CDC7-Inhibiting Purine Derivatives and their use for the Treatment of Neurological Conditions

Abstract

The present invention relates to the use of a series of purine derivatives having the following general formula:

##STR00001##

(I) and which are capable of inhibiting CDC7 kinase activity and, therefore, suitable for use in the treatment of neurological diseases such as, inter alia, Alzheimer's disease, amyotrophic lateral sclerosis or frontotemporal dementia, which involve hyperphosphorylation of TDP-43 and the subsequent formation of clusters, induced by CDC7.

Claims

1. A method of treatment and/or prevention of pathologies related to the TDP-43 protein in a subject, comprising administering to said subject an effective amount of a compound of formula (I) ##STR00012## wherein: X is selected from CH.sub.2, CO; n is 1; Ar is selected from the following groups: ##STR00013## wherein R.sub.1 to R.sub.7 are independently selected from H, NH.sub.2, —O(C.sub.1-C.sub.4 alkyl), NH(C.sub.1-C.sub.4 alkyl) or halogen and represents the binding site to the rest of the molecule; or wherein n is 0; Y is CH; and Ar is the following group: ##STR00014## wherein R.sub.1 to R.sub.5 are independently selected from H, NH.sub.2, —O(C.sub.1-C.sub.4 alkyl), NH(C.sub.1-C.sub.4 alkyl) or halogen and represents the binding site to the rest of the molecule; or any of the pharmaceutically acceptable salts, solvates or isomers thereof; with the condition that the compound of formula (I) is not the following compound: ##STR00015##

2. The method according to claim 1, wherein Ar is the following group: ##STR00016##

3. The method according to claim 2, wherein n is 1.

4. The method according to claim 3, wherein X is CO and Y is N.

5. The method according to claim 3, wherein X is CH.sub.2 and Y is N.

6. The method according to any of claim 2, wherein at least one of R.sub.1 to R.sub.5 is —O(C.sub.1-C.sub.4 alkyl).

7. The method according to claim 6, wherein at least one of R.sub.1 to R.sub.5 is —O-methyl.

8. The method according to any of claim 2, wherein at least one of R.sub.1 to R.sub.5 is halogen.

9. The method according to claim 1, wherein Ar is the following group: ##STR00017##

10. The method according to claim 9, wherein X is CO and Y is N.

11. The method according to claim 9, wherein R.sub.1 to R.sub.7 is H.

12. The method according to claim 1, wherein the compound is selected from the list consisting of: 1-(4-methoxyphenyl)-2-((9H-purin-6-yl) thio)-ethan-1-one 1-(naphthalen-2-yl)-2-((9H-purin-6-yl)thio)-ethan-1-one 1-(3-methoxyphenyl)-2-((9H-purin-6-yl)thio)-ethan-1-one 1-(4-iodophenyl)-2-((9H-purin-6-yl)thio)-ethan-1-one 6-((4-chlorophenethyl)thio)-9H-purine 6-((3-chlorophenethyl)thio)-9H-purine 6-((2-chlorophenethyl)thio)-9H-purine 6-((4-fluorophenethyl)thio)-9H-purine, and 4-(benzylthio)-7H-pyrrolo[2,3-d]pyrimidine.

13. The method according to claim 1, wherein the disease related to the TDP-43 protein is a neurological disease.

14. The method according to claim 13, wherein the disease related to the TDP-43 protein is a neurological disease selected from amyotrophic lateral sclerosis, frontotemporal dementia, Alzheimer's disease, age-related cognitive decline and chronic traumatic encephalopathy.

15. The method according to claim 14, wherein the disease related to the TDP-43 protein is selected from amyotrophic lateral sclerosis, frontotemporal dementia and Alzheimer's disease.

16. A compound selected from: 6-((4-Chlorophenethyl)thio)-9H-purine 6-((3-Chlorophenethyl)thio)-9H-purine 6-((2-Chlorophenethyl)thio)-9H-purine 6-((4-Fluorophenethyl)thio)-9H-purine, and 4-(benzylthio)-7H-pyrrolo[2,3-d]pyrimidine.

17. A pharmaceutical composition comprising a compound according to claim 16 together with a pharmaceutically acceptable vehicle.

18. (canceled)

Description

BRIEF DESCRIPTION OF THE FIGURES

[0060] FIG. 1 shows the linear correlation between the described permeability and the experimental permeability of 10 commercial compounds using the PAMPA methodology.

[0061] FIG. 2 shows the neuroprotective effect of the CDC7 inhibitor (compound 3) in SH-SY5Y human neuroblastoma cells previously treated with ethacrynic acid (EA, 20 μM) for 12 hours in the presence or absence of the inhibitor at 10 μM and two reference compounds (GSK-3 inhibitors). The data represents the mean of four different experiments ±SEM (*p<0.05).

EXAMPLES

[0062] Next, the invention will be illustrated by means of assays carried out by the inventors which demonstrate the effectiveness of the product of the invention.

Example 1: Synthesis of the New Compounds of the Invention

1-(4-Bromophenyl)-2((9H-purin-6-yl)thio)-ethan-1-one (1)

[0063] Product 1 has been synthesised according to the procedure described in Chemische Berichte, 1989, 122(5), 919-924.

1-(4-Methoxyphenyl)-2((9H-purin-6-yl)thio)-ethan-1-one (2)

[0064] Product 2 has been synthesised according to the procedure described in Asian Journal of Chemistry, 2010, 22(1), 689-698.

1-(Naphthalen-2-yl)-2((9H-purin-6-yl)thio)-ethan-1-one (3)

[0065] (Commercially available CAS Registry Number 312281-93-9) 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 2 h at room temperature. Then 2-bromo-2′-acetonaphthone (439.2 mg, 1.76 mmol) is added and it is stirred for 78 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. It is not necessary to purify it by means of column chromatography. In this manner, product 3 is obtained in the form of a yellow solid (158.6 mg, 28%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.57 (s, 1H), 8.87 (s, 1H), 8.56 (s, 1H), 8.46 (s, 1H), 8.16 (dd, J=8.0, 4.0 Hz, 1H), 8.10-7.98 (m, 3H), 7.75-7.60 (m, 2H), 5.18 (s, 2H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 193.9 (1C), 158.2 (1C), 152.0 (1C), 149.9 (1C), 143.8 (1C), 135.8 (1C), 133.8 (1C), 132.8 (1C), 131.1 (1C), 130.3 (2C), 129.5 (1C), 129.1 (1C), 128.4 (1C), 127.8 (1C), 124.5 (1C), 37.0 (1C). HPLC: Purity=95%. MS (ES): m/z 321 [M+1]. M.p. 202-204° C. Elemental analysis (C.sub.17H.sub.12N.sub.4OS) Calculated: C, 63.73%, H, 3.78%, N, 17.49%, S, 10.01%. Found: C, 63.56%, H, 4.02%, N, 17.21%, S, 9.89%.

1-(3-Methoxyphenyl)-2((9H-purin-6-yl)thio)-ethan-1-one (4)

[0066] (Commercially available CAS Registry Number 1458134-70-7) 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 2 h at room temperature. Then 2-bromo-3′-methoxyacetophenone (403.2 mg, 1.76 mmol) is added and it is stirred for 20 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. It is not necessary to purify it by means of column chromatography. In this manner, product 4 is obtained in the form of a brown solid (297.0 mg, 55%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.53 (s, 1H), 8.54 (s, 1H), 8.44 (s, 1H), 7.66 (dd, J=8.0, 4.0 Hz, 1H), 7.52 (s, 1H), 7.46 (dd, J=8.0, 4.0 Hz, 1H), 7.23 (dd, J=8.0, 4.0 Hz, 1H), 5.01 (s, 2H), 3.80 (s, 3H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 193.8 (1C), 160.1 (1C), 158.2 (1C), 151.9 (1C), 149.9 (1C), 143.8 (1C), 137.9 (1C), 130.7 (2C), 121.5 (1C), 120.2 (1C), 113.4 (1C), 56.1 (1C), 37.0 (1C). HPLC: Purity=98%. MS (ES): m/z 301 [M+1]. M.p. 190-192° C. Elemental analysis (C.sub.14H.sub.12N.sub.4O.sub.2S) Calculated: C, 55.99%, H, 4.03%, N, 18.65%, S, 10.68%. Found: C, 55.87%, H, 4.00%, N, 18.52%, S, 10.39%.

1-(4-Iodophenyl)-2((9H-purin-6-yl)thio)-ethan-1-one (5)

[0067] (Commercially available CAS Registry Number 1460254-51-6) 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 2 h at room temperature. Then 2-bromo-4′-iodoacetophenone (571.9 mg, 1.76 mmol) is added and it is stirred for 17 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. The crude product is purified by means of an automatic column using CH.sub.2Cl.sub.2/MeOH (9:1) as eluent. In this manner, product 5 is obtained in the form of a yellow solid (61.8 mg, 9%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.53 (s, 1H), 8.52 (s, 1H), 8.42 (s, 1H), 7.98 (dd, J=8.0, 4.0 Hz, 2H), 7.85 (dd, J=8.0, 4.0 Hz, 2H), 4.96 (s, 2H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 193.7 (1C), 157.2 (1C), 153.7 (1C), 151.7 (1C), 143.9 (1C), 138.3 (2C), 135.6 (1C), 130.5 (3C), 102.5 (1C), 36.5 (1C). MS (ES): m/z 396 [M+1]. M.p. 192-194° C. Elemental analysis (C.sub.13H.sub.9IN.sub.4OS) Calculated: C, 39.41%, H, 2.29%, N, 14.14%, S, 8.09%. Found: C, 39.20%, H, 2.19%, N, 13.90%, S, 7.74%.

6-((4-Chlorophenethyl)thio)-9H-purine (6)

[0068] 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 1 h at room temperature. Then 4-chlorophenethyl bromide (387.0 mg, 1.76 mmol) is added and it is stirred for 2 h and 30 min at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. The crude product is purified by means of a chromatographic column using CH.sub.2Cl.sub.2/MeOH (10:1) as eluent. In this manner, product 6 is obtained in the form of a white solid (315.4 mg, 62%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.51 (s, 1H), 8.71 (s, 1H), 8.44 (s, 1H), 7.40-7.29 (m, 4H), 3.59 (t, J=7.5 Hz, 2H), 3.02 (t, J=7.5 Hz, 2H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 158.2 (1C), 151.5 (1C), 149.6 (1C), 143.3 (1C), 139.1 (1C), 131.0 (1C), 130.5 (2C), 130.0 (1C), 128.3 (2C), 34.4 (1C), 29.0 (1C). M.p. 190-192° C. Elemental analysis (C.sub.13H.sub.11ClN.sub.4S) Calculated: C, 53.70%, H, 3.81%, N, 19.27%, S, 11.03%. Found: C, 53.27%, H, 3.79%, N, 19.03%, S, 10.85%.

6-((3-Chlorophenethyl)thio)-9H-purine (7)

[0069] 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 1 h at room temperature. Then 3-chlorophenethyl bromide (387.0 mg, 1.76 mmol) is added and it is stirred for 3 h and 30 min at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. The crude product is purified by means of a chromatographic column using CH.sub.2Cl.sub.2/MeOH (20:1) as eluent. In this manner, product 7 is obtained in the form of a white solid (402.2 mg, 78%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.51 (s, 1H), 8.71 (s, 1H), 8.42 (s, 1H), 7.39 (t, J=1.6 Hz, 1H), 7.37-7.24 (m, 3H), 3.62 (t, J=7.5 Hz, 2H), 3.04 (t, J=7.5 Hz, 2H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 158.5 (1C), 151.5 (1C), 149.2 (1C), 142.9 (1C), 142.6 (1C), 133.0 (1C), 130.4 (1C), 130.2 (1C), 128.5 (1C), 127.4 (1C), 126.4 (1C), 34.7 (1C), 28.8 (1C). M.p. 143-145° C. Elemental analysis (C.sub.13H.sub.11ClN.sub.4S) Calculated: C, 53.70%, H, 3.81%, N, 19.27%, S, 11.03%. Found: C, 53.34%, H, 3.80%, N, 19.09%, S, 11.00%.

6-((2-Chlorophenethyl)thio)-9H-purine (8)

[0070] 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 1 h at room temperature. Then 2-chlorophenethyl bromide (387.0 mg, 1.76 mmol) is added and it is stirred for 5 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. The crude product is purified by means of a chromatographic column using CH.sub.2Cl.sub.2/MeOH (10:1) as eluent. In this manner, product 8 is obtained in the form of a white solid (250.4 mg, 49%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.49 (s, 1H), 8.70 (s, 1H), 8.43 (s, 1H), 7.44 (dd, J=6.9, 2.5 Hz, 1H), 7.43 (dd, J=6.9, 2.4 Hz, 1H), 7.33-7.22 (m, 2H), 3.62 (dd, J=8.3, 6.6 Hz, 2H), 3.17 (dd, J=8.2, 6.7 Hz, 2H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 157.6 (1C), 151.4 (1C), 150.3 (1C), 143.3 (1C), 137.3 (1C), 133.1 (1C), 131.1 (1C), 129.2 (2C), 128.4 (1C), 127.2 (1C), 32.7 (1C), 27.6 (1C). M.p. 154-156° C. Elemental analysis (C.sub.13H.sub.11ClN.sub.4S) Calculated: C, 53.70%, H, 3.81%, N, 19.27%, S, 11.03%. Found: C, 53.60%, H, 3.86%, N, 19.14%, S, 10.85%.

6-((4-Fluorophenethyl)thio)-9H-purine (9)

[0071] 6-mercaptopurine monohydrate (300.0 mg, 1.76 mmol) and K.sub.2CO.sub.3 (243.7 mg, 1.76 mmol) are dissolved in DMF (25 ml). The reaction mixture is stirred for 1 h at room temperature. Then 4-fluorophenethyl bromide (358.0 mg, 1.76 mmol) is added and it is stirred for 5 h at room temperature. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml) by adding a little NaCl. It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. The crude product is purified by means of a chromatographic column using CH.sub.2Cl.sub.2/MeOH (10:1) as eluent. In this manner, product 9 is obtained in the form of a white solid (285.0 mg, 59%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 13.51 (s, 1H), 8.70 (s, 1H), 8.42 (s, 1H), 7.45-7.26 (m, 2H), 7.22-6.94 (m, 2H), 3.59 (t, J=7.6 Hz, 2H), 3.02 (t, J=7.5 Hz, 2H). .sup.13C-NMR (75 MHz, DMSO-d.sub.6): δ 160.9 (d, J=241.7 Hz, 1C), 158.6 (1C), 151.5 (1C), 149.2 (1C), 142.9 (1C), 136.3 (d, J=2.7 Hz, 1C), 130.4 (d, J=7.9 Hz, 2C), 130.4 (1C), 115.0 (d, J=21.0 Hz, 2C), 34.3 (1C), 29.2 (1C). HPLC: Purity >99%, t.r.=6.99 min. MS (ES): m/z 275 [M+1]. M.p. 184-186° C. Elemental analysis (C.sub.13H.sub.11FN.sub.4S) Calculated: C, 56.82%, H, 4.04%, N, 20.42%, S, 11.69%. Found: C, 56.38%, H, 4.01%, N, 20.04%, S, 11.43%.

4-(benzylthio)-7H-pyrrolo[2,3-d]pyrimidine (10)

[0072] Under an argon atmosphere and at 0° C., a mixture of 6-mercaptopurine (0.49 g, 3.9 mmol) and NaH (47 mg, 1.96 mmol) is added in DMF on a solution of 4-chloro-7H-pyrrolo[2,3-d]pyrimidine (0.15 g, 1.0 mmol) in DMF. The reaction mixture is stirred at room temperature under an argon atmosphere overnight. After the reaction is complete, the mixture is neutralised with methanol. The solvent is evaporated under reduced pressure and AcOEt (100 ml) is added. The organic phase is washed with distilled water (3×100 ml). It is dried over anhydrous Mg.sub.2SO.sub.4, filtered and concentrated until it is dry. The crude product is purified by means of a chromatographic column using Hexane/AcOEt (10:1) as eluent. In this manner, product 10 is obtained in the form of a white solid (164 mg; 70%). .sup.1H-NMR (300 MHz, DMSO-d.sub.6): δ 4.68 (s, 2H, CH.sub.2), 6.52 (dd, J=4.0, 2.0 Hz, 1H), 7.26-7.50 (m, 6H), 8.69 (s, 1H), 12.25 (s, 1H, NH). .sup.13C-NMR (DMSO-d.sub.6): δ 31.6, 98.2, 114.7, 125.8, 127.0, 128.4, 128.9, 138.0, 149.0, 150.2, 158.7. M.p. 167-168° C. Elemental analysis (C.sub.13H.sub.11N.sub.3S) Calculated: C, 64.71; H, 4.59; N, 17.41; S, 13.29. Found: C, 64.59; H, 4.64; N, 17.28; S, 13.22.

Example 2: Measurement of CDC7 Inhibition

[0073] The LanthaScreen Eu Kinase Inhibition Assay uses an Alexa Fluor™ marker which binds to a kinase and is detected by the addition of an Eu-labelled antibody. The binding of the marker and the antibody to the kinase results in a high degree of FRET, whereas the displacement of the marker by an inhibitor results in a loss of FRET. Unlike many other kinase activity assays, this is a simple mix and read assay, without stages of development. This assay method has been developed by “Life Technologies” and they identify competitive ATP kinase inhibitors, making them suitable for the detection of any compound which binds to the ATP site.

[0074] The compounds are evaluated in 1% DMSO (final) in the well. A mixture of recombinant human Cdc7/DBF4 (0.5 nM), Eu-anti-GST antibody (2 nM) and Alexa Fluor marker (1 nM) in a buffer of 50 mM HEPES pH 7.5, 0.01% BRIJ-35, 10 mM MgCl2, 1 mM EGTA was used. In white plates with 348 wells, with a low volume, and coded (Greiner cat. #784207), 160 nL (100×compound in 100% DMSO), 3.84 μl (buffer with Cdc7/DBF4), 8.0 μl (antibody), 4.0 μl (marker) are added. It is stirred for 30 s and incubated at room temperature for 60 min. Next, the fluorescence is measured in the plate reader and the data is analysed (Table 1).

TABLE-US-00001 TABLE 1 Inhibition values in Cdc7 of the compounds of formula (I): Comp. [00008] [00009] X Z Y Cdc7 CI.sub.50 (μM)  1* 4-Br — CO S N 2.120 2 4-OMe — CO S N 1.080 3 — H CO S N 0.698 4 3-OMe — CO S N 1.710 5 4-I — CO S N 0.380 6 4-Cl — CH.sub.2 S N 0.142 7 3-Cl — CH.sub.2 S N 0.305 8 2-Cl — CH.sub.2 S N 0.191 9 4-F — CH.sub.2 S N 0.397 10  H — — S CH 0.234 *Comp. (1): 1-(4-Bromophenyl)-2-((9H-purin-6-yl)thio)-ethan-1-one, included for comparison.

Example 3: Prediction of the Passage Across the Blood-Brain Barrier

[0075] An essential requirement that must be met by drugs for the treatment of neurodegenerative diseases is the ability to cross the blood-brain barrier (BBB) since, otherwise, they could not act on the target of interest. Therefore, for the compounds that are non-permeable or located in the zone of uncertainty, it could be necessary for them to be suitably conveyed in a pharmaceutical formulation by means of methods known by those skilled in the art, such as by means of encapsulation. This ability can be predicted in vitro by using a method known by the acronym PAMPA (Parallel Artificial Membrane Permeability Assay) described by Di et al. (Di, L.; Kerns, E. H.; Fan, K.; McConnell, O. J.; Carter, G. T. Eur. J. Med. Chem., 2003, 38 (3), 223-232) and that has subsequently been fine-tuned in our research group. Said method enables the effective permeability through artificial membranes to be predicted by means of a passive diffusion process.

[0076] First, it is necessary to validate the method, for which reason 10 commercial compounds, whose ability to penetrate the central nervous system (CNS) is known, are used and will be specified below, obtaining in this case good linear correlation between the experimental permeability values (Pe) and those described (FIG. 1). This correlation line obtained following the pattern described in the literature makes it possible to establish the limits for predicting whether a compound can cross the blood-brain barrier or not. Thus, a compound is considered to be permeable to the BBB (CNS+) if it has a permeability >4.48×10.sup.−6 cm.Math.s.sup.−1.

[0077] For the procedure, between 3-5 mg of caffeine, desipramine, enoxacin, hydrocortisone, ofloxacin, piroxicam and testosterone, 12 mg of promazine and 25 mg of atenolol and verapamil were taken and dissolved in EtOH (1000 μl). 100 μl of these solutions were taken and EtOH (1400 μl) and phosphate buffer (PBS) pH=7.4 (3500 μl) were added in order to reach a final EtOH concentration of 30% v/v in solution. Lastly, the solutions were filtered.

[0078] Moreover, a solution of PBS/EtOH (70:30) was added to each well of the acceptor plate (180 μl). The donor plate was impregnated with a solution of porcine brain lipid (4 μl) dissolved in dodecane (20 mg.Math.ml.sup.−1). After 5 min, a solution of each compound was added on this plate (180 μl).

[0079] Of the compounds evaluated, between 1-2 mg were taken and dissolved in EtOH (1500 μl) and phosphate buffer (PBS) pH=7.4 (3500 μl), filtered and added to the donor plate. With these solutions, the wavelengths at which they absorb the compounds are determined and the initial absorbance levels at these wavelengths are measured by using a UV absorbance reader. Each sample was analysed at two to five wavelengths, in three wells and in two independent experiments.

[0080] Next, the donor plate was deposited on the acceptor plate forming a sort of “sandwich” and they were left to incubate for 2 h and 30 min at 25° C. Thus, the compounds will pass from the donor plate to the acceptor plate through the porcine brain lipid by means of passive diffusion. After that time, the donor plate is carefully removed and the final concentration and absorbance of both the commercial and the synthesised compounds are determined. The results obtained are expressed as the mean of the measurements [standard deviation (SD)] of the different experiments performed and are recorded in Table 2.

TABLE-US-00002 TABLE 2 Permeability values (Pe 10.sup.-6 cm s.sup.-1) in the PAMPA-BBB experiment and prediction of the penetration into the central nervous system (CNS) of the compounds of formula (II) as they are also described in Table 1: Comp. [00010] [00011] X Z Y Pe (10.sup.-6 cm s.sup.-1) PAMPA prediction 2 4-OMe — CO S N  2.5 ± 0.1 CNS+/− 3 — H CO S N  9.6 ± 0.8 CNS+ 4 3-OMe — CO S N 2.4 ± 1  CNS+/− 5 4-I — CO S N 10.5 ± 1.2 CNS+ 6 4-Cl — CH.sub.2 S N 25.1 ± 1.3 CNS+ 7 3-Cl — CH.sub.2 S N 10.6 ± 0.5 CNS+ 8 2-Cl — CH.sub.2 S N 11.6 ± 0.5 CNS+ 9 4-F — CH.sub.2 S N 10.2 ± 0.5 CNS+ 10 H — — S CH  4.5 ± 1.2 CNS+

Example 4: Neuroprotective Effect of the Cdc7 Inhibitors Against Ethacrynic Acid

[0081] The SH-SY5Y human neuroblastoma cell line was grown at 37° C. with 5% CO.sub.2 in DMEN (Dulbecco's Modified Eagle Medium) enriched with L-glutamine (2 mM), 1% non-essential amino acids, 1% Penicillin/Streptomycin and 10% foetal bovine serum. In the state of semi-confluence, the cells were treated with the Cdc7 inhibitor (compound 3) at different concentrations for 1.30 hours post-addition of the causative agent of the TDP-43 phosphorylation; ethacrynic acid (20 μM) (Sigma). At 24 hours, the cellular viability was evaluated with MTT ([3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) following a described procedure (Denizot F, Lang R. J Immunol Methods. 1987; 89:271-7). FIG. 2 shows how the cells have a 40% mortality due to the excess TDP-43 phosphorylation (ethacrynic acid) and how this death is reversed with the addition of the reference standards, as well as with the CDC7 inhibitor.