COMPOUNDS USEFUL IN PREVENTION AND/OR TREATMENT OF SENESCENT CELL-RELATED PATHOLOGIES AND DISEASES

Abstract

A compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof:

##STR00001## wherein R is chosen from phenyl, a 6-membered heteroaryl group, cyclohexyl, a 5-membered heteroaryl group; a bicyclo [3.1.0] hexanyl group; a C.sub.2-C.sub.5 alkynyl group; and a cubanyl group; wherein R1 and R2, independently, are chosen from H; C.sub.1-C.sub.6 alkyl (optionally substituted with one or more halogens); C.sub.1-C.sub.6 alkyl-S(O).sub.n; CO.sub.2H (or C.sub.1-C.sub.6 alkyl esters thereof or C.sub.1-C.sub.6 alkyl amides thereof); halogen; C.sub.1-C.sub.6 alkoxy; CN; NO.sub.2; and NR.sub.7R.sub.8; wherein R.sub.7 and R.sub.8, independently, each represent H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, arylsulphonyl, heteroarylsulphonyl, heterocyclosulphonyl, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, or C.sub.1-C.sub.6 alkylsulphonyl, or R.sub.7, R.sub.8 and the nitrogen to which they are attached form a 5 or 6 membered heterocyclic ring (such as morpholine or piperidine); and wherein n represents 0-2.

Claims

1. A compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof: ##STR00015## wherein R is chosen from phenyl, a 6-membered heteroaryl group, cyclohexyl, a 5-membered heteroaryl group; a bicyclo [3.1.0] hexanyl group; a C.sub.2-C.sub.5 alkynyl group; and a cubanyl group; wherein R.sup.1 and R.sup.2, independently, are chosen from H; C.sub.1-C.sub.6 alkyl (optionally substituted with one or more halogens); C.sub.1-C.sub.6 alkyl-S(O).sub.n; CO.sub.2H (or C.sub.1-C.sub.6 alkyl esters thereof or C.sub.1-C.sub.6 alkyl amides thereof); halogen; C.sub.1-C.sub.6 alkoxy; CN; NO.sub.2; and NR.sub.7R.sub.8; wherein R.sub.7 and R.sub.8, independently, each represent H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, arylsulphonyl, heteroarylsulphonyl, heterocyclosulphonyl, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, or C.sub.1-C.sub.6 alkylsulphonyl, or R.sub.7, R.sub.8 and the nitrogen to which they are attached form a 5 or 6 membered heterocyclic ring (such as morpholine or piperidine); and n represents 0-2; for use as a medicament.

2. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, wherein, in R, the 6-membered heteroaryl group or 5-membered heteroaryl group has 1 or 2 heteroatoms, preferably independently selected from N and S.

3. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, wherein R1 and R2 are independently chosen from H, CH.sub.3 and CH.sub.2CH.sub.3, more preferably R1 and R2 are both H.

4. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, wherein R is chosen from the following groups of formula (a), (b), (c), (d), (e), (f), (g), (h), (i), (l): ##STR00016## preferably R is the group of formula (a): ##STR00017##

5. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, wherein the compound of formula (I) is the compound of formula (Ia): ##STR00018##

6. A pharmaceutical composition comprising, as active ingredient, at least one compound of formula (I) of claim 1, or pharmaceutically acceptable salts or hydrates thereof, and at least one pharmaceutically acceptable excipient.

7. The pharmaceutical composition of claim 6, for use as a medicament.

8. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, for use as HSP90 inhibitor, more preferably HSP90 inhibitor.

9. The pharmaceutical composition of claim 6, for use as HSP90 inhibitor, more preferably HSP90 inhibitor.

10. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, for use in selectively killing senescent cells.

11. The pharmaceutical composition of claim 6, for use in selectively killing senescent cells.

12. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, for use in delaying ageing in a subject.

13. The pharmaceutical composition of claim 6, for use in delaying ageing in a subject.

14. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, for use in treatment and/or prevention of a senescence-associated disease or disorder.

15. The pharmaceutical composition of claim 6, for use in treatment and/or prevention of a senescence-associated disease or disorder.

16. The compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, wherein the senescence-associated disease or disorder is chosen from a tumor, a metabolic disease, an inflammatory disease or disorder, including an autoimmune disease or disorder, a neurological or neurodegenerative disease or disorder, a pulmonary or respiratory disease or disorder, a proliferative disorder, a renal disorder or disease, a liver disorder or disease, an eye disease or disorder, a cardiovascular disease or disorder, a fibrotic disease or disorder, a viral infection, a dermatological disorder or disease, a geriatric disease or disorder, and side effects associated with chemotherapy or radiotherapy.

17. A compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof: ##STR00019## wherein R is chosen from phenyl, a 6-membered heteroaryl group, cyclohexyl, a 5-membered heteroaryl group; a bicyclo [3.1.0] hexanyl group; a C.sub.2-C.sub.5 alkynyl group; and a cubanyl group; wherein R1 and R2, independently, are chosen from H; C.sub.1-C.sub.6 alkyl (optionally substituted with one or more halogens); C.sub.1-C.sub.6 alkyl-S(O).sub.n; CO.sub.2H (or C.sub.1-C.sub.6 alkyl esters thereof or C.sub.1-C.sub.6 alkyl amides thereof); halogen; C.sub.1-C.sub.6 alkoxy; CN; NO.sub.2; and NR.sub.7R.sub.8; wherein R.sub.7 and R.sub.8, independently, each represent H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, arylsulphonyl, heteroarylsulphonyl, heterocyclosulphonyl, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, or C.sub.1-C.sub.6 alkylsulphonyl, or R.sub.7, R.sub.8 and the nitrogen to which they are attached form a 5 or 6 membered heterocyclic ring (such as morpholine or piperidine); and n represents 0-2; excluding a compound of formula (Ia): ##STR00020##

18. A method for preparing the compound of formula (I) or pharmaceutically acceptable salts or hydrates thereof of claim 1, the method comprising the steps of synthesis scheme 3: ##STR00021##

19. A cosmetic use of a compound of formula (I) or cosmetically acceptable salts or hydrates thereof: ##STR00022## wherein R is chosen from phenyl, a 6-membered heteroaryl group, cyclohexyl, a 5-membered heteroaryl group; a bicyclo [3.1.0] hexanyl group; a C.sub.2-C.sub.5 alkynyl group; and a cubanyl group; wherein R1 and R2, independently, are chosen from H; C.sub.1-C.sub.6 alkyl (optionally substituted with one or more halogens); C.sub.1-C.sub.6 alkyl-S(O).sub.n; CO.sub.2H (or C.sub.1-C.sub.6 alkyl esters thereof or C.sub.1-C.sub.6 alkyl amides thereof); halogen; C.sub.1-C.sub.6 alkoxy; CN; NO.sub.2; and NR.sub.7R.sub.8; wherein R.sub.7 and R.sub.8, independently, each represent H, C.sub.1-C.sub.6 alkyl, C.sub.1-C.sub.6 alkylcarbonyl, C.sub.1-C.sub.6 alkoxycarbonyl, arylsulphonyl, heteroarylsulphonyl, heterocyclosulphonyl, arylcarbonyl, heteroarylcarbonyl, heterocyclocarbonyl, or C.sub.1-C.sub.6 alkylsulphonyl, or R.sub.7, R.sub.8 and the nitrogen to which they are attached form a 5 or 6 membered heterocyclic ring (such as morpholine or piperidine); and n represents 0-2; to prevent, reduce, and/or treat signs of ageing of skin.

20. The cosmetic use of claim 19, wherein the compound of formula (I) is a compound of formula (Ia): ##STR00023##

21. A cosmetic composition comprising at least one compound of formula (I) or cosmetically acceptable salts or hydrates thereof of claim 19 and at least one cosmetically acceptable excipient.

22. A cosmetic use of the cosmetic composition of claim 21 to prevent, reduce, and/or treat the signs of ageing of the skin.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0267] FIG. 1 shows: (A) The images showing early-passage cells (P15, left) and advanced-passage cells (P19, right), on which the -galactosidase assay was performed, according to the supplier protocol (Senescence -Galactosidase Staining Kit, #9860S, Cell Signaling Technology, Inc., MA, USA). (B) The graph representing the mean quantification of the colorimetric signal of -galactosidase product, comparing experiments on P15-P17 cells and P19-P21 cells (representative of senescent cells). Microscopy images were obtained using a Leica DMil microscope (Leica Microsystems, IL, USA), at magnification 5 and analyzed by ImageJ software, normalized to the number of cells, n=6. The error bars represent the standard error, ** p<0.005. The data were analyzed using the Mann-Whitney test.

[0268] FIG. 2 shows: (A) The graph representing the average of the number of cells surviving treatment with 13 compounds, including K5, on the two groups of cell populations P15-P17 and P19-P21, at 1 M concentration, counted manually by burker chamber, n=3; and (B) The graph representing the average of the fold changes of the signal emitted by the DAPI, quantified by spectrophotometer, vs. solvent. K5 was compared with the two known inhibitors at a concentration of 0.5 M, n=3. The error bars represent the standard error, the data were analyzed using two-way ANOVA, followed by Sidak post-hoc test. DMSO (dimethyl sulfoxide) is the control.

[0269] FIG. 3 is a graph representing the HSP90 activity inhibition curves of K5, compared with the compound 17-DMAG, n=3. Data are reported as residual activity in response to increasing dose of the inhibitor. The data has been analyzed using GraphPad Prism 7 Software, which allowed to extrapolate the sigmoidal curve.

DETAILED DESCRIPTION OF THE INVENTION

[0270] The invention is further described by way of the following non-limiting examples.

Materials and Methods

[0271] Cell culture and replicative senescence. Fetal pulmonary fibroblasts, IMR90, were provided by the European Collection of Authenticated Cell Culture (ECACC) and grown in a complete medium consisting of Minimum Essential Medium (MEM), 10% Fetal Bovine Serum (FBS), 1% penicillin-streptomycin (antibiotic), 1% L-glutamine. Cells with passages from P15 to P21 were used; cells were considered incrementally senescent from passage P19 to passage P21. As can be seen in FIG. 1, at P19, the cells were bigger and the beta-galactosidase activity is higher. FIG. 1B shows that the ratio of the intensity of the beta-gal (signal) to the number of cells, increases with the number of passages of the cells. -galactosidase assay (Itahana et al., (2007) Methods to Detect Biomarkers of Cellular Senescence. In: Tollefsbol T. O. (eds) Biological Aging. Methods in Molecular Biology, vol 371. Humana Press. https://doi.org/10.1007/978-1-59745-361-5_3). The cells were fixed in paraformaldehyde 2% and incubated with the substrate, X-gal, in a solution at pH 6, at 37 C., overnight (16 h).

[0272] The images were acquired using an optical microscope (Leica) with 5 magnification. The images were analyzed using the ImageJ software, quantifying the colorimetric signal emitted by each cell. The mean signal intensity was then normalized for the total number of cells. Survival curves of the compounds. To perform survival curves, cells with passages from P15 to P21 were tested at increasing concentrations of 0.5 M, 1 M, 10 M, 25 M over 24 and 48 h to evaluate the compounds' efficacy and cytotoxicity compared to the solvent (DMSO). The cells were then counted manually, in triplicate, using disposable burker chambers.

[0273] For the DAPI staining (4,6-diamidin-2-phenylindol) assay, each compound was tested at increasing concentrations 0.1 M, 0.5 M, 1 M, 10 M, for 48 h, the reference compounds 17-DMAG and Ganetespib were also used as control of the experiment (Fuhrmann-Stroissnigg et al. Identification of HSP90 inhibitors as a novel class of senolytics. Nat Commun. 2017 8:422 doi: 10.1038/s41467-017-00314-z). Following treatment, the cells were seeded in 96mw. The adherent cells were then fixed with 70% ethanol, incubated for 30 min with a 3 M DAPI solution; the fluorimetric signal was measured using a multiplate reader (Victor Nivo-Perkin Elmer) set to 350/460 nm (Ligasov A, Koberna K. Quantification of fixed adherent cells using a strong enhancer of the fluorescence of DNA dyes. Sci Rep. 2019 Jun. 18; 9(1):8701. doi: 10.1038/s41598-019-45217-9. PMID: 31213648; PMCID: PMC6581942.).

[0274] HSP90 inhibition activity assay. HSP90-directed inhibitory activity was assessed employing fluorescence polarization measuring competitive binding of fluorescein isothiocyanate-labeled geldanamycin (FITC-geldanamycin) to recombinant HSP90 as a function of test compound concentration using the commercially available HSP90 N-Terminal Domain Assay Kit (BPS Bioscience, Catalog #50293, CA, USA) according to the manufacturer's instructions. Briefly, the compounds were diluted at different concentrations between 1 pM to 10 M and incubated for 2 h with the enzyme and substrate at RT while stirring. The fluorescence intensity was measured with a multiplate reader (Victor Nivo-Perkin Elmer) set at 485/530 nm. Data have been represented as the percentage of residual enzyme activity relative to the solvent (DMSO). Data were analyzed by GraphPad Prism 7 Software which allowed to extrapolate the sigmoidal curve and calculate the IC50.

[0275] Statistical analysis. The results were analyzed using GraphPad Prism 7 statistical software to generate graphs showing the mean values of at least three independent experimentsstandard error (SE); for each analysis, a specific comment has been added. The following tests determined significance between experimental groups: non-parametric Mann Whitney and two-way ANOVA, followed by Sidak post-hoc tests to compare the various conditions; The p-value indicates statistical significance: *p<0.05, **p<; 0.005.

Example 1

Characterisation of the Biological Properties of 1-benzyl-3-(2-methylphenyl)-2,3,6,7-tetrahydro-1H-purine-2,6-dione (K5)

[0276] 13 compounds including K5, with different structures, were assayed using an in vitro senescence model, that was developed utilizing serial passages of primary human fetal lung cells IMR90. In this context, the first signs of senescence were found between the nineteenth and twenty-first passage. Senescence was detected using the Senescence -Galactosidase Staining Kit (#9860S, Cell Signaling Technology, Inc., MA, USA). Briefly, cells at different passages were fixed and incubated with the -Galactosidase substrate at 37 C., overnight in a dry incubator. Then the 13 compounds were tested for their potential senolytic activity on cells between passages 19 and 21, considering cells around passage 15 as control reference (FIG. 2A).

[0277] The 13 compounds were purchased from MolPort (MolPort, SIA, Riga, Latvia). In particular, K5 was purchased online from the website www.molport.com using identification code MolPort-002-908-949.

[0278] K5 has the following structure:

##STR00013##

[0279] Of the 13 compounds examined, K5 (1-benzyl-3-(2-methylphenyl)-2,3,6,7-tetrahydro-1H-purine-2,6-dione) revealed a marked senolytic capacity, reducing the senescent population by about 50% in the first 48 hours of exposure to the compound (see FIG. 2A).

[0280] More in detail, growth curves were initially performed, testing each compound in a concentration range from 0.5 to 25 M for 48 h. FIG. 2A shows the differences in cells treated at different passages at 1 M concentration, respectively. In particular, K5 appeared to act selectively and significantly, in cells at passages between P19 and P21, without showing particular cytotoxic effects when compared to cells at early passages (P15-P17). The reduced cytotoxic effect of K5 is shown by the fact that the younger cells (P15-P17) are not killed by the compounds. The compounds instead selectively kill only senescent cells (P19-P21).

[0281] To confirm data obtained by traditional cell counting by a single operator, a quantification method based on UV-visible spectrophotometry was also implemented, exploiting the ability of the DAPI to mark cell nuclei. The cells that survived the action of senolytics were fixed and incubated with the fluorescent indicator. The specific signal was measured using the microplate multimode reader, Victor NIVO (PerkinElmer Tech. GmbH, Germany). FIG. 2B shows the effect of the two selected compounds, at 0.5 M, quantified using this technique and compared with known senolytics such as 17-DMAG and Ganetespib, which in the cellular model IMR90 seem to have cytotoxic effects already at low concentrations and also in early passages. The compounds were tested in a concentration range between 0.1 and 10 M for 48 h, appreciating a significant effect starting from the concentration of 0.5 M. At higher concentrations (>10 M), the variability increases and the differences between the two cell populations.

[0282] For K5, the in vitro affinity to the target protein HSP90 was evaluated using a geldanamycin-competitive binding fluorimetric assay (HSP90 N-Terminal Domain Assay Kit, #50293, BPS Bioscience, Inc., CA, USA). K5 was incubated in a concentration range between 1 pM and 10 M for 2 h, with a FITC-labeled geldanamycin and the recombinant HSP90 enzyme to produce a change in fluorescent polarization, measured by the microplate multimode reader, Victor NIVO (PerkinElmer Tech. GmbH, Germany). The resulting data allowed the calculation of the compound IC50s, both in the nanomolar order (K5=731 nM); 17-DMAG was used as the reference molecule (IC50=11 nM).

[0283] IC50 or 50 inhibiting concentration is the concentration of a compound required to inhibit by 50% the target activity under examination with respect to the values measured in the absence of inhibitor. IC50 is a parameter used to evaluate the effectiveness of a substance in inhibiting the target and it is one of the methods commonly used in the pharmacological research to measure the power of an antagonist.

[0284] A test was carried out to compare the binding affinities for HSP90 of the compounds of formula (Ia) to (Il) according to the invention.

[0285] Table 2 below summarises the results.

TABLE-US-00002 TABLE 2 Binding affinities for HSP90 of the compounds of formula (Ia) to (Il) Binding affinity Compound (Kcal/mol) Ia (K5) 4.964 Ib 7.029 Ic 6.607 Id 5.675 Ie 5.502 If 5.083 Ig 5.041 Ih 4.974 Ii 4.872 Il 4.857

[0286] Binding affinities for HSP90 for all compounds of formula (I) were calculated by the program Glide (Schrdinger, Inc.)

Example 2

Synthesis of the Compound 1-benzyl-3-(2-methylphenyl)-2,3,6,7-tetrahydro-1H-purine-2,6-dione(K5)

[0287] The synthesis took place according to the following scheme:

##STR00014##

[0288] Ac.sub.2O is acetic anhydride; EtOH is ethanol.

[0289] Compounds 1 and 2 were purchased from Sigma-Aldrich (US) (www.sigmaaldrich.com) (CAS numbers 95-53-4 and 3173-56-6, respectively).

[0290] The condensation reaction of o-toluidine (1) (CAS number 95-53-4) with benzyl isocyanate (2) (CAS number 3173-56-6) gives urea (3) whose acylation with cyanoacetic acid, provides acylurea (4). Hydrolysis of the cyano group, followed by in situ cyclization, yields aminouracyl (5) (see Papesch V, Schroder E. F., J. Org. Chem. 1951, 16, 1879; Ohtsuka Y., Bull. Soc. Chim. Jap. 1973, 46, 506). Nitrosation of (5) and reduction (sodium dithionite/formamide) of the corresponding nitroso derivative to amine and reaction of the resulting diamine intermediate with formic acid, gives the compound of formula (Ia) (K5) (see Brodereck H., Edenhofer A., Chem. Berichte 1955, 1306; Montana J. G. et al., Bioorg. Med. Chem. 1998, 8, 2925).