Tocopherol and tocopheryl quinone derivatives as correctors of lysosomal storage disorders

Abstract

The subject invention relates to improved tocopheryl quinine derivatives and tocopherol derivatives having improved pharmacokinetics in vivo that can, in some embodiments, be useful in the treatment of Lysosomal Storage Disorder, restoration of normal mitochondrial ATP production, modulation of intracellular calcium ion concentration and other treatments or therapies. The tocopheryl quinone derivatives and tocopherol derivatives have side chains that have terminally halogenated carbon atoms.

Claims

1. A tocopheryl quinone derivative of formula (I): ##STR00015## wherein R.sub.1, R.sub.2, and R.sub.3 independently are hydrogen, halogen, alkyl, or fluoroalkyl; R.sub.a and R.sub.b independently are hydrogen or absent in each repeat; X is halogen; and the dotted line bonds indicate single or double bonds.

Description

BRIEF DESCRIPTION OF THE DRAWINGS

(1) The following Detailed description of the Invention, given by way of Examples, but not intended to limit the invention to specific embodiments described, may be understood in conjunction with the accompanying figures, in which:

(2) FIG. 1A shows a reduction of cholesterol levels in wild type cells upon treatment with delta tocopherol and analogues. FIG. 1B shows a reduction of cholesterol levels in NPC fibroblasts upon treatment with delta tocopherol and analogues. FIG. 1C provides the structures of a number of compounds (tocopherol analogues) tested (assayed in the NPC phenotypic assay).

(3) FIG. 2 shows the results of a Filipin staining assay for the evaluation of cholesterol levels in wild type and in NPC fibroblast upon treatment with delta tocopherol and NCGC00250218 (X-analogue).

(4) FIG. 3A shows a table providing the results of a study of brain and plasma pharmacokinetics upon oral administration of X-analogue to Balb/c mice. FIG. 3B depicts, in graph form, brain and plasma pharmacokinetics upon oral administration of NCGC00250218 (X-analogue).

(5) FIG. 4A depict, in graph form, a mean plasma concentration-time profile comparison of different administration routes and doses for of NCGC00250218 (X-analogue). FIG. 4B depicts, in graph form, a mean brain concentration-time profile comparison of different administration routes and doses for of NCGC00250218 (X-analogue). FIG. 4C shows the mean plasma and brain concentration-time profiles of NCGC00250218-01 after a per os (P.O.) (by mouth) does of 250 mg/kg in male C5BL/6 mice.

(6) FIG. 5A shows a table providing the levels of CF3-tocopherol NCGC00250218 in the brain of NPC mice at 72 h after 300 mg/kg single dose IP administration. FIG. 5B shows a table providing the levels of cholesterol esters in the brain of NPC mice before and after 72 h exposure to NCGC00250218 upon 300 mg/kg single dose IP administration.

DETAILED DESCRIPTION OF THE INVENTION

(7) Reference will now be made in detail to representative embodiments of the invention. While the invention will be described in conjunction with the enumerated embodiments, it will be understood that the invention is not intended to be limited to those embodiments. On the contrary, the invention is intended to cover all alternatives, modifications and equivalents that may be included within the scope of the present invention as defined by the claims.

(8) One skilled in the art will recognize many methods and materials similar or equivalent to those described herein, which could be used in and are within the scope of the practice of the present invention. The present invention is in no way limited to the methods and materials described herein.

(9) Unless defined otherwise, technical and scientific terms used herein have the same meaning as commonly understood by one of ordinary skill in the art to which this invention belongs. Although any methods, devices, and materials similar or equivalent to those described herein can be used in the practice or testing of the invention, the preferred methods, devices and materials are now described.

(10) All publications, published patent documents, and patent applications cited in this application are indicative of the level of skill in the art(s) to which the application pertains. All publications, published patent documents, and patent applications cited herein are hereby incorporated by reference in their entirety and to the same extent as though each individual publication, published patent document, or patent application was specifically and individually indicated as being incorporated by reference in their entirety.

(11) As used in this application, including the appended claims, the singular forms a, an, and the include plural references, unless the content clearly dictates otherwise, and are used interchangeably with at least one and one or more.

(12) As used herein, the term about represents an insignificant modification or variation of the numerical value such that the basic function of the item to which the numerical value relates is unchanged.

(13) As used herein, the terms comprises, comprising, includes, including, contains, containing, and any variations thereof, are intended to cover a non-exclusive inclusion, such that a process, method, product-by-process, or composition of matter that comprises, includes, or contains an element or list of elements does not include only those elements but may include other elements not expressly listed or inherent to such process, method, product-by-process, or composition of matter.

(14) In the description that follows, a number of terms used in chemistry, medicine and biotechnology are used. In order to provide a clear understanding of the terms, the following definitions are provided.

(15) As used herein, Lysosomal Storage Disorders or LSDs include, but are not limited to, the following disorders and diseases: Glycogen storage disease type II (Pompe disease), Mucopolysaccharides (MPS) (e.g., MPS types I-IV and VI-VII), Mucolipidoses (e.g., I-IV), Oligosaccharidoses (e.g., Schindler disease/Kanzaki disease, and alpha- and beta-mannosidoses), Lipidoses (e.g., Niemann-Pick disease types C as to D, and Wolman disease), Sphingolipdoses (e.g., Niemann-Pick disease types A and B, Gaucher disease types I, II, and III, and GM1 and GM2 gangliosidoses including Tay-Sachs disease), and Lysosomal Transport diseases (e.g., Sialic acid storage disease).

(16) Also contemplated for treatment employing the compounds of the invention are mitochondrial disorders and neurodegeneration. There are more than 40 distinct mitochondrial cytopathies including, without limitation, Friedreich's ataxia, Kearns-Sayre syndrome (KSS), Myoclonus epilepsy with ragged-red fibers (MERRF), Mitochondrial encephalomyopathy with lactic acidosis and stroke-like episodes (MELAS), Leber hereditary optic neuropathy (LHON), Leigh syndrome, Myoneurogenic gastrointestinal encephalopathy (MNGIE), Pearson syndrome, Neuropathy, ataxia, and retinitis pigmentosa (NARP).

(17) An individual refers to a test subject or patient. The individual can be a mammal or a non-mammal. In various embodiments, the individual is a mammal. A mammalian individual can be a human or non-human. In various embodiments, the individual is a human. A healthy or normal individual is an individual in which the LSD disease is not detectable by conventional diagnostic methods.

(18) NCGC00250218, X analogue, and CF.sub.3-tocopherol are used interchangeably herein.

(19) A pharmaceutical composition means a composition comprising a pharmaceutical agent, a pharmaceutically acceptable vehicle and optionally other components such as stabilizers, preservatives, pharmaceutically acceptable carriers and the like. The pharmaceutical agent is the tocopheryl quinone derivative and/or the tocopherol derivative described herein.

(20) The pharmaceutical compositions of the subject invention can be prepared by known methods of combination of compounds in admixture with a pharmaceutically acceptable vehicle. Suitable vehicles and their formulation in pharmaceutical compositions are described in Remington's Pharmaceutical Sciences (16th ed. Osol, E. ed., Mack Easton Pa. (1980)). Essentially pure or pure pharmaceutical agents can be admixed with a pharmaceutically acceptable vehicle and other components to produce a pharmaceutical composition using current Good Manufacturing Practices.

(21) An essentially purified agent is one that is substantially free from matter that is not of interest. With increasing preference, an essentially purified molecule is at least 80% pure, at least 90% pure, at least pure 95% pure, at least 97% pure, at least 98% pure, and at least 99% pure. A pure agent is one that has been purified to homogeneity, i.e., is 100% pure.

(22) The invention includes essentially purified or pure tocopherol quinone derivative and/or tocopherol derivative, and their inclusion in pharmaceutical compositions.

(23) Additionally, stabilizers that can increase shelf life can be included in the pharmaceutical composition. Suitable stabilizers can include monosodium glutamate and 2-phenoxyethanol. Further, preservatives can be added so as to prevent contamination with bacteria and permit multidose vials. Suitable preservatives can include phenoxyethanol and formaldehyde.

(24) A pharmaceutically acceptable carrier is slowly metabolized macromolecule including, without limitation, proteins, polysaccharides, polyglycolic acids, amino acid copolymers, and like carriers well known in the art.

(25) Pharmaceutically acceptable vehicle refers to the water, saline, glycerol, ethanol, etc. used for dissolution, suspension, or mixing of components in the pharmaceutical composition.

(26) The term effective amount or therapeutically acceptable amount for therapeutic treatment refers to an amount of agent sufficient to substantially improve lysosomal exocytosis in affected cells or tissues, and/or substantially improve clinical symptoms of the LSD at issue. It is believed that the effective amount(s) can be found within a relatively large, non-critical range. Routine experimentation can be used to determine appropriate effective amounts.

(27) Methods of administration of a pharmaceutical composition of the subject invention to an individual can be carried out by any suitable means, including parenteral, e.g., intravenous, intradermal, intramuscular, subcutaneous, intranasal, intrathecal, transdermal (topical), transmuccosal; and rectal and oral administration. In a preferred embodiment, the pharmaceutical composition is administered orally. In another embodiment, the pharmaceutical composition administered via infusion. In another embodiment, the pharmaceutical composition is injected directly into the central nervous system (CNS) to bypass the blood brain barrier.

(28) The dosage administered depends on the route of administration. Generally, however, a dosage range of about 5 to about 300 mg/kg body weight of a subject can be useful. In one embodiment, the dosage range is about 10 to about 250 mg/kg. These dosage ranges (and others listed herein) include all of the dosage amounts around and between the stated values, including decimal values. Appropriate dosages can be determined using experimentation known to the technically skilled research scientist or clinician. Such determination may identify dosages outside of the stated ranges, which are also contemplated herein. For example, a single dosage may be about 1 mg/kg body weight.

(29) Cyclodextrins are cyclic oligosaccharides used in pharmaceutical, drug delivery, food, agricultural and chemical industries, and environmental engineering. They are typically 6, 7 and 8-membered rings respectively denoted as -, - and -cyclodextrins. Cyclodextrin assists in moving cholesterol out of lysosomes in LSDs. Examples cyclodextrins that could be suitable in the subject invention include, without limitation, hydroxypropyl -cyclodextrin (HPCD) and methyl--cyclodextrin (MCD). In the subject invention, the hydrophobic cholesterol molecule lodges within the cyclodextrin ring which is then removed from the cell.

(30) Biological sample means a fluid or tissue of an individual that commonly contains cells with impaired lysosomal function as evidenced by accumulation of substrate, enlarged liposomes and reduced mitochondrial ATP production. Biological samples include, without limitation, blood, plasma, serum, white blood cells, cerebral spinal fluid (CSF), myelomas, tears, saliva, milk, urine, lymph fluid, respiratory secretions, and genitourinary or intestinal tract secretions.

(31) The subject invention provides, in one embodiment, a tocopheryl quinone derivative comprising a compound having structure [1]:

(32) ##STR00001##
wherein the dotted line bonds indicate single or double bonds; R.sub.1, R.sub.2 and R.sub.3=alkyl, fluoroalkyl, halogen or H; the side chain is an alkyl, alkenyl, or alkynyl chain, straight or branched, R.sub.7H or a single or double bond and X=a halogen.

(33) The subject invention also includes a pharmaceutical compositions that comprises the structure [1] compound and a pharmaceutically acceptable vehicle. The pharmaceutical composition comprising structure [1] can be used in a method of treating LSDs. In such method, the composition of administered to an individual in need thereof in a therapeutically effective amount.

(34) In another aspect of the invention, the quinone derivative has structure [2]:

(35) ##STR00002##
wherein R.sub.1, R.sub.2 and R.sub.3=alkyl, fluoroalkyl, halogen or H; n=20; R.sub.4, R.sub.5=alkyl, fluoroalkyl, hydroxyl, alkoxy, or H; the side chain is an alkyl, alkenyl, or alkynyl chain, straight or branched and X=a halogen.

(36) The subject invention also includes a pharmaceutical composition that comprises the structure [2] compound and a pharmaceutically acceptable vehicle. The pharmaceutical composition comprising structure [2] can optionally also include a cyclodextrin. The pharmaceutical composition can be used in a method of treating LSDs. In such method, the composition is administered to an individual in need thereof in a therapeutically effective amount.

(37) Compounds having structures [1] and [2] can be used to improve mitochondrial ATP production. An improvement in ATP production can ameliorate the symptoms of certain LSDs. The functions of the different complexes within isolated mitochondria and the final ATP production can be measured using, e.g., the XF CELL MITO STRESS TEST KIT (Seahorse Bioscience).

(38) In both of the compounds having structures [1] and [2], the terminal carbon has been halogenated. Typically, two branching tri-halogenated methyl groups are provided at the end of the side chain to substantially improved pharmacokinetics. Such compounds have been found to avoid hydrolysis and oxidation in the liver by the P450 isoform, CYP4F2.

(39) In a further embodiment, the invention includes a tocopherol derivative having the structure [3]:

(40) ##STR00003##
wherein R.sub.1-R.sub.3=alkyl, fluoroalkyl, halogen or H; R.sub.6=alkyl, fluoroalkyl, alkenyl or H; R.sub.8=a lower alkyl having C.sub.1 to C.sub.6; and the side chain is an alkyl, alkenyl, or alkynyl chain, straight or branched, wherein R.sub.7H or a single or double bond and X=a halogen.

(41) The subject invention also includes a pharmaceutical composition that comprises the structure [3] compound and a pharmaceutically acceptable vehicle. The pharmaceutical composition comprising structure [3] can optionally also include a cyclodextrin. The pharmaceutical composition can be used in a method of treating LSDs. In such method, the composition is administered to an individual in need thereof in a therapeutically effective amount.

(42) In a further aspect, the tocopherol derivative has structure [4]:

(43) ##STR00004##
wherein R.sub.1-R.sub.3=alkyl, fluoroalkyl, halogen or H; n=1-20, the side chain is an alkyl, alkenyl, or alkynyl chain, straight or branched, and X=a halogen.

(44) The subject invention also includes a pharmaceutical composition that comprises the structure [4] compound and a pharmaceutically acceptable vehicle. The pharmaceutical composition comprising structure [4] can optionally also include a cyclodextrin. The pharmaceutical composition can be used in a method of treating LSDs. In such method, the composition is administered to an individual in need thereof in a therapeutically effective amount.

(45) In both of the compounds designated by structures [3] and [4], the terminal carbon has been halogenated. Typically, two branching tri-halogenated methyl groups are provided on the side chain to substantially improved pharmacokinetics. Such compounds have been found to avoid hydrolysis and oxidation in the liver by the P450 isoform, CYP4F2.

(46) Treatment with the composition comprising the tocopheryl quinone derivative or tocopherol derivative comprising structures [1], [2], [3] or [4] can result in improvements that include, e.g., a substantial increase in Ca.sup.2+ influx to the cells, lysosomal size reduction and/or an increment in cholesterol exocytosis from the cells of the patent, or otherwise provide improved properties as described herein.

(47) Pharmaceutical Compositions

(48) Once prepared, a compound of the invention in a pharmaceutically appropriate form and optionally including pharmaceutically acceptable carriers, excipients, diluents, complexation agents, or additives, will be administered to the patient requiring therapy and/or prophylaxis. Administration to patients can, for example, be via oral and/or parenteral administration routes. In certain embodiments, the compounds of the invention can be administered in any manner or employing any mode that will achieve effective concentrations of the compounds in the brain or central nervous system of the patient.

(49) Thus, in one embodiment, the compound is formulated into a stable, safe pharmaceutical composition for administration to a patient. The composition can be prepared according to conventional methods by dissolving or suspending an amount of the compound ingredient in a diluent. The amount might be between about 0.1 mg and about 1000 mg per ml of different of the compound. A buffer may be added; optionally, a carbohydrate or polyhydric alcohol tonicifier and/or a preservative may also be added. Other excipients may also be present, if desired, to maintain the overall tonicity of the tocopherol derivative or other contemplated compound.

(50) The terms buffer, buffer solution, and buffered solution, when used with reference to hydrogen-ion concentration or pH, refer to the ability of a system, particularly an aqueous solution, to resist a change of pH on adding acid or alkali, or on dilution with a solvent. Characteristic of buffered solutions, which undergo small changes of pH on addition of acid or base, is the presence either of a weak acid and a salt of the weak acid, or a weak base and a salt of the weak base. The stability of a formulation may be enhanced by maintaining the pH of the formulation in a range of approximately 5.0 to approximately 9.5. The buffer may, for example, be selected from an acetate buffer, a phosphate buffer, or a glutamate buffer.

(51) Of greater relevance to a formulation comprising a compound of the invention for longer-term storage (i.e., with shelf-life in mind) is minimizing the Oxygen concentration. In one embodiment, Oxygen is essentially excluded from the formulation, so that reaction over time of the compound with oxygen cannot occur.

(52) Carriers or excipients can also be used to facilitate administration of a compound according to the invention. Examples of carriers and excipients include, without limitation, calcium carbonate, calcium phosphate, various sugars such as lactose, glucose, or sucrose, or types of starch, cellulose derivatives, gelatin, polyethylene glycols, and physiologically compatible solvents.

(53) A stabilizer may also be included in a pharmaceutical composition according to the invention. Exemplary stabilizers include carbohydrates and polyhydric alcohols.

(54) A preservative may be added to a pharmaceutical composition described herein to inhibit microbial growth to avoid consequent spoilage of the composition by microorganism. The amount of the preservative is not great, however, as it may affect the overall stability of the inventive compound. Preservatives, as well as each of the other components of pharmaceutical compositions are known in the art and are, for example, described in Remington's Pharmaceutical Sciences as well as Pharmaceutical Dosage Forms: Parenteral Medications, Vol. 1, 1992, Avis et al., Mercel Dekker, New York, N.Y. 1992, which is incorporated by reference in its entirety herein.

(55) Pharmaceutically acceptable carriers, excipients, diluents, complexation agents, and/or additives may be chosen to enhance the stability of a compound according to the invention, facilitate synthesis or formulation of a pharmaceutical composition comprising the compound, and/or to enhance the bioavailability of the compound.

(56) For example, carrier molecules such as cyclodextrin and derivatives thereof are well known in the art for their potential as complexation agents capable of altering the physiochemical attributes of drug molecules. For example, cyclodextrins may stabilize (both thermally and oxidatively), reduce the volatility of, and alter the solubility of, active agents with which they are complexed. Accommodation of one molecule within another is known as complexion and the resulting product is referred to as an inclusion complex.

(57) Alternatively, the pharmaceutically appropriate form of the inventive tocopherol derivative compound may be formulated so as to enhance the stability and bioavailability of the compound.

(58) One oral controlled release structure is enteric coating of a solid dosage form. Enteric coatings promote the compounds remaining physically incorporated in the dosage form for a specified period when exposed to gastric juice, yet the enteric coatings are designed to distintegrate in intestinal fluid for ready absorption. Delay of absorption is dependent on the rate of transfer through the gastrointestinal tract, and so the rate of gastric emptying is an important factor. For some administrations, a multiple-unit type dosage form, such as granules, may be useful.

(59) Examples of preferred tocopherol derivative compounds of the present invention and/or compositions and/or complexes thereof exhibit advantageous pharmaceutical properties: they may be readily formulatable, are chemically and physically stable, readily water soluble, have low hygroscopicity, and/or exhibit good shelf life.

(60) Aspects and embodiments of the present invention will now be described in the Examples. Further aspects and embodiments will be apparent to those skilled in the art.

EXAMPLES

Example 1Synthesis of a Tocopherol Derivative

(61) Step 1

(62) ##STR00005##
Reaction Procedure:

(63) To a 0 C. cooked solution of NaH (5.75 g, 143.67 mmol, 1 eq.) in dry DMF 100 ml), was slowly added compound 1 (25 g, 143.67 mmol, 1 eq.) in dry DMF (150 mL) at 0 C. and the resulting mixture was stirred 1 h at 0 C. After 1 h, benzyl bromide (20.62 ml, 172.41 mmol, 1.2 eq.) was added to the reaction mixture slowly drop-wise over a period of 20 min at 0 C. Then the reaction mixture was stirred at room temp for 12 h. After the completion of the reaction was checked by TLC, the reaction mixture was quenched in ice water and the aqueous layer was extracted with ether (230 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4, filtered and concentrated under reduced pressure to obtain the crude product. The crude was purified by flash column chromatography over silica gel (100-200 mesh) by using 10% ethyl acetate in pet ether as eluent to obtain (23 g, yield-60%) of pure compound.

(64) NMR data verified characteristics or properties of the step 1 compound (data not shown).

(65) Step 2

(66) ##STR00006##
Reaction Procedure:

(67) To a solution of compound 2 (25 g, 94.69 mmol, 1 eq.) in diethyl ether and acetonitrile (3:1 ratio) at 0 C., imidazole (9.02 g, 132.57 mmol, 1.4 eq.) and triphenyl phosphine (29.77 g, 113.63 mmol, 1.2 eq.) were added at 0 C. After 5 min stirring, iodine (31.26 g, 123.10 mmol, and 1.3 eq.) was added to the reaction mixture at 0 C. and the reaction mixture was stirred for 45 min at 0 C. After the completion of reaction was checked by TLC, the reaction mixture was filtered through celite bed and washed with diethyl ether. The filtrate was concentrated under reduced pressure to obtain the crude product. The crude was purified by flash column chromatography eluting with 2-3% ethyl acetate in pet ether to obtain (32.5 g, yield-92%) pure compound.

(68) NMR data verified characteristics or properties of the step 2 compound (data not shown).

(69) Step 3

(70) ##STR00007##
Reaction Procedure:

(71) To a solution of compound 3 (25 g, 66.84 mmol, 1 eq.) in dry THF, triphenyl phosphine (22.76 g, 86.89 mmol, and 1.3 eq.) was added. Then the reaction mixture was refluxed for 12 h. After the completion of reaction checked by TLC, the reaction mixture was distilled under reduced pressure and the crude compound was purified through flash column chromatography by using 5% methanol in dichloromethane as eluent to obtain (35.2 g, yield-83%) pure compound.

(72) NMR data verified characteristics or properties of the step 3 compounds (data not shown).

(73) Step 4

(74) ##STR00008##
Reaction Procedure:

(75) Compound 4 (50 g, 78.54 mmol, 1 eq) was taken in dry THF and cooled to 78 C., n-BuLi (103 ml, 164.9 mmol, 2.1 eq) was added drop-wise over a period of 1 h at 78 C. Hexafluoroacetone trihydrate (46 ml, 314.1 mmol, 4 eq) is added drop wise to conc.H.sub.2SO.sub.4. The gas hexafluoroacetone released was slowly purging into the above solution 78 C. The reaction mixture left for 12 h at room temperature. After completion of reaction, it was quenched with 2N HCl and the aqueous layer was extracted with ethyl acetate (2300 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtain the crude product. The crude was purified by flash column chromatography eluting with 5% ethyl acetate in pet ether to obtain (23.3 g, yield-75%) pure compound.

(76) NMR date verified characteristics or properties of the step 4 compounds (data not shown).

(77) Step 5

(78) ##STR00009##
Reaction Procedure:

(79) Compound 5 (25 g, 63.13 mmol, 1 eq) was taken in MeOH, Pd/C (10 g) was added portion wise under N.sub.2. It was heated to 50 C., under 80 psi in par shaker apparatus for 24 h. After completin of reaction it was filtered through Celite, the filtrate was concentrated under reduced pressure to obtain the crude product. The crude was purified by flash column (100-200 silica gel) chromatography eluting with 2-3% MeOH in DCM to obtain (11 g, yield-85%) pure compound.

(80) MMR data verified characteristics or properties of step 5 compounds (data not shown).

(81) Step 6

(82) ##STR00010##
Reaction Procedure:

(83) Compound 6 (20 g, 64.93 mmol, 1 eq) was taken in DCM and was cooled to 0 C., DMP (38.5 g, 90.90 mmol, 1.4 eq) was added portion-wise. The reaction mixture was left for 2 h at RT. After completion of reaction DCM was distilled off. The residue was dissolved in Diethyl ether and was filtered through celite. The filtrate was washed with sodium bicarbonate (2300 ml). Organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtain (17.5 g, yield-88%) the crude product. The crude was itself taken to next step without further purification.

(84) HPLC and NMR data verified characteristics or properties of the step 6 compounds (data not shown).

(85) Step 7

(86) ##STR00011##
Reaction Procedure:

(87) NaH (2.1 g, 86.192 mmol, and 2.2 eq) was added portion wise in THF at 0 C., then Wittig reagent (11.4 g, 43.095 mmol, 1.1 eq) was added drop-wise, and it was left for 1 h. Then HMPA (14 ml, 78.356 mmol, and 2 eq) and compound 7 (12 g, 39.178 mmol, 1 eq) in THF were added drop-wise at 0 C. The reaction mixture was left for 2 h at RT. After completion, the reaction was quenched with ice, extracted twice with diethyl ether and extracted with ether (230 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtain the crude product. The crude was purified by flash column chromatography eluting with 10% ethyl acetate in pet ether to obtain (1.85 g, yield-65%) pure compound.

(88) LCMS, HPLC and NMR data verified characteristics or properties of the step 7 compounds (data not shown).

(89) Step 8

(90) ##STR00012##
Reaction Procedure:

(91) To a 78 C. cooled solution of Compound 8 (1 g, 2.4 mmol, 1 eqiv) in dry THF (20 mL), was added LAH (4.8 ml, 1M solution, 2 equiv.) at 78 C., and the resulting mixture was stirred 3 h at 30 C. After the completion of the reaction checked by TLC, a saturated solution of NH.sub.4Cl was added to the reaction mixture and the aqueous layer was extracted with ether (310 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure to obtained 0.8 g of compound 8 as colorless syrup. The obtained (0.75, yield-83%) crude was taken to next step without purification.

(92) HPLC data verified characteristics or properties of the step 8 compound (data not shown).

(93) Step 9

(94) ##STR00013##
Reaction Procedure:

(95) To a solution of Compound 9 (1 g, 2.67 mmol, 1 eq.) in EtOAc (20 mL) were added hydroquinone (0.29 g, 2.67 mmol, 1 eq.), zinc chloride (0.286 g, 2.13 mmol, 0.8 eq.), and HCl 37% aq (0.2 equiv) at 0 C. and the resulting mixture temperature was allowed to reach RT and maintained for 12 h at RT. After completion of the reaction was checked by TLC, a saturated solution of NaHCO.sub.3 (10 mL) was added to the reaction mixture and the aqueous layer was extracted with ether (310 mL). The combined organic extracts were dried over Na.sub.2SO.sub.4 and concentrated under reduced pressure. The residue was purified by flash column chromatography on silica gel (hexane:EtOAc: 85:15) to give 0.25-0.3 g (75% LS-MS) of a brown syrup.

(96) LCMS and NMR data verified characteristics or properties of step 9 compounds (data not shown).

(97) Step 10

(98) ##STR00014##
Reaction Procedure:

(99) To a solution of Compound 10 (1 g. 2.14 mmol.) in methanol (20 mL), was added 10% Pd/C (0.25 g) and the resulting reaction mixture was stirred 12 h at hydrogen (balloon pressure) atmosphere. After completion of reaction was checked by mass spectrometry, the reaction mixture was filtered through celite bed, washed with methanol. The filtrate was concentrated under reduced pressure to obtained 0.8 g of compound GVK-NCI-02 as colorless syrup. The obtained crude was purified by prep HPLC to give (0.3 g, yield-30%) (95% HPLC) of light brown syrup.

(100) HPLC and NMR data verified characteristics or properties of step 10 compounds (data not shown).

Example 2Reduction of Cholesterol Levels in Wild Type and in NPC Fibroblast Upon Treatment with Delta Tocopherol and Analogues

(101) The half maximal effective concentration (EC-50) for delta-tocopherol and five analogues was measured in wild type and NPC 3123 cells. FIG. 1A illustrates that as delta-tocopherol and analogue concentrations increased in wild type cells, serum cholesterol levels dropped. Further, in NPC cells (FIG. 1B), the cholesterol level drop was substantial relative to the control (no delta-tocopherol). The IC50 for phenotypic cholesterol elimination in NCP fibroblasts upon exposure to NCGC00250218 was in the range of 10 M. The most significant change was seen with the X-analogue or NCGC00250218. Indeed, CF.sub.3-tocopherol was more potent that delta-tocopherol (-T).

Example 3Filipin Staining Assay for the Evaluation of Cholesterol Levels in Wild Type and NPC Fibroblast Cells Upon Treatment with Delta-Tocopherol and NCGC00250218 (X-Analogue)

(102) Filipin, a histochemical dye for cholesterol, was used to detect cholesterol in wild type fibroblasts and NPC fibroblasts. Control fibroblasts, i.e., wild type and NPC fibroblasts, were treated only with filipin and no delta-tocopherol or analogues. Experimental samples were NPC cells that received either the X-analogue or delta-tocopherol. FIG. 2 shows that NPC cells treated with filipin and X-analogue (20 M) have less cholesterol than NPC cells treated with filipin and delta-tocopherol (40 M). Thus, treatment of NPC cells with X-analogue significantly reduces cholesterol level relative to cells treated with delta-tocopherol, even though the latter was used at double the concentration as the former.

Example 4Brain and Plasma Pharmacokinetics Upon Oral Administration of X-Analogue to Balb/c Mice

(103) The X-analogue was administered to female Balb/c mice so as the study time-concentration curves in brain and plasma. The FIG. 3A tables set forth the results. The peak mean concentration of X-analogue in serum occurs at approximately 2-4 hours; while the peak concentration in brain occurs at or after 72 hours. These results are also illustrate in the graph of FIG. 3B.

Example 5Effect of Different Administrations Routes and Doses for X-Analogue

(104) The X-analogue was administered per os (PO) or intraperitoneal (IP) to female Balb/c mice so as to observe the effect in brain and plasma of administration route and dose. The results are set forth in FIGS. 4A, 4B, and 4C. In plasma (FIG. 4A), the PO route was seen to be more productive in generating a higher level of X-analogue. Surprisingly, the lower dose (10 mg/kg) of administered X-analogue produce a higher plasma concentration (ng/mL) than the higher dose (250 mg/kg). In brain (FIG. 4B), the PO route was again observed to be more productive in generating a higher level of X-analogue. The different doses (10 mg/kg and 250 mg/kg) for PO administration produce approximately the same brain concentration outcome. FIG. 4C shows the comparison of plasma vs. brain at 250 mg/kg. Thus, synthetic modulation of the aliphatic chain including CF.sub.3-functional groups yielded molecules with improved pharmacokinetics. Of note, natural enzymes do not have the capacity to oxidize fluoroaliphatic chains.

(105) Initial in vivo studies indicated that a single administration of CF.sub.3-tocopherol (NCGC00250218) in Niemann Pick C mice elevates the production of cholesterol esters in the brain, which constitutes a sign of restoration of cholesterol homeostasis. Furthermore, a long-term pharmacokinetic study (up to 42 days after single I.P. dose of 100 mg/kg) has been completed (data not shown).

Example 6Exposure to the CF3-Tocopherol NCGC00250218 Activates Cholesterol Metabolism

(106) Like other natural occurring tocopherols, NCGC00250218 is most likely to have good bioavailability, but, for easy handling of NPC mice and for comparison of brain levels with previous pharmacokinetics experiments, I.P. administration was carried out. Based on previous experience with HPCD, compound and cholesterol-ester levels were measured after 72 hours exposure. It was found that exocytosis of lysosomal trapped cholesterol activates cholesterol metabolism increasing levels of cholesterol esters in brain and 24-hydroxycholesterol in plasma. A dose of NCGC00250218 was selected that would allow reaching brain concentrations similar to its IC50 in fibroblast (10 M). The results are shown in FIGS. 5A and 5B.

(107) Additional studies with NCGC00250218 can be carried out to evaluate the kinetics of cholesterol turnover, measuring the elevation of cholesterol esters at 24 and 48 hours after 300 mg/kg single dose IP administration. Furthermore, the dose can be reduced to determine the minimum dose of NCGC00250218 that will produce cholesterol turnover. These results, together with the long term pharmacokinetic studies described, allow neurodegenerative and survival studies.

(108) Studies described herein, thus, show that CF.sub.3-tocopherol: decreases cholesterol accumulation in NPC patient cells (fibroblast and neurons); reduces enlarged lysosome in NPC cells; increases intracellular Ca.sup.2+ and enhances lysosomal exocytosis; shows cholesterol metabolism turnover in NPC mice upon single administration; and has good brain penetration and an exceptionally long half-life (about 20 to 30 days). Indeed, the long half-life of CF.sub.3-tocopherol allows the compound to reach the brain. It also allows for the possibility of administration over timevia infusion, for example, or low (micromolar range) daily, weekly, or even monthly oral concentrations. Thus, CF.sub.3-tocopherol can be orally bioavailable and safe (based on large daily dosing in a rat model, up to 5 g/kg, without measurable or observable side effects). It is not metabolized as quickly.

(109) CF.sub.3-tocopherol (and CF.sub.3-tocopheryl quinone) have potential applications in mitochondrial disorders and lysosomal storage diseases and neurodegeneration.