MODIFIED AMINO ACID DERIVATIVES FOR THE TREATMENT OF NEUROLOGICAL DISEASES AND SELECTED PSYCHIATRIC DISORDERS

Abstract

2-(2,5-dioxopyrrolidin-1-yl)propanamide and 2-(2-oxopyrrolidin-1-yl)propanamide derivatives with R-configuration at the stereogenic center are disclosed, showing broad-spectrum protective activity in animal models of epileptic seizures, pain, depression and anxiety that are simultaneously devoid of undesirable sedative effects. Additionally, the disclosed derivatives have neuroprotective effects in the in vitro and in vivo studies.

##STR00001##

Claims

1. A compound of formula (I) ##STR00007## wherein: X is hydrogen or N(CH.sub.3).sub.2, Y is CH.sub.2 or C═O, R is hydrogen or halogen, or pharmaceutically acceptable salts thereof.

2. The compound of claim 1, wherein the compound is selected from: (2R)—N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide, (2R)-2-(2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide, (2R)—N-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)—N-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)—N-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide, and (2R)-2-(3-(dimethylamino)-2,5-dioxo-pyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide.

3. The compound of claim 1, wherein the compound is selected from: (2R)—N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide and (2R)-2-(2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide.

4. The compound of claim 1, wherein the compound is selected from: (2R)—N-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)—N-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide, (2R)—N-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide and (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide.

5. The compound of claim 1, wherein the compound is in the form of a pharmaceutically acceptable salt selected from: hydrochloride, sulfate, methanesulfonate, toluenesulfonate, succinate, fumarate or lactate.

6. A method for the treatment or prevention of epilepsy, epilepsy with concomitant depression and anxiety disorder, depression, anxiety, neurological pain, inflammatory pain or neurodegenerative disease, wherein the method comprises administering the compound of claim 1 to an animal.

7. The method of claim 6, wherein the neurodegenerative disease is Parkinson's disease or Alzheimer's disease or amyotrophic lateral sclerosis.

8. The compound of claim 1, wherein R is F.

9. The compound of claim 1, wherein the compound is (2R)—N-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide or (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide.

10. The method of claim 6, wherein the animal is a human or a mouse.

Description

[0042] FIG. 1 shows analgesic activity of compound 1 in the first and second phase of the formalin test. The results are presented as paw licking time in the first phase of the test (0-5 minutes after formalin injection) and in the second phase of the test (15-30 minutes after formalin injection). The values represent mean±SEM for a group of 8-10 animals. Statistically significant difference in comparison to the control group given only vehicle (Tween 80). One-way analysis of variance (ANOVA) followed by post-hoc Dunnett's test: *p<0.05, **p<0.01, ***p<0.001, ****p<0.0001. C—control group.

[0043] FIG. 2 shows the analgesic activity of compound 1 in the oxaliplatin-induced neuropathic pain model. The results show the pain threshold (stimulus at which the animal withdraws its paw) 30 minutes after administration of the compound. The values represent mean±SEM for a group of 10 animals. A statistically significant difference compared to the group given only oxaliplatin (one-way repeated-measures analysis of variance (ANOVA), Dunnett's post hoc test): *p<0.05, ***p<0.001, ****p<0.0001.

[0044] FIG. 3 shows the analgesic activity of compound 1 in the streptozotocin-induced model of painful diabetic neuropathy. The results show the pain threshold (stimulus at which the animal withdraws its paw) 30 minutes after administration of the compound. The values represent the mean±SEM for a group of 10 animals. A statistically significant difference compared to the group given the vehicle (1% Tween 80) (one-way repeated-measures analysis of variance (ANOVA), Dunnett's post hoc test): *p<0.05, ***p<0.001, ****p<0.0001.

[0045] FIG. 4 shows the antidepressant activity of compound 1 in the forced swim test. The results show the total immobility time during 4 minutes of observation of mice placed in a cylinder filled with water. The values represent mean±SEM for a group of 8-10 animals. A statistically significant difference compared to the control group given the vehicle alone (1% Tween 80) (one-way analysis of variance (ANOVA), Dunnett's post hoc test): ***p<0.001.

[0046] FIG. 5 shows the anxiolytic activity of compound 1 in the four plate test. The results show the total number of electrically punished plate crossings during 60 seconds of observation of mice placed in specialized cages. The values represent mean±SEM for a group of 8-10 animals A statistically significant difference compared to the control group given the vehicle alone (1% Tween 80) (one-way analysis of variance (ANOVA), Dunnett's post hoc test): ***p<0.001.

[0047] FIG. 6 shows the effect of compound 1 and 2 on the spontaneous locomotor activity of animals. The results show the number of infrared light beams interruptions during 30 minutes of measurement. The values represent mean±SEM for a group of 10 animals. A statistically significant difference compared to the control group (one-way repeated-measures analysis of variance (ANOVA), Dunnett's post hoc test): *p<0.05, **p<0.01.

[0048] FIG. 7 shows the neuroprotective properties of test compound 1 after PILO-induced SE (A—control mouse+PILO, B-F— compound 1+PILO).

[0049] FIG. 8A shows the effect of the reference inhibitor ketoconazole (KE) and 1 on CYP3A4 activity. FIG. 8B shows the effect of the reference inhibitor quinidine (QD) and 1 on CYP2D6 activity. FIG. 8C shows the effect of the reference inhibitor sulfafenazole (SE) and 1 on CYP2C9 activity. Statistical significance was calculated by one-way analysis of variance (ANOVA) and the Bonferroni method (*p<0.05, ***p<0.001, compounds tested in triplicate).

[0050] FIG. 9 shows a chromatogram of a reaction mixture after 120 min incubation of compound 1 with human liver microsomes.

[0051] FIG. 10 shows the effect of the reference cytostatics doxorubicin (DX) and compound 1 on HEK-293 cell viability after 72 hours of incubation. Statistical significance was calculated by one-way analysis of variance (ANOVA) and the Bonferroni method (***p<0.001, compounds tested in four replications).

[0052] FIG. 11 shows the effect of the reference cytostatics—doxorubicin (DX), mitochondrial toxin CCCP and 1 on HepG2 cell viability after 72 hours of incubation. Statistical significance was calculated by one-way analysis of variance (ANOVA) and the Bonferroni method (***p<0.001, compounds tested in four replications).

[0053] FIG. 12 shows the neuroprotective properties of compounds 1 and 2 against human neurons derived from SH-SY5Y neuroblastoma cells under trophic stress. Viability of neurons was determined by MTT test after 48 hours exposure to test compounds under standard conditions and trophic stress induced by removal of the B27 supplement from the culture medium. The results are presented as the mean±SD of at least 6 measurements. Statistically significant differences compared to controls at ***p<0.001; compared to cells grown in medium without B27 *p<0.05, **p<0.01, ***p<0.001. Statistical analysis was performed by one-way ANOVA with Tukey's post-hoc test.

[0054] The following are examples of embodiments of the invention.

EXAMPLE 1. PREPARATION OF COMPOUNDS ACCORDING TO THE INVENTION

Analytical Methods:

[0055] Proton magnetic resonance (.sub.1H NMR) and carbon nuclear magnetic resonance (.sub.13C NMR) spectra were recorded using a JEOL-500 spectrometer (JEOL USA, Inc. MA, USA), at 500 MHz and 126 MHz, respectively. Chemical shifts are reported in δ values (ppm) relative to TMS δ=0 (.sub.1H) as an internal standard. The J values are expressed in hertz (Hz). Deuterated chloroform (CDCl.sub.3) was used as the solvent. The following signal abbreviations have been used in the description of the spectra: s (singlet), br s (broad singlet), d (doublet), t (triplet), q (quartet), m (multiplet). The UPLC/MS analysis system consisted of a Waters ACQUITY® UPLC® apparatus (Waters Corporation, Milford, Mass., USA) coupled with a Waters TQD mass spectrometer operating in electrospray ionization (ESI) mode. Chromatographic separations were carried out using an Acquity UPLC BEH C18 (2.1×100 mm, 1.7 μm) column. The column was maintained at 40° C. and eluted with a gradient of 95% to 0% of eluent A over 10 min, with a flow rate of 0.3 mL/min. Eluent A: water/formic acid (0.1%, v/v); eluent B: acetonitrile/formic acid (0.1%, v/v). Chromatograms were recorded using a Waters eλ PDA detector. Spectra were analyzed in the 200-700 nm range with a resolution of 1.2 nm and a sampling rate of 20 points/s. Enantiomeric purity of compounds 1-4 was determined using a chiral HPLC chromatograms analysis on a Shimadzu Prominence and LC-2030C SD Plus apparatus (Shimadzu Corporation, Kyoto, Japan) equipped with an Amylose-C (250×4.6 mm) chiral column. The analysis was performed under the following conditions: column temperature: 20° C., eluent mixture: hexane/i-PrOH=85/15 (v/v), flow rate: 0.7 mL/min, detection at λ=209 nm. For intermediate VII, the analysis was performed under the following conditions: column temperature: 33° C., eluent mixture hexane/i-PrOH/TFA=93.4/6.4/0.2 (v/v/v), flow rate: 0.75 mL/min, detection at λ=206 nm. Specific rotation ([α].sub.20D) of compounds was tested on a Jasco p-2000 polarimeter (Jasco Inc. Easton, Md., USA). Thin layer chromatography (TLC) was performed on aluminum plates precoated with silica gel 60 F.sub.254 (Macherey-Nagel, Duren, Germany), using solvent systems with the following composition: DCM:MeOH (9:0.3; v/v), DCM:MeOH (9:0.5; v/v). Spot detection—UV light (λ=254 nm). Melting points (m.p.) were determined using open capillaries in a Büchi 353 apparatus (Büchi Labortechnik, Flawil, Switzerland). Absolute configuration was confirmed by crystallographic method using a SuperNova diffractometer (Rigaku—Oxford Diffraction, UK). The names of the chemical compounds described below as exemplary embodiments of the invention were obtained using the ChemBioDraw Ultra 12.0 program.

[0056] The preparation of compounds according to the invention is illustrated in the following examples. The synthesis presented in the examples were not optimized in terms of yield, amount of reagents used or the final form of the compounds obtained.

Abbreviations used: AcOEt—ethyl acetate, DCM—dichloromethane, DCC—N,N′-dicyclohexylcarbodiimide, Et.sub.2O—diethyl ether, HCl—hydrochloric acid, HMDS—hexamethyldisilazane, MeOH—methanol, NaCl—sodium chloride, NaH—sodium hydride, NH.sub.4OH—ammonium hydroxide, Na.sub.2SO.sub.4—sodium sulfate, TFA—trifluoroacetic acid, TEA—triethylamine, ZnCl.sub.2—zinc chloride.

Examples of Synthesis as Well as Physicochemical and Spectral Data of Intermediate Products (II-IV) According to Scheme 1

Intermediate II (R═H): Tert-butyl-(R)-(1-(benzylamino)-1-oxopropan-2-yl)carbamate

[0057] Boc-D-alanine (5.1 g, 27 mmol, 1 eq) was dissolved in 20 mL of DCM, then DCC (6.68 g, 32.4 mmol, 1.2 eq) was added, the mixture was stirred and after 30 minutes benzylamine (2.89 g, 27 mmol, 1 eq) was added dropwise. The reaction evaporated to dryness. Intermediate II was purified by column chromatography using a DCM:MeOH (9:0.3; v/v) eluent system. Intermediate II was obtained as a light oil. Yield: 91% (6.95 g); TLC:R.sub.f=0.43 (DCM:MeOH (9:0.3; v/v)); C.sub.15H.sub.22N.sub.2O.sub.3 (278.35), monoisotopic mass: 278.16. UPLC (100% purity): t.sub.R=5.44 min. (M+H)+279.3.

Intermediate III (R═H): (R)-2-amino-N-benzylpropanamide

[0058] 10 ml of TFA was added to a solution of tert-butyl-(R)-(1-(benzylamino)-1-oxopropan-2-yl)carbamate (6.95 g, 25 mmol, 1 eq) (II) in DCM (40 mL) and the whole reaction mixture was stirred for 2 hours. TFA was then neutralized with a 25% NH.sub.4OH solution, followed by extraction with DCM (3×50 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4, then DCM was evaporated to dryness. (R)-2-Amino-N-benzylpropanamide (III) was obtained as a light oil. Yield: 89% (3.9 g); TLC:R.sub.f=0.21 (DCM:MeOH (9:0.5; v/v)); C.sub.10H.sub.14N.sub.2O (178.24), monoisotopic mass: 178.11. UPLC (96.8% purity): t.sub.R=2.11 min. (M+H)+179.2.

Intermediate IV (R═H): (R)-4-((1-(benzylamino)-1-oxopropan-2-yl)amino)-4-oxobutanoic acid

[0059] Succinic anhydride (2.19 g, 21 mmol, 1 eq) was added to a solution of (R)-2-amino-N-benzylpropanamide (3.9 g, 21 mmol, 1 eq) (III) in AcOEt (40 mL) and the whole mixture was stirred for 30 minutes. After this time, AcOEt was distilled off to dryness. The compound was obtained in solid form after washing with Et.sub.2O. White solid. Yield: 95% (5.80 g); m.p. 129.8-131.4° C.; TLC:R.sub.f=0.34 (DCM:MeOH (9:0.5; v/v)); C.sub.14H.sub.15N.sub.2O.sub.4 (278.31), monoisotopic mass: 278.13. UPLC (98.4% purity): t.sub.R=3.23 min. (M+H)+279.2.

Synthesis and Physicochemical and Spectral Data of Final Compounds 1 and 2

Compound 1: (2R)—N-benzyl-2-(2,5-dioxopyrrolidin-1-yl)propanamide

[0060] ZnCl.sub.2 (1.36 g, 10 mmol, 1 eq) was added to the suspension of (R)-4-((1-(benzylamino)-1-oxopropan-2-yl)amino)-4-oxobutanoic acid (2.78 g, 10 mmol, 1 eq) (IV, R═H) in dry benzene (40 mL) and the whole mixture was heated to 80° C. with stirring. Then a solution of HMDS (2.42 g, 3.14 ml, 15 mmol, 1.5 eq) in dry benzene (15 mL) was added dropwise over 30 minutes. The reaction was continued with stirring at reflux for about 24 hours and then concentrated under reduced pressure. After distilling off the solvent, the oily residue was dissolved in DCM (50 mL) and extracted with 0.1 M HCl (3×50 mL), water (3×50 mL) and saturated NaCl solution (3×50 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and then evaporated to dryness. The crude product was purified by column chromatography using DCM:MeOH (9:0.3; v/v) eluent system. The compound was obtained as a solid after washing with Et.sub.2O. White solid. Yield: 90% (2.34 g); m.p. 138.2-138.9° C.; Chiral HPLC>99% ee (t.sub.R=22.649 min); [α].sub.20D+51.52° (c 0.1%, DCM); TLC:R.sub.f=0.39 (DCM:MeOH (9:0.3; v/v)); C.sub.14H.sub.16N.sub.2O.sub.3 (260.29), monoisotopic mass: 260.12. UPLC (100% purity): t.sub.R=3.94 min. (M+H)+261.1. .sub.1H NMR (500 MHz, CDCl.sub.3) δ 1.56 (d, J=7.5 Hz, 3H), 2.66 (s, 4H), 4.39 (d, J=5.7 Hz, 2H), 4.76 (q, J=7.3 Hz, 1H), 6.45 (br s, 1H), 7.22-7.26 (m, 3H), 7.30-7.32 (m, 2H). .sub.13C NMR (126 MHz, CDCl.sub.3) δ 14.5, 24.9, 25.6, 28.3, 33.7, 43.8, 49.8, 127.6, 127.7, 128.8, 137.9, 168.6, 177.0.

Compound 2: (2R)-2-(2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl)propanamide

[0061] The compound was prepared using the procedure analogous to that described for the synthesis of compound 1. (R)-4-((1-((2-fluorobenzyl)amino)-1-oxopropan-2-yl)amino)-4-oxobutanoic acid (2.96 g, 10 mmol, 1 eq) (IV, R═F), ZnCl.sub.2 (1.36 g, 10 mmol, 1 eq) and HMDS (2.42 g, 3.14 ml, 15 mmol, 1, 5 eq) were used in the reaction. The crude product was purified by column chromatography using DCM:MeOH (9:0.3; v/v) eluent system. White solid. Yield: 89% (2.48 g); m.p. 115.1-115.8° C.; Chiral HPLC>99% ee (t.sub.R=24.859 min); [α].sub.20D+27.90° (c 0.1%, DCM); TLC:R.sub.f=0.43 (DCM:MeOH (9:0.3; v/v)); C.sub.14H.sub.15FN.sub.2O.sub.3 (278.28), monoisotopic mass: 278.11. UPLC (100% purity): t.sub.R=4.08 min, (M+H)+279.2. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.56 (d, J=7.5 Hz, 3H), 2.68 (s, 4H), 4.43 (t, J=6.0 Hz, 2H), 4.73-4.76 (m, 1H), 6.50 (br s, 1H), 7.00 (t, J=9.1 Hz, 1H), 7.08 (t, J=7.5 Hz, 1H), 7.21-7.28 (m, 1H), 7.29-7.31 (m, 1H). 13C NMR (126 MHz, CDCl.sub.3) δ 14.5, 28.2, 37.9, 37.9, 49.8, 115.3, 115.5, 124.5, 124.5, 124.8, 124.9, 129.4, 129.4, 130.2, 130.2, 160.0, 161.9, 168.8, 176.9.

Example of Synthesis as Well as Physicochemical and Spectral Data of Intermediate (V) According to Scheme 2

Intermediate V (R═H) (R)—N-(1-(benzylamino)-1-oxopropan-2-yl)-4-chlorobutanamide

[0062] 4-Chlorobutanoic acid chloride (0.59 g, 4.2 mmol, 1.5 eq) and TEA (0.85 g, 8.4 mmol, 3 eq) were added to a solution of (R)-2-amino-N-benzylpropanamide (0.50 g, 2.8 mmol, 1 eq) (III) in DCM (20 mL) and the whole reaction mixture was stirred for 0.5 hour. Then DCM was evaporated to dryness. Intermediate V was purified by column chromatography using DCM:MeOH (9:0.5; v/v) eluent system. Intermediate V was obtained as a light oil. Yield: 82% (0.65 g); TLC:R.sub.f=0.53 (DCM:MeOH (9:0.5; v/v)); C.sub.14H.sub.19ClN.sub.2O.sub.2 (282.77), monoisotopic mass: 282.11. UPLC (97.8% purity): t.sub.R=4.52 min. (M+H)+283.2.

Synthesis and Physicochemical and Spectral Data of Final Compounds 3 and 4

Compound 3: (2R)—N-benzyl-2-(2-oxopyrrolidin-1-yl)propanamide

[0063] NaH (0.106 g, 4.4 mmol, 2 eq) was added to a solution of (R)—N-(1-(benzylamino)-1-oxopropan-2-yl)-4-chlorobutanamide (0.63 g, 2.2 mmol, 1 eq) (V, R═H) in anhydrous THF, the hole reaction mixture was stirred for 4 hours and then concentrated under reduced pressure. After distilling off the solvent, the oily residue was dissolved in 0.1 M HCl (50 mL) and extracted with DCM (3×50 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and then evaporated to dryness. The crude product was purified by column chromatography using DCM:MeOH (9:0.5; v/v) eluent system. The compound was obtained as a solid after washing with Et.sub.2O. White solid. Yield: 86% (0.47 g); m.p. 96.7-97.5° C.; Chiral HPLC>99% ee (t.sub.R=10.623 min); TLC:R.sub.f=0.42 (DCM:MeOH (9:0.5; v/v)); C.sub.14H.sub.15N.sub.2O.sub.2 (246.31), Monoisotopic Mass: 264.13. UPLC (purity: >99.9%): t.sub.R=3.92 min, (M+H)+247.2. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.36 (d, J=7.2 Hz, 3H), 1.95-1.99 (m, 2H), 2.28-2.36 (m, 2H), 3.36-3.43 (m, 2H), 4.38 (dd, J=5.9, 2.2 Hz, 2H), 4.65-4.74 (m, 1H), 6.75 (br s, 1H), 7.19-7.22 (m, 2H), 7.23-7.25 (m, 1H), 7.27-7.31 (m, 2H). .sub.13C NMR (126 MHz, CDCl.sub.3) δ 13.8, 18.1, 31.1, 43.5, 43.8, 50.3, 127.5, 127.6, 128.7, 138.3, 170.6, 175.8.

Compound 4: (2R)—N-(2-fluorobenzyl)-2-(2-oxopyrrolidin-1-yl)propanamide

[0064] The compound was prepared using the procedure analogous to that described for the synthesis of compound 3. (R)-4-chloro-N-(1-((2-fluorobenzyl)amino)-1-oxopropan-2-yl)butanamide (0.57 g, 1.9 mmol, 1 eq) (V, R═F) and NaH (0.091 g, 3.8 mmol, 2 eq) were used in the reaction. The crude product was purified by column chromatography using DCM:MeOH (9:0.5; v/v) eluent system. White solid. Yield: 88% (0.44 g); m.p. 92.5-93.1° C.; Chiral HPLC>99% ee (t.sub.R=8.959 min); TLC:R.sub.f=0.39 (DCM:MeOH (9:0.5; v/v)); C.sub.14H.sub.17FN.sub.2O.sub.2 (264.30), Monoisotopic Mass: 264.13. UPLC (purity: >99.9%): t.sub.R=4.04 min, (M+H)+265.9. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.34 (d, J=7.2 Hz, 3H), 1.96-2.00 (m, 1H), 2.27-2.44 (m, 2H), 3.30-3.34 (m, 1H), 3.39-3.42 (m, 1H), 4.39 (dd, J=14.9, 5.7 Hz, 1H), 4.47 (dd, J=15.0, 6.2 Hz, 1H), 4.64-4.75 (m, 1H), 4.69 (d, J=7.2 Hz, 1H), 6.69 (br s, 1H), 7.01 (t, J=9.1 Hz, 1H), 7.07 (t, J=7.4 Hz, 1H), 7.21-7.27 (m, 2H). .sub.13C NMR (126 MHz, CDCl.sub.3) δ 13.7, 18.1, 31.1, 37.6, 37.7, 43.7, 50.3, 115.4, 115.5, 124.3, 124.3, 125.1, 125.3, 129.3, 129.4, 130.0, 130.1, 160.0, 162.0, 170.6, 175.8.

Examples of Synthesis as Well as Physicochemical and Spectral Data of Intermediate Products (VI, VII) According to Scheme 3

Intermediate VI (R═H) (R)-4-((1-(benzylamino)-1-oxopropan-2-yl)amino)-4-oxobut-2-enoic acid

[0065] Maleic anhydride (0.33 g, 3.4 mmol) was added to a solution of (R)-2-amino-N-benzylpropanamide (0.6 g, 3.4 mmol, 1 eq) (III) in AcOEt (40 mL, 1 eq) and the whole mixture was stirred for 30 minutes. After this time, AcOEt was evaporated to dryness. The compound was obtained in solid form after washing with Et.sub.2O. White solid. Yield: 98% (0.91 g); TLC:R.sub.f=0.28 (DCM:MeOH (9:0.1; v/v)); C.sub.14H.sub.16N.sub.2O.sub.4 (276.29), monoisotopic mass: 276.11. UPLC (purity >99.9%): t.sub.R=3.72 min. (M+H).sub.+ 278.2.

Intermediate VII (R═H) (R)—N-benzyl-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide

[0066] ZnCl.sub.2 (0.39 g, 10 mmol, 1 eq) was added to the suspension of (R)-4-((1-(benzylamino)-1-oxopropan-2-yl)amino)-4-oxobut-2-enoic acid (0.80 g, 2.9 mmol, 1 eq) (VI, R═H) in dry benzene (20 mL) and heated to 80° C. with stirring. Then a solution of HMDS (0.70 g, 0.91 mL, 4.35 mmol, 1.5 eq) in dry benzene (8 mL) was added dropwise over 30 minutes. The reaction was continued with stirring at reflux for about 24 hours and then concentrated under reduced pressure. After distilling off the solvent, the oily residue was dissolved in DCM (50 mL) and extracted with 0.1 M HCl (3×50 mL), water (3×50 mL) and saturated NaCl solution (3×50 mL). The organic layer was dried over anhydrous Na.sub.2SO.sub.4 and then evaporated to dryness. The crude product was purified by column chromatography using DCM:MeOH (9:0.5; v/v) eluent system. The compound was obtained as a solid after washing with Et.sub.2O. White solid. Yield: 80% (0.60 g); m.p. 98.3-98.8° C.; Chiral HPLC>99% ee (t.sub.R=50.631 min); TLC:R.sub.f=0.34 (DCM:MeOH (9:0.5; v/v)); C.sub.14H.sub.14N.sub.2O.sub.3 (258.28), monoisotopic mass: 258.10. UPLC (100% purity): t.sub.R=4.41 min. (M+H).sub.+ 259.2.

Synthesis and Physicochemical and Spectral Data of Final Compounds 5 and 6

Compound 5: (2R)—N-benzyl-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)propanamide hydrochloride

[0067] 2M solution of dimethylamine (0.105 g, 2.3 mmol, 1 eq) in THF was added to a solution of (R)—N-benzyl-2-(2,5-dioxo-2,5-dihydro-1H-pyrrol-1-yl)propanamide (0.60 g, 2.3 mmol, 1 eq) (VII, R═H) in dry benzene (30 mL). The crude product was purified by column chromatography using DCM:MeOH (9:0.5; v/v) eluent system. The compound was then converted into the hydrochloride salt by treating the compound with a 2M methanolic hydrochloric acid solution. White solid. Yield: 88% (0.62 g); m.p. 115.8-116.9° C.; TLC:R.sub.f=0.36 (DCM:MeOH (9:0.5; v/v)); C.sub.16H.sub.22ClN.sub.3O.sub.3 (339.82), Monoisotope mass (calculated for: C.sub.16H.sub.21N.sub.3O.sub.3): 303.16. UPLC (purity: >99.9%): t.sub.R=2.79 min, (M+H).sub.+ 304.2. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.52 (d, J=7.2 Hz, 3H), 2.78 (s, 6H), 2.85 (br s, 3H), 4.34 (d, J=6.0 Hz, 2H), 4.74-4.77 (m, 1H), 7.18-7.24 (m, 2H), 7.25-7.29 (m, 3H), 7.59 (br s, 1H), 7.73 (br s, 1H). .sub.13C NMR (126 MHz, CDCl.sub.3) δ 14.2, 14.2, 31.0, 32.1, 32.2, 41.8, 42.1, 43.6, 49.9, 50.0, 61.0, 61.3, 127.3, 127.4, 127.6, 127.8, 128.6, 128.6, 138.5, 138.6, 167.9, 168.1, 171.8.

Compound 6: (2R)-2-(3-(dimethylamino)-2,5-dioxopyrrolidin-1-yl)-N-(2-fluorobenzyl) propanamide hydrochloride

[0068] The compound was prepared using the procedure analogous to that described for the synthesis of compound 5. 2-(2,5-Dioxo-2,5-dihydro-1H-pyrrol-1-yl)-N-(2-fluorobenzyl)propanamide (0.64 g, 2.3 mmol, 1 eq) (VII, R═F) and a 2M solution of dimethylamine (0.105 g, 2.3 mmol, 1 eq) in THF were used in the reaction. The crude product was purified by column chromatography using DCM:MeOH (9:0.5; v/v) eluent system. The compound was then converted into the hydrochloride salt by treating the compound with a 2M methanolic hydrochloric acid solution. White solid. Yield: 85% (0.63 g); m.p. 118.4-119.6° C.; TLC:R.sub.f=0.45 (DCM:MeOH (9:0.5; v/v)); C.sub.16H.sub.21ClFN.sub.3O.sub.3 (357.81), Monoisotope mass (calculated as: C.sub.16H.sub.20FN.sub.3O.sub.3): 321.15. UPLC (purity: >99.9%): t.sub.R=2.91 min, (M+H).sub.+ 322.2. .sup.1H NMR (500 MHz, CDCl.sub.3) δ 1.52 (dd, J=11.5, 7.5 Hz, 3H), 2.94 (s, 6H), 3.08-3.36 (m, 3H), 4.38-4.44 (m, 2H), 4.76 (dd, J=15.0, 7.30 Hz, 1H), 6.93-7.01 (m, 1H), 7.03-7.10 (m, 1H), 7.19 (dd, J=4.7, 2.2 Hz, 1H), 7.26-7.33 (m, 1H), 7.61 (br s, 1H), 7.8 (br s, Hz, 1H). .sub.13C NMR (126 MHz, CDCl.sub.3) δ 14.2, 31.0, 32.2, 32.4, 37.5, 50.0, 50.0, 60.9, 61.2, 115.2, 115.3, 115.3, 124.3, 129.1, 129.2, 129.8, 130.0, 159.8, 161.7, 167.9, 168.0, 171.4.

EXAMPLE 2. BIOLOGICAL ACTIVITY OF COMPOUNDS ACCORDING TO THE INVENTION

In Vivo Studies

[0069] The study was conducted on male Swiss albino mice (CD-1) weighing 18-26 g. All procedures were carried out in accordance with applicable Polish and international guidelines on the ethics of animal testing, after obtaining appropriate institutional approval. The substances 1-2 were administered intraperitoneally (i.p.), after suspension in a 1% aqueous Tween 80 solution, as single injections with a volume of 10 ml/kg of body weight, 30 minutes before the given test. Anticonvulsant activity and the effect on motor coordination in the rotarod test after intragastric (p.o.) administration to mice were also evaluated for compound 1. In these studies, compound 1 was administered in a volume of 10 ml/kg by oral gavage after its dissolution in a mixture of DMSO, PEG 400, water for injection (10/40/50, v/v/v), 60 minutes before the given test. Compound 1 was also tested for anticonvulsant activity as part of the American search program for new antiepileptic drugs—ETSP (Epilepsy Therapy Screening Project), which is implemented by the National Institute of Neurological Disorders and Stroke, National Institutes of Health (NIH, Bethesda, Md., USA). These studies were carried out on individuals of male C57 black mice (Charles River) and individuals of male Sprague-Dawley rats. All tests were carried out based on the procedures described in the specialist literature.

Determination of In Vivo Anticonvulsant Activity

[0070] Screening was performed on groups of 4-8 mice or 4-8 rats. The median effective dose (ED.sub.50) in a given test was estimated based on the results obtained in 3-4 groups of animals consisting of at least 6 individuals. All tests were carried out based on the procedures described in the specialist literature: maximal electroshock seizure test (Kamiński et al. Bioorg. Med. Chem. 2015, 23, 2548-2561; Castel-Branco et al. Methods Find. Exp. Clin. Pharmacol. 2009, 31, 101-106, Riban et al. Neurosci. 2002, 112, 101-111); 6 Hz (32 mA and/or 44 mA) seizure model (Barton et al. Epilepsy Res. 2001, 47, 217-227; Wojda et al. Epilepsy Res. 2009, 86, 163-174); subcutaneous pentylenetetrazole (scPTZ) seizure test (Ferreri et al. Pharmacol. Biochem. Behav. 2004, 77, 85-94), corneal kindled seizure model (https://panache.ninds.nih.gov/Model2A.aspx), lamotrigine resistant amygdala kindled seizure model (https://panache.ninds.nih.gov/Model2B.aspx).

Determination of In Vivo Antinociceptive Activity in Mice

[0071] All tests/models were carried out based on the procedures described in the specialist literature: formalin test (Beirith et al. Eur. J. Pharmacol. 1998, 345, 233-245), a model of oxaliplatin-induced neuropathic pain—von Frey test (Salat et al. Pharmacol. Biochem. Behav. 2014, 122, 173-181), streptozotocin-induced model of painful diabetic neuropathy (Salat et al. Neuropharmacology 2017, 125, 181-188; Tanabe et al. J. Pharmacol. Sci. 2008, 107, 213-220). The study group consisted of 8-10 animals.

Forced Swim Test (Porsolt Test) in Mice

[0072] To assess the potential antidepressant effect, a forced swim test was carried out in accordance with the methodology described in the scientific literature (Pytka et al. Behav Brain Res. 2017, 333, 54-66). The study group consisted of 8-10 animals.

Four Plates Test (Aron Test) in Mice

[0073] The potential anti-anxiety activity of the compounds tested was assessed using a four plates test according to the methodology described in the specialist literature (Pytka et al. Front. Pharmacol. 2018, 9, 627-13). The study group consisted of 8-10 animals.

Spontaneous Locomotor Activity Test in Mice

[0074] The assessment of the influence of the tested compounds on the spontaneous locomotor activity of animals (assessment of the sedative or activating effect) was carried out in accordance with the methodology described in the scientific literature (Mogilski et al. Inflamm. Res. 2017, 66, 79-95). The study group consisted of 10 animals.

Assessment of the Influence on the Motor Coordination of Mice in the Rotarod Test

[0075] The effect of test compounds on the motor coordination in mice was assessed in a rotarod test (the apparatus—May Commat, RR 0711 Rota Rod, Turkey was used) according to the procedure described in the literature (Dunham et al. J. Am. Pharm. Assoc. 1957, 46, 64-66). These studies were also carried out in rats according to the methodology described by ETSP (https://panache.ninds.nih.gov/Model3B.aspx). The toxic dose (TD.sub.50) in the rotarod test was estimated based on the results obtained in 3-4 groups of animals consisting of at least 6 individuals.

Neuroprotective Activity in the Pilocarpine-Induced Seizures

[0076] To assess the neuroprotective properties of compound 1, studies using pilocarpine (PILO) as a neurodegenerative agent were carried out. The dose of compound 1 determined in the 6 Hz (32 mA) test was used for the study. At the peak of compound 1 activity, the animals received PILO at a dose of 300 mg/kg (intraperitoneally) to induce status epilepticus (SE). 30 min before PILO administration, the mice received methylscopolamine (1 mg/kg) to reduce the PILO peripheral cholinergic effect. 72 hours later, animals were transcardially perfused (0.9% saline, followed by freshly prepared 4% paraformaldehyde) for brain collection for further analysis using the Fluoro-Jade B (FJB) immunofluorescence method (Schmued et al. Brain Res. 2000, 874, 123-130). Qualitative analysis was performed using a Nikon AIR confocal microscope (Tokyo, Japan).

Statistical Analysis

[0077] The ED.sub.50 (effective dose) and TD.sub.50 (toxic dose) values along with the corresponding 95% confidence limits were calculated based on the Litchfield and Wilcoxon method. To perform a statistical evaluation of the results, one-way analysis of variance (ANOVA) followed by Dunnett's post hoc test (multiple comparison test) were used. The values were considered statistically significant if p<0.05.

In Vitro Studies

Effects on Cytochrome P-450 Isoforms CYP3A4, CYP2D6 and CYP2C9

[0078] The studies were conducted using commercial luminescence tests CYP3A4 P450-Glo™, CYP2D6 P450-Glo™ and CYP2C9 P450-Glo™ from Promega (Madison, Wis., USA). Detailed methodology is described in the literature (Socala et al. ACS Chem. Neurosci. 2019, 10, 636-648, Kamiński et al. Neurotherapeutics 2020, 17, 309-328).

Plasma Protein Binding

[0079] Determination of binding parameters to two major plasma proteins—α1-glycoprotein (AGP) and human albumin (HSA) was performed using the TRANSIL-XL PPB Assay kit (Sovicell, Leipzig, Germany). Detailed methodology is described in the literature (Lubelska et al. Molecules 2019, 24, pii: E4472).

Metabolic Stability

[0080] The metabolic stability study was performed using human liver microsomes purchased from Sigma-Aldrich (St. Louis, Mo., USA). Detailed methodology is described in the literature (Kamiński et al. Neurotherapeutics 2020, 17, 309-328).

Assessment of Cytotoxic Activity Against the HaCaT Cell Line

[0081] Cell culture. The research material was the human HaCaT cell line (immortalized keratinocyte line) from the American Type Culture Collection (ATCC, Rockville, USA). Cells were cultured in DMEM medium (Dulbecco's Modified Eagle's Medium, Biowest SAS, France) with the addition of 10% fetal bovine serum, penicillin (100 U/ml), streptomycin (100 ug/ml) and HEPES (20 mM) at 37° C. with 5% CO.sub.2 until 80% confluency is reached. Cells were then harvested using 0.25% trypsin-0.02% EDTA (Gibco Life Technologies, USA) and plated into 96-well plates (1×10.sub.4 per well) to check the cytotoxicity of test compounds by MTT assay.

[0082] MTT assay. The MTT assay (3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide) is based on the metabolic reduction of the tetrazole salt (MTT) to formazan, which occurs under the influence of mitochondrial succinate dehydrogenase active in living cells. Test cells were preincubated for 24 hours at 37° C. with 5% CO.sub.2, then the medium was removed and the medium with the addition of test compounds in the concentration range from 140 to 20 μM was added. HaCaT cells cultured in the appropriate medium without the test compound were a control. After 72 h incubation, the medium was removed and MTT solution (0.5 mg/mL) dissolved in serum-free medium was added in a volume of 200 μL/well. After a 4-hour incubation at 37° C., the cells were washed with a 1:1 mixture of DMSO (dimethyl sulfoxide) and isopropanol to release and dissolve the formazan crystals. The optical density of dissolved formazan crystals was measured with a UVM 340 reader (ASYS Hitech GmbH, Austria) at 570 nm. The IC.sub.50 value was estimated using CompuSyn version 1.0.

Assessment of Cytotoxic Activity Against the HEK-293 Cell Line

[0083] Human embryonic kidney cell line HEK-293 and ATCC CRL-1573 were also used for safety tests. The HEK-293 line was cultured in DMEM medium (Dulbecco's Modified Eagle's Medium) with the addition of 10% bovine serum (FBS) from Gibco (Carlsbad, Calif., USA) at 37° C. and an atmosphere containing 5% CO.sub.2. Prior to testing, cells were plated onto Thermo Scientific Nunc™ 96-well transparent culture plates (Waltham, Mass., USA) at a concentration of 1.5×10.sub.4 cells per well and incubated for 24 hours. Then a 10 mM stock solution of the test compound was diluted in culture medium and added to the cells at final concentrations in the range of 0.1-100 μM (DMSO concentration in all wells was 1%). The reference compound—doxorubicin (DX) was applied at a concentration of 1 μM. After 72 hours of incubation at 37° C. and an atmosphere containing 5% CO.sub.2, the medium with the compound was removed, and then fresh medium with diluted MTS reagent (CellTiter 96® AQueous Non-Radioactive Cell Proliferation Assay) supplied by Promega (Madison, Wis., USA) was added. Plates were again incubated for 2 hours, followed by absorbance measurement at 490 nm with an Perkin Elmer EnSpire reader (Waltham, Mass. USA). Statistical significance was calculated by one-way analysis of variance (ANOVA) and the Bonferroni method. The compounds were tested in four replications.

Assessment of Hepatotoxicity Against the HepG2 Cell Line

[0084] HepG2 cell line, ATCC HB-8065 was used for potential hepatotoxicity studies. Detailed methodology is described in the literature (Socala et al. ACS Chem. Neurosci. 2019, 10, 636-648, Kamiński et al. Neurotherapeutics 2020, 17, 309-328).

Assessment of Neurotrophic Activity Against Human Neurons

[0085] The study was carried out with culture of neurons derived from human neuroblastoma SH-SY5Y cells (from the European Collection of Cell Cultures, Salisbury, UK). The neuroprotective properties of compounds 1 and 2 under trophic stress conditions, induced by removal of the B27 supplement from the culture medium, were determined by MTT assay after 48 hours exposure of neurons to test compounds at concentrations from 0.1 to 100 μg/mL. Statistical significance was tested using the one-way ANOVA with Tukey's post-hoc test. A detailed protocol of neuronal differentiation and assessment of the neuroprotective properties of a substance is described in the literature (Lemieszek et al. Carbohydr. Polym. 2018, 197, 598-607).

In Vivo Test Results

Anticonvulsant Activities

[0086] Compounds 1 and 2 according to the invention exhibit broad anticonvulsant activity by acting effectively in the MES, 6 Hz (32 mA/44 mA) and scPTZ tests after intraperitoneal administration to mice. The obtained results revealed that the substances with R-absolute configuration (compounds (R)-1 and (R)-2, Table 1), being embodiments of the invention, showed stronger anticonvulsant activity in the epileptic seizure models used in comparison to the S-enantiomers (compounds (S)-1 and (S)-2, Table 1) and the corresponding racemic mixtures (compounds (RS)-1 and (RS)-2, Table 1)). Data for R and S enantiomers and racemic mixtures are presented in Table 1.

TABLE-US-00001 TABLE 1 ED.sub.50 (MES, scPTZ, 6 Hz test) and TD.sub.50 (rotarod test) parameters values for compounds 1 and 2 (R configuration), S enantiomers and racemic mixtures (RS), and the reference antiepileptic drug (valproic acid) after intraperitoneal administration to Swiss albino mice (CD-1). ED.sub.50 [mg/kg] TD.sub.50 6 Hz 6 Hz [mg/kg] PI Compound MES (32 mA) (44 mA) scPTZ Rotarod test TD.sub.50/ED.sub.50 (R)-1 66.3 15.6 41.6 36.3 >500 >7.5 (MES) (53.6-82.0)  (9.1-26.9) (32.8-52.7) (15.5-73.5) >32.0 (6 Hz, 32 mA) >12.0 (6 Hz, 44 mA) >13.8 (scPTZ) (S)-1 87.5 28.8 115.1 52.7 >500 >5.7 (MES)  (69.5-110.2) (16.9-48.9) (107.9-122.7) (37.7-85.0) >17.4 (6 Hz, 32 mA) >4.3 (6 Hz, 44 mA >9.5 (scPTZ) (RS)-1* 67.6 24.6 75.41 42.8 347.6 5.1 (MES) (56.3-81.2) (18.1-33.5) (63.60-89.42) (24.4-74.9) (307.5-392.8) 14.1 (6 Hz, 32 mA) 8.1 (scPTZ) (R)-2 33.0 14.1 37.2 33.2 298.8 9.0 (MES) (22.5-48.2)  (8.4-23.5) (23.0-60.4) (29.3-37.5) (265.3-315.5) 21.2 (6 Hz, 32 mA) 8.0 (6 Hz, 44 mA (S)-2 49.9 62.9 ND 78.8 310.2 9.0 (scPTZ) (44.7-55.8) (45.7-86.6) (53.2-95.3) (275.5-342.8) 6.2 (MES) 4.9 (6 Hz, 32 mA) 3.9 (scPTZ) (RS)-2* 54.9 33.8 ND 50.3 300.9 5.5 (MES) (48.3-62.3)  (11.0-103.7) (34.7-72.6) (256.7-352.6) 8.9 (6 Hz, 32 mA) 6.0 (scPTZ) VPA 252.7 130.6 183.1 239.4 430.7 1.7 (MES) (220.1-290.2) (117.6-145.2) (143.5-233.7) (209.2-274.1) (407.9-454.9) 3.3 (6 Hz, 32 mA) 2.4 (6 Hz, 44 mA 1.8 (scPTZ) The compounds were tested 30 min after intraperitoneal administration; MES - maximal electroshock seizure test; 6 Hz (32 mA) and 6 Hz (44 mA) - test of psychomotor seizures induced by low frequency 6 Hz electrical stimulus oapplying current intensity of 32 mA or 44 mA; scPTZ - subcutaneous pentylenetetrazole seizure test; Rota rod test - rotating rod test; PI - protective index (TD.sub.50/ED.sub.50). ND - no data available. *The results for (RS)-1 and (RS)-2 were disclosed in publications: Kamiński, et al. Bioorg. Med. Chem. 2015, 23, 2548-2561; Rapacz, et al. Naunyn Schmiedeberg's Arch. Pharmacol. 2017,6, 567-579. VPA - valproic acid.

[0087] The obtained results confirmed that compounds 1 and 2 (with R configuration) have a stronger anticonvulsant effect and more favorable protective indexes (PI) compared to S-enantiomers and racemic mixtures (RS). Surprisingly, in the case of compound 2 in the MES test, the racemic mixture (RS)-2 is characterized by the weakest activity. Importantly, compounds 1 and 2 exhibited significantly higher activity and a better safety profile (PI values) than valproic acid, which is a model antiepileptic drug with a wide range of therapeutic indications (generalized seizures: myoclonic seizures, tonic-clonic seizures, atonic seizures, absence seizure); focal onset seizures: simple or complex seizures, secondary generalized seizures, Lennox-Gastaut syndrome; treatment of manic episodes in bipolar disorder; migraine).

[0088] Further studies carried out for compound 1 revealed its strong anticonvulsant activity in the MES, 6 Hz (32 mA/44 mA) and scPTZ tests after their intragastric (per os) administration to mice. The substance given in this way is also characterized by weak acute neurotoxicity in the rotating bar test (rotarod), which translates into very favorable safety margins expressed by the protective index (PI) (Table 2).

TABLE-US-00002 TABLE 2 ED.sub.50 (MES, scPTZ, 6 Hz test), TD.sub.50 (rotarod test) and PI parameters values for compound 1 after intragastric (per os) administration to Swiss albino mice (CD-1). ED.sub.50 [mg/kg] 6 Hz 6 Hz TD.sub.50 [mg/kg] PI Compound MES (32 mA) (44 mA) scPTZ Rotarod test TD.sub.50/ED.sub.50 (R)-1 48.6 40.3 73.2 83.5 473.7 9.7 (MES) (42.4-55.8) (33.9-47.8) (57.4-93.4) (65.9-105.7) (454.7-493.4) 11.7 (6 Hz, 32 mA) 6.5 (6 Hz, 44 mA) 5.7 (scPTZ) The compound was tested 60 min after intragastric (per os) administration; MES - maximal electroshock seizure test; 6 Hz (32 mA) and 6 Hz (44 mA) - test of psychomotor seizures induced by low frequency 6 Hz electrical stimulus oapplying current intensity of 32 mA or 44 mA; scPTZ - subcutaneous pentylenetetrazole seizure test; Rotarod test - rotating rod test; PI - protective index (TD.sub.50/ED.sub.50).

[0089] Data obtained in mice after intragastric (per os) administration indicate good absorption of compound 1 from the gastrointestinal tract, passage through the liver and the possibility of achieving effective concentration in the central nervous system.

[0090] Compound 1, which is an embodiment of the present invention, is additionally characterized by strong protective activity in black C57 mice (Charles River) in the 6 Hz (44 mA) test, and the corneal kindled seizure model (Table 3). This substance was also effective in the MES test and lamotrigine resistant amygdala kindled seizure model in rats after intraperitoneal administration (Table 4).

TABLE-US-00003 TABLE 3 ED.sub.50 (th e6 Hz, 44 mA test and the corneal kindled seizure model), TD.sub.50 (rotarod test) and PIs for compound 1 after intraperitoneal administration to black C57 mice (Charles River). ED.sub.50 [mg/kg] TD.sub.50 Corneal [mg/kg] PI 6 Hz kindled Rotarod Compound (44 mA) seizure model test TD.sub.50/ED.sub.50 (R)-1 69.9 38.1 >150 >2.1 (6 Hz, 44 mA) (45.2-93.6) (34.2-42.8) >3.9 (Corneal kindled seizure model) The compound was tested 60 min after intraperitoneal administration; 6 Hz (44 mA)—test of psychomotor seizures induced by low frequency 6 Hz electrical stimulus oapplying current intensity of 44 mA; Rotarod test—rotating rod test; PI—protective index (TD50/ED50).

TABLE-US-00004 TABLE 4 Protective effect of compound 1 in the MES test and lamotrigine resistant amygdala kindled seizure model in rats after intraperitoneal administration ED.sub.50 [mg/kg] TD.sub.50 [mg/kg] PI Compound MES Rotarod test TD.sub.50/ED.sub.50 (R)-1 34.9 (27.1-44.7) 253.8 (220.7-294.5) 7.3 (MES) Lamotrigine resistant amygdala kindled seizure model (R)-1 Dosage: Number of protected rats: 3 Number of rats 70 mg/kg (protection—60%) tested: 5 The compound was tested 30 min after intraperitoneal administration. PI—protective index (TD50/ED50).

[0091] The data presented in Tables 3 and 4 show the potential efficacy of compound 1 according to the invention in human generalized tonic-clonic seizures (MES test), focal onset seizures (corneal kindled seizure model) and drug-resistant seizures (6 Hz, 44 mA model and lamotrigine resistant amygdala kindled seizure model).

[0092] Summing up, the data obtained in the tests/models used in the preclinical assessment of anticonvulsant activity indicate that the compounds according to the invention, and in particular compound 1, is a promising candidate for antiepileptic drug with potential for efficacy in many types of human epilepsy, including drug-resistant epilepsy. The good tolerance of compound 1 expressed in the weak influence on the motor coordination of animals in the rotarod test and the very high divergence of the active dose to the toxic dose suggests the usefulness of the compounds according to the invention, in particular compound 1, in special populations including pediatric patients (children and adolescents) and elderly patients.

Antinociceptive Activity

[0093] Formalin test: Compound 1 according to the invention showed distinct analgesic activity in both phases of the test. The mean nociceptive response time in the control group was 92.25±9.46 seconds and 200.60±18.28 seconds in the first and second phase of the test, respectively. Compound 1 reduced the nociceptive response time in the first phase of the formalin test, corresponding to acute pain, at all doses, with a statistically significant effect observed at the two highest doses—60 and 90 mg/kg. In the second phase of the test, corresponding to tonic inflammatory pain, compound 1 statistically significantly shortened the time of nociceptive response at doses of 30, 60 and 90 mg/kg (FIG. 1).

[0094] Oxaliplatin-induced neuropathic pain model—von Frey test: A single administration of oxaliplatin resulted in a lowering of the pain threshold in animals in response to a mechanical stimulus. The reaction was observed both 3 hours after administration of oxaliplatin (early phase) and 7 days after its administration (late phase). Test compound 1 administered at doses of 60 mg/kg and 90 mg/kg led to an increase in pain threshold at a dose-dependent manner, compared to the measurement made before the compound was administered. This compound at a dose of 30 mg/kg increased the pain threshold only in the early phase. The obtained results indicate that compound 1 has analgesic activity (reduces mechanical allodynia) in the oxaliplatin-induced neuropathic pain model (FIG. 2).

Streptozotocin-Induced Model of Diabetic Neuropathy—Von Frey Test

[0095] A single administration of streptozotocin resulted in the development of hyperglycemia (plasma glucose concentration exceeded 300 mg/dl) and a decrease in the pain threshold in animals in response to a mechanical stimulus (mechanical allodynia). The reaction was tested 3 weeks after streptozotocin injection. Test compound 1 administered at doses of 30 mg/kg, 60 mg/kg and 90 mg/kg led to an increase in the pain threshold in a statistically significant and dose-dependent manner, compared to the measurement made before the compound was administered. The compound at a dose of 90 mg/kg increased the pain threshold to the values observed before the induction of streptozotocin diabetes, and thus completely abolished the symptoms of developing sensory neuropathy. The obtained results indicate that compound 1 has analgesic activity (reduces mechanical allodynia) in the streptozotocin-induced model of diabetic neuropathy (FIG. 3).

Forced Swim Test (Porsolt Test)—Assessment of Antidepressant Effect

[0096] Test compound 1 administered at doses of 30 mg/kg, 45 mg/kg and 60 mg/kg statistically significantly shortened the immobility time in the forced swim test. The obtained results clearly indicate the potential antidepressant activity of the tested compound (FIG. 4).

Four Plates Test (Aron Test)—Evaluation of Anxiolytic Effect

[0097] Test compound 1 administered at doses of 60 mg/kg and 90 mg/kg statistically significantly increased the number of electrically punished plate crossings. Administration of the test compound at a dose of 30 mg/kg did not result in a statistically significant difference compared to the control group given a vehicle. The results obtained indicate the anxiolytic effect of the compound. The effects of administration of 60 mg/kg and 90 mg/kg do not differ significantly, suggesting that the anxiolytic effect of the compound has a ceiling nature (FIG. 5).

Assessment of an Effect on Spontaneous Locomotor Activity of Mice

[0098] Compound 1 administered at 30 mg/kg, 60 mg/kg and 90 mg/kg doses statistically significantly increased the number of light beams interruptions in actometers during 30 minutes of measurement. The obtained result indicates that the tested compound increases the spontaneous locomotor activity of animals in the aforementioned dose range. For compound 2, a statistically significant increase in spontaneous locomotor activity of the mice was observed at dose of 90 mg/kg (FIG. 6). These data confirm the lack of sedative effects of the disclosed compounds, which confirms their usefulness as drugs, including the special population, in children and adolescents.

Qualitative Analysis of Neuroprotective Properties in the Pilocarpine-Induced (PILO) Status Epilepticus

[0099] PILO-induced SE (300 mg/kg) caused strong neurodegenerative changes in the control animals group (FIG. 7A), while in the test group with compound 1 a protective effect was obtained for four mice (B-E), whereas changes were observed for only one mouse (F), as shown in FIG. 7B-F. The obtained results indicate a strong neuroprotective effect of compound 1 in pilocarpine-induced status epilepticus model in mice.

In Vivo Test Results

Effects on Cytochrome P-450 Isoforms CYP3A4, CYP2D6 and CYP2C9

[0100] The studies were conducted using commercial luminescence tests CYP3A4 P450-Glo™, CYP2D6 P450-Glo™ and CYP2C9 P450-Glo™ from Promega (Madison, Wis., USA). The CYP isoforms selected for study are responsible for the metabolism of approximately 60-70% of the drugs available and their stimulation or inhibition determines the majority of metabolic drug interactions. The results obtained indicate no effect of Compound 1 on CYP3A4 and CYP2D6 activity at a concentration of 10 μM. In contrast, a small, statistically significant (p<0.05) inhibition of the CYP2C9 isoform was observed at a high concentration of 10 μM. In summary, the results obtained indicate a low probability of potential metabolic interactions induced by compound 1 (FIG. 8)

Plasma Protein Binding

[0101] The distribution parameter of compound 1 was determined by incubating the compound with two major plasma proteins—α1-glycoprotein and human albumin. Then the bound fraction of compound (f.sub.b) and the dissociation constant (K.sub.D) were calculated, which were f.sub.b=23.8%, K.sub.D=1970 μM, respectively. For the reference compound warfarin exhibiting high plasma protein binding, these parameters were f.sub.b=98.5% and K.sub.D=9.50 μM. On the basis of the results obtained, it is shown that low binding of compound 1 to plasma proteins is advantageous, which translates into favorable distribution parameters.

Metabolic Stability

[0102] Prediction of compound 1 metabolism in the human body was carried out by incubating this compound for 120 minutes with human liver microsomes in the presence of the NADPH cofactor. Based on the resulting UPLC chromatogram of the reaction mixture, no metabolites appeared (FIG. 9). This suggests a very high stability of this compound and resistance to any metabolic transformations by human liver enzymes.

Evaluation of Cytotoxic Activity Against the HaCaT Cell Line in the MTT Test

[0103] The cytotoxic activity of compound 1 was estimated by determining the half-maximal inhibitory concentration IC.sub.50 (concentration of compound that corresponds to 50% cell viability compared to control). Doxorubicin, a drug with confirmed cytotoxicity, was used as a reference compound. Independent experiments were carried out three times in triplicate. Test compound 1 showed significantly lower toxicity compared to doxorubicin, which confirms its very low cytotoxic potential (Table 5).

TABLE-US-00005 TABLE 5 Cytotoxic activity (IC.sub.50, μM) of test compounds in the MTT test. Compound IC.sub.50 [μM] HaCaT cells* 1 239.8 ± 0.6 Doxorubicin  0.23 ± 0.03 *Data are expressed as mean ± SD; IC.sub.50 (μM)—compound concentration that corresponds to 50% inhibition of cell line growth (compared to control) after incubating the cells for 72 hours with the individual compound; Human immortalized keratinocyte cell line (HaCaT); Doxorubicin—a reference drug exhibiting cytotoxic effect, commonly used in the treatment of cancer.

Evaluation of Cytotoxic Activity Against the HEK-293 Cell Line in the MTS Test

[0104] The safety of compound 1 was also assessed using a human embryonic kidney cell line (HEK-293). The MTS colorimetric assay from Promega (Madison, Wis., USA) was used to test the effect of compound 1 on cell viability and proliferation. The compound was tested at four concentrations in the range of 0.1-100 μM. Doxorubicin at a concentration of 1 μM was used as a reference cytostatics. MTS analysis performed after 72 hours of incubation of the HEK-293 line with compound 1 did not show a statistically significant effect of this compound on cell viability in the concentration range of 1-100 μM (FIG. 10).

Assessment of Hepatotoxicity on the HepG2 Cell Line

[0105] Hepatotoxicity of compound 1 was tested using the same procedure as for the toxicity assessment against the HEK-293 line. HepG2 human hepatoma cell line and the CCCP mitochondrial respiratory toxin as reference compound were used in the test. MTS analysis performed after 72 hours of incubation of the HepG2 line with compound 1 did not show a statistically significant effect of this compound on cell viability in the concentration range of 1-100 μM (FIG. 11).

Assessment of Neurotrophic Properties of Compounds 1 and 2 Against Human Neuron Cultures—MTT Test

[0106] Compounds 1 and 2 in the whole range of concentrations analyzed (from 0.1 to 100 μg/ml) did not affect the viability of human neurons under standard conditions. Removal of trophic factor B27 (trophic stress) from the culture medium reduced the viability of neurons by an average of 15%. Compound 1 at concentrations from 1μg/ml, and compound 2 at concentrations from 0.1 μg/ml acted trophically on nerve cells—eliminating the negative effect of the lack of B27 supplement in culture medium (FIG. 12). The observed effect indicates the in vitro neuroprotective effect of the analyzed compounds.