹ is one of the following groups: or a pharmaceutically acceptable salt or a solvate thereof, possibly except for the following compounds:

Preferably, said compound has been selected among of following compounds:

The synthesis pathway for compounds according to Formula I is defined by the following general approach:

The chloroquinoline derivative 1 was hydrolyze in acidic condition by refluxing in cone. HCI during 12 h. Pirydone 2b was treated with heteroarylmethylamine and subsequently reduced with sodium borohydride to amine 3 in one pot procedure. Amine 3 reacts with appropriate isothiocyanate to produce final product 5. (Scheme below)

The synthesis pathway for compounds according to Formula II is defined by the following general approach: The acid 1 was protected on amino group with Di-tert-butyl pyrocarbonate, and when activated with TBTU and coupled with amine H2N-R1 on standard procedure, to obtain amide 3. Amide 3 was deported with TFA in DCM to produce compound 4. In last step 4 was treated with aliphatic aldehyde (possessing HetAr moiety) and subsequently reduced with sodium borohydride to obtain final product 5. (Scheme below)

The compounds of the present disclosure invention may also be present in the form of pharmaceutically acceptable salts. For use in medicine, the salts of the compounds of this invention refer to non-toxic " pharmaceutically acceptable salts" (Ref International J. Pharm., 1986, 33, 201-217 ; J. Pharm.Sci., 1997 (Jan), 66, 1 , 1 ). Other salts well known to those in the art may however be useful in the preparation of the compounds according to this disclosure or of their pharmaceutically acceptable salts. Representative organic or inorganic acids include, but are not limited to, hydrochloric, hydrobromic, hydriodic, perchloric, sulfuric, nitric, phosphoric, acetic, propionic, glycolic, lactic, succinic, maleic, fumaric, malic, tartaric, citric, benzoic, mandelic, methanesulfonic, hydroxyethanesulfonic, benzenesulfonic, oxalic, pamoic, 2- naphthalenesulfonic, p-toluenesulfonic, cyclohexanesulfamic, salicylic, saccharinic or trifluoroacetic acid. Representative organic or inorganic bases include, but are not limited to, basic or cationic salts such as benzathine, chloroprocaine, choline, diethanolamine, ethylenediamine, meglumine, procaine, aluminum, calcium, lithium, magnesium, potassium, sodium and zinc.

The compounds according to the subject invention may be present in the pharmaceutical composition in the form used for parenteral, oral, rectal, replacement or transdermal administration. Thus, they will be presented in the form of injectable solutions or suspensions or multiple dose vials in the form of ordinary or coated tablets, dragees, wafer capsules, gel capsules, pills, cachets, powders, suppositories, or rectal capsules, for transdermal use in polar solvents, or for fixed use.

Suitable excipients for this application are cellulose or microcrystalline cellulose derivatives, alkaline earth metal carbonates, magnesium phosphates, starches, modified starches and lactose in solid form.

For rectal use, cocoa butter or polyethylene glycol stearate is a preferred excipient. For parenteral use, water, aqueous solutions, physiological saline and isotonic solutions are the most suitable carriers for use.

It is also apparent to one skilled in the art that the therapeutically effective dose for active compounds of the disclosure or a pharmaceutical composition thereof will vary according to the desired effect. Therefore, optimal dosages to be administered may be readily determined and will vary with the particular compound used, the mode of administration, the strength of the preparation, and the advancement of the disease condition. In addition, factors associated with the particular subject being treated, including subject age, weight, diet and time of administration, will result in the need to adjust the dose to an appropriate therapeutic level. The above dosages are thus exemplary of the average case. There can, of course, be individual instances where higher or lower dosage ranges are merited, and such are within the scope of this disclosure.

Compounds of the present disclosure may be administered in any of the foregoing compositions and dosage regimens or by means of those compositions and dosage regimens established in the art whenever use of the compounds of the present disclosure as it is required for a subject in need thereof.

The present disclosure also provides a pharmaceutical or veterinary pack or kit comprising one or more containers filled with one or more of the ingredients of the pharmaceutical and veterinary compositions of the disclosure. Optionally associated with such container(s) can be a notice in the form prescribed by a governmental agency regulating the manufacture, use or sale of pharmaceuticals or biological products, which notice reflects approval by the agency of manufacture, use or sale for human administration. The compounds of the present disclosure may be used to treat or prevent the cancer in warm-blooded animals such as humans by administration of an anticancer effective dose. The dosage range would be from about 0.1 mg to about 15,000 mg, in particular from about 50 mg to about 3500 mg or, more particularly from about 100 mg to about 1000 mg of active ingredient in a regimen of about 1 to 4 times per day for an average (70 kg) human; although, it is apparent to one skilled in the art that the therapeutically effective amount for active compounds of the present disclosure will vary as will the types of cancer being treated.

For oral administration, a pharmaceutical composition is preferably provided in the form of tablets containing 0.01 , 10.0, 50.0, 100, 150, 200, 250, and 500 milligrams of the active ingredient for the symptomatic adjustment of the dosage to the subject to be treated. Examples of cancer as mentioned in the present invention include, but are not limited to, breast and lung cancer.

DESCRIPTION OF THE FIGURES

Figure 1 presents on upper panel Compound A822 (Asinex ASN 03579822) and compound B327 (Biofocus 179_0115_0327) - lower panel.

Figure 2 presents SPR analysis of A822 binding to TRF1 protein and its influence on TRF1-TIN2 interaction. (A) The interaction of A822 with TRF1 protein was analyzed when TRF1 protein was immobilized on a surface of CM4 sensor chip using the amine coupling method. Then the increasing concentrations of A822 (20, 30, 60 pM) were run over the surface of a sensor chip with immobilized TRF1 protein. (B) The interaction of TRF1 protein with TIN2 peptide was analyzed after the immobilization of biotinylated TIN2 peptide was on a surface of SA sensor chip. Next, the increasing concentrations of TRF1 protein (3, 6, 12.5, 25, 50, 100, 250, 500 nM) were run over the surface of a sensor chip with an immobilized peptide. (C) The biotinylated TIN2 peptide was immobilized on the SA sensor chip Surface. Next 100nM TRF1 protein, 5pM A822, or a mixture of both were injected, and the obtained responses were detected. In all analyses, the results are presented as sensorgrams obtained after subtracting the background response signal from a reference flow cell and a control experiment with buffer injection. At least three kinetic experiments were performed for kinetic analysis (A and B). In C, the sensorgrams show the average from six independent experiments. (D) Kinetic constants calculated from SPR’s data for analytes (TIN2 peptide and A822) interacting with TRF1 protein. Constants were calculated with Biacore T200 Evaluation Software using data from at least two separate titration analysis. The 1 :1 binding model was applied. SD - standard deviation.

Figure 3 presents SPR analysis of B327 binding to TRF2 protein and its influence on TRF2-TIN2 interaction. (A) The interaction of 327 with TRF2 protein was analyzed when TRF2 protein was immobilized on a surface of CM4 sensor chip using the amine coupling method. Then the increasing concentrations of 327 (20, 30, 60 pM) were run over the surface of a sensor chip with immobilized TRF2 protein. (B) The interaction of TRF2 protein with TIN2 peptide was analyzed after the immobilization of biotinylated TIN2 peptide was on a surface of SA sensor chip. Next, the increasing concentrations of TRF2 protein (3, 6, 12.5, 25, 50, 100, 250, 500 nM) were run over the surface of a sensor chip with an immobilized peptide. (C) The biotinylated TIN2 peptide was immobilized on the SA sensor chip Surface. Next 100nM TRF2 protein, 5pM 327, or a mixture of both were injected, and the obtained responses were detected. In all analyses, the results are presented as sensorgrams obtained after subtracting the background response signal from a reference flow cell and a control experiment with buffer injection. At least three kinetic experiments were performed for kinetic analysis (A and B). In C, the sensorgrams show the average from six independent experiments. (D) Kinetic constants calculated from SPR’s data for analytes (TIN2 peptide and 327) interacting with TRF2 protein. Constants were calculated with Biacore T200 Evaluation Software using data from at least two separate titration analysis. The 1 :1 binding model was applied. SD - standard deviation.

Figure 4 presents determination of p-galactosidase activity in the population of cells of the MCF7 cell line after exposure to the following compounds: A822 (10 pM) or B327 (100 pM) after 5-day incubation. Left panel - representative fields of view 20x objective lens; area delineated with white rectangular outline were magnified and presented in the right panel. Scale bars correspond to 20 pm.

Figure 5 presents qPCR amplification curves using compounds. Cell lines - MCF7. Telomere length - sampled without treatment =yellow, samples after treatment = brown, negative control =silver SCR length - sampled without treatment =green, samples after treatment = blue, negative control = red.

Figure 6 presents: A) Immunostaining images of MCF7 cells showing co-localization between TRF1 (green) and TIN2 (red) proteins (control - 10pM DMSO and cells after treatment of 10pM A822 compound). Telomeres were identified by immunostaining using a mix of anti-TRF1 and anti-TIN2 antibodies. DNA was stained by DAPI (blue). Incubation time - 72h;

B) Quantification of TRF1 and TIN2 co-localization in MCF7 cells - control (75pM DMSO) and after treatment of A822 compound (10 pM, incubation time - 72h) with used Pearson's correlation coefficient (r). Plot shows the average number of TRF1- TIN2 co-localization per nucleus. All quantifications were carried out blindly. Each point on the plot represents a value obtained from one image (cells' nuclei). Mean values are indicated in red.

Figure 7 presents A) Immunostaining images of MCF7 cells showing co-localization between TRF2 (green) and TIN2 (red) proteins (control - 75pM DMSO and cells after treatment of 75pM B327 compound) . Telomeres were identified by immunostaining using a mix of anti-TRF2 and anti-TIN2 antibodies. DNA was stained by DAPI (blue). Incubation time - 72h;

B) Quantification of TRF2 and TIN2 co-localization in MCF7 cells - control (75pM DMSO) and after treatment of B327 compound (75 pM, incubation time - 72h) with used Pearson's correlation coefficient (r). Plot shows the average number of TRF2- TIN2 co-localization per nucleus. All quantifications were carried out blindly. Each point on the plot represents a value obtained from one image (cells' nuclei). Mean values are indicated in red.

DETAILED DESCRIPTION OF THE INVENTION

The present invention stems from the previous in silica molecular dynamics studies of TRF proteins as potential targets for the small molecules inhibition of TIN2 binding to TRF1/2 [30, 31]. Based on these simulations an internal pharmacophore model for in silica high-throughput-screening (HTS) was elaborated and about 20 small molecules have been selected as potential inhibitors of TRF1/2-TIN2 interactions. Asinex and Biofocus databases were selected for this HTS study [32, 33]. Two of these 20 molecules (Fig. 1) exhibited interesting TRF1/2 modulation and anticancer properties. A822 was selected as TRF1 inhibitor and B327 as TRF2 inhibitor.

Examples

Example 1. Obtaining compounds according to the invention

Compounds according to Formula I Step 1. Synthesis of 6,7-dimethyl-2-oxo-1 H-quinoline-3-carbaldehyde.

Into the 10mL round bottom flask was weighted 2-chloro-6,7-dimethyl-1 ,2- dihydroquinoline-3-carbaldehyde in amount of 150 mg (0.683mmol) swollen by 5mL of acetic acid and 0.3mL of distilled water. The reaction was performed under reflux conditions (80°C) and under argon pressure, for 4-5 hours. The progress of the reaction was monitored by thin-layer chromatography(TLC). After the reaction the product was crystallized, filtered under lower pressure, washed three times with 2 mL of AcOH, and dried under vacuum pump.

Step 2a Method A. Synthesis of 6,7-dimethyl-3-[(2-thienylmethylamino)methyl]-1 H- quinolin-2-one.

Into the 10mL round bottom flask was weighted 6,7-dimethyl-2-oxo-1 H-quinoline-3- carbaldehyde in amount of 100mg (0.496mmol, 1 Eq) swollen by 5mL acidic acid. Following that, was added 2-thienylmethanamine in amount of 112mg (0.992mmol, 2Eq), subsequently the reaction mixture was refluxed at 100° C for 4 hours, the progress of the reaction was checked by TLC.

The next step was acetic acid evaporation from the intermediate product under lower pressure in rotavapor to dry, and then swollen with ethanol. After that, to the intermediate product, was added sodium cyanoborohydride (683 mg, 9.92mmol), stirred at room temperature, under argon pressure, overnight. Reaction was monitored by thin-layer chromatography, after completion of the reaction, the product was dried, by rotavapor, and washed from by-products by extraction. The procedure for extraction was performed as following: dichloromethane : 0.5M hydrochloric acid (1 :1 v/v), in volume of 5mL, repeated three times, following that, the organic layer was neutralize by 3 x 5mL of sodium hydroxide. The organic layer was collected, dried with magnesium (VI) sulfate, filtered and dried in vacuum pump.

Step 2a Method B. Synthesis of 6,7-dimethyl-3-[(2-thienylmethylamino)methyl]-1 H- quinolin-2-one.

Into the 10mL round bottom flask was weighted 6,7-dimethyl-2-oxo-1 H-quinoline-3- carbaldehyde in amount of 101 mg (0.5mmol, 1 Eq) swollen by 5mL iPrOH. Following that, was added 2-thienylmethanamine in amount of 79,1mg (0.7mmol, 1.4Eq), subsequently the reaction mixture was refluxed at boiling point for 8 hours, the progress of the reaction was checked by TLC. Resulting solid was filtred and dried. 1 H NMR spectrum was performed to confirm structure of product

Crude imine was suspended in 5mL od iPrOH and sodium borohydride (91 mg, 2.4 mmol) was added. Reaction mixture was stirred at room temperature, under argon pressure, overnight. Reaction was monitored by thin-layer chromatography, after completion of the reaction, solvents was removed under reduced pressure. Water was added to the residue and stirred for 2 h, precipitate was filterd and dried in vacuum. 1 H NMR spectrum was performed to confirm structure of product

Step 2b. Method A Synthesis of 6,7-dimethyl-3-[(2-pyridylmethylamino)methyl]-1 H- quinolin-2-one.

Into the 10mL round bottom flask was weighted 6,7-dimethyl-2-oxo-1 H-quinoline-3- carbaldehyde in amount of 100mg (0.496mmol, 1 Eq) swollen by 5mL acidic acid. Following that, was added 2-pyridylmethanamine in amount of 107mg (0.992mmol, 2Eq), subsequently the reaction mixture was refluxed at 100° C for 4 hours, the progress of the reaction was checked by TLC. The next step was acetic acid evaporation from the intermediate product under lower pressure in rotavapor to dry, and then swollen with ethanol. After that, to the intermediate product, was added sodium cyanoborohydride (683 mg, 9.92mmol), stirred at room temperature, under argon pressure, overnight. Reaction was monitored by thin-layer chromatography, after completion of the reaction the product was dried, by rotavapor, and washed from by-products by extraction. The procedure for extraction was performed as following: dichloromethane : 0.5M hydrochloric acid (1 :1 v/v), in volume of 5mL, repeated three times, following that, the organic layer was neutralize by 3 x 5mL of sodium hydroxide. The organic layer was collected, dried with magnesium (VI) sulfate, filtered and dried in vacuum pump.

Step 2b Method B. Synthesis of 6,7-dimethyl-3-[(2-pyridylmethylamino)methyl]-1 H- quinolin-2-one.

Into the 10mL round bottom flask was weighted 6,7-dimethyl-2-oxo-1 H-quinoline-3- carbaldehyde in amount of 101 mg (0.5mmol, 1 Eq) swollen by 5mL iPrOH. Following that, was added 2-pyridylmethanamine in amount of 75,6mg (0.7mmol, 1.4 Eq), subsequently the reaction mixture was refluxed at boiling point for 8 hours, the progress of the reaction was checked by TLC. Resulting solid was filtred and dried.

Crude imine was suspended in 5mL od iPrOH and sodium borohydride (91 mg, 2.4 mmol) was added. Reaction mixture was stirred at room temperature, under argon pressure, overnight. Reaction was monitored by thin-layer chromatography, after completion of the reaction, solvents was removed under reduced pressure. Water was added to the residue and stirred for 2 h, precipitate was filterd and dried in vacuum.

Step 2c. Method A. Synthesis of 3-[(2-furylmethylamino)methyl]-6,7-dimethyl-1 H- quinolin-2-one. Into the 10mL round bottom flask was weighted 6,7-dimethyl-2-oxo-1 H-quinoline-3- carbaldehyde in amount of 100mg (0.496mmol, 1 Eq) swollen by 5mL acidic acid. Following that, was added 2-furylmethanamine in amount of 96mg (0.992mmol, 2Eq), subsequently the reaction mixture was refluxed at 100° C for 4 hours, the progress of the reaction was checked by TLC.

The next step was acetic acid evaporation from the intermediate product under lower pressure in rotavapor to dry, and then swollen with ethanol. After that, to the intermediate product, was added sodium cyanoborohydride (683 mg, 9.92mmol), stirred at room temperature, under argon pressure, overnight. Reaction was monitored by thin-layer chromatography, after completion of the reaction, the product was dried, by rotavapor, and washed from by-products by extraction. The procedure for extraction was performed as following: dichloromethane : 0.5M hydrochloric acid (1 :1 v/v), in volume of 5mL, repeated three times, following that, the organic layer was neutralize by 3 x 5mL of sodium hydroxide. The organic layer was collected, dried with magnesium (VI) sulfate, filtered and dried in vacuum pump.

Step 2c Method B. Synthesis of 3-[(2-furylmethylamino)methyl]-6,7-dimethyl-1 H- quinolin-2-one.

Into the 10mL round bottom flask was weighted 6,7-dimethyl-2-oxo-1 H-quinoline-3- carbaldehyde in amount of 101 mg (0.5mmol, 1 Eq) swollen by 5mL iPrOH. Following that, was added 2-pyridylmethanamine in amount of 67,9mg (0.7mmol, 1.4 Eq), subsequently the reaction mixture was refluxed at boiling point for 8 hours, the progress of the reaction was checked by TLC. Resulting solid was filtred and dried.

Crude imine was suspended in 5mL od iPrOH and sodium borohydride (91 mg, 2.4 mmol) was added. Reaction mixture was stirred at room temperature, under argon pressure, overnight. Reaction was monitored by thin-layer chromatography, after completion of the reaction, solvents was removed under reduced pressure. Water was added to the residue and stirred for 2 h, precipitate was filterd and dried in vacuum. Step 3a. 1 -((6,7-dimethyl-2-oxo-1 ,2-dihydroquinolin-3-yl)methyl)-3-(3-

(dimethylamino)propyl)-1-(thiophen-2-ylmethyl)thiourea

6,7-dimethyl-3-[(2-thienylmethylamino)methyl]-1 H-quinolin-2-one (54mg, 0.18mmol) was weighted and poured into 10mL round bottom flask and swollen with 2mL of dioxane. Following that, 3-isothiocyanato-N,N-dimethylpropan-1-amine was weighted in amount of 38 mg(0.27mmol, 1.5 Eq) and added to the mixture, the reaction was conducted at 80°C in oil bath, under argon pressure. The progress of the reaction was monitored by TLC, after 1 h reaction was completed. After cooling to RT precipitate was formed. Precipitate was filtered washed with dithyl ether and dried under rediced pressiure.

1 H NMR spectrum was performed to confirm structure of product.

Step 3b. 1 -((6,7-dimethyl-2-oxo-1 ,2-dihydroquinolin-3-yl)methyl)-3-(3-

(dimethylamino)propyl)-1-(pyridin-2-ylmethyl)thiourea

6,7-dimethyl-3-[(2-pyridylmethylamino)methyl]-1 H-quinolin-2-one (53mg, 0.18mmol) was weighted and poured into 10mL round bottom flask and swollen with 2mL of dioxane. Following that, 3-isothiocyanato-N,N-dimethylpropan-1 -amine was weighted in amount of 38 mg (0.27mmol, 1.5 Eq) and added to the mixture, the reaction was conducted at 80°C in oil bath, under argon pressure. The progress of the reaction was monitored by TLC, after 1 h reaction was completed. After cooling to RT precipitate was formed. Precipitate was filtered washed with dithyl ether and dried under rediced pressiure. Step 3c. 1-((6,7-dimethyl-2-oxo-1 ,2-dihydroquinolin-3-yl)methyl)-3-(3- (dimethylamino)propyl)-1-(furan-2-ylmethyl)thiourea

3-[(2-furylmethylamino)methyl]-6,7-dimethyl-1 H-quinolin-2-one (51 mg, 0.18mmol) was weighted and poured into 10mL round bottom flask and swollen with 2mL of dioxane. Following that, 3-isothiocyanato-N,N-dimethylpropan-1 -amine was weighted in amount of 38 mg (0.27mmol, 1.5 Eq) and added to the mixture, the reaction was conducted at 80°C in oil bath, under argon pressure. The progress of the reaction was monitored by TLC, after 1 h reaction was completed. After cooling to RT precipitate was formed. Precipitate was filtered washed with dithyl ether and dried under rediced pressiure. Step 3d. Synthesis of 1-[(6,7-dimethyl-2-oxo-1 H-quinolin-3-yl)methyl]-3-(2- morpholinoethyl)-1-(2-thienylmethyl)thiourea.

6,7-dimethyl-3-[(2-thienylmethylamino)methyl]-1 H-quinolin-2-one (54mg, 0.18mmol) was weighted and poured into 10mL round bottom flask and swollen with 2mL of dioxane. Following that, 4-(2-isothiocyanatoethyl)morpholine was weighted in amount of 46 mg (0.27mmol, 1.5 Eq) and added to the mixture, the reaction was conducted at 80°C in oil bath, under argon pressure. The progress of the reaction was monitored by TLC, after 1 h reaction was completed. After cooling to RT precipitate was formed. Precipitate was filtered washed with dithyl ether and dried under reduced pressure Step 3f. Synthesis of 1-[(6,7-dimethyl-2-oxo-1 H-quinolin-3-yl)methyl]-3-(2- morpholinoethyl)-1-(2-pyridylmethyl)thiourea.

6,7-dimethyl-3-[(2-pyridylmethylamino)methyl]-1 H-quinolin-2-one (53mg, 0.18mmol) was weighted and poured into 10mL round bottom flask and swollen with 2mL of dioxane. Following that, 4-(2-isothiocyanatoethyl)morpholine was weighted in amount of 46 mg (0.27mmol, 1.5 Eq) and added to the mixture, the reaction was conducted at 80°C in oil bath, under argon pressure. The progress of the reaction was monitored by TLC, after 1 h reaction was completed. After cooling to RT precipitate was formed. Precipitate was filtered washed with dithyl ether and dried under rediced pressiure. Step 3g. 1-[(6,7-dimethyl-2-oxo-1 H-quinolin-3-yl)methyl]-1-(2-furylmethyl)-3-(2- morpholinoethyl)thiourea.

3-[(2-furylmethylamino)methyl]-6,7-dimethyl-1 H-quinolin-2-one (51 mg, 0.18mmol) was weighted and poured into 10mL round bottom flask and swollen with 2mL of dioxane. Following that, 4-(2-isothiocyanatoethyl)morpholine was weighted in amount of 46 mg (0.27mmol, 1.5 Eq) and added to the mixture, the reaction was conducted at 80°C in oil bath, under argon pressure. The progress of the reaction was monitored by TLC, after 1 h reaction was completed. After cooling to RT precipitate was formed. Precipitate was filtered washed with dithyl ether and dried under rediced pressiure. Compounds according to Formula

Step 1. Synthesis of 4-[1-[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4- yl]benzoic acid. (Z)-1 -[4-chloro-5-(methyleneamino)pyrazol-1 -yl] prop- 1 -en-1 -amine was weighted 250 mg (1.35mmol), in round bottom flask, was swollen in ACN (5mL) with 0.5M sodium carbonate, subsequently was added 3-carboxyphenylboronic acid (225 mg, 1 ,35mmol) and tetrakis (triphenylphosphine) palladium. The reaction was conducted at 90°C under reflux conditions and argon pressure, the progress was monitored by TLC. After completion of the reaction, the mixture was left to cool down, later was added 1 M HCI (15mL) and mixed with ethyl acetate and extracted. The procedure was repeated three times. Next step included washing the organic layer with brine (3x15mL) and drying with magnesium sulfate(VI). The product was dried under vacuum pump.

Step 2a. Synthesis of 4-[1-[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]- N-[2-(dimethylamino)ethyl]benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]-

N[2(dimethylamino)ethyl]benzamide was weighted (100mg, 0.370mmol, 1 Eq) and then swollen with 10mL of DMF and stirred. The reaction was cooled and TBTU

(130mg, 0.407 mmol, 1.1 Eq) was added. Following by TEA (56pL, 0.407 mmol, 1.1 Eq) and N',N'-dimethylethane-1,2-diamine was weighted (32.5mg, 0.370mmol, 1 Eq) and added to the reaction flask. The progress of the reaction was monitored with TLC and leaved for 24 hours, after completing the reaction, the flask was left for cooling down. Last step included solvent evaporation and product purification with column chromatography.

Step 2b. Synthesis of 4-[1-[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]- N-(2-morpholinoethyl)benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]- N[2(dimethylamino)ethyl]benzamide was weighted (100mg, 0.370mmol, 1 Eq) and then swollen with 10mL of DMF and stirred. The reaction was cooled and TBTU (130mg, 0.407 mmol, 1.1 Eq) was added. Following by TEA (56pL, 0.407 mmol, 1.1 Eq) and 2-morpholinoethanamine was weighted (48mg, 0.370mmol, 1 Eq) and added to the reaction flask. The progress of the reaction was monitored with TLC and leaved for 24 hours, after completing the reaction, the flask was left for cooling down. Last step included solvent evaporation and product purification with column chromatography.

Step 3a. Synthesis of 4-[5-(methyleneamino)-1-[(Z)-1-[2-(2-pyridyl)ethylamino]prop -1- enyl]pyrazol-4-yl]-N-(2-morpholinoethyl)benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]-N-(2- morpholinoethyl)benzamide derivative was weighted (50mg, 0.130mmol, 1 Eq) and then swollen with 5mL of THF and stirred under reflux conditions. Then 2-(2- chloroethyl)pyridine was added in amount of 36mg (0.259mmol, 2Eq) and checked the reaction progress with TLC, the reaction was conducted for 4-5 hours. After completing the reaction, the product was evaporated under vacuum pump, and chromatographically purified.

Step 3b. Synthesis of 4-[5-(methyleneamino)-1-[(Z)-1-[2-(2-pyridyl)propylamino]pro p-

1-enyl]pyrazol-4-yl]-N-(2-morpholinoethyl)benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]-N-(2- morpholinoethyl)benzamide derivative was weighted (50mg, 0.130mmol, 1 Eq) and then swollen with 5mL of THF and stirred under reflux conditions. Then 2-(3- chloropropyl)pyridine was added in amount of 40mg (0.259mmol, 2Eq) and checked the reaction progress with TLC, the reaction was conducted for 4-5 hours. After completing the reaction, the product was evaporated under vacuum pump, and chromatographically purified.

Step 3c. Synthesis of 4-[5-(methyleneamino)-1-[(Z)-1-[2-(2-thienyl)ethylamino]prop -1- enyl]pyrazol-4-yl]-N-(2-morpholinoethyl)benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]-N-(2- morpholinoethyl)benzamide derivative was weighted (50mg, 0.130mmol, 1 Eq) and then swollen with 5mL of THF and stirred under reflux conditions. Then 2-(2- chloroethyl)thiophene was added in amount of 37.9mg (0.259mmol, 2Eq) and checked the reaction progress with TLC, the reaction was conducted for 4-5 hours. After completing the reaction, the product was evaporated under vacuum pump, and chromatographically purified.

Step3d. 4-[1 -[(Z)-1 -[2-(2-furyl)ethylamino]prop-1 -enyl]-5-(methyleneamino)pyrazol-4- yl]-N-(2-morpholinoethyl)benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]-N-(2- morpholinoethyl)benzamide derivative was weighted (50mg, 0.130mmol, 1 Eq) and then swollen with 5mL of THF and stirred under reflux conditions. Then 2-(2- chloroethyl)furan was added in amount of 33.8 mg (0.259mmol, 2Eq) and checked the reaction progress with TLC, the reaction was conducted for 4-5 hours. After completing the reaction, the product was evaporated under vacuum pump, and chromatographically purified.

Step 3e. Synthesis of N-[2-(dimethylamino)ethyl]-4-[1-[(Z)-1-[2-(2- furyl)ethylamino]prop-1-enyl]-5-(methyleneamino)pyrazol-4-yl ]benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]-

N[2(dimethylamino)ethyl]benzamide derivative was weighted (50mg, 0.147mmol, 1 Eq) and then swollen with 5mL of THF and stirred under reflux conditions. Then 2-(2- chloroethyl)furan was added in amount of 38.2 mg (0.294mmol, 2Eq) and checked the reaction progress with TLC, the reaction was conducted for 4-5 hours. After completing the reaction, the product was evaporated under vacuum pump, and chromatographically purified.

Step 3f. Synthesis of N-[2-(dimethylamino)ethyl]-4-[5-(methyleneamino)-1-[(Z)-1-[2 - (2-thienyl)ethylamino]prop-1-enyl]pyrazol-4-yl]benzamide.

4-[ 1 -[(Z)-1 -aminoprop-1 -enyl]-5-(methyleneamino)pyrazol-4-yl]- N[2(dimethylamino)ethyl]benzamide derivative was weighted (50mg, 0.147mmol, 1 Eq) and then swollen with 5mL of THF and stirred under reflux conditions. Then 2-(2- chloroethyl)thiophene was added in amount of 43mg (0.294mmol, 2Eq) and checked the reaction progress with TLC, the reaction was conducted for 4-5 hours. After completing the reaction, the product was evaporated under vacuum pump, and chromatographically purified.

Example 2. Biological activity

Surface Plasmon Resonance (SPR)

In order to show in vitro specific TRF-ligand interaction SPR technique were used. Both the hTRF1 and hTRF2 recombinant proteins were biosynthesized in E.coli ArcticExpress DE3 [BF ^_ ompT hsdS(rB ^_ mBj dcm ⁺ TetR gal A(DE3) endA Hte [cpn10 cpn60 GentR] (Agilent Technologies, St. Clara, CA, USA), in fusion with the Fh8 tag at the N terminus [34] and His-tag at the C terminus.

In Fig. 2A the interaction of A822 with TRF1 protein was recorded. As a reference interaction between TRF1 and TIN2 was confirmed in Fig. 2B. In Fig. 2C it is shown that when A822 is present interactions between TRF1 and TIN2 is lower what directly indicates that A822 interact within the same binding area of TRF1 occupied by TIN2. The values of ka (association constant) kd (dissociation constant) and KD (dissociation constant in equilibrium state) are given in Fig. 2D.

Moreover, in Fig. 3A the interaction of B327 with TRF2 protein was recorded. As a reference interaction between TRF1 and TIN2 was confirmed in Fig. 3B. In Fig. 3C it is shown that when B327 is present interactions between TRF1 and TIN2 is lower what directly indicates that B327 interact within the same binding TRF2 area occupied by TIN2. The values of ka (association constant) kd (dissociation constant) and KD (dissociation constant in equilibrium state) are given in Fig. 3D.

In vitro and ex vivo anticancer cytotoxicity

Cell viability data using MTT assay were measured as described below in methodology (MTT assay). The mean of the control was standardized and defined as 100% cell activity. The difference in cell viability between reference compound (doxorubicin) and cells after treatment with tested compounds were analyzed using one-way ANOVA corrected by Tukey's test.

The first stage of research has tested both compounds, A822 or B0327, in 10 mammalian cell lines including: including (i) six breast cancer cell lines: MCF7, MDA- MB-231 , BT-474, SK-BR-3, T47D and BT20; (ii) one multidrug-resistant breast cancer cell line - MCF7/Adr; (iii) one non-cancer cell line MCF 10A; (iv) human mammary epithelial cells (HMEC) and (v) human endothelial cell (HUVEC). All cells were treated with different concentrations of tested compounds in range 0.78-1 OOpM and doxorubicin in range 0.01-12.5pM for 72 h.

As demonstrated in Tab. 1 and Tab. 2, the strongest cytotoxic effect was obtained after the application of compounds: A822 for all cell lines (range of IC50: 2-20pM) and for primary cell cultures from breast tissues (range of IC50: 2-22pM) and B327 for most cell lines (range of IC50: 13-35pM) and primary cell cultures from breast tissues (range of IC50: 4-40pM). Importantly, the application of A822 appeared effective to the multidrug-resistant breast cancer cell line (MCF7/Adr) towards which A822 evoked even higher cytotoxicity than doxorubicin.

Tab. 1. IC50 values for compounds: A822 and 0327 (for cell lines: MCF7, MDA-MB- 231 , MCF 10A, BT-474, SK-BR-3, T47D,BT20 MCF7/Adr and HUVEC, HMEC). Doxorubicin used as a cytotoxic reference drug. The data shown are means [pM] ± SEM obtained from three independent experiments.

Tab. 2. IC50 values for compounds: B327 and A822 tested in primary cell cultures derived from normal and cancer breast tissues (ex vivo experiments). Doxorubicin was used as a cytotoxic reference drug. The data shown are means ± SEM. N - denotes normal breast tissues and T- denotes breast cancer tissues. The tissue were delivered as a postoperational material from the Gdynia Oncology Center of the Polish Red Cross Maritime Hospital. The human material was sampled according to the local bioethical commission guidelines and the informed consent of the patient was obtained each time.

Method: MTT assay MTT is a colorimetric non-clonogenic assay that measures cell viability in culture by its metabolic activity (Stockert et al., 2012). MCF7, MDA-MB-231 , MCF 10A, BT474, SK- BR-3, T47D, BT20, MCF7/Adr, HMEC, HUVEC and cell cultures from normal and cancer breast tissues were cultured as mentioned above. Once cell lines were about 80% confluent, cells were counted by a hemacytometer (Hausser Scientific, Horsham, PA, USA) and plated in 96-well microtiter plates at concentrations: 4000cells/well for MCF7 and MDA-MB-231 , 3000 cells/well for MCF10A, 40 000 cells/well for BT-474 , 4500 cells/well for SK-BR-3, 8000 cells/well for T47D, 6000 cells/well for BT20 and HMEC, 5000 cells/well for MCF7/Adr, 2000 cells/well for HUVEC and 4500 cells/well for primary cell cultures derived from normal and cancer breast tissues. Cells were allowed to attach overnight. Tested compounds were dissolved and diluted in serial concentrations (100 - 0.78 pM), in cell culture medium and added to wells in 100 pl aliquots, in triplicates. Doxorubicin was used as a reference drug with dissolved and serially diluted (12.5 - 0.1 pM). The final concentration of DMSO was ensured to be around 1 % in all experiments. Cells were incubated with studied compounds for 72 h at 37 °C and 5% CO2 or 10% CO2 optimal depending on cell lines. After incubation, 20 pL aliquots of MTT solution (3-[4,5-dimethylthiazol-2-yl]-2,5- diphenyltetrazolium bromide) in PBS (4 mg/ml) were added to all wells and incubated further for 3h at 37 °C. Formazan crystals formed were dissolved in 150 pL DMSO aliquots and absorbance was measured using an Asys UVM340 multiwell plate reader at A=540 nm. Cytotoxicity was determined for tested compounds and compared to drug-free control. All biological experiments were performed in triplicates. GraphPad Prism 8.0.1 was used to statistically analyze the results.

Cellular Senescence

To identify the possible senescence-involving cytotoxic activity of studied compounds, SAp-gal assay was performed. MCF7 cells were plated on glass slides at low density in a 35 mm Peri dish to achieve the logarithmic phase of cell growth. After 24 hours the cells were treated with A822 compound at 10pM concentration or B327 compound at 100pM concentration, respectively, for five days. The large senescent cells with strong blue stain (SApgal-assay-positive) became visible in the case of treatment of either compounds, A822 or B327, while negative control cells demonstrated none or minimal stain at background levels (Fig. 4).

Method: Senescence-Associated-p-Galactosidase (SA-p-gal) staining Limited capacity to replicate allowing the cell to pass no more than 50-55 divisions in its entire life-span - the Hayflick limit - is a fundamental security mechanism preventing excessive accumulation of genetic errors and chromosomal instability. Normally reaching Hayflick limit culminates in senescence, an arrested state in which the cell remains viable however not dividing any longer. State of senescence is detected by measuring of activity of p-galactosidase, a pH-dependent enzymatic assay that characterizes senescent cells.

In this study, MCF7 cells were cultured on a 0.15mm thin glass slide, placed in a 35 mm Petri dish and treated with the cells were treated with A822 compound at 10pM concentration and B0327 compound at 100pM concentration for five days for 120 h. Treated and control cells were then washed in PBS and fixed with 2% formaldehyde/0.2% glutaraldehyde for 5 min at room temperature. The cells were washed twice with PBS ,beta-Gal staining solution was added, were incubated at 37°C for 15 h in the dark. After incubation, citric acid/sodium phosphate buffer was used to detect beta-Gal’s activity product. Cells on the glass slides were observed in the bright field mode using an Olympus Fluorescence Microscope BX60 equipped with 20x DIC lens.

Telomere length

Comparative AACq (Quantification Cycle Value) method demonstrated that the use of A822 and B0327 compounds caused a reduction in telomere length compared to the control by 1.15 times for A822 and by 1 .42 times after the application of B327 (Fig. 5).

Method

ScienCell's Relative Human Telomere Length Quantification qPCR Assay Kit (RHTLQ) was used to directly compare the average telomere length of the control sample and samples treated with compounds: A822 and B327. In this study, we used MCF7 cells after 10 cell passages. The difference in the mean telomere length of the tested samples was assessed using the comparative AACq (Quantification Cycle Value) method.

Colocolacallsatlon of TRF1/2 proteins and TIN2

Colocalisation was made using immunostaining fluorescent technique in order to see if addition of A822 or B327 can change location of TRF1 and Trf2 relative to TIN2. The experiment confirmed that both compounds has ability to increase delocalization of TRF1/2 with regard to TIN2 what indicate that they disrupt complex formation between TRF1/2 and TIN2. This delocalization for TRF1-TIN2-A822 is presented in Fig. 6, and for TRF2-TIN2-B327 in Fig. 7.

Summary

As presented studies shows, proposed lead compounds which were obtained by inventors with the symbols A822 and B327 (Fig. 1), exhibit interesting biological properties. In particular, these compounds have been shown to interact with the telomeric proteins, namely A822 of TRF1 and B327 of TRF2, respectively. This interaction at the molecular level leads to the blocking of the binding of these proteins to the TIN2 protein, which generally disrupts the formation of the shelterin complex. These compounds also show cytotoxicity to neoplastic cells in vitro and ex vivo. It is not a high activity, but it is related to the mechanism of its action as these compounds induce cellular aging in neoplastic cells, which in the long run leads to the death of these cells anyway. In addition, as shown by additional studies involving the determination of telomere length, the tested compounds lead to telomere shortening, which is an effect of disruption of the shelterin complex. On the other hand, the results of studies on the co-localization of TRF1/2 and TIN2 proteins (immunofluorescence methods using confocal microscopy) show that this co-localization is blurred, which confirms that the action of the compounds leads to the breakdown of TRF1/2-TIN2 complexes. The disclosed compounds are therefore a kind of modulators of the functions of the TRF1/2 telomeric proteins and at the same time can be considered as interesting model anticancer compounds acting on the basis of a completely new mechanism.

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