Neurosteroid compounds and methods for their preparation and use in treating central nervous system disorders
11542296 · 2023-01-03
Assignee
Inventors
Cpc classification
C07J3/00
CHEMISTRY; METALLURGY
C07J41/005
CHEMISTRY; METALLURGY
C07J21/00
CHEMISTRY; METALLURGY
C07J3/005
CHEMISTRY; METALLURGY
C07J41/0038
CHEMISTRY; METALLURGY
C07J51/00
CHEMISTRY; METALLURGY
International classification
C07J43/00
CHEMISTRY; METALLURGY
C07J5/00
CHEMISTRY; METALLURGY
Abstract
Described herein is the chemical structure of neurosteroid derivative compounds, methods of synthesizing the derivatives, and their uses in treating disorders, including those of the central nervous system. These compounds are readily synthesized and have improved pharmaceutical properties, including water solubility, compared to known neurosteroids.
Claims
1. A method for treating epilepsy comprising administering to a subject in need thereof a therapeutically effective amount of a compound, a pharmaceutically acceptable salt of said compound, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient and said compound, said compound or said pharmaceutically acceptable salt being selected from: ##STR00060##
2. The method of claim 1, wherein the compound is administered to a patient by oral, parenteral, intravenous, transdermal, inhalation, intracerebral or topical administration in a suitable formulation.
3. A method for treating epilepsy comprising administering to a subject in need thereof a therapeutically effective amount of a compound, a pharmaceutically acceptable salt of said compound, or a pharmaceutical composition comprising a pharmaceutically acceptable excipient and said compound, said compound or pharmaceutically acceptable salt being selected from: ##STR00061##
4. The method of claim 3, wherein the compound is given to a patient by oral, parenteral, intravenous, transdermal, inhalation, intracerebral or topical administration in a suitable formulation.
5. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00062##
6. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00063##
7. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00064##
8. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00065##
9. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00066##
10. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00067##
11. The method of claim 1, wherein said compound or said pharmaceutically acceptable salt is ##STR00068##
12. The method of claim 3, wherein said compound or said pharmaceutically acceptable salt is ##STR00069##
13. The method of claim 3, wherein said compound or said pharmaceutically acceptable salt is ##STR00070##
14. The method of claim 3, wherein said compound or said pharmaceutically acceptable salt is ##STR00071##
15. The method of claim 3, wherein said compound or said pharmaceutically acceptable salt is ##STR00072##
16. The method of claim 3, wherein said compound or said pharmaceutically acceptable salt is ##STR00073##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
(1) In order that the manner in which the above-recited and other enhancements and objects of the disclosure are obtained, a more particular description of the disclosure briefly described above will be rendered by reference to specific embodiments thereof which are illustrated in the appended drawings. Understanding that these drawings depict only typical embodiments of the disclosure and are therefore not to be considered limiting of its scope, the disclosure will be described with additional specificity and detail through the use of the accompanying drawings in which:
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DETAILED DESCRIPTION
(26) The particulars shown herein are by way of example and for purposes of illustrative discussion of the preferred embodiments of the present disclosure only and are presented in the cause of providing what is believed to be the most useful and readily understood description of the principles and conceptual aspects of various embodiments of the disclosure. In this regard, no attempt is made to show structural details of the disclosure in more detail than is necessary for the fundamental understanding of the disclosure, the description taken with the drawings making apparent to those skilled in the art how the several forms of the disclosure may be embodied in practice.
(27) The following definitions and explanations are meant and intended to be controlling in any future construction unless clearly and unambiguously modified in the following examples or when application of the meaning renders any construction meaningless or essentially meaningless. In cases where the construction of the term would render it meaningless or essentially meaningless, the definition should be taken from Webster's Dictionary 3.sup.rd Edition.
(28) The term “halogen” means a fluorine, a chlorine, a bromine or an iodine.
(29) The term “halo” means fluoro, chloro, bromo, or iodo, preferably fluoro and chloro.
(30) The term “alkyl group” means a saturated, linear or branched, aliphatic group. Examples of an alkyl group include the methyl, ethyl, n-propyl, n-butyl, n-pentyl, n-hexyl, n-heptyl, 1-methylethyl, 1-methylpropyl, 2-methylpropyl, 1,1-dimethylethyl, 1-methylbutyl, 2-methylbutyl, 3-methylbutyl, 1,1-dimethylpropyl, 1,2-dimethylpropyl, 2,2-dimethylpropyl, 1-methylpentyl, 2-methylpentyl, 3-methylpentyl, 4-methylpentyl, 1,1-dimethylbutyl, 1,2-dimethylbutyl, 1,3-dimethylbutyl, 2,2-dimethylbutyl, 2,3-dimethylbutyl, 3,3-dimethylbutyl, 1,1,2-trimethylpropyl, 1,2,2-trimethylpropyl, 1-ethyl-1-methyl-propyl, 1-ethyl-2-methylpropyl, 1-ethylbutyl, 2-ethylbutyl, 1-methylhexyl, 2-methylhexyl, 3-methylhexyl, 4-methylhexyl, 5-methylhexyl, 1,1-dimethylpentyl, 1,2-dimethylpentyl, 1,3-dimethylpentyl, 1,4-dimethylpentyl, 2,2-dimethylpentyl, 2,3-dimethylpentyl, 2,4-dimethylpentyl, 3,3-dimethylpentyl, 3,4-dimethylpentyl, 4,4-dimethylpentyl, 1,1,2-trimethylbutyl, 1,1,3-trimethylbutyl, 1,2,2-trimethylbutyl, 1,2,3-tri-methylbutyl, 1,3,3-trimethylbutyl, 2,2,3-trimethylbutyl, 2,3,3-trimethylbutyl, 1,1,2,2-tetramethylpropyl, 1-ethylpentyl, 2-ethylpentyl, 3-ethylpentyl, 1-ethyl-1-methylbutyl, 1-ethyl-2-methylbutyl, 1-ethyl-3-methylbutyl, 2-ethyl-1-methylbutyl, 2-ethyl-2-methylbutyl, 2-ethyl-3-methylbutyl, 1-propylbutyl, 1-(1-methylethyl)butyl or 1-(1-methylethyl)-2-methylpropyl groups.
(31) The term “lower alkyl” means an alkyl group having 1 to 6 carbons linear or branched.
(32) The term “alkenyl group” means a mono- or polyunsaturated, linear or branched, aliphatic group comprising, for example, one or two ethylenic unsaturations.
(33) The term “alkynyl group” means a mono- or polyunsaturated, linear or branched, aliphatic group comprising, for example, one or two acetylenic unsaturations.
(34) The term “cycloalkyl group” means cyclopropyl, cyclobutyl, cyclopentyl, cyclohexyl, cycloheptyl, bicyclo[2.2.1]heptyl, cyclooctyl, bicyclo[2.2.2]octyl, bicyclo[3.2.1]octyl or adamantyl.
(35) The term “acyl” means a radical —C(O)R′, where R′ is hydrogen, alkyl, alkenyl, cycloalkyl, cycloalkylalkyl, phenyl or phenylalkyl wherein alkyl, cycloalkyl, cycloalkyl-alkyl, and phenyl-alkyl are as defined herein. Representative examples include, but are not limited to formyl, acetyl, cylcohexylcarbonyl, cyclohexylmethylcarbonyl, benzoyl, benzylcarbonyl, and the like.
(36) The term “alkoxyl” means a radical —OR where R is an alkyl as defined above. Representative examples include, but are not limited to methoxy, ethoxy, propoxy, butoxy, t-butoxyl and the like.
(37) The term “aryl” means a monovalent monocyclic or polycyclic aromatic hydrocarbon radical; it includes, but is not limited to, phenyl and naphthyl.
(38) The term “heteroaryl” means a monovalent monocyclic or bicyclic radical of 5 to 12 ring atoms having at least one aromatic ring containing one, two, or three ring heteroatoms independently selected from N, O, or S, the remaining ring atoms being C, with the understanding that the attachment point of the heteroaryl radical will be on an aromatic ring. More specifically the term heteroaryl includes, but is not limited to, pyridyl, furanyl, thienyl, thiazolyl, isothiazolyl, triazolyl, imidazolyl, isoxazolyl, pyrrolyl, pyrazolyl, pyrimidinyl, benzofuranyl, tetrahydrobenzofuranyl, isobenzofuranyl, benzothiazolyl, benzoisothiazolyl, benzotriazolyl, indolyl, isoindolyl, benzoxazolyl, quinolyl, tetrahydroquinolinyl, isoquinolyl, benzimidazolyl, benzisoxazolyl or benzothienyl, imidazo[1,2-a]-pyridinyl, and imidazo[2,1-b]thiazolyl.
(39) The term “heterocyclic ring” means a saturated or unsaturated non-aromatic cyclic radical of 3 to 8 ring atoms in which one or two ring atoms are heteroatoms independently selected from N, O, or S(O).sub.e (where e is an integer from 0 to 2).
(40) The term “substituent” means hydrogen, halogen, alkyl, alkoxyl, cycloalkyl, cyano (CN), OH, acyl, haloalkyl, heteroaryl, C(halogen).sub.3, CN, C(═O)CH.sub.3, C(═O)NQ.sub.1Q.sub.2, N.sub.3, NCS, NO.sub.2, NQ.sub.1Q.sub.2 wherein Q.sub.1 and Q.sub.2 each is independently selected from H and alkyl.
(41) Methods for Using Neurosteroid Derivatives
(42) The disclosure further relates to derivative neurosteroid compounds of ganaxolone (GX) and its 17-isooxazole analog (UCI-50027), and CNS selective GABA-A receptor modulators, to the preparation thereof and to the therapeutic use thereof, wherein said compounds are of general formulas (A). These compounds are potentially useful in treating brain disorders including, but not limited to seizures, epilepsy, anxiety, depression, cognition, behavioral and neurological effects, and central nervous system dysfunction.
(43) In a further aspect, the present disclosure provides methods for using neurosteroid derivatives. In an embodiment, the Formula (A) neurosteroid derivatives can be used as therapeutic agents, such as for treating seizures, epilepsy, and neurobehavioral effects and dysfunction.
(44) In an embodiment, the neurosteroid derivatives of the disclosure can be used for the treatment of disorders associated with the central nervous system, which include, but are not limited epilepsy, refractory epilepsy, status epilepticus, catamenial epilepsy; Alzheimer's disease, chronic pain, alcohol dependence, alcohol withdrawal, drug addiction, infantile spasm, traumatic brain injury, post-traumatic epilepsy, Fragile-X syndrome, chemical neurotoxicity, smoking cessation, bipolar disorder, anxiety, generalized anxiety syndrome, panic attacks, depression, postpartum depression, premenstrual disorder, essential tremor, rare epilepsies including Rett's syndrome, Dravet syndrome and PHD19 condition.
(45) In an embodiment, for therapeutic applications, the neurosteroid derivatives of the disclosure can be formulated with a pharmaceutically acceptable carrier suitable for the desired method of administration. Pharmaceutically acceptable carriers are known in the art. Pharmaceutically acceptable carriers include, but are not limited to a cream, emulsion, gel, liposome, nanoparticle, ointment, polymeric micelle, a protein, and microspheres. In an embodiment, the neurosteroid derivatives of the disclosure can be formulated with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients include, but are not limited to antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles. In an embodiment, the neurosteroid derivatives of this disclosure can be administered systemically by oral, intravenous, parenteral injections, inhalation, transdermal, intracerebral or topical administration.
(46) Methods for Using Neurosteroid Derivatives
(47) The disclosure further relates to derivative neurosteroid compounds of ganaxolone (GX) and its 17-isooxazole analog (UCI-50027), and CNS selective GABA-A receptor modulators, to the preparation thereof and to the therapeutic use thereof, wherein said compounds are of general formulas (A). These compounds are potentially useful in treating brain disorders including, but not limited to, seizures, epilepsy, cognition, behavioral and neurological effects, and central nervous system dysfunction.
(48) In a further aspect, the present disclosure provides methods for using neurosteroid derivatives. In an embodiment, the Formula (A) neurosteroid derivatives can be used as therapeutic agents, including, but not limited to use for treating seizures, epilepsy, and neurobehavioral effects and dysfunction.
(49) In an embodiment, the neurosteroid derivatives of the disclosure can be used for the treatment of disorders associated with central nervous system, which include, but are not limited to epilepsy, refractory epilepsy, status epilepticus, catamenial epilepsy; Alzheimer's disease, chronic pain, alcohol dependence, alcohol withdrawal, drug addiction, infantile spasm, traumatic brain injury, post-traumatic epilepsy, Fragile-X syndrome, chemical neurotoxicity, smoking cessation, bipolar disorder, anxiety, generalized anxiety syndrome, panic attacks, depression, postpartum depression, premenstrual disorder, essential tremor, rare epilepsies including Rett's syndrome, Dravet syndrome and PHD19 condition.
(50) In an embodiment, for therapeutic applications, the neurosteroid derivatives of the disclosure can be formulated with a pharmaceutically acceptable carrier suitable for the desired method of administration. Pharmaceutically acceptable carriers are known in the art. Pharmaceutically acceptable carriers include, but are not limited to a cream, emulsion, gel, liposome, nanoparticle, ointment, polymeric micelle, a protein, and microspheres. In an embodiment, the neurosteroid derivatives of this disclosure can be administered systemically by oral, intravenous, parenteral injections, inhalation, transdermal, intracerebral or topical administration. In an embodiment, the neurosteroid derivatives of the disclosure can be formulated with a pharmaceutically acceptable excipient. Pharmaceutically acceptable excipients are known in the art. Pharmaceutically acceptable excipients include, but are not limited to antiadherents, binders, coatings, colors, disintegrants, flavors, glidants, lubricants, preservatives, sorbents, sweeteners, and vehicles.
(51) Ganaxolone and its analog (UCI-17-isooxazole) are neurosteroid analogs and CNS-selective GABA-A receptor modulators that act on well-characterized targets in the brain. They both have demonstrated the anti-seizure and anxiolytic effect; however, their bioavailability are extremely low due to their high lipophilicity and low aqueous solubility. Thus, a prodrug is a viable approach to improve their bioavailability. Various types of analogs or prodrugs of ganaxolone and UCI-17-isooxazole have been synthesized in >250 mg quantity with >95% purity. Their aqueous solubility and stability were also measured. In general, great improvements were observed over the parent compounds.
(52) ##STR00025## ##STR00026## ##STR00027## ##STR00028##
EXAMPLES
(53) The following examples are provided for the purpose of illustrating, not limiting the disclosure.
Example 1. Preclinical Efficacy Studies of Neurosteroid Derivatives in Seizure Models
(54) Many new derivatives of neurosteroid molecules were synthesized and their effect in three standard seizure models was demonstrated: (i) hippocampus kindling seizures; (ii) 6-Hz limbic-seizures; and (iii) pilocarpine status epilepticus. For comparison, parallel experiments were conducted with ganaxolone. These results are summarized in Table 1.
(55) TABLE-US-00001 TABLE 1 Anticonvulsant and protective (ED50 values, mg/kg, s.c.) of neurosteroid derivatives. Kindling 6-Hz Pilocarpine Compound model model model 21-OH-ganaxolone 3.8 mg/kg 2.6 mg/kg 6 mg/kg 21-OH-ganaxolone phosphate 3.5 mg/kg 2.8 mg/kg 6 mg/kg 21-OH-ganaxolone succinate 3.5 mg/kg 2.1 mg/kg 6 mg/kg 21-OH-ganaxolone valine 3.3 mg/kg 2.6 mg/kg 6 mg/kg Ganaxolone 3.5 mg/kg .sup. 2 mg/kg 6 mg/kg
(56) Kindling model: As shown in
(57) 6-Hz seizure model: As shown in
(58) Pilocarpine SE model: As shown in Table 1, all four neurosteroid derivatives protected pilocarpine-induced SE in a dose-dependent fashion. Also, test compounds improved the survival rate from SE-related mortality.
(59) Neurosteroid derivatives were tested for their effect on synaptic and extrasynaptic GABA-A receptors in dentate gyrus granule cells (DGGCs) in the hippocampus. Since the expression of δ-subunit is higher in DGGCs than in other neurons, DG neurons were utilized for testing the effect of neurosteroid analogs on whole-cell and extrasynaptic tonic currents. To determine the modulatory effects of neurosteroid analogs on whole-cell GABA-gated currents, neurosteroid analogs were studied in native neurons using patch-clamp electrophysiology. GABA-A receptor currents were recorded from acutely dissociated, voltage-clamped DGGCs or CA1PCs from adult male mice in whole-cell mode. 3 μM GABA was utilized, which was within the range of EC.sub.10 response for both DGGCs to determine a baseline response and allosteric activation by tested compounds. A test neurosteroid analog produced allosteric potentiation of GABA currents in a concentration-dependent manner (
(60) Test compounds were studied for their effect on tonic current in DGGCs in brain slices. Voltage-clamp electrophysiology was used to record enhancement of GABAergic tonic currents by neurosteroid analogs. Baseline tonic currents were derived in a bath perfusion with 1 μM GABA. Test neurosteroid analog was co-applied with 1 μM GABA and at the end of each recording, 50 μM GBZ was perfused in order to determine the total tonic current shift (
(61) In PK studies, the plasma pharmacokinetics of ganaxolone analogs following an intravenous (iv) or intramuscular (im) dose administration. Male Sprague Dawley rats were administered a single dose of the neurosteroid analog. Rats received a single iv or im dose of 21-OH-GX, 21-OH-GX phosphate, 21-OH-GX lysine, or 21-OH-GX valine at 10 mg/kg. The pharmacokinetic parameters of four ganaxolone analogs were assessed by measuring plasma levels of the active form, 21-OH-GX, which was administered directly or as three different prodrugs by the iv and im routes. The plasma drug concentration versus time profiles for the four neurosteroid analogs were shown in
(62) Neurosteroid analogs were tested for the anticonvulsant efficacy in the DFP model in rats. The organophosphate pesticide DFP is widely used as a model of organophosphate intoxication, which causes seizures, status epilepticus (SE) and brain injury. Test compounds were given intramuscularly (im) 40 min after DFP. Test analogs (10 mg/kg, im) produced a striking protection against DFP-induced seizures (
(63) Neurosteroid analogs were tested for neuroprotectant efficacy in the DFP model in rats. Test compounds were given intramuscularly (im) 40 min after DFP. The brains were perfused and processed for histology 72 h after DFP exposure. Neurosteroid compounds significantly reduced neuronal injury assessed by FJB(+) stereology counts in the hippocampus (
(64) Neurosteroid analogs were tested for anticonvulsant efficacy in the nerve agent soman model in rats. The nerve agent soman causes seizures, status epilepticus (SE) and brain injury. Test compounds were given intramuscularly (im) 40 min after soman exposure. Neurosteroid analogs (10 mg/kg, im) produced a striking protection against soman-induced behavioral seizures (
(65) Neurosteroid analogs were tested for neuroprotectant efficacy in the nerve agent soman model in rats. Test compounds were given intramuscularly (im) 40 min after soman. The brains were perfused and processed for histology 24 h after soman exposure. In untreated control group, soman caused extensive neuronal injury in the hippocampus and other brain regions as revealed by FJB staining. Neurosteroid analogs significantly reduced neuronal injury assessed by FJB(+) stereology counts in the hippocampus (
(66) Many of the neurosteroid derivatives of ganaxolone described herein are active with powerful protective activity in multiple seizure models. The neurosteroid derivatives produced a rapid and dose-dependent suppression of behavioral and electrographic seizures, with nearly complete seizure protection within 15-min after administration. The anticonvulsant ED50 doses were comparable to that of ganaxolone. Since ganaxolone is highly effective in organophosphate (OP) models, these neurosteroid derivatives were determined to be similarly active in the DFP and Sarin model. Thus, these compounds are clinically useful for epilepsy, chemical neurotoxicity and other brain disorders.
(67) While the preferred embodiment of the disclosure has been illustrated and described, it will be appreciated that various changes can be made therein without departing from the spirit and scope of the disclosure.
Example 2. Methods for Making Neurosteroid Derivatives
(68) .sup.1H NMR was recorded on a Varian Utility 300 or 400 MHz spectrometer, in deuterated chloroform (CDCl.sub.3) or dimethylsulfoxide (DMSO-d.sub.6) solvent. Chemical shifts were reported as ppm from solvent reference. Coupling constants (J values) were measured in hertz. Low resolution electrospray mass spectra (ES-MS) were collected on a Finnigan liquid chromatography quadrupole (LCQ) Duo liquid chromatography tandem mass spectrometer (LC-MS-MS) (Thermoquest). Crude products were purified by column chromatography and recrystallization. Purity was assessed by HPLC and .sup.1H NMR. HPLC method used a reverse phase XBridge C18 5 m 4.6×250 mm column from Waters running a binary gradient with water (with 0.1% TFA) and acetonitrile (with 0.1% TFA). Peaks were detected using the wavelength MAX reading from a photodiode array detector scanning from 200-600 nm on a 20 min gradient running from 10% to 95% acetonitrile/TFA in H.sub.2O/TFA at 1 mL/min on a Waters Alliance HPLC.
(69) In one aspect, the present disclosure provides methods for making neurosteroid derivatives.
(70) In an embodiment, the disclosure provides a method for using pregnenolone to make 21-OH ganaxolone and other intermediary compounds which are useful for preparing neurosteroid derivatives. The method of making 21-OH ganaxolone is shown below in Route 1.
(71) Route 1
(72) ##STR00029## ##STR00030##
Referring to Route 1, Synthesis of 1-((3S,8R,10S,13S,14S,17S)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethenone
(73) ##STR00031##
Pregnenolone (3.17 g, 10 mmol) was dissolved in 30 mL of THF and 5 mL of acetic acid. To it, 10% Pd/C (0.3 g) was added. The resulting mixture was shaken under 60 psi hydrogen at 60° C. overnight. It was filtered through a CELITE® pad and concentrated to give 3.2 g of the desired product (100%). .sup.1H NMR (400 MHz, CDCl3) δ 3.58 (tt, J=11.0, 4.8 Hz, 1H), 2.50 (t, J=9.0 Hz, 1H), 2.19-2.11 (m, 2H), 2.09 (s, 3H), 2.06-1.93 (m, 2H), 1.85-1.75 (m, 1H), 1.74-1.50 (m, 6H), 1.47-1.04 (m, 9H), 1.04-0.82 (m, 2H), 0.79 (s, 3H), 0.72-0.61 (m, 1H), 0.58 (d, J=2.4 Hz, 3H).
Synthesis of (8R,10S,13S,14S,17S)-17-acetyl-10,13-dimethyltetradecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one
(74) ##STR00032##
(75) To a solution of the above product (1-((3S,8R,10S,13S,14S,17S)-3-hydroxy-10,13-dimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethanone, 3.2 g, 10 mmol) in 40 mL of THF and 10 mL of acetic acid was added NaBr (1.03 g, 0.1 eq.). It was cooled in an ice bath and was followed by the dropwise addition of NaOCl (82 mL, 10-15%, 18 eq.) at such a rate that the internal temperature was maintained <40° C. After addition, it was stirred at room temperature for 2 h. Thin layer chromatography (TLC) indicated it was complete. The mixture was diluted with dichloromethane and layers were separated. The organic layer was washed with Na.sub.2S.sub.2O.sub.3 (10% aq.), H.sub.2O, NaHCO.sub.3 (sat.) and NaCl (sat.). Drying over Na.sub.2SO.sub.4 and concentration afforded 3.8 g of the crude product, which was recrystallized from CH.sub.2Cl.sub.2/Hex to give 2.57 g of the desired product (81%). .sup.1H NMR (400 MHz, CDCl3): 2.51 (t, 1H), 2.2-2.4 (m, 3H), 2.1-2.2 (m, 1H), 2.10 (s, 3H), 1.98-2.01 (m, 2H), 1.6-1.7 (m, 4H), 1.55-1.6 (m, 1H), 1.3-1.4 (m, 7H), 1.1-1.2 (m, 2H), 0.99 (s, 3H), 0.95-0.98 (m, 1H), 0.75-0.78 (m, 1H), 0.62 (s, 3H).
Synthesis of 1-((2′R,8R,10S,13S,14S,17S)-10,13-dimethylhexadecahydrospiro[cyclopenta[a]phenanthrene-3,2′-oxiran]-17-yl)ethanone
(76) ##STR00033##
(77) Under argon, trimethylsulfoxonium iodide (2.6 g, 1.7 eq.) and sodium t-butoxide (1.18 g, 1.75 eq.) in DMSO (20 mL) was heated at 65° C. for 2 h. After it was cooled to RT, the above di-ketone ((8R,10S,13S,14S,17S)-17-acetyl-10,13-dimethyltetradecahydro-1H-cyclopenta[a]phenanthren-3(2H)-one, 2.2 g, 7 mmol) was added scoop-wise so that the internal temperature was maintained between 25-35° C. The resulting mixture was stirred at RT for 2 h. After TLC indicated it was complete, it was quenched with 30 mL of H.sub.2O, stirred for 10 min and was kept in fridge overnight. The precipitate was filtered, washed with 20 mL of (4:1 of H.sub.2O/MeOH), dried to give 94% of the desired product (W=2.17 g). .sup.1H NMR (400 MHz, CDCl3) δ 2.63 (s, 2H), 2.53 (t, J=8.9 Hz, 1H), 2.20-2.13 (m, 1H), 2.11 (s, 3H), 2.10-1.95 (m, 2H), 1.87 (dd, J=13.9, 13.1 Hz, 1H), 1.76-1.59 (m, 4H), 1.58-1.48 (m, 1H), 1.48-1.24 (m, 5H), 1.24-1.07 (m, 3H), 1.02-0.87 (m, 2H), 0.86 (dd, J=3.7, 2.2 Hz, 1H), 0.84 (s, 3H), 0.81-0.74 (m, 1H), 0.61 (s, 3H).
Synthesis of 1-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethanone (ganaxolone)
(78) ##STR00034##
(79) To a solution of the above epoxide (1.5 g, 4.56 mmol) in 15 mL of THF and 15 mL of MeOH were added NaI (1.02 g, 1.5 eq.) and HOAc (0.6 mL, 2.2 eq.). The resulting mixture was heated at 65° C. for 2 h. After TLC indicated that the epoxide was completely converted to an iodo compound, it was cooled to RT. Sodium acetate (1.02 g, 2.7 eq.) and 150 mg of 10% Pd/C were added and the mixture was transferred to a hydrogenation bottle with the aid of MeOH (10 mL) and was hydrogenated under 50 psi hydrogen over the weekend. It was filtered through CELITE® and the filtrate was concentrated. The residue was then partitioned between dichloromethane and water. The aqueous solution was extracted twice with CH.sub.2Cl.sub.2 and the combined organic layers were washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. The Biotage flash purification with 10-35% EtOAc in hexane to give 0.5 g of the desired product (33%).
(80) The synthesis was repeated with 1.1 g of the epoxide and 1 g of the product was obtained (90%).
(81) Both lots of product were combined and recrystallized with CH.sub.2Cl.sub.2 and hexane to give 0.522 g of the product with 96.6% purity by HPLC. .sup.1H NMR (400 MHz, Chloroform-d) δ 2.51 (t, J=8.9 Hz, 1H), 2.18-2.10 (m, 1H), 2.09 (s, 3H), 2.01-1.93 (m, 1H), 1.72-1.57 (m, 4H), 1.57-1.41 (m, 5H), 1.41-1.30 (m, 3H), 1.30-1.20 (m, 3H), 1.18 (s, 3H), 1.17-1.09 (m, 2H), 1.00-0.85 (m, 1H), 0.78 (ddd, J=10.6, 7.7, 5.4 Hz, 1H), 0.73 (d, J=0.6 Hz, 3H), 0.58 (s, 3H). UV: Absorbances at 206.2 nm. TLC: (Silica Gel plates) 20% EtOAc/Hexane; Rf=0.50. HPLC: Sunfire C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 80% Acetonitrile in H.sub.2O (0.1% formic acid) over 30 min; retention time 8.24 min; 96.6%.
Synthesis of 2-bromo-1-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethan-1-one (21-Bromo-ganaxolone)
(82) ##STR00035##
(83) To a solution of ganaxolone (0.33 g, 1 mmol) in 20 mL of methanol were added 2 drops of HBr (48% in H.sub.2O) and 0.13 mL of bromine (2.5 eq.) dropwise. The resulting mixture was stirred at RT for 1.25 h, during which precipitate formed. TLC indicated the complete consumption of the starting material. The mixture was poured into icy water and the product was extracted with dichloromethane. The organic layer was washed with Na.sub.2S.sub.2O.sub.3 (10% aqueous solution), H.sub.2O, NaCl (saturated) and dried (Na.sub.2SO.sub.4). Evaporation of solvents gave 0.4 g of the desired product (100% yield). .sup.1H NMR (400 Hz, CDCl3): δ 3.89 (d, J=3.0 Hz, 2H), 2.80 (t, J=8.9 Hz, 1H), 2.21-2.10 (m, 1H), 1.89 (dt, J=11.8, 3.4 Hz, 1H), 1.79-1.58 (m, 4H), 1.57-1.43 (m, 5H), 1.42-1.30 (m, 2H), 1.30-1.20 (m, 4H), 1.18 (s, 3H), 1.16-1.11 (m, 2H), 0.94 (qd, J=12.2, 5.6 Hz, 1H), 0.78 (ddd, J=12.9, 10.6, 4.5 Hz, 1H), 0.73 (s, 3H), 0.61 (s, 3H).
Synthesis of 2-hydroxy-1-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)ethan-1-one (21-OH-ganaxolone)
(84) ##STR00036##
(85) To a solution of trimethylamine (13.2 mL, 95 mmol, 13 eq.) in 180 mL of acetone at 0° C. was added trifluoroacetic acid (5.4 mL, 73 mmol, 10 eq.). The mixture was stirred at RT after the addition for 10 min. To this mixture was then added 21-bromo-ganaxolone (3.0 g, 7.3 mmol). After the resulting mixture was heated in 65° C. oil bath for 1 hour, CF.sub.3COONa (15 eq. 109 mmol, 14.9 g) was added. The mixture was heated at 65° C. overnight. It was cooled to RT and was concentrated. The residue was partitioned between dichloromethane and water. The aqueous layer was extracted with DCM. The combined organic layers were washed with brine and dried over MgSO.sub.4. The product was purified on a Biotage column with 5-80% EtOAc in hexane to give 2.09 g of the desired product (82% yield). .sup.1H NMR (400 MHz, DMSO-d6) δ 4.87 (dd, J=5.9, 0.7 Hz, 1H), 4.01 (d, J=5.9 Hz, 2H), 3.83 (s, 1H), 2.55 (t, J=8.8 Hz, 1H), 2.00 (dd, J=21.0, 10.3 Hz, 1H), 1.80 (d, J=12.5 Hz, 1H), 1.66-1.42 (m, 6H), 1.39-1.24 (m, 4H), 1.24-1.07 (m, 9H), 1.04 (s, 3H), 0.97-0.78 (m, 1H), 0.67 (s, 3H), 0.50 (s, 3H). UV: 201, 280 nM. TLC: (Silica Gel plates) 20% EtOAc/Hexane; Rf=0.25. HPLC: Sunfire C18 5 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 50% Acetonitrile in H.sub.2O (0.1% TFA) over 30 min; retention time 14.36 min; 98.84%.
(86) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone Valine. The method of making 21-OH Ganaxolone Valine from 21-Bromo Ganaxolone is shown below in Route 2.
(87) Route 2
(88) ##STR00037##
Referring to Route 2, Synthesis of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl valinate hydrochloride
(89) To a suspension of 21-Bromo-ganaxolone (5.0 g, 12.2 mmol) and BOC L-valine (26.5 g, 10 eq.) in 500 mL of acetone was added trimethylamine (20 mL, 13 eq.). The resulting mixture was heated at 65° C. for 5.5 h. It was then cooled to RT and was concentrated. The residue was partitioned between dichloromethane and water. The aqueous layer was extracted twice with dichloromethane. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. Biotage purification 20-80% EtOAc in hexane provided 5.59 g of the desired product (84% yield).
(90) The above product was dissolved in 200 mL of methanol and 34 mL of 3N HCl in methanol was added. The mixture was stirred at RT for 72 h and was concentrated. The product was purified by reverse phase Biotage C-18 column with 10-90% CH.sub.3CN in 0.1% TFA in H.sub.2O. Pure fractions were collected and 7 mL of 3N HCl in MeOH was added. Lyophilization afforded 3.46 g of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl valinate hydrochloride. .sup.1H NMR (400 MHz, CD3OD): δ 5.00 (d, J=17.0 Hz, 1H), 4.76 (d, J=16.9 Hz, 1H), 4.02 (d, J=4.4 Hz, 1H), 2.60 (t, J=8.8 Hz, 1H), 2.37 (pd, J=7.0, 4.4 Hz, 1H), 2.13 (q, J=11.6, 10.7 Hz, 1H), 2.06-1.97 (m, 1H), 1.80-1.58 (m, 4H), 1.58-1.28 (m, 5H), 1.22 (dd, J=10.8, 3.2 Hz, 4H), 1.15 (d, J=4.2 Hz, 3H), 1.14 (t, J=2.1 Hz, 7H), 1.10-0.78 (m, 1H), 0.76 (s, 4H), 0.62 (s, 3H). MS: M+1, 448.32; 2M+1, 895.13. UV: 201, 280 nM. HPLC: Sunfire C18 5 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 40% Acetonitrile in H.sub.2O (0.1% TFA) over 30 min; retention time 6.31 min; 94.4% pure. Solubility: 2 mg/mL in pH=7 phosphate buffer.
(91) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone Lysine. The method of making 21-OH Ganaxolone Lysine from 21-Bromo Ganaxolone is shown below in Route 3.
(92) Route 3
(93) ##STR00038##
Referring to Route 3, Synthesis of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 2,5-diaminopentanoate dihydrochloride
(94) To a suspension of 21-Bromo-ganaxolone (5.5 g, 13.4 mmol) and bis-BOC L-lysine (23.4 g, 5 eq.) in 500 mL of acetone was added trimethylamine (11 mL, 6.5 eq.). The resulting mixture was heated at 65° C. for 3 h. It was then cooled to RT and was concentrated. The residue was partitioned between dichloromethane and water. The aqueous layer was extracted twice with dichloromethane. The combined organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. Biotage purification 20-80% EtOAc in hexane provided 8.8 g of the desired product (97% yield).
(95) The above product was dissolved in 300 mL of methanol and 43 mL of 3N HCl in methanol (10 eq.) was added. The mixture was heated at 55° C. for 2.5 h. It was cooled and was concentrated. The product was purified by reverse phase Biotage C-18 column with 10-90% CH3CN in 0.1% TFA in H.sub.2O. Pure fractions were collected and 9 mL of 3N HCl in MeOH was added. Lyophilization afforded 3.5 g (57%). NMR: 1H (400 MHz, Methanol-d4) δ 5.06 (d, J=17.3 Hz, 1H), 4.75 (d, J=17.3 Hz, 1H), 4.17 (t, J=6.2 Hz, 1H), 3.02-2.93 (m, 2H), 2.62 (t, J=8.7 Hz, 1H), 2.05 (dddd, J=29.6, 18.1, 13.1, 6.8 Hz, 4H), 1.84-1.60 (m, 8H), 1.60-1.27 (m, 5H), 1.22 (dt, J=12.4, 5.0 Hz, 4H), 1.14 (d, J=1.0 Hz, 3H), 1.06-0.92 (m, 1H), 0.83 (dd, J=14.5, 10.8 Hz, 1H), 0.76 (d, J=1.1 Hz, 3H), 0.61 (s, 3H). MS: M+1, 477.36; 2M+1, 953.08. UV: 201, 281 nM. HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 207 nm; solvent 10-90% Acetonitrile in H.sub.2O (0.1% TFA) over 20 min; retention time 11.08 min; 96.1% pure. Solubility: 9.7 mg/mL. Stability: 75% prodrug remain after 24 h in PBS (pH=7) at the solubility (9.7 mg/mL) concentration.
(96) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone Phosphate Disodium. The method of making 21-OH Ganaxolone Phosphate Disodium from 21-Bromo Ganaxolone is shown below in Route 4.
(97) Route 4
(98) ##STR00039##
Referring to Route 4, Synthesis of bis-sodium 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl phosphate
(99) The mixture of 21-bromo-ganaxolone (0.82 g, 1.99 mmol), dibenzyl phosphate (1.66 g, 3 eq.) and trimethylamine (0.83 mL, 3.3 eq.) in 26 mL of THF was heated at 80° C. for 3 h. It was cooled and was concentrated. Biotage purification (10-80% EtOAc in hexane) afforded the desired product (0.675 g, 56% yield). LCMS (M+1, 609.38). 1H NMR (400 MHz, Chloroform-d3) δ 7.41-7.27 (m, 10H), 5.21-4.98 (m, 4H), 4.57-4.32 (m, 2H), 2.44 (t, J=8.8 Hz, 1H), 2.15 (q, J=11.6, 10.7 Hz, 1H), 1.81 (d, J=9.0 Hz, 1H), 1.66 (dd, J=11.4, 6.0 Hz, 3H), 1.52 (d, J=18.7 Hz, 7H), 1.42-1.22 (m, 3H), 1.20 (d, J=1.4 Hz, 3H), 1.18-1.01 (m, 1H), 0.94 (qd, J=11.7, 11.2, 5.0 Hz, 1H), 0.83-0.75 (m, 1H), 0.74 (s, 3H), 0.59 (s, 3H).
(100) The above product (0.675 g, 1.11 mmol) was dissolved in 18 mL of MeOH and 10 mL of THF. To it was added 118 mg of 10% Pd/C (0.1 eq.). The mixture was degassed and was stirred under H2 balloon for 2.5 h at RT. It was filtered through CELITE®, washed with dichloromethane and concentrated to give 0.48 g of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl phosphate, which was dissolved in 100 mL of MeOH and 30 mL of H.sub.2O and cooled in an ice bath. 2 eq. of NaHCO3 (0.19 g) in 10 mL was dropwise added and pH was monitored for the change of 3 to 7. It was stirred for 10 min after addition. Volatiles were evaporated and the aqueous solution was lyophilized to give 0.52 g of bis-sodium 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl phosphate. NMR: 1H (400 MHz, Methanol-d4) δ 4.53 (dd, J=20.1, 14.2 Hz, 2H), 2.57-2.75 (m, 1H), 2.22 2.07 (m, 1H), 2.02 (d, J=11.7 Hz, 1H), 1.77-1.59 (m, 5H), 1.59-1.27 (m, 7H), 1.23 (d, J=10.7 Hz, 6H), 1.14 (s, 3H), 1.00 (q, J=10.8, 9.6 Hz, 1H), 0.86-0.79 (m, 1H), 0.77 (s, 3H), 0.65 (d, J=1.8 Hz, 3H). MS: M−1, 427.27; 2M−1, 855.21; M+1, 429.11; 2M+1, 857.18. UV: 201, 280 nM. HPLC: Sunfire C18 5 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 40% Acetonitrile in H.sub.2O (0.1% TFA) over 60 min; retention time 8.3 min; 95% pure. Solubility: >10 mg/mL in PBS (pH=7.4). Stability: No decomposition observed at 26 h in PBS (pH=7.4).
(101) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone Succinate Sodium. The method of making 21-OH Ganaxolone Succinate Sodium from 21-Bromo Ganaxolone is shown below in Route 5.
(102) Route 5
(103) ##STR00040##
Referring to Route 5, synthesis of sodium 4-(2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-4-oxobutanoate
(104) 21-Bromoganaxolone (0.6 g, 1.46 mmol) and succinic acid (2.76 g, 16 eq.) were suspended in 65 mL of acetone and 2.4 mL of triethyl amine (13 eq.). The mixture was heated at 65° C. for 2 h. Volatiles were removed and the residue was partitioned between water and DCM. The aqueous layer was extracted twice and the combined organic layer was washed with brine and dried (Na.sub.2SO.sub.4). Biotage purification with 1-10% MeOH in (45% EtOAc in DCM) provided 0.44 g of product (96% pure), which was dissolved in 20 mL of methanol at 0° C. To it was added 0.079 g of NaHCO.sub.3 in 5 mL of H.sub.2O also cooled at 0° C. dropwise until pH reached to 7. Freeze-dry afforded 0.39 g of sodium 4-(2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-4-oxobutanoate.
(105) NMR: 1H (400 MHz, Methanol-d4) δ 4.75 (dd, J=17.1, 0.9 Hz, 1H), 4.56 (dd, J=17.0, 1.7 Hz, 1H), 2.68-2.58 (m, 2H), 2.46 (dd, J=7.6, 6.5 Hz, 2H), 2.16-1.97 (m, 2H), 1.66 (ddt, J=18.3, 14.5, 4.6 Hz, 5H), 1.58-1.26 (m, 9H), 1.20 (td, J=9.2, 8.1, 2.7 Hz, 5H), 1.13 (s, 3H), 0.98 (ddd, J=23.4, 18.5, 10.7 Hz, 1H), 0.83-0.78 (m, 1H), 0.76 (s, 3H), 0.61 (s, 3H).
(106) MS: Negative ion mode: 2M−1, 895.44; positive ion mode: M−18+1, 431.12; 2M+23, 919.18. UV: 201, 280 nM. HPLC: Sunfire C18 5 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 50% Acetonitrile in H.sub.2O (0.1% TFA) over 60 min; retention time 15.5 min; 94% pure. Solubility: 0.96 mg/mL. Stability: 98% remaining after 24 h in pH=7 phosphate buffer.
(107) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone Malic Acid. The method of making 21-OH Ganaxolone Malic Acid from 21-Bromo Ganaxolone is shown below in Route 6.
(108) Route 6
(109) ##STR00041##
Referring to Route 6, synthesis of 2-hydroxy-4-(2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)-4-oxobutanoic acid
(110) The mixture of 21-bromoganaxolone (300 mg, 0.73 mmol), (R)-(−)-2,2-dimethyl-5-oxo-1,3-dioxolane-4-acetic acid (1.3 g, 7.3 mmol, 10 eq.) and triethyl amine (1.2 mL, 13 eq.) in 30 mL of acetone was heated at 65° C. for 3 h. It was then concentrated and partitioned between water and DCM. The aqueous layer was extracted with DCM three times and the combined organic layer was washed with brine and dried (Na.sub.2SO.sub.4). Biotage purification with 20-80% EtOAc in hexane afforded 345 mg of the protected product, which was dissolved in 20 mL of HOAc, 5 mL of THF and 10 mL of water. The resulting mixture was heated at 45 degree for 5 h. Volatiles were removed and water was evaporated by freeze-drying. Purification on a reverse phase Biotage with MeCN and 0.1% TFA in H.sub.2O gave 251 mg of the final product.
(111) NMR: 1H (400 MHz, Methanol-d4) δ 4.83 (d, J=17.1 Hz, 1H), 4.63 (d, J=17.1 Hz, 1H), 4.52 (dd, J=8.0, 4.1 Hz, 1H), 2.95 (dd, J=16.1, 4.0 Hz, 1H), 2.78 (dd, J=16.0, 7.9 Hz, 1H), 2.63 (t, J=8.9 Hz, 1H), 2.14 (q, J=11.0, 10.3 Hz, 1H), 2.03 (dt, J=12.1, 3.3 Hz, 1H), 1.68 (ddd, J=21.9, 10.2, 4.0 Hz, 5H), 1.60-1.27 (m, 8H), 1.22 (td, J=9.5, 7.7, 2.8 Hz, 5H), 1.15 (s, 3H), 1.06-0.87 (m, 1H), 0.82 (dd, J=11.7, 4.1 Hz, 1H), 0.78 (s, 3H), 0.63 (s, 3H).
(112) MS: M+NH4, 482.22; 2M+1, 929.17.
(113) UV: 205, 284 nM
(114) HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 207 nm; solvent 10-90% Acetonitrile in H.sub.2O (0.1% TFA) over 20 min; retention time 16.5 min; 96.6% pure for batch 1 & 96.0% for batch 2. Solubility: 0.406 mg/mL in PBS (pH=7.4). Stability: 100% remaining after 24 h in PBS (pH=7.4).
(115) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone Piperdine Diol. The method of making 21-OH Ganaxolone Piperdine Diol from 21-Bromo Ganaxolone is shown below in Route 7.
(116) Route 7
(117) ##STR00042##
Referring to Route 7, synthesis of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 1-(2,3-dihydroxypropyl)piperidine-4-carboxylate hydrochloride
(118) Synthesis of lithium 1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)piperidine-4-carboxylate was accomplished in two steps. Step 1: to a solution of (2,2-dimethyl-1,3-dioxolan-4-yl)methanol (2.84 g, 21.5 mmol) in 50 mL of dichloromethane and 5.4 mL of triethylamine (2 eq.) at 0 degree was added methanesulfonyl chloride (1.2 eq. 2 mL). The resulting mixture was stirred at 0° C. for 5 min and at RT for 1 h. Volatiles were removed and the residue was dissolved in 30 mL of MeCN. To this solution were also added triethylamine (5.4 mL, 39 mmol) and ethyl 4-piperidinecarboxylate (3 mL, 19.5 mmol). The resulting mixture was heated at 80 degree for 2 h 15 min. It was cooled to RT and concentrated. Ethyl 1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)piperidine-4-carboxylate was purified on a Biotage column using 2-20% (10% NH4OH in MeOH) in DCM to afford 2.57 g (49% overall yield); Step 2: 2.57 g of ethyl 2-(2,2-dimethyl-5-oxo-1,3-dioxolan-4-yl)acetate (9.5 mmol) was stirred with LiOH—H.sub.2O (250 mg, 1.1 eq.) in 6 mL of THF, 6 mL of water and 3 mL of MeOH at RT for 5 h. Volatiles were removed and the residue was dried under high vacuum to give 2.8 g of lithium 1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)piperidine-4-carboxylate.
(119) 21-Bromoganaxolone (590 mg, 1.44 mmol) and 1.75 g (5 eq.) of lithium 1-(2,2-dimethyl-1,3-dioxolan-4-yl)methyl)piperidine-4-carboxylate were dissolved in 30 mL of acetone and heated at 65° C. for 10 h. Volatiles were removed and the residue was partitioned between water and ethyl acetate. The aqueous layer was extracted with ethyl acetate and the combined organic layer was washed with brine and dried over Na.sub.2SO.sub.4. Purification with 1-10% MeOH in DCM on a Biotage column afforded 481 mg of the desired product, 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)piperidine-4-carboxylate. 481 mg of the above product, 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 1-((2,2-dimethyl-1,3-dioxolan-4-yl)methyl)piperidine-4-carboxylate (0.84 mmol) was dissolved in 60 mL of MeOH. To it was added p-toluenesulfonic acid (2 eq. 319 mg). The resulting mixture was heated at 40° C. for 2 h. The mixture was neutralized with NaHCO.sub.3 (2 eq.) and was concentrated. Purification on a Biotage column with 2-10% MeOH in 1% NH.sub.4OH in DCM gave 384 g of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 1-(2,3-dihydroxypropyl)piperidine-4-carboxylate, which was mixed with 2 eq. 3N HCl in methanol and concentrated under vacuum. The final product, 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl 1-(2,3-dihydroxypropyl)piperidine-4-carboxylate hydrochloride, 410 mg was obtained in 97.4% purity.
(120) NMR: 1H (400 MHz, Methanol-d4) δ 7.77-7.65 (m, 4H), 7.23 (d, J=8.1 Hz, 4H), 4.68 (d, J=17.1 Hz, 1H), 4.58 (s, 2H), 4.01 (dtd, J=10.1, 5.1, 3.2 Hz, 1H), 3.60-3.36 (m, 2H), 3.24-2.97 (m, 3H), 2.81 (s, 1H), 2.61 (t, J=8.9 Hz, 1H), 2.37 (s, 5H), 2.24-1.97 (m, 6H), 1.79-1.58 (m, 4H), 1.58-1.28 (m, 6H), 1.24 (dt, J=10.6, 2.7 Hz, 4H), 1.15 (s, 3H), 0.98 (dt, J=17.7, 11.6 Hz, 1H), 0.83 (dd, J=13.0, 8.7 Hz, 1H), 0.78 (s, 3H), 0.63 (s, 3H). MS: M+1, 534.5 for free base parent compound. UV: 204, 282 nM. HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 207 nm; solvent 10-90% Acetonitrile in H.sub.2O (0.1% TFA) over 20 min; retention time 13.5 min; 97.4%. Solubility: 0.48 mg/mL in pH=7 phosphate buffer. Stability: 100% remaining after 24 h in pH=7 phosphate buffer.
(121) In another embodiment, the disclosure provides a method for using 21-Bromo ganaxolone to make 21-OH Ganaxolone methyl phosphite sodium salt. The method of making 21-OH Ganaxolone methyl phosphite sodium salt from 21-Bromo Ganaxolone is shown below in Route 8.
(122) Route 8
(123) ##STR00043##
Referring to Route 8, synthesis of dibenzyl ((2-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)methyl)phosphonate
(124) The mixture of dibenzyl phosphite (4.11 g, 15.7 mmol), paraformaldehyde (0.517 g, 1.1 eq.) and triethyl amine (0.2 mL, 0.1 eq.) was heated in a capped vial at 130° C. for 25 min. The mixture was then cooled and purified on a Biotage column with 5-70% EtOAc in DCM to give 3.27 g of dibenzyl (hydroxymethyl)phosphonate (71% yield).
(125) Dibenzyl (hydroxymethyl)phosphonate (0.99 g, 3.39 mmol) was dissolved in 7 mL of THF and cooled to −75° C. NaH (60% in oil, 203 mg, 1.5 eq.) was added and the mixture was stirred at −75° C. for 4 h. 21-Bromoganaxolone (2.79 g, 6.78 mmol, 2 eq.) was then added. The mixture was allowed to warm to RT slowly overnight while stirring. The reaction was quenched with NH.sub.4Cl (sat.) and was diluted with EtOAc. The organic layer was washed with brine and was dried over Na.sub.2SO.sub.4. Biotage purification with 2-80% EtOAc in hexane gave 1 g of the product, dibenzyl ((2-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)methyl)phosphonate (47%).
(126) .sup.1H NMR (400 MHz, DMSO-d6) δ 7.33 (q, J=3.9, 3.4 Hz, 10H), 5.13-4.94 (m, 4H), 4.27-4.04 (m, 2H), 4.04-3.90 (m, 2H), 3.84 (s, 1H), 2.51 (d, J=9.0 Hz, 1H), 2.02-1.86 (m, 1H), 1.76 (d, J=11.7 Hz, 1H), 1.64-1.39 (m, 6H), 1.38-1.04 (m, 13H), 1.02 (s, 3H), 0.91-0.68 (m, 1H), 0.64 (s, 3H), 0.44 (s, 3H).
(127) LCMS: M+1, 623.32.
Synthesis of bis-sodium ((2-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)methyl)phosphonate
(128) Dibenzyl ((2-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethoxy)methyl)phosphonate (0.627 g, 1 mmol) was stirred under H2 balloon in 11 mL of MeOH and 107 mg of 10% Pd/C (0.1 eq.) at RT for 2 h. The mixture was diluted with MeOH and filtered through CELITE®. The filtrate was concentrated to give 417 mg of white solid, which was dissolved in 5 mL of MeOH and cooled in an ice bath. NaHCO.sub.3 (0.1 M, 20 mL, 2 eq.) was dropwise added at 0° C. until pH=7. MeOH was removed under rotary evaporator and water was lyophilized to give 451 mg of the product was sodium salt.
(129) NMR: 1H (400 MHz) (CD3OD): δ 4.26 (s, 2H), 3.52 (dd, J=9.2, 2.6 Hz, 2H), 3.31 (d, J=12.9 Hz, 1H), 2.55 (t, J=8.7 Hz, 1H), 2.12 (t, J=10.6 Hz, 1H), 1.92 (d, J=11.2 Hz, 1H), 1.78-1.15 (m, 17H), 1.13 (s, 3H), 0.83-0.71 (m, 1H), 0.97 (t, J=9.5 Hz, 1H), 0.76 (s, 3H), 0.69-0.51 (m, 1H), 0.63 (s, 3H). UV: Absorbances at 207 nm. MS: 443.1 (M+1) as free acid. HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 212 nm; solvent 50% Acetonitrile in H.sub.2O (0.1% formic acid) over 15 min; retention time 5.21 min in; 100% purity. Solubility: 7.3 mg/mL in pH=7.4 PBS. Stability: no decomposition observed over 24 h in pH=7.4 PBS.
(130) In another embodiment, the disclosure provides a method for using 21-OH ganaxolone to make 21-OH Ganaxolone Carbamate. The method of making 21-OH Ganaxolone Carbamate from 21-OH Ganaxolone is shown below in Route 9.
(131) Route 9
(132) ##STR00044##
Referring to Route 9, Synthesis of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl (4-nitrophenyl) carbonate
(133) ##STR00045##
(134) 21-Hydroxyganaxolone (0.5 g, 1.49 mmol) was dissolved in 24 mL of THF. To it were added p-nitrophenol chloroformate (349 mg, 1.2 eq.) and DMAP (228 mg, 1.3 eq.). The resulting mixture was stirred at RT overnight and was quenched with water. EtOAc was used to extract and the organic layer was washed with water, brine and dried (Na.sub.2SO.sub.4). Biotage purification (20-80% EtOAc in hexane) gave 545 mg of the product (74% yield).
(135) 1H NMR (400 MHz, Chloroform-d) δ 8.40-8.16 (m, 2H), 7.49-7.35 (m, 2H), 4.85 (d, J=16.8 Hz, 1H), 4.69 (d, J=16.8 Hz, 1H), 2.51 (t, J=8.9 Hz, 1H), 2.28-2.15 (m, 1H), 2.02-1.93 (m, 1H), 1.78-1.59 (m, 3H), 1.59-1.43 (m, 8H), 1.43-1.32 (m, 2H), 1.32-1.22 (m, 3H), 1.20 (s, 3H), 1.19-1.10 (m, 2H), 1.03-0.90 (m, 1H), 0.85-0.76 (m, 1H), 0.74 (s, 3H), 0.66 (s, 3H).
Synthesis of 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)-2-oxoethyl (2-(piperidin-1-yl)ethyl)carbamate
(136) ##STR00046##
(137) 2-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta [a]phenanthren-17-yl)-2-oxoethyl (4-nitrophenyl) carbonate (300 mg, 0.585 mmol) and 1-(2-Aminoethyl)piperidine (0.25 mL, 3 eq.) were stirred in 15 mL of THF at RT for 2 h. Volatiles were removed and Biotage purification with 10% (10% NH.sub.4OH in MeOH)) in DCM (isocratic) gave 220 mg of the product.
(138) NMR: 1H (400 MHz) (CD3OD): δ 5.47 (s, 2H), 4.51 (d, J=9.6 Hz, 1H), 4.12 (d, J=10 Hz, 1H), 3.47 (d, J=16 Hz, 1H), 3.25 (m, 1H), 2.69 (br. 1H), 2.5 (m, 2H), 2.25-2.4 (m, 4H), 1.95 (t, J=9.6 Hz, 1H), 1.2-1.7 (m, 19H), 1.13 (s, 3H), 0.95 (m, 1H), 0.77 (s, 3H), 0.72 (s, 3H). MS: M+1, 503.45. UV: 202, 283 nM. HPLC: Sunfire C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 40% Acetonitrile in H.sub.2O (0.1% TFA) over 60 min; retention time 7.37 min; 100% pure. Solubility: 0.12 mg/mL at pH=7.4; 1.1 mg/mL at pH=6 of PBS. Stability: Decomposition at pH=6 was visible, but not quantified.
(139) In one embodiment, the disclosure provides a method for using ganaxolone to make UCI-50027 and other intermediary compounds which are useful for preparing UCI-50027 derivatives. The method of making UCI-50027 is shown below in Route 10.
(140) Route 10:
(141) ##STR00047##
Referring to Route 10, Synthesis of (3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carboxylic acid
(142) ##STR00048##
(143) 21-OH-ganaxolone (4.38 g, 12.6 mmol) and NaIO4 (13.44 g, 5 eq.) were heated in 47 mL of H.sub.2O and 160 mL of THF at 50° C. for 18 h. It was acidified with 1 N HCl (160 mL) and the product was extracted with ethyl acetate three times. The organic layer was washed with brine and dried over Na.sub.2SO.sub.4. Concentration gave a yellow solid, which was washed with hexane and DCM. An off-white solid was obtained in 88% yield. 1H NMR (400 MHz, DMSO-d6) δ 11.85 (s, 1H), 3.83 (s, 1H), 2.24 (t, J=9.2 Hz, 1H), 1.98-1.83 (m, 2H), 1.73-1.40 (m, 4H), 1.40-1.05 (m, 14H), 1.03 (s, 3H), 0.96-0.78 (m, 1H), 0.73-0.62 (m, 1H), 0.68 (s, 3H), 0.59 (s, 3H).
Synthesis of (3R,8R,10S,13S,14S,17S)-17-(hydroxymethyl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol
(144) ##STR00049##
(145) To a solution of (3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carboxylic acid (3.70 g, 11.1 mmol) in 220 mL of THF at 0° C. was added LAH (0.842 g, 22.2 mmol, 2 eq.) slowly. The mixture was stirred and was allowed to warm to RT, then was heated at 65° C. for 3 h. After it was cooled to RT, the reaction was quenched by adding 0.84 mL of H.sub.2O, 0.84 mL of 15% NaOH and 2.52 mL of H.sub.2O. The resulting suspension was diluted with EtOAc and stirred at RT for 30 min. It was filtered through a CELITE® pad and washed with EtOAc. The filtrate was concentrated to give quantitative amount of (3R,8R,10S,13S,14S,17S)-17-(hydroxymethyl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (3.68 g).
(146) 1H NMR (400 MHz, DMSO-d6) δ 4.19-4.09 (m, 1H), 3.82 (s, 1H), 3.48-3.36 (m, 1H), 3.29-3.19 (m, 1H), 1.86-1.77 (m, 1H), 1.77-1.63 (m, 1H), 1.63-1.38 (m, 5H), 1.38-1.05 (m, 14H), 1.03 (d, J=0.9 Hz, 3H), 1.00-0.76 (m, 1H), 0.74-0.61 (m, 1H), 0.68 (s, 3H), 0.54 (s, 3H).
Synthesis of (3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde
(147) ##STR00050##
(148) (3R,8R,10S,13S,14S,17S)-17-(hydroxymethyl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (0.64 g, 2 mmol) was dissolved in 30 mL of DCM. To it were added PCC (0.646 g, 1.5 eq.) and 2 g of CELITE®. After the resulting mixture was stirred at RT for 3 h, it was filtered through a silica-gel pad and washed with DCM and EtOAc. The filtrate was concentrated to give 0.623 g of (3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde in 98% yield.
(149) 1H NMR (399 MHz, Chloroform-d) δ 9.76 (s, 1H), 2.29 (t, J=8.9 Hz, 1H), 2.16-1.90 (m, 2H), 1.79-1.32 (m, 11H), 1.33-1.07 (m, 7H), 1.20 (s, 3H), 1.07-0.88 (m, 1H), 0.86-0.76 (m, 1H), 0.75 (s, 3H).
Synthesis of (3R,8R,10S,13S,14S,17S)-17-(5-(hydroxymethyl)isoxazol-3-yl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (UCI-50027)
(150) ##STR00051##
(151) The mixture of (3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde (0.623 g, 1.96 mmol), Na.sub.2CO.sub.3 (1.5 eq. 312 mg) and NH.sub.2OH—HCl (204 mg, 1.5 eq.) in 30 ml of EtOH and 10 ml of H.sub.2O were stirred at RT overnight. The mixture was then partitioned between water and EtOAc. The organic layer was washed with brine and dried over Na.sub.2SO.sub.4. Concentration gave 0.584 g of solid, (E)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde oxime in 89%.
(152) The mixture of (E)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde oxime (1.69 g, 5.07 mmol), NCS (N-Chlorosuccimide, 0.71 g, 1.05 eq.) and pyridine (20 drops) in 100 mL of DCM was stirred at RT for 1.5 h. Propyargyl alcohol (1.6 mL, 5.4 eq.) and diisopropylethylamine (0.97 mL 1.1 eq.) were then added. The resulting mixture was stirred at RT overnight. The reaction mixture was diluted with DCM, washed with NaHCO.sub.3 (sat.), brine and dried over Na.sub.2SO.sub.4. Biotage purification with 0-80% EtOAc in hexane gave (3R,8R,10S,13S,14S,17S)-17-(5-(hydroxymethyl)isoxazol-3-yl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (UCI-50027) 1.13 g of in 58% yield.
(153) 1H NMR (400 MHz, DMSO-d6) δ 6.20 (d, J=0.8 Hz, 1H), 5.54 (tt, J=6.0, 0.9 Hz, 1H), 4.49 (dd, J=6.0, 0.8 Hz, 2H), 3.84 (s, 1H), 2.65 (t, J=9.7 Hz, 1H), 2.07-1.97 (m, 1H), 1.96-1.84 (m, 1H), 1.66 (dd, J=15.8, 10.8 Hz, 4H), 1.52 (d, J=13.3 Hz, 2H), 1.33 (t, J=7.0 Hz, 4H), 1.30-1.08 (m, 8H), 1.04 (s, 3H), 0.91 (dt, J=21.4, 11.2 Hz, 1H), 0.78-0.71 (m, 1H), 0.68 (s, 3H), 0.47 (s, 3H). UV: Absorbances at 214.2 nm.
(154) TLC: (Silica Gel plates) 40% EtOAc/Hexane; Rf=0.35. HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 210 nm; solvent 55% Acetonitrile in H.sub.2O (0.1% formic acid) over 30 min; retention time 12.45 min; 99.6%. Solubility: <1 μg/mL
(155) In one embodiment, the disclosure provides a method for using UCI-50027 to make UCI-50027 Valine Citric salt. The method of making UCI-50027 Valine Citric salt is shown below in Route 11.
(156) Route 11:
(157) ##STR00052##
Referring to Route 11, synthesis of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl L-valinate 2-hydroxypropane-1,2,3-tricarboxylic acid
(158) To a solution of (3R,8R,10S,13S,14S,17S)-17-(5-(hydroxymethypisoxazol-3-yl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (UCI-50027) (285 mg, 0.735 mmol) in 8 mL of THF and triethyl amine (0.15 mL, 1.5 eq.) at 0° C. was added methanesulfonyl chloride (0.063 mL, 1.1 eq.). After the resulting mixture was stirred at 0° C. for 1 h, it was diluted with EtOAc. The organic layer was washed with brine and dried over Na.sub.2SO.sub.4. The residue after concentration was dissolved in 8 mL of acetone. To it were added BOC-L-valine (1.60 g, 10 eq.) and triethyl amine (1.33 mL, 13 eq.). The resulting mixture was heated at 65° C. for 2.5 h. It was cooled and the volatiles were removed. The residue was diluted with DCM and was washed with brine and dried over Na.sub.2SO.sub.4. Biotage purification with 0-60% EtOAc in hexane gave 316 mg of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl (tert-butoxycarbonyl)-L-valinate (73%).
(159) The above product (316 mg) was stirred with 3M of HCl in 5 mL of MeOH (10 eq.) at RT for 24 h. The mixture was purified on a C18 reverse phase Biotage column with 0-50% and 50-100% MeOH in 0.1% HCOOH in H.sub.2O to give 161 mg of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl L-valinate, which was dissolved in 2 mL of MeOH and mixed with 1 eq. of 2M citric acid solution (0.166 ml). Lyophilization provided 208 mg of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl L-valinate 2-hydroxypropane-1,2,3-tricarboxylic acid. 1H NMR (400 MHz, DMSO-d6) δ 6.48 (s, 1H), 5.38 (d, J=13.6 Hz, 1H), 5.30 (d, J=13.7 Hz, 1H), 3.87 (d, J=4.7 Hz, 1H), 3.84 (s, 1H), 2.69 (t, J=9.7 Hz, 1H), 2.62 (d, J=15.3 Hz, 2H), 2.53 (d, J=15.3 Hz, 2H), 2.12-1.97 (m, 2H), 1.97-1.80 (m, 1H), 1.76-1.58 (m, 4H), 1.58-1.41 (m, 3H), 1.40-1.07 (m, 8H), 1.04 (s, 3H), 0.99-0.76 (m, 5H), 0.89 (dd, J=8.9, 6.9 Hz, 6H), 0.85-0.78 (m, 1H), 0.68 (s, 3H), 0.45 (s, 3H). UV: Absorbances at 213 nm. MS: 487.44 (M+1), 973.29 (2M+1). HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 222 nm; solvent 50% Acetonitrile in H.sub.2O (0.1% formic acid) over 16 min; retention time 8.10 min in; 99.9% purity. Solubility: 58 μg/mL. Stability: 100% parent remaining after 24 h in PBS (pH=7.4).
(160) In one embodiment, the disclosure provides a method for using UCI-50027 to make UCI-50027 Lysine bis-citric acid salt. The method of making UCI-50027 Lysine bis-citric acid salt is shown below in Route 12.
(161) Route 12:
(162) ##STR00053##
Referring to Route 12, synthesis of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl L-lysinate bis-2-hydroxypropane-1,2,3-tricarboxylic acid
(163) To a solution of (3R,8R,10S,13S,14S,17S)-17-(5-(hydroxymethyl)isoxazol-3-yl)-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-3-ol (UCI-50027) (392 mg, 1.01 mmol) in 10 mL of THF and triethyl amine (0.21 mL, 1.5 eq.) at 0° C. was added methanesulfonyl chloride (0.086 mL, 1.1 eq.). After the resulting mixture was stirred at 0° C. for 1 h, it was diluted with EtOAc. The organic layer was washed with brine and dried over Na.sub.2SO.sub.4. The residue after concentration was dissolved in 10 mL of acetone. To it were added bis-BOC-L-lysine (3.50 g, 10 eq.) and triethyl amine (1.8 mL, 13 eq.). The resulting mixture was heated at 65° C. for 3 h. It was cooled and the volatiles were removed. The residue was diluted with DCM and was washed with brine and dried over Na.sub.2SO.sub.4. Biotage purification with 0-60% EtOAc in hexane gave 639 mg of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazole-5-yl)methyl N2,N6-bis(tert-butoxycarbonyl)-L-lysinate (88%).
(164) The above product (639 mg) was stirred with 3M of HCl in 15 mL of MeOH (50 eq.) at RT for 24 h. The mixture was purified on a C18 reverse phase Biotage column with 0-50% and 50-100% MeOH in 0.1% HCOOH in H.sub.2O to give 110 mg of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl L-lysinate, which was dissolved in 2 mL of MeOH and mixed with 2 eq. of 2M citric acid solution (0.181 ml). Lyophilization provided 144 mg of (3-((3R,8R,10S,13S,14S,17S)-3-hydroxy-3,10,13-trimethyl hexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl L-lysinate bis-2-hydroxypropane-1,2,3-tricarboxylic acid. NMR: 1H NMR (400 MHz, DMSO-d6) δ 6.50 (s, 1H), 5.32 (d, J=1.7 Hz, 2H), 3.95 (d, J=6.6 Hz, 1H), 3.85 (s, 1H), 2.75-2.67 (m, 3H), 2.62 (d, J=15.2 Hz, 4H), 2.53 (d, J=15.2 Hz, 4H), 2.10-1.82 (m, 3H), 1.80-1.59 (m, 6H), 1.57-1.43 (m, 4H), 1.43-1.07 (m, 13H), 1.04 (s, 3H), 1.00-0.69 (m, 2H), 0.68 (s, 3H), 0.46 (s, 3H). UV: Absorbances at 214 nm. MS: 516.47 (M+1). HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 222 nm; solvent 38% Acetonitrile in H.sub.2O (0.1% formic acid) over 15 min; retention time 6.14 min in; 100% purity. Solubility: 5.2 mg/mL. Stability: 94.5% prodrug remaining after 24 h in PBS (pH=7) at RT.
(165) In one embodiment, the disclosure provides a method for using ganaxolone to make UCI-50027 MethylPhosphite Disodium. The method of making UCI-50027 MethylPhosphite Disodium is shown below in Route 13.
(166) Route 13:
(167) ##STR00054##
Referring to Route 13, synthesis of dibenzyl (((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methoxy)methyl)phosphonate
(168) ##STR00055##
(169) To a suspension of NaH (567 mg, 14.2 mmol) in 8 mL of THF at −78° C. was added dibenzyl (hydroxymethyl)phosphonate (2.76 g, 9.44 mmol) in 3 mL of THF dropwise. The resulting mixture was stirred at −78° C. for 5 h. Propargyl bromide (80% in toluene, 2.4 mL, 3 eq.) was then added and the mixture was stirred and was allowed to warm to RT overnight. It was quenched with NH4Cl (sat.) and diluted with EtOAc and water. The organic layer was washed with brine, dried over Na.sub.2SO.sub.4 and concentrated. Biotage purification with 0-80% EtOAc in hexane gave 1.49 g of dibenzyl ((prop-2-yn-1-yloxy)methyl)phosphonate in 48% yield.
(170) ##STR00056##
(171) The mixture of (E)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde oxime (346 mg, 1.04 mmol), NCS (N-Chlorosuccimide, 146 mg, 1.05 eq.) and pyridine (5 drops) in 21 mL of DCM was stirred at RT for 1 h. Dibenzyl ((prop-2-yn-1-yloxy)methyl)phosphonate (1.49 g, 4.3 eq. in 2 mL of DCM) and diisopropylethylamine (0.2 mL 1.1 eq.) were then added. The resulting mixture was stirred at RT overnight. The reaction mixture was diluted with DCM, washed with NaHCO.sub.3 (sat.), brine and dried over Na.sub.2SO.sub.4. Biotage purification with 0-80% EtOAc in hexane gave 318 mg of dibenzyl (((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthr en-17-yl)isoxazol-5-yl)methoxy)methyl)phosphonate in 46% yield. 1H NMR (400 MHz, DMSO-d6) δ 7.44-7.17 (m, 10H), 6.33 (s, 1H), 5.09-4.97 (m, 4H), 4.64 (s, 2H), 3.96 (d, J=8.5 Hz, 2H), 3.84 (s, 1H), 2.66 (t, J=9.6 Hz, 1H), 2.06-1.98 (m, 1H), 1.90 (dd, J=9.7, 5.5 Hz, 1H), 1.64 (dd, J=18.8, 8.4 Hz, 4H), 1.50 (d, J=12.1 Hz, 3H), 1.39-1.06 (m, 11H), 1.04 (s, 3H), 1.00-0.70 (m, 2H), 0.68 (s, 3H), 0.43 (s, 3H).
(172) ##STR00057##
(173) Dibenzyl (((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methoxy)methyl)phosphonate (318 mg, 0.481 mmol) was dissolved in 7 mL of THF. To it was added 10% Pd/C (51 mg, 0.1 eq.). The mixture was degassed under vacuum and flushed with H2. After it was stirred under H2 balloon for 2 h at RT, it was filtered through a CELITE® pad and washed with MeOH. The filtrate was concentrated and the residue was purified on a C18 reverse phase Biotage column using 0-100% MeOH in 0.1% formic acid water to give 216 mg (0.449 mL) of methylphosphonic acid, which was dissolved 2.5 mL of MeOH and cooled to 0° C. To it was added 2 eq. of NaHCO.sub.3 (0.1 M, 9 mL) and stirred (pH=7). The final sodium salt was obtained by lyophilization (250 mg). NMR: 1H NMR (400 MHz, Methanol-d4) δ 6.41 (s, 1H), 4.68 (s, 2H), 3.60 (d, J=9.0 Hz, 2H), 2.71 (t, J=9.7 Hz, 1H), 2.20-1.91 (m, 2H), 1.66-1.18 (m, 18H), 1.15 (s, 3H), 1.09-0.80 (m, 2H), 0.78 (s, 3H), 0.57 (s, 3H). UV: Absorbances at 214 nm. MS: 468.23 (M+1); 935.29 (2M+1). HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 222 nm; solvent 50% Acetonitrile in H.sub.2O (0.1% formic acid) over 15 min; retention time 6.38 min in; 99.3% purity. Solubility: 4.3 mg/mL. Stability: 98.3% prodrug remaining after 23 h in PBS (pH=7) at room temperature.
(174) In one embodiment, the disclosure provides a method for using ganaxolone to make UCI-50027 Diisopropyl Carbonate. The method of making UCI-50027 Diisopropyl Carbonate is shown below in Route 14.
(175) Route 14:
(176) ##STR00058##
Referring to Route 14, Synthesis of (((((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methoxy)methyl)phosphoryl)bis(oxy))bis(methylene) diisopropyl bis(carbonate)
(177) The mixture of (((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methoxy)methyl)phosphonic acid (260 mg, 0.54 mmol), tetrn-butylammonium bromide (174 mg, 1 eq.), triethyl amine (0.3 mL, 4 eq.) and chloromethyl isopropyl carbonate (0.34 mL, 5 eq.) in 0.64 mL of NMP was heated at 60° C. for 3 h. After it was cooled to RT, it was diluted with EtOAc and was washed with brine and dried over Na.sub.2SO.sub.4. Biotage purification with 0-70% EtOAc in hexane gave the desired product, (((((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methoxy)methyl)phosphoryl)bis(oxy))bis(m ethylene) diisopropyl bis(carbonate), 342 mg, 63%. NMR: 1H NMR (400 MHz, DMSO-d6) δ 6.38 (s, 1H), 5.58 (dd, J=12.6, 2.4 Hz, 4H), 4.79 (q, J=6.2 Hz, 2H), 4.64 (s, 2H), 3.99 (dd, J=16.6, 8.1 Hz, 2H), 3.83 (s, 1H), 2.65 (t, J=9.4 Hz, 1H), 2.07-1.77 (m, 2H), 1.74-1.26 (m, 8H), 1.26-1.20 (m, 10H), 1.21 (d, J=6.4 Hz, 12H), 1.02 (s, 2H), 0.98-0.62 (m, 2H), 0.66 (s, 3H), 0.44 (s, 3H). UV: Absorbances at 210 nm. MS: 714.31 (M+1). HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 212 nm; solvent 10%-100% Acetonitrile in H.sub.2O (0.1% formic acid) over 20 min; retention time 13.4 min in; 97.1% purity. Solubility: 6.6 μg/mL in pH=7.4 buffer. Stability: cannot be accurately measured due to the extremely low intensity of the peak, which is the result of extremely low solubility in PBS.
(178) In one embodiment, the disclosure provides a method for using ganaxolone to make UCI-50027 MethylPhosphite Disodium. The method of making UCI-50027 MethylPhosphite Disodium is shown below in Route 15.
(179) Route 15
(180) ##STR00059##
Referring to Route 15, Synthesis of bis-sodium (3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl phosphate
(181) Dibenzyl phosphonate (1.72 g, 6.56 mmol) and NCS (N-chlorosuccinimide, 0.963 g, 1.1 eq.) were stirred together in 35 mL of toluene at RT for 2.5 h. It was filtered and the filtrate was concentrated to give 2.15 g of dibenzyl phosphorochloridate in quantitative yield. It was used in the next step without any further purification.
(182) At 0° C., dibenzyl phosphorochloridate (1.94 g, 6.56 mmol, 1.3 eq.) was added to a solution of propargyl alcohol (0.29 mL, 5.05 mmol) and DMAP (81 mg, 0.1 eq.) in 65 mL of DCM. It was followed by the addition of triethyl amine (1.1 mL, 1.2 eq.). The mixture was stirred and was allowed to warm to RT overnight. It was then quenched with NH4Cl (sat.) and was diluted with DCM. The organic layer was washed with brine and dried (Na.sub.2SO.sub.4). Biotage purification with 0-60% EtOAc in hexane gave 0.553 g of dibenzyl prop-2-yn-1-yl phosphate (B) in 19% yield.
(183) The mixture of (E)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthrene-17-carbaldehyde oxime (181 mg, 0.543 mmol), NCS (N-Chlorosuccimide, 76 mg, 1.05 eq.) and pyridine (3 drops) in 11 mL of DCM was stirred at RT for 1 h. Dibenzyl prop-2-yn-1-yl phosphate (B) (553 mg, 2.2 eq. in 3 mL of DCM) and diisopropylethylamine (0.1 mL 1.1 eq.) were then added. The resulting mixture was stirred at RT overnight. The reaction mixture was diluted with DCM, washed with NaHCO.sub.3 (sat.), brine and dried over Na.sub.2SO.sub.4. Biotage purification with 0-70% EtOAc in hexane gave 240 mg of dibenzyl ((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl) phosphate in 68% yield.
(184) The mixture of 240 mg of dibenzyl ((3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl) phosphate (0.37 mmol) and 10% Pd/C (39 mg, 0.1 eq.) in 5 mL of MeOH was degassed under house vacuum and was flushed with hydrogen. After the mixture was stirred at RT under H2 balloon for 2 h, it was filtered through a CELITE® pad. The filtrate was concentrated to give 173 mg of dihydro-phosphate, which was converted to sodium salt by dissolving in 5 mL of MeOH and reacted with 2 eq. of NaHCO.sub.3 (0.1M, 7.4 mL) at 0° C. Lyophilization afforded 188 mg of bis-sodium (3-((3R,10S,13S,17S)-3-hydroxy-3,10,13-trimethylhexadecahydro-1H-cyclopenta[a]phenanthren-17-yl)isoxazol-5-yl)methyl phosphate. NMR: 1H NMR (400 MHz, DMSO-d6) δ 6.21 (s, 1H), 4.63 (d, J=6.6 Hz, 2H), 2.68-2.60 (m, 1H), 2.19-1.77 (m, 2H), 1.77-1.60 (m, 2H), 1.61-1.41 (m, 2H), 1.41-1.07 (m, 13H), 1.04 (s, 3H), 1.00-0.70 (m, 2H), 0.69 (s, 3H), 0.48 (s, 3H). UV: Absorbances at 214 nm. MS: 468.23 (M+1); 935.29 (2M+1). HPLC: X-Bridge C18 5μ 250×4.6 mm; flow 1.0 mL/min; Waters 996 PDA detection at 222 nm; solvent 50% Acetonitrile in H.sub.2O (0.1% formic acid) over 15 min; retention time 6.38 min in; 99.3% purity. Solubility: 4.3 mg/mL. Stability: 98.3% prodrug remaining after 23 h in PBS (pH=7) at room temperature.
(185) The following tables summarizes the amount, the purity, solubility and stability of the compounds described in these routes of synthesis.
(186) TABLE-US-00002 TABLE 2 # Name Amount Purity Solubility 1 Ganaxolone (GX) 522 mg 96.6% <BQL* 2 21-OH GX 290 mg 98.8% <BQL* 3 UCI-50027 356 mg 99.6% <1 μg/mL *BQL = below the quantification level
(187) TABLE-US-00003 TABLE 3 Stability.sup.2 # Name Amount Purity Solubility.sup.1 (24 h) 1 21-OH Gx Phosphate Disodium 545 mg .sup. 95% >10 mg/mL 100% 2 21-OH Gx Valine 335 mg 94.4% .sup. 2 mg/mL 75% (6 h) 3 21-OH Gx Lysine 305 mg 96.1% 9.7 mg/mL 75% 4 21-OH Gx Succinate Sodium 268 mg .sup. 94% 0.96 mg/mL 98% 5 21-OH Gx Malic Acid 300 mg 96.0% 0.41 mg/mL 100% 6 21-OH Gx Piperidine diol 393 mg 97.4% 0.48 mg/mL 100% 7 UCI-50027 Phosphate Disodium 120 mg 100% .sup. 5 mg/mL 100% 110 mg 100% 8 UCI-50027 Valine 170 mg 99.1% 58 μg/mL 100% 190 mg 99.9% 9 UCI-50027 Lysine 211 mg 100% 5.2 mg/mL 94.5% 10 UCI-50027 methylphosphite 210 mg 99.3% 4.3 mg/mL 98.3% sodium 11 UCI-50027 methylphosphite 243 mg 97.1% 6.6 μg/mL N.A..sup.3 diisopropyl carbonate 12 21-OH GX methylphosphite 368 mg 100% 7.3 mg/mL 100% sodium 13 21-OH GX carbamate 201 mg 100% 118 μg/mL N.D..sup.4 (pH = 7.4) .sup.1Solubility was measured in pH = 7 phosphate buffer. .sup.2Stability was measured in pH = 7 phosphate buffer at room temperature over 24 h time period and percentage was reported as how much a drug was remaining in the buffer solution at 24 h time point or otherwise specified time point. .sup.3Not available due to weak signal. .sup.4Not determined.
(188) It is apparent that comparison of Table 2 and Table 3 indicate that there is a significant improvement in solubility for the neurosteroid derivatives compared to GX, 21-OH GX and UCI-50027. Among all the compounds, bis-sodium phosphate or phosphite salts show the best solubility 4 to >10 mg/mL (entry 1, 7, 10). Lysine esters have good solubility as well, being associated with 2HCl in entry 3 or with 2 citric acid in entry 9, provided 9.7 mg/mL and 5.2 mg/mL solubility, respectively. Another case in point is entry #13 in Table 3. When the basic nitrogen was free base, the 21-OH GX carbamate has low solubility and the solubility increases by 10-fold when the nitrogen was protonated at pH=6.
(189) In summary, novel compounds from the pharmacophore template described in Formula (A) were prepared in various multi-step reactions from easily accessible pregnenolone, 21-Bromo GX, and 21-OH GX. The 21-hydroxyl group allows for simple substituents, such as amino analogs, alkyl ether derivatives, and other functional groups. Additionally, the 21-bromo-GX derivative provides a variety of options for modifying GX.
(190) It will be appreciated that the neurosteroid derivatives of the disclosure can be provided in the form of a base or an acid addition salt prepare from a pharmaceutically acceptable salt including those known in the art, or in the form of a hydrate or solvate.
(191) All of the compositions and methods disclosed and claimed herein can be made and executed without undue experimentation in light of the present disclosure. While the compositions and methods of this disclosure have been described in terms of preferred embodiments, it will be apparent to those of skill in the art that variations may be applied to the compositions and methods and in the steps or in the sequence of steps of the methods described herein without departing from the concept, spirit and scope of the disclosure. More specifically, it will be apparent that certain agents which are both chemically related may be substituted for the agents described herein while the same or similar results would be achieved. All such similar substitutes and modifications apparent to those skilled in the art are deemed to be within the spirit, scope and concept of the disclosure as defined by the appended claims.