3ALPHA, 5BETA-NEUROACTIVE STEROIDS FOR THE TREATMENT OF EPILEPSY AND SEIZURE DISEASES

Abstract

3α5β-steroid compounds of general formula I is disclosed. These compounds are useful in the treatment of epilepsy or comorbidities associated with epilepsy or conditions associated with convulsions, such as seizures associated with hypoxia, seizures associated with traumatic brain damage, seizures associated with intoxication, pathological changes caused by hyperexcitation, or in treatment of conditions accompanying epilepsy, such as affective disorders, depression, post-traumatic stress disorder (PTSD) and stress-related diseases, anxiety, schizophrenia and psychotic disorders, related ischemic CNS damage, neurodegenerative changes and disorders, multiple sclerosis. The compounds of general formula I also show age-specific efficacy.

Claims

1. A compound of general formula I, ##STR00002## wherein, R.sup.1 represents methyl, and then R.sup.2 is a hydrogen atom or linear or branched C.sub.1-C.sub.4 alkyl, or R.sup.1 and R.sup.2 form together a group —(CH.sub.2).sub.p—, where p=2 or 3, which forms a five- or six-membered ring together with carbon atoms 1 and 3 and with the nitrogen atom of the general formula I, R.sup.3 is —O—(CH.sub.2).sub.n—O.sub.m—, wherein n=0, 1 or 2 and m=0 or 1, R.sup.4 is a hydrogen atom or hydroxyl group, R.sup.5 is a hydrogen atom, and then R.sup.6 is selected from a group consisting of a hydrogen atom, acetyl group, cyano group, C.sub.1-C.sub.2 cyanoalkyl group, 1,1-difluoroethyl group and linear or branched C.sub.1-C.sub.4 alkyl, or R.sup.5 and R.sup.6 together form a structure selected from a group consisting of C.sub.1-C.sub.2 alkylidene, cyanomethylene group, atom of oxygen, two atoms of fluorine.

2. The compound of general formula I according to claim 1, selected from a group consisting of: (3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]fenantren-3-yl 5-oxopyrrolidine-2-carboxylate (1), (3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenantren-3-yl 6-oxopiperidine-2-carboxylate (2) (3R,5R,8R,9S,10S,13S,14S,17S)-17-Cyano-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl5-oxopyrrolidine-2-carboxylate (3), (3R,5R,8S,9S,10S,11R,13S,14S,17S)-17-Acetyl-11-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (4), (3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (5), (3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (6), (3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 6-oxopiperidine-2-carboxylate (7), (3R,5R,8R,9S,10S,13S,14S,17S)-17-Cyano-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 6-oxopiperidine-2-carboxylate (8), (3R,5R, 8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl acetylglycinate (9), (3R,5R, 8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl acetylleucinate (10), (3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (11), (3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl5-oxopyrrolidine-2-carboxylate (12), 2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)ethyl 5-oxopyrrolidine-2-carboxylate (13), 2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)ethyl 6-oxopiperidine-2-carboxylate (14), 2-((3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl 5-oxopyrrolidine-2-carboxylate (16), 2-((3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl 6-oxopiperidine-2-carboxylate (17).

3. A method of treatment, comprising the step of administering a medicament comprising the compound of general formula I according to claim 1 to a subject in need thereof.

4. A method of treatment of a condition, comprising the step of administering the compound of general formula I according to claim 1 to a subject in need thereof, wherein the condition is selected from the group consisting of epilepsy and conditions associated with convulsions selected from the group consisting of seizures associated with hypoxia; seizures associated with traumatic brain damage; seizures associated with intoxication; and pathological changes caused by hyperexcitation.

5. A method of treatment of a condition, comprising the step of administering the compound of general formula I according to claim 1 to a subject in need thereof, wherein the condition is a condition accompanying epilepsy selected from the group consisting of affective disorders, depression, post-traumatic stress disorder (PTSD) and stress-related diseases, anxiety, schizophrenia and psychotic disorders, related ischemic CNS damage, neurodegenerative changes and disorders, and multiple sclerosis.

6. (canceled)

7. A pharmaceutical composition, characterized in that it contains, as an active ingredient, at least one compound of the general formula I according to claim 1, and at least one pharmaceutically acceptable excipient.

8. A method of treatment of epilepsy and comorbidities associated with epilepsy or conditions associated with convulsions, such as seizures associated with hypoxia; seizures associated with traumatic brain damage; seizures associated with intoxication; pathological changes caused by hyperexcitation; or in treatment of conditions accompanying epilepsy, such as affective disorders, depression, post-traumatic stress disorder (PTSD) and stress-related diseases, anxiety, schizophrenia and psychotic disorders, related ischemic CNS damage, neurodegenerative changes and disorders, multiple sclerosis, comprising the step of administering the pharmaceutical composition according to claim 7 to a subject in need thereof.

9. A method of treatment of epilepsy and comorbidities associated with it or other conditions associated with convulsions, such as seizures associated with hypoxia; seizures associated with traumatic brain damage; seizures associated with intoxication; pathological changes caused by hyperexcitation; or for the treatment of conditions that may accompany epilepsy, such as affective disorders, depression, post-traumatic stress disorder (PTSD) and stress-related diseases, anxiety, schizophrenia and psychotic disorders, related ischemic CNS damage, neurodegenerative changes and disorders, multiple sclerosis, said method comprising the step of administering at least one compound of general formula I according to claim 1 to a patient in need of such treatment.

10. (canceled)

11. A method of improving responses of individual parts of the body to convulsion-related diseases, comprising the step of administering a food supplement or cosmetic product comprising the compound of general formula I according to claim 1 to a subject in need thereof.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

[0054] FIGS. 1A-1C show anticonvulsant effect of substances from Example 1, 2, and 3 in a model of PTZ-induced seizures in zebrafish (Danio rerio). Substances from Example 1 (FIG. 1A), 2 (FIG. 1B) and 3 (FIG. 1C) were used in doses of 1, 3, and 5 mg/kg. Individual graphs demonstrate an effect on spontaneous locomotor activity (left graph), swimming velocity (middle graph) and number of turns (specific unpredictable movements, right graph).

[0055] FIGS. 2A-2C show anticonvulsant effect of substances from Example 1, 2, and 3 in a model of PTZ-induced seizures in zebrafish (Danio rerio). Substances from Example 1 (FIG. 2A), 2 (FIG. 2B) and 3 (FIG. 2C) were used in doses of 1, 3, and 5 mg/kg. Individual graphs demonstrate an effect on spontaneous locomotor activity and dark/light behavioral patterns.

[0056] FIGS. 3A-3C show anticonvulsant effect of a substance from Example 1 in a model of PTZ-induced seizures in 12- and 25-day-old rats. Doses of 1, 5, and 10 mg/kg i.p. were used (x-axis). Graph 3A (y-axis in %)—incidence of generalized seizures with a tonic phase (GTCS) and incomplete seizures without the tonic phase (GCS); graph 3B (y-axis in s)—latencies to onset of generalized seizures; graph 3C (y-axis is score)—severity of seizures evaluated by means of a 5-point scale; each animal was classified according to the most severe event. Scale: 0—no activity; 1—isolated myoclonic jerks; 2—isolated elements of minimal clonic seizures and/or epileptic automatisms; 3—minimal clonic seizures with preserved righting ability; 4—incomplete generalized seizures (GCS); 5—complete generalized tonic-clonic seizures (GTCS). Abbreviations: P12—12-days old animals; P25—25-days old animals. Asterisks denote a significant difference in comparison with controls; 0 or 1—seizures were abolished or present in one rat only.

[0057] FIGS. 4A-4C show anticonvulsant effect of a substance from Example 2 in a model of PTZ-induced seizures in 12- and 25-day-old rats. Doses of 1, 5, and 10 mg/kg i.p. were used (x-axis). Graph 4A (y-axis in %)—incidence of generalized seizures with a tonic phase (GTCS) and incomplete seizures without the tonic phase (GCS); graph 4B (y-axis in s)—latencies to onset of generalized seizures; graph 4C (y-axis is score)—severity of seizures evaluated by means of a 5-point scale; each animal was classified according to the most severe event. Scale: 0—no activity; 1—isolated myoclonic jerks; 2—isolated elements of minimal clonic seizures and/or epileptic automatisms; 3—minimal clonic seizures with preserved righting ability; 4—incomplete generalized seizures (GCS); 5—complete generalized tonic-clonic seizures (GTCS). Abbreviations: P12—12-days old animals; P25—25-days old animals. Asterisks denote a significant difference in comparison with controls; 0 or 1—seizures were abolished or present in one rat only; nt—not tested.

[0058] FIGS. 5A-5C show anticonvulsant effect of a substance from Example 3 in a model of PTZ-induced seizures in 12- and 25-day-old rats. Doses of 1, 5, and 10 mg/kg i.p. were used (x-axis). Graph 5A (y-axis in %)—incidence of generalized seizures with a tonic phase (GTCS) and incomplete seizures without the tonic phase (GCS); graph 5B (y-axis in s)—latencies to onset of generalized seizures; graph 5C (y-axis is score)—severity of seizures evaluated by means of a 5-point scale; each animal was classified according to the most severe event. Scale: 0—no activity; 1—isolated myoclonic jerks; 2—isolated elements of minimal clonic seizures and/or epileptic automatisms; 3—minimal clonic seizures with preserved righting ability; 4—incomplete generalized seizures (GCS); 5—complete generalized tonic-clonic seizures (GTCS). Abbreviations: P12—12-days old animals; P25—25-days old animals. Asterisks denote a significant difference in comparison with controls; 0 or 1—seizures were abolished or present in one rat only; nt—not tested.

[0059] FIGS. 6A-6C show anticonvulsant effect of a substance from Example 1 in a model of seizures elicited by 6 Hz transcorneal electrical stimulation in young adult (P60) male rats. Steroid substance in dose 1, 5, or 10 mg/kg i.p. (x-axis) was administered 20 min before the first stimulation. Graph 6A (y-axis in %)—incidence of complete (GTCS) and incomplete, i.e. without the tonic phase (GCS) generalized seizures; graph 6B (y-axis in %)—incidence of minimal clonic seizures; graph 6C (y-axis in s)—latencies of generalized seizures.

[0060] FIGS. 7A-7C show anticonvulsant effect of a substance from Example 1 in a model of seizures elicited by 6 Hz transcorneal electrical stimulation in 15- and 25-day-old male rats. X-axis—current intensities used (60 and 80 mA in P15 and 40 and 60 mA in P25 rats). Y-axis—incidence of seizures in % (FIG. 7A); duration of seizures in s (FIG. 7B), and seizure severity expressed as a score (FIG. 7C). Seizure severity evaluated by means of a 5-point scale; each animal was classified according to the most severe event. Scale: 0—no activity; 1—isolated myoclonic jerks; 2—isolated elements of minimal clonic seizures and/or epileptic automatisms; 3—minimal clonic seizures with preserved righting ability; 4—incomplete generalized seizures (GCS); 5—complete generalized tonic-clonic seizures (GTCS). Asterisks denote a significant difference in comparison with controls. Abbreviations: P12—12-days old animals; P25—25-days old animals.

[0061] FIG. 8 shows anticonvulsant effect of a substance from Example 1 in a model of seizures elicited by 6 Hz transcorneal electrical stimulation in young adult (P60) male rats presented as box plots (median with 25 and 75%) and maximal and minimal values (y-axis); doses in mg/kg (x-axis). Symbols in individual boxes show individual values for animals.

[0062] FIGS. 9A-9C shows histological damage, respectively neuroprotective effect of Compound from Example 1 at dose 1 mg/kg in the model of NMDA lesion, a model of the glutamate-induced excitotoxicity. The tissue damage is induced by infusing NMDA (25 mmol.L.sup.−1) into the dorsal hippocampus in the male rats. The figures in panel (FIG. 9A) show representative images of brain slices of control animals, in panel (FIG. 9B) the group of animals which were administered the solutions containing NMDA into dorsal hippocampus (NMDA lesion) and i.p. treated by (2-hydroxypropyl)-β-cyclodextrin (CDX) and in panel (FIG. 9C) brain slices of the animals, with NMDA lesions of the dorsal hippocampus and i.p. treated by the Compound of Example 1 at a dose of 1 mg/kg dissolved in CDX.

[0063] The present invention will be further illustrated by Examples, which should not be construed as limiting the scope of the invention.

LIST OF ABBREVIATIONS

[0064] CHCl.sub.3 chloroform [0065] CDX (2-hydroxypropyl)-β-cyclodextrin [0066] DCM dichloromethane [0067] DMAP 4-dimethylaminopyridine (IUPAC: N,N-Dimethylpyridin-4-amine) [0068] DMF dimethylformamide (IUPAC: N,N-Dimethylformamide) [0069] DMSO dimethyl sulfoxide [0070] EDCI 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride [0071] ESI electrospray ionization [0072] HPLC high-performance liquid chromatography) [0073] HRMS high resolution mass spectrometry [0074] IC infrared spectroscopy [0075] MS mass spectrometry [0076] NMDA N-methyl-D-aspartic acid [0077] NMR nuclear magnetic resonance [0078] PBS phosphate buffered saline

[0079] Experimental Part—Chemistry

[0080] The reactions which require anhydrous conditions were always carried out in a pre-dried apparatus and under an inert atmosphere. Samples for analysis were dried over phosphorous pentoxide at 50° C. and a pressure of 100 mbar. Solvents were removed from the solution by rotary evaporator (0.25 kPa) at 50° C. bath. Thin layer chromatography (TLC) was performed on plates coated with a thin layer of silica gel (ICN Biochemicals). Preparative column chromatography was performed on silica gel Fluka (60 microns). Melting points were measured at Hund Wetzlar H-600 (Helmut Hund, Germany). Optical rotation was measured in chloroform Autopol IV polarimeter (Rudolph Research Analytical, Flanders, USA), [α].sub.D values are shown in [10.sup.−1.deg.cm.sup.2.g.sup.−1], concentration values as [g 100 ml.sup.−1] and were compensated to a standard temperature of 20° C. Infrared spectra were measured in chloroform using a Nicolet 6700 (Thermo Scientific, USA). NMR spectra were measured in FT mode on Bruker AVANCE III™ 400 MHz with tetramethylsilane (TMS) as internal standard. Chemical shifts are given in ppm (δ-scale), coupling constants (J) are given in Hz. Signal multiplicities are designated as follows: s—singlet, d—doublet, t—triplet, q—quartet, m—multiplet, br denotes broad. Mass spectra were measured on a LTQ Advantage Thermo spectrometer with ESI or EI ionization (10 eV) in positive or negative mode.

General Procedures

[0081] General Procedure A

[0082] A mixture of 3-hydroxy steroid (1.0 mmol), 4-dimethylaminopyridine (DMAP, 0.25 mmol), 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.7 mmol), and hydroxybenzotriazole (HOBt (2.2 mmol) was dried under vacuum at room temperature for 1 hour. Then, dry dimethylformamide (10 mL) was added under inert atmosphere, followed by slow dropwise addition of particular carboxylic acid (1.5 mmol) in dry DMF (5 mL). The reaction mixture was allowed to stir overnight at room temperature. After solvent evaporation, the residue was purified by a column chromatography.

[0083] General Procedure B

[0084] A mixture of 3-hydroxy steroid (1.0 mmol), 4-dimethylaminopyridine (DMAP, 0.25 mmol), and particular carboxylic acid (1.5 mmol) was dried under vacuum at room temperature for 1 hour. Then, dry dichloromethane (DCM, 2 mL) was added under inert atmosphere, followed by slow dropwise addition of 1-ethyl-3-(3-dimethylaminopropyl)carbodiimide hydrochloride (EDCI, 2.7 mmol) in dry DCM (10 mL). The reaction mixture was allowed to stir overnight at room temperature. After 18 h, it was poured into water. The combined extracts were washed with aqueous HCl (5%), aqueous NaHCO.sub.3, brine, and dried over anhydrous Na.sub.2SO.sub.4. After solvent evaporation, the residue was purified column chromatography on silica gel.

EXAMPLE 1

(3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]fenantren-3-yl 5-oxopyrrolidine-2-carboxylate (1)

[0085] Compound 1 was prepared according to the General Procedure A (DMF). Starting from 20-oxo-5β-pregnan-3α-ol (318 mg, 1.0 mmol), using L-pyroglutamic acid (194 mg, 1.5 mmol), compound 1 (370 mg, 89%) was obtained by column chromatography on silica gel (5% acetone/chloroform): mp 167-168° C. (chloroform, diethyl ether), [α].sub.D.sup.20 +105.6 (c 0.3, CHCl.sub.3). NMR (400 MHz, CDCl.sub.3): δ 0.60 (3H, s, H-18), 0.94 (3H, s, H-19), 2.11 (3H, s, H-21), 4.16-4.24 (1H, m, H-C2′), 4.80 (1H tt, J=11.4, 4.8 Hz, H-3), 5.86 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 209.7, 177.6, 171.5, 75.9, 64.0, 56.8, 55.6, 44.5, 41.9, 40.6, 39.3, 35.9, 35.1, 34.7, 32.2, 31.7, 29.3, 26.9, 26.7, 26.4, 25.1, 24.6, 23.4, 23.1, 21.0, 13.6. IR spectrum (CHCl.sub.3): 1734, 1702 (C═O), 1230 (C—O). MS: ESI m/z 452.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.26H.sub.39NO.sub.4Na [M+Na] calcd, 452.27713; found, 452.26742. For C.sub.26H.sub.39NO.sub.4 (429.6) calcd: 72.69%, C; 9,15%, H; 3.26%, N. Found: 72.29%, C; 9,15%, H; 3.11%, N.

EXAMPLE 2

(3R,5R,8R,9 S,10S,13 S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenantren-3-yl 6-oxopiperidine-2-carboxylate (2)

[0086] Compound 2 was prepared according to the General Procedure B (DCM). Starting from 20-oxo-5β-pregnan-3α-ol (318 mg, 1.0 mmol), using 6-oxo-L-pipecolic acid (213 mg, 1.5 mmol), compound 2 (142 mg, 32%) was obtained by column chromatography on silica gel (3% acetone/chloroform): mp 139-141° C. (acetone/n-heptane), [α].sub.D.sup.20 +103.2 (c 0.3, CHCl.sub.3). NMR (400 MHz, CDCl.sub.3): δ 0.60 (3H, s, H-18), 0.94 (3H, s, H-19), 4.05 (1H, m, H-C2′), 4.81 (1H, m, H-3), 6.14 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 209.7, 171.4, 170.5, 76.1, 64.0, 56.8, 55.1, 44.5, 42.0, 41.0, 39.3, 35.9, 35.0, 34.7, 32.2, 31.7, 31.2, 27.0, 26.7, 26.4, 25.6, 24.6, 23.4, 23.1, 21.0, 19.7, 13.6. IR spectrum (CHCl.sub.3): 3402 (NH); 1734, 1698, 1663 (C═O). MS: ESI m/z 466.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.27H.sub.41NO.sub.4Na [M+Na] calcd, 466.29278; found, 466.29283.

EXAMPLE 3

(3R,5R,8R,9 S,10S,13 S,14S,17 S)-17-Cyano-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (3)

[0087] Compound 3 was prepared according to the General Procedure B (DCM). Starting from 3α-hydroxy-5β-androstan-17β-carbonitrile (250 mg, 0.83 mmol), using L-pyroglutamic acid (139 mg, 1.07 mmol), compound 3 (180 mg, 53%) was obtained by column chromatography on silica gel (20% acetone/chloroform): mp 188-189° C. (chloroform/diethyl ether), [α].sub.D.sup.20 +75.5 (c 0.2, CHCl.sub.3). NMR (400 MHz, CDCl.sub.3): δ 0.91 (3H, s, H-18), 0.96 (3H, s, H-19), 4.25 (1H, ddd, J=8.8, 5.1, 0.7 Hz, H-C2′), 4.80 (1H tt, J=11.3, 4.8 Hz, H-3), 5.92 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 177.7, 171.5, 121.4, 75.8, 55.6, 54.5, 44.7, 41.8, 40.5, 40.5, 37.4, 36.3, 35.1, 34.8, 32.2, 29.3, 26.8, 26.8, 26.6, 26.4, 25.0, 24.7, 23.3, 20.6, 14.8. IR spectrum (CHCl.sub.3): 2237 (CN), 1735, 1705 (C═O), 1229 (C—O). MS: ESI m/z 435.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.25H.sub.36N.sub.2O.sub.3Na [M+Na] calcd, 435.26181; found, 345.26135. For C.sub.25H.sub.36N.sub.2O.sub.3 (412.6) calcd: 72.78%, C; 8.80%, H; 6.79%, N. Found: 72.39%, C; 8.64%, H; 6.33%, N.

EXAMPLE 4

(3R,5R,8S,9S,10S,11R,13S,14S,17S)-17-Acetyl-11-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (4)

[0088] Compound 4 was prepared according to the General Procedure A (DMF). Starting from 20-oxo-5β-pregnan-3α,11α-diol (334 mg, 1.0 mmol), using L-pyroglutamic acid (193 mg, 1.5 mmol), compound 4 (213 mg, 48%) was obtained by column chromatography on silica gel (30% acetone in CHCl.sub.3): mp 183-185° C. (chloroform/diethyl ether), [α].sub.D.sup.20 +86.4 (c 0.2, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.62 (3H, s, H-18), 1.06 (3H, s, H-19), 2.13 (1H, s, H-21), 3.91 (1H, s, H-11), 4.20 (1H, ddd, J=8.7, 5.2, 0.7 Hz, H-C2′), 4.85 (1H, tt, J=10.9, 5.1 Hz, H-3), 5.83 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 209.1, 177.7, 171.5, 76.3, 69.1, 63.5, 55.7, 55.6, 50.8, 47.3, 44.3, 43.5, 38.0, 35.9, 34.8, 32.7, 31.6, 29.3, 27.6, 27.4, 26.3, 25.0, 24.5, 23.7, 23.1, 14.6. IR spectrum (CHCl.sub.3): 1734, 1702 (C═O). MS: ESI m/z 468.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.26H.sub.39NO.sub.5Na [M+Na] calcd, 468.27219; found, 468.27204. For C.sub.26H.sub.39NO.sub.5(445.6) calcd: 70.08%, C; 8.82%, H; 3.14%, N. Found: 69.91%, C; 8.68%, H; 2.87%, N.

EXAMPLE 5

(3R,5R,8 S,9S,10S,13 S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (5)

[0089] Compound 5 was prepared according to the General Procedure A (DFM). Starting from 3α-hydroxy-5β-androstane (300 mg, 0.94 mmol), using L-pyroglutamic acid (190 mg, 1.5 mmol), compound 5 (210 mg, 58%) was obtained by column chromatography on silica gel (5% acetone/chloroform): mp 136-137 ° C. (chloroform/diethyl ether), [α].sub.D.sup.20 +29.5 (c 0.2, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.69 (3H, s, H-18), 0.94 (3H, s, H-19), 4.16-4.24 (1H, m, H-C2′), 4.79 (1H tt, J=11.3, 4.8 Hz, H-3), 5.89 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 177.5, 171.4, 76.0, 55.5, 54.5, 41.9, 40.9, 40.7, 40.5, 39.0, 36.2, 35.0, 34.7, 32.1, 29.2, 27.0, 26.7, 26.5, 25.5, 24.9, 23.3, 20.8, 20.6, 17.5. IR spectrum (CHCl.sub.3): 1734, 1704 (C═O). MS: ESI m/z 386.3 (100%, M−H). HR-MS (ESI) m/z: for C.sub.24H.sub.36NO.sub.3 [M−H] calcd, 386.27007; found, 386.26962. For C.sub.24H.sub.37NO.sub.3 (387.3) calcd: 74.38%, C; 9.62%, H; 3.61% N. Found: 74.20%, C; 9.61%, H; 3.26%, N.

EXAMPLE 6

(3R,5R,8R,9S,10S,13S,14S)-10,13-Dimethyl-17-oxohexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (6)

[0090] Compound 6 was prepared according to the General Procedure A (DMF). Starting from 17-oxo-5β-androstan-3α-ol (290 mg, 1.0 mmol), using L-pyroglutamic acid (194 mg, 1.5 mmol), compound 6 (197 mg, 49%) was obtained by column chromatography on silica gel (5% acetone/chloroform): mp 114-116° C. (acetone/n-heptane), [α].sub.D.sup.20 +98.5 (c 0.2, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.86 (3H, s, H-18), 0.97 (3H, s, H-19), 4.20 (1H, m, H-C2′), 4.80 (1H, m, H-3), 5.91 (1H, s, N—H). .sup.13C NMR (101 MHz, CHCl.sub.3): δ 221.3, 177.7, 171.5, 75.8, 55.6, 51.6, 47.9, 41.9, 40.9, 36.1, 35.5, 35.0, 34.9, 32.2, 31.8, 29.3, 26.8, 26.6, 25.4, 25.0, 23.3, 21.9, 20.3, 13.9. IR spectrum (CHCl.sub.3): 3439 (NH); 1703, 1706 (C═O); 1060 (C—O). MS: ESI m/z 424.2 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.24H.sub.35NO.sub.4Na [M+Na] calcd, 424.24583; found, 424.24548. For C.sub.24H.sub.35NO.sub.4 (401.2) calcd: 71.79%, C; 8.79%, H; 3.49%, N. Found: 71.40%, C; 8.93%, H, 3.32%, N.

EXAMPLE 7

(3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 6-oxopiperidine-2-carboxylate (7)

[0091] Compound 7 was prepared according to the General Procedure A (DMF). Starting from 5β-androstan-3α-ol (276 mg, 1.0 mmol), using 6-oxo-L-pipecolic acid (213 mg, 1.5 mmol), compound 7 (189 mg, 47%) was obtained by column chromatography on silica gel (5% acetone/chloroform): mp 109-111° C. (diethyl ether), [α].sub.D.sup.20 +18.3 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.68 (3H, s, H-18), 0.94 (3H, s, H-19), 4.04 (1H, m, H-C2′), 4.81 (1H, m, H-3), 6.13 (1H, s, N—H). .sup.13C NMR (101 MHz, CHCl.sub.3): δ 171.4, 170.6, 76.3, 55.1, 54.7, 42.0, 41.1, 40.9, 40.6, 39.1, 36.3, 35.2, 34.9, 32.3, 31.2, 27.1, 26.8, 26.7, 25.7, 25.6, 25.6, 23.4, 21.0, 20.7, 19.7, 17.6. IR spectrum (CHCl.sub.3): 3402 (NH); 1734, 1665 (C═O); 1062 (C—O). MS: ESI m/z 424.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.25H.sub.39NO.sub.3Na [M+Na] calcd, 424.28222; found, 424.28258. For C.sub.25H.sub.39NO.sub.3 (401.3) calcd: 74.77%, C; 9.79%, H; 3.49%, N. Found: 74.97%, C; 9.92%, H; 3.43%, N.

EXAMPLE 8

(3R,5R,8R,9S,10S,13S,14S,17S)-17-Cyano-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 6-oxopiperidine-2-carboxylate (8)

[0092] Compound 8 was prepared according to the General Procedure A (DMF). Starting from 3α-hydroxy-5β-androstan-17β-carbonitrile (112 mg, 0.37 mmol), using 6-oxo-L-pipecolic acid (80 mg, 0.56 mmol), compound 8 (76 mg, 48%) was obtained by column chromatography on silica gel (3% acetone/chloroform): mp 69-71° C. (ethyl acetate/n-heptane), [α].sub.D.sup.20 +65.4 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.91 (3H, s, H-19), 0.95 (3H, s, H-18), 2.29 (1H, t, J=8.8 Hz, H-17), 4.04 (1H, m, H-C2′), 4.81 (1H, m, H-3), 6.12 (1H, s, N—H). .sup.13C NMR (101 MHz, CHCl.sub.3): δ 171.4, 170.5, 121.4, 75.9, 55.1, 54.5, 44.7, 41.8, 40.5, 40.5, 37.4, 36.3, 35.0, 34.8, 32.2, 31.2, 26.8, 26.8, 26.7, 26.4, 25.6, 24.7, 23.3, 20.7, 19.7, 14.5. IR spectrum (CHCl.sub.3): 3403 (NH); 2237 (CN); 1734, 1664, 1418 (C═O); 1062 (C—O). MS: ESI m/z 449.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.26H.sub.38N.sub.2O.sub.3Na [M+Na] calcd, 449.27746; found, 449.27789. For C.sub.26H.sub.38N.sub.2O.sub.3 (426.6) calcd: 73.20%, C; 8.98%, H; 6.57%, N. Found: 73.22%, C; 9.29%, H; 6.25%, N.

EXAMPLE 9

(3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl acetylglycinate (9)

[0093] Compound 9 was prepared according to the General Procedure A (DMF). Starting from 20-oxo-5β-pregnan-3α-ol (318 mg, 1.0 mmol), using N-acetylglycine (176 mg, 1.5 mmol), compound 9 (338 mg, 81%) was obtained by column chromatography on silica gel (3% acetone/chloroform): mp 111-113° C. (ethyl acetate/n-heptane), [α].sub.D.sup.20 +92.0 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.60 (3H, s, H-18), 0.93 (3H, s, H-19), 2.04 (3H, s, H-C5′), 2.11 (3H, s, H-21), 2.53 (1H, t, J=8.8 Hz, H-17), 4.00 (2H, d, J=5.0 Hz, H-C5′), 4.80 (1H, m, H-3), 6.00 (1H, m, N—H). .sup.13C NMR (101 MHz, CHCl.sub.3): δ 209.7, 170.2, 169.7, 75.9, 63.9, 56.8, 44.4, 41.9, 41.8, 40.6, 39.3, 35.9, 35.1, 34.8, 32.3, 31.7, 27.0, 26.7, 26.4, 24.6, 23.4, 23.2, 23.0, 21.0, 13.6. IR spectrum (CHCl.sub.3): 3463, 1516 (NH); 1734, 1697, 1676 (C═O); 1192, 1063 (C—O). MS: ESI m/z 440.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.25H.sub.39NO.sub.4Na

[0094] [M+Na] calcd, 440.27713; found, 440.27753. For C.sub.25H.sub.39NO.sub.4 (417.6) calcd: 71.91%, C; 9.41%, H; 3.35%, N. Found: 72.03%, C; 9.36%, H; 3.39%, N.

EXAMPLE 10

(3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopentatalphenanthren-3-yl acetylleucinate (10)

[0095] Compound 10 was prepared according to the General Procedure A (DMF). Starting from 20-oxo-5β-pregnan-3α-ol (318 mg, 1.0 mmol), using N-acetyl-L-leucine (260 mg, 1.5 mmol), compound 10 (155 mg, 33%) was obtained by column chromatography on silica gel (15% ethyl acetate/petroleum ether) as a mixture of diasteromers, followed by HPLC purification (20% acetone/hexane) affording C3′-R/S (unidentified) diastereomer as an oily product: [α].sub.D.sup.20 +93.3 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.60 (3H, s, H-18), 0.93 (3H, s, H-19), 0.95 (6H, dd, J=6.3, 4.5 Hz, H-C5′ and C6′), 2.02 (3H, s, H-acetyl-L-leucinate), 2.11 (3H, s, H-21), 2.55 (1H, t, J=8.8 Hz, H-17), 4.58 (1H, m, H-C2′), 4.76 (1H, m, H-3), 5.83 (1H, m, NH). .sup.13C NMR (101 MHz, CHCl.sub.3): δ 209.8, 173.0, 169.9, 75.6, 64.0, 56.8, 51.1, 44.5, 42.0, 40.6, 39.3, 35.9, 35.1, 34.8, 32.2, 31.7, 27.0, 26.7, 26.5, 25.0, 24.6, 23.4, 23.4, 23.0, 23.0, 22.3, 21.0. IR spectrum (CHCl.sub.3): 3436 (NH); 2959, 2872 (CH.sub.3); 1727, 1697 (C═O); 1193, 1021 (C—O). MS: ESI m/z 496.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.29H.sub.48NO.sub.4 [M+H] calcd, 474.35779; found, 474.35753. For C.sub.29H.sub.47NO.sub.4 (473.7) calcd: 73.53%, C; 10.00%, H; 2.96%, N. Found: 73.37%, C; 10.35%, H; 2.69%, N.

EXAMPLE 11

(3R,5R,8S,9S,10S,13R,14S,17S)-10,13,17-Trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (11)

[0096] Compound 11 was prepared according to the General Procedure A (DMF). Starting from 17β-methyl-5β-androstan-3α-ol (102 mg, 0.35 mmol), using L-pyroglutamic acid (112 mg, 0.525 mmol), compound 11 (124 mg, 60%) was obtained as a white amorphous solid by column chromatography on silica gel (acetone/chloroform, 1:50 to 1:6): mp 157-158° C. (acetone/n-heptane), [α].sub.D.sup.20 +31.0 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.52 (3H, s, H-18), 0.82 (3H, d, J=6.8 Hz, 17-Me), 0.94 (3H, s, H-19), 4.19 (dd, J=8.6, 5.2 Hz, H-C2′), 4.78 (1H, tt, J=11.4, 4.8 Hz, H-3), 6.19 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 177.9, 171.6, 76.1, 55.9, 55.7, 45.3, 42.3, 42.2, 40.9, 37.8, 36.2, 35.2, 34.8, 32.3, 30.4, 29.4, 27.2, 26.7, 26.6, 25.0, 24.8, 23.5, 20.7, 13.9, 12.2. IR spectrum (CHCl.sub.3): 3206, 3118 (NH), 2951, 2867 (CH.sub.2), 1736 (C═O), 1716 (C═O), 1448 (CH.sub.2), 1203, 1022 (C-0). MS: ESI m/z 424.3 (100%, M+Na), 402.3 (24%, M+H). HR-MS (ESI) m/z: for C.sub.25H.sub.39NO.sub.3Na [M+Na] calcd, 424.2823, found, 424.2822; for C.sub.25H.sub.40NO.sub.3 [M+H] calcd, 402.3003, found, 402.3003. For C.sub.25H.sub.39NO.sub.3 (401.6) calcd: 74.77%, C; 9.79%, H; 3.49%, N. Found: 74.77%, C; 9.94%, H; 2.94%, N.

EXAMPLE 12

(3R,5R,8R,9S,10S,13S,14S,Z)-17-Ethylidene-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl 5-oxopyrrolidine-2-carboxylate (12)

[0097] Compound 12 was prepared according to the General Procedure A (DMF). Starting from 5β-pregnan-17-ethylidene-3α-ol (302 mg, 1.0 mmol), using L-pyroglutamic acid (194 mg, 1.5 mmol). The crude product was pre-purified by column chromatography on silica gel (8% acetone/chloroform). Compound 12 (190 mg, 46%) was obtained by HPLC separation in the following setting. High Pressure Pump (model 361, Gilson), Inject Valve Rheodyne, preparative ELSD Detector (Gilson) connected with PC (software Trilution LC, Gilson). Flow rate 17 mL/min, acetone/hexane 20/80, elution time 48 min. Column Luna 5 μm Si(2); Axia Packed 250×21.2 mm. Loading: 1 mL of dichloromethane: mp 155-157° C. (acetone/hexane), [α]D.sup.20 +57.1 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.86 (3H, s, H-18), 0.95 (3H, s, H-19), 4.20 (1H, ddd, J=8.7, 5.4, 0.7 Hz, H-C2′), 4.79 (1H, tt, J=11.4, 4.8 Hz, H-3), 5.11 (1H, qt, J=7.1, 2.0 Hz, H-20), 5.86 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 177.6, 171.6, 150.4, 113.4, 76.1, 56.4, 55.6, 44.6, 42.0, 40.7, 37.5, 35.5, 35.0, 34.8, 32.3, 31.6, 29.3, 27.1, 26.7, 26.3, 25.0, 24.5, 23.4, 21.2, 17.0, 13.3. IR spectrum (CHCl.sub.3): 3438, 1706 (oxopyrrolidine); 1734 (C═O); 1244, 1022 (C—O); 1678, 828 (C═C). MS: ESI m/z 827.6 (100%, 2M), 414.3 (65%, M+H). HR-MS (ESI) m/z: for C.sub.26H.sub.40NO.sub.3 [M+H] calcd, 414.30027; found, 414.29987. For C.sub.26H.sub.39NO.sub.3 (413.6) calcd: 75.50%, C; 9.50%, H; 3.39%, N. Found: 75.24%, C; 9.51%, H; 3.02%, N.

EXAMPLE 13

2-(((3R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)ethyl 5-oxopyrrolidine-2-carboxylate (13)

[0098] Compound 13 was prepared according to the General Procedure A (DMF). Starting from 345(3-androstan-3α-yl)oxy)ethan-1-ol (320 mg, 1.0 mmol), using L-pyroglutamic acid (194 mg, 1.5 mmol). The crude product was pre-purified by column chromatography on silica gel (8% acetone/chloroform). Compound 13 (180 mg, 42%) was obtained by HPLC separation in the following setting. High Pressure Pump (model 361, Gilson), Inject Valve Rheodyne, preparative ELSD Detector (Gilson) connected with PC (software Trilution LC, Gilson). Flow rate 17 mL/min, acetone/hexane 20/80, elution time 45 min. Column Luna 5 μm Si(2); Axia Packed 250×21.2 mm. Loading: 1 mL of dichloromethane: mp 110-111° C. (acetone/hexane), [α].sub.D.sup.20 +11.4 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.67 (3H, s, H-18), 0.92 (3H, s, H-19), 3.28 (1H, tt, J=11.1, 4.6 Hz, H-3), 3.48 (1H, q, J=7.0 Hz, H-C2′), 3.64-3.74 (2H, m, OCH.sub.2CH.sub.2O-ster), 4.18-4.42 (2H, m, OCH.sub.2CH.sub.2O-ster), 5.92 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 177.6, 172.1, 79.9, 65.7, 65.3, 55.4, 54.7, 42.3, 41.1, 40.8, 40.6, 39.2, 36.4, 35.6, 35.2, 33.3, 29.2, 27.4, 27.3, 26.9, 25.7, 25.0, 23.6, 21.0, 20.7, 17.6. IR spectrum (CHCl.sub.3): 3437, 1705 (oxopyrrolidine); 1744 (C═O); 1240, 1033 (C—O). MS: ESI m/z 454.3 (87%, M+Na), 432.3 (58%, M+H). HR-MS (ESI) m/z: for C.sub.26H.sub.42NO.sub.4 [M+H] calcd, 432.31084; found, 432.31049. For C.sub.26H.sub.41NO.sub.4 (431.6) calcd: 72.35%, C; 9.58%, H; 3.25%, N. Found: 71.99%, C; 9.93%, H; 3.13%, N.

EXAMPLE 14

2-(43R,5R,8S,9S,10S,13S,14S)-10,13-Dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)oxy)ethyl 6-oxopiperidine-2-carboxylate (14)

[0099] Compound 14 was prepared according to the General Procedure A (DMF). Starting from 3-(5β-androstan-3α-yl)oxy)ethan-1-ol (320 mg, 1.0 mmol), using 6-oxo-L-pipecolic acid (214 mg, 1.5 mmol). The crude product was pre-purified by column chromatography on silica gel (5% acetone/chloroform). Compound 14 (170 mg, 38%) was obtained by HPLC separation in the following setting. High Pressure Pump (model 361, Gilson), Inject Valve Rheodyne, preparative ELSD Detector (Gilson) connected with PC (software Trilution LC, Gilson). Flow rate 17 mL/min, acetone/hexane 20/80, elution time 42 min. Column Luna 5 μm Si(2); Axia Packed 250×21.2 mm. Loading: 1 mL of dichlormethane: mp 117-119° C. (acetone/hexane), [α].sub.D.sup.20 +8.1 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.68 (3H, s, H-18), 0.92 (3H, s, H-19), 3.27 (1H, tt, J=11.1, 4.6 Hz, H-3), 3.69 (2H, ddd, J=5.5, 4.2, 1.3 Hz, OCH.sub.2CH.sub.2O-ster), 4.12 (1H, ddd, J=8.3, 5,1, 1,6 Hz, H-C2′), 4.25-4.36 (2H, m, OCH.sub.2CH.sub.2O-ster), 6.11 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 171.0, 170.9, 79.6, 65.3, 65.1, 54.6, 54.4, 42.0, 40.8, 40.5, 40.3, 38.9, 36.0, 35.2, 34.8, 33.0, 30.9, 27.1, 26.9, 26.6, 25.4, 25.3, 23.2, 20.7, 20.4, 19.4, 17.3. IR spectrum (CHCl.sub.3): 3400, 1666 (oxopiperidine); 1743 (C═O); 1294, 1244 (C—O). MS: ESI m/z 468.3 (100%, M+Na), 446.3 (88%, M+H). HR-MS (ESI) m/z: for C.sub.27H.sub.44NO.sub.4 [M+H] calcd, 446.32649; found, 446.32614. For C.sub.27H.sub.43NO.sub.4 (445.6) calcd: 72.77%, C; 9.73%, H; 3.14%, N. Found: 72.63%, C; 9.65%, H; 3.03%, N.

EXAMPLE 15

14(3R,5R,8R,9S,10S,13S,14S,17S)-3-(2-Hydroxyethyl)-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethan-1-one (15)

[0100] A solution of 20-oxo-5β-pregnan-3a-acetic acid (5.9 g, 16.0 mmol) in THF (20 mL) was added dropwise to the refluxing solution of LiAlH.sub.4 (1.4 g, 36.8 mmol) in THF (580 mL). After 2 hrs of reflux, the reaction mixture was cooled to 0° C., quenched with saturated aqueous solution of Na.sub.2SO.sub.4 and solids were filtered off. The filtrate was concentrated in vacuo and the residue was diluted with ethyl acetate. The combined extracts were washed with aqueous solution of hydrochloric acid (10%; 3×40 mL), water (3×40 mL), and saturated solution of NaHCO.sub.3 (3×40 mL). Solvents were dried over Na.sub.2SO.sub.4 and evaporated. Purification by column chromatography on silica gel (4% ethyl acetate/petroleum ether) gave hydroxyl derivative (3.1 g, 54%) as a mixture of 20R and 20S-isomers that was used for the next reaction step. Aqueous solution of NaOCl (5.5%, 30 mL) was added into a solution of hydroxy derivative (3 g, 8.6 mmol) in acetic acid (75 mL). After stirring at room temperature for 1 hour, isopropanol was added (45 mL) and the reaction mixture was stirred for additional 30 min. Then, it was diluted with water and extracted with chloroform (3×30 mL). Combined extracts were washed with brine, dried over anhydrous Na.sub.2SO.sub.4 and the solvents evaporated. Column chromatography on silica gel (3-5% acetone/hexane) was followed by HPLC separation in the following setting. High Pressure Pump (model 361, Gilson), Inject Valve Rheodyne, preparative ELSD Detector (Gilson) connected with PC (software Trilution LC, Gilson). Flow rate 10 mL/min, acetone/hexane 20/80, elution time 36 min. Column Luna 5 μm Si(2); Axia Packed 250×21.2 mm. Loading: 3 mL of dichloromethane. Compound 15 (850 mg, 25%) was used in the next reaction step: .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.59 (3H, s, H-18), 0.92 (3H, s, H-19), 2.11 (3H, s, H-21), 2.53 (1H, t, J=9.0 Hz, H-17), 3.66 (2H, t, J=6.9 Hz, O—CH.sub.2—CH-ster). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 209.6, 63.8, 61.7, 56.7, 44.2, 39.9, 39.2, 37.3, 35.6, 35.3, 34.6, 31.4, 31.1, 30.4, 29.5, 27.0, 26.2, 24.6, 24.3, 24.0, 22.7, 20.8, 13.3.

EXAMPLE 16

24(3R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl 5-oxopyrrolidine-2-carboxylate (16)

[0101] Compound 16 was prepared according to the General Procedure A (DMF). Starting from 20-oxo-5β-pregnan-3α-ethanol (347 mg, 1 mmol), using L-pyroglutamic acid (194 mg, 1.5 mmol). The crude product was pre-purified by column chromatography on silica gel (8% acetone/chloroform). Compound 16 (200 mg, 44%) was obtained by HPLC separation in the following setting. High Pressure Pump (model 361, Gilson), Inject Valve Rheodyne, preparative ELSD Detector (Gilson) connected with PC (software Trilution LC, Gilson). Flow rate 10 mL/min, acetone/hexane 40/80, elution time 42 min. Column Luna 5 μm Si(2); Axia Packed 250×21.2 mm. Loading: 3 mL of dichloromethane: low melting point solid, [α].sub.D.sup.20 +66.3 (c 0.3, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.59 (3H, s, H-18), 0.93 (3H, s, H-19), 2.11 (3H, s, H-21), 4.18 (2H, t, J=6.9 Hz, O—CH.sub.2—CH-ster), 4.26-4.21 (1H, m, H-C2′), 5.90 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3): δ 209.8, 177.6, 172.1, 65.1, 64.1, 57.0, 55.5, 44.5, 40.2, 39.5, 37.6, 35.9, 35.6, 31.7, 31.3, 30.5, 30.2, 29.3, 27.2, 26.5, 25.0, 25.0, 24.7, 24.6, 24.3, 23.0, 21.1, 13.6. IR spectrum (CHCl.sub.3): 3438, 1739, 1702 (oxopyrrolidine); 2924, 2864 (CH.sub.2). MS: ESI m/z 480.3 (100%, M+Na), 458.3 (12%, M+H). HR-MS (ESI) m/z: for C.sub.28H.sub.44NO.sub.4 [M+H] calcd, 458.32649; found, 458.32611. For C.sub.28H.sub.43NO.sub.4 (457.7) calcd: 73.49%, C; 9.47%, H; 3.06%, N. Found: 73.08%, C; 9.43%, H; 2.79%, N.

EXAMPLE 17

2-43R,5R,8R,9S,10S,13S,14S,17S)-17-Acetyl-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-yl)ethyl 6-oxopiperidine-2-carboxylate (17)

[0102] Compound KK-17 was prepared according to the General Procedure A (DMF). Starting from 20-oxo-5β-pregnan-3α-ethanol (347 mg, 1.0 mmol), using 6-oxo-L-pipecolic acid (214 mg, 1.5 mmol). The crude product was pre-purified by column chromatography on silica gel (5% acetone/chloroform). Compound KK-17 (175 mg, 37%) was obtained by HPLC purification in the following setting. High Pressure Pump (model 361, Gilson), Inject Valve Rheodyne, preparative ELSD Detector (Gilson) connected with PC (software Trilution LC, Gilson). Flow rate 12 mL/min, acetone/hexane 40/60, elution time 37 min. Column Luna 5 μm Si(2); Axia Packed 250×21.2 mm. Loading: 1 mL of dichlormethane: mp oil, [α].sub.D.sup.20 +61.5 (c 0.2, CHCl.sub.3). .sup.1H NMR (400 MHz, CDCl.sub.3): δ 0.59 (3H, s, H-18), 0.93 (3H, s, H-19), 2.11 (3H, s, H-21), 4.10-4.03 (2H, m, OCH.sub.2CH.sub.2O-ster), 4.30-4.11 (1H, m, H-C2′), 6.11 (1H, s, N—H). .sup.13C NMR (101 MHz, CDCl.sub.3) δ 209.8, 171.3, 171.2, 164.4, 65.2, 64.1, 57.0, 55.0, 44.5, 40.2, 39.5, 37.5, 35.9, 35.6, 31.7, 31.3, 31.2, 30.5, 30.2, 27.9, 27.2, 26.5, 25.6, 24.6, 24.3, 23.0, 21.1, 19.7, 13.6. IR spectrum (CHCl.sub.3): 3102 (oxopiperidine); 1739, 1697 (C═O); 1265, 1011 (C-0). MS: ESI m/z 494.3 (100%, M+Na). HR-MS (ESI) m/z: for C.sub.29H.sub.45NO.sub.4Na [M+Na] calcd, 494.32408; found, 494.32371.

[0103] Experiments in Vivo

[0104] Adults wild-type (strain AB) zebrafish (Danio rerio) were maintained at 28-29° C. on a light cycle of 14 h light:10h dark (lights on at 7 am; lights off at 9 pm) at ZeClinics animal facility (Barcelona). All procedures involving animals and their care were conducted accordingly to CEA-OH/9421/2 authorization from Government of Catalonia. Embryos obtained by pairwise mating were raised in E3 media at the controlled temperature of 28.5° C. until 7 days post-fertilization (dpf) when the experiments were performed.

[0105] Anticonvulsant effect of compounds was evaluated in two models of epileptic seizures in immature rats (PTZ model and 6 Hz model). PTZ convulsions were tested in 12- and 25-day-old rats, 6 Hz-induced seizures in 15- and 25-day-old rats. Young adult rats (P60) were also used in either test. Day of birth was taken as P0. Experiments evaluating precognitive effect were performed in adult male rats of the Wistar and Long Evans strain (3 months old, b.w. 300-400 g). All animals were obtained from a breeding facility of Institute of Physiology CAS (certificate No. 1396/2014-MZE-17214) Animals were held in standard conditions (21±1° C., humidity 50-60%, light regime 12/12) with unrestricted access to food and water. All manipulations with animals were performed according to the https://nc3rs.org.uk/arrive-guidelines and to Czech Animal Protection Law 246/1992 Sb. And international directives (EU Directive 2010/63/EU for animals' experiments).

[0106] Compound from Example 1 was selected to test anticonvulsant and neuroprotective effects in relation to an ethical demand for reduction of number of laboratory animals in experiments.

[0107] Anticonvulsant effect of steroid compounds in the model of pentetrazol-induced seizures in zebrafish (Danio rerio)

[0108] The anticonvulsant activity of the claimed compounds on the nervous system of zebrafish (Danio rerio). This test is the modern alternative to tests in adult animals as Danio rerio is very versatile organism that has a number of physiological similarities to mammals, including humans (Expert Opin. Drug Metab. Toxicol. 2009, 5, 393). Steroidal compounds were tested in three concentrations (1, 3 and 5 μmol.L.sup.−1) on 7 days post-fertilization (dpf) wild-type larvae. As a negative control, sibling larvae were incubated with DMSO 1%, which allows us to detect behavioral alterations of PTZ treated larvae. Seizures were induced by treatment of pentetrazol (PTZ) at the dose 5 mmol.L.sup.−1. Commercially available anti-convulsant drug Topiramate was used as comparator. Results were analyzed with GraphPad Prism software and statistical significance is assessed by the One-way ANOVA statistical analysis followed by Tukey's multiple comparisons test. The zebrafish larvae locomotion and response to visual stimuli are traced and analyzed by the EthoVision XT 12 software and the DanioVision device from Noldus Information Technologies, Wageningen, The Netherlands.

[0109] This closed system consists of a camera placed above a chamber with circulating water and a temperature sensor that is set at 28° C. Individualized larvae in a 48-wells plate are placed in the chamber, which can provide different stimuli (light/dark environment, tapping, sound) controlled by the software. Prior to each experiment, larvae were left for 10 minutes in dark for acclimation, then predetermined series of alternating dark and light environment are presented to the larvae. The final experimental protocol is divided in three main part: first a 15 minutes step with light on, then a series of 5 short flashes of light to induce epileptic seizures and finally a 25 minutes dark/light alternating environments phase. Different sets of information can be extrapolated from the different phases: while the first step is useful to detect changes in the total larvae locomotion, the second part allows us to analyze the larval response to a seizure-inducing visual stimulus and measure the seizure characteristics: the maximum velocity and the number of angles turns (specific of seizure erratic movement). The final phase is useful to detect anomalies in larval movement and deviations from the stereotyped behavior (natural locomotor behavior of zebrafish is active in dark and immobile in light). The anticonvulsant effect of compounds was studied at doses of 1, 3 and 5 mg/kg for the compound shown in Example 1, Example 2 and Example 3. The results (FIGS. 1A-1C and 2A-2C) indicate that all substances significantly reduced the locomotor activity epileptiform induced by PTZ in the three evaluated parameters—spontaneous locomotor activity, maximum velocity and the angle turns. All substances also decreased epileptiform locomotor activity in the dark phase/light test without causing sedation.

[0110] Anticonvulsant Effects of Steroid Derivatives in Rats

[0111] Two age groups of immature male Wistar rats were used in either experiment. In PTZ model P12 corresponding by a maturation of the brain to early postnatal human babies, i.e. to the period where children epileptic encephalopathy mostly resistant to present pharmacotherapy start to appear. The other group—P25 animals corresponds with school-age children. Generalized tonic-clonic seizures were elicited by a subcutaneous injection of PTZ in a dose of 100 mg/kg. The 6 Hz model was tested in P15 (at this age eyes are open and it is not necessary to surgically distract lids). Biphasic pulses with 1-s duration were applied transcorneally for 3 s by a constant current stimulator of Ugo Basile company. PTZ induced generalized seizures represent a model of human generalized tonic-clonic seizures, seizures elicited by 6 Hz stimulation are generally taken as a model of temporal (psychomotor, complex partial) seizures. Differences in latencies and seizure severity were evaluated by ANOVA with a subsequent pairwise comparison by Holm-Sidak test. Incidence of seizures were evaluated by Fischer exact test. Critical p value was set at p=0.05.

[0112] Effects of Steroid Derivatives in PTZ-Induced Seizures

[0113] Anticonvulsant effect was studied in 12- and 25-day-old male Wistar rats. Steroid derivatives were administered at doses of 1, 5 and 10 mg/kg i.p. 20 min before the administration of PTZ (100 mg/kg s.c.).

[0114] Anticonvulsant effect of the compound from Example 1 is presented at FIGS. 3A-3C. Twelve-day-old rats: The 1-mg/kg dose suppressed the tonic phase of GTCS, the 5-mg/kg dose significantly decrease the incidence of seizures and the 10-mg/kg dose completely suppressed the seizures. The 1- and 5-mg/kg doses significantly prolonged latencies to GTCS. Twenty-five-day-old rats: The 5- and 10-mg/kg doses decrease the incidence of GTCS without a selective effect against the tonic phase. Latencies of seizures were not changed after the 1-mg/kg dose, the two higher doses elicited seizures in one animal only. Seizure severity copied the presence of GTCS. Anticonvulsant effect of the compound from Example 2 is presented in FIGS. 4A-4C. Compound from Example 2 in doses of 5 and 10 mg/kg suppressed significantly incidence of GTCS and seizure severity in both age groups. Anticonvulsant effect of compound from the Example 3 is shown in FIGS. 5A-5C. This substance in the 5- and 10-mg/kg doses suppressed incidence of GTCS in 25-day-old rats. The 12-day-old group was significantly affected only by the 5-mg/kg dose.

[0115] Anticonvulsant effect of the compound from Example 1 in adult rats is demonstrated in FIGS. 6A-6C. The 10-mg/kg dose significantly decreased the incidence of generalized tonic-clonic seizures (GTCS), whereas minimal clonic seizures remained unaffected. Latency to GTCS was significantly decreased by the 5-mg/kg dose in both animals exhibiting these seizures. GTCS were present in only one rat after the 10-mg/kg dose. Seizure severity was significantly decreased after either 5- and 10-mg/kg dose.

[0116] Efficiency of Steroid Derivatives in the 6 Hz Seizure Model in Rats

[0117] Younger group was formed by 15-day-old animals—the animals at this age have open eyes and it is not necessary to distract eyelids surgically. The other group was 25 days old. Sensitivity of these two age groups is different, therefore we used current intensities of 40, 60, and 80 mA in 15-day-old rats and 20, 40, and 60 mA in the 25-day-old animals. Stimulations were made in 20-min intervals, 10 min before each stimulation a drop of mesocaine into either eye was used as a local anesthetic. Steroid derivatives in a dose of 10 mg/kg i.p. were administered 20 min before the first stimulation. Anticonvulsant effect of the compound from Example 1 is presented in FIGS. 7A-7C. Seizures elicited by 60-mA intensity of stimulation current were suppressed, those elicited by 80 mA not. Duration of seizures was significantly shortened at both current intensities; seizure severity was not affected. Older age group exhibited a tendency to a decreased incidence of seizures. Seizures were significantly shorter and exhibited lower intensity. FIG. 8 demonstrates anticonvulsant effect of the substance from Example 1 in adult rats. Changes of the threshold intensity did not reach the level of significance—this tendency was seen after the high dose of 10 mg/kg i.p. Comparison of data from immature and adult rats clearly demonstrate higher efficiency and broader spectrum of effects in pediatric models.

[0118] Efficacy of Compound from Example 1 in the Models of Affective Disorder (Epilepsy Comorbidity)

[0119] Behavioral models of the affective disorders are based on exposure to stress. Two behavioral tests were performed: the Elevated plus maze test (EPM, Nat. Protoc. 2007, 2, 322) and the Novelty suppressed feeding model (NSF, Interdiscip. Toxicol. 2017, 10, 40).

[0120] In the EPM test, the Compound from Example 1 was administered at doses of 1, 3 and 10 mg/kg, i.p. 30 minutes before maze testing. Time spent in open arms, closed arms, and central platform were recorded during a 10-min test session. Administration of the Compound from Example 1 at a dose of 10 mg/kg resulted into significant reduction of anxiety parameters compare to the control group. The EPM test demonstrated the anxiolytic properties of the Compound from Example 1.

[0121] In the NSF test, the Compound from Example 1 was administered i.p. 30 min before the test at doses 0.1; 0.3; 1; 3 and 10 mg/kg. The results showed the significant decrease in latency to feed (anxiolytic parameter) at 0.3 mg/kg. In doses 1 and 3 mg/kg, there was a trend to decrease the latency of feed intake.

[0122] Efficacy of the Compound from Example 1 in the Models of Schizophrenia-Like Behavior and Procognitive Effect

[0123] Schizophrenia-like behavior was induced by i.p. administration of dizocilpine (MK-801) at a dose of 0.1 mg/kg. The effect of the Compound from Example 1 was tested in Passive avoidance test (Psychopharmacology (Berl). 2016, 233, 2077). This test allows to study memory and learning ability. The Compound from Example 1 was applied for 30 min i.p. before the task in doses of 0.1; 1 and 3 mg/kg. The results show that administration of the Compound from Example 1 to intact animals does not impair memory one hour after application of the aversive stimulus. The effect on reducing the latency to entry was showed for latency measured 24 h after association at a dose of 0.1 mg/kg. Application of dizocilpine resulted in memory impairment, measured as latency to entrance at 1 and 24 h after application of the aversive stimulus. Administration of the Compound of Example 1 at a dose of 0.1 mg/kg prevented memory damage measured 1 h after the inverse stimulus. A similar trend was observed at other doses and for a longer period of time. Thus, the application of the Compound from Example 1 shows a pro-cognitive effect, particularly in short-term memory.

[0124] The Neuroprotective Effect of the Compound of Example 1 in the Model of Excitotoxic CNS Injury

[0125] The neuroprotective effect of the Compound from Example 1 was tested in the model of bilateral excitotoxic lesion of the dorsal hippocampus. The procedure was performed according to literature (Neuropharmacology 2011, 61, 61). This model simulates the overstimulation of NMDA receptors that occurs in the number of pathophysiological states, leading to calcium flow into the neuron's and, consequently, to apoptosis almost necrosis. Clinically, this phenomenon is manifested by neurodegeneration and CNS damage. Rats were randomly divided into three groups. Control animals were those operated animals that were administered phosphate buffer pH 7.4 into the hippocampus. The second group, called NMDA, represented the animals that had been induced NMDA lesions of the hippocampus. Animals in the third group received Compound from Example 1 at dose 1 mg/kg i.p. 5 minutes after NMDA lesion. NMDA lesion was induced by infusing excitotoxic NMDA (25 mmol.L.sup.−1, volume 1 μl) into the dorsal hippocampus, control animals received sterile PBS (10 mmol.L.sup.−1). Compounds from Example 1 (1 mg/kg) or vehicle was administered 5 minutes after the end of the NMDA infusion. Transcardial perfusion was performed 24 h after induction of excitotoxic damage to the hippocampus. Brain tissue was then post-fixed overnight in 4% PFA followed by 10%, 20%, 30% (v/v) sucrose (for cryoprotection) for histological evaluation. Histological damage, e.g. the neuroprotective effect of Compound from Example 1 was evaluated by staining damaged neurons with Fluoro Jade B (Merck Millipore, Catalog Number AG310-30MG), and evaluated areas included: hippocampus-DG, hilus, CA3, CA1. The results in FIGS. 9A-9C show a significant reduction in the dorsal hippocampus damage following administration of the Compound of Example 1. These results clearly demonstrate the neuroprotective effect of the Compound from Example 1.

INDUSTRIAL APPLICABILITY

[0126] The compounds from the submitted invention will be useful for treating central nervous system diseases, particularly epilepsy and seizure conditions in children. In addition, they can also be used to influence associated comorbidities. These include depression, anxiety, schizophrenia-like behavior, neurodevelopmental disorders, affective disorders and stress disorders. The claimed substances can be used separately in therapy, but also as adjuvant treatment to medicines currently approved for therapy.