Classifications

• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61K—PREPARATIONS FOR MEDICAL, DENTAL OR TOILETRY PURPOSES
  • A61K31/00—Medicinal preparations containing organic active ingredients
  • A61K31/56—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids
  • A61K31/565—Compounds containing cyclopenta[a]hydrophenanthrene ring systems; Derivatives thereof, e.g. steroids not substituted in position 17 beta by a carbon atom, e.g. estrane, estradiol
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P25/00—Drugs for disorders of the nervous system
  • A61P25/28—Drugs for disorders of the nervous system for treating neurodegenerative disorders of the central nervous system, e.g. nootropic agents, cognition enhancers, drugs for treating Alzheimer's disease or other forms of dementia
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P43/00—Drugs for specific purposes, not provided for in groups A61P1/00-A61P41/00

Definitions

• the present invention relates to a method of treating Alzheimer's Disease (AD) with 6-substituted estradiol compounds and their pharmaceutically acceptable salts or prodrugs.
• the compounds provide for up-regulation of lipoprotein lipase (LPL) and/or apoliprotein C2 (ApoC2), the protein that activates LPL.
• LPL lipoprotein lipase
• ApoC2 apoliprotein C2
• AD Alzheimer's Disease
• Early-onset AD is rare, strikes susceptible individuals as early as the third decade, and is frequently associated with mutations in a small set of genes.
• Late onset, or spontaneous AD is common, strikes in the seventh or eighth decade, and is a multifactorial disease with many genetic risk factors.
• Late-onset AD is the leading cause of dementia in persons over the age of 65.
• An estimated 7-10% of the American population over 65, and up to 40% of the American population greater than 80 years of age is afflicted with AD.
• patients experience loss of memory and orientation. As the disease progresses, additional cognitive functions become impaired, until the patient is completely incapacitated.
• ⁇ amyloid-beta peptides
• APP amyloid precursor protein
• gamma-secretase cleavage results in ⁇ proteins of 40 amino acids in length ( ⁇ 40) and 42 amino acids in length ( ⁇ 42), the latter being the predominant species in senile plaques (Iwatsubo, T., et al, 1994 Neuron 13, 45-53).
• the ⁇ levels are determined by the balance between its production and degradation/clearance, and an attenuated ⁇ catabolism is suggested to cause ⁇ accumulation in aging brains (Tanzi, R. E., et al, 2004 Neuron 43, 605-608).
• Previous studies have shown that astrocytes and microglia directly take up and degrade ⁇ 42 (Wyss-Coray, T., et al, 2003 Nat. Med.
• LPL Lipoprotein lipase
• GAGs sulfated glycosaminoglycans
• Sulfated GAGs are side chains of proteoglycans normally found in the extracellular matrix and on the cell surface in the peripheral tissues, e.g. adipose, heart and skeletal muscle tissue, and brain. The role of LPL in the brain is, to date, unknown.
• LPL is accumulated in senile plaques of AD brains (Rebeck, G. W., et al, 1995 Ann. Neurol. 37, 21 1-217).
• SNPs single nucleotide polymorphisms in the coding region of the LPL gene are associated with disease incidence in clinically diagnosed AD subjects, LPL mRNA expression level, brain cholesterol level, and the severity of AD pathologies, including neurofibrillary tangles and senile plaque density (Blain, J. F., et al, 2006 Eur. J. Neurosci. 24, 1245- 1251).
• SNPs single nucleotide polymorphisms
• cyclin-dependent kinase 5 (CDK5) activity contributed to LPL up-regulation and promoted ⁇ phagocytosis in microglia, whereas inhibition of CDK5 reduced LPL expression and ⁇ internalization.
• a viable treatment for AD is the up-regulation of LPL (and/or ApoC2, or apoliprotein C2, the protein that activates LPL).
• compounds such as statins have been shown to stimulate LPL activity and therefore perhaps could play a role in the pathogenesis of AD (Schoonjans, K., et al, 1999 FEBS Lett 452, 160-164; Mead, J., et al, 2002 J. Mol. Med. 80, 753- 769).
• estradiol derivatives that provide for the up-regulation of LPL (and/or ApoC2).
• estrogen itself has previously been reported to markedly decrease the amounts of fat accumulation and LPL mRNA as well as triglyceride accumulation in genetically manipulated 3T3-L1 adipocytes stably expressing the estrogen receptor (ER) (Homma, H., et al, 2000 J. Biol. Chem. 275(15), 1 1404-1 141 1.
• ER estrogen receptor
• an object of the present invention to provide a method of treating a patient with Alzheimer's Disease comprising administering to a patient in need thereof a therapeutically effective amount of a 6-substituted estradiol derivative.
• the 6-substituted estradiol derivatives of the invention provide for the up- regulation of lipoprotein lipase (LPL) and/or apoliprotein C2 (ApoC2).
• LPL lipoprotein lipase
• AdoC2 apoliprotein C2
• the 6-substituted estradiol derivatives used in the methods disclosed herein are a com ound of the formula I:
• Ri, R 2 , R3 and R4 are independently hydrogen, Ci-C 6 alkyl, halo, a sulfate, a glucuronide, -OH, a bulky group, aryl, cycloalkyl, heteroaryl, heterocycloalkyl, -N(CH 2 ) n ; a phosphate group, and a phosphinate group;
• X is selected from the group consisting of alkyl, C 2 -Ci 2 alkenyl, C
• the method specifically provides for compounds that bind to one or both of estrogen receptor-a (ER-a) and estrogen receptor- ⁇ (ER- ⁇ ).
• ER-a estrogen receptor-a
• ER- ⁇ estrogen receptor- ⁇
• Such a method can comprise initiating, enhancing or increasing gene transcription for RNA encoding genes involved in key signaling pathways for the expression of LPL and/or ApoC2.
• a corresponding salt of the active compound for example, a pharmaceutically-acceptable salt.
• a pharmaceutically-acceptable salt examples are discussed in Berge et al, 1977, "Pharmaceutically Acceptable Salts," J. Pharm. Sci., Vol. 66, pp. 1-19, and discussed herein.
• treatment refers generally to treatment and therapy of a mammalian subject, whether of a human or a non-human animal (e.g., in veterinary applications), in which some desired therapeutic effect is achieved, for example, the inhibition of the progress of the condition, and includes a reduction in the rate of progress, a halt in the rate of progress, amelioration of the condition, and/or cure of the condition.
• Treatment as a prophylactic measure is also included.
• Treatment includes combination treatments and therapies, in which two or more treatments or therapies are combined, for example, sequentially or simultaneously.
• treatments and therapies include, but are not limited to, chemotherapy (the administration of active agents, including, e.g., drugs, antibodies (e.g., as in immunotherapy), anti-inflammatory, prodrugs (e.g., employing protecting groups including phosphoric acid derivatives and phosphinates at suitable positions such as position 3 or 17, other compounds used for photodynamic therapy, GDEPT, ADEPT, etc.), surgery, radiation therapy, and gene therapy.
• active agents including, e.g., drugs, antibodies (e.g., as in immunotherapy), anti-inflammatory, prodrugs (e.g., employing protecting groups including phosphoric acid derivatives and phosphinates at suitable positions such as position 3 or 17, other compounds used for photodynamic therapy, GDEPT, ADEPT, etc.)
• suitable positions such as position 3 or 17, other compounds used for photodynamic therapy, GDEPT, ADEPT, etc.
• stereochemical isomer refers to isomers that differ from each other only in the way the atoms are oriented in space.
• the two stereoisomers particularly of importance in the instant invention are enantiomers and diastereomers depending on whether or not the two isomers are mirror images of each other.
• the claimed formulations comprise such compounds that isolated, resolved and are "substantially free of other isomers.”
• terapéuticaally-effective amount pertains to that amount of an active compound, or a material, composition or dosage form comprising an active compound, which is effective for producing some desired therapeutic effect, commensurate with a reasonable benefit/risk ratio.
• effective amount is meant an amount that can bring about a detectable effect, generally.
• patient or “subject” refers to animals, including mammals, preferably humans.
• tissue refers generally to specialized cells which may perform a particular function.
• tissue may refer to an individual cell or a plurality or aggregate of cells, for example, membranes, blood or organs.
• tissue also includes reference to an abnormal cell or a plurality of abnormal cells.
• Exemplary tissues include breast tissue, including breast cells, membranous tissues, including endothelium and epithelium, laminae, connective tissue, including interstitial tissue, brain tissue and tumors.
• alkyl in the present invention is meant a straight or branched chain alkyl radical having 1-20, and preferably from 1-12, carbon atoms. Examples include but are not limited to methyl, ethyl, propyl, isopropyl, n-butyl, sec-butyl, tert-butyl, pentyl, 2- pentyl, isopentyl, neopentyl, hexyl, 2-hexyl, 3-hexyl, and 3-methylpentyl. Each alkyl group may be optionally substituted with one, two or three substituents such as, for example, a halo, cycloalkyl, aryl, alkenyl or alkoxy group and the like.
• aryl is meant an aromatic carbocylic radical having a single ring (e.g. phenyl), multiple rings (e.g. biphenyl) or multiple fused rings in which at least one is aromatic (e.g. 1,2,3,4-tetrahydronaphthyl).
• the aryl group can also be optionally mono-, di-, or trisubstituted with, for example, halo, alkyl, alkenyl, cycloalkyl or alkoxy and the like.
• heteroaryl is meant one or multiple fused aromatic ring systems of 5-, 6- or 7-membered rings containing at least one and up to four heteroatoms selected from nitrogen, oxygen or sulfur. Examples include but are not limited to furanyl, thienyl, pyridinyl, pyrimidinyl, benzimidazolyl and benzoxazolyl.
• the heteroaryl group can also be optionally mono-, di-, or trisubstituted with, for example, halo, alkyl, alkenyl, cycloalkyl or alkoxy and the like.
• cycloalkyl is meant a carbocylic radical having a single ring (e.g. cyclohexyl), multiple rings (e.g. bicyclohexyl) or multiple fused rings (e.g. ).
• the cycloalkyl group can optionally contain from 1 to 4 heteroatoms.
• the cycloalkyl group may have one or more double bonds.
• the cycloalkyl group can also be optionally mono-, di-, or trisubstituted with, for example, halo, alkyl, alkenyl, aryl or alkoxy and the like.
• alkoxy is meant an oxy-containing radical having an alkyl portion. Examples include, but are not limited to, methoxy, ethoxy, propoxy, butoxy and tert- butoxy.
• the alkoxy group can also be optionally mono-, di-, or trisubstituted with, for example, halo, aryl, cycloalkyl or alkoxy and the like.
• alkenyl is meant a straight or branched hydrocarbon radical having from 2 to 20, and preferably from 2-6, carbon atoms and from one to three double bonds and includes, for example, ethenyl, propenyl, l-but-3-enyl, l-pent-3-enyl, l-hex-5-enyl.
• the alkenyl group can also be optionally mono-, di-, or trisubstituted with, for example, halo, aryl, cycloalkyl or alkoxy and the like.
• Halo or "halogen” is a halogen radical of fluorine, chlorine, bromine or iodine.
• glucuronide is meant a glycoside radical of glucuronic acid.
• sulfate refers to a radical having the general formula -OS(0)2-OR', wherein R' is hydrogen, a metal or an alkyl group.
• phosphate refers to a radical having the general formula - OP(0)(OR')2, wherein each R is independently hydrogen, a metal or an alkyl group.
• phosphinate refers to a radical having the general formula - OP(0)(R) 2 , wherein each R' is independently hydrogen, a metal or an alkyl group.
• bulky group is meant a substituent that produces steric hindrance about the space to which it is attached, e.g. a t-butyl group.
• amino alkyl refers to an alkyl group with an amino group on it, for example, H 2 N-CH 2 -, H 2 N-CH 2 CH 2 -, Me 2 NCH 2 -, etc., wherein the point of attachment is a carbon of the alkyl chain; and the term "alkyl amino” as used herein refers to an amino group with an alkyl group attached to the nitrogen atom, for example, CH3NH-, EtNH-, iPr-NH-, etc., wherein the point of attachment is via the nitrogen atom of the amino group. All other terms wherein successive radicals are employed will adhere to a similar rule.
• a method of treating or preventing Alzheimer's Disease in a patient comprising administering to a patient in need thereof a therapeutically effective amount of a 6-substituted estradiol derivative of Formula I is described.
• the method provides up-regulation of LPL and/or ApoC2 functional activity in a mammal, said method comprising administering to said mammal an effective amount of a compound of Formula I.
• amyloid plaques can also be treated by the compounds of Formula I, and include but are not limited to, for example, Lewy body dementia, inclusion body myositis, and cerebral amyloid angiopathy.
• Y is (5)-OH;
• R 4 is selected from hydrogen or alkyl;
• R 2 is hydrogen;
• R 3 is hydrogen; and
• X is selected from C ⁇ -Cn alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m - 0-CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , and -(CH 2 ) m -S-(CH 2 ) n CH 3 ;
• m is an integer from 1-12;
• n is an integer from 0-4; and the C-13 methyl is in the (S) configuration.
• Yet another embodiment of the present invention is directed to methods using compounds of a Formula (lb):
• Ri R 2 , R 3 , R 4 and X are as defined above for Formula (I). Even more preferably, Ri is selected from hydrogen, -OH and halo; R 4 is selected from hydrogen, halo and Ci- C 6 alkyl; R 2 is selected from hydrogen and halo; R 3 is selected from hydrogen, halo and - OH; and X is selected from C 1 -C 12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m COOCH 3 , -(CH 2 ) m -0- CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , -(CH 2 ) m -S-(CH 2 ) n CH 3 , -(CH 2 ) m -N- (CH 2 ) n CH 3 , -C 2 -C 8 alkenyl-0-(CH 2 ) )
• Ri is hydrogen;
• R 4 is selected from hydrogen or alkyl;
• R 2 is hydrogen;
• R 3 is hydrogen; and
• X is selected from alkyl, C 2 -C 12 alkenyl, -(CH 2 ) m -0-CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , and -(CH 2 ) m -S- (CH 2 ) n CH 3 ;
• m is an integer from 1-12;
• n is an integer from 0-4; and both the C-13 methyl and C-17 hydroxyl are in the (S) configuration.
• Still another embodiment of the invention is directed to methods using a compound of a Formula (Ic):
• Rn is hydrogen or Ci-C 6 alkyl
• R 4 is selected from hydrogen, halo and Ci-C 6 alkyl
• R 2 is selected from hydrogen and halo
• R 3 is selected from hydrogen, halo and - OH
• X is selected from C 1 -C 12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m COOCH 3 , -(CH 2 ) m -0- CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , -(CH 2 ) m -S-(CH 2 ) n CH 3 , -(CH 2 ) m -N- (CH 2 ) n CH 3 , -C 2 -C 8 alkenyl-0-(CH 2 )
• Rn is hydrogen;
• R 4 is selected from hydrogen or alkyl;
• R 2 is hydrogen;
• R 3 is hydrogen; and
• X is selected from alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m -0-CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , and -(CH 2 ) m -S- (CH 2 ) n CH 3 ;
• m is an integer from 1-12;
• n is an integer from 0-4; and both the C-13 methyl and C-17 hydroxyl are in the (S) configuration.
• Yet another embodiment of the present invention is directed to methods using a compound of a Formula (Id):
• R ls R 2 , and X are as defined above for Formula (I).
• Ri is selected from hydrogen, -OH and halo
• R 2 is selected from hydrogen and halo
• X is selected from C 1 -C 12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m COOCH 3 , -(CH 2 ) m -0-CH 3 , -(CH 2 ) m - 0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , -(CH 2 ) m -S-(CH 2 ) n CH 3 , -(CH 2 ) m -N-(CH 2 ) n CH 3 , -C 2 -C 8 alkenyl-0-(CH 2 ) n CH 3 , -C 2 -C 8 alkenyl-S-(CH 2 ) n CH 3 , -
• Yet another embodiment of the present invention is directed to methods using a compound of a Formula (Ie):
• m, n, R l s R 2 , R 3 and R4 are as defined above for Formula (I), and Z is selected from -0-, -S- and -NH-. Even more preferably, m is 1-12, n is 0-4, Ri is selected from hydrogen, -OH and halo; R4 is selected from hydrogen, halo and Ci-C 6 alkyl; R 2 is selected from hydrogen and halo; R 3 is selected from hydrogen, halo and -OH; Z is selected from -O- and -S-; and both the C-13 methyl and C-17 hydroxyl are in the (S) configuration.
• Still another embodiment of the present invention is directed to methods using a compound of a Formula (If):
• R ls R 2 , R3, R 4 and X are as defined above for Formula (I).
• Ri is selected from hydrogen, -OH and halo
• R 4 is selected from hydrogen, halo and Ci-C 6 alkyl
• R 2 is selected from hydrogen and halo
• R 3 is selected from hydrogen, halo and -OH
• X is selected from C1-C12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m COOCH 3 , -(CH 2 ) m -0-CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , -(CH 2 ) m -S- (CH 2 ) n CH 3 , -(CH 2 ) m -N-(CH 2 ) n CH 3 , -C 2 -C 8 alkenyl-0-(CH 2 )
• X is selected from C1-C12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m -0-CH 3 , -(CH 2 ) m -0- (CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , and -(CH 2 ) m -S-(CH 2 ) n CH 3 ;
• m is an integer from 1-20; and n is an integer from 0-8.
• Ri, R 2 , R 3 and R 4 are hydrogen; m is an integer from 1-12; and n is an integer from 0-4.
• Still another embodiment of the present invention is directed to methods using a compound of a Formula (Ig):
• R ls R 2 , R 3 , R 4 , Rn and X are as defined above for Formula (I).
• Ri is selected from hydrogen, -OH and halo
• R 4 is selected from hydrogen, halo and Ci-C 6 alkyl
• R 2 is selected from hydrogen and halo
• R 3 is selected from hydrogen, halo and -OH
• X is selected from C]-C 12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m COOCH 3 , -(CH 2 ) m -0-CH 3 , -(CH 2 ) m -0-(CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , -(CH 2 ) m -S- (CH 2 ) n CH 3 , -(CH 2 ) m -N-(CH 2 ) n CH 3 , -C 2 -C 8 alkeny
• X is selected from C 1 -C 12 alkyl, C 2 -Ci 2 alkenyl, -(CH 2 ) m -0-CH 3 , -(CH 2 ) m -0- (CH 2 ) n CH 3 , (CH 2 ) m -S-CH 3 , and -(CH 2 ) m -S-(CH 2 ) n CH 3 ;
• m is an integer from 1-20;
• Ri, R 2 , R 3 and R 4 are hydrogen; m is an integer from 1-12; and n is an integer from 1 from
• Embodiment compounds of the present invention can be used in a pharmaceutical composition.
• a composition can comprise one or more compounds selected from those discussed above, illustrated below or otherwise inferred herein, and combinations thereof.
• such a composition can comprise a pharmaceutically-acceptable carrier component.
• such a composition can comprise a racemic mixture of compounds.
• such a compound can be present as the S and R enantiomer, preferably its isolated and purified form which is substantially free of the other isomer.
• the compounds of the present invention may have asymmetric centers and may occur as a racemate, a racemic mixture or as individual and purified diastereomers or enantiomers such as (named via ChemDraw Ultra, Version 11.0(3) or 12.0) (6S,8R,9S, 135, 145)-3-hydroxy-6-(methoxymethyl)- 13-methyl-7,8,9, 11,12,13,15,16- octahydro-6H-cyclopenta[a]phenanthren-17(14H)-one (compound 1);
• the compounds of the present invention can also be prepared by a method comprising such steps as: (i) protecting an estrodial compound, (ii) acylating the protected estradiol compound at the benzylic 6-position with LIDAKOR/Butyl-Lithium/Diisopropylamine/potassium tert-amylate, (iii) reducing the position 6 aldehyde with lithium aluminum hydride, (iv) deprotecting the protected regions of the estradiol compound.
• a reaction scheme for preparing estradiol derivatives is given below in Scheme 2.
• alkyloxyalkyl derivatives involve selection of alkylating agents. Such derivatives would be understood by those skilled in art made aware of this invention, and is available through synthetic procedures of the sort described herein. Accordingly, without limitation, various Ci to C 6 alkyl and substituted alkyl reagents can be used as described herein to prepare the corresponding alkyloxyalkyl derivatives.
• methods of making 6-amino derivatives of the estradiol are disclosed in reaction schemes below. Accordingly, 6-methoxylated estradiols described in Schemes 1-2 are employed and converted to their respective amino derivatives.
• a method for initiating, enhancing or increasing gene transcription for RNA encoding the LPL protein gene and/or the ApoC2 protein gene in a cell comprising contacting the cell with an effective amount of a 6-substituted estradiol derivative selected from Formulas (I) and (la) to (If). It is to be understood that such initiating, enhancing or increasing of gene transcription can occur for one or more of these genes.
• salts of the compounds of this invention refer to non-toxic “pharmaceutically acceptable salts.” Other salts may, however, be useful in the preparation of the compounds according to the invention or of their pharmaceutically acceptable salts.
• salts encompassed within the term "pharmaceutically acceptable salts" refer to non-toxic salts which are generally prepared by reacting the free base with a suitable organic or inorganic acid. Representative salts include any such salt known in the art.
• suitable pharmaceutically acceptable salts thereof may include alkali metal salts, e.g., sodium or potassium salts; alkaline earth metal salts, e.g., calcium or magnesium salts; and salts formed with suitable organic ligands, e.g., quaternary ammonium salts.
• alkali metal salts e.g., sodium or potassium salts
• alkaline earth metal salts e.g., calcium or magnesium salts
• suitable organic ligands e.g., quaternary ammonium salts.
• the compounds of the present invention can be used in combination with other agents or other agents which will enhance the treatment regime for the mammalian subject.
• the compounds of the methods could be used in combination with other estrogen receptor- ⁇ modulators.
• the individual components of such combinations can be administered separately at different times during the course of therapy or concurrently in divided or single combination forms to patients or regions of such patients in need of such therapy.
• the instant invention is therefore to be understood as embracing all such regimes of simultaneous or alternating treatment and the term "administering" is to be interpreted accordingly.
• the scope of combinations of the compounds of this invention with other agents useful to treat the targeted disease includes in principle any combination with any pharmaceutical composition useful for treating disorders related to estrogen functioning.
• the compounds of the methods are prepared according the procedures disclosed in U.S.S.N. 12/627,874 and U.S. S.N. 13/232,798.
• the preparation of compound 21 is provided in Example 1.
• estradiol derivatives compounds 3 and 4 are synthesized in the following manner.
• the protected estradiol compound is prepared by reaction of ⁇ -estradiol with dihydropyran in THF, using toluenesulfonic acid or camphorsulfonic acid as catalyst. As one of ordinary skill in the art can appreciate, this reaction is an equilibrium reaction and does not go to completion under such conditions. Thus, both the mono-protected estradiols can be found in the reaction mixture.
• Such crude reaction mixture undergoes a trituration step with acetonitrile causing the desired bis-THP estradiol to crystallize in approximately 70 % yield.
• the key intermediate is obtained via acylation at the benzylic 6-position with the strong base mixture referred to as LiDAKOR: butyl lithium, diisopropylamine, and potassium tert-amylate.
• LiDAKOR strong base mixture
• the intermediate is then purified by column chromatography to give a syrup in approximately 50 % yield, still containing minor impurities and column solvents. Reduction of the aldehyde with an excess of lithium aluminum hydride results in high yields of the racemic hydroxymethyl estradiol compound.
• the methoxymethyl compound is prepared by methylation of hydroxymethyl estradiol compound with sodium hydride and methyl iodide.
• the methoxymethyl compound is purified by column chromatography to give a glassy foam. Deprotecting the protected groups give racemic 6 -methoxymethyl estradiol compound. Separation of the enantiomers is performed using chiral preparative HPLC to give the compounds 3 and 4.
• a chiral purity of >95:5 R:S is realized.
• a chiral purity of 86: 14 S:R is realized. It is well within the level of one of ordinary skill in the art to employ NMR for determination of the absolute stereochemistry of the 6-position, where the 4-and 6-protons are diagnostic.
• the study includes three human tumor cell lines: A549, Panc-1, and SK-OV-3.
• the lines are each grown in two flasks cultured to roughly 40% confluence.
• One of the flasks is treated by addition of compound to the culture media at a various concentrations, i.e. 10 ⁇ , 20 ⁇ 50 ⁇ , or 100 ⁇ .
• the other, mock treated, flask is treated only with the vehicle used to solubilize and deliver the drug.
• R A extracted from the pairs of treated and untreated samples is subjected to microarray analysis on Agilent Whole Human Genome Microarrays (G4112F). Each analysis reports the difference in abundance of messenger R As for each of the 41,000 specific mRNA detectors on the array.
• This direct comparison of the treated versus untreated samples for each cell line provides extremely sensitive detection of changes in mRNA abundance resulting from the drug treatment. As each cell line comparison is self-normalized, the results can be compared across the samples with high confidence.
• Three human tumor cell lines are each grown in two flasks cultured to roughly 40% confluence.
• One of the flasks is treated by addition of compound to the culture media at concentrations of 10 ⁇ , 50 ⁇ and 100 ⁇ .
• the other, mock treated, flask is treated only with the vehicle used to solubilize and deliver the drug. All flasks are cultured for a further 24 hours, and then the cells are scraped free and washed in ice-cold PBS, then collected by centrifugation. The harvested cells are immediately frozen, and stored at -80°C or colder.
• Total RNA is prepared from the frozen tissue samples using Trizol-based cell lysis followed by 65°C hot phenol extraction and RNeasy chromatography purification. The purified RNA samples are analyzed spectrophotometrically. The concentration of RNA is determined by measuring the absorbance at 260 nm (A260). Given an absorbance of 1 unit at 260 nm corresponds to 35 ⁇ g of RNA per ml when measured at pH 11. RNA Quality Assessment - A260/A280 Absorbance Ratios
• RNA has a theoretical A260/A280 ratio (10 mM Tris-Cl, pH 7.5) of approximately 2.1. Extracted RNAs having an A260/A280 ratio of 1.8 or greater provide excellent results in this assay.
• RNA is tested for relative integrity by determining the ratio of intact 28S and 18S ribosomal RNAs, using capillary electrophoresis (Agilent Bio Analyzer). Completely intact RNA has a 28S/18S ratio of 2.2. All RNAs accepted for array analysis have ratios exceeding 1, the minimal 28S/18S ratio for reliably reproducible microarray results as determined by review of internal reproducibility among samples with varying 28S/18S ratios.
• RNAs are labeled using 1 microgram of RNA as input to an Agilent Low Input Labeling reaction.
• Test RNA is labeled with Cy5 (650 nm emitter) and reference RNA is labeled with Cy3 (550 nm emitter) nucleotides. Labeling, hybridizations and subsequent washings are carried out on Agilent HlAv2 human expression chips. The resulting hybridized chips are scanned on an Agilent microarray scanner, and intensity information for each detector spot is extracted from the scanned image using Agilent feature extraction software.
• the key data for all three hybridizations is collected in a FileMaker Pro relational database to allow for easy formulation of searches that can identify genes that exhibit particular transcriptional patterns.
• the data reported are the red (treated) and green (untreated) background-subtracted signals. This is the least modified form of the data.
• a background "surface" is estimated across the slide, based on numerous probes that are not complementary to human DNA. These serve as estimators of both nonspecific binding of labeled cR A to array surfaces and non-specific binding of labeled cRNA to the immobilized DNA oligomers.
• This threshold reduces the number of expected false positives to a reasonable level given the -40,000 ratios that are being surveyed in each assay.
• a field that indicates significant change and the direction of the change relative to the untreated sample reduces the result of the assay to a trinary categorical; 1, up regulated relative to untreated, 0, unchanged relative to untreated and -1, down regulated relative to untreated (Tri). Using this representation, one easily constructs searches that identify genes that have changed in any single or multiple sets of experiments.

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• 2015
  • 2015-03-19 JP JP2016556841A patent/JP2017512769A/ja active Pending
  • 2015-03-19 AU AU2015231172A patent/AU2015231172A1/en not_active Abandoned
  • 2015-03-19 EP EP15765290.0A patent/EP3119402A4/en not_active Withdrawn
  • 2015-03-19 SG SG11201607693WA patent/SG11201607693WA/en unknown
  • 2015-03-19 CN CN201580014623.7A patent/CN106255501A/zh active Pending
  • 2015-03-19 US US14/663,043 patent/US20150265630A1/en not_active Abandoned
  • 2015-03-19 KR KR1020167028986A patent/KR20160136363A/ko not_active Withdrawn
  • 2015-03-19 MX MX2016012045A patent/MX2016012045A/es unknown
  • 2015-03-19 BR BR112016021408A patent/BR112016021408A2/pt not_active IP Right Cessation
  • 2015-03-19 CA CA2940536A patent/CA2940536A1/en not_active Abandoned
  • 2015-03-19 WO PCT/US2015/021538 patent/WO2015143201A2/en not_active Ceased
• 2018
  • 2018-02-12 AU AU2018201003A patent/AU2018201003A1/en not_active Abandoned

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