Classifications

• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
  • C07J1/0051—Estrane derivatives
  • C07J1/0062—Estrane derivatives substituted in position 17 alfa not substituted in position 17 beta
• • A—HUMAN NECESSITIES
  • A61—MEDICAL OR VETERINARY SCIENCE; HYGIENE
  • A61P—SPECIFIC THERAPEUTIC ACTIVITY OF CHEMICAL COMPOUNDS OR MEDICINAL PREPARATIONS
  • A61P5/00—Drugs for disorders of the endocrine system
  • A61P5/24—Drugs for disorders of the endocrine system of the sex hormones
  • A61P5/30—Oestrogens
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J1/00—Normal steroids containing carbon, hydrogen, halogen or oxygen, not substituted in position 17 beta by a carbon atom, e.g. estrane, androstane
  • C07J1/0051—Estrane derivatives
  • C07J1/0066—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa
  • C07J1/007—Estrane derivatives substituted in position 17 beta not substituted in position 17 alfa the substituent being an OH group free esterified or etherified
• • C—CHEMISTRY; METALLURGY
  • C07—ORGANIC CHEMISTRY
  • C07J—STEROIDS
  • C07J31/00—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring
  • C07J31/006—Normal steroids containing one or more sulfur atoms not belonging to a hetero ring not covered by C07J31/003

Definitions

• estrogenic compositions of substantial purity and low toxicity such as PREMARIN (conjugated equine estrogens) has become a preferred medical treatment for alleviating the symptoms of menopausal syndrome, osteoporosis/osteopenia in estrogen deficient women and in other hormone related disorders.
• the estrogenic components of the naturally occurring estrogenic compositions have been generally identified as sulfate esters of estrone, equilin, equilenin, 17- ⁇ -estradiol, dihydroequilenin and 17- ⁇ -dihydroequilenin (U.S. Patent 2,834,712).
• the estrogenic compositions are usually buffered or stabilized with alkali metal salts of organic or inorganic acids at a substantially neutral pH of about 6.5 to 7.5.
• Urea has also been used as a stabilizer (U.S. 3,608,077).
• the incorporation of antioxidants to stabilize synthetic conjugated estrogens and the failure of pH control with tris(hydroxymethyl)aminomethane (TRIS) to prevent hydrolysis is discussed in U.S. 4,154,820.
• 8,9-Dehydroestrone is a known compound useful as an intermediate in the synthetic production of estrone by isomerization to 9,11 unsaturation (U.S. Patent 3,394,153) and as an intermediate in the production of 3-cyclopentyloxy-17-ethynyl derivatives of the hormone (U.S. Patent 3,649,621).
• 8,9- dehydroestrone is known to possess estrogenic activity and to lower blood lipid levels (U.S. Patent 3,391,169).
• alkah metal salts of 8,9-dehydroestrone, 8,9- dehydroestrone-3-sulfate ester and its alkali metal salts, and their use in estrogen replacement therapy, atherosclerosis, and senile osteoporosis are disclosed in U.S. Patents 5,210,081 and 5,288,717.
• 17 ⁇ , ⁇ 8,9- dehydroestradiol or a pharmaceutically acceptable salt of its 3-sulfate ester and 17 ⁇ , ⁇ 8,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-sulfate ester.
• 17o ⁇ 8,9-dehydroestradiol and 17 ⁇ , ⁇ 8,9-dehydroestradiol are shown below as compounds I and II, respectively.
• the compounds of this invention can also be named 17 ⁇ , ⁇ 8,9-dehydroestrone and 17 ⁇ , ⁇ 8,9-dehydroestrone; and 17 ⁇ -estra-l,3,5(10),8-tetraene-3,17-diol and 17 ⁇ - estra-l,3,5(10),8-tetraene-3,17-diol, depending on which nomenclature system is used.
• salts of 17 ⁇ , ⁇ 8,9-dehydroestradiol 3-sulfate ester or 17 ⁇ , ⁇ 8,9-dehydroestradiol 3-sulfate ester include, but are not limited to, the alkali metal salts, alkaline earth metal salts, ammonium salts, alkylammonium salts containing
• 17 ⁇ , ⁇ 8,9-dehydroestradiol-3-sodium sulfate and 17 ⁇ , ⁇ 8,9-dehydroestradiol- 3-sodium sulfate are metabolites of ⁇ 8,9-dehydroestrone, that are formed following the administration of ⁇ 8,9-dehydroestrone.
• This invention therefore also provides 17 ⁇ , ⁇ 8,9-dehydroesrradiol or a pharmaceutically acceptable salt of its 3-sulfate ester in greater than one percent purity, and 17 ⁇ , ⁇ 8,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-sulfate ester in greater than one percent purity.
• treating covers treatment of an existing condition, ameliorating the condition, or providing palliation of the condition and inhibiting includes inhibiting or preventing the progress or development of the condition.
• the compounds of this invention can be prepared either by the jn vivo metabolism of ⁇ 8,9-dehydroestrone, as shown in Example 5, or can be prepared synthetically from ⁇ 8,9-dehydroestrone as outlined in Schemes I and ⁇ .
• Scheme I outlines the preparation of 17 ⁇ , ⁇ 8,9-dehydroestradiol and salts of its 3-sulfate ester beginning with the reduction of the 17-ketone of ⁇ 8,9-dehydroestrone with a suitable reducing agent, such as sodium borohydride to produce 17 ⁇ - ⁇ 8,9- dehydroestradiol.
• a suitable reducing agent such as sodium borohydride
• Other hydride reducing agents can be readily used such as sodium cyanoborohydride or lithium aluminum hydride.
• the 3-hydroxyl group can be selectively acylated with a suitable acylating agent, such benzoyl chloride or acetyl chloride. Inversion of stereochemistry at the 17-position can be accomplished using a Mitsunobu reaction.
• the diol is acylated with a suitable acylating reagent such as acetic anhydride, and the 3-acyl group is selectively cleaved using mild basic conditions, such as potassium carbonate in methanol.
• a suitable acylating reagent such as acetic anhydride
• the 3-acyl group is selectively cleaved using mild basic conditions, such as potassium carbonate in methanol.
• Formation of the 3-sulfate ester can be accomplished with a sulfur trioxide -ammonia, -alkylamine, -dialkylamine, or -trialkyamine reagent, such as sulfur trioxide-triethylamine or sulfur trioxide-pyridine.
• the resultant ammonium, monoalkylammonium, dialkylammonium or trialkylammonium salt of the 3-sulfate ester can be converted into another salt by exchange of cations, optionally via the acid.
• conversion to the 3-sulfate metal salt can be accomplished with a metal hydroxide solution.
• the preparation of 17 ⁇ , ⁇ 8,9-dehydroestradiol and the sodium and triethylammonium salts of its 3-sulfate ester is provided in Example 1.
• 17 ⁇ , ⁇ 8,9-dehydroestradiol and salts of its 3-sulfate ester can be prepared as shown in Scheme II, starting from ⁇ 8,9-dehydroestrone, except that the inversion of stereochemistry at the 17-position is not needed.
• 17 ⁇ , ⁇ 8,9-dehydroestradiol and the sodium and triethylammonium salts of its 3-sulfate ester are produced.
• Other salts of the 3-sulfate ester can be formed by varying the bases used.
• the preparation of 17 ⁇ , ⁇ 8,9- dehydroestradiol and the sodium and triethylammonium salts of its 3-sulfate ester is provided in Example 2.
• the compounds of Examples 1 and 3 were found to degrade over time.
• the preparation of TRIS stabihzed complexes of the compounds of Examples 1 and 3 are shown in Examples 2 and 4, respectively.
• the compounds of this invention are estrogenic, as shown in an in vivo standard pharmacological test procedure which measured uterine growth in immature female mice and ovariectomized rats as an evaluation of estrogenicity. 17 ⁇ , ⁇ 8,9- Dehydroestradiol-3-sodium sulfate and 17 ⁇ , ⁇ 8,9-dehydroestradiol-3-sodium sulfate were evaluated as representative compounds of this invention. Estrone and ⁇ 8,9- dehydroestrone were also evaluated for the purpose of comparison. The compounds to be evaluated were prepared as suspensions in corn oil. Com oil alone was used as a control.
• mice Intact immature Charles River CD female mice (23 days of age) were used.
• the compounds to be evaluated were administered subcutaneously or orally at total doses administered over 3 days of 1 to 1000 ⁇ g subcutaneously and 3 to 1000 ⁇ g orally.
• Estrone was administered subcutaneously at total doses of 0.03 to 1 ⁇ g over 3 days.
• the mice were sacrificed approximately 24 hours after the last dose was administered, and the uteri was removed and weighed. The results obtained are summarized in the following table.
• ⁇ 8,9 ⁇ 8,9-dehydroestrone
• 17 ⁇ 17 ⁇ , ⁇ 8,9-dehydroestradiol
• 17 ⁇ 17 ⁇ , ⁇ 8,9- dehydroestradiol.
• b Minimal effective dose given over 3 days required to produce a significant increase in uterine weight over that of vehicle.
• c Total dose given over 3 days required to produce a 100% increase in uterine weight over that of vehicle.
• a value of dose of 16.6 ⁇ g would be obtained if the results from an isolated strong uterine response to the 10 ⁇ g dose are deleted.
• the following procedure describes the evaluation of estrogenicity in ovariectomized rats. Immature Charles River CD rats were ovariectomized at 30 days of age and allowed 12 days for uterine regression. Administration of compounds to be evaluated was initiated 13 days post ovariectomy and was continued for 7 days. The compounds to be evaluated were administered at daily subcutaneous or oral doses of 3 to 1000 ⁇ g/rat/day. The rats were sacrificed approximately 24 hours after the last dose was administered. The uteri were removed and weighed. The results obtained are summarized in the following table.
• ⁇ 8,9 ⁇ 8,9-dehydroestrone
• 17 ⁇ 17 ⁇ , ⁇ 8,9-dehydroestradiol
• 17 ⁇ 17 ⁇ , ⁇ 8,9- dehydroestradiol.
• the compounds of this invention are antioxidants.
• the antioxidant properties of 17 ⁇ , ⁇ 8,9-dehydroestradiol and 17 ⁇ , ⁇ 8,9-dehydroestradiol were established in a standard pharmacological test procedure that measured the its ability to inhibit the formation of oxidatively modified low density lipoprotein (LDL) induced by exposure to either Cu++ ions or cultured endothelial cells (Parthasarathy S, Proc Natl Acad Sci USA 86:1046-1050 (1989)) by the TBARS (thiobarbituric acid reactive substances) method for analysis of free aldehydes (Yagi K., Biochem Med 15:212-216 (1976)).
• LDL low density lipoprotein
• J774 macrophages which express scavenger lipoprotein receptors which bind oxidatively modified-LDL, were exposed to the treated H ⁇ l-LDL.
• the results of this experiment demonstrate that binding of the LDL that was oxidized in the presence of 2.5 ⁇ M concentration of 17 ⁇ , ⁇ 8,9-dehydroestradiol or 17 ⁇ , ⁇ 8,9-dehydroestradiol, was reduced by 48% and 54%, respectively, and at a concentration of 0.25 uM was reduced by 34% and 28%, respectively.
• the same concentrations of estrone reduced the binding of LDL that was oxidized by 39% and 0%, respectively.
• porcine aortic endothelial cells were exposed to LDL that had been modified as above, by exposure to Cu++ in the presence and absence of 17 ⁇ , ⁇ 8,9-dehydroestradiol or 17 ⁇ , ⁇ 8,9-dehydroestradiol,.
• Oxidized LDL has been demonstrated to be cytotoxic to endothelial cells, and this process has also been strongly implicated in the atherogenic process.
• an MTT assay was performed to assess cytotoxicity (Hansen MB, J Immu Methods 119:203-210 (1989)).
• 17 ⁇ , ⁇ 8,9-dehydroestradiol or a pharmaceutically acceptable salt of its 3-sulfate ester are useful in replacement therapy in estrogen deficiency.
• the compounds of this invention are therefore useful in providing estrogen replacement therapy following ovariectomy or menopause, and in relieving symptoms related to estrogen deficiency, including vasomotor symptoms, such as hot flushes, and other menopausal related conditions, such as vaginal atrophy, vaginitis, and atrophic changes of the lower urinary tract which may cause increased urinary frequency, incontinence, and dysuria.
• the compounds of this invention are useful in preventing bone loss and in the inhibition or treatment of osteoporosis.
• the compounds of this invention are cardioprotective and they are useful in the treatment of atherosclerosis. These cardiovascular protective properties are of great importance when treating postmenopausal patients with estrogens to prevent osteoporosis and in the male when estrogen therapy is indicated.
• the compounds of this invention are also antioxidants are therefore useful in treating or inhibiting free radical induced disease states.
• antioxidant therapy is indicated to be warranted are with cancers, central nervous system disorders, Alzheimer's disease, bone disease, aging, inflammatory disorders, peripheral vascular disease, rheumatoid arthritis, autoimmune diseases, respiratory distress, emphysema, prevention of reperfusion injury, viral hepatitis, chronic active hepatitis, tuberculosis, psoriasis, systemic lupus erythematosus, adult respiratory distress syndrome, central nervous system trauma and stroke. Additionally, the compounds of this invention are useful in the suppression of lactation, and in the prophylaxis and treatment of mumps orchids. The compounds of this invention can be formulated neat.
• a pharmaceutical composition comprising a compound of this invention in association or combination with a pharmaceutically acceptable carrier.
• the proportion of the pharmaceutical carrier may be determined by the solubility and chemical nature of the compound, chosen route of administration and standard pharmacological practice.
• the pharmaceutical carrier may be solid or liquid.
• a solid carrier can include one or more substances which may also act as flavoring agents, lubricants, solubilizers, suspending agents, fillers, glidants, compression aids, binders or tablet-disintegrating agents; it can also be an encapsulating material.
• the carrier is a finely divided solid which is in admixture with the finely divided active ingredient.
• the active ingredient is mixed with a carrier having the necessary compression properties in suitable proportions and compacted in the shape and size desired. The powders and tablets preferably contain up to 99% of the active ingredient.
• Suitable solid carriers include, for example, calcium phosphate, magnesium stearate, talc, sugars, lactose, dextrin, starch, gelatin, cellulose, methyl cellulose, sodium carboxymethyl cellulose, polyvinylpyrrolidine, low melting waxes and ion exchange resins.
• Liquid carriers are used in preparing solutions, suspensions, emulsions, syrups, elixirs and pressurized compositions.
• the active ingredient can be dissolved or suspended in a pharmaceutically acceptable liquid carrier such as water, an organic solvent, a mixture of both or pharmaceutically acceptable oils or fats.
• the liquid carrier can contain other suitable pharmaceutical additives such as solubilizers, emulsifiers, buffers, preservatives, sweeteners, flavoring agents, suspending agents, thickening agents, colors, viscosity regulators, stabilizers or osmo-regulators.
• suitable examples of liquid carriers for oral and parenteral administration include water (partially containing additives as above, e.g.
• cellulose derivatives preferably sodium carboxymethyl cellulose solution
• alcohols including monohydric alcohols and polyhydric alcohols, e.g. glycols) and their derivatives, lethicins, and oils (e.g. fractionated coconut oil and arachis oil).
• the carrier can also be an oily ester such as ethyl oleate and isopropyl myristate.
• Sterile hquid carriers are useful in sterile hquid form compositions for parenteral administration.
• the hquid carrier for pressurized compositions can be halogenated hydrocarbon or other pharmaceutically acceptable propellant.
• Liquid pharmaceutical compositions which are sterile solutions or suspensions can be utilized by, for example, intramuscular, intraperitoneal or subcutaneous injection. Sterile solutions can also be administered intravenously.
• the compounds of this invention can also be administered orally either in hquid or solid composition form.
• the compounds of this invention may be administered rectally or vaginally in the form of a conventional suppository.
• the compounds of this invention may be formulated into an aqueous or partially aqueous solution, which can then be utilized in the form of an aerosol.
• the compounds of this invention may also be administered transdermally through the use of a transdermal patch containing the active compound and a carrier that is inert to the active compound, is non toxic to the skin, and allows delivery of the agent for systemic absorption into the blood stream via the skin.
• the carrier may take any number of forms such as creams and ointments, pastes, gels, and occlusive devices.
• the creams and ointments may be viscous liquid or semisolid emulsions of either the oil-in-water or water-in-oil type. Pastes comprised of absorptive powders dispersed in petroleum or hydrophilic petroleum containing the active ingredient may also be suitable.
• occlusive devices may be used to release the active ingredient into the blood stream such as a semipermiable membrane covering a reservoir containing the active ingredient with or without a carrier, or a matrix containing the active ingredient.
• Other occlusive devices are known in the literature.
• the compounds of this invention may be employed as a solution, cream, or lotion by formulation with pharmaceutically acceptable vehicles containing 0.1 - 5 percent, preferably 2%, of active compound which may be administered to a fungally affected area.
• the dosage requirements vary with the particular compositions employed, the route of administration, the severity of the symptoms presented and the particular subject being treated. Based on the results obtained in the standard pharmacological test procedures, projected daily dosages of active compound would be 0.02 ⁇ g/kg - 500 ⁇ g/kg. Treatment will generally be initiated with small dosages less than the optimum dose of the compound. Thereafter the dosage is increased until the optimum effect under the circumstances is reached; precise dosages for oral, parenteral, nasal, or intrabronchial administration will be determined by the administering physician based on experience with the individual subject treated.
• the pharmaceutical composition is in unit dosage form, e.g. as tablets or capsules.
• the composition is sub-divided in unit dose containing appropriate quantities of the active ingredient;
• the unit dosage forms can be packaged compositions, for example, packeted powders, vials, ampoules, prefilled syringes or sachets containing liquids.
• the unit dosage form can be, for example, a capsule or tablet itself, or it can be the appropriate number of any such compositions in package form.
• a 2 L multi-neck flask was equipped with a mechanical stirrer, a condenser with a nitrogen inlet, a pressure equalizing graduated addition funnel and a thermometer.
• the flask was charged with 17 ⁇ -estra-l,3,5(10),8-tetraene-3,17-diol (60.00 g, 0.2219 mol.), tetrahydrofuran (500 mL) and triethylamine (46.4 mL, 0.3329 mol., 1.5 molar excess) and the mixture was stirred under nitrogen until a solution was obtained.
• the addition funnel was charged with benzoyl chloride (30.98 mL, 0.2669 mol., 1.203 molar excess) and tetrahydrofuran up to the 100 mL mark.
• the benzoyl chloride solution was added dropwise over 25 minutes, under nitrogen, while maintaining the reaction temperature between 15 and 20° using a water/ice bath. The cooling was removed and the stirred suspension (E.3N.HCI ppt) was allowed to warm to room tempetature (23°).
• the reaction mixture was chilled in ice and a mixture of saturated brine (204 mL), water (68 mL) and concentrated hydrochloric acid (34 mL) was added rapidly.
• the aqueous phase was separated from the organic phase and extracted with tetrahydrofuran (1 x 200 mL and 1 x 100 mL).
• the combined organic phases were washed with saturated brine (2 x 100 mL) and thrice with a mixture of saturated brine (70 mL) and 5% sodium bicarbonate (30 mL).
• a further wash with saturated brine (100 mL) did not completely remove residual benzoyl chloride and so the solution was dried by magnetic stirring with anhydrous magnesium sulfate for 15 minutes.
• the drying agent was removed by filtration and the filter pad washed with tetrahydrofuran.
• the crude benzoate (5.0 g) was stirred and heated in 95% ethanol (65 mL) to give a solution which was filtered through a fluted, fast filter paper.
• the filter was rinsed with hot 95% ethanol and the solution was distilled to 50 mL volume.
• the solution was allowed to cool and the resulting slurry of crystals was refrigerated. After chilling to -10° the product was collected on a filter and washed with cold (-10°) 95% ethanol. Drying in a vacuum oven at 65° overnight gave purified product (3.82 g, 76.40%).
• Spectral (ir, pmr & ms) data were appropriate and elemental analyses were acceptable.
• the product (38.60 g) was dissolved in hot methanol (120 mL) and the hot solution was heated briefly with charcoal. The charcoal was removed by filtration through Celite and the filter pad was washed with hot methanol. The product started to crystallize in the filtrate. The filtrate and washings were reheated to dissolve the product and the solution was distilled to approximately 120 mL volume. The solution was allowed to cool to near room temperature and the resulting slurry of crystals was chilled to 0-5°. The crystals were collected on a filter and washed with cold (0-5°) methanol (2 x 35 mL). Drying in a vacuum oven at room temperature gave the title compound (31.86 g, 82.39%).
• a second crop (172 g, 6.57%) was obtained from the mother liquor. This was combined with material remaining from the first crop (22.27 g) and the total (22.99 g) was stirred magnetically and heated in methanol (230 mL) to produce a yellow solution. The solution was filtered through a fluted, fast filter paper and rinsed through with 20 mL of hot methanol. The solution was distilled to a volume of 125 mL, allowed to cool to room temperature and then refrigerated. The resulting crystals were collected on a filter and washed with cold (0°) methanol (3 x 15 ml).
• the mixture was transferred to a separatory funnel with a mixture of 20 mL n-butanol, 10 mL of saturated brine and 10 mL of water. The layers separated slowly.
• the upper organic phase was washed consecutively with saturated brine (120 mL, 2 x 100 mL, 3 x 75 mL and 1 x 50 mL) until the pH of the final wash was 7.
• the cloudy organic phase was filtered through a small pad of solka Floe (prewashed with n-butanol) on a 7 cm Buchner funnel and rinsed through with n-butanol (2 x 25 mL).
• the solution was transferred to a 2 L round bottom flask with a 50 mL rinse of n-butanol. The solution was evaporated at 35-40° under oil pump vacuum until a thick slurry of crystals was obtained and 300 mL of distillate had been collected. Ether (500 mL) was added and mixed with the slurry. The white crystals were collected on a 10 cm filter and washed with ether (4 x 80 mL). The filter cake was transferred to a 1 L Erlenmeyer and magnetically stirred with 400 mL of ether for 5 minutes. The crystals were collected once more and washed with ether (5 x 70 mL). The wet cake was dried in a vacuum oven with a nitrogen bleed for 6 days to give 17.17 g (96.49% based on a hydrate) of white solid.
• the solution was transferred to a tray at -40° in a large Virtis Freeze-dryer using a 500 mL water rinse.
• the solution was frozen and the solid was freeze dried for 5 days.
• the resulting white, soft, flaky material was pressed, scraped, and mixed well in a bottle.
• the material was dried for a further 4 days at room temperature in the freeze-drier.
• the white product weighed 20.93 g (98.54%).
• the solution was distilled to approximately 500 mL and 404 mL of distillate was collected.
• the resulting slurry of crystals was allowed to cool to 35° and then cooled to -10°.
• the crystals were collected on a filter and washed with cold (-10°) methanol (3 x 50 mL).
• the wet crystals (30.14 g) were dried in a vacuum oven at 65° for 4 days to provide 25.15 g (89.24%) of white, crystalline 3, 17 ⁇ -diacetate, having consistent spectral (ir, pmr and ms) data and acceptable elemental (C & H) analyses.
• the white crude material was stirred magnetically with 300 mL of water and collected once more on a filter. Washing with water (9 x 40 mLO removed traces of acid and the pH of the last wash was 5.5. Drying the wet cake (37.06 g) in a vacuum desiccator over phosphorus pentoxide overnight provided 20.02 g (94.03%) of crude 17 ⁇ -monoacetate.
• crude product (20.02 g), methanol (100 mL) and dichloromethane (200 mL).
• the pH (wetted pH paper) of the solution was 8; 5 drops of glacial acetic acid were added to bring the pH to 5.
• the solution was distilled at atmospheric pressure until crystallization was copious (volume of distillate 220 mL).
• the slurry was cooled to 40° and then to -10° with a dry ice-acetone bath.
• the product was collected on a filter and washed with cold (-10°) methanol (3 x 25 mL).
• the wet cake (21.65 g) was dried at 70° overnight and then at 85° for 5.5 hours in a vacuum oven.
• the resulting white crystalline material (18.07 g, 90.25%) had appropriate specral (ir, pmr and ms) data.
• the wet cake (32.63 g) was dried overnight in a vacuum oven at room temperature to give 23.25 g (85.64%) of purified material having appropraite spectral (ir, pmr and ms) data and acceptable elemental (QH and N) analyses.
• the mixture was transferred to a separatory funnel along with 20 mL of n-butanol, 10 mL of saturated brine and 10 mL of water. The separation of phases was slow so a further 60 mL of brine was added. The lower aqueous phase ( ⁇ 126 mL) was separated and the organic phase was washed with saturated brine (1 x 40 mL, followed by 3 x 30 mL, final wash pH 13-14). Solid product started to form. n-Butanol (500 mL), saturated brine (100 mL) and water (20 mL) were added. The product redissolved on mixing and the pH of the wash was 12.
• the resulting slurry was stirred for 10 minutes at room temperature and then filtered.
• the filter cake was washed with ether ( 5 x 100 mL.)
• the resulting white, powdery solid (17.28 g) still smelled of ethanol and so it was magnetically stirred with ether (300 mL) for 10 minutes and collected once more on a filter.
• the filter cake was washed with ether (4 x 50 mL) and the wet cake (25.18 g) was dried at room temperature overnight in a vacuum oven.
• the white product (15.50 g, 86.51%) had appropriate spectral (ir, pmr and ms) data.
• Urinary metabolites were determined by analyzing 0-24 hr samples after enzyme hydrolysis. To hydrolyze the conjugates, an aliquot (1 ml) of urine was combined with 1 ml of 0.05 M sodium acetate buffer, pH 5.0, and incubated overnight at 37°C with
• the sample (supernatant) was applied and the cartridges were washed with 2 x 1 ml H 2 O, 2 x 1 ml 0.1 N acetic acid, 2 x 1 ml H 2 O and 2 x 1 ml hexane.
• the metabohtes were then eluted in 2 x 1 ml acetone.
• the extracts were dried under N and reconstituted in 1 ml of mobile phase [60% buffer (0.05 M KH 2 PO 4 , pH 3.0), and 40% organic (2:1 acetonitrlie; methanol)].
• Plasma samples were also analyzed by HPLC after hydrolysis. Hydrolysis of the conjugates was achieved by incubating the samples with Glusulase as described for the urine samples above. Following centrifugation, the plasma samples were extracted using C-18/N+ cartridges by the above described procedure and analyzed by HPLC- electrochemical detection.
• the type(s) of conjugates present in the plasma and urine samples were determined by conducting the hydrolysis of all samples in the presence or absence of 200 ⁇ M of saccharolactone, an inhibitor of ⁇ -glucuronidase.
• the hydrolysis, extraction and HPLC analyses were performed as described above.
• the structures of the metabolites were identified by EI-GC/MS analysis of TMS derivatives of extracts of hydrolyzed urine and plasma samples. The structures of the metabohtes were confirmed by analyzing and comparing the mass spectra of authentic reference standards. 1. GC/MS analysis of hydrolyzed urine samples
• Each extract or reference standard was then reconstituted in 80 ⁇ l of toluene; then 10 ⁇ l of BSTFA and 5 ⁇ l of pyridine were added and reacted at 65 °C for 1 hr to form TMS derivatives of the metabolites.
• the derivatized samples were analyzed using a Finnigan-MAT 8230 high resolution mass spectrometer directly interfaced to a Varian 3400 gas chromatograph. The data were acquired on an SS-300 data system and printed on a Vionix. The source ionization mode was positive electron impact.
• the column used was a J&W DB-5MS 30 M x 0.32 mm ID. The initial column was 80°C with a -1 minute hold programmed to 260°C at 10°C/min. The injection temperature was 250°C. The injection volume was 2 ⁇ l.
• Two ml of plasma was mixed with 2 ml of pH 5.0 sodium acetate buffer (0.05 M) and hydrolyzed with 2000 units Glusulase (DuPont). The mixture was incubated overnight at 37°C in a shaking water bath and extracted as described previously.
• Plasma Analysis of plasma samples after hydrolysis also showed the presence of the same three peaks as in the urine samples at all time points examined.
• addition of saccharolactone inhibited the hydrolysis of the conjugates, indicating that most of the metabohtes were in glucuronide forms.

Landscapes

• Chemical & Material Sciences (AREA)
• Organic Chemistry (AREA)
• Health & Medical Sciences (AREA)
• General Health & Medical Sciences (AREA)
• Chemical Kinetics & Catalysis (AREA)
• Nuclear Medicine, Radiotherapy & Molecular Imaging (AREA)
• Diabetes (AREA)
• Endocrinology (AREA)
• Engineering & Computer Science (AREA)
• General Chemical & Material Sciences (AREA)
• Medicinal Chemistry (AREA)
• Bioinformatics & Cheminformatics (AREA)
• Pharmacology & Pharmacy (AREA)
• Life Sciences & Earth Sciences (AREA)
• Animal Behavior & Ethology (AREA)
• Public Health (AREA)
• Veterinary Medicine (AREA)
• Steroid Compounds (AREA)
• Pharmaceuticals Containing Other Organic And Inorganic Compounds (AREA)

Applications Claiming Priority (3)

Publications (1)

Family

ID=21840614

Family Applications (1)

Country Status (13)

Families Citing this family (1)

Family Cites Families (5)

• 1997
  • 1997-10-06 AR ARP970104601A patent/AR014096A1/es not_active Application Discontinuation
  • 1997-10-09 BR BR9712628-4A patent/BR9712628A/pt not_active IP Right Cessation
  • 1997-10-09 CN CN97180394A patent/CN1239967A/zh active Pending
  • 1997-10-09 WO PCT/US1997/018306 patent/WO1998016544A1/en not_active Application Discontinuation
  • 1997-10-09 HU HU9903369A patent/HUP9903369A3/hu unknown
  • 1997-10-09 JP JP10518462A patent/JP2001502328A/ja active Pending
  • 1997-10-09 NZ NZ335183A patent/NZ335183A/xx unknown
  • 1997-10-09 AU AU51459/98A patent/AU743530B2/en not_active Ceased
  • 1997-10-09 ZA ZA979069A patent/ZA979069B/xx unknown
  • 1997-10-09 CA CA002268330A patent/CA2268330A1/en not_active Abandoned
  • 1997-10-09 KR KR1019990703126A patent/KR20000049055A/ko not_active Application Discontinuation
  • 1997-10-09 EP EP97946247A patent/EP0934334A1/en not_active Withdrawn
  • 1997-10-09 IL IL12902997A patent/IL129029A0/xx unknown

Non-Patent Citations (1)

Also Published As

Similar Documents

Legal Events