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WO1996013258A1 - Analogues c-glycosidiques de n-(4-hydroxyphenyle) retinamide-o-glucuronide - Google Patents

Analogues c-glycosidiques de n-(4-hydroxyphenyle) retinamide-o-glucuronide Download PDF

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WO1996013258A1
WO1996013258A1 PCT/US1995/012258 US9512258W WO9613258A1 WO 1996013258 A1 WO1996013258 A1 WO 1996013258A1 US 9512258 W US9512258 W US 9512258W WO 9613258 A1 WO9613258 A1 WO 9613258A1
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retinamide
glucuronide
retinamido
mmol
mixture
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PCT/US1995/012258
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English (en)
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Robert W. Curley, Jr.
Michael J. Robarge
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The Ohio State Research Foundation
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Priority claimed from US08/316,140 external-priority patent/US5516792A/en
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Priority to JP8511156A priority Critical patent/JPH09509680A/ja
Priority to EP95935115A priority patent/EP0732921A4/fr
Publication of WO1996013258A1 publication Critical patent/WO1996013258A1/fr

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    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • A—HUMAN NECESSITIES
    • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
    • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
    • A61P35/00—Antineoplastic agents
    • C—CHEMISTRY; METALLURGY
    • C07—ORGANIC CHEMISTRY
    • C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
    • C07H13/00—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids
    • C07H13/02—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids
    • C07H13/04—Compounds containing saccharide radicals esterified by carbonic acid or derivatives thereof, or by organic acids, e.g. phosphonic acids by carboxylic acids having the esterifying carboxyl radicals attached to acyclic carbon atoms
    • C07H13/06—Fatty acids

Definitions

  • retinoic acid a vitamin A metabolite, and certain retinoic acid analogues
  • retinoic acid appear to be necessary for the maintenance of normal epithelial tissue differentiation and can reverse the metaplastic condition of hamster tracheal in vitamin A deficient epithelial tissue.
  • retinoic acid and certain amide analogues have been proposed as cancer chemopreventive agents, and display cancer preventive activity (Moon et al . Cancer Res . (1979) 3_9., 1339-1346) .
  • retinoic acid analogues such as retinyl acetate, 13 -cis- retinoic acid, glucuronide analogues of retinoic acid also display cancer preventive activity, including breast cancer preventative activity (Hill, D.L. et al. Ann. Rev. Nutrition (1992) 12., 161-181 and Mehta, R.G. et al . Oncology (1991) _____.1505-1509).
  • the glucuronide N- (4-hydroxyphenyl)retinamide-O-glucuronide (hereinafter "4-HPR-O-glucuronide”) has a greater antiproliferative activity and less toxicity in both MCF-7 human mammary cell tumor culture than the 4-HPR. (Bhatnagar, R. et al . Biochem. Pharmacol . (1991) 11, 1471-1477.)
  • the 4-HPR-O-glucuronide is unstable; it is hydrolyzed in acidic media and also by the enzyme 0-glucuronidase.
  • the propensity to acid hydrolysis may limit the clinical usefulness of 4-HPR-O- glucuronide since oral administration of the drug may reduce the total available concentration of the active drug.
  • /3-glucuronidase is present in virtually all mammalian cells, including the blood, liver and intestine, the in vivo half life is quite limited. It is desirable to have stable chemopreventive drugs, for the prevention and treatment of breast cancer, which resist acid and 3-glucuronidase hydrolysis.
  • the present invention provides breast cancer chemopreventive and treatment arylamide analogues of retinoic acid, more particularly C- glycoside analogues of N- (4-hydroxyphenyl)retinamide-O-glucuronide and N-glycoside analogues of retinoyl ⁇ -glucuronide that resist both ⁇ - glucuronidase mediated enzymatic hydrolysis as well as acid catalyzed hydrolysis.
  • the drugs include 4- (retinamido)phenyl-C- glucuronide; 4- (retinamido)phenyl-C-glucoside; 4- (retinamido)benzyl-C- xyloside; 4- (retinamido)benzyl-C-glucoside; 4- (retinamido)benzyl- C-glucuronide; 4- (retinamido)phenyl-C-xyloside, 1- (B-D-glucopyranosyl) retinamide and 1- (D-glucopyranosyluronosyl) retinamide.
  • Figure 1 is a graph showing competition for human retinoic acid receptors/BCV between [ 3 H] -retinoic acid and: unlabeled retinoic acid, represented by the squares, and 1- (B-D-glucopyranosyl) retinamide, represented by the triangles or 1- (D-glucopyranosyluronosyl) retinamide represented by the diamonds. Values represent the means of three determinations;
  • Figure 2 is a graph showing competition for human retinoic acid receptors alpha/BCV between [ 3 H] -retinoic acid and: unlabeled retinoic acid, represented by squares, and 1- (B-D-glucopyranosyl) retinamide, represented by the triangles, or l- (D-glucopyranosyluronosyl) retinamide represented by the diamonds.
  • Figure 3 is a graph showing competition for human retinoic acid receptors beta/BCV between [ 3 H] -retinoic acid and: unlabeled retinoic acid, represented by closed squares, and 1- (B-D-glucopyranosyl) retinamide, represented by the triangles, or 1- (D- glucopyranosyluronosyl) retinamide represented by the diamonds. Values represent the means of three determinations.
  • the present invention provides breast cancer chemopreventive drugs that may also be used for the treatment of cancer, that are arylamide analogues of retinoic acid, more particularly C-glycoside analogues of N- (4-hydroxyphenyl)retinamide-O-glucuronide and analogues of retinoyl ⁇ -glucuronide that are not hydrolysed by acid nor the enzyme 3-glucuronidase.
  • the drugs include 4- (retinamido)phenyl-C-glucuronide; 4- (retinamido)phenyl-C-glucoside; 4- (retinamido)benzyl-C-xyloside; 4- (retinamido)benzyl-C-glucoside; 4- (retinamido)benzyl-C-glucuronide; 4- (retinamido)phenyl-C-xyloside.
  • the structure of the N- (4-hydroxyphenyl) retinamide-O-glucuronide analogues is represented below:
  • the molecule is 4- (retinamido)phenyl-C- glucuronide.
  • the molecule is 4- (retinamido)benzyl-C- xyloside.
  • the molecule is 4- (retinamido)benzyl-C-glucoside.
  • the structure of the retinoyl ⁇ -glucuronide analogues is represented below:
  • R' is H, COOH or CH 2 OH.
  • the drugs may be prepared according to the following examples. PREPARATION OF THE DRUGS Example 1: 4- (Retinamido)phenyl-C-glucuronide
  • acetobromoglucose (12.1 mmol) was dissolved in 125 ml of ether and added over 1 hour to phenylmagnesium bromide prepared from 5.6 g (0.23 g atom, 19 equiv.) of magnesium turnings and 25 ml (237 mmol, 19 equiv.) of bromobenzene in 250 ml of ether.
  • the mixture was heated at reflux for 6 hours then poured into 400 ml H 2 0.
  • the 20 ml glacial acetic acid was added to dissolve the magnesium salts. The mixture was shaken and the layers were separated.
  • aqueous layer was concentrated to dryness and the residue was reacted with 70 ml of acetic anhydride and 100 ml of pyridine overnight.
  • the mixture was poured into 750 ml H 2 0 and the precipitate was isolated by filtration and recrystallized from 2-propanol, to yield 2.236 g (45%), of tetra-0- acetylphenyl-C-glucoside.
  • Retinoyl chloride was prepared from 264 mg (0.88 mmol) retinoic acid, and 73 ⁇ L of pyridine, 65 ⁇ h (0.88 mmol) of thionyl chloride in 10 ml of ether at 0°C. The mixture was allowed to warm to room temperature over 1 hour. Then 360 mg (0.88 mmol) of 4- (methyltri-O- acetyl) aminophenyl-C-glucuronide was added in 15 ml of benzene containing 100 ⁇ L of pyridine.
  • Example 2 4- (Retinamido)phenyl-C-glucoside First, 14 g of glucose pentaacetate, from Sigma Chemical Company, was converted to 10.88 g of acetobromoglucose as described in Example 1.
  • aqueous layer was concentrated to dryness and the residue was reacted with 70 ml of acetic anhydride and 100 ml of pyridine overnight.
  • the mixture was poured into 750 ml H 2 0 and the precipitate was isolated by filtration and recrystallized from 2-propanol, to yield 2.236 g (45%), of tetra-O-acetylphenyl-C-glucoside.
  • 5.63 g. (13.8 mmol) tetra-O-acetyl phenyl-C-glucoside was dissolved in 150 ml of acetic anhydride and then 25.58 g (110.4 mmol, 8.0 equiv.) copper (II) nitrate was added.
  • retinoyl chloride was prepared by dissolving 192 mg (0.64 mmol) of retinoic acid in 10 ml of ether containing 100 ml of pyridine and cooled to 0°C. Then 47 ⁇ l_ (0.64 mmol) thionyl chloride was added. The mixture was allowed to come to room temperature over 90 minutes and then 270 mg (0.64 mmol) of the 4- (tetra-O-acetyl) aminophenyl-C- glucoside was added in 9 ml benzene and 1 ml pyridine.
  • xylose from Sigma Chemical Company
  • 12.5 g of sodium acetate (anhydrous) and 200 ml of acetic anhydride were heated to reflux for 1 hour and poured into 1 L of ice water.
  • Xylose tetraacetate was isolated by filtration and recrystallized from 95% ethanol to yield 56.75 g (54%).
  • the crude acetochloroxylose was dissolved in 100 ml ether and added to a refluxing solution of phenylmagnesium bromide prepared from 18 ml (169 mmol, 10.8 equiv.) of bromobenzene and 4.12 g (0.169 g atom, 10.8 equiv) of magnesium turnings in 250 ml of ether.
  • the mixture was heated at reflux for 5 hours and poured into 150 ml H 2 0 containing 15 ml of glacial acetic acid.
  • the aqueous layer was concentrated to dryness and the residue was reacted with 75 ml acetic anhydride and 75 ml of pyridine for 20 hours.
  • the mixture was poured into 300 ml H 2 0.
  • the product was isolated by filtration and recrystallized from 2-propanol, to yield 1.28 g (24%), tri-O-acetylphenyl-C-xyloside having a melting point of 161-162°C
  • the ether extracts were washed twice with 150 ml with water then washed twice with 50 ml saturated NaHC0 3 , then washed with 50 ml brine, dried using MgS0 4 and concentrated to yield 1.347 g (99%) of a 1:1 mixture of o- and p-tri-0- acetyl nitrophenyl-C-xylosides.
  • Retinoyl chloride was prepared by dissolving 320 mg (1.07 mmol) retinoic acid suspended in 10 ml ether containing 200 ⁇ l of pyridine. The mixture was cooled to 0°C. and 77 ⁇ l (1.07 mmol) of thionyl chloride was added. The mixture was allowed to warm to room temperature over 1.5 hours. The acetylated aminophenyl-C-xyloside was suspended in 16 ml benzene and 800 ⁇ l pyridine was added.
  • the mixture was shaken and separated.
  • the ether layer was washed twice with 50 ml saturated NaHC0 3 , once with 50 ml brine, dried using MgS0 4 , and concentrated.
  • the residue was dissolved in 80 ml glacial acetic acid containing 0.1 g of 10% palladium on carbon and shaken under 40 PSI of hydrogen for 19 hours.
  • the catalyst was removed by filtration and the filtrate was concentrated to dryness.
  • the residue was acetylated with 30 ml of pyridine and 30 ml of acetic anhydride over 16 hours.
  • the reaction was poured into 100 ml of water and extracted twice with 50 ml of ether.
  • the ether extracts were washed twice with 50 ml water, once with 50 ml 5% HC1, once with 50 ml saturated NaHC0 3 , once with 50 ml brine.
  • the extracts were dried using MgS0 4 , concentrated, and crystallized from 2- propanol to yield 194 mg (16%) , tetra-O-acetyl benzyl-C-glucoside having a melting point of 118-119°C.
  • the crude nitrobenzyl-C-glucoside was dissolved in 30 ml of methanol and 340 mg of potassium carbonate was added. The mixture was stirred at room temperature overnight, concentrated, and dissolved in 100 ml of water containing 1 g of freshly reduced platinum (IV) oxide. The mixture was heated to 80°C and oxygen was bubbled in for 10 hours. The catalyst was removed by filtration and the filtrate concentrated to dryness. The residue was heated to reflux in 100 ml of 2% H 2 S0 4 in methanol for 1 hour. Excess H 2 S0 4 was neutralized with 10 ml of pyridine.
  • the mixture was concentrated to dryness and the residue was reacted with 30 ml of pyridine and 60 ml of acetic anhydride for 16 hours.
  • the mixture was poured into 300 ml of ice water and extracted twice with 100 ml ether.
  • the ether layers were washed twice with 200 ml with water, once with 100 ml saturated NaCH0 3 , dried using MgS0 4 , and concentrated to yield 1.107 g (66%) of a 1 to 1 mixture ortho and para methyl tri-O-acetyl nitrobenzyl-C-glucuronides.
  • the 1.107 g (2.44 mmol) of the 4-nitrobenzyl-C-glucuronide mixture was dissolved in 100 ml of methanol and 100 mg of 10% palladium on carbon was added. The mixture was shaken under 40 PSI of hydrogen for 1 hour, the catalyst was removed by filtration, and the filtrate was concentrated and chromatographed on silica gel using 1:1 ethyl acetate/hexanes as elutant to yield 310 mg (30%) of methyl tri-O- acetyl-4-aminobenzyl-C-glucuronide.
  • the retinoylated benzyl-C-glucuronide was prepared as in Example 1. From 219 mg (0.73 mmol) of retinoic acid and 310 mg (0.73 mmol) of the protected 4-aminobenzyl-C-glucuronide, 303 mg (59%) of the protected retinamidobenzyl-C-glucuronide having a melting point 80-82°C was obtained after chromatography.
  • the crude nitro benzyl-C-glucoside was dissolved in 25 ml of methanol containing 50 mg of 10% palladium on carbon and was shaken under 40 PSI of hydrogen for 1 hour. The catalyst was removed by filtration. The filtrate was concentrated and chromatographed on silica gel using 1:1 ethyl acetate/hexanes as elutant, to yield 173.2 mg (33%), 4- (tetra-O- acetyl) aminobenzyl-C-glucoside having a melting point of 145-146°C.
  • the retinamide was prepared as in Example 2.
  • xylose from Sigma Chemical Company
  • xylose tetracetate as described in Example 3.
  • 2.07g (6.5 mmol) of the xylose tetracetate was dissolved in 125 ml dry chloroform and 3.3 g (24.8 mmol, 3.8 equiv.) aluminum chloride was added.
  • the mixture was stirred at room temperature for 1.5 hours then washed once with 50 ml saturated NaHC0 3 , once with 50 ml water, and once with 50 ml brine, then dried using MgS0 , and the solvent removed.
  • the product from multiple runs of a synthesis step may have been pooled to obtain the quantities used in subsequent steps.
  • Example 7 preparation of 1- ( ⁇ -D-glucopyranosyl) retinamide Preparation of (2,3,4,6-tetra-0-acetylglucopyranosyl azide)
  • 2,3,4,6-tetra-0-acetylglucopyranosyl bromide was prepared from glucose pentaacetate by dissolving 50 gm of glucose pentaacetate, available from Sigma Chemical Company, in 200 mL of 30% HBr/acetic acid; the mixture was allowed to stand overnight in the refrigerator. The solvent was removed under reduced pressure, the residue dissolved in CHCI 3 , and the CHC1 3 solution was washed with H 2 0, saturated NaHC0 3 , saturated NaCl, and dried over Na 2 S0 4 . The drying agent was removed by filtration and the residue crystallized from ethanol. The acetobromoglucose has a melting point of 89°C.
  • the residue was recrystallized from ethanol to yield 6.1 g, that is, 82% of 2,3,4,6-tetra-0-acetylglucopyranosyl azide.
  • Retinoyl chloride was prepared from 3.24 g (10.8 mmol) retinoic acid, 1. 1 ml of pyridine, and 0.79 ml (10.8 mmol) of thionyl chloride in 60 ml of ether at 0°C. The mixture was allowed to warm to room temperature over 1 hour, and 3.82g, 11 mmol of 2, 3,4,6-tetra-0- acetylglucopyranosyl amine was added in 50 ml of benzene containing 3 ml of pyridine. The mixture was stirred at room temperature for 72 hours, diluted with 200 ml of ethyl acetate, and extracted three times with 150 ml H 2 0 and brine.
  • 1- (B-D-glucopyranosyl) retinamide has the following characteristics: melting point of 166°C (decomposition); IR (KBr) 3369, 2927, 1662 cm “1 ; UV (CH 3 0H) ⁇ , ⁇ 349 nm ( ⁇ 24043); ⁇ H NMR (DMS0-d s ) ⁇ 1.00 (s, 6H, C(CH 3 ) 2 ), 1.42 (m, 2H, retinoid H-2) 11 , 1.55 (m, 2H, retinoid H-3) , 1.70 (s, 3H, retinoid 5-CH 3 ) , 1.95 (br s, 5H, retinoid 9CH 3 and H-4) , 2.28 (s, 3H, retinoid 13-CH 3 ) , 3.15 (br s, 4H, -OH), 3.4-3.7 (m, 6H, pyranose H's), 4.75 (br t, 1H, J-9Hz
  • Example 8 l- (D-glucopyranosyluronosyl) retinamide: First methyl-1,2,3,4-tetra-0-acetylglucuronate was prepared from glucurono-6,3-lactone by dissolving 40 gm of glucurono-6,3-lactone, available from Aldrich Chemical Co., in 300 mL of CH 3 OH containing 100 mg NaOH and allowed to stand one hour. The solvent was removed under reduced pressure and the residue dissolved in 100 mL pyridine and 150 mL acetic anhydride; the mixture was stored in the refrigerator.
  • the resulting solid methyl-1,2,3,4-tetra-0-acetylglucuronate was filtered and recrystallized from 95% ethanol.
  • the methyl-1,2,3,4-tetra-0- acetylglucuronate has a melting point of 178°C.
  • Methyl 1-bromo-2,3,4-tetra-0-acetylglucuronate was prepared from methyl-1,2,3,4-tetra-0-acetylglucuronate. 50 gmethyl-1,2,3,4-tetra-0- acetylglucuronate was dissolved in 200 mL of 30% HBr/acetic acid; the mixture was allowed to stand overnight in the refrigerator.
  • the solvent was removed under reduced pressure, the residue was dissolved in CHC1 3 and the CHC1 3 solution was washed with H 2 0, saturated NaHC0 3 , saturated NaCl and dried over Na 2 S0 4 .
  • the drying agent was removed by filtration and the residue crystallized from ethanol.
  • the methyl 1- bromo-2,3,4-tetra-0-acetylglucuronate has a melting point of 107°C.
  • Methyl 2,3,4-tri-0-acetylglicopyranuronosyl bromide (7.94 g, 20 mmol) was dissolved in 250 ml of dimethylformamide and 2.6 g, that is 40 mmol sodium azide was added. The mixture was stirred at room temperature for 24 hours and then poured into 500 ml of H 2 0. The organic soluble material was extracted three times with 150 ml ethyl acetate. The combined ethyl acetate extracts were washed twice with 100 ml H 2 0, brine, dried over MgS0 4 , and concentrated to dryness.
  • Retinoyl chloride was prepared by reacting 4.5 g, 15 mmol retinoic acid , 2 ml of pyridine, and 1.09 ml, that is, 15 mmol of thionyl chloride in 85 ml of ether at 0°C. The mixture was cooled to - 15°C and 5.66 g, that is, 17 mmol methyl 2,3,4-tri-0- acetylglucopyranuronosyl amine was added in 70 ml of benzene containing 3.3 ml of pyridine.
  • the ethyl acetate extracts were dried over MgS0 4 and concentrated to yield 1.58 g of 1- (D-glucopyranosyluronosyl) retinamide, (83%) .
  • the 1- (D-glucopyranosyluronosyl) retinamide was purified by chromatography on RP-18 with 80% MeOH/H 2 0.
  • the 1- (D- glucopyranosyluronosyl) retinamide so obtained, is a mixture of ⁇ and ⁇ epimers in approximately a 4 to 1 ratio.
  • 1- (D-glucopyranosyluronosyl) retinamide and RAG were treated with 0.1 N HCl at 37°C for two hours.
  • the RAG had undergone approximately 50% solvolysis to liberate retinoic acid as determined by HPLC.
  • the 1- (D-glucopyranosyluronosyl) retinamide remained intact.
  • the 4-HPR-O-glucuronide is hydrolysed to glucuronic acid and the aglycone by /S-glucuronidase, the 4- (retinamido)phenyl-C- glucuronide and the 4- (retinamido)benzyl-C-glucuronide are not. This indicates that the 4- (retinamido)phenyl-C-glucuronide and the 4- (retinamido)benzyl-C-glucuronide would not be hydrolyzed by the enzyme in vivo.
  • the 4- (retinamido)phenyl-C-glucuronide and 4- ⁇ retinamido)benzyl-C-glucuronide act as inhibitors of 0-glucuronidase mediated hydrolysis of N-4-hydroxyphenylretinamide-O-glucuronide to N- 4-hydroxyphenylretinamide and glucuronic acid with 4- (retinamido)benzyl-C-glucuronide being slightly more effective than the 4- (retinamido)phenyl-C-glucuronide.
  • the Km value of 3-glucuronidase for 4-HPR-O-glucuronide under experimental conditions was determined to be 184.5 ⁇ M, using the method of Beinfeld, P. et al . , J.
  • the concentration of the 4- HPR-O-glucuronide in the inhibition study was selected to be below 184.5 ⁇ M.
  • the IC 50 values were determined according to the method of Lang, J.H. et. al . J. Med. Chem. (1971), 14, 223-236, using 10% methanol as a co-solvent, for 4- (retinamido)phenyl-C-glucuronide and 4- (retinamido)benzyl-C-glucuronide.
  • the IC 50 values which represent the concentration of the drug that inhibits the cleavage of 50% of the 4- HPR-O-glucuronide, were found to be 267 ⁇ M for the 4- (retinamido)phenyl-C-glucuronide and 236 ⁇ M for the 4- (retinamido)benzyl-C-glucuronide.
  • the IC 50 values for the C-phenyl and C-benzyl glycosides indicate that they significantly inhibit /S-glucuronidase.
  • 1- (D-glucopyranosyluronosyl) retinamide and RAG were evaluated for their stability toward 3-glucuronidase medicated hydrolysis under the same conditions as described above.
  • the antiproliterative activity of the drugs in MCF-7 human mammary tumor cell culture models were determined.
  • the MCF-7 cells were obtained originally from the Michigan Cancer Foundation. The cells were routinely grown as monolayers in T-25 plastic tissue culture flasks from Falcon, in Oxnard, CA containing 5.0 mL of B-10 medium consisting of Minimum Essential Medium (MEM, Earle's Base) supplemented with 1.0 mM MEM non-essential amino acid, 2.0 mM 1- glutamine, 10% fetal bovine serum (FBS) , and antibiotics. Culture conditions included a humidified atmosphere of 5% C0 2 at 37°C. One-half of the medium was replenished twice a week. Cells were harvested by trypsinization upon confluency. All variables were evaluated in triplicate.
  • MEM Minimum Essential Medium
  • FBS fetal bovine serum
  • the MCF-7 cells were plated in 24-well plates at a density of 1.5 x 10 4 cells/cm 2 . After a 24 hour period and again on day 4, the medium was replaced with treatment medium containing the specified drugs at 10" s to 10'* M in absolute ethanol and added to the cell cultures so that the ethanol concentration did not exceed 0.1%. Control cultures were treated with an equivalent volume of absolute ethanol. On day 7 the cells were harvested by trypsinizatio ⁇ and viability was assessed by trypan blue exclusion according to Ueda, H. et al . Cancer 1980, 46, 2203-2209. Total cell counts were obtained using a hemocytometer as well as a the Coulter Counter. Growth inhibition, that is, cell survival, was calculated for the various drug concentrations using the equation: % inhibition ⁇ 100 - R/C X 100 where R and C are the number of cells in the drug-treated and control cultures, respectively.
  • the ED 50 for growth inhibition of the MCF-7 cells by the drugs has been evaluated in three separate assays as shown in Table I.
  • the 4- (retinamido)phenyl-C-glucuronide appears to show a potency consistent with its glucoside analogue, retinamidophenyl-C-glucoside.
  • the 4- (retinamido)benzyl-C-glucuronide and its glucoside analogue, 4- (retinamido)benzyl-C-glucose exhibit the best growth inhibition of the cells.
  • the glucuronides and glycosides drugs of the present invention are more effective than the xylosides of the present invention and the benzyl drugs are more active than the phenyl drugs. Although the mechanism of the drugs is not well understood, it has been shown that active drugs in this class compete with retinoic acid for nuclear receptors for retinoic acid.
  • Retinoic acid receptors, (RAR) a, ⁇ , and ⁇ were expressed using a baculovirus expression system in Spodoter fr giperda insect cells. DNA, coding for each receptor was introduced into the genome of the baculovirus which was then introduced into the cultured insect cells.
  • Whole cell extracts were prepared by homogenizing cells in 50 mM Tris buffer at pH 7.4 containing 1.5 mM EDTA, 5 mM dithiothreitol, 300 mM KC1 and the protease inhibitors, 1 mM phenylmethylsulfonyl fluoride and 5 ug/ml soybean trypsin inhibitor.
  • the homogenate was subjected to centrifugation at 45,000 rpm for 45 minutes at 4 ⁇ C in a Ti 70.1 Beckman rotor. The supernatant fraction was stored at -70°C until use.
  • 500 ⁇ l portions of the retinoic acid receptor-containing protein extracts were incubated on ice for 3 hours in the presence of 5 nM [ 3 H] trans-retinoic acid, from DuPont-NEN, 50.6 Ci/mmole) (RA) ⁇ 1 ⁇ M of each of the drugs listed in Table II or the equivalent volume of ethanol. Ethanol comprised 3.8% of the final volume in each tube.
  • the drugs of this invention exert their chemopreventative effect by binding to receptors for retinoic acid, then the data supports the preference for 4- (retinamido)phenyl-C-glucuronide, 4- (retinamido)benzyl-C-glucosideandparticularly4- (retinamido)benzyl-C- glucuronide, which significantly compete with retinoic acid for the receptors.
  • the human retinoic acid receptor cDNA was obtained by reverse transcription/PCR amplification using RNA from MCF-7 cells. The coding region of the cDNA was subcloned into the baculovirus vector pVL1393. Production of the receptor protein, human retinoic acid receptor ⁇ /BCV, followed conventional procedures.
  • Generation of retinoic acid receptors or and ⁇ , and competition binding methodology are described in Proc. Nat'l Acad. Sci. USA, 1993, 90, 7293.
  • l- (B-D-glucopyranosyl) retinamide, and 1- (D-glucopyranosyluronosyl) retinamide were 1500- and -500-fold less effective than retinoic acid in competing for [ 3 H] -retinoic acid binding to retinoic acid receptor ⁇ .
  • the Ki constant was 0.5 nM for retinoic acid, 710 nM for 1- (B-D-glucopyranosyl) retinamide, and 280 nM for 1- (D-glucopyranosyluronosyl) retinamide.
  • the drugs of the present invention while described as cancer preventatives and cancer treatments, may also be used in the study of cancer, particularly breast cancer. Evaluation in-vivo
  • the first tumors were not observed at until 64 days post DMBA intubation in the rats that received 1- (B-D-glucopyranosyl) retinamide, and 1- (D- glucopyranosyluronosyl) retinamide. 58% of the rats that received l- (B-D-glucopyranosyl) retinamide and 50% of the rats that received 1-(D- glucopyranosyluronosyl) retinamide had developed tumors by 110 days post intubation.
  • 1- (D-glucopyranosyluronosyl) retinamide caused the largest reduction in multiple mammary tumors.
  • the control rats had an average of 1.5 tumors per rat compared to 0.83 tumors per rat in animals receiving 1- (D- glucopyranosyluronosyl) retinamide, to 0.92 tumors per rat in animals receiving 1- (B-D-glucopyranosyl) retinamide.
  • 1- (B-D-glucopyranosyl) retinamide, and 1- (D-glucopyranosyluronosyl) retinamide show a greater mammary tumor chemopreventive effect than does retinoic acid.
  • 1- (B-D- glucopyranosyl) retinamide, and 1- (D-glucopyranosyluronosyl) retinamide inhibit DMBA induced mammary tumor development.

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Abstract

L'invention concerne des analogues arylamides d'acide rétinoïque chimiopréventifs du cancer du sein, plus particulièrement des analogues C-glycosidiques de N-(4-hydroxyphényl) rétinamide-O-glucuronide et un analogue N-glycosidique de β-glucuronide de rétinoyle résistant à la fois à l'hydrolyse enzymatique induite par β-glucuronidase et à l'hydrolyse catalysée par acide. Spécifiquement, les médicaments comprennent 4-(rétinamido)phényl-C-glucuronide; 4-(rétinamido)phényl-C-glucoside; 4-(rétinamido)benzyl-C-xyloside; 4-(rétinamido)benzyl-C-glucoside; 4-(rétinamido)benzyl-C-glucuronide; 4-(rétinamido)phényl-C-xyloside, 1-(B-D-glucopyranosyl)rétinamide et 1-(D-glucopyranosyluronosyle). L'invention concerne également un procédé de production de ces médicaments.
PCT/US1995/012258 1994-09-30 1995-09-26 Analogues c-glycosidiques de n-(4-hydroxyphenyle) retinamide-o-glucuronide WO1996013258A1 (fr)

Priority Applications (2)

Application Number Priority Date Filing Date Title
JP8511156A JPH09509680A (ja) 1994-09-30 1995-09-26 Ｎ−（４−ヒドロキシフェニル）レチンアミド−ｏ−グルクロニドのｃ−グリコシド類似物
EP95935115A EP0732921A4 (fr) 1994-09-30 1995-09-26 Analogues c-glycosidiques de n-(4-hydroxyphenyle) retinamide-o-glucuronide

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
US08/316,140 US5516792A (en) 1992-11-13 1994-09-30 C-glycoside analogues of N-(4-hydroxyphenyl)retinamide-O-glucuronide
US08/316,140 1994-09-30
US08/321,207 US5574177A (en) 1992-11-13 1994-10-07 Arylamide analogues of N-(4-hydroxyphenyl) retinamide-o-glucuronide
US08/321,207 1994-10-07

Publications (1)

Publication Number Publication Date
WO1996013258A1 true WO1996013258A1 (fr) 1996-05-09

Family

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PCT/US1995/012258 WO1996013258A1 (fr) 1994-09-30 1995-09-26 Analogues c-glycosidiques de n-(4-hydroxyphenyle) retinamide-o-glucuronide

Country Status (3)

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EP (1) EP0732921A4 (fr)
JP (1) JPH09509680A (fr)
WO (1) WO1996013258A1 (fr)

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117845A (en) * 1999-06-11 2000-09-12 Ohio State University Research Foundation C-linked analogs of N-(4-hydroxyphenyl) retinamide
WO2003018598A3 (fr) * 2001-08-30 2003-11-27 Univ Dublin Derives de monosaccharides
US9024009B2 (en) 2007-09-10 2015-05-05 Janssen Pharmaceutica N.V. Process for the preparation of compounds useful as inhibitors of SGLT
US9035044B2 (en) 2011-05-09 2015-05-19 Janssen Pharmaceutica Nv L-proline and citric acid co-crystals of (2S, 3R, 4R, 5S,6R)-2-(3-((5-(4-fluorophenyl)thiopen-2-yl)methyl)4-methylphenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol
US9056850B2 (en) 2008-10-17 2015-06-16 Janssen Pharmaceutica N.V. Process for the preparation of compounds useful as inhibitors of SGLT
US9174971B2 (en) 2009-10-14 2015-11-03 Janssen Pharmaceutica Nv Process for the preparation of compounds useful as inhibitors of SGLT2
US10544135B2 (en) 2011-04-13 2020-01-28 Janssen Pharmaceutica Nv Process for the preparation of compounds useful as inhibitors of SGLT2
US10617668B2 (en) 2010-05-11 2020-04-14 Janssen Pharmaceutica Nv Pharmaceutical formulations
US11576894B2 (en) 2009-07-08 2023-02-14 Janssen Pharmaceutica Nv Combination therapy for the treatment of diabetes

Families Citing this family (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
AR056195A1 (es) * 2005-09-15 2007-09-26 Boehringer Ingelheim Int Procedimientos para preparar derivados de (etinil-bencil)-benceno sustituidos de glucopiranosilo y compuestos intermedios de los mismos

Family Cites Families (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
EP0671953A4 (fr) * 1992-11-13 1996-01-10 Univ Ohio State Res Found Analogues arylamide de n-(4-hydroxyphenyl)retinamide-o-glucuronide.

Non-Patent Citations (2)

* Cited by examiner, † Cited by third party
Title
JOURNAL OF LIPID RESEARCH, Volume 26, issued 1985, ARUN B. BARUA et al., "Chemical Synthesis of All-Trans Retinoyl Beta-Glucuronide", pages 1277-1282. *
See also references of EP0732921A4 *

Cited By (9)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
US6117845A (en) * 1999-06-11 2000-09-12 Ohio State University Research Foundation C-linked analogs of N-(4-hydroxyphenyl) retinamide
WO2003018598A3 (fr) * 2001-08-30 2003-11-27 Univ Dublin Derives de monosaccharides
US9024009B2 (en) 2007-09-10 2015-05-05 Janssen Pharmaceutica N.V. Process for the preparation of compounds useful as inhibitors of SGLT
US9056850B2 (en) 2008-10-17 2015-06-16 Janssen Pharmaceutica N.V. Process for the preparation of compounds useful as inhibitors of SGLT
US11576894B2 (en) 2009-07-08 2023-02-14 Janssen Pharmaceutica Nv Combination therapy for the treatment of diabetes
US9174971B2 (en) 2009-10-14 2015-11-03 Janssen Pharmaceutica Nv Process for the preparation of compounds useful as inhibitors of SGLT2
US10617668B2 (en) 2010-05-11 2020-04-14 Janssen Pharmaceutica Nv Pharmaceutical formulations
US10544135B2 (en) 2011-04-13 2020-01-28 Janssen Pharmaceutica Nv Process for the preparation of compounds useful as inhibitors of SGLT2
US9035044B2 (en) 2011-05-09 2015-05-19 Janssen Pharmaceutica Nv L-proline and citric acid co-crystals of (2S, 3R, 4R, 5S,6R)-2-(3-((5-(4-fluorophenyl)thiopen-2-yl)methyl)4-methylphenyl)-6-(hydroxymethyl)tetrahydro-2H-pyran-3,4,5-triol

Also Published As

Publication number Publication date
EP0732921A1 (fr) 1996-09-25
EP0732921A4 (fr) 1998-09-02
JPH09509680A (ja) 1997-09-30

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