Senescence tracers

Abstract

The instant invention relates to novel compounds useful for visualizing cell senescence, their preparation and use. In particular, this invention relates to novel fucose and amino-quinoline derivatives useful as senescence traces and their preparation.

Claims

1. Compound of formula
A-L-M  (I) in which, A represents ##STR00036## wherein, * represents the binding site to the residue represented by L, L represents *Y-(CH.sub.2).sub.n-.sup.# or ##STR00037## wherein, Y is selected from the group consisting of O, N, S, SO and SO.sub.2, n is an integer from 2 to 6, * represents the binding site to the residue represented by A, and .sup.# represents the binding site to the residue represented by M, and M represents a fluorophore, a residue comprising at least one atom selected from the group consisting of .sup.76Br, .sup.75Br, .sup.18F, .sup.13C and .sup.11C or a combination thereof, and the salts thereof, the solvates thereof and the solvates of the salts thereof.

2. Compound according to claim 1, wherein n is 2, 3 or 4.

3. Compound according to claim 1, wherein M represents a fluorophore.

4. Compound according to claim 3, wherein the fluorophore is selected from the group consisting of the xanthene derivatives, fluorescein, rhodamine, Oregon green, eosin, and Texas red, the cyanine derivatives, cyanine, indocarbocyanine, oxacarbocyanine, thiacarbocyanine, and merocyanine, the naphthalene derivatives, a dansyl or derivatives thereof, the coumarin derivatives, the oxadiazole derivatives, pyridyloxazole, nitrobenzoxadiazole and benzoxadiazole, the pyrene derivatives, the oxazine derivatives, Nile red, Nile blue and cresyl violet, the acridine derivatives, proflavin, acridine orange and acridine yellow, the arylmethine derivatives, auramine, crystal violet and malachite green, and the tetrapyrrole derivatives, porphin, phthalocyanine and bilirubin.

5. Compound according to claim 1, wherein M is selected from .sup.76Br, .sup.75Br, .sup.18F, ##STR00038## wherein, X is selected from the group consisting of Br, .sup.76Br, .sup.75Br, Cl, F and .sup.18F, R.sub.1 represents hydrogen or (C.sub.1-C.sub.6)-alkyl, whereby alkyl is substituted with one to three substituents selected independently from the group consisting of hydroxy, amino, cyano, nitro, halogen, carbonyl, (C.sub.1-C.sub.4)-alkoxy, mono (C.sub.1-C.sub.4)-alkylamino and di-(C.sub.1-C.sub.4)-alkylamino, whereby R.sub.1 represents an optionally substituted (C.sub.1-C.sub.6)-alkyl comprising at least one .sup.76Br, .sup.75Br, .sup.18F or .sup.11C if X is not .sup.76Br, .sup.75Br or .sup.18F, R.sub.2 and R.sub.3 independently from one another represent hydrogen or (C.sub.1-C.sub.6)-alkyl, whereby alkyl is substituted with one to three substituents selected independently form the group consisting of, hydroxy, amino, cyano, nitro, halogen, carbonyl, (C.sub.1-C.sub.4)-alkoxy, mono (C.sub.1-C.sub.4)-alkylamino and di-(C.sub.1-C.sub.4)-alkylamino, whereby at least one of R.sub.2 and R.sub.3 represents a (C.sub.1-C.sub.6)-alkyl comprising at least one .sup.11C or being substituted with a residue comprising at least one .sup.76Br, .sup.75Br, .sup.18F or .sup.11C, and * represents the binding site to the residue represented by L.

6. Medicament comprising a compound according to claim 1 in combination with at least one inert, non-toxic, pharmaceutically suitable excipient.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) FIG. 1: FACS-analysis data of the compound of Example 5 in HCT116 cells (colorectal carcinoma cell line) and MCF7 cells (breast cancer cell line) with and with out 250 nM Doxorubicin treatment.

(2) FIG. 2: Light microscope pictures of β-gal-stained HCT116 and MCF7 cells with and with out 250 nM Doxorubicin treatment. Treated cells, mainly MCF7 cells, showed strong β-gal expression.

(3) FIG. 3: Graph of staining signals obtained for HCT116 and MCF7 cells stained with LysoTracker® both in the presence and absence of Doxorubicin.

(4) FIG. 4: FACS-analysis data for the compound of Example 2 in HCT116 and

(5) MCF7 cells with (senescence +) and without 250 nM Doxorubicin treatment. The cells were incubated 20 min in 37° C. with 5% CO2. Cells were labeled with 40 ng/ml of the compound of Example 2.Both HCT116 and MCF7 cells treated with Doxorubicin showed enhanced enrichment of the labeled molecule without the need for permiablization.

DESCRIPTION OF PREFERRED EMBODIMENTS

(6) General Methods

(7) All LCMS data (unless otherwise specified) was obtained using an Agilent ESI/APCI HPLC-ms system with a Phenomenex (Luna C-18, 250×4.60 mm, 5 micron) column. The samples were run using a gradient of 5% CH.sub.3CN in water (+0.1% AcOH) for 2 minutes and then increased to 100% CH.sub.3CN over 26 minutes, after which the solvent was maintained at 100% CH.sub.3CN for a further 2 minutes.

(8) Starting Materials and Intermediates

EXAMPLE 1A

(9) ##STR00014##

(10) A solution of 4,7-dichloroquinoline (5.0 g, 25.2 mmol) in ethylenediamine (15 g, 250 mmol) was heated to 90° C., stirring overnight under Argon. TLC indicated the complete consumption of the starting quinoline, with a spot-to-spot conversion into a more polar product. 6M NaOH (30 mL) was added and the organic material was extracted into DCM (3×100 mL). After washing with brine (2×100 mL), drying over MgSO.sub.4, the solvents were removed under vacuum, yielding 2.0 g of a colourless crystalline solid, which was not purified further. The identity and purity were confirmed using LCMS, revealing a single uv active peak (3.47 minutes, m/z 221.7 [M].sup.+).

EXAMPLE 2A

(11) ##STR00015##

(12) To a boiling solution of acetic anhydride (35 mL) and NaOAc (2.5 g, 30.5 mmol) was added L-fucose (5 g, 30.5 mmol) in small portions. Once the solution became translucent, it was removed from the heat and partially solidified upon cooling. The residues were poured over crushed ice (50 g) and solid sodium bicarbonate was added until the evolution of gas ceased. The organic material was extracted into EtOAc (3×100 mL), washed with sodium bicarbonate (100 mL) and brine (100 mL) and dried over MgSO.sub.4. Removing the solvents under vacuum, resulted in a yellow syrup (10.1 g, 99.6%).

EXAMPLE 3A

(13) ##STR00016##

(14) L-fucose (2.0 g, 12.2 mmol) was dissolved in pyridine (15 mL) and cooled to 0° C. in an ice bath. Benzoyl chloride (7.1 mL, 60.1 mmol) was added though a dropping funnel over 5 minutes, after which the ice bath was removed and the reaction was allowed to stir at RT for 3 hours. The reaction mixture was poured into ice-water (100 mL) and extracted into EtOAc (3×100 mL). The residues were washed with ice-cold 1 M HCl (2×100 mL) and brine (100 mL), followed by drying over MgSO.sub.4. After removal of solvents under vacuum, the resulting semi-solid was purified using silica-gel column chromatography (EtOAC:PE=3:7), yielding a crystalline colourless solid (6.9 g, 93%).

EXAMPLE 4A

(15) ##STR00017##

(16) To a solution of HBr (10 mL, 33% in AcOH) was added the compound of Example 2A (2.1 g, 6.3 mmol). The reaction was allowed to stir for 2 hours at RT, after which it was poured over crushed ice (50 g). After extraction into DCM (3×30 mL), the combined organic phases were washed repetitively with portions of sodium bicarbonate (3 to 7×50 ml) until neutral. Drying over MgSO.sub.4 and removal of solvents under vacuum, afforded the bromide as a thick orange syrup. Due to its instability, it was not purified further but used directly in the following reactions.

EXAMPLE 5A

(17) ##STR00018##

(18) The compound of Example 3A (1.1 g, 1.9 mmol) in dry DCM (6 mL) was cooled to 0° C. in a RBF fitted with a rubber septum. After degassing and charging the vessel with Argon, PBr.sub.3 (0.285 mL, 3.1 mmol), followed by water (0.190 mL, 10.5 mmol) were added dropwise through separate disposable syringes. After 15 minutes of stirring at 0° C., the ice bath was removed and the reaction was allowed to stir at RT for 3 hours. DCM (50 mL) was added, followed by water (50 mL). The residues were further extracted into DCM (2×30 mL), washed with sodium bicarbonate (2×50 mL), brine (50 mL) and dried over MgSO.sub.4. After the solvent was removed under vacuum, the resulting colourless solid, shown pure by TLC, remained (1.0 g, 98%).

EXAMPLE 6A

(19) ##STR00019##

(20) The compound of Example 4A (4.0 g, 11.3 mmol), ethylene glycol (7.0 g, 112.7 mmol) and Ag.sub.2CO.sub.3 (4.7 g, 16.9 mmol) in dry CH.sub.3CN (25 mL) were stirred for 24 hours at RT under a calcium chloride drying tube. The solution was filtered through a pad of celite to remove all of the solid material, after which sodium bicarbonate (150 mL) was added and the organic material extracted into DCM (3×100 mL). After drying over MgSO.sub.4 and removing the solvents under vacuum, the resulting orange oil was purified using silica-gel column chromatography (EtOAC:PE=2:5), affording the title compound as an orange oil (1.5 g, 40%).

EXAMPLE 7A

(21) ##STR00020##

(22) Tosyl chloride (1.1 g, 3.0 mmol) was added to a solution of the compound of Example 6A (1.0 g, 3.0 mmol) and pyridine (1 mL) in DCM (15 mL), stirring at 0° C. for 30 minutes. The reaction was then allowed to warm to RT and stirred overnight, after which sodium bicarbonate (50 mL) was added and the organic material extracted into DCM (3×50 mL). After washing with brine (2×30 mL) and aqueous 10% copper sulphate, the crude product was dried over MgSO.sub.4, concentrated under vacuum and was purified using silica-gel column chromatography (EtOAC:PE=2:5) to afford the title compound as a crystalline white solid (0.43 g, 29%).

EXAMPLE 8A

(23) ##STR00021##

(24) 4,7-dichloroquinoline (11.6 g, 58.6 mmol) in 2-aminoethanol (45 mL, 1.37 mol) was refluxed under Argon overnight. After cooling to RT, 1M NaOH (100 mL) was added and allowed to stir for 10 minutes. The resulting yellow solid material was collected using a Büchner funnel, washed with water (5×100 mL) and dried thoroughly under vacuum, affording the title compound as a light yellow solid (12.27 g, 94%).

EXAMPLE 9A

(25) ##STR00022##

(26) A solution containing the compound of Example 2A (2.0 g, 6.0 mmol), the compound of Example 8A (2.0 g, 9.0 mmol) and mol-sieve 4 Å (5 g) in dry DCM (15 mL) was prepared, degassed and allowed to stir under argon for 10 minutes at 0° C. in an ice bath. BF.sub.3.OEt.sub.2 (2.6 g, 18.2 mmol) was added through a rubber septum and the reaction was maintained at 0° C. for a further 30 minutes, after which the ice bath was removed and the reaction was allowed to stir at RT overnight. The next day, the reaction mixture was filtered through a pad of celite and washed thoroughly with DCM (3×20 mL). The organic residue was washed until neutral with sodium bicarbonate (3×50 mL), after which it was further washed with brine (50 mL), dried over MgSO.sub.4 and the solvent removed under vacuum. The crude mixture was purified using silica gel column chromatography (MeOH:EtOAc:NEt3=10:90:1), which afforded the title compound as a yellow semi-solid (0.6 g, 20%). The identity was confirmed by LCMS (10.58 minutes, m/z 494.6 [M].sup.+).

EXAMPLE 10A

(27) ##STR00023##

(28) A solution containing the compound of Example 3A (3.3 g, 5.7 mmol), the compound of Example 8A (1.9 g, 8.3 mmol) and mol-sieve 4 Å (10 g) in dry DCM (30 mL) was prepared, degassed and allowed to stir under argon for 10 minutes at 0° C. in an ice bath. BF3.OEt.sub.2 (4.8 g, 35.06 mmol) was added through a rubber septum and the reaction was maintained at 0° C. for a further 30 minutes, after which the ice bath was removed and the reaction was allowed to stir at RT overnight. The next day, the reaction mixture was filtered through a pad of celite and washed thoroughly with DCM (3×30 mL). The organic residue was washed until neutral with sodium bicarbonate (3×50 mL), after which it was further washed with brine (50 mL), dried over MgSO.sub.4 and the solvent removed under vacuum. The crude mixture was purified using silica gel column chromatography (MeOH:EtOAc:NEt3=10:90:1), which afforded the title compound as a yellow semi-solid (0.9 g, 23%). The identity was confirmed by LCMS (13.29 minutes, m/z 680.6 [M].sup.+).

EXAMPLE 11A

(29) ##STR00024##

(30) To a solution of the compound of Example 11A (107 mg, 0.17 mmol) and K.sub.2OC.sub.3 (40 mg, 0.29 mmol) in DMF (5 mL), stirring at RT under argon, was added CH.sub.3I (50 mg, 0.35 mmol). The reaction was heated to 90° C. for 1 hour. The reaction mixture was poured into a saturated solution sodium bicarbonate (50 mL), extracted into DCM (3×50 mL) and washed with brine (50 mL). Drying over MgSO.sub.4 and removing the solvent under vacuum, afforded the intermediate as an yellow oil. No purification was attempted and the crude reaction mixture was carried over the next step. The identity was confirmed by LCMS (13.64 minutes, m/z 695.7 [M].sup.+).

EXAMPLE 12A

(31) ##STR00025##

(32) A solution of the compound of Example 8A (1.0 g, 4.5 mmol) and pyridine (1 mL, 12.4 mmol) in DCM (13 mL) was prepared and cooled with stirring in an ice-bath. Tosyl chloride (0.9 g, 4.7 mmol) was added and the reaction was allowed to warm to RT. Saturated aqueous sodium bicarbonate (100 mL) was added and the organics extracted into DCM (3×50 mL), followed by washings with more sodium bicarbonate (2×50 mL) and brine (50 mL). The organic material was dried over MgSO.sub.4, the solvents removed under vacuum and purified using silica gel column chromatography (MeOH:EtOAc:NEt.sub.3=5:95:1-MeOH:EtOAc:NEt.sub.3=10:90:1), affording the title compound as a yellow solid (0.55 g, 33%).

EXAMPLE 13A

(33) ##STR00026##

(34) A solution containing 3-diethylaminophenol (15.0 g, 90.7 mmol) in concentrated HCl (100 mL) was prepared and cooled to 0° C. in an ice-bath. NaNO.sub.2 (6.9 g, 100.0 mmol) in water (50 mL) was added slowly over 40 minutes such that no brown NO.sub.x vapours were observed. The reaction was left to stir for 2 hours, after which the thick precipitate was filtered using a Büchner funnel and washed with small portions of water (3×50 mL). After drying the solid for 1 hour on the Büchner funnel, the solid material was dissolved into EtOH (70 mL), Et.sub.2O (35 mL) was added and the solution stored at −20° C. overnight to allow for crystallization. The next day, the solid material was collected by vacuum filtration, using a Büchner funnel and air dried. The product was an orange/red solid (8.8 g, 50%).

EXAMPLE 14A

(35) ##STR00027##

(36) The compound of Example 13A (7.0 g, 36.0 mmol) and 1,6-dihydroxynaphthelene (5.9 g, 36.1 mmol) were refluxed in DMF (100 mL) for 3 hours. After the solvents were removed under vacuum, the residue was redissolved in methanol and adsorbed onto silica and purified using silica gel column chromatography (MeOH:NEt.sub.3=50:1) to afford the title compound as a dark purple solid (11.1 g, 92%).

EXAMPLE 15A

(37) ##STR00028##

(38) A solution containing the compound of Example 14A (4.2 g, 12.6 mmol), K.sub.2CO.sub.3 (2.5 g, 18.1 mmol) and 1,3-dibromopropane (25.2 g, 126 mmol) in DMF (30 mL) was refluxed for 2 hours. The solvent was removed under vacuum and the crude solid purified using silica gel column chromatography (EtOAc:Pet Ether:NEt.sub.3=40:60:1), affording the title compound as a purple solid (1.1 g, 19%). LCMS confirmed the product with a uv-peak (21.40 minutes, m/z 454.6 [M+H].sup.+).

EXAMPLE 16A

(39) ##STR00029##

(40) The compound of Example 4A (1.5 g, 4.3 mmol) and thiourea (0.4 g, 5.5 mmol) were refluxed in dry degassed acetonitrile (20 mL) for 1 hour, after which the flask was cooled for 30 minutes in an ice bath. The solid material was collected with a Büchner funnel, washed with cold acetonitrile and air dried for 30 minutes (0.9 g). The isothiouronium salt (0.9 g, 2.5 mmol) in DCM (10 mL) was added to a 3-necked flask containing thoroughly degassed water (20 mL). To this was added Na.sub.2S.sub.2O.sub.5 (0.6 g, 2.5 mmol), after which the solution was refluxed at 50° C. under argon for 30 minutes and then allowed to cool for a further 10 minutes. An extraction using DCM (3×50 mL), drying over Na.sub.2SO.sub.4, followed by removal of solvents under vacuum afforded the title compound as a translucent solid (0.5 g, 37%).

EXAMPLE 17A

(41) ##STR00030##

(42) The compound of Example 15A (450 mg, 0.99 mmol), the compound of Example 16A (470 mg, 1.53 mmol) and K.sub.2CO.sub.3 (260 mg, 1.88 mmol) were refluxed for 1 hour in acetonitrile (20 mL). TLC revealed a spot-to-spot conversion of the Nile red-bromide to a more polar fluorescent product. After purification using silica gel column chromatography (EtOAc:Pet Ether:NEt.sub.3=1:1), the title compound was afforded as a purple solid (450 mg, 67% mmol).

(43) Exemplary Compounds

EXAMPLE 1

(44) ##STR00031##

(45) The unpurified compound of Example 12A was dissolved in dry methanol (5 mL), after which NaOMe (30-40% in MeOH, 0.1 mL) was added and the reaction was left to stir under a drying tube for 90 minutes. AcOH (30% in methanol) was added dropwise to lower the pH to 4-6. After quenching the NaOMe, the solvents were removed under vacuum to give the title compound. The identity was confirmed by LCMS (8.91 minutes, m/z 382.7 [M].sup.+).

(46) This compound does not fall within the scope of claim 1 but is provided as a model e.g. for the .sup.11C-labeled compounds of the invention.

EXAMPLE 2

(47) ##STR00032##

(48) The compound of Example 1A (0.5 g, 2.3 mmol) was dissolved in DCM (10 mL) containing NEt.sub.3 (1 mL, 6.9 mmol) and the solution was cooled to 0° C. in an ice bath. dansyl chloride (0.6 g, 2.3 mmol) was added as a solid and the reaction was allowed to warm to RT, while stirring under a calcium chloride packed drying tube. Sodium bicarbonate (10 mL) was added and the organic residue extracted into DCM (3×15 mL). The combined organic material was washed with water (10 mL) and then brine (10 mL), dried over MgSO.sub.4 and the solvents removed under vacuum. Purification using silica gel column chromatography (MeOH:EtOAc:NEt.sub.3=10:90:1) afforded the title compound as a yellow solid (1.0 g, 96%). The identity was confirmed by LCMS (10.94 minutes, m/z 454.7 [M].sup.+).

EXAMPLE 3

(49) ##STR00033##

(50) A suspension containing the compound of Example 12A (0.83 g, 3.75 mmol) in DCM (15 mL) was prepared. This suspension was added to a 3 necked flask containing Deoxofluor (50% in THF, 1.79 mL, 4.1 mmol) at RT, stirring under argon. Saturated aqueous sodium bicarbonate (50 mL) was added and the residues extracted into DCM (3×30 mL), washed with more sodium bicarbonate until neutral. The organic material was dried over MgSO.sub.4, the solvents removed under vacuum and the crude material purified using silica gel column chromatography (MeOH:EtOAc:NEt.sub.3=10:90:1) to afford the title compound as a yellow solid (0.37 g, 44%). The identity was confirmed by LCMS (9.29 minutes, m/z 224.7 [M].sup.+).

(51) This compound does not fall within the scope of claim 1 but is provided as a model for the .sup.18F-labeled compounds of the invention.

EXAMPLE 4

(52) ##STR00034##

(53) 4,7-dichloroquinoline (3.0 g, 15.1 mmol) and N,N-dimethylethylenediamine (10.0 g, 113.4 mmol) were heated to 115° C. over night. 6M NaOH (20 mL) was added and the organics extracted into DCM (3×50 mL). After washing with brine (2×100 mL) and drying over MgSO.sub.4, the solvents were removed under vacuum, affording the title compound as a light brown solid (3.7 g, 98%). The product shown to be pure by LCMS (2.83 minutes, m/z 249.7 [M].sup.+).

(54) This compound does not fall within the scope of claim 1 but is provided as a model e.g. for the .sup.11C-labeled compounds of the invention.

EXAMPLE 5

(55) ##STR00035##

(56) The compound of Example 17A (450 mg, 0.7 mmol) was dissolved in dry methanol (10 mL), to which was added sodium methoxide (30-40% in methanol, 0.1 mL). The reaction was allowed to proceed at RT for 5 hours, after which acetic acid (0.5 mL) was added, the solvents removed under vacuum and the crude residue purified using silica gel column chromatography (EtOAc:methanol=4:1), affording JC106 as a black oil (356 mg, 96% mmol).

(57) Evaluation of the Pharmacological Activity

(58) The pharmacological activity of the compounds of the invention is described below in and in reference to the enclosed drawings.

(59) To show the capability of the compounds of the invention to mark and visualise senescent cells, HCT116 cells (colorectal carcinoma cell line) and MCF7 cells (breast cancer cell line) were incubated 20 min at 37° C. with 5% CO.sub.2 in the presence and absence of Doxorubicin. The cells were then labeled with 40 ng/ml of the compound of Example 5.

(60) MCF7 cells treated with Doxorubicin showed enhanced enrichment of the compound of Example 5 inhibitor indicated by a shift in the direction of PerCP-A. This demonstrates the ability of the compound of Example 5 to bind to and visualize senescent cells.

(61) To stimulate HCT116 (colorectal carcinoma cell line) cells and MCF7 (breast cancer cell line) cells for senescence β-galactosidase staining, the cells were treated with 250nM Doxorubicin for 24 hours. Doxorubicin interacts with DNA by intercalation and inhibits the progression of the enzyme topoisonerase II. FIG. 2 shows representative light microscope pictures of β-gal-stained HCT116 and MCF7 cells with and with out 250 nM Doxorubicin treatment. Doxorubicin treated cells enlarge in senescence and, mainly MCF7 cells, showed strong expression of β-gal (blue), indicating the presence of senescent cells (see FIG. 2).

(62) In FIG. 3 a graph is displayed indicating the signal obtained from a lysosome staining of HCT116 (colorectal carcinoma cell line) cells and MCF7 (breast cancer cell line) cells, both in the presence and absence of Doxorubicin. This graph again shows an enhanced positive lysosome staining compared to cells without Doxorubicin treatment (untreated cells), indicating the presence of senescent cells.

(63) To show the capability of the compounds of the invention to mark and visualise senescent cells, HCT116 cells (colorectal carcinoma cell line) and MCF7 cells (breast cancer cell line) were incubated 20 min in 37° C. with 5% CO.sub.2. The cells were then labeled with 40 ng/ml of the compound of Example 2. Both HCT116 and MCF7 cells treated with Doxorubicin showed enhanced enrichment of the labeled molecule without the need for permeabilisation (see FIG. 4). This demonstrates the ability of the compound of Example 2 to bind to and visualize senescent cells even in non-permeabilised cells.

(64) Summary

(65) The data displayed in FIG. 2 and FIG. 3 clearly demonstrates the presence of senescent cells in the cell lines treated with Doxorubicin. FIG. 1 and FIG. 4 show clearly that the compounds of the invention accumulate in senescent cells and produce a measurable signal. This shows that the compounds of the invention can serve as markers for senescent cells and can be detected e.g. by optical imaging methods.