Patent Application: US-43548189-A

Abstract:
this invention provides novel cyclic hydrocarbons of the formula : ## str1 ## wherein r is selected from the group consisting of ch 2 ═ ch -- ch 2 --, ho -- ch 2 ch 2 ch 2 --, and ch 3 ; wherein r 1 is selected from the group consisting of m - trifluoromethylphenylmethyl , 2 - thienylmethyl , and p - aminosulfonylphenylethyl ; wherein r 2 and r 3 are methyl or hydrogen ; wherein r 4 is hydrogen or -- oh ; a compound of the formula ## str2 ## wherein r is 2 nch 2 ch 2 ch 2 -- or nh 2 ch 2 ch 2 ch 2 --; or a compound of the formula ## str3 ## wherein the dashed line indicates that the 2 - 3 bond is saturated or unsaturated and , wherein r is hydrogen or methyl . these compounds are useful for inhibiting adverse physiological symptoms associated with phospholipase a 2 and for treating hyperglycemia associated diseases such as diabetes and obesity .

Description:
the dosage regimen for preventing or treating phospholipase mediated conditions , pmc , by the compounds represented by formula ii are selected in accordance with a variety of factors , including the type , age , weight , sex , medical condition of the mammal , severity of the pmc and the particular compound employed . an ordinarily skilled physician or veterinarian will readily determine and prescribe the effective amount of the compound to prevent or arrest the progress of the condition . typically , the physician or veterinarian employs relatively low dosages at first and then increases the dose until a desired or maximum response is obtained . because the diseases or conditions caused by the arachidonic acid cascade are varied , methods of administering these compounds to patients must depend on the particular pmc to be treated . preferred patients to be treated are domesticated animals and humans , the most preferred patients are humans . regardless of the route of administration selected , the compounds used are formulated into pharmaceutically acceptable dosage forms by conventional methods known to the pharmaceutical art . thus , the compounds can be administered orally in forms such as pills , capsules , solutions or suspensions ; rectally or vaginally in forms such as suppositories or bougies ; parenterally , either subcutaneously , intravenously , or intramuscularly using sterile injectable forms known to the pharmaceutical art . for treatment of conditions such as erythema the compounds of this invention may also be administered topically in the form of ointments , creams , gels , or the like . initial dosages of the compounds of this invention are from about 0 . 003 to 3 . 0 g per 70 kg mammal per 6 - 8 hours orally . when other forms of administration are employed , equivalent doses are administered . when dosages beyond 45 mg / kg are employed , care should be taken with each subsequent dose to monitor possible toxic effects . the phospholipase a2 inhibitory compounds of the present invention ameliorates the cellular damage resulting from the degradation of cell membranes by phospholipase a2 after hypoxic states such as coronary infarcts , ligation of the aorta during surgery for aortic aneurysms ( resulting in kidney damage ), and the like , see zalewski . et al ., clinical research 31 , p . 227 ( 1983 ). preferred patients to be treated are domesticated animals and humans , the most preferred patients are humans . this is a preferred use of these compounds . the phospholipase a2 inhibitory compounds are useful in the treatment of asthma . asthma is a lung disease in which a wide variety of stimuli can result in an asthmatic attack . these stimuli could be damp cold air or allergens in the environment . the asthmatic response is initially characterized by constriction of the bronchioles leading to increased airway resistance . this early constrictive phase is due to mast cell release of histamine and other modulators such as peptides . after the constrictive phase , a late sustained phase occurs which , in human beings , may reach a maximum in 6 - 8 hours . this phase is slower in onset and disappearance and is due to metabolites of the arachidonic acid cascade such as thromboxanes , prostaglandins , and leukotrienes , see &# 34 ; corticosteroid treatment in allergic airway diseases &# 34 ;, proceedings of a symposium in copenhagen oct . 1 - 2 , 1981 ( editors : t . h . clark , n . myginfd , and o . selroos , munksgaard / copenhagen 1982 ). inhibition of phospholipase a2 at a physiologically acceptable level will prevent formation of these products in the lung thought to be responsible for the &# 34 ; 2nd wave &# 34 ; of airway resistance . preferred patients to be treated are domesticated animals and humans , the most preferred patients are humans . for these uses , the compounds are administered in a variety of dosage forms : orally in the form of tablets , capsules , or liquids ; rectally in the form of suppositories ; parenterally , subcutaneously , intradermally , or intramuscularly , with intravenous administration being preferred in emergency situations , by inhalation in the form of aerosols or solutions for nebulizers ; or by insufflation in the form of powder . these compounds are effectively administered to human asthma patients by oral or aerosol inhalation . doses in the range of about 0 . 01 to 50 mg per kg of body weight are used 1 to 4 times a day , the exact dose depending on the age , weight , condition of the patient and frequency and route of administration . for the above use these compounds can be combined advantageously with other anti - asthmatic agents , such as sympathomimetics ( isoproterenol , phenylephrine , ephedrine ); xanthine derivatives ( theophylline , aminophylline ); and corticosteroids ( prednisolone ). administration by oral inhalation with conventional nebulizers or by oxygen aarosolization conveniently provides the active ingredient in dilute solution . preferably at concentrations of about 1 part of medicament to from about 100 to 200 parts of weight of total solution . entirely conventional additives may be employed to stabilize these solutions or to provide isotonic media , for example , sodium chloride , sodium citrate , citric acid , sodium bisulfite , and the like can be employed . a self propelled dosage unit suitable for inhalation therapy for administering the active ingredient in aerosol form comprises the active ingredient suspended in an inert propellant such as a mixture of dichlorodifluoromethane and dichlorotetrafluoroethane together with a co - solvent such as ethanol , flavoring materials and stabilizers . instead of a co - solvent , a dispensing agent such as oleyl alcohol can also be used . suitable means to employ the aerosol inhalation therapy technique are described fully in u . s . pat . no . 2 , 868 , 691 . the compounds represented by formula ii also control spasm and facilitate breathing in conditions such as bronchial asthma , bronchitis , bronchiectasis , pneumonia and emphysema . to treat a patient with these symptoms , the compounds would be administered to the patient in the same manner as described above . this is a preferred use of compounds represented by formula ii . these novel phospholipase a2 inhibitory compounds are also useful as anti - inflammatory agents in mammals , especially humans , and for this purpose , are administered systemically and preferably orally . for oral administration , a dose range of 0 . 05 to 50 mg per kg of human body weight is used to give relief from pain associated with inflammatory disorders such as rheumatoid arthritis . they are also administered intravenously in aggravated cases of inflammation , preferably in a dose range 0 . 01 to 100 g per kg per min until relief from pain is attained . when these novel compounds are administered orally , they are formulated as tablets , capsules , or as liquid preparations , with the usual pharmaceutical carriers , binders , and the like . for intravenous use , sterile isotonic solutions are preferred . these novel phospholipase a2 inhibitory compounds are useful whenever it is desired to inhibit platelet aggregation , reduce the adhesive character of platelets , or remove or prevent the formation of thrombi in mammals . for example , these compounds are useful to prevent myocardial infarcts , to prevent post - operative thrombosis to promote patency of vascular grafts following surgery , or to treat conditions such as atherosclerosis , arteriosclerosis , blood clotting defects due to lipemia , and other clinical conditions . preferred patients to be treated are domesticated animals and humans , the most preferred patients are humans . for these uses , these compounds are administered intravenously , subcutaneously , intramuscularly , or in the form of sterile implants for prolonged action . for rapid response especially in emergency situations , the intravenous route of administration is preferred . doses in the range about 0 . 005 to about 20 mg per kg of body weight per day are used , the exact dose depending on the age , weight , condition of the patient , and frequency and route of administration . phospholipase a2 inhibitors of the present invention are useful in the treatment or prevention of conditions due to increased phospholipase activity observed after central nervous system ( cns ) trauma such as brain and spinal cord injury , see , e . p . wei , et al ., j . neurosurg ., 56 , pp . 695 - 698 ( 1982 ) and e . d . hall and j . m . braughler , surgical neurology , 18 , pp . 320 - 327 ( 1982 ). preferred patients to be treated are domesticated animals and humans , the most preferred patients are humans . the compounds represented by formula ii are useful to treat pmc symptoms which have already occurred in the mammal or to prevent pmc symptoms in particularly susceptible mammals . use of these compounds before the development of pmc symptoms prevents the formation of the prostaglandins and similar products causing such conditions . thus , phospholipase inhibitors of the present invention are used to prevent edema and erythema from sunburn by administering these compounds prior to exposure to sunlight . these compounds are also administered to persons suffering from hayfever or similar allergies prior to exposure to the allergenic substances to which hayfever sufferers are sensitive . it is most preferred to use the compounds of this invention in the treatment or prevention of asthma and in the treatment or prevention of cellular death resulting from hypoxic states . the preferred route in most cases is to systemically administer the compounds in order to allow them to enter the mammal &# 39 ; s bloodstream and thus be distributed throughout the mammal &# 39 ; s system . in certain cases , where the pmc is of a localized nature ( sunburn or psoriasis ), topical administration is employed in order that the phospholipase a2 inhibition is confined to the afflicted area . while conventional treatment for hyperglycemic conditions may include restriction of carbohydrate intake and insulin injection , one important means of treating hyperglycemic patients is with oral hypoglycemic agents . compounds of this invention represented by formulas i and iii are useful to treat niddm and its complications in mammals , including human beings because these compounds lower the serum glucose levels when administered to kka y mice with spontaneous diabetes . accordingly , a patient to be treated with certain of the novel hypoglycemic compounds of this invention is first diagnosed as a diabetic by conventional means , usually by the persistence of elevated serum glucose levels , and a treatment regimen with compounds of this invention is established so that the elevation in a patient &# 39 ; s serum glucose level is either significantly reduced or eliminated . the precise therapeutic endpoint of treatment , elimination or merely reduction in hyperglycemia , is readily determined by the attending physician based upon the clinical presentation and concomitantly employed treatment . for example , certain of the novel hypoglycemic compounds of this invention may be employed to significantly reduce hyperglycemia in a patient , with a carbohydrate - restricted diet providing a further measure of control . preferred patients to be treated are domesticated animals and humans , the most preferred patients are humans . while the novel hypoglycemic compounds of this aspect of the invention may be administered by any convenient route ; orally , subcutaneously , intravenously , intramuscularly , topically , or rectally , these compounds are most significantly and usefully employed as oral hypoglycemic agents , particularly in solid dosage form such as capsules and tablets . alternatively , liquid oral dosage forms , such as syrups and elixirs , are alternatively employed . the solid , oral pharmaceutical compositions in accordance with the present invention are all prepared by methods known in the art for preparing other oral antidiabetic compositions . since an individual patient response to treatment with compounds in accordance with the present invention may vary , effective dosages of the compounds of the instant invention will vary from patient to patient . ordinarily , an oral dosage of from 0 . 1 to 10 mg / kg of these compounds will be adequate to significantly reduce hyperglycemia in a patient being treated . repeated dosages , every 4 - 12 hr , may be required during the day to maintain the antihyperglycemic effect . accordingly , dosages from about 0 . 1 mg / kg / dose to about 10 mg / kg / dose , depending upon the patient , frequency of treatment , and observed response . an attending physician may at first prescribe a relatively small amount of a novel hypoglycemic compound of this invention and later increase this dosage as necessary to achieve the desired level of control . novel hypoglycemic compounds of the present invention are also useful to treat and / or prevent obesity in mammals including human beings . for this purpose , the novel compounds of this invention are formulated and administered as described above for hyperglycemia . all compounds of the present invention may be formulated into pharmaceutical compositions , employing a pharmaceutically acceptable carrier . pharmaceutical formulations include pharmaceutical compositions suitable for oral , parenteral , vaginal , topical , and rectal use , such as tablets , powder packets , cachets , dragees , suppositories , bougies , and the like . suitable diluents or carriers such as carbohydrates ( lactose ), proteins , lipids , calcium phosphate , cornstarch , stearic acid , methylcellulose and the like may be used as carriers or for coating purposes . coconut oil , sesame oil , safflower oil , cottonseed oil , peanut oil may be used for preparing solutions or suspensions . sweetening , coloring and flavoring agents may be added . in general the preferred route of administration depends on the condition being treated . for asthma , oral or aerosol inhalation is preferred . for most other conditions the preferred mode of administration is oral . the phospholipase a2 inhibitory compounds of this invention are useful in any mammal whose metabolic system includes the phospholipase . induced arachidonic acid cascade . the mammals which are preferred . are generally domesticated animals and humans . humans are the most preferred mammals to be treated . the utility of the compounds of this invention represented by formula ii is demonstrated in the following laboratory tests which determine phospholipase inhibition . weigh sufficient thioglycolate medium , usp grade , to prepare a 5 % w / v solution in sterile water . heat the solution for 10 minutes on a boiling water bath . remove and allow the solution to cool to 20 °- 25 °. inject 10 ± 0 . 5 ml intraperitoneally into sprague - dawley rats as described below . six ( 6 ) sprague - dawley , pathogen free , female rats ( 230 - 270 grams ) are injected intraperitoneally with 10 . 0 ± 0 . 5 ml thioglyco . late broth . 5 % w / v 16 . 18 hours prior to sacrifice . after sacrifice by cervical dislocation , leukocytes accumulated in the peritoneal cavity are collected by injecting 30 ml of sterile 0 . 9 % w / v sodium chloride intraperitoneally and vigorously massaging the abdomen to assure uniform dispersion of the cells within the carcass . use a pasteur pipet to remove approximately 20 ml of fluid with suspended cells from a small incision through the abodominal wall . collect the cell suspension in plastic culture tubes . centrifuge the cell suspensions isolated above for 10 minutes at 1000 rpm ( sorvall rc . 3 , hg - 4l rotor , 25 ° c .). discard the supernatant . resuspend the cells evenly in 0 . 9 % nacl to original volume , centrifuge a second time for 10 minutes at 1000 rpm . discard the supernatant . resuspend the cells evenly in hanks buffer . transfer 10 μl of leukocyte suspension into a plastic cell counting cup . add 15 . 0 ml of isoton ˜ diluent for cell counting . determine the cell count with a model zbi coulter counter or equivalent . a . add 0 . 5 ml of rat leukocyte ( neutrophil ) suspension to each channel of a payton dual channel aggregometer . cuvettes 45 mm × 4 mm i . d ., are used . cell suspensions at 37 ° c . are stirred ( 400 rpm ). b . add 5 μl test compound ( 0 . 01m in absolute ethanol ) to cell and evaporate to dryness under nitrogen . add 0 . 5 ml cell suspension ( 37 ° c . 400 rpm ). incubate for 2 minutes , then add 1 μl of the agonist . 10 - 4 m fmlp . c . record the aggregation trace (% transmitted light ) on a potentiometric recorder . both soybean lipoxidase and hog pancreas phospholipase a 2 are obtained commercially from sigma . the soybean lipoxidase is dis . solved at a concentration of 5 mg / ml in 0 . 033 m ammediol - hcl buffer ph 8 . 5 with 1 × 10 - 4 m ca ++ . the hog pancreas enzyme is added at the rate of approximately 350 units per ml of final mixture . thus , 0 . 025 ml is equivalent to 9 units of phospholipase and 0 . 125 mg of lipoxidase . the substrate is phosphatidyl choline . the material has a fatty acid composition upon saponification , of 2 % of 16 : 0 , 1 % of 18 : 0 , 3 % of 18 : 1 , 18 % of 18 : 2 , and 12 % of 18 : 3 fatty acids with the largest fraction being linoleic acid . the estimated molecular weight is 780 . 78 mg of this substrate is put in a 10 ml volumetric flask containing 100 mg of deoxycholic acid . a &# 34 ; pill &# 34 ; magnetic stirrer is added along with 7 - 8 ml of water , and the whole stirred rapidly until all the lecithin is dissolved . the &# 34 ; pill &# 34 ; is then removed and the flask contents are made up to 10 ml with water . to three oxygraph cells equipped with magnetic stirrers is added 2 . 5 ml of 0 . 033m ammediol - hcl buffer , ph 8 . 5 , containing 1 × 10 - 4 m ca ++ . this is followed by 0 . 1 ml of the inhibitors made up at an initial concentration of 0 . 01m in methanol . where controls are run , 0 . 1 ml of methanol is added to each cell . the cells are then put in the oxygraph apparatus and the contents are stirred briefly . 0 . 025 ml of the enzyme mixture is then added and the electrodes are inserted in each cell , care being taken to exclude all air bubbles . with the stirrer and water bath pump on , the contents of each cell are stirred for 2 . 5 minutes at 37 . 5 ° c . the reaction is initiated by adding to the cells 0 . 05 ml of 0 . 01m lecithin substrate . the reaction is monitored by continuous measurement of rates of oxygen depletion from the medium as a consequence of unsaturated fatty acids ( linoleic acid ) being released from esterified form by the phospholipase . these fatty acids immediately become substrates for the soybean lipoxidase which forms the 15 - hydroxy acids , with consequent oxygen utilization . the initial rates of oxygen consumption are recorded using a sargeant - welch recorder set at 5 mv full scale . the &# 34 ; air &# 34 ; setting and medium chart speed are used . the slopes of oxygen consumption are then determined in triplicate , and these are compared with the methanol controls to determine the degree of inhibition . if complete inhibition is seen at the first concentration , appropriate dilutions are made to bring the inhibition percentages down to at least 3 concentrations of inhibitor where partial inhibition is observed . the i 50 can then be calculated for that particular inhibitor , using linear regression slopes . all compounds for which i 50 value is shown are tested for inhibitory activity on the soybean lipoxidase . none inhibit at the test concentration . the utility of the compounds of this invention represented by formulas i and iii is demonstrated in the following laboratory tests which determine serum glucose levels in mice . testing for blood glucose lowering in the kka y mouse all kka y mice used for screening are produced and selected by methods previously outlined , t . fujita et al ., diabetes , 32 804 - 10 ( 1983 ). groups of 6 animals each are employed . pre - treatment nonfasting blood glucose ( nfbg ) samples are measured 5 days prior to the start of a screening run by previously described methodologies . these blood sugar values are used to place animals into groups with equal mean blood glucose concentrations and to eliminate any mice with a nfbg value & lt ; 250 mg / dl . on day 0 , compounds chosen to be run are incorporated into ground mouse chow ( purina 5015 ). compounds are included at a rate of 1 mg / gram of chow . generally , 300 g of drugs containing diet is prepared for each group . mice receiving ground chow only are the negative control . each screening run also uses ciglitazone ( t . fujita , et al ., supra ) as a positive control ( 0 . 5 to 1 . 0 mg / gram chow ). initial body and food weights are taken on day 1 . food is placed in a crock which contains an adequate amount to last for the length of the study . in order to acclimate the mice from pelleted mouse chow to ground mouse chow , they are fed the ground chow for 9 days prior to use in the screen . on day 4 of treatment , a nfbg sample is again measured , as well as food and body weights . food consumption measurements are used to determine an average mg / kg dose the mice received over the testing period , and to evaluate the compound &# 39 ; s effect on food consumption . this group must not show a significant change ( p & lt ; 0 . 05 ) from pre - to post - treatment . if there is a significant decrease in blood sugar the run is not valid . this group must show a significant depression in blood sugar mean levels from pre - to post - treatment . a lack of activity in this group would also invalidate the run . this contrast must be significant . it is a further assurance that both control groups performed as expected . ( 1 ) a significant decrease in blood sugar mean levels from pre - to post - treatment . ( 2 ) negative control vs . compound : this contrast allows one to determine if these groups are dissimilar , which is required for the compound to be considered active . general syntheses of compounds similar to those of the present invention are set forth in pct application pct / us86 / 02116 , filed 7 october 1986 , which is incorporated herein by reference . a bond indicated as &# 34 ;˜&# 34 ; includes both the α and β configurations . compounds that have been found to have a phospholipase a 2 inhibitory or antidiabetic effect as determined by at least one of the above assays are indicated in the examples and preparations which follow by the notation &# 34 ; pla2 &# 34 ; and / or &# 34 ; diabetes &# 34 ; respectively . a 250 ml , 3 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 10 g of 17β - hydroxy - 5α - estran - 3 - one dissolved in 150 ml of dimethylformamide . the solution was treated with 3 . 7 g of imidazole and the solution was cooled to 0 ° c . the solution was then treated with 6 . 5 g of t - butyldimethylsilylchloride and allowed to stir at room temperature for 48 hours . the reaction mixture was diluted with water and extracted twice with hexane / ethyl acetate ( 9 : 1 ). the combined organic layers were washed with brine , dried over anhydrous magnesium sulfate and concentrated in vacuo . the crude product ( 14 . 6 g ) was flash chromatographed on 90 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 91 : 9 ) hexane / ethyl acetate with ( no column volume eluted ) 30 ml fractions collected . based on their tlc homogeneity , fractions 4 - 13 were combined affording 14 g of the title compound , m . p . 103 °- 104 ° c . a 2000 ml , 3 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 14 g of the title compound from preparation 1 dissolved in 850 ml of methanol / methylene chloride ( 15 : 2 ). the solution was treated with 7 g of sodium borohydride in small portions . the reaction mixture was allowed to stir for 15 minutes . the reaction mixture was quenched with 2m nahso 4 , diluted with water and extracted with ethyl acetate . the organic layer was washed with water , washed with brine , dried over anhydrous magnesium sulfate , and concentrated in vacuo . the crude product ( 15 . 3 g ) was chromatographed on 1000 g of 230 . 400 mesh silica gel . the column was packed and eluted with ( 98 : 2 ) methylene chloride / acetone . an initial fraction of 1200 ml was collected followed by 17 ml fractions . based on their tlc homogeneity , fractions 280 - 430 were combined affording 10 . 9 g ( 77 % of theory ) of the title compound ( βisomer ). m . p . 135 °- 138 ° c . a 250 ml , 3 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 8 . 0 g of 3β alcohol steroid from preparation 2 dissolved in 150 ml of benzene . the solution was treated with 2 . 2 ml of freshly distilled acrylonitrile followed by 0 . 37 ml of triton b and the reaction mixture was stirred at room temperature for 72 hours . the reaction mixture was washed with dilute hcl , washed with water , dried over anhydrous magnesium sulfate and concentrated in vacuo . the crude product ( 10 . 9 g ) was chromatographed on 1070 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 83 : 17 ) hexane / ethyl acetate . an initial fraction of 1500 ml was collected followed by 18 ml fractions . based on their tlc homogeneity , fractions 81 - 110 were combined affording 7 . 0 g of the title compound , nmr ( cdcl 3 , tms ): δ3 . 8 - 3 . 4 ( m ), 3 . 4 - 3 . 0 ( br ). 2 . 7 - 2 . 45 ( t ), 2 . 2 - 0 . 4 ( m ), 0 . 9 ( s ) and 0 . 75 ppm ( s ). a 500 ml , 3 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 4 . 4 g ( 9 . 9 mmol ) of 3β - cyano ether from preparation 3 dissolved in 500 ml of methylene chloride and 200 ml of methanol . the solution was treatad with 32 ml ( 102 mmol ) of a 3 . 2m hcl in methanol solution . the reaction mixture was allowed to stir at room temperature for 30 min . the reaction mixture was diluted with water and extracted with ethyl acetate . the organic layer was washed again with water , washed with brine , dried over anhydrous magnesium sulfate and concentrated in vacuo . the crude product ( 3 . 73 g ) was flash chromatographed on 90 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 98 : 2 ) methylene chloride / acetone ( no column volume eluted ) with 25 ml fractions collected . based on their tlc homogeneity , fractions 8 - 25 were combined affording 3 . 1 g of the title compound . nmr ( cdcl 3 , tms ): δ 3 . 75 - 3 . 4 ( m ), 3 . 4 - 3 . 0 ( br , 2 . 6 - 2 . 3 ( t ), 2 . 15 - 0 . 75 ( m ) and 0 . 75 ppm ( s ). a 50 ml , 3 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 1 . 0 g of 3β - 17 - hydroxy steroid from preparation 4 dissolved in 20 ml of acetone and the solution was cooled to 0 ° c . the solution was treated with 1 . 0 ml of jones reagent reaction and the mixture was stirred for 15 minutes . the reaction mixture was quenched with 5 ml of 2 - propanol . the reaction mixture was diluted with water and extracted with ethyl acetate . the organic layer was washed with water , washed with brine , dried over anhydrous magnesium sulfate , and concentrated in vacuo , to give 1 . 1 g of the title compound . nmr ( cdcl 3 , tms ): δ 3 . 8 - 3 . 55 ( t , 2h ), 3 . 5 - 3 . 1 ( br , 1h ), 2 . 8 - 2 . 45 ( t , 2h ), 2 . 45 - 0 95 ( m ) and 0 . 9 ppm ( s , 3h ). a 50 ml , 2 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 0 . 36 ml of 1 , 3 - diaminopropane dissolved in 10 ml of methanol . the ph of the solution was adjusted to 6 . 0 with glacdial acetic acid . the solution was then treated with 282 mg of 3β - steroid from preparation 5 and followed by 75 mg of sodium cyanoborohydride . the reaction mixture was refluxed for 18 hours . the reaction mixture was basified with concentrated ammonium hydroxide and then concentrated in vacuo . the crude product ( 3 . 29 g ) was chromatographed on 100 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 92 . 3 : 7 . 0 : 0 . 7 ) chloroform / methanol / ammonia . an initial fraction of 150 ml was collected , followed by 6 ml fractions . based on their tlc homogeneity , fractions 45 - 180 were combined affording 250 mg of the title compound . nmr ( cdcl 3 , tms ) δ 3 . 75 - 3 . 55 ( t , 2h ), 3 . 4 - 3 . 0 ( br , 1h ), 2 . 8 - 0 . 75 ( m ) and 0 . 65 ppm ( s , 3h ). a 250 ml , 3 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 105 mg of lithium aluminum hydride slurried in 30 ml of diethyl ether . the solution was treated with 230 mg of 3β - ether - 17 - hydroxy steroid from preparation 4 dissolved in 50 ml of diethyl ether and the reaction mixture was refluxed for 2 hours . the reaction mixture was quenched with 0 . 21 ml of water followed by 0 . 17 ml of a 10 % sodium hydroxide solution and the reaction mixture was allowed to stir at room temperature for 18 hours . the reaction mixture was filtered and the solids washed several times with hot chloroform . the filtrate was concentrated in vacuo . the crude product ( 350 mg ) was flash chromatographed on 90 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 92 . 3 : 7 . 0 : 0 . 7 ) chloroform / methanol / ammonia . an initial 5 - 15 ml fractions were collected followed by 30 ml fractions . based on their tlc homogeneity , fractions 8 - 14 were combined affording 175 mg of the title compound nmr ( cdcl 3 , tms ): δ 3 . 56 - 3 . 51 ( t , 2h ), 3 . 25 - 3 . 1 ( br , 1h ), 2 . 8 - 2 . 77 ( t , 2h ), 2 . 05 - 0 . 92 ( m and 0 . 74 ppm ( s , 3h ). a 25 ml , 2 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 170 ml of 3β - aminoether - 17 - hydroxy steroid from preparation 7 dissolved in 5 ml of acetone and then cooled to 0 ° c . the solution was treated with 27 μl ( 0 . 51 mmol ) of concentrated sulfuric acid followed by 0 . 31 ml of jones reagent and was allowed to stir for 1 hour . the reaction mixture was quenched with 2 ml of 2 - propanol followed by 0 . 6 g of sodium citrate dihydrate and a small piece of amalgamated zinc ( org . syn . coll ., vol . iv , p . 696 ( 1963 ). the reaction mixture was allowed to stir for 30 minutes at room temperature . the reaction mixture was basified with 3 mkoh ( ph 10 ) and the aqueous layer saturated with sodium chloride . the aqueous layer was extracted 5 times with methylene chloride , 2 times with chloroform and once with diethyl ether . the aqueous layer was then stirred vigorously with methylene chloride . the combined organic extracts were dried over anhydrous magnesium sulfate and concentrated in vacuo . the crude product ( 85 mg ) was chromatographed on 8 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 92 . 3 : 7 . 0 . : 0 . 7 ) chloroform / methanol / ammonia . an initial fraction of 5 ml was collected followed by 0 . 8 ml fractions . based on their tlc homogeneity , fractions 23 - 40 were combined affording 45 mg of the title compound . infrared : λ max ( chloroform solution ) 2950 , 2850 and 1740 cm - 1 ( pla2 ) a 10 ml , 2 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 44 mg of 3β - aminoether - 17 - keto - steroid from preparation 8 dissolved in 1 ml of dioxane . the solution was treated with 1 . 3 ml of a 1 . 0 m monosodium phosphorous acid solution followed by 98 μl of 37 % formaldehyde . the reaction mixture was then heated at 60 ° c . for 2 hours . the reaction mixture was diluted with methylene chloride and water . the aqueous layer was basified with 3m koh to a ph of 11 . the organic layer was separated and the aqueous layer was re - extracted 2 times with methylene chloride . the combined organic layers were washed with brine , dried over anhydrous magnesium sulfate and concentrated in vacuo . the crude product ( 36 mg ) was not chromatographed . based on tlc , crude product was determined to be relatively pure title compound . nmr ( cdcl 3 , tms ): δ 3 . 6 - 3 . 4 ( m ), 3 . 3 - 2 . 8 ( m ), 2 . 7 ( s ), 2 . 6 - 0 . 4 ( m ) and 0 . 8 ppm ( s ). a solution of 7 . 96 g of 2 - thiophsnemethylamine in 50 ml of meoh and 150 ml of thf was acidified with 6 ml ( 6 . 29 g ) of glacial acetic acid . then 10 g of estrone methyl ether was added . the mixture was heated until a solution was obtained and then stirred at ambient temperature for 1 hour . sodium cyanoborohydride ( 2 . 18 g ) was added . the resulting solution was stirred for 5 hours . an additional 2 . 18 g of sodium cyanoborohydride was added . the stirring was continued for 17 hours . the solvent was evaporated . the residue was treated with 200 ml of h 2 o and basified with a 50 % naoh solution . the mixture was extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left 17 . 54 g of oil . the oil was chromatographed on a 1100 g column of silica gel . the column was eluted with 7 . 5 % meoh - ch 2 cl 2 and 200 ml fractions were collected . the fractions were assayed by silica gel tlc ( 1 × 4 &# 34 ;) ( 5 % meoh - ch 2 cl 2 ). fractions 14 - 22 were combined and crystallized from ch 2 cl 2 - hexane affording 9 . 0 g of the title compound as clusters of pale yellow needles . m . p . 85 . 5 - 5 . 87 ° . ( diabetes ) a solution of 4 g of 3 -( trifluoromethyl ) benzylamine in 25 ml of meoh and 75 ml of thf was acidified with 3 ml ( 3 . 15 g ) of acetic acid . then 5 g of 3 - allyloxyestrone was added . after a solution was obtained , 1 . 2 g of sodium cyanoborohydride was added . the resulting solution was stirred for 3 hours . an additional 1 . 2 g of nacnbh 3 was added . the stirring was continued for 66 bours . the solvent was evaporated . the residue was treated with 200 al of h 2 o basified with 50 % naoh solution , and extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left a pale yellow oil . the oil was chromatographed on a 700 g column of silica gel . the column was eluted with 10 % acetone ch 2 cl 2 and 200 ml fractions were collected . the fractions were assayed by silica gel tlc ( 1 × 4 &# 34 ;)( 10 % acetone . ch 2 cl 2 ). fractions 9 - 18 were combined giving 6 . 83 g of a pale yellow oil . a solution of the 6 . 83 g of oil in 100 ml of acetone was added to a solution of 1 . 68 g ( 14 . 47 mmoles ) of fumaric acid in 30 ml of etoh . the solution was concentrated and then hexane was added . cooling gave 7 . 56 of the title compound as s white solid , m . p . 171 °- 174 °. ( diabetes ) a solution of 2 . 5 g of 3 -( trifluoromethyl ) benzylamine in 25 ml of meoh and 75 ml of thf was acidified with 3 ml ( 3 . 15 g ) of acetic acid . then 2 . 37 g of 3 -( 3 - hydroxypropoxy )- estra - 1 , 3 , 5 ( 10 ) trien - 17 - one was added . after a solution was obtained , 1 . 2 g of sodium cyanoborohydride was added . the resulting solution was stirred for 4 hours . an additional 1 . 2 g of nacnbh 3 was added . the stirring was continued for 18 . 5 hours . the solvent was evaporated . the residue was treated with 200 ml of h 2 o basified with 50 % naoh solution , and extracted in the ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left a pale yellow oil . the oil was chromatographed on a 400 g column of silica gel . the column was eluted with 25 % acetone - ch 2 cl 2 and 100 ml fractions were collected . the fractions were assayed by silica gel tlc ( 1 × 4 &# 34 ;)( 25 % acetone - ch 2 cl 2 ). fractions 16 - 23 were combined giving 2 . 94 g of a pale yellow oil . a solution of the 2 . 94 g ( 6 . 03 mmoles ) of oil in 75 ml of acetone was added to a solution of 0 . 7 g ( 6 . 03 mmoles ) of fumaric acid in 20 ml of etoh . the solution obtained was concentrated and the hexane was added . cooling gave 2 . 54 g ( 70 %) of the title compound as a white solid , m . p . 117 °- 121 °. ( diabetes ) a solution of 3 . 32 g of 3 -( trifluoromethyl ) benzylamine in 25 ml of meoh and 75 ml of thf was acidified with 3 ml ( 3 . 15 g ) of acetic acid . then 2 . 83 g of 7α - methylestrone methyl ether was added . after a solution was obtained , 1 . 2 g of sodium cyanoborohydride was added . the resulting solution was stirred for 6 hours . an additional 1 . 2 g of nacnbh 3 was added . the stirring was continued for 17 hours . the solvent was evaporated . the residue was treated with 200 ml of h 2 o , basified with 50 % naoh solution , and extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left a pink oil . the oil was chromatographed on a 400 g column of silica gel . the column was eluted with 10 % acetone - ch 2 cl 2 and 100 ml fractions were collected . the fractions were assayed by silica gel tlc ( 1 × 4 &# 34 ;)( 10 % acetone - ch 2 cl 2 ). fractions 10 - 19 were combined giving 3 . 93 g of pale yellow oil . a solution of the 3 . 93 g of oil in 75 ml of acetone was added to a solution of 0 . 5 g ( 4 . 31 mmoles ) of fumaric acid in 15 ml of etoh . the mixture was reduced in volume to 40 ml . then 100 ml of hexane was added . cooling gave 2 . 79 g of the title compound as a white solid . m . p . 189 °- 190 °. ( diabetes ) a solution of 2 . 47 g of 3 -( trifluoromethyl ) benzylamine in 25 ml of meoh and 75 ml of thf was acidified with 3 ml ( 3 . 15 g ) of acetic acid . then 2 . 12 g ( 7 . 06 mmoles ) of 11β - hydroxyestrone methyl ether was added . after a solution was obtained , 1 . 2 g of sodium cyanoborohydride was added . the resulting solution was stirred for 6 hours . an additional 1 . 2 g of nacnbh 3 was added . the stirring was continued for 18 hours . the solvent was evaporated . the residue was treated with 200 ml of h 2 o , basified with 50 % naoh solution , and extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left an oil . the oil was chromatographed on a 400 g column of silica gel . the column was eluted with 10 % meoh - ch 2 cl 2 and 100 ml fractions were collected . the fractions were assayed by silica gel tlc ( 1 × 4 &# 34 ;)( 10 % meoh - ch 2 cl 2 ). fraction 11 was crystallized from ch 2 cl 2 - hexane giving 0 . 39 g of white needles . the 0 . 39 g was combined with fraction 12 and crystallized from ch 2 cl 2 - hexane giving 1 . 06 g of the title compound as white needles . m . p . 122 °- 123 °. ( diabetes ) to a stirred solution of 3 . 45 g ( 7 . 52 mmoles ) of crude 17β -[ 2 -( 4 - aminosulfonylphenyl ) ethyl ) amino ]- 5α - androstane in 175 ml of thf and 125 ml of acetonitrile was added 3 ml ( 40 mmoles ) of 37 % formaldehyde solution and 1 g ( 15 . 91 mmoles ) of sodium cyanoborohydride followed by 1 ml ( 1 . 05 g , 17 . 47 mmoles ) of acetic acid . the mixture was stirred for 24 hours . the solvent was evaporated . the residue was treated with 200 ml of h 2 o , basified with 50 % naoh solution , and extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left 0 . 66 g of oil . the combined aqueous phases , which were milky , were acidified with acetic acid . the mixture was extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left 3 . 3 g of solid . the 3 . 3 g appeared to be the acetate salt . a mixture of the 3 . 3 g . 100 ml of ch 2 cl 2 , and 150 ml of 10 % nahco 3 solution was stirred for 17 hours . the layers were separated . the ch 2 cl 2 layer was dried over mgso 4 . evaporation of the solvent left 2 . 5 g of foamy solid . the aqueous layer was extracted with 100 ml of ch 2 cl 2 . the extract was dried over mgso 4 . evaporation of the solvent left 0 . 05 g of oil which was discarded . the 0 . 66 g and the 2 . 58 g were combined , dissolved in 50 ml of 1 : 1 acetone . ch 2 cl 2 , and applied to a 400 g column of silica gel ( packed in 1 : 1 acetone . ch 2 cl 2 ). the column was eluted with 1 : 1 acetone - ch 2 cl 2 and 100 ml fractions were collected . fraction 13 was crystallized from ch 2 cl 2 skelly b giving 0 . 39 g of solid . the 0 . 39 g was recrystallized from acetone skelly b giving 0 . 16 g of solid . the 0 . 16 g was combined with fractions 14 - 22 and crystallized from ch 2 cl 2 - hexane ( cooled at room temperature overnight ) giving a white solid . the solid was dried in a vacuum oven at 55 ° for 21 hours giving 1 . 46 g of the title compound , m . p . 168 °- 172 ° ( diabetes ) a mixture of 2 . 54 g ( 8 . 51 mmoles ) of 2 - methyl - 3 - methoxyestra - 1 , 3 , 5 ( 10 )- trien - 17 - one , 3 . 41 g ( 17 . 03 mmoles ) of 4 . ( 2 . aminoethyl ) benzenesulfonamide ( interchem . corp ), and 180 ml of meoh was heated until a solution was obtained . then 0 . 6 g ( 9 . 55 mmoles ) of sodium cyanoborohydride was added . the resulting solution was stirred for 3 hours . then 1 ml ( 1 . 05 g , 17 . 47 mmoles ) of acetic acid was added . the mixture was stirred and refluxed for 44 hours . the solvent was evaporated . the residue was treated with a solution of 5 g of nahco 3 in 200 ml of h 2 o and extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left 4 . 03 g of solid . the solid was dissolved in 50 ml of 10 % meoh - ch 2 cl 2 and applied to a 400 g column of silica gel ( packed in 10 % meoh - ch 2 cl 2 ). the column was eluted with 10 % meoh - ch 2 cl 2 and 100 ml fractions were collected . the fractions contained some solid , m . p . ca 230 , which was insoluble in ch 2 cl 2 and h 2 o but was soluble in meoh . the solid was saved . fractions 15 - 23 were combined in ch 2 cl 2 and filtered to remove insoluble material . the filtrate was concentrated and hexane was added . cooling gave 2 . 80 g of white solid . the 2 . 80 g was recrystallized from acetone - hexane giving a white solid . the solid was dried in a vacuum oven at 54 ° for 17 hours giving 2 . 60 g of the title compound , m . p . 170 - 173 . ( diabetes ) a mixture of 4 . 32 g ( 15 . 86 mmoles ) of 5α - androst - 2 - en - 17 - one , 4 . 76 g ( 23 . 17 mmoles ) of 4 . ( 2 . aminoethyl ) benzenesulfonamide ( interch em corp . ), and 180 ml of meoh was heated until a solution was obtained . then 1 g ( 15 . 91 mmoles ) of sodium cyanoborohydride was added followed by 1 . 5 ml ( 1 . 57 g , 26 . 2 mmoles ) of acetic acid . the resulting solution was stirred and refluxed for 42 hours . the solvent was evaporated . the residue was treated with a solution of 5 g of nahco 3 in 200 ml of h 2 o and extracted with ch 2 cl 2 ( 3 × 100 ml ). the combined extracts were washed with 50 ml of brine and dried over mgso 4 . evaporation of the solvent left 6 . 67 of solid . the solid was dissolved in 50 ml of 10 % meoh - ch 2 cl 2 and applied to a 400 g column of silica gel ( packed in 10 % meoh - ch 2 cl 2 ). the column was eluted with 10 % meoh - ch 2 cl 2 and 100 ml fractions were collected . fractions 15 - 25 were combined and dissolved in meoh . triethylamine ( 5 ml ) was added followed by h 2 o . the mixture was cooled in an ice bath . the somewhat gelatinous solid which separated was collected by filtration , washed with h 2 o , and dried in a vacuum oven at 55 ° for 16 hours giving 6 . 1 g . the 6 . 1 g was crystallized from acetone - hexane giving a white solid . the solid was crushed and then dried in a vacuum oven at 54 ° for 65 hours giving 4 . 97 of the title compound , m . p . 158 °- 159 . 5 °. ( diabetes ) a 500 ml parr flask was charged with 195 mg of 3β - ether - 17 - aminopropyl steroid n -[( 3β , 5α )- 3 -(( 3 - propanenitrile ) oxy ) estran - 17 - yl ]- 1 , 3 - propanediamine dissolved in 25 ml of a 2 . 5m nh 3 in ethanol solution . the solution was treated with 100 mg of 5 % rhodium on alumina and the reaction mixture was placed on the parr hydrogenation apparatus maintained at 50 psi for 4 hrs . the reaction mixture was filtered through celite and the solids were washed with 100 ml of ethanol . the filtrated was concentrated in vacuo . the crude product ( 176 mg ) was chromatographed on 60 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 90 0 : 9 . 1 : 0 . 9 ) chloroform / methanol / ammonia . an initial fraction of 150 ml was collected followed by 6 ml fractions . based on their tlc homogeneity . fractions 160 - 290 were combined giving 117 mg of the title compound . nmr ( cdcl 3 , tms ): δ 3 . 6 - 3 . 4 ( t ), 3 . 35 . 2 9 ( br ), 2 . 85 - 2 . 2 ( m ). 2 . 2 - 0 . 75 ( m ) and 0 . 65 ppm ( s ). a 10 ml , 2 - necked flask , equipped with a magnetic stirrer , was flame dried and then cooled in a nitrogen atmosphere . the flask was charged with 41 μl ( 0 . 5 mmol ) of 1 , 3 - diaminopropane dissolved in 1 . 5 ml of methanol . the ph of the solution was adjusted to 6 . 0 with glacial acetic acid . the solution was then treated with 35 ml of 3β - dimethyloaminoether - 17 - keto - steroid from preparation 9 followed by 9 mg of sodium cyanoborohydride and the reaction mixture was refluxed for 5 hours . the reaction mixture was concentrated in vacuo . the crude product ( 135 mg ) was chromatographed on 8 g of 230 - 400 mesh silica gel . the column was packed and eluted with ( 85 . 7 :- 12 . 9 : 1 . 4 ) chloroform / methanol / ammonia . an initial fraction of 5 ml was collected , followed by 0 . 9 ml fractions . based on their tlc homogeneity , fractions 35 - 54 were combined affording 14 mg of the title compound . nmr ( cdcl 3 , tms ): δ 3 . 6 - 3 . 45 ( t , 2h ), 3 . 3 - 3 . 1 ( br , 1h ), 2 . 8 - 2 . 65 ( m ), 2 . 6 - 2 . 25 ( m ), 2 . 4 - 2 . 3 ( m ), 2 . 2 ( s ), 2 . 10 . 8 ( m ) and 0 . 7 ppm ( s , 3h ). 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