Deuterated compounds for treating Fabry, Gaucher, Parkinson's and related diseases and conditions, and compositions and methods thereof
10519157 ยท 2019-12-31
Assignee
Inventors
- Hui Joyce Li (Westborough, MA, US)
- Shuhao Wen (Andover, MA, US)
- Changfu Cheng (Northborough, MA, US)
- Xiao Chen (Princeton, NJ, US)
- Gao Shang (Doylestown, PA, US)
Cpc classification
C07B2200/05
CHEMISTRY; METALLURGY
International classification
C07D453/00
CHEMISTRY; METALLURGY
Abstract
Novel deuterium-containing chemical compounds are provided. They are glucosylceramide (GSC) synthase inhibitors and are useful for treating various types of lysosomal storage diseases including Fabry's disease, Gaucher's disease and Parkinson's disease, or related diseases and conditions. Also described are pharmaceutical compositions, and methods of preparation and use thereof.
Claims
1. A compound of the following Formula (II): ##STR00014##
2. A compound of the following Formula (III): ##STR00015##
3. A compound of the following Formula (IV): ##STR00016##
Description
BRIEF DESCRIPTION OF THE DRAWINGS
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DETAILED DESCRIPTION OF THE INVENTION
I. Definitions
(14) Unless otherwise noted, technical terms are used according to conventional usage.
(15) As used herein, a or an may mean one or more. As used herein when used in conjunction with the word comprising, the words a or an may mean one or more than one. As used herein another may mean at least a second or more. Furthermore, unless otherwise required by context, singular terms include pluralities and plural terms include the singular.
(16) As used herein, about refers to a numeric value, including, for example, whole numbers, fractions, and percentages, whether or not explicitly indicated. The term about generally refers to a range of numerical values (e.g., +/5-10% of the recited value) that one of ordinary skill in the art would consider equivalent to the recited value (e.g., having the same function or result). In some instances, the term about may include numerical values that are rounded to the nearest significant figure.
(17) The term subject refers to any animal (e.g., a mammal), including non-human primates, rodents, and the like, which is to be the recipient of a particular treatment. Typically, the terms subject and patient are used interchangeably herein in reference to a human subject.
(18) The term effective as used in connection with an amount of an active agent, refers to an amount sufficient to elicit the desired biological response. As will be appreciated by those of ordinary skill in this art, the effective amount of a compound of the invention may vary depending on such factors as the desired biological endpoint, the pharmacokinetics of the compound, the disease being treated, the mode of administration, and the patient.
(19) The terms treating, reducing, or preventing a condition refer to ameliorating such a condition before or after it has occurred. As compared with an equivalent untreated control, such reduction or degree of prevention is at least 5%, 10%, 20%, 40%, 50%, 60%, 80%, 90%, 95%, or 100% as measured by any standard technique.
(20) The term pharmaceutically acceptable excipient, carrier, or diluent, as used herein, refers to a pharmaceutically acceptable material, composition or vehicle, such as a liquid or solid filler, diluent, excipient, solvent or encapsulating material, involved in carrying or transporting the subject pharmaceutical agent from one organ, or portion of the body, to another organ, or portion of the body. Each carrier must be acceptable in the sense of being compatible with the other ingredients of the formulation and not injurious to the patient. Some examples of materials which can serve as pharmaceutically-acceptable carriers include: sugars, such as lactose, glucose and sucrose; starches, such as corn starch and potato starch; cellulose, and its derivatives, such as sodium carboxymethyl cellulose, ethyl cellulose and cellulose acetate; dextrin and cyclodextrin (alpha, beta and gamma); powdered tragacanth; malt; gelatin; talc; excipients, such as cocoa butter and suppository waxes; oils, such as peanut oil, cottonseed oil, safflower oil, sesame oil, olive oil, corn oil and soybean oil; glycols, such as propylene glycol; polyols, such as glycerin, sorbitol, mannitol and polyethylene glycol; esters, such as ethyl oleate and ethyl laurate; agar; buffering agents, such as magnesium hydroxide and aluminum hydroxide; alginic acid; pyrogen-free water; isotonic saline; Ringer's solution; ethyl alcohol; phosphate buffer solutions; and other non-toxic compatible substances employed in pharmaceutical formulations. Wetting agents, emulsifiers and lubricants, such as sodium lauryl sulfate, magnesium stearate, and polyethylene oxide-polypropylene oxide copolymer as well as coloring agents, release agents, coating agents, sweetening, flavoring and perfuming agents, preservatives and antioxidants can also be present in the compositions.
II. The Present Invention
(21) The invention provides novel chemical compounds that may be used to treat lysosomal storage diseases (e.g., Gaucher, Fabry diseases). These compounds are biochemically potent and physiologically active, with improved pharmacokinetic, therapeutic and toxicological properties over ibiglustat. For comparison, ibiglustat was synthesized, tested and analyzed parallel to the compounds described in this invention. The mass spectrometric analytical results of ibiglustat are demonstrated in
(22) The compounds disclosed herein are deuterium-substituted versions of ibiglustat, where one or more hydrogen atoms are substituted with deuterium at strategic locations of the molecule. Three examples of such substitutions are shown in
(23) First-generation GCS inhibitor miglustat was approved by the FDA in 2003 to treat type I Gaucher disease when ERT was not an option and later for treating Niemann-Pick disease type C, another rare neurodegenerative, autosomal recessive lipid storage disease with mutations in the NPC1 gene that is involved in intracellular lipid trafficking (Platt, Boland, and van der Spoel 2012; Madra and Sturley 2010; Rosenbaum and Maxfield 2011). It is a small water soluble iminosugar molecule derived from the naturally occurring glucosidase inhibitor deoxynojirimycin (Lachmann and Platt 2001; Platt et al. 1994) that reversibly inhibits glycosphingolipid synthesis (GCS). Patients with type 1 Gaucher disease normally show good initial responses to oral Miglustat treatment with improved biochemical outcomes, and lowered spleen and liver volumes (Cox et al. 2000; Heitner et al. 2002). Most patients who respond to therapy, however, suffered severe diarrhea and weight loss in about 6 to 12 months of treatment. Side effects have also been identified including neurological symptoms, and reproduction toxicity. Thus, its less favorable efficacy, safety and tolerability profile limit its clinical usage ((Cox et al. 2012); (http://www.ema.europa.eu/docs/en_GB/document_library/EPAR_-_product_information/human/000435/WC500046726.pdf (2013)).
(24) Two-thirds of lysosomal storage disorders are accompanied with progressive neurological manifestations. Traditional enzyme replacement therapy failed to improve those neurological complications due to the blood-brain barrier. Miglustat did not show much improvement on this aspect as well. Second generation GSC inhibitor Eliglustat has been approved by FDA in 2017 as a first-line oral drug for treating adult type 1 Gaucher disease. It is a selective, more potent, enzyme-specific small molecular inhibitor of GCS (McEachern et al. 2007). Oral eliglustat is as effective as ERT in treating Gaucher disease (Cox et al. 2015; Mistry et al. 2015). Compared with miglustat, eliglustat showed stronger inhibitory potency (IC50=0.024 uM for eliglustat, IC50=5-50 uM for miglustat) and less gastrointestinal manifestation side effect because eliglustat does not inhibit intestinal enzyme (Smid et al. 2016; Balwani et al. 2016). However, it does not traverse the blood brain barrier (Shayman 2013), limiting its usage in treating neuronopoathic forms of Gaucher disease or other LSDs.
(25) Therefore, current substrate reduction therapies for LSDs are quite limited. A significant unmet medical need exists for treating LSDs with neurological disorders. To this end, Sanofi-Genzyme has developed a promising novel GCS antagonist, ibiglustat, for the treatment of Fabry, Gaucher and other lipid storage diseases. Ibiglustat is a selective and potent GCS inhibitor with central nervous system (CNS) access (Ashe et al. 2015; Marshall et al. 2016), leading to improved clinical effectiveness with reduced side effects.
(26) Compared to ibiglustat, the compounds disclosed herein are potent, selective inhibitors of GCS that show increased pharmacokinetic profiles.
(27) In addition to use as a monogenic SRT therapy, compounds of this invention may also be used in combination with ERT, that may confer both complementary and additive therapeutic benefits in lipid storage diseases including Fabry disease, Gaucher diseases.
(28) In the present invention, all compounds disclosed herein have the general structure of Formula (I):
(29) ##STR00003##
wherein each of R.sup.1 and R.sup.2 is independently selected from H, or D, and at least one of R.sup.1 and R.sup.2 is D.
(30) In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the Formula (I), or a pharmaceutically acceptable salt or ester thereof, effective to treat Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(31) In another aspect, the invention relates to a dosage form comprising a compound of the Formula (I), wherein the dosage form is suitable for administrating to a subject, animal or human, that suffers from Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(32) In another aspect, the invention relates to a method of making or using a compound of the Formula (I).
(33) In certain embodiments, each of R.sup.1 in the compound is D and each of R.sup.2 is H, having the structural Formula (II) (ibiglustat-d6). The mass spectrometric analytical results of ibiglustat-d6 are demonstrated in
(34) ##STR00004##
(35) In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the Formula (II), or a pharmaceutically acceptable salt or ester thereof, effective to treat Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(36) In another aspect, the invention relates to a dosage form comprising a compound of the Formula (II), wherein the dosage form is suitable for administrating to a subject, animal or human, that suffers from Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(37) In another aspect, the invention relates to a method of making or using a compound of the Formula (II).
(38) In certain embodiments, each of R.sup.2 in the compound is D and each of R.sup.1 is H, having the structural Formula (III) (ibiglustat-d12). The mass spectrometric analytical results of ibiglustat-d12 are demonstrated in
(39) ##STR00005##
(40) In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the Formula (III), or a pharmaceutically acceptable salt or ester thereof, effective to treat Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(41) In another aspect, the invention relates to a dosage form comprising a compound of the Formula (III), wherein the dosage form is suitable for administrating to a subject, animal or human, that suffers from Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(42) In another aspect, the invention relates to a method of making or using a compound of the Formula (III).
(43) In certain embodiments, each of R.sup.1 and R.sup.2 is D, having the structural formula (IV) (ibiglustat-d18). The mass spectrometric analytical results of ibiglustat-d18 are demonstrated in
(44) ##STR00006##
(45) In another aspect, the invention relates to a pharmaceutical composition comprising a compound of the Formula (IV), or a pharmaceutically acceptable salt or ester thereof, effective to treat Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(46) In another aspect, the invention relates to a dosage form comprising a compound of the Formula (IV), wherein the dosage form is suitable for administrating to a subject, animal or human, that suffers from Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(47) In another aspect, the invention relates to a method of making or using a compound of the Formula (IV).
(48) In another aspect, the invention relates to a pharmaceutical composition comprising at least one compound selected from the group consisting of the compounds of Formulas (I) to (IV) or a pharmaceutically acceptable salt or ester thereof.
(49) In yet another aspect, the invention relates to a unit dosage form comprising the pharmaceutical composition disclosed herein. The unit dosage is suitable for administration to a subject suffering Fabry or Gaucher diseases and other related diseases including Parkinson's disease.
(50) In yet another aspect, the invention relates to a method for treating, reducing, or preventing a disease or disorder. The method includes: administering to a subject in need thereof a pharmaceutical composition comprising at least one compound selected from the group consisting of the compounds of Formulas (I) to (IV), or a pharmaceutically acceptable salt or ester thereof.
(51) In certain preferred embodiments, the method of treatment includes administering to a subject in need thereof a pharmaceutical composition comprising a compound of any of the Formulas (I) to (IV), or a pharmaceutically acceptable salt or ester thereof.
IV. Examples
(52) Unless defined otherwise, all technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art. Although any methods and materials similar or equivalent to those described herein can also be used in the practice or testing of the present disclosure, the preferred methods and materials are now described.
Example 1
Synthetic Scheme for the Synthesis of Compound 1
(53) ##STR00007##
Synthesis of Compound 3
(54) 4-Fluorophenyl thioamide 2 (20 g, 128.9 mmol, 1 equiv) was dissolved in ethanol (100 mL) by heating to 50 C. Ethyl 4-chloroacetoacetate (23.3 g, 141.8 mmol, 1.1 equivalent) was added dropwise. After 3 hours of reflux, the dark-brown solution was concentrated to dryness. To the residue, hexanes (100 mL) was added to get yellow solid. The solid was filtered and washed with hexanes (20 mL). The solid was subsequently neutralized with saturated bicarbonate aqueous solution (100 mL) and extracted with MTBE (100 mL2). The combined organic layer was dried over Na.sub.2SO.sub.4 and concentrated to give compound 3 as a brown oil (25.8 g, 75% yield).
Synthesis of Compound 4
(55) Compound 3 (8 g, 30.2 mmol, 1 equivalent) and CD.sub.3I (17.5 g, 120.6 mmol, 4 equivalent) were mixed in DMSO (60 mL). Cooled by ice bath, 50% NaOH aqueous solution (4.8 g, 120.6 mmol, 4 equivalent) was added dropwise. The mixture was stirred at room temperature overnight. The mixture was neutralized by 2 N HCl aqueous solution, then poured into water (150 mL) and extracted with MTBE (50 mL3). The organic layer was dried over Na.sub.2SO.sub.4 and concentrated to give an oil (12.6 g). The residue was dissolved in THF (50 mL). A solution of lithium hydroxide monohydrate (3.6 g, 85.9 mmol) in water (50 mL) was added. The solution was heated at reflux overnight. The THF was removed on Rotavap to give a solution. The solution was extracted by MTBE (25 mL2) and then acidified to pH=2-3 by 2 N HCl. The acidic aqueous solution with the gummy solid was extracted by MTBE (50 mL3). The combined MTBE layer was dried over Na.sub.2SO.sub.4 and evaporated to get a solid. The solid was recrystallized by MTBE-hexanes (1:3, 75 mL) to afford an off-white solid 4 (5.1 g, yield 42%).
Synthesis of Compound 4a
(56) ##STR00008##
(57) Repeat the synthetic procedure of compound 4 by using compound 3 and methyl iodide, to get compound 4a.
Synthesis of Compound 5
(58) Compound 4 (3.0 g, 11.3 mmol, 1 equiv.) was dissolved in dry THF (20 mL) under nitrogen. Cooled by ice-bath, CDI (2.2 g, 13.5 mmol, 1.2 equivalent) was added. The ice-bath was removed, and the solution was stirred at room temperature for 1 h. The solution was again cooled in an ice-water bath, and hydroxylamine hydrochloride (1.6 g, 22.6 mmol, 2.0 equivalent) was added. The reaction mixture was stirred under nitrogen at room temperature overnight. The THF was then evaporated in vacuo and the residue was dissolved in ethyl acetate (50 mL). The ethyl acetate layer was washed with saturated bicarbonate solution (30 mL) and brine (30 mL) successively, dried over Na.sub.2SO.sub.4, and the solvent was evaporated in vacuo to obtain a pale-yellow solid. The solid was recrystallized with MTBE-hexane (4:1) to get compound 5 as an off-white solid (2.4 g, 76% yield).
Synthesis of Compound 5a
(59) ##STR00009##
(60) Repeat the synthetic procedure of compound 5 by using compound 4a, to get compound 5a.
Synthesis of Compound 1
(61) To a stirred solution of compound 5 (0.64 g, 2.2 mmol, 1 equivalent) in MeCN (5 mL) at room temperature was added carbonyl diimidazole (0.42 g, 2.57 mmol, 1.15 equivalent). The reaction was stirred for 2 h and then concentrated to dryness. To the residue, toluene (10 mL) was added and the mixture was heated at reflux for 3 h. (S)-(+)-quinuclidinol (0.34 g, 2.67 mmol, 1.2 equivalent) was added and the reaction was heated at reflux for 16 hrs. The solution was cooled to room temperature and washed with water (10 mL). The aqueous phase was extracted with toluene (10 mL). The combined organic layer was washed with 1 N HCl (15 mL2). The combined acidic aqueous layers were basified to pH=10-11 by 2 N NaOH. The formed gummy solid was extracted with DCM (15 mL3). The DCM layer was washed with brine (20 mL), dried over Na.sub.2SO.sub.4 and concentrated to obtain a beige solid (0.53 g). The solid was recrystallized with MTBE to get compound 1 as an off-white solid (0.32 g, 45% yield).
Synthesis of Compound 1a
(62) ##STR00010##
(63) Repeat the synthetic procedure of compound 1 by using compound 5 and compound 6, to get the compound 1a as off-white solid.
Synthesis of Compound 1b
(64) ##STR00011##
(65) Repeat the synthetic procedure of compound 1 by using compound 5a and compound 6, get the compound 1b as off-white solid.
Synthetic Scheme for the Synthesis of Compound 6
(66) ##STR00012## ##STR00013##
Synthetic Scheme for the Synthesis of Compound 7
(67) To the pentachloropyridine (160 g, 0.63 mol, 1 equivalent) in ether solution (1.44 L) under N.sub.2 below 55 C., n-BuLi (2.5 M in hexane. 270 mL, 1.06 equivalent) was added dropwise. The mixture was stirred below 55 C. for 30 min. Dry CO.sub.2 was bubbled into the mixture and the temperature rose to room temperature. The mixture was acidified by adding conc. HCl (70 mL). The organic phase was separated and dried over Na.sub.2SO.sub.4, concentrated to dryness. The residue was suspended in refluxing DCM for 30 min, cooled down and filtered to get compound 7 as an off-white solid (80 g, 48% yield).
Synthesis of Compound 8
(68) To a 1 L autoclave, compound 7 (38 g, 0.145 mol, 1 equiv) was mixed with 10% Pd/C (3.8 g) and K.sub.2CO.sub.3 (50.0 g, 0.362 mol, 2.5 equivalent in D.sub.2O (380 mL). After three nitrogen purges, the mixture was heated to 80 C. under D.sub.2 (200 psi) for 24 hours. The mixture was filtered through a celite and was concentrated to dryness. The dryness was refluxing in toluene (200 mL) to remove the remaining D.sub.2O by Dean-Stark trap and the obtained solid 8 was directly used in the next step.
Synthesis of Compound 9
(69) Compound 8 was suspended in EtOH (250 mL). Cooled by ice-bath, thionyl chloride (26.1 g, 0.22 mol) was added dropwise. The mixture was stirred at room temperature overnight and then heated and refluxed for 4 hours. The mixture was concentrated to give compound 9 and directly used in the next step.
Synthesis of Compound 10
(70) Compound 9 was mixed with chloroacetate (27.8 g, 0.22 mol) in MeCN (150 mL). Cooled by ice-bath, K.sub.2CO.sub.3 (78.6 g, 0.57 mol) was added in portions. The mixture was stirred overnight and filtered through a celite pad. The filter cake was washed with MeCN (100 mL). The filtrate was concentrated to give compound 10 as a yellow oil (25.9 g, 71% yield from compound 7).
Synthesis of Compound 11
(71) Compound 10 (14.8 g, 58.6 mmol, 1 equivalent) was refluxed with NaOMe (0.63 g, 11.7 mmol, 20% equiv) in MeOD (60 mL) for 20 hours. The mixture was concentrated to dryness. The residue was refluxed in MeOD (40 mL) for another 20 hours. After the solvent was evaporated, the residue was dissolved in DCM, washed with brine, dried over Na.sub.2SO.sub.4, and concentrated to give compound 11 as a yellow oil (12.2 g, 91% yield).
Synthesis of Compound 12
(72) Potassium tert-butoxide (15.1 g, 134.8 mmol, 2.5 equivalent) was suspended in dry toluene (150 mL) and heated to reflux under N.sub.2. Compound 11 (12.2 g, 53.9 mmol, 1 equivalent) in dry toluene (30 mL) was added dropwise to the above suspension. The mixture was kept refluxing for 3 hours and then cooled by ice-bath. DCl aqueous solution (30 g conc. DCl with 30 mL D.sub.2O) was added to the reaction mixture. The aqueous phase was separated. The toluene phase was washed with diluted DCl (20 g conc. DCl with 10 mL D.sub.2O). The combined aqueous phase was heated and refluxed for 18 hours. Cooled by ice-bath, the reaction solution was basified to pH=10-11 by K.sub.2CO.sub.3, extracted with DCM (80 mL3). The DCM layer was dried over Na.sub.2SO.sub.4 and concentrated to give compound 12 as a yellow oil (4 g, 54% yield).
Synthesis of Compound 13
(73) Compound 12 (4 g, 29.3 mmol, 1 equivalent) was dissolved in D.sub.2O (20 mL). Cooled by ice-bath, NaBD.sub.4 (0.61 g, 14.6 mmol, 0.5 equivalent) was added to the solution. The mixture was stirred for 20 min at room temperature and then quenched by conc. DCl (2 g). The solution was basified by NaOH to pH=12-13 and concentrated to dryness. The residue was extracted with CHCl.sub.3 (30 mL2). The chloroform layer was dried over Na.sub.2SO.sub.4 and concentrated to give compound 13 as a yellow solid (3 g, 75% yield).
Chiral Resolution of Compound 13 to Obtain Compound 6
(74) A mixture of dibenzoyl-L-(+)-tartaric acid monohydrate (4.4 g, 11.8 mmol), ethanol (20 mL) and racemic compound 13 (3 g, 23.6 mmol) was heated to reflux and maintained at reflux for 1 hour. After the solution was cooled to 0-5 C., the precipitating solid was filtered, washed with ethanol (10 mL), dried under vacuum to give a white solid (2.4 g). The obtained solid (2.4 g) was recrystallized three times in EtOH to give 1.6 g (S)-3-quinuclidinol dibenzoyl-L-(+)-tartrate salt. The salt was dissolved in water (5 mL). To the solution, 2 N HCl aqueous solution was added to get white solid. The solid was extracted with Ethyl acetate (15 mL). The EA layer was washed with water (3 mL). The combined aqueous layer was basified by NaOH to pH=12-13, extracted with chloroform (30 mL3). The chloroform was dried over Na.sub.2SO.sub.4 and concentrated to give compound 6 as a white solid (0.3 g, yield 10%). The chiral purity of benzoylate of compound 6 was >99.5% ee.
NMR Analysis of Compounds 1, 1a and 1b
(75)
(76)
(77)
Example 2
Incubation of Ibiglustat Verses Compounds of the Present Invention in Animal and Human Microsomes and in Vitro Sample Analysis by LC-MS/MS
(78) Reagents
(79) Dimethyl sulfoxide (DMSO) and acetonitrile (ACN) were obtained from VWR (Radnor, Pa.). 10PBS buffer pH 7.4 was obtained from Thermo-Fischer Scientific (Waltham, Mass.). Ethylenediaminetetraacetic acid (EDTA) was obtained from Sigma-Aldrich (St. Louis, Mo.). Type I water was obtained from a reverse osmosis filtration system from MilliporeSigma (Burlington, Mass.). Dextromethorphan, ibiglustat, ibiglustat-d6, ibiglustat-d12, and ibiglustat-d18 were provided by Ascendex Scientific (Levittown, Pa.). Sodium hydroxide (NaOH) was obtained from Fisher Scientific (Hampton, N.H.). Human, beagle, rat and mouse microsomes, as well as NADPH regenerating system Solutions A and B, were obtained from Corning (Corning-Gentest, Tewksvury, Mass.).
Preparation of Primary Stock Solution and Working Solution
(80) Each of the compounds presented in this disclosure (ibiglustat, ibiglustat-d6, ibiglustat-d12, and ibiglustat-d18) was weighed out in powder form and dissolved in DMSO to create primary stock solutions at concentrations of 10 mM. An aliquot of each primary solution was subsequently diluted in sample dilution buffer to a concentration of 100 M. The four 100 M solutions were then combined equally and diluted using sample dilution buffer to create a 4-in-1 solution in which each compound's concentration was 100 nM. Dextromethorphan was weighed out in powder form and dissolved in DMSO to create a primary stock solution with a concentration of 10 mM. The 10 mM stock was serially diluted in sample dilution buffer to create a working solution with a concentration of 100 nM. Each solution, upon dilution, was vortexed for at least two minutes to ensure homogeneity.
Preparation of Sample Dilution Buffer
(81) To prepare 0.5 M EDTA (pH8.0) solution, disodium EDTA (Na.sub.2EDTA.2H.sub.2O) was dissolved in Type 1 water and pH to 8.0 was adjusted using sodium hydroxide. 1PBS buffer (pH7.4) was prepared by diluting 10PBS buffer pH 7.4 with Type 1 water. Sample dilution buffer was made by adding 1 mL of 0.5 M EDTA (pH8.0) solution to 99 mL of 1PBS buffer pH 7.4.
Preparation of Microsome Solution
(82) Rat and Dog pooled liver microsomes were purchased from Corning-Gentest (Tewksbury, Mass.) at a concentration of 20 mg protein/mL. They were diluted on ice to 5 mg/mL by combining three parts of 1PBS with one part of 20 mg/mL microsome right before each experiment.
Preparation of Reaction Mixture
(83) The reaction mixtures with or without microsomes were prepared as indicated in Table 1. As a negative control, microsome-free samples were prepared by substituting the microsome solution in the reaction mixture with an equal volume of 1Phosphate Buffer Solution pH 7.4. The reaction mixture was prepared in triplicates. In addition, dextromethorphan was used in this assay as a positive control.
(84) TABLE-US-00001 TABLE 1 Preparation of Reaction Mixture with or without Microsome Preparation of Reaction Mixture of Sample with Microsome Test Compounds at 100 nM in Sample Dilution Buffer 90 L (25 nM final concentration during incubation) Solution A 13 L Solution B 3 L 1 Phosphate Buffer Solution, pH 7.4 220 L 5 mg/mL Microsome Solution 34 L (1.25 mg/mL final concentration during incubation) Preparation of Reaction Mixture of Sample without Microsome Test Compounds at 100 nM in Sample Dilution Buffer 90 L (25 nM final concentration during incubation) Solution A 13 L Solution B 3 L 1 Phosphate Buffer Solution, pH 7.4 254 L
Incubation and Processing of Reaction Mixture
(85) The reaction mixture was incubated in a water bath incubator at 37 C. At predetermined time points (0, 15, 30, 45, 60 and 120 minutes), an aliquot of 50 L reaction mixture was removed from the incubation tube to a clean tube containing 250 L of ice cold acetonitrile to terminate the reaction. The terminated aliquots were mixed well by vortexing and centrifuged for five minutes at 13,000 RPM. A portion of supernatant (100 L) was transferred to an autosampler vial and analyzed using liquid chromatography-mass spectrometry (LC-MS).
LC-MS/MS Analysis
(86) Reversed-phase liquid chromatographic separation was done using ACE 3 C18 column (502.1 mm id, 3 m). Mobile phase A was water with 0.1% formic acid and mobile phase B was acetonitrile with 0.1% formic acid with a combined flow rate of 0.3 mL/min. The applied gradient elution was: 10% B at 0 min, 90% B at 2 min, 90% B at 2.5 min, and 10% B at 2.6 min. Five compounds were monitored in positive mode over five-minute acquisition time by a triple quadrupole mass spectrometer. The sample run and data processing were done by Analyst version 1.6.2. Table 2 indicates multiple-reaction monitoring conditions of five analytes.
(87) TABLE-US-00002 TABLE 2 MRM conditions of ibiglustat, ibiglustat-d6, ibiglustat-d12, ibiglustat-d18, and dextromethorphan Compounds Q1 > Q3 (m/z) DP EP CP CXP ibiglustat 390.2 > 220.1 70 14 43 12 ibiglustat-d6 396.4 > 226.1 90 10 45 12 ibiglustat-d12 402.5 > 220.0 90 12 45 13 ibiglustat-d18 408.5 > 226.0 90 11 45 11 Dextromethorphan 272.2 > 147.2 80 10 42 10
Results from Co-Incubation Studies
(88) The compounds of the present invention showed higher stability over ibiglustat when they were 1:1 co-incubated in microsomes. As shown in
(89) The representative examples are intended to help illustrate the invention, and are not intended to, nor should they be construed to, limit the scope of the invention. Indeed, various modifications of the invention and many further embodiments thereof, in addition to those shown and described herein, will become apparent to those skilled in the art from the full contents of this document, including the examples and the references to the scientific and patent literature included herein. The examples contain important additional information, exemplification and guidance that can be adapted to the practice of this invention in its various embodiments and equivalents thereof.
REFERENCES
(90) References and citations to other documents, such as patents, patent applications, patent publications, journals, books, papers, web contents, have been made in this disclosure. All such documents are hereby incorporated herein by reference in their entirety for all purposes. Any material, or portion thereof, that is said to be incorporated by reference herein, but which conflicts with existing definitions, statements, or other disclosure material explicitly set forth herein is only incorporated to the extent that no conflict arises between that incorporated material and the present disclosure material. In the event of a conflict, the conflict is to be resolved in favor of the present disclosure as the preferred disclosure. The following is the list of references: 1. Ashe, K. M., E. Budman, D. S. Bangari, C. S. Siegel, J. B. Nietupski, B. Wang, R. J. Desnick, R. K. Scheule, J. P. Leonard, S. H. Cheng, and J. Marshall. 2015. 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