Patent Application: US-201213602239-A

Abstract:
the invention relates to the fields of the chemical and pharmaceutical industry and medicine and concerns a novel compound which can be used as an antiulcer agent . the compound is 9 -- 2 - p - ethoxyphenylethyl - 4 , 5 , 6 , 7 , 8 , 9 , 10 , 11 - octahydropyrido pyrazindione - 1 , 4 of formula , or pharmaceutically acceptable complex derivatives thereof . the compound can be used as an antiulcer agent .

Description:
the parent substance for synthesis is a chromene derivative ( ii ) which is converted by chloroacetyl chlorine to the corresponding chloroacetyl derivative ( iii ). the action on the latter of β ( p - ethoxyphenyl ) ethylamine have caused the opening of pyran cycle and formation of piperazinedione ring . subsequent oxidation of hydroquinone fragment in the intermediate iv have led to the formation of compound i . ( synthesis of chromene ii is described by us in the paper : b . m . lubchanskaya , l . m . alexeeva , s . a . savina , a . s . shashkov , v . g . granik , academy of sciences izv ., chem .., 2002 , n10 , p . 1736 - 1743 ). 7 . 7 ml ( 95 mmol ) of chloroacetyl chloride are added , stirring continuously , into a suspension of 4 . 23 g ( 19 mmol ) chromene in 35 ml of toluene , boiled for 1 hour , cooled down , filtered 5 . 47 g iii ( yield 98 %), m . p . 162 ° c . ( etoh ). 1 h nmr ( dms - d 6 , δ ) 2 . 01 ( q , 2h ), 2 . 89 ( t , 2h ), 3 . 71 ( br . s , 2h ), 4 . 38 ( s , 2h ), 7 . 03 ( m , 2h ), 7 . 26 ( d , 1h ), 9 . 60 ( s , 1h ). 12 mmol β -( p - ethoxyphenyl ) ethylamine is added to the solution of 0 . 88 g ( 3 mmol ) iii in 6 ml of dimethylformamide , stirred for 1 hour under 20 ° c ., diluted by water , filtered 1 g iv , yield 93 %, m . p . 169 - 171 ° c . ( mecn ). a solution of 0 . 14 sodium bicarbonate in 2 . 5 ml of water and oxidizing mixture of 0 . 6 g k 3 [ fe ( cn ) 6 ], 0 . 14 g nahco 3 and 0 . 18 g k 2 co 3 in 6 ml water are added into a suspension of 1 mmol iv in 10 ml chloroform . the mixture is stirred for 1 hour under 20 ° c ., the organic layer is separated and evaporated , the residue is grinded with ether , yielding 85 % i , m . p . 137 - 140 ° c . found : c 68 . 36 , h 6 . 12 , n 6 . 57 . calcd for c 24 h 24 n 2 o 4 : c 68 . 56 , h 5 . 75 , n 6 . 66 . 1 h nmr ( dms - d 6 , δ ) 1 . 32 , 4 . 25 ( t and q , 3h and 2h , ch 3 ch 2 , j = 7 . 5 hz ), 1 . 86 ( quint , 2h , 7 - ch 2 , j = 7 . 5 hz ), 2 . 34 ( br . s , 2h , 8 - ch 2 ), 2 . 68 3 . 40 ( both t , 2h , 2 - ch 2 ch 2 , j = 7 . 2 hz ), 4 . 04 ( s , 2h , 3 - ch 2 ), 6 . 68 ( d , 1h , j = 2 . 8 hz ), 6 . 78 ( quint , 1h , 4 ′- h ), 6 . 89 ( quint , 1h , 5 ′- h , j = 8 . 4 hz ), 6 . 80 - 7 . 10 ( aa ′ bb ′, 4h , c 6 h 4 ). compounds containing , for example , cyclodextrins may be used as pharmaceutically acceptable complex compounds . new compounds possess antisecretory and gastroprotective activity . 1 . 1 . gastric mucosa damage in mice , induced by the administration of 0 . 6 n hydrochloric acid and indomethacin [ 13 ]. male mice ( body mass 23 - 24 g ) were deprived of food for 24 hours while allowing water . the studied compound was administered in 25 , 50 and 100 mg / kg doses by a tube into the stomach 1 , 3 and 6 hours before the administration of 0 . 6 n hydrochloric acid ( 5 ml / kg doses ) and indomethacine ( 20 mg / kg doses ), after an hour the animals were sacrificed , their stomachs were extracted , the number of ulcers was studied . famotidine in 25 and 50 mg / kg doses and omeprazole in 50 mg / kg dose were chosen as drugs of comparison . 1 . 2 . model of gastric mucosa damage , induced by the administration of absolute ethanol [ 14 ]. male rats ( body mass 20 - 23 g ) were deprived of food 24 hours before the experiment and of water 18 hours before the experiment . the studied compound was administered in 25 , 50 and 100 mg / kg doses by a tube into the stomach 1 , 3 and 6 hours before the administration of absolute ethanol ( 0 , 3 ml ), after that the animals were sacrificed , their stomachs were extracted and the length of ulcerations was measured in millimeters . the experiments also used control animals that received only 0 . 3 ml of saline . famotidine in 25 and 50 mg / kg doses and omeprazole in 50 mg / kg dose were chosen as drugs of comparison . 1 . 3 . model of gastric mucosa damage , induced by the administration of the compound 48 / 80 [ 15 ]. the studied compound was administered in 25 and 100 mg / kg by stomach tube 1 and 6 hours before the intraperitoneal administration of compound 48 / 80 , the damage to gastric mucous membranes was observed 3 hours afterwards by sacrificing animals , extracting their stomachs and measuring the length of ulcers in millimeters . famotidine in 25 and 50 mg / kg doses and omeprazole in 50 mg / kg dose were chosen as drugs of comparison . 1 . 4 . model of chronic stomach ulcer , induced by the administration of 0 . 025 ml 20 % acetic acid to rats [ 16 ]. chronic gastric ulcer was induced in rats by administering 0 . 025 ml of 20 % acetic acid under the stomach serous membrane into the border between the antrum and fundus . 24 hours after the operation the studied compound in 50 mg / kg dose and drugs of comparison ( omeprazole - 50 mg / kg and famotidine — 50 mg / kg ) started to be administered by a tube into the stomach once a day during 21 days . every 7 days 6 animals from control and test groups were sacrificed and the surface of stomach ulceration was measured in mm 2 . 2 . 1 . studying the influence of the compound on basal secretion in rats [ 17 ]. the study was conducted using male rats weighting 180 - 200 g . the animals were deprived of food for 24 hours , water access was not limited . using ether anesthesia their abdomens were opened and a ligature put on the pylorus . 4 hours after the operation stomach contents were studied : secretion volume per 100 g of animal mass , gastric acid ph , free hydrochloric acid and general acidity of gastric acid . the studied compound was administered in 100 mg / kg dose as water suspension , with the addition of tween - 80 , 60 minutes before the ligature of pylorus . control animals were given distilled water with the addition of tween - 80 . omeprazole in 50 mg / kg dose and famotidine in 25 and 50 mg / kg doses were used as drugs of comparison . the study was conducted using male rats weighting 180 - 200 g . the animals were deprived of food for 24 hours , water access was not limited . before putting a ligature on the pylorus , one group of animals was administered histamine ( 2 . 5 mg / kg ) subcutaneously , second group was administered pentagastrin ( 10 mg / kg ) subcutaneously , third group was administered 1 - aminopyrene 1 mg / kg . the studied compound was administered in 100 mg / kg dose as water suspension , with the addition of tween - 80 , 1 and 3 hours before the ligature of pylorus . control animals were given distilled water with the addition of tween - 80 . omeprazole in 50 mg / kg dose and famotidine in 25 and 50 mg / kg doses were used as the drugs of comparison . 4 hours after the operation stomach contents were studied : secretion volume per 100 g of animal mass , gastric acid ph , free hydrochloric acid and general acidity of gastric acid . the studied compounds were administered to male mice ( body weight 22 - 24 g ) by a tube into the stomach 60 minutes before the administration of activated charcoal ( 0 . 3 ml of 10 % suspension into the stomach ). after 2 hours the animals were sacrificed by co2 and the length of small intestine filled with charcoal was measured in cm . control group animals were administered 0 . 3 ml of water , cerucal in 25 mg / kg dose was chosen as the drug of comparison . studying the toxicity ( ld50 ) of studied compounds following single administration [ 19 ] 1 . it was shown that piperazindione derivatives exhibit gastroprotective action towards ulcers induced by absolute ethanol , voltaren , compound 48 / 80 , and also exhibits antiulcer action by enhancing ulcer healing in the model of chronic gastric ulcer induced by acetic acid . they are more effective than famotidine and are as potent as omeprazole . their time of action is equal to omeprazole and famotidine , no prolonged action was noted . 2 . studies of antisecretory action have shown that omeprazole inhibits basal secretion by 25 % one hour after administration and by 40 % after three hours ; famotidine : by 56 % after 1 hour and by 80 % after 3 hours . unlike known antiulcer drugs ( proton pump inhibitors , h 2 receptor antagonists ) the new compound does not inhibit basal gastric acid secretion level . thus , famotidine severely inhibits basal secretion , omeprasol — moderately . the new compound does not inhibit basal secretion level at all . 3 . studies of stimulated gastric acid secretion have shown that omeprazole inhibits histamine - induced secretion by 70 % one hour after administration , and by 80 % after 3 hours ; pentagastrin - induced and 1 - aminopyrene - induced secretion — by about 90 %. famotidine inhibits histamine - induced secretion by 90 % one hour after administration , and by 95 % after 3 hours ; pentagastrin - induced secretion by 40 % and 1 - aminopyrene - induced secretion — by 36 % after 1 hour and by 40 % after 3 hours . the new compound inhibits histamine - induced secretion by 40 % one hour after administration , and by 57 % after 3 hours ; pentagastrin - induced secretion by about 54 % and 1 - aminopyrene - induced secretion by 50 %. thus , omeprazole inhibits gastric acid secretion induced by all three substances ( histamine , pentagastrin , 1 - aminopyrene ), famotidine inhibits only histamine - induced secretion , and weakly inhibits secretion caused by two other substances , the studied compound , like omeprazole , inhibits all three types of stimulated secretion , but less potently . 4 . peristalsis study has shown that omeprazole slows down intestinal peristalsis by 35 %, famotidine does not influence peristalsis , the new compound does not influence peristalsis as well . thus , only omeprazole inhibits intestinal peristalsis . this effect may be detrimental to its antiulcer activity , especially during long - term use , because inhibition of peristalsis causes stomach contents to exhibit additional deleterious effect on ulcers . general toxicity studies have shown that the new compound and its salts are low - toxic , like omeprazole . comparison of ld50 has shown that new compounds are less toxic than famotidine and omeprazole . study of compound i action on enzyme activity of h , k - atpase of rabbit gastric mucosa , its reversibility and potency in the presence of k + ions microsome preparation , enriched with h +, k +- atpase , was produced from the rabbit stomach mucosa using a method proposed by farley and faller . gastric mucosa was produced from a decapitated rabbit . all operations were carried out in ice . the stomach was cut at the greatest curve line , washed and scrubbed . obtained mucosa was frozen and kept at − 80 ° c . temperature . to obtain a microsome preparation enriched with h , k - atpase , gastric mucosa was defrozen and homogenized in extraction medium ( 0 . 25 mol sucrose , 5 mmol pipes , 20 mmol tris ( tham ), ph 7 . 4 ) by a polytron - class homogenizer ( max rotations 30 / sec ). mass ratio of tissue to extraction medium — 1 : 20 . the homogenate was then centrifuged for 10 min at 10000 g . the sediment was discarded and supernatant was them centrifuged for 1 hour at 100000 g . the produced sediment was re - suspended in extraction medium ( using the minimal volume necessary for re - suspension ). the suspension was layered above ficoll 400 , prepared in the extraction medium : 12 % ficoll solution , 4 % ficoll solution . it was further centrifuged for 180 min at 100000 g . after centrifuging , a white layer was seen on the borderline of 4 % and 12 % ficoll that was a microsome fraction , enriched with h , k - atpase . the fraction was collected , divided into aliquots and kept at − 80 ° c . the effect was measured by the growth of inorganic phosphate that is produced during atp hydrolysis . the concentration was measured using ratbun and betlah method . the incubation medium contained : 30 mmol imidazole , ph 7 . 4 , 3 mmol mgcl2 , 130 mmol nacl , 20 mmol kcl , 3 mmol atp , 2 mmol ouabain , 0 . 1 mmol egta , 2 . 25 μmol valinomycin , 5 mmol nan3 , 5 μmol of cccp ( carbonyl cyanide m - chlorophenyl hydrazone ). incubation medium and defrozen and homogenized microsome fraction were added into incubation sample . the sample was incubated for 20 min at 37 ° c ., then the reaction was stopped by a cold stop - solution ( 3 mol sodium acetate , 3 . 7 % formaldehyde , ph 4 . 3 ). to evaluate the content of phosphate , produced during enzymatic hydrolysis of atp , 2 % ammonium molybdate solution and tin chloride solution ( 3 mg / ml ) was into the sample . after the appearance of blue color , after 15 minutes , the samples were studied by colorimetry using wavelength of 650 nm the h , k - atpase activity was determined as activity inhibited by 0 . 1 mmol sch - 28080 . this inhibitor inhibited about 30 % of h , k - atpase activity . the concentration of the studied compound was varied in range of 10 − 8 to 10 − 4 mol . compound i exhibits inhibitory action on h , k - atpase activity of rabbit gastric mucosa with i50 = 10 − 6 m . its effect on h , k - atpase is reversible , it develops instantly and stops after the dilution of enzyme - inhibitor complex . possible competition between compound i and potassium ions was studied . to achieve this , the relation of h , k - atpase and kcl concentration was studied , and then the inhibitory action of compound i on this enzyme in saturated ( 20 μmol ) and unsaturated ( 2 μmol ) concentrations of kcl was studied . it was shown that compound i does not compete with k + ions , on the contrary , its inhibitory effect of rabbit mucosa h , k - atpase became more potent in the presence of k + ions : i 50 was 10 − 5 mol at 2 μmol kcl and 10 − 6 at 20 μmol kcl . 1 . compound i reversibly inhibits the hydrolytic activity of h , k - atpase , i 50 is 20 μmol kcl . 2 . inhibitory effect of compound i becomes more potent in high k + concentrations . i 50 goes down from 10 − 5 mol at 2 μmol kcl to 10 − 6 mol at 20 μmol kcl . this property of compound i is an advantage in living organism use . results show that compound i is an effective reversible h , k - atpase inhibitor . but , unlike the known reversible inhibitor sch - 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